The Dinerentiai Migration oCBlood andLymph Lymphoeytos

Timothy James Seabrook

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy

Graduate Department of Laboratory Medicine and Pathobiology

University of Toronto, 2000

O Copyright by Timothy James Seabrook 2000

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Abstmct

Ih sheep, a pool of lymphocytes resides in the bIood that does not recirculate as

efficiencly as lymph lymphocytes. However, there is limited information on the

differential migration of blood and lymph lymphocytes into wnlymphoid tissues or

during infiammation. Therefore, experïments in this thesis were designed to investigate

the migration of blood and Lymph Lymphocytes after splenectorny, antigen challenge to a

single lymph node, into n o d cerebral spinal fluid (CSR, and aftet TNFa induced

inflammation-

Following neoaatal splenectomy, no merence in the number or phenotype of

lymphocytes was observed, However, splenectorny did sesuit in an increased migration

of lymph lymphocytes into lymph nodes and a trend towards a longer residence time in

blood for the blood pool of lymphocytes. Whüe splenectomy has little effect on the

development or distribution of lymphocyte subsets in blood and lyrnph, evidence was

obtahed that it affects the rate of Lymphocyte rec~cdation.

Lymph node shutdown was induced by PPD in a BCG immunised sheep. Both

lymph and blood pool CD4 lymphocytes increased in efferent lymph dwing Lymph node

shutdo wn. IFN-y and IL-6 IeveIs were uicreased in efferent lymph plasma during l p p h

node shutdown and may have a role in both the recruitment and retention of lymphocytes-

Lymph lymphocytes preferenwy migrate h to CSF and &erent lymph under

normal conditions, implying that iymph lymphocytes have a greater role in immune

surveillance as compared to blood lymphocytes. After the intracerebrovenaicular

infusion of TNF-a, blood lymphocytes are found in a greater percentage as compared to

l p p h lymphocytes. In both CSF and the perivascuiar spaces of the brain, an

innammatory innltrate composed of CD4, CD8 and y6 lymphocytes was observed.

Together these data demonstrate that under iaflammatory conditions and

fobwing splenectomy there are dinerences in the migration of blood and lymph

lymphocytes. The redts in this thesis provide some basic data into the mi-gration of the

blood and lymph pools of lymphocytes and provides the background for further

investigations into the migration and functional differences between blood and lymph

lymphocytes.

Dedication

This thesis is dedicated to my grandparents

Floyd and Gladys Seabrook

Ferman and Alice Hanes

Firstly, 1 must thank Dr* Jack Hay for hisencouragement and guidance over the

Iast 5 years- He is a wonderfiil mentor, in both science and mee 1 feeI privileged to have

had the opportunîty to work with him,

Dr. Miles Johnston has helped with many discussions on various papers,

experiments and letters of reference. 1 must also thank the rest of my commîttee for their

pidance over the years including Dr. M- Cybulsky, Dr. L. Becker and Dr. R. Midha.

Dr. Hay has fiiIed the lab with many incredible people over the iast 5 years and 1

have benefited fkom working with them- Without the assistance of WiU Andrade, Binh

Au, Jodi Dickstein, Bill Ristevski and Me1 Boulton, this thesis wodd not have k e n

completed. Catherine Munroe deserves special thanks for her help with figures,

references and editing. Diana Armstrong gave mmy hours of her time helping me with

swgeries.

In addition, 1 must thank Dr. A Young and Mk. Lisbeth Dudler at the Basel

Institute for Immunology. Because of hem, 1 had the chance to experïence not o d y a

wonderfiil research institute but also Switzerland.

Cheryl Smith was responsible for teaching me flow cytometry. But even more

important she shared coffee with me every morning at the MSB.

Frank, Ranier, Angela, Wendy and aii of the other people in the animal facitity

took excellent care of my animals.

Lastly, 1 must thank my family for their support over the years. Most of ail

without Darren this thesis would have k e n impossile. His understanding and patience

with my long nights in the lab and talkùig about science over dinner is incredible. Thank

you.

Table of contents

INTRODUCTION ......................................................................................................... 1 1-1 HISTOWCALBACKGROUND .............................................................................. 1 1.2 SIZE OFTHE LYMPHOCYTE POOL ............................................................................. 2

1.3 LYMPHOCYTE SUBSET DISTRIBU~ON OF LYMPHOCYTES IN SWEEP. ...................... ......3

1.4 MOLECULES WOLVED IN LYMPHOCYTE MIGRATION: AN OVERViEW ....................... 3

1.5 LYMPHOCYTE MIGRATION INTO LYMPH NODES ........................................................ 5

1.6 LYMPH~~YTEMIGRATIoN INTO THE SP LEEN .......................................................... 10 1 -7 LYMPHOCVTE MIGRAIION INTO THE CNS AND CSF ...............................-............ - 1 1

1.8 LYMPHOCYTE SUBPOPZ~LATIONS: PHENOTYPE vs . MIGRATION PATERBIS ............... 12

.................... 1.9 DEFIMTION OF BLOOD LYMPHOCYTES AND LYMPH LYMPHOCYTES .,... 14 1-10 RATIONALEFORTHEEXPERIMENTS~THISTHESIS .............................................. 17 1- 1 1 EXPERIMENTAL SYSTEM ................................................................................ 1 8

1.12 MAIN HYPOTEESIS ............................................................................................. 20

1.13 S T R U ~ OFTHETHESIS AND STATEMENT ..................................................... 2 1

2.1 ANIMALS ............................................................................................................. 22

2.2 S ~ G E R Y ............................................................................................................. 23

2.3 CELL LASELLJNG ................................................................................................. 24

2.3. 1 11 1-h .................................................. ,.. ................................................ -24

.......................................................................................................... 2.3.2 Sl.Cr... 25

2.3.3 FlTC labelling of blood ................................................................................. 25 2.3.4 FITC labelling of lymph lymphocytes ......... ... .......................................... 26 2-35 CFSE labeiiing of lymphocytes ...................................................................... 26 2.3.6 DiI-DS Iabelling of efferent lymphocytes ...................................................... 26

2.4 ANTIBODIES USED IN THIS TfIESIS .......................................................................... 28 ............................................................. 2.5 IMMUNOPHENOTYPING OF LYMPHOCYTES 28

2.6 CrTOMETRY ............................................................................................... 29

.................................................................................... 2-7 ~ O E ~ T O C H E M I S T R Y 29

2.8 REAGENTS ........................................................................................................... -30

2.8.1 Hank's balanced salt solution (EIBSS) ........................................................... 30

.............................. 2.8 -2 Phosphate buffered saiïne (PBS) without divalent cations 31

2.8.3 Dulbecco's phosphate buffered saline ............................................................ 31

2.8.4 PBSflween buffer .................................................................................. 3 1

2.8.5 ParaformaIdehyde solution ............................................................................ 31

2.8.6 Erythrocyte lysis solution ............................................................................. .32

2.8.7 Acid Citrate Dextrose ..................................................................................... 32

INVESTIGATIONS INTO THE MIGRATIONPA'ITERN OF BL AND LL INTO

AFFERENT LYMPH AND AETER SPLENECTOMY weoeeew.moe.~eew~woeeeweeo.eweowewoeeeee35

3.1 A~STRACT ........................................................................................................... 35

3.2 INTRODUCTION ..................................................................................................... 36

.................................................................................. 3 -3 MATE RIAL^ AND M~THODS 37

3.3.1 Animals and surgery ..................................................................................... 38

3.3 -2 Lymphocyte labeiiing ......................... ..... ............................................... 38

3.3.3 Sarnphg of blood and lymph for trackùig fluorescent labeUed celis .............. 39

3.3.4 Statistical analysis .............................................................*........................... 39

3.4 RESULTS .............................................................................................................. 40 3.4.1 Repetitive sampling of blood and lymph ........................................................ 40

3.4.2 Aflerent lymph .............................................................................................. 40

3.4.3 Splenectomized sheep migration .................................................................... 41

.................. 3.4.4 Immunophenotyping of lymphocytes in splenectomized sheep .... 4 L

3.5 DISCUSSION ......................................................................................................... 54

4.3.3 Ceilcollection andphenotyping ............................................................... ......60

.............................................................................. 4.3 -4 hunohistochemistry 6 1

4.4.4 Cytokine EUS& .......................................................................................... 61 . . 4.4.5 Stawtics ......................................................................................................... 62

4-5 RESULT~ .............................................................................................................. 62

4.5.1 Lymphocyte Migration ............................................................~..................... 62

4.5.2 Cytokine Levels ............................................................................................. 63

4.5.3 'Immunohistochemistry .................................................................................. 63

........................................................................................................ . 4.6 D~scvss~o~ 70

LYMPHOCYTE IN CEREBROSPINAL FLUID ARE PART OF THE

............................................................ RECIRCULATING LYMPHOCYTE POOL 74

5.3.1 Animais and surgery ...................................................................................... 76

5-3-2 Determining normal CSF ceii counts ............................................................. 76

................................................................................................. 5.3.3 Ceil l abehg 77

5.3.4 Sample collection of blood, lymph, CSF and lymph nodes .............................. 77

5.3.4 Intracerebroventricular infusions of 1 11-In labeiled lymphocytes ................... 78

..................... 5.3.5 Intracerebroventricuiar infusions of J3TC labeiled lymphocyte -78

5.3.6 Flow cytometry .............................................................. ... 5.4 m m .. .............................................-...................................... ................. 79

5.4.1 Cells in normal CSF of sheep ......................................................................... 79

5.4.2 Repeated infusion of labelled lymphocytes .................................................... 79

5.4.3 Singe bolus of labelled efferent Lymphocytes ................................ ............... 80

5.4.4 Kuietics of FITC labelleci ce& in CSF and efferent lymp h. ............................ 84

5.4.5 Lymphocyte egress fkomCSF ........................................................................ 84

......................................... 5.4.6 LL and BL migration into CSF and afferent Lymph 85

......................................................................................................... 5.5 Drscussro~ 93

TNF-a INJECTIONS INM CSF. BUT NOT THE BRAIN PARENCHYMA.

RESULTS IN LEUKOCYTE RECRUiTMENT ....................................................... 98

........................................................................................................... 6.1 A~STRACT 98

6.2 INTRODUCTION ..................................................................................................... 98

6-3 MATERIALS AND MEIHODS ................................................................................. 100

6.3.1 Animais and surgery ................................................................................... 100

............................................................................................... 6.3.2 Cell labeiling 100

6.3.3 TNF-a injections ..................................................................................... 1 0 0

6.3.4 CSF collection, differential and phenotyping ............................................... 101 . . 6.3.5 Bram tissue collection .................................................................................. 101

6.3.6 Immunohistochemistry ............................................................................. 101

6-4 RESULTS ............................................................................................................ 102

6.4.1 Leukocyte number, differential and phewtype after the intracerebroventrïcular

infusion of TNF-a ............................................................................................... 102

6.4.2 CSF leukocyte numbers after parenchyma injections of TNF-a ................... 102

6.4.3 BL and LL migration înto CSF after TNT-a injection .................................. 103

................................................................................ 6.4.4 Immunohistochemistry 103

. 6.4.5 The effect of intracerebrovenvicular injection of TNF-a on cervical Lymph .. 1 19

6.5 DISCUSSION ....................................................................................................... 119

GENERAL DISCUSSION ........................................................................................ 125

7-2 ~ T I G A ~ ~ N S INTO T H E M I W O N PA- OFBL AND LL NCO AEFERENT

LYMPH AND AFER SPLENECTOMY ............................................................................. 126

7-2-1 Repetitive samphg ..................................................................................... 126

............................................................................................ 7.2.2 Merent lymph 127

7.2.3 Splenectomized sheep ............................................................................... 128

7-3 THE MIGRATION OF BL AND LL TEiRoUGH ANTIGEN STIMULATED LYMPH NODES . 130

..................................................... 7 -4 LYMPHOCYTES IN CSF ARE PART OF THE RLP 132

7.4 TNF-a IJSDUCED CSF LEUKOCYTOSIS ................................................................. 133

7-5 FUllJRE EXPE~UMENTS TO D- THE EMS'ENCE OF A RAPIDLY RECIRCULATING

............................................................................................. EQOL OF LYMPHOCYTES 135

7.6 SUMMARY- ......................................................................................................... 139

REFERENCE LIST ............................................................... w........................~......... 140

LET OF FIGURES

F~GURE 1 NUMBER OF LYMPH~~YTES IN VARIOUS TISSUES AND POOLS IN S~EEP ........... --.-.6

~ G U R E 2 P H E N o ~ E OF S- L Y M P H ~ INLYMPH, BLOOD AND LYMPH NODES ..... -8

FIGURE 3 ~RESENTATIVE EXPERTMENT OFREPETLTIVE SAMPLING OVER A ~ ~ H R P E R I O D ~ ~

4 RATIOS OF BL AND LL LABELLED CELLS IN = E L Y M P H AND BUX)D OVER

A 27 HR P m O D ..................................................-..............o...... 9,.-.-..------.----..---.--.----45

FIGURE 5 SHORT-TERM MIGRATiON OFLABELLED LYMPHOCYTES IN SPLENECTOMIZED

SHEEP ....................................o................................................................-.......... --..--48

FIGURE 6 TISSUE LOCAUSATION OF RADIOLABELED LL AND BL LYMPHOCYTES. ,. .- .,. .---- 50

FIGURE 7 PPD INDUCED LYMPH NODE SHUTDOWN -,*.,,-----.,--..-,. ~~~..~~~----.---.--..-------.--------65

FIGURE 8 PHENOTYPE OF LYMPHOCYTES IN LYMPH DURING LYMPH NODE

SHUTDOWN ..................................................................................... ---.-------..-.--.----.--66

FIGURE 9 C Y T O ~ LEVELS DURTNG LWH NODE SHUTDOWN ...................................... 69 RGURE 10 PERCENTAGE OF LABELT.ED c m IN nssms AFIER CONSTANT REINFUSION -8 1

FIGURE 1 1 PERCENTAGE OF LABELLED LYMPHoCXES 2 4 ~ ~ AFïER A SINGLE INFUSION OF

c m .....-........................................................................................................ -.--.--Ai6

FIGURE 12 APPEARANCE OF FIII'C LABET.T.ED LYMPHOCYTES IN CSF AND LYMPH ,,.,.,-.,--88

FIGURE 13 ~~RACEREBROVENTRICULAR NEcïEJ3 1 1 1-1. LAB- CELLS MIGRATE TO

LYMPH NODES KNOWN TO DRAIN CSF ....................................o.............................--... 89

FIGURE 14 CSF C E U ~ ~CREGSES m THE INJECTION OF TNF-a .................. 105

FIGURE 15 BOTH BL AND LL INCREASE AFïER THE INTRACEREBROVENTRTCULAR

INJECTION OFTNF-a ......................... -, ................................................................ 108

FIGURE 16 CNS PARENCHYMA AFER AN INTRACEREBROENTRICULAR INJECTION OF

................................................................................................................. TNF-a.. 1 1 1

FIGURE 17 CHOROID PLEXUS A F E R AN INTRACEREBROVENTRICULAR INJECIION OF

--a .............................................................................................................. 113

FIGURE 18 CNS PARENCHYMA AETERTHE INTRACEREBRAL INJECITON OF m - a 1 15

~ G U R E 19 ~ C E R E B R O V E N T R I C U L A R INJECIION OF --a HAS NO EFFECT ON

CERVICAL LYMPH FLOW OR CELLULARITY ......................................... . . . . . . . 1 1 7

- fi-

- - -

List of Tables

List of ab breviations

Ag

ANOVA

APC

BCG

BBB

Bn:

BL

BLC

CD

CFSE

CM-DiI

C N S

CSF

ELISA

DiI-DS

DMSO

EAE

FITC

GlyCAM

HBSS

HEV

antigen

analysis of variance

diop hycocyanin

Bacillus Calmette- gué^

blood brain barrier

Basel Institute for lmmunology

blood lymphocyte

B-lymphocyte chemoattractant

cluster of differentiation antigen

carboxy-îiuorescein diacetate succinimidyl ester

CeliTracker carboc yanine fluorescent dye

central nervous system

cerebral spinal fluid

enzyme iinked immunoassay

lipophilic, carbocyanine fluorescent dye

dimet hylsulfoxide

experimental autoimune encep halo myelitis

fluorescein isothiocyanate

glycosylation-dependent ceU adhesion molecule

Hank's balanced salt solution

high endothelia1 venule

ICAM-i

IFN-Y

IL

LFA- 1

LL

mAb

MAdCAM

MHC

MS

OCT

PBS

PKH

PPD

rhTNF-a

RLP

SLC

TNF-a

VCAM- 1

interceiIular adhesion molecule

interferon gamma

interleukin

leukocyte hinction antigen- 1

lymph Lymphocytes

monoclonai antiïdy

mucosal addressin ceU adhesio n mo lecule

major histocompatibility complex

multiple sclerosis

optimal cooling temperature

phosphate bUnered saline

lipophiiïc, carbocyanine fluorescent dye

purified protein derivative

recombinant human tumour necrosis factor-a

recirculating Lymphocyte pool

secondary lymphoid tissue chemokine

tumour necrosis factor alpha

vascular cell adhesion moiecule-1

Chapter 1 Introduction

Lymphocytes are unique in their ability to continuously recircuiate fkom blood,

into tissues and retum to blood via the Ipphatic system (Young et aI., 1993a). This

process is important for the dissemination of immunological memory and immune

suweiiiance. Lymphocytes recirculate through Lymph wdes thereby allowing the

presentation of antigen (Ag) to a large number of Lymphocytes. Once a lymphocyte

recognises its Ag and receives the relevant costimulatory signals, an Ag specsc h.mune

response begins. Effector celis exit the lymph node via the efferent lymphatic and

migrate to sites of inflammation (Picker and Siegelman, 1993). Once the immune

response has been resolved, a subset of Lymphocytes become memory cells which

concinuously recirculate through the body ailowing for a rapid secondary immune

response (Sprent, 1994; Butcher and Picker, 1996).

1.1 Historical Background

Gowans, in a series of seminal experiments elucidated the fùndamentals of

lymphocyte recirculation. He and colieagues demonstrated that cannulating the thoracic

duct in rats and diverting the Lymph resulted in a decrease in the number of lymphocytes

in both blood and lyrnph (Gowaas and Knight, 1964). When lymphocytes were Iabelled

with radioisotopes, and intravenously injected, labelled celis could be found in l p p h

(Gowans, 1959). Taken together these experiments demonstrated that the lymphatic

system was responsible for the retum of lymphocytes to blood. Morris and severai

collaborators developed surgical protocols that alio wed the cannulation of Lymphatics and

the continuous collection of lymph in sheep (LasceIles & Mom-s 1961; Smith et al.

1970). This development permïtted the quantincation of lymphocyte t r a c through an

isolated lymph node under normal and inflammatocy conditions. This led to severaï

important discoveries including the preferentiai recirculation of memory lymphocytes

through tissues (Mackay et al., 1990) and the quantincation of lymphocytes produced and

exported de novo in a lymph node during an immune response (Hall and Morris7 196%).

1.2 Site of the lymphocyte pool

There is approximately 1 kg of lymphoid tissue in an adult sheep, containing

approximately 10" lymphocytes (Chin et ai., 1985). %y canndating the thoracic duct

lymphatic, it was demonstrated that approximately 10% of the total lymphocyte

population, or approximately 10'' cek , compose the recirculating lymphocyte pool

(RLP) (Schnappauf and Schnappauf. 1968) (Figure 1). The RLP is comprised of

Lymphocytes that conùnuously recircuiate through the body, while the remainder may be

fixed in tissues or do not recircuiate under normal conditions.

One defi t ion of "pool" is a common supply of a commodity for sharing amongst

a group e.g. a pool of money for a department. Pool in this thesis refers to a population

of lymphocytes that preferentially localise, migrate or are retained in a specific tissue

cornpartment. For example, ali Lymphocytes that recirculate are part of the RLP but this

pool is further subdivided based on preferential tissue migration includuig a lymph pool

of lymphocytes (discussed in detail in section 1.8).

Blood contains approximately 1% of the total lymphocyte pool at any one time

(Figure 1) (Schnappauf and Schnappauf U, 1968). When arterial blood passes through a

lymph node approxmiately one quarter of aü Lymphocytes are extracted by the poa

capiUary vendes B a y and Hobbs, 1977). These Lymphocytes migrate into the lymph

node and exit via the eEerent Lymphatic. The Lymph node also receives Lymphocytes

fiom the tissues via afferent lymphatics, which contain approximatefy 1 x 106 ceWmL

Efferent lymph bas ten times the amount of Lymphocyteslml. Previous studies by Hail

and Morris have demonstrated that fewer then 4% of Lymphocytes present in efferent

lymph are produced de novo in the lymph node (HaU and Morris, 1965a). Therefore,

approximately 90% of lymphocytes present in efferent Iymph migrate directly from the

blood-

1.3 Lymphocyte subset distribution of lymphocytes in sheep

In sheep, there are differences not only in the number of Lymphocytes but also the

phenotype of lymphocytes in the blood, efferent and afferent lymph (Figure 2) (Mackay

et al., 1988). Approximately 10% of afferent lymph in sheep is composed of dendritic

ceiis and other ceiIs of monocytic origin (Haig et aL, 1999). Efferent lymph is virtually

1 0 % lymphocytes, the majority king small resting ceils. In sheep, lymphocytes rnake

up approximately 5060% of the Ieukocytes present in blood (BluntJ975 and my own

observations). The topic of lymphocyte phenotypes is fbrther discussed in section 1.8.

1.4 Molecules involved in lymphocyte migration: an ovewiew

Lymphocytes migrate into tissues fkom the vascular system ushg various

adhesion molecules includïng selectins, integruis and their ligands in a CO-ordinated

series of events (Butcher et ai., 1999; SpMger, 1994). Broadly speaking this can be

divided into discrete steps ùicluding, primary adhesion ( r o b g and tethering). integrin

activation, firm adhesion and tran.smïgration.

Primary adhesion is a reversible process in which lymphocytes transientiy adhere

to endothelial ceiis, allowing lymphocytes to be acted upon by chemokines and other

activating substances. Selectins (Gaiiaùn et ai., 1983) and a4 containing integrins

(Berlin et al., 1995) are responsible for primary adhesion. Selectins are membrane bound

Iectins present on the surface of both endothem ceUs and lymphocytes and bind to

mucin like proteins (Vestweber and Blanks, 1999). L selectin is expressed on the

micovilli of lymphocytes (Stein et ai., 1999) and binds to endothelial cells via several

Ligands, including CD34 and glycosylation-dependent celi adhesion molecule (GlyCAM)

(Vestweber and Blanks, 1999). E and P-selectin are expressed on endothelid celis and

have an important role in the recruitment of Lymphocytes into inflmed skin (Austmp et

ai., 1997).

Integrin activation is a critical step in lymphocyte migration that involves

chemokines bound to glycosaminoglycans on endothelial cells (Koopmann et al., 1999)

and other activating substances including platetet activating factor (Kim and Broxmeyer,

1999). Chemokines are small chernotactic proteins produced by a number of c e k

(Campbell et ai., 1996). They interact with G protein coupled receptors, causing the

activation of integrins on the Lymphocyte surface (Taub and Mqhey, 1997). Ligation of

L-selectin after binding its ligand also activates integrins (Hwang et al., 1996). htegrins

are composed of noncovaiently associated a and chahs and are critical in the firm

adhesio n and transmigration of lymphocytes. Upon activation integrins undergo a

conformational change, thereby increasing their amty for Iigands which are members

of the immunoglobuiin superfamily (Springer, 1995). Lenkocyte fimctionai antigen- 1

(a&) which binds to intracellular adhesion mlecuie-1. 2 and 3, is important in

lymphocyte m*gration into ineammatory sites and Iymph nodes (Butcher et ai.. 1999)

Transmigration involves the lymphocytes migrating between endothelial ceiis and

adhering to structural proteins. This step involves CD31 and some cytoskelatai

rearrangements on the part of the endothelial cells (Zocchi et al, 1996; Allport et al-,

1997). The lymphocyte now migrates into the tissue underlying the endothelid ceiL

1.5 Lymphocyte migration into lymph nodes

Migration of lymphocytes into lymph nodes is dso highly regulated-

Lymphocytes continuously recirculate through Lymp h nodes using various adhesion

molecules but there are several ciifferences fiom the general pattern as discussed above.

Firstly, in most species Lymphocytes migrate into lymph nodes using high endothelial

venules (HEV) which are h e d by cuboidal endothelial ceiis that are n o d l y present

only in iymph nodes (Girard and Springer, 1995). Recent studies using molecular

biology techniques have demonstrated that these endothelial cells preferentiaIly express

unique genes (Izawa et al., 1999; Girard et al., L999). L-selectin is a critical adhesion

molecule for the normal migration of lymphocytes into lymph nodes as demonstrated by

the impaked traffic in L-selectin kwckout mice (Asbones et aL, 1994). GlyCAM and

CD34 are expressed on the HEV of peripheral lymph nodes and are ligands for L-selech

(Vestweber and BI&, 1999).

Figure 1 Number of lyrnphocfles in various tissues and poois in sheep

The data to construct this figure carne fiom several different sources as outluied in the

discussion. Data representing bone rnarrow is not available. Although some data has

k e n descriid in young Iambs for thymic output (Cahili, personal communication) it has

k e n excluded here because older animals were used.

? nurnber of lymphocytes Non HEV containing tissue

Blood 1 x 10 El 0 total lymphocytes in blood

Recirculating lymphocyte pool approx 1 x lOEll

Total lymphocytes in 30kg sheep approx. 1 x 10E12

1 ? Number of lymphocytes

Spleen

5 x 10E10 in total

afferent ly mphatics

2 x 1 0E9 per hour migrate into the lyrnph node via HEV

1

70 g in total

efferent lymphatics - Thoracic duct

2 X 10E9 per hour

Figure 2 Phenotype of sheep lymphocytes in lymph, blwd and lymph c d e s

The phenotypic data in this diagram was obtained nom this thesis and firom the literature

referenced in the text above-

BIood CD4 CDS GD B CD2 1 sIgM L seIectin CDLlb

Afferent Lymph

CD4 50% CD8 13% GD 30% B 7%

15% 12% 10% 57% 24% 38% ubcataneous L p p h Node 31% 40% CD4 39%

CD8 19% GD 5% B 44% CD21 36%

CD4 47% CD8 19%

CDL1b 1% L selectin 89%

Pemissis toxin inhibits the migration of lymphocytes into Lymph nodes, thereby

implicating a G protein coupled receptor in the migration of Lymphocytes into lymph

nodes (B argatze and Butcher, 1993). Recently a chemokine, secondary lymp hoid tissue

chemokine (SLC), was shown to be expressed in E V and T ceil areas within the lymph

node (Warnock et aL, 2000): This chemokuie, with others, may CO-ordhate the migrafion

of Lymphocytes into and their position within the lymph w d e

To date little is known about the mechanismi and adhesion molecules involved in

the migration of lymphocytes within Lymph nodes. AdditionaUy, the signals that are

responsible for the exit of Lymphocytes are relatively unknown.

1.6 Lymphocyte migration into the spleen

The spleen receives fiom and rehuns to the blood more lymphocytes than any

other organ in the body (Pabst and Westermann, 1991). Lymphocytes migrate directly

out of the blood into the white puip without crossing HEV. Unlike lymph nodes

lymphocytes migrate directly back into the blood with only a srnail proportion entering

splenic lymphatics (Pellas and Weiss. 1990). Therefore, lymphocyte migration into the

spleen and their retum to blood is not considered recirculation as defmed in this thesis-

SLC, B-Lymphocyte cheumattractant (BLC) and other chemokùies act in concert

to regulate the migration of lymphocytes nom blood into the white pulp of the spleen

(Liadhut et al, 1999). Chemokines also CO-ordinate the migration of lymphocytes into

their respective niches, for example BLC attracts B celis into follicles (GUM et al., 1998).

1.7 Lymphocyte migration into the CNS and CSF

There is Iittle normai migration of nonactivated Iymphocytes h o the centrai

nervous system (CM) across the intact blood brain barrier (BBB) (Hickey, 1991;

Wekerle et al., 1986). T'bis has been examined using a variety o f experlmentai protocols

including immunohistochernistry (Hickey et ai., 199 1) and radiolabeled (Raine et aL,

1990a) ceus, both of which demonstrate few lymphocytes within the CNS parenchyma.

However, there are a smaU number of Iymphocytes present in normal cerebrai spinal fluid

(CSFI-

During most infiammatory conditions there is a ciramatic increase in lymphocytes

and monocytes within the C N S (Raine, 1994; Anthony et al., 1998). In both multiple

sclerosis and its animal model experimental autoimmune encephdomyeiîtis (EAE),

lymphocytes and monocytes accumulate in perïvascuiar spaces of the brain. The initial

migration of Lymphocytes across the idamed BBB uulises the same adhesion molecules

as seen in the periphery with the exception of selectins (Cannella and Raine, 1995;

Engelhardt et ai., 1994). To date only one putative BBB specifc endothelial adhesion

molecule, recognised by the 4A2 antibody, has been demonstrated (Male et al., 1995).

Leukocyte entry into the CSF has not been as extensively studied as entry into the

CNS. Indeed, the exact pathway by which lymphocytes enter the CSF has not been W y

eluc idated. Under inflammatory conditio os, it appears that Lymphocytes c m directly

enter the subarachnoid space by traverskg venules in this area Another potential route is

across the BBB in the CNS parenchyma into the perivascular sheath of fluid that

surrounds the vessels and eventually joins the CSF (Weller, 1998). Another pathway

may be via the c~cumventricular orgaos and the choroid plexus, which lack a typical

BBB (Engelhardt and Risau, 1995)-

The adhesion moIecdes required for Lymphocyte migration into CSF may wt be

the same as those required for CNS migratioa It has been demnstrated that in EAE,

selectuis are not important (Engelhardt et al-, 1997), however in meningitis the blocking

of selectins attenuates the number of cells in CSF (Tang et ai., 1996). Injections of

cytokines into the CSF causes an influx of ceus (Sankkonen et al., 1990; Quagliareiio et

al., 1991; RamiIo et aL, 199û), but when the same cytokines are injected into the CNS

parenchyma few leukocytes are recruited (Scbneil et aL, 1999; Andersson et al., 1992)-

Taken together these data demonstrate that the CSF and the rest of the CNS differ in their

response to infiammation,

1.8 Lymphocyte subpopulations: phenotype vs. migration patterns

Lymphocytes can be divided into subpopulations based on different attributes

inc luding the tissue or organ of extraction, phenotype, funct ion, migration patterns,

expression of adhesion molecules, etc. Immunophenotyping is a common method that

uses the expression of specific surface markers to classify lymphocytes by using

monoclonal anubodies against these marken. This method is used in the cluster of

differentiation (CD) classification of surface markers. Ho wever, heterogeneity ofien

exists amongst Lymphocytes defhed on the basis of a single phenotypic marker.

Combinations of attniutes are often used to subdivide lymphocytes based on multiple

characteristics.

The function of lymphocytes is utiiised to classify cells and is often used in

conjunction with their phenotypic profile, an example is the Thl, Th2 systern This

system is based on the heterogeneity of cytokuie production by T helper c e h and their

effect on the immune response. The CD4 antigen is the e s t defrnlng charactenstic and

then fuaher subdivision is based on functioa For exaniple, Th1 CD4 c e k are

Lymphocytes that express CD4 and influence other immune ceiIs by producing various

cytokines including IFN-y. This cytokine production has a role in skewhg the immune

response to wards a ceU-mediated immune response (Sailusto et al., 1999). Th2 CD4 cells

have a role in inducing a humoral respoose.

Tissue specific is another marner to subdivide lymphocytes and is

based on the observation that Lymphocyte recirculation is not random Lymphocytes

isolated fiom the efferent lymph of lymph nodes draining specific tissues preferentiaiiy

r e m to those lymph nodes. There are populations (or pools) of gut and skin migrating

lymphocytes (Cam et al., L977; C6in and Hay, 1980). W e this type of lymphocyte

migration has been studied more extensively in sheep than in other species (Abemethy

and Hay, 1992), results fkom mice confirmed these hdings. It has been detennuied that

most of the organ specifc migration is due to memory T ceus (Williams and Butcher,

1997)-

Several papers have used the expression of adhesion molecules to d e h e

migration patterns (Mackay et al., 1992a; Abitorabi et al. 1996)- Investigators have

identifïed the a4B7 integrin as being cntical for the ability of Lymphocytes to migrate

through gut associated lymph nodes, whilst L selectin is important for their abiüty to

migrate into nibcutaoeous lymph nodes (Mackay et al., 1996 Abitorabi et al-, 1996)-

Thus, these studies have further defïned tissue fioming using adhesion molecules.

However, few studies bave examined if lymphocytes are 'fiozen' with these adhesion

molecules or if they can change.

There are reports to suggest that in rats the expression of specific adhesion

molecules may not be an absolute predictor for tissue or lymph node homing

(Westermann et ai-, 1994a; Wdter et al., 1995;). The dlscrepancy in findings maybe due

to the different species (rat vs. mice and sheep), the source of ceils (Lymph nodes vs.

lymph), the age of the animel (young vs. aged) and living conditions (pathogen free vs.

normal housing).

Taken together these data demonstrate that there is tissue specific migration of

lymghocytes in both mice and sheep. Much of this migration is the property of memory

T ceils, which display specific adhesion molecuIes, One hypthesis States that rnemory T

celis home to the tissue in which they 6rst encountered their antigen, dowing for a rapid

secondary immune response (Williams and Butcher, 1997). Ho wever others have

questioned this hypothesis (Westemiann and Pabst, 1996). Nevertheless, the

preponderauce of data in mice and sheep demonstrates that there is a population of

lymphocytes that experiences tissue specific homing and that adhesion molecuIes have a

role in this phenornenon.

1.9 Definition of blood lymphocytes and lymph lymphocytes

Previous studies using sheep have demonstrated a population of lymphocytes

present in blood that does not recirculate as efficientiy through lymph nodes as compared

to efferent lymph lymphocytes. This pooriy recirculating blood pl of lymphocytes

(BL) was detected by labelhg blood and lymph lymphocytes with different fluorescent

dyes and then reinfushg them simultaneously (Young et aL, 1997a; Andrade et al,

1998). These experknents demonstrated that iabeiIed Iymph lymphocytes (LL) were

e ~ c h e d in efferent lymph as compared to bIood, whilst Iabelled BL were concentrated in

blood. Based on these data it was concluded that blood contains a pool of lymphocytes

that does not recirculate as competently through lymph nodes as do LL.

Further studies demonstrated that BL are found in the spleen in greater numbers

than LL (Young et al., 1997a). Phenotypicaiiy, this pool is composed mainly, but not

exclusively, of B c e k that are CD21- and L selectin low, with smaller populations of

CD4, CDS and perhaps y6 T ceiis (Young et ai., 1997a). Recently m e r studies have

demonstrated that BL B ceiis are aiso CDS+, CDLlb+ and surface IgM high (Chevallier

et al., 1998; Gupta et al., 1998). To date, no unique cell surface antigen common to BL

has been found, though this is an area of active research in other laboratories.

Nor has a unique function been found for BL Lymphocytes. It has been speculated

that the B ceiis in BL are BL iïke cells and therefore produce low aff.iinity anti'bodies to

some bacteria (Chevallier, et aI., 1998). Others have theorised that they may have a role

in the imrnunity against blood borne infections (Andrade, 1996). Neither of these

hypotheses has been proven and pnor to the studies in this thesis, any functional

difference between BL and LL remained to be determined-

BLood contains not only BL but also LL in transit. When the experiments in this

thesis were designed various methods were considered to isolate or enrich BL to enable

more definitive experiments. One isolation method examined was panning or magnetic

bead separation protocols. It was decided that this could introduce several problems that

are inherent with ex vivo manipulation of ceiis, such as inadvertent activation of

lymphocytes- As weii, it is difîïcuit to isolate suscient lymphocytes necessary for

effective tracking studies in sheep. Additiondy, isolathg the B ceils alone wodd have

ignored the other subsets present in the BL. Since dead or riamaged lymphocytes do not

recirculate (Andrade 1996), it is important that the isolation procedures are not

excessively long or damagkg to the lymphocytes.

Therefore, similar protocols were foUowed as were used in earlier experïments

(Young et ai., 1997a; Andrade et aL, 1998). S e e s of blood and lymph were coiIected,

Iabeiled with different fluorescent dyes, reinfused intravenously and then subsequently

identified using flow cytometry. This allowed for the examination of many of the subsets

of lymphocytes present within the BL. This approach has been successhilly used in rats

to e x m e the effect of IFN-y on the migration of various lymphocyte subsets

(Westermann et ai., 1994b). However, the extensive number of shared properties

between BL and LL often complicates the simple interpretation of the data

Therefore, in this thesis, the dennition of the blood pool of lymphocytes (BL) is:

Those lymphocyresfkom blood rhat when labelled and reinfused intravenously do not

recirculate a s efficiently as a sample of Iymph lymphocytes simultaneously CO-infused-

The definition of the l p p h pool of lymphocytes (LL) is: Those lymphocytes, that

when labelled and reinfused intravenously c m recirculute and be found in Herent

lymph.

These dennitions are based on migration patterns seen after Iabelling ce& fiom

blood and lymph, reinfushg them and t r achg their migration. This is similar to tissue

specific homing patterns previously described for gut and subcutaneous Lymph node

lymphocytes (a; and Hay, 1980; Cahili et ai., 1977).

1.1 0 Rationak for the expewiments in this thesis

In a clinical setting* blood is ofien sampied to determhe the immunologicd health

of an individual. However, it has been shown that this is not always an accurate

reflection of the immune system as a whole (Westermann and Pabst, 1990). In human

immunodeficiency virus-affected patients, the blood shows a decrease in the number of

CD4+ lymphocytes but in lymph nodes, no decrease in this subset is found (Pabst and

Rosenberg, 1998). Blood contaios only 1% of the total lymphocytes in the body*

therefore the loss of blood CD4 cek may be £imctionally insignificant. Nonetheless,

blood WU continue to be sampled in the foreseeable future due to its ease of collection.

The data fiom this thesis may help in the interpretation of blood samples by detahg

some of the differences between lymphocytes found in blood and the rest of the body.

Using sheep it is possible to retrieve sarnples fiom several different tissues

including spleen, blood, lymph nodes, CSF and both afferent and efferent lymph-

DifTerences may exist in the migration of BL and LL into these tissues, however io date

this migration has not been examined In sheep there is a ciifference in the migration of

lymphocytes obtained fkom iyrnph nodes as compared to efferent lymph Lymphocytes

(Reynolds et al., 1982). Indeed Williams and Butcher (1997) argue that the different

migration pattern of Lpph node and efferent lymph lymphocytes may be partially

responsble for the controversy surroundhg tissue specific homing.

As weIi, several investigators, including our laboratory, are interested in labelling

lymphocytes with contrast media and using them in a clinical setting (Sipe et aL, 1999;

Bulte et al, 1992). This would aliow for the non-invasive trackuig of lymphocytes and

may have a roIe in revealing infIammatory lesions such as early plaques in multiple

sclerosis. However, basic migration patterns of blood derived lymphocytes need to be

elucidated to allow this tracking of blood lymphocytes to pmceed.

Specific studies in this thesis examine the effects of splenectomy on BL-

Splenectomy is known to have an effect on Lymphocyte number and function in patients

(Ferrante et al.. 1987; Sieber et al., 1985). nie experïments in this thesis tested if these

abnormalities maybe a result of changes in BL lymphocyte numbea or migration.

The migration of lymphocytes into the CSF may have a role in the immune

surveillance of the C N S under normal and idammatory conditions, such as menhgitis.

Therefore, experiments were performed to determine the migration of both BL and LL

into this important tissue-

The experiments in this thesis were designed to investigate ciifferences in the

m i m o n of blood and lymph lymphocytes under a variety of conditions. These

experiments were designed to explore both the basic biology of BL and how this may

impact on patients in a clinical setting.

1.1 1 Experimental System

Sheep were utilised in all of the experiments reported in this thesis for a number

of reasons. Foremost amongst them is the ability to chronicdy cannulate lymphatics and

couect lymph tkom an anùnal with a relatively intact lymphatic system (Smith et d,

L970; Cahili et aL, 1974; Young et al., 1997b). This ability allows for experiments that

examine the dynamic nature of lymphocyte recirculation. Lymphocytes c m be re-infused

into a sheep and their migration monitored in severaI co~artrnents both sequentially and

simultaneously, which can not be pedonned in smailer animais-

Mice, rats and rabbits have ail been used to investigate lymphocyte migration into

CSF, but because of their size, limited numbers of cells are avaiiable for examination.

Sheep are large enough that snfficient CSF can be collected and examuied for labeiïed

cells. Lastly, there is a large body of literature examuiing lymphocyte migration in sheep

upon which the present experiments are based (Mackay, 1988; Mackay, et al., 1992a;

Young, et al- 1993b: Seabrook et al-, 1999)-

Fluorescent labehg of cells and their detection using flow cytornetry was chosen

for several reasons. Fluorescent labelled Lipophilic dyes including CM-Da, PKH and

DiI-DS, are retained within the cytoplasm of Lymphocytes for weeks (Salvato et aL 1996;

Andrade et al., L996a; Young and Hay, 1995) and do not affect the migration of labelled

cells from blood into lymph (Teare et al., 199 1). Therefore, these labels allow the Iong-

term tracking of lymphocytes in vivo. Fluorescein isothïocyanate (FITC) has been used

for several years to label lymphocytes and does not impair the migration of Lymphocytes

(Butcher et ai., 1980). Unfortunately, this dye is not amenable for experiments longer

than approximately 2 weeks as its intensity decreases and it can no longer be detected. A

method in which whole blood is labelied with FITC (Andrade et al., 1996b) was selected

and used in this thesis as it allows the rapid labelling of large numbers of BL with little

manipulation.

Radioisotopes have been used in the past as whole tissues can be easily assessed

for the number of labelled tek (Issekutz et aL, 1981). However, the isotopes leach fiom

cells Iuniting the duration of experiments (Issekutz et al., 1980). Radioisotope labels are

superior to fluorescent labels for investigatùig lymphocyte migration into several tissues

simultaneousLy at necropsy and those tissue that are difficult to isoiate lymphocytes ftom

such as skin and Peyerrs patches.

Fluorescent dyes have the advantage that immunophenotyping can be carrïed out

on labeiied ceiis in blood and lymph. However, care must be taken in the labelling of

lymphocytes with fluorescent compounds as prolonged incubation will overlabel the cells

and affect their ability to migrate. Additionaii~~ the PKH class of compounds must be

used with diluents that can aggregate Iymphocytes if the incubation is prolonged (Salvato

et al., 1996). To ensure that the i a b e h g protocol does not adversely affect the migration

of lymphocytes, recovery data is obtained. Recovery data is based on the number of cells

infused and the number which subsequently is recovered fkom a specifïc tissue. This is

often expressed as the percentage injected and ailows the comparisoa between different

labels and experiments. Ali labehg procedures in this thesis were previously published

and/or validated to ensure that they had no effect on the migration of labelled

lymphocytes fiom blood to Iymph.

1.1 2 Main Hypothesis

Published reports have demonstrated that, in sheep, a pool of lymphocytes exists

in blood that does not migrate into the lymphatic system as efficiently as the lymph pool

of lymphocytes. We hypothesised that dinerences in this migration may be exaggerated

by experimental manipulation and therefore could Iead to a better description of the

functional significance of these ciifferences, if any, between these two pools. Therefore,

experiments using splenectomy, antigen challenge of a single Lymph node, examhation

of CSF and r e c r u i e n t with TNF-a were perfomied to test this hypothesis.

1.1 3 Structure of the thesis and statement

Chapter 2 descn i s the methods and materials common to several of the

experiments in this thesis. This limits some of the redundancy fi-om subsequent chapters

but some methods are repeated as 1) there were some minor modifications of standard

protocols and 2) some chapters are presented in manuscript f o m Chapter 3 addresses

some of the basic questions of BL migration including migration into Serent lymph and

the effect of splenectomy. Lymph node shutdown and its affect on BL migration is

discussed in Chaper4. Chapters 5 and 6 investigate the migration of BL and LL into

normal and innamed CSF. Finaily a general discussion, including experiments to

examine the existence of a rapidly recirculating lymphocyte pool is included in Chapter

7.

The experiments in this thesis were performed at Sumybrook Health Science

Centre, Medical Sciences Building, University of Toronto and the Basel Institute for

Immunology. Ail experiments in this thesis were perfonned by myself. However,

surgeries conducted at the Basel Institute for Immunology were perfomied by Dr. W.

Hein, Dr. A- Young and Ms. L. Dudler.

Chapter 2 Methods and Materials

Several methods and techniques are common to many of the experiments and are

discussed in this chapter. Methods that are specific for panicular experiments are

included in subsequent chapters.

Outbred femaie sheep of between 30 and 35 kg were used for ai i experiments

performed in this thesis. Exceptions to this were the older sheep used in the

splenectomized experiments and a single maIe sheep in the antigen stimulation

experiments. Sheep were obtained iÏom 3 sources depending on the site at which the

experiments were performed. Suppiiers included Ledo Farms (Oshawa ON), Bowood

Farms (London ON) and Versuchsbetrieb Semweid (Olsberg, S witzerland). AU s heep

had fiee access to hay, pellets and water at aL times except for the 24 hows imrnediately

preceeding surgery.

Experiments conducted at the Division of Comparative Medicine, University of

Toronto, were approved by the Animal Care Committee of the Faculty of Medicine- The

experiments in Chapter 5 were approved by the Animal Care Committee of Sunnybrook

Heaith Science Centre. Al1 were in accordance with the Canadian Council on Animal

Care and the Anùnals for Research Act of Ontario. For those conducted at the Basel

Institute, Switzerland, handihg and treatment of the animais was accordùig to protocols

approved by the regional government authority, the Kantonales Veteriniiramt.

2.2 Surgery

AU surgicd procedures were pedormed uader steriie conditions. Animais were

anaesthetised with sodium pentothd to effect and were intubated with an endotracheal

tube. A surgical plane of anaesthesia was maintained with either halothane or isoflwane

in oxygen with the aid of a respirator. AU surgical techniques have k e n previously

described (Young et al., 1997b)- Briefly, a catheter attached to a 3-way stopcock, was

surgically placed into one of the jugulas vehs for blood sampluig. Lymphatic vessels

canuiated included the prescapular, prefemoral and cervical efferent lymphatics and

hindlimb merent lymphatics. Anatomicaily these are distinct lymph nodes but the

surgical manipulation required is the same for alI lymph nodes. The efferent lymphatic

was exposed with a minimum of trauma, and a section approximately 3 cm in length

stripped of adherent fascia A silk suture was used to Ligate the efferent lymphatic

downstream fiom the proposed incision site, aiiowing the lymphatic to &te. A second

suture was then loosely placed around the vessel upstream and a srnail incision made in

the vessel. A length of polyvinyl tubing of the appropriate size, previously flushed with a

heparin saline solution, was then gently inserted into the lymphatic and secured in place.

The catheter was carefully extenorised and the wound closed. A bottle holder was

sutured to the animals' skin, a bottie containing approximately 300 IU of heparin saline

was attached and the end of the catheter placed in the bottle allowing the collection of

lymp h.

In those experiments that examined CSF, the following surgeries were performed

at least 5 days prior to lymphatic cannulations. In some experiments, access to the CSF

was required, therefore a laminectomy was performed on vertebrae S2 or S3. A midline

incision was made in the overlying skin and the muscle gently dissected away hrom the

spinous process uncü the vertebral arch was exposed Using a high-speed d d I equipped

with a burr bit the laminae was removed and the dura exposed. An incision was made in

the dura and a catheter then inserted. To aUow the infiision of ceiis and cytokines into the

laterai venuic-les two bilateral 0.3 cm b m holes were made approximately 1.5 cm

anterior and posterior to the posterior fontanelle. A guide screw was then inserted into

the burr holes and a 16 or 2 1 gauge iv, catheter was fed through the guide screw-

Animals were given buprenorphrine (0.005 mglkg i.rn) during surgery and as

required thereafter. Experiments were not carried until the following day to d o w the

animai to recover-

2.3 Cell Labelling

I L 1 -Indium oxine (Amersbarn Coq, Baie d'Urfe. Que.) labelling was performed

as previously descriid (Issekutz et ai., 1980). Briefly lymph was coiiected. Lymphocytes

were then harvested by cenuifugation at 400 g for 10 min and washed twice with HBBS

or PBS. After the finai wash, the c e k were resuspended in either buffer at a

concentration of 1 x 10* ceiis/ml. Ten pCi of 11 1-In was then added, the c e k gentiy

mïxed and incubated at room temperature for 10 min. Ten ml of autologous lymph

plasma was added, the celis suspension washed hvice with either PBS or HBSS and

resuspended in an appropriate volume of saline for infusion.

2.3.2 51-Cr s

Lymphocytes were prepared as above except 50 pCi of ~ a ? ' Cr04 (rm, Costa

Mesa, CA) was added aad the ceils incubated for 30 min at 37°C These ce& were

suspended in saline for reinfûsion-

2.3.3 FITC labelling of blood

A saturated solution of FLTC (Sigma, Oakville, ON) was prepared by adding

0.05g to 500 ml of PBS or HBSS and stimng overnight at 4°C. The solution was then

filtered through a 0.2 micron fiIter immediately prior to Iabelling celis-

A whole blood method developed by Andrade et al. (1996) was used. Briefly,

approxllnately 300 ml of blood was withdrawn fkom the jugular catheter. This represents

approximately 15% of the total blood volume and is well tolerated by sheep. The blood

was added to a sterile beaker containing approximately 70 ml of acid citrate

anticoagulant. The blood was then divided into 2 large centrifuge tubes and cenVifuged

at 400g for 15 min. The plasma was carefüily removed, taking care wt to disturb the

bu- coat and the ce& resuspended in either PBS or HBSS. The c e k were split

between 4 tubes and washed twice to ensure the remval of all plasma. The blood was

then resuspended in saturated FITC and incubated at 4OC for 30 min, after which the ceus

were washed twice with either buffer, suspended in saline and reinfused intravenously.

2.3.4 FlTC labelling of lymph lymphocytes

Efferent lymph was collecte& harvested by cenaifugation at 400 g for 10 min,

washed twice with HBSS or PBS, resuspended in either b a e r and the concentration

adjusted to 1 x IO* ceWml. Seventy pl of saturated FITC was added for every log cells

and încubated at 37OC for 15 min. After this the cell suspension were washed twice with

either buffer and resuspended in saiîne for reinjection.

2.3.5 CFSE labelling of lymphocytes

Approxïmately 300 mi of blood was withdrawn via the indwelhg catheter into 60

cc syringes containing a small amount of EDTA in saline. Mononuclear celis were

harvested by centrihgation over Percoii gradients as previously descrïbed (Young et al.,

1997)-

CFSE (Molecular Probes, Eugene, OR) was diluted to 500 pghl in DMSO and

kept at -20 O C untii required. Mononuclear cells were resuspended at a concentration of 5

x 10'/ml in 37OC PBS containing 1 pglml CFSE for 15 min. The ceils were washed

twice with ice cold PBS contairung 1 8 foetal caif senim. The cells were resuspended in

s terile saline and reinfiised intravenously.

2.3.6 DiI-DS labelling of efferent lymphocytes

Efferent lymph was collected and ceiis harvested by cenaifugation at 400g for 10

min. The ceiis were washed twice with rmm temperature PBS and resuspended at a

concentration of 2 x log ceWd in 37°C Isocoves Modified Dulbecos media (Giko Life

Technologies, Burhgton, ON). The tells were then placed in a 37°C water bath for 10

mis DS-DS (Molecular Probes) was used at a concentration of 12 ~lgl10' c e k and was

weighed and diluted in 300 pl of DMSO immediately pnor to use. The DZ-DSDMSO

solution was added to the s a m ~ amount of tissue culture media as the celIs and gently

mixed with the cells to give a final concentration of log ceWmL The ceiis were aliowed

to incubate at 37OC for 30 min after which the cells were washed twice with room

temperature PBS and resuspended in stede saline for rehfbsion,

As this was a new application for this label it was determined by the author that

lymphocyte subsets were not adversely affected by comparing the phenotype of both the

beginning population and labelled ceiis (data not shown)- Using the above procedure

labeiied lymphocytes were obsecved to migrate fiorn blood to lymph, which dead or

overlabefled cells can not do. Previous work in our Iaboratory bas demonstrated that this

family of dyes efficiently labels c e k with w ceiL to cell transfer. Therefore, this method

of labeliing large numbers of lymphocytes is relatively easy and cost effective. A recent

report has used DiI-DS to label T c e k in mice and found that after intravenous infusion

DiI-DS labelled lymphocytes are found in lymph nodes (Dittel et al., 1999).

For al i of the above labelhg procedures aiiquots of ceiis were retained for

viabiüty staining using 0.4% trypan blue, ceU enurneration and labeIiing efficiency.

most of the experiments the c e k were >95% viable. Additionally the c e k were brightly

labelled with the CFSE and FïïC labelhg procedure. The DZ-DS did not label celis as

brightly as FITC and CFSE but usuaiiy greater than 85% of cells were labelled.

2.4 Antibodies used in this thesis

Most a n t i i e s and ceii h e s were generously shared by Dr. A Young of the

Basel Institute for hunology. These antiidies have k e n extensively characteriseci

and the results published (Table 1). For the fist senes of experiments, antiidy

supernatants were provided by Dr. Young. In 1998 we received the cell h e s and began

to culture the hybrïdomas ourselves using standard tissue culture techniques. AU

antibodies are antiovine muruie aatlbodies and used as ceU cuiture supemtants-

Exceptions to this was the antiovine CD25 (VMRD) and antiVCAM (gift fiom T-

Tedder), both of which were ascites.

2.5 lmmunophenotyping of lymphocytes

Blood samples were collected fkom the jugular catheter in a syrïnge containhg a

s m d amount of heparin (approximately 50U). Erythrocytes were lysed with either

distilled water or Tris:N&CI Iysis solution. The Ieukocytes were then pelleted by

centrifugation at 400g and washed twice with either PBS or HBSS. Samples of lymph

were washed twice with either buffer. The ceil count was determined using a ZN mode1

(Coulter Electronics, Hialeah, Fl) and 2 x 1o6 ceiis added per weii of a 96 weli U bottom

tissue culture plate (Becton Dickinson, San Jose. CA). The volume was adjusted to 100

pl with ice cold buffer and 50 fl of primary antiidy added. A 10 min incubation on ice

was foiiowed by centrifugation at 450 g. The supernatant was decanted and the cells

washed twice. Fifty pl of appropriately diluted secondary (see Table 1) antifbody was

added, the volume adjusted to 150 pi and incubated for 10 min on ice. After this fiaai

incubation the cells were washed twice with buffer and resuspended in 1%

paraformaldehyde. If flow cytometcy was not perf'ioffned immediately the plates were

wrapped in tin foil and stored at 4°C.

Celis were examined by fiow cytomeq wïthia 1 day of staining- In some

experiments, the buffer had 1% bovine aibumin added and the cells were preincubated

with goat IgG as a b l o c h g agent. These steps did not make a significant ciifference in

the background staining. In ai i experiments negative celis and ceus incubated with

secondary mAb and/or m u s e IgG were used as controis,

2.6 Flow cytometry

Either a FACScan or FACScahbur (Becton Dickinson) with Celiquest software

was used to perform the flow cytometry analysis. The instruments were checked with

quality convol beads by an operator every day to ensure its proper function.

Additionaily, if the second laser was required, calibrating beads were immediately nui

before the instrument was used. An eiectronic gate was drawn around the lymphocyte

population based on their typical side and forward light scatter properties. Lymphocytes

were then examuied using the relevant detector depending on the expeeen t and the

secondary anti'body used.

2.7 lmmunohistochemistry

Tissue was harvested and as soon as possible placed in a medium size cryomold,

embedded in OCT and fiozen in liquid nitrogen. Blocks were wrapped in aluminium foil

and stored at -70°C. Eight pm sections were cut using a cryostat and placed on

~ i ~ a n i z e d siides. The slides were dried ovedght, fixed in cold acetone for 5 mùi. air

dried and stored at -20°C

Siides were allowed to corne to m m temperature in the slide box and then

endogenous peroxidases were blocked with a solution of PBS, 0.1% hydrogen peroxide

and 1% sodium azide. AU incubations were at room temperature in a humid container.

The slides were rinsed in PBS, biotin and avidin blocked using a commercial blocking kit

(Vector Laboratones, Burluigton, ON) and incubated with 10% foetal calf senun for 30

min. The undiluted primary antibody was added (Table 1) and the slides incubated for 2

hr. PBSî'ïween was used to wash the siides twice, biotin labelIed goat antirnouse

antibody added and incubated for 30 min, The siides were washed twice, the commercial

avidin/biotin complex (Vector Laboratories) added and incubated for 30 min- The slides

were rinsed twice and DAB solution (Vector Laboratories) added for 5 min. Several

rinses of distüied water were used and a Light nuclear counterstain of haemotxylin

applied. The slides were dried overnight and pemianently coverslipped with mounting

media (Perrnount),

2.8 Reagents

2.8.1 HanKs balanced salt solution (HBSS)

For imrnunophenoîyping, sterile 1X Ca and Mg containhg buffer without phenol

red was used (Gibco Canada, Burhgton, ON). For a general wash buffer 1OX

concentrated buffer without divatent cations was used and reconstituted with stede

distilied water.

2.8.2 Phosphate buffered saline (PBS) without divalent cations

This was made using the foUowuig protocol; 2L d&O. 400g NaCl, log KCl,

57-58 N a m 4 and log L(H2 PO4. The LOX-concentrated buffer was prepared in 4 L

batches, autoclaved and stored a m m temperature until use. It was then reconstituted

using distilled water.

2.8.3 Dulbecco's phosphate buffered saline

This was prepared ushg the foliowing protocol;

solution 1 - 8.0g NaCl, O.2g KCI, 1.15g Na2HP04, 0.2g KH2P04 and 800 mi Hfi

solution II- O. lg CaC12 and LOO mi Hfl

solution III- O. lg MgC12 *6 H20 and 100 ml H20

The three solutions were made separately, autoclaved and mixed immediately

pnor to use. This buffer was used for immunophenotyping and to dilute secondary

antibodies-

2.8.4 PBSrrween buffer

10 X PBS was reconstituted with distilied Hfl and 0.05% Tween added-

2.8.5 Parafonnaldehyde solution

100 ml of PBS without divalent cations was preheated to 53-57OC in a fumehood

using a hotplate equipped with a stirrer. One gram of paraformaldehyde was added and

ailowed to stir until dissolved. The solution was then cooled and filtered through a 0.2

micron filter to remove any undissolved particles. The solution was then aliquoted and

stored fiozen until required.

2-8.6 Erythrocyte lysis solution

solution 1 - Tris O- 17M was dissolved in dH20 with continuous s t idg

solution II - O.83g NE&CL was dissolved in 1OOmls dEF20

The solutions were stored at 4°C mtiI needed- When the solution was required

they were mixed at a ratio of 19, (HI) and prewarmed to 37°C. The solution was then

added at a ratio of 4:L blood

2.8.7 Acid Citrate Dextrose This was prepared as followed C&&as07 2Sg, C&Nag 1.5g and -1206 2-08 were

added to LOO ml H20- It was used at a ratio of 1:6 (acid citrate dextrose to blood).

Table 1 Antibodies used in this thesis

Antihdies obtained fiom the Basel Institute for Immunology @II) were either cell

culture supernatants or hybridomas. Severai of the hybridoma hes were grown in

Toronto by TS- Cederlane= Cederlane, Hornby, ON

Antigen Distribution Clone# Supplier Reference

Recognised

PrÏrnarv Anhibodies

CD4

CD8

YS B cell

CD2 1

CD25

CD45 RA

L selectin

SIEN VCAM

Secondarv

Antibodies

FITC-GAM IgG

PE-CAM-Ifi

APC-GAM-I,@

T ce11 subset

T ce11 subset

T cell subset

Ail B cells

Absent from T celk

some B c e k &

monocytes

Subset B cells

Activated T celis

B celldnaive T cells

recirculatïng

lymehocyt=

B cells

endothelial cells

DU2-128 BII

VMRD

p z 0 BII

DU2945 BII

13-30 BII

HAE-2 T- Tedder

Cederlane

Cederlane

Cederlane

(Mackay et a1, 1986)

(Mackay et al., 1986)

(Mackay et al, L99 1)

(Young et al., 1997a)

unpublished

(Young et al., 1997a)

(Mackay et al., 1990)

(Spertini et al, 199 1)

unpublished

(Mackay et al, 1992b)

Chapter 3 Investigations into the migration pattern of BL and LL

into afferent lymph and after splenectomy

3.1 Abstract

Previous data demonstrate a relative preponderance of BL to localise in the spleen

and blood as compared to LL (Young et al. 1997). Sequentki repeitive sampling of

blood and lymph was performed to test the stability of both pools in blood and lymph-

Normal afferent lymph was also examined for labe1Ied BL and LL to determine if either

preferentially migrated through tissues. Experiments were performed to investigate the

effect of splenectomy on the maintenance and migration of BL. The relative proportions

of labeUed BL and LL remained constant in both blood and efferent lymph over the 27 hr

samphg period- LL are found in afferent lyrnph in greater numbers than BL, and one

may postulate that LL have a greater role in immune surveillance of peripheral tissues-

Neonatal splenectomy did not result in a change in the number or phenotype of

lymphocytes in either lyrnph or blood indicating that the spleen is not necessary for the

long-term maintenance of BL. However, its removai resulted in an increase in the

migration of LL into peripheral lymph nodes and a retention of labelled BL in blood as

compared to intact sheep. Together these data provide information requirrd for the

interpretation and design of subsequent experiments in this thesis.

3.2 Introduction

Previous experiments using fiuorescent labels and flow cytometry demonstrated

the existence of the BL pool (Young, et al., 1997a; Andrade, et aL, 1998). This pool does

not migrate into lymph nodes as efficiently as LL, but instead localises in the spleen

(Chevallier et al., 1998). The majority of BL are B cells that do not express either CD21

or L selectin, however CD4 CD8 and perhaps y6 T cek are also present in the BL pool

(Young et al., 1997a). Two recent publications have demonsaated tbat BL B cells are B 1

like ceils and express CDS, CDllb and high levels of surface IgM (Chevallier et ai.,

1998; Gupta, et al.. 1998). Separating these ceils and perfomiing tracking studies

confirmed that BL B cells are excluded fiom the lymphatic system (Gupta, et al., 1998).

Investigations into the existence of BL were begun in 1994 by Young (Young,

1994). However Iittle data exist about these cells beyond their anatomic location,

phenotype and some preliminary data on their behaviour (Chevallier, et aL, 1998). TO

Leam more about BL, the present experiments were designed to investigate the migration

of BL into afferent lyrnph, and after splenectomy. Additionally, repetitive sampling of

blood and lymph was perforrned to examine the stability of the two pools.

To date BL have not k e n examined in normal afferent lymph. There are

significant ciifferences in the cellular composition of blood, afferent and efferent lpph.

This led to the speculation that BL and LL may daer in their migration into afferent

lymph. The appearance of labelled cells in aEerent lymph implies migration through

tissue and hence immune surveillance (Schieiffenbaum and Fehr, 1996).

The spleen is an important site of lymphocyte migration, with BL localising in

this tissue to a greater extent than LL (Young et al., 1997a). Previous snidies have

demonstrated that in splenectomized rats a lymphocytosis, composed oCB celis and CD8

T celis, developed (Westernmm et al, 1990). Using FïïC hbe1Ied thoracic duct ce&

Westennana et ai. (1989) demnstrated that splenectomy caused an hcrease in labelied B

cells as compared to T c e k in blood and Lymph nodes. Based on these data it was

concluded that splenectomy changed the migration pattern of both B and T ceiis.

However, no experiments have been perfomied that examines the migration of the BL

and LL in splenectomized animals.

The increased B ceils in the blood of splenectomùed subjects may possibly be

due to an accumulation of BL (Sieber et aL, 1985). If splenectomy increases BL in

blood, this could be a method to selectively expand thk pool for hiture experiments.

Therefore, the eexperiments in this chapter were performed to gain a better

understanding of the basic biobgy of BL. These data are important for the design and

interpretation of subsequent experiments.

3.3 Materials and Methods

The experiments involving the splenectomized sheep were performed at the Basel

Institute for Immunology. The repetitive sampling and afferent lymph experiments were

performed at the University of Toronto.

Lymphocyte migration in splenectomized sheep was £irst investigated ushg

fluorescent labeiled celis and immunophenotyping. After 4 days this was foliowed by

tissue distribution e x p e e n t s using radiolabeled cells. Sheep used in the repetitive

samphg experiments had prescapular and prefemord efferent lymphatics cannulated.

Both BL and LL were labelled with fluorescent dyes, intravenously infused and then

blood and lymph were repetitively sampled

3.3.1 Anirnals and surgery

Sheep used in the splenectomy experiments had their spleens removed between

the ages of 19 and 2L days by Dr. W. Heia The animals were then retumed to the fann

for the foIlowing 2 years. At the tïme of the experiments the animals were of a n o d

size and weight and did not appear to s&er fkom disease. The control sheep were fiom

the same supplier and were approximately the same age but were not splenectomïsed.

For aii experiments, prescapular and prefemoral lymphatics were canulated as

described in Chapter 2. Afferent lymphatics were canulated in some experiments- A

jugular catheter was also surgically placed at the same time to d o w access for blood

samphg.

3.3.2 Lymphocyte labelling

For the splenectomized sheep experiments peripheral blood mownuclear cells

were labelled with CFSE. For aii other experiments, the whole blood labelling procedure

using FITC was used. In al l experiments, lymph Lymphocytes were labelled with DiI-DS.

Radiolabeling of lymphocytes with 111-lk and 51-Cr was as outiined in Chapter 2.

Blood lymphocytes were isolated using Percoli and labeled with 11 1-In, while lymph

lymphocytes were labeled with 5 1-Cr. The radiolabeled cells were infused intravenously

and allowed to migrate for 8 hours before tissues were harvested. The tissues were

3.3.3 Sampling of blood and lymph for tracking fluorescent labelled cells

Samples of blood were cokcted and the erythrocytes lysed. The samples of

lymph and blood were washed twice with buffer and resuspended in paraformaldehyde.

A FACScalibur was used to determine the percentage of IabeIIed cek in the samples.

Lymphocytes were examined by using thek typical side and forward Light-scattering

properties.

3.3.4 Statistical analysis

To ailow pooling of the data two methods were employed. In the experïtnents

involving the repetitive sampluig of blood and lymph the average percentage of FITC+

and DiI-DS+ was determined for aII samples. AU values were divided by this number to

give a ratio and these ratios fiom al l experiments poded. The second method was used

for splenectomized sheep, in which the percentage injected was calcuIated and the results

pooled. GraphPad Instat software was used to perform one way ANOVA and appropriate

post-hoc tests. Student t-tests were used were appropriate-

3.4 Results

3.4.1 Repetitive sampling of blood and lymph

Labeiled ceiis were infbsed and allowed to equilibrate for 20 hours and blood and

Lymph were then sampled houriy. As previously docamented lymphocytes isolated nom

lymp h, labelled and reinfused intravenously are found in higher concentrations in lymp h

as compared to blood Figure 3 is a representative experiment fiom 5 experiments

performed and shows that FïïC labelled blood Lymphocytes were approximately 3 times

enriched in blood as compared to lymph. Conversely, DiI-DS labeiied Lymph

lymphocytes were approximately 1.5 times enriched in lymph as compared to blood

Pooling of the data fkom the 5 separate experiments demonstrated that there was no

significant change in the concentration of BL and LL over t h e in either blood or lymph

(Fi-gure 4).

3.4.2 Afferent lymph

Samples of afferent and efferent lymph and blood were collected in 3 normal

anhals. The percentage of labelied ceUs in afferent and efferent lymph was divided by

their percentage in blood, Blood was chosen as the denominator as cells were migrating

kom this tissue. As previously reported there is an encichment of FLTC+ lymphocytes in

blood and DiI-DS+ cells in efferent lymph (Table 2). In afferent lymph FlTC labelled

BL were not e ~ c h e d as the ratio was approxirnately 1. Ln contrast, the ratio of Da-DS

labelled lymphocytes was approximately 3, indicatuig a selective migration of LL into

afferent lymph.

3.4.3 Splenectomized sheep migration

In 4 splenectomized sheep the migration of BL and LL was examined using both

fluorescent and radioiabeled lymphocytes. Using fluorescent c e k the short-term

migration (4 hr) was examined and compared to control animals (n=3). In the blood of

intact sheep DiI-DS labeiied LL ceiis decreased to approximately 0.00246 injected by 1

hour p o s ~ i o n , whilst the CFSE iabeIied BL were retained in the blood (Figure 5)- In

splenectomized sheep, there was no significant difference in the migration pattern as

cornpared to control sheep. However, there was a trend towards BL king retained withui

the blood in greater numbers as compared to intact sheep 0.004 % injected vs. 0.002 %

injected-

Radiolabeled cells were used to determine which tissues BL and LL migrated into

after splenectomy. No tissue had a higher concentration of labelied BL as compared to

LL; therefore, no tissue appeared to be a preferential site of BL homing (Figure 6). When

the data is compared to that previously reported (Young et ai., 1997a) for intact sheep it is

seen that LL enter lymph nodes and tiver in signincantly higher amounts and BL are

partiaiiy excluded fiom the lungs (Table 3).

3.4.4 lmmunophenotyping of lymphocytes in splenectomized sheep

There was no lymphocytosis or difference in blood and lymph phenotypes in

splenectomized sheep (Table 4) as compared to contml sheep.

As seen in intact animais there is an enrichment of CFSE labeiied BL In blood and

Da-DS IabeUed LL in lymph. Table 4 summarises the phewtypic data of IabeIIed cek

and udabeled c e h at 24 hours pst-infusion. The resuits are sunüar to those previously

reported by Andrade (1996) in intact animals, There is an enrichment of sIgM and

CD1 Lb+ aad B ceils in the CFSE labelled lymphocytes in blood. CD4 and CD8 CFSE

labeiled celis are present in a lower number in blood. In Iymph, y6 T cek are enriched in

both populations as compared to uniabeIed tells-

Figure 3 Representative expehent of repetitive snmpling over a 27 br pend

One representative experiment is shown. FiTC labelled BL are e ~ c h e d in blood as

cornpared to lymph, whilst DE-DS Iabeiled LL are concentrated in lymph.

A - Flow cytoxnetric detection of labelled ceiis in blood

B - Flow cytometric detection of IabeIled ceh in efferent lymph

-% FITC

Time (hours)

B Efferent lymph

-% FITC -cl- % DiI-DS

Wgure 4 Ratios of BL and LL lnbeiied c e k in efferent lymph and blood over a 27 hr period

There is little variation of FITC labelled blood lymphocytes in blood or efferent lymph

over 27 hr. SimilarIy, DE-DS Iabeiied lymph lymphocytes exhibit little variation. Ratios

were determined by dividing the percentage of labelied ce& at each tirnepoint by the

average percentage obtained during the entire experiment (n=5)-

A - ratio of Iabeiied cells in efferent lymph

B - ratio of labeiled c e k in blood

A Efferent lymph

Time (hours)

0 . 5 0 ! , 1 I 1 l I l I L 1 l I I 1 1 1 1 I L L L t i i '

O 2 4 6 8 10 12 14 16 18 20 22 24 26

T ime (hours)

Table 2 Perrentage and ratio oPIabeIled ceL in bbod, efferent and aerent lymph

The percentage of labeiled cells is shown with the ratios in parenthesis. A ratio was

determined by dividing the percentage in lymph by the value in blood DS-DS labelleci

LL is enriched in both afferent and efferent lymph as compared to FITC labeiïed BL.

Experiment % btood % efferent (ratio) % afferent (ratio)

HTC DIT-DS FITC DiI-DS FITC DiI-DS

Expt. #l 1.22 0.30 0.28 (0.2) 0.54 (1.8) 0.54 (0.4) 0.80 (2.7)

Expt- #2 0.48 0.09 0.20 (0.4) 0.30 (3.3) 0.62 (1.3) 0.44 (4.9)

Expt. #3 1-15 0.88 0.49 (0.4) 0.90 (1.0) 0.87 (0.7) 1.41 (1.6)

average ratio f SEM 0.33 k 0.07 2.0 f: 0.67 0.8 t 0.26 3.1 & 0.96

Figure 5 Short-tenn migration oflpbelled lymphocytes in the blood of splenectomized sheep

Blood was sampled every 15 min during the E s t 4 hr d e r the infusion of fluorescentfy

labelied cells. In splenectomized sheep (A) (n4) there was a trend for Da-DS IabelIed

LL to disappear fiom the blood at a faster rate than in control sheep (B) (n=3). CFSE

labeiied BL were enriched in blood in both splenectomized (A) and control (B) sheep- A

trend for splenectomized sheep to retain BL in blood is seea

A Splemectodzeà sheep

1 -Avg CFSE

+Avg DiI-DS

O 15 30 45 60 7s 90 105 120 135 150 165 T80 195210 225280

T ime (min)

B Control sheep

-Avg CFSE

+ Avg D il-OS

1 ime (min)

Tissue

Figure 6 T'me localisation of radiokbeied LL and BL lymphocytes

AU lymphoid and control tissues in splenectorrüzed sheep contain greater amounts of LL

than BL (n=4),

LL - 5 1 -Cr labeiied Lymphocytes (solid bacs)

BL - 1 1 1-In iabeiied Lymphocytes (open bars)

Table 3 Tissue localisation h splenectornized sheep

Peripherai blood lymphocytes and efferent lymph lymphocytes were labeiled with

radioisotopes (see figure 6) and their migration into ciifferent tissues determined. There is

a signincant increase in the amount of LL in prescapular Lymph nocies of splenectomized

sheep (n=4) as compared to intact sheep. The lung has fewer BL in the splenectomized

sheep as compared to the controI subject-

* = percentage of injected c e k I gram of tissue t SEM

# = significantly different (pc 0.05) fiom the correspondhg ceii migration in the control

animal

S denectodzed shee~ Contrai s h e e ~

Tissue BL LL BL LL

PrescapuIar LN .04 f .()os* -204 t -03" .O24 tOO8 -043 & .O1 1

Mesenteric LN .O2 + -005 . 107 + -0 19 .O21 f -008 -065 I -020

Lung -03 ,t ,005' , 106 + -007 ,065 I .O 12 -075 t .O 16

Peyer's patch ,008 f ,004 .O12 k -002 .O05 & .O02 .O20 I .O05

Liver .O12 t .O04 -020 I .w# .O14 + .O03 ,011 t .O01

Blood cells .006 f .O01 ,002 f .O003 .004+.001 .O02 I .O01

Table 4 Immunophenotypic data h m splenectomized sheep

S plenectomized s heep ( n 4 ) demonstrate no merence in the phenotype of Lymphocytes

in blood and lymph as compared to intact sheep. BIood Lymphocytes were labelled with

CFSE and LL with DiI-DS,

A = p henotypes in splenectomized and intact animals

B = phenotypes of CFSE and DI[-DS labelled ceils in blood

C = phenotypes of CFSE and DiI-DS Iabelled cells in lymph

* = average + SEM

7 = significantly different (p < 0.05) than total (unlabeiied) Lymphocytes

§ = ~i~onificantly different (p < 0.05) than CFSE labeiled B L

A Cornparison o€lymphocytes in bbod and lympb

S~lenectom*zed sheee htact s h e e ~ Cell Type Blood Efferent Lymph Blood Efferent Lymph £3 cells 56.7 4.L* 23.4 * 3.2 43.8 I 7.5 24.4 I 3.5 CD4 14.6 * 1-7 42.8 * 1.5 18.0 & 4-1 35.0 * 3-4 CD8 12-2 & 1.3 14-0 I 1.3 14.2 I 3.3 15.1 i3.8 y5 9.9 k2.1 L 1.3 * 1.0 12.3 I 7.5 13.5 * 2.1

B Phenotype O€ IabeUed cells in blood

Subset Unlabeled C'SE ~abemed CD4 15 I 1.7 4 t 0.8' CD8 12 t 1-3 2 k 0.2~

10 t2.1 7 + 1-3 B 57 & 4-1 75 I 6.5

CDllb 40 t 4.7 75 I 3.2' CD21 24 t 6.3 25 -t 8.9

L selectin 31 I3-3 16 t 2.7' sIgM 38 t 2.6 63 + 5.4'

C Phenotype of kbeUed ceL in lymph

Subset CD4 CD8 YS B

CD1 lb CD2 1

L selectin

SI@

DZ-DS labeiled

Lvm~hocvte ~ o ~ u h t i o n s in lvmah Unlabeled CFSE IabeLled DiI-DS labeiied 43 k 1.5 16 I 1.5' 38 t 3.4' 14 I 1.3 4 & 0.3' 8 + 0-9'' 11 I 1-0 40 I 3.4' 3 1 + 2.7': 23 I 3.2 28 f 1.4 16 k 0.9~ 1 k 1.0 5 + 1-9 1.2 I0.3 23 33.2 31 13.2 19 & 2.7 89 I 1.4 89 ,t 1.8 90 k 1.3 22 f 2.7 31 12.9 17 t 2.2"

3.5 Oiscussion

The data presented in this chapter confïrm and extend previous hdings of BL as

compared to LL. These basic data are also important in the interpretation of the

expeaents in subsequent chapters.

Previous investigators have used fluorescentiy labelled Lymphocytes to examine

the migration of lymphocytes obtained nom blood and lymph (Young et al., 1997a;

Andrade et A, 1998). There are several advantages to labehg the whole population of

blood and efferent lymph and reinfusing all cells. Fkstly, both blood and lymph

lymphocytes undergo Little in vitro manipulation, thereby reducing any chance of

inadvertent activation or change in adhesion molecules. However, the main advantage is

that the whole population is retucned to the blood and the subpopulations of lymphocytes

can migrate into their respective compartments (Westemiann et al., 1993).

Repetitive samplîng of both blood and lymph confirmed that a pool of

lymphocytes exists that is preferentially retained in blood as compared to LL, as seen in

Figure 3. The BL are found in blood at a percentage 3 times that in simultaneously

coiiected efferent lymph. Figure 4 demonstrates that there is Little variation of the two

populations in either blood or lymph during the entire 27 hr sampiing period.

Immune surveillance is a putative function of recirculating lymphocytes

(Schleiffenbaum and Fehr, 1996; Butcher et al., 1999). In afferent lymph, the ratio of

labelled LL is 3 times higher than in blood but BL are found in a similar percentage

(Table 2). The appearance of IabelIed cells in aiferent lymph impiies that they have

migrated through tissues. However, Lymphocytes may migrate into tissues and be

retained or deleted, therefore not entering afferent lymph. Prior snidies using

radiolabeled BL and LL have demonstrated greater numbers of LL in slcin (Young et al.,

1997)- Taken together these data demonstrate that LL m i t e into nonlymphoid tissues

in larger numbers than BL. Fiutber experiments are required to investigate transit times

and quanti@ the trafGc of these pools into tissues.

In splenectomized sheep, no Iymphocytosis or differences in the phenotype of

blood and Lymph lymphocytes was observed as compared to control sheep (Table 4).

These results disagree with previous reports in rats (Westemiann et ai., 1990) and humaos

(Ferrante et aL, 1987). One explanation for this discrepancy maybe that the effects of

splenectomy are species specific. However, 1 believe a more plausible explanation is that

the animals in this study were s p l e n e c t o ~ d at approximately 3 weeks of age as

compared to the other studies, which used adult subjects splenectomized as adults.

Tracking studies reveal no statisticd diffferences between the disappearance of the

two pools in intact and splenectomized sheep, either in the short-tenn @ours) or up to 4

days af3er the infusion of ceils (Figure 5). As weil, there were no differences in the

phenotype of labelied ceils in blood and lymph (Table 4) as compared to previously

reported data (Gupta et al., 1998). Those CFSE labelled Lymphocytes retaioed in the

blood were mainly B celis, CD L Lb+ and L selectin-, whilst those in lymph were CD1 Lb-

and L selectin+.

In agreement with a previous report (Young et al., 1997a), both CFSE and DiI-DS

labelled cells were enriched for y6 ceils in Lymph 40% f 3.4 and 3 LI + 2.7 respectively

as compared to 11% t 1.0 for unlabelled Lymph. In blood there is no enrichment of

labeiled y6 BL as compared to unlabelled lymphocytes, 7% f 1.3 vs. 10% f 2.1, but LL

y6 are signifcantly increased (29% k 3.2). Thecefore, y6 celis may rapidly recirculate

nom blood into lymph. This conclusion disagrees with previous results by Andrade

(1998) and others (Witherden et aL, 1990), who suggest that a pool of wnrecircuIating ya

T ceus is present in blood. An explanation for this discrepancy maybe the different ages

of the sheep used in these experiments as Young et aL (personal communication) have

demonstrated a difference in the migration and number of y6 T cells in young and adult

sheep.

Experiments using radioactive IabeIled BL and LL were executed to determine the

migration pattern of the two pools in splenectomized sheep. No examuled tissue in

splenectomized sheep had a hîgher concentration of BL as compared to LL 6).

This is in contrat to intact sheep in which BL preferentiaiIy home to the spleen (Young

et al., 1997a). Peripherd lymph nodes and Iiver had a greater percentage of injected LL

as compared to control sheep. In splenectoniized sheep significantly fewer BL were

detected in the lung compared to control sheep, 0.03 k 0.005 and 0.065 + 0.012 %

injected (Table 3).

These radioactive and fluorescent tracking data demo as trate a ciifference in the

circulation of BL and LL in splenectomized sheep. An explanation of these data is that in

splenectomized sheep Li, spend a shorter time in blood aad quickly enter the lymphatic

system and iïver. This is shown by the decrease in labelied cells in the short term

t r a c h g studies and the higher numbers of radiolabeled ceils in peripheral lymph nodes.

The spleen therefore appears to act as an organ of localisation of the BL but retards the

migration of the LL in its migration into lymph nodes.

In conclusion, these experiments contriiute to the increasing amount of data that

demonstrates the existence of a pool of lymphocytes in blood that does not recirculate as

efficiently as LL. The spleen is not necessary for the maintenance of these BL but its

removai does have an effect on the migration of both pools, resulting in enhanced

migration of LL Ïnto perip heral lymph nodes. In afferent lymph, there is a preponderance

of LL as compared to BL. This in combination with previous resdts using radioisotopes

implies that LL have a greater role in the immune surveillance of tissues as compared to

BL. The BL and LL exhiiit littie variation in their concentration in blood and Iyrnph

over time. Taken together these data provided the basis for the design and anaiysis of

subsequent experiments presented in this thesis.

Chapter 4 Lymph and blood CD4 cells increase in efferent

lymph during antigen induced lymph node shutdown

4.1 Abstract

Lymph node shutdown is a period of decreased cell output in efferent Iymph after

the introduction of certain antigens. Previous studies have demoostrated that CD4 ceUs

are not retained within the Iymph node to the same extent as other Lymphocyte subsets

during shutdown. However, the effect of shutdown on the migration of LL and BL has

not been investigated. Cytokines may mediate Lymphocyte retention within the lymph

node by upregulating adhesion molecules; therefore, IL-1P, IL-6, IL-8, IFN-y and TNF-a

were measured in efferent Lymph during shutdown. The percentage of CD4 Lymphocytes

f?om both pools increased in efferent Lymph during lymph node shutdown. IFN-y and IL-

6 increased in efferent lymph when tell output reached its nadir, implying they have a

role in the recruitment &or retention of Lymphocytes within the Lymph node d u ~ g

shutdown.

4.2 Introduction

Certain antigens, when introduced into lymph nodes, uivoke a penod of decreased

cellularity in efferent lymph, which has been called "lyrnph node shutdown" (Bujdoso et

al, 1989). Hall and Morris (1065b) reported that antigens ranging from viruses to

globulins induced Lymph node shutdown for various Iengths of tirne. This is fiequently

foliowed by a period of increased lymphocyte output, including antigen specinc blast

ceus (Cahill et aL, 1974). TNFa (Young et ai, 2000) and IFN-a (Hein and Supersaxo,

1988; E;alaaji e t aL, 1989) also invoke a period of shutdown demnstrating that this

phenornenon does not require antigen. The physiological relevance o f lymph node

shutdown is unknown but one speculation is that it aiIows the presentation of Ag to large

numbers of recruited lymphocytes in a shoa time (Haii and Morris, 1965b).

Previous studies have investigated lymphocyte output and subsets during

shutdown. (Bujdoso et ai., 1989; Mackay et al., 1992b) but the migration of BL and LL

through an antigen s h l a t e d Lymph node has not been examioed The present

experiments may begin to detennine if different fiuictions exist for the two pools.

Various cytokines can increase adhesion molecules on the blood vascuiar

endotheiium, thereby contributing to lymphocyte migration (Juda et ai, 1989; Raine et al,

1990b). Cytokines produced during lymph w d e shutdown may increase the t r a c of

lymphocytes into Lymp h nodes and t heû nibsequent retention. Lymphocyte retention

may be due to the upregulation of adhesion molecules on lymph node stroma1 ceus,

lymp hatic endothelium and other lymphocytes. Interleukin- 1 f3, IL-6, IL-8, IFN-y and

TNF-a levels were measured as they are produced during BCG induced grandomas and

intravesical BCG infusions (Sugisalci et al, 1998; O'DonnelI et al, 1999). These same

cytokines are known to increase adhesion molecules on endotheliai ceiis and may have a

role in lymph node shutdown,

4.3 Material and Methods

4.3.1 Animals and Surgeries

These experiments were performed at the Basel Lnstinite for Immunology and aJI

surgeries were performed by Dr. A Young and Ms. Lisbeth Dudler. Prescapular efferent

Lymphatics were canulated as descriid in Chapter 2.

The sheep had been immunised with 5 times the nomial human dose of BCG

(Institut Serotherapique et Vaccinal Suisse, Berne) at least 21 days prior to nirgev For

antigen stimulation 50 pg PPD (Staîens Seniminstitut, BaseI) was suspended in 1 mi of

salùie with 2% Evan's blue and htradermally injected in several sites located in the

drainage area of the canulated lymph node. The appearance of Evan's blue in the efferent

lyrnph allowed the confirmation that PPD had reached the lymph node. Evan7s blue has

been used in our laboratory and others for several years to locate lymph nodes and does

not have a detrimental effect on lymphocyte output-

4.3.2 Cell labelling

Twenty-four hours after surgery, blood cells were coiiected and labelled with

CFSE and lymph Lymphocytes labelled with DiI-DS as in Chapter 2 Both celi

suspensions were intravenously reinfused.

4.3.3 Cell collection and phenotyping

M e r 48 hours sampies of efferent Lymph were collected every 4 hours for 16

houn to obtain baseline Ievels of A) lymphocyte subsets, B) percentage iabeiled ceff s and

C) cytokine levels. Lymph plasma for cytokine levels was harvested after cenuifugation

and fiozen at -80°C until required. Lymphocytes were ïmmunophenonlped and analysed

ushg APC as a secondary antibody. After the injection of PPD, samples of lymph were

collecced at vai5ous t h e s over the foIlowing 48 hours.

After the last collection of Lymph, PPD and saluie were separately injected i'to

the drainage area of contralateral prefemoral lymph nodes in two sheep. Sixteen hours

later, during the period of maximum shutdown, the sheep were sacrificed and the lymph

nodes excised. They were bisected and half was miaced in coM media, fikered, washed

twice with PBS and immunophenotyping performed as previously outlined- The other

haif was fiozen in OCT and stored at -80°C until immunofiistochemistry was performed-

4.3.4 Imrnunohistochemistry

Murine antiovine MF-a, IL-1$, IL-6 and IL-8 were used to stah the excised

lymph nodes for cytokine production. Optunal concentrations were determined and the

procedure in Chapter 2 was followed. A lymph node fiom a normal sheep was used as a

control.

4.4.4 Cytokine ELlSAs

ELISAs for ovine cytokines were performed as previously descnbed with minor

modifications. Briefly, matched pairs of antiovine cytokine antibdies for L I $ , IL-6,

IL-8 (Serotec, Oxford, U.K) and TNF-a (Centre for Aaimal Biotechnology, Melbourne

Aus.) were obtained. Each pair consisted of a monoclonal murine antihdy and

polyclonal rabbit senun. 0.1% alpha casein was used as the blocking agent and

PBSflween as the washing buffer. The muruie anti idy was used as the capture

antibody, followed by efferent lymph plasma, then rabbit senun, which was detected by

horse radish peroxidase-conjugated goat anti rabbh IgG (Southern Biotechnology,

Birmingham, AL) and visualised using tetramthylbenzidlne iiquid substrate (Sigma,

Basel, S witzerland),

IFN-y was detected using the BOVIGAM commercial kit obtained fÎom CSL

Vete~ary (Australia) and the instructions were foilo wed exactly, This kit cross-reacts

with ovine IFN-y (BOVIGAM insert and ourown unpublished observations)

Duplicate samples £iom each experiment were nui on the same day and each nin

Ïncluded a standard curve or controis. The Iowest amount of cytokine that could be

reliably detected was 0.1 nglml, therefore this was used as the cut-off point. No W - a

of a known concentration was available, therefore a standard cuve could not be

constnicted. This cytokine was therefore measured as a change in absorbance over the.

A sample of tissue culture supernatant fiom a TNF-ût producing ceiI line (gift of J

Hopkins) was used a positive control,

4.4.5 Statistics A Students two taiied paired t-test with a signiticance of pe0.05 and a repeated

measures ANOVA using a Dumets p s t test were used to determine statistical

4.5 Results

4.5.1 Lymphocyte Migration

Lymph node shutdown was detected as previously reported (n=3), with efferent lymph

ceii output decreasing to approximately 20% of baseline vahes at 16 hour postinjection

of PPD (Figure 7). These experiments were terminated at 48 hours posîk.jection,

therefore, the subsequent typical increase in Iymphocyte output was not observed.

The number of CD4 CDS, y5 T celis and CD2h B ce& ail decreased during

lymph node shutdown to at least 50% of basehe, However, when the percentage of c e k

exiting the lymph node was determiaed CD4 cek did not decrease as compared to the

other subsets. Using three-colour flow cytornetry, CD4 ceiis fkom both pools appear to

be retained less efficientiy. This clifference was not st atistically significant . Nonetheless.

it appears that BL and LI, CD4 celIs exit the lymph node easier then the other subset

du~511g lymph node shutdown (Figure 8).

PPD ïnjected lymph nodes excised during shutdown (n=2) did not statistically

m e r from saline injected lymph nodes with respect to the percentage of CD4, CD8 y6 T

cells and B celis (Table 5)-

4.5.2 Cytokine Levels

IL-lp, IL-6, IL-8 and IFN=y could not be detected in efferent lymph during the

pre-injection baseline period Both IFN-y and IL-6 levels had significantly increased in

concentration at 9 and 12 hr postinjection (Figure 9), occurring just prior to the period of

rnïnïxnal ceil output in the efferent lymph. TNF-a, IL-lp and IL-8 levels were not

increased over baseline measurements.

IL-6 was found in both PPD and saline injected lymph nodes but not in normal

lymph node. The IL-6 appeared to be Iocated near endothelid and smooth muscle ceh,

both of which have been reporteci previously. IL-@, IL-8 and TNF-a were not found in

the PPD or saline injected nodes,

Time (hours)

Figure 7 PPD induced lymph node shutdown

There was a substantial decrease in the ceiiularity of efferent lymph after the injection of

PPD hto the drainage area of a prescapular Lymph node (n=3). Ratios for individual

experiments were determined by dividing aU values by the average obtained during the

baseLine period. These ratios were then pooled for all three experiments.

Figure 8 Phenotype of lymphocytes in efferent lyrnph during lymph n d e shutdown

There is a decrease in the number of all measured subsets during lymph node shutdown

(A). The percentage of CD4 c e k demonstrate a trend to an increase (B), whilst other

subsets decrease. Both the CFSE iabeiied blood lymphocytes CD4 ceils (C) and DiI-DS

IabeiIed CD4 lymph lymphocytes (D) increase-

Ratios were produced by d e t e e g the average value obtained during the preinjection

baseline and dividing a i i values by this average. Therefore, a value of greater then 1

indicates an increase. (n=3)

A = number of ceiis as compared to baseline (ratio)

B, C, D = percentage of ceiis as compared to baseline(ratio)

+- CD4

A - y 5

m- CD8

O- CD21

Table 5 Immunophenotype of PPD and saline hjected Iymph nodes

There is no merence between lymph nodes excised 16 hours after the injection of saline

or PPD. (n=2)

* average percent t SEM

Subset PPD injected Saiine injected

CD4 41.5 f 7.0* 37.1 & 2.9

CD8 L8.9 t 0.6 18.8 t 0-6

T i e (hourd

Figure 9 Cytokine levels during lymph node shutdown

IL6 and IFN-y significantly increase (p4.05) during lymph node shutdown, but TNF-ot,

IL-1P and IL-8 are not increased above baseline levels. The average i SEM of 3

experiments is shown

4.6. Discussion

The exact mechanïsm(s) and reason(s) for lymph node shutdown remains obscure.

Ic may have a role in the propagation of an efficient immune response by presenting Ag

to large numbers of lymphocytes (Mackay et al., 1992b; HaU and Morris, 196%)-

However, it does appear to be a valid p hysiological response to certain antigeas based on

the reproducibb shutdown observed by independent researchers.

Durhg shutdown, the lymph node output of aii measured lymphocyte subsets

decreased to at Ieast 50% of the^ basehe measurements (Figure 8). However, when the

data were examùied using the percentage of subsets as compared to baseline. it was found

that CD4 lymphocytes increased whilst CD8, y5 and B cells decreased. That is, the

overaii number of lymphocytes decreased, but of those present, the percentage of CD4

ceus increased by approximately 50%. This is not the fkst report of an increase in C m

cells during shutdown. Bujdoso et al (1989) reported that 24 hours after the injection of

PPD, CD4 cells increased and then seturned to normal after 3 days. Mackay et al (1992)

also noted an increase in the percentage of CD4 ceiis in efferent lymph during shutdown

and a decrease in the number of CD4 celis exiting the lymph node, similar to the present

report. Therefore, in a l l three studies a change in the output of CD4 lymphocytes in

efferent Lymph during lymph node shutdown was observed.

Two explanations are possible for the finding that CD4 ceils are less efficiently

retained during shutdown: A) CD4 ceils are migrating into the lymph node faster and

therefore present in a greater percentage than other subsets or B) non CD4 lymphocytes

are k i n g retained in the lymph node in a greater percentage. To determine if the tranic

of CD4 cells was increased, lymph nodes were excised dwing shutdown. There was no

difference in the percentage of CD4 cells between lymph nodes injected with saline or

PPD (Table S), arguhg that CD4 ceils were not migrating into Lymph nodes in a greater

percentage. Therefore, it was concluded that the change in CD4 ceiis was due to

regulation within the Lymph node.

Three-colour flow cytometry was used to examine the subsets of BL and LL in

the efferent lymph during shutdown. An increase the percentage in CFSE and Da-DS

CD4 cells was seen (Figure 8). This trend towards an increase in CD4 cells was

nonsignificant but was observed for both labeiled and nonlabelled cek. Though the

difference was not statisticaiiy significant there maybe a biological basis for the increase.

Therefore, it appears CD4 lymphocytes may be retaioed within the lymph node less

efficiently then other measured subsets,

Only efferent lymph was examined in these experiments. By dennition, ail

lymphocytes present in efferent lymph are part of the RLP, including blood derived

lymphocytes labelied with CFSE. The increase in both CFSE and Dit-DS labelled CD4

cells may indicate a population of celis that can quickly recirculate. pool would be

found in lymph and in transit through the blood, therefore it would be labelied with both

dyes. Under inflammatory conditions, this population of rapidly recirculating celis

enter the lymph node. If this specuiation is correct, these cells may have different

adhesio n rnolecule profiles thus facilitating more rapid recirculation. This population

may be quite small and therefore wouldn't be detectable in the recirculation of

lymphocytes under normal conditions. This population may be composed of memory T

cells as reported by others (Mackay et al., 1992b). However, this explanation does not

account for the increased migration of BL CD4 cells as the number of memory T ce& in

blood is similar to that in efferent Iymph @a@ et aL, 1999). A possible expianation is

that blood memory ce& have a greater prepouderance to migrate into infiamed lymph

nodes. This is fùrther discussed in section 7.5.

A Iess Likely explanation is that labelled c e k are preferentially recruited. This is

the least Likely of the possibilities as several studies have demonstrated no difference

between labelled and uniabeled ceh (Davenpeck et ai, 1995; Samlowski et al, 1991).

This was determined by performing flow cytomeuy to measure the amount of various

adhesion and activation molecules and kding no ciifference between unlabeled and

labelled ceils.

There is evidence in the Literature to support the specdation of multiple

populations of lymphocytes with varying degrees of migratory abilities. Mita and

colieagues have demonstrated a subset of y6 T cells in calves that can preferentially

localise in lymph nodes after the injection of TNF-a (Wilson et al, 1998). In other

experiments, this same group demonstrated an inabiliry of a subset of y6 T cells to

migrate into a skh site of idammation (Wilson et al, 1999). The y6 T cells were found

to express varying amounts of L-selectin and E-selectin ligands, resulting in differing

abilities to migrate into tissues. These data indicate that calves have multiple subsets of

y6 T ceils that Vary in their migratory capacity. Therefore, it is conceivable that sheep

also contain similar subsets, varying in their degree to migrate into sites of inflammation.

Some cytokines have an effect on the migration of lymphocytes when injected

into the drainage areas of lymph nodes (Young et aL, 2000; Hein and Supersaxo. 1988)-

IL-@, IL-6, IL-8, IFN-y and TNF-a levels were measured in efferent lymph aiter the

injection of PPD. IL-6 and IFN-y increased reaching maximum Levels at 12 hr, just prior

to the ceii output attaining its nadir at 16 hr. (Figure 9) EN-y has been demonstrated to

have a proadhesive effect for Lymphocytes on cultured skep lymphatic and bIood vesse1

endothelia1 ceils (Borron, 199 1). Therefore, it may increase adhesion molecules on p s t

capillary venules and lymphatic endothelid ceiis withui the lymph node, causing an

increase in both lymphocyte migration and retention. IL-6 has been demonstrated to be

important in the migration of lymphocytes into areas of inflammation by increasing the

expression of chemokines (Romano et al, 1997)- It is reasonabIe to assume that it has a

similar role in the infiamed lymph node and increases the amount of chemokines thereby

enhancing the entry of Lymphocytes. Conceivably, wndetected cytokines, iacluding

W-CX, could be produced in the lymph node but not secreted into efferent Lymph.

Immunohistochemistry on excised lymph nodes provided no evidence for their

production.

Taken together these data demonstrate that there is a decrease in Lymphocqtte

output of CD4 CD8, y6 T cells and CD21+ B cells. However, CD4 c e h may be the Ieast

affected, indicating that a subset of c e k may rapidly respond to the antigenic stimulation

of lymph nodes. IFN-y and IL6 are increased in lymph plasma and may have a role in

the increased migration and retention of lymphocytes during lymph node shutdown.

Chapter 5 Lymphocytes in cerebrospinal f luid are part of the

recirculating lymphocyte pool

5.1 Abstract

CSF contains a small number of lymphocytes under normal circumstances. To

determine if these lymphocytes are part of the normal recirculating pool of iymphocytes

efferent lymph was labelled, reinfused and samples of blood, lymph and CSF obtained.

Similar concentrations of IabelIed ce& were found in all three tissues. Based on these

data, it was concluded that wnactivated lymphocytes migrate into the CSE TO

determine if lymphocytes egress from the CSF, labeiled ceUs were infused h o the lateral

ventricle. Retropharyngeal lymph nodes contaiaed higher numbers of 1 1 1 -In labelled

lymphocytes as compared to other lymph nodes. Further experiments using labelied BL

and LL conclusively demonstrated that CSF lymphocytes belong to the RLP. This may

have implications for the immune surveillance of the CNS.

5.2 Introduction

The CNS was thought to be an immunologicaiy pnvileged site based on the

paucity of lymphocytes present in the brain parenchyma, lack of a lymphatic system and

the delayed rejection of transplanted tissue (Selmaj, 1996). However, studies have

demonstrated that there is immunological surveillance of the CNS, albeit at a lower level

than other tissues (W-iams and Hickey, 1995; Hickey, 199 1).

Activated T c e k enter the CNS parenchyma regardless of their antigen specincity

( Bauer et aI, 1998; aickey et aL, 1991). Activated Lymphocytes that encounter their

antigen in the CNS are retained and initiate an immune respome causing a non-specific

migration of Lymphocytes (Knopf et al 1998; Ludowyk et ai, 1992; Mor and Cohen,

L992;Cross et al, L99 1). Most investigations have not examined the entry of lymphocytes

into the CSF but rather have concentrated on the brain parenchyma, especially during

inflanimatory conditions such as multiple sclerosk (MS) (Hickey, 1991). Studies have

revealed that CSF and brain parenchyma dBer in their immunological potentid

therefore, resuks fiom one tissue can not be extrapolated to the other (Matyszak and

Perry, 1996).

The CSF under normal physiologicai situations contains a small number of

lymphocytes, with the majoricy behg CD4 T c e k (Vrethem et al, 1998; Svenningsson et

ai, 1995). Previous reports suggest CSF Lymphocytes are not a static population but may

enter and exit the CSF, irnplying that they are part of the RLP. Hafler and Weiner (1987)

in a study of MS patients, found that in vivo 1abeiIed CD4 Lymphocytes enter the CSF.

Lymphocytes and other cells injected into CSF and brain parenchyma migrate into

cervical iymph nodes (Carson et al, 1999; Oehmichen et al, 1979)- These same lymph

nodes drain fluids and proteins fiom the CNS (Boulton et ai, 1997; Cserr and Knopf,

1992), therefore, it is possible that Lymphocytes also use these pathways to exit CSE

Therefore, experiments were performed to detemine if lymphocytes in m~mal

CSF belong to LL. As well, cervical lymphatics were canulated and monitored for the

appearance of IabeiIed ceiis infused into CSF.

5.3 Materials and methods

The £irst experirnents examine the normal ceU concentraiion in CSF. Experiments

were then performed to determine if FïTC labelled lymphocytes migrate into the CSF and

detected ushg fiow cytomtry. The next experiments investigated the migration kioetics

of lymphocytes into the CSF and their concentrations as compared to efferent lympb. As

well, the migration of lymphocytes out of the CSF iato Lymph nodes and cervical lymph

was detennined,

5.3.1 Anirnals and surgery

Jugular veùis, prescapular, prefewral and ceMcal lymphatics were canulated as

described in Chapter 2. As weii, catheters were placed into the lateral ventricles to allow

the infusion of cells. Two sheep had successful laminectomies perfonned and catheters

introduced into the subdural space to allow seriai collections of CSF-

5.3.2 Detemiining normal CSF cell counts

The normal oumber and differential of ceIIs in the CSF of sheep was the first

experiment performed. This involved coilecting CSF ftom sheep involved in other

nonrelated studies as weii as this snidy. A lumbar puncture was performed by inserting a

needle between lumbar vertebrae 3 and 4 and aiiowing approximately 2 mi of CSF to drip

into a sterile tube. The CSF was immediately placed on ice and a ceU count performed

using a Neubaur chamber. Slides were prepared using a cytocentrifuge (S handon),

stained with a modified Wright stain and a differential perforrned. Samples contaminated

with red blood cells were discarded.

Efferent lymph ceiis were collected and labeiied with FITC as d e s c n i d in

chapter 2 and reinfused intravenously. In al1 experiments, greater than 80% of c e k were

viable using trypan blue exclusion and greater than 95% of the celis were labeiled with

FITC.

In some experiments, lymph lymphocytes were labeiIed with I l 1-Ih and infused

into the lateral ventricles. h experiments in which LL and BL lymphocytes were king

simultaneously investigated, blood was labelled with FITC and lymp h Lymphocytes wit h

DiI-DS.

5.3.4 Sample collection of blood, lymph, CSF and lymph nodes

Samples of blood, lymph and if required CSF were coiiected as needed. If

anùnals did not have catheters in the subdural space, CSF was coilected by performiag a

lumbar puncture. For kinetic studies, in which serial samples of CSF were required,

samples of between L and 2 ml were removed. For terminal sampling at the end of

experiments 2 to 10 ml of CSF was collected.

Samples of blood and lymph were prepared as descnid in Chapter 2. CSF

samples required more care, as few lymphocytes were present. To avoid loss and

maintain the integrity of cells, CSF was immediately placed on ice and an equal volume

of paraformaldehyde added after an aliquot was removed for a cell count. Cenaifugation

was avoided if less then 2 ml of CSF was coliected, instead the whole sample was

analysed by flow cytometry.

Prescapular, prefemoral and popliteal lymph nodes were harvested at the end of

severai experiments. They were immediately bisected, wmpped in saIine soaked gauze

and placed on ice. To harvest Lymphocytes, the tissues were muiced in cold media,

fdtered and washed twice in PBS. These ceUs were mtered, paraformaidehyde added and

processed using identical methods as for lymph samples.

5.3.4 Intracerebroventricular infusions of 1 1 1 -In labelled lymphocytes

To determine if labelied lymphocytes can migrate from CSF hto cenical lymph

nodes, 11 1-In labelled c e k were infused into the lateral ventricIes in 3 sheep. Between

2-5 x 10' ceils were infused. At 24 hrs (nd) and 48 hrs (n=2) the sheep were sacriuced

and various lymph nodes and control tissues excised. These tissues were weighed,

counted on a y-spectrometer and the counts per minutefgram of tissue deterrnined TO

ensure that the radioactivity was cell associated, the retropharyngeal lymph nodes were

minced, washed with PBS and the radioactivity associated with the cells detemiined. The

majority of the radioactivity (>go%) was celi associated.

5.3.5 Intracerebroventricular infusions of FlTC labelled lymphocyte

To determine if cervical lymphatics are a path of exit for CSF Lymphocytes, these

lymp hatics were canulated. Lymphocytes were coilected labelled wit h FJTC, suspended

in 1 ml of saline and infbsed into the lateral verticais. Samples of cervical lymph were

collected for flow cpometry. To ensure that the celi infusions were sterile, agar was

inoculated with samples of celi solution. No samples tested grew bacteria after 4 days.

5.3.6 Flow cytometry

AU samples were analysed using a FACScan (Becton Dickinson). Srnail

lymphocytes were analysed based on their olpicai forward and side Light scatter

properties. Aliquots of iabeiied and unlabeled lymphocytes were used as controis.

The percentage of FKC, and if required, DiI-DS labeiled tek in each sample

were determined For blood and lymph, at least 1 6 lymphocytes were analysed but

because of low numben of ceils present in CSF, oniy 1o3 lymphocytes codd be

examined.

5.4 Results

5.4.1 Cells in normal CSF of sheep

Samples of CSF were coliected from 7 sheep and a cell count deterrnined. The

mean of these counts was 3.0 + 0.3 x 10~/ml, No red blood cells were seen in the those

samples used to determine this count, therefore the samples were not contaminate with

blood. The differential showed that the majority of these cells were small lymphocytes

but, because of the low number of ceils present, an accurate differential could not be

performed.

5 A.2 Repeated infusion of labelled lymphocytes

The next series of experiments was carried out to determine if FITC labelied

lymphocytes could migrate into CSF and be detected using flow cytometry. Ail coliected

efferent lymph was iabelled with HTC and intravenously reinfused at regular intervals

(approximately 12 hr) for a minimum of 3 days in 3 sheep. Between 5-10 x 10' ce&

were infused. Samples of blood, lymph, CSF and lymph nodes were coiiected 24 hotus

after the last infusion and the percentage of FITC labeiled c e k determined. Slmilit~~

concentrations of labelled cells were found in all examined tissues within the same

experiment. Because of the large differences between the nmber of injected ceh. these

data could w t be pooled. Representative histograms h m one experiment c m be seen in

Figure 10. These data demonstrate that JTC labelled lymphocytes migrate into and are

detected in CSF. Next experiments using a single bolus of labeiied lymphocytes were

performed, allowing kinetic studies.

5.4.3 Single bolus of labelled efferent lymphocytes

To determine the percentage of FXTC labelled efferent lymphocytes in tissues of

interest after a single infusion of ceiis, samples of CSF, blood. lymph and lymph nodes

were collected 24 hours postinfusion. The percentage of labelled cells was as follows

CSF 0.6 1 % I 0.26. efferent Lymph 0.50% I O. L6 and lymph nodes 0.35% f 0.13. There

was no signifrcant ciifference between any of the tissues sampled both in the same

experiment and in the Gnal average (Table 6). Fiepre LI is composed of representative

histograms fIom one experiment. Subcutaneous efferent lymph and CSF appear to share

the same population of lymphocytes.

Figure 10 Percentage of IabeMed ceiis in tissues afkr constant reinfusion

Representative histograms nom one of three experiments in which lymphocytes were

coliected and intravenously infüsed over three days. LabeUed cells are clearly seen in the

CSF, blood, efferent lymph and lyrnph node,

A - FITC labded ceils and negative control

B - CSF before the infiision of cells

C - efferent subcutaneous lymph

D - CSF

E - blood

F - popLiteai Lymph node

Log fluorescence intensity

Table 6 Percentage FITC lnbelled efferent lymphocytes in CSF, Iymph and lymph nodes after a single uifiision

There is no signifïcant merence (p < 0.05) in the percentage of FïTC hbeiled cek 24 hr

after a single intravenous infusion of ceils. ND = not determined

* = percentage labeiied cells & SEM

Exp. number CSF Lymph Lymph node 1 O. 16 + 0-04 0.23 I 0.05 0.17 + 0.04 2 0.75 + 0-05 0.75 t 0.05 0.60 + 0.05 3 0.5 L t 0.08 0.49 I 0.02 0.58 t 0.02 4 0.1 1 t 0-02 0.07 f 0.02 0.05 + 0-0 I 5 1.55 t 0.11 0.97 f 0.03 ND

Average 0.6 1 + 0-26 0.50 f O- L6 0-35 k O. 13

5.4.4 Kinetics of FlTC labelled cells in CSF and efferent Lymph

Multiple samples of CSF and eEerent lymph were collected over a 24 hr sampling

period. In both sheep, in which successful Iaminectomies were performed, labened

lymphocytes appeared in approoximately the same concentrations and at similar times in

CSF and efferent lymph (Figure 12). Using regession analysis the correlation between

appearance of lymphocytes in lymph and CSF in the separate experiments was 0.6L and

0.98. These data indicate that subcutaneous efferent lymph and CSF share the same pool

of recirculating Lymphocytes.

5.4.5 Lymphocyte egress from CSF

If Lymphocytes in the CSF are part of the recirculating Lymphocyte pool then they

must be able to exit the CSF- To investigate this, Ill-In labelled cells were Uifused into

the laterai ventricles and radioactivity determined in tissues, In 3 experiments, lymph

nodes known to drain fluids and proteins fiom CSF had greater radioactivity than distal

lymph nodes or control tissues (Figure 13). These data were not pooled as different

amounts of IabeUed cells were infirsed and tissues harvested at 24 and 48 hr-

Lymphocytes appear to migrate fiom the CSF and enter retropharyngeal lymph nodes,

perhaps using the same pathways descn id for proteins.

To determine if labelled c e k migrate from the CSF using cervical lymphatics,

FITC labelled ce& were infused into the lateral ventricles and cervical lymph mnitored

for their appearance. In only 2 of 6 experiments codd ETïC labelled cells be detected in

cervical lymph (data not shown). However, retropharyngeal lymph nodes hamsted at

the termination of experïments, contained FITC IabeIIed ceiis. Therefore, E7ITC Iabeiied

c e k were able to migrate from the CSF. Experïments were perforxned to confiml that

the canulated cervicaI lymphatics were communicating with the CNS. In 2 experiments

in which iabelled c e k couid not be detected in cervical lymph radioactive albumin was

infused into the lateral ventricles. In both experlments, radioactive albumin was detected

in cervical lymph, connrming that the correct lymphatics were canulated.

5.4.6 LL and BL migration hto CSF and afferent lymph

Table 7 shows that LL (0.52% f 0.15) are present in CSF at greater

concentrations than BL (0.036 k 0.02). There was no significant dinerence in the

percentage of labeiied LL in CSF (0.52% + 0.19, blood (0.454 & 0.10) and efferent

lymph (0.79% + 0.19). However, labelled BL were found to be ~ign~caatly lower in

CSF (0.03% + 0.02) as compared to blood (0.72% f 0.13)-

Expressing the data as percentage injected ceils corrects for the daerence of

infused cells. Using this method LL are present at 1.07 x 106% injected I 0.44 and BL

1-15 x 10-~% injected & 0.86 (n=S)(Figure 15). This coafïrms that LL enter CSF in

approxbmtely 10X greater numbers than BL.

Both LL and BL are present in Herent lymph in higher concentrations than CSF.

Nevertheless, the RLP is present in both tissues at a higher percentage than BL.

Figure 11 Percentage of iabeüed lymphocytes 24 hr &et a single inhision of cells

Representative histograms 24 hr after a single intravenous uihision of FITC iabeiied

subcutaneous efferent lymphocytes. Similar concenaations of FïïC labelled cells are

seen in di tissues examined-

A - FITC labeiied and negative lymphocytes

B - efferent lymph

C - CSF D - blood

E - prefemoral lymph node

Log fluorescence intensity

8 14 19 24

Time (hrs)

Figure 12 Appeanuiee of EïïC iabeUed lymphocytes in CSF and lymph

In two experiments, CSF and lymph were monitored for the appearance of labeiled ceik

after a single infusion of FITC labelled lymphocytes. Labeiied cells were detected in

sirnilar concentrations and the in both compartrnents. One experiment is s hown above.

Figure 13 Intracerebmventcic~~r injectecl Ill-In bbelled ceUs migmte to lymph nodes known to drain CSF

After the intracerebroventricular infusion of 11 1-In iabelled lymphocytes, they are found

at higher concentrations in lymph nodes known to drain CSF.

Retro : retropharyngeal lymph node

Mand: submandibdar lymph node

Prefem: prefemord lymph node

Mesent: mesenteric lymph node

Table 7 Percentage labelled LL and BL in CSF, blood, and lymph

A The Di[-DS IabeUed LL were aiways present in higher concentrations than FITC

labeiied BL in CSE The percentage of FiTC labeiied cek in blood was sigoificantly

different than in CSF (pd.0 1). No significant difference (pM.05) was seen when the

percentage DiI-DS labeiled ceils was compared between tissues.

B In normal CSF and afferent lymph the DiI-DS labelled RLP is present in a higher

percentage. There is no signincant ciifference (p>0.05) between the percentage of

labeiied ceiis in afferent lymph and CSF,

A Percentage iabelled in blood, CSF andefferent lymph

Percenta~e FITC labelIed Percentaee DiI-DS labelled Exp. number Lymph Blood CSF Lymph Blood CSF

1 0.33 0.58 0.09 0.79 0.60 0.79

6 0. 15 0.4 1 O 1.56 0.58 1-10 Avg- t SEM 0.25 f 0.02 0.72 I0.13 0.03 k0.02 0.79 i0.19 0.45 &0.10 0.52 I0.15

B Percentage Iabeiied in Pnerent lymph and CSF

Afferent lvm~h - CSF Exp. number 9% FiTC % Dïi-DS % FITC % DiI-DS

4 0.54 0.80 O O. 17 5 0.62 0.44 O 0.59 7 0.87 1-41 0.44 0.44

Avg. t SEM 0.67 t 0.10 0.88 +, 0.28 0-15 2-14 0.38 +0.11

5.5 Discussion

Tissues are continuously patroiled by Lymphocytes, but it was believed that the

CNS was spared this surveiiiance (Selmaj, 1996). However, it has become increasingly

obvious that activated Lymphocytes migrate into the CNS, albeit at lower nurnbers than

most tissues (Hîckey, 1999). Normal CSF contains a srnail number of lymphocytes

(Svenningsson et ai., 1995), but brain parenchyma is vùnially devoid of Lymphocytes.

Matyszak and Perry (1996) injected BCG into the brain parenchyma and the CSF and

demonstrated that these two compartments dBer in their immune response. The

inflammatory response was Iimited in the parenchyma but the CSF immune response was

similar to that seen in the periphery. These data together illustrate that the CSF and brain

parenchyma can not be cousidered as the same tissue with respect to their interactions

with the immune system,

Several connections exist between the CNS and immune system, It has been

clearly demonstrated that antigeos infused into the CNS drain Uito cervical Lymph nodes

and stimulate an immune response (Knopf et al, 1995)- Cytokines in the periphery can

enter the CNS (Pan et al, 1997a), while those in the CSF exit via the cervicai lymphatics

(Dickstein et al, 1999). Recent experiments have demonstrated that tbe removal of

cervical Lymph nodes lessens the impact of EAE in rats (Philiïps et al, 1997). Taken

together these data prove that communication exists between the CNS and the immune

systern.

If CSF lymphocytes belong to the RLP, this would be yet another connection

between the CNS and the immune system. Therefore, experiments were desigoed and

perfomd to test if this connection is present. First, the normal concentration of cells

within the CSF of sheep was determuied to be 3.0 x 1o3 celWrnl f 0.3 which is similar to

humans (Svennhgsson et al., 1995). The major* of celis are srnail, nomial appearing

lymphocytes.

M e r repeated intravenous infusions of FITC Iabelled efferent LL over severai

days, labelled ceiis were identifîed in CSF- In 3 separate experiments, the percentage of

1abeIIed ce& was similar in blood, CSF, efferent lymph and Lymph nodes (Figure 10).

These preliminary experiments were required to eosure that iabelled cells could be

detected using our methodologies and to confirm that lymphocytes migrate into the CSF.

In agreement with previous reports @LUC et aL, 1994)- it was critical to place CSF

samples on ice and avoid prolonged cenvifugation to maintain the integrïty of the ceh.

Next, experiments using a singie bolus of labelied cells were performed allowing

for kinetic studies (Table 6). Efferent lymph and CSF were found to contain similar

percentages of labelled cek, 0.50% f 0.16 and 0.618 k 0.26 respectively (n=S)-

Additionally, in 2 experiments, multiple samples of CSF and efferent lymph were

coiiected over a 24 hr period and found to contain sunilar percentages of labelied cells

(Figure 12). Taken together these data ïmply that lymphocytes in CSF are part of the

EUP. Further experiments labelling both blood and efferent lymph lymphocytes

confirmed this (Table 7). Di[-DS labeiled LL were found in higher percentage than FI'ïC

labelied BL, (0.52% f 0.15 vs. 0.03% +, 0.02). The few FLTC labelled cells detected in

CSF rnay belong to the RLP but were labelled as they were in transit through blood- A

siagpSicznt ciifference (pc0.01) in the percentage of FITC labelied cells in blood and CSF,

0.72% f 0.13 vs. 0.03% I0.02. This implies that BL do not migrate weii fiom blood into

CSF. Expressing the data as percentage of injected ceiis codirmed that U are found in

greater numbers than BL (Figure 17)- arefore, m normal CSF LL m-grate into the CSF

in greater numbers than BL.

These data imply that lymphocytes are wt reqoired to be activated to migrate into

n o r d CSE Previous studies have examined the phenotype of Ipphocytes in CSF and

concluded that the majority of celis in CSF are activated CD4 celis (Vrethem et ai.,L998;

Scoloui et ai., 1992; Mix et aL, 1990). Others have disputed these kdings and argue

that CSF cells do not need to be activated (Svenningsson et ai., 1995; Kleine et al., 1999).

Several reasons may account for these discrepancies including the selection of patients.

Often subjects are recruited fiom patients seen in hospitals and cluiics and have

underlying non-inflammatory neurological problems but are considered as 'cnormai".

However, Svenningsson et ai. (1995) examined healthy volunteers without a history of

neurologicai disease and did not îind that Lymphocytes isolated from CSF expressed

activation markers. Another explanation for these discrepancies is the variations in

critena that different investigators use to determine activation, Vrethem et ai. (1998)

w d the expression of CD45RA+ to assign activation status, whilst Svenningsson et aL

(1995) utilise CD25 (IL-2 receptor). The results repted here support the findiags that

nonactivated cells migrate into CSF. This statement is based on the fact that normal

efferent lymph contains approdtely 5% activated celis as measured by CD25

expression (Haig et al., 1999). As weii, the majority of lymphocytes in efferent lymph

are small noncycling lymphocytes, thereby implying a low level of activation- Therefore,

if oniy activated lymphocytes migrated into CSF, we would expect to fhd Iow ievels of

k h d e d ce&. W e fouad sirnilar levels of labeiled cells in both lymph and CSF implying

that nonactivated lymphocytes do migrate into CSF.

If these Lymphocytes are tculy part of the RLP, they must be able to exit the CSF.

Experiments using 111-In labelied lymphocytes infused into the lateral ventricles found

amounts of radioactivity in retmpharyngeal lymph nodes that was 5 times higher than

other lymph nodes (Figure 13). Previous reports have also demonstrated this (Carson et

ai., 1999; Oehmichen et al., 1979).

Boulton and colleagues (1997) demonstrated that cervical lymphatics in sheep are

responsible for transporting approximately 50% of a protein tracer infused into CSF-

These same pathways may be used by lymphocytes to migrate into the retropharyngeai

lymph node. To inveaigate this, cervical lymphatics were canulated and monitored for

intracerebroventricular infused FITC labeiied cells. In only 2 of 6 experiments labelled

celis were detected in cervical Lymph, nonetheless, in every experiment labelled cefi

were found in retropharyngeal lymph nodes at necropsy. These data demonstrate that

labeiled cells appear to migrate using the cervical lymphatics and enter retmpharyngeal

lymph nodes. There are severai reasons that iabelled cells were not consistently found in

cervical lyrnph. First, the number of labelled c e k would have ken a minor population

of lymphocytes in cervical lymph and possibly below our of detection. Secondly,

the infusion of a large number of lymphocytes (approximately 2 - 5 x 108j, initiated an

inIlammatory response as seen by an increase in neutrophils in the CSE Lastly, the large

number of celis may cause a physical obstruction of the pathways used by the

lymphocytes to exit, as under normal conditions oniy thousands of cells would migrate

every day.

hmrine surveiiIance of CSF occurs under normal conditions as shown by the

ingress and egress of lymphocytes. The data presented in this chapter are the moa

complete nirvey of this dynamic process to date and demonstrates that U are found in

CSF in greater numbers than BL. hdeed LL are also found in a greater percentage in

&erent lymph, indicating that this pool has a larger role in tissue surveillance, includuig

the CSF. Percentages of labelied ceiis in tissues is not a perfect method to determine

migration, as differing amounts of infûsed cells will impact this measmement but

measuring percentage of injected ceIls accounts for this varïability. Expressing the data

with this method contirms that LL are found in a greater percentage than BL in CSF.

Unfortunately, the ce11 concentration of afferent lymph was not measured, so the direct

cornparison of these two tissues can not be made. Nevertheless, the data clearly shows

that U migrate into CSF in a greater numbers then BL,

Taken together, these data demonsuate that those lymphocytes present in CSF

under normal physiological conditions are mainly from the RLP. Lymphocytes do

recirculate through the CSF and perhaps have a role in the immune surveillance of the

CNS -

Chapter 6 TNFa injections into CSF, but not the brain parenchyma, results in leukocyte reccuitment

No tissue outside of the Lymphatic system has been investigated for the migration

of LL and BL under idlammatory conditions. Under normal conditions, lymphocytes

belonging to the RLP are present in CSF. htracerebroventricular injections of TNF-a

were used to recnùt lymphocytes into CSF and labeiled ceils fiom both pools were

quantified. TM-a induced a . inEIammatory response in CSF, which by 48 hours

postinfusion was predominately composed of CDS+ lymphocytes. Lymphocytes fiom

blood were present in the infiamed CSF in greater percentage than LL. Injections of the

same amount of TNF-a into the parenchyma of the brain did not hcrease CSF cell counts

and resdted in minimal leukocyte recruitment. These data provide evidence that BL

respond to inflammation in the CSF in a greater percentage as compared to LL. As well,

these data provide fiirther evidence that the CSF and CNS respond differently to

inflammatory stimuli.

6.2 Introduction

During meningitis and sorne infl-tory reactions within the CNS the number

of leukocytes in CSF hcreases. These ieukocytes may be a mixture of neutrophils,

monocytes or lymphocytes dependhg on the stimuli (Schoning et ai, 1999; Bamborschke

et 1990)- Cytokines, chemokines and adhesion molecules orchestrate tk migration of

leukocytes into CSF (Spelierberg and Tuornuien, 1994).

The CSF and brain parenchyma differ in their immuw Iogical response to antigen.

BCG injected ioto the CSF resuits in a robust immune response, but has a minimai effect

when injected into the parenchyma (Matyszak and Perry, 1996). Injections of TNF-a

into CSF resdts in increases in leukocytes (Schoning et aL, 1999) but has v h & y no

effect when injected into the brain parenchyma (Andersson et aL, 1992). This maybe due

to varying concentrations of neuropeptides, immunosuppressant factors or ciifferences in

vasculature (Phillips and Lampson, 1999). Therefore, the CSF and the brain parenchyma

must be considered as separate compartments with respect to inflammation and leukocyte

migration.

Dickstein et al. (1999) have shown that radioactive labelled TNF-a can be

transported to the retropharyngeal lymph nodes and cem-cd lymph. The function of this,

if any, bas not been elucidated. The sanie lymph nodes are important in the immune

response to CNS infused antigen (Harling-Berg et al 1999; Harhg-Berg et al., 1989). It

is conceivable that TNF-a kv ing in the lymph node with antigen may potentiate the

immune response by increasing lymphocyte tranic into the lymph node.

Studies were performed to determine if inuacerebrovenuicular injections of Tb?F-

a înduced inflammatory conditions in sheep. Additionally, the effect of TNF-a on BL

and LL migration into the CSF, lymphocyte subsets in the idammatory infiltrate and its

effect on cervical lymph was investigated. The migration of lpphocytes into CSF

injections of TNFa into brain parenchyma was also examined.

6.3 Materials and methods

6.3.1 Animais and surgery

AU experiments were carrïed out in the Division of Comparative Medicine at the

University of Toronto. Sheep of approximately 6-8 months and approxbately 30 kg

were obtained from Boxwood famis. Surgeries were performed as outlined in Chapter 2

including cenical, prescapular and prefemoral efferent Iymphatic cannuiation- Guide

screws were irnplanted at least 5 days prior to the lymphatic cannulations to allow

adequate healing.

6.3.2 Cell labelling

Blood c e k were labelled with FITC and lymph lymphocytes with DiI-DS as

explained in chapter 2, Cells were reinfused and allowed to equili'brate for at least 24 hrs.

6.3.3 TNF-a injections

Five hundred ng of recombinant human TNF-a (Cederiane Hornby ON) was

diluted in 1 ml of sterile saline and infiised into the lateral ventricles using the implmted

guide screws. Two Werent lots of TNF-a were used, both giving similar results-

Previous studies have shown human TNF-a WU induce an inflammatory response in

sheep (Kallaaji et al., 1989).

If the cytokine was to be injected into the brain parenchyma, 500 ng was diluted

in 25 pl of saline. It was injected approximately 1 cm into the cortex overlying the lateral

ventricles using a Hamilton syringe. The syringe was slowly removed after king left in

place for a few minutes to prevent backaow dong the needle track The opposite side of

the brain was lnjected with saline to serve as a control-

6.3.4 CSF collection, differential and phenotyping

CSF was coiiected by lumbar puncture as previously descnibed in Chapter 5. A

cell count was determined and a slide made by cytosph This slide was stained with a

modified Wright stain and a differential performed. CSF ceiis were washed twice with

PBS and at l e s t 20 000 cells immunophenotyped as per Chapter 2.

6.3.5 Brain tissue collection

Sheep were sacrficed and the braia immediately removed- If TNF-a had been

hjected ïnto the brain, the areas under the guide screws were exckd dong with control

areas. If the cytokine had been infused into the CSF, representative areas of cortex,

choroid plexus and areas surroundhg the ventricles were removed. Brains fiom control

animais were obtained which had never experïenced any CNS surgery and therefore

served as normal controls.

The tissue was placed in saline soaked gauze and placed on ice as soon as it was

removed It was then embedded in OCT, fiozen in liquid nitrogen and stored at -80°C.

6.3.6 lmmunohistochemistry

Blocks of brain and control tissues were cut at 6-8 pm using a cryostat and ptaced

on poly-L Lysine coated slides. These slides were ailowed to dry overnight and stored at -

20°C until use. hunohistochemistry was perfonned as outlined in Chapter 2.

6.4 Results

6.4.1 Leukocyte number, differential and phenotype after the

intracerebroventricular infusion of TNF-a

M e r the infusion of 500 ng of TNF-a into the iateral ventricle, there was a ciramatic

bcrease in leukocyte numben. At 24 and 48 hm postinfusion the leukocyte count was

5.7 x 10' t 1.4 (n=8) and 1.3 x 106 + 0.5 (n=7) respectively as compared to the n o d

count of 3 -0 x ld/ml (p c 0.05) (Figure 14).

Under normal circumstances, the leukocytes present in CSF are predominately

smdl Lymphocytes. Twenty-four hours after the infusion of TNF-a the majonty of

leukocytes in the CSF were neutrophils and monocytes with Lymphocytes king a s d

population (Table 8). However, by 48 hrs postinjection lymphocytes composed

approximately 70% of the leukocytes and neutrophils had decreased.

Using flow cytometry the lymphocyte phenotype was determîned. At 24 hr (nd)

CD4 cells were the predominate lymphocyte subset present (38.5% f 8.0) and CD8 celis

were lower (14.6% k 4.0). However 48 hr postinhision CD8 cells had increased to 37.2%

t 6.0 and CD4 c e k had decreased to 14.6% + 4.0 (n=3). B c e k and y6 T ceils were

minor populations of approximately 15 4b at both tirnes (Table 9).

6.4.2 CSF leu kocyte numbers after brain parenchymal injections of TNF-a

Forty-eight hours after the parencymal injection of 500 ng of rhTNF-a there was

no increase in the CSF cellulanty (2.0 x lo3/ml f 0.5 (n=3)) as compared to normal CSF

(3 -0 x 10' f 0.3 cells/d. Because of this low ceil number no phenotype or dBerentia.1

couid be performed.

6.4.3 BL and LL migration into CSF after TNF-a injection

After the infusion of T M - a into the CSF, FKC labelled BL increased and

surpassed the percentage injected of Dit-DS IabelIed LL, 2.7 x 10-~% I 1.3 vs. 81) x 10-

4 % f 3 (n=6). The ciifference between the two pools was not statisticaiiy signincant

(Figure 15). Forty-eight houn was chosen as the samphg t h e as previous experiments

had shown that this was the tune of maximum Lymphocyte recruitment. h 5 of 6

experiments, the percentage of m C labeued celk was higher in CSF as compared to

lymph but was aiways lower than blood (Table 10). DI[-DS labeiied LC in 5 of 5

experiments were lower in CSF as compared to lymph but varied as compared to blood.

Due to various technical difficulties including poor labelling of ceils and blood

contaminated CSF collections, the perceotages of the two pools could not be determined

after the parenchpal injections of TNF-a.

6.4.4 Irnmunohistochemistry

The ~unohistochernical analysis of the brain removed after the CSF infusion of

500 ng of TNF-a demonstrated a widespread infiammatory infiltrate surroundhg vessels

(n4). The phenotype of this appeared to mirror that found in the CSF, that is CD8

lymphocytes predominated but populations of CD4 and y6 T ceils were present (Figure

16). B cells and CD25+ cells were rarely detected The anti-CD25 aotibody used in

these studies has not k e n previously characterised for immunohistochemïstr~ and

therefore the results should be interpreted with caution. Lectin stalliing ushg GSA 1-B4

was perfonned for the detection of macrophages, wbich were located in small numbers in

the infiltrate. EndotheIIal ceils were &O stained with this Iectin as previousiy reported

(Kmeger et al, 1995).

The choroid plexus was examuied 48 hours after the intracerebroventricular

infusion of TNF-a. In control choroid plexus, macrophages were seen as were CD4

CD8 and y6 T ceiis (Figure 17). M e r the intracerebroventcicular injection of TNF-a the

numbers of ceiis appeared to increase and were located around the vendes and in the

stroma of the choroid plexus. However, without quantitative measures, the results must

be interpreted with caution, but it appears that TNF-a injections increase the number of

lymphocytes in the choroid plexus,

In contrast, the injection of TNF-a into the cortex of 3 sheep demonstrated very

different results. At the site of injection there was iïttle increase in the number of

leukocytes over that found in the saline control injection site (Figure 18)- In control areas

distant fkom the site of injection, no Lymphocyte infiltrate was seen.

There appeared to be an increase in the amount of VCAM on small vesseis in the

brain and the choroid plexus afier the CSF infusion of TNF-a. However, it was diffcult

to quantify.

Control brain tissue removed from sheep with no CNS surgery had vimidy no

CD4, y6, or B ceus present but some CD8+ stainuig ceiis were scattered throughout the

parenchyma. It seems unlikely that CD8 lymphocytes are present in the normal sheep

brain as lymphocytes are rarely seen in the <3NS of other animals but there are no prier

studies in sheep for cornparison. Ail tissuas with normal muse IgG or without primary

anti'body exhibited minimal background staining.

normal 24 hours 48 hours

Figure 14 CSF celiularity increases after the injection of TNF-a

Mer the intracerebrovenuicu1ar injection of 500 ng of rhTNF-a the number of

leukocytes increases significantly (@.OS) at both 24 ( 0 ~ 8 ) and 48 hr (n=7).

Table 8 Leukoryte difEereotia1 M e r the intracerebroventricular injection of TNF-a

Twenty-four hours after the injection of TNF-a the leukocyte idtrate is dominated by

neutrophils and monocytes (n=9). However, at 48 hours lymphocytes predominate (n=!5)-

Leukocyte type 24 hours 4û ~OULS

neu trophils 68 t4 10t4

monocytes 2 L I l 17f 6

lymphocytes 10tL 72 & 10

Table 9 Lymphocyte subsets present after the intracerebmventriculr hjection of TNF-a

There is a change in the subsets of lymphocytes present in the CSF after the injection of

TNF-a. At 24 hours postinjection (UA) CD4 c e k are the predominate lymphocyte

present, but by 48 hours (n=3) CD8 ce& predominate.

Note that due to the s d amount of CSF coilected only 1 sample was

immunophenotyped for y8 T ceiis at 48 hours. There is no signifcant ciifference (p

0.05) between any of the subset percentages at 24 and 48 hr-

Lymphocyte Subset 24 hours 48 houls

Figure 15 Both BL and LL increase after the intracerebroventricular injection of TNF-a

DZ-DS Iabeiled LL are present in a greater amount in normal CSF as compaced to FIT%

labeiied BL (n=5). Both DiI-DS IabeIled and FlTC labeiled lymphocytes increase

significantly after the injection of 500 ng rhTNF-a (pc0.05). FITC labeiied BL increase

to a greater extent than Da-DS IabeUed LL after the injection of TNF-a (nt@. The

percentage of iojected celis was used to quantify the ce& as it accounts for the clifferhg

amounts of infUsed IabelIed lymphocytes.

Samples of CSF were coliected 48 hours &er the infusion of 500 ng of rhTNF-a into the

lateral ventricIe.

A - normai CSF

B - CSF after the rhTNF-a injection

% injected DiI-OS O h injected FlTC

B CSF 48 hr &ter a TNF-a injection

% injected Dii-DS % iniected FKC

Table 10 Percentage of iabeiïed c e b in CSF, biood and efferent lymph 48 hr after the intracerebroventcicular îqjection of TNF-a

After the intracerebr~vent~cular uijection of TNF-a FITC labeiied blood Lymphocytes

increase as compared to LL. As compared to nomial CSF, FïTC labeiied c e k c m be

found in aU sarnples of inflamed CSF. There is no signincant difference (p>O-05) when

the percentage of labeiied ceils in CSF is compared to blood or Lymph-

ND = not determined

Percentage FLTC labeiled ceüs Percentage DiI-DS iabelled ceUs

E ~ P # B l d L Y ~ P ~ CSF Blood L Y ~ P ~ CSF

1 0.42 0, 19 0.25 0.33 0.47 0.4

Figure 16 CNS parenchyma after an intr~rerebroventn*calar injection of TNF-OC

An idammatory infiltrate is seen in the perivascular spaces throughout the CNS 48 h .

after the intracerebroventricular infusion of rhTNF-cm CD4 ceiIs are present in the

infiltrate (A) but CD8t cells predominate (B). y6 T c e k (C) are also present. MHC II is

upre,oulated on what appear to be endotheliai ceils (D), even in areas with few leukocytes

in the perîvascular space. There is a scattering of unidentined ceiis in the b r d

parenchyma that express CD8 (E) but there are no y6 T cek (F) in normal sheep. AU

photos were printed at 200x mgnification-

A - CD4 ceiis

B - CD8 ceils

C - y6 T ceiis

D-MHCII

E - CD8 staining in control (normal) brain

F - y6 staîning in control ( n o d ) brain

- ILL-

Figure 17 Choroid plexus Pfter an intracerebroventricular iqjection of rhTNF-a

In n o d choroid plexus, CD8 stalliing c e k are present in the stroma (A), but after 48 hr

after the injection of TNF-a there appears to be a slight increase in numbers (B). MHC II

staining is also present in stromal ceiis under normal conditions (C). ikely due to resident

macrophages. Forty-eight hours after the injection of T3JF-a the number of ceh appears

to increase, but this may be due to an upregulation of the amount of MHC II on celis @)-

There is a s d amount of VCAM stainuig on seromal cells (E), which appears to

increase after the injection of TNF-a.

A - CD8 normal choroid plexus (2ûûx magnification)

B - CD8 alter the injection of TNFa (2ûûx magnification)

C - MHC II normal choroid plexus (400~ magnification)

D - MHC II post TNF-a injection (400x magnification)

E - VCAM normal choroid plexus (400x magnification)

F - VCAM after TNF-a injection (200x magnification)

Figure 18 CNS pareachyma d e r the intmcerebral iqiection of rbTNF-a

- - There is minrmal hfiammatory infiltrate 48 hr after the injection of rhTNF-a into the

parenchyma as compared to the injection of saline. Few CD4+ ceils are seen in the area

after TNF-a (A) or saline (B). TNF-a (C) and saluie @) sites of injection attract

minimal numbers of CD8+ cek or y6 T cek. AU photos were p ~ t e d at 400x

magnific atio n,

A - CD4 cells afier a parenchymai injection ofTNF-a

B - CD4 ceiis after a control injection of saLine

C - CDS+ ceUs post TNF-a injection

D - CD8+ cek d e r a saline injection

E - y6 T ceils after TNFa

F - y6 T ceiis post saline injection

Figure 19 Iatracerebroventncuiar injection ofrhTNF-a has no effect on cervical lymph flow or ce11uiarity

Five hundred ng of rhTNF-a injected at O hr did not significantly affect the Lymph flow

or ceilularity of cervical lymph (n=3). Ratios were determined by dividing al i values by

the average value obtained during the 6 hr baseline.

A - cervical lymph flow

B - cervical lymph ceii concentration

A Cervical Iymph flow

Time (hr)

B Cervical lymph cell concentration

4 - 2 1 3 5 7 9 1 1 1 3 2 0 2 4

Time (hr)

6.4.5 The effect of intracerebroventncular injection of TNF-a on ceMcal lymph

In 3 sheep, the cervical Lymph was sampled after the injection of TNF-a into CSF.

The percentage of CD4, CD8, y6 T cek, B cells, CD45 and L selectin positive

lymphocytes were monitored. There was no significant change in any of the subsets (not

shown), nor in Lymph flow or ceii output (Figure 19). Lymph flow did decrease at

approximately 2 hours afier the injection but this may be due to the animai k i n g in a

recumbent position due to increased sleep as been previously demonstrated by Dickstein

et al, (1999).

6.5 Discussion

TNF-cf is present in a number of inflammatory diseases in the CNS includuig

menïngitis and MS. Previous reports have demonstrated that an infusion of =-a into

CSF causes a leukocytosû (Paris et al., 1995; RamiIo et al., 1990). Many of these studies

were performed under anaesthesia (Saukkonen et al., 1990) and/or the experhents were

limited to 24 hours or less (Quagliareilo et al., 1991). As mice and rats were used in

previous studies, immunopheno typing of recruited lymphocytes was not possible (Tang et

al., 1996). Therefore, experiments were perfonned to determine which pool of

lymphocytes responds to TNF-a and the subsets recruited. Additionally, the ciifference

between parenchymal and ventricular injections of TNF-a was investigated with respect

to Lymphocytes recruited and CSF ceilularity.

The infusion of 500 ng of rhTNF-a caused a 1000 fold iocrease in the number of

CSF leukocytes at both 24 and 48 hours. M o u s snidies dexnonstrated a peak in

leukocyte numbers within 12 hr after the infusion of TNF-a Cparis et al-, 1995; RamiIo et

al., 1990) with a decrease by 24 hr. In contrast, we found a sustained CSF ceudarity,

which lasted unti148 hr postinntsion (Figure 14). There are several explmations for this

discrepancy. Firstly, other midies used rabbits, rats and mice, whilst we used sheep. AS

w e k human recombinant TNF-a was used in our experiments instead of species specific

cytokine. Prior studies have demonstrated that human TNF-a recruits Lymphocytes into

skin sites in sheep (Kalaaji et al., 1989) and induces lymph node shutdown (Young et ai.

2000). Therefore, this cytokine appears to cross react in sheep and is unlikely to be the

cause of the dflerences observed. The dosage of injected TNF-a varied widely between

different studies. We used a bolus injection of 500 ng of TNF-a, whilst QuagliareUo et

al. (1992) used up to 20 pg in a rats and Angstwurm (1998) used injections ranging fiom

5 pg to 280 pg in rats. Similar results to our study were obtained by others using a

continuous infusion of TNF-a over 48 hrs (Schoning et aL, 1999). There is evidence to

suggest that TNF-a acts as an anti-inflammatory cytokine during EAE (Liu et al., 1998).

Therefore, it is conceivable that the larger doses of TNF-a resulted in a decreased

recruitment of leukocytes into CSF.

A study to examine the effiects of various TNF-a doses on leukocyte migration in

sheep was not perforrned. Five hundred ng of TNF-a was injected in di experiments

based on previous work by Dickstein (1999), which demonstrated that this dose of

cytokine induced sleep in sheep. Additionally, this dose was chosen to mimic levels

found in CNS infiammatory conditions. Udortunately, there was no method to measure

the levels of CSF TM-a afier injectioa Theceforefore, we can not ascertain if the levek

were comparable to those found in CNS inflammatory conditions.

Few snidies have examined the type of Ieukocytes present in CSF afier TNF-a

infusion. Tang et al. (1996) demonstrated an increase in neutrophils in response to TNF-

a - L p injections at eariy tirnepoints (2-6 hr) and mownuclear celis at 8 hr. In sheep

neutrophils and monocytes predominate in the h s t 24 hrs after TNF-a. By 48 hrs, these

Ieukocytes were a minor population with lymphocytes predominating amongst recmited

ceiis (Table 8). CD4 cells are the most nunmerous subset at 24 hrs but CD8 lymphocytes

predominate by 48 hrs (Table 9). A previous in vitro snidy suggested that CD8 ce& do

not migrate across brain e n d o t h e h as quickiy as the CD4 subset of Lymphocytes (Pryce

et al., 1994). These in vivo data reported here confirm this, as CW ceils predominate

early but are surpassed by CD8 Lymphocytes at 48 hr.

Lymph Lymphocytes are present in CSF in a greater percentage under normal

ckumstances therefore; 1 hypothesised that this pool would aiso migrate in response to

TNF-a in a greater percentage as compared to BL. However, cornparing the percentage

injected of labelied BL to labelled LL, 2.7 x 10-~ & 1.3 vs. 8.0 x 1 0 ~ t 3 (Figure 15), it îs

clear that BL migrate in greater percentage. The merence between BL and LL was not

statistically significant, however the increased percentage of BL in the infiamed CSF may

have biological signifïcance. As well, FïïC Iabelled BL are present in a higher

percentage in CSF as compared to lymph (0.61 f 0.14 vs. 0.34 10.3) (Table 10). This

indicates that BL were not non-specificaiiy migrating into aii tissues but were selectively

recruited into the ïnflamed CSF. This could be accomplished in one of two ways.

Firstly, there may be a pool of rapidly recirculating lymphocytes recruited into the CSF as

in the antigen stimulated Lymph node (Chapter 4). An alternative explanation is that the

idamed CSF preferentially attracts BL. Perhaps blood lymphocytes have higher levels

of adhesion molecules or chemokiw receptors ailowing them to enter the CSF- This

should be tested in funire experiments.

Due to smaU numbers of labelled ceh, immunophenotyphg of the recruited FJTC

and DiI-DS Lymphocytes was not performed. This would have allowed the direct

cornparison between the antigen stimulated lymph node experiments and these

experiments.

Intracerbrovenuicular TNF-a injections resulted in a widespread infiammatory

infiltrate within the perivascular spaces of the brain. The recmited lymphocytes did not

appear to migrate into the parenchyma of the CNS. This may be due to lymphocytes

undergo ing apo ptosis outside of the perivascular space as previously reported (Bauer et

al., 1998). Imrnunohistochemistry demonstrated that the perivascular infiitrate was

similar to that seen in the CSF with respect to lymphocyte subsets, that is

CD8>CD4>ybB cells. There were rare CD2k lymphocytes in the infiltrate indicating

that the majority of celis were not fully activated. This is similar to other studies that

investïgated MS (Hofinan et aL, 1986), EAE (Korner et aL, 1997) and toxopiasmosis

infection of the brain (Deckert-Schluter et al., 1994). If few activated lymphocytes are

present in these autoimmune and parasitic diseases it wodd be expected that few

lymphocytes would be activated after a cytokine injection. MKC II is present in small

amounts in nomial control brauis but is upregulated after the intracerebroventnfular

injection of TNF-a, simiiar to results obtained in rats after IFN-y injections (Vass and

Lassrnaan, 1990). The infT1trate included MHC II positive cells, which may be

macrophages, either recruited fkom blood or activated CNS macrophages. The

endotheIium of blood vesseis also appeared to be positive for MAC II as previously

descnibed in visna Wus infected sheep (Torsteiosdottir et al., L992).

Choroid plexus samples were taken to determine if Lymphocytes migrate dicectiy

into the CSF fiom blood as suggested by others (Engeihardt, 1997). The data obtained

was not dennitive but there appeared to be an increase in lymphocytes within the choroid

plexus and VCAM was expressed on choroid plexus bbod vessels. These data m e r

kom previous reports tbat demonstrated an increase of VCAM on the epitheiial c e h of

the choroid plexus but not on the endothelia1 cells (Wolburg et al, 1999; Steffen et al,

1996). This maybe due to diffierences in experimental protocois (EAE vs. TNF-a

injections) and animai species (mice vs. sheep). Further experiments are required to

determine if choroid plexus can support leukocyte extravasation into the CSF.

Afier an injection of 500 ng ofTNF-a into the cortex of the braïn, no h e a s e in

CSF cellularity was seen. There was Little inaammatory ianltrate at the site of injection

as compared to saline injected controls. This is in contrast to other reports (WdIenborg et

al., 1993; Wright and Merchant, 1992), which demonstrated that TNF-a injections

resulted in leukocyte innltratioe The discrepancy maybe due to a species ciifference or

arnounts of TNF-a injected. The dose of TNF-a used in the study by Wright and

Merchant was 5 tirnes the dose used in the present study. Schnell and colleagues (1999)

demonstrated that TNF-a injected into the spioal cord of rats resulted in an inflammatory

infiltrate, but the brain parenchyma was resistant. WiUenborg et al. (1993) used the

spinal cord and Wright and Merchant (1992) the parietal lobe, while the present snidy

injected TNF-a into the cortex overlying the lateral ventricles.

TNF-a has been shown to into the rettopharyngeal Lymph nodes but the

function of this is stili unknown (Dickstein et aL, 1999). Its injection into CSF did not

have a discernible effect on cemecai Lpph with respect to Lymphocyte subset, ffow or

cellularity. Perhaps the parameters exûmuied in this series of experiments were not able

to detect subtle changes that may be present. TNF-a causes lymph node shu tdom

which may have a role in the immune response to some Ag (Young et al., 2000). It is

conceivable that TNF-a draining lÏom the CSF may increase the immune response to Ag

arrïving fiom the CSF (Harhg-Berg et al, 1989). The drainage of proinfiamatory

cytokines into retropharyngeal lymph nodes may assist in the activation of T c e b

specifc for antigens sequestered in the CNS. This is speculation at this point and fuaher

studies are required.

In conclusion, the data in this chapter fuaher demonstrates that a pool of

lymphocytes present in blood may be able to rapidly enter sites of uinarnmation.

Intracerebroventricular TNF-a injections resulted in a rapid increase in ieukocytes b ~ t h

in CSF and brain parench- Simi1a.r injections into the cortex did not increase

ieukocyte numbers in either cornpartment.

Chapter 7 General Discussion

7.1 Introduction

The recirculation of lymphocytes aliows for the dissemination of immuno1ogica.l

memory and immune surveillance (Butcher et ai., 1999). Recently, in sheep, a pool of

blood lymp hocpes has been descnibed whic h recirculates poorly through lymp hatic tissue

(Andrade et al., 1998; Chevallier et al., 1998; Young et ai., 1997a). The main objective

of this thesis was to investigate the migration of the blood pool of Lymphocytes in

cornparison to the lymph pool of lymphocytes in a variety of compartments under n o n d

and hfhnmatory conditions. Experiments were conducted to compare the migration of

LL and BL into CSF under both normal and TNF-a induced inflammation- As well,

experiments were performed to determine the migration of the two pools after

splenectomy and through an antigen stimulated lymph node. Together these data extend

earlier studies and show that a functional dinrerence may exist between BL and LL.

Sheep were used in aii experiments in this thesis, as they permit chronic

collections of lyrnph. Additionaiiy, a large body of Iiterature exists on lymphocyte

migration in this animal, allowing a critical cornparison of my data to others. Indeed it

has been said that more is kno wn about the in vivo physio logicai dynarnics of lymphocyte

recirculation in sheep than in any other animal (Abemethy and Hay, 1992)

In this discussionT the main conclusion fkom each major study will be re-

examined, the limitations of each wiU be considered and funher experiments suggested-

At the end of ehis chapter, a series of experiments to investigate the existence of a rapidly

recirculating pool of lymphocytes is proposed.

7.2 Investigations into the migration pattern of BL and LL into afferent

lymph and after splenectomy

The main objectives of this senes of experiments was to extend our basic

knowledge of BL migration. The h t report of BL appeared in 1994 (Young, 1994) but

Little was kaow about these Lymphocytes beyond thek retention in blood (Andrade et al.,

1998) and that B ce& compose the largest population of BL (Gupta et aL, 1998; Young

et al., 1997a).

Repetitive sequential sarnpling of blood and lymph was performed to gain insight

into the stability of the relative proportion of BL and LL. The migration and cornparison

of LL and BL into normal afferent lymph was determined adding an additional

cornpartmentai analysis. Since there are significant ciifferences in the cellular

composition of blood and aEerent lymph (Haig et al. 1999)- it was thought that the two

pools may differ in their migration. Additionally, prior studies had demonstrated changes

in lymphocyte subpopulations in blood d e r splenectomy (Sieber et ai. 1985) and it was

hypothesised that this maybe due to changes in the migration or number of BL-

7.2.1 Repetitive sampling

The relative proportions of the labeiied BL and LL remained constant in both

blood and efferent Lymph over the 27 hour sampling period (Figure 4).

A Iunitation of the repetitive sampiing experiments was the method used to

coilect sarnples as the animal was disturbed hourly during the expriment. This =y

result in stress due to alterations in the normal sleep pattern of the sheep, which

affect lymphocyte migratioa A remote sampling technique to which the sheep was

oblivious, as used by Dickstein (1999), wodd have avoided this problem Nonetheless,

these experiments confkmed previoas fïndings that BL and U are enriched in blood and

Lymph, respectively (Andrade et aL, 1998; Young et al., 1997a). Based on these hdings

the tirne of day of sampling was not considered a confounding factor ùi subsequent

experiments.

LL IabelIed with the fluorescent compouod, DiI-DS, recirculated fiom blood into

efferent lymph in percentages comparable to c e k labelied with other Lipophilic dyes

(Andrade et al., 1996a). This was considered a validation for the subsequent use of this

tracking label in sheep.

7.2.2 Afferent lymph

In afferent lymph, a ratio was determined by dividing the percentage of labelied

lymphocytes in afferent lymph by the percentage in blood Lymph lymphocytes are

enriched in aeren t lymph as compared to BL (3.1 + .96 vs. 0.8 & 0.26) (Table 2) The

appearance of labeiled cells in aerent lymph implies that they have migrated fiom blood

through tissues. Therefore, the population of lymphocytes in afferent lymph may reflect

that present in tissues.

A limitation of this study is that these samples were acquired at varyiag times

after the iofusion of labelled ceils. However, ail samples were obtained at least 3 days

postiofusion at which t h e previous studies (Andrade et al. 1998) have demonstrated that

the BL and LL have equihibrated between blood and efferent Lymph. Experiments to

examine the kiwtics of migration and standardised recoveries are required to compare the

two pools in afferent Lymph. As weli, the appearance of labeUed ceils in afferent Lymph

implies that lymphocytes have migrated into tissues but tbir is wt a direct measure-

Further experiments are required to detennine if BL migrating into tissues become

resident ceils or are deleted in greater numbers as compared to U.

7.2.3 Splenectomized sheep

In splenectornized sheep, no differences were detected in the relative proportions

of the analysed lymphocyte mbsets (Table 4)- Furthemore, the concentration of

lymphocytes was not different ikom intact sheep. The migration experiments

demonstrated that LL exit blood quicker as compared to intact sheep and enter lymph

nodes and liver in significantly greater numbers. This is demonstrated by the trend

towards fluorescently labelied LL to exit the blood quicker in splenectomized sheep

(Figure 5) and by a greater number of radioisotope labeiled celis localising in lymph

nodes and liver (Table 3). Blood lymphocytes demonstrated a trend to an increase in

blood and are found in Lesser numbers in the lung of splenectomized sheep. This may

indicate that the marginating pool in the lung is saturated and excluding BL fiom entering

in the normal m e r , thereby enriching this population in blood.

However, these experiments should be repeated in sheep that undergo

splenectomy at a later age. Previous reports have demonstrated that there is extensive

replacement and/or expansion of foetal T celis during the fkst week of life (Cahill et aL,

1997). The sheep in these experiments were splenectomized between 19 and 21 days

after birth, which may allow the newly forrned lymphocytes to adapt to the absence of the

spleen. Therefore, the data fiom these studies may not refiect the changes in migration of

BL and LL that result after adult splenectomy. As well, fkther snidies are required to

investigate B ceii function as there are abnofmalities in humorai immunïty in humans

folio wing spIenectorny.

Conclusion

The cepetitive sampling experiments demonstrated that BL and LL are maintained

at steady levels after equiii'brium and that the time of day did not &ect their

concentrations,

There are no other reports comparing BL and LL in aEerent lymph. Lymph

lymphocytes. which belong to the weii-characterised RLP (Young, 1999), are found in a

greater ratio in afferent lymph as compared to BL. These results indicate that LL migrate

through tissues in greater numbers than BL and therefore may have a greater role in

immune surveilIance.

The BL pool is present in normal numbers 2 years after neonatal splenectomy

ïmplying that the spleen is not necessary for its maintenance. DBerences are seen in the

migration of bo th BL and LL in splenectomized sheep, with LL migrating into peripheral

lymph nodes and the liver in greater numbers than in intact sheep. These data indicate

that after splenectomy LL may exit the blood in greater numbers to maintain lymphocyte

homeostasis,

These data extend previous reports regarding the migration of BL and provide a

foundation for subsequent experiments-

7.3 The migration of BL and LL through antigen stimulated lymph nodes

These were the first experiments to examine the m-gration of BL and LL

lymphocytes during ï p p h wde shutdown. They were part of a larger series examining

lymphocyte retention during lymph node shut down but much of that data was wt

included in this thesis as it was performed by collaborators (manuscript in preparation).

In agreement with previous studies (Mackay et al, 1992b; Bujdoso et al., 1989) CD4 T

cells increased over baseline in their migration through inflamed lymph nodes (Figure 8).

An increase in IFN-y and IL-6 in efferent Lymph during Lymph node shutdown was

observed (Figure 9) and may have a role in the recniitment and retention of Lymphocytes.

IFN-y can recruit lymphocytes when injected into the skin (Colditz and Watson, 1992)

and has been shown to retain naive lymphocytes withui the lymphatic tissue of rats

(Westermann et al., 1994b). IL-6 induces the production of chemokines in skin

infiammatory conditions (Romano et al., 1997) and may have a similar role in the lymph

node.

An increase in both CFSE (blood) and Di[-DS (lymph) iabelIed CD4 cells was

observed (Figure 8), indicating that both BL and LL contributed to the increase in CD4

cells. However, BL CD4 ceils increased to a greater extent than LL. One explanation is

that memory T cells are increasing as previously shown durhg antigen induced lymph

node shutdown (Mackay et ai., 1992b). However, this does not account for the greater

hcrease amongst CD4 BL unless blood memory CD4 cells preferentidy migrate into

inflamed lymph nodes. An alternative explmation is the existence of a rapidly

rec irculating poo 1 of lymphocytes. This hypothesis is M e r explored and experiments

proposed in section 7.5.

Previous experiinents have demonstrated that labelbg and infirsing a whole

population of lymphocytes is an effective method to study subset migration in rats

(Westernmm et al., 1993) and sheep (Andrade et ai., 1998). This avoids the separation of

subsets using various in vitro methods such as anh'body/compiement, magnetic bead

separation etc- Our methodology minimises Ln vitro rnaIumaIupulation of cells thereby

decreasing my chance of inadvertent activation and allows ail subsets to be examined

after in vivo migration. Using these techniques we observed several subsets present Ï n

BL and demonstrate that CD4 ceiis are not retained within the l p p h node to the same

extent as other measured subsets.

A Limitation of the present studies is that only one antigen, PPD, has been

examined for its effect on LL and BL migration. Other antigens including vinises and

bacteria should be investigated to ensure that this phenomenon is not unique to PPD-

Additionaiiy, the direct effect of cytokines including IFN-y and IL-6 on the retention of

lymphocytes within a single lymph node should be examined Previous reports have

demonstrated that both EN-a (Hein and Supersaxo, 1988; Kalaaji et aL, 1988)and N-

a (Young et al., 2000) induce lymph node shutdown.

Conclusion

These experiments were undertaken to examine BL and LL under infiammatory

conditions. Both pools appeared to contriiute to the increase in CD4 celIs during lymph

node shutdown but BL CD4 ceus increased to a greater extent. As weil, these data

suggest the existence of a rapidy recirculating pool of lymphocytes.

7.4 Lymphocytes in CSF are part of the RLP

CSF is the fîrst noniymphoid tissue to k examined for the differentiai migration

of BL and LL. Using FïïC Iabeiied efferent LL it was found that labelled cens appeared

in similar concentrations and times in both CSF and efferent Lymph (Table 6 & Figure

12). This impLies that CSF lymphocytes belong to LL or the RLP-

The data fiom this series of experiments argue that activation is not required for

lymphocytes to enter n o d CSF as previously reported (Vrethem et aL, 1998; ScoIozzi

et al., 1992; Mïx et al, L990). Less than 5% of efferent LL express the activation marker

CD25 (IL-2 receptor) (Haig et al., 1999). Therefore, ifonly activated T ceils migrate into

CSF it would be expected that the number of labeiied ce& wouId be Iow. Svenningsson

et al. (1995) argues that activation is not required for lymphocyte migration into CSF

based on a series of phenotypic markers on Lymphocytes obtained £tom normal human

CSF. They did not find any upregulation of CD25 or HLA-DR, both markers indicating

lymphocyte activation. Reasons for the discrepancy between snidies maybe due to

staining protocols or subject selection as often patients with tension headaches or other

neurologicai abnormalities are used as normal controls. A more likely explanation is that

the definition for activated T c e k is different in a i l studies- For example, Vrethem et al-

(1998) use the expression of CD45RO as the sole indicator of activation, whilst

Svenningson et al. use CD25+ and HLA-DR to determine activation of T cells. Based on

these ciifferhg dennitions of activation it is not surpriskg that fidings confiict. The data

in this thesis irnply that Lymphocytes are not required to be activated to migrate into the

CSF.

Studies were undertaken to examine the migration of LL and BL into CSF. AS

measured by percentage of injected cek , LL are present in greater numbers in normal

CSF as compared to BL, confinning that CSF lymphocytes are part of the RLP (Figure

15)- These results are similar to that found in affierent lymph (Chapter 3), implying that

LL have a greater role in immune surveillance of tissues as compared to BL.

A limitation to these studies is that due to problems m a i n t d g a patent catheter

in the subdural space, multiple samples of CSF over a 24 br period were not obtaiwd to

determine kinetic data for BL. As weil, fluorescently conjugated primary antibdies were

not avaïlable to perform immunophenotyping of Lymphocytes present in normal CSF- If

activation markers were examined it may ~ ~ p p o a the fbdings that nonactivated T c e k

migrate into CSF-

Conclusion

These data support previous reports that lymphocytes do not need to be activated

to migrate into the CSF- Lymph lymphocytes migrate into the CSF in greater numbers as

compared to BL. These data in conjunction with data from the afferent lymph

experiments, imply that LL have a greater role in immune surveiüance of tissues.

7.4 TNFa induced CSF leukocytosis

Several other studies have examined the proidlamatory effects of

intracerebroventriculat injections of TNF-a but in most of these studies, cytokine was

injected in amounts several times higher than the 500 ng injected in the present snidy.

For example, Angstwurm et al. (1998) injected up to 280 pg of TNF-a in adult rats, while

QuagliareIlo et aL (1991) used 20 ug. Both of these injected amounts are thousands of

fold higher than levels of TNF-a measured during CNS inflainmation. Glimaker e t ai.

(1993) reported levels of 1.16 n g h i of CSF during purulent meningitis, whilst in MS

values of 10-1 pg/ml of CSF are found (Druuiovic et ai. 1997). Previous work In our

laboratory demonstrated that an intracerebroventricular injection of 500 ng of rhTNFa

induced sleep in sheep (Dickstein et al., 1999) and produced infiammation in CSF.

Therefore, th% amount was used as it was determlned to cause inflammation in

preliminary experiments and had a physiological effect in vivo. As welI, it may be

similar to TNF-a levels measured during meningitis.

The concentration of leukocytes in CSF after the infusion of TNF-a was similar

to those reported in bacterial rneningitis 5.75 x ld celldml vs. 3.40 x 1o3 cellslml

(Ghaker et al., 1993). The phenotypic subset of recnUted lymphocytes changed over

the 48 hr observation period, with CD4 ceiis king the largest subpopulation at 24 hr but

by 48 hr, CD8 cells predominated (Table 9). These rii vivo data confirm a previous in

vitro report that CD4 ceiis migrate across cultured cerebral endothelhm more rapidly

than CD8 lymphocytes (Pryce et al., 1994)-

An intracerebroventricular infision of TNF-or was used to induce inflammation in

the CSF. This resulted in a 1000 fold increase in both BL and LL into the inflamed CSF

as compared to normal (Figure 15). However, BL were present in 4 times the

concentration as LL. At les t two explanatioas for the increase in FITC labeiled blood

c e k in the infiamed CSF are possible. 1) a rapidly recirculating pool of T cells is present

that enters sites of inflammation 2) blood CD8 cells are better able to migrate into CSF as

compared to other lymphocytes. Neither of these expianations can be discounted nom

these data. E x p e k n t s are proposed in section 7.5 to test between these alternatives.

CSF inflammation was induced with TNF-a in these studies but to ensure that

these fhdings are not unique to this cytokine fuaher experiments are required-

Intracerebroventricular injections of other cytokines should be examllied for their ability

to attract BL and LL hto CSF, As welI, viruses and bacteria can be used to mimic

meningitis to determine if these conditions cause the preferential migration of BL-

Conclusion

TNF-a induced inflammation in the CSF results in FITC labelied BL migrahg in

a greater percentage as coqared to Da-DS labelled LL. The FLTC Iabelied cells may be

part of a rapidly recirculating pool or a BL subset may be preferentially recnùted to the

inflamed CSF. Further experiments are required to determine which is correct.

7.5 Future experiments to determine the existence of a rapidly recirculating

pool of lymphocytes

Findings fio m the experiments examining Lymphocyte migration into the inflamed

CSF and during lymph node shutdown may be due to either of two explanations; A)

increased migration of memory T ceils. B) a pool of Lymphocytes may exist which

rapidly recirculates as compared to the majority of the RLP.

The increased CD4 and CD8 cells, in lyrnph and CSF respectively, d-g

inflammation may be memory T cells. They have been demonstrated to have different

migration kinetics through antigen stimulated lymph nodes (Mackay et al., 1992b) and

after the infusion of IFN-y pestermiann et &, 1994~). The increase in BL may indicate *

that blood memory T c e k respond to inflammation and migrate into antigen stimuiated

l p p h nodes or uiflamed CSF in greater numbers than LL memory T cek.

An alternative hypothesis is that a population of Lymphocytes exists which rapidly

recirculates and is found in transit through blood and lymph. As this population may be

present in both blood and lymph, the labelling protocols Ï n this thesis would label it with

either fluorescent dye. This population of c e k may be functionaily different fiom both

BL and LL and/or may express different adhesion moIecdes or chemokuie receptors.

There are data in the Literature to both support and refute this hypothesis of a population

of lymphocytes that rapidly recirculates.

In sheep, 11 1-In labelied lymphocytes are detected in efferent and afferent Lymph

within 5 hours of intravenous injection (Issekutz et al. 1981). As weil, in rats

Westennann et al. (1994b) demonstrated that FITC Iabeiied Lymphocytes are found in

thoracic duct lymph within 12 hours after intravenous injection. Both the sheep and rat

studies detect labelied lymphocytes in lymph preceding the t h e of maximum levels of

detected ceiis, 2 1 - 23 and 24 - 36 hours respectively. Fuahermore, there is a population

of lymphocytes in rats, that is rapidiy mobilised after thoracic duct lymphatic cannulation

(Westermann et al. 1994b). These studies hply that a population of rapidly recirculating

lymphocytes is present within the RLP,

However, Ford and Simmoads (1972) reported that in rats RLP lymphocytes

could not be aivded into "fast" and "slow" populations. This was based on a series of

experiments in which labelied lymphocytes were obtained either 12 or 36 hours &es

thoracic duct cannulatioa The two populations were then differentially labelied and

injected into syngeneic recipients. No merence in the kinetic appeacance of the two

popdations in thoracic duct lymph of the recipients was found. However, the rats

underwent a splenectomy less than 24 hours before the experiment was begun.

Splenectomy may affect the normal migration patterns of lymphocytes in adult subjects

by removing this important lymphoid orgaa Additionaüy, the shidy was performed

before the use of monocIonal antiidies to detect lymphocyte phenotypes- Westermann et

aL (1993) using monoclonai antiidies reponed rbat CDS lymphocytes recirculated with

slower kinetics than CD4 celfs. Therefore, there is data to suggest that the RLP is

composed of several populations of lymphocytes with varying migration khetics and

may include a rapidiy recirculating population of ce&.

Experiments to determine the existence of thû putative recirculating population

with rapid migratory properties are as foiiows;

1) Label blood and lymph ceils with fluorescent dyes and intravenously reinfuse.

Coilect efferent lymph hourly and phenotype the c e k for adhesion molecules includuig L

selectin, CD44 and a4 integrin. Phenotyping for activation and memory markers such as

CD45R.A would demonstrate if the labelled ceffs that first appear in lymph were memory

ceiis or activated. I hypothesise that the c e h recovered in lymp h on the ascending part of

a recovery curve may have higher levels of adhesion molecules than those recovered

during the descendhg phase.

2) It remains to be determined if the labeiied BL seen in efferent lymph d m g

Lymph node shutdown and those in CSF after =-a induced inflammation both belong

to a rapidly recirculating pool. 1 propose the following two experimental protocols to

examine this question. The h t series of experiments wouid require inaadermal

injections TNF-a and PPD in BCG primed sheep. Both have been used in separate

experiments in this thesis but have not been dïrectly compared to determine if they resdt

in the preferentiai recruïtment of BL. Prior midies by Waaji and coiieagues (1989)

demonstrated that both PPD and TNF-a recruit LL into the skin of BCG primed sheep.

Therefore, this protocol wiii aliow the direct comparison of the ability of TNF-a and PPD

to recruït BL into slon. Blood and lymph Lymphocytes can be labelled with different

radioisotopes, such as 11 1-In and 5 1-Cr, and infüsed intravenously. The sites of injection

cari be removed and the anmount of radioactivity determined. These results WU indicate if

the BL is preferentiaily recded into the skui by both PPD and TNF-a.

A second method w u examine the detailed kinetics of the two Iabeiied pools in

the aerent lymph drainlog a delayed type hypersensitivity site. ABerent lymphatics will

be canulated, blood and efferent lymph cells fluorescentiy IabeUed and reinhised. PPD

injections into the drainage area of the &erent lymphatic c m then be performed and the

Lymph monitored for labelled ceils- The colIected afferent lymph lymphocytes can then

be immunophenotyped for several adhesion molecules and activation markers. Thû

would al10 w the direct comparison of BL and LL labelled cells, which was not possible in

the CSF inaarnmation experiments.

The proposed experiments might document a rapidly recirculating population of

lymphocytes, if the hypothesis is correct. Alternatively, if the increase is due to the

increased tr&c of memory T celis these proposed experiments would also determine if

this is correct,

7.6 Summary

Tbe experiments in this thesis were designed to investigate the migration of the

LL and BL under normal and infiammatory conditions. It appears that LL may have a

greater role in immune su~veiliance as compared to BL. TNF-a induces inflammation in

sheep CSF with an infiammatory infiltrate that changes in composition over time. The

experiments in this thesis provide the first in vivo experimental evidence that BL and LL

may have Heren t functions under both normal and infiammatory conditions. Further

experiments are required to determine if these differences c m be -pulated to gain

m e r insight into their roles in immune system.

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