immunology of the female reproductive tract
TRANSCRIPT
Immunology of the Female ReproductiveTract
Charles R. WiraDepartment of PhysiologyDartmouth Medical School
1 Medical Center DriveLebanon, NH 03756
USA
Mucosal Immunity In The Human FemaleReproductive Tract
• Immune cells in the female reproductive tract.
l Antigen presentation by uterine epithelial cells.
l Localization of HIV receptors and co-receptorsin the uterus.
l Innate Immune responses in the uterus:bactericidal activity and secretory leukocyteprotease inhibitor (SLPI) production byepithelial cells.
Leukocytes in The Human FemaleReproductive Tract
0
10
20
30
% C
D45
Pos
itive
ALL PROLIF. SECRET. INACTIVE
*
*
**
%CD45-POSITIVE CELLSOF TOTAL DISPERSED CELLS
12
0
2
4
6
8
10
Num
ber o
f CD
45 X
106 p
er g
ram
ALL PROLIF. SECRET. INACTIVE
VMECXCXUTFT
*
*
*
NUMBER OF CD45-POSITIVE CELLSPER GRAM OF TISSUE (x 106)
Givan et. al., 1997. Am. J. Reprod. Immunol. 38:350
T Cells (CD3), Neutrophils (Cd66b), B Cells (CD19) and Monocytes(CD14) at Different Stages of the Cycle
FT EM CX ECX VM
B: SECRETORY
CD3
CD66b
CD14
CD190
20
40
60
% O
F C
D45
-PO
SITI
VE C
ELLS
FT UT CX ECX VM
A. PROLIFERATIVE
0
20
40
60
FT UT CX ECX VM
C. INACTIVE
CD3
CD3
CD66b
CD66b
CD14 CD14
CD19 CD19
*
* **
* * *
*
*
*
*
Givan et. al., 1997. Am. J. Reprod. Immunol. 38:350
Mucosal Immunity In The Human FemaleReproductive Tract
• Immune cells in the female reproductive tract.
l Antigen presentation by uterine epithelial cells.
l Localization of HIV receptors and co-receptorsin the uterus.
l Innate Immune responses in the uterus:bactericidal activity and secretory leukocyteprotease inhibitor (SLPI) production byepithelial cells.
Experimental Design: Antigen Presentation,Human Studies
250 µm
40 µm
Enzymaticdigestion ofhuman uterito obtainepithelial &stromal cells
Epithelial cells
3[H]-thymidineProliferation
TT
Purified T cells
Ficoll & T cellenrichmentcolumn
• Confirm PBMCpresentation of TT
• Check T cell purity• Determine if EC
present TT
1. 2.
3.
Antigen Presentation by Mixed Uterine Cellsto Tetanus Toxoid Primed T Cells
#789 #861 #839 #771 #783 #808 #8160
5000
10000
15000
20000
45000
55000
RT+T+TT
RT+T
Proliferative Secretory PostmenopausalMenstrual Cycle (stage)
33 33 H-H- H-H-Th
ymid
ine
Thym
idin
eTh
ymid
ine
Thym
idin
e ( ( ( (cp
mcp
mcp
mcp
m/w
ell)
/wel
l)/w
ell)
/wel
l)
UTERUS
Fahey et. al. Am. J. Reprod. Immunol. 42:49-57,1999
Antigen Presentation By Cervical And VaginalCells To Tetanus Toxoid Primed T Cells
780 771 698 783 8840
500
1000
1500
2000
2500
3000
CX+T+TTCX+T+TTCX+T+TTCX+T+TTCX+TCX+TCX+TCX+T
Menstrual Cycle (stage)Menstrual Cycle (stage)Menstrual Cycle (stage)Menstrual Cycle (stage)
H-t
hym
idin
e (c
pm
/wel
l)3
CERVIXCERVIXCERVIXCERVIX
****
****
latelatelatelateproliferativeproliferativeproliferativeproliferative
earlyearlyearlyearlysecretorysecretorysecretorysecretory secretorysecretorysecretorysecretory postmenopausalpostmenopausalpostmenopausalpostmenopausal
----------------------------------------------------------------------------------------780 771 884
0
300
600
900
1200
1500
VM+T+TTVM+T+TTVM+T+TTVM+T+TTVM+TVM+TVM+TVM+T
Menstrual Cycle (stage)Menstrual Cycle (stage)Menstrual Cycle (stage)Menstrual Cycle (stage)
H-t
hym
idin
e (c
pm
/wel
l)
VAGINAVAGINAVAGINAVAGINA
****
********
latelatelatelate proliferative proliferative proliferative proliferative earlyearlyearlyearly secretory secretory secretory secretory postmenopausalpostmenopausalpostmenopausalpostmenopausal
3
Fahey et. al. Am. J. Reprod. Immunol. 42:49-57,1999
Presentation of Tetanus Toxoid by UterineEpithelial cells
EpithelialCells
T Cells Epithelial& T Cells
0
1000
2000
3000
4000
5000
6000
0 g/ml
10 g/ml
100 g/ml
3 H-T
hym
idin
e (c
pm/w
ell)
Wallace, P.K., Yeaman, G.R., Kristy Johnson, K., Collins, J.E., Guyre, P.M. and Wira, C.R.J.Steroid Biochem. Mol. Biol. . 76(1-5):203-11, 2001
Mucosal Immunity In The HumanFemale Reproductive Tract
• Immune cells in the female reproductive tract.
l Antigen presentation by uterine epithelial cells.
l Localization of HIV receptors and co-receptorsin the uterus.
l Innate Immune responses in the uterus:bactericidal activity and secretory leukocyteprotease inhibitor (SLPI) production byepithelial cells.
Time Course of HIV Infectivity of UterineEpithelial Cells
1 5 15 30 60 120 240 360 7200
5
10
15
20
25
Time of exposure of PHA blasts to HIV-1 (min)
p24
conc
entr
atio
n on
day
10
(ng/
ml)
From: Howell et. al. J. Virol. 71: 3498-3506, 1997.
3 Color Immuno-fluorescent staining.Typically mAbsconjugated to FITC,Cy3 and Cy5
Hysterectomy Tissue.
Prepare vibratome orFrozen Sections.
Fix and mount
Confocal Analysis.Simultaneousacquisition of opticalsections (minimimthickness 0.7µm).
Preparation of Viable Tissue Sections andConfocal Analysis.
1. Uterine epithelial cells express CD4, CCR5 and CXCR4.
• Expression of these receptors varies with the stageof the menstrual cycle.
3. These studies suggest that the uterus may be a site of HIV entry.
Yeaman et al. Unpublished observations
Analysis of CD4, CCR5 and CXCR4CD4, CCR5 and CXCR4CD4, CCR5 and CXCR4CD4, CCR5 and CXCR4 on Viable Tissue Sections by Confocal Analysis
Conclusions
Mucosal Immunity in the Human FemaleReproductive Tract
• Immune cells in the female reproductive tract.
l Antigen presentation by uterine epithelial cells.
l Localization of HIV receptors and co-receptorsin the uterus.
l Innate Immune responses in the uterus:bactericidal activity and secretory leukocyteprotease inhibitor (SLPI) production byepithelial cells.
Effect of Menstrual Cycle Stage and Menopause on Anti-bacterial Activity by Human Uterine Epithelial Cells
ProliferativeProliferativeProliferativeProliferative SecretorySecretorySecretorySecretory InactiveInactiveInactiveInactive0000
20202020
40404040
60606060
80808080
100100100100
120120120120
Menstrual Status
% In
hibi
tion
(+/
- SD)
% In
hibi
tion
(+/
- SD)
% In
hibi
tion
(+/
- SD)
% In
hibi
tion
(+/
- SD)
N=6N=6N=6N=6N=6N=6N=6N=6
N=4N=4N=4N=4
****
Fahey and Wira.. J. Infect. Diseases 185:1606-1613, 2002.
SLPI Concentrations in Apical rinses ofUterine Epithelial Cells
1111 4444 242424240000
4444
8888
12121212
16161616
20202020
24242424
Incubation Time (hrs)Incubation Time (hrs)Incubation Time (hrs)Incubation Time (hrs)
SLPI
(ng
/ml)
SLPI
(ng
/ml)
SLPI
(ng
/ml)
SLPI
(ng
/ml)
Patient 1407Patient 1407Patient 1407Patient 1407
Fahey and Wira.. J. Infect. Diseases 185:1606-1613, 2002.
SLPI Concentrations in Water Rinses ofUterine Epithelial Cell Monlayers
1226122612261226 1365136513651365 1407140714071407 1308130813081308 1341134113411341 13971397139713970
3
6
9
12
15
18
21
24
Patient #
SLPI
(ng
/ml)
SLPI
(ng
/ml)
SLPI
(ng
/ml)
SLPI
(ng
/ml)
PPPP LSLSLSLS PPPP IIII IIII IIII
Menstrual statusMenstrual statusMenstrual statusMenstrual status
******** ****
Fahey and Wira.. J. Infect. Diseases 185:1606-1613, 2002.
Correlation Between Anti-bacterial Activity and SLPIProduction by Human Uterine Epithelial Cells in Culture
ControlControlControlControl 1111 4444 242424240000
40404040
80808080
120120120120
160160160160
200200200200Colony Forming Units (CFU)SLPI (ng/ml)
Incubation Time (hrs)Incubation Time (hrs)Incubation Time (hrs)Incubation Time (hrs)
Colo
ny F
orm
ing
Units
(CF
U)Co
lony
For
min
g Un
its (
CFU)
Colo
ny F
orm
ing
Units
(CF
U)Co
lony
For
min
g Un
its (
CFU)
SLPI
(ng
/ml)
SLPI
(ng
/ml)
SLPI
(ng
/ml)
SLPI
(ng
/ml)
20202020
16161616
12121212
8888
4444
0000
Fahey and Wira.. J. Infect. Diseases 185:1606-1613, 2002.
Neutralization of S. Aureus Anti-Bactericidal Activity inUterine Epithelial Cell Apical Secretionsby anti-human SLPI Antibody
ControlControlControlControl UT SECUT SECUT SECUT SEC UT SECUT SECUT SECUT SEC UT SECUT SECUT SECUT SEC0
20
40
60
80
100
Colo
ny F
orm
ing
Units
(CF
U)Co
lony
For
min
g Un
its (
CFU)
Colo
ny F
orm
ing
Units
(CF
U)Co
lony
For
min
g Un
its (
CFU)
++++ ++++IsotypeIsotypeIsotypeIsotype anti-SLPIanti-SLPIanti-SLPIanti-SLPI
******** ********
Fahey and Wira.. J. Infect. Diseases 185:1606-1613, 2002.
ConclusionsConclusionsConclusionsConclusions
• The female reproductive tract (FRT) contains a full spectrum ofimmune cells.
• Antigen presenting cells in the uterus, cervix and vagina are able topresent antigen. Epithelial cells are able to present antigen toautologous T cells.
• Uterine epithelial cells express CD4, CCR5 and CXCR4. Expression ofthese receptors varies with the stage of the menstrual cycle. Thesestudies suggest that the uterus may be a site of HIV entry.
• Uterine epithelial cells produce SLPI, which appears to be underhormonal control, to protect pre-menopausal women from potentialbacterial pathogens.
• Overall, these studies demonstrate that immune cell function in theFRT varies with stage of the menstrual cycle and menopause.
Acknowledgments
Alice GivanAlice GivanAlice GivanAlice GivanPaulPaulPaulPaul Wallace Wallace Wallace Wallace
Grant YeamanGrant YeamanGrant YeamanGrant YeamanJohn FaheyJohn FaheyJohn FaheyJohn FaheyAlix HowellAlix HowellAlix HowellAlix HowellAlice GivanAlice GivanAlice GivanAlice Givan
andandandandGabriellaGabriellaGabriellaGabriella
Dartmouth Medical SchoolLebanon, NH
CLINICAL ACKNOWLEDGMENTS
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