www.elsevier.com/locate/jneuroim

Journal of Neuroimmunolo

IL-12 driven upregulation of P-selectin ligand on myelin-specific T cells

is a critical step in an animal model of autoimmune demyelination

Pratima Deshpande a, Irah L. King b, Benjamin M. Segal c,*

a Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry,

601 Elmwood Avenue, Box 605, Rochester, NY, 14642, USAb Interdepartmental Graduate Program in Neuroscience, University of Rochester School of Medicine and Dentistry,

601 Elmwood Avenue, Box 605, Rochester, NY, 14642, USAc Departments of Neurology, Microbiology and Immunology and the Cancer Center, University of Rochester School of Medicine and Dentistry,

601 Elmwood Avenue, Box 605, Rochester, NY, 14642, USA

Received 20 August 2005; accepted 18 November 2005

Abstract

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system. IL-12p40

monokines play a critical role in the generation of EAE-inducing CD4+Tcells. Here we show that IL-12 directly upregulates the expression of

the adhesion molecule, P-selectin glycoprotein ligand (PSGL-1), on B10.PL MBP-TCR transgenic T cells during their initial encounter with

antigen. Pre-incubation of IL-12-stimulated myelin-reactive CD4+T cells with a blocking antibody against PSGL-1 reduced the incidence and

severity of EAE. We conclude that IL-12-driven PSGL-1 expression can facilitate the development of autoimmune demyelination.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Experimental autoimmune encephalomyelitis; Interleukin-12; Cytokines; Th1 cells; P-selectin ligand; Adhesion molecules; Multiple sclerosis

1. Introduction

Experimental autoimmune encephalomyelitis (EAE) is

an inflammatory demyelinating disease of the central

nervous system (CNS) that is induced in laboratory animals

by immunization with myelin antigens in combination with

adjuvants (Rao and Segal, 2004). It is widely used as an

animal model of multiple sclerosis (MS). Adoptive transfer

studies have shown that myelin-specific CD4+ T cells

mediate EAE (Pettinelli and McFarlin, 1981). These cells

accumulate in the brain and spinal cord prior to the onset of

clinical deficits and trigger the formation of perivascular and

meningeal myeloid/ lymphoid infiltrates, demyelination and

axonal damage (Hickey, 1991; Skundric et al., 1993).

Expression of IL-12p40 monokines is a critical step in

the differentiation of encephalitogenic CD4+ T cells and the

development of clinical EAE. Hence, IL-12p40 deficient

0165-5728/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.jneuroim.2005.11.016

* Corresponding author. Tel.: +1 585 275 7854; fax: +1 585 275 9953.

E-mail address: benjamin_segal@URMC.Rochester.edu (B.M. Segal).

mice and wildtype mice treated with neutralizing antibodies

against IL-12p40 are resistant to disease induction (Leonard

et al., 1995; Segal et al., 1998). The IL-12p40 monokine

family consists of IL-12p70 and IL-23, each of which is a

heterodimer composed of a common IL-12p40 chain and a

unique chain (IL-12p35 and IL-23p19, respectively) (Opp-

mann et al., 2000). Recent studies using panels of single

chain deficient mice have demonstrated that IL-23, but not

IL-12p70, plays a non-redundant role in the development of

EAE in C57BL/6 mice induced by active immunization with

myelin oligodendrocyte glycoprotein (MOG) peptide

(Langrish et al., 2005). Nonetheless, there is unequivocal

evidence that IL-12p70 itself has disease promoting

properties. For example, C57BL/6 IL-12p40�/� mice only

develop full blown EAE, that approximates the clinical

severity of wildtype mice, following reconstitution with

both IL-12p70 and IL-23p19 (Langrish et al., 2005).

Furthermore, ordinarily innocuous myelin-specific T cells

(for example, those derived from EAE-resistant strains or

from donors that have been primed with myelin antigen

gy 173 (2006) 35 – 44

P. Deshpande et al. / Journal of Neuroimmunology 173 (2006) 35–4436

emulsified in IFA rather than CFA) acquire encephalitogenic

properties following reactivation in vitro in the presence of

recombinant IL-12p70 (Ichikawa et al., 2002; Segal et al.,

2000; Segal and Shevach, 1996). The mechanism by which

IL-12p70 confers encephaltiogenicity to myelin-specific T

cells remains to be fully elucidated.

IL-12p70 programs myelin-specific T cells to differentiate

into Th1 cells that are committed to IFNg production.

However, the ability of myelin-specific T cells to secrete

IFNg does not strictly correlate with their ability to induce

EAE. IFNg�/�mice, by contrast to IL-12p40�/�mice,

readily succumb to EAE (Ferber et al., 1996; Segal, 1998;

Willenborg et al., 1996). These mice are protected from the

disease by treatment with anti-IL-12p40 antibodies, support-

ing the contention that IL-12p40 monokines contribute to

autoimmune pathogenesis via IFNg-independent pathways

(Segal et al., 1998). Myelin-specific T cells that have

differentiated in the absence of IL-12 fail to accumulate in

the CNS or trigger demyelination following adoptive transfer.

Nevertheless, the transferred cells survive in peripheral

lymphoid tissues and mount robust proliferative and cytokine

recall responses upon ex vivo challenge (Segal et al., 2000;

Segal and Shevach, 1996). On the other hand, the same

myelin-specific T cells readily accumulate in the CNS and

induce demyelination within 5–10 days of adoptive transfer

following reactivation with antigen and recombinant IL-

12p70 (Segal et al., 2000; Segal and Shevach, 1996).

Collectively, these observations have lead us to postulate

that IL-12p70 plays a role in the trafficking of myelin-specific

T cells to the CNS and/ or in the early stages of lesion

development prior to the establishment of stable perivascular

infiltrates and the onset of clinical deficits. In previous

publications we demonstrated that IL-12p70 directly upre-

gulates CCR5, the chemokine receptor for CCL2, CCL3 and

CCL5, on myelin-specific CD4+ T cells (Bagaeva et al.,

2003). In the current manuscript we expand those findings by

examining the effect of the cytokine on adhesion molecule

expression.

2. Materials and methods

2.1. Mice

Mice that express transgenic TCRVß8.2 and Va4 chains

specific for myelin basic protein (MBP) Ac1-17 in the

context of I-Au (MBP-TCR Tg+) have been previously

described and were originally provided by Dr. Charles A.

Janeway (Yale University School of Medicine, New Haven,

CT) (Hardardottir et al., 1995). Coisogenic B10.PL mice

were obtained from Jackson Laboratories. MBP-TCR Tg+

and wildtype B10.PL mice were paired for breeding and

maintained in University of Rochester Medical Center

animal facilities under specific pathogen free conditions.

Off spring were typed by flow cytometric analysis of blood

samples for Vß8.2 and CD4 expression.

2.2. Antibodies

The following antibodies/ fusion proteins used for FACS

and Whole-Mount Immunoflourescence were purchased

from Pharmingen: P selectin-human IgG fusion protein,

anti-P selectin-PE (anti-CD62p; clone RB40.34), anti-CD16/

CD32 (Fc block; 2.4G2), anti-CD11a-PE (anti-a L integrin;

2D7), anti-CD49d-PE (anti-a 4 integrin; SG31), anti-CD162-

PE (anti-PSGL-1; 2PH1), anti-CD25-PE (PC61), anti-Vh8.2 -

FITC (MR5-2), and anti-CD69 (H1.2F3). Anti-human IgG-

PE was obtained from Jackson Immunoresearch Lab and

anti-CD4-APC (RM4-5) from eBioscience. For in vivo

blocking experiments purified rat monoclonal anti-CD162

antibody (NA/LE) and the isotype matched control antibody

rat IgG1, n were obtained from Pharmingen.

2.3. Cell culture

Pooled splenocytes and lymph node (LN) cells from

naı̈ve MBP-TCR Tg+ mice were washed twice and then

resuspended in RPMI 1640 supplemented with 10% heat-

inactivated FCS, penicillin (100 Ag/ml), streptomycin

(100 Ag/ml), L-glutamine (2 mM), Hepes (100 mM), non-

essential amino acids (1 mM), sodium pyruvate (10 mM) and

2- Mercaptoethanol (0.055 mM) (all obtained from Invi-

trogen, NY, USA). Cells were cultured at 5�106 cells/ml

either in 6 well plates (5 ml/well) for FACS analysis or

250 ml flasks for adoptive transfer (all plasticware from

Costar, Corning, NY). The following reagents were added

where indicated: MBPAc1-17 (50 Ag/ml; Macromolecular

Resources, Fort Collins, CO); recombinant murine IL-12

(10 ng/ml, R&D systems); anti-IL-12p40 monoclonal anti-

body (10 Ag/ml; clone C17.8; the hybridoma was originally

obtained from G. Trinchieri), anti-IFNg mAb (10 Ag/ml;

clone XMG 1.2) or rat IgG (10 Ag/ml; Sigma). In preliminary

experiments the bioactivity of anti-IL-12 and anti-IFNg at the

above concentrations was demonstrated by suppression of

IFNg and CXCL9 production, respectively, in cultures of

anti-CD3 stimulated splenocytes (data not shown). After 4

days, cells were washed and used either for adoptive transfer

into naı̈ve recipients or RT-PCR and FACS analysis.

2.4. Induction and evaluation of EAE

B10.PL Tg negative mice (Tg�) were injected i.p. with

35�106 IL-12/MBPAc1-17 activated MBP-TCR Tg+ cells

suspended in 200 Al of PBS. Recipients were observed for

the signs of EAE daily and graded on the following scale:

0—no deficits; 1—limp tail; 2—mild hind limb weakness;

3—moderate hind limb weakness; 4—hind limb paralysis;

5—forelimb and hindlimb paralysis or moribund.

2.5. Proliferation assay

Cells were seeded in round-bottom 96-well plates

(5�105 cells/well in 0.2 ml tissue culture media) and

P. Deshpande et al. / Journal of Neuroimmunology 173 (2006) 35–44 37

cultured for 72 h in the presence of MBPAc1-17, over a

range of concentrations, and either IL-12 or anti-IL-

12p40 monoclonal antibody. For the last 18 h wells were

pulsed with [3H]-thymidine (1 ACi/well; Amersham).

Incorporated radioactivity was measured using a Beta-

plate scintillation counter. Assays were performed in

triplicate.

2.6. Real time RT-PCR

MBP-TCR+ cells were harvested after 48 h of culture for

RNA extraction (Trizol, Invitrogen, Carlsbad, CA), DNase

treatment (Invitrogen Life Technologies) and reverse tran-

scription with oligo-dT and M-MLV reverse transcriptase

(Invitrogen). cDNAwas amplified using the iCycler iQ Real-

Time PCR system (Bio-Rad, Hercules, CA) with the

following primer and Taqman probe sequences (Integrated

DNA Technologies, Coralville, IA): Fucosyltransferase

VII (FucT-VII): 5V-AATTCCAGAAGGCTCCAGATG-3V(forward), 5V-AGTGTGGACTGAGGCACAG-3V (reverse)

and 5V-6-Fam-CACTTCCAGGAGCTGATCCCCACA-

BHQ-1-3V (probe); Core 2 glucosaminyl transferase 1

(C2GnT-I): 5V-AATATTCCCTCTGAGCAAGTACA-3V, 5V-GGCCTTGAATGCCAATGATG-3 V and 5 V-6-Fam-

TGTCACCAGGAGTCAGAGCCTCAA-BHQ-1-3V; and

CD4 : 5V-GTGTCTACTGAGTGAAGGTGATAAGG-3V,5V-GGAAACCCAGAAAGCCGAAGG-3V and 5V-6-FAM-

ACCCAGCACGCAAGCCAGGAACACT-BHQ-1-3V.Samples were amplified over 40 cycles according to the

following protocol: 30 s at 95 -C, 30 s at 55 -C, 30 s at 72 -C.Each sample was run in triplicate. C2GnT and FucT-VII

levels were normalized to CD4.

2.7. Flow cytometry

Cells were harvested from in vitro cultures and incubated

with Fc block for 10 min on ice. They were then incubated

with P selectin-IgG fusion protein and anti-Vh8.2-FITC or

the relevant isotype matched control antibodies for an

additional 30 min. Cells were washed with 3% FBS in PBS,

incubated with PE-conjugated anti-human IgG for 30 min,

washed twice and fixed in 1% paraformaldehyde. Analysis

was performed on a BD Flow Cytometer using Cell Quest

software.

2.8. Whole-mount immunofluorescence

Whole-Mount Immunofluorescence was performed on

the spinal cords of adoptive transfer recipients using

previously described methodologies (Gerber et al., 2003).

In brief, each spinal cord was flushed out of its vertebral

column and dissected into sections of 2–3 mm thickness

and 3 mm length. The pieces were then incubated with Fc

Block (10 Ag/ ml in 200 Al of PBS with 1% BSA) in 6 ml

polypropylene tubes for 30 min over ice. Fluorochrome-

conjugated antibodies specific for CD4 (APC), CD162

(PE), CD62P (PE) and CD31 (FITC) or flourochrome-

conjugated isotype matched control antibodies were added

directly to the tubes at a predetermined concentration and

left on ice for 1 h. After staining, samples were washed

twice in PBS with 3% FBS and placed on glass slides with 2

drops of PBS containing 1% bovine serum albumin. A

cover slip was placed on top of each piece of spinal cord

tissue. Slides were viewed under a fluorescent microscope

(Olympus BX40F; Olympus Optical Co. Ltd., Japan). In all

cases, background staining with isotype control antibodies

was minimal.

2.9. Incubation of MBP-TCR Tg cells with anti-PSGL-1 Ab

prior to adoptive transfer

MBP-TCR Tg+ cells obtained after 4 day cultures with

antigen and IL-12 were incubated with either an anti-

CD162 blocking antibody (anti-PSGL1; NA/LE; 5 Ag/ml)

or rat IgGn isotype matched control antibody (5 Ag/ml)

for 1 h on ice. Cells were washed twice with PBS prior

to transfer into syngeneic wildtype hosts as described

above.

2.10. IL-2 and IFNc elispot assays

96 well filtration plates (MAIP N4550; Millipore, Bed-

ford, MA) were coated with purified anti-IFNg (clone AN-

18) or anti-IL-2 (clone JES6-1A12) at 3 Ag/ml in 50 Al of PBSfor 2 h at room temperature. They were then washed three

times with washing buffer (1% Tween-20 in PBS). Spleen

and LN cells from adoptive transfer recipients were plated

in triplicate across 2 fold dilutions starting at 5�105 cells/

well. Lower cell concentrations were supplemented with

naive syngeneic splenocytes to keep the total cell number/

well constant at 5�105 /well in a final volume of 200 Al.MBPAc1-17 (50 Ag/ml) was added to some of the wells. After

48 h of incubation at 37 -C, plates were washed prior to the

addition of biotinylated anti-IFNg (clone XMG-1.2) or anti-

IL-2 (clone JES6-5H4) at 3 Ag/ml dissolved in PBS-TB

buffer (1% Tween-20 and 2% BSA; 50 Al/well). After a

2 h incubation at RT, plates were washed, loaded with 5

streptavidin– alkaline phosphatase conjugate (diluted

1 :1000 in PBS-TB; 50 Al/ well), and incubated for 45 min

at RT. Plates were washed 6 times and developed using the

alkaline phosphatase substrate Vector Blue (Vector Labora-

tories, Burlingame, CA). Spots were counted using an

automated ELISPOT counter (CTL ImmunoSpot Analyzer

with ImmunoSpot software, version 2.08) from Cellular

Technology (Cleveland, OH). Counts are shown as the

meanTSD for each set of triplicate wells.

2.11. Statistical analyses

Clinical scores, elispot data and mRNA levels were

compared between experimental groups using the unpaired

Student’s t test assuming unequal variance.

Table 1

Effect of IL-12 on the expression of cell surface molecules on MBP-

stimulated T lymphocytes

Culture conditions Unstimulated MBP MBP+

IL-12

MBP+

anti-IL-12

CD25 3.8 (3) 61 (45) 60 (60) 59 (41)

CD69 1.8 (3) 54 (43) 56 (52) 54 (41)

CD11a (LFA-1) 24 (4) 83 (39) 81 (44) 86 (40)

CD49d (a4 integrin) 59 (21) 42 (18) 41.5 (22) 40 (17)

CD162 (PSGL-1) 5.5 (1) 10 (9) 39.4 (66) 6.7 (7)

Effects of IL-12 on the expression of a panel of activation markers and

adhesion molecules on MBP-stimulated T lymphocytes. Pooled LN cells

and splenocytes from MBP-TCR Tg+ mice were cultured in the presence of

MBPAc1-17 with or without IL-12 or anti-IL-12 antibody. After 24 h (for

measurement of activation markers) or 96 h (for measurement of adhesion

molecules), the cells were harvested, washed, double stained with

fluorochrome-conjugated Abs against CD4 or Vh8.2 and the molecule

indicated (or isotype matched controls) and analyzed by flow cytometry.

The data shown represents the percentage of CD4+/ Vh8.2+T cells that were

positive for the relevant marker. Gates were selected based on background

staining with isotype matched control antibodies. MFI values are shown in

parentheses. The results are representative of four independent experiments.

P. Deshpande et al. / Journal of Neuroimmunology 173 (2006) 35–4438

3. Results

3.1. The role of IL-12p70 in priming encephalitogenic MBP-

TCR transgenic cells

In order to directly investigate the effects of IL-12p70 on

myelin-specific T cells during their initial encounter with

antigen, we stimulated lymph node cells from MBP-TCR

transgenic mice with either antigen alone or in combination

with recombinant IL-12p70 or a neutralizing antibody

against IL-12p40. MBP-TCR Tg+ cells primed under

neutral conditions occasionally transferred EAE to naı̈ve

syngeneic recipients, but at a low incidence (1 out of 14

transfer recipients succumbed in a typical experiment). By

contrast, the same cells activated in the presence of IL-

12p70 transferred a severe form of disease in 100% of

recipients. In repeated experiments, none of the wildtype

mice injected with MBP-TCR Tg+ cells that had been

stimulated with antigen and anti-IL-12p40 developed signs

of EAE (Fig. 1). We considered the possibility that IL-

12p70 was required for the activation and/ or clonal

expansion of MBP-TCR Tg+ cells. However, MBP-trans-

genic cells upregulated activation markers and proliferated

in response to antigenic challenge to an equivalent extent in

the presence or absence of IL-12p70 (Table 1; Fig. 2).

3.2. IL-12p70 dependent induction of adhesion molecules

on MBP-TCR transgenic cells

Next we analyzed MBP-TCR Tg+ cells for adhesion

molecule expression following culture according to the

conditions described above. IL-12p70 costimulation had no

significant effect on expression of LFA-1 or a4 integrin

(Table 1). By contrast, PSGL-1 was expressed at high levels

on the majority of MBP-specific T cells stimulated with IL-

12p70 and antigen, but not on cells activated with either

antigen or IL-12p70 alone or antigen plus anti-IL-12p70

Days

Mea

n cl

inic

al s

core

0

0.5

1

1.5

2

2.5

3

3.5

1 2 3 4 5 6 7 8 9 10 11 12 13

MBP+ IL-12 stimulated cells

MBP stimulated cells

MBP+ anti-IL-12 stimulated cells

Fig. 1. IL-12 dictates the encephalitogenicity of MBP reactive T cells.

B10.PL mice were injected with MBP-TCR Tg+ cells following culture in

the presence of MBPAc1-17 (50 Ag/ml) plus or minus recombinant murine

IL-12 (10 ng/ml) or anti-IL-12p40 neutralizing antibody (10 Ag/ml). The

mean clinical score of each group was determined on a daily basis post

transfer. The data shown is representative of three independent experiments

with 4–7 mice/ group.

(Table 1; Fig. 3A). The data shown was generated by

staining with a P-selectin-IgG fusion protein and, therefore,

represents expression of functional PSGL-1. PSGL-1

upregulation was a direct result of IL-12p70 signaling and

not secondary to IFNg induction since the addition of an

anti-IFNg neutralizing antibody had no significant effect on

PSGL-1 levels (Fig. 3B).

3.3. Effects of IL-12p70 on the expression of enzymes

involved in the post-translational modification of PSGL-1

Expression of functional PSGL-1 is regulated, in large

part, at the level of post-translational modification. Core 2 h-1,6-N-acetylglucosaminyltransferase (C2GnT-I) and a (1,3)-

fucosyltransferase-VII (FucT-VII) are glycosylation enzymes

that play critical roles in this process; both are required for

[MBPAc1-17] μg/mL

Thy

mid

ine

inco

rpor

atio

n

0

50000

100000

150000

200000

250000

300000

50 0

MBP+ IL-12

MBP+ anti-IL-12

Fig. 2. IL-12 does not enhance the proliferation of MBP-specific T cells.

Pooled LN cells and splenocytes from naı̈ve MBP-TCR Tg+ mice were

cultured in the presence or absence of MBPAc1-17 (50 Ag/ml) plus or minus

recombinant IL-12 (10 ng/ml) or anti-IL-12 neutralizing antibody (10 Ag/ml) for 3 days. Wells were pulsed with [3 H]-thymidine (1 ACi/well) for thefinal 18 h of culture. The data shown is representative of three independent

experiments.

Fig. 3. IL-12 upregulates PSGL-1 expression on MBP-specific T cells. Pooled LN cells and splenocytes from MBP-TCR Tg+ mice were cultured in the

presence of IL-12 alone or MBPAc1-17 either alone, with IL-12 or with anti-IL-12 antibody (A). MBP-TCR Tg+ cells were cultured with MBPAc1-17, IL-12 and

either anti-IFNg or isotype matched control antibody (B). After 4 days cells were washed, stained with fluorochrome conjugated antibodies and analyzed for

expression of PSGL-1 on Vh 8.2+ -gated cells by flow cytometry. The thin lines in the histogram plots represent isotype matched control antibody staining and

the thick lines represent PSGL-1 staining. The median fluorescence intensity of PSGL-1 stained cells is shown in the upper right hand corner of each histogram.

These results are representative of three independent experiments.

P. Deshpande et al. / Journal of Neuroimmunology 173 (2006) 35–44 39

high affinity P-selectin binding (Smithson et al., 2001;

Sperandio et al., 2001). We measured the effects of IL-

12p70 on transcription of these enzymes in antigen-stimu-

lated MBP-TCR CD4+ T cells by real time RT-PCR. MBP-

TCR Tg+ cells cultured with a combination of recombinant

IL-12p70 and MBPAc1-17 reproducibly expressed higher

levels of C2GnT-I than cells cultured with antigen alone or

antigen plus anti-IL-12p40 (Fig. 4). By contrast, IL-12p70

0

10

20

30

40

50

* ***

Rel

ativ

e C

2GnT

mR

NA

exp

ress

ion

MBP IL-12 MBP+IL-12 MBP+ αIL-12

Fig. 4. IL-12 increases the expression of C2GnT mRNA in MBP-specific T

cells. Pooled LN cells and splenocytes from MBP-TCR Tg+ mice were

cultured as described in Fig. 3 for 3 days. Total RNA was extracted using

Trizol and cDNA was prepared using standard procedures as described in

Materials and Methods. C2GnT and CD4 mRNA transcripts were

quantified by real-time PCR. The data represents the ratio of C2GnT to

CD4 mRNA levels. The experiment was repeated five times with similar

results. (*P�0.01; ** P�0.001).

costimulation did not result in higher expression of FucT-VII

mRNA on a consistent basis (data not shown).

3.4. Expression of P-selectin and PSGL-1 in the CNS during

EAE induced by transfer of IL-12p70 stimulated MBP-TCR

Tg+ cells

Next we examined PSGL-1 and P-selectin expression in

the CNS of adoptive transfer recipients. Whole mount

immunofluoresence studies revealed perivascular clusters of

PSGL-1+ CD4+ T cells in the spinal cords of afflicted mice

(Fig. 5A). Functional PSGL-1 was also detected on CD4�

cells in the infiltrates, which might include activated

microglia, CD8+ T cells and/ or neutrophils. Analysis of

spinal cords harvested at serial time points demonstrated

that P-selectin is upregulated on CD31+ CNS blood vessels

during the preclinical phase, 4–6 days following the

adoptive transfer of MBP-TCR Tg+ cells (Fig. 5B). P-

selectin expression was sporadic and tended to occur on

those blood vessels associated with infiltrating leukocytes.

These results were corroborated by RT-PCR analysis of

spinal cord tissues which showed induction of P-selectin

mRNA over the same time frame (Fig. 5C).

3.5. The role of PSGL-1/P-selectin interactions in the

clinical manifestation of EAE mediated by IL-12p70

stimulated MBP-TCR Tg+ cells

In order to determine whether PSGL-1 expression on

myelin-reactive T cells is important for the clinical

25μm 25μm 50μm 50μm

25μm 25μm 50μm 50μm

HPRT

CD62p

1 3 42 5 6C

PSGL-1 CD4A

CD31 P selectin / CD31 overlay

Rat IgG1, κ Rat IgG2a, κ Rat IgG2a, κ Rat IgG1, λ

B

Fig. 5. P-selectin and PSGL-1 are expressed in the spinal cords of mice injected with IL-12-stimulated MBP-reactive cells during preclinical and acute stages of

EAE. Spinal cords harvested from adoptive transfer recipients on day 8 (A) or day 4 (B) post transfer were sectioned and stained for CD4, PSGL-1, CD31, and/

or P selectin (CD62p) prior to analysis by whole-mount Immunofluorescence. Clusters of CD4 (red) and PSGL-1 (yellow) positive cells were detectable in

symptomatic mice (A). The arrows point to selected double positive cells. The upper right panel (B) shows CD31+CNS blood vessels (green) co-expressing P-

selectin (orange). P-selectin was only detectable in cords harvested between 2–6 days of transfer but not at later time points. The lower panels show

background staining with isotype matched control antibodies. No staining was observed for CD4, PSGL-1 or P-selectin in spinal cords of naive mice (data not

shown). Original magnification of the images is �40. P-selectin was also detected by mRNA analysis of the spinal cords of transfer recipients (C). Each lane

represents an individual cord. Lanes 1–4 correspond to cords harvested from adoptive transfer recipients on day 4 post transfer; lanes 5–6 correspond to cords

from naive mice. Similar results were obtained in 4 independent experiments with 4–6 cords per group.

Table 2

Pre-incubation of myelin-activated T cells with a blocking antibody against

PSGL-1 reduces the severity of tEAE

Experimental

group

Incidence Mean day of

onset TS.D.

Mean peak

score

Mean cumulative

score

Anti-PSGL-1

antibody

10/15 7.3T1.5 0.8T0.47* 4.7T4.3 **

Isotype antibody 15/15 7T1.7 2.3T0.47 15.8T4.3

Pre-incubation of myelin-activated cells with blocking antibody against

PSGL-1 reduces the severity and incidence of adoptively transferred EAE.

MBP-TCR Tg+ cells were cultured for 4 days in the presence of MBPAc1-17and IL-12. Cells were harvested, washed and then incubated with either

anti-PSGL-1 or isotype matched control (rat IgG) antibodies (10 Ag/ml) for

1 h prior to transfer into naı̈ve B10.PL Tg-recipients. Mice were graded

daily based on a 5 point scale (as described in Materials and Methods) from

the day of immunization onward. Mean cumulative scores were calculated

from day 0 post immunization to day 15 (the day of sacrifice). The table

shows data pooled from three separate experiments each with 5 mice/

group. (* P <0.006; ** P <0.02 by comparison to the control group).

P. Deshpande et al. / Journal of Neuroimmunology 173 (2006) 35–4440

manifestation of EAE, we incubated MBP-TCR Tg+ cells

with anti-PSGL-1 monoclonal antibodies or isotype

matched control antibodies for 1 h following a 96 h culture

with antigen and IL-12, and then transferred them into

naı̈ve coisogenic recipients. Mice that were injected with

cells incubated with anti-PSGL-1 antibody experienced a

significantly milder course than their counterparts that

were injected with cells incubated with isotype matched

control antibody (Table 2). Lymph node cells and

splenocytes, harvested from both groups of hosts on day

10 post-transfer, mounted comparable IL-2 and IFNg

responses upon ex vivo challenge, indicating that pretreat-

ment of donor cells with anti-PSGL-1 antibodies did not

result in their depletion or inactivation in the host

following adoptive transfer (Fig. 6). Consistent with these

findings, incubation of B10.PL splenocytes with anti-

PSGL-1 antibody, as opposed to rat IgG or media alone,

did not enhance their susceptibility to complement

mediated lysis in vitro (data not shown). Collectively our

results suggest that anti-PSGL-1 blocks CNS infiltration

and/ or demyelination by IL-12 stimulated MBP-TCR Tg+

cells.

4. Discussion

We as well as others have previously demonstrated that IL-

12p70 can unmask latent encephalitogenic properties of

0

20

40

60

80

100

120

140

160Isotype treated group

Anti-PSGL-1 treated group

0

50

100

150

200

250

300

5*105 cells/well 2.5*105 cells/well 1.25*105 cells/well

5*105 cells/well 2.5*105 cells/well 1.25*105 cells/well

Isotype treated group

Anti-PSGL-1 treated group

IL-2

spo

ts /

wel

lIF

Nγ

spot

s / w

ell

A

B

Fig. 6. Adoptive transfer recipients of MBP-TCR Tg+ effector cells,

injected following incubation with either anti-PSGL-1 or isotype matched

control antibodies, mount comparable cytokine responses upon ex-vivo

challenge. Pooled splenocytes and LN cells from adoptive transfer

recipients (n =3/group) were assessed for IFNg (A) and IL-2 (B) production

by ELISPOT assay. The X-axis represents the total number of cells added

per well and the Y-axis, the number of cytokine producing cells detected per

well. Antigen-specific responses were determined by subtracting the

number of spots in unstimulated wells (which ranged between 1–5/well)

from the number in peptide-pulsed wells. The average number of spleen

and LN cells harvested per mouse was comparable between the groups.

Hence, in the experiment shown, 124�106 cells were recovered per mouse

in the anti-PSGL1 treated group versus 130�106 cells in the control

antibody treated group. Splenocytes and LN cells from naı̈ve B10.PL mice

that were not injected with MBP-TCR Tg+ effector cells failed to mount

MBP-specific IL-2 or IFNg responses above background levels (data not

shown). This experiment was repeated twice with similar results.

P. Deshpande et al. / Journal of Neuroimmunology 173 (2006) 35–44 41

ordinarily innocuous myelin-specific CD4+ Tcells. However,

the molecular pathways underlying this effect remain to be

fully clarified. IL-12p70 directly stimulates myelin-reactive

T cells to express the chemokine receptor CCR5, potentially

facilitating CNS homing during EAE (Bagaeva et al., 2003).

We recently reported that IL-12p70 induces CD25�CD4+ T

cell activation and expansion in the presence of CD4+CD25+

T regulatory cells (King and Segal, 2005). This suggests that,

under certain circumstances, IL-12 might assist myelin-

specific T cells to escape immunoregulatory constraints and

initiate CNS inflammation. The current manuscript illustrates

yet another mechanism by which the cytokine could promote

encephalitogenicity, namely via induction of bioactive

PSGL-1 on autoimmune effector cells.

Expression of functional PSGL-1 is controlled, in large

part, at the level of post-translational modification. Synthe-

sis of bioactive PSGL-1 requires fucosylation, tyrosine-

sulfation and attachment of branched carbohydrate side

chains (McEver and Cummings, 1997). These events are

catalyzed by a series of dynamically regulated enzymes

including C2GnT-I and FucT-VII (Smithson et al., 2001;

Sperandio et al., 2001). It is likely that IL-12 dependent

expression of functional PSGL-1 on MBP-TCR Tg+ cells

results from enhanced transcription of C2GnT-I, which

catalyzes the addition of branched O-glycan side chains to

PSGL-1. The attachment of O-glycan side chains is critical

for P-selectin binding (Sperandio et al., 2001). By contrast,

IL-12 did not up-regulate expression of FucT-VII. These

results are consistent with a previous publication in which it

was demonstrated that induction of C2GnT-1, but not FucT-

VII, is dependent on IL-12/STAT-4 signaling in Th1

polarized ovalbumin-specific T cells (Lim et al., 2001). In

fact, several laboratories have found that, while IL-12

costimulation can prolong FucT-VII mRNA expression,

TCR activation alone is sufficient to induce FucT-VII

transcription (Lim et al., 2001; Blander et al., 1999).

Pre-incubation of IL-12/antigen stimulated MBP-TCR

Tg+ cells with anti-PSGL-1 antibodies suppressed their

encephalitogenicity, demonstrating the importance of

PSGL-1 for autoimmune pathogenesis in our experimental

system. It has previously been shown that foreign antigen-

specific or polyclonally activated Th1, as opposed to Th2,

cells preferentially express PSGL-1 that, in turn, influences

their migration patterns in vivo and facilitates their

recruitment to sites of inflammation in the skin and lamina

propia (Austrup et al., 1997; Borges et al., 1997; Haddad et

al., 2003). The current manuscript expands upon those

studies by demonstrating that IL-12 can directly stimulate

autoreactive, myelin-specific T cells to express PSGL-1 and

that this step can have a crucial impact on the ability of

those cells to trigger inflammatory demyelination in the

CNS. In future experiments we plan to assess whether active

immunization with myelin peptides in CFA is sufficient to

induce PSGL1 expression on MBP-specific T cells in vivo

by an IL-12 dependent pathway.

PSGL-1 has been extensively characterized as an adhesion

molecule that mediates leukocyte rolling on inflamed blood

vessels by binding P-and/ or E-selectin on endothelial cells.

Consistent with our findings, several investigators have

detected P-selectin protein on CNS endothelial cells during

the presymptomatic phase of EAE induced either by active

immunization or by the adoptive transfer of encephalitogenic

T cells (Carrithers et al., 2000; Kerfoot and Kubes, 2002;

Piccio et al., 2002). Bioactive PSGL-1 has been repeatedly

detected on myelin specific T cells used for EAE induction as

well as on CNS-infiltrating T cells harvested from symptom-

P. Deshpande et al. / Journal of Neuroimmunology 173 (2006) 35–4442

atic mice (Engelhardt et al., 2005, 1998, 1997). Therefore, all

of the components are in place for PSGL-1-mediated

tethering and rolling of autoreactive T cells in CNS vessels

early in EAE pathogenesis. However, there is conflicting data

regarding the actual importance of PSGL-1/P-selectin inter-

actions for the passage of activated Tcells (including myelin-

specific Th1 cells) across the blood–brain-barrier.

Piccio, et al. used intravital microscopy to show that

antibodies specific for either PSGL-1 or E and P selectins

inhibit the rolling and arrest of encephalitogenic T cells and

Th1-polarized CD4+ T cells on inflamed brain endothelium

(Piccio et al., 2005). Furthermore, Th1 cells from Fucosyl-

transferase VII-deficient mice, that are unable to synthesize

functional PSGL-1, were impaired in their ability to roll and

firmly adhere within CNS blood vessels. Using a similar

approach, the same investigators found that CD8+, though not

CD4+, cells from patients with multiple sclerosis roll and

tether in inflamed mouse cerebrovasculature via a PSGL-1

dependent mechanism (Battistini et al., 2003). Similarly,

Kerfoot and colleagues completely inhibited leukocyte roll-

ing and dramatically inhibited leukocyte adhesion on brain

microvasculature by in vivo administration of anti-P-selectin

antibodies to MOG-immunized C57BL/6 mice during pre-

symptomatic, acute and chronic phases of EAE (Kerfoot and

Kubes, 2002). In yet another study, anti-P selectin antibody

blocked early migration of labeled myelin-specific T cells

into the CNS (Carrithers et al., 2000). On the other hand,

Engelhardt and colleagues reported that in vivo administra-

tion of anti-E and anti-P selectin antibodies did not inhibit the

development of CNS infiltrates or clinical EAE in SJL mice

actively immunized with spinal cord homogenate (Engel-

hardt et al., 1997). Furthermore, treatment of SJL hosts with

anti-PSLG1 antibodies did not prevent the transfer of EAE

by established PLP-specific T cell lines (Engelhardt et al.,

2005). These apparent discrepancies might be explained, at

least in part, by differences in the experimental protocol

(including antibody dosing schedule), the characteristics of

monoclonal antibodies (affinity, pharmacokinetics, bioavail-

ability) and the inbred murine strains employed. Carrithers

and colleagues found significant differences between inbred

mouse strains with respect to constitutive and inducible P-

selectin expression in the CNS vasculature, which could

conceivably translate into inter-strain differences in the

relative contribution of PSGL-1/P-selectin interactions to

CNS trafficking (Carrithers et al., 2002).

Two recent studies showed that PSGL-1-deficient C57BL/

6 mice are susceptible to EAE induced by active immuniza-

tion with MOG peptide (Engelhardt et al., 2005; Osmers et

al., 2005). The knock-out mice resembled their wildtype

counterparts with respect to clinical course and immunohis-

tological characteristics of the inflammatory infiltrates.

However, PSGL-1 has been implicated in a wide range of

trafficking activities during homeostasis as well as inflam-

mation, including the migration of T cell precursors from

bone marrow to thymus (Rossi et al., 2005). Therefore, the

genetically engineered mice might have developed compen-

satory pathways that are not representative of immune

interactions in wildtype mice. Furthermore, in one of the

above studies PSGL-1-deficient mice exhibited a trend

towards delayed disease onset and had significantly lower

clinical scores during the early acute phase of EAE (days 10–

14 post immunization) (Osmers et al., 2005). Hence, although

PSGL-1/ P-selectin interactions might not be an absolute

requirement for CNS homing during the course of MOG-

induced EAE in C57BL/6 mice, such interactions could

enhance the efficiency and rate of disease induction.

In our studies, pre-incubation of MBP-TCR Tg+ cells

with anti-PSGL-1 antibodies had a dramatic effect in

reducing the severity as well as the incidence of EAE.

However, PSGL-1 blockade was not 100% effective in

preventing clinical EAE. This suggests that under some

circumstances circulating cells gain access to the brain and

spinal cord via PSGL-1-independent pathways. Indeed, in a

number of experimental paradigms a4 integrin indepen-

dently induced tethering, rolling and adhesion (or even

adhesion without prerequisite rolling) thereby bypassing the

need for selectins (Berlin et al., 1995). The existence of such

pathways does not exclude a role for PSGL-1/Pselectin

mediated rolling in boosting or optimizing CNS infiltration

by autoreactive T cells during the normal progression of

EAE. It is possible that anti-PSGL-1-treated MBP-TCR Tg+

cells are relatively compromised in their ability to roll and,

hence, adhere to cerebrovascular endothelium, decreasing

the probability that a given donor cell would penetrate the

BBB while still in an activated state. This might have lead to

a reduction in the total number of CNS inflammatory foci

established and/ or a reduction in the number of myelin-

reactive T cells that accumulate at individual foci following

adoptive transfer. It is also possible that PSGL-1/Pselectin

interactions play a role further downstream in the pathologic

process, beyond the point of BBB passage, perhaps during

the reactivation of effector T cells that have penetrated into

the target organ. Previous studies have demonstrated that

PSGL-1 ligation induces signaling events in T cells as well

as neutrophils and monocytes. For example, P-SGL1

ligation triggers phosphorylation of focal adhesion kinase

and Syk, promotes LFA-1 clustering and augments GM-

CSF production in T cells (Haller et al., 1997; Urzainqui et

al., 2002; Damle et al., 1992; Atarashi et al., 2005). Hence,

PSGL-1 blockade might directly inhibit myelin-specific T

cells from manifesting effector functions important for

induction of demyelination in the CNS.

Autoimmune demyelinating syndromes are driven by

complex immune–nervous system interactions. The specific

nature of these interactions might differ between experi-

mental models of EAE as well as between subsets of MS

patients, nonetheless resulting in a common histopatholog-

ical endpoint. The current study demonstrates that IL-12-

driven PSGL-1 expression can facilitate the development of

EAE and raises the possibility of blockade of PSGL-1/P-

selectin interactions which might be therapeutic in some

patients with MS.

P. Deshpande et al. / Journal of Neuroimmunology 173 (2006) 35–44 43

Acknowledgements

This work was supported by grants from the National

Multiple Sclerosis Society (JF2098A1/1) and the National

Institutes of Health (NS41562 and NS147687-0A1/1). BMS

is a Harry Weaver Neuroscience Scholar of the National

Multiple Sclerosis Society. The authors thank Dr. John

Olschowka for critically reading of the manuscript.

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