Gender-Specific Expression of b1 Integrin of VLA-4 inMyelin Basic Protein-Primed T Cells: Implications forGender Bias in Multiple Sclerosis
Saurav Brahmachari and Kalipada Pahan
Susceptibility to multiple sclerosis is higher in females than males. However, the underlying mechanism behind this gender dif-
ference is poorly understood. Because the presence of neuroantigen-primed T cells in the CNS is necessary to initiate the neuro-
inflammatory cascade of multiple sclerosis, we first investigated how these T cells interacted with astroglia, major resident glial cells
of the CNS. Interestingly, we found that myelin basic protein (MBP)-primed T cells from female and castrated male mice, but not
from male mice, produced proinflammatory molecules, such as NO, IL-1b, and IL-6 in astroglia, and these responses were purely
via contact between T cells and astroglia. Because T cell:glia contact requires several integrin molecules, we examined the
involvement of integrins in this process. Both a4 and b1, subunits of VLA-4 integrin, were found to be necessary for T cell
contact-induced generation of proinflammatory molecules in astroglia. Interestingly, the expression of b1, but not a4, was absent
in male MBP-primed T cells. In contrast, female and castrated male MBP-primed T cells expressed both a4 and b1. Similarly, we
also detected b1 in spleen of normal young female, but not male, mice. Furthermore, we show that male sex hormones (testos-
terone and dihydrotestosterone), but not female sex hormones (estrogen and progesterone), were able to suppress the mRNA
expression of b1 in female MBP-primed T cells. These studies suggest that b1, but not a4, integrin of VLA-4 is the sex-specific
molecule on T cell surface, and that the presence or absence of b1 determines gender-specific T cell contact-mediated glial
activation. The Journal of Immunology, 2010, 184: 6103–6113.
Multiple sclerosis (MS) is the most common humanautoimmune demyelinating disease of the CNS. It hasbeen known for decades that a female is twice as likely
as a male to be affected from MS. This is evident from the fact that~66% of MS patients are female (1, 2). Female prevalence is notonly observed in MS, but also in other autoimmune diseases, suchas Addison’s, rheumatoid arthritis, pernicious anemia, Sjogren’s,systemic lupus erythematosus, and thyroiditis (3). The corre-sponding animal models of these diseases, including experimentalallergic encephalomyelitis (EAE), an animal model of MS, alsoexhibit female bias (4–7). Although hormonal and genetic factorshave been postulated (8), the molecular mechanism behind thisgender bias is still poorly understood.The hallmark of brain inflammation in MS is the activation of
glial cells that express and produce a variety of proinflammatoryand neurotoxic molecules, including inducible NO synthase(iNOS) and proinflammatory cytokines (9–13). Semiquantitative
RT-PCR for iNOS mRNA in MS brains shows markedly higherexpression of iNOS mRNA in MS brains than in normal brains(14, 15). Hooper et al. (16) have reported that uric acid, a scav-enger of peroxynitrite (a highly reactive derivative of NO),markedly inhibits the appearance of EAE in mice, and that theincidence of MS is very rare among gout patients having higherlevels of uric acid. Among proinflammatory cytokines, primaryinflammatory cytokines, such as IL-1ba, TNF-ab, and IL-6, playa predominant role because they are involved at multiple levels ofneuroimmune regulation (13, 17, 18). Analysis of cerebrospinalfluid from MS patients has shown increased levels of proin-flammatory cytokines compared with normal control, and levels ofthose cytokines in the cerebrospinal fluid of MS patients alsocorrelate with disease severity (19). Consistently, blockade ofproinflammatory cytokine synthesis or function by signaling in-hibitors or neutralizing Abs or gene knockout can also prevent thedevelopment of EAE (18, 20). However, the mechanisms bywhich these proinflammatory molecules are produced in the CNSof MS patients are poorly understood.Recently, we have observed that neuroantigen-specific T cells
induce microglial expression of iNOS and proinflammatorycytokines (IL-1b, IL-1a, TNF-a, and IL-6) through VLA-4-me-diated cell-to-cell contact (21). Activation of both NF-kB and C/EBPb was involved in T cell contact-mediated microglial acti-vation (21). However, VLA-4-mediated contact was responsiblefor microglial activation of C/EBPb, but not NF-kB (21). Whenwe examined the gender dependency of this response, we foundthat myelin basic protein (MBP)-primed T cells isolated fromfemale and castrated male mice, but not male mice, induced theexpression of proinflammatory molecules (iNOS, IL-1b, IL-1a,IL-6, and TNF-a) in microglia via cell-to-cell contact (22). In-terestingly, T cell contact-mediated microglial activation of C/EBPb, but not NF-kB, was gender sensitive (22). Taken together,these results suggest that VLA-4 integrin on T cell surface could
Department of Neurological Sciences, Rush University Medical Center, Chicago, IL60612
Received for publication December 29, 2008. Accepted for publication March 24,2010.
This work was supported by grants from National Institutes of Health (NS39940,NS39940-10S1, and NS48923). The University of Rush Flow facility was supportedin part by the James B. Pendleton Charitable Trust.
Address correspondence and reprint requests to Dr. Kalipada Pahan, Department ofNeurological Sciences, Rush University Medical Center, Cohn Research Building, Suite320, 1735 West Harrison Street, Chicago, IL 60612. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this paper: DHT, dihydrotestosterone; EAE, experimentalallergic encephalomyelitis; ER, endoplasmic reticulum; ET, estrogen; GFAP, glialfibrillary acidic protein; iNOS, inducible NO synthase; MBP, myelin basic protein;MS, multiple sclerosis; PT, progesterone; TT, testosterone.
Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
be the gender-specific molecule regulating gender-specific acti-vation of microglial C/EBPb by T cell contact.Due to the facts that astroglia constitute the majority of resident
glial cells outclassing neuron and microglia by huge margin ofpopulation and that astroglial activation also contributes signifi-cantly to overall CNS inflammation (23–26), we tried to unravelthe mystery further behind gender bias of neuroantigen-specificT cell contact-mediated glial activation using primary mouse as-troglia. In this study, we report that female and castrated male, butnot male, MBP-specific T cells induce the expression of proin-flammatory molecules in astroglia via cell-to-cell contact. VLA-4is a heterodimer of a4 and b1 integrins. Interestingly, MBP-primed T cells of female, male, and castrated male mice expresseda4 integrin of VLA-4. In contrast, MBP-primed T cells of femaleand castrated male mice, but not that of male mice, expressed b1integrin. Furthermore, we demonstrate that male (testosterone[TT] and dihydrotestosterone [DHT]), but not female, sex hor-mones (estrogen [ET] and progesterone [PT]) are capable ofsuppressing the expression of b1 in MBP-specific T cells. Thesestudies identify b1 integrin of VLA-4 as a gender-specific mole-cule on T cell surface dictating the gender-specific T cell function.
Materials and MethodsReagents
FBS, HBSS, DMEM/F-12, RPMI 1640, L-glutamine, and 2-ME were fromMediatech (Washington, DC). Assay systems for IL-1b and IL-6 werepurchased from BD Pharmingen (San Diego, CA). Bovine MBP waspurchased from Invitrogen. Functional blocking Abs and FITC-labeledAbs to CD49d (the a4 chain of VLA-4) and CD29 (the b1 chain of VLA-4) were obtained from BD Pharmingen. PE-labeled Ab to CD3 was pur-chased from eBioscience (San Diego, CA). Multigene-12 RT-PCR pro-filing kits for mouse integrin gene family I and II were purchased fromSuperArray Bioscience (Frederick, MD). Annexin V-PE apoptosis de-tection kit was obtained from Biovision (Mountain View, CA). b-estradiol,PT, TT, and DHT (5a-androstan-17b-ol-3-one) were purchased fromSigma-Aldrich (St. Louis, MO).
Isolation of MBP-primed T cells
Specific pathogen-free female, male, and castrated male SJL/J mice (4–6wk old) were purchased from Harlan Sprague-Dawley (Indianapolis, IN).MBP-primed T cells were isolated and purified, as described earlier (21,22). Briefly, mice were immunized s.c. with 400 mg bovine MBP and 60mg Mycobacterium tuberculosis (H37RA; Difco Laboratories, Detroit, MI)in IFA (Calbiochem, San Diego, CA). Lymph nodes and spleens werecollected from these mice, and single-cell suspension was prepared inRPMI 1640 medium containing 10% FBS, 2 mM L-glutamine, 50 mM 2-ME, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells were culturedat a concentration of 4–5 3 106 cells/ml in 12-well plates. Cells isolatedfrom MBP-immunized mice were incubated with 50 mg/ml MBP for 4 d.The nonadherent cells were used to stimulate astroglial cells.
Passive transfer of MBP-primed T cells
Donor mice were immunized s.c. with 400 mg bovine MBP and 60 mg M.tuberculosis in IFA (16). Animals were killed 10–12 d postimmunization,and the draining lymph nodes were harvested. Single-cell suspensionswere treated with RBC lysis buffer (Sigma-Aldrich), washed, and culturedat a concentration of 4–5 3 106 cells/ml in six-well plates in RPMI 1640supplemented with 10% FBS, 50 mg/ml MBP, 50 mM 2-ME, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. On day 4,cells were harvested and resuspended in HBSS. A total of 2 3 107 viablecells in a volume of 200 ml was injected into the tail vein of naive mice.Pertussis toxin (150 ng/mouse; Sigma-Aldrich) was injected once via i.p.route on 0 d posttransfer of cells. Cells isolated from donor mice immu-nized with CFA or IFA alone were not viable after 4 d in culture with MBP,and therefore, were not transferred.
Isolation of mouse primary astroglia
Astroglia were isolated from mixed glial cultures following the procedure ofGiulian and Baker (27), as described previously (28). Briefly, cerebra takenfrom2- to 3-d-oldmousepupswere chopped, triturated, passed throughmesh,and trypsinized for the isolation ofmixed glial cells. On day 9, themixed glial
cultures were washed three times with DMEM/F-12 and subjected to a shakeat 240 rpm for 2 h at 37˚C on a rotary shaker to remove microglia. Similarly,on day 11, cells were shaken at 180 rpm for 18 h to remove oligodendroglia.Then, attached cells, primarily the astroglia, were trypsinized, subcultured,and plated accordingly to our experimental requirements.
Preparation of plasma membrane
Plasmamembranes of MBP-primed T cells were prepared by sonication andcentrifugation. Briefly, the cells were broken up by sonication, and thenuclear fraction was discarded after centrifugation for 10 min at 4,0003 g.The supernatant was centrifuged for 45 min at 100,000 3 g. The pellet ofT cell membranes was resuspended at 50 3 106 cell equivalents/ml bysonication in HBSS containing 20 mM EDTA and 5 mM iodoacetamide.
Stimulation of mouse primary astroglia by MBP-primed T cells
Astroglial cells were stimulated with different concentrations of MBP-primed T cells under serum-free condition. After 1 h of incubation, culturedishes were shaken and washed thricewith HBSS to lower the concentrationof T cells. Earlier, by FACS analysis of adherent microglial cells usingFITC-labeled anti-CD3 Abs, we demonstrated that more than 80% T cellswere removed from microglial cells by this procedure (21). Then astroglialcells were incubated in serum-free media for different periods of timedepending on the experimental requirements.
Assay for NO synthesis
Synthesis of NOwas determined by assay of culture supernatants for nitrite,a stable reaction product of NO with molecular oxygen. Briefly, super-natants were centrifuged to remove cells, and 400 ml of each supernatantwas allowed to react with 400 ml Griess reagent (29, 30) and incubated at
FIGURE 1. MBP-primed T cells from female and castrated male, but not
male, SJL/J mice induce the expression of proinflammatory molecules in
primarymouse astroglia. Primary astroglia received different concentrations
of MBP-primed T cells of female, male, and castrated male mice in direct
contact under serum-free condition. After 1 h of incubation, culture dishes
were shaken and washed thrice with HBSS to remove burden of T cells. A,
Then adherent astroglial cells were incubated in serum-free media for 5 h,
and expressions of iNOS, IL-1b, and IL-6were analyzed by semiquantitative
RT-PCR.After removal of T cells, adherent astroglial cells were incubated in
serum-free media for 23 h and supernatants were used to assay nitrite (B),
IL-1b (C), and IL-6 (D), as described in Materials and Methods. Data are
room temperature for 15 min. The OD of the assay samples was measuredspectrophotometrically at 570 nm. Fresh culture media served as the blank.Nitrite concentrations were calculated from a standard curve derived fromthe reaction of NaNO2 in the assay.
Assay for IL-1b and IL-6 synthesis
Concentrations of IL-1b and IL-6 were measured in culture supernatantsby a high-sensitivity ELISA (BD Pharmingen), according to the manu-facturer’s instruction, as described earlier (31).
Semiquantitative RT-PCR analysis
Total RNA was isolated from cells by using RNeasy mini kit (Qiagen,Valencia, CA) and from spleen by using Ultraspec-II RNA reagent (BiotecxLaboratories, Houston, TX), following manufacturer’s protocol. To removeany contaminating genomic DNA, total RNA was digested with DNase.SemiquantitativeRT-PCRwas carried out, as described earlier (32, 33), usinga RT-PCR kit from Clonetech (Mountain View, CA). Briefly, 1 mg total RNAwas reverse transcribed using oligo(dT)12–18 as primer and Moloney murineleukemia virus reverse transcriptase (BD Clontech, Palo Alto, CA) in a 20 mlreaction mixture. The resulting cDNAwas appropriately diluted, and dilutedcDNA was amplified using Titanium Taq DNA polymerase and followingprimers. Amplified products were electrophoresed on a 1.8% agarose gels andvisualized by ethidium bromide staining. iNOS, sense, 59-CCCTTCCGA-AGTTTCTGGCAGCAGC-39 and antisense, 59-GGCTGTCAGAGCCTC-GTGGCTTTGG-39; IL-1b, sense, 59-CTCCATGAGCTTTGTACAAGG-39and antisense, 59-TGCTGATGTACCAGTTGGGG-39; IL-6, sense, 59-GA-CAACTTTGGCATTGTGG-39 and antisense, 59-ATGCAGGGATGATGT-TCTG-39; integrin b1, sense, 59-GAGACATGTCAGACCTGCCTTGGCG-39 and antisense, 59-GGGATGATGTGGGGACCAGTAGGAC-39; integrina4, sense, 59-AACCGGGCACTCCTACAACCTGGAC-39 and antisense,59-ACCCCCAGCCACTGGTTATCCCTCT-39; integrin b2, sense, 59-CT-GCTGTGTCCCAGGAATGCACC-39 and antisense, 59-CCCGCCCAGC-TTCTTGACGTTGT-39; integrin b7, sense, 59-CTGAACTTCACTGCCT-
CGGGAGAGG-39 and antisense, 59-CTAGCTGGCGCACACGTTCCA-AGTC-39; andGAPDH, sense, 59-GGTGAAGGTCGGTGTGAACG-39 andantisense, 59-TTGGCTCCACCCTTCAAGTG-39.
Real-time PCR analysis
It was performed using the ABI-Prism7700 sequence detection system(Applied Biosystems, Foster City, CA), as described earlier (32). All primersand FAM-labeled probes for mouse genes and GAPDH were obtained fromApplied Biosystems. The mRNA expressions of respective genes werenormalized to the level of GAPDH mRNA. Data were processed by theABI Sequence Detection System 1.6 software and analyzed by ANOVA.
Flow cytometry
Surface expression of a4 and b1 and the surface expression of b1 alongwith CD3 on MBP-primed T cells or apoptosis of MBP-primed T cellswere monitored by single-color and two-color flow cytometry, re-spectively, as described previously (34, 35). Approximately 1 3 106 cellssuspended in RPMI 1640 medium-FBS were incubated in the dark withappropriately diluted FITC-labeled Abs to CD49d (integrin a4 chain) orCD29 (integrin b1 chain) for single color at 4˚C for 1 h. For two-color, 13106 cells suspended in 13 binding buffer were incubated under the samecondition with appropriately diluted FITC-labeled Abs to b1 and PE-la-beled CD3 or annexin V-PE. Following incubation, cell suspension wascentrifuged, washed three times, and resuspended in 500 ml RPMI 1640medium-FBS for single color or 13 PBS for two color. The cells were thenanalyzed through FACS (BD Biosciences, San Jose, CA) present in theUniversity of Rush Flow facility. A minimum of 10,000 cells was acceptedfor FACS analysis. Cells were gated based on morphological character-istics. Apoptotic and necrotic cells were not accepted for FACS analysis.
Analysis of mouse integrin a and b gene families by gene array
Expression of different integrins was analyzed in MBP-primed T cells bya RT-PCR-based gene array kit (GEArray) from SuperArray Bioscience,following manufacturer’s protocol. Briefly, the lyophilized component of
FIGURE 2. MBP-primed, but not naive, T cells
induce the expression of proinflammatory molecules
in primary mouse astroglia via cell-to-cell contact.
Primary astroglia received different concentrations
of naive or MBP-primed T cells of female mice in
direct contact under serum-free condition. After 1 h
of incubation, culture dishes were shaken and
washed thrice with HBSS to remove burden of
T cells. After 5-h incubation with serum-free media,
astroglia were analyzed for expression of iNOS and
IL-1b by semiquantitative RT-PCR (A), and after
24 h of incubation, supernatants were used for nitrite
(B) and ELISA assay (C). Primary astroglia received
different concentrations of conditioned supernatants
of female MBP-primed T cells under serum-free
condition. After 6 h of incubation, adherent astroglia
were analyzed for expression of iNOS by semi-
quantitative RT-PCR (D), and after 24 h of in-
cubation, supernatants were used to assay nitrite (E).
Astroglia received different concentrations of female
MBP-primed T cells within insert under serum-free
condition. After 6 h of incubation, adherent astroglia
were analyzed for expression of iNOS by semi-
quantitative RT-PCR (F), and after 24 h of in-
cubation, supernatants were used to assay nitrite (G).
After cell counting, female MBP-primed T cells
were subjected to plasma membrane preparation, as
mentioned in Materials and Methods. Then astroglia
were incubated with plasma membranes of MBP-
primed T cells under serum-free condition. After 6 h
of incubation, astroglia were analyzed for expression
of iNOS, IL-1b, and IL-6 by semiquantitative RT-
PCR (H), and after 24 h of incubation, supernatants
HotStart Sweet PCR master mix was resuspended in 300 ml double-distilled water. Then 20 ml of each cDNA synthesis reaction product wastransferred to separate tube of master mix. A total of 25 ml of a single PCRmixture was then dispensed to each of the 12 PCR tubes of the sameMultigene-12 Primer Strip. Strips were next placed in the thermal cyclerblock, and the appropriate program was run. Amplified products wereelectrophoresed on 4% agarose gels and visualized by ethidium bromidestaining.
Immunofluorescence analysis
Immunofluorescence analysis was performed, as described earlier (24).Briefly, mice were perfused intracardially with PBS (pH 7.4), and thenwith 4% (w/v) paraformaldehyde solution in PBS. Dissected spleens andcerebellum were postfixed in 4% formaldehyde/PBS for 2–5 d and cry-oprotected in 20% sucrose/PBS overnight at 4˚C. Tissues were then em-bedded in OCT (TissueTek, Elkhart, IN) at 250˚C, and processed forconventional cryosectioning to obtain frozen longitudinal sections (8 mm)and stored at 280˚C. Frozen sections were then allowed to cool at roomtemperature for 1.5–2 h, washed six times each for 5 min in 13 PBS,blocked in 2% BSA in 13 PBS with 0.5% Triton X-100 at room tem-perature, and incubated with rat anti-integrin b1 (1:400; Chemicon) andgoat anti-CD3 (1:100; eBioscience) Abs overnight at room temperature fordual immunohistochemistry. Sections were then washed six times in 13PBS and further incubated with Cy2 and Cy5 (Jackson ImmunoResearchLaboratories, West Grove, PA) for 1.5 h at room temperature, followed byovernight drying. Next, the sections were rinsed in distilled water, dehy-drated successively in ethanol and xylene, and mounted and observedunder an Olympus fluorescence microscope using a 403 objective.
ResultsMBP-primed T cells isolated from female and castrated male,but not male, SJL/J mice induced the expression of iNOS, IL-1b,and IL-6 in mouse primary astroglia via cell-to-cell contact
Earlier we have noticed that MBP-primed T cell contact-inducedexpression of proinflammatory molecules in microglia is gendersensitive (21, 22). Because astroglia are major glial cells in theCNS, we investigated whether astroglial production of proin-flammatory molecules via T cell contact also has gender speci-ficity. As described in earlier studies (21, 22), MBP-primed T cellswere washed and added to mouse primary astroglia in directcontact. After 1 h of contact, culture dishes were shaken andwashed thrice to remove MBP-primed T cells. We found thatastroglia also responded in a similar fashion like microglia toMBP-primed T cells (21, 22). MBP-primed T cells of female micemarkedly induced the expression of proinflammatory molecules(iNOS, IL-1b, and IL-6) in astroglia at different ratios of T cell/glia with the maximum increase found at 0.5:1 or 1:1 of T cell/glia(Fig. 1A). Nitrite estimation and ELISA of supernatants also showthat female MBP-primed T cells induced the production of NO(Fig. 1B), IL-1b (Fig. 1C), and IL-6 (Fig. 1D) in mouse primaryastroglia. However, unlike female MBP-primed T cells, MBP-primed T cells isolated from male mice were unable to induce theexpression of proinflammatory molecules (Fig. 1A) and the pro-duction of NO, IL-1b, and IL-6 (Fig. 1B–D) in astroglia, sug-gesting the possible involvement of male sex hormone in disablingmale MBP-primed T cells from contact-mediated activation ofastroglia. To further establish this hypothesis, we isolated MBP-primed T cells from castrated male mice. Interestingly, aftercastration, MBP-primed T cells from male mice behaved similarlyto female MBP-primed T cells and induced the mRNA expressionof iNOS, IL-1b, and IL-6 (Fig. 1A) and the production of NO, IL-1b, and IL-6 proteins (Fig. 1B–D) in astroglia.To examine whether priming of T cells with Ag is necessary to
induce proinflammatory molecules in astroglia, different doses ofnaive and MBP-primed T cells from female mice were added tomouse primary astroglia in direct contact. Our results (Fig. 2A–C)clearly demonstrate that only MBP-primed, but not naive, T cellswere able to induce the mRNA expressions of iNOS and IL-1b
(Fig. 2A), and produce NO (Fig. 2B) and IL-1b (Fig. 2C), sug-gesting that Ag priming of T cells is necessary to elicit proin-flammatory responses in astroglia.Next, we examined whether contact was necessary for the in-
duction of proinflammatory molecules in astroglia. Therefore, atfirst, we used conditioned supernatants of T cells to investigate therole of soluble factors released from T cells. It is clear from Fig. 2Dand 2E that different amount of supernatants was unable to inducethe expression of iNOS and the production of NO in astroglia. Wemust mention that 50 ml of supernatant was equivalent to T cells for0.5:1 of T cell/astroglia. Then T cells were put in inserts, so that thecells were maintained in close proximity to astroglia, but the actualcontact between them was shut off. In contrast to marked inductionof iNOS, IL-1b, and IL-6 by T cell:astroglia contact (Fig. 1), nosignificant increase either in the expression of iNOS (Fig. 2F) or inthe production of NO (Fig. 2G) was observed. These results sug-gest that direct contact between T cells and astroglia is essential forthe induction of iNOS and proinflammatory cytokines in astroglia.
FIGURE 3. Functional blocking Abs against either a4 or b1 chain of
VLA-4 inhibit the ability of female MBP-primed T cells to induce the
expression of proinflammatory molecules in primary mouse astroglia via
cell-to-cell contact. MBP-primed T cells were mixed with either different
concentrations of Abs against the a4 (A–D) or b1 (E–H) chain of VLA-4
or control IgG and rocked gently for 1 h at room temperature. Cells were
centrifuged, washed twice, and added to astroglia at a ratio of 0.5:1 T cell/
glia. After 1 h of stimulation, culture dishes were shaken and washed to
lower T cell concentration. A and E, Adherent astroglia were incubated in
serum-free media for 5 h, and expression of iNOS was analyzed by
semiquantitative RT-PCR (A, E). After removal of T cells, adherent as-
troglia were incubated in serum-free media for 23 h and supernatants were
used to assay nitrite (B, F), IL-1b (C, G), and IL-6 (D, H), as described in
Materials and Methods. Plasma membranes of MBP-primed T cells were
mixed with different concentrations of Ab against the b1 chain of VLA-4
or control IgG for 1 h, washed, and added to astroglia. After 6 h of in-
cubation, expression of iNOS in astroglia was analyzed by semi-
quantitative RT-PCR (I), and after 24 h of incubation, supernatants were
used to assay nitrite (J) and IL-1b (K). Data are mean 6 SD of three
different experiments. ap , 0.001 versus MBP-primed T cells only; bp ,0.05 versus MBP-primed T cells only.
To show that direct contact is sufficient to induce the expressionof these proinflammatory molecules in astroglia, membranes ofMBP-primed T cells were prepared and added on astroglia inequivalent amounts of T cell/astroglia. As expected, there wasgradual increase in the expression of iNOS, IL-1b, and IL-6mRNAs (Fig. 2H) as well as the production of NO (Fig. 2I) byplasma membrane of female MBP-primed T cells. These ob-servations strongly suggest that MBP-primed T cell contact issufficient to induce the expression of proinflammatory moleculesin astroglia.
Functional blocking Abs against subunits of VLA-4 inhibitedthe ability of female MBP-primed T cells to inducecontact-mediated production of proinflammatory molecules inmouse primary astroglia
Earlier we have shown that a4 integrin of VLA-4 on the surface ofMBP-primed T cells plays an important role in T cell contact-mediated activation of microglia (21, 22). Therefore, we examinedwhether this molecule was also involved in T cell contact-medi-ated induction of proinflammatory factors in astroglia. We blockeda4 and b1 subunits one at a time by using functional blocking Absagainst these subunits. Although not very effective at low con-centration (25 mg/ml), blocking of a4 chain by Abs at the con-centration of 50 and 75 mg/ml significantly inhibited theexpression of iNOS mRNA (Fig. 3A) and the production ofproinflammatory molecules (NO, IL-1b, and IL-6) (Fig. 3B–D) in
mouse primary astroglia. Interestingly, functional blocking of b1was more effective than that of a4 in negating the contact activityof MBP-primed T cells. A concentration of 5 mg/ml anti-b1 Abwas sufficient to inhibit the T cell contact-mediated expression ofiNOS mRNA (Fig. 3E) and the production of proinflammatorymolecules (Fig. 3F–H) in astroglia. Almost complete inhibition ofproinflammatory molecule production was observed at a concen-tration of 25 mg/ml anti-b1 Ab (Fig. 3E–H). These observationssuggest that both a4 and b1 chain of VLA-4 is necessary forcontact-mediated induction of proinflammatory molecules in as-troglia. Similar result was observed when b1 subunit was blockedin membrane fraction instead of whole cells (Fig. 3I–K), con-firming that VLA-4 is essential for contact-mediated induction ofproinflammatory molecules in astroglia.
b1, but not a4, integrin was differentially expressed in female,male, and castrated MBP-primed T cells of SJL/J mice
Because a4 and b1 integrins play a vital role in T cell contact-mediated expression of proinflammatory molecules in glial cellsand male MBP-primed T cells are incapable of inducing thesemolecules, we were prompted to investigate the expression patternof these integrins in male and female mice. Apart from forminga heterodimer with b1, the a4 integrin forms heterodimers withother integrins like b7. Therefore, we decided to include b7 in thestudy as well. It is evident from semiquantitative RT-PCR in Fig.4A and real-time PCR in Fig. 4B that MBP-primed T cells of
FIGURE 4. Expression of a4 and b1 integrins in
male, female, and castrated male MBP-primed T cells
and spleens. MBP-primed T cells of female, male, and
castrated male mice were analyzed for the expression of
a4, b1, and b7 integrins by semiquantitative RT-PCR
(A). The mRNA expression of a4 and b1 in female,
male, and castrated male MBP-primed T cells was
further analyzed by quantitative real-time PCR (B).
Data are mean 6 SD of three different experiments.
Female, male, and castrated male MBP-primed T cells
were treated with appropriately diluted FITC-labeled
Abs against a4 or b1 or b1 and CD3 for 30 min, fol-
lowed by FACS analysis (C, D). Splenic cross sections
of MBP-immunized female and male mice were double
immunolabeled with Abs against CD3 and b1 (E).
Setting of the microscope was strictly unaltered during
the whole study. Figures are representative of three in-
dependent experiments. Original magnification340. F,
Cells positive for CD3, b1, or CD3 and b1 were counted
in five splenic sections (three images per slide) of each
female, male, and castrated male mice expressed a4 and b7mRNAs. In contrast, MBP-primed T cells of female and castratedmale, but not male, mice expressed b1 mRNA (Fig. 4A, 4B). Real-time PCR analysis shows that the mRNA expression of b1 infemale and castrated male MBP-primed T cells was ~40-foldhigher than that in male T cells (Fig. 4B).Because these integrins are surface molecules, we confirmed our
result by FACS analysis. Consistent to mRNA expression, we didnot find any significant difference in the surface expression of a4integrin among female, male, and castrated male MBP-primedT cells (Fig. 4C, middle panel). However, the surface expressionof b1 integrin was much higher in female and castrated maleMBP-primed T cells than male MBP-primed T cells (Fig. 4C,bottom panel). We examined the pattern of surface expression ofb1 integrin in CD3-positive cells. Dual FACS analysis for CD3and b1 (Fig. 4D) clearly indicates significant increase in the ex-pression of b1 in CD3-positive cells following immunization withMBP in female mice. It is also evident from our FACS data thatapart from T cells, other nonadherent cells also express b1 atsignificant level (Fig. 4D). To further substantiate the fact, double-label immunofluorescence studies with anti-CD3 and anti-b1 Absin the splenic cross sections of MBP-immunized mice were per-formed and also revealed dramatic decrease in expression of b1 inmale compared with female, whereas there was no difference inCD3 expression (Fig. 4E). To quantitatively estimate the numberof cells expressing CD3 and/or b1, the absolute numbers of cellswere counted. Consistently, the results in Fig. 4F clearly indicatemarked decrease in b1-producing cells in the spleen of malecompared with female. Interestingly, apart from T cells that areCD3+, other splenic cells also expressed b1 integrin, as evidentfrom our results (Fig. 4F). The CD3-negative b1-producingsplenic cells are likely to be macrophages, which are the majorAPCs in spleen. However, further studies are needed to confirmthis fact. Taken together, our results suggest that inability of maleMBP-primed T cells to induce the expression of iNOS, IL-1b, andIL-6 in astroglia is probably due to the absence of b1 integrin inthese cells, and that the expression of this may be negativelyregulated by male sex hormone.
Are other integrins also absent in male MBP-primed T cells?
To examine whether b1 is the only integrin expressed differen-tially in female and male MBP-primed T cells, we analyzed geneexpression profiles of mouse integrin a and b family of genes byRT-PCR gene array analysis. We found that there was no signifi-cant difference in the expression of a4, a7, a8, a9, a10, a2b, aL,aM, aX, b2, b3, b4, b5, b6, and b7 between male and femaleMBP-primed T cells (Fig. 5, Supplemental Material). In contrast,the expression of a2, a3, a5, and aV was higher in male MBP-primed T cells than female MBP-primed T cells (Fig. 5, Supple-mental Material). Interestingly, b1 is the only integrin that wasfound to be almost missing from male MBP-primed T cellscompared with female T cells (Fig. 5). Because b1 integrin playsan important role in MBP-primed T cell contact-induced expres-sion of proinflammatory molecules in astroglia (Fig. 4), our datastrongly suggest that incapability of male T cells to induceproinflammatory molecules in astroglia is probably due to theabsence of b1 integrin.
Is the expression of b1 integrin in normal young female micehigher than normal young male mice?
We were further interested to see whether there was any differencein the expression of b1 integrin between normal young female andmale SJL/J mice. Interestingly, both semiquantitative and real-time RT-PCR analysis showed that spleen of naive female SJL/J
expressed significantly higher level of b1 compared with theirmale counterpart (Fig. 6A, 6B). The b1 integrin was dramaticallylow in male spleen (Fig. 6A, 6B). In contrast, the expression of b2integrin was same in both male and female MBP-primed T cells(Fig. 6A), suggesting the specificity of our observation. Becauseintegrins are surface molecules, to strengthen our finding, proteinlevel of the b1 was analyzed by FACS. Consistently, we observedmarked reduction in expression of b1 in naive splenocytes of malecompared with female, but there was no difference in expressionof CD3 (Fig. 6C).To further substantiate these findings, dual immunohistochemical
studies for CD3 and b1 were performed on splenic sections of naivemouse. In this study, it also showed dramatic decrease in expressionof b1 in the spleen of naive young male in comparison with femalewithout any alteration of expression of CD3 (Fig. 6D). The quan-titative estimation of CD3- and/or b1-positive cells further con-formed to our immunohistochemical studies (Fig. 6E). Interestingly,majority of the b1-positive cells were found to be CD3 negative,suggesting that in naive spleen, T cells do not express significantamount of b1. Therefore, it is likely that majority of the b1-pro-ducing cells in naive spleen are macrophages, which are the majorAPCs, but it requires further studies to establish this fact. However,irrespective of cell type, the level ofb1 is abruptly low in naivemalespleen, which clearly indicates that male-specific reduction in b1expression is not only limited to T cells, but also includes othersplenic cells expressing the integrin b1. Taken together, these
FIGURE 5. Gene array analysis of mouse integrin a and b gene fami-
lies. A, MBP-primed T cells of female and male mice were analyzed for a
and b integrin gene families by Multigene-12 PCR (SuperArray), as de-
scribed in Materials and Methods. B, Densitometric analysis was per-
formed to show comparative expressions of a and b integrin genes
between female and male MBP-primed T cells relative to GAPDH. Data
observations suggest that the difference in expression ofb1 betweenmale and female SJL/J mice is not a result of MBP immunization;rather, it exists normally, and therefore, could be described as anintrinsic sex-related phenomenon.
TT and DHT, but not ET and PT, selectively inhibited theexpression of b1 in female MBP-primed T cells in adose-dependent manner
Because castration of male SJL/J resulted in marked increase in theexpression of b1 at a level same as female MBP-T cells, we in-vestigated whether androgens had any effect on the expression ofthis integrin. Female MBP-primed T cells were treated withphysiologic doses of male and female sex hormones during MBPpriming. As evident from RT-PCR and quantitative real-time PCRanalysis, both TT (Fig. 7C) and DHT (Fig. 7D) dose dependentlyinhibited the expression of b1 integrin in female MBP-primedT cells. However, at the same condition, TT and DHT had noeffect on the expression of a4 integrin, the partner of b1 in VLA-4(Fig 7C, 7D). This was consistent with our observation that cas-tration of male did not alter the expression level of a4 integrin(Fig. 4A). These results suggest that the inhibitory effect of malesex hormones is specific for b1 integrin of VLA-4. In contrast, asevident from Fig. 7A and 7B, female-specific hormones (ET andPT) at physiologic doses had minimal or no effect on the ex-pression of either b1 or a4 integrin. To confirm the results further,
we also performed FACS analysis. Two-color FACS analysis usingPE-labeled annexin V and FITC-labeled b1 revealed that therewas no significant apoptotic cell death of T cells after MBP and/orhormone treatments (Fig. 8A, upper right quadrants). The FACSanalysis further showed that both TT and DHT, but not ET or PT,significantly reduced the proportion of b1 integrin-positive cells inthe MBP-primed T cells (Fig. 8A, lower right quadrants). Al-though there was substantial reduction in b1+ MBP-primed T cellsby TT or DHT treatment, still ~50% of MBP-primed T cellstreated with TT or DHT were found to be b1+. On the contrary,mRNA level of b1 was almost completely absent in TT- or DHT-treated female MBP-primed T cells (Fig. 7C, 7D). This apparentdiscrepancy could be explained by the fact that t1/2 of the in-tegrins, which are surface molecules, is usually relatively longerthan other molecules. Therefore, although mRNA expression wasinhibited completely, protein level was still there.To further confirm whether there was significant downregulation
of integrin b1 in MBP-primed T cells following hormone treat-ment, we also analyzed the expression level of b1 per cell bycalculating mean fluorescence intensity of b1+ cells. Expectedly,we found that both TT and DHT, but not ET or PT, markedlydownregulated the expression of b1 in MBP-primed T cells (Fig.8B). Interestingly, DHT appeared to have stronger effect than TTon the expression of b1 (Figs. 7D, 8A, 8B). This could be becauseof aromatase activity, which is capable of converting some TT to
FIGURE 6. Expression of b1 integrin in spleens of
naive young female and male mice. RNA isolated
from spleen of naive young female and male SJL/J
mice (4–6 wk old) was analyzed for the mRNA ex-
pression of b1 and b2 integrins by semiquantitative
RT-PCR (A). The mRNA expression of b1 integrin
was also confirmed by quantitative real-time PCR (B).
Data are mean 6 SD of three different experiments. a
p , 0.001 versus male. Naive female and male
splenocytes after isolation were treated with appro-
priately diluted FITC-labeled Abs against b1 or PE-
labeled Abs against CD3 for 30 min, followed by
FACS analysis (C). Splenic cross sections of naive
young female and male mice were double im-
munolabeled with Abs against CD3 and b1 (D).
Setting of the microscope was strictly unaltered dur-
ing the whole study. Figures are representative of
three independent experiments. Original magnifica-
tion 340. E, Cells positive for CD3, b1, or CD3 and
b1 were counted in five splenic sections (three images
ET, whereas DHT, the active metabolite of TT, remains un-affected. These findings suggest that male- but not female-specifichormones are capable of downregulating the b1 subunit of VLA-4integrin at physiological doses.
TT and DHT, but not ET and PT, inhibited the ability of femaleMBP-primed T cells to induce contact-mediated expression ofproinflammatory molecules in mouse primary astroglia
Because of our findings that TT and DHT treatment of femaleMBP-primed T cells resulted in the inhibition of b1 and that b1was found to be necessary for T cell contact-mediated induction ofproinflammatory molecules in mouse primary astroglia, we wereinterested to examine whether androgens downregulated thiscontact activity of MBP-primed T cells. Female MBP-primedT cells were treated with TT, DHT, ET, and PT, followed by ad-dition of hormone-treated T cells to astroglia in direct contact.Consistent with the inhibition of b1 integrin, both TT and DHTsuppressed the ability of female MBP-primed T cells to inducecontact-mediated expression of proinflammatory molecules (iNOSand IL-1b) (Fig. 9A) and production of NO (Fig. 9B) and IL-1b(Fig. 9C) in astroglia. Expectedly, female-specific hormones (ETand PT) were unable to inhibit this contact activity of femaleMBP-primed T cells (Fig. 9A–C). In parallel experiments, we alsoexamined whether these male and female sex hormones had anyeffect on the missing proinflammatory contact activity of maleMBP-primed T cells. As evident from Fig. 9D–F, all four sexsteroids had no effect on the missing contact activity in maleMBP-primed T cells.
DHT, but not ET, inhibited the ability of female MBP-primedT cells to induce iNOS in vivo in the cerebellum of adoptivelytransferred mice
To confirm our findings in vivo, we examined the expression ofiNOS and glial fibrillary acidic protein (GFAP), the marker ofastroglial activation, in the cerebellum of female SJL/J mice that
received adoptive transfer of hormone-treated or untreated femaleor male MBP-primed T cells. Consistent with our in vitro results,mice transferred with female MBP-primed T cells or ET-treatedfemale MBP-primed T cells showed significant increase in the levelof iNOS in astroglial cells compared with control (Fig. 10A, secondand third rows). Parallel increase in GFAP in iNOS-producingastroglia indicates astroglial activation, which is consistent withour previous studies (24). Expectedly, DHT treatment of MBP-primed T cells markedly reduced the level of iNOS and GFAP inthe cerebellum, thereby further confirming the consequence ofhormonal regulation of b1 integrin in T cells under in vivo con-dition (Fig. 10A, bottom row). Similar to our in vitro results, micereceiving male MBP-primed T cells did not show any significantlevel of iNOS (Fig. 10B, first row) and hormone treatment of maleMBP-primed T cells almost had no effect (Fig. 10B).
DiscussionT cell-mediated autoimmune response is believed to cause damagein the CNS ofMS patients. Because lymph node and spleen, the twoprimary activation site T cells, express MBP mRNA and protein inrat, mouse, and human (36), it is widely believed that neuro-antigen-specific autoimmune T cells are activated at those sitesand thereby infiltrate into CNS after crossing blood brain barrier.In the CNS microenvironment, these T cells recognize their Agsand interact with resident glial cells, and subsequent glial acti-vation triggers a broad-spectrum inflammatory cascade, whichultimately results in oligodendrocyte death and demyelination. Itmay be likely that as females are more susceptible to MS thanmales, the neuroantigen-specific T cells do more severe CNSdamage in female than in male. However, the exact molecular
FIGURE 7. Effect of ET, PT, TT, and DHT on the expression of a4 and
b1 integrins in female MBP-primed T cells. MBP-primed T cells treated
with different concentrations of ET (A), PT (B), TT (C), or DHT (D) for
72 h during MBP priming were analyzed for the mRNA expression of a4
and b1 integrins by semiquantitative RT-PCR (upper panels) and real-time
PCR (lower panels). Data are mean 6 SD of three different experiments.ap , 0.001 versus MBP only.
FIGURE 8. Effect of ET, PT, TT, and DHT on the surface expression of
b1 integrin in female MBP-primed T cells. Female MBP-primed T cells
treated with ET, PT, TT, and DHT for 72 h during MBP priming were
incubated with appropriately diluted FITC-labeled anti–VLA-4 b1 and PE-
labeled annexin V for 1 h, followed by two-color FACS analysis. Figures
represent three independent experiments (A). Mean fluorescence intensity
of b1 in FITC-positive cells was calculated by using CellQuest software
(B). Data are 6 SD of three independent experiments.
mechanism behind the sexual dimorphism of CNS neuro-degeneration in MS is unknown.We have previously reported that MBP-primed T cell contact-
mediated activation of microglia is gender sensitive (22). Becauseastroglia constitute majority of resident glial cells, astroglial ac-tivation could also play a vital role in the pathogenesis of MS andEAE. In this manuscript, we have presented substantial evidencethat supports that MBP-primed T cell contact-mediated astroglialactivation is also gender sensitive. First, female MBP-primedT cells dose dependently induced the expression of iNOS andproinflammatory cytokines (IL-1b and IL-6) as well as the pro-duction of these proinflammatory molecules in primary mouseastroglia. Either T cell in direct contact or T cell membrane wascapable of inducing proinflammatory molecules in astroglia. Incontrast, T cells placed on inserts and supernatants of T cells wereunable to induce the same proinflammatory molecules in astroglia,suggesting that this induction was purely because of direct contactbetween T cells and astroglia. Second, female and castrated maleMBP-primed T cells, but not male MBP-primed T cells, were ableto induce the expression of proinflammatory molecules (iNOS, IL-1b, and IL-6) in astroglia, clearly suggesting the gender specificityof astroglial activation driven by MBP-primed T cell contacts.We next investigated the underlying mechanism behind the
inability of male MBP-primed T cells to activate astroglia viacontact. Because VLA-4, according to our previous report (21),plays an important role in contact-mediated induction of proin-flammatory molecules in microglia, and as VLA-4 is a hetero-dimer of a4 and b1, we examined the role of a4 and b1 integrinsin contact-mediated induction of proinflammatory molecules in
astroglia. Impairing the function of either a4 or b1 integrin ofVLA-4 of female MBP-primed T cells significantly inhibited theirability to induce the expression of proinflammatory molecules inmouse astroglia, suggesting an essential role of each of the sub-units of VLA-4 integrin in contact-mediated activation of as-troglia. However, how VLA-4 leads to astroglial activation is notunderstood yet. The contact molecule for VLA-4 in astroglia isprobably the VCAM-1. Activated T cells secrete various proin-flammatory molecules, such as IFN-g and TNF-a, and thesemolecules are capable of upregulating VCAM-1 in astrocytes (37).Blocking of VLA-4 or VCAM-1 has been shown to prevent T celladhesion to astrocytes, suggesting that VLA-4-VCAM-1 in-teraction is necessary for T cell adhesion to astrocytes (37).Moreover, induction of VCAM-1 has been found to be associatedwith astroglial activation in the spinal cord of EAE, a model ofMS (38). However, the downstream signaling events leading toastroglial activation are yet to be investigated. As we have pre-viously reported that VLA-4-mediated microglial activation byT cell contact involves activation of C/EBPb (21, 22), it can bespeculated that this molecule may also be involved in astroglialactivation by T cell contact.Requirement of VLA-4 subunits in T cell contact-mediated
induction of proinflammatory molecules in astrocytes prompted usto examine whether there was any gender bias in the expression ofindividual subunits of VLA-4 in T cells. Surprisingly, the ex-pression of only b1 integrin, but not a4 and others, was found
FIGURE 9. Effect of ET, PT, TT, and DHT on the ability of female and
male MBP-primed T cells to induce contact-mediated expression of
proinflammatory molecules in primary mouse astroglia. Female (A–C) and
male (D–F) MBP-primed T cells treated with respective concentrations of
ET, PT, TT, and DHT for 72 h during MBP priming were added to astroglia
at a ratio of 1:1 T cell/astroglia. After 1 h of stimulation, culture dishes
were shaken and washed to lower T cell concentration. Then adherent
astroglia were incubated in serum-free media for 5 h, and the expression of
iNOS mRNA was analyzed by semiquantitative RT-PCR (A, D). Adherent
astroglia were incubated in serum-free media for 23 h, and supernatants
were used to assay nitrite (B, E) and IL-1b (C, F). Data are mean 6 SD of
three different experiments. ap , 0.001 versus MBP-primed T cells only.
FIGURE 10. Effect of ET and DHT on the ability of female and male
MBP-primed T cells to induce expression of iNOS in the cerebellum of
adoptively transferred mice. Female (A) and male (B) MBP-primed T cells,
treated with appropriate concentrations of ET or DHT for 96 h during MBP
priming, were adoptively transferred to female SJL/L mice. On day 5
postimmunization, cerebellar sections were double immunolabeled with
Abs against iNOS and GFAP. Setting of the microscope was strictly un-
altered during the whole study. Figures are representative of three in-
significantly less in male MBP-primed T cells compared withfemales. However, after castration, castrated male MBP-primedT cells expressed b1 at a level comparable to female MBP-primedT cells. These studies strongly suggest that inability of male MBP-primed T cells to activate astroglia is attributed by diminishedlevel of b1 subunit of VLA-4.We next asked why the male SJL/J has defective b1. Comparable
level of b1 in castrated male MBP-primed T cells suggested thatsex hormones might play an important role in the regulation of b1.Sex steroids are important modulators of disease processes of MSand EAE. Sex hormones can have both immunomodulatory andneuroprotective effect on EAE (39–42). Both TT and ET havebeen found to be protective in EAE (40, 42, 43). The immuno-modulatory effect of male sex hormones on EAE is mostly exertedthrough reduction of Th1 cytokines. However, immunomodulatoryand neuroprotective effects of ET are specifically mediated byendoplasmic reticulum (ER) a, but not ERb (44). Therefore, it islikely that distribution of ER as well as the ET level may playa critical role in determining the beneficial role of ET in MS andEAE. It is probably the higher level of ET than the normalphysiological level that protects MS and other autoimmune dis-eases. Probably because of this reason, MS patients as well aspatients of other autoimmune diseases experience reduced clinicalsymptoms during pregnancy when ET level goes up (45, 46).In our current study, we found that TT and DHT dose de-
pendently downregulated expression of b1, but not a4, of femaleMBP-primed T cells, whereas ET and PT had no significant effect.Effect of DHT was relatively stronger than TT. It is probablybecause of aromatase activity that could convert some TT, but notDHT, to ET (47). We further demonstrated that contact-mediatedinduction of proinflammatory molecules in astroglia was signifi-cantly inhibited by TT- or DHT-treated female MBP-primedT cells, but the same T cells when treated with ET or PT wereincapable of inhibiting the induction of proinflammatory mole-cules in astroglia. Our in vivo results further substantiated thisfinding, in which we noticed that mice adoptively transferred withDHT-treated, but not ET-treated, female MBP-primed T cellsshowed dramatically low level of iNOS and GFAP, the markerprotein of astroglial activation. Therefore, inability of female-de-rived MBP-primed T cells treated with TT or DHT to expressspecifically b1 and, subsequently, the inability of those cells toproduce proinflammatory molecules in mouse primary astrogliavia contact clearly suggest that specifically the b1 subunit ofVLA-4 is negatively regulated by male sex hormones and, thereby,these hormones inhibit T cell contact-mediated astroglial activa-tion. Thus, our results explore a novel mechanistic aspect ofprotective role of male sex hormone that emphasizes the impor-tance of T cell contact-mediated CNS inflammation in the gendersusceptibility of EAE.In summary, we have delineated a possible mechanism of sexual
dimorphism in MS. Because even the naive young male mice didnot express b1, this integrin may turn out to be a susceptibilitymarker for MS and, thus, it may be considered as a possiblegender-specific factor for increased incidence of MS in females.Our study also reveals a possible new direction for MS therapy.Tysabri, as a new avatar of antegrin, which is a functional blockingAb against a4 integrin of VLA-4, is rocking the headlines asa new treatment for MS. However, Tysabri increases the risk ofprogressive multifocal leukoencephalopathy, an opportunistic vi-ral infection of the brain that usually leads to death or severedisability. In contrast, if our mouse results are extrapolated tohuman, young males should have lower level of b1. However,despite the deficiency of b1, young males do fine because otherintegrins are present in male T cells at a comparable level or at
a level higher than female T cells. According to our unpublishedobservation, like female MBP-primed T cells, the male MBP-primed T cells were also capable of infiltrating into the CNS. Thisobservation suggests that function of VLA-4 was compensated bysome other molecule(s) that facilitated the extravasation of maleT cells as efficiently as that of females. Although b1 null mice areembryonic lethal (48–50), this is because the integrin b1 is criti-cally required for embryogenesis. In contrast, our experimentalfindings suggest that absence or low amount of b1 in male isprobably due to the regulation by male-specific sex hormones. Assex hormones can play a role only during puberty and onward, it islikely that during embryogenesis, the level of b1 integrin in maleis normal. Therefore, developing functional blocking Abs againstb1 may provide a safer handle than Tysabri to contain MS andother autoimmune disorders.
DisclosuresThe authors have no financial conflicts of interest.
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