P-Selectin Glycoprotein Ligand 1 Is Not Required for the Development of Experimental Autoimmune Encephalomyelitis in SJL and C57BL/6 Mice1
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免疫学杂志 2005年第14期
Abstract
In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the endothelial blood-brain barrier and gain access to the CNS. The involvement of P-selectin glycoprotein ligand 1 (PSGL-1) and of its major endothelial ligand P-selectin in this process have been controversial. In this study we demonstrate that although encephalitogenic T cells express functional PSGL-1, which can bind to soluble and immobilize P-selectin if presented in high concentrations, PSGL-1 is not involved T cell interaction with P-selectin expressing brain endothelial cells in vitro. Furthermore, neither anti-PSGL-1 Abs nor the lack of PSGL-1 in PSGL-1-deficient mice inhibits the recruitment of inflammatory cells across the blood-brain barrier or the development of clinical EAE. Taken together, our findings demonstrate that PSGL-1 is not required for the pathogenesis of EAE.
Introduction
The CNS is considered an immuneprivileged site where the endothelial blood-brain barrier (BBB)3 tightly controls lymphocyte entry into the CNS. Under physiological conditions lymphocyte traffic into the CNS is low whereas during inflammatory diseases of the CNS, such as multiple sclerosis or in the animal model experimental autoimmune encephalomyelitis (EAE), a large number of circulating lymphocytes readily gain access to the CNS. EAE is a CD4+ T cell-mediated autoimmune disease of the CNS, which is initiated by autoaggressive T cells activated outside the CNS. These encephalitogenic T cell blasts enter the CNS parenchyma across the healthy BBB. Upon encounter of Ag, autoaggressive T cells initiate inflammation and the recruitment of inflammatory effector cells across the inflamed BBB into the CNS leading to edema and demyelination. Thus, the interaction of autoaggressive and inflammatory effector cells with the BBB is a critical step in the pathogenesis of EAE.
In general, lymphocyte recruitment across the vascular wall is regulated by the sequential interaction of different adhesion or signaling molecules on lymphocytes and endothelial cells lining the vessel wall (1). An initial transient contact of the circulating leukocyte with the vascular endothelium, generally mediated by adhesion molecules of the selectin family or alternatively by 4 integrins and their respective ligands, slows down the leukocyte in the bloodstream. With greatly reduced velocity the leukocyte rolls along the vascular wall allowing it to recognize chemokines displayed on the endothelial surface. Binding of a chemokine to its G protein-coupled receptor on the leukocyte surface results in a pertussis toxin-sensitive activation of integrins on the leukocyte surface. Activated integrins mediate the firm adhesion of the leukocytes to the vascular endothelium by binding to their endothelial ligands, which belong to the Ig superfamily. This ultimately leads to the extravasation of the leukocyte. Successful recruitment of circulating leukocytes into the tissue depends on the productive leukocyte/endothelial interaction during each of these sequential steps.
The mechanisms mediating leukocyte recruitment into the CNS are not completely understood. mAbs directed against 4 integrin have been shown to successfully interfere with the development of EAE in different animal models (2, 3, 4, 5) and moreover to reverse the ongoing disease process (6). As the anti-4 integrin treatment proved to block adhesion of inflammatory cells to inflamed cerebral venules in vitro (2, 7) and to substantially decrease the recruitment of inflammatory cells across the BBB in vivo, there is general agreement that there is a pivotal role for 4 integrins in leukocyte/BBB interaction during EAE (2, 5).
The predominant involvement of 4 integrins in leukocyte recruitment across the BBB during EAE does not exclude the contribution of other adhesion molecules in this process. The potential involvement of selectins in particular in T lymphocyte interaction with the BBB has been controversial. We have reported a lack of E- and P-selectin expression in CNS microvessels during EAE and demonstrated that anti-E- and anti-P-selectin Abs do not inhibit the development of EAE (8). More recent work of other laboratories using intravital microscopy demonstrated the involvement of E- and P-selectin and their ligand P-selectin protein ligand 1 (PSGL-1) in tethering and rolling of leukocytes or encephalitogenic T cells in superficial brain microvessels in vivo (9, 10). In contrast, when performing intravital microscopy of the spinal cord white matter, we found a lack of rolling and a predominant role of 4 integrins in the initial capture and the firm adhesion of autoaggressive T cells within the spinal cord white matter microvasculature (11).
These apparent discrepancies prompted us now to investigate the functional expression of the homodimeric sialomucin PSGL-1 on encephalitogenic T cells and its potential involvement in inflammatory cell recruitment across the BBB during EAE. In this study we demonstrate that although encephalitogenic T cells express functional PSGL-1 on their surface as exemplified by the ability to bind purified P-selectin, PSGL-1 is not involved in interactions of these T cells with brain endothelium in vitro. Furthermore, PSGL-1 is not required for the development of EAE in SJL or C57BL/6 mice.
Results
To find out whether PSGL-1 expression can be detected in the brains and spinal cords of mice afflicted with EAE, we performed immunohistochemistry using three different anti-PSGL-1 Abs. During EAE immunostaining for PSGL-1 was observed on the majority of inflammatory cells localized within perivascular cuffs (Fig. 1A). Additionally immunostaining for PSGL-1 was observed in the choroid plexus and the CNS parenchyme resembling immunostaining for CD45 (Fig. 1B). PSGL-1-positive parenchymal cells were characterized as Mac-1-positive microglial cells by double-immunofluorescence staining (Fig. 1B). Thus, PSGL-1 might be involved in inflammatory cell recruitment into the CNS, whereas its up-regulated expression on microglial cells within the CNS parenchyme suggests additional functions for PSGL-1 in the pathogenesis of EAE.
Encephalitogenic T cells express functional PSGL-1
To define whether encephalitogenic T cells express PSGL-1 on their surface we performed immunofluorescence stainings using three different mAbs directed against murine PSGL-1 (4RA10, 4RB12, 2PH1). Encephalitogenic PLP-specific T cells were found to display surface expression levels of PSGL-1 comparable to those observed on mPC7 cells or on bone marrow-derived murine neutrophils (Fig. 2). Surface expression levels of PSGL-1 did not change depending on the activation status of the T cells (data not shown). Selectin ligands including PSGL-1 require posttranslational modifications for selectin binding. To determine whether encephalitogenic T cells bind P-selectin we studied the binding of P-selectin IgG to encephalitogenic T cells as compared with mPC7 cells or bone marrow-derived neutrophils by FACS analysis. Binding of P-selectin IgG to encephalitogenic T cells could only be observed if P-selectin IgG was offered in high concentrations (30–50 μg/ml). Under these experimental conditions only a minor population of encephalitogenic T cell blasts (15%) were found to bind P-selectin IgG in high amounts whereas binding of P-selectin IgG to the majority of T cells was found to be very low (Fig. 2A). In contrast, P-selectin binding to mPC7 cells or bone marrow-derived neutrophils was already found to be optimal at lower concentrations of P-selectin IgG fusion protein (10–25 μg/ml) with 100% of the cells binding P-selectin homogeneously (Fig. 2, B and C). Dependence of P-selectin binding on the presence of divalent cations was confirmed by the loss of P-selectin binding to all three investigated cell populations in the presence of 2 mM EDTA, whereas detection of PSGL-1 protein surface expression remained unaffected (Fig. 2). Thus, although encephalitogenic T cells can bind soluble P-selectin, it is less efficient than P-selectin binding to mPC7 cells or polymorphonuclear granulocytes (PMNs).
Encephalitogenic T cells can bind to high concentrations of immobilized P-selectin in vitro
To determine whether P-selectin binding to encephalitogenic T cells is mediated via PSGL-1, we analyzed their binding to P-selectin IgG immobilized on glass slides. Optimal binding of mPC7 cells to immobilized P-selectin IgG could already be observed when 20 μl of 25 μg/ml fusion protein was used for coating (data not shown). T cell binding to immobilized P-selectin IgG was only observed at concentrations of 50 μg/ml and reached an optimum at 100 μg/ml. Under these experimental conditions encephalitogenic T cells were found to bind to P-selectin, which could be inhibited by the anti-P-selectin Ab RB40 (Fig. 3). PSGL-1 was found to be the major P-selectin ligand on encephalitogenic T cells as the PSGL-1 blocking mAbs 4RA10 and 2PH1 almost completely inhibited T cell binding. At the same time, the nonblocking mAb 4RB12 or the anti-LFA-1 Ab FD441.8 had no effect on T cell binding to immobilized P-selectin. Thus, provided P-selectin is available in high density, encephalitogenic T cells can bind via PSGL-1 to immobilized P-selectin.
PSGL-1 is not involved in adhesive interactions of T cells with brain endothelium in vitro
We next asked whether encephalitogenic T cells can use PSGL-1 to adhere to P-selectin on activated brain endothelial cells in vitro. Therefore we performed adhesion assays with freshly activated PLP-specific T cell blasts and the brain endothelioma cell line bEnd5, which upon 3 h of stimulation with LPS or TNF- expresses P-selectin on its surface (22). PLP-specific T cell blasts readily bound to stimulated bEnd5. Pretreatment of T cell blasts with a mAb directed against PSGL-1 had no effect on T cell adhesion to stimulated bEnd5 (Fig. 4). Supporting a lack of involvement of PSGL-1/P-selectin interaction in T cell adhesion to brain endothelium after 3 h of stimulation, T cell binding to E/P-selectin double-deficient bEndEP.5 was found to be indistinguishable from T cell adhesion to wild-type bEnd5 when compared within the same assay (Fig. 4). At the same time blocking LFA-1 significantly inhibited T cell adhesion to both, stimulated bEnd5 and bEndEP.5 (Fig. 4).
Comparing T cell migration across stimulated bEnd5 in the presence or absence of PSGL-1 blocking Abs confirmed a lack of involvement of PSGL-1 in this process (data not shown). Finally, the pretreatment of bEnd5 with the P-selectin blocking Ab RB40.34 had no influence on T cell adhesion to and migration across stimulated brain endothelium confirming the lack of involvement of PSGL-1/P-selectin binding during these processes (data not shown).
Anti-PSGL-1 Abs do not inhibit PLP-induced T cell proliferation or IFN- production
Adhesion molecules on autoaggressive T cells could be involved in the pathogenic processes during EAE other than T cell migration into the CNS. Therefore we asked whether PSGL-1 is involved in the Ag-dependent activation of encephalitogenic T cells. PLP-specific T cells were restimulated with their specific PLP peptide using irradiated syngeneic splenocytes as APCs in the presence or absence of anti-PSGL-1 Abs. Blocking PSGL-1 did neither influence Ag-induced T cell proliferation (Fig. 5A) nor IFN- production (Fig. 5B).
Anti-PSGl-1 Abs do not inhibit the development of tEAE in the SJL mouse
To investigate whether, regardless of the lack of PSGL-1 contribution to T cell activation or T cell interaction with brain endothelium in vitro, PSGL-1 could still be involved in the pathogenesis of EAE, we investigated the effect of the PSGL-1 blocking mAb 4RA10 on the development of EAE in the SJL mouse. Only 30 μg of 4RA10 injected into a mouse were previously shown to be sufficient to completely block PSGL-1-mediated rolling of leukocytes in vivo (26). EAE was transferred by the injection of 3 x 106 syngeneic PLP-specific T cell blasts and animals were monitored daily for the development of clinical disease. In seven different experiments, repeated infusions of 4RA10 injected at dosages up to 400 μg/injection failed to reproducibly interfere with the onset of clinical EAE or the recruitment of inflammatory cells into the CNS when compared with animals treated with control Abs (Fig. 6). Quantitative assessment of the number of perivascular inflammatory cuffs present in CD45-immunostained frozen brain and spinal cord sections of mice treated with anti-PSGL-1 Abs in comparison to control EAE animals did not reveal any difference between the different treatment groups (Table I). Also, no difference was found in the composition of the CD45-positive inflammatory infiltrates, which consisted mainly of CD4+ T cells, Mac-1+ macrophages, some B220+ B cells and rare CD8+ T cells (data not shown). Gr-1-positive granulocytes were not observed. Thus, inhibition of the interaction of PSGL-1 with its respective selectin ligands in vivo did not significantly alter the development of tEAE in the SJL mouse. Taken together, these observations demonstrate that PSGL-1 is not required for the development of EAE in SJL mice.
PSGL-1-deficient C57BL/6 mice develop EAE
To confirm the lack of requirement of PSGL-1 in EAE pathogenesis, PSGL-1-deficient mice were backcrossed into the C57BL/6 background. Active EAE was induced by immunization with the MOG peptide (aa 35–55) in CFA and mice were monitored daily for clinical symptoms of EAE. In five different experiments PSGL-1-deficient C57BL/6 mice did not show any significant difference in the onset of actively induced EAE when compared with C57BL/6 wild-type mice (Fig. 7). Both groups of mice exhibited chronic disease, which is typical of MOG-induced EAE in the C57BL/6 model. Quantitative assessment of the number of perivascular cellular infiltrates in CD45-immunostained frozen brain and spinal cord sections of wild-type and PSGL-1-deficient mice did not reveal any significant difference between both groups (Table I). Immunohistological analysis of the cellular composition of the inflammatory infiltrates confirmed the presence of mainly Mac-1+ macrophages, CD4+ T cells, and scattered Gr-1-positive granulocytes in the brain and spinal cord of both wild-type and PSGL-1-deficient mice (Fig. 8). Thus, as absence of PSGL-1 neither influences the recruitment of different inflammatory cell subsets across the BBB during EAE nor inhibits the development of clinical EAE, PSGL-1 is not required for the development of clinical EAE in the C57BL/6 mouse.
Discussion
In this study we show that although encephalitogenic T lymphocytes display surface expression of PSGL-1, their PSGL-1-mediated binding to P-selectin is apparently poor as it was only observed when P-selectin was available at high concentration or density. Furthermore, PSGL-1 was not involved in the interaction of encephalitogenic T cells with P-selectin on stimulated brain endothelial cells in vitro, which was dominated by LFA-1-mediated adhesion. In accordance with our in vitro findings we demonstrate that PSGL-1 is not required for the development of EAE in two different mouse models. Neither blocking PSGL-1 with anti-PSGL-1 mAbs in the transfer EAE model in the SJL mouse nor deficiency of PSGL-1 in the C57BL/6 actively induced EAE model resulted in an obvious impact on leukocyte infiltration of the CNS or the development of the clinical disease.
Our observations are in apparent contradiction to the observations made by Piccio et al. (10). By performing intravital microscopy of the brain surface through the intact skull of young mice they demonstrated that autoreactive T lymphocytes rolled and arrested on LPS- or TNF--stimulated endothelium within the observed CNS microvessels. Abs blocking PSGL-1 or its endothelial ligands P- and E-selectin blocked tethering and rolling of the observed T lymphocytes, suggesting that PSGL-1/selectin interactions are critical for the recruitment of encephalitogenic T cells in inflamed brain venules. Using the same experimental approach the research group also demonstrated that CD8+ but not CD4+ T cells from patients with acute multiple sclerosis tether and roll in inflamed brain venules of mice via PSGL-1 (27).
PSGL-1-mediated rolling requires the presence of P-selectin or E-selectin on the observed brain endothelium. Performing immunohistology and in situ hybridizations, we failed to detect expression of E- and P-selectin in parenchymal vessels of the brain and spinal cord during preclinical or clinical EAE in the SJL mouse (8). In contrast, i.v. injection of fluorescently labeled anti-E- and anti-P-selectin Abs documented the presence of both selectins within superficial brain microvessels during the preclinical phase of EAE, and E-selectin but not P-selectin during EAE (10). Quantifying P-selectin expression in brain and spinal cord homogenates from mice during preclinical or clinical EAE after injection of radiolabeled P-selectin Abs revealed increased P-selectin expression in the brains and spinal cords of mice before the onset of EAE with elevated levels remaining until at least 2 wk post EAE symptoms (9). The overall levels of P-selectin expression detected in the CNS of mice after induction of EAE were, however, extremely low. Kerfoot and Kubes (9) argue that comparable levels of P-selectin have been shown in other organs such as muscle and skin to suffice for basal leukocyte rolling. As the latter technique does not allow localization of the P-selectin expression within the CNS the apparent discrepancies of the individual studies might be explained such that in EAE low levels of E- and P-selectin can be induced within the CNS and expression might be restricted to superficial CNS vessels within the meninges or the superficial brain cortex where the PSGL-1/P-selectin-mediated rolling of lymphocytes was observed by intravital microscopy (9, 10). Expression of P-selectin in murine brains restricted to the meningeal sites and the choroid plexus was in fact reported by Carrithers et al. (29) performing immunohistochemistry with a polyclonal P-selectin Ab.
As PSGL-1/P-selectin-mediated rolling of inflammatory T cells so far was only observed in superficial brain microvessels (9, 10), its occurrence elsewhere in the CNS remains to be shown. It should be noted though that leukocyte infiltration of the meninges can always be observed in tissue sections of brains and spinal cords taken from mice afflicted with EAE. Infiltration of leukocytes across meningeal vessels might be regulated by mechanisms distinct of those directing inflammatory cells across microvessels in the CNS parenchyme. Also, intravital microscopy of the brain only allows investigation of leukocyte interaction with microvessels of the CNS gray matter. In the mouse, EAE starts in the lower spinal cord ascending to the brain with inflammatory cuffs localized within the CNS white matter. Performing intravital microscopy of the spinal cord white matter we have failed to detect rolling of encephalitogenic T cells in the white matter microvessels and rather observed their 4 integrin-mediated capture to endothelial VCAM-1 (11). Complementing these findings, blocking 4 integrin has been shown to successfully inhibit the development of EAE (2, 5).
Although the cell surface expression levels of PSGL-1 on the encephalitogenic T cells were found to be similar to that on PMNs or mPC7 cells, only PSGL-1 on PMNs and mPC7 cells mediated efficient binding to P-selectin. Binding of soluble P-selectin or immobilized P-selectin to encephalitogenic T cells could only be observed at concentrations that were 2- to 4-fold above those allowing maximal P-selectin binding to PMNs or mPC7 cells. Therefore a lack of a detectable contribution of PSGL-1 in T cell adhesion to stimulated brain endothelium is not too surprising given the predominant involvement of LFA-1 and 4 integrin in T cell adhesion to brain endothelium (17, 22).
PSGL-1 requires certain posttranslational modifications for binding to P-selectin, such as fucosylation, tyrosine-sulfation, and branched carbohydrate side chains generated by the core2 -1,6-N-acetylglucosaminyltransferase (core-2 enzyme) (reviewed in Ref. 30). Naive CD4+ T cells do not express functional PSGL-1, which is only induced by the up-regulated expression of fucosyltransferase (FucT) VII and core-2 enzyme upon activation and differentiation in effector/memory T cells (31). Skin homing effector/memory T cells have been demonstrated to use PSGL-1 for their immigration into inflamed skin (32). Thus, based on the observation that encephalitogenic T cells fail to efficiently bind to P-selectin it is tempting to speculate that they fail to up-regulate the functional expression of the enzymes required to decorate PSGL-1 with the relevant sugars necessary for P-selectin binding.
In agreement with our in vitro observations we found that the anti-PSGL-1 Ab 4RA10, which has been used successfully by us and others to inhibit the interactions of leukocytes in areas of inflammation in other animal models (14, 26, 32), fails to inhibit adoptively tEAE in the SJL mouse model. Supporting our Ab inhibition studies development of actively induced EAE in PSGL-1-deficient C57BL/6 mice was indistinguishable from the disease course induced in wild-type C57BL/6.
Taken together, this investigation demonstrates that PSGL-1 is not required for the development of cellular infiltrates within the CNS and the development of clinical EAE. PSGL-1/P-selectin-mediated rolling of inflammatory T lymphocytes in superficial brain microvessels might therefore have little clinical relevance. Accordingly, therapeutic targeting of PSGL-1 to inhibit leukocyte infiltration of the CNS during multiple sclerosis does not seem promising.
Acknowledgments
We acknowledge the Münster Team Gabriele Verberk and Barbara Waschk and the Bern Team GraceGordon and Andrea Blaser for expert technical assistance.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This study was funded by the Max-Planck Society and by the Deutsche Forschungsgemeinschaft SFB 293 (Germany) and grants from the National Institutes of Health.
2 Address correspondence and reprint requests to Dr. Britta Engelhardt, Professor for Immunobiology, Theodor Kocher Institute, University of Bern, Freiestrasse 1, CH-3012 Bern, Switzerland, E-mail address: bengel@tki.unibe.ch
3 Abbreviations used in this paper: BBB, blood-brain barrier; PSGL, P-selectin glycoprotein ligand; EAE, experimental autoimmune encephalomyelitis; tEAE, transferred EAE; PLP, proteolipid protein; MOG, myelin oligodendrocytic glycoprotein; PMN, polymorphonuclear granulocyte.
Received for publication December 21, 2004. Accepted for publication March 10, 2005.
References
Butcher, E. C., M. Williams, K. Youngman, L. Rott, M. Briskin. 1999. Lymphocyte trafficking and regional immunity. Adv. Immunol. 72: 209-253.
Yednock, T. A., C. Cannon, L. C. Fritz, F. Sanchez-Madrid, L. Steinman, N. Karin. 1992. Prevention of experimental autoimmune encephalomyelitis by antibodies against 41 integrin. Nature 356: 63-66.
Baron, J. L., J. A. Madri, N. H. Ruddle, G. Hashim, C. A. Janeway, Jr. 1993. Surface expression of 4 integrin by CD4 T cells is required for their entry into brain parenchyma. J. Exp. Med. 177: 57-68
Kent, S. J., S. J. Karlik, C. Cannon, D. K. Hines, T. A. Yednock, L. C. Fritz, H. C. Horner. 1995. A monoclonal antibody to 4 integrin suppresses and reverses active experimental allergic encephalomyelitis. J. Neuroimmunol. 58: 1-10.
Engelhardt, B., M. Laschinger, M. Schulz, U. Samulowitz, D. Vestweber, G. Hoch. 1998. The development of experimental autoimmune encephalomyelitis in the mouse requires 4-integrin but not 47-integrin. J. Clin. Invest. 102: 2096-2105.
Keszthelyi, E., S. J. Karlik, S. Hyduk, G. P. A. Rice, G. Gordon, T. Yednock, H. Horner. 1996. Evidence for a prolonged role of 4 integrin throughout active experimental allergic encephalomyelitis. Neurology 47: 1053-1059.(Britta Engelhardt2,*,, Bi)
In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the endothelial blood-brain barrier and gain access to the CNS. The involvement of P-selectin glycoprotein ligand 1 (PSGL-1) and of its major endothelial ligand P-selectin in this process have been controversial. In this study we demonstrate that although encephalitogenic T cells express functional PSGL-1, which can bind to soluble and immobilize P-selectin if presented in high concentrations, PSGL-1 is not involved T cell interaction with P-selectin expressing brain endothelial cells in vitro. Furthermore, neither anti-PSGL-1 Abs nor the lack of PSGL-1 in PSGL-1-deficient mice inhibits the recruitment of inflammatory cells across the blood-brain barrier or the development of clinical EAE. Taken together, our findings demonstrate that PSGL-1 is not required for the pathogenesis of EAE.
Introduction
The CNS is considered an immuneprivileged site where the endothelial blood-brain barrier (BBB)3 tightly controls lymphocyte entry into the CNS. Under physiological conditions lymphocyte traffic into the CNS is low whereas during inflammatory diseases of the CNS, such as multiple sclerosis or in the animal model experimental autoimmune encephalomyelitis (EAE), a large number of circulating lymphocytes readily gain access to the CNS. EAE is a CD4+ T cell-mediated autoimmune disease of the CNS, which is initiated by autoaggressive T cells activated outside the CNS. These encephalitogenic T cell blasts enter the CNS parenchyma across the healthy BBB. Upon encounter of Ag, autoaggressive T cells initiate inflammation and the recruitment of inflammatory effector cells across the inflamed BBB into the CNS leading to edema and demyelination. Thus, the interaction of autoaggressive and inflammatory effector cells with the BBB is a critical step in the pathogenesis of EAE.
In general, lymphocyte recruitment across the vascular wall is regulated by the sequential interaction of different adhesion or signaling molecules on lymphocytes and endothelial cells lining the vessel wall (1). An initial transient contact of the circulating leukocyte with the vascular endothelium, generally mediated by adhesion molecules of the selectin family or alternatively by 4 integrins and their respective ligands, slows down the leukocyte in the bloodstream. With greatly reduced velocity the leukocyte rolls along the vascular wall allowing it to recognize chemokines displayed on the endothelial surface. Binding of a chemokine to its G protein-coupled receptor on the leukocyte surface results in a pertussis toxin-sensitive activation of integrins on the leukocyte surface. Activated integrins mediate the firm adhesion of the leukocytes to the vascular endothelium by binding to their endothelial ligands, which belong to the Ig superfamily. This ultimately leads to the extravasation of the leukocyte. Successful recruitment of circulating leukocytes into the tissue depends on the productive leukocyte/endothelial interaction during each of these sequential steps.
The mechanisms mediating leukocyte recruitment into the CNS are not completely understood. mAbs directed against 4 integrin have been shown to successfully interfere with the development of EAE in different animal models (2, 3, 4, 5) and moreover to reverse the ongoing disease process (6). As the anti-4 integrin treatment proved to block adhesion of inflammatory cells to inflamed cerebral venules in vitro (2, 7) and to substantially decrease the recruitment of inflammatory cells across the BBB in vivo, there is general agreement that there is a pivotal role for 4 integrins in leukocyte/BBB interaction during EAE (2, 5).
The predominant involvement of 4 integrins in leukocyte recruitment across the BBB during EAE does not exclude the contribution of other adhesion molecules in this process. The potential involvement of selectins in particular in T lymphocyte interaction with the BBB has been controversial. We have reported a lack of E- and P-selectin expression in CNS microvessels during EAE and demonstrated that anti-E- and anti-P-selectin Abs do not inhibit the development of EAE (8). More recent work of other laboratories using intravital microscopy demonstrated the involvement of E- and P-selectin and their ligand P-selectin protein ligand 1 (PSGL-1) in tethering and rolling of leukocytes or encephalitogenic T cells in superficial brain microvessels in vivo (9, 10). In contrast, when performing intravital microscopy of the spinal cord white matter, we found a lack of rolling and a predominant role of 4 integrins in the initial capture and the firm adhesion of autoaggressive T cells within the spinal cord white matter microvasculature (11).
These apparent discrepancies prompted us now to investigate the functional expression of the homodimeric sialomucin PSGL-1 on encephalitogenic T cells and its potential involvement in inflammatory cell recruitment across the BBB during EAE. In this study we demonstrate that although encephalitogenic T cells express functional PSGL-1 on their surface as exemplified by the ability to bind purified P-selectin, PSGL-1 is not involved in interactions of these T cells with brain endothelium in vitro. Furthermore, PSGL-1 is not required for the development of EAE in SJL or C57BL/6 mice.
Results
To find out whether PSGL-1 expression can be detected in the brains and spinal cords of mice afflicted with EAE, we performed immunohistochemistry using three different anti-PSGL-1 Abs. During EAE immunostaining for PSGL-1 was observed on the majority of inflammatory cells localized within perivascular cuffs (Fig. 1A). Additionally immunostaining for PSGL-1 was observed in the choroid plexus and the CNS parenchyme resembling immunostaining for CD45 (Fig. 1B). PSGL-1-positive parenchymal cells were characterized as Mac-1-positive microglial cells by double-immunofluorescence staining (Fig. 1B). Thus, PSGL-1 might be involved in inflammatory cell recruitment into the CNS, whereas its up-regulated expression on microglial cells within the CNS parenchyme suggests additional functions for PSGL-1 in the pathogenesis of EAE.
Encephalitogenic T cells express functional PSGL-1
To define whether encephalitogenic T cells express PSGL-1 on their surface we performed immunofluorescence stainings using three different mAbs directed against murine PSGL-1 (4RA10, 4RB12, 2PH1). Encephalitogenic PLP-specific T cells were found to display surface expression levels of PSGL-1 comparable to those observed on mPC7 cells or on bone marrow-derived murine neutrophils (Fig. 2). Surface expression levels of PSGL-1 did not change depending on the activation status of the T cells (data not shown). Selectin ligands including PSGL-1 require posttranslational modifications for selectin binding. To determine whether encephalitogenic T cells bind P-selectin we studied the binding of P-selectin IgG to encephalitogenic T cells as compared with mPC7 cells or bone marrow-derived neutrophils by FACS analysis. Binding of P-selectin IgG to encephalitogenic T cells could only be observed if P-selectin IgG was offered in high concentrations (30–50 μg/ml). Under these experimental conditions only a minor population of encephalitogenic T cell blasts (15%) were found to bind P-selectin IgG in high amounts whereas binding of P-selectin IgG to the majority of T cells was found to be very low (Fig. 2A). In contrast, P-selectin binding to mPC7 cells or bone marrow-derived neutrophils was already found to be optimal at lower concentrations of P-selectin IgG fusion protein (10–25 μg/ml) with 100% of the cells binding P-selectin homogeneously (Fig. 2, B and C). Dependence of P-selectin binding on the presence of divalent cations was confirmed by the loss of P-selectin binding to all three investigated cell populations in the presence of 2 mM EDTA, whereas detection of PSGL-1 protein surface expression remained unaffected (Fig. 2). Thus, although encephalitogenic T cells can bind soluble P-selectin, it is less efficient than P-selectin binding to mPC7 cells or polymorphonuclear granulocytes (PMNs).
Encephalitogenic T cells can bind to high concentrations of immobilized P-selectin in vitro
To determine whether P-selectin binding to encephalitogenic T cells is mediated via PSGL-1, we analyzed their binding to P-selectin IgG immobilized on glass slides. Optimal binding of mPC7 cells to immobilized P-selectin IgG could already be observed when 20 μl of 25 μg/ml fusion protein was used for coating (data not shown). T cell binding to immobilized P-selectin IgG was only observed at concentrations of 50 μg/ml and reached an optimum at 100 μg/ml. Under these experimental conditions encephalitogenic T cells were found to bind to P-selectin, which could be inhibited by the anti-P-selectin Ab RB40 (Fig. 3). PSGL-1 was found to be the major P-selectin ligand on encephalitogenic T cells as the PSGL-1 blocking mAbs 4RA10 and 2PH1 almost completely inhibited T cell binding. At the same time, the nonblocking mAb 4RB12 or the anti-LFA-1 Ab FD441.8 had no effect on T cell binding to immobilized P-selectin. Thus, provided P-selectin is available in high density, encephalitogenic T cells can bind via PSGL-1 to immobilized P-selectin.
PSGL-1 is not involved in adhesive interactions of T cells with brain endothelium in vitro
We next asked whether encephalitogenic T cells can use PSGL-1 to adhere to P-selectin on activated brain endothelial cells in vitro. Therefore we performed adhesion assays with freshly activated PLP-specific T cell blasts and the brain endothelioma cell line bEnd5, which upon 3 h of stimulation with LPS or TNF- expresses P-selectin on its surface (22). PLP-specific T cell blasts readily bound to stimulated bEnd5. Pretreatment of T cell blasts with a mAb directed against PSGL-1 had no effect on T cell adhesion to stimulated bEnd5 (Fig. 4). Supporting a lack of involvement of PSGL-1/P-selectin interaction in T cell adhesion to brain endothelium after 3 h of stimulation, T cell binding to E/P-selectin double-deficient bEndEP.5 was found to be indistinguishable from T cell adhesion to wild-type bEnd5 when compared within the same assay (Fig. 4). At the same time blocking LFA-1 significantly inhibited T cell adhesion to both, stimulated bEnd5 and bEndEP.5 (Fig. 4).
Comparing T cell migration across stimulated bEnd5 in the presence or absence of PSGL-1 blocking Abs confirmed a lack of involvement of PSGL-1 in this process (data not shown). Finally, the pretreatment of bEnd5 with the P-selectin blocking Ab RB40.34 had no influence on T cell adhesion to and migration across stimulated brain endothelium confirming the lack of involvement of PSGL-1/P-selectin binding during these processes (data not shown).
Anti-PSGL-1 Abs do not inhibit PLP-induced T cell proliferation or IFN- production
Adhesion molecules on autoaggressive T cells could be involved in the pathogenic processes during EAE other than T cell migration into the CNS. Therefore we asked whether PSGL-1 is involved in the Ag-dependent activation of encephalitogenic T cells. PLP-specific T cells were restimulated with their specific PLP peptide using irradiated syngeneic splenocytes as APCs in the presence or absence of anti-PSGL-1 Abs. Blocking PSGL-1 did neither influence Ag-induced T cell proliferation (Fig. 5A) nor IFN- production (Fig. 5B).
Anti-PSGl-1 Abs do not inhibit the development of tEAE in the SJL mouse
To investigate whether, regardless of the lack of PSGL-1 contribution to T cell activation or T cell interaction with brain endothelium in vitro, PSGL-1 could still be involved in the pathogenesis of EAE, we investigated the effect of the PSGL-1 blocking mAb 4RA10 on the development of EAE in the SJL mouse. Only 30 μg of 4RA10 injected into a mouse were previously shown to be sufficient to completely block PSGL-1-mediated rolling of leukocytes in vivo (26). EAE was transferred by the injection of 3 x 106 syngeneic PLP-specific T cell blasts and animals were monitored daily for the development of clinical disease. In seven different experiments, repeated infusions of 4RA10 injected at dosages up to 400 μg/injection failed to reproducibly interfere with the onset of clinical EAE or the recruitment of inflammatory cells into the CNS when compared with animals treated with control Abs (Fig. 6). Quantitative assessment of the number of perivascular inflammatory cuffs present in CD45-immunostained frozen brain and spinal cord sections of mice treated with anti-PSGL-1 Abs in comparison to control EAE animals did not reveal any difference between the different treatment groups (Table I). Also, no difference was found in the composition of the CD45-positive inflammatory infiltrates, which consisted mainly of CD4+ T cells, Mac-1+ macrophages, some B220+ B cells and rare CD8+ T cells (data not shown). Gr-1-positive granulocytes were not observed. Thus, inhibition of the interaction of PSGL-1 with its respective selectin ligands in vivo did not significantly alter the development of tEAE in the SJL mouse. Taken together, these observations demonstrate that PSGL-1 is not required for the development of EAE in SJL mice.
PSGL-1-deficient C57BL/6 mice develop EAE
To confirm the lack of requirement of PSGL-1 in EAE pathogenesis, PSGL-1-deficient mice were backcrossed into the C57BL/6 background. Active EAE was induced by immunization with the MOG peptide (aa 35–55) in CFA and mice were monitored daily for clinical symptoms of EAE. In five different experiments PSGL-1-deficient C57BL/6 mice did not show any significant difference in the onset of actively induced EAE when compared with C57BL/6 wild-type mice (Fig. 7). Both groups of mice exhibited chronic disease, which is typical of MOG-induced EAE in the C57BL/6 model. Quantitative assessment of the number of perivascular cellular infiltrates in CD45-immunostained frozen brain and spinal cord sections of wild-type and PSGL-1-deficient mice did not reveal any significant difference between both groups (Table I). Immunohistological analysis of the cellular composition of the inflammatory infiltrates confirmed the presence of mainly Mac-1+ macrophages, CD4+ T cells, and scattered Gr-1-positive granulocytes in the brain and spinal cord of both wild-type and PSGL-1-deficient mice (Fig. 8). Thus, as absence of PSGL-1 neither influences the recruitment of different inflammatory cell subsets across the BBB during EAE nor inhibits the development of clinical EAE, PSGL-1 is not required for the development of clinical EAE in the C57BL/6 mouse.
Discussion
In this study we show that although encephalitogenic T lymphocytes display surface expression of PSGL-1, their PSGL-1-mediated binding to P-selectin is apparently poor as it was only observed when P-selectin was available at high concentration or density. Furthermore, PSGL-1 was not involved in the interaction of encephalitogenic T cells with P-selectin on stimulated brain endothelial cells in vitro, which was dominated by LFA-1-mediated adhesion. In accordance with our in vitro findings we demonstrate that PSGL-1 is not required for the development of EAE in two different mouse models. Neither blocking PSGL-1 with anti-PSGL-1 mAbs in the transfer EAE model in the SJL mouse nor deficiency of PSGL-1 in the C57BL/6 actively induced EAE model resulted in an obvious impact on leukocyte infiltration of the CNS or the development of the clinical disease.
Our observations are in apparent contradiction to the observations made by Piccio et al. (10). By performing intravital microscopy of the brain surface through the intact skull of young mice they demonstrated that autoreactive T lymphocytes rolled and arrested on LPS- or TNF--stimulated endothelium within the observed CNS microvessels. Abs blocking PSGL-1 or its endothelial ligands P- and E-selectin blocked tethering and rolling of the observed T lymphocytes, suggesting that PSGL-1/selectin interactions are critical for the recruitment of encephalitogenic T cells in inflamed brain venules. Using the same experimental approach the research group also demonstrated that CD8+ but not CD4+ T cells from patients with acute multiple sclerosis tether and roll in inflamed brain venules of mice via PSGL-1 (27).
PSGL-1-mediated rolling requires the presence of P-selectin or E-selectin on the observed brain endothelium. Performing immunohistology and in situ hybridizations, we failed to detect expression of E- and P-selectin in parenchymal vessels of the brain and spinal cord during preclinical or clinical EAE in the SJL mouse (8). In contrast, i.v. injection of fluorescently labeled anti-E- and anti-P-selectin Abs documented the presence of both selectins within superficial brain microvessels during the preclinical phase of EAE, and E-selectin but not P-selectin during EAE (10). Quantifying P-selectin expression in brain and spinal cord homogenates from mice during preclinical or clinical EAE after injection of radiolabeled P-selectin Abs revealed increased P-selectin expression in the brains and spinal cords of mice before the onset of EAE with elevated levels remaining until at least 2 wk post EAE symptoms (9). The overall levels of P-selectin expression detected in the CNS of mice after induction of EAE were, however, extremely low. Kerfoot and Kubes (9) argue that comparable levels of P-selectin have been shown in other organs such as muscle and skin to suffice for basal leukocyte rolling. As the latter technique does not allow localization of the P-selectin expression within the CNS the apparent discrepancies of the individual studies might be explained such that in EAE low levels of E- and P-selectin can be induced within the CNS and expression might be restricted to superficial CNS vessels within the meninges or the superficial brain cortex where the PSGL-1/P-selectin-mediated rolling of lymphocytes was observed by intravital microscopy (9, 10). Expression of P-selectin in murine brains restricted to the meningeal sites and the choroid plexus was in fact reported by Carrithers et al. (29) performing immunohistochemistry with a polyclonal P-selectin Ab.
As PSGL-1/P-selectin-mediated rolling of inflammatory T cells so far was only observed in superficial brain microvessels (9, 10), its occurrence elsewhere in the CNS remains to be shown. It should be noted though that leukocyte infiltration of the meninges can always be observed in tissue sections of brains and spinal cords taken from mice afflicted with EAE. Infiltration of leukocytes across meningeal vessels might be regulated by mechanisms distinct of those directing inflammatory cells across microvessels in the CNS parenchyme. Also, intravital microscopy of the brain only allows investigation of leukocyte interaction with microvessels of the CNS gray matter. In the mouse, EAE starts in the lower spinal cord ascending to the brain with inflammatory cuffs localized within the CNS white matter. Performing intravital microscopy of the spinal cord white matter we have failed to detect rolling of encephalitogenic T cells in the white matter microvessels and rather observed their 4 integrin-mediated capture to endothelial VCAM-1 (11). Complementing these findings, blocking 4 integrin has been shown to successfully inhibit the development of EAE (2, 5).
Although the cell surface expression levels of PSGL-1 on the encephalitogenic T cells were found to be similar to that on PMNs or mPC7 cells, only PSGL-1 on PMNs and mPC7 cells mediated efficient binding to P-selectin. Binding of soluble P-selectin or immobilized P-selectin to encephalitogenic T cells could only be observed at concentrations that were 2- to 4-fold above those allowing maximal P-selectin binding to PMNs or mPC7 cells. Therefore a lack of a detectable contribution of PSGL-1 in T cell adhesion to stimulated brain endothelium is not too surprising given the predominant involvement of LFA-1 and 4 integrin in T cell adhesion to brain endothelium (17, 22).
PSGL-1 requires certain posttranslational modifications for binding to P-selectin, such as fucosylation, tyrosine-sulfation, and branched carbohydrate side chains generated by the core2 -1,6-N-acetylglucosaminyltransferase (core-2 enzyme) (reviewed in Ref. 30). Naive CD4+ T cells do not express functional PSGL-1, which is only induced by the up-regulated expression of fucosyltransferase (FucT) VII and core-2 enzyme upon activation and differentiation in effector/memory T cells (31). Skin homing effector/memory T cells have been demonstrated to use PSGL-1 for their immigration into inflamed skin (32). Thus, based on the observation that encephalitogenic T cells fail to efficiently bind to P-selectin it is tempting to speculate that they fail to up-regulate the functional expression of the enzymes required to decorate PSGL-1 with the relevant sugars necessary for P-selectin binding.
In agreement with our in vitro observations we found that the anti-PSGL-1 Ab 4RA10, which has been used successfully by us and others to inhibit the interactions of leukocytes in areas of inflammation in other animal models (14, 26, 32), fails to inhibit adoptively tEAE in the SJL mouse model. Supporting our Ab inhibition studies development of actively induced EAE in PSGL-1-deficient C57BL/6 mice was indistinguishable from the disease course induced in wild-type C57BL/6.
Taken together, this investigation demonstrates that PSGL-1 is not required for the development of cellular infiltrates within the CNS and the development of clinical EAE. PSGL-1/P-selectin-mediated rolling of inflammatory T lymphocytes in superficial brain microvessels might therefore have little clinical relevance. Accordingly, therapeutic targeting of PSGL-1 to inhibit leukocyte infiltration of the CNS during multiple sclerosis does not seem promising.
Acknowledgments
We acknowledge the Münster Team Gabriele Verberk and Barbara Waschk and the Bern Team GraceGordon and Andrea Blaser for expert technical assistance.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This study was funded by the Max-Planck Society and by the Deutsche Forschungsgemeinschaft SFB 293 (Germany) and grants from the National Institutes of Health.
2 Address correspondence and reprint requests to Dr. Britta Engelhardt, Professor for Immunobiology, Theodor Kocher Institute, University of Bern, Freiestrasse 1, CH-3012 Bern, Switzerland, E-mail address: bengel@tki.unibe.ch
3 Abbreviations used in this paper: BBB, blood-brain barrier; PSGL, P-selectin glycoprotein ligand; EAE, experimental autoimmune encephalomyelitis; tEAE, transferred EAE; PLP, proteolipid protein; MOG, myelin oligodendrocytic glycoprotein; PMN, polymorphonuclear granulocyte.
Received for publication December 21, 2004. Accepted for publication March 10, 2005.
References
Butcher, E. C., M. Williams, K. Youngman, L. Rott, M. Briskin. 1999. Lymphocyte trafficking and regional immunity. Adv. Immunol. 72: 209-253.
Yednock, T. A., C. Cannon, L. C. Fritz, F. Sanchez-Madrid, L. Steinman, N. Karin. 1992. Prevention of experimental autoimmune encephalomyelitis by antibodies against 41 integrin. Nature 356: 63-66.
Baron, J. L., J. A. Madri, N. H. Ruddle, G. Hashim, C. A. Janeway, Jr. 1993. Surface expression of 4 integrin by CD4 T cells is required for their entry into brain parenchyma. J. Exp. Med. 177: 57-68
Kent, S. J., S. J. Karlik, C. Cannon, D. K. Hines, T. A. Yednock, L. C. Fritz, H. C. Horner. 1995. A monoclonal antibody to 4 integrin suppresses and reverses active experimental allergic encephalomyelitis. J. Neuroimmunol. 58: 1-10.
Engelhardt, B., M. Laschinger, M. Schulz, U. Samulowitz, D. Vestweber, G. Hoch. 1998. The development of experimental autoimmune encephalomyelitis in the mouse requires 4-integrin but not 47-integrin. J. Clin. Invest. 102: 2096-2105.
Keszthelyi, E., S. J. Karlik, S. Hyduk, G. P. A. Rice, G. Gordon, T. Yednock, H. Horner. 1996. Evidence for a prolonged role of 4 integrin throughout active experimental allergic encephalomyelitis. Neurology 47: 1053-1059.(Britta Engelhardt2,*,, Bi)