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Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by disruption of the blood-brain barrier (BBB). This breach in CNS immune privilege allows undeterred trafficking of myelin-specific lymphocytes into the CNS where they induce demyelination. Although the mechanism of BBB compromise is not known, the chemokine CXCL12 has been implicated as a molecular component of the BBB whose pattern of expression is specifically altered during MS and which correlates with disease severity. The inflammatory cytokine IL-1β has recently been shown to contribute not only to BBB permeability, but also to the development of IL-17-driven autoimmune responses. Using the rodent model of MS, experimental autoimmune encephalomyelitis (EAE), we demonstrate that IL-1β mediates pathologic relocation of CXCL12 during the induction phase of the disease, prior to the development of BBB disruption. We also show that CD4, CD8 and, surprisingly, γδ T cells are all sources of IL-1β. In addition, γδ T cells are also targets of this cytokine, contributing to IL-1β mediated production of IL-17. Finally, we show that the level of CNS IL-1R determines the clinical severity of EAE. These data suggest that T cell-derived IL-1β contributes to loss of immune privilege during CNS autoimmunity via pathologic alteration in the expression of CXCL12 at the BBB.
Multiple sclerosis (MS) is believed to be an autoimmune disease in which myelin-specific CD4+ T cells gain excessive entry to the central nervous system (CNS) parenchyma and induce demyelinating disease (1, 2). The increased trafficking of mononuclear cells during MS exacerbations is accompanied by BBB disruption, as evidenced by the appearance of gadolinium (gd)-enhancing lesions on magnetic resonance (MR) imaging (3). However, serial radiologic studies of MS patients indicate that normal appearing white matter may exhibit altered water diffusion prior to the development of gd enhancement (4). Thus, alterations in BBB biology may occur prior to the onset of severe disruption. Understanding how changes in BBB function lead to loss of leukocyte restriction is essential for developing therapies that limit inflammation during MS.
CXCL12 is a secondary lymphoid chemokine that is constitutively expressed by the central nervous system (CNS) (5). Under normal conditions, CXCL12 is detected on the basolateral surface of the CNS endothelium where it serves to restrict the entry of infiltrating leukocytes (6-8). This polarized expression is altered in individuals with CNS autoimmune diseases with loss of perivascular CXCL12 expression and relocation of the chemokine to the luminal side of the microvasculature (7, 8). This pathologic pattern of CXCL12 expression is associated with enhanced activation of the CXCL12 receptor CXCR4 on infiltrating leukocytes and is unique to MS and its murine model, experimental autoimmune encephalomyelitis (EAE) (8). Thus, the altered expression of CXCL12 at the BBB could promote inappropriate leukocyte trafficking and contribute to disease pathogenesis.
Several molecules, including TNF-α, IL-1β, and CD40, can affect CXCL12 expression. In vitro, TNF-α and IL-1β increase CXCL12 in osteoblasts (9) and astrocytes (10), respectively, while IL-1β decreases CXCL12 expression in dermal fibroblasts (11). Studies have shown that, in vivo, CXCL12 expression is increased by CD40/CD40L in the joint synovium during arthritis (12) and by macrophage-produced IL-1β astrocytes during HIV encephalitis (13).
IL-1β is an important component of disease pathogenesis in MS (14). IL-1β is found in MS lesions (15) and individuals with a relatively high ratio of IL-1β to the naturally occurring receptor antagonist are genetically predisposed to MS (16). Studies have indicated that IL-1β induces BBB plasticity by inducing angiogenic transcription factors (17) and chronic expression of IL-1β leads to leukocyte accumulation in the otherwise homeostatic CNS (18). BBB endothelial cells (19) and astrocytes (20) express the IL-1 receptor (IL-1R) and recent studies have shown that the IL-1R deficient mouse is protected from EAE induced by active immunization with myelin peptide (21). Additionally, IL-1β is required for the proper development of Th17 cells in mice (21, 22) and humans (23). In this study, we utilized EAE to evaluate the kinetics of and identify cytokines and their cellular sources responsible for the pathologic relocation of CXCL12 at the BBB. We demonstrate that relocation of CXCL12 occurs at pre-clinical time-points in mice with EAE, prior to the development of spinal cord gd-enhancement as demonstrated by MRI and is mediated by IL-1β derived from IL-17-expressing γδT cells during the induction phase of disease. In addition, via generation of chimeric mice, we determined that CNS levels of IL-1R determine the severity of EAE.
Either C57BL/6 wild-type, IL-1R deficient, or TNFR1 deficient mice (Jackson Laboratories, Bar Harbor, ME) were used for all experiments. All mice were maintained under pathogen free conditions (Department of Comparative Medicine, Washington University, St. Louis, MO) and studies were performed in compliance with the guidelines of the Washington University School of Medicine Animal Safety Committee.
Active EAE was induced in 8- to 12-wk-old mice by s.c. immunization with a murine myelin oligodendroglial glycoprotein (MOG) peptide (MOGp35-55) (Sigma Genosys) in PBS emulsified 1:1 with IFA supplemented with 500μg/ml inactivated Mycobacterium tuberculosis for a total 50μg immunizing dose of MOGp35-55.Mice received 300ng of pertussis toxin (List Laboratories) i.v. at the time of immunization and 48 h later, as previously described (7). For adoptive transfer studies, MOG-specific cell lines, from C57BL/6 or IL-1R deficient C57BL/6 mice, were generated from animals immunized s.c. with 50 μg MOG35-55 peptide (Sigma-Aldrich) emulsified in IFA and supplemented with 500 μg/ml Mycobacterium tuberculosis. CD4+ cells were isolated from the spleen as previously described (24). Cells were then stimulated at a concentration of 106 cells/ml in the presence of 5 × 106 cells/ml of irradiated C57BL/6 splenocytes, 10 μg/ml MOG35-55 peptide, 10 U/ml IL-12, and 10 U/ml IL-2, in 10% FCS containing RPMI complete media to polarize cells to a Th1 phenotype. Cells were isolated 7 d later by Histopaque 1.077 (Sigma-Aldrich) and then restimulated in the absence of IL-12 under the other conditions outlined earlier in the section. Cells underwent two rounds of stimulation before transferred by retroorbital i.v. injection to 6–8-wk-old mice, which also received 300 ng pertussis toxin on the day of transfer and two days later. The grading scale used for scoring clinical manifestations of EAE in both models was the following: 1, tail weakness; 2, difficulty righting; 3, hind limb paralysis; 4, forelimb weakness or paralysis; 5, moribund or dead.
Systemic administration of recombinant murine IL-1β (100 ng), TNFα (500 ng) and CD40L (500 ng) (R&D Systems) was accomplished via tail-vein injection of reconstituted protein in normal saline. Administration of TNF-α (10 ng/ml) directly into the spinal cord was also performed. For these experiments, isofluorane-anesthetized animals were immobilized in a steel harness and the entire length of spine exposed via incision and retraction of the skin. Individual injection sites were initiated at sections of the spinal cord separated by two vertebrae of space and carried out via spot reduction of the bone covering the target injection sites. TNF-α was injected at a volume of 1 μl per injection point using a calibrated microinjector. Individual mice received three injections at different sections of the spine, with interspersed sections of uninjected spinal cord acting as internal controls for unactivated tissue. Injections were made using microscopic needle-points produced by melting capillary tubes. Following injections, skin was sutured and animals monitored until awake and ambulating.
The following antibodies were used in this study. CXCL12 rabbit polyclonal antibody (Peprotech, Rocky Hills, NJ); IgG isotype control antibodies (Jackson ImmunoResearch); monoclonal rat anti-mouse-CD31 (PECAM) and CD11b (BD Pharmingen); CD3 (Dako Cytomation); IL-1β (R&D Systems); and fluorescently-conjugated antibodies against CD4, CD8, CD3, (BD Pharmingen), IL-17, and γδTCR (eBioscience).
Frozen sections were permeabilized and stained as described (7). For detection of CXCL12, additional blocking with image-iT Fx signal enhancer (Molecular Probes, Eugene, O.R.) solution was used according to the manufacturer's instructions. Primary antibodies were used at the following dilutions: anti-CXCL12 (1:20), anti-mPECAM-1 (2ug/mL), anti-CD11b (1:25), anti-CD3 (1:500 dilution) and anti-IL-1β (1:66 dilution). Primary antibodies were detected with secondary goat anti-rabbit and anti-rat IgG conjugated to Alexa 555 or Alexa 488 and donkey anti-goat IgG conjugated to Alexa 568 (Molecular Probes Inc.) for immunofluorescence and nuclei were counterstained with ToPro3. Control sections were incubated with isotype-matched IgG. Sections were analyzed using a Zeiss LSM 510 laser scanning confocal microscope and accompanying software (Zeiss).
Cells were isolated from the spinal cords of mice with EAE as previously described (7) and stained with fluorescently conjugated antibodies to CD4, CD8, CD3, IL-17, and γδTCR (see above) or with unconjugated primaries (IL-1β) which were then detected with anti-goat fluorescently conjugated secondary antibodies. Data collection and analysis were conducted using a FACScaliburTM flow cytometer using CellQuestTM software (Becton Dickinson). For fluorescence analysis, a bivariate dot plot of forward versus side scatter was generated, and cells were analyzed through a live cell gate.
Cells isolated as above from the spinal cords of mice were briefly restimulated in vitro for 2 hours at 37° C with 50ng/mL PMA add 1uM ionomycin and cytokines were concentrated with 1μg/ml Brefeldin A. Cells were then washed in PBS, fixed with 0.4% PFA, and permeablized with 0.1% saponin, in PBS supplemented with 0.5% BSA and 0.1% azide. Cells were incubated with antibodies in the PBS-saponin buffer, washed, resuspended in PFA, and analyzed immediately.
Bone marrow harvested from the femur and tibula was depleted of differentiated lymphocytes by negative selection using anti-CD4 and anti-CD8 magnetic beads (Dynal Biotech, Oslo, Norway) according to manufacture's instructions. After 45 minutes rocking at 4° C, lymphocytes bound to beads were removed and extensively washed prior to being resuspended. 2×10^6 cells were then transferred retro-orbitally to sex-matched recipients that had received a lethal irradiation dose of 1,000 rads 24 hours prior. Irradiation control mice did not received transferred cells and served as an irradiation control in each experiment. Transferred bone marrow was allowed 6 weeks for full engraftment prior to MOG immunization as described above.
EAE mice underwent Gd-MRI either before the onset of clinical signs, during the acute phase, or in the chronic phase. Unimmunized mice (n=2) served as controls. Mice were anesthetized using 5% isoflurane in oxygen, fitted with a custom nose cone to deliver 1% isoflurane in oxygen for maintenance, and placed in a custom holder and surface coil as described previously (25). The tail vein of the mouse was cannulated with a 28 G catheter (Strategic Applications, Inc., Libertyville, IL) prior to placement of the entire preparation in a 4.7 T MRI (Varian Inc, Palo Alto, CA). T1-weighted spin-echo images were collected before and 5 minutes after the administration of 0.02 ml bolus of 17.2 mg/ml gadolinium-diethylenetriamine pentaacetic acid (DTPA; Omniscan, GE Healthcare, Princeton, NJ) with the following parameters: TR/TE 700/14 ms, 128 × 128 matrix (zero filled to 256 × 256), 1 cm2 FOV, 1 mm slice thickness, 4 averages. Data analysis was accomplished via the creation of enhancement maps, which were created on a pixel-by-pixel basis using the equation: % Enhancement = (Spost - Spre) / Spre × 100, where Spre and Spost are the signal intensities of the pre- and post-contrast images, respectively. Regions of interest were manually traced along the ventrolateral white matter on the pre-contrast images and applied to the enhancement maps.
A Student's t-test was used to evaluate statisitical significance in the following studies: enhancement between mice imaged during different phases of EAE and control mice, flow cytometric analyses, and Q-RT-PCR. Statistical significance was assigned to p values less than 0.05 and a single asterisk indicates p<0.05, double asterisk indicates p<0.005, and triple asterisk indicates p<0.0005.
Initiation of inflammation within the CNS during MS and EAE is classically attributed to the entrance of myelin-specific CD4+ T cells (26, 27), which leads to demyelination. CXCL12 redistribution at the BBB occurs at the peak of EAE (7) and in active MS lesions (8). In order to define the kinetics of CXCL12 redistribution with regard to leukocyte entry, we evaluated CXCL12 expression in the spinal cords of mice during preclinical (days 11-13) and peak (days 14-16) stages of active immunization EAE (Figure 1a). Alterations in the CXCL12 expression pattern were observed in tissues collected from preclinical mice on day 11 post-immunization when there was little to no obvious inflammatory lesion formation and at the peak of EAE, as previously described (7). Loss in CXCL12 polarity at peak of disease was highly associated with the presence of classic inflammatory cuffs (Figure 1a) and analysis of 132 vessels in low-power spinal cord images from four mice revealed that 88.2% of inflamed (> 10 perivascular leukocytes) versus 0% of uninflamed (<10 perivascular leukocytes) vessels exhibited loss in CXCL12 polarity, defined as loss of basolateral expression of CXCL12 with co-localization with CD31 (Figure 1a). To examine whether CXCL12 redistribution is associated with immune cell entry into the CNS, serial spinal cord sections collected from preclinical EAE mice were examined for perivascular associated CD3+ and CD11b+ cells via immunohistochemical labeling. In vessels where loss of CXCL12 polarity was evident (Figure 1b, left panel), 100% of the serial sections analyzed had detectable T cells (Figure 1b, middle panel) whereas no macrophages were detected (Figure 1b, right panel and graph). As observed in human studies (8), arterioles retain polarized expression of CXCL12 (Figure 1b, left panel) and do not exhibit leukocytes within the perivascular space (Figure 1b, middle panel).
Among the possibilities for the mechanism of CXCL12 relocation would be nonspecific BBB disruption. One way to examine this in vivo, is via Gd-enhanced magnetic resonance imaging (MRI). Gd is a paramagnetic contrast agent that enters the CNS parenchyma when there is disruption of the BBB (28). Gd-MRI imaging is used in patients with neurologic diseases to detect inflammation and/or BBB compromise within the CNS (3). We performed Gd-MRI of spinal cords in unimmunzed mice and in mice at pre-clinical, peak, and chronic phases of EAE. Examination of Gd uptake revealed no enhancement in spinal cords of unimmunized mice or in mice at preclinical or chronic time-points after MOG immunization (Figure 2). These data suggest that CXCL12 relocalization detected during preclinical time-points is not due to BBB disruption but is instead likely to be occurring in response to the presence of molecules expressed by the myelin-specific T cells that initiate autoimmune inflammation.
Activated T cells that traffic across the CNS endothelium have been shown to produce inflammatory cytokines such as TNF-α and IL-1β (29, 30), both of which have been observed to regulate CXCL12 expression in other contexts (9, 10, 31, 32). To determine if these cytokines play a role in the relocation of CXCL12 at the BBB, we administered IL-1β, TNF-α, and PBS intravenously to wild-type mice. Spinal cords were collected at 3, 6, and 24 hours post-treatment and analyzed by immunohistochemical staining. At 3 hours post-treatment CXCL12 remained expressed at the basolateral surface of the endothelium in all treatment groups. By 6 hours post-treatment there was a decrease in basolateral CXCL12 staining in the mice given IL-1β, without associated relocation to the lumen (data not shown). Twenty-four hours after cytokine administration, analysis revealed that IL-1β, but not TNF-α, induced CXCL12 relocation in 90.1% of vessels. The CXCL12 expression pattern induced by IL-1β was similar to that seen in vessels from MOG-immunized wild-type mice in the induction phase of EAE (Figure 3a). Administration of an equal volume of PBS did not lead to any change in the pattern of CXCL12 expression (Figure 3a). In addition, direct administration of TNF-α to the spinal cord via stereotactic injection did not induce alterations in the pattern of expression of CXCL12 at the BBB (data not shown).
We confirmed that TNF-α was not required for CXCL12 relocation by examining MOG-immunized mice deficient in TNFR1, which were previously reported to be resistant to EAE (33), at the time-point where their wild-type controls reached the peak of disease. Immunostaining of spinal cord sections from TNFR1 deficient mice showed that basolateral expression of CXCL12 was lost in 92.3% of vessels analyzed while 100% of the vessels analyzed in immunized IL-1R deficient mice, which are also protected from EAE (21), had retained the basolateral expression (Figure 3b). Although TNFR1-deficient mice exhibited relocation of CXCL12 such that expression was no longer polarized, the pattern of CXCL12 staining in these mice was not identical to that observed at the peak of EAE in wild-type mice with increased co-localization with CD31 (Figure 3b). This suggests that although TNF-α is not required for CXCL12 relocation, it may be required for the establishment of the appropriate binding sites for this chemokine during the relocation process. Thus, while lack of basolateral CXCL12 expression is not sufficient to induce disease, there may be additional CXCL12 binding sites regulated by TNF-α that allow relocation of CXCL12 to sites that impact on expression of EAE.
IL-1β is produced by macrophages and activated microglia (34, 35); however CXCL12 relocation occurs during the induction of EAE when initial T cell entry occurs and macrophages and activated microglia are yet not detected. To determine if T cells are the source of IL-1β, we performed extensive analyses of IL-1β production during the induction of EAE. In spinal cord sections selected from mice at the preclinical time-point, double-label immunofluorescent staining revealed that a subset of T cells was IL-1β positive (Figure 4a). Cells were isolated from the spinal cords of MOG-immunized mice at the first sign of EAE for flow cytometric analysis. Onset of disease was chosen as the earliest time-point at which adequate numbers of cells for analysis can be isolated from the spinal cord. Flow cytometric analysis of cells recovered from the spinal cord of EAE mice also showed that amongst the CD3+ T cell population, an average of 67.9% of cells were positive for IL-1β (Figure 4b). Flow cytometric analysis of CD3+ lymphocytes showed that 72.2% to 94.2% of cells in the CD8+, CD4+, and γδ+ T cell populations were IL-1β+ (Figure 4c).
MOG-immunized TNFR1 deficient mice, in which CXCL12 is relocated, have relatively robust trafficking of T cells into the perivascular space, given the absence of clinical disease, with entry of few or no CD11b+ or GR1+ cells (36). Thus, the use of these mice was ideal for determining which IL-1β-producing T cell subtype was associated with CXCL12 relocation. Cells were isolated from the spinal cords of MOG-immunized TNFR deficient mice when their wild-type controls began to show signs of disease and analyzed by flow cytometry. Almost 50% of CD3+ T cells expressed IL-1β and an analysis of the T cell subsets showed that 77% of T cells isolated were γδT cells (Figure 5a). Depletion of γδT cells has been shown to decrease the levels of IL-1β at the induction of EAE (37) and IL-17+ γδT cells have been implicated in the induction of autoimmune joint inflammation (38). We therefore further analyzed the γδT cell population isolated from wild-type mice with EAE and found a population of γδT cells that produced significantly more IL-17 than the γδ- T cells. An average of 95.1% of these γδ IL-17+ cells also expressed IL-1β (Figure 5b). In accordance with previously published data suggesting a role for IL-1β in the promotion of IL-17 during CNS autoimmunity, no IL-17 mRNA was detectable in the spinal cords recovered from MOG-immunized IL-1R deficient mice (Figure 5c).
While both IL-1 (22) and IL-1R (21) deficient mice are resistant to EAE induced by MOG-immunization, adoptive transfer of wild-type myelin-specific T cells into IL-1R deficient mice was not protective from EAE (21), suggesting a requirement of IL-1R within the adoptively transferred T cell population. In order to address the T cell role of IL-1R on CXCL12 relocation, CXCL12 expression patterns were evaluated in the spinal cords of mice with EAE induced by adoptive transfer of wild-type or IL-R deficient T cells derived from MOG-immunized mice into wild-type or IL-1R deficient recipients (Figure 6a). Spinal cords were harvested at the onset of disease and, of the inflamed vessels, 73% exhibited CXCL12 relocation in wild-type mice adoptively transferred with wild-type MOG-specific T cells (incidence 80%), while 51% had CXCL12 relocation in IL-1R-deficient mice adoptively transferred with wild-type MOG-specific T cells (incidence 57%) and only 20% of inflamed vessels of wild-type mice adoptively transferred with IL-1R-deficient, MOG-specific T cells (incidence 33%) exhibited relocation (Figure 6a). These data suggest that expression of IL-1R in recipient mice determines the extent of CXCL12 relocation. Because endogenous T cells also participate in the induction of adoptive transfer EAE (39), we wished to more definitively determine if the effect of IL-1R deficiency on disease severity and CXCL12 relocation was localized to the leukocyte population or the CNS. Thus, we also generated bone marrow chimeras using IL-1R deficient and wild-type mice. Chimeric mice lacking the IL-1R and reconstituted with wild-type bone marrow or vice-versa were both susceptible to EAE, although the group of IL-1R deficient mice receiving wild-type bone marrow exhibited a significant decrease in severity of disease compared to either wild-type mice receiving IL-1R deficient bone marrow or wild-type into wild-type controls (Figure 6b). Additionally, chimeras with IL-1R deficiency on irradiation-protected CNS resident cells had a reduced mean-maximal disease score compared to wild-type into wild-type controls and a delayed onset compared to both other disease groups (Table 1). As expected, IL-1R deficient into IL-1R deficient controls exhibited minimal clinical disease that was significantly decreased compared with all groups and a delay in onset compared with the wild-type into wild-type group. As in the adoptive transfer experiments, we evaluated the CXCL12 expression within spinal cord tissues of both types of chimera after MOG immunization at the first signs of clinical disease and obtained similar results (Figure 6c). Of the inflamed vessels, 42% had CXCL12 relocation in IL-1R-deficient mice reconstituted with wild-type bone marrow whereas only 17% of inflamed vessels of wild-type mice reconstituted with IL-1R-deficient bone marrow exhibited relocation. Identical staining protocols using spinal cord tissue collected from naïve IL-1R deficient mice showed that 100% of vessels analyzed displayed normal, basolateral expression of CXCL12 indicating that IL-1R deficiency does not affect the pattern of CXCL12 expression in the absence of EAE (data not shown). Although the immunohistochemical data suggest that IL-1R expression on recipient CNS cells, including the endothelial cells, determine the severity of EAE and the extent of CXCL12 relocation, the clinical disease severity data also indicate a role for IL-1R expression on infiltrating hematopoietic cells. Flow cytometric analysis of cells isolated from the spinal cords of mice with EAE indicate that the percentage of γδ T cells expressing IL-1R (83.3%) is significantly higher than the percentages of CD8 (21.7%) and CD4 (11.2%) cells expressing the IL-1R (Figure 6d).
IL-1β has historically been associated with the induction of autoimmune neuroinflammatory diseases but few studies have uncovered down-stream effects of IL-1R signaling at the BBB. Because prior studies have demonstrated that pathologic alteration in CXCL12 expression at the BBB occurs specifically during autoimmune diseases (8) and that IL-1β can regulate CXCL12 expression in neural cells (31), we hypothesized that the inflammatory cytokine IL-1β might play a role in regulating the relocation of CXCL12 at the CNS endothelium. Using EAE, a murine model of MS, we found that CXCL12 relocation occurs at early, preclinical time-points, prior to overt BBB disruption and is associated with the perivascular infiltration of IL-1β-expressing T cells that include a population of γδ T cells that also express IL-17. We also observed that IL-1β can directly induce relocation of CXCL12 at the BBB and that decreased levels of CNS IL-1R diminish the extent of this relocation and of clinical disease severity.
In most diseases and animal models, activated microglia and macrophages are considered to be the predominant source of IL-1β in the CNS (14, 40). Because of its unique regulation and inflammatory potential, IL-1β is usually considered to be an innate cytokine as initial tissue insult leads to activation and release of this potent molecule (41). Gamma-delta T cells participate in the innate arm of the immune response via recognition of non-peptide ligands (32, 33). These cells have been detected within EAE and MS lesions (42, 43) and numbers of activated γδT cells are elevated in the peripheral blood of MS patients compared to control patients (44). Depletion of γδ T cells in mice with EAE leads to a reduction in CNS IL-1β and decreased clinical disease severity (37, 42, 45). We directly measured levels of IL-1β in T cells derived from the CNS of mice with EAE and observed that the γδ T cell fraction expressed the highest levels of this cytokine. We also observed that the majority of T cells present in the CNS of MOG-immunized TNFR1 deficient mice, which exhibit loss of basolateral expression of CXCL12, are γδT cells. However, given that TNFR1-deficient mice are resistant to EAE (33, 36), the presence of IL-1β-expressing γδT cells and CXCL12 relocation may be necessary but insufficient to induce clinical disease. Because CXCL12 expression at the BBB of MOG-immunized TNFR1-deficient mice displayed increased co-localization with CD31, it is possible that TNF-α regulates only a subset of possible CXCL12 binding sites. For example, the in vitro attachment of CXCL12 to heparan sulphate proteoglycans on cultured rheumatoid arthritis endothelial cells was recently shown to be enhanced during exposure to inflammatory cytokines (46). It is therefore possible that multiple T cell-derived cytokines act in concert to promote the perivascular egress of leukocytes during induction of autoimmunity.
Prior studies indicated that CXCL12 relocation at the BBB correlates with disease severity in MS patients and may contribute to both leukocyte entry into the CNS and egress from perivascular spaces (6, 8). The loss of CXCL12 polarity at the induction of EAE, prior to the onset of BBB disruption and clinical disease suggests this phenomenon is a precursor to the development of lesions. Indeed, use of Gd-MRI demonstrated that the time-point of CXCL12 relocation does not correlate with detectable levels of Gd enhancement. Understanding the molecular mechanism of CXCL12 relocation might therefore reveal novel therapeutic targets for the prevention of leukocyte entry early during the course of an MS exacerbation. We observed a population of γδT cells producing relatively high levels of both IL-1β and IL-17. IL-17 has been detected in both inflammatory lesions (47) and CSF (48) of MS patients and has recently been shown to be important for the induction of EAE (49). In addition, adoptive transfer of myelin-specific CD4+ Th17 cells has been shown to induce the selective upregulation of the chemokines CXCL1 and CXCL2 within the spinal cords of recipient mice (50). CXCL1 and CXCL2 are ELR+ chemokines found in EAE lesions that are potent chemoattractants for PMN (51). In EAE, the expression of these molecules was shown to be essential for the early infiltration of PMN and induction of EAE. In our study, γδ T cells were found to be a source of IL-17 and could therefore also play a role in the recruitment of PMN and eventual BBB disruption that allow the infiltration of nonspecific leukocytes during clinical onset of EAE.
Although IL-1R-deficient mice were shown to be resistant to EAE (21), these studies did not identify cellular targets of IL-1β. Induction of EAE using chimeric mice suggest that IL-1R-expressing cells within the irradiation resistant CNS determine the severity of clinical disease. This is consistent with the reduction in disease seen in mice with over-expression of IL-1β antagonists within the CNS (52). Since IL-1β, which induces CXCL12 relocation, is produced by IL-17-producing γδ T cells, it is possible that these molecules interact to promote CNS inflammation, suggesting that IL-1β and its receptor might be targets for the treatment of MS. Further studies examining the effects of IL-1β and IL-17 on the expression of CXCL12 binding receptors are currently underway. As γδ T cells represent only a portion of the T cell population identified within the CNS of mice with early EAE, they may not be required for inducing CXCL12 relocation. Studies in γδ T cell-deficient mice and/or adoptive transfer experiments utilizing IL-1β-deficent γδ T cells, will be required to directly demonstrate that these cells mediate CXCL12 relocation via their production of IL-1β.
Studies have implicated a number of different molecules in the trafficking of leukocytes at the BBB including the interaction between endothelial CD40 and CD40L expressed on T cells. Lack of CD40 activations leads to decreased parenchymal penetration of T cells within the CNS during both EAE and encephalitis due to West Nile virus with extensive perivascular localization of T cells noted in both models (53-55). CD40: CD40L interactions have also been shown to regulate cell trafficking via CXCL12 expression during joint inflammation (12). These studies suggest that CD40: CD40L interactions could potentially contribute to CXCL12 expression patterns at the BBB. Although the current study does not directly address this possibility, administration of soluble CD40 ligand did not induce CXCL12 redistribution and analysis of CXCL12 expression within the spinal cords of MOG-immunized CD40-deficient mice did not reveal alteration in CXCL12 polarity at the BBB (all our unpublished data). Thus, it is unlikely that CD40 participates in the relocation of CXCL12 during EAE.
To date, very little is known about the mechanisms involving CXCL12 regulation within the CNS or about the molecules contributing to the relocalization of CXCL12 at the BBB, which appears to be an important feature of MS. This study indicates that the early entrance of IL-1β-expressing, autoreactive T cells leads to the pathologic relocation of CXCL12 and that disruption of IL-1R signaling on CNS cells may ameliorate the extent of autoimmune disease. While further studies are needed to determine the contribution of other molecules in the relocation of CXCL12 at the BBB, the current study suggests that IL-1β and the IL-1R could potentially be targets for the prevention of new lesions during MS.
The authors would like to thank Dr. John Russell for advice in experimental design, critical feedback, and gifts of reagents. Additional thanks to Julia Sim, Dawn Koch and Mickey Croyle for technical assistance.