The scavenger receptor that binds phosphatidylserine and oxidized lipoprotein (SR-PSOX)/CXCL16 is a chemokine expressed on macrophages and dendritic cells, while its receptor expresses on T and NK T cells. We investigated the role of SR-PSOX/CXCL16 on acute and adoptive experimental autoimmune encephalomyelitis (EAE), which is Th1-polarized T cell-mediated autoimmune disease of the CNS. Administration of mAb against SR-PSOX/CXCL16 around the primary immunization decreased disease incidence of acute EAE with associated reduced infiltration of mononuclear cells into the CNS. Its administration was also shown to inhibit elevation of serum IFN-γ level at primary immune response, as well as subsequent generation of Ag-specific T cells. In adoptive transfer EAE, treatment of recipient mice with anti-SR-PSOX/CXCL16 mAb also induced not only decreased clinical disease incidence, but also diminished traffic of mononuclear cells into the CNS. In addition, histopathological analyses showed that clinical development of EAE correlates well with expression of SR-PSOX/CXCL16 in the CNS. All the results show that SR-PSOX/CXCL16 plays important roles in EAE by supporting generation of Ag-specific T cells, as well as recruitment of inflammatory mononuclear cells into the CNS.

Multiple sclerosis (MS)3 and its animal model, experimental autoimmune encephalomyelitis (EAE), are type I (Th1)-polarized T cell-mediated autoimmune diseases of the CNS (1, 2). In both diseases, Th1 autoreactive T cells for self-Ags in the CNS are initially generated (3, 4), and then circulating leukocytes including the autoreactive T cells penetrate the blood brain barrier. Finally, the penetrated leukocytes induce damage of myelin, resulting in impaired nerve conduction and paralysis (2). However, the mechanisms of generation of autoreactive Th1 T cells, recruitment of leukocytes into the CNS, and accumulation of the leukocytes in the CNS before and during clinical disease are not well understood.

Chemokines are a family of cytokines exhibiting selective chemoattractant properties for target leukocytes. Based on the motif of the first two cysteines, chemokines have been classified into four highly conserved but distinct subfamilies: CC, CXC, C, and CX3C chemokines (5). Chemokines play an important role in recruitment of leukocytes at the site of initial immunoreactions induced by such as infection as well as infiltration of leukocytes into the site of inflammation during the T cell-mediated inflammatory conditions (3, 6). Various chemokine members have been implicated as candidates involved in the immunopathology of EAE. CCL2/MCP-1, CCL3/MIP-1α, and CXCL1/IFN-γ-inducible protein-10 (IP-10) were reported to be expressed in the CNS in acute rat and murine EAE models (7, 8). T cell clones, which could induce adoptive transfer EAE, were reported to express CCR5, a receptor for CCL3/MIP-1α.

MIP-1α was demonstrated to play a functionally significant role in pathogenesis of mouse EAE by analyses that administration of neutralizing polyclonal Ab for MIP-1α suppressed severity of clinical EAE through partial inhibition of recruitment of inflammatory mononuclear cells including Th1 T cells into the CNS (7). In the case of IP-10, two contradictory results were reported (9, 10). Administration of neutralizing Ab against IP-10 partially decreased or increased clinical and histological disease incidence and severity, as well as infiltration of mononuclear cells into the CNS. As for MCP-1, gene-disrupted mice of MCP-1 and its receptor, CCR2, were shown to be completely resistant to development of EAE by inability of monocyte to be recruited into the CNS (9, 11, 12). Recruitment of monocytes into the CNS by MCP-1 must play an essential rule in EAE, while it has not been clear whether chemokines other than MIP-1α and IP-10 are involved in the infiltration of autoreactive T cells into the CNS.

Recently, polyclonal Ab against CCL20/MIP-3α was reported to partially suppress clinical EAE by inhibiting sensitization of naive lymphocytes to myelin Ags through inhibition of naive dendritic cells (DCs) trafficking or by inhibiting exit of sensitized lymphocytes from the draining lymph nodes (13). However, it has not been clear what chemokines are involved in the generation of Th1-polarized autoreactive T cells in primary immune response in EAE.

Recently, we and others identified a novel transmembrane protein that was designated as SR-PSOX (scavenger receptor that binds phosphatidylserine and oxidized lipoprotein) and CXCL 16, respectively (14, 15, 16). Interestingly, SR-PSOX/CXCL16 was shown to possess two different biological activities, scavenger receptor and chemokine activities. SR-PSOX/CXCL16 is the ligand for Bonzo/CXCR6 expressed on naive and active CD8 T cells, Th1-polarized activated CD4, and naive and activated NK T cells (14, 15). SR-PSOX/CXCL16 was shown to have chemoattractant activity for activated T cells, but not naive CD8 T cells (15). Cell surface-anchored SR-PSOX/CXCL16 with a transmembrane domain shows not only scavenger receptor activity but also cell adhesion activity against CXCR6-expressing cells, while membrane metalloprotease-cleaved soluble SR-PSOX/CXCL16 shows chemokine activity for CXCR6-expressing cells (17, 18). Bonzo/CXCR6, a receptor of SR-PSOX/CXCL16, was reported to be expressed on a subset of Th1 T cells but not on Th2 T cells, and its expression has been regarded as a differential marker of Th1-polarized T cells (19). Furthermore, expression of Bonzo/CXCR6 was confirmed in myelin basic protein-reactive T cell lines with IFN-γ-producing activity (20).

In this study, we investigated in vivo effects of neutralizing anti-SR-PSOX/CXCL16 mAb in both acute and adoptive transfer EAE. SR-PSOX/CXCL16 was clearly shown to play an important role in different two phases of EAE: IFN-γ-production at primary immune response followed by generations of myelin basic protein-specific T cells and recruitment of mononuclear cells into the CNS.

C57BL/6J mice were purchased from CLEA Japan (Tokyo, Japan) and housed under the specific pathogen-free condition. Myelin oligodendrocyte glycoprotein peptide (MOG35–55; MEVGWYRSPFSRVVHLYRNGK), which was used for Ags inducing acute EAE, was synthesized by Toray Research Center (Kamakura, Japan), and purity was determined to be >95% by reversed-phase HPLC.

We generated anti-SR-PSOX/CXCL16 mAb IgG1 12-81 as described previously (18). This Ab did not cross any other chemokines, which could be expected. Anti-human SR-PSOX mAb IgG1 22-19-12 was generated and characterized as described previously (17). Both mAbs were provided by Sankyo (Tokyo, Japan).

Chemotaxis assay using transwell plates with 5-μm pore size membrane (Corning Japan, Tokyo, Japan) and calcium mobilization assays were performed as described previously (18, 21).

CXCR6-expressed L1.2 cells (17) were incubated for 1 h on ice with mouse SR-PSOX/CXCL16-Fc (1 μg/ml), which was preincubated with anti-SR-PSOX mAbs 12-81 (5 μg/ml) or control rat IgG for 30 min on ice. For determining the quantity of cell-bound SR-PSOX/CXCL16-Fc, cells were stained with PE-labeled goat anti-human-Fc and analyzed by flow cytometry using Epics XL (Beckman Coulter, Fullerton, CA).

Spleen cells were prepared from mice on day 4 after immunization of MOG35–55, and cultured for 4 days in the presence or absence 25 μg/ml MOG35–55. Cells were incubated for 1 h on ice with mouse SR-PSOX/CXCL16-Fc (1 μg/ml), followed by staining with PE-labeled goat anti-human-Fc, together with FITC-labeled anti-mouse CD4 mAb or FITC-labeled anti-mouse CD8 mAb (BD Biosciences, San Diego, CA) as described previously (18), and then analyzed by flow cytometry.

Six to 9-wk-old female mice were immunized by s.c. injection into thighs of bilateral hind feet with 150 μg/mouse of MOG peptides in 0.15 ml of sterilized PBS emulsified with an equal volume of CFA containing 4 mg/ml Mycobacterium tuberculosis (BD Diagnostic Systems, Sparks, MD) (22, 23). Two hours before and 2, 4, and 7 days after the immunization, mice were injected with 500 μg anti-SR-PSOX/CXCL16 mAb or control rat IgG. The mice were i.v. injected with pertussis toxin (List Biological Laboratories, Campbell, CA) at day 0 and 2 after immunization. Animals were scored daily for 5 wk for clinical signs of EAE using the following criteria: 0, no clinical signs; 1, limp tail (tail paralysis); 2, complete loss of tail tonicity or abnormal gait; 3, partial hind limb paralysis; 4, complete hind limb paralysis; 5, forelimb paralysis or moribund; and 6, death (24).

Spleen-cell suspension was prepared from mice 4 days after the immunization with MOG35–55 as described in acute EAE and cultured in RPMI 1640 containing 10% FCS and 25 μg/ml MOG35–55 for 4 days at 37°C. Cells were harvested, washed three times with PBS and transferred i.v. into normal C57BL/6 recipient mice (1–2 × 107 viable cells/mouse). Animals were scored daily for 5 wk for signs of EAE using the above-described criterion (8, 13).

Spleen-cell suspension (5 × 106/ml) was prepared from mice on day 6 after immunization of MOG35–55, and cultured in triplicate in 96-well flat-bottom plates (IWAKI, Tokyo, Japan) in the presence or absence of MOG35–55. Cells were pulsed with 0.5 μCi/well [3H]thymidine (Valeant Pharmaceuticals, Costa Mesa, CA) at 72 h after cultivation, and incubated further for 12 h. Cells were then harvested and incorporated radioactivity was measured using a MicroBeta PLUS liquid scintillation counter with software v3.3 (PerkinElmer Wallac, Gaithersburg, MD).

IFN-γ in serum, which was prepared from MOG35–55-immunized mice on 36, 48, 60, and 84 h after immunization, was quantified using a mouse IFN-γ ELISA kit (GE Medical Systems, Milwaukee, WI), and the data were measured using Wallac 1420 ARVO fluoroscan (PerkinElmer Wallac).

Spleen-cell suspension (5 × 106/ml) was prepared from mice on day 6 after immunization of MOG35–55, and cultured in triplicate in 96-well flat-bottom plates (IWAKI) in the presence or absence of MOG35–55 for 48 h. IFN-γ in the culture supernatants was quantified using a mouse IFN-γ ELISA kit (Amersham Biosciences), and the data were measured using Wallac 1420 ARVO fluoroscan (PerkinElmer Wallac).

Total RNA was extracted from PBS-perfused and snap-frozen spinal cords using a total RNA Purification System (Invitrogen Life Technologies, Carlsbad, CA). cDNA was synthesized and RNA was amplified using Ready-To-Go RT-PCR Beads (GE Medical Systems). CXCR6 was detected by using the forward primer, 5′-CACTCTGGAACAAAGCTACTGGGCT-3′, and reverse primer, 5′-AGGTGAGAGTGAGCATGGACA-3′, and CXCL16 was detected by using primers as previously described (14).

Spinal cords from EAE-induced mice were dissected on day 15 after immunization and fixed in 4% neutral buffered formalin in PBS. Paraffin-embedded sections of 4-μm thickness were cut from the spinal cords and stained with H&E or with the myelin-specific Bodian and Luxol fast blue (25). For in situ hybridization, paraffin-embedded sections of spinal cords from EAE-induced mice were sliced and fixed on glass slides precoated with 3-aminopropyltriethoxysilane. Antisense and sense 35S-labeled cDNA probes specific for mouse SR-PSOX/CXCL16 were prepared by in vitro transcription with T3/T7 RNA polymerase (Stratagene, La Jolla, CA). SR-PSOX/CXCL16 mRNA-positive cells were examined under fluorescence microscope. Data were collected in several independent visual fields (24).

To characterize rat anti-mouse SR-PSOX/CXCL16 mAb 12-81, we examined inhibitory activity of the mAb against the chemotactic activity of soluble SR-PSOX/CXCL16 for CXCR6-expressing cells using the standard transwell assay. The number of cells that migrated into bottom wells was shown to decrease in accordance with the concentration of anti-SR-PSOX/CXCL16 mAb (Fig. 1,A). Then, we examined whether the mAb inhibits direct binding of SR-PSOX/CXCL16 to its receptor, CXCR6. Direct binding of soluble SR-PSOX/CXCL16-Fc to CXCR6-expressing L1.2 cells was quantified by flow cytometry. Anti-SR-PSOX/CXCL16 mAb 12-81 was clearly shown to specifically inhibit direct binding of SR-PSOX/CXCL16-Fc to CXCR6-expressing cells (Fig. 1 B). These results indicate that anti-SR-PSOX/CXCL16 mAb 12-81 can inhibit SR-PSOX/CXCL16-induced migration as well as direct binding of SR-PSOX/CXCL16 to CXCR6-expressing cells.

FIGURE 1.

Characterization of anti-SR-PSOX/CXCL16 mAb 12-81. A, Effect on SR-PSOX/CXCL16-induced chemotaxis. Migration of CXCR6-expressing L1.2 cells to recombinant soluble SR-PSOX/CXCL16 (100 ng/ml) was quantified by chemotaxis assay using transwell plate as described previously (21 ). Neutralizing activity of anti-SR-PSOX/CXCL16 was analyzed by using CXCR6-expressing cells pretreated with indicated amounts of anti-SR-PSOX/CXCL16 mAb 12-81 or control rat IgG. Values in the absence of Ab were set as 100%. The data shown represent the mean ± SD (n = 3). B, Effect of SR-PSOX/CXCL16-binding to CXCR6-expressing cells. Binding of control Fc (dotted line) or soluble SR-PSOX/CXCL16-Fc (solid line) against CXCR6-expressing L1.2 cells was analyzed by flow cytometry. SR-PSOX/CXCL16-Fc was preincubated with control rat IgG (thin line) or anti-SR-PSOX/CXCL16 mAb 12-81 (bold line), as described in Materials and Methods.

FIGURE 1.

Characterization of anti-SR-PSOX/CXCL16 mAb 12-81. A, Effect on SR-PSOX/CXCL16-induced chemotaxis. Migration of CXCR6-expressing L1.2 cells to recombinant soluble SR-PSOX/CXCL16 (100 ng/ml) was quantified by chemotaxis assay using transwell plate as described previously (21 ). Neutralizing activity of anti-SR-PSOX/CXCL16 was analyzed by using CXCR6-expressing cells pretreated with indicated amounts of anti-SR-PSOX/CXCL16 mAb 12-81 or control rat IgG. Values in the absence of Ab were set as 100%. The data shown represent the mean ± SD (n = 3). B, Effect of SR-PSOX/CXCL16-binding to CXCR6-expressing cells. Binding of control Fc (dotted line) or soluble SR-PSOX/CXCL16-Fc (solid line) against CXCR6-expressing L1.2 cells was analyzed by flow cytometry. SR-PSOX/CXCL16-Fc was preincubated with control rat IgG (thin line) or anti-SR-PSOX/CXCL16 mAb 12-81 (bold line), as described in Materials and Methods.

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To determine the roles of SR-PSOX/CXCL16 on development of clinical EAE, we examined the in vivo activity of neutralizing anti-mouse SR-PSOX/CXCL16 mAb 12-81 against induction of clinical acute EAE. Mice, immunized with the encephalitogenic peptide MOG35–55 on day 0, were administrated with anti-SR-PSOX/CXCL16 mAb on day 0, 2, 4, and 7, and scored daily for signs of disease (Fig. 2 A). As expected, mice treated with control rat IgG instead of anti-SR-PSOX/CXCL16 mAb developed clinical EAE with a mode incidence of 100%. In contrast, the clinical disease severity was dramatically decreased in anti-SR-PSOX/CXCL16 mAb-treated mice. Although the control mice developed severe EAE with a mean clinical score of 3.8 on day 18 after immunization, anti-SR-PSOX/CXCL16 mAb-treated mice developed disease with a significantly decreased mean clinical score of 1.3 on day 19 with associated delayed onset of the clinical disease. Interestingly, administration of anti-SR-PSOX/CXCL16 mAb on only day 0 and 2 after immunization of MOG35–55 was shown to be enough to induce delayed onset of clinical EAE while reduction of the disease severity was lower than administration on day 0, 2, 4, and 7 after immunization (data not shown). Administration of anti-SR-PSOX/CXCL16 mAb on day 4 and 7 after immunization did not affect clinical EAE. All the results indicate that in vivo neutralization of SR-PSOX/CXCL16 before the onset of clinical EAE significantly reduced the severity as well as early onset of acute EAE.

FIGURE 2.

Effects of anti-SR-PSOX/CXCL16 mAb 12-81 on development of clinical disease in MOG35–55-induced acute EAE. A, Development of MOG35–55-induced clinical EAE. Mice were immunized with MOG35–55 and then received pertussis toxin as described in Materials and Methods. Results of mice receiving control rat IgG (n = 7) or anti-SR-PSOX/CXCL16 mAb (n = 7) are expressed as mean disease score ± SD. The data are representative of three independent experiments with essentially similar results; ∗, p < 0.02; ∗∗, p < 0.01 (Mann-Whitney U test). BE, Histological examination. Spinal cords from EAE-induced mice treated with control rat IgG (B and D) or anti-SR-PSOX/CXCL16 mAb (C and E) were analyzed. Serial tissue sections were stained by Bodian and Luxol fast blue (B and C) and H&E (D and E) in each mouse. Demyelinating lesions (B) and lesions with infiltrated mononuclear cells (D) are indicated in control IgG-treated mice, while little demyelinating lesion (C) and lesions with only some extent of perivascular mononuclear cell infiltration (E) are indicated in anti-SR-PSOX/CXCL16 mAb-treated mouse. Scale bars, B and C, 150 μm; D and E, 200 μm.

FIGURE 2.

Effects of anti-SR-PSOX/CXCL16 mAb 12-81 on development of clinical disease in MOG35–55-induced acute EAE. A, Development of MOG35–55-induced clinical EAE. Mice were immunized with MOG35–55 and then received pertussis toxin as described in Materials and Methods. Results of mice receiving control rat IgG (n = 7) or anti-SR-PSOX/CXCL16 mAb (n = 7) are expressed as mean disease score ± SD. The data are representative of three independent experiments with essentially similar results; ∗, p < 0.02; ∗∗, p < 0.01 (Mann-Whitney U test). BE, Histological examination. Spinal cords from EAE-induced mice treated with control rat IgG (B and D) or anti-SR-PSOX/CXCL16 mAb (C and E) were analyzed. Serial tissue sections were stained by Bodian and Luxol fast blue (B and C) and H&E (D and E) in each mouse. Demyelinating lesions (B) and lesions with infiltrated mononuclear cells (D) are indicated in control IgG-treated mice, while little demyelinating lesion (C) and lesions with only some extent of perivascular mononuclear cell infiltration (E) are indicated in anti-SR-PSOX/CXCL16 mAb-treated mouse. Scale bars, B and C, 150 μm; D and E, 200 μm.

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Then, we histologically examined spinal cords of acute EAE-induced mice treated with or without anti-SR-PSOX/CXCL16 mAb (Fig. 2, BE). Anti-SR-PSOX/CXCL16 mAb-treated mice showed no signs of histological EAE with demyelination and mononuclear cell infiltration, while there were numerous inflammatory and demyelinating lesions in the perivascular areas and parenchymal in spinal cord of control rat IgG-received mice that showed severe clinical EAE. These inflammatory lesions contain infiltrated mononuclear cells, while spinal cords of anti-SR-PSOX/CXCL16 mAb-treated mice lacked mononuclear cell infiltration and showed significantly decreased number of demyelinating lesions per section compared with the control mice. These results of histological analyses in anti-SR-PSOX/CXCL16 mAb-received mice correlate well with the absence of clinical EAE. Thus, inhibition of SR-PSOX/CXCL16 activities by anti-SR-PSOX/CXCL16 mAb 12-81 is efficient to suppress both clinical and histological EAE.

Because anti-SR-PSOX/CXCL16 mAb-treated mice were resistant to development of both clinical and histological EAE, we examined which phase was inhibited by administration of anti-SR-PSOX/CXCL16 mAb 12-81, generation of MOG35–55-specific Th1-polarized T cells, or infiltration of MOG35–55-specific T cells into the CNS. We analyzed generation of Ag-specific T cells in spleens of MOG35–55-immunized mice. Spleen cells were prepared from mice on day 6 after immunization with MOG35–55, and their MOG35–55-specific in vitro proliferation and in vitro IFN-γ production were quantified by [3H]thymidine incorporation assay (Fig. 3,A) and by ELISA (Fig. 3,B), respectively. Spleen T cells from control rat IgG-received mice showed robust and dose-dependent proliferative response in accordance with the amounts of added MOG35–55. In contrast, spleen T cells from anti-SR-PSOX/CXCL16 mAb-treated mice did not show MOG35–55-dependent increase of cell proliferative response in vitro (Fig. 3,A). Moreover, MOG35–55-dependent increase of in vitro IFN-γ production was not induced in spleen cells from anti-SR-PSOX/CXCL16 mAb-received mice, although MOG35–55-induced dramatic increase of IFN-γ production was observed in spleen cells from control rat IgG-received mice (Fig. 3 B). These results suggest that generation of MOG35–55-specific T cells was inhibited by anti-SR-PSOX/CXCL16 mAb in primary in vivo immune response, because mice received MOG35–55 only once on day 0.

FIGURE 3.

Effects of anti-SR-PSOX/CXCL16 mAb 12-81 on MOG35–55-specific immune responses. A and B, Six days after administration of MOG35–55, splenocytes were isolated from mice treated with anti-SR-PSOX/CXCL16 mAb (anti-SR-PSOX/CXCL16) or control rat IgG (vehicle). A, Incorporation of [3H]thymidine by the splenocytes was measured after in vitro stimulation with MOG35–55-peptide as described in Materials and Methods; ∗, p < 0.002; ∗∗, p < 0.02. B, Production of IFN-γ by the splenocytes was measured after in vitro stimulation with MOG35–55-peptide as described in Materials and Methods; ∗, p < 0.03. C, Production of IFN-γ was quantified by ELISA in serum of MOG35–55-injected mice treated with anti-SR-PSOX/CXCL16 mAb (anti-SR-PSOX/CXCL16) or rat IgG (vehicle). Serum was harvested at indicated hours after the immunization with MOG35–55; ∗, p < 0.002; ∗∗, p < 0.02.

FIGURE 3.

Effects of anti-SR-PSOX/CXCL16 mAb 12-81 on MOG35–55-specific immune responses. A and B, Six days after administration of MOG35–55, splenocytes were isolated from mice treated with anti-SR-PSOX/CXCL16 mAb (anti-SR-PSOX/CXCL16) or control rat IgG (vehicle). A, Incorporation of [3H]thymidine by the splenocytes was measured after in vitro stimulation with MOG35–55-peptide as described in Materials and Methods; ∗, p < 0.002; ∗∗, p < 0.02. B, Production of IFN-γ by the splenocytes was measured after in vitro stimulation with MOG35–55-peptide as described in Materials and Methods; ∗, p < 0.03. C, Production of IFN-γ was quantified by ELISA in serum of MOG35–55-injected mice treated with anti-SR-PSOX/CXCL16 mAb (anti-SR-PSOX/CXCL16) or rat IgG (vehicle). Serum was harvested at indicated hours after the immunization with MOG35–55; ∗, p < 0.002; ∗∗, p < 0.02.

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Because EAE is a Th1-dominant autoimmune disease model, we also examined the amount of proinflammatory Th1 cytokine IFN-γ secreted in serum of mice immunized with MOG35–55. IFN-γ is known to be involved in the pathogenesis of EAE at different points in the course of disease, including primary immune response (26, 27). At 48 and 60 h after primary immunization with MOG35–55, amount of secreted IFN-γ was elevated in serum of mice receiving control rat IgG. In contrast, production of IFN-γ was vigorously suppressed in mice receiving anti-SR-PSOX/CXCL16 mAb (Fig. 3 C). These observations suggest that anti-SR-PSOX/CXCL16 mAb inhibits generation of MOG35–55-specific Th1 T cell by suppression of IFN-γ production in primary immune response.

Next, we examined whether anti-SR-PSOX/CXCL16 mAb 12-81 could inhibit migration of MOG35–55-specific activated T cells into the spinal cord. Then, effect of administrated anti-SR-PSOX/CXCL16 mAb was analyzed in adoptive transfer EAE, which is developed in recipient mice receiving MOG35–55-specific activated T cells. Anti-SR-PSOX/CXCL16 mAb-treated mice receiving MOG35–55 peptide-specific T cells were indicated to manifest significantly delayed onset of clinical EAE at day 21 on average, while control rat IgG-treated mice showed onset of clinical EAE at day 13 on average (Fig. 4). In addition, anti-SR-PSOX/CXCL16 mAb-treated mice showed milder neurological impairment in histological EAE (data not shown). These results suggest that SR-PSOX/CXCL16 is relevant to onset of adoptive transfer EAE by inducing migration of MOG35–55-specific T cells into CNS, because CXCR6, a receptor of SR-PSOX/CXCL16, was reported to be expressed on activated CD4+, and naive and activated CD8+ T cells (14, 15).

FIGURE 4.

Effects of anti-SR-PSOX/CXCL16 mAb 12-81 on onset and development of adoptive transferred clinical EAE. Mice were immunized with MOG35–55 as described in Fig. 2, and splenocytes of the mice 4 days after the immunization were cultured with MOG35–55 for 4 days, and then, obtained MOG35–55-specific T cell blasts were transferred into recipient B6 mice as described in Materials and Methods. Two hours before and 2 days after the transfer of encephalitogenic T cells, recipient mice were injected i.p. with 0.2 ml/mouse of PBS containing 500 μg of anti-SR-PSOX/CXCL16 mAb or control rat IgG (arrows). The data of mice receiving anti-SR-PSOX/CXCL16 mAb (n = 6) or control rat IgG (n = 6) are expressed as the mean clinical disease score ± SD; ∗, p < 0.05; ∗∗, p < 0.002. The data are representative of three independent experiments with similar results.

FIGURE 4.

Effects of anti-SR-PSOX/CXCL16 mAb 12-81 on onset and development of adoptive transferred clinical EAE. Mice were immunized with MOG35–55 as described in Fig. 2, and splenocytes of the mice 4 days after the immunization were cultured with MOG35–55 for 4 days, and then, obtained MOG35–55-specific T cell blasts were transferred into recipient B6 mice as described in Materials and Methods. Two hours before and 2 days after the transfer of encephalitogenic T cells, recipient mice were injected i.p. with 0.2 ml/mouse of PBS containing 500 μg of anti-SR-PSOX/CXCL16 mAb or control rat IgG (arrows). The data of mice receiving anti-SR-PSOX/CXCL16 mAb (n = 6) or control rat IgG (n = 6) are expressed as the mean clinical disease score ± SD; ∗, p < 0.05; ∗∗, p < 0.002. The data are representative of three independent experiments with similar results.

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Then, we analyzed whether CXCR6 was actually expressed on activated T cells used in adoptive transfer EAE (Fig. 5). Although a small part of CD4+ spleen T cells from unimmunized mice expressed increased amounts of CXCR6 after in vitro cultivation with MOG35–55, the number of CXCR6-positive CD4+ T cells was significantly larger in splenocytes of MOG35–55-immunized mice than in unimmunized mice after in vitro cultivation with MOG35–55. However, in CD8+ spleen T cells, in vitro cultivation with MOG35–55 similarly increased expression of CXCR6 between MOG35–55-immunized and -unimmunized mice. Thus, in vitro cultivation with MOG35–55 specifically augments expression of CXCR6 on CD4+ T cells from MOG35–55-immunzed mice, which may be relevant to onset and development of transfer EAE.

FIGURE 5.

Expression of CXCR6 on MOG35–55-specific activated CD4-positive and CD8-positive T cells. Mice were immunized with MOG35–55 as described in Fig. 2, and spleen cells of mice 4 days after immunization (EAE) or without immunization (normal) were analyzed before (−) and after (+) in vitro cultivation with MOG35–55 for 4 days. Cells were stained with SR-PSOX/CXCL16-Fc, followed by PE-labeled anti-human-Fc mAb together with anti-FITC-labeled CD4 mAb (A and C) or anti-FITC-labeled CD8 mAb (B and D), and then analyzed by two-dimensional flow cytometry (A and B). CD4-positive and CD8-positive cells in A and B, respectively, were gated, and the expression of CXCR6 was analyzed (C and D). The data are representative of three independent experiments with similar results.

FIGURE 5.

Expression of CXCR6 on MOG35–55-specific activated CD4-positive and CD8-positive T cells. Mice were immunized with MOG35–55 as described in Fig. 2, and spleen cells of mice 4 days after immunization (EAE) or without immunization (normal) were analyzed before (−) and after (+) in vitro cultivation with MOG35–55 for 4 days. Cells were stained with SR-PSOX/CXCL16-Fc, followed by PE-labeled anti-human-Fc mAb together with anti-FITC-labeled CD4 mAb (A and C) or anti-FITC-labeled CD8 mAb (B and D), and then analyzed by two-dimensional flow cytometry (A and B). CD4-positive and CD8-positive cells in A and B, respectively, were gated, and the expression of CXCR6 was analyzed (C and D). The data are representative of three independent experiments with similar results.

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Then, we ascertained the expression of SR-PSOX/CXCL16 in the CNS of EAE mice that might induce chemotaxis of MOG35–55-specific activated T cells into the CNS. Expression of SR-PSOX/CXCL16 was analyzed in the spinal cords of EAE mice 14 days after immunization by in situ hybridization. SR-PSOX/CXCL16-expressing cells were observed around in the white matter of the spinal cord, while significant expression of SR-PSOX/CXCL16 was not detected in control mice (Fig. 6, B and C). Next, we confirmed the expression of SR-PSOX/CXCL16 by RT-PCR in spinal cords of EAE mice. SR-PSOX/CXCL16 mRNA was shown to express at low level in spinal cords of mice 2, 4, and 6 days after immunization with MOG35–55, while at detectable high level after onset of acute clinical EAE 14 days after immunization (Fig. 6 D). The results suggested that a small increase of SR-PSOX/CXCL16 mRNA in the spinal cords preceded the development of clinical disease symptoms, which might have an affect on the disease onset, and its high expression was induced after disease onset.

FIGURE 6.

Expression of SR-PSOX/CXCL16 in the CNS of mice with EAE. AC, Expression of SR-PSOX/CXCL16 mRNA was analyzed in spinal cord of EAE-induced mice 14 days after immunization or of control mice without immunization (normal) by in situ hybridization. D, mRNA of SR-PSOX/CXCL16 and CXCR6/Bonzo in spinal cord from EAE mice were measured by RT-PCR 2, 4, 6, and 14 days after immunization. Expression of elongation factor 1-α (EF1-α) mRNA was also measured as control.

FIGURE 6.

Expression of SR-PSOX/CXCL16 in the CNS of mice with EAE. AC, Expression of SR-PSOX/CXCL16 mRNA was analyzed in spinal cord of EAE-induced mice 14 days after immunization or of control mice without immunization (normal) by in situ hybridization. D, mRNA of SR-PSOX/CXCL16 and CXCR6/Bonzo in spinal cord from EAE mice were measured by RT-PCR 2, 4, 6, and 14 days after immunization. Expression of elongation factor 1-α (EF1-α) mRNA was also measured as control.

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From the observation that SR-PSOX/CXCL16 was highly produced in the CNS at the time of clinical EAE onset, we predicted that T cells in the CNS after induction of EAE would express CXCR6, the receptor for SR-PSOX/CXCL16. To ascertain this possibility, we analyzed CXCR6 mRNA level by RT-PCR in the spinal cords of EAE-induced mice. CXCR6 was shown to express in the CNS of acute clinical EAE 14 days after immunization, while expression of CXCR6 was not detected in mice 2, 4, and 6 days after immunization of MOG35–55 before onset of clinical EAE (Fig. 6 D). Taken together, both SR-PSOX/CXCL16 and its receptor CXCR6 were expressed in the CNS during the development of acute EAE.

EAE is a Th1-polarized T cell-mediated autoimmune disease of the CNS in appropriate strains of laboratory animals with a relapsing-remitting course that serves as a model for human MS. Th1-poralized T cells that are reactive to autoantigen in the CNS play an essential role in development of EAE (1, 2, 4). In EAE induced in this manuscript, such autoreactive Th1-poralized T cells were generated in peripheral lymphoid organ by s.c. immunization with MOG35–55 peptides, and EAE was caused by infiltration of mononuclear cells including the autoreactive T cells into the CNS. The present study demonstrates that administration of neutralizing mAb against transmembrane chemokine SR-PSOX/CXCL16 suppresses the development of both acute and adoptive transfer EAE by inhibiting the generation of the MOG35–55-reactive T cells in periphery and the recruitment/accumulation of mononuclear cells, including MOG35–55-reactive T cells, into the CNS, respectively. Thus, SR-PSOX/CXCL16 was suggested to play an important role in the development of EAE at different two phases: 1) generation of autoreactive Th1-polarized T cells and 2) its recruitment into and/or accumulation in the CNS.

In acute EAE, it was shown that SR-PSOX/CXCL16 was involved in generation of MOG35–55-reactive T cells as well as production of IFN-γ in primary immune response against MOG35–55 peptides used as immunogen (Fig. 3). Because SR-PSOX/CXCL16 has been expressed on APCs such as DCs and macrophages (14, 15, 17), which play an important role in primary immune response, SR-PSOX/CXCL16 may be involved in generation of Th1-polarized T cells through supporting IFN-γ production in primary immune response. However, it has not been clarified which activity of SR-PSOX/CXCL16, scavenger receptor or chemokine activity, plays a role in generation of the MOG-reactive T cells, although it is possible that both activities coordinately function on the generation of the MOG-reactive T cells. As a scavenger receptor, SR-PSOX/CXCL16 on APCs may be able to support uptake and presentation of various Ags including MOG peptide. As a transmembrane chemokine, SR-PSOX/CXCL16 exerts chemotaxis-inducing activity against CXCR6-expressing cells after cleaving by membrane metalloprotease (18). CXCR6 was shown to express on naive CD8 T cells, Th1-polarized activated CD4 T cells, and naive and activated NK T cells (15). SR-PSOX/CXCL16 on DCs may play a role to induce chemotaxis of T and/or NK T cells. We are now analyzing a role of NK T cells in generation of the MOG-reactive T cells, because NK T cells with high expression of CXCR6 have been reported to support primary Th1-poralized immune response by producing high amounts of IFN-γ (28, 29). Involvement of NK T cells in the generation of Th1-inclined autoreactive T cells in EAE must be intimately examined in the near future.

In adoptive transfer EAE, SR-PSOX/CXCL16 was indicated to induce mononuclear cell traffic into the CNS. In addition, our data indicate that clinical disease severity of both acute and adoptive transfer EAE correlates well with expression levels of both SR-PSOX/CXCL16 and its receptor CXCR6 in spinal cords (Fig. 6,D). Because 1) CXCR6 was expressed on both CD4+ and CD8+ T cells used in transfer EAE (Fig. 5); 2) CXCR6 was reported to express on a subset of Th1-polarized T cells but not on Th2 T cells or monocytes/macrophages (19); 3) SR-PSOX/CXCL16 was reported to have chemoattractant activity for activated T cells but not for monocytes/macrophages (15, 17); and 4) EAE has been regarded as Th1 T cell-mediated autoimmune disease (1, 2), SR-PSOX/CXCL16 may induce chemotaxis of MOG35–55-specific Th1-polarized activated CD4 T cells and/or CD8 T cells into the CNS, although we cannot deny the possibility that SR-PSOX-CXCL16 is also involved in accumulation, activation, and/or proliferation of MOG35–55 peptide-specific, Th1-poralized, activated CD4 T cells in the CNS.

Previous reports clearly indicated the essential role of MCP-1/CCL2 in adoptive transfer EAE (11, 13), and MCP-1/CCL2-attracted monocyte/macrophage may induce traffic of activated T cell into the CNS by the function of SR-PSOX/CXCL16. SR-PSOX/CXCL16 that induces chemotaxis and accumulation of MOG35–55-specific T cells expressing CXCR6 (Figs. 5 and 6,D) might be expressed on infiltrated monocyte/macrophage into the CNS. Actually, SR-PSOX/CXCL16 was shown to be expressed on activated macrophages (14) and was suggested to be expressed in infiltrated cells into the CNS of EAE mice (Fig. 6,B). However, we could not detect significant expression of SR-PSOX/CXCL16 in the spinal cords before immunization by in situ hybridization (Fig. 6, B and C). These data suggest that adoptively transferred MOG-reactive T cells infiltrate into the spinal cords of unimmunized mice without using CXCR6 and SR-PSOX/CXCL16, although administration of anti-SR-PSOX/CXCL16 mAb into the recipient mice inhibits onset of transfer EAE, probably through inhibiting direct infiltration of the transferred MOG-reactive T cells into spinal cord. Low level expression of SR-PSOX/CXCL16, which could be slightly detected by RT-PCR (Fig. 6,D) but not by in situ hybridization (Fig. 6 C) in the spinal cords of unimmunized mice, might induce traffic of the transferred MOG-reactive T cells, although we cannot deny the possibility that SR-PSOX/CXCL16 does not play a role in infiltration of the adoptively transferred T cells into spinal cord.

MIP-1α/CCL3 and IP-10/CXCL10, which possess similar functions to SR-PSOX/CXCL16 against activated T cells, might be also involved in activated T cell traffic into the CNS in EAE (7, 8). However, two contradictory results were reported on the function of IP-10/CXCL10 for EAE, which indicated that administration of neutralizing Abs decreased or increased clinical disease incidence and severity, as well as infiltration of mononuclear cells into the CNS in transfer EAE, while anti-IP-10/CXCL10 did not show any effects on acute EAE (8, 10). SR-PSOX/CXCL16 and MIP-1α/CCL3 may coordinately and/or complementarily function in EAE together with or without IP-10/CXCL10 by inducing chemotaxis of activated Th1 T cells into the CNS, and/or accumulation of the activated T cells in the CNS.

Our findings in this report open the possibility that mAb-induced in vivo inhibition of biological activities of SR-PSOX/CXCL16, such as generation of Ag-specific T cells in primary immune response in acute EAE and traffic of Th1-poralized activated T cells into the CNS in transfer EAE, may be useful for clinical therapy of autoimmune diseases including MS.

We thank H. Tomimoto, K. Togi, K. Okamoto, K. Sakamaki, and K. K. Lee for their generous help.

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 work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government. N.F. was supported by the 21st Century Center of Excellence Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan to Graduate School of Biostudies and Institute for Virus Research, Kyoto University.

3

Abbreviations used in this paper: MS, multiple sclerosis; DC, dendritic cell; EAE, experimental autoimmune encephalomyelitis; IP-10, IFN-γ-inducible protein-10; MOG, myelin oligodendrocyte glycoprotein; SR-PSOX, scavenger receptor that binds phosphatidylserine and oxidized lipoprotein.

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