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-Inducible Protein of 10 kDa (CXC Chemokine Ligand (CXCL) 10) but Not Monokine Induced by IFN- (CXCL9) Imprints a Pattern for the Subsequent Development of Autoimmune Disease 1
* Department of Developmental Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121;
Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037; and
Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129
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Abstract |
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-inducible protein of 10 kDa (IP-10), in contrast with monokine induced by IFN- and IFN-inducible T cell-
chemoattractant, is strongly expressed within 24 h postinfection. Blocking of IP-10, but not monokine induced by IFN-, aborts severity of Ag-specific injury of pancreatic 
cells and abrogates type 1 diabetes. Mechanistically, IP-10 blockade impedes the expansion of peripheral Ag-specific T cells and hinders their migration into the pancreas. IP-10 expression was restricted to viruses infecting the pancreas and that are capable of causing diabetes. Hence, virus-induced organ-specific autoimmune diseases may be dependent on virus tropism and its ability to alter the local milieu by selectively inducing chemokines that prepare the infected tissue for the subsequent destruction by the adaptive immune response. |
Introduction |
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Based on the dominance of Th1/Tc1 cells in driving various immunopathological diseases, we questioned how Th1/Tc1 chemokines expressed during an initial inflammatory process induced by a virus influenced the development of immune-mediated diseases. As a model system, we used acute lymphocytic choriomeningitis virus (LCMV) infection of transgenic mice that expressed the glycoprotein (GP) of LCMV in the cells of the islets of Langerhans (rat insulin promotor (RIP)-GP mice) (8, 9). LCMV was chosen because it does not lyse the cells it infects; it generates of an impressive antiviral immune response, and disease associated with LCMV infection is exclusively mediated by the antiviral immune response. RIP-GP transgenic mice were selected as the host because the expression of the target Ag (LCMV-GP transgene) is restricted to the islets of Langerhans in the pancreas, which facilitates measurement and quantification of the biochemical and pathologic phenotype. RIP-GP mice do not develop type 1 diabetes spontaneously; however, upon infection with LCMV, >95% of the RIP-GP mice develop a rapid disease onset as determined by a blood glucose concentration of >300 mg/dl, massive T cell infiltration restricted to the islets, and destruction of cells with resultant low to absent pancreatic insulin levels (9, 10). Concurrent with LCMV-mediated immunopathological diabetes is a profound Th1/Tc1-type response characterized by the release of the proinflammatory cytokines IFN- and TNF- in the target organ (11, 12) and a cell destructive process that is dependent on CD8 T cells specific for LCMV-GP (9, 10).
After i.v. or i.p. LCMV infection, a modest replication of virus is found in the pancreas (13). We now document that within the first 18–24 h after virus inoculation, CXCR3 chemokines are differentially expressed. IFN--inducible protein of 10 kDa (IP-10; CXC chemokine ligand (CXCL) 10), in contrast with the other CXCR3 chemokines, monokine induced by IFN- (Mig; CXCL9) and IFN-inducible T cell- chemoattractant (I-TAC; CXCL11), is highly expressed and plays a dominant role in programming the ensuing autoimmune disease. The onset and kinetics of IP-10 expression observed in the pancreas are unique and markedly contrast in three respects with previous reports of IP-10 expression after infection with a variety of viruses of the CNS (14, 15, 16). First, in CNS infection a battery of chemokines is elevated without a clear preference to any one. Second, chemokine expression occurs later at days 5–7. Third, there is no dominance for IP-10 expression. Our data demonstrate that IP-10 was selectively enhanced in infected RIP-LCMV mice and that neutralization of IP-10, but not Mig, blocked the virus-induced immune-mediated disease.
Using a novel assay with LCMV-GP-specific CD8 T cells expressing green fluorescent protein (GFP), we could determine the mechanism(s) by which blockade of IP-10 significantly lowered the incidence of autoimmune type 1 diabetes. First, a decrease in the clonal expansion of Ag-specific CD8 T cells was observed and, second, the recruitment of such autoaggressive T cells into the islets of Langerhans was found to be significantly lowered. Hence, IP-10 is a critical determinant for the migration of Ag-specific CD8 T cells into the target organ and is required to initiate the subsequent development of immune-mediated disease. In addition, IP-10 expression in the pancreas is a general phenomenon, in that infection of mice with viruses that are tropic for the pancreas and are known to cause diabetes, such as Coxsackievirus B4 (CVB) and encephalomyocarditis virus variant B (EMC-B), resulted in high-level pancreatic expression of IP-10 at days 1–4 postinfection. In contrast, infection with Theiler’s murine encephalomyelitis virus (TV), a virus without pancreas tropism that does not cause diabetes, did not result in the expression of significant levels of IP-10 in pancreas.
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Materials and Methods |
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Generation of H-2b RIP-LCMV-GP transgenic mice has been described previously (9, 10). The presence of transgenic RIP-GP sequences was determined by performing standard PCR with genomic DNA obtained from mouse tails as described (12). Generation of GFP x TCR(GP33–41) double transgenic mice has been described (17). Briefly, C57BL/6-TgN(ACTbEGFP)1Osb mice (GFP mice; The Jackson Laboratory, Bar Harbor, ME) that express GFP under the control of the -actin promoter (18) were crossed with B6;D2-TgN(TcRLCMV)327Sdz mice that express a Db(GP33–41)-specific TCR (19). The Armstrong clone 53b strain of LCMV was used for all experiments. LCMV was plaque purified three times on Vero cells, and stocks were prepared by a single passage on hamster kidney fibroblast cell line BHK-21 (ATCC CCL-10, American Type Culture Collection, Manassas, VA) cells. Mice were infected with a single i.p. dose of 5 x 103 PFU. CVB and EMC-B (20) were kindly provided by Dr. J. L. Whitton (The Scripps Research Institute, La Jolla, CA) and Dr. A. L. Notkins (National Institutes of Health, Bethesda, MD), respectively. The hamster anti-murine IP-10 mAb (1F11) was generated and purified as described (21) and was administered at 100 µg in 100 µl of PBS per injection 6 h before and then at days 1, 2, 4, and 6 after LCMV infection. The hamster anti-murine Mig hybridoma (2A6.9.9) was a kind gift from Dr. R. Schreiber (Washington University School of Medicine, St. Louis, MO). This anti-Mig mAb specifically neutralized murine Mig function (A. D. Luster, unpublished observations) and was produced and purified in parallel using the same conditions as the anti-IP-10 mAb (21). As a control, a group of four mice were injected with an isotype-matched hamster IgG Ab (Southern Biotechnology Associates, Birmingham, AL). For blocking of RANTES, a neutralizing monoclonal anti-RANTES Ab from R&D Systems (Minneapolis, MN) was used. The polyclonal rabbit anti-CXCR3 Ab was purchased from Zymed Laboratories (South San Francisco, CA) and the anti-IFN- mAb was obtained from BD PharMingen (San Diego, CA).
Blood glucose (BG) measurements
Blood samples were obtained from the retro-orbital plexus and plasma glucose concentration was determined using a ONE TOUCH Ultra glucometer (LifeScan, Milpitas, CA). Mice with BG values of 300 mg/dl or higher were considered diabetic (22).
RNase protection assay (RPA)
Total RNA was isolated from whole pancreas homogenates using Tri-Reagent (Molecular Research Center, Cincinnati, OH). RNA was extracted with chloroform followed by isopropanol precipitation and washing with ethanol. Twenty micrograms of total pancreatic RNA was used for hybridization with a 32P-UTP-labeled multitemplate set containing specific probes for various chemokines (Riboquant, mCK-5; BD PharMingen). Specific probes for IP-10, Mig, I-TAC, and CXCR3 were kindly provided by Iain Campbell and Valerie Asensio (The Scripps Research Institute). The RPA was conducted according to the manufacturer’s guidelines. The resulting analytical acrylamide gel was scanned using a STORM-860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and the intensity of bands corresponding to protected mRNA was quantified using ImageQuant image analysis software (Molecular Dynamics) and L32 as a reference gene.
Immunohistochemistry
Organs were harvested, immersed in Tissue-Tek OCT (Bayer, Elkhart, IN), and quick frozen on dry ice. Six- to ten-micrometer tissue sections were cut using a cryomicrotome and placed onto sialin-coated Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA). Sections were fixed with 90% EtOH at -20°C, and after washing in PBS an avidin-biotin blocking step was performed (Vector Laboratories, Burlingame, CA). Primary and biotinylated secondary Abs (Vector Laboratories) were incubated on the sections for 30 min each, and the color reaction was obtained by sequential incubation with an avidin-peroxidase conjugate (Vector Laboratories) and diaminobenzidine-hydrogen peroxide. Primary Abs were rat anti-mouse CD8a (Ly2), rat anti-mouse CD8b (Ly3), rat anti-mouse CD4 (BD PharMingen), and rat anti-mouse F4/80 (Serotec, Raleigh, NC).
Adoptive transfer of GFP-(GP33–41) T cells
GFP-Db(GP33–41) T cells were purified from the spleens of GFP x TCR(GP33–41) double transgenic mice by negative selection (StemCell Technologies, Vancouver, BC, Canada). After the enrichment procedure, it was determined that 98% of the GFP-positive cells were CD8+. A total of 104 GFP-Db(GP33–41) T cells in 100 µl of PBS were injected i.v. into naive RIP-GP mice. Two days later, the mice were infected with 5 x 103 PFU of LCMV. Anti-IP-10 mAb was administered 6 h before infection and then at days 1, 2, 4, and 6 postinfection. At day 8 postinfection, mice were sacrificed, and blood and spleen were removed for analysis of GFP-Db(GP33–41)-specific T cells by flow cytometry. After removal of the blood and spleen, mice received an intracardiac perfusion with 30 ml of 4% paraformaldehyde (in PBS). Pancreata were removed, weighed, incubated in 4% paraformaldehyde (4°C, 24 h), and then incubated for an additional 24 h (4°C) in 30% sucrose. After freezing tissues in OCT, a single 6-µm frozen section was cut from each pancreas, stained with 4',6'-diamidino-2-phenylindole (1 µg/ml, 5 min, room temperature; Sigma-Aldrich, St. Louis, MO) to visualize nuclei, and analyzed by fluorescence microscopy to obtain the total number of infiltrating GFP-Db(GP33–41) T cells. Reconstructions of pancreas sections were accomplished using an Axiovert S100 immunofluorescence microscope (Zeiss, Oberkochen, Germany) fitted with an automated xy stage, an Axiocam color digital camera, and a 5x objective. Registered two-color images (4',6'-diamidino-2-phenylindole and GFP) were captured for each field on pancreas sections, and reconstructions were performed using the MosaiX function in the K300 image analysis software (Zeiss). The total number of GFP-Db(GP33–41) T cells per pancreas was calculated based on the organ weight. In the KS300 image analysis program, reconstructions of pancreas sections were used to calculate the percentage of tissue occupied by GFP signal. This percentage was multiplied by the organ volume and then divided by the average volume of a single T cell to obtain the total number of GFP-Db(GP33–41) T cells per pancreas. As a background control, the same procedure was performed on a mouse that did not receive an adoptive transfer of GFP-Db(GP33–41) T cells. Analysis of the GFP signal in the control pancreas resulted in the detection of no cells. Statistical analyses of flow cytometry and pancreas data were performed using a Student’s t test.
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Results |
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To examine the kinetics and pattern of pancreatic chemokine expression, we isolated total RNA from pancreata of either RIP-GP or wild-type C57BL/6 mice at different times after i.p. infection with 5 x 103 PFU of LCMV-Armstrong. Analysis of 10 different chemokine mRNA species, including the three known CXCR3 chemokines, by RPA revealed a prominent and extensive expression of IP-10 after infection. IP-10 mRNA was detected as early as day 1 after infection, and quantification of the data revealed an elevation by >400-fold (Fig. 1). At day 2, IP-10 expression decreased to less than half of that amount and thereafter dropped rapidly to levels found in an uninfected animal (Fig. 1). In support of these data, expression of CXCR3 was detected as early as day 7 after LCMV infection (Fig. 1). Because CXCR3 is expressed predominantly on activated T cells, it is most likely that CXCR3 expression in the pancreas is due to the infiltration of activated T cells that occurs around days 7–10 after LCMV infection (13). Indeed, LCMV-specific CD8 T cells express high levels of CXCR3 at their cell surface. As determined by flow cytometry using a polyclonal anti-CXCR3 Ab, CXCR3 was expressed in 
60% of splenic CD8 T cells of uninfected RIP-GP mice and in 80% of all CD8 T cells at day 7 after infection of RIP-GP mice with LCMV (Fig. 2A). Among activated LCMV-specific CD8 T cells, the percentage of CXCR3high cells was further elevated to 93% (Fig. 2A). There was no significant difference in the percentage of CXCR3high CD8 T cells between transgenic RIP-GP and wild-type C57BL/6 mice (Fig. 2A).
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30-fold between days 2 and 7 after LCMV infection and decreased to baseline levels by day 21 (Fig. 1). I-TAC was expressed with a kinetics similar to that of IP-10 but was only up-regulated threefold over levels expressed in uninfected controls (Fig. 1). The levels and kinetics of IP-10, Mig, and I-TAC expression induced by an acute LCMV infection were equivalent in both C57BL/6 wild-type and RIP-GP transgenic mice. Thus, the chemokine expression observed was a direct result of the viral infection and not the presence of the transgene in the cells or the adaptive immune response to the virus that is first detected at 5 days after viral inoculation (9, 10). Other chemokines, such as RANTES (CCL5), showed a kinetic profile different from those of the CXCR3 chemokines. Expression of RANTES peaked between days 7 and 10 postinfection, a time corresponding to maximal CD8 T cell infiltration into the pancreas (Fig. 1A). Only modest increases of CCL2 (monocyte chemoattractant protein-1), CCL11 (Eotaxin), and CXCL1 (Lymphotactin) were noted (Fig. 1A), with no detectable expression of CCL1 (TCA-3), CCL4 (macrophage-inflammatory protein-1 (MIP-1)), and CXCL2 (MIP-2). Neutralization of IP-10, but not of Mig, abrogates the development of type 1 diabetes
Because both IP-10 and Mig showed an elevated expression early after LCMV infection (Fig. 1), we next addressed the role of these CXCR3 chemokines in the subsequent development of autoimmune diabetes. To this end, we injected mAbs to either IP-10 or Mig into RIP-GP mice that were infected with 5 x 103 PFU of LCMV. These Abs neutralize IP-10 or Mig, respectively, and were used at concentrations exceeding their neutralizing capacity (see Materials and Methods) (21). Ab to either IP-10 or Mig (100 µg of each) was administered i.p. 6 h before LCMV infection and then at days 1, 2, 4, and 6 postinfection. The incidence of diabetes (BG >300 mg/dl) was significantly reduced to 31% in RIP-GP mice that received anti-IP-10 mAb compared with control animals, which were injected with an unspecific isotype-matched hamster Ab or with PBS (incidence of 100%) (Fig. 2B). In contrast with IP-10 neutralization, no significant reduction in diabetes incidence or onset could be detected for animals that were treated with the anti-Mig mAb (Fig. 2B). In addition, when anti-IP-10 mAb and anti-Mig mAb were coinjected, the incidence of diabetes was equivalent to that observed after administration of anti-IP-10 mAb alone (Fig. 2B). The data from these studies indicate that IP-10, but not Mig, is an essential component in the initiation of the process that results in immune-mediated disease. Blockade of RANTES at the time of its highest expression by administration of 100 µg of a neutralizing anti-RANTES Ab at days 4, 7, and 10 postinfection had no effect on the incidence of diabetes (data not shown).
Neutralization of IP-10 results in decreased cellular infiltration into islets and preserved insulin production
To determine the mechanism(s) by which IP-10 neutralization protects against T cell-mediated immunopathology, we first analyzed the degree of cellular infiltration into the islets of Langerhans. Upon receiving Ab to IP-10, RIP-GP mice were sacrificed at 5, 10, and 23 days postinfection. Pancreata were collected and stained by immunohistochemistry for insulin content using an Ab to insulin B chain. At day 5 after LCMV infection, islets from both treated and untreated mice appeared normal, showing no lymphocyte infiltration and robust insulin staining (Fig. 3). By day 10 postinfection, significant differences between these two groups became apparent, as the islets from mice receiving Ab to IP-10 were still morphologically normal, displayed minimal lymphoid cell infiltrates, and retained the ability to produce insulin (Fig. 3). In contrast, age- and sex-matched littermates that did not receive anti-IP-10 therapy showed substantial lymphocyte infiltration and impaired cell function, as evidenced by both a marked reduction in islet insulin staining and blood glucose concentrations >300 mg/dl (Fig. 3A). The cells infiltrating the islets were predominantly of the CD8 (Fig. 3B) and CD4 (data not shown) phenotypes, whereas macrophages showed primarily a peri-insular localization (data not shown). By day 23 postinfection, the islets of mice not receiving Ab to IP-10 were destroyed, leaving islet scars consisting mainly of clumped infiltrating cells (CD8/CD4 T cells and macrophages). Negligible to minimal cells were found that contained insulin (Fig. 3, A and B). At this same time point, mice treated with Ab to IP-10 showed a marked decrease in CD8/CD4 T cell and macrophage infiltrates as well as morphologically viable islets with insulin expression in the cells. These findings, concurrent with normal BG levels, indicated that the islets remained functional in the regulation of BG (Fig. 3, A and B).
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Previous studies using RIP-GP transgenic mice either genetically or Ab depleted of CD4 and CD8 T cells showed that virus-induced diabetes depends primarily on the expansion and migration of LCMV-specific CD8 T cells into the islets of Langerhans followed by their interactions with cells expressing the LCMV-GP transgene (10, 23). In C57BL/6 mice (H-2b background), one of the immunodominant CD8 T cell epitopes residing in the LCMV-GP was identified as amino acids 33–41 (24, 25, 26). Furthermore, the GP33–41 peptide can be administrated to tolerize and prevent immune-mediated diabetes after LCMV infection (27, 28).
Because CD8 T cells specific for GP33–41 are required to cause diabetes in H-2b RIP-GP transgenic mice, we next asked whether IP-10 neutralization interfered with the expansion of Ag-specific CD8 T cells and/or their migration into the pancreas. Two complementary approaches were used to visualize and enumerate H-2Db restricted GP33–41-specific CD8 T cells in target tissues infected by LCMV (17). First, using in situ MHC class I tetramer staining (17, 29, 30), it was determined that the frequency of GP33–41-specific CD8 T cells in the islets of RIP-GP mice during the effector phase of the disease (days 7 and 10) was surprisingly only 1% of the total infiltrating CD8 T cell population (D. B. McGavern, U. Christen, M. G. von Herrath, and M. B. A. Oldstone, manuscript in preparation). The second approach involved adoptive transfer of Db(GP33–41)-specific TCR transgenic cells genetically tagged with GFP (17). This technique has an advantage over the in situ tetramer staining, because it allows one to increase the frequency of Ag-specific T cells in the islets to >1%, thus facilitating quantification and comparisons between experimental and control groups. In addition, the number of specific CD8 T cells that have migrated into the pancreas can be easily visualized and quantified because of the GFP tag (see Materials and Methods for details) (17). A total of 104 GFP-Db(GP33–41)-specific T cells were adoptively transferred i.v. into naive RIP-GP mice, which were infected 2 days later with 5 x 103 PFU of LCMV. Ab to IP-10 was administered as described above. At day 8 after infection, we first harvested blood and spleen for the assessment of GFP-Db(GP33–41)-specific T cell frequencies and second performed a cardiac perfusion to analyze the number of infiltrating GFP-Db(GP33–41)-specific T cells in the pancreas of the same individual animal. We found that neutralization of IP-10 significantly reduced the numbers of GFP-Db(GP33–41)-specific T cells in the spleen and blood at day 8 after infection. In untreated RIP-GP mice, GFP-Db(GP33–41)-specific T cells expanded to account for 51% and 49% of all CD8 T cells in the blood and spleen, respectively. In contrast, there was a reduction of GFP-Db(GP33–41)-specific CD8 T cells by 31% in the blood and 33% in the spleen of mice treated with Ab to IP-10 (Fig. 4A) (p < 0.05). Next, the total number of GFP-Db(GP33–41)-specific CD8 T cells in the pancreas were enumerated using fluorescence microscopy and an image analysis program (see Materials and Methods for details). At day 8 postinfection, GFP-Db(GP33–41)-specific CD8 T cells were found in the islets as well as the exocrine pancreas of both Ab to IP-10 treated and untreated mice (Fig. 4, C and D). However, the RIP-GP mice that received Ab to IP-10 displayed a statistically significant reduction (p < 0.05) in the total number of GP33–41-specific CD8 T cells in the pancreas. Untreated RIP-GP mice had an average of 2.1 x 106 GP33–41-specific CD8 T cells in their pancreas, whereas only 1.2 x 106 GP33–41-specific CD8 T cells were found in the pancreata of mice treated with Ab to IP-10 (Fig. 4B). Interestingly, the reduction in migration of Ag-specific CD8 T cells into the pancreas (45% reduction) exceeded the reduction observed in the blood (31% reduction) and the spleen (33% reduction) (Fig. 4B).
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The last series of experiments compared the ability of viruses that were tropic for the pancreas and cause diabetes with a virus that is not tropic for the pancreas and does not induce diabetes for early pancreatic expression of IP-10. At day 1 after infection, high levels of IP-10 could be detected in mice infected with the pancreas tropic viruses: LCMV strains Armstrong and Pasteur as well as EMC-B (Fig. 5). In addition, infection with a sublethal dose of the pancreas tropic CVB showed a massive expression of IP-10 at day 4 postinfection (Fig. 5). In contrast, infection with TV, which is not tropic for the pancreas, did not result in the expression of significant pancreatic IP-10 levels at days 1 or 4 after infection (Fig. 5). These data indicate that enhanced IP-10 expression in the pancreas is a general phenomenon for viruses associated with causing diabetes. Furthermore, the data suggest that virus tropism for the pancreas and alteration of that target tissue’s local milieu by induction of selected chemokines, such as IP-10, may be an initial step that imprints a pattern for the subsequent development of tissue/organ-specific autoimmune diseases caused by viruses.
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Discussion |
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Previous studies reported CXCR3 chemokine expression after infection of the CNS with viruses such as LCMV (15), mouse hepatitis virus (14, 31), and TV (16). In contrast with the findings we report here, that CXCR3 chemokines are maximally expressed during the first few days after infection of the pancreas with LCMV and precede expression of other chemokines, these same chemokines were expressed later (days 3–11) in the CNS and were made in concert with several other chemokines (e.g., monocyte chemoattractant protein-1, RANTES, MIP-1, and MIP-1) (14, 15, 16). Because of the unique and elevated pancreatic expression of CXCR3 chemokines (IP-10 and Mig) early after LCMV infection and the high expression of CXCR3 on the surface of LCMV-specific CD8 T cells, we hypothesized that neutralization of IP-10 and/or Mig would limit the development of an LCMV-specific CD8 T cell response that ultimately causes autoimmune diabetes. Indeed, injection of a neutralizing anti-IP-10 mAb during the time of maximal IP-10 expression significantly reduced the anti-LCMV specific CD8 T cell response as well as the incidence of type 1 diabetes in RIP-GP mice. In addition, a unique and nonredundant role for IP-10 was revealed, because it was not possible to abrogate diabetes by treatment with a neutralizing anti-Mig mAb. Moreover, coadministration of anti-Mig and anti-IP-10 mAbs did not decrease further the incidence of diabetes observed with anti-IP-10 mAb treatment alone. These data indicate distinct roles for IP-10 and Mig in the initiation of a virus-induced immune-mediated diabetes.
Although both IP-10 and Mig were expressed at an early stage after LCMV infection, expression of Mig followed a different kinetic pattern than did IP-10 expression. Peak expression of Mig occurred later, at a time concurrent with the expression of other chemokines (RANTES; see Fig. 1) and proinflammatory cytokines (TNF- and IFN-) (11, 12). Hence, it appears that the timing of immunotherapy will be critical for the successful prevention of immune-mediated disease, and possibly in contrast with early IP-10 neutralization, blockade of Mig might inhibit just one of many factors that influence the local inflammatory profile at a later time point. Importantly, despite the multiplicity of chemokine and cytokine release during inflammation and the likely redundancy in the system, we show here that neutralization of just one chemokine, IP-10, is able to abort an immune-mediated disease.
The dominant role of CXCR3 chemokines after microbial infection is not unexpected, because CXCR3 is expressed predominantly on activated T cells (32, 33), which function to eliminate virus-infected cells and to control viral infection. Furthermore, there is considerable evidence that CXCR3 chemokines are important mediators of Th1/Tc1-type inflammation processes. For example, neutralization of IP-10 inhibited the massive migration of T cells into the liver and spleen that normally follows infection of mice with Toxoplasma gondii (21). Impaired Ag-specific T cell effector function was also observed, indicating that IP-10 plays a central role in localization and function of effector T cells at sites of Th1/Tc1 inflammation (21). Similar results have been obtained using IP-10-deficient (IP-10-/-) mice (34). After infection with a neurotropic mouse hepatitis virus, IP-10-/- mice had a decreased recruitment of CD4 and CD8 T cells to the brain and reduced numbers of virus-specific IFN--secreting CD8 T cells in the spleen. In addition, it has been demonstrated that IP-10 is a critical factor in the pathogenesis of experimental autoimmune encephalomyelitis (35, 36, 37), mouse hepatitis virus-induced encephalomyelitis (38), and adjuvant arthritis (39). Recently, it was also reported that CXCR3 is required for the infiltration of the islets of Langerhans and the subsequent development of diabetes in RIP-LCMV mice (40). It was shown that the onset of diabetes was delayed in CXCR3-deficient mice and that cells may be responsible for the CXCR3-mediated infiltration of autoaggressive lymphocytes. Our studies extend and clarify this observation by showing that neutralization of just one particular CXCR3 chemokine, namely IP-10, is responsible for abrogation of type 1 diabetes in RIP-LCMV mice. Furthermore, we revealed that the mechanism by which this occurs is through a reduction in the expansion of Ag-specific CD8 T cells in the spleen and their subsequent migration into the pancreas. Thus, neutralization of one component in the innate immune response can impair the programming of the Ag-specific adaptive immune response and therefore can prevent a virus infection from causing an autoimmune disease.
Recent interest has refocused on the hypothesis that distressed or injured cells release alarm signals that in turn activate the resting immune system (41). Our results support this hypothesis and indicate that IP-10 is an early alarm signal for LCMV infection in the pancreas. Others reported that CVB infection of the pancreas initiated and potentiated diabetes in the BDC2.5 transgenic nonobese diabetic mouse by bystander damage as a result of local inflammation (42, 43). However, the precise factors contributing to the acceleration of diabetes were not determined. As an extension of these findings, we found a massive elevation of IP-10 expression in the pancreas of C57BL/6 mice 4 days after infection with CVB. Furthermore, secondary infection of RIP-LCMV-NP mice (10) with CVB 1 mo after an initial infection with LCMV significantly accelerated and enhanced the course of diabetes. However, CVB alone was unable to initiate type 1 diabetes in the RIP-LCMV model and CVB does not contain an epitope that mimics an LCMV T cell epitope. These results suggest, like the report by Horwitz et al. (42), that once autoaggressive (anti-LCMV) T cells are formed after the primary LCMV infection, a later infection by an unrelated virus with tropism for the pancreas results in the release of additional alarm signals, such as IP-10. As shown here, IP-10 can then contribute to the expansion of autoaggressive T cells and enhance their migration into the islets. Blocking IP-10 is associated with a reduction in the number of Ag-specific T cells required to cause diabetes. Hence, our findings with IP-10 noted in the RIP-LCMV mouse may be relevant for understanding the pathogenesis of human type 1 diabetes and perhaps other autoimmune diseases.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Urs Christen, Department of Developmental Immunology, DI-3, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address: christen{at}liai.org
3 Abbreviations used in this paper: Tc, cytotoxic T lymphocyte; LCMV, lymphocytic choriomeningitis virus; GP, glycoprotein; RIP, rat insulin promotor; IP-10, IFN--inducible protein of 10 kDa; Mig, monokine induced by IFN-; I-TAC, IFN-inducible T cell- chemoattractant; GFP, green fluorescent protein; CVB, Coxsackievirus B4; EMC-B, encephalomyocarditis virus variant B; TV, Theiler’s murine encephalomyelitis virus; RPA, RNase protection assay; MIP, macrophage-inflammatory protein.
Received for publication June 20, 2003. Accepted for publication October 7, 2003.
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References |
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J. E. Christensen, S. Simonsen, C. Fenger, M. R. Sorensen, T. Moos, J. P. Christensen, B. Finsen, and A. R. Thomsen Fulminant Lymphocytic Choriomeningitis Virus-Induced Inflammation of the CNS Involves a Cytokine-Chemokine-Cytokine-Chemokine Cascade J. Immunol., January 15, 2009; 182(2): 1079 - 1087. [Abstract] [Full Text] [PDF]
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 J. Menke, G. C. Zeller, E. Kikawada, T. K. Means, X. R. Huang, H. Y. Lan, B. Lu, J. Farber, A. D. Luster, and V. R. Kelley CXCL9, but not CXCL10, Promotes CXCR3-Dependent Immune-Mediated Kidney Disease J. Am. Soc. Nephrol., June 1, 2008; 19(6): 1177 - 1189. [Full Text] [PDF]
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J. J. Hooks, C. N. Nagineni, L. C. Hooper, K. Hayashi, and B. Detrick IFN-{beta} Provides Immuno-Protection in the Retina by Inhibiting ICAM-1 and CXCL9 in Retinal Pigment Epithelial Cells J. Immunol., March 15, 2008; 180(6): 3789 - 3796. [Abstract] [Full Text] [PDF]
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S. B. J. Wong, R. Bos, and L. A. Sherman Tumor-Specific CD4+ T Cells Render the Tumor Environment Permissive for Infiltration by Low-Avidity CD8+ T Cells J. Immunol., March 1, 2008; 180(5): 3122 - 3131. [Abstract] [Full Text] [PDF]
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A. P. Martin, M. G. Grisotto, C. Canasto-Chibuque, S. L. Kunkel, J. S. Bromberg, G. C. Furtado, and S. A. Lira Islet Expression of M3 Uncovers a Key Role for Chemokines in the Development and Recruitment of Diabetogenic Cells in NOD Mice Diabetes, February 1, 2008; 57(2): 387 - 394. [Abstract] [Full Text] [PDF]
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 E. M. Erin, G. R. Jenkins, O. M. Kon, A. S. Zacharasiewicz, G. C. Nicholson, H. Neighbour, R. C. Tennant, A. J. Tan, B. R. Leaker, A. Bush, et al. Optimized Dialysis and Protease Inhibition of Sputum Dithiothreitol Supernatants Am. J. Respir. Crit. Care Med., January 15, 2008; 177(2): 132 - 141. [Abstract] [Full Text] [PDF]
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K. A. Smith, S. Efstathiou, and A. Cooke Murine Gammaherpesvirus-68 Infection Alters Self-Antigen Presentation and Type 1 Diabetes Onset in NOD Mice J. Immunol., December 1, 2007; 179(11): 7325 - 7333. [Abstract] [Full Text] [PDF]
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 M. Rotondi, L. Chiovato, S. Romagnani, M. Serio, and P. Romagnani Role of Chemokines in Endocrine Autoimmune Diseases Endocr. Rev., August 1, 2007; 28(5): 492 - 520. [Abstract] [Full Text] [PDF]
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M. M. Martinic, A. E. Juedes, D. Bresson, D. Homann, K. Skak, C. Huber, E. Ling, M. Ejrnaes, T. Wolfe, L. Togher, et al. Minimal Impact of a De Novo-Expressed {beta}-Cell Autoantigen on Spontaneous Diabetes Development in NOD Mice Diabetes, April 1, 2007; 56(4): 1059 - 1068. [Abstract] [Full Text] [PDF]
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A. P. Martin, J. M. Alexander-Brett, C. Canasto-Chibuque, A. Garin, J. S. Bromberg, D. H. Fremont, and S. A. Lira The Chemokine Binding Protein M3 Prevents Diabetes Induced by Multiple Low Doses of Streptozotocin J. Immunol., April 1, 2007; 178(7): 4623 - 4631. [Abstract] [Full Text] [PDF]
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 M. D. Wareing, A. Lyon, C. Inglis, F. Giannoni, I. Charo, and S. R. Sarawar Chemokine regulation of the inflammatory response to a low-dose influenza infection in CCR2-/- mice J. Leukoc. Biol., March 1, 2007; 81(3): 793 - 801. [Abstract] [Full Text] [PDF]
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L. N. Stiles, J. L. Hardison, C. S. Schaumburg, L. M. Whitman, and T. E. Lane T Cell Antiviral Effector Function Is Not Dependent on CXCL10 Following Murine Coronavirus Infection J. Immunol., December 15, 2006; 177(12): 8372 - 8380. [Abstract] [Full Text] [PDF]
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G. S. V. Campanella, J. Grimm, L. A. Manice, R. A. Colvin, B. D. Medoff, G. R. Wojtkiewicz, R. Weissleder, and A. D. Luster Oligomerization of CXCL10 Is Necessary for Endothelial Cell Presentation and In Vivo Activity J. Immunol., November 15, 2006; 177(10): 6991 - 6998. [Abstract] [Full Text] [PDF]
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T. Raine, P. Zaccone, P. Mastroeni, and A. Cooke Salmonella typhimurium Infection in Nonobese Diabetic Mice Generates Immunomodulatory Dendritic Cells Able to Prevent Type 1 Diabetes J. Immunol., August 15, 2006; 177(4): 2224 - 2233. [Abstract] [Full Text] [PDF]
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 R. A. Colvin, G. S. V. Campanella, L. A. Manice, and A. D. Luster CXCR3 Requires Tyrosine Sulfation for Ligand Binding and a Second Extracellular Loop Arginine Residue for Ligand-Induced Chemotaxis Mol. Cell. Biol., August 1, 2006; 26(15): 5838 - 5849. [Abstract] [Full Text] [PDF]
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T. Shigihara, A. Shimada, Y. Oikawa, H. Yoneyama, Y. Kanazawa, Y. Okubo, K. Matsushima, E. Yamato, J.-i. Miyazaki, A. Kasuga, et al. CXCL10 DNA Vaccination Prevents Spontaneous Diabetes through Enhanced {beta} Cell Proliferation in NOD Mice J. Immunol., December 15, 2005; 175(12): 8401 - 8408. [Abstract] [Full Text] [PDF]
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A. Rhode, M. E. Pauza, A. M. Barral, E. Rodrigo, M. B. A. Oldstone, M. G. von Herrath, and U. Christen Islet-Specific Expression of CXCL10 Causes Spontaneous Islet Infiltration and Accelerates Diabetes Development J. Immunol., September 15, 2005; 175(6): 3516 - 3524. [Abstract] [Full Text] [PDF]
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K. Chen, Y. Wei, A. Alter, G. C. Sharp, and H. Braley-Mullen Chemokine expression during development of fibrosis versus resolution in a murine model of granulomatous experimental autoimmune thyroiditis J. Leukoc. Biol., September 1, 2005; 78(3): 716 - 724. [Abstract] [Full Text] [PDF]
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 C. A. Gysemans, A. K. Cardozo, H. Callewaert, A. Giulietti, L. Hulshagen, R. Bouillon, D. L. Eizirik, and C. Mathieu 1,25-Dihydroxyvitamin D3 Modulates Expression of Chemokines and Cytokines in Pancreatic Islets: Implications for Prevention of Diabetes in Nonobese Diabetic Mice Endocrinology, April 1, 2005; 146(4): 1956 - 1964. [Abstract] [Full Text] [PDF]
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M. Ejrnaes, N. Videbaek, U. Christen, A. Cooke, B. K. Michelsen, and M. von Herrath Different Diabetogenic Potential of Autoaggressive CD8+ Clones Associated with IFN-{gamma}-Inducible Protein 10 (CXC Chemokine Ligand 10) Production but Not Cytokine Expression, Cytolytic Activity, or Homing Characteristics J. Immunol., March 1, 2005; 174(5): 2746 - 2755. [Abstract] [Full Text] [PDF]
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J. Morimoto, H. Yoneyama, A. Shimada, T. Shigihara, S. Yamada, Y. Oikawa, K. Matsushima, T. Saruta, and S. Narumi CXC Chemokine Ligand 10 Neutralization Suppresses the Occurrence of Diabetes in Nonobese Diabetic Mice through Enhanced {beta} Cell Proliferation without Affecting Insulitis J. Immunol., December 1, 2004; 173(11): 7017 - 7024. [Abstract] [Full Text] [PDF]
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N. Sarween, A. Chodos, C. Raykundalia, M. Khan, A. K. Abbas, and L. S. K. Walker CD4+CD25+ Cells Controlling a Pathogenic CD4 Response Inhibit Cytokine Differentiation, CXCR-3 Expression, and Tissue Invasion J. Immunol., September 1, 2004; 173(5): 2942 - 2951. [Abstract] [Full Text] [PDF]
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