Leucine-Rich Repeat Kinase 2 (Lrrk2) Deficiency Diminishes the Development of Experimental Autoimmune Uveitis (EAU) and the Adaptive Immune Response.

BACKGROUND: Mutations in LRRK2 are related to certain forms of Parkinson's disease and, possibly, to the pathogenesis of Crohn's disease. In both these diseases inflammatory processes participate in the pathogenic process. LRRK2 is expressed in lymphoid cells and, interestingly, Lrrk2 (-/-) mice were reported to develop more severe experimental colitis than their wild type (WT) controls. Here, we examined the possible involvement of LRRK2 in the pathogenesis of experimental autoimmune uveitis (EAU), an animal model for human uveitis, by testing Lrrk2 (-/-) mice for their capacity to develop this experimental eye disease and related immune responses. METHODS: Lrrk2 (-/-) mice and their WT controls (C57Bl/6) were immunized with interphotoreceptor retinoid-binding protein (IRBP) and compared for their development of EAU, delayed type hypersensitivity (DTH) by skin tests, production of cytokines in culture, and expression of interferon (IFN)-γ, interleukin (IL)-17 and FoxP3 by spleen cells, using flow cytometry. Peritoneal macrophages were examined for their production of cytokines/chemokines in culture following stimulation with LPS or the oligodeoxynucleotide CpG. The Lrrk2 (-/-) and WT mice were also compared for their response to bovine serum albumin (BSA). RESULTS: The Lrrk2 (-/-) mice developed lower levels of EAU, DTH responses and cytokine production by lymphocytes than did their WT controls. Intracellular expression of IFN-γ and IL-17, by spleen cells, and secretion of cytokines/chemokines by activated peritoneal macrophages of Lrrk2 (-/-) mice trended toward diminished levels, although variabilities were noted. The expression levels of FoxP3 by Lrrk2 (-/-) spleen cells, however, were similar to those seen in WT controls. Consistent with their low response to IRBP, Lrrk2 (-/-) mice responded to BSA less vigorously than their WT controls. CONCLUSIONS: Lrrk2 deficiency in mice diminished the development of EAU and the related adaptive immune responses to IRBP as compared to the WT controls.

Glucocorticoid-resistant Th17 cells are selectively attenuated by cyclosporine A.

Glucocorticoids remain the cornerstone of treatment for inflammatory conditions, but their utility is limited by a plethora of side effects. One of the key goals of immunotherapy across medical disciplines is to minimize patients' glucocorticoid use. Increasing evidence suggests that variations in the adaptive immune response play a critical role in defining the dose of glucocorticoids required to control an individual's disease, and Th17 cells are strong candidate drivers for nonresponsiveness [also called steroid resistance (SR)]. Here we use gene-expression profiling to further characterize the SR phenotype in T cells and show that Th17 cells generated from both SR and steroid-sensitive individuals exhibit restricted genome-wide responses to glucocorticoids in vitro, and that this is independent of glucocorticoid receptor translocation or isoform expression. In addition, we demonstrate, both in transgenic murine T cells in vitro and in an in vivo murine model of autoimmunity, that Th17 cells are reciprocally sensitive to suppression with the calcineurin inhibitor, cyclosporine A. This result was replicated in human Th17 cells in vitro, which were found to have a conversely large genome-wide shift in response to cyclosporine A. These observations suggest that the clinical efficacy of cyclosporine A in the treatment of SR diseases may be because of its selective attenuation of Th17 cells, and also that novel therapeutics, which target either Th17 cells themselves or the effector memory T-helper cell population from which they are derived, would be strong candidates for drug development in the context of SR inflammation.

Induced regulatory T-cells (iTregs) generated by activation with anti-CD3/CD28 antibodies differ from those generated by the physiological-like activation with antigen/APC.

Regulatory T-cells (Tregs) are responsible for homeostasis of the immune system, as well as for inhibition of pathogenic autoimmune processes. Induced-(i)-Tregs, can be generated in vitro by activation of CD4 cells in the presence of TGF-ß. A commonly used activation mechanism is by antibodies against CD3 and CD28. The physiological-like activation of T-cells, however, is with the specific target antigen presented by antigen-presenting cells (APC). The two modes of activation have been considered to yield the same populations of iTregs. Here, we compared between iTreg populations generated by either one of the two methods and found differences between their capacities to inhibit T-lymphocyte proliferative response, their expression of cell surface antigens and particularly, in their transcript expression profiles of certain chemokines and chemokine receptors. Our data thus indicate that iTregs generated by activation with anti-CD3/CD28 antibodies cannot be considered identical to iTregs generated by antigen/APC.

Phenotypes of Th lineages generated by the commonly used activation with anti-CD3/CD28 antibodies differ from those generated by the physiological activation with the specific antigen.

T-helper (Th) lineages have been generated in vitro by activating CD4 cells with anti-CD3/CD28 antibodies during polarization. Physiologically, however, the generation of Th lineages is by activation with the specific antigen presented by antigen-presenting cells (APC). Here, we used T-cell receptor (TCR)-transgenic mice to compare the phenotypes of Th1, Th9 and Th17 lineages when generated by either one of the two activation modes. Lineage Th cells specific against hen egg lysozyme (HEL), were adoptively transferred into recipient mice transgenically expressing HEL in their lens. Remarkable differences were found between lineages of Th1, Th9 or Th17, generated by either one of the two modes in their capacities to migrate to and proliferate in the recipient spleen and, importantly, to induce inflammation in the recipient mouse eyes. Substantial differences were also observed between the lineage pairs in their transcript expression profiles of certain chemokines and chemokine receptors. Surprisingly, however, close similarities were observed between the transcript expression profiles of lineages of the three phenotypes, activated by the same mode. Furthermore, Th cell lineages generated by the two activation modes differed considerably in their pattern of gene expression, as monitored by microarray analysis, but exhibited commonality with lineages of other phenotypes generated by the same activation mode. This study thus shows that (i) Th lineages generated by activation with anti-CD3/CD28 antibodies differ from lineages generated by antigen/APC; and (ii) the mode of activation determines to a large extent the expression profile of major transcripts.

ITE, a novel endogenous nontoxic aryl hydrocarbon receptor ligand, efficiently suppresses EAU and T-cell-mediated immunity.

PURPOSE: Ligands for aryl hydrocarbon receptor (AHR), such as dioxins, are highly toxic. One such ligand, TCDD, was found to exert potent immunosuppressive capacities in mice developing pathogenic autoimmune processes, including EAU, but its toxicity makes it unusable for humans. A recently identified endogenous AHR ligand, ITE, is also immunosuppressive, but is nontoxic and could therefore be useful for therapy in humans. Here, we tested ITE for its capacity to inhibit EAU and related immune responses. METHODS: EAU was induced in B10.A mice by immunization with interphotoreceptor retinoid-binding protein (IRBP; 40 µg) in CFA. Treatment with ITE was by daily intraperitoneal injection of 0.2 mg. Disease severity was assessed by both fundoscopy and histological examination. Draining lymph node cells were tested for proliferation by thymidine uptake and for cytokine production and release by ELISA. In addition, the intracellular expression of cytokines and Foxp3 was determined by flow cytometry. Serum antibodies were measured by ELISA. RESULTS: Treatment with ITE efficiently inhibited the development of EAU in mice, as well as the cellular immune responses against IRBP and PPD. ITE treatment inhibited the expansion of both Th1 and Th17 subpopulations, as well as their release of the signature cytokines, IFN-gamma and IL-17. The treatment moderately increased, however, the proportion of Foxp3 expressing T-regulatory cells. Antibody production was not affected by the treatment. CONCLUSIONS: ITE, an endogenous AHR ligand, efficiently inhibits EAU development and related cellular immune responses. Being nontoxic, ITE may be considered for treatment of pathogenic immunity in humans.

Environmental factors determine DAP12 deficiency to either enhance or suppress immunopathogenic processes.

DNAX-activation protein 12 (DAP12), a transmembrane adapter, plays a major role in transducing activation signals in natural killer cells and various myeloid cells. Quantitative RT-PCR detected in normal mouse eyes considerable levels of DAP12 and multiple DAP12-coupled receptors, in particular TREM-1, Clec5a and SIRPb1. The role of DAP12 and its receptors in experimental autoimmune diseases has been controversial. Here, we analysed the effect of DAP12 deficiency on the capacity of mice to mount immunopathogenic cellular responses to the uveitogenic ocular antigen and interphotoreceptor retinoid-binding protein (IRBP), and to develop experimental autoimmune uveitis (EAU). Surprisingly, sequential analysis of EAU in mice deficient in DAP12 in two different animal facilities at first revealed enhanced disease as compared with wild-type mice, but when these mice were re-derived into a second, cleaner, animal facility, the response of control mice was essentially unchanged, whereas the DAP12 null mice were markedly hyporesponsive relative to controls in the new facility. Accordingly, when stimulated in vitro with IRBP, lymphocytes from the DAP12-deficient mice housed in the two facilities proliferated and produced opposite profiles of pro-inflammatory and anti-inflammatory cytokines, compared with their controls. These findings therefore demonstrate that the effects of DAP12 deficiency on development of autoimmune disease are dramatically affected by environmental factors.

Differential involvement of Th1 and Th17 in pathogenic autoimmune processes triggered by different TLR ligands.

The interaction between TLRs and their cognate ligands triggers both the innate and adaptive immune systems, and thus can play a pivotal role in the defense against pathogen invasion. This work investigates the differentiation of naive CD4 cells into Th1 or Th17 phenotypes in mice treated with different TLR ligands. We use a model system in which naive transgenic cells specific to hen egg lysozyme are adoptively transferred into recipients that express hen egg lysozyme in the lens of the eye. The transferred naive T cells induce ocular inflammation only in recipients treated with TLR ligands. Treatment with LPS preferentially stimulated IL-17 production, whereas CpG oligodeoxynucleotide and polyinosinic:polycytidylic acid primarily stimulated Th1 cells. Peptidoglycan stimulated the two Th subpopulations equally. The preferential induction of Th1 or Th17 by the four ligands was detected in the spleen (where a major portion of the adoptively transferred cells homed) and in the eyes, where activated Th cells initiate inflammation. Analysis of the cytokines present in recipient mice suggests that Th1 induction is elicited by IL-12 and/or IFN-α, whereas Th17 generation is preferentially mediated by IL-6. Importantly, we show in this article that treatment with LPS selectively promoted in the recipient mice the generation of IL-6-producing activated B cells. An inverse correlation was found between the level of regulatory T cells and severity of inflammation induced by the donor cells. Taken together, our data show that specific TLR ligands differentially activate the immune system as evidenced by the generation of distinct Th phenotypes from naive CD4 cells.

SLAT/Def6 plays a critical role in the pathogenic process of experimental autoimmune uveitis (EAU).

PURPOSE: SWAP 70-like adaptor of T cells (SLAT; aka Def6) is a recently discovered guanine nucleotide exchange factor for Rho guanosine triphosphate (GTP)ases that has been previously shown to play a role in cluster of differentiation(CD)4+ T cell activation, T-helper (Th)1/Th2/Th17 differentiation and development of experimental autoimmune encephalomyelitis. Here, we investigated the role of SLAT/Def6 in the development of experimental autoimmune uveitis (EAU), an animal model for several uveitic conditions in humans. METHODS: SLAT/Def6 deficient ("KO") mice and C57BL/6 controls were immunized with interphotoreceptor retinoid-binding protein (IRBP), along with pertussis toxin. The development of ocular inflammation was determined by both fundoscopy and histological examination. Lymphoid cells from draining lymph nodes were cultured with IRBP to measure lymphocyte proliferation and release of cytokines. Purified dendritic cells were tested for their capacity to present antigen to responding lymphocytes. In addition, the lymphoid cells were tested for the expression of forkhead box P3 (FoxP3), using conventional methods, and the activity of T-regulatory cells was determined by their capacity to inhibit in vitro proliferative responses. Serum anti -IRBP antibody levels were measured by enzyme-linked immunosorbant assay (ELISA). quantitative polymerase chain reaction (qPCR) was used to determine the transcript levels of cytokines in inflamed eyes. RESULTS: SLAT/Def6 KO mice had significantly reduced EAU compared to controls. Cells isolated from draining lymph nodes of SLAT/Def6 KO mice exhibited impaired proliferation and production of Th1 and Th17 signature cytokines (interferon [IFN]-γ and interleukin [IL]-17, respectively) when compared with cells isolated from control mice. qPCR of inflamed eyes detected similar levels of IFN-γ transcript in control and SLAT/Def6 KO mice, whereas the IL-17 transcript levels in eyes of the SLAT/Def6 KO mice were lower than in eyes of the controls. The SLAT/Def6 KO mice resembled their wild type (WT) controls, however, in the levels of their serum antibody against IRBP, the antigen presenting capacity of their dendritic cells, the proportion of cells expressing Foxp3 and the immunosuppressive activity of their T-regulatory cells. CONCLUSIONS: SLAT/Def6 KO mice exhibit reduced capacity to develop ocular inflammation and cellular activity when immunized with IRBP. Our study provides new data showing that SLAT/Def6 plays a major role in the T cell-mediated autoimmune processes that bring about the inflammatory eye disease, EAU.

Cell-cell interaction with APC, not IL-23, is required for naive CD4 cells to acquire pathogenicity during Th17 lineage commitment.

Subpopulations of pathogenic or nonpathogenic Th17 cells were reported to develop when presensitized CD4 cells were activated with their target Ag during polarization by either IL-23 or IL-6 and TGF-ß, respectively. In this study, we generated two Th17 subpopulations by using a system in which naive CD4 cells from TCR transgenic mice specific to hen egg lysozyme (HEL) are polarized with IL-6/TGF-ß and, concurrently, are activated either with HEL presented by APCs, or with anti-CD3/CD28 Abs. Only the former cells were pathogenic, inducing inflammation in eyes expressing HEL. Naive CD4 cells activated by the anti-CD3/CD28 Abs acquired pathogenicity, however, when cocultured with HEL/APC. Importantly, the naive CD4 cells did not acquire pathogenicity when cocultured with APCs stimulated with LPS or when separated from the HEL-presenting cells by a semipermeable membrane. Unlike with presensitized Th17, soluble IL-23 does not participate in pathogenicity acquisition by naive CD4 cells; no pathogenicity was induced by adding IL-23 to cultures activated with anti-CD3/CD28 Abs. Furthermore, Abs against IL-23 or IL-23R did not inhibit acquisition of pathogenicity in cultures of naive CD4 cells activated by HEL/APC. Our data thus show that, unlike presensitized CD4 cells, naive CD4 cells polarized toward Th17 phenotype acquire pathogenicity only by direct interaction with APCs presenting the Ag, with no apparent involvement of soluble IL-23. We suggest that the Th17 lymphocytes derived from naive CD4 cells participate in pathogenic and other immune processes, along with the IL-23-dependent Th17 cells.

Inflammation-inducing Th1 and Th17 cells differ in their expression patterns of apoptosis-related molecules.

Th1 cells are remarkably more susceptible to activation induced cell death than Th17. Here, we compared cultures of these two cell subpopulations for their expression of apoptosis-related molecules when re-exposed to their specific antigen. We also compared the expression of apoptosis-related molecules in the mouse eye with inflammation induced by Th1 or Th17 cells. Using qPCR we found that the mRNA transcript levels of the majority of tested apoptosis-related molecules were higher in the Th1 cultures, and in eyes with Th1-induced inflammation. Apoptotic intrinsic pathway molecules played minor roles in the processes in vitro or in vivo, whereas extrinsic pathway molecules, as well as PD-1, its ligands and Tim3, were heavily involved.

Antigen-specific Th9 cells exhibit uniqueness in their kinetics of cytokine production and short retention at the inflammatory site.

Recently reported lines of Th9 cells, producing IL-9 and IL-10, were generated by polarization with IL-4 and TGF-ß and activation with Abs against CD3 and CD28. In this paper, we analyzed features of Th9 lines similarly polarized but activated by the "natural mode" (i.e., exposure of CD4 cells to their target Ag, hen egg lysozyme [HEL] and APCs). Main observations are the following: 1) both IL-9 and IL-10 were expressed by the line cells, but with strikingly different kinetics, with IL-9 being produced rapidly, reaching a peak on day 3 in culture and declining sharply thereafter, whereas IL-10 production increased gradually, resembling IL-4 and IL-17 production by their corresponding lineage cells; 2) reactivation of Th9, following expansion, triggered faster and higher production of both IL-9 and IL-10; 3) incubating Th9 cells in polarizing media specific for other phenotypes stimulated moderate levels of phenotype switching to Th1 or Th17 but a massive switching to Th2; 4) Th9 cells induced moderate inflammation in HEL-expressing recipient eyes but only when producing high levels of IL-9; and 5) IL-9-producing donor cells were detected in the blood of Th9 recipients but not in their inflamed eyes, suggesting that similar to findings in culture, exposure to HEL in these eyes arrested the IL-9 production in Th9 cells. Collectively, these data provide new information concerning Th9 cells and reveal their uniqueness, in particular with regard to the unusual production kinetics of IL-9 and the short retention of these cells in affected target tissues.

Unlike Th1, Th17 cells mediate sustained autoimmune inflammation and are highly resistant to restimulation-induced cell death.

Both Th1 and Th17 T cell subsets can mediate inflammation, but the kinetics of the pathogenic processes mediated by these two subsets have not been investigated. Using an experimental system in which TCR-transgenic Th1 or Th17 cells specific for hen egg lysozyme induce ocular inflammation in recipient mice expressing eye-restricted hen egg lysozyme, we found important differences in the in vivo behavior of these two subsets. Th1 cells initially proliferated considerably faster and invaded the eye more quickly than their Th17 counterparts, but then disappeared rapidly. By contrast, Th17 cells accumulated and remained the majority of the infiltrating CD4(+) cells in the eye for as long as 25 days after transfer, mediating more long-lasting pathological changes. Unlike Th1, Th17 cells were highly resistant to restimulation-induced apoptosis, a major pathway by which autoimmune and chronically restimulated Th1 cells are eliminated. Th17 cells had reduced Fas ligand production and resistance to Fas-induced apoptosis, relative to Th1 cells, despite similar surface expression of Fas. Th17-induced ocular inflammation also differed from Th1-induced inflammation by consisting of more neutrophils, whereas Th1-induced disease had higher proportions of CD8 cells. Taken together, our data show that pathogenic processes triggered by Th17 lag behind those induced by Th1, but then persist remarkably longer, apparently due to the relative resistance of Th17 cells to restimulation-induced cell death. The long-lasting inflammation induced by Th17 cells is in accord with these cells being involved in chronic conditions in humans.

Inhibition of experimental autoimmune uveitis by amino acid copolymers.

Glatiramer acetate (GA), a synthetic random amino acid copolymer, poly(Y, E, A, K)n, is widely used for treatment of multiple sclerosis. It inhibits experimental autoimmune encephalomyelitis (EAE) in mice by competition with the antigen and by induction of regulatory T cells. A novel copolymer, poly (F, Y, A, K)n , designated FYAK, was more effective than GA in its immunomodulatory activity in EAE. Here, FYAK and GA were compared in the amelioration of another disease model in mice, experimental autoimmune uveoretinitis (EAU). When tested by co-immunization with an uveitogenic antigen, FYAK was superior to GA in its capacity to inhibit EAU induction, as well as immune processes related to this condition. Further, regulatory T-cell lines specific to FYAK were more immunosuppressive than GA-specific lines in the EAU model. The superiority of FYAK-specific lines was accompanied by higher production of Th2 cytokines. These data thus demonstrate that FYAK, a novel copolymer, is superior to GA in its capacity to inhibit immunopathogenic processes in a non-central nervous system tissue.

Phenotype switching by inflammation-inducing polarized Th17 cells, but not by Th1 cells.

Th1 and Th17 cells are characterized by their expression of IFN-gamma or IL-17, respectively. The finding of Th cells producing both IL-17 and IFN-gamma suggested, however, that certain Th cells may modify their selective cytokine expression. In this study, we examined changes in cytokine expression in an experimental system in which polarized Th1 or Th17 cells specific against hen egg lysozyme induce ocular inflammation in recipient mice expressing hen egg lysozyme in their eyes. Whereas only IFN-gamma was expressed in eyes of Th1 recipient mice, substantial proportions of donor cells expressed IFN-gamma or both IFN-gamma and IL-17 in Th17 recipient eyes. The possibility that nonpolarized cells in Th17 preparations were responsible for expression of IFN-gamma or IFN-gamma/IL-17 in Th17 recipient eyes was contradicted by the finding that the proportions of such cells were larger in recipients of Th17 preparations with 20-25% nonpolarized cells than in recipients of 35-40% preparations. Moreover, whereas incubation in vitro of Th1 cells with Th17-polarizing mixture had no effect on their phenotype, incubation of Th17 with Th1-polarizing mixture, or in the absence of cytokines, converted most of these cells into IFN-gamma or IFN-gamma/IL-17-expressing cells. In addition, Th17 incubated with the Th1 mixture expressed T-bet, whereas no ROR-gamma t was detected in Th1 incubated with Th17 mixture. Thus, polarized Th1 cells retain their phenotype in the tested systems, whereas Th17 may switch to express IFN-gamma or IFN-gamma/IL-17 following activation in the absence of cytokines, or exposure to certain cytokine milieus at the inflammation site or in culture.

Both Th1 and Th17 are immunopathogenic but differ in other key biological activities.

The role of Th17 lymphocytes in immunopathogenic processes has been well established, but little is known about their basic cell features. In this study, we compared polarized Th1 and Th17 for key biological activities related to pathogenicity and trafficking. Th1 and Th17 lineages were derived from TCR-transgenic CD4 murine cells specific against hen egg lysozyme. When adoptively transferred into mice expressing hen egg lysozyme in their eyes, both Th1 and Th17 induced ocular inflammation but with slight differences in histological pathology. PCR analysis revealed selective expression of IFN-gamma or IL-17 in eyes of Th1 or Th17 recipients, respectively. Additionally, Th1 and Th17 were found to differ in three other key activities: 1) Th17 cells were inferior to Th1 cells in their capacity to trigger massive lymphoid expansion and splenomegaly; 2) the proportion of Th1 cells among infiltrating cells in inflamed recipient eyes declined rapidly, becoming a minority by day 7, whereas Th17 cells remained in the majority throughout this period; and 3) remarkable differences were noted between Th1 and Th17 cells in their expression of certain surface markers. In particular, reactivated Th1 expressed higher levels of CD49d and alpha(4)beta(7) (mucosal homing) in vitro and higher levels of CXCR3 (Th1 trafficking) in vivo. Reactivated Th17, however, expressed higher levels of alpha(E)beta(7) (epithelial tissue homing) and CD38 (activation, maturation and trafficking) in vitro, but in vivo Th17 expressed higher levels of alpha(4)beta(7) and CCR6 (lymphocyte trafficking). These data reveal that Th1 and Th17 cells differ in several key biological activities influencing migration and pathogenic behavior during inflammatory disease.

Pertussis toxin is superior to TLR ligands in enhancing pathogenic autoimmunity, targeted at a neo-self antigen, by triggering robust expansion of Th1 cells and their cytokine production.

Microbial products are assumed to play a major role in triggering pathogenic autoimmunity. Recently accumulated data have shown that these products stimulate the immune system by interacting with TLRs, expressed on APCs. To examine the capacity of various TLR ligands to trigger pathogenic autoimmunity, we used a system in which naive CD4 cells, specific against hen egg lysozyme (HEL), are injected into recipient mice expressing HEL in their eyes. Only when stimulated, the naive cells acquire pathogenic capacity and induce ocular inflammation. Seven TLR ligands were tested in this system: lipoteichoic acid/peptidoglycan, zymosan, poly (I:C), LPS, pertussis toxin (PTX), flagellin, and CpG oligodeoxynucleotide. Treatment of recipient mice with HEL alone stimulated proliferation of the transferred cells, but no disease, whereas ocular inflammation did develop in recipient mice coinjected with HEL and any one of the seven TLR ligands. Inflammation induced by PTX surpassed by its severity those induced by all other tested TLR ligands and was accompanied by a dramatic increase in number of the transferred cells that acquired features of effector Th1 lymphocytes. Ocular inflammation and number of transferred cells in recipients injected with PTX and HEL were substantially reduced by treatment with Abs against IFN-gamma or IL-12, thus indicating the role of these cytokines in the PTX effect. Overall, our observations demonstrate that various TLR ligands are capable of triggering pathogenic autoimmunity and that PTX surpasses other microbial products in this activity, by stimulating excessive proliferation and polarization toward Th1 of naive T cells.

Osteopontin is proinflammatory in experimental autoimmune uveitis.

PURPOSE: Osteopontin (OPN) has been implicated in inflammatory and wound-healing processes. Increased OPN mRNA levels have been reported in experimental autoimmune uveitis (EAU), but the function of OPN in the inflamed eye is unknown. The purpose of this study was to investigate the role of OPN in the pathogenesis of EAU. METHODS: EAU was induced in OPN-null and wild-type (WT) mice by immunization with interphotoreceptor retinoid-binding protein (IRBP). Immunofluorescence experiments were performed to identify OPN-positive cells in WT mice. Disease incidence, serum IRBP antibody levels, vitreous infiltrates, retinal granulomas, and lymphocyte proliferation were assessed in OPN-null and WT mice. To determine whether OPN could induce an EAU-like condition, purified OPN and OPN fragments were injected intraocularly into WT mice and vitreous infiltrates were characterized and quantified. RESULTS: In WT EAU-positive eyes, cell types with increased OPN immunoreactivity were identified as F4/80-positive macrophages/microglia and CD4-positive T cells. OPN-null mice manifested attenuated disease with decreased vitreous infiltrates, fewer granulomas, less lymphocyte proliferation, and lower serum IRBP antibody levels. Exogenous full-length OPN, as well as N- and C-terminal fragments, induced leukocyte infiltration and retinal folding, with some similarities to EAU. CONCLUSIONS: The results demonstrate that OPN is proinflammatory in EAU and may be important for recruitment and activation of leukocytes in retinal inflammation.

Active participation of antigen-nonspecific lymphoid cells in immune-mediated inflammation.

The pathogenic process of tissue-specific autoimmune disease depends to a large extent on recruitment of Ag-nonspecific cells into the target tissue. Little is known, however, about the recruitment process and the features that characterize the recruited cells. In this study, we analyzed the recruitment of Ag-nonspecific lymphoid cells into an inflammatory site by using an experimental system in which TCR-transgenic Th1 cells are adoptively transferred to induce ocular inflammation in recipient mice that express the target Ag in their eyes. A sharp increase in number of all host cell populations was observed in the recipient spleen, reaching a peak on day 4 postcell transfer and declining thereafter. A large portion of the host's spleen CD4 cells underwent phenotypic changes that facilitate their migration into the target organ, the eye. These changes included increased expression of the chemokine receptor CXCR3, and the adhesion molecule CD49d, as well as a decline in expression of CD62L. The host lymphocytes migrated into the recipient mouse eye more slowly than the donor cells, but became the great majority of the infiltrating cells at the peak of inflammation on day 7 postcell injection. Interestingly, the mass migration of host T cells was preceded by an influx of host dendritic cells, that reached their peak on day 4 postcell injection. The eye-infiltrating host CD4 lymphocytes underwent additional changes, acquiring a profile of activated lymphocytes, i.e., up-regulation of CD25 and CD69. Our results thus provide new information about the active participation of Ag-nonspecific lymphoid cells in immune-mediated inflammation.

Inflammatory processes triggered by TCR engagement or by local cytokine expression: differences in profiles of gene expression and infiltrating cell populations.

Immune cell-mediated inflammatory responses are triggered by TCR engagement with the target antigen, the initial event that brings about the complex sequence of events of the inflammatory process. Another form of inflammation is induced by local expression of certain cytokines. Unlike the former form of inflammation, little is known about the basic features of the cytokine-induced responses. Here, we analyzed tissue morphology, the infiltrating cells, and up-regulated, inflammation-related genes in mouse eyes in which inflammation is triggered by local transgenic (Tg) expression of cytokines and compared these features with those in eyes with experimental autoimmune uveitis (EAU), in which inflammation is initiated by engagement of TCR on sensitized T cells with their target antigen, followed by the well-defined, subsequent cytokine production. Eyes of IFN-gamma Tg mice exhibited severe, morphological changes but essentially no inflammation, and intense inflammation was found in eyes of interleukin (IL)-1 or IL-7 Tg mice. The cellular infiltration in eyes of these latter two lines of Tg mice resembled that in eyes with EAU by including many CD4 cells, but unlike in EAU, the infiltration in Tg eyes contained large proportions of B cells and only small numbers of macrophages. Real-time PCR analysis of eye RNA revealed differences among the disease models in the expression profiles of various inflammation-related genes. It is interesting that a bias toward T helper cell type 1 immunity (high IFN-gamma, RANTES/CCL5, MIG/CXCL9, and T-bet but low IL-4, IL-5, and GATA-3 transcripts) was found in EAU eyes but not in eyes of IL-1 and IL-7 Tg mice. The results thus show that similar to TCR engagement, local expression of certain cytokines triggers a complex, subsequent production of numerous inflammation-related molecules, but features of the ensued inflammatory process are determined by the triggering mechanism.

A unique pattern of up- and down-regulation of chemokine receptor CXCR3 on inflammation-inducing Th1 cells.

Chemokine receptor CXCR3 and its CXC ligands play major roles in Th1 cell-induced inflammatory processes. Here, we examined the expression of CXCR3 by TCR-transgenic Th1 lymphocytes that induce ocular inflammation in mice expressing the target antigen in their lenses. The essential role of CXCR3 in this model was indicated by the observation that the ocular inflammation was significantly blocked by an antibody against this receptor. CXCR3 expression by Th1 cells was elevated during their initial activation in culture and further increased during the consecutive incubation with IL-2. However, CXCR3 expression declined dramatically during the ensuing antigenic reactivation, in parallel with down-regulation of its mRNA. Yet, reactivated Th1 cells exhibited the highest degree of pathogenicity when adoptively transferred into recipients. Transferred reactivated Th1 cells proliferated vigorously and re-expressed CXCR3 while residing in the spleen of recipient mice, reaching approximately 85% positivity 4 days post cell transfer when their massive migration to the target eyes began. Importantly, infiltrating Th1 cells underwent profound phenotypic changes in the eye that closely resembled those seen during reactivation of Th1 cells in vitro and included down-regulation of CXCR3. These observations thus show that expression of CXCR3, a major participant in Th1-induced inflammation, fluctuates profoundly during cell activation and migration and is down-regulated upon re-exposure of these cells to the antigen, in vitro or in the target organ.