Antigenic mimicry of infectious agents and autoantigens is a proposed pathomechanism for autoimmune diseases. Here, we describe antigenic mimicry of a peptide from rotavirus, a nutritional protein from bovine milk ( § s2-casein) and a peptide thereof as well as a highly pathogenic peptide from retinal S-antigen (PDSAg), a major autoantigen in experimental autoimmune uveitis in Lewis rats. Immunization of rats with the peptides and the casein protein induced uveitis, an intraocular inflammation leading to decreased vision and even blindness. The peptides elicited cross-reactive T cell responses and uveitis in rats and were also recognized by lymphocytes and sera from uveitis patients. Oral tolerization with PDSAg, but not with rotavirus-and casein-derived peptides or casein protein, prevented PDSAginduced uveitis in rats. Cofeeding of casein with cholera toxin induced uveitis in rats, suggesting that breaking oral tolerance to casein during gastrointestinal infections might also be able to initiate uveitis in humans.

Leukocyte recruitment from the blood into injured tissues during inflammatory diseases is the result of sequential events involving chemokines binding to their GPC receptors as well as to their glycosaminoglycan (GAG) co-receptors. The induction and the crucial role of MCP-1/CCL2 in the course of diseases that feature monocyte-rich infiltrates have been validated in many animal models, and several MCP-1/CCL2 as well as CCR2 antagonists have since been generated. However, despite some of them being shown to be efficacious in a number of animal models, many failed in clinical trials, and therapeutically interfering with the activity of this chemokine is not yet possible. We have therefore generated novel MCP-1/CCL2 mutants with increased GAG binding affinity and knocked out CCR2 activity, which were designed to interrupt the MCP-1/CCL2-related signaling cascade. We provide evidence that our lead mutant MCP-1(Y13A/S21K/Q23R) exhibits a 4-fold higher affinity toward the natural MCP-1 GAG ligand heparan sulfate and that it shows a complete deficiency in activating CCR2 on THP-1 cells. Furthermore, a significantly longer residual time on GAG ligands was observed by surface plasmon resonance. Finally, we were able to show that MCP-1(Y13A/S21K/Q23R) had a mild ameliorating effect on experimental autoimmune uveitis and that a marginal effect on oral tolerance in the group co-fed with Met-MCP-1(Y13A/S21K/Q23R) plus immunogenic peptide PDSAg was observed. These results suggest that disrupting wild type chemokine-GAG interactions by a chemokine-based antagonist can result in anti-inflammatory activity that could have potential therapeutic implications.Chemokines are small secreted proteins that function as messengers by orchestrating activation and directional migration of specific subtypes of leukocytes from the blood stream into injured tissues (1-3). They exert their specific functions through interactions with G protein-coupled receptors on the one hand and with cell-surface proteoglycans on the other hand, which we therefore call chemokine co-receptors to differentiate them from the "classical" chemokine GPC receptors. Proteoglycans are commonly defined as silent receptors, although evidence is accumulating that shows that proteoglycans binding chemokines exhibit non-classical downstream signaling in endothelial cells.2 Within the CC family of chemokines, MCP-1/CCL2 (monocyte chemoattractant protein-1) specifically recruits and activates CCR2-positive cells. Thus, MCP-1 is highly induced in a variety of diseases that feature monocyterich cellular infiltrates, such as in atherosclerosis (4, 5), congestive heart failure (6, 7), rheumatoid arthritis (8 -10), inflammatory bowel disease (11), and uveitis (12-14). Unlike many other chemokines, MCP-1/CCL2 binds exclusively to CCR2, and the importance of this chemokine-receptor pair in mouse models of inflammatory diseases, including multiple sclerosis, atherosclerosis, and uveitis, has been confirmed (12-17). It has, for example, been shown that knock-out mice lacking MCP-1/ ...

PurposeThe retinal pigment epithelium (RPE) is a main target for complement activation in age-related macular degeneration (AMD). The anaphylatoxins C3a and C5a have been thought to mostly play a role as chemoattractants for macrophages and immune cells; here, we explore whether they trigger RPE alterations. Specifically, we investigated the RPE as a potential immunoregulatory gate, allowing for active changes in the RPE microenvironment in response to complement.DesignIn vitro and in vivo analysis of signaling pathways.MethodsIndividual activities of and interaction between the two anaphylatoxin receptors were tested in cultured RPE cells by fluorescence microscopy, western blot, and immunohistochemistry.Main outcome measuresIntracellular free calcium, protein phosphorylation, immunostaining of tissues/cells, and multiplex secretion assay.ResultsSimilar to immune cells, anaphylatoxin exposure resulted in increases in free cytosolic Ca2+, PI3-kinase/Akt activation, FoxP3 and FOXO1 phosphorylation, and cytokine/chemokine secretion. Differential responses were elicited depending on whether C3a and C5a were co-administered or applied consecutively, and response amplitudes in co-administration experiments ranged from additive to driven by C5a (C3a + C5a = C5a) or being smaller than those elicited by C3a alone (C3a + C5a < C3a).ConclusionWe suggest that this combination of integrative signaling between C3aR and C5aR helps the RPE to precisely adopt its immune regulatory function. These data further contribute to our understanding of AMD pathophysiology.

A major limitation of most animal models of autoimmune diseases is that they do not reproduce the chronic or relapsing-remitting pattern characteristic of many human autoimmune diseases. This problem has been overcome in our rat models of experimentally induced monophasic or relapsing-remitting autoimmune uveitis (EAU), which depend on the inducing antigen peptides from retinal S-Antigen (monophasic EAU) or interphotoreceptor retinoid-binding protein (relapsing EAU). These models enable us to compare autoreactive and regulatory T cell populations. Intraocular, but not peripheral T cells differ in their cytokine profiles (IFN-γ, IL-17 and IL-10) at distinct time points during monophasic or relapsing EAU. Only intraocular T cells concomitantly produced IFN-γ, IL-17 and/or IL-10. Monophasic EAU presented rising numbers of cells expressing IFN-γ and IL-17 (Th1/Th17) and cells expressing IL-10 or Foxp3. During relapsing uveitis an increase of intraocular IFN-γ+ cells and a concomitant decrease of IL-17+ cells was detected, while IL-10+ populations remained stable. Foxp3+ cells and cells expressing IL-10, even in combination with IFN-γ or IL-17, increased during the resolution of monophasic EAU, suggesting a regulatory role for these T cells. In general, cells producing multiple cytokines increased in monophasic and decreased in relapsing EAU. The distinct appearance of certain intraocular populations with characteristics of regulatory cells points to a differential influence of the ocular environment on T cells that induce acute and monophasic or relapsing disease. Here we provide evidence that different autoantigens can elicit distinct and differently regulated immune responses. IFN-γ, but not IL-17 seems to be the key player in relapsing-remitting uveitis, as shown by increased, synchronized relapses after intraocular application of IFN-γ. We demonstrated dynamic changes of the cytokine pattern during monophasic and relapsing-remitting disease with strongly increasing IL-10 expression in intraocular T cells during monophasic uveitis.

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