Mimicry epitope from Ehrlichia canis for interphotoreceptor retinoid-binding protein 201-216 prevents autoimmune uveoretinitis by acting as altered peptide ligand.

We report here identification of novel mimicry epitopes for interphotoreceptor retinoid-binding protein (IRBP) 201-216, a candidate ocular antigen that causes experimental autoimmune uveoretinitis (EAU) in A/J mice. One mimicry epitope from Ehrlichia canis (EHC), designated EHC 44-59, induced cross-reactive T cells for IRBP 201-216 capable of producing T helper (Th)1 and Th17 cytokines, but failed to induce EAU in A/J mice. In addition, animals first primed with suboptimal doses of IRBP 201-216 and subsequently immunized with EHC 44-59 did not develop EAU; rather, the mimicry epitope prevented the disease induced by IRBP 201-216. However, alteration in the composition of EHC 44-59 by substituting alanine with valine at position 49, similar to the composition of IRBP 201-216, enabled the mimicry epitope to acquire uveitogenicity. The data provide new insights as to how microbes containing mimicry sequences for retinal antigens can prevent ocular inflammation by acting as naturally occurring altered peptide ligands.

Posttranslational modification of differentially expressed mitochondrial proteins in the retina during early experimental autoimmune uveitis.

PURPOSE: Posttranslational modification of proteins plays an important role in cellular functions and is a key event in signal transduction pathways leading to oxidative stress and DNA damage. In this study, we used matrix-assisted laser desorption/ionization- time of flight (MALDI-TOF) to investigate the posttranslational modifications of the differentially expressed proteins in the retinal mitochondria during early experimental autoimmune uveitis (EAU). METHODS: EAU was induced in 18 B10RIII mice with 25 µg of inter-photoreceptor retinoid-binding protein (IRBP) emulsified with complete Freund's adjuvant (CFA); 18 mice treated with CFA without IRBP served as controls. Retinas were removed from the experimental and control groups on day 7 post immunization; mitochondrial fractions were extracted and subjected to 2 dimentional-difference in gel electrophoresis (2D-DIGE); and the protein spots indicating differential expression were subjected to MALDI-TOF for protein identification and indication of any posttranslational modifications. RESULTS: Of the 13 proteins found to be differentially expressed by 2D-DIGE (including upregulated aconitase, mitochondrial heat shock protein (mtHsp) 70, lamin-1, syntaxin-binding protein, αA crystallin, ßB2 crystallin, along with downregulated guanine nucleotide-binding protein and ATP synthase) nine were found to undergo posttranslational modification. Oxidation was a common modification found to occur on aconitase, mtHsp 70, ATP synthase, lamin-1, ßB2-crystallin, guanine nucleotide-binding protein, and manganese superoxide dismutase (MnSOD). In addition, aconitase hydratase, mtHsp 70, guanine nucleotide-binding protein, ATP synthase, syntaxin-binding protein, ßB2-crystallin, and lamin-1 were also modified by carbamidomethylation. αA-crystallin had a pyro-glu modification. CONCLUSIONS: Several proteins present in the retinal mitochondria are posttranslationally modified during early EAU, indicating the presence of oxidative stress and mitochondrial DNA damage. The most common modifications are oxidation and carbamidomethylation. A better understanding of the proteins susceptible to posttranslational modifications in the mitochondria at the early stage of the disease may serve to advance therapeutic interventions to attenuate disease progression.

CpG-containing oligodeoxynucleotide 1826 converts the weak uveitogenic rat interphotoreceptor retinoid-binding protein peptide 1181-1191 into a strong uveitogen.

Aberrant activation of autoreactive T cells is one of the major causes of autoimmune disease. Autoantigens are sequestered and in many cases weak immunogens. For example, in experimental autoimmune uveitis, immunization of naive rats with autologous interphotoreceptor retinoid-binding protein (IRBP) fails to induce intraocular inflammation or a strong T cell response, whereas bovine IRBP is a strong inducer of experimental autoimmune uveitis. Such observations challenge the view that the autoantigen alone is responsible for the development of autoimmunity. Here, we demonstrate that autologous rat IRBP is converted to a strong immunogen in the presence of a small dose of CpG-containing oligodeoxynucleotides. Our results indicate that specific CpG-containing oligodeoxynucleotides may play an important role in the activation and expansion of autoreactive T cells in vivo, leading to autoimmune disease.

Pertussis toxin enhances Th1 responses by stimulation of dendritic cells.

Pertussis toxin (PTX) has been widely used as an adjuvant to induce Th1-mediated organ-specific autoimmune diseases in animal models. However, the cellular and molecular mechanisms remain to be defined. In this study, we showed that dendritic cells (DC) stimulated with PTX (PTX-DC) were able to substitute for PTX to promote experimental autoimmune uveitis (EAU). EAU induced by PTX-DC revealed a typical Th1 response, characterized by high uveitogenic retinal Ag interphotoreceptor retinoid-binding protein (IRBP)-specific IFN-gamma and IL-12 production in the draining lymph nodes, as well as increased levels of anti-IRBP IgG2a and decreased levels of anti-IRBP IgG1 in the serum of IRBP-immunized mice. Furthermore, PTX-DC preferentially induced T cells to produce the Th1 cytokine, IFN-gamma. After being stimulated with PTX, DC exhibited up-regulation of MHC class II, CD80, CD86, CD40, and DEC205. PTX-DC had also increased allostimulatory capacity and IL-12 and TNF-alpha production. Serum IL-12 was increased in naive mice that received PTX-DC i.p. In addition, PTX activated extracellular signal-regulated kinase in DC. Following the inhibition of extracellular signal-regulated kinase signaling, the maturation of PTX-DC was reduced. Subsequently, the ability of PTX-DC to promote IFN-gamma production by T cells in vitro and to induce EAU in vivo was blocked. The results suggest that PTX might exert an adjuvant effect on DC to promote their maturation and the production of proinflammatory cytokines, thereby eliciting a Th1 response.

Uveitis in horses induced by interphotoreceptor retinoid-binding protein is similar to the spontaneous disease.

Equine recurrent uveitis (ERU) is an inflammatory eye disease with high similarity to uveitis in man. It is the only spontaneous animal model for uveitis and the most frequent eye disease in horses affecting up to 10% of the population. To further investigate the pathophysiology of ERU we now report the establishment of an inducible uveitis model in horses. An ERU-like disease was elicited in seven out of seven horses by injection of interphotoreceptor retinoid-binding protein (IRBP) in complete Freund's adjuvant. Control horses did not develop uveitis. The disease model is characterized by a highly reproducible disease course and recurrent episodes with an identical time course elicited in all horses by repeated IRBP injections. The histology revealed the formation of lymphoid follicle-like structures in the eyes and an intraocular infiltration dominated by CD3(+) lymphocytes, morphological patterns typical for the spontaneous disease. Antigen-specific T cell proliferation of PBL was monitored prior to clinical uveitis and during disease episodes. An initial T cell response to IRBP-derived peptides was followed by epitope spreading to S-antigen-derived peptides in response to subsequent immunizations. Thus, horse experimental uveitis represents a valuable disease model for comparative studies with the spontaneous disease and the investigation of immunomodulatory therapeutic approaches after onset of the disease.

Experimental autoimmune uveitis in HLA-B27 transgenic mice.

The major histocompatibility complex (MHC) gene, HLA-B27 is strongly associated with auto-immune uveitis and spondyloarthropathies in humans. Experimental mouse models of autoimmune uveitis involve systemic immunization with the retinal autoantigen interphotoreceptor retinoid binding protein (IRBP). To assess possible roles of HLA-B27 in autoimmune uveitis, as well as to investigate a possible new animal model of human uveitis, inbred strains of C57BL/6 and C57BL/6 possessing the human HLA-B27 or HLA-A2 transgene were immunized with IRBP emulsified in complete Freund's adjuvant (CFA). Dilated eye examinations were performed to assess the timing and clinical course of any ensuing uveitis. Mice were sacrificed 3 to 4 weeks postinjection and the eyes submitted for histopathologic analysis. CFA alone did not produce any clinical uveitis. Fifty percent of eyes from the background C57BL/6 strain developed uveitis as early as 10 days postinjection. Of the eyes demonstrating uveitis, an average clinical score of 2.5 was present. Pathologically, a moderate scleritis and anterior uveitis was present. Fifty percent of A2 transgenic eyes developed uveitis as early as 14 days postinjection with an average clinical score of 2.0. Pathologically, a mild vitritis was present. Uveitis developed in only 20% of B27 transgenic mice and reached a peak on day 28. The average EAU score in diseased animals was 4.5. A dense retinitis and panuveitis was associated with severe vitritis. We conclude that the presence of the B27 gene is associated with a decreased incidence and slower rate of onset of EAU following immunization with IRBP; however, EAU may be more severe in the HLA-B27 expressing animals who do develop disease.

Interleukin-2 treatment potentiates induction of oral tolerance in a murine model of autoimmunity.

The present study addresses the feasibility of potentiating oral tolerance by immunomanipulation, using the murine model of experimental autoimmune uveoretinitis (EAU) induced by immunization with the retinal antigen interphotoreceptor retinoid binding protein (IRBP). Three feedings of 0.2 mg IRBP every other day before immunization did not protect against EAU, whereas a similar regimen of five doses was protective. However, supplementing the nonprotective 3x regimen with as little as one injection of 1,000 U of human recombinant interleukin-2 (IL-2) resulted in disease suppression that was equal to that of the protective 5x regimen. The protective effect was maintained across a range of IL-2 doses and times of administration; none of the IL-2 regimens tested resulted in disease enhancement. Peyer's Patch cells of 3x-fed and IL-2-treated mice showed greatly increased production of TGF-beta, IL-4, and IL-10 compared with animals given the nonprotective 3x regimen and to animals given the protective 5x regimen. We propose that IL-2 treatment enhances protection from EAU at least in part by stimulating production of antiinflammatory cytokines by regulatory cells in Payer's Patches. Moreover, the observed lymphokine production patterns suggest that whereas protection induced by the 3x + IL-2 regimen is likely to involve antiinflammatory cytokines, protection induced by the 5x regimen might involve anergy or deletion of the uveitogenic T cells. These results could have practical implications for use of IL-2 as a safe and effective way of potentiating oral tolerance.

Immunomodulation of experimental autoimmune uveoretinitis by intravenous injection of uveitogenic peptides.

Intravenous (IV) injection of antigenic proteins induces specific unresponsiveness, as shown by the diminished response to a challenge with these proteins in complete Freund's adjuvant. This study examined the effect of IV treatment with uveitogenic peptides on the development of experimental autoimmune uveoretinitis (EAU). The peptides used were derived from the sequence of bovine interphotoreceptor retinoid-binding protein (IRBP) and included R16 (sequence, 1177-1191), which is immunodominant and highly uveitogenic, and R4 (sequence, 1158-1180), which is nondominant and weakly uveitogenic. The efficacy of this treatment was found to depend on both the dose used for the IV injection and that used for the challenge. Thus, EAU induced by R16 at a dose of 0.2 nmol/rat was inhibited completely in all rats treated with the peptide at doses of 400 or 133 nmol and partially by the low dose of 5 nmol/rat. However, the EAU induced by a R16 challenge of 40 nmol/rat was inhibited only partially by the high treatment dose of 400 nmol/rat. The IV treatment was found to be effective in inhibiting the EAU induced by peptide R4. A large dose of R4 was needed to induce EAU (40 nmol/rat), and the disease was inhibited completely in all rats treated IV with this peptide at doses of 800, 400, or 133 nmol. In most animals injected with the 44-nmol dose, also, inhibition was complete. These data show that there is a correlation between the doses needed for achieving inhibition and those used for the challenge. The ratios between these doses in all experiments were found within the range 1-20.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental autoimmune uveoretinitis in mice. Induction by a single eliciting event and dependence on quantitative parameters of immunization.

Experimental autoimmune uveoretinitis (EAU) in the mouse is a recently developed model of ocular autoimmunity. Dependence of disease induction on qualitative and quantitative parameters of immunization was studied in B10.A mice immunized with interphotoreceptor retinoid-binding protein (IRBP). It was found that use of Bordetella pertussis adjuvant as well as its mode of preparation was of critical importance for disease induction; no disease was induced if pertussis adjuvant was omitted. The minimal effective protocol for EAU induction when the vaccine form of B. pertussis adjuvant was used consisted of pretreatment with cyclophosphamide, two divided doses of IRBP in complete Freund's adjuvant (CFA), and two divided doses of B. pertussis vaccine. Any reduction in the immunization schedule resulted in reduced incidence of disease. In contrast, substituting purified B. pertussis toxin (PTX) for the vaccine allowed reduction of the immunization schedule to a single dose of IRBP in CFA and omission of the cyclophosphamide pretreatment. Severity and incidence of disease could be quantitatively controlled by varying the respective doses of IRBP and PTX. In addition, a chronic or an acute clinical course of EAU could be obtained by using either a low-dose or a high-dose immunization, respectively. Establishment of a single dose induction protocol and the quantitation of the immunopathogenic response as a function of the variables of immunization lay the foundation for the further development and utilization of this promising model of ocular autoimmunity.