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Purpose: Optic neuritis (ON) is a condition involving primary inflammation, demyelination, and axonal injury in the optic nerve and leads to apoptotic retinal ganglion cell (RGC) death, which contributes to the persistence of visual loss. Currently, ON has no effective treatment. The goal was to determine the effectiveness of immunotherapy with recombinant T-cell receptor ligand (RTL) in preventing ON in humanized HLA-DR2 transgenic mice., Methods: Experimental autoimmune encephalomyelitis (EAE) was induced with myelin oligodendrocyte glycoprotein in humanized HLA-DR2 (DRβ1*1501) transgenic mice. Five consecutive doses of RTL342M were administrated at the onset of ON. The development of autoimmune ON was assessed by histopathology at different time points. The levels of myelin loss, axonal loss, and RGC damage were examined by immunofluorescence., Results: HLA-DR2 mice developed chronic ON 2 days before EAE characterized by progressive neurodegeneration in both organs. RTL342M significantly suppressed inflammation in the optic nerve and spinal cord and provided protection for at least 30 days. Examination of myelin loss showed a marked suppression of demyelination and an increase in myelin recovery in the optic nerve. Moreover, RTL342M treatment revealed a neuroprotective effect on optic nerve axons and RGCs in retinas at postimmunization (PI) day 62., Conclusions: RTL342M suppressed clinical and histologic signs of EAE/ON by preventing the recruitment of inflammatory cells into the optic nerve and showed neuroprotective effects against ON. However, to achieve full therapeutic benefit, more doses may be needed. These findings suggest a possible clinical application of this novel class of T-cell-tolerizing drugs for patients with optic neuritis.

Purpose: T cells are essential for the development of autoimmune uveitis. Although the costimulatory molecule OX40 promotes T-cell function and expansion, it is unclear whether OX40 is implicated in ocular inflammation. The purpose of this study was to examine the role of OX40 in uveitis., Methods: Experimental autoimmune uveitis (EAU) was induced in B10.RIII mice by subcutaneous injection of interphotoreceptor retinoid-binding protein peptide 161-180 (IRBP(161-180)). Some mice received an intravenous administration of OX40-activating antibody on days 0 and 4 after IRBP(161-180) sensitization or on days 10 and 14 of uveitis onset. The severity of EAU was evaluated by histology at different time points. In addition, ocular inflammatory cytokine expression was determined by real time-PCR, and peripheral activated CD4(+)CD44(+)CD62L(-) T cells and IL-7Rα expression were analyzed by flow cytometry. The activated CD4(+)CD44(+) lymphocytes were rechallenged with IRBP(161-180) in vitro to assess their antigen recall response., Results: The authors demonstrated a marked OX40 expression by infiltrating lymphocytes in enucleated human eyes with end-stage inflammation. In addition, the administration of OX40-activating antibody prolonged and exacerbated the disease course of EAU. Moreover, activation of OX40 not only increased CD4(+)CD44(+)CD62L(-) lymphocyte number, it upregulated IL-7Rα expression in the activated T-cell population. Lastly, these cells exhibited a stronger interferon-γ response to IRBP(161-180) restimulation in vitro., Conclusions: The results reveal a pathogenic role of OX40 in uveitis. Furthermore, the upregulation of IL-7R in CD4(+)CD44(+) lymphocytes suggests that the activation of OX40 promotes the generation or expansion of uveitogenic memory T cells.

Purpose: To determine protective properties of recombinant TCR ligands (RTLs) as a new treatment for experimental autoimmune anterior uveitis (AU). RTLs comprise the rat RT1.B beta1alpha1 domains, linked either to the guinea pig MBP69-89 peptide (RTL201), to the corresponding rat MBP69-89 peptide (RTL200), or to the cardiac myosin peptide CM-2 (RTL203)., Methods: AU associated with experimental autoimmune encephalomyelitis (EAE) was actively induced in Lewis rats by injection of myelin basic protein emulsified in complete Freund's adjuvant (CFA) or passively by the transfer of pathogenic T cells. Rats received five daily doses each of 300 microg RTL201 in saline, intravenously. Control rats received the same dose of RTL203 or an "empty" beta1alpha1 protein (no peptide). The rats were evaluated for the suppression of clinical and histologic signs of AU., Results: RTL201 prevented active and passive AU and reduced the clinical symptoms of established AU. RTL201 completely prevented clinical and histologic AU in the treated rats, compared with disease progression in the untreated rats or those treated with an "empty" construct. The suppression of clinical AU correlated with a significant reduction in inflammatory cells infiltrating the eyes of the RTL201-treated rats. Furthermore, RTL201 inhibited T cell proliferation, DTH responses, and cytokine mRNA expression in the eye, in contrast to the untreated rats. In comparison with RTL201, RTL200 was less effective in protecting the eye from AU. RTL203 also significantly inhibited clinical AU, but not EAE., Conclusions: RTL constructs suppressed clinical and histologic AU by inhibiting the systemic activation of specific T cells and preventing the recruitment of inflammatory cells into the eye. These findings suggest a possible clinical application of this novel class of peptide/MHC class II constructs in patients with AU that is mediated by T-cell responses to known antigenic peptides.

Purpose: Lewis rats immunized with myelin basic protein (MBP) develop experimental autoimmune encephalomyelitis (EAE) and associated anterior uveitis (AU), which recurs. The goal was to analyze cellular activation markers and adhesion molecules of T cells that infiltrate the eyes and spinal cord during acute and recurrent AU in actively and passively induced diseases simultaneously in the same animals., Methods: EAE-AU was induced in Lewis rats by immunization with MBP in CFA, or by adoptive transfer of MBP-specific T-cell lines, and the signs of clinical EAE and AU was scored. Cells isolated from the iris-ciliary body were tested by flow cytometry for expression of CD4, CD8, CD45RC, T-cell receptor (TCR) Vbeta8.2, alpha4 integrin, L-selectin, CD44, and CD134., Results: Ocular T cells showed a significantly higher expression of CD62L (l-selectin) than did T cells in the spinal cord. In addition, a much lower percentage of infiltrating CD8(+) T cells was found in the eyes during AU. In passive transfer experiments, T-cell lines derived from acute and recurrent uveitis showing similar phenotypes differing in specificities but possessed the capacity of inducing both AU and EAE. Pretreatment of rats with effector CD4(+) T cell before MBP immunization did not induce suppression of EAE or AU. However, pretreatment with regulatory CD8(+) T cells significantly reduced the severity and duration of both EAE and AU., Conclusions: T cells recruited into the inflamed eyes or central nervous system (CNS) are mainly activated/memory T cells expressing different levels of L-selectin. Regulatory CD8(+) T cells may contribute to the susceptibility of the eye to recurrent AU. The differences in phenotypes of T cells recruited simultaneously to two different organs suggest that microenvironment also plays a role in determining lymphocyte homing.

Purpose: To determine the pattern of expression of CC chemokines and their receptors in the eyes of Lewis rats and to establish their role in autoimmune anterior uveitis (AU) associated with experimental autoimmune encephalomyelitis (EAE)., Methods: EAE/AU was induced in Lewis rats with myelin basic protein in complete Freund's adjuvant (CFA). The rats were scored for the development of clinical EAE and AU. The expression of CCL5/regulated on activation normal T-cell expressed and secreted (RANTES), CCL2/monocyte chemotactic protein (MCP)-1, CCL3/macrophage inflammatory protein (MIP)-1alpha, and CCL4/MIP-1beta and their receptors was examined at the preclinical stage, onset, peak, and recovery by RT-PCR and ELISA. EAE/AU rats were treated with neutralizing polyclonal antibodies against CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL2/MCP-1, and CCL5/RANTES and tested for the suppression of onset of clinical AU and EAE. The control group received normal rabbit IgG at the same dose., Results: The gene expression of those chemokines was upregulated concurrently with symptom onset of EAE/AU and correlated with the intensity of inflammatory changes in the eye and central nervous system (CNS). The highest expression of CCL4/RANTES, CCL2/MCP-1, and CCL3/MIP-1alpha in the eye was detected at onset of clinical uveitis, whereas CCL4/MIP-1beta was elevated at the peak of AU. The expression of chemokine receptors associated with T-helper (Th)1-type response, CCR1 and CCR5, correlated with their appropriate ligands and was the highest at the peak of AU, whereas CCR2, the receptor for CCL2/MCP-1, was present before the onset of the disease. Treatment of anti-MIP-1beta and anti-MCP-1 significantly delayed the onset and shortened the duration of AU and EAE. Anti-MIP-1alpha treatment had no effect on clinical EAE but inhibited the clinical signs of AU. Although CCL5/RANTES expression was observed during the entire course of the disease, anti-RANTES treatment had no effect on clinical disease progression., Conclusions: The data suggest that CCL2/MCP-1, CCL3/MIP-1alpha, and CCL4/MIP-beta contribute to the recruitment of inflammatory cells into the eye and CNS and to disease activity.

Purpose: Recoverin has been identified as a target autoantigen for antirecoverin antibodies found in the sera of some patients with cancer-associated retinopathy. The aim of this study was to investigate the role of antirecoverin antibodies in cancer-associated retinopathy., Methods: Human, rat, and rabbit antirecoverin antibodies were purified using a recoverin-affinity column. Purified biotinylated antibodies were cultured with recoverin-positive rat retinal cells E1A.NR3. Antibody uptake by retinal cells in vitro was analyzed by immunocytochemistry. Cytotoxic effect of antibodies on retinal cells was measured by the MTT colorimetric method. Apoptosis was shown by the ladder DNA fragmentation method and by fluorescent dye chromatin fragmentation analysis., Results: Antirecoverin antibodies obtained either from sera from five cancer-associated retinopathy patients or from sera of immunized animals were internalized by E1A.NR3 cells. Only specific, antirecoverin antibodies produced destruction of the cells in a dose- and time-dependent manner. Normal immunoglobulin G did not have such effects on retinal cells. No additional cell destruction was observed in the presence of complement as compared with cultures incubated with antirecoverin antibodies alone. Internucleosomal DNA fragmentation and presence of apoptotic cells was observed throughout the culture treated with recoverin specific antibodies but not with normal antibodies. Cells not expressing recoverin (Y79, PC12, and GH3) were not susceptible to cell destruction because of antirecoverin antibody action., Conclusions: These studies showed that antibodies specific to recoverin are able to enter and cause death of cells expressing recoverin. In humans, autoantibodies originally elicited against recoverin expressed in tumor cells may damage retinal photoreceptors and play a role in the pathogenesis of cancer-associated retinopathy. Results suggest that autoantibody to recoverin, when given access to recoverin in the retina through the blood-retina barrier, could initiate photoreceptor degeneration leading to blindness. Such mechanism may be common for other paraneoplastic disorders or autoimmune diseases where antibodies interfere with the normal cell physiology.

Purpose: To examine the retina and test the serum of a patient with cancer-associated retinopathy syndrome who was diagnosed with small cell carcinoma of the lung and experienced unexpected visual loss., Methods: Proteins from normal human retina were extracted, separated by one- and two-dimensional gel electrophoresis, transferred to PVDF membrane, and used for immunostaining. Antibody specificity was determined by use of solid-phase peptides in a solid-phase immunoassay., Results: Histologic examination of the retina showed loss of the photoreceptor cell layer. This finding correlated with the results of clinical (loss of vision) and electrophysiologic (abnormal electroretinograph [ERG]) tests. The patient's serum antibodies specifically recognized recoverin, a protein predominantly found in retinal photoreceptor cells. The patient's serum also labeled some higher molecular weight proteins present in normal lung and other normal tissues, as well as in lung cell carcinoma cell lines. The only other tissue in which immunoreactivity against p23 could be found was the optic nerve. Our data revealed a lack of cross-reactivity between specific anti-recoverin antibodies and lung proteins. The results indicate that the patient serum contains more than one type of antibody activity. The autoantibodies were tested for fine immunospecificity by use of solid-phase peptides in a solid-phase immunoassay. Patient's antibodies reacted with a major determinant located in the recoverin sequence 62-68 (PKAYAQH) and with several minor ones., Conclusion: Based on the fact that the recoverin appears to be distributed in several different cell types, we suggest that this protein may be present in cancer cells and may play a role in the pathogenesis of some cancer-associated retinopathies.