A suppressive oligodeoxynucleotide inhibits ocular inflammation.

Synthetic oligodeoxynucleotides (ODN) expressing 'suppressive' TTAGGG motifs down-regulate a variety of proinflammatory and T helper type 1 (Th1)-mediated pathological immune responses. The ability of the archetypal suppressive ODN A151 to inhibit ocular inflammation was examined in two murine models: experimental autoimmune uveitis, induced by immunization with a retinal antigen, interphotoreceptor retinoid-binding protein (IRBP) and adoptively transferred ocular inflammation, induced by transferring Th1 cells specific to hen egg lysozyme (HEL) into recipient mice that express HEL in their eyes. A151 treatment suppressed the inflammation in both models. In addition, A151 inhibited IRBP-specific cytokine production and lymphocyte proliferation in mice immunized with IRBP. These findings suggest that suppressive ODN affects both afferent and efferent limbs of the immunopathogenic process and may be of use in the treatment of autoimmune ocular inflammation.

Induction of suppressors of cytokine signaling (SOCS) in the retina during experimental autoimmune uveitis (EAU): potential neuroprotective role of SOCS proteins.

Suppressors of cytokine signaling (SOCS) are implicated in immunopathogenic mechanisms of autoimmune diseases. We show here that SOCS expression in retina is temporarily correlated with progression of experimental autoimmune uveitis (EAU), an organ-specific autoimmune disease that serves as model of human uveitis. Peak of EAU correlates with highest SOCS genes expression while disease resolution coincides with their down-regulation. Surprisingly, SOCS5 is constitutively expressed in retina. SOCS5 expression increases significantly during EAU and remains elevated even after disease resolution. Our data suggest that cytokine-inducible SOCS members may be involved in negative regulation of inflammatory cytokines activities during EAU, while constitutively expressed SOCS5 may have neuroprotective functions.

A novel inflammatory eye disease induced by lymphocytes from knockout mice sensitized against the deleted ocular antigen.

Lens-associated uveitis (LAU), a severe inflammatory eye disease, is thought to be mediated by autoimmunity against lens crystallins. Previously described animal models for this disease are antibody-mediated, since no cellular response to self crystallins could be induced in experimental animals. Here, we describe a new model for LAU, in which lymphocytes from knockout mice deficient in alphaB-crystallin are sensitized against the deleted protein and induce severe ocular inflammation when adoptively transferred into wild type recipients. Similar to LAU, the experimental disease developed only following rupture of the lens capsule, produced in this study by capsulotomy; no disease was detected in recipient eyes with no capsulotomy, or in those treated with cautery, or in eyes affected by systemic treatment with sodium iodate, lipopolysaccharide or X-irradiation. The ocular changes in affected eyes included heavy cellular infiltration and proteinaceous exudate in both the anterior and posterior segments of the eye, that reached their peak on day 4 following cell transfer and subsided quite rapidly thereafter.

Immune response to retinal antigens in patients with gyrate atrophy and other hereditary retinal dystrophies.

PURPOSE: Gyrate atrophy (GA) is a rare hereditary disease that causes retinal destruction. Retinal damage in GA and other heredodegenerative diseases such as retinitis pigmentosa (RP) releases sequestered antigens and may trigger immune response to these molecules. Here, we studied the immune response to retinal antigens in patients with GA and RP and compared it with that of patients with inactive posterior uveitis and normal volunteers. PATIENTS AND METHODS: Peripheral blood was collected from 24 patients with RP, 10 patients with GA, 10 patients with inactive posterior uveitis, and 16 normal volunteers. Cell-mediated immune responses to human S-antigen (HS-Ag), bovine S-antigen (BS-Ag), and interphotoreceptor retinoid-binding protein (IRBP) were investigated by lymphocyte proliferation assay. In addition, serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) were studied by ELISA. Immunologic data were correlated with clinical and electrophysiological findings. RESULTS: Patients with GA or RP responded to HS-Ag and BS-Ag more vigorously than patients with uveitis or healthy controls, as shown by higher mean stimulation indices and larger proportions of responders. Unlike S-Ag, IRBP stimulated low lymphocyte responses in only a small proportion of RP patients. The mean sVCAM-1 levels were significantly higher in the sera from patients with GA than in that from normal controls. CONCLUSION: An elevated cellular immune response to S-Ag is common in patients with GA and RP. This elevated cellular immune response to S-Ag may exacerbate retinal destruction in patients with GA and RP.

Skewed abrogation of tolerance to a neo self-antigen in double-transgenic mice coexpressing the antigen with interleukin-1beta or interferon-gamma.

Transgenic (Tg) mice expressing hen egg lysozyme (HEL) under the control of the alphaA-crystallin promoter exhibit tolerance to HEL by both their T- and B-cell compartments. Here, we show that double-Tg mice, coexpressing HEL with either interleukin-1beta or interferon (IFN)-gamma, demonstrated unresponsiveness to HEL by their T-cell compartment, but most of them developed antibodies against HEL following a challenge with the antigen. The abrogation of humoral tolerance was more pronounced in the HEL/IL-1 double-Tg mice than in the HEL/IFN-gamma mice. Unlike their controls, double-Tg mice exhibited remarkable levels of variability in their antibody levels. The skewed abrogation of tolerance in the double-Tg mice is proposed to be due to the cytokines' capacity to rescue from clonal deletion small numbers of T cells, which provide help to antibody producing B cells. This notion is supported by the finding that adoptive transfer of small numbers of Th1 or Th2 cells into HEL-Tg mice made possible antibody production similar to that seen in the double-Tg mice.

Xenopus IRBP, a phylogenetically remote protein, is uveitogenic in Lewis rats.

Mammalian interphotoreceptor retinoid-binding proteins (IRBPs) are highly uveitogenic in Lewis rats. Xenopus laevis IRBP resembles mammalian IRBP in its four-fold structure, and has approximately 70% amino acid sequence identity with the bovine protein. This study investigated the uveitogenicity of recombinant Xenopus IRBP and two of its derived peptides in Lewis rats. Rats immunized with Xenopus IRBP developed uveoretinitis as well as pineal inflammation. The Xenopus molecule was, however, less immunopathogenic than the bovine IRBP. Of the two Xenopus IRBP peptides tested, 1180-1191 was remarkably uveitogenic, whereas sequence 521-540 exhibited low activity. It is assumed, therefore, that as with bovine IRBP, peptide 1180-1191 is the major uveitogenic sequence in Xenopus IRBP. The role individual residues of these peptides play in the immunopathogenic process is discussed. Our data thus demonstrate that despite its being phylogenetically remote, Xenopus IRBP is uveitogenic in Lewis rats

Recurrent intraocular inflammation in endotoxin-induced uveitis.

PURPOSE: Endotoxin-induced uveitis (EIU) in rats and mice peaks 24 hours after endotoxin injection and is commonly assumed to be a monophasic disease. This study examined intraocular inflammation at later time points to determine whether endotoxin injection can induce recurrent intraocular inflammation in strains of mice with high or moderate levels of susceptibility to EIU. METHODS: EIU was elicited in two mouse strains with high (C3H/HeN) and moderate (FVB/N) susceptibility, by means of intraperitoneal injections of Salmonella typhimurium endotoxin. Inflammatory cells in the anterior and posterior segments of the eye were counted by a masked observer on histologic sections of eyes from 1 to 17 days after endotoxin injection. RESULTS: A bimodal distribution of inflammatory cell infiltration was noted in eyes from C3H/HeN mice. As previously reported, inflammation peaked at 24 hours after endotoxin injection. However, a second, more pronounced peak of intraocular inflammation occurred approximately 5 days after endotoxin injection. FVB/N mice had a single peak of intraocular inflammation 4 days after injection. CONCLUSIONS: Endotoxin injection in C3H/HeN elicits recurrent intraocular inflammation. The previously unrecognized second peak of inflammation is more severe than the initial inflammatory disease. Studies on this second inflammatory peak may be useful in determining the pathogenesis of recurrent uveitis in humans.

Breakdown of tolerance to a neo-self antigen in double transgenic mice in which B cells present the antigen.

Transgenic (Tg) mice expressing a foreign Ag, hen egg lysozyme (HEL), under control of the alphaA-crystallin promoter ("HEL-Tg" mice) develop immunotolerance to HEL attributed to the expression of HEL in their thymus. In this paper we analyzed the immune response in double (Dbl)-Tg mice generated by mating the HEL-Tg mice with Tg mice that express HEL Abs on their B cells ("Ig-Tg" mice). The B cell compartment of the Dbl-Tg mice was unaffected by the HEL presence and was essentially identical to that of the Ig-Tg mice. A partial breakdown of tolerance was seen in the T cell response to HEL of the Dbl-Tg mice, i.e., their lymphocyte proliferative response against HEL was remarkably higher than that of the HEL-Tg mice. T-lymphocytes of both Dbl-Tg and Ig-Tg mice responded to HEL at concentrations drastically lower than those found stimulatory to lymphocytes of the wild-type controls. Cell mixing experiments demonstrated that 1) the lymphocyte response against low concentrations of HEL is due to the exceedingly efficient Ag presenting capacity of the Ab expressing B cells and 2) breakdown of tolerance in Dbl-Tg mice can also be attributed to the APC capacity of B cells, that sensitize in vivo and stimulate in vitro populations of T cells with low affinity toward HEL, assumed to be escapees of thymic deletion. These results thus indicate that T cell tolerance can be partially overcome by the highly potent Ag presenting capacity of Ab expressing B cells.

Copolymer 1 inhibits experimental autoimmune uveoretinitis.

Copolymer 1 (Cop 1) inhibits experimental allergic encephalomyelitis induced by a variety of myelin proteins, but has been found ineffective so far in inhibiting other experimental autoimmune diseases such as diabetes or arthritis. Here, we report for the first time that Cop I inhibits the development of experimental autoimmune uveoretinitis, induced in mice by interphotoreceptor retinoid-binding protein (IRBP). Pooled data of three experiments showed that treatment with Cop 1, at 0.5 mg/mouse, reduced the disease severity by 53% ( p = 0.0002). Cop 1 treatment also inhibited the proliferation and the production of cytokines by lymph node cells in response to IRBP and moderately reduced the antibody response to this antigen. The possible mechanisms of EAU inhibition by Cop 1 are discussed.

Vasoactive intestinal peptide (VIP) exacerbates endotoxin-induced uveitis (EIU) in mice.

PURPOSE: We have previously shown that inhibition of the proinflammatory cytokine tumor necrosis factor- (TNF)-alpha exacerbates the inflammatory process of EIU. To further examine this paradoxic phenomenon, we investigated here the effect on EIU of VIP, a neuropeptide that inbibits TNF-alpha production. METHODS: VIP was injected concurrently with endotoxin at doses that induce EIU or lethality in mice. Severity of EIU was measured by counting infiltrating cells in eye sections, at 1 or 5 days post endotoxin injection. Survival of mice was monitored periodically, while serum levels of TNF-alpha, interleukin-(IL)-1beta and IL-10 were determined by caputure ELISA. RESULTS: Treatment with VIP exacerbated EIU but provided partial protection from the lethal endotoxin effect. VIP treatment also reduced serum levels of TNF-alpha and IL-1beta, but increased levels of IL-10. CONCLUSION: This study further established the paradoxical observation that EIU is exacerbated by lowering the levels of circulating pro-inflammatory cytokines, in particular TNF-alpha.

Multiformic modulation of endotoxin effects by linomide.

Linomide is a potent immunomodulator that either enhances or suppresses certain immunological processes. Of particular interest is this compound's capacity to inhibit a variety of organ-specific autoimmune diseases. Here, we report on the effects of linomide on several immunological reactions elicited by endotoxin (LPS), both in vivo and in vitro. In rats and mice linomide inhibited the elicitation of endotoxin-induced uveitis (EIU), an acute inflammatory eye disease that develops within 24 h following footpad injection of LPS. Linomide also inhibited the production of TNF-alpha and IL-6 by LPS-stimulated rat and mouse macrophage monolayers. On the other hand, treatment with linomide significantly increased the levels of IL-1beta (mice and less in rats), IL-6 (rats), and TNF-alpha (mice) in serum samples collected 2 h following injection with LPS. The increased production of proinflammatory cytokines in linomide-treated mice was also indicated by the enhanced lethal effect of LPS in these mice. The finding of elevated levels of these cytokines in animals with suppressed EIU is also in line with previous observations of an inverse relationship between EIU severity and levels of TNF-alpha. Data recorded here underscore the unique capacity of linomide to both enhance and suppress the immune system.

Uveitis induced by lymphocytes sensitized against a transgenically expressed lens protein.

PURPOSE: Previously established experimental models for lens-associated uveitis (LAU) are all mediated by antibodies. The present study analyzed the features of a novel experimental intraocular inflammatory eye disease that is mediated by lymphocytes targeted at a lens antigen. METHODS: Conventional technologies were used to generate three lines of transgenic (Tg) mice, expressing hen egg lysozyme (HEL) under the control of the alphaA-crystallin promoter. To induce intraocular inflammation, these Tg mice were injected with lymphocytes from syngeneic wild-type donors sensitized against HEL. Before their injection, the cells were stimulated in culture with HEL. To release lenticular material, some eyes were capsulotomized. Ocular histopathologic changes were examined by routine methods. Levels of HEL antibody were measured by enzyme-linked immunosorbent assay, whereas cellular immunity was determined by the lymphocyte proliferation assay. RESULTS: Intraocular inflammation developed in HEL-Tg mice injected with syngeneic lymphocytes sensitized against HEL. The severity of inflammation was directly related to the number of injected cells, as well as to the accessibility of HEL. The most intense inflammation was seen in Tg mice in which the lens was disintegrated due to high production of HEL. In mice with no apparent lenticular changes the inflammation was enhanced by capsulotomy. The inflammation affected all segments of the eye and persisted for at least 39 days after adoptive transfer of cells. Four days after cell injection, the inflammation consisted of subacute infiltration, with both mononuclear and polymorphonuclear leukocytes, whereas more chronic infiltration was seen at later times. Vigorous cellular immunity but no antibody to HEL was found in recipient mice, thus demonstrating the exclusive participation of cellular immunity in the pathogenesis of this experimental disease. CONCLUSIONS: Transgenic mice expressing HEL in their lenses develop intraocular inflammation after injection of syngeneic lymphocytes sensitized against HEL. This experimental disease is a novel cell-mediated model for LAU.

Immunotolerance against a foreign antigen transgenically expressed in the lens.

PURPOSE: To extend our knowledge concerning immunotolerance against autologous lens crystallins, transgenic (Tg) mice that express a foreign antigen in their lens were generated, and the immune response against the antigen in these mice was analyzed. METHODS: Conventional techniques were used to generate lines of Tg mice that express soluble (S-) or membrane-bound (M-) hen egg lysozyme (HEL) under the control of the alphaA-crystallin promoter. The presence of HEL in various organs was determined by the particle concentration fluorescence immunoassay (PCFIA), and reverse transcription-polymerase chain reaction technique was used to detect mRNA transcripts of the molecule. To examine the development of immunity (or tolerance), Tg mice and their wild-type controls were immunized with HEL (25 microg) in Freund's complete adjuvant and 14 days later were tested for immune response against the antigen. Cellular immunity was measured by the lymphocyte proliferation assay and cytokine production, and humoral immunity was determined by enzyme-linked immunosorbent assay. RESULTS: Eyes of the high copy number M-HEL Tg mice were dystrophic, with disrupted lens, whereas no morphologic changes were detected in the eyes of the other Tg mouse lines. All Tg mice exhibited tolerance to HEL by their cellular and humoral immune compartments. The state of immunotolerance to HEL was retained in the Tg mice for as long as 10 months after removal of the main depot of this protein, by enucleation. Measurable amounts of HEL were found in the eyes of all Tg mice, but the protein could not be detected in the serum or in other organs by the sensitive PCFIA (with a threshold of 1 ng/ml). Yet, HEL mRNA was found in the thymus of the Tg mice, suggesting that minute amounts of the protein are expressed in this organ. CONCLUSIONS: The unresponsiveness to HEL in the Tg mice seems to be due to a "central" mechanism of tolerance, mediated by a minuscule amount of HEL in the thymus. Conversely, the much larger amounts of HEL in the peripheral depot, the eyes, play a minor role if any in the tolerogenic process. It is further proposed that a similar mechanism of central tolerance is responsible for the immunotolerance against autologous lens crystallins.

Expression of ocular autoantigens in the mouse thymus.

UNLABELLED: PURPOSE. The occurrence of eye diseases of autoimmune nature, as well as experimental models of these diseases, has been attributed to the sequestration of ocular antigens from the immune system, that prevents the development of tolerance against these antigens. Here, we tested this assertion by examining whether transcripts of certain ocular antigens are constitutively expressed in the thymus, the site of central tolerance induction. METHOD: RNA was isolated from the eyes and thymi of two mouse strains and analyzed for the expression of genes encoding four retinal and three lens proteins by reverse transcribed-polymerase chain reaction. Southern blot and DNA sequence analyses. RESULTS: We detected gene transcripts of S-Antigen (S-Ag), interphotoreceptor retinoid-binding protein, opsin, recoverin, lens major intrinsic protein (MIP), alphaA-, alphaA(-ins)- and gamma-crystallins in the thymi of BALB/c and FVB/N mouse strains. DNA sequence analysis of the thymic MIP and S-Ag transcripts confirmed their identity to the lens and retinal proteins, respectively. CONCLUSIONS: Our results reveal that transcripts of several ocular-specific proteins are expressed in the thymus and suggest that the commonly held view that ocular-specific antigens are sequestered from the immune system should be modified.

Recoverin-associated retinopathy: a clinically and immunologically distinctive disease.

PURPOSE: To compare the immune response to retinal antigens in a patient with a clinical condition resembling cancer-associated retinopathy with the immune responses of patients with other retinal degenerations or uveitis. METHODS: Cellular and humoral immune responses to retinal S-antigen and recoverin were determined in one patient with disease resembling cancer-associated retinopathy, three patients with other retinal degenerations, and eight patients with uveitis. RESULTS: A cellular immune response against recoverin was found only in the patient with the condition resembling cancer-associated retinopathy. Elevated levels of antibody against recoverin were found in this patient and in one of the three patients with a retinal degeneration, but in none of the eight patients with uveitis. In contrast, moderate lymphocyte responses to retinal S-antigen were found in most of the patients studied, and this response did not distinguish among the patient groups. Levels of serum antibodies against retinal S-antigen were also similar in all patients tested. Serum from the patient with disease resembling cancer-associated retinopathy produced strong immunostaining of the rods, cones, outer plexiform layer, and some cone bipolar cells, but serum from the patients with uveitis or other retinal degenerations did not show specific reactivity with the retina. CONCLUSIONS: We propose that this immunologically and clinically distinctive condition be termed recoverin-associated retinopathy, and we suggest that a cellular immune response against recoverin may be a distinguishing feature of the disorder.

Thymic expression of autoantigens correlates with resistance to autoimmune disease.

Experimental autoimmune uveoretinitis (EAU), an animal model for human intraocular inflammation (uveitis), is induced by immunization with retinal proteins such as S-Ag or interphotoreceptor retinoid binding protein. Marked differences exist among different animal species and strains in their susceptibility to EAU induction, but the cause of these differences is not completely clear. Here we show for the first time a correlation between constitutive expression of ocular autoantigens in the thymus (mRNA and protein) and resistance to EAU. This correlation was noted both at the species (mice vs rats or monkeys) and the subspecies (differences among strains) level. The data thus provide a novel mechanistic explanation for the differences in susceptibility to autoimmune diseases, suggesting that resistance to an organ-specific autoimmune disease may be regulated at least in part by capacity to establish central tolerance to the relevant autoantigen.

Immunomodulatory effects of linomide in animals immunized with immunopathogenic retinal antigens: dissociation between different immune functions.

Linomide (LS-2616, quinoline-3-carboxamide) has been reported to exert a diverse range of effects on the immune system. On one hand, this drug was found to stimulate the immune system and to enhance activities such as DTH or allograft rejection. On the other hand, linomide was shown to inhibit the induction of experimental autoimmune encephalomyelitis and myasthenia gravis, as well as the development of diabetes in non-obese diabetic (NOD) mice. Here we report the effects of linomide in animals immunized with uveitogenic retinal antigens. Treatment with linomide completely inhibited the development of experimental autoimmune uveoretinitis (EAU) in mice immunized with interphotoreceptor retinoid-binding protein and markedly suppressed EAU in rats immunized with S-antigen (S-Ag). In addition, linomide-treated rats exhibited reduced antibody production and lymphocyte proliferative response to S-Ag. In contrast to these suppressive activities, linomide treatment did not affect the development of adoptively transferred EAU in rats and moderately enhanced the DTH reactions to S-Ag in immunized rats in which EAU and other immune responses to this antigen were suppressed.

Immunogenicity and immunopathogenicity of an autoimmune epitope are potentiated by increasing MHC binding through residue substitution.

We have previously shown that in Lewis rats, peptide 273-283 (TWEGSGVLPCV) of rat interphotoreceptor retinoid-binding protein (IRBP) serves as a "surrogate" epitope for pathogenic lymphocytes sensitized against peptide 1181-1191 (SWEGVGVVPDV) of bovine IRBP. Yet, peptide 273-283 itself causes experimental autoimmune uveoretinitis (EAU) only at 200 nmol/rat, whereas peptide 1181-1191 is pathogenic even at 0.2 nmol. This difference was attributed to the higher affinity of 1181-1191 to MHC molecules. Here we demonstrate that substitution of putative MHC binding-residues of peptide 273-283 with the corresponding ones of 1181-1191 results in increased binding to MHC and in remarkably elevated immunologic capacities. Analogs of 273-283 were synthesized, in which residues 277 and 282 were substituted with one or both the corresponding amino acids of peptide 1181-1191, V and D, respectively. The main findings were: 1) substitutions drastically increased MHC affinity, namely, 273-283(V277,D282) >> 273-283(D282) >>> 273-283; 2) the substituted analogs were much more immunogenic than the native peptide, inducing cellular responses at much lower doses; 3) the analogs were more antigenic in vitro than the native peptide; 4) the analogs were exceedingly pathogenic; peptide 273-283(V277,D282) caused disease even at 0.02 nmol/rat; and 5) the analogs were superior to the native peptide in their capacity to stimulate production of IFN-gamma by sensitized lymphocytes. Thus, enhancement of affinity of an autoimmune peptide for MHC molecules increases both immunogenicity and pathogenicity.