Ocular Inflammatory Diseases: Molecular Pathogenesis and Immunotherapy.

Uveitis is a diverse group of potentially sight-threatening intraocular inflammatory diseases of infectious or autoimmune etiology and accounts for more than 10% of severe visual handicaps in the United States. Pathology derives from the presence of inflammatory cells in the optical axis and sustained production of cytotoxic cytokines and other immuneregulatory proteins in the eye. The main therapeutic goals are to down-regulate the immune response, preserve the integrity of the ocular architecture and eventually eliminate the inciting uveitogenic stimuli. Current therapy is based on topical or systemic corticosteroid with or without second line agents and serious adverse effects of these drugs are the impetus for development of less toxic and more specific therapies for uveitis. This review summarizes the pathophysiology of uveitis, molecular mechanisms that regulate the initiation and progression of uveitis and concludes with emerging strategies for the treatment of this group of potentially blinding diseases.

Expression of SOCS1 and SOCS3 genes is differentially regulated in breast cancer cells in response to proinflammatory cytokine and growth factor signals.

DNA-hypermethylation of SOCS genes in breast, ovarian, squamous cell and hepatocellular carcinoma has led to speculation that silencing of SOCS1 and SOCS3 genes might promote oncogenic transformation of epithelial tissues. To examine whether transcriptional silencing of SOCS genes is a common feature of human carcinoma, we have investigated regulation of SOCS genes expression by IFNgamma, IGF-1 and ionizing radiation, in a normal human mammary epithelial cell line (AG11134), two breast-cancer cell lines (MCF-7, HCC1937) and three prostate cancer cell lines. Compared to normal breast cells, we observe a high level constitutive expression of SOCS2, SOCS3, SOCS5, SOCS6, SOCS7, CIS and/or SOCS1 genes in the human cancer cells. In MCF-7 and HCC1937 breast-cancer cells, transcription of SOCS1 is dramatically up-regulated by IFNgamma and/or ionizing-radiation while SOCS3 is transiently down-regulated by IFNgamma and IGF-1, suggesting that SOCS genes are not silenced in these cells by the epigenetic mechanism of DNA-hypermethylation. We further show that the kinetics of SOCS1-mediated feedback inhibition of IFNgamma signaling is comparable to normal breast cells, indicating that the SOCS1 protein in breast-cancer cells is functional. We provide direct evidence that STAT3 pathways are constitutively activated in MCF-7 and HCC1937 cells and may drive the aberrant persistent activation of SOCS genes in breast-cancer cells. Our data therefore suggest that elevated expression of SOCS genes is a specific lesion of breast-cancer cells that may confer resistance to proinflammatory cytokines and trophic factors, by shutting down STAT1/STAT5 signaling that mediate essential functions in the mammary gland.

Interferon-gamma induces regression of epithelial cell carcinoma: critical roles of IRF-1 and ICSBP transcription factors.

We have developed an epithelial cell carcinoma model for studying efficacy of IFNgamma gene therapy and have identified components of IFNgamma-signaling pathway responsible for its direct anti-tumor actions. The tumor results from ectopic expression of SV40 Large T-Antigen (SV40 T-Ag) oncogene in lens of transgenic mouse (alphaT3) and complete regression of the tumor is induced by targeting expression of IFNgamma into malignant lens cells. Inflammatory cells are absent in lens of alphaT3 or DT (co-expressing IFNgamma and SV40-T-Antigen) mice and the transformed lens cells are non-immunogenic, suggesting non-involvement of immunologic cells. We show that IFNgamma has direct growth-inhibitory effects on tumor cells, induces death of tumor cells by apoptosis and that these effects are mediated by two transcription factors, IRF-1 (interferon-regulatory factor-1) and ICSBP (interferon-consensus sequence-binding protein) induced by IFNgamma. Furthermore, stable transfection with ICSBP or IRF-1 construct inhibits lens carcinoma cell growth by upregulating Caspase-1, p21(WAF1) and p27 expression. In contrast, tumor progression in alphaT3 lens correlates with inhibition of IRF-1 and ICSBP expression. Our results suggest that IFNgamma gene therapy maybe effective in malignant diseases for which DNA tumor viruses are etiologic agents and that antitumor actions of IRF-1/ICSBP can be exploited therapeutically to circumvent adverse clinical effects associated with IFN therapy.

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.

Detection of Toxoplasma gondii DNA in primary intraocular B-cell lymphoma.

Primary intraocular lymphoma, a variant of primary central nervous system lymphoma with ocular involvement, is a large B-cell non-Hodgkin's lymphoma. Some cases of primary intraocular lymphoma have been reported to be associated with microorganisms including Epstein-Barr virus (EBV) and human herpes virus-8 (HHV-8), but not parasites. We analyzed 10 cases of primary intraocular lymphoma using microdissection and PCR. Tumor and normal cells were microdissected from ocular tissue on slides and subjected to PCR for genes from Toxoplasma gondii, EBV, and HHV-8. We detected Toxoplasma gondii, not HHV-8 or EBV, DNA in the lymphoma but not in normal cells of two cases that resembled ocular toxoplasmosis clinically. We speculate that Toxoplasma gondii may play a role in some forms of primary intraocular B-cell lymphoma.

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.

Interferon regulatory transcription factors are constitutively expressed and spatially regulated in the mouse lens.

Interferon regulatory factors (IRFs) are a family of transcription factors involved in regulation of cell growth and immunological responses. Nine IRFs have been described and they are expressed in a variety of cells, except for ICSBP and LSIRF/Pip, which are thought to be expressed exclusively in immune cells. Here, we show that IRF-1, IRF-2, ICSBP, and LSIRF/Pip are constitutively expressed in the mouse lens. These IRFs are present in both the cytoplasm and the nuclei of lens cells. However, the nuclear and cytoplasmic proteins exhibit distinct mobilities on SDS/PAGE. We further show that in the developing mouse lens, IRF-1 and IRF-2 are expressed at high levels in differentiated lens fiber cells with very low and barely detectable levels in undifferentiated lens epithelial cells. Although the level of ICSBP expression is very low in the normal mouse lens, in transgenic mice with constitutive expression of interferon gamma in the lens, its level is markedly elevated and ICSBP expression is detected exclusively in the nuclei of undifferentiated lens cells. Taken together, our data suggest that expression of IRF transcription factors is spatially regulated in the lens and that distinct IRFs may contribute to differential gene regulation in the epithelial and fiber compartments of the vertebrate lens.

Expression of interferon-gamma in the lens exacerbates anterior uveitis and induces retinal degenerative changes in transgenic Lewis rats.

Interferon-gamma (IFN-gamma) is a pleiotropic cytokine that has been implicated in immunopathogenic mechanisms of a number of inflammatory diseases of autoimmune or infectious disease etiology. However, its exact role is still a matter of debate. In experimental mouse models, IFN-gamma has been shown to exacerbate autoimmune thyroiditis, insulin-dependent diabetes mellitus, and autoimmune neuritis while it confers protection against experimental allergic encephalomyelitis and experimental uveitis. In this study, we generated transgenic rats with constitutive expression of IFN-gamma in the eye to study its paracrine effects and to investigate whether local production of IFN-gamma also confers protection against uveitis in the rat species. We show here that chronic exposure of ocular cells to IFN-gamma results in apoptotic death of retinal ganglion cells, development of chronic choroiditis, formation of retinal in-foldings, and activation of proinflammatory genes. In contrast to its protective systemic effect in the mouse, constitutive secretion of IFN-gamma in the rat eye was found to predispose the development of severe anterior uveitis and induction of retinal degenerative processes that impair visual acuity. Our data underscore the danger in extrapolation of cytokine effects in the mouse to humans without corroborating evidence in other species.

Interferon-gamma signaling in human retinal pigment epithelial cells mediated by STAT1, ICSBP, and IRF-1 transcription factors.

PURPOSE: Studies have shown that interferon (IFN)-gamma stimulates expression of intercellular adhesion molecule-1 (ICAM-1), major histocompatibility complex (MHC) class II, interleukin (IL)-6, and inducible nitric oxide synthase and inhibits replication of Toxoplasma gondii in human retinal pigment epithelial (HRPE) cells. The present study was undertaken to investigate the molecular mechanisms of IFN-gamma action. METHODS: RNA, whole-cell extracts, and nuclear extracts were prepared from HRPE cells cultured in the presence or absence of IFN-gamma. Activation of IFN-gamma-responsive genes was analyzed by electrophoretic mobility shift assay, reverse transcription-polymerase chain reaction (RT-PCR), western blot analysis, and immunoprecipitation. RESULTS: HRPE cells constitutively expressed two members of the IFN regulatory factor (IRF) family of transcription factors, IRF-1 and IRF-2. After exposure to IFN-gamma, transcription of IRF-1 and IFN consensus sequence binding protein (ICSBP) genes were induced; IRF-2 gene transcription was not upregulated. Activation of IFN-gamma-responsive genes was mediated by tyrosine phosphorylation of the signal transducer and activator of transcription (STAT)-1 factor. CONCLUSIONS: This study characterized the IFN-gamma signaling pathway in HRPE cells and identified IRF-1, ICSBP, and tyrosine-phosphorylated STAT1 as mediators of IFN-gamma action in these cells. ICSBP is thought to be exclusively used in immunologic responses and has previously been detected only in lymphoid cells. However, the current study shows that ICSBP expression is inducible in HRPE cells, suggesting that it may regulate gene transcription in RPE cells and possibly in other nonimmunologic cell types.

Interferon consensus sequence-binding protein is constitutively expressed and differentially regulated in the ocular lens.

Interferon signaling is mediated by STATs and interferon regulatory factor (IRF) families of transcription factors. Ten distinct IRFs have been described and most are expressed in a variety of cells except for interferon consensus sequence-binding protein (ICSBP) and lymphoid-specific IRF/Pip that are thought to be exclusively expressed in lymphoid cells. We show here for the first time that ICSBP is constitutively and inducibly expressed in the mouse lens. In contrast to lymphoid cells with exclusive expression of ICSBP in the nucleus, ICSBP is present in both the cytoplasm and nucleus of the lens cell. However, ICSBP in the nucleus is of lower apparent molecular weight. We further show that the ICSBP promoter is constitutively bound by lens nuclear factors and that its activation requires binding of additional factors including STAT1. Furthermore, transcriptional activation of ICSBP gene by interferon gamma is accompanied by selective nuclear localization of ICSBP in proliferating epithelial cells but not in the nuclei of nondividing cells in the lens fiber compartment. Constitutive and inducible expression of ICSBP in the ocular lens and differential regulation of its subcellular localization in the developing lens suggest that ICSBP may have nonimmunity related functions and that the commonly held view that it is lymphoid-specific be modified.

IFN-gamma increases the severity and accelerates the onset of experimental autoimmune uveitis in transgenic rats.

Experimental autoimmune uveitis (EAU) is a predominantly Th1-mediated intraocular inflammatory disease that serves as a model for studying the immunopathogenic mechanisms of uveitis and organ-specific autoimmune diseases. Despite the well-documented role of IFN-gamma in the activation of inflammatory cells that mediate autoimmune pathology, recent studies in IFN-gamma-deficient mice paradoxically show that IFN-gamma confers protection from EAU. Because of the implications of these findings for therapeutic use of IFN-gamma, we sought to reexamine these results in the rat, another species that shares essential immunopathologic features with human uveitis and is the commonly used animal model of uveitis. We generated transgenic rats (TR) with targeted expression of IFN-gamma in the eye and examined whether constitutive ocular expression of IFN-gamma would influence the course of EAU. We show here that the onset of rat EAU is markedly accelerated and is severely exacerbated by IFN-gamma. In both wild-type and TR rats, we found that the disease onset is preceded by induction of ICAM-1 gene expression and is characterized by selective recruitment of T cells expressing a restricted TCR repertoire in the retina. In addition, these events occur 2 days earlier in TR rats. Thus, in contrast to the protective effects of IFN-gamma in mouse EAU, our data clearly show that intraocular secretion of IFN-gamma does not confer protection against EAU in the rat and suggest that IFN-gamma may activate distinct immunomodulatory pathways in mice and rats during uveitis.

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.

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.

Congenital toxoplasmosis transmitted from an immunologically competent mother infected before conception.

Congenital transmission of Toxoplasma gondii from a mother who was apparently immunologically competent and who had toxoplasmic lymphadenitis 2 months before conception is described. Since no T. gondii-specific serological data were available for this mother from the time her lymph node biopsy specimen was obtained, the specimen was studied by polymerase chain reaction (PCR) to determine whether the T. gondii B1 gene was present. The predictive diagnostic value of histologic findings previously considered to be classic signs of T. gondii lymphadenitis also was studied. This was done by correlation of serological tests diagnostic of acute acquired T. gondii infection and presence of characteristic findings in biopsy specimens from persons without known immunocompromise. Both PCR and review of the characteristic features of her lymph node biopsy specimen confirmed the diagnosis of preconceptual infection in the mother. We also discuss two other cases in which apparently immunologically competent mothers with preconceptually acquired infection transmitted this parasite to their fetuses.

Synthesis and analysis of the enantiomers of calmidazolium, and a 1H NMR demonstration of a chiral interaction with calmodulin.

Calmidazolium [R24571, 1-[bis(4-chlorophenyl)methyl]-3-[2-(2,4-dichlorophenyl)-2-[(2,4- dichlorophenyl)methoxy]ethyl]-1H-imidazolium chloride] is a potent calmodulin inhibitor. This paper describes the synthesis and properties of the enantiomers of calmidazolium from the enantiomers of miconazole [1(N)-(2-(2,4-dichlorobenzyloxy)-2-(2,4 dichlorophenyl))-ethyl imidazole], prepared from the racemate by chiral preparative scale high performance liquid chromatography. Overlap between ligand and protein resonances in the aromatic region of the 1H NMR spectrum of the calmidazolium-calmodulin complexes has been obviated by preparation of the protein with all of its nine phenylalanine rings deuterated (Phe-d5 calmodulin). This has been accomplished by the overexpression of calmodulin derived from Trypanosoma brucei rhodiesiense in E. coli in a medium supplemented with ring-deuterated phenylalanine. The kinetics of binding of each enantiomer are slow on the 1H NMR time scale as judged by the behaviour of the H2 resonance of Histidine-107, which is clearly visible under the sample conditions used. The aromatic spectral regions of the protein-bound (+) and (-) enantiomers contrast strikingly, reflecting differences in bound environment and/or conformation.

Usage of TCR Vγ2(+) T Lymphocytes in Experimental Autoimmune Uveoretinitis.

Experimental autoimmune uveoretinitis (EAU) is a T cell mediated autoimmune disease that serves as a model of human intraocular inflammatory disease (uveitis). It is initiated in susceptible animals by immunization with retinal antigens, such as interphotoreceptor retinoid binding protein (IRBP) and S-Antigen (SAg) or by adoptive transfer of ocular Ag-specific uveitogenic T cells. Previous studies of T cell receptor (TCR) usage by uveitogenic T cells have implicated Vß8(+) -expressing T cells in the pathogenesis of EAU. Here, the authors have analyzed the TCR Vγ repertoire in the retinas of Lewis rats with and without EAU as well as the repertoire of several SAg- or IRBP-specific T cell lines. They detected Vγ2 transcripts in all four pathogenic lines and in the retinas of Lewis rats with EAU but not in the two non-pathogenic lines nor in the retinas of naive rats. Vγ7 transcripts were detected in RNAs obtained from the retina, regardless of whether the rat had EAU or not. However, the authors could not detect Vγ4, Vγ5 or Vγ6 TCR transcripts in any of the samples analyzed. Taken together, their data suggests a correlation between recruitment of Vγ2(+) T cells and EAU pathogenesis.