Corneal stem cells: bridging the knowledge gap.

Stem cell research offers hope to countless patients whose conditions have heretofore been deemed incurable. Better understanding of stem cell behaviors and functions will lead to insights into biological mysteries encompassing the fields of angiogenesis, development, tissue homeostasis, wound healing, and carcinogenesis. Clarity of vision requires smooth ocular surface on which the corneal epithelial cells undergo continuous turnover every 3 to 10 days. Tragically, many patients are blinded and devastated by severe ocular surface diseases due to limbal stem cell deficiency even though, besides opaque cornea, their eyes are otherwise healthy. Corneal stem cell transplantation offers hope by creating clear windows for these eyes; unfortunately, the long-term successful outcome remains limited. The nature of corneal epithelial stem cell is poorly understood, but many circumstantial evidences suggest the presence of "source cells" in the limbal region of the eye. Nonetheless, the precise biomarker of corneal stem cell remains elusive. The stem cell puzzle can be solved with application of the fundamental scientific method-asking salient questions at the right time and finding answers using keen observations and proper tools. Readily accessibility and structural simplicity of the cornea lend themselves to study of the stem cell biology. The ability to identify and isolate corneal stem cell will be a gateway to meaningful investigation into its biology. This advance will also have direct impact on improving the efficacy of promising stem-cell-based therapies, including limbal stem cell transplantation.

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.

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.