Santa Jeremy Ono

Biology and genetics of atopic disease

Several immunological disorders including allergic rhinitis, bronchial asthma, atopic dermatitis, food allergies, urticaria, nonhereditary angioedema, systemic anaphylaxis, and allergic conjunctivitis are associated with a positive family history, and share a positive response in the Prausnitz-Kuster (wheal and flare) reaction. Studies have shown that 20-30% of the population has a strong genetic predisposition for this condition, termed atopy, whose hallmark is a greatly elevated serum IgE concentration. A great deal is known about the cellular interactions that mediate the sensitization, immediate and late-phase reactions that follow encounters with allergen, as well as about the cell surface and signaling events that result in mediator release from inflammatory cells. Less is known of the genes that confer genetic predisposition for atopy; however, a worldwide effort to identify atopy genes is making significant progress.

Macrophage inflammatory protein–1α as a costimulatory signal for mast cell–mediated immediate hypersensitivity reactions

Regulation of the immune response requires the cooperation of multiple signals in the activation of effector cells. For example, T cells require signals emanating from both the TCR for antigen (upon recognition of MHC/antigenic peptide) and receptors for costimulatory molecules (e.g., CD80 and CD60) for full activation. Here we show that IgE-mediated reactions in the conjunctiva also require multiple signals. Immediate hypersensitivity reactions in the conjunctiva were inhibited in mice deficient in macrophage inflammatory protein-1alpha (MIP-1alpha) despite normal numbers of tissue mast cells and no decrease in the levels of allergen-specific IgE. Treatment of sensitized animals with neutralizing antibodies with specificity for MIP-1alpha also inhibited hypersensitivity in the conjunctiva. In both cases (MIP-1alpha deficiency and antibody treatment), the degranulation of mast cells in situ was affected. In vitro sensitization assays showed that MIP-1alpha is indeed required for optimal mast cell degranulation, along with cross-linking of the high-affinity IgE receptor, FcepsilonRI. The data indicate that MIP-1alpha constitutes an important second signal for mast cell degranulation in the conjunctiva in vivo and consequently for acute-phase disease. Antagonizing the interaction of MIP-1alpha with its receptor CC chemokine receptor 1 (CCR1) or signal transduction from CCR1 may therefore prove to be effective as an antiinflammatory therapy on the ocular surface.

Circulating anti‐retinal antibodies as immune markers in age‐related macular degeneration

Age‐related macular maculopathy (ARM) and age‐related macular degeneration (AMD) are the leading causes of blindness in the Western world. Despite the magnitude of this clinical problem, very little is known about the pathogenesis of the disease. In this study, we analysed the sera (using indirect immunohistochemistry and Western blot analysis) from a very large cohort of such patients and normal age‐matched controls to detect circulating anti‐retinal antibodies. Patients with bilateral drusen (n = 64) and with chorioretinal neovascularization (CNV) (n = 51) were recruited in addition to age‐matched control subjects (n = 39). The sera were analysed for anti‐retinal immunoglobulins on retinal sections. The data were then correlated with the clinical features graded according to the International Classification and Grading System of ARM and AMD. The sera of patients with drusen (93·75%) and CNV (82·27%) were found to have a significantly (P = 0·02) higher titre of autoantibodies to the retina in comparison with controls (8·69%), indicating significant evidence of involvement of the immune process in early stages of AMD. Subsequent statistical analysis of the drusen group showed significant progressive staining (P = 0·0009) in the nuclei layers from early to late stages of ARM. Western blotting confirmed the presence of anti‐retinal immunoglobulins to retinal antigens. As anti‐retinal immunoglobulins are present in patients with bilateral drusen and exudative AMD, these antibodies could play a significant role in the pathogenesis of AMD. Whilst we do not have evidence that these antibodies precede disease onset, the possibility that their presence might contribute to disease progression needs to be investigated. Finally, the eventual identification of the target antigens detected by these antibodies may permit the future development of new diagnostic methods for ARM and AMD.

Retinal transplantation: progress and problems in clinical application

There is currently no real treatment for blinding disorders that stem from the degeneration of cells in the retina and affect at least 50 million individuals worldwide. The excitement that accompanied the first studies showing the potential of retinal cell transplantation to alleviate the progress of blindness in such diseases as retinitis pigmentosa and age‐related macular degeneration has lost some of its momentum, as attempts to apply research to the clinic have failed so far to provide effective treatments. What these studies have shown, however, is not that the approach is flawed but rather that the steps that need to be taken to achieve a viable, clinical treatment are many. This review summarizes the course of retinal transplant studies and points to obstacles that still need to be overcome to improve graft survival and efficacy and to develop a protocol that is effective in a clinical setting. Emphasis is given particularly to the consequences of introducing transplants to sites that have been considered immunologically privileged and to the role of the major histocompatibility complex classes I and II molecules in graft survival and rejection.

Autoimmunity in retinal degeneration: Autoimmune retinopathy and age-related macular degeneration

Autoantibody production is associated with a variety of ocular disorders, including autoimmune retinopathy (AIR) and age-related macular degeneration (AMD). A breakdown of immunologic tolerance (ocular immune privilege), including the blood-retinal barrier, anti-immune and anti-inflammatory proteins, and anterior chamber-associated immune deviation may play important roles in these disorders. Although the exact triggers for ocular autoimmunity are unknown, autoimmune targeting of retinal tissue is clearly associated with and may contribute to the pathogenesis of both AIR and AMD. Autoantibody production has long been associated with AIR, a collection of disorders that includes cancer-associated retinopathy, melanoma-associated retinopathy and non-paraneoplastic autoimmune retinopathy. A growing body of evidence indicates that AMD pathogenesis, too, involves ocular inflammation and autoimmunity. Identification and quantification of autoantibodies produced in patients with AIR and AMD may assist with diagnosis, prognosis, and choice of treatments. Animal models that allow investigation of ocular autoimmunity will also be needed to better understand the disease processes and to develop novel therapies. In this review we discuss ocular immune privilege and potential mechanisms of autoimmunity in the eye. We describe how autoimmunity relates to the pathogenesis of AIR and AMD. We explain how the antigen microarray technique is used to detect autoantibodies in patient serum samples, and discuss how current animal models for AMD can be used to investigate autoimmune pathogenesis. Finally, we outline unanswered questions and exciting areas of future study related to autoimmune retinal degeneration.

Genomics and proteomics of allergic disease

The world‐wide effort to identify susceptibility genes for allergic diseases is motivated by the conviction that the identification of disease genes may permit the design of new classes of anti‐inflammatory compounds. Molecules concerned with the allergic reaction, such as cytokines, chemokines, their receptors, major histocompatibility complex molecules, and transcription factors, could provide the candidate genes of the allergic diseases. On the basis of genetic studies, multiple research groups have attempted to identify a susceptibility gene for allergy using the candidate gene approach and/or genome‐wide screening. Both of these approaches suggest genetic heterogeneity of allergic diseases. Many variants of candidate genes are or are not associated with particular diseases in different ethnic groups and the function of variants is now being investigated. Based on the information accumulated thus far and the information on the human genome sequence, future advances in research on genetic factors for allergic diseases will be likely lead to the establishment of more effective prophylaxis and therapy for these diseases.

How are class II MHC genes turned on and off

Fragments of foreign antigen are detected by CD4+ helper T cells via the T cell receptor for antigen in the context of major histocompatibility complex (MHC) class II molecules. Very few cells normally express class II MHC molecules, and these cells play critical roles in antigen presentation and in the thymic selection of T lymphocytes before their exit into the periphery. Because of the central role the class II MHC molecules play in immune system function, it is not surprising that the lack of expression of these molecules results in a severe combined immunodeficiency disorder (called bare lymphocyte syndrome) and that the aberrant expression of the molecules is frequently observed in the target organs of various autoimmune disorders (e.g., multiple sclerosis and rheumatoid arthritis). Because both classes of disease could conceivably be treated by molecular approaches targeted at either restoring or inhibiting expression of class II MHC genes, there has been an intense effort during the past decade to elucidate the regulatory mechanisms of class II MHC genes. An analysis of recent advances in this effort is provided in this reviewr article.—Abdulkadir, S. A., Ono, S. J. How are class II MHC genes turned on and off? FASEBJ. 9. 1429‐1435 (1995)

Functional domains of the cone-rod homeobox (CRX) transcription factor.

The paired-like homeodomain transcription factor CRX (cone-rod homeobox) is involved in regulating photoreceptor gene expression and rod outer segment development. Mutations in CRX have been associated with several retinal degenerative diseases. These conditions range from Leber congenital amaurosis (a severe cone and rod degeneration of childhood onset) to adult onset cone-rod dystrophy and retinitis pigmentosa (an adult onset condition that primarily affects rods). The goal of this study is to better understand the molecular basis of CRX function and to provide insight into how mutations in CRX cause such a variety of clinical phenotypes. We performed deletion analysis in conjunction with DNA binding and transient transfection-based transactivation studies to identify the functional domains within CRX. DNA binding requires a complete homeodomain. Furthermore, truncated proteins that did not contain an intact homeodomain failed to demonstrate detectable expression in tissue culture upon transfection. Transactivation analysis indicated that both the OTX tail and the WSP domain are important for controlling positive regulatory activity of CRX. Interestingly, the mapped CRX transactivation domains were also critical when coexpressed with NRL. Specifically, the synergy between CRX and NRL was constant regardless of which CRX variant was used.

Identification of anti-retinal antibodies in patients with age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in industrial counties. Recent findings indicate that the autoimmunity is involved in the pathogenesis of the disease. However, there is no autoantibody biomarker applied in a clinical setting for diagnosis and prognosis of AMD. In order to reveal retinal antigens targeted by serum IgG from AMD patients, mouse retinal tissue proteins were separated by 2-dimensional electrophoresis and the proteins in the immunoblots that were specific for dry and wet AMD patients IgG were identified by LC-MS/MS. Retinol-binding protein 3 and aldolase C (ALDOC) were mainly recognized by IgG form wet AMD patients. Pyruvate kinase M2 (PKM2) was targeted by both dry and wet AMD and level of anti-PKM2 IgG antibody was correlated best with the stage of AMD. Expression of ALDOC and PKM2 was decreased in mouse retina from aging whereas PKM2 deposit on RPE was increased in aged mice. Our data demonstrate that sera of AMD patients contain autoantibodies against retinal proteins and anti-PKM2 IgG serves as a biomarker for diagnosis and prognosis of AMD. Further investigation of the association of anti-retinal antibody level with expression level of antigens in retina will be needed to reveal the disease pathogenesis.

Identification and Characterization of a Critical CP2-binding Element in the Human Interleukin-4 Promoter

Expression of cytokine genes in T cells is thought to result from a complex network of antigen- and mitogen-activated transcriptional regulators. CP2, a factor homologous to Drosophila Elf-1 and previously found to be a critical regulator of several viral and cellular genes in response to developmental signals, is rapidly activated in T helper (Th) cells in response to mitogenic stimulation. Here we show that overexpression of CP2 enhances interleukin (IL)-4 promoter-driven chloramphenicol acetyltransferase expression, while repressing IL-2 promoter activity, in transiently transfected Jurkat cells. A CP2-protected element, partially overlapping the nuclear factor of activated T cell-binding P2 sequence, was required for IL-4 promoter activation in CP2-overexpressing Jurkat cells. This CP2-response element is the site of a cooperative interaction between CP2 and an inducible heteromeric co-factor(s). Mutation of conserved nucleotide contacts within the CP2-response element prevented CP2 binding and significantly reduced constitutive and induced IL-4 promoter activity. Expression of a CP2 mutant lacking the Elf-1-homology region of the DNA-binding domain inhibited IL-4 promoter activity in a dominant negative fashion in transiently transfected Jurkat cells. Moreover, overexpressed CP2 markedly enhanced, while its dominant negative mutant consistently suppressed, expression of the endogenous IL-4 gene in the murine Th2 cell line D10. Taken together, these findings point to CP2 as a critical IL-4 transactivator in Th cells.