Background: Lupus nephritis is a leading cause of morbidity and mortality for lupus patients. Although current immunosuppressive regimens have dramatically improved its prognosis they are not uniformly effective and are associated with significant toxicities. Elucidation of its pathogenesis and improvement of available theurapeutic regimens are of paramount importance. Production of pathogenic autoantibodies to a limited number of nuclear and cytoplasmic antigens and formation of immune complexes are the hallmarks of the disease. Autoantibody production is antigen driven and T cell dependent. Studies in animals and humans have demonstrated the essential role of interactions involving the CD40 - CD40L pair of molecules in this process. CD40L (ligand) is a 33Kda, type II membrane glycoprotein that is transiently expressed primarily on the surface of activated CD4+ T cells. Interaction of CD40L with its receptor CD40, which is constitutively expressed by B cells and macrophages, is involved in both humoral and cellular immune responses CD40L is expressed on <1% of human CD4+ T cells from normal controls, but on >17% of CD4+ T cells from active SLE patients. Persistent expression of CD40L has been reported on T cells of lupus patients by several goups. Upregulation of CD40 expression has also been observed in renal biopsy specimens from patients with proliferative but not membranous lupus nephritis. Antibodies to CD40L ameliorate nephritis in several animal models of lupus. Tissue injury in lupus is mediated predominantly by deposition of immune complexes (IC) in tissues such as kidneys. Lupus prone mice deficient for g-chain of Fcg receptor (FcgR) are protected from severe nephritis in spite of extensive deposition of IC in the kidneys and complement activation suggesting that signaling through the FcgR represents an important effector pathway in this disease. The functional domain of the g chain, the ITAM, has two fundamental attributes, namely signal transduction and internalization, and mutational analysis has shown that these two functions can be dissociated. FcgRIIIa is expressed on mononuclear phagocytes and renal mesangial cells and is coded by a polymorphic gene located in a region on chromosone 1. This region has been linked to lupus by microsatellite-based scanning of the genome of lupus patients. Mutations that affect IC internalization may affect IC clearance and thus may lead to prolonged stimulation of cells bearing Fcg receptors. Objectives1. To further delineate the role of CD40L-CD40 interaction between T cells and macrophages/mesangial/tubular epithelial cells in the pathogenesis of lupus nephritis. 2. To examine the therapeutic activity of anti-CD40L therapy in patients with lupus nephritis. 3. To examine the impact of anti-CD40L therapy on cellular interactions between T cells and other cells, and autoantibody production. 4. To study the natural history of lupus nephritis treated with different immunosuppressive regimens and to examine their long-term toxicity and efficacy.5. To develop improved outcome criteria and/or surrogate markers to be used for future trials of lupus nephritis. 6. To collect DNA from patients and their parents for studies of lupus nephritis susceptibility and severity genetic markers. 7. To study the biology of FcgR in mononuclear phagocytes and renal mesangial cells in lupus nephritis by:a) examining the binding, endocytosis and processing of immune complexes as well as the biologic response of mononuclear and mesangial cells, and b) correlating binding/response with polymorphisms of the FcRg-chain.Results. 1. Role of CD40-CD40L Interactions In co-culture experiments, activated peripheral blood T cells stimulated human renal tubular epithilial cells to produce chemotactic cytokines (MCP-1, IP-10,RANTES). Both soluble factors (TNF-a, IL-1b) and cell contact via b-2 integrins (LFA-1 and Mac-1)and CD40 are involved in this process. In on-going experiments normal monocytes are being compared to monocytes from lupus patients. In contrast to previous findings suggesting deteective monocyted function, monocytes from lupus patients produce normal amounts of IL-12 and IL-18 two cytokines known to promote Th1 immune responses. 2. Anti-CD40L Therapy A multicenter study sponsored by Biogen, Inc. has been initiated with NIH as one of the 3 leading centers. The study will involve 30 patients and is expected to be completed in 1 year. Thus far we have evaluated over 30 patients and have enrolled 6 patients in this study. In addition to clinical parameters immune responses before and after treatment will also be analyzed in detail. These will also involve both T and B cell responses and urine chemokine production. 3. Natural History and Outcome Criteria. The traditional end points of lupus nephritis (end-stage renal disease, doubling of serum creatinine or remission) require long periods of follow-up and a large number patients to demonstrate differences among treatment groups. Our current efforts involve analysis of data obtained during previous studies to improve outcome criteria and/or identify surrogate markers. Patients participating in our most recent randomized trial of immunosuppressive drugs have participated in a comprehensive evaluation of long term morbidity (associated with both disease and with therapy) and efficacy. In a preliminary analysis, we have examined the prevalence of renal flares after immunosuppressive therapy and have introduced a new classification for therapeutic and prognostic purposes. 4. Genetic Markers for Susceptibility/Severity. We have collected DNA from over 140 patients with lupus nephritis as well as from 26 parents of these patients. These samples will be used for studies to identify the role of putative genetic markers in determining susceptibility to disease, its severity and its response to therapy. 5. Role of Fcg Receptors. To date, we have collected elutriated monocytes from 16 patients with lupus nephritis and 15 normal donors. In addition, we have established mesangial cell lines from 4 lupus patients and 4 non-lupus nephrectomy specimens obtained from anatomic pathology. We have detected tyrosine phosphorylation of multiple intracellular proteins in these cell lines when stimulated with human IC. This is associated with production of cytokines which is our biologic read out assay. In addition, we have sequenced the FcRg gene in genomic DNA obtained from 24 patients with lupus nephritis and 24 race-matched normal controls. No polymorphisms / mutations were found in the coding regions of the gene. However, we have identified novel polymorphisms in the intron between exons 2 and 3 and exons 4 and 5 present in 4 patients and in 3 normal controls. Exploration of the impact of this polymorphisms in FcRg expression is in progress. Lay SummaryInvolvement of the kidneys (nephritis) is the main cause of morbidity and mortality in patients with systemic lupus erythematosus. In this so called autoimmune disease, the body turns against itself producing antibodies directed to molecules found in body tissue (antigens). In lupus nephritis complexes of antigens with antibodies (immune complexes) deposit in the kidney and cause inflammation. Our studies are aimed at a) identifying the role of a molecule called CD40 ligand in injuring the kidney of patients with lupus and b) the response of kidneys to deposition of immune complexes. To this end, we use molecules which interfere with function of CD40 ligand (anti-CD40 ligand antibodies) and examine their effects in patients with lupus nephritis. Moreover, we grow cells from kidney biopsies of these patients and examine their ?functions? in the test tube. Finally, we monitor a group of over 150 lupus patients that have participated in studies on the treatment of lupus nephritis in an effort to better understand the course of the disease and the long-term side effects of treatment.
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