Marko Z. Radic - US grants

Antigen selection and affinity maturation of autoantibodies leave a genetic imprint indicative of the type of antigen that is bound in vivo. Therefore, a detailed investigation of genetic mechanisms that generate the immunological diversity of autoantibodies should provide crucial information about the induction of autoimmunity. The principle of antigen selection has been tested with murine anti-DNA antibodies and recent studies suggest that it also applies to a wide range of autoantibodies that arise in human autoimmune disease. This application proposes to analyze by in vitro mutagenesis three autospecificities that are typical of Systemic Lupus Erythematosus: anti- double stranded DNA, anti-histone, and anti-snRNP. In particular, the effects of V gene use, rearrangement, and somatic mutation will be evaluated in three antibodies that have dual specificity and bind either histones or snRNP in addition to DNA. The focus of the studies will be to learn: a) the relative contributions of the different diversity-generating mechanisms, and b) the order in which the three specificities arise. It is hoped that conclusions from these studies will be relevant for the etiology and possible treatment of Systemic Lupus Erythematosus. The experiments described have the following aims: Specific Aim 1: Clone and express anti-DNA V genes. The heavy (H) and light (L) chain V genes used by three hybridomas 3H9, LG8-1, and 1-42 will be cloned and manipulated in vitro, in order to determine the structural basis for dsDNA, nucleosome, and SmD binding. These experiments will involve chain recombination, VHCDR3 grafting, and site-directed mutagenesis. Specific Aim 2: Test antibody specificity for dsDNA conformation. Purified mutant anti-dsDNA antibodies will be used to establish whether DNA curvature improves DNA binding or not. If conformational preference is found, then: a) The size and location of bound DNA sequences will be established using Exonuclease III and ethylation interference footprinting. b) Single chain antibody fragments (scFv) will be constructed to distinguish whether preference is intrinsic to each combining site or is a consequence of IgG structure. Specific Aim 3: Explore antibody specificity for dsDNA sequence. In order to evaluate the potential for DNA sequence specificity, oligonucleotides of initially random composition will be processed through several cycles of affinity enrichment and PCR purification. Affinity chromatography will be on anti-dsDNA antibodies or anti-dsDNA scFv linked to functional domains from the transcriptional factor GCN4 and from protein A. Substrates containing a palindromic core sequence will be used to reduce the lateral displacement of bound antibodies.

Macrophage participate or play a leading role in all stages of atherosclerosis. Therefore, identifying and regulating genes that code for macrophage functions in atherosclerotic lesions has the potential of preventing many aspects of this vascular disease. The present application proposes to adapt anti-DNA antibodies for the purpose of repressing genes that promote the atherosclerotic process and depend on the AP-1 transcription factor for expression. Preliminary data suggest that the AP-1 dependent transcription of the interstitial collagenase (MMP-1) gene is effectively repressed by intracellular fusion proteins consisting of a single-chain anti-DNA Fv fragment (scFv) and leucine zipper (Lz) domains derived from c-Jun or c-Fos. The goal of the present proposal is to extend these studies to other AP-l activated genes expressed by macrophage that are implicated in aspects of atherosclerosis. Expression vectors for anti-DNA scFv-LZ fusion proteins will be transfected into the human U937 and THP-1 monocytic cell lines as well as into purrried monocytes from peripheral blood. The expression of the genes for the following proteins will be investigated to establish the extent to which they are susceptible to AP-1 inhibition: 1. The beta2 integrin family adhesion molecule CD11c (p150,95) that is predominantly expressed on monocytes and macrophage and contributes to their adhesion to activated endothelial cells; 2. The matrix metalloprotease MMP-1 that is expressed in a cell tppe selective manner by monocytic cells and contributes to extracellular matrix (ECM) degradation and fibrous plaque rupture; 3. The scavenger receptor that becomes expressed as monocytes differentiate into foam cells and that mediates oxidized low density lipoprotein absorption; 4. Tissue factor, an enzyme expressed on the surface of foam cells in atherosclerotic lesions that acts as a cofactor for Factor VIIa and thus participates in the first reaction of the extiiinsic pathway of coagulation. There is preliminary evidence that each of these genes requires AP-1 for trnscription and therefore should be repressed by AP-1 inhibition. It is hoped that the proposed experiments will lay the foundation for the future testing of these treatments in animal models of atherosclerosis.

DESCRIPTION( provided by the applicant): Compelling evidence links the initiation and progression of Systemic Lupus Erythematosus (SLE) and Anti-Phospholipid Syndrome (APS) to aberrant immune responses to apoptotic cells. However, the circumstances that lead to the production of anti-nuclear autoantibodies, the hallmark of SLE, and of autoantibodies to complexes between serum proteins and membrane phospholipids, the serologicalI markers for APS, remain poorly understood. B cells with Ig receptors for apoptotic cells may be in a position to gain access to relevant autoantigens. Following positive selection by autoreactive T cells, such B cells may be induced to secrete autoantibodies to apoptotic cells (ATAC), and disrupt normal clearance mechanisms, thus perpetuating and even expanding the autoimmune response. Alternatively, ATAC may arise secondary to defects in clearance, or an overabundance of apoptotic cell remnants. To distinguish between these possibilities, more detailed information on the genetics, regulation, and function of ATAC is needed. This proposal aims to derive ATAC from mice with defects in phagocytosis of apoptotic cells. The specific aims are: 1) Test sera of Mer, Axl, Tyro3 triply nullizygous mice for the expression of ATAC, and construct hybridomas from positive mice; 2) Screen hybridomas for ATAC specificity by flow cytometry and confocal microscopy and survey their relationship to other autoreactive hybridomas from the same fusions; 3) Determine the genetic basis for ATAC specificity and compare the contributions of immunoglobulin heavy and light chain V gene use, junctional diversity, and somatic mutations to the recognition of apoptotic cells. Future studies will explore the contribution of ATAC-bearing B cells to autoimmune responses against nuclear and cell surface antigens, the effect of soluble ATAC on the clearance of apoptotic cells by macrophage, and the regulation of ATAC-bearing B cells. The results of these studies will allow a fuller assessment of the role of apoptotic cells in the induction of B cell tolerance or autoimmunity.