Date on Master's Thesis/Doctoral Dissertation

5-2007

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Committee Chair

Bodduluri, Haribabu

Author's Keywords

Leukotriene; Inflammatory arthritis; Arthritis

Subject

Leukotrienes; Inflammation; Arthritis

Abstract

Inflammation is now recognized as an important factor in several age-related diseases such as arthritis, atherosclerosis, multiple sclerosis and diabetes. In each case, sub-clinical chronic inflammation occurs over years and leads to progressive destruction of the tissue until the symptoms become clinically apparent. Eicosanoids such as prostaglandins and leukotrienes play an important role in inflammation. Among these, leukotriene B 4 (LTB 4 ) has had a long history of being associated with numerous inflammatory diseases. A high affinity receptor for LTB 4 , LTB 4 receptor 1 (BLT 1) has been well-characterized in human and murine tissues. While the molecular mechanisms remain unclear, its importance in diverse inflammatory diseases was demonstrated in mice lacking BLT1. More recently, a second highly conserved low affinity LTB 4 receptor, LTB 4 receptor 2 (BLT2) was identified, but its functional significance remains completely unknown. Both BLT1 and BLT2 are seven-transmembrane G protein coupled receptors transducing signals through heterotrimeric G-proteins. Chapter II of this thesis describes the generation and characterization of monoclonal antibodies to BLT1. Immunization of BLT1-deficient mice with 300.19, a murine pre-B cell line expressing high levels of either human or murine BLT1 allowed isolation of highly species specific anti-BLT1 antibodies. Using an approach involving a series of human/murine BLT1 chimeric receptors the monoclonal antibody binding sites were mapped to extracellular loop-2. Extensive characterization of BLT1 in murine primary cells revealed high levels of BLT1 in neutrophils and eosinophils and a previously unsuspected regulation of BLT1 in macrophages. While bone marrow derived macrophages expressed high levels of BLT1 its expression in peripheral tissue macrophages was significantly reduced suggesting a potential novel mechanism, and a role for BLT1 in inflammation-induced egress of bone marrow derived cells. The antibodies also allowed demonstration of normal BLT1 expression in the recently generated BLT2 deficient mice. Future studies could explore the therapeutic potential of these antibodies. In Chapter III, we investigated the role of BLT1 and BLT2 in inflammatory arthritis in mouse models. Collagen induced arthritis (CIA) is a model where mice are immunized with chicken collagen type II in complete freund's adjuvant and leads to a polyarthritis in the distal joints. BLT1 deficient mice on the DBA/1 background were completely protected from the development of CIA which confirms the previous observations with BLT1 antagonists and BLT1 deficient mice on the C57B1/6 background. Arthritis was also induced by transfer of K/BxN serum to naÏve mice. K/BxN is a T cell receptor transgenic mouse (KRN) crossed with a NOD mouse. These mice spontaneously develop arthritis due to auto-antibody production against glucose-6-phosphate isomerase, a ubiquitous enzyme. In this model, we report for the first time that BLT2 deficient mice are completely protected from inflammatory arthritis. Histopathologic examination revealed a massive inflammatory cell influx in wild-type mice that was completely absent in BLT2 deficient mice. Further analysis of these mice using bone marrow transplantation studies demonstrated development of arthritis requires BLT2 expression on bone marrow-derived cells. Wild-type mice which received bone marrow from BLT2 -/- mice showed little sign of disease on histological analysis, while BLT2-/- mice receiving wild-type bone marrow showed intense inflammation leading to severe destruction in cartilage and bone. When BLT1/BLT2 double deficient mice were transplanted with a mixture of BLT1 deficient and BLT2 deficient bone marrow, a gain of clinical disease was observed. All these data demonstrate that BLT2 has a critical role in arthritis and it is non-redundant with the role played by BLT1. Further study is needed to determine the function of BLT2 and in which cell types it is importa

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