Development and application of monoclonal antibodies in studies of the human leukotriene B4 receptor, BLT1

Abstract: Infection and inflammation are characterized by the release of chemotactic substances that activate cells of the immune system. One of these important mediators is LTB4, an arachidonic acid derivate that attracts e.g. monocytes and granulocytes to inflammatory sites and induces phagocytosis and degranulation. LTB4 binds to BLT1 which is a 7-transmembrane receptor that belongs to the superfamily of G protein-coupled receptors (GPCRs). BLT1 and LTB4 are not only important in physiological processes but also in several pathological conditions such as psoriasis, arthritis and atherosclerosis. Monoclonal antibodies recognizing BLT1 were developed using the hybridoma technique and they were extensively characterized with regard to specificity and applicability. The resulting two antibodies were then applied in a flow cytometric mapping of human peripheral blood and bone marrow where the highest expression of BLT1 was seen in myeloid cells. One of the antibodies was found to inhibit LTB4 binding to BLT1 while both antibodies could inhibit receptor activation and chemotaxis towards LTB4. These findings demonstrated that one antibody was a competitive antagonist, and the other a non-competitive antagonist. Myeloid expression of BLT1 was further investigated in monocytes using real-time PCR and flow cytomtery. Pro- and anti-inflammatory mediators were shown to modulate both mRNA and protein levels. A closer analysis of IFN-g-mediated effects was conducted and this mediator down-modulated BLT1 mRNA in a time and concentration dependent manner as well as inhibited LTB4 mediated chemotaxis.