Phagocytosis is the engulfment of large particles by eukaryotic cells. As an integral part of the immune system in animals, it plays an essential role in the uptake and destruction of microbes as well as in the clearance of apoptotic and necrotic cells. Phagocytosis of microbes and necrotic cells generally leads to inflammation, whereas apoptotic cells are cleared in a non-inflammatory manner. In mammals, phagocytosis is primarily carried out by certain cell types that are specialized for phagocytosis; these ''professional phagocytes,'' of myeloid origin (''white blood cells''), include polymorphonuclear leukocytes(PMNs, also sometimes called granulocytes), monocytes and macrophages. Until recently, it was thought that, in fish, the same was true, i.e., that granulocytes and macrophages were the main phagocytic cells in fish. Contrary to this belief, studies carried out in Dr. Sunyer''s laboratory with prior NSF support led to the exciting finding that B cells (immune cells that are the source of antibodies, or immunoglobulins) in fish have potent phagocytic abilities and comprise a significant fraction of the ''professional phagocytes'' in these species. These surprising findings were reported in Nature Immunology and made the cover of the 2006 October issue. This renewal project builds on three key observations made during the study of phagocytic B cells in rainbow trout: 1) gene expression and antibody staining analysis suggesting that B cells of teleosts can be divided at least into two major populations, one expressing IgM and the other expressing IgT (an immunoglobulin unique to these fish; 2) The finding that all IgM+ B cells express significant levels of Il-1, TNF-a, C3aR and C5aR, a group of pro-inflammatory molecules that in mammals is restricted to innate inflammatory leukocytes of myeloid origin (i.e., macrophages); 3) The identification and expression of the phosphatidylserine receptor (PSR) in trout IgM+ B cells, a finding that suggests the involvement of these lymphocytes in the removal of apoptotic cells (this role in mammalian leukocytes is restricted to macrophages and dendritic cells). Based on the aforementioned observations and because of the unusually high numbers of B cells in fish, it is hypothesized that B cells in these species represent an essential component of the inflammatory response and contribute to homeostasis by participating in the clearance of apoptotic cells. Thus, the aims of this project are: 1, to analyze phenotypic, phagocytic and bactericidal differences between IgM+ and IgT+ lymphocyte populations. Serological methods will be used to separate IgM+ B cells from IgT+ B cells, in order to investigate and compare the phagocytic and microbicidal activities of these two lymphocyte populations. 2, to determine the contribution by phagocytic B cells to the inflammatory response. To address the hypothesis that B cells in fish respond to inflammatory stimuli, the expression of various pro-inflammatory mediators (e.g., interleukins, TNFs, COX-1 and COX-2) in vivo an in vitro will be analyzed after stimulation of fish or cells with endogenous (i.e., C5a, C3a) or exogenous (i.e., microbial compounds, bacteria) stimuli. 3, to determine the contribution of phagocytic B cells to the clearance of apoptotic and necrotic cells. To test the hypothesis that trout IgM+ B cells may be involved also in the non-inflammatory clearance of apoptotic cells, the capacity of IgM+ and IgT+ B cell subsets to phagocytose trout apoptotic and necrotic cells will be assessed and compared to that of trout granulocytes and macrophages.
Broader Impacts: The PI will make available purified IgT to the US Veterinary Reagent Network with the goal of generating monoclonal antibodies that will be freely distributed, thus benefiting the entire immunology research community. This project will involve the training of a postdoctoral fellow and a graduate student. In addition, the PI will continue his annual participation in the Comparative Immunology Symposium held at Florida International University (FIU), a minority serving institution where most of the attending students are primarily from underrepresented groups. On a broader level, the studies proposed here will benefit society by contributing to the aquaculture industry, since a better knowledge of the fish immune system is essential to the development of new therapeutic agents and vaccines to prevent fish diseases. The PI will continue presenting the results of his research in national and international conferences.
