Monocyte And Dendritic Cell Development And Trafficking

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Abstract

The ongoing investigations in my laboratory are focused on delineating immunomodulatory mechanisms of innate and adaptive immune responses. We use the p47phox gene knockout mouse as the primary model for our in vivo, ex vivo and in vitro investigations. We also use a novel ex vivo system we developed for differentiating Sca-1+Lin- hematopoietic progenitor cells into functional myeloid precursor, immature and mature dendritic cells. The interrelated objectives of our research focuses on distinguishing phagocyte NADPH oxidase p47phox derived reactive oxygen species (ROS) dependent and ROS independent regulation of dendritic cell, macrophage and T cell function. Work and progress in our group over the past year can be defined in both of the interrelated objectives: (I) NADPH oxidase p47phox ROS independent mediated survival of cytotoxic T cells, (II) Phagocyte NADPH oxidase p47phox-/- dendritic cell regulated T cell function, and (III) Spontaneous systemic and organ specific autoreactive disease in phagocyte NADPH oxidase p47phox-/- mice. We previously reported that T cell receptor (TcR) stimulated T lymphocyte blasts generate NADPH oxidase dependent hydrogen peroxide (H2O2), and that cytokine secretion is skewed towards a T helper 1 phenotype in NADPH oxidase deficient T lymphocytes. This year we expanded on our observations to define a role for p47phox as a critical regulator for CD8 lymphocyte survival. Using in vitro cytokine and antigen stand-in models we found that NADPH oxidase p47phox-/- T cells have a previously unrecognized survival defect that is only partially corrected with exogenous pro-survival cytokines and restoration of H2O2. CD8+ p47phox-/- lymphocytes undergo a rapid and profound apoptosis in vitro and using an adoptive transfer model we found that CD8+ p47phox-/- lymphocytes rapidly die ex vivo. Ongoing investigations indicate that CD8+ p47phox-/- lymphocytes have a defect of an intrinsic survival pathway. The NADPH oxidase enzyme complex is present in all professional phagocytes (macrophages, neutrophils, eosinophils) and also in dendritic cells. Using tissue derived and ex vivo generated myeloid dendritic cells we have characterized p47phox-/- dendritic cell maturation, antigen processing and presentation, and T cell activation. Results from in vitro antigen specific co-cultures using p47phox-/- dendritic cells to activate ovalbumin specific transgenic CD4+ T cells indicate that p47phox-/- dendritic cells significantly enhanced CD4+ T cell proliferation and T helper I cytokine secretion. We intend to further explore this enhancement by investigating immunological synapse interactions, and cytokine regulated pathways in p47phox-/- dendritic cells. A current focus of this project is to characterize the aberrant function of dendritic cells, macrophages and T cells from p47phox-/- mice to understand the consequences in (1) host defense against infection, and (2) autoimmune disease. In addition to our investigations to systematically characterize p47phox-/- immune cell function, we have worked collaboratively with the NIAID comparative medicine branch to examine p47phox-/- mice for evidence of spontaneous systemic and organ specific autoreactive and autoimmune disease. Gross and histological analysis of 1 tp 14 month old p47phox-/- mice showed that aging p47phox-/- mice develop a severe diffuse progressive macrophage pneumonia that is 100% fatal in mice that remain free of infection. The predominant lung lesion is the development of macrophage hyperplasia with intracellular eosinophilic crystals and Chitinase-like Ym1/Ym2 protein expression. In addition spleen, LN and bone marrow macrophages also develop atypical macrophage foci. We have also found that aging p47phox-/- mice develop a membranoproliferative glomerulonephritis with CD3+ infiltrates independent of the macrophage pneumonia.