Cytotoxicity is a crucial effector mechanism for anti-microbial control and for tumor surveillance. Perforin-1, a pore forming protein expressed and secreted through granule exocytosis by NK and CTL, is a central player for cytotoxicity. Unexpectedly, we discovered that Perforin-1 also has homeostatic functions, since its absence together with Fas-L causes spontaneous lethal disease in mice (cdd - cytotoxic double deficient - disease) which is associated with organ invasion by macrophages and CD8 T cells. The subsequent discovery of human Perforin deficiency and its association with the lethal FHL (familial hemophagocytic lympho-histiocytosis) syndrome in children underlined the importance of Perforin in homeostatic regulation. Competing models have been proposed to explain the mechanisms by which Perforin controls macrophage and CD8 T cell-homeostasis. CD8-CTL fratricide has been proposed as a homeostatic control mechanism for CD8 cells by others; we have proposed a model of homeostatic control in which CTL-perforin is required to kill antigen presenting cells (including macrophages) thereby turning off continued CD8 CTL activation. We propose to further study this negative feed back hypothesis in specific aim 1. ? ? A macrophage expressed mRNA predicting a protein with a membrane attack complex/perforin domain has been identified by several groups. However no information about the identity of the predicted protein has been available. We have finally succeeded to conclusively show that the macrophage expressed mRNA encodes a novel pore forming protein with striking similarities, but also with differences, to Perforin-1. We designated the novel macrophage protein as Perforin-2 (P2) in distinction to classical Perforin-1 (P1) expressed by NK and CTL. P2 is a membrane anchored (tethered) protein with a long phylogenetic history going back to early metazoans such as sponge and sea anemone. There is evidence that sponge P2 has anti bacterial activity and that anemones use it to catch prey. We propose the hypothesis that macrophage P2 is a bactericidal protein and may also have anti tumor activity. In addition macrophage P2 mRNA is endowed with fascinating regulatory controls for protein translation while posttranslational mechanisms appear to control P2-protein polymerization and pore-formation. These mechanisms will be studied in specific aim 2. ? ? ?
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