Analysis of the non-self recognition mechanism of ascidian hemocytes

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1) Analysis of gene expression and signal transduction pathway of ascidian hemocytes : Hemocyte aggregation is an early inflammatory response upon injury in the ascidian Halocynthia roretzi. We have already reported that the monoclonal antibody A74 strongly inhibits hemocyte aggregation in H.roretzi. The A74 antigen protein is a novel membrane glycoprotein, and it has two immunoreceptor tyrosine-based activation motifs (ITAMs) and several motifs that have been proposed to play a role in signal transduction. To identify the proteins that function downstream of ITAMs in H.roretzi hemocytes, we carried out a differential display analysis to evaluate mRNAs differentially expressed before and after hemocyte aggregation. Four amplicons (16A, 18A, 20A and 20G-1), the expression levels of which increased after induction of hemocyte aggregation, were isolated. Their expressions were found to be induced by treatment with A74 antibody, but not by control mouse IgG, and were strongly inhibited by … More treatment with BAPTA-AM, Wortmannin and cyclosporin A.From the results of cDNA cloning, it was revealed that 18A-1 clone encoded a glutathione S-transferase omega (GSTO) protein, and 20G-1 clone encoded a novel protein that had little similarity to other proteins. We also found that GSTO gene expression was specifically induced in mouse T-cell hybridoma by treatment with anti-TCR antibody.2) Analyses of opsonic factors in H.roretzi plasma and complement 3 receptor of H.roreti hemocytes : H.roretzi plasma contains two opsonic factors, galactose-specific lectin (Gal-lectin) and complement 3. Using their antibodies and a fluorescence-activated-cell sorter, we found that Gal-lectin bound only to SRBC, while C3 bound only to yeast cells. Furthermore, we found that H.roretzi hemocytes had an integrin α subunit protein and that its antibody inhibited C3-dependent phagocytosis of H.roretzi hemocytes. These results suggest that H.roretzi hemocytes have an integrin-type receptor that functions as a C3 receptor. Less