The granular exocytosis pathway is one mechanism by which natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) induce cytolysis of target cells. Adhesion molecules such as CD 2 and LFA-1, as well as F_<cγ> RIII (CD 16), are believed to be involved in this pathway. Here we report that crosslinking of CD 2 as well as CD 16 by immobilized antibodies enhances granular exocytosis in an NK cell line, NK 3.3. Herbimycin, a protein tyrosine kinase (PTK) inhibitor, or wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (Pl 3-K), either completely inhibited, or essentially abrogated, CD 2-and CD 16-mediated granular exocytosis, suggesting the involvement of PTKs and Pl 3-K in both CD 2-and CD 16-mediated granular exocytosis. We also observed that crosslinking of CD 2 as well as CD 16 enhanced p 72^syk tyrosine kinase activity, and that this enhancement correlated well with the increased tyrosine phosphorylation of several cellular proteins, including the adapter protein, Shc. Furthermore, we observed that crosslinking of CD 2 as well as CD 16 enhanced the Pl 3-K activity associated with the tyrosine phosphorylated cellular proteins and Shc. These results provide insight into the signaling pathways by which triggering of CD 2 and CD 16 on NK cells leads to cytolysis of target cells.

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Natural killer (NK) cells have cytolytic activity without prior antigenic stimulation, and IL-2 activated NK cells are used for treatment of melanoma and renal cell carcinoma. However, the advantage of this immunotherapy is limited by the side effects of vascular damege and insufficient accumulation in the target tumor cells. Chemokines were initially reported as chemoattractant cytokines synthesized at inflammation sites. Monocyte chemoattractant protein-1 (MCP-1) is one of the CC-chemokine family and is reported to induce chemotaxis of monocytes, T cells and NK cells. We investigated the effect of MCP-1 on degranulation and cytotoxicity of the NK cell line, NK 3.3 cell. Expression of CD 16 and non-expression of CD 3 in the NK 3.3 cells were remarkably similar to the phenotype of fresh NK cells. In addition, cross-link stimulation of CD 2 and CD 16 induced degranulation from NK 3.3 cells. IL-2, but not MCP-1 or IL-12, markedly enhanced proliferation of NK 3.3 cells. MCP-1, IL-2 and IL-12 also induced degranulation from NK 3.3 cells. Kinetic studies revealed that MCP-1 mediated enhancement of degranulation occurred within one hour, and was sustained throughout the assay. MCP-1 also enhanced the cytotoxic activity of NK 3.3 cells in a dose-dependant manner. These results suggest that MCP-1 may enhance the cytotoxicity of NK cells as a result of granular exocytosis and produce an improvement in the immunotherapy.

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We studied the rearrangement and amplification of the myc (c-myc, N-myc, L-myc) and ras (H-ras, K-ras) gene families, and the c-erbB2 gene in rat free-cell type tumor (CTS) cells derived from Tawa sarcoma. Southern-blotting analysis showed that CTS cells presented a different pattern from normal rat fibroblast cells in c-myc and c-erbB2 probe hybridization, indicating that the molecular weights of c-myc and c-erbB2 fragments in CTS cells differed from those in normal cells. This implies that the c-myc and c-erbB2 in CTS cells had been rearranged. Slot-blotting analysis showed that the myc and ras gene families, and the c-erbB2 gene in CTS cells had the same intensity as those in the control, indicating an absence of amplification of these genes in CTS cells. These observations may contribute to our understanding of the characteristics of gene expression in CTS cells.

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Changes in cerebral blood flow and brain temperature during transient global cerebral ischemia are the primary factors influencing the expression of delayed neuronal death. To further define this phenomenon, we experimentally induced transient global cerebral ischemia in Mongolian gerbils by temporarily blocking the carotid arteries bilaterally in order to restrict carotid circulation to the cerebrum. Carotid blood flow was temporarily blocked for 5 minutes during which both temperature of the dura and blood flow in the cerebral cortex were continually monitored. Animals were sacrificed at 3 and 7 days after the 5 minute induced global ischemia, and subsequently fixed by perfusion of a 4% paraformaldehyde solution. The cerebrum was then enucleated and processed for preparation of both hematoxylin and eosin stained paraffin sections and sections suitable for electron microscopic observation. Examination of sections using TdT-mediated dUTP nick end labeling (TUNEL) revealed apoptosis. TUNEL-positive reactions were observed within pyramidal cells of the CA 1 site of the hippocampus. Cerebral tissue processed for electron microscopy revealed characteristic ultrastructural changes of apoptosis. Results suggest that transient global ischemia induces delayed neuronal death through apoptotic change.

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We investigated the localization of MMP-3 and TIMP-2 in the temporomandibular joint of MRL/lpr/lpr mice with rheumatoid arthritis using histological and immunohistochemical methods. There was a strong MMP-3 reaction in the proliferating synovial tissue originating from the pannus, and around the cells gathering in the surface cartilage at 32 weeks. TIMP-2 negative domains were found in the same places. Since MMP-3 was positive where TIMP-2 was negative, this seems to indicate a complimentary reration between the two. MMP-3 was intensely positive in the thinnest area of the articular disc during the growth process. We observed MMP-3 positive cartilage-like cells in the disc at an advanced age. Bone reaction was negative through the entire time of observation. These results suggest that the synovium, disc and cartilage create MMP-3 in the rheumatic temporomandibular joint and explain why the concentration of MMP-3 is high in rheumatoid synovial fluid.

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