calpain and 3-3--4-5--tetrahydroxystilbene

We previously showed that LFA-1-dependent in vitro invasion and in vivo migration of a T cell hybridoma was blocked in cells overexpressing a truncated dominant-negative zeta-associated protein (ZAP)-70. The truncated ZAP-70 also blocked LFA-1-dependent chemotaxis through ICAM-1-coated filters induced by 1 ng/ml stromal cell-derived factor-1, but not LFA-1-independent chemotaxis induced by 100 ng/ml stromal cell-derived factor-1. This suggested that LFA-1 engagement triggers a signal that amplifies a weak chemokine signal and that dominant-negative ZAP-70 blocks this LFA-1 signal. Here we show that cross-linking of part of the LFA-1 molecules with Abs causes activation of free LFA-1 molecules (not occupied by the Ab) on the same cell, which then bind to ICAM-2 on other cells. This causes cell aggregation that was also blocked by dominant-negative ZAP-70. Thus, an LFA-1 signal involving ZAP-70 activates other LFA-1 molecules, suggesting that the chemokine signal can be amplified by multiple cycles of LFA-1 activation. The chemokine and the LFA-1 signal were both blocked by a phospholipase C inhibitor and a calpain inhibitor, suggesting that one of the amplified signals is the phospholipase C-dependent activation of calpain. Finally, we show that both Src-homology 2 domains are required for inhibition of invasion, chemotaxis, and aggregation by the truncated ZAP-70, suggesting that ZAP-70 interacts with a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) sequence. Remarkably, this is not an ITAM in the TCR/CD3 complex because this is not expressed by this T cell hybridoma.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Binding Sites, Antibody; Calpain; Cell Adhesion; Cell Adhesion Molecules; Cell Aggregation; Cell Movement; Dose-Response Relationship, Immunologic; Enzyme Inhibitors; Hybridomas; Immunoglobulin Fab Fragments; Isoenzymes; Lymphocyte Function-Associated Antigen-1; Mice; Peptide Fragments; Phospholipase C gamma; Protein-Tyrosine Kinases; Rats; Signal Transduction; Stilbenes; T-Lymphocytes; Type C Phospholipases; ZAP-70 Protein-Tyrosine Kinase