INTEGRIN REGULATION OF POLYMORPHONUCLEAR LEUKOCYTE APOXIS DURING HYPOXIA IS PRIMARILY DEPENDENT ON VERY LATE ACTIVATION ANTIGEN-3 AND ANTIGEN-5

Background. Apoptosis is thought to be a central mechanism that leads to resolution of the inflammatory response. The regulation of polymorp honuclear leukocyte (PMN) apoptosis during hypoxia has not been previo usly characterized, and we hypothesized that integrin signaling by mat rix proteins (laminin) would regulate PMN apoptosis. Methods. PMNs at 1 x 10(5)/ml were adhered on plastic or laminin for 12 hours during no rmoxia or hypoxia. Apoptosis was determined both by cellular histologi c evaluation and the TUNEL assays (Tdt). Phagocytosis in apoptotic PMN s was determined with two-color flow cytometric analyses with rhodamin e-labeled heat-killed Escherichia coli (511 nm) and the Tdt reagent (5 63 nm). Western blot analyses were performed on nine apoptotic regulat ory proteins with monoclonal antibodies directed against each protein, and tyrosine phosphorylation was assessed after integrin receptor cro ss-linkage. Results. Adherence of PMNs to laminin reduced apoptosis by cellular histologic evaluation and the Tdt method (%apoptosis = 19 +/ - 1.0 versus 63 +/- 4.2 by histologic evaluation, 38 +/- 3.8 versus 60 +/- 10.5 by flow cytometry +/- adherence to laminin). Apoptosis-posit ive PMNs exhibited significantly greater phagocytosis than apoptosis-n egative PMNs +/- laminin. Western blot analyses demonstrated increased p53 expression after 2 and 4 hours of hypoxia. Cross-linkage of very late activation antigen-3 (alpha(3)/beta(1)) resulted in the phosphory lation of 53 kd, 44 kd, and 39 kd proteins at 30 seconds. Conclusions. (1) Chemotaxis of PMNs into the interstitium during hypoxia not only provides a means of ensuring PMN-pathogen contact but also provides a mechanism for improved survival by reducing apoptosis. (2) The reducti on of apoptosis is mediated primarily by very late activation antigen- 3, which leads to a subsequent increase in the intracellular expressio n of p53 and increased bacterial phagocytosis.