The atypical zeta (zeta) isoform of protein kinase C regulates CD11b/CD18 activation in human neutrophils

Objective: The objective of this study was to examine the role of protein kinase C zeta (PKC zeta) in interleukin (IL)-8-mediated activation of Mac-1 (CD11b/CD18) in human neutrophils. Materials and Methods: Neutrophils were stimulated with IL-8 in the presence or absence of pharmacologic inhibitors of PKC or a myristoylated PKC zeta pseudosubstrate. The resulting changes in Mac-1 surface expression, affinity, and avidity, as measured by clustering, were determined by using a combination of flow cytometry and immunofluorescence (IF). Colocalization of Mac-1 with PKC zeta was also probed using IF. Finally, neutrophil adhesion to matrix proteins was examined under static conditions and adhesion to tumor necrosis factor-alpha-stimulated human umbilical vein endothelial cells was examined under flow conditions, using a parallel-plate flow chamber. Results: PKC zeta and Mac-1 colocalized following stimulation with IL-8. Blocking PKC zeta prevented IL-8-induced Mac-1 clustering while simultaneously increasing Mac-1 affinity. To determine the relative contribution of affinity versus avidity in neutrophil adhesion, we examined adhesion under both static and flow conditions, and found that blocking PKC zeta prevented neutrophil adhesion, despite increased affinity of Mac-1. Conclusions: These data suggest that PKC zeta is a negative regulator of Mac-1 affinity and a positive regulator of Mac-1 avidity. Further, Mac-1 avidity is more important than increased affinity alone in regulating neutrophil firm adhesion.