Abstract
Objective: The objective of this study was to examine the role of protein kinase C zeta (PKCζ) 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 PKCζ 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 PKCζ 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: PKCζ and Mac-1 colocalized following stimulation with IL-8. Blocking PKCζ 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 PKCζ prevented neutrophil adhesion, despite increased affinity of Mac-1.
Conclusions: These data suggest that PKCζ 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.
ACKNOWLEDGMENTS
The authors thank Dr. Pina Colarusso for her critical reading of the manuscript and suggestions on image analysis, Evelyn Lailey for her excellent technical support, the Canadian Institutes of Health Research (CIHR) Group Grant in Leukocyte Trafficking, and the Live Cell Imaging Facility. Dr. K.D. Patel is a Canada Research Chair and an Alberta Heritage Foundation for Medical Research (AHFMR) Senior Scholar. Subhadeep Chakrabarti is supported by a full-time graduate studentship from AHFMR.
This work was funded by the Canadian Institutes of Health Research (CIHR)