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 (PKCzeta) in interleukin (IL)-8-mediated activation of Mac-1 (CD11b/CD18) in human neutrophils.


METHODS
Neutrophils were stimulated with IL-8 in the presence or absence of pharmacologic inhibitors of PKC or a myristoylated PKCzeta 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 PKCzeta was also probed using IE 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
PKCzeta and Mac-1 colocalized following stimulation with IL-8. Blocking PKCzeta 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 PKCzeta prevented neutrophil adhesion, despite increased affinity of Mac-1.


CONCLUSIONS
These data suggest that PKCzeta 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.
Chakrabarti
Subhadeep
S
Department of Physiology and Biophysics, Institute of Infection, Immunity and Inflammation, Hotchkiss Brain Institute, University of Calgary, Alberta, Canada.
Patel
Kamala D
KD
D039481:CD11b Antigen / Q000378:metabolism*; D018821:CD18 Antigens / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D018920:Coculture Techniques; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism* / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000099221:Protein Kinase C zeta
D039481:CD11b Antigen; D018821:CD18 Antigens; D016209:Interleukin-8; D007527:Isoenzymes; D011493:Protein Kinase C; D000099221:Protein Kinase C zeta; C508606:CXCL8 protein, human; C509295:ITGAM protein, human