Effects of a monoclonal anti-calpain antibody on responses of stimulated human neutrophils. Evidence for a role for proteolytically modified protein kinase C.
A monoclonal antibody directed against the Ca2+-requiring proteinase (calpain) of human neutrophils was employed to assess the role of this proteinase in mediating the responses to stimuli such as phorbol 12-myristate 13-acetate or fMet-Leu-Phe. In the presence of either phorbol 12-myristate 13-acetate or fMet-Leu-Phe the antibody is taken up by the neutrophils, and a marked inhibition of intracellular calpain is observed. The decreased calpain activity is accompanied by (a) a significant decrease in the proteolytic conversion of native protein kinase C (Ca2+/phospholipid-dependent enzyme) to the soluble form that does not require Ca2+ or phospholipids for activity; (b) a marked increase in the production of superoxide anion; and (c) a decrease in the exocytosis of granule contents. The increase in superoxide production can be attributed to a more prolonged association of native protein kinase C with the plasma membrane, thus enhancing the phosphorylation of membrane proteins that precedes O(2-) production (Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Michetti, M., Sacco, O., and Horecker, B. L. (1986), Biochem. Biophys. Res. Commun. 140, 1121-1126). The decreased exocytosis can be attributed to a decreased phosphorylation of certain cytoskeletal proteins, catalyzed by the soluble form of protein kinase C (Pontremoli, S., Melloni, E., Michetti, M., Sparatore, B., Salamino, F., Sacco, O., and Horecker, B. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3604-3608); the subsequent reorganization of the cytoskeleton appears to be related to degranulation. These effects of the monoclonal anti-calpain provide direct evidence for an essential role for calpain in the activation of human neutrophils.
Pontremoli
S
S
Institute of Biological Chemistry, University of Genoa, Italy.
Melloni
E
E
Damiani
G
G
Salamino
F
F
Sparatore
B
B
Michetti
M
M
Horecker
B L
BL
D000818:Animals; D000911:Antibodies, Monoclonal; D002154:Calpain / Q000276:immunology*; D051379:Mice; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D010447:Peptide Hydrolases / Q000097:blood; D011493:Protein Kinase C / Q000378:metabolism*; D013481:Superoxides / Q000096:biosynthesis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine; D011493:Protein Kinase C; D010447:Peptide Hydrolases; D002154:Calpain; D013755:Tetradecanoylphorbol Acetate