The control of neutrophil chemotaxis by inhibitors of cathepsin G and chymotrypsin.
Neutrophil chemotaxis plays an important role in the inflammatory response and when excessive or persistent may augment tissue damage. The effects of inhibitors indicated the involvement of one or more serine proteinases in human neutrophil migration and shape change in response to a chemoattractant. Monospecific antibodies, chloromethylketone inhibitors, and reactive-site mutants of alpha 1-antitrypsin and alpha 1-antichymotrypsin were used to probe the specificity of the proteinases involved in chemotaxis. Antibodies specific for cathepsin G inhibited chemotaxis. Moreover, rapid inhibitors of cathepsin G and alpha-chymotrypsin suppressed neutrophil chemotaxis to the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and zymosan-activated serum in multiple blind well assays and to fMLP in migration assays under agarose. The concentrations of antichymotrypsin mutants that reduced chemotaxis by 50% would inactivate free cathepsin G with a half-life of 1.5-3 s, whereas the concentrations of chloromethylketones required to produce a similar inhibition of chemotaxis would inactivate cathepsin G with a half-life of 345 s. These data suggest different modes of action for these two classes of inhibitors. Indeed the chloromethylketone inhibitors of cathepsin G (Z-Gly-Leu-Phe-CMK) and to a lesser extent of chymotrypsin (Cbz-Gly-Gly-Phe-CMK) mediated their effect by preventing a shape change in the purified neutrophils exposed to fMLP. Antichymotrypsin did not affect shape change in response to fMLP even at concentrations that were able to reduce neutrophil chemotaxis by 50%. These results support the involvement of cell surface proteinases in the control of cell migration and show that antichymotrypsin and chloromethylketones have differing modes of action. This opens the possibility for the rational design of anti-inflammatory agents targeted at neutrophil membrane enzymes.
Lomas
D A
DA
Department of Medicine, University of Cambridge, United Kingdom.
Stone
S R
SR
Llewellyn-Jones
C
C
Keogan
M T
MT
Wang
Z M
ZM
Rubin
H
H
Carrell
R W
RW
Stockley
R A
RA
D000590:Amino Acid Chloromethyl Ketones / Q000737:chemistry / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D002401:Cathepsin B / Q000037:antagonists & inhibitors; D056649:Cathepsin G; D002403:Cathepsins / Q000037:antagonists & inhibitors*; D016764:Cell Polarity / Q000187:drug effects; D002634:Chemotaxis, Leukocyte / Q000187:drug effects*; D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects* / Q000502:physiology; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D012697:Serine Endopeptidases; D014361:Trypsin Inhibitors / Q000235:genetics / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D009842:Oligopeptides; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D009240:N-Formylmethionine Leucyl-Phenylalanine; D002403:Cathepsins; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase; D002401:Cathepsin B