NSAIDs upregulate beta 2-integrin expression on human neutrophils through a calcium-dependent pathway.
BACKGROUND
Margination of circulating neutrophils (PMN) into the gastric microcirculation is an early and critical event in the pathogenesis of non-steroidal antinflammatory drug (NSAID)-induced gastropathy. This effect is mediated through the upregulation of beta 2 integrins on the PMN surface.


OBJECTIVE
To investigate whether indomethacin modulates: (1) Mac-1 expression; (2) Ca2+ mobilization ([Ca2+]i), protein kinase C and nitric oxide accumulation; and (3) mitogen-associated protein kinase phosphorylation in human PMN.


METHODS
Human PMN were isolated by centrifugation through a double Ficoll gradient. [Ca2+]i was measured in PMN loaded with fura-2 and Mac-1 expression by flow cytometry.


RESULTS
Indomethacin caused a concentration- and time-dependent upregulation of CD11b and CD18 expression and PMN adhesion to endothelial cells. Maximal upregulation of Mac-1 expression (40-50%) occurred after a 30-min incubation with 0.1mM indomethacin. The effect was prevented by removing the Ca2+. Ionomycin and thapsigargin caused a 7-10-fold increase in [Ca2+]i and a 2-4-fold increase in Mac-1 expression. Indomethacin induced a concentration-dependent phosphorylation of a 41-kDa mitogen-associated protein kinase. Tyrosine kinase inhibitors prevented the effect of indomethacin on Mac-1 expression and Ca2+ mobilization. Indomethacin and ionomycin increased superoxide generation, myeloperoxidase secretion and PMN adherence to endothelial cells and stimulated nitric oxide production. Indomethacin-induced Mac-1 upregulation was prevented by a nitric oxide synthase inhibitor.


CONCLUSIONS
Indomethacin-induced upregulation of Mac-1 is mediated by changes in [Ca2+]i and nitric oxide. Phosphorylation of the 41-kDa mitogen-associated protein isoform is a previously unreported target of NSAID action. These effects might help to explain the ability of indomethacin to cause gastric neutrophil margination.
Fiorucci
S
S
Dipartimento di Medicina Clinica, Patologia e Farmacologia, Università degli Studi di Perugia, Italy.
Santucci
L
L
Gerli
R
R
Brunori
P M
PM
Federici
B
B
Ugolini
B
B
Fabbri
C
C
Morelli
A
A
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D018821:CD18 Antigens / Q000235:genetics*; D002118:Calcium / Q000378:metabolism*; D002452:Cell Count / Q000187:drug effects; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology; D016177:Macrophage-1 Antigen / Q000235:genetics / Q000378:metabolism*; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
D000894:Anti-Inflammatory Agents, Non-Steroidal; D018821:CD18 Antigens; D016177:Macrophage-1 Antigen; D013481:Superoxides; D009569:Nitric Oxide; D009195:Peroxidase; D011493:Protein Kinase C; D002118:Calcium; D007213:Indomethacin