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NSAIDs upregulate beta 2-integrin expression on human neutrophils through a calcium-dependent pathway
S Fiorucci1, L Santucci, R Gerli
1Dipartimento di Medicina Clinica, Patologia e Farmacologia, Università degli Studi di Perugia, Italy.
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.
Aims:
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.
Insights
Non-steroidal anti-inflammatory drug (NSAID) use increases neutrophil adhesion in the stomach. Indomethacin upregulates Mac-1 expression via calcium and nitric oxide pathways, impacting neutrophil margination in NSAID-induced gastropathy.
Area of Science:
- Gastroenterology
- Immunology
- Pharmacology
Background:
- Neutrophil (PMN) margination in gastric microcirculation is key in NSAID-induced gastropathy.
- This process involves the upregulation of beta-2 integrins on PMN surfaces.
Purpose of the Study:
- To investigate indomethacin's modulation of Mac-1 expression in human PMNs.
- To examine indomethacin's effects on intracellular calcium ([Ca2+]i), protein kinase C, nitric oxide, and mitogen-associated protein kinase phosphorylation.
Main Methods:
- Human PMNs were isolated using Ficoll gradient centrifugation.
- Intracellular calcium ([Ca2+]i) was measured using fura-2.
- Mac-1 expression and PMN adhesion were assessed via flow cytometry.
Main Results:
- Indomethacin caused dose- and time-dependent upregulation of CD11b/CD18 (Mac-1) and PMN adhesion.
- Maximal Mac-1 upregulation occurred at 0.1mM indomethacin after 30 minutes; this was calcium-dependent.
- Indomethacin induced mitogen-associated protein kinase phosphorylation and increased nitric oxide production, superoxide generation, and PMN adherence.
Conclusions:
- Indomethacin-induced Mac-1 upregulation is mediated by intracellular calcium ([Ca2+]i) and nitric oxide.
- Phosphorylation of a 41-kDa mitogen-associated protein kinase is a novel NSAID target.
- These findings elucidate mechanisms contributing to indomethacin-induced gastric neutrophil margination.