BACKGROUND AND PURPOSE: Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to induce PG-independent anti-inflammatory actions. Here, we investigated the role of three different NSAIDs (naproxen, ibuprofen and oxaprozin) on neutrophil responses to CXCL8 and C5a. EXPERIMENTAL APPROACH: Human neutrophils were isolated from healthy volunteers by dextran and Ficoll-Hypaque density gradients. Neutrophils were pre-incubated with different concentrations (1-100 µM) of NSAIDs or kinase inhibitors. Neutrophil degranulation into supernatants was tested by elisa and zymography. Neutrophil chemotaxis was determined using Boyden chambers. F-actin polymerization was determined by Alexa-Fluor 488-conjugated phalloidin fluorescent assay. Integrin expression was assessed by flow cytometry. The phosphorylation of intracellular kinases was studied by Western blot. KEY RESULTS: Pretreatment with NSAIDs did not affect neutrophil degranulation, but inhibited neutrophil migration and polymerization of F-actin, in response to CXCL8 and C5a. Pretreatment with different NSAIDs prevented C5a-induced integrin (CD11b) up-regulation, while only ibuprofen reduced CXCL8-induced CD11b up-regulation. Pre-incubation with naproxen or oxaprozin, but not ibuprofen, inhibited the PI3K/Akt-dependent chemotactic pathways. Both endogenous (released in cell supernatants) or exogenous (added to cell cultures) PGE2 did not affect C5a- or CXCL8-induced activities. Short-term incubation with NSAIDs did not affect neutrophil PGE2 release. CONCLUSION AND IMPLICATIONS: Treatment with NSAIDs reduced C5a- and CXCL8-induced neutrophil migration and F-actin polymerization via different mechanisms. Inhibition by ibuprofen was associated with integrin down-regulation, while naproxen and oxaprozin blocked the PI3K/Akt pathway. Both NSAID actions were independent of COX inhibition and PGE2 release.

Neutrophil migration towards C5a and CXCL8 is abrogated by non-steroidal anti-inflammatory drugs (NSAIDs) via the inhibition of different pathways.

BERTOLOTTO, MARIA BIANCA;CONTINI, PAOLA;PENDE, ALDO;DALLEGRI, FRANCO;MONTECUCCO, FABRIZIO
2014

Abstract

BACKGROUND AND PURPOSE: Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to induce PG-independent anti-inflammatory actions. Here, we investigated the role of three different NSAIDs (naproxen, ibuprofen and oxaprozin) on neutrophil responses to CXCL8 and C5a. EXPERIMENTAL APPROACH: Human neutrophils were isolated from healthy volunteers by dextran and Ficoll-Hypaque density gradients. Neutrophils were pre-incubated with different concentrations (1-100 µM) of NSAIDs or kinase inhibitors. Neutrophil degranulation into supernatants was tested by elisa and zymography. Neutrophil chemotaxis was determined using Boyden chambers. F-actin polymerization was determined by Alexa-Fluor 488-conjugated phalloidin fluorescent assay. Integrin expression was assessed by flow cytometry. The phosphorylation of intracellular kinases was studied by Western blot. KEY RESULTS: Pretreatment with NSAIDs did not affect neutrophil degranulation, but inhibited neutrophil migration and polymerization of F-actin, in response to CXCL8 and C5a. Pretreatment with different NSAIDs prevented C5a-induced integrin (CD11b) up-regulation, while only ibuprofen reduced CXCL8-induced CD11b up-regulation. Pre-incubation with naproxen or oxaprozin, but not ibuprofen, inhibited the PI3K/Akt-dependent chemotactic pathways. Both endogenous (released in cell supernatants) or exogenous (added to cell cultures) PGE2 did not affect C5a- or CXCL8-induced activities. Short-term incubation with NSAIDs did not affect neutrophil PGE2 release. CONCLUSION AND IMPLICATIONS: Treatment with NSAIDs reduced C5a- and CXCL8-induced neutrophil migration and F-actin polymerization via different mechanisms. Inhibition by ibuprofen was associated with integrin down-regulation, while naproxen and oxaprozin blocked the PI3K/Akt pathway. Both NSAID actions were independent of COX inhibition and PGE2 release.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11567/690993
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