Immunopharmacology and InflammationDifferential involvement of cyclooxygenase isoforms in neutrophil migration in vivo and in vitro
Introduction
Leukocyte recruitment is a prominent feature of inflammatory processes. The sequence involved in this phenomenon is a multistep process that includes an initial endothelium–leukocyte interaction (known as a tethering and rolling process), firm adhesion and subsequent transmigration out of the blood vessels (Kubes et al., 1991). After diapedesis, leukocytes migrate toward the site of the lesion in response to locally derived chemotactic factors. The molecules present in leukocytes and the endothelial lining that orchestrates this process belong to different families, which include selectins (E, P and L series) and α4-integrins (Kelly et al., 2007), among others. Selectins are important at the rolling stage, while integrins (mainly β2-integrins), lymphocyte function-associated antigen-1 (LFA-1) and macrophage associated chemokine (MAC-1) promote firm adhesion that is important for the subsequent transmigration event (Issekutz and Issekutz, 1992). Infiltrated neutrophils are first line defense cells and the importance of these cells in the initiation and resolution phases of the inflammatory process is well documented (Serhan, 2007).
Despite the importance of the presence of leukocytes at the site of lesions and infection in relation to defending the host, the accumulation of these cells in tissues and organs is also a hallmark of the development of inflammatory conditions, such as atherosclerosis, severe pulmonary and renal diseases and acute myocardial infarction (Alvarez et al., 2001). In the last case, patients submitted to surgical reperfusion can present extensive lesioning of the cardiac muscle due the massive inflammatory process mediated by neutrophils, which are attracted by mediators released under hypoxic conditions (Abdel-Rahman et al., 2007). In this paradigm, modulation of the recruitment of these cells could provide a new way to regulate the inflammatory response.
Nonsteroidal antiinflammatory drugs (NSAIDs) are widely used to control pain, fever and edematogenic conditions (Ferreira et al., 1973). The most accepted mechanism of action of these drugs is the inhibition of cyclooxygenase (COX) isoenzymes, which reduce the biosynthesis of eicosanoids, including prostaglandins and tromboxane (Vane, 1971). Prostaglandins are responsible for the majority of the primary symptoms of the inflammatory process (Ferreira et al., 1973, Moncada et al., 1973) and among these, PGF2α has been shown to trigger neutrophil migration (Arnould et al., 2001, Menezes et al., 2005, Sandig et al., 2006). Moreover, nonselective COX inhibitors (indomethacin) and selective COX-2 inhibitor (celecoxib) can reduce leukocyte recruitment (Menezes et al., 2003), an effect observed in various experimental inflammation models (Diaz-Gonzalez and Sanchez-Madrid, 1998, García-Vicuña et al., 1997, Maltos et al., 2004, Queiroz-Junior et al., in press). Given the pattern of leukocyte recruitment inhibition demonstrated in vivo by selective COX-2 inhibitors and considering the lack of effect of selective COX-1 inhibitors in the same model, this study aimed to investigate the role played by selective COX-1 and COX-2 inhibitors in various steps of the leukocyte–endothelial interaction using in vitro and in vivo models of leukocyte recruitment.
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Animals
Holtzman female rats and male Swiss mice were purchased from the CEBIO/UFMG. Rats and mice weighing 140–180 g and 20–25 g, respectively, were used in the experiments. All animal protocols were approved by the Animal Care Committee of the Federal University of Minas Gerais (protocol 120/05). The rodents were provided with food ad libitum and free water access.
Neutrophil isolation
Rats were anesthetized by i.p. injection of a mixture of 10 mg/kg xylazine and 200 mg/kg ketamine hydrochloride and blood (~ 5 ml) was
Celecoxib and indomethacin inhibited neutrophil chemotaxis to FMLP
To study the effect of celecoxib on a leukocyte chemotaxis assay, neutrophils were purified from the blood of rats that were pretreated or not with celecoxib. FMLP induced neutrophil chemotaxis under control conditions and pretreatment of the rats with 12–30 mg/kg celecoxib revealed a dose-dependent reduction in ex vivo neutrophil migration under these conditions, as shown in Fig. 1A. To study the direct effect of celecoxib on these cells, neutrophils were incubated (30 min before chemotactic
Discussion
Neutrophil recruitment to tissues is an important feature of an acute inflammatory process. Indeed, they can play a key role in the healing and resolution of inflammation, besides the obvious defense function of these cells (Serhan, 2007). However, to exert these functions, neutrophils produce oxygen radicals and proteolytic enzymes stimulated by bacterial products (Brown and Treacher, 2006) and mediators released by hypoxic cells (Abdel-Rahman et al., 2007). In situations where an uncontrolled
Acknowledgements
The authors would like to thank Mr. Webster Pimenta for his excellent technical assistance. This project was granted by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-Brazil). Gustavo B Menezes and JN Francischi received a CNPq fellowship during this study. Part of these results was accepted for presentation in The IX World Conference on Clinical Pharmacology and Therapeutics, Québec, Canada. A British-born scientific text editor revised entire text.
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