Monocyte diluent (RPMI 1640 with 5% FBS), alone or with hrIL-8 (4 ng/ml), was used as negative and positive control, respectively

Monocyte diluent (RPMI 1640 with 5% FBS), alone or with hrIL-8 (4 ng/ml), was used as negative and positive control, respectively. that in the murine air-pouch model, extracellular nucleotides were instrumental in LPS-induced neutrophil migration. Altogether, these data imply that LPS induces the release of nucleotides from monocytes and that by autocrine stimulation, the latter molecules regulate neutrophil migration caused by Gram-negative bacteria, suggesting a proinflammatory role of extracellular nucleotides in innate immunity. O111:B4, potato apyrase grade VII, nucleotides (ATP, ADP, UTP, and UDP), ,-methyleneadenosine-5-diphosphate (,-meADP), pyridoxal-phosphate-6-azophenyl-2, 4-disulfonate (PPADS), and suramin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Adenosine deaminase (ADA) was provided by Roche Diagnostics (Indianapolis, IN, USA), MRS2578 by Tocris Bioscience (Bristol, UK), and reactive blue 2 (RB-2) by ICN Biochemicals Inc. (Aurora, OH, USA). Human recombinant (hr)IL-8 was purchased from Medicorp (Montreal, Canada), IL-8 neutralizing antibody MAB208 from Ribitol (Adonitol) R&D Systems (Minneapolis, MN, USA), and RPMI-1640 medium from Wisent (St-Bruno, Canada). A matching isotype mouse IgG1 antibody to DNP was used as a control in Boyden chamber transmigration assays. LPS and apyrase were reconstituted in an endotoxin-free saline (Sigma Chemical Co.). Nucleotides, ,-meADP and P2 receptor antagonists (RB-2, PPADS, and suramin) were dissolved in tissue-culture, endotoxin-free water purchased from Sigma Chemical Co. Prior to cell stimulation, LPS was sonicated for 10 min in a water bath sonicator. Before use, ADA was dialyzed against sterile 0.9% NaCl and 10 mM Hepes, pH 7.4, in a Slide-A-Lyser? cassette (MWCO 3500, Pierce, Rockford, IL, USA) to remove ammonium sulfate present in the commercial preparation that induces calcium mobilization in cells. MRS2578 was dissolved in DMSO at the concentration of 0.1 M and diluted further with RPMI 1640 plus 5% FBS to the working concentration of 10 M, which therefore contained 0.01% DMSO. Isolation Ribitol (Adonitol) of monocytes and neutrophils Human monocytes and neutrophils were isolated as described originally [18] with some modifications. Briefly, venous blood of healthy volunteers was collected on isocitrate anticoagulant solution and centrifuged (250 test was performed using Excel software (Microsoft? Office OneNote? 2003). RESULTS Extracellular nucleotides regulate neutrophil transmigration in vitro To determine if extracellular nucleotides are involved in neutrophil migration toward the media of LPS-stimulated monocytes, freshly isolated human monocytes were stimulated with LPS in the presence or absence of apyrase, an enzyme that breaks down tri (e.g., ATP, UTP)- and diphosphonucleosides (e.g., ADP, UDP), the Ribitol (Adonitol) natural agonists of P2 receptors. The conditioned media of monocytes were subsequently tested for neutrophil migration in a modified Boyden chamber assay, where neutrophils were allowed to migrate across a membrane coated with a monolayer of endothelial cells (HUVEC). As shown in Figure 1, the media of LPS-stimulated monocytes were more chemotactic than the media where apyrase was added simultaneously with LPS. No decrease in neutrophil migration was observed with heat-inactivated apyrase (95C, 5 min; Fig. 1). Open in a separate window Fig. 1 Neutrophil transmigration to the media of LPS-stimulated monocytes Pik3r2 is dependent on extracellular nucleotides. Freshly isolated human monocytes (1106) were stimulated with LPS (0.1 g/ml) in the absence or presence of apyrase (2 U/ml), as described in Materials and Methods. The supernatants of treated monocytes Ribitol (Adonitol) were passed through a polymyxin B column to remove the endotoxin and were subsequently tested as neutrophil chemoattractants in a Boyden chamber transmigration assay. The media of untreated monocytes designated as medium were also tested. Monocyte diluent (RPMI 1640 with 5% FBS), alone or with hrIL-8 (4 ng/ml), was used as negative and positive control, respectively. The media of monocytes incubated with 100 M UDP were also tested for neutrophil migration. These data represent the mean SEM of four independent experiments with cells of eight blood donors. One hundred percent was set for the media of LPS-treated monocytes and depending on experiment, ranged from 0.22 to 0.53 106 neutrophils (*, [5]. Altogether, these data suggest a general implication of nucleotides in neutrophil migration toward bacterial infections. Acknowledgments This work was supported by grants to J. S. from the Canadian Institutes of Health Research (CIHR; MOP-68957) and from The Arthritis Society (TAS; 01/0078). F. K. was a recipient of a fellowship from the CIHR/Wyeth Pharmaceuticals, F. B. Y. of a scholarship from Fonds de la Recherche sur lArthrite et les Maladies Rhumatismales (FRAMR), J. L. of a scholarship from the CIHR, P. A. T. of a scholarship from the Fonds de la Recherche en Sant du.