Neutrophils have a dual affect on epithelial pIgR/SC the critical receptor for transcellular routing of mucosal IgA but mechanisms of pIgR/SC upregulation remain elusive. TGF-production. Exogenous TGF-stimulated SC production with a maximal effect at 48?hrs and both TGF-alone were incubated (from 1.25 to 5 × 106 cells) in complete medium for 48?hrs to determine the production of cytokines including TGF-were cultured for 48?hrs with or without TGF-To analyse the dose-response effect of neutrophils on SC production by bronchial epithelial cells confluent monolayers of Calu-3 cells were incubated for 48?hrs with increasing Acetanilide numbers of activated neutrophils (from 0.3 to 15 × 106 cells) in the presence or not of 2.5?(from 0.2 to 80?ng/ml) with CSE (from 0.1% to 10%) and with MAPKinases inhibitors (PD SP and SB) for 48?hrs. After incubation cells were washed with PBS. 100?concentration was determined in culture Acetanilide supernatants and cell lysates from epithelial cells by sandwich ELISA as previously reported [8] by using affinity purified goat antihuman SC polyclonal antibody (developed in our laboratory recognizing both soluble SC and membrane pIgR/SC) as capture and detection (biotinylated) antibody. The reaction was revealed by streptavidine-HRP followed by tetramethylbenzidine (TMB) in the presence of hydrogen peroxide. OD were recorded at 450?nm using a microplate spectrometer Rabbit polyclonal to PACT. (Biorad). Sensitivity of the immunoassay was ~0.2?ng/ml. production was measured in supernatants from Calu-3 cells neutrophils or Calu-3/neutrophils cocultures by ELISA. A step of sample extraction (acidification) was carried out to allow for the release of TGF-immunoassay kit Biosource Nivelles Belgium). The sensitivity of TGF-of a confluent monolayer in a 12-well plate Calu-3 cells were lyzed in 150?values <.05 were considered as statistically significant. 3 Results 3.1 Neutrophils Have a Dual Effect on pIgR/SC Production To set up a model to examine effects of neutrophils on SC production by bronchial epithelial cells confluent epithelial layers of Calu-3 cells were incubated for 48?hrs with increasing numbers of activated neutrophils (Figure 1(a)) with evaluation of cytotoxicity. Neutrophil concentrations from 0.3 × 106 Acetanilide to 5 × 106 did not significantly affect epithelial cell viability according to MTT assay (viability ≥ 80% compared to control). However cytotoxicity was observed for ≥10 × 106 neutrophils (cell viability decreased by < .01 Figure 1(a)). At further increased neutrophil numbers (>5 × 106) SC production was downregulated and tended to decrease at 15 × 106 neutrophils as compared to control (= .08 Figure 1(a)) consistent with cytotoxicity (MTT assay) and/or proteolytic cleavage [8]. Elastase activity evaluated in supernatants from the same Calu-3/neutrophils cocultures increased as expected with the number of neutrophils (Figure 1(b)). These results indicated that this system allowed to address the two opposite effects of neutrophils on epithelial SC and experiments were carried out to explore the mechanisms of SC upregulation by “nontoxic” numbers of neutrophils releasing elastase in a concentration range that could be observed in chronic airway neutrophilic diseases for example during exacerbations. 3.2 Neutrophil-Driven SC Upregulation Is Modulated by Serine Proteinase Activity To evaluate the role of serine proteinases on the regulation of SC production by neutrophils Calu-3 cells Acetanilide were cultured with increasing neutrophil numbers in the presence or not of secretory leukocyte protease inhibitor (SLPI) a natural inhibitor of neutrophil elastase at a concentration (2.5?< .05 at 2.5 × 106 neutrophils) was further increased. In addition the presence of SLPI promoted an increase in pIgR/SC in epithelial lysates (< .05 at 0.6 × 106 neutrophils) while no effect was observed in this cell Acetanilide compartment in the absence of SLPI (Figure 2) or with SLPI alone without neutrophils (data not shown). Figure 2 Effect of Secretory Leucoproteinase Inhibitor (SLPI) on pIgR/SC production. Confluent Calu-3 monolayers were cultured with increasing numbers of neutrophils (0.6 × 106 to 10 × 106 cells) in the presence (dashed line) or in the absence ... 3.3.