[PubMed] [Google Scholar] 29
[PubMed] [Google Scholar] 29. against a bacterial stimulus, a toll-like receptor (TLR) 4 agonist, SERPINA3 in healthy human airways. We also demonstrated that IL-26 enhances the chemotactic response of neutrophils toward a bacterial stimulus and toward IL-8, while at the same time inhibiting the chemokinesis in these innate effector cells (9C11). Moreover, we demonstrated that alveolar macrophages constitute a prominent source of IL-26, in addition to Th17 cells and other lymphocyte subsets. Based upon these findings, we proposed that IL-26 serves to focus neutrophil mobilization toward sites of bacterial infection and inflammation during activation of pulmonary host defense. In line with our proposal, Meller test was utilized to compute comparisons between paired data sets unless otherwise stated. The MannCWhitney test was used to compute differences between the human samples. Correlation analyses were performed using the Spearman rank correlation test. 0.05 were considered statistically significant. RESULTS Primary Bronchial Epithelial Cells Produce IL-26 Protein in Response to TLR3 Stimulation The primary bronchial epithelial cells were exposed (24 h) to viral stimulation and we found that these cells contain the mRNA that was increased approximately 3 fold after stimulation with poly-IC (1g/mL) (Figure 1A). Furthermore, we found that increasing concentrations of poly-IC caused a corresponding increase in IL-26 protein release in the cell-free DGAT-1 inhibitor 2 conditioned media (Figure 1B). Using western blot, we found that intracellular IL-26 protein was also increased in response to poly-IC (1 ug/mL) (Figure 1C, ?,D).D). Notably, we found that only the dimeric form of IL-26 (36kDa) was detectable in the bronchial epithelial cells. Similar to the viral stimulus poly-IC, stimulation with other viral stimuli, the TLR7 agonist Imiquimod (1 g/mL) and the TLR8 agonist ssRNA (1 g/mL) (16), also increased IL-26 protein concentrations in the conditioned media (Figure 1E). Moreover, we also verified that the bronchial epithelial cells contain mRNA for and (Figure 1F), here presented as fold differences, with a similar magnitude of transcription. Open in a separate window Figure 1. Primary bronchial epithelial cells produce IL-26 enhanced by viral-related stimuli. DGAT-1 inhibitor 2 Cells were stimulated (24 h) with different viral stimuli (TLR3 agonist poly-IC, TLR7 agonist imiquimod and TLR8 agonist ssRNA). Extracellular concentrations in cell-free conditioned media as well as intracellular expression of IL-26 protein were measured using ELISA and western blot, respectively, and levels of mRNA using real time. (A) mRNA levels after stimulation with poly-IC (n = 11). (B) Extracellular concentrations of IL-26 in cell-free conditioned media in response to poly-IC at different concentrations (n = 8). (C) Intracellular IL-26 protein (representative western blot). (D) the average protein expression (fold difference) after stimulation with poly-IC (1ug/mL) during 24 h. (E) Extracellular concentrations of IL-26 in cell-free conditioned media in response to imiquimod or ssRNA (n = 8). (F) and mRNA levels (fold) (n = 5). The values indicated are according to the Student paired test. 0.05 is considered significant. The Release of IL-26 Protein in Response to Poly-IC Involves TRIF, MAP Kinases and NF-B The adaptor protein TRIF and the MAP kinases p38, JNK1C3 and ERK1/2 and NF-B are generic molecules involved in signal transduction downstream of TLR3 (28C32). Given the lack of specific knowledge for IL-26 release in this respect, we determined the involvement of these signaling molecules in the release of IL-26. First, TRIF was inhibited (25 mol/L) in cultures in the presence of a suboptimal concentration of poly-IC (0.05g/mL) to render any TRIF-inhibitory effect noticeable. We found that IL-26 release was almost completely blocked by the TRIF inhibitor (Figure 2A). Notably, given that an unaltered number of cells were stimulated in half the volume of culture media (0.5 mL), the IL-26 concentrations became higher. We then investigated the intracellular levels of IL-26 at the gene and protein level using RT-PCR and western blot respectively. To induce an optimal mRNA level and protein expression, cells were stimulated with 1 ug/mL poly-IC. We then found that inhibition of TRIF did not alter the mRNA (Figure 2B) nor did it alter the intracellular protein levels (Figure 2C, ?,DD). Open in a separate window Figure 2. The adaptor protein TRIF is involved in poly-IC-induced release of IL-26. Primary bronchial epithelial cells were preincubated (5 h) DGAT-1 inhibitor 2 with TRIF inhibitor and vehicle (25 mol/L). Extracellular concentrations of IL-26 in cell-free conditioned media as well as intracellular levels were measured using ELISA and western blot respectively and mRNA levels using real time. (A) Extracellular concentrations of IL-26 in cell-free conditioned in response to poly-IC (0.05 g/mL) in the presence of the TRIF inhibitor (n = 7). (B) Level of mRNA in response to poly-IC (1g/mL) in the presence of the TRIF inhibitor (n = 8). (C) Intracellular IL-26 protein (representative western blot).