Effect Of Human Neutrophil Defensins And Nucleotides On Regulation Of Innate Immunity In Tracheal Epithelial Cells

OBJCTIVE: To study IL-8 expression and its mechanisms in airway epithelial cells stimulated by human neutrophil defensins and extracellular nucleotides, clone human neutrophil defensin gene cDNA and examine the production of human neutrophil defensin in transfected tracheal epithelial cells, providing the insight into the pathogenesis of respiratory tract infection and inflammation and some valuable reference of remedy for these disease. METHODS: The human neutrophil defensins was isolated from the patients of cystic fibrosis through Biogel p-10 molecular filtration technique. The purified products were identified and characterized by Mass spectrometry and capillary electrophoresis. The antimicrobial activity was detected by agar diffusion assay. The cytotoxicity and synergetic cytotoxity of neutrophil defensins, Suramin and reactive blue (inhibitor of p2y receptors) were determined by lactate dehydrogenase(LDH) release. The content of human 10 cytokines, which is related to inflammation, in A549 cell exposed to neutrophil defensins and nucleotides were measured by LiquiChip reader. The cell-free culture supernatants of A549 cells were collected after inducers (neutrophil defensins, nucleotides) or inhibiters (suramin, reactive blue, Apyrase BAPTA-AM) stimulation. IL-8 content inA549 cells culture supematants were measured by ELISA. P2Y2, P2Y4 and P2Y6 receptors mRNA expression was detected by RT-PCR in A549 cells stimulated by neutrophil defensins. Antisense oligonucleotides of P2Y receptors was transferred into A549 cells by liposome and stimulated by neutrophil defensins. The IL-8 concentration of the culture supematants was assay by ELISA. ATP concentration in culture supernants of A549 cells stimulated by neutrophil defensin was measured by bio-luminescence assays method. 125I or FITC labeled neutrophil defensins was used for binding assay of neutrophil defensins on A549 cells. IL-8 reporter gene expression in A549 cells that transferred with recombinant plasmid EGFP1-IL-8 and stimulated with ATP was observed under laser confocal microscopy. Recombinant plasmid pBabe-Neo- HNP1 was used to transfect the human tracheal epithelial cells cultured in serum-free medium, by liposome. The specific primer for HNP1 gene of neutrophil defensins was designed. Total RNA was extracted from the transfected cells and RT-PCR detected the HNP1 mRNA expression. The Neutrophil defensins antiserum was prepared by immunizing rabbit with the BSA/neutrophil defensins conjugate via intrademic injection. Immunocytochemistry staining detected the expression of HNP1 in human tracheal epithelial cells transferred with pBabe-Neo- HNP1. The cDNA fragment of HNP1 was amplified by PCR from pBabe-Neo- HNP1, purified PCR products was digested by BamH I and Sal I and cloned into pBK CMV vector, transferred to E.coli and positive recombinants was selected by white-blue screening. RESULTS: The molecular masses of purified neutrophil defensins were 3373.01,3444.29, 3488.38. Agar diffusion assay showed that the purified HNP1-3 inhibited the E.coli ATCC 25922 growth in the does-dependent manner. Cytotoxicity of neutrophil defensins in lOOug/ml against A549 cells was 3.5% and the synergetic cytotoxicity of defensins(100(J.g/ml) with suramin or reactive in blue (12.5uM /L) were 1.5% and 2.3%, respectively. Neutrophil defensin, ATP and UDP could induce IL-8 releases. Meanwhile, P2Y receptor inhibitors suramin and reactive blue inhibited IL-8 release of defensin- or ATP-stimulated A549 cells in does-dependent manner. Antisense oligonucleotides to P2Y2, P2Y4, P2Y6 receptors inhibited the 11-8 production in A549 cell stimulated by neutrophil defensins, and the inhibitory rate were 44%,11% and 46% respectively. Suramin (12. 5^M/L) inhibitory rate in the defensins-stimulated IL-8 release was 100% and 49.6% in ATP stimulation. Ayprase(4U/ml) also completely inhibited the IL-8 release in ATP stimulated A549 cells. ATP could activate IL-8 report gene expression in A549 cells transfected with recombinant plasmid EPGF1-IL-8. The bio-luminescence assay showed that neutrophil defensins might induce ATP secretion, and the maximum release was at 10 minute and graduatedly reduced extending to 8 hours at least, neutrophil defensins only induced IL-8 increase in A549 cells, whereas, IL-6 also increased when stimulated by ATP. The other detected cytokines were not significantly increased by LiquiChip reader assay. RT-PCR results indicated that A549 cell strongly expressed P2Y2 and P2Y6 mRNAs, and weakly expressed P2Y4 gene. There was no differences in the density of RT-PCR products to be observed between normal and defensins-stimulated A549 cells. IL-8release was not inhibited when A549 cells exposed to various concentrations of Ca++ chelator BAPTA-AM. i25I labeling analysis demonstrated that nutrophil defensins could bind to A549 cells, and the binding was does-dependent. The competition assay with non-labeled neutrophil defensins confirmed that the binding was specific. Laser confocal microscopy observation showed that the nutrophil defensins could bind to the surface of A549 cells.In order for demonstrating the in vivo role of neutrophil defensins in the regulation of airway innate immunity. Preliminary gene transfer experiments were performed in this study. RT-PCR detected a 319 bp HNP-1 mRNA in pBabe-Neo-HNP-1-transfected primary cultured human tracheal epithelial cells. To determine the HNP-1 expression in the gene transfected cells, the rabbit antiserum against HNP-1 was prepared. Its titer was 1:12800 measured by ELISA. irnrnunocytochemistry showed positive staining in transfected cells. CONCLUSION: Human neutrophil defensins could induce release of IL-8 and ATP in the A549 cells, and the release was not due to TNF-cc and IL-8P action. A549 cells expressed P2Y2, P2Y4, and P2Y6 mRNAs. IL-8 release decreased when the p2y receptors was blocked, suggesting that p2y receptor subfamily mediated IL-8 production of A549 cells in response to human neutrophil defensins. Among them, P2Y2 and P2Y6 might play a major role in this process. Intracellular Ca++ seemed to be not important for regulating IL-8 production in the response of A549 cells to human neutrophil defensins.Extracellular nucleotides ATP and UDP enhanced release of IL-8 inA549 cell. ATP also could stimulate IL-6 production of A549 cells. P2Y receptors mediated ATP-induced IL-8 gene transcription and IL-8 production.Recombinant plasmid pBabe-Neo-HNPl could be transferred into human tracheal cells and expressed at mRNA and protein level. Another recombinant eukaryotic expression vector pBK CMV-HNP1 was constructed successfully.