Analysis of the induction of cytokine production by Pseudomonas aeruginosa pilin in the lung of mice

众所周知，铜绿假单胞菌的菌毛对于粘附上皮细胞具有重要作用。在体外实验中，一些研究发现毛菌可诱导上皮细胞产生IL-8。在本研究中，我们研究了气管内接种毛毛成分蛋白毛素对小鼠模型的影响。经蔗糖梯度离心，从SDS-PAGE凝胶中提取皮林。将纯化的铜绿假单胞菌、BSA或脂多糖（LPS）经气管内接种于6周龄Balb/c小鼠。处死小鼠，行支气管肺泡灌洗（BAL）。分析BAL液的细胞计数和细胞因子（TNF-α、MIP-2、IL-4、IFN-γ）浓度。此外，为了研究炎症细胞浸润的关键表位，我们合成了13个肽段（残基为1-20,11-30,21-40,31-50,41-60,51-70,61-80,71-90,81-100,91-110,101-120,111-130,121-143，基于PAO-1氨基酸序列），并接种这些肽段，然后进行BAL检测白细胞计数和细胞因子浓度。与接种bsa的对照组小鼠相比，接种pilin的小鼠BAL液中白细胞在接种6小时后增加。接种后72hr接种匹林BAL液中白细胞峰值计数。超过90%的白细胞是中性粒细胞。气管内接种匹林诱导BAL液中TNF-α和MIP-2浓度与BSA接种组比较，但匹林接种组和BSA接种组对BAL液中IL-4和IFN-γ浓度无显著差异。另一方面，所有13种多肽均不显著影响BAL液中炎症细胞和细胞因子浓度的反应，但部分多肽可轻微诱导BAL液中的中性粒细胞和TNF-α。提示铜绿假单胞菌的匹林是铜绿假单胞菌呼吸道感染中诱导炎症的重要因素之一。少

It is well known that pili of Pseudomonas aeruginosa has important role for adherence to epithelial cell. Some studies have been revealed that pili induced IL-8 production from epithelial cell in vitro experiments. In this study, we investigated that the affects of intratracheal inoculation of pilin, which is a component protein of pili, in murine model. Pilin was extracted from SDS-PAGE gel after sucrose gradient centrifugation. Purified pilin of P.aeruginosa, BSA or lipopolysaccharide(LPS) were inoculated to 6 weeks old Balb/c mice intratracheally. The mice were sacrificed and performed bronchoalveolar lavage(BAL). BAL fluid were analysed for cell counts and cytokines(TNF-α, MIP-2, IL-4, IFN-γ) concentrations. Additionally, to investigate the critical epitope for infiltration of inflammatory cells, we sythesized thirteen peptides(residues 1-20,11-30,21-40,31-50,41-60,51-70,61-80,71-90,81-100,91-110,101-120,111-130,121-143 based on PAO-1 amino acids sequence) and inoculated these pept … More ides intratracheally followed by BAL for examination of WBC counts and cytokine concentration. White blood cells in BAL fluid whose mice were inoculated pilin increased after 6 hr inoculation compared with the BSA-inoculated control mice. Peak counts of WBC in BAL fluid of pilin inoculated at 72hr after inoculation. More than ninety percent of WBC were neutrophils. Intratracheal inoculation of pilin induced TNF-α and MIP-2 concentrations in BAL fluid compared with BSA inoculated group although there was no significant difference between pilin inoculated and BSA inoculated groups in the concentrations of IL-4 and IFN-γ in BAL fluid. On the other hand, all thirteen peptides did not affect any response of inflammatory cell and cytokine concentration in BAL fluid significantly although some peptides slightly induced neutrophils and TNF-α in BAL fluid. These findings suggest that pilin of P.aeruginosa is one of the important factors' for induction of inflammation in the P.aeruginosa respiratory infection. Less