Fazal Nadeem, Shelip Alla, Siddiqui Erum, Ali Ashraf, Azim Anser C, Al-Ghoul Walid M
Department of Pharmaceutical Sciences, College of Pharmacy, Chicago State University, Chicago, IL 60628, USA.
FEMS Immunol Med Microbiol. 2012 Mar;64(2):191-204. doi: 10.1111/j.1574-695X.2011.00881.x.
Recently we found that superimposition of Enterococcus faecalis infection on burn injury caused an eruption of host mortality not seen with either individual challenge. We hypothesized that the Enterococcus bacteria, and/or factors related to these organisms, aggravate burn-induced modulations in host defense by neutrophils. Our study focuses on alterations in neutrophils' oxidative, proteolytic, and adhesive functions and transendothelial migration of neutrophils in burn rats inoculated with E. faecalis. Rats were subjected to burn (30% total body surface area) and then intra-abdominally inoculated with E. faecalis (10(4)CFU kg(-1) b.w). Polymorphonuclear neutrophils (PMNs) were harvested from circulating/blood and tissue/peritoneal cavity at day-2 post injury. Extracellular release of O(-)(2) anion production was determined by luminometry, and intracellular production of reactive oxygen species was measured by digital imaging technique. Fluoroscan analysis and confocal microscopy determined intracellular elastase production. The expression of adhesion molecule CD11b/CD18 was performed by flow cytometry. Calcein AM-labeled PMNs were co-cultured with TNF-α-stimulated rat lung microvascular endothelial cells, and their ability to adhere was assessed by fluorometry and digital imaging and finally, chemotaxis was measured by neutrophil transmigration assays. The results showed differential effector responses by circulatory and/or tissue PMNs. Tissue/peritoneal PMNs produced more O(-)(2), less intracellular elastase, and increased expression of CD11b/CD18 accompanied with increased adhesivity of MIP-2-stimulated PMNs to endothelial cells as compared to circulatory/blood PMNs. This differential effect was more pronounced following burn plus E. faecalis infection, indicating that the combined injury changed neutrophil functions.
最近我们发现，粪肠球菌感染叠加在烧伤之上会导致宿主死亡率急剧上升，而单独的任何一种损伤都不会出现这种情况。我们推测，粪肠球菌及其相关因素会加重烧伤诱导的中性粒细胞对宿主防御的调节作用。我们的研究聚焦于接种粪肠球菌的烧伤大鼠中性粒细胞的氧化、蛋白水解和黏附功能的改变以及中性粒细胞的跨内皮迁移。大鼠遭受烧伤（全身表面积的30%），然后腹腔内接种粪肠球菌（10⁴CFU kg⁻¹体重）。在损伤后第2天从循环血液和组织/腹腔中采集多形核中性粒细胞（PMN）。通过发光法测定O₂⁻阴离子的细胞外释放量，通过数字成像技术测量活性氧的细胞内生成量。荧光扫描分析和共聚焦显微镜检测细胞内弹性蛋白酶的生成。通过流式细胞术检测黏附分子CD11b/CD18的表达。用钙黄绿素AM标记的PMN与肿瘤坏死因子-α刺激的大鼠肺微血管内皮细胞共培养，通过荧光测定法和数字成像评估其黏附能力，最后通过中性粒细胞迁移试验测量趋化性。结果显示循环和/或组织PMN的效应反应存在差异。与循环血液PMN相比，组织/腹腔PMN产生更多的O₂⁻，细胞内弹性蛋白酶更少，CD11b/CD18的表达增加，同时MIP-2刺激的PMN与内皮细胞的黏附性增强。烧伤加粪肠球菌感染后，这种差异效应更为明显，表明联合损伤改变了中性粒细胞的功能。
