Effect Of Cytokines In Host Defense And Inflammation

今年，我们重新建立了一个模型无菌炎性渗出物在人类捐赠者使用皮肤吸水泡。在过去的6个月里，我们招募了9名捐赠者，并建立了基线细胞因子反应，部分重现了该实验室之前的结果，但也发现了以前未报道的在水疱形成过程中上调的其他细胞因子。我们计划使用这种水疱技术来探索免疫功能障碍患者的炎症反应，以及局部（在水疱部位）使用免疫调节剂治疗的异常患者。我们同时收集了渗出细胞和外周细胞的RNA，并将使用微阵列技术来鉴定由足底沉降诱导的基因组调控。(Kol Zarember)

This year we reestablished a model of sterile inflammatory exudates in human donors using skin suction blisters. Over the past six months we have enrolled 9 donors and have established a baseline cytokine response in part reproducting previous results from this lab, but also identifying additional cytokines not previously reported to be upregulated during blister formation. We are planning on using this blister technique to explore inflammatory responses in patients with immune dysfunction but also innormal patients treated topically (at the blister site) with immunomodulatoryagents. We have collected RNA concurently with exudate cells and from peripheral cells and will be using microarray technology to identify genomic regulation induced by diapediesis.(Kol Zarember) We continued our studies of fibrinogen as a regulator of IL-8 production. In previous work we showed that fibrinogen amplifies IL-8 synthesis in neutrophils stimulated with other chemoattractants such as fmet-leu-phe and LTB4. We extended this studies to human monocytes and showed that addition of physiological concentrations of fibrinogen amplified IL-8 production by monocytes as well as increased IL-6 and TNF alpha production. In contrast, fibrinogen had no effect on monocyte chemoattractant protein-1 (MCP-1), inteferon-beta, or interferon inducible protein-10 (IP-10). Treatment of monocytes with fibrinogen (less than 2 mg/ml) and complement 5 fragment, C5a, resulted in a 100% increase in both IL-8 and IL-6 production, compared to fibrinogen treatment alone. This was associated with a transient increase in monocyte IL-8 mRNA and NF-kB activity. Monocytes from patients with defective LPS and IL-1 signaling through the Toll-like receptor pathway (NEMO deficiency and IRAK-4 deficiency)had 80% reduced IL-8 response to fibrinogen compared with normal monocytes. Moreover, normal monocyte responses to fibrinogen were blocked by antibody that blocks CD14, a subunit of the LPS receptor that transduces signal throug TLR 4. MY4 had no effect on cytokine production induced by PMA and ionomycin. (Dough Kuhns) This year we have utilized our patient with a disorder of the Toll-like receptor pathway and recurrent bacterial infections (IRAK-4 deficiency), to study neutrophil priming for superoxide production by endotoxin. We have shown that IRAK-4 deficiency has dminished priming of superoxide production in response to endotoxin and diminished translocation of cytosolic NADPH oxidase proteins p47phox and p67phox to the plasma membrane. Similar results were obtained by blocking toll-like receptor 4(TLR-4) signalling in normal cells using the TLR-4 blocking antibody made by Imgenix #IMG-417E). These studies indicate an importrant role for the TLR pathway in endotoxin priming of NADPH oxidase pathway. (Anjali Singh)