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)
