Effect Of Cytokines In Host Defense And Inflammation

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Abstract

This year we continued to use 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 in normal patients treated topically (at the blister site) with immunomodulatory agents. 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) [unreadable] [unreadable] We continued our studies of fibrinogen as a regulator of IL8 production. In previous work we showed that fibrinogen amplifies IL8 synthesis in neutrophils stimulated with other chemoattractants such as fmetleuphe and LTB4. We extended this studies to human monocytes and showed that addition of physiological concentrations of fibrinogen amplified IL8 production by monocytes as well as increased IL6 and TNF alpha production. In contrast, fibrinogen had no effect on monocyte chemoattractant protein1 (MCP1), inteferonbeta, or interferon inducible protein10 (IP10). Treatment of monocytes with fibrinogen (less than 2 mgml) and complement 5 fragment, C5a, resulted in a 100% increase in both IL8 and IL6 production, compared to fibrinogen treatment alone. This was associated with a transient increase in monocyte IL8 mRNA and NFkB activity. Monocytes from patients with defective LPS and IL1 signaling through the Tolllike receptor pathway (NEMO deficiency and IRAK4 deficiency)had 80% reduced IL8 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)[unreadable] [unreadable] We continue to study the molecular basis for priming and activation of the NADPH oxidase and superoxide in patients with disorders of the Toll like receptor pathway and recurrent bacterial infections (IRAK4 deficiency and NEMO deficiency). This year we focused these studies on neutrophil priming for superoxide production by endotoxin (LPS). In normal subjects incubation of neutrophils with LPS alone induced NADPH oxidase cytosolic factors p47phox and Rac2, and the membrane integrated subunit gp91phox translocation to the membrane. Addition of fMLP to LPS treated neutrophils augmented the translocation of NADPH oxidase regulatory comonents (p47phox, p67phox, p40phox, Rac2 and gp91phox) compared with LPS or fMLP alone. LPS mediated phosphorylation of p47phox ran parallel to the priming effect of LPS on p47 phox translocation and superoxide production, though this was significantly inhibited by the phosphatidylinositol 3kinase (PI3K)inhibitor LY2940002. We also observed impaired superoxide generation by LPS in neutrophils from patients with IRAK4 deficiency that correlated with the inefficient translocation of cytosolic factors (p47phox, p67phox, p40phox, Rac2) and membrane integrated flavocytochrome b558 subunit gp91phox. However, neutrophils from patients with NEMO deficiency were much more responsive to the LPS priming than IRAK4 deficient cells, with normal levels of translocation for the oxidase modulatory subunits. Compared to the normal cells and NEMO deficient cells, we noted the incomplete phosphorlyation of p47phox in neutrophils of IRAK4 deficient cells. However, unlike the diferential priming and activation in the neutrophils from patients with TLR signaling defects, the levels of TLR expression remained unchanged in both IRAK4 and NEMO deficient cells. The use of LY2940002, as with IRAK4 deficient cells, led to the sub normal levels of superoxide generation and minimalphosphorylation and translocation of p47phox. (Anjali Singh)