Effect Of Cytokines In Host Defense And Inflammation

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This year we continued our investigations of the basis for excessive granuloma formation in CGD. We showed that CGD neutrophils make two to four-fold more IL-8 chemoattractant and a sustained IL-8 mRNA response compared with normal neutrophils. Moreover, normal neutrophils treated with catalase to scavange extracellular hydrogen peroxide, or treated with diphenyleneiodonium chloride (DPI) to inhibit NADPH oxidase, exhibit IL-8 responses comparable to CGD neutrophils. In contrast, there is no difference in the fMLF-induced transient increases in IL-1a protein and IL-1a mRNA in CGD vs. normal neutrophils. In addition, fMLF fails to induce increases in IL-1a, TNF-a, IL-6, IL-1ra or other chemokines in normal neutrophils treated with either catalase or DPI. Addition of hydrogen peroxide or a hydrogen peroxide-generating system (hypoxanthine plus xanthine oxidase) suppresses the sustained IL-8 mRNA and increased protein production observed in CGD neutrophils. These results indicate that effectors downstream of the activation of NADPH oxidase negatively regulate IL-8 mRNA in normal neutrophils and their absence in CGD cells results in prolonged IL-8 mRNA transcription and enhanced IL-8 levels. This abnormal regulation of IL-8 may contribute to the excess granuloma formation in CGD. Reactive oxygen species may play a critical role in regulating inflammation through this mechanism. 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 fibrinogen below normal plasma concentrations (less than 2 mg/ml) 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 prod8uction, 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. Concentrations of fibrinogen above 2mg/ml inhibited these responses.