Effect Of Cytokines In Host Defense And Inflammation

今年，我们继续研究CGD中过度肉芽肿形成的基础。我们发现，与正常中性粒细胞相比，CGD中性粒细胞产生的IL-8趋化因子和持续的IL-8信使核糖核酸反应增加了两到四倍。此外，用过氧化氢酶处理的正常中性粒细胞清除胞外过氧化氢，或用二苯基碘氯(DPI)处理以抑制NADPH氧化酶，表现出与CGD中性粒细胞类似的IL-8反应。相反，在FMLF诱导的CGD中性粒细胞IL-1a蛋白和IL-1a mRNA的瞬时升高方面与正常中性粒细胞相比没有差异。此外，在用过氧化氢酶或DPI处理的正常中性粒细胞中，FMLF未能诱导IL-1a、TNF-a、IL-6、IL-1ra或其他趋化因子的增加。加入过氧化氢或过氧化氢产生系统(次黄嘌呤加黄嘌呤氧化酶)可抑制CGD中性粒细胞持续的IL-8mRNA和蛋白质产量的增加。这些结果表明，NADPH氧化酶激活下游的效应物对正常中性粒细胞的IL-8mRNA具有负性调节作用，而在CGD细胞中它们的缺失会导致IL-8mRNA转录延长和IL-8水平升高。IL-8的这种异常调节可能是CGD过度肉芽肿形成的原因之一。通过这一机制，活性氧可能在调节炎症反应中发挥重要作用。 我们继续研究纤维蛋白原作为IL-8产生的调节因子。在以前的工作中，我们发现纤维蛋白原可以在其他化学诱导剂如FMET-Leu-Phe和LTB4的刺激下增强中性粒细胞IL-8的合成。我们将这项研究扩展到人类单核细胞，结果表明，低于正常血浆浓度(低于2 mg/ml)的纤维蛋白原可促进单核细胞产生IL-8，并增加IL-6和肿瘤坏死因子α的产生。纤维蛋白原对单核细胞趋化蛋白-1(MCP-1)、干扰素-β和干扰素诱导蛋白-10(IP-10)无影响。单核细胞经纤维蛋白原(<2 mg/ml)和补体5片段C5a处理后，IL-8和IL-6的产生均比单用纤维蛋白原增加100%。这与单核细胞IL-8mRNA和核因子-kB活性的一过性增加有关。与正常单核细胞相比，通过Toll样受体途径(Nemo缺陷和IRAK-4缺陷)产生的内毒素和IL-1信号缺陷患者的单核细胞对纤维蛋白原的反应降低了80%。此外，正常单核细胞对纤维蛋白原的反应可被阻断CD14的抗体阻断，CD14是脂多糖受体的一个亚单位，通过TLR4传递信号。MY4对PMA和离子霉素诱导的细胞因子的产生没有影响。高于2 mg/ml的纤维蛋白原浓度可抑制这些反应。

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.