Effect Of Cytokines In Host Defense And Inflammation

细胞因子在宿主防御和炎症中的作用

• 批准号: 7192860
• 负责人: JOHN I GALLIN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7192860
• 关键词: RNase protection assay; chemotaxis; chronic granulomatous disease; clinical research; enzyme linked immunosorbent assay; fibrinogen; human subject; hydrogen peroxide; immune response; immunoregulation; inflammation; interleukin 8; monocyte; neutrophil; northern blottings

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.

今年我们继续研究CGD中过度肉芽肿形成的基础。我们发现，CGD中性粒细胞比正常中性粒细胞产生2 - 4倍的IL-8化学引诱物和持续的IL-8 mRNA应答。此外，用过氧化氢酶处理以清除细胞外过氧化氢，或用氯化二苯碘铵（DPI）处理以抑制NADPH氧化酶的正常嗜中性粒细胞表现出与CGD嗜中性粒细胞相当的IL-8应答。相比之下，在CGD与正常中性粒细胞中，fMLF诱导的IL-1a蛋白和IL-1a mRNA的瞬时增加没有差异。此外，在用过氧化氢酶或DPI处理的正常嗜中性粒细胞中，fMLF不能诱导IL-1 α、TNF-α、IL-6、IL-1ra或其他趋化因子的增加。添加过氧化氢或过氧化氢生成系统（次黄嘌呤加黄嘌呤氧化酶）抑制持续的IL-8 mRNA和CGD中性粒细胞中观察到的蛋白质产生增加。这些结果表明，NADPH氧化酶活化下游的效应物负调节正常中性粒细胞中的IL-8 mRNA，并且它们在CGD细胞中的缺失导致IL-8 mRNA转录延长和IL-8水平提高。这种IL-8的异常调节可能有助于CGD中过度肉芽肿的形成。活性氧可能通过这种机制在调节炎症中发挥关键作用。 我们继续研究纤维蛋白原作为IL-8产生的调节剂。在以前的工作中，我们表明，纤维蛋白原放大IL-8的合成与其他化学引诱剂，如fmet-leu-phe和LTB4刺激的中性粒细胞。我们将这项研究扩展到人单核细胞，并表明加入低于正常血浆浓度（低于2 mg/ml）的纤维蛋白原可扩增单核细胞产生IL-8，并增加IL-6和TNF α的产生。相反，纤维蛋白原对单核细胞趋化蛋白-1（MCP-1）、干扰素-β或干扰素诱导蛋白-10（IP-10）没有影响。与单独的纤维蛋白原处理相比，用纤维蛋白原（小于2mg/ml）和补体5片段C5a处理单核细胞导致IL-8和IL-6产物增加100%。这与单核细胞IL-8 mRNA和NF-κ B活性的瞬时增加有关。与正常单核细胞相比，LPS和IL-1通过Toll样受体途径（NEMO缺陷和IRAK-4缺陷）信号传导缺陷患者的单核细胞对纤维蛋白原的IL-8反应降低了80%。此外，正常单核细胞对纤维蛋白原的反应被阻断CD14的抗体阻断，CD14是LPS受体的亚单位，其转导信号转导TLR4。MY4对PMA和离子霉素诱导的细胞因子产生无影响。纤维蛋白原浓度高于2mg/ml时，抑制上述反应。