EXPRESSION OF LPS-INDUCIBLE CYTOKINES BY HUMAN MONOCYTES--PRIMING EFFECTS

细菌脂多糖(LPS)是一种有效的刺激剂 单核细胞和巨噬细胞。内毒素诱导的功能对 有效的宿主防御，但内毒素也可诱导病理 当反应继续不受控制时，情况会变得复杂。的长期目标是 这个项目是为了阐明与内毒素有关的分子事件。 对人单核细胞的刺激。这些研究的重点是 调节有效的生物活性细胞因子的产生(如 肿瘤坏死因子、干扰素、白介素1或白介素12) 对内毒素的反应。我们目前的具体兴趣在于机制(S) 通过T细胞衍生的细胞因子启动单核细胞，从而导致 随后的内毒素反应显著增强。引爆发生在以下位置 干扰素-γ或粒-巨噬细胞培养 集落刺激因子(GM-CSF)。单核细胞产生的两种细胞因子 绝对依赖于启动：干扰素-α和白细胞介素12。 这两种细胞因子都在积极研究抗病毒作用。 和抗肿瘤特性。引爆也会导致大量的 促进肿瘤坏死因子的产生。对这些病毒的特异性RNA进行分析 细胞因子表明它们在预置的单核细胞中的表达增强 发生在RNA水平上。有一些证据表明，对肿瘤坏死因子的影响 MRNA的表达是转录的；核连续分析表明 内毒素诱导转录，这通过启动而增强，但是 这还不足以解释信使核糖核酸积累的巨大增长。 启动对核因子-kB转录因子也有显著影响 激活；核因子-kB参与介导内毒素诱导 转录反应。此外，启动显著地影响 细胞因子mRNA稳定性的调节。正在研究的细胞因子 (肿瘤坏死因子、干扰素)在拥有3‘-侧翼序列方面是独特的 被认为与信使核糖核酸不稳定有关。对…的详尽研究 肿瘤坏死因子的信使核糖核酸半衰期表明，启动导致显著的 增加了肿瘤坏死因子mRNA的稳定性。白介素12的研究进展 生产已经证明，这种细胞因子的表达是 受p35亚基的严格调控(以前 被认为是符合宪法的表达，并且相对不受监管)。这个 只有当细胞被干扰素-伽马激活时，p35的表达才被诱导。 然后进行内毒素刺激。仅靠刺激是不够的，而且 在同等水平上启动肿瘤坏死因子表达的GM-CSF是一种糟糕的 IL-12的诱导物p35或活性异源二聚体(p35/p40)。这些研究 进一步揭示了IL-12表达的严格时间要求 亚单位。我们最近分离出了IL-12p35的人类基因组克隆。 这些克隆的使用表明，p35 mRNA的转录是 从紧邻单核细胞下游的一个不同的位置开始 TATA盒，位于最初从EBV转化的cDNA中 淋巴母细胞。

Bacterial lipopolysaccharide (LPS) is a potent stimulatory agent for monocytes and macrophages. The functions induced by LPS are critical to effective host defense, but LPS can also induce pathological complications when responses continue unchecked. The long-term goal of this project is to elucidate molecular events that are involved in LPS stimulation of human monocytes. The focus of these studies is the regulation of the production of potent, bioactive cytokines (such as tumor necrosis factor, interferon, interleukin-1, or interleukin-12) in response to LPS. Our current specific interest is in the mechanism(s) of priming of monocytes, by T cell-derived cytokines, which results in marked enhancement of subsequent LPS responses. Priming occurs following culture in either interferon-gamma (IFN-gamma) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Two cytokines produced by monocytes are absolutely dependent upon priming: IFN-alpha and interleukin-12. Both of these cytokines are under active investigation for the anti-viral and anti-tumor properties. Priming also results in substantial enhancement of TNF production. Analysis of specific RNA for these cytokines indicated that their enhanced expression in primed monocytes occurs at the RNA level. There is some evidence that the effects on TNF mRNA expression are transcriptional; nuclear run-on analysis suggested that LPS induces trancription and that this is enhanced by priming, but not to a degree that explains the vast increase in mRNA accumulation. Priming also has dramatic effects on NF-kB transcription factor activation; NF-kB has been implicated in mediating LPS-induced transcriptional responses. In addition, priming significantly affects the regulation of cytokine mRNA stability. The cytokines under study (TNF, IFN) are unique with respect to possessing 3'-flanking sequences thought to be associated with mRNA instability. Exhaustive studies of mRNA half-life of TNF demonstrated that priming results in a substantial increase in TNF mRNA stability. Studies of interleukin 12 (IL-12) production have demonstrated that expression of this cytokine is controlled by the stringent regulation of the p35 subunit (previously thought to be constitutively expressed and relatively unregulated). The expression of p35 is induced only when cells are primed with IFN-gamma, followed by LPS stimulation. Neither stimulus alone is sufficient, and GM-CSF, which primes for TNF expression at equivalent levels, is a poor inducer of IL-12 p35 or the active heterodimer (p35/p40). These studies further revealed the strict temporal requirements for expression of IL-12 subunits. We have recently isolated human genomic clones for IL-12 p35. Use of these clones has revealed that transcription of p35 mRNA is initiated from a distinct site in monocytes immediately downstream of a TATA box that is within the cDNA originally cloned from EBV-transformed lymphoblastoid cells.