Iwagaki A, Porro M, Pollack M
Department of Medicine, Uniformed Services University of the Health Sciences, F. Edward Hebert School of Medicine, Bethesda, Maryland 20814, USA.
Infect Immun. 2000 Mar;68(3):1655-63. doi: 10.1128/IAI.68.3.1655-1663.2000.
Lipopolysaccharides (LPS) are proinflammatory bacterial products implicated in the pathogenesis of gram-negative sepsis and septic shock. Polymyxin B (PMB), a cyclic, cationic peptide antibiotic, inhibits biological activities of LPS through high-affinity binding to the lipid A moiety. Small synthetic peptides have been designed to mimic the primary and secondary structures of PMB to determine structural requirements for binding and detoxification of lipid A and to assess possible therapeutic potential. The purpose of this study was to compare and contrast the endotoxin-neutralizing activities of two synthetic antiendotoxin peptides (SAEP-2 and SAEP-4), PMB, and an LPS core-specific monoclonal antibody (MAb), WN1 222-5, based on their abilities to inhibit CD14-mediated target cell uptake of fluorescein isothiocyanate (FITC)-conjugated LPS, detected by flow cytometry and confocal microscopy, and LPS-induced production of the proinflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha), as measured by bioassays. PMB and SAEP-4 produced dose-dependent inhibition of FITC-LPS uptake by CD14-transfected Chinese hamster ovary fibroblasts (CHO-CD14 cells) and by human peripheral blood mononuclear cells. The anti-LPS MAb, WN1 222-5, also blocked LPS uptake by these cells and synergized with PMB and SAEP-4. LPS-induced IL-6 release was inhibited by PMB, SAEP-4, and MAb WN1 222-5, and these inhibitory activities were additive or synergistic. LPS-induced TNF-alpha release by PBMC was also inhibited by PMB and SAEP-4 alone and in combination with anti-LPS MAb. SAEP-2, in contrast, produced comparatively minor decrements in cellular uptake of LPS and LPS-induced cytokine responses, and did so only in the absence of serum, while a nonsense peptide exerted no discernible inhibitory effect on LPS uptake or LPS-induced cytokine expression in the presence or absence of serum. Thus, PMB and SAEP-4, like the LPS-reactive MAb, WN1 222-5, block proinflammatory activities of LPS in part by preventing LPS recognition by membrane-bound CD14-expressing target cells. Differences in peptide structure, however, like those exemplified by SAEP-2 and SAEP-4, may differentially affect the endotoxin-neutralizing potency of these peptides despite similar binding activity against lipid A, reflecting possible differences in peptide solubility or peptide regulation of intracellular signal transduction.
脂多糖（LPS）是促炎性细菌产物，与革兰氏阴性菌败血症和感染性休克的发病机制有关。多粘菌素B（PMB）是一种环状阳离子肽抗生素，通过与脂质A部分高亲和力结合来抑制LPS的生物学活性。人们设计了小的合成肽来模拟PMB的一级和二级结构，以确定脂质A结合和解毒的结构要求，并评估其可能的治疗潜力。本研究的目的是比较和对比两种合成抗内毒素肽（SAEP-2和SAEP-4）、PMB和一种LPS核心特异性单克隆抗体（MAb）WN1 222-5的内毒素中和活性，基于它们抑制CD14介导的异硫氰酸荧光素（FITC）偶联LPS的靶细胞摄取的能力（通过流式细胞术和共聚焦显微镜检测），以及LPS诱导的促炎细胞因子白细胞介素-6（IL-6）和肿瘤坏死因子α（TNF-α）的产生（通过生物测定法测量）。PMB和SAEP-4对CD14转染的中国仓鼠卵巢成纤维细胞（CHO-CD14细胞）和人外周血单核细胞摄取FITC-LPS产生剂量依赖性抑制。抗LPS单克隆抗体WN1 222-5也阻断这些细胞摄取LPS，并与PMB和SAEP-4协同作用。LPS诱导的IL-6释放受到PMB、SAEP-4和单克隆抗体WN1 222-5的抑制，并且这些抑制活性是相加或协同的。单独的PMB和SAEP-4以及与抗LPS单克隆抗体联合使用时，也抑制PBMC中LPS诱导的TNF-α释放。相比之下，SAEP-2仅在无血清时对LPS的细胞摄取和LPS诱导的细胞因子反应产生相对较小的降低，而无义肽在有或无血清时对LPS摄取或LPS诱导的细胞因子表达均无明显抑制作用。因此，PMB和SAEP-4与LPS反应性单克隆抗体WN1 222-5一样，部分通过阻止膜结合的表达CD14的靶细胞识别LPS来阻断LPS的促炎活性。然而，肽结构的差异，例如SAEP-2和SAEP-4所体现的差异，尽管对脂质A具有相似的结合活性，但可能会不同程度地影响这些肽的内毒素中和效力，这反映了肽溶解度或细胞内信号转导的肽调节方面可能存在的差异。
