Golenbock D T, Hampton R Y, Raetz C R, Wright S D
Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, New York 10021.
Infect Immun. 1990 Dec;58(12):4069-75. doi: 10.1128/iai.58.12.4069-4075.1990.
Bacterial lipopolysaccharide (LPS) is a potent stimulus of cells, yet a target protein for LPS has not been defined. We used two approaches to define LPS-binding sites on cell surfaces: one assay measured binding of LPS-coated erythrocytes (ELPS) to cultured human cells, and a second measured binding of a radiolabeled probe, [32P]lipid IVA, to intact leukocytes. The first approach identified the CD11-CD18 family of integrins as lipid A-binding sites in human phagocytes, and the latter approach demonstrated saturable lipid A binding to intact murine macrophages, as well as to an approximately 95-kDa binding protein in purified membrane preparations. Because CD18 has a known molecular mass of 95 kDa, we sought to determine whether the [32P]lipid IVA-binding site was CD18. Binding of ELPS and [32P]lipid IVA to human macrophages was found to differ with respect to temperature, divalent cation dependence, cellular specificity, and susceptibility to competition by polyanions. To determine whether the previously described 95-kDa lipid A-binding protein was CD18, nitrocellulose-immobilized RAW264.7 membrane proteins were probed with [32P]lipid IVA and subsequently immunoblotted with a monoclonal antibody to murine CD18. The lipid A-binding protein has an electrophoretic mobility slightly different from that of CD18. Moreover, monoclonal antibodies and polyclonal antiserum to the CD11-CD18 family of proteins did not inhibit lipid IVA binding to intact human macrophages. Finally, mononuclear cells from two patients with CD18 deficiency failed to form rosettes with ELPS but bound [32P]lipid IVA normally. Thus, different LPS preparations may bind to cells in a CD18-dependent or -independent manner. Since ELPS is particulate and lipid IVA is a fine dispersion, the identity of the binding site may depend on the physical state of the LPS.
细菌脂多糖（LPS）是细胞的一种强效刺激物，但LPS的靶蛋白尚未明确。我们采用两种方法来确定细胞表面的LPS结合位点：一种测定方法是检测LPS包被的红细胞（ELPS）与培养的人细胞的结合，另一种测定方法是检测放射性标记探针[32P]脂质IVA与完整白细胞的结合。第一种方法确定整合素的CD11 - CD18家族是人吞噬细胞中的脂质A结合位点，后一种方法证明脂质A可饱和结合完整的小鼠巨噬细胞以及纯化膜制剂中的一种约95 kDa的结合蛋白。由于已知CD18的分子量为95 kDa，我们试图确定[32P]脂质IVA结合位点是否为CD18。发现ELPS和[32P]脂质IVA与人巨噬细胞的结合在温度、二价阳离子依赖性、细胞特异性以及对多阴离子竞争的敏感性方面存在差异。为了确定先前描述的95 kDa脂质A结合蛋白是否为CD18，用[32P]脂质IVA探测硝酸纤维素固定的RAW264.7膜蛋白，随后用抗小鼠CD18的单克隆抗体进行免疫印迹。脂质A结合蛋白的电泳迁移率与CD18略有不同。此外，针对CD11 - CD18蛋白家族的单克隆抗体和多克隆抗血清均不抑制脂质IVA与完整人巨噬细胞的结合。最后，两名CD18缺陷患者的单核细胞不能与ELPS形成花环，但能正常结合[32P]脂质IVA。因此，不同的LPS制剂可能以依赖或不依赖CD18的方式与细胞结合。由于ELPS是颗粒状的，而脂质IVA是精细分散的，结合位点的身份可能取决于LPS的物理状态。
