Stolpen A H, Golan D E, Pober J S
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
J Cell Biol. 1988 Aug;107(2):781-9. doi: 10.1083/jcb.107.2.781.
Vascular endothelial surface-related activities may depend on the lateral mobility of specific cell surface macromolecules. Previous studies have shown that cytokines induce changes in the morphology and surface antigen composition of vascular endothelial cells in vitro and at sites of immune and inflammatory reactions in vivo. The effects of cytokines on membrane dynamic properties have not been examined. In the present study, we have used fluorescence photobleaching recovery (FPR) to quantify the effects of the cytokines tumor necrosis factor (TNF) and immune interferon (IFN-gamma) on the lateral mobilities of class I major histocompatibility complex protein, of an abundant 96,000 Mr mesenchymal cell surface glycoprotein (gp96), and of a phospholipid probe in cultured human endothelial cell (HEC) membranes. Class I protein and gp96 were directly labeled with fluorescein-conjugated monoclonal antibodies; plasma membrane lipid mobility was examined with the phospholipid analogue fluorescein phosphatidylethanolamine (Fl-PE). In untreated, confluent HEC monolayers, diffusion coefficients were 30 x 10(-10) cm2 s-1 for class I protein, 14 x 10(-10) cm2 s-1 for gp96, and 80 x 10(-10) cm2 s-1 for Fl-PE. Fractional mobilities were greater than 80% for each probe. Cultures treated at visual confluence for 3-4 d with either 100 U/ml TNF or 200 U/ml IFN-gamma did not exhibit significant changes in protein or lipid mobilities despite significant changes in cell morphology and membrane antigen composition. In HEC cultures treated concomitantly with TNF and IFN-gamma, however, diffusion coefficients decreased by 71-79% for class I protein, 29-55% for gp96, and 23-38% for Fl-PE. Fractional mobilities were unchanged. By immunoperoxidase transmission electron microscopy, plasma membranes of untreated and cytokine-treated HEC were flat and stained uniformly for class I antigen. "Line" FPR measurements on doubly treated HEC demonstrated isotropic diffusion of class I protein, gp96, and Fl-PE. Finally, although TNF and IFN-gamma retarded the growth of HEC cultures and disrupted the organization of cell monolayers, the slow diffusion rates of gp96 and Fl-PE in confluent doubly treated monolayers were not reproduced in sparse or subconfluent untreated monolayers. We conclude that the slowing of protein and lipid diffusion induced by the combination of TNF and IFN-gamma is not due to plasma membrane corrugations, to anisotropic diffusion barriers, or to decreased numbers of cell-cell contacts.(ABSTRACT TRUNCATED AT 400 WORDS)
血管内皮表面相关活性可能取决于特定细胞表面大分子的侧向流动性。先前的研究表明，细胞因子在体外以及体内免疫和炎症反应部位可诱导血管内皮细胞形态和表面抗原组成发生变化。细胞因子对膜动态特性的影响尚未得到研究。在本研究中，我们使用荧光光漂白恢复技术（FPR）来量化细胞因子肿瘤坏死因子（TNF）和免疫干扰素（IFN-γ）对人内皮细胞膜（HEC）中I类主要组织相容性复合体蛋白、一种丰富的96,000 Mr间充质细胞表面糖蛋白（gp96）以及磷脂探针侧向流动性的影响。I类蛋白和gp96直接用荧光素偶联的单克隆抗体标记；用磷脂类似物荧光素磷脂酰乙醇胺（Fl-PE）检测质膜脂质流动性。在未处理的汇合HEC单层中，I类蛋白的扩散系数为30×10⁻¹⁰ cm² s⁻¹，gp96为14×10⁻¹⁰ cm² s⁻¹，Fl-PE为80×10⁻¹⁰ cm² s⁻¹。每种探针的分数流动性均大于80%。用100 U/ml TNF或200 U/ml IFN-γ在视觉汇合时处理3 - 4天的培养物，尽管细胞形态和膜抗原组成发生了显著变化，但蛋白质或脂质的流动性没有显著变化。然而，在同时用TNF和IFN-γ处理的HEC培养物中，I类蛋白的扩散系数降低了71 - 79%，gp96降低了29 - 55%，Fl-PE降低了23 - 38%。分数流动性没有变化。通过免疫过氧化物酶透射电子显微镜观察，未处理和细胞因子处理的HEC的质膜是平的，I类抗原染色均匀。对双重处理的HEC进行“线”FPR测量表明，I类蛋白、gp96和Fl-PE呈各向同性扩散。最后，尽管TNF和IFN-γ抑制了HEC培养物的生长并破坏了细胞单层的组织，但在稀疏或未汇合的未处理单层中并未重现汇合的双重处理单层中gp96和Fl-PE的缓慢扩散速率。我们得出结论，TNF和IFN-γ联合诱导的蛋白质和脂质扩散减慢不是由于质膜褶皱、各向异性扩散屏障或细胞间接触数量减少所致。（摘要截短至400字）
