Odekon L E, Frewin M B, Del Vecchio P, Saba T M, Gudewicz P W
Department of Physiology and Cell Biology, Albany Medical College, New York.
Immunology. 1991 Sep;74(1):114-20.
We have recently shown that monolayer cultures of calf pulmonary artery endothelial (CPAE) cells pretreated with phorbol myristate acetate (PMA) generate a conditioned medium that is chemotactic for human polymorphonuclear leucocytes (PMNL). Fibronectin (Fn) is a multidomain protein found in the plasma and subendothelial extracellular matrix that induces attachment and migration of a variety of cell types. The present study was designed to evaluate the role of Fn or fragments of Fn present in conditioned medium from phorbol ester-stimulated endothelial cells as potential chemotactic factors for human PMNL. A large number of Fn fragments were revealed by Western immunoblotting of serum-free conditioned medium 4 hr after treatment of CPAE monolayers with PMA. Gelatin-Sepharose affinity chromatography of 4-hr conditioned medium demonstrated chemotactic activity for PMNL in both gelatin-binding and non-gelatin-binding fractions. The addition of bovine Fn antiserum to the conditioned medium inhibited PMNL chemotaxis in a dose-dependent manner while having no effect on PMNL chemotaxis generated by zymosan-activated serum. One site on the Fn molecule known to interact with phagocytic cells is the cell-binding domain containing the Arg-Gly-Asp (RGD) sequence. Pretreatment of PMNL with a RGD-containing peptide (1 mM GRGDSPK) for 10 min completely inhibited the expression of chemotactic activity present in conditioned medium and in the gelatin-binding and non-gelatin-binding fractions. PMNL chemotaxis was not stimulated by either intact Fn or the RGD-containing septapeptide tested over a wide concentration range. However, incubation of PMNL with a purified 120,000-MW fragment of Fn containing the cell-binding domain stimulated chemotaxis in a dose-dependent manner. In contrast, a purified 45,000 MW fragment of Fn containing the gelatin-binding domain was not chemotactic for PMNL. When a monoclonal antibody directed against the cell-binding domain of Fn was incubated with conditioned medium, a significant reduction in PMNL chemotaxis was observed. These results demonstrate that phorbol ester-stimulated pulmonary artery endothelial cells release Fn fragments and suggest an important role for Fn fragments containing the cell-binding domain in stimulating the migration of PMNL.
我们最近发现，用佛波酯肉豆蔻酸乙酸酯（PMA）预处理的小牛肺动脉内皮（CPAE）细胞单层培养物能产生一种对人多形核白细胞（PMNL）具有趋化作用的条件培养基。纤连蛋白（Fn）是一种存在于血浆和内皮下细胞外基质中的多结构域蛋白，可诱导多种细胞类型的附着和迁移。本研究旨在评估Fn或佛波酯刺激的内皮细胞条件培养基中存在的Fn片段作为人PMNL潜在趋化因子的作用。用PMA处理CPAE单层4小时后，通过无血清条件培养基的Western免疫印迹法发现了大量的Fn片段。对4小时的条件培养基进行明胶-琼脂糖亲和层析，结果表明在明胶结合和非明胶结合部分均对PMNL具有趋化活性。向条件培养基中添加牛Fn抗血清可剂量依赖性地抑制PMNL趋化作用，而对酵母聚糖激活血清产生的PMNL趋化作用无影响。已知Fn分子上与吞噬细胞相互作用的一个位点是含有精氨酸-甘氨酸-天冬氨酸（RGD）序列的细胞结合结构域。用含RGD的肽（1 mM GRGDSPK）预处理PMNL 10分钟可完全抑制条件培养基以及明胶结合和非明胶结合部分中存在的趋化活性的表达。在很宽的浓度范围内，完整的Fn或含RGD的七肽均未刺激PMNL趋化作用。然而，用含有细胞结合结构域的纯化的120,000-MW Fn片段孵育PMNL可剂量依赖性地刺激趋化作用。相反，含有明胶结合结构域的纯化的45,000 MW Fn片段对PMNL没有趋化作用。当将针对Fn细胞结合结构域的单克隆抗体与条件培养基一起孵育时，观察到PMNL趋化作用显著降低。这些结果表明佛波酯刺激的肺动脉内皮细胞释放Fn片段，并提示含有细胞结合结构域的Fn片段在刺激PMNL迁移中起重要作用。
