Guzhova I V, Arnholdt A C, Darieva Z A, Kinev A V, Lasunskaia E B, Nilsson K, Bozhkov V M, Voronin A P, Margulis B A
State University of Northern Fluminense, Campos RJ, Brazil.
Cell Stress Chaperones. 1998 Mar;3(1):67-77. doi: 10.1379/1466-1268(1998)003<0067:eoespo>2.3.co;2.
The presence of antibodies against the major stress protein, Hsp70, in patients with autoimmune diseases led us to hypothesize that Hsp70 may occur extracellularly, and could exert chaperoning and regulatory effects on various cells. We examined the action of pure Hsp/Hsc70 on the main physiological functions of human promonocytic U-937 cells. The protein was isolated from calf muscle and was shown to be a mixture of inducible Hsp70 (60%) and constitutive Hsc70 (40%) isoforms. It was observed that the addition of the protein up-regulated two major monocyte/macrophage differentiation markers, CD11c and CD23, by 20-35%, while it had no effect on CD14. The experiments performed to investigate the influence of Hsp/Hsc70 on the reaction of U-937 cells to differentiation stimuli demonstrated that the addition of the protein prior to PMA was able to inhibit binding of proper transcription factors to double-symmetry and cAMP-response elements of the c-fos early response gene promoter. Administration of exogenous Hsp/Hsc70 prior to treatment with the tumor necrosis factor-alpha significantly lowered the number of apoptotic and necrotic cells. In no case did the control protein, ovalbumin, taken in the same concentration give a comparable effect on U-937 cells. Since the Hsp/Hsc70 effects occurred within the first hour of co-incubation, and therefore they might be explained by its interaction with the cell surface, we assayed binding of the biotinylated protein to U-937 cells by immunoenzyme assay, flow cytometry and indirect immunofluorescence. Using these three techniques we were able to detect Hsp/Hsc70 bound to cells after a 20 min incubation. According to flow cytometry data, at this time 32% of cells were positively stained with streptavidin-FITC. Immunofluorescence studies demonstrated Hsp/Hsc70 bound to the cell surface after a 20 min incubation followed by induction of patch and cap-like structures. One hour later, the majority of the protein had been internalized by U-937 cells.
自身免疫性疾病患者体内存在针对主要应激蛋白Hsp70的抗体，这使我们推测Hsp70可能存在于细胞外，并可能对各种细胞发挥伴侣和调节作用。我们研究了纯Hsp/Hsc70对人原单核细胞U-937细胞主要生理功能的作用。该蛋白从小牛肌肉中分离得到，显示为诱导型Hsp70（60%）和组成型Hsc70（40%）异构体的混合物。据观察，添加该蛋白可使两种主要的单核细胞/巨噬细胞分化标志物CD11c和CD23上调20%-35%，而对CD14没有影响。为研究Hsp/Hsc70对U-937细胞分化刺激反应的影响而进行的实验表明，在加入佛波酯（PMA）之前添加该蛋白能够抑制相关转录因子与c-fos早期反应基因启动子的双对称和cAMP反应元件的结合。在用肿瘤坏死因子-α处理之前给予外源性Hsp/Hsc70可显著降低凋亡和坏死细胞的数量。相同浓度的对照蛋白卵清蛋白对U-937细胞从未产生类似的作用。由于Hsp/Hsc70的作用在共孵育的第一小时内就出现了，因此可能是由于它与细胞表面的相互作用所致，我们通过免疫酶测定、流式细胞术和间接免疫荧光法检测了生物素化蛋白与U-937细胞的结合。使用这三种技术，我们能够在孵育20分钟后检测到与细胞结合的Hsp/Hsc70。根据流式细胞术数据，此时32%的细胞被链霉亲和素-异硫氰酸荧光素（streptavidin-FITC）阳性染色。免疫荧光研究表明，孵育20分钟后Hsp/Hsc70与细胞表面结合，随后诱导形成斑块和帽状结构。一小时后，大部分蛋白已被U-937细胞内化。
