EFFECTS OF EXOGENOUS STRESS PROTEIN-70 ON THE FUNCTIONAL-PROPERTIES OF HUMAN PROMONOCYTES THROUGH BINDING TO CELL-SURFACE AND INTERNALIZATION

The presence of antibodies against the major stress protein, Hsp70, in patients with autoimmune diseases led us to hypothesize that Hsp70 ma y occur extracellularly, and could exert chaperoning and regulatory ef fects on various cells. We examined the action of pure Hsp/Hsc70 on th e 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 o bserved that the addition of the protein up-regulated two major monocy te/macrophage differentiation markers, CD11c and CD23, by 20-35%, whil e it had no effect on CD14. The experiments performed to investigate t he influence of Hsp/Hsc70 on the reaction of U-937 cells to differenti ation stimuli demonstrated that the addition of the protein prior to P MA was able to inhibit binding of proper transcription factors to doub le-symmetry and cAMP-response elements of the c-fos early response gen e promoter. Administration of exogenous Hsp/Hsc70 prior to treatment w ith the tumor necrosis factor-alpha significantly lowered the number o f apoptotic and necrotic cells. In no case did the control protein, ov albumin, 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 intera ction with the cell surface, we assayed binding of the biotinylated pr otein to U-937 cells by immunoenzyme assay, flow cytometry and indirec t immunofluorescence. Using these three techniques we were able to det ect Hsp/Hsc70 bound to cells after a 20 min incubation. According to f low cytometry data, at this time 32% of cells were positively stained with streptavidin-FITC. Immunofluorescence studies demonstrated Hsp/Hs c70 bound to the cell surface after a 20 min incubation followed by in duction of patch and cap-like structures. One hour later, the majority of the protein had been internalized by U-937 cells.