Iv. Guzhova et al., EFFECTS OF EXOGENOUS STRESS PROTEIN-70 ON THE FUNCTIONAL-PROPERTIES OF HUMAN PROMONOCYTES THROUGH BINDING TO CELL-SURFACE AND INTERNALIZATION, Cell stress & chaperones, 3(1), 1998, pp. 67-77
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.