HSP70-mediated phagocytosis is specific, titratable, actin dependent, and independent of protein synthesis and HSP-peptides. (A) Macrophages were treated with HSP70 or non-HSP controls (as indicated) to examine their specific effects on phagocytosis of Alexafluor-labeled yeast (S cerevisiae) at the concentration of 40 particles/macrophage. ^*P < .05 when compared to medium. (B) Macrophages were treated with HSP70 (doses indicated) and subsequently tested for their ability to phagocytose Alexafluor-labeled yeast (S cerevisiae) at the concentration of 40 particles/macrophage. (C) Representative microphotographs (× 10 magnification) of macrophages (treated for minutes as indicated). At 15 and 30 minutes the macrophages show ongoing phagocytic activity (elongated cells with cytoskeletal alterations), whereas at 60 minutes the cells are round and appear quiescent. Images were visualized under a Nikon Optiphot microscope (Nikon, Melville, NY) equipped with objective lenses ranging from 10 ×/2.5 to 40 ×/16.0. Images were captured with a Kodak DC 120-zoom camera (Kodak, Rochester, NY) and processed with Adobe Photoshop 6.0 software (Adobe Systems, San Jose, CA). (D) Macrophages were pretreated with HSP70 (100 μg/mL), as indicated, and washed free of residual HSP70. Macrophages were then administered yeast and tested in a phagocytosis assay. For comparison, macrophages were with HSP70 and administered the yeast at the same time. (E) HSP70-coated yeast was prepared by coincubating Alexafluor-labeled S cerevisiae (Sc) with HSP70 and washed until free of unbound HSP70. Experimental groups (as indicated) were tested for their ability to enhance phagocytosis. (F) Macrophages were treated with HSP70 (100 μg/mL) under (1) conditions that prevent actin-mediated cytoskeletal changes (low temperature and cytochalasin D as indicated) or (2) in the presence or absence of cycloheximide (as indicated) and subjected to a phagocytosis assay or (3) phagocytosis-enhancing effects of ATP-treated HSP70 (peptide free) was compared with that of ADP-purified HSP70 (with peptides). The error bar represents 1 SD. The results shown are a cumulative analysis of 3 experiments, 3 wells/group.