The receptor for heat shock protein 60 on macrophages is saturable, specific, and distinct from receptors for other heat shock proteins.
Previous studies have shown that human heat shock protein (hsp) 60 elicits a strong proinflammatory response in cells of the innate immune system with CD14, Toll-like receptor (TLR) 2, and TLR4 as mediators of signaling, but probably not of binding. In the present study, we directly demonstrate binding of hsp60 to the macrophage surface and find the binding receptor for hsp60 different from the previously described common receptor for several other heat shock proteins, including hsp70, hsp90, and gp96. Fluorescence-labeled human hsp60 bound to cell surfaces of the murine macrophage lines J774 A.1 and RAW264.7 and to mouse bone marrow-derived macrophages. By flow cytometry, we could demonstrate for the first time that hsp60 binding to macrophages occurred at submicromolar concentrations, is saturable, and can be competed by unlabeled hsp60, but not by unrelated proteins, thus confirming the classic characteristics of specific ligand-receptor interactions. Binding of hsp60 at 4 degrees C was followed by endocytosis at 37 degrees C. Hsp60 binding to macrophages could not be competed by excess hsp70, hsp90, or gp96, all of which share the alpha(2)-macroglobulin receptor as binding site. Hsp60 binding occurred in the absence of surface TLR4. However, no cytokine response was induced by hsp60 in TLR4-deficient macrophages. We conclude that hsp60 binds to a stereo-specific receptor on macrophages, and that different surface molecules are engaged in binding and signal transduction. Furthermore, the binding site for hsp60 is separate from the common receptor for hsp70, hsp90, and gp96, which suggests an independent role of hsp60 as danger Ag and in immunoregulation.
Habich
Christiane
C
German Diabetes Research Institute at the Heinrich-Heine-University of Düsseldorf, Düsseldorf, Germany. christiane.habich@ddfi.uni-duesseldorf.de
Baumgart
Karina
K
Kolb
Hubert
H
Burkart
Volker
V
D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism; D001665:Binding Sites / Q000276:immunology; D002460:Cell Line; D018834:Chaperonin 60 / Q000032:analysis / Q000378:metabolism* / Q000502:physiology; D029721:Drosophila Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008264:Macrophages / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000032:analysis / Q000378:metabolism / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D009569:Nitric Oxide / Q000096:biosynthesis; D011485:Protein Binding / Q000276:immunology; D011956:Receptors, Cell Surface / Q000032:analysis / Q000378:metabolism / Q000502:physiology; D011971:Receptors, Immunologic / Q000032:analysis / Q000378:metabolism* / Q000502:physiology; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D000951:Antigens, Neoplasm; D018834:Chaperonin 60; D029721:Drosophila Proteins; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008024:Ligands; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; C052646:sarcoma glycoprotein gp96 rejection antigens; D009569:Nitric Oxide