BINDING OF ACTIVATED PROTEIN-C TO A SPECIFIC RECEPTOR ON HUMAN MONONUCLEAR PHAGOCYTES INHIBITS INTRACELLULAR CALCIUM SIGNALING AND MONOCYTE-DEPENDENT PROLIFERATIVE RESPONSES

Upon activation, mononuclear phagocytes (M<empty set>) play key roles in the development of septic shock and multiple host immune responses, but details of the regulation of M<empty set> activation are little u nderstood. We recently showed that the physiologic anticoagulant molec ule, activated protein C (APC), blocks responses of human blood M<empt y set>, alveolar M<empty set>, or THP-1 cells induced by LPS, IFN-gamm a, or PMA, including TNF-alpha production and down-regulation of sever al LPS binding-related proteins. We now report a possible mechanism of action through inhibition of the rapid intracellular calcium signalin g that occurs at the onset of M<empty set> activation, and characteriz ation of a specific M<empty set> receptor for APC. Flow cytometry stud ies using Fluo-3 showed that M<emptyset> activation by Fc-receptor cro ss-linking or rIFN-gamma caused a rapid increase in free intracellular calcium, a primary event in multiple signal transduction pathways, wh ich was blocked by pretreatment with APC. Consistent with this, additi on of APC inhibited PHA-induced T cell proliferation in a dose- and ti me-dependent manner. Peak suppression (>70%) required addition of APC within the first hour of 72 hr cocultures of M<empty set> and lymphocy tes, and proliferative responses were not restored by addition of IL-2 or TNF-alpha. Biochemical studies showed that I-125-labeled APC bound specifically to M<empty set> in a time-dependent and saturable manner . Scatchard analysis indicated there were 180,690 binding sites for AP C per cell, which were of high affinity (Kd value of 12.9 nM). Binding of I-125-APC was doubled by activation of M<empty set> with LPS, and bound APC was not displaced by the zymogen, protein C (PC), or by enzy matically inactive (diisopropyl fluorophosphate- or PPACK-treated) APC , indicating an absolute requirement for the active site of APC in its binding to M<empty set>. APC binding was blocked by a polyclonal Ab t o human PC/APC, but not by protein S, factor Va or Xa, or a polyclonal antithrombomodulin antibody. When I-125-APC was crosslinked to its re ceptor, immunoprecipitated and analyzed by SDS-PAGE under reducing con ditions, a covalent complex (110-115 kD) of I-125-APC (62 kD) and its receptor was seen. In addition, a M<empty set> membrane protein of 50- 55 kD, as determined by SDS-PAGE, was affinity-purified using an APC-A ffigel column, and confirmed by ligand binding. Taken together, our fi ndings document the presence of a M<empty set> surface receptor for AP C, which appears distinct from a recently described endothelial recept or for PC and APC, and which may be involved in the inhibitory effects of APC on activation of human M<empty set>, including M<empty set>-de pendent T cell proliferation.