Bidirectional signaling between stromal and hemopoietic cells regulates interleukin-1 expression during human osteoclast formation

Interleukin-1 (IL-1) has been shown to promote osteoclast (OC) differentiat ion, in addition to acting as a survival factor for mature osteoclasts. In this study, we investigate the expression of IL-1 during human osteoclast f ormation, taking advantage of a recently reported in vitro culture system t hat generates human OC from precursors in the peripheral blood mononuclear cell (PBMC) fraction, in the presence of murine stromal cells. This system enabled us to use species-specific probes and immunoassays to determine the respective cytokine contributions of the stromal cell and hemopoietic cell populations. Formation of functional osteoclasts occurred in cocultures of human PBMC and ST-2 cells for up to 21 days in the presence of 1 alpha,25( OH)(2)-vitamin D-3 dexamethasone, and recombinant human macrophage colony-s timulating factor (rhM-CSF). Total RNA was prepared at intervals during the cocultures and reverse transcriptasepolymerase chain reaction (RT-PCR) was performed using primers designed to amplify specifically the mRNA species corresponding to the respective murine or human IL-1 alpha and IL-1 beta is oforms, Using human-specific primers, it was found that the hemopoietic cel l component expressed both IL-1 alpha and IL-1 beta mRNA, Specific measurem ent of secreted human IL-1 beta protein showed greatly augmented levels in coculture compared with hemopoietic cells grown in the absence of ST-2 cell s, consistent with the known signaling from stromal cells to hemopoietic ce lls during osteoclastogenesis. Specific detection of mouse mRNA products sh owed that the ST-2 stromal cells in the coculture also expressed mRNA corre sponding to IL-1 alpha and IL-1 beta, The expression of both mouse and huma n IL-1 mRNA was found to decline over the course of the coculture, although the level of IL-1 alpha mRNA relative to IL-1 beta mRNA remained constant, indicating that the two isoforms were coregulated in both cell populations under these conditions. Importantly, the hemopoietic cells were found to i nfluence strongly the IL-1 mRNA levels in ST-2 cells, such that mouse IL-1 alpha and IL-1 beta mRNA levels were greatly enhanced in coculture, compare d with ST-2 cells alone, Secreted mouse IL-1 beta protein was upregulated i n coculture in parallel with mRNA levels. However, the absolute levels of m ouse IL-1 beta achieved were more than 20-fold lower than the human IL-1 be ta levels. Prostaglandin estradiol (PGE(2)) levels were measured and found to be greatly increased in the coculture compared with ST-2 cells or hemopo ietic cells alone, consistent with evidence that IL-1 action in osteoclasto genesis is mediated by PGE(2). These results provide novel evidence that bi directional signaling between stromal and hemopoietic cells may be importan t in the generation of human osteoclasts. (C) 1999 by Elsevier Science Inc. All rights reserved.