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) differentiation, in addition to acting as a survival factor for mature osteoclasts. In this study, we investigate the expression of IL-1 during human osteoclast formation, taking advantage of a recently reported in vitro culture system that 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-stimulating 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 isoforms, Using human-specific primers, it was found that the hemopoietic cell component expressed both IL-1 alpha and IL-1 beta mRNA, Specific measurement of secreted human IL-1 beta protein showed greatly augmented levels in coculture compared with hemopoietic cells grown in the absence of ST-2 cells, consistent with the known signaling from stromal cells to hemopoietic cells during osteoclastogenesis. Specific detection of mouse mRNA products showed that the ST-2 stromal cells in the coculture also expressed mRNA corresponding to IL-1 alpha and IL-1 beta, The expression of both mouse and human 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 influence 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, compared with ST-2 cells alone, Secreted mouse IL-1 beta protein was upregulated in coculture in parallel with mRNA levels. However, the absolute levels of mouse IL-1 beta achieved were more than 20-fold lower than the human IL-1 beta levels. Prostaglandin estradiol (PGE(2)) levels were measured and found to be greatly increased in the coculture compared with ST-2 cells or hemopoietic cells alone, consistent with evidence that IL-1 action in osteoclastogenesis is mediated by PGE(2). These results provide novel evidence that bidirectional signaling between stromal and hemopoietic cells may be important in the generation of human osteoclasts. (C) 1999 by Elsevier Science Inc. All rights reserved.