Interleukin 4, a cytokine produced by activated T cells and mast cells, affects a wide variety of cells of hematopoietic lineage including B and T lymphocytes, monocytes, and mast cells as well as pluripotent hematopoietic progenitors. In bone, IL-4 has been shown to inhibit calcium release by organ cultures stimulated by different osteolytic factors including peptides, steroid hormones, and prostaglandins. Recently, these results have been extended to the intact animal where IL- 4 was shown to antagonize osteolysis caused by tumors and infused bone resorption-inducing stimuli. Because IL-4 has been shown to inhibit osteoclast formation in vitro, this project is focussed on defining the potential role of IL-4 as a modulator of skeletal metabolism through its effects on osteoclast progenitors and osteoblasts. Using an in vitro model of osteoclastogenesis requiring bone marrow cell/stromal cell cocultures, the cell population, and any potentially induced inhibitory factors affected by IL-4 treatment will be defined. As preliminary evidence indicates that bone marrow macrophages are proficient osteoclast progenitors, the influence of IL-4 on these cells will be the initial focus. Particular attention will be directed at the macrophage population, the possibility that IL-4 impacts a non-adherent bone marrow cell, which will be fractionated into B, T and non-B, non-T cells, will be examined. In addition to identifying the responsible cell, the mechanism of action will be explored including the existence of IL-4- induced soluble mediators and the requirement, if any, for cell contact. Because E series prostaglandins antagonize IL-4's inhibitory effects on osteoclast formation and enhanced Ia expression, the mechanism of this effect will also be characterized. Because osteoblasts are intermediaries targeted by stimuli that cause increased resorptive activity, the second major focus is defining IL-4's modulation of osteoblast (using a non-transformed osteoblastic cell line) secretion of factors known to modulate osteoclast formation, in particular, interleukin 6 (IL-6) and transforming growth factor beta 1 (TGF beta1). In the case of IL-6, an excess of which has been implicated in osteoporosis and Paget's disease of bone, IL-4's antagonism of IL-1 induced-IL-6 production will be the focus of our studies. In addition to defining the role of IL-6 mRNA regulation in this effect, these experiments will focus on the possibility that IL-4 impacts IL-1-induced IL-6 production at the level of the IL-1 receptor. Preliminary evidence suggests that IL-4, in contrast to its effects on IL-6, enhances TGF beta1 expression at the message level. This phenomena could contribute to the inhibitory effects that IL-4 exerts on osteoclast formation, if increased amounts of TGF beta1 were also secreted. Therefore, the impact of IL-4 on TGF beta1 expression both at the level of mRNA regulation and protein synthesis will be characterized. Understanding how IL-4 inhibits osteoclast formation may result in unique therapeutic approaches for common adult skeletal diseases characterized by excess osteoclastic activity (osteoporosis and hypercalcemia of malignancy).
