Poster presentation. Abstract: Although it is known that macrophage content in visceral adipose tissue is significantly increased in obese humans and mice, the crosstalk between macrophages and adipocytes and its impact on the expression of genes involved in inflammation that contributes to insulin resistance have not been well elucidated. In this study, we incubated human monocytes, macrophages, or U937 mononuclear cells with adipocytes in a transwell system and studied how cell interaction regulated the expression of osteopontin, a matrix glycoprotein and proinflammatory cytokine that mediates obesity-induced adipose tissue macrophage infiltration and insulin resistance. Results showed that coculture of human monocytes, macrophages or U937 cells with adipocytes led to a 3- to 5-fold increase in osteopontin production when compared to that released by independent cultures of monocytes, macrophages or U937 cells and adipocytes. Moreover, LPS or palmitic acid, which activates Toll-like receptor (TLR)4, and high glucose augmented the coculture-stimulated osteopontin secretion. Real-time PCR studies showed that coculture of U937 cells and adipocytes led to increased osteopontin mRNA in U937 cells, but not adipocytes, suggesting that adipocyte-derived soluble factors may stimulate osteopontin expression in U937 cells. This notion was supported by the observation that osteopontin expression by U937 cells was increased by 2-fold by treatment with adipocyte-conditioned medium. In our studies to explore the mechanism involved in the coculture-stimulated osteopontin expression, we found that the neutralizing antibodies against IL-6 and IL-1b, but not those against IL-1a and TNFa, effectively blocked coculture-augmented osteopontin expression, indicating that IL-6 and IL-1b may stimulate osteopontin expression in the coculture. In support of this finding, experiments showed that IL-6 and IL-1b stimulated osteopontin expression. In conclusion, this study demonstrated that the crosstalk between adipocytes and U937 cells stimulated osteopontin expression via IL-6 and IL-1b and that TLR4 activation and high glucose further enhanced the adipocyte/U937 cell interaction-stimulated osteopontin expression.