Regulation of IL-15-stimulated TNF-alpha production by rolipram.

Agents that increase intracellular cAMP have been shown to reduce joint inflammation in experimental arthritis, presumably by lowering the release of proinflammatory cytokines, such as TNF-alpha. Recent studies suggest that, in joints of patients with rheumatoid arthritis, TNF-alpha release from macrophages is triggered by their interaction with IL-15-stimulated T lymphocytes. In this report, we analyze the effect of rolipram, a cAMP-specific phosphodiesterase inhibitor, on TNF-alpha production in this experimental system. Cocultures of U937 cells with IL-15-stimulated T cells, but not control T cells, resulted in increased release of TNF-alpha. Pretreatment of T cells with rolipram or cAMP analogues inhibited the IL-15-stimulated increases in proliferation, expression of cell surface molecules CD69, ICAM-1, and LFA-1, and release of TNF-alpha from macrophages. Addition of PMA to T cells dramatically increased the expression of cell surface molecules, but had little or no effect on TNF-alpha release from either T cells or from cocultures, suggesting that other surface molecules must also be involved in T cell/macrophage contact-mediated production of TNF-alpha. Addition of PMA synergistically increased the proliferation of IL-15-stimulated T cells and the secretion of TNF-alpha from IL-15-stimulated T cell/macrophage cocultures. Rolipram and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) blocked these increases. Measurement of protein kinase A (PKA) activity and the use of inhibitory cAMP analogues (RpCPT-cAMP) confirmed that rolipram worked by stimulating PKA. These data suggest that PKA-activating agents, such as rolipram, can block secretion of TNF-alpha from macrophages by inhibiting T cell activation and expression of surface molecules.

Positive regulatory role of cAMP on alkaline phosphatase activity and proliferation of mitogen stimulated B lymphocytes.

Effect of dibutyryl cyclic adenosine monophosphate (dbt cAMP) on alkaline phosphatase (APase) of mitogen stimulated murine B lymphocytes was studied. Addition of dbtcAMP to lipopolysaccharide (LPS) stimulated B cells enhanced APase activity in a dose dependent and synergistic manner. dbtcAMP also stimulated the proliferative response of LPS treated B lymphocytes. On the other hand, when B lymphocytes stimulated with anti-immunoglobulin (anti-Ig) were treated with dbtcAMP neither DNA synthesis nor APase activity was enhanced. These results suggest that cAMP is a potent synergistic activator of APase in B lymphocytes committed to proliferation.

Alkaline phosphatase activity is expressed only in B lymphocytes committed to proliferation.

Alkaline phosphatase (APase) activity was measured in murine splenic lymphocytes stimulated with the T lymphocyte mitogens phytohemagglutinin (PHA) and Concanavalin A (Con A) and the B lymphocyte mitogens lipopolysaccharide (LPS) and anti-immunoglobulin (anti-Ig). APase activity was found to be enhanced specifically in mitogen-stimulated B lymphocytes, but not in T lymphocytes. This enhancement starts around 8 h after stimulation with a mitogen. With soluble anti-Ig it was observed that the B cells enter G1 phase as assessed by RNA synthesis and blast transformation. However, these cells fail to synthesize DNA and also do not show any increase in APase activity. When the same anti-Ig coupled to Sepharose was used as a stimulator, cells synthesized DNA and also showed significant increase in APase activity. When hydroxyurea was added, the enhancement in APase activity by the mitogen was not diminished although the cells failed to synthesize DNA. These observations indicate that APase activity is enhanced only in activated B cells committed to proliferation.