We have previously shown that cultured RBL-2H3 cells, a mast cell line, generate physiologically significant concentrations of the cytokines, TNFalpha and IL-6 in response to antigen and other stimulants. Both production and release of these cytokines are dependent on cell stimulation and both appear to be separately regulated processes. Production of TNFalpha, for example, is dependent on mobilization of Ca2+, activation of protein kinase C (PKC) and a third synergising signal that is particularly strong in antigen-stimulated cells. Secretion of the newly formed TNFalpha via Golgi is dependent on mobilization of Ca2+ and activation of PKC and is readily blocked by inhibitors of PKC. We have now tentatively identified the Mitogen Activated Protein (MAP) kinase pathway as the third synergising signal for production of TNFalpha on the following basis. Of all stimulants tested, antigen is by far the most potent stimulant of this pathway (see report ZO1 HL 00937-14 LMI). The MAP kinase pathway (see last years report ZO1 HL 00937-13) and production of TNFalpha are both inhibited by nanomolar concentrations of the glucocorticoid, dexamethasone, or concentrations that have minimal effects on other stimulatory events in RBL-2H3 cells. Expression of dominant negative Raf, a MAP-kinase-regulatory protein in RBL-2H3 cells, results in markedly reduced activation of MAP kinase and production of TNFalpha. Expression of this protein, however, does not impede secretion of the small amounts of TNFalpha that are formed, consistent with previous indications that a third synergising signal is not required for release of TNF alpha. In contrast to the TNFalpha, the anti-inflammatory cytokine, TGFbeta, is constitutively produced and secreted via Golgi in RBL-2H3 cells and these processes were not inhibited by dexamethasone of inhibitors of PKC. A small increase in production and secretion of TGFbeta could be induced by antigen and this increase could be blocked by PKC inhibitors and dexamethasone. Therefore, although both types of cytokines were processed by Golgi, only TNFalpha and the inducible component of TGFbeta production were protein kinase C or steroid-regulated processes. These findings suggested that constitutive and inducible pathways exist for production and release of cytokines and that the inducible pathways can be selectively suppressed by pharmacologic agents.
Showing the most recent 10 out of 13 publications
