TNF-alpha induced CD38 expression in human airway smooth muscle cells: role of MAP kinases and transcription factors NF-kappaB and AP-1.

In human airway smooth muscle (HASM) cells, the expression of CD38, which synthesizes the calcium-mobilizing molecule cyclic ADP-ribose, is augmented by TNF-alpha, a cytokine implicated in asthma. We determined the role of mitogen-activated protein kinase (MAPK) in the activation of NF-kappaB and AP-1 in the regulation of CD38 expression in HASM cells. In HASM cells exposed to TNF-alpha (40 ng/ml), the inhibitors of extracellular signal-regulated kinase (ERK), p38, or c-Jun NH(2)-terminal kinase (JNK) decreased CD38 expression and ADP-ribosyl cyclase activity. Transfection of HASM cells with a dominant negative MEK decreased while a wild-type ERK increased TNF-alpha-induced CD38 expression. Electrophoretic mobility shift assays (EMSAs) were performed using nuclear proteins and consensus sequences to detect the effect of the MAPKs on NF-kappaB and AP-1 activation. EMSAs confirmed the role of p38 and JNK in mediating NF-kappaB and AP-1 activation. Transfection of a dominant negative c-Jun decreased TNF-alpha-induced CD38 expression indicating involvement of AP-1. Stability of TNF-alpha-induced CD38 transcripts were determined in the presence of MAPK inhibitors after arresting the transcription with actinomycin D. Transcript stability decreased in the presence of ERK and p38 MAPK, but not the JNK, inhibitors. These results indicate that regulation of CD38 expression through p38 and JNK MAPKs involves NF-kappaB and AP-1 activation, and ERK and p38 MAPKs also regulate expression posttranscriptionally through message stability.

Regulation of apoptosis by somatostatin and substance P in peritoneal macrophages.

Recent studies have shown that somatostatin (SOM) inhibits interleukin 6 (IL-6) and interferon gamma (IFNgamma) production by lymphocytes and peritoneal macrophages, whereas substance P (SP) enhances these cytokines production. To define the mechanism of the cytokine production enhancements and inhibitions by SOM and SP, we examined the expression of apoptosis modulator, p53, Bcl-2, Bax, inducible nitric oxide synthase (iNOS), Fas, caspase-8 and nitric oxide (NO) in thioglycolate-elicited peritoneal macrophages. SOM caused up-regulation of p53, Bcl-2, Fas and caspase-8 activities, and down-regulation of iNOS expression and NO production. On the other hand, SP slightly induces p53 and highly induces Bcl-2, iNOS expression and NO production. These data suggest that apoptosis by SOM may occur by a Bax- and NO-independent p53 accumulation, and through Fas and caspase-8 activation pathways, and that the inducible expression of Bcl-2 and NO production by SP may contribute to prevent the signals of apoptosis by Bax, and via Fas and caspase-8 activation.

Modulation of transferrin synthesis, transferrin receptor expression, iNOS expression and NO production in mouse macrophages by cytokines, either alone or in combination.

Iron, an essential element for all living organisms, is central importance in a number of crucial metabolic pathways, including the regulation of immune function. Iron delivery to cells is accomplished by the complexing of iron to transferrin (Tf), a monomeric iron-binding protein in the plasma, followed by specific binding of Tf to cell-surface receptors, endocytosis of the receptor-ligand complexes and ultimately, release of iron from endosomal vesicles to the cytoplasm. The purpose of this study was to evaluate the effect of cytokines, alone and in combination, on the factors that can affect the iron delivery in thioglycollate-elicited macrophages. In this study, IFN gamma induced a marked increase in Tf synthesis by macrophages, while IL-1, IL-6 and TNF alpha produced a more modest increase. Combinations of these cytokines were shown to be less effective in promoting macrophage Tf synthesis than the cytokines by themselves. IFN gamma alone and in combination with other cytokines was effective in inducing nitrite (NO) production and inducible nitric oxide synthetase (iNOS) expression in macrophages, while IL-1, TNF alpha and IL-6 individually, as well as in various combinations, were not. While all tested cytokines individually and in combination inhibited the expression of the transferrin receptor (TfR) on macrophages, IFN gamma alone and in combination with other cytokines most strongly repressed the TfR expression. TfR localization in macrophages after IFN gamma stimulation showed that TfR fluorescence was most intense in the perinuclear region after 6 hours and scattered diffusely throughout the cytoplasm after 24 hours. This data suggests that IFN gamma may enhance iron uptake during the early phase of macrophage activation, and in later phases, down-regulate TfR expression by inducing NO, thus contributing to intracellular oxidative stress reduction.

Somatostatin and substance P induced in vivo by lipopolysaccharide and in peritoneal macrophages stimulated with lipopolysaccharide or interferon-gamma have differential effects on murine cytokine production.

We have investigated whether lipopolysaccharide (LPS) induces substance P (SP) and somatostatin (SOM) in popliteal lymph nodes in vivo and whether macrophages are a source of SP and SOM in vitro. We have also investigated the effect of SP and SOM treatment on the production of cytokines. SP reached a maximum 3 days after injection of LPS (100 microg/footpad) and then declined. SOM expression after LPS injection reached a maximum at 5-7 days. Stimulation of thioglycolate-elicited peritoneal macrophages with LPS (20 microg/ml), recombinant interferon-gamma (rIFN-gamma, 100 U/ml), and LPS plus rIFN-gamma induced SOM and SP. Thioglycolate-elicited, unstimulated peritoneal macrophages also synthesized these peptides. SOM (10(-12)-10(-8) M) significantly inhibited IL-6 and IFN-gamma production, whereas SP at those concentrations enhanced cytokine production by activated lymphocytes and macrophages. These findings suggest that neuropeptides which originate from macrophages and nerve fibers act as immunomodulators to mediate changes in the pattern of cytokine production.