Role of cytokines in the modulation of nitric oxide production by cyclic AMP

Enhanced levels of cyclic AMP (cAMP), resulting from stimulation of adenylyl cyclase through activation of distinct pharmacological receptor systems, have a remarkable impact on the activity of the immune system. Among other responses, production of nitric oxide (NO) is also affected. The effects of cAMP range from stimulation to inhibition (or no effect) of immune-stimulated biosynthesis of NO, with a preponderance of stimulatory interference. cAMP has been shown to be a potent, dual modulator of cytokine expression. It dose-dependently suppresses secretion of major NO up-regulatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). On the other hand, production of IL-10, which is known to regulate the inducible NO synthase (iNOS) activation in both a positive and negative direction, is inversely enhanced. It is suggested that the dual effects of cAMP on NO formation are likely to result from the differences in the concentration ratio of these cytokines. The value of this parameter depends on the type and concentration of cAMP-stable derivatives and cAMP-enhancing agents, such as prostaglandins, beta-adrenoceptor agonists, phosphodiesterase inhibitors, forskolin and cholera toxin. The cytokine ratio may be influenced by dynamically developing multiple down- and up-regulatory feedback circuits among cytokines, NO, and cAMP.     

cAMP inhibits cytokine-induced biosynthesis of tetrahydrobiopterin in human umbilical vein endothelial cells

We studied the effects of cAMP on cytokine (interferon-gamma plus tumor necrosis factor-alpha)-induced stimulation of tetrahydrobiopterin (BH4) synthesis in human umbilical vein endothelial cells (HUVEC). The cytokine mixture caused a marked increase in the biosynthesis and release of BH4 by HUVEC. Dibutyryl-cAMP produced a dose-dependent inhibition of this cytokine-induced stimulation of synthesis and release of BH4 by these cells. 8-Bromo-cAMP also caused a significant inhibition, although the effects were less marked than those of dibutyryl-cAMP. Both forskolin and the stable analog of prostacyclin, iloprost, caused cAMP accumulation and a concomitant diminution of the cytokine-induced BH4 synthesis in HUVEC. Dibutyryl-cAMP and iloprost also significantly inhibited the cytokine-induced stimulation of GTP cyclohydrolase I (GCHI) activity and mRNA production. We concluded that the suppression by the cAMP messenger system of cytokine-induced stimulation of synthesis and release of BH4 by HUVEC can be attributed to the inhibition of the activity of GCHI, the rate-limiting enzyme in BH4 biosynthetic pathway, in HUVEC. The data also suggest that the cAMP-mediated reduction in the GCHI mRNA level may at least partially explain the decline in GCHI activity. It is reasoned that under inflammatory conditions, cAMP-elevating agents such as prostacyclin exert regulatory effects on circulation by inhibiting cytokine-induced synthesis and release of BH4 by HUVEC.     

Lipopolysaccharide and cytokines induce a macrophage-type of nitric oxide synthase in bovine retinal pigmented epithelial cells.

The present study demonstrates that bovine retinal pigmented epithelial cells, which are neuroectodermal in origin, produce nitric oxide (NO) upon treatment with interferon-gamma in the presence of lipopolysaccharide or tumor necrosis factor-alpha. NO production was measured by the accumulation of the stable endproduct NO2-. The biosynthesis of NO requires an induction period of approximately 12 hours and continues for at least 96 hours. The synthesis was abolished by the stereoselective inhibitors of NO synthase, NG-monomethyl-L-arginine and NG-nitro-L-arginine-benzylester. Cycloheximide and dexamethasone blocked cytokine-induced NO production. The results indicate that endotoxin and cytokines are capable of inducing NO synthase of the macrophage type, in retinal pigmented epithelial cells.     

Cytotoxicity of IFN-gamma and TNF-alpha for vascular endothelial cell is mediated by nitric oxide

Endothelial cell injury is a critical event in tissue damage accompanying inflammation, in which both inflammatory cytokines and reactive oxygen species may play pivotal roles, although the exact mechanism has not yet been clarified. We found that combined stimulation with interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) induced both cytotoxicity to murine vascular endothelial cell line F-2 and an increase in nitric oxide (NO). Therefore, in the present study, the implication of NO in cytotoxicity was examined. A potent iNOS-specific inhibitor ONO-1714 completely blocked both cytokine-induced cytotoxicity and NO production. NO scavengers such as carboxy-PTIO and hemoglobin blocked cytotoxicity. Moreover, exogenous NO from NOC 18 also caused cytotoxicity. These results together demonstrated that cytotoxicity of IFN-gamma and TNF-alpha for endothelial cell F-2 was mediated by NO, suggesting a pathogenic role of cytokine-induced NO production in endothelial damage under inflammatory conditions.     

Differential effects of cyclic AMP on induction of nitric oxide synthase in 3T3-L1 cells and brown adipocytes

The aim of this study was to determine whether cyclic AMP (cAMP) pathways alter the nitric oxide (NO) production mediated by inducible NO synthase (iNOS) in adipocytes. The treatment of 3T3-L1 cells, a model of white adipocytes, with the combination of lipopolysaccharide (L), tumor necrosis factor-alpha (T), and interferon-gamma (I) synergistically induced iNOS, leading to the production of NO. Enhancers of intracellular cAMP (dibutyryl cAMP, forskolin, and IBMX) inhibited the NO production elicited by LTI, whereas H89, a specific inhibitor of PKA, stimulated the NO production in 3T3-L1 cells. In rat brown adipocyte cell line, the combined treatment with LT synergistically elicited the NO production, and the cAMP analogues further enhanced it. Forskolin inhibited the NO production in 3T3-L1 cells, but enhanced it in brown adipocytes, in a dose-dependent manner. The changes in NO production paralleled the change in iNOS mRNA and protein level in both cell types. The activation of NF-kappaB by LTI/LT was blocked in 3T3-L1 cells, but enhanced in brown adipocytes, by the co-treatment with cAMP analogues. The protein level of 1-kappaBalpha, a NF-kappaB stabilizer, changed reciprocally to that of NF-kappaB activity in each cell type. These results suggest that cAMP regulates iNOS expression in adipocytes through modulating NF-kappaB activity. The differential regulation of iNOS in 3T3-L1 cells from that in the brown adipocytes indicates that intracellular signal pathways activated by cAMP are different between the cell types.     

Down-regulation of myelin-associated glycoprotein on Schwann cells by interferon-gamma and tumor necrosis factor-alpha affects neurite outgrowth.

To investigate the influence of inflammatory cytokines on the potential of peripheral nerves to regenerate, we analyzed the effect of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on the ability of immortalized Schwann cells to mediate outgrowth of neurites from primary DRG neurons. We found that IFN-gamma and TNF-alpha synergistically inhibited the neurite outgrowth-promoting properties of the Schwann cells by specifically down-regulating myelin-associated glycoprotein (MAG) at the levels of mRNA and cell surface protein by approximately 60%. Antibodies to MAg inhibited the outgrowth of neurites on Schwann cells to the same extent as treatment with the two cytokines. Since MAG appears to be involved in both neurite outgrowth and myelination, our findings may provide evidence for a mechanism, by which inflammatory cytokines interfere with Schwann cell-neuron interactions.     

PGE2 enhances cytokine-elicited nitric oxide production in mouse cortical collecting duct cells.

It has been documented that arginine vasopressin (AVP) and prostaglandin E(2) (PGE(2)) regulate water reabsorption in renal tubular cells. The present study was attempted to delineate the downstream signaling of AVP and PGE(2) in a cortical collecting duct cell line (M-1 cell). Using RT-PCR, we detected mRNA for V2 and VACM-1 but not for V1a and AII/AVP receptors of AVP. Furthermore, neither AVP nor V2 receptor agonist and antagonist alter cellular cAMP. These together with unchanged cellular Ca(2+) by AVP suggested that AVP pathway was not operating in M-1 cells. All four classical PGE(2) receptors with EP3 and EP4 as the most prominent were detected in M-1 cells. PGE(2), 11-deoxy-PGE(1) (EP2 and EP4 agonist), and 17-phenyl-trinor-PGE(2) (EP1 agonist) increased cellular concentration of cAMP. There was no effect of PGE(2) or EP1 agonist on cellular Ca(2+). These findings provide evidence of the involvement of PGE(2) cascade in M-1 cells. M-1 cells were capable of synthesizing nitric oxide (NO). Although individual cytokines did not affect NO production, a mixture of tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma elevated NO concentration to 4.5-fold of the control. Addition of PGE(2) and db-cAMP to the cytokine mixture further increased NO production to 7.0- and 9.8-fold, respectively, of that seen in non-treated cells. PGE(2) or db-cAMP alone, however, had no effect on NO production. The results of the study led us to speculate that enhanced production of cAMP via PGE(2) signaling pathway in M-1 cells could either stimulate or attenuate water reabsorption in renal tubule. While an increase in cAMP alone may enhance water reabsorption, a concomitant increase in cAMP and cytokines may inhibit water reabsorption in renal tubule.     

Calcitonin gene-related peptide inhibits interleukin 2 production by murine T lymphocytes.

Evidence from the literature suggests that the nervous and the immune systems closely interact via neuromediators, which affect the immune system, and cytokines, which control nerve cell growth and activity. Calcitonin gene-related peptide (CGRP) is a neuropeptide that has been identified in numerous tissues including immune organs and inhibits the proliferation of spleen cells. We investigated whether CGRP altered the function of T lymphocytes. We present evidence that CGRP induces a dose-dependent cAMP accumulation in interleukin 2-producing TH1 cells and inhibits their production of interleukin 2. These effects are prevented by CGRP8-37, a CGRP antagonist that is missing the first 7 amino acids. This CGRP-mediated inhibition of interleukin 2 production is accompanied by a decrease in interleukin 2 mRNA accumulation. CGRP also inhibits the accumulation of mRNA coding for tumor necrosis factor-alpha and -beta and interferon-gamma. Thus, we have identified one mechanism by which CGRP inhibits the proliferation of spleen cells.     

The adenylate cyclase activator forskolin partially protects L929 cells against tumour necrosis factor-alpha-mediated cytotoxicity via a cAMP-independent mechanism

Recent reports indicate that cAMP-elevating agents can protect against cell death induced by many stimuli, including tumour necrosis factor-alpha (TNF-alpha). We investigated the ability of cAMP-elevating agents to modulate TNF-alpha-mediated cytotoxicity in L929 cells. Using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) reduction assay and a DNA fragmentation assay as indicators of cell survival, we have shown that forskolin confers partial protection against TNF-alpha-mediated cytotoxicity and inhibits TNF-alpha-induced internucleosomal DNA fragmentation in L929 cells. The protection conferred by forskolin is cAMP-independent since 1,9-dideoxyforskolin (an adenylate cyclase-inactive analog) also protected against TNF-alpha, while both dibutyryl-cAMP and the cAMP-phosphodiesterase inhibitor theophylline were not protective. This is the first example (that we know of) of cAMP-independent cytoprotection by forskolin. We conclude that forskolin acts in a cAMP-independent manner, potentially at a site upstream of caspase-3 activation, to protect against TNF-alpha-mediated cytotoxicity in L929 cells, and that cAMP elevation, in general, does not confer protection against TNF-alpha-induced death in L929 cells. In addition, we observed that Cyclosporin A, a mitochondrial permeability transition (MPT) inhibitor, protected L929 cells against TNF-alpha, underlining the importance of mitochondria in the cytotoxic process induced by TNF-alpha in L929 cells.     

Platelet-derived growth factors and fibroblast growth factors are mitogens for rat Schwann cells

Rat sciatic nerve Schwann cells in culture respond to a limited range of mitogens, including glial growth factor, transforming growth factors beta-1 and beta-2 (TGF-beta 1, TGF-beta 2), some cell membrane-associated factors, and to agents such as cholera toxin and forskolin which raise intracellular levels of cAMP. These responses require the presence of FCS, which exhibits little or no mitogenic activity in the absence of other factors. However, we recently found that forskolin greatly potentiates the mitogenic signal from TGFs-beta 1 and beta 2, raising the possibility that cAMP might couple other factors to mitogenesis. We have therefore screened a range of candidate mitogens using DNA synthesis assays. Other than TGFs-beta and glial growth factor, none of the factors tested were mitogenic in the presence of 10% serum alone. With the addition of forskolin, however, porcine PDGF, human PDGF, acidic and basic FGF were potent mitogens for rat Schwann cells, stimulating DNA synthesis and increasing cell number. Cholera toxin and dibutyrylcyclicAMP, but not 1,9-dideoxyforskolin, can substitute for forskolin indicating that the mitogenic effect is mediated via adenylyl cyclase activation. Porcine PDGF gave half-maximal stimulation at 15 pM, and human PGDF an equivalent response at 1 nM. Basic FGF was half maximal at 5 pM, acidic FGF at 1 nM. The recognition of PDGFs and FGFs as mitogens for Schwann cells has many implications for the study of Schwann cell proliferation in the development and regeneration of nerves, and in Schwann cell tumorigenesis.     

Inhibition of the proliferation of cultured immortalized schwann cells by forskolin with a decreased basal level of diacylglycerol

The repetitive passages of a Schwann cell culture results in the appearance of immortalized cells. In order to investigate the direct effects of cyclic AMP (cAMP) on Schwann cell proliferation, we used the immortalized Schwann cells because the responses of a short-term Schwann cell culture to agents increasing the intracellular cAMP are more complicated and it does not seem that all of them are due to the direct effects of cAMP. By adding up to 200 M of forskolin, an adenylate cyclase activator, to the culture medium, Schwann cell proliferation was inhibited and the intracellular 1,2-diacylglycerol (DG) level was decreased in a dose-dependent manner to 44 and 53% of the control values, respectively. The protein phosphorylation activity in the cytosol from the cell treated with 100 M forskolin, assayed with myelin basic protein as the acceptor, decreased to 78% and this inhibition was then reversed by the addition of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeable DG, to the assay mixture. The cell proliferation inhibited by forskolin was also restored by the addition of OAG. These data suggest that cAMP inhibits both the activity of protein kinase C (PKC) and consequently cell proliferation through suppression of intracellular DG level, an activator of PKC. Since the inositol 1,4,5-triphosphate level and the hydrolysis of phosphatidylcholine to DG and phosphorylcholine were not affected, forskolin therefore appears to suppress the de novo synthesis of DG.     

Cytokines in the central nervous system of mice during chronic relapsing experimental allergic encephalomyelitis

Clinical disease phases of chronic relapsing experimental allergic encephalomyelitis (CREAE) in the Biozzi AB/H mouse model are associated with extensive cellular infiltration of the central nervous system, principally the spinal cord. The activation of these cells is further suggested by the immunocytochemical demonstration of cytokines (migration inhibition factor, interferon-gamma, tumour necrosis factor-alpha, and interleukins 1, 2, and 3) within these infiltrates. The in vitro functions attributed to these cytokines indicate their potential role in cell recruitment, activation, and differentiation of the ongoing immune response which could contribute to the pathogenesis of disease.     

Cytokine-activated endothelial cells express an isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent, calmodulin-independent and inhibited by arginine analogs with a rank-order of potency characteristic of activated macrophages.

We have previously reported that cultured murine brain endothelial cells (MBE) produce large quantities of nitric oxide (NO) after activation with interferon-gamma in combination with any of several immunoactivators including: bacterial endotoxin, tumor necrosis factor and interleukin-1. Since endothelial cells are the first example of a cell-type which may possess both a constitutive and an inducible type of NO synthase, it was of interest to compare the requirements of these two enzyme activities. Induction of NO synthesis in MBE by cytokines was abolished by the protein synthesis inhibitor, cycloheximide, and by 2,4-diamino-6-hydroxypyridine (DAHP), a selective inhibitor of GTP cyclohydrolase I, the rate-limiting enzyme for de novo synthesis of tetrahydrobiopterin (THB). In the presence of DAHP, NO synthesis was restored by sepiapterin (SEP), a substrate for the alternative pathway of THB synthesis occurring via pterin salvage. Moreover, SEP increased NO synthesis to greater than 150% of control values, suggesting that THB availability is rate-limiting for NO synthesis by cytokine-induced MBE. Methotrexate, an inhibitor of the pterin salvage pathway of THB synthesis, completely reversed the stimulation of NO synthesis by sepiapterin. Thus, cytokine-induced MBE NO synthase appears to have an absolute requirement for THB as cofactor. In additional studies we found that NO synthesis by cytokine-activated MBE was inhibited by NG-monosubstituted arginine analogs with a rank-order of potency NH2 greater than CH3 greater than NO2, in contrast with the rank-order of NO2 greater than NH2 greater than CH3 previously described for inhibition of the constitutive endothelial cell enzyme. Using a kinetic assay for NO synthase activity, based on oxidation of myoglobin heme-iron, we have found that these rank orders of potency also apply to cytosol preparations of cytokine-induced and untreated endothelial cells, respectively. Further differences between constitutive and cytokine-induced NO synthase were observed with regard to calmodulin requirements. Whereas constitutive NO synthase was potently inhibited by the calmodulin antagonists mellitin and trifluoperazine, cytokine-induced NO synthase was unaffected. In summary, NO synthesis by cytokine-activated MBE is THB-dependent, calmodulin-independent and inhibited by NG-substituted arginine analogs with a rank-order profile distinct from that for untreated endothelial cells but identical to that for cytokine-activated macrophages.     

Nitric oxide synthesis and TNF-alpha secretion in RAW 264.7 macrophages: mode of action of a fermented papaya preparation

Macrophage inducible nitric oxide synthase is able to generate massive amounts of nitric oxide (NO) which contributes to the host immune defense against viruses and bacteria. Monocyte-macrophages stimulated with the bacterial wall component lipopolysaccharide (LPS) and cytokines such as interferon-gamma (IFN-gamma) express the inducible form of nitric oxide synthase (iNOS). Furthermore, tumor necrosis factor-alpha (TNF-alpha) is one of the central regulatory cytokines in macrophage antimicrobial activity and synergizes with IFN-gamma in the induction of NO synthesis. Because of its pivotal role in both antimicrobial and tumoricidal activities of macrophages, a significant effort has focused on developing therapeutic agents that regulate NO production. In the present study fermented papaya preparation (FPP) is shown to exert both immunomodulatory and antioxidant activity in the macrophage cell line RAW 264.7. Interestingly, a low and a high molecular weight fraction (LMF and HMF, respectively) of FPP exhibited different activity patterns. FPP fractions alone did not affect NO production. However in the presence of IFN-gamma, both LMF and HMF significantly increased iNOS activity and nitrite as well as nitrate accumulation. NO radical formation measured in real-time by electron paramagnetic resonance spectroscopy was higher in the presence of LMF and IFN-gamma. On the contrary, iNOS mRNA levels were enhanced further with HMF than with LMF. Moreover, LMF displayed a stronger superoxide anion scavenging activity than HMF. In the presence of IFN-gamma, both FPP fractions stimulated TNF-alpha secretion. However in non-stimulated macrophages, TNF-alpha secretion was enhanced by HMF only. Since water-soluble FPP fractions contained no lipid A, present data indicate that FPP is a macrophage activator which augments nitric oxide synthesis and TNF-alpha secretion independently of lipopolysaccharides.     

Calcitonin gene-related peptide promotes Schwann cell proliferation

Schwann cells in culture divide in response to defined mitogens such as PDGF and glial growth factor (GGF), but proliferation is greatly enhanced if agents such as forskolin, which increases Schwann cell intracellular cAMP, are added at the same time as PDGF or GGF (Davis, J. B., and P. Stroobant. 1990. J. Cell Biol. 110:1353-1360). The effect of forskolin is probably due to an increase in numbers of PDGF receptors (Weinmaster, G., and G. Lemke. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:915-920. Neuropeptides and beta-adrenergic agonists have been reported to have no effect on potentiating the mitogenic response of either PDGF or GGF. We show that the neuropeptide calcitonin gene- related peptide (CGRP) increases Schwann cell cAMP levels, but the cells rapidly desensitize. We therefore stimulated the cells in pulsatile fashion to partly overcome the effects of desensitization and show that CGRP can synergize with PDGF to stimulate Schwann cell proliferation, and that CGRP is as effective as forskolin in the pulsatile regime. CGRP is a good substrate for the neutral endopeptidase 24.11. Schwann cells in vivo have this protease on their surface, so the action of CGRP could be terminated by this enzyme and desensitization prevented. We therefore suggest that CGRP may play an important role in stimulating Schwann cell proliferation by regulating the response of mitogenic factors such as PDGF.     

A selective adenosine A2A receptor agonist, ATL-146e, prevents concanavalin A-induced acute liver injury in mice

BACKGROUND AND AIMS: Concanavalin A (Con A) activates T lymphocytes and induces CD4+ T cell-mediated hepatic injury in mice. Pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and interleukin-6 (IL-6), are critical mediators in this experimental model. Activation of adenosine A2A receptors reduces the production of various pro-inflammatory cytokines and suppresses T cell activation. A selective adenosine A2A receptor agonist (ATL-146e) has been shown to be a potent inhibitor of inflammation by increasing intracellular cyclic AMP (cAMP) in leukocytes. The aim of the present study was to determine whether ATL-146e could ameliorate Con A-induced hepatic injury, reduction of pro-inflammatory cytokine production. METHODS: Balb/c mice were injected with 25mg/kg Con A with or without a single injection of ATL-146e (0.5-50 microg/kg), 5 min prior to Con A administration. Liver enzymes, histology, and serum levels of tumor necrosis factor-alpha, interferon-gamma, and interleukin-6 were examined. We also assessed the effects of ATL-146e on pro-inflammatory cytokine production with CD4+ T cell. RESULTS: Pretreatment with ATL-146e significantly reduced serum levels of liver enzymes (P<0.001). The serum pro-inflammatory cytokines were all increased after Con A administration and reduced to near normal levels by ATL-146e. ATL-146e also inhibited CD4+ T cell pro-inflammatory cytokine production. CONCLUSION: A selective adenosine A2A receptor agonist, ATL-146e, can prevent concanavalin A-induced hepatic injury that is presumably mediated by its anti-inflammatory properties.