Ethanol inhibition of the chemiluminescent response of stimulated macrophages.

The effect of ethanol on the luminol-dependent chemiluminescence and cAMP level in mice peritoneal macrophages was investigated. Ethanol was shown to inhibit the chemiluminescence of macrophages by acting both as a "trap" for active radicals and as a suppressor of the cellular functional activity. A short preincubation of macrophages with ethanol results in a dose-dependent decrease of the chemiluminescent response to the stimulatory agent (opsonized zymosan). Ethanol was also shown to induce a peakwise rise of the intracellular cAMP after a 2-min incubation. The observed effects are correlated both in time and concentration, which allows the presumption that inhibition of the functional activity of macrophages is mediated by the increase of the intracellular cAMP level.     

The effect of serotonin on the functional activity of monocytes]

The treatment of human peripheral blood monocytes with serotonin at concentrations 10(-3) and 10(-5) M over 20 minutes decreases a zymosan-induced luminol-dependent chemiluminescence of cells, whereas a 5 minutes treatment with serotonin at the concentration of 10(-5) M increases the chemoluminescence. The correlated change in monocyte capacity of secreting hydrogen peroxide has been registered. Serotonin activates, to a little extent, the monocyte capacity of phagocytizing the opsonised sheep erythrocyte. The maximum increase (2-3 times) of intracellular cAMP content and the decrease in cytosol cAMP-binding capacity are registered after a 5 minutes incubation. The lowering of the share of irreversibly bound in vitro cAMP under the influence of serotonin may suggest a preferable binding of cyclic nucleotide in vivo by regulatory subunits of cAMP-dependent protein kinase I.     

Tumor necrosis factor alpha stimulates mycobactericidal/mycobacteriostatic activity in human macrophages by a protein kinase C-independent pathway.

Tumor necrosis factor (TNF) is a 17-kDa protein produced by endotoxin-stimulated macrophages. We have demonstrated that recombinant human TNF activates human macrophages to kill intracellular bacteria of the Mycobacterium avium complex (MAC) in a dose-related manner. TNF also primed macrophages to produce superoxide anion (O2-) following treatment with phorbol esther PMA (0.1 micrograms/ml). To investigate the intracellular pathway involved in the TNF-mediated activation of mycobacteriostatic/mycobactericidal activity in macrophages, we used two different protein kinase C (PKC) inhibitors: H7 (10(-5)-10(7) M) and staurosporine (10(-7)-10(-9) M). Mellitin (1 and 100 mM) was used as a calmodulin inhibitor. Human peripheral blood-derived macrophages cultured for 7 days were treated with H7, mellitin, or staurosporine for 1 hr prior to incubation with TNF (10(3) U/ml). Twenty-four hours after treatment with TNF the O2- release was measured spectrophotometrically following exposure to PMA. Macrophages were infected with MAC and the viable intracellular bacilli were quantitated following 4 days of treatment with TNF. All PKC inhibitors suppressed O2- production after incubation with PMA. However, treatment with either PKC or calmodulin inhibitors did not influence the intracellular killing of M. avium by TNF-stimulated macrophages. Exposure of the macrophages to cGMP inhibitor but not to cAMP inhibitor significantly impaired the response to the stimulation with TNF. In contrast, incubation of macrophages with protein kinase A (PKA) had no effect on TNF-mediated mycobacteriostatic/mycobactericidal activity. These results suggest that the TNF-mediated mycobactericidal activity in cultured macrophages probably occurs by a PKC-independent mechanism.     

Effect of cAMP modifiers on the cytotoxic activity of macrophages

The effects of various cAMP modifiers on the changes in the intracellular cAMP level and on the coupling of the cAMP system with realization of macrophage cytotoxicity depending on their functional activity were studied. Nonactivated and activated by E. coli polysaccharides peritoneal macrophages of BALB/c mice and macrophage-like cells of J744 mice were incubated in the presence of cAMP modifiers and further assayed for cytolytic and cytostatic activities. Cells of tetraploid strain of Ehrlich carcinoma G10 were used as target cells. Among other modifiers only dibutyryl-cAMP caused a steady increase of the intracellular nucleotide content, whereas methylisobutylxanthine and isoproterenol in combination with methylisobutylxanthine caused only a temporary increase of the cAMP level. Isoproterenol did not induce any appreciable changes in the intracellular cAMP level. All modifiers under study suppressed the cytotoxic activity of macrophages irrespective of the nature of changes in the intracellular cAMP content. It was assumed that cAMP accomplishes a triggering function in the regulation of the cytotoxic activity of macrophages and that the cAMP system is universal in the regulation of cytotoxicity at various functional states of macrophages.     

Uptake of cyclic adenosine-3,5-monophosphate by cultured mammalian cells and frog muscles

Uptake of 3H-cAMP by isolated frog sartorii muscles and cultured mouse 3T3 and 3T6 cells was studied. It was shown that after 1-2 hours of incubation the intracellular level of cAMP in frog muscles reached 10-20% of its concentration in the incubation medium. About 50% of intracellular radioactivity was represented by chromatographically pure cAMP which testifies to the insignificant cAMP metabolism rate. In the experiments with 3T3 and 3T6 cells the influence of possible admixtures and degradation products on 3H-cAMP uptake was revealed. To avoid these influences it is necessary to measure the uptake of cAMP in the presence of theophylline and with abundance of adenosine. In such experimental conditions the intracellular level of cAMP after 1 hour of incubation did not exceed 10% of its extracellular level, which is similar to values obtained on frog muscles. Permeability coefficient of cAMP for membrane of frog muscles and 3T3 and 3T6 cells was about 10(-9)-10(-8) cm X sec-1.     

The effect of exogenous peroxidase on the intensity of luminol-dependent macrophage chemiluminescence

The influence of exogenous horse-radish peroxidase on the capacity of mouse peritoneal macrophages to luminol-dependent chemiluminescence induced by zymosan was investigated. It was revealed that peroxidase (1-50 mg/ml) increased the chemiluminescence in a dose-dependent manner. The maximum increase of the response (4-6 times) was obtained with the enzyme concentration being 10 mg/ml. It is found that peroxidase acts as a co-oxidant of the peroxide-dependent extracellular luminol oxidation. The including of the enzyme into macrophages makes it possible to register the intracellular chemiluminescence.     

Effect of cAMP-elevating drugs on Ca2+ efflux and actin polymerization in peritoneal macrophages stimulated with N-formyl chemotactic peptide

To investigate intracellular cAMP inhibitory mechanisms related to migration of guinea-pig peritoneal macrophages, we examined the effects of cAMP-elevating drugs on the Ca²⁺ efflux and actin polymerization in macrophages stimulated with fMet-Leu-Phe, a chemotactic peptide. The stimulation with 1·10^?8 M fMet-Leu-Phe enhanced the Ca²⁺ efflux, and induced actin polymerization. Dibutyryl cAMP, theophylline and papaverine, which continuously increased the levels of intracellular cAMP, inhibited the enhancement of Ca²⁺ efflux and induction of actin polymerization by fMet-Leu-Phe. On the other hand, isoproterenol, which transiently increased the cAMP level, inhibited only the early phase of Ca²⁺ efflux and not the actin polymerization. As additions of both cAMP and cAMP-dependent protein kinase did not modify the Ca²⁺ uptake of phagocytic vesicles, the inhibition of Ca²⁺ efflux by these drugs may be due to the inhibition of the Ca²⁺ release from the intracellular store site(s). The cAMP-elevating drugs increased the monomeric actin content without change in the total actin content, indicating an induction of the depolymerization of filamentous actin. From these findings, we conclude that the inhibition of macrophage migration induced by cAMP may be due to the inhibition of both the increase of intracellular Ca²⁺ concentration and actin polymerization. Furthermore, the intracellular levels of cAMP probably play a role in regulating actin states in the macrophages.     

Formation of cyclic adenosine-3',5'-monophosphate in phagocytosis]

The content of cAMP in the phagocytizing macrophages increased, especially in the phagocytosis of live microbes. cAMP formed in phagocytosis was determined in the incubation medium, whereas in the cells its content remained practically unchanged. In the administration of E. coli 055 to the germ-free guinea pigs the concentration of cAMP was found to be increased in the mucosa cells of the small intestine and in the blood serum; this fact indicated that adenylcyclase system participated in the reactions of the interrelations of the microorganisms with the epithelial cells of the smal intestiine.     

Effect of vitamin E on the oxidative metabolism of macrophages

The oxidative metabolism of macrophages in vitamin E deficiency was studied on Aug-Lac strain rats. Vitamin E deficiency was shown to enhance luminol-dependent chemiluminescence of macrophages stimulated by opsonized zymosan. There was also an increase in microviscosity of macrophage membrane lipid phase, that was estimated with a fluorescent probe. The incubation of macrophages with dl-alpha-tocopherol led to the inhibition of macrophage chemiluminescence. Superoxide dismutase, glutathione peroxidase and glutathione reductase activity was not affected by vitamin E deficiency.     

Effect of insulin and isoproterenol on macrophage phagocytic activity

To reveal the influence of cyclic adenosine monophosphate (cAMP) on the completion of the phagocytosis of salmonellae, the influence of insulin and isoproterenol on the phagocytic activity of peritoneal macrophages obtained from mice infected with S. typhimurium strains differing in virulence was studied in vitro. The study showed that isoproterenol, while increasing the intracellular content of cAMP, suppressed the bactericidal properties of macrophages with respect to salmonellae, whereas insulin decreased the level of cAMP in the cells and thus facilitated more rapid and complete digestion of ingested bacteria irrespective of their virulence.     

Involvement of the CDC25 gene product in the signal transmission pathway of the glucose-induced RAS-mediated cAMP signal in the yeast Saccharomyces cerevisiae.

Addition of glucose or related fermentable sugars to derepressed cells of the yeast Saccharomyces cerevisiae triggers a RAS-protein-mediated cAMP signal, which induces a protein phosphorylation cascade. Yeast strains without a functional CDC25 gene were deficient in basal cAMP synthesis and in the glucose-induced cAMP signal. Addition of dinitrophenol, which in wild-type strains strongly stimulates in vivo cAMP synthesis by lowering intracellular pH, did not enhance the cAMP level. cdc25 disruption mutants, in which the basal cAMP level was restored by the RAS2val19 oncogene or by disruption of the gene (PDE2) coding for the high-affinity phosphodiesterase, were still deficient in the glucose- and acidification-induced cAMP responses. These results indicate that the CDC25 gene product is required not only for basal cAMP synthesis in yeast but also for specific activation of cAMP synthesis by the signal transmission pathway leading from glucose to adenyl cyclase. They also show that intracellular acidification stimulates the pathway at or upstream of the CDC25 protein. When shifted to the restrictive temperature, cells with the temperature sensitive cdc25-5 mutation lost their cAMP content within a few minutes. After prolonged incubation at the restrictive temperature, cells with this mutation, and also those with the temperature sensitive cdc25-1 mutation, arrested at the 'start' point (in G1) of the cell cycle, and subsequently accumulated in the resting state G0. In contrast with cdc25-5 cells, however, the cAMP level did not decrease and normal glucose- and acidification-induced cAMP responses were observed when cdc25-1 cells were shifted to the restrictive temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanism of action of soluble lymphocyte mediators. IV. Effect of migratio inhibitory factor (MIF) on macrophage cyclic AMP and on responsiveness to adenylate cyclase stimulators.

Intracellular levels of cyclic 3′,5′-adenosine monophosphate (cAMP) in purified guinea pig peritoneal macrophages were elevated following incubation with the adenylate cyclase stimulators prostaglandins E₁ and E₂ (PGE₁, PGE₂), isoproterenol, and cholera toxin. Exposure of macrophages to antigen-stimulated lymphocyte culture supernatants, containing migration inhibitory factor (MIF), resulted in a moderate but consistent decrease in the cAMP level, which was best expressed after 1–2 hr of incubation. Incubation of macrophages with MIF-containing supernatants or partially purified MIF for 1–2 hr resulted in reduced cAMP accumulation in response to PGE₁, PGE₂, isoproterenol, and cholera toxin (nonspecific refractoriness). These findings indicate that MIF-induced inhibition of macrophage migration is not due to an increase in the cellular level of cAMP and that the reduction in cAMP concentration, caused by MIF, is probably a secondary phenomenon unrelated to the inhibition of cellular motility.     

Suppression of Programmed Cell Death by Intracellular cAMP Is not Mediated by Expression of Genes Encoding an Inhibitor of Apoptosis

The elevation of intracellular cAMP content is accompanied by expression of genes whose promoter contains a Ca²⁺-cAMP responsive element. In vascular smooth muscle cells (VSMC), activation of cAMP signaling blocks apoptosis triggered by serum deprivation. In the present study we investigated the role of gene expression in the inhibition of apoptosis by cAMP. In VSMC transfected with E1A adenovirus, incubation in the absence of serum for 6 h led to 20-fold elevation of chromatin fragmentation and 10-fold activation of caspase-3 activity, these being employed as markers of apoptosis. Forskolin induced activation of cAMP signaling was accompanied by 50% elevation of RNA synthesis and completely abolished the development of apoptosis during the initial 6 h incubation in growth factor-free medium. In 12 h apoptosis in forskolin-treated VSMC was slowly developed and after 24 h the content of chromatin fragments was 2-fold less than in control cells. Addition of actinomycin D and cycloheximide completely blocked RNA synthesis and decreased protein synthesis by 80%, respectively. Neither compound affected baseline apoptosis or its inhibition by forskolin. More than 70 newly phosphorylated proteins were observed by 2D-electrophoresis of VSMC after incubation with forskolin for 3 h; in 24 h the number of phosphoproteins triggered by forskolin was decreased by 2-3-fold. These results show that suppression of VSMC apoptosis under activation of cAMP signaling is mediated via posttranslational modification of pre-existing intermediates of the apoptotic machinery rather than by de novo synthesis of inhibitors of programmed cell death.     

Regulation by PGE2 of the production of oxygen intermediates by LPS-activated macrophages

The regulation by prostaglandin E2 (PGE2) of production of oxygen radicals by bacterial lipopolysaccharide-(LPS) activated macrophages was studied in vitro. A 48-hr incubation of murine thioglycollate-elicited macrophages with LPS (0.1 micrograms/ml) resulted in an enhanced ability of these cells to produce oxygen radicals when challenged with phorbol myristate acetate (PMA). Macrophages incubated for 48 hr without LPS did not produce measurable amounts of oxygen radicals when exposed to this triggering stimulus. Thus, PMA-triggered production of oxygen radicals was the result of macrophage activation by LPS. The PMA-triggered production of oxygen radicals by the LPS-activated macrophages was inhibited when PGE2 (10(-5) to 10(-9) M) was present during the incubation with LPS. Inhibition by PGE2 occurred during the early stages of macrophage activation, since the addition of PGE2 24 hr after LPS no longer inhibited the production of oxygen radicals by the macrophages. This inhibitory effect of PGE2 on the LPS-induced activation of macrophages could be reproduced by cyclic-adenosine-monophosphate (cAMP) agonists, such as isoproterenol and cholera toxin as well as by the cAMP analog dibutyryl-cAMP, suggesting a cAMP-mediated mechanism for the inhibitory effect of PGE2 on macrophage activation by LPS. Previous reports have implicated prostaglandins as mediators of destructive processes associated with chronic inflammation. Our findings suggest that PGE2 may, on the other hand, reduce tissue damage in a chronic inflammatory site by inhibiting the production of oxygen radicals by macrophages activated in the sera.     

Anti-leptin receptor antibody mimics the stimulation of lipolysis induced by leptin in isolated mouse fat pads

An anti-leptin receptor polyclonal antibody (receptor antibody), as well as leptin, stimulated the release of free fatty acids from isolated mouse fat pads in a time-dependent manner. Following a 90-min incubation, maximal lipolysis was observed at 6 microg/ml receptor antibody and 0.1 nM leptin. The receptor antibody did not show any additive effect to the stimulation of lipolysis induced by leptin, suggesting that they exert their actions through a similar mechanism involving the leptin receptor. N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), quin 2-AM, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and neomycin sulfate (neomycin) all potently inhibited the stimulation of lipolysis by the receptor antibody and leptin. Short-term incubation of the fat pads with the receptor antibody or leptin showed a transient increase in the cellular content of cAMP and myo-inositol 1,4,5-trisphosphate (IP3) in similar concentrations to the free fatty acid release. Quin 2-AM and W-7 also inhibited the increase in cAMP content, suggesting that a Ca(2)+/calmodulin-dependent process may be involved in a part of the mechanism in which the receptor antibody and leptin exert their effects. The increase in cellular IP3 content via phosphoinositide-specific phospholipase C (PLC) sensitive to neomycin appears to be a primary step to initiate intracellular events. Both the receptor antibody and leptin may stimulate the lipolysis through mechanisms involving a transient increase in the cellular IP3 content followed by cAMP production, which leads to the activation of cAMP-dependent protein kinase.     

Activation of phospholipase A(2) by low levels of fluoride in THP1 macrophages via altered Ca(2+) and cAMP concentration

Phospholipases (PLA's) participate in the regulation of physiological and pathological processes in the cell, including the release of pro-inflammatory mediators and stimulation of inflammatory processes. It is also well known that fluoride can increase the inflammatory reactions. Therefore we decided to examine the effect of fluorides in concentrations determined in human serum on cPLA(2) and sPLA(2) activity. The incubation of macrophages in fluoride solutions significantly increased the amount of synthesized cellular cAMP, intracellular calcium and sPLA(2) activity in a dose-dependent pattern. The cPLA(2) activity, estimated by the amount of released arachidonic acid, increased significantly when 10 μM NaF was used. The results of our study suggest that fluoride may change the activity of phospholipases in macrophage cells. Probably, increased cAMP concentration activates protein kinase C (PKC) and thus stimulates PLA(2). cAMP also regulates the passage of Ca(2+) through ion channels, which additionally influence PLA(2) throughout Ca(2+)-calmodulin dependent protein kinase.     

Release of tumor necrosis factor-alpha from macrophages. Enhancement and suppression are dose-dependently regulated by prostaglandin E2 and cyclic nucleotides

PGE2 has previously been shown to suppress various leukocyte functions. In this study, we examined whether PGE2 would affect release of TNF-alpha from rat resident peritoneal macrophages. Two different, dose-dependent effects were observed: low PGE2 concentrations (0.1 to 10 ng/ml) stimulated, whereas higher concentrations (greater than 10 ng/ml) suppressed TNF-alpha release. PGE2-stimulated TNF-alpha production was dependent on de novo protein synthesis and was associated with an intracellular rise of cGMP. The importance of cGMP as an intracellular messenger for PGE2 was confirmed by the following evidence: (1) low PGE2 concentrations preferentially increased cGMP and not cAMP and (2) cGMP, either exogenously added or endogenously generated by sodium nitroprusside, were efficient stimulators of TNF-alpha production. In contrast, agents increasing intracellular cAMP concentrations such as PGE1, higher PGE2 doses, isoproterenol, and theophylline, all suppressed TNF-alpha synthesis. Only resident, but not casein-elicited or Corynebacterium parvum-activated macrophages, were stimulated by low PGE2 concentrations to increase TNF-alpha production. In tumor cytotoxicity assays, PGE2-activated macrophages were active only against TNF-alpha-sensitive target cells. These findings demonstrate that TNF-alpha synthesis in macrophages is up-regulated by cGMP and down-regulated by cAMP, which indicates that cyclic nucleotides act as intracellular messengers for extracellular signals of macrophage activation.     

Cytokines and oxygen radicals after hyperoxia in preterm and term alveolar macrophages

To determine if the alveolar macrophage inflammatory cytokine response to oxygen differs in premature cells, macrophages were obtained from litters of premature (27 days) and term (31 days) rabbits. The majority of these cells were nonspecific esterase positive and actively phagocytosed latex particles. The cells that expressed cytokines also reacted with a monoclonal antibody against rabbit macrophages. After incubation overnight in 5 or 95% oxygen, the amount of interleukin (IL)-1beta and IL-8 mRNA was assessed by RT-PCR and the amount of cytokine protein by quantitative immunofluorescence microscopy. The preterm macrophage showed a significant increase in cytokine mRNA and protein after overnight incubation in 95% oxygen. This response was not seen in the term cells. Only premature macrophages had a significant increase in intracellular oxygen radical content, measured by 2',7'-dichlorofluorescin analysis, after incubation in 95% oxygen. This enhanced inflammatory cytokine response to oxygen may be one mechanism involved in the early development of chronic lung disease in premature infants.     

Antiviral activity of lambda-, beta-, and gamma-interferons in the presence of theophylline: involvement of 2',5'-oligo(A) synthetase

Theophylline, an inhibitor of cAMP phosphodiesterase, induces in human ovary carcinoma cells (CaOv) a 2-2.5-fold elevation of intracellular cAMP. This rise in the cAMP level is followed by an increase of the activity of 2',5'-oligo(A) synthetase in CaOv cells -insignificant (1.5-fold) after 16 hr incubation, and substantial (3.7-fold) after 30 hr incubation, as well as the development of antiviral resistance. Once CaOv cells have been incubated with the mixtures containing theophylline (2 mM) and lambda-, beta-, and gamma-interferon preparations (0.5-13 IU/ml), the total antiviral effect of the mixtures exceeds that generated by interferon or theophylline separately; the action of the above agents being additive. These data agree with the previously obtained results and support the suggestion that cAMP phosphodiesterase inhibitors partially mimic the antiviral action of interferon.     

Inhibition of prostaglandin E2-stimulated cAMP accumulation by lipopolysaccharide in murine peritoneal macrophages.

Treatment of murine peritoneal macrophages with 100 nM prostaglandin E2 (PGE2) produced a rapid biphasic increase in intracellular cAMP that was maximal at 1 min and sustained through 20 min. Pretreatment of macrophages with 100 ng/ml of lipopolysaccharide (LPS) for 60 min prior to PGE2 decreased the magnitude of cAMP elevation by 50%, accelerated the decrease of cAMP to basal levels, and abolished the sustained phase of cAMP elevation. The effect of LPS was concentration-dependent, with maximal effect at 10 ng/ml in cells incubated in the presence of 5% fetal calf serum and at 1 microgram/ml in the absence of fetal calf serum. LPS also inhibited cAMP accumulation in cells treated with 100 microM forskolin, but the decrease was about half that seen in cells treated with PGE2. LPS concentrations that inhibited cAMP accumulation produced a 30% increase in soluble low Km cAMP phosphodiesterase activity while having no effect on particulate phosphodiesterase activity. The nonspecific phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, as well as the more specific inhibitors rolipram and Ro-20-1724 were effective in inhibiting soluble phosphodiesterase activity in vitro, producing synergistic elevation of cAMP in PGE2-treated cells, and blocking the ability of LPS to inhibit accumulation of cAMP. Separation of the phosphodiesterase isoforms in the soluble fraction by DEAE chromatography indicated that LPS activated a low Km cAMP phosphodiesterase. The enzyme(s) present in this peak could be activated 6-fold by cGMP and were potently inhibited by low micromolar concentrations of Ro-20-1724 and rolipram. Using both membranes from LPS-treated cells and membranes incubated with LPS, no decrease in adenylylcyclase activity could be attributed to LPS. Although effects of LPS on the rate of synthesis of cAMP cannot be excluded, the present evidence is most consistent with a role for phosphodiesterase activation in the inhibitory effects of LPS on cAMP accumulation in murine peritoneal macrophages.