Effect of irradiation on cytokine secretion and nitric oxide production by inflammatory macrophages

This study explored the effects of low-dose and high-dose irradiation on inflammatory macrophage cells, specifically inflammatory cytokine secretion and nitric oxide (NO) production after irradiation. To elucidate the effect of irradiation on active and inactive macrophages, we exposed LPS-treated or untreated murine monocyte/macrophage RAW 264.7 cell lines to low-dose to high-dose radiation (0.01–10 Gy). We analyzed the effects of irradiation on RAW 264.7 cell proliferation by MTT assays and analyzed cytokine secretion and NO production related to inflammation by ELISA assays. Low-to-high doses of radiation did not significantly affect the proliferation of LPS-treated or untreated RAW 264.7 cells. Pro-inflammatory cytokine IL-1ß was generally increased in RAW 264.7 cells at 3 days after radiation. Especially, IL-1ß was significantly increased in only high dose-irradiation (2 and 10 Gy irradiation) groups in LPS-untreated RAW 264.7 cells but increased in both low and high dose-irradiation groups (0.01–10 Gy) in LPS-treated RAW 264.7 cells at 3 days after irradiation. Whereas, the expression of IL-1ß was prolonged in high-dose irradiation group at 5 days after irradiation. The production of anti-inflammatory cytokine IL-10 did not change significantly at 3 days after radiation but was significantly reduced at 5 days after 10 Gy radiation. The effect of irradiation on the secretion of IL-1ß and IL-10 was not significantly different between RAW 264.7 cells treated or not treated with LPS. The effect of irradiation on NO secretion by RAW 264.7 cells showed a specific pattern. NO was produced after low-dose irradiation but reduced in a high-dose irradiation group at 3 days after irradiation. However, NO production was not changed after low-dose irradiation and reduced at 5 days after high-dose irradiation. These results showed that irradiation affected the inflammatory system and regulated NO production in both activated and inactivated macrophages through different regulation mechanisms, depending on irradiation dose.     

Evaluation of inflammatory cytokine secretion by human alveolar macrophages

The alveolar macrophage (AM) secretes interleukin 1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-8 (IL-8), all of them inflammatory cytokines involved in the pathogenesis of many lung diseases. The aim of the present work was to evaluate the basal and stimulated secretion of these cytokines by human AMs. Human AMs were collected by bronchoalveolar lavage (BAL) from four healthy controls and 13 patients with diffuse interstitial lung disease (five cases of sarcoidosis, three of hypersensitivity pneumonitis and five of idiopathic pulmonary fibrosis). AMs were cultured in the presence or absence of different concentrations of lipopolysaccharide (LPS), phorbolmyristate and gamma-interferon. IL-1beta, TNF-alpha, IL-6 and IL-8 levels were measured in BAL fluid and culture supernatant using specific enzyme-linked immunosorbent assays. The substance found to stimulate the secretion of inflammatory cytokines to the greatest extent was LPS at a concentration of 10 microg/ml. Regarding the secretion of IL-1beta, four observations were of interest: basal secretion was very low; LPS exerted a potent stimulatory effect; considerable within-group variability was observed; and there were no significant differences in the comparisons among groups. With respect to TNF-alpha secretion, the results were similar. The only striking finding was the higher basal secretion of this cytokine with respect to that of IL-1beta. Regarding the secretion of IL-6, the same pattern followed by TNF-alpha was found. However, it should be stressed that the increase induced by LPS was smaller than in the two previous cytokines. Regarding the secretion of IL-8, three findings were patent: the strong basal secretion of this cytokine; the moderate increase induced by LPS; and the existence of significant differences among the different groups with respect to the stimulated secretion of this cytokine, which reached maximum values in patients with idiopathic pulmonary fibrosis. Finally, it should be noted that the pattern of cytokines observed in the BAL fluid was similar to that found in cultured AM supernatants. The pattern of inflammatory cytokine secretion by AMs differs from that of other cells of the mononuclear phagocyte system (MPS). In this sense. AMs secrete low amounts of IL-1, moderate amounts of TNF-alpha and IL-6, and high quantities of IL-8. Adherence is an important stimulus in the secretion of these molecules and LPS elicits an increased secretion inverse to the basal secretion. There is considerable individual variability in the secretion of inflammatory cytokines by the AMs of patients with interstitial lung disease and the AMs of these patients are primed in vivo for the secretion of these cytokines. The results of our study, carried out in vitro, can be extrapolated to the in vivo setting.     

Analysis of the effects of nitric oxide and oxygen on nitric oxide production by macrophages

The interactions between NO and O(2) in activated macrophages were analysed by incorporating previous cell culture and enzyme kinetic results into a novel reaction-diffusion model for plate cultures. The kinetic factors considered were: (i) the effect of O(2) on NO production by inducible NO synthase (iNOS); (ii) the effect of NO on NO synthesis by iNOS; (iii) the effect of NO on respiratory and other O(2) consumption; and (iv) the effects of NO and O(2) on NO consumption by a possible NO dioxygenase (NOD). Published data obtained by varying the liquid depth in macrophage cultures provided a revealing test of the model, because varying the depth should perturb both the O(2) and the NO concentrations at the level of the cells. The model predicted that the rate of NO(2)(-) production should be nearly constant, and that the net rate of NO production should decline sharply with increases in liquid depth, in excellent agreement with the experimental findings. In further agreement with available results for macrophage cultures, the model predicted that net NO synthesis should be more sensitive to liquid depth than to the O(2) concentration in the headspace. The main reason for the decrease in NO production with increasing liquid depth was the modulation of NO synthesis by NO, with O(2) availability playing only a minor role. The model suggests that it is the ability of iNOS to consume NO, as well as to synthesize it, that creates very sensitive feedback control, setting an upper bound on the NO concentration of approximately 1 microM. The effect of NO consumption by other possible pathways (e.g., NOD) would be similar to that of iNOS, in that it would help limit net NO production. The O(2) utilized during enzymatic NO consumption is predicted to make the O(2) demands of activated macrophages much larger than those of unactivated ones (where iNOS is absent); this remains to be tested experimentally.     

Effect of bismuth subgallate on nitric oxide and prostaglandin E2 production by macrophages

Bismuth subgallate (BSG) is used widely in clinics, including Vincent's angina, syphilis, and adenotonsillectomy. This study examined the effects of BSG on nitric oxide (NO) and prostaglandin E2 (PGE2) production in activated RAW 264.7 cells. BSG suppressed production of NO and PGE2 in a dose-dependent manner. BSG could increase TGF-beta1 production, which in turn might promote degradation of iNOS mRNA, thus inhibiting NO production. Additionally, BSG inhibited mPGES protein expression and COX-2 activity in activated RAW 264.7 cells. Exogenous addition of SNP reversed the inhibition effect of PGE2 production by BSG. This behavior indicates that PGE2 inhibition by BSG exerts an indirect effect through NO inhibition.     

Fatty acid control of nitric oxide production by macrophages

Modulation of macrophage functions by fatty acids (FA) has been studied by several groups, but the effect of FA on nitric oxide production by macrophages has been poorly examined. In the present study the effect of palmitic, stearic, oleic, linoleic, arachidonic, docosahexaenoic and eicosapentaenoic acids on NF-kappaB activity and NO production in J774 cells (a murine macrophage cell line) was investigated. All FA tested stimulated NO production at low doses (1-10 microM) and inhibited it at high doses (50-200 microM). An increase of iNOS expression and activity in J774 cells treated with a low concentration of FA (5 microM) was observed. The activity of NF-kappaB was time-dependently enhanced by the FA treatment. The inhibitory effect of FA on NO production may be due to their cytotoxicity, as observed by loss of membrane integrity and/or increase of DNA fragmentation in cells treated for 48 h with high concentrations. The results indicate that, at low concentrations FA increase NO production by J774 cells, whereas at high concentrations they cause cell death.     

Effect of a combination of deoxynivalenol and nivalenol on lipopolisaccharide-induced nitric oxide production by mouse macrophages

Deoxynivalenol (DON) and nivalenol (NIV) are trichothecene mycotoxins produced by Fusarium fungi as secondary metabolites. Both compounds have the immunotoxic effects that the productions of inflammatory mediators by activated macrophages is disturbed. Co-contamination with DON and NIV can occur; however, the effects of simultaneous contamination are not well known. The present study investigated the combined effects of DON and NIV on nitric oxide (NO) production by mouse macrophages stimulated with lipopolisaccharide (LPS). The inhibitory effect of DON and NIV on NO release from activated macrophages has already been reported as an appropriate indicator of immunotoxic effect of the both compounds. LPS-induced NO production in macrophages was inhibited by both of these toxins individually in a dose-dependent manner, and toxin mixtures at the same concentration inhibited NO production in the same manner. In addition, there were no unique inhibitory effects on LPS-induced NO production in macrophages in the presence of mixtures of various molar ratios. These results suggest that the combined effects of DON and NIV can be predicted based on addition of each compound alone.     

Effect of nitric oxide donor and gamma irradiation on MAPK signaling in murine peritoneal macrophages

Irradiation (IR) of cells is known to activate enzymes of mitogen activated protein kinase (MAPK) family. These are known to be involved in cellular response to stress and are determinants of cell death or survival. When radiotherapy is delivered to malignant cells, macrophages, being radioresistant, survive, get activated, and produce large amounts of nitric oxide. As a result of activation they recognize and phagocytose tumor and normal cell apoptotic bodies leading to tumor regression. In this study, the MAPK signaling in peritoneal macrophages was investigated which plays an important role in its various functions, in an environment which is predominantly nitric oxide, as is after IR. The behavior of macrophages in such an environment was also looked at. The three MAPK (ERK1/2, p38, and JNK) respond differently to Sodium nitroprusside (SNP) alone or IR alone. All the three were activated following IR but only JNK was activated following SNP treatment. Surprisingly, when both the stresses were given simultaneously or one after the other, this differential response was lost and there was a complete inhibition of phosphorylation of all the three MAPKs, irrespective of the order of the two insults (IR and SNP). The noteworthy observation was that despite the complete inhibition of MAPK signaling there was no effect on either the viability or the phagocytic efficiency of peritoneal macrophages.     

Effect of naturally occurring organosulfur compounds on nitric oxide production in lipopolysaccharide-activated macrophages

Excessive nitric oxide (NO) production is involved in cellular injury and possibly in the multistage process of carcinogenesis. In this study, we investigated the effect of organosulfur compounds (S-allyl cysteine, allyl sulfide, diallyl disulfide, allyl isothiocyanate, phenyl isothiocyanate, and benzyl isothiocyanate) that are found in allium or cruciferous vegetables on NO production in J774.1 macrophages activated with lipopolysaccharide (LPS). Diallyl disulfide, allyl, phenyl, and benzyl isothiocyanates inhibited NO production, as evaluated by nitrite formation at 25 microM. Allyl and benzyl isothiocyanates, the most active of the six organosulfur compounds, exhibited dose-dependent inhibition and had IC(50) values of 1.6 and 2.7 microM, respectively. Western blot analysis suggested that suppression of the induction of inducible NO synthase (iNOS) expression is responsible for the inhibition of NO production by allyl and benzyl isothiocyanates. In contrast, these isothiocyanates increased LPS-stimulated tumor necrosis factor alpha (TNF-alpha) release, suggesting their selective action on genes activated by LPS. Our results demonstrate that certain organosulfur compounds inhibit NO synthesis in LPS-activated macrophages, and the inhibitory effect may be a significant component of their anticarcinogenic activity.     

Quantitative nitric oxide production by rat, bovine and porcine macrophages

The aim of this work was to compare in vitro nitric oxide (NO) production by rat, bovine and porcine macrophages. NO production was induced by lipopolysaccharide (LPS) or by phorbol 12-myristate 13-acetate (PMA) with ionomycin or recombinant interferon gamma (rIFN-γ) and was assessed by Griess reaction. NO synthase type II (NOS II) expression was quantified by immunocytochemistry, Western blot and real-time polymerase chain reaction (RT-PCR). There were differences in NO production by pulmonary alveolar macrophages (PAM) in all species tested. The largest amounts of NO were produced by rat PAM. Less NO was produced by bovine PAM. Moreover, PAM in rats and cows differed in their abilities to respond to various stimulators. Neither porcine PAM nor Kupffer cells produced NO. Stimulation of porcine PAM with alternative concentrations of LPS did not lead to inducing NO production. Stimulation of porcine PAM with rIFN-γ together with LPS led to a significant increase in the expression of NOS II mRNA, albeit without detectable NO production or NOS II expression on the protein level.     

Stimulation of cytokine and nitric oxide production by acyclic nucleoside phosphonates.

Several antiviral acyclic nucleotide analogues activate expression of genes for cytokines, such as TNF-alpha, IL-10 in macrophages and IFN-gamma in splenocytes. This is an underlying mechanism for substantially enhanced production of nitric oxide generated by IFN-gamma. More lipophilic prodrugs, bis-POM-PMEA and bis-POC-PMPA, are cytocidal for macrophages and thus inhibit nitric oxide formation.     

No effect of exposure to static and sinusoidal magnetic fields on nitric oxide production by macrophages

The effects of exposure to static (1–100 mT) or sinusoidal (1 Hz, 1.6 mT) magnetic fields on the production of nitric oxide (NO) by murine BCG-activated macrophages were investigated. In these cells, the inducible isoform of NO synthase is present. No significant differences were observed in nitrite levels among exposed, sham-exposed, or control macrophages after exposure for 14 h to static fields of 1, 10, 50, and 100 mT and to sinusoidal 1.6 mT, 1 Hz magnetic fields. © 1996 Wiley-Liss, Inc.     

Regulation of nitric oxide production from macrophages by lipopolysaccharide and catecholamines.

Catecholamines are elaborated in stress responses to mediate vasoconstriction, and elevate systemic vascular resistance and blood pressure. They are elaborated in disorders such as sepsis, cocaine abuse, and cardiovascular disease. The aim of the study was to determine whether catecholamines affect nitric oxide (NO) production, as NO is a vasodilator and counteracts the harmful effects of catecholamines. RAW264.7 macrophage cells were cultured with lipopolysaccharide (LPS)+/-epinephrine, norepinephrine, and dopamine at 5x10(-6)M concentrations for 24h. Supernatants were harvested for measuring NO by spectrophotometry using the Greiss reagent and cells were harvested for detecting inducible NO synthase (iNOS) by Western blot. NO production in RAW 264.7 macrophages was increased significantly by addition of LPS (0.5-10ng/ml) in a dose-dependent fashion. The NO production induced by LPS was further enhanced by epinephrine and norepinephrine, and to a lesser extent by dopamine. These increases in NO correlated with expression of iNOS protein in these cells. The enhancing effect of iNOS synthesis by epinephrine and norepinephrine on LPS-induced macrophages was down regulated by beta-adrenoceptor antagonist, propranolol, and dexamethasone. The results suggest that catecholamines have a synergic effect on LPS in induction of iNOS synthesis and NO production, and this may mediate some of the vascular effects of infection. These data support a novel role for catecholamines in disorders such as septic shock and cocaine use, and indicate that beta-adrenoceptor antagonists and glucocorticoids may be used therapeutically for modulation of the catecholamine-NO axis in disease states.     

Apoptotic cells can deliver chemotherapeutics to engulfing macrophages and suppress inflammatory cytokine production

Immunosuppression via cell-cell contact with apoptotic cells is a well studied immunological phenomenon. Although the original studies of immune repression used primary cells, which undergo spontaneous cell death or apoptosis in response to irradiation, more recent studies have relied on chemotherapeutic agents to induce apoptosis in cell lines. In this work, we demonstrate that Jurkat cells induced to die with actinomycin D suppressed inflammatory cytokine production by macrophages, whereas cells treated with etoposide did not. This immune repression mediated by actinomycin D-treated cells did not require phagocytosis or cell-cell contact and thus occurs through a different mechanism from that seen with primary apoptotic neutrophils. Moreover, cells induced to die with etoposide and then treated for a short time with actinomycin D also suppressed macrophage responses, indicating that suppression was mediated by actinomycin D independent of the mechanism of cell death. Finally, phagocytosis of actinomycin D-treated cells caused apoptosis in macrophages, and suppression could be blocked by inhibition of caspase activity in the target macrophage. Together, these data indicate that apoptotic cells act as "Trojan horses," delivering actinomycin D to engulfing macrophages. Suppression of cytokine production by macrophages is therefore due to exposure to actinomycin D from apoptotic cells and is not the result of cell-receptor interactions. These data suggest that drug-induced death may not be an appropriate surrogate for the immunosuppressive activity of apoptotic cells. Furthermore, these effects of cytotoxic drugs on infiltrating immune phagocytes may have clinical ramifications for their use as antitumor therapies.     

Chitinous materials inhibit nitric oxide production by activated RAW 264.7 macrophages

Chitinous materials have been studied in wound healing and artificial skin substitutes for many years. Nitric oxide (NO) has been shown to contribute to cytotoxicity in cell proliferation during inflammation of wound healing. In this study, we examined the effect of chitin and its derivatives on NO production by activated RAW 264.7 macrophages. Chitin and chitosan showed a significantly inhibitory effect on NO production by the activated macrophages. Hexa-N-acetylchitohexaose and penta-N-acetylchitopentaose also inhibited NO production but with less potency. However, N-acetylchitotetraose, -triose, -biose, and monomer of chitin, N-acetylglucosamine and glucosamine had little effect on NO production by the activated cells. These results suggest that the promotive effect of chitinous material on wound healing be related, at least partly, to inhibit NO production by the activated macrophages.     

Apolipoprotein E acts to increase nitric oxide production in macrophages by stimulating arginine transport

Previous studies have shown that apolipoprotein E (apoE) plays a role in immune function by modulating tissue redox balance. Using a mouse macrophage cell line (RAW 264.7), we have examined the mechanism by which apoE regulates nitric oxide (NO) production in macrophages. ApoE potentiates NO production in immune activated RAW cells in combination with lipopolysaccharide or polyinosinic:polycytidylic acid (PIC), agents known to induce expression of inducible nitric oxide synthase mRNA and protein. The effect is not observed with apolipoprotein B or heat-inactivated apoE. The combination of PIC plus apoE produced more NO than the level expected from an additive effect of PIC and apoE alone. Furthermore, this increase was observed at submaximal extracellular arginine concentrations, suggesting that apoE altered arginine (substrate) availability. Examination of [(3)H]arginine uptake across the cell membrane demonstrated that arginine uptake was increased by PIC but further increased by PIC plus apoE. Treatment of RAW cells with apoE was associated with an increased apparent V(max) and decreased affinity for arginine as well as a switch in the induction of mRNA for subtypes of cationic amino acid transporters (CAT). Treatment of RAW cells with PIC plus apoE resulted in the loss of detectable CAT1 mRNA and expression of CAT2 mRNA. Regulation of arginine availability is a novel action of apoE on the regulation of macrophage function and the immune response.     

Exogenous nitric oxide modulates cytokine production in human leukocytes

Exogenous nitric oxide was found to modify the pattern of cytokine secretion from human leukocytes, with similar outcome in 11 different healthy blood donors. Peripheral blood mononuclear cells (PBMC) were stimulated with phytohaemagglutinin (PHA) in the presence of increasing amounts of the NO donor S-nitroso-N-acetyl-penicillamine (SNAP). The NO donor dose-dependently enhanced IL-4 secretion into the supernatant (p<0.01). In contrast, IFNgamma production was not affected while IL-10 levels were slightly decreased. Comparable changes were observed when analysing cytokine mRNA levels by semiquantitative RT-PCR. The differential effect of the NO donor on IL-4 versus IL-10 and IFNgamma gene expression suggests an immunomodulatory potential of NO, which may serve to limit inflammatory responses.     

Concomitant production of nitric oxide and superoxide in human macrophages

Many harmful effects of nitric oxide are caused by the reaction of NO with superoxide anion. The present study was carried out to find out the concomitant production of superoxide and to investigate a suitable inhibitor of NO, which is produced by iNOS. THP-1 cells were differentiated into macrophages by PMA and cytokine. Addition of L-NAME showed decrement in superoxide production. Addition of apocynin, aminoguanidine or ONO 1714 brought about a significant reduction in superoxide production. The expressions of p67 and p47(phox) were reduced by the addition of apocynin, aminoguanidine or ONO 1714 whereas xanthine oxidase and cyclooxygenase did not have a major role in superoxide production. The results of the present study show that iNOS and NADPH oxidase play an important role in superoxide release. It suggests that addition of iNOS inhibitor together with apocynin may be more effective in case of therapeutic application in disease conditions like atherosclerosis.     

Effect of nitric oxide donor and gamma irradiation on modifications of ERK and JNK in murine peritoneal macrophages

Mitogen activated protein kinases (MAPKs) play an important role in activation, differentiation and proliferation of macrophages. Macrophages, upon activation, produce large amounts of nitric oxide that inhibit the growth of variety of microorganisms and tumor cells. This nitric oxide which is known to interfere with tyrosine phosphorylation may result in changes in the pattern of activation of MAPKs. In a previous study we have found that tyrosine phosphorylation of MAPKs was completely abolished in the presence of nitric oxide donor and radiation but this did not affect the function of macrophages. In this study the other post translational modifications namely nitration and ubiquitination of JNK and ERK have been looked at. Both ERK and JNK were found to be nitrated. However, there was no increase in ubiquitination of ERK and JNK, indicating that ubiquitination, in this case was not a natural consequence of nitration and may serve in signaling. Additionally, when the nitration was extensive, phosphorylation was also inhibited. The activation of substrates of ERK and JNK were looked at to determine the consequences of such modifications. Inhibition of phosphorylation and extensive nitration of JNK did not prevent activation of its substrate, c-jun. This study indicates that ERK and JNK may be under regulation by different type of modifications in macrophages.     

Effect of nitric oxide donor and gamma irradiation on modifications of ERK and JNK in murine peritoneal macrophages

Mitogen activated protein kinases (MAPKs) play an important role in activation, differentiation and proliferation of macrophages. Macrophages, upon activation, produce large amounts of nitric oxide that inhibit the growth of variety of microorganisms and tumor cells. This nitric oxide which is known to interfere with tyrosine phosphorylation may result in changes in the pattern of activation of MAPKs. In a previous study we have found that tyrosine phosphorylation of MAPKs was completely abolished in the presence of nitric oxide donor and radiation but this did not affect the function of macrophages. In this study the other post translational modifications namely nitration and ubiquitination of JNK and ERK have been looked at. Both ERK and JNK were found to be nitrated. However, there was no increase in ubiquitination of ERK and JNK, indicating that ubiquitination, in this case was not a natural consequence of nitration and may serve in signaling. Additionally, when the nitration was extensive, phosphorylation was also inhibited. The activation of substrates of ERK and JNK were looked at to determine the consequences of such modifications. Inhibition of phosphorylation and extensive nitration of JNK did not prevent activation of its substrate, c-jun. This study indicates that ERK and JNK may be under regulation by different type of modifications in macrophages.