Modulation by drugs of leukotriene and prostaglandin production from mouse peritoneal macrophages

Leukotriene and prostaglandin production by mouse peritoneal macrophages was investigated. It could be shown that the tumour promoter 12-O-tetradecanoylphorbol-13-acetate, despite initiating the release of prostaglandin E2, had little effect on the release of leukotriene C4-like immunoreactivity. The divalent cation ionophore A 23187 at concentrations between 10(-6) and 10(-8) mol/l initiated prostaglandin as well as leukotriene release. This prostaglandin and leukotriene release could be modulated by drugs. Non-steroidal anti-inflammatory drugs inhibited prostaglandin release but enhanced leukotriene production. The experimental compound BW 755C inhibited prostaglandin and leukotriene production, whereas the antithrombotic compound nafazatrom inhibited the production of leukotriene C4-like immunoreactivity but enhanced the prostaglandin E2 production. Nordihydroguaiaretic acid inhibited prostaglandin and leukotriene production. The results show that the metabolism of arachidonic acid in macrophages via the cyclooxygenase or the lipoxygenase pathway is dependent on the stimulus applied. Both pathways can be inhibited conjointly or selectively by drugs. The experimental system described may be used for assessing the potency of drugs to inhibit the lipoxygenase and the cyclooxygenase pathway of arachidonic acid metabolism.     

Pharmacological control of leukotriene and prostaglandin production from mouse peritoneal macrophages

Leukotriene and prostaglandin production by mouse peritoneal macrophages was investigated. It could be shown that the tumour promoter 12-O-tetradecanoylphorbol-13-acetate initiated the release of prostaglandin E₂ but had little effect on the release of leukotriene C₄-like immunoreactivity. The divalent cation ionophore A 23187 at concentrations between 10^–6 and 10^–8 mol/l initiated prostaglandin as well as leukotriene release. This prostaglandin and leukotriene release could be modulated by drugs. Non-steroidal anti-inflammatory drugs including benoxaprofen inhibited prostaglandin release but simultaneously enhanced leukotriene production. The analgesics paracetamol and 4-methylaminoantipyrine had similar effects at high concentrations. The experimental compound BW 755 c inhibited prostaglandin and leukotriene production while the antithrombotic compound nafazatrom inhibited the production of leukotriene C₄-like immunoreactivity but enhanced prostaglandin E₂ production. Nordihydroguaiaretic acid inhibited prostaglandin and leukotriene production. The results show that the metabolism of arachidonic acid in macrophages via the cyclooxygenase or the lipoxygenase pathway is dependent on the stimulus applied. Both pathways can be inhibited conjointly or selectively by drugs. Our results do not provide evidence that differences in anti-inflammatory activity claimed for some of the drugs tested can be explained by differential inhibition of either pathway. The experimental system described may be used for assessing the potency of drugs to inhibit the lipoxygenase and the cyclooxygenase pathway of arachidonic acid metabolism.     

Differential infiltration of macrophages and prostaglandin production by different uterine leiomyomas

BACKGROUND: The association between uterine myoma and infertility is still controversial. The anatomical defect of endometrium by uterine fibroids could be a factor for reducing pregnancy rates and increasing miscarriage rates. However, pregnancy and implantation rates were found to be significantly lower in women with intramural myomas (IMMs), when there was no deformity of uterine cavity. This could be due to other biological factors such as increased accumulation of inflammatory cells within fibroid tissue and corresponding endometrium that might impair fertility. Therefore, we tried to investigate the pattern of macrophage (Mvarphi) accumulation in different uterine fibroids and the production of chemokine and prostaglandin (PG) by these tissues. METHODS: The selection criteria of uterine fibroids were based on the classification of European Society of Hysteroscopy. Biopsy specimens were collected from respective nodules and autologous endometrium of 20 women with submucosal myoma (SMM), 29 women with IMM and 18 women with subserosal myoma (SSM). CD68 immunoreactive Mvarphis were identified in these tissues by immunohistochemistry. A fraction of corresponding tissues were homogenized, and levels of monocyte chemotactic protein-1 (MCP-1) and PGF(2alpha) were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Mvarphi infiltration in the myoma nodule and corresponding endometrium of women with SMM and IMM was significantly higher than that of women with SSM or control women (P<0.01 and P<0.05, respectively). This tissue accumulation of inflammatory cells was independent of the sizes of the myoma nodules and phases of menstrual cycle. The tissue concentration of MCP-1 corresponded to increased Mvarphi infiltration and was significantly higher in women with SMM and IMM than that in women with SSM (P<0.05 for each). A positive correlation was observed between MCP-1 concentration and accumulated Mvarphi numbers in the endometrium of women with SMM and IMM but not in women with SSM. The tissue levels of PGF2alpha were also significantly higher in the nodule and corresponding endometrium of women with SMM and IMM than that in SSM or control women (P<0.05 for each). CONCLUSIONS: Higher production of MCP-1 could be responsible for the increased accumulation of Mvarphi in women with SMM and IMM. The augmented inflammatory reaction in endometrium and increased PGF2alpha levels might be detrimental to reproductive outcome in women with SMM or IMM.     

Nitric oxide production by Leishmania-infected macrophages and modulation by prostaglandin E2

Nitric oxide (NO), produced by the nitric oxide synthase (iNOS) enzyme, is the most-important molecule responsible for the killing of Leishmania parasites by macrophages. In previous work we have demonstrated that, after activation with recombinant human interferon-γ and/or bacterial lipopolysaccharide, human macrophages infected with Leishmania infantum are able to produce nitric oxide and to express nitric oxide synthase. The arachidonate derivative prostaglandin E₂ has been shown to modulate various macrophage activities, and in particular nitric oxide production, sometimes with opposite effects, related to experimental conditions. In this work we have evaluated nitric oxide release and parasite killing by peripheral blood-derived L. infantum-infected human macrophages in vitro stimulated with lipopolysaccharide and simultaneously treated with prostaglandin E₂. Experiments were also performed in the presence of the nitric oxide synthase inhibitor l-N ^G monomethylarginine (l-NMMA) and of the cyclooxygenase inhibitor indomethacin. Nitric oxide release in supernatants of macrophage cultures was measured by the Griess reaction for nitrites. Parasite killing was microscopically evaluated by fluorescent dyes. Results demonstrated that macrophages stimulated with lipopolysaccharide and treated with prostaglandin E₂ exhibited increased nitric oxide producation and parasite killing, which were significantly reduced by either l-NMMA or indomethacin. In indomethacin-treated macrophages, nitric oxide production and leishmanicidal ability were partially restored by the addition of exogenous prostaglandin E₂. Taken together, these results indicate that prostaglandin E₂ may be involved in nitric oxide production, and possibly in the host-protective immune response against Leishmania. Moreover, the demonstration of a stimulatory role of prostaglandin E₂ on nitric oxide production induced by intracellular pathogens in humans is interesting in the light of a possible pharmacological regulation of nitric oxide by modulation of prostaglandin E₂ synthesis. Received: 27 March 2001 / Accepted: 24 August 2001     

Lectins modulate prostaglandin E2 production by rat peritoneal macrophages

The effect of Aloctin A (Alo A), a lectin having anti-inflammatory activities, on prostaglandin (PG) E₂ production by activated rat peritoneal macrophages was compared with that of concanavalin A (Con A), wheat germ agglutinin (WGA), plsum sativum agglutinin (PSA) and soybean agglutinin (SBA). Alo A, WGA, Con A and PSA at 10 g per ml inhibited PG E₂ production. But SBA, even at a dose of 1 g per ml, stimulated PG E₂ production. The inhibition by Alo A treatment of the release of radioactivity from (³H)arachidonic acid-labeled macrophages and the stimulation of this release by SBA treatment were observed. The uptake of (⁵¹Cr)-labeled sheep red blood cells by the macrophage was inhibited by Alo A, Con A, and PSA, all at 10 g per ml and SBA at 1 g per ml, however, WGA at 10 g per ml stimulated the uptake of the sheep red blood cells. The mechanism of the anti-inflammatory properties of Alo A was discussed.To whom correspondence should be addressed.     

African trypanosomiasis alters prostaglandin production by murine peritoneal macrophages.

Sera from 39 patients with systemic sclerosis were examined for a cytotoxic effect on human umbilical vein endothelium. Although none of the sera produced direct cytotoxicity of 51Cr-labelled endothelial cells, even with added complement, nine sera did produce increased 51Cr release when co-cultured with endothelial cells and normal human peripheral blood mononuclear cells. The effector cells involved in this cytotoxicity possessed Fc receptors but were non-T and non-adherent while the responsible serum factor(s) was present in IgG containing fractions. This cytotoxicity tended to occur in patients with both circulating immune complexes and precipitating antibodies to nuclear and cytoplasmic antigens who, as a group, had more severe and extensive visceral disease than those without such serological abnormalities. Control studies using sera from both 27 normal controls and 19 patients with either diabetes or extensive athero-sclerotic vascular disease failed to reveal any similar cytotoxicity.     

Differential prostaglandin production by unfractionated and density-fractionated human monocytes and alveolar macrophages

Mononuclear phagocyte elaboration of E series prostaglandins (PGE) may be important in the regulation of inflammatory and fibrotic reactions. Mononuclear phagocytes are morphologically and functionally heterogeneous cells. To further understand the processes controlling inflammation and fibrosis, in particular that in the human lung, we characterized the ability of unfractionated and density-fractionated human alveolar macrophages and blood monocytes to elaborate PGE. Alveolar macrophages and blood monocytes constitutively elaborated small amounts of PGE, and their elaboration of PGE was increased with lipopolysaccharide (LPS) stimulation. Monocytes elaborated more PGE than autologous alveolar macrophages. In addition, denser monocytes (specific gravity greater than 1.055) and denser alveolar macrophages (specific gravity greater than 1.044) elaborated more PGE than less dense monocytes and alveolar macrophages, respectively. When monocytes were incubated in vitro, their constitutive PGE elaboration decreased with time. However, in vitro incubation did not cause monocytes to lose their capacity to elaborate PGE in response to LPS. Thus, mononuclear phagocyte populations differ in their ability to elaborate PGE. These differences can be only partially attributed to differences in cell maturation.     

Regulation of alveolar macrophage production of chemoattractants by leukotriene B4 and prostaglandin E2.

Alveolar macrophages (AM) appear to influence the recruitment of neutrophils into the lung by the elaboration of both lipid and peptide chemotactic molecules for neutrophils. Little is known about the mechanisms that regulate production or release of chemotactic molecules by AM or the interaction between these classes of chemotactic molecules. We investigated the hypothesis that the lipid mediator leukotriene B4 (LTB4) has an in vitro regulatory action on the production of chemotactic proteins by AM. In these experiments, the chemotactic activity in AM culture supernatants was measured in a modified Boyden chamber. LTB4 treatment increased AM production of chemotactic activity in excess of what might be attributed to the amount of LTB4 measured in the culture supernatant after the incubation period. This effect was magnified by in vivo administration of endotoxin prior to AM harvesting. Pretreatment with LTB4 caused a sustained 250% increase in AM production of chemotactic activity, yet only negligible amounts of LTB4 were measured by gas chromatography/mass spectrometry in the LTB4-pretreated AM culture supernatants, indicating that LTB4 alone did not account for the chemotactic activity observed in our studies. A chemotactic peptide in LTB4-treated AM culture supernatant could be isolated and separated from LTB4 by molecular sieve chromatography. Purified column fractions contained 80% of the chemotactic activity of endotoxin-stimulated AM culture supernatant and had a molecular mass of 10,000 D. In contrast to LTB4, prostaglandin E2 (PGE2) suppressed chemotactic activity production by endotoxin-stimulated AM by 70%. Pretreatment with PGE2 was not effective; PGE2 had to be present in the AM culture medium during endotoxin exposure in order to exert a suppressive effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nerve growth factor regulation and production by macrophages in osteoarthritic synovium

Nerve growth factor (NGF) functions to modulate osteoarthritis (OA)‐associated pain. Although recent studies suggest that tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β mediate NGF activity in human synovial fibroblasts, the regulation of NGF expression in human synovial macrophages remains unclear. Here, we examined the role of macrophages in the production and regulation of synovial (SYN) NGF in osteoarthritic knee joints by examining the mRNA expression of TNF‐α and IL‐1β in freshly isolated CD14‐positive (macrophage‐rich fraction) and CD14‐negative cells (fibroblast‐rich fraction) in synovial tissue from OA patients by quantitative polymerase chain reaction. We also examined the effects of IL‐1β and TNF‐α on NGF mRNA expression in cultured CD14‐positive (macrophage‐rich fraction) and CD14‐negative cells (fibroblast‐rich fraction). In addition, to examine the contribution of macrophages to NGF, TNF‐α and IL‐1β expression, we injected clodronate liposomes systemically into STR/Ort mice, an osteoarthritis animal model, to deplete macrophages. TNF‐α and IL‐1β mRNA levels in CD14‐positive cells from the SYN of OA patients was significantly higher than that in CD14‐negative cells, while NGF expression did not differ markedly between the two cell fractions. In addition, treatment of human cultured CD14‐positive and ‐negative cells with IL‐1β and TNF‐α enhanced NGF mRNA and protein levels. Expression of NGF, IL‐1β and TNF‐α was also reduced significantly in STR/Ort mice upon macrophage depletion. These findings suggest that IL‐1β and TNF‐α regulate NGF expression and production in synovial macrophages and fibroblasts in osteoarthritic joints.     

Modulation of antitumour activity of macrophages by regulation of eicosanoids and cytokine production

Production and release of arachidonic acid (AA) compounds (eicosanoids: prostaglandins-cycloxygenase and leukotrienes-lipoxygenase) and monokines (TNF-α, IL-1 and others) play an essential role in the expression of antitumour activity of macrophages (MO). We investigated the possibility of inducing the antitumour activity of peritoneal murine and human MO by regulating their production of eicosanoids and monokines. The antitumour activity of MO was inversely correlated to production of PGE₂ and directly correlated to production of leukotrienes (LTC₄ and LTD₄). Thus, indomethacin rendered murine MO cytostatic against tumour cells and enhanced the antitumour activity of human peritoneal macrophages from renal patients on CAPD (continuous ambulatory peritoneal dialysis), and leukotriene inhibitors (NDGA-nordihydroguaiaretic acid and AA861) prevented antitumour cystostatic activity of MO. Human peritoneal MO collected during periods of inflammation (infectious peritonitis) were more active against tumour cells, especially when cultured in the presence of LPS, and their activity was correlated to increase with the release of TNF and of IL-1β. Human peritoneal MO from inflammation-free patients reacted against a human tumour cell line if cultured with LPS and TPA (phorbol-myristate acetate) and were therapeutically effective against the same palpable s.c. tumours implanted in nude mice.     

Pneumolysin potentiates production of prostaglandin E(2) and leukotriene B(4) by human neutrophils

Exposure to pneumolysin (8.37 and 41.75 ng/ml) caused a calcium-dependent increase in the generation of prostaglandin E(2) and leukotriene B(4) by both resting and chemoattractant-activated human neutrophils in vitro. These interactions of pneumolysin with neutrophils may result in dysregulation of inflammatory responses during pneumococcal infection.     

Reactive-oxygen formation and its relationship to prostaglandin and cyclic AMP production by zymosan-treated rat peritoneal macrophages

Addition of zymosan (20 particles/cell) to suspensions of resident rat peritoneal macrophages caused an increase in the concns of prostaglandins and cyclic AMP. Preincubation of the cells with inhibitors of arachidonate metabolism led to inhibition of prostaglandin, but not of cyclic AMP, formation, which suggested that the two processes may occur independently of each other in phagocytosing cells. The luminol-dependent chemiluminescence associated with the addition of zymosan to the cells consisted of a minor, Ca²⁺-dependent, glucose-independent component and a major, glucose-dependent, Ca²⁺-independent component. Only the minor, Ca²⁺-dependent component appeared to be related to the lipoxygenation of arachidonic acid. Close examination of the production of prostaglandins and cyclic AMP and of chemiluminescence after zymosan addition, indicated that the expanded pool of endogenous cyclic AMP was probably not a negative modulator of the other two processes, although they remained susceptible to inhibition by exogenously-added cyclic AMP analogues or PGE₂, The events induced by zymosan may be relevant to the physiological roles of prostaglandins during the inflammatory response.     

Interferon-alpha enhances the production of leukotriene B4 in murine peritoneal macrophages stimulated by opsonized zymosan.

Murine peritoneal macrophages pretreated with interferon (IFN)-alpha and then stimulated by opsonized zymosan produced two to three times more LTB4 than untreated macrophages. However, PGE2 production was not changed by IFN-alpha. Meanwhile, IFN-gamma did not affect the production of LTB4 and PGE2. From the results it is considered that IFN-alpha can modulate inflammation or host defence through the production of LTB4.     

Chemoattractant and leukotriene B4 production from rat alveolar macrophages exposed to nitrogen dioxide

The present study examined the hypothesis that exposure of alveolar macrophages to nitrogen dioxide (NO2) resulted in enhanced production of a lipophilic chemotactic agent for neutrophils, possibly leukotriene B4 (LTB4). Neutrophil migration was significantly increased in response to the reconstituted ethyl acetate extract of the medium surrounding macrophages exposed for 1 h to 5 or 20 ppm NO2. Compared with air-treated macrophages, production of LTB4 was found to be significantly increased by exposure to 5 ppm NO2, but unchanged by exposure to 20 ppm NO2. Treatment of macrophages with the calcium ionophore A23187 at a concentration of 2 microM for 15 min following a 1-h exposure to 5 ppm NO2 led to a significant increase in the production of LTB4 compared with A23187-treated air controls; however, LTB4 production in response to the calcium ionophore was unchanged following exposure to 20 ppm NO2. Thus, while increased neutrophil migration in response to products from macrophages exposed to 5 ppm NO2 correlated with the increased production of LTB4, increased migration in response to products from macrophages exposed to 20 ppm NO2 suggested the presence of another chemotactic lipid. Lipid peroxidation processes induced by NO2 at 5 ppm may lead to the formation of hydroperoxides that enhance the formation of LTB4; yet at 20 ppm, significantly higher concentrations of hydroperoxides may be responsible for impaired LTB4 formation. Phorbol ester-stimulated macrophage superoxide production was significantly inhibited in a dose-dependent manner following exposure to NO2 concentrations of 1, 5, or 20 ppm.     

内毒素对巨噬细胞和内皮细胞前列腺素合成的影响

内毒素刺激小鼠腹腔巨噬细胞和牛主动脉内皮细胞合成前列腺素I2、F2α、E2和血栓素A2。这种刺激作用有剂量和时间依赖性。对于预行色[^3H]花生四烯酸标记的内皮细胞，内毒素增加放射性花生四烯酸的释放和前列腺素I2的合成，多粘菌素B，放线菌酮和地塞米松抑制内素素对前列腺素合成的刺激作用。结果提示内毒素可能通过影响花生四烯酸由细胞脂质的释放过程刺激前列腺素合成。     

Up-regulation of prostaglandin biosynthesis by leukotriene C4 in elicited mice peritoneal macrophages activated with lipopolysaccharide/interferon-{gamma}

Leukotrienes (LT) and prostaglandins (PG) are proinflammatory mediators generated by the conversion of arachidonic acid via 5-lipoxygenase (5-LO) and cyclooxygenase (COX) pathways. It has long been proposed that the inhibition of the 5-LO could enhance the COX pathway leading to an increased PG generation. We have found that in in vitro models of inflammation, such as mice-elicited peritoneal macrophages activated with lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma), the deletion of the gene encoding for 5-LO or the enzyme activity inhibition corresponded to a negative modulation of the COX pathway. Moreover, exogenously added LTC(4), but not LTD(4), LTE(4), and LTB(4), was able to increase PG production in stimulated cells from 5-LO wild-type and knockout mice. LTC(4) was not able to induce COX-2 expression by itself but rather potentiated the action of LPS/IFN-gamma through the extracellular signal-regulated kinase-1/2 activation, as demonstrated by the use of a specific mitogen-activated protein kinase (MAPK) kinase inhibitor. The LT-induced increase in PG generation, as well as MAPK activation, was dependent by a specific ligand-receptor interaction, as demonstrated by the use of a cys-LT1 receptor antagonist, although also a direct action of the antagonist used, on PG generation, cannot be excluded. Thus, the balance between COX and 5-LO metabolites could be of great importance in controlling macrophage functions and consequently, inflammation and tumor promotion.     

Negative regulation by SHPS-1 of Toll-like receptor-dependent proinflammatory cytokine production in macrophages

SHPS-1 is a transmembrane protein predominantly expressed in macrophages. The possible role of SHPS-1 in regulation of Toll-like receptor (TLR)-dependent production of proinflammatory cytokines by macrophages has remained unknown, however. We now show that expression either of a mutant version of mouse SHPS-1 (SHPS-1–4F) in which the four tyrosine phosphorylation sites in the cytoplasmic region are replaced by phenylalanine or of a chimeric protein comprising the extracellular and transmembrane regions of human CD8 fused to the cytoplasmic region of SHPS-1–4F (CD8–4F) markedly promoted the production of tumor necrosis factor-α (TNF-α) or interleukin-6 (IL-6) induced by lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid [poly(I : C)] in RAW264.7 macrophages. In contrast, expression of a mutant form of SHPS-1 that lacks most of the cytoplasmic region did not promote such responses. Expression of SHPS-1–4F promoted the LPS- or poly(I : C)-induced activation of NF-κB. LPS and poly(I : C) each induced the tyrosine phosphorylation of SHPS-1 through a Src family kinase and the association of SHPS-1 with SHP-1 and SHP-2. These results suggest that LPS or poly(I : C) induces tyrosine phosphorylation of SHPS-1 and the association of SHPS-1 with SHP-1 and SHP-2 in a manner dependent on a Src family kinase. SHPS-1 then negatively regulates TLR4- or TLR3-dependent cytokine production through inhibition of NF-κB-dependent signaling.     

Inhibition of prostaglandin and leukotriene generation by the plant alkaloids tetrandrine and berbamine

We compared the effects of two bisbenzylisoquinoline compounds on leukotriene and prostaglandin generation by human monocytes and neutrophils. The results show that tetrandrine had a much greater effect than berbamine on leukotriene generation. However, both compounds were equally potent in suppression of prostaglandin generation. This inhibitory effect on prostaglandin generation can be overcome by exogenous arachidonic acid (AA), suggesting that the site of inhibition is not on the cyclooxygenase enzyme complex, but more proximally on the phospholipase-mediated release of AA from the cell membrane, similar to the action of corticosteroids. These results, together with previous findings of inhibitory effects on other inflammatory mediators such as histamine, platelet-activating-factor (PAF) and interleukin 1 (IL-1) indicate that these plant alkaloids may be useful lead compounds for the development of a new class of anti-inflammatory drugs.