Eicosanoid production by density-defined human peritoneal macrophages during inflammation

Density-defined macrophages isolated from fluids of patients with liver cirrhosis mainly generated the 5-lipoxygenase products leukotriene B₄ (LTB₄, 16%) and 5-hydroxy-eicosatetraenoic acid (5-HETE, 24%) and the cyclooxygenase products 12-hydroxyheptadecatrienoic acid (HHT, 22%) and thromboxane B₂ (TXB₂, 18%). The synthesis of eicosanoids was linear with the maturity of the macrophage subpopulations, suggesting that eicosanoid production is increased inin-vivo activated macrophages.     

Eicosanoid production by peritoneal and splenic macrophages in mice depleted of bone marrow by 89Sr

Previous studies showed that the prostaglandin-forming macrophages (M phi) induced in the spleens of CBA/J mice by intraperitoneal administration of Corynebacterium parvum (CP) could not be demonstrated following the depletion of bone marrow and blood monocytes with 89Sr. The present study compares prostaglandin E2 (PGE2), leukotriene C4 (LTC4), and LTB4 release by splenic and resident peritoneal M phi in 89Sr-treated mice and 88Sr controls following in vivo CP and in vitro incubation with zymosan, calcium ionophore A23187, or phorbol ester (PMA). Intraperitoneal administration of CP resulted in the appearance of PGE2- and LTB4-releasing M phi in the spleens of control but not 89Sr mice. The incorporation and quantitative distribution of 3H-arachidonic acid into membrane lipids, however, were comparable in test and control mice. Neither zymosan nor any of the other stimulatory agents was able to effect significant release of PGE2 in vitro. No release of LTC4 by splenic M phi was detectable under experimental or control conditions. In contrast, the capacity of resident peritoneal M phi to release PGE2, LTC4, and LTB4 was apparently unaffected by 89Sr-induced bone marrow and monocyte depletion with virtually no demonstrable elicitation. Resident peritoneal M phi removed after CP in such mice showed a dramatic decrease in PGE2 release when incubated in vitro with zymosan, A23187, or PMA. These results, taken with earlier findings, demonstrate characteristically different phenotypic expression of metabolism of certain eicosanoids by splenic M phi from the spleen and the peritoneal cavity and suggest in addition that the induction of PGE2-synthesizing M phi in the spleen by CP is dependent on either an immigrant cell originating in the bone marrow or a regulatory agent derived from a bone marrow cell.     

Eicosanoid production and phospholipase A2 secretion by peritoneal macrophages from rats with adjuvant-induced arthritis

The molecular mechanism of action of several non-immunological histamine releasers has been investigated using pertussis toxin which interfers, via ADP-ribosylation, with some G-proteins. Pertussis toxin (100 ng/ml) inhibited histamine release induced by compound 48/80, substance P, mastoparan, peptide 401, bradykinin and spermine showing that a G-protein sensitive to pertussis toxin was involved in the non-immunological histamine release. All these compounds directly activate purified G-proteins. The sensitivity to pertussis toxin of this direct stimulatory effect was demonstrated for compound 48/80, mastoparan and substance P. Altogether these results suggest that a direct activation of G-protein might be the molecular mechanism of action of histamine secretagogues acting through a pertussis toxin sensitive G-protein and in this way mimic agonist-ligand receptor interaction.     

Eicosanoid production by mouse peritoneal macrophages during Toxoplasma gondii penetration: role of parasite and host cell phospholipases.

The metabolism of endogenous arachidonic acid by mouse resident peritoneal macrophages infected in vitro with Toxoplasma gondii was studied. Prelabeling of macrophages with [5,6,8,9,11,12,14,15-3H]arachidonic acid and challenge with tachyzoites for 15 min resulted in a high mobilization of free labeled arachidonic acid (178%) in the culture medium. The parasites also triggered the synthesis of 6-keto-prostaglandin F1 alpha (47%), prostaglandin E2 (44%), leukotrienes C4 and D4 (33%) and 5-, 12-hydroxyeicosatetraenoic acids (155%). The study indicated that during the intracellular development phase of the parasites, 6-keto-prostaglandin F1 alpha (38%), prostaglandin E2 (31%) leukotrienes C4 and D4 (15%), hydroxyeicosatetraenoic acids (43%), and free arachidonic acid (110%) were secreted into the culture medium. Pretreatment of tachyzoites with phospholipase A2 inhibitors (4-p-bromophenacyl bromide and quinacrine) and no calcium in the culture medium resulted in inhibition of tachyzoite penetration into the macrophages and a decrease of the arachidonic acid metabolism. The triggering of the arachidonic acid cascade by T. gondii was dependent on the active penetration of the parasites into the macrophages, whereas preincubation of the macrophages with phospholipase A2 inhibitors did not affect penetration or free arachidonic acid release, thereby supporting a role for parasite phospholipase in the penetration process and in arachidonic acid mobilization from macrophage membrane phospholipids. Moreover, treatment of macrophages with phospholipase A2 inhibitors decreased the activities of the cyclooxygenase and lipoxygenase pathways, also suggesting an activation of host cell phospholipase A2 by the parasite.     

Arginase production by peritoneal macrophages: A new assay

The arginase activity of murine mononuclear cells was assessed with a new highly sensitive radioassay. Adherent mononuclear cells obtained by peritoneal lavage from normal mice contain significant quantities of enzyme. This was released in culture and continued synthesis could be blocked with cycloheximide. Arginase activity was not found in adherent or non-adherent mononuclear cells from the spleen. Peritoneal cells obtained from mice infected with P. berghei or from mice injected with LPS intraperitoneally had higher arginase activity than cells from control mice.     

Tryptophan consumption and indoleamines production by peritoneal cavity macrophages

Melatonin has been shown to regulate several immune functions, and some authors showed that leukocytes are also able to produce the indolamine. In fact, it seems to take part in some immunoregulatory axis, including that related to interferon (IFN) production. So, we evaluated the rate of tryptophan consumption and melatonin and serotonin production in peritoneal cavity-isolated macrophages and the effect of IFN-alpha and -gamma, lipopolysaccharide (LPS), and phorbol myristate acetate (PMA) on such parameters. Our results indicate that macrophages obtained from the peritoneal cavity of normal rats when incubated with tryptophan show an increase in arylalkylamine N-acetyltransferase activity that corresponds to an increased melatonin production, as determined in the incubation medium. This process is regulated by IFN-alpha and -gamma, PMA, LPS, and the serum from tumor-bearing rats, opening the possibility of speculation about different immunoregulatory loops acting through the balance of melatonin/serotonin production by such cells.     

Hyaluronan and myeloperoxidase in human peritoneal fluid during genital inflammation

The changes in concentration of hyaluronan (HYA) and myeloperoxidase in peritoneal fluid (PF) were studied during genital intraperitoneal inflammation. PF were collected from 111 women undergoing laparatomy for adhesiolysis and reconstructive surgery of the fallopian tubes, or laparoscopy in search of causes of infertility or low abdominal pain. When the number of leukocytes in the PF had been counted, the fluid samples were centrifuged and the supernatants analyzed for the concentrations of HYA and of myeloperoxidase. During genital inflammation, whether postoperative or postinfectious, leukocytosis and elevated levels of HYA and myeloperoxidase were found in the PF. Concentrations of these substances in the PF may be usable as clinical markers for genital inflammation.Supported by grants from the Swedish Medical Research Council (03X-4, 17X-3495, 03X-4954) and from Förenade Liv Mutual Group Life Insurance Company, Stockholm, Sweden.     

Hyaluronan and myeloperoxidase in human peritoneal fluid during genital inflammation

The changes in concentration of hyaluronan (HYA) and myeloperoxidase in the peritoneal fluid (PF) were studied during genital intraperitoneal inflammation. PF were collected from 111 women undergoing laparotomy for adhesiolysis and reconstructive surgery of the fallopian tubes or laparoscopy in search of causes of infertility or low abdominal pain. When the number of leukocytes in the PF had been counted, the fluid samples were centrifuged and the supernatants analyzed for the concentrations of HYA and myeloperoxidase. During genital inflammation, whether postoperative or postinfectious, leukocytosis and elevated levels of HYA and myeloperoxidase were found in the PF. Concentrations of these substances in the PF may be usable as clinical markers for genital inflammation.Supported by grants from the Swedish Medical Research Council (03X-4, 17X-3495, 03X-4954) and from Förenade Liv Mutual Group Life Insurance Company, Stockholm, Sweden.     

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.     

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.     

The production of ceroid by mouse peritoneal macrophages in vitro.

Murine resident peritoneal macrophages accumulated lipid droplets and subsequently insoluble, ceroid-like material when cultured in vitro in a medium containing 33% fetal calf serum. At least some of this insoluble lipid was membrane-bound and by light microscopy it often appeared as ''rings'' with a hollow centre. It is suggested that the production of ceroid may be the consequence of the uptake of lipids from the extracellular medium and the activity of the macrophage''s membrane-bound oxidative microbicidal mechanisms. The results indicate that macrophages are capable of rendering lipids insoluble, supporting the suggestion that this might occur in the atherosclerotic plaque.     

Interleukin-4 production by human alveolar macrophages

BACKGROUND: IL-4 is a key factor for T helper type 2 (Th2) differentiation and Ig class switching to IgE and IgG(4) during the development of immune responses. IL-4 is produced by T cells, mast cells, basophils, and eosinophils. However, there is also evidence suggesting that rat alveolar macrophages (AMs) produce IL-4. OBJECTIVE: Given the importance of AMs and Th2-related diseases in the lung, we investigated the production of IL-4 by human AMs. METHODS: Human AMs were isolated from bronchoalveolar lavage, purified, and IL-4 production was investigated at mRNA and protein levels using real-time PCR, flow cytometry, immunocytochemistry, and ELISA. The presence of IL-4 was investigated in subjects with asthma or asymptomatic airway hyper-responsiveness, and in normal non-smokers. RESULTS: IL-4 and IL-4delta2 (a splice variant found in other IL-4 producing cells) mRNAs were found in all these subjects, but IL-4 expression could not be correlated with a particular disease. Protein production was verified by immunocytochemistry and flow cytometry analysis demonstrating, respectively, up to 69% and 59% positive AMs, regardless of the subject condition. Furthermore, phorbol-12-myristate-13-acetate and calcium ionophore stimulated the release of IL-4 after 48 h treatment in the presence of anti-IL-4 receptor antibody. CONCLUSION: Our results show for the first time that IL-4 and IL-4delta2 mRNA are expressed and IL-4 protein produced and released by human AMs, suggesting a contribution of these cells in the modulation of Th2 immune response.     

Down-regulation of cyclooxygenase product generation by human peritoneal macrophages.

Isolated human peritoneal macrophages under resting conditions synthesize and release significant quantities of the cyclooxygenase products prostaglandin E2 (PGE2) and thromboxane B2 (TXB2). These increased linearly with time and were dependent on de novo protein synthesis. Following the addition of calcium ionophore A23187 there was a marked decrease in total generation of immunoreactive cyclooxygenase products. In contrast, there was a concomitant increase in the release of 5-lipoxygenase products. The down-regulation of both PGE2 and TXB2 was not due to diversion of substrate from the cyclooxygenase pathway to the 5-lipoxygenase pathway nor was it due to inhibitory effects of products of the 5-lipoxygenase pathway on the generation of cyclooxygenase products. Instead, the inhibitory effects of A23187 were thought to be due to a down-regulation of cyclooxygenase itself, a hypothesis supported by the finding that TXA2 synthase proved to be unaltered and Western analysis of crude peritoneal macrophage (PM phi) membranes demonstrated lesser quantities of cyclooxygenase in membranes from A23187-treated PM phi than in those prepared from control cells.     

IL-4 suppresses IL-1 beta, TNF-alpha and PGE2 production by human peritoneal macrophages.

Human interleukin-4 (IL-4) down-regulates IL-1 and tumour necrosis factor-alpha (TNF-alpha) production by monocytes stimulated in vitro. In contrast, in studies of activation of murine macrophages, both stimulatory and inhibitory functions of murine IL-4 have been documented. To investigate whether opposing activities of IL-4 reflect a difference in the target cell studied, due either to cell maturation or the site from which the cells were isolated, we examined the effect of IL-4 on human peritoneal macrophage production of IL-1 beta, TNF-alpha and prostaglandin E2 (PGE2). Human peritoneal macrophages stimulated with lipopolysaccharide (LPS) produced levels of these mediators that were at least as great as those previously reported for monocytes. Similarly, IL-4 was inhibitory for peritoneal macrophage mediator production after in vitro stimulation. Thus, IL-4 has effects on human peritoneal macrophages similar to those on blood monocytes. In addition, as it down-regulates mediator production by cells that have left the circulation, it may be important in controlling the immune response in tissues.     

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.     

Enhanced production of interleukin 1 by mouse peritoneal macrophages after aclacinomycin administration

The peritoneal cells of mice injected with aclacinomycin (ACM), an oncostatic drug of the anthracyclin family, were found to secrete more interleukin (IL-1), after two successive 24-h periods of in vitro LPS stimulation than those of control mice. This measured IL-1 production is one of the signs of enhanced macrophage activity. The cells of ACM-injected mice also contained more intracellular IL-1 than those of controls. In contrast, macrophages from ACM-injected mice only increased their IL-1 production after the first 24-h incubation with PMA, and not after the second 24-h incubation. The response to ACM was dose- and time-dependent. We have also compared the IL-1 production by macrophages from mice injected with other anthracyclins, at doses equimolar to that of 4 mg/kg ACM and we have observed that adriamycin, 4'-epiadriamycin and aclacinomycin had similar activity, while THP-adriamycin an daunorubicine were slightly more active. Exploitation of this increased IL-1 production by macrophages could be beneficial in the design of tumor treatment protocols.     

Nitric oxide production by human peritoneal mesothelial cells

Nitric oxide (NO) has multiple actions, ranging from immunomodulation to regulation of vascular tone and capillary flow. Thus NO generation within the peritoneum could potentially affect peritoneal transport by increasing capillary vasodilatation, and regulate the response to bacterial invasion. Peritoneal mesothelial cells have a common embryological derivation with endothelial cells. As mesothelial cells are the predominant cell type lining the peritoneal cavity, they could potentially be a major source of locally produced nitric oxide. Nitric oxide was measured using the Griess reaction, as total nitrite and nitrate, in fresh unused and spent dialysate effluent (SPDE) from both healthy peritoneal dialysis patients, and during episodes of bacterial peritonitis. Whereas fresh CAPD dialysate was nitrite free (5 +/- 0.1 microM), SPDE from a standard 4 h day time exchange contained 10.2 +/- 0.6 microM/L/h, and that from the overnight dwell 9.1 +/- 0.7 microM/L/h. During an episode of peritonitis, dialysate nitrite and nitrate increased significantly from 9.0 +/- 1.0 microM/L/h, when not infected to 17.5 +/- 2.4, from the first CAPD bag drained at presentation, and 15.2 +/- 1.8 for the second and 16.0 +/- 2.5 for the third exchange (p<0.01). By the following day nitrite levels had returned to baseline, 7.0 +/- 1.0 microM/L/h. Human peritoneal mesothelial cells (HPMC) were cultured and found to produce nitric oxide (261 nmol/mg cell protein), which increased in a dose dependent manner with the addition of spent uninfected CAPD dialysate. The addition of L-arginine, a NO substrate resulted in a 10% increase in nitric oxide production, whereas the addition of the blocker L-NMMA produced a 10% reduction. RNA for inducible nitric oxide synthase (iNOS) was sought using northern blotting technique following combination stimulation with lipopolysaccharide and cytokines (IL-1beta, TNFalpha and gamma-INF, and/or spent dialysate from patients with bacterial peritonitis). However, we could not demonstrate RNA production for iNOS. Peritoneal mesothelial cells may be an important source of locally generated nitric oxide within the peritoneal cavity under basal conditions, but as they do not contain iNOS, the markedly increased NO production observed with episodes of acute bacterial peritonitis is more likely due to a combination of increased NO production by peritoneal macrophages and endothelial cells.     

GTP-related difference in cyclic AMP production between resident and inflammatory human peritoneal macrophages.

In macrophages cyclic AMP (c-AMP) plays an important role in regulating many activities such as phagocytosis, migration and tumoricidal activity. High intracellular levels of c-AMP are negatively correlated with these activities. In earlier studies we have shown that c-AMP levels in inflammatory human peritoneal macrophages (IM) were markedly lower when compared to levels in resident macrophages (RM). This is in line with the fact that c-AMP down-regulates macrophage activity. To our knowledge no data are available on the mechanism underlying the difference in c-AMP production between RM and IM. In this study the difference in c-AMP production between RM and IM has been investigated on the level of receptor and G-protein-related mechanisms. Macrophage membranes were incubated with different agents i.e. prostaglandin E2 (PGE2), prostacyclin I2 (PGI2), isoprenalin (ISO) and sodium fluoride (SF). Additionally, the capacity of IM and RM to hydrolyse quanosine triphosphate (GTP) was measured. Only in the presence of GTP (10(-4) M) could the c-AMP difference be detected (RM = 51 +/- 4.4 pmol/mg protein/min +/- S.E., n = 22, IM = 32.8 +/- 5.2 pmol/mg protein/min +/- S.E., n = 10, p less than 0.01). After receptor stimulation with PGE2, PGI2 and ISO, c-AMP levels increased to the same extent in both IM and RM with no effect on the GTP-related difference. After SF stimulation, c-AMP levels in RM and IM increased to the same level (RM = 63 +/- 8 pmol/mg protein/min, n = 14, IM = 58 +/- 11 pmol/mg protein/min, n = 13).(ABSTRACT TRUNCATED AT 250 WORDS)     

Resident peritoneal macrophages and mast cells are important cellular sites of COX-1 and COX-2 activity during acute peritoneal inflammation

Introduction  

Cyclooxygenases (COXs) play important roles during inflammation. While reports on COX-2 function in inflammation preceded those on COX-1, it is now well established that both isoforms participate in this process. During inflammation, COX expression was reported in inflammatory leukocytes, but much less is known about their presence in tissue- resident leukocytes. The aim was thus to verify the expression and activity of the COX isoforms in resident peritoneal mast cells and macrophages during acute peritonitis.