Peritoneal mast cell degranulation differently affected thioglycollate-induced macrophage phenotype and activity in Dark Agouti and Albino Oxford rats

Aims

Macrophages are heterogeneous population of inflammatory cells and, in response to the microenvironment, become differentially activated. The objective of the study was to explore macrophage effector functions during different inflammatory conditions in two rat strains.

Main methods

We have investigated the effects of in vivo treatment with mast cell-degranulating compound 48/80 and/or thioglycollate on peritoneal macrophage phagocytosis and capacity to secrete hydrogen peroxide (H₂O₂), tumor necrosis factor-α (TNF-α) and nitric oxide (NO) in Dark Agouti (DA) and Albino Oxford (AO) rat strains. Besides, fresh peritoneal cells were examined for the expression of ED1, ED2 and CD86 molecules.

Key findings

In thioglycollate-elicited macrophages, increased proportion of ED1 + cells was accompanied with elevated phagocytosis of zymosan (DA strain), whereas increased expression level of CD86 molecule on ED2 + macrophages matched elevated secretory capacity for H₂O₂, TNF-α and NO (AO rats). Although mast cell degranulation induced by compound 48/80 increased the percentages of ED2 + macrophages in both rat strains, the proportion of ED2 + cells expressing CD86 molecule was decreased and increased in DA and AO rats, respectively. Furthermore, in DA strain compound 48/80 diminished macrophage secretion of NO, but stimulated all macrophage functions tested in AO strain. If applied concomitantly, the compound 48/80 additively increased macrophage activity induced by thioglycollate in AO rats.

Significance

Macrophages from DA and AO rat strains show different susceptibility to mediators released from mast cells, suggesting that strain-dependant predisposition(s) toward particular activation pattern is decisive for the macrophage efficacy in response to inflammatory agents.     

Identification and macrophage-activating activity of glycolipids released from intracellular Mycobacterium bovis BCG

Intracellular mycobacteria release cell wall glycolipids into the endosomal network of infected macrophages. Here, we characterize the glycolipids of Mycobacterium bovis BCG (BCG) that are released into murine bone marrow‐derived macrophages (BMMØ). Intracellularly released mycobacterial lipids were harvested from BMMØ that had been infected with ¹⁴C‐labelled BCG. Released BCG lipids were resolved by thin‐layer chromatography, and they migrated similarly to phosphatidylinositol dimannosides (PIM₂), mono‐ and diphosphatidylglycerol, phosphatidylethanolamine, trehalose mono‐ and dimycolates and the phenolic glycolipid, mycoside B. Culture‐derived BCG lipids that co‐migrated with the intracellularly released lipids were purified and identified by electrospray ionization mass spectrometry. When delivered on polystyrene microspheres, fluorescently tagged BCG lipids were also released into the BMMØ, in a manner similar to release from viable or heat‐killed BCG bacilli. To determine whether the released lipids elicited macrophage responses, BCG lipid‐coated microspheres were delivered to interferon gamma‐primed macrophages (BMMØ or thioglycollate‐elicited peritoneal macrophages), and reactive nitrogen intermediates as well as tumour necrosis factor‐alpha and monocyte chemoattractant protein‐1 production were induced. When fractionated BCG lipids were delivered on the microspheres, PIM2 species reproduced the macrophage‐activating activity of total BCG lipids. These results demonstrate that intracellular mycobacteria release a heterogeneous mix of lipids, some of which elicit the production of proinflammatory cytokines from macrophages that could potentially contribute to the granulomatous response in tuberculous diseases.     

Inhibition of Nitric Oxide Production by Macrophages in Chromoblastomycosis: A Role for Fonsecaea pedrosoi Melanin

Chromoblastomycosis is a chronic and progressive deep mycosis that is usually found in tropical and subtropical areas. Fonsecaea pedrosoi is considered its most frequent etiologic agent and causes a typical granulomatous inflammatory response, whose degree reflects the immune status of the host. Since macrophages play a fundamental role in the control of the infection, this study aimed at investigating the production of oxygen reactive specimens, the phagocytic capacity and the production of nitric oxide (NO) by macrophages employing in vitro assays and an in vivo model of chromoblastomycosis. Our results demonstrated that, during the infection, peritoneal macrophages show an increased phagocytic capacity and H₂O₂ production, but also a reduced ability to produce NO. Moreover, F. pedrosoi stimulated H₂O₂ production in vitro but not the synthesis of NO. The incubation of IFNγ and LPS-stimulated macrophages with melanin, obtained from the fungus, inhibited NO production. Examination of the liver and spleen of infected animals, at day 30 or 60 following inoculation, showed a progressive increase in the number and size of granulomas, indicating that macrophages are properly mobilized and activated. Our data suggest that the inability of the host to clear F. pedrosoi, leading to a chronic disease, is due, at least in part, to the inhibition of NO synthesis by macrophages by fungus-produced melanin.     

Peptides from Paracoccidioides brasiliensis GP43 inhibit macrophage functions and inflammatory response

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by Paracoccidioides brasiliensis (Pb), a thermal dimorphic fungus. Its major antigen is a 43-kDa glycoprotein. Gp43 embodies different functions: it participates in evasion mechanisms during the installation of primary infection, stimulates granuloma-like formation in vitro and presents T-cell epitopes that induce protective response against the fungus. Here, we investigated epitopes from gp43 inhibitory of both, macrophage functions and inflammatory reaction. Different gp43 peptides, spanning the entire sequence of the molecule, were added to cultures of bone marrow-derived macrophages. After challenge with zymosan or Pb cells, phagocytic indexes were measured. Peptides expressed on the molecule surface were determined by graphic analysis using the Protean module; DNAstar Inc. Two peptides which decreased phagocytic index and were expressed at the surface of the molecule, P4 and P23, were selected for further studies. It was shown that both inhibited the release of NO by zymosan stimulated macrophages while enhanced release of H₂O₂. The release of TNF-α in culture supernatants from in vitro phagocytic tests showed different response depending of P4 concentration (data not shown). In vivo assays with Mycobacterium bovis – bacillus Calmette–Guérin (BCG) or Pb cells demonstrated that these peptides presented non-specific and specific anti-inflammatory properties.     

Production of arachidonic acid metabolites by operationally defined macrophage subsets

The antitumor activity and arachidonic acid metabolism of operationally defined macrophage populations was examined. Macrophages from mice injected with (strain BCG) or with pyran-copolymer were cytotoxic for tumor cells. The major arachidonic acid metabolite of these cells was PGE₂. Neither resident nor elicited macrophages were cytotoxic. However, elicited macrophages as well as macrophages from BCG injected mice inhibited tumor cell growth. The production of arachidonic acid metabolites by elicited cells, while low initially, was followed by a rapid increase in PGE₂. The major metabolites of resident cells were PGE₂ and prostacyclin. The cAMP:cGMP ratio correlated with the metabolic activity of the cells.     

The macrophage adherence phenomenon: Its relationship to prostaglandin E2 and superoxide anion production and changes in transmembrane potential

Mononuclear phagocytes are undoubtedly the sine qua non of chronic inflammatory reactions. This is demonstrated by their unique ability to function as phagocytic, secretory, or effector cells during the course of an immune event. Although macrophages can perform a variety of immune tasks, their ability to function appropriately is dependent upon the mode of elicitation, the stimulus under investigation, the source of the macrophages (peritoneal, alveolar, etc.), and whether the macrophages are monolayers or in suspension. We have examined the relationship between adherent and non-adherent elicited peritoneal macrophages in terms of prostaglandin E₂ (PGE₂) and superoxide anion (O₂) production; in addition, we hae studied these elicited macrophages in suspension for their ability to undergo transmembrane potential changes in response to several stimuli. Non-adherent, elicited peritoneal macrophages demonstrated an increase in basal PGE₂ production, and were refractory to particulate stimulus. After monolayer formation, basal PGE₂ levels dropped and the cell could respond to both soluble and particulate stimuli. Only adherent macrophages could respond to a specific challenge and synthesize O₂. Both O₂ production and depolarization of the transmembrane potential were suppressed in cell in suspension. Furthermore, both exogenous PGE₂ and supernatant from macrophages in suspension could modulate O₂ production by PMA challenged macrophages monolayers. These studies indicate that PGE₂ may modulate macrophage function and dictate activity as macrophages go from the non-adherent to adherent state.     

A continuous alveolar macrophage cell line: Comparisons with freshly derived alveolar macrophages

Summary Responses of a recently developed rat alveolar macrophage cell (NR8383.1) line were compared to those of freshly derived alveolar macrophages in vitro. Marked inter- and intraspecies heterogeneity in levels of phagocytosis of unopsonizedPseudomonas aeruginosa or zymosan was noted among freshly derived alveolar macrophages from rats, rabbits, and baboons. In contrast, phagocytic responses of alveolar macrophage cell line were predictable and highly reproducible. Similar results were obtained in measuring oxidative burst, as indicated by the production of H₂O₂ and luminol-enhanced chemiluminescence. Responses were again highly variable in freshly derived alveolar macrophages stimulated with zymosan or phorbol myristic acetate; moreover, freshly derived alveolar macrophages exhibited a wide range of chemiluminescence activity in unstimulated cultures. Results strongly suggest that data derived from the continuous alveolar macrophage culture NR8383.1 can be extrapolated to freshly derived alveolar macrophages of various species, and in many experiments will be useful in avoiding the significant animal-to-animal variance observed among freshly derived cell preparations. This work was supported in part by grant A119811 and SCOR HL23578, from the National Institutes of Health, Bethesda, MD. Portions of these studies appeared as a poster presentation at the American Society for Microbiology, Atlanta, GA, 1987.     

Redox imbalance induces remodeling of glucose metabolism in Rhipicephalus microplus embryonic cell line

Carbohydrate metabolism not only functions in supplying cellular energy but also has an important role in maintaining physiological homeostasis and in preventing oxidative damage caused by reactive oxygen species. Previously, we showed that arthropod embryonic cell lines have high tolerance to H₂O₂ exposure. Here, we describe that Rhipicephalus microplus tick embryonic cell line (BME26) employs an adaptive glucose metabolism mechanism that confers tolerance to hydrogen peroxide at concentrations too high for other organisms. This adaptive mechanism sustained by glucose metabolism remodeling promotes cell survival and redox balance in BME26 cell line after millimolar H₂O₂ exposure. The present work shows that this tick cell line could tolerate high H₂O₂ concentrations by initiating a carbohydrate-related adaptive response. We demonstrate that gluconeogenesis was induced as a compensation strategy that involved, among other molecules, the metabolic enzymes NADP-ICDH, G6PDH, and PEPCK. We also found that this phenomenon was coupled to glycogen accumulation and glucose uptake, supporting the pentose phosphate pathway to sustain NADPH production and leading to cell survival and proliferation. Our findings suggest that the described response is not atypical, being also observed in cancer cells, which highlights the importance of this model to all proliferative cells. We propose that these results will be useful in generating basic biological information to support the development of new strategies for disease treatment and parasite control.     

Macrophage Activation by Tetrahymena pyriformis. I. Active Subcellular Fractions of Tetrahymena

Experiments were carried out to determine what subcellular fractions of Tetrahymena pyriformis could, after inoculation into mice, activate macrophages to kill Toxoplasma gondii in vitro. Peritoneal macrophages from mice inoculated intraperitoneally with cilia, pellicles, mitochondria, and microsomes exhibited strong toxoplasmacidal activity and had an enhanced capacity to release hydrogen peroxide (H₂O₂) by stimulation of a membrane-active agent as compared with resident macrophages. In contrast, macrophages from mice inoculated with macronuclei and postmicrosomal supernatant showed no toxoplasmacidal activity and a low level of H₂O₂ release. Similar dose response was observed on the active subcellular fractions with regard to the degree of macrophage activation. Treatment of the active subcellular fractions with heating and trypsin markedly reduced their activity.     

Effect of adrenaline on lymphocyte metabolism and function. A mechanism involving cAMP and hydrogen peroxide

This study examined the effect of adrenaline on lymphocyte metabolism and function. The following parameters were addressed: cell proliferation, glucose and glutamine metabolism as indicated by the measurement of enzyme activities, the utilization of metabolites and production and oxidation of substrates. We also evaluated the involvement of beta-receptors in this response as well as the possible effect of cAMP and hydrogen peroxide in the process of lymphocyte activation by adrenaline. The results indicated that adrenaline is able to induce metabolic changes in lymphocytes that are related to enhanced proliferative capacity, but under physiological conditions fails to initiate the process, the catecholamine could, increase cell proliferation via increased production of H₂O₂ by macrophages, since this reactive oxygen intermediate can act as a trigger for lymphocyte activation. The results also showed that distinct populations of lymphocytes present different responses to adrenaline activation, as demonstrated by cells obtained from the same site but exposed to different mitogens such as LPS and ConA. © 1997 John Wiley & Sons, Ltd.     

Effect of hypo- and hyperthyroidism on the function and metabolism of macrophages in rats

The effect of thyroid hormones on monocyte migration, phagocytic capacity and hydrogen peroxide production by macrophages and the effect of these hormones on glutamine and glucose metabolism was investigated. The experiments were performed on resident, thioglycollate- and BCG-stimulated cells from hypo- and hyperthyroid rats. High plasma levels of thyroid hormones suppressed the migration of monocytes and hydrogen peroxide production, whereas hypothyuroidism did not affect cell migration but rasied the phagocytic capacity and the hydrogen peroxide production. Hyperthyroidism increased the activities of glutaminase and hexokinase and the rates of decarboxylation of [U-¹⁴C]-glutamine and [U-¹⁴C]-glucose in inflammatory and activated cells. Hypothyroidism stimulated glucose metabolism and had only a slight effect on glutaminolysis. The activity of the TCA cycle was, however, diminished in the presence of high plasma levels of thyroid hormones and enhanced by the hypothyroid state. These findings suggest that the functional changes observed are more likely to be related to the activity of the TCA cycle rather than to glutaminolysis and glycolysis.     

Nitroarachidonic acid prevents NADPH oxidase assembly and superoxide radical production in activated macrophages

Nitration of arachidonic acid (AA) to nitroarachidonic acid (AANO₂) leads to anti-inflammatory intracellular activities during macrophage activation. However, less is known about the capacity of AANO₂ to regulate the production of reactive oxygen species under proinflammatory conditions. One of the immediate responses upon macrophage activation involves the production of superoxide radical (O₂^•−) due to the NADPH-dependent univalent reduction of oxygen to O₂^•− by the phagocytic NADPH oxidase isoform (NOX2), the activity of NOX2 being the main source of O₂^•− in monocytes/macrophages. Because the NOX2 and AA pathways are connected, we propose that AANO₂ can modulate macrophage activation by inhibiting O₂^•− formation by NOX2. When macrophages were activated in the presence of AANO₂, a significant inhibition of NOX2 activity was observed as evaluated by cytochrome c reduction, luminol chemiluminescence, Amplex red fluorescence, and flow cytometry; this process also occurs under physiological mimic conditions within the phagosomes. AANO₂ decreased O₂^•− production in a dose- (IC₅₀=4.1±1.8 μM AANO₂) and time-dependent manner. The observed inhibition was not due to a decreased phosphorylation of the cytosolic subunits (e.g., p40^phox and p47^phox), as analyzed by immunoprecipitation and Western blot. However, a reduction in the migration to the membrane of p47^phox was obtained, suggesting that the protective actions involve the prevention of the correct assembly of the active enzyme in the membrane. Finally, the observed in vitro effects were confirmed in an in vivo inflammatory model, in which subcutaneous injection of AANO₂ was able to decrease NOX2 activity in macrophages from thioglycolate-treated mice.     

Hydrogen Peroxide-Induced Inhibition of Vasomotor Activity: Evaluation of Single and Combined Treatments With Vitamin A and Insulin in Streptozotocin-Diabetic Rats

A positive correlation has been established between increased oxidative stress and cardiovascular diseases in diabetes mellitus. We evaluated the effects of single or combined treatments with vitamin A (retinol acetate, 30 mg/kg/day, for 12-weeks) and insulin (8-10 IU/rat/day for the final 6-week) on vasomotor activity, oxidative stress and retinol metabolism in 12-week streptozotocin diabetic rats. The vasomotor activity was determined by measuring in vitro responsiveness of aorta rings to phenylephrine (PE) and acetylcholine (ACh) in the absence or in the presence of hydrogen peroxide (H₂O₂). Preincubation with H₂O₂ (10 μM) produced a significant decrease in PE (1 mM)-induced contraction in untreated-diabetic but not in control rats. Single treatment with insulin counteracted this effect of H₂O₂ and also reversed the increased contractile response of diabetic aorta to PE, while vitamin A was found to be ineffective. H₂O₂ (10 μM) also inhibited ACh (1 mM)-stimulated endothelium- dependent relaxation two fold more in diabetic than in control aorta. In the prevention of H₂O₂-induced inhibition of vascular relaxation to ACh, vitamin A alone was markedly effective while insulin alone was not. The combination of vitamin A plus insulin removed the inhibitory action of H₂O₂ in diabetic aorta. Diabetic animals displayed an increased level of aorta thiobarbituric acid reactive substance (TBARS) in association with decreased levels of plasma retinol and retinol-binding protein (RBP). Single treatment with insulin, in spite of allowing recovery of normal growth rate and improved glucose and retinol metabolism in diabetic rats, was unable to control TBARS production to the same extent as vitamin A alone. Our findings suggest that the maintenance of ACh-stimulated endothelium-dependent vasorelaxant tone in normal physiological levels depends largely on the prevention and/or inhibition of peroxidative stress, which is achieved by combined treatment with vitamin A plus insulin. The use of vitamin A together with insulin provides a better metabolic control and more benefits than use of insulin alone in the reduction of diabetes-induced vascular complications.     

Insulin increases H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced pancreatic beta cell death

Insulin resistance results, in part, from impaired insulin signaling in insulin target tissues. Consequently, increased levels of insulin are necessary to control plasma glucose levels. The effects of elevated insulin levels on pancreatic beta (β) cell function, however, are unclear. In this study, we investigated the possibility that insulin may influence survival of pancreatic β cells. Studies were conducted on RINm, RINm5F and Min-6 pancreatic β-cells. Cell death was induced by treatment with H₂O₂, and was estimated by measurements of LDH levels, viability assay (Cell-Titer Blue), propidium iodide staining and FACS analysis, and mitochondrial membrane potential (JC-1). In addition, levels of cleaved caspase-3 and caspase activity were determined. Treatment with H₂O₂ increased cell death; this effect was increased by simultaneous treatment of cells with insulin. Insulin treatment alone caused a slight increase in cell death. Inhibition of caspase-3 reduced the effect of insulin to increase H₂O₂-induced cell death. Insulin increased ROS production by pancreatic β cells and increased the effect of H₂O₂. These effects were increased by inhibition of IR signaling, indicative of an effect independent of the IR cascade. We conclude that elevated levels of insulin may act to exacerbate cell death induced by H₂O₂ and, perhaps, other inducers of apoptosis.     

Functional transitions in macrophages during in vivo infection with Mycobacterium bovis bacillus Calmette-Guérin

Macrophage activation during the immune response to intracellular bacteria is critical for resolution of the infection. We have investigated the pathway of macrophage activation during murine Mycobacterium bovis bacillus Calmette-Guérin (BCG) infection. Three distinct phenotypes of macrophages were identified and compared: resident peritoneal macrophages, day 2 postinfection macrophages, and 12-day postinfection macrophages. Compared with resident peritoneal macrophages, day 2 BCG macrophages expressed intermediate levels of the cell surface receptors Mac1 and F4/80 and low levels of MHC class II molecules. These cells were highly phagocytic and produced large amounts of mRNA encoding the chemokine IP-10. In addition, day 2 BCG macrophages did not generate reactive nitrogen intermediates, though they were primed to do so, and did not have increased levels of TNF-alpha mRNA. Blockade of monocyte influx into the peritoneal cavity using Abs to platelet endothelial cell adhesion molecule 1 had no effect on the appearance of day 2 BCG macrophages, suggesting this cell can differentiate from resident peritoneal macrophages. In contrast to day 2 BCG macrophages, day 12 BCG macrophages were poorly phagocytic, but produced high levels of reactive nitrogen intermediates, IP-10 and TNF-alpha mRNA, and class II MHC molecules. We propose that day 2 BCG macrophages are specialized for phagocytic uptake of pathogens from the extracellular space, whereas day 12 BCG macrophages are specialized for killing of the internalized pathogens. This functional transition during activation is reminiscent of that seen during maturation/activation of the related dendritic cell lineage induced by bacterial or inflammatory stimuli.     

Comparative biochemical and cytochemical studies on superoxide and peroxide in mouse macrophages

Maximal rates of superoxide (O) release, and the cytochemical locales of peroxide staining in resident, elicited, and activated macrophages have been determined. Macrophages elicited into the peritoneum with either casein (1.2% w/v) or proteose-peptone (10.0% w/v) release about twice as much O as macrophages activated by infection of the animals with either Listeria monocytogenes, or Bacille Calmette-Guerin (BCG) followed by immune boosting with Purified Protein Derivative (PPD) (i.e., about 35 vs. 14–18 nmol O/min/10⁷ cells). Macrophages elicited with thioglycollate (3.0% w/v) and resident macrophages produce negligible amounts of O upon stimulation with PMA. These data are compared with those reported by other investigators who used different procedures. A cytochemical procedure for localizing peroxide has been modified for use with murine macrophages. No production of H₂O₂ by macrophages is detected cytochemically in the absence of stimulation. Upon exposure to PMA, resident macrophages are still largely unresponsive. Approximately 20% of the casein elicited macrophages and BCG-PPD activated macrophages exhibit H₂O₂ staining, which is largely restricted to the cytoplasmic vesicles and channels induced by PMA in these cells. The only exception to this staining pattern is a small population (about 2%) of activated macrophages which exhibits H₂O₂ staining in the cytoplasmic vesicles and channels and on the plasmalemma as well.     

Functional tuftsin binding sites on macrophage-like tumor line P388DI and on bone marrow cells differentiated in vitro into mononuclear phagocytes

Summary Mouse bone marrow cells, differentiated in vitro into mononuclear phagocytes (BMDMP), possess functional tuftsin binding sites, i.e. both tuftsin binding capacity and augmented phagocytic response related to tuftsin binding. The binding capacity of BMDMP was shown to be higher by a factor of about three than that exhibited by mouse monocytes and by normal, thioglycollate and Corynebacterium parvum induced peritoneal macrophages. The relatively high binding capacity did not depend on experimental variations in the in vitro culture of these populations (i.e. length of in vitro cultivation, source of serum or presence of conditioned medium leading to cell proliferation).The macrophage-like line, P388D1, was also shown to possess functional tuftsin binding sites and its binding capacity was comparable to that of the peritoneal macrophage populations.     

Protease inhibitors antagonize the activation of polymorphonuclear leukocyte oxygen consumption.

We report that the burst of oxygen consumption, as well as the resultant production of O₂^?? and H₂O₂, occurring in activated human polymorphonuclear leukocytes is inhibited by various compounds which have in common the ability to antagonize the effects of proteolytic enzymes. This effect of protease inhibitors was observed with a variety of stimuli, both phagocytic and non-phagocytic, used to activate O₂^?? production in human polymorphonuclear leukocytes. Inhibition was also noted in rat polymorphonuclear leukocytes and alveolar macrophages. The results indicate that proteolysis may be involved in activating the burst of oxygen consumption following stimulation of phagocytic cells.