Orchestration of metabolism by macrophages

Metabolic adaptation is a key component of macrophage plasticity and polarization, instrumental to their function in homeostasis, immunity, and inflammation. Macrophage products also impact metabolism, as illustrated by obesity-associated pathologies. Defining the mechanisms regulating macrophage metabolic activity and orchestration of metabolism by macrophages is crucial to pathology and therapeutic intervention.     

Activation of macrophages by peroxidases

Peritoneal macrophages from C57BL/6 mice were activated in vitro with various peroxidases and their cytotoxic activity toward 3T12 cells was determined. Destruction of 3T12 cells by macrophages stimulated with horseradish peroxidase, lactoperoxidase, and microperoxidase was observed at peroxidase concentrations as low as 9, 1.6, and 200 nM, respectively. A 50% cytotoxic effect was obtained at peroxidase concentrations of 0.9, 1.6, and 1.5 microM, respectively. The macrophage-stimulating activity of horseradish peroxidase was not destroyed by boiling. This, together with the high activity of microperoxidase, indicates that the macrophage-stimulating activity of the peroxidases is probably associated with the heme portion of the enzymes. On a molar basis the peroxidases are much less potent macrophage activators than interferon (alpha + beta) and endotoxin. Nevertheless, our data clearly indicate that peroxidases are a group of enzymes capable of inducing macrophage activation, resulting in cytostatic and/or cytocidal activity.     

Leukotriene C release by macrophages

Leukotriene C (LTC) and its metabolites leukotriene D and leukotriene E collectively make up the biological activity known as slow-reacting substance of anaphylaxis. Murine macrophages are potent sources of LTC (5(S)-hydroxy-6(R)-gamma-glutamylcysteinylglycyl-7,9-trans-11,14-cis-eicosatetr aenoic acid). Peritoneal and pulmonary tissue macrophages synthesize LTC and other arachidonic acid (20:4) metabolites in response to inflammatory stimuli such as unopsonized zymosan and IgG immune complexes. Peritoneal macrophages, in addition, release 20:4 when challenged with IgE immune complexes. These results suggest that macrophages may be a major source of leukotrienes in acute inflammation and also in immediate-type hypersensitivity reactions.     

Modulation of lymphocyte proliferation by macrophages and macrophages loaded with arachidonic acid

Arachidonic acid (AA) is incorporated and exported by macrophages. This fatty acid is also transferred from macrophages (Mphi) to lymphocytes (LY) in co-culture. This observation led us to investigate the effect of macrophages pre-loaded with AA on concanavalin A (Con A)-stimulated lymphocyte proliferation. The experiments were performed in co-culture. This condition reproduces the in vivo microenvironment in which the modulation of lymphocyte proliferation is dependent on the interaction with macrophages. Lymphocytes obtained from untreated rats or from intraperitoneally thioglycolate-injected rats (THIO-treated) were co-cultured with macrophages from the same rats. Firstly, macrophages were co-cultured for 48 h with Con A-stimulated lymphocytes in different proportions: 0.5, 1, 2.5, 5, 10, 20 and 30% of 5 x 10(5) lymphocytes per well. At 1% proportion, macrophages caused maximum stimulation of lymphocyte proliferation; a four- to five-fold increase, for cells from both thioglycolate-treated and untreated rats, respectively, whereas at 20% it caused maximum inhibition. In addition, 1 or 20% macrophages were pre-loaded with several AA concentrations during a period of 6 h and co-cultured with lymphocytes. At 180 microM AA and 1% macrophages, lymphocyte proliferation was inhibited (by 25%), whereas at 20% macrophages, proliferation was increased, by 25- and three-fold, respectively, for cells from untreated and THIO-treated rats. AA added directly to the medium reduced lymphocyte proliferation, also being toxic to these cells at 100 microM. No toxic effects of AA were observed on macrophages. Additional evidence suggests that nitric oxide production is involved in the modulation of lymphocyte proliferation by AA-pre-loaded macrophages. These findings support the proposition that AA can directly modulate lymphocyte proliferation and the interaction between macrophages and lymphocytes.     

Phagocytosis by macrophages in zinc-deficient rats

Phagocytosis by polymorphonuclear cells has been found to be significantly reduced in zinc-deficient patients and this finding was confirmed in animal experiments. In order to find out whether phagocytosis by macrophages is similarly altered, experiments were conducted in three groups of 18 rats. Control, zinc-deficient and pair-fed rats were given ^99mTc nanocolloid intravenously. In ten other experiments (5 experimental and 5 control rats) ^99mTc-sulfur colloid was injected intravenously. The biodistribution was determined by a well-type gamma counter and the results were evaluated statistically. The greatest amount of radioactivity was taken up by the liver, followed by the spleen, lung and kidney. In both series of experiments however the zinc-deficient animals appeared to take up a greater amount of the radiotracer (P < 0.05).     

Androgen metabolism by human peritoneal macrophages

Peritoneal macrophages (PM) were obtained by peritoneal dialysis from a regularly menstruating woman with renal failure. Macrophages (10(6) cells) were incubated at 37 degrees C for various periods of time (0-4 hr) in the presence of 14C-androstenedione or 3H-androstenedione and various concentrations (0.06-5.06 microM) of nonradiolabeled androstenedione (A). Testosterone (T) formed was purified by column chromatography, thin layer chromatography, acetylation, and recrystalization to constant 3H:14C ratios. The rate of formation of T from A was linear for nearly 2 hr. Conversion of A to T was linear at cell numbers in the incubation up to 1 x 10(6). The formation of T from A followed Michaelis-Menten kinetics at concentrations of A between 0.06 and 5.06 microM. The apparent Km of the enzyme for A was 0.75 microM and the Vmax for T formation from A in these cells was 33.9 pmol x hr-1 x 10(6) cells-1. PM were obtained also from normal patients (n = 6) and patients with endometriosis (n = 5). The rate of T synthesis from A in PM obtained from patients with endometriosis [527 +/- 263 pmol x hr-1 x 10(6) cells-1 (mean +/- SEM, n = 5)] was similar to that observed in PM obtained from normal patients [518 +/- 226 pmol x hr-1 x 10(6) cells-1 (mean +/- SEM, n = 6)]. We observed a near 30-fold variation in the rate of formation of T from A by PM obtained from different individuals (range 54 to 1580 pmol x hr-1 x 10(6) cells-1). Further study is needed to elucidate the physiologic significance of PM androgen metabolism and its relationship to reproductive function.     

Activation of peritoneal macrophages by lysophosphatidylcholine

Lysophosphatidylcholine (lyso-PC), a product of inflammation induced by infectious and other agents, is able to stimulate mouse peritoneal macrophages to ingest target cells coated with IgG but not IgM regardless of the presence of complement. In vitro treatment of mouse resident peritoneal macrophages (adherent cells) alone with lyso-PC stimulated spreading activity but did not enhance ingestion activity of macrophages. However, when mixed cultures of adherent and nonadherent (lymphocytes) cells were treated with lyso-PC, macrophage ingestion activity of IgG-coated target cells (i.e., via Fc-mediated ingestion) was markedly enhanced. Analysis of lyso-PC activation process of macrophages for ingestion activity suggests that nonadherent (lymphocytes) cells are required for the induction of the manifestation of ingestion capacity. This requirement was also met by addition of untreated nonadherent cells to treated adherent cells. Thus, the activation mechanism of macrophages by lyso-PC for ingestion requires contribution of lymphocytes to promote enhanced ingestion activity. Since lyso-PC is a metabolite of a representative membrane phospholipid, we propose that lyso-PC and other lysophospholipids are mediators for activation of macrophages regardless of the type of inflammation-causative agent.     

Vimentin is secreted by activated macrophages

Vimentin is a widely expressed intermediate filament protein thought to be involved mainly in structural processes, such as wound healing. We now demonstrate that activated human macrophages secrete vimentin into the extracellular space. The maturation of blood-derived monocytes into macrophages involves several signalling pathways. We show that secretion of vimentin, which is phosphorylated at serine and threonine residues, is enhanced by the phosphatase inhibitor okadaic acid and blocked by the specific protein kinase C inhibitor GO6983. These findings are consistent with previous observations that phosphorylation of vimentin affects its intracellular localization and that vimentin is a substrate for protein kinase C (PKC). We also show that the anti-inflammatory cytokine interleukin-10 (IL-10), which inhibits PKC activity, blocks secretion of vimentin. In contrast, the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-alpha) can trigger secretion of vimentin. Finally, we found that extracellular vimentin is involved in bacterial killing and the generation of oxidative metabolites, two important functions of activated macrophages. These data establish that vimentin is secreted by macrophages in response to pro-inflammatory signalling pathways and is probably involved in immune function.     

Immune opsonin-independent phagocytosis by pulmonary macrophages

The uptake of albumin-coated latex particles by hamster pulmonary macrophages (PM) in vitro was investigated by using a new technique that combined flow cytometry and fluorescence microscopy to differentiate and quantitate bound vs ingested particles. In the absence of serum, PM avidly bound and ingested particles, whereas phagocytosis by hamster polymorphonuclear leukocytes (PMN) was less marked. In the presence of serum, phagocytosis by PM was slightly depressed, whereas phagocytosis by PMN was stimulated more than 10-fold. The binding of particles to PM in the absence of serum was pH, temperature, and trypsin sensitive and was dependent on the presence of extracellular Ca++ but not Mg++. The ingestion of particles by this immune opsonin-independent pathway was also temperature sensitive but was not affected by either pH or extracellular Ca++. Particle ingestion, but not binding, was inhibited by cytochalasin D and the divalent cation ionophore A23187.     

Superoxide generation by pig alveolar macrophages

Pig alveolar macrophages generate superoxide anions at a rate of 1.8 nanomoles/1 X 10(6) cells/min. The intracellular value of ATP in resting cells was 4.0 +/- 0.1 X 10(-16) mole/cell; in contrast the value in cells generating superoxide anions was 2.0 +/- 0.6. Superoxide generation was increasingly inhibited by exposing cells to adenosine from 0.1 to 1.0 mM. Unlike human macrophages, pig cell production of superoxide anions was not inhibited by exposure to the adenosine analog, 2-Cl-adenosine.     

Modulation of lymphocyte motility by macrophages

Motility of lymphocytes plays a significant role in their functions. Because macrophages frequently associate with lymphocytes in lymphoid tissues and inflammatory sites, they are likely to be important in regulating lymphocyte motility. In this study, we identified a chemokinetic activity in macrophage culture supernatants. Interestingly, this activity could be detected by the capillary migration assay but not by the more commonly used Boyden chamber chemotaxis assay. Colchicine, on the other hand, was chemokinetic for lymphocytes in the Boyden chamber chemotaxis assay but not in the capillary migration assay. Both these observations and previous studies on the morphology of motile lymphocytes on two-dimensional (2-D) surfaces (capillary migration assay) and in 3-D matrices (Boyden chamber chemotaxis assay) suggest that lymphocytes possess more than one motility mechanism--one for 2-D surfaces and one for 3-D matrices. We propose that the macrophage-derived chemokinetic activity described herein only affected the motility mechanism on 2-D surfaces. In addition, we also observed that the chemokinetic activity was produced by "resting" macrophages and could not be augmented by further activation. Finally, the effect was greatest on mature T cells. We propose that this factor plays an important role in facilitating cell interactions within lymphoid tissues and inflammatory sites.     

Hapten-specific T lymphocyte activation by glutaraldehyde-treated macrophages: an argument against antigen processing by macrophages.

In this communication the effects of glutaraldehyde treatment of trinitrophenyl-(TNP) modified macrophages on their ability to stimulate TNP-specific guinea pig T lymphocyte proliferation were studied. TNP-modified macrophages briefly treated with glutaraldehyde retained much of their ability to stimulate TNP-primed T cells. In contrast, similar treatment of allogeneic macrophages or soluble protein antigen-pulsed syngeneic macrophages completely eliminated their ability to stimulate a mixed leukocyte reaction or protein antigen-specific proliferation, respectively. TNP-modification did not appear to interfere with glutaraldehyde reactivity since macrophages treated with glutaraldehyde before or after TNP-modification stimulated equivalent T cell responses. However, glutaraldehyde treatment of TNP-modified macrophages that had been cultured overnight dramatically reduced their ability to stimulate TNP-specific T cells. Glutaraldehyde-treated TNP-modified macrophages also expressed the same genetic restrictions of T cell activation as untreated stimulators. Thus, T cells primed with syngeneic TNP-modified macrophages were restimulated only by glutaraldehyde-treated TNP-modified syngeneic, but not by allogeneic, macrophages. These results are discussed with respect to the nature of the TNP-specific immunogen recognized by T cells.     

Antigen processing and presentation by macrophages

The classical macrophage is one of the most important cells involved in presenting antigen to helper T cells, because of its ability to regulate its expression of Ia molecules and to encounter and process particulate and soluble antigens. We have summarized in this report studies examining the handling by macrophages of two different antigens, the bacteria Listeria monocytogenes and the protein hen egg white lysozyme (HEL). The purpose was to identify potential sources of immunogenic peptides. Presentation of Listeria required an intracellular processing stage sensitive to lysosomotropic drugs. The Listeria required internalization and processing, after which immunogenic molecules were recognized by T cells on the macrophage surface. Metabolic studies showed that Listeria-derived peptides were released by macrophages that had phagocytosized the bacteria. The release of these peptides was a temperature-dependent process, unaffected by inhibiting lysosomal catabolism by treatment with chloroquine. Listeria-derived peptides were also detected on the surface of the macrophage. These peptides behaved like integral membrane proteins, some of which persisted for at least 24 hr at the macrophage surface. When tested for immunogenicity, the released peptides were very weakly immunogenic. The membrane-associated peptides alone could not stimulate Listeria-specific T cells, but could be reprocessed by additional macrophages and subsequently stimulate the T cells. A defined antigen system using HEL-specific T-cell hybridomas was used to examine the processing of HEL. Presentation of HEL required a chloroquine-sensitive intracellular processing stage. In examining two T-cell hybridomas, a differential requirement for antigen processing was determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of interferon by alveolar macrophages

Acton, Jean D. (Bowman Gray School of Medicine, Winston-Salem, N.C.), and Quentin N. Myrvik. Production of interferon by alveolar macrophages. J. Bacteriol. 91:2300-2304. 1966.-Rabbit alveolar macrophages inoculated with parainfluenza-3 virus in vitro produce a viral inhibitor which possesses the properties of interferon. The interferon is nondialyzable, is stable at pH 4, is not sedimented at 100,000 x g, exhibits species specificity, and can passively protect other alveolar macrophages from infection with virulent rabbitpox virus. The possible significance of alveolar macrophage-produced interferon is discussed.     

Decreased prostaglandin production by cholesterol-rich macrophages

The regulation of prostaglandin production by macrophages enriched in cholesterol was examined. Mouse peritoneal macrophages were incubated for 18 h with 25 micrograms/ml of human acetyl-LDL (low density lipoprotein) and trace amounts of labeled arachidonic acid. After cholesterol enrichment, the cells were incubated with phorbol 12-myristate 13-acetate (PMA), calcium ionophore, or zymosan to stimulate endogenous arachidonic acid metabolism. A high performance liquid chromatography profile of the eicosanoids released revealed no qualitative differences between unmodified and modified macrophages. Cholesterol-rich cells, however, released less prostacyclin (PGI2) and prostaglandin E2 (PGE2) compared to unmodified cells, and products from the lipoxygenase pathway became the predominant metabolites. A decrease in the synthesis of PGI2 and PGE2 by cholesterol-rich macrophages was confirmed by radioimmunoassay and radiolabeled experiments. The activity of prostaglandin synthetase was modestly increased in the cholesterol-modified macrophages compared to controls. As an estimation of phospholipase activity, the release of labeled arachidonic acid from membrane phospholipids, however, was significantly decreased in cholesterol-rich macrophages. The phosphatidylinositol fraction was particularly resistant to arachidonate release in response to calcium ionophore and PMA in the modified cells. The measurement of membrane phospholipid fatty acid composition before and after calcium ionophore supported the observation that less arachidonate was released by cholesterol-enriched cells in response to the ionophore. Based on these observations, we propose that prostaglandin synthesis from endogenous arachidonate stores is decreased in the cholesterol-rich macrophage. A decrease in agonist-induced activation of the phospholipase activity is proposed as a mechanism for this effect.     

Metabolism of oxidized LDL by macrophages

Oxidation products of lipids and proteins are found in atherosclerotic plaque and in macrophage foam cells. Macrophages avidly endocytose in-vitro oxidized LDL and accumulate sterols. What is the evidence that such a process is involved in in-vivo foam cell formation? The present review surveys current knowledge on the metabolism of oxidized LDL by macrophages, and the types, amounts and location of oxidation products that accumulate in these cells. Comparable studies of lesion lipoproteins and foam cells indicate that limited extracellular lipoprotein oxidation, perhaps followed by more extensive intracellular oxidation subsequent to uptake by macrophages, is a more likely scenario in vivo.