Abilities of activated macrophages to manifest tumoricidal activity and to generate reactive nitrogen intermediates: a comparative study in vitro and ex vivo

The abilities of lymphokines and heat-killed bacteria to induce and to maintain tumoricidal activity and/or the secretion of reactive nitrogen intermediates (RNI) were comparatively assessed in bone marrow-derived mononuclear phagocytes (BMM phi) in vitro and in adherent peritoneal cells (APC) ex vivo. In showing that the kinetics of tumoricidal activity and of secretion of RNI induced by macrophage-activating agents in BMM phi and/or in peritoneal cells do largely parallel each other, the present findings provide evidence for a role of RNI in tumor cell killing by activated macrophages both in vitro and in vivo.     

Induction of nitric oxide synthase is a necessary precondition for expression of tumor necrosis factor-independent tumoricidal activity by activated macrophages.

Various bacteria and bacterial products induce in pure, lymphocyte-free bone marrow-derived mononuclear phagocytes (BMM?) the generation of tumor necrosis factor, nitric oxide (NO) synthase, NO and nitrite (NO2-), the flow of L-arginine to citrulline, and tumoricidal activity. The flow of L-arginine to citrulline and formation of NO/NO2- on the one hand and expression of tumoricidal activity were not always closely related; however, these parameters were suppressed in a dose-dependent manner by the flavoprotein inhibitor, diphenyleneiodonium (DPI) and the L-arginine analogue, NG-monomethyl-L-arginine (NMMA). The findings support the concept of a central role of the NO synthase pathway in the generation of tumor necrosis factor-independent tumoricidal activity by activated macrophages but the exact conditions which enable the transfer of the lytic principle from the effector to the target cell remain to be elucidated.     

The macrophage response to bacteria: flow of L-arginine through the nitric oxide and urea pathways and induction of tumoricidal activity

The consequences of the interaction of heat-killed bacteria and lipopolysaccharide (LPS) with a pure population of bone marrow-derived mononuclear phagocytes (BMM?) were investigated, utilizing changes in the flow of L-arginine and expression of tumoricidal activity as parameters of macrophage (m?) function. Gram negative bacteria and LPS proved potent in inducing the flow of L-arginine through the nitric oxide and the urea pathways but were mostly poor in eliciting tumoricidal activity. Gram positive bacteria affected the metabolism of L-arginine only little but were often efficient in triggering tumoricidal activity. The findings show that the m? response to bacteria, which may determine the outcome of their interaction with the host, may differ considerably depending on the type of bacteria.     

The interaction of macrophages and bacteria: Escherichia coli species, bacterial lipopolysaccharide, and lipid A differ in their ability to induce tumoricidal activity and the secretion of reactive nitrogen intermediates in macrophages.

The ability of nine Escherichia coli strains, and of bacterial lipopolysaccharide (LPS)3 and lipid A preparations, to elicit in a pure population of bone marrow-derived mononuclear phagocytes (BMM phi) tumoricidal activity and/or the generation of reactive nitrogen intermediates (RNI) was compared. Generally, low concentrations of E. coli organisms were able to trigger the generation of RNI: however, for induction of tumoricidal activity, higher concentrations were required. Nonisogenic E. coli species exhibited different ability; isogenic E. coli organisms that differed only in the expression of K antigen exhibited similar ability to elicit the macrophage activities. LPS proved to be highly efficient in triggering the secretion of reactive nitrogen intermediates; lipid A was clearly less potent, but evidence is presented to suggest that this was due to the diminished solubility of these reagents. On the other hand, all LPS and lipid A samples were very poor inducers of tumoricidal activity. Although RNI secretion and expression of tumoricidal activity are both strongly dependent on L-arginine, various evidence suggests that the two functions are not closely correlated and are induced by different bacterial structures.     

The interaction of macrophages and bacteria: a comparative study of the induction of tumoricidal activity and of reactive nitrogen intermediates

The abilities of various bacteria to induce in a pure population of bone marrow-derived mononuclear phagocytes (BMM phi) tumoricidal activity and/or the generation of reactive nitrogen intermediates (RNI) were comparatively assessed. Interaction of BMM phi with bacteria led to expression of these functional activities, indicating that the organisms were recognized as foreign. As the majority of bacteria elicited in BMM phi either tumoricidal activity (that is maintained for days) or the production of RNI, measured by the release of nitrite (that is short-lived), it appears that the two functions are under separate control. However, both functions are inhibited or even abrogated by arginase or the L-arginine analogue, NG-monomethyl-L-arginine, suggesting that their expression is dependent on L-arginine.     

Bone marrow-derived IFN-producing killer dendritic cells account for the tumoricidal activity of unpulsed dendritic cells

The CD11c(int)B220(+)NK1.1(+)CD49(+) subset of cells has recently been described as IFN-producing killer dendritic cells (IKDC), which share phenotypic and functional properties of dendritic cells and NK cells. Herein we show that bone marrow-derived murine dendritic cell preparations contain abundant CD11c(int)B220(+)NK1.1(+)CD49(+) cells, the removal of which results in loss of tumoricidal activity of unpulsed dendritic cells in vivo. Moreover, following s.c. injection, as few as 5 x 10(3) highly pure bone marrow-derived IKDC cells are capable of shrinking small contralateral syngeneic tumors in C57BL/6 mice, but not in immunodeficient mice, implying the obligate involvement of host effector cells in tumor rejection. Our data suggest that bone marrow-derived IKDC represent a population that has powerful tumoricidal activity in vivo.     

Identification of a macrophage-activating factor in granules of the RNK large granular lymphocyte leukemia

Recent work from our laboratory has shown that NK cells rapidly release preformed factor(s) that stimulate monocyte oxidative metabolism and microbicidal activity. We have hypothesized that such factors could also activate macrophage (M phi) tumor lysis and might be stored in the cytoplasmic granules. Granules were isolated from the RNK large granular lymphocyte leukemias by nitrogen cavitation and Percoll fractionation of the cell homogenate. Utilizing CSF-1 differentiated murine bone marrow-derived M phi and P815 tumor target cells, a M phi-activating factor (MAF) was found. The MAF activity was identified in two peaks, the first was coincident with dense granule enzymes and was 60 times more concentrated per mg protein than a second peak in the cytosol fractions. Solubilization in 2 M NaCl was necessary to recover activity from both peaks. Granule NK-MAF required the simultaneous presence of LPS in order to induce tumoricidal activity. Kinetics of NK-MAF activation peaked after 12 h of exposure. The NK-MAF was short lived in the solubilized granules; however, its heat resistance allowed us to prepare enriched and stable preparations. Treatment of NK-MAF with pepsin but not trypsin completely abrogated its activity. The NK-MAF passed through an ultrafiltration membrane with a nominal cut-off of 10 kDa. This work indicates that NK cell granules contain a small heat-stable peptide capable of activating M phi tumoricidal activity.     

Beta-galactosidase--an indicator of the maturational stage of mouse and human mononuclear phagocytes

Resident, elicited, and activated mouse peritoneal macrophages exhibit a differential expression of the activity of the enzyme beta-galactosidase; freshly harvested resident macrophages express a remarkably high activity whereas the latter two populations are almost void of enzymic activity. During in vitro cultivation there is an enhancement in the level of the enzyme in the three populations, and a significant proportion of both thioglycollate-elicited and Corynebacterium parvum-activated macrophages acquire beta-galactosidase activity. Cells within in vitro differentiated bone marrow-derived mononuclear phagocyte colonies are heterogeneous with respect to expression of beta-galactosidase activity. The percentage of cells expressing medium to intense enzymic activity is augmented with time in culture. Essentially the same pattern is observed in colonies differentiated from bone marrows of mice bearing acute or chronic inflammation. Freshly isolated human peripheral blood monocytes are essentially void of detectable beta-galactosidase activity. Eighty to ninety percent of the monocytes acquire medium to intense activity during a 7-day cultivation period. The data support the suggestion that beta-galactosidase expression in mononuclear phagocytes is a correlate of their maturational stage both in vivo and in vitro and does not reflect the state of elicitation or activation of these cells.     

Production of C3 as a marker of lymphokine-mediated macrophage activation

C3 production was assayed using an enzyme-linked immunosorbent assay (ELISA) in cell-free supernatants harvested from thioglycollate-elicited macrophages exposed to a variety of macrophage stimulating and activating agents. Macrophage monolayers treated with the stimulating agents starch, glycogen, and zymosan secreted three- to four-fold less C3 (mean 12 ng/10(5) cells/12 hr) than macrophages exposed to lymphokines containing macrophage-activating factor (MAF) (mean C3 production 44 ng/10(5) cells/12 hr). The increased production of C3 in macrophages exposed to MAF parallels the ability of these macrophages to acquire tumoricidal capacity as monitored in an in vitro 72 hr tumor cell cytotoxicity assay using B16 melanoma cells. Macrophages previously rendered tumoricidal by exposure to MAF and which are refractory to further challenge by MAF following decay of their tumoricidal properties, do not produce C3 on rechallenge with MAF. Exposure of refractory macrophages to liposome-encapsulated MAF overcomes the refractory state and induces re-expression of the tumoricidal phenotype and C3 production. We conclude that quantitative detection of macrophage-generated C3 antigen provides a useful biochemical marker for monitoring the acquisition of tumoricidal properties in macrophages exposed to MAF and offers a sensitive assay for screening novel agents that activate macrophages via mechanisms similar to MAF.     

Dissociation between macrophage tumoricidal capacity and suppressive activity: analysis with macrophage-defective mouse strains

Macrophages (M phi diameter) from three mouse strains with genetically distinct M phi diameter deficits (C3H/HeJ, A/J, and P/J) were unable to develop high cytolytic and cytotoxic activity against tumor cells in vitro when exposed to agents (MAF and IFN-beta) that strongly increased the tumoricidal capacity of M phi diameter from nondefective C3H/HeN mice. Nevertheless, the tumoricidal deficits of M phi diameter from the defective strains did not affect their suppressive capacity on Con A-induced lymphoproliferation, nor their ability to react to IFN-beta by decreasing suppressive activity. In fact, natural suppressive activity and IFN-beta-induced changes in the suppression of M phi diameter from C3H/HeJ, A/J, and P/J mice were highly comparable to those of C3H/HeN M phi diameter, thus stressing the dissociation between the mechanisms governing M phi diameter suppression and M phi diameter tumoricidal activity. Analysis of the modulation by MAF and IFN-beta of M phi diameter ability to release the oxygen metabolites O2- and H2O2, molecules possibly involved in the effector mechanism of both M phi diameter cytotoxicity and suppression, revealed a close correlation with the patterns of suppressive activity in both nondefective and defective strains. In contrast, no correlation between the production of oxygen-reactive species and M phi diameter tumoricidal activity was observed. The ability of MAF- and IFN-beta-treated M phi diameter to produce PGE, a molecule of major importance in M phi diameter-mediated suppression and possibly involved also in the regulation of M phi diameter tumoricidal activity, again paralleled M phi diameter suppressive capacity. Thus, the mechanisms controlling M phi diameter antitumor activity appeared to be clearly distinct from those involved in M phi diameter suppression.     

Surfactant protein A suppresses lipopolysaccharide-induced IL-10 production by murine macrophages

Upon LPS exposure, mononuclear phagocytes produce TNF-alpha and IL-10, two cytokines with pro- and anti-inflammatory activities, respectively. We previously described that murine resident alveolar macrophages, which play a central role in the immunosurveillance of the lung alveoli, do not synthesize IL-10 in vivo or in vitro when exposed to LPS. In the present report we demonstrate that during lung inflammation induced by the intranasal administration of LPS, bronchoalveolar cells collected between days 3 and 5 are able to synthesize IL-10 when exposed to LPS. We also show that depletion of resident alveolar macrophages by an intratracheal instillation of liposome-encapsulated clodronate is followed by subsequent replenishment of the airspaces by mononuclear phagocytes. This is accompanied by the transient competence of cells for IL-10 production. The cell capacity to produce IL-10 is evident up to 3 days and then decreases. This led us to hypothesize that the alveolar environment contains a down-regulator of LPS-induced IL-10 synthesis by recently emigrating mononuclear phagocytes. We show that the surfactant protein A, an airspace protein that has known immunomodulatory activities, dramatically inhibits LPS-induced IL-10 formation by bone marrow-derived macrophages. These data show a difference between resident and inflammatory macrophages with respect to IL-10 synthesis. Moreover, this study highlights for the first time the inhibitory role of surfactant protein A in the anti-inflammatory activity of macrophages through inhibition of IL-10 production.     

Induction by immunomodulatory agents of a macrophage antigen recognized by monoclonal antibody 158.2 and correlation with macrophage function

MA158.2, a rat monoclonal antibody with binding specificity for cells of the monocyte-macrophage lineage, reacts with an antigen (158.2) whose expression is enhanced on mononuclear cells activated to the tumoricidal phenotype by treatment with lymphokine supernatant containing macrophage activating factor (MAF). The functional relevance of enhanced expression of this antigen has been examined in mouse peritoneal macrophages treated with a variety of immunomodulatory agents and assayed for augmented macrophage-mediated defense reactions, including O-2 production, microbicidal, and tumoricidal activity. An interferon-gamma (IFN-gamma) preparation produced by recombinant DNA technology induced a dose-dependent increase in expression of the 158.2 antigen in inflammatory macrophages which was accompanied by acquisition of microbicidal activity against Listeria monocytogenes. However, these cells did not express tumoricidal activity and induction of this property required concomitant exposure to lipopolysaccharide (LPS). Similar results were obtained using macrophages elicited with pyran copolymer. Exposure to LPS alone induced enhanced expression of antigen 158.2 but did not elicit microbicidal activity. Macrophages challenged with IFN-alpha, IFN-beta, MDP, and bestatin did not exhibit increased 158.2 and also failed to acquire tumoricidal activity when treated concomitantly with LPS. Collectively, these data indicate that the MA 158.2 antibody recognizes an antigen expressed by macrophage populations displaying the so-called primed phenotype in which microbicidal activity is expressed but in which induction of tumoricidal activity requires the addition of a second signal such as LPS.     

Effect of myelopeptide-1 on the functional activity of phagocytes from mice treated with cyclophosphane

The capacity of the bone marrow-derived myelopeptide-1 (MP-1) to affect in vivo and in vitro the functional activity of phagocytes of intact mice and mice treated with a cytostatic agent (cyclophosphane) has been studied. It was found that MP-1 produces a correcting effect on the functional activity of bone marrow and peripheral blood phagocytes. An optimal scheme of the injection of MP-1 to mice with the cyclophosphane-induced immunodeficiency was developed, which provides a maximum immunocorrecting action. MP-1 had the most pronounced effect on the quantitative characteristics and the functional activity of phagocytes of different localization when introduced prior to the cytostatic; under these conditions, the pep tide affects peripheral blood neutrophils. The results obtained enable one to consider MP-1 as a preparation protecting the peripheral blood phagocytes from the damaging action of cyclophosphane.     

Cutting edge: VacA, a vacuolating cytotoxin of Helicobacter pylori, directly activates mast cells for migration and production of proinflammatory cytokines

Mucosal mast cells strategically located at the optimal site interact with invading bacteria. Presence of VacA, the virulent Helicobacter pylori cytotoxin, is correlated with the severity of H. pylori-induced gastritis. To examine the mechanisms of inflammation in H. pylori-induced gastritis, we administered VacA to the mice. Inoculation of VacA resulted in epithelium vacuolization and marked infiltrations of mast cells and mononuclear cells into the mucosal epithelium within 24 h. In an in vitro study using bone marrow-derived mast cells, VacA directly bound and showed a chemotactic activity to the mast cell. In addition, VacA induced bone marrow-derived mast cells to produce proinflammatory cytokines, TNF-alpha, macrophage-inflammatory protein-1alpha, IL-1beta, IL-6, IL-10, and IL-13 in a dose-dependent manner without causing degranulation. The present study suggests that early activation of mast cells by VacA may be the host early response to clear the bacteria and also may contribute to the pathogenesis of H. pylori-induced gastritis.     

Opposing effects of dexamethasone on the clonal growth of granulocyte and macrophage progenitor cells and on the phagocytic capability of mononuclear phagocytes at different stages of differentiation

Dexamethasone, a synthetic glucocorticosteroid, was shown to modulate the colony-stimulating factor-dependent clonal growth of myeloid progenitor cells in semisolid agar cultures, enhancing the formation of granulocyte colonies (50–100%) and suppressing the formation of macrophage colonies (75–97%). Modulation of the pattern of myeloid colony formation by dexamethasone (12–125 nM) was brought about when the steroid was administered to 6-day cultures at the time of culture initiation and up to 72 hr later. Dexamethasone inhibited myeloid cell proliferation when administered to 5-day liquid cultures at culture initiation and up to 96 hr later. Dexamethasone (12–250 nM) also enhanced the phagocytic activity of bone marrow-derived mononuclear phagocytes toward heat-killed (HK) yeast cells (up to 100%) and IgG-coated sheep red blood cells (up to 60%). Enhancement of the phagocytic capability depended critically on the stage in culture at which dexamethasone was administered. Exposure to dexamethasone for 28 hr up to 96 hr of 96-hr cultures of bone marrow cells did not lead to a modulation of phagocytic activity of the developing mononuclear phagocytes. The presence of dexamethasone during the critical period of 96 hr to 120 hr after culture initiation led to an enhanced phagocytic capability, which was statistically significant already 12 hr after the administration of the glucocorticoid. Dexamethasone induced an enhanced phagocytic activity when administered at any time after culture initiation provided that it was in culture during this critical period. When added at 120 hr of culture, dexamethasone no longer enhanced the phagocytic capability of mononuclear phagocytes and when added later than 156 hr of culture suppressed it. Dexamethasone also suppressed (up to 68%) the phagocytic capability of resident and elicited peritoneal macrophages. The results suggest that glucocorticoids shift the balance of granulocyte vs. macrophage formation at early stages of precursor cell differentiation. Reduction in mononuclear phagocyte growth and enhancement of its phagocytic capability might reflect accelerated differentiation/maturation steps. The inhibitory effect of dexamethasone on macrophage formation and on the phagocytic capability of mature mononuclear phagocytes and peritoneal macrophages might be a relevant aspect of the in vivo immune suppression encountered after glucocorticoid administration.     

Distinct bone marrow precursors for mononuclear phagocytes expressing high and low 5' -nucleotidase activity

Using a cytochemical assay we were able to show that the peritoneal macrophage population of normal nontreated mice (resident peritoneal macrophages) exhibits a heterogeneity with regard to the expression of the activity of the ecto-enzyme 5′-nucleotidase (5′-N). About 75% of the macrophages express high enzymic activity whereas the remaining 25% express low 5′-N activity. Macrophages accumulating in the peritoneum as a result of an inflammatory response are predominantly of the low activity type. In vitro activation of resident peritoneal macrophages by lymphokines does not result in a decrease in the number of macrophages expressing high enzymic activity though the level of the enzymic activity of these cells is reduced by about 36%. Bone marrow derived mononuclear phagocyte colonies developing in vitro, under liquid culture conditions, from bone marrows of normal mice can be divided into three types with respect to their expression of 5′-N activity: (1) high activity colonies–relatively small colonies in which all the cells express high 5′-N activity (about 20% of the colonies); (2) low activity colonies – relatively large colonies in which all the cells express low 5′-N activity (about 70% of the colonies); and (3) mixed colonies–relatively large colonies in which all the cells express low enzymic activity except for about 8% of cells located at the periphery of the colonies which express high enzymic activity (about 10% of the colonies). During an inflammatory response the frequency of the high activity colonies is significantly reduced. Our results provide evidence for distinct bone marrow precursors for mononuclear phagocytes expressing high and low 5′-N activity and suggest that (1) the resident macrophages derive from a subpopulation of bone marrow precursor cells developing in vitro into high 5′-N activity mononuclear phagocytes, and (2) during an inflammatory response there is a preferential expansion of clones of the low enzymic activity phenotype.     

Macrophage growth inhibitors derived from the murine peritoneal cavity

Summary The murine peritoneal cavity contains factors that inhibit the in vitro growth and colony formation of macrophages. The inhibition of macrophage growth is not due to cell death. In the presence of inhibitors, the growth of colony-forming macrophages is suppressed, and small clusters are formed as a result of limited proliferation. The more mature mono-nuclear phagocytes (blood monocytes and peritoneal exudate macrophages) are more sensitive to the overall inhibitory effect of the peritoneal inhibitors than the less mature bone marrow mononuclear phagocytes. Furthermore, using dialysis and Amicon ultrafiltration, at least two inhibitors with differential inhibitory effects can be demonstrated. The colony formation of bone marrow mononuclear phagocytes is suppressed mainly by a protease-resistant, small molecular weight (<1,000) dialyzable inhibitor. In contrast, peritoneal exudate macrophages are sensitive to both the small molecular weight inhibitor and a protease-sensitive, large molecular weight (>12,000), nondialyzable inhibitor. The data suggest a possible existence of a dual inhibitor control on the proliferation of mononuclear phagocytes in vivo. In addition, the in vitro cultured peritoneal exudate cells are capable of producing inhibitors that mimic the activity of the in vivo inhibitors. This investigation was supported by Grants CA 09 11(SY) and AI15563(CCS) from the National Institutes of Health, Bethesda, MD     

Discrimination between macrophage-and NK-type tumoricidal activities via anti-asialo GM1 antibody

The usefulness of asialo GM1, a glycolipid surface marker, to define the effector cell types involved in tumor resistance in vitro and in vivo was assessed. Pretreatment of rat effector cells with anti-asialo GM1 antibody plus complement in vitro either abrogated or markedly diminished NK activity; in contrast, macrophage-type cytocidal activity was not diminished by such pretreatment. Similarly, systemic inoculation of anti-asialo GM1 antibody selectively eliminated NK activity, leaving macrophage-type tumoricidal reactivity intact. Finally, such pretreatment did not diminish host resistance in an in vivo tumor model in which the available evidence suggests a critical role for macrophages. The asialo GM1 marker may thus be useful in delimitating the tumoricidal capacity of cells exhibiting NK activity from that mediated by other cell types.     

Effects of parathyroid hormone and calcitonin on adenylate cyclase in murine mononuclear phagocytes

Peritoneal mononuclear phagocytes elicited by thioglycollate demonstrate responsiveness to parathyroid hormone (PTH) and calcitonin (CT) which differs from that seen in the normal resident population. PTH causes a twofold stimulation of adenylate cyclase activity in elicited cells but inhibits this activity in resident cells. CT causes a greater stimulation of adenylate cyclase in elicited than in resident cells. Both CT and PTH cause an increase in cyclic AMP accumulation in cultures of elicited mononuclear phagocytes. These results indicate that cells of the mononuclear phagocyte lineage have functional receptors for both PTH and CT. This is the first biochemical evidence to support the hypothesis that mononuclear phagocytes are precursors of the bone resorbing osteoclast.     

Effect of mononuclear phagocytes on the differentiation of syngeneic, allogeneic and xenogeneic hematopoietic stem cells

The colony formation in spleen of lethally irradiated syngeneic or hybrid recipients was studied after transplantation of bone marrow cells, with or without macrophages from lymph nodules or from peritoneal cavity of mice, cells of macrophage-like cell line J-774, and monocytes from peripheral blood of healthy donors. The direction of stem cell differentiations in the presence of all the types of mononuclear phagocytes was seen to change from mainly erythroid to mainly myeloid one. The ratio of erythroid to myeloid colonies became equal to 0.5-0.9 instead of 2.0, when bone marrow cells were injected with equivalent quantity of mononuclear phagocytes. This new regulatory function of mononuclear phagocytes is discussed.