Dolph O. Adams

Molecular transductional mechanisms by which IFN gamma and other signals regulate macrophage development.

Mononuclear phagocytes constitute a major, host regulatory and efTector system (Adams & Hamilton 1984, 1987, Nathan & Cohn 1980). These highly versatile cells, which populate all tissues in the body, are not only phagocytic but are avid secretory cells and have potent destructive potential toward microbes and neoplastic cells. It has long been appreciated that the function of these phagocytes is not static but rather can be dramatically changed in a variety of circumstances. The modulation of macrophage function by interaction with products from Tlymphocytes has long been considered the prototypic example of such alteration in function. Over the past 5 yr, IFNy has become recognized as the major product from T-cells that alters the function of macrophages. Specially, it has been identified as a principal macrophage activating factor responsible for initiating enhanced competence of macrophages to destroy facultative and obligate intracellular parasites and tumor cells. Actions of IFNy on macrophages are not, however, limited to these effects, since it can also induce competence to present antigen to T-cells, mount an effective respiratory burst, and either enhance or reduce the expression of a wide variety of macrophage proteins. The macrophage, after having entered the tissues, often becomes a relatively quiescent resident cell unless confronted with one or more stimulatory signals in its local environment (Adams & Hamilton 1984, 1987, Nathan & Cohn 1980). In the latter case, it may undergo dramatic changes in functional potential. Mononuclear phagocytes are muitipotential cells and can develop increased function along a variety of disparate paths. Some of these paths represent mutually exclusive routes, such that enhanced competence for one function is usually gained at the expense of diminished competence for another. It is useful to distinguish between complex

Molecular mechanisms in tumor-cell killing by activated macrophages

Macrophages kill tumor cells with and without the aid of antibody and evidence suggests that secreted cytotoxic substances are at work in each system. Here Dolph Adams and Carl Nathan discuss the likely involvement in both pathways of several such substances including cytolytic protease and hydrogen peroxide.

Molecular interactions in macrophage activation

Macrophage activation, broadly defined as the acquisition of competence to execute a complex function, continues to intrigue biologists, because of its fundamental importance to the host and the fascinating scientific challenges it poses. A recent meeting* emphasized the considerable progress made in this area since the topic was last considered in such a workshop. The workshop focused sharply on one aspect of the field: the molecular basis of macrophage activation, and covered the multiple, specific signals that induce and suppress activation, how these signals are transduced and thus affect macrophage behaviour and how induced function, in turn, is effected.

Regulation of tumor necrosis factor (TNF) expression: Interferon-γ enhances the accumulation of mRNA for TNF induced by lipopolysaccharide in murine peritoneal macrophages☆

The secretion of tumor necrosis factor (TNF) by macrophages is initiated by lipopolysaccharide (LPS); considerable evidence indicates that such secretion can be potentiated by interferon-gamma (IFN-gamma). The present studies show that accumulation of mRNA for tumor necrosis factor, which represents an important regulatory focus for controlling secretion of TNF, is enhanced by physiologic doses of IFN-gamma (20 units/ml of purified recombinant IFN-gamma). mRNA for TNF induced by LPS, which was maximal 2 hr after LPS was applied to the cells, was enhanced 5- to 8-fold by IFN-gamma as determined by Northern blot analysis. Interferon did not change the kinetics of accumulation but did change the dose effects of LPS in that increasing amounts of LPS led to increasing amounts of TNF mRNA in IFN-gamma-treated macrophages. IFN-gamma itself, however, did not induce expression of TNF mRNA. These studies document that IFN-gamma potentiates the cytoplasmic accumulation of mRNA for TNF induced in murine peritoneal macrophages by LPS.

Immunosuppression following 7,12-dimethylbenz[a]anthracene exposure in B6C3F1 mice-II. Altered cell-mediated immunity and tumor resistance

We have previously demonstrated that the polycyclic aromatic hydrocarbons benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) produce a marked decrease in spleen weight, spleen and bone marrow cellularity and the number of IgM plaque forming cells generated in response to a T-dependent antigen. Exposure to DMBA, but not B[a]P, increased susceptibility to challenge with PYB6 tumor cells and Listeria monocytogenes suggesting that DMBA produces immune impairment involving cell-mediated immunity (CMI) and tumor resistance mechanisms. In this study, female B6C3F1 mice received total doses of 5, 50 and 100 micrograms DMBA/g of body weight in ten subcutaneous injections of 0.5, 5, or 10 micrograms/g over a 2 week period and CMI and tumoricidal functions were examined 3-5 days following the final injection of DMBA. DMBA exposed mice exhibited suppressed splenic cellularity (decreased 62%) and decreased numbers of resident peritoneal cells (down to 47% of control), although the proportion of T cell and T cell subsets, B cells and macrophages in spleens from exposed mice was not altered. Lymphocyte blastogenesis in response to mitogens was suppressed up to 49% with PHA, 48% with Con A and 76% with LPS. The response to alloantigens in unidirectional mixed lymphocyte culture was depressed as much as 73% following exposure to DMBA. Tumor cytolysis mediated by cytotoxic T cells (CTL) was impaired at doses of 50 and 100 micrograms DMBA/g body weight (88-95% suppressed respectively) as was natural killer cell (NK)-mediated tumor cytolysis (24% and 55% suppressed). Antibody-dependent cytotoxicity was significantly depressed in the highest exposure group. Peritoneal macrophage accumulation was decreased in DMBA-treated mice, but the macrophages present were pushed towards activation. The ability of DMBA-exposed mice to eliminate intravenously injected B16F10 tumor cells from the lungs was not impaired. Since NK- and M phi-mediated tumor cytotoxicity are thought to be primarily responsible for pulmonary elimination of B16F10 melanoma cells, the extent of NK suppression observed following DMBA exposure appeared to be insufficient to alter in vivo B16F10 pulmonary elimination. In contrast, the loss of the CTL tumoricidal response correlated with an increased frequency of tumors following challenge with PYB6 tumor cells.

Interaction of Bacillus Calmette-Guérin-activated macrophages and neoplastic cells in vitro: I. Conditions of binding and its selectivity

Abstract The initial interaction in vitro between Bacillus Calmette-Guerin-activated, peritoneal macrophages from C57B1/6J mice and two nonadherent neoplastic targets (P815 and EL-4) was found to represent firm physical binding of the targets to the macrophages. Binding between the Bacillus Calmette-Guerin macrophages and the EL-4 or P815 targets was greater than that between these two targets and inflammatory macrophages elicited by thioglycollate broth or between lymphocytes and either type of macrophage. Bacillus Calmette-Guerin MΦ also selectively bound three other neoplastic targets (P388, L1210, and RBL-5). The binding, which rose progressively for 60 min of cocultivation at 37 °C, was linear with respect to both the number of interacting targets and macrophages and required the presence of divalent cations and trypsin-sensitive structures on the macrophages. Binding was temperature dependent and required living, metabolically active macrophages. H-2 differences between targets and activated MΦ were not required for binding and did not prevent it. Finally, binding of the P815 targets to the Bacillus Calmette-Gueŕin MΦ could be saturated by the addition of excess targets.

Gene regulation in macrophage activation: differential regulation of genes encoding for tumor necrosis factor, interleukin-1, JE, and KC by interferon-gamma and lipopolysaccharide.

Although macrophage activation is induced in a complex manner by signals such as Interferon‐γ(IFNγ) and bacterial lipopolysaccharide (LPS) and depends on alterations in levels of specific proteins due to differences in gene expression, the complexity of gene regulation during macrophage activation in regard to multiple signals is not fully appreciated. To probe this question, we selected four model genes encoding for tumor necrosis factor (TNF), interleukin‐1 (IL‐1), and the immediate early genes JE and KC. After analyses of Northern blots for specific mRNA, LPS was found to enhance levels of mRNA for IL‐1, TNF, JE, and KC. IFNγ initialed heightened mRNA levels for JE but did not alter specific mRNA for IL‐1, TNF, or KC. When IFNγ and LPS were combined, additive effects on levels of specific mRNA for JE, enhancement of mRNA for TNF, suppressed mRNA for KC, and no effect on mRNA for IL‐1 were observed. When transcription of these genes was assessed by nuclear “run on” experiments, LPS increased transcription of KC and TNF but not of IL‐1 or JE, implying that the increased levels of mRNA for JE and IL‐1 were attributable to increased stability of mRNA. Likewise, IFNγ did not initiate transcription of JE. When IFNγ and LPS were given together, IFNγ enhanced the LPS‐induced transcription of TNF and KC, suggesting decreased stability of mRNA for KC. A distinct pattern of regulation for each of the four genes was thus observed. Taken together, the data suggest that gene regulation in macrophage activation represents a complex response of enhanced and suppressed transcription and mRNA stability, the precise pattern of which depends on the stimuli given to the macrophages and the gene examined.

Expression of the transferrin receptor on murine peritoneal macrophages is modulated by in vitro treatment with interferon gamma

The expression of transferrin receptors on murine peritoneal macrophages has been shown to be down regulated during functional activation in vivo. This observation suggested that the level of transferrin receptor expression varies in response to discrete extracellular signals known to induce macrophage activation. We have tested this concept directly and have shown that decreased transferrin receptor expression can be reproduced in vitro by treatment of inflammatory macrophages with preparations of interferon gamma derived from a T cell hybridoma supernatant. The ability of this agent to down regulate the expression of the transferrin receptor exhibited dose and time dependencies similar to those required for development of other macrophage functions in vitro. The addition of LPS produced no further decrease in receptor expression. Furthermore, murine gamma interferon, produced by recombinant DNA technology also caused a downshift in transferrin receptor expression at doses similar to those which have been shown previously to induce activation. The changes in receptor activity were the result of altered numbers of binding sites and the receptor:ligand affinity remained unaffected. These results indicate that altered expression of the transferrin receptor is one element of the pleiotypic change which macrophages undergo in response to IFN gamma. This system may, therefore, provide a useful model in which to study the biochemical basis of IFN gamma action in mononuclear phagocytes.

Effects of oxidized LDL on mononuclear phagocytes: inhibition of induction of four inflammatory cytokine gene RNAs, release of NO, and cytolysis of tumor cells.

A critical step in development of atherosclerosis is the interaction of oxidized low‐density lipoprotein (LDL) with mononuclear phagocytes. Oxidized LDL, as well as acetyl‐LDL, is rapidly taken up into macrophages via a family of scavenger receptors. We report that macrophages treated with oxidized LDL have markedly lower levels of mRNA specific for the genes MCP‐1, TNF‐α, IL‐1α, and KC as measured by Northern blot analyses of lipopolysaccharide (LPS)‐stimulated macrophages. By contrast, acetyl‐LDL does not inhibit these genes at the doses at which oxidized‐LDL is effective. Similar effects are observed whether the LDL is oxidized in the presence of Cu2+ or of Fe2+. Such inhibition also occurs when maleylated bovine serum albumin (BSA), which also clears by one or more scavenger receptors on macrophages, is used as the stimulant. Fe2+ or Cu2+ oxidized LDL inhibits release of nitric oxide when triggered by LPS and direct cytolysis of tumor cells when triggered by maleylated BSA or LPS. Taken together, the data presented indicate that oxidized LDL inhibits induction of several important gene RNAs as well as functional markers that characterize the development of inflammatory and fully activated macrophages. J. Leukoc. Biol. 57: 427–433; 1995.

Interaction of bacillus calmette-guerin-activated macrophages and neoplastic cells in vitro. II. The relationship of selective binding to cytolysis.

Abstract The selective binding in vitro between BCG-activated peritoneal macrophages from C57B1/6J mice and five nonadherent neoplastic targets (P815, EL-4, P388, RBL-5, and L1210) was found to lead to cytolysis of the targets. Cytolysis was selective, in that interaction between BCG macrophages and the neoplastic targets led to significant lysis, while interaction between these targets and inflammatory macrophages or between lymphocytes and either type of macrophage did not. Cytolysis of the targets was respectively decreased or increased by treatments which decreased (chelators or trypsinization of the macrophages) or increased (Con A or neuraminadase plus galactose oxidase) binding. Addition of unlabeled P815 or EL-4 cells but not lymphocytes reduced binding and cytolysis of subsequently added EL-4 or P815 targets. El-4 or P815 targets bound to the BCG-activated macrophages were lysed in preference to targets not bound to the macrophages. Interposition of a mechanical barrier between the macrophages and the targets blocked both binding and cytolysis. The data suggest that the spontaneous binding of EL-4 or P815 targets to BCG-activated peritoneal macrophages is an initial and necessary step in cytolytic destruction of those targets.