Thomas A. Hamilton

Molecular mechanisms of signal transduction in macrophages

The mechanisms by which extracellular signals are received and transduced across the cell membrane are being illuminated by studies in macrophages. In this review Tom Hamilton and Dolph Adams discuss mechanisms of signal transduction which either initiate rapid execution of function in macrophages or alter the cells potential for taking such action.

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

Tissue-specific expression of murine IP-10 mRNA following systemic treatment with interferon γ

We have examined the tissue distribution of 10‐kd inflammatory protein (IP‐10) mRNA expression in C57Bl/6 mice injected intravenously (i.v.) with various inflammatory stimuli. IP‐10 mRNA was strongly induced by interferon‐γ (IFN‐γ) in liver and kidney but only poorly in skin, heart, and lung. IFN‐γ had nearly equivalent access to these tissues as indicated by the distribution of radiolabeled recombinant IFN‐γ 1 h after injection. The time course of IP‐10 mRNA appearance was rapid and transient in both liver and kidney; maximal expression in the liver (2 h) preceded that in the kidney (3 h) and declined rapidly thereafter in both tissues. Expression of IP‐10 mRNA in the liver and kidney was highly sensitive to IFN‐γ treatment; nearly maximal stimulation occurred with injection of 500 U of IFN‐γ per mouse. Comparable stimulation of IP‐10 mRNA expression in splenic macrophages required 10,000 U of IFN‐γ administered i.v., indicating that liver and kidney responses are 10‐ to 20‐fold more sensitive. IP‐10 mRNA expression in both tissues was not restricted to stimulation by IFN‐γ but was also seen with injection of lipopolysaccharide (LPS) (25 μg/mouse) or IFN‐β (100,000 U/mouse). Two other members of the IP‐10 gene family, KC (gro) and JE (MCP‐1), were expressed at lower levels under similar treatment conditions. Analysis of IP‐10 mRNA distribution in the liver and kidney by in situ hybridization indicated that expression in both tissues was most prominent in the reticuloendothelial cell system, particularly in the endothelial lining of the microvascular circulation. Although the function of the IP‐10 gene product has not been defined, these results suggest that it may play an important role in the response of both the liver and kidney to systemic inflammation.

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.

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.

Biochemical models of interferon-γ-mediated macrophage activation: Independent regulation of lymphocyte function associated antigen (LFA)-1 and I-A antigen on murine peritoneal macrophages

IFN-gamma can induce the expression of both class II histocompatibility antigens (Ia) and the lymphocyte function associated (LFA)-1 antigen on murine peritoneal macrophages. We have examined the molecular changes which lead to altered expression of these two cell surface proteins to determine whether they are regulated by similar or independent mechanisms. While I-A antigen expression can be induced or enhanced by treatment of macrophages with either phorbol diesters and/or the Ca2+ ionophore A23187, these agents had no effect upon expression of LFA-1 under similar conditions. Macrophages from the A/J strain mouse exhibit a deficiency in their sensitivity to IFN-gamma which is seen in our studies as an inability of IFN-gamma to elevate I-A antigen expression. However, expression of I-A could be modulated in these cells by treatment with either phorbol diesters or A23187. In contrast, IFN-gamma could induce LFA-1 antigen on A/J derived macrophages and this was not affected by either phorbol or A23187. Thus these two antigens, despite coordinate expression in response to IFN-gamma in normal mouse strains, are clearly regulated independently. These results suggest that IFN-gamma generates at least two independent molecular events in macrophages which ultimately modulate the expression of cell surface proteins important to the performance of activated functions.

Quiescent lymphocytes express intracellular transferrin receptors

Both quiescent and concanavalin A stimulated murine splenic lymphocytes were examined for the expression of surface and intracellular binding sites for the serum glycoprotein transferrin. Transferrin binding activity was observed on the surface of mitogen stimulated cells only. When soluble detergent extracts of both populations were studied, quiescent lymphocytes were shown to contain a significant pool of non-surface exposed, intracellular receptors which was approximately 20% of the total receptor complement of proliferating cells. Because the ratio of surface to intracellular binding sites was dramatically increased following mitogen stimulation, the regulation of transferrin receptor expression during this process may involve a substantial alteration in its subcellular distribution in addition to the well documented increase in number of binding sites.

The early competence genes JE and KC are differentially regulated in murine peritoneal macrophages in response to lipopolysaccharide.

Treatment of murine peritoneal macrophages with bacterial lipopolysaccharide (LPS) has been previously documented to induce accumulation of mRNA for the early or competence genes JE and KC; the data further suggested that multiple pathways existed for the transduction of the LPS signal, since induction of mRNA for JE was related to breakdown of polyphosphoinositides while induction of KC was not (Introna et al. 1987 J. Immunol. 138, 3891). This study provides analysis of the regulation of the expression of these genes by using the nuclear transcription assay. We present evidence that LPS enhanced transcriptional activity of the KC gene, but not of the JE gene. By contrast, serum stimulation of quiescent BALB/c-3T3 fibroblasts induced transcription of the JE and KC genes. The data imply that expression of the KC gene in LPS-treated macrophages is regulated transcriptionally, while that of the JE gene is regulated post-transcriptionally. Furthermore, there appear to be two mechanistic pathways for the induction of JE mRNA depending upon the stimulus and upon the cell type: one involving transcriptional and one post-transcriptional control.

Respiratory burst in murine peritoneal macrophages: Differential sensitivity to phorbol diesters by macrophages in different states of functional activation

Activation of macrophages either in vivo or in vitro can modulate the capacity to generate and secrete reactive oxygen intermediates including H2O2 and O2-. Thus, the cellular and biochemical components requisite for execution of the respiratory burst must be regulated during the activation process. In the present report, we have examined murine peritoneal macrophages in different stages of activation for their sensitivity to stimulants of respiratory burst known to activate protein kinase c (i.e., phorbol dibutyrate or diacylglycerol). The results demonstrated that more highly activated macrophages showed, in addition to greater magnitude of H2O2 or O2- production, a two- to fourfold greater sensitivity to these stimuli. While more active macrophages also exhibited a higher rate of H2O2 secretion, the time at which secretion was measured did not account for or modulate the heightened sensitivity. The increased sensitivity to stimulation was dependent upon the stage of activation and not on the agent used to elicit the macrophages. Increased sensitivity of the more active macrophage populations was also seen when physiologic stimuli (i.e., insoluble immune complexes or unopsonized zymosan) were used. These findings indicate that macrophage activation for H2O2 secretion modulates the sensitivity to stimulation such that more H2O2 is produced in a shorter time and at a lower concentration of stimulus, thereby heightening the inflammatory response in several independent ways. Because all the stimuli employed in the present study have in common the ability to activate protein kinase c (either directly or indirectly), the data also suggest that this form of macrophage activation may involve, at least in part, modulation of the stimulus-response coupling mechanisms which utilize this enzyme.