Search

Although macrophage activation is induced in a complex manner by signals such as interferon-gamma (IFN gamma) 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. IFN gamma initiated heightened mRNA levels for JE but did not alter specific mRNA for IL-1, TNF, or KC. When IFN gamma 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, IFN gamma did not initiate transcription of JE. When IFN gamma and LPS were given together, IFN gamma 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.

The surface expression of class II major histocompatibility molecules (immune associated or Ia antigens) is an acquired property of macrophages, essential to their ability to interact effectively with T lymphocytes. Surface expression of Ia is induced by stimulants such as interferon-gamma and is suppressed by agents such as lipopolysaccharide (LPS). Recent studies on several cultured cell lines indicate that interferon-gamma can heighten cellular levels of mRNA encoding Ia, and the level of such mRNA may represent an important regulatory focus for controlling expression of surface Ia. Murine peritoneal macrophages were treated with interferon-gamma and/or LPS and expression of Ia mRNA determined by Northern blot analysis with a probe specific for the murine beta-chain of I-A. mRNA specific for I-A beta was not detectable in explanted macrophages obtained from sites of sterile inflammation but was induced by treatment of purified recombinant interferon-gamma. This effect was dose dependent and was optimal by 24 hr after stimulation. Ia-specific mRNA preceded the surface expression of Ia as monitored by a radioimmunoassay using a monoclonal antibody specific for I-A beta. When a physiologic dose of LPS was added concomitantly with the interferon-gamma, the time course of induction if Ia-specific mRNA was not altered, but the amount of such mRNA detected was suppressed 40 to 80%. This effect was dependent on the dose of LPS, and the levels of mRNA correlated closely with subsequent surface expression of Ia. The ability of LPS to suppress both mRNA and cell surface Ia expression required that the suppressive agent be added within 12 hr of the inducing stimulus. This is the time frame during which accumulation of mRNA occurs. Thus the data demonstrates that accumulation of specific mRNA is a major regulatory focus governing expression of Ia both by interferon-gamma and LPS.

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.

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.

A cDNA library from LPS-treated murine peritoneal macrophages has been screened by differential hybridization with radiolabeled cDNA from untreated and LPS-treated macrophages. Six clones hybridizing with mRNA sequences present in LPS-treated cells but not in controls were selected for further characterization. When the recombinant bacteriophage DNA from each clone was used as a probe in Northern analysis of total RNA from LPS-treated macrophages, inducible mRNA ranging from 1.45 to 6.4 kb were seen. In five of six cases, the mRNA expression was undetectable in untreated macrophage cultures. All but one clone identified mRNA that were inducible even in the presence of cycloheximide, indicating the independence of such gene expression from protein synthesis; none of the genes were superinduced by this treatment. The time course of expression differed among the individual genes. Four were induced transiently, whereas two showed stable increasing accumulation through an 8-h period after stimulation. In addition, four of the genes were seen within 30 min of stimulation, whereas two were seen only after 2 to 4 h. Two genes were induced only by treatment with LPS, whereas four were also induced in response to other agents, including IFN-gamma, macrophage CSF, and PMA. The insert sequences from these recombinant clones did not hybridize with a set of cDNA encoding other inducible gene products, including TNF, IL-1, ornithine decarboxylase, c-myc, c-fos, JE, or KC. Thus, these six cDNA appear to encode inducible macrophage genes that are distinct from one another as well as from a selection of previously described early genes. Although their functional identity remains indeterminate, they may encode previously described early proteins induced in macrophages treated with LPS.