Lipopolysaccharide induces Egr-1 mRNA and protein in murine peritoneal macrophages.

Bacterial LPS has diverse effects on the function of immune cells, in general, and macrophages, in particular. The intracellular molecular events that mediate the effects of LPS are unclear. We undertook a series of studies in thioglycollate-elicited murine peritoneal macrophages to evaluate the effect of LPS on expression of Egr-1, a member of the immediate early response gene family. Egr-1 may function as an intranuclear "third messenger" because it is rapidly induced in a variety of cell types and encodes a 75- to 80-kDa nuclear phosphoprotein that activates transcription of genes containing the DNA consensus sequence GCGGGGGCG. LPS from Salmonella minnesota Re595 induced a maximal increase in Egr-1 mRNA in macrophages after 30 to 60 min of incubation that returned to baseline level by 120 min. LPS increased Egr-1 mRNA at 0.01 to 0.1 ng/ml with a maximal effect at 10 to 100 ng/ml. LPS markedly increased the transcription rate of Egr-1 by 10 min of incubation using nuclear run on analysis. Using a polyclonal anti-Egr-1 antibody, nuclear staining for Egr-1 protein was prominent after 1 to 2 h of incubation with LPS and declined to baseline by 4 h. Inasmuch as protein kinase C (PKC) has been implicated in mediating the effects of LPS, we determined whether PKC was required for LPS to increase Egr-1 mRNA. Two pharmacologic approaches were used to deplete PKC, PMA pretreatment, and H-7. The induction of Egr-1 mRNA by LPS was markedly reduced in PKC-depleted macrophages. These data reveal that LPS induces transcriptional activation of Egr-1 and increases Egr-1 protein in peritoneal macrophages. In addition, these findings support further study of the potential role of Egr-1 in mediating the effects of LPS in peritoneal macrophages.

Recombinant granulocyte-macrophage colony-stimulating factor increases adenylate cyclase activity in murine peritoneal macrophages.

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotrophic cytokine which stimulates the function and proliferation of macrophage populations. Although the effects of GM-CSF are diverse and GM-CSF has entered into clinical trials, relatively little is known about signal transduction pathways utilized by GM-CSF. In view of previous studies which have suggested that some of the effects of GM-CSF on monocyte-macrophages can be mimicked by agents which increase intracellular cAMP, we investigated the effect of rGM-CSF on adenylate cyclase (AC) activity in murine peritoneal macrophages. Adenylate cyclase activity was quantitated in macrophage membrane preparations and in intact cells. In seven separate experiments, GM-CSF (50 U/ml) increased AC activity by 61(6)% relative to macrophages treated with carrier medium alone. A dose-dependent increase in AC activity was observed (10 to 100 U/ml) which peaked within 1 to 5 min after the addition of GM-CSF and returned to basal levels by 10 to 20 min. Lineweaver-Burk analysis revealed that the Vmax of macrophage AC was increased from 0.40 to 0.52 pmoles cAMP/min by GM-CSF but the Km was unchanged. Intracellular cAMP was increased by GM-CSF to 129(27)% of control values by 1 min of treatment (n = 6). Under similar experimental conditions, GM-CSF did not increase the activity of PK C (n = 14) or phospholipase A2 (n = 3) in peritoneal macrophages. These data show that macrophage adenylate cyclase activity is rapidly stimulated by GM-CSF. Moreover, these findings support further study of the role of cAMP in transmitting the intracellular signals initiated by GM-CSF in tissue macrophages.

Granulocyte-macrophage colony-stimulating factor enhances selective effector functions of tissue-derived macrophages.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is produced by a variety of cells at sites of exposure to antigens. GM-CSF has a stimulatory effect on a number of neutrophil functions, but the effect on macrophage function is less clear. We investigated the effect of purified murine recombinant GM-CSF on murine peritoneal macrophage oxidative metabolism, Fc-dependent phagocytosis, anti-Toxoplasma activity, and expression of class II major histocompatibility antigen (Iad). GM-CSF significantly increased phorbol myristate acetate- and zymosan-elicited H2O2 release by resident and thioglycollate-elicited macrophages after 48 hours in vitro. The effect of recombinant GM-CSF was blocked by polyclonal anti-GM-CSF antibody and was not altered by lipopolysaccharide (0.01 to 1.0 microgram/mL). GM-CSF also stimulated Fc-dependent phagocytosis by peritoneal macrophages, although the stimulation of resident macrophages (1.4-fold) was less dramatic than that of thioglycollate-elicited cells (2.1-fold). GM-CSF (at doses up to 100 U/mL) had no effect on macrophage anti-Toxoplasma activity or on expression of Iad. In addition to stimulating macrophage growth, GM-CSF selectively promotes the functional capacity of tissue-derived macrophages.

Stimulation of macrophage H2O2 release by resident thymocytes: effect of a soluble factor distinct from interferon-gamma.

Activated T cells are known to stimulate macrophage oxidative metabolism and antimicrobial activity through release of interferon-gamma (IFN-gamma). In contrast, the role of nonactivated T cells in regulating macrophage effector functions is less well defined. We have previously reported that a low molecular weight soluble factor derived from resident (nonactivated) thymocytes enhances macrophage receptor-mediated phagocytosis. In the present study, we examined the capacity of resident murine thymocytes to stimulate the respiratory burst and microbicidal activity of peritoneal macrophages. Macrophages cultured for 1-2 days with cell-free thymocyte supernatant (TS) released two to three times more H2O2 in response to PMA or opsonized zymosan than did control macrophages. The H2O2-stimulating factor in TS was distinguished from IFN-gamma by its heat stability (100 degrees C, 20 min), approximate MW of 2400 Da (gel filtration high-pressure liquid chromatography), and absence of interferon activity in both antiviral and enzyme-linked immunosorbent assays. TS-treated macrophages, however, did not exhibit a greater capacity to kill or inhibit the intracellular growth of Toxoplasma gondii, indicating that the thymocyte factor did not fully activate macrophage microbicidal mechanisms. These data suggest that thymocytes can increase the respiratory burst capacity of macrophages in the absence of antigen-specific immune responses.