IFN-gamma mediates increased glucocorticoid receptor expression in murine macrophages.

Exposure of the murine macrophage cell line, RAW 264.7, to murine rIFN-gamma resulted in a significant increase in the number of glucocorticoid receptors (GcR). A doubling in the number of GcR was observed as early as 24 h after rIFN-gamma treatment, and receptor number was maximal by 36 h after rIFN-gamma treatment and represented approximately a fourfold increase. Scatchard analysis indicated that a twofold increase in GcR affinity was concomitant with the rIFN-gamma-induced increase in GcR number in RAW 264.7 cells. Increased GcR numbers were induced after exposure of RAW 264.7 cells to as little as 0.1 U/ml rIFN-gamma, and optimal expression was observed at 5 U/ml. Treatment of peritoneal exudate macrophages from C3H/OuJ mice and the LPS hyporesponsive mouse strain, C3H/HeJ, with rIFN-gamma induced an approximately twofold increase in the GcR with no concomitant change in receptor affinity. These results suggest that IFN-gamma may be essential not only for macrophage activation, but also for increasing macrophage sensitivity to feedback inhibition by glucocorticoids by increasing the number and/or affinity of available GcR.     

Correlation between DNA methylation and murine IFN-gamma and IL-4 expression

In order to determine the possible role of DNA methylation as a regulatory mechanism for the restricted pattern of lymphokine production among differentiated Th(1)and Th(2)cells, we examined the extent of methylation of the interferon gamma (IFN-gamma) and the interleukin 4 (IL-4) genes in fresh activated murine Th(0), Th(1)and Th(2)cells, unstimulated naive T cells, B cells, bone marrow derived non-B non-T cells, thymocytes and liver. All of the CpG dinucleotides examined in the IL-4 and the IFN-gamma genes, were fully methylated over the body of the gene in all of the examined cells. However, analysis of the promoter regions of these genes revealed a different pattern. While the IL-4 promoter is fully methylated in all of the examined cells, two adjacent CpG dinucleotides near the initiation point of the IFN-gamma gene were unmethylated in all T cells, including 17-day-old fetal thymocytes. In contrast, B cells, bone marrow non-B non-T cells and liver cells displayed a full methylated profile of the IFNgamma promoter. These results suggest that the mutually exclusive pattern of IFNgamma and IL-4 production in Th(1)and Th(2)cells is not regulated by differential demethylation of these two genes.     

Characterization of IL-2 receptor expression and function on murine macrophages

This study was designed to examine the expression and function of IL-2R on murine macrophages. We used a model system of murine macrophage cell lines (ANA-1 and GG2EE) that was established by infecting normal murine bone marrow-derived cells with the J2 (v-raf/v-myc) recombinant murine retrovirus. ANA-1 macrophages did not constitutively express detectable levels of mRNA for the p55, IL-2R alpha. However, a brief exposure to IFN-gamma was sufficient to induce IL-2R alpha mRNA in ANA-1 macrophages. Flow cytometric analysis indicated that ANA-1 macrophages expressed low constitutive levels of IL-2R alpha on their cell surface that were augmented after treatment of the cells with IFN-gamma. Affinity binding and cross-linking of [125I]IL-2 to ANA-1 macrophages demonstrated that IL-2R alpha and the p70-75, IL-2R beta were both present on ANA-1 macrophages constitutively. IFN-gamma increased the expression of IL-2R alpha on ANA-1 macrophages but did not increase the expression of IL-2R beta on these macrophages. Although IL-2 alone did not induce the tumoricidal activity of ANA-1 macrophages, IL-2 acted synergistically with IFN-gamma to induce macrophage tumoricidal activity. These data demonstrate the expression of IL-2R on murine macrophage cell lines and establish the role of IL-2 as a costimulator of macrophage-mediated tumoricidal activity.     

CpG oligodeoxynucleotides induce murine macrophages to up-regulate chemokine mRNA expression

Intramuscular injection of synthetic oligodeoxynucleotides (ODN) expressing unmethylated CpG motifs trigger the rapid development of a local inflammatory response. In vitro studies demonstrate that macrophages exposed to CpG ODN up-regulate expression of mRNA encoding the chemokines MIP-1alpha, MIP-1beta, MIP-2, RANTES, JE/MCP-1, and IP-10. Within 6 h of in vivo administration, CpG ODN induce a significant increase in chemokine mRNA levels at the site of injection and draining lymph nodes. These chemokines may contribute to the migration and stimulation of inflammatory cells that contribute to the development of CpG ODN-induced immune responses.     

IL-4-dependent up-regulation of IL-4 receptor expression in murine T and B cells

This study examined mRNA levels and cell surface expression of IL-4 receptor (IL-4R) in murine T and B cells after incubation with IL-4. Northern blot analysis of mRNA levels of T cells isolated from mesenteric lymph nodes and spleen revealed that IL-4 induced a transient augmentation of IL-4R mRNA in a dose-dependent manner. Maximal levels of mRNA were detected as early as 5 h after initiation of culture. These data were complemented by studies examining the cell surface expression of IL-4R using an anti-IL-4R mAb. Resting T and B lymphocytes express IL-4R (T greater than B) and incubation of these cells with exogenous IL-4 increased IL-4R expression to a maximum after 24 h. This effect was abolished after addition of anti-IL-4 antibody. Continuous incubation of T cells in the presence of high concentrations of IL-4 resulted in a down-regulation of IL-4R expression. Addition of the protein synthesis inhibitor cycloheximide blocked the induced increases in IL-4R expression, indicating the requirement for de novo protein synthesis. Both the levels of mRNA and cell surface expression of IL-4R were not affected by addition of exogenous IL-2, and IL-4 regulation of IL-4R expression was not influenced by the immunosuppressive drug cyclosporin A. These data demonstrate that in T and B cells, IL-4 induces a transient up-regulation of IL-4 mRNA levels that is subsequently reflected in increased numbers of IL-4R displayed on the cell surface. This regulation of IL-4R expression by IL-4 provides an important mechanism for amplification of IL-4-dependent activation pathways.     

T cell substance P receptor governs antigen-elicited IFN-gamma production

Substance P (SP) enhances antigen-dependent T cell IFN-gamma production. It was determined if a T cell neurokinin-1 receptor (NK-1R) was critical for IFN-gamma regulation. T cells from schistosome-infected mice were mixed with splenocytes from uninfected NK-1R knockout (KO) animals. Thus only the schistosome egg antigen-specific T cells expressed NK-1R. The cells were cultured 18 h with or without SP. SP enhanced antigen-induced IFN-gamma production fourfold without affecting IL-4 or IL-5 secretion. NK-1R inhibitor blocked this stimulation. Neither purified T cells nor naive KO splenocytes cultured alone responded to antigen. To further define the importance of T cell NK-1R, we developed a T cell-selective NK-1R KO mouse by reconstituting T cell-deficient Rag mice with NK-1R KO T cells. These mice challanged with schistosomiasis developed abnormal liver granulomas. Granuloma size was smaller in T cell-selective NK-1R KO mice compared with granulomas in Rag reconstituted with normal T cells. Splenocytes and granuloma cells from NK-1R KO mice made less IFN-gamma. The mice also made less IgG2a. Thus T cell NK-1R is important for IFN-gamma regulation.     

Regulation of apoptosis by somatostatin and substance P in peritoneal macrophages

Recent studies have shown that somatostatin (SOM) inhibits interleukin 6 (IL-6) and interferon gamma (IFNgamma) production by lymphocytes and peritoneal macrophages, whereas substance P (SP) enhances these cytokines production. To define the mechanism of the cytokine production enhancements and inhibitions by SOM and SP, we examined the expression of apoptosis modulator, p53, Bcl-2, Bax, inducible nitric oxide synthase (iNOS), Fas, caspase-8 and nitric oxide (NO) in thioglycolate-elicited peritoneal macrophages. SOM caused up-regulation of p53, Bcl-2, Fas and caspase-8 activities, and down-regulation of iNOS expression and NO production. On the other hand, SP slightly induces p53 and highly induces Bcl-2, iNOS expression and NO production. These data suggest that apoptosis by SOM may occur by a Bax- and NO-independent p53 accumulation, and through Fas and caspase-8 activation pathways, and that the inducible expression of Bcl-2 and NO production by SP may contribute to prevent the signals of apoptosis by Bax, and via Fas and caspase-8 activation.     

IL-1 and IL-4 modulate IL-1 receptor expression in a murine T cell line

The combination of IL-1 and IL-4 stimulates the proliferation of certain murine T cell populations. Although this effect has been best characterized for a number of murine type 2 Th cell (Th2) clones, the mechanism(s) by which these cytokines effect this response is unclear. We have examined the effects of IL-1 and IL-4 on IL-1R expression by MD10 cells, and IL-1-responsive murine T cell line. These cells bear specific IL-1R, which bind human and murine IL-1 alpha and -beta. The measured apparent IL-1R dissociation constant ranged from 41 to 255 pM using 125I-HrIL-1 alpha. Cross-linking studies demonstrated two different 125I-HrIL-1 alpha binding complexes having Mr of 70,000 and 130,000 to 156,000. When removed from passage conditions and placed in non-growth factor-supplemented media, MD10 IL-1R expression spontaneously increased two- to fourfold over the first 11 to 12 h of culture followed by a decline. This phenomenon is partially inhibitable by cycloheximide suggesting that protein synthesis is involved. In agreement with other reports, HrIL-1 alpha down-regulated the expression of its own receptor with an ED50 of between 1 and 10 pM HrIL-1 alpha for this effect. In most experiments, low amounts of HrIL-1 alpha (1.0, 0.1 pM) significantly augmented IL-1R expression. Scatchard analysis of data obtained with all HrIL-1 alpha treatment conditions showed that the effects were due to a change in receptor number, not affinity. Significantly, purified murine IL-4 (MpIL-4) augmented MD10 IL-1R expression in both a time- and dose-dependent fashion. In the presence of 50 U/ml MpIL-4, MD10 IL-1R expression increased two- to threefold after 24 h without a change in receptor affinity. When MpIL-4 (50 U/ml) and various amounts of HrIL-1 alpha (.01-1000 pM) were co-added, the down-regulatory effect of high levels of HrIL-1 alpha was significantly antagonized. When added to cultures after 24 h of HrIL-1 alpha (100 pM) treatment, MpIL-4 reversed the IL-1R down-regulatory effect induced by high levels of HrIL-1 alpha. Finally, when combined in MD10 proliferation assays, MpIL-4 synergistically enhanced the proliferation of MD10 cells treated with suboptimal levels of HrIL-1 alpha.(ABSTRACT TRUNCATED AT 400 WORDS)     

Maleylated-BSA suppresses IFN-gamma-mediated Ia expression in murine peritoneal macrophages

Maleylated bovine serum albumin (maleyl-BSA) and other polyanionic polymers that are recognized by cell surface receptors on macrophages have been shown to induce chemotaxis, protease secretion, and tumoricidal function in this cell type. In this paper the effect of maleyl-BSA on Ia antigen expression has been evaluated. In a fashion similar to LPS, maleyl-BSA suppressed IFN-gamma-induced expression of Ia in a time- and dose-dependent manner. Also like LPS, maleyl-BSA stimulated the production and secretion of substantial amounts of PGE2 over a 24-hr period. This did not, however, appear to be the primary mechanism by which expression of Ia was suppressed, because co-treatment of the cells with indomethacin, which totally inhibited the production of PGE2, only minimally affected the suppressive activity. Surprisingly, the suppressive activity of both maleyl-BSA and LPS could be largely abrogated by co-treatment of the cells with cyclohexamide during the time period when Ia expression was sensitive to suppression. This effect was selective in that PGE2- or dibutyryl cyclic AMP-induced suppression of Ia expression was not affected by cyclohexamide treatment. The data support the concept that there are multiple molecular mechanisms involved in the negative regulation of IFN-gamma-induced Ia expression in macrophages. Such mechanisms may include, in addition to the synthesis of PGE2 and consequent elevation in intracellular levels of cyclic AMP, one or more proteins made early after treatment with either maleyl-BSA or LPS. Thus the function of some of these early gene products may be to regulate expression of functional genes such as that encoding Ia antigen.     

IL-12 induction of mRNA encoding substance P in murine macrophages from the spleen and sites of inflammation

Substance P (SP), a neuropeptide, interacts with the neurokinin 1 receptor (NK-1R) on immune cells to help control IFN-gamma production. In murine schistosomiasis mansoni, schistosome worms produce ova that incite focal Th2-type granulomatous inflammation within the liver and intestines. Normal gut is characterized by a controlled state of inflammation. IL-10 knockout mice develop chronic Th1-type colitis spontaneously. Both schistosome granulomas and gut mucosa display an SP immune regulatory circuit. However, the origin and regulation of SP production at these sites of inflammation are poorly understood. Macrophages are a potential source of SP. We therefore studied macrophages (F4/80(+)) from these models of inflammation. SP mRNA (preprotachykinin A (PPT A)) was detected within the schistosome granuloma, spleen, and lamina propria macrophages. Compared with those from wild-type mice, granuloma macrophages from STAT6(-/-) mice had 10-fold higher PPT A mRNA expression, whereas in STAT4(-/-) animals, PPT A mRNA expression was nearly abolished. IL-12 signals via STAT4 to induce Th1-type inflammation. It was demonstrated that IL-12, but not IL-18, induces SP mRNA expression in resting splenic macrophages from Schistosoma-infected mice and in wild-type lamina propria mononuclear cells. Thus, macrophages are a source for SP at these sites of chronic inflammation, and IL-12 and STAT4 are regulators of macrophage SP mRNA expression.