Regulation of aminopeptidase-N (CD13) and Fc epsilon RIIb (CD23) expression by IL-4 depends on the stage of maturation of monocytes/macrophages.

IL-4 has multiple biologic activities and it has been shown to have effects on B and T lymphocytes, mast cells, NK cells, and monocytes. We studied the influence of IL-4 on the expression of cell membrane determinants, in particular aminopeptidase-N (CD13) and Fc epsilon RIIb (CD23), on human peripheral blood monocytes. We compared the response of monocytes with the response of human alveolar macrophages and monocytic cell lines (U937 and THP1), as mature and more immature representatives of the mononuclear phagocyte system, respectively. A dose-dependent increase of the expression of CD13 Ag was observed when monocytes were cultured with IL-4. Kinetic analyses revealed that this induction was maximal after 2 to 3 days of culture and resembled the kinetics of IL-4-induced expression of Fc epsilon RIIb on monocytes. This IL-4-induced increase was absent when monocytes were cultured with IL-4 and an anti-IL-4 antiserum. Concomitantly, an IL-4-induced increase in leucine-aminopeptidase activity could be observed. Northern blot analysis showed that incubation of monocytes with IL-4 induced a marked increase in CD13 mRNA. Alveolar macrophages also exhibited an increase in CD13 Ag expression when exposed to IL-4. Surprisingly, IL-4 was unable to induce expression of Fc epsilon RIIb on alveolar macrophages. U937 and THP1 cells did not show an induction of CD13 Ag when cultured in the presence of IL-4. However, IL-4 did induce the expression of Fc epsilon RIIb on both cell lines, suggesting the presence of functional IL-4R. Our data demonstrate that IL-4 increases the expression of CD13 Ag on monocytes. This IL-4-induced increase can also be observed in more mature monocytic cells such as alveolar macrophages, but is absent in immature cells such as U937 or THP1 cells. This is functionally accompanied by an increase in leucine-aminopeptidase activity and may be part of the general activation of monocytes/macrophages by IL-4. In conclusion, the data suggest that IL-4 responsiveness, in particular the induction of CD13 Ag and Fc epsilon RIIb expression, may be dependent on the stage of maturation of monocytes/macrophages.     

Switch of histamine receptor expression from H2 to H1 during differentiation of monocytes into macrophages

It is known that histamine suppresses gene expression and synthesis of tumor necrosis factor alpha (TNF-alpha) induced by lipopolysaccharide (LPS) in human peripheral blood mononuclear monocytes (HPM) or alveolar macrophages via histamine H2 receptors. We investigated the effect of histamine and differentiation in macrophages on the expression and secretion of TNF-alpha, TNF-alpha-converting enzyme (TACE), and histamine H1 and H2 receptors by use of a leukemia cell line, U937, and HPM. Differentiation of U937 and HPM cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced the H1 receptor expression and rather suppressed the H2 receptor, resulting in up-regulation of the histamine-induced expression and secretion of TNF-alpha, modulated via TACE. Therefore, histamine failed to inhibit up-regulated expression of TNF-alpha induced by LPS in macrophages. The switch from H2 to H1 receptors during differentiation in the monocyte/macrophage lineage could participate in the pathogenic processes of atherosclerosis and inflammatory reactions in the arterial wall.     

The role of histamine in the intracellular survival of Mycobacterium bovis BCG

The course and outcome of infection with mycobacteria are determined by a complex interplay between the immune system of the host and the survival mechanisms developed by the bacilli. Histamine plays an important role in various processes, including cell division, metabolism, and apoptosis, and it modulates innate and adaptive immune responses. In the present study we investigated the intracellular survival of Mycobacterium bovis BCG in murine bone-marrow macrophages isolated from wild-type (WT) and histidine-decarboxylase knock-out [HDC (-/-)] mice. Mycobacterial titers were significantly higher in the HDC (-/-) macrophages as compared with the WT cells. M. bovis BCG growth in WT macrophages could be enhanced by pyrilamine and cimetidine. Exogenously added histamine decreased the intracellular counts of M. bovis BCG in HDC (-/-) macrophages. Infection of activated macrophages with M. bovis BCG elicited apoptosis, but there was no significant difference between the WT and the HDC (-/-) cells. These bacilli induced comparable levels of tumor necrosis factor-alpha production in the WT and the HDC (-/-) macrophages. M. bovis BCG stimulated interleukin-18 (IL-18) production in the macrophages from WT mice, but not in the HDC (-/-) cells. Exogenously added IL-18 decreased the titers of intracellular mycobacteria in HDC (-/-) cells. In conclusion, these data implicate histamine in the intracellular survival of M. bovis BCG. The cellular control mechanisms restricting the growth of M. bovis BCG are complex and involve H1 and H2 receptor-mediated events. Histamine might be an important mediator of M. bovis BCG-induced IL-18 production, which in turn contributes to immune protection.     

The role of the chemokines MCP-1, GRO-alpha, IL-8 and their receptors in the adhesion of monocytic cells to human atherosclerotic plaques

Monocyte adhesion to the arterial endothelium and subsequent migration into the intima are central events in the pathogenesis of atherosclerosis. Previous experimental models have shown that chemokines can enhance monocyte–endothelial adhesion by activating monocyte integrins. Our study assesses the role of chemokines IL-8, MCP-1 and GRO-α, together with their monocyte receptors CCR2 and CXCR2 in monocyte adhesion to human atherosclerotic plaques. In an adhesion assay, a suspension of monocytic U937 cells was incubated with human atherosclerotic artery sections and the levels of endothelial adhesion were quantified. Adhesion performed in the presence of a monoclonal antibody to a chemokine, chemokine receptor or of an isotype matched control immunoglobulin, shows that antibodies to all chemokines tested, as well as their receptors, inhibit adhesion compared to the control immunoglobulins. Immunohistochemistry demonstrated the expression of MCP-1, GRO-α and their receptors in the endothelial cells and intima of all atherosclerotic lesions. These results suggest that all these chemokines and their receptors can play a role in the adhesion of monocytes to human atherosclerotic plaques. Furthermore, they suggest that these chemokine interactions provide potential targets for the therapy of atherosclerosis.     

Differential regulation of chemokine gene expression by 15-deoxy-delta 12,14 prostaglandin J2

Ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), such as 15-deoxy-Delta(12,14)PGJ2 (15d-PGJ2) have been proposed as a new class of antiinflammatory compounds with possible clinical applications. As there is some controversy over the inhibitory effects of 15d-PGJ2 on chemokine gene expression, we investigated whether 15d-PGJ2 itself affected chemokine gene expression in human monocytes/macrophages and two monocytic cell lines. Here we demonstrate that the 15d-PGJ2 can induce IL-8 gene expression. In contrast, monocyte chemoattractant protein-1 gene expression was suppressed by 15d-PGJ2, while the expression of RANTES was unaltered. Furthermore, concomitant treatment of monocytes/macrophages with 15d-PGJ2 (2.5 x 10(-6) M) potentiated LPS-induced gene expression of IL-8 mRNA, but suppressed PMA-induction of IL-8 mRNA. In addition, treatment of U937 and THP-1 cells with 15d-PGJ2 also resulted in induction of IL-8 gene expression. Further studies demonstrated that 15d-PGJ2 regulated IL-8 gene expression via a ligand-specific and PPARgamma-dependent pathway. Our observations revealed a previous unappreciated function and mechanism of 15d-PGJ2-mediated regulation of cytokine gene expression in monocytes/macrophages.     

Human monocyte differentiation stage affects response to arachidonic acid

AA-induced cell death mechanisms acting on human monocytes and monocyte-derived macrophages (MDM), U937 promonocytes and PMA-differentiated U937 cells were studied. Arachidonic acid induced apoptosis and necrosis in monocytes and U937 cells but only apoptosis in MDM and U937D cells. AA increased both types of death in Mycobacterium tuberculosis-infected cells and increased the percentage of TNFα+ cells and reduced IL-10+ cells. Experiments blocking these cytokines indicated that AA-mediated death was TNFα- and IL-10-independent. The differences in AA-mediated cell death could be explained by high ROS, calpain and sPLA-2 production and activity in monocytes. Blocking sPLA-2 in monocytes and treatment with antioxidants favored M. tuberculosis control whereas AA enhanced M. tuberculosis growth in MDM. Such evidence suggested that AA-modulated effector mechanisms depend on mononuclear phagocytes’ differentiation stage.     

Participation of beta-adrenergic receptors on macrophages in modulation of LPS-induced cytokine release

For several years it is known that beta-adrenergic receptor agonists have anti-inflammatory effects. However, little is known about the role of beta-adrenergic receptors on macrophages in the modulation of cytokine production by beta-agonists during inflammation. In this study, the presence of beta-receptors on PMA-differentiated U937 human macrophages, and the participation of these receptors in the modulation of LPS-mediated cytokine production by beta-agonists was investigated. Total beta-receptor expression on undifferentiated (monocyte) and PMA-differentiated U937 cells was established using receptor binding studies on membrane fractions with a radio ligand. The expression of beta-receptors proved to be significantly lower on monocytes than on macrophages, additionally a predominant expression of beta 2-receptors was found. Production of the cytokines TNF-alpha, IL-6, and IL-10 by LPS-stimulated differentiated U937 cells was measured in time. Peak concentrations for TNF-alpha, IL-6 and IL-10 occurred at 3, 12 and 9 hrs, respectively. When differentiated U937 cells were incubated with both LPS and the beta-agonist clenbuterol the production of TNF-alpha and IL-6 was significantly reduced. However the production of IL-10 was increased. To study the mechanism of modulation of cytokine production in more detail, U937 macrophages were incubated with LPS/clenbuterol in combination with selective beta 1- and beta 2-antagonists. These results indicated that the beta 2- and not the beta 1-receptor is involved in the anti-inflammatory activity of clenbuterol.     

In vitro increase of histidine decarboxylase activity and release of histamine by peritoneal resident cells of mast cell-deficient W/Wv mice; possible involvement of macrophages

When peritoneal resident cells (PRCs) of genetically mast cell-deficient WBB6F1-W/Wv mice were cultured in vitro for 5 h at 37 degrees C, their histidine decarboxylase [HDC, L-histidine carboxylase, E.C. 4.1.1.22] activity increased 10-fold. Since inhibitors for energy production and mRNA and protein syntheses inhibited this increase of HDC activity, it appeared to represent de novo synthesis of the enzyme, i.e., induction. This increase was followed by an increase in the amount of histamine in the culture medium of the cells, indicating that histamine synthesized by the induced HDC was not stored in the cells but was quickly released. Mast cells were not involved in the HDC induction, because the extents of HDC induction in PRCs of W/Wv and wild type +/+ mice were similar. The removal of T cells with anti-Thy-1,2 antibody and complement from the PRCs did not affect the HDC induction, but the removal of phagocytes decreased the induction to one-tenth in spite of a 2-fold increase in the proportion of B cells in the PRCs. After separation of the PRCs into adherent and non-adherent fractions, the increase in HDC activity was found to be associated with the adherent fraction that was mostly positive to esterase staining. These results suggest that HDC was induced in peritoneal macrophages.     

Macrophages can produce factors capable of inducing histidine decarboxylase, a histamine-forming enzyme, in vivo in the liver, spleen, and lung of mice

Injection into mice of culture supernatant of P388D1 cells, a murine macrophage cell line, produced a rapid increase in histidine decarboxylase (HDC) activities in the liver, spleen, and lung. Factors in the culture supernatant capable of inducing the HDC elevation were purified by gel filtration and chromatofocusing. Throughout these procedures, the HDC-inducing activity accompanied the mitogenic activity for thymocytes or interleukin 1 (IL-1) activity. Although, because of low purity of the preparations, it is not confirmed whether the HDC inducer is IL-1 itself or not, the present results indicate that P388D1 cells can produce a factor(s) capable of inducing HDC in mouse tissues in vivo. After the injection of the HDC-inducing factor into mice, HDC induction in the tissues occurred within 2 hr and peaked at 2 to 4 hr, resulting in the increase in histamine levels 1 to 10 nmol/g tissue. These results provide important information concerning the source of endogenous histamine that might be involved in inflammatory reactions in delayed-hypersensitivity reactions or in the immune regulation observed in many in vitro systems.     

Activation of apoptosis, but not necrosis, during Mycobacterium tuberculosis infection correlated with decreased bacterial growth: role of TNF-alpha, IL-10, caspases and phospholipase A2

Monocyte/macrophage cell death is an important event during mycobacterial infection. To get insights about the influence of mononuclear phagocyte maturation in this event we compared the response to Mycobacterium tuberculosis (Mtb) infection of fresh isolated monocytes and monocyte-derived macrophages (MDM) from healthy tuberculin positive individuals. Both monocytes and MDM underwent apoptosis, however, there was a higher numbers of apoptotic macrophages with active Caspases 8 and 9. We also compared Mtb-induced cell death in U937 pro-monocytes and PMA-differentiated cells (U937D). In response to Mtb infection, U937D cells underwent apoptosis and promonocytes both apoptosis and necrosis. There were high number of U937D cells producing TNF-α and high number of IL-10⁺ promonocytes. These evidences suggest that U937 could be a valid model to study the mechanisms that rule Mtb-induced cell death. Experiments with the cell line and fresh isolated mononuclear cells with pharmacological inhibitors showed that induction of necrosis involved calcium and cAMP signals resulting in IL-10 production. Necrosis also correlated with Caspase 3, PLA2 activity and bacterial growth. In U937D cells and monocytes from healthy donors there was activation of calcium, TNF-α and Caspase 8 activation and decreased bacterial load. Understanding the mechanisms that control the dichotomy events between apoptosis and necrosis/oncosis associated with cell maturity might open new strategies to better control the course of mycobacterial infections.     

Regulation of monocyte/macrophage C2 production and HLA-DR expression by IL-4 (BSF-1) and IFN-gamma

IL-4 was originally described on the basis of its ability to co-stimulate the proliferation of resting B cells treated with anti-IgM. Recently, this cytokine has been shown to have other effects on mast cells, T cells, B cells, and macrophages. We studied the ability of IL-4 to regulate the production of C2 by human monocytes and monocytic cell lines and compared this with stimulation of HLA-DR expression, another recently described activity of IL-4. Responses to IL-4 were compared to IFN-gamma, a cytokine with both activities. IL-4 up-regulated C2 production by human monocytes and this effect was not inhibited by neutralizing anti-IFN-gamma antibody. IL-4 also stimulated C2 production by HL-60 cells that had been pre-treated with vitamin D3 to induce monocytic differentiation. IL-4 did not stimulate C2 production by U937 cells. IFN-gamma, in contrast to IL-4, stimulates C2 production by all three cell types. Although IL-4 increased C2 production by HL-60 cells we could not detect C2 mRNA by Northern blotting. However, co-stimulation of these cells with IL-4 and low concentrations of IFN-gamma resulted in an additive effect on C2 production and a greater increase in C2 mRNA than was seen with IFN-gamma alone. As reported by others, IL-4-stimulated HLA-DR expression by monocytes. In contrast to our findings regarding C2 production, stimulation of HLA-DR expression was inhibited by neutralizing anti-IFN-gamma mAb and IL-4 did not stimulate HLA-DR expression by U937 or HL-60 cells. IFN-gamma stimulated HLA-DR expression by all three cell types. These results identify IL-4 as an additional cytokine able to directly stimulate C2 production by human monocytes and by a monocytic cell line whereas IL-4 stimulation of HLA-DR expression by monocytes appears to be IFN-gamma dependent.     

IL-6 augments Fc IgE receptor (Fc epsilon RII/CD23) expression on human monoblastic/monocytic cell lines U937, THP-1, and Mono-Mac-6 but not on blood monocytes. Regulatory effects of IL-4 and IFN-gamma.

Human monoblastic/monocytic leukemia cell lines U937, THP-1, Mono-Mac-6, and blood monocytes were incubated with various concentrations of human rIL-6 and other cytokines and analyzed for their capacity to bind several anti-Fc epsilon RII/CD23 mAb. A marked and dose-dependent increase in the percentage of CD23+ cells, as well as in the mean channel fluorescence intensity, as demonstrated by FACS analysis, was noted after 8- to 72-h incubation of U937 cells with 1 to 1000 U/ml of human rIL-6. Furthermore, rIL-4 synergized with rIL-6 and rIFN-tau in augmenting the Fc epsilon RII expression on U937 cells, whereas rIFN-tau and rIL-6 showed rather additive effects. The enhancement of CD23 expression on IL-6-treated U937 cells was blocked by anti-IL-6 antibodies. Northern blot analysis, employing cDNA probes for Fc epsilon RII, showed that U937 cells contain Fc epsilon RII-specific mRNA. The level of Fc epsilon RII-encoding mRNA was evidently increased by treatment of U937 cells with human rIL-6, rIL-4, or with rIL-6 + rIL-4. The expression of CD23 on THP-1 and Mono-Mac-6 cells was increased slightly by rIL-6 and markedly by rIL-4, rIFN-tau, or a mixture of them. Approximately 14% of blood monocytes, isolated from apparently healthy donors, constitutively possess Fc epsilon RII. In contrast to the cell lines, the Fc epsilon RII density and the percentage of blood monocytes bearing Fc epsilon RII was not augmented by IL-6. Furthermore, rIL-6, and more evidently rIFN-tau, down-regulate rIL-4-driven Fc epsilon RII expression on monocytes but not on monocytic cell lines. Our findings point to differences in the capability of mononuclear phagocytes to respond to cytokine treatment, which may be differentiation dependent, and suggest separate regulatory pathways.     

Increase of histidine decarboxylase activity in murine myelomonocytic leukemia cells (WEHI-3B) in parallel to their differentiation into macrophages

When cells of mouse myelomonocytyc leukemia cell line, WEHI-3B, were cultured in the presence of actinomycin D plus the serum which was obtained from mice injected with bacterial endotoxin, i.e., lipopolysaccharide, their histidine decarboxylase (l-histidine carboxy-lyase, EC 4.1.1.22) (HDC) activity increased about 100-fold with a peak at 48 h. According to the increase in HDC activity, the expression of surface antigens associated with macrophages, such as Mac II, Mac III and Ia, increased markedly on WEHI-3B cells as well as their morphological changes to macrophages. Histamine levels in the culture medium increased concomitantly with the increase in the HDC activity in WEHI-3B cells, whereas the histamine contents inside the cells did not increase remarkably. Furthermore, the addition of lipopolysaccharide to the culture medium caused an additional 2-fold increase in the HDC activity of WEHI-3B cells. These results indicate that the increase in HDC activity in WEHI-3B cells may represent an event in the process of the differentiation to macrophages.     

Increase of histidine decarboxylase activity in murine myelomonocytic leukemia cells (WEHI-3B) in parallel to their differentiation into macrophages

When cells of mouse myelomonocytic leukemia cell line, WEHI-3B, were cultured in the presence of actinomycin D plus the serum which was obtained from mice injected with bacterial endotoxin, i.e., lipopolysaccharide, their histidine decarboxylase (L-histidine carboxy-lyase, EC 4.1.1.22) (HDC) activity increased about 100-fold with a peak at 48 h. According to the increase in HDC activity, the expression of surface antigens associated with macrophages, such as Mac II, Mac III and Iad, increased markedly on WEHI-3B cells as well as their morphological changes to macrophages. Histamine levels in the culture medium increased concomitantly with the increase in the HDC activity in WEHI-3B cells, whereas the histamine contents inside the cells did not increase remarkably. Furthermore, the addition of lipopolysaccharide to the culture medium caused an additional 2-fold increase in the HDC activity of WEHI-3B cells. These results indicate that the increase in HDC activity in WEHI-3B cells may represent an event in the process of the differentiation to macrophages.     

Histamine and histidine decarboxylase are hallmark features of ECL cells but not G cells in rat stomach

The oxyntic mucosa of the rat stomach is rich in ECL cells which produce and secrete histamine in response to gastrin. Histamine and the histamine-forming enzyme histidine decarboxylase (HDC) have been claimed to occur also in the gastrin-secreting G cells in the antrum. In the present study, we used a panel of five HDC antisera and one histamine antiserum to investigate whether histamine and HDC are exclusive to the ECL cells. By immunocytochemistry, we could show that the ECL cells were stained with the histamine antiserum and all five HDC antisera. The G cells, however, were not stained with the histamine antiserum, but with three of the five HDC antisera. Thus, histamine and HDC coexist in the ECL cells (oxyntic mucosa) but not in G cells (antral mucosa). Western blot analysis revealed a typical pattern of HDC-immunoreactive bands (74, 63 and 54 kDa) in oxyntic mucosa extracts with all five antisera. In antral extracts, immunoreactive bands were detected with three of the five HDC antisera (same as above); the pattern of immunoreactivity differed from that in oxyntic mucosa. Food intake of fasted rats or treatment with the proton pump inhibitor omeprazole raised the HDC activity and the HDC protein content of the oxyntic mucosa but not of the antral mucosa; the HDC activity in the antrum was barely detectable. We suggest that the HDC-like immunoreactivity in the antrum represents a cross-reaction with non-HDC proteins and conclude that histamine and HDC are hallmark features of ECL cells but not of G cells.     

Regulation of the expression of IL-6 in human monocytes

IL-6 is a cellular regulatory molecule with various cell-dependent functions. We have studied the control of IL-6 expression in human monocytes because they play a key role in the production of this molecule. The effects of adherence and different cytokines including CSF-1, IFN-gamma, IL-1 alpha, and granulocyte-macrophage-CSF were tested on IL-6 expression. IL-6 mRNA was usually not detected in the starting population of PBMC. Adherence induced IL-6 gene expression in monocytes in less than 2 h and subsequently IL-6 secretion. Priming of monocytes by adherence was more efficient for IL-6 overinduction by CSF-1. In contrast, high level induction of IL-6 by IFN-gamma in unfractionated PBMC did not require adherence and in situ hybridization revealed that IL-6 mRNA was present in monocytes but not in lymphocytes. A similar phenomenon was observed for IL-1 alpha and granulocyte-macrophage-CSF. Two cell lines, HL-60 and U937, in which monocytic differentiation occurs after induction by PMA, were subsequently investigated. IL-6 was not constitutively detectable in these two cell lines, whereas PMA treatment induced IL-6 expression. This effect was rapid (30 min) and transitory in HL-60, whereas IL-6 mRNA was still detected after 72 h of induction in U937. Addition of human rIL-6 on U937 and HL-60 cells inhibited their proliferation and enhanced expression of HLA class I Ag.