Angiopoietin like protein 4 expression is decreased in activated macrophages

Angiopoietin like protein 4 (ANGPTL4) inhibits lipoprotein lipase (LPL) activity. Previous studies have shown that Toll-like Receptor (TLR) activation increases serum levels of ANGPTL4 and expression of ANGPTL4 in liver, heart, muscle, and adipose tissue in mice. ANGPTL4 is expressed in macrophages and is induced by inflammatory saturated fatty acids. The absence of ANGPTL4 leads to the increased uptake of pro-inflammatory saturated fatty acids by macrophages in the mesentery lymph nodes due to the failure of ANGPTL4 to inhibit LPL activity, resulting in peritonitis, intestinal fibrosis, weight loss, and death. Here we determined the effect of TLR activation on the expression of macrophage ANGPTL4. LPS treatment resulted in a 70% decrease in ANGPTL4 expression in mouse spleen, a tissue enriched in macrophages. In mouse peritoneal macrophages, LPS treatment also markedly decreased ANGPTL4 expression. In RAW cells, a macrophage cell line, LPS, zymosan, poly I:C, and imiquimod all inhibited ANGPTL4 expression. In contrast, neither TNF, IL-1, nor IL-6 altered ANGPTL4 expression. Finally, in cholesterol loaded macrophages, LPS treatment still decreased ANGPTL4 expression. Thus, while in most tissues ANGPTL4 expression is stimulated by inflammatory stimuli, in macrophages TLR activators inhibit ANGPTL4 expression, which could lead to a variety of down-stream effects important in host defense and wound repair.     

Expression of macrophage-selective markers in human and rodent adipocytes

CD14, CD68 and/or mouse F4/80 or human epidermal growth factor module-containing mucin-like receptor 1 (EMR1) are widely used as macrophage-specific markers. Since macrophages infiltrate several tissues during inflammatory processes, CD14, CD68 and EMR1-F4/80 have been employed to discriminate between tissue-containing macrophages, like adipose tissue (AT), and other cells. Using real-time PCR experiments, we show that isolated adipocytes from humans and mice AT express high levels of CD14 and CD68 mRNA, whereas EMR1-F4/80 is mainly present in the macrophage-containing stroma-vascular fraction. Furthermore, fibroblasts-like cells (adipoblasts), preadipocytes and adipocytes from the murine cell lines, 3T3-F442A and BFC-1, express CD14 and CD68 mRNA and protein as determined by fluorescence-activated cell sorter, but not F4/80 which, as expected, is strongly expressed in the macrophage cell line RAW264.7. These results reinforce the view that EMR1-F4/80 is the best macrophage marker to date and show that CD14 and CD68 are not macrophage-specific proteins.     

Molecular cloning of F4/80-like-receptor, a seven-span membrane protein expressed differentially by dendritic cell and monocyte-macrophage subpopulations

A novel dendritic cell (DC) surface molecule termed F4/80-like-receptor (FIRE) has been selected based on its differential expression between DC subsets. The gene encoding FIRE has been cloned and sequenced, and mAbs specific for FIRE have been produced. FIRE is a seven-transmembrane-spanning molecule with two epidermal growth factor-like domains in the extracellular region. It is a novel member of the epidermal growth factor/transmembrane-7 protein subfamily and shows similarity to the macrophage marker F4/80. FIRE is expressed by CD8- DC, but not by CD8+ DC, and it is down-regulated on DC activation. It is expressed by blood monocytes and by some tissue macrophages, but not by most macrophage cell lines or by lymphoid cells. FIRE is a useful marker of myeloid cells with a DC developmental potential.     

Repeated social defeat increases the bactericidal activity of splenic macrophages through a Toll-like receptor-dependent pathway

Phagocytes of the innate immune system, such as monocytes/macrophages, represent a first line of defense against invading microorganisms. Psychological stress is often thought to suppress the functioning of these cells, in part due to the immunosuppressive activity of stress-induced glucocorticoid hormones. However, exposure to the stressor social disruption (SDR) has been shown to increase cytokine production by monocytes/macrophages and to reduce their sensitivity to corticosterone. Thus, it was hypothesized that splenic monocytes/macrophages from socially stressed mice would be primed to be more physiologically active than cells from nonstressed controls. Flow cytometry was used to demonstrate that exposure to SDR significantly increased the expression of Toll-like receptors (TLR) 2 and 4 on the surface of splenic macrophages. In a follow-up experiment, exposure to SDR also increased the ability of these macrophages to kill Escherichia coli ex vivo and in vivo. However, SDR failed to increase the bactericidal activity of splenic macrophages from C3H/HeJ mice, which lack functional TLR4. In mice with functional TLR4, the stress-induced increase in bactericidal activity was associated with a significant increase in macrophage gene expression for inducible nitric oxide synthase and subunits of the NADPH oxidase complex, which are responsible for generating reactive nitrogen and oxygen intermediates, respectively. This stress-induced increase in gene expression was not evident in the TLR4-deficient mice. These data indicate that SDR increases TLR expression, which in turn enhances the bactericidal activity of splenic macrophages, in part by increasing pathways responsible for reactive oxygen and nitrogen intermediate production.     

Diversity in phenotype and steroid hormone dependence in dendritic cells and macrophages in the mouse uterus

The dendritic cells and related antigen-presenting cells (APCs) that activate lymphocytes for acquired immunity in the female reproductive tract are not well characterized. The aim of the present study was to examine heterogeneity among uterine APCs in mice and, specifically, to determine whether phenotypically and functionally distinct subpopulations of dendritic cells and macrophages can be identified. Using immunohistochemistry, abundant cells expressing APC-restricted molecules major histocompatibility complex (MHC) class II, F4/80, class A scavenger receptor, macrosialin, and sialoadhesin were evident in estrous mice. Cells expressing the costimulatory molecule B7-2 were rarely observed. Flow cytometric analysis revealed three subpopulations of uterine APCs. Undifferentiated macrophages were F4/80-positive (+), MHC class II-negative (-) cells, of which 70-80% expressed CD11b, but few expressed class A scavenger receptor, macrosialin, or sialoadhesin. Mature macrophages were F4/80+/MHC class II+ cells, of which approximately 50% expressed CD11b, class A scavenger receptor, macrosialin, and sialoadhesin. Uterine dendritic cells were F4/ 80-/MHC class II+ cells, with stimulatory immunoaccessory function relative to uterine macrophages and heterogeneous expression of dendritic markers 33D1, DEC205, CD11c, and CD1. Experiments in ovariectomized mice showed that undifferentiated macrophages were steroid hormone dependent but that mature macrophages and dendritic cells persisted after depletion of ovarian steroid hormones, although with altered phenotypes. In summary, our findings identify three discrete populations of APCs inhabiting the murine uterus and suggest that both mature macrophages and dendritic cells differentiate from undifferentiated macrophage precursor cells. Plasticity in the ontogenetic and functional relationships between uterine dendritic cells and macrophages likely is critical in regulating immune responses conducive to reproductive success.     

Macrophages driven to a novel state of activation have anti-inflammatory properties in mice

Recurrent episodes of inflammation underlie numerous pathologies, notably those of inflammatory bowel diseases. In this study, we describe a population of macrophages in a novel state of activation that mitigates colitis in mice. The cells responsible for this effect, called IFN-gamma-stimulated monocyte-derived cells (IFNgamma-MdC), derive from mouse spleen, blood, and bone marrow monocytes and are distinguished from known macrophage populations by mode of generation, cell surface phenotype, and function. IFNgamma-MdC only arise when macrophages are cultivated in the presence of CD40L-expressing CD4+ T cells, M-CSF, and IFN-gamma. IFNgamma-MdC express markers including F4/80, CD11b/c, CD86, and CD274; they are negative for CD4, CD8, Gr1, CD19, CD80, and CD207. Functionally, IFNgamma-MdC are defined by their capacity to enrich cocultured T cell populations for CD4+CD25+Foxp3+ regulatory cells; this enrichment, constituting up to 60% or more of residual lymphocytes, is attributed to an expansion, but also to a cell contact and caspase-dependent depletion of activated T cells. In mice, IFNgamma-MdC delivered i.v. traffic to gut-associated peripheral lymphoid tissues, including the mesenteric lymph nodes, Peyer's patches, and colonic mucosa, and promote the clinical and histological resolution of chronic colitis. We conclude that IFNgamma-MdC represent macrophages in a novel state of activation, possessing multiple T cell-suppressive effects with therapeutic potential for the treatment of autoimmune inflammation.     

Protective roles of CX3CR1-mediated signals in toxin A-induced enteritis through the induction of heme oxygenase-1 expression

The injection of Clostridium difficile toxin A into the ileal loops caused fluid accumulation with the destruction of intestinal epithelial structure and the recruitment of neutrophils and macrophages. Concomitantly, intraileal gene expression of CX3CL1/fractalkine (FKN) and its receptor, CX3CR1, was enhanced. When treated with toxin A in a similar manner, CX3CR1-deficient (CX3CR1(-/-)) mice exhibited exaggerated fluid accumulation, histopathological alterations, and neutrophil recruitment, but not macrophage infiltration. Mice reconstituted with CX3CR1(-/-) mouse-derived bone marrow cells exhibited exacerbated toxin A-induced enteritis, indicating that the lack of the CX3CR1 gene for hematopoietic cells aggravated toxin A-induced enteritis. A heme oxygenase-1 (HO-1) inhibitor, tin-protoporphyrin-IX, markedly increased fluid accumulation in toxin A-treated wild-type mice, indicating the protective roles of HO-1 in this situation. HO-1 expression was detected mainly in F4/80-positive cells expressing CX3CR1, and CX3CR1(-/-) mice failed to increase HO-1 expression after toxin A treatment. Moreover, CX3CL1/FKN induced HO-1 gene expression by isolated lamina propria-derived macrophages or a mouse macrophage cell line, RAW264.7, through the activation of the ERK signal pathway. Thus, CX3CL1/FKN could induce CX3CR1-expressing macrophages to express HO-1, thereby ameliorating toxin A-induced enteritis.     

IL-4 inhibits vasoactive intestinal peptide production by macrophages

In schistosomiasis, eggs induce granulomas that have a vasoactive intestinal peptide (VIP) immunoregulatory circuit. This study explored the regulation of VIP production at sites of inflammation. Splenocytes from uninfected C57BL/6 mice expressed VIP mRNA and protein, which stopped following egg deposition. Eggs induce a Th2 response, suggesting that Th2 cytokines like interleukin (IL)-4 can regulate VIP. To address this issue, splenocytes from uninfected mice were incubated for 4 h with or without recombinant IL-4. IL-4 inhibited VIP mRNA expression. F4/80+ macrophages were the source of constitutively expressed VIP, subject to IL-4 regulation. In IL-4 knockout mice, splenic VIP production did not downmodulate during schistosome infection, suggesting that IL-4 is a critical cytokine regulating VIP production in wild-type mouse spleen. IL-4-producing granulomas in schistosomiasis made VIP. Experiments showed that granuloma VIP derived from F4/80- (nonmacrophage) cell populations, explaining this paradox. Granuloma F4/80+ cells from IL-4 knockout mice expressed VIP. Thus macrophages can make VIP, which is subject to IL-4 regulation. However, in the Th2 granulomas, other cell types produce VIP, which compensates for loss of macrophages as a source of this molecule.     

Preadipocyte conversion to macrophage. Evidence of plasticity

Preadipocytes are present throughout adult life in adipose tissues and can proliferate and differentiate into mature adipocytes according to the energy balance. An increasing number of reports demonstrate that cells from adipose lineages (preadipocytes and adipocytes) and macrophages share numerous functional or antigenic properties. No large scale comparison reflecting the phenotype complexity has been performed between these different cell types until now. We used profiling analysis to define the common features shared by preadipocyte, adipocyte, and macrophage populations. Our analysis showed that the preadipocyte profile is surprisingly closer to the macrophage than to the adipocyte profile. From these data, we hypothesized that in a macrophage environment preadipocytes could effectively be converted into macrophages. We injected labeled stroma-vascular cells isolated from mouse white adipose tissue or 3T3-L1 preadipocyte cell line into the peritoneal cavity of nude mice and investigated changes in their phenotype. Preadipocytes rapidly and massively acquired high phagocytic activity and index. 60-70% of preadipocytes also expressed five macrophage-specific antigens: F4/80, Mac-1, CD80, CD86, and CD45. These values were similar to those observed for peritoneal macrophages. In vitro experiments showed that cell-to-cell contact between preadipocytes and peritoneal macrophages partially induced this preadipocyte phenotype conversion. Overall, these results suggest that preadipocyte and macrophage phenotypes are very similar and that preadipocytes have the potential to be very efficiently and rapidly converted into macrophages. This work emphasizes the great cellular plasticity of adipose precursors and reinforces the link between adipose tissue and innate immunity processes.     

Myocardial injury after ischemia-reperfusion in mice deficient in Akt2 is associated with increased cardiac macrophage density

Akt2 protein kinase has been shown to promote cell migration and actin polymerization in several cell types, including macrophages. Because migrating macrophages constitute an important inflammatory response after myocardial ischemia, we determined cardiac macrophage expression after ischemia-reperfusion (I/R) injury and cryo-injury in mice lacking Akt2 (Akt2-KO). At 7 days post-I/R, Akt2-KO cardiac tissues showed an increase in immunohistochemical staining for macrophage markers (Galectin 3 and F4/80) compared with wild-type (WT) mice, indicating macrophage density was increased in the injured Akt2-KO myocardium. This change was time dependent because macrophage density was similar between WT and Akt2-KO myocardium at 3 days post-I/R, but by 7 and 14 days post-I/R, macrophage density was significantly increased in Akt2-KO myocardium. Concomitantly, infarct size was larger and cardiac function was reduced in Akt2-KO mice subjected to I/R. However, when cryo-infarction produced similar infarct sizes in the anterior wall in both WT and Akt2-KO mice, macrophage density remained higher in Akt2-KO mouse myocardium, suggesting Akt2 regulates myocardial macrophage density independent of infarct size. Consistently, bone marrow from Akt2-KO mice enhanced myocardial macrophage density in both C57/B6 WT and Akt2-KO recipient mice. Finally, reciprocal ex-vivo coculturing of macrophages and cardiac myocytes showed that activated Akt2-KO peritoneal macrophages had reduced mobility and adhesion when compared with WT littermate controls. Thus, although Akt-2 KO mice did not affect the initial inflammation response after injury and Akt2 deficiency has been shown to impair cell migration or motility in macrophages, our data suggested a novel mechanism in which increasing retention of Akt2-KO macrophages resulted in increasing cardiac Akt2-KO macrophage density in the myocardial space.     

Natural Helicobacter infection modulates mouse intestinal muscularis macrophage responses

Helicobacter species are common laboratory pathogens which induce intestinal inflammation and disease in susceptible mice. Since in vitro studies indicate that Helicobacter products activate macrophages, we hypothesized that in vivo Helicobacter infection regulates the inflammatory response of intestinal muscularis macrophages from C57Bl/6 mice. Helicobacter hepaticus infection increased surface expression of macrophage markers F4/80, CD11b and MHC-II within whole intestinal muscle mounts. However, constitutive cytokine and chemokine production by macrophages isolated from infected mice significantly decreased compared to macrophages from uninfected mice despite no detectable bacterial products in the cultures. In addition, muscularis macrophages from infected mice up-regulated FIZZ-1 and SK-1 gene expression, suggesting the macrophages had an anti-inflammatory phenotype. Corresponding with increased anti-inflammatory gene expression, macrophages from infected mice were more phagocytic but did not produce cytokines after stimulation with LPS and IFN-γ or immune complexes and IL-4. Therefore, the presence of Helicobacter infection matures intestinal muscularis macrophages, modulating the constitutive macrophage response to become more anti-inflammatory and resistant to secondary stimulation.     

IL-1 gene expression in lymphoid tissues

We examined the expression of IL-1 mRNA in vivo by in situ hybridization. RNA probes for murine IL-1 alpha and IL-1 beta were used to detect IL-1 mRNA in frozen sections of spleen, lymph node, and thymus of mice injected with Salmonella typhi LPS or SRBC. No IL-1 was detected in lymphoid tissues from un-injected mice. This lack of expression correlated with the absence of IL-1 biologic activity. However, after LPS injection, IL-1 alpha and beta mRNA expression was found in macrophages of the red pulp and marginal zone of the spleen. The periarteriolar lymphoid sheath contained cells that only expressed IL-1 beta mRNA. These cells were not lymphocytes and did not stain with the macrophage marker F4/80. A similar cellular response was found after SRBC injection. Scattered macrophages in lymph nodes and thymus were positive, but only after LPS or SRBC injection. The spleens of mice injected with LPS had megakaryocytes containing IL-1 mRNA.     

Polymorphism of class A scavenger receptors in C57BL/6 mice

Scavenger receptors class A (SR-A) have been hypothesized to regulate the development of atherosclerotic lesions through recognition of modified low density lipoprotein (LDL) and macrophage adhesion to substrata. Supporting data have been collected from studies using the monoclonal antibody 2F8, an antibody developed from the BALB/c strain-derived macrophage cell line, RAW.264. Although 2F8 immunostained both cultured peritoneal macrophages (MPM) and thymic macrophages from Swiss, BALB/c, and DBA/2 mice, no immunostaining was detected in cells and tissues from C57BL/6 mice, one of the most commonly used atherosclerosis-susceptible mouse strains. Similarly, 2F8 detected SR-A protein in MPM by Western blotting in all strains except C57BL/6. However, a guinea pig antiserum developed to a fusion protein of the extracellular SR-A domain detected appropriately sized bands in all strains. Incubation with 2F8 antagonized acetylated low-density lipoprotein (AcLDL)-induced cholesterol esterification in MPM from BALB/c, Swiss, and DBA/2 strains but had no effect on MPM from C57BL/6 mice. Sequencing of SR-A cDNA from C57BL/6 mice demonstrated complete identity with published sequence in the collagen-like domain. However, four single-residue substitutions were noted in the alpha-helical coiled-coil domain. Site-directed mutagenesis demonstrated that a single substitution (L168S) in this domain accounted for the loss of 2F8 immunoreactivity. Differing reactivities toward a commonly used monoclonal antibody were used to identify polymorphism of SR-A in C57BL/6 mice.     

Loss of PPAR gamma in immune cells impairs the ability of abscisic acid to improve insulin sensitivity by suppressing monocyte chemoattractant protein-1 expression and macrophage infiltration into white adipose tissue

Abscisic acid (ABA) is a natural phytohormone and peroxisome proliferator-activated receptor gamma (PPARgamma) agonist that significantly improves insulin sensitivity in db/db mice. Although it has become clear that obesity is associated with macrophage infiltration into white adipose tissue (WAT), the phenotype of adipose tissue macrophages (ATMs) and the mechanisms by which insulin-sensitizing compounds modulate their infiltration remain unknown. We used a loss-of-function approach to investigate whether ABA ameliorates insulin resistance through a mechanism dependent on immune cell PPARgamma. We characterized two phenotypically distinct ATM subsets in db/db mice based on their surface expression of F4/80. F4/80(hi) ATMs were more abundant and expressed greater concentrations of chemokine receptor (CCR) 2 and CCR5 when compared to F4/80(lo) ATMs. ABA significantly decreased CCR2(+) F4/80(hi) infiltration into WAT and suppressed monocyte chemoattractant protein-1 (MCP-1) expression in WAT and plasma. Furthermore, the deficiency of PPARgamma in immune cells, including macrophages, impaired the ability of ABA to suppress the infiltration of F4/80(hi) ATMs into WAT, to repress WAT MCP-1 expression and to improve glucose tolerance. We provide molecular evidence in vivo demonstrating that ABA improves insulin sensitivity and obesity-related inflammation by inhibiting MCP-1 expression and F4/80(hi) ATM infiltration through a PPARgamma-dependent mechanism.     

The development of the resident pattern of endogenous peroxidatic activity in mouse peritoneal macrophages coincides with the expression of the differentiation antigen F4/80. A combined method for immunoperoxidase labeling and endogenous peroxidase cytochemistry

In the mouse the maturation of mononuclear phagocytes was followed by comparing the ultrastructural pattern of endogenous peroxidatic activity (PA) at different time points during an acute peritonitis induced with newborn calf serum (NCS). Exudate macrophages demonstrate PA only in lysosomes, whereas resident macrophages have reaction product in the nuclear envelope (NE) and rough endoplasmic reticulum (RER). Transitional cells called "exudate-resident" macrophages have PA in the NE, RER, and some virginal lysosomes. In addition, peroxidase-negative macrophages were also present. A monoclonal antibody, F4/80, that specifically recognizes a mouse macrophage differentiation antigen (Austyn and Gordon, 1981) was used in this study. To compare the indirect immunoperoxidase labeling of this antigen and the endogenous peroxidase cytochemistry on the cellular level, a combined method was developed. Finally, the method was applied to the peritoneal cells at different time points after intraperitoneal injection of NCS in mice. The relative numbers of cells demonstrating the different patterns of endogenous PA and the proportions of each subpopulation expressing F4/80 antigen were estimated. It appeared that the expression of the antigen F4/80 coincides with the development of the resident pattern of PA. It is therefore concluded that the macrophages with the resident pattern of endogenous peroxidase are derived from monocyte-like exudate macrophages. In addition, the results indicate that both exudate-resident macrophages and at least a part of the peroxidase-negative macrophages are transitional forms.     

Immunocytochemical analyses and targeted gene disruption of GTPBP1

We previously identified a gene encoding a putative GTPase, GTPBP1, which is structurally related to elongation factor 1alpha, a key component of protein biosynthesis machinery. The primary structure of GTPBP1 is highly conserved between human and mouse (97% identical at the amino acid level). Expression of this gene is enhanced by gamma interferon in a monocytic cell line, THP-1. Although counterparts of this molecule in Caenorhabditis elegans and Ascaris suum have also been identified, the function of this molecule remains to be clarified. In the present study, our immunohistochemical analyses on mouse tissues revealed that GTPBP1 is expressed in some neurons and smooth muscle cells of various organs as well as macrophages. Immunofluorescence analyses revealed that GTPBP1 is localized exclusively in cytoplasm and shows a diffuse granular network forming a gradient from the nucleus to the periphery of the cells in smooth muscle cell lines and macrophages. To investigate the physiological role of GTPBP1, we used targeted gene disruption in embryonic stem cells to generate GTPBP1-deficient mice. The mutant mice were born at the expected Mendelian frequency, developed normally, and were fertile. No manifest anatomical or behavioral abnormality was observed in the mutant mice. Functions of macrophages, including chemotaxis, phagocytosis, and nitric oxide production, in mutant mice were equivalent to those seen in wild-type mice. No significant difference was observed in the immune response to protein antigen between mutant mice and wild-type mice, suggesting normal function of antigen-presenting cells of the mutant mice. The absence of an eminent phenotype in GTPBP1-deficient mice may be due to functional compensation by GTPBP2, a molecule we recently identified which is similar to GTPBP1 in structure and tissue distribution.     

小鼠胸腺巨噬细胞的免疫组织化学研究

目的研究小鼠胸腺巨噬细胞的形态、分布及其独特亚型。方法利用巨噬细胞的四种单克隆抗体（F4／80、Mac-2、CD32／16、I-A）,通过免疫组化双重染色来鉴定小鼠胸腺巨噬细胞表型、分布及形态学的多样性。结果形态学上,我们鉴定出三种类型巨噬细胞：树突状、圆形、扁平形,它们在胸腺具有不同的分布特征。树突状巨噬细胞被四种抗体标记;圆形巨噬细胞在皮髓交界至髓质为F4／80阴性,Mac-2阳性,CD32／16阴性,I-A阳性,与此相反,位于皮质的表达F4／80;扁平形巨噬细胞F4／80和CD32／16阳性,但是Mac-2阴性。结论在胸腺的不同位置,巨噬细胞具有形态学多样性,且具有不同的表型变化,提示在胸腺细胞的发育过程中,不同类型的巨噬细胞可能在胸腺的不同区域起着不同的作用。     

Interleukin-6 plays a critical role in aldosterone-induced macrophage recruitment and infiltration in the myocardium

Macrophages play an important role in aldosterone-induced myocardial fibrosis, in which the first key steps are macrophage recruitment and infiltration. We hypothesized that IL-6 may be a key mediator of aldosterone-induced macrophage recruitment and infiltration. To test this hypothesis, we designed cell studies with a human monocytic cell line THP-1 that with monocyte/macrophage functions to explore the signaling pathway of aldosterone-induced macrophage infiltration, and further investigated the phenomenon and consequent pathway in aldosterone-infused mice studies. The results showed that aldosterone induced the expression of IL-6 via mineralocorticoid receptors, and enhanced THP-1 cell migration and infiltration. Further experiments using a protease array and siRNA revealed that expressions of MMP-1 and MMP-9 were associated with aldosterone-induced macrophage infiltration. In addition, aldosterone-induced MMP-1 and MMP-9 expressions were mediated via cyclooxygenase-II and prostaglandin E2/EP-2 and EP-4 receptors. In aldosterone-infused mice, mRNA expressions of MMP-1, MMP-9 and COX-2 in peripheral blood monocytic cells were significantly increased. Moreover, the number of mouse macrophage-restricted F4/80 protein-positive cells in the myocardium was significantly higher in the aldosterone-infused mice compared with control mice. The increase in F4/80-positive cells in the myocardium was suppressed in the aldosterone-infused mice with the aldosterone antagonist eplerenone or anti-IL-6 antibody treatment. In conclusion, interleukin-6 played an important role in aldosterone-induced macrophage recruitment and infiltration in the myocardium.     

The nuclear IkappaB protein IkappaBNS selectively inhibits lipopolysaccharide-induced IL-6 production in macrophages of the colonic lamina propria

Macrophages play an important role in the pathogenesis of chronic colitis. However, it remains unknown how macrophages residing in the colonic lamina propria are regulated. We characterized colonic lamina proprial CD11b-positive cells (CLPMphi). CLPMphi of wild-type mice, but not IL-10-deficient mice, displayed hyporesponsiveness to TLR stimulation in terms of cytokine production and costimulatory molecule expression. We compared CLPMphi gene expression profiles of wild-type mice with IL-10-deficient mice, and identified genes that are selectively expressed in wild-type CLPMphi. These genes included nuclear IkappaB proteins such as Bcl-3 and IkappaBNS. Because Bcl-3 has been shown to specifically inhibit LPS-induced TNF-alpha production, we analyzed the role of IkappaBNS in macrophages. Lentiviral introduction of IkappaBNS resulted in impaired LPS-induced IL-6 production, but not TNF-alpha production in the murine macrophage cell line RAW264.7. IkappaBNS expression led to constitutive and intense DNA binding of NF-kappaB p50/p50 homodimers. IkappaBNS was recruited to the IL-6 promoter, but not to the TNF-alpha promoter, together with p50. Furthermore, small interference RNA-mediated reduction in IkappaBNS expression in RAW264.7 cells resulted in increased LPS-induced production of IL-6, but not TNF-alpha. Thus, IkappaBNS selectively suppresses LPS-induced IL-6 production in macrophages. This study established that nuclear IkappaB proteins differentially regulate LPS-induced inflammatory cytokine production in macrophages.