ABSTRACT
Alternatively activated (M2) macrophages regulate steady state-, cancer-, and inflammation-related tissue remodeling. They are induced by Th2-cytokines and glucocorticoids (GC). The responsiveness of mature macrophages to TGF-beta, a cytokine involved in inflammation, cancer, and atherosclerosis, is currently controversial. Recently, we demonstrated that IL-17 receptor B is up-regulated in human monocyte-derived macrophages differentiated in the presence of Th2 cytokines IL-4 and TGF-beta1. In this study, we show that mature human macrophages differentiated in the presence of IL-4, and dexamethasone (M2(IL-4/GC)) but not M2(IL-4) responds to TGF-beta1 which induced a gene expression program comprising 111 genes including transcriptional/signaling regulators (ID3 and RGS1), immune modulators (ALOX5AP and IL-17 receptor B) and atherosclerosis-related genes (ALOX5AP, ORL1, APOC1, APOC2, and APOE). Analysis of molecular mechanism underlying GC/TGF-beta cooperation revealed that surface expression of TGF-betaRII was high in M2(GC) and M2(IL-4/GC), but absent from M2(IL-4), whereas the expression of TGF-betaRI/II mRNA, TGF-betaRII total protein, and surface expression of TGF-betaRIII were unchanged. GC dexamethasone was essential for increased surface expression of functional TGF-betaRII because its effect was observed also in combination with IL-13, M-CSF, and GM-CSF. Prolonged Smad2-mediated signaling observed in TGF-beta1-treated M2(IL-4/GC) was due to insufficient activity of negative feedback mechanism what can be explained by up-regulation of SIRT1, a negative regulator of Smad7, and the retention of TGF-betaRII complex on the cell surface. In summary, mature human M2 macrophages made permissive to TGF-beta by GC-induced surface expression of TGF-betaRII activate in response to TGF-beta1, a multistep gene expression program featuring traits of macrophages found within an atherosclerotic lesion.
Subject(s)
Gene Expression , Glucocorticoids/metabolism , Macrophage Activation/genetics , Macrophages/metabolism , Receptors, Transforming Growth Factor beta/biosynthesis , Transforming Growth Factor beta/metabolism , Cell Differentiation/immunology , Cell Membrane/metabolism , Flow Cytometry , Gene Expression Profiling , Humans , Interleukin-4/metabolism , Macrophage Activation/immunology , Macrophages/cytology , Macrophages/immunology , Oligonucleotide Array Sequence Analysis , RNA, Messenger/analysis , Receptors, Interleukin/biosynthesis , Receptors, Interleukin-17 , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Macrophages (Mphi) represent a dynamic cell population that develops and operates within a changing microenvironment. In parallel to Th1/Th2 cells, primary Mphi may undergo classical (Mphi1) or alternative (Mphi2) activation. Here, we investigated whether Mphi1/Mphi2 may be re-polarized by a secondary stimulation by Th1 or Th2 cytokines or by exogenous danger signals. We established that Mphi1IFNgamma respond to alternative activation by IL-4 and IL-10 by de novo secretion of Th2 cytokines AMAC-1 and IL-1ra, and by an increase in phagocytic capacity and a decrease in bactericidal activity. Vice versa, Mphi2 responded to classical activation by IFNgamma exhibiting reduced phagocytosis and significantly increased bacterial killing while being refractory regarding secretion of TNFalpha, IL-1beta and IL-12. In response to the bacterial danger signals LPS and MDP, both Mphi1 and Mphi2 produced IL-1beta and TNFalpha; in addition Mphi2 expressed the Th1-inducing cytokine IL-12. The ability of Mphi to be re-polarized and to react to exogenous danger signals is a precondition to down-regulate an outdated immune reaction and to retain the capacity to mount an adequate anti-bacterial response. Selective refractoriness of Mphi1 and Mphi2 to IFNgamma- and LPS-induced cytokine secretion may contribute to prevent autoimmunity.
Subject(s)
Cytokines/physiology , Macrophages/immunology , Th1 Cells/immunology , Th2 Cells/immunology , Cells, Cultured , Humans , Lipopolysaccharides/immunology , Macrophages/metabolism , Receptors, Cytokine/biosynthesis , Receptors, Cytokine/genetics , Th1 Cells/physiology , Th2 Cells/physiologyABSTRACT
MIM/MTSS1 was initially described as a gene missing in invasive bladder cancer cell lines. Functional analysis revealed that MIM is an actin binding protein involved in the regulation of actin cytoskeleton dynamics. MIM was shown to be sonic hedgehog (Shh) signaling dependent and synergizes with the effects of Gli transcription factors. Overexpression of MIM in cell lines leads to the inhibition of cell proliferation. In this study, we showed that the inhibition of cell growth by MIM is anchorage independent. We identified and cloned the promoter region of MIM and located the main promoter activity to 276 bp of 5' flanking sequence sited within a CpG island. Analysis of DNA methylation using bisulphite sequencing revealed that MIM promoter is methylated in its 5' region in cells and tissue samples with reduced endogenous MIM expression. Using luciferase reporter assay, we demonstrated that nonmethylated MIM promoter has a similar activity in cell lines with different endogenous MIM expression. Inhibition of DNA methylation by 5-Aza-2'-deoxycytidine led to upregulation of MIM expression in a low expressing cell line. In conclusion, we clearly demonstrate here that the expression of metastasis suppressor MIM is regulated by DNA methylation of a CpG island within its promoter region.
