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. 2010 Jun 18;285(25):19593-604.
doi: 10.1074/jbc.M109.069955. Epub 2010 Apr 13.

Inflammation anergy in human intestinal macrophages is due to Smad-induced IkappaBalpha expression and NF-kappaB inactivation

Lesley E Smythies  1 , Ruizhong Shen, Diane Bimczok, Lea Novak, Ronald H Clements, Devin E Eckhoff, Phillipe Bouchard, Michael D George, William K Hu, Satya Dandekar, Phillip D Smith
Affiliations

Inflammation anergy in human intestinal macrophages is due to Smad-induced IkappaBalpha expression and NF-kappaB inactivation

Lesley E Smythies et al. J Biol Chem. .

Abstract

Human intestinal macrophages contribute to tissue homeostasis in noninflamed mucosa through profound down-regulation of pro-inflammatory cytokine release. Here, we show that this down-regulation extends to Toll-like receptor (TLR)-induced cytokine release, as intestinal macrophages expressed TLR3-TLR9 but did not release cytokines in response to TLR-specific ligands. Likely contributing to this unique functional profile, intestinal macrophages expressed markedly down-regulated adapter proteins MyD88 and Toll interleukin receptor 1 domain-containing adapter-inducing interferon beta, which together mediate all TLR MyD88-dependent and -independent NF-kappaB signaling, did not phosphorylate NF-kappaB p65 or Smad-induced IkappaBalpha, and did not translocate NF-kappaB into the nucleus. Importantly, transforming growth factor-beta released from intestinal extracellular matrix (stroma) induced identical down-regulation in the NF-kappaB signaling and function of blood monocytes, the exclusive source of intestinal macrophages. Our findings implicate stromal transforming growth factor-beta-induced dysregulation of NF-kappaB proteins and Smad signaling in the differentiation of pro-inflammatory blood monocytes into noninflammatory intestinal macrophages.

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Figures

FIGURE 1.
FIGURE 1.
Intestinal macrophages are down-regulated for pro-inflammatory cytokine release. A, intestinal macrophage LPS responsiveness is not restored in the presence of sCD14. Intestinal macrophages (2 × 106/ml) incubated with increasing concentrations of smooth LPS in the presence or absence of sCD14 did not release detectable IL-6, but sCD14 potently enhanced IL-6 release from autologous LPS-stimulated blood monocytes and caused inducible IL-6 release from human umbilical vein endothelial cells (HU-VEC). B, intestinal macrophages do not release TNF-α in response to stimulation with LPS or H. pylori urease. Intestinal macrophages (2 × 106/ml) cultured with increasing concentrations of rough LPS, smooth LPS, or H. pylori urease did not release detectable TNF-α, in sharp contrast to autologous blood monocytes, which released high levels of TNF-α in response to each stimulus. C, intestinal macrophage cytokine mRNA levels are sharply reduced compared with the levels in blood monocytes. Cytokine mRNA levels, determined by Affymetrix gene array analysis, were significantly lower in resting intestinal macrophages compared with autologous, unstimulated monocytes (C, left panel); mRNA levels did not increase in intestinal macrophages following LPS treatment but increased sharply in LPS-stimulated blood monocytes (C, right panel). Data are from representative experiments (n = 3) (***, p < 0.001; **, p < 0.01; *, p < 0.05).
FIGURE 2.
FIGURE 2.
MD-2 gene transfection does not restore LPS-responsiveness to intestinal macrophages. Intestinal macrophages, which are exclusively CD14−, expressed markedly reduced levels of the transcription factors involved in activation of the CD14 promoter (CEBPα, CEBPβ, and Sp1) (A) and low levels of TLR4 protein and no detectable MD-2 protein or MD-2 mRNA (B). MD-2-transfected intestinal macrophages (C, inset) did not release TNF-α, IL-6, or IL-8 after culture for 24 h with rough (R) LPS (1 μg/ml) (C, left panel), whereas a mixture of intestinal macrophages plus 4% blood monocytes released high levels of inducible TNF-α, IL-6, and IL-8 (C, right panel) (values represent mean ± S.E. of triplicate wells). Data are from representative experiments (n = 3) (*, p < 0.005).
FIGURE 3.
FIGURE 3.
Intestinal macrophages express TLRs but do not respond to TLR ligation. A, intestinal macrophages and blood monocytes analyzed by fluorescence-activated cell sorter expressed TLR1 and TLR3–9 but not TLR2. Data shown are from one representative experiment (n = 6). B, blood monocytes but not intestinal macrophages (2 × 106/ml) incubated 18 h with TLR ligands produced TNF-α, IFN-α, IL-8, and IL-10. Values represent mean ± S.E. of triplicate wells (n = 4).
FIGURE 4.
FIGURE 4.
Intestinal macrophages do not phosphorylate NF-κB p65 or translocate NF-κB p50 into the nucleus. A, intestinal macrophages and autologous blood monocytes were stimulated with smooth LPS (1 μg/ml) for 5, 15, 30, or 60 min, and NF-κB p65 phosphorylation was assessed by flow cytometry. B, nuclear transport of NF-κB p50 was determined by comparison of NF-κB p50 levels in nuclear cell extracts by ELISA after exposure to LPS (1 μg/ml). Levels of nuclear NF-κB p50 were significantly lower in intestinal macrophages than blood monocytes at each time point, p < 0.006. C, intestinal macrophages and autologous blood monocytes (10 × 106/ml) were incubated for 0 or 30 min with smooth LPS (1 μg/ml), and extracts were analyzed for MyD88-dependent and -independent NF-κB from signal proteins by Western blot (A). D, macrophages were also analyzed for Smad signal proteins by Western blot. Data are from a representative experiment (n = 3) (**, p < 0.01; *, p < 0.05).
FIGURE 5.
FIGURE 5.
Gene expression for MyD88 signal pathway proteins in intestinal macrophages. Affymetrix gene array analysis showed undetectable or markedly reduced levels of mRNA for MyD88, the NF-κB components (p105 and p65), and Bruton agammaglobulinemia tyrosine kinase, IRAK1, and IRAK4 genes in intestinal macrophages compared with autologous blood monocytes but increased mRNA levels for SARM, SOCS1, and TRAF6 in the macrophages (***, p < 0.001; *, p < 0.05).
FIGURE 6.
FIGURE 6.
Stromal factors down-regulate NF-κB translocation and IκBα phosphorylation in blood monocytes. A, intestinal macrophages and blood monocytes (2 × 106/ml), the latter cultured in the presence or absence of S-CM (500 μg protein/ml), were incubated with or without specific TLR ligands for 18 h, and culture supernatants were assayed for TNF-α and RANTES (inset). Values are the means ± S.E. (n = 4). B, blood monocytes and intestinal macrophages incubated in media alone, in media plus smooth LPS (1 μg/ml, 1 h), or treated with S-CM (500 μg/ml, 1 h) and then LPS were analyzed for NF-κB nuclear translocation by immunofluorescence and confocal microscopy (upper panels) and fluorescence intensity (lower panels: green line, NF-κB p65; blue line, nucleus). NF-κB localized predominantly to the cytoplasm of untreated monocytes (left panel) and translocated into the nucleus after LPS treatment (middle panel) but did not translocate when the cells were treated with S-CM prior to the LPS (right panel). NF-κB remained exclusively in the cytoplasm of intestinal macrophages (insets) in each condition. Histograms show distribution of NF-κB (green line) in relation to the nucleus (blue line). C, anti-TGF-β antibodies block S-CM down-regulation of stimulus-driven nuclear translocation of NF-κB in blood monocytes. Blood monocytes preincubated with M-CSF (10 ng/ml) (or LPS, H. pylori urease, or IFN-γ, data not shown) display nuclear NF-κB (left panel), whereas preincubation of M-CSF-pulsed monocytes with S-CM (100 μg/ml) inhibited nuclear translocation of NF-κB (middle panel). However, preincubation of M-CSF-pulsed monocytes with S-CM plus anti-TGF-β antibodies (25 μg/ml) reversed S-CM down-regulation of the stimulus-driven nuclear translocation of NF-κB (right panel). Data are representative of a single experiment for each stimulus (n = 4). Histograms depict NF-κB distribution, as described in B. D, expression and phosphorylation of IκBα in intestinal macrophages and monocytes by flow cytometry. Intestinal macrophages, unlike blood monocytes, did not phosphorylate IκBα after stimulation with LPS, and monocyte phosphorylation of IκBα was inhibited by pretreatment of the cells with S-CM (250 μg/ml). E, expression and phosphorylation of IκBα and Smad2 in intestinal macrophages and blood monocytes. Intestinal macrophages expressed constitutive IκBα but did not phosphorylate IκBα. Blood monocytes also expressed constitutive IκBα but did phosphorylate IκBα after LPS stimulation, and phosphorylation was inhibited by pretreatment of the cells with S-CM. However, S-CM inhibition of inducible IκBα phosphorylation in blood monocytes was reversed when the S-CM (250 μg/ml) was preincubated (1 h) with anti-TGF-β antibodies (25 μg/ml). Coincident with blockade of inducible IκBα phosphorylation in monocytes, exposure of the monocytes to S-CM prior to stimulation caused a sharp increase in IκBα, which also was reversed when the S-CM was preincubated with anti-TGF-β antibodies. Intestinal macrophages expressed, but did not phosphorylate, Smad2. Blood monocytes expressed Smad2 but did not express phosphorylated Smad2 in medium alone, or after LPS stimulation, but in the presence of TGF-β alone (100 ng/500 μl), S-CM alone (250 μg/500 μl), or S-CM + LPS, monocytes expressed phosphorylated Smad2. The S-CM induction of phosphorylated Smad2 was blocked by pretreatment of S-CM with anti-TGF-β antibodies. Data are representative of three independent experiments (n = 3). rh, recombinant human.
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