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.