Human intestinal macrophages display profound inflammatory anergy despite avid phagocytic and bacteriocidal activity

Abstract

Intestinal macrophages, which are thought to orchestrate mucosal inflammatory responses, have received little investigative attention compared with macrophages from other tissues. Here we show that human intestinal macrophages do not express innate response receptors, including the receptors for LPS (CD14), Fcalpha (CD89), Fcgamma (CD64, CD32, CD16), CR3 (CD11b/CD18), and CR4 (CD11c/CD18); the growth factor receptors IL-2 (CD25) and IL-3 (CD123); and the integrin LFA-1 (CD11a/CD18). Moreover, resident intestinal macrophages also do not produce proinflammatory cytokines, including IL-1, IL-6, IL-10, IL-12, RANTES, TGF-beta, and TNF-alpha, in response to an array of inflammatory stimuli but retain avid phagocytic and bacteriocidal activity. Thus, intestinal macrophages are markedly distinct in phenotype and function from blood monocytes, although intestinal macrophages are derived from blood monocytes. To explain this paradox, we show that intestinal stromal cell-derived products downregulate both monocyte receptor expression and, via TGF-beta, cytokine production but not phagocytic or bacteriocidal activity, eliciting the phenotype and functional profile of intestinal macrophages. These findings indicate a mechanism in which blood monocytes recruited to the intestinal mucosa retain avid scavenger and host defense functions but acquire profound "inflammatory anergy," thereby promoting the absence of inflammation characteristic of normal intestinal mucosa despite the close proximity of immunostimulatory bacteria.

Figure 1

Ultrastructure and purity of intestinal macrophages and blood monocytes. A representative intestinal macrophage and blood monocyte show typical eccentric nuclei, villous processes (especially on the intestinal macrophage), and in the macrophage, primary and secondary lysosomes and phagocytic vacuoles (magnification, ×10,000). FACS profiles for intestinal macrophages and blood monocytes show that both populations express the mononuclear phagocyte markers HLA-DR and CD13, but not markers for T cells (CD3), B cells (CD20), NK cells (CD69), or DCs (CD83). Insets show control cells stained with CD3 (PBLs), CD20 (PBLs), CD69 (PBLs), and CD83 (monocyte-derived DCs). Both populations also did not express CD33, CD34, or CD103, markers for bone marrow macrophage precursors, follicular DCs and intestinal lymphocytes, respectively.

Figure 2

Downregulated cytokine production by LPS-stimulated intestinal macrophages. Blood monocytes and intestinal macrophages (2 × 10⁶/ml) were incubated with or without LPS for 24, 48, and 72 hours, and supernatants were assayed for IL-1, IL-6, TNF-α, and IL-8. Values are mean + SD (n = 3).

Figure 3

Stimulus-independent down-regulation of intestinal macro-phage cytokine production. Blood monocytes and intestinal macrophages (2 × 10⁶/ml) were incubated with and without H. pylori urease (10 μg/ml), HK S. aureus (10⁷ CFU/ml), IFN-γ (100 U/ml), and PMA (40 ng/ml) for 24 hours, and supernatants were assayed for IL-1, IL-6, TNF-α, and IL-8. Values are mean + SD (n = 3).

Figure 4

S-CM downregulation of monocyte surface antigen expression. (A) Blood monocytes cultured for 4 days in the absence or presence of S-CM, E-CM, or MNL-CM at the indicated concentrations were analyzed by FACS for HLA-DR, CD13, CD14, and CD16 (open histograms). Cells were also stained with FITC IgG1 and PE IgG2a irrelevant Abs (solid histograms). FACS insets show the flow-cytometric analysis of intestinal macrophages for the same surface marker. Data are from a representative experiment (n = 3). Electron micrograph inset shows a representative monocyte after 24-hour culture with S-CM (150 μg/ml total protein). (B) Blood monocytes incubated with S-CM (150 μg/ml total protein) and in the absence or presence of protease inhibitors, including trypsin, chymotrypsin, pronase, thermolysin, papain, and pancreas extract, as described in Methods, were analyzed by FACS as above.

Figure 5

S-CM downregulation of monocyte cytokine production. Blood monocytes were incubated in the presence or absence of H. pylori urease and either S-CM, E-CM, or MNL-CM at the indicated concentrations for 24 hours, and supernatants were assayed for IL-1, IL-6, TNF-α, IL-10 and RANTES. Values are mean + SD (n = 7).

Figure 6

Phagocytic and bacteriocidal activity of blood monocytes and intestinal macrophages. (A) Blood monocyte and intestinal macrophage phagocytosis in the absence or presence of S-CM. Phagocytosis was measured as the percentage of cells that contained FITC-labeled beads after 1-hour incubation. Values are mean + SD (n = 3). (B) Phagocytosis-induced cytokine production by blood monocytes and intestinal macrophages. Blood monocytes and intestinal macrophages were incubated with latex beads for 2 hours, washed, cultured for 24 hours, and the supernatants assayed for IL-1 (black bars), IL-6 (dark gray bars), TNF-α (light gray bars), and IL-8 (white bars). Values are mean + SD (n = 4). Inset: S-CM downregulation of phagocytosis-induced cytokine release by monocytes. Blood monocytes were incubated with latex beads in the absence or presence of increasing concentrations of S-CM, washed, and cultured for 24 hours in the same concentrations of S-CM. Culture supernatants were harvested and assayed for IL-1 and TNF-α. Values are mean + SD (n = 3). (C and D) Killing of Gram-negative bacteria by blood monocytes and intestinal macrophages. Intestinal macrophages, blood monocytes, and blood monocytes plus S-CM 500 μg/ml (2 × 10⁵ cells/250 μl) were incubated with (C) S. typhimurium (8 × 10⁶ CFU/ml) or (D) E. coli (4 × 10⁶ CFU/ml), and intracellular killing was determined as described in Methods. The 3 populations of cells killed the vast majority of the bacteria within 30–60 minutes.

Figure 7

S-CM TGF-β downregulates monocyte cytokine production. (A) S-CM induces decreased TNF-α and increased TGF-β release by monocytes. Blood monocytes were incubated in the absence or presence of increasing concentrations of S-CM for 1 hour and then for an additional 24 hours with LPS (1 μg/ml). The harvested supernatants were analyzed for TNF-α and TGF-β protein. Values are mean + SD (n = 3). Parallel cultures of similarly treated monocytes (2 hours) were analyzed by RT-PCR for TNF-α and GAPDH mRNA. (B) TGF-β Abs block S-CM–induced downregulation of LPS-stimulated TNF-α release by monocytes. Blood monocytes were incubated with increasing concentrations of anti–TGF-β Abs and S-CM (150 μg/ml) and stimulated with LPS (1 μg/ml) for 24 hours. Supernatants were analyzed for TNF-α protein. Neither anti–TGF-β nor anti–IL-10 Abs (200 μg/ml each) in the absence of S-CM significantly affected monocyte production of TNF-α or IL-1. Values are mean + SD (n = 3). Parallel cultures were analyzed for TNF-α and GAPDH mRNA as in A.

Figure 8

Jejunal tissue TGF-β downregulates monocyte cytokine production. (A) Culture supernatants from explants of jejunal mucosa cause dose-dependent downregulation of TNF-α and reciprocal upregulation of TGF-β release by monocytes. Monocytes were incubated with culture supernatant from explants of jejunal mucosa of increasing wet weight for 1 hour and then for 24 hours with LPS (1 μg/ml). Culture supernatants were harvested and analyzed for TNF-α and TGF-β protein. Values are mean ± SD (n = 2). (B) TGF-β Abs reverse the ability of jejunal mucosa culture supernatant to inhibit monocyte TNF-α release. Monocytes were incubated with increasing concentrations of anti–TGF-β Abs and jejunal mucosa culture supernatant and stimulated with LPS (1 μg/ml) for 24 hours. Supernatants were analyzed for TNF-α. Values are mean ± SD (n = 2).

Figure 9

Detection of TGF-β. (A–D) TGF-β was detected (arrows) in (A) lamina propria cells that stained with (B) mast cell marker c-kit but not (C) macrophage marker HAM56 and in (D) epithelial cells (magnification, ×40). (E) Soluble TGF-β protein was detected in S-CM but not E-CM or MNL-CM (each normalized to 2 mg/ml total protein), which were generated from lamina propria stroma, intestinal epithelial cells, and intestinal mononuclear cells, respectively (mean values for CMs from 3 separate donors). (F) TGF-β mRNA was present in whole tissue, stroma and epithelial cells, but not lamina propria (L.P.) macrophages from a representative donor (n = 3). Insets show a TGF-β⁺ (A), c-kit⁺ (B), and HAM56^– (C) cell at high magnification (×100).