Abstract

Background & Aims: Dendritic cells (DCs) recognize and respond to microbial structures using pattern recognition receptors, including Toll-like receptors (TLRs). In the intestine, DCs are pivotal in tolerance induction and direct the differentiation of T cells. We aimed to identify changes in intestinal DCs that may underlie the dysregulated immune response to enteric bacteria that occurs in patients with inflammatory bowel disease (IBD). Methods: DCs were identified in freshly isolated lamina propria mononuclear cells by multicolor flow cytometry in patients with IBD and controls. Expression of TLR2, TLR4, and the activation/maturation marker CD40 was assessed by cell surface labeling. Production of cytokines (interleukin [IL]-12, IL-6, and IL-10) was assessed in the absence of exogenous stimulation by intracellular staining of permeabilized cells. Results: In healthy controls, few intestinal DCs expressed TLR2 or TLR4, in contrast to blood DCs. DC expression of both TLRs was significantly enhanced in Crohn’s disease and ulcerative colitis. DCs from inflamed tissue of patients with Crohn’s disease expressed significantly higher levels of the maturation/activation marker CD40. Elevated levels of CD40 on DCs were decreased after treating patients with anti-tumor necrosis factor α. In Crohn’s disease, but not ulcerative colitis, more colonic DCs produced IL-12 and IL-6. The number of IL-10-producing DCs did not differ significantly between patients with IBD and controls. Conclusions: In IBD, DCs are activated, their expression of microbial recognition receptors is up-regulated, and more DCs produce pathologically relevant cytokines. Intestinal DCs are likely to be key initiators or perpetuators of the inflammatory response that characterizes IBD. Background & Aims: Dendritic cells (DCs) recognize and respond to microbial structures using pattern recognition receptors, including Toll-like receptors (TLRs). In the intestine, DCs are pivotal in tolerance induction and direct the differentiation of T cells. We aimed to identify changes in intestinal DCs that may underlie the dysregulated immune response to enteric bacteria that occurs in patients with inflammatory bowel disease (IBD). Methods: DCs were identified in freshly isolated lamina propria mononuclear cells by multicolor flow cytometry in patients with IBD and controls. Expression of TLR2, TLR4, and the activation/maturation marker CD40 was assessed by cell surface labeling. Production of cytokines (interleukin [IL]-12, IL-6, and IL-10) was assessed in the absence of exogenous stimulation by intracellular staining of permeabilized cells. Results: In healthy controls, few intestinal DCs expressed TLR2 or TLR4, in contrast to blood DCs. DC expression of both TLRs was significantly enhanced in Crohn’s disease and ulcerative colitis. DCs from inflamed tissue of patients with Crohn’s disease expressed significantly higher levels of the maturation/activation marker CD40. Elevated levels of CD40 on DCs were decreased after treating patients with anti-tumor necrosis factor α. In Crohn’s disease, but not ulcerative colitis, more colonic DCs produced IL-12 and IL-6. The number of IL-10-producing DCs did not differ significantly between patients with IBD and controls. Conclusions: In IBD, DCs are activated, their expression of microbial recognition receptors is up-regulated, and more DCs produce pathologically relevant cytokines. Intestinal DCs are likely to be key initiators or perpetuators of the inflammatory response that characterizes IBD. Crohn’s disease and ulcerative colitis are chronic relapsing inflammatory diseases of the gastrointestinal tract. The immunopathology of these diseases relates to an inappropriate and exaggerated mucosal immune response to constituents of the intestinal flora in genetically predisposed individuals. Antigen-presenting cells such as dendritic cells (DCs) are likely to play a central role in the host response to intestinal flora, both in innate responses to bacteria and by shaping the character of the host’s adaptive immune response. In healthy mice, lamina propria DCs in the distal ileum show evidence of bacterial sampling.1Becker C. Wirtz S. Blessing M. Pirhonen J. Strand D. Bechthold O. Frick J. Galle P.R. Autenrieth I. Neurath M.F. Constitutive p40 promoter activation and IL-23 production in the terminal ileum mediated by dendritic cells.J Clin Invest. 2003; 112: 693-706Crossref PubMed Scopus (303) Google Scholar In mice with genetic abnormalities involving the function of antigen-presenting cells, intestinal inflammation occurs. Myeloid-specific Stat3-deficient animals that have a defect in the response of their macrophages and DCs to Stat3-dependent cytokines such as interleukin (IL)-10 develop intestinal inflammation characterized by enhanced production of proinflammatory IL-12, IL-6, and tumor necrosis factor (TNF)-α.2Kobayashi M. Kweon M.N. Kuwata H. Schreiber R.D. Kiyono H. Takeda K. Akira S. Toll-like receptor-dependent production of IL-12p40 causes chronic enterocolitis in myeloid cell-specific Stat3-deficient mice.J Clin Invest. 2003; 111: 1297-1308Crossref PubMed Scopus (225) Google Scholar, 3Takeda K. Clausen B.E. Kaisho T. Tsujimura T. Terada N. Forster I. Akira S. Enhanced Th1 activity and development of chronic enterocolitis in mice devoid of Stat3 in macrophages and neutrophils.Immunity. 1999; 10: 39-49Abstract Full Text Full Text PDF PubMed Scopus (1028) Google Scholar Furthermore, studies in murine adoptive transfer models of colitis suggest that DCs are important in the initiation4Leithauser F. Trobonjaca Z. Moller P. Reimann J. Clustering of colonic lamina propria CD4(+) T cells to subepithelial dendritic cell aggregates precedes the development of colitis in a murine adoptive transfer model.Lab Invest. 2001; 81: 1339-1349Crossref PubMed Scopus (59) Google Scholar and perpetuation of intestinal inflammation.5Malmstrom V. Shipton D. Singh B. Al Shamkhani A. Puklavec M.J. Barclay A.N. Powrie F. CD134L expression on dendritic cells in the mesenteric lymph nodes drives colitis in T cell-restored SCID mice.J Immunol. 2001; 166: 6972-6981PubMed Google Scholar In Crohn’s disease, a subgroup of patients possesses variants of the NOD2 protein. NOD2 is expressed by myeloid cells, including DCs, and is involved in innate bacterial recognition and regulation of the inflammatory cascade,6Hampe J. Cuthbert A. Croucher P.J. Mirza M.M. Mascheretti S. Fisher S. Frenzel H. King K. Hasselmeyer A. Macpherson A.J. Bridger S. Van Deventer S. Forbes A. Nikolaus S. Lennard-Jones J.E. Foelsch U.R. Krawczak M. Lewis C. Schreiber S. Mathew C.G. Association between insertion mutation in NOD2 gene and Crohn’s disease in German and British populations.Lancet. 2001; 357: 1925-1928Abstract Full Text Full Text PDF PubMed Scopus (1009) Google Scholar, 7Hugot J.P. Chamaillard M. Zouali H. Lesage S. Cezard J.P. Belaiche J. Almer S. Tysk C. O’Morain C.A. Gassull M. Binder V. Finkel Y. Cortot A. Modigliani R. Laurent-Puig P. Gower-Rousseau C. Macry J. Colombel J.F. Sahbatou M. Thomas G. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease.Nature. 2001; 411: 599-603Crossref PubMed Scopus (4653) Google Scholar, 8Ogura Y. Bonen D.K. Inohara N. Nicolae D.L. Chen F.F. Ramos R. Britton H. Moran T. Karaliuskas R. Duerr R.H. Achkar J.P. Brant S.R. Bayless T.M. Kirschner B.S. Hanauer S.B. Nunez G. Cho J.H. A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease.Nature. 2001; 411: 603-606Crossref PubMed Scopus (4143) Google Scholar suggesting that altered responses to the microflora by DCs may contribute to the inflammatory response in Crohn’s disease. DCs are present in the intestine in the gut-associated lymphoid tissue and the lamina propria and lie in close proximity to the large and dynamic antigenic load in the gut lumen.9Bell S.J. Rigby R. English N. Mann S.D. Knight S.C. Kamm M.A. Stagg A.J. Migration and maturation of human colonic dendritic cells.J Immunol. 2001; 166: 4958-4967PubMed Google Scholar, 10Iwasaki A. Kelsall B.L. Freshly isolated Peyer’s patch, but not spleen, dendritic cells produce interleukin 10 and induce the differentiation of T helper type 2 cells.J Exp Med. 1999; 190: 229-239Crossref PubMed Scopus (550) Google Scholar, 11Liu L.M. MacPherson G.G. Lymph-borne (veiled) dendritic cells can acquire and present intestinally administered antigens.Immunology. 1991; 73: 281-286PubMed Google Scholar, 12Maric I. Holt P.G. Perdue M.H. Bienenstock J. Class II MHC antigen (Ia)-bearing dendritic cells in the epithelium of the rat intestine.J Immunol. 1996; 156: 1408-1414PubMed Google Scholar DCs in Peyer’s patches sample commensal bacteria,13Macpherson A.J. Uhr T. Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria.Science. 2004; 303: 1662-1665Crossref PubMed Scopus (1158) Google Scholar but this is not the only site at which antigen uptake occurs. Lamina propria DCs pass their dendrites between epithelial tight junctions and interact directly with luminal antigens14Rescigno M. Urbano M. Valzasina B. Francolini M. Rotta G. Bonasio R. Granucci F. Kraehenbuhl J.P. Ricciardi-Castagnoli P. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria.Nat Immunol. 2001; 2: 361-367Crossref PubMed Scopus (2013) Google Scholar and can sample luminal antigens that have passed through the epithelium. Intestinal DCs have properties distinct from their nonmucosal counterparts, probably as a result of their association with the external environment. They are involved in both nonresponsiveness or tolerance induction and responsiveness to antigens in the gut.15Viney J.L. Mowat A.M. O’Malley J.M. Williamson E. Fanger N.A. Expanding dendritic cells in vivo enhances the induction of oral tolerance.J Immunol. 1998; 160: 5815-5825PubMed Google Scholar, 16Williamson E. Westrich G.M. Viney J.L. Modulating dendritic cells to optimize mucosal immunization protocols.J Immunol. 1999; 163: 3668-3675PubMed Google Scholar For example, DCs isolated from murine Peyer’s patches produce more IL-10 than splenic DCs and have a tendency to induce Th2/3 responses.10Iwasaki A. Kelsall B.L. Freshly isolated Peyer’s patch, but not spleen, dendritic cells produce interleukin 10 and induce the differentiation of T helper type 2 cells.J Exp Med. 1999; 190: 229-239Crossref PubMed Scopus (550) Google Scholar DCs sense microbes by a series of surface receptors, including Toll-like receptors (TLRs), that recognize structural elements displayed on the surface of microbes.17Kadowaki N. Ho S. Antonenko S. Malefyt R.W. Kastelein R.A. Bazan F. Liu Y.J. Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens.J Exp Med. 2001; 194: 863-869Crossref PubMed Scopus (1673) Google Scholar TLR4 is required for recognition of lipopolysaccharide from Escherichia coli, and TLR2 recognizes peptidoglycan and lipoteichoic acid from gram-positive bacteria and lipoproteins from both gram-positive and gram-negative organisms.18Chow J.C. Young D.W. Golenbock D.T. Christ W.J. Gusovsky F. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction.J Biol Chem. 1999; 274: 10689-10692Crossref PubMed Scopus (1609) Google Scholar, 19Michelsen K.S. Aicher A. Mohaupt M. Hartung T. Dimmeler S. Kirschning C.J. Schumann R.R. The role of toll-like receptors (TLRs) in bacteria-induced maturation of murine dendritic cells (DCS). Peptidoglycan and lipoteichoic acid are inducers of DC maturation and require TLR2.J Biol Chem. 2001; 276: 25680-25686Crossref PubMed Scopus (245) Google Scholar, 20Morath S. Stadelmaier A. Geyer A. Schmidt R.R. Hartung T. Synthetic lipoteichoic acid from Staphylococcus aureus is a potent stimulus of cytokine release.J Exp Med. 2002; 195: 1635-1640Crossref PubMed Scopus (208) Google Scholar, 21Schwandner R. Dziarski R. Wesche H. Rothe M. Kirschning C.J. Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by toll-like receptor 2.J Biol Chem. 1999; 274: 17406-17409Crossref PubMed Scopus (1424) Google Scholar, 22Takeuchi O. Hoshino K. Akira S. Cutting edge TLR2-deficient and MyD88-deficient mice are highly susceptible to Staphylococcus aureus infection.J Immunol. 2000; 165: 5392-5396PubMed Google Scholar, 23Yoshimura A. Lien E. Ingalls R.R. Tuomanen E. Dziarski R. Golenbock D. Cutting edge recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2.J Immunol. 1999; 163: 1-5PubMed Google Scholar DCs control microbial-driven T-cell polarization in part through the ligation of TLRs.24Kaisho T. Akira S. Regulation of dendritic cell function through Toll-like receptors.Curr Mol Med. 2003; 3: 373-385Crossref PubMed Scopus (159) Google Scholar After interaction with microbial products or other maturation stimuli such as cytokines, immature DCs in peripheral tissues change their pattern of chemokine receptors and migrate to the draining lymphoid tissue.25Banchereau J. Steinman R.M. Dendritic cells and the control of immunity.Nature. 1998; 392: 245-252Crossref PubMed Scopus (12189) Google Scholar During this process, DCs down-regulate their antigen acquisition machinery, up-regulate the cell surface expression of major histocompatibility complex/peptide antigen complexes and maturation/costimulatory molecules such as CD40, and acquire their characteristic ability to stimulate naive T cells. The type of effector T-cell response is influenced by the cytokines produced by the activating DCs. For example, production of IL-12 by DCs polarizes a Th1 response,26Macatonia S.E. Hosken N.A. Litton M. Vieira P. Hsieh C.S. Culpepper J.A. Wysocka M. Trinchieri G. Murphy K.M. O’Garra A. Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells.J Immunol. 1995; 154: 5071-5079PubMed Google Scholar production of IL-10 by DCs influences a regulatory response,10Iwasaki A. Kelsall B.L. Freshly isolated Peyer’s patch, but not spleen, dendritic cells produce interleukin 10 and induce the differentiation of T helper type 2 cells.J Exp Med. 1999; 190: 229-239Crossref PubMed Scopus (550) Google Scholar and production of IL-6 plays a role in overcoming the suppressive effect of regulatory T cells.27Pasare C. Medzhitov R. Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells.Science. 2003; 299: 1033-1036Crossref PubMed Scopus (1797) Google Scholar We hypothesized that alterations in gut DCs may contribute to the dysregulated immune response that underlies human inflammatory bowel disease (IBD). In particular, we hypothesized that an abnormal pattern of bacterial recognition by DCs through TLRs and altered DC activation and cytokine production may underlie chronic inflammatory processes. Therefore, we analyzed TLR2 and TLR4 expression on DCs, maturation status of DCs, and production of the cytokines IL-12, IL-6, and IL-10 by DCs present in the lamina propria of patients with IBD and healthy controls. Intestinal specimens were obtained at colonoscopy from patients with Crohn’s disease, patients with ulcerative colitis, or controls. The Crohn’s disease group (n = 31) consisted of 15 men and 16 women, ranging from 18 to 69 years of age. The diagnosis for each patient was made using clinical parameters, radiographic studies, and histologic criteria. At the time of sample collection, 10 patients had a new diagnosis of Crohn’s disease and were on no medication, 6 patients were receiving corticosteroids, 6 patients were receiving azathioprine, and 9 patients were receiving an oral sulfasalazine preparation. There were 7 patients with Crohn’s disease who received anti-TNF-α treatment, and biopsy samples were taken before and 2 weeks after infusion. The ulcerative colitis group (n = 24) consisted of 21 men and 3 women, ranging from 18 to 65 years of age. The diagnosis for each patient was made using clinical parameters, radiographic studies, and histologic criteria. In the ulcerative colitis group, 1 patient was receiving corticosteroids, 3 patients were receiving azathioprine, 21 patients were receiving an oral sulfasalazine preparation, and 2 patients were on no medication. The control group consisted of 39 patients with macroscopically and histologically normal intestine who had been referred with rectal bleeding or a change in bowel habit. There were 17 men and 22 women in the control group, ranging from 22 to 85 years of age. Informed consent was obtained from all patients, and the protocol was approved by the local ethics committee. Antibodies with the following specificities and fluorochrome labels were used: CD11c-FITC (KB90) from Dako (Ely, England); CD3-PC5 (UCHT-1), CD14-PC5 (MIP9), CD16-PC5 (B73.1), CD19-PC5 (4G7), CD56-PC5 (N901), and CD8-PC5 (B9.11) from Beckman Coulter (High Wycombe, England); CD34-CyChrome (581), CD40-PE (LOB7/6), TLR2-FITC (TL2.1), and TLR4-FITC (HTA125) from Serotec (Oxford, England); CD11c-PE (B-ly6), CD14-PE (MϕP9), CD16-PE (B73.1), CD19-PE (4G7), CD34-PE (8G12), HLA-DR-APC (G46-6), HLA-DR-PE (L243), and CD8-FITC/PE/APC (SK1) from BD Biosciences (Oxford, England); and unconjugated TLR2 (TL2.1) and TLR4 (HTA125) from Imgenex (San Diego, CA). Intracellular cytokine staining used IL-10-PE (JES3-9D7; Serotec), IL-12p40/p70-PE (C11.5; BD Biosciences), and IL-6 PE (#1936; R&D, Abingdon, England). Fluorescein isothiocyanate (FITC)-conjugated anti-goat F[ab′]2 was purchased from Dako. Isotype-matched controls were obtained from the same manufacturers. The method used was described in detail by Bell et al.9Bell S.J. Rigby R. English N. Mann S.D. Knight S.C. Kamm M.A. Stagg A.J. Migration and maturation of human colonic dendritic cells.J Immunol. 2001; 166: 4958-4967PubMed Google Scholar Approximately 10 mucosal biopsy specimens (approximately 60 mg) were taken per patient. Biopsy specimens were collected in RPMI 1640 Dutch modification (Sigma-Aldrich, Dorset, England) supplemented with 10% fetal calf serum, 25 μg/mL gentamicin, and 100 U/mL penicillin/streptomycin (complete medium). Mucus and feces were removed from the tissue using 1 mmol/L dithiothreitol (Sigma-Aldrich) in Hank’s balanced salt solution (Gibco BRL, Paisley, Scotland) for 20 minutes in T25 flasks. The epithelium was removed using two 30-minute treatments with 1 mmol/L EDTA in calcium- and magnesium-free Hank’s balanced salt solution at 37°C with gentle agitation. The biopsy samples were washed in Hank’s balanced salt solution between each treatment. Tissue was digested with 1 mg/mL collagenase D (Roche Diagnostics Ltd, Lewes, England) in RPMI 1640/HEPES (Sigma-Aldrich Co Ltd, Poole, England) containing 2% fetal calf serum and 20 μg/mL deoxyribonuclease I (Roche Diagnostics Ltd). Tissue was digested using approximately 10 mL collagenase digestion medium per 200 mg of tissue in T25 flasks by agitating for 90–120 minutes at 37°C. After incubation, lamina propria mononuclear cells (LPMCs) released from the tissue samples were passed through a cell strainer and washed in complete medium. The cells were assessed for viability by their ability to exclude trypan blue. Cells were labeled in diluted whole blood (50 μL blood and 50 μL RPMI medium per tube) or, for peripheral blood mononuclear cells and LPMCs, in phosphate-buffered saline (PBS) containing 1 mmol/L EDTA and 0.02% sodium azide (fluorescence-activated cell sorter [FACS] buffer). A minimum of 50,000 LPMCs or peripheral blood mononuclear cells was used per antibody labeling. Antibodies were added at predetermined optimal concentrations. For staining involving only directly conjugated reagents, all antibodies were added simultaneously. For experiments involving an unconjugated antibody, cells were initially labeled with the unconjugated antibody, washed by centrifugation, and then labeled with goat anti-mouse FITC-conjugated secondary antibody. Unoccupied binding sites were blocked with normal mouse serum before addition of the directly conjugated antibodies. Labeling of whole blood was performed at room temperature for 15 minutes per step, and then red cells were lysed using Optilyse C (500 μL; Beckman Coulter) for 15 minutes at room temperature. Labeling of LPMCs and peripheral blood mononuclear cells was performed on ice for 20 minutes, and the cells were then washed twice by centrifugation in FACS buffer (300g, 10 minutes, 4°C). Paraformaldehyde (500 μL of a 1.0% wt/vol solution in .85% saline) was added, and the samples were stored at 4°C until acquisition on the flow cytometer within 24 hours. For assessment of intracellular TLRs, cells were initially surface labeled for identification of cell populations (see following text). They were then fixed with Leucoperm A (100 μL; Serotec) and permeabilized with Leucoperm B (100 μL; Serotec) before addition of FITC-conjugated anti-TLR antibodies. LPMCs were placed in wells at 2.5 × 105 cells per well in complete medium (96-well, U-bottom, Falcon; BD Biosciences). Paired cultures, one incubated with monensin (3 μmol/L) to maintain cytokine within the cells and the other incubated without monensin, were cultured for 4 hours at 37°C in a humidified atmosphere of 5% co2 in air. Cells were then labeled for surface markers, fixed with Leucoperm A (100 μL), and permeabilized with Leucoperm B (100 μL). The anti-cytokine antibodies IL-10-PE, IL-12-PE, or IL-6-PE (5 μL) were added for 30 minutes, and the cells were washed and finally resuspended in 1% paraformaldehyde. To confirm specificity of cytokine labeling, competition experiments were performed on blood DCs using unlabeled antibodies of clones identical to those used for cytokine staining. Cells were then stained with an antibody mixture containing PC5-conjugated monoclonal antibodies against CD3, CD14, CD16, CD19, CD34, and CD56 (lineage markers), an anti-HLA-DR-APC conjugate, and an anti-CD11c-FITC or anti-CD11c-PE conjugate. LPMCs were extracted as previously described, washed for 5 minutes, and resuspended in MiniMacs buffer (PBS supplemented with 2 mmol/L EDTA and 0.5% bovine serum albumin). To enrich DCs before sorting, cells were labeled with HLA-DR-PE antibody (40 μL) and incubated on ice for 20 minutes. After washing twice in FACS buffer, cells were incubated with anti-PE beads (50 μL) for 20 minutes before washing twice. Separation of HLA-DR+ cells was performed by positive selection using a MiniMacs magnetic cell sorting system (Miltenyi Biotec, Sunnyvale, CA). HLA-DR+ enriched cells were then incubated for 20 minutes on ice with an antibody mixture containing PC5-conjugated CD3, CD14, CD16, CD19, CD34, and CD56 (lineage markers) and CD11c-APC before sorting on a Becton Dickinson FACSCalibur machine (Oxford, England) as a CD11c+ HLA-DR+ lineage− population. Sorted cells were then cytocentrifuged, and slides were air dried overnight. In addition, as a positive control for TLR2 and TLR4 staining, blood monocytes were sorted and cytospins prepared. Cytospin cells were prepared by positive selection of CD14+ monocytes from anticoagulated whole blood. CD14+ cells were enriched using magnetic cell sorting with CD14 microbeads. Sorted cells were then cytocentrifuged, and cytospin slides were air dried overnight. Indirect immunofluorescence labeling was performed on CD11c+ HLA-DR+ lineage− DCs and CD14+ monocytes using cytospins. Slides were washed in PBS for 5 minutes and fixed in 1:1 acetone/methanol (vol/vol) for 4 minutes at room temperature. After permeabilization with 0.1% Triton X-100/PBS for 4 minutes at 4°C, cells were washed twice and nonspecific binding was blocked by 5% normal goat serum diluted in PBS for 30 minutes at room temperature in a humidified chamber. Cytospins were then incubated with anti-TLR2 (1:50) and anti-TLR4 (1:100) diluted in 1% bovine serum albumin/PBS in a humidified chamber for 16 hours at 4°C. After washing 3 times in PBS, cells were incubated with FITC-conjugated goat anti-mouse immunoglobulin G (1:100) diluted in 1% bovine serum albumin/PBS for 1 hour at room temperature in the dark. Slides were then rinsed in PBS for 5 minutes and mounted under coverslips. Control experiments were performed in parallel with the omission of one of the primary antibodies or by using the appropriate isotype controls instead of the primary antibodies. Data were acquired using a FACSCalibur flow cytometer (Becton Dickinson, Oxford, England). Using multicolor analysis, DCs were identified as an HLA-DR+ lineage− (CD3−, CD14−, CD16−, CD19−, CD34−, CD56−) population, and within this gate the CD11c+ population was assessed. CD40 expression was analyzed using CellQuest software (Becton Dickinson). The level of CD40 expression on gated populations was determined by geometric mean fluorescence intensity (MFI), with subtraction of values for isotype-matched controls. The analysis of TLR-positive and cytokine-positive cell populations used WinList version 5.0 flow cytometry software (Verity, Topsham, ME).28Panoskaltsis N. Reid C.D. Knight S.C. Quantification and cytokine production of circulating lymphoid and myeloid cells in acute myelogenous leukaemia.Leukemia. 2003; 17: 716-730Crossref PubMed Scopus (63) Google Scholar TLR-positive CD11c+ DCs were quantified by comparing a normalized cumulative histogram of anti-TLR staining of the gated cell population with a similar histogram of staining with an isotype-matched control antibody. The proportion of TLR-positive cells was determined by superenhanced Dmax (SED) normalized subtraction of the isotype control histogram from the histogram of anti-TLR staining. Thus, SED analysis uses the cumulative normalized histograms and is not equivalent to channel number subtraction. It allows positive cells to be resolved in situations where distribution histograms overlap and is a development of the enhanced normalized subtraction method29Bagwell B. A journey through flow cytometric immunofluorescence analyses—finding accurate and robust algorithms that estimate positive fraction distributions.Clin Immunol Newslett. 1996; 16: 33-37Crossref Google Scholar in which errors in estimation of the positive fraction have been reduced. The intensity of staining with anti-TLR reagents was determined as the ratio of the linearized median value of the TLR-positive cells, determined by SED, to the linearized median of the total gated DC population stained with the isotype control reagent. The proportion of cytokine-positive cells was also determined using SED normalized subtraction. In this case, normalized cumulative histograms of the staining of cells cultured in the absence of monensin were subtracted from histograms of the staining of cells cultured in the presence of monensin, giving a measure of ongoing cytokine production.28Panoskaltsis N. Reid C.D. Knight S.C. Quantification and cytokine production of circulating lymphoid and myeloid cells in acute myelogenous leukaemia.Leukemia. 2003; 17: 716-730Crossref PubMed Scopus (63) Google Scholar The use of the same antibody to label cells from both monensin-treated and untreated cultures gives this technique a high degree of sensitivity for detecting small changes in antibody binding. Specificity of antibody labeling was confirmed in competition experiments with unlabeled antibodies. Two-tailed t tests were used to compare proportions of cells. Data were paired where appropriate. Values of P < .05 were regarded as significant. Myeloid DCs were identified in whole blood as a CD11c+ HLA-DR+ lineage− population (Figure 1A). An example of the subtraction technique used to quantify labeling with anti-TLR antibodies is shown in Figure 1B, which illustrates detection of TLR2 on the surface of myeloid but not plasmacytoid (CD11c−) DCs. Overall, a high proportion of these DCs expressed surface TLR2 (mean ± SEM, 92.1% ± 0.6%, n = 8; Figure 1C). A smaller proportion of myeloid DCs expressed surface TLR4 (25.4% ± 5.3%, n = 8; Figure 1C). The CD11c− plasmacytoid DC subset expressed neither TLR2 nor TLR4. A CD11c+ HLA-DR+ lineage− population was identified in LPMCs obtained from colonic biopsy specimens by rapid collagenase digestion (Figure 2A). These cells have been extensively characterized in our earlier study9Bell S.J. Rigby R. English N. Mann S.D. Knight S.C. Kamm M.A. Stagg A.J. Migration and maturation of human colonic dendritic cells.J Immunol. 2001; 166: 4958-4967PubMed Google Scholar and classified as myeloid DCs on the basis of morphology, phenotype, and function (stimulatory capacity, endocytic activity, adherence properties, and the ability to undergo “maturation”). In contrast to the blood myeloid DC population, only a small proportion of colonic myeloid DCs from healthy controls expressed either TLR2 (19.0% ± 5.0%, n = 16; Figure 2B) or TLR4 (7.8% ± 4.3%, n = 16; Figure 2B) at the cell surface. Given the anatomic variation in microbial flora along the axis of the intestine, we compared expression of TLR2 and TLR4 in ileal and colonic tissue. In 6 healthy controls with endoscopically and histologically normal mucosa, paired samples were taken from the ileum and the colon and TLR expression was assessed. There was no significant difference in the proportion of DCs expressing either TLR2 or TLR4 in the ileum compared with the colon (Figure 3A). To rule out the possibility that exposure to EDTA, dithiothreitol, or collagenase/deoxyribonuclease removes TLRs from the surface of DCs, expression of TLR2 and TLR4 was examined on blood DCs that had been exposed to the isolation procedure used for colonic tissue. Expression of TLR2 and TLR4 by DCs was unaffected by exposure to the colonic tissue isolation procedure (data not shown). To test whether low levels of TLR2 and TLR4 on colonic DCs from healthy tissue could be explained by redistribution to intracellular compartments, labeling of permeabilized cells was examined. There was no increase in levels of st