characteristics of intestinal dendritic cells in inflammatory bowel diseases

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GASTROENTEROLOGY 2005;129:50–65

haracteristics of Intestinal Dendritic Cells in Inflammatoryowel Diseases

ILSA L. HART,*,‡ HAFID OMAR AL–HASSI,* RACHAEL J. RIGBY,* SALLY J. BELL,*,‡

NTON V. EMMANUEL,‡ STELLA C. KNIGHT,* MICHAEL A. KAMM,*,‡ and ANDREW J. STAGG*Antigen Presentation Research Group, Imperial College London, Northwick Park Campus; and ‡St Mark’s Hospital, Harrow,
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ackground & Aims: Dendritic cells (DCs) recognize andespond to microbial structures using pattern recogni-ion receptors, including Toll-like receptors (TLRs). In thentestine, DCs are pivotal in tolerance induction andirect the differentiation of T cells. We aimed to identifyhanges in intestinal DCs that may underlie the dysregu-ated immune response to enteric bacteria that occursn patients with inflammatory bowel disease (IBD).ethods: DCs were identified in freshly isolated laminaropria mononuclear cells by multicolor flow cytometry

n patients with IBD and controls. Expression of TLR2,LR4, and the activation/maturation marker CD40 wasssessed by cell surface labeling. Production of cyto-ines (interleukin [IL]-12, IL-6, and IL-10) was assessed

n the absence of exogenous stimulation by intracellulartaining of permeabilized cells. Results: In healthy con-rols, few intestinal DCs expressed TLR2 or TLR4, inontrast to blood DCs. DC expression of both TLRs wasignificantly enhanced in Crohn’s disease and ulcerativeolitis. DCs from inflamed tissue of patients with Crohn’sisease expressed significantly higher levels of the mat-ration/activation marker CD40. Elevated levels ofD40 on DCs were decreased after treating patientsith anti–tumor necrosis factor �. In Crohn’s disease,ut not ulcerative colitis, more colonic DCs producedL-12 and IL-6. The number of IL-10–producing DCs didot differ significantly between patients with IBD andontrols. Conclusions: In IBD, DCs are activated, theirxpression of microbial recognition receptors is up-reg-lated, and more DCs produce pathologically relevantytokines. Intestinal DCs are likely to be key initiators orerpetuators of the inflammatory response that charac-erizes IBD.

rohn’s disease and ulcerative colitis are chronic re-lapsing inflammatory diseases of the gastrointestinal

ract. The immunopathology of these diseases relates ton inappropriate and exaggerated mucosal immune re-ponse to constituents of the intestinal flora in geneti-ally predisposed individuals.

Antigen-presenting cells such as dendritic cells (DCs)
re likely to play a central role in the host response to
ntestinal flora, both in innate responses to bacteria andy shaping the character of the host’s adaptive immuneesponse. In healthy mice, lamina propria DCs in theistal ileum show evidence of bacterial sampling.1 Inice with genetic abnormalities involving the function

f antigen-presenting cells, intestinal inflammation oc-urs. Myeloid-specific Stat3-deficient animals that have aefect in the response of their macrophages and DCs totat3-dependent cytokines such as interleukin (IL)-10evelop intestinal inflammation characterized by en-anced production of proinflammatory IL-12, IL-6, andumor necrosis factor (TNF)-�.2,3 Furthermore, studiesn murine adoptive transfer models of colitis suggest thatCs are important in the initiation4 and perpetuation of

ntestinal inflammation.5

In Crohn’s disease, a subgroup of patients possessesariants of the NOD2 protein. NOD2 is expressed byyeloid cells, including DCs, and is involved in innate

acterial recognition and regulation of the inflammatoryascade,6–8 suggesting that altered responses to the mi-roflora by DCs may contribute to the inflammatoryesponse in Crohn’s disease.

DCs are present in the intestine in the gut-associatedymphoid tissue and the lamina propria and lie in closeroximity to the large and dynamic antigenic load in theut lumen.9–12 DCs in Peyer’s patches sample commen-al bacteria,13 but this is not the only site at whichntigen uptake occurs. Lamina propria DCs pass theirendrites between epithelial tight junctions and interactirectly with luminal antigens14 and can sample luminalntigens that have passed through the epithelium. Intes-inal DCs have properties distinct from their nonmucosal

Abbreviations used in this paper: DC, dendritic cell; FACS, fluores-ence-activated cell sorter; FITC, fluorescein isothiocyanate; IL, inter-eukin; LPMC, lamina propria mononuclear cell; MFI, mean fluorescentntensity; SED, superenhanced Dmax; TLR, Toll-like receptor; TNF, tumorecrosis factor.

© 2005 by the American Gastroenterological Association0016-5085/05/$30.00

doi:10.1053/j.gastro.2005.05.013

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July 2005 INTESTINAL DENDRITIC CELLS IN IBD 51

ounterparts, probably as a result of their associationith the external environment. They are involved inoth nonresponsiveness or tolerance induction and re-ponsiveness to antigens in the gut.15,16 For example,Cs isolated from murine Peyer’s patches produce more

L-10 than splenic DCs and have a tendency to induceh2/3 responses.10

DCs sense microbes by a series of surface receptors,ncluding Toll-like receptors (TLRs), that recognizetructural elements displayed on the surface of mi-robes.17 TLR4 is required for recognition of lipopoly-accharide from Escherichia coli, and TLR2 recognizeseptidoglycan and lipoteichoic acid from gram-positiveacteria and lipoproteins from both gram-positive andram-negative organisms.18–23 DCs control microbial-riven T-cell polarization in part through the ligation ofLRs.24 After interaction with microbial products orther maturation stimuli such as cytokines, immatureCs in peripheral tissues change their pattern of chemo-ine receptors and migrate to the draining lymphoidissue.25 During this process, DCs down-regulate theirntigen acquisition machinery, up-regulate the cell sur-ace expression of major histocompatibility complex/pep-ide antigen complexes and maturation/costimulatoryolecules such as CD40, and acquire their characteristic

bility to stimulate naive T cells. The type of effector-cell response is influenced by the cytokines producedy the activating DCs. For example, production of IL-12y DCs polarizes a Th1 response,26 production of IL-10y DCs influences a regulatory response,10 and produc-ion of IL-6 plays a role in overcoming the suppressiveffect of regulatory T cells.27

We hypothesized that alterations in gut DCs mayontribute to the dysregulated immune response thatnderlies human inflammatory bowel disease (IBD). Inarticular, we hypothesized that an abnormal pattern ofacterial recognition by DCs through TLRs and alteredC activation and cytokine production may underlie

hronic inflammatory processes. Therefore, we analyzedLR2 and TLR4 expression on DCs, maturation status ofCs, and production of the cytokines IL-12, IL-6, and

L-10 by DCs present in the lamina propria of patientsith IBD and healthy controls.

Materials and Methods

Patients

Intestinal specimens were obtained at colonoscopyrom patients with Crohn’s disease, patients with ulcerativeolitis, or controls. The Crohn’s disease group (n � 31) con-isted of 15 men and 16 women, ranging from 18 to 69 years
f age. The diagnosis for each patient was made using clinical c
arameters, radiographic studies, and histologic criteria. Athe time of sample collection, 10 patients had a new diagnosisf Crohn’s disease and were on no medication, 6 patients wereeceiving corticosteroids, 6 patients were receiving azathio-rine, and 9 patients were receiving an oral sulfasalazinereparation. There were 7 patients with Crohn’s disease whoeceived anti–TNF-� treatment, and biopsy samples wereaken before and 2 weeks after infusion. The ulcerative colitisroup (n � 24) consisted of 21 men and 3 women, rangingrom 18 to 65 years of age. The diagnosis for each patient wasade using clinical parameters, radiographic studies, and his-

ologic criteria. In the ulcerative colitis group, 1 patient waseceiving corticosteroids, 3 patients were receiving azathio-rine, 21 patients were receiving an oral sulfasalazine prepa-ation, and 2 patients were on no medication. The controlroup consisted of 39 patients with macroscopically and his-ologically normal intestine who had been referred with rectalleeding or a change in bowel habit. There were 17 men and2 women in the control group, ranging from 22 to 85 yearsf age. Informed consent was obtained from all patients, andhe protocol was approved by the local ethics committee.

Antibodies

Antibodies with the following specificities and fluoro-hrome labels were used: CD11c-FITC (KB90) from DakoEly, England); CD3-PC5 (UCHT-1), CD14-PC5 (MIP9),D16-PC5 (B73.1), CD19-PC5 (4G7), CD56-PC5 (N901),nd CD8-PC5 (B9.11) from Beckman Coulter (High Wy-ombe, England); CD34-CyChrome (581), CD40-PE (LOB7/), TLR2-FITC (TL2.1), and TLR4-FITC (HTA125) fromerotec (Oxford, England); CD11c-PE (B-ly6), CD14-PEM�P9), CD16-PE (B73.1), CD19-PE (4G7), CD34-PE8G12), HLA-DR-APC (G46-6), HLA-DR-PE (L243), andD8-FITC/PE/APC (SK1) from BD Biosciences (Oxford, En-land); and unconjugated TLR2 (TL2.1) and TLR4 (HTA125)rom Imgenex (San Diego, CA). Intracellular cytokine stainingsed IL-10-PE (JES3-9D7; Serotec), IL-12p40/p70-PE (C11.5;D Biosciences), and IL-6 PE (#1936; R&D, Abingdon, En-land). Fluorescein isothiocyanate (FITC)-conjugated anti-oat F[ab=]2 was purchased from Dako. Isotype-matched con-rols were obtained from the same manufacturers.

Isolation of Lamina PropriaMononuclear Cells

The method used was described in detail by Bell et al.9

pproximately 10 mucosal biopsy specimens (approximately0 mg) were taken per patient. Biopsy specimens were col-ected in RPMI 1640 Dutch modification (Sigma-Aldrich,orset, England) supplemented with 10% fetal calf serum, 25g/mL gentamicin, and 100 U/mL penicillin/streptomycin

complete medium). Mucus and feces were removed from theissue using 1 mmol/L dithiothreitol (Sigma-Aldrich) inank’s balanced salt solution (Gibco BRL, Paisley, Scotland)

or 20 minutes in T25 flasks. The epithelium was removedsing two 30-minute treatments with 1 mmol/L EDTA in
alcium- and magnesium-free Hank’s balanced salt solution at

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52 HART ET AL GASTROENTEROLOGY Vol. 129, No. 1

7°C with gentle agitation. The biopsy samples were washedn Hank’s balanced salt solution between each treatment.issue was digested with 1 mg/mL collagenase D (Rocheiagnostics Ltd, Lewes, England) in RPMI 1640/HEPES

Sigma-Aldrich Co Ltd, Poole, England) containing 2% fetalalf serum and 20 �g/mL deoxyribonuclease I (Roche Diag-ostics Ltd). Tissue was digested using approximately 10 mLollagenase digestion medium per 200 mg of tissue in T25asks by agitating for 90–120 minutes at 37°C. After incu-ation, lamina propria mononuclear cells (LPMCs) releasedrom the tissue samples were passed through a cell strainer andashed in complete medium. The cells were assessed foriability by their ability to exclude trypan blue.

Cell Surface and Intracellular Labeling

Cells were labeled in diluted whole blood (50 �L bloodnd 50 �L RPMI medium per tube) or, for peripheral bloodononuclear cells and LPMCs, in phosphate-buffered saline

PBS) containing 1 mmol/L EDTA and 0.02% sodium azidefluorescence-activated cell sorter [FACS] buffer). A minimumf 50,000 LPMCs or peripheral blood mononuclear cells wassed per antibody labeling. Antibodies were added at prede-ermined optimal concentrations. For staining involving onlyirectly conjugated reagents, all antibodies were added simul-aneously. For experiments involving an unconjugated anti-ody, cells were initially labeled with the unconjugated anti-ody, washed by centrifugation, and then labeled with goatnti-mouse FITC-conjugated secondary antibody. Unoccupiedinding sites were blocked with normal mouse serum beforeddition of the directly conjugated antibodies. Labeling ofhole blood was performed at room temperature for 15 min-tes per step, and then red cells were lysed using Optilyse C500 �L; Beckman Coulter) for 15 minutes at room temper-ture. Labeling of LPMCs and peripheral blood mononuclearells was performed on ice for 20 minutes, and the cells werehen washed twice by centrifugation in FACS buffer (300g, 10inutes, 4°C). Paraformaldehyde (500 �L of a 1.0% wt/vol

olution in .85% saline) was added, and the samples weretored at 4°C until acquisition on the flow cytometer within4 hours. For assessment of intracellular TLRs, cells werenitially surface labeled for identification of cell populationssee following text). They were then fixed with Leucoperm A100 �L; Serotec) and permeabilized with Leucoperm B (100L; Serotec) before addition of FITC-conjugated anti-TLR

ntibodies.

Cytokine Production by Lamina Propria DCs

LPMCs were placed in wells at 2.5 � 105 cells per welln complete medium (96-well, U-bottom, Falcon; BD Bio-ciences). Paired cultures, one incubated with monensin (3mol/L) to maintain cytokine within the cells and the other

ncubated without monensin, were cultured for 4 hours at7°C in a humidified atmosphere of 5% CO2 in air. Cells werehen labeled for surface markers, fixed with Leucoperm A (100L), and permeabilized with Leucoperm B (100 �L). The

nti-cytokine antibodies IL-10-PE, IL-12-PE, or IL-6-PE (5 i

L) were added for 30 minutes, and the cells were washed andnally resuspended in 1% paraformaldehyde. To confirm spec-ficity of cytokine labeling, competition experiments wereerformed on blood DCs using unlabeled antibodies of clonesdentical to those used for cytokine staining. Cells were thentained with an antibody mixture containing PC5-conjugatedonoclonal antibodies against CD3, CD14, CD16, CD19,D34, and CD56 (lineage markers), an anti–HLA-DR-APConjugate, and an anti–CD11c-FITC or anti–CD11c-PEonjugate.

Sorting of CD11c� DCs From LPMCs

LPMCs were extracted as previously described, washedor 5 minutes, and resuspended in MiniMacs buffer (PBSupplemented with 2 mmol/L EDTA and 0.5% bovine serumlbumin). To enrich DCs before sorting, cells were labeledith HLA-DR-PE antibody (40 �L) and incubated on ice for0 minutes. After washing twice in FACS buffer, cells werencubated with anti-PE beads (50 �L) for 20 minutes beforeashing twice. Separation of HLA-DR� cells was performedy positive selection using a MiniMacs magnetic cell sortingystem (Miltenyi Biotec, Sunnyvale, CA). HLA-DR� enrichedells were then incubated for 20 minutes on ice with anntibody mixture containing PC5-conjugated CD3, CD14,D16, CD19, CD34, and CD56 (lineage markers) andD11c-APC before sorting on a Becton Dickinson FACSCali-ur machine (Oxford, England) as a CD11c� HLA-DR� lin-age� population. Sorted cells were then cytocentrifuged, andlides were air dried overnight. In addition, as a positiveontrol for TLR2 and TLR4 staining, blood monocytes wereorted and cytospins prepared. Cytospin cells were prepared byositive selection of CD14� monocytes from anticoagulatedhole blood. CD14� cells were enriched using magnetic cell

orting with CD14 microbeads. Sorted cells were then cyto-entrifuged, and cytospin slides were air dried overnight.

Immunofluorescence Microscopy

Indirect immunofluorescence labeling was performedn CD11c� HLA-DR� lineage� DCs and CD14� monocytessing cytospins. Slides were washed in PBS for 5 minutes andxed in 1:1 acetone/methanol (vol/vol) for 4 minutes at roomemperature. After permeabilization with 0.1% Triton X-100/BS for 4 minutes at 4°C, cells were washed twice andonspecific binding was blocked by 5% normal goat serumiluted in PBS for 30 minutes at room temperature in aumidified chamber. Cytospins were then incubated with anti-LR2 (1:50) and anti-TLR4 (1:100) diluted in 1% bovine

erum albumin/PBS in a humidified chamber for 16 hours at°C. After washing 3 times in PBS, cells were incubated withITC-conjugated goat anti-mouse immunoglobulin G (1:100)iluted in 1% bovine serum albumin/PBS for 1 hour at roomemperature in the dark. Slides were then rinsed in PBS for 5inutes and mounted under coverslips. Control experimentsere performed in parallel with the omission of one of therimary antibodies or by using the appropriate isotype controls
nstead of the primary antibodies.

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Flow Cytometry

Data were acquired using a FACSCalibur flow cytom-ter (Becton Dickinson, Oxford, England). Using multicolornalysis, DCs were identified as an HLA-DR� lineage�

CD3�, CD14�, CD16�, CD19�, CD34�, CD56�) popula-ion, and within this gate the CD11c� population was as-essed.

CD40 expression was analyzed using CellQuest softwareBecton Dickinson). The level of CD40 expression on gatedopulations was determined by geometric mean fluorescencentensity (MFI), with subtraction of values for isotype-matchedontrols.

The analysis of TLR-positive and cytokine-positive cell pop-lations used WinList version 5.0 flow cytometry softwareVerity, Topsham, ME).28 TLR-positive CD11c� DCs wereuantified by comparing a normalized cumulative histogramf anti-TLR staining of the gated cell population with aimilar histogram of staining with an isotype-matched controlntibody. The proportion of TLR-positive cells was deter-ined by superenhanced Dmax (SED) normalized subtraction of

he isotype control histogram from the histogram of anti-TLRtaining. Thus, SED analysis uses the cumulative normalizedistograms and is not equivalent to channel number subtrac-ion. It allows positive cells to be resolved in situations whereistribution histograms overlap and is a development of thenhanced normalized subtraction method29 in which errors instimation of the positive fraction have been reduced.

The intensity of staining with anti-TLR reagents was de-ermined as the ratio of the linearized median value of theLR-positive cells, determined by SED, to the linearizededian of the total gated DC population stained with the

sotype control reagent.The proportion of cytokine-positive cells was also deter-ined using SED normalized subtraction. In this case, nor-alized cumulative histograms of the staining of cells cultured

n the absence of monensin were subtracted from histograms ofhe staining of cells cultured in the presence of monensin,iving a measure of ongoing cytokine production.28 The use ofhe same antibody to label cells from both monensin-treatednd untreated cultures gives this technique a high degree ofensitivity for detecting small changes in antibody binding.pecificity of antibody labeling was confirmed in competitionxperiments with unlabeled antibodies.

Statistical Analysis

Two-tailed t tests were used to compare proportions ofells. Data were paired where appropriate. Values of P � .05ere regarded as significant.

Results

TLR2 and TLR4 Are Expressed on BloodMyeloid DCs

Myeloid DCs were identified in whole blood as a
D11c� HLA-DR� lineage� population (Figure 1A). p
n example of the subtraction technique used to quan-ify labeling with anti-TLR antibodies is shown in Fig-re 1B, which illustrates detection of TLR2 on theurface of myeloid but not plasmacytoid (CD11c�) DCs.verall, a high proportion of these DCs expressed surfaceLR2 (mean SEM, 92.1% 0.6%, n � 8; FigureC). A smaller proportion of myeloid DCs expressedurface TLR4 (25.4% 5.3%, n � 8; Figure 1C). TheD11c� plasmacytoid DC subset expressed neitherLR2 nor TLR4.

In Healthy Controls, Intestinal DCs ExpressLess TLR2 and TLR4 Than Blood DCs

A CD11c� HLA-DR� lineage� population wasdentified in LPMCs obtained from colonic biopsy spec-mens by rapid collagenase digestion (Figure 2A). Theseells have been extensively characterized in our earliertudy9 and classified as myeloid DCs on the basis oforphology, phenotype, and function (stimulatory ca-

acity, endocytic activity, adherence properties, and thebility to undergo “maturation”).

In contrast to the blood myeloid DC population, onlysmall proportion of colonic myeloid DCs from healthy

ontrols expressed either TLR2 (19.0% 5.0%, n � 16;igure 2B) or TLR4 (7.8% 4.3%, n � 16; Figure 2B)t the cell surface.

Given the anatomic variation in microbial flora alonghe axis of the intestine, we compared expression ofLR2 and TLR4 in ileal and colonic tissue. In 6 healthyontrols with endoscopically and histologically normalucosa, paired samples were taken from the ileum and

he colon and TLR expression was assessed. There was noignificant difference in the proportion of DCs expressingither TLR2 or TLR4 in the ileum compared with theolon (Figure 3A).

To rule out the possibility that exposure to EDTA,ithiothreitol, or collagenase/deoxyribonuclease removesLRs from the surface of DCs, expression of TLR2 andLR4 was examined on blood DCs that had been exposed

o the isolation procedure used for colonic tissue. Expres-ion of TLR2 and TLR4 by DCs was unaffected byxposure to the colonic tissue isolation procedure (dataot shown).To test whether low levels of TLR2 and TLR4 on

olonic DCs from healthy tissue could be explained byedistribution to intracellular compartments, labeling ofermeabilized cells was examined. There was no increasen levels of staining for either TLR2 or TLR4 on DCs ifhe cells were permeabilized before labeling (Figure 3B).n contrast, staining for the positive control intracellular
rotein -actin was increased in permeabilized cells.


Figure 1. Identification of blood DCs and expression of TLR2 andTLR4. (A) DCs were identified in blood by multicolor flow cytometry asHLA-DR� lineage� (CD3�, CD14�, CD16�, CD19�, CD34�, CD56�)cells. Within this DC gate, CD11c� and CD11c� DC subsets wereidentified. (B) Labeling of gated DC subsets with anti-TLR antibodieswas quantified using SED normalized subtraction. In this example,1-parameter histograms for staining with anti-TLR2 or its isotype-matched control are shown. The proportion of TLR2-positive DCs wasdetermined by subtracting normalized cumulative histograms of label-ing with the control antibody from similar histograms of labeling withanti-TLR2. In the histograms with subtraction percentages, the outlineof the histogram indicates staining with anti-TLR2 and the shadedarea represents the proportion of TLR2-positive cells after subtractionof isotype control binding. (C) The proportion of blood CD11c� DCsexpressing TLR2 and TLR4 in healthy controls (n � 8) was assessed
using the SED normalized subtraction facility in WinList 5.0.

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hus, we did not find evidence for a major intracellularool of TLR2 or TLR4 in colonic DCs.

In IBD, More DCs Express TLR2 and TLR4

Compared with DCs from healthy controls, moreolonic DCs from patients with active IBD expressedLR2 and TLR4 (Figure 2B). The increase in DCsxpressing TLR was similar in ulcerative colitis androhn’s disease. In 8 patients with subtotal ulcerativeolitis in which there was a clear demarcation betweennflamed and noninflamed tissue, paired samples wereaken (Figure 4). A representative pair of antibody stain-ng for TLR2 and TLR4 is shown in Figure 4A; com-ined data for all 8 patients are shown in Figure 4B. Theroportion of myeloid DCs expressing TLR2 was highern inflamed versus noninflamed tissue. More myeloid

igure 2. Identification of co-onic DCs and expression ofLR2 and TLR4. (A) DCs weredentified in colonic tissue by mul-icolor flow cytometry as HLA-DR�

ineage� (CD3�, CD14�, CD16�,D19�, CD34�, CD56�). Within

his gate, CD11c� DCs weredentified. (B) The proportion ofD11c� DCs staining positive forLR2 and TLR4 in tissue fromealthy controls (n � 13), in-amed tissue from patients withlcerative colitis (n � 15), and

nflamed tissue from patientsith Crohn’s disease (n � 7) ishown.

Cs also expressed TLR4. There was no significant dif- p

erence in binding of the isotype control reagent to DCsrom inflamed and noninflamed tissue. The proportion ofCs expressing TLRs did not differ significantly betweenoninflamed tissue from patients with ulcerative colitisnd tissue from healthy controls.

For the patients with ulcerative colitis, there was noorrelation between the proportion of TLR-positive DCsnd the severity of the intestinal inflammation as assessedy the Ulcerative Colitis Disease Activity Index.30 Thereas also no correlation between the proportion of TLR-ositive DCs and whether or not the patients were takinghe immunosuppressive drug azathioprine.

To confirm expression of TLR2 and TLR4 on colonicCs from patients with intestinal inflammation, DCsere purified by cell sorting (Figure 5A) and cytospins
repared. Immunofluorescent labeling with anti-TLR

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ntibodies showed expression of TLR2 and TLR4 (FigureB). Labeling of colonic DCs was similar to that ofurified peripheral blood monocytes used as a positiveontrol for TLR expression.

Enhanced Expression of CD40 on ColonicDCs in IBD

CD40 is expressed at low levels on immature DCs

igure 3. TLR2 and TLR4 expression on intestinal DCs from healthyontrols. (A) In 6 healthy controls, paired samples were taken fromleal and colonic tissue and the proportion of intestinal DCs express-ng TLR2 and TLR4 was assessed. (B) Labeling of permeabilized andonpermeabilized colonic DCs from healthy controls with antibodieso TLR2, TLR4, and -actin. Intensity of staining was determined byubtraction using WinList, and data are presented as the ratio ofabeling of permeabilized cells (surface plus intracellular staining) toabeling of nonpermeabilized cells (cell surface staining only). Values1 indicate the presence of intracellular antigen.

rom nonintestinal sites but is up-regulated when the t

Cs undergo maturation or activation.31 Colonic DCsrom healthy controls consistently expressed low butetectable levels of CD40 (Figures 6 and 7A). Levels ofD40 on DCs from Crohn’s disease tissue were moreariable but were significantly greater on DCs fromnflamed Crohn’s disease tissue than on DCs from bothontrol and noninflamed Crohn’s disease tissue. Thereas no significant difference between the level of CD40n DCs from noninflamed Crohn’s tissue or tissue fromealthy controls. Expression of CD11c was used as part ofhe gating strategy to define the DC population undertudy. The level of CD11c did not differ between healthyontrols and patients with Crohn’s disease, suggestinghat there is not a generalized up-regulation of surfaceakers in intestinal inflammation (data not shown).

Treatment of Patients With Crohn’sDisease With Anti–TNF-� Reduces ColonicDC Expression of CD40

In 7 patients with Crohn’s disease receiving treat-ent with anti–TNF-�, levels of CD40 expression on

olonic DCs were evaluated before and after infusion.here was a significant reduction of CD40 expression onCs after treatment with anti–TNF-� (Figure 7B).ean levels of expression decreased from an MFI of 201

efore infusion to an MFI of 92 after infusion. Theecrease in CD40 level occurred irrespective of resolutionf inflammation; in 5 of the 7 patients, the tissue ob-ained on biopsy after infusion was inflamed yet DCxpression of CD40 was reduced (Figure 7B).

Increased Production of IL-12 and IL-6 byColonic DCs in Crohn’s Disease

Production of IL-12, IL-6, and IL-10 by colonicCs was assessed in the absence of exogenous stimulationsing intracellular cytokine staining. We chose this ap-roach because it enabled cytokine production by DCs toe assessed specifically; cytokines measured in superna-ants of cell mixtures may not have reflected productiony this numerically small population. Representativetaining of DCs from inflamed and noninflamed tissues ishown in Figure 8A, and pooled data from all patientsre shown in Figure 8B. In cells from control tissue, theroportion of IL-12–producing DCs was variable andow; overall, it did not reach statistical significance.here were no detectable IL-6–producing DCs. How-ver, a significant proportion of colonic DCs fromealthy tissue produced IL-10.Compared with colonic DCs from healthy controls,ore DCs from patients with active Crohn’s disease

roduced IL-12 and IL-6 (Figure 8). In contrast, produc-
ion of IL-12 and IL-6 by DCs from patients with

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lcerative colitis did not differ from that of control DCs.L-10–producing DCs were detected in both diseaseroups, but the proportion of IL-10–positive DCs didot differ among patients with ulcerative colitis, patientsith Crohn’s disease, and healthy controls.

DiscussionThis study shows that DCs in the human colonic

igure 4. TLR2 and TLR4 ex-ression on inflamed and nonin-amed tissue from patientsith ulcerative colitis. In 8 pa-

ients with ulcerative colitis inhom there was a demarcationetween inflamed and nonin-amed tissue, biopsy speci-ens were taken from bothoninflamed and inflamed sec-ions. The proportion of colonicCs expressing TLR2 and TLR4as assessed. (A) A represen-

ative example of the staining ofated DC from 1 patient. Openistograms show staining withn anti-TLR antibody or with an

sotype-matched control as indi-ated. To the right of each ofhese pairs of histograms is ahird histogram showing the re-ult of the subtraction analysis.ere, the open histogram is thetaining with the anti-TLR re-gent and the shaded area rep-esents the fraction of positiveells after subtraction of iso-ype control binding using SEDormalized subtraction. Numer-

cal values are the percentagef positive cells. (B) Pooled dataor all 8 patients.

ucosa are altered in IBD. DCs from diseased tissue a

how enhanced expression of TLR2 and TLR4, whichay contribute to altered microbial recognition; the DCs

re activated, and an increased proportion of them releaseroinflammatory cytokines. These altered DCs are likelyo contribute to the initiation or perpetuation of intes-inal inflammation, either as a local effector cell popula-ion active in innate immunity or by modifying theesponse of lymphocytes that the DCs activate as part of
n adaptive immune response.


Figure 5. TLR2 and TLR4 ex-pression demonstrated by im-munofluorescence on sorted co-lonic DCs from inflamed tissue.(A) The 2-parameter histogramsof HLA-DR and lineage (CD3,CD14, CD16, CD19, CD34,CD56) indicate the cells beforeand after the sorting process.The single-parameter histogramof CD11c indicates the expres-sion of CD11c on these cellsbefore and after the sort. (B)Indirect immunofluorescencewas performed on cytospins ofsorted CD11c� HLA-DR� lin-eage� cells derived from in-flamed tissue of a patient withCrohn’s disease. The right pan-els show immunofluorescentstaining, and the left panelsshow the equivalent bright field
images.

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Although originally characterized by their potent abil-ty to activate T cells, it is now clear that DCs have moreubtle immunoregulatory roles.32 They can determinehether or not a response is initiated to a particular

ntigenic challenge and can influence whether type 1,ype 2, or regulatory cells predominate when a responses initiated. The different outcomes are in turn influencedy the subpopulation of DCs involved, its state of acti-ation, and exposure of the DCs to microbial products,hich interact with pattern recognition receptors onCs, including TLRs and C-type lectins such as DC-

IGN. These interactions are likely to be of crucialmportance to immune regulation in the microbe-richnvironment of the intestine.

To study DCs in the human colon, we used multicolorow cytometry to identify CD11c� HLA-DR� lineage�

ells in LPMCs. What is the evidence that these cells areCs? In our earlier study, these cells were extensively

haracterized for their stimulatory capacity, endocyticctivity, adherence properties, and ability to undergomaturation.”9 In all regards, they had properties consis-ent with those of myeloid DCs. That the CD11c�

LA-DR� lineage� population comprises DCs ratherhan monocytes is strongly suggested by 2 properties:hey stimulate a primary mixed leukocyte reaction onaturation in vitro, and they display intermediate levels

f endocytic activity when FITC/dextran is used as a

igure 6. Flow cytometric datahowing CD40 expression onCs from patients with Crohn’sisease and controls. DCs were

dentified as HLA-DR� lineage�

ells in LPMCs (upper panel). Inhe lower panel, solid histo-rams represent staining ofD11c� DCs with anti-CD40;pen histograms show stainingith the isotype control. Numer-

cal values represent the MFI ofnti-CD40 staining with the iso-ype control subtracted. Repre-entative examples of DCs fromatients with Crohn’s diseasend controls are shown. Pooledata are presented in Figure 7.

racer.9 c

Here, we examined DC cell surface expression ofLR4, a receptor for lipopolysaccharide from gram-neg-tive bacteria,18 and TLR2, which interacts with pepti-oglycan and lipoproteins from gram-positive bacte-ia.19–21 Consistent with previous analyses of messengerNA from purified blood DC subsets that found message

or TLR2 and in some cases TLR4 in myeloid DCs,17,33

e detected TLR2 on the majority and TLR4 on a subsetf myeloid DCs in blood. In healthy controls, colonicCs showed little or no surface expression of TLR2 orLR4, in contrast to blood DCs, and control experimentsstablished that this finding was not an artifact of dif-erent tissue processing. Permeabilization of colonic DCsefore labeling did not increase staining with the anti-LR antibodies, arguing against significant intracellularmounts of these receptors and suggesting that the lowurface expression is not due to receptor redistribution. Aumber of mechanisms, including regulation of the levelf receptor expression and the activity of inhibitoryignaling molecules, such as Toll-interacting proteinTollip) or IL-1R–associated kinase (IRAK), serve toimit recognition and signaling through TLRs.34–36

hus, the lack of TLR2 and TLR4 on colonic DCs mayerve to limit recognition of the commensal flora in theealthy gut. Our data do not permit us to determinehether the relative absence of TLRs on the surface of

olonic DCs is due to redistribution to intracellular
ompartments. However, previous reports showing an

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verall paucity of TLR2- and TLR4-expressing cells inealthy colonic tissue by either immunohistologic oreal-time polymerase chain reaction methods35,37,38 indi-ate an absence of TLRs from both the surface andntracellular compartments of intestinal DCs. Studies onhe response of highly purified colonic DCs to definedLR ligands would test the relationship between levelsf TLR expression and response to microbial products byolonic DCs, but the low frequency of these cells inissues and the difficulty in obtaining large amounts oformal intestinal tissue make such experiments veryifficult to perform.In cells from IBD tissue, there were increased numbers

f TLR2-positive and TLR4-positive DCs. A generalizedncrease in TLR2 and TLR4 in inflamed intestinal tissueas been previously reported in the dextran sodiumulfate animal model of colitis.39 In human IBD, in-reased TLR2 and TLR4 expression has been reported onpithelial cells, and 10%–20% of cells in the laminaropria of both patients with ulcerative colitis and pa-ients with Crohn’s disease stained positive for TLR2 andLR4, with TLR-positive cells accumulating subepithe-

ially and close to the crypts.38 Thus, increased expressionf TLRs by DCs and other cells interacting with theicrobial flora may lead to increased recognition of

™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™igure 8. Cytokine production by DCs in patients with IBD and conttaining and SED normalized subtraction (see Materials and Methodsrom inflamed and noninflamed tissue. In each case, pairs of histogran the presence of monensin and the second histogram shows stainistogram showing the result of the subtraction analysis. Here the ophaded area represents the fraction of cytokine-positive DCs after sualues show the percent cytokine-positive DCs. (B) Pooled data showi
ith Crohn’s disease, patients with ulcerative colitis, and healthy controls
acterial products and enhanced responses to them. Aecent study suggests that TLR-mediated recognition oficrobial components by DCs triggers intestinal inflam-ation in an animal model of colitis driven by produc-

ion of IL-12.2

The increase in TLR-positive DCs in IBD was con-ned to areas of inflamed tissue. Cytokines, bacterialroducts, and necrotic cells have all been reported toodulate TLR expression, and one or more of these

actors may be responsible for the up-regulation of TLR2nd TLR4 on DCs in inflamed tissue.33,40–43 A numberf cytokines, including IL-6, TNF-�, and interferonamma, that are present in the inflamed mucosa ofatients with IBD44–47 caused increased expression ofLR2 and/or TLR4 in several experimental systems.acterial products may act directly via TLRs to up-

egulate further TLR expression on DCs, or they may actndirectly via cytokine production. Increased intestinalermeability may therefore lead to greater exposure toicrobial products and bacterially driven increases inLR expression. Recognition of bacterial structures byC-SIGN can inhibit signaling through TLRs, raising

he possibility that local changes in the microflora inBD may alter the balance of positive and negativeignals received by mucosal DCs.48

Figure 7. CD40 expression onDCs from patients with Crohn’sdisease and controls. (A) CD40expression on CD11c� colonicDCs. Net MFI represents theMFI of anti-CD40 staining withisotype control binding sub-tracted. DCs from healthy con-trols and patients with Crohn’sdisease, both inflamed (I) andnoninflamed (NI), were as-sessed. (B) Expression of CD40by colonic DCs assessed by netMFI before and after treatmentof patients with Crohn’s dis-ease with anti–TNF-�. Solidsymbols represent DCs from in-flamed tissue, and open sym-bols represent DCs from nonin-flamed tissue.

™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™3Cytokine production by DCs was assessed by intracellular cytokineRepresentative intracellular staining for IL-12, IL-6, and IL-10 in DCsre presented in which one histogram shows staining of cells culturedf DCs cultured without monensin. To the right of each pair is a thirdstogram shows staining of DCs in the presence of monensin and thetion of staining of cells from the “no monensin” cultures. Numericalduction of IL-12, IL-6, and IL-10 by DCs are shown for 5–10 patients

™™™rols.). (A)ms aing oen hibtracng pro

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An alternative explanation for the increase in TLR-ositive DCs may lie in the recruitment of DC popula-ions into the mucosa. Both TLR2 and TLR4 wereetected on blood DCs, raising the possibility that theseirculating TLR-positive DCs are abnormally recruitednto intestinal tissue under the influence of inflammatoryediators. To test this concept experimentally, studies in

nimal models will probably be required. Irrespective ofhich pathway(s) lead to increased TLR expression byCs in IBD, a consequent alteration in microbial recog-ition may be central to the disease process.DCs from the inflamed colonic tissue of patients with

rohn’s disease also expressed increased levels of CD40.n animal models, interactions between CD40 and itsigand, CD154, promote the development of intestinalnflammation.49–51 In human IBD, CD154 is expressedt increased levels in the inflamed mucosa,52–54 indicat-ng the local availability of both members of the recep-or-ligand pair and the potential for elevated mutualtimulation in interactions between CD40� andD154� cells. The levels of CD40 expressed by DCs

rom inflamed tissue did not approach the high levelsxpressed by mature DCs (data not shown), suggestingimited local activation of DCs rather than the presencef a population of fully mature DCs nucleating inflam-ation. These findings are in agreement with our pre-

ious report in which we examined expression of CD80,D86, and CD83 by colonic DCs and concluded that

here was no evidence of fully mature DCs in the in-amed mucosa of patients with IBD.9 It remains to beetermined in future, larger studies if other DC matu-ation markers such as CD80, CD86, and CD83 alsohow evidence of limited up-regulation in IBD or if thisffect is restricted to CD40.

CD40 expression on DCs is increased by TLR trigger-ng with microbial products such as lipopolysaccharidend by cytokines including TNF-�. In our study, levelsf CD40 on colonic DCs were reduced 2–3 weeks fol-owing treatment of patients with Crohn’s disease withnti–TNF-�. The post-treatment reduction in CD40evel was observed even when the tissue from which theCs were obtained was still macroscopically inflamed.his indicates that the reduction of CD40 on DCs can beue to a direct action of the anti–TNF-� drug ratherhan an indirect consequence of resolving inflammation.xtrapolating to the pathologic process, these findingsay suggest that activated DCs are involved early in the

nflammatory pathway in IBD.In vitro, studies on cells from nonintestinal tissues

ave shown that stimulation via CD40 results in thectivation of DCs and the production of cytokines,
ncluding IL-12, IL-6, and TNF-�.55–58 We therefore p
sed intracellular staining to examine “spontaneous”roduction of cytokines by DCs from the intestinalucosa. These studies were made possible by the use

f a newly developed, highly sensitive technique forata analysis28 that we have adapted and validated fornalysis of DC populations in blood and mucosalpecimens.59 This approach permits the proportion ofositively staining cells to be accurately quantifiedven in circumstances in which labeling levels are lownd the staining distributions of positive and negativeell populations overlap. The specificity of low levelsf staining detected in this way has been confirmed inompetition experiments with unlabeled antibodies.59

n cells from the healthy colonic mucosa, there wereo IL-6 –positive DCs and few, if any, IL-12–positiveCs; IL-10 –producing DCs were the major popula-

ion in healthy tissue. In contrast, DCs making IL-12nd IL-6 were prominent in cells from active Crohn’sisease tissue. Unlike the observations on TLR andD40 expression described previously, the pattern ofC cytokine production appeared to be disease spe-

ific; DCs producing IL-12 and IL-6 did not differignificantly between patients with ulcerative colitisnd healthy controls.

By using intracellular staining approaches, we wereble to focus specifically on which cytokines DCs wereaking. Analysis of supernatants from cell mixturesould not have allowed the contribution of DCs to beetermined because it would have measured cytokineroduction by a mixed population of cells in which DCsere a numerically minor component.DC-derived cytokines are likely to be particularly

mportant in immune regulation, but their contribu-ion to the total production of cytokines (for instance,easured in supernatants of LPMCs) is unknown.owever, it is notable that elevated mucosal IL-6 in

BD,47,60 elevated IL-12 in Crohn’s disease,61 and noifference between IBD and control tissue with regardo IL-10 levels62 have been reported. These observa-ions parallel our findings with DCs, perhaps suggest-ng that DCs are major contributors to the productionf some cytokines.

Enhanced production of IL-12 and IL-6 by DCs mayontribute to the pathogenesis of Crohn’s disease in aumber of ways. Cytokines derived from DCs may havedirect local effect, regulating survival or function ofucosal populations, or may act indirectly by redirecting-cell differentiation. Increased production of IL-12 maynderlie the predominance of Th1 cells and productionf interferon gamma in Crohn’s disease.45,63,64 Interferonamma may also be a cofactor for CD40-dependent IL-12
roduction.65 Modulation of antigen presentation by

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Cs by autocrine IL-12 may also contribute to thencreased response to microbial antigens.66,67 IL-6 pro-uction by antigen-presenting cells has recently beeneported to render effector T cells insensitive to thection of regulatory T cells27 and change the repertoire ofntigens presented to DCs.68 Therefore, the local produc-ion of high levels of IL-6 by DCs in IBD may overcomehe regulatory T-cell population specific for commensalntigens or allow responses to new specificities tomerge. In addition, IL-6 may contribute to T-cell re-istance against apoptosis in Crohn’s disease.69,70 The rolef IL-6 in IBD has been further suggested by recenttudies showing that antibodies to IL-6R amelioratective Crohn’s disease.71

This study describes the characteristics of DCs presentn noninflamed and inflamed human colon. The dataupport the importance of microbial/TLR interactionsnd CD40/CD40L interactions in the pathogenesis ofntestinal inflammation and identify DCs as importantD40� cells that produce enhanced levels of proinflam-atory cytokines, including IL-12 and IL-6. DCs in the

ntestinal mucosa are likely to play a fundamental role inBD, initiating and/or perpetuating the inflammatoryascade.

References1. Becker C, Wirtz S, Blessing M, Pirhonen J, Strand D, Bechthold

O, Frick J, Galle PR, Autenrieth I, Neurath MF. Constitutive p40promoter activation and IL-23 production in the terminal ileummediated by dendritic cells. J Clin Invest 2003;112:693–706.

2. Kobayashi M, Kweon MN, Kuwata H, Schreiber RD, Kiyono H,Takeda K, Akira S. Toll-like receptor-dependent production ofIL-12p40 causes chronic enterocolitis in myeloid cell-specificStat3-deficient mice. J Clin Invest 2003;111:1297–1308.

3. Takeda K, Clausen BE, Kaisho T, Tsujimura T, Terada N, ForsterI, Akira S. Enhanced Th1 activity and development of chronicenterocolitis in mice devoid of Stat3 in macrophages and neutro-phils. Immunity 1999;10:39–49.

4. Leithauser F, Trobonjaca Z, Moller P, Reimann J. Clustering ofcolonic lamina propria CD4(�) T cells to subepithelial dendriticcell aggregates precedes the development of colitis in a murineadoptive transfer model. Lab Invest 2001;81:1339–1349.

5. Malmstrom V, Shipton D, Singh B, Al Shamkhani A, Puklavec MJ,Barclay AN, Powrie F. CD134L expression on dendritic cells in themesenteric lymph nodes drives colitis in T cell-restored SCIDmice. J Immunol 2001;166:6972–6981.

6. Hampe J, Cuthbert A, Croucher PJ, Mirza MM, Mascheretti S,Fisher S, Frenzel H, King K, Hasselmeyer A, Macpherson AJ,Bridger S, Van Deventer S, Forbes A, Nikolaus S, Lennard-JonesJE, Foelsch UR, Krawczak M, Lewis C, Schreiber S, Mathew CG.Association between insertion mutation in NOD2 gene andCrohn’s disease in German and British populations. Lancet2001;357:1925–1928.

7. Hugot JP, Chamaillard M, Zouali H, Lesage S, Cezard JP, BelaicheJ, Almer S, Tysk C, O’Morain CA, Gassull M, Binder V, Finkel Y,Cortot A, Modigliani R, Laurent-Puig P, Gower-Rousseau C, Macry
J, Colombel JF, Sahbatou M, Thomas G. Association of NOD2
leucine-rich repeat variants with susceptibility to Crohn’sdisease. Nature 2001;411:599–603.

8. Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, Ramos R,Britton H, Moran T, Karaliuskas R, Duerr RH, Achkar JP, Brant SR,Bayless TM, Kirschner BS, Hanauer SB, Nunez G, Cho JH. Aframeshift mutation in NOD2 associated with susceptibility toCrohn’s disease. Nature 2001;411:603–606.

9. Bell SJ, Rigby R, English N, Mann SD, Knight SC, Kamm MA,Stagg AJ. Migration and maturation of human colonic dendriticcells. J Immunol 2001;166:4958–4967.

0. Iwasaki A, Kelsall BL. Freshly isolated Peyer’s patch, but notspleen, dendritic cells produce interleukin 10 and induce thedifferentiation of T helper type 2 cells. J Exp Med 1999;190:229–239.

1. Liu LM, MacPherson GG. Lymph-borne (veiled) dendritic cells canacquire and present intestinally administered antigens. Immunol-ogy 1991;73:281–286.

2. Maric I, Holt PG, Perdue MH, Bienenstock J. Class II MHC antigen(Ia)-bearing dendritic cells in the epithelium of the rat intestine.J Immunol 1996;156:1408–1414.

3. Macpherson AJ, Uhr T. Induction of protective IgA by intestinaldendritic cells carrying commensal bacteria. Science 2004;303:1662–1665.

4. Rescigno M, Urbano M, Valzasina B, Francolini M, Rotta G,Bonasio R, Granucci F, Kraehenbuhl JP, Ricciardi-Castagnoli P.Dendritic cells express tight junction proteins and penetrate gutepithelial monolayers to sample bacteria. Nat Immunol 2001;2:361–367.

5. Viney JL, Mowat AM, O’Malley JM, Williamson E, Fanger NA.Expanding dendritic cells in vivo enhances the induction of oraltolerance. J Immunol 1998;160:5815–5825.

6. Williamson E, Westrich GM, Viney JL. Modulating dendritic cellsto optimize mucosal immunization protocols. J Immunol 1999;163:3668–3675.

7. Kadowaki N, Ho S, Antonenko S, Malefyt RW, Kastelein RA,Bazan F, Liu YJ. Subsets of human dendritic cell precursorsexpress different toll-like receptors and respond to different mi-crobial antigens. J Exp Med 2001;194:863–869.

8. Chow JC, Young DW, Golenbock DT, Christ WJ, Gusovsky F.Toll-like receptor-4 mediates lipopolysaccharide-induced signaltransduction. J Biol Chem 1999;274:10689–10692.

9. Michelsen KS, Aicher A, Mohaupt M, Hartung T, Dimmeler S,Kirschning CJ, Schumann RR. The role of toll-like receptors (TLRs)in bacteria-induced maturation of murine dendritic cells (DCS).Peptidoglycan and lipoteichoic acid are inducers of DC matura-tion and require TLR2. J Biol Chem 2001;276:25680–25686.

0. Morath S, Stadelmaier A, Geyer A, Schmidt RR, Hartung T. Syn-thetic lipoteichoic acid from Staphylococcus aureus is a potentstimulus of cytokine release. J Exp Med 2002;195:1635–1640.

1. Schwandner R, Dziarski R, Wesche H, Rothe M, Kirschning CJ.Peptidoglycan- and lipoteichoic acid-induced cell activation ismediated by toll-like receptor 2. J Biol Chem 1999;274:17406–17409.

2. Takeuchi O, Hoshino K, Akira S. Cutting edge: TLR2-deficient andMyD88-deficient mice are highly susceptible to Staphylococcusaureus infection. J Immunol 2000;165:5392–5396.

3. Yoshimura A, Lien E, Ingalls RR, Tuomanen E, Dziarski R, Golen-bock D. Cutting edge: recognition of Gram-positive bacterial cellwall components by the innate immune system occurs via Toll-like receptor 2. J Immunol 1999;163:1–5.

4. Kaisho T, Akira S. Regulation of dendritic cell function throughToll-like receptors. Curr Mol Med 2003;3:373–385.

5. Banchereau J, Steinman RM. Dendritic cells and the control ofimmunity. Nature 1998;392:245–252.

6. Macatonia SE, Hosken NA, Litton M, Vieira P, Hsieh CS, Culpep-
per JA, Wysocka M, Trinchieri G, Murphy KM, O’Garra A. Dendritic

2

2

2

3

3

3

3

3

3

3

3

3

3

4

4

4

4

4

4

4

4

4

4

5

5

5

5

5

5

5

5

5


cells produce IL-12 and direct the development of Th1 cells fromnaive CD4� T cells. J Immunol 1995;154:5071–5079.

7. Pasare C, Medzhitov R. Toll pathway-dependent blockade ofCD4�CD25� T cell-mediated suppression by dendritic cells.Science 2003;299:1033–1036.

8. Panoskaltsis N, Reid CD, Knight SC. Quantification and cytokineproduction of circulating lymphoid and myeloid cells in acutemyelogenous leukaemia. Leukemia 2003;17:716–730.

9. Bagwell B. A journey through flow cytometric immunofluorescenceanalyses—finding accurate and robust algorithms that estimatepositive fraction distributions. Clin Immunol Newslett 1996;16:33–37.

0. Schroeder KW, Tremaine WJ, Ilstrup DM. Coated oral 5-amino-salicylic acid therapy for mildly to moderately active ulcerativecolitis. A randomized study. N Engl J Med 1987;317:1625–1629.

1. Verhasselt V, Buelens C, Willems F, De Groote D, Haeffner-Cavaillon N, Goldman M. Bacterial lipopolysaccharide stimulatesthe production of cytokines and the expression of costimulatorymolecules by human peripheral blood dendritic cells: evidencefor a soluble CD14-dependent pathway. J Immunol 1997;158:2919–2925.

2. Steinman RM, Hawiger D, Nussenzweig MC. Tolerogenic den-dritic cells. Annu Rev Immunol 2003;21:685–711.

3. Muzio M, Bosisio D, Polentarutti N, D’Amico G, Stoppacciaro A,Mancinelli R, van’t Veer C, Penton-Rol G, Ruco LP, Allavena P,Mantovani A. Differential expression and regulation of toll-likereceptors (TLR) in human leukocytes: selective expression ofTLR3 in dendritic cells. J Immunol 2000;164:5998–6004.

4. Kobayashi K, Hernandez LD, Galan JE, Janeway CA Jr, MedzhitovR, Flavell RA. IRAK-M is a negative regulator of Toll-like receptorsignaling. Cell 2002;110:191–202.

5. Melmed G, Thomas LS, Lee N, Tesfay SY, Lukasek K, MichelsenKS, Zhou Y, Hu B, Arditi M, Abreu MT. Human intestinal epithelialcells are broadly unresponsive to Toll-like receptor 2-dependentbacterial ligands: implications for host-microbial interactions inthe gut. J Immunol 2003;170:1406–1415.

6. Zhang G, Ghosh S. Negative regulation of toll-like receptor-medi-ated signaling by Tollip. J Biol Chem 2002;277:7059–7065.

7. Cario E, Podolsky DK. Differential alteration in intestinal epithelialcell expression of Toll-like receptor 3 (TLR3) and TLR4 in inflamma-tory bowel disease. Infect Immun 2000;68:7010–7017.

8. Hausmann M, Kiessling S, Mestermann S, Webb G, Spottl T,Andus T, Scholmerich J, Herfarth H, Ray K, Falk W, Rogler G.Toll-like receptors 2 and 4 are up-regulated during intestinalinflammation. Gastroenterology 2002;122:1987–2000.

9. Ortega-Cava CF, Ishihara S, Rumi MA, Kawashima K, Ishimura N,Kazumori H, Udagawa J, Kadowaki Y, Kinoshita Y. Strategiccompartmentalization of Toll-like receptor 4 in the mouse gut.J Immunol 2003;170:3977–3985.

0. Shuto T, Imasato A, Jono H, Sakai A, Xu H, Watanabe T, RixterDD, Kai H, Andalibi A, Linthicum F, Guan YL, Han J, Cato AC, LimDJ, Akira S, Li JD. Glucocorticoids synergistically enhance non-typeable Haemophilus influenzae-induced Toll-like receptor 2 ex-pression via a negative cross-talk with p38 MAP kinase. J BiolChem 2002;277:17263–17270.

1. Tamandl D, Bahrami M, Wessner B, Weigel G, Ploder M, Furst W,Roth E, Boltz-Nitulescu G, Spittler A. Modulation of Toll-like re-ceptor 4 expression on human monocytes by tumor necrosisfactor and interleukin-6: tumor necrosis factor evokes lipopoly-saccharide hyporesponsiveness, whereas interleukin-6 en-hances lipopolysaccharide activity. Shock 2003;20:224–229.

2. Wolfs TG, Buurman WA, van Schadewijk A, de Vries B, DaemenMA, Hiemstra PS, van’t Veer C. In vivo expression of Toll-likereceptor 2 and 4 by renal epithelial cells: IFN-gamma and TNF-alpha mediated up-regulation during inflammation. J Immunol
2002;168:1286–1293.
3. Zarember KA, Godowski PJ. Tissue expression of human Toll-likereceptors and differential regulation of Toll-like receptor mRNAsin leukocytes in response to microbes, their products, and cyto-kines. J Immunol 2002;168:554–561.

4. Breese EJ, Michie CA, Nicholls SW, Murch SH, Williams CB,Domizio P, Walker-Smith JA, MacDonald TT. Tumor necrosis fac-tor alpha-producing cells in the intestinal mucosa of children withinflammatory bowel disease. Gastroenterology 1994;106:1455–1466.

5. Fuss IJ, Neurath M, Boirivant M, Klein JS, de la Motte C, StrongSA, Fiocchi C, Strober W. Disparate CD4� lamina propria (LP)lymphokine secretion profiles in inflammatory bowel disease.Crohn’s disease LP cells manifest increased secretion of IFN-gamma, whereas ulcerative colitis LP cells manifest increasedsecretion of IL-5. J Immunol 1996;157:1261–1270.

6. Hosokawa T, Kusugami K, Ina K, Ando T, Shinoda M, Imada A,Ohsuga M, Sakai T, Matsuura T, Ito K, Kaneshiro K. Interleukin-6and soluble interleukin-6 receptor in the colonic mucosa of in-flammatory bowel disease. J Gastroenterol Hepatol 1999;14:987–996.

7. Reimund JM, Wittersheim C, Dumont S, Muller CD, Baumann R,Poindron P, Duclos B. Mucosal inflammatory cytokine productionby intestinal biopsies in patients with ulcerative colitis andCrohn’s disease. J Clin Immunol 1996;16:144–150.

8. van Kooyk Y, Geijtenbeek TB. DC-SIGN: escape mechanism forpathogens. Nat Rev Immunol 2003;3:697–709.

9. Clegg CH, Rulffes JT, Haugen HS, Hoggatt IH, Aruffo A, DurhamSK, Farr AG, Hollenbaugh D. Thymus dysfunction and chronicinflammatory disease in gp39 transgenic mice. Int Immunol1997;9:1111–1122.

0. Cong Y, Weaver CT, Lazenby A, Elson CO. Colitis induced byenteric bacterial antigen-specific CD4� T cells requires CD40-CD40 ligand interactions for a sustained increase in mucosalIL-12. J Immunol 2000;165:2173–2182.

1. Stuber E, Strober W, Neurath M. Blocking the CD40L-CD40 inter-action in vivo specifically prevents the priming of T helper 1 cellsthrough the inhibition of interleukin 12 secretion. J Exp Med1996;183:693–698.

2. Battaglia E, Biancone L, Resegotti A, Emanuelli G, Fronda GR,Camussi G. Expression of CD40 and its ligand, CD40L, in intes-tinal lesions of Crohn’s disease. Am J Gastroenterol 1999;94:3279–3284.

3. Liu Z, Colpaert S, D’Haens GR, Kasran A, de Boer M, RutgeertsP, Geboes K, Ceuppens JL. Hyperexpression of CD40 ligand(CD154) in inflammatory bowel disease and its contribution topathogenic cytokine production. J Immunol 1999;163:4049–4057.

4. Vogel JD, West GA, Danese S, de la Motte C, Phillips MH, StrongSA, Willis J, Fiocchi C. CD40-mediated immune-nonimmune cellinteractions induce mucosal fibroblast chemokines leading toT-cell transmigration. Gastroenterology 2004;126:63–80.

5. Caux C, Massacrier C, Vanbervliet B, Dubois B, Van Kooten C,Durand I, Banchereau J. Activation of human dendritic cellsthrough CD40 cross-linking. J Exp Med 1994;180:1263–1272.

6. Cella M, Scheidegger D, Palmer-Lehmann K, Lane P, Lanzavec-chia A, Alber G. Ligation of CD40 on dendritic cells triggersproduction of high levels of interleukin-12 and enhances T cellstimulatory capacity: T-T help via APC activation. J Exp Med1996;184:747–752.

7. Gardella S, Andrei C, Costigliolo S, Poggi A, Zocchi MR, RubartelliA. Interleukin-18 synthesis and secretion by dendritic cells aremodulated by interaction with antigen-specific T cells. J LeukocBiol 1999;66:237–241.

8. McDyer JF, Dybul M, Goletz TJ, Kinter AL, Thomas EK, BerzofskyJA, Fauci AS, Seder RA. Differential effects of CD40 ligand/trimer
stimulation on the ability of dendritic cells to replicate and trans-

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6

6

6

6

6

6

6

6

6

6

7

7

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mit HIV infection: evidence for CC-chemokine-dependent and-independent mechanisms. J Immunol 1999;162:3711–3717.

9. Hart AL, Lammers K, Brigidi P, Vitali B, Rizzello F, Gionchetti P,Campieri M, Kamm MA, Knight SC, Stagg AJ. Modulation ofhuman dendritic cell phenotype and function by probiotic bacte-ria. Gut 2004;53:1602–1609.

0. Reinecker HC, Steffen M, Witthoeft T, Pflueger I, Schreiber S,MacDermott RP, Raedler A. Enhanced secretion of tumour necro-sis factor-alpha, IL-6, and IL-1 beta by isolated lamina propriamononuclear cells from patients with ulcerative colitis andCrohn’s disease. Clin Exp Immunol 1993;94:174–181.

1. Monteleone G, Biancone L, Marasco R, Morrone G, Marasco O,Luzza F, Pallone F. Interleukin 12 is expressed and activelyreleased by Crohn’s disease intestinal lamina propria mononu-clear cells. Gastroenterology 1997;112:1169–1178.

2. Schreiber S, Heinig T, Thiele HG, Raedler A. Immunoregulatoryrole of interleukin 10 in patients with inflammatory bowel dis-ease. Gastroenterology 1995;108:1434–1444.

3. Parronchi P, Romagnani P, Annunziato F, Sampognaro S, BecchioA, Giannarini L, Maggi E, Pupilli C, Tonelli F, Romagnani S. Type1 T-helper cell predominance and interleukin-12 expression in thegut of patients with Crohn’s disease. Am J Pathol 1997;150:823–832.

4. Strober W, Kelsall B, Fuss I, Marth T, Ludviksson B, Ehrhardt R,Neurath M. Reciprocal IFN-gamma and TGF-beta responses reg-ulate the occurrence of mucosal inflammation. Immunol Today1997;18:61–64.

5. Snijders A, Kalinski P, Hilkens CM, Kapsenberg ML. High-levelIL-12 production by human dendritic cells requires two signals.Int Immunol 1998;10:1593–1598.

6. Bianchi R, Grohmann U, Vacca C, Belladonna ML, Fioretti MC,Puccetti P. Autocrine IL-12 is involved in dendritic cell modulation
via CD40 ligation. J Immunol 1999;163:2517–2521. T
7. Kelleher P, Maroof A, Knight SC. Retrovirally induced switch fromproduction of IL-12 to IL-4 in dendritic cells. Eur J Immunol1999;29:2309–2318.

8. Drakesmith H, O’Neil D, Schneider SC, Binks M, Medd P, Sercarz E,Beverley P, Chain B. In vivo priming of T cells against cryptic deter-minants by dendritic cells exposed to interleukin 6 and native anti-gen. Proc Natl Acad Sci U S A 1998;95:14903–14908.

9. Atreya R, Mudter J, Finotto S, Mullberg J, Jostock T, Wirtz S,Schutz M, Bartsch B, Holtmann M, Becker C, Strand D, Czaja J,Schlaak JF, Lehr HA, Autschbach F, Schurmann G, Nishimoto N,Yoshizaki K, Ito H, Kishimoto T, Galle PR, Rose-John S, Neurath MF.Blockade of interleukin 6 trans signaling suppresses T-cell resis-tance against apoptosis in chronic intestinal inflammation: evi-dence in Crohn disease and experimental colitis in vivo. Nat Med2000;6:583–588.

0. Ito H, Hirotani T, Yamamoto M, Ogawa H, Kishimoto T. Anti-IL-6receptor monoclonal antibody inhibits leukocyte recruitment andpromotes T-cell apoptosis in a murine model of Crohn’s disease.J Gastroenterol 2002;37(Suppl 14):56–61.

1. Ito H, Takazoe M, Fukuda Y, Hibi T, Kusugami K, Andoh A,Matsumoto T, Yamamura T, Azuma J, Nishimoto N, Yoshizaki K,Shimoyama T, Kishimoto T. A pilot randomized trial of a humananti-interleukin-6 receptor monoclonal antibody in active Crohn’sdisease. Gastroenterology 2004;126:989–996.

Received May 12, 2004. Accepted March 23, 2005.Address requests for reprints to: Andrew J. Stagg, PhD, Antigen

resentation Research Group, Imperial College London, Northwickark Campus, Watford Road, Harrow, HA1 3UJ, England. e-mail:[email protected]; fax: (44) 2088693532.Supported by the Medical Research Council (UK) and Wellcome

rust.

characteristics of intestinal dendritic cells in inflammatory bowel diseases

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