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Am J Physiol Gastrointest Liver Physiol 293: G1315-G1324, 2007. First published October 18, 2007; doi:10.1152/ajpgi.00142.2007
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INFLAMMATION/IMMUNITY/MEDIATORS

Expression of IL-12-related molecules in human intestinal microvascular endothelial cells is regulated by TLR3

Jan Heidemann,1 Christoph Rüther,1 Moritz Kebschull,1 Wolfram Domschke,1 Matthias Brüwer,2 Stefan Koch,1 Torsten Kucharzik,1 and Christian Maaser1

Departments of 1Medicine B and 2General Surgery, University of Münster, Münster, Germany

Submitted 3 April 2007 ; accepted in final form 17 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Members of the interleukin (IL)-12 family constitute subunits of IL-12, -23, and -27. These ILs represent pivotal mediators in the regulation of cell-mediated immune responses and in animal models of human inflammatory bowel disease. Recent work has suggested that intestinal endothelial cells might serve as a second line of defense in bacterial sensing of invading pathogens. The purpose of this study was to examine the production of IL-12 family members in intestinal endothelial cells (HIMEC). HIMEC were stimulated with proinflammatory agents (TNF-{alpha}, IFN-{gamma}, IL-1β) and microbial antigens [LPS, lipoteichoic acid, peptidoglycan, CpG-DNA, flagellin, poly(I:C)]. Expression of IL-12 family members and of Toll-like receptor (TLR)3 in HIMEC was assessed by real-time RT-PCR, immunostaining, flow cytometry, and immunoblot analysis. HIMEC display an induction of Epstein-Barr virus-induced gene 3 (EBI3), IL-12p35, and IL-23p19, whereas no expression of IL-12p40 and IL-27p28 was detectable. The strongest induction was induced by proinflammatory factors known to utilize the NF-{kappa}B pathway, and expression of EBI3 and IL-23p19 was diminished by an NF-{kappa}B inhibitor. HIMEC display regulated expression of TLR3. Adhesion and transmigration assays showed proinflammatory responses after HIMEC stimulation. HIMEC are capable of producing IL-12 family members as a response to microbial stimuli. The TLR3 agonist, poly(I:C), was shown to enhance leukocyte adhesion in vitro in HIMEC. Our data suggest that the intestinal microvasculature is responsive to ligands of TLR3 expressed on intestinal endothelial cells, thereby adding to the regulation of adaptive immunity and leukocyte recruitment.

Epstein-Barr virus-induced gene 3; inflammatory bowel disease; Toll-like receptor 3

MEMBERS OF THE FAMILY OF INTERLEUKIN (IL)-12-related molecules have recently been attributed a critical role in Th1-mediated immune disorders (17), including Crohn's disease (CD) (3, 11, 33). IL-12-related molecules include a protein encoded by Epstein-Barr virus-induced gene 3 (EBI3), as well as IL-12p35 and p19 that associate with IL-12p40 to form IL-12 (IL-12p70) and IL-23, respectively. EBI3 protein, which is structurally related to the IL-12 subunit IL-12p40, was shown to associate with p28 to form IL-27, which has been demonstrated to possess complex pro- and anti-inflammatory functions (40). For example, IL-27 is believed to exert important initiator functions in the Th1-differentiation of Th0 naive CD4+ T cells (6).

CD is widely regarded as a granulomatous Th1-mediated immune disease. Likewise, high mucosal expression levels of IL-12 (29) and EBI3 (26) have been observed in active CD. In addition, a high expression of the IL-12-related cytokine, IL-27, has been observed in granulomatous Th1-mediated diseases, such as sarcoidosis and CD (17).

Intestinal microvascular endothelial cells have recently been recognized as a cell population actively involved in the pathogenesis of inflammatory bowel diseases (IBD) and IBD-associated microvascular dysfunction (13). Mucosal microvascular endothelial cells, which constitute the final anatomical barrier between the blood circulation and subepithelial mucosal compartments, were shown to function as major histocompatibility complex (MHC) class II antigen-presenting cells in vitro (12). In addition, activated monocytes (35) and dendritic cells (38, 39), which are both key antigen-presenting cells in the pathogenesis of IBD, are known to produce IL-12-related molecules in response to microbial antigens.

Both disruption of the intestinal epithelial barrier and subsequent mucosal translocation of enteric microbial antigens (32) are considered major pathophysiological events in the initiation and chronic perpetuation of CD (36) and ulcerative colitis (UC) (34).

Microbial antigens, including LPS and flagellin from flagellated Enterobacteriaceae spp., were shown to elicit rapid innate immune responses, including upregulation of endothelial leukocyte adhesion molecules and enhanced in vitro transendothelial leukocyte migration, in human intestinal microvascular endothelial cells (HIMEC) (21, 25). Furthermore, initial gene array analyses were conducted in our laboratory to characterize the proinflammatory response patterns in primary cultures of HIMEC. Using this assay, we were able to document a marked upregulation of EBI3, compared with the reference microvascular endothelial cell line derived from human skin, HMEC-1. For these reasons, we set about characterizing how specific microbial antigens would induce IL-12-related molecules in primary cultures of HIMEC.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cells and reagents. The immortalized dermal human microvascular endothelial cell line, HMEC-1, was maintained as described earlier (22). For HIMEC isolation, macroscopically normal colonic specimens were obtained from patients undergoing scheduled colonic resection. The use of human tissues for immunohistochemistry and isolation of endothelial cells was approved by the ethical committee of the University of Münster. HIMEC were isolated as previously described (5). In brief, mucosal strips from resected normal colon were washed, minced, and digested in collagenase type II solution (Worthington, Lakewood, NJ; 2 mg/ml). Endothelial cells were extruded by mechanical compression and plated onto tissue culture dishes coated with collagen type I from rat tail (Upstate Biotechnology, Waltham, MA) in Endothelial Cell Growth Medium MV (PromoCell, Heidelberg, Germany) containing antibiotic/antimycotic solution at 1x concentration (Calbiochem). Following 7–10 days of culture, microvascular endothelial cell clusters were physically isolated and a pure culture was obtained. HIMEC cultures were recognized by microscopic phenotype and expression of factor VIII-associated antigen. All experiments were carried out using HIMEC cultures between passages 4 and 8. U937 cells (ATCC, Manassas, VA), a human monocytic cell line derived from monocytic leukemia, were maintained in RPMI 1640 medium supplemented with 10% vol/vol FBS and 2 mM glutamine. Peripheral blood mononuclear cells (PBMC) were purified from whole blood obtained from healthy donors by density gradient centrifugation using Ficoll (Biochrom, Berlin, Germany). PBMC were washed in PBS, plated in supplemented RPMI medium and stimulated with concanavalin A (10 µg/ml) and LPS from Escherichia coli (1 µg/ml) for 16 h. PBMC were subjected to RNA extraction and used as positive control for RT-PCR.

Unless otherwise indicated, all chemicals were obtained from Sigma Aldrich (Steinheim, Germany). The monoclonal EBI3 mAb (clone 2H4G6) was a kind gift by O. Devergne, University of Paris, France. Polyclonal goat anti-human Toll-like receptor (TLR)3 antibody was purchased from R&D Systems. Polyclonal rabbit-anti IL-12p35 was obtained from Santa Cruz Biotechnologies (Santa Cruz, CA).

Gene array analysis. Intestinal (HIMEC) and dermal microvascular endothelial cells (HMEC-1) were stimulated with recombinant human TNF-{alpha} (20 ng/ml) for 4 h and 24 h, respectively. Endothelial cell monolayers were briefly rinsed with PBS and subjected to total RNA extraction as indicated above. Total RNA was quantitated and checked for purity by photometry (BioPhotometer; Eppendorf, Hamburg, Germany). RNA was purified from buffer salt remnants and small molecular contaminants by use of the QIAquick Nucleotide Removal Kit (Qiagen, Hilden, Germany). After reverse transcription, second-strand synthesis and in vitro transcription, biotinylated RNA fragments were hybridized for 16 h onto HG-U133A gene chips (Affymetrix) according to the manufacturer's protocol. Data obtained were processed by using the MicroArraySuite software, version 5.0 (Affymetrix). Expression level changes threefold from baseline were considered significant, whereas lesser values were excluded from further analysis. Sample number was n = 3 for each condition.

Reverse transcription-polymerase chain reaction (RT-PCR). Endothelial cells were stimulated for 8 h with TNF-{alpha} (20 ng/ml), IFN-{gamma} (40 ng/ml), LPS from E. coli O111:B4 (1 µg/ml), IL-1β (20 ng/ml), or combined TNF-{alpha}/LPS. Control cells remained without any stimulation. In some experiments, the NF-{kappa}B inhibitor MG132 was added to the monolayers 30 min before proinflammatory stimulation. Total cellular RNA was extracted from endothelial cells using an acid guanidinium-phenol-chloroform method (TRIzol Reagent; GIBCO-BRL Life Technologies, Grand Island, NY) and treated with ribonuclease-free deoxyribonuclease (Stratagene, La Jolla, CA). Reverse transcription and PCR were performed as described before. Primers and PCR conditions were used as described earlier (20). Experiments were performed three times, and representative results are shown.

Real-time RT-PCR. Real-time PCR was performed using an ABI Prism 5700 Sequence Detection System (PE Applied Biosystems, Foster City, CA). Each reaction contained 25 µl of 2x SYBR Green Master Mix (comprising 200 nM dATP, dGTP, and dCTP; 400 nM dUTP; 2 mM MgCl2, 0.25 units uracil N-glycosylase, and 0.625 units Amplitaq Gold DNA polymerase), 25 pmol each of sense and antisense primers, and 2 µl of cDNA in a final volume of 50 µl. Amplification of the expected single products was confirmed on 1% agarose gels stained with ethidium bromide. Data analysis used sequence-detection system software provided by the manufacturer where change in fluorescence signal ({Delta}Rn) was calculated by the equation {Delta}Rn = (Rn+) – (Rn–) with Rn+ being the fluorescence signal of the product and Rn– the fluorescence signal of the baseline emission, where Rn is the normalized reporter, the fluorescence emission intensity of the reporter dye divided by the fluorescence emission intensity of the passive reference dye. The threshold cycle (CT) is the cycle number at which the {Delta}Rn crosses threshold. Fold changes in target mRNA expression were determined as fold change = 2Formula, where

Formula
Data shown are representative of three independent experiments. Each condition was assessed in triplicate.

Indirect immunofluorescence. For immunohistochemistry, resected specimens of normal colon were fixed in 4% wt/vol paraformaldehyde in PBS, saturated in 20% wt/vol sucrose in PBS overnight, embedded in optimal cutting temperature compound (Sakura, Japan), and snap frozen in isopentane-liquid nitrogen. Sections 5 µm in thickness were prepared, rehydrated in PBS, and blocked in blocking buffer (PBS containing 10% vol/vol of donkey serum) for 1 h at room temperature. Tissue samples were then incubated with the respective antibody (concentrations: 1:1,000 for rabbit anti-human IL-12p35; 14 µg/ml for anti-EBI3 mAb) in blocking buffer for 16 h at 4°C in a humid chamber. Sections were washed 3 x 5 min in PBS, and immunodetection was performed using polyclonal Cy3-labeled donkey anti-rabbit (IL-12p35 and ICAM-1) or anti-mouse (EBI3) antibodies (Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature in the dark. Sections were washed and nuclei were counterstained with 4',6'-diamidino-2-phenylindole (DAPI; Hoechst, Frankfurt, Germany).

For immunocytochemistry, HIMEC were seeded on collagen-coated glass chamber slides (LabTek; Nalgen Nunc International, Naperville, IL), cultured to subconfluence in growth medium, and stimulated as indicated in RESULTS. After brief fixation with 4% wt/vol paraformaldehyde in PBS, cells were washed with PBS, permeabilized with Triton X-100 (0.1% vol/vol in PBS) for 5 min, and blocked in blocking buffer for 1 h at room temperature. Endothelial cells were incubated with the respective primary Ab diluted in blocking buffer at 4°C overnight. After extensive washing with PBS, immunodetection and counterstaining of nuclei were performed as described above. Staining experiments were repeated at least three times, and representative results were shown.

Flow cytometry. HIMEC monolayers were stimulated with TNF-{alpha} (20 ng/ml) or polyinosinic-polycytidylic acid [poly(I:C); 100 µg/ml] for 24 h as indicated. Cells were washed and detached from the cell culture dishes using ice-cold PBS containing 10 mmol EDTA (pH = 8.0) as a cation chelator. After centrifugation, cells were resuspended and permeabilized in FACS buffer (PBS containing 3% vol/vol fetal calf serum, 0.1% wt/vol sodium azide, and 0.1% wt/vol saponin) on ice. Cells were then incubated at 4°C for 1 h with the polyclonal TLR3 antibody. After washing, immunodetection of bound antibody was achieved by incubation with a biotinylated secondary antibody (Pharmingen) and streptavidin-horseradish peroxidase (Pharmingen). Signals were analyzed on a FACSCalibur cytometer (Becton Dickinson, Mountain View, CA) using CellQuest and WinMDI (version 2.8) software. Experiments were performed three times, and representative results are shown.

Immunoblotting. Stimulated HIMEC monolayers were lysed in modified RIPA buffer (50 mM Tris·HCl, pH 7.6, 1 mM EDTA, 150 mM NaCl, 0.25% wt/vol sodium deoxycholate, 1% vol/vol Igepal CA-630, 0.1% wt/vol SDS; all Sigma Chemical, St. Louis, MO) containing Protease Inhibitor Cocktail III (Calbiochem, San Diego, CA) on ice. Lysates were cleared by centrifugation, and total protein concentration was determined by Bradford assay (Bio-Rad, Hercules, CA). Thirty micrograms of total cellular protein were size separated on a 4–20% gradient SDS-PAGE gel (NOVEX, Invitrogen) blotted onto nitrocellulose membranes (Amersham Pharmacia Biotech, Arlington Heights, IL) and blocked in PBS containing 0.1% vol/vol Tween-20, 10% wt/vol nonfat dry milk, and 1% wt/vol BSA. Blots were incubated with EBI3 mAb overnight at 4°C. Immunodetection was performed using horseradish peroxidase-conjugated rabbit anti-mouse antibody (Sigma) and enhanced chemiluminescence reagents (ECL, Amersham Pharmacia Biotech). Blots were stripped and reprobed by use of mouse anti-actin monoclonal antibody (Sigma) followed by the appropriate secondary reagents. Bands were analyzed via NIH imager software, and signal intensities were compared with β-actin of the respective sample. Experiments were performed three times, and representative results are shown.

Leukocyte adhesion and transmigration assays. Static leukocyte adhesion assays were performed using HIMEC monolayers growing on 96-well plates. HIMEC were stimulated with TNF-{alpha} (20 ng/ml) or poly(I:C) at doses ranging from 1 to 100 µg/ml for 24 h. U937 cells were suspended in RPMI 1640 medium containing 10% vol/vol of FBS and 1% wt/vol of glutamine and fluorescence-labeled with calcein AM (Molecular Probes, Eugene, OR) at 37°C for 30 min. Labeled cells (2 x 106 cells/ml) were added on top of the stimulated HIMEC and were allowed to adhere for 20 min at 37°C. The cell culture plates were then briefly rinsed with PBS and centrifuged upside down (5 min, 500 g) to remove any nonadherent U937 cells. Fluorescence was quantified by using the FLUOstar OPTIMA (BMG Labtech, Offenburg, Germany) fluorometer with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. A standard curve was determined by using known numbers of fluorescence-labeled cells.

Endothelial transmigration assays were performed as described earlier (21). In brief, HIMEC were plated onto collagen-coated Transwell polycarbonate filter inserts (24-well format, pore size 5 µm) in growth medium and allowed to grow to confluence. Monolayer integrity was assessed in parallel inserts by crystal violet staining followed by light microscopy. Forty-eight hours after reaching confluence, endothelial monolayers were stimulated with growth medium containing poly(I:C) at differing concentrations or TNF-{alpha} as a positive control. U937 cells were labeled with calcein AM as described above. For each well, 4 x 106 labeled U937 cells/ml were added on top of the endothelial monolayers. U937 cells were allowed to migrate for 4 h at 37°C/5% vol/vol CO2, and transmigrated cells (lower well) and cells remaining in the upper well were quantified by fluorescence reading as described above. Equal volumes and counting intervals were applied. Each condition was assessed in triplicate.

Statistical analysis. Values are expressed as means ± SE. The data were analyzed by one-way ANOVA followed by Dunnett's post hoc test for equal sample sizes (SigmaStat v. 3.5, Systat Software, San Jose, CA). P values <0.05 were considered significant. All experiments were performed at least three times.


   RESULTS
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Gene array analysis reveals expression of EBI3 in HIMEC. To determine the intestine-specific gene expression patterns of proinflammatory stimulated human intestinal endothelial cells in vitro, we conducted gene array experiments with skin-derived HMEC-1 cells as a reference cell line. Among others, the expression of EBI3 was specifically enhanced in HIMEC after proinflammatory stimulation with TNF-{alpha} (20 ng/ml, 4 h and 24 h), a prototypical cytokine involved in the pathogenesis of IBD. In contrast, TNF-{alpha} stimulation did not lead to any significant upregulation, defined as more than threefold induction of EBI3 gene expression in the reference cell line, HMEC-1 (Table 1).


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  		Table 1. Comparative gene array analysis of EBI3-mRNA expression in HIMEC and HMEC-1 cells after proinflammatory stimulation with TNF-{alpha}

 
Proinflammatory induction of IL-12-related molecules in HIMEC vs. HMEC-1. Because TNF-{alpha} appears to be a potent stimulus for the induction of EBI3 expression in HIMEC, we aimed to determine how other IL-12-related molecules are regulated in HIMEC and HMEC-1 after proinflammatory stimulation. Using an array of established proinflammatory stimuli, including TNF-{alpha} (20 ng/ml), IFN-{gamma} (40 ng/ml), LPS from E. coli (1 µg/ml), IL-1β (20 ng/ml), or combined TNF-{alpha}/LPS for 8 h, we assessed the expression of IL-12p35, EBI3, p19, p28, and IL-12p40 mRNA by semiquantitative RT-PCR. Control cells remained without any stimulation, and PBMC from a healthy donor stimulated with TNF-{alpha}/concanavalin A served as a positive control (not shown). Using this assay, a robust induction of EBI3, IL-12p35, and IL-23p19 was detected in HIMEC after stimulation with agents activating the NF-{kappa}B pathway (LPS and IL-1β). TNF-{alpha} was less effective on the expression of IL-12p35. In contrast, HIMEC did not respond to a high concentration of IFN-{gamma} (40 ng/ml), suggesting that the expression of IL-12-related molecules is less likely dependent on activation of the IFN-associated signaling pathways, which include signal transducer and activator of transcription (STAT) and Janus kinase (JAK) (Fig. 1). Of note, no mRNA transcripts for IL-12p40 and IL-27p28 were detectable in HIMEC. This is consistent with a prior report that indicated that endothelial expression of IL-12p40 appears to be dependent on CD154 (CD40 ligand) stimulation, because sole stimulation with TNF-{alpha} was not sufficient to induce IL-12p40 mRNA (18). In the dermal reference endothelial cell line (HMEC-1), IL-12p35 and EBI3 were expressed at constitutively high levels, and only minimal regulation occurred in the expression of EBI3 after stimulation with IL-1β and LPS. IL-23p19 was not expressed constitutively, but induction occurred after stimulation with LPS, but not IL-1β, IFN-{gamma}, and TNF-{alpha}. As observed in HIMEC, both IL-12p40 and IL-27p28 were neither inducible nor expressed at constitutive levels (not shown).


Figure 1
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  		Fig. 1. Semiquantitative RT-PCR of IL-12-related molecules expressed in human microvascular endothelial cells HMEC-1 in response to proinflammatory stimuli. Stimuli signaling through the NF-{kappa}B pathway (IL-1β, TNF-{alpha}, and LPS) strongly upregulate Epstein-Barr virus-induced gene 3 (EBI3), IL-12p35, and IL-12p19 mRNA, whereas stimulation with IFN-{gamma} is not effective. Both constitutive and regulated expression of IL-12-related molecules IL-12p40 and IL-27p28 was not detectable in human intestinal microvascular endothelial cells (HIMEC) (not shown).

 
Expression of EBI3 and IL-23p19 in HIMEC is NF-{kappa}B dependent. Having detected a marked upregulation of specific IL-12-related molecules, we were interested in how intestinal endothelial cells would regulate the expression of these gene products. To a large extent, the biological action of TNF-{alpha} is dependent on activation of the transcription factor NF-{kappa}B. Using an inhibitor of NF-{kappa}B activation, MG132, the expression of the IL-12-related molecules EBI3 and IL23p19 induced by TNF-{alpha} (20 ng/ml, 8 h) in HIMEC was diminished, as shown by RT-PCR (Fig. 2). This effect is attributable to MG-132, because the solvent control (DMSO+TNF-{alpha}) was not effective as an inhibitor. However, the expression of IL-12p35 appeared to be enhanced after pretreatment with MG132, and TNF-{alpha} was again not effective im upregulating IL-12p35 in HIMEC.


Figure 2
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  		Fig. 2. Inhibition of TNF-{alpha}-induced expression of IL-12-related molecules in HIMEC by the NF-{kappa}B inhibitor MG-132. Pretreatment with the NF-{kappa}B inhibitor MG-132 30 min prior to TNF-{alpha} stimulation diminishes expression of EBI3 and IL-23p19, compared with the MG-132 solvent control (DMSO+TNF-{alpha}). β-Actin served as loading control (bottom). In contrast, the high constitutive expression of IL-12p35 was not further enhanced by TNF-{alpha} stimulation, but MG132 appeared to upregulated IL-12p35 mRNA. Representative results of 3 independent experiments are shown, and values stated indicate relative expression levels as assessed via NIH imager software.

 
The TLR3 ligand poly(I:C) is a potent stimulus for the expression of EBI3 in HIMEC. Having learned that specific IL-12-related molecules are expressed in HIMEC after proinflammatory stimulation by both TNF-{alpha} and LPS, we sought to characterize how other TLR-specific microbial ligands would induce the expression of EBI3. Using HIMEC and a variety of established TLR agents at validated concentrations, we found that EBI3 mRNA was effectively regulated by stimulation with the TLR3 ligand, poly(I:C), in a dose-dependent fashion, as depicted according to real-time RT-PCR data (Fig. 3). In strong contrast, ligands for the TLR heterodimers 1/2 (peptidoglycan) and 2/6 (lipoteichoic acid), and the homodimer TLR9 (bacterial CpG-DNA; ODN2006), all used at established concentrations, were not effective in enhancing the expression of EBI3 mRNA after 16 h of stimulation. As expected from previous studies indicating endothelial expression of TLR4 and TLR5, both LPS from E. coli (TLR4 ligand) and flagellin from Salmonella typhimurium (TLR5 ligand) stimulated EBI3 expression to some extent (Fig. 3).


Figure 3
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  		Fig. 3. Regulated expression of EBI3 in HIMEC in response to various Toll-like receptor (TLR) agonists. Established agonists of the TLR were used to stimulate EBI3 mRNA expression in HIMEC, as assessed quantitatively by real-time RT-PCR. The TLR3 agonist poly(I:C) leads to a marked and dose-dependent upregulation of EBI3 mRNA in HIMEC, comparable to that obtained by stimulation with LPS (TLR4 agonist) and Salmonella dublin flagellin (TLR5 agonist). In contrast, stimulation with bacterial CpG DNA (TLR9 agonist), lipoteichoic acid (LTA), and peptidoglycan (PGN) (TLR2 agonists) at established concentrations was ineffective in regulating expression of EBI3 mRNA in HIMEC. Bacterial GpC DNA served as control for CpG DNA. All results are expressed at relative arbitrary units, compared with unstimulated constitutive EBI3 mRNA expression in HIMEC. All experiments were assessed in triplicate, and error bars indicate standard deviations.

 
The TLR3 ligand poly(I:C) enhances expression of TLR3 in HIMEC. The finding that in HIMEC poly(I:C) stimulation led to an upregulation of IL-12-related molecules leads to the assumption that HIMEC are expressing the receptor for viral dsRNA, TLR3. Using HIMEC stimulated with proinflammatory stimuli for 16 h as indicated, we saw a marked regulation of TLR3 by semiquantitative RT-PCR in HIMEC, compared with unstimulated control cells. Interestingly and in contrast to the observations made with IL-12-related molecules, IFN-{gamma} appeared to be a more potent induction stimulus than IL-1β or LPS (Fig. 4A). Notably, stimulation with the TLR3 agonist poly(I:C), as well as with TNF-{alpha}, was effective in upregulating TLR3 immunofluorescence in HIMEC, as shown by flow cytometry analysis (Fig. 4B). Finally, high-magnification immunofluorescence staining revealed perinuclear upregulation of TLR3 in permeabilized HIMEC after stimulation with poly(I:C) (100 µg/ml, 16 h) (Fig. 4C).


Figure 4
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  		Fig. 4. Regulated expression of TLR3 in HIMEC. Expression of TLR3 mRNA appears to upregulated after stimulation with TNF-{alpha} (20 ng/ml) and to a lesser extent IFN-{gamma} (40 ng/ml). Interestingly, both LPS and IL-1β were ineffective in upregulating TLR3 in HIMEC, as assessed by semiquantitative RT-PCR (A). By flow cytometry, an appreciable upregulation of TLR3 immunofluorescence in permeabilized HIMEC was detectable after stimulation with the TLR3 agonist, poly(I:C) (100 µg/ml). TNF-{alpha} served as positive control, and unstimulated cells (unstim.) served as background control (B). By indirect immunofluorescence staining, TLR3-specific fluorescence (red) was strongly enhanced after stimulation with poly(I:C) (100 µg/ml). Note the characteristic speckled fluorescence pattern indicative of membrane-associated immunoreactivity (C). Representative results of 3 independent experiments are shown.

 
Protein expression of EBI3 and IL-12p35 after stimulation with poly(I:C). We next assessed the protein expression of EBI3 and IL-12p35 in human endothelial cells. Whole cell lysates of HIMEC stimulated with poly(I:C) were subjected to immunoblotting using an EBI3-specific monoclonal antibody (8). Unstimulated cells served as control. As expected, stimulation of HIMEC with various doses of poly(I:C) (0, 1, 10, 50, and 100 µg/ml) for 16 h (Fig. 4B) led to a robust and dose-dependent increase of EBI3 expression, as shown by immunoblot analysis. Results are shown as relative values compared with constitutive EBI3 expression, as analyzed with NIH imager software (Fig. 5).


Figure 5
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  		Fig. 5. Protein expression of IL-12-related molecules in HIMEC. By immunoblotting using EBI3 mAb, poly(I:C) at various concentrations was found to upregulate EBI3 protein in HIMEC. Band intensities were measured with NIH imager software and compared with the respective β-actin content. Values indicate relative EBI3 expression levels compared with unstimulated control cells. Peripheral blood mononuclear cells (PBMC) served as positive control.

 
By indirect immunofluorescence, we examined the expression of EBI3 and IL-12p35 in HIMEC in vitro. HIMEC grown on glass coverslips were stimulated with TNF-{alpha} (20 ng/ml) or poly(I:C) (100 µg/ml) for 16 h or were left untreated. Permeabilized and fixed cells were then stained with monoclonal EBI3 or IL-12p35 pAb, respectively. Appropriate Cy3-labeled secondary antibodies were used to visualize bound primary Abs. In unstimulated cells, EBI3-specific immunofluorescence was mainly located in the perinuclear cytosol. After proinflammatory stimulation with poly(I:C), cytosolic EBI3-specific immunofluorescence was slightly upregulated. TNF-{alpha} stimulation appeared to slightly increase immunofluorescence of IL-12p35, compared with poly(I:C) (arrows), whereas the negative control was lacking any specific immunoreactivity (Fig. 6).


Figure 6
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  		Fig. 6. Immunofluorescence staining of EBI3 and IL-12p35 in HIMEC. Stimulation with both poly(I:C) (100 µg/ml) and TNF-{alpha} (20 ng/ml) leads to a slight upregulation of EBI3 and IL-12p35 associated immunofluorescence (red) in permeabilized HIMEC (arrows). Unstimulated cells remaining without primary antibody were used as negative control. Representative results of 3 independent experiments are shown.

 
EBI3 and IL-12p35 are expressed in human intestinal microvessels in vivo. We next assessed expression of the IL-12-related molecules EBI3 and IL-12p35 in normal human intestinal mucosal microvessels in vivo. By using indirect immunofluorescence technique on frozen sections, EBI3 and IL-12p35-specific immunofluorescence was confined to the microvascular endothelial cell lining. EBI3 immunoreactivity was present most of the microvessels examined, whereas some of the microvessels were devoid of IL-12p35 immunoreactivity. Specific endothelial immunoreactivity was undetectable in the negative control (Fig. 7).


Figure 7
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  		Fig. 7. Constitutive expression of the IL-12-related molecules IL-12p35 and EBI3 in normal intestinal mucosal microvessels. Fresh frozen sections of normal human colon were stained with IL-12p35 pAb and EBI3 mAb. Both IL-12-related molecules appear to be expressed in intestinal microvascular endothelial cells, as indicated by red immunofluorescence (arrows). Nuclei were counterstained using DAPI (blue). In both antigens examined the staining was detectable in a proportion of microvessels only, whereas some microvessels showed low or lacking immunoreaction. Frozen sections remaining without primary antibody were used as negative control. Representative results of 3 independent experiments are shown.

 
IL-12-related molecules are differentially regulated after stimulation with poly(I:C). The differentiation of Th1 cells from naive CD4+ T cells is believed to occur as a sequential process involving IL-12-related cytokines at different stages. For this reason, we were interested in how HIMEC would respond to poly(I:C) stimulation in a time-dependent manner. HIMEC were stimulated for 2, 4, 8, 16, and 24 h with a single dose of poly(I:C) and the expression of IL-12-related molecules was assessed by real-time RT-PCR. Interestingly, the expression of IL-12-related molecules appeared to be regulated in a time-dependent manner. IL-23p19 was upregulated as early as 2 h after poly(I:C) stimulation in HIMEC, whereas the expression of EBI3 appeared to be less rapid, reaching its maximum after 16 h of continuous stimulation. There was a slight increase in IL-12p35 expression at 4 h of poly(I:C) stimulation, but significance was not reached (#P = 0.052) (Fig. 8).


Figure 8
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  		Fig. 8. Time-dependent mRNA expression of IL-12-related molecules in response to the TLR3 agonist poly(I:C). HIMEC were stimulated with a fixed dose of the TLR3 agonist poly(I:C) (100 µg/ml) for 2, 4, 8, 16, and 24 h. Expression of IL-12-related molecules was assessed by real-time RT-PCR, compared with unstimulated control cells. IL-12p19 expression peaks comparably early at 2 h after stimulation, whereas EBI3 shows maximal upregulation at 16 h. All experiments were assessed in triplicate, and error bars indicate standard deviation. Of note, IL-12p35 was not significantly upregulated by poly(I:C) stimulation, as shown by a minimal P value of 0.052 at 4 h (#).

 
Poly(I:C) enhances leukocyte adhesion in HIMEC in vitro. TLR stimulation frequently leads to an upregulation of leukocyte adhesion molecules in endothelial cells. Therefore, we next examined the influence of poly(I:C) on the regulation of endothelial leukocyte adhesion molecules in HIMEC. Apart from its functions as an inducer of IL-12-related molecules in HIMEC, poly(I:C) is also effective in the regulation of the leukocyte adhesion molecules VCAM-1, ICAM-1, and E-selectin, as shown by semiquantitative RT-PCR. These findings were paralleled by a functional adhesion assay. In a static adhesion assay using fluorescently labeled U937 monocytic leukemia cells, stimulation with poly(I:C) leads to a dose-dependent increase in endothelial leukocyte adhesion, compared with unstimulated cells (Fig. 9A). TNF-{alpha} enhanced transendothelial leukocyte migration, as shown by comparative quantification of transmigrated cells and cells remaining in the upper compartment of the transmigration well (Fig. 9B). Poly(I:C) stimulation led to an observable increase in transmigrated cells, but statistical significance was not reached. These findings indicate that stimulation with a TLR3 agonist potentially activates intestinal endothelial cells to induce proinflammatory tissue infiltration. Concordant with this hypothesis, stimulation with poly(I:C) (100 µg/ml, 16 h) strongly upregulated expression of the endothelial leukocyte adhesion molecules, ICAM-1 and VCAM-1, as assessed by RT-PCR (Fig. 9C). Both of these molecules are indicators of proinflammatory endothelial cell activation.


Figure 9
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  		Fig. 9. Leukocyte adhesion and transendothelial leukocyte migration in response to the TLR agonist poly(I:C). U937 monocytic cells were fluorescently labeled and subjected to a static adhesion assay using HIMEC monolayers stimulated with various doses of poly(I:C). Mean fluorescence of adhering U937 cells was measured with a fluorescence reader. Cells stimulated with TNF-{alpha} (20 ng/ml) served as positive control, whereas unstimulated cells were used as negative control (A). Transendothelial migration was assessed by an assay in which labeled U937 cells were allowed to transmigrate through HIMEC monolayers seeded onto polycarbonate pore filters. Proinflammatory stimulation with TNF-{alpha} shifts the ratio from cells residing in the supernatant to cells transmigrated to the lower compartment (B). Although a trend to enhanced transmigration was observed after poly(I:C) (100 µg/ml) stimulation, significance was not reached in this assay. All experiments were assessed in triplicate, and error bars indicate standard deviations. Regulated expression of the prototypical endothelial cell adhesion molecules VCAM-1 and ICAM-1 in response to TNF-{alpha} and poly(I:C), as assessed by semiquantitative RT-PCR. Note the strong upregulation of both adhesion factors after stimulation of HIMEC. β-Actin served as loading control (C) for PCR.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
There is a growing body of evidence that disruption of intestinal barrier integrity is a central event in the setting of human IBD. Accordingly, enteric microbia residing in the bowel lumen are allowed to invade and traverse the epithelial layer, gaining access to underlying mucosal tissue. Local immune cell populations, including tissue macrophages and dendritic cells, sample microbia-associated antigens, which leads to antigen presentation, secretion of soluble immune mediators, and expression of costimulatory molecules. The mechanisms by which immune cells sense and identify microbial antigens include activation of pathogen-associated molecular pattern receptors, including TLRs (7), which have been shown to serve as important sentinels in immediate innate immune responses. TLRs have also been identified as a potent response arsenal against microbial challenge in other so-called "nonimmune cell populations," including intestinal myofibroblasts (28), which with regard to their relative numbers have to be considered as a major component of the intestinal mucosa. In accordance, these cells were found to be involved in the process of fibrogenesis and stenosis in CD (23).

TLRs are members of a large family of genetically conserved pattern-recognition receptors, which are readily activated upon ligation of specific microbial ligands. The human TLR family is comprised of 10 members, all of which are expressed by antigen-presenting cells at constitutive amounts. TLRs were shown to sense and respond to specific pathogen-associated molecular patterns (PAMPs).

Intestinal mucosal microvascular endothelial cells have been recognized as an important anatomical barrier, protecting against invading bacteria and enterotoxins (2), preventing systemic inflammatory responses, as others have shown for the pathogenesis of hemolytic-uremic syndrome (4, 15).

As demonstrated in previous projects, human intestinal microvascular endothelial cells express functional TLRs 4 and 5 (21, 25), which both have to be considered as TLRs of central relevance to the enteric microflora (1). The major ligand of TLR4 was shown to be LPS from gram-negative bacteria, including E. coli, whereas flagellin, an antigen derived from flagellated bacteria such as Salmonella and Shigella spp., has been identified as the only ligand for TLR5. Both above-mentioned antigens are believed to be dominant antigens in the pathogenesis of human IBD (10, 19). In addition, both were shown to elicit rapid and sustained proinflammatory responses in HIMEC, including upregulation of ICAM-1, as well as increased static and dynamic leukocyte adhesion and enhanced transendothelial leukocyte transmigration (21, 25).

Given that TNF-{alpha} is considered one of the most important cytokines driving exaggerated immune responses and anti-TNF-{alpha} strategies represent an established treatment in human IBD, we were interested in which genes would be specifically expressed in HIMEC after proinflammatory stimulation with this cytokine. Compared with the microvascular reference endothelial cell line, EBI3 mRNA was markedly enhanced after stimulation with TNF-{alpha} (4 h and 24 h, 20 ng/ml) whereas no significant increase in signal was observed in HMEC-1 cells (Table 1). Preincubation with the NF-{kappa}B inhibitor MG132 inhibited the upregulation of EBI3 and IL-23p19, but not Il-12p35. These results suggest that expression of specific IL-12-related molecules in HIMEC is dependent on activation of the transcription factor NF-{kappa}B. Similar observations have been made for the expression of EBI3 in murine colitis models, where EBI3 expression was tightly dependent on intact NF-{kappa}B, as measured by reporter gene constructs (42).

By semiquantitative RT-PCR, we were able to detect stimulated expression of the IL-12-related molecules IL-23p19, IL-12p35, and EBI3 in HIMEC, whereas IL-12p40 and IL-27p28 could not be detected. In contrast, the dermal reference endothelial cell line (HMEC-1) did not show a pronounced response to TNF-{alpha} stimulation (Fig. 1). These findings indicate that HMEC-1 as an immortalized cell line may have lost TNF-{alpha} sensitivity.

In HIMEC, stimuli generally known to activate the NF-{kappa}B pathway appeared to be potent inducers of IL-12-related molecules. Prior data have suggested that upregulation of IL12p40 in endothelial cells is mainly dependent on stimulation with CD154, which is the CD40 ligand (18). In accordance with our data, this group was unable to stimulate IL-12p40 expression in endothelial cells using TNF-{alpha} and other stimuli. Our findings leave room for speculation that additional stimuli not yet identified might regulate the expression of these genes in HIMEC. Furthermore, these gene products might be synthesized as monomers, forming IL-12 and IL-23 extracellularly. Interestingly, stimulation of endothelial cells with IFN-{gamma}, a proinflammatory stimulus known to utilize the Janus kinase (JAK) as well as signal transducer and activation of transcription (STAT) pathways, did not lead to any regulation of IL-12-related molecules, as assessed by semiquantitative RT-PCR.

There are emerging data supporting a role of endothelial cells in the setting of innate immune responses. Likewise, our group has shown that HIMEC express the PAMP receptors for bacterial LPS and microbial flagellin, TLR4 and TLR5, respectively (21, 25). These data have sparked a hypothesis by which mucosal endothelial cells might be able to sense invading microbia, serving as a second line of mucosal defense (14).

For this reason, we were interested in which PAMP would induce the expression of IL-12-related molecules in HIMEC. Using a variety of well-characterized TLR agonists at established doses, we were intrigued to see that HIMEC appeared to strongly upregulate EBI3 expression after stimulation with the viral antigen analog poly(I:C). As expected, stimulation with the TLR4 agonist LPS and the TLR5 agonist flagellin also appeared to be, to a lesser extent, regulators of this IL-12-related molecule. Using HIMEC stimulated with poly(I:C), we were able to show upregulation of EBI3 on the protein level, as assessed by immunoblotting using the monoclonal antibody specific for EBI3 (8). Expression of EBI3 was further assessed by indirect immunofluorescence, where HIMEC stimulated with poly(I:C) showed a striking increase in cytosolic EBI3 immunoreactivity.

Having learned that the TLR3 agonist poly(I:C) appears to be a regulator of specific IL-12-related molecules in HIMEC, we were interested in whether HIMEC would express TLR3. As expected, HIMEC displayed both constitutive and regulated expression of TLR3, as assessed by semiquantitative RT-PCR. These findings were further corroborated by flow cytometry and immunofluorescence experiments from which we learned that endothelial expression of TLR3 is potently controlled by the ligand poly(I:C) itself.

According to what is known about the differentiation of naive CD4+ T cells, IL-12-related cytokines such as IL-17, IL-23, and IL-27 appear to act in a sequential and time-dependent manner (6) to induce Th1, Th2, and TH17 differentiation in naive CD4+ T cells (Th0 cells) (16). Using quantitative real-time RT-PCR, we assessed the time course of IL-12-related molecule expression in HIMEC in response to continuous stimulation with a fixed dose of poly(I:C). Interestingly, IL-23p19 was upregulated as early as 2 h after stimulation, whereas EBI3 expression levels peaked at later time points. These findings may reflect sequential innate immune functions elicited by mucosal endothelial cells upon activation of TLR3 by viral antigens.

IL-12 was found to be expressed at high levels in actively inflamed mucosa of both CD and UC (24). A recent study published by Stallmach et al. (37) was suggestive of a therapeutic effect of the IL-12p40-IgG2b dimeric fusion protein in experimental murine colitis models.

IL-23 is a heterodimer of the subunit p19, which is structurally related to IL-12p35, and IL-12p40. In contrast to IL-12, IL-23 was found to stimulate the proliferation of memory (CD4+CD45RB low) T cells (27). Recently, IL-23 was shown to stimulate differentiation of naive T cells into a novel subset of T cells termed Th17 or Th IL-17 cells. Th17 cells, which represent a major source of IL-17 upon stimulation with IL-23, are believed to exert proinflammatory effects of secondary local cell populations, including macrophages/monocytes and fibroblasts as well as endothelial cells. In these cells, IL-17 derived from Th17 cells is believed to set off a paracrine and autocrine secretion of proinflammatory effector mediators, including IL-1, IL-6, IL-8, and TNF-{alpha}.

The crucial proinflammatory role of IL-23 is evident, because mice ubiquitously overexpressing the IL-23 subunit p19 were found to suffer from generalized and lethal inflammatory disease (41). Current knowledge suggests constitutive expression of IL-23 in dendritic cells in the terminal ileum of mice (3).

Recent data have shown a beneficial effect brought about by neutralization of IL-23 biological activity rather than of IL-12 in experimental models of murine IBD (43). In these experimental series, antibody treatment neutralizing the biological activity of anti-IL-23p19 was superior to anti-p40 (subunit of both IL-12 and IL-23) in preventing the onset of and treating established murine experimental colitis (43). These findings are suggestive of a previously underestimated role of IL-23 in regulating intestinal inflammation. Interestingly, a new study supports a role of distinct IL23R mutations as a predisposing factor in the development of IBD (9).

The role of IL-27 in IBD remains to be identified as of yet. In vitro studies have shown a marked upregulation of IL-27 in activated monocytes and dendritic cells, which led to the idea that antigen-presenting cells might represent the main source of secreted IL-27 in vivo. However, biological functions of IL-27 are poorly understood. IL-27 was found to specifically bind to the receptor T cell cytokine receptor (TCCR) [synonomous with WSXWS type I receptor (WSX-1)], which is highly expressed by naive T cells (30). IL-27 induces the proliferation of CD4+ naive T cells and acts synergistically with IL-12 to induce expression of IFN-{gamma} (30). In addition, IL-27 appears to play an important role in the regulation of Th1-mediated immune responses (31). Microvascular endothelial cells derived from human gut (HIMEC) were demonstrated to possess antigen-presenting functions in vivo (12).

In conclusion, regulated production of IL-12-related molecules by mucosal endothelial cells might reflect adaptive immune functions in response to specific microbial antigens translocated into deeper mucosal layers. These immune functions may involve both secreted factors and direct interactions between mucosal endothelial cells and circulating and tissue-bound mononuclear cells.


   GRANTS
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG, to C. Maaser), Innovative Medizinische Forschung at the University of Münster (IMF, to J. Heidemann), and Deutsche Morbus Crohn/Colitis ulcerosa Vereinigung (DCCV e.V., to J. Heidemann).


   ACKNOWLEDGMENTS
 
The EBI3 mAb was generously supplied by Prof. O. Devergne, University of Paris, France. The authors are grateful for expert technical support by E. Weber.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Heidemann, Dept. of Medicine B, Univ. of Münster, Albert-Schweitzer-Str. 33, D-48129 Münster, Germany (e-mail: Jan_Heidemann{at}hotmail.com)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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