Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Psychoneuroimmunology wikipedia , lookup
12-Hydroxyeicosatetraenoic acid wikipedia , lookup
Lymphopoiesis wikipedia , lookup
Polyclonal B cell response wikipedia , lookup
Adaptive immune system wikipedia , lookup
Molecular mimicry wikipedia , lookup
Cancer immunotherapy wikipedia , lookup
Innate immune system wikipedia , lookup
Am J Physiol Gastrointest Liver Physiol 280: G710–G719, 2001. Regulated MIP-3␣/CCL20 production by human intestinal epithelium: mechanism for modulating mucosal immunity ARASH IZADPANAH, MICHAEL B. DWINELL, LARS ECKMANN, NISSI M. VARKI, AND MARTIN F. KAGNOFF Laboratory of Mucosal Immunology, Department of Medicine, University of California, San Diego, La Jolla, California 92093-0623 Received 13 July 2000; accepted in final form 19 October 2000 of the human intestine forms a physical barrier that separates the internal milieu of the host from luminal contents. During the course of intestinal inflammation or microbial infection, intestinal epithelial cells can, in addition to their normal absorptive and secretory functions, develop additional characteristics usually attributed to classic inflamma- tory cell types. Thus, after stimulation with proinflammatory mediators such as tumor necrosis factor (TNF)-␣ or interleukin (IL)-1␣ or in response to infection with enteric pathogens, intestinal epithelial cells upregulate a program of genes whose products can signal the onset of an acute mucosal inflammatory response characterized by an influx of neutrophils and monocytes (9, 10, 21, 23, 29, 32, 37, 42). Many of the genes, including several of the chemokine genes that are activated in intestinal epithelial cells in response to agonist stimulation or bacterial infection, are target genes of the transcription factor nuclear factor (NF)-B (13, 19, 20, 32). In this regard, NF-B can be viewed as a central regulator of the intestinal epithelial cell response to a set of signals, activated by proinflammatory stimuli and bacterial infection, that are thought to be important for the initiation of mucosal innate immune responses and acute mucosal inflammatory responses (13). Whereas intestinal epithelial cells do not produce the cytokines interferon (IFN)-␥, IL-2, IL-4, and IL-5, which are essential components of host adaptive immune responses (9, 21), recent studies have shown that they do produce three IFN-inducible T cell chemoattractants that may play a role in physiological inflammation characteristic of the normal intestinal mucosa and in the chemoattraction of T helper (Th)1-type CD4⫹ T cells within the intestinal mucosa (8). Chemokines are low-molecular-weight chemotactic cytokines that have a diverse set of activities after binding and signaling through their cognate receptors on target cells (2, 30). Chemokines play a key role in the directional trafficking of leukocytes and dendritic cells (DCs) and may play a further role in angiogenesis, hematopoiesis, organogenesis, and viral pathogenesis (2, 25, 30). Chemokines signal target cells through G protein-coupled seven-transmembrane-spanning receptors that, in many cases, are promiscuous in that a single chemokine receptor frequently binds several different chemokines. In addition, several known chemokines can bind to more than one chemokine receptor (2, 26, 30). The chemokine superfamily can be divided into four groups based on the number and spacing of the Address for reprint requests and other correspondence: M. F. Kagnoff, Laboratory of Mucosal Immunology, Dept. of Medicine, Univ. of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0623. 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. chemokines; dendritic cells; infectious immunity; inflammation; T lymphocytes THE EPITHELIAL CELL LINING G710 0193-1857/01 $5.00 Copyright © 2001 the American Physiological Society http://www.ajpgi.org Downloaded from http://ajpgi.physiology.org/ by 10.220.33.3 on June 17, 2017 Izadpanah, Arash, Michael B. Dwinell, Lars Eckmann, Nissi M. Varki, and Martin F. Kagnoff. Regulated MIP-3␣/CCL20 production by human intestinal epithelium: mechanism for modulating mucosal immunity. Am J Physiol Gastrointest Liver Physiol 280: G710–G719, 2001.—Human intestinal epithelial cells secrete an array of chemokines known to signal the trafficking of neutrophils and monocytes important in innate mucosal immunity. We hypothesized that intestinal epithelium may also have the capacity to play a role in signaling host adaptive immunity. The CC chemokine macrophage inflammatory protein (MIP)-3␣/CCL20 is chemotactic for immature dendritic cells and CD45RO⫹ T cells that are important components of the host adaptive immune system. In these studies, we demonstrate the widespread production and regulated expression of MIP-3␣ by human intestinal epithelium. Several intestinal epithelial cell lines were shown to constitutively express MIP-3␣ mRNA. Moreover, MIP-3␣ mRNA expression and protein production were upregulated by stimulation of intestinal epithelial cells with the proinflammatory cytokines tumor necrosis factor-␣ or interleukin-1␣ or in response to infection with the enteric bacterial pathogens Salmonella or enteroinvasive Escherichia coli. In addition, MIP-3␣ was shown to function as a nuclear factor-B target gene. In vitro findings were paralleled in vivo by increased expression of MIP-3␣ in the epithelium of cytokine-stimulated or bacteria-infected human intestinal xenografts and in the epithelium of inflamed human colon. Mucosal T cells, other mucosal mononuclear cells, and intestinal epithelial cells expressed CCR6, the cognate receptor for MIP-3␣. The constitutive and regulated expression of MIP-3␣ by human intestinal epithelium is consistent with a role for epithelial cell-produced MIP-3␣ in modulating mucosal adaptive immune responses. INTESTINAL EPITHELIAL CELLS PRODUCE MIP-3␣ MATERIALS AND METHODS Reagents. Recombinant human (rh)IL-1␣, IFN-␥, MIP-3␣, and TNF-␣ were from PeproTech (Rocky Hill, NJ). Biotinconjugated affinity-purified goat anti-human MIP-3␣, murine monoclonal antibody (MAb) to human CCR6 (clone 53103.111), and murine MAb to human MIP-3␣ (clone 67310.111) were from R&D Systems (Minneapolis, MN). Mouse IgG1 and IgG2b as well as MG-132 were from Sigma Chemical (St. Louis, MO). Rabbit anti-human CD3 was from Dako (Carpenteria, CA). Alexa 488-conjugated goat antirabbit IgG was from Molecular Probes (Eugene, OR), and Cy3-conjugated goat anti-mouse IgG was from Jackson ImmunoResearch Laboratories (West Grove, PA). Cell culture and stimulation protocols. The human colon adenocarcinoma cell lines HT-29, HCA-7, LS174T, HCT-8, and I-407 were grown in DMEM supplemented with 10% heat-inactivated FCS and 2 mM L-glutamine as described previously (7), and the Caco-2 cell line was grown in DMEM supplemented with 15% heat-inactivated FCS. T84 human colon carcinoma cells were grown in 50% DMEM-50% Ham’s F12 medium supplemented with 5% newborn calf serum and 2 mM L-glutamine (21). HT-29, LS174T, HCT-8, I-407, and Caco-2 were from the American Type Culture Collection, HCA-7 colony 29 was a gift from S. C. Kirkland (Royal Postgraduate Medical School, London, UK), and T84 was initially obtained from K. Dharmsathaphorn (UCSD). Cells were cultured at 37°C under 5% CO2-95% air. For agonist stimulation, confluent monolayers of HT-29, Caco-2, or T84 cells grown in six-well plates (Costar, Cambridge, MA) or Caco-2 cells grown in Transwell cultures (24-mm diameter, 0.4-m pore size; Costar) were stimulated with TNF-␣ (20 ng/ml), IL-1␣ (20 ng/ml), or IFN-␥ (40 ng/ml). For bacterial infections, confluent HT-29 or T84 monolayers in six-well plates were incubated with Salmonella dublin or enteroinvasive Escherichia coli O29:NM at a multiplicity of infection (MOI) of 100 and 500 for 1 h as described previously (10, 11), after which the medium was removed and cells were washed and incubated with fresh gentamicin (50 g/ml)containing medium to kill remaining extracellular bacteria. Human fetal intestinal xenografts. Human fetal intestine (obtained from Advanced Biosciences Resources, Alameda, CA), gestational age 12–18 wk, was transplanted subcutaneously onto the backs of C57BL/6 severe combined immunodeficiency (SCID) mice as described previously (11, 17, 23). Human fetal intestinal xenografts were allowed to develop for 10 wk after implantation, at which time the epithelium, which is strictly of human origin, is fully differentiated (35). Littermate SCID mice were injected subcutaneously with ⬃106 HT-29 cells, and tumors were allowed to develop for 5 wk. Mice carrying mature xenografts or HT-29 tumors were injected intraperitoneally with 1 g of human IL-1␣ in 200 l of PBS or with 200 l of PBS alone. Intestinal xenografts and HT-29 tumors were removed 5 h later, and adjacent segments of intestine and HT-29 tumors were frozen in liquid nitrogen for RNA isolation or were embedded in optimum cutting temperature compound (TissueTek, Torrance, CA) and frozen in isopentane-dry ice or fixed in 10% neutral buffered formalin for immunohistochemical analysis. In additional experiments, intestinal xenografts were infected with ⬃5 ⫻ 107 of an attenuated aroA aroC S. typhi, in DMEM-F12 medium at a 100-l volume, injected intraluminally by subcutaneous injection (17, 27). Those xenografts were removed 6 h after infection, after which mucosal scrapings were prepared and immediately frozen in liquid nitrogen. These studies were approved by the University of California, San Diego Human and Animal Subjects Committees. Adenovirus constructs and adenovirus infection. Recombinant adenovirus 5 (Ad5) containing an IB␣-AA superrepressor (Ad5IB-A32/36) or the E. coli -galactosidase gene (Ad5LacZ) was constructed as described previously (13, 20). Ad5IB-A32/36 expresses a hemagglutinin (HA) epitopetagged mutant form of IB␣ in which serine residues 32 and 36 are replaced by alanine residues. The mutant IB␣ cannot be phosphorylated at positions 32 and 36 and acts as a Downloaded from http://ajpgi.physiology.org/ by 10.220.33.3 on June 17, 2017 amino-terminal cysteines. In CC chemokines, two aminoterminal cysteines are adjacent, whereas in CXC chemokines, the two amino-terminal cysteines are separated by an intervening amino acid (2, 30). Macrophage inflammatory protein (MIP)-3␣/CCL20 (31), also known as liver and activation-regulated chemokine (LARC) (15) or Exodus (16), is a member of the CC chemokine subfamily initially noted to be expressed in human liver, lung, appendix, and tonsillar crypts (5, 6, 15, 16, 38). MIP-3␣ is selectively chemotactic for CD34⫹ bone marrow cell-derived immature DCs and CD45RO⫹ memory T cells that express the cognate receptor CCR6 (1, 14, 24, 28). MIP-3␣ produced at sites of inflammation may chemoattract CCR6-expressing immature DCs to the subepithelial region of mucosal surfaces (5, 18, 34). As immature DCs capture antigen at mucosal surfaces, they undergo a functional and phenotypic change that includes a decrease in CCR6 expression and a concomitant increase in expression of CCR7, the receptor for secondary lymphoid tissue chemokine (SLC) and MIP-3. This change enables DCs to traffic out of the tissues, where they encounter antigen and migrate to the blood and secondary lymphoid organs (5, 34, 40). Almost all of the memory T cells in the circulation that express the ␣47 integrin characteristic of mucosal homing lymphocytes in humans (3) also coexpress CCR6 (24), suggesting that MIP-3␣ may also be important for chemoattracting mucosal T cells. The orthologue of human MIP-3␣ in mice, termed mLARC, was recently cloned and shown to have a selective distribution in follicle-associated epithelium overlying murine Peyer’s patches and other mucosal lymphoid follicles (38). Unlike human MIP-3␣, mLARC is not expressed in liver or lung (38), and mLARC differs functionally from human MIP-3␣ in that mLARC, but not human MIP-3␣, is chemotactic for CCR6-expressing B cells (24). mRNA for MIP-3␣ has been demonstrated in epithelial cells in human appendix and in inflamed epithelial crypts of tonsils (5, 38), but little is known regarding the overall distribution and regulation of MIP-3␣ production in the normal or inflamed human intestinal tract. We hypothesized that human intestinal epithelial cells might have the capacity to link mucosal innate and acquired immunity through the regulated production of MIP-3␣, a chemokine capable of signaling immature DCs and CD45RO⫹ T cells. The studies herein describe the constitutive and regulated expression and production of MIP-3␣ mRNA and protein by human intestinal epithelial cells, using in vitro and in vivo models, and the presence of cells in the intestinal mucosa that express CCR6, the cognate receptor for MIP-3␣. G711 G712 INTESTINAL EPITHELIAL CELLS PRODUCE MIP-3␣ tory bowel disease were embedded in optimum cutting temperature compound and snap frozen in isopentane-dry ice as described previously (7, 27). Serial cryostat sections (5 m) were prepared, fixed in acetone for 10 min, and blocked in PBS-1% BSA for 1 h at room temperature. For MIP-3␣ and CCR6 immunostaining, sections were incubated overnight at 4°C with 5 g/ml mouse MAb to human MIP-3␣ or control mouse IgG1 or 5 g/ml mouse MAb to human CCR6 or control mouse IgG2b, and sections were subsequently stained with Cy3-conjugated goat anti-mouse IgG antibody. The isotypematched control antibodies were used at the same concentrations as the MIP-3␣ and CCR6 antibodies, respectively. For CD3 immunostaining, sections were incubated as above with rabbit anti-human CD3 or normal rabbit serum and subsequently stained with Alexa 488-conjugated goat anti-rabbit IgG. RESULTS MIP-3␣ expression by epithelium of normal and inflamed adult human colon. MIP-3␣ is a chemokine known to chemoattract populations of immature DCs and CD45RO⫹ T cells. To test whether epithelial cells are the major source of MIP-3␣ production in human colon, sections of normal and inflamed human colon were immunostained for MIP-3␣. As shown in Fig. 1B, MIP-3␣ is minimally expressed by normal colon epithelium. In contrast, epithelial MIP-3␣ immunostaining was markedly increased in sections of inflamed human colon and the epithelium was the major site of MIP-3␣ production (Fig. 1A). No immunostaining was seen in sections of inflamed human colon incubated with an isotype-matched control antibody. Constitutive and regulated MIP-3␣ mRNA expression in human intestinal epithelial cell lines. To better characterize the expression of MIP-3␣ seen in vivo, we used several human colon epithelial cell lines. As shown in Fig. 2A, several human colon epithelial cell lines, namely, HT-29, Caco-2, LS174T, and, to a lesser extent, I-407, constitutively expressed MIP-3␣ mRNA. In contrast, none of the cell lines expressed mRNA for MIP-3, a chemoattractant for CCR7-expressing mature DCs. As shown in Fig. 2B, MIP-3␣ mRNA levels in HT-29 and Caco-2 cells were upregulated after stimulation with the proinflammatory mediators TNF-␣ or IL-1␣, which are cytokines produced by mononuclear cells in the intestinal mucosa during the course of mucosal inflammation. MIP-3␣ mRNA levels were not upregulated by stimulation of the human colon epithelial cell lines with IFN-␥, granulocyte-macrophage colony-stimulating factor (GM-CSF), or IL-4 (data not shown). None of the cytokines tested (i.e., TNF-␣, IL-1␣, IFN-␥, GM-CSF, or IL-4) upregulated expression of the mature DC chemoattractant MIP-3 (data not shown). Regulated production of MIP-3␣ by human intestinal epithelial cell lines. We next assessed whether the constitutive and regulated expression of MIP-3␣ mRNA by the epithelial cell lines was paralleled by protein production, using an ELISA to measure secreted levels of MIP-3␣. As shown in Fig. 3, unstimulated HT-29, Caco-2, and T84 cell lines secreted 2.1, 18.6, and 1.4 ng/ml MIP-3␣, respectively, as determined after 18-h incubation. Stimulation of those cells Downloaded from http://ajpgi.physiology.org/ by 10.220.33.3 on June 17, 2017 superrepressor of NF-B activation (13, 20). The HA epitope tag enables identification of the exogenous superrepressor with anti-hemagglutinin antibodies. Viral titers were determined by plaque assay. Recombinant virus was stored in PBS containing 10% (vol/vol) glycerol at ⫺80°C. HT-29 cells grown to confluence in six-well tissue culture plates were infected with Ad5IB-A32/36 or Ad5LacZ in serum-free medium at a MOI of 100 for 16 h as described previously (13, 27). At this MOI, Ad5IB-A32/36 or Ad5LacZ infected ⬎80% of HT-29 cells and infected cells expressed IB␣-A32/36 and -galactosidase, respectively, at high levels as assessed by staining for -galactosidase and immunostaining for HA-tagged IB␣-A32/36 (data not shown). After infection, adenovirus was removed by washing, fresh medium containing serum was added, and cells were incubated for an additional 12 h before bacterial infection or IL-1␣ or TNF-␣ stimulation. The IB␣-AA superrepressor inhibited NF-B activation in HT-29 cells, as assessed by electrophoretic mobility shift assay, and inhibited cytokine-induced and bacteria-induced upregulation of IL-8 and intercellular adhesion molecule-1 expression in HT-29 cells but did not alter -actin mRNA levels in the same cells (data not shown; see Ref. 13). RNA isolation. Total cellular RNA was extracted using an acid guanidinium-phenol-chloroform method (TRIzol Reagent, Gibco Life Technologies, Grand Island, NY) and treated with RNase-free DNase (Stratagene, La Jolla, CA) (7, 21). For RT-PCR, 1 g of total cellular RNA was reverse transcribed and cDNA was amplified as described previously (21). The primers for human MIP-3␣ (sense 5⬘-ACC ATG TGC TGT ACC AAG AGT TTG-3⬘ and antisense 5⬘-CTA AAC CCT CCA TGA TGT GCA AGT GA-3⬘) and MIP-3 (sense 5⬘-CAG CCT GCT GGT TCT CTG GAC TTC-3⬘ and antisense 5⬘-GCC CCT CAG TGT GGT GAA CAC TAC-3⬘) were designed from available sequences from GenBank (NM004591 and AJ223410) and yielded PCR products of 390 bp and 276 bp, respectively. The amplification profile was 30 cycles of 45-s denaturation at 95°C and 2.5-min annealing and extension at 60°C for cell lines and tissues. Negative control reactions had no added RNA in the RT and subsequent PCR amplification. Peripheral blood mononuclear cells stimulated overnight with 10 g/ml phytohemagglutinin-P and 2 g/ml lipopolysaccharide (E. coli O111:B4) were used as a source of cDNA for positive controls. The MIP-3␣ primers did not amplify sequences in murine or human genomic DNA or murine cDNA. After amplification, aliquots of the PCR reaction were size separated on a 1.0% agarose gel containing ethidium bromide and photographed. ELISA. Polystyrene 96-well plates (Immulon-4, Dynex Technologies, Chantilly, VA) were coated with murine MAb to human MIP-3␣ diluted in carbonate buffer as the capture antibody. Affinity-purified biotinylated goat anti-human MIP-3␣ diluted in PBS, 1.0% BSA, and 0.1% Tween-20 was used as the detection antibody. The second step reagent was horseradish peroxidase (HRP)-conjugated streptavidin. Bound HRP was visualized with 3,3⬘,5,5⬘-tetramethylbenzidine dihydrochloride and H2O2 diluted in sodium acetate buffer (pH 6.0), the color reaction was stopped by addition of 1.2 M H2SO4, and absorbance was measured at 450 nm. MIP-3␣ concentration was calculated from a standard curve using rhMIP-3␣. The ELISA was sensitive to 50 pg/ml. Immunohistochemistry. Segments of human colon from the histologically normal-appearing resection margin of a surgical specimen from an individual undergoing partial colectomy for diverticular disease, mucosal biopsies from four healthy individuals obtained during screening colonoscopy, and mucosal biopsies from three individuals with inflamma- INTESTINAL EPITHELIAL CELLS PRODUCE MIP-3␣ G713 Fig. 2. Constitutive and regulated expression of MIP-3␣ mRNA by human intestinal epithelial cell lines. A: total RNA was isolated from the indicated cell lines, and MIP-3␣ and MIP-3 mRNA were amplified by RT-PCR using 30 amplification cycles. Peripheral blood mononuclear cells (PBMC) were stimulated with lipopolysaccharide and phytohemagglutinin. Negative controls had no added RNA. B: total RNA isolated from control HT-29 and Caco-2 cells or HT-29 and Caco-2 cells stimulated for 3–12 h with tumor necrosis factor (TNF)-␣ and interleukin (IL)-1␣ was amplified as in A. with IL-1␣ or TNF-␣ markedly increased MIP-3␣ secretion within 3 h, with MIP-3␣ levels ranging as high as 200–500 ng/ml at 18 h after stimulation. Consistent with the lack of effect on mRNA expression, stimulation of the same cell lines with IFN-␥, GM-CSF, or IL-4 did not increase MIP-3␣ secretion (data not shown). Consistent with the data for T84, the cell lines HCA-7 and HCT-8, which did not constitutively express MIP-3␣ mRNA, could also be induced to upregulate the production of MIP-3␣ protein in response to IL-1␣ or TNF-␣ stimulation (data not shown). The human intestinal epithelium is polarized into phenotypically and functionally distinct apical and basolateral domains. For MIP-3␣ produced by epithelial cells to imprint a chemotactic gradient and act as a chemoattractant for mucosal T cells or DCs, it predictably would be secreted by epithelial cells in the physiologically relevant basolateral direction rather than apically into the intestinal lumen. To determine whether MIP-3␣ was vectorially secreted by intestinal epithelial cells, we used Caco-2 cells grown on microporous filter supports as a polarized monolayer in Transwell cultures. Polarized Caco-2 cells were stimulated with IL-1␣ added to the basal chamber, as intestinal epithelium in vivo would normally be exposed to IL-1 produced in the intestinal mucosa at the basolateral membrane. These experiments revealed that polarized intestinal epithelial cells mainly secreted MIP-3␣ into the basal rather than the apical Transwell Downloaded from http://ajpgi.physiology.org/ by 10.220.33.3 on June 17, 2017 Fig. 1. Epithelial macrophage inflammatory protein (MIP)-3␣ immunostaining of normal and inflamed human colon. Sections of inflamed (A) and normal (B) human colon were immunostained for MIP-3␣. Adjacent sections from inflamed (C) and normal (D) human colon were stained with hematoxylin and eosin. As shown, MIP-3␣ immunostaining was heterogeneous and was most marked in the inflamed colon (A) and restricted to epithelial cells. No immunostaining was seen in sections stained with an isotype-matched mouse IgG1 antibody at the same concentration used as a control (data not shown). The nonspecific background staining was seen in sections incubated with either specific or nonspecific antibodies. Original magnification ⫻200. G714 INTESTINAL EPITHELIAL CELLS PRODUCE MIP-3␣ Fig. 3. Secretion of MIP-3␣ by HT-29, Caco-2, and T84 cells stimulated with TNF-␣ or IL-1␣. Confluent HT-29 (A), Caco-2 (B), or T84 (C) cells were stimulated with TNF-␣ (E) or IL-1␣ () for 3–18 h, whereas control cultures (F) remained unstimulated. MIP-3␣ in culture supernatants was assayed by ELISA. Results are means ⫾ SE of 3–5 repeated experiments. Table 1. MIP-3␣ secretion in HT-29 and T84 cells infected with Salmonella dublin or Escherichia coli O29:NM MIP-3␣ Secretion, ng/ml ⫹ S. dublin No bacteria ⫹ E. coli O29:NM Cell Line 6h 12 h 6h 12 h 6h 12 h HT-29 T84 0.9 ⫾ 0.2 0.3 ⫾ 0.07 2.8 ⫾ 0.03 0.6 ⫾ 0.1 42.3 ⫾ 4.2 36.1 ⫾ 2.0 65.2 ⫾ 5.8 41.6 ⫾ 2.9 16.9 ⫾ 1.9 ND 59.6 ⫾ 7.8 ND Values are means ⫾ SE of 3–5 repeated experiments. MIP, macrophage inflammatory protein; ND, not done. Downloaded from http://ajpgi.physiology.org/ by 10.220.33.3 on June 17, 2017 chamber. Thus MIP-3␣ secretion in the IL-1␣-stimulated Caco-2 cultures was 173.0 ⫾ 12.2 ng in the basal chamber compared with 25.1 ⫾ 2.9 ng in the apical chamber. This contrasts with 6.9 ⫾ 0.1 ng in the basal chamber and 0.5 ⫾ 0.2 ng in the apical chamber of unstimulated cultures. Values are means ⫾ SD from four or five replicate wells in a single representative experiment. Similar results were obtained in two repeated experiments. We next sought to determine whether infection of intestinal epithelial cells with enteric bacterial pathogens that are known to upregulate the expression of epithelial cell proinflammatory genes whose products chemoattract neutrophils and monocytes increases epithelial cell MIP-3␣ secretion. For these studies, HT-29 and T84 cells were infected with S. dublin or enteroinvasive E. coli O29:NM. As shown in Table 1, MIP-3␣ secretion increased as much as 20- to 120-fold in bacteria-infected cultures. IB␣ superrepressor blocks inducible MIP-3␣ production in HT-29 cells. Many of the genes upregulated in response to bacterial infection of intestinal epithelial cells, or after stimulation of those cells with TNF-␣ or IL-1␣, are NF-B target genes (13, 20), although the role, if any, of NF-B in the transcriptional regulation of the CC chemokine MIP-3␣ has not previously been reported. To determine whether MIP-3␣ in intestinal epithelial cells functions as an NF-B target gene, we first assessed whether blocking NF-B activation with a proteasome inhibitor, MG-132, decreased MIP-3␣ secretion in response to TNF-␣ or IL-1␣ stimulation. Treatment of HT-29 cells with 50 M of MG-132 before stimulation with TNF-␣ decreased MIP-3␣ secretion by 90% compared with cells not pretreated with MG-132 (data not shown). Because pharmacological agents are not always completely specific, we also used an additional approach to block NF-B activation. In this approach, cells were infected with a recombinant adenovirus expressing a mutant IB␣ protein that has serine-to-alanine substitutions at positions 32 and 36 (Ad5IB-A32/36). Ad5IB-A32/36 acts as a superrepressor of NF-B activation by preventing signal-induced IB␣ phosphorylation (13, 20). After infection with this recombinant adenovirus, HT-29 cells were stimulated with TNF-␣ or IL-1␣ or infected with S. dublin or enteroinvasive E. coli. As shown in Table 2, MIP-3␣ secretion was markedly inhibited in agoniststimulated or bacteria-infected HT-29 cells infected with recombinant adenovirus expressing the super- INTESTINAL EPITHELIAL CELLS PRODUCE MIP-3␣ Table 2. MIP-3␣ is a NF-B target gene MIP-3␣ Secretion, ng/ml Stimulus ⫹Ad5IB-A32/36 superrepressor ⫹Ad5 LacZ control virus None IL-1␣ TNF-␣ S. dublin E. coli O29:NM 0.8 3.4 5.2 7.3 5.4 2.4 23.7 40.7 57.5 30.9 G715 with human IL-1␣. As shown in Fig. 4, MIP-3␣ mRNA expression increased in the xenografts of IL-1␣-injected mice, although constitutive background levels of MIP-3␣ varied from xenograft to xenograft. MIP-3␣ Downloaded from http://ajpgi.physiology.org/ by 10.220.33.3 on June 17, 2017 Values are means of replicate wells from a representative experiment. Similar results were obtained in 2 repeated experiments. HT-29 cells were infected at a multiplicity of infection of 100 with a recombinant adenovirus (Ad5IB-A32/36) expressing a mutant IB␣ protein that acts as a superrepressor of nuclear factor-B activation or the same recombinant adenovirus expressing the LacZ gene (Ad5LacZ). After infection, cells were stimulated for 6 h with tumor necrosis factor (TNF)-␣ or interleukin (IL)-1␣ or infected with enteroinvasive bacteria as described in MATERIALS AND METHODS. repressor but not in cells infected with the same recombinant adenovirus expressing the LacZ gene (Ad5LacZ). MIP-3␣ expression is upregulated in human intestinal xenografts in response to IL-1␣ stimulation or Salmonella infection. To determine whether regulated MIP-3␣ expression by the human intestinal epithelial cell lines was paralleled in vivo, expression of MIP-3␣ was assessed in human intestinal xenografts. As shown in Fig. 4, we first showed that HT-29 cells, which respond to cytokine stimulation in vitro, also do so in vivo when implanted subcutaneously in SCID mice. We next used human intestinal xenografts implanted subcutaneously in SCID mice and cytokine treatment to assess regulated MIP-3␣ production by normal human intestinal epithelium. For these studies, we first assessed MIP-3␣ mRNA expression in xenografts after intraperitoneal injection of SCID mice Fig. 4. Expression of MIP-3␣ mRNA in human intestinal xenografts. Total RNA isolated from intestinal xenografts or HT-29 tumors implanted subcutaneously in severe combined immunodeficiency (SCID) mice was amplified by RT-PCR using 30 amplification cycles. Data in A, B, and C are from three different stimulated or infected xenografts. Although constitutive expression of MIP-3␣ by the xenografts varied from experiment to experiment, MIP-3␣ expression was consistently upregulated in the xenografts in response to cytokine stimulation or bacterial infection. Fig. 5. Epithelial cell expression of MIP-3␣ in response to IL-1␣ stimulation of human intestinal xenografts. Sections of human intestinal xenografts from SCID mice injected with human IL-1␣ (A and B) were immunostained for MIP-3␣ (A) or stained with Hoechst dye 33258 to reveal nuclear morphology (B). Note that MIP-3␣ immunostaining is restricted to the intestinal epithelium. C: xenograft section of the same donor fetal intestine implanted in parallel in a SCID mouse that was not injected with IL-1␣ before xenograft harvesting. Adjacent sections stained with isotype-matched control mouse IgG1 revealed no immunostaining (data not shown). Original magnification ⫻400. G716 INTESTINAL EPITHELIAL CELLS PRODUCE MIP-3␣ mRNA expression was also increased in xenografts infected with an aroA aroC mutant of S. typhi. We note that IL-1␣ is not species specific and may have stimulated additional murine mediators that potentially can act on human cells in the xenografts. Nonetheless, the approach used herein allows the assessment of early changes in epithelial cell chemokine gene expression and regulation in response to a limited set of stimuli in human intestine in vivo, which is not possible when studying naturally infected human subjects or individuals with ongoing acute or chronic intestinal mucosal inflammation. To show that increased chemokine mRNA expression was accompanied by epithelial protein production, adjacent segments of the intestinal xenografts were immunostained for MIP-3␣. As shown in Fig. 5, there Downloaded from http://ajpgi.physiology.org/ by 10.220.33.3 on June 17, 2017 Fig. 6. CCR6 and CD3 expression in normal human colon. Adjacent sections of normal adult human colon were immunostained for CCR6 (A), for CD3 (C), or with an isotype control antibody for CCR6 (D). An additional section of normal human colon, not associated with a lymphoid follicle, was also immunostained for CCR6 (B). As shown in A, numerous CCR6-staining cells are present in the T cell-rich marginal zone, with fewer in the B cell-containing germinal center (GC), of a lymphoid follicle and in the adjacent lamina propria. The colonic epithelium also stained positively for CCR6 as shown in the top and left of A and in B. As shown in C, CD3 immunostaining is seen in mononuclear cells in the lamina propria and T cell-abundant marginal zone of the lymphoid follicle and only scattered CD3-positive cells are present in the GC. Colocalization studies showed that some CCR6-positive cells in the lamina propria were CD3 positive, whereas others were CD3 negative. The intestinal lumen is oriented to the top in A, C, and D and on the right in B. Original magnification ⫻200. was little MIP-3␣ immunostaining in unstimulated xenografts but a marked increase in epithelial cell MIP-3␣ immunostaining that was localized in the intestinal epithelium of the human xenografts from IL-1␣-injected mice. The relatively low level of epithelial MIP-3␣ immunostaining in unstimulated xenografts is consistent with the minimal constitutive expression seen in normal human colon (compare with Fig. 1B). CCR6, the cognate receptor for MIP-3␣, is expressed by mononuclear cells and epithelial cells in human colon mucosa. The normal human intestinal mucosa contains an abundant population of T cells and DCs. Because CCR6 is the only currently known cellular receptor for MIP-3␣, we sought to determine whether cells in human colon mucosa express CCR6. As shown in Fig. 6A, cells within the lamina propria and a lym- INTESTINAL EPITHELIAL CELLS PRODUCE MIP-3␣ DISCUSSION We report here that the CC chemokine MIP-3␣ is widely expressed and regulated in human intestinal epithelium. Moreover, epithelial expression of MIP-3␣ is upregulated by stimulation with the proinflammatory cytokines IL-1␣ and TNF-␣, cytokines that can be produced by mononuclear cells in the intestinal mucosa during acute inflammation, or in response to bacterial infection. Intestinal epithelial cells activated by proinflammatory stimuli, infected with enteric pathogens, or stimulated with bacterial products have been shown to produce signals for the chemoattraction of neutrophils and monocytes, cells important in host innate mucosal immunity (22, 29, 32, 37, 39, 42). Because MIP-3␣ is known to chemoattract CCR6-expressing memory T cells and immature DCs (6, 24), our data extend that current view and suggest that intestinal epithelial cells also have the capacity to play a role in orchestrating antigen-specific acquired mucosal immune responses. The pattern of expression of MIP-3␣ throughout the epithelium of human intestine and its expression in other sites such as human lung and liver contrasts with its more localized expression in follicleassociated epithelium in mice and suggests that the functional activity of this chemokine goes beyond a proposed role in the genesis and function of mucosal lymphoid follicles (5, 6, 15, 16, 18, 38). In addition, the upregulated expression of human MIP-3␣ in response to TNF-␣ or IL-1 stimulation of human intestinal epithelial cells contrasts with the lack of response of MIP-3␣ production in J774 mouse monocytes to those stimuli (38), indicating that there may be differences in the signal transduction pathways important for activating MIP-3␣ in different cell types and/or species. Our finding of upregulated epithelial cell MIP-3␣ in cytokine-stimulated or bacteria-infected human intestinal xenografts, and in inflamed human colon, indicates that human intestinal epithelial cells regulate the expression of a chemokine whose major known function is to chemoattract cells important for antigen presentation and the development of the host adaptive immune response. Thus, in response to inflammatory stimuli, intestinal epithelial cells may develop the capacity to chemoattract immature DCs and memory T cells in close proximity to the single layer of intestinal epithelium that separates the intestinal lumen from the host’s internal milieu. After capturing antigen, immature DCs at sites of inflammation undergo a phenotypic change and migrate to regional lymph nodes. As part of this process, they downregulate surface expression of several chemokine receptors (i.e., CCR1, CCR5, and CCR6), concurrently lose their responsiveness to the chemokine ligands for those receptors (i.e., MIP-1␣, MIP-1, RANTES, and MIP-3␣) (33, 34, 36), upregulate the expression of CXCR4, CCR4, and CCR7, and become responsive to chemokines expressed in blood vessels and secondary lymphoid organs [e.g., SLC, MIP-3, stromal cell-derived factor (SDF-1), macrophage-derived chemokine, thymus- and activation-regulated chemokine] (5, 33, 34, 36). Consistent with this phenotypic and functional change in DCs, intestinal epithelial cells do not express the mature DC chemoattractant MIP-3. In addition to highlevel MIP-3␣ production after stimulation, it is possible that constitutive low levels of epithelial expression of MIP-3␣ may serve to maintain immature DCs and memory T cells in close proximity to the epithelial surface, the first site of host contact with antigen in the intestinal lumen. MIP-3␣ is shown to function as a NF-B target gene in human intestinal epithelial cells, and its expression is upregulated by TNF-␣ or IL-1␣ stimulation or infection with enteric bacteria. The same is true of IL-8 and growth-related protein-␣ (GRO-␣) that, like MIP-3␣, are upregulated in the intestinal epithelium by IL-1 or TNF-␣ stimulation or infection with enteric bacterial pathogens (13, 19, 22, 42). However, MIP-3␣ mainly signals immature DC and CD45RO⫹ T cells important in host adaptive immune responses, whereas IL-8 and GRO-␣ signal neutrophils that can play a role in mucosal innate immune defense. We recently described (8) the expression of three additional T cell chemoattractants expressed by human intestinal epithelium, the CXC chemokines IP-10, Mig, and I-TAC, which, in contrast to MIP-3␣, are known to signal activated/ memory CD4⫹ CD45RO⫹ T cells that express the receptor CXCR3. Unlike MIP-3␣, those chemokines are preferentially upregulated by the Th1 cytokine IFN-␥ and are only minimally, if at all, regulated by TNF-␣, IL-1, or bacterial infection in the absence of IFN-␥. Together, these data suggest that the intestinal epithelium can play a role in regulating both innate and acquired mucosal immune responses and demonstrate differential regulation of epithelial cell-produced chemokines that act on different target populations of T cells. The human intestinal epithelial cell lines tested in our studies secreted up to 200–500 ng/ml of MIP-3␣ in response to IL-1␣ stimulation. It is not currently possible to determine the effective concentrations of MIP-3␣ in the microenvironment of the intestinal mu- Downloaded from http://ajpgi.physiology.org/ by 10.220.33.3 on June 17, 2017 phoid follicle, most markedly in the marginal zone rather than the germinal center, expressed CCR6. In addition, human colon epithelial cells expressed CCR6 (Fig. 6, A and B), consistent with our prior report (7) of the constitutive expression of CCR6 mRNA by several human intestinal epithelial cell lines. Whereas many CCR6-positive mononuclear cells also stained for the T cell marker CD3, a number of CCR6-expressing cells in the lamina propria, some of which were in close proximity to the epithelium, did not stain for CD3 (compare Fig. 6C with Fig. 6A). This finding is consistent with the reported expression of CCR6 by both T cells and immature DCs (5, 24). In contrast to MIP-3␣ mRNA expression and protein production, CCR6 mRNA expression was not significantly increased in intestinal epithelial cell lines infected with Salmonella, as we reported previously (12), and epithelial CCR6 immunostaining was not increased in sections of inflamed human colon (data not shown). G717 G718 INTESTINAL EPITHELIAL CELLS PRODUCE MIP-3␣ We thank J. Leopard and D. McCole for helpful assistance and R. Lara for final preparation of the manuscript. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-35108. A. Izadpanah is a Howard Hughes Medical Institute Medical Student Research Training Fellow; M. B. Dwinell was supported by a Research Career Development Award from the Crohn’s and Colitis Foundation of America (CCFA) and NIDDK Grant K01-DK-02808; and L. Eckmann was supported, in part, by a research grant from the CCFA. REFERENCES 1. Baba M, Imai T, Nishimura M, Kakizaki M, Takagi S, Hieshima K, Nomiyama H, and Yoshie O. Identification of CCR6, the specific receptor for a novel lymphocyte-directed CC chemokine LARC. J Biol Chem 272: 14893–14898, 1997. 2. Baggiolini M, Dewald B, and Moser B. Human chemokines: an update. Annu Rev Immunol 15: 675–705, 1997. 3. Butcher EC, Williams M, Youngman K, Rott L, and Briskin M. Lymphocyte trafficking and regional immunity. Adv Immunol 72: 209–253, 1999. 4. Colotta F, Dower SK, Sims JE, and Mantovani A. The type II “decoy” receptor: a novel regulatory pathway for interleukin 1. Immunol Today 15: 562–566, 1994. 5. Dieu MC, Vanbervliet B, Vicari A, Bridon JM, Oldham E, Ait-Yahia S, Briere F, Zlotnik A, Lebecque S, and Caux C. Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J Exp Med 188: 373–386, 1998. 6. Dieu-Nosjean MC, Vicari A, Lebecque S, and Caux C. Regulation of dendritic cell trafficking: a process that involves the participation of selective chemokines. J Leukoc Biol 66: 252–262, 1999. 7. Dwinell MB, Eckmann L, Leopard JD, Varki NM, and Kagnoff MF. Chemokine receptor expression by human intestinal epithelial cells. Gastroenterology 117: 359–367, 1999. 8. Dwinell MB, Lügering N, Eckmann L, and Kagnoff MF. Regulated production of interferon-inducible T cell chemoattractants by human intestinal epithelial cells. Gastroenterology 120: 49–59, 2001. 9. Eckmann L, Jung HC, Schurer-Maly C, Panja A, Morzycka-Wroblewska E, and Kagnoff MF. Differential cytokine expression by human intestinal epithelial cell lines: regulated expression of interleukin 8. Gastroenterology 105: 1689–1697, 1993. 10. Eckmann L, Kagnoff MF, and Fierer J. Epithelial cells secrete the chemokine interleukin-8 in response to bacterial entry. Infect Immun 61: 4569–4574, 1993. 11. Eckmann L, Stenson WF, Savidge TC, Lowe DC, Barrett KE, Fierer J, Smith JR, and Kagnoff MF. Role of intestinal epithelial cells in the host secretory response to infection by invasive bacteria. Bacterial entry induces epithelial prostaglandin H synthase-2 expression and prostaglandin E2 and F2␣ production. J Clin Invest 100: 296–309, 1997. 12. Eckmann L, Smith JR, Housley MP, Dwinell MB, and Kagnoff MF. Analysis by high-density cDNA arrays of altered gene expression in human intestinal epithelial cells in response to infection with the invasive enteric bacteria Salmonella. J Biol Chem 275: 14084–14094, 2000. 13. Elewaut D, DiDonato JA, Kim JM, Truong F, Eckmann L, and Kagnoff MF. NF-B is a central regulator of the intestinal epithelial cell innate immune response induced by infection with enteroinvasive bacteria. J Immunol 163: 1457–1466, 1999. 14. Greaves DR, Wang W, Dairaghi DJ, Dieu MC, Saint-Vis B, Franz-Bacon K, Rossi D, Caux C, McClanahan T, Gordon S, Zlotnik A, and Schall TJ. CCR6, a CC chemokine receptor that interacts with macrophage inflammatory protein 3␣ and is highly expressed in human dendritic cells. J Exp Med 186: 837–844, 1997. 15. Hieshima K, Imai T, Opdenakker G, Van DJ, Kusuda J, Tei H, Sakaki Y, Takatsuki K, Miura R, Yoshie O, and Nomiyama H. Molecular cloning of a novel human CC chemokine liver and activation-regulated chemokine (LARC) expressed in liver. Chemotactic activity for lymphocytes and gene localization on chromosome 2. J Biol Chem 272: 5846–5853, 1997. 16. Hromas R, Gray PW, Chantry D, Godiska R, Krathwohl M, Fife K, Bell GI, Takeda J, Aronica S, Gordon M, Cooper S, Downloaded from http://ajpgi.physiology.org/ by 10.220.33.3 on June 17, 2017 cosa because this depends on several factors, including the biological half-life of MIP-3␣ in the mucosa, the extent to which it binds proteoglycans and is available for binding to CCR6 on target cells, and its effective diffusion distance from the epithelium. Nevertheless, the quantity of MIP-3␣ secreted by human intestinal epithelial cell lines is within the range shown in vitro to be chemotactic for lymphocytes. Thus 1 g/ml recombinant MIP-3␣ was maximally chemotactic for freshly isolated human peripheral blood lymphocytes, and significant chemotaxis was observed at concentrations of 100 ng/ml (1). The present studies report on the regulated expression of MIP-3␣ by human intestinal epithelium. It will be important for future studies to assess the functional activity of this chemokine on target cells in the complex microenvironment of the human intestinal mucosa. Relevant to MIP-3␣ function in the intestinal tract, a recent study in mice indicated that a specific subset of mucosal DC within the subepithelial region of Peyer’s patches express CCR6 and migrate in response to MIP-3␣ (18). However, we also note that CCR6, the cognate receptor for MIP-3␣, was abundantly expressed by epithelial cells in normal human colon. This is consistent with our prior report (7) that several human intestinal epithelial cell lines express CCR6 mRNA as well as functional receptors for several other CC and CXC chemokines, most notably CXCR4, which binds the CXC chemokine SDF-1, and CCR5, which binds the CC chemokines MIP-1␣, MIP-1, and RANTES. Like CXCR4 and CCR5, CCR6 on intestinal epithelial cells acts as a functional signaling receptor after interaction with its ligand MIP-3␣ (unpublished data). Thus, in addition to paracrine effects on DCs and T cells, MIP-3␣, like several other mediators produced by intestinal epithelial cells, may mediate autocrine effects on the intestinal epithelium. In contrast to MIP-3␣, CCR6 was not upregulated by intestinal epithelial cells in inflamed colon. Finally, we note that CCR6 has been reported to act as a functional receptor for a second epithelial cellproduced ligand, the antimicrobial peptide human  defensin-2 (hBD-2) (41). In addition, we have reported (27) the upregulated expression of hBD-2 by human intestinal epithelial cells in vitro and in vivo in response to proinflammatory cytokine stimulation or bacterial infection. However, we found (present study and Ref. 27) that MIP-3␣ secretion by agonist-stimulated or bacteria-infected human intestinal epithelial cell lines was ⬃100-fold greater than that of hBD-2 in response to identical proinflammatory stimuli. Coupled with a lower affinity of hBD-2 binding to CCR6 compared with MIP-3␣ (41), hBD-2 produced by intestinal epithelium may not compete effectively with MIP-3␣ for binding to CCR6 on immature DC or mucosal T cells or to CCR6 expressed by intestinal epithelium. INTESTINAL EPITHELIAL CELLS PRODUCE MIP-3␣ 17. 18. 19. 20. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. gene expression in intestinal epithelial cells and inflammatory bowel disease mucosal. Gastroenterology 108: 40–50, 1995. Rollins BJ. Chemokines. Blood 90: 909–928, 1997. Rossi DL, Vicari AP, Franz-Bacon K, McClanahan TK, and Zlotnik A. Identification through bioinformatics of two new macrophage proinflammatory human chemokines: MIP-3␣ and MIP-3. J Immunol 158: 1033–1036, 1997. Savkovic DS, Koutsouris A, and Hecht G. Activation of NF-B in intestinal epithelial cells by enteropathogenic Escherichia coli. Am J Physiol Cell Physiol 273: C1160–C1170, 1997. Sallusto F and Lanzavecchia A. Mobilizing dendritic cells for tolerance, priming, and chronic inflammation. J Exp Med 189: 611–614, 1999. Sallusto F, Schaerli P, Loetscher P, Schaniel C, Lenig D, Mackay CR, Qin S, and Lanzavecchia A. Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation. Eur J Immunol 28: 2760–2769, 1998. Savidge TC, Morey AL, Ferguson DJ, Fleming KA, Shmakov AN, and Phillips AD. Human intestinal development in a severe-combined immunodeficient xenograft model. Differentiation 58: 361–371, 1995. Sozzani S, Allavena P, D’Amico G, Luini W, Bianchi G, Kataura M, Imai T, Yoshie O, Bonecchi R, and Mantovani A. Differential regulation of chemokine receptors during dendritic cell maturation: a model for their trafficking properties. J Immunol 161: 1083–1086, 1998. Steiner TS, Nataro JP, Poteet-Smith CE, Smith JA, and Guerrant RL. Enteroaggregative Escherichia coli expresses a novel flagellin that causes IL-8 release from intestinal epithelial cells. J Clin Invest 105: 1769–1777, 2000. Tanaka Y, Imai T, Baba M, Ishikawa I, Uehira M, Nomiyama H, and Yoshie O. Selective expression of liver and activation-regulated chemokine (LARC) in intestinal epithelium in mice and humans. Eur J Immunol 29: 633–642, 1999. Thorpe CM, Hurley BP, Lincicome LL, Jacewicz MS, Keusch GT, and Acheson DW. Shiga toxins stimulate secretion of interleukin-8 from intestinal epithelial cells. Infect Immun 67: 5985–5993, 1999. Willimann K, Legler DF, Loetscher M, Roos RS, Delgado MB, Clark-Lewis I, Baggiolini M, and Moser B. The chemokine SLC is expressed in T cell areas of lymph nodes and mucosal lymphoid tissues and attracts activated T cells via CCR7. Eur J Immunol 28: 2025–2034, 1998. Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J, Anderson M, Schroder JM, Wang JM, Howard OM, and Oppenheim JJ. -defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 286: 525–528, 1999. Yang SK, Eckmann L, Panja A, and Kagnoff MF. Differential and regulated expression of C-X-C, C-C, and C-chemokines by human colon epithelial cells. Gastroenterology 113: 1214– 1223, 1997. Downloaded from http://ajpgi.physiology.org/ by 10.220.33.3 on June 17, 2017 21. Broxmeyer HE, and Klemsz MJ. Cloning and characterization of exodus, a novel -chemokine. Blood 89: 3315–3322, 1997. Huang GT, Eckmann L, Savidge TC, and Kagnoff MF. Infection of human intestinal epithelial cells with invasive bacteria upregulates apical intercellular adhesion molecule-1 (ICAM-1) expression and neutrophil adhesion. J Clin Invest 98: 572–583, 1996. Iwasaki A and Kelsall BL. Localization of distinct Peyer’s patch dendritic cell subsets and their recruitment by chemokines macrophage inflammatory protein (MIP)-3␣, MIP-3, and secondary lymphoid organ chemokine. J Exp Med 191: 1381–1393, 2000. Jobin C, Holt L, Bradham CA, Streetz K, Brenner DA, and Sartor RB. TNF receptor-associated factor-2 is involved in both IL-1 and TNF-␣ signaling cascades leading to NF-B activation and IL-8 expression in human intestinal epithelial cells. J Immunol 162: 4447–4454, 1999. Jobin C, Panja A, Hellerbrand C, Iimuro Y, DiDonato J, Brenner DA, and Sartor RB. Inhibition of proinflammatory molecule production by adenovirus-mediated expression of a nuclear factor B super-repressor in human intestinal epithelial cells. J Immunol 160: 410–418, 1998. Jung HC, Eckmann L, Yang SK, Panja A, Fierer J, Morzycka-Wroblewska E, and Kagnoff MF. A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion. J Clin Invest 95: 55–65, 1995. Kagnoff MF and Eckmann L. Epithelial cells as sensors for microbial infection. J Clin Invest 100: 6–10, 1997. Laurent F, Eckmann L, Savidge TC, Morgan G, Theodos C, Naciri M, and Kagnoff MF. Cryptosporidium parvum infection of human intestinal epithelial cells induces the polarized secretion of C-X-C chemokines. Infect Immun 65: 5067–5073, 1997. Liao F, Rabin RL, Smith CS, Sharma G, Nutman TB, and Farber JM. CC-chemokine receptor 6 is expressed on diverse memory subsets of T cells and determines responsiveness to macrophage inflammatory protein 3␣. J Immunol 162: 186–194, 1999. Locati M and Murphy PM. Chemokines and chemokine receptors: biology and clinical relevance in inflammation and AIDS. Annu Rev Med 50: 425–440, 1999. Mantovani A. The chemokine system: redundancy for robust outputs. Immunol Today 20: 254–257, 1999. O’Neil DA, Porter EM, Elewaut D, Anderson GM, Eckmann L, Ganz T, and Kagnoff MF. Expression and regulation of the human -defensins hBD-1 and hBD-2 in intestinal epithelium. J Immunol 163: 6718–6724, 1999. Power CA, Church DJ, Meyer A, Alouani S, Proudfoot AE, Clark-Lewis I, Sozzani S, Mantovani A, and Wells TN. Cloning and characterization of a specific receptor for the novel CC chemokine MIP-3␣ from lung dendritic cells. J Exp Med 186: 825–835, 1997. Reinecker HC, Loh EY, Ringler DJ, Mehta A, Rombeau JL, and MacDermott RP. Monocyte-chemoattractant protein 1 G719