Regulated MIP-3alpha /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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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)-3alpha /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-3alpha by human intestinal epithelium. Several intestinal epithelial cell lines were shown to constitutively express MIP-3alpha mRNA. Moreover, MIP-3alpha mRNA expression and protein production were upregulated by stimulation of intestinal epithelial cells with the proinflammatory cytokines tumor necrosis factor-alpha or interleukin-1alpha or in response to infection with the enteric bacterial pathogens Salmonella or enteroinvasive Escherichia coli. In addition, MIP-3alpha was shown to function as a nuclear factor-kappa B target gene. In vitro findings were paralleled in vivo by increased expression of MIP-3alpha 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-3alpha . The constitutive and regulated expression of MIP-3alpha by human intestinal epithelium is consistent with a role for epithelial cell-produced MIP-3alpha in modulating mucosal adaptive immune responses.

chemokines; dendritic cells; infectious immunity; inflammation; T lymphocytes


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE EPITHELIAL CELL LINING 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 inflammatory cell types. Thus, after stimulation with proinflammatory mediators such as tumor necrosis factor (TNF)-alpha or interleukin (IL)-1alpha 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)-kappa B (13, 19, 20, 32). In this regard, NF-kappa 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)-gamma , 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 amino-terminal cysteines. In CC chemokines, two amino-terminal 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)-3alpha /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-3alpha 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-3alpha 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-3beta . 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 alpha 4beta 7 integrin characteristic of mucosal homing lymphocytes in humans (3) also coexpress CCR6 (24), suggesting that MIP-3alpha may also be important for chemoattracting mucosal T cells.

The orthologue of human MIP-3alpha 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-3alpha , mLARC is not expressed in liver or lung (38), and mLARC differs functionally from human MIP-3alpha in that mLARC, but not human MIP-3alpha , is chemotactic for CCR6-expressing B cells (24). mRNA for MIP-3alpha 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-3alpha 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-3alpha , a chemokine capable of signaling immature DCs and CD45RO+ T cells. The studies herein describe the constitutive and regulated expression and production of MIP-3alpha 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-3alpha .


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. Recombinant human (rh)IL-1alpha , IFN-gamma , MIP-3alpha , and TNF-alpha were from PeproTech (Rocky Hill, NJ). Biotin-conjugated affinity-purified goat anti-human MIP-3alpha , murine monoclonal antibody (MAb) to human CCR6 (clone 53103.111), and murine MAb to human MIP-3alpha (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 anti-rabbit 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-µm pore size; Costar) were stimulated with TNF-alpha (20 ng/ml), IL-1alpha (20 ng/ml), or IFN-gamma (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 µg/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 µg of human IL-1alpha in 200 µl of PBS or with 200 µl 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-µl 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 Ikappa Balpha -AA superrepressor (Ad5Ikappa B-A32/36) or the E. coli beta -galactosidase gene (Ad5LacZ) was constructed as described previously (13, 20). Ad5Ikappa B-A32/36 expresses a hemagglutinin (HA) epitope-tagged mutant form of Ikappa Balpha in which serine residues 32 and 36 are replaced by alanine residues. The mutant Ikappa Balpha cannot be phosphorylated at positions 32 and 36 and acts as a superrepressor of NF-kappa 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 Ad5Ikappa B-A32/36 or Ad5LacZ in serum-free medium at a MOI of 100 for 16 h as described previously (13, 27). At this MOI, Ad5Ikappa B-A32/36 or Ad5LacZ infected >80% of HT-29 cells and infected cells expressed Ikappa Balpha -A32/36 and beta -galactosidase, respectively, at high levels as assessed by staining for beta -galactosidase and immunostaining for HA-tagged Ikappa Balpha -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-1alpha or TNF-alpha stimulation. The Ikappa Balpha -AA superrepressor inhibited NF-kappa 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 beta -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 µg of total cellular RNA was reverse transcribed and cDNA was amplified as described previously (21). The primers for human MIP-3alpha (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-3beta (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 µg/ml phytohemagglutinin-P and 2 µg/ml lipopolysaccharide (E. coli O111:B4) were used as a source of cDNA for positive controls. The MIP-3alpha 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-3alpha diluted in carbonate buffer as the capture antibody. Affinity-purified biotinylated goat anti-human MIP-3alpha 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-3alpha concentration was calculated from a standard curve using rhMIP-3alpha . 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 inflammatory 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 µm) were prepared, fixed in acetone for 10 min, and blocked in PBS-1% BSA for 1 h at room temperature. For MIP-3alpha and CCR6 immunostaining, sections were incubated overnight at 4°C with 5 µg/ml mouse MAb to human MIP-3alpha or control mouse IgG1 or 5 µg/ml mouse MAb to human CCR6 or control mouse IgG2b, and sections were subsequently stained with Cy3-conjugated goat anti-mouse IgG antibody. The isotype-matched control antibodies were used at the same concentrations as the MIP-3alpha 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MIP-3alpha expression by epithelium of normal and inflamed adult human colon. MIP-3alpha 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-3alpha production in human colon, sections of normal and inflamed human colon were immunostained for MIP-3alpha . As shown in Fig. 1B, MIP-3alpha is minimally expressed by normal colon epithelium. In contrast, epithelial MIP-3alpha immunostaining was markedly increased in sections of inflamed human colon and the epithelium was the major site of MIP-3alpha production (Fig. 1A). No immunostaining was seen in sections of inflamed human colon incubated with an isotype-matched control antibody.


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Fig. 1.   Epithelial macrophage inflammatory protein (MIP)-3alpha immunostaining of normal and inflamed human colon. Sections of inflamed (A) and normal (B) human colon were immunostained for MIP-3alpha . Adjacent sections from inflamed (C) and normal (D) human colon were stained with hematoxylin and eosin. As shown, MIP-3alpha 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.

Constitutive and regulated MIP-3alpha mRNA expression in human intestinal epithelial cell lines. To better characterize the expression of MIP-3alpha 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-3alpha mRNA. In contrast, none of the cell lines expressed mRNA for MIP-3beta , a chemoattractant for CCR7-expressing mature DCs. As shown in Fig. 2B, MIP-3alpha mRNA levels in HT-29 and Caco-2 cells were upregulated after stimulation with the proinflammatory mediators TNF-alpha or IL-1alpha , which are cytokines produced by mononuclear cells in the intestinal mucosa during the course of mucosal inflammation. MIP-3alpha mRNA levels were not upregulated by stimulation of the human colon epithelial cell lines with IFN-gamma , granulocyte-macrophage colony-stimulating factor (GM-CSF), or IL-4 (data not shown). None of the cytokines tested (i.e., TNF-alpha , IL-1alpha , IFN-gamma , GM-CSF, or IL-4) upregulated expression of the mature DC chemoattractant MIP-3beta (data not shown).


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Fig. 2.   Constitutive and regulated expression of MIP-3alpha mRNA by human intestinal epithelial cell lines. A: total RNA was isolated from the indicated cell lines, and MIP-3alpha and MIP-3beta 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)-alpha and interleukin (IL)-1alpha was amplified as in A.

Regulated production of MIP-3alpha by human intestinal epithelial cell lines. We next assessed whether the constitutive and regulated expression of MIP-3alpha mRNA by the epithelial cell lines was paralleled by protein production, using an ELISA to measure secreted levels of MIP-3alpha . 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-3alpha , respectively, as determined after 18-h incubation. Stimulation of those cells with IL-1alpha or TNF-alpha markedly increased MIP-3alpha secretion within 3 h, with MIP-3alpha 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-gamma , GM-CSF, or IL-4 did not increase MIP-3alpha secretion (data not shown). Consistent with the data for T84, the cell lines HCA-7 and HCT-8, which did not constitutively express MIP-3alpha mRNA, could also be induced to upregulate the production of MIP-3alpha protein in response to IL-1alpha or TNF-alpha stimulation (data not shown).


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Fig. 3.   Secretion of MIP-3alpha by HT-29, Caco-2, and T84 cells stimulated with TNF-alpha or IL-1alpha . Confluent HT-29 (A), Caco-2 (B), or T84 (C) cells were stimulated with TNF-alpha (open circle ) or IL-1alpha (black-down-triangle ) for 3-18 h, whereas control cultures () remained unstimulated. MIP-3alpha in culture supernatants was assayed by ELISA. Results are means ± SE of 3-5 repeated experiments.

The human intestinal epithelium is polarized into phenotypically and functionally distinct apical and basolateral domains. For MIP-3alpha 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-3alpha 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-1alpha 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-3alpha into the basal rather than the apical Transwell chamber. Thus MIP-3alpha secretion in the IL-1alpha -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-3alpha 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-3alpha secretion increased as much as 20- to 120-fold in bacteria-infected cultures.

                              
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Table 1.   MIP-3alpha secretion in HT-29 and T84 cells infected with Salmonella dublin or Escherichia coli O29:NM

Ikappa Balpha superrepressor blocks inducible MIP-3alpha 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-alpha or IL-1alpha , are NF-kappa B target genes (13, 20), although the role, if any, of NF-kappa B in the transcriptional regulation of the CC chemokine MIP-3alpha has not previously been reported. To determine whether MIP-3alpha in intestinal epithelial cells functions as an NF-kappa B target gene, we first assessed whether blocking NF-kappa B activation with a proteasome inhibitor, MG-132, decreased MIP-3alpha secretion in response to TNF-alpha or IL-1alpha stimulation. Treatment of HT-29 cells with 50 µM of MG-132 before stimulation with TNF-alpha decreased MIP-3alpha 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-kappa B activation. In this approach, cells were infected with a recombinant adenovirus expressing a mutant Ikappa Balpha protein that has serine-to-alanine substitutions at positions 32 and 36 (Ad5Ikappa B-A32/36). Ad5Ikappa B-A32/36 acts as a superrepressor of NF-kappa B activation by preventing signal-induced Ikappa Balpha phosphorylation (13, 20). After infection with this recombinant adenovirus, HT-29 cells were stimulated with TNF-alpha or IL-1alpha or infected with S. dublin or enteroinvasive E. coli. As shown in Table 2, MIP-3alpha secretion was markedly inhibited in agonist-stimulated or bacteria-infected HT-29 cells infected with recombinant adenovirus expressing the superrepressor but not in cells infected with the same recombinant adenovirus expressing the LacZ gene (Ad5LacZ).

                              
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Table 2.   MIP-3alpha is a NF-kappa B target gene

MIP-3alpha expression is upregulated in human intestinal xenografts in response to IL-1alpha stimulation or Salmonella infection. To determine whether regulated MIP-3alpha expression by the human intestinal epithelial cell lines was paralleled in vivo, expression of MIP-3alpha 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-3alpha production by normal human intestinal epithelium. For these studies, we first assessed MIP-3alpha mRNA expression in xenografts after intraperitoneal injection of SCID mice with human IL-1alpha . As shown in Fig. 4, MIP-3alpha mRNA expression increased in the xenografts of IL-1alpha -injected mice, although constitutive background levels of MIP-3alpha varied from xenograft to xenograft. MIP-3alpha mRNA expression was also increased in xenografts infected with an aroA aroC mutant of S. typhi. We note that IL-1alpha 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.


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Fig. 4.   Expression of MIP-3alpha 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-3alpha by the xenografts varied from experiment to experiment, MIP-3alpha expression was consistently upregulated in the xenografts in response to cytokine stimulation or bacterial infection.

To show that increased chemokine mRNA expression was accompanied by epithelial protein production, adjacent segments of the intestinal xenografts were immunostained for MIP-3alpha . As shown in Fig. 5, there was little MIP-3alpha immunostaining in unstimulated xenografts but a marked increase in epithelial cell MIP-3alpha immunostaining that was localized in the intestinal epithelium of the human xenografts from IL-1alpha -injected mice. The relatively low level of epithelial MIP-3alpha immunostaining in unstimulated xenografts is consistent with the minimal constitutive expression seen in normal human colon (compare with Fig. 1B).


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Fig. 5.   Epithelial cell expression of MIP-3alpha in response to IL-1alpha stimulation of human intestinal xenografts. Sections of human intestinal xenografts from SCID mice injected with human IL-1alpha (A and B) were immunostained for MIP-3alpha (A) or stained with Hoechst dye 33258 to reveal nuclear morphology (B). Note that MIP-3alpha 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-1alpha before xenograft harvesting. Adjacent sections stained with isotype-matched control mouse IgG1 revealed no immunostaining (data not shown). Original magnification ×400.

CCR6, the cognate receptor for MIP-3alpha , 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-3alpha , we sought to determine whether cells in human colon mucosa express CCR6. As shown in Fig. 6A, cells within the lamina propria and a lymphoid 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-3alpha 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).


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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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We report here that the CC chemokine MIP-3alpha is widely expressed and regulated in human intestinal epithelium. Moreover, epithelial expression of MIP-3alpha is upregulated by stimulation with the proinflammatory cytokines IL-1alpha and TNF-alpha , 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-3alpha 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-3alpha 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 follicle-associated 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-3alpha in response to TNF-alpha or IL-1 stimulation of human intestinal epithelial cells contrasts with the lack of response of MIP-3alpha production in J774 mouse monocytes to those stimuli (38), indicating that there may be differences in the signal transduction pathways important for activating MIP-3alpha in different cell types and/or species.

Our finding of upregulated epithelial cell MIP-3alpha 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-1alpha , MIP-1beta , RANTES, and MIP-3alpha ) (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-3beta , 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-3beta . In addition to high-level MIP-3alpha production after stimulation, it is possible that constitutive low levels of epithelial expression of MIP-3alpha 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-3alpha is shown to function as a NF-kappa B target gene in human intestinal epithelial cells, and its expression is upregulated by TNF-alpha or IL-1alpha stimulation or infection with enteric bacteria. The same is true of IL-8 and growth-related protein-alpha (GRO-alpha ) that, like MIP-3alpha , are upregulated in the intestinal epithelium by IL-1 or TNF-alpha stimulation or infection with enteric bacterial pathogens (13, 19, 22, 42). However, MIP-3alpha mainly signals immature DC and CD45RO+ T cells important in host adaptive immune responses, whereas IL-8 and GRO-alpha 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-3alpha , are known to signal activated/memory CD4+ CD45RO+ T cells that express the receptor CXCR3. Unlike MIP-3alpha , those chemokines are preferentially upregulated by the Th1 cytokine IFN-gamma and are only minimally, if at all, regulated by TNF-alpha , IL-1, or bacterial infection in the absence of IFN-gamma . 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-3alpha in response to IL-1alpha stimulation. It is not currently possible to determine the effective concentrations of MIP-3alpha in the microenvironment of the intestinal mucosa because this depends on several factors, including the biological half-life of MIP-3alpha 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-3alpha secreted by human intestinal epithelial cell lines is within the range shown in vitro to be chemotactic for lymphocytes. Thus 1 µg/ml recombinant MIP-3alpha 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-3alpha 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-3alpha 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-3alpha (18). However, we also note that CCR6, the cognate receptor for MIP-3alpha , 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-1alpha , MIP-1beta , and RANTES. Like CXCR4 and CCR5, CCR6 on intestinal epithelial cells acts as a functional signaling receptor after interaction with its ligand MIP-3alpha (unpublished data). Thus, in addition to paracrine effects on DCs and T cells, MIP-3alpha , like several other mediators produced by intestinal epithelial cells, may mediate autocrine effects on the intestinal epithelium. In contrast to MIP-3alpha , 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 cell-produced ligand, the antimicrobial peptide human beta  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-3alpha 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-3alpha (41), hBD-2 produced by intestinal epithelium may not compete effectively with MIP-3alpha for binding to CCR6 on immature DC or mucosal T cells or to CCR6 expressed by intestinal epithelium.


    ACKNOWLEDGEMENTS

We thank J. Leopard and D. McCole for helpful assistance and R. Lara for final preparation of the manuscript.


    FOOTNOTES

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.

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.

Received 13 July 2000; accepted in final form 19 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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