AJP - GI Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 287: G1062-G1069, 2004. First published July 8, 2004; doi:10.1152/ajpgi.00162.2004
0193-1857/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/5/G1062    most recent
00162.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ogawa, H.
Right arrow Articles by Kagnoff, M. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ogawa, H.
Right arrow Articles by Kagnoff, M. F.

INFLAMMATION/IMMUNITY/MEDIATORS

Regulated production of the chemokine CCL28 in human colon epithelium

Hiroyuki Ogawa,1,3 Mitsutoshi Iimura,1,3 Lars Eckmann,1 and Martin F. Kagnoff1,2,3

1Department of Medicine, 2Department of Pediatrics, and the 3Laboratory of Mucosal Immunology, University of California at San Diego, La Jolla, California 92903

Submitted 12 April 2004 ; accepted in final form 30 June 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The chemokine CCL28 is constitutively expressed by epithelial cells at several mucosal sites and is thought to function as a homeostatic chemoattractant of subpopulations of T cells and IgA B cells and to mediate antimicrobial activity. We report herein on the regulation of CCL28 in human colon epithelium by the proinflammatory cytokine IL-1, bacterial flagellin, and n-butyrate, a product of microbial metabolism. In vivo, CCL28 was markedly increased in the epithelium of pathologically inflamed compared with normal human colon. Human colon and small intestinal xenografts were used to model human intestinal epithelium in vivo. Xenografts constitutively expressed little, if any, CCL28 mRNA or protein. After stimulation with the proinflammatory cytokine IL-1, CCL28 mRNA and protein were significantly increased in the epithelium of colon but not small intestinal xenografts, although both upregulated the expression of another prototypic chemokine, CXCL8, in response to the identical stimulus. In studies of CCL28 regulation using human colon epithelial cell lines, proinflammatory stimuli, including IL-1, bacterial flagellin, and bacterial infection, significantly upregulated CCL28 mRNA expression and protein production. In addition, CCL28 mRNA expression and protein secretion by those cells were significantly increased by the short-chain fatty acid n-butyrate, and IL-1- or flagellin-stimulated upregulation of CCL28 by colon epithelial cells was synergistically increased by pretreatment of cells with n-butyrate. Consistent with its upregulated expression by proinflammatory stimuli, CCL28 mRNA expression was attenuated by pharmacological inhibitors of NF-{kappa}B activation. These findings indicate that CCL28 functions as an "inflammatory" chemokine in human colon epithelium and suggest the notion that CCL28 may act to counterregulate colonic inflammation.

human intestinal xenografts; HCA7 cells; butyrate; interleukin-1; Salmonella; flagellin

INTESTINAL EPITHELIAL CELLS produce mediators that can signal the chemoattraction of leukocyte populations important in mucosal innate and acquired immunity. For example, in response to stimulation with the proinflammatory mediators IL-1{alpha} or TNF-{alpha} or following infection with enteric pathogens, intestinal epithelial cells activate a group of proinflammatory genes, many of which are targets of the transcription factor NF-{kappa}B (6, 13, 20–22, 39). The epithelial cell products of these genes can, in turn, activate the mucosal influx of neutrophils, monocytes, and dendritic cells (11, 20, 23, 35, 42).

Chemokines are low-molecular-weight chemotactic cytokines that play a key role in leukocyte trafficking (4, 5, 32) and can be categorized into subfamilies based on the number and spacing of the NH2-terminal cysteines (2, 5). For example, in the C-C chemokine subfamily, the first two cysteines are adjacent (3). Chemokines are also classified based on functional criteria into those that are constitutively expressed (variably termed constitutive, homeostatic, or homing chemokines) and generally play a role in the homeostatic migration of leukocytes into and through different tissue compartments, e.g., CXCL13 (BCA-1), CXCL12 (SDF-1), CCL25 (TECK) (4, 25, 26, 32), and inducible chemokines (also termed inflammatory chemokines) that are produced in response to microbial, inflammatory, and immune signals, e.g., CXCL8 (IL-8), CXCL1 (GRO{alpha}), CCL2 (MCP-1), CXCL10 (IP-10) (3, 4, 32).

CCL28 (also termed mucosal epithelial chemokine or MEC) is a member of the C-C chemokine subfamily and is constitutively expressed in different mucosal sites, including salivary and mammary glands, the trachea, as well as in the colon, and to a substantially lesser extent in the small intestine (34, 40). CCL28 has been categorized as a constitutive chemokine important in homeostatic lymphocyte trafficking (25, 26). The cognate receptor for CCL28 is CCR10 (40).

CCL28 can chemoattract populations of CD4 and CD8 T cells that express CCR10 (40). However, in the intestinal mucosa, few if any T cells are reported to express CCR10 (28), suggesting that CCL28 may play another role in that site. Relative to other B cell populations, CCR10 appears to be expressed selectively by IgA plasmablasts and IgA-secreting cells (i.e., plasma cells) (28, 31). Such studies indicate a possible role for CCL28 as a chemoattractant of IgA-committed B cells in the intestinal mucosa. As the most abundant isotype in human intestinal secretions, IgA is thought to increase intestinal mucosal defense through binding and preventing the mucosal entry of microbes, toxins, and other antigens and by neutralizing viruses (29). IgA can also play a direct anti-inflammatory role by preventing bacterial LPS-induced NF-{kappa}B activation in epithelial cells (14), by neutralizing viruses within intestinal epithelial cells (7), and possibly by excreting antigens from the subepithelial mucosal compartment across the epithelium into the intestinal lumen (24, 37). To the extent that intestinal epithelial cell production of CCL28 influences mucosal IgA B cell migration and, similar to several other chemokines, mediates antimicrobial activity (8, 18), CCL28 could contribute to the regulation of host mucosal defense and inflammation.

The proposed functional activities of CCL28 as a chemoattractant and antimicrobial protein led us to hypothesize that the production of CCL28 in human colon might not simply be constitutive, but might also manifest a more novel regulation compared with other intestinal epithelial chemokines. We report herein that CCL28 is significantly upregulated in inflamed colon epithelium, and its production is upregulated in human colon epithelial cells by proinflammatory stimuli alone or in combination with n-butryate, a product of bacterial metabolism. Moreover, CCL28 expression is regulated, in part, by the transcription factor NF-{kappa}B.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. Recombinant human (rh) IL-1{alpha}, IL-6, IFN-{gamma}, and TNF-{alpha} were from Peprotech (Rocky Hill, NJ). Mouse IgG1 and sodium butyrate were from Sigma (St. Louis, MO). rhCCL28, biotin-conjugated affinity-purified goat anti-human CCL28, and murine MAb to human CCL28 (clone 62705) were from R&D Systems (Minneapolis, MN). Cy3-conjugated goat anti-mouse IgG was from Jackson ImmunoResearch Laboratories (West Grove, PA). BAY 11–7082 and N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) were from Calbiochem (La Jolla, CA). Purified H7 flagellin from Escherichia coli serotype O157:H7 strain 86–24 was a gift of Y. Miyomoto (University of California San Diego).

Human colon epithelial cell lines. The human colon cell lines HCA-7, HT-29, HCT-8, and Caco-2, and the intestinal epithelial cell line I-407 were grown in DMEM supplemented with 10 or 15% (Caco-2) heat-inactivated FBS (9). HT-29.19A cells were grown in McCoy's 5A medium with 10% FBS, and T84 cells were grown in 50% DMEM/50% Ham's F-12 medium with 5% newborn calf serum (23). Media were supplemented with 2 mM L-glutamine. Cells were maintained in 95% air-5% CO2 at 37°C.

Cell stimulation and infection. Cells in six-well tissue culture plates were stimulated with IL-1{alpha}, IL-6, and TNF-{alpha} (each at 20 ng/ml) or IFN-{gamma} (40 ng/ml). For bacterial infections, cells were cultured in serum-free DMEM with 1 x 108 colony-forming units of Salmonella serovar Dublin for 1 h, after which the bacteria were removed by washing and gentamicin (50 µg/ml) was added to kill any remaining extracellular bacteria (11, 23). To inhibit NF-{kappa}B activation, cells were preincubated with 10 µM BAY 11–7082 or 10 µM of the calpain inhibitor ALLN for 45 min before the addition of IL-1{alpha}.

RNA extraction and RT-PCR analysis. Total cellular RNA was extracted using an acid guanidinium-phenol-chloroform method (TRIzol reagent; GIBCO-BRL Life Technologies, Grand Island, NY). Reverse transcription and PCR amplification were performed as described before (16). Primers for CCL28 were designed from sequences available from GenBank (AF220210); sense 5'-AGC CAT ACT TCC CAT TGC CTC C-3' and antisense 5'-GCC CTG TTA CTG TTC CTC TTG CC-3' and yielded a 276-bp product. Primers for IL-8 and {beta}-actin were as described before (9, 23). After a hot start, the amplification profile was 45 s of denaturation at 95°C and 2.5 min of annealing and extension at 62°C for 32 cycles for CCL28, 2 min of annealing and extension at 60°C for 30 cycles for IL-8, and 2 min of annealing and extension at 72°C for 28 cycles for {beta}-actin. Negative control reactions contained no added RNA in the RT reaction and no added cDNA in the PCR amplification. Positive control reactions used RNA from peripheral blood mononuclear cells.

Real-time PCR. Real-time PCR was performed using an ABI Prism 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA). Each reaction mixture contained 12.5 µl 2x SYBRgreen Master Mix (containing 200 µM each of dATP, dGTP, and dCTP, 400 µM dUTP, 2 mM MgCl2, 0.125 U uracil N-glycosylase, and 0.313 U Amplitaq Gold DNA polymerase; PE Biosystems), 6.25 pmol each of sense and antisense primer, and 1 µl cDNA in a 25-µl final volume. The CCL28 primers were as described above. Primer sequences for IL-8 were sense 5'-ATG ACT TCC AAG CTG GCC GTG GCT-3' and antisense 5'-TCT CAG CCC TCT TCA AAA ACT TCT C-3'. The {beta}-actin primers were sense 5'-CAA AGA CCT GTA CGC CAA CAC-3' and antisense 5'-CAT ACT CCT GCT TGC TGA TCC-3'. Reaction mixtures were incubated at 50°C for 2 min followed by 95°C for 10 min to activate Amplitaq Gold DNA polymerase after which the amplification profile was 40 cycles of 15 s of denaturation at 95°C and 1 min of annealing at 60°C for CCL28 and {beta}-actin. Amplification of the expected single products was confirmed on 1% agarose gels stained with ethidium bromide. Data analysis was done using Sequence Detection System software (PE Biosystems) provided by the manufacturer as described before (6, 16). Fold changes in CCL28 mRNA expression were determined as 2{Delta}Ct, where {Delta}Ct = (CtCCL28 control – CtCCL28 stimulated) – (Ct{beta}-actin control – Ct{beta}-actin stimulated) (6, 16).

Human intestinal xenografts. Human colon and small intestinal xenografts were generated as described before (12, 16, 20). Briefly, human fetal colon or small intestine (Advanced Biosciences Resources, Alameda, CA) of 16- to 20-wk gestational age was transplanted subcutaneously onto the backs of C57BL/6 severe combined immunodeficient (SCID) mice and allowed to develop for at least 10 wk (12, 16, 20, 30, 38). Mice with mature xenografts were injected intraperitoneally with 1 µg human IL-1{alpha} in 200 µl of PBS or PBSalone as a control. Colon or small intestinal xenografts were removed 5 h later, and adjacent segments were frozen in liquid nitrogen for RNA isolation, or for immunohistochemical analysis, they were embedded in optimal cutting temperature compound (TissueTek, Torrance, CA) and frozen in isopentane-dry ice or fixed in 10% neutral buffered formalin. All studies in this report using human and animal subjects were approved by the University of California, San Diego Human and Animal Subjects Committees.

Immunohistochemistry. Cultured epithelial cells were detached with 0.25% trypsin/0.1 mM EDTA, washed in serum-free DMEM, and resuspended in PBS. Aliquots were cytocentrifuged onto positively charged glass slides, which were air dried and fixed in acetone for 10 min at –20°C. For immunostaining, cells were rehydrated in PBS, blocked for 1 h with 1% BSA in PBS, and incubated overnight at 4°C with 10 µg/ml mouse MAb to human CCL28 or control mouse IgG1. Cy3-conjugated goat anti-mouse antibody was added as secondary antibody at a 1:200 dilution for 1 h. Nuclei were counterstained with Hoechst 33258 dye (100 ng/ml).

Cryostat sections (5 µm) were prepared from human intestinal xenografts and from human colon biopsies obtained at colonoscopy from three normal individuals, five patients with ulcerative colitis, and two subjects with colon inflammation not due to ulcerative colitis (1 with Crohn's disease and 1 with inflammation in an area adjacent to a colon cancer). Sections were fixed in ice-cold acetone for 10 min, blocked in PBS-1% BSA with 10% goat serum for 1 h at room temperature, and immunostained for CCL28.

CCL28 enzyme-linked immunospot assay. 96-well polyvinylidene difluoride membrane plates (MAIP S4510, Millipore, Bedford, MA) were coated overnight at 4°C with 50 µl of 5 µg/ml mouse anti-human CCL28 MAb or isotype-matched control mouse mIgG1 MAb in carbonate buffer, pH 9.3 (15 mM Na2CO3, 35 mM NaHCO3), washed with PBS, and then incubated for 1 h at 37°C with 50 µM {beta}-mercaptoethanol, 2 mg/ml NaHCO3, 10% FCS, 2 mM L-glutamine, and 50 µg/ml gentamicin in 100 µl DMEM to block remaining free binding sites. After being washed in PBS, HCA-7 cells were added (1 x 105/well) and incubated for 24 h in 95% air-5% CO2 at 37°C, after which IL-1{alpha} (20 ng/ml) and n-butyrate (2 mM) or IL-1{alpha} plus n-butyrate were added and the plates were further incubated for an additional 24 h. Wells were washed with PBS plus 0.1% Tween 20 (PBST) and then incubated with 50 µl of biotin-conjugated affinity-purified goat anti-human CCL28 (0.5 µg/ml) in PBST containing 1% BSA overnight at 4°C. Reactions were developed using streptavidin-alkaline phosphatase (Roche, Indianapolis, IN; 100 µl of a 1:2,000 dilution in PBST containing 1% BSA) for 1 h at room temperature followed by 1 h incubation at room temperature with 100 µl of a 1:50 dilution of Nitro blue tetrazolium chloride/5-Bromo-4-chloro-3-indolyl phosphate (Roche) in 100 mM Tris buffer, pH 9.5, and 50 mM MgSO4. Plates were air-dried overnight and photographed.

ELISA. HCA-7 cell supernatants were assayed for CCL28 using a solid-phase ELISA (Quantikine, R&D systems). The ELISA was sensitive to 10 pg/ml.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Increased CCL28 expression in inflamed adult human colon. CCL28 mRNA is constitutively expressed in the human colon and in several other mucosal sites leading to the concept that this chemokine controls the homeostatic trafficking of lymphocytes, which express its cognate receptor, to and within those sites (34, 40). As a "homeostatic" chemokine, little to no attention has been focused on its possible regulated expression. During the course of characterizing chemokines produced by human colon epithelium, we made the novel finding that CCL28 was markedly increased in the epithelium of mucosal biopsies from inflamed compared with normal colon (Fig. 1). This suggested that CCL28 production is not strictly constitutive in human colon epithelium, but rather, it is subject to regulation by proinflammatory stimuli.



View larger version (49K):
[in this window]
[in a new window]
  		Fig. 1. CCL28 immunostaining of normal and inflamed human colon. A–C: sections of normal human colon. D–F: inflamed colon from a patient with ulcerative colitis. G–I: inflamed colon adjacent to a colon cancer. A, D, and G were immunostained for CCL28, and C, F, and I are adjacent sections stained with hematoxylin and eosin (H&E). CCL28 immunostaining was most marked in the inflamed colon (D, G) and restricted to epithelial cells. No immunostaining was seen in sections stained with the isotype-matched control antibody at same concentration (B, E, I). Original magnification, x200. Similar results with no or minimal immunostaining for CCL28 were noted in normal colon from 3 additional healthy subjects. Ulcerative colitis is shown as a model of intestinal inflammation, and each of 5 patients examined with that disease had a marked increase in colon epithelial CCL28 similar to that shown, although such increases are not specific for ulcerative colitis and were also seen in other causes of colon inflammation as shown and in Crohn's colitis.

 
Regulated CCL28 expression in human colon xenografts. To further define the regulated expression of CCL28 in intact human epithelium in vivo, we used a human intestinal xenograft model (12, 38). In this model, human fetal colon or small intestine is transplanted subcutaneously onto the backs of SCID mice and allowed to mature for 10 wk. After maturation, these xenografts contain an epithelium that is strictly of human origin (38). CCL28 mRNA was constitutively expressed at low levels in human colon xenografts, and little, if at all, by small intestinal xenografts (Fig. 2A). To determine whether CCL28 mRNA expression in the xenografts was upregulated by stimulation with a proinflammatory mediator, mice carrying either colon or small intestinal xenografts were injected with rhIL-1{alpha} or, as a control, PBS. IL-1{alpha} resulted in a marked upregulation of CCL28 mRNA in colon but not small intestinal xenografts as assessed by real-time PCR (Fig. 2B). In contrast, human IL-8, which was assayed as a control inflammatory chemokine, was not constitutively expressed by the xenografts but was upregulated in both colon and small intestinal xenografts following IL-1{alpha} injection (Fig. 2, A and B). Consistent with the low levels of constitutively expressed CCL28 mRNA in xenografts and upregulated expression after IL-1 injection, we found little, if any, CCL28 immunostaining in unstimulated xenografts but a marked increase in CCL28 immunostaining that was strictly localized to the colon epithelium in xenografts from IL-1{alpha}-injected mice (Fig. 2C).



View larger version (27K):
[in this window]
[in a new window]
  		Fig. 2. Expression of CCL28 mRNA in human intestinal xenografts. A: mice carrying human colon or small intestinal (S.I.) xenografts were injected with PBS or IL-1{alpha} (1 µg); the xenografts were harvested 5 h later, and total RNA was isolated from the colon xenografts. CCL28, IL-8, and {beta}-actin transcripts were amplified by RT-PCR. B: CCL28 and IL-8 mRNA levels were assessed by real-time PCR in human colon and small intestinal xenografts from the PBS or IL-1{alpha}-injected mice. Values are means ± SD of 3 or more xenografts under each set of conditions. C: sections of human colon xenografts from the severe combined immunodeficient mice injected with human IL-1{alpha} (D–F) or PBS (A–C) were immunostained for CCL28 (A, D), stained with an isotype-matched control mouse IgG1 (B, E), or stained with H&E (C, F). Original magnification, x200.

 
CCL28 regulation in human intestinal epithelial cell lines. To study the putative role of specific agonists in the regulated expression of CCL28, we used established human colon epithelial cell lines. Several of these lines constitutively expressed CCL28 mRNA (Fig. 3A). Because HCA-7 cells had the lowest background levels of constitutive CCL28 mRNA expression and thereby most closely resembled the low levels of CCL28 mRNA in the epithelium of human colon xenografts, HCA-7 colon epithelial cells were used for further studies of CCL28 regulation in vitro. As shown by qualitative RT-PCR in Fig. 3B, IL-1{alpha} (20 ng/ml) significantly increased CCL28 mRNA expression in those cells and, to a lesser extent, in two other cell lines tested (Caco-2, HT-29; not shown). In contrast, CCL28 mRNA expression was not significantly increased by TNF-{alpha} (20 ng/ml), IFN-{gamma} (40 ng/ml; Fig. 2B), or IL-6 (20 ng/ml) stimulation. This was also the case when cells were stimulated for 6, 12, or 24 h with TNF-{alpha}, IFN-{gamma}, or IL-6 at titrated concentrations ranging from 1 to 100 ng/ml, and CCL28 mRNA levels were as assessed by real-time PCR, although those cytokines markedly upregulated the expression of other chemokines assayed as positive controls (e.g., CXCL8, CCL22, CCL10). By real-time RT-PCR, CCL28 mRNA levels increased by ~10-fold by 18 h after IL-1{alpha} stimulation (Fig. 4A), but no further increase was seen over the same time course when cells were stimulated with IL-1{alpha} in combination with IFN-{gamma} (40 ng/ml) or TNF-{alpha} (20 ng/ml), and no significant increase in CCL28 mRNA levels above background were noted when cells were stimulated with IFN-{gamma} (40 ng/ml) plus TNF-{alpha} (20 ng/ml) in combination. In further studies, HCA-7 cells were infected with the enteroinvasive bacterial pathogen S. dublin, which is another potent proinflammatory stimulus for intestinal epithelial cells (11, 13, 23). S. dublin infection increased CCL28 mRNA expression over a time course similar to that of IL-1, with maximal CCL28 mRNA levels being approximately twofold higher than those induced by IL-1 stimulation (Fig. 4A).



View larger version (43K):
[in this window]
[in a new window]
  		Fig. 3. Constitutive and regulated CCL28 mRNA expression by human intestinal epithelial cell lines. A: total RNA isolated from the indicated cell lines was amplified by RT-PCR. RNA from peripheral blood mononuclear lymphocytes (PBMC) was used as a positive control. Negative control reactions had no added RNA. B: HCA-7 cells were left unstimulated (control) or were stimulated with IFN-{gamma} (40 ng/ml), TNF-{alpha} (20 ng/ml), or IL-1{alpha} (20 ng/ml). RNA isolation and amplification methods were as in A.

 


View larger version (12K):
[in this window]
[in a new window]
  		Fig. 4. Regulated expression of CCL28 mRNA. A: HCA-7 cells were stimulated with IL-1{alpha} (20 ng/ml; {square}) or infected with S. dublin ({blacksquare}). Cells were harvested at the indicated times. CCL28 mRNA levels were assessed by real-time PCR and normalized to {beta}-actin, which was nearly constant in all groups throughout the experiments. Data are expressed as the fold change in mRNA levels in stimulated or infected cultures relative to control cultures incubated for the same duration. Values are means ± SE of 3 repeated experiments. *P < 0.05 compared with control cultures. B: HCA-7 cells were left unstimulated (control) or stimulated with IL-1{alpha} (20 ng/ml) for 18 h or with 2 mM butyrate for 24 h or were preincubated with 2 mM butyrate for 6 h, after which IL-1{alpha} (20 ng/ml) was added and cultures were incubated for an additional 18 h. CCL28 mRNA expression assessed by real-time PCR is expressed as in A. Values are means ± SD from a representative experiment. Similar data were obtained in 2 repeated experiments. *P < 0.05 compared with control; **P < 0.05 compared with butyrate or IL-1{alpha}. C: HCA-7 cells were left unstimulated (control), stimulated with IL-1{alpha} (20 ng/ml) or bacterial flagellin (100 ng/ml) for 18 h, or preincubated with 2 mM butyrate for 6 h, after which bacterial flagellin (100 ng/ml) was added and cultures were incubated for an additional 18 h. CCL28 mRNA expression was assessed as in A. Values are means ± SD from a representative experiment.

 
Butyrate is a short-chain fatty acid that is generated in the colon by bacterial digestion of complex carbohydrates. It is known to regulate the expression of several epithelial cell genes and products that are important for host innate and acquired immune defense (17, 19, 36) and to increase the differentiation of cultured human colon epithelial cell lines, including HCA-7, Caco-2, and HT-29 (16). Because initial reports noted that CCL28 mRNA was preferentially expressed in the colon, relative to the small intestine (34, 40), and we found little, if any, constitutive CCL28 expression in colon xenografts, which lack a microbial flora (16), we hypothesized that exposure to bacterial products such as butyrate may have a role in regulating epithelial CCL28 expression. HCA-7 cells were incubated with butyrate, with or without IL-1{alpha}. As shown in Fig. 3B, CCL28 expression was significantly increased when HCA-7 cells were incubated with butyrate. Moreover, pretreatment of HCA-7 cells with butyrate before IL-1 stimulation resulted in an ~100-fold synergistic increase in IL-1{alpha}-stimulated CCL28 mRNA levels (Fig. 4B).

IL-1 and bacterial flagellin share a common intracellular signaling pathway that is activated by signaling through the IL-1 receptor or human toll-like receptor 5 (1). Because CCL28 mRNA levels were markedly upregulated by stimulation with IL-1{alpha}, but not by stimulation with TNF-{alpha}, although both of these proinflammatory cytokines are potent activators of the transcription factor NF-{kappa}B, we asked whether activation of human colon epithelial cells by bacterial flagellin, like IL-1{alpha} also activates CCL28. Bacterial flagellin used at optimal concentrations (100 ng/ml) upregulated CCL28 mRNA levels, and, similar to IL-1{alpha} stimulation, CCL28 mRNA levels were synergistically increased in flagellin-stimulated cells pretreated with butyrate (Fig. 4C).

Consistent with the mRNA results, CCL28 protein was markedly increased in HCA-7 cells stimulated with IL-1{alpha} (Fig. 5A). Furthermore, another potent proinflammatory stimulus, Salmonella infection, significantly increased CCL28 secretion (Fig. 5A). This increase was further enhanced by butyrate treatment of cells subsequently stimulated with IL-1{alpha} (Fig. 5B). As shown by enzyme-linked immunospot assay (Fig. 5C), agonist stimulation resulted in both greater numbers of cells producing CCL28 as well as more CCL28 production per cell, although there was marked cell-to-cell heterogeneity.



View larger version (36K):
[in this window]
[in a new window]
  		Fig. 5. CCL28 secretion by agonist stimulated or S. dublin infected HCA-7 cells. A: HCA-7 cells were left unstimulated or stimulated with IL-1{alpha} (20 ng/ml; {square}) or infected with S. dublin ({blacksquare}). Culture supernatants were harvested at the indicated times and assayed for CCL28 by ELISA. Values are means ± SD from a representative experiment. Similar results were obtained in 2 repeated experiments. *P < 0.05 compared with unstimulated or uninfected control cultures. CCL28 secretion in unstimulated cultures was 17 pg/ml at 6 h and 25 pg/ml at 24 h (not shown). B and C: HCA-7 cells were left unstimulated (control) or were stimulated with IL-1{alpha} (20 ng/ml) for 18 h or 2 mM butyrate for 24 h or were preincubated with 2 mM butyrate for 6 h, after which IL-1{alpha} (20 ng/ml) was added and cultures were incubated for an additional 18 h. CCL28 levels in supernatants were assayed by ELISA (B), and production of CCL28 by individual cells was determined by enzyme-linked immunospot assay (C). *P < 0.05 compared with control; **P < 0.05 compared with butyrate or IL-1{alpha}.

 
Finally, we hypothesized that CCL28 expression might, in part, be regulated by the transcription factor NF-{kappa}B in light of its upregulated expression in response to IL-1{alpha} stimulation and Salmonella infection, both of which can activate NF-{kappa}B through a similar intracellular signal-transduction pathway (13). As shown in Fig. 6, IL-1{alpha}-stimulated CCL28 expression was significantly abrogated by two different pharmacological inhibitors of NF-{kappa}B activation, BAY 11–7082 and ALLN, suggesting that NF-{kappa}B has a role in regulating CCL28 expression.



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 6. Pharmacological inhibitors of NF-{kappa}B activation abrogate IL-1-stimulated upregulation of CCL28. HCA-7 cells were incubated in the presence or absence of BAY 11–7082 (10 µM) or N-acetyl-leucinyl-leucinyl-norleucinal (ALLN; 10 µM) after which IL-1{alpha} was added. Growth medium containing BAY 11–7082 and IL-1{alpha} was changed every 3 h. Cells were harvested after 12 h and CCL28 mRNA levels were determined by real-time PCR. Values are means ± SD from a representative experiment. *P < 0.05 compared with IL-1{alpha}-stimulated cultures without inhibitors. Similar results were obtained in 2 repeated experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
CCL28 has been regarded as a constitutively expressed homeostatic chemokine involved in the migration of CCR10-expressing lymphocyte populations to mucosal sites. We demonstrate here that CCL28 production by colon epithelial cells is, in fact, upregulated by proinflammatory stimuli including microbial pathogens, microbial products, and proinflammatory cytokines. However, CCL28 differs from that of several other chemokines produced by the intestinal epithelium with respect to its regulated expression and its regional distribution in the intestinal tract. Although CXCL1, CXCL8, CCL2, and CCL20 are produced by intestinal epithelial cells, are upregulated by proinflammatory stimuli, and function as NF-{kappa}B target genes (13, 20), in contrast to CCL28, they are rapidly upregulated in colon epithelial cells within a few hours by either TNF-{alpha} or IL-1 (6, 20, 23, 42). Several other chemokines produced by colon epithelial cells (e.g., CXCL9, CXCL10, CXCL11, CCL22) are upregulated by IFN-{gamma} alone or in synergy with TNF-{alpha} or IL-1 (6, 10), but this was not the case for CCL28. The upregulated expression of CCL28 in colon epithelium, but not in small intestinal epithelium, also differs from that of other inducible chemokines, which, in contrast, are abundantly produced by both colon and small intestinal epithelium (6, 20). The selective production of CCL28 by colon relative to small intestinal epithelium also differs from that of CCL25, a chemokine that governs the homeostatic trafficking of CCR9-expressing lymphocytes, and is produced by small intestinal but not colonic epithelium (27, 41). These findings, taken together, indicate that the regulation and distribution of CCL28 differ in several respects from those of previously described intestinal epithelial cell chemokines.

The short-chain fatty acid butyrate induced CCL28 production by cultured colon epithelial cells and synergistically increased colon epithelial cell CCL28 production in response to IL-1 or flagellin stimulation. The cathelicidin hCAP18/LL37 is an antimicrobial protein that also manifests chemoattractant activity. hCAP18/LL37, similar to CCL28, is preferentially produced by colonic compared with small intestinal epithelium, and we found its expression is also upregulated by butyrate (16). Unlike CCL28, however, hCAP/LL37 was abundantly expressed by the epithelium of human colon xenografts in the absence of IL-1 stimulation (16). Whereas butyrate increased IL-1-stimulated production of the neutrophil chemoattractant CXCL8 by a human colon epithelial cell line and MIP-2 secretion by a rat small intestinal epithelial cell line (15, 33), in contrast to CCL28, butyrate stimulation by itself did not substantially upregulate the production of those chemokines. Taken together, these data suggest that butyrate, or other products generated by intestinal microbes, may have a role in regulating the constitutive expression of CCL28 by colonic epithelium in vivo. We note, however, that higher concentrations of butyrate than those we used to stimulate HCA7 cells are present in the colon, yet only low levels of CCL28 were expressed by colon epithelium in vivo in the absence of inflammation. Because butyrate has multiple effects on cellular functions, the marked effects of butyrate on CCL28 expression, as revealed in the cell line studies, may, in part, be masked by its other effects in vivo. Alternatively, results obtained with butyrate stimulation of colon epithelial cell lines may not be paralleled in normal colon epithelium in vivo.

Prior studies (18, 26, 28, 31, 34, 40) considered CCL28 to be a constitutively expressed mucosal chemokine and focused on its possible role in the homeostatic trafficking of CCR10-expressing T cells and/or populations of CCR10-expressing IgA B cells and its antimicrobial activity. Those studies did not consider that its expression in sites such as the colon may be regulated. As shown in vivo and in vitro, CCL28 production can be upregulated by specific proinflammatory signals, bacterial infection, and products of bacterial metabolism. To the extent that the targets of CCL28 predictably mediate anti-inflammatory functions, our data suggest that CCL28 could have an important role in counterregulating mucosal inflammation in the colon.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-35108 and DK-58960 (to M. F. Kagnoff).

H. Ogawa was supported, in part, by a research fellowship from the Yamada Science Foundation.


   ACKNOWLEDGMENTS
 
Present address of H. Ogawa: Dept. of Molecular Medicine 2-2, Yamada-oka, Suita-city, Osaka University Medical School, Osaka, 565-0871 Japan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. F. Kagnoff, Laboratory of Mucosal Immunology, Dept. of Medicine and Pediatrics Mail Code 0623D, Univ. of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093–0623 (E-mail: mkagnoff{at}ucsd.edu)

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


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Akira S and Sato S. Toll-like receptors and their signaling mechanisms. Scand J Infect Dis 35: 555–562, 2003.[CrossRef][ISI][Medline]
  2. Bacon K, Baggiolini M, Broxmeyer H, Horuk R, Lindley I, Mantovani A, Maysushima K, Murphy P, Nomiyama H, Oppenheim J, Rot A, Schall T, Tsang M, Thorpe R, Van Damme J, Wadhwa M, Yoshie O, Zlotnik A, and Zoon K. Chemokine/chemokine receptor nomenclature. J Interferon Cytokine Res 22: 1067–1068, 2002.[CrossRef][ISI][Medline]
  3. Baggiolini M. Chemokines and leukocyte traffic. Nature 392: 565–568, 1998.[CrossRef][Medline]
  4. Baggiolini M. Chemokines in pathology and medicine. J Intern Med 250: 91–104, 2001.[CrossRef][ISI][Medline]
  5. Baggiolini M, Dewald B, and Moser B. Human chemokines: an update. Annu Rev Immunol 15: 675–705, 1997.[CrossRef][ISI][Medline]
  6. Berin MC, Dwinell MB, Eckmann L, and Kagnoff MF. Production of MDC/CCL22 by human intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 280: G1217–G1226, 2001.[Abstract/Free Full Text]
  7. Burns JW, Siadat-Pajouh M, Krishnaney AA, and Greenberg HB. Protective effect of rotavirus VP6-specific IgA monoclonal antibodies that lack neutralizing activity. Science 272: 104–107, 1996.[Abstract]
  8. Cole AM, Ganz T, Liese AM, Burdick MD, Liu L, and Strieter RM. Cutting edge: IFN-inducible ELR-CXC chemokines display defensin-like antimicrobial activity. J Immunol 167: 623–627, 2001.[Abstract/Free Full Text]
  9. Dwinell MB, Eckmann L, Leopard JD, Varki NM, and Kagnoff MF. Chemokine receptor expression by human intestinal epithelial cells. Gastroenterology 117: 359–367, 1999.[CrossRef][ISI][Medline]
  10. Dwinell MB, Lugering N, Eckmann L, and Kagnoff MF. Regulated production of interferon-inducible T-cell chemoattractants by human intestinal epithelial cells. Gastroenterology 120: 49–59, 2001.[CrossRef][ISI][Medline]
  11. 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.[Abstract/Free Full Text]
  12. 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{alpha} production. J Clin Invest 100: 296–309, 1997.[ISI][Medline]
  13. Elewaut D, DiDonato JA, Kim JM, Truong F, Eckmann L, and Kagnoff MF. NF-{kappa}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.[Abstract/Free Full Text]
  14. Fernandez MI, Pedron T, Tournebize R, Olivo-Marin JC, Sansonetti PJ, and Phalipon A. Anti-inflammatory role for intracellular dimeric immunoglobulin a by neutralization of lipopolysaccharide in epithelial cells. Immunity 18: 739–749, 2003.[CrossRef][ISI][Medline]
  15. Fusunyan RD, Quinn JJ, Ohno Y, MacDermott RP, and Sanderson IR. Butyrate enhances interleukin (IL)-8 secretion by intestinal epithelial cells in response to IL-1{beta} and lipopolysaccharide. Pediatr Res 43: 84–90, 1998.[ISI][Medline]
  16. Hase K, Eckmann L, Leopard JD, Varki N, and Kagnoff MF. Cell differentiation is a key determinant of cathelicidin LL-37/human cationic antimicrobial protein 18 expression by human colon epithelium. Infect Immun 70: 953–963, 2002.[Abstract/Free Full Text]
  17. Heerdt BG, Houston MA, and Augenlicht LH. Potentiation by specific short-chain fatty acids of differentiation and apoptosis in human colonic carcinoma cell lines. Cancer Res 54: 3288–3293, 1994.[Abstract/Free Full Text]
  18. Hieshima K, Ohtani H, Shibano M, Izawa D, Nakayama T, Kawasaki Y, Shiba F, Shiota M, Katou F, Saito T, and Yoshie O. CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity. J Immunol 170: 1452–1461, 2003.[Abstract/Free Full Text]
  19. Inan MS, Rasoulpour RJ, Yin L, Hubbard AK, Rosenberg DW, and Giardina C. The luminal short-chain fatty acid butyrate modulates NF-{kappa}B activity in a human colonic epithelial cell line. Gastroenterology 118: 724–734, 2000.[CrossRef][ISI][Medline]
  20. Izadpanah A, Dwinell MB, Eckmann L, Varki NM, and Kagnoff MF. Regulated MIP-3{alpha}/CCL20 production by human intestinal epithelium: mechanism for modulating mucosal immunity. Am J Physiol Gastrointest Liver Physiol 280: G710–G719, 2001.[Abstract/Free Full Text]
  21. Jobin C, Holt L, Bradham CA, Streetz K, Brenner DA, and Sartor RB. TNF receptor-associated factor-2 is involved in both IL-1{beta} and TNF-{alpha} signaling cascades leading to NF-{kappa}B activation and IL-8 expression in human intestinal epithelial cells. J Immunol 162: 4447–4454, 1999.[Abstract/Free Full Text]
  22. 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 {kappa}B super-repressor in human intestinal epithelial cells. J Immunol 160: 410–418, 1998.[Abstract/Free Full Text]
  23. 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.[ISI][Medline]
  24. Kaetzel CS, Robinson JK, Chintalacharuvu KR, Vaerman JP, and Lamm ME. The polymeric immunoglobulin receptor (secretory component) mediates transport of immune complexes across epithelial cells: a local defense function for IgA. Proc Natl Acad Sci USA 88: 8796–8800, 1991.[Abstract/Free Full Text]
  25. Kunkel E and Butcher E. Homeostatic chemokines and the targeting of regional immunity. Adv Exp Med Biol 512: 65–72, 2002.[ISI][Medline]
  26. Kunkel EJ and Butcher EC. Chemokines and the tissue-specific migration of lymphocytes. Immunity 16: 1–4, 2002.[CrossRef][ISI][Medline]
  27. Kunkel EJ, Campbell JJ, Haraldsen G, Pan J, Boisvert J, Roberts AI, Ebert EC, Vierra MA, Goodman SB, Genovese MC, Wardlaw AJ, Greenberg HB, Parker CM, Butcher EC, Andrew DP, and Agace WW. Lymphocyte CC chemokine receptor 9 and epithelial thymus-expressed chemokine (TECK) expression distinguish the small intestinal immune compartment: epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity. J Exp Med 192: 761–768, 2000.[Abstract/Free Full Text]
  28. Kunkel EJ, Kim CH, Lazarus NH, Vierra MA, Soler D, Bowman EP, and Butcher EC. CCR10 expression is a common feature of circulating and mucosal epithelial tissue IgA Ab-secreting cells. J Clin Invest 111: 1001–1010, 2003.[CrossRef][ISI][Medline]
  29. Lamm ME. Interaction of antigens and antibodies at mucosal surfaces. Annu Rev Microbiol 51: 311–340, 1997.[CrossRef][ISI][Medline]
  30. 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.[Abstract]
  31. Lazarus NH, Kunkel EJ, Johnston B, Wilson E, Youngman KR, and Butcher EC. A common mucosal chemokine (mucosae-associated epithelial chemokine/CCL28) selectively attracts IgA plasmablasts. J Immunol 170: 3799–3805, 2003.[Abstract/Free Full Text]
  32. Mantovani A. The chemokine system: redundancy for robust outputs. Immunol Today 20: 254–257, 1999.[CrossRef][ISI][Medline]
  33. Ohno Y, Lee J, Fusunyan RD, MacDermott RP, and Sanderson IR. Macrophage inflammatory protein-2: chromosomal regulation in rat small intestinal epithelial cells. Proc Natl Acad Sci USA 94: 10279–10284, 1997.[Abstract/Free Full Text]
  34. Pan J, Kunkel EJ, Gosslar U, Lazarus N, Langdon P, Broadwell K, Vierra MA, Genovese MC, Butcher EC, and Soler D. A novel chemokine ligand for CCR10 and CCR3 expressed by epithelial cells in mucosal tissues. J Immunol 165: 2943–2949, 2000.[Abstract/Free Full Text]
  35. Reinecker HC, Loh EY, Ringler DJ, Mehta A, Rombeau JL, and MacDermott RP. Monocyte-chemoattractant protein 1 gene expression in intestinal epithelial cells and inflammatory bowel disease mucosa. Gastroenterology 108: 40–50, 1995.[CrossRef][ISI][Medline]
  36. Rickard KL, Gibson PR, Wilson NJ, Mariadason JM, and Phillips WA. Short-chain fatty acids reduce expression of specific protein kinase C isoforms in human colonic epithelial cells. J Cell Physiol 182: 222–231, 2000.[CrossRef][ISI][Medline]
  37. Robinson JK, Blanchard TG, Levine AD, Emancipator SN, and Lamm ME. A mucosal IgA-mediated excretory immune system in vivo. J Immunol 166: 3688–3692, 2001.[Abstract/Free Full Text]
  38. 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.[CrossRef][ISI][Medline]
  39. Savkovic SD, Koutsouris A, and Hecht G. Activation of NF-{kappa}B in intestinal epithelial cells by enteropathogenic Escherichia coli. Am J Physiol Cell Physiol 273: C1160–C1167, 1997.[Abstract/Free Full Text]
  40. Wang W, Soto H, Oldham ER, Buchanan ME, Homey B, Catron D, Jenkins N, Copeland NG, Gilbert DJ, Nguyen N, Abrams J, Kershenovich D, Smith K, McClanahan T, Vicari AP, and Zlotnik A. Identification of a novel chemokine (CCL28), which binds CCR10 (GPR2). J Biol Chem 275: 22313–22323, 2000.[Abstract/Free Full Text]
  41. Wurbel MA, Philippe JM, Nguyen C, Victorero G, Freeman T, Wooding P, Miazek A, Mattei MG, Malissen M, Jordan BR, Malissen B, Carrier A, and Naquet P. The chemokine TECK is expressed by thymic and intestinal epithelial cells and attracts double- and single-positive thymocytes expressing the TECK receptor CCR9. Eur J Immunol 30: 262–271, 2000.[CrossRef][ISI][Medline]
  42. 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.[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 Infect. Immun.Home page
M. Hansson, M. Hermansson, H. Svensson, A. Elfvin, L.-E. Hansson, E. Johnsson, A. Sjoling, and M. Quiding-Jarbrink
CCL28 Is Increased in Human Helicobacter pylori-Induced Gastritis and Mediates Recruitment of Gastric Immunoglobulin A-Secreting Cells
Infect. Immun., July 1, 2008; 76(7): 3304 - 3311.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/5/G1062    most recent
00162.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ogawa, H.
Right arrow Articles by Kagnoff, M. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ogawa, H.
Right arrow Articles by Kagnoff, M. F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.