Expression of IL-10 receptors on epithelial cells from the murine small and large intestine

Timothy L. Denning1,3, Nicola A. Campbell1, Fei Song1, Roberto P. Garofalo1,3, Gary R. Klimpel3, Victor E. Reyes1,3 and Peter B. Ernst1,2,3

1 Department of Pediatrics,
2 Sealy Center for Molecular Sciences and
3 Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA

Correspondence to: P. B. Ernst, Department of Pediatrics, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0366, USA


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The appearance of chronic intestinal inflammation in IL-10 knockout mice suggests IL-10 may inhibit adverse responses to luminal antigen. Moreover, this inflammation is associated with an increase in class II MHC molecule expression on intestinal epithelial cells. Thus, the role of IL-10 regulation in epithelial cell function was investigated. Using RT-PCR, it was shown that intestinal epithelial cells express mRNA for both subunits of the IL-10 receptor-signaling complex. In addition, biotinylated IL-10 was shown to bind to both cultured and freshly isolated intestinal epithelial cells prepared from the small or large intestine. This binding appeared specific as it was blocked by neutralizing antibodies to IL-10 but not the isotype control. Moreover, an excess of native IL-10 also inhibited the binding of radiolabeled IL-10. To evaluate whether IL-10 mediated any functions through this receptor, epithelial cells were cultured with IL-10 alone or with IFN-{gamma} plus IL-10. IL-10 alone had no detectable effects on epithelial cell growth or their expression of class II MHC molecules but it did antagonize the effect of IFN-{gamma} on the viability of cultured cells. In addition, IL-10 blocked the IFN-{gamma}-induced expression of class II MHC molecules on cultured epithelial cells. These results suggest that IL-10 binds to a specific receptor on intestinal epithelial cells and may regulate the contribution of epithelial cells to the inflammatory and immune response in the digestive tract.

Keywords: class II MHC, cytokines, IL-10, intestine


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Intestinal epithelial cells can regulate local immune and inflammatory processes in several ways. For example, they constitute the initial barrier that limits the influx of proinflammatory materials from the lumen to the immune and inflammatory cells in the underlying tissue. Furthermore, after exposure to infection (14), bacterial toxins (5,6), or pro-inflammatory cytokines including IL-1, tumor necrosis factor (TNF)-{alpha} or IFN-{gamma} (1), epithelial cells produce pro-inflammatory cytokines that can recruit and activate inflammatory cells. In addition to producing soluble factors, epithelial cells express class I and class II MHC molecules (711) as well as some accessory molecules (1215) that contribute to the adherence and activation of immune and inflammatory cells including T cells. Thus, epithelial cells regulate the host response by transducing signals from luminal pathogens to adjacent immune and inflammatory cells.

It has been proposed that the expression of class II MHC molecules on intestinal epithelial cells allows them to stimulate CD4+ T cells during inflammation (16,17). The expression of class II MHC molecules by intestinal epithelial cells increases in response to infection with parasites (7) and the introduction of bacterial flora to germ-free mice (18). In addition, class II MHC expression is increased on epithelial cells in IL-10-deficient mice that develop a severe and progressive chronic enterocolitis (19). These findings suggest that IL-10 may regulate the pro-inflammatory effects of intestinal epithelial cells in IL-10-deficient mice.

IL-10 has been shown to mediate anti-inflammatory activity in cells of different lineages. For example, mice deficient in IL-10 (19) or its receptor (20) spontaneously develop colitis that is characterized by the production of TNF-{alpha} and IFN-{gamma} (21). These cytokines can modulate epithelial cell function by altering electrolyte transport (22,23), compromising barrier function (24,25) as well as increasing the expression of cytokines (26) and class II MHC molecules (27). The role of the epithelium in gating the access of inflammatory cells to microbial antigens has been proposed in view of the fact that colitis does not develop in germ-free IL-10-deficient mice (28,29). While the epithelium may be the target of adjacent immune cells that are normally controlled by IL-10 (30), changes in the function of intestinal epithelial cells are at least partly accounted for by a direct, anti-inflammatory effect of IL-10 (31). Thus, the purpose of this study was to determine if intestinal epithelial cells express a functional IL-10 receptor and if IL-10 could directly modulate the function of these cells.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Epithelial cell lines
Mode-K is an intestinal epithelial cell line derived from C3H/HeJ mice (32) that was kindly provided by Dr D. Kaiserlian (Lyon, France). Mode-K cells were grown in media consisting of DMEM low glucose supplemented with 2 mM sodium pyruvate (Sigma, St Louis, MO), 10% heat-inactivated FCS (Gibco, Grand Island, NY) and 10 ml/l non-essential amino acids (Gibco). MCA 38 is a murine colonic epithelial cell line (33) that was kindly provided by Dr C. Elson (Birmingham, AL). MCA 38 cells were maintained in RPMI 1640 media containing 10% FCS. Both cell lines were cultured at 37°C in a humidified, 5% CO2 incubator.

Isolation of mouse intestinal epithelial cells
To validate observations made in vitro, epithelial cells were studied immediately after isolation from C3H/HeJ mice using a slightly modified procedure that has been described previously (34). Mice were anesthetized and exsanguinated, and the entire small intestine was removed and placed in calcium/magnesium-free HBSS containing 5% FCS. The intestinal lumen was gently rinsed to remove chyme, and subsequently cannulated using polyethylene tubing (Becton Dickinson, Sparks, MD), tied at one end and turned inside out. The inverted intestine was then submerged in HBSS/FCS and shaken for 1 min to remove weakly attached epithelial cells. Small intestinal tissue was cut into pieces 2.5 cm long and placed in a 50 ml conical tube filled with 45 ml HBSS/FCS containing 1 mM DTT. Large intestinal tissue was cut into 0.5 cm segments and placed in a 50 ml tube filled with 45 ml HBSS/FCS containing 1 mM DTT and 0.5 mM EDTA, pH 8.0. All tubes were shaken at 37°C in an orbital shaker for 40 min. Histological examination of the intestinal fragments after EDTA treatment has been shown to strip epithelial cells from the entire villous crypt axis (unpublished observations). The supernatant was filtered through cotton gauze and then centrifuged for 5 min at 1500 r.p.m. The pellet was re-suspended and epithelial cells were collected by centrifugation through a 25%/40% discontinuous Percoll gradient at 600 g for 10 min. Predominantly epithelial cells were collected from the interface between the two layers and resuspended in 100% FCS. At this point, the cell number, viability and purity was assessed after staining with Trypan blue, and the cells were used in the various assays described below. Typically, epithelial cells were >95% pure, and viability was >90% for the small intestine and 80% for the large intestine. Purity was confirmed by flow cytometry showing that <1% of isolated cells were CD3+.

Detection of IL-10 receptor mRNA
Total cellular RNA from cultured epithelial cells was extracted using the RNA-zol kit according to manufacturer's instructions (Biotecx, Houston, TX). Equivalent amounts of RNA were monitored by absorption at 260 nm, and by monitoring the density of the 28S and 18S RNA detected following electrophoresis and staining with propidium iodide. The RNA was reverse transcribed and the cDNA product used as the template for the PCR. This template was amplified using a kit according to manufacturer's instructions (Cetus, Emeryville, CA). IL-10 receptor cDNA was amplified in a 50 µl reaction in a Perkin-Elmer thermocycler (Cetus) for 30 cycles with 50 pmol of each primer. Primers were as follows: mouse IL-10R1 sense, 5'-AGG CAG AGG CAG CAG GCC CAG CAG AAT GCT-3', antisense, 5'-TGG AGC CTG GCT AGC TGG TCA CAG TAG GTC-3'; mouse IL-10R2 (CRF2-4) sense, 5'-GCC AGC TCT AGG AAT GAT TC-3', antisense, 5'-AAT GTT CTT CAA GGT CCA C-3'. These primers generate fragments of 508 (IL-10R1) and 400 (IL-10R2) bp. To confirm that the PCR product was the region of DNA corresponding to the IL-10R1, the initial product was digested using an ACC-1 restriction enzyme. Based on the DNA sequence, this should yield two fragments of 324 and 184 bp. To ensure that amplification of genomic DNA was not responsible for generation of the product, RT-PCR was performed in the absence of reverse transcriptase. The products of the RT-PCR and restriction endonuclease digest were separated by 1.5% agarose gel electrophoresis and stained with ethidium bromide.

Evaluation of IL-10 receptors on the surface of epithelial cells by flow cytometry
Mode-K cells or freshly isolated epithelial cells were suspended in PBS/0.1% BSA to a concentration of 1x107 cells/ml. Then 100 µl of the cell suspension was added to Eppendorf tubes, the cells were washed once more in PBS/0.1% BSA and centrifuged at 300 g for 10 min. To these cell pellets, 50 µl of biotinylated human IL-10 followed by streptavidin-conjugated FITC were added according to the manufacturers instructions (R&D, Minneapolis, MN). Some tubes were also treated with a neutralizing antibody to IL-10, again according to the manufacturer's instructions. Cells treated with biotinylated soybean trypsin inhibitor were used as a control for non-specific fluorescence. In some experiments, freshly isolated epithelial cells were also fixed, permeabilized and stained with a FITC-conjugated antibody recognizing cyto-keratin to confirm the purity of the epithelial cells. After a final wash, the cells were fixed in 1 ml of fresh 1.0% paraformaldehyde. Subsequently, cells were examined by flow cytometry using a Becton Dickinson FACScan (San Jose, CA) and data analysis was performed using the CellQuest program.

Receptor binding studies
As previously reported, human IL-10 binds specifically to both the human and murine IL-10 receptor expressed on MC/9 cells (murine mast cell line) (3537). Thus, Mode-K cells were tested for specific binding using iodinated IL-10 and an excess of cold ligand. Samples of 4x106 MC/9 cells (positive control) or Mode-K cells were incubated with varying concentrations of 125I-labeled human IL-10 (Boehringer Mannheim Biochemica, Indianapolis, IN) for 4 h at 4°C as previously described. Bound iodinated IL-10 was separated from the free IL-10 by rapid filtration. Whatman 2.5 cm2 glass-fiber discs (GF/C) were inserted into a filtration manifold and pre-wet in ice cold wash buffer before the filtration of the labeled cells. After two subsequent rinses with buffer, the filter was removed, dried and the c.p.m. measured using a {gamma}-counter. In order to determine if the binding of the IL-10 was specific, 200-fold excess of cold murine IL-10 was incubated with the 125I-labeled human IL-10 and Mode-K cells and assayed as described above.

Regulation of epithelial cell recovery and function by IL-10
Epithelial cell recovery
Mode-K cells were seeded with ~2x105 cells/ml and stimulated with varying doses of murine IFN-{gamma} (Boehringer Mannheim Biochemica, Indianapolis, IN) for 5–7 days before cells were harvested and viability was assessed by Trypan blue staining. In other cultures, a previously determined optimal dose (10 U/ml) of recombinant murine IL-10 (kindly provided by Dr Narula, Schering-Plough) was added to Mode-K cells alone or in combination with the IFN-{gamma}. To confirm the specificity of any effect of IL-10, a neutralizing murine monoclonal antibody (PharMingen, San Diego, CA; clone JES5-2A5) was added and its effects compared to equivalent amounts of an isotype control. In some experiments, the effect of rIL-10 was compared to lipopolysaccharide (LPS) or thermally inactivated IL-10. This procedure was carried out at various time points in relation to IFN-{gamma} stimulation. After stimulation of the cells with varying concentrations of cytokines, recovery and viability was assessed after staining the cells with Trypan blue.

Expression of class II MHC molecules
Treated or control Mode-K cells were collected, washed in PBS/0.1% BSA and labeled with mAb IgG2b (10.2-16; ATCC, Rockville, MD) recognizing a polymorphic determinant on class II MHC molecules. Some cells were stained with equivalent amounts of an isotype control. Subsequently cells were labeled with an optimal concentration of FITC-conjugated goat anti-mouse IgG (Tago Immunological, Camarillo, CA), fixed in 1 ml of fresh 1.0% paraformaldehyde and examined by flow cytometry as described above.

Statistical analysis
The mean values for the different parameters were compared using Student's t-test.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IL-10R mRNA expression and binding of IL-10 by intestinal epithelial cell lines
Epithelial cell lines derived from the small (Mode-K) and large (MCA 38) intestine were incubated with biotinylated IL-10 or biotinylated soybean trypsin inhibitor (negative control) and analyzed using flow cytometry. As shown in Fig. 1Go, 75% of the Mode-K cells and virtually 100% of the MCA 38 bound IL-10. This binding was blocked with neutralizing antibodies to the biotinylated IL-10. To provide additional evidence for the specificity of the binding of IL-10 to the intestinal epithelial cells, the Mode-K cell line was examined in more detail. As shown in Fig. 1Go(A), native IL-10 competitively inhibited the binding of [125I]IL-10 suggesting that the binding was specific. Using RT-PCR, it was shown that Mode-K cells expressed a product of the predicted size for the IL-10 binding moiety of the IL-10R (IL-10R1). Moreover, this PCR product was digested by a restriction endonuclease to yield two products of the size corresponding to the location of the restriction site (Fig. 1BGo).



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Fig. 1. Demonstration of IL-10 binding by cultured epithelial cells using flow cytometry. To examine the ability of intestinal epithelial cell lines to bind IL-10, Mode-K or MCA 38 cells were incubated with biotinylated IL-10 or with biotinylated soybean trypsin inhibitor (negative control) and analyzed using flow cytometry. The main figure is a representative histogram showing the staining of Mode-K cells (left) and MCA 38 cells (right). Binding was blocked when the biotinylated IL-10 was incubated with anti-IL-10 mAb. Additional evidence for the specificity of the binding was obtained by labeling ~5x106 Mode-K cells with 200 pM [125I]IL-10 alone (filled bar) or in combination with a 300-fold excess of native IL-10 (shaded bar). Duplicate binding assays were performed as described in the Methods. As shown in (A), native IL-10 can competitively inhibit the binding of [125I]IL-10 to intestinal epithelial cells. Further, RT-PCR analysis of IL-10 receptor (IL-10R1) mRNA in Mode-K cells was performed (B). Lane 1, positive control; lane 2, expression of the 508 bp product corresponding to the size of the mRNA fragment for IL-10R; lane 3, products obtained after digestion of a sample of the material used in lane 2 with Acc-1 producing two bands of the predicted size corresponding to 184 and 324 bp.

 
IL-10 binds specifically to freshly isolated and cultured intestinal epithelial cells
To determine if the binding of IL-10 to cell lines reflected the phenotype of the intestinal epithelial cell in vivo, epithelial cells were isolated from normal murine (C3H) small and large intestine, labeled and examined. Based on the evaluation of forward and side scatter, isolated epithelial cell preparations contained 95% epithelial cells with no contaminating intraepithelial lymphocytes, as validated by <1% of the cells staining positive for CD3 (data not shown). After gating on viable epithelial cells, a comparable percentage of epithelial cells isolated from the small and large intestine were shown to bind the biotinylated IL-10 (Fig. 2Go). Again, neutralizing antibodies to IL-10 but not the isotype control blocked binding of IL-10.



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Fig. 2. Freshly isolated intestinal epithelial cells exhibit specific binding of biotinylated rhIL-10. Small (left column) and large (right column) intestinal epithelial cells were isolated, labeled and examined by flow cytometry as described in Methods. This figure shows representative histograms of the frequency and staining intensity after labeling cells with isotype control (top panels), biotinylated IL-10 (middle panels) or biotinylated IL-10 + neutralizing anti-IL-10 mAb (lower panels). Binding was blocked by anti-IL-10 but not by isotype control antibodies. This figure reflects representative results from two to five separate experiments.

 
Expression of the IL-10R2 subunit by both cultured and freshly isolated epithelial cells
While the IL-10R1 subunit is sufficient for binding IL-10, it has been reported that cells must express the IL-10R2 subunit in order that the IL-10 signal to the host cell (38). Therefore, total RNA was extracted, reverse transcribed and amplified by PCR with specific primers for IL-10R2. As shown in Fig. 3Go, RT-PCR of template RNA extracted from small or large intestinal epithelial cells (cultured or freshly isolated) yielded a product of the predicted size (400 bp). To confirm that the observed products were not due to amplification of genomic DNA, samples prepared in the absence of a reverse transcriptase enzyme were run in parallel and yielded no PCR product. Results presented here support the notion that murine intestinal epithelial cells express mRNA for an essential subunit of the IL-10R, IL-10R2, and may transmit signals via this receptor.



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Fig. 3. Reverse transcription PCR analysis of IL-10 receptor (IL-10R2) mRNA in intestinal epithelial cells. Total RNA from freshly isolated, or cultured, intestinal epithelial cells was extracted, reverse transcribed and amplified by PCR with specific primers for IL-10R2 spanning a 400 bp sequence within the murine IL-10R2 gene. Lane 1, Mode-K cell line; lane 2, MCA 38 cell line; lane 3, freshly isolated small intestinal epithelial cells; lane 4, freshly isolated large intestinal epithelial cells; lane 5, Mode-K minus reverse transcriptase; lane 6, MCA 38 minus reverse transcriptase.

 
Modulation of epithelial cell growth and viability by IL-10
IFN-{gamma} is known to modulate the viability and recovery of a variety of cells maintained in vitro. Therefore, Mode-K cells were grown in the presence of IFN-{gamma} alone or in combination with IL-10. Treatment of Mode-K cells with IFN-{gamma} decreased their recovery and viability from 50 to 75% compared to media alone (Fig. 4Go). Although IL-10 alone had no effect on cell growth or viability (data not shown, P > 0.05), IL-10 abrogated the effects of IFN-{gamma}, in this case, its effects on cell growth and viability (P < 0.05, Fig. 4Go).



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Fig. 4. Effect of IL-10 on IFN-{gamma}-mediated changes in the viability of cultured intestinal epithelial cells. Mode-K cells were grown in the presence or absence of IFN-{gamma} and compared to similar cultures exposed to 10 U/ml of IL-10, in terms of percent viable cells. This figure shows a representative experiment (one of three) indicating that IFN-{gamma} significantly decreased the viability of the cultured cells (P < 0.05) and neutralizing antibodies to IL-10 but not the isotype control reversed this effect. Once again, IL-10 enhanced the viability of IFN-{gamma}-treated Mode-K cells, which was blocked by neutralizing antibodies to IL-10 but not the isotype control.

 
To determine that the antagonistic effect of IL-10 was specific, rat anti-mouse antibodies were used to block the effects of the IL-10. Mode-K cells were seeded at identical concentrations in the presence of IFN-{gamma}, IL-10, IFN-{gamma} + IL-10 or IFN-{gamma} + IL-10 + anti-IL-10 mAb. IL-10 inhibited IFN-{gamma}-mediated effects on cell viability and this inhibition was blocked by anti-IL-10 but not the isotype control antibodies.

Regulation of the expression of class II MHC molecules on intestinal epithelial cells
In order to evaluate the anti-inflammatory effect of IL-10 on epithelial cells, surface class II MHC molecule expression was evaluated on Mode-K cells before or after exposure to IFN-{gamma}. Subsequently, class II MHC expression was measured on IFN-{gamma}-treated Mode-K cells in the presence or absence of varying doses of IL-10. Based on the dose–response curve (data not shown), 10 U/ml of IL-10 was used as the optimal dose and at this concentration it almost completely blocked the IFN-{gamma}-induced increase of class II MHC expression on cultured intestinal epithelial cells (Fig. 5Go). IL-10 alone had no effect on the expression of class II MHC by Mode-K cells. In addition, LPS, a potential contaminant of rhIL-10, and thermally inactivated IL-10 had no effect on blocking the effects of IFN-{gamma} on class II MHC molecule expression (data not shown).



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Fig. 5. Effect of IL-10 on reversing the IFN-{gamma}-induced increase in class II MHC molecules. In order to evaluate the anti-inflammatory effect of IL-10 on epithelial cells, surface class II MHC molecule expression was evaluated on Mode-K cells before or after exposure to IFN-{gamma}. IFN-{gamma} induced class II MHC expression and this induction was antagonized in the presence of 10 U/ml of IL-10. In addition, LPS, a contaminant of rIL-10, and thermally inactivated IL-10 did not antagonize the effects of IFN-{gamma} (data not shown) .

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The epithelial cell has been shown to promote inflammation and as well as become the target of activated leukocytes or cytokines. While, many reports have described the effects of cytokines on epithelial cells, relatively few have associated these activities with specific receptors. Some reports have shown that growth factors, such as transforming growth factor (TGF)-{alpha}, TGF-ß and cytokines, including IL-1, IL-2, IL-6 or IL-15, can bind to epithelial cells via specific receptors (3942). In this report, evidence is provided demonstrating that epithelial cells express both subunits of the IL-10 receptor, thereby enabling IL-10 to bind specifically and to modulate cell function.

Murine mast cells, B cells and fibroblasts as well as human peripheral blood leukocytes (3537) express the IL-10R1 gene. Although several recent studies have identified an essential subunit for the IL-10 receptor termed IL-10R2 or CRF2-4 (20,38), our report is the first to assess the expression of this molecule in the digestive tract. While expression of the receptor for IL-10 differed between cells lines from the small and large intestine, our results indicate that freshly isolated small and large intestinal epithelial cells bind the ligand to a comparable degree. Thus, differences in binding observed in the cell lines appears to be attributable to properties of the lines that are not identified using freshly isolated cells from the small or large intestine. In addition, cell lines expressed more IL-10R than freshly isolated cells suggesting that expression differs throughout the villous–crypt axis. Using isolation techniques that differentially enrich for epithelial cells from the villous or crypt, preliminary reports have suggested that IL-10R expression is decreased at the villous tips (43). Panja and colleagues have also suggested that IL-10R expression is greater using crypt cells compared to villous cells (44). However, the variation in expression along the villous–crypt axis throughout the bowel remains to be determined definitively when more direct assays become available.

Many of the cells that express receptors for IL-10 also express receptors for IFN-{gamma}. Given that IL-10 receptors structurally resemble the receptors for IFN-{gamma} (20,35), as well as the fact that IL-10 antagonizes many of the inflammatory events induced by IFN-{gamma} (45), it should not be surprising that these receptors are expressed in parallel. The effect of IL-10 on the intestinal epithelial cells fits the paradigm that IL-10 competes functionally with IFN-{gamma}. While IL-10 had little effect on intestinal epithelial cells alone, it did antagonize several changes that were induced by IFN-{gamma}. For example, the effects of IFN-{gamma} on growth, viability as well as the expression of class II MHC molecules, were all antagonized by IL-10. This is consistent with the report that IL-10 antagonizes the effect of IFN-{gamma} on epithelial cell permeability (46).

It has been known for several years that epithelial cells in the intestine express class II MHC molecules (7,8). Since the epithelial cells are in direct contact with intraepithelial lymphocytes and may also contact T cells from the lamina propria that reach the epithelial surface, it is possible that epithelial cells can present antigen to CD4+ T cells. In fact, several laboratories have shown that epithelial cells do, in fact, stimulate helper T cells (11,47,48). However, other reports suggest that epithelial cells may preferentially stimulate CD8+ T cells (11,49,50). In addition, intestinal epithelial cells may lack the expression of key accessory molecules, such as B7-1 or B7-2 that facilitate T cell activation. These observations have led to the concept that epithelial cells play an important role in inhibiting immune responses through the induction of suppressor cells or by inducing anergy in T cells. The fact that epithelial cells responded to IL-10 with a decrease in class II MHC molecules also suggests that their ability to stimulate T cells and modify the functional phenotype of Th cells may be regulated by cytokines including IL-10. However, it should be pointed out that similar studies in human cell lines have recently reported that IL-10 may have no effect on these antigen presentation-associated molecules (51). Future studies will have to resolve the relative importance of IL-10 in its direct effects on the immune functions of epithelial cells.

Functional studies have shown that IL-10 also inhibits cytokine production by various cells including macrophages (52), endothelial cells, T cells (53) and fibroblasts (37). Based on the model that IL-10 deficiency leads to colitis (19), others have shown that IL-10 can directly inhibit the pro-inflammatory activity of mononuclear cells isolated from the intestine of patients with inflammatory bowel disease (54) or the effect of activated T cells on epithelial cell function in vitro (30). The necessity for IL-10 to balance pro-inflammatory cytokines is illustrated further by the reports describing the ability of IL-10 to limit the inflammatory response to endotoxin (37,5558) and the subsequent development of endotoxic shock. Since intestinal epithelial cells can produce various cytokines that promote inflammation, it is possible that a broad panel of pro-inflammatory molecules produced by the epithelial cells is regulated by IL-10. However, results to date suggest that IL-10 cannot block the induction of chemokines in either the human (5) or murine system (29).

IL-10 may have other effects on epithelial cell function and the regulation of inflammation. For example, cytokines such as IL-10 may play a role in the restitution of the epithelial layer which, in itself, would provide an anti-inflammatory function due to the exclusion of luminal contents. This notion is supported by the observations suggesting that IL-10 may also correct the degeneration of the epithelial barrier function that is induced by immune stimulation (30,31). As IFN-{gamma} can impair epithelial barrier function (24,25) this would be yet another example of the antagonism between IL-10 and IFN-{gamma} at the level of the intestinal epithelial cell.

In summary, previous studies have shown that epithelial cells can respond to infection or other insults allowing them to modulate local immune and inflammatory responses. This report shows that abnormal inflammatory responses within the intestinal epithelium, such as those observed in mice deficient in IL-10 (19) or its receptor (20), may be regulated directly by IL-10 and are not simply due to the loss of IL-10 activity on inflammatory cells adjacent to the epithelium. Future studies will determine how broadly IL-10 can function in the control of inflammation that is modulated by epithelial cells.


    Acknowledgments
 
Recombinant murine IL-10 was kindly provided by Dr S. Narula, Schering Plough Research Institute. This work was supported by NIH DK 50980, CHD 35741, and by the Crohn's and Colitis Foundation of America. T. L. D. is a recipient of pre-doctoral support from the McLaughlin Foundation. F. S. is a recipient of post-doctoral support from the Sealy Center on Aging.


    Abbreviations
 
LPS lipopolysaccharide
TGF transforming growth factor
TNF tumor necrosis factor

    Notes
 
The first two authors contributed equally to this work

Transmitting editor: C. Terhorst

Received 18 February 1999, accepted 21 September 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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