Intestinal epithelial cells both express and respond to interleukin 15

Intestinal epithelial cells both express and respond to interleukin 15

GASTROENTEROLOGY 1996;111:1706–1713 RAPID COMMUNICATIONS Intestinal Epithelial Cells Both Express and Respond to Interleukin 15 HANS–CHRISTIAN REINEC...

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GASTROENTEROLOGY 1996;111:1706–1713

RAPID COMMUNICATIONS Intestinal Epithelial Cells Both Express and Respond to Interleukin 15 HANS–CHRISTIAN REINECKER,* RICHARD P. MACDERMOTT,*,‡ SILVIA MIRAU,*,‡ AXEL DIGNASS,§ and DANIEL K. PODOLSKY* *Gastrointestinal Unit, Department of Medicine, Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts; §Department of Medicine, University of Essen, Essen, Germany; and ‡Lahey Clinic, Burlington, Massachusetts

Background & Aims: Interleukin (IL)-15 exerts functional effects on lymphocytes similar to those of IL2. IL-15 is expressed by nonlymphoid cells and may integrate these cells into classical immune responses. The aim of this study was to characterize the expression of IL-15 by intestinal epithelial cells and determine the functional roles of IL-15 within the mucosal immune system. Methods: Rat IL-15 was cloned from a rat jejunal library. Expression of IL-15 in rat and human intestinal epithelial cells was assessed by Northern and Western blotting. Tyrosine kinase activation in response to IL-15 in intestinal epithelial cells was determined by immunoprecipitation. Results: Rat and human intestinal epithelial cells express IL-15 messenger RNA. IL15 activates Stat3 and stimulates the proliferation of intestinal epithelial cells. The relevance of the observations for intestinal epithelial cell function in vivo was supported by the demonstration of transcripts for IL15 in primary human intestinal epithelial cells. Conclusions: IL-15 is expressed by intestinal epithelial cells and may be able to regulate intestinal epithelial cell function. These experiments suggest that IL-15 is an important mediator that could integrate intestinal epithelial cell function with the intestinal immune system.

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espite lack of a significant sequence homology, interleukin (IL)-15 and IL-2 have a similar four-helix bundle structure1 and share the requirement of the common gc receptor and the IL-2 receptor b subunits for signal transduction.1,2 As a result, IL-15 exerts functional effects on T and B cells similar to those exerted by IL-2. These include stimulation of the proliferation of primed peripheral blood mononuclear cells and T-cell lines1 and induction of alloantigen-specific cytotoxic T lymphocytes and nonantigen-specific lymphokine-activated killer cells.1 IL-15 also activates human natural killer (NK) cells3 and induces B-cell proliferation and differentiation in vitro.4 Previous experiments have shown that rat and human / 5e14$$0071

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intestinal epithelial cells express functional cytokine receptors that share the common gc receptor chain, including the receptors for IL-2.5 In contrast to IL-2, which is produced by activated T cells, IL-15 seems to be expressed in a variety of tissues, including skeletal muscles, kidney, and activated monocytes and macrophages. Because IL-15 and IL-2 seem to be produced by different cell types, it is likely that these two cytokines have overlapping functional roles in vivo that are mediated at distinct anatomical sites. To assess the role of IL-15 in the mucosal immune system, we have cloned the rat IL-15 and characterized the expression of IL-15 in intestinal epithelial cells. This report shows that rodent and human intestinal epithelial cells are able to express IL-15. Expression of IL-15 may enable intestinal epithelial cells to regulate lymphocyte development and differentiation within the lamina propria. In addition, IL-15 is able to regulate intestinal epithelial cell function. The ability of intestinal epithelial cells to express IL-15 in conjunction with the previously reported ability to express functional receptors sharing the gc receptor subunit may constitute an important ligand-receptor system that integrates intestinal epithelial cells into the mucosal immune system.

Materials and Methods Cloning of Rat IL-15 IL-15 primers, 5* ATT TCC ATC CAG TGC TAC CT and 3* CTT CAT TGC TGT TAC CTT GC, were designed based on the simian IL-15 sequence (Genbank, U03099) and used to amplify a 211–base pair (bp) fragment of the human IL-15 complementary DNA (cDNA) from reverse-transcribed Abbreviations used in this paper: DMEM, Dulbecco’s modified Eagle medium; IFN-g, interferon gamma; IL, interleukin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; TGF, transforming growth factor. q 1996 by the American Gastroenterological Association 0016-5085/96/$3.00

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messenger RNA (mRNA) isolated from differentiated Caco-2 cells. This polymerase chain reaction (PCR) fragment was cloned into Bluescript SK/ (Stratagene, La Jolla, CA) and sequenced by standard techniques and found to have a 94% homology with the simian IL-15 sequence. The PCR fragment was radioactively labeled with [32P]deoxycytidine triphosphate using a random labeling kit (Promega, Madison, WI) and used to screen a rat jejunal cDNA library (Stratagene). Phage plaques, 1 1 106, were screened as described.6 Filters were prehybridized for 2 hours and hybridized with 1 1 109 cpm of probe and 100 mg/mL salmon sperm DNA (Life Technologies, Gaithersburg, MD) for additional 24 hours at 427C in 40% formamide, 51 Denhardt’s, 51 SSPE, 0.1% sodium dodecyl sulfate (SDS), and 10% dextran sulfate.

Cell Culture Caco-2, HT-29, T-84, HL-60, and Jurkat cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured as recommended by the ATCC. Before assessment of IL-15 transcripts, Caco-2 cells were stimulated by replacement with fresh media containing 100 ng/mL interferon gamma (IFN-g) (R&D Systems, Minneapolis, MN) after they had reached confluence for 2 weeks. IEC-6 were cultured in Dulbecco’s modified Eagle medium (DMEM), 10% fetal calf serum, 5 mg/mL insulin, and 4 mmol/L L-glutamine.

Isolation of Rodent Intestinal Epithelial Cells Intestinal epithelial cells of 500-g Wistar rats were isolated by ethylenediaminetetraacetic acid (EDTA) perfusion.7 Rats were anesthetized with ether, and the intestine was flushed in situ with Ca2/- and Mg2/-free phosphate-buffered saline (PBS) at 377C. After transsection of the vena cava, the intestine was perfused with 10 mmol/L EDTA in Ca2/- and Mg2/-free PBS injected into the left ventricle for 10 minutes. Subsequently, the whole intestine was resected and separated into functional different parts from the duodenum to the colon. The intestinal sections were further incubated in 10 mmol/L EDTA in Ca2/- and Mg2/-free PBS with inverted mucosa until complete removal of intestinal epithelial cells. The viability of cells was ú90%, as assessed by 0.1% trypan blue exclusion. Cells prepared by this method contain õ 5% contaminating nonepithelial cells.8

Proliferation Assay Subconfluent Caco-2 cells maintained in 24-well multiwell plates were changed to fresh media containing 0.1% fetal calf serum 18 hours before addition of cytokines. [3H]Thymidine (2 mCi/well) was added after a 20-hour incubation period with cytokines and incubated for 4 hours, and incorporation was determined using standard techniques.9 Data from three independent experiments were compared by Student’s t test.

Isolation of Primary Human Intestinal Epithelial Cells Intestinal epithelial cells were isolated from colonic biopsy specimens as previously described.10 Intact epithelial

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cell crypts were first removed from the biopsy specimen by incubation for 30 minutes at 377C with 1 mmol/L EDTA in Ca2/-and Mg2/- free PBS. Crypts were then further dissociated into single epithelial cells by incubation with 0.15 mol/L NaCl containing 3 mmol/L sodium tetraphenyl borate (Sigma Chemical Co., St. Louis, MO). Separated epithelial cells were resuspended in DMEM media, and 50 mL of the suspension was placed on caved glass slides and overlaid with mineral oil. Single epithelial cells were then transferred to a second slide with a micropipette. RNA from 20 to 40 primary epithelial cells was isolated after addition of 50 mg carrier ribosomal RNA from Escherichia coli strain W (Sigma Chemical Co.). After removal of intestinal epithelial cells, the remaining mucosal tissue was used to assess the expression of IL-15 in lamina propria mononuclear cells.

PCR Amplification Primers and PCR conditions for amplification of transcripts encoding IL-2, CD45, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been described previously.5,11 Primers specific for IL-15 were as described above. The PCR reaction was performed on a Perkin–Elmer thermal reactor (Perkin–Elmer, Norwalk, CT) with 30 cycles (947C, 557C, and 727C each for 1 minute) in the presence of 1.5 mmol/ L Mg2/ for the PCRs using reverse-transcribed RNA from intestinal epithelial cell lines or controls and with 40 cycles for the PCRs with RNA obtained from primary intestinal epithelial cells. In addition, PCRs were performed with RNA alone to rule out amplification products derived from genomic DNA.

Northern Blot Analysis RNA was isolated using acid guanidinium isothiocyanate–phenol–chloroform extraction,12 and Northern blotting was performed as described previously.13 Poly(A)/ mRNA was obtained by oligo(dT) chromatography, and 5 mg mRNA per sample was electrophoresed on a denaturing 1% agarose gel and blotted onto nitrocellulose paper (MSI, Westboro, MA). Probes were radioactively labeled with [32P]deoxycytidine triphosphate using a random prime labeling kit (Promega). Probes for the detection of rat IL-15 transcripts were generated from a 335-bp Dpn1 fragment representing part of the coding region of rat IL-15; probes specific for the detection of the human IL-15 were generated by PCR as described above. The transforming growth factor (TGF)-b1 probe was obtained from ATCC (no. 59954). Hybridizations were performed with QuikHyb solution (Stratagene) at 687C. Blots were exposed overnight to film and analyzed on a Densitometer and Image Quant Software (Molecular Dynamics, Sunnyvale, CA).

Western Blotting and Immunoprecipitations For the detection of IL-15, cells were lysed in 1 mL buffer containing 0.0625 mol/L Tris ( pH 6.8), 2% SDS, 3% b-mercaptoethanol, 10% glycerol, 100 mmol/L sodium fluoride, 10 mg/mL aprotinin, 10 mg/mL leupeptin, 10 mg/mL

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manufacturer’s recommendations and by addition of proteinase and phosphatase inhibitors as previously described.5 The immunoprecipitates were resolved by SDS-PAGE (7.5%). Activated Stat3 was detected with an antibody specific for Stat3 activated by phosphorylation at tyrosine 705 (New England Biolabs, Beverly, MA) by Western blotting according to the manufacturer’s recommendations. To confirm the equal loading of each lane, the blot was stripped and reprobed with phosphorylation-independent anti-Stat3 antibodies.

Results Molecular Cloning of Rat IL-15

Figure 1. Rat (rIL-15; U69272), mouse (mIL15; U14332), simian (sIL15; U03099), and human (hIL-15; U14407) multiple IL-15 protein sequence alignment. In the alignment, the shaded residues are conserved among rodents and primates. Cysteines predicted to form disulfide cross-links are highlighted in open boxes, and putative linking is indicated. The arrowhead points to a predicted cleavage point of signal peptide.

pepstatin, and 1 mmol/L phenylmethylsulfonyl fluoride (all from Sigma Chemical Co.). Then 300 mg of cell lysate proteins and 200 ng of recombinant IL-15 (Genzyme, Cambridge, MA) were separated on a 15% SDS–polyacrylamide gel and electrophoretically transferred onto Immobilon-P membrane (Millipore, Bedford, MA). IL-15 protein was detected after incubation with 5 mg of anti–IL-15 antibodies (Genzyme) and subsequent incubation with horseradish peroxidase–conjugated goat anti-mouse immunoglobulin (Ig) G (H/L) monoclonal antibody (Amersham, Arlington Heights, IL) diluted 1:5000 in 10 mmol/L Tris (pH 7.5), 100 mmol/L NaCl, and 0.1% Tween 20 (TBST) containing 0.1% bovine serum albumin. Antibody reaction was detected with a chemiluminescence detection kit (DuPont/NEN, Boston, MA). Assessment of cytokine-induced tyrosine phosphorylation was performed as described previously.5 Caco-2 cells were grown in full media in six-well plates until they reached confluence for 1 week, serum deprived for 18 hours, and stimulated with 100 U/mL IL-2 (R&D Systems), 250 ng/mL IL-15 (Genzyme), or 100 ng/ mL epidermal growth factor (R&D Systems) for 5 minutes. Then 50 mL of cell lysates was separated by SDS–polyacrylamide gel electrophoresis (PAGE) (7.5%) and electrophoretically transferred onto Immobilon-P membrane (Millipore). Equal loading and transfer of proteins was determined by ponceau S staining. Phosphotyrosine containing proteins were stained with the mouse monoclonal antiphosphotyrosine antibody PY-20 (Transduction Laboratories, Lexington, KY) diluted 1:500 in TBST–1% bovine serum albumin for 1 hour and visualized as described above. Immunoprecipitation for Stat3 was performed with a phosphorylation-independent anti-Stat3 antibody raised against amino acid 1–178 of rat Stat3 and goat anti-mouse IgG (Transduction Laboratories, Lexington, KY) on protein A– Sepharose beads (Pharmacia, Piscataway, NJ) according to the

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The screening of the rat jejunal cDNA library with a 211-bp IL-15 PCR fragment generated from reverse-transcribed RNA of Caco-2 cells yielded two identical full-length cDNA clones encoding rat IL-15. The full-length cDNA and deduced amino acid sequence of the rat IL-15 are available from Genbank (U69272). The rat IL-15 encodes a 162–amino acid protein, including a 48–amino acid putative leader sequence. Sequence analysis showed an overall homology of rat IL-15 with the published simian and human IL-15 of 75% and 79% within the translated 489-bp region on the nucleotide level and 81% on the amino acid level. The homology of rat and mouse IL-15 is 92% at the nucleotide level. As shown in Figure 1, at the protein level, rat and mouse IL-15 differ in only 5 residues of 162 (97% homology). Intestinal Expression of IL-15 Northern blot analysis showed that isolated rat intestinal epithelial cells express IL-15 constitutively. IL15 mRNA expression in rat intestinal epithelial cells increased along the longitudinal axis of the small intestine from the jejunum to the terminal ileum (Figure 2, lanes 1–3). The nontransformed, undifferentiated rat intestinal epithelial cell line IEC-6 expressed less IL-15 mRNA (Figure 2, lane 5) than differentiated primary

Figure 2. Northern blot analysis of IL-15 mRNA expression in isolated intestinal epithelial cells from rat jejunum (lane 1), ileum (lane 2), terminal ileum (lane 3), colon (lane 4), and RNA isolated from IEC-6 cells preconfluent (lane 5) and 10 days after confluence (lane 6). IL-15 mRNA was detected as a single transcript of 1400 bp. Five micrograms of poly(A)/ RNA was analyzed in each lane. Blots were subsequently hybridized with cDNA specific for GAPDH to assess loading. One representative Northern blot of three independent experiments is shown.

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contamination was confirmed by the detection of transcripts for CD45 in a preparation of isolated epithelial cells (Figure 3A, lane 3), which was intentionally contaminated with two lymphocytes. As shown in Figure 3A, two representative RNA isolations from primary intestinal epithelial cells were free of contamination with leukocyte RNA (Figure 3A, lanes 1 and 2). These two primary intestinal epithelial cell isolations contained transcripts for the human IL-15 (Figure 3A, lanes 1 and 2) but not IL-2. In addition to intestinal epithelial cells, lamina propria mononuclear cells also expressed transcripts for IL-15 (Figure 3A, lane 4). IL-15 Expression in Human Intestinal Epithelial Tumor–Derived Cell Lines

Figure 3. (A ) Determination of IL-15 and IL-2 expression in isolated intestinal epithelial cells from three different human colonic biopsy specimens (1–3) by reverse-transcription PCR and lamina propria mononuclear cells (LPMNC) from normal mucosa. Lane 3* shows reverse-transcription PCRs performed with RNA from intestinal epithelial cells that were intentionally contaminated with two lymphocytes to test sensitivity of the PCR for CD45. (B ) Detection of IL-15 transcripts in human intestinal epithelial tumor–derived cell lines by PCR. Cell lines with enterocyte-like phenotype (Caco-2, HT-29, and T-84) as well as goblet cell–like phenotype (LS-174) express IL-15 transcripts (F X174-DNA Ladder; Promega). One representative PCR of three independent experiments is shown.

Several human intestinal epithelial tumor–derived cell lines including HT-29, Caco-2, T-84, and LS174 were tested for the presence of IL-15 transcripts. Figure 3B shows that all tested intestinal epithelial cell lines contained transcripts for IL-15. The human intestinal epithelial cell line Caco-2 has the ability to acquire an enterocyte-like phenotype with continued culture after reaching a confluent state. These features include formation of tight junctions, expression of brush border enzymes, and alteration in cell shape to resemble the columnar form of primary intestinal epithelial cells.14 The expression of IL-15 mRNA in 2-week confluent Caco-2 cells could be up-regulated 10-fold within 4 hours by addition of 100 ng/mL IFN-g (Figure 4, lane 2). IL-15 Protein Expression in Intestinal Epithelial Cells To determine if intestinal epithelial cells are able to produce IL-15 protein, Western blot analysis was per-

intestinal epithelial cells, although the expression levels increased 10 days after the cells reached confluence (Figure 2, lane 6). IL-15 mRNA Expression in Primary Human Epithelial Cells To determine whether IL-15 expression was a feature shared by human intestinal epithelial cells, primary human intestinal epithelial cells were isolated from normal colonic mucosal biopsy specimens. Isolated intestinal epithelial cells seemed to be free of contamination with lymphocytes and monocytes, as reflected by the inability to detect transcripts for CD45, which were detectable in RNA from lamina propria mononuclear cells (Figure 3A). The sensitivity of this method to detect even minimal / 5e14$$0071

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Figure 4. IL-15 mRNA expression in Caco-2 cells is regulated by IFNg. Five micrograms of poly(A)/ RNA was analyzed in each lane. Expression of IL-15 mRNA in Caco-2 cells confluent for 2 weeks increased 10-fold within 4 hours after stimulation with IFN-g as assessed by densitometric analysis after overnight exposure of the blot. One representative Northern blot of four independent experiments is shown.

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formed on total cell lysates from intestinal epithelial cell lines and leukocyte cell lines. Intestinal epithelial cell lines HT-29 and Caco-2 contained a 14–15-kilodalton protein that was stained by an anti–IL-15 monoclonal antibody (Figure 5, lanes 4 and 5). The promyelomonocytic cell line HL-60 and the T-cell line Jurkat expressed very little IL-15 protein in comparison with the epithelial cell lines (Figure 5, lanes 2 and 3). Anti–IL-15 antibodies also detected a larger protein with a molecular size of 18 kilodaltons, which may represent IL-15 propeptide including the signal peptide sequence. The recombinant IL-15 was generated from a cDNA lacking the signal peptide sequence and therefore corresponds in size to the cleaved IL-15 protein. IL-15 Is a Regulator of Intestinal Epithelial Cells Intestinal epithelial cell lines and primary intestinal epithelial cells are able to express intermediate affinity receptors for IL-2, composed of the common gc and IL2 receptor b subunits.5,15 On lymphocytes, IL-15 has been shown to use the common gc and the IL-2 receptor b subunit for signal transduction.3 We therefore assessed the capacity of IL-15 to modulate intestinal epithelial cell function. To determine whether IL-15 is able to induce signal transduction in intestinal epithelial cells,

Figure 5. Western blot analysis of IL-15 protein expression in human intestinal epithelial cell lines. One representative Western blot of three independent experiments is shown. Anti–IL-15 antibodies detected a protein corresponding to the size of recombinant IL-15 (lane 1) in cell lysates prepared from HT-29 (lane 4) and Caco-2 (lane 5) cells as indicated by the arrow. Very low levels of IL-15 were found in HL-60 (lane 2) and Jurkat (lane 3) cells. Sizes of protein standards are given in kilodaltons.

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Caco-2 cells were incubated with 250 ng/mL IL-15 or IL-2 and 100 ng/mL epidermal growth factor as a positive control. Protein tyrosine phosphorylation was assessed by Western blotting. Lane 3 in Figure 6 shows that IL-15 stimulated a pattern of protein tyrosine phosphorylation in Caco-2 cells within 5 minutes similar to but not identical to that induced by IL-2 (lane 2). One of the proteins phosphorylated in response to IL15 in intestinal epithelial cells could be identified as Stat3. IL-15 rapidly stimulated the activation of Stat3 in Caco-2 cells within 5 minutes (Figure 7, lane 2). Phosphorylation levels of Stat3 returned to baseline within 20 minutes (lane 4). After confirmation of ligand-induced protein phosphorylation in response to IL-15, the effect of this cytokine on intestinal epithelial cell proliferation was assessed. IL-15 was able to stimulate the proliferation of Caco-2 cells as determined by [3H]thymidine uptake over a time period of 24 hours (Figure 8). Stimulation reached significance for IL-15 (P õ 0.05) and IL-2 (P õ 0.01) at a concentration of 100 ng/mL of cytokine.

Discussion The development of classical immune cells results from the expansion and differentiation of committed lymphoid precursor cells under the influence of the bone

Figure 6. IL-15–induced tyrosine phosphorylation of proteins in the human intestinal epithelial cell line Caco-2. Differentiated monolayers of Caco-2 cells were serum deprived for 18 hours and subsequently stimulated for indicated periods of time with IL-2 (lane 2), IL-15 (lane 3), or epidermal growth factor (EGF) (lane 4). Equal amounts of proteins per lane were analyzed by SDS-PAGE (7.5%) as described in Materials and Methods. Arrows indicate major tyrosine phosphorylated proteins. Sizes of protein standards are given in kilodaltons. One representative Western blot of three independent experiments is shown.

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Figure 7. Activation of Stat3 in the human intestinal epithelial cell line Caco-2. Stat3 was immunoprecipitated with an antibody recognizing total Stat3, resolved by SDS-PAGE (7.5%), and transferred onto a polyvinylidene membrane. Activated Stat3 was detected by incubating the blot with an antibody that recognizes only Stat3 phosphorylated at tyrosine 705. Equal loading of the gel was confirmed by stripping and reprobing the blot with phosphorylation-independent anti-Stat3 antibodies. Sizes of protein standards are given in kilodaltons.

marrow, thymic, and gastrointestinal tract microenvironments. Increasing evidence shows that intestinal epithelial cells are important regulators of the development of mucosal immunity. Preliminary data suggest that the mechanisms integrating intestinal epithelial cell function are coupled with classical immune cells present in the intestinal mucosa through the action of cytokines. In this report we show the ability of intestinal epithelial cell lines and primary intestinal epithelial cells from both rodent and human to express IL-15. Molecular cloning of the rat IL-15 from a jejunal cDNA library together with demonstration of specific epithelial cell expression by Northern blot confirms intestinal epithelial cells as a source of IL-15. IL-15 shares several overlapping functional roles in the immune system with IL-2. IL-2 has a central role in both antigen-driven and nonspecific immune responses. Therefore, intestinal epithelial cells may direct lymphocyte development and differentiation within the intestinal mucosa through the production of IL-15. Cell surface receptors for IL-15 have been shown on a variety of T cells, including murine antigen-dependent T-cell clones and human and murine T-cell lines as well as peripheral blood monocytes.2 The IL-2 receptor b and the gc chains of the IL-2 receptor complex are necessary for the signal transduction of IL-151,2 but not the IL2 receptor a chain.2 More recently, an a subunit that specifically binds IL-15 as a heterodimeric complex with the b and gc chains was cloned from endothelial cells that lack the IL-2–specific a chain.16 In contrast to the IL-2 receptor a chain, IL-15 receptor a subunit is expressed by nonlymphoid cells, including stromal cells from bone marrow, fetal liver, or thymic epithelium,2 suggesting that IL-15 may affect biological functions differently than IL-2. / 5e14$$0071

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Targets for IL-15 produced by intestinal epithelial cells may include antigen-dependent T cells and B cells within the lamina propria that have been shown to be regulated by IL-15 in vitro.1,2,4 In contrast to IL-2, IL15 is able to stimulate mast cell proliferation.17 Furthermore, IL-15 has been shown to be chemotactic for T lymphocytes.18 IL-15 may therefore enable intestinal epithelial cells to influence the lymphocyte composition within the lamina propria. The expression of IL-15 in differentiated Caco-2 cells could be up-regulated by IFN-g. IFN-g is one of the major cytokines involved in inflammation and the development of immunity. IFN-g has been shown to up-regulate major histocompatibility complex class I and class II expression by intestinal epithelial cells.19 In the intestine, both epithelial cells and lamina propria mononuclear cells seem to produce IL-15. In addition to its ability to provide intestinal epithelial regulation of lamina propria cell populations, the present study shows that IL-15 may regulate intestinal epithelial cell function itself. Our previous studies showed that human intestinal epithelial cells express transcripts for cytokine receptors that share the gc subunit in their receptor complexes, including receptors for IL-2, IL-4, IL7, and IL-9.5 The receptors for IL-2 on intestinal epithelial cells consist of the IL-2 receptor b and gc subunits, which have been shown to be functional on NK cells20 and intestinal epithelial cells.5 Furthermore, IL-2 is able

Figure 8. Proliferation of Caco-2 cells in response to IL-2 ( ) and IL15 (j) as determined by [3H]thymidine uptake. Error bars indicate standard deviations for three different experiments. Stimulation reached significance for IL-2 (*P õ 0.01) and IL-15 (**P õ 0.05) at a concentration of 100 ng/mL of cytokine.

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to regulate rat intestinal epithelial cell function, as shown by the modulation of intestinal epithelial cell proliferation and intestinal epithelial cell restitution.9 IL-15 may use the IL-2 receptor b and gc chains expressed on intestinal epithelial cells for signaling, as shown by an increase in tyrosine kinase activity in intestinal epithelial cells after stimulation with IL-15. The response of intestinal epithelial cells to IL-15 is mediated through the activation of Stat3 in a manner similar to that found in T cells.21 As shown in this study, IL-15 is able to stimulate intestinal epithelial cell proliferation in a manner similar to that observed for IL-2.15 Similar to IL-2, IL-15 was also able to up-regulate the expression of TGF-b (data not shown). TGF-b is able to stimulate epithelial cell migration, promoting recovery of epithelial integrity after wounding.22 TGF-b produced by intestinal epithelial cells also regulates the expression of the integrin aEb7 on intestinal intraepithelial lymphocytes. This integrin anchors the intraepithelial lymphocyte to intestinal epithelial cells through its interaction with E-cadherin.23 Mechanisms coordinating intestinal epithelial cells and classical immune cells within the lamina propria are essential for the preservation of mucosal integrity. We have identified a ligand-receptor system that may be pivotal for the interaction of intestinal epithelial cells and intestinal immune cells. The ability of intestinal epithelial cells to express functional ‘‘IL-2’’ receptors in conjunction with the expression of IL-15 constitute an important ligand-receptor system that may be critical for the regulation of intestinal epithelial cell function as well as the development of intestinal lymphocytes within the intestinal mucosa. The current data show that intestinal epithelial cells are able to express mRNA for IL-15 and that intestinal epithelial tumor–derived cell lines are able to express IL-15 protein. Identification of cell populations expressing mature IL-15 and the exact mechanisms of its secretion in vivo remain to be determined. Further characterization of the function of IL-15 produced by intestinal epithelial cells may provide insight into previously unrecognized pathways integrating intestinal epithelial cell function with classical immune cells in the intestinal mucosa.

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3. Carson WE, Giri JG, Lindemann MJ, Linett ML, Ahdieh M, Paxton R, Anderson D, Eisenmann J, Grabstein K, Caligiuri MA. Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor. J Exp Med 1994;4:1395–1403. 4. Armitage RJ, Macduff BM, Eisenman J, Paxton R, Grabstein KH. IL-15 has stimulatory activity for the induction of B cell proliferation and differentiation. J Immunol 1995;154:483–490. 5. Reinecker HC, Podolsky DK. Human intestinal epithelial cells express functional cytokine receptors sharing the common gc chain of the interleukin-2 receptor. Proc Natl Acad Sci USA 1995; 92:8353–8357. 6. Seidman JG, Quertermous T, Weis JH, Straus WM. Screening of recombinant DNA libraries. In: Ausuble FM, ed. Current protocols in molecular biology. Volume 1, Chapter 6. New York: Wiley, 1995:6.0.3–6.3.5. 7. Bjerknes M, Cheng H. Methods for the isolation of intact epithelium from the mouse intestine. Anat Rec 1981;199:565–574. 8. Meijssen MAC, Deraney K, Bhan AK, Podolsky DK. Altered cytokine and CD14 gene expression by intestinal epithelial cells of interleukin-2 deficient mice (abstr). Gastroenterology 1996;110:A966. 9. Ciacci C, Lind SE, Podolsky DK. Transforming growth factor b regulation of migration in wounded rat intestinal epithelial monolayers. Gastroenterology 1993;5:93–101. 10. Harrison DD, Webster HL. The preparation of isolated intestinal crypt cells. Exp Cell Res 1969; 55:257–260. 11. Brenner CA, Tam AW, Nelson PA, Engleman EG, Suzuki N, Fry KE, Larrick JW. Message amplification phenotyping (MAPPing): a technique to simultaneously measure multiple mRNAs from small number of cells. Biotechniques 1989;7:1096–1103. 12. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal Biochem 1987;162:156–159. 13. Maniatis T, Fritsch EF, Sambrock J. In: Sambrock J, ed. Molecular cloning: a laboratory manual. Chapter 7.37. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982:113. 14. Chantret I, Barbat A, Dussauix E, Brattain MG, Zweibaum A. Epithelial polarity, villin expression and enterocytic differentiation of cultured hunab colon carcinoma cells: survey of twenty cell lines. Cancer Res 1988;48:1932–1942. 15. Ciacci C, Mahida YR, Dignass A, Koizumi M, Podolsky DK. Functional interleukin-2 receptors on intestinal epithelial cells. J Clin Invest 1993;92:527–532. 16. Giri JG, Kumaki S, Ahdieh M, Friend DJ, Loomis A, Shanebeck K, DuBose R, Cosman D, Park LS, Anderson DM. Identification and cloning of a novel IL-15 binding protein that is structurally related to the alpha chain of the IL-2 receptor. EMBO J 1995;14:3654–3663. 17. Tagaya Y, Bamford RN, DeFilippis AP, Waldmann TA. IL-15: a pleiotropic cytokine with diverse receptor/signaling pathways whose expression is controlled at multiple levels. Immunity 1996;4:329–336. 18. Wilkinson PC, Liew FY. Chemoattraction of human blood T lymphocytes by interleukin-15. J Exp Med 1995;181:1255–1259. 19. Colgan SP, Parkos CA, Matthews JB, D’Andrea L, Awtrey CS, Lichtman AH, Delp-Archer C, Madara JL. Interferon-gamma induces a cell surface phenotype switch on T84 intestinal epithelial cells. Am J Physiol 194;267:402–410. 20. Voss SD, Robb RJ, Weil-Hillman G, Hank JA, Sugamura K, Tsudo M, Sondel PM. Increased expression of the interleukin 2 (IL-2) receptor b chain (p70) on CD56/ natural killer cells after in vivo IL-2 therapy: p70 expression does not alone predict the level of intermediate affinity IL-2 binding. J Exp Med 1990;172:1101–1114. 21. Lin JX, Migone TS, Tsang M, Friedmann M, Weatherbee JA, Zhou L, Yamauchi A, Bloom ET, Mietz J, John S, Leonard WJ. The role of shared receptor motifs and common Stat proteins in the generation of cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13, and IL-15. Immunity 1995;2:331–339. 22. Dignass A, Lynch-Devaney K, Kindon H, Thim L, Podolsky DK. Trefoil

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peptides promote epithelial migration through a transforming growth factor b–independent pathway. J Clin Invest 1994;94:376–383. 23. Cepek KL, Shaw SK, Parker CM, Rusell GJ, Morrow JS, Rimm DL, Brenner MB. Adhesion between epithelial cells and T lymphocytes mediated by E-cadherin and the aEb7 integrin. Nature 1994; 372:190–193.

Received July 8, 1996. Accepted September 18, 1996. Address requests for reprints to: Daniel K. Podolsky, M.D., Gastro-

/ 5e14$$0071

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gasa

intestinal Unit, Jackson 7, Massachusetts General Hospital, 32 Fruit Street, Boston, Massachusetts 02114. Fax: (617) 724-2136. Supported by National Institutes of Health grants DK 41557 and DK 43351 (to D.K.P.), DK 21474 (to R.P.M), and DK 51003 and a research grant from the Crohn’s and Colitis Foundation of America (to H.C.R.). Additional support was provided by the K.C. Irving Family Research Fund. Presented in part at the annual meetings of the American Gastroenterological Association in New Orleans in 1995 (Gastroenterology 1995;108:A749) and San Francisco in 1996 (Gastroenterology 1996;110:A110).

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