Human intestinal epithelial cells express functional cytokine receptors sharing the common yc chain of the interleukin 2 receptor HANS -

Interleukin (IL) 2 signaling requires the dimerization of the IL-2 receptor f3 (IL-2Rf3) and common y ('yc) chains. The yc is also a component of the receptors for IL-4, IL-7, and IL-9. To assess the extent and role of the receptor signal transducing system utilizing the yc chain on human intestinal epithelial cells, the expression of yc, IL-2Rj3, and receptor chains specific for IL-4, IL-7, and IL-9 was assessed by reverse transcription-coupled PCR on human intestinal epithelial cell lines and on isolated primary human intestinal epithelial cells. Caco-2, HT-29, and T-84 cells were found to express transcripts for the yc and IL-4R chains constitutively. IL-2Rj8 chain expression was demonstrated in Caco-2 and HT-29 but not in T-84 cells. None of the cell lines expressed mRNA for the IL-2Ra chain. After stimulation with epidermal growth factor for 24 h Caco-2, HT-29, and T-84 cells expressed transcripts for IL-7R. In addition, Caco-2 and HT-29 cells expressed mRNA for the IL-9R. Receptors for IL-2, IL-4, IL-7, and IL-9 on intestinal epithelial cells lines appeared to be functional; stimulation with these cytokines caused rapid tyrosine phosphorylation of proteins. The relevance of the observations in intestinal epithelial cell lines for intestinal epithelial function in vivo was supported by the demonstration of transcripts for yc, IL-2Rg3, IL-4R, IL-7R, and IL-9R in primary human intestinal epithelial cells. Interleukin (IL) 2 appears to play a pivotal role in regulating immune cell populations, exerting its effects through binding to specific cell surface receptors. The IL-2 receptor (IL-2R) consists of three membrane proteins: the IL-2Ra chain, the IL-2R13 chain, and the common y (yc) chain (1). Different combinations of these receptors on human cells give rise to high-, intermediate-, and low-affinity binding sites for IL-2 (1). The IL-2Rf3 and yc chains are required for ligand internalization and signal transduction (2). The yc chain has been shown to dimerize also with the IL-4R (3), IL-7R (4), and IL-9R (5) chains to form signal transducing receptors for these cytokines. The cytokines utilizing the yc chain in their receptor complexes have been identified (1-5) through their regulatory effects on T and B lymphocytes. Little is known about the function of these cytokine receptors and their intracellular signaling pathways in nonhematopoetic cells. In this report we demonstrate that intestinal epithelial cells are able to utilize the yc receptor signal transducing system. Both human intestinal epithelial cell lines and primary human intestinal epithelial cells are able to express transcripts for zc, IL-2R,B, IL-4R, IL-7R, and IL-9R chains. These receptors are functional, as demonstrated by the initiation of tyrosine phosphorylation of proteins within intestinal epithelial cells by IL-2, IL-4, IL-7, and IL-9. Intestinal epithelial cells may be integrated into the mucosal immune system by their ability to respond to cytokines produced by classical immune cells. MATERIALS AND METHODS Cell Culture. Caco-2, HT-29, and T-84 cells were obtained from the American Type Culture Collection and used for experiments after they had reached confluence for 1 week. Caco-2 and HT-29 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol/vol) fetal calf serum and 4 mM L-glutamine (Sigma) in tissue culture plates (Costar). T-84 cells were cultured in DMEM/ F-12 medium (GIBCO) containing 10% fetal calf serum. For the detection of cytokine transcripts, cells were stimulated by replacement with fresh medium with or without recombinant epidermal growth factor (EGF) (R & D Systems; 100 ng/ml). Isolation of Primary Intestinal Epithelial Cells. Intestinal epithelial cells were isolated from colonic biopsies by modifications of a technique described by Harrison and Webster (6). Separated epithelial cells were resuspended in DMEM and 50 ,ul of cell suspension was placed on a caved glass slide and overlayed with mineral oil. Single epithelial cells were then transferred to a second slide with a micropipette (see Fig. 2). RNA from 20 to 40 primary epithelial cells was isolated after addition of 50 jig of carrier rRNA from Escherichia coli W (Sigma). Reverse Transcription. Total cellular RNA was isolated by a modified acid guanidinium isothiocyanate/phenol/ chloroform extraction (7). Total sample RNA was reversetranscribed by using 200 units of Moloney murine leukemia virus reverse transcriptase (GIBCO), 20 units of RNasin (Promega), 1 ,LM dGTP, 1 ,uM dATP, 1 ,uM dTTP, and 1 ,uM dCTP, and 1 ,ug of hexanucleotide random primer (Boehringer Mannheim) in 50 mM Tris HCl, pH 8.3/75 mM KCl/3 mM MgCl2/10 mM dithiothreitol for 1 h at 37°C. PCR Amplification. Primers for amplification of transcripts encoding the IL receptor subunits were designed by using OLIGO 4.0 software and synthesized on a PCR-Mate DNA synthesizer (Applied Biosystems) (Table 1). The PCR mixture contained 1x PCR buffer (Perkin-Elmer/Cetus, with 1.5 mM Mg), all four dNTPs (each at 50 ,uM), each 5' and 3' primer at 1 ,uM, and 1 unit of Taq polymerase (Perkin-Elmer/Cetus), in a total volume of 100 ,ul. The PCR specific for IL-7R transcripts was carried out with the addition of 10 ,uCi [a-32P]dATP (Amersham; 1 Ci = 37 GBq) per reaction. IL-9R transcripts were detected by using a nested PCR, which was carried out for 20 cycles with the first set of primers, diluted 1:100, and continued with the second set of primers for an additional 30 cycles. Amplification was carried out on a Abbreviations: IL, interleukin; IL-nR, IL-n receptor; RT-PCR, reverse transcription-coupled PCR; PTK, protein tyrosine kinase; yc chain, common y chain; EGF, epidermal growth factor; BSA, bovine serum albumin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 8353 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 8354 Cell Biology: Reinecker and Podolsky Table 1. PCR primers for the detection of cytokine receptor transcripts and control transcripts used in this study