Increased expression of keratinocyte growth factor messenger RNA associated with inflammatory bowel disease

GASTROENTEROLOGY 1996;110:441–451

Increased Expression of Keratinocyte Growth Factor Messenger RNA Associated With Inflammatory Bowel Disease PAUL W. FINCH,* VICTOR PRICOLO,‡ ANDREW WU,§ and SYDNEY D. FINKELSTEINx *Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York; Departments of ‡Surgery and §Clinical Neurosciences, Rhode Island Hospital and Brown University, Providence, Rhode Island; and 㛳Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania

Background & Aims: Alterations in intestinal epithelial cell function are common in inflammatory bowel disease (IBD). Keratinocyte growth factor (KGF) is an epithelial cell–specific mitogen. The aim of this study was to examine IBD tissue for altered KGF expression. Methods: Expression levels of the KGF and KGF receptor transcripts were analyzed by ribonuclease protection assay. The cellular localization of each transcript was determined using in situ hybridization. Results: KGF messenger RNA levels were increased in inflamed IBD tissue in comparison with control tissues. In normal tissue, KGF messenger RNA was localized to the mesenchymal cells at the tip of the villi in the small intestine and directly underlying the mature enterocytes in the colon, whereas in IBD it was present throughout the lamina propria, although distinct from the germinal centers. The topographic distribution of the KGF in situ hybridization signal in IBD was similar to that observed for T lymphocytes. In contrast, KGF receptor transcripts were localized to the cryptal region of the mucosal epithelium in both normal and IBD tissue, with no apparent differences in the level of expression. Conclusions: The increased expression of KGF in IBD suggests that it may be involved in mediating the altered regulatory functions of intestinal epithelial cells in this disease.

I

nflammatory bowel disease (IBD) encompasses at least two forms of intestinal inflammation: ulcerative colitis (UC) and Crohn’s disease (CD). Although these forms of inflammation are probably distinct in their initial pathogenic events, they share some clinical and pathological manifestations.1,2 Alterations in intestinal epithelial cell (IEC) phenotype and function are common in both UC and CD. Epithelial proliferation and turnover is more rapid in IBD than in the normal gut.3,4 There are cytological alterations in IECs in IBD that are characteristic of the disease stage.5 Epithelial permeability to ions, small solutes, and macromolecules is increased in IBD.6,7 There is also an altered antigen presentation by IECs in IBD. In vitro antigen presentation by normal IECs stimulates the proliferation of suppressor T cells, indicating that / m4787$0030

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they play a central role in down-regulating normal mucosal immune responses.8 – 10 In contrast, antigen presentation by IECs in IBD leads primarily to the activation of T cells with potent helper activity.2 The normally downregulated inflammatory response would no longer be suppressed, and the resulting production of inflammatory mediators by highly activated effector cells would lead to prolonged and intense damage to the mucosal epithelial layer. That such alterations are important in vivo is suggested by observations that the expression of certain class II antigens, which bind processed peptides that have been taken up exogenously and present them to T lymphocytes, is increased in IECs in IBD.11,12 Despite this evidence for the central role of epithelial regulation of intestinal function and mucosal immune responses, very little is known about the normal mechanisms that underlie these processes or how they are altered in IBD. Keratinocyte growth factor (KGF) is a fibroblast-derived member of the fibroblast growth factor (FGF) family, which has potent mitogenic activity on epithelial cells but no corresponding activity on fibroblasts, endothelial cells, or other nonepithelial targets of FGF action.13,14 The KGF receptor (KGFR) is a transmembrane tyrosine kinase, which is a splice variant of the FGFR2/bek gene.15,16 The KGFR binds KGF and acidic FGF with high affinity and basic FGF at a lower affinity17 and is expressed only by epithelial cells, whereas FGFR-2 is present in a variety of different cell types.16 Expression of KGF and KGFR messenger RNA (mRNA) has been detected throughout the gastrointestinal tract in adult rats, suggesting a possible role for endogenous KGF in the maintenance of mucosal epithelial populations.18 Indeed, the ability of systemically administered KGF to rapidly induce the proliferation of Abbreviations used in this paper: FGF, fibroblast growth factor; FGFR, FGF receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GC, germinal centers; IEC, intestinal epithelial cells; ISH, in situ hybridization; KGF, keratinocyte growth factor; KGFR, KGF receptor; RNase, ribonuclease. 䉷 1996 by the American Gastroenterological Association 0016-5085/96/$3.00

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Figure 1. RNase protection assay of KGF and KGFR mRNA in IBD. Total cellular RNA (10 mg) extracted from normal, neoplastic, and IBD tissue samples was hybridized to 32P-labeled antisense KGF, KGFR, and GAPDH probes. After digestion with RNase, protected hybrids were resolved by electrophoresis through denaturing polyacrylamide gels. RNA extracted from the human embryonic lung fibroblast cell line, M426, and an immortalized human keratinocyte cell line were included as controls for KGF and KGFR expression, respectively. N, normal tissue; T, tumor tissue; U, uninvolved UC or CD tissue; I, involved UC or CD tissue. Exposure times were as follows: KGF, 24 hours; KGFR, 72 hours; GAPDH, 6 hours.

epithelial cells from the foregut to the colon, with continued treatment leading to the selective induction of mucin-producing cells in a dose-dependent fashion,18 provides functional evidence for the ability of KGF to activate gastrointestinal epithelial populations in vivo. In addition, it has recently been reported that expression of the KGF gene can be induced by interleukin 1 and interleukin 6.19,20 Given that these proinflammatory cytokines are likely to play critical roles in the pathogenesis of IBD21 – 23 and the fact that the gastrointestinal tract appears to be one of the major targets of endogenous KGF action in vivo, we examined IBD tissue for evidence of altered KGF expression associated with this disease.

Materials and Methods Collection of Tissue Fourteen surgical specimens of UC and 9 of CD, comprising full-thickness samples of the uninvolved and involved regions of the bowel, were collected directly from the operating room. The specimens were inspected by a pathologist (S.D.F.) for histological features consistent with acute and chronic stages of IBD. Acute aspects included mucosal ulceration, crypt abscess formation, and acute inflammatory infiltration of the lamina propria. Chronic histological findings included crypt distortion, architectural branching, and mucosal atro-

phy. Control tissues included 1 specimen from a patient with a premalignant colorectal lesion and 3 specimens of sporadic colorectal cancer. Tissue taken from these patients, at least 10 cm away from the tumor, served as normal controls. In addition, 1 case of diverticulitis was included as a control for non-IBD inflammatory disease. All human tissues collected for this study were from surplus remnants in excess of that required for adequate pathological examination. The collection and use of these tissues for this study were approved by the Mount Sinai and Rhode Island Hospital Institutional Review Boards. For RNA extraction, the tissues were snap-frozen in liquid nitrogen and stored at 070⬚C. Total cellular RNA was isolated as described by Chirgwin et al.24 For in situ hybridization (ISH), tissues were fixed for 8 hours in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4⬚C. After fixation, tissues were dehydrated through graded alcohols, cleared with toluene, and embedded in paraffin. Sections were cut at 6 mm and mounted on aminoalkylsilane-treated slides.

Generation of KGF and KGFR Complementary DNA Probes A 145–base pair (bp) BamHI/EcoRI fragment of the human complementary DNA (cDNA) sequence (nucleotides 1–145) served as a probe for ribonuclease (RNase) protection, and a 324-bp EcoRI/BamHI fragment (nucleotides 145–469) was used for ISH. A 148-bp polymerase chain reaction fragment specific for exon K of the human bek gene16 served as a KGFR probe for RNase protection and ISH. These fragments were cloned into pGEM3Zf(0) (Promega, Madison, WI). Transcription with T7-RNA polymerase generated antisense transcripts from the 145-bp KGF and 148-bp KGFR constructs, and SP6-RNA polymerase generated the antisense strand transcript from the 324-bp KGF probe. The human vimentin probe was a 396-bp BamHI/PstI fragment from cHuViml25 (purchased from the American Type Culture Collection, Rockville, MD) subcloned into pGEM3Zf(0) so that transcription with SP6 polymerase generated the antisense transcript. For the RNase protection assay, transcription reactions were performed using 32P-labeled uridine triphosphate (800 Ci/mmol; New England Nuclear, Boston, MA) as the labeled nucleotide, whereas for ISH experiments transcripts were labeled using [33P]uridine triphosphate (1000–3000 Ci/ mmol; New England Nuclear). Labeled transcripts were prepared as described.26

RNase Protection Assay Ten micrograms of total cell RNA was hybridized overnight at 43⬚C with 1 1 105 cpm of a gel-purified probe. Hybrids were digested with 0.1 U RNase A and 20 U RNase

䉴 Figure 2. ISH of KGF mRNA expression in a normal human intestine. (A and C ) Bright-field and (B and D ) corresponding dark-field micrographs of intestinal tissue sections. (A and B ) KGF mRNA expression. Note KGF signal in tips of the villi. (C and D ) Vimentin mRNA expression. Note strong signal throughout the lamina propria consistent with expression from primarily mesenchymal cells. No signal is present in the intestinal epithelial cells (toluidine blue; original magnification 781).

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T1 (Ambion Inc., Austin, TX) for 60 minutes at 35⬚C. Protected fragments were resolved on 6% polyacrylamide and 8 mol/L urea gels and visualized by autoradiography.

ISH ISH was performed on deparaffinized tissue sections essentially as described by Wilkinson et al.27 using 33P-labeled RNA probes at a concentration of 0.2 ngrmL01rkb length01 of a cloned fragment in hybridization buffer (50% deionized formamide; 0.3 mol/L NaCl; 20 mmol/L Tris-HCl, pH 8.0; 5 mmol/L ethylenediminetetraacetic acid [EDTA]; 10% dextran sulfate; 11 Denhardt’s buffer; and 0.5 mg/mL yeast RNA). Control sections received similar concentrations of the corresponding labeled sense strand RNA probe. Tissue sections were hybridized at 55⬚C for 16–18 hours and then washed under high stringency conditions (21 standard saline citrate, 50% formamide at 65⬚C) for 30 minutes. Nonhybridized probe was digested with 20 mg/mL RNase A for 30 minutes at 37⬚C. After further washing under high stringency conditions, slides were dehydrated through graded alcohols containing 0.3 mol/L ammonium acetate. Sections were dipped in NTB-2 emulsion (Eastman Kodak, Rochester, NY), diluted 1:1 in H2O, airdried, and stored desiccated at 4⬚C. After appropriate exposure times, slides were developed in Kodak D-19 developer and counterstained with 0.02% toluidine blue.

Immunohistochemistry Indirect staining with specific antibodies was conducted using a single-step immunoperoxidase technique in which the primary antibody was linked to horseradish peroxidase by using a flexible polymer backbone (Dako Corp., Carpinteria, CA). The specific antibodies used were anti-CD3 (rabbit polyclonal), anti-CD20 (clone L26), and anti-CD68 (clone PG-M1), indicating T cells, B cells, and macrophage origin, respectively. Mouse immunoglobulins and horseradish peroxidase coupled to the same inert polymer backbone were used as a negative control (Dako Corp.). Endogenous peroxidase activity was blocked by preincubation of tissue sections with 3% hydrogen peroxide in distilled water for 5 minutes. After incubation with the appropriate antibodies, tissue sections were washed with PBS, incubated with the chromogenic substrate solution (diaminobenzidine tetrachloride) according to the manufacturer’s protocol (Dako Corp.), rinsed with water, counterstained with hematoxylin, and mounted with coverslips.

Results Increased Expression of KGF mRNA in IBD RNA extracted from IBD tissue specimens was screened for KGF and KGFR transcript expression by RNase protection assay. Control tissues included 1 specimen from a patient with a premalignant colorectal lesion (specimen 2T), 3 specimens of sporadic colorectal cancer (specimens 3T, 5T, and 6T), and 1 case of diverticulitis that was included as a control for non-IBD inflammatory / m4787$0030

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disease (specimen 10U and I). Examples of the expression data are shown in Figure 1. Increased KGF expression was found in all cases of IBD in comparison with histologically uninvolved tissue removed from the same specimen, with the exception of specimen CD4. In some cases (UC5, CD5, and CD8), transcript levels were at least as high as that detected in the positive control M426 embryonic lung fibroblast cell line. This cell line, which was used to collect the conditioned medium from which KGF was originally purified, contains high levels of KGF mRNA and protein.13,14 In contrast, KGF transcript levels were unaltered in the premalignant cancer lesion and markedly decreased in all the specimens of colorectal cancer, relative to normal colon tissue. No change in KGF transcript expression was observed in the 1 case of diverticulitis. These results indicate that increased expression of the KGF message is frequently associated with inflamed IBD tissue in comparison with uninvolved and normal tissue. Furthermore, such alterations were not detected in neoplastic or non-IBD inflammatory disease. The expression of the KGFR was also examined. Although KGFR expression was detected in all of the samples analyzed, there appeared to be an overall trend for decreased expression of the transcript in inflamed tissue in comparison with that present in uninvolved tissue. Analysis of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcript in the same specimens showed the integrity of the RNA used in each assay (Figure 1). ISH Analysis of KGF/KGFR Expression in the Normal Intestine To fully evaluate the pathological implications of KGF/KGFR expression in IBD, it was necessary to first determine the cellular basis of expression of these genes in the normal intestine using ISH. Six-micrometer-thick paraffin-embedded sections were hybridized to 33P-labeled antisense cRNA hybridization probes complementary to the primary KGF and KGFR transcripts, and the resulting hybrids were detected by nuclear-track emulsion autoradiography. The localization of silver grains over the tissue section showed that the KGF signal was present in the mesenchymal cells at the tip of the villi in the small intestine (Figure 2A and B) and directly underlying the mature enterocytes in the colon. In contrast, the KGFR transcript was localized to the mucosal epithelial layer, with most signal being present in the more mitotically active crypt regions (Figure 3A and B). Hybridization of serial sections with the 33P-labeled sense strand cRNA KGF or KGFR probes resulted in sparse background labeling of cells (results not shown). ISH of equivalent areas with an antisense probe for vimentin, which served as a positive control, resulted in a strong WBS-Gastro

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Figure 3. ISH of KGFR mRNA expression in a normal human intestine. (A and C ) Bright-field and (B and D ) corresponding dark-field micrographs of intestinal tissue sections. (A and B ) KGFR mRNA expression in intestinal epithelium. Signal is present in the mucosal epithelial cells, with most grains present in the crypt regions (straight arrows) and relatively few grains in the mature enterocytes (curved arrows). (C and D ) Vimentin mRNA expression (toluidine blue; original magnification 781).

signal throughout the lamina propria and deeper levels of the bowel wall, consistent with expression from primarily mesenchymal cells (Figure 2C and D and Figure 3C and D). Intestinal epithelial cells were not positive for a vimentin signal. ISH Analysis of KGF/KGFR Expression in IBD ISH was then used to investigate KGF/KGFR expression in IBD. There was a close relationship between the degree of ongoing inflammation and the elevated KGF expression. For example, in cases showing only a moderate degree of inflammation, the KGF signal was still present in the mesenchymal cells directly underlying the mature enterocytes. However, the KGF mRNA expression was also present overlying a subpopulation of pericryptal cells (Figure 4A and B). In specimens with severe inflammation, this pattern was much more exaggerated, with high levels of KGF mRNA expression present in a subset of lamina propria cells throughout the upper mucosa but distinct from the germinal centers / m4787$0030

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(Figure 4C and D). Furthermore, these cells had the histological appearance of lymphocytes (Figure 4E). This relationship between increased KGF expression and the degree of inflammation was found in both UC and CD specimens. Specimens with quiescent IBD showed a reduced KGF signal compared with inflamed tissue. Normal-appearing mucosae had the patterns of expression described in the previous section. In contrast to the KGF expression, the KGFR transcript was localized to the mucosal epithelial cell layer. As was seen with normal tissue, most of the KGFR signal was present in the crypt regions of the mucosal epithelial layer, with no apparent change in the level of KGFR mRNA expression between normal and IBD tissue specimens or between IBD tissues having different levels of inflammation (Figure 5A–D). Further Investigation of the Cellular Origin of KGF Synthesis in IBD KGF mRNA was not uniformly expressed throughout the lamina propria in IBD but was limited to a subset of cells situated in the upper mucosa away from the germinal WBS-Gastro

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Figure 4. ISH of KGF mRNA in IBD. (A, C, and E ) Bright-field and (B and D ) corresponding dark-field micrographs of IBD tissue sections. (A and B ) Tissue section showing moderate inflammatory infiltration of the lamina propria. KGF signal is present in the mesenchymal cells underlying the mature enterocytes (large arrows) as well as in pericryptal cells (small arrows) (toluidine blue; original magnification 781). (C and D ) Tissue section showing acute inflammatory infiltration of the lamina propria. There is a strong KGF signal in the upper mucosa but not associated with the germinal centers (GC) (toluidine blue; original magnification 12.51). (Continued on next page.)

centers, which had the morphological characteristics of lymphocytes (Figure 4C–E). To further investigate which cell population was associated with increased KGF expression in IBD, immunohistochemistry was performed using anti/ m4787$0030

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bodies for the major lymphocyte populations. Sections were stained using anti-CD3, -CD20, and -CD68 antibodies, which indicate T-cell, B-cell, and macrophage origin, respectively, and then compared with KGF ISH sections WBS-Gastro

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Figure 4 (cont’d.). (E ) High-power field of section shown in C showing localization of KGF mRNA to pericryptal lymphoid-like cells (toluidine blue; original magnification 1561).

taken from the same specimen. The topographical distribution of pericryptal lymphocytes and lymphocytes outside of the germinal centers, which showed a T-cell immunohistochemical marking, closely corresponded to that of the KGF ISH signal (Figure 6A–C).

Discussion In this study, we used an RNase protection assay to show that increased expression of KGF mRNA is associated with inflamed IBD tissue but not with normal, uninflamed IBD, non-IBD inflammatory, or neoplastic intestinal tissue. ISH was used to further investigate the cellular basis of KGF and KGFR expression in normal and IBD tissue specimens. In the normal small intestine and colon, KGF was localized to the mesenchymal cells directly underlying the mature enterocytes, corresponding to the region in which mature cells are lost. In contrast, the KGFR mRNA signal was localized to the mucosal epithelial layer and was most prominent in the / m4787$0030

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crypt cells. Presumably, mechanisms must exist to balance proliferative activity in the crypt with subsequent differentiation and loss of mature enterocytes.28 These patterns of KGF and KGFR expression raise the possibility of two mechanisms whereby KGF could help to mediate these processes. First, KGF diffusion down from the villus tip could stimulate the proliferation and subsequent differentiation of cryptal epithelial cells. Alternatively, there may be an additional KGFR species that mediates the gradient of increasing IEC differentiation along the longitudinal axis of the villus. The KGFR is encoded by an alternative exon of the FGFR2 gene, known as the IIIb exon.16 Corresponding IIIb exons exist in the FGFR1 and FGFR3 genes,29,30 which are 78% and 40% homologous, respectively, to the IIIb exon of the FGFR2 gene. Although the FGFR3 IIIb isoform is associated with the human colonic epithelium,30 it has only been shown to bind acidic FGF with high affinity, not KGF.31 Therefore, it seems unlikely that this receptor isoform mediates mucosal epithelial responses to KGF. In comparison with normal tissue, increased KGF mRNA was observed in inflamed IBD tissue. Furthermore, ISH analysis showed a strikingly different pattern of KGF transcript synthesis. Instead of highly localized expression to mesenchymal cells directly beneath the mature enterocytes (Figure 2), there was a widespread and intense signal throughout the upper mucosa (Figure 4). These observations suggest that increased levels of KGF may play a role in mediating the altered growth and regulation of IBD IECs. Supporting this hypothesis is a recent study showing that systemic administration of KGF to adult rats induced the proliferation of epithelial cells throughout the gastrointestinal tract.18 In the small and large intestines, KGF treatment resulted in crypt hyperplasia after crypt elongation and an increased number of separated (bifurcated and trifurcated) crypts. Hyperplasia and increased separation of crypts are common histological features of IBD, both of which become more prominent with increasing inflammation. In addition, KGF treatment caused a specific increase in the differentiation of mucus-producing goblet cells on the villi.18 Undifferentiated epithelial stem cells are located in the midcrypts and produce daughter cells that continuously migrate upward to the crypt mouth and downward to the crypt base. These cells differentiate into absorptive cells and goblet cells above and Paneth’s cells and enteroendocrine cells below. Our ISH results, showing that the KGFR mRNA is localized predominantly in the cryptal region, would therefore be consistent with a role for KGF in stimulating the proliferation of goblet cells, through stimulation of either stem cells or specific goblet-cell precursors. Goblet-cell depletion is a further defining WBS-Gastro

Figure 5. ISH of KGFR mRNA in IBD. (A and C ) Bright-field and (B and D ) corresponding dark-field micrographs of toluidine blue–stained IBD tissue sections. Tissue sections showing (A and B ) moderate or (C and D ) acute inflammatory infiltration of the lamina propria. In each case, the KGFR mRNA is predominantly localized to the mucosal epithelial cells lining the colonic crypts (toluidine blue; original magnification 1561 [A and B ] and 781 [C and D ]).

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Figure 6. Comparison of the KGF ISH signal and T-cell immunohistochemical staining in IBD. (A ) Bright-field and corresponding (B ) dark-field micrographs of IBD tissue sections after ISH for KGF mRNA. Note strong signal for KGF evident throughout the lamina propria (toluidine blue; original magnification 62.51). (C ) CD3 immunohistochemical staining for T cells of the colonic mucosa. The topographical distribution of lymphocytes showing T-cell immunohistochemical staining closely corresponds to that of the KGF ISH signal (hematoxylin; original magnification 62.51).

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histopathologic feature of inflamed UC tissue. It is an intriguing possibility that increased KGF expression represents a key mechanism in mediating changes in the proliferation and physiology of this epithelial cell lineage in IBD. These in vivo results suggest a functional role for KGF in mediating mucosal epithelial proliferation in the gastrointestinal tract. The fact that systemic KGF administration results in the proliferation of one of the key epithelial cell lineages affected during inflammation and common histological changes (crypt hyperplasia and branching) associated with IBD suggests that increased KGF expression may result in similar changes in the mucosal epithelium of inflamed tissue. Our results show that increased expression of the KGF gene is frequently associated with inflamed IBD tissue. KGFR transcripts were also detected. However, using RNase protection assay, there appeared to be decreased transcript levels in inflamed tissue compared with normal and noninflamed IBD tissue. This may reflect downregulation of KGFR mRNA synthesis in IBD or the destruction of the mucosal layer in chronically inflamed tissue, resulting in fewer KGFR-containing epithelial cells present in these specimens. Qualitative comparison of KGFR mRNA expression between normal and inflamed tissue specimens using ISH indicated that KGFR expression in IBD IECs was not appreciably different than that in normal IECs, both in terms of the overall intensity and the distribution of the signal. This suggests that mucosal destruction may be responsible for the decrease in KGFR-transcript expression in the RNA that was isolated from IBD tissue specimens as detected by RNase protection assay. The use of freshly isolated populations of normal and IBD IECs from surgical specimens will allow a quantitative assessment of KGFR mRNA expression. An examination of the increased KGF ISH signal in IBD tissue sections indicated that it was associated with a subpopulation of pericryptal cells with the histological characteristics of lymphocytes (Figure 4E). Comparison of the topographic distribution of the KGF ISH signal with the staining pattern for an anti-CD3 antibody suggested the possibility that these cells were lymphocytes of T-cell origin (Figure 6). There has been a recent report of KGF-transcript synthesis by activated intraepithelial gd T cells obtained from skin and intestinal tissues, but not intraepithelial ab T cells or peripheral ab or gd Tcell populations.32 It was suggested that the restricted nature of the antigen receptor expression by gd T cells residing in epithelia makes it probable that the function of these cells differs from other T cells, perhaps through the ability to recognize and orchestrate the repair of damaged epithelium.32 However, our ISH data provide / m4787$0030

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no evidence that KGF mRNA is associated with this cell population in IBD. Clearly, further work is required to confirm whether KGF is indeed synthesized by T lymphocytes in inflamed tissue or, alternatively, whether the similarity in the distributions of the KGF ISH signal and the T-cell immunohistochemical marking is caused by increased KGF expression by lamina propria mesenchymal cells after induction by the local release of inflammatory mediators from neighboring activated T cells. Resolution of this important question, and a demonstration of functional responses to KGF by normal and IBD IECs will be required to fully appreciate the implications of increased KGF expression in this disease.

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Received August 17, 1995. Accepted October 23, 1995. Address requests for reprints to: Paul W. Finch, Ph.D., Derald H. Ruttenberg Cancer Center, Box 1130, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, New York 10029-6574. Fax: (212) 987-2240. Supported by grants from the Crohn’s and Colitis Foundation of America and Amgen Inc. and by National Institutes of Health grant DK47102 (to P.W.F.). The authors thank Dr. Lisa Glantz for help in collecting some of the tissue specimens used in this study and Drs. M. Babyatsky and L. Mayer of the Mount Sinai School of Medicine for careful reading of this manuscript.

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