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. 1999 Dec;45(6):848–855. doi: 10.1136/gut.45.6.848

Analysis of the cellular basis of keratinocyte growth factor overexpression in inflammatory bowel disease

P Finch 1, A Cheng 1
PMCID: PMC1727748  PMID: 10562583

Abstract

BACKGROUND—Keratinocyte growth factor (KGF) stimulates gastrointestinal epithelial cells in vivo, and is protective against gastrointestinal injury and colitis. Endogenous KGF is increased in inflammatory bowel disease (IBD), and may be an important mediator of mucosal repair. KGF is expressed by mesenchymal cells and activated intraepithelial lymphocytes (IEL).
AIMS—To investigate the relative contributions of these cellular sources of KGF expression in IBD.
METHODS—IELs and lamina propria lymphocytes (LPL) were isolated from inflamed and uninflamed IBD tissues. mRNA expression was determined by ribonuclease protection assay. In situ hybridisation was combined with immunohistochemistry to determine whether KGF mRNA was expressed by specific cell types in vivo.
RESULTS—Low levels of KGF mRNA expression were detected in three of five IEL samples derived from inflamed tissue, but not in two IEL samples from uninflamed tissue. No KGF expression was detected in LPLs from either inflamed or uninflamed tissue. In contrast, KGF was expressed by primary cultures of human intestinal fibroblasts, and was induced by treatment with interleukin 1.
CONCLUSIONS—The major source of KGF expression in IBD was lamina propria cells of non-immune origin, most likely fibroblasts and/or smooth muscle cells. Compared with these cell types, relatively little KGF synthesis was associated with IELs in inflamed IBD tissue.


Keywords: fibroblasts; intestinal inflammation; intraepithelial lymphocytes; keratinocyte growth factor; lamina propria lymphocytes

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Figure 1  .

Figure 1  

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Analysis of keratinocyte growth factor (KGF), CD45, and α2(I) type I procollagen transcripts in lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) isolated from uninvolved and inflamed inflammatory bowel disease tissues. RNAs (2 µg samples) were hybridised to 32P-labelled antisense probes and then digested with RNAse. Protected hybrids were resolved by electrophoresis through denaturing polyacrylamide gels. RNAs extracted from the embryonic lung fibroblast cell line, M426, and from three primary cultures of human intestinal fibroblasts isolated from normal (N), ulcerative colitis (UC), and Crohn's disease (CD) tissue, were included as positive controls for KGF and α2(I) type I procollagen expression. RNA isolated from the MOLT-4 T cell line was used as a positive control for CD45 expression. Total RNAs isolated from an uninvolved (U) and inflamed (I) region of ulcerative colitis were included for comparison. Exposure times were as follows: KGF, 72 h; CD45, 72 h; α2(I) type I procollagen, 24 h.

Figure 2  .

Figure 2  

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Joint localisation of keratinocyte growth factor (KGF) transcript expression and T cell, B cell, and macrophage immunohistochemical staining in uninvolved and inflamed inflammatory bowel disease tissues. Sections were processed for in situ hybridisation using an antisense KGF cRNA probe. The immunohistochemical label (brown) was visualised after incubation with antibodies directed against the CD3 (A and D), CD20 (B and E), or CD68 epitopes (C and F), which are specific for T cells, B cells, and macrophages, respectively. Following exposure to NTB-2 emulsion, slides were developed and counterstained with haematoxylin. Autoradiographic grain clusters show cells expressing KGF mRNA (arrows). (A-C) uninvolved tissue, original magnification × 156; (D-F) inflamed submucosa, original magnification × 187. Representative immunohistochemically labelled cells in each section are indicated by red arrows, and KGF expressing cells, identified by autoradiographic silver grain clusters, are indicated by black arrows.

Figure 3  .

Figure 3  

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Induction of keratinocyte growth factor (KGF) mRNA by interleukin (IL) 1α in human intestinal fibroblasts (HIFs). Primary HIFs were derived from normal (N), and inflamed ulcerative colitis (UC) and Crohn's disease (CD) tissue. Cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum until 90% confluent, serum starved for 24 hours, and incubated with 20 ng/ml IL-1α for six hours (+) or left untreated (−). RNA was prepared and 10 µg samples were analysed by ribonuclease protection assay for KGF and α2(I) type I procollagen. Exposure times were 24 hours for the KGF autoradiograph, and six hours for α2(I) type I procollagen.

Figure 4  .

Figure 4  

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Induction of keratinocyte growth factor (KGF) RNA by interleukin (IL) 1α in 7-17 γδ dendritic epidermal T cells. Serum starved 7-17 cells were treated for 12 hours with IL-1α (20 ng/ml) in the presence or absence of ibuprofen (120 µM), dexamethasone (100 nM), 6-mercaptopurine (10 µM), or cyclosporin (1 µM). The control culture received vehicle alone. Total RNA was prepared and 10 µg samples were analysed by ribonuclease protection assay. Exposure times were 36 hours for the KGF autoradiograph, and six hours for glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Figure 5  .

Figure 5  

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In situ hybridisation (ISH) analysis of keratinocyte growth factor (KGF) RNA expression in inflammatory bowel disease tissue of patients not receiving steroidal anti-inflammatory medication. Sections were processed for ISH using an antisense KGF cRNA probe, and then for immunohistochemistry using an anti-CD3 monoclonal antibody. Following exposure to NTB-2 emulsion, slides were developed and counterstained with haematoxylin. (A) Ulcerative colitis; (B) Crohn's disease; (C) ischaemic colitis. Intraepithelial lymphocytes are indicated by grey arrows, and representative KGF expressing cells by black arrows. Original magnification × 187.    

Figure 6  .

Figure 6  

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In situ hybridisation (ISH) analysis of keratinocyte growth factor (KGF) RNA expression in ischaemic colitis, shown in bright field (A and C) and corresponding dark field (B and D) micrographs of inflamed human intestinal tissue. Sections were processed for ISH using an antisense KGF cRNA probe. In (A), the mucosal epithelial layer is still intact, whereas in (C) the mucosal layer has been destroyed, and is absent. Numerous KGF expressing cells are present in the lamina propria of both tissue sections. Representative KGF expressing cells are indicated by arrows in (B) and (D). Stain: haematoxylin; original magnification × 62.5. 

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