Specificity of the Localization of Transforming Growth Factor-α Immunoreactivity in Colon Mucosa

Abstract

SUMMARY Transforming growth factor-␣ (TGF-␣ ) plays an important role in gastrointestinal pathophysiology. However, the exact location of its expression in the intestine is still controversial. This study systematically compared the localization of TGF-␣ immunoreactivity in frozen or fixed human colon using three different antibodies and examined specificity of antibodies by using tissues from TGF-␣ knockout mice and by Western blotting. Consistent with the mRNA distribution revealed by in situ hybridization, a similar staining pattern was obtained in frozen sections by all three antibodies, localizing on the surface and along the crypt epithelium. In paraffin sections, although the polyclonal antibodies (raised against recombinant human or rat TGF-␣ ) gave minimal staining, the monoclonal antibody (against C-terminal peptide of human TGF-␣ ) still gave intense staining on the surface and upper crypt epithelium. By using specimens from TGF-␣ knockout mice in immunostaining and Western blotting, the polyclonal antibodies were shown to be specific. In contrast, specificity of the monoclonal antibody was in doubt in rodent tissues because it gave similar detection between wild-type and knockout mice in both analyses, indicating its crossreaction to non-TGF-␣ molecules. In conclusion, frozen sections and antibodies raised from recombinant TGF-␣ should be used for TGF-␣ immunohistochemistry in the colon. (J Histochem Cytochem 47:949-957, 1999)

Effects of TGF‐α gene knockout on epithelial cell kinetics and repair of methotrexate‐induced damage in mouse small intestine

“…Dunn (Ludwig Institute of Cancer Research, Melbourne, Australia). The homozygous TGF-a null mutation resulted in a complete lack of TGF-a peptide synthesis in vivo as demonstrated previously (Mann et al, 1993;Xian et al, 1999b). To compare intestinal crypt cell production between the TGF-a wild type and knockout mice, the vincristine metaphase arrest technique was employed (Wright and Appleton, 1980).…”

Intestinal macrophages: differentiation and involvement in intestinal immunopathologies

“…A variety of intestinal cell types including epithelial cells, subepithelial myofibroblasts, fibroblasts, lamina propria lymphocytes, and intraepithelial lymphocytes are all able to produce soluble factors, particularly, TGFβ and IL-10, which may affect the phenotypic and functional properties of intestinal macrophages [57][58][59][60]. The importance of stromal cell-derived factors in shaping the functional capacities of intestinal macrophages is illustrated by the effects induced in blood monocytes upon exposure to intestinal stromal cell-conditioned media.…”

Cloning of Rat Betacellulin and Characterization of its Expression in the Gastrointestinal Tract

“…In the colon, we have also detected BTC expression in both crypt and surface epithelium. Previously, TGFa has been observed to be expressed throughout the crypt epithelium and surface epithelium of the colon (Babyatsky et al, 1996;Xian et al, 1999a).…”

“…BTC was shown to be a potent mitogen for epithelial and smooth muscle cells and is unique amongst the EGF ligands by virtue of its preferential dominant expression in human pancreas and small intestine Masaharu etal., 1996). The receptor for BTC, EGF-R, and other EGF family ligands, notably TGFa, amphiregulin and HB-EGF, have previously been found to be expressed in the gastrointestinal tract (Barnard et al, 1995;Beauchamp etal., 1989;Xian et al, 1999a). Indeed TGFa amphiregulin and HB-EGF have all been postulated to play important roles in the development, maintenance and defence of the gastrointestinal tract (Barnard et al, 1995).…”