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Expression and Regulation of the Human β-Defensins hBD-1 and hBD-2 in Intestinal Epithelium

Deborah A. O’Neil, Edith Martin Porter, Dirk Elewaut, G. Mark Anderson, Lars Eckmann, Tomas Ganz and Martin F. Kagnoff
J Immunol December 15, 1999, 163 (12) 6718-6724;
Deborah A. O’Neil
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Edith Martin Porter
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Dirk Elewaut
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G. Mark Anderson
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Lars Eckmann
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Tomas Ganz
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Martin F. Kagnoff
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  • FIGURE 1.
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    FIGURE 1.

    hBD-1 mRNA is constitutively expressed by colon epithelial cells, whereas expression of hBD-2 mRNA is up-regulated by IL-1 and enteroinvasive bacteria. RNA from Caco-2 and HT-29 cells was amplified by RT-PCR with primers specific for hBD-1, hBD-2, and β-actin as detailed in Materials and Methods. The first lane contains no RNA. PCR products from control Caco-2 and HT-29 cells left untreated are shown in the second lane, and those from cells following stimulation with optimal concentrations of agonist or bacterial infection are as follows: IL-1α stimulated (20 ng/ml), TNF-α stimulated (20 ng/ml), IFN-γ stimulated (40 ng/ml), TPA stimulated (100 ng/ml), LPS stimulated (10 μg/ml), S. dublin infected, and E. coli O29:NM infected.

  • FIGURE 2.
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    FIGURE 2.

    Detection of hBD-1 and hBD-2 in culture supernatants from unstimulated and IL-1α-stimulated Caco-2 cells in the absence or presence of IL-1α. Cationic peptides from culture supernatants were subjected to electrophoresis on acetic acid urea gels, transferred to polyvinylidene difluoride membrane, probed with anti-hBD-1 (top) or anti-hBD-2 Abs (bottom), and developed with alkaline phosphatase conjugate. The positions of hBD-1 or hBD-2 standards of the indicated length in amino acids are depicted on the right of each panel. Specific staining is marked by arrows on the left.

  • FIGURE 3.
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    FIGURE 3.

    hBD-2 is an NF-κB target gene. A, Caco-2 and HT-29 cells were either left untreated, treated with MG-132 (25 μM), or infected with 75 MOI of an adenovirus (Ad5IκB-A32/36) expressing an IκBα superrepressor before stimulation with IL-1α (20 ng/ml) for 6 h, after which RNA was extracted and amplified by RT-PCR with primers specific for hBD-1, hBD-2, and β-actin. B, Caco-2 cells were either left uninfected (no virus); infected with a control adenovirus, Ad5LacZ; or infected with Ad5IκB-A32/36, which expresses a mutant IκBα protein that acts as a superrepressor of NF-κB activation. Cultures then either were left uninfected (no bacteria) or were infected with S. dublin or enteroinvasive E. coli O29:NM. RNA was extracted and amplified by RT-PCR for hBD-1, hBD-2, and β-actin. Parallel results were obtained using HT-29 cells (not shown).

  • FIGURE 4.
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    FIGURE 4.

    α- and β-defensin mRNA expression in Salmonella-infected human intestinal xenografts. RNA from uninfected control xenografts and S. typhi aroA aroC-infected human small-intestinal xenografts was amplified by RT-PCR for the β-defensins, hBD-1 and hBD-2; the α-defensins, HD-5 and HD-6; and β-actin.

  • FIGURE 5.
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    FIGURE 5.

    Immunohistochemical detection of hBD-1 expression in human colon and small intestine. Human colon and small-intestinal tissue was analyzed by immunohistochemistry for hBD-1 expression. Uninflamed normal colon (A and B) and small intestine (E and F) as well as inflamed colon tissue obtained at surgery from an ulcerative colitis patient (C and D) and inflamed duodenal tissue obtained from a patient with acid-peptic disease (G and H) was examined. The sections shown in A, C, E, and G were immunostained with an hBD-1-specific antiserum. Adjacent sections (B, D, F, and H) were stained with matched preimmune serum. hBD-1 was expressed in surface and crypt epithelium in uninflamed and inflamed colon and in crypt and villous cells in uninflamed and inflamed small intestine. All sections are shown at a magnification of ×200. Similar results were noted in inflamed and uninflamed tissue from three additional patients.

  • FIGURE 6.
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    FIGURE 6.

    Immunohistochemical detection of hBD-2 expression in human colon. Human colon was analyzed by immunohistochemistry for hBD-2 expression. Uninflamed normal colon tissue (A and B) and inflamed colon tissue obtained at surgery from two patients with ulcerative colitis (C and D, E and F) was examined. The sections shown in A, C, and E were immunostained with an hBD-2-specific antiserum. Adjacent sections (B, D, and F) were stained with matched preimmune serum. hBD-2 was only expressed in surface and crypt epithelium of inflamed colon. All sections are shown at a magnification of ×200. Similar results were noted in inflamed and uninflamed colon tissue from three additional patients.

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The Journal of Immunology: 163 (12)
The Journal of Immunology
Vol. 163, Issue 12
15 Dec 1999
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Expression and Regulation of the Human β-Defensins hBD-1 and hBD-2 in Intestinal Epithelium
Deborah A. O’Neil, Edith Martin Porter, Dirk Elewaut, G. Mark Anderson, Lars Eckmann, Tomas Ganz, Martin F. Kagnoff
The Journal of Immunology December 15, 1999, 163 (12) 6718-6724;

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Expression and Regulation of the Human β-Defensins hBD-1 and hBD-2 in Intestinal Epithelium
Deborah A. O’Neil, Edith Martin Porter, Dirk Elewaut, G. Mark Anderson, Lars Eckmann, Tomas Ganz, Martin F. Kagnoff
The Journal of Immunology December 15, 1999, 163 (12) 6718-6724;
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