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. 2001 Oct;49(4):481–487. doi: 10.1136/gut.49.4.481

Expression of human β-defensin 2 (hBD-2) in Helicobacter pylori induced gastritis: antibacterial effect of hBD-2 against Helicobacter pylori

Y Hamanaka 1, M Nakashima 1, A Wada 1, M Ito 1, H Kurazono 1, H Hojo 1, Y Nakahara 1, S Kohno 1, T Hirayama 1, I Sekine 1
PMCID: PMC1728463  PMID: 11559643

Abstract

BACKGROUND—Human β-defensin 2 (hBD-2) plays a role in the innate defence system at mucosal surfaces. Colonisation of Helicobacter pylori in the stomach is an important pathological factor in gastrointestinal illnesses, including gastritis, peptic ulcer, and gastric adenocarcinoma.
AIMS—To evaluate the antibacterial role of hBD-2 against H pylori infection in the gastric mucosa.
SUBJECTS—Biopsied gastric mucosa specimens from H pylori positive (n=6) and H pylori negative (n=6) individuals were used. H pylori was determined by the presence of urease activity and microscopic examination.
METHODS—The specimens were examined for hBD-2 expression by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and in situ hybridisation. The antibacterial effect of hBD-2 against H pylori was evaluated by the number of colony forming units of H pylori after incubation with 0, 10−9, 10−8, 10−7, 10−6, or 10−5 M of hBD-2 peptide.
RESULTS—All six H pylori positive specimens expressed a high level of hBD-2 mRNA while hBD-2 mRNA was not detected in the H pylori negative specimens by RT-PCR. Immunohistochemistry using anti-hBD-2 antiserum revealed that hBD-2 was expressed in the surface epithelium of H pylori infected specimens. In gastric specimens obtained after H pylori eradication, hBD-2 immunoreactivity had dramatically decreased. In situ hybridisation confirmed that hBD-2 transcripts were localised in the epithelium of H pylori infected gastric specimens. Incubation with hBD-2 reduced the growth rate of cultured H pylori in a dose dependent manner, and incubation with 10−5 M hBD-2 completely inhibited the proliferation of H pylori.
CONCLUSIONS—H pylori infection induces hBD-2 expression in the human gastric epithelium. hBD-2 inhibited the growth of H pylori in vitro, suggesting that hBD-2 plays an antibacterial role in H pylori induced gastritis.


Keywords: human β-defensin 2; Helicobacter pylori; gastritis; antimicrobial peptide

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Figure 1  .

Figure 1  

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Reverse transcription-polymerase chain reaction (RT-PCR) analysis of human β-defensin 2 (hBD-2) expression in gastric mucosa specimens. (A) Lanes 1-12 correspond to gastric specimens obtained from cases 1-12, respectively, in table 1. Cases 1-6 were not infected with Helicobacter pylori and cases 7-12 were infected with H pylori. (B) Expression of hBD-2 mRNA in gastric mucosa specimens obtained from cases 7-9 before and after H pylori eradication. Cases 7-9 in table 1 were applicable in this experiment. +, before H pylori eradication; −, after H pylori eradication; M, 100 bp DNA ladder as a marker.

Figure 2  .

Figure 2  

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Immunohistochemical analysis of human β-defensin 2 (hBD-2) expression in gastric mucosa specimens with (case 9) (A) or without (case 3) (B) Helicobacter pylori colonisation. Both (A) and (B) have the same magnification. The scale bar in (A) represents 200 µm. (C) May-Giemsa stained section adjacent to (D) and demonstrating colonisation of H pylori at the mucosal surface (case 9). (D) Immunoreactivity for hBD-2 in gastric epithelium at a high magnification can be seen. The scale bar in (C) represents 800 µm.

Figure 3  .

Figure 3  

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Immunohistochemical analysis of human β-defensin 2 (hBD-2) expression in gastric mucosa specimens obtained before (A) and after (B) Helicobacter pylori eradication in case No 7. (A) and (B) have the same magnification. The scale bar in (A) represents 200 µm.

Figure 4  .

Figure 4  

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In situ hybridisation of human β-defensin 2 (hBD-2) mRNA in Helicobacter pylori infected gastric mucosa specimens. (A) A hybridisation signal with the hBD-2 antisense probe was predominantly observed in the surface epithelium and pits, and also in the inflammatory cells scattered in the interstitium (case 12). The full length hBD-2 antisense probe may cross react with hBD-1 as a leucocytic defensin because the open frame of hBD-2 cDNA shows 39% identity with hBD-1.34 (B) No signals were detected on hybridisation with the hBD-2 sense probe as a negative control (case 12). (A) and (B) have the same magnification. The scale bar in (A) represents 100 µm.

Figure 5  .

Figure 5  

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Antimicrobial effect of synthetic human β-defensin 2 (hBD-2) peptides on Helicobacter pylori colony formation in an in vitro assay. Each bar represents mean (SEM). CFU, colony forming units.

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