High level expression of human epithelial beta-defensins (hBD-1, 2 and 3) in papillomavirus induced lesions

Abstract

Background: Epithelial defensins including human beta-defensins (hBDs) and alpha-defensins (HDs) are antimicrobial peptides that play important roles in the mucosal defense system. However, the role of defensins in papillomavirus induced epithelial lesions is unknown.

Results: Papilloma tissues were prospectively collected from 15 patients with recurrent respiratory papillomatosis (RRP) and analyzed for defensins and chemokine IL-8 expression by quantitative, reverse-transcriptase polymerase chain reaction (RT-PCR) assays. HBD-1, -2 and -3 mRNAs were detectable in papilloma samples from all RRP patients and the levels were higher than in normal oral mucosal tissues from healthy individuals. Immunohistochemical analysis showed that both hBD-1 and 2 were localized in the upper epithelial layers of papilloma tissues. Expression of hBD-2 and hBD-3 appeared to be correlated as indicated by scatter plot analysis (r = 0.837, p < 0.01) suggesting that they were co-inducible in papillomavirus induced lesions. Unlike hBDs, only low levels of HD5 and HD6 were detectable in papillomas and in oral mucosa.

Conclusion: Human beta-defensins are upregulated in respiratory papillomas. This novel finding suggests that hBDs might contribute to innate and adaptive immune responses targeted against papillomavirus-induced epithelial lesions.

Figure 1

RT-PCR analysis of hBD-1, hBD-2 and hBD-3 mRNA expression in RRP papilloma samples from 15 different individuals ((Left Panel, Lanes 1–15) and normal oral mucosa tissue from 10 different individuals (Right Panel, Lanes 1–10). A housekeeping gene, β-actin (a), was detected as a 450 bp PCR product; hBD-1 (b), hBD-2 (c) and hBD-3 (d) expressions were detected as 108, 172 and 98 bp PCR products, respectively. Samples were separated by 2% agarose gel electrophoresis and stained with ethidium bromide. 100 bp-ladder molecular-weight markers are presented on the left of the panel. Negative controls including amplification without reverse transcriptase or with cDNA sample replaced by DNase, RNase – free, distilled water were performed in each experiment but are not shown due to limited gel space.

Figure 2

hBD-1, hBD-2, hBD-3, HD5, HD6 and IL-8 mRNA expressions were analyzed by real-time RT-PCR. Bars represent defensins expression normalized to β-actin and relative to normal mucosa using the 2^-ΔΔCT analysis as described in Materials and Methods. Error bars represent the standard error of the mean of triplicate analysis.

Figure 3

Immunostaining of a representative frozen section of respiratory papilloma with antibody preparations to hBD-1 or hBD-2. Defensins were detectable as strong perinuclear staining of hBD-2 (A) and cytoplasmic immunostaining of hBD-1 (B) in all papilloma sections. Tissue section was counterstained with DAPI (C), and a merged picture was shown in (D). Arrows indicate areas of negative staining for hBD-1 and hBD-2 immunostaining. Tissue sections exposed to antibody preparations derived from preimmune or unrelated immunogens showed no reactivity (data not shown).