hBD-2 Protein Research Articles

ENDOGENOUS ANTIMICROBIAL PEPTIDES AND SKIN INFECTIONS IN ATOPIC DERMATITIS

Ong PY, Ohtake T, Brandt C, et al. N Engl J Med. 2002;347:1151–1160The immune system of human skin contains several antimicrobial peptides, particularly cathelicidins (LL-37) and β-defensins. Although these peptides are negligible in normal skin, they accumulate in skin affected by inflammatory diseases such as psoriasis. The purpose of this study was to compare the levels of expression of LL-37 and human β-defensin 2 (HBD-2) in inflamed skin from patients with atopic dermatitis and from those with psoriasis.Patients with atopic dermatitis or psoriasis were compared with normal controls with no skin disease.The expression of LL-37 and HBD-2 protein in skin biopsy specimens was determined by immunohistochemical analysis. The amount of antimicrobial peptides in extracts of skin samples was also analyzed by immunodot blot analysis (for LL-37) and Western blot analysis (for HBD-2). Reverse transcriptase-polymerase chain reaction (RT-PCR) assays were used to confirm the relative expression of HBD-2 and LL-37 messenger RNA (mRNA) in the skin-biopsy specimens. These peptides were also tested for antimicrobial activity against Staphylococcus aureus with the use of a colony-forming assay.Immunohistochemical analysis confirmed the presence of abundant LL-37 and HBD-2 in the superficial epidermis of all patients with psoriasis. In comparison, immunostaining for these peptides was significantly decreased in acute and chronic lesions from patients with atopic dermatitis (P = .006 and P = .03, respectively). RT-PCR showed significantly lower expression of HBD-2 mRNA and LL-37 mRNA in atopic lesions than in psoriatic lesions (P = .009 and P = .02, respectively). The combination of LL-37 and HBD-2 showed synergistic antimicrobial activity by effectively killing S aureus.A deficiency in the expression of antimicrobial peptides may account for the susceptibility of patients with atopic dermatitis to skin infection with S aureus.It has long been recognized that patients with atopic dermatitis have an enormous predilection for cutaneous infections with S aureus, as well as viral pathogens. Although this could be attributable in part to damage to the skin through excessive scratching, it has also been presumed that specific immunologic mechanisms must also play a role. This study elegantly unravels at least part of the immunologic basis for this common clinical problem.

Neutrophil elastase up-regulates human β-defensin-2 expression in human bronchial epithelial cells

Human β-defensin-2 (HBD-2) gene expression is induced by tumour necrosis factor-α, interleukin-1β and lipopolysaccharide. The objective of this study was to investigate the effect of neutrophil elastase (NE), a major pro-inflammatory protease, on HBD-2 expression. HBD-2 gene expression was assessed by reverse transcription polymerase chain reaction in the human bronchial epithelial cell line 16HBE14o− and primary normal human bronchial epithelial (NHBE) cells. Optimal HBD-2 expression was induced with 100 nM NE. Using a HBD-2-luciferase reporter construct, luciferase activity increased significantly in 16HBE14o− cells following incubation with NE. An increase in HBD-2 protein expression was observed in primary NHBE cells after incubation with NE as assessed by laser scanning cytometry. In conclusion, NE up-regulates HBD-2 expression in bronchial epithelial cells.

Defensin expression by the cornea: multiple signalling pathways mediate IL-1beta stimulation of hBD-2 expression by human corneal epithelial cells.

To investigate the expression of human beta-defensins (hBDs) by human corneal epithelium and determine the effects of proinflammatory cytokines on expression of human beta-defensin (hBD)-2 by human corneal epithelial cells (HCECs) in culture. RNA was extracted from corneal epithelial cells scraped from cadaveric corneas and from cultured HCECs, and RT-PCR was performed to detect hBD-1, -2, and -3 mRNA. To study the effects of proinflammatory cytokines on expression of defensin, HCECs were cultured and then exposed to interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha for up to 36 hours, with a range of concentrations (0.01-100 ng/mL). In some experiments, cells were pretreated with various cell signaling pathway inhibitors before the addition of IL-1beta. At the end of the incubations, the cells were harvested for RT-PCR and the culture media collected for the detection by immunoblot analysis of secreted defensin peptide. All epithelial tissue collected from cadaveric corneas expressed mRNA for hBD-1. hBD-2 was detectable in two of eight donors corneas, whereas hBD-3 was detected in five. All primary cultures of HCECs expressed hBD-1 and -3. A faint band for hBD-2 was detectable in three of eight cultures. Cultures of simian virus (SV)40-transformed HCECs always expressed hBD-1 and -3, but did not express hBD-2 under control conditions. IL-1beta and TNFalpha each stimulated the expression of hBD-2 in HCECs and were more effective in combination than alone. The effects of IL-1beta were concentration- (maximal at 10 ng/mL) and time-dependent (maximal at 12 hours and 24 hours for hBD-2 mRNA expression and protein secretion, respectively). The upregulation of hBD-2 mRNA persisted for at least 24 hours after removal of IL-1beta. The NFkappaB inhibitors pyrrolidinedithiocarbamate (PDTC; 100 microM), caffeic acid phenethyl ester (CAPE; 90 microM), and MG-132 (25 microM), blocked IL-1beta-stimulated expression of hBD-2. The p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 (5 microM) and the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125 (25 microM) partially blocked (by 47% and 59%, respectively) the effect of IL-1beta. However, PD98059, an ERK inhibitor, had no effect. Genistein (50 microM) and dexamethasone (1 microM) also partially blocked (by 26% and 28%, respectively) the effect of IL-1beta. Human corneal epithelium expresses hBD-1 and -3. hBD-2 is not typically present, but its expression can be stimulated by proinflammatory cytokines such as IL-1beta, acting through mitogen-activated protein (MAP) kinase and nuclear factor (NF)-kappaB pathways. Because IL-1 is known to be increased at the ocular surface after injury, the current observations provide a mechanism to explain the previous finding that hBD-2 is upregulated in regenerating corneal epithelium. Cytokine stimulation of hBD-2 expression most likely provides additional protection against infection and raises the possibility that this defensin in particular may be involved in the wound-healing response, per se.

Human beta-defensin-2 induction in Helicobacter pylori-infected gastric mucosal tissues: antimicrobial effect of overexpression.

The objective of this study was to understand more of the innate immune response to Helicobacter pylori by determining the expression of human beta-defensin-2 (hBD-2) in various gastric mucosal tissues and MKN45 gastric cancer cells with or without H. pylori. Semi-quantitative TaqMan RT-PCR and immunohistochemistry were carried out. The antimicrobial effects of a transfected hBD-2 gene against H. pylori were also evaluated. The results showed that hBD-2 was expressed in inflamed gastric mucosal tissues with H. pylori infection, but not in the absence of H. pylori infection. Expression was also detected in gastric cancers in patients with H. pylori infection. Expression was induced in the MKN45 gastric cancer cell line by H. pylori in a manner dependent on the abundance of bacteria. hBD-2-transfected 3T3J2-1 cells secreted hBD-2 protein into the culture medium and this protein inhibited growth of H. pylori completely. The results suggest that hBD-2 may be involved in the pathophysiology of H. pylori-induced gastritis.

Endogenous Antimicrobial Peptides and Skin Infections in Atopic Dermatitis

The innate immune system of human skin contains antimicrobial peptides known as cathelicidins (LL-37) and beta-defensins. In normal skin these peptides are negligible, but they accumulate in skin affected by inflammatory diseases such as psoriasis. We compared the levels of expression of LL-37 and human beta-defensin 2 (HBD-2) in inflamed skin from patients with atopic dermatitis and from those with psoriasis. The expression of LL-37 and HBD-2 protein in skin-biopsy specimens from patients with psoriasis, patients with atopic dermatitis, and normal subjects was determined by immunohistochemical analysis. The amount of antimicrobial peptides in extracts of skin samples was also analyzed by immunodot blot analysis (for LL-37) and Western blot analysis (for HBD-2). Quantitative, real-time reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assays were used to confirm the relative expression of HBD-2 and LL-37 messenger RNA (mRNA) in the skin-biopsy specimens. These peptides were also tested for antimicrobial activity against Staphylococcus aureus with the use of a colony-forming assay. Immunohistochemical analysis confirmed the presence of abundant LL-37 and HBD-2 in the superficial epidermis of all patients with psoriasis. In comparison, immunostaining for these peptides was significantly decreased in acute and chronic lesions from patients with atopic dermatitis (P=0.006 and P=0.03, respectively). These results were confirmed by immunodot blot and Western blot analyses. Real-time RT-PCR showed significantly lower expression of HBD-2 mRNA and LL-37 mRNA in atopic lesions than in psoriatic lesions (P=0.009 and P=0.02, respectively). The combination of LL-37 and HBD-2 showed synergistic antimicrobial activity by effectively killing S. aureus. A deficiency in the expression of antimicrobial peptides may account for the susceptibility of patients with atopic dermatitis to skin infection with S. aureus.

Induction of human beta-defensin-2 expression in human astrocytes by lipopolysaccharide and cytokines.

Defensins are cationic peptides with broad-spectrum antimicrobial activity. They are members of a supergene family consisting of alpha and beta subtypes and each subtype is comprised of a number of different isoforms. For example, human alpha-defensin (HAD) has six isoforms, which are expressed by polymorphonuclear leukocytes and Paneth cells. In contrast, human beta-defensin (HBD) has two isoforms that are expressed by epithelial cells of the skin, gut, respiratory and urogenital tracts. Recently, HBD-1 was detected in human brain biopsy tissue. However, little is known about the expression of HBD-1 or HBD-2 in the CNS and whether neural cells can secrete these peptides. For the present study, human astrocyte, microglial, meningeal fibroblast and neuronal cultures were probed for the expression of HBD-1 and HBD-2 mRNA and protein. Each cell type was either maintained in tissue culture medium alone or in medium containing lipopolysaccharide (LPS) at concentrations ranging from 0.1 to 1 microgram/mL, interleukin-1 beta (IL-1beta) at 1-50 ng/mL, or tumor necrosis factor alpha (TNF-alpha) at the same concentrations. The expression of HBD-1 and HBD-2 mRNAs was monitored by RT-PCR. The cDNA products were sequenced to characterize the gene product. HBD-2 protein was detected by immunoblot, immunoprecipitation and immunocytochemistry. Results of these studies showed that HBD-1 mRNA was detected in all cell cultures except in those enriched for neurons. In contrast, HBD-2 mRNA was detected only in astrocyte cultures that were treated with LPS, IL-1beta or TNF-alpha. The detection of the respective proteins correlated positively with the mRNA results. As such, these data represent the first demonstration of HBD-2 expression by astrocytes and suggest that this peptide may play a role in host defense against bacterial CNS pathogenesis.