Production of β-Defensin Antimicrobial Peptides by the Oral Mucosa and Salivary Glands

beta-Defensins are cationic peptides with broad-spectrum antimicrobial activity that are produced by epithelia at mucosal surfaces. Two human beta-defensins, HBD-1 and HBD-2, were discovered in 1995 and 1997, respectively. However, little is known about the expression of HBD-1 or HBD-2 in tissues of the oral cavity and whether these proteins are secreted. In this study, we characterized the expression of HBD-1 and HBD-2 mRNAs within the major salivary glands, tongue, gingiva, and buccal mucosa and detected beta-defensin peptides in salivary secretions. Defensin mRNA expression was quantitated by RNase protection assays. HBD-1 mRNA expression was detected in the gingiva, parotid gland, buccal mucosa, and tongue. Expression of HBD-2 mRNA was detected only in the gingival mucosa and was most abundant in tissues with associated inflammation. To test whether beta-defensin expression was inducible, gingival keratinocyte cell cultures were treated with interleukin-1beta (IL-1beta) or bacterial lipopolysaccharide (LPS) for 24 h. HBD-2 expression increased approximately 16-fold with IL-1beta treatment and approximately 5-fold in the presence of LPS. Western immunoblotting, liquid chromatography, and mass spectrometry were used to identify the HBD-1 and HBD-2 peptides in human saliva. Human beta-defensins are expressed in oral tissues, and the proteins are secreted in saliva; HBD-1 expression was constitutive, while HBD-2 expression was induced by IL-1beta and LPS. Human beta-defensins may play an important role in the innate defenses against oral microorganisms.     

Pathophysiological role of human beta-defensins 2 in gastric mucosa

Human beta-defensins (HBDs) recognized in the stomach include HBD1, which is the constitutional human beta-defensin (HBD), and HBD-2 and HBD-4, which are inducible HBDs. HBD-2 is an antimicrobial peptide that is involved in host defences against bacterial infections, such as Helicobacter pylori (H.pylori) in infection of the gastric mucosal epithelium. We examined the pathophysiological role of HBD-2, besides their roles as antimicrobial peptides. The materials used for the study consisted of gastric mucosal tissue specimens collected endoscopically from patients with conditions such as chronic gastritis associated with H. pylori infection, and gastric ulcers and gastritis due to non-steroidal anti-inflammatory drugs (NSAIDs) with or without H. pylori infection. We investigated the expression of HBD-2 and NF-kappaB by RT-PCR and immunoblotting, and the relation between the localization of HBD-2 and follicular dendritic cells (FDCs) by immunohistochemistry. Expression of HBD-2 was recognized in all the mucosal tissue specimens, irrespective of the presence or absence of H. pylori infection. All of the mucosal specimens expressing HBD-2 also revealed expression of NF-kappaB. In consecutive immunohisto-chemical staining, while expression of HBD-2 was observed in the gastric mucosal epithelium, FDCs were found to be localized in the lamina propria mucosae under the epithelial cell layer. These data suggested that in addition to being antimicrobial peptides, HBD-2 may also have a pathophysiological role as proinflammatory mediators, and that the HBD may act as proinflammatory mediators in concert with the dendritic cells (DC) by transmitting a signal from the mucosal surface to the lamina propria mucosae, which seems to be the original site of gastric mucosal damage.     

Expression and regulation of antimicrobial peptides in the gastrointestinal tract

The gastrointestinal (GI) tract is exposed to a wide range of microorganisms. The expression of antimicrobial peptides has been demonstrated in different regions of the GI tract, predominantly in epithelial cells, which represent the first host cells with which the microorganisms have to interact for invasion. The intestinal epithelial monolayer is complex, consisting of different cell types, and most have a limited lifespan. Of the GI antimicrobial peptides, alpha- and beta-defensins have been studied the most and are expressed by distinct types of epithelial cells. Enteric alpha-defensin expression is normally restricted to Paneth and intermediate cells in the small intestine. However, there are important differences between mice and humans in the processing of the precursor forms of enteric alpha-defensins. Parasite infection induces an increase in the number of enteric alpha-defensin-expressing Paneth and intermediate cells in the murine small intestine. In the chronically inflamed colonic mucosa, metaplastic Paneth cells (which are absent in the normal colon) also express enteric alpha-defensins. Epithelial expression of beta-defensins may be constitutive or inducible by infectious and inflammatory stimuli. The production of some members of the beta-defensin family appears to be restricted to distinct parts of the GI tract. Recent studies using genetically manipulated rodents have demonstrated the likely in vivo importance of enteric antimicrobial peptides in innate host defense against microorganisms. The ability of these peptides to act as chemoattractants for cells of the innate- and adaptive-immune system may also play an important role in perpetuating chronic inflammation in the GI tract.     

Defensins: key players or bystanders in infection, injury, and repair in the lung?

Antimicrobial peptides have been identified as key elements in the innate host defense against infection. Recent studies have indicated that the activity of antimicrobial peptides may be decreased in cystic fibrosis, suggesting a major role for these peptides in host defense against infection. One of the most intensively studied classes of antimicrobial peptides are defensins. Defensins comprise a family of cationic peptides that in human subjects can be divided into the alpha- and beta-defensin subfamilies. The alpha-defensins are produced by neutrophils and intestinal Paneth's cells, whereas beta-defensins are mainly produced by epithelial cells. Although studies on beta-defensins have so far focused on their antimicrobial activity, studies on alpha-defensins have suggested a role of these peptides in inflammation, wound repair, and specific immune responses. alpha-Defensins, which accumulate in airway secretions of patients with various chronic inflammatory lung disorders, were shown to be cytotoxic toward airway epithelial cells and to induce chemokine secretion in several cell types. Furthermore, the capacity of alpha-defensins to promote bacterial adherence to epithelial cells in vitro further supports a role for these peptides in the pathogenesis of chronic obstructive pulmonary disease and cystic fibrosis. Increased numbers of neutrophils are also present in the airways of patients with asthma, suggesting that neutrophils are involved in the pathogenesis of this disease. Because defensins are able to induce histamine release by mast cells and increase the airway hyperresponsiveness to histamine, it is tempting to speculate that defensins may also contribute to the inflammatory processes in asthma. Besides these proinflammatory effects, alpha-defensins may also display anti-inflammatory activities, including regulation of complement activation and proteinase inhibitor secretion. Finally, defensins may be involved in wound repair because defensins increase epithelial cell proliferation. Thus recent defensin research has revealed potential links between the innate and acquired immune system.     

beta-Defensins chemoattract macrophages and mast cells but not lymphocytes and dendritic cells: CCR6 is not involved

beta-Defensins are natural peptide antibiotics whose immunomodulatory functions are poorly understood. In the present study, macrophages were found to migrate to human beta-defensins (HBD)-1 to -4 using Galpha(i) proteins as well as MAPK ERK, p38 and JNK as signal transducers. In addition, mast cells responded to HBD-1 to -4 with calcium fluxes as well as chemotaxis, which increased upon stimulation with IgE plus antigen or ionomycin. In contrast, human beta-defensins were unable to induce migration of memory lymphocytes and dendritic cells (DC). Similar to HBD, the murine beta-defensin (mBD)-8 mobilized macrophages and lacked the ability to recruit memory T cells. These findings were unexpected as CCR6 on memory T cells and DC has been previously observed to be a receptor for human beta-defensins. In support of our findings, however, RBL-2H3 as well as 300.19 cells stably expressing CCR6 proved to be unresponsive to HBD-2 and -3. Intriguingly, our observation of a PKC-independent homologous desensitization between HBD-1 to -4 suggests a common receptor for HBD. In summary, chemoattraction of macrophages and mast cells is evolutionary conserved within the beta-defensin family despite a considerable sequence variation and distinct antimicrobial activities. However, CCR6 is not a functional receptor for beta-defensins.     

Intestinal innate immunity to Campylobacter jejuni results in induction of bactericidal human beta-defensins 2 and 3

Campylobacter jejuni is the most prevalent cause of bacterial diarrhea worldwide. Despite the serious health problems caused by this bacterium, human innate immune responses to C. jejuni infection remain poorly defined. Human beta-defensins, a family of epithelial antimicrobial peptides, are a major component of host innate defense at the gastrointestinal mucosal surface. In this study, the effect of two different C. jejuni wild-type strains on human intestinal epithelial innate responses was investigated. Up-regulation of beta-defensin gene and peptide expression during infection was observed and recombinant beta-defensins were shown to have a direct bactericidal effect against C. jejuni through disruption of cell wall integrity. Further studies using an isogenic capsule-deficient mutant showed that, surprisingly, the absence of the bacterial polysaccharide capsule did not change the innate immune responses induced by C. jejuni or the ability of C. jejuni to survive exposure to recombinant beta-defensins. This study suggests a major role for this family of antimicrobial peptides in the innate immune defense against this human pathogen.     

Human Toll-like receptor 2 mediates induction of the antimicrobial peptide human beta-defensin 2 in response to bacterial lipoprotein.

Recognition of pathogens by Drosophila Toll or human Toll-like receptors results in translocation of Dorsal or its human homologue NF-kappaB, respectively; in Drosophila, this is followed by the production of antimicrobial peptides serving as antimicrobial effector system of the innate immune response. We investigated whether human Toll-like receptors also mediate induction of the synthesis of antimicrobial peptides. We found that HEK293 cells transfected with Toll-like receptor 2, but not wild-type cells responded to stimulation with bacterial lipoprotein by production of human beta-defensin 2. Furthermore, the human lung epithelial cell line A549 was found to constitutively express Toll-like receptor 2 and to produce beta-defensin 2 in response to bacterial lipoprotein. This response was abrogated by blocking the signaling pathway activated through Toll-like receptors by transfecting the A549 cells with a dominant-negative form of IRAK-2. Thus, exposure of human cells to bacterial lipoprotein elicits production of the antimicrobial peptide beta-defensin 2 through Toll-like receptor 2.     

Defensins and acne

Acne a disease of the pilosebaceous unit is characterised by hypercornification and hyperkeratosis of outer root sheath (ORS) and sebaceous duct and perilesional infiltrate. Lesions may be characterised as "non"-inflammatory versus inflammatory. Hypercornification of the distal ORS and the pilosebaceous duct in concert with increased sebum production and abnormalities of the microbial flora are considered to be major factors in the pathogenesis of acne vulgaris. However, the basic mechanisms involved in the development of inflammation during acne vulgaris remain unclear. We have investigated the expression patterns of two antimicrobial peptides, human beta-defensin 1 (hBD1) and human beta-defensin 2 (hBD2) in healthy human hair follicles as well as in peri- and intralesional skin of acne vulgaris lesions such as comedones, papules and pustules. Strong hBD1 and hBD2 immunoreactivity was found in all suprabasal layers of the epidermis, and all permanent compartments of the hair follicle including the distal ORS of the hair follicle and the pilosebaceous duct. Moreover, marked hBD1 and hBD2 expression was also detected in the hair follicle stem cell compartment. In contrast, the proximal follicle bulb which undergoes apoptotic regression and is also able to regenerate following injury did not express hBD1 or hBD2. The majority of acne biopsies displayed a marked upregulation of hBD2 IR in the lesional and perilesional epithelium; in particular in pustules, and a less marked upregulation of hBD1 IR. The upregulation of beta-defensins expression in acne vulgaris lesions when compared to controls suggests that beta-defensins may be involved in the pathogenesis of acne vulgaris.     

Current Aspects of Innate and Adaptive Immunity in Atopic Dermatitis

Atopic dermatitis (AD) is a highly pruritic, chronic, multifactorial skin disease predisposing to bacterial and viral infections based on abnormalities of the innate and acquired immune system. The innate system quickly mobilizes an inflexible, standardized first-line response against different pathogens. Epidermal barrier dysfunction results in increased protein allergen penetration through the epidermis and predisposes to secondary skin infections. Two loss-of-function mutations in the epidermal filaggrin gene are associated with AD. Langerhans cells and inflammatory dendritic epidermal cells (IDEC) express high affinity IgE receptors, which are functional in IgE-mediated antigen presentation. Inducible antimicrobial peptides including the antiviral cathelicidin and the antibacterial beta-defensins show defective upregulation in lesional AD skin. The desmosomal protein nectin-1 is unmasked in AD lesions, thus becoming a relevant herpes simplex virus (HSV) entry receptor. Type I IFN-producing plasmacytoid dendritic cells are decreased and dysfunctional in AD skin, predisposing the patients to viral skin infections. Molluscum contagiosum virus produces a unique IL-18 binding protein to evade antiviral defense mechanisms. Innate and adaptive immunity do not simply coexist but are linked to one another in a complex network of skin immunobiology.     

In human epidermis,beta-defensin 2 is packaged in lamellar bodies

The skin presents a mechanical, as well as an immunological barrier to infection, and displays considerable innate immune capacity. Recently, cultured human keratinocytes were described to produce and export a microbicidal peptide human beta-defensin 2 (HBD-2). Immunogold was used to label ultrathin cryosections of stimulated, cultured human epidermis. HBD-2 was found to be stored in the lamellar bodies (LBs) of the stimulated keratinocytes of the spinous layer of the epidermis. HBD-2 was also found in the intercellular space. These findings suggest that HBD-2 is released with the contents of the LBs. Along with other investigations, our findings indicate that the lipid "permeability" barrier of the skin contains antimicrobial substances.     

Antimycobacterial agent based on mRNA encoding human beta-defensin 2 enables primary macrophages to restrict growth of Mycobacterium tuberculosis

Human macrophages are hosts for Mycobacterium tuberculosis, the causative agent of tuberculosis, which killed approximately 1.87 million people in 1997. Human alveolar macrophages do not express alpha- or beta-defensins, broad-spectrum antimicrobial peptides which are expressed in macrophages from other species more resistant to infection with M. tuberculosis. It has been previously reported that M. tuberculosis is susceptible to killing by defensins, which may explain the difference in resistance. Defensin peptides have been suggested as a possible therapeutic strategy for a variety of infectious diseases, but development has been hampered by difficulties in their large-scale production. Here we report the cellular synthesis of human beta-defensin 2 via highly efficient mRNA transfection of human macrophages. This enabled mycobactericidal and mycobacteristatic activity by the macrophages. Although human macrophages are difficult to transfect with plasmid vectors, these studies illustrate that primary macrophages are permissive for mRNA transfection, which enabled expression of a potentially therapeutic protein.