Keratinocytes: innate immune cells in atopic dermatitis.

The skin is a unique immune organ that constitutes a complex network of physical, chemical and microbiological barriers against external insults. Keratinocytes are the most abundant cell type in the epidermis. These cells form the physical skin barrier and represent the first line of the host defense system by sensing pathogens via innate immune receptors, initiating anti-microbial responses and producing various cytokines, chemokines and anti-microbial peptides, which are important events in immunity. A damaged epidermal barrier in atopic dermatitis allows the penetration of potential allergens and pathogens to activate keratinocytes. Among the dysregulation of immune responses in atopic dermatitis, activated keratinocytes play a role in several biological processes that contribute to the pathogenesis of atopic dermatitis. In this review, we summarize the current understanding of the innate immune functions of keratinocytes in the pathogenesis of atopic dermatitis, with a special emphasis on skin-derived anti-microbial peptides and atopic dermatitis-related cytokines and chemokines in keratinocytes. An improved understanding of the innate immunity mediated by keratinocytes can provide helpful insight into the pathophysiological processes of atopic dermatitis and support new therapeutic efforts.

Current insights into the role of human ß-defensins in atopic dermatitis.

Anti-microbial peptides or host defence peptides are small molecules that display both anti-microbial activities and complex immunomodulatory functions to protect against various diseases. Among these peptides, the human ß-defensins (hBDs) are localized primarily in epithelial surfaces, including those of the skin, where they contribute to protective barriers. In atopic dermatitis skin lesions, altered skin barrier and immune dysregulation are believed to be responsible for reduced hBD synthesis. Impaired hBD expression in the skin is reportedly the leading cause of increased susceptibility to bacterial and viral infection in patients with atopic dermatitis. Although hBDs have considerable beneficial effects as anti-microbial agents and immunomodulators and may ameliorate atopic dermatitis clinically, recent evidence has also suggested the negative effects of hBDs in atopic dermatitis development. In the current review, we provide an overview of the regulation of hBDs and their role in the pathogenesis of atopic dermatitis. The efforts to utilize these molecules in clinical applications are also described.

The role of human ß-defensins in allergic diseases.

Antimicrobial peptides (AMPs), also referred to as host defence peptides (HDPs), comprise a large family of small molecules broadly distributed throughout the animal and plant kingdom, historically serving as natural antibiotics. In mammals, there are two major families of AMPs/HDPs, the defensins and the cathelicidins. These peptides have evolved to protect against a wide range of infections from bacteria, viruses, fungi and some parasites. However, in addition to their broad-spectrum killing activities, AMPs/HDPs also possess various biological functions. They activate a variety of cell types, such as keratinocytes, airway epithelial cells and mast cells, among others, and regulate cytokine/chemokine production, cell migration, proliferation, differentiation, angiogenesis, the wound healing process and maintenance of the skin barrier function. Recently, it has become clear that alterations in the level of AMPs/HDPs are associated with the initiation and development of various inflammatory and allergic diseases. In this review, we will discuss the regulation and functions of human ß-defensins and outline the current evidence supporting the role of these peptides in the pathogenesis of allergic diseases, including atopic dermatitis, allergic rhinitis, asthma and chronic rhinosinusitis. Understanding the functions and mechanisms of human ß-defensins may aid in the development of novel therapeutic strategies for allergic diseases.

The antimicrobial protein S100A7/psoriasin enhances the expression of keratinocyte differentiation markers and strengthens the skin's tight junction barrier.

BACKGROUND: S100A7/psoriasin is a member of the S100 protein family and is encoded in the epidermal differentiation complex, which contains genes for markers of epidermal differentiation. S100A7/psoriasin is overexpressed in hyperproliferative skin diseases, where it is believed not only to exhibit antimicrobial functions, but also to induce immunomodulatory activities, including chemotaxis and cytokine/chemokine production. OBJECTIVES: To evaluate the effect of S100A7/psoriasin on keratinocyte differentiation and regulation of the tight junction (TJ) barrier. METHODS: Expression of differentiation markers and TJ proteins in human keratinocytes was determined by real-time polymerase chain reaction and Western blot. The changes in TJ barrier function were assessed by transepithelial electrical resistance and paracellular permeability assays. Glycogen synthase kinase-3 (GSK-3) and mitogen-activated protein kinase (MAPK) activation was analysed by Western blot, whereas ß-catenin and E-cadherin activation was evaluated by Western blot and immunofluorescence. RESULTS: S100A7/psoriasin enhanced the expression of several differentiation markers and selectively increased the expression of TJ proteins (e.g. claudins and occludin), which are known to strengthen the TJ barrier. Furthermore, S100A7/psoriasin increased ß-catenin and E-cadherin accumulation at cell-cell contact, and enhanced transepithelial electrical resistance while reducing the paracellular permeability of keratinocyte layers. The data suggest that S100A7/psoriasin-mediated regulation of the TJ barrier was via both the GSK-3 and MAPK pathways, as evidenced by the inhibitory effects of inhibitors for GSK-3 and MAPKs. CONCLUSIONS: Our finding that S100A7/psoriasin regulates differentiation and strengthens TJ barrier function provides novel evidence that, in addition to antimicrobial and immunoregulatory activities, S100A7/psoriasin is involved in skin innate immunity.

The human antimicrobial peptide dermcidin activates normal human keratinocytes.

BACKGROUND: The skin has evolved an epithelial defence mechanism which is characterized by antimicrobial peptides that inactivate various microorganisms and exhibit stimulatory activities bridging innate and adaptive immunity. Dermcidin (DCD) is a newly isolated antimicrobial peptide produced by the eccrine sweat glands in the skin. Recently, the DCD peptides DCD-1 and DCD-1L have been shown to display in vitro microbicidal activities against bacteria and viruses. OBJECTIVES: Because some skin-derived antimicrobial peptides activate keratinocytes, we investigated whether DCD-1L would also trigger keratinocyte activation. METHODS: Normal human keratinocytes were used in this study. The ability of DCD-1L to induce the production of cytokines/chemokines by keratinocytes was determined by enzyme-linked immunosorbent assay, and various inhibitors were used to investigate the stimulatory mechanism of DCD-1L. Mitogen-activated protein kinase (MAPK) phosphorylation and NF-kappaB activation were analysed by Western blotting. RESULTS: DCD-1L stimulated keratinocytes to generate cytokines and chemokines including tumour necrosis factor-alpha, interleukin-8 (CXCL8), interferon-inducible protein 10 (CXCL10) and macrophage inflammatory protein-3alpha (CCL20). To determine the molecular mechanism involved, we showed that DCD-1L-mediated cytokine/chemokine production was controlled by both G-protein and MAPK pathways, as evidenced by the inhibitory effects of pertussis toxin and specific inhibitors for p38 and ERK, but not for JNK, on DCD-1L-induced keratinocyte activation. Furthermore, we confirmed that DCD-1L could induce phosphorylation of p38 and ERK, and noticeably upregulated NF-kappaB activation. CONCLUSIONS: Taken together, the new activity of DCD-1L to stimulate the production of cytokines/chemokines by keratinocytes provides novel evidence for the implication of DCD, beyond its microbicidal ability, in skin immunity.

Cathelicidin LL-37 induces the generation of reactive oxygen species and release of human alpha-defensins from neutrophils.

BACKGROUND: Psoriasis is characterized by epidermal infiltration of neutrophils that destroy invading microorganisms via a potent antimicrobial arsenal of oxidants and antimicrobial agents. In contrast to atopic dermatitis, psoriasis exhibits low levels of skin infections due to the presence of antimicrobial agents, including cathelicidin LL-37. LL-37 kills a broad spectrum of microbes, and activates neutrophil chemotaxis. OBJECTIVE: To determine whether or not LL-37 could regulate additional neutrophil functions such as production of cytokines/chemokines, reactive oxygen species and release of neutrophil antimicrobial peptides. METHODS: Human peripheral blood neutrophils were used in this study. The production of interleukin (IL)-8 and release of alpha-defensins were analysed by enzyme-linked immunosorbent assay, and real-time polymerase chain reaction (PCR) was used to quantify alpha-defensin gene expression. Phosphorylation of mitogen-activated protein kinase (MAPK) was determined by Western blotting. The generation of reactive oxygen species was examined using flow cytometry, and intracellular Ca(2+) mobilization was measured using a calcium assay kit. RESULTS: LL-37 enhanced the production of IL-8 under the control of MAPK p38 and extracellular signal regulated kinase (ERK), as evidenced by the inhibitory effects of p38 and ERK1/2 inhibitors on LL-37-mediated IL-8 production. Furthermore, LL-37 induced phosphorylation of p38 and ERK. We also revealed that LL-37 stimulated the generation of reactive oxygen species dose- and time-dependently, most probably via NADPH oxidase activation and intracellular Ca(2+) mobilization. Finally, LL-37 induced both mRNA expression and protein release of alpha-defensins, known as human neutrophil peptide 1-3. CONCLUSION: Taken together, we suggest that in addition to its microbicidal properties, LL-37 may contribute to innate immunity by enhancing neutrophil host defence functions at inflammation and/or infection sites.

Cathelicidin family of antibacterial peptides CAP18 and CAP11 inhibit the expression of TNF-alpha by blocking the binding of LPS to CD14(+) cells.

Mammalian myeloid and epithelial cells express several kinds of antibacterial peptides (alpha-/beta-defensins and cathelicidins) that contribute to the innate host defense by killing invading micro-organisms. In this study we evaluated the LPS-neutralizing activities of cathelicidin peptides human CAP18 (cationic antibacterial proteins of 18 kDa) and guinea pig CAP11 using the CD14(+) murine macrophage cell line RAW264.7 and the murine endotoxin shock model. Flow cytometric analysis revealed that CAP18 and CAP11 inhibited the binding of FITC-conjugated LPS to RAW264.7 cells. Likewise, Northern and Western blot analyses indicated that CAP18 and CAP11 suppressed LPS-induced TNF-alpha mRNA and protein expression by RAW264.7 cells. Interestingly, CAP18 and CAP11 possessed LPS-binding activities, and they strongly suppressed the interaction of LPS with LPS binding protein that mediates the transport of LPS to CD14 to facilitate the activation of CD14(+) cells by LPS. Moreover, when CAP18 and CAP11 were preincubated with RAW264.7 cells, they bound to the cell surface CD14 and inhibited the binding of FITC-LPS to the cells. Furthermore, in the murine endotoxin shock model, CAP18 or CAP11 administration inhibited the binding of LPS to CD14(+) cells (peritoneal macrophages) and suppressed LPS-induced TNF-alpha expression by these cells. Together these observations indicate that cathelicidin peptides CAP18 and CAP11 probably exert protective actions against endotoxin shock by blocking the binding of LPS to CD14(+) cells, thereby suppressing the production of cytokines by these cells via their potent binding activities for LPS and CD14.

Evaluation of the effects of peptide antibiotics human beta-defensins-1/-2 and LL-37 on histamine release and prostaglandin D(2) production from mast cells.

Antimicrobial peptides, human beta-defensins (hBD-1/-2), and LL-37 (a peptide of human cathelicidin CAP18) are predominately expressed at epithelial tissues, where they participate in the innate host defense by killing invading microorganisms. In this study, to investigate the interactions between epithelial cell-derived antimicrobial peptides and mast cells, we evaluated the effects of hBD-1/-2 and LL-37 on mast cell functions using rat peritoneal mast cells. hBD-2 and LL-37 but not hBD-1 induced histamine release and intracellular Ca(2+) mobilization, and hBD-2 was more potent than LL-37. Interestingly, histamine release and intracellular Ca(2+) mobilization elicited by hBD-2 and LL-37 were markedly suppressed by BAPTA-AM (an intracellular Ca(2+) chelating agent), pertussis toxin and U-73122 (a phospholipase C inhibitor). In addition, among the peptides examined, only hBD-2 significantly induced PGD(2) production, which was abolished by indomethacin (cyclooxygenase-1/-2 inhibitor) but not NS-398 (cyclooxygenase-2 inhibitor), suggesting that hBD-2-induced PGD(2) production is mediated by cyclooxygenase-1. Likewise, the PGD(2) production was suppressed by pertussis toxin and U-73122. These observations suggest that hBD-2 and LL-37 stimulate mast cells to mobilize intracellular Ca(2+) and release histamine or generate PGD(2) in a G protein-phospholipase C-dependent manner. Thus, hBD-2 and LL-37 may have modulatory effects on inflammatory reactions.