Antimicrobial peptides human beta-defensins and cathelicidin LL-37 induce the secretion of a pruritogenic cytokine IL-31 by human mast cells.

In addition to their microbiocidal properties, human beta-defensins (hBDs) and cathelicidin LL-37 stimulate a number of mammalian cell activities, including migration, proliferation, and cytokine/chemokine production. Because hBDs and LL-37 cause mast cells to release pruritogens such as histamine and PGs, we hypothesized that these peptides would stimulate the secretion of a novel pruritogenic mediator IL-31, predominantly produced by T cells. hBDs and LL-37 enhanced IL-31 gene expression and IL-31 protein production and release in the human mast cell line LAD2, as well as in peripheral blood-derived cultured mast cells, suggesting that mast cells are another source of IL-31. Moreover, the expression of IL-31 was elevated in psoriatic skin mast cells, and hBD-2-4 and LL-37, but not hBD-1, enhanced its expression in vivo in rat skin mast cells. hBDs and LL-37 also induced the release of other pruritogenic mediators, including IL-2, IL-4, IL-6, GM-CSF, nerve growth factor, PGE(2), and leukotriene C(4), and increased mRNA expression of substance P. hBD- and LL-37-mediated IL-31 production/release was markedly reduced by pertussis toxin and wortmannin, inhibitors of G-protein and PI3K, respectively. As evidenced by the inhibitory effects of MAPK-specific inhibitors, hBD-2-4 and LL-37 activated the phosphorylation of MAPKs p38, ERK, and JNK that were required for IL-31 production and release. The ability of hBDs and LL-37 to stimulate the production and release of IL-31 by human mast cells provides a novel mechanism by which skin-derived antimicrobial peptides/proteins may contribute to inflammatory reactions and suggests a central role of these peptides in the pathogenesis of skin disorders.

Crucial role for autophagy in degranulation of mast cells.

BACKGROUND: Autophagy plays a crucial role in controlling various biological responses including starvation, homeostatic turnover of long-lived proteins, and invasion of bacteria. However, a role for autophagy in development and/or function of mast cells is unknown. OBJECTIVE: To investigate a role for autophagy in mast cells, we generated bone marrow-derived mast cells (BMMCs) from mice lacking autophagy related gene (Atg) 7, an essential enzyme for autophagy induction. METHODS: Bone marrow-derived mast cells were generated from bone marrow cells of control and IFN-inducible Atg7-deficient mice, and morphologic and functional analyses were performed. RESULTS: We found that conversion of type I to type II light chain (LC3)-II, a hallmark of autophagy, was constitutively induced in mast cells under full nutrient conditions, and LC3-II localized in secretory granules of mast cells. Although deletion of Atg7 did not impair the development of BMMCs, Atg7(-/-) BMMCs showed severe impairment of degranulation, but not cytokine production on FcεRI cross-linking. Intriguingly, LC3-II but not LC3-I was co-localized with CD63, a secretory lysosomal marker, and was released extracellularly along with degranulation in Atg7(+/+) but not Atg7(-/-) BMMCs. Moreover, passive cutaneous anaphylaxis reactions were severely impaired in mast cell-deficient WBB6F1-W/W(V) mice reconstituted with Atg7(-/-) BMMCs compared with Atg7(+/+) BMMCs. CONCLUSION: These results suggest that autophagy is not essential for the development but plays a crucial role in degranulation of mast cells. Thus, autophagy might be a potential target to treat allergic diseases in which mast cells are critically involved.

Catestatin, a neuroendocrine antimicrobial peptide, induces human mast cell migration, degranulation and production of cytokines and chemokines.

Catestatin, a neuroendocrine peptide with effects on human autonomic function, has recently been found to be a cutaneous antimicrobial peptide. Human catestatin exhibits three single nucleotide polymorphisms: Gly364Ser, Pro370Leu and Arg374Gln. Given reports indicating that antimicrobial peptides and neuropeptides induce mast cell activation, we postulated that catestatin might stimulate numerous functions of human mast cells, thereby participating in the regulation of skin inflammatory responses. Catestatin and its naturally occurring variants caused the human mast cell line LAD2 and peripheral blood-derived mast cells to migrate, degranulate and release leukotriene C(4) and prostaglandins D(2) and E(2). Moreover, catestatins increased intracellular Ca(2+) mobilization in mast cells, and induced the production of pro-inflammatory cytokines/chemokines such as granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein-1/CCL2, macrophage inflammatory protein-1α/CCL3 and macrophage inflammatory protein-1ß/CCL4. Our evaluation of possible cellular mechanisms suggested that G-proteins, phospholipase C and the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) are involved in catestatin-induced mast cell activation as evidenced by the inhibitory effects of pertussis toxin (G-protein inhibitor), U-73122 (phospholipase C inhibitor) and U0126 (ERK inhibitor), respectively. We also found that human mast cells express the α7 subunit of the nicotinic acetylcholine receptor at both the mRNA and protein levels. Given that silencing the α7 receptor mRNA and an α7-specific inhibitor did not affect catestatin-mediated activation of mast cells, however, we concluded that this receptor is not likely to be functional in human mast cell stimulation by catestatins. Our finding that the neuroendocrine antimicrobial peptide catestatin activates human mast cells suggests that this peptide might have immunomodulatory functions, and provides a new link between neuroendocrine and cutaneous immune systems.

Cupressaceae pollen grains modulate dendritic cell response and exhibit IgE-inducing adjuvant activity in vivo.

Pollen is considered a source of not only allergens but also immunomodulatory substances, which could play crucial roles in sensitization and/or the exacerbation of allergies. We investigated how allergenic pollens from different plant species (Japanese cedar and Japanese cypress, which belong to the Cupressaceae family, and birch, ragweed, and grass) modulate murine bone marrow-derived dendritic cell (DC) responses and examined the effect of Cupressaceae pollen in vivo using mice. DCs were stimulated with pollen extracts or grains in the presence or absence of LPS. Cell maturation and cytokine production in DCs were analyzed by flow cytometry, ELISA, and/or quantitative PCR. Pollen extracts suppressed LPS-induced IL-12 production and the effect was greatest for birch and grass. Without LPS, pollen grains induced DC maturation and cytokine production without IL-12 secretion and the response, for which TLR 4 was dispensable, was greatest for the Cupressaceae family. Intranasal administration of Cupressaceae pollen in mice induced an elevation of serum IgE levels and airway eosinophil infiltration. Coadministration of ovalbumin with Cupressaceae pollen grains induced ovalbumin-specific IgE responses associated with eosinophil infiltration. The results suggest that modulation of DC responses by pollen differs among the plant families via (1) the promotion of DC maturation and cytokine production by direct contact and/or (2) the inhibition of IL-12 production by soluble factors. The strong DC stimulatory activity in vitro and IgE-inducing activity in mice support the clinical relevance of Cupressaceae pollen to allergies in humans.

Synergistic augmentation of inflammatory cytokine productions from murine mast cells by monomeric IgE and toll-like receptor ligands.

Simultaneous activation of murine mast cells by monomeric IgE and toll-like receptor (TLR) ligands was examined. Inflammatory cytokine production elicited by the binding of IgE in the absence of antigen, was further enhanced by the addition of lipopolysaccharide (LPS) or peptidoglycan (PGN). Enhancement by LPS or PGN on cytokine production was mediated by TLR4 and TLR2, respectively, since TLR4- and TLR2-deficient mast cells did not show synergistic activation by monomeric IgE and LPS/PGN. Synergistic activation of mast cells was obtained via phosphorylation of several mitogen-activated protein kinases (MAPK). Furthermore, MAPK inhibitors, significantly attenuated the augmentation of inflammatory cytokine production by monomeric IgE and LPS or PGN. Altogether, these results suggest that simultaneous TLR activation of mast cells with IgE molecules, particularly highly cytokinergic (HC) IgE, might contribute to the exacerbation of allergic diseases associated with infection even in the absence of a specific antigen.

Cytokine milieu modulates release of thymic stromal lymphopoietin from human keratinocytes stimulated with double-stranded RNA.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) plays a key role in allergic diseases, such as atopic dermatitis (AD) and asthma. TSLP is highly expressed by keratinocytes in skin lesions of patients with AD, but environmental triggers for its release from keratinocytes with endogenous factors are not well understood. Patients with AD, in whom allergic sensitization is already established, are susceptible to viral dissemination. OBJECTIVES: We investigated TSLP's release from primary human keratinocytes stimulated with a Toll-like receptor (TLR) 3 ligand, polyinosinic-polycytidylic acid, which mimics viral double-stranded RNA (dsRNA), and its modulation by cytokines. METHODS: Primary human keratinocytes were stimulated with TLR ligands, cytokines, or both. TSLP released into culture supernatants was measured by means of ELISA. RESULTS: Stimulation of keratinocytes with dsRNA induced release of TSLP and upregulated gene expression of TSLP and other cytokines and chemokines. The release of TSLP was enhanced by the addition of IL-4, IL-13, and/or TNF-alpha. With or without the T(H)2/TNF cytokines, the dsRNA-induced release of TSLP was upregulated by IFN-alpha and IFN-beta and suppressed by IFN-gamma, TGF-beta, or IL-17. CONCLUSIONS: The effect of the TLR3 ligand on keratinocytes suggests contribution of viral dsRNA to skin inflammations under the influence of a cytokine milieu. The results imply that viral dsRNA and a T(H)2 cytokine milieu might promote T(H)2-type inflammation through an induction of TSLP expression, suggesting that a vicious cycle exists between AD with T(H)2-type inflammation and viral infections and a possible blockade of this cycle by other cytokine milieus provided by cells, such as T(H)1, regulatory T, and T(H)17 cells.

Expression and function of fibroblast growth factor-inducible 14 in human corneal myofibroblasts.

The interaction of fibroblast growth factor-inducible 14 (Fn14) and, its ligand tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is known to be important in wound healing of tissues. However, to our knowledge, expression and function of Fn14 in corneal myofibroblasts, which have a crucial role in wound healing of corneal stroma, has not been investigated. In this study, we investigated the expression and function of Fn14 in corneal myofibroblasts. Expression of Fn14 protein was assessed by flow cytometry. Corneal myofibroblasts showed strong expression of Fn14 protein, while keratocytes did not. TGF-beta(1) promoted the differentiation of keratocytes into corneal myofibroblasts, and induced Fn14 expression. These data reveal that keratocytes phenotype determines the level of Fn14 expression. ELISA was used to detect chemokines and matrix metalloproteinases in the supernatant of corneal myofibroblasts cultured with or without stimulation by TWEAK and/or TGF-beta(1). TWEAK increased the production of IL-8, MCP-1, and RANTES by corneal myofibroblasts via Fn14. TGF-beta(1) augmented the TWEAK-induced production of these chemokines. TWEAK also increased the production of MMP-1 and -3 by corneal myofibroblasts via Fn14, while TGF-beta(1) inhibited this effect of TWEAK on MMP production. TWEAK-induced phosphorylation of NF-kappaB and MAP kinase in corneal myofibroblasts. Furthermore, TWEAK partially inhibited the differentiation of keratocytes into corneal myofibroblasts promoted by TGF-beta(1). These data suggest that the Fn14/TWEAK system may have several roles in wound healing by corneal myofibroblasts. In the future, modulation of the TWEAK/Fn14 system may become a novel approach for control corneal wound healing.

Lipid-soluble components of honeybee-collected pollen exert antiallergic effect by inhibiting IgE-mediated mast cell activation in vivo.

It was shown previously that bee-collected pollen (bee pollen, BP), inhibited in vitro murine mast cell activation. This study further analysed the antiallergic effect of BP in vivo by measuring cutaneous mast cell activation using a passive cutaneous anaphylaxis reaction. Daily oral administration of BP to mice, dose-dependently reduced the cutaneous mast cell activation elicited by IgE and specific antigens. Administration of BP also reduced the plasma concentration of malondialdehyde (MDA), an indicator of lipid peroxidation. The inhibitory effect of BP was mostly in a lipid- but not in water-soluble fraction. The HPLC analysis of isoflavones in BP revealed that genistein was a major isoflavone. However, administration of genistein alone at the concentration found in BP, did not show an inhibitory effect as observed in whole BP, suggesting that component(s) other than genistein would be responsible for the inhibitory effect of BP. These results first reveal that lipid-soluble components of BP exert an antiallergic action by inhibiting the FcåRI-mediated cutaneous mast cell activation.

Microbicidal protein psoriasin is a multifunctional modulator of neutrophil activation.

As effector cells in host defence, neutrophils actively destroy invading microorganisms via a potent antimicrobial arsenal composed of oxidants and antimicrobial peptides. Psoriasin, an Escherichia coli-cidal antimicrobial protein, has been found to be overexpressed in psoriasis, a skin disease characterized by infiltration of neutrophils. In addition to its microbicidal activities and chemotaxis of neutrophils reported previously, we hypothesized that psoriasin might regulate other neutrophil functions such as cytokine and chemokine production, reactive oxygen species generation, and release of antimicrobial peptides. In the current study, we demonstrate that psoriasin activates neutrophils to produce a range of cytokines and chemokines including interleukin-6 (IL-6), IL-8/CXCL8, tumour necrosis factor-alpha, macrophage inflammatory protein-1alpha (MIP-1alpha)/CCL3, MIP-1beta/CCL4 and MIP-3alpha/CCL20. Furthermore, psoriasin induces phosphorylation of mitogen-activated protein kinase p38 and extracellular signal-regulated kinase (ERK), but not c-Jun N-terminal kinase (JNK), both of which are required for the production of cytokines and chemokines as evidenced by the inhibitory effects of p38 and ERK inhibitors on psoriasin-mediated neutrophil activation. Moreover, psoriasin stimulates the generation of reactive oxygen species from neutrophils, most likely via nicotinamide adenine dinucleotide phosphate oxidase activation. Finally, we demonstrate that psoriasin enhances messenger RNA expression of alpha-defensins, termed human neutrophil peptides (HNP) 1 to 3, and induces their extracellular release. Besides its antimicrobial properties, therefore, psoriasin may contribute to innate immunity through enhancing neutrophil host defence functions at sites of inflammation or infection.

Monomeric IgE and lipopolysaccharide synergistically prevent mast-cell apoptosis.

The apoptosis of bone marrow-derived mast-cells (BMMCs) after growth factor withdrawal was significantly prevented by a high concentration of IgE in the absence of antigen, and further enhanced by the presence of Toll-like receptor4 (TLR4) ligand, lipopolysaccharide (LPS). The effect of LPS was mediated by TLR4, since TLR4-deficient BMMCs did not show synergistic effects with IgE. The neutralizing amount of anti-IL-3 did not reverse the anti-apoptotic effects of both IgE and combination with LPS. LPS treatment with monomeric IgE synergistically prevented the loss of mitochondrial membrane potentials and was associated with an enhanced expression of anti-apoptotic protein, Bcl-xL, or with a reduced expression of proapoptotic protein, Puma, and Bim, respectively. Altogether, these results suggest that LPS, in a TLR4-dependent manner, together with IgE, synergistically prevent mast-cell apoptosis and may contribute to regulate the tissue mast-cell number.

The extra domain A of fibronectin stimulates murine mast cells via toll-like receptor 4.

The activation of mast cells by extra domain A of fibronectin (FN-EDA), an endogenous ligand of TLR4, and its contribution to the pathogenesis of rheumatoid arthritis (RA) in vivo were examined. FN-EDA, but no other domain of the fibronectin fragment, III(11) (FN-III(11)) and III(12) (FN-III(12)), stimulated bone marrow-derived murine mast cells (BMMCs) dose-dependently to secret cytokines (TNF-alpha, IL-6, and IL-1beta), similar to the pattern produced by LPS. FN-EDA-induced cytokine production was mediated by TLR4, as cytokine production by FN-EDA was absent in TLR4-deficient (TLR4-/-) BMMCs. We examined the roles of TLR4-mediated mast cell activation by this form of fibronectin fragment in the pathogenesis of RA in vivo. The injection of FN-EDA, but not FN-III(11)and FN-III(12), to joints resulted in joint swelling of mice in vivo. Genetically mast cell-deficient WBB6F(1)-W/W(v) mice exhibited significantly less swelling and cytokine production compared with mast cell-sufficient +/+ mice, suggesting that swelling and inflammatory cytokine production were partially dependent on tissue mast cells. Reduced swelling and cytokine production were recovered by the reconstitution of tissue mast cells by the injection of BMMCs from wild-type mice but not from TLR4-/- mice. Altogether, these results suggest that the TLR4-mediated activation of mast cells by endogenous ligand FN-EDA might contribute to the pathogenesis of RA through proinflammatory cytokine production.

Inhibitory effect of honeybee-collected pollen on mast cell degranulation in vivo and in vitro.

Bee-collected pollen (bee pollen [BP]) has been used as a folk medicine for centuries against various diseases, including allergy. There is no study elucidating how BP exerts such an anti-allergic effect. Since mast cells play a central role in the pathogenesis of various allergic diseases, we investigated the effect of BP on mast cell activation elicited by the Fc immunoglobulin E (IgE) receptor (Fc epsilon RI)-mediated pathways. The in vivo effect of orally administered BP on cutaneous mast cell activation was examined by passive cutaneous anaphylaxis reaction. In vitro mast cell degranulation and IgE binding to mast cells and the status of protein tyrosine phosphorylation were examined using bone marrow-derived mast cells. Daily oral administration of BP to mice significantly reduced the cutaneous mast cell activation elicited by IgE and specific antigens. BP also reduced in vitro mast cell degranulation and tumor necrosis factor-alpha production by inhibiting IgE binding to Fc epsilon RI on mast cells. The inhibitory effect of BP on mast cell degranulation by preventing IgE binding was confirmed by the reduced levels of protein tyrosine phosphorylation, which occurred as downstream events in activated mast cells via Fc epsilon RI. These results first revealed that the anti-allergic action of BP was exerted by inhibiting the Fc epsilon RI-mediated activation of mast cells, which plays important roles, not only in the early phase, but also in the late phase of allergic reactions.

Differential responses of mast cell Toll-like receptors 2 and 4 in allergy and innate immunity.

Toll-like receptor 2 (TLR2) and TLR4 play important roles in the early innate immune response to microbial challenge. To clarify the functional roles of TLRs 2 and 4 in mast cells, we examined bone marrow-derived mast cells (BMMCs) from TLR2 or TLR4 gene-targeted mice. Peptidoglycan (PGN) from Staphylococcus aureus stimulated mast cells in a TLR2-dependent manner to produce TNF-alpha, IL-4, IL-5, IL-6, and IL-13, but not IL-1beta. In contrast, LPS from Escherichia coli stimulated mast cells in a TLR4-dependent manner to produce TNF-alpha, IL-1beta, IL-6, and IL-13, but not IL-4 nor IL-5. Furthermore, TLR2- but not TLR4-dependent mast cell stimulation resulted in mast cell degranulation and Ca2+ mobilization. In a mast cell-dependent model of acute sepsis, TLR4 deficiency of BMMCs in mice resulted in significantly higher mortality because of defective neutrophil recruitment and production of proinflammatory cytokines in the peritoneal cavity. Intradermal injection of PGN led to increased vasodilatation and inflammation through TLR2-dependent activation of mast cells in the skin. Taken together, these results suggest that direct activation of mast cells via TLR2 or TLR4 by respective microligands contributes to innate and allergic immune responses.

Expression and function of toll-like receptor-3 and -9 in human corneal myofibroblasts.

PURPOSE: To investigate the expression and function of toll-like receptor (TLR)-3 and -9 in corneal myofibroblasts. METHODS: Two types of human keratocytes were used, which were freshly isolated keratocytes from donor corneas and cultured keratocytes. Expression of the mRNAs for various molecular markers was analyzed in these cells by RT-PCR, and TLR-2, -3, -4, and -9 mRNAs were also analyzed by RT-PCR. Expression of TLR-3 and -9 at the protein level was assessed by flow cytometry. In addition, an antibody array and ELISA were used to detect chemokines and cytokines in the supernatant of cultured keratocytes, with or without stimulation by poly inosine-polycytidylic acid (poly (I:C)) or CpG-DNA. Furthermore, a phagocytosis assay was performed to evaluate whether signaling via TLR-3 and -9 enhances phagocytosis. RESULTS: Keratocytes cultured for three passages underwent differentiation into corneal myofibroblasts. TLR-3 and -9 were detected in corneal myofibroblasts at the mRNA and protein levels, but not in freshly isolated keratocytes. Stimulation of corneal myofibroblasts with poly (I:C) or CpG-DNA enhanced the production of IL-6, IL-8, GRO, ENA-78, and RANTES compared with that by untreated cells. Phagocytic activity of myofibroblasts was upregulated by signaling via TLR-3 and -9. CONCLUSIONS: This is the first report on the in vitro expression and function of TLR-3 and -9 in corneal myofibroblasts. The findings suggest that the keratocyte phenotype determines the expression of TLR-3 and -9 and that corneal myofibroblasts may have an important role in bacterial and viral clearance.

Synergistic effect of antibacterial agents human beta-defensins, cathelicidin LL-37 and lysozyme against Staphylococcus aureus and Escherichia coli.

BACKGROUND: The antimicrobial properties of the skin are attributed to several agents including human beta-defensins (hBDs), cathelicidin LL-37 and skin lysozyme. Although these antibacterial agents reside in the skin to protect it against infection, it is not well known whether the total analysis of all combinations of these agents may result in synergistic effect to enhance their antibacterial activities against invading microorganisms. OBJECTIVE: To elucidate the interactions between keratinocyte-derived antibacterial agents in the extracellular milieu, we investigated the individual and synergistic activities of hBDs, LL-37 and lysozyme against Staphylococcus aureus and Escherichia coli in neutral and acidic milieus. METHODS: The colorimetric method using alamarBlue was employed to assess the antibacterial activities of hBD-1, -2, -3, LL-37 and lysozyme and the viability of bacteria was read spectrophotometrically. RESULTS: In both neutral and acidic pH milieus, hBD-1, -2, -3, LL-37 and lysozyme exhibited antibacterial activity against S. aureus and E. coli in a dose-dependent manner. Interestingly, the antibacterial activity of hBD-1, -2, -3 and lysozyme but not LL-37 was significantly enhanced in acidic milieu (pH 4.6). Furthermore, various combinations of above agents resulted in a synergistic or additive antibacterial effect against S. aureus and E. coli in neutral milieu. The synergistic effect of hBDs, LL-37 and lysozyme against S. aureus was further significantly enhanced in acidic milieu. In contrast, above antibacterial agents exhibited mainly additive rather than synergistic effect on antibacterial activity against E. coli in acidic milieu. CONCLUSION: Taken together, these results provide a novel evidence of antimicrobial mechanism of natural human skin-derived antibacterial agents against bacterial infection, and their involvement in innate immunity.

Human ß-defensin-3 increases the expression of interleukin-37 through CCR6 in human keratinocytes.

BACKGROUND: Interleukin (IL)-37, a new member of the IL-1 family, is characterized as a fundamental inhibitor of innate immunity: it dampens the production of proinflammatory cytokines, protects against inflammatory and autoimmune diseases, and plays a potent immunosuppressive role in the pathogenesis of psoriasis. IL-37 is highly expressed in psoriatic skin, in which human ß-defensins (hBDs) have been detected. Although hBDs enhance the production of cytokines, including IL-1 cytokines, whether they stimulate the production of IL-37 remains unclear. OBJECTIVES: To assess the ability of hBDs to stimulate IL-37 expression/production by human keratinocytes and to determine the mechanism involved. METHODS: Real-time PCR and Western blotting were used to evaluate IL-37 expression. Caspase activities were assessed using colorimetric assay kits. A CCR6 antibody, siRNA, and caspase, Smad3, MAPK and NF-κB inhibitors were used to investigate the signaling mechanism of hBDs. RESULTS: Among the four hBDs used, only hBD-3 up-regulated the mRNA and protein expression of IL-37. The combination of TNF-α, EGF and poly (I:C) with hBD-3 synergistically enhanced the mRNA but not the protein expression of IL-37. Furthermore, hBD-3 increased the release of IL-37 into the culture supernatants. Evaluation of the signaling mechanism of hBD-3 suggested that caspases 1 and 4, Smad3, CCR6, MAPKs and NF-κB were required for hBD-3-mediated IL-37 expression. CONCLUSIONS: The finding that hBD-3 stimulates IL-37 expression, a novel target for the pathogenesis and therapy of cutaneous inflammatory diseases, provides evidence that hBDs contribute to the suppression of inflammatory and innate immune responses through the regulation of IL-37 expression.

Roles of retinoic acid-inducible gene-I-like receptors (RLRs), Toll-like receptor (TLR) 3 and 2'-5' oligoadenylate synthetase as viral recognition receptors on human mast cells in response to viral infection.

To investigate the anti-viral responses of human mast cells, we performed PCR array analysis of these cells after infection with vesicular stomatitis virus (VSV). PCR array analysis revealed that human mast cells up-regulated several anti-viral genes, including melanoma differentiation-associated gene 5, retinoic acid-inducible gene-I, and Toll-like receptor 3, together with type I interferons and chemokines, upon VSV infection. Additionally, we found that 2'-5' oligoadenylate synthetase, which also works as a virus recognition receptor by activating the latent form of RNase L, leading to viral RNA degradation, was up-regulated in human mast cells upon VSV infection. Moreover, small interfering RNA analysis to identify the receptors responsible for mast cell activation by VSV revealed that these receptors reciprocally cooperate to produce anti-viral cytokines and chemokines, inhibiting VSV replication. Our findings suggest that human mast cells produce cytokines and chemokines using several viral recognition receptors, leading to the inhibition of viral replication. These data provide novel information that improves our understanding of the roles of human mast cells in immune responses against viruses.

Up-regulation of interleukin-13 receptor alpha1 on human keratinocytes in the skin of psoriasis and atopic dermatitis.

BACKGROUND: Interleukin (IL)-13 is a pleiotropic cytokine, which shares many biological functions with IL-4. The receptor subunits of IL-13 consist of IL-4Ralpha, IL-13Ralpha1 and IL-13Ralpha2. The regulatory mechanisms of the IL-13Ralpha expression in the keratinocytes of certain skin disease have not been known. OBJECTIVE: To clear the roles of IL-13 and the regulatory mechanisms of its receptor in atopic dermatitis (AD) and psoriasis. METHOD: The expression of IL-13Ralpha1 in the skin of AD and psoriasis was investigated by immunohistochemistry. The regulation of IL-13Ralpha mRNA in the skin and human primary keratinocyte (HPK) was investigated by quantitative PCR. The secretion of IL-6 and RANTES from HPK was measured by ELISA. RESULTS: The expression of IL-13Ralpha1 was more prominent on the suprabasal keratinocytes in the skin of AD and striking increase of staining was observed on all layers of keratinocyte in the skin of psoriasis. The mRNA of IL-13Ralpha1, but not of IL-13Ralpha2 was overexpressed in both skin of AD and psoriasis. In vitro experiment using HPK demonstrated that IFN-gamma, IL-13 but not IL-4 could up-regulate the mRNA expression of IL-13Ralpha1. In contrast, IL-13Ralpha2 mRNA expression was up-regulated by IFN-gamma plus IL-4. Furthermore, the stimulation of HPK with IFN-gamma plus IL-13 and/or IL-4 resulted in significant enhancement of IL-6 and RANTES secretion. CONCLUSION: These findings indicate that IL-4 and IL-13 have different regulatory effects on the expression of IL-13Ralpha1 and alpha2, and the overexpression of IL-13Ralpha1 may play some roles in the pathogenesis of chronic stage of AD or psoriasis.

Involvement of p300 in TGF-beta/Smad-pathway-mediated alpha2(I) collagen expression in mouse mesangial cells.

BACKGROUND: Transforming growth factor beta 1 (TGF-beta1) induces alpha2(I) collagen gene (COL1A2) expression in mesangial cells through physical and functional cooperation of Smad proteins and Sp1. A transcriptional coactivator, p300, is also suggested to play an important role in TGF-beta1/Smad signal transduction. However, the role of p300 in TGF-beta1/Smad-pathway-mediated transcriptional activation of the COL1A2 gene in mesangial cells is still obscure. METHODS: Endogenous p300 expression and its modulation by TGF-beta1 were evaluated by Western blotting and immunofluorescence. The physical interaction of p300 with Smad2/3 was examined by immunoprecipitation followed by Western blotting. The functional role of p300 in TGF-beta1/Smad-pathway-mediated COL1A2 transcription was investigated in cotransfection experiments using a COL1A2 promoter-luciferase reporter gene construct and p300 expression plasmids. RESULTS: TGF-beta1 induced COL1A2 gene expression in cultured mouse mesangial cells which was blocked by overexpression of inhibitory Smad7. In addition, TGF-beta1-induced nuclear export of endogenous Smad7 was observed in mouse mesangial cells. Endogenous p300 was expressed in the nucleus of the cells. TGF-beta1 induced interaction of endogenous p300 with Smad2/3, and a dominant negative construct of p300 inhibited the TGF-beta1-induced COL1A2 expression in cultured mouse mesangial cells. CONCLUSIONS: p300 may be involved in TGF-beta1/Smad-pathway-mediated type I collagen gene transcription in mouse mesangial cells. Our findings would reveal a molecular basis of TGF-beta1-induced type I collagen gene transcription in mouse mesangial cells.

The human cathelicidin LL-37 host defense peptide upregulates tight junction-related proteins and increases human epidermal keratinocyte barrier function.

Both psoriasis and atopic dermatitis (AD) are not only associated with an impaired stratum corneum barrier, but also with abnormal expression of the tight junction (TJ) proteins. Because host defense peptides, including LL-37, are overexpressed in lesional psoriatic skin but are downregulated in lesional AD skin, we hypothesized that LL-37 might regulate the TJ function in keratinocytes. We demonstrated that LL-37 selectively increased the expression of several claudins and occludin, and enhanced their membrane distribution. Furthermore, LL-37 elevated the transepithelial electrical resistance while reducing the paracellular permeability of keratinocyte layers, and this activity was weakened by the claudin inhibitor ochratoxin A. A characterization of the molecular mechanism underlying the regulation of the TJ barrier by LL-37 revealed that LL-37 induced the activation of the Rac1, atypical PKC, glycogen synthase kinase-3 and PI3K pathways, and the specific inhibition of these pathways reversed the LL-37-mediated regulation of TJ function. In addition, LL-37 enhanced the expression of differentiation markers under the control of ochratoxin A, suggesting an association between LL-37-induced TJ function and keratinocyte differentiation. These data provide novel evidence that, in addition to its antimicrobial and other immunoregulatory functions, LL-37 contributes to cutaneous immunity by strengthening the skin's barrier function.