Sadatoshi Maeda

House dust mite major allergen Der f 1 enhances proinflammatory cytokine and chemokine gene expression in a cell line of canine epidermal keratinocytes.

House dust mite (HDM) allergens are the most common allergens involved in the induction of IgE-mediated hypersensitivity. Recently, epicutaneous sensitization with HDM allergens has been emphasized in the development of atopic dermatitis (AD); however, direct stimulation of canine keratinocytes by mite allergens has not been well investigated. In the present study, we investigated the effects of Der f 1, a major allergen of Dermatophagoides farinae, on cytokine and chemokine gene expression in a canine keratinocyte cell line, CPEK. CPEK constitutively expressed mRNA for TNF-alpha, IL-12p35, IL-18, GM-CSF, TGF-beta, IL-8/CXCL8, TARC/CCL17, CTACK/CCL27 and MEC/CCL28. Of all the cytokines and chemokines investigated in CPEK, transcription levels of GM-CSF, IL-8/CXCL8 and TNF-alpha mRNA were significantly enhanced by stimulation with Der f 1. The present results suggest that Der f 1 can directly augment inflammatory cytokine and chemokine production from keratinocytes, and may initiate allergic inflammation independently of Type-I hypersensitivity.

Augmentation of CCL17 and CCL28 gene expression by TNF-α, IL-1β, or IFN-γ in cultured canine keratinocytes.

Keratinocytes produce inflammatory mediators that are involved in the pathogenesis of skin disorders such as atopic dermatitis (AD). In particular, the CC chemokines, thymus and activation regulated chemokine (TARC)/CCL17 and mucosae-associated epithelial chemokine (MEC)/CCL28 are considered to play an important role in the lesional infiltration of lymphocytes in canine AD. However, there have been no reports on the regulatory mechanisms of CCL17 and CCL28 transcription in canine keratinocytes. In this study, we investigated whether CCL17 and CCL28 transcription in cultured keratinocytes is induced by TNF-alpha, IL-1beta, or IFN-gamma. It was found that CCL17 mRNA transcription is augmented by TNF-alpha only, whereas the CCL28 mRNA level could be increased by TNF-alpha, IL-1beta, or IFN-gamma. The present study suggests that pro-inflammatory cytokines are important inducing factors for the production of CCL17 and CCL28 in the lesional skin of dogs with AD.

Molecular cloning of canine interleukin-31 and its expression in various tissues

The newly discovered cytokine, interleukin-31 (IL-31), belongs to the short-chain cytokine group. It was reported that transgenic expression of IL-31-induced pruritus, similar to atopic dermatitis, in mice, further, excessive amounts of IL-31 was also expressed in the skin from human patients with atopic dermatitis as compared to that from normal people. In this study, canine IL-31 was molecularly cloned from concanavalin A-stimulated canine peripheral blood mononuclear cells (PBMCs), and its nucleotide sequence was determined. Canine IL-31 contains 4 alpha-helix structures characteristic of the IL-31 family, and the amino acid identity of canine IL-31 with those of human or mouse is 54% and 28%, respectively. Furthermore, we detected low levels of canine IL-31 in the thymus, testis, spleen, and kidneys, but not in the skin of atopic dogs.

Protease-activated receptor-2 induces proinflammatory cytokine and chemokine gene expression in canine keratinocytes.

Although the molecular basis of the allergenicity remains to be fully elucidated, the ability of allergens to elicit allergic responses is at least partly attributed to their proteolytic activity. Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is activated by site-specific proteolysis by serine proteases and is known to mediate inflammatory processes in various tissues. In this study, we investigated the effects of trypsin, a major serine protease, and a human PAR-2 agonist peptide (SLIGKV-NH2) on proinflammatory cytokine and chemokine gene expression in the canine keratinocyte cell line CPEK. The expression of PAR-2 mRNA and protein in CPEK cells was detected by RT-PCR and Western blotting, respectively. The localization of PAR-2 in CPEK was examined by immunofluorescence. The mRNA expression levels of proinflammatory cytokines and chemokines were quantified by real-time RT-PCR. The free intracellular Ca(2+) concentration was measured using the Ca(2+)-sensitive fluorescent dye. CPEK cells constitutively expressed PAR-2 mRNA and protein. Stimulation of CPEK cells with trypsin induced significant upregulation of the mRNA expression levels of tumor necrosis factor alpha (TNF-α, P<0.05), granulocyte-macrophage colony-stimulating factor (GM-CSF, P<0.01), thymus and activation regulated chemokine (TARC/CCL17, P<0.01), and interleukin 8 (IL-8/CXCL8, P<0.01). Similarly, the PAR-2 agonist peptide increased the mRNA expression levels of TNF-α (P<0.05), GM-CSF (P<0.05), TARC/CCL17 (P<0.05), and IL-8/CXCL8 (P<0.05) in CPEK cells. Both trypsin and the PAR-2 agonist peptide increased the intracellular Ca(2+) concentration and PAR-2 internalization. These results suggest that PAR-2 activation can augment inflammatory cytokine and chemokine expression in canine keratinocytes, and it may initiate allergic inflammation through the proteolytic activity of allergens in canine atopic dermatitis.

Presence of anti-insulin natural autoantibodies in healthy cats and its interference with immunoassay for serum insulin concentrations

A substance interfering with the enzyme-linked immunosorbent assay (ELISA) for feline insulin concentration was investigated in healthy cats. An insulin-binding substance isolated from feline serum showed 2 bands at 25 and 50 kDa in SDS-PAGE, suggesting the presence of immunoglobulin G (IgG). Insulin-binding IgG from healthy cats indeed reduced insulin immunoreactivity in the ELISA for determining insulin concentration. The insulin-binding IgG was polyclonal/polyreactive and showed certain specificity, high affinity, and high binding capacity, which was evaluated by liquid-phase radioimmunoassay with Scatchard plot analysis. Epitope analysis revealed that the insulin-binding IgG showed significant binding at residues A1-5 and B20-30 of the insulin molecule. Removal of the antibodies from serum enabled the determination of serum insulin concentrations by ELISA. Our data indicated that serum from healthy cats contained substantial amounts of natural autoantibodies combined with insulin, and that the antibodies interfered with the heterologous immunoassay for serum insulin concentration.

Gene transcription analysis in lesional skin of canine epitheliotropic cutaneous lymphoma using quantitative real-time RT-PCR.

Canine epitheliotropic cutaneous lymphoma (cECL) is characterized by infiltration of neoplastic lymphocytes in the skin with a specific tropism for the epidermis. Migration of lymphocytes is strictly controlled by interactions between chemokines and chemokine receptors, which may be involved in the pathogenesis of cECL. In this study, we investigated mRNA transcription levels of several chemokines (CCL17, CCL19, CCL21, CCL22, CCL27, CCL28 and CXCL10) and chemokine receptors (CCR4, CCR7, CCR10 and CXCR3) in lesional skin of cECL by quantitative real-time RT-PCR. To examine the subsets of accumulating neoplastic lymphocytes, we also investigated transcription levels of type-1 (IFN-γ, IL-12p35, IL-12p40 and LT-α) and type-2 (IL-4 and IL-13) cytokines and cytotoxic markers (perforin and granzyme B). We found that the lesional skin had higher mRNA transcription of CCL19, CXCL10, CCR4, CCR7, CCR10 and CXCR3 and lower transcription of CCL27 than healthy dog skin (p<0.05). In addition, transcription levels of type-1 cytokine and cytotoxic markers in lesional skin were significantly higher than those in healthy dog skin. These results indicate that the transcription of some chemokines and chemokine receptors, which are necessary for skin-homing, epitheliotropism and peripheral segregation of T-cells, is upregulated in the lesional skin of cECL. In addition, our results also indicate that the subset of neoplastic lymphocytes in cECL is most likely type-1 cytotoxic T-cells.

Transcription of thymic stromal lymphopoietin via Toll-like receptor 2 in canine keratinocytes: a possible association of Staphylococcus spp. in the deterioration of allergic inflammation in canine atopic dermatitis.

BACKGROUND Colonization, overgrowth and subsequent infection by Staphylococcus spp. is frequently observed in canine atopic dermatitis (CAD), where it contributes to the intensity of cutaneous inflammation. The mechanisms by which staphylococci contribute to the pathogenesis of CAD are unclear. Studies suggest that thymic stromal lymphopoietin (TSLP), a cytokine induced by a cell wall component of Staphylococcus spp., may play a critical role in Th2 responses including the pathogenesis of CAD. HYPOTHESIS/OBJECTIVE To determine if synthetic triacylated lipopeptide (TLR1/2 ligand), a cell wall component of Staphylococcus spp., induces the transcription of TSLP via TLR2 in canine keratinocytes. METHODS Transcription of TSLP was quantified in a canine keratinocyte cell line after stimulation with synthetic triacylated lipopeptide, and again after inhibition of TLR2 by a targeted small interfering RNA. RESULTS The transcription of TSLP was enhanced 6 h after stimulation with the synthetic triacylated lipopeptide; it was completely suppressed by knockdown of TLR2. CONCLUSIONS AND CLINICAL IMPORTANCE The results demonstrated that a synthetic cell wall component of Staphylococcus spp. induced transcription of TSLP via TLR2 in canine keratinocytes. Additional studies will be required to investigate whether Staphylococcus spp. contributes to Th2 responses in CAD through TLR2-mediated TSLP production.

Involvement of nuclear factor of activated T cells in granulocyte–macrophage colony-stimulating factor production in canine keratinocytes stimulated with a cysteine protease

BACKGROUND A previous study demonstrated that the cysteine protease of Dermatophagoides farinae induced production of granulocyte-macrophage colony-stimulating factor (GM-CSF) in a canine epidermal keratinocyte progenitor cell line (CPEK); however, the molecular mechanism has not been elucidated. HYPOTHESIS/OBJECTIVES Given that the transcription of GM-CSF mRNA in human lymphocytes is mainly regulated by the nuclear factor of activated T cells (NFAT), it is hypothesized that NFAT also contributes to GM-CSF production in canine keratinocytes stimulated with a cysteine protease. METHODS Nuclear translocation of NFAT was evaluated in CPEK cells in the absence or presence of the cysteine protease papain. We also investigated whether blockade of NFAT could inhibit GM-CSF production. RESULTS Papain-induced nuclear translocation of NFAT, producing GM-CSF, was partly inhibited by ciclosporin. CONCLUSIONS AND CLINICAL IMPORTANCE The results suggest that GM-CSF production mediated by the cysteine protease is regulated not only by NFAT but also by unknown signalling pathways in canine keratinocytes.

Identification of the signaling pathway of TNF-α-induced CCL17/TARC transcription in a canine keratinocyte cell line.

A CC chemokine, CCL17/TARC, has been shown to be a factor in the immunopathogenesis of canine atopic dermatitis (cAD). In canine keratinocytes, the transcription of CCL17 mRNA is preferentially induced by tumor necrosis factor-alpha (TNF-α); however, its regulatory mechanism has not been elucidated. The aim of the present study is to clarify the regulatory mechanism of TNF-α-induced CCL17 mRNA transcription in canine keratinocytes leading to the development of a chemokine-targeted therapy for cAD. In a cell line of canine epidermal keratinocyte, CPEK, stimulation with TNF-α induced not only the activation of nuclear factor-kappa B (NF-κB) but also the phosphorylation of c-Jun-N-terminal kinase (JNK) and mitogen-activated protein kinase p38 (p38). Extracellular signal-regulated kinase (ERK) was found to be constitutively phosphorylated, which was temporarily augmented by TNF-α. Results of the inhibition assay indicated that the CCL17 mRNA transcription level was significantly decreased by p38 inhibitors but was not altered by either JNK or NF-κB inhibitors. Surprisingly, the ERK inhibitor increased the transcription level of CCL17 mRNA. Stimulation with epidermal growth factor (EGF), an ERK activator, suppressed the transcription of CCL17 mRNA. The present results suggest that TNF-α-induced CCL17 mRNA transcription in CPEK is positively regulated by p38 but negatively controlled by ERK.

Effect of recombinant canine interferon-γ on granulocyte–macrophage colony-stimulating factor, transforming growth factor-β and CC chemokine ligand 17 mRNA transcription in a canine keratinocyte cell line (CPEK)

Recombinant canine interferon-γ (rCaIFN-γ) produced by a baculovirus expression system has therapeutic efficacy against atopic dermatitis in dogs. Although the mechanism of action of rCaIFN-γ is not completely understood, rCaIFN-γ is thought to downregulate the activity of interleukin-4- and interleukin-5-producing T helper 2 cells. However, rCaIFN-γ may also act directly on canine keratinocytes by inhibiting the release of inflammatory mediators. In this study, we investigated the effects of rCaIFN-γ on cytokine and chemokine mRNA transcription in a canine keratinocyte cell line, CPEK. It was found that granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA transcription was significantly inhibited after treatment with rCaIFN-γ (P<0.001), whereas transforming growth factor-β and CC chemokine ligand 17 mRNA levels were unchanged. This study suggests that rCaIFN-γ may suppress GM-CSF production from canine keratinocytes, although further studies are required to confirm this.