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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., (© 2013 The Authors. Veterinary Dermatology © 2013 ESVD and ACVD.)

House dust mite (HDM) allergens are the most common allergens for induction of IgE-mediated hypersensitivity. Recently, epicutaneous sensitization with HDM allergens has been emphasized in the development of atopic dermatitis (AD) by producing various soluble factors in keratinocytes. Among the soluble factors, GM-CSF is a key molecule that activates Langerhans cells, antigen-presenting cells in the epidermis. In the present study, we investigated the effects of Dermatophagoides farinae (Der f) on GM-CSF production in a canine keratinocyte cell line, CPEK. CPEKs were found to produce GM-CSF upon stimulation by Der f. The GM-CSF production was suppressed by addition of a cysteine protease inhibitor. The present results suggest that cysteine protease-derived Der f may be an initiator of allergic inflammation by inducing the production of GM-CSF in keratinocytes.

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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., (© 2010 The Authors. Journal compilation © 2010 ESVD and ACVD.)

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., (Copyright 2009 Elsevier Ltd. All rights reserved.)

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.