Transcription profile of chemokine receptors, cytokines and cytotoxic markers in peripheral blood of dogs with epitheliotropic cutaneous lymphoma.

BACKGROUND: Mycosis fungoides (MF) is the most common form of canine epitheliotropic cutaneous lymphoma, which is characterized by the accumulation of neoplastic CD8(+) T cells. Given that multifocal skin lesions are commonly seen in MF, neoplastic lymphocytes may actively migrate into the blood circulation. HYPOTHESIS/OBJECTIVES: Cytotoxic T cells with a skin-homing phenotype could be increased in the blood circulation of dogs with MF. ANIMALS: Ten dogs with MF and 10 age-matched healthy dogs were included. METHODS: The transcription levels of chemokine receptors, cytokines and cytotoxic markers in peripheral blood of dogs with MF were quantified by real-time RT-PCR. RESULTS: The dogs with MF had lower transcription levels of chemokine receptors associated with skin homing (CCR4), epitheliotropism (CXCR3), lymph node homing (CCR7), a type-1 cytokine (LT-α) and cytotoxic markers (perforin and granzyme B) in the circulation than healthy control dogs (P < 0.05). CONCLUSIONS AND CLINICAL IMPORTANCE: The present results suggest that the number of peripheral cytotoxic T cells with a skin-homing phenotype could be decreased in the peripheral blood of dogs with MF, which might be due to the sequestration of cytotoxic T cells in the lesional skin.

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.

Production of GM-CSF mediated by cysteine protease of Der f in canine keratinocytes.

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.

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.

Effect of recombinant canine interferon-γ on granulocyte-macrophage colony-stimulating factor, transforming growth factor-ß 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-ß 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.

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.

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.