Expression analysis of CCL27 and CCL28 mRNA in lesional and non-lesional skin of dogs with atopic dermatitis

Chemokines are important regulators of the selective recruitment of inflammatory cells into sites of allergic inflammation. Since canine atopic dermatitis (AD) shares many clinical features of human AD, patterns of chemokine production in dogs may also be similar with those in humans. The aim of this study was to examine mRNA expression of CCL27 and CCL28 in lesional skin of dogs with AD to demonstrate similarity of chemokine production with human counterparts. RNA was extracted from skin biopsy specimens of 12 dogs with AD. The mRNA expression of CC chemokines (CCL4, CCL19, CCL20, CCL21, CCL24, CCL27 and CCL28) was analyzed by quantitative real-time PCR and was compared between lesional and non-lesional skin. Seven types of chemokines examined were constitutively expressed in both lesional and non-lesional skin. It was found that mRNA expression levels of CCL27 and CCL28 among the chemokines were significantly different between lesional and non-lesional skin (P<0.05). Expression level of CCL27 mRNA in lesional skin was significantly lower than that in non-lesional skin. On the other hand, CCL28 mRNA expression in lesional skin was found to be higher than that in non-lesional skin. These results suggest that CCL28 but not CCL27 may play important roles in immunopathogenesis of canine AD, indicating that experimental canine study may provide additional information that can be extrapolated to human AD.     

Evaluation of the expression of P-selectin, ICAM-1, and TNF-alpha in bacteria-free lesional skin of atopic dogs with low-to-mild inflammation

Canine atopic dermatitis (AD) is a pruritic skin condition that shares many clinical and pathophysiological features with its human counterpart. A major therapeutic challenge of AD is the control of the skin inflammatory process. A detailed knowledge of the pro-inflammatory molecules involved in cell recruitment in AD would allow for a better control of the disease. We thus have studied the protein expression of P-selectin, ICAM-1 and TNF-alpha in the lesional and non-lesional skin of atopic dogs that had been treated for bacterial infections. Despite a low-to-mild inflammatory process, P-selectin protein was clearly upregulated in the lesional skin areas when compared with non-lesional skin (four-fold average increase). This P-selectin upregulation was accompanied by signs of functional changes such as increased cell margination, and membrane-associated protein expression. Although the expression of ICAM-1 and TNF-alpha was not enhanced in the lesional versus the non-lesional skin, there was a trend towards a correlated upregulation of both molecules. Further studies will help elucidate the significance of the substantial overexpression of P-selectin in canine AD, in particular in a scenario where bacterial antigens are not contributing as pro-inflammatory stimuli.     

Expression of CC chemokine receptor 4 (CCR4) mRNA in canine atopic skin lesion

CC chemokine receptor 4 (CCR4) is a G protein-coupled seven transmembrane receptor that is selectively expressed on Th2 cells and plays an important role in the trafficking of Th2 cells into inflammatory sites. In this study, a full-length canine CCR4 cDNA was cloned and characterized in order to examine the potential role of CCR4 in allergic responses that produce skin lesions in canine atopic dermatitis (AD). The canine CCR4 cDNA reported in this study contained an open reading frame of 1083 nucleotides encoding 360 amino acids. The predicted amino acid sequence of canine CCR4 showed 91.9, 85.3 and 84.5% similarity with those of the human, mouse and guinea pig counterparts, respectively. Expression of CCR4 mRNA was detected in various tissues including thymus, spleen, heart, small intestine and lymph node. Furthermore, it was found that CCR4 mRNA was preferentially expressed in lesional skin of dogs with AD, together with the mRNA of thymus and activation-regulated chemokine (TARC), which is a ligand for CCR4. The present study demonstrates that CCR4 contributes strongly to the immunopathogenesis of canine AD.     

Allergen-induced production of IL-31 by canine Th2 cells and identification of immune,skin, and neuronal target cells

The canine cytokine IL-31 induces pruritus in dogs and can be detected in dogs with atopic dermatitis; however very little is understood around its interactions with specific canine cells. We hypothesize that IL-31 is involved in the progression of allergic skin disease by coordinating the interaction between the immune system with skin and neuronal systems. The goal of the following work was to identify cells that produce IL-31 as well as cells that may respond to this cytokine. Peripheral blood mononuclear cells (PBMCs) were collected from naïve and house dust mite (HDM) allergen-sensitized beagle dogs and used for ex vivo characterization of cytokine production assessed using ELISpot and quantitative immunoassay. Sensitization to HDM allergen induced a T-helper type 2 (Th2) cell phenotype characterized by an increase in the production of IL-4 protein. Interestingly, repeated allergen challenge over time also resulted in an increase in IFN-γ. Further evaluation showed that co-stimulation of Th2 polarized cells with antigen and the bacterial component Staphylococcus enterotoxin B (SEB) produced higher levels of IL-31 compared to either stimulant alone. Production of IL-31 when PBMCs were stimulated by T cell mitogens suggests T cells as a source of IL-31. Quantitative real-time PCR was utilized to determine expression of the IL-31 receptor alpha chain in canine cell lines and tissue. Canine monocytic cells, keratinocytes, and dorsal root ganglia were shown to express the IL-31 receptor alpha chain mRNA. In a multifaceted disease such as canine atopic dermatitis, the combination of Th2 polarization and microbial presence may lead to IL-31 mediated effects driving inflammation and pruritus by immune cells, keratinocytes, and direct neuronal stimulation.     

Reference genes for canine skin when using quantitative real-time PCR

Quantitative real-time PCR (qPCR) facilitates the quantification of mRNA expression. Accurate qPCR analysis of gene expression requires the normalisation of data using a reference or housekeeping gene which is expressed at a similar level in all tissues tested. GAPDH is the most well known and most widely used reference gene but many papers have demonstrated that it is not stably expressed in different tissues. The aim of this study was to measure reference gene stability in canine skin using real-time qPCR. Skin samples from healthy control dogs (n=7) and dogs with atopic dermatitis (lesional skin n=7 and non-lesional skin n=7) were used to quantify seven reference genes (IMP, CG14980, S7, HIRA, GAPDH, RPL13A and SDHA) in canine whole skin. Three different statistical programs (Bestkeeper, GeNorm and Normfinder) were used to assess the stability of the reference genes. The results confirmed that GAPDH is not a stably expressed reference gene in canine skin; this finding may influence interpretation of previous qPCR studies on canine skin using this as a reference gene. RPL13A and CG14980 were found to be the most stably expressed genes in canine whole skin and would be more suitable as reference genes in future studies.     

Dermatophagoides farinae-specific IgG subclass responses in atopic dogs undergoing allergen-specific immunotherapy

Atopic dermatitis (AD) is thought to be caused by immunologic abnormalities expressed as a Th1/Th2 cytokine imbalance in both humans and dogs. Several studies have focused on the therapeutic effects of IFNγ in human AD with successful results; however, the mechanism of action of IFNγ is not fully understood. We investigated the effect of recombinant canine interferon gamma (rCaIFNγ) on 10 dogs with AD and evaluated the ratio of IL-4 mRNA to IFNγ mRNA in peripheral blood mononuclear cells, serum total IgE levels, and histological changes in skin. After six injections of rCaIFNγ over a span of 2 weeks, seven of the 10 dogs showed improvement, and six of these seven dogs exhibited decreased IL-4:IFNγ mRNA ratios. Two of the three cases that did not improve had increased IL-4:IFNγ mRNA ratios. Total serum IgE levels were significantly decreased in nine of 10 cases. The number of IgE-positive cells detected by immunostaining and the number of mast cells in skin biopsy samples were decreased. A reduction of epidermal cell layers was demonstrated by histopathology after treatment. These results demonstrated that rCaIFNγ may be a novel safe and effective therapeutic option for the treatment of canine AD, and the mechanism of action of rCaIFNγ may be related to the modulation of Th2 cytokines to Th1 cytokines with the reduction of serum IgE production.
Funding: Self-funded.     

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.