Effect of recombinant canine IFNγ on canine atopic dermatitis evaluated by clinical signs,histopathology and expression of Th1/Th2 cytokine mRNA

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.     

Inhibitory effects of interleukin-10 plasmid DNA on the development of atopic dermatitis-like skin lesions in NC/Nga mice

Interleukin (IL)-10 exerts potent anti-inflammatory effects by suppression of both T-help (Th) 1 and Th2 cells. Previous studies have reported that IL-10 can ameliorate various inflammatory disorders. The present study was performed to examine whether IL-10 plasmid DNA could suppress development of atopic dermatitis (AD)-like skin lesions in NC/Nga mice, as an initial step towards the development of an appliance for use in dogs with AD. Intradermal injection of IL-10 plasmid DNA markedly inhibited the development of AD-like skin lesions, as evidenced by a marked decrease in skin symptoms and reduced inflammation within the skin lesions. Efficacy was confirmed by significant decreases in eosinophil ratio and serum IgE concentration, and a reduction in the number of Staphylococcus aureus recovered from the ear. Moreover, relative mRNA expression levels of IL-4 and interferon-γ in the skin lesions of mice injected with IL-10 plasmid DNA were also decreased compared with those of control mice. Of note, higher serum IL-10 levels in mice injected with IL-10 plasmid DNA were maintained compared with those in control mice. Taken together, the results indicate that IL-10 plasmid DNA can suppress the development of AD-like skin lesions by suppressing both Th1 and Th2 cell responses. Beneficial effects of IL-10 plasmid DNA may be expected in dogs with AD.     

FC-35 Real-time evaluation of cytokine and protease expression in flea-allergic and nonallergic dogs

Atopic dermatitis (AD) is very common in dogs, but its pathogenesis is not yet fully understood. It has been suggested that a Th2-dominant status may be associated with the occurrence of canine AD. IL-12 is thought to be important for the differentiation of Th1 cells. The IL-12 receptor β2 (IL-12Rβ2) gene is considered to play a critical role in signal transduction and is attracting attention as one of the causative genes of AD in humans. The purpose of this study was to investigate the relationship between IL-12Rβ2 gene expression and canine AD. The canine IL-12Rβ2 gene was cloned by RT-PCR and its nucleotide sequences were determined. Canine IL-12Rβ2 showed 76.8% homology at the amino acid level with human IL-12Rβ2, and its structural motifs were well conserved. cDNA with a 91 bp deletion including the transmembrane region was also cloned, which consequently produced a frame shift and an early stop codon. The deletion region corresponded to exon 14 of the human IL-12Rβ2 gene on chromosome 1. The expression of deleted canine IL-12Rβ2 mRNA in phytohemagglutinin-stimulated peripheral blood mononuclear cells was examined in seven healthy dogs and 11 AD dogs. Both deleted and intact mRNAs were expressed at constant ratios in healthy and AD dogs. The results indicate that the deletion of the transmembrane region is not associated with the occurrence of AD, and that the expression of the deleted mRNA may be constitutive and produced by alternative splicing.
Funding: Self-funded.     

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.     

Semi-quantitative analysis of cytokine expression in asymptomatic canine leishmaniasis

The dog is the main reservoir of Leishmania infantum, the parasite responsible for visceral leishmaniasis in Mediterranean countries. The infection in dogs shows different clinical presentations, from subclinical/asymptomatic to a fully developed disease, depending on the host's immune responses. The Th1/Th2 dichotomy is not clear in the different forms of canine leishmaniasis, since the data available from studies of immunity response in canine leishmaniasis are scarce and fragmented. The present work describes the cytokine expression in peripheral blood mononuclear cells (PBMC) obtained from asymptomatic dogs experimentally infected with L. infantum that present a cellular protective immune response. The results obtained from freshly isolated PBMC showed expressions of TNF-alpha, IL-2, IFN-gamma, IL-10 and IL-18 mRNA, similar to those from non-infected dogs. However, there was almost no expression of IL-4 mRNA detected in the asymptomatic infected dogs compared to the control dogs. Unspecific stimulation with ConA promoted the expression in a greater or lower degree of all the cytokines studied. In vitro stimulation of PBMC with soluble leishmanial antigen (SLA) promoted the expression of IL-2, IFN-gamma, TNF-alpha, IL-18, IL-4, IL-6 and IL-10 mRNA, with the two first being specifically induced. Although both Th1 and Th2 cytokines are produced, cell mediated immunity observed in these L. infantum-infected asymptomatic dogs depended on the preferential expression of Th1 cytokines.     

FC-38 Efficacy of conjugated linoleic acid and black currant seed oil in the treatment of canine atopic dermatitis: a double-blinded, randomized, placebo-controlled study

The purpose of this study was to evaluate a combination of immunostimulatory bacterial DNA sequences and allergen-specific immunotherapy for the treatment of canine atopic dermatitis. Seven dogs with nonseasonal atopic dermatitis diagnosed by history, clinical signs and exclusion of differential diagnoses were included. All dogs had been on allergen-specific immunotherapy for at least 12 months with incomplete responses, were on additional antipruritic therapy and showed residual pruritus. Pruritus was marked by the owner on a visual analogue scale, lesions were determined by a clinician using the Canine Atopic Dermatitis Extent and Severity Index (CADESI), and concurrent medications were recorded before entering the study and after 14 weeks of treatment. Peripheral blood mononuclear cells were isolated and cultured; canine cytokine message for IFNγ, IL-4, TNF and IL-10 was quantitated using RT-PCR. A mixture of allergen extract and liposome-DNA complexes was injected intradermally at the beginning of the study and after 2, 4, 6, 10 and 14 weeks. CADESI, pruritus and medication scores, and cytokine messages at the beginning and end of the study were compared with a paired t -test. There were significant improvements in pruritus scores ( P  = 0.0277). Reductions in medication scores and CADESI were not statistically significant. IL-4 production decreased significantly ( P  = 0.0428); decreases in other cytokines were not significant. Although the number of dogs in this pilot study was small, the results warrant further investigation of a combination of immunostimulatory bacterial DNA sequences and allergen-specific immunotherapy for the treatment of canine atopic dermatitis.
Funding: Self-funded.     

Immune dysregulation in flea allergy dermatitis--a model for the immunopathogenesis of allergic dermatitis

BACKGROUND: Flea allergy dermatitis (FAD) is a common skin disease in dogs and can be induced experimentally. It often coexists with other allergic conditions. So far no studies have investigated the quantitative production of cytokine mRNA in skin biopsies and peripheral blood mononuclear cells (PBMC) in flea allergic dogs. OBJECTIVE: The aim of our study was to improve the understanding of the immunopathogenesis of allergic dermatitis as a response to fleabites. MATERIAL AND METHODS: Allergic and non-allergic dogs were exposed to fleas. Before and after 4 days of flea exposure mRNA was isolated from biopsies and PBMC. Production of chymase, tryptase, IL-4, IL-5, IL-13, TNF-alpha and IFN-gamma mRNA was measured by real-time RT-PCR. The inflammatory infiltrate in the skin was scored semi-quantitatively. The number of eosinophils, mast cells (MC) and IgE+ cells/mm2 was evaluated to complete the picture. RESULTS: FAD was associated with a higher number of MC before flea exposure and with a significant increase of eosinophils after flea exposure as compared to non-allergic dogs. The number of IgE+ cells was higher in allergic dogs before and after flea exposure. In allergic dogs mRNA for most cytokines and proteases tested was higher before flea exposure than after flea exposure. After exposure to fleas an increased mRNA production was only observed in non-allergic dogs. In vitro stimulation with flea antigen resulted in a decreased expression of most cytokines in allergic dogs before flea exposure. In contrast, in PBMC, only increased levels of IL-4 and IL-5 mRNA were observed in allergic dogs before flea exposure. However, after flea exposure and additional stimulation with flea antigen the production of mRNA for all cytokines tested was significantly increased in allergic dogs. CONCLUSION: We demonstrated that the response in biopsies and PBMC is different and that FAD is associated with a TH2 response.     

Lack of detection of circulating skin-specific IgE autoantibodies in dogs with moderate or severe atopic dermatitis

Human patients with atopic dermatitis (AD) commonly exhibit IgE reactivity to cutaneous self-antigens. The presence of serum IgE autoantibodies appears to correlate with disease severity, and it is suspected to reflect or contribute to tissue damage. The objective of this study was to determine whether IgE autoantibodies specific for cutaneous antigens could be detected in the serum of dogs with AD. Serum was collected from 19 dogs with untreated moderate to severe AD and four specific-pathogen free (SPF) dogs. Indirect immunofluorescence was performed using normal canine skin collected at four different locations (concave ear, nose, medial thigh and lateral thorax), while Western immunoblotting was done using normal canine ear pinna epidermal and dermal extracts and reducing conditions. In both methods, IgE was detected using a monoclonal antibody specific for heat stable epitopes of canine IgE. At 1:10 dilution, specific IgE autoantibodies against cutaneous autoantigens were not detected, with either method, in AD and SPF canine sera. Either IgE autoreactivity is not associated with moderate to severe AD in dogs, or the methods employed herein were not sensitive enough to permit IgE autoantibody detection.     

FC-56 Nodular and non-nodular focal alopecia related to drug injections: a retrospective study of 32 dogs

IgE-mediated late-phase reactions can be induced in the skin of normal and atopic dogs by intradermal injections of anti-IgE antibody. The histology of these reactions is very similar to that of naturally occurring atopic dermatitis. To characterize the cellular, cytokine and chemokine responses in the skin of placebo- and prednisolone-treated dogs, normal beagles received either placebo or 0.5 mg/kg prednisolone twice daily for three days prior to intradermal injection of polyclonal rabbit anti-canine IgE. Eight-millimetre punch biopsy skin samples were taken before injection and at the injection sites after 6, 24 and 48 h. Histological and immunohistochemical examination revealed a rapid cellular influx. Eosinophil and neutrophil numbers increased from <1 to 61.4 ± 14.1, and from 7 to 62.2 ± 10.8 cells/mm², respectively, within 6 h after injection , and remained moderately elevated 48 h later. The numbers of CD1c+, CD3+ and CD4+ mononuclear cells were also increased by 6 h. Taqman analysis demonstrated 2.5- to 72-fold increases in mRNA expression for IL-13, IL-5, MCP (CCL2), RANTES (CCL5) and TARC (CCL17). Levels of mRNA for IL-2, IL-4, IL-6 , and IFNγ remained negligible. Prednisolone administration suppressed the influx of neutrophils and eosinophils, and the expression of IL-13, CCL2, CCL5 and CCL17 (33, 97, 58, 86, 73 and 90%, respectively), as well as the influx of CD1c+ and CD3+ cells. These data document the cytokine and chemokine response to anti-IgE injection and demonstrate the anti-inflammatory effect of prednisolone.
Funding: Schering-Plough Animal Health.     

Quantitation of canine regulatory T cell populations, serum interleukin-10 and allergen-specific IgE concentrations in healthy control dogs and canine atopic dermatitis patients receiving allergen-specific immunotherapy

Canine atopic dermatitis (AD) shares many clinical and immunological similarities with human AD. Regulatory T cells (Treg) are a distinct lineage of T lymphocytes with various immunosuppressive properties including the down-regulation of allergic inflammation associated with IgE production. Antigen-induced Treg typically regulate immune homeostasis via productions of cytokines such as interleukin-10. Given the immunological similarities with human AD, it is likely that Tregs and the cytokines they produce play an important role in diseases of dogs as well. A cross-reactive FoxP3 antibody was used to identify a subset of CD4(+) T cells in the blood of both healthy dogs and dogs with atopic dermatitis undergoing immunotherapy over a year period. There was no significant difference in the Treg percentage over time in the healthy dogs. The immunotherapy group showed a significant increase in Treg percentage at 6, 9, and 12 months when compared to the healthy dogs. For the immunotherapy group, the mean Treg percentage at the beginning of the study was 4.94+/-0.71 and 10.86+/-2.73 at the completion. A commercially available ELISA kit was also used to quantitate the concentration of IL-10 in the serum of the same subsets of dogs. There was no significant difference in the IL-10 concentrations over time in the healthy dogs. The immunotherapy group showed a significant increase in serum IL-10 concentrations at 6, 9, and 12 months when compared to the control group. The mean serum IL-10 concentration at the initiation of immunotherapy was 20.40+/-3.52ngL(-1) and 37.26+/-15.26ngL(-1) at the completion of the study. The immunotherapy group also showed a significant decrease in serum IgE levels over the 1-year treatment period for specific allergens identified during ASIT. We conclude from these studies that similar to humans undergoing immunotherapy, increasing Treg populations likely play a significant role in the success of this particular type of therapy for atopic dermatitis and other allergic conditions.     

FC‐33 Expression of IL‐12 receptor β2 gene in atopic dogs

Atopic dermatitis (AD) is very common in dogs, but its pathogenesis is not yet fully understood. It has been suggested that a Th2‐dominant status may be associated with the occurrence of canine AD. IL‐12 is thought to be important for the differentiation of Th1 cells. The IL‐12 receptor β2 (IL‐12Rβ2) gene is considered to play a critical role in signal transduction and is attracting attention as one of the causative genes of AD in humans. The purpose of this study was to investigate the relationship between IL‐12Rβ2 gene expression and canine AD. The canine IL‐12Rβ2 gene was cloned by RT‐PCR and its nucleotide sequences were determined. Canine IL‐12Rβ2 showed 76.8% homology at the amino acid level with human IL‐12Rβ2, and its structural motifs were well conserved. cDNA with a 91 bp deletion including the transmembrane region was also cloned, which consequently produced a frame shift and an early stop codon. The deletion region corresponded to exon 14 of the human IL‐12Rβ2 gene on chromosome 1. The expression of deleted canine IL‐12Rβ2 mRNA in phytohemagglutinin‐stimulated peripheral blood mononuclear cells was examined in seven healthy dogs and 11 AD dogs. Both deleted and intact mRNAs were expressed at constant ratios in healthy and AD dogs. The results indicate that the deletion of the transmembrane region is not associated with the occurrence of AD, and that the expression of the deleted mRNA may be constitutive and produced by alternative splicing. Funding: Self‐funded.     

A functional comparison of canine and murine bone marrow derived cultured mast cells

Disorders involving mast cells are extremely common in dogs, ranging from allergic diseases to neoplastic transformation resulting in malignant mast cell tumors. Relatively little is known regarding the basic biologic properties of normal canine mast cells, largely due to the difficulty in reliably purifying large numbers from canine skin. In vitro generated bone marrow derived cultured mast cells (BMCMCs) are routinely used in both human and murine studies as a ready source of material for in vitro and in vivo studies. We previously developed a technique to generate canine BMCMCs from bone marrow derived CD34+ cells and demonstrated that these cells exhibit the phenotypic properties characteristic of mast cells and release histamine in response to IgE cross-linking. The purpose of the following study was to characterize the functional properties of these canine BMCMCs and contrast these with the functional properties of murine BMCMCs. Our work demonstrates that both IL-4 and IL-10 promote canine BMCMC proliferation, possibly through upregulation of Kit expression, while TGFbeta inhibits proliferation. The canine BMCMCs produce a variety of cytokines and chemokines in response to IgE cross-linking and chemical stimulation including IL-3, IL-4, IL-13, GM-CSF, RANTES, and MIP1alpha. Interestingly, the canine BMCMCs released significantly larger amounts of MCP-1 and tryptase and significantly smaller amounts of IL-6 following chemical stimulation and IgE cross-linking when compared to murine BMCMCs. Lastly, the canine BMCMCs produced larger amounts of active MMP9 than their murine counterparts. In summary, canine BMCMCs exhibit unique functional properties that distinguish them from murine BMCMCs and provide insight into the contribution of these cells to mast cell disorders in the dog.     

Gene expression of selected signature cytokines of T cell subsets in duodenal tissues of dogs with and without inflammatory bowel disease

Inflammatory bowel disease (IBD) is a common cause of chronic diarrhoea in dogs. In people, specific cytokine patterns attributed to T cell subsets, especially T helper cell [Th]1, Th17 and regulatory T(reg) cells have emerged in IBD. In contrast, no specific involvement of a distinct T cell subset has been described so far in canine IBD. Thus, the aim of the present study was to assess gene expression of signature cytokines in duodenal tissues from 18 German shepherd dogs with IBD (group 1), 33 dogs of other breeds with IBD (group 2) and 15 control dogs (group 3). Relative quantification of IL-17A, IL-22, IL-10, IFNy and TGFβ was performed. Expression of IL-17A was significantly lower in groups 1 and 2 compared to group 3 (p=0.014), but no difference in the expression of IL-22 (p=0.839), IFNγ (p=0.359), IL-10 (p=0.085) or TGFβ (p=0.551) across groups was detected. Thus, no clear evidence for the involvement of Th-17 signature cytokines in canine IBD at the mRNA level could be shown. The contribution of specific T cell subsets to the pathogenesis of canine IBD warrants further investigation.     

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.     

Evaluation of cytokine messenger RNA expression in peripheral blood mononuclear cells from dogs with canine demodicosis

Using RT-PCR and semi-quantitative PCR, mRNA expression for canine interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-5, IL-10, tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta in peripheral blood mononuclear cells (PBMCs) was examined in dogs with or without demodicosis. mRNA expression for IFN-gamma as well as TNF-alpha in dogs with demodicosis (localized (LD) and generalized (GD)) was slightly lower than those in dogs without demodicosis (healthy controls). Expression of IL-5 mRNA in dogs with demodicosis was higher than that in control dogs, but there were no significant differences in IL-4 and IL-10 mRNA expression levels among the three groups. On the other hand, expression levels of TGF-beta mRNA in dogs with GD were higher than those in control dogs and dogs with LD. The expression levels of IL-5 and TGF-beta mRNA decreased in all three dogs with GD which showed resolution of the clinical signs. Taken together, these results suggest that the Th2-like response in PBMCs from dogs with demodicosis is up-regulated, and that subsequent increased expression of IL-5 and TGF-beta mRNA in dogs with GD is reversible after treatment. Therefore, these cytokines, particularly IL-5, might be a useful clinical index of the clinical course in demodicosis. Also, increased TGF-beta mRNA expression might be a key factor for revealing the difference in the mechanism of onset between LD and GD.