Further studies on the immunopathogenesis of canine atopic dermatitis

Abstract

Various aspects of the pathogenesis of canine atopic dermatitis have been elucidated such as the role of certain allergens, antigen presenting cells, allergen-specific T lymphocytes, IgE and mast cells. However, gaps in our understanding still remain, such as the role of antigen-specific IgG and cytokine subsets. Canine atopic dermatitis is commonly treated using allergen-specific immunotherapy, but the mechanisms of its action are still incompletely understood. The studies in this thesis investigate further some aspects of the immunopathogenesis of canine atopic dermatitis and the changes that occur during allergen-specific immunotherapy.To investigate IgG responses to D.farinae antigens, a semi-quantitative, Western blot, digital image analysis system was developed and validated. Both healthy and atopic dogs mounted D. /armae-specific total IgG, IgGl and IgG4 responses to separated antigens. D. farinae-specific IgG2 and IgG3 responses were difficult to detect. The profile of IgG binding was similar in the two groups, both in terms of the number of bands recognised and their molecular weights. The most commonly recognised band in both groups was a 98 kDa antigen, most likely to be the major allergen Der f 15. These results indicate that D. farinae antigens are recognised by the canine immune system regardless of whether or not a dog is atopic. They also demonstrate that antibody class switching to IgE in atopic dogs does not appear to inhibit IgG production. The IgG antibody response does not appear to be protective against the development of clinical atopic disease, but whether or not it plays any role in the pathogenesis of the disease remains to be determined.In atopic dogs undergoing allergen-specific immunotherapy (ASIT) against D. farinae with alum-precipitated vaccines, there was no significant increase in D. farinae-specific total IgG, IgGl or IgG4, even in dogs showing apparent clinical improvements. In contrast, ASIT using aqueous vaccines resulted in significant increases in D. farinae-specific IgG responses. These results suggest that aqueous ASIT may elicit the production of IgG blocking antibodies in atopic dogs, an effect not detected using alum-precipitated vaccines.To investigate the cytokine milieu in dogs with atopic dermatitis, real-time quantitative RT-PCR was used to detect the expression of mRNA transcripts of the Thl cytokine IFN-y, the Th2 cytokine IL-4, the Treg cytokine TGF-|3 and inducible NO synthase (iNOS) as a measure of the innate immune response. The housekeeping gene for 18S ribosomal RNA (rRNA) was chosen as internal control to normalise variations in the amount of starting material between samples and between individuals. The expression of TGF-p and iNOS were lower in lesional skin compared to non-lesional skin in atopic dogs, whilst IFN-y was expressed in a significantly higher level in lesional skin compared to healthy controls. IL-4 expression did not differ between the groups. These cytokine profiles show distinct differences to those reported using non real-time, semi-quantitative RT-PCR. These differences may reflect the various methodological approaches used and illustrate the limitations inherent in such techniques for the quantification of cytokine expression.