Breed differences in development of anti-insulin antibodies in diabetic dogs and investigation of the role of dog leukocyte antigen (DLA) genes

Abstract

Administration of insulin for treatment of diabetes mellitus in dogs can stimulate an immune response, with a proportion of animals developing anti-insulin antibodies (AIA). For an IgG antibody response to occur, this would require B cell presentation of insulin peptides by major histocompatibility complex (MHC) class II molecules, encoded by dog leukocyte antigen (DLA) genes, in order to receive T-cell help for class switching. DLA genes are highly polymorphic in the dog population and vary from breed to breed. The aim of the present study was to evaluate AIA reactivity in diabetic dogs of different breeds and to investigate whether DLA genes influence AIA status.

Indirect ELISA was used to determine serological reactivity to insulin in diabetic dogs, treated with either a porcine or bovine insulin preparation. DLA haplotypes for diabetic dogs were determined by sequence-based typing of DLA-DRB1, -DQA1 and -DQB1 loci. Significantly greater insulin reactivity was seen in treated diabetic dogs (n = 942) compared with non-diabetic dogs (n = 100). Relatively few newly diagnosed diabetic dogs (3/109) were found to be AIA positive, although this provides evidence that insulin autoantibodies might be involved in the pathogenesis of the disease in some cases. Of the diabetic dogs treated with a bovine insulin preparation, 52.3% (182/348) were AIA positive, compared with 12.6% (75/594) of dogs treated with a porcine insulin preparation, suggesting that bovine insulin is more immunogenic. Breeds such as dachshund, Cairn terrier, miniature schnauzer and Tibetan terrier were more likely to develop AIA, whereas cocker spaniels were less likely to develop AIA, compared with crossbreed dogs. In diabetic dogs, DLA haplotype DRB1*0015--DQA1*006--DQB1*023 was associated with being AIA positive, whereas the haplotype DLA-DRB1*006--DQA1*005--DQB1*007 showed an association with being AIA negative. These research findings suggest that DLA genes influence AIA responses in treated diabetic dogs.

Introduction

Diabetes mellitus is one of the most common endocrine disorders in dogs, with an estimated prevalence of 0.32% in the UK (Davison et al., 2005). It is a disease of middle to late age, with the majority of dogs diagnosed between 7 and 12 years old. It has been proposed that there are several potential underlying causes of diabetes in dogs, including immune mediated destruction of the beta cells of the pancreas, chronic pancreatitis and insulin resistance due to hormonal antagonism (Hoenig, 2002, Rand et al., 2004). Certain breeds of dog are predisposed to developing diabetes, which strongly suggests that there is a genetic component to disease susceptibility (Catchpole et al., 2008). Breeds such as the Samoyed, Tibetan terrier and Cairn terrier have an increased risk of developing diabetes, whereas other breeds, such as the Boxer and German Shepherd Dog have a reduced risk (Catchpole et al., 2005).

Dog leukocyte antigen (DLA) genes, which encode MHC class II molecules, demonstrate considerable inter-breed variability (Kennedy et al., 2002) and have been linked with susceptibility to diabetes mellitus (Catchpole et al., 2008, Kennedy et al., 2006). Three DLA haplotypes in particular, DLA-DRB1*009--DQA1*001--DQB1*008, DRB1*015--DQA1*006--DQB1*023 and DRB1*002--DQA1*009--DQB1*001, have been shown to be associated with susceptibility to diabetes and are prevalent in the Samoyed, Tibetan terrier and Cairn terrier breeds.

Virtually all diabetic dogs require insulin by injection to control their hyperglycaemia, but this can stimulate an immune response and some dogs develop anti-insulin antibodies (AIA) following initiation of therapy (Davison et al., 2003). A previous study showed that around 60% of dogs treated with a bovine insulin preparation, developed AIA, whereas only around 10% of dogs treated with a porcine insulin preparation, developed AIA (Davison et al., 2008). This suggests that bovine insulin is more immunogenic than porcine insulin, which is likely due to variation of the insulin sequence between species (Fineberg et al., 2007); the amino acid sequence is the same comparing porcine and canine insulin, but bovine insulin differs from canine insulin by two amino acids in the A chain (Davison et al., 2003). Although bovine insulin seems to be more immunogenic in dogs, the AIA that develop are not species specific as they react with both porcine and bovine insulin in ELISA and typically recognise conformational, rather than linear epitopes (Davison et al., 2003).

Generation of an immunoglobulin (Ig) G antibody response to a foreign protein requires B cells to process antigen and present digested peptide fragments, bound to MHC class II molecules, to recruit T cell help. Polymorphisms in MHC class II genes influence the structure of the peptide-binding groove and therefore the repertoire of antigenic peptides that can be presented to the immune system. Considering the small size of the insulin molecule, it is likely that there are limited peptide epitopes available for presentation, suggesting that MHC class II genes could play a major role in whether an anti-insulin response is initiated or not (Fineberg et al., 2007). In humans, differences in AIA production have been associated with particular human leukocyte antigen (HLA) types (Reeves et al., 1984, Schernthaner et al., 1979, Sklenar et al., 1982) and in mice the presence of specific H-2 linked immune response genes for insulin has been demonstrated (Kapp and Strayer, 1978).

The aim of the present study was to further evaluate AIA in diabetic dogs, treated with different insulin preparations, to determine whether there were breed differences in AIA reactivity and to evaluate whether there was any evidence for a DLA genetic influence on AIA responses in diabetic dogs.