New Approaches to Immunosuppressive Therapy for Autoimmune Diseases
ACVIM 2008
Steven Dow, DVM, PhD
Dept of Clinical Sciences and Dept of Microbiology, Immunology, & Pathology
Ft. Collins, CO, USA

Introduction

Autoimmune diseases in dogs range in severity from nuisance problems to acutely life-threatening illnesses. Our understanding of the immunology underlying these diseases has increased substantially over the past 20 years, though unfortunately our ability to manage the diseases has not kept pace. The most serious autoimmune disease in dogs continues to be immune-mediated hemolytic anemia (IMHA), which appears to be increasing in severity in recent years and is now the leading cause of mortality due to autoimmune disease in dogs.1,2 Therefore, this proceeding will focus primarily on the management of IMHA in dogs, which continues to remain controversial.2,3 We will briefly discuss the pathogenesis of IMHA, and then discuss new developments in the management of the disease, including a novel macrophage depletion approach, the best strategies for prevention of thromboembolic complications, the generation of immunosuppression through the use of intravenous immune globulin, and combinations of newer immune suppressive drugs, including mycophenolate.

Current Understanding of Pathogenesis of IMHA in Dogs

The spontaneous production of autoantibodies directed against RBC antigens is responsible for some, though not all of the features of IMHA in dogs. Several target antigens on the surface of RBC that are recognized by RBC autoantibodies have been identified, including glycophorins and RBC membrane antigens. Several of these antigens are also recognized by CD4+ T cells from dogs that have recovered from IMHA.4 To date, no cause for IMHA has been identified and most cases remain idiopathic. It is likely that there are strong genetic contributions to the disease susceptibility, based on the fact that Cocker Spaniels and Schnauzer dogs are over-represented in disease-association studies.5,6 In addition, an association between certain MHC class II alleles in the dog and the risk of development of RBC autoantibodies and IMHA has been reported.6 The results of this report suggest that CD4+ T cell and B cell responses to unknown antigens presented by MHC class II molecules may strongly influence the development of disease. Though there is one previous report of a link between vaccination and IMHA, that finding was not been confirmed in a follow-up study.5,7 Thus, it is questionable whether vaccination is truly linked to IMHA in dogs.

Though severe and rapid onset anemia is the hallmark of acute IMHA, it is likely that more dogs actually succumb to IMHA-associated thromboembolic diseases than to anemia per se. The association between IMHA and thromboembolism was first noted by our group and subsequently confirmed by others.8-10 In fact, the major prognostic findings for IMHA appear in many cases to be related to the effects of thromboembolism.10 The thromboemboli that develop in IMHA typically develop in the venous circulation and are often very widespread, involving the heart, spleen, liver, kidneys and brain, in addition to pulmonary emboli.10

The major negative prognostic factors associated with a high risk of thromboembolism are hyperbilirubinemia, thrombocytopenia, leukocytosis with left-shift, and hypoalbuminemia.5,10 At present, it is not clear what mechanisms associated with IMHA actually trigger thromboembolic disease, though hemostatic abnormalities are common.11 It has been proposed that anti-endothelial antibodies may trigger endothelial injury and clotting in dogs with IMHA, but in a recent study we failed to find anti-endothelial antibodies in dogs with IMHA (Wells, R, et al; manuscript submitted). Studies investigating a possible association with anti-phospholipid antibodies are currently underway.

Preventing Thromboembolic Disease in Dogs with IMHA

Given the high risk of thromboembolic disease in dogs with severe IMHA, strategies designed to prevent development of thromboemboli are likely to have a significant effect on reducing morbidity and mortality from IMHA. Currently, the two recommended approaches are heparinization or treatment with ultra-low dose aspirin therapy. The benefits of either approach have not been established in carefully controlled randomized clinical trials. However, a retrospective study of dogs treated with a combination of corticosteroids with azathioprine and ultra-low dose aspirin therapy found that there was a significant improvement in survival to discharge and 30 day survival, compared to treatment with steroids and azathioprine alone and other treatments evaluated.12 In the same study, there was no benefit found for combining heparin therapy with prednisone and azathioprine therapy.

Macrophage Depletion to Suppress RBC Destruction: The Liposomal Clodronate Approach

An alternative to conventional therapeutics for treatment of IMHA is to stop the clearance of opsonized RBC by eliminating macrophages, the cells primarily responsible for destruction of antibody coated RBC. Previous studies in rodents have shown that when the bisphosphonate drug dichloroethylene bisphosphonate (clodronate) is incorporated within liposomes and injected intravenously in mice, the treatment can lead to the rapid elimination of macrophages in the spleen and liver. In a mouse model of IMHA, injection of liposomal clodronate was shown to be effective in markedly reducing RBC destruction.13

In a recent study, we evaluated the effectiveness of liposomal clodronate therapy in normal dogs and in dogs with IMHA.14 In that study, we found that liposomal clodronate therapy was well-tolerated by healthy dogs and by dogs with severe IMHA. We also found that a single liposome clodronate treatment rapidly suppressed elimination of opsonized RBC (Figure 1). For example, clearance of opsonized RBC was rapid in untreated healthy dogs (LC-), where clearance was almost completely suppressed in dogs treated with liposomal clodronate (LC+).

Figure 1.
Figure 1.

 

In another portion of the study, we used infusion of fluorescently labeled autologous RBC to investigate the turnover kinetics of RBC in dogs with active IMHA. Remarkably, we found that the turnover of RBC was dramatically increased in 7 dogs with severe IMHA, resulting in virtually complete destruction of all infused RBC within 24 hours of infusion (Figure 2). This finding illustrates dramatically the magnitude of RBC destruction that develops in dogs with severe IMHA. However, following liposomal clodronate treatment, the destruction of infused labeled RBC was decreased in a subset of treated dogs. Perhaps more importantly, both the short-term and long-term survival of 7 dogs with IMHA treated with liposomal clodronate was significantly increased compared to that of a control population of dogs matched for disease severity. The effectiveness of liposomal clodronate as a treatment for IMHA in dogs is currently being evaluated in a multi-center clinical trial at Colorado State University.

Figure 2.
Figure 2.

 

Adjunctive Drug Therapy for Rapid Immunosuppression in Dogs with IMHA

Though most dogs with IMHA are currently treated with corticosteroids and azathioprine, a number of other drugs are often used in an attempt to increase the potency and the speed of onset of immunosuppression. Among the adjunctive treatments administered include cyclophosphamide, cyclosporine, and leflunomide. With most of these adjunctive therapies, there have been few appropriately designed clinical trials to assess the benefit gained from adding these other immunosuppressive drugs. In the case of cyclophosphamide, the evidence is relatively clear that the drug adds no benefit and in fact may lead to poorer outcomes in dogs with IMHA.15,16 Cyclosporine has not been evaluated carefully for its benefit in IMHA and there are reasons to question the rationale for use of this drug in the acute IMHA setting. For example, cyclosporine is primarily active in suppressing cytokine secretion by memory T cells and therefore has a slow onset of action and no direct activity in suppressing antibody production by B cells.

Mycophenolate is an attractive alternative for immunosuppression in dogs, which we have used clinically in a number of dogs with IMHA, though carefully controlled clinical studies have not been conducted yet. This drug is being used increasingly for immunosuppression in humans undergoing bone marrow transplants and in some patients with autoimmune disease. In fact, mycophenolate has also been widely used experimentally in dogs undergoing bone marrow transplantation.17-19 The mechanism of activity is very similar to that of azathioprine, which is to competitively inhibit purine biosynthesis and suppress B and T cell proliferation. However, mycophenolate is much more potent than azathioprine and also stimulates the production of regulatory T cells, which would be beneficial in IMHA. Because of the more rapid onset of action than cyclosporine, mycophenolate is an attractive option for the treatment of IMHA, though the effectiveness of mycophenolate has yet to be proven in a randomized trial.

Other Options for the Acute Management of IMHA

Currently, the most effective treatment for IMHA in children with acute, severe IMHA is the use of monoclonal antibody therapy to deplete B cells.20,21 This can be accomplished with antibodies to CD20 (e.g., rituximab), which is expressed on most B cells. Unfortunately, to date it has not been shown that these antibodies cross-react with the homologous CD20 molecule on dog B cells. However, if such a cross-reactive antibody were identified, it would be a potential candidate for the acute management of IMHA in dogs.

Treatment with intravenous human gamma globulin (IVIG) is also effective in managing IMHA in humans and has been evaluated in dogs as well, though again not in carefully controlled clinical trials.22-25 It is difficult, therefore, to determine whether the expense of IVIG therapy is justified. The drug exerts at least two potential effects in suppressing clearance of opsonized RBC. Though it was originally thought that IVIG acted primarily by competing for Fc receptors, it now appears that the primary effect is by suppressing macrophage function through negative signaling via a certain type of Fc receptor. The major drawbacks to IVIG therapy are the high cost and the inability to obtain the drug because of the high demand for treatment of human immune-mediated diseases.

Conclusions

At present, the best approach to the management of IMHA in dogs appears to be combination therapy with steroids, azathioprine, and possibly ultra low-dose aspirin therapy. However, for severe cases, the addition of either IVIG or possibly mycophenolate may provide some benefit, though proof of efficacy awaits clinical trials. Liposomal clodronate may also prove to be an effective new treatment, pending the results of the current randomized clinical trial.

References

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Speaker Information
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Steven Dow, DVM, PhD
Colorado State University
Ft. Collins, CO


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