Pathogenesis

The hallmark of all forms of lupus is the development of autoantibodies against many different nuclear structures, including nucleic acids, ribonucleoproteins, and chromatin. These antinuclear antibodies (ANAs) are found in 97% to 100% of dogs with lupus compared with 16% to 20% of normal control animals. About 16 different nuclear antigens have been described in humans. Dogs differ from humans in that they mainly develop autoantibodies against nuclear proteins such as histones and ribonucleoproteins. These antinuclear antibodies cause tissue injury by several mechanisms. They can combine with free antigens to form immune complexes that are deposited in glomeruli, causing a membranoproliferative glomerulonephritis (Chapter 30). They may be deposited in arteriolar walls, where they cause fibrinoid necrosis and fibrosis, or in synovia, where they provoke arthritis. They may bind to Fc receptors on immune cells, leading to cell activation. The immune responses in SLE are associated with the production of interferon- α (IFN-α) so that the level of this cytokine correlates with disease activity. IFN-α is produced by plasmacytoid dendritic cells. It is believed that in SLE, FcR-, and TLR-mediated uptake of immune complexes and nucleic acids activates plasmacytoid dendritic cells. The IFN promotes inflammatory responses, activating macrophages and autoreactive T cells. ANAs also bind to the nuclei of degenerating cells to produce round or oval structures called hematoxylin bodies in the skin, kidney, lung, lymph nodes, spleen, and heart. Within the bone marrow, opsonized nuclei may be phagocytosed, giving rise to lupus erythematosus (LE) cells (Figure 36-3).

In addition to making antinuclear antibodies, many patients with lupus and related diseases make autoantibodies against two ribonucleoproteins called SSA/Ro and SSB/La. There appear to be significant and consistent clinical differences between patients producing these two types of autoantibody. These antibodies have been detected in dogs.

The production of ANAs in lupus may result from several possible defects. One possibility is that the intracellular TLRs, notably TLR7 and TLR9, lose the ability to discriminate between microbial and self-DNA. If, at the same time, some of their B cells undergo somatic mutation that enables their B cell receptors (BCRs) to bind self-DNA, the ingredients necessary for a profound antibody response to mammalian DNA come together.

Bacterial DNA is a potent antigen and immune stimulant. Since mammalian DNA and bacterial DNA have a conserved backbone structure, it is possible that patients with lupus may respond to bacterial infection by producing cross-reactive antibodies that react with mammalian DNA. For example, the NZB/NZW mouse strain spontaneously develops a lupus-like syndrome when immunized with bacterial DNA. This induces antibodies that bind mammalian double-stranded DNA. These anti-DNA antibodies may form immune complexes and cause arthritis, skin rashes, and vascular disease. Antibody-DNA immune complexes may bind to TLR9 and activate autoreactive B cells by triggering both the TLRs and antigen receptors.

A related defect in some lupus patients appears to be the impaired clearance of apoptotic cells. Normally, apoptotic cells are removed by phagocytosis by macrophages without causing inflammation (Chapter 5). Macrophages from SLE patients, however, show defective phagocytosis of apoptotic cells, which as a result accumulate in tissues. Nuclear fragments from these cells may be trapped and processed by dendritic cells, thereby triggering autoantibody formation. The defect is most obvious in the skin of affected animals, where ultraviolet (UV) radiation leads triggers apoptotic cell death. Nucleic acids from these cells may then act as autoantigens and trigger autoantibody formation. These autoantibodies in turn lead to immune complex deposition and tissue damage. Complement components mediate the efficient clearance of apoptotic cells, so complement deficiencies are associated with the development of lupus-like syndromes. Although a failure of apoptosis leading to activation of autoimmune B cells and multiple autoimmune disorders is a feature of lupus, its initiating cause remains obscure. Although ANAs are characteristic of lupus, many other autoantibodies are produced, suggesting that affected animals may have grossly abnormal B cell function. Affected animals show abnormalities in B cell signaling and migration, overexpression of CD154 (CD40L), and enhanced production of interleukin-6 (IL-6) and IL-10. Some experimental mouse models show overexpression of B cell stimulatory molecules by T cells and dendritic cells. It is therefore possible that the production of multiple autoantibodies in lupus is a combined result of defective apoptosis, overstimulation of B cells, and a failure to eliminate self-reactive B cells.

Autoantibodies to red cells induce a hemolytic anemia. Antibodies to platelets induce a thrombocytopenia. Antilymphocyte antibodies may interfere with immune regulation. About 20% of dogs with lupus produce antibodies to IgG (RFs). Antimuscle antibodies may cause myositis, and antimyocardial antibodies may provoke myocarditis or endocarditis. Antibodies to skin basement membrane cause a dermatitis characterized by changes in the thickness of the epidermis, focal mononuclear cell infiltration, collagen degeneration, and immunoglobulin deposits at the dermoepidermal junction. These deposits form a “lupus band,” seen in many other autoimmune skin diseases in addition to lupus (Figure 36-4). In humans, lupus skin lesions are commonly restricted to the bridge of the nose and the area around the eyes since apoptosis is triggered by UV radiation in sunlight. The results of this excessive immune reactivity are also reflected in a polyclonal gammopathy, enlargement of lymph nodes and spleen, and thymic enlargement with germinal center formation. As described in Chapter 7, some lupus patients have a deficiency of the complement receptor CD35. As a result, immune complexes are not bound to red cells or platelets and therefore are not effectively removed from the circulation. These immune complexes may then be deposited in the glomeruli or in joints.

The great variety of autoantibodies produced in lupus can cause an equally great variety of clinical symptoms. Polyarthritis, fever, proteinuria, anemia, and skin diseases are the most common abnormalities, but pericarditis, myocarditis, myositis, lymphadenopathy, and pneumonia have also been reported.

Equine Lupus

Equine lupus presents as a generalized skin disease (alopecia, dermal ulceration, and crusting), accompanied by an antiglobulin-positive anemia. The disease is remarkable insofar as affected horses may be almost totally hairless (Figure 36-5). Affected horses are ANA positive, although LE cell tests are equivocal in this species. Skin biopsies show basement membrane degeneration and immunoglobulin deposition typical of lupus. Affected horses may also have glomerulonephritis, synovitis, and lymphadenopathy. Treatment of reported cases has been unsuccessful.

Canine Lupus

Lupus affects middle-aged dogs (between 2 and 12 years of age) and affects males more than females. The disease is commonly seen in Collies, German Shepherds, Nova Scotia Duck Tolling Retrievers, and Shetland Sheepdogs, but Beagles, Irish Setters, Poodles, and Afghan Hounds are also affected. Lupus (or positive lupus serology) may occur in related animals, supporting the importance of genetic factors. For example, dogs possessing the MHC class I antigen DLA-A7 are at increased risk and those possessing DLA-A1 and -B5 are at decreased risk for developing disease (Figure 36-6). When dogs affected by lupus are bred, the number of affected offspring is higher than can be accounted for genetically, suggesting that the disease may be vertically transmitted. A type C retrovirus has been suggested as a potential trigger of lupus.

Dogs may present with one or more signs of disease. However, the disease is progressive, so the severity of the lesions and the number of organ systems involved gradually increases in untreated cases. The most characteristic presentation is a fever accompanied by a symmetrical, nonerosive polyarthritis. Indeed, as many as 90% of dogs with lupus may develop arthritis at some stage. Other common presenting signs include renal failure (65%), skin disease (60%), lymphadenopathy or splenomegaly (50%), leukopenia (20%), hemolytic anemia (13%), and thrombocytopenia (4%). Dogs may also show myositis (8%) or pericarditis (8%) and neurological abnormalities (1.6%). The leukopenia involves a major loss of CD8+ T cells with a somewhat smaller loss of CD4+ T cells so that the CD4/CD8 ratio may climb as high as 6, compared with a normal value of about 1.7. The skin lesions are highly variable but are commonly restricted to areas exposed to sunlight. With this great variety of clinical presentations to choose from, it is not surprising that lupus is so difficult to diagnose.

Several unique variants of lupus have been described in dogs. All are very rare, and many are associated with specific breeds, strongly suggesting a genetic predisposition. For example, vesicular systemic lupus is seen in Shetland Sheepdogs and Rough Collies. It is characterized by vesicular erosive and ulcerative skin lesions, subepidermal vesicles, and immunoglobulin deposition at the dermal-epidermal junction. Affected animals have antibodies against type VII collagen as well as ANAs. It may be treated with aggressive immunosuppressive therapy.

Exfoliative lupus dermatitis has been described in German Short-Haired Pointers. Young adult dogs develop scaling and alopecia on the muzzle, pinnae, and dorsum. Some dogs may exhibit signs of pain and arthritis. Others may develop anemia and thrombocytopenia. Histopathology shows hyperkeratosis with a lymphocytic interface dermatitis similar to that seen in human lupus. IgG is deposited in the epidermal and follicular basement membranes. These dogs have circulating autoantibodies to epidermal basement membranes. Affected animals respond poorly to immunosuppressive therapy. This disease is inherited in an autosomal recessive manner, and the responsible gene is DFP2 on chromosome 18.

Another lupus-related disease has been described in Gordon Setters. These dogs developed a symmetrical onychodystrophy, malformations, and loss of the claws. As a result, affected animals show lameness, severe discomfort, and acute pain. Some develop ANAs. A related disease of Gordon Setters is possibly black hair follicular dysplasia. In this disease, dogs begin to shed their black hair without normal regrowth. The remaining black hair is either short and stiff or is thin and easily removed. Many affected dogs had positive ANA titers. These two diseases, occurring in the same breed and often in the same individual, may be closely related.

Diagnosis

A simple diagnostic rule for lupus could be stated as follows: Suspect lupus in an animal with multiple disorders such as those described previously and either a positive test for ANA or a positive test for LE cells (Box 36-1).

ANAs are normally demonstrated by immunofluorescence. Cultured cells or frozen sections of mouse or rat liver on a microscope slide are used as a source of antigen. Dilutions of a patient’s serum are applied to this, and the slide is incubated and then washed off. Binding of ANA to the cell nuclei is revealed by incubating the tissue in a fluorescein-labeled antiserum to canine or feline immunoglobulins and then rewashing. Several different nuclear staining patterns have been described for humans and their clinical correlations identified. In animals, staining patterns have been less thoroughly investigated, and their significance is less clear. Evidence suggests that a homogeneous staining pattern or staining of the nuclear rim is of greatest diagnostic significance but that nucleolar fluorescence is not (Figure 36-7). Dogs whose serum generates a speckled fluorescence pattern tend to have autoimmune diseases other than lupus. Some normal dogs, dogs undergoing treatment with certain drugs (griseofulvin, penicillin, sulfonamides, tetracyclines, phenytoin, procainamide), and some dogs with liver disease or lymphosarcoma may have detectable ANAs. ANAs are also found in a significant proportion of dogs infected with Bartonella vinsonii subsp. berkhoffii, Ehrlichia canis, and Leishmania infantum. Dogs infected with multiple vector-borne organisms are especially likely to be ANA positive.