Canine-canine Pemphigus Complex-diagnosis And Therapy

Vol. 22, No. 7 July 2000

CE

Refereed Peer Review

FOCAL POINT ★ A rigorous approach to the diagnosis of pemphigus is important because of the waxing and waning nature of the disease and the severity of adverse effects associated with immunosuppressive therapy.

KEY FACTS ■ Diagnosis of canine pemphigus is made by histopathology. ■ Because acantholytic cells are not exclusive to pemphigus, other diseases that could cause acantholytic cells must be ruled out. ■ Direct immunofluorescence testing and immunoperoxidase staining are not very reliable in veterinary medicine. ■ Dogs with pemphigus rarely have circulating autoantibodies. ■ Therapy for pemphigus consists of a combination of immunosuppressive agents, which are typically required for life. ■ Affected dogs should be monitored frequently at the beginning of treatment to decrease the incidence of life-threatening adverse effects.

Canine Pemphigus Complex: Diagnosis and Therapy* University of Florida

Rosanna Marsella, DVM ABSTRACT: Canine pemphigus can easily be confused with other diseases (e.g., dermatophytosis, superficial bacterial pyoderma) that may have a similar clinical presentation and occasionally result in acantholytic cells. Biopsy samples must be collected before immunosuppressive therapy is initiated because the drugs may mask histopathologic changes suggestive of pemphigus. This article reviews the diagnosis (including the histopathologic characteristics of the various forms of pemphigus) and treatment of affected animals.

P

emphigus is one of the most common autoimmune skin diseases in dogs. It occurs in middle-aged animals and has a waxing and waning course. Most animals have a secondary bacterial infection at the time of initial presentation. Diagnosis is usually postponed until secondary infections are cleared because they may interfere with the interpretation of diagnostic tests (e.g., cytology, histopathology).

DIAGNOSIS Pemphigus is diagnosed based on clinical history (waxing and waning course), physical examination findings (e.g., pustular to crusting dermatitis starting on the face for pemphigus foliaceus [PF]), cytology, and histopathology.1 Appropriate samples should be obtained from intact primary lesions before any antiinflammatory or immunosuppressive therapy is initiated. When histopathology is inconclusive, pemphigus autoantibody detection may be attempted by immunofluorescence testing or immunoperoxidase staining. Cytology Cytology of an intact pemphigus pustule (Tzanck preparation) reveals nondegenerate neutrophils (unless a secondary infection is present), eosinophils, and numerous acantholytic keratinocytes (i.e., epithelial cells from the stratum granulosum and stratum spinosum that have lost their cohesion because of lysis of intercellular bridges; Figure 1). Acantholytic keratinocytes may form in clusters. *A companion article entitled “Canine Pemphigus Complex: Pathogenesis and Clinical Presentation” appeared in the June 2000 issue (Vol. 22, No. 6) of Compendium.

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However, formation of small to moderate numbers of acantholytic keratinocytes has also been reported with other diseases, including superficial bacterial pyoderma and dermatophytosis.2 Thus cytologic examination should be used only as a preliminary test and not as a substitute for histopathology.

Hematology The hematologic modifications associated with pemphigus are nonspecific. They may include neutrophilia, mild to moderate normocytic normochromic nonregenerative anemia, hypoalbuminemia, and hyperglobulinemia.1

Figure 1A

Histopathology Histopathologic examination of skin samples in cases of pemphigus vulgaris (PV) reveals intraepidermal suprabasal vesicles with acantholytFigure 1B ic cells that may extend to the outer root sheath of the hair Figure 1—Cytology from a pemphigus pustule. (A) Note follicle.3 Basal cells remain at- the acantholytic keratinocytes and nondegenerate neutrophils. (Tzanck preparation; original magnification, tached to the basement mem- ×100) (B) Acantholytic keratinocytes. (Tzanck preparabrane and resemble a row of tion; original magnification, ×1000) tombstones. Fully developed vesicles contain clusters of epithelial cells that have lost coherence with their neighboring cells (acantholytic cells). These cells are rounded and have a large nucleus. An eosinophilic, superficial perivascular to interstitial infiltrate is often seen. In cases of PF, acantholysis occurs below the stratum corneum and in the granular layer3 (Figure 2). Spongiosis and acantholysis within the Figure 2—Histopathology of a pustular lesion in dog with stratum spinosum of the epi- pemphigus foliaceus. Note the large subcorneal pustule dermis and outer root sheath containing acantholytic keratinocytes and neutrophils. are common. Acantholytic, dyskeratotic granular epidermal cells (grains) may be seen munoperoxidase and the at the surface of erosions. In a comparative study of the sections).

histopathologic features of PF and bacterial folliculitis in dogs, several characteristics were found to be associated with PF significantly more frequently than with bacterial pyoderma; these characteristics included a greater density of acantholytic cells, recornification of the pustule base and re-formation of the pustule, and a longer pustule (bridging several hair follicles).4 In pemphigus vegetans (PVeg), intraepidermal pustules, spongiosis, and acantholysis are seen at all levels of the stratified squamous epithelium and the infundibular outer root sheath. 3 Marked papillated hyperplasia and pustules containing numerous eosinophils are present. The inflammatory infiltrate of plasma cells and lymphocytes is superficial perivascular to lichenoid. In pemphigus erythematosus (PE), acantholysis is subcorneal or intragranular with lichenoid cellular infiltrate of lymphocytes, plasma cells, neutrophils, and eosinophils.3 Scattered hydropic basal cells and apoptotic epidermal cells are also characteristic. Pigmentary incontinence is common. Grains may be seen at the surface of erosions. In paraneoplastic pemphigus (PP), features of both pemphigus and erythema multiforme are found. 5 These include suprabasal acantholysis, apoptotic keratinocytes, vacuolar changes of the basilar epidermis, and interface dermatitis. In additional, specific antigens are targeted in dogs with PP (see the Direct Immunofluorescence and ImIndirect Immunofluorescence

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monly used in human mediDirect cine to detect circulating auImmunofluorescence toantibodies. In humans, IIF and Immunoperoxidase is positive for IgG in 80% to Direct immunofluorescence 85% of cases.6 IIF tends to be (DIF) testing and immunonegative in patients with earperoxidase staining are used ly, localized disease and in pato detect deposition of antitients that are in remission. body in tissue. In humans, a Antibodies IIF is less sensitive than is positive DIF for IgG is obtained in all active cases of Figure 3—In pemphigus erythematosus, antibodies are di- DIF. Circulating autoantipemphigus.6 Other antibodies rected against both the basement membrane and des- bodies are always of the IgG moglein 1, which is prevalent in the upper part of the epi(IgA, IgM, C3 complement) dermis (below the stratum corneum and in the stratum class. A positive correlation between titers and the extent may be detected in up to 50% granulosum). of the disease exists in huof patients. In humans, a difmans.6 Antibodies in cases of fuse intercellular fluorescence endemic pemphigus are of is common with all types of the IgG4 subtype against despemphigus. The presence of a moglein 1.13 lupus band in the superficial In veterinary medicine, IIF dermis in addition to interceltesting is neither sensitive nor lular pemphigus antibodies specific. 8,9 When detected, (Figure 3) in the epidermis is titers are usually low. Dogs found in 81% of patients with Antibodies rarely have high titers of cirPE.7 culating antibodies; sensitiviIn dogs, sensitivities for DIF range from 45% to 100%.1,8,9 Figure 4—In pemphigus foliaceus, antibodies are directed ty is variable (0% to 64.3%). Antibodies are not species speLimitations of DIF testing in- against desmoglein 1. cific, and canine squamous clude the need for frozen samepithelium is an excellent ples or the use of a special substrate for human antibodies. In one study,14 fewer medium (Michel’s fixative). If Michel’s fixative is used, than 5% of dogs with PF had circulating antibodies; the pH must be maintained between 7.0 and 7.2 to more recently, 62.5% of canine serum samples were preserve the immunoglobulins.10 Morphologic characpositive when bovine esophagus substrate was used, teristics are poorly preserved and future reexaminations suggesting that this might be the optimal substrate for are not allowed with this method. In addition, canine IIF in dogs.15 The substrate used therefore seems to innose and footpad samples may give false-positive results 8 fluence the sensitivity of this test. Canine PF antibodies (IgM); lip samples are more reliable. False-negative reare directed against desmoglein 116 (Figure 4). This sults may also be caused by previous glucocorticoid molecule has been cloned and has high identity to its therapy, improper pH of Michel’s fixative, or failure to bovine and human counterparts.17 Antibodies in pasample a primary lesion. Because immunoglobulin detients with PV are directed against desmoglein 3 (130 position may be the result of tissue damage, false-posikD; Figure 5).18,19 tive reactions have been reported in dogs with epitheAntibody response in human patients with spontaliotropic lymphoma, demodicosis, sarcoptic mange, neous pemphigus is similar to that associated with and dermatomycosis.9 Patchy staining or staining of the drug-related disease. 20 Whether this also applies to cytoplasm rather than the keratinocyte membrane indidogs is currently unknown. In human PP, serum anticates nonspecific percolation of immunoglobulins withbodies are usually directed against high-molecularin the epidermis. weight proteins (210, 190, 170, and 130 kD).21 A negThe main advantage of immunoperoxidase over DIF 11 ative IIF does not exclude the possibility of PP, is that formalin-fixed samples can be used. Reported however, and immunochemical techniques (e.g., imsensitivity of immunoperoxidase for canine pemphigus munoprecipitation) must be performed. PP has recentvaries from 59% to 100%.12 Immunosuppressive theraly been reported in a dog.22 In this case, deposition of py may induce false-negative results. IgG and C3 complement (positive DIF) was detected in the epidermis and at the basement membrane zone; Indirect Immunofluorescence circulating antibodies were measured against the same Indirect immunofluorescence (IIF) testing is comANTIBODIES ■ MICHEL’S FIXATIVE ■ SUBSTRATE

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antigens targeted by human Commonly used drugs inPP, which suggests a common clude azathioprine and chlopathogenesis.23 rambucil. Azathioprine is Pemphiguslike antibodies usually the first choice for have been recognized in huimmunosuppressive therapy mans.24 They are distinguishin dogs (but is contraindicated from true antibodies by ed in cats); a dose of 1.5 to their failure to bind in vivo 2.5 mg/kg every 48 hours is Antibodies (negative DIF). They are usucommonly used.25 The dose of azathioprine is usually not ally present at low titers and are complement fixing. They Figure 5—In pemphigus vulgaris, antibodies are directed tapered unless severe bone maragainst desmoglein 3, which is prevalent in the lower part row suppression occurs. A are associated with thermal of the epidermis. common maintenance protoburns, drug eruptions, and col is to administer azathioleprosy. Pemphiguslike antiprine on days when glucocorticoids are not given. bodies have been reported in dogs with demodicosis.8,9 Azathioprine is an antimetabolite that interferes with THERAPY synthesis of nucleic acid and is cytotoxic to T cells. It Therapy for pemphigus includes the use of glucocorhas slow onset of action, taking 4 to 6 weeks to proticoids in combination with other immunosuppressive duce clinical effects. Adverse effects include myelosupagents.25 Glucocorticoids are used because of their abilipression, pancreatitis, and hepatotoxicity. Complete ty to inhibit leukocyte phagocytosis, chemotaxis, and blood cell and platelet counts should initially be moniplasminogen activator synthesis. They are seldom used tored every 2 weeks. Once the disease is in remission, as the sole therapy. Adverse effects include secondary the frequency of monitoring can be decreased to every hyperadrenocorticism, diabetes mellitus, gastroduode2 to 3 months. Humans who are deficient in thiopurine nal ulcers, hepatopathy, nephropathy, hypertension, methyltransferase have an increased risk of developing electrolyte disturbances, and secondary infections. myelosuppression,26 and a recent study revealed that a similar situation may exist in dogs.27 Owners should be warned about the possibility of ulcer Chlorambucil (0.1 to 0.2 mg/kg every 48 hours) may development; careful monitoring of bowel movements be used as adjunctive immunosuppressive therapy in is recommended. dogs that cannot tolerate azathioprine.25 It is the slowAn induction dose of 2.2 to 4.4 mg/kg/day of oral est-acting alkylating agent suppressing B-cell function prednisone is used in dogs. Induction lasts 10 to 14 and thus is not considered a first choice for dogs. Addays, depending on the severity of the case. When the verse effects include hepatotoxicity and bone marrow disease is in remission (i.e., no new lesions are found on suppression.25 Monitoring is similar to that described physical examination), the dose is slowly tapered to an for azathioprine. alternate-day administration schedule. Once the alterGold salts (chrysotherapy) are used as adjunctive nate-day regimen is achieved, the dose should continue therapy25 in combination with glucocorticoids because to be tapered until the maintenance dose is reached. they inhibit phagocytosis, prostaglandin synthesis, The maintenance dose of prednisone ranges from 0.5 complement fixation, and epidermal lysosomal ento 1.1 mg/kg every 48 hours for most dogs. Tapering to zymes. Intramuscular injection is the most effective the maintenance dose is best achieved over an 8- to 10route of administration. In dogs, aurothioglucose (1 week period. For an 80-lb dog, the dose is initially tamg/kg intramuscularly) is used once weekly until repered at a rate of 10 to 20 mg/week until an alternatemission occurs, then once every 2 weeks for several day regimen is achieved and then more slowly (5 to 10 treatments, and finally once monthly. If no improvemg/week) until the maintenance dose is reached. ment is seen after 12 weeks of therapy, the dose may be Glucocorticoid pulse therapy is reserved for very seincreased to 1.5 to 2 mg/kg. Adverse effects include vere cases. In dogs, intravenous methylprednisolone nephrotoxicity, bone marrow suppression, thrombocy(sodium succinate; 1.1 mg/kg/day) is administered over topenia, and cutaneous drug eruptions (e.g., erythema a 3-hour period for 1 to 3 days. When remission is obmultiforme). Eosinophilia may precede the occurrence tained, intermediate- to low-dose oral glucocorticoid of cutaneous drug eruptions. Because of its particularly therapy is initiated in conjunction with other immunoslow onset of activity, moderate efficacy, high potential suppressive drugs. for severe drug eruptions, and prolonged half-life, gold Combination therapy may prolong remission and reis usually not a first-choice therapy and is instead reduce side effects associated with glucocorticoid therapy. PREDNISONE ■ AZATHIOPRINE ■ CHLORAMBUCIL ■ CHRYSOTHERAPY

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served for cases that do not respond to other treatment modalities. Oral tetracycline and niacinamide have been used with variable success in dogs and humans with pemphigus.28,29 The rationale for this therapy is that tetracycline inhibits chemotaxis, complement activation, prostaglandin synthesis, lipases, and collagenases and niacinamide inhibits mast-cell degranulation and phosphodiesterase. Adverse effects in dogs include vomiting, diarrhea, anorexia, and increased liver enzyme activity. In dogs weighing more than 10 kg, 500 mg of tetracycline and 500 mg of niacinamide are administered three times daily, whereas dogs weighing less than 10 kg should receive 250 mg of each. Dogs that show improvement with this therapy do so within the first 1 or 2 months. After remission has occurred, the dose may gradually be tapered to once-daily administration. Cyclophosphamide is another alkylating agent that can be used in combination with glucocorticoids. The suggested dose in dogs is 1.5 mg/kg every 48 hours. However, cyclophosphamide is used infrequently because of its potential for bone marrow suppression and hemorrhagic cystitis. Cyclosporine also has limited success in treating canine pemphigus.25 It is an immunosuppressive polypeptide that inhibits interleukin-2 (T-cell growth factor) and thus suppresses antigen presentation and T-cell proliferation. It is used in conjunction with glucocorticoids. The recommended induction dose is 20 mg/ kg/day. After remission occurs, the dose is tapered to 10 mg/kg every 48 hours. Adverse effects include gastrointestinal signs, nephrotoxicity, gingival hyperplasia, and papillomatous dermatitis. Dapsone (1 mg/kg three times daily) has been used in combination with glucocorticoids in a few cases of PF.30 This agent decreases complement activation, antibody production, lysosomal enzyme synthesis, and neutrophil chemotaxis. Adverse effects include anemia, neutropenia, thrombocytopenia, hepatotoxicity, gastrointestinal signs, neuropathies, and cutaneous drug eruptions. Clinical improvement should be seen in 4 to 6 weeks. Finally, heparin has been tried with limited success in one case of canine PV.31 The rational for heparin use is that it inhibits serine proteases and thus might inhibit intraepidermal proteolysis responsible for acantholysis.

PROGNOSIS AND CONCLUSION Mortality in human cases of pemphigus is primarily related to complications of therapy. As a general rule, the prognosis becomes excellent after 5 years of survival. The mortality rate of PV is 100% without treatment and 10% with therapy. The mortality rate of PF

is 60% without treatment and less than 10% with therapy. The risk for adverse effects in dogs is usually highest in the first months of immunosuppressive therapy. PE and PF have a better prognosis than does PV; in the latter, the therapy required to induce remission is rarely tolerated. Careful monitoring of complete blood cell and platelet counts is recommended to prevent the occurrence of irreversible adverse effects. If sudden worsening of the condition is noted, the possibility of other concurrent diseases (e.g., superficial bacterial pyoderma, demodicosis, dermatophytosis) must be considered before making any adjustments to the immunosuppressive regimen.

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14. Scott DW, Walton DK, Slater MR, et al: Immune mediated dermatoses in domestic animals: Ten years after—Part I. Compend Contin Educ Pract Vet 9(4):424–435, 1987. 15. Iwasaki T, Shimizu M, Obata H, et al: Effect of substrate on indirect immunofluorescence test for canine pemphigus foliaceus. Vet Pathol 33:332–336, 1996. 16. Iwasaki T, Shimizu M, Obata H, et al: Detection of canine pemphigus foliaceus autoantigens by immunoblotting. Vet Immunol Immunopathol 59(1–2):1–10, 1997. 17. Suter MM, Ziegra CJ, Cayatte SM, et al: Identification of canine pemphigus antigens, in Ihrke PJ, Mason IS, White SD (eds): Advances in Veterinary Dermatology, Oxford, Pergamon Press, 1993, pp 367–380. 18. Suter MM, De Bruin A, Wurm S, et al: Bullous autoimmune skin diseases in animals. Proc Annu Meet Am Acad Vet Dermatol Am Coll Vet Dermatol:100–103, 1997. 19. Suter MM, Wilkinson JE, Dougherty EP, et al: Ultrastructural localization of pemphigus vulgaris antigen on keratinocytes in vivo and in vitro. Am J Vet Res 51(4):507–511, 1990. 20. Brenner S, Bialygolan A, Anhalt GJ: Recognition of pemphigus antigens in drug induced pemphigus vulgaris and pemphigus foliaceus. J Am Acad Dermatol 36(6 part 1):919–923, 1997. 21. Anhalt GJ: Paraneoplastic pemphigus. Adv Dermatol 12:77– 97, 1997. 22. Lemmens P, De Bruin A, Meulemeester J, et al: Paraneoplastic pemphigus in a dog. Vet Dermatol 9:127–134, 1998. 23. Debruin A, Muller E, Wyder M, et al: Periplakin and envoplakin are target antigens in canine and human paraneoplastic pemphigus. J Am Acad Dermatol 40:682–685, 1999. 24. Robinson ND, Hashimoto T, Amagai M, et al: The new pemphigus variants. J Am Acad Dermatol 40(5):649–671, 1999.

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25. Rosenkrantz WS: Pemphigus foliaceus, in Griffin CE, Kwochka KW, MacDonald JM (eds): Current Veterinary Dermatology. St. Louis, Mosby, 1993, pp 141–148. 26. Snow JL, Gibson LE: A pharmacological basis for the safe and effective use of azathioprine and other thiopurine drugs in dermatologic patients. J Am Acad Dermatol 32:114–116, 1995. 27. White SD, Rosychuck RAW, Scott KV: Investigation into the role of thiopurine methyltransferase in the use of azathioprine in dogs: Phase one. Proc Annu Meet Am Acad Vet Dermatol Am Coll Vet Dermatol:111–112, 1998. 28. White SD, Rosychuk RA, Reinke SI, Paradis M: Use of tetracycline and niacinamide for the treatment of autoimmune skin disease in 20 dogs. JAVMA 200(10):1497–1500, 1992. 29. Berk MA, Lorincz AL: The treatment of bullous pemphigoid with tetracycline and niacinamide. Arch Dermatol 122: 670–674, 1986. 30. Scott DW: Sulfones and sulfonamides in canine dermatology, in Kirk RW (ed): Current Veterinary Therapy IX. Philadelphia, WB Saunders Co, 1980, pp 606–609. 31. Olivry T, Ihrke PJ, Atlee BA: Pemphigus vulgaris lacking mucosal involvement in a German Shepherd dog: Possible response to heparin. Vet Dermatol 3:79–84, 1992.

About the Author Dr. Marsella is affiliated with the Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville. She is a Diplomate of the American College of Veterinary Dermatology.