Effect of Substrate on Indirect Immunofluorescence Test for Canine Pemphigus Foliaceus

Transcription

1 Vet Pathol33: (1996) Effect of Substrate on Indirect Immunofluorescence Test for Canine Pemphigus Foliaceus T. IWASAKI, M. SHIMIZU, H. OBATA, M. OGATA, M. NAGATA, T. YANAI, H. KITAGAWA, AND Y. SASAKI Veterinary Medical Teaching Hospital, Gifu University, Gifu, Japan (TI, MS, HO, TY); Veterinary Medical Teaching Hospital, Azabu University School of Veterinary Medicine, Sagamihara, Japan (MO); Department of Clinical Pathology, Nippon Zoological and Veterinary Medical School, Musashino, Japan (MN); and Department of Internal Medicine, Faculty of Veterinary Medicine, Gifu University, Gifu, Japan (HK, YS) Abstract. The effect of substrate on indirect immunofluorescence (IIF) tests for the detection of circulating autoantibodies was studied by examining sera from 14 canine pemphigus foliaceus patients, six sera with nonpemphigus dermatoses and ten normal dog sera against five different substrates from three species. These substrates included bovine esophagus, bovine nose, bovine tongue, monkey esophagus, and canine nose skin. Nine out of 14 (64.3%) sera from patients with canine pemphigus foliaceus showed intercellular space staining by indirect immunofluorescence using bovine esophagus as substrate. However, sera from nonpemphigus dermatoses and normal dog did not react with bovine esophagus. In other substrates, only bovine tongue showed 1/8 (12.5%) positive reaction at the intercellular space by sera from canine pemphigus foliaceus. Dog nose skin showed the intercellular space staining against ten of ten (1%) normal dog serum. Monkey esophagus showed the fluorescent deposit at the intercellular space in four of nine (44.4%) of pemphigus foliacues dog sera, however, four of ten (4%) of normal dog sera revealed nonspecific intercellular staining. These results indicate that the sensitivity and the specifity of IIF test in canine pemphigus foliaceus depend on the substrate. The best substrate for detecting circulating autoantibody in canine pemphigus foliaceus patients among five different substrates was bovine esophagus because of its sensitivy and high specifity. The diagnosis of canine pemphigus foliaceus should be made on the basis of a combination of clinical signs, histopathology, direct immunofluorescence, and the detection of circulating autoantibody. Key words: Autoantibodies; bullous diseases; dogs; indirect immunofluorescence; pemphigus foliaceus; skin. Human pemphigus foliaceus (PF) is an autoimmune blistering skin disease characterized by intraepidermal blister frmatin.l~ It has been reported that an IgG autoantibody binds to a desmosomal plaque protein, desmoglein I, in human PF,I and the activation of plasminogen activator by antigenantibody reaction may play an important role in the pathogenesis of PF.6 Canine PF was first described as a scaling eruptive dermatitis with bulla formation and showed circulating autoantibody by indirect immunofluorescence (IIF) in the first reported case.5 Clinical findings of canine PF in reported cases are characterized by crusting, scaling, and erosions of face, ears, and foot pad.2,7,11,15.19 Histopathologically, canine PF patients show the bulla or pustules within the upper part of the epidermis with numerous acantholytic keratino~ytes.~.~,~~ Immunohistologically, canine PF shows immunofluorescent deposits of immunogloblins in the intercellular space of epidermis by direct immunofluorescence.7" Human PF patients revealed Circulating autoantibodies against intercellular substances demonstrated by IIF and immunoblottings. However, the demonstra 332 tion of the antigen targeted by the circulating autoantibody has not been well characterized in canine patients with PF.22 Moreover, the detection of the autoantibody by IIF is not regarded as a diagnostic procedure in canine PF because of the low sensitivity of IIF.13 However, studies have not been conducted to obtain an improved method using alternative substrates in canine PF. This study was performed to find better, optimal substrates for the immunological diagnosis of canine PF. It is suggested that IIF using bovine esophagus is a useful procedure for the detection of autoantibody in canine PF. Materials and Methods Sera from 14 patients with canine pemphigus foliaceus (PFJ were used for indirect immunofluorescence (IIF). The diagnosis of canine PF was established by clinical and histopathologic findings. The clinical findings included ulcer, erasion, hyperkeratosis, and crusting of muzzle, pinna, foot pad, and/or t~nk ,1s,19 The histopathologic findings were the existence of intraepidermal bulla in upper epidermis with numerous and active acantholytic cells without bacteria^.^,^,^^

2 Vet Pathol 33:3, 1996 Indirect Immunofluorescence Test for Canine Pemphigus Foliaceus 333 The patients were free from corticosteroids at least 21 days prior to obtaining sera and skin samples. The skin samples were biopsied by 6mm punch under local or systemic anesthesia. Skin samples were divided into two pieces, one for histopathology and one for direct immunofluorescence test (DIF). One half of the sample was snapfrozen and stored at 7 C until DIF was performed. Sera from six patients with nonpemphigus dermatoses, including discoid lupus erythematoses, atopic dermatitis, bullous pemphigoid, erythema multiforme, seborrhic dermatitis, and generalized demodicosis, were used for the negative controls in IIF. Biopsied skin from six patients with nonpf dermatoses were also processed for histopathology and DIF. Sera from ten clinically normal dogs were used as the negative control for IIF. The human serum from a patient with PF, a generous gift from Dr. T. Hashimoto, Department of Dermatology, Keio University School ofmedicine, was used as a positive control for IIF. The following substrates were used for IIF bovine esophagus, bovine nose, bovine tongue, monkey esophagus, and dog nose skin. These samples other than monkey esophagus were obtained from each animal immediately in the order of euthanatize, snapfrozen, embedding in OCT compound (Miles, Elkhart, IN), and storing at 7 C until IIF was performed. Preparation slides of monkey esophagus were purchased from MeDiCa (Carlsbad, CA). IIF studies were performed as previously described.8 Briefly, 4pm cryosections were obtained from each sample and airdried for 3 minutes. Sections were first incubated with canine (1 : 1 and 1 : 2) or human sera (1 : 16 and 1 : 32) for 6 minutes at room temperature in a moist chamber, and then incubated with fluorescence isothiocyanate (F1TC)conjugated goat antidog (1 : 4, gamma chainspecific; Kirkegaard & Perry Laboratories, Gaithersburg, MD) or antihuman IgG (1 : 4, Cappel, Durham, NC) for 2 minutes at room temperature. The slices were washed with phosphatebuffered saline and observed by fluorescence microscope (Carl Zeiss, Thornwood, NY). DIF was performed as previously described.8 The fluorescent conjugates used in this study included: FITCconjugated antidog IgG (Kirkegaard & Perry Laboratories), antidog IgA (Bethyl Laboratories, Montgomery, TX), antidog IgM (Bethyl Laboratories), and antidog C3 (The Binding Site, San Diego, CA). Antinuclear antibody test was performed using rat liver as substrate. The 4pm sections of rat liver were prepared by cryostat and airdried for 3 minutes. Sections were incu Fig. 1. Clinical appearance of canine pemphigus foliaceus in this study. Heavy crusting, erythema, and erosion are seen on the face. Fig. 2. Histopathology of canine pemphigus foliaceus. Numerous acantholytic keratinocytes and infiltrating granulocytes are seen in superficial pustules. Bar = 25 pm. Fig. 3. Indirect immunofluorescence of canine pemphigus foliaceus using patient serum and bovine esophagus. IgG class antibodies deposit at the intercellular space. Bar = 1 fin.

3 334 Iwasaki, Shirnizu, Obata, Ogata, Nagata, Yanai, Kitagawa, and Sasaki Vet Pathol 33:3, 1996 Table 1. Positive rate of indirect immunofluorescence in dogs with pemphigus foliaceus, nonpemphigus dermatoses, and normal dogs using various substrates. GroupISpecies Organ Bovine Bovine Bovine Monkey Dog ANA* Esophagus Tongue Nose Esophagus Nose Test Pemphigus foliaceus (n)t 64.3% n.d.s 1. Nonpemphigus dermatoses (n) (14) (8) (1 1) (9) n.d. (1) Normal dog (n) (5) (3) (3) (2) 4. 1 (1) (1) (1) (1) (1) (1) (1) * ANA: antinuclear antigen. t n = Number of dogs. f n.d. = Not done. 5 Linear IgG deposition at basement membrane zone. bated with serum from each dog for 45 minutes at room temperature, washed with phosphatebuffered saline, and then reacted with FITCconJugated antidog IgG (1 : 4, gamma chainspecific; Kirkegaard & Perry Laboratories) for 2 minutes. Results finding was blister or pustule formation with eosinophilic acantholytic keratinocytes within upper epidermis (Fig. 2). Eosinophils and neutrophils often infiltrated blisters. Direct staining using antidog IgG, IgM, IgA, or C3 revealed immunoglobulin deposition in the intercellular spaces in 84.6% (11/13) of PF patients. Results of indirect immunofluorescence (IIF) using Dogs diagnosed with PemPhiWs foliaceus (PF) showed erosion, ulcer, crusting on face (Fig. pinna, several substrates are summarized in Table 1. When foot pad, and/or trunk. The typical histopathologic the bovine esophagus was used as substrate and the sera from human or canine PF patients were used as Table 2. The results of the individual indirect immuprimary antibodies, IIF revealed fluorescence deposits nofluorescent testing using different substrates in patients at the intercellular spaces throughout the epidermis with pemphigus foliaceus nonpemphigus dermatoses, and (Fig. 3). Sera from PF patients had a 64.3% (9/14) normal dogs. positive reaction at the intercellular space of the bovine esophagus epithelium. On the other hand, bovine No. Diagnosis DIF* BE BT BN ME ANA tongue, which is reported to be enriched in hemides 1 PF mosomes, showed only a 12.5% (118) positive reaction 2 PF nd against sera from PF patients. When the bovine nose 3 PF was used as substrate, there was no positive intercel 4 PF lular staining by PF sera. When the monkey esophagus 5 PF was used as substrate, PF serum showed 44.4% (4/9) 6 PF positive reaction at the intercellular space in epidermis. 7 PF nd nd However, normal dog sera also revealed 4% (4/1) 8 PF nd nd nd 9 PF positive reaction. Because IIF using dog nose skin nd nd 1 PF yielded a positive intercellular reaction in all of ten 11 PF nd nd normal dog sera, we did not perform IIF with PF pa 12 PF nd nd nd tients sera using dog nose skin. 13 PF nd nd nd The sera from nonpemphigus patients did not show 14 PF nd nd nd fluorescent depositions in the intercellular spaces in 1 DLE BM nd nd nd bovine esophagus. The patient sera from discoid lupus 2 AtOPY nd erythematosus and bullous pemphigoid had linear flu 3 Demodicosis nd orescent deposition at the basement membrane zone 4 BP BM BM nd nd by DIF. These sera showed basement membrane zone 5 EM nd nd nd nd staining by IIF when bovine esophagus or bovine tongue 6 Seborrhea nd nd nd nd was used as substrate. However, the sera did not reveal * DIF = direct imrnunofluorescence, BE = bovine esophagus, BT the intercellular deposition of fluorescence by either = bovine tongue, BN = bovine nose, ME = monkey esophagus, DIF or IIF. The sera from normal dogs did not show ANA = antinuclear test, PF = pemphigus foliaceus, nd = not done, DLE = discoid lupus erythernatous, BM = basement membrane the intercellular or basement membrane zone staining zone, BP = bullous pemphigoid, EM = erythema multiforme. with bovine esophagus, bovine tongue, and bovine nose.

4 Vet Pathol 33:3, 1996 Indirect Immunofluorescence Test for Canine Pemphigus Foliaceus 335 The human patient serum with PF revealed very intense fluorescence in the intercellular space at the dilution of 1 : 32 in bovine esophagus and 1 : 16 in other substrate. Individually, one case with a positive antinuclear antibody test showed negative DIF and IIF, three cases were DIF positive and IIF negative, and one case with negative DIF had positive IIF by bovine esophagus (Table 2). Discussion Attempts have been made to demonstrate circulating autoantibodies in canine pemphigus foliaceus (PF) patients, but the results of these studies have been controversial. Halliwell and Goldschmidt reported the first case of canine PF with positive indirect immunofluorescence (IIF) using normal dog buccal mucosa as ~ubstrate.~ Manning et al. reported a 33.3% positive rate in three canine PF cases using normal dog tongue.' ' In 1981, Scott and Lewis described six cases of PF with one case of positive IIF.15 Scott et al. also reported 26 cases of canine PF, in which 2 of the 26 cases were positive in direct immunofluorescence (DIF), but only one case revealed a positive reaction by IIF.19 On the other hand, Ihrke et al. demonstrated six cases with intercellular space staining by IIF among nine selected PF cases using canine lip epitheli~m.~ Another report indicated that none of 14 PF cases showed the circulating autoantibody by IIF using healthy dog lip.12 According to the results of these studies, Muller et al. found IIF to be unreliable for diagnosis of canine pemphigus because it is rarely positive in true canine pemphigus and may even be positive in nonpemphigus diseases.13 However, the specificity of IIF in canine PF in reported cases and in the present study seems acceptable. Only one case had intercellular space staining in IIF among 1 nonpemphigus canine dermatoses,18 and no positive intercellular space staining was detected among 75 nonpemphigus cats.17 Based on a personal communication from Stannard, Ihrke et al. stated that no intercellular space stainings were seen among a total of 5 nonpemphigus dog sera.' Moreover, in this study, ten normal dog sera and six selected canine dermatoses including discoid lupus erythematose bullous pemphigoid, seborrheic dermatitis, demodicosis, and superficial bacterial dermatitis, were examined by IIF using bovine esophagus, bovine tongue, and bovine nose, and no positive intercellular staining was seen in these control sera. Immunoperoxidase is a sensitive procedure for the demonstration of epidermal intercellular immunoglobulin depo~its.~,~' The immunoperoxidase method using formalinfixed, paraffinembedded sections is not appropriate for detecting the circulating autoantibody in PF because of the low stability of PF antigen (desmoglein I) in formalin fixatives and high temperature during paraffin embedding.i3 Many studies have been performed to obtain optimal substrate for detecting autoantibodies in these human PF patients.' Fclr the diagnosis of human autoimmune skin blistering diseases such as pemphigus, bullous pemphigoid (BP), and epidermolysis bullosa acquisita, the monkey esophagus and the guinea pig esophagus have been used for the detection of circulating autoantibody by IIF as well as human skin because these substrates are easy to obtain, well prepared, and show a better positive rate.' For the diagnosis of canine PF, canine buccal mucosa and lip epithelium have served as substrate for IIF. Studies aimed at detecting the circulating autoantibodies of canine PF using these substrates usually failed to demonstrate the circulating antibodies. However, no studies for detecting a better, optimal substrate with canine PF have been conducted. The sensitivity of IIF for human autoimmune blistering diseases is strongly influenced by the substrate used for the test.' One study reported that the bovine tongue is enriched in hemidesmosomes, and that it can be used for the IIF of human patients with BP.9 In canine BP, Iwasaki et al. reported intense fluorescence deposition in the basement membrane zone when bovine tongue was used as a substrate, compared to canine skin.8 In this study, five substrates from three different species were used for the detection of intercellular space staining in canine patients with PF. Among them, only bovine esophagus showed a high percentage of fluorescent deposition as well as DIF without nonspecific staining by sera from normal dogs and six nonpemphigus dermatoses. On the other hand, bovine nose and tongue showed a very low percentage of positive staining. Because canine nose skin was consistently positive even when normal dog sera were used as primary antibodies, it was not thought to be the suitable substrate for IIF. Although serum from a human patient with PF revealed a positive reaction at the dilution of 1 : 16 or 1 : 32, canine PF sera had a positive reaction at the dilution of 1 : 1 or 1 : 2 on the same substrate. This result indicates that the low titer of circulating autoantibody in canine PF may contribute to the low percentage of positive IIF. In conclusion, bovine esophagus is suggested to be the better, optimal substrate for detection of circulating antibody in canine PF patients, and the diagnosis of canine pemphigus foliaceus should be made from the combination of clinical signs, histopathologic findings, DIF, and the detection of circulating autoantibodies by IIF. References 1 Bystryn JC, Sabolinski M: Effect of substrate on indirect immunofluorescence tests for intercellular and basement

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