CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

Vol. 65, No. 2, November, pp. 110-118, 1992

Immunologic Abnormalities in Canine Juvenile Polyarteritis Syndrome: A Naturally Occurring Animal Model of Kawasaki Disease’ PETER J. FELSBURG,*,~

HARM

*Department

of Pathobiology,

RICHARD L. SOMBERG,*

HOGENRSCH,t AND

T.

LAWRENCE

School of Veterinary Medicine, Experimental Gerontology, P.O.

Purdue University, West Lafayette, Indiana Box 5815,228O HV Ri$swijk, The Netherlands

This study describesthe immunologic abnormalities during the acute phaseof juvenile polyarteritis syndrome (JPS), a multisystem necrotizing vasculitis of young dogs with a predilection for the coronary arteries. JPS has striking clinical, laboratory, and pathologic similarities to Kawasaki disease(KD), the most common cause of acquired heart diseasein children in the United States. The immunologic abnormalities include an increase in serum IgA, an increase in the percentage of peripheral B cells and a decrease in the percentage of total peripheral T cells, a marked suppression of the blastogenic responseto mitogenic stimulation, an inability to generate immunoglobulin-secreting plasma cells following polyclonal activation, the presenceof antineutrophi1 cytoplasmic antibodies, and evidence of monocyte/ macrophage activation. These immunoregulatory abnormalities are similar to those observed in children during the acute phaseof KD. This unique, naturally occurring animal model of necrotizing vasculitis may prove useful for investigating novel therapeutic interventions in the treatment of necrotizing vasculitis and may yield insight into the immunopathology and etiology of KD. Q 1992 Academic press. I~C. INTRODUCTION

Over the past decade, isolated clinical and pathologic reports have appeared in the veterinary literature describing a febrile disease characterized histopathologically by a necrotizing vasculitis in young beagle dogs used for toxicity studies (l-6). Although the majority of cases have been reported in beagle dogs, the disease also occurs spontaneously in both pet and kennel dogs. This disease has been referred to under a variety of names including pain syndrome, beagle pain syndrome and canine pain syndrome because of the severe generalized pain in the acute stages of the disease. Due to i Supported by grants from the National Institutes of Health (RR 06224) and the American Heart Association Indiana Affiliate, and the Canine Disease Research Fund of Purdue University. ’ To whom correspondence should be addressed at Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104. 110 0090-1229/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

PAUL

W.

SNYDER,*

GLICKMAN” 47907;

and

+TNO

Institute

fur

its characteristic pathologic features, we have chosen to call the disease canine juvenile polyarteritis syndrome. Canine juvenile polyarteritis syndrome (JPS) is an acute, systemic necrotizing vasculitis of unknown etiology affecting primarily small and medium-sized arteries with special predilection for the coronary arteries. It occurs primarily in young dogs, usually between 3 and 18 months of age. Dogs with JPS present with a fever of greater than 104°F that is unresponsive to antibiotic therapy and persists for more than 72 hr. The major clinical signs are pain with guarding and a characteristic hunched stance with the neck held in rigid extension that persists for 3 to 7 days. During the febrile period, JPS dogs are anorexic and may appear painful in the abdomen. The pharyngeal mucosa may be inflammed. The ciinical signs may have a remitting and relapsing course with regular cycling at 15. to 45. day intervals. The major clinical laboratory findings include a marked leukocytosis (>25,000 WBC/mm3) with a neutrophilia (>15,000/mm3), elevated acute phase proteins as determined by serum protein electrophoresis, hypoalbuminemia, elevated platelet counts, mild anemia, and an increased erythrocyte sedimentation rate (7, 8). Although JPS is a systemic vasculitis involving small to medium-sized muscular arteries, the primary arteries involved are the extramural coronary arteries of the heart and the meningeal vessels. At first, intimal thicking occurs associated with a mononuclear cell infiltrate that progresses to involve all layers of the arterial wall. A mixed inflammatory infiltrate subsequently surrounds the artery. The intima and media are disrupted by fibrinoid necrosis that leads to luminal thrombosis. The vessels heal with fibrous scarring and intimal thickening. Immune complex deposition has not been observed in JPS (7, 8). Burns et al. (7) have recently reported on the marked clinical, laboratory, and histopathologic similarities between JPS and KD which is currently the most common cause of acquired heart disease in children in the

IMMUNOLOGIC

ABNORMALITIES

United States (9-11). The purpose of this study was to describe the immunologic abnormalities observed in JPS dogs and compare them to those observed in children with KD. MATERIALS

AND

METHODS

Dogs and Samples

The source of dogs with JPS was a large, commercial beagle breeding kennel. The control dogs were clinically normal, age-matched, unrelated beagles from the same kennel. A diagnosis of JPS was based upon the criteria established by our laboratory (7, 8). This consists of clinical signs of JPS including a hunched stance, cervical pain, stiff gait, decreased movement, and/or resistance to manipulation of the head and neck; an acute onset of fever greater than 104°F that persists for more than 72 hr; a leukocytosis of greater than 25,000 WBC/mm3 and a neutrophilia of greater than 15,000/mm3; an elevated 01-2 globulin; and a hypogammaglobulinemia. Blood samples for collection of serum and heparinized whole blood were collected by venipuncture during the acute phase of the disease, 3 to 5 days following onset of clinical signs. Quantitation

of Serum Immunoglobulins

Serum IgG, IgM, and IgA concentrations were measured by single radial immunodiffusion (12, 13) using affinity-purified heavy-chain specific antisera to each class of immunoglobulin supplied by Dr. Robert Schwartzman of the University of Pennsylvania. Identification Lymphocyte

and Quantitation Subpopulations

of

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized whole blood by centrifugation over a discontinuous density gradient of Hypaque-Ficoll(14). Identification and enumeration of B lymphocytes was performed by direct immunofluorescence using affinity-purified FITC-labeled F(ab’), anticanine polyvalent immunoglobulin (Cappell, West Chester, PA). Identification and enumeration of total T cells and T helper cells were performed by indirect immunofluorescence using monoclonal antibodies F320-7 (Bioproducts for Science, Indianapolis, IN) and DT-2 (a gift from Warren Ladiges, Fred Hutchison Cancer Center, Seattle, WA), respectively. Monoclonal antibody F3-20-7 recognizes canine Thy-l and labels all mature circulating T cells (151, whereas, monoclonal antibody DT-2 recognizes a subpopulation of canine helper T cells (16). Analysis of lymphocyte subpopulations was performed on an EPICS C flow Cytometer (Coulter Electronics, Hialeah, FL).

111

IN JPS

Proliferation

Assays

The response of peripheral blood lymphocytes to in stimulation was evaluated by lymphocyte transformation. Briefly, isolated PBMC were resuspended in RPM1 1640 (Mediatech, Fisher Scientific, Chicago, IL) supplemented with 10% FCS (Sigma, St. Louis, MO) and cultured in 96-well flat-bottom microplates (Costar, Cambridge, MA) at a concentration of 2 x lo5 cells/well in the presence or absence of optimal and suboptimal concentrations of mitogens. The mitogens used were phytohemagglutinin-P (PHA-P; Sigma), concanavalin A (Con A; Sigma), and pokeweed mitogen (PWM; Sigma). Each sample was run in triplicate. The cultures were incubated for 72 hr at 37°C in a 5% COs atmosphere. Sixteen hours before the end of the culture period, 0.5 $X3Hlthymidine (2 Ci/mmol; New England Nuclear, Boston, MA) was added to each well. At the end of the incubation period, the cells were harvested onto glass fiber filters and the incorporation of radioactivity was measured by liquid scintillation spectrometry. The results are expressed as counts per minute. To study the effect of indomethacin on the proliferative response of JPS PBMC, cultures were established as above with a suboptimal concentration of PHA-P (0.5 p,g/ml) in the absence and presence of indomethatin (Sigma) at a final concentration of 1 kg/ml (17, 18). To examine the effect of exogenous interleukin-2 (IL2) on the proliferative response, PHA-P (2.5 Kg/ml) activated cultures were established in the absence or presence of a 25% final concentration of an IL-2 containing supernatant from EL-4 thymoma cells stimulated with phorbol ester (19) that has been shown by us and others to support the growth of canine IL-2dependent cell lines (20, 21). vitro mitogenic

Polyclonal

Activation

of B Cells

Polyclonal activation of canine B cells and determination of immunoglobulin secreting plasma cells by reverse hemolytic plaque assay were performed according to the technique of Felsburg et al. (12, 13, 22). Essentially, PBMC were cultured in RPM1 1640 supplemented with 10% FCS at a concentration of 1 x lo6 cells/ml in the absence (unstimulated) and in the presence of PWM (stimulated). The cultures were incubated for 5 days at 37°C in a 5% COs atmosphere. Following incubation, the lymphocytes were washed and added to protein A-coated sheep red blood cells. A monolayer was created in a liquid phase in 96-well flat-bottom microplates, and the class-specific immunoglobulin secreting plaque forming cells were determined using heavy chain-specific antisera supplied by Dr. Robert Schwartzman of the University of Pennsylvania.

112 Detection

FELSBURG

of Anti-neutrophil

Cytoplasmic

of Autoantibodies

Anti-nuclear antibody (ANA) and rheumatoid factor were determined using routine procedures for evaluating these autoantibodies in the dog (12). ANA was detected by indirect immunofluorescence using monkey kidney cells as a substrate and rheumatoid factor by the Rose-Waaler Passive hemagglutination test. RESULTS

We have studied the occurrence ring JPS in the largest commercial in the United States (approximately

80

Antibodies

Antibodies against neutrophil cytoplasmic components were determined according to techniques previously described for man (23,241. Freshly isolated buffy coat cells were resuspended in RPM1 1640 supplemented with 10% FCS. Buffy coat cells were used to control for any lymphocyte or monocyte staining (23). Air-dried cytocentrifuged preparations were fixed in chilled ethanol for 5 min at 4°C. Test or control sera were diluted in phosphate-buffered saline (PBS) and incubated on the slides for 1 hr at room temperature in a humid chamber. The slides were then washed three times in PBS and incubated with FITC-labeled affinity-purified goat anti-dog IgG (Sigma) for 1 hr at room temperature. Finally, the slides were washed and mounted in 90% glycerol in PBS containing 1,4diazobicyclo-(2,2,2)-octane as an “anti-fade” agent (25). A sample was considered positive if the neutrophils revealed bright cytoplasmic or perinuclear fluorescence and negative if no staining was present. Detection

ET AL.

of naturally occurbeagle dog kennel 20,000 pups/year