Anti-Human Cardiac Myosin in Kawasaki Syndrome Madeleine W. Cunningham, H. Cody Meissner, Janet S. Heuser, Biagio A. Pietra, David K. Kurahara and Donald Y. This information is current as M. Leung2 of September 29, 2021. J Immunol 1999; 163:1060-1065; ; http://www.jimmunol.org/content/163/2/1060 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Anti-Human Cardiac Myosin Autoantibodies in Kawasaki Syndrome1

Madeleine W. Cunningham,* H. Cody Meissner,† Janet S. Heuser, * Biagio A. Pietra,§ David K. Kurahara,‡ and Donald Y. M. Leung2¶

Kawasaki syndrome (KS) is the major cause of acquired disease in children. Although acute is observed in most patients with KS, its pathogenesis is unknown. Because antimyosin autoantibodies are present in autoimmune myocarditis and rheumatic , the purpose of the current study was to determine whether anticardiac myosin Abs might be present during the acute stage of KS. Sera from KS patients as well as age-matched febrile controls and normal adults were compared for reactivity with human cardiac myosin in ELISAs and Western blot assays. A total of 5 of 13 KS sera, as compared with 5 of 8 acute sera, contained Ab titers to human cardiac myosin that were significantly higher than those found in control sera.

Both cardiac and skeletal myosins were recognized in the ELISA by KS sera, although stronger reactivity was observed to human Downloaded from cardiac myosin. Only IgM antimyosin Abs from KS sera were significantly elevated relative to control sera. KS sera containing antimyosin Abs recognized synthetic peptides from the light meromyosin region of the human cardiac myosin molecule and had a different pattern of reactivity than acute rheumatic fever sera, further supporting the association of antimyosin Ab with KS. These Abs may contribute to the pathogenesis of acute myocarditis found in patients with KS. The Journal of Immunology, 1999, 163: 1060–1065. http://www.jimmunol.org/ awasaki syndrome (KS)3 is an acute febrile illness that the appearance of circulating Abs that are cytotoxic against vas- occurs primarily in infants and young children (1, 2). KS cular endothelial cells prestimulated with IL-1␤ (10), TNF-␣ (11), K and rheumatic fever are the two leading causes of ac- or IFN-␥ (12) but not against unstimulated endothelial cells. quired heart disease in children (3). Myocarditis occurs in over half Therefore, it has been postulated that immune reactivity to vascu- the patients with acute KS (4, 5). In a review of endomyocardial lar endothelium may contribute to the vasculitis associated with biopsies from 201 patients with KS obtained 1 mo to 11 years after acute KS. onset of disease, Yutani et al. reported changes consistent with The etiology of immune activation in acute KS is not certain, myocarditis (6). Manifestations may include: mild mitral regurgi- although evidence supports an infectious etiology. Microbiologic tation, depressed myocardial function, and increased end systolic data suggest a role for staphylococcal and streptococcal superan- by guest on September 29, 2021 and end diastolic dimensions, which do not correlate with the pres- tigens (13, 14). Furthermore, the clinical symptoms of KS share a ence of coronary artery lesions (7). The myocarditis of KS is as- number of features with staphylococcal and streptococcal diseases, sociated with clinical features of tachycardia disproportionate to such as toxic shock syndrome and scarlet fever. Superantigens the degree of fever, gallop rhythm, murmur, and electrocardiogram have also been reported to induce the production of autoantibodies changes, including prolonged PR, QT intervals and diffuse low such as rheumatoid factor (15). voltage, ST-T wave changes, as well as the described echocardio- Autoantibodies against myosin have been implicated in the graphic findings. Although myocarditis is a well-known feature of pathogenesis of rheumatic carditis and autoimmune myocarditis, acute KS, little is known about its pathogenesis (7, 8). Immune diseases which also affect tissue and myocardium (16, activation associated with polyclonal B cell activation as well as an 17). Furthermore, cardiac myosin is known to induce myocarditis increased number of activated T cells and macrophages is found in animal models (18). To date, there have been no studies exam- during the acute phase of KS (9). Acute KS is also associated with ining the occurrence of anticardiac myosin autoantibodies in acute KS. Sera were also obtained from eight children presenting with acute rheumatic fever (ARF). The purpose of the current study was *Department of Microbiology and Immunology, University of Oklahoma Health Sci- ences Center, Oklahoma City, OK 73190; †Department of Pediatrics, New England to seek evidence for antimyosin Abs in patients with acute KS. Medical Center, Boston, MA 02111; ‡Department of Tropical Medicine and Medical Microbiology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96822; §Department of Pediatrics, University of Colorado Health Sciences Center, Denver, CO 80262; and ¶Department of Pediatrics, National Jewish Medical and Materials and Methods Research Center, Denver, CO 80206 Sera Received for publication October 29, 1998. Accepted for publication May 10, 1999. The study was conducted on sera from 13 children in the acute phase of The costs of publication of this article were defrayed in part by the payment of page KS. These patients fulfilled the established clinical criteria for KS (19). charges. This article must therefore be hereby marked advertisement in accordance These criteria included a fever for Ն5 days and at least four of the five with 18 U.S.C. Section 1734 solely to indicate this fact. following symptoms: nonexudative conjunctival injection; changes in the 1 This work was supported by Grants HL35280 and HL56267 (to M.W.C.) and Grants oral pharynx, including mucosal erythema, dry fissured lips, and “straw- HL37260 and AR41256 (to D.Y.M.L.) from the National Institutes of Health. berry tongue”; changes in the extremities, characteristically erythema of 2 Address correspondence and reprint requests to Dr. Donald Y. M. Leung, Depart- the palms and soles, induration of the hands and feet, or perungual des- ment of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson quamation in the subacute phase of the disease; polymorphous rash; and Street, Room K926, Denver, CO 80206. E-mail address: [email protected] cervical adenopathy (one or more nodes of Ն1.5 cm in diameter). A total 3 Abbreviations used in this paper: KS, Kawasaki syndrome; ARF, acute rheumatic of 11 of the 13 KS patients had clinical evidence of myocarditis during the fever; LMM, light meromyosin. acute phase of their illness. Sera were also obtained from 12 age-matched

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 1061

Table I. Sequences of LMM peptides

Peptide Sequence Peptide Sequence

LMM-1 KEALISSLTRGKLTYTQQ LMM-26 EGDLNEMEIQLSHANRMA LMM-2 TYTQQLEDLKRQLEEEVK LMM-27 ANRMAAEAQKQVKSLQSL LMM-3 EEEVKAKNALAHALQSAR LMM-28 SLQSLLKDTQIQLDDAVR LMM-4 LQSARHDCDLLREQYEEE LMM-29 RANDDLKENIAIVERRNN LMM-5 EQYEEETEAKAELQRVLSK LMM-30 IAIVERRNNLLQAELEEL LMM-6 RVLSKANSEVAQWRTKYE LMM-31 ELEELRAVVEQTERSRKL LMM-7 RTKYETDAIQRTEELEEA LMM-32 RSRKLAEQELIETSERVQ LMM-8 ELEEAKKKLQRLQEAEE LMM-33 SERVQLLHSQNTSLINQK LMM-9 QEAEEAVEAVNAKCSSLE LMM-34 LINQKKKMDADLSQLQTE LMM-10 CSSLEKTKHRLQNEIEDL LMM-35 TEVEEAVQESRNAEEKAKK LMM-11 EIEDLMVDVERSNAAAAA LMM-36 RNAEEKAKKAITDAAMMA LMM-12 AAAAALDKKQRNFDKILA LMM-37 AAMMAEELKKEQDTSAHL LMM-13 DKILAEWKQKYEESQSEL LMM-38 TSAHLERMKKNMEQTIKDL LMM-14 SQSELESSQKEARSLSTE LMM-39 TIKDLQHRLDEAEQIALK LMM-15 SLSTELFKLKNAYEESLE LMM-40 EQIALKGGKKQLQKLEARV LMM-16 EESLEHLETFKRENKNLQ LMM-41 LEARVRELENELEAEQKR LMM-17 NKNLQEEISDLTEQLGSS LMM-42 AEQKRNAESVKGMRKSER

LMM-18 EQLGSSGKTIHELEKVRKQ LMM-43 RKSERRIKELTYQTEEDR Downloaded from LMM-19 KVRKQLEAEKMELQSALE LMM-44 TEEDRKNLLRLQDLVDKL LMM-20 LQSALEEAEASLEHEEG LMM-45 LVDKLQLKVKAYKRQAEE LMM-21 EEGKILRAQLEFNQIKAE LMM-46 RQAEEAEEQANTNLSKFR LMM-22 NQIKAEIERKLAEKDEEME LMM-47 LSKFRKVQHELDEAEERA LMM-23 DEEMEQEKRNHLRVVDSL LMM-48 AEERADIAESQVNKLRAK LMM-24 VVDSLQTSLDAETRSRNE LMM-49 KLRAKSRDIGTKGLNEE LMM-25 RSRNEALRVKKKMEGDLN http://www.jimmunol.org/

febrile children, 10 normal adults who had no known illness, and 14 chil- lent Igs conjugated with HRP. The blot was developed using chloronaph- dren presenting with ARF. All ARF patients demonstrated positive anti- thol as indicator with hydrogen peroxide as substrate as described previ- streptolysin O titers and fulfilled the revised Jones criteria for diagnosis of ously (17). The serum reaction with the human cardiac myosin heavy chain ARF. Informed consent was obtained from each patient and/or their parents was scored as 0–4ϩ as compared with a 4ϩ reaction by the positive before the study. control anti-rat human cardiac myosin and a PBS-conjugated secondary Ab control that was negative or 0 reactivity. 51

Cr release assay by guest on September 29, 2021 Human cardiac myosin was purified as described previously (20); the light meromyosin (LMM) peptides of human cardiac myosin were synthesized A primary rat heart cell line (no. CRL-1446, American Type Culture Col- using a fluorenylmethoxycarbonyl strategy (21) and purified by HPLC. lection, Manassas, VA) was plated into 96-well tissue culture plates at 1 ϫ Sequences of the LMM peptides are indicated in Table I. 104 cells/well in IMDM with 20% FBS. The heart cells were incubated at ␮ 51 Purified rabbit skeletal myosin was obtained from Sigma (St. 37°C and in 5% CO2. A total of 5 Ci of Cr was added per well and Louis, MO). incubated at 37°C for 2 h. The cells were washed in the IMDM containing 20% FBS, and 100 ␮l of IMDM plus 20% FBS was added to each well and ELISA and Western immunoblot technique incubated at 37°C for 1 h. Sera from patients with KS and control sera were diluted 1/5 in IMDM. Plates were washed with serum-free IMDM three Microtiter plates were coated at 4°C overnight with purified human cardiac times, and 100 ␮l of diluted serum was added to wells in triplicate. Neg- ␮ myosin or LMM peptides at 10 g/ml in 0.1 M carbonate-bicarbonate ative control wells received 100 ␮l of IMDM; maximum release wells coating buffer (pH 9.6). The plates were washed with PBS Tween 20 and received 100 ␮l of 1N HCL. Positive control wells received human and blocked with 1% BSA for1hat37°C. The plates were washed again with mouse mAbs that were cytotoxic for the heart cell line. Once the test sera ␮ PBS Tween 20, and 50- l serum dilutions beginning at 1/250 were added were added, plates were incubated at 37°C for 45 min and 100 ␮l of freshly to the microtiter wells. Sera were diluted 2-fold in PBS. Serum dilutions prepared guinea pig complement was added. After a 1-h incubation at were incubated overnight with Ag at 4°C, and all tests were performed in 37°C, the supernatants were harvested by a Skatron harvester (Lier, Nor- duplicate. The plates were washed again with PBS Tween 20, and goat way) and counted on an LKB compugamma counter (Uppsala, Sweden). ␮ ␥ ␣ anti-human IgM ( -chain-specific), IgG ( -chain-specific), and IgA ( - The formula used to calculated percent lysis is as follows: ([test cpm Ϫ chain-specific) conjugated with alkaline phosphatase (1/250 dilution) was spontaneous release cpm]/[maximum release cpm Ϫ spontaneous release ␮ added in 50 l amounts to the microtiter plates and incubated at 37°C for cpm]) ϫ 100. Spontaneous release was determined as cpm from the IMDM 1 h. Conjugated Igs were obtained from Sigma. Next, the plates were negative control samples without complement. washed with PBS Tween 20, and 50 ␮l of the substrate para-phenyl-phos- phate (Sigma 104) in diethanolamine buffer was added to the wells. OD Statistics was measured at 410 nm in an ELISA plate reader (Dynatech, Chantilly, VA). Titers were calculated from the 0.30-OD endpoint in the ELISA. Means with SDs were calculated for the KS and control sera groups; the Western blots were performed as described previously (17). Briefly, 8 means of the sera groups were compared by the unpaired Student’s t test to ␮g of purified human cardiac myosin was loaded into wells of an SDS determine significance, which was calculated as two-tailed p values. polyacrylamide gel and electrophoresed until the tracking dye reached the bottom of the gel. The gel was blotted onto a nitrocellulose membrane Results using a blotting apparatus (Bio-Rad, Hercules, CA). The blot was blocked Anti-human cardiac myosin Abs in KS sera: ELISA titers and with 3% milk for1hat37°C. Sera from control subjects and KS patients Western blot were diluted 1/1000 in PBS. A section of the blot was used to stain the cardiac myosin heavy chain with a molecular mass of 200 kDa. Nitrocel- To investigate the possibility that immune responses to cardiac lulose strips containing the human cardiac myosin protein band were cut myosin were elevated in KS, sera from 13 patients with KS, 12 and reacted separately with each of the patient sera and with rat anti-human cardiac myosin sera at 1/1000. The control sera recognized the 200-kDa age-matched febrile controls, and 10 normal adults were reacted myosin heavy chain. The strips were washed five times with PBS Tween with purified human cardiac myosin by ELISA. Fig. 1 illustrates 20 and reacted with the secondary Ab conjugate goat anti-human polyva- the stronger reactivity of a group of KS sera with human cardiac 1062 ANTICARDIAC MYOSIN AUTOANTIBODIES AND KAWASAKI SYNDROME

FIGURE 1. Scattergram of anti-human cardiac myosin Ab titers of sera from acute KS patients and age-matched febrile controls as well as normal FIGURE 2. . Reactivity of 8 ARF sera and 2 normal control sera with adult sera. Endpoints were calculated at an OD of 0.3. Means and p values human cardiac myosin. The ELISA antimyosin Ab titers as shown in the were calculated using the unpaired Student’s t test. There was a significant figure are, from left to right, 512,000; 32,000; 16,000; 8,000; 32,000; difference between the means of the KS and control groups (p Ͻ 0.05). 32,000; 64,000; 8,000; and 8,000 for ARF sera and 8,000 and 2,000 for control (Cont) sera. Downloaded from myosin in the ELISA as compared with sera from age-matched controls and normal adults. The figure shows a scattergram of in- dividual serum titers when reacted with human cardiac myosin as from 2ϩ to 3ϩ, and the control sera reactivity was negative. The Ag. Greater than 50% of KS sera had titers greater than the mean Western blot is shown in Fig. 3. The KS sera were strongly pos- antimyosin titers in febrile control patients (Fig. 1). However, only itive in either the blot or the ELISA or both. Further analysis of 5 of 13 KS sera tested had higher titers. Titers of normal sera four additional KS sera demonstrated that they were all 2ϩ myosin ranged from 500 to 8,000, whereas Ab titers against human cardiac blot positive. Western blot results did not always correlate with the http://www.jimmunol.org/ myosin in KS patients ranged from 1000 to 64,000 (Fig. 1). The ELISA titers of the KS or control sera (Table II). The control sera KS titers to human cardiac myosin as a group were significantly were negative in the blot in which serum titers of 8000 were not different from age-matched febrile controls ( p ϭ 0.047) and nor- positive in the blot. The Western blot (Fig. 3) indicated the spec- mal adult controls ( p ϭ 0.04). Only antimyosin IgM Ab titers are ificity of the KS sera for human cardiac myosin, whereas the nor- shown, because there was little or no difference observed between mal sera at a 1/1000 dilution were negative. All of the normal sera KS and age-matched control sera in their IgG reactivity to cardiac tested in the blot had detectable antimyosin titers (1000, 1000, or skeletal myosins (data not shown). No antimyosin IgA Abs were 1000, and 8000) in the ELISA (Table II) but were not reactive in

detected in any of the sera. KS sera, some of which had high titers the Western blot. These data suggest that the reactivity of the KS by guest on September 29, 2021 to human cardiac myosin, also reacted with skeletal myosin but sera was highly specific for human cardiac myosin heavy chain, consistently with lower titers. Thus, there is a group of KS patients but that the reactivity of the normal control sera in the ELISA and that responds strongly to human cardiac myosin. not in the Western blot was due to 1) recognition of highly con- For comparison, antimyosin Ab ELISA titers of eight ARF sera formational epitopes, 2) cross-reactivity, or 3) background in the are shown in Fig. 2. Five of eight sera demonstrated significantly test system. elevated titers over two controls that had titers of 2000 and 8000. The individual ARF sera titers are, from left to right in Fig. 2, Specificity of KS sera for peptides in the LMM region of human 512,000; 32,000; 16,000; 8,000; 32,000; 32,000; 64,000; and cardiac myosin 8,000. ARF sera have been shown previously to react in the West- To determine whether the KS sera that were highly reactive with ern immunoblot with human cardiac myosin (16, 22) human cardiac myosin would recognize peptide epitopes in the To further demonstrate the antimyosin specificity of the KS sera, human cardiac myosin heavy chain, eight KS sera with elevated the sera were reacted with purified human cardiac myosin (200 titers (titers ϭ 64,000; 32,000; 32,000; 32,000; 16,000; 8,000; kDa) in a Western immunoblot. Table II shows the reactivity of 8,000; and 2,000) to myosin were pooled and reacted with 49 each of the sera with the cardiac myosin heavy chain as compared LMM peptides in the ELISA. In addition, febrile age-matched con- with strongly positive control rat anti-human cardiac myosin poly- trol sera (titers ϭ 1000; 1000; 1000; and 8000) were also pooled clonal sera (4ϩ). The KS sera reactivity (1/1000 dilution) ranged and reacted with the LMM peptides. The reactivity of the KS sera

Table II. Reactivity of KS sera with human cardiac myosin heavy chain in the Western immunoblot

Reactivity Reactivity

KS Sera Blot ELISA titerControl Sera Blot ELISA titer

KS-1 3ϩ 32,000 C-1 — 1000 KS-2 2ϩ 32,000 C-2 — 1000 KS-3 2ϩ 64,000 C-3 — 1000 KS-4 2ϩ 8,000 C-4 — 8000 KS-5 3ϩ 1,000 Anticardiac myosin 4ϩ sera (ϩ control) KS-6 2ϩ 2,000 KS-7 2ϩ 1,000 KS-8 3ϩ 8,000 The Journal of Immunology 1063

FIGURE 3. Reactivity of KS and control sera with human cardiac myosin in the Western immu- noblot. Sera were tested at a 1/1000 dilution.

group with the LMM peptides is shown in Fig. 4A. LMM peptides Table III (one of nine for many LMM peptides not recognized by 1, 18, 32, 34, 47, and 49 were the most reactive with KS sera after the other eight KS sera). Features for this patient included a gallop normal age-matched control sera reactivity was subtracted to ob- rhythm, a murmur tricuspid regurgitation, and sinus tachycardia. In tain the final reactivity of the KS sera shown in Fig. 4A. The Downloaded from reactivity of the control sera ranged from an OD of 0.2 to 0.5 and was subtracted. These data are compared with the reactivity of a group of eight ARF sera reacted with the LMM peptides (Fig. 4B). The reactivities of the KS and ARF sera with the LMM peptides have similarities, but are also clearly different. The greatest dif-

ferences are observed for LMM peptides 13 and 25, which are http://www.jimmunol.org/ positive only in ARF, whereas peptide 47 was twice as reactive with KS sera. Because results using pooled sera may not reflect the individual serum reactivities, we tested nine KS and six ARF sera individually. Table III shows the results of these tests. Table III compares the reactivity of both KS and ARF sera with the LMM peptide panel. As underlined in the Table III, peptides positive with three or more sera in each group were underlined to highlight the reactivities of multiple sera with a particular peptide. The KS sera reacted with LMM-1, -4, -7, -16, -18, -32, and -43, whereas by guest on September 29, 2021 the ARF sera reactive with LMM-6, -7, -18, -19, -25, -29, -32, -36, and -47. However, normal sera did not react positively with any of the LMM peptides as shown in Table III. Comparison of data from pooled KS sera in Fig. 4A with the individual serum study indi- cated that the peptides identified on the pooled sera were similar to those identified in individual sera. Comparison of the ARF sera reacted with LMM peptides (Table III) and with the pooled sera (Fig. 4B) suggested that the data were quite similar. Some of the other LMM peptides recognized by two of six ARF sera and not considered mainstream were LMM-1, -4, -8, -16, -22, -28, -34, -35, -39, -40, -43, -45, and -49, which were also seen in Fig. 4B. The conclusion from these data is that both KS and ARF sera recognize different but some overlapping epitopes. In individual and pooled serum studies, LMM-6, -19, and -25 were unique to or seen more often in ARF than in KS. The LMM peptides seen frequently in both diseases were LMM-7, -18, -32, and -47.

Cytotoxicity of KS sera for heart cells To further characterize the sera containing antimyosin Abs, the sera were tested for cytotoxicity against a rat heart cell line. KS-8 (ELISA titer ϭ 8000) and KS-11 (ELISA titer ϭ 32,000 and my- osin blot 2ϩ) sera produced 31% and 63% cytotoxicity, respec- tively. The cytotoxicity of normal sera ranged from 2 to 14%. FIGURE 4. A, Reactivity of KS sera with synthetic peptides of human These results were highly reproducible and did not necessarily cardiac LMM in the ELISA. A pool of eight anti-human cardiac myosin- positive KS sera and a pool of four age-matched control sera were reacted correlate with anti-human cardiac myosin titers. However, the two with the 49 synthetic LMM peptides. Sera were tested at a 1/250 dilution, patients with cytotoxic Abs had titers above the normal mean (Fig. and control serum reactivity with LMM peptides ranged from 0.2 to 0.5. 1). The strong cytotoxicity of KS-11 was particularly interesting in Control OD (ϳ0.2–0.5) was subtracted from the KS sera reactivity with that this patient was documented to have features of myocarditis each individual LMM peptide. B, Reactivity of a pool of eight ARF sera clinically at the time the acute sample was obtained. In addition, with synthetic LMM peptides under the same conditions as described in this patient recognized many of the LMM peptides as shown in Fig. 3A. 1064 ANTICARDIAC MYOSIN AUTOANTIBODIES AND KAWASAKI SYNDROME

Table III. Comparison of KS with ARF in reactivity of sera with multiple severe episodes of acute rejection. To our knowlege, she peptides of human cardiac LMMa is the only reported death posttransplant for a KS patient (23).

Peptide Normal Serum KS ARF Discussion LMM-1 0/3 3/9 2/6 LMM-2 0/3 1/9 1/6 The current study was performed to gain further insight into po- LMM-3 0/3 1/9 1/6 tential mechanisms of acute myocarditis in KS. Our results indi- LMM-4 0/3 4/9 2/6 cate that, by ELISA, acute KS is associated with an abnormally LMM-5 0/3 0/9 1/6 high level of circulating anti-human cardiac myosin. Western blot LMM-6 0/3 1/9 3/6 LMM-7 0/3 4/9 5/6 analysis confirmed the reactivity of acute KS sera to purified human LMM-8 0/3 1/9 2/6 cardiac myosin. Interestingly, Ab reactivity to cardiac myosin in LMM-9 0/3 0/9 1/6 acute KS sera was localized to the IgM fraction and was not present LMM-10 0/3 1/9 1/6 in the IgG fraction. This contrasts with rheumatic carditis, which is LMM-11 0/3 1/9 0/6 LMM-12 0/3 1/9 1/6 associated with both IgG and IgM antimyosin reactivity (20). LMM-13 0/3 1/9 1/6 Myosin-reactive sera from acute KS patients were also exam- LMM-14 0/3 0/9 1/6 ined for reactivity with a panel of 49 overlapping peptides that LMM-15 0/3 0/9 1/6 span the human LMM subfragment of cardiac myosin. Sera from LMM-16 0/3 3/9 2/6 acute KS patients had a different pattern of reactivity than sera LMM-17 0/3 1/9 1/6 LMM-18 0/3 6/9 3/6 from ARF patients, although some LMM peptides were recognized Downloaded from LMM-19 0/3 1/9 3/6 by both diseases. These data suggest that acute KS is associated LMM-20 0/3 0/9 1/6 with a specific Ab response to certain epitopes of human cardiac LMM-21 0/3 0/9 1/6 LMM. Strong LMM reactivity by a serum reflected a pattern of LMM-22 0/3 0/9 2/6 LMM-23 0/3 0/9 1/6 autoimmune heart disease (either rheumatic heart disease in ARF LMM-24 0/3 1/9 1/6 or myocarditis in KS). This LMM reactivity may indicate epitope

LMM-25 0/3 1/9 3/6 spreading throughout the myosin molecule. Epitope spreading http://www.jimmunol.org/ LMM-26 0/3 0/9 1/6 within and among autoantigens has been suggested in other auto- LMM-27 0/3 0/9 1/6 immune states (22). It is possible that the more peptides recog- LMM-28 0/3 0/9 2/6 LMM-29 0/3 0/9 3/6 nized, the greater the risk of developing pathogenic autoantibodies LMM-30 0/3 0/9 1/6 reactive not only with epitopes within the myosin molecule but LMM-31 0/3 0/9 1/6 with cross-reactive epitopes present at the cell surface extracellular LMM-32 0/3 3/9 3/6 matrix, or basement membrane. The presence of cytotoxic Ab in LMM-33 0/3 0/9 1/6 LMM-34 0/3 0/9 2/6 some of the KS sera suggests that this possibility exists. As for the LMM-35 0/3 0/9 2/6 diagnostic potential of the antimyosin or anti-LMM Abs in KS or LMM-36 0/3 2/9 3/6 ARF, we believe that they at least represent a relative risk factor by guest on September 29, 2021 LMM-37 0/3 1/9 1/6 and could be important in identifying patients predisposed to au- LMM-38 0/3 0/9 0/6 toimmune heart disease. LMM-39 0/3 2/9 2/6 LMM-40 0/3 2/9 2/6 Antimyosin Abs have been associated with a number of auto- LMM-41 0/3 0/9 1/6 immune states, including rheumatic fever and chronic autoimmune LMM-42 0/3 0/9 1/6 myocarditis (16, 18, 20, 24–27). It is not clear what role these LMM-43 0/3 3/9 2/6 antimyosin Abs play in the pathogenesis of disease. However, it is LMM-44 0/3 1/9 0/6 LMM-45 0/3 3/9 2/6 known that antimyosin Abs deposit in the heart of susceptible an- LMM-46 0/3 0/9 1/6 imals that develop myocarditis (26), and antimyosin/antistrepto- LMM-47 0/3 5/9 5/6 coccal mAbs are cytotoxic for heart cells in culture (28, 29). In LMM-48 0/3 0/9 1/6 addition, antimyosin Abs recognize bacterial Ags such as the LMM-49 0/3 3/9 2/6 group A streptococcal M protein and N-acetyl-glucosamine, the a Positive sera were Ͼ0.4 OD at 410 nm; negative sera were Ͻ0.4 OD at 410 nm. dominant epitope of the group A streptococcal carbohydrate (17, Sera were tested by ELISA at a 1250 dilution. 20, 24, 29–32) as well as viral Ags such as the Coxsackie viral capsid proteins (29, 33). Although antimyosin Ab can be present in the sera of normal individuals (34), it is seen usually at low levels addition, the patient had clinical features of congestive heart fail- compared with disease (16). ure and required diuretic therapy. Clinical laboratory evidence in- Although depressed myocardial function and wall motion ab- cluded electrocardiogram changes of low voltage and T wave in- normalities may be a manifestation of coronary injury and isch- version that resolved in convalescence. The initial two emia, it is unlikely that this is the sole mechanism; only 20% of echocardiograms demonstrated biventricular dysfunction, pericar- untreated KS patients develop coronary artery lesions, yet over dial effusion, and normal coronary arteries. Subsequent echocar- half have evidence clinically for myocarditis. In addition, the myo- diograms demonstrated the development of diffuse aneurysms of cardial depression seen is reversible with i.v. ␥-globulin in the the left main, left anterior descending, and right coronary artery. absence of coronary artery involvement and does not correlate The myocardial function of this patient improved and returned to with the presence of coronary artery lesions (7). Cytokines such as normal, as did her clinical features of myocarditis. She developed TNF-␣ and IL-6 have both been reported to depress myocardial worsening ischemic heart disease over the next 6 mo. The patient function (35, 36). They as well as other cytokines are known to be went on to cardiac transplantation, and the explanted heart had elevated in acute KS and could also contribute to the acute de- evidence of coronary stenosis and patchy vasculitis as well as pressed myocardial function. myocyte damage consistent with previous features of myocarditis The mechanism by which anticardiac myosin may contribute to and ischemic damage. This patient died 9 mo posttransplant after acute myocarditis in KS is not known. Preliminary studies suggest The Journal of Immunology 1065 that some of these sera may cause myocardial injury via their cy- creting Staphylococcus aureus with Kawasaki syndrome complicated by coro- totoxic activity against heart cells. Cytotoxic Abs against vascular nary artery disease. Pediatr. Res. 42:268. 15. He, X., J. Goronzy, and C. Weyand. 1992. Selective induction of rheumatoid endothelium have been found previously in KS sera, and antimyo- factors by superantigens and human helper T cells. J. Clin. Invest. 89:673. sin mAbs that recognize cell surface epitopes or Ags such as lami- 16. Cunningham, M. W., J. M. McCormack, L. R. Talaber, J. B. Harley, E. M. Ayoub, R. S. Muneer, L. T. Chun, and D. V. Reddy. 1988. Human mono- nin are cytotoxic for the rat heart cell line used in this study (24). clonal reactive with antigens of the group A Streptococcus and human However, currently there is no definitive explanation for the cy- heart. J. Immunol. 141:2760. totoxicity of KS sera. Further studies are required to determine 17. Cunningham, M. W., and R. A. Swerlick. 1986. Polyspecificity of antistrepto- coccal murine monoclonal antibodies and their implications in . whether antimyosin Abs are able to depress myocardial function J. Exp. Med. 164:998. without causing frank cytolysis of cells. Nevertheless, our current 18. Neu, N., N. R. Rose, K. W. Beisel, A. Herskowitz, G. Gurri-Glass, and studies open up a new avenue for investigation into the mecha- S. W. Craig. 1987. Cardiac myosin induces myocarditis in genetically predis- posed mice. J. Immunol. 139:3630. nisms of myocarditis in KS, one of the most common causes of 19. 1990. American Heart Association Committee on Rheumatic Fever, , acquired heart disease in childhood. and Kawasaki Disease. Diagnostic Guidelines for Kawasaki Disease. J. Am. Med. Assoc. 44:1218. 20. Adderson, E. E., A. R. Shikhman, K. E. Ward, and M. W. Cunningham. 1998. Acknowledgments Molecular analysis of polyreactive monoclonal antibodies from rheumatic cardi- tis: human anti-N-acetyl-glucosamine/anti-myosin V region genes. We thank Dr. Mark Hemric for purification of the human cardiac myosin, J. Immunol. 161:2020. Dr. Kenneth Jackson and the W.K. Warren Molecular Biology Resource 21. Carpino, L. A., and G. Y. Han. 1972. The 9-fluorenylmethoxy-carbonyl amino Facility at the University of Oklahoma Health Sciences Center for synthe- protecting group. J. Org. Chem. 37:3404. 22. Lehmann, P. V., T. Forsthuber, A. Miller, and E. E. Sercarz. 1992. Spreading of sis and purification of the LMM peptides, and Maureen Sandoval for her T-cell autoimmunity to cryptic determinants of an autoantigen. Nature 358:155. assistance in the preparation of this manuscript. 23. Checchia, P. A., E. Pahl, R. E. Shaddy, and S. T. Shulman. 1997. Cardiac trans- Downloaded from plantation for Kawasaki disease. Pediatrics 100:695. 24. Cunningham, M. W., J. M. McCormack, P. G. Fenderson, M. K. Ho, References E. H. Beachey, and J. B. Dale. 1989. Human and murine antibodies cross-reactive 1. Kawasaki, T. 1967. Acute febrile mucocutaneous syndrome with lymphoid in- with streptococcal M protein and myosin recognize the sequence GLN-LYS- volvement with specific desquamation of the fingers and toes in children: clinical SER-LYS-GLN in M protein. J. Immunol. 143:2677. observations of 50 cases. Jpn. J. Allergol. 16:178. 25. McCormack, J. M., C. A. Crossley, E. M. Ayoub, J. B. Harley, and 2. Meissner, H. C., and D. Y. M. Leung. 1995. 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