Amercan Journal of Pathology, Vol. 138, No. 2, FebruaY 1991 Copfrigbt © Amerian Association of Pathologists

Immunoreactivity of Anti-streptococcal Monoclonal Antibodies to Human Heart Valves Evidence for Multiple Cross-reactive Epitopes

James M. Gulizia,* Madeleine W. Cunningham,t and Bruce M. McManus* From the Cardiovascular Regishty, Department of Pathology and Microbiology, University of Nebraska Medical Center,* Omaha, Nebraska; and the Department of Microbiology and Immunology, University of Oklaboma Health Sciences Center, t Oklahoma City, Oklahoma

Association of group A streptococci with acute rheumatic fever and valvular heart disease is well established; however the basis of valve injury remains unclear. In this study, anti-streptococcal monoclonal antibodies (MAbs) cross-reactive with myocardium were reacted with sections from 22 rheumatic valves, nine normal five endocarditic, one floppy,' and one Marfan valve. In immunohistochemical studies, MAb reactivity was observed with cardiac myocytes, smooth muscle cells; cell surface and cytoplasm of endothelial cells lining valves, and valvular interstitial cells Endothelial basement membrane and elastin fibrils reacted with the MAbs whereas collagen was unreactive. Similar reactivity was seen with sera from acute rheumatic fever patients. The antistreptococcal MAbs reacted with intravalvular myosin and vimentin in Western blots, and purified elastin competitively inhibited the binding of the antistreptococcal MAbs to whole group A streptococci. The data show that human heart valves have numerous sites of immunoreactivity with anti-streptococcal MAbs and acute rheumatic fever sera of potential importance in the pathogenesis of rheumatic valvular injury. (Am JPathol 1991, 138:285-301)

Classical studies by Kaplan,1" Zabriskie,45 Dale and Beachey,67 and Cunningham and colleagues-12 have documented the significant role of autoimmunity in the induction of postinfectious sequelae related to group A

streptococcal (Streptococcus pyogenes) infections. Humoral responses after streptococcal infection result in the recognition of epitopes shared between the offending organism itself and human tissues.1-12 The findings are the basis for the current hypothesis suggesting immunologic cross-reactivity between the group A streptococcus and myosin as the basis for rheumatic heart disease.10I 1 The search for the specific protein structures shared between the streptococcus and human tissues has led to characterization of a large number of monoclonal antibodies (MAbs) that cross-react with the streptococcal virulence determinant, the M protein, and host ax-helical, coiled-coil proteins, such as myosin and tropomyosin.-1 1 The demonstration of antibodies present in acute rheumatic fever sera with cross-reactivities between streptococcal M protein and human cardiac myosin7,13 further establishes the relationship of these antibodies with the disease. Furthermore affinity-purified antisera from rabbits immunized with streptococcal pepsindigested M 5 protein have shown cross-reactivity with human cardiac myosin.14 Recently, a specific pentapeptide in the M protein11 has been identified as an amino acid sequence that is immunologically cross-reactive with human proteins, specifically, cardiac myosin. The pathogenetic relevance of M protein-myosin crossreactivity is supported also by the well-described model of myosin-induced, autoimmune murine myocarditis.15 This model lends credence to the concept that an immuSupported in part by National Institutes of Health grants, HL35280 and HL01913 to Madeleine W. Cunningham. James M. Gulizia is a recipient of a fellowship awarded through the Nebraska Govemor's Research Initiative in Biotechnology. Madeleine W. Cunningham is a recipient of a Research Career Development Award from the National Heart, Lung, and Blood Institute. Accepted for publication September 6, 1990. Address reprint requests to Bruce M. McManus, MD, PhD, Director, Cardiovascular Registry, Department of Pathology and Microbiology, University of Nebraska Medical Center, 600 South 42nd St., Omaha, NE 68198-6495.

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nologic response against an epitopes on myosin may lead to heart injury; however valve injury has not been studied in this model. In addition to myosin, the intermediate filament, vimentin, found in a variety of mesenchymal cells in vivo, and in some epithelial cell lines in vitro1'18 may represent an important target involved in poststreptococcal autoimmune injury. Like the streptococcal M protein, vimentin also contains regions composed of an a-helical, coiled-coil motif.19 Recently Kraus, Beachey, and colleagues-'21 studied autoantigens putatively involved in autoimmune acute poststreptococcal glomerulonephritis. These studies demonstrated that NH2-terminal M protein amino acid residues of so-called 'nephritogenic' group A streptococci, M types 1 and 12, contain distinctive epitopes that cross-react immunologically with glomerular mesangial vimentin. Furthermore, Cunningham and Swerlick9 have shown that cytoskeletal vimentin in fibroblasts is among the a-helical molecules recognized by a MAb raised against the 'rheumatogenic' M type 5 group A streptococcus. Site-specific rheumatic injury and repair of human heart valves has remained a conundrum in the myosinheart injury paradigm. Indeed, in the late period of disease after acute rheumatic fever, the myocardium typically is unscathed, with only residual, connective tissuebased, healed Aschoff nodules and no apparent decrement in function. In contrast, heart valves, particularly the mitral valve, may undergo distortive, fibrotic thickening, with degenerative calcific changes and severe clinical malfunction. Plausible targets for initiation or perpetuation of valve injury in rheumatic heart disease might be inferred from knowledge of normal histology.' Certainly a wide variety of cellular and extracellular components could serve as crucial autoantigens. Recently Filip and colleagues23 reported details of fine structural and functional studies of a prominent cell population in mammalian atrioventricular valves that may be pathogenetically important in rheumatic injury. These valvular interstitial cells (VICs) appear to have properties intermediate to fibroblasts and smooth muscle cells. In particular, they have contractile activity, Golgi, and endoplasmic reticula, suggestive of the capability of elaborating extracellular matrix proteins. Thus VICs and other normal valvular constituents must be considered potentially pivotal to poststreptococcal heart

valve injury. In efforts to investigate further the basis of heart valve injury after acute rheumatic fever, we studied a series of operatively excised or autopsy heart valve specimens. We used MAbs raised against streptococcal cell membrane proteins in immunoperoxidase evaluation of formaldehyde-fixed, paraffin-embedded or frozen tissue sections. Likewise valve sections were also stained with sera

from patients with acute rheumatic fever or normal individuals. Specifically an effort was made to identify the location, degree, and comparability of the immunoreactivities of the MAbs and sera in normal and diseased human heart valves. The anti-streptococcal MAbs were used to identify further the relevant cross-reactive antigens in human heart valves by Western immunoblotting and competitive inhibition enzyme-linked immunosorbent assays (ELISAs).

Materials and Methods Sources and Types of Valve Specimens Mitral (22 specimens), aortic (4), and tricuspid (1) valves were obtained at operation or autopsy from 22 patients with rheumatic heart disease. The mean age of rheumatic patients was 54 years (range, 22 to 74 years), including 17 women and five men. Fourteen patients had known congestive heart failure and nine had documented pulmonary hypertension. Four patients had specific histories of acute rheumatic fever or scarlet fever. Valvular dysfunction included mitral stenosis in 17 patients, mitral regurgitation in 16, aortic stenosis in 5, aortic regurgitation in 5, and tricuspid stenosis in 2. Mitral stenosis and regurgitation were concurrent in 11 patients. Four mitral valves and one aortic valve from patients with infective (2) or noninfective (2) endocarditis were studied. Also a floppy mitral valve from a 76-year-old man and a mitral valve from a 37-year-old man with Marfan's syndrome were examined. Diseased valves were considered in comparison with anatomically and functionally normal mitral and tricuspid valves from nine autopsy patients with a mean age of 59 years (range, 25 to 81 years), including four women and five men. These patients died of conditions unrelated to the heart. All specimens were accessioned in the Cardiovascular Registry of the University of Nebraska Medical Center and were fixed in either 10% neutral buffered formaldehyde solution (for paraffin embedding) or snap frozen before further processing.

Test Antibody Characteristics The test MAbs used were raised in mice immunized against purified, group A (type 5) streptococcal membranes.10 In general, the prototypic gamma M immunoglobulin (IgM) MAbs, 36.2.2, 49.8.9, and 54.2.8 were classified into three families, based on characteristic reactivities with human tissues. Monoclonal antibody 54.2.8 has been shown to react with group A streptococcal M protein, myosin (heavy meromyosin), vimentin,

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DNA, and structurally related molecules, such as cardiolipin and synthetic nucleotide homopolymers, polyinosinic acid, and polydeoxythymidylic acid.8924 Whereas MAb 36.2.2 reacted with M protein, myosin (light meromyosin), actin, laminin (Antone SM and Cunningham MW: Manuscript in preparation), and other a-helical, coiled-coil proteins, such as keratin and cardiac tropomyosin. 12'24 Of the three anti-streptococcal MAbs, 36.2.2 is the most highly cytotoxic to primary heart and fibroblast cell lines in vitro in the presence of complement.25'26 The reactivities of MAb 49.8.9 are less well characterized and, although the MAb does react with extracts of human myocardium, kidney, and skeletal muscle, its reactivity with purified myosin is more uncertain.9'24 All three MAbs reacted in indirect immunofluorescence with cytoskeletal components of fibroblasts.9 Also this fibroblast cytoskeletal reactivity of MAb 54.2.8 was eliminated by pretreatment of the fixed cells with anti-vimentin antisera.9 However none of the MAbs react with calf skin collagen, nor do they appear to be rheumatoid factors, as mouse and human IgG fail to inhibit the MAbs in competitive binding assays.9 The anti-muscle actin IgG MAb, HHF-35,27 was obtained from Enzo Biochemicals, Inc. (New York, NY). The anti-myosin IgG MAb, CCM-52,28 was a generous gift from Dr. William A. Clark, Jr. (Northwestern University Medical School, Chicago, IL). The anti-elastin IgG MAb (No. MM872) was obtained from Elastin Products Company, Inc. (Owensville, MO). The anti-vimentin IgG MAb (Clone V9)29 was obtained from Boehringer-Mannheim Biochemicals (Indianapolis, IN). Acute rheumatic fever antisera (ASO more than 160 Todd units) was a gift of Drs. L. T. Chun and D. V. Reddy, Kapiolani Women's and Children's Medical Center (Honolulu, HI). Normal human sera used had an ASO of less than 125 Todd units.

Histochemistry and Immunohistochemistry Formalin-fixed tissue was processed routinely and embedded in paraffin for 4- to 5-,u thick sections on gelatincoated slides. For frozen sections, tissue was snap frozen in Tissue-Tek O.C.T compound (Miles Laboratories, Elkhart, IN) and 4-, sections were mounted on gelatincoated slides. Serial sections of each specimen were stained with hematoxylin and eosin or Movat's pentachrome. Immunoperoxidase staining by the avidinbiotin-peroxidase complex method was performed as previously described.30 Briefly, after deparaffinizing in xylene, the slides were rehydrated in graded ethanol and washed with 0.1 mol/l (molar) phosphate-buffered saline (PBS) at a pH of 7.2. Then a 1 5-minute preincubation in a humidified chamber was performed with normal horse serum (diluted 1:100 in

1% bovine serum albumin [BSAJ/PBS) to minimize nonspecific binding of the test MAbs. The sections were then incubated with the MAbs for 30 minutes at 24°C. Hybridoma culture supernatants of MAbs 36.2.2, and 54.2.8 in culture medium containing 10% horse serum, were used undiluted at IgM concentrations of 3.4, 7.4, and 13.3 ,ug/ml, respectively. The IgM isotype control MAb 58.2.312 was used at 39 ,ug/ml. Subsequently the slides were washed with PBS, then biotinylated horse antimouse immunoglobulin (Vector Laboratories, Burlingame, CA) was applied for a 30-minute incubation. After washing in PBS, endogenous peroxidase activity was quenched with a 5-minute incubation in 3% hydrogen peroxide substrate. The slides were again washed in PBS, then incubated for 30 minutes in avidin DH horseradish peroxidase H complex (Vector Laboratories). Antibody binding was visualized with the chromogen diami-

nobenzidine against a light hematoxylin counterstain. On selected valve specimens, staining with MAbs specific for myosin, vimentin, muscle actin, or elastin was carried out for comparison with the anti-streptococcal MAbs. Dilutions or concentrations (in PBS) of the additional test MAbs used were as follows: anti-myosin, 20 ,ug/ml; anti-vimentin, 3 jig/ml; anti-actin (ascites fluid, 1:500); and anti-elastin (ascites fluid, 1:200). As well, comparison of MAb staining and tissue morphology was made on 4-,u frozen sections, versus paraffin sections. A similar staining technique was applied in the frozen tissue as was used on paraffin sections. In addition, valve sections were stained with sera (diluted 1:400 in PBS) from acute rheumatic fever patients or normal individuals, using the above procedure for MAbs, with two modifications. The blocking step was done with normal goat serum at 1:80 in 1% BSA/PBS. The secondary MAb used was biotinylated goat anti-human immunoglobulin from Zymed Laboratories, Inc. (San Francisco, CA) at 1:500 in PBS. In immunoinhibition studies, the anti-streptococcal MAbs were mixed with equal volumes of purified soluble elastin (1 mg/ml in PBS; Elastin Products Company) or Heparin (10 USP U/ml; SoloPak Laboratories, Franklin Park, IL). After a 6-hour incubation at 40C, the solutions were used in immunoperoxidase studies as above.

Scoring Algorithm for Immunoreactivity Glass slides were reviewed on a light microscope by two investigators blinded to source of valve tissue and scored semiquantitatively for degree (intensity) of immunoreactivity in various sites on a scale of zero (0) = no staining, 1 + = minimal, 2 + = weak, 3 + = intermediate, and 4 + = strong. Cellular and extracellular sites of MAb reactivity were noted, and their geographic patterns re-

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corded. The site-specific and apparent background reactivity of each anti-streptococcal MAb was also recorded. Observations on immunoperoxidase-stained slides were referenced to valvular characteristics defined by hematoxylin and eosin and Movat's pentachrome stains.

Laemmli.31 Western blots of the extracted proteins were reacted with anti-streptococcal MAbs 36.2.2 (3 ,ug/ml), 49.8.9 (20 jig/ml), 54.2.8 (6 jxg/ml), and 654.1.1 (10 jig/ml) and with anti-myosin (10 ,ug/ml) and anti-vimentin (3 ,ug/ml) MAbs, according to the method of Towbin and colleagues.32

Preparation of Triton X-100-Extracted Mitral Valvular Antigens

Competitive Inhibition Enzyme-linked Immunosorbent Assay

A normal adult human mitral valve was obtained from autopsy and frozen at - 700C, within 12 hours postmortem. Valvular antigen preparation was performed as previously described,24 with the following modifications: Valve leaflets were cut into small fragments at 0.2 g (wet weight) per ml of 0.15 mol/A PBS (pH = 7), containing 4% Triton X-1 00, 3 mmol/l (millimolar) phenyl methyl sulfonyl fluoride, 3 mmol/l ethylenediaminetetra-acetate, and 3 mmol/I N-alpha-p-tosyl-L-lysine chloromethyl ketone. Homogenization was performed by two, 20-second bursts at 00C with the Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, NY). The homogenate was added to an equal volume of 6% sodium dodecyl sulfate (SDS), then boiled for 5 minutes. The sample was then centrifuged at 1 2,000g in a Sorvall RC-2B centrifuge at 40C for 15 minutes. The supernatant was used for sodium do-

Competitive inhibition ELISAs were performed according to previously described methods.8 Briefly, the antistreptococcal MAbs were mixed 1:2 with increasing concentrations (125 ,g/ml to 2 mg/ml in neutral PBS) of inhibitors. Inhibitors included purified, soluble bovine neck ligament elastin or a and 3 elastin (Elastin Products Co.), which are the partial hydrolysis products of insoluble elastin.33 The final IgM concentrations for MAbs 36.2.2, 49.8.9, and 54.2.8 were 3.8, 3.2, and 4.4 ,xg/ml, respectively. The mixtures were incubated at 370C for 1 hour, then overnight at 40C. Fifty microliters of the MAbfinhibitor mixtures were placed in triplicate onto M type 5 group A streptococci, glutaraldehyde-fixed to polyvinyl chloride microtiter plates. Enzyme-linked immunosorbent assays were performed as previously described,34 using alkaline phosphatase-conjugated anti-mouse immunoglobulins (Sigma Chemical Co., St. Louis, MO) to measure antibody reactivity. One hundred percent reactivity was de-

decyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) with a 7.5% acrylamide gel, using the methods of

Figure 1. Schematic longitudinal section of the anterior leaflet of the mitral valve. The subendothelial, free margin area (FM) contains large, branching mesenchymal cells (VICs) (A) embedded in a glycosaminoglycan matrix The mid-valvular region contains a thin layer of elastin microfibrils (B) and a discontinuous layer of subendothelial smooth muscle cells (C), sometimes organized in tight aggregates (D), while the underlying ventricularis contains a dense layer of laminar collagen (COL). The valve base of attachment to the myocardium contains bundles of cardiac myocytes (MYO), several small blood vessels (E), and a prominent elastic layer (F) in association with smooth muscle cells (G). Chordae tendineae (CT), attached to the ventricular surface, contain a central collagenous core, surrounded by a thin connective tissue membrane and covering endothelium.

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termined from MAb controls diluted 1:2 with PBS and reacted with whole streptococci in the ELISA. Percent inhibition was calculated by comparison with these controls.

Results General Normal Histologic Features Normal valvular histology seen in the heart valves was similar to that described in classical studies,22 and as illustrated in a schematic longitudinal diagram of an anterior leaflet of a normal mitral valve (Figure 1). Mitral valve features in both the anterior and posterior leaflets are summarized as follows: The leaflets varied from approximately 0.5 to 1.5 mm in thickness, the thickest region being at the point of leaflet apposition, the free margin. The base of leaflet attachment was invested in bundles of cardiac myocytes. The atrialis began basally with a prominent, discontinuous layer of smooth muscle cells arranged in tight aggregates. Within this region there were Figure 2. Atrialis margin of the base of attachment of the anterior leaflet of a normal mitral valve. a, b, c, and d were stained with Movat'spentachrome, or MAbs 54.2.8, 49.8.9, or 58.2.3, respectively. Many elastinfibrils (arrows in a) are reactive with the antistreptococcal antibodies in b and c (double arrowheads in c). Also cardiac myocytes (M) are reactive with MAbs 49.8.9 and 54.2.8, and most intensely with MAb 49.8.9. Valvular collagen (C) is unreactive with the MAhs, while many spindle cells embedded in the collagenous stroma are reactive cytoplasmically (open arrow in c). No reactivity is seen with the isotype control, MAb 58.2.3 (d). Photomicrographs, x 125.

also prominent elastic fibrils, which diminished in density toward the midportion of the leaflet, and were even less dense and less organized toward the free margin. In addition, there was a membranelike band of connective tissue underlying the surface endothelium of the atrialis, which was thicker than a typical basement membrane. This elastin-deficient band appeared to separate the overlying endothelium from the underlying elastotic region of the atrialis. In the region of thickening, related to leaflet apposition, variable swirls of dense collagenized tissue and loose cellular zones were present. The spongiosa was narrow, relative to the thickness of the atrialis or the ventricularis. The region had less dense connective tissue elements with slightly more mesenchymal cells, most of which were consistent with smooth muscle cells. The swirl of expanded tissue in the appositional region appeared similar to spongiosa. Large, branching mesenchymal cells with large oval nuclei, consistent with valvular interstitial cells (VICs),23 were found in normal leaflets. The ventricularis was largely a well-organized, sheathlike layer of laminar collagen. Over the basal half of the leaflets, there was a rather discrete subendothelial

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Figure 3. Atrialis aspect of the base of the anterior leaflet of a normal mitral valve stained with Movat (a), MAb 54.2.8 (b), or MAb 49.8.9 (c). Staining within the musculoelastic (M) airialis, the central collagenous (C) junction of the spongiosa and ventriculars, and the deeper glycosaminoglycan-rich (G) region is observed. Immunoreactivity offibrillar material in the elastic zone as well as ofapparent smooth muscle cells throughout the leaflet (arrowhead in c), and of glycosaminoglycan-rich areas (arrows in C) exemplifies localization of staining. As illustrated in Figure 2, the collagenous zone lacks reactivity with the MAbs. Photomicrographs, x 125.

Figure 4. Ventricularis aspect of base of the anterior leaflet of a normal mitral valve. Staining with MAbs 49.8.9 and 54.2.8 in a and b, respectively, empbasizes the affinity of the antibodiesfor areas ofglycosaminoglycan (as seen in Figure 3). Staining is denoted by arrowheads Surface staining of the elastica on the ventricularis surface is also evident. Photomicrographs, x 125.

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Figure 5. Free margin of the anterior leaflet of a rheumatic mitral valve. Prominent nodular protuberance of the valve (a and b) includes numerous spindle-shaped mesenchymal cells in a loose ground substance. The cells are illustrated at x 125 (a and b) and X500 (c and d) with Movat (a and c) and MAb 54.2.8 (b and d) staining. Cytoplasmic staining of cells with Ahb 54.2.8 is prominent in surface endothelium (b) (arrowheads) and in large (large arrowhead) and small (arrows) (d) mesencdxymal cells. Tentacular cytoplasmic processes of mesencbymal cells also stain positively.

elastin membrane on the ventricularis aspect. This elastin membrane was much more dense and condensed in appearance than that seen on the atrialis. Rare small vessels were found at the base of attachment in the normal valve; some were muscular arteries, while others were venous and lymphatic structures. Chordae had a typical dense collagenized structure with thin, overlying loose connective tissue and covering endothelium. The superficial connective tissue layer of chordae contained a small amount of elastin.

Immunoreactivity of Normal and Rheumatic Mitral Valves Several sites of immunoreactivity were demonstrable with the three anti-streptococcal MAbs studied. These immu-

noreactive sites are illustrated in the schematic longitudinal diagram of the anterior leaflet of the mitral valve (Figure 1). Reactive sites included cytoplasmic staining of cardiac myocytes (Figure 2), smooth muscle cells (Figures 2, 3), undefined mesenchymal cells (Figures 4 to 8), valve-lining endothelial cells (Figures 5,6), and endothelium of small intravalvular blood vessels (Figure 9). Basement membrane staining was evident (Figure 9), and there was staining within regions of prominent elastic fibrils (Figures 2 to 4, 8, 10), as well as similar, slightly less prominent, apparent staining within glycosaminoglycanrich areas (Figures 3, 4). Valvular interstitial cells (VICs) also reacted strongly with the anti-streptococcal MAbs (Figures 1 1, 12). Valvular interstitial cells were strikingly more visible in frozen sections than in paraffin-embedded samples. Although

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Figure 6. Airialis aspect of the free margin of the anterior leaflet of a rheumatic mitral valve. Prominent staining of the cytoplasm of the endothelial cells by MAb 54.2.8 is evident (arrows). Also, deeper cells in the stroma reveal vivid cytoplasmic staining (arrowhead) (X500).

their reactivity with the anti-streptococcal MAbs was less intense and more diffusely cytoplasmic in frozen valves, freezing allowed better visualization of the unusual tentacular structure of these cells and of the large cytoplasmic:nuclear ratio as compared with typical smooth muscle cells (Figures 11, 12). Vivid staining of the same cells was seen with the anti-vimentin MAb (Figure 12). In addition, as demonstrated with anti-streptococcal MAbs, the long tentacular processes of VICs were exceptionally more visible in frozen sections than after paraffin embedding. These cells failed to stain with MAbs specific for myosin, actin, or elastin (data not shown). Also the IgM isotype control, MAb 58.2.3, did not stain any valvular structures (Figure 2). Together these results suggest that the intermediate filament, vimentin, may be a prominent, intracellular target of the anti-streptococcal MAbs. Staining intensity was generally greatest for smooth muscle cells and VICs (3 to 4+/4 +) (Figures 3, 9, 11, 12). The next most intense staining was found in some regions of endothelium and basement membrane (2-3 +) (Figures 6,9), with a comparable degree of staining of cardiac myocytes (Figure 2), and elastic regions (Figure 10). Endothelial staining was more visible on normal valve surfaces than diseased, perhaps because the endothelial lining was more widely intact on normal valves. It has been reported that endothelium is lost consistently from the surface of rheumatic heart valves.35 Endothelial staining in intraleaflet blood vessels was also

vivid (Figure 9). Staining intensity of different cell types was generally uniform throughout the length of valve leaflets. Tightly aggregated bundles of smooth muscle cells in the proximal atrialis stained comparably to dispersed smooth muscle cells and VICs, which were more visible in the spongiosa layer and in expanded connective tissue of the free margin (Figures 2, 3, 5, 12). Patterns and intensities of staining of valvular components were somewhat similar for each of the antistreptococcal MAbs evaluated (Table 1). Although MAb 54.2.8 has shown antinuclear staining,9 cytoplasmic staining far exceeded any apparent nuclear reactivity in valve sections at all times. No reactivity was seen with any of the antibodies against sheaths of collagen in the valve leaflets or cusps proper or in chordal structures (Figures 2, 3). Staining of normal valve leaflets with MAbs against muscle actin or myosin generally disclosed more exclusive reactivity to apparent muscular cells, including cardiac myocytes and smooth muscle cells (data not shown), than was demonstrable with the three streptococcus-derived MAbs. Endothelia, basement membrane, elastin, and collagen did not stain with MAbs against myosin or actin (data not shown). The anti-elastin MAb exclusively stained subendothelial areas rich in elastin microfibrils (data not shown) in a pattern similar to that seen with Movat's pentachrome and antistreptococcal MAbs (Figure 10). Valvular elastin fibril reactivity with the anti-streptococcal MAbs, however, was

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Figure 7. Area of calcification and proliferative repair in the anterior leaflet of a rheumatic mitral valve. Staining oftheproliferative cellular region with Movat's stain (a and c; x 125 and x500, respectively) as well as by MAb 54.2.8 (b and d; x 125 and X500, respectively) of the same fields reveals moderate cytoplasmic reactivity in mesenchymal cells of the healing zone (arrows in d).

not inhibited by preincubation of the MAbs with purified soluble elastin (data not shown). This finding may be explained by the fact that solubilization of elastin requires rigorous hydrolysis in a series of hot oxalic acid treatments.i6 It is plausible that a significant number of the appropriate elastin epitopes cross-reactive with the streptococcus are destroyed during this process. Staining with Movat's pentachrome allowed visualization of areas rich in glycosaminoglycans. The basis of staining in these areas with anti-streptococcal MAbs is not known. Preincubation of anti-streptococcal Mabs with heparin, however, failed to inhibit the apparent reactivity in these areas. Thus, it is probable that the MAbs recognized protein core determinants of valvular proteoglycans. It may be important to note that reactivity in these areas was significantly less prominent than cytoplasmic staining of VICs, smooth muscle cells, and cardiac myocytes. As observed with hematoxylin and eosin or Movat's pentachrome staining, the features of classical, chronic

rheumatic valvular disease were present (data not shown). Thus the greatly thickened valve leaflets had diffuse expansion of all three valvular layers by dense, collagenized connective tissue. This connective tissue was not uniformly layered, as in normal valves, but rather in disorganized and unpredictable nodular thickenings, as well as in layers parallel to the leaflet axis. The proximal regions of the leaflets were much less thickened than distal regions. In some rheumatic valves an apparent 'onlay' of connective tissue was present on the atrial surface of the distal half of the valve leaflet (data not shown). The base of attachment was altered with an increased thickness of the elastotic zone of the atrialis and a concomitant smudginess to the new and diffuse elastin fibrils. The elastotic layer did not continue uninterrupted to the free margin. Rather the connective tissue expansion at the free margin was characterized by diffuse elastin proliferation more on the ventricular aspect of the mitral leaflets, in association with chordal origin. Also the more discrete elastin layer on the basal half of the ventricularis of the

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Figure 8. Transleaflet longitudinal section in mid-region of anterior mitral leaflet of a normal mitral valve. The atrialis (arrowhead in a) and ventricularis margins (arrow in a) are denoted. In the central region of the leaflet an aggregate of smooth muscle cells shown at low (a, x 125) and high (b, x 500) power has prominent cytoplasmic staining with anti-streptococcal MAb 49.8.9. Slight staining of the elic region in the atialis also is appreciated in comparison to lack of staining in the collagenous zone of the ventricukris in a.

mitral leaflets was expanded and more intense on Movat's pentachrome than in normal leaflets (data not shown). The spongiosa contained discontinuous areas of loose connective tissue in association with widely proliferated neovascular channels and an apparent increase in number and density of mesenchymal cells, consistent with smooth muscle cells. Numbers of VICs were variably, but notably, increased in the spongiosa of diseased valves. Reactivity was more prominent in the spongiosa for smooth muscle cell and VIC aggregates in diseased valves as compared with normal valves. Also endothelial cytoplasm of intravalvular neovascular vessels appeared to stain intensely, similar to surface valvular endothelium (Figures 6, 9). Sites and general intensity of immunoreactivity with the three streptococcal-derived MAbs were similar in rheumatic valves to those in normal valves.

macrophagelike mononuclear cells were present in the base of attachment to the myocardium. Tricuspid valve leaflets were, in essence, a delicate rendition of mitral valve leaflets. Thus the general structures of the three layers, namely, the atrialis, spongiosa, and ventrcularis were comparable to those of the mitral valve, although tricuspid leaflets were approximately one third to one half as thick as mitral valve leaflets (data not shown). Distortive morphologic changes, similar to those in diseased mitral valves, were observed in rheumatic aortic and tricuspid valves. Similar sites, intensities, and comparabilities of anti-streptococcal MAb staining were observed (data not shown). In addition, at the root of attachment of aortic valve cusps, occasional clusters of cells with trace, diffuse cytoplasmic staining (2+) and apparent nuclear membrane rimming (2 +) were observed on the ventricular aspect of the cusps (data not shown). These cells appeared morphologically reminiscent of monocytemacrophages or a secretory type of smooth muscle cells.

Normal and Rheumatic Nonmitral Valves Normal aortic valve cusps had similar architectural features to the mitral valve leaflets (data not shown). The elastotic layer, however, was more prominent on the ventricular aspect of the cusps, and small aggregates of

Immunoreactivity with Acute Rheumatic Fever or Normal Sera On comparison of the immunoreactivity of sera from acute rheumatic fever patients with that of normal sera,

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Figure 9. Proliferative small arteries in the anterior leaflet of a rheumatic mitral valve. Staining with Movat's (a and c; x125 and X500, respectively) and MAb 54.2.8 (b and d; x 125 and X500, respectively) reveal moderate smooth muscle cell staining within walls of small blood vessels The cytoplasm of mesencymrl cells within the stroma, surrounding the arteries, stains more deeply (arrow in d) than arterial smooth muscle cells (arrowhead in d). In addition, basement membrane staining was seen in small arteries (open arrows in d) similar to staining found in basement membrane underlying endothelia of valve surfaces.

staining intensity of sites also recognized by anti-streptococcal MAbs is much greater with acute rheumatic fever sera than with normal sera (Figure 13). This reactivity is concordant with other evidence we present, suggesting that humoral responses in acute rheumatic fever do involve recognition of heart valve epitopes that are shared with streptococcal proteins.

we found that

Nonrheumatic Diseased Valves Evaluation of valves from patients with infective or noninfective endocarditis, floppiness, or Marfan's syndrome allowed education of different inflammatory or metabolic injurious processes, with respect to epitope expression. Lesions of healing or healed infective or noninfective en-

Figure 10. Elastic zone of the atrialis in a normal mitral valve. Tbe elastic fibrils (arrows) seen in a (Movat's stain) are as visible as fibillar staining with MAb 54.2.8 in b. Photomicrographs, X330.

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Figure 1 1. Loose cellular zone in the deepfree margin of the anterior leaflet of a normal mitral valve stained with MAb 49.8.9. Staining of mesenc*ymal cell cytoplasm (arrowhead in b) is observed at low (a, x 125) and high (b, X500) power. Also tentacular processes of these mesenchymal cells are visible in both panels. Large and rather variable nuclear contours and irregular cell processes are typical of valvular interstitial cells (VICs). While cell processes of VICs are not as clearly visualized in paraffin sections as in frozen sections, intensity of cytoplasmic staining proximate to nuclei is greater

docarditis were localized to regions of valvular damage and were characterized by typical tissue destruction and repair, with variable foci of granulation tissue, neovascularity, and surface thrombosis. Immunoreactivity of endocarditis valve lesions was similar to that of normal and rheumatic valves, insofar as types and intensities of tissue and cellular reactivity (data not shown). The particular reactivity within spindle cells of 'active' granulation tissue strongly suggested that they were primarily smooth muscle cells. Such prominence of immunoreactive spindle cells was similar to smaller, less frequent regions in the free margins of normal valves and in the expanded spongiosa and free margin tissue of rheumatic valves. As is typical of the cardiac valves in connective tissue syndromes, an excess of ground substance, consistent with glycosaminoglycan accumulation, was observed (data not shown). Immunoreactivity in the floppy valve

and in that from the Marfan's patient was observed in areas rich in glycosaminoglycans, as well as in muscle cells, endothelia, and elastic regions.

Western Immunoblotting of Extracted Valvular Proteins

Anti-streptococcal MAbs 36.2.2, 49.8.9, 54.2.8, or 654.1.1, or MAbs specific for myosin or vimentin were reacted with solubilized valvular proteins in Western immunoblots (Figure 14). Monoclonal antibodies 36.2.2 and 54.2.8 recognized a high molecular weight protein of 205 kd, identified as myosin by anti-myosin MAb, CCM-52. The three smaller bands, between 1 16 and 200 kd, recognized by MAbs 36.2.2 and 54.2.8, are probably myosin degradation products, which have been seen in pre-

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grapbs, X500.

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Table 1. Immunoreactivity of Anti-streptococcal MAbs 362.2, 49.8.9, and 54.2.8 in Human Mitral Valve Leaflets 36.2.2 49.8.9 54.2.8 Sites of reactivity* 3+ 2-4+ 2-4+ Cardiac myocytes 3-4+ 3-4+ 4+ Smooth muscle cells 2-3+ 3-4+ 3+ Endothelial cells 3+ 3-4+ 3+ Valvular interstitial cells 2+ 3+ 2-3+ Basement membrane 2 2+ 2-3+ Elastin fibrils 0 0 0 Collagen *

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vious studies.1'113 Monoclonal antibody 49.8.9 did not recognize the 205-kd band, suggesting that the cardiac myosin epitope recognized by the MAb in tissue sections is labile to SDS treatment. Monoclonal antibody 49.8.9 reacted strongly with a band of approximately 57 kd, however, corresponding to the vimentin band recognized by the anti-vimentin MAb. Also, MAbs 36.2.2 and 54.2.8 reacted to a lesser degree with this band. The IgM isotype control, MAb 654.1.1, failed to react with valvular proteins in these studies. Therefore, the data suggest that valvular myosin and vimentin contain epitopes crossreactive with the group A streptococcus.

Characterization of Elastin Reactivity To investigate elastin fibril reactivity of the antistreptococcal MAbs, as seen by immunohistochemistry, purified soluble elastin, or the two partial hydrolysis products of insoluble elastin, a and I elastin, were reacted with anti-streptococcal MAbs or an anti-elastin MAb in Western immunoblots and competitive inhibition ELISAs. Although not shown, the MAbs failed to react with elastin in blots, while the MAb specific for elastin reacted strongly with the purified elastins. To test whether anti-streptococcal MAbs recognized soluble elastin, we evaluated the ability of soluble elastins

to competitively inhibit binding of the MAbs to whole, group A streptococci on microtiter plates by ELISA. We found inhibition of MAbs 36.2.2 and 54.2.8 by either soluble elastin or a elastin at high concentrations (Figure 15). The elastins failed to inhibit binding of MAb 49.8.9 to group A streptococci. p elastin did not inhibit binding of any of the anti-streptococcal MAbs tested in these studies (data not shown); therefore, most inhibition appeared associated with a elastin, the larger molecular weight partial hydrolysis fraction of insoluble elastin. Thus the anti-streptococcal MAbs recognized, and could be inhibited by, native elastin in ELISAs, while not binding elastin under denatured conditions. These data help explain elastin reactivity seen in cardiac valvular sections in our immunohistochemical studies and suggest that antigenic cross-reactivity between group A streptococci and valvular elastin may be due in part to elastin conformation.

Discussion An attractive hypothesis explaining the genesis of tissue injury in rheumatic heart disease is that in susceptible individuals autoreactive lymphocytes may be stimulated by streptococcal components, such as M protein. Such sensitized lymphocytes may recognize and respond to host cell-surface determinants containing sequential, structural, or conformational homology with a protein such as myosin, as a means of inducing inflammatory injury. Murine anti-streptococcal MAbs that cross-react with human tissue antigens provide a useful tool with which to study the antigens potentially involved in disease. Multiple anti-streptococcal MAb reactivities observed in valve sections may be explained partially by previous evidence of dual specificity of the cross-reactive MAbs, such as MAb 36.2.2, which recognized streptococcal M protein, actin, laminin, cardiac myosin, and other a-helical coiled-coil proteins, such as keratin and

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atrialis of a normal mitral valve stained with sera from a patient with acute rheumatic fever (a) or a normnal individual (b). The intensity of immunoreactivit is approximately two times greater with rheumatic sera than with nornal. Photomicrographs, x500

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Figure 14. Western immunoblot ofextracted proteins from a normal human mitral valve. Valvular extracts (V) and molecular weight standards (S) were stained for protein with Amido black. Anti-streptococcal MAbs 362.2 (36), 49.8.9 (49), 54.2.8 (54), and 654.1.1 (654), and the anti-myosin MAb, CCM-52 (CCM), or anti-vimentin MAb (VIM) were reacted with solubilized valvuklr proteins. No reactivity is detected with the conjugate control (PBS) or with the IgM isotype control MAb

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tropomyosin.8-1224 Conversely, MAb 54.2.8 crossreacted with streptococcal M protein, myosin, and DNA.6924 As an a-helical molecule, streptococcal M protein exhibits significant sequential and structural homology with the myosin rod region and with tropomyosin.3710 These data are consistent with observed cross-reactivity of MAbs 36.2.2 and 54.2.8 with muscle components of the valve and with other valvular structures containing a-helical, coiled-coil proteins. The dual specificities of these anti-streptococcal MAbs implies their lower avidity and recognition of structural epitopes shared between a-helical proteins and the streptococcal M protein. These shared epitopes may not have an amino acid sequence identical to the defined M protein epitope (GLN-LYS-SER-LYS-GLN) shared with myosin or other a-helical molecules.11 Epitopes involved in cross-reactivity were found to be most likely conformational in nature, based on our peptide studies.11 The less well-defined specificity of MAb 49.8.9 may be explained by the recognition of highly conformational epitopes in tissues. Although MAb 49.8.9 has been less well characterized molecularly than the other MAbs studied herein, it was shown to react strongly with vimentin and it may be most useful for characterizing streptococcal-human heart valve cross-reactivity in tissue sections. The specificity of staining appeared greatest for MAb 49.8.9, because of minimal background reactivity. The reaction of MAb 49.8.9 with VICs in the valve, however, was certainly rea

markable. Valvular interstitial cells may play a significant role in the genesis of fibrotic injury in disease, based on the high numbers of these cells found in diseased valves and on studies suggesting their capability of elaborating extracellular matrix.23 The present study defined several sites of immunoreactivity for anti-streptococcal MAbs in normal and diseased human heart valve sections. Smooth muscle cells, in tight aggregates or loosely distributed in the spongiosa and thickened free margin, generally had the greatest reactivity of all valvular constituents. The MAbs also reacted with cardiac myocytes, endothelial cells, VICs, and elastic regions. While the degree of reactivity was somewhat different for each of the anti-streptococcal MAbs, the general patterns of cellular and extracellular staining were similar. Also binding patterns and intensities observed with acute rheumatic fever sera were similar to those seen with anti-streptococcal MAbs (Figure 13). Consistent with our immunohistochemical findings of anti-streptococcal MAb reactivity with myosin-containing smooth muscle cells and cardiac myocytes, myosin was also recognized in Western immunoblots by MAbs 36.2.2 and 54.2.8 (Figure 14). In addition, remarkably similar VIC staining patterns were seen when the anti-streptococcal MAbs were compared with the anti-vimentin MAb. Valvular vimentin was also recognized in Western immunoblots most strongly by MAb 49.8.9 and to a lesser degree by MAbs 36.2.2 and 54.2.8, underscoring the possibility of

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vimentin sharing an epitope(s) with the group A streptococcus. These data agree with recent findings of immunologic cross-reactivity between the streptococcal M protein (types 1 and 12) and renal glomerular mesangial vimentin.21 The data also agree with a previous finding suggesting recognition of fibroblast vimentin by MAb

54.2.8.9 Although the mechanisms of autoimmune injury involving vimentin are still unknown, anti-vimentin specificity has also been found in the context of other autoimmune disorders. Andre-Schwartz and colleagues41 developed IgM MAbs, from mice and humans with systemic lupus erythematosus, which recognized DNA and vimentin. Also an IgM MAb developed from splenocytes of an autoimmune thrombocytopenic purpura patient has recently been shown to recognize vimentin in platelets.42 Reactivity of the anti-streptococcal MAbs with elastic regions of valves suggested binding to elastin. Interestingly, high concentrations of purified, soluble elastin inhibited the binding of MAbs 36.2.2 and 54.2.8 to whole streptococci significantly, but failed to inhibit MAb 49.8.9 in competitive inhibition ELISAs (Figure 15). However, all three MAbs reacted with elastic regions in valve tissue sections, again suggesting that MAb 49.8.9 recognized conformational epitopes that were lost on solubilization of elastin. Reactivity of elastin was limited to a elastin, because p elastin did not inhibit the reaction of any of the MAbs. These data certainly suggest that valvular elastin may be an important autoantigen that cross-reacts with the group A streptococcus. Whereas the mechanisms leading to valvular injury in rheumatic heart disease remain an enigma, myosin or other proteins, such as laminin or vimentin, may be involved in immunologic cross-reactions with the streptococcal M protein.910'20'21 Considering that cardiac myosin is a potent autoantigen that, when injected into genetically predisposed mice, leads to fulminant autoimmune myocarditis,15 it may be of relevance that myosin is a fairly ubiquitous, contractile protein, found in muscle, epithelioid cells, fibroblasts, and endothelial cells.43'" Also, some have suggested the presence of myosin on the surface of cultured fibroblasts.43 Moreover laminin, a large glycoprotein, recognized by MAb 36.2.2 (Antone SM, Cunningham MW: Manuscript in preparation), is a major component of basal laminae, underlying all epithelia and surrounding individual muscle cells.' Interestingly laminin is an antigenically cross-reactive molecule between human heart and Trypanosoma cruzi, and is thought to play a role in the immunopathology of

Chagas' disease.46 Recent studies by Cunningham and colleagues have strengthened the argument in favor of a direct role of heart-reactive, antibody-mediated cytotoxicity in rheu-

matic heart disease.25 In these in vitro studies, incubations with low concentrations of MAb 36.2.2 lead to complement-mediated, target cell cytotoxicity in 51Cr-release assays. Cytotoxicity was unique to cardiac myocytes and fibroblasts, but not to hepatocytes. Importantly, identification of cell-surface, or cardiac valve, constituent(s) recognized by these cross-reactive MAbs may shed light on possible mechanisms of pathogenesis of valvular injury in rheumatic disease. The fact that anti-streptococcal MAbs bind valvular surface endothelium (Figures 5, 6) and immediate subendothelial structures, such as elastin microfibrils (Figures 2 to 4, 8, 10) and VICs (Figures 1 1, 12), may be of pathogenetic relevance. It is plausible that initial sites of inflammatory injury could result from autoantibodymediated endothelial cell destruction, exposing subendothelial structures and cells, such as smooth muscle cells and VICs, potentially capable of responding to such injury by aberrant proliferation and elaboration of connective tissue elements. Electron microscopic studies of cardiac valves damaged by chronic rheumatic disease demonstrate consistent loss of the integrity of valvular surface endothelium with exposure of subendothelial basement membrane, collagen, and elastin.3 Whether the critical factor in the pathogenesis of rheumatic heart valve injury is one of initiation or of response to injury is unresolved. Our findings, however, present some important clues for understanding the potential of heartcross-reactive streptococcal antibodies in valvular injury and subsequent development of rheumatic heart disease.

Acknowledgments The authors thank Dr. Yuling Ye, visiting scientist, University of Nebraska Medical Center, for technical assistance, and Carol Crossley, technician, University of Oklahoma Health Sciences Center. They also thank Dr. William A. Clark, Jr. for supplying monoclonal antibody CCM-52, Drs. L. T. Chun and D. V. Reddy for supplying acute rheumatic fever sera, and Michelle Williams for clerical help.

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with human heart tissue. J Immunol 1963, 90:595-606 2. Kaplan MH, Bolande R, Rakita L, Blair J: Presence of bound immunoglobulins and complement in the myocardium in acute rheumatic fever. Association with cardiac failure. N

Engl J Med 1964, 271:637-645 3. Kaplan MH: Cross-reaction of group A streptococci and

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heart tissue: Varying serologic specificity of cross-reactive antisera and relation to carrier-hapten specificity. Transplant Proc 1969, 1 (suppl 4):976-980 4. Zabriskie JB, Freimer EH: An immunological relationship between the group A streptococcus and mammalian muscle. J Exp Med 1966,124:661-678 5. Zabriskie JB: The relationship of streptococcal crossreactive antigens to rheumatic fever. Transplant Proc 1969, 1:968-975 6. Dale JB, Beachey EH: Protective antigenic determinants of streptococcal M protein shared with sarcolemmal membrane protein of human heart. J Exp Med 1982, 156:11651176 7. Dale JB, Beachey EH: Epitopes of streptococcal M proteins shared with cardiac myosin. J Exp Med 1985,162:583-591 8. Cunningham MW, Hall NK, Krisher KK, Spanier AM: A study of anti-group A streptococcal monoclonal antibodies crossreactive with myosin. J Immunol 1986, 136:293-298 9. Cunningham MW, Swerlick RA: Polyspecificity of antistreptococcal murine monoclonal antibodies and their implications in autoimmunity. J Exp Med 1986, 164:998-1012 10. Krisher K, Cunningham MW: Myosin: A link between streptococci and heart. Science 1985, 227:413-415 11. Cunningham MW, McCormack JM, Fenderson PG, Ho M, Beachey EH, Dale JB: Human and murine antibodies crossreactive with streptococcal M protein and myosin recognize the sequence GLN-LYS-SER-LYS-GLN in M protein. J Immunol 1989,143:2677-2683 12. Fenderson PG, Fischetti VA, Cunningham MW: Tropomyosin shares immunologic epitopes with group A streptococcal M proteins. J Immunol 1989,142:2475-2481 13. Cunningham MW, McCormack JM, Talaber LR, Harley JB, Ayoub EM, Muneer RS, Chun LT, Venu Reddy D: Human monoclonal antibodies reactive with antigens of the group A streptococcus and human heart. J Immunol 1988,

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Immunoreactivity of anti-streptococcal monoclonal antibodies to human heart valves. Evidence for multiple cross-reactive epitopes.

Association of group A streptococci with acute rheumatic fever and valvular heart disease is well established; however the basis of valve injury remai...
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