Molecular Analysis of Polyreactive Monoclonal Antibodies from Rheumatic Carditis: Human Anti-N-Acetylglucosamine/Anti-Myosin Antibody V Region Genes

Elisabeth E. Adderson, Alexander R. Shikhman, Kent E. Ward and Madeleine W. Cunningham
J Immunol August 15, 1998, 161 (4) 2020-2031;

Abstract

Anti-myosin Abs are associated with inflammatory heart diseases such as rheumatic carditis and myocarditis. In this study, human cross-reactive anti-streptococcal/anti-myosin mAbs 1.C8, 1.H9, 5.G3, and 3.B6, produced from peripheral blood lymphocytes of patients with rheumatic carditis, and mAb 10.2.5, produced from a tonsil, were characterized, and the nucleotide sequences of their VH and VL genes were analyzed. Human mAbs 1.C8, 1.H9, 10.2.5, and 3.B6 reacted with human cardiac myosin while mAb 5.G3 did not. The mAbs were strongly reactive with N-acetyl-β-d-glucosamine, the dominant epitope of the group A streptococcal carbohydrate. mAb 1.H9 was moderately cytotoxic to rat heart cells in vitro in the presence of complement. The anti-myosin mAbs from rheumatic carditis were found to react with specific peptides from the light meromyosin region of the human cardiac myosin molecule. Anti-streptococcal/anti-myosin mAbs from normal individuals reacted with distinctly different light meromyosin peptides. The mAbs were encoded by VH3 gene segments V3-8, V3-23, and V3-30 and by the VH4 gene segment V4-59. The variable region genes encoding the anti-streptococcal/anti-myosin repertoire were heterogeneous and exhibited little evidence of Ag-driven somatic mutation.

Polyreactive autoantibody responses against streptococci, myosin, and other α-helical proteins are characteristically found in streptococcal diseases and sequelae such as acute rheumatic fever (ARF)3 (1, 2, 3, 4). Abs that recognize Ags shared between Streptococcus pyogenes and heart tissues may play a role in the autoimmune cardiac injury associated with ARF. Previous studies of murine cross-reactive anti-myosin/anti-streptococcal Abs have classified the Abs into three groups. The groups include: 1) those anti-streptococcal/anti-myosin mAbs reacting with α-helical molecules including myosin, tropomyosin, and vimentin (5, 6); 2) anti-nuclear Abs (5); and 3) mAbs that react with myosin and the carbohydrate N-acetylglucosamine (7, 8, 9). In humans with ARF, anti-nuclear Abs are not characteristically found in the serum. Furthermore, human anti-streptococcal/anti-myosin mAbs produced from ARF do not have anti-nuclear reactivity but demonstrate strong reactivity with the group A carbohydrate epitope, N-acetylglucosamine, as shown in this report. In ARF Abs and complement were shown to be deposited in hearts of patients who died from the disease. In our studies, murine anti-streptococcal/anti-myosin mAb 36.2.2 was shown to be cytotoxic and to react with extracellular molecules on the surface of heart cells (10). Therefore, anti-myosin Abs capable of recognizing surface molecules may play a role in development of disease manifestations observed in ARF. Only one of the human mAbs reported herein was cytotoxic for heart cells in culture. Cytotoxic Abs have the potential for deposition in tissues and initiation of inflammation.

Anti-myosin Abs have also been associated with chronic autoimmune heart disease such as myocarditis (11, 12). It was reported that anti-myosin Abs were present in animals developing coxsackieviral-induced or cardiac myosin-induced myocarditis (11, 12, 13, 14, 15). Studies have shown that coxsackievirus, streptococcal M protein, and cardiac myosin share epitopes that contribute to antigenic cross-reactivity as well as disease production (15, 16, 17, 18). Cardiac myosin was shown to play a major role in myosin-induced myocarditis models such as the A/J mouse strain (19, 20). In this model, myocarditis was dependent on the presence of CD4+ lymphocytes (20). However, in the DBA/2 mouse model of myocarditis, it was shown that passive administration of mouse IgG anti-myosin mAb produced myocarditis and concomitant deposition of the Ab (13). Ab deposition in the extracellular matrix of the cardiac tissues was explained by the expression of a 200-kDa molecule in the susceptible tissue. The expression of myosin or a myosin-like molecule was believed to be the susceptibility factor in the DBA/2 animals. Other animals not expressing the molecule did not develop myocarditis after passive administration of the mouse anti-myosin Ab (13).

In this report, we identify the autoantigens recognized by the human anti-myosin Abs from patients with rheumatic carditis and correlate this information with the nucleotide sequences utilized for their Ig genes. We recently reported that an anti-streptococcal/anti-myosin cross-reactive human mAb (10.2.3) derived from normal human tonsil was encoded by V3-23 VH and L9 Vκ variable segments (21) and that cytotoxic anti-streptococcal/anti-myosin mouse mAb 36.2.2 was encoded by VH7183 and Vκ8 (10). In our report, human IgM anti-streptococcal/anti-myosin mAbs 1.C8, 1.H9, 5.G3, 3.B6, and 10.2.5 are shown to react strongly with the N-acetylglucosamine determinant of the group A streptococcal carbohydrate, with human cardiac myosin, other related self Ags, and streptococcal M proteins. Elevated reactivity of ARF sera with the group A carbohydrate has been reported to be associated with persistent valvular heart disease (22). The human anti-streptococcal mAbs 1.C8, 1.H9, and 5.G3 have been characterized for their reactivity with keratin and keratin peptides, a feature of Abs reactive with N-acetylglucosamine (8). The nucleotide sequences of their Ig VH and VL region genes now reveal that the five mAbs analyzed are encoded by a heterogeneous group of preferentially expressed VH and VL genes. Rearranged Ig VH and Ig VL genes exhibit little evidence of Ag-driven somatic mutation irrespective of germline origin and pathogenic potential.

Materials and Methods

Patients

Patients were treated in the department of pediatric cardiology at the University of Oklahoma Children’s Heart Center, and ARF was diagnosed on the basis of the Jones criteria. All patients had carditis with heart murmur, and in one instance a patient had both carditis and arthritis. In addition, serology revealed elevated anti-streptolysin O titers of ≥600 in all cases. Two of the hybridomas, 1.C8 and 1.H9, were generated from the same patient, while hybridomas 5.G3 and 3.B6 were derived from two other patients. The hybridoma secreting mAb 10.2.5 was derived from a tonsil of a normal individual.

Human hybridomas

Human hybridomas utilized in this study were produced as described by Shikhman and Cunningham (2, 3, 8). Human PBL were isolated using Histopaque-1077 Hybri-Max (Sigma, St. Louis, MO) and were stimulated for 1 week at 37°C with streptococcal peptidoglycan-polysaccharide complexes (10 μg/ml) (mAbs 1.C8, 1.H9, 5.G3, and 3.B6) as previously described (8) or with streptococcal M5 membranes (mAb 10.2.5) (3). After 1 week, Iscove’s modified Dulbecco’s medium (IMDM) containing the peptidoglycan-polysaccharide complexes and 10% human AB serum was replaced with IMDM containing 10% serum and pokeweed mitogen (1 μg/ml). The cells were stimulated for an additional week. Cells were then collected by centrifugation, washed three times in IMDM without serum, and fused by standard methods as described with either HMMA2.11TG/O, a rat-human heterohybridoma purchased from Dr. M. R. Posner, Brown University, Providence, RI, or with WI-L2-729HF2, a human lymphoblastoid cell line purchased from Dr. R. Lundak, Techniclone International, Santa Ana, CA (3, 8). Hybridomas were selected on the basis of reactivity with group A streptococci, myosin, and N-acetylglucosamine-BSA with no reactivity with BSA as previously described (7, 8). Positive hybridomas were cloned three times by limiting dilution in 96-well tissue culture plates. Hybridomas were grown in IMDM plus 20% FBS.

Synthetic peptides

The light meromyosin (LMM) tail region of the human cardiac myosin heavy chain was synthesized in overlapping 18-mer peptides with a 5-amino acid overlap. The sequence of human cardiac myosin β-chain was taken from the sequence published by Vosberg et al. (23). In some cases, the peptides were synthesized longer or shorter to facilitate purification of the peptide by HPLC or to prevent cyclization. All peptides were chemically synthesized on a Dupont RAMPS manual synthesizer using the f-moc strategy (24) and purified by HPLC. The synthetic LMM peptides have been reported previously (21), and their amino acid sequences are shown in Results. The synthetic peptides were tested for reactivity with the human mAbs in immunodot blots as previously described (3).

Enzyme-linked immunosorbent assay

A variety of streptococcal and self Ags were tested against the mAbs in the ELISA. Purified human cardiac myosin was prepared according to a procedure described previously (25). Skeletal myosin from rabbit muscle, tropomyosin from rabbit muscle, vimentin from bovine lens, LMM from rabbit muscle, heavy meromyosin from rabbit muscle, actin from rabbit muscle, keratin from human epidermis, mouse laminin, BSA, ss-DNA and ds-DNA were obtained from Sigma. Synthetic polynucleotides were obtained from Pharmacia (Piscataway, NJ), and elastin from bovine neck ligament was obtained from Elastin Products. Streptococcal recombinant M5 and M6 proteins were gifts from Dr. James B. Dale (VA Medical Center, Memphis, TN) and Dr. Vincent A. Fischetti (Rockefeller University, New York, NY). Phosphorylcholine conjugated with BSA was provided by Dr. David Briles (University of Alabama, Birmingham, AL). N-Acetyl-β-d-glucosamine (GlcNAc) conjugated to BSA was produced by a two-step reaction as previously described (14). Ags were tested at a concentration of 10 μg/ml in 0.1 M sodium carbonate buffer, pH 9.0, adsorbed to 96-well Immulon-4 microtiter plates (Dynatech, Alexandria, VA). The plates were coated with Ag overnight at 4°C and then blocked with 1% BSA in PBS containing 0.5% Tween 20. The ELISA was performed according to standard protocol. The secondary Ab was peroxidase labeled goat anti-human IgM. H2O2 and o-phenylenediamine (Sigma) were added finally as the peroxidase substrate to detect Ab binding. Results were calculated from triplicate measurements. Assays were performed several times with the human mAbs in the ELISA.

Western immunoblots

Human cardiac myosin as well as other proteins were separated by discontinuous SDS-PAGE by the method described by Laemmli (26). The stacking gel contained 4% acrylamide and the separating gel contained 7.5% acrylamide. Proteins were separated by SDS-PAGE electrophoresis using the SE 280 11-cm vertical slab gel unit (Hoeffer Scientific Instruments, San Francisco, CA) with 1.5-mm-thick gel. After electrophoresis, the gel was overlayed with a sheet of Immobilon-NC (Millipore, Bedford, MA), and the proteins were transferred overnight at 50 mA current in the transfer unit TE22 (Hoeffer Scientific Instruments). Efficiency of the protein transfer was determined by the transfer of prestained m.w. standards (Sigma). In addition, a portion of the blot was stained with amido black, and the transferred bands were observed after destaining. The blot was blocked with PBS containing 2% Tween and 2% BSA and was then washed and subsequently incubated with the human mAbs overnight at 4°C. After washing, a peroxidase-labeled goat anti-human IgM (Kierkegaard and Perry Laboratories, Gaithersburg, MD) was incubated with the blot, and after washing a TMB membrane substrate system (Kierkegaard and Perry) was used to develop the blots.

51Cr release assay for cytotoxicity

A primary rat heart cell line (ATCC-CRL-1446) was cultured overnight in sterile 96-well microtiter plates in IMDM containing 20% FBS. Cells were cultured initially at 1 × 104 cells/well at 37°C and 5% CO2. The cells were labeled with Na251CrO4 (Dupont) at 5 μCi/well (1 Ci = 37 GBq) for 2 h at 37°C. Culture medium was removed and the attached cells were washed 3 times with IMDM plus 20% fetal bovine serum and incubated for 1 h in IMDM with serum. Ab was added to the cells at 50 μl/well for 1 h at 37°C. An equal volume of guinea pig complement (Whittaker Bioproducts, Walkersville, MD) was added, and the mixture was incubated for 1 h at 37°C. Supernatant fluid was harvested from the 96-well plate using a Skatron harvester system (Sterling, VA) and 51Cr release was measured in an LKB gamma-counter (LKB-1282 Compugamma; LKB Instruments, Gaithersburg, MD). Minimum lysis was calculated from measurements of supernatants from cells treated with culture medium alone, and 100% lysis was calculated from measurements of supernatants taken from cells treated with 1 M HCl for maximum 51Cr release. Percent lysis was determined by calculating [(test sample release − minimum release)/(maximum release − minimum release)] × 100. Tests were performed in quadruplicate, and the average and SD determined for each sample tested.

Cloning and sequencing of Ig variable region genes

Ig variable region genes were cloned and sequenced following amplification by PCR. Total RNA was prepared from 106 to 108 hybridoma cells by the phenol-guanidine isothiocyanate method (TRIzol Reagent; Life Technologies, Gaithersburg, MD) according to the manufacturer’s protocol. A 5-μg sample of total RNA was primed for first-strand synthesis with 10 pmol of an antisense human JH or antisense human Cκ or Cλ primer as previously described, or by using commercially available human Cμ and Cλ primers (Ig-Prime; Novagen, Madison, WI) (27, 28). First-strand cDNA synthesis was performed with recombinant Moloney murine leukemia virus reverse transcriptase (Superscript II; Life Technologies) using the manufacturer’s protocol, except that DMSP (6%) was added to each reaction. Variable region genes were amplified as previously described, using sense primers corresponding to each human heavy chain variable region (VH) family leader sequence or degenerate primers homologous to κ or λ variable region (Vκ/Vλ) leader sequences and antisense JH or internal Cκ/Cλ primers, respectively (27, 28).

Amplification products were ligated into pBS phagemid vectors (Stratagene, La Jolla, CA) using standard protocols and both strands of inserts sequenced by the dideoxy technique (29). A minimum of two identical sequences from two different PCR reactions were obtained for all variable region genes except for the 1.H9 Vλ segment. Variable region gene sequences were compared with those entered into the GenBank database.

Results

Characterization of the Ag-binding specificities of the human cross-reactive anti-streptococcal mAbs

Human mAbs 1.C8, 1.H9, 5.G3, and 3.B6, derived from patients at the onset of ARF, and human mAb 10.2.5, derived from a human tonsil, reacted with N-acetylglucosamine, a dominant epitope of the group A carbohydrate, and with α-helical structures such as myosin, keratin, or streptococcal M protein (Fig. 1⇓). Only mAb 3.B6 reacted with tropomyosin (Fig. 1⇓D). Western blots demonstrated the strong reactivity of mAbs 1.C8, 1.H9, 3.B6, and 10.2.5 with purified human cardiac myosin in the Western blot, while mAb 5.G3 did not react with purified human cardiac myosin in the Western blot. Figure 2⇓ shows the reactivity of mAbs 1.C8 and 1.H9 with purified human cardiac myosin in the Western blot. mAbs 3.B6 and 10.2.5 reacted similarly with human cardiac myosin heavy chain in the immunoblots (data not shown). Differences in reactivity of the two mAbs 1.C8 and 1.H9 was observed when the Abs were compared for reactivity with vimentin in the Western blot as shown in Figure 3⇓ and with keratin and human skin as previously reported (8). mAb 1.C8 reacted with vimentin in the Western blot (Fig. 3⇓) and also with tissue sections whereas mAb 1.H9 did not (8). Despite these differences, mAbs 1.C8 and 1.H9 reacted strongly with human cardiac myosin in Western blots, and the specificity of the two mAbs for human cardiac LMM synthetic peptides was nearly identical in an immunodot blot (Table I⇓). Rheumatic carditis mAbs 1.C8 and 1.H9 reacted most strongly with LMM 21, 28, 33, and 46, and rheumatic carditis mAb 3.B6 reacted strongly with LMM 1 and 33. All three rheumatic carditis mAbs reacted with LMM peptide 33. However, mAb 10.2.5 from a normal tonsil reacted most strongly with LMM 10, 23, 27, and 30. Two other mAbs produced from a streptococcal carrier reacted with the same peptides recognized by the mAb 10.2.5. Although this is a small number of mAbs to compare, the data suggest that the peptides recognized by the Abs from rheumatic heart disease were distinctly different from those mAbs isolated from normal individuals. Among the different peptides binding a single Ab, none contained identities in amino acid sequence. However, structural or conformational similarities may exist among the peptides due to positions of hydrophobic and hydrophilic amino acid residues characteristic of α helices. The conjugate control was negative with all of the LMM peptides in the immunoassay. Differences in reactivity of mAbs 1.C8 and 1.H9 were also observed in their abilities to bind to GlcNAc:BSA conjugates containing different numbers of GlcNAc residues conjugated to BSA. Figure 4⇓ shows that mAb 1.H9 was more efficient at binding lower numbers of GlcNAc molecules on BSA than 1.C8. In additional studies, the 51Cr release assay was used to determine whether any of the human mAbs were cytotoxic for heart cells. mAb 1.H9 was moderately cytotoxic for rat heart cells in culture in the presence of complement, as shown in Figure 5⇓. The other human mAbs were not cytotoxic for rat heart cells.

FIGURE 1.
FIGURE 1.

Ag binding profiles of human mAbs 1.C8, 1.H9, 5.G3, 3.B6, and 10.2.5 are shown in A through E, respectively. The Abs were tested (10 μg/ml) in the ELISA against Ags coated onto microtiter plates at 10 μg/ml. Optical density at 405 nm is shown in the bar graph representing the reactivities of the mAbs. PC, phosphorylcholine; HMM heavy meromyosin.

FIGURE 2.
FIGURE 2.

Reaction of human mAbs 1.C8, 1.H9, and 5.G3 with purified human cardiac myosin (200 kDa) in the Western immunoblot. Both mAbs 1.C8 and 1.H9 (10 μg/ml) reacted strongly with myosin, while mAb 5.G3 did not react (data not shown). An IgM isotype Ab control and conjugate control (C) were also negative. S shows the stained band of human cardiac myosin heavy chain.

FIGURE 3.
FIGURE 3.

Reaction of human mAb 1.C8 with vimentin in the Western blot. mAbs 1.H9 and 5.G3 were not reactive with vimentin in the blot. Abs (IgM) were reacted with the blot at 10 μg/ml. The Brilliant Blue G stain (S) of vimentin demonstrates a 57-kDa protein. The conjugate control (C) as shown is negative.

FIGURE 4.
FIGURE 4.

Binding of human mAbs 1.C8 and 1.H9 to GlcNAc-BSA conjugates with varied GlcNAc density in the ELISA. mAb 1.H9 (A) reacted with lower epitope densities of GlcNAc residues on BSA, suggesting a higher binding efficiency for GlcNAc than mAb 1.C8. In B, mAb 1.C8 reacted strongly with GlcNAc-BSA conjugates 50:1 and 20:1 with 100% binding, while lower binding was observed with GlcNac-BSA conjugates containing GlcNAc densities of 10:1, 5:1, and 1:1. Optical densities at 405 nm for 100% binding were 1.0. The 50:1 conjugate was considered 100% at an optical density of 1.0. mAb 1.C8 reacted at <30% binding to lower GlcNAc epitope densities (B). In A, 1.H9 is shown to react with 100% binding to the 50:1 and 20:1 conjugates and to also bind with 40 to 65% efficiency to 10:1 and 5:1 conjugates.

FIGURE 5.
FIGURE 5.

Cytotoxicity of mAb 1.H9 in chromium release assays on rat heart cells with complement. The graph shows the cytotoxicity of human mAbs 1.H9, 1.C8, 5.G3, 3.B6, 10.2.5, and positive control mouse mAb 36.2.2 (+Control), an anti-streptococcal/anti-myosin mAb known to be highly cytotoxic for rat heart cells. Error bars are shown for the average of quadruplicate assays.

View this table:
Table I.

Reaction of human anti-myosin mAbs 1.C8, 1.H9, 3.B6, and 10.2.5 with overlapping synthetic peptides of human cardiac LMM in immunodot blot

View this table:
Table II.

Cross-reactive anti-myosin anti-streptococcal VH mAbs

View this table:
Table III.

Cross-reactive anti-myosin anti-streptococcal VL mAbs

Variable region genes encoding cross-reactive anti-streptococcal/anti-myosin mAbs

Germline genes most closely homologous to those encoding mAb IgVH and IgVL and the nucleic acid and amino acid sequence homology to these genes are noted in Tables II and III. In Figure 6⇓A, the nucleotide sequence and the translated amino acid sequence of the mAb 3.B6 heavy chain variable region gene is shown. The sequence is identical with the germline 4–59 VH, DXP′1, and JH4 genes (30, 31). In Figure 6⇓B, the nucleotide sequence and the translated amino acid sequence of the mAb 3.B6 light chain variable region gene is shown. The sequence is compared with the germline A27 and Jκ3 (32).

FIGURE 6.
FIGURE 6.

A, Nucleotide sequence and translated amino acid sequence of the mAb 3.B6 heavy chain variable region gene. The sequence is compared with the germline 4-59 VH, DXP′1, and JH4 genes (30, 31). Data are listed with the GenBank database under accession number U96389. B, Nucleotide sequence and translated amino acid sequence of the mAb 3.B6 light chain variable region gene. The sequence is compared with the germline A27 and Jκ3 (32). Data are listed with the GenBank database under accession number U96390.

mAb 5.G3 VH is 99% homologous to the VH3-8 germline segment obtained from a patient with systemic lupus erythematosus and is 99% homologous to rearranged VH segments cloned from cord blood (N1p1) and from a patient with X-linked agammaglobulinemia (LE-315) (Fig. 7⇓A) (33, 34, 35). Recently, two laboratories have cloned the entire VH locus (30, 36). A germline VH segment identical with VH3-8 was not identified in these studies, but the VH3-8 germline gene and each of these rearranged genes are also 97% homologous to the V3-11 (DP-35/22-2B). VH3-8 may, therefore, represent an allele of V3-11. Alternatively, like certain other closely related VH segments that appear to have arisen through duplication events (notably V3-30, V3-30b, and V30.3), VH3-8 may be located on an insertion polymorphism present in only certain haplotypes (36, 37). In Figure⇑ 7B, the nucleotide sequence and the translated amino acid sequence of the mAb 5.G3 light chain variable region gene is shown. The sequence is compared with the germline 012/02 VH and Jκ4 (38).

FIGURE 7.
FIGURE 7.

A, Nucleotide sequence and translated amino acid sequence of the mAb 5.G3 heavy chain variable region gene. The sequence is compared with the germline VH3-8, DXP′1, and JH6 genes (31, 35). Data are listed with the GenBank database under accession number U96395. B, Nucleotide sequence and translated amino acid sequence of the mAb 5.G3 light chain variable region gene. The sequence is compared with the germline 012/02 VH and Jκ4 (38). Data are listed with the GenBank database under accession number U96396.

Both mAbs 1.H9 and 1.C8, obtained from the same subject, have VH segments highly homologous to the V3-30 germline gene and have identical VDJ joints (Fig. 8⇓A) (30). The 1.C8 Vλ segment is 98% homologous to the DPL11 germline Vλ gene segment and uses a Jλ2 or 3 gene (Fig. 8⇓B) (39, 40). A total of 12 clones from 4 amplification reactions were obtained for the 1.H9 Vλ gene, the variable segment of which shared 99% homology with the germline DPL3 gene (39). In each clone, we observed a single base pair deletion, resulting in a frame shift at the VJ joint (Fig. 8⇓B). This deletion was present on sequencing both sense and antisense strands and with alternative sequencing methods (PCR). No other λ rearrangement was detected in any amplification reaction. Unfortunately, this cell line was unstable, and we were unable to examine heavy or light chain rearrangements by Southern blotting (data not shown). It is not clear, therefore, whether this transcript represents the functional rearrangement with an unusual sequencing artifact, whether it is a highly expressed nonproductive rearrangement, or whether this deletion may represent an acquired mutation in the 1.H9 cell line (which might also affect the stability of the line).

FIGURE 8.
FIGURE 8.

A, Nucleotide sequences and translated amino acid sequences of the mAb 1.C8 and 1.H9 heavy chain variable region genes. The sequence is compared with the germline 3-30 VH, DXP′4, and JH4 genes (30, 31). Data are listed with the GenBank database under accession numbers U96392 and U96391, respectively. B, Nucleotide sequence and translated amino acid sequence of the mAb 1.C8 light chain variable region gene. The sequence is compared with the germline DPL11 and Jλ2/3 (39, 40). Data are listed with the GenBank database under accession number U96393. C, nucleotide sequence and translated amino acid sequence of the mAb 1.H9 light chain variable region gene. The sequence is compared with the germline DPL3 and Jλ2/3 (39, 40). Data are listed with the GenBank database under accession number U96394.

The 10.2.5 mAb has minor differences in Ag binding from the previously described mAb 10.2.3 (21). Both mAb are encoded by identical Ig VH, with variable segments 99% homologous to the V3-23 germline VH segment (30, 36). Both IgVκ genes are encoded by one of the duplicated L9 germline Vκ segments and Jκ1 genes; however, the 10.2.5 Vκ segment has three nucleotide differences from the 10.2.3 gene, including a single coding change in complement-determining region (CDR) 2 that may be responsible for the very subtle changes in reactivity between the two mAbs (Fig. 9⇓) (21).

FIGURE 9.
FIGURE 9.

Nucleotide sequence and translated amino acid sequence of the mAb 10.2.5 light chain variable region gene. The sequence is compared with the germline L9 and Jκ1 (67). Also shown is the light chain variable region gene of the clonally related mAb 10.2.3. Data are listed with the GenBank database under accession number U96397.

Discussion

It is well known that Abs deposit in hearts of patients with rheumatic carditis (41), and studies have shown that myosin is an important target Ag recognized by anti-streptococcal Abs in ARF (2, 3). Furthermore, in our studies we have found that ARF sera contains both IgM and IgG anti-myosin Abs at levels of ∼50 μg/ml IgM and 300 μg/ml IgG, while sera from normal subjects and uncomplicated streptococcal infection contain ∼12 μg/ml IgM and IgG anti-myosin Ab as acertained from human cardiac myosin affinity column calculations (M. W. Cunningham, unpublished data). In this report, we investigated anti-streptococcal/anti-myosin mAbs, produced from patients with rheumatic carditis, for their reactivity and specificity for various streptococcal and tissue Ags, myosins, and LMM peptides. In addition, we analyzed the nucleotide sequence of their variable region VH and VL genes.

All of the human anti-streptococcal/anti-myosin mAbs were strikingly reactive with the group A streptococcal carbohydrate epitope N-acetylglucosamine. This is an important feature since elevated and persistent levels of Abs against group A streptococcal carbohydrate were previously reported in cases of chronic rheumatic valvulitis (22). In addition, the mAbs were highly reactive with myosin and its fragments as well as other α-helical coiled-coil proteins. The mAbs did not react with DNA as might be expected, since patients with ARF do not characteristically develop antinuclear Abs. The most interesting mAb was 1.H9 which demonstrated moderate cytotoxic activity for rat heart cells in the presence of complement. These Abs were polyreactive with nonidentical peptide structures of human cardiac myosin and the carbohydrate N-acetylglucosamine. Although the peptides contained no common sequences, the Ab combining site may accommodate these Ags due to its flexibility. Recently, it was proposed that a germline antibody may be polyreactive due to conformational rearrangement and configurational change, permitting binding of diverse molecules such as carbohydrates and peptides (42).

Human anti-streptococcal/anti-myosin Abs were encoded by a heterogeneous population of Ig variable region genes. This repertoire, however, was striking in that it was encoded by a group of variable region gene segments, including V3-23, V4-59, V3-11, and V3-30, which are preferentially expressed beginning in early life, and in protective and autoimmune responses (34, 43, 44, 45, 46).

DXP family members are highly represented among the variable region D genes in the human anti-streptococcal mAbs. In the adult repertoire, the DXP family is used almost twice as frequently as expected based on its germline complexity (47). Two of the mAbs, 5.G3 and 3.B6, could not be assigned. The mAb 5.G3 D segment shares some homology with DXP′1, and the short D segment of mAb 3.B6 shares homology with an inverted DIR2 element; however, the germline origin of these D segments is not certain (47, 48). In the case of mAb 5.G3, which has a relatively long CDR-3, the VDJ may have resulted from a complex recombination event such as D-D fusion or inversion. Three of five of the anti-streptococcal mAbs were encoded by JH4, and none by the infrequently rearranged JH1 and JH2 germline joining segments. In all human developmental stages, the JH4 segment is markedly overutilized, encoding 45 to 65% of heavy chain rearrangements (47, 49). The mean length of the anti-streptococcal mAb CDR-3 was 44 nucleotides, with a range of 27 to 63 nucleotides, and in each gene in which the germline D segment is identifiable, potential “N” and “P” segments are present. The molecular characteristics of these cross-reactive anti-streptococcal Ig VH are more like the Ig VH of the normal adult repertoire than the early human Ab repertoire.

Considerably less is known about human light chain expression than that of heavy chains, but in mice Vκ gene usage appears nonstochastic (50, 51). A recently reported compilation of expressed human Vκ genes also suggests that some Vκ segments, conspicuously A27 and 012/02, are highly expressed in random libraries and in protective and autoimmune responses (52). Vκ genes for A27- and 012/02-encoded VL chains for mAbs 3.B6 and 5.G3, respectively.

The early Ab repertoire is characterized by striking poly- and autoreactivity (53). Like the fetal repertoire, the variable region genes that we have identified in the human cross-reactive anti-streptococcal/anti-myosin repertoire were encoded by a small group of preferentially expressed germline genes. Each of these segments also encodes other autoantibodies, such as anti-DNA, RF, anti-i, and others (54, 55, 56). This report of cross-reactive Abs directed against both foreign and self Ags and studies of other autoimmune and physiologic immune responses suggest that ligand binding may be an important force shaping the expressed Ab repertoire (2, 21, 57, 58). Ab molecules encoded by preferentially expressed germline genes may have the potential to reconfigure and bind to a wide array of three-dimensionally similar ligands (“molecular mimicry”) such as streptococcal M protein, myosin, other α-helical proteins, and carbohydrate epitopes (7, 8, 9, 59).

A disadvantage of a polyreactive repertoire is the potential for the emergence of autoimmune disease. Despite extensive study, the relationship among variable region gene usage, “natural” polyreactive Abs, and pathogenic autoantibodies is unclear. Conventionally, “natural” autoreactive Abs are of the IgM isotype, are polyspecific, have low affinity interactions with Ag, and are encoded by variable region genes identical with or very close to germline configurations, whereas Abs implicated in the pathogenesis of autoimmune disease are of the IgG or IgA isotype, are monospecific, have high affinity, and have evidence of Ag-driven somatic mutation. The anti-streptococcal/anti-myosin mAbs reported herein are of the IgM isotype and are encoded by variable region genes closely homologous to or identical with germline sequences. Overall R:S ratios range from 0:0 to 10:5, CDR R:S ratios range from 0:0 to 8:5, and FR R:S ratios range from 0:0 to 2:1. Only genes encoding mAbs 1.C8 and 1.H9 show significant mutation, and only the mAb1.C8 Vλ segment has some evidence suggestive of Ag selection, with an overall R:S ratio of 4:1 (but CDR and framework ratios 2:0 and 2:1, respectively).

Numerous exceptions to generalizations about pathogenic and natural autoantibodies have been reported. Low affinity autoantibodies may be pathogenic, and Ag-selected responses need not demonstrate the molecular characteristics of Ag-driven selection (60). It is possible that the IgM anti-myosin Abs reported from rheumatic carditis could be part of the natural autoantibody population and quite distinct from those involved in disease. However, in Table I⇑, the mAbs 1.C8, 1.H9, and 3.B6 from rheumatic carditis patients demonstrated a distinctly different peptide binding pattern from mAb 10.2.5 and other normal anti-myosin mAbs, which all reacted with the same set of peptides. In this study, mAb 1.H9 appeared to be cytotoxic for rat heart cells in culture. There were no obvious features other than heavy-light chain pairing to distinguish this Ab from the noncytotoxic Abs. We have also characterized a murine anti-streptococcal/anti-myosin mAb (36.2.2), which like the human mAbs described here reacts with myosin and other structurally similar proteins and is cytotoxic to rat heart cells (3, 16). The IgVH of the murine mAb 36.2.2 is encoded by a member of the preferentially expressed 7183 VH family and is highly homologous to genes encoding protective and anti-carbohydrate immune responses (10). The development of anti-myosin Abs recognizing cell surface molecules may be important in the development of disease pathogenesis.

Autoimmune cardiac injury in most animal models occurs by a more complex mechanism than direct Ab-mediated cytotoxicity. Autoimmune myocarditis models induced by cardiac myosin immunization or by coxsackieviral infection have been shown to be dependent on T lymphocytes (20, 61, 62). In mice, myocarditis is strain specific, suggesting that genetic factors influence susceptibility to disease (63). In ARF, the relatively low frequency of disease in individuals exposed to rheumatogenic strains of S. pyogenes and the increased prevalence of ARF in related individuals support the hypothesis that genetic susceptibility plays a role in development of the disease (64, 65, 66). The deposition of Ab and complement in the heart in ARF supports a pathogenic role for Ab in the disease. Passively administered anti-myosin (IgG) Abs were shown to deposit in the hearts of DBA/2 mice which then developed cardiac lesions (13). Only DBA/2 mice expressed a 200-kDa protein in the extracellular matrix of the heart, which was suggested to bind the anti-myosin Ab and to deposit in hearts in the susceptible strain. Anti-myosin Abs of the IgM isotype did not deposit or produce cardiac lesions in the heart tissues of the mice. Ab deposition in the heart may depend on the IgG isotype. Autoantibodies like 1.H9 could potentially bind to epitopes in tissues of susceptible individuals. What is most clear from the studies relating gene expression to normal and pathogenic Ab repertoires is that given the great potential and cross-reactivity between protective and autoimmune Ab repertoires, the most important contributors to Ab-mediated autoimmune disease must be the inability to delete or suppress the expression of pathogenic Abs and the presence of the antigenic epitope in the target tissues. In the absence of the appropriate development of tolerance or clonal deletion, humoral immune responses elicited by streptococcal infection may have the potential to precipitate or exacerbate autoimmune cardiac disease in susceptible individuals.

Acknowledgments

We thank Dr. Kenneth Jackson and the W. K. Warren Molecular Biology Resource Facility at the University of Oklahoma Health Sciences Center for synthesis and purification of the LMM peptides and Drs. Vincent A. Fischetti and James B. Dale for recombinant streptococcal M proteins.

Footnotes

  • ↵1 This work was supported by Grant AI01251 from the National Institute of Allergy and Infectious Diseases and the Primary Children’s Medical Center Research Foundation (E.E.A.) and Grant HL35280 from the National Heart, Lung, and Blood Institute to M.W.C.

  • ↵2 Address correspondence and reprint requests to Dr. Madeleine Cunningham, Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, OK 73190. E-mail address: madeleine-cunningham{at}ouhsc.edu

  • ↵3 Abbreviations used in this paper: ARF, acute rheumatic fever; IMDM, Iscove’s modified Dulbecco’s medium; LMM, light meromyosin; GlcNAc, N-acetyl-β-d-glucosamine; CDR, complement-determining region.

  • Received January 5, 1998.
  • Accepted April 9, 1998.

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The Journal of Immunology
Vol. 161, Issue 4
15 Aug 1998
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