ASSOCIATION BETWEEN HUMAN IgM AUTOANTIBODIES AND κ CHAIN ALLOTYPES

The relationship between the immunoglobulin kappa light chain allotypes and autoantibodies was studied in a series of seven human monoclonal kappa-bearing IgM antibodies with Rheumatoid Factor (RF) activity, two IgM anti-low density lipoprotein (LDL) antibodies, and one IgM anti-intermediate filament (IF) antibody. Residues at amino acid positions 153 and 191 related to the Km allotypes in human kappa chains were determined by an HPLC tryptic fingerprint and corroborated by amino acid sequence analysis. All the autoantibodies shared similar variable regions derived from the V gene(s). The seven RF and the anti IF were associated with the Km(3) constant region allotype whereas the two antiLDL were associated with the Km(1,2) allotype. Thus, monoclonal autoantibodies showed the same Km allotypic distribution as the normal population. However, although the number of samples is small, it seems likely that a preferential association may exist between particular V, genes and Km alleles in the generation of autoantibodies with different specificities.     

A new assay uses monoclonal anti-Rh(D) antibodies to determine rheumatoid factor specificity: reactivity to a monoclonal antibody of the Gm allotype G3m(21) is more frequent in rheumatoid patients negative for G3m(21)

A new method has been developed to determine the specificities of polyclonal rheumatoid factors (naturally occurring antibodies which react with human Fc gamma) (RF) found in sera from patients with rheumatoid arthritis. In this method, monoclonal anti-Rh(D) antibodies of known IgG isotype and allotype are bound to erythrocytes and then act as the target IgG antigen for RF in a direct haemagglutination test. Using two monoclonal anti-D antibodies of the IgG3 isotype and G3m(21) allotype, which were cloned from different donors, we found that a large number of rheumatoid sera reacted with both these G3m(21) proteins. In contrast reactivity of rheumatoid sera with polyclonal anti-D of the G3m(21) allotype in the direct haemagglutination test was rare. A strong correlation was found between reactivities to both G3m(21) monoclonal anti-D antibodies but not with a monoclonal anti-D antibody carrying the alternative allele, namely G3m(5). Haemagglutination inhibition experiments using human paraproteins of known IgG isotype and allotype provided some additional evidence that this method can detect RF with specificity for the G3m(21) allotypic determinant or a related allotypic determinant in polyclonal rheumatoid sera. When each patient's autoantibody response was related to their Gm phenotype, we found that the frequency of reactivity for G3m(21) monoclonal anti-D antibodies was significantly greater in patients negative for G3m(21) than in patients positive for the G3m(21) allotype. IgM preparations from patients' sera were dissociated at acid pH but no 'hidden' antibodies were found. We suggest trans-placental sensitization as one of several possible interpretations of this finding.     

Molecular analysis of the V kappa III-J kappa junctional diversity of polyclonal rheumatoid factors during rheumatoid arthritis frequently reveals N addition.

Much interest was stirred in recent years by the evidence that rheumatoid factors (RF) variable regions are encoded by a restricted set of V genes, with little or no somatic mutations, that are often overexpressed in the fetal repertoire. This is reminiscent of what has been observed for natural autoantibodies. However, these data come from studies of monoclonal RF (mRF) isolated from patients with lymphoproliferative disorders who usually do not present autoimmune symptoms. The molecular characterization of RF during autoimmune diseases such as rheumatoid arthritis (RA) has been hampered for some time because of their polyclonality; recently using the polymerase chain reaction method, we have demonstrated that RF kappa variable regions from a patient with RA were encoded by V kappa III genes known to code for mRF but that these genes had undergone somatic mutations with a pattern suggesting an antigen-driven maturation. Because an important role of the light chain third complementarity-determining region (CDR3) in anti-IgG reactivity and idiotype expression has already been suspected for RF, we now report the molecular characterization of the junction regions of these rearranged V kappa gens. Surprisingly, our data show that in 55% of the cases there is addition of a proline and/or glycine amino acid residue at the recombination site between V kappa and J kappa. The sequence analysis of our patients' germ-line Vg and J kappa 4 genes segments and their flanking regions demonstrates that the additional codons are not readily explicable by recombination between germ-line sequences and probably result from an N addition process. Since we could not find such an additional codon in 15 previously published mRF kappa chains we suggest that "pathogenic" RF during RA and mRF derive from different, although overlapping, B cell subsets. Moreover, since additional codons at the recombination site of V kappa and J kappa seem exceptional in expressed human kappa chains and because the resulting amino acid residue is a proline in most cases, we think that RF kappa chain CDR3 is under a very strong selective pressure during RA.     

Autoantibody production by severe combined immunodeficient mice reconstituted with synovial cells from rheumatoid arthritis patients

In an attempt to characterize the heterogeneity of the human autoantibody response, mice with severe combined immunodeficiency were reconstituted with synovial or blood lymphocytes from patients with rheumatoid arthritis (RA). Mononuclear cells extracted from synovial fluid or tissue (SMC) were a greatly enriched source of IgM rheumatoid factor (RF)-producing cells compared to the peripheral blood mononuclear cells (PBMC) of rheumatoid arthritis patients or normal donors. Six to nine weeks after reconstitution of mice with synovial mononuclear cells, 0%-39.3% (mean = 11.4%) of total IgM consisted of IgM RF compared to 0%-0.15% (mean = 0.02%) in mice given RA PBMC and 0%-1.2% (mean = 0.34%) in mice given normal PBMC. Detectable levels of IgM RF were maintained in some mice for as long as 20 weeks after transfer. Mice reconstituted with synovial membrane or synovial fluid lymphocytes produced a heterogeneous mixture of immunoglobulins. These included other autoantibodies, such as anti-nuclear and anti-cytoplasmic antibodies, and antibodies to exogenous antigens such as the Epstein-Barr virus nuclear antigen-1 (EBNA-1). This heterogeneity is further illustrated by the demonstration that the sera from mice given synovial cells also contained IgG antibodies possessing all three major VH families (VH1, VH3 and VH4) and the four major V kappa families (V kappa 1 to V kappa 4). Autoantibody production gradually decreased with time even under circumstances where total immunoglobulin levels increased, and elevated production could not be induced by antigenic stimulation. These findings describe a new model for the analysis of human autoantibody production.     

Immunoglobulin allotypes in systemic lupus erythematosus--results of a central European multicenter study. SLE Study Group

Immunoglobulin heavy chains (G1m, G2m, G3m, A2m) and kappa light chain (Km) allotype and phenotype frequencies were examined in 323 central European Caucasian patients with systemic lupus erythematosus (SLE). No significant differences were found between the different allotype or phenotype frequencies of the SLE patients and a control group of healthy individuals. Our results indicate that Gm, A2m and Km allotypes do not represent susceptibility factors for SLE in Caucasians.     

The V kappa IIIb light chain sub-subgroup. II. Isotype expression in acquired hypogammaglobulinaemia and as a function of age.

The V kappa IIIb sub-subgroup of the human immunoglobulin V kappa III light chain subgroup has at least two unique properties. First, some IgM monoclonal autoantibodies, in particular cryoprecipitable anti-IgG (Kunkel et al., 1974; Ledford et al., 1983), anti-low density lipoprotein (Ledford et al., 1983) and certain cold agglutinin (Feizi et al., 1976) antibodies predominantly contain V kappa IIIb light chains. Second, in normal adult human serum, V kappa IIIb comprises approximately 25% of the IgM but less than 0.4% of the IgG or IgA kappa-chain pools (Moynihan, Looney & Abraham, 1985). Studies have suggested an altered regulation of IgM, IgG, and IgA-V kappa IIIb in some patients with acquired hypogammaglobulinaemia, a heterogeneous group of immunodeficiencies with low serum levels of Ig. An increase in the serum levels of V kappa IIIb light chains has been noted in some of these patients (Solomon & Mclaughlin, 1969) which is apparently due to elevated levels of light chains of the V kappa IIIb sub-subgroup (Greenstein & Abraham, 1984). However, the isotype association of V kappa IIIb in these patients has not been determined. In order to clarify whether the noted increase in V kappa IIIb levels is due to its selective expression with IgM or to an abnormality of immunoregulation, the heavy chain isotypes associated with V kappa IIIb light chains have been determined in a previously studied group of adults with acquired hypogammaglobulinaemia (Greenstein & Abraham, 1984). Further, the isotype distribution of V kappa IIIb light chains in another group of functional, albeit transient, hypogammaglobulinaemics (i.e. normal neonates) has also been studied by measuring IgM-V kappa IIIb levels in cord blood, and compared to the V kappa IIIb serum levels found in aged adults (mean age 75 years).     

CD5-positive B-cell malignancies frequently express cross-reactive idiotypes associated with IgM autoantibodies.

Using monoclonal antibodies (MAb) specific for cross-reactive idiotypes (CRIs) associated with human monoclonal IgM autoantibodies, we examined 57 biopsy specimens that previously had been noted to have immunohistologic features of CD5-positive B-cell small lymphocytic (SL) non-Hodgkin's lymphoma (NHL). Twenty-five lymphoma specimens were noted to be from patients with chronic lymphocytic leukemia (CLL). Eight of thirty-four (24%) immunoglobulin (Ig) kappa light-chain expressing lymphomas reacted with 17.109, a MAb specific for a major CRI encoded by a conserved Ig kappa variable region gene (Vk gene) of the VkIIIb sub-subgroup. All 17.109-reactive tissues and two 17.109-negative specimens were recognized by another MAb specific for VkIIIb framework determinant(s). Seven of all fifty-six (13%) Ig-expressing tumors bound G6, a MAb specific for an autoantibody heavy-chain-associated CRI that is encoded by a conserved antibody heavy chain variable region gene(s) (VHgene) of the VH1 subgroup. All seven G6-positive lymphomas and two G6-negative tumors reacted with Cc1, another MAb specific for a rheumatoid factor heavy-chain-associated CRI. A third autoantibody-heavy-chain-associated CRI, termed Lc1, was expressed by seven (13%) other lymphomas. Finally, a fourth MAb specific for RF heavy-chain-associated CRI, named B6, detected two additional tumors. The expression frequencies of autoantibody-associated CRIs among SL NHL patients without peripheral lymphocytosis did not differ from those noted among patients with CLL but were significantly higher than those observed among patients with NHL of follicular center-cell origin. These data imply that the malignant B cells of patients with either CD5-positive B-cell SL NHL or CLL express a restricted set of Ig V genes that have not substantially diversified from the germline DNA.     

Association of KM genotype with bullous pemphigoid

Association of kappa light chain immunoglobulin allotypes with bullous pemphigoid was examined in 101 Caucasian patients. Km alleles were determined by polymerase chain reaction amplification followed by restriction enzyme digestion. The frequency of Km(3)/Km(1,2)kappa light-chain genotype was found to be significantly associated with the disease, while that of the Km(3)homozygous genotype was significantly higher in patients with both anti-BPAG1 and anti-BPAG2 autoantibodies.     

Molecular interactions between human IgG, IgM rheumatoid factor and streptococcal IgG Fc receptors

Group A streptococci type M15 were previously shown to bind both human IgG via the Fc component and a purified monoclonal IgM kappa rheumatoid factor (IgM RF). Using 125I-labelled IgG and 125I-labelled IgM RF, the present study gave association constants of 2.2 x 10(7) and 2.9 x 10(8) M-1, respectively. The binding of 125I-IgG to the streptococci was inhibited by unlabelled IgG, IgG Fc and fragment D of staphylococcal protein A but not by the IgM RF or F(ab')2 of anti-idiotype antibodies to RF (anti-Id RF). Inversely, unlabelled IgM RF and anti-Id RF inhibited the binding of 125I-IgM RF markedly and unlabelled human IgG and IgG Fc only slightly or moderately, respectively. Thus, group A streptococci type M15 showed different binding sites for IgG Fc and the antibody combining sites of a human monoclonal RF. The findings were still more complex on a background of previous reports showing that streptococcal IgG Fc receptors and RFs bind to the same amino acids on the Fc molecule. This complex pattern may play a role in the pathogenesis of rheumatoid arthritis.     

Human monoclonal rheumatoid factors derived from the polyclonal repertoire of rheumatoid synovial tissue: incidence of cross-reactive idiotopes and expression of VH and V kappa subgroups.

A panel of 14 human monoclonal and monoreactive IgM rheumatoid factors (RF) derived from the synovial tissue of rheumatoid arthritis (RA) patients was studied for the expression of cross-reactive idiotopes (CRI) and variable heavy (VH) and variable kappa (V kappa) subgroups. Four of the twelve kappa RF expressed light chains of the V kappa III subgroup. Three of these were characterized as belonging to the V kappa IIIb sub-subgroup, and expressed the V kappa IIIb associated CRI, 17.109. None of the RF expressed the V kappa IIIa-associated CRI, 6B6.6. One of the fourteen RF expressed the VH-associated CRI, G6, and five expressed either or both the VHIII-associated CRI, B6 and D12. Seven RF bound to protein A (SpA), which indicates the expression of VHIII subgroup V regions. Together the data indicated that 9/11 RF express VHIII regions and 2/11 express VHI regions. There was no obvious correlation between V region usage or CRI expression and fine specificity of the RF for human IgG subclasses. These data indicate a difference in V gene usage by RF derived from RA patients compared with RF paraproteins derived from non-RA patients. There is not a bias towards variable chains of the V kappa III subgroup, but a marked preference for variable heavy chains of the VHIII subgroup is seen. Further studies may elucidate the pathological significance of these findings in RA.     

Complete sequence of the genes encoding the VH and VL regions of low- and high-affinity monoclonal IgM and IgA1 rheumatoid factors produced by CD5+ B cells from a rheumatoid arthritis patient.

We have characterized the VH and VL genes of three low-affinity polyreactive and two high-affinity monoreactive IgM and IgA1 rheumatoid factor (RF) mAb generated using circulating CD5+ B cells from a single rheumatoid arthritis patient. We found that four and one RF mAb utilized genes of the VHIV and VHIII families, respectively. The VHIV gene usage by these RF mAb differs from the preferential VHIII, VHI, and, to a lesser extent, VHII gene usage by the IgM with RF activity found in patients with mixed cryoglobulinemia, Waldenstrom's macroglobulinemia, and other monoclonal gammopathies. In addition, in contrast to the preponderant kappa L chain usage by the RF in these patients, a lambda L chain was utilized by all RF mAb from our rheumatoid arthritis patient. Two RF mAbs utilized V lambda I, two V lambda IV, and one V lambda III L chains. The VH genes of the two low-affinity polyreactive IgM RF mAb were in germline configuration. When compared with the deduced amino acid sequence of the putatively corresponding genomic segment, the VH gene of the high-affinity monoreactive IgM RF mAb displayed five amino acid differences, all of which are in the complementarity determining regions (CDR), possibly the result of a process of somatic point mutation and clonal selection driven by Ag. The unavailability of the corresponding genomic VH segment sequences made it impossible to infer whether the VH genes utilized by the two IgA1 RF were in a germline or somatically mutated configuration. Sequencing of the genes encoding the H chain CDR3 (D segments) revealed that all three low-affinity polyreactive RF mAb displayed a much longer D segment (36-45 bases) than their high-affinity monoreactive counterparts (15-24 bases), raising the possibility that a long D segment may be one of the factors involved in antibody polyreactivity.     

A common idiotope on human rheumatoid factors identified by a hybridoma antibody

Human monoclonal and polyclonal anti-IgG autoantibodies [rheumatoid factors (RFs)] are composed primarily of kappa light chains, and may display cross-reactive idiotypes. However, the nature of the shared idiotope(s) has remained unclear. We have prepared a murine hybridoma antibody (17-109) that recognizes an idiotope present on 30% (3/10) of human IgM-RF paraproteins, and absent on immunoglobulins without RF activity. The idiotope was measurable on isolated, intact kappa light chains, but not on light-chain tryptic peptides, nor on isolated heavy chains. A comparison of the binding to 17-109 of five IgM-RF paraproteins, with known kappa chain amino acid sequences, suggested a relationship between the idiotope recognized by the hybridoma and the complementarity-determining regions. The serum of patients with rheumatoid arthritis contained idiotope positive material that bound specifically to a 17-109 immunoadsorbent column. Moreover, the 17-109 anti-idiotope antibody partially inhibited the binding to IgG of IgM-RF and IgA-RF in serum, but did not effect the binding to antigen of IgM and IgA anti-tetanus toxoid antibodies. These results suggest that a significant proportion of IgM-RF paraproteins share an idiotope located at or near the complementarity-determining regions of the kappa light chain. Human serum RFs include a kappa light chain family that is idiotopically related to the kappa chains on IgM-RF paraproteins.     

Complete Amino Acid Sequence of the Variable Regions of Two Highly Related Heavy and Light Chains of Human IgG (SFL) and IgM (RIV) Rheumatoid Factors

The complete amino acid structure of the variable regions of two monoclonal human rheumatoid factors (RF), antibodies that bind to the Fc portion of IgG, is presented. Although these RFs are of different isotypes, IgG (SFL) and IgM (RIV), they are highly related. They probably derive from the same KIII light chain variable region used, but the heavy chains are derived from genes of the VHIII family that are probably evolutionarily related. An analysis of the level of somatic mutation reveals that antigen selection was probably involved in the maturation of these clones. These antibodies, although highly related, are not merely IgM to IgG switch variants which occurred independently in different individuals. Little is currently known about the structure of IgG RFs and this study indicates that the level of somatic mutation of SFL is similar to other autoantibodies or antiviral antibodies of the IgG isotype.     

Autoantibodies and immunoglobulin allotypes in healthy North American Blacks of different age groups

A population of 291 healthy North American Black subjects of different ages was studied for immunoglobulin (Ig) allotypes and the prevalence of autoantibodies, to determine possible associations between Ig allotypes and age, autoantibodies and age, and Ig allotypes and autoantibodies. Indirect immunofluorescence was used to detect anti-gastric parietal cell, anti-smooth muscle, anti-thyroid microsomal, anti-nuclear, and anti-mitochondrial antibodies. The sera were typed for the Ig allotypes Gm(1, 2, 3, 5, 6, 13, 14, 17, and 21) and Km(1) with a hemagglutination-inhibition assay. A significant association between advanced age and an increased prevalence of anti-nuclear antibodies was observed in females. There was no significant association between Ig allotypes and the autoantibodies tested. The results suggest that Ig allotypes are not involved in the development of autoantibodies in healthy Blacks.     

A highly conserved determinant on human rheumatoid factor idiotypes defined by a mouse monoclonal antibody

Different human IgM rheumatoid factor (IgM RF) idiotypes have been described defined by polyclonal rabbit anti-idiotypic antibodies. These antisera do not allow clear genetic analysis of the idiotypic determinants, be they cross-reactive or private. Therefore, we tried to obtain a set of monoclonal anti-idiotypic antibodies directed against RF idiotypes. Purified IgM RF serum from a patient with classical rheumatoid arthritis was used to immunize BALB/c mice. The spleen cells were fused with Sp 2/0 Ag 14, a nonsecreting mouse myeloma cell line, and a hybrid producing monoclonal anti-idiotypic antibody was selected. The mouse antibody, an IgG1 kappa, reacts with an identical or similar determinant located on (or close to) the binding site of all tested monoclonal or polyclonal IgM RF from totally unrelated patients with Waldenstr?ms's macroglobulinemias or rheumatoid arthritis. The monoclonal antibody also reacts with 2 rheumatoid arthritis patients' IgG RF and with a low proportion of normal polyclonal IgM without detectable RF activity. An hypothesis is proposed to explain the existence of a such highly conserved determinant on RF idiotypes.     

Variable region primary structures of monoclonal anti-DNA autoantibodies from NZB/NZW F1 mice

VH and VL region primary structures of five NZB/NZW F1 derived monoclonal anti-DNA autoantibodies were determined from cloned cDNA. Comparative analysis of VH genes showed that except for two VH genes that shared complete identity the overall VH gene usage was diverse. Comparison of VH genes with those utilized in a variety of antibody responses showed they were generally unique to the autoanti-DNA response although framework homologies allowed assignment of four of five VH genes to existing murine heavy chain gene families. Only one out of five D segments shared homology to existing germline D segments, and all were rearranged to JH3. V kappa genes showed restriction for four of five light chains to the V kappa 1 subgroup. The V kappa 1 subgroup has been shown previously to be utilized in several anti-DNA autoantibodies as well as a variety of antibodies to exogenous antigens. H and L chain amino acid residues associated with the active site of a ssDNA specific autoantibody, 04-01, are discussed based on recently obtained crystallographic data.     

Expression of a germline human kappa chain-associated cross-reactive idiotype after in vitro and in vivo infection with Epstein-Barr virus

The mouse monoclonal antibody 17.109 recognizes a cross-reactive idiotype (CRI) associated with kappa IIIb light chains of human IgM-rheumatoid factor (RF) paraproteins. The 17.109 idiotypic determinant is encoded by one or a group of closely related V kappa genes. The association of the idiotype with IgM- and IgA-rheumatoid factors in certain autoimmune diseases necessitates an understanding of how human B lymphocytes can be induced to express the idiotype. To investigate the cellular expression of the 17.109 CRI, peripheral blood lymphocytes from normal donors were stimulated in vitro with Epstein-Barr virus (EBV) and pokeweed mitogen (PWM). EBV induced greater expression of IgM-associated 17.109 CRI than did PWM. The 17.109 CRI was preferentially associated with IgM rather than with IgG. In vivo EBV infection was studied in college students with infectious mononucleosis and displayed similar elevation of IgM-associated 17.109 CRI in sera obtained at presentation of clinical illness. Later, IgM levels declined while IgG-associated 17.109 CRI rose. The 17.109 idiotype was unrelated to antibodies against the Epstein-Barr virus nuclear antigen and the viral capsid antigen and was probably due to generalized activation of early B cells. These observations support the hypothesis that the 17.109 CRI is expressed by in vitro and in vivo EBV-infected cells. The 17.109 idiotype identifies a highly conserved V kappa gene product, which is expressed preferentially after EBV infection, but not exclusively with RF autoantibodies.     

Discrepancy in the expression of autoantibodies in healthy aged individuals

Sera from 50 healthy old subjects and from 51 young controls were tested by ELISA assays for a panel of autoantibodies, including IgM RF, anti-DNA, anti-F(ab')2, antithyroglobulin, anti-human albumin, anti-human hemoglobin, anti-secretory component from human IgA, and anti-gliadin. In vitro production of anti-DNA as well as anti-F(ab')2 antibodies were measured after stimulation of PBMC by pokeweed mitogen (PWM) in 12 healthy elderly subjects and 11 young controls. Sera from elderly donors contained threefold higher amounts of IgM RF than young controls (P less than 0.001). On the contrary, the levels of anti-DNA as well as anti-F(ab')2 antibodies were similar in both groups (P less than 0.3 for the two determinations). Anti-DNA and anti-F(ab')2 antibodies were also measured in supernates of PWM-stimulated glass nonadherent PBMC cultures from both old and young healthy donors without finding any significant difference between the two groups. Additional ELISA tests were also performed in both elderly and young control sera to detect antibodies against six other different antigens. No significant difference was found in the percentages of positive sera between the two groups. This discrepancy in production of IgM RF compared to other autoantibodies in healthy elderly subjects does not provide support for a general increase of autoantibodies with aging. The biological significance of an increase in IgM RF with aging remains to be determined.     

Immunoglobulin kappa light chain gene alleles are not associated with primary Sjögren's syndrome

The immunoglobulin kappa (Km) light chain gene is polymorphic and is believed to play a role in the pathology of infectious and autoimmune diseases. Polymorphisms within the constant region of the Km gene encode three alleles designated Km1, Km1,2 and Km3. Previous studies using serological detection of Km allotypes reported associations between specific Km allotypes, systemic lupus erythematosus and the presence of anti-La antibodies, yet these findings were not confirmed in other studies. In order to more precisely define any associations between Km alleles and anti-Ro/La antibodies we used the polymerase chain reaction and restriction fragment length polymorphisms for Km genotyping in a large cohort of patients with primary Sj?gren's syndrome (SS). No associations were observed between specific Km alleles and primary SS when compared with a control population, nor within serologically defined subsets of SS patients. We conclude that Km alleles are not associated with primary SS or the Ro/La autoantibody response.