Monoclonal antibody 6B6.6 defines a cross-reactive kappa light chain idiotope on human monoclonal and polyclonal rheumatoid factors.

Mouse monoclonal antibody (MAb) 6B6.6 was raised against a cross-reactive idiotope (CRI) present on the light chains of 2 human IgM paraproteins with rheumatoid factor (RF) activity. The MAb inhibited the IgG-binding activity of these proteins, and thus appears to react with an epitope located at or near the RF-binding site. Enzyme-linked immunosorbent assay (ELISA) and Western immunoblotting studies indicate that the 6B6.6 CRI is associated with kappa IIIa sub-subgroup light chains, is not related to the Wa, Po, and Bla RF cross-idiotypic specificities, and is clearly distinct from the kappa IIIb-associated CRI detected by MAb 17.109. Using an ELISA, we detected 6B6.6 CRI in 59% of 107 sera and 48% of 50 synovial fluids from patients with seropositive rheumatoid arthritis (RA). However, the quantities of CRI-positive RF were small, and the amount of CRI-positive RF did not correlate with the amount of IgM-RF. The 6B6.6 CRI was shown to occur primarily in the IgM fraction of RA sera by both chromatographic studies and isotype-specific ELISA, although small quantities appeared to be associated with IgA and IgG in some sera. The presence of 6B6.6 CRI on both monoclonal and polyclonal RF is consistent with the view that both are derived, at least in part, from a common gene pool. However, its occurrence in relatively low levels suggests that the number of germline genes encoding for RF is large or that extensive mutation occurs in the course of RF expression in RA.     

Structural characterization of the second major cross-reactive idiotype group of human rheumatoid factors. Association with the VH4 gene family

Rheumatoid factors (RF) are the most common type of functional antibodies among naturally occurring human monoclonal IgM proteins. A large subset of these autoantibodies use structurally homologous light chains of the kappa III subgroup, which bear the 6B6.6 cross-reactive idiotype (CRI). Although antibody binding activity requires both heavy and light chains, information about the heavy chains used by these autoantibodies is limited. To investigate these proteins, the murine monoclonal antibodies, 5-14 and 6-10, were generated by immunization with the heavy chains of the 6B6.6 CRI-positive RF, COR and LEW. These antiidiotypic antibodies reacted with 8 of 11 autoantibodies that coexpressed the 6B6.6 CRI. All 8 RF had heavy chains from the VH4 gene family, as assessed by reactivity with a VH4-specific primary sequence-dependent antibody. The same RF were also identified by the previously described murine monoclonal antiidiotype, LC1. Further experiments revealed that the LC1 antibody delineates a subfamily of VH4 heavy chains that is preferentially used in kappa III-6B6.6 CRI-positive IgM-RF. The cumulative data suggest that 13-22% of RF express both the kappa III-6B6.6 and VH4-LC1 CRI. These findings document that RF autoantibody activity requires specific VL-VH pairing, and that a subset of idiotypically related VH4 heavy chains is commonly expressed in disease-associated monoclonal IgM-RF.     

Qualitative and quantitative expression of VHI associated cross reactive idiotopes within IgM rheumatoid factor from patients with early synovitis.

Monoclonal rheumatoid factors (RFs) of the major Wa cross reactive idiotype group have been shown to express exclusively VKIII subgroup light chains and VHI subgroup heavy chains. A VKIII associated cross reactive idiotope (CRI) (17-109), however, was shown not to be exclusively expressed on IgM paraproteins having rheumatoid factor activity or to be present at increased levels in the sera of patients with rheumatoid arthritis (RA). Three VHI associated CRIs have been defined with monoclonal antibodies and quantitative studies of their representation are reported, together with VKIII, in IgM and IgM RF isolated from the sera of patients with early synovitis, some of whom progressed to classical RA. The results show (a) the probed CRIs were expressed predominantly on IgM RF rather than on non-RF IgM; (b) 5-10% of IgM RFs from patients with classical RA expressed the CRIs, but this represented a lower proportion of IgM RFs than observed for normal individuals or patients with self limiting synovitis; (c) VKIII light chains were highly associated with IgM RFs rather than non-RF IgM (75% and 25% respectively). It is suggested that the CRIs probed are markers for germline gene encoded antibodies or sequences resulting from minimal mutation of germline genes. The lowered proportion of RFs expressing CRIs in RA may therefore be evidence of polyclonal activation or specific antigenic stimulation, or both, resulting in maturation of the RF response with recruitment of further VH genes or extensive mutation of germline genes. These studies show that monoclonal RFs are relevant models of RF produced in RA and that the repertoire of RF autoantibodies may be encompassed within a small number of CRI expressing families.     

Occurrence of a rheumatoid factor cross-reactive kappa light-chain idiotope in rheumatoid arthritis families

Summary The occurrence of IgM rheumatoid factor (RF) and RF-associated-III light-chain idiotope identified by monoclonal antibody 6B6.6 in the serum from 22 patients with rheumatoid arthritis (RA) and 68 relatives without connective tissue diseases in 15 families was determined by solid-phase enzyme-linked immunosorbent assays. Serum IgM RF was present in 19 RA patients from 12 families and 12 arthritis-free relatives of 4 families. It was not found in any of 9 spouses included in the study or in 44 of 45 unrelated healthy adult controls. RF-associated 6B6.6 idiotope was detected in 42% of the IgM RF(+) RA patients and in 50% of the IgM RF(+) arthritis-free relative, but not in the adult controls, spouses, and IgM RF(–) RA patients and relatives. It was present in one RA serum from each of 8 families and 6 sera from arthritis-free relatives of 2 families (5 of whom were from one family). Where present, the idiotope-positive RF represented only a small fraction of the serum IgM RF of the RA patients (0.1–2.1%) and relatives (1.5–14%). The increased frequency of IgM RF(+) individuals, with and without RA, in family groups suggests a genetic predisposition for expression of RF. The small proportion of RF bearing the 6B6.6 idiotope in both RA patients and unaffected family members supports the view that the number of germline genes encoding for RF is large or that extensive mutation occurs in the course of RF expression, whether idiopathic or associated with RA. In addition, nonuniform expression of the idiotope in RF within family groups indicates that the various clones of RF producing cells are to a large extent independently regulated.     

A sensitive radioimmunoassay for quantitation of igm rheumatoid factor

A solid-phase radioimmunoassay capable of detecting nanogram quantities of human IgM rheumatoid factor (RF) in biologic fluids has been developed. Binding curves for monoclonal IgM RF and polyclonal IgM rheumatoid factors were similar under the conditions utilized for the assay. Human IgG did not interfere with the detection of IgM RF by this method. Small quantities (≤ 0.2%) of nonspecific binding by nonRF IgM to the human IgG coated tubes utilized in the assay were corrected for by assaying samples in parallel bovine serum albumin coated control tubes. As expected, patients with seropositive rheumatoid arthritis (RA) had significantly higher concentrations of IgM RF than seronegative RA patients (mean ± 1 SD = 652 ± 553 μg/ml versus 11.3 ± 13.3 μg/ml, P < 0.001). In contrast, all normal control sera assayed to date contained < 0.1 μg/ml of IgM RF. The capacity of the assay to detect nanogram quantities of IgM RF should permit investigation of the cellular mechanisms underlying RF production.     

Increased IgA rheumatoid factor and V(H)1 associated cross reactive idiotype expression in patients with Lyme arthritis and neuroborreliosis

OBJECTIVE: To investigate whether autoreactive mechanisms occur in Lyme disease (LD) by determining IgA, IgG and IgM rheumatoid factor (RF) concentrations and RF associated cross reactive idiotype (CRI) expression in the serum of LD patients, with comparison to patients with rheumatoid arthritis (RA). METHODS: The RF isotype profiles were determined in 59 patients with LD; erythema migrans (EM) (n=19), neuroborreliosis (NB) (n=20) and Lyme arthritis (LA) (n=20). Mouse monoclonal antibodies (mAbs) G6 and G8 (V(H)1 gene associated), D12 (V(H)3 gene associated) and C7 (V(kappa)III gene associated) were then used to determine the RF associated CRI expression on IgM antibodies in 16 of these LD patients (eight seropositive for RF); (EM (n=3), NB (n=6), LA (n=7)). RESULTS: Seven (18%) patients with either NB or LA had increased concentrations of IgA RF compared with none with EM. Significant differences in the number of patients with raised concentrations of IgG RF or IgM RF were not found between the LD patient groups. Five (3NB, 1LA and 1 EM) (31%) and three (2NB and 1LA) (19%) of LD patients had raised concentrations of the CRIs recognised by mAbs G6 and G8, respectively. These CRIs were detected in LD sera both with and without raised concentrations of RF and were not demonstrated on anti-Borrelia burgdorferi antibodies using ELISA. No LD sera tested had raised concentrations of the determinants recognised by mAbs C7 or D12. CONCLUSION: Significantly raised concentrations of IgA RF and increased use of V(H)1 germline gene associated CRIs are found on IgM antibodies in the serum of LD patients. These data indicate the recruitment of autoreactive B lymphocytes in some patients with the later stages of LD.     

Complement mediated inhibition of immune precipitation in rheumatoid arthritis: studies on interaction of heat aggregated IgG with IgM rheumatoid factor.

Serum samples from patients with seropositive rheumatoid arthritis contain an inhibitor of complement mediated inhibition of immune precipitation (CMIP). This inhibitory effect can be produced by the addition of either purified monoclonal or polyclonal IgM rheumatoid factor (RF) to human serum. The specificity of the rheumatoid factor influences the degree of inhibition, and when precipitation occurs the rheumatoid factor coprecipitates with the antigen-antibody complex. In rheumatoid sera there was a significant positive correlation between IgM RF concentration and inhibitory activity, though the range of inhibitory activity seen for the same concentration of rheumatoid factor was considerable. Small quantities of heat aggregated IgG (HAGG) had a much greater effect on the measurement in an enzyme linked immunosorbent assay (ELISA) of IgM RF than they did on the inhibitory activity of IgM RF in the CMIP assay. Larger quantities of HAGG initiated complement activation and increased the precipitation of immune complexes. IgM RF reduced the complement activating properties of HAGG by reducing the amount of Clq which bound to the aggregate. The mechanisms by which IgM RF overcomes CMIP in rheumatoid sera may involve its inhibitory effects on the binding of Cl to the antigen-antibody complex.     

A highly conserved conformational idiotope on human IgM rheumatoid factor paraproteins of the Wa cross-reactive idiotype family defined by a monoclonal antibody

Summary Human IgM rheumatoid factors have been classified into major (Wa) and minor (Po) idiotypically cross-reactive families on the basis of reactivity with polyclonal anti-idotypic antisera. Extensive structural studies have revealed that RFs from the Wa CRI family have homologous light chains. The structural basis of this CRI however is not known. In this study we have defined a conformational idiotope requiring the association of the V_H and V_L of a number of RF from the Wa CRI family for its expression and which thus comprise part of the Wa CRI. A murine monoclonal antibody, G8 specific for the idiotope reacted with 67% of RFs from the Wa CRI family. G8 also reacted with one IgG₂K paraprotein with unknown specificity suggesting that the idiotope per se does not confer RF reactivity. Comparative studies of G8 expression relative to previously described sequence-dependent heavy and light chain associated idiotopes suggest that G8 recognises a separate determinant. Furthermore, the G8 idiotope is found on RFs expressing the V_HI subgroup of heavy chain. Study of G8 expression in polyclonal IgM in normal sera demonstrated that the idiotope is widely expressed in the population. However, significantly higher levels of IgM bearing G8 were detected in the sera of patients with rheumatoid arthritis.     

Seronegative rheumatoid arthritis, rheumatoid factor cross reactive idiotype expression, and hidden rheumatoid factors.

The major rheumatoid factor cross reactive idiotype (RCRI), defined by prototypic monoclonal rheumatoid factors (RFs), is expressed as a dominant idiotype by pokeweed mitogen induced plasma cells obtained from seropositive (RF+) patients with rheumatoid arthritis (RA). Some patients who meet clinical diagnostic criteria for RA set by the American Rheumatism Association fail to express RFs at any time during their clinical course. To determine if seronegative (RF-) patients with RA, so designated by the latex fixation, Rose-Waaler classic binding assays, or a RF enzyme linked immunosorbent assay (ELISA), express the RCRI in the absence of detectable RFs we examined pokeweed mitogen plasma cells from these patients by indirect immunofluorescence. In addition, we used an inhibition ELISA to detect RCRI bearing molecules in the sera of RF- patients with RA. Five of 10 RF- patients with RA had a high prevalence of RCRI+ plasma cells (16-49% of total pokeweed mitogen plasma cells in culture). Six of 20 RF- patients with RA had high serum concentrations of molecules marked by the RCRI, equivalent to 21-110 micrograms/ml of RCRI+ reference monoclonal IgM RF. Four of five patients who expressed the RCRI in high prevalence in pokeweed mitogen plasma cells, also demonstrated high concentrations of RCRI in their sera detected by inhibition ELISA. There was significant concordance of RCRI expression determined by the two different assays. Four RF- patients with RA who expressed RCRI in their whole sera had hidden RFs detected in their 19S and, in one case, 7S serum fraction. Detection of RF related molecules in whole sera by the expression of RCRI in RF- patients with RA identifies a subgroup of RF- patients with RA who possess hidden RFs. Some RF- patients with RA can express the major RCRI in pokeweed mitogen plasma cells and in their sera and therefore are related to patients with prototypic Waldenstrom's macroglobulinaemia, who produce RCRI+ 19S IgM monoclonal RFs.     

Isotypes of rheumatoid factors in rheumatoid arthritis and chronic liver diseases

We studied isotype-specific rheumatoid factors (RFs) to clarify their significance in rheumatoid arthritis (RA) and to verify the difference in RF isotypes between RA and chronic liver diseases (CLD). Isotype-specific RFs in RA and in CLD were measured by enzyme-linked immunosorbent assay (ELISA). Most sera (n = 51, 94.1%) from RA patients contained some kind of RF isotypes (92.1% for IgM RF, 76.4% for IgG RF, and 43.1% for IgA RF), and seronegative RA by ELISA was seen in only 11.8% (n = 6). The most characteristic combination of RF isotypes in active RA was IgG, IgA, and IgM. This combination of RF isotypes changed to IgG plus IgM, according to the diminution of RA activity; then, we found only IgM RF in inactive RA. The titers of each RF isotype also decreased in parallel with the activity of RA. IgA RF seemed to be the most sensitive factor for evaluating the activity of RA. In CLD, almost the same high frequency (n = 49, 89.8% for IgM RF, 59.2% for IgG RF), with the same titer levels seen in RA, was observed. On the other hand, IgA RF was significantly lower in frequency (n = 9, 18.4%) and in titer, compared with the finding in RA. Surprisingly, even in CLD, true seronegativity by ELISA was also found in very few patients (n = 4, 8.1%). In CLD, positive RFs detected by agglutination assay were seen more often in chronic hepatitis than in liver cirrhosis. In RA patients, significant associations of IgA RF and the serum concentration of IgA, and IgG RF and the serum concentration of IgG, were observed. On the other hand, in CLD patients, significant associations of IgG RF and the serum IgG concentration, and of IgM RF and the serum IgM concentration, were observed. These results indicated that IgA RF in active RA is the most characteristic RF isotype distinguishing it from other nonrheumatic diseases, as well as from inactive RA. RF isotypes reflected the background polyclonal B-cell activation in different manners in both diseases. In CLD, RF isotypes seemed to be disease-related immunological disorders reflecting disease progression. Received: February 17, 2000 / Accepted: July 5, 2001     

Rheumatoid factors in rheumatoid arthritis and vasculitis

Summary To study the occurrence of rheumatoid factors (RF) in relation to the activity of rheumatoid arthritis and the occurrence of vasculitis, RF of IgM, IgA, and IgG classes were measured in sera from 35 patients with definite or classic rheumatoid arthritis (RA) using ELISA. For 26 patients, the RF levels were studied longitudinally and compared with changes in the articular index. Although IgM RF was occasionally found in patients without RA, IgA and/or IgG RF were almost exclusively associated with RA. The titers of IgM, IgA, and IgG RF were significantly higher in sera from patients with clinically diagnosed rheumatoid vasculitis than in sera from patients without vasculitis. No significant correlation between changes in the articular index and changes in titer of any class-specific RF could be found for the group of RA patients as a whole. However, in individual patients, increases or decreases in IgM and IgG RF titer were significantly correlated with an increase or decrease in the articular index.     

The quantitative determination of the IgM rheumatoid factor using a solid-phase radioimmunoassay. Comparison with the agglutination test and the radioimmuno-polyethyleneglycol precipitation test

A solid-phase radioimmunoassay capable of detecting nanogram quantities of human IgM rheumatoid factor using a monoclonal anti-mu-chain antibody is described. Human IgG did not interfere with the detection of IgM RF by this method. The small nonspecific binding of nonRF IgM to the human IgG coated tubes utilized in the assay must be corrected for by assaying samples in parallel bovine serum albumin coated control tubes only in cases of deviation of IgM from normal range. 69 coded and randomly arranged sera from patients with rheumatoid arthritis (RA), nonrheumatic joint diseases and healthy adult control subjects were investigated by this method, agglutination techniques as well as RIPEGA. A good correlation between solid-phase radioimmunoassay and agglutination techniques was found. Patients with seropositive RA had significantly higher concentrations of IgM RF than seronegative RA patients or control subjects (mean +/- 1 SD = 133,3 +/- 187,2 micrograms/ml versus 4,7 +/- 6,5 micrograms/ml and 2,2 +/- 4,0 micrograms/ml; resp.).     

Enhanced in vitro synthesis of igm rheumatoid factor in rheumatoid arthritis

Peripheral blood mononuclear leukocytes (MNL) from 42 patients with classic/definite seropositive rheumatoid arthritis (RA) and 24 healthy adult controls were tested for their capacity to produce IgM rheumatoid factor (RF) in vitro in the presence and absence of pokeweed mitogen (PWM). In no instance was spontaneous elaboration of IgM RF from control MNL observed. In contrast, MNL from 16 of the 42 RA patients spontaneously synthesized IgM RF (22 ± 43 ng/ culture) which constituted a substantial fraction of the total IgM in these culture fluids (48 ± 26%). Pokeweed mitogen induced detectable quantities of IgM RF in MNL culture supernatants from 10 of 24 controls (12 ± 11 ng/culture) and 33 of 42 RA patients (60 ± 82 ng/ culture, p = 0.008). IgM RF constituted a significantly higher proportion of the total IgM in RA MNL supernatants than in control supernatants (11 ± 11% versus 1.01 ± 1.03%; P = 0.013). IgM RF production (spontaneous and PWM-induced) was dependent upon de novo protein synthesis. The results indicate that B cells programmed to produce IgM RF are present in both normal and RA B cell repertoires, but are preferentially expressed in RA.     

Rheumatoid factors react with Fab fragments of monoclonal antibodies to herpes simplex virus types 1 and 2 Fc gamma-binding proteins.

Human polyclonal IgM rheumatoid factors (RF) were tested in an enzyme-linked immunosorbent assay with monoclonal antibodies (MAb) (II-481 and B10/A8) to glycoprotein E (gE), the Fc gamma-binding protein of herpes simplex virus type 1 (HSV-1), as well as with MAb 88-S to gE of HSV-2. Most of the RF reacted with II-481 and 88-S. Positive reactions were recorded for RF reacting with whole MAb II-481 and 88-S, as well as with their Fab, but not their Fc, fragments. Human monoclonal IgM RF isolated from mixed cryoglobulins showed a similar profile, with reactivity for both whole MAb II-481 and 88-S and for their Fab fragments. Reactivity with MAb to gE was observed regardless of the Gm specificity of the polyclonal RF and the cross-reactive idiotypes (6B6, 17.109, or G6) of the monoclonal RF. No positive reactions were noted between protein A and Fab fragments of any of the anti-gE MAb. These findings indicate that many RF may bear the internal image of the Fc gamma-binding regions of 2 different herpesviruses: HSV-1 and HSV-2.     

7S IgM IN THE SERA OF PATIENTS WITH ARTHRITIS

Using radial immunodiffusion in 7% agarose, 7S IgM was quantifiedin the sera of 45 normal individuals, 37 patients with rheumatoidarthritis (RA), 18 patients with psoriatic arthritis and 11patients with ankylosing spondylitis. 7S IgM was only foundin the sera of patients with RA, 43% of whom had detectablelevels of 7S IgM (median 47.5 µg/ml). The patients with7S IgM had significantly higher IgM rheumatoid factor (IgM RF)and C-reactive protein levels in their sera (p < 0.005).There was a strong correlation between 7S IgM and IgM RF levelsin the sera of these patients. These data demonstrate that patientswith more active and severe disease have 7S IgM present in theirsera but the absence of 7S IgM from the sera of some patientswith high levels of IgM RF and CRP suggest that additional factorsmay influence the synthesis and secretion of 7S IgM by B cellsin RA. KEY WORDS: Rheumatoid arthritis, IgM RF, C-reactive protein, 7S IgM, B lymphocytes     

A QUANTITATIVE ELISA FOR MEASUREMENT OF RHEUMATOID FACTOR ASSOCIATED CROSS-REACTIVE IDIOTYPES IN SERUM FROM PATIENTS WITH RHEUMATIC DISEASES

A sensitive ELISA for the quantitation of RF associated cross-reactiveidiotypes (CRI) has been developed. CRI bearing IgM was capturedby immobilized F(ab')2 fragments of monoclonal anti-CRI antibodiesand revealed with isotype specific conjugates. Factors affectingsensitivity, specificity and reproducibility of the assay wereassessed. The sensitivity limit was less than 10 ng/ml and thelinear assay range for the CRI standard curve was from 80 to500 ng/ml. Using this ELISA system we have quantitated serumlevels of the CRI in different groups of patients with RA, earlysynovitis and normal individuals. Our results indicate a significantincrease in the levelsof the CRI in the patient groups comparedto the normal subjects tested, and suggest involvement of adiverse repertoire of Ig variable region germline genes and/orsomatic mutation in production of polyclonal RF in RA. KEY WORDS: ELISA, Rheumatoid factor, Rheumatoid arthritis, Cross-reactive idiotypes ^*Present address: Department of Immunology, School of PublicHealth, University of Medical Sciences of Tehran, Tehran, Iran      

Autoantibody-associated cross-reactive idiotypes expressed at high frequency in chronic lymphocytic leukemia relative to B-cell lymphomas of follicular center cell origin

Using murine monoclonal antibodies (MoAbs) specific for immunoglobulin (Ig) cross-reactive idiotypes (CRI), we performed immunohistochemical analyses on frozen tissue sections and cytocentrifuge preparations of Ig-expressing malignant cells from patients with chronic lymphocytic leukemia (CLL) and B-cell non-Hodgkin's lymphomas (NHL) of follicular center cell origin. Twenty percent (4/20) of the Ig kappa light chain- expressing CLL cells reacted with 17.109, a MoAb against a major CRI on human IgM autoantibodies that is encoded by a conserved Ig variable- region gene (V gene) of the V kappa IIIb sub-subgroup. Another MoAb specific for V kappa IIIb framework determinant(s) reacted exclusively with all the 17.109-reactive CLL cells. Only one of 20 kappa light- chain-expressing CLL cells reacted with 6B6.6, a monoclonal antibody specific for a CRI commonly found on rheumatoid factor (RF) paraproteins with light-chain variable regions of the V kappa IIIa sub- subgroup. Finally, greater than 20% (8/34) of all CLL reacted with G6, a MoAb specific for an Ig heavy chain-associated CRI present on several RF paraproteins. In contrast, these CRIs were expressed at significantly lower frequencies in NHL of follicular center cell origin. Only one of 30 NHL expressing kappa light chains reacted with the 17.109 MoAb. Also, in contrast to the concordance between the 17.109-CRI and V kappa IIIb framework determinant(s) in CLL, two lymphomas in addition to the 17.109-reactive lymphoma were recognized by the anti-V kappa IIIb framework MoAb. None of the NHL reacted with either the 6B6.6 or the G6 MoAbs. These results are the first to demonstrate that CLL and NHL differ with respect to the expression of autoantibody-associated CRIs. The data support the notion that NHL of follicular center cell origin differs from CLL in its utilization and/or somatic mutation of Ig variable-region genes. The physiological and immunotherapeutic implications of these findings are discussed.     

Low molecular weight IgM and CD5 B lymphocytes in rheumatoid arthritis.

OBJECTIVES--To evaluate the role of low molecular weight (LMW) IgM and CD5 B cells in rheumatoid arthritis (RA) and to explore the possibility that LMW IgM is derived selectively from this subset of B cells. METHODS--LMW IgM in sera and culture supernatants was detected by a sensitive immunoblot technique with an enhanced chemiluminescence detection system. CD5 B cells were determined by FACScan cytometry. In vitro studies were established in culture plates containing pokeweed mitogen with or without 2-mercaptoethanol (2-ME). Supernatants were obtained from CD5 positive hybridomas and CD5 negative hybridomas. Other immunological indices were measured by laser nephelometry. RESULTS--Circulating LMW IgM was detected in all rheumatoid patients with significantly higher levels being observed in sero-positive patients. LMW IgM correlated significantly with total IgM and RF. Peripheral blood mononuclear cells (PBMC) from the majority of the patients with RA secreted LMW IgM in vitro as did mononuclear cells from a synovial fluid sample. The addition of low concentrations of 2-ME to the culture medium enhanced the proportions of secreted monomeric IgM. In contrast, PBMC from healthy subjects secreted only trace quantities of LMW IgM. In RA no significant correlations were observed between CD5 B cells and LMW IgM and RF. LMW IgM could be detected in the supernatants from both CD5+ and CD5- B cell lines. Finally, CD5 B cells were not significantly elevated in RA and levels remained constant over time. CONCLUSION--LMW IgM exists in high concentrations in RA sera and synovial fluid. Serum level correlates with RF and IgM. In vitro studies have suggested that the occurrence of LMW IgM may be due to an intrinsic defect(s) in the assembly of the IgM pentameric molecule. LMW IgM is unlikely to be derived solely from CD5 B cells.     

Autoantibodies in rheumatoid arthritis: association with severity of disease in established RA

Introduction: Autoantibodies in rheumatoid arthritis (RA) are useful both for diagnosis and prognosis. Antibodies directed against citrullinated antigens have recently been shown to predict development of RA as well as poor outcome in early arthritis. Data on their role in established RA is limited. We studied the association of various autoantibodies in RA with its severity. Materials and methods: A total of one hundred and twenty nine-patients with established RA was enrolled and sera were collected and stored at −70°C. Data regarding erosions, deformities, and extra-articular features were collected. IgM rheumatoid factor (RF) was measured using nephelometry and value above 20 U was considered positive. IgA RF was measured by enzyme-linked immunosorbent assay (ELISA) and value above the mean±2 SD of normal healthy control was taken as positive. Anti-keratin antibody (AKA) was detected by indirect immunofluorescence assay using rat esophagus as substrate. Anti-cyclic citrullinated peptide (CCP) antibodies were measured by commercial ELISA and a value above 5 U was considered as positive. Results: The prevalence of various autoantibodies was: IgM RF 82.2%, anti-CCP antibodies 82.2%, AKA 51.9%, and anti IgA RF 45%. The concordance rate of anti-CCP antibodies with IgM RF was 83%, with AKA 68%, and with IgA RF 60.5%. All but one patient positive for AKA were positive for anti-CCP antibodies. The presence of IgM RF, AKA, and anti-CCP antibody was associated with joint erosions and deformities. None of the antibodies had any association with presence of extra-articular features. No association of IgA RF was seen with erosions, deformities, or extra-articular features. Among 23 seronegative RA patients, 11 were positive for anti-CCP antibodies and 6 were AKA positive. The presence of anti-CCP antibodies was associated with presence of deformities (p<0.05). Conclusion: Anti-CCP antibodies are present in majority of patients with established RA including seronegative patients. Both anti-CCP and AKA, in addition to conventional marker like IgM RF, are associated with severe erosive disease.     

Differences in the relationship of specificity to titre and functional affinity between circulating Ga- and pan-reactive IgM rheumatoid factors in rheumatoid arthritis

OBJECTIVE: To determine if there are the differences in titre and functional affinity for immunoglobulin (Ig) G subclasses and glycoforms between the Ga- and pan-specific IgM rheumatoid factors (RFs) present in the sera of patients with rheumatoid arthritis (RA), and to determine whether these two broad specificities have different functional roles in RA. METHODS: We used direct ELISA and modified ELISA to study the binding of IgM RF in the sera of 32 patients with RA with a range of RF titres to a panel of 14 IgG paraproteins of all four subclasses, some allotypes and different glycosylation patterns. RESULTS: Pan-specific RFs were mostly found in RA sera with high RF titres, and these RFs generally had higher avidity. A trend towards higher avidity of RFs with higher titre was observed for pan-specific, but not for Ga-specific RFs. With increasing titre, pan-specific RFs tended to react strongly with fucosylated and bisected variants of hypogalactosylated IgG3 of G3m(b1) allotype and hypergalactosylated IgG4 of 4a allotype. CONCLUSION: Among high-titred pan-specific IgM RFs, there is a subpopulation responsible for strong anti-IgG activity in RA. The possible mechanisms of production of pan- and Ga-specific RFs are discussed.