Activity of rheumatoid factors of different molecular sizes: comparison of autologous monomeric and polymeric monoclonal IgA rheumatoid factors

Differences in rheumatoid factor (RF) activity among the molecular species of IgA were investigated with the use of monomeric and polymeric monoclonal IgA RF paraprotein from the serum of a patient (PS) with idiopathic hyperviscosity syndrome. After fractionation by gel chromatography in acidic buffer, RF activity as determined by latex fixation and solid-phase radioimmunoassay (RIA) specific for IgA RF was confined to the high m.w. (greater than 7S) fractions. However, after adsorption into polystyrene wells, fractions containing monomeric (7S) IgA, as well as those containing polymeric IgA, bound 125I-labeled heat-aggregated human IgG. These observations were confirmed after further purification of the IgA fractions by passage through a protein A-Sepharose CL-4B column followed by precipitation of the IgA proteins with ammonium sulfate at 50% saturation. After "cross-linkage" by a hybridoma anti-human alpha-chain antibody, the activity of the monomeric IgA preparation in the IgA RF RIA approached that of the polymeric IgA preparation. Gel filtration studies verified that this activity was not due to contamination by polymeric IgA RF. Further, classic RF specificity was confirmed for both the monomeric and polymeric IgA RF by reaction with human Fc-coated but not Fab-coated wells. A control monomeric IgA myeloma protein and normal serum IgA did not react in the RF RIA when analyzed in the presence or absence of the hybridoma anti-alpha-chain antibody. Moreover, the activity of the polymeric IgA RF preparation from patient PS in the RIA was minimally influenced by the hybridoma antibody. These results indicate that IgA RF can coexist in both polymeric and monomeric forms, demonstrate that monomeric IgA RF may escape detection by previously described RIA techniques, and suggest an approach for its detection.     

Routine assay for detection of IgG and IgM antiglobulins in seronegative and seropositive rheumatoid arthritis.

A convenient technique suitable for the routine estimation of IgM and IgG antiglobulins has been devised. The assay involves the binding of antiglobulins (rheumatoid factors) to rabbit immunoglobulin linked to the surface of plastic tubes; the amount of antiglobulin bound is then determined by adding radiolabelled antihuman IgG or IgM. Both antiglobulins were raised in virtually all seropositive rheumatoid arthritics, and 19 out of 22 seronegative patients had raised values for either IgM or IgG rheumatoid factors. The test should prove valuable in diagnosis and the results further emphasize autosensitization to IgG as a dominant immunological characteristic of different forms of rheumatoid arthritis.     

Rheumatoid factors may bear the internal image of the Fc gamma-binding protein of herpes simplex virus type 1

Affinity-purified rheumatoid factors (RF) from 20 patients with rheumatoid arthritis were tested for their reactivity with the mAb II-481 against glycoprotein E (gE), the Fc gamma-binding protein of HSV-1, as well as with a panel of mAb against human Fc gamma R. All RF bound to mAb II-481 in preference to mAb IV.3 (anti-human Fc gamma RII) or MOPC 141 (control mAb) which belong to the same IgG2b subclass. Five RF showed strong reactivity with II-481. No significant reactivity was observed between RF and mAb against human Fc gamma R. Non-RF human IgM did not react with any of the mAb. Clear-cut binding to II-481 was also seen with monoclonal IgM-RF derived from MRL/1 mice (mRF-2). The reaction between RF and II-481 was completely inhibited by human IgG. It was also inhibited by BHK cell extract infected with HSV-1, and with purified gE. II-481 inhibited the binding of human IgG Fc to the infected cell extract, confirming that II-481 recognizes the Fc-binding site on gE. II-481 did not react directly with human IgG or Fc of IgG. mAb to human IgG2 showed stronger binding to II-481 than to MOPC 141, suggesting II-481 has conformational similarity to human IgG H chain. These results suggest that at least some RF bear the "internal image" of HSV-1 Fc gamma-binding protein and support the hypothesis that some RF may be generated as anti-idiotype antibodies against antiviral antibodies.     

Heteroclitic polyclonal and monoclonal anti-Gm(a) and anti-Gm(g) human rheumatoid factors react with epitopes induced in Gm(a-), Gm(g-) IgG by interaction with antigen or by nonspecific aggregation. A possible mechanism for the in vivo generation of rheumatoid factors.

Heteroclitic rheumatoid factors (RF) are specific for allotypic determinants, e.g., Gm(a) or Gm(g) on allogeneic, but not autologous IgG. All polyclonal RF we isolated from nine rheumatoid arthritis patients with circulating Gm(a-), (b+), (g-), (f+) IgG displayed dual heteroclitic activity for the Gm(a) and Gm(g) allotypes, as shown by using appropriate RBC agglutination assays and affinity columns bearing Gm(a+) or Gm(g+) IgG. To investigate possible mechanisms underlying the in vivo generation of heteroclitic RF, we tested the ability of nonspecifically and immune-specifically aggregated Gm(a-), (g-) IgG to function as targets for RF from Gm(a-), (g-) patients with rheumatoid arthritis. Heat aggregation (63 degrees C for 20 min) or binding to Ag (as in tetanus toxoid-antitetanus toxoid complexes) induced a "functional" Gm(a+) and/or (g+) phenotype in Gm(a-), (g-) IgG from five healthy subjects and five rheumatoid patients, as suggested by the ability of these altered IgG to function as efficient targets for six heteroclitic RF in direct binding and competitive inhibition experiments. That heterocliticity and dual Gm(a), Gm(g) specificity can be features of a single antibody molecule was formally demonstrated by analysis of a monoclonal RF (IgM mAb 61) generated from a Gm(a-), (g-) rheumatoid patient. RF mAb 61 displayed a high affinity (Kd, 10(-7) M) for IgG Fc fragment of Gm(a+) and (g+) IgG or aggregated autologous Gm(a-), (g-) IgG but did not bind to native autologous IgG. To investigate the molecular basis of the acquired Gm(a) phenotype, PBMC from five Gm(a-) patients with rheumatoid arthritis and two Gm(a-) normal subjects arthritis and two Gm(a-) normal subjects were cultured in vitro after activation with PWM. In most instances, these PBMC produced IgG that behaved as Gm(a+) in sensitive ELISA. Application of the polymerase chain reaction (PCR), using probes specific for the nucleotide sequence coding for the Gm(a) tetrapeptide, to the amplification of DNA from the in vitro-stimulated Gm(a-) normals or rheumatoid patients' PBMC provided no evidence for Gm(a) nucleotide sequences. The present data suggest that acquisition of the Gm(a) determinant by Gm(a-) IgG may result from subtle changes in the CH2-CH3 RF-binding region. Such changes would occur when Gm(a) IgG are complexed with Ag or nonspecifically altered, thereby providing a possible explanation for the induction of heteroclitic RF in Gm(a-) rheumatoid arthritis patients.     

Rheumatoid factors from patients with rheumatoid arthritis react with beta 2-microglobulin.

IgM rheumatoid factors (RF) from 18 sera of rheumatoid arthritis (RA) patients isolated from monomeric IgG affinity columns showed strongly positive ELISA reactions with human beta 2-microglobulin (beta 2m), as well as with recombinant beta 2m. When the same RA sera were adsorbed to beta 2m-Sepharose affinity columns, eluted material showed predominant IgM anti-Fc of IgG and anti-beta 2m reactivity. Inhibition reactions with "RF" obtained from IgG affinity columns showed slightly higher reactivity of RF for Fc over beta 2m; however, when RF from the same RA serum had been adsorbed to and eluted from beta 2m affinity columns, beta 2m showed greater inhibition than Fc for RF reacting with either beta 2m or Fc on ELISA plates. Thus two overlapping populations of RF were identified in RA sera showing reactivity with both beta 2m and Fc of IgG. When RF were isolated from IgG columns, affinity was slightly higher for Fc than beta 2m. Conversely, RF eluted from beta 2m Sepharose reacted slightly more with beta 2 m than Fc. Trypsin digests of a polyclonal RA IgM RF showed no beta 2m reactivity in Fc mu 5 fragments. Fab mu RF retained slight anti-Fc IgG but no residual anti-beta 2m activity. Monoclonal human IgM, IgG, or IgA RF either from mixed cryoglobulins or EBV-stimulated RA lymphoid cell lines showed negative or occasional weakly positive anti-beta 2m activity. Overlapping 7-mer peptide ELISA analysis of the entire 99-amino acid sequence of beta 2m showed a major RF-reactive linear hydrophilic sequence at positions 56-60 which included a 3-amino acid exact homology to positions 401, 403, and 404 of the C gamma 3 domain. A peptide encompassing this sequence produced 90% inhibition of RF binding to whole beta 2m. Substitution of neutral glycines for each amino acid throughout the reactive epitope at positions 56-66 indicated that lysine at position 58 aspartic acid at 59, and tryptophane at 60 represented major portions of the RF-reactive epitope. These findings indicate that human RF derived from patients with RA react with other epitopes besides those present on IgG Fc, including epitopes on human beta 2m. For many years serum RF3 found in patients with RA have been regarded as premier examples of autoantibodies to autologous IgG.(ABSTRACT TRUNCATED AT 400 WORDS)     

Polymeric IgA rheumatoid factor in idiopathic IgA mesangial nephropathy (Berger's disease)

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was used to detect IgA rheumatoid factor (RF) in sera from 88 patients with IgA nephropathy (IgA GN), a disease characterized by abnormalities of IgA production. Significantly higher levels of IgA antiglobulins were demonstrated in IgA GN patients than in normal healthy controls and patients with other forms of chronic primary glomerulonephritis (mean +/- SEM 28.4 +/- 6.6 vs 6.0 +/- 0.4 and 8.3 +/- 1.2 micrograms/ml respectively; p less than 0.002). Interestingly, in contrast to rheumatoid arthritis, IgA RF activity was not associated with IgM antiglobulins. Analysis of sera fractionated by gel chromatography at acid pH revealed that anti-IgG activity resided predominantly in the polymeric fractions of IgA as confirmed by the ability to bind "free" secretory component. Several findings in patients with IgA GN suggest that the IgA deposited in the glomeruli is polymeric, and levels of circulating macromolecular IgA are increased. Our findings confirm a general perturbation of IgA metabolism in this disease. Although the polymeric nature of the IgA RF is suggestive of a mucosal origin, additional evidence is needed to confirm this hypothesis.     

类风湿关节炎患者RF、ANA、CCP、Ig及炎症因子的水平测定及临床意义

目的:探讨类风湿关节炎(RA)患者血清类风湿性因子(RF)、抗核抗体(ANA)、抗环瓜氨酸肽(CCP)抗体、免疫球蛋白(Ig)、补体(C3、C4)以及炎症因子的水平及临床意义。方法:收集2016年9月至2017年4月我院收治的165例RA患者为RA组,其中RA活动期患者93例(RA活动组),RA缓解期患者72例(RA缓解组),并于同期随机选取30例健康体检者为对照组。采用免疫散射比浊法检测各组血清RF、IgM、IgG、IgA、C3、C4水平,采用酶联免疫吸附法(ELISA)检测各组血清CCP抗体,电化学发光法检测白介素-6(IL-6)、化学发光法检测白介素-8(IL-8)。免疫荧光法检测ANA。比较不同组别各检测指标水平,并分析RA患者RF、ANA、CCP抗体、Ig、C3、C4与炎症因子的相关性。结果:RA活动组、RA缓解组血清RF、ANA、CCP抗体、IgM、IgG、IgA、IL-6、IL-8水平高于对照组,且RA活动组血清RF、ANA、CCP抗体、IgM、IgG、IgA、IL-6、IL-8水平高于RA缓解组,差异均有统计学意义(P0.05)。RA活动组、RA缓解组血清C3、C4水平低于对照组,且RA活动组血清C3、C4水平低于RA缓解组,差异均有统计学意义(P0.05)。经Pearson积矩相关分析,RA活动期和缓解期患者血清RF、ANA、CCP抗体、IgM、IgG、IgA与炎症因子IL-6、IL-8呈正相关关系(P0.05),血清C3、C4与炎症因子IL-6、IL-8呈负相关关系(P0.05)。结论:RA患者体内RF、ANA、CCP抗体、Ig及IL-6、IL-8水平明显较高,C3,C4水平明显较低,活动期RA患者更为显著,联合检测可早期辅助诊断RA及判断病情进展,在临床上有重要的参考意义。     

Serum C-X-C motif chemokine 13 is elevated in early and established rheumatoid arthritis and correlates with rheumatoid factor levels

Introduction

We hypothesized that serum levels of C-X-C motif chemokine 13 (CXCL13), a B-cell chemokine, would delineate a subset of rheumatoid arthritis (RA) patients characterized by increased humoral immunity.

Methods

Serum from patients with established RA (the Dartmouth RA Cohort) was analyzed for CXCL13, rheumatoid factor (RF) levels, anticitrullinated peptide/protein antibody (ACPA) and total immunoglobulin G (IgG); other parameters were obtained by chart review. A confirmatory analysis was performed using samples from the Sherbrooke Early Undifferentiated PolyArthritis (EUPA) Cohort. The Wilcoxon rank-sum test, a t-test and Spearman’s correlation analysis were utilized to determine relationships between variables.

Results

In both the Dartmouth and Sherbrooke cohorts, CXCL13 levels were selectively increased in seropositive relative to seronegative RA patients (P = 0.0002 and P < 0.0001 for the respective cohorts), with a strong correlation to both immunoglobulin M (IgM) and IgA RF levels (P < 0.0001). There was a weaker relationship to ACPA titers (P = 0.03 and P = 0.006, respectively) and total IgG (P = 0.02 and P = 0.14, respectively). No relationship was seen with regard to age, sex, shared epitope status or inclusion high-sensitivity C-reactive protein (hsCRP) in either cohort or regarding the presence of baseline erosions in the Sherbrooke Cohort, whereas a modest relationship with Disease Activity Score in 28 joints CRP (DAS28-CRP) was seen in the Dartmouth cohort but not the Sherbrooke cohort.

Conclusion

Using both established and early RA cohorts, marked elevations of serum CXCL13 levels resided nearly completely within the seropositive population. CXCL13 levels exhibited a strong relationship with RF, whereas the association with clinical parameters (age, sex, DAS28-CRP and erosions) or other serologic markers (ACPA and IgG) was either much weaker or absent. Elevated serum CXCL13 levels may identify a subset of seropositive RA patients whose disease is shaped by or responsive to RF production.     

Analysis of paraprotein transport into the saliva by using anti-idiotype antibodies

To determine the extent of clonal involvement of the secretory immune system and the origin of salivary immunoglobulins (Ig) in monoclonal gammopathy patients, saliva and serum samples were collected from five affected individuals (two IgA myelomas, one IgG myeloma, one IgG benign monoclonal gammopathy, and one IgM lymphoma) and were assayed for the presence of monoclonal Ig. Purified polyclonal or monoclonal anti-idiotype (Id) antibodies were prepared against each of the isolated serum paraproteins. In all five individuals, the patient saliva samples inhibited the binding of 125I-labeled homologous Ig to the corresponding anti-Id antibodies, but normal saliva did not. The concentration of Id in patients' saliva varied from 1 to 400 micrograms/ml; i.e., 0.004 to 1.0% of the corresponding serum values. Saliva of a lymphoma patient whose IgM kappa protein exhibited rheumatoid factor (RF) activity also contained RF. The salivary Id-bearing molecules were found to have the same Ig isotype as the serum paraproteins. The myeloma IgA represented a minor component (0.4 and 3.9%) of the total salivary IgA. The salivary IgA myeloma proteins were associated at least in part with secretory component, but the salivary IgG paraproteins were not. In an IgA myeloma patient, a minority (17%) of the IgA+ plasma cells found in the lacrymal gland biopsy specimen were Id+, whereas the great majority (98%) of bone marrow IgA plasma cells were Id+. The results suggest active transport rather than passive transudation of myeloma IgA into the patients' saliva, and the integrity of the secretory immune system was not compromised by the neoplastic process.     

Rheumatoid arthritis–associated autoantibodies in non–rheumatoid arthritis patients with mucosal inflammation: a case–control study

IntroductionRheumatoid arthritis–associated autoantibodies (RA-AAB) can be present in serum years before clinical onset of rheumatoid arthritis (RA). It has been hypothesized that initiation of RA-AAB generation occurs at inflamed mucosal surfaces, such as in the oral cavity or lungs. The aim of this study was to assess systemic presence of RA-AAB in patients without RA who had oral or lung mucosal inflammation.MethodsThe presence of RA-AAB (immunoglobulin A [IgA] and IgG anti-cyclic citrullinated peptide 2 antibodies (anti-CCP2), IgM and IgA rheumatoid factor (RF), IgG anti-carbamylated protein antibodies and IgG and IgA anti-citrullinated peptide antibodies against fibrinogen, vimentin and enolase) were determined in sera of non-RA patients with periodontitis (PD, n = 114), bronchiectasis (BR, n = 80) or cystic fibrosis (CF, n = 41). Serum RA-AAB levels were compared with those of periodontally healthy controls (n = 36). Patients with established RA (n = 86) served as a reference group. Association of the diseases with RA-AAB seropositivity was assessed with a logistic regression model, adjusted for age, sex and smoking.ResultsLogistic regression analysis revealed that IgG anti-CCP seropositivity was associated with BR and RA, whereas the association with PD was borderline significant. IgA anti-CCP seropositivity was associated with CF and RA. IgM RF seropositivity was associated with RA, whereas the association with BR was borderline significant. IgA RF seropositivity was associated with CF and RA. Apart from an influence of smoking on IgA RF in patients with RA, there was no influence of age, sex or smoking on the association of RA-AAB seropositivity with the diseases. Anti-CarP levels were increased only in patients with RA. The same held for IgG reactivity against all investigated citrullinated peptides.ConclusionAlthough overall levels were low, RA-AAB seropositivity was associated with lung mucosal inflammation (BR and CF) and may be associated with oral mucosal inflammation (PD). To further determine whether mucosal inflammation functions as a site for induction of RA-AAB and precedes RA, longitudinal studies are necessary in which RA-AAB of specifically the IgA isotype should be assessed in inflamed mucosal tissues and/or in their inflammatory exudates.     

Molecular analysis of IgM rheumatoid factor binding to chimeric IgG.

To localize regions on IgG bound by rheumatoid factors (RF), we studied IgM RF binding to chimeric IgG antibodies consisting of murine V regions fused to human constant regions. Using a modified RF ELISA, we showed that polyclonal RF from rheumatoid arthritis patients bound IgG1, 2, and 4 strongly; IgG3 was also bound, although less well. The majority of 18 monoclonal RF from patients with Waldenstrom's macroglobulinemia bound IgG1, 2, and 4 only. In contrast to RF from RA, 14 of 18 monoclonal RF did not react with IgG3. Only 3 of 18 monoclonal RF bound IgG3 well. By shuffling C region domains between IgG3 and IgG4, we showed that sequence variation in the CH3 domain is responsible for the differential binding of monoclonal RF to IgG3 and IgG4. Hybrid IgG3/IgG4 antibodies containing the CH3 domain of IgG4 were bound by monoclonal RF, whereas those containing the CH3 domain of IgG3 were not. To evaluate the contribution of the N-linked carbohydrate moiety at Asn-297 to RF binding sites on IgG, we measured RF binding to aglycosylated IgG antibodies produced by mutating Asn-297 to another amino acid. Glycosylated and aglycosylated IgG1, 2, and 4 were bound identically by monoclonal and polyclonal RF. Aglycosylated IgG3, however, was bound better than glycosylated IgG3 by polyclonal RF and by IgG3-reactive monoclonal RF.     

IgM rheumatoid factors in mice injected with bacterial lipopolysaccharides.

Bacterial lipopolysaccharides (LPS) induced the formation of IgM rheumatoid factors (RF) in several strains of mice including athymic C57BL/6 nude mice, but not in the LPS-resistant C3H/HeJ mice. The RF induced by LPS reacted not only with murine IgG but also with IgG from cows, goats, guinea pigs, and humans. The kinetics of this RF response to injection of LPS were similar to those of antibody response against DNA and a hapten, dinitrophenyl (DNP), and to those of total IgM production. In addition, the RF activity of individual serum samples correlated significantly with levels of anti-DNA and anti-DNP antibodies and of IgM. Therefore, it is concluded that the induction of RF results from polyclonal antibody synthesis by B cells stimulated with LPS. This observation suggests that LPS or LPS-like substances may help to generate RF in patients with rheumatoid arthritis or with some infectious diseases.     

Evaluation of the usefulness for the latex agglutination test for serodiagnosis of Mycoplasma pneumoniae infections

The usefulness of latex agglutination test prepared in our laboratory for the diagnosis of M. pneumoniae infections was assessed. A total of 628 serum samples obtained from patients with respiratory tract infections were tested by complement fixation test and by latex test, from among them 274 serum samples were additionally tested by ELISA--Ig A/--IgG/--IgM and by immunoelectroprecipitation test. The highest sensitivity and specificity was displayed by the latex test in relation to ELISA when determining mycoplasmal antibodies of IgM class (respectively 82.1% and 89.6%) and to the complement fixation test (81.0% and 89.0%). Positive latex test results in our investigations were associated only with the presence of IgM antibodies and were not dependent on the IgA and IgG antibody classes. The latex agglutination test may be used in routine serodiagnosis of mycoplasmosis under condition that the results obtained in this test will be confirmed by the complement fixation test or ELISA.     

Antigenic specificities of human monoclonal and polyclonal IgM rheumatoid factors. The C gamma 2-C gamma 3 interface region contains the major determinants

The binding site specificity of 12 monoclonal and 11 polyclonal IgM rheumatoid factors (RF) isolated from human plasma or serum has been studied. All IgM RF bound best to sites on IgG and intact Fc. The monoclonal IgM RF did not bind at all to fragments lacking the C gamma 2 or C gamma 3 domains. In contrast, low level binding to the pFc' fragment, composed of the C gamma 3 domain, was seen with seven IgM RF, mainly from patients with rheumatoid arthritis (RA). IgG1 binding appeared to be a requisite specificity of all human IgM RF. IgM RF binding to IgG3 subclass was common among the monoclonal IgM RF. Most RA polyclonal IgM RF but only 2 of the monoclonal IgM RF possessed the IgG1, 2 and 4 binding pattern. Monoclonal IgM RF which bound best to histidine-modified IgG also bound well to IgG3. The 7-kDa fragment D of staphylococcal protein A inhibited the IgG binding of most monoclonal and to a lesser degree polyclonal IgM RF. Thus, the results indicate that the C gamma 2-C gamma 3 interface region of IgG contains the predominant determinants for monoclonal and polyclonal IgM RF. For some monoclonal IgM RF the binding site, even though at the interface of the C gamma 2 and C gamma 3 domains, is not the staphylococcal protein A site. Furthermore, polyclonal IgM RF possess specificities not encountered among the monoclonal IgM RF. These specificities may have special     

The subclass distribution of human IgG rheumatoid factor

The subclass distribution of IgG rheumatoid factor (RF) was determined by a sensitive ELISA assay in sera from patients with rheumatoid arthritis and from normal controls. In both instances, the most important subclasses were IgG1 and IgG4. The IgG4 RF was directed against the Fc region of IgG, and recognized human as well as rabbit IgG. Although human IgG4 myeloma proteins bound to rabbit IgG better than did myelomas of other IgG subclasses, the IgG4 RF activity in rheumatoid sera showed an additional specificity, because the fraction of IgG4 RF/total IgG4 for rheumatoid arthritis sera was far greater than for myelomas. This inference was supported by the observation that there was persistent, albeit diminished, IgG RF activity in pepsin-digested, RF-containing sera (but not myeloma proteins), indicating that a critical component of IgG4 RF activity was contained within the Fab region of the IgG4 molecule. The finding of large quantities of IgG4 RF was not due to a bias of the assay, because the preponderance of IgG4 did not extend to the subclass distribution of antibodies directed against other antigens. The demonstration of an important role for IgG4 as a RF is of special interest because of the relative inability of this subclass to fix complement or to bind to Fc receptors, and because of its potential role as a mediator of increased vascular permeability.     

A rheumatoid factor paradox: inhibition of rituximab effector function

Introduction

Rituximab (RTX) therapy of rheumatoid arthritis (RA) exhibits enhanced effectiveness in seropositive patients. Using patient sera, we tested if this improved efficacy was associated with enhanced RTX mediated complement-dependent cytotoxicity (RTX-CDC).

Methods

We developed an in vitro assay for RTX-CDC using patient sera and the Daudi human B cell line. Using propidium iodide uptake and flow cytometry, we compared RTX-CDC with rheumatoid factor (RF)+ sera relative to normal volunteer, non-RA and RF- sera. Additional studies examined mixing studies of RF+ and RF- sera, as well as the effect of monoclonal IgA or IgM RF. Finally, the effect of RF on RTX mediated trogocytosis of normal B cells was evaluated.

Results

Using human sera, addition of RTX resulted in rapid and profound (> 50%) Daudi cell death that was complement dependent. Surprisingly, RF+ patient sera exhibited reduced RTX-CDC relative to RF- sera, with an inverse relationship of RTX-CDC and RF titer. Mixing studies indicated the presence of an inhibitor of RTX-CDC in RF+ sera. The addition of monoclonal IgM or IgA RF to RF- sera markedly inhibited RTX-CDC. This effect was specific for RF binding to the Fc portion of RTX as it was not apparent with the F(ab)'' domains of RTX engineered onto IgG3 heavy chain. RF also modestly inhibited RTX mediated trogocytosis.

Conclusions

Contrary to expectations, RF+ sera exhibits reduced RTX-CDC due to the presence of RF. The enhanced efficacy of RTX in seropositive RA patients cannot be attributed to improved B cell depletion through CDC. This result indicates that high RF levels may potentially modulate the efficacy of any therapeutic monoclonal antibody dependent on Fc effector function.     

Clonally related IgM rheumatoid factors undergo affinity maturation in the rheumatoid synovial tissue.

Using hybridoma technology we established a panel of human monoclonal rheumatoid factors (RF) from the synovial tissues of two patients with rheumatoid arthritis (RA), and one patient with polyarticular juvenile RA. Nucleotide sequence analysis of the V regions of these RF indicates that two independently derived antibodies from one of the RA patients are clonally related. One of these antibodies appears to be close to germ-line configuration, whereas the other has accumulated a total of 36 substitutions in both H and L chains. Measurements of the affinity for human IgG of the two RF show that the extensively mutated RF has 100-fold higher affinity for IgG than the RF close to germline. These findings indicate that IgM RF in RA can undergo affinity maturation and suggest that certain RF may be the product of an Ag-driven immune response.     

Restrictive IgG antibody response against mutated citrullinated vimentin predicts response to rituximab in patients with rheumatoid arthritis

IntroductionAntibodies against mutated citrullinated vimentin (AMCV) represent a useful diagnostic marker with correlation to disease activity in patients with rheumatoid arthritis (RA). Since seropositivity for citrullinated autoantibodies was predictive for response to B-cell depleting therapy (BCDT) with rituximab (RTX), we investigated whether differences in antibody fine reactivity and immunoglobulin (Ig) isotype kinetics among AMCV-positive patients could provide additional information about outcome.MethodsA total of 50 AMCV IgG-positive RA patients (RTX responders (RRs) n = 37 and non-responders (NRRs) n = 13) were analyzed for reactivity against MCV epitopes and co-existent AMCV isotypes IgM and IgA. Antibody titers were determined by enzyme-linked immunosorbent assay at baseline and 24 weeks after the first cycle of RTX, and compared to kinetics of rheumatoid factor (RF) and antibodies against cyclic citrullinated peptide (ACCP).ResultsRecognized MCV epitopes by AMCV IgG of RRs and NRRs showed similar baseline patterns, with reducing reactivity in RRs and unchanged or even expanding reactivity in NRRs upon RTX treatment. At baseline, RRs were more frequently negative for AMCV subtypes, especially for IgA (68 %), compared to NRRs (31 %). Being AMCV IgA-negative at baseline indicated a good treatment response to RTX (negative predictive value = 0.86). Co-existence of AMCV IgA and IgG with stable titers upon treatment were associated with poorer responses to RTX. Furthermore, reductions of AMCV IgA levels upon RTX correlated with the improvement of 28-joint Disease Activity Score (DAS28). In comparison, subtypes of RF and ACCP were not of additional value for prediction of RTX response.ConclusionsRestrictive IgG seropositivity against MCV with treatment-associated decline in fine reactivity and titers was predictive for response to RTX. Double-positivity for AMCV IgG and IgA was associated with failure to respond to BCDT, suggesting a pathogenetic and less sensitive IgA-producing B-cell subset in NRRs.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0717-z) contains supplementary material, which is available to authorized users.     

Antibody-bearing liposomes improve agglutination of latex particles used in clinical diagnostic assays

Ligand-bearing liposomes are used to enhance the agglutination ‘signal’ of a typical latex assay for the detection of human rheumatoid factor. Heat-denatured IgG, the antigen to which rheumatoid factor binds naturally, was covalently attached to latex spheres. The liposomes were covalently coated with a ‘second ligand’ which also recognizes rheumatoid factor, anti-human IgM Fab′ fragments. In the present test configuration, rheumatoid factor present in a patient's serum binds to the IgG attached to the latex particles. The liposomes, in turn, bind rapidly to rheumatoid factor-sensitized latex, via the second ligand, promoting the formation of large, clearly visible latex aggregates. When latex spheres bearing the same type and density of second ligand were used to replace the liposomes they failed to improve agglutination, suggesting that multivalent presentation of the second ligand is not sufficient to insure the improvement. These results suggest that fluidity of the liposomal membrane is a requirement for the effect. Sensitivity as well as ‘readability’ are improved by the liposomes while specificity remains unaffected. The principle of using ligand-bearing liposomes to enhance particle agglutination is applicable to a wide range of other diagnostic assays.     

Detection of anti-HIV IgA in tears of children born to seropositive mothers is highly specific

In order to improve the diagnosis of HIV infection in children born to seropositive mothers, 86 children were previously tested by Western blotting for anti-HIV IgA in tears and IgG in serum, at a median age of 9.2 months. To determine the exact value of the assay, 68/86 children of the same cohort were retested 9 months later.Nine children (13.4 %) were seropositive and all had anti-HIV IgA in tears. Eight of them had possessed lachrymal antibodies 9 months earlier. The ninth child was seronegative when 9 months old and then seroconverted. Four children (6 %), known to be seronegative, had an indeterminate Western blot pattern and no HIV IgA in tears. Fifty four (80.6 %) were seronegative at 18 months; none of them had ever had anti-HIV IgA in tears. This highlights the fact that only the children without lachrymal HIV IgA at the age of 9 months became seronegative at the age of 18 months.Our results clearly show that the detection of anti-HIV IgA in tears is a highly specific and reliable diagnostic test in children aged less than 15 months, born to seropositive mothers.