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.     

Protein microarray signature of autoantibody biomarkers for the early detection of breast cancer

Cancer patients spontaneously generate autoantibodies (AAb) to tumor-derived proteins. To detect AAb, we have probed novel high-density custom protein microarrays (NAPPA) expressing 4988 candidate tumor antigens with sera from patients with early stage breast cancer (IBC), and bound IgG was measured. We used a three-phase serial screening approach. First, a prescreen was performed to eliminate uninformative antigens. Sera from stage I-III IBC (n = 53) and healthy women (n = 53) were screened for AAb to all 4988 protein antigens. Antigens were selected if the 95th percentile of signal of cases and controls were significantly different (p < 0.05) and if the number of cases with signals above the 95th percentile of controls was significant (p < 0.05). These 761 antigens were screened using an independent set of IBC sera (n = 51) and sera from women with benign breast disease (BBD) (n = 39). From these, 119 antigens had a partial area under the ROC curve (p < 0.05), with sensitivities ranging from 9-40% at >91% specificity. Twenty-eight of these antigens were confirmed using an independent serum cohort (n = 51 cases/38 controls, p < 0.05). Using all 28 AAb, a classifier was identified with a sensitivity of 80.8% and a specificity of 61.6% (AUC = 0.756). These are potential biomarkers for the early detection of breast cancer.     

Proteolysis activity of IgM antibodies from rheumatoid arthritis patients' sera: evidence of atypical catalytic site

The IgM antibodies from rheumatoid arthritis (RA) patients' sera were screened for peptide hydrolyzing activity. Recovery of structurally intact IgM antibodies (Abs), in a single step, was achieved using a weak anion-exchange methacrylate monolith disk. The IgM Abs from patients' sera hydrolyzed the Pro-Phe-Arg-4-methyl-coumaryl-7-amide (PFR-MCA) substrate appreciably compared to the healthy donors. The apparent K(m) values of IgM Abs from patients' sera were between 0.4 and 0.7 mM. Furthermore, IgM Abs displayed 5 to 10-folds greater proteolysis activity than IgG Abs, recovered from the same pathological serum. The proteolysis activity, as a function, was found to be independent of IgM-RF titer value. Affinity labeling approach targeted at the catalytic site histidine was studied, using a specific irreversible inhibitor, N-α-tosyl-L-lysine chloromethyl ketone (TLCK). Despite modification of catalytic His, observation of serine protease like activity suggest presence of an atypical catalytic framework in a few pathological IgM Abs.     

Antiglobulin factor in sera from patients with liver disease.

(1) An antiglobulin factor, non-neutralizable by human gamma-globulin, was demonstrated in sera of two patients with liver disease. (2) By absorption and elution techniques, two fractions were differentiated in a serum: one is reactive to rabbit antibody and the other seems cross-reactive to rabbit and human antibodies. (3) In double immunodiffusion test, the antiglobulin factor formed a precipitation band with heat-aggregated human IgG as did rheumatoid arthritis serum. (4) While the antiglobulin activity to rabbit antibody was demonstrated in both the IgM and IgG fractions, the reactivity to human antibody was localized in the IgM fraction. (5) From its selective reactivity to individual anti-D sera (sensitizers), at least a part of the specificity of the antiglobulin factor must be related to anti-Gm (1), and consequently can be regarded as auto-reactive.     

Autoantibody activity of IgG rheumatoid factor increases with decreasing levels of galactosylation and sialylation

The occurrence of N-linked oligosaccharides lacking galactose is significantly higher than normal in serum IgG of patients with rheumatoid arthritis (RA) in whom rheumatoid factor (RF), an autoantibody against autologous IgG, has been detected. In the present study, IgGs with and without RF activity (IgGRF and non-RF IgG, respectively) were prepared from sera of RA patients, and their oligosaccharide structures were characterized in order to investigate the relationship between RF activity and glycosylation. Three IgGRF fractions and a non-RF IgG fraction were obtained based on their ability to bind to an IgG-Sepharose column. The specific RF activity, as measured by immunoassays, was highest in the IgGRF fraction, which bound most avidly to the IgG-Sepharose. When the oligosaccharides were released by hydrazinolysis, and analyzed by MALDI-TOF mass spectrometry and HPLC, in combination with sequential exoglycosidase treatment, all the IgG samples were found to contain a series of biantennary complex-type oligosaccharides. The incidence of galactose-free oligosaccharides was significantly higher in both IgGRFs and non-RF IgG from RA patients compared with IgG from healthy individuals. In all IgGRFs, the levels of sialylation and galactosylation were lower than those in non-RF IgG from RA patients; the sialylation of non-RF IgG was the same as that of IgG from healthy individuals. In addition, the decreases in galactosylation and sialylation of oligosaccharides in IgGRF correlated well with the increase in RF activity. These findings could contribute to our understanding of the mechanisms of IgG-IgG complex formation and the pathogenicity of these complexes in RA patients.     

Immune complexes present in the sera of autoimmune mice activate rheumatoid factor B cells

The fate of an autoreactive B cell is determined in part by the nature of the interaction of the B cell receptor with its autoantigen. In the lpr model of systemic autoimmunity, as well as in certain human diseases, autoreactive B cells expressing rheumatoid factor (RF) binding activity are prominent. A murine B cell transgenic model in which the B cell receptor is a RF that recognizes IgG2a of the j allotype (IgG2aj), but not the b allotype, was used in this study to investigate how the form of the autoantigen influences its ability to activate B cells. We found that sera from autoimmune mice, but not from nonautoimmune mice, were able to induce the proliferation of these RF+ B cells but did not stimulate B cells from RF- littermate controls. The stimulatory factor in serum was found to be IgG2aj, but the IgG2aj was stimulatory only when in the form of immune complexes. Monomeric IgG2aj failed to stimulate. Immune complexes containing lupus-associated nuclear and cytoplasmic autoantigens were particularly potent B cell activators in this system. Appropriate manipulation of such autoantibody/autoantigen complexes may eventually provide a means for therapeutic intervention in patients with certain systemic autoimmune disorders.     

Assay of rheumatoid factors by the indirect immunofluorescence method

Sera from 69 patients affected with rheumatoid arthritis were examined for IgM, IgG and IgA rheumatoid factors (RF) by a indirect immunofluorescence method. The results were compared with those obtained from the classical rheumatoid factor latex test. By this technique we have demonstrated antigammaglobulin activity in a high proportion (23%) of sera from latex test seronegative rheumatoid patients. Moreover, by fractionated antisera it was possible to detect also IgG and IgA factors. Indirect immunofluorescence results to be a simple and available technique for detection of RF, also in many "seronegative" patients.     

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.     

A monoclonal anti-idiotype specific for human polyclonal IgM rheumatoid factor.

One of the hallmarks of rheumatoid arthritis (RA) is the production of high titers of rheumatoid factor (RF) antibody directed against the Fc portion of IgG. Anti-Id that recognize the majority of monoclonal RF from patients with B cell dyscrasias are reactive with only 1 to 2% of these polyclonal RF from RA patients. We describe a new monoclonal anti-Id, 4C9, that recognizes a L chain determinant on polyclonal IgM RF from patients with RA but does not recognize a panel of monoclonal RF from patients with B cell malignancies. 4C9 reactivity is found in the serum of 34/43 RF-positive RA patients and in 12/12 RF-positive synovial fluids, but in only 1/14 RF-negative sera from RA patients and 1/22 sera containing monoclonal IgM RF. 4C9 reactivity is highly enriched in purified IgM RF from nine RA patients and represents a variable percentage of total IgM RF up to a maximum of 23%. Furthermore, 4C9 reactivity is enriched in the synovial fluid of three of five RA patients compared with serum, suggesting that 4C9-reactive IgM RF are synthesized within the joint. IgG RF from RA synovial fluids are not 4C9 reactive, indicating either that different genes are used to encode IgM and IgG RF in RA patients, or that IgG RF have somatically mutated away from idiotypic reactivity.     

Mapping rheumatoid factor binding sites using genetically engineered, chimeric IgG antibodies.

We are using chimeric IgG antibodies consisting of murine variable regions joined to human constant regions as rheumatoid factor (RF) binding substrates to localize and map IgM RF binding sites on IgG. Using chimeric antibodies in a modified RF ELISA, we showed that RFs from rheumatoid arthritis (RA) and Waldenstrom's macroglobulinemia (WMac) patients differ in their binding specificities for IgG3, although some of these RFs share common specificity for IgG1, IgG2, and IgG4. By shuffling constant region domains between IgG3 and IgG4, we showed that sequence variation in the CH3 domain is responsible for WMac-derived RF differentiation of IgG3 and IgG4. By making site-directed mutations in the wild-type IgG3 or IgG4 human gamma constant genes, we showed that His-435 is an essential residue in RF binding to IgG for most WMac RFs. The allotypic polymorphism in IgG3 at 436 is not responsible for differences in previous reports of high-frequency IgG3 binding by WMac RFs. A amino acid loop in the CH2 domain of IgG4 proximal to the CH2-CH3 interface is important in WMac RF binding to IgG; a more distal CH2 loop in CH2 has a more variable effect on WMac RF binding. 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 the glycosylation signal Asn-297 to another amino acid. Of all four IgG subclasses, only aglycosylated IgG3 was a better RF binding substrate than its glycosylated subclass counterpart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cryoglobulinemia induced by monoclonal immunoglobulin G rheumatoid factors derived from autoimmune MRL/MpJ-lpr/lpr mice

A MRL strain bearing the autosomal recessive mutant gene, lpr (lymphoproliferation), spontaneously develops, in addition to a lupus-like syndrome, unique serological and pathological manifestations. Production of high titers of IgG rheumatoid factors (RF) may be related to the formation of extremely large amounts of cryoglobulins and the development of tissue lesions such as necrotizing polyarteritis, arthritis, and glomerulonephritis. To analyze more directly the relationship of IgG RF to the development of cryoglobulins and tissue injuries, we have established four monoclonal IgG RF secreting hybridomas from unimmunized MRL-lpr/lpr mice and determined their pathogenic effects in normal strains of mice. All the monoclonal IgG RF obtained in this study were of the IgG3 subclass and generated cryoglobulins. However, the fact that not only IgG3 Rf monoclonals but also four of five non-RF IgG3 monoclonals were able to form cryoglobulins, which were composed exclusively of each IgG3 monoclonal, indicates that the IgG3 molecule has a unique physicochemical property to self-associate via nonimmunological interaction and the ability to form cryoglobulins. When the in vivo pathogenic activities of these IgG3 RF and non-RF monoclonals were examined, three of IgG3 RF monoclonals with the specificity to IgG2a were able to induce extensive pathologic manifestations including peripheral vasculitis and glomerulonephritis characteristic of patients with cryoglobulinemia. Our results indicate that the IgG3 itself, independently of its specificity, could be a potential source of cryoglobulins and IgG3 RF, combined with its activity of cryoglobulin formation, may play a significant role in the development of glomerulonephritis and cutaneous vascular lesions of ears and foot pads observed frequently in aged MRL-lpr/lpr mice.     

Kinetic analysis of interaction of different types of rheumatoid factors with immobilized IgG using surface plasmon resonance

Rheumatoid factors (RFs) are autoantibodies, which recognize antigens on a constant region of immunoglobulin G (IgG). Among various RF classes, RF of the IgG class (IgGRF) forms immune complexes in rheumatoid joints and is implicated in the pathogenesis of rheumatoid arthritis (RA). To characterize the formation of IgGRF immune complexes, in the present study, IgGRF was isolated from sera of RA patients, and its interaction with immobilized IgG was analyzed and compared to that of IgMRF or IgARF by means of surface plasmon resonance. On gel filtration, the IgGRF was eluted as a single peak corresponding to IgG, excluding the possible formation of self-associating IgGRF complexes in solution. Sensorgrams of the interaction of IgGRF with immobilized IgG revealed that it clearly bound to the IgG at 6 degrees C, but not at 30 degrees C. The degree of interaction decreased inversely with an increase in temperature, suggesting that IgGRF is much more reactive at lower temperatures. In contrast, the interaction of IgARF and IgMRF with IgG at 6 degrees C was similar to that at 30 degrees C. The association rate constant (k(a)) of IgGRF decreased with an increase in temperature, while those of IgARF and IgMRF were similar under various thermal conditions. The dissociation rate constant (k(d)) of IgGRF was greatly reduced at 25 degrees C, but those of IgARF and IgMRF slightly increased with an increase in temperature. These results suggested that the mode of interaction of IgGRF with IgG differed from in the cases of IgMRF and IgARF. The kinetic properties of the IgGRF-IgG interaction may facilitate elucidation of the IgGRF immune complex formation in rheumatoid joints.     

DNA-Hydrolyzing Antibodies from Sera of Autoimmune-Prone MRL/MpJ-lpr Mice

Catalytically active antibodies (abzymes) hydrolyzing proteins, polysaccharides, ATP, DNA, and RNA have been detected in the sera of patients with various autoimmune and some viral diseases, but abzymes from the sera of animals are practically unstudied. The development of lupus-like autoimmune disease of MRL/MpJ-lpr mice is an experimental model for study of autoimmune pathologies and immunopathogenesis. In this work, homogeneous IgG preparations were isolated from the sera of MRL/MpJ-lpr mice. These antibodies (Abs), their Fab-fragments, and isolated light chains were shown to possess catalytic activity in DNA hydrolysis, whereas Abs from the sera of control healthy mice did not hydrolyze DNA. The data demonstrate that DNA hydrolyzing activity is an intrinsic property of Abs from MRL/MpJ-lpr mice. It was shown that various markers of autoimmune pathologies (level of total protein concentration in urea (proteinuria), Abs titers to native and denatured DNA, and DNA-hydrolyzing activity of IgG) increased in animals with aging, but they noticeably increased (2-22 times) only after appearance of obvious indicators of pathology independently of age. The highest increase in proteinuria (25-fold), anti-DNA Abs titers (12-19-fold), and abzyme activity (120-fold) was found in mice after their immunization with DNA–protein complex.     

Possible Relationship between Bacterial or Viral Fcγ-Binding Proteins and Rheumatoid Factor

Bacterial Fc-binding proteins (FcBPs) such as staphylococcal protein A and streptococcal protein G possess IgG fine specificity strikingly similar to that of rheumatoid factors (RFs) derived from patients with rheumatoid arthritis (RA). They were shown to bind to the C_H2-C_H3 interface region of IgG. It has also been shown that peripheral blood lymphocytes can be selectively induced to produce RF by protein A. Several hypotheses, including idiotypic mimicry, have been proposed to explain the relationship of RF and bacterial FcBPs. Although convincing evidence for the involvement of bacterial infection in the etiology of RA has not been available, viral infection has frequently been strongly suspected as the agent possibly triggering RA. Herpes family viruses possess FcBPs reacting with the same C_H2-C_H3 interface region of IgG. Fab fragments of monoclonal antibodies (II-481, 88-S) to the IgG-bindlng site of glycoprotein E (gE), the FcBP glycoprotein of herpes simplex viruses, showed strong binding to RF. The epitope on gE reacting with mAb II-481 showed significant overlap with the IgG Fc-binding site. Antibodies to cytomegalovirus FcBP have been detected in a substantial proportion of sera from patients with RA. These observations may imply that some RFs may be produced as anti-idiotype antibodies to anti-viral FcBP antibodies. Thus, bacterial or viral FcBP could provide a link between RF production and a possible infectious etiology of RA.     

Affinity and catalytic heterogeneity of polyclonal myelin basic protein‐hydrolyzing IgGs from sera of patients with multiple sclerosis

Human myelin basic protein (hMBP)‐hydrolyzing activity was recently shown to be an intrinsic property of antibodies (Abs) from multiple sclerosis (MS) patients. Here, we present the first evidence demonstrating a significant diversity of different fractions of polyclonal IgGs (pIgGs) from MS patients in their affinity for hMBP and in the ability of pIgGs to hydrolyze hBMP at different optimal pHs (3–10.5). IgGs containing λ‐ and κ‐types of light chains demonstrated comparable relative activities in the hydrolysis of hMBP. IgGs of IgG1–IgG4 sub‐classes were analyzed for catalytic activity. IgGs of all four sub‐classes were catalytically active, with their contribution to the total activity of Abzs in the hydrolysis of hMBP and its 19‐mer oligopeptide increasing in the order: IgG1 (1.5–2.1%) < IgG2 (4.9–12.8%) < IgG3 (14.7–25.0%) < IgG4 (71–78%). Our findings suggest that the immune systems of individual MS patients generate a variety of anti‐hMBP abzymes with different catalytic properties, which can attack hMBP of myelin‐proteolipid shell of axons, playing an important role in MS pathogenesis.     

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)     

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.     

One-step separation of IgG subclasses of DNA hydrolyzing autoantibodies: a study on sera of patients with systemic lupus erythematosus and primary antiphospholipid syndrome by histidine ligand affinity chromatography

A method based on histidine ligand affinity chromatography has been utilized for the separation of DNA hydrolyzing autoantibodies from sera of patients suffering from systemic lupus erythematosus and primary antiphospholipid syndrome using the gel, histidyl-aminohexyl-sepharose. The separation of autoantibodies was carried out under mild chromatographic conditions. Human IgG subclass distribution in the different fractions separated on the column was studied by enzyme-linked immunosorbent assay. The purified DNA hydrolyzing autoantibodies were shown to hydrolyze plasmid DNA.     

Human monoclonal IgG rheumatoid factor has structural homology with bacterial Fc receptor proteins

A cloned lymphoblast cell line, hRF-1, that secreted human monoclonal IgG4 rheumatoid factor autoantibody was produced by Epstein-Barr virus transformation of lymphocytes from rheumatoid arthritis synovium. The binding of hRF-1 rheumatoid factor to IgG globulins of different mammalian species was similar to the binding specificity of Staphylococcus aureus protein A (SpA) and to antibodies found in the sera from patients with rheumatoid arthritis. hRF-1 also had the same binding pattern to human IgG subclasses as SpA. Direct competition was observed between SpA and hRF-1 in binding IgG Fc. These results provide evidence for structural homology between a bacterial Fc receptor protein (SpA) and the monoclonal IgG rheumatoid factor.     

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.