Anti-Fas2 IgM antibodies in Fasciola hepatica infected patients with positive IgG serology.

Background: Fascioliasis is an infectious disease caused by parasites Fasciola hepatica and F. gigantica. Humans are infected by the consumption of vegetables and water contaminated with the infective form of the parasite. Materials and Methods: In this study, an IgM-ELISA with the cysteine proteinase Fas2 antigen was evaluated with sera from 76 patients infected with F. hepatica, 24 patients with other parasite infections and 84 healthy volunteers. Results: IgM-ELISA resulted in 43% positives in F. hepatica patients with positive serology to Fas2-ELISA, but no positives resulted from testing healthy volunteers and individuals infected with other parasites. The IgM-ELISA diagnostic parameters showed a sensitivity of 43.4% (95% CI 0.321-0.553), specificity of 100% (95% CI 0.957-1), and no cross-reactivity with other parasitic infection. Interference by rheumatoid factor in the IgM immunoassay was controlled by treating sera with rheumatoid factor absorbent before testing. Conclusions: Fas2 antigen is detected by circulating IgM in patients infected with F. hepatica and IgM-ELISA using Fas2 appears as a specific immunoassay to detect the acute phase of the acute phase of F. hepatica infection in humans.

IgM and IgA rheumatoid factors purified from rheumatoid arthritis sera boost the Fc receptor- and complement-dependent effector functions of the disease-specific anti-citrullinated protein autoantibodies.

Rheumatoid factors (RF) and the disease-specific anti-citrullinated protein autoantibodies (ACPA) coexist in the joints of rheumatoid arthritis (RA) patients where they probably contribute to synovitis. We investigated the influence of IgM and IgA RF on the FcR- and complement-dependent effects of ACPA immune complexes (ACPA-IC). When stimulated by ACPA-IC formed in the presence of IgM RF or IgA RF fractions purified from RA serum pools, M-CSF-generated macrophages skewed their cytokine response toward inflammation, with increases in the TNF-α/IL-10 ratio and in IL-6 and IL-8 secretion, and decreases in the IL-1Ra/IL-1ß ratio. In the IgM RF-mediated amplification of the inflammatory response of macrophages, the participation of an IgM receptor was excluded, notably by showing that they did not express any established receptor for IgM. Rather, this amplification depended on the IgM RF-mediated recruitment of more IgG into the ACPA-IC. However, the macrophages expressed FcαRI and blocking its interaction with IgA inhibited the IgA RF-mediated amplification of TNF-α secretion induced by ACPA-IC, showing its major implication in the effects of RF of the IgA class. LPS further amplified the TNF-α response of macrophages to RF-containing ACPA-IC. Lastly, the presence of IgM or IgA RF increased the capacity of ACPA-IC to activate the complement cascade. Therefore, specifically using autoantibodies from RA patients, the strong FcR-mediated or complement-dependent pathogenic potential of IC including both ACPA and IgM or IgA RF was established. Simultaneous FcR triggering by these RF-containing ACPA-IC and TLR4 ligation possibly makes a major contribution to RA synovitis.

IgM rheumatoid factor amplifies the inflammatory response of macrophages induced by the rheumatoid arthritis-specific immune complexes containing anticitrullinated protein antibodies.

OBJECTIVES: Anticitrullinated protein antibodies (ACPA) are specifically associated with rheumatoid arthritis (RA) and produced in inflamed synovial membranes where citrullinated fibrin, their antigenic target, is abundant. We showed that immune complexes containing IgG ACPA (ACPA-IC) induce FcγR-mediated tumour necrosis factor (TNF)-α secretion in macrophages. Since IgM rheumatoid factor (RF), an autoantibody directed to the Fc fragment of IgG, is also produced and concentrated in the rheumatoid synovial tissue, we evaluated its influence on macrophage stimulation by ACPA-IC. METHODS: With monocyte-derived macrophages from more than 40 healthy individuals and different human IgM cryoglobulins with RF activity, using a previously developed human in vitro model, we evaluated the effect of the incorporation of IgM RF into ACPA-IC. RESULTS: IgM RF induced an important amplification of the TNF-α secretion. This effect was not observed in monocytes and depended on an increase in the number of IgG-engaged FcγR. It extended to the secretion of interleukin (IL)-1ß and IL-6, was paralleled by IL-8 secretion and was not associated with overwhelming secretion of IL-10 or IL-1Ra. Moreover, the RF-induced increased proinflammatory bioactivity of the cytokine response to ACPA-IC was confirmed by an enhanced, not entirely TNF-dependent, capacity of the secreted cytokine cocktail to prompt IL-6 secretion by RA synoviocytes. CONCLUSIONS: By showing that it can greatly enhance the proinflammatory cytokine response induced in macrophages by the RA-specific ACPA-IC, these results highlight a previously undescribed, FcγR-dependent strong proinflammatory potential of IgM RF. They clarify the pathophysiological link between the presence of ACPA and IgM RF, and RA severity.

Rheumatoid factors induce signaling from B cells, leading to Epstein-Barr virus and B-cell activation.

B-cell antigen receptor signaling is initiated upon binding of the antigen to membrane-bound immunoblobulin (Ig), and the anti-Ig antibody (Ab) mimics this signaling. In B cells latently infected with Epstein-Barr virus (EBV), the same signals induce virus activation. We examine here whether rheumatoid factors (RFs), autoantibodies directed against the Fc portion of IgG, induce EBV and B-cell activation. As a source of RFs, RF-producing lymphoblastoid cell line (LCL) clones were isolated from peripheral blood mononuclear cells (PBMC) and synovial cells from patients with rheumatoid arthritis (RA) by EBV transformation. Burkitt's lymphoma-derived Akata cells, which are highly responsive to EBV activation by anti-Ig Abs, were used for the assay of EBV activation. Akata cells expressed IgG3 as membrane-bound Ig. RFs from a synovium-derived LCL were directed to IgG3 and induced EBV activation in 16 to 18% of Akata cells, whereas RFs from another synovium-derived LCL were directed to IgG1 and did not induce EBV activation. Pretreatment of RFs with the purified Fc fragment of human IgG completely abolished EBV activation. Furthermore, B-cell activation was assessed by incorporation of [3H]thymidine. RFs from synovium-derived LCLs efficiently induced B-cell activation, and the addition of CD40 ligand had a synergistic effect. On the other hand, RFs from PBMC-derived LCLs were polyreactive, had a lower affinity to IgG, and did not induce EBV and B-cell activation. The present findings imply a possible role for RFs as EBV and B-cell activators.

Human monoclonal rheumatoid factors augment arthritis in mice by the activation of T cells.

In order to investigate the in vivo role of rheumatoid factor (RF), the effects of the administration of human monoclonal (m) IgM-RF and IgG-RF on the development of arthritis in mice were examined. The administration of human mRFs into mice immunized with type II collagen (CII) markedly enhanced the clinical score and paw swelling. The severity of arthritic joint disease with a marked infiltration of lymphoid cells, proliferation of synovial membrane, pannus formation and destruction of articular cartilage was significantly enhanced in both groups receiving RF (RF-enhanced arthritis). Skin ulcers were also observed in some of these RF-enhanced arthritis mice, whereas no such signs were observed in CII-immunized mice without mRFs. Both IgM-RF and IgG-RF increased CII-specific IgG antibodies in circulation, and the severity of arthritis correlated with the production of high titres of anti-CII antibodies. In vivo treatment of RF-enhanced arthritis mice with an anti-CD4 MoAb or an anti-CD8 MoAb inhibited the induction and progression of arthritis in these mice. Administration of RF to severe combined immunodeficient (SCID) mice with arthritis developed by the transfer of spleen cells from CII-immunized mice, prolonged the arthritis and enhanced the severity. This murine model of RF-enhanced arthritis may provide a useful tool for analysing the pathogenesis of rheumatoid arthritis in RF-positive patients.

An evaluation of the effectiveness of three immunoglobulin G (IgG) removal procedures for routine IgM serological testing.

Three procedures for the removal of immunoglobulin G (IgG) from human serum were evaluated for their effectiveness in eliminating false-positive results caused by rheumatoid factor and in removing IgG from serum to reduce competing-IgG interference in IgM enzyme-linked immunosorbent assay (ELISA) testing. The procedures investigated employed two anti-human IgG diluents and a recombinant protein G-filled tube. The anti-human IgG was more effective than the protein G method in eliminating false-positive results caused by rheumatoid factor and removed 5.4% more IgG from serum samples in the normal range (< 1,700 mg/dl) and up to 16.4% more of the IgG from samples with elevated levels (> 1,700 mg/dl). The recombinant protein G removed less IgM than the anti-human IgG diluents; however, this difference did not affect the results of the ELISA. For these reasons, the in-house-developed anti-human IgG diluent proved to be the most effective and economical for IgM serological testing.

Production of IgM rheumatoid factor by normal lymphocytes after stimulation with preparations containing IgM rheumatoid factor from patients with juvenile arthritis.

Preparations containing IgM rheumatoid factor (RF) and hidden IgM RF were isolated from the serum samples of nine patients with juvenile rheumatoid arthritis. Six of these preparations stimulated lymphocytes from normal donors to produce IgG and IgM, of which up to 11% had IgM RF activity. In contrast, the polyclonal activator pokeweed mitogen also stimulated IgM production, but only 1% had IgM RF activity. A relation between the activator and IgM RF or hidden IgM RF is suggested. This is based on the positive correlation between IgM RF concentration in these preparations and their ability to stimulate lymphocytes to produce IgG, IgM, and IgM RF. These data indicate that preparations from patients with juvenile rheumatoid arthritis containing IgM RF and hidden IgM RF are potent stimulants of lymphocytes from normal donors, with specific production of IgM RF.

Measurement of complement activation products in patients with chronic rheumatic diseases.

Measurement of the complement activation products C1s:C1-inh, C3bP and C5b-9 by ELISA in plasma samples from normals, rheumatoid arthritis (RA) and systemic lupus erythematosis (SLE) patients showed significantly elevated levels in the two patient groups (P less than 0.0001 for C1s:C1-inh, C3bP and C5b-9) compared to normals. In seropositive RA patients there were significant correlations between the levels of the three complement activation complexes and IgM-RF, IgG-RF and IgA-RF. However, IgM-RF did not interfere with any of the ELISA systems. Mean levels of C1s:C1-inh, C3bP and C5b-9 were the same in paired plasma and synovial fluids; however, C3bP levels in the paired samples did not correlate with one another by rank. Our conclusions are that: (a) elevated plasma levels of these complement activation products are detectable in rheumatic diseases; (b) plasma levels of these complement activation products are related to Rheumatoid factor (RF) levels in seropositive RA patients; and (c) IgM-RF does not influence these solid-phase ELISA procedures.

Rheumatoid factor modulation of neutrophil superoxide generation enhancing activity of preformed immune complexes.

Heat aggregated human (HAG) IgG pretreated with total rheumatoid factors isolated from the serum of rheumatoid arthritis patients showed decreased superoxide generation enhancing activity as compared with HAG pretreated with buffer alone. Similarly, monoclonal IgM rheumatoid factor isolated from the serum of a patient with macroglobulinemia complicated by rheumatoid arthritis inhibited superoxide generation enhancing activity of HAG. On the other hand, superoxide generation enhancing activity of BSA-antiBSA immune complexes was not affected by preincubation with rheumatoid factors isolated from the sera of either rheumatoid arthritis patients or the macroglobulinemia patient. Rheumatoid factors isolated from rheumatoid arthritis serum were fractionated by high performance liquid chromatography and IgM-class and IgG-class rheumatoid factors were obtained. IgG-class rheumatoid factor significantly enhanced the superoxide generation enhancing activity of HAG, whereas IgM rheumatoid factor inhibited it. Rheumatoid arthritis sera showed significantly higher superoxide generation enhancing activity than normal sera. HAG preincubated with rheumatoid arthritis sera showed significantly lower superoxide generation enhancing activity than HAG preincubated with normal sera. These results suggest that factors inhibiting superoxide generation enhancing activity of HAG are present in rheumatoid arthritis sera, and that the responsible is IgM rheumatoid factor, whereas IgG rheumatoid factor enhances it. The factors that express superoxide generation enhancing activity in rheumatoid arthritis sera are suggested to be intermediate size immune complexes.

Generation of superoxide by immunologically stimulated normal human neutrophils and possible modulation by intracellular and extracellular SOD and rheumatoid factors.

Rheumatoid synovial fluids generated significantly greater amounts of superoxide, lysosomal enzymes, and superoxide dismutase from neutrophils into extracellular fluid than osteoarthritic synovial fluids. Rheumatoid factors isolated from serum suppressed superoxide-generating activity of performed immune complexes, but did not suppress that of intermediate-sized immune complexes isolated from RA serum. Synovial fluid neutrophils has a greater capacity to generate superoxide and lower intracellular superoxide dismutase activity, compared with peripheral neutrophils of the corresponding patients. These results suggest that neutrophil superoxide release may be modulated, both by rheumatoid factor and by intracellular and extracellular superoxide dismutase.

Detection of measles-specific IgM antibodies: comparison of 2-mercaptoethanol treatment, density gradient centrifugation, protein A-sepharose affinity chromatography, ion-exchange chromatography and haemadsorption techniques.

The sensitivity, specificity and effectivity of 5 methods for detection of measles-specific IgM antibodies were compared. A total of 371 sera from non-immunized as well as immunized measles patients was included into the study. The highest positive rate was achieved by the haemadsorption-immunosorbent (HIST) test. The density gradient (DG) centrifugation and the ion-exchange chromatography (IERCR) were 10-15% less effective. Protein A-Sepharose affinity chromatography (ACR) and 2-mercaptoethanol (2-ME) treatment showed positive results in the half of samples which had been positive in the HIST. The titres were in significant correlation (r = 0.90 to r = 0.8793 at P = 0.01). Specificity and reproducibility of the tests were good. Rheumatoid factor did not influence the results. In general, HIST was found as most sensitive and effective.

Complement activation by 19S IgM rheumatoid factor: relationship to disease activity in rheumatoid arthritis.

19S IgM rheumatoid factor (RF) in rheumatoid arthritis (RA) are polyclonal autoantibodies directed against the Fc piece of IgG. Rheumatoid patients with RF tend to have aggressive synovitis, nodules, and extraarticular manifestations. Although RF titer does not correlate with disease activity, RF activates complement (C) by the classical pathway. Thus, we postulated that selective stimulation of cell clones producing efficient C activating RF molecules might be associated with disease flares, independent of changes in serum RF concentration. To address the question, 42 patients with RA were evaluated prospectively. Serum RF concentration was measured by radioimmunoassay (RIA) and C activating activity by hemolytic assay. We then calculated the mean hemolysis (MH) of sensitized sheep erythrocytes (SRC) produced/ml of RF serum (MH/ml) and MH/microgram of RF as an expression of RF C activating properties (CAP). The following observations were made: RF CAP varied among the patients studied; RF CAP varied over time in individual patients; RF CAP differences varied in both groups independently from RF concentration; RF CAP correlated with both systemic and articular disease activity; and total RF concentration correlated with articular findings and nodules but less well with systemic disease activity.

Selective reactivity of antibodies to human immunoglobulins G, M, and A with rubella virus proteins.

Proteins of purified rubella virus were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with human sera and immunoglobulin class heavy-chain-specific peroxidase conjugates. The levels of rubella antibodies in these sera were predetermined by the radial hemolysis test, the density gradient centrifugation method for immunoglobulin M (IgM) antibodies, and IgG-, IgM-, and IgA-specific enzyme immunoassays. In immunoblotting, rubella-specific IgG antibodies reacted with both envelope glycoproteins (E1 and E2) and the capsid protein (C). In contrast, rubella IgM antibodies reacted predominantly with E1, whereas the specific reactivity of IgA antibodies was directed mainly to the capsid protein. Purified IgM rheumatoid factor added to IgG-positive, IgM-negative serum did not give false-positive reactivity in the immunoblotting test as it did in solid-phase enzyme immunoassays. The immunoglobulin class-specific reactivities with the different viral proteins are expected to have diagnostic applications.

Cellular myeloperoxidase activity in human monocytes stimulated by hyposialylated immunoglobulins and rheumatoid factors.

When hyposialylated , immunoglobulins become immunogenic and tend to form aggregates. In pursuit of the possibility that hyposialylated immunoglobulins (hs-Ig) can trigger human mononuclear phagocytic cells, we have investigated the effects of such hs-Ig on the myeloperoxidase (MPO) activity of these cells. The incubation of human monocytes with aggregated hs-Ig leads to the decrease of intracellular MPO activity. This decrease is dependent on the incubation time, on the amount of hs-Ig added, and on the degree of aggregation. Incubation with unaggregated hs-Ig has a similar effect, thus providing evidence that the loss of sialic acid residues per se is enough to render these molecules capable of decreasing the MPO content of phagocytic cells. Furthermore, human rheumatoid factors, isolated from the sera of rheumatoid arthritis patients, and previously characterized as hyposailylated Ig, interact in the same way with monocytes in triggering the MPO decrease. These observations imply that hs-Ig may be considered as active stimuli in the induction of inflammatory processes, through the initiation of oxidative reactions.

The detection of circulating immune complexes containing immunoglobulin G.

An immune complex assay using radiolabelled immunospecific antibodies against human IgG and polyethylene glycol precipitation (IgG-PEG assay) is described. The material reactive in this assay was evaluated using aggregated immunoglobulins, immune complexes prepared in vitro, sera of patients with a variety of disorders and normal human serum. Sucrose density gradient ultracentrifugation showed that only large-sized immune complexes (greater than 25 S) were detected. Comparison of the results of the IgG-PEG assay with those of the C1q binding assay showed a highly significant positive correlation (p less than 0.001). It was found that rheumatoid factors do not influence the results of the IgG-PEG assay. The method described in this study detects specifically immune complexes containing IgG and might be extended to the detection of other constituents of circulating immune complexes.

Effect of rheumatoid factors and normal human sera on immune complex-Fc gamma R interaction.

The results of this study demonstrate that inhibition of antibody-dependent cell-mediated cytotoxicity (ADCC) of human peripheral blood mononuclear cells (PBMC) by ovalbumin (OA)-IgG anti-OA immune complexes (IC) can be affected by rheumatoid factors (RF). Thus, pre-incubation of IC with IgM RF reduced the blocking effect of IC on IgG-Fc receptors (FC gamma R) measured by ADCC activity. However, the opposite effect could be observed when IgM RF was added after IC-Fc gamma R interaction. IgG RF did not modify these interactions significantly. In a previous report it was demonstrated that normal human sera (NHS) lead to recovery of the ADCC activity of PBMC that had been previously blocked by IC. IgM RF enhanced the recovery of ADCC by NHS, while IgG RF did not alter, or slightly decreased the ability of NHS to restore ADCC. These effects correlated with the different capacities of RF to activate complement (C). The results obtained show a new effect of RF on the regulation of immune mechanisms.

Self-associating IgG rheumatoid factors stimulate monocytes to release prostaglandins and mononuclear cell factor that stimulates collagenase and prostaglandin production by synovial cells.

Self-associating IgG rheumatoid factors isolated from three patients with rheumatoid arthritis activated human monocytes to release prostaglandin E2 as well as a previously described mononuclear cell factor, which stimulates human synovial cells to release additional prostaglandin E2 and collagenase. Thus, the immune complexes composed of self-associating IgG rheumatoid factors can contribute to the processes that destroy cartilage and bone in patients with rheumatoid arthritis.

Human complement activation by self-associated IgG rheumatoid factors.

IgG rheumatoid factors (IgG-RFs) undergo concentration-dependent self-association into dimers and higher polymers, as previously reported. The interactions of purified IgG-RF from the plasma of 3 patients with rheumatoid arthritis, with guinea pig and human complement were studied. Self-associating IgG-RFs were isolated by affinity columns and gel filtration. These preparations contained no detectable IgM and were composed only of IgG subclasses known to fix complement. Complement utilization of IgG-RF was compared with that of monomeric IgG, heat-aggregated IgG, and soluble rabbit IgG immune complexes. Although incubation of IgG-RF or monomeric IgG with 3 units of guinea pig or human complement resulted in decreased hemolysis of sheep erythrocytes sensitized with IgM hemolysin, these substances were less than 100 times as effective as heat-aggregated IgG or soluble immune complexes. The ability of human or guinea pig complement that had been incubated with IgG-RF to restore hemolytic activity to C4-deficient guinea pig serum served to distinguish Clq binding from complement cascade activation. IgG-RFs and monomeric IgG did not activate guinea pig complement cascade in contrast to aggregated IgG. IgG-RFs, however, activated human complement cascade; monomeric IgG only bound human Clq. These results indicate that self-associated IgG-RFs can activate human complement in fluid phase, but less effectively than aggregated IgG or large-latticed immune complexes.

Studies of phagocytic functions in rheumatoid arthritis. II. Effects of serum factors on phagocytic and metabolic activities of neutrophils.

A previous article has shown a diminished phagocytic and intracellular metabolic activity of the polymorphonuclear cells (PMN's) from patients with rheumatoid arthritis (RA). No correlation was found between PMN's functions with either clinical or laboratory expression of disease activity. In the present study the effects of rheumatoid sera on PMN's functions were investigated and the results revealed significant inhibition of phagocytic activity (PA) and nitroblue tetrazolium (NBT) dye reduction capacity of cells from RA as well as from a normal (N) control group. The number of PMN's showing aggregated latex particles on their surfaces was significantly higher in RA patient groups in the presence of rheumatoid sera. PMN's from N and RA groups were lysed in the presence of rheumatoid sera, with more significant destruction of RA-PMN's. No correlation was found between the PMN's function and the amount of circulating immune complexes (CIC's) or rheumatoid factor (RF). Significantly lower concentration of complement (C4) levels and higher IgA mean values were observed in the patient group compared to the standard values. The mechanism(s) underlying the impaired PA and NBT reduction may be due to defective intrinsic cell function as well as the extra cellular effects of blocking or lytic factors present in rheumatoid sera.

[The production of superoxide anion (O2-) by polymorphonuclear cells in patients with rheumatoid arthritis]. / La produzione di superossido anione (O2-) da parte dei polimorfonucleati di pazienti con artrite reumatoide.

Superoxide anion production has been determined in controls and RA patients PMNs stimulated with zymosan, rheumatoid synovial membrane, nodule and synovial fluid, rheumatoid factor, aggregated gamma-globulins, Mycoplasma and Epstein-Barr virus. Patients with RA showed an increased production of O2- after incubation with zymosan and rheumatoid tissue extracts or synovial fluid in comparison to normal controls. The findings indicate that rheumatoid PMNs become activated by different stimuli to produce an excess of O2- which can contribute to chronic inflammatory process.