Soluble immune complexes binding to human monocytes and polymorphonuclear leucocytes.

Soluble human serum albumin anti-albumin immune complexes (IC) have been shown to bind to freshly isolated human peripheral blood monocytes and polymorphonuclear leucocytes (PMN) in vitro. Binding sites on both cell types were saturable and specifically inhibited by heat aggregated IgG1 and IgG3 subclasses. PMN contained a greater number of binding sites than monocytes although the affinity was similar for both cell types. The enhanced binding of IC by both cell types observed after incubation at low pH (pH 6.0) was a consequence of increased affinity of the PMN binding site and an increase in the number of sites in monocytes. Binding of IC by both cell types was inhibited by normal human serum. Enhanced IC binding associated with enhanced affinity and number of sites was observed in PMN and monocytes preincubated in suspension with trypsin. However, monocytes exposed to trypsin while adherent to microexudate coated flasks demonstrated a marked increase in affinity without any change in the number of sites.     

A simple radiolabelled rheumatoid factor binding assay for the measurement of circulating immune complexes.

A simple assay for circulating immune complexes has been developed and the optimal conditions for reaction defined. Partially purified radiolabelled polyclonal or monoclonal rheumatoid factor was incubated with aggregated IgG or soluble immune complexes and the resultant macromolecule precipitated with 3% polyethylene glycol. Monoclonal rheumatoid factor was much the better reagent, allowing the detection of approx. 0.35microgram/ml or more of aggregated IgG. Soluble immune complexes formed in vitro at between x2--x20 antigen excess were detectable over a wide range of molecular size. Clinical studies indicated that the assay is a useful addition to the currently available techniques for measuring circulating immune complexes.     

Circular dichroism of immune complexes with rheumatoid factor activity

The circular dichroism (CD) of IgG complexes with RF activity and that of monomeric IgG without this activity, and isolated from the same individual, were compared. The IgG complexes had a significantly deviant CD spectrum in the near u.v. region, both before and after dissociation, whereas the monomeric IgG had a normal spectrum. Immunoglobulins were isolated from the same serum with the use of specific antiserum against unique determinants in some IgG complexes with RF activity. Both before and after dissociation the CD spectrum in the far u.v. region of these immunoglobulins differed significantly from that of the above-mentioned preparations. The results confirmed that the structure of IgG in the RF-active complexes differed from that of normal IgG. The immunoglobulins with the unique determinants had, in turn, a structure that was not found in the pool of the RF-active IgG molecules or in normal IgG.     

IgG rheumatoid factor in subacute bacterial endocarditis: relationship to IgM rheumatoid factor and circulating immune complexes.

With recently developed radioimmunoassays, we have been able to study the levels and properties of IgG rheumatoid factor (IgG RF) and IgM rheumatoid factor (IgM RF) in patients with subacute bacterial endocarditis (SBE), as well as the relationship of these autoantibodies to circulating immune complexes. We found significantly elevated amounts of IgG RF and IgM RF in SBE sera. The IgG RF chromatographed on Sepharose 6B as an intermediate complex, indistinguishable from the pattern seen in rheumatoid arthritis. RF levels peaked later in the course of SBE than did levels of circulating immune complexes. With antibiotic treatment RF levels declined, although not as fast nor as completely as circulating immune complexes. These results suggest that both IgG RF and IgM RF in SBE may be part of a polyvalent antibody response to elevated levels of circulating immune complexes which do not themselves contain RF.     

Suramin inhibits the binding of complement-fixing antibody/double-stranded DNA immune complexes to CR1

The effects of varying concentrations of heparin and suramin on the complement-mediated binding of antibody/double-stranded DNA immune complexes to red blood cells (RBCs) and Raji cells have been investigated. If the immune complexes are briefly opsonized with complement, suramin can block binding to both cell types, and heparin can block binding to RBCs. In addition, if these complexes are first allowed to bind to RBCs or Raji cells, relatively brief incubations in suramin are sufficient to cause release of the complexes from the cells' C3b receptors. The potential clinical and diagnostic implications of these findings are discussed.     

Histidine-rich glycoprotein prevents the formation of insoluble immune complexes by rheumatoid factor

In previous studies we have shown that histidine-rich glycoprotein (HRG), a relatively abundant plasma protein, can bind to immunoglobulin G (IgG) and inhibit the insolubilization of IgG-containing immune complexes (IC). It was of interest, therefore, to determine whether HRG can inhibit the formation of insoluble IC (IIC) resulting from the interaction of rheumatoid factor (RF) with human IgG-containing IC. Light scattering techniques were used to examine the effect of HRG on the formation of IIC between RF and IC containing human IgG according to three different models. In all three models physiological concentrations of HRG could block the formation of IIC induced by RF. Optical biosensor studies of the RF-IgG interaction also revealed that HRG can mask the epitopes on IgG recognized by RF. Additional studies examined whether HRG can solubilize already formed IIC and demonstrated that HRG can, in fact, partially solubilized IIC. These data indicate that HRG can regulate the formation of IIC induced by RF at three levels: namely by inhibiting the initial recognition of IgG containing IC by RF, by inhibiting the subsequent insolubilization of IgG containing IC by RF and by solubilizing already formed IIC. Collectively, these findings suggest that HRG may be an important inhibitor of the formation of pathogenic IC in diseases such as systemic lupus erythematosus and rheumatoid arthritis.     

A technique for the purification of immune complexes using rheumatoid factor.

A method for the purification of immune complexes (IC) from human serum is described, using as a model complexes of tetanus toxoid with human antitoxoid antibodies (TAT). The technique is based on the ability of IC to bind to tubes coated with rheumatoid factor (RF). Small amounts of TAT IC were added to human serum and the mixtures were rotated overnight in tubes coated with RF. The tubes were then washed and the bound material was eluted with sodium dodecyl sulphate and iodinated with 125I. Analysis of the labeled preparation by electrophoresis in polyacrylamide gels revealed the presence of the toxoid component. The technique should be useful in isolating small amounts of IC for analytical purposes.     

The stimulation of IgM rheumatoid factor from human B lymphocytes by rheumatoid arthritis complement-activating immune complexes.

Immune complexes from rheumatoid arthritis (RA IC) and Hodgkin's disease (HD IC) sera were separated on an anti-C3g affinity column and their ability to stimulate the production of IgM and IgM RF by normal and RA B lymphocytes tested in a culture system in vitro. RA IC stimulated IgM production of which up to 91.3% had IgM RF activity. HD IC were incapable of stimulating the production of IgM and IgM RF. The stimulation of IgM and IgM RF production by RA IC required de novo protein synthesis. Both RA IC and HD IC were capable of significantly inhibiting (from 47.6 to 72.0%) pokeweed mitogen (PWM)-induced and goat F(ab)2 antihuman mu-induced B lymphocyte proliferation. Thus it is proposed that IC present in human pathological sera may regulate immunoglobulin production by an effect on B lymphocyte proliferation while some may, in addition, be capable of inducing IgM RF production from such cells.     

A reappraisal of the monoclonal rheumatoid factor test for circulating immune complexes: a comparison of two monoclonal rheumatoid factor reagents

Assays involving monoclonal rheumatoid factor (mRF) reagents are frequently used for the detection of circulating immune complexes, particularly in the rheumatic diseases. A study has been performed to investigate the interaction of two purified mRF reagents with normal sera, sera and synovial fluid from patients with rheumatic diseases and with heat-aggregated human IgG used as a model of immune complexes. Interaction has been measured by a simple laser nephelometric technique and a sensitive radiolabelled mRF precipitation assay. Both mRF reagents showed little reactivity with normal sera but reacted strongly with many of the pathological specimens. Similarly both mRFs reacted with large molecular sized heat aggregates of IgG while a variable reactivity was found with uncomplexed or monomeric IgG. However in pathological sera, both mRFs reacted predominantly with monomeric IgG and a significant correlation was found between the two reagents. This reactivity with monomeric IgG remained after separation of pathological sera in low pH sucrose gradients suggesting it was not due to the presence of small immune complexes of the classical type. In addition no reactivity was found with either reagent with IgG-RF intermediate complexes. It is concluded that mRF reagents are not specific for IgG containing immune complexes. They also react with monomeric IgG and this reactivity is particularly prominent in certain pathological sera. The possible nature of this reactive monomeric IgG is discussed.     

Studies on rheumatoid factor: I. The effect of rheumatoid factor on the clearance of preformed immune complexes in mice.

The blood clearance of passively transferred immune complexes (IC), preformed at different antigen-antibody ratios, was measured in mice with LPS-induced rheumatoid factor (RF) and in normal controls. RF had a differential effect on IC clearance, significantly enhancing clearance of complexes formed in antibody excess and slight antigen excess but inhibiting the clearance of complexes formed in moderate (x10) antigen excess. The results are discussed with regard to the mechanisms of IC clearance and suggest that RF may play an important role in governing the behaviour of immune complexes in vivo.     

Circulating immune complexes and complement in rheumatoid arthritis

Rheumatoid arthritis is characterized immunologically by the detection of rheumatoid factor (RF) and circulating immune complexes (CIC). The pathogenesis role played by these CIC has been discussed a long time. A part of this theoretical question, it could be of interest to know if these technics could help the clinician in the diagnosis or in the follow up of the patients with RA.     

An enzyme-immunoassay method for detecting circulating immune complexes by inhibition of polyclonal rheumatoid factor.

An enzyme-immunoassay for circulating immune complexes (IC-EIA is described which depends on inhibition of binding of rheumatoid factor (RF) to aggregated IgG. The assay involves 3 steps: (1) sera are pretreated with an aggregated IgG sorbent in order to remove any endogenous RF; (2) absorbed sera are than incubated with a polyclonal RF of known titre and an aggregated IgG sorbent; (3) after washing, the amount of RF on the solid phase is revealed by an IgG-glucose oxidase conjugate.Results are expressed as per cent inhibition of RF fixation. The assay range extends from 1 to 100 mg/l. Intra- and inter-assay coefficients were 6 and 9% respectively.Specificity has been assessed by various tests: dose-response curve with aggregated monomeric or denatured IgG and human albumin localization of the inhibitory fraction of sera after Sephadex G-200 chromatography, absence of interference of IgG, and use of artificial IC.Poor correlation was obtained between IC-EIA and other techniques.The method demonstrated the occurence of IC in a large proportion of patients with viral hepatitis, schistosomiasis and rheumatoid arthritis and their absence in normal subjects.     

Influence of serum complement and rheumatoid factor on detection of immune complexes by the C1q and monoclonal rheumatoid factor solid-phase assay

Soluble purified monoclonal and polyclonal rheumatoid factor, total serum complement, and soluble C1q all inhibit the detection of model tetanus toxoid/anti-toxoid immune complexes in the solid-phase C1q assay. The binding of these immune complexes to solid-phase monoclonal rheumatoid factor is less inhibited by soluble C1q and by total serum complement, but clearly decreased by soluble monoclonal or polyclonal rheumatoid factor. Serum complement does not reduce the size of these model complexes. We recommend the use of low ionic strength EDTA (10 mM) to partly neutralize the complement-mediated inhibition. This procedure is shown to be superior to currently used higher EDTA concentrations and to the use of IgG-Sepharose.     

IgEb immune complexes activate macrophages through FcgammaRIV binding

Because functional analysis of Fc receptors (FcRs) relies heavily on mouse models, the identification of another Fcgamma receptor is particularly noteworthy. We demonstrate that FcgammaRIV, identified here as the mouse ortholog of primate FcgammaRIII, required association of the FcR gamma-chain for optimal expression and function on myeloid cells; its signaling potential was also enhanced by a cytoplasmic 'YEEP' motif that was able to recruit the adaptor molecule Crk-L and phosphatidylinositol-3-OH kinase. Unexpectedly, FcgammaRIV 'preferentially' bound immunoglobulin E antibodies of the 'b' allotype (IgE(b)) as well as IgG2a and IgG2b antibodies. Ligation of FcgammaRIV by antigen-IgE(b) immune complexes promoted macrophage-mediated phagocytosis, presentation of antigen to T cells, production of proinflammatory cytokines and the late phase of cutaneous allergic reactions. IgE(b) antibody-mediated modification of macrophage responses may therefore influence mouse asthma models and strain-dependent differences in parasite susceptibility.     

IgG rheumatoid factor, complement and immune complexes in rheumatoid synovitis and vasculitis: comparative and serial studies during cytotoxic therapy.

IgG and IgM rheumatoid factors (IgG-RF and IgM-RF), complement and three assays for immune complexes were measured in 22 patients with rheumatoid arthritis (RA) complicated by either chronic active synovitis or vasculitis. Patients with vasculitis had relatively inactive arthritis but had higher titres of rheumatoid factors, especially IgG-RF, anticomplementary activity (ACA) and lower levels of C4 than those with synovitis. Clq-binding and platelet aggregation (PA) levels were similar in both groups. Serial measurements during cytotoxic therapy showed a close temporal relationship between the clinical features of vasculitis and levels of IgG-RF, ACA and C4 both with remission and with relapse. We suggest that immune complexes containing IgG-RF which activate complement and are detected by ACA are useful markers of rheumatoid vasculitis and may be important in its pathogenesis.     

In vitro IgG rheumatoid factor production by CD5-negative murine B cells in response to immune complexes of lipopolysaccharide.

We studied the in vitro production of rheumatoid factor (RF) by spleen cells of normal adult mice. IgG RF cross-reactive with rabbit IgG was produced in response to immune complexes of TNP-lipopolysaccharide (LPS) with murine IgG anti-TNP antibody in an Fc-specific manner, but not to a mixture of IgG and LPS. Antibody-uncomplexed LPS induced little IgG RF production, but suppressed the subsequent IgG RF response to antibody-complexed LPS, whereas IgM RF was induced by either LPS or antibody-complexed LPS. The IgG RF production followed as rapid a time course as IgM RF production; the rate of IgG RF production reached its maximum soon after a lag period of 1 day and declined after 5 days. Treatment of splenic B cells from BALB/c mice with anti-Ly-1.2 antibody and rabbit complement resulted in a selective reduction of IgM RF production by 90%, with little effect of IgG RF production. These results suggest that IgG RF is derived primarily from CD5- memory B cells which have been developed in normal mice by an unknown mechanism. Unlike the CD5+ precursor cells for IgM RF, these memory cells are unresponsive to polyclonal stimulation by LPS but are activated by simultaneous stimulation by aggregated Fc epitopes and the mitogenic stimulus from LPS.     

Correlation of antibody to rheumatoid arthritis associated nuclear antigen and immune complexes to disease activity in patients with rheumatoid arthritis

Twenty-seven patients with rheumatoid arthritis (RA) maintained on drug regimens were studied monthly for 6-10 months. Disease activity was assessed and levels of anti-RA associated nuclear antigen (RANA) and immune complexes were determined. Anti-RANA generally paralleled disease activity in 64% of cases. Immune complex levels paralleled disease activity in 56% of cases and paralleled anti-RANA in 52% of patients. Immune complexes paralleled anti-RANA together with disease activity in only 33% of patients. Anti-RANA and/or immune complex levels paralleled disease activity indices in 82% of cases. Significant fluctuations (greater than or equal to four-fold) in anti-RANA were frequently found (65%) and were associated with concordant changes in immune complex levels 50% of the time and with changes in disease activity indices 59% of the time. The data suggest that levels of anti-RANA and immune complexes may be important in RA and warrant further investigation.     

Molecular analysis of complement-fixing rheumatoid synovial fluid immune complexes.

The characteristics of the solid-phase conglutinin method for the isolation of C3-containing complexes from the synovial effusions of rheumatoid arthritis patients were assessed. All major proteins in such complexes were identified and shown to be either immunoglobulin or complement components. The high proportion of IgM and the association between complexed IgM and latex agglutination titre suggest that IgM rheumatoid factor, probably binding to self-associated IgG antiglobulins, is of major importance in the formation of complement-fixing complexes. A minority of samples contained unidentified trace components and these differed from one fluid to another. The levels of complexed immunoglobulins were closely correlated to the titres of synovial fluid antiglobulins. The data accords with the view that autosensitization to IgG plays the primary role in the development of immunopathological features of rheumatoid arthritis.     

Rheumatoid factors in immune complexes of patients with rheumatoid arthritis

Conclusions Substantial evidence indicates that humoral immunity through antigen-antibody complex formation contributes to the pathogenesis of RA. Several studies have examined the composition of immune complexes present in the synovial fluid of patients with RA. The constituents of these immune complexes have been immunoglobulins and complement components. Only a few polypeptides in these immune complexes have not been identified. These unidentified components account for only a few percent of total proteins present. These results suggest that rheumatoid factors and their antigens are quantitatively the important ingredients of immune complexes in synovial fluid of patients with RA. The immune deposits in the superficial layers of articular cartilage from patients with RA contain RFs and antibodies to type II collagen.IgG RFs are unique autoantibodies in that they can form immune complexes with pathogenic potential by self-association and without the presence of separate antigen molecules. Self-association becomes possible when the antigenic specificity of IgG RFs is directed to antigenic determinants present on the Fc region of the same molecule. Self-association of IgG RFs is enhanced in a milieu of high concentration of these antibodies and in relative paucity of normal IgG — i. e., conditions that exist in the synovial tissue where IgG RFs are synthesized.Relatively little attention has been devoted to antigenic specificity of RFs in relation to disease. The self-associating IgG RFs illustrate how the antibody specificity and presence of antigenic determinants can contribute to formation of unique immune complexes. One of the major antigenic determinants for IgM RFs is in the C2-C3 domain interface and involves some of the same amino acids that constitute the site for binding SPA. The specificity for self-associating IgG RFs is directed to the same antigenic determinant. Fc binding proteins from Streptococci and the Fc binding proteins induced by herpes type 1 virus bind to the same or overlapping areas of human IgG. The reasons for this similarity of binding to IgG between microbial Fc binding proteins and RFs is not apparent. This relationship, however, may stimulate the synthesis of some RFs by an internal image autoantiidiotype mechanism.