IgG3 Cryoglobulin Research Articles

Acute onset small joint affliction in ankylosing spondylitis: A red herring

Glomerulopathy induced by IgG3 anti-trinitrophenyl monoclonal cryoglobulins derived from non-autoimmune mice. We have previously shown that murine IgG3 monoclonal autoantibodies with cryoglobulin activity, derived from lupus-prone mice, are able to induce glomerular lesions resembling the “wire-loop” lesion typically described for human lupus nephritis. In the present study, we have further assessed the nephritogenic potential of four IgG3 anti-hapten, trinitrophenyl (TNP), monoclonal antibodies (mAb) obtained from non-autoimmune mice immunized with TNP-conjugated foreign antigens. Our results showed that two of four IgG3 anti-TNP monoclonal cryoglobulins were capable of inducing glomerular lesions, characterized by voluminous intracapillary thrombi and mesangial deposition of PAS-positive materials, which differed from “wire-loop” lesions generated by IgG3 monoclonal cryoglobulins with autoantibody activities. These anti-TNP monoclonal cryoglobulins, however, failed to induce glomerular lesions when mice were kept at 37°C after the mAb administration. This finding formally proves that the cryoglobulin activity is critically involved in the development of glomerular lesions induced by IgG3 anti-TNP mAb. In addition, we have demonstrated a remarkable difference in the nephritogenic activities of two IgG3 anti-TNP mAb, which exhibit a marked sequence homology in the variable regions of their heavy and light chains (91.5% and 99.1% at the amino acid level, respectively) and an identical isoelectric point. Our results indicate first, that IgG3 monoclonal cryoglobulins are able to generate two different kinds of glomerular lesions, and second, that a subtle difference in variable region sequences may determine not only the nephritogenic activities, but also the type of glomerular lesions mediated by IgG3 cryoglobulins.

Induction of IgA Deposits and Glomerulonephritis by IgA Rheumatoid Factor

Induction of IgA Deposits and Glomerulonephritis by IgA Rheumatoid Factor

The Role of Complement in Cryoglobulin-Induced Immune Complex Glomerulonephritis

Many forms of glomerulonephritis are triggered by Ab localization in the glomerulus, but the mechanisms by which this induces glomerular inflammation are not fully understood. In this study we investigated the role of complement in a mouse model of cryoglobulin-induced immune complex glomerulonephritis. Several complement-deficient mice on a C57BL/6 and BALB/c genetic background were used and compared with strain-matched, wild-type controls. Cryoglobulinemia was induced by i.p. injection of 6-19 hybridoma cells producing an IgG3 cryoglobulin with rheumatoid factor activity against IgG2a of allotype a present in BALB/c, but not C57BL/6, mice. Thus, the cryoprecipitate in C57BL/6 mice consisted of the IgG3 cryoglobulin only (type I cryoglobulinemia) compared with IgG3-IgG2a complexes in BALB/c (type II cryoglobulinemia). The survival of mice was not affected by complement deficiency. Glomerular influx of neutrophils was significantly less in C3-, factor B-, and C5-deficient mice compared with wild-type and C1q-deficient mice. It did not correlate with C3 deposition, but did correlate with the amount of C6 deposited. Deficiency of CD59a, the membrane inhibitor of the membrane attack complex, did not induce an increase in neutrophil infiltration, suggesting that the generation of C5a accounts for the effects observed. There was no apparent difference between cryoglobulinemia types I and II regarding the role of complement. Our results suggest that in this model of cryoglobulin-induced glomerulonephritis the neutrophil influx was mediated by C5 activation with the alternative pathway playing a prominent role in its cleavage. Thus, blocking C5 is a potential therapeutic strategy for preventing renal injury in cryoglobulinemia.

A Critical Role for Sialylation in Cryoglobulin Activity of Murine IgG3 Monoclonal Antibodies

Cryoprecipitating IgG3 autoantibodies have been shown to play a significant role in the development of murine lupus-like autoimmune syndrome. However, the structural basis of IgG3 cryoprecipitation still remains to be defined. In view of the implication of positively charged amino acid residues present in variable regions in IgG3 cryoglobulin activity, we explored the role of terminal sialic acids in oligosaccharide side chains for the cryogenic activity of IgG3 mAb. Comparative oligosaccharide structural analysis of different cryogenic and non-cryogenic IgG3 mAb showed an inverse correlation between the extent of sialylation and cryogenic activity. The inhibitory role of sialylation was further confirmed by the demonstration of enrichment of less and more sialylated IgG3 in cryoprecipitated and noncryoprecipitated fractions, respectively, separated from four different cryogenic IgG3 mAb. Significantly, the sialic acid contents of the latter fraction became comparable to those of non-cryogenic IgG3 mAb. Finally, we observed that highly sialylated non-cryogenic IgG3 mAb was more potent in the inhibition of cryoprecipitation of cryogenic IgG3 mAb. Our results thus suggest that the content of negatively charged sialic acids in oligosaccharide side chains is one of the critical factors to determine IgG3 cryoglobulin activity, along with amino acid sequences of the IgG3 variable regions.

Level of galactosylation determines cryoglobulin activity of murine IgG3 monoclonal rheumatoid factor

AbstractAutoantibodies of the cryoprecipitating IgG3 isotype have been shown to play a significant role in the development of murine lupus–like autoimmune syndrome. At present, the structural basis of IgG3 cryoprecipitation and its role in autoantibody pathogenicity remain to be defined. Using molecular variants of an IgG3 monoclonal rheumatoid factor, 6-19, derived from an autoimmune MRL-Faslpr mouse, we have investigated the implication of charged residues in the heavy-chain variable (VH) region, potential CH3-linked oligosaccharides, and galactosylation of CH2-linked oligosaccharides in its cryoglobulin activity. The cryoglobulin activity of the IgG3 6-19 mutant bearing more negatively charged residues at VH 6 and 23 was found to be reduced but still highly significant, whereas that of the mutant lacking a potential CH3 glycosylation site remained unchanged. In marked contrast, IgG3 6-19 variants obtained from 6-19 heavy-chain transgenic mice displayed barely detectable cryoglobulin activity associated with an increased level of galactosylation in the CH2 oligosaccharide side chains. Thus, our data strongly suggest that the cryoglobulin activity of IgG3 6-19 autoantibody is critically determined by levels of galactosylation in the CH2 oligosaccharide side chains, whereas VH residues play a secondary role in 6-19 IgG3 cryoglobulin activity.

Modulation of renal disease in MRL/lpr mice genetically deficient in the alternative complement pathway factor B.

In systemic lupus erythematosus, the renal deposition of complement-containing immune complexes initiates an inflammatory cascade resulting in glomerulonephritis. Activation of the classical complement pathway with deposition of C3 is pathogenic in lupus nephritis. Although the alternative complement pathway is activated in lupus nephritis, its role in disease pathogenesis is unknown. To determine the role of the alternative pathway in lupus nephritis, complement factor B-deficient mice were backcrossed to MRL/lpr mice. MRL/lpr mice develop a spontaneous lupus-like disease characterized by immune complex glomerulonephritis. We derived complement factor B wild-type (B+/+), homozygous knockout (B-/-), and heterozygous (B+/-) MRL/lpr mice. Compared with B+/- or B+/+ mice, MRL/lpr B-/- mice developed significantly less proteinuria, less glomerular IgG deposition, and decreased renal scores as well as lower IgG3 cryoglobulin production and vasculitis. Serum C3 levels were normal in the B-/- mice compared with significantly decreased levels in the other two groups. These results suggest that: 1) factor B plays an important role in the pathogenesis of glomerulonephritis and vasculitis in MRL/lpr mice; and 2) activation of the alternative pathway, either by the amplification loop or by IgA immune complexes, has a prominent effect on serum C3 levels in this lupus model.

IFN-gamma is essential for the development of autoimmune glomerulonephritis in MRL/Ipr mice.

MRL/lpr mice develop lymphoproliferation and accelerated autoimmune glomerulonephritis from which they ultimately die. To investigate the role of IFN-gamma in the manifestation of the disease, we generated MRL/lpr mice lacking the IFN-gamma receptor (MRL/lpr gammaR -/-). The absence of IFN-gamma signaling had no effect on generalized lymphoproliferation, expansion of CD4- CD8- double-negative T cells, or hypergammaglobulinemia. By contrast, glomerulonephritis as detected by proteinuria and histology was absent in MRL/lpr gammaR -/- mice. While serum IgG1 anti-dsDNA Abs were increased in all three strains of MRL/lpr mice (gammaR +/+, +/-, -/-), those of the IgG2a and IgG3 isotypes were low in MRL/lpr gammaR -/- mice. Immune complexes and C3 deposition were dramatically reduced in the glomerular capillaries of MRL/lpr gammaR -/- mice compared with MRL/lpr gammaR +/+ and +/- mice. Therefore, IFN-gamma plays a key regulatory role in the development of nephritis in MRL/lpr mice. Low levels of IFN-gamma-dependent IgG2a and IgG3 autoantibodies in MRL/lpr gammaR -/- mice might protect them from the pathogenic features of IgG3 cryoglobulins and complement-activating IgG2a and IgG3.

The effect of VH residues 6 and 23 on IgG3 cryoprecipitation and glomerular deposition.

MRL/lpr mice spontaneously develop a lupus-like autoimmune syndrome characterized by immunopathologic manifestations such as necrotizing vasculitis of the skin and glomerulonephritis. A feature of this autoimmune syndrome is the production of extremely large amounts of monoclonal IgG3 cryoglobulins. The structural basis of IgG3 cryoprecipitation is not well understood. Although the IgG3 isotype is necessary for cryoprecipitation, not all IgG3 antibodies cryoprecipitate. It has been postulated that electrostatic charge may be influential in cryoprecipitation. To investigate this problem, the VH and VL sequences of a panel of IgG3 cryoglobulins and non-cryoglobulins were compared, with particular attention to charged amino acid differences. At VH residues 6 and 23 the cryoglobulins were more positively charged than their non-cryoglobulin counterparts. To analyze further the effect of charge on cryoprecipitation, the sequence of an IgG3 monoclonal cryoprecipitating rheumatoid factor was modified by site-directed mutagenesis. The more positive residues at VH 6 and 23 present in some of the cryoglobulin antibodies were mutated to the more negative residues found in the non-cryoglobulins. The results show that VH residue 6 affects cryoprecipitation while residue 23 does not. When injected into normal BALB/c mice, the unmutated antibody produced glomerular immune deposits and focal glomerulonephritis, whereas loss of cryoprecipitability by mutating residue 6 completely abrogated glomerular immune deposition and glomerular injury. In contrast, the mutation at residue 23 which retains cryoprecipitability reduced glomerular immune deposition and prevented glomerular injury.

Glomerulopathy induced by IgG3 anti-trinitrophenyl monoclonal cryoglobulins derived from non-autoimmune mice

We have previously shown that murine IgG3 monoclonal autoantibodies with cryoglobulin activity, derived from lupus-prone mice, are able to induce glomerular lesions resembling the "wire-loop" lesion typically described for human lupus nephritis. In the present study, we have further assessed the nephritogenic potential of four IgG3 anti-hapten, trinitrophenyl (TNP), monoclonal antibodies (mAb) obtained from non-autoimmune mice immunized with TNP-conjugated foreign antigens. Our results showed that two of four IgG3 anti-TNP monoclonal cryoglobulins were capable of inducing glomerular lesions, characterized by voluminous intracapillary thrombi and mesangial deposition of PAS-positive materials, which differed from "wire-loop" lesions generated by IgG3 monoclonal cryoglobulins with autoantibody activities. These anti-TNP monoclonal cryoglobulins, however, failed to induce glomerular lesions when mice were kept at 37 degrees C after the mAb administration. This finding formally proves that the cryoglobulin activity is critically involved in the development of glomerular lesions induced by IgG3 anti-TNP mAb. In addition, we have demonstrated a remarkable difference in the nephritogenic activities of two IgG3 anti-TNP mAb, which exhibit a marked sequence homology in the variable regions of their heavy and light chains (91.5% and 99.1% at the amino acid level, respectively) and an identical isoelectric point. Our results indicate first, that IgG3 monoclonal cryoglobulins are able to generate two different kinds of glomerular lesions, and second, that a subtle difference in variable region sequences may determine not only the nephritogenic activities, but also the type of glomerular lesions mediated by IgG3 cryoglobulins.

Nephritogenic antibodies in lupus nephritis.

A critical discussion of data on the possible role of IgG3 cryoglobulins, cross-reactive anti-DNA antibodies and anti-DNA idiotypes in the pathogenesis of lupus nephritis is included. A further possibility involving cationic nuclear autoantigens is presented in detail. Histone was employed as a model antigen, representative of this group. Histones were shown to have high affinity for the rat GBM; they could also act as planted antigen and induce IC formation; furthermore they were able to mediate the binding of highly anionic DNA. The demonstration of histones in glomerular deposits in both lupus mice and patients with SLE is important evidence, linking such molecules with the kidney lesions.

IgG3 cryoglobulins in autoimmune MRL-lpr/lpr mice: immunopathogenesis, therapeutic approaches and relevance to similar human diseases.

MRL-lpr/lpr mice spontaneously develop an autoimmune disease resembling systemic lupus erythematosus and rheumatoid arthritis. One of the unique serological abnormalities in this strain is remarkably high concentrations of cryoglobulins. Analysis of immunoglobulin components in their cryoglobulins has shown selective enrichment of a particular IgG subclass, IgG3. As IgG3 enrichment is also found in two other cryoglobulins, which are induced after injection with bacterial lipopolysaccharides or infection with malaria, IgG3 apparently represents a major source of murine cryoglobulins. Studies on murine IgG3 monoclonal antibodies (mAbs) have clearly shown that murine IgG3 have the unique physiochemical property to self associate through non-specific IgG3 Fc-Fc interaction, and that most of them can generate monoclonal cryoglobulins. Most strikingly, IgG3 monoclonal cryoglobulins with rheumatoid factor (RF) activity induce extensive pathological manifestations: skin vascular purpura and glomerulonephritis with 'wire loop' lesions. Although the cryoglobulin activity of IgG3 RF mAb is solely responsible for the generation of glomerular lesions (both RF and cryoglobulin activities are necessary for skin vascular lesions), the absence of nephritogenic activity by some IgG3 cryoglobulins supports the idea that qualitative features of cryoglobulins are critical to determine their pathogenic activity. The demonstration of a positive correlation between the production of IgG3 cryoglobulins and the development of lupus nephritis in MRL-lpr/lpr mice further substantiates the pathological importance of cryogenic autoantibodies. On the other hand, it should be emphasised that non-cryogenerating IgG3 autoantibodies may not be harmful, but even protective, as a result of their interaction with pathogenic IgG3 cryoglobulins. Finally, the development of an experimental model of cryoglobulinaemia associated with vascular and glomerular disease certainly represents an invaluable opportunity to study the molecular mechanisms responsible for the generation of cryoglobulins and their associated tissue lesions, and also to assess various therapeutic approaches. Our demonstration that anti-idiotypic mAb can prevent the pathogenic effects of the cryoprecipitable IgG3 RF mAb suggests strongly that such a therapeutic approach might be successful in similar diseases in man.

Induction of "wire-loop" lesions by murine monoclonal IgG3 cryoglobulins.

We have recently demonstrated that an IgG3 rheumatoid factor (RF) monoclonal antibody (mAb), clone 6-19, derived from unmanipulated autoimmune MRL/MpJ-lpr/lpr mice, is able to generate cryoglobulins via a non-immunological IgG3 Fc interaction, and to induce an acute glomerulonephritis associated with cryoglobulinemia. Using this experimental model, we have characterized the glomerular lesions induced by the 6-19 RF monoclonal cryoglobulin, in particular the ultrastructural localization of the cryoglobulin deposits. Although their initial localization was confined to the mesangium, the 6-19 cryoglobulins were progressively accumulated in the subendothelial spaces of glomerular capillary walls, leading to the formation of glomerular lesions resembling the "wire-loop" lesion characteristically described for lupus nephritis. In addition, we have found that identical glomerular "wire-loop" lesions were induced by the 6-19-J558 hybrid antibody, composed of the 6-19 gamma 3 heavy chain and J558 lambda 1 light chain, which loses the RF activity, but retains the cryoglobulin activity. These results strongly suggest that the direct deposition of IgG3 cryoglobulins by itself, without involvement of immune complex formation, results in the generation of the classical "wire-loop" lesion characteristic of lupus nephritis. In addition, we have found that similar "wire-loop" lesions were generated by one anti-DNA mAb derived from (NZB x NZW)F1 hybrid mice, and two of four IgG3 mAb of unknown specificities, derived from MRL/MpJ-lpr/lpr mice. The absence of significant glomerular lesions, in spite of large amounts of cryoglobulins, in mice receiving two IgG3 mAb suggests the importance of physicochemical property of cryoglobulins to provoke glomerular lesions.

IgG3 is the major source of cryoglobulins in mice.

A total of 20 of 23 IgG3 mAb derived from unmanipulated autoimmune MRL/MpJ-lpr/lpr mice was shown to generate cryoglobulins which were composed exclusively of IgG3. Although three IgG3 mAb failed to develop cryoglobulins, they were able to bind nonspecifically to any IgG3 molecules as efficiently as cryoprecipitable IgG did. The direct role of the gamma 3 constant region for the generation of cryoglobulins was demonstrated by the following findings: 1) the cryoglobulin activity was independent of the specificity of the IgG3 mAb, 2) no mAb other than those of the IgG3 subclass, including IgM rheumatoid factors (RF), generated cryoglobulins, and 3) the cryoglobulin activity was gained after the Ig class switch of mAb from IgM to IgG3. Analysis of Ig components in three different sources of cryoglobulins, either induced by the injection of bacterial LPS or by the infection with Plasmodium yoelii in BALB/c mice or developed spontaneously in MRL/MpJ-lpr/lpr mice, revealed the selective concentration of IgG3 in these cryoglobulins; greater than 99%, 73% and 58% of IgG recoverable from these three cryoglobulins, respectively, were IgG3. This further attests to the major role of IgG3 in the generation of cryoglobulins in mice. In addition, the enhanced formation and even induction of IgG3 cryoglobulins in the presence of IgM anti-IgG3 RF mAb, and the enrichment of IgM RF in LPS- or malaria-induced cryoglobulins indicated that IgM RF can be involved in the generation of cryoglobulins by interacting with noncryoprecipitable IgG3 as well as cryoprecipitable IgG3.

Temperature dependent activation of the alternate complement pathway by an IgG cryoglobulin

A patient with chronic membranoproliferative glomerulonephritis is presented whose serum contains a monoclonal IgG3 cryoglobulin. The presence of persistent hypocomplementemia suggested the possibility that the cryoglobulin, upon cold-induced precipitation, was capable of activating the complement system. Because visible cryoprecipitation commenced in vitro at 30 degrees C, the patient's serum and normal serum had been added the isolated cryoglobulin were repeatedly cooled to 30 degrees C and rewarmed to 37 degrees C. This reproduction of the in vivo counterpart of blood circulating through an extremity exposed to the cold resulted in activation of C3-proactivator (properdin factor B), C3 cleavage, and a 78% reduction in total hemolytic complement. This study demonstrates that IgG is capable of activating complement via the alternate pathway and reveals a mechanism through which this can occur in vivo; namely, by means of temperature dependent polymerization. In addition, we postulate that episodic complement activation initiated by the cryoglobulin contributed to the development of glomerulonephritis in this patient.

The Unique Molecular Weight of the Heavy Chain from Human IgG3

Abstract Human IgG consists of four subclasses (IgG1, IgG2, IgG3 and IgG4) which are known to differ from each other in certain antigenic, structural and biologic properties (1–5). In particular, the IgG3 subclass has many unique properties and it is catabolized faster than the other three subclasses (6, 7). Most monoclonal IgG cryoglobulins with anti-IgG activity belong to this subclass (8) as do IgG myeloma proteins that exhibit marked concentration- and temperature-dependent aggregation (9). In addition, IgG3 is the only subclass which does not react with staphyloccal protein A (10). One interesting structural feature unique to IgG3 is five inter-heavy chain disulfide bonds (11). Recently we found that gel filtration of a IgG3 cryoglobulin on a Sephadex (Pharmacia Fine Chemicals, Piscataway, N. J.) G-200 column at pH 3.9 yielded an elution position ahead of human Cohn fraction II (FII).