Differences in the relationship of specificity to titre and functional affinity between circulating Ga- and pan-reactive IgM rheumatoid factors in rheumatoid arthritis

N. Milosevic-Jovcic, D. Ciric, L. Hajdukovic-Dragojlovic1 and V. Mircetic2

Institute for Medical Research, 1 Institute for the Application of Nuclear Energy (INEP) and 2 Institute of Rheumatology, Clinical Center of Serbia, Belgrade, Serbia and Montenegro

Correspondence to: N. Milosevic-Jovcic, Institute for Medical Research, Dr Subotica 4, 11129 Belgrade, Serbia and Montenegro. E-mail: nadamj{at}imi.bg.ac.yu


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. To determine if there are the differences in titre and functional affinity for immunoglobulin (Ig) G subclasses and glycoforms between the Ga- and pan-specific IgM rheumatoid factors (RFs) present in the sera of patients with rheumatoid arthritis (RA), and to determine whether these two broad specificities have different functional roles in RA.

Methods. We used direct ELISA and modified ELISA to study the binding of IgM RF in the sera of 32 patients with RA with a range of RF titres to a panel of 14 IgG paraproteins of all four subclasses, some allotypes and different glycosylation patterns.

Results. Pan-specific RFs were mostly found in RA sera with high RF titres, and these RFs generally had higher avidity. A trend towards higher avidity of RFs with higher titre was observed for pan-specific, but not for Ga-specific RFs. With increasing titre, pan-specific RFs tended to react strongly with fucosylated and bisected variants of hypogalactosylated IgG3 of G3m(b1) allotype and hypergalactosylated IgG4 of 4a allotype.

Conclusion. Among high-titred pan-specific IgM RFs, there is a subpopulation responsible for strong anti-IgG activity in RA. The possible mechanisms of production of pan- and Ga-specific RFs are discussed.

KEY WORDS: Pan and Ga RFs, Avidity, Titre, IgG glycoforms, Rheumatoid arthritis


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Rheumatoid factors (RFs) are tolerance-resistant autoantibodies that bind epitopes located in the Fc region of IgG molecules. They may have a physiological immunoregulatory function in healthy individuals, and their transient increase in various chronic inflammatory diseases may reflect a natural response to immune complexes. Continual production of RFs at a high level is typical of rheumatoid arthritis (RA), in which they may have a pathological role and contribute to disease progression [1]. It has been speculated that disease-specific RFs directed against a disease-relevant antigen are present in patients with RA [2]. In particular, the pathogenic potential has been ascribed to a select subgroup of RFs reactive with agalactosylated (G0) immunoglobulin (Ig) G [3, 4], although the gross specificity profiles of these RFs for IgG subclasses remain uncertain. Bonagura et al. [5] refocused attention on the pan- and Ga-specificity of human RFs in RA, when they found that 29% of synovial IgM RFs from patients with RA were pan-specific (reactive with all four subclasses of IgG), while only 2% of RFs from individuals without joint disease showed this reactivity pattern. In contrast, Ga-specificity, i.e. reactivity with IgG1, IgG2 and IgG4, was commonly found in patients with RA and individuals without joint disease. Although these findings have indicated that potentially pathogenic RFs may be present within the pan- rather than the Ga-reactive pattern, no information has appeared so far regarding the properties of these two types of RFs, such as titre and avidity, which are likely to be important in determining the degree of the RF pathogenicity in RA.

We report here on the presence of high-titre pan-specific RFs in RA sera, which, with increasing titre, tend to react more intensely with fucosylated and bisected variants of hypogalactosylated IgG3 of G3m(b1) allotype and hypergalactosylated IgG4 of 4a alotype.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
RF-positive sera originated from 32 patients with RA (ARA criteria for RA diagnosis). The titre, as measured by the latex agglutination test, ranged from 1:40 to 1:1280.

IgG antigens
Fourteen highly purified IgG paraproteins of known subclass, allotype and glycosylation status were selected for analyses. IgGs were assigned to an IgG glycoform on the basis of their reactivity with sugar-specific lectins in competitive lectin-binding ELISA (ELBA), lectin-immunoblotting assay and cross-affinity immunoelectrophoresis [6]. Subclass-dominant glycosylation profiles were G0 (IgG2), G0f (IgG1), G0fb (IgG3), G2f and G2fb (IgG4).

RF quantitation
A direct ELISA (dELISA) was used as described [2]. To minimize possible interference due to IgG and IgA RF, RF-positive sera were diluted 1:200, and reactions giving an optical density (405 nm) greater than 0.300 were considered positive.

RF avidity measurement
Functional affinity (avidity) of rheumatoid factors was measured by modified ELISA (mELISA) as described [4]. mELISA was performed in the same manner as dELISA up to the IgM detection stage, before which 2 M guanidine HCL was added. The percentage of residual RF binding after treatment with guanidine to the total RF binding in the absence of guanidine was calculated and considered a measurement of avidity.

Statistical analysis
Statistical analyses were performed on 306 different RF–IgG interactions of measurable avidity. For all statistical determinations, including linear regression analysis, Prism Pad software (version 3.0; San Diego, CA, USA) was used. Confidence intervals for the difference between the slopes of the regression lines were calculated.

This study was performed according to the standards and rules for clinical investigations set by the Ethics Committee of the Institute of Rheumatology of Belgrade (which comply with international standards), and thereby received its approval. The patients were informed that their sera would be used for scientific purposes. All patients gave informed consent.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pan and Ga RF specificity and RF titre
IgM RF in the majority of sera (17/32) reacted with all four IgG subclasses (pan-specific RF). Ten of 32 RFs reacted with IgG1, 2 and 4 (Ga-specific). Pan-specific RFs were mostly found in sera with high RF titre. Among RFs with the highest titre (1:1280), 83% were pan-specific and 16% were Ga-specific. Among RA RFs with the lowest titre (1:40), 75% were Ga-specific and 25% were pan-specific.

Pan and Ga RF specificity and RF avidity
The mean RF avidity was 10.84% (range 0–40.7%), as calculated from a total of 306 interactions in which, after treatment with dissociating agent, RFs remained attached (% residual binding) to one or more of 14 IgGs. In relation to the mean value, the pan-specific RFs generally had high avidity (12.47%) and Ga-specific RFs had low avidity (7.59%) (Fig. 1A). Pan-specific RFs showed very high average avidity for IgG subclasses which, regardless of their galactosylation profile, were fucosylated or fucosylated and bisected. These RFs had the highest avidity (mean 17%) for hypogalactosylated, fucosylated, bisected (G0fb) IgG3 of G3m(b1) allotype. Ga-specific RFs had low avidity for either of the G0 IgG glycoforms (Fig. 1B).



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FIG. 1. Avidities of pan- and Ga-specific RFs. (A) Avidity, calculated as the mean value of binding of all pan- and all Ga-specific RFs to 14 IgGs. (B) Avidity for IgG subclasses/glycoforms.

 
RF titre and RF avidity
RFs with the same titre were considered as one RF for statistical comparisons. Although not statistically significant, a tendency towards higher avidity of RFs with higher titre was observed for the pan-specific but not for the Ga-specific RFs, which had a slightly negative trend (Fig. 2A). IgG3 of G3m(b1) allotype and IgG4 of 4a allotype contributed most to the positive trend of pan-specific RFs. Linear regression analysis of association of RF titre and RF avidity for individual subclass/glycoforms, done for the pan-specific RFs, showed (Fig. 2B) that the slopes of the hypogalactosylated, fucosylated, bisected (G0fb) IgG3 (r = 0.754; P = 0.083) and hypergalactosylated, fucosylated, bisected (G2fb) IgG4 glycoforms (r = 0.657; P = 0.156) were the most positive slopes. The slope of hypogalactosylated IgG1 was slightly positive (r = 0.355; P = 0.490), and a negative slope was obtained for hypogalactosylated IgG2 (r = –0.614; P = 0.195). However, at the level of 95% confidence intervals there was no significant difference between the slopes of the regression lines.



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FIG. 2. (A) Linear correlation between RF titre and RF avidity of pan- and Ga-specific RFs. (B) The effect of IgG subclasses/glycoforms on titre/avidity changes within the pan-reactivity pattern.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although RFs have been studied extensively, the origin, pathogenic potential and target immunogen(s) of these autoantibodies continue to be debated. Most studies have addressed the Ga-specific RFs and heterogeneity in their fine specificity [7–9]. However, disease-related RFs [2] do not appear to belong to this reactivity pattern. The pan-specific RFs have received little attention, although their importance could have been predicted because of their reactivity with IgG3 subclass. It has been assumed that this subclass, particularly its G3m(b1) allotypic variant, has a role in the sustained production of pathogenic synovial RFs in RA [10]. From our study, it is apparent that higher titres and higher avidities of IgM RFs in the sera of RA patients are associated with pan-specificity, not with Ga-specificity. In addition, a statistical trend towards higher avidity of RFs with higher titre was observed for pan-specific but not for Ga-specific RFs. It appears from these findings that the high-avidity RF subpopulation, which may bind preferentially to the G0 glycoforms of IgG, belongs to the pan-specific RFs, but also that hypogalactosylated IgG is a better ligand for RFs when it contains sugars which are not a part of the biantennary chains. This may be of interest in view of the speculation that polyclonal RFs that bind to the G0 glycoform with high avidity may contribute to disease pathogenesis in RA [4].

It is difficult to ascertain changes in the specificity of polyclonal IgM RFs during the disease in RA, since other RF isotypes, such as IgG and IgA RFs, may occur in RA sera. Although the possibility that these isotypes were present in the samples we analysed cannot be excluded, we think that they had little impact on the binding of IgM RFs to solid-phase IgG in our assay, due to their level becoming excessively low in highly diluted serum samples. Generally, the serum levels of IgG and IgA RFs are lower than that of IgM RFs, since patients with RA continue to produce high levels of IgM RFs despite the disease chronicity and the possible isotypic switch. Thus, it is highly likely that estimated avidities in our study reflect the binding strengths of IgM RFs. The fact that in the majority of RA sera the RFs of this isotype were pan- or Ga-specific suggests that the fluctuations in specificity and avidity are to be expected mainly within these two reactivity patterns. It is questionable, however, whether the change from Ga- to pan-specificity is due to the generation of a real pan-specific IgM RF or to increased production of RF with specificity for IgG3 in sera with high IgM RF titres. It is almost impossible to ascertain this for polyclonal IgM RFs. However, the latter possibility could perhaps be neglected since RFs which bind only IgG3 (IgG3-specific RA RFs) are rarely present in serum. Such RFs were found mainly in the synovial tissue [11], and they bound more strongly to completely aglycosylated IgG3 than to glycosylated IgG3 [9].

The mechanisms controlling the level and distribution of RFs of different specificities and avidities in autoimmune response are poorly understood. We can only speculate on the origin of the pan- and Ga-specific RFs in RA. These RFs may be two parallel repertoires, or the pan-specific RFs may appear over the course of the disease as a result of an intraclonal shift in specificity and, depending on the degree of expansion of the clone which produces them and the avidity they express for a subclass and glycoform of IgG which dominates quantitatively, may acquire a pathogenic capacity in RA. Pan-specificity with strong anti-IgG3 G3m(b1) reactivity may predispose RFs to a pathological rather than to a physiological role. The qualitative characteristics of the Ga-specific RFs show them to be similar to the so-called natural RFs. The fact that Ga-specificity has been found both among arthritic and non-arthritic RFs [5], as well as the fact that it dominates IgM RFs appearing after the immunization of normal healthy individuals [2], indicates that Ga-specific RFs are less relevant to the RA disease process than the RFs of other specificities. The hypothetical mechanism of competitive tolerance, proposed to solve the paradox of coexistence of pathological and normal RFs in diseased individuals [12] is, in our opinion, able to explain the presence of both Ga- and pan-specific RFs in RA patients. Large amounts of low-avidity Ga-specific RFs (probably always present in diseased individuals as physiological RFs) might, by competing for IgG Fc, operate so as to prevent the activation of higher-affinity RF B cells. Chronic antigenic stimulation with an antigen that is immune-complexed with IgG3 antibodies, particularly with the G3m(b1) allotype variant, may prevent the Ga-specific RFs from competing because IgG3, as opposed to the other three subclasses, does not bear the classic Ga epitope. It has been observed in MRL/lpr mice that the RF response evolves towards high affinity for a distinct epitope (IgG2a), but retains some of the characteristics of the natural RF response (for IgG1) [13].


    Acknowledgments
 
This work was supported by a Basic Science Grant from the Ministry of Science and Technology of Serbia. The authors are grateful to Professor Royston Jefferis, University of Birmingham, UK, for providing a set of IgG standards of known %G0.

The authors have declared no conflicts of interest.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 20 December 2003; revised version accepted 28 May 2004.



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