Prospective evaluation of the frequency and clinical significance of antineutrophil cytoplasmic and anticardiolipin antibodies in community cases of patients with rheumatoid arthritis

O. Vittecoq1,2,, F. Jouen-Beades2, K. Krzanowska1, I. Bichon-Tauvel3, J. F. Menard4, A. Daragon1,2, D. Gilbert2, F. Tron2 and X. Le Loët1,2

1 Service de Rhumatologie,
2 INSERM U 519 et Institut Fédératif de Recherche Multidisciplinaire sur les Peptides,
3 Collège des Rhumatologues de Haute-Normandie and
4 Unité de Biométrie-Biostatistique, Centre Hospitalier Universitaire de Rouen, 76031 Rouen Cedex, France


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objectives. To evaluate the frequencies of antineutrophil cytoplasmic (ANCA), anticardiolipin (aCLA) and anti-ß2-glycoprotein 1 antibodies (aß2-GP1A) in rheumatoid arthritis (RA) of limited duration in patients recruited primarily from private practitioners (80%), and to attempt to correlate the presence of these antibodies with certain clinical and/or biological criteria.

Patients and methods. Patients (n = 102) with RA evolving for <5 yr (mean 2.2 yr) were recruited. A home evaluation collected clinical data [Ritchie articular index, Health Assessment Questionnaire (HAQ) index, extra-articular manifestations] and blood for biological analyses [C-reactive protein (CRP), rheumatoid factor, ANCA, aCLA, aß2-GP1A]. ANCA were detected by indirect immunofluorescence on neutrophils and their specificity was determined by enzyme-linked immunosorbent assay (ELISA) and confirmed by immunoblotting; aCLA and aß2-GP1A were detected by ELISA.

Results. Patients had mild RA (Ritchie = 11/78 ± 9.6; HAQ = 0.79/3 ± 0.7), probably due to the recruitment procedure. ANCA, aCLA and aß2-GP1A frequencies were 18.5, 7 and 0%, respectively. Titres of ANCA and aCLA were low. A perinuclear ANCA staining pattern was exclusively observed and lactoferrin was shown to be the major antigen recognized. No relationship was found between ANCA and aCLA and/or rheumatoid factor, or any clinical manifestations. ANCA were more common in RA of longer duration (cut-off: 4 yr; P = 0.05) and aCLA were correlated with the CRP level (P = 0.05).

Conclusions. In RA of recent onset, ANCA and aCLA were detected at low titres and frequencies, and were not associated with any clinical manifestations. A longitudinal study is needed to determine whether their early appearance is predictive of subsequent disease severity.

KEY WORDS: Rheumatoid arthritis, ANCA, Anticardiolipin antibodies, Anti-ß2-glycoprotein 1 antibodies, Antilactoferrin antibodies.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is a chronic inflammatory rheumatic disease whose frequency is estimated to be between 0.3 and 0.8% of the adult Caucasian population [1]. RA evolution varies widely from one patient to another and is classically considered unpredictable. Attempts to find markers predictive of disease evolution have identified some immunological molecules, notably IgA rheumatoid factor (RF), whose high titre at the onset of disease is considered predictive of early bone erosion [2], and antifilaggrin antibodies, whose prognostic value seems limited [3, 4]. Other markers are genetic, for example, certain high-risk alleles of the HLA DRB1 genes [5, 6]. However, these studies were generally retrospective and based on hospital recruitment that is not representative of daily rheumatological practice.

During RA, inflammatory angiitis can occur. Vessels of all sizes may be involved and lesions of all types can be observed in this disease; sometimes the vascular lesions are similar to those seen in polyarteritis nodosa [7]. The pathogenetic role of antineutrophil cytoplasmic antibodies (ANCA) in the genesis of vascular lesions is strongly suspected in Wegener's granulomatosis and microscopic polyarteritis [8]; the role of anticardiolipin antibodies (aCLA) is likely in primary antiphospholipid syndrome and in systemic lupus erythematosus [9]. Nevertheless, in RA, the relationship between these antibodies and vessel involvement is unclear. Using an indirect immunofluorescence (IIF) assay on polymorphonuclear neutrophil (PN) cytospin preparations, ANCA give two staining patterns: a granular cytoplasmic pattern defines cANCA, which recognize proteinase 3 (PR3); and a perinuclear fluorescence characterizes pANCA, which are directed against different proteins, primarily myeloperoxidase (MPO) and lactoferrin (LF). Among aCLA, some recognize ß2-glycoprotein 1 (ß2-GP1) which may bind to some other phospholipids in vivo and be involved in different stages of haemostasis [10]; however, the involvement of ß2-GP1 in haemostasis remains unsolved. The discordant reported results concerning the frequency and clinical significance of these antibodies in RA may be explained by the different clinical and biological procedures [recruitment, duration of RA evolution, assays to detect antibodies and identify their target antigen(s)] and their prognostic value remains to be determined.

Therefore, we set up a clinicobiological study to evaluate prospectively the frequencies of ANCA and aCLA and to attempt to correlate their presence with clinical and/or biological characteristics in a cohort of 102 patients with RA evolving for <5 yr and primarily recruited from private practice. The specific objectives were: (1) to evaluate the frequency of each of the antibody populations and determine the antigenic targets of ANCA and aCLA; (2) to look for an association between the presence of these antibodies and the major clinical and biological criteria; and (3) to determine whether or not these antibodies are independent immunological markers during the course of the disease.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
Patient recruitment.
One hundred and twenty-seven patients with RA evolving for <5 yr, fulfilling at least four of the seven American College of Rheumatology (ACR) criteria [11], were enrolled prospectively between 1 February 1996 and 1 July 1996. This recruitment was made possible by the major contribution (80%) of the Collège des Rhumatologues de Haute-Normandie and 120 general practitioners, and that of regional hospital rheumatologists (20%). These patients were enlisted within the framework of the programme ‘Therapeutic strategies for RA: impact on the quality of life’. After verification of the criteria of inclusion and exclusion (handicap prior to RA onset; presence of another chronic, invalidating disease), the evaluation was conducted by two investigators from the Department of Rheumatology of the Rouen University Hospital at the patient's home and not at the time of a requested consultation for a functional complaint. This study considered clinical data and laboratory blood analyses. The different biological markers were analysed for the 102 patients who had blood drawn after giving their written consent.

Criteria investigated.
The following information was recorded: clinical characteristics: demographic data, number of ACR criteria met, date of appearance of first symptoms, duration of RA determined as the time separating the date at which the patient first met at least four of the seven ACR criteria and the date of the evaluation, presence or not of extra-articular manifestations, Ritchie index, the validated French version of the Health Assessment Questionnaire (HAQ) [12]; biological analyses : C-reactive protein (CRP; N < 10 mg/l), search for autoantibodies and latex test; drugs prescribed to patients at the time of the evaluation and during the preceding year.

Autoantibodies
IIF on PN cytospin preparations.
IgG ANCA were detected on ethanol-fixed normal human PN according to the recommendations of the First International Congress on ANCA [13]. Two immunofluorescence patterns were considered: pANCA and cANCA. Each serum sample positive on ethanol-fixed PN was re-tested in an IIF assay on formaldehyde-fixed PN (Biomedical Diagnostics, Marne-la-Vallée, France) and HEp-2 cells to distinguish between perinuclear and antinuclear reactivities, respectively.

Enzyme-linked immunosorbent assay (ELISA) using leucocyte proteins as antigens.
All sera were tested. Pre-saturated microtitre plates (Virion, Roche, France) were coated with MPO (5 µg/ml) (EPC, Owensville, MI, USA) or LF extracted from milk (1 µg/ml) (Sigma, St Louis, MO, USA) in 0.1 M carbonate–bicarbonate buffer, pH 9.6, or elastase (0.002 U/ml) (Calbiochem, La Jolla, CA, USA) in phosphate-buffered saline (PBS). After three washes with PBS containing 0.1% Tween 20 (PBST), the wells were saturated for 1 h at 37°C with 2% bovine serum albumin (BSA)–PBS for LF-coated plates or with 5% fetal calf serum (FCS)–PBS for elastase-coated plates; MPO-coated plates were not saturated. After three washes with PBST, test sera, diluted 1:50 in 2% BSA–PBST (for anti-LF and anti-elastase activities) and diluted 1:100 in 10% goat serum–PBST (anti-MPO activity), were incubated for 2 h at 37°C. All sera were also tested on uncoated wells. After washing, F(ab)'2 fragments of goat antihuman IgG conjugated to alkaline phosphatase (Caltag Laboratories, San Francisco, CA, USA), diluted 1:2000 in PBST were added and incubated for 90 min at 37°C. After washing, 100 µl of a solution containing 1 mg/ml p-nitrophenyl phosphate (Sigma) dissolved in 1 M Tris–HCl, pH 9.8, and 1.5 M NaCl were added. The optical density (OD) of the different wells was determined with a Titer-Tek spectrophometer (Flow Laboratories, Les Ulis, France) set at an absorbance of 405 nm. The threshold of positivity was three standard deviations (S.D.) above the mean value obtained with the sera of 100 blood donors. The results were expressed in arbitrary units and positivity cut-off was 60 units.

aCLA.
IgG and IgM classes of aCLA were detected by ELISA, according to internationally recommended procedures [14]. The results were expressed in standardized GPL-U and MPL-U (IgG- and IgM-antiphospholipid units) for IgG and IgM aCLA. Positivity cut-offs were 15 units for the two assays.

2-GP1A.
An ELISA was used to detect aß2-GP1A. In half the wells of a 96-well microtitre plate (Nunc Maxisorp, Polylabo, Strasbourg, France), PBS containing 10 µg/ml of purified ß2-GP1 (Diagnostica Stago, Asnières, France) was deposited; the remaining wells received 100 µl of PBS. After incubation for 18 h at 4°C, the wells were washed and then saturated with 2% BSA–PBS for 2 h at 37°C. After washing, individual patients' sera, diluted 1:100 in 2% BSA–PBS, were added to antigen-coated and uncoated wells for 18 h at 4°C. After washing, goat antihuman IgG (H + L) conjugated to alkaline phosphatase (Biosys, Compiègne, France), diluted 1:2000 in PBS, was added to every well and incubated for 1 h at 37°C. Subsequent steps were the same as those described above. To calculate the concentration of aß2-GP1A, the OD measured in uncoated wells was subtracted from that in coated wells and this value was then compared with a standard curve obtained with a reference serum; the threshold of positivity was 3 S.D.

Determination of RF isotypes.
Isotypes were identified by ELISA run in Virion plates coated with purified rabbit IgG (Jackson Immunoresearch, Westgrove, USA) concentrated to 10 µg/ml in 0.1 M carbonate– bicarbonate buffer, pH 9.6, overnight at 4°C.

After three washes, individual patients' sera, diluted 1:100 in 5% FCS–PBST, were added and incubated for 2 h at 37°C. After washing, F(ab)'2 fragments of goat antihuman IgM, IgA or IgG coupled to alkaline phosphatase (Sigma), diluted 1:10 000 in 5% FCS–PBST, were added and incubated for 1 h at 37°C. Labelling was detected as described above for the ELISA. The threshold of positivity was 3 S.D. for IgM, and 6 S.D. for IgA and IgG.

Immunoblotting on LF.
Boiled LF (10 µg) (Sigma) was deposited in each lane of a 4–12% Tris-glycine–polyacrylamide minigel (Novex, San Diego, CA, USA). After electrophoresis, the proteins were transferred on to nitrocellulose filters (HybondTM-C extra, Amersham Life Sciences, Buckingham, UK) for 1.5 h at 500 mA in 0.01 M 3-cyclohexylamino-1-propanesulphonic acid, pH 11.5. The nitrocellulose bands were saturated with PBST containing 0.5% gelatin (PBSTG) and incubated for 2 h with individual RA patients' sera shown to have anti-LF activity in the ELISA or normal control sera diluted 1:25 in PBSTG. After washing, goat antihuman IgG (H + L) coupled to alkaline phosphatase (Caltag), diluted 1:1000 in PBSTG, was added and incubated for 1 h at 37°C. Bound antibody was detected by reaction with the alkaline phosphatase substrate, 5-bromo-4-chloro-3-indoyl-ß-galactopyranoside (BCIP), and then nitroblue tetrazolium salt (NBT) (Sigma). A solution containing different standard molecular mass markers (SeeBlueTM, Novex) was used as the reference.

Statistical analyses.
Correlations between the presence of antibody and clinical and/or laboratory findings were assessed using Fisher's exact test for two qualitative variables, Spearman's correlation coefficient for quantitative parameters, and the Mann–Whitney U-test for the comparison of one qualitative variable and one quantitative variable (equivalent to Student's t-test comparing means). A P <= 0.05 was considered to be significant.


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Demographic and clinicobiological characteristics of the study population
The main findings are reported in Table 1Go. The cohort was comprised mostly of women (75%; F/M sex ratio = 2.9). RA was mainly of recent onset, evolving for lt;3 yr for 71% of the patients and lt;1 yr for 19% of them, and only mildly active and invalidating (Ritchie articular index and CRP level, and HAQ index, respectively). One-quarter of the patients had at least one rheumatoid nodule. Slightly more than one-third (36%) had other minor extra-articular manifestations, primarily mucocutaneous: sicca syndrome, n = 32; anaemia, n = 3; serous inflammation, n = 2 (one pleurisy and one pericarditis). No rheumatoid vasculitis was observed.


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TABLE 1. Demographic, clinical and biological characteristics of the RA population studied

 
Almost all patients (96%) were being treated (Table 1Go) at the time of the evaluation, which, it must be kept in mind, the patient had not requested for treatment. Oral prednisone was prescribed at a mean dose of 6.5 mg/day (range: 0–40 mg/day). One or several disease-modifying anti-rheumatic drugs (DMARDs) were prescribed to 95% of the patients, with more than half of them taking methotrexate at a mean dose of 10 mg/week (range: 5–20 mg/week).

Half the patients were RF positive with the latex test; when combined with ELISA, this rate rose to 62%. All sera with RF IgG activity also had RF IgM activity; four patients had exclusively RF IgA activity. When the results obtained with the various assays were combined, 65% of the patients had RF reactivity.

ANCA frequency evaluated with the IIF assay
Forty-two sera (41% of the patients) gave a perinuclear and/or nuclear staining pattern on ethanol-fixed PN cytospin preparations. No cANCA were observed. To determine whether the antigen recognized by the antibody was perinuclear or nuclear, the 42 sera were re-tested on formaldehyde-fixed PN and HEp-2 cells: 19 (18.5%) sera were considered to have a perinuclear reactivity (pANCA) and among them, three had perinuclear and nuclear reactivities; 23 (22.3%) gave an antinuclear labelling pattern and were classified as antinuclear antibodies (ANA) (Table 2Go).


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TABLE 2. Reactivity of the 102 RA sera by IIF on PN and on HEp-2 cells and by ELISA using different leucocyte proteins as antigens

 

pANCA antigenic specificities
Because no cytoplasmic staining was observed in the IIF assay, PR3 reactivity was not evaluated. ANCA activity was tested against MPO, LF and elastase, antigens that have a perinuclear distribution in ethanol-fixed PN. No anti-MPO activity was detected. The ELISA detected anti-LF activity in the sera of 14/102 (13.7%) patients. Surprisingly, these anti-LF-positive sera gave different IIF patterns on neutrophils: 4/19 (21%) were pANCA positive, 3/23 (13%) were ANA positive and 7/60 (12%) were negative. Thus, there was a discordance between the results obtained with ELISA and IIF (Table 2Go). The anti-LF titres were low or moderate (mean: 103 U; N < 60 U), with the exception of one serum (Fig. 1Go). All sera that reacted with solid-phase LF were subsequently evaluated by immunoblotting. They all recognized a polypeptide band of 78 kDa, which corresponds to the molecular mass of LF (Fig. 2Go). In addition, ELISA detected anti-elastase reactivity in only one serum that showed no IIF activity in PN; it was not subjected to immunoblotting.



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FIG. 1. Levels of aCLA and anti-LF antibodies of the IgG isotype detected by ELISA. The horizontal bars represent the thresholds of positivity.

 


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FIG. 2. Immunoblotting analysis of anti-LF activity in the sera of patients with RA. Purified LF, prepared under denaturing and reducing conditions, was used as the antigen. Lane 1, polyclonal rabbit antihuman LF antibody (Sigma); lanes 2–10, patients' sera with anti-LF activity detected by ELISA; lane 11, normal serum; lane 12, buffer. Molecular weight markers are indicated on the left.

 

aCLA and aß2-GP1A frequencies
IgG aCLA were detected in 7% of the patients' sera, but only one sample had a high titre (100 GPL/ml); the mean titre for the other sera was low (22 GPL/ml; N < 15 GPL/ml) (Fig. 1Go). Only one serum also had IgM aCLA activity. No aß2-GP1 reactivity was observed in any of the 102 sera tested.

Statistical analyses
No statistically significant relationship was found between the different antibody populations. However, a significant positive correlation was established between pANCA and an RA duration of >= 4 yr (P = 0.05) (Table 3Go). A statistically significant positive correlation was found between aCLA and CRP concentrations (P = 0.05) (Table 4Go). Analysis of the effect of treatment on antibody titres showed that anti-LF titres were lower in patients taking hydroxychloroquine (P = 0.02) and that every RF isotype concentration was lower in patients on prednisone (P = 0.01).


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TABLE 3. ANCA frequency and serological and clinical correlations reported in the literature and this study

 

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TABLE 4. Frequency of aCLA and correlations between their presence and clinical and/or biological criteria during RA reported in the literature and this study

 


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
This prospective study of immunological markers usually associated with vascular lesions (ANCA, aCLA and aß2-GP1A) was conducted from a population-based recruitment of 102 patients with RA evolving for a mean of 2.2 yr. Because of the type of recruitment, it is appropriate to underline that this group of patients is probably highly representative of RA in the Normandy region of France. In this respect, this cohort of RA patients differs from those previously subjected to the same analysis which were either RA of recent onset and hospital recruitment [17, 24, 31], of long duration (mean: 13 yr) and private recruitment [25] or, in most cases, RA of long duration and hospital recruitment (Tables 3Go and 4Go). Our approach probably accurately reflects the characteristics of RA at the time of the evaluation which, it must be remembered, was not instigated at the patient's request for treatment: mild clinical status, minor functional disability and few mild extra-articular manifestations. These features could also be explained by the high rate of RA patients treated with both steroids (72%) and DMARDs (95%). In addition, because this was a prospective study, the different clinical criteria taken into consideration could be evaluated precisely.

ANCA frequency, detected by IIF assay, first on ethanol-fixed PN and confirmed on formaldehyde-fixed PN preparations and HEp-2 cells, was low (18.5%), but previously reported frequencies vary widely (Table 3Go). However, it should be noted that, in documented RA with a mean duration of 12 yr, complicated or not by extra-articular manifestations and/or vasculitis, such as Felty's syndrome [20], the frequency was often rather high, whereas those observed in cohorts of RA of recent onset [24] or private recruitment [25] were comparable to ours. Recruitment from private practitioners often includes benign forms and disease of recent onset, two factors that probably influence the frequency of ANCA. This frequency may also depend upon the interpretation of the perinuclear fluorescent staining pattern observed on ethanol-fixed PN which is not always clear cut. In particular, it can be difficult to distinguish true pANCA reactivity from antinuclear activity. Re-testing the sera in IIF assays on formaldehyde-fixed PN and HEp-2 cells often enables the distinction to be made, but was only rarely previously carried out [17, 21, 25]. And even when this approach is applied, the interpretation of all the staining patterns observed with the different assays is not always decisive. For example, some authors [21, 32] introduced the notion of granulocyte-specific ANA (GS-ANA) that recognize a nuclear component specific to PN but their existence remains controversial. Others have described a homogeneous cytoplasmic fluorescence, defining xANCA of unknown significance, that was predominant in some series of RA patients [16, 17, 23, 24]; we never saw such a pattern. In this study and others [17, 21, 22, 25], all ANCA gave a perinuclear staining pattern. Indeed, cANCA are rarely observed in this context and are usually associated with PR3 identification by ELISA. In such cases, RA is usually complicated by vasculitis [16].

The fine specificities of the pANCA, identified in the IIF assay, were determined with an ELISA using the antigens generally recognized by these antibodies during the course of RA, i.e. MPO, LF and elastase (Table 5Go). As in previous studies, the structure most frequently recognized by pANCA was LF (Table 5Go) and anti-LF antibody titres were usually low [16, 19, 22, 33]. However, because of the lack of a standardized ELISA for the identification of ANCA target antigens [13] and to demonstrate the specificity of our assay (commercially purified antigens can be contaminated with irrelevant proteins according to the variety of protein purification methods available) [34], we thought it was necessary to verify any ELISA-detected specificity by immunoblotting. In all cases, this procedure allowed the confirmation of anti-LF activity detected by ELISA. Furthermore, the sensitivities of ELISA and immunoblotting seem to be superior to that of IIF [17, 2325]. In our study, only 28.6% of the sera showing anti-LF reactivity in ELISA gave pANCA staining of PN. These discrepancies are probably attributable to methodological weaknesses specific to the IIF assay: poor fixation of PN preparations, loss of the target antigen during ethanol fixation (membrane solubilization), variation of PN granule contents from one donor to another. Thus, the IIF assay does not seem to be the most reliable one for detecting pANCA reactivity. In contrast, for some sera exhibiting a pANCA staining pattern on IIF, the specificity was not identified by ELISA in our study (Table 2Go). It has recently been demonstrated that most RA sera recognize more than one neutrophil component. Indeed, other antigens than LF, MPO and elastase may be recognized by RA sera, in particular lysozyme, cathepsin G and bactericidal permeability-increasing protein (BPI) [22, 25, 35]. Thus, although LF is the major antigen recognized by pANCA in RA, study of their fine specificities should be extended to several neutrophil components.


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TABLE 5. Staining patterns observed in an IIF assay on PN reported in the literature and this study

 
During the course of RA, the clinical significance of ANCA has not been clearly established. Indeed, the association of pANCA and rheumatoid vasculitis remains highly controversial [15, 17, 18, 36]. As previously reported [20], the frequency of ANCA seemed to be higher in our patients with RA of longer duration. We thought that an analysis of the antigenic specificities of pANCA might provide additional clinical input. For example, anti-MPO antibody could be an immunological marker of renal and/or pulmonary involvement during RA [21, 22, 25, 33]. It is not surprising that no such antibodies were detected in our population with RA of recent onset and without these extra-articular manifestations. Anti-LF antibodies were reported to be associated with cutaneous and/or neurological vasculitides [16] and anti-elastase activity with severe vasculitis [24]. As our patients had no known vascular complications at the time of the evaluation, we cannot confirm these findings.

The frequency of aCLA in our patients was 7%, a value lower than those found in most of the other studies (12–48%) (Table 4Go). In contrast to our data and those of Mustila et al. [25], who used similar recruitment and aCLA detection methodologies, the other reported analyses were conducted exclusively on long-lasting RA and utilized non-standard tests. Here again, these differences may explain the divergent results. In earlier studies [2631] and ours, aCLA titres were low and weaker than those measured in the sera of systemic lupus erythematosus patients. During the course of RA, the clinical significance of these antibodies remains unclear. In the literature, this antibody population is associated with clinical manifestations and/or biological abnormalities that differ from one study to another (Table 4Go). However, like others [15, 29, 30, 37], we also found a significant positive correlation between the presence of aCLA and elevated CRP levels. Despite some reports suggesting the occurrence of thromboses during RA [26, 3840], we, like others, found no relationship between this antibody population and repeated thrombotic events. The absence of such a relationship probably reflects the finding, by us and Kaburaki et al. [41], that aCLA activity in RA appears to be independent of ß2-GP1. Indeed, it is widely accepted that ß2-GP1-dependent aCLA, mainly detected during primary antiphospholipid syndrome and systemic lupus erythematosus, are associated with thrombotic events [41]. However, the rate of thrombosis seems to be higher in RA patients compared with healthy subjects. For some authors, the presence of aCLA but also of other factors, particularly an altered lipid profile such as increased lipoprotein(a), could represent an important risk factor [40].

In our study, ANCA positivity was not correlated to aCLA positivity. In addition, we noted no relationship between the presence of each of these antibody populations and that of the different RF isotypes, in agreement with all but one [27] previous study. The only association consistently reported in the literature is that between aCLA and ANA [26, 29]. This relationship is not surprising, as some aCL monoclonal antibodies derived from (NZW x BXSB)F1 mice, selected for their abilities to recognize CL, bind to single- and/or double-stranded deoxyribonucleic acid [42, 43]. Such a relationship between aCLA and ANA was not found in our study.

This study shows that, in RA of recent onset, ANCA and aCLA were detected at low titres and frequencies. These results could be explained, in part, by the high rate of RA patients treated with steroids and DMARDs that decrease the immune response and therefore the production of such antibodies. Moreover, these antibodies were not associated with any particular clinical manifestations. So, their significance remains speculative. On the one hand, they can be considered as a consequence of the sustained stimulation of the immune system observed in RA and the expansion of natural autoantibodies present in the B cell repertoire of healthy individuals. In this regard, the absence of aß2-GP1A in RA patients with aCLA and the recent demonstration that ANCA are present in the peripheral repertoire of healthy individuals [44] speak for this hypothesis. On the other hand, the observations that ANCA production is not associated with that of aCLA and that the presence of both antibody populations does not parallel that of RF in our RA patients suggest that other mechanisms may account for their production. Finally, we can hypothesize that the presence of these antibody populations predisposes RA patients to the pathological manifestations, i.e. vasculitis and thrombosis, respectively, associated with ANCA and aCLA. Follow-up of the patients enrolled in this prospective study will permit the clarification of the predictive value of aCLA and ANCA in RA.


    Acknowledgments
 
The authors would like to thank the group ‘Polyarthrite rhumatoïde et consommation de soins' for its contribution. The authors would like to thank Janet Jacobson for correcting the manuscript and Ms Maquin for typing it. The study was funded by the Société Française de Rhumatologie (SFR) and The Institut National de la Santé et de la Recherche Médicale (INSERM).


    Notes
 
Correspondence to: O. Vittecoq. Back


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

  1. Spector TD. Rheumatoid arthritis. Rheum Dis Clin North Am1990;16:513–37.[ISI][Medline]
  2. Van Zeben D, Hazes JMW, Zwinderman AH, Cats A, van der Voort EAM, Breedveld FC. Clinical significance of rheumatoid factors in early rheumatoid arthritis: results of a follow-up study. Ann Rheum Dis1992;51:1029–35.[Abstract]
  3. Aho K, Palusuo T, Kurki P. Marker antibodies of rheumatoid arthritis: diagnostic and pathogenetic implications. Semin Arthritis Rheum1994;23:379–87.[ISI][Medline]
  4. Vittecoq O, Jouen-Beades F, Delpech A, Gilbert D, Le Loët X, Tron F. Autoantibodies in rheumatoid arthritis. Rev Rhum [Engl. Ed]1995;62:195S–200S.
  5. Combe B, Eliaou JF, Daurès JP, Meyer O, Clot J, Sany J. Prognostic factors in rheumatoid arthritis. Comparative study of two subsets of patients according to severity of articular damage. Br J Rheumatol1995;34:529–34.[ISI][Medline]
  6. Wagner U, Kaltenhauser S, Sauer H et al. HLA markers and prediction of clinical course and outcome in rheumatoid arthritis. Arthritis Rheum1997;40:341–51.[ISI][Medline]
  7. Wattiaux MJ, Kahn MF, Bourgeois P, Vinceneux Ph, Grossin M, Kaplan G. Analyse anatomique et nosologique de 100 cas d'angéites diffuses vues en Rhumatologie. Rev Rhum Mal Osteoartic1985;52:599–603.[Medline]
  8. Schultz DR, Tozman EC. Anti-neutrophil cytoplasmic antibodies: major autoantigens, pathophysiology, and disease associations. Semin Arthritis Rheum1995;25:143–59.[ISI][Medline]
  9. Lockshin MD. Pathogenesis of the antiphospholipid antibody syndrome. Lupus1996;5:404–8.[ISI][Medline]
  10. McNeil HP, Simpson RJ, Chesterman CN, Krilis SA. Antiphospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation ß2-glycoprotein 1 (apolipoprotein H). Proc Natl Acad Sci USA1990;87:4120–4.[Abstract]
  11. Arnett FC, Edworthy SM, Bloch DA et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum1998;31:315–24.[Medline]
  12. Guillemin F, Briançon S, Pourel J. Mesure de la capacité fonctionnelle dans la polyarthrite rhumatoide: adaptation française du Health Assessment Questionnaire. Rev Rhum1991;58:459–65.[ISI][Medline]
  13. Wiik A. Delineation of a standard procedure for indirect immunofluorescence detection of ANCA. APMIS1989;97(Suppl. 6):12–3.
  14. Harris EN. Special report. The second international anti-cardiolipin standardization workshop/the Kingston anti-phospholipid antibody study (KAPS) group. Am J Clin Path1990;94:476–84.[ISI][Medline]
  15. Nässberger L, Sjöholm AG, Sturfelt G. Absence of circulating antineutrophil cytoplasmic antibodies (ANCA) in severe vasculitis associated with rheumatoid arthritis. Scand J Rheumatol1990;19:189–92.[ISI][Medline]
  16. Coremans IEM, Hagen EC, Daha MR et al. Antilactoferrin antibodies in patients with rheumatoid arthritis are associated with vasculitis. Arthritis Rheum1992;35:1466–75.[ISI][Medline]
  17. Mulder AHL, Horst G, van Leeuwen MA, Limburg PC, Kallenberg CGM. Antineutrophil cytoplasmic antibodies in rheumatoid arthritis. Characterization and clinical correlations. Arthritis Rheum1993;36:1054–60.[ISI][Medline]
  18. Nässberger L, Hultquist R, Sturfelt G. Occurrence of anti-lactoferrin antibodies in patients with systemic lupus erythematosus, hydralazine-induced lupus, and rheumatoid arthritis. Scand J Rheumatol1994;23:206–10.[ISI][Medline]
  19. Cambridge G, Williams M, Leaker B, Corbett M, Smith CR. Anti-myeloperoxidase antibodies in patients with rheumatoid arthritis: prevalence, clinical correlates, and IgG subclass. Ann Rheum Dis1994;53:24–9.[Abstract]
  20. Helsloot J, Virgo S, McGuigan L, Sturgess A. Antineutrophil cytoplasmic antibodies in inflammatory arthritis—Potential for misdiagnosis? Br J Rheumatol1995;34:820–4.[ISI][Medline]
  21. Bosch X, Llena J, Collado A et al. Occurrence of antineutrophil cytoplasmic and antineutrophil (peri)nuclear antibodies in rheumatoid arthritis. J Rheumatol1995;22:2038–45.[ISI][Medline]
  22. Braun MG, Csernok E, Schmitt WH, Gross WL. Incidence, target antigens, and clinical implications of antineutrophil cytoplasmic antibodies in rheumatoid arthritis. J Rheumatol1996;23:826–30.[ISI][Medline]
  23. Afeltra A, Sebastiani GD, Galeazzi M et al. Antineutrophil cytoplasmic antibodies in synovial fluid and in serum of patients with rheumatoid arthritis and other types of synovitis. J Rheumatol1996;23:10–5.[ISI][Medline]
  24. de Bandt M, Meyer O, Haim T, Kahn MF. Antineutrophil cytoplasmic antibodies in rheumatoid arthritis patients. Br J Rheumatol1996;35:38–43.[ISI][Medline]
  25. Mustila A, Korpela M, Mustonen J et al. Perinuclear antineutrophil cytoplasmic antibody in rheumatoid arthritis. A marker of severe disease with associated nephropathy. Arthritis Rheum1997;40:710–7.[ISI][Medline]
  26. Fort JG, Cowchock FS, Abruzzo RL, Smith JB. Anticardiolipin antibodies in patients with rheumatic diseases. Arthritis Rheum1987;30:752–60.[ISI][Medline]
  27. Keane A, Woods R, Dowding V, Roden D, Barry C. Anticardiolipin antibodies in rheumatoid arthritis. Br J Rheumatol1987;26:346–50.[ISI][Medline]
  28. Buchanan RR, Wardlaw JR, Riglar AG, Little John GO, Miller MH. Antiphospholipid antibodies in the connective tissue diseases: their relation to the antiphospholipid syndrome and forme fruste disease. J Rheumatol1989;16:757–61.[ISI][Medline]
  29. Seriolo B, Cutolo M, Fasciolo D, de Cesari F, Accardo S. Anticardiolipin antibodies in rheumatoid arthritis. Ann Rheum Dis1992;51:1100.[ISI][Medline]
  30. Wolf P, Gretler J, Aglas F, Auer-Grumbach P, Rainer F. Anticardiolipin antibodies in rheumatoid arthritis: their relation to rheumatoid nodules and cutaneous vascular manifestations. Br J Dermatol1994;131:48–51.[ISI][Medline]
  31. Merkel PA, Chang YC, Pierangeli SS, Convery K, Harris N, Polisson RP. The prevalence and clinical associations of anticardiolipin antibodies in a large inception cohort of patients with connective tissue diseases. Am J Med1996;101:576–83.[ISI][Medline]
  32. Wiik A. Granulocyte-specific antinuclear antibodies. Allergy1980;35:263–89.[ISI][Medline]
  33. Schnabel A, Hauschild S, Gross WL. Anti-neutrophil cytoplasmic antibodies in generalized autoimmune diseases. Int Arch Allergy Appl Immunol1996;109:201–6.
  34. Audrain MAP, Baranger AR, Lockwood CM, Esnault VLM. High immunoreactivity of lactoferrin contaminating commercially purified myeloperoxidase. J Immunol Methods1994;176:23–31.[ISI][Medline]
  35. Brimnes J, Halberg P, Jacobsen S, Wiik A, Heegard NHH. Specificities of anti-neutrophil antibodies in patients with rheumatoid arthritis (RA). Clin Exp Immunol1997;110:250–6.[ISI][Medline]
  36. Savige JA, Gallichio MC, Stockman A, Cunningham TJ, Rowley MJ. Antineutrophil cytoplasmic antibodies in rheumatoid arthritis. Clin Exp Immunol1991;86:92–8.[ISI][Medline]
  37. Rabee SA, Sallam AM, Ayuoub NM, Kholy EE. Anticardiolipin antibodies in rheumatoid arthritis. J Egyptian Public Health Assoc1994;69:47–63.[Medline]
  38. Monteagudo M, Montalban J, Alvarez J, Lima J, Garcia-Bragado F, Barquinero J. Stroke and anticardiolipin antibodies in a patient with rheumatoid arthritis and large granular lymphocyte proliferation. J Rheumatol1988;15:1589–90.[Medline]
  39. Voisin L, Dérumeaux G, Borg JY et al. Catastrophic antiphospholipid syndrome with fatal acute course in rheumatoid arthritis. J Rheumatol1995;22:1586–8.[ISI][Medline]
  40. Seriolo B, Accardo S, Fasciolo D, Sulli A, Bertolini D, Cutolo M. Lipoprotein (a) and anticardiolipin antibodies as risk factors for vascular disease in rheumatoid arthritis. Thromb Haemostasis1995;74:799–800.[ISI][Medline]
  41. Kaburaki J, Kuwana M, Yamamoto M, Kawai S, Matsuura E, Ikeda Y. Disease distribution of beta 2-glycoprotein 1-dependent anticardiolipin antibodies in rheumatic diseases. Lupus1995;4:27S–31S.
  42. Monestier M, Kandiah DA, Kouts S et al. Monoclonal antibodies from NZW x BXSB F1 mice to ß2-glycoprotein 1 and cardiolipin. J Immunol1996;156:2631–41.[Abstract]
  43. Hashimoto Y, Kawamura M, Ichkawa K et al. Anticardiolipin antibodies in NZW x BXSB F1 mice. A model of antiphospholipid syndrome. J Immunol1992;149:1063–8.[Abstract/Free Full Text]
  44. Finnern R, Bye JM, Dolman KM et al. Molecular characteristics of anti-self antibody fragments against neutrophil cytoplasmic antigens from human V gene phage display libraries. Clin Exp Immunol1995;102:566–74.[ISI][Medline]
Submitted 15 October 1998; revised version accepted 23 November 1999.