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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 carbonatebicarbonate 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% BSAPBST (for anti-LF and anti-elastase activities) and diluted 1:100 in 10% goat serumPBST (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 TrisHCl, 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.
aß2-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% BSAPBS for 2 h at 37°C. After washing, individual patients' sera, diluted 1:100 in 2% BSAPBS, 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% FCSPBST, 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% FCSPBST, 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 412% Tris-glycinepolyacrylamide 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 MannWhitney 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 2).
|
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 2). The anti-LF titres were low or moderate (mean: 103 U; N < 60 U), with the exception of one serum (Fig. 1
). 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. 2
). In addition, ELISA detected anti-elastase reactivity in only one serum that showed no IIF activity in PN; it was not subjected to immunoblotting.
|
|
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. 1). 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 3
). A statistically significant positive correlation was found between aCLA and CRP concentrations (P = 0.05) (Table 4
). 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).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 3). 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 5). As in previous studies, the structure most frequently recognized by pANCA was LF (Table 5
) 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 2
). 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.
|
The frequency of aCLA in our patients was 7%, a value lower than those found in most of the other studies (1248%) (Table 4). 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 4
). 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 |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|