Increased Ed-B fibronectin plasma levels in spondyloarthropathies: comparison with rheumatoid arthritis patients and a healthy population
P. Claudepierre,
Y. Allanore,
L. Belec1,
B. Larget-Piet,
L. Zardi2 and
X. Chevalier
Department of Rheumatology, Hôpital Henri Mondor, 94010 Créteil,
1 Department of Virology, Hôpital Broussais, Paris, France and
2 Institute for Cancer Research, Genoa, Italy
Correspondence to:
P. Claudepierre, Service de Rhumatologie, Hôpital Henri Mondor, 51 Avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France.
 |
Abstract
|
---|
Objective. To determine, for the first time, plasma levels of general fibronectin (Fn) and two spliced isoforms, Ed-A and Ed-B, in patients with spondyloarthropathy (SpA) in comparison with rheumatoid arthritis (RA) patients and healthy volunteers (HV).
Methods. Plasmas (EDTA) as well as clinical data, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels were collected in two groups of 10 patients fulfilling the European Spondylarthropathy Study Group criteria for SpA or the 1987 American College of Rheumatology criteria for RA. Plasmas of 21 blood donors served as controls. Plasma levels of Fns were determined by using an in-house immunocapture ELISA, using monoclonal antibodies (MAbs) against general Fn and its isoforms.
Results. Total Fn plasma levels were significantly higher in the SpA group (mean±S.D.=1387±569 mg/l) than in the RA group (684±196 mg/l; P=0.02) and in HV (303±211 mg/l; P<0.0001). Ed-A Fn levels appeared higher in SpA (23±10.4 mg/l) and RA (32.5±16.5 mg/l) groups than in the HV group (2.8±0.9 mg/l; P=0.0003 and P<0.0001, respectively), without a significant difference between SpA and RA groups. Ed-B Fn levels were higher in SpA (6.9±2.1 mg/l) than in RA (3.2±1.9 mg/l; P=0.02) and HV (1.1±0.8 mg/l; P=0.0003) groups. No significant correlation was observed in SpA patients between each Fn level and clinical activity, ESR or CRP levels.
Conclusions. This study showed an increase in plasma levels of Fn and Ed-B Fn in SpA patients compared with RA patients and HV, which could not be attributed solely to systemic inflammation. It may be hypothesized that Ed-A and Ed-B Fn might reflect local turnover in inflamed tissues, and that Ed-B Fn might be particularly involved in the musculoskeletal inflammatory process of SpA.
KEY WORDS: Spondyloarthropathy, Ankylosing spondylitis, Fibronectin
 |
Introduction
|
---|
Fibronectin (Fn), a large extracellular glycoprotein, is involved in a wide variety of cellular activities such as differentiation, adhesion and migration [1]. These activities result from the ability of the Fn dimer to interact with numerous molecules, such as cell surface proteins (integrins), collagen, fibrin and heparin [1]. Therefore, Fn primarily functions as a connecting molecule in the extracellular matrices of tissues by mediating both cellmatrix and matrixmatrix interactions [1]. Variability in function of the Fn molecule may partially result from the heterogeneous structure of the Fn monomer [1]. Indeed, the alternative splicing of the primary transcript of the Fn single gene generates different Fn isoforms [1]. Extra domains A and B (Ed-A and Ed-B) and the IIICS regions are such alternatively spliced regions [1]. While the IIICS region can be spliced partially, Ed-A and Ed-B are spliced in or out completely [1]. Exon Ed-A is present in cell-associated Fn, in particular in fibroblasts and endothelial cells of larger blood vessels [2]. It has also been found to be expressed by synoviocytes of rheumatoid synovial membrane [3], and Ed-A Fn has been observed in cartilage of rheumatoid arthritis (RA) and, to a lesser extent, osteoarthritis (OA) [4]. Exon Ed-B is highly expressed in Fn of transformed human cells and during embryogenesis [5, 6]. In normal adult human cartilage, only low amounts of Ed-A and Ed-B Fn have been found [4, 7], although there is an increase in Ed-B Fn content in osteoarthritic cartilage [7, 8]. Thus, this specific isoform, Ed-B, might be involved in matrix remodelling of tissues containing chondrocytes [5].
Fn in plasma is produced primarily by hepatocytes [1]. However, exon Ed-A is not transcribed in hepatocytes [2], and plasmas of healthy subjects contain very small quantities of Ed-A Fn [9]. Increased plasma levels of Ed-A Fn have been reported in patients with vascular injury, probably resulting from a stimulation of Ed-A Fn production by endothelial cells [9]. To the best of our knowledge, Ed-B plasma levels in healthy subjects or in patients have never been determined.
Spondyloarthropathy (SpA) constitutes a group of inflammatory rheumatic diseases in which the major target of the inflammatory process is the enthesis, rather than the synovium as in RA [10]. The enthesis represents the interface between tendons, capsules or ligaments and bone [10].
Because Ed-A and particularly Ed-B Fn were found in tissues with chondrocytes, we wondered whether circulating levels of such isoforms may be elevated in SpA, owing to the presence of chondrocyte-like cells in the enthesis. We therefore measured plasma levels of these molecules in patients with SpA, and compared them with RA patients and healthy volunteers (HV).
 |
Patients and methods
|
---|
Patients
Two groups of patients were included: SpA patients fulfilling the European Spondylarthropathy Study Group criteria [11] and RA patients fulfilling the American College of Rheumatology criteria [12]. In- or out-patients seen in our department of rheumatology between January and June 1996, and fulfilling these criteria, were enrolled in the study when venous blood sampling was necessary for medical follow-up. Ten patients were included in each group. The mean age and disease duration of SpA patients (seven men) were 37±8 and 8±8 yr, respectively. Six of these patients had ankylosing spondylitis, one patient had Crohn's disease with inflammatory articular symptoms and psoriasis, one patient had psoriatic arthritis and two patients had a reactive arthritis. Six were HLA B27 antigen positive. The overall assessment of clinical activity (see below) classified six patients as `active' and four as `inactive'. Seven patients were under non-steroidal anti-inflammatory drug (NSAID) treatment, but none of them had current corticosteroid treatment. Their mean erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels were 38±28 mm/h and 22±30 mg/l, respectively. The mean age and disease duration of RA patients (two men) were 64±13 and 13.8±14.7 yr, respectively. All RA patients had clinically active disease at the time of study (synovitis), seven of them being radiologically erosive and eight with serum rheumatoid factor (RF); none had vasculitis. Seven patients were on a low-dose steroid regimen and four an NSAID treatment. Their mean ESR and CRP level were 58±49 mm/h and 77±112 mg/l, respectively. Plasmas of 21 healthy volunteers obtained from the transfusion centre of Henri Mondor Hospital served as controls. Six of them were men, and the mean age of this group was 45±13.2 yr.
Data and plasma collection
Ten millilitres of blood were collected in a plasma monovette (containing EDTA) by venous puncture during blood sampling for routine biological tests, including an ESR and CRP. Blood samples were immediately centrifuged (10 min at 1500 rv/min), and plasmas were aliquoted and stored at -80°C until used. Data concerning diagnostic criteria, age, sex and current treatment were collected. An overall assessment of disease activity in spondyloarthropathy patients was made by the physician, before knowledge of any laboratory results; the patient was classified as `active' if symptoms were present and `inactive' in the absence of symptoms, whatever the current treatment.
Fibronectin plasma level determinations
Plasma levels of Fns were determined by using an in-house immunocapture ELISA using monoclonal antibodies (MAbs) against each Fn isoform. The characterization and specificity of these MAbs have been described previously [13]. They came from hybridoma conditioned medium: BC-1 for Ed-B Fn, IST-9 for Ed-A Fn and IST-4 for all the Fn isoforms [13]. Microplate wells (Maxisorp, Nunc, Denmark) were coated overnight (+4°C) with MAbs diluted 1:1500 in phosphate-buffered saline (PBS) (200 µl/well). After three washings (PBSTween 20 0.1%), non-specific binding was prevented by incubation with 300 µl/well of skim milk powder at 4% concentration in PBS, for 3 h, at 37°C. After three washings with PBSTween 20 (0.1%), 200 µl/well of plasma samples were added, for 1.5 h, at 37°C. For general Fn (namely soluble plasma Fn), plasma samples were diluted at 1:10 and 1:50 in PBS. For spliced isoforms, plasma samples were used non-diluted or at 1:10 dilution in PBS. After three washings with PBSTween 20, the conjugate [rabbit polyclonal anti-human Fn (Sigma Chemical Co., St Louis, MO, USA)] was added, at 1:2500 dilution, for 1 h, at 37°C (200 µl/well). After three washings with PBSTween 20, peroxidase-linked goat anti-rabbit Fc
globulin was added at 1:5000, for 1 h, at 37°C. After three further washings, the substrate, 12 phenylenediamine dichloride (100 µl/well) and 20 µl H2O2 (30%) in 10 ml citrate buffer (0.15 M , pH 5) were added. The reaction was stopped by adding 50 µl HCl (2 M). Optical density was read at 492 nm on a Titertek Multiscan spectrophotometer. Purified antigens were used for standard curves: general Fn (Sigma), Ed-A Fn (100 µg/ml) (Sigma) and recombinant Ed-B Fn (from L. Zardi) (100 µg/ml). All measurements were performed during the same experiment and in triplicate.
Statistical analysis
Non-parametric tests were used. Comparisons of Fn plasma levels between pairs of groups were made using the MannWhitney test. Correlations between Fn levels and ESR or CRP levels were sought by using Spearman's rank correlation coefficient. A P value of <0.05 was considered significant. The PCSM computer program was used.
 |
Results
|
---|
Plasma levels of total Fn, Ed-A and Ed-B Fn
Plasma levels of total Fn and of each isoform are shown in Figs 13

. Total Fn plasma levels were significantly higher in the SpA group (median=1504 mg/l, mean±S.D.=1387±569 mg/l) than in the RA group (688; 684±196; P=0.02) and HV (290; 303±211; P<0.0001), without any significant difference between the RA and HV groups. Ed-A Fn levels were higher in SpA (22.1; 23±10.4) and RA (33.9; 32.5±16.5) groups than in the HV group (2.9; 2.8±0.9; P=0.0003 and P<0.0001, respectively), without a significant difference between SpA and RA groups. Ed-B Fn levels were higher in SpA (6.7; 6.9±2.1) than in RA (2.6; 3.2±1.9; P=0.02) and HV (1.1; 1.1±0.8; P=0.0003), and higher in RA than in HV. The proportion of plasma Fn containing the Ed-A sequence (Ed-A Fn/Fn) was significantly increased in RA patients (5.3±2.5%) compared with SpA (1.5±0.2%; P=0.02) and HV (1.9±0.4%; P<0.05). The proportion of Ed-B Fn was similar in the three groups, between 0.36 and 0.48%.

View larger version (8K):
[in this window]
[in a new window]
|
FIG. 1. Plasma levels of fibronectin in patients with spondyloarthropathy or rheumatoid arthritis and in healthy volunteers. SpA, spondyloarthropathy; RA, rheumatoid arthritis; HV, healthy volunteer. The median is presented in each group. Comparisons between pairs of groups are made by using the MannWhitney test. *Significantly different in the SpA group from the RA group; #significantly different in the SpA group from the HV group; **significantly different in the RA group from the HV group.
|
|

View larger version (10K):
[in this window]
[in a new window]
|
FIG. 2. Plasma levels of Ed-A in patients with spondyloarthropathy or rheumatoid arthritis and in healthy volunteers. SpA, spondyloarthropathy; RA, rheumatoid arthritis; HV, healthy volunteer. The median is presented in each group. Comparisons between pairs of groups are made by using the MannWhitney test. #Significantly different in the SpA group from the HV group; **significantly different in the RA group from the HV group.
|
|

View larger version (11K):
[in this window]
[in a new window]
|
FIG. 3. Plasma levels of Ed-B fibronectin in patients with spondyloarthropathy or rheumatoid arthritis and in healthy volunteers. SpA, spondyloarthropathy; RA, rheumatoid arthritis; HV, healthy volunteer. The median is presented in each group. Comparisons between pairs of groups are made by using the MannWhitney test. *Significantly different in the SpA group from the RA group; #significantly different in the SpA group from the HV group; **significantly different in the RA group from the HV group.
|
|
Relationships between plasma levels of total Fn, Ed-A, Ed-B Fn and disease activity
In the SpA group, levels of Fn, Ed-A Fn and Ed-B Fn did not differ significantly according to the overall assessment of clinical activity (`active' vs `inactive'; data not shown). However, there was a trend towards an increase of Ed-B Fn in `active' patients (8.2 vs 4.9 mg/l; P=0.06). The clinical picture of the disease, e.g. axial or peripheral, did not seem to influence the levels of Fn and of each isoform. Indeed, comparing the six patients with ankylosing spondylitis (sacroiliitis and lumbar stiffness) with the others, it appeared that Fn levels (median 1622 vs 1318 mg/l, respectively), Ed-A Fn levels (median 20.3 vs 28.9 mg/l, respectively) and Ed-B Fn levels (median 6.3 vs 7.9 mg/l, respectively) did not differ significantly. Furthermore, it should be noted that the two SpA patients who had Fn levels within the range of RA patients did not appear to be distinguishable from the other patients in any point.
No significant correlation was observed in any group of patients between the level of each Fn and the ESR or CRP levels.
 |
Discussion
|
---|
Our study showed increased plasma levels of Fn and Ed-B Fn in SpA patients compared with RA patients and HV. It also revealed an increased proportion of plasma Fn containing the Ed-A sequence in RA patients compared with the two other groups.
Despite many reports on plasma Fn levels in RA and OA (in part in ref. [14]), there are no previous reports in SpA. The mechanism underlying the 2-fold increase in plasma levels of Fn we observed in SpA patients compared with RA patients remains unclear. Indeed, we observed no correlation with the ESR and CRP, suggesting that it does not, or that it does not merely, reflect systemic inflammation as indicated by other acute-phase proteins.
The determination of plasma levels of Ed-B Fn is reported here for the first time, using an in-house immunocapture ELISA and recombinant Ed-B Fn. As expected, these results show that the major part of circulating Fn does not express either Ed-A or Ed-B sequence. To date, the Ed-B exon appears to be expressed during embryogenesis and in transformed cells [5, 6], namely tissues and cells which originate from tissues with high turnover of their matrix. Furthermore, the content in Ed-B has been reported to be increased in human osteoarthritic cartilage compared with normal human cartilage [7, 8], and is also increased in wound healing in rats [15]. Taken together, these data suggest that Ed-B Fn is involved in matrix remodelling, and perhaps particularly in the repair process. In SpA, the main inflammatory site is the enthesis, rather than articular cartilage or synovium [10]. The enthesis is a transitory zone between tendons, ligaments or capsules and bone. It is composed of cells which vary from tendon fibroblasts to chondrocytes, in an extracellular matrix which becomes mineralized in deeper layers [10]. The inflammatory process first gives rise to a transient focal erosive lesion, followed by the repair process, leading to fibrous tissue and then woven bone [10]. Afterwards, this new bone is reorganized, leading to normal bone, in excess compared with the initial state [10]. A new enthesis exists which can be the target of a new inflammatory cycle [10]. Because increased levels of Fn and Ed-B Fn in SpA were not related to systemic inflammation, we may hypothesize that these circulating levels might reflect local turnover in the enthesis matrix.
Another interesting finding of this study concerns plasma Ed-A Fn levels in RA. Our data showing increased levels are in agreement with results of the previous reported study on this issue [3]. However, since we did not extract RF before performing the ELISA test, we cannot exclude an overestimation of the different levels in the RA group by a potential interaction between the multimeric RF and the `capture' and `conjugated' antibodies. We feel that this bias is unlikely here since the two patients without RF had their different Fn levels in the range of the eight RF+ patients. The earlier study of Peters et al. [9] also reported plasma Ed-A Fn levels, but in a group of patients with mixed collagen vascular diseases including some RA patients. These data are in line with the significant expression of Ed-A Fn which has been reported in joints of RA patients, both in synoviocytes [3] and in cartilage specimens [4]. Taken together, these results suggest a role of this Fn isoform in the pathogenesis of RA. However, it cannot be confirmed that excess plasma Ed-A Fn originates from synovium and cartilage, since it may also be derived from endothelial cells or another site of production.
These data led us to conclude that, first, plasma Ed-A and Ed-B Fn might reflect local turnover in inflamed tissues, and, secondly, Ed-B Fn might be particularly involved in the musculoskeletal inflammatory process of SpA.
 |
Acknowledgments
|
---|
Supported by `Association pour la Recherche dans la Polyarthrite'.
 |
References
|
---|
-
Hynes RO. Fibronectins. New York: Springer-Verlag, 1990.
-
Vartio T, Laitinen L, Narvanen O, Cutolo M, Thornell LE, Zardi L et al. Differential expression of the ED sequence-containing form of cellular fibronectin in embryonic and adult human tissues. J Cell Sci 1987;88:41930.[Abstract]
-
Hino K, Shiozawa S, Kuroki Y, Ishikawa H, Shiozawa K, Sekiguchi K et al. EDA-containing fibronectin is synthesized from rheumatoid synovial fibroblast-like cells. Arthritis Rheum 1995;38:67883.[Medline]
-
Chevalier X, Claudepierre P, Groult N, Zardi L, Hornebeck W. Presence of ED-A containing fibronectin in human cartilage from patients with osteoarthritis and rheumatoid arthritis. J Rheumatol 1996;23:102230.[ISI][Medline]
-
ffrench-Constant C, Hynes RO. Alternative splicing of fibronectin is temporally and spatially regulated in the chicken embryo. Development 1989;106:37588.[Abstract]
-
Carnemolla B, Balza E, Siri A, Zardi L, Nicotra MR, Bigotti A et al. A tumor-associated fibronectin isoform generated by alternative splicing of messenger RNA precursors. J Cell Biol 1989;108:113948.[Abstract]
-
Chevalier X, Groult N, Hornebeck W. Increased expression of the Ed-B-containing fibronectin (an embryonic isoform of fibronectin) in human osteoarthritic cartilage. Br J Rheumatol 1996;35:40715.[ISI][Medline]
-
Burton-Wurster N, Lust G, Wert R. Expression of the ED B fibronectin isoform in adult human articular cartilage. Biochem Biophys Res Commun 1989;165:7827.[ISI][Medline]
-
Peters JH, Maunder RJ, Woolf AD, Cochrane CG, Ginsberg MH. Elevated plasma level of ED1+ (`cellular') fibronectin in patients with vascular injury. J Lab Clin Med 1989;113:58697.[ISI][Medline]
-
Ball J. Enthesopathy of rheumatoid and ankylosing spondylitis. Ann Rheum Dis 1971;30:21323.[ISI][Medline]
-
Dougados M, Van der Linden S, Juhlin R, Huitfeldt B, Amor B, Calin A et al. The European Spondylarthropathy Study Group preliminary criteria for the classification of spondyloarthropathy. Arthritis Rheum 1991;34: 121827.[ISI][Medline]
-
Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:31524.[ISI][Medline]
-
Borsi L, Carnemolla B, Castellani P, Rosellini C, Vecchio D, Allemanni G et al. Monoclonal antibodies in the analysis of fibronectin isoforms generated by alternative splicing of mRNA precursors in normal and transformed human cells. J Cell Biol 1987;104:595600.[Abstract]
-
Scott DL, Walton KW. The significance of fibronectin in rheumatoid arthritis. Semin Arthritis Rheum 1984;13: 24454.[ISI][Medline]
-
ffrench-constant C, Van der Watter L, Dvark HF, Hynes RO. Reappearance of embryonic pattern during wound healing in the adult rat. J Cell Biol 1989;109:90913.
Submitted 28 January 1999;
revised version accepted 30 April 1999.