Matrix metalloproteinase 9 (MMP-9) gene polymorphism and MMP-9 plasma levels in primary Sjögren's syndrome
J. Hulkkonen,
M. Pertovaara1,
J. Antonen1,
A. Pasternack1,
M. Hurme1,
P. Pöllänen and
T. Lehtimäki1
University of Tampere Medical School and 1 Tampere University Hospital, Tampere, Finland.
Correspondence to: J. Hulkkonen, Department of Microbiology and Immunology, Medical School, FIN-33014, University of Tampere, Finland. E-mail: bljahu{at}uta.fi
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Abstract
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Objectives. To determine whether plasma matrix metalloproteinase 9 (MMP-9) and MMP9 (1562C
T) polymorphism have an effect on the disease phenotype in primary Sjögren's syndrome (pSS).
Methods. Plasma MMP-9 concentrations and polymorphism of the MMP9 gene were analysed in 66 patients with pSS. These data were studied in relation to the clinical data of the patients. The genetic data of patients were compared with the data of 66 healthy subjects.
Results. Plasma MMP-9 was higher in patients with definite pSS than in patients with possible pSS. This association was principally caused by higher plasma MMP-9 in patients with a positive Schirmer test and keratoconjunctivitis sicca. pSS patients with purpura, SS-A autoantibodies and RF had significantly lower plasma MMP-9 than patients without these characteristics. The overall MMP9 (1562C
T) allele frequencies were similar in patients and control subjects. The frequency of the allele T was higher in patients without Raynaud's phenomenon than in the control group.
Conclusions. MMP9 (1562C
T) could not be used for risk assessment in pSS. The presence of the rarer allele T may decrease the risk of Raynaud's phenomenon in pSS. High plasma MMP-9 is indicative of definite pSS but may paradoxically have a preventive effect on the eruption of purpura and on the development of autoantibody reaction in pSS.
KEY WORDS: Polymorphism, Sjögren's syndrome, Gene, Matrix metalloproteinase, Gelatinase B, Plasma, Risk, Raynaud's phenomenon, Purpura
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Introduction
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Matrix metalloproteinase 9 (MMP-9, gelatinase-B, collagenase type IV-B) is a proteinase capable of degrading a broad range of extracellular matrix components and basement membrane compounds. In addition to its role in physiological tissue remodelling, MMP-9 has a potential role in autoimmunity as it may promote the development of autoimmune neo-epitopes [1]. Moreover, MMP-9 mediates inflammation by converting inflammatory cytokine interleukin (IL)-1ß into its active, secreted form [1].
The regulation of MMP-9 gene (MMP9, chromosomal location 20q13.2) expression is not definitely known, but there is evidence that the promoter region polymorphism of MMP9 partly explains the heterogeneity in MMP-9 transcription. The single-nucleotide polymorphism (SNP) caused by cytosine (C) to thymine (T) base exchange 1562 bases upstream of the MMP9 transcription initiation site (MMP9 1562C
T) has been associated with the transcription rate of MMP9 [2]. The allele C construct has been associated with low and the allele T construct with high MMP9 promoter activity in a reporter gene assay of transiently transfected macrophages [2].
In recent studies the local activity of MMP-9 has been found to be elevated in the saliva and tear fluid of patients with Sjögren's syndrome (SS) [35]. Moreover, high activity of MMP-9 has been detected in the labial salivary glands of patients with severe, active primary SS (pSS), and high MMP-9 expression was correlated with structural and functional glandular changes in these patients [6]. High serum activity of MMP-9 has been found in patients with SLE and particularly with its manifestations such as discoid rash, Raynaud's phenomenon, pneumonitis, mucosal ulcers and anti-phospholipid antibodies [7]. Moreover, excess MMP-9 activation and expression have been implicated in the accelerated breakdown of connective tissue in RA, tumour metastasis and multiple sclerosis [810].
In this study we explored circulating MMP-9 levels and the MMP9 (1562C
T) SNP in well-characterized patients with pSS (n = 66). The potential genetic risk was evaluated by comparing the genetic data with data from healthy control subjects (n = 66). Moreover, the MMP9 allele status and plasma MMP-9 levels were compared with clinical and laboratory parameters in order to evaluate the association of MMP-9 with distinctive phenotypes of pSS.
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Subjects and methods
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Patients
All patients fulfilling three or more modified [11] Californian criteria for primary Sjögren's syndrome [12] were selected from the records of patients with sicca symptoms examined in the Department of Internal Medicine, Section of Rheumatology, at Tampere University Hospital, Finland, during the years 19771992 (n = 111). All patients also fulfilled the European criteria for pSS [13]. Histological findings were graded on the ChisholmMason scale; grades 3 and 4 were regarded as diagnostic [14]. For frequency analyses, patients were assigned to low-grade (grade 02, n = 17) and high-grade (grades 3 and 4, n = 49) groups according to their labial salivary gland histology. Patients were invited for cytokine and gene polymorphism determinations by letter, and specimens were obtained after informed consent from 66 pSS patients. A careful clinical examination and an in-depth interview with the patients had recently been conducted [11]. Clinical methods and classification criteria for the characterization of glandular and extraglandular symptoms of patients have been presented in detail in this journal [11, 15]. Clinical data and patient characteristics at study entry are summarized in Table 1. The study protocol was approved by the ethics committee of Tampere University Hospital.
Control subjects
Sixty-six healthy Finnish Red Cross transfusion service blood donors who were matched for sex (64 female, two male), ethnic origin (Finnish Caucasian) and area of residence (Tampere, Finland) served as a control group for the DNA studies of pSS patients. The mean age of the control subjects was 53 (S.D. 7) years. The DNA sampling of the control group was approved by the Finnish Red Cross transfusion service ethics committee.
Laboratory tests
Rheumatoid factor (RF) was determined by an immunoturbidimetric assay and antinuclear antibodies by indirect immunofluorescence test using Hep-2-cells. Antibodies against ribonuclear antigens SS-A (Ro) and SS-B (La) were detected by enzyme-linked immunosorbent assay (ELISA).
MMP-9 determination
Plasma MMP-9 concentrations were determined using commercially available ELISA (Quantikine human total MMP-9 ELISA kit; RD Systems, MN, USA). The optical density of individual wells was determined with a Multiscan Biochromatic 348 (Labsystems, Helsinki, Finland) spectrophotometer. The detection limit of the MMP-9 assay was 0.156 ng/ml.
DNA isolation and polymorphism analysis
DNA was isolated from citrated blood samples of 65 pSS patients and from leucocyte-rich buffy coats of 66 control subjects using the salting out method [16]. The genetic polymorphism of the MMP9 gene was analysed with nested PCR followed by restriction enzyme digestion with BbuI and 2.5% agarose gel electrophoresis. The method is described in detail elsewhere [2, 17].
Statistical analysis
Group means were compared using KruskalWallis analysis of variance and the MannWhitney U test. The
2 test of 3 x 2 contingency tables for genotype frequencies and 2 x 2 contingency tables for allele frequencies and allele carrier frequencies were carried out as recently suggested [18]. Yates correction was applied when appropriate (n<10). Odds ratios and 95% confidence intervals were calculated using CIA software (v. 1.1, copyrighted by M. J. Gardner and the British Medical Journal, 1989). Findings were considered statistically significant at P<0.05.
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Results
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MMP-9 plasma levels and disease phenotype
The median plasma MMP-9 concentration in patients was 30.9 (mean 39.9, range 12.5134.3, quartile range 21.355.6) ng/ml. The plasma MMP-9 was higher in patients with definite pSS (four Californian criteria, n = 30) than in patients with possible pSS (three criteria, n = 36, P = 0.017; Fig. 1). This association was principally caused by higher plasma MMP-9 in patients with a positive Schirmer test (median 32.8, quartile range 22.859.2 ng/ml, n = 54) compared with patients who were negative for the test (median 24.0, quartile range 17.532.6 ng/ml, n = 12, P = 0.032). When patients having a positive Schirmer test and abnormal rose bengal test at least in one eye were regrouped under the diagnosis keratoconjunctivitis sicca (KCS), the patients with KCS appeared to have higher plasma MMP-9 (median 32.8, quartile range 22.859.2, n = 45) than patients without KCS (median 24.4, quartile range 18.132.9 ng/ml, n = 14, P = 0.057). In contrast to the above, plasma MMP-9 was lower in patients with purpura (n = 12) than in those without (n = 54, P = 0.043; Fig. 1) and markedly lower in patients who had SS-A autoantibodies (n = 45) when compared with SS-A-negative patients (n = 19, P = 0.001; Fig. 1). Plasma MMP-9 was also lower in RF-positive patients (n = 47) than in RF-negative ones (n = 17, P = 0.049; Fig. 1). High plasma MMP-9 was also observed in patients with high (
3) lymphocyte infiltration grade in minor salivary gland biopsy but the difference compared with the low-grade (<3) group did not reach statistical significance (P = 0.154).

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FIG. 1. (A) Plasma MMP-9 concentrations (ng/ml) in pSS patients with possible and definite pSS. (B) Plasma MMP-9 in patients with or without purpura. (C and D) Plasma MMP-9 in patients with or without SS-A autoantibody or RF. Values are median (box) and quartile range (whiskers). Dashed horizontal lines indicate the overall median value in all patients (n = 66).
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MMP9 (1562C
T) allele association analysis
No association between MMP9 (1562C
T) polymorphism and pSS was observed. The percentages of MMP9 (1562C
T) allele C homozygotes, C/T heterozygotes and allele T homozygotes were 62, 34 and 4.6%, respectively, in patients, and 73, 26 and 1.5% in controls (P = 0.372). The overall frequency of allele T was 0.215 for patients and 0.144 for control subjects (P = 0.132). The frequency of MMP9 allele T was higher (0.276) in patients without Raynaud's phenomenon than in control subjects (P = 0.0308, odds ratio 2.27, 95% CI 1.14.8) as the patients with Raynaud's phenomenon had a frequency of 0.167. We did not observe any other differences in the MMP9 genotype or allele frequency distributions when these parameters were compared with the other clinical and immunological characteristics presented in Table 1. The plasma MMP-9 appeared to be higher in allele T homozygous patients than in patients with the C/T or C/C genotype, but this difference was not statistically significant (P = 0.31).
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Discussion
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In the present study, we observed for the first time that the circulating levels of MMP-9 were higher in patients with definite pSS than in patients with possible pSS. This result was principally caused by the association of high plasma MMP-9 with decreased tear fluid flow and KCS. Our results are in line with the observations made by Perez et al. [6] in the labial salivary glands of patients with active, severe pSS. They observed that high local MMP-9 activity correlated with structural and functional glandular impairment in pSS patients. Several other reports suggest that MMP-9 may have a role in glandular destruction and in the development of glandular dysfunction in SS [35, 19]. MMP-9 is also a potential mediator of corneal matrix degeneration in humans [20] and studies on MMP-9-deficient mice suggest that MMP-9 inhibits the rate of corneal epithelial regeneration [21]. As was the case here with circulating MMP-9 and lymphocyte infiltration grade, Perez et al. [6] could not establish any statistically significant correlation with local MMP-9 expression and the number of salivary gland infiltrating lymphocytes. Collectively, these data suggest that high circulating and local MMP-9 are associated with disruption of exocrine gland function, with distinct objective and subjective pSS symptoms such as KCS, and with a high diagnostic score for pSS independent of the rate of lymphocyte infiltration of the glandular tissue.
In addition to glandular dysfunction, we observed that plasma MMP-9 was associated with some extraglandular manifestations of pSS. Low plasma MMP-9 was associated with the presence of purpura in pSS patients. It is interesting that in our previous study the presence of purpura was also associated with low plasma IL-6 [15]. As MMP-9 is one of the key mediators in angiogenesis, endothelial cell morphogenesis and capillary regression, and as IL-6 plays an important role in megakaryocyte maturation, these associations appear plausible [22]. However, within the limitations of our cross-sectional study, the pathogenetic role of low MMP-9 in the development of purpura remains to be elucidated in further studies.
In the previous literature, high MMP-9 has been associated with the development of autoimmunological neo-epitopes [1]. In our study SS-A autoantibodies and RF positivity were associated with low plasma MMP-9. This is interesting as SS-A is a nuclear antigen and the theories concerning the neo-epitope formation all concern the development of extracellular neo-epitopes by extracellular MMPs [1, 23]. The present study is the first to report an association of MMP-9 with autoantibody reaction against an intracellular antigen.
In the present study, we did not observe any significant difference in MMP9 (1562C
T) allele distribution in pSS patients and that in control subjects. It can be concluded from these data that MMP9 (1562C
T) polymorphism does not substantially increase the risk of pSS and that factors other than allelic imbalance of MMP9 (1562C
T) cause the high MMP-9 expression observed in the labial salivary glands, saliva and tear fluid of SS patients [46]. In context of a previous report in SLE [7], which linked high MMP-9 activity with Raynaud's phenomenon, it was a surprise for us to find that the MMP9 (1562) allele T, which has been associated with high MMP-9 expression, seemed to protect pSS patients from Raynaud's phenomenon in pSS. This contrast may be explained by the fact that only eight SLE patients with Raynaud's phenomenon were studied in this previous report, while in our study, there were 36 pSS patients with Raynaud's phenomenon. These associations of MMP-9 with Raynaud's phenomenon, purpura and atherosclerosis are all characterized by endothelial damage, procoagulant activity and vasospasm [2, 24], making MMP-9 an interesting candidate for further endothelial cell studies.
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Acknowledgments
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This study was supported by grants from the Medical Research Fund of Tampere University Hospital and The Pirkanmaa Regional Fund of the Finnish Cultural Foundation. We thank Sinikka Repo-Koskinen and Marita Koli for technical assistance.
The authors have declared no conflicts of interest.
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Submitted 1 April 2004;
revised version accepted 13 July 2004.