Activity of matrix metalloproteinase-9 is elevated in sera of patients with systemic lupus erythematosus

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the increased production of autoantibodies and by systemic clinical manifestations and damage to multiple organs. The aim of the present study was to analyse matrix metalloproteinase (MMP)-9 activity in sera of patients with active and inactive SLE in order to evaluate its role in the pathogenesis and course of the disease, as well as its diagnostic value. We measured activity levels of MMP-9 and MMP-2, using both gel zymography and activity assay kits, in sera of 40 SLE patients and of 25 healthy controls. We found that MMP-9 activity, but not MMP-2 activity, is significantly elevated in the sera of SLE patients compared with sera samples of healthy controls. High activity levels of MMP-9 were determined in sera of 68% of the SLE patients. Elevated levels of MMP-9 were correlated with the presence of discoid rash, Raynaud phenomenon, pneumonitis, mucosal ulcers and anti-phospholipid antibodies. Changes in activity levels of MMP-9, but not of MMP-2, were observed in sera of the same patient at different periods of the disease course. High levels of MMP-9 did not correlate with disease activity index (SLEDAI, BILAG) in female patients, but correlated with SLE activity in the group of male patients. The results of the present study suggest that MMP-9 plays a role in the pathogenesis of SLE.

INTRODUCTION

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that exhibits various clinical manifestations, including immune complex depositions in the kidneys and other organs [1]. The cause of the disease has not yet been defined yet. However, it involves the production of a broad spectrum of autoantibodies against nuclear, cytoplasmic and cell-surface antigens, and different impairments of B-and T-cell functions [1,2]. Proinflammatory cytokines, especially TNF-α and IL-1, were shown to play an important role in the pathogenesis of SLE, both in the human disease [3] and in murine models [4–6]. The latter cytokines were shown to directly induce matrix metalloproteinases (MMPs) in several cell types, including rheumatoid synovial fibroblasts [7] and monocytes [8]. Thus, MMPs may also play a role in the pathogenesis of SLE.

MMPs constitute a family of zinc-containing endo-proteinases [9–11] that not only play an important role in the remodelling of extracellular matrix in normal tissues, but also contribute to pathological processes. They share structural domains but differ in substrate specificity, cellular sources and inducibility. The MMPs are synthesized as zymogen-like latent precursors and are converted subsequently to an active form. MMP-2 and MMP-9, which are both gelatinases, can degrade type IV collagen, denatured collagens, types V, VII, X and XII collagens, vitronectin aggrecan, galectin-3 and elastin. MMP-2 is the most widely expressed MMP. It is produced by a variety of cells and is frequently elevated in malignant tumour metastasis [9–11]. In terms of protein and domain structure, MMP-9 is the largest and most complex member identified so far. MMP-9 expression is characterized by a complex regulation with a tight control at the levels of gene transcription and protein secretion (such as cytokines, chemokines, eicosanoids), inflammatory mediators, and action of tissue inhibitors of metalloproteinases (TIMPs). In addition, MMP-9 activity is modulated by the activation of pro-MMP-9 by components of the plasminogen activation system or other MMPs [12]. The involvement of MMPs in autoimmune diseases was demonstrated in multiple sclerosis [13] and its experimental model experimental autoimmune encephalomyelitis [14], rheumatoid arthritis [15], experimental bullous pemphigoid [16], Guillain–Barré syndrome [17] and experimental autoimmune neuritis [18]. Serum levels of MMP-3 [15, 19–21] and tissue inhibitor of metalloproteinases (TIMP)-2 in patients with lupus nephritis were reported to be significantly higher than those of healthy controls.

The potential importance of the many activities of MMPs in inflammatory responses has been suggested by the inhibitory effects of MMP inhibitors in several animal models of autoimmune diseases. Specific inhibition of MMPs in vivo suppresses oedema, pathologic tissue proliferation, and damage to specialized tissue structures in several inflammatory and autoimmune diseases [14,22,23].

In the present study, we determined the levels of MMP-9 and MMP-2 in sera of 40 patients with SLE, and we demonstrate that MMP-9, but not MMP-2, activity is significantly elevated in sera of SLE patients compared with healthy controls. High MMP-9 activity correlated with the presence of discoid rash, Raynaud phenomenon, pneumonitis, mucosal ulcers and the presence of anti-phospholipid antibodies (APLA). In addition, elevated levels of MMP-9 correlated with SLE activity in the group of male patients.

MATERIALS AND METHODS

Patients

Forty patients, 32 females and eight males with SLE participated in this study. All patients revealed at least four of the revised diagnostic criteria of the American College of Rheumatism (ACR) for the diagnosis of SLE [2]. Twenty-five sex-and age-matched healthy volunteers served as a control group in our studies. The mean age of patients at diagnosis was 29 ± 9·7 (range 15–48) years and the mean follow-up period was 11 ± 10 (range 1–32) years. Disease activity was determined according to the SLEDAI lupus activity index [24] and by the BILAG index [25]. The study was approved by the ethic committee of the Kaplan Medical Center.

Measurement of MMP-2 and MMP-9 by activity assay kits

Activities of MMP-2 and MMP-9 were measured by specific Biotrak MMP-2 or MMP-9 activity assay kits (Amersham Pharmacia Biotech UK Limited, Little Chalfont, UK) according to the manufacturer’s instructions. Sera were diluted 1:100 and 1:32 for the determination of MMP-2 and MMP-9 activities, respectively. The appropriate standards were added in each assay. In order to measure the total content of the MMPs, activation of the pro form of the MMPs was performed using p-aminophenylmercuric acetate (APMA).

Measurement of MMP-2 and MMP-9 activities by gel zymography

MMP-2 and MMP-9 activities were tested by gelatin zymography. A 5 μl sample of serum was separated by an 8% SDS-PAGE gel polymerized with 1 mg/ml gelatin. Gels were washed once for 30 min in 2·5% Triton X-100 to remove the SDS, and once for 30 min in the reaction buffer containing 50 mm Tris-HCl, 200 mm NaCl, 10 mm CaCl₂ and 0·02% (w/v) Brij 35 (pH 7·5). The reaction buffer was changed to a fresh one, and the gels were incubated at 37°C for 24 h. Gelatinolytic activity was visualized by staining the gels with 0·5% Coomassie brilliant blue and quantified by densitometry.

Statistical analyses

The data were evaluated using chi-square or Fisher exact tests, unpaired t-test and two-tailed P-values. Pearson, Spearman and multivariate analyses were also used.

RESULTS

Activity of MMP-9 but not of MMP-2 is elevated in SLE

MMP-9 was shown to be involved in several autoimmune diseases [13,16,17] as well as in animal models of SLE (A. Faber-Elmann et al. unpublished results). Thus, we were interested in studying whether MMP-9 is also elevated in sera of SLE patients. For this purpose, we examined sera of 40 SLE patients and 25 healthy controls by gel zymography, in which both MMP-9 and MMP-2 activities can be visualized. A representative gel is shown in Fig. 1. As can be seen in this figure, levels of MMP-9 are elevated in the sera of SLE patients compared with healthy controls. Densitometric analysis of the zymograms of sera of 40 SLE patients and 25 healthy controls indicated that the mean MMP-9 activity for SLE patients was 109 ± 5·6 densitometry units and for the healthy controls, 76·5 ± 4·2 densitometry units (P = 0·0001). Activity values of above 85 densitometry units (mean of healthy controls + 2 s.e.) were considered high. The results demonstrated high activity levels of MMP-9 in 68% of the SLE patients. Only 3% of healthy controls exhibited high MMP-9 activity (P = 0·001). Densitometric analysis of MMP-2 levels in the same serum samples revealed that the differences in MMP-2 activity between sera of SLE patients and of healthy controls were not significant. Thus, values of 109 ± 7 and 123 ± 5 (mean activity densitometry units ±s.e.) were determined for healthy controls and SLE patients, respectively (P = 0·0531). To quantify the activity levels of MMP-9 and MMP-2 in the serum further, we used activity assay kits. Figure 2 shows that the activity of MMP-9 is elevated threefold in sera of SLE patients compared with sera of healthy controls, and this elevation is statistically significant (P = 0·0302). In contrast, the differences in the levels of MMP-2 between the two groups are not significant (P = 0·1254).

Figure 1.

Activity of MMP-2 and MMP-9 in sera of SLE patients and healthy controls. Sera (5 μl) of 40 individual SLE patients and 25 healthy controls were analysed for their MMP-2 and MMP-9 activities by gel zymography. The figure shows representative results with serum samples of the two groups.

Figure 2.

Quantitative analysis of MMP-2 and MMP-9 activities in the sera of SLE patients ▪ and healthy controls (□). Thirty-six serum samples of SLE patients and 15 serum samples of healthy controls were tested for MMP-2 or MMP-9 activity using specific activity assay kits. Results are expressed as the mean ± s.e.m. *P = 0·0302.

Since we, as well as others [26,27], detected high MMP-9 levels in sera of patients with non-SLE chronic renal failure (e.g. diabetes mellitus, hypertension) probably due to the retention of the enzyme, we analysed the correlation between levels of MMP-9 and kidney function in the group of SLE patients tested. No correlation was observed between creatinine levels and MMP-9 levels (r² = 0·01), indicating that the elevated levels of MMP-9 in SLE patients were not the result of retention of the enzyme due to renal impairment.

Correlation of MMP-9 activity with clinical and laboratory parameters

The elevation in the activity levels of MMP-9 in sera of SLE patients prompted us to look for possible correlation between clinical and laboratory parameters, and serum MMP-9 levels. Statistical analysis (chi-square or Fisher exact tests) was performed by investigating the number of patients with high and normal MMP-levels for each clinical manifestation (Table 1), as well as by taking into consideration the actual mean activity levels of MMP-9 for patients with or without a certain clinical symptom. The results were similar by both analyses. It is noteworthy that for all clinical symptoms, the percentage of patients with elevated MMP-9 levels is much higher than that in the group of healthy controls. Levels of MMP-9 did not correlate with gender, duration of disease or the age of its onset (Pearson, Spearman). Table 1 shows the clinical and laboratory characteristics of the SLE patients according to their MMP-9 activity levels (lower or equal to healthy controls = normal). High levels of MMP-9 correlated significantly with the presence of Raynaud phenomenon (P = 0·0138) and APLA (P = 0·041). A strong correlation could be observed with pneumonitis, discoid rash, neurological disorders and mucosal ulcers. However, the number of patients with the latter manifestations was too small to perform a statistical analysis. Multivariate analysis revealed that Raynaud phenomenon and low complement (C3, C4) levels are positively correlated with high MMP-9 levels (P = 0·0001 and 0·0137, respectively). In contrast, photosensitivity, arthritis and haematological disorders are negatively correlated with MMP-9 activity levels (P = 0·0381, 0·0014 and 0·0065, respectively).

Table 1.

Clinical characteristics of SLE patients with high and normal MMP-9 activities according to their MMP-9 levels

		MMP-9 levels (%)
		High	Normal
Number of patients (%)	40 (100)	27 (68)	13 (32)
Photosensitivity	13	8 (62)	5 (38)
Mucosal ulcers	9	8 (89)	1 (11)
Malar rash	9	7 (78)	2 (22)
Discoid rash	5	5 (100)	0 (0)
Raynaud phenomenon	8	8 (100)	0 (0)
Vasculitis	18	14 (78)	4 (22)
Arthritis	31	21 (68)	10 (32)
Serositis	9	7 (78)	2 (22)
Pneumonitis	4	4 (100)	0 (0)
Neurological disorders	4	4 (100)	0 (0)
Renal disorder	16	11 (69)	5 (31)
Haematological disorders	29	18 (62)	11 (38)
ANA	40	27 (68)	13 (32)
αds-DNA	36	24 (67)	12 (33)
APLA	25	20 (80)	5 (20)
Low complement (C3, C4)	30	21 (70)	9 (30)

Clinical involvement was defined according to the ACR revised criteria [2]. Anti-nuclear antibodies (ANA) and anti-ds DNA antibodies were determined using Hep2 cells and Crithidia luciliae, respectively. Anti-phospholipid antibodies (APLA) were defined as reactivity with one or more of the following assays: false positive VDR, lupus anticoagulant (LAC) or ELISA for anticardiolipin antibodies.

We also looked for a possible correlation between SLEDAI and MMP-9 activity in male (Fig. 3a) and female patients (Fig. 3b). Interestingly, the correlation coefficient was significant and positive for men (r² = 0·6333) but insignificant and negative for women (r² = 0·0571). Similar results were obtained using the BILAG scoring system. Thus, a positive correlation coefficient between MMP-9 activity and BILAG scores was observed for men (r² = 0·6442) and an insignificant one for women.

Figure 3.

MMP-9 activity levels and disease activity indices (SLEDAI) in patients with SLE. Thirty-five serum samples from eight males (a) and 27 females (b) SLE patients were tested for MMP-9 activity by a specific activity assay kit. The distribution of MMP-9 activity according to the SLEDAI of the patients is presented. The dashed line represents the activity of MMP-9 in healthy controls.

It was also of interest to determine whether a correlation exists between the use of various treatment modalities by the patients and MMP-9 activity. As can be seen in Table 2A, there was no significant correlation between the current treatment of the patients and MMP-9 activity. However, when we looked at treatment of patients at any time during their disease course (Table 2B), high MMP-9 levels were associated with usage of cytotoxic agents (82%).

Table 2.

Treatment modalities of SLE patients according to their MMP-9 levels

		MMP-9 Levels (%)
	Total nuber of patients	High	Normal
A. Current treatment
Cytotoxic agents	8	6 (75)	2 (25)
Steroids	23	17 (74)	6 (26)
Anti-malarial	21	14 (67)	7 (33)
NSAID	7	5 (71)	2 (29)
B. Treatment during the follow-up period
Cytotoxic agents	17	14 (82)	3 (18)
Steroids	29	19 (66)	10 (34)
Anti-malarial	26	16 (62)	10 (38)
NSAID	18	12 (67)	6 (33)

The anti-malarial agent hydroxychloroquine was used at dose of 200–400 mg/day. Steroid treatment was defined as a daily dose ≥5 mg of prednisone. Cytotoxic agents used were cyclophosphamide (0·5–1 g/m² monthly) or azathioprine (100–150 mg/day).

Variations in MMP-9 activity in serum samples taken from individual SLE patients at different time points

Since disease activity varies over time, we measured the activity levels of MMP-9 and MMP-2 in the serum of individual patients that were sampled during 4–6 years of follow-up. Sera of nine patients taken at different time points were analysed. Levels of MMP-2 did not vary significantly between patients and healthy controls. In five out of the nine patients tested, variations in MMP-9 activity in serum samples of individual patients could be observed with time. The results for two representative SLE patients are shown in Fig. 4. As can be seen, MMP-9 activity, but not MMP-2 activity, has been changing with time in the same patients. These changes were not associated with disease activity indices as determined by either the SLEDAI or BILAG systems. Changes in MMP-9 activity were not detected in sera of five healthy controls that were sampled at different time points (data not shown). In four other SLE patients, no substantial changes in MMP-9 or MMP-2 activity were observed with time, and MMP-9 activity levels remained either high or low, depending on the individual patient.

Figure 4.

Pattern of (○) MMP-2 and (•) MMP-9 activities in sera of two SLE patients sampled during 4–6 years of disease. The sera were tested for MMP-2 or MMP-9 activities by specific activity assay kits. The assays were performed in duplicate.

DISCUSSION

The present study demonstrates for the first time the involvement of MMP-9 in human SLE. We show that the activity of MMP-9, but not MMP-2, is significantly elevated in sera of 68% of SLE patients compared with healthy controls. High MMP-9 levels correlated with Raynaud phenomenon, pneumonitis, neurological disorders, discoid rash and the presence of APLA. Changes in MMP-9 activity were observed in serum of the same patient at different periods of the disease. MMP-9 activity levels did not correlate with disease activity index (SLEDAI, BILAG) in female patients, but correlated with SLE activity in the group of male patients.

The present study shows that activity levels of MMP-2 are not elevated significantly in sera of SLE patients. These results are compatible with those reported by Zucker [20], that MMP-2 levels were not increased in SLE. Levels of MMP-2 were also constitutive and unchanged in other pathological conditions (like optic neuritis and multiple sclerosis) in which levels of MMP-9 were elevated relative to the healthy controls [28,29]. Elevated levels of MMP-2 were determined in patients with membranous nephropathy [30] and in psoriatic epidermis [31].

Involvement of an additional MMP, namely MMP-3, was suggested in the pathogenesis of SLE, since it was significantly increased in sera of patients with SLE [32]. The frequency of SLE patients with elevated MMP-9 activity (68%) shown in the present report, resembles the frequencies reported [32] for high MMP-3 levels in SLE (76%) and in RA (82%) patients. Furthermore, the MMP-3 transcript was shown to increase significantly with the progression of nephritis in (NZB × NZW) F1 mice [33]. It is noteworthy that we have recently shown elevated levels of MMP-3 in sera and kidneys of mice afflicted with SLE in induced and spontaneous models of SLE (A. Faber-Elmann et al., unpublished results).

The origin of the elevated MMPs in sera of SLE patients is not known. MMP-9 has been shown to be secreted by peripheral blood cells such as T cells, neutrophils and macrophages (for review see [10]). It has also been demonstrated that MMP-9 is produced by glomerular epithelial [34] and mesangial cells [35], as well as by pleuritic cells [36]. The fact that no correlation was found between MMP-9 activity levels and the number of peripheral blood cells in the patients may suggest that MMP-9 was not secreted by peripheral blood immune cells but rather, by SLE-affected organs like kidneys or lungs/pleura. The observation that all SLE patients with pneumonitis exhibited high MMP-9 activity levels may suggest the diseased lung as a source of high MMP-9 levels. Moreover, the association between cytotoxic treatment, which represents the severity of SLE-related organ impairment, and high levels of MMP-9 in the sera may also support the notion that the diseased organs are the source of MMP-9 activity in SLE patients. Nevertheless, the possibility still exists that fewer peripheral blood lymphocytes secreted higher activity levels of MMP-9.

Elevated levels of MMP-9 activity correlated significantly with disease activity indices in male but not in female SLE patients (Fig. 3). It is possible that these differences are due to the small number of male patients. However, sex hormones, especially progesterone, were shown to regulate the production and activity of MMP-9 [37,38]. Thus, the lack of correlation between disease activity indices and MMP-9 activity levels in the group of SLE female patients might be attributed to the influence of sex hormones.

TNF-α and IL-1 were shown to play an important role in the pathogenesis of SLE both in the human disease [3] and in murine models [4–6]. It has been shown in several systems that these cytokines induce MMP-9 production [12] and thus, it is possible that the induction of the latter MMPs is part of the pathogenic effect of these cytokines in SLE. It has been reported that levels of MMP-9, that are secreted spontaneously by peripheral blood monocytes of healthy individuals, were up-regulated upon exposure to TNF and IL-1 [39]. TNF and IL-1 can also stimulate an additional metalloproteinase, namely PUMP-1, which is excreted by mesangial cells [40]. In addition, MMPs of both T cells and macrophages facilitate secretion of TNF-α by cleavage of the membrane-bound form [41]. Thus, these examples demonstrate the mutual regulatory effects of MMP on the proinflammatory cytokines and vice versa. Nevertheless, the fact that in the sera of some of the patients the activity levels of MMP-9 remained within the normal range during the follow-up period, whereas high activity levels of MMP-9 were measured in the sera of most patients, may suggest the involvement of genetic factors in the regulation of the latter.

The results of the present report suggest that MMP-9 might play a role in the pathogenesis of SLE, and that measurement of plasma/serum activity levels of this metalloproteinase may provide important information when monitoring patients treated with drugs that interfere with MMP-9 activity.