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Clin Exp Immunol. Feb 2002; 127(2): 283–288.
PMCID: PMC1906340

Increased levels of serum tissue inhibitor of metalloproteinase-1 but not metalloproteinase-3 in atopic dermatitis

Abstract

Matrix metalloproteinases and their specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs), contribute to inflammation-induced tissue destruction and subsequent remodeling for maintenance of tissue homeostasis. Since the production of these enzymes and their inhibitors is regulated by mediators such as proinflammatory cytokines and growth factors, elevated levels of serum TIMPs and/or MMPs have been documented in patients with several inflammatory disorders. In this study, we examined the role of TIMPs and MMPs in the pathogenesis of atopic dermatitis (AD) by evaluating the serum levels of TIMP-1 and MMP-3 in 40 patients with AD and 20 control subjects by ELISA. The serum TIMP-1 levels were significantly higher in AD patients in exacerbation status than in nonatopic subjects, whereas serum MMP-3 levels were not significantly different between them. As a result, AD patients revealed significantly elevated TIMP-1/MMP-3 ratios. The levels of serum TIMP-1 were significantly reduced in AD patients following conventional treatments. Significantly higher values of peripheral eosinophil counts, serum levels of IgE and lactate dehydrogenase, eruption score, and eruption area were noted in the AD patients with elevated TIMP-1 levels when compared with those with normal values. Moreover, the points of chronic eruptions such as lichenification and prurigo were significantly higher in the patients with elevated TIMP-1 levels than those with normal TIMP-1, while those of acute lesions such as oozy/microvesicles and oedema were not different between these groups. Serum TIMP-1 level may be a useful marker to estimate the long-term disease activity of AD.

Keywords: TIMP-1, MMP-3, atopic dermatitis, tissue remodeling, chronicity

INTRODUCTION

Atopic dermatitis (AD) is a chronically relapsing inflammatory skin disease frequently seen in individuals with a genetic predisposition to develop an IgE response to common environmental allergens [13]. Eruption of AD begins as acute lesions characterized clinically by oedema, seropapules, vesicles, oozing and crust. Irritable skin associated with a decreased epidermal barrier, repeated exposure to food and inhalant allergens, persistent scratching and excoriating, and concomitant bacterial infections are all responsible for the development and chronicity of the lesions in AD. As inflammation comes and goes, eruptions reveal a chronic type of lesion recognized clinically as lichenified plaques and/or pruriginous nodules, which demonstrate increased collagen as fibrosis in the upper dermis. Proinflammatory cytokines such as IL-1, IL-6, TNF-α and GM-CSF produced by keratinocytes, lymphocytes and macrophages play important roles in the initiation and perpetuation of the eruption [4].

Inflammation-induced tissue destruction is followed by its remodeling for maintenance of tissue homeostasis. Matrix metalloproteinases (MMPs) are a family of zinc- and calcium-dependent endopeptidases that degrade the various components of the connective tissue matrix [5]. In contrast, tissue inhibitors of metalloproteinases (TIMPs) are secreted proteins that block the activities of MMPs with a one-to-one stoichiometry [5,6]. Appropriate regulation of MMPs and their inhibitors TIMPs during the tissue repair processes is important for tissue remodeling, because any imbalance between the enhanced matrix synthesis and diminished breakdown of connective tissue proteins results in increased depositions of extracellular matrix. Elevated levels of serum TIMPs over MMPs have been documented in patients with disorders associated with fibrosis such as systemic sclerosis [79], corticosteroid resistant asthma [10], and liver fibrosis [11]. In AD, it may also be the case that increased production of TIMPs and/or decreased production of MMPs would contribute to the formation of chronic eruptions such as lichenified plaque and prurigo.

The production of TIMPs and MMPs is regulated by pro- and anti-inflammatory cytokines in several inflammatory conditions [5]. Increased production and elevated serum concentration of MMPs and/or TIMPs have been shown to be associated with several clinical markers of disease activity in inflammatory diseases such as rheumatoid arthritis and asthma [1214]. This suggests that increased production of TIMPs and MMPs may also predict the disease activity in AD.

In this report, we examined the serum levels of MMP-3, which is capable of degrading a broad range of extracellular components including type I and III collagens after initial cleavage by a collagenase [5], and its inhibitor TIMP-1 in patients with AD. We evaluate whether these values are correlated with the disease activity of AD. Special attention was paid to the relationship between the serum levels of TIMP-1/MMP-3 and the type of eruptions: acute or chronic.

MATERIALS AND METHODS

Subjects

Forty patients with AD (history of AD: median 18 years, range 2–36) diagnosed according to the criteria of Hanifin and Rajka [15] (age: median 25 years old, range 11–55) were selected for this study. Peripheral blood was obtained when their eruptions were exacerbated. The median serum IgE level of the patients was 4121IU/ml (range: 34–53 300; reference value: <380IU/ml). Patients were then treated with conventional therapy including systemic antihistamine and topical steroids. No patients had been treated with systemic steroids or immune suppressive agents. Serum was obtained once more from 31 of these patients after treatment of AD for an average of 185 ± 104 days. The activity of AD was calculated using our scoring system reported previously [16]. Briefly, it consisted of scoring types of lesions (erythema, oozy/microvesicles, excoriation, lichenification, oedema, prurigo) in 22 anatomical areas: face, scalp, right and left sides of the chest, abdomen, back and buttocks, anterior and posterior aspects of both arms, thighs and lower legs. We estimated the clinical score as the sum of the number of types of lesions in all areas, giving a maximum score of 154. Scratch marks were also evaluated in a similar manner. Patients with moderate psoriasis (n = 10) and healthy controls (n = 20) without any history of atopic diseases including AD, asthma or allergic rhinitis also participated in this study. Informed consent was obtained from all subjects or from their parents.

ELISA

Serum levels of TIMP-1 and MMP-3 (Fuji Chemical Industries, Toyama, Japan) were measured with commercially available kits for sandwich ELISA, according to the manufacturer’s instructions. All serum samples, stored at –80°C until use, were assayed in duplicate. Commercially available kits were used for assaying serum levels of soluble CD25 (Yamanouchi Pharma, Tokyo, Japan) and eosinophil chemotactic protein (Pharmacia & Upjohn AB, Uppsala, Sweden). The levels of major basic protein were also estimated as previously described [17].

Statistical analysis

Data are expressed as mean ± s.d. unless otherwise indicated. Wilcoxon signed-rank tests were performed for comparison analysis between before and after treatment. Other comparisons between two groups were carried out using the Mann–Whitney U-tests, when not otherwise specified. To compare more than two groups, the Kruskal–Wallis test was initially used. All statistical analyses were assessed using StatView Ver. 5 (SAS Institute Inc. NC) on a Macintosh™ computer. A P-value less than 0·05 was regarded as significant.

RESULTS

Serum TIMP-1 and MMP-3 levels in patients with AD

We examined the serum levels of TIMP-1 and MMP-3 in AD patients when their eruptions were aggravated. The serum TIMP-1 levels were significantly higher in AD patients (201 ± 64 ng/ml; range, 93–504 ng/ml) than in control subjects (152 ± 28 ng/ml; range, 110–204 ng/ml) (P = 0·023) but not in the patients with psoriasis (172 ± 24 ng/ml; range, 130–210 ng/ml) (P = 0·493) (Fig. 1a). As shown in Fig. 1b, the increase in the levels of TIMP-1 was still significant (P = 0·0263, Kruskal–Wallis test) when the patients were subdivided into mild (176 ± 32 ng/ml), moderate (182 ± 46 ng/ml), and severe AD (216 ± 72 ng/ml) according to the eruption score. In contrast, the values of serum MMP-3 were not significantly different between AD patients (50 ± 38 ng/ml) and control subjects (52 ± 36 ng/ml) (P = 0·633), or among the patients with each severity (data not shown). The serum MMP-3 levels in patients with psoriasis (133 ± 80 ng/ml) were significantly higher than those in the patients with AD (P = 0·0169) and in the control individuals (P = 0·0089). The serum levels of MMP-3 and TIMP-1 correlated significantly in the control individuals (r = 0·470, P = 0·0353) but not in AD patients (r = 0·195, P = 0·2554, Fig. 2a) or in the patients with psoriasis (r = – 0·113, P = 0·7637). The TIMP-1/MMP-3 ratio was significantly higher in AD patients (5·59 ± 4·46) compared with controls (2·87 ± 1·20, P = 0·0404) and with the patients with psoriasis (1·73 ± 1·28, P = 0·0066). The TIMP-1/MMP-3 ratio in the patients with psoriasis was significantly lower than that in the control subjects (P = 0·0155).

Fig. 1
(a) Serum levels of TIMP-1 in patients with atopic dermatitis (AD, n = 40), with psoriasis (Pso, n = 10), and nonatopic healthy subjects (NA, n = 20). (b) Serum levels of TIMP-1 in control groups and in the patients subdivided by disease severity according ...
Fig. 2
(a) Correlation of serum TIMP-1 and MMP-3. (b) Transition of serum TIMP-1 and MMP-3 levels in association with exacerbation (Pre) followed by treatment (Post) in atopic dermatitis patients. Concentrations of serum TIMP-1 (•) and MMP-3 (□) ...

Next, we analysed whether the values of serum TIMP-1 and MMP-3 would change in association with the treatment of AD eruption. The mean value of the eruption score was reduced from 35·9 to 25·3 after treatment with oral antihistamine and topical steroid. The serum levels of TIMP-1 were significantly reduced in AD patients following conventional treatments (178 ± 55 ng/ml) (P = 0·045), whereas those of MMP-3 showed no significant decrease (52 ± 36 ng/ml) (P = 0·8418) (Fig. 2b). The change of TIMP-1/MMP-3 ratio was not significant (P = 0·0618).

Relation between serum TIMP-1/MMP-3 concentration and other predictors of disease activity

In order to determine the relationship between the clinical backgrounds of the patients whose values of serum TIMP-1 and TIMP-1/MMP-3 ratio were elevated, the patients were subdivided into two groups: patients with AD with elevated levels of TIMP-1 or TIMP-1/MMP-3 ratio and those with normal TIMP-1 or TIMP-1/MMP-3. The upper limit of the normal TIMP-1-values was set as the mean +2s.d. of the values in the control group. As shown in Table 1, significant differences were shown between the patients with elevated TIMP-1 (n = 15) and those with normal TIMP-1 levels (n = 25) in peripheral eosinophil counts (P = 0·0436), serum levels of lactate dehydrogenase (LDH) (P = 0·0066), eruption score (P = 0·0014), and the area of eruption (P = 0·0017). Serum total IgE levels were also significantly higher in the elevated TIMP-1 group than in the normal TIMP-1 group (P = 0·0249). No significant difference was demonstrated between these groups in serum levels of soluble CD25, eosinophil chemotactic protein, or major basic protein (data not shown).

Table 1
The values of the predictors of disease activity in atopic dermatitis

Concerning TIMP-1/MMP-3 ratio, we could not find any significant differences between the patients with elevated TIMP-1/MMP-3 ratio (n = 14) and normal TIMP-1/MMP-3 ratio (n = 26) in the clinical predictors mentioned above (data not shown).

Relation between serum TIMP-1/MMP-3 levels and types of lesions

Finally, we analysed the relationship between serum TIMP-1 titres and types of eruptions in order to estimate in which eruption TIMP-1 was involved. Among the six types of lesions mentioned in Materials and Methods, oozy/microvesicles and oedema were regarded as representative of acute lesion, and lichenification and prurigo as chronic. Scratch marks were also evaluated as representative of itching. The points of lichenification and prurigo were significantly higher in the patients with elevated TIMP-1 levels than in those with normal TIMP-1-values (P = 0·045 and 0·021, respectively, Table 2). No significant differences were demonstrated between these groups in the points of oozy/microvesicles, oedematous erythema, and scratch marks.

Table 2
The points of the eruptions in atopic dermatitis

In the patients with elevated TIMP-1/MMP-3 ratio, only the points of lichenification were shown to be significantly higher than in those with normal TIMP-1/MMP-3-values (P = 0·0326).

DISCUSSION

In this study, well-known predictors of disease activity in AD such as peripheral eosinophil counts and serum LDH levels were shown to be significantly higher in AD patients with elevated serum TIMP-1 values compared with those with normal TIMP-1 levels. The dermal infiltrate in AD consists mainly of lymphocytes, eosinophils, mast cells and macrophages. Although the sources of TIMP-1 in AD are not yet clear, most of the cell types existing in the AD lesions are reported to be able to produce TIMP-1 in other pathological conditions, that is, keratinocytes in wound healing [18], monocytes/macrophages in bronchoalveolar lavage [19], dermal fibroblasts in scleroderma and normal dermis [7,8,20], eosinophils [21], and T lymphocytes [22]. The infiltrating cells in AD produce various kinds of proinflammatory cytokines and growth factors. These mediators and the cells stimulate each other in an autocrine and a paracrine fashion for activation and proliferation, resulting in amplification of allergic inflammation. Moreover, these factors promote signals activating the surrounding keratinocytes and fibroblasts. Proinflammatory cytokines and growth factors such as IL-6, tumour necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor and transforming growth factor-beta have been shown to enhance TIMP-1 production by these cells [2325]. It is therefore suggested that progress of disease activity in AD would be correlated with enhanced release of these mediators, resulting in increased production of TIMP-1 by the cells presenting at the inflammatory site.

Despite the elevation of serum TIMP-1 levels in our AD patients, the serum values of MMP-3 in AD patients were not different from those in nonatopic individuals. As a result, TIMP-1/MMP-3 ratios were significantly higher in sera from the patients with AD when compared with those from control subjects. Similar results were presented in asthmatics, where high serum TIMP-1/MMP-9 ratios were correlated with a poor response to systemic steroid therapy [10]. In another study, Mautino et al. demonstrated that the molar ratio of TIMP-1 to MMP-9 was close to a one-to-one stoichiometry in control individuals and steroid- or cyclosporin A-treated asthmatics, whereas this was not the case in untreated asthmatics [24]. The mechanism by which these enzymes and inhibitors are differentially regulated in some pathological conditions is not fully understood. One of the possible explanations is that the productions of MMPs and TIMPs are differentially regulated by Th1 and Th2 cytokines. Th2-type cytokines IL-10 [19] and IL-13 [20] are reported to enhance TIMP-1 production by macrophages and fibroblasts, respectively. In addition, IL-4 [26] and IL-13 [20] have been shown to inhibit MMP-3 production by fibroblasts. Several reports using tissue samples and sera from AD patients have demonstrated that acute exacerbation of AD eruption is driven by Th2 cells, while maintenance of chronic lesions is associated with both Th1 and Th2 cells [16,2730]. In this study, we examined the serum TIMP-1/MMP-3 levels of the chronic AD patients when their eruptions were agravated. It is thus speculated that Th2-type cytokines may play some role in the elevation of serum TIMP-1/MMP-3 ratio in exacerbation status.

In this study, the points of lichenification and prurigo were significantly higher in the patients with elevated TIMP-1 levels compared to those with normal TIMP-1-values. The points of lichenification were also higher in the patients with elevated TIMP-1/MMP-3 ratio. Excess production of TIMPs over MMPs would prevent the proteolytic degradation and cause deposition of matrix components and subepithelial fibrosis. In addition to inhibiting the enzymatic activity of MMPs, TIMP-1 has been shown to induce proliferation of skin fibroblasts in an autocrine fashion, suggesting that TIMPs themselves have fibrogenic activities [7]. It is therefore suggested that long-term enhanced production of TIMPs compared with MMPs induces thickening of the basement membrane and dermal fibrosis of the lesion in AD, and may lead to steroid-resistant and refractory eruptions.

To conclude, our data demonstrated enhanced production of TIMP-1 in AD and, more interestingly, its significant correlation with disease activity and chronicity. The serum TIMP-1 level may be a useful marker to estimate the long-term disease activity of AD.

Acknowledgments

This work was supported in part by a grant from the Japanese Ministry of Education, Science, Sports and Culture.

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