The Role of Metalloproteinases & Tissue Inhibitors of Metalloproteinases in the Pathogenesis of Atop

Metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play a role in degradation of extracellular matrix and subsequent tissue remodeling during inflammatory process. Elevated levels of serum TIMPs and /or MMPs have been documented in patients with several inflammatory disorders. Skin inflammation and remodeling are supposed to be pathophysiological features of atopic dermatitis.

The aim of this study was to evaluate the role of TIMPS and MMPs in the pathogenesis of atopic dermatitis (AD) by estimation of serum levels of MMP-3 and TIMP-1 in atopic dermatitis patients before and after treatment.

This study was carried out on 40 atopic dermatitis patients. 20 age and sex matched healthy normal volunteers were chosen as a control group. They were selected from outpatient clinic of Dermatology and Venereology Department of Benha Teaching Hospital. Activity of AD was calculated using the SCORAD index. Patients and controls were subjected to measurement of esoinophil counts from blood film and serum LDH by kinetic assay. Serum IgE, MMP-3 and TIMP-1 were measured by ELISA technique. Patients were given treatment for 4 – 8 weeks in the form of oral antihistamines and topical steroids. Same parameters were measured after treatment.

Results of the study revealed significantly high serum levels of TIMP-1, MMP-3 and TIMP-1/MMP-3 ratio in AD patient before treatment versus controls. After treatment TIMP-1 serum levels and TIMP-1/MMP3 ratio were significantly reduced, but MMP-3 serum levels showed non-significant difference. TIMP-1 serum levels showed positive significant correlation with predictors of disease activity. Patients presented with lichenification, edema, papules, vesicles, oozing, crusts and scratch marks showed significantly higher score in the group of high TIMP-1 serum levels versus the group of normal TIMP-1 serum levels.

The Role of Metalloproteinases & Tissue Inhibitors of Metalloproteinases in the Pathogenesis of Atopic Dermatitis

Maisa M Mokhtar*, Manal I Helmy*, Eman A Eissa** and Lubna Y Ibrahim***

Departments of Dermatology & Venereology*, Clinical Pathology** and Biochemistry ***, Benha
Teaching Hospital

Abstract

Metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play a role in degradation of extracellular matrix and subsequent tissue remodeling during inflammatory process. Elevated levels of serum TIMPs and /or MMPs have been documented in patients with several inflammatory disorders. Skin inflammation and remodeling are supposed to be pathophysiological features of atopic dermatitis.

The aim of this study was to evaluate the role of TIMPS and MMPs in the pathogenesis of atopic dermatitis (AD) by estimation of serum levels of MMP-3 and TIMP-1 in atopic dermatitis patients before and after treatment.

This study was carried out on 40 atopic dermatitis patients. 20 age and sex matched healthy normal volunteers were chosen as a control group. They were selected from outpatient clinic of Dermatology and Venereology Department of Benha Teaching Hospital. Activity of AD was calculated using the SCORAD index. Patients and controls were subjected to measurement of esoinophil counts from blood film and serum LDH by kinetic assay. Serum IgE, MMP-3 and TIMP-1 were measured by ELISA technique. Patients were given treatment for 4 – 8 weeks in the form of oral antihistamines and topical steroids. Same parameters were measured after treatment.

Results of the study revealed significantly high serum levels of TIMP-1, MMP-3 and TIMP-1/MMP-3 ratio in AD patient before treatment versus controls. After treatment TIMP-1 serum levels and TIMP-1/MMP3 ratio were significantly reduced, but MMP-3 serum levels showed non-significant difference. TIMP-1 serum levels showed positive significant correlation with predictors of disease activity. Patients presented with lichenification, edema, papules, vesicles, oozing, crusts and scratch marks showed significantly higher score in the group of high TIMP-1 serum levels versus the group of normal TIMP-1 serum levels.

In conclusion: MMP-3 and TIMP-1 were found to be expressed in enhanced manner in AD patients with significant high TIMP-1/MMP3 ratio. Serum TIMP-1 was significantly correlated with disease activity and chronicity. Serum TIMP-1 level may be a useful marker to estimate long term disease activity.

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^(1-3).   

As inflammation comes and goes, eruptions reveal a chronic type of lesions recognized clinically as lichenified plaques and/or pruriginous nodules which demonstrate increased collagen as fibrosis in the upper dermis⁽⁴⁾. Proinflammatory cytokines such as IL1, IL6, TNF-a and GM-CSF produced by keratinocytes, lymphocytes and macrophages play important roles in the initiation and perpetuation of the eruption⁽⁵⁾. 

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⁽⁶⁾. 

In contrast, tissue inhibitors of metalloproteinases (TIMPs) are secreted proteins that block the activities of MMPs^(6,7). 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^(8-10).

In atopic dermatitis, 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⁽⁴⁾.

The production of TIMPs and MMPs is regulated by pro-and anti-inflammatory cytokines. 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^(11-13). This suggests that increased production of TIMPs and MMPs may also predict the disease activity in atopic dermatitis⁽⁴⁾.

Aim of the work

The aim of this work was to study 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 AD patients before and after treatment. We evaluated whether these values were correlated with disease activity of AD or not. The relationship between serum levels of TIMP-1 / MMP-3 and the type of eruption (acute or chronic) was also determined.

Subjects and Methods

This study was conducted on 40 AD patients (51.35% were females and 48.65% were males). Their ages ranged between 4 and 14 years, with mean ± SD was 9.2 ± 2.758 years. They were selected from outpatient clinic of Dermatology and Venereology Department of Benha Teaching Hospital. 20 healthy normal volunteers were chosen as a control group (had no history of allergic diseases), 46.67% were females and 53.33% were males with their ages ranged between 1.3 and 12 years, mean ± SD was 5.03 ± 3.35 years. All individuals gave informed consent for the study.

Diagnosis of AD patients was made according to criteria proposed by Hanifin and Rajka⁽¹⁴⁾.  

Patients were subjected to the following:

·         Full history taking including onset, course and duration of the disease. Assessment of subjective symptoms (pruritus and sleep loss).

·         Complete clinical examination including assessment of objective signs (extent and intensity).

·         Clinical scoring system:  

Activity of AD was calculated using the scoring atopic dermatitis (SCORAD) index⁽¹⁵⁾. This included assessment of objective signs (extent and intensity) and subjective symptoms (pruritus and sleep loss), the last value depends on an analogue scale by the parents.

Extent was calculated using the "rule of nine" and expressed the skin surface area involved.

Intensity items were erythema, edema / papulation, oozing / crusts, excoriations, lichenification and dryness of uninvolved skin (0 to 3 points for each item). The final score was then calculated by using the following equation: A/5 + 7 B/ 2 +C.

A= extent, B = intensity, C = subjective symptoms.

An objective SCORAD index range lies between 0 and 83.

All patients were treated with topical corticosteroids and systemic antihistamines. No systemic steroids were used. Treatment was continued for 4 – 8 weeks according to the response to treatment. The activity of AD after treatment was calculated using SCORAD index.

Venous blood samples were obtained from patients at 2 occasions: the first was on exacerbation of the disease and the second was after improvement. Blood samples were obtained from controls once. Patients and controls were subjected to the following laboratory investigations: Assessment of peripheral eosinophil counts, serum lactate dehydrogenase (LDH), serum total IgE, serum TIMP-1 and serum MMP-3.

Blood sampling

Six milliliters of venous blood were collected under aseptic precautions. Two milliliters were collected on EDTA for eosinophil counts, whereas the remaining four milliliters were collected in plain tubes, then allowed to clot, followed by centrifugation at 1600 g with subsequent prompt separation of serum. An aliquot of serum was used for immediate assay of serum LDH. The remaining amount was stored at -70°C for the subsequent assay of IgE, TIMP-1 and MMP-3.

A- Analytical methods:

·      Principle of LDH measurement⁽¹⁶⁾: LDH was determined by kinetic assay from a commercial kit available from Centronic GmbH-Germany. The principle of the assay depends on the conversion of pyruvate to lactate with subsequent oxidation of NADH to NAD. The rate of NADH decrease is directly proportional to the LDH activity.

·      Principle of IgE measurement⁽¹⁷⁾: IgE was determined by solid phase enzyme – linked immunosorbent assay technique produced by Clinotech. Diagnostics & Pharmaceuticals.

·      Principle of serum TIMP-1⁽¹⁸⁾ and MMP-3⁽¹⁹⁾ measurement: Serum TIMP-1 and MMP-3 were determined by solid phase sandwich Enzyme linked - Immuno-sorbent Assay (ELISA) technique produced by BioSource International, Inc. A monoclonal antibody specific for TIMP-1 or MMP-3 has been coated on to the wells of the microtiter strips provided. Samples, including standards of known TIMP-1 or MMP-3 content, control specimens, and unknowns, were pipetted into these wells, followed by the addition of a biotinylated monoclonal second antibody.

During the first incubation, the TIMP-1 or MMP-3 antigen bound simultaneously to the immobilized (capture) antibody on one site, and to the solution phase biotinylated antibody on a second site.

After removal of excess second antibody, streptavidin – Peroxidase (enzyme) was added.

This binded to the biotinylated antibody to complete the four member sandwich. After a second incubation and washing to remove all the unbound enzyme, a substrate solution was added, which was acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of TIMP-1 or MMP-3 present in the original specimen.

Statistical analysis⁽²⁰⁾

Data were expressed as mean values, mean ± standard deviation (SD). Student's t test was used to assess the difference between the studied parameters in two groups. Correlation coefficient "r" test was used to evaluate the relation between studied parameters in the same group. The probability (P) value was considered significant if less than 0.05, highly significant if less than 0.01 and very highly significant if less than 0.001. 

Results

This study was carried out on 40 atopic dermatitis patients, 26 females (65%) and 14 males (35%) with their ages ranged between 4 – 14 years and mean ± SD was 9.2 ± 2.758 years. The duration of the disease ranged from 1-6 years with mean ± SD was 3.2 ± 1.9. Twenty healthy volunteers, 9 females (45%) and 11 males (55%) with their ages ranged between 2 – 12 years and mean ± SD was 5.03 ± 3.35 (Table 1).

SCORAD index was very highly significantly higher in AD patients before treatment compared to that after treatment (Table 2).

Laboratory markers of disease activity (serum LDH, serum IgE and peripheral eosinophil counts) were very highly significantly higher in AD patients before treatment versus controls, patients after treatment compared to controls and AD patients before treatment compared to those after treatment (Tables 3, 4, 5).

Serum levels of TIMP-1 were significantly higher in AD patients before treatment compared to controls, patients after treatment versus controls and patients before treatment versus those after treatment. Same results were revealed regarding TIMP1 / MMP-3 ratio (Tables 3, 4, 5).   

MMP-3 serum levels showed significant elevations in AD patients before treatment compared to controls and in AD patients after treatment versus controls (Table 3, 4). But non-significant difference was revealed between serum levels of MMP-3 in AD patients before and after treatment (Table 5).

Comparing the values of predictors of disease activity in atopic dermatitis patients before treatment between the group of high serum levels of TIMP-1 and the group of normal serum levels of TIMP-1 we found the following:

Significant elevations of SCORAD index and serum IgE in the group of high TIMP-1 versus the group of normal TIMP-1. Non-significant elevation of serum LDH and peripheral eosinophil counts in the group of high TIMP-1 (Table 6).

Serum MMP-3 and TIMP-1/MMP-3 showed significant elevation in the group of high TIMP1 levels versus the group of normal TIMP-1 levels (Table 6).

Patients presented with lichenification, edema, papules, vesicles, oozing, crusts and scratch marks showed significantly higher score in the group of high TIMP-1 compared to the group of normal TIMP-1. Patients presented with erythema showed insignificant elevated score in the group of high TIMP-1 (Table 7).

TIMP-1 serum levels showed positive very highly significant correlation with SCORAD index and laboratory predictors of disease activity (LDH, IgE & eosinophil counts) in AD patients before treatment. MMP-3 serum levels showed positive significant correlation with SCORAD only (Table 8).

TIMP-1 serum levels were positively significantly correlated with TIMP-1/MMP-3 ratio while they were insignificantly correlated with serum levels of MMP3 in AD patients before treatment (Table 9).

Table 1.    Demographic data of studied cases versus controls.

Table 2.    Severity index of atopic dermatitis (SCORAD) in patients before and after treatment.

Table 3.    Laboratory data of studied parameters in AD patients before treatment versus controls.

Table 4.    Laboratory data of studied parameters in AD patients after treatment versus controls

Table 5.    Laboratory data of studied parameters in AD patients before and after treatment.

Table 6.    The values of predictors of disease activity and other studied parameters in atopic dermatitis patients before treatment in the group of high TIMP-1 (n=30) versus those with normal TIMP-1 (n=10). The upper limit of the normal TIMP-1 values was set as the mean +2 s.d. of the values in the control group.

Table 7.    Comparison of the points of the eruptions in the group with high TIMP-1 versus those with normal TIMP-1.

Table 8.    Correlation coefficient between TIMP-1 & MMP-3 serum levels and predictors of disease activity in AD patients group before treatment.

Table 9.    Correlation coefficient between TIMP-1 serum levels and MMP-3 serum levels & TIMP-1/MMP-3 in AD patients before treatment and control group.

Discussion

Tissue remodeling of the extracellular matrix (ECM) is an essential and dynamic process associated with physiological and pathological responses. Remodeling involves both degradation and clearance of ECM components as well as the production and deposition of newly synthesized components, the balance of these processes results in either preservation or alteration of the structure and functions of the supported tissue⁽²¹⁾.  

MMPs and TIMPs play a role in degradation of extracellular matrix and subsequent tissue remodeling during inflammatory process⁽²²⁾.

Resorption of ECM is medicated predominantly by the matrix metalloproteinases (MMPs)⁽²³⁾. In contrast, tissue inhibitors of metalloproteinases (TIMPs) are secreted proteins that block the activities of MMPs^(6,7).

Skin inflammation and remodeling are important pathophysiological features of chronic eczymatous skin diseases such as allergic contact dermatitis and atopic dermatitis (AD). MMPs have been described to influence tissue remodeling and to facilitate cell migration through their ability to proteolyse the extracellular matrix^(24,25).

Skin lesion in such eczymatous disorders are characterized by the recruritment of inflammatory cells, especially cutaneous lymphocyte- associated antigen – positive activated memory / effector T – lymphocytes, monocytes and mast cells^(26,27).  

In this study we have determined the role of TIMPs and MMPs in the pathogenesis of atopic dermatitis. We evaluated the serum levels of TIMP-1 and MMP-3 in AD patients before and after treatment.

Our study revealed that serum levels of TIMP-1, MMP-3 and TIMP-1 / MMP-3 ratio were significantly higher in AD patients before treatment compared to controls.

Katoh et al.⁽⁴⁾reported that the serum TIMP-1 levels were significantly higher in AD patients in exacerbation status than in non atopic subjects, whereas serum MMP-3 levels were not significantly different between them. As a result AD patients revealed significantly elevated TIMP-1 /MMP-3 ratio.

Matrix metalloproteinases and their inhibitors were found to be expressed in enhanced manner in allergic diseases⁽²⁸⁾.   

In the skin compartment, eosinophils, mast cells, langerhans cells (LCs) and keratinocytes express MMP-9 and MMP-3^{(29,30,31,32,33)}. MMP-3 known as stromelysin is an effective activator of proMMP-9, but the broad substrate specificity of MMP-3 also includes laminin, collagen type IV, fibronectin and proteoglycans⁽³⁴⁾. 

Plasma MMP-9 levels were found to be significantly higher in AD patients compared to controls⁽³⁵⁾. So serum levels of MMP-3 is supposed to be significantly higher in AD patients versus controls as MMP-3 is the effective activator of proMMP-9⁽³⁴⁾ as proved by our study. 

The results of Devillers et al.⁽³⁵⁾ supported the role of MMP-9 in the pathogenesis of AD. Similarly our results supported the role of MMP-3 in the pathogenesis of AD.

Migration of inflammatory cells into the epidermis and mobilization of langerhans cells (LCs) in and out of the epidermis involve the complex regulation of cell – cell and cell – matrix interaction by means of adhesion molecules, MMPs and cytokines⁽³⁶⁾.

It has been demonstrated that MMP-9 enables the emigration of LCs from basal membrane to the lymph node and migration of inflammatory cells into the epidermis⁽³⁷⁾.

In spite of different MMP-3 serum levels in our study compared to the results of Katoh et al.⁽⁴⁾,the net result and the most important point was the highly significant elevation of TIMP-1 / MMP-3 ratio in AD patients before treatment in both studies.

Our study revealed that TIMP-1 levels showed positive insignificant correlations with MMP-3 serum levels in AD patients before treatment and in controls. TIMP-1 serum levels showed positive significant correlations with TIMP-1 /MMP-3 ratio in AD patients before treatment. Katoh et al.⁽⁴⁾proved that serum levels of MMP-3 and TIMP-1 correlated significantly in control individuals but not in AD patients.

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 autocrine fashion suggesting that TIMPs themselves have fibrogenic activities⁽⁸⁾. Longterm 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⁽⁴⁾. This will explain the results of our study that the points of chronic eruptions such as lichenification was significantly high in patients with elevated TIMP-1 compared to those with normal TIMP-1 values. Same results were proved by Katoh et al.⁽⁴⁾.     

A high number of IL13 expressing CD₄⁺ T cells in peripheral blood and in the skin compartment have been observed in AD patients^(38,39).

Keratinocytes (KCs) respond to IL13 with increased MMP-9 expression in acute eczyme. IL13 in KCs may contribute to the initiation and chronification of skin inflammation also by means of degradation of the basement membrane and thus facilitating migration of inflammatory cells into the epidermis which is a hallmark feature of eczyma⁽²⁵⁾. This could explain our results regarding the significantly higher points of acute lesions such as edema, papules, oozing and crusts in AD patients with elevated TIMP-1 levels than those with normal TIMP-1. Our results revealed significantly higher MMP3 serum levels in cases with high TIMP-1 levels compared to those with normal levels. Most of our patients presented with acute exacerbation on top of chronic lichenification. But Katah et al.⁽⁴⁾ found that the points of acute lesions were not different between these groups.

T cells are key regulators of immunological disease parameters. However their contribution to the process of tissues remodeling is ill defined⁽²⁸⁾. The mechanisms by which MMPs and TIMPs are differentially regulated in some pathological conditions is not fully understood⁽⁴⁾. One of the possible explanation is that the production of MMPs and TIMPs are differentially regulated by Th1 and Th₂ cytokines⁽⁴⁾. IL13 is produced by Th1 and Th₂ cells^(40,41). Th₂ – type cytokines IL10⁽⁴²⁾ and IL13⁽⁴³⁾ are reported to enhance TIMP-1 production by macrophages and fibroblasts respectively. In addition IL4⁽⁴⁴⁾ and IL13⁽⁴³⁾ have been shown to inhibit MMP-3 production by fibroblasts. Decreased MMP activity is associated with upregulation of TIMP-1⁽⁴⁵⁾. IL13 has been implicated in the pathogenesis of a number of diseases associated with fibrosis including atopic dermatitis, asthma and systemic sclerosis^{(46,38,47,48,49)}. IL13 (and IL4) may inhibit collagen degradation through inhibition of MMP-1 and MMP-3, enhancing collagen deposition⁽⁴³⁾.   

Several reports using tissue sample 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^{(50,51,38,52,53)}. Katoh et al.⁽⁴⁾ reported that Th₂ – type cytokine might play some role in the elevation of serum TIMP-1 / MMP-3 ratio in exacerbation status. Our study confirmed the same result.

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^{(54,42,8,9,43,30,55)}. 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 factors alpha, granulocyte – macrophage colony – stimulating factor and transforming growth factor beta have been shown to enhance TIMP-1 productions by these cells^(56,57,58). 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⁽⁴⁾.    

Evaluating the predictors of disease activity in AD patients before treatment in the group with high TIMP-1 levels in relation to the group with normal levels, our study proved that SCORAD and serum total IgE levels were significantly higher in the group with high TIMP-1 levels than those with normal levels. Katoh et al.⁽⁴⁾ revealed that peripheral eosinophil counts, serum levels of LDH, serum total IgE levels and eruption score were significantly higher in AD patients with elevated TIMP-1 values compared with those with normal TIMP-1 levels.

Our study revealed that TIMP-1 serum levels in AD patients before treatment showed positive significant correlation with SCORAD, serum levels of LDH, total IgE and peripheral eosinophil counts. MMP-3 serum levels showed positive significant correlation with SCORAD and insignificant correlation with serum levels of LDH, total IgE and peripheral eosinophil counts in AD patients before treatment. So our study revealed that TIMP-1 serum levels were correlated with disease activity.

The mean value of eruption score was reduced significantly after treatment of our patients with oral antihistamines and local steroids. The serum levels of TIMP-1 were significantly reduced in AD patients following conventional treatments, whereas those of MMP-3 showed insignificant decrease. The change of TIMP-1 / MMP-3 ratio was significant. Same results were reported by Katoh et al.⁽⁴⁾ except that TIMP-1 /MMP3 showed insignificant change after treatment. Our study revealed that serum levels of TIMP-1, MMP-3 and TIMP-1 /MMP-3 ratio were significantly higher in AD patients after treatment compared to controls. This means that the pathological changes of AD remained after treatment but reduced to some extent.

In conclusion our study revealed that MMP-3 and TIMP-1 were found to be expressed in enhanced manner in AD patients with significant high TIMP-1/MMP3 ratio. TIMP-1 serum levels correlated significantly with all predictors of disease activity. MMP-3 may have a role in the pathogenesis of acute phase of AD. Our data demonstrated significant correlation of serum TIMP-1 levels with disease activity and chronicity. The most important parameter is TIMP-1/MMP3 ratio. Serum TIMP-1 level may be a useful marker to estimate long term disease activity of AD and the occurrence of steroid resistant and refractory eruptions.

Recommendations

The consideration of changes of MMPs and their inhibitors in AD patients may help the evolution of new therapeutic interventions that can be further investigated. New therapeutic interventions are greatly needed for steroid resistant and refractory eruptions. They may selectively regulate aberrant pathophysiological mechanisms in AD.   

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