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Year : 2014  |  Volume : 6  |  Issue : 1  |  Page : 25-30

Expression of matrix metalloproteinase-2 and 9 with their inhibitors, tissue inhibitors of metalloproteinase-1 and 2 in oral lichen planus

1 Department of Oral and Craniofacial Health Sciences, College of Dentistry, University of Sharjah, UAE
2 Department of Oral Pathology, College of Dentistry, University of Anbar, Iraq
3 Department of Oral diagnosis, College of Dentistry, University of Baghdad, Iraq

Date of Web Publication15-May-2014

Correspondence Address:
Natheer Hashim Al-Rawi
College of Dentistry, University of Sharjah, UAE

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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0975-8844.132576

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Objectives: The study aims to explore the expression of the matrix metalloproteinases (MMP-2, -9) and their inhibitors (tissue inhibitors of metalloproteinase [TIMP]-1, -2) as well as the correlation between them in the different morphological forms of oral lichen planus (OLP). Materials and Methods: 28, formalin-fixed, paraffin-embedded specimens of OLP and six specimens of squamous cell carcinoma, which served as the positive control and another six normal oral tissue specimens, which served as the negative control were used in this study. All the 40 samples were investigated immunohistochemically for the expression of MMP-2, -9 with their tissue inhibitors TIMP-1, -2. Results: The immune reactivity of MMP-2, -9, TIMP-1 and TIMP-2 were detected at significant levels within the keratinocytes and lymphocytes of the OLP cases when compared with the normal control group (P < 0.0001) while the MMP-2, -9 immune reactivity was found to be about 1/5-1/10 of that detected in the positive control group. The TIMP-1, -2 were strongly expressed in both keratinocytes and lesional lymphocytes. However, no significant relationship was discovered between the expressions of MMP-2, MMP-9, TIMP-1 and TIMP-2 and the morphological types of OLP. Conclusion: A distinct imbalance is observed between the MMP and TIMP activities when the normal mucosa and OLP sample are compared. The TIMP intensities, on the other hand, were found to be very high, suggesting an endogenous protection of the basement membrane against the degrading effect of the MMP enzymes.

Keywords: Matrix metalloproteinase, matrix metalloproteinase-2, matrix metalloproteinase-9, oral lichen planus, tissue inhibitors of metalloproteinase-1, tissue inhibitors of metalloproteinase-2

How to cite this article:
Al-Rawi NH, Al-Kassam TK, Majeed AH. Expression of matrix metalloproteinase-2 and 9 with their inhibitors, tissue inhibitors of metalloproteinase-1 and 2 in oral lichen planus. J Orofac Sci 2014;6:25-30

How to cite this URL:
Al-Rawi NH, Al-Kassam TK, Majeed AH. Expression of matrix metalloproteinase-2 and 9 with their inhibitors, tissue inhibitors of metalloproteinase-1 and 2 in oral lichen planus. J Orofac Sci [serial online] 2014 [cited 2018 Oct 23];6:25-30. Available from:

  Introduction Top

Oral lichen planus (OLP) is a relatively common disorder affecting about 2% of the general population. [1] It takes several forms; of which; the reticular form is the most common type and appears as a lace-like keratotic mucosal pattern. One variant of reticular OLP is the plaque-like form, which clinically resembles leukoplakia, albeit with multifocal distribution. Both the reticular form and its plaque-like variant are usually asymptomatic. [2] Erosive lichen planus is the second most common type occurring in the oral cavity. Patients with this form of lichen planus usually complain of sore mouth. OLP occurs more frequently than the cutaneous form [2] and could be premalignant, although the malignant transformations were under view. [3],[4]

Matrix metalloproteinases (MMPs) are a structurally related group including at least 25 Ca +2 and Zn−2 -dependent endopeptidases collectively capable of degrading practically all the extracellular matrix (ECM) and basement membrane components. [5] MMPs are known to influence several chronic tissue destructive inflammatory and autoimmune diseases like rheumatoid arthritis, osteoarthritis and periodontitis. [6],[7] In the inflammatory process, the MMPs are upregulated by the cytokines and other proinflammatory mediators. [8] MMPs and mast cell chymase were known to cause basement membrane damage in OLP. Zhou et al. suggested that this damage is usually due to cleavage of collage IV, stromeolysin and laminin by various forms of MMPs like MMP-2 and -9 beside MMP-3 and -10. [9] However, the proteolytic action of these enzymes will be balanced by tissue inhibitors of metalloproteinase (TIMPs) such as TIMP-2 and -2. [10] . The increase in the physiological levels of theTIMP-2 can also promote MMP-2 activation and invasion of certain cancer cells. [11] Therefore, the TIMPs may play paradoxical roles in the progression of several diseases. Zhou et al. suggested that lesional T-cells are found to upregulates MMP-9, which cleaves collagen IV resulting in the basement membrane disruption. [9] The disrupted basement membrane in OLP no longer delivers the keratinocyte survival signals, which, in turn may trigger keratinocytes apoptosis. Based on the effects of the MMPs and their inhibitors mentioned above, the present study was designed to investigate the distribution of some MMPs and their inhibitors TIMPs among the epithelial and inflammatory cells in the different types of OLP patterns.

  Materials and Methods Top

In all 28-formalin fixed paraffin embedded blocks of histologically confirmed OLP were retrieved from the archives of the oral pathology laboratory specimen files of the Dental School in Baghdad University. Six blocks of histologically confirmed oral squamous cell carcinoma (OSCC) were also retrieved and used as the positive control. Another six samples of healthy tissues obtained from the cheek and gingiva surrounding the surgically extracted teeth were used as the negative control. All the 40 paraffin blocks sectioned to 4 µm thickness and mounted on poly-L-lysine-coated slides. The sections were deparaffinized and stained using a standard immunohistochemical technique in a high temperature water bath for antigen enhancement. A peroxidase based system was used to identify the antigen-antibody conjugates (US Biological Co, Massachusetts, USA). The sections were then incubated with primary antibodies against MMP-2 and MMP-9 (mouse monoclonal anti MMP-2 clone 0.N.430, USB M2420). The remaining sections were incubated with primary anti TIMP-2 (mouse monoclonal anti TIMP-2 clone ZQ672, USB TSS85-27) and anti TIMP-1 (rabbit anti TIMP-1 polyclonal antibody, Chemicon AB 8228). The incubation time for all the primary antibodies was extended to 30 min at room temperature for optimum results. The immunostaining procedure was performed using a labeled streptavidin-biotin system (LSAB system-HRP DakoCytomation, Carpinteria, California, USA). Next, the biotinylated secondary antibodies were applied for 60 min. The presence of the antigen was detected by the streptavidin conjugated to the horseradish peroxidase. DAB (3, 3'-diaminobenzidine, DakoCytomation) was used as a chromogen. Mayer's hemotoxylin counter stain was then applied to all the sections for 2 min, dehydrated in graduated alcohol concentrations and finally mounted with distrene, plasticizer, xylene and cover slip. Signal specificity was demonstrated by the absence of immunostaining in the negative control and by the presence of immunostaining in positive control. The positive slides had the typical brown cytoplasm of the keratinocytes and lymphocytes. All the slides were examined using a light microscope (Olympus BX41) with a digital camera at 10, 40 and 100×. The MMP-1, -2 and TIMP-1 and 2 staining reactions were quantitatively evaluated in five fields of each slide at ×40 magnification and recorded as a mean. All the data were analyzed using the statistical package for the social sciences statistical package (SPSS version 18, Chicago, IL, USA). The data were then expressed as mean ± standard deviation. The differences between the groups were analyzed for significance using one-way ANOVA. Statistical significance was defined as P < 0.05.

  Results Top

Among the 28 cases of OLP selected in this study, 15 (53.6%) were of the reticular type, 8 (28.6%) of the plaque type and 5 (17.8%) of the erosive type [Table 1]. Oral keratinocytes in the OLP expressed moderate MMP-2 cytoplasmic intensity (mean: 7.85 ± 14.22), which is about 1/5 of that expressed in the positive control (OSCC samples) (36.5 ± 17.61). Non-significant statistical difference in the mean values of the MMP-2 was seen among the three clinical subtypes of OLP, although the reticular type had a higher MMP-2 score than the others [Table 1], [Figure 1]. MMP-9 was also expressed in the oral keratinocytes, which is about 1/10 of that expressed in the positive control (2.50 ± 3.06 versus 23.17 ± 6.67). The erosive type of OLP revealed the highest MMP-9 score when compared with the other clinical subtypes, although without reaching the statistical level of significance [Figure 2]. Regarding the TIMP-1, the oral keratinocytes showed slightly stronger reactivity when compared with the positive control (27.78 ± 14.49 vs. 25.0 ± 11.4). The erosive type showed the highest mean value among the other subtypes [Table 1], [Figure 3]. However, TIMP-2, was strongly expressed in the oral keratinocytes and its mean value was significantly greater than that of the positive control (40.68 ± 22.4 vs. 20.83 ± 9.17). The reticular type demonstrated the least TIMP-2 reactivity among the other subtypes [Table 2] and [Table 3], [Figure 4]. The lesional lymphocytic infiltrate was also assessed for the immune reaction of the MMPs and TIMPs. The MMP-2 was found to be significantly lower than that of the positive control (7.84 ± 7.72 vs. 45.0 ± 0.55). The reticular type showed the highest MMP-2 immune reaction among the other patterns [Figure 5]. The MMP-9 expression among the lymphocytes was also significantly lower than that of the positive control (7.14 ± 5.12 vs. 35.33 ± 11.55). The erosive type showed the lowest MMP-9 value [Figure 6]. Regarding the expression of the TIMPs, TIMP-1 in all the OLP subtypes was strongly expressed in the lymphocytes and was significantly greater than that in the positive control (31.28 ± 18.33 vs. 25.0 ± 6.77) [Figure 7]. The TIMP-2 expression in the lymphocytes of the reticular type revealed the lowest value among the other types, while the mean values were closer to that of the positive control (31.55 ± 18.91 vs. 32.00 ± 8.24) [Table 4] and [Table 5]. A statistically significant direct relationship was seen between the MMP-2 activity in the lymphocytes and keratinocytes (r = 0.735; P = 0.0001). The TIMP-1 and TIMP-2 were also directly related to each other in both the lymphocytes and keratinocytes [Table 6]. However, no significant relationship was seen between TIMP-1 and MMP-9 or between TIMP-2 and MMP-2 either in the keratinocytes or lymphocytes. In order to assess the state of homeostasis between the MMPs and their tissue inhibitors, the ratio of the MMP-9 to TIMP-1 was measured among the entire sample studied. It is worth mentioning that the negative control sample had a ratio of 1.5:0.33 for keratinocytes and a 1:0.83 ratio for the lymphocytes, whereas for the positive control sample (OSCC) it was 23.17:25.00 for the keratinocytes and 35.33:25.00 for the lymphocytes. The OLP keratinocytes had an MMP-9 to TIMP-1 ratio of about 2.5:27.78 and 7.14:31.28 for the lesional lymphocytes. Regarding the MMP-2 toTIMP-2 ratio for the negative control sample, it was 0.67:1 for the keratinocytes and 1:0.85 for the lymphocytes, for the positive control sample it was 36.5:20.83 for the keratinocytes and 45:32 for the lymphocytes whereas for the OLP it was 7.85:40.68 for the keratinocytes and 4.84:31.55 for the lesional lymphocytes.
Figure 1: Matrix metalloproteinase-2 expression in the basal
keratinocytes of oral lichen planus (×40)

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Figure 2: Matrix metalloproteinase-9 expression in the keratinocytes in oral lichen planus (×40)

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Figure 3: Tissue inhibitors of metalloproteinase-1 expression in the keratinocytes of oral lichen planus (×10)

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Figure 4: Tissue inhibitors of metalloproteinase-2 expression in the keratinocytes in oral lichen planus (×10)

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Figure 5: Expression of matrix metalloproteinase-2 in the lymphocytes of oral lichen planus (×10)

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Figure 6: Matrix metalloproteinase-9 expression in the lymphocytes (×40)

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Figure 7: Tissue inhibitors of metalloproteinase-1 expression in the lymphocytes and keratinocytes (×40)

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Table 1: Morphological types of OLP

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Table 2: MMP-2, -9 and TIMP-1, -2 expressions in the oral keratinocytes

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Table 3: MMP-2, -9 and TIMP-1, -2 expression in the keratinocytes of the different morphological types of OLP

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Table 4: MMP-2, -9 and TIMP-1, -2 immune reaction in the lymphocytes

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Table 5: MMP-2, -9 and TIMP-1, -2 immune reaction in the lymphocytes of the different morphological types of OLP

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Table 6: Correlation coefficient among the parameters studied

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  Discussion Top

In the present investigation, the MMP-9 was upregulated in the lesional T-cells of OLP with very little expression on the keratinocytes, whereas the MMP-2 which digests collagen Type I, II and III was up regulated in both the lesional T-cells and keratinocytes particularly in the reticular form. The present finding was in accordance to that of Zhou et al. [9] who found that MMP-9 and TIMP-1 mRNA levels were greater in the OLP lesional T-cells when compared with the healthy control subjects. They concluded that the T-cell derived MMP-9 may be involved in the pathogenesis of OLP and the relative over expression of the MMP-9 (compared with the TIMP-1) may cause the basement membrane disruption and facilitate migration of the intraepithelial T-cells in OLP. The MMP-9-mediated breakdown of the basement membrane could trigger keratinocyte apoptosis and facilitate the migration of the CD8 + cytotoxic T-cells into the OLP epithelium where they too trigger apoptosis of the keratinocytes. Zhou et al. [9] suggested that the T-cell derived MMP-9 could perhaps play a central role in the disease pathogenesis and new therapies might include blocking the MMP-9 activity in OLP. MMPs proteolysis is regulated by the action of the endogenous inhibitors (TIMPs), which form stable active enzyme-inhibitor complexes with active MMPs or pro MMPs. [3] In this context, either the TIMP-1 and TIMP-2 were over expressed either in the lesional T-cells or oral keratinocytes and their immune scores were even higher than those recorded in the positive control (OSCC). Such changes in the TIMP activities are considered as an important because they directly affect the degree of MMP activity. Zhang et al. [12] suggested that the increase in both the MMP and TIMP levels represented a physiological attempt by the cells to control the MMP activity and maintain a balanced ratio between the two proteins. Maintenance of this equilibrium is essential and any disturbance in this balance will probably result in tissue damage due to increased proteolysis.

This study identified the immune reactivity of the MMP-2, -9 and TIMP-1, -2 in the oral epithelial and lesional T-cells. MMP-2 was found both in the oral epithelia and T-cells in equal proportions, whereas MMP-9 was expressed mainly with the inflammatory infiltrate in the lamina propria with occasional positive cells in the epithelium. The reason for this altered distribution is still unknown. The over expression of the TIMP-1 (compared with the MMP-9) detected in this study may maintain the basement membrane integrity and prevent the migration of the intra-epithelial T-cells. This finding, however, is contradictory to that of Zhou et al. [9] who found that the MMP-9 is over expressed in favor of the TIMP-1 and this might facilitate the intraepithelial T-cell migration in OLP. The MMPs cooperate in a parallel and/or cascade-type of fashion to achieve effective and targeted ECM degradation. These enzymes are not expressed constitutively in vivo but are induced temporarily at the transcriptional level in response to the exogenous signals such as the various cytokines and growth factors, [13] cell matrix interactions [14],[15] and cell-to-cell interaction. [16] The TIMPs are specific inhibitors that bind MMPs in a stoichiometric ratio of 1:1. Under pathologic conditions associated with an imbalance in the MMP activities, changes occurring in the TIMP levels are considered to be significant because they directly affect the degree of MMP activity. The altered ratios of MMP-9 to TIMP-1 and MMP-2 to TIMP-2 measured in this study, in both the lesional T-cells and oral keratinocytes were in favor of the TIMP-1 and TIMP-2 which to suggests that the over expression of theTIMP-1and TIMP-2 might lead to ECM preservation and prevention of the gelatinolytic activity of the MMP-2, -9 in the epithelial basement membrane of OLP. Besides, both TIMP-1 and TIMP-2 possess anti apoptotic activity, [17],[18] which may prolong the keratinocytes and T-cell survival in OLP. Gomez et al. [19] suggested that the TIMPs could be reciprocally expressed with the MMPs; this expression may depend on the release of the endogenous cytokines such as interleukin-1 α [20] and tumor necrosis factor-α [21] from the lesional T-cells or from the oral keratinocytes.

  Conclusion Top

In OLP, an imbalance between the MMP and TIMP activities is clearly observed. The intensities of the TIMPs, on the other hand, were very high suggesting an endogenous protection of the basement membrane against the degrading effect of the gelatinases. The different expression rates between the MMPs and TIMPs could suggest that these enzymes and their inhibitors might be regulated independently and reciprocally. More research is required to unveil the effect of the different inflammatory cytokines and growth factors and their role in the release of the MMPs and TIMPs in OLP.

  References Top

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18.Valente P, Fassina G, Melchiori A, Masiello L, Cilli M, Vacca A, et al. TIMP-2 over-expression reduces invasion and angiogenesis and protects B16F10 melanoma cells from apoptosis. Int J Cancer 1998;75:246-53.  Back to cited text no. 18
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20.Al-Mosawi H. Evaluation of serum and salivary IL-1-α and IL-8 as potential biomarkers in lichen planus diagnosis. M.Sc Thesis. Iraq: College of Dentistry, University of Baghdad; 2004.  Back to cited text no. 20
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]

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