Expression and Roles of MMP-2, MMP-9, MMP-13, TIMP-1, and TIMP-2 in Allergic Nasal Mucosa

Purpose

Allergic rhinitis (AR) and asthma share many characteristics, but structural changes are observed far less often in AR. Matrix metalloproteinases (MMPs) constitute a family of Zn-dependent endopeptidases that can decompose the extracellular matrix and basement membrane, and regulate cell infiltration. We analyzed the expression of MMPs and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), in allergic nasal mucosa after nasal allergen challenge (NAC) and determined their relationship to inflammatory cells.

Methods

Nasal mucosa specimens were obtained at surgery performed for hypertrophied turbinates. We performed NAC with house dust mite (HDM) allergen disks and control disks, and took biopsies at 30 minutes, 6 hours, and 12 hours after NAC. Cells expressing MMP-2, MMP-9, MMP-13, TIMP-1, and TIMP-2, as well as eosinophils and mast cells, were analyzed immunohistochemically. The MMPs and TIMPs in allergic nasal mucosa were quantified using enzyme-linked immunosorbent assays.

Results

At 30 minutes post-NAC, HDM-exposed nasal mucosa exhibited significantly more MMP-2+, MMP-9+, MMP-13+, TIMP-1+, and TIMP-2+ cells compared with control mucosa, and the numbers of MMP-9+ and TIMP-1+ cells correlated strongly with the number of mast cells. At 6 hours post-NAC, the numbers of MMP+ and TIMP+ cells did not differ significantly between HDM-exposed mucosa and control mucosa, but the ratios of MMP+ cells to TIMP+ cells were higher in HDM-exposed mucosa. At 12 hours post-NAC, the number of MMP-13+ cells tended to be higher in HDM-exposed mucosa and was strongly correlated with the number of eosinophils. Quantitatively, the levels of MMP-2 and MMP-13 were significantly higher than the MMP-9 level, and the TIMP-2 level was significantly higher than the TIMP-1 level in allergic nasal mucosa.

Conclusions

We demonstrated increased expression of MMP-2, MMP-9, and MMP-13 in allergic nasal mucosa, high MMPs-to-TIMP-1 ratios, and a strong correlation between MMP-9 and mast cells and between MMP-13 and eosinophils. The imbalance between MMPs and TIMPs may contribute to the migration of inflammatory cells such as eosinophils and mast cells to the nasal mucosa of AR patients, suggesting a possible active role of MMPs in AR.     

Increased release of matrix metalloproteinase-9 in the plasma of acute severe asthmatic patients.

BACKGROUND: Matrix metalloproteinases (MMPs) are likely to be relevant mediators of the extracellular matrix (ECM) degradation and airway remodelling. OBJECTIVE: We have compared the levels of MMPs, eotaxin and soluble interleukin 2 receptor (IL-2R) in the plasma of healthy subjects, atopic patients and asthmatic patients. METHODS: The asthmatic patients were separated into two groups, either well controlled on inhaled therapy or acute severe asthma. Patients with acute severe disease had all received systemic corticosteroids from 12 to 48 h before the blood was taken. Blood was recovered in EDTA tubes, incubated with either f MLP, PMA or vehicle for 10 min and centrifuged. MMP-9, TIMP-1, IL-2R and eotaxin levels were measured in the plasma by ELISA. Moreover, the activity of MMPs was also evaluated by zymography. RESULTS: An increased basal level of MMP-9 and IL2-R was observed in acute severe asthma. Following stimulation with f MLP and PMA there was an enhanced production of MMP-9 in the plasma of all groups of patients. However, the MMP-9 level was significantly enhanced in acute severe asthma, compared with the others. No difference was found for the TIMP-1 level between the patients. The eotaxin level in plasma was found to be significantly lower in acute severe asthmatics compared with the others groups. Zymography technique showed a significant increased activity of MMP-9 (92 kDa) but not MMP-2 (66 kDa) in the plasma of patients with acute asthma. CONCLUSION: The increased in MMP-9 production and activity observed in the present study suggests a process of extracellular matrix degradation in acute severe asthmatic patients and proposes MMP-9 as a non-invasive systemic marker of inflammation and airway remodelling in asthma.     

Implication of extracellular matrix metalloproteinases in the course of chronic inflammatory airway diseases

Matrix metalloproteinases (MMPs) are major proteolytic enzymes that are involved in extracellular matrix (ECM) turn over. MMP-2 (gelatinase A) and MMP-9 (gelatinase B) cleave type IV collagen, which is an important constituent of basement membrane. These enzymes play an important role in normal tissue homeostasis, but imbalance between MMPs and their tissue inhibitors (TIMPs) is thought to be a critical factor in regulating tissue remodeling. MMP-2 is produced by fibroblasts, endothelial, and epithelial cells, while MMP-9 is mainly produced by inflammatory cells. The role of MMPs was investigated through biochemical analysis or in situ expression, in the pathogenesis of two chronic inflammatory airway diseases, asthma and nasal polyposis. Both are characterized with the accumulation of active inflammatory cells, matrix remodeling and epithelial changes. Increased levels of MMP-9 and TIMP-1 were found in asthmatic subjects and NP. In NP, MMP-9 expression was detected in epithelial, endothelial and inflammatory cells. In this setting, MMP-9 could play a crucial role in the transmigration of basement membrane components by inflammatory cells leading to inflammatory cell accumulation and maintenance of inflammation in airway. Moreover, MMP-9 may contribute to cell migration, an important mechanism involved in the repair of the respiratory epithelium.     

Eosinophil inflammation of nasal polyp tissue: relationships with matrix metalloproteinases,tissue inhibitor of metalloproteinase-1, and transforming growth factor-beta1

Eosinophil and mast cell infiltrations are consistent findings in nasal polyp tissue. Previous studies have shown that matrix metalloproteinases (MMPs) may be involved in eosinophil infiltration in airway mucosa of asthmatic patients, and that transforming growth factor-beta1 (TGF-beta1) induces extracellular matrix deposition in nasal polyp tissue. The aim of this study was to evaluate the role of MMPs and tissue-inhibitor of metalloproteinase-1 (TIMP-1) in association with TGF-beta1, eosinophils and mast cell activation in nasal polyp tissue. Nasal polyp tissues from 20 patients who underwent polypectomies were collected and prepared into tissue homogenate. Eosinophil cationic protein (ECP) and tryptase levels were measured by CAP system (Pharmacia, Sweden). MMP-2, MMP-9, TIMP-1 and TGF-beta1 levels were measured by enzyme-liked immunosorbent assay. MMP-2 was the predominant form of MMPs, followed by MMP-9 and TIMP-1. There were significant correlations between ECP, and MMP-9, MMP-2, TGF-beta1 and tryptase, but not with TIMP-1. Significant correlations were noted between tryptase, and MMP-2, MMP-9, and TGF-beta1, but not with TIMP-1. Close correlations were noted between TGF-beta1, and MMP-9 and MMP-2, but not with TIMP-1. MMP-2, MMP-9, and TGF-beta1 may contribute to eosinophil and mast cell migrations into nasal polyp tissue.     

Promoted activation of matrix metalloproteinase (MMP)-2 in keloid fibroblasts and increased expression of MMP-2 in collagen bundle regions: implications for mechanisms of keloid progression

Aims:  Keloid is characterized by excessive deposition of collagen, resulting from aberrant extracellular matrix (ECM) production and degradation. The aim was to investigate the role of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) in pathological wound healing in keloids.
Methods and results:  Semiquantitative analysis of 60 keloid tissue samples and 25 mature scar tissue samples demonstrated significantly increased expression of MMP-2, TIMP-2 and TIMP-3 in keloids compared with mature scars. Within keloid regions, MMP-2 expression was significantly higher in collagen bundle regions than in non-collagen bundle regions. Double immunofluorescence revealed that keloid fibroblasts between collagen bundles exhibited MMP-2, TIMP-2 and membrane-type 1 MMP (MT1-MMP) co-expression, whereas only MMP-2 expression was evident on the edge of collagen bundles. Western blot analysis and gelatin zymography of 13 keloid-derived fibroblasts (KFbs) and six normal skin dermal-derived fibroblasts (NFbs) demonstrated that unstimulated KFbs exhibited significantly increased MMP-2 activity and expression compared with NFbs under the same conditions.
Conclusions:  These results together indicate that MMP-2 activity can be promoted in keloid fibroblasts between collagen bundles in cooperation with TIMP-2 and MT1-MMP. This could contribute to remodelling of collagen bundle regions and invasion of fibroblasts into peripheral normal regions through promoted degradation of ECM.     

Metalloproteinases in juvenile angiofibroma--a collagen rich tumor

Matrix metalloproteinases (MMPs) act in diverse physiological and pathological conditions such as tumor growth and angiogenesis by cleaving extracellular matrix and nonmatrix substrates. MMPs with gelatinase/collagenase activity have not yet been studied in juvenile angiofibroma, a unique fibrovascular tumor with prominent collagen expression. Quantitative real-time polymerase chain reaction studies, Western blot analysis, immunofluorescence studies, gel zymography, and in situ zymography were used to analyze MMP-1, MMP-2, MMP-9, MMP-13, MMP-14, TIMP-1, and TIMP-2 in 9 juvenile angiofibromas and 2 inferior nasal turbinate specimens. Quantitative real-time polymerase chain reaction found significantly elevated expression of MMP-2, MMP-9, and MMP-14 (P < .05) in tumor tissue compared with the inferior nasal turbinate specimens. Western blot analysis detected more prominent MMP-1, MMP-2, and MMP-9 protein levels in juvenile angiofibromas compared with inferior nasal turbinates, but not MMP-13, MMP-14, TIMP-1, and TIMP-2. Immunofluorescent staining proved a mainly stromal localization of the analyzed MMPs. Only MMP-9 and MMP-14 were also detected in vessel walls. MMP-1, MMP-2, and MMP-13 also stained mast cells. Gel zymography indicated increased MMP-2 and MMP-9 gelatinase activity in juvenile angiofibromas compared with inferior nasal turbinates. Finally, in situ zymography detected very high stromal gelatinase/collagenase activity. This study indicates significant expression of MMPs with gelatinase/collagenase activity in juvenile angiofibromas with evidence of a disturbed balance of MMPs to TIMPs toward enhanced MMP activity. These MMPs are assumed to be involved in tumor pathology with an influence on tumor growth and angiogenesis.     

Metalloproteinases and their Tissue Inhibitors in Multiple Sclerosis

Matrix metalloproteinases (MMPs) comprise a family of proteolytic enzymes. MMPs are capable of disrupting the blood-brain barrier (BBB), mediating the destruction of extracellular matrix and myelin components. MMPs are also involved in the processing of a variety of cell surface molecules, including the proinflammatory cytokine TNF-alpha. Each of these mechanisms are thought to be important in the pathogenesis of multiple sclerosis (MS). We investigated mRNA expression of MMP-3, MMP-9 and two tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in parallel in blood mononuclear cells (MNC) from patients with MS and controls, using in situ hybridization. Numbers of MMP-9 mRNA-expressing cells in blood were higher in patients with MS compared to other neurological diseases (OND), other inflammatory neurological diseases (OIND) and healthy subjects (P<0.0001 for all comparisons). Patients with MS had also higher levels of MMP-3 and TIMP-1 mRNA expressing blood MNC compared to patients with OND and healthy subjects. A positive correlation was observed for MMP-9 and TIMP-1 mRNA expression in MS. These results demonstrate that MMPs and TIMPs are upregulated in MS and may contribute to the pathogenesis of the disease.     

Thrombin and TNF-alpha/IL-1beta synergistically induce fibroblast-mediated collagen gel degradation

Degradation of preexisting and newly synthesized extracellular matrix is thought to play an important role in tissue remodeling. The current study evaluated whether thrombin and TNF-alpha/IL-1beta could collaboratively induce collagen degradation by human fetal lung fibroblasts (HFL-1) and adult bronchial fibroblasts cultured in three-dimensional collagen gels. TNF-alpha/IL-1beta alone induced production of matrix metalloproteinases (MMPs)-1, -3, and -9, which were released in latent form. With the addition of thrombin, the latent MMPs were converted into active forms and this resulted in collagen gel degradation. Part of the activation of MMPs by thrombin resulted from direct activation of MMP-1, MMP-2, MMP-3, and MMP-9 in the absence of cells. In addition, tissue inhibitor of metalloproteinase-1 production was inhibited by the combination of thrombin and TNF-alpha/IL-1beta. These results suggest that thrombin and TNF-alpha/IL-1beta synergize to induce degradation of three-dimensional collagen gels through increasing the production and activation of MMPs, and that this effect is mediated through both direct activation of MMPs by thrombin and indirectly by thrombin activation of fibroblasts. Through such mechanisms, thrombin could contribute to many chronic lung disorders characterized by tissue remodeling.     

Comparison of localized versus systemic levels of Matrix metalloproteinases (MMPs), its tissue inhibitors (TIMPs) and cytokines in tuberculous and non-tuberculous pleuritis patients

The interaction of Matrix metalloproteinases (MMPs), its tissue inhibitors (TIMPs) and pro-inflammatory cytokines in response to Mycobacterium tuberculosis (MTB) infection is important to understand the immune response at the site of infection. We compared the levels of MMPs, TIMPs and cytokines in plasma (BL) and pleural fluid (PF) of tuberculosis (TB) and non tuberculosis (NTB) patients. Comparison between BL and PF showed significantly higher levels of MMP-1, TIMP-1 and -3 in TB PF; of MMP-7, -8, -9 in BL of both groups. Also, levels of MMP-1,-8,-9 and TIMP-3 were significantly higher in TB PF compared to NTB. Cytokines INF-γ, TNF-α, and IL-6 significantly increased in PF of both groups. A positive correlation of MMPs with TIMPs in TB, MMP-1 and -9 with IL-6 in TB PF and MMP-9 with IFN-γ in NTB PF was observed. This study implicates the possible usage of MMPs as bio-markers aiding diagnosis in TB pleuritis.     

The distribution of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the lungs of congenital diaphragmatic hernia patients and age-matched controls

AIMS: In congenital diaphragmatic hernia (CDH), the pathogenesis of abnormal pulmonary morphology is still incompletely understood. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are known to play an important role in the turnover of the extracellular matrix (ECM) during development and in remodelling of tissue. The aim of this study was to investigate differences in the expression of MMPs and TIMPs between CDH lungs and controls, against the background of the abnormal pulmonary vasculature in CDH. METHODS: We studied 12 lungs of term CDH patients who died < 24 h after birth and 11 normal age-matched control lungs, by immunohistochemistry with antibodies against human MMP-1, -2, -9, TIMP-1 and -2. RESULTS: There was a clear increase in the number of MMP-1-reactive capillaries and fibroblasts in CDH lungs compared with controls. In contrast, TIMP-2 reactivity in these structures was decreased in CDH lungs. The arterial endothelium and medial smooth muscle expressed MMP-2, -9 and TIMP-2 in both CDH and control lungs. In small arteries (< 100 microm in diameter), the positive surface area of MMP-2, -9 and TIMP-2 was significantly larger in CDH lungs than in controls. There was no difference in the distribution and expression of TIMP-1 between CDH lungs and normal controls. CONCLUSION: The differences in staining pattern of MMPs and TIMPs between normal and CDH lungs suggest that these enzymes might play a role in the abnormal remodelling of the interstitium and the pulmonary arteries in CDH lungs. This could contribute to our understanding of the abnormal lung morphology and the occurrence of pulmonary hypertension, which forms one of the major obstacles to the successful treatment of these patients.     

Vascular smooth muscle cells and pericytes express MMP-1, MMP-9, TIMP-1 and type I procollagen in inflammatory bowel disease

AIMS: Matrix metalloproteinases (MMPs) are involved in tissue remodelling, which is one of the important aspects of inflammatory disease. To assess the balance between the matrix degradation and production, we analysed the in situ expression of MMP-1, -3, -8 and -9, tissue inhibitor of metalloproteinases (TIMP)-1 and -2, and type I procollagen (PC-I) in inflammatory bowel disease. Methods and results: Immunohistochemistry using frozen sections was performed in 17 patients with ulcerative colitis (UC) and 16 with Crohn's disease (CD). In both UC and CD, MMPs and TIMPs were expressed by inflammatory cells as well as by fibroblastic cells most prominently in actively inflamed areas in ulcer bases, but sparsely in intact inflamed mucosa in both UC and CD. In UC, inflamed mucosa with erosions expressed these substances focally. Fibroblasts also expressed PC-I. We identified that vascular smooth muscle cells of venules in ulcer bases expressed MMP-1 and -9, TIMP-1 and PC-I. These venules also expressed E-selectin, a cell adhesion molecule to facilitate the leucocyte extravasation, and vascular endothelial growth factor (VEGF) receptor 2, consistent with their property of newly formed vessels. CONCLUSIONS: Our results suggest that MMPs are involved in the tissue remodelling, angiogenesis and promotion of leucocyte extravasation in the actively inflamed area in the ulcer base in both UC and CD. MMP-1 expression in the mucosa may be related to the initial step of ulceration in UC. Therapeutic manipulation of extracellular matrix turnover would be an effective therapy to alleviate active inflammation and accelerate ulcer healing.     

咪达普利拉对人心脏成纤维细胞基质金属蛋白酶-2和抑制剂-2的作用及机制研究

目的：研究血管紧张素转化酶抑制剂（angiotensin converting enzyme inhibitor，ACEI）咪达普利活性代谢物咪达普利拉对白细胞介素－1β（interleukin -1β，IL-1β）诱导的心脏成纤维细胞基质金属蛋白酶（matrix metalloproteinases，MMPs）和基质金属蛋白酶2型抑制剂（type 2 tissue inhibitor of matrix metalloproteinase，TIMP-2）表达的影响及其可能机制。方法:原代人心脏成纤维细胞从美国细胞应用所购买。用RT-PCR检测心脏成纤维细胞MMP-2、MMP-9和TIMP-2 mRNA水平的变化；用凝胶酶谱法分析心脏成纤维细胞中MMP-2、MMP-9的活性；用Griess方法检测培养上清一氧化氮（nitric oxide，NO）水平。结果:通过凝胶酶谱分析，RT-PCR和Griess方法发现IL-1β能显著增加MMP-2基因转录以及活性(P<0.05)，同时也显著增加了细胞培养上清中NO的产生(P<0.05)。IL-1β的这些效应可以被咪达普利拉和外源性NO合酶抑制剂L-单甲基精氨酸（NG-methyl L-Arginine，L-NMMA）所抑制(P<0.05)，而外源性NO供体亚硝基铁氰化钠(sodium nitroprusside，SNP）可以取消咪达普利拉对于NO、MMP-2的有效作用(P<0.05)。实验中发现IL-1β以及咪达普利拉对TIMP-2基因转录无明显作用(P>0.05)。结论:咪达普利拉能通过NO途径抑制IL-1β诱导的心脏成纤维细胞MMP-2的合成和活性；提示ACEI抗心肌重塑以及心力衰竭的部分作用可能是通过MMPs途径实现的。     

Matrix metalloproteinases and their inhibitors in the nasal mucosa of patients with perennial allergic rhinitis.

BACKGROUND: Allergic rhinitis and asthma show many similarities in their epithelial and inflammatory responses to allergens. However, one notable difference is that disruption and desquamation of the epithelium is a characteristic feature of asthma, whereas in perennial allergic rhinitis the epithelium is intact and thickened. One reason for this might be differing expression of matrix metalloproteinases (MMPs) or their inhibitors (TIMPs). There are few published data on the presence of MMPs or TIMPs in the nasal mucosa in rhinitis. OBJECTIVE: The purpose of this study was to investigate MMP and TIMP mRNA and protein in nasal mucosa from subjects with perennial allergic rhinitis and from nonrhinitic control subjects. METHODS: Biopsy specimens of nasal mucosa were taken from 10 well-characterized subjects with perennial allergic rhinitis and 10 nonrhinitic control subjects. MMP and TIMP mRNA was quantified through use of competitive RT-PCR, and protein was detected by means of Western blotting and ELISA. RESULTS: TIMP-1 mRNA and TIMP-2 mRNA were present in nasal samples, but there was no significant difference between the 2 groups. Only small amounts of MMP-1, -2, -3, and -9 mRNA were detected in the same samples. The corresponding proteins were detected by means of Western blotting. TIMP-1 protein and TIMP-2 protein were quantified in tissue homogenates; there was no significant difference between the 2 groups. CONCLUSION: Our studies have demonstrated the presence of large amounts of TIMP-1 and TIMP-2 mRNA and protein in nasal mucosa. There is no upregulation of MMPs or changes in TIMP expression in the nasal mucosa of patients with allergic rhinitis.     

TNF-alpha-mediated expression of membrane-type matrix metalloproteinase in rheumatoid synovial fibroblasts.

Degradation of the extracellular matrix plays an important role in rheumatoid articular destruction. Rheumatoid synovial fibroblasts secrete a large amount of matrix-degrading metalloproteinases (MMPs), which initiate tissue damage by proteolytic degradation of collagens and proteoglycans. Cytokines, such as interleukin-1 alpha, -1 beta or tumour necrosis factor (TNF)-alpha, are potent inducers of MMPs in rheumatoid synovial fibroblasts, MMPs are synthesized and secreted as latent pro-enzymes and their activation is achieved by proteolytic cleavage or the propeptide domain at the N-terminus of the molecule. Thus, the interaction of the pro-enzymes with specific activators determines the enzymatic activity in the extracellular space. In the present study, we identified a novel mechanism for the activation of pro-MMP-2, which can be achieved through the interaction of the inflammatory cytokine, TNF-alpha, with synovial fibroblasts. Although MMP-2 is constitutively secreted by synovial fibroblasts as a pro-enzyme, stimulation of fibroblasts by TNF-alpha-induced secretion of MMP-2 in an active form. In support of this result, TNF-alpha stimulation-induced membrane-type matrix metalloproteinase (MT-MMP), a newly identified MMP-2-specific activator on synovial fibroblasts. Cycloheximide analysis demonstrated that protein synthesis may be required for TNF-alpha-mediated MT-MMP expression on synovial fibroblasts. Our results suggest that TNF-alpha induces MMP-2 activation in part by up-regulating MT-MMP expression, thus representing a new mechanism for cytokine-mediated articular destruction in rheumatoid arthritis (RA).     

Matrix metalloproteinases MMP-7, MMP-9 and their tissue inhibitor TIMP-1: expression in chronic sinusitis vs nasal polyposis

BACKGROUND: Nasal polyps (NP) are characterized by pseudocyst formation, whereas the mucosa in chronic sinusitis (CS) only shows a limited oedema. Matrix metalloproteinases (MMPs) are a family of endopeptidases able to degrade the extracellular matrix. Differences in histological features between CS and NP might be related to the respective expression of MMPs and their tissue inhibitors (TIMPs). OBJECTIVE: The aim of this study was to investigate MMP-7, MMP-9 and TIMP-1 proteins in NP and CS in comparison with normal mucosa. METHODS: Nasal samples, obtained from controls (n = 10), from NP (n = 8) and from CS (n = 10), were analysed by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). RESULTS: In NP, compared with controls, staining for MMP-9 and MMP-7 appeared in blood vessels. Matrix metalloproteinase-9-positive inflammatory cells could be detected in increased numbers in pseudocyst formations. Concentrations of MMP-9 protein was found significantly increased in both CS and NP compared with controls, while MMP-7 was significantly increased in NP compared with controls and CS. Tissue inhibitors of metalloproteinase-1 protein was significantly increased in CS and NP when compared with controls. CONCLUSIONS: Chronic sinusitis and NP show different pattern of MMP-7/-9 and TIMP-1 expression. We suggest that this difference in regulation of enzymes is related to the respective tissue remodelling observed in both diseases.     

CD40 engagement modulates the production of matrix metalloproteinases by gingival fibroblasts

Chronic periodontitis is a destructive inflammatory disease linked with unbalanced production between matrix metalloproteinases (MMPs), such as interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3) and their endogenous tissue inhibitors of MMPs (TIMPs). In addition to aberrant MMP-1 and MMP-3 expression, periodontal lesions are characterized by dense infiltrations of activated T lymphocytes which may interact with CD40-expressing gingival fibroblasts in the connective tissue via the CD40L-CD40 pathway. In this study we investigated whether CD40 cross-linking influenced MMP production by gingival fibroblasts. Therefore, we analysed the CD40L-induced MMP production by these fibroblasts in the presence of cytokines that are increased in periodontal lesions, such as IL-1β, tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). We show that CD40 ligation on gingival fibroblasts resulted in a decrease of their MMP-1 and MMP-3 production, while MMP-2 and TIMP-1 production were unaffected as determined by Western blot. This down-regulatory effect of CD40 engagement on MMP-1 and MMP-3 production by gingival fibroblasts was also present when MMP production was up-regulated by IL-1β and TNF-α or down-regulated by IFN-γ. These results suggest that CD40 ligation on gingival fibroblasts leads to a restraining of MMP-1 and MMP-3 production by gingival fibroblasts and thereby may be an important mechanism in the retardation of further periodontal tissue damage.