The role of IL-18 and IL-12 in the modulation of matrix metalloproteinases and their tissue inhibitors in monocytic cells

Michal Abraham1,4, Sarah Shapiro2, Nitza Lahat2,4 and Ariel Miller1,3,4

1 Neuroimmunology and 2 Immunology Research Units, Carmel Medical Center, and 3 Rappaport Institute for Research in the Medical Sciences, and 4 Faculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel

Correspondence to: A. Miller, Neuroimmunology Unit, Department of Neurology, Carmel Medical Center, 7 Michal Street, Haifa 34362, Israel.E-mail: millera{at}tx.technion.ac.il
Transmitting editor: D. Wallach


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The matrix metalloproteinases (MMP) are enzymes crucial for the physiological patrol as well as pathological chemotaxis of immune cells to target tissues. The present study examined differential effects of pro-inflammatory [IL-18, IL-12 and tumor necrosis factor (TNF)-{alpha}] versus anti-inflammatory (IL-4) cytokines on the modulation of MMP and their endogenous tissue inhibitors (TIMP) expression in the U937 cell line. IL-18 and IL-12 separately and synergistically enhanced MMP-2, while TNF-{alpha} led to the elevation of MMP-9. All pro-inflammatory cytokines enhanced MT1-MMP expression and IL-4 suppressed TNF-{alpha}-induced MMP-9 expression. This study demonstrated that elevated IL-18 and IL-12, and related pro-inflammatory activity, may be associated with aberrant MMP activity, suggesting modulation of MMP expression using IL-12 and IL-18 antagonists as future therapeutic strategies to attenuate inflammatory and autoimmune disorders.

Keywords: autoimmunity, cytokines, gelatinases, IL-4, immunomodulation, tumor necrosis factor-{alpha}


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The complex process of circulating immune cell transmigration to target tissues involves focal degradation of the vascular basement membrane and extracellular matrix (ECM). In this process a central role is played by the matrix metalloproteinases (MMP), a family of proteolytic enzymes secreted by a variety of cells, amongst them monocytes/macrophages (1). The MMP are divided into subgroups according to their structure and substrate specificities (2,3). The main subgroups include the collagenases, gelatinases, stromelysins and membrane-anchored MMP [membrane-type (MT)-MMP]. The gelatinases, MMP-2 and MMP-9, which cleave collagen type IV, laminin, fibronectin and gelatin, are key participators in the transmigratory process (1,4).

Elevated expression of MMP has been suggested to contribute to excessive immune cell infiltration and target tissue destruction, characteristic of autoimmune processes as observed in multiple sclerosis (MS) (59). In addition, different studies (1015) have provided supporting evidence for the specific involvement of MMP-2 and MMP-9 in blood–brain barrier eruption, enabling the influx of monocytes and T cells into the CNS, and ensuing brain edema, characteristic of immune-mediated CNS disorders such as MS and its animal model, experimental autoimmune encephalomyelitis (EAE).

The potentially deleterious activity of MMP is tightly controlled at transcriptional as well as post-transcriptional levels (2,3). Post-transcriptional regulation includes their secretion as inactive zymogens and stepwise activation, generally in the extracellular milieu, through interactions with other MMP, adhesion molecules or serum proteases. On the other hand, inhibition of MMP activity is achieved by their binding to serum proteins and endogenous tissue inhibitors of MMP (TIMP) (2,16). Of the four members of the TIMP family identified, TIMP-1 and TIMP-2 also inhibit activation of pro-MMP-9 and pro-MMP-2 respectively. The balance between MMP and TIMP activity in a particular microenvironment seems to determine whether physiological homeostatic ECM remodeling or excessive proteolysis and possible pathological consequences occur.

Cytokines, which are key mediators of inflammatory and autoimmune processes, play a critical role in the regulation of both MMP and TIMP expression in different cell types. It has recently been suggested that the cytokine profile associated with Th cell subgroups may differentially influence the profile of MMP expressed and thus the ability of monocytes to degrade the ECM (17,18). The studies conducted regarding the effect of cytokines on the expression of gelatinases by monocytes have mainly demonstrated up-regulation of MMP-9 by the pro-inflammatory cytokines, TNF-{alpha} and IL-1 (19,20). Recent studies have also shown induction of MMP-9 protein expression by IL-18 (21,22). Anti-inflammatory Th2 cytokines, such as IL-4 and IL-10, which suppress Th1 responses and autoimmune disorders, are reported to down-regulate MMP-9 (17,21).

An important role in driving and determining Th1 responses has been delegated to IL-18 [previously named ‘IFN-{gamma}-inducing factor’ (IGIF)] and IL-12, the recently described pro-inflammatory pleiotropic cytokines (2325). The IL-12-induced up-regulation of IL-18 receptors on Th1 cells is part of the initiation of the pro-inflammatory cascade that leads to the transcription of the IFN-{gamma} gene (26). Additional biological properties have been attributed to IL-18 and IL-12, through their effects on multiple cellular targets, including monocytes/macrophages. The IL-18 effects include the induction of TNF-{alpha}, Fas ligand, chemokines, adhesion molecules, NO and prostaglandins (23,27).

In light of the recent studies describing, in MS as well as other autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus, uveitis or arteriosclerosis, elevated IL-18 and IL-12 levels (22,2833), as well as the accumulating evidence supporting a pathogenic role played by MMP (22,28,3436) in these inflammatory disorders, the present study examined the effects of IL-18 and IL-12 on the mRNA and protein expression of MMP and TIMP in the U937 cell line. The in vitro influence of these Th1-promoting cytokines on the expression of MMP-2, MMP-9, MT1-MMP (the physiological activator of MMP-2) as well as TIMP-1 and TIMP-2, were compared to the effects of TNF-{alpha}, the classical pro-inflammatory cytokine as well as to the effects of IL-4, the characteristic anti-inflammatory Th2 cytokine.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell cultures
Human monocytic line cells (U937) were cultured in RPMI 1640 supplemented with 10% FCS, glutamine and antibiotics (Biological Industries, Bet Haemek, Israel). Cells were cultured at 37°C in a humidified incubator with 5% CO2. For zymography assay cells were cultured in serum-free medium containing 0.1% BSA.

Exposure to cytokines
Cells cultured in microtiter plates (1.86 x 105 cells/well) for protein assays and in 6-cm culture dishes (8 x 106 cells/dish) for RNA assays were incubated with phorbol 12-myristate 13-acetate 10 ng/ml for 48 h (37), washed and exposed to cytokines for 24 h. Cytokines used were recombinant TNF-{alpha} (0.1–20 ng/ml) (R & D Systems, Minneapolis, MN), recombinant IL-18 (10–200 ng/ml) (a gift from Professor Menachem Rubinstein, Weizmann Institute of Science, Rehovot, Israel), in the presence or absence of recombinant IL-12 (1–10 ng/ml) or recombinant IL-4 (1–10 ng/ml) (R & D Systems). Non-cytotoxic concentrations of cytokines were chosen for each assay, determined by XTT assay according to the manufacturer’s instructions (Biological Industries).

Northern blot analysis
Total cellular RNA was extracted from U937 cells with the use of Tri-Reagent (Medical Research Center, Cincinnati, OH) according to the manufacturer’s instructions. The RNA (20 µg) was then separated by electrophoresis on 1% agarose gels containing formaldehyde, and integrity was visualized following ethidium bromide staining. The RNA was capillary transferred to Hybond membranes (Amersham, Little Chalfont, UK), and hybridized to end-labeled MMP and TIMP oligomer probes (Biognostik, Gottingen, Germany) at 65°C in 50 mM Tris–HCl (pH 7.5), 10% dextran sulfate, 1 N NaCl, 1% SDS and 100 µg/ml sheared salmon sperm DNA. Following autoradiography, membranes were stripped and exposed to G3PDH probe (Clontech, San Diego, CA) to normalize for loading errors. Relative levels of MMP or TIMP to G3PDH level were determined following computerized densitometric assessment (BioImaging Gel Documentation System, Dinco and Renium, Jerusalem, Israel) and TINA software (Raytest, Straubenhardt, Germany).

Gelatin zymography
Gelatinase (MMP-2 and MMP-9) activity was determined by SDS–PAGE. Proteins in supernatant were separated on gels containing 0.1% gelatin (Sigma, St Louis, MO). Gels were then re-natured by incubation in 2.5% Triton X-100 for 30 min, and incubated overnight in substrate buffer (50 mM Tris–HCl, pH 7.5) containing 10 mM CaCl2 and 0.05% Brij (Sigma) at 37°C then stained with Coomassie brilliant blue (0.5%). Clear areas in the blue background of gels demonstrated the presence of gelatinase activity. Molecular weight markers (Bio-Rad, Hercules, CA), and commercial positive controls for MMP-2 and MMP-9 (Chemicon, Temecula, CA) were run with each gel. Computerized densitometry was used to evaluate relative enzymatic activity.

Flow cytometric analysis
The level of membrane MT1-MMP expressed by the U937 cells was evaluated following 1 h of incubation with anti-MT1-MMP rabbit polyclonal antibody (Chemicon) in PBS containing 1% FCS at 4°C. Cells were washed with PBS and then exposed for 30 min to FITC-labeled second antibody (Jackson ImmunoResearch, West Grove, PA) as described above. Cells were washed and re-suspended in 0.1% formaldehyde PBS. Within 24 h, the level of fluorescence for 10,000 cells was quantified with a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA).

Statistical evaluation
Statistical significance of results was determined using ANOVA and the Tukey–Kramer multiple comparisons test. P < 0.05 was considered significant. Each experiment was repeated 3–5 times, with good agreement between the results of individual experiments. The data shown are the mean ± SD.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
TNF-{alpha} but not IL-12 and IL-18 induce MMP-9 expression in the U937 cell line
Typical results obtained for mRNA expression, evaluated by Northern Blot, and protein expression, assessed by zymography analysis, are demonstrated in Fig. 1(A and B respectively). MMP-9 mRNA and protein were constitutively expressed by the monocytes (U937 cells). Exposure to TNF-{alpha} induced a prominent dose-dependent elevation of MMP-9 mRNA expression, which at the concentration of 20 ng/ml was 3-fold higher than the baseline level (P < 0.0001) (Fig. 1C). Although TNF-{alpha} also induced a significant (P = 0.004) elevation in the expression of MMP-9 protein (Fig. 1D), this elevation was less pronounced (1.4-fold, at 20 ng/ml TNF-{alpha}, compared to baseline) than that induced in mRNA expression. In contrast, IL-18, IL-12 and their combination did not affect either the MMP-9 mRNA or protein expression (Fig. 1E and F).



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Fig. 1. Modulation of MMP-9 mRNA and protein by inflammatory cytokines. Monocytes (U937 cells) were exposed to TNF-{alpha} (0.1–20 ng/ml), IL-12 (1–10 ng/ml) and/or IL-18 (10–200 ng/ml) for 24 h. Northern blot analysis was used to assess mRNA expression (A, C and E) and zymography was employed to assess protein expression (B, D and F) of MMP-9. (A and B) Results for Northern blot and zymography assessment respectively. Commercially available gelatinases were used as positive control (P.C.) in electrophoresis. Histograms demonstrate the mean densitometric intensity (± SD) as assessed in five separate experiments. *P < 0.05 versus control.

 
IL-18 and TNF-{alpha} induce MMP-2 expression in the U937 cell line
TNF-{alpha} induced a moderate though significant (P = 0.0009) dose-dependent elevation of MMP-2 mRNA expression, which at the highest TNF-{alpha} concentration was 1.3-fold compared to baseline level (Fig. 2A), in accordance with results observed for MMP-2 protein expression (Fig. 2B). IL-12 alone did not significantly influence MMP-2 mRNA or protein expression, whereas IL-18 significantly (P < 0.0001) enhanced the mRNA expression of MMP-2, although only the highest concentration of IL-18 (200 ng/ml) led to an elevation of MMP-2 protein. The combination of IL-18 and IL-12 led to a synergistic effect on the expression of MMP-2 mRNA, which reached significance (P < 0.05) at the concentrations of 100 and 1 ng/ml respectively. However, it did not influence the level of MMP-2 protein, beyond that induced by IL-18 alone (Fig. 2C and D).



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Fig. 2. Modulation of MMP-2 mRNA and protein by inflammatory cytokines. Monocytes (U937 cells) were exposed to TNF-{alpha} (0.1–20 ng/ml), IL-12 (1–10 ng/ml) and/or IL-18 (10–200 ng/ml) for 24 h. Northern blot analysis was used to assess mRNA expression (A and C) and zymography was employed to assess protein expression of MMP-2 (B and D). Histograms demonstrate the mean densitometric intensity (± SD) as assessed in five separate experiments. *P < 0.05 versus control.

 
Pro-inflammatory cytokines induced MT1-MMP expression in the U937 cell line
Exposure of the U937 cells to TNF-{alpha} at concentrations >5 ng/ml induced a prominent dose-dependent elevation of MT1-MMP mRNA (P < 0.0001) (Fig. 3A). Nevertheless, TNF-{alpha} at a concentration as low as 0.1 ng/ml led to up-regulation of MT1-MMP molecules on monocyte cell surfaces. This elevated level remained constant despite the increase in cytokine concentration (Fig. 3B). Incubation of cells with IL-12 or IL-18 separately did not influence the level of MT1-MMP mRNA expression (Fig. 3C), although at the protein level, each cytokine separately led to an increase in the surface expression of this molecule compared to the baseline level (Fig. 3D). Only the combined exposure to IL-12 and IL-18 led to the elevation of MT1-MMP mRNA expression, compared to both the baseline level and the levels induced by each cytokine separately (Fig. 3C), although it did not affect the elevated level of MT1-MMP protein beyond that induced by each cytokine separately (Fig. 3D).



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Fig. 3. Modulation of MT1-MMP mRNA and protein by inflammatory cytokines. Monocytes (U937 cells) were exposed to TNF-{alpha} (0.1–20 ng/ml), IL-12 (1–10 ng/ml) and/or IL-18 (10–200 ng/ml) for 24 h. Northern blot analysis was used to assess mRNA expression (A and C) and fluoresceinated flow cytometry was employed to assess the level of membranal MT1-MMP expression (B and D). Histograms demonstrate the mean fluorescence intensity or densitomeric intensity (± SD) as assessed in five separate experiments. *P < 0.05 versus control.

 
IL-18 induced TIMP-1, while all cytokines examined induced TIMP-2 expression
Neither TNF-{alpha} nor IL-12 significantly influenced the constitutive level of TIMP-1 mRNA expression, whereas IL-18, both at the concentration of 100 ng/ml and in combination with IL-12, led to a moderate elevation in its mRNA expression (Fig. 4A and B). TIMP-2 mRNA expression was slightly, albeit significantly (P = 0.0004), affected by exposure of cells to TNF-{alpha} (1.3-fold at the highest TNF-{alpha} concentration; Fig. 4C). Combined IL-12 and IL-18 also led to a significant (P = 0.008) elevation in the mRNA expression of TIMP-2 (Fig. 4D).



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Fig. 4. Modulation of TIMP-1 and TIMP-2 mRNA by inflammatory cytokines. Monocytes (U937 cells) were exposed to TNF-{alpha} (0.1–20 ng/ml) (A and C) or IL-12 (1–10 ng/ml) with/without IL-18 (10–200 ng/ml) (B and D) for 24 h. Northern blot analysis was used to assess mRNA expression for TIMP-1 (A and B) and for TIMP-2 (C and D). Histograms demonstrate the mean densitometric intensity (± SD) as assessed in five separate experiments. *P < 0.05 versus control.

 
IL-4 down-regulated expression of TNF-{alpha}-induced MMP-9 and elevated TIMP-1 mRNA expression in the U937 cell line
The levels of MMP-2, MT1-MMP and TIMP-2 mRNA expression were not affected by exposure of cells to IL-4, whether alone or together with TNF-{alpha} (data not shown). In contrast, IL-4 (1–10 ng/ml) inhibited both the constitutive and TNF-{alpha}-induced expression of MMP-9 mRNA in a dose-dependent manner (P < 0.001; Fig. 5A). IL-4 alone did not influence the constitutive level of TIMP-1 mRNA expression. However, IL-4 combined with TNF-{alpha} (10 ng/ml) led to the enhancement of TIMP-1 mRNA expression (P < 0.0001; Fig. 5B).



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Fig. 5. Modulation of MMP-9 and TIMP-1 mRNA by IL-4. Monocytes (U937 cells) were exposed to IL-4 (0.1–10 ng/ml) in the absence or presence of TNF-{alpha} (10 ng/ml) for 24 h. Northern blot analysis was used to assess mRNA expression of MMP-9 (A) and TIMP-1 (B). Histograms demonstrate the mean densitometric intensity (± SD) as assessed in three separate experiments. {Delta}P < 0.05 versus TNF-induced effect.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The present study examined the influence mediated by distinct members of the cytokine network on the expression of MMP and TIMP by the U937 cell line. An important finding of this study is the IL-18-mediated-up-regulation of MMP-2 expression in the cells studied, while having no influence on MMP-9 expression. MMP-2 is considered to be a housekeeping protease generally constitutively expressed and resistant to most activators (2,3). Hence, the IL-18 modulation of MMP-2 may be of importance to the understanding of factors or signaling pathways related to the transcription of this MMP. IL-18 also led to the elevation of MT1-MMP, the prominent activator of MMP-2 (2,3,38), thus possibly contributing also to the enhanced MMP-2 activity. To date, the available reports relating to the influence of IL-18 on MMP expression in monocytes demonstrated induction of MMP-9 in murine peritoneal macrophages (21) and induction of MMP-9 as well as the interstitial collagenases (MMP-1 and MMP-13) in human monocytes (22). These observations contrasting to the findings of the present study may be dependent on the different pre-stimulus of the monocytes and need to be clarified in future studies. As to IL-12, previous studies have shown that this cytokine down-regulates MMP-9 and up-regulates TIMP-1 in stromal cells (39), while failing to induce MMP-9 in peripheral blood monocytes or T cells (21,40). The present study demonstrated that IL-12 alone did not significantly modulate the mRNA expression of the gelatinases, although together with IL-18 an additive effect was noted in the induction of MMP-2 and MT1-MMP mRNA.

IL-18 and IL-12 are known to act synergistically in enhancing IFN-{gamma} production by monocytes (23,26), although they are reported to employ distinct signal transduction pathways in the attainment of this effect (41,42). The findings in our laboratory (43) that IFN-{gamma} up-regulates MMP-2 mRNA expression, and the report by Shapiro et al. (44) that IFN-{gamma} has no effect on MMP-9 mRNA and protein expression, are in line with the results observed for IL-18 in the current study, i.e. that IL-18 induces elevation of MMP-2 while having no influence on MMP-9 expression. Recent studies have shown that monocytes are also capable of producing IFN-{gamma} (22,45). Therefore, the presently observed IL-12- and IL-18-mediated modulation of MMP-2 in the U937 cell line may be, at least partially, secondary to the effects of IFN-{gamma} induced by these inflammatory cytokines.

The observed TNF-{alpha}-induced elevation of MMP-9 mRNA and protein in the present study is in agreement with previous in vitro findings in peripheral blood monocytes (19,20). However, the present study did not observe TNF-{alpha}-induced modulation of TIMP-1 mRNA expression, in contrast to a previous report (20). Our findings regarding the up-regulation of MT1-MMP protein and mRNA levels by TNF-{alpha} are supported by a preliminary report on the elevation of MT1-MMP protein in monocytes (39). The post-transcriptional activation of MMP-2 by TNF-{alpha} may be achieved through enhancement of its activator, MT1-MMP, while not affecting the level of TIMP-2 expression. Interestingly, low levels of TIMP-2, the pro-MMP-2 inhibitor, paradoxically are necessary for the MT1-MMP-mediated activation of MMP-2, wherein TIMP-2 serves as an adaptor molecule that binds pro-MMP-2 to membranal MT1-MMP (38,46). The ‘MT1-MMP/TIMP-2/pro-MMP-2’ complex enables MMP-2 activation by additional free MT1-MMP surface molecules. At high TIMP-2 levels complete blocking of surface MT1-MMP molecules may occur, consequently preventing MMP-2 activation. Thus, the present results support a role for TNF-{alpha}, not only in the transcriptional modulation of MMP-9 expression, but also in the post-transcriptional activation process of MMP-2.

IL-18, in addition to its ability to enhance IFN-{gamma}, is reported to induce gene expression and synthesis of TNF-{alpha} (23,27). However, due to the different pattern observed in the MMP/TIMP responses to IL-18 and TNF-{alpha}, the present results suggest that the IL-18-mediated effects on MMP and TIMP expression in the U937 cell line are not secondary to IL-18 induction of TNF-{alpha}. As a result, it may be presumed that TNF-{alpha} and IL-18 induce independent regulatory pathways that may lead to differential modulation of MMP and consequently different degradation patterns of ECM components. The finding that the IL-18-mediated effects appear to differ from those of the classical pro-inflammatory cytokine, TNF-{alpha}, further emphasizes the complexity of signals mediated by distinct members of the cytokine network in the modulation of MMP/TIMP expression in immune cells.

The present study also demonstrated IL-4 inhibition of both baseline and TNF-{alpha}-induced MMP-9 mRNA expression, possibly due to inhibition of MMP-9 transcription or its enhanced mRNA degradation. Additionally, this study demonstrates that IL-4 in the presence of TNF-{alpha} enhances TIMP-1 mRNA expression. In agreement with the present results, others have described that the anti-inflammatory Th2 cytokines IL-10 and IL-4 mediate suppression of protein expression of MMP in monocytes (21,4749). However, Zhang et al. (20) reported suppression of TIMP-1 expression by IL-4, in contrast to the present findings. These seemingly conflicting findings may be due to the fact that our study examined the modulation of TIMP-1 at the mRNA level, as opposed to the previous study that was conducted at the protein level. The finding of IL-4-mediated suppression of MMP-9 and increased TIMP-1 support the possibility that the immune-suppressive role of Th2 cytokines may also include the suppression of immune cell migratory capacity in inflammatory and autoimmune processes.

The differences between the modulation of the mRNA and protein expression observed in the present study support regulation of MMP at both transcriptional and post-transcriptional levels (2,3). Possible mechanisms that may explain these differences include the existence of intracellular reservoirs of protein, as well as the possibility of cytokine-induced modulation of molecules involved either in the process of MMP secretion or in their cell-surface expression (3).

Inflammatory reactions and autoimmune processes necessitate extravasation of immune cells from the circulation to target organs. This process is coordinated by a complex cascade of events involving both cytokines and MMP. Improved understanding of the cytokine–MMP interactions is of vital importance to promote tissue repair in physiological as well as pathological processes. Elevated levels of cytokines such as IL-18, IL-12 and TNF-{alpha}, found in autoimmune disorders such as MS, RA, uveitis and others (22,2833), may contribute to high MMP activity associated with these conditions (49,28,3436,50). In parallel, MMP influence the inflammatory process by activating or degrading cytokines [transforming growth factor (TGF)-ß and IL-1ß respectively] (51,52), as well as by releasing cell-surface cytokines such as TNF-{alpha} (53), or shedding cytokine receptors such as IL-6 and TGF-{alpha} (54). It is thus conceivable that MMP, together with cytokines such as IL-18 and IL-12 (28,30,55), may contribute to immunological cascade of some of the autoimmune disorders. Up-regulation of MMP in relation to TIMP activity by pro-inflammatory cytokines may contribute to disease activity, while anti-inflammatory cytokines may down-regulate deleterious proteolytic activity, promoting disease remission and allowing repair. It may be presumed that the balance between the Th1 and Th2 cytokines, known to have critical effect on inflammatory activity, may affect disease processes also by modulating the balance between MMP and TIMP expression and activity in the inflammatory microenvironment.

The present study employed a monocytic cell line to evaluate the influence of pro- and anti-inflammatory cytokines on the expression of MMP and TIMP. Reservations often exist regarding conclusions obtained from studies of cell lines and the applicability of the results to primary cells in an in vivo environment. Nevertheless, the U937 cell line used has been extensively studied and widely described. Most reports demonstrate its function and phenotype to be similar to peripheral blood monocytes.

In summary, the IL-18- and IL-12-mediated modulation of MMP and TIMP, observed in the present study, suggests that these inflammatory cytokines may have a potential role as pathological enhancers of MMP in immune-mediated diseases. Recent studies have already demonstrated the successful use of IL-12 or IL-18 as therapeutic targets in Th1-mediated autoimmune disease models such as EAE (5658). Findings from the current study suggest that amongst the mechanism underlying the positive effect of such therapeutic strategies may be inhibition of the pathological induction of MMP mediated by these inflammatory cytokines. Thus, the present findings, once confirmed by similar experiments and functional assays utilizing peripheral blood cells, may suggest the implementation of IL-12 and IL-18 inhibitors (59) as well as specific MMP antagonists (60), capable of attenuating deleterious inflammatory and proteolytic activity, as future therapies for autoimmune diseases.


    Acknowledgements
 
We thank Professor Menachem Rubinstein (Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel) for the generous gift of IL-18. This work was supported by funds provided by Serono Pharmaceuticals Ltd (Geneva, Switzerland), the Rappaport Institute for Research in the Medical Sciences and the Technion—Israel Institute of Technology, Haifa (Israel).


    Abbreviations
 
EAE—experimental autoimmune encephalomyelitis

ECM—extracellular matrix

MMP—matrix metalloproteinase

MS—multiple sclerosis

MT—membrane type

RA—rheumatoid arthritis

TIMP—tissue inhibitor of matrix metalloproteinase

TGF—transforming growth factor

TNF—tumor necrosis factor


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
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