matrix metalloproteinases as modulators of inflammation and ... - Nature

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MATRIX METALLOPROTEINASES AS MODULATORS OF INFLAMMATION AND INNATE IMMUNITY William C. Parks*‡, Carole L. Wilson§ and Yolanda S. López-Boado|| As their name implies, matrix metalloproteinases are thought to be responsible for the turnover and degradation of the extracellular matrix. However, matrix degradation is neither the sole nor the main function of these proteinases. Indeed, as we discuss here, recent findings indicate that matrix metalloproteinases act on pro-inflammatory cytokines, chemokines and other proteins to regulate varied aspects of inflammation and immunity.

*University of Washington, Harborview Medical Center, Department of Medicine, Box 359640, 325 9th Avenue, Seattle, Washington 98104, USA. ‡ Departments of Medicine and §Pathology, University of Washington School of Medicine, Seattle, Washington 98195, USA. || Department of Internal Medicine (Molecular Medicine), Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA. Correspondence to W.C.P. e-mail: [email protected] doi:10.1038/nri1418

If inflammation and immunity are defined broadly, including all of the processes that are associated with defence against microorganisms and other environmental insults, then a wide variety of cell types and proteins participate in these processes. Although leukocytes are the paradigmatic inflammatory cell type, they are not the only cells that function in defence and immunity. For example, epithelial cells, although specialized to have distinct functions in different tissues, respond similarly to injury and infection, and regulate leukocyte influx by equivalent mechanisms. Overall, the programmes that regulate repair, defence, inflammation and immunity, regardless of the cell type invoked, might have co-evolved, particularly with respect to the type of protein that functions in all of these processes. As discussed in this review, new views on the function of matrix metalloproteinases (MMPs) indicate that this family of enzymes regulates various inflammatory and repair processes and therefore might represent an early step in the evolution of the immune system. The MMP family

The MMP family currently comprises 25 related, but distinct, vertebrate gene products, of which 24 are found in mammals (TABLE 1). MMPs are secreted or anchored to the cell surface, thereby confining their catalytic activity to membrane proteins and proteins in the secretory pathway or extracellular space. To be classified as an MMP, a protein needs to have at least the conserved PRO-DOMAIN and CATALYTIC DOMAIN (FIG. 1). The pro-domain of a typical

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MMP is ~80 amino acids and contains the consensus sequence PRCXXPD (where X denotes any amino acid). The exception is MMP23, in which the crucial cysteine residue is found in a distinct amino-acid sequence1. The catalytic domain of a typical MMP contains a zinc ion (Zn2+) in the active site (the reason for the prefix ‘metallo’) that is ligated to three conserved histidine residues in the sequence HEXXHXXGXXH. The glutamic acid residue (E) in this catalytic motif provides the nucleophile that severs peptide bonds. The backbone structures of the MMP catalytic domain, including a characteristic MET TURN that is caused by a conserved methionine residue downstream of the zinc-binding site2, are similar to those of the astacin-, reprolysin- (also known as ADAM) and serralysin-family metalloproteinases. Together, these four families comprise the metzincins CLAN of the metalloendopeptidase superfamily. Similar to the diverse functions of MMPs, these other metalloproteinases also participate in a range of processes, such as matrix synthesis, cytokine activation and ligand shedding. With the exception of MMP7, -23 and -26, MMPs have a flexible proline-rich HINGE REGION and a carboxy (C)-terminal HEMOPEXIN-LIKE DOMAIN, which functions in substrate recognition (FIG. 1). Other domains found in MMPs are restricted to subgroups of enzymes. For example, four membrane-type MMPs (MMP14, -15, -16 and -24) have transmembrane and cytosolic domains, whereas MMP17 and MMP25 have C-terminal hydrophobic extensions that function as GLYCOSYLPHOSPHATIDYLINOSITOL (GPI)-ANCHORING SIGNALS. The two gelatinases VOLUME 4 | AUGUST 2004 | 6 1 7

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Table 1 | Mammalian matrix metalloproteinases Designation*

Common name

Other name(s)

Substrates‡

References§

MMP1

Collagenase-1

Fibroblast collagenase, interstitial collagenase

Type I and II fibrillar collagens?||

ColA¶ ColB¶

115 115

MMP2

Gelatinase A

72-kDa gelatinase, 72-kDa type IV collagenase

CCL7 CXCL12

MMP3

Stromelysin-1

Transin-1

E-cadherin Laminin, type IV collagen Latent TGF-β1

100 101 81

MMP7

Matrilysin

PUMP

Pro-α-defensins FAS ligand (CD95L) Latent TNF Syndecan-1 E-cadherin Elastin

51 53 102 66 46 35

MMP8

Collagenase-2

Neutrophil collagenase

Mouse CXCL5

MMP9

Gelatinase B

92-kDa gelatinase, 92-kDa type IV collagenase

Zona occludens 1 α1-Antiproteinase Latent TGF-β1 Latent VEGF Fibrin NG2 proteoglycan

MMP10

Stromelysin-2

Transin-2

ND

MMP11

Stromelysin-3

MMP12

Metalloelastase

MMP13

Collagenase-3

Rat collagenase

Type I and II fibrillar collagens?||

111–114

MMP14

MT1-MMP

Membrane-type MMP

ProMMP2 Fibrillar collagens Fibrin Syndecan-1 γ2-Subunit of laminin-5

32 31,33,34 34,107 108 109

MMP15

MT2-MMP

Fibrin

34,107

MMP16

MT3-MMP

Fibrin Syndecan-1

34,107 108

MMP17

MT4-MMP

ND

Enamelysin

Amelogenin

PRO-DOMAIN

The matrix metalloproteinase (MMP) pro-peptide region (or pro-domain) contains ~80 amino acids, typically with a hydrophobic residue at the amino terminus. It also contains the highly conserved sequence PRCXXPD, where X denotes any amino acid. The thiol group of the cysteine residue in this sequence ligates with the zinc ion that is held by the histidine residues in the catalytic domain of the MMP. In this state, the enzyme is stable and inactive and is known as a zymogen. CATALYTIC-DOMAIN

The typical matrix metalloproteinase catalytic domain contains ~160–170 residues, including the binding sites for the structural (calcium and zinc) and catalytic (zinc) metal ions. The 50–54 residues at the carboxyl terminus of the catalytic domain include a highly conserved HEXXHXXGXXH sequence (where X denotes any amino acid), which includes a glutamic acid residue (E) that provides the nucleophile that severs peptide bonds and histidine residues that coordinate the zinc ions. MET TURN

On the carboxy side of the zinc active site, matrix metalloproteinases have a methionine residue that is always conserved. This residue is part of a 1,4-β-turn that loops the polypeptide chain beneath the catalytic zinc ion and forms a hydrophobic base for the zinc-binding site. ADAM

A disintegrin and metalloproteinase family of proteases. They contain disintegrin-like and metalloproteinase-like domains and are involved in the regulation of developmental processes, cell–cell interactions and protein processing, including ectodomain shedding.

618

111–114

Latent TNF

106

ND

MMP21

110

ND

MMP22

ND

MMP23

CA-MMP

ND

MMP24

MT5-MMP

ND

MMP25

Leukolysin

MT6-MMP

ND

MMP26

Endometase

Matrilysin-2

ND

MMP27** MMP28

68 103 99 23 75 104 105

ND

MMP19# MMP20

27 67

ND Epilysin

ND

*Matrix metalloproteinase (MMP)-4, -5 and -6 were found to be identical to either MMP2 or MMP3 and, therefore, are not unique. MMP18 has only been cloned from Xenopus laevis; a mammalian homologue has not been found. ‡This list of substrates is limited to proteins that have been shown to be cleaved by an MMP by either a gain- or loss-of-function approach or both. For some substrates, only indirect evidence has been provided, but, importantly, the substrate is known to be cleaved. Excluded from this list are the many proteins that have been shown to be cleaved by an MMP only in a defined in vitro setting. In addition, proteins that were identified only by the treatment of cells with exogenous MMP or by overexpression of a particular MMP have also been omitted. §References are reports of identification of substrates only. ||Various assays and parameters, such as the formation of neoepitopes in vivo111–113, indicate that MMP1 and MMP13 probably act on fibrillar collagens (type I and type II). However, data that compare the collagenolytic activity of these MMPs with that of MMP14 (REF. 114) question which enzymes created these neoepitopes. ¶|Collagenase-like protein A (ColA) and ColB are probably the murine homologues of MMP1 (REF. 115). On the basis of its chromosomal position and enzymatic activity, McolA is a strong candidate to be the murine orthologue of human MMP1. #In the initial cloning paper116, human MMP19 was called MMP18, but this designation was already assigned to X. laevis MMP18 (REF. 117). **In addition to being identified in a large scale bioinformatic search of secreted proteins118, full-length cDNAs for MMP27 from humans, rats and northern tree shrews have been submitted to GenBank. CA, cysteine array; CCL, CC-chemokine ligand; CXCL, CXC-chemokine ligand; E-cadherin, epithelial cadherin; ND, not determined; PUMP, putative metalloproteinase; TGF, transforming growth factor; TNF, tumour-necrosis factor; VEGF, vascular endothelial growth factor.

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Basic

MMP1, -3, -8, -10, -12, -13, -18, -19, -20, -22, -27 ColA, ColB

Minimal

Zn2+ Hinge

Pro

MMP7, -26

SP

Pro

Furinactivated

MMP11, -28

SP

Pro

Fr

Catalytic

Hemopexin-like

Membraneanchored

TM: MMP14, -15, -16, -24 GPI: MMP17, -25

SP

Pro

Fr

Catalytic

Hemopexin-like

Gelatinbinding

MMP2, -9

SP

Pro

Type II membrane

MMP23

SA

Pro

CLAN

The superfamily of metalloenzymes includes more than 200 members. It has been divided into eight clans based on the similarity of protein folding characteristics and ~40 families according to evolutionary relationships. The matrixmetalloproteinase family belongs to clan MB, the members of which have three histidine residues as zinc-binding ligands in the consensus sequence HEXXHXXGXXH (where X denotes any amino acid).

SH

SP

Catalytic

Hemopexin-like

Catalytic

TM Cs

Catalytic Fn Fn Fn

HINGE REGION

A domain that is typically ~75 residues and links the catalytic domain to the hemopexin-like domain of most matrix metalloproteinases. HEMOPEXIN-LIKE DOMAIN

This matrix-metalloproteinase domain comprises ~200 residues and contains four repeats that resemble hemopexin and vitronectin. It is not essential for catalytic activity but does modulate substrate specificity and binding to tissue inhibitors of metalloproteinases. GLYCOSYLPHOSPHATIDYLINOSITOL (GPI)-ANCHORING SIGNALS

A glycolipid modification that is usually located at the carboxyl terminus and anchors proteins to the external surface of the plasma membrane. GELATIN-BINDING DOMAINS

These domains contain three fibronectin-like modules (also known as fibronectin type II modules) and are present in the catalytic domain of both matrix metalloproteinase 2 and -9. CYSTEINE-SWITCH MECHANISM

The pro-peptide maintains a matrix metalloproteinase (MMP) in an inactive state. When the interaction between the conserved cysteine residue in the pro-domain and the active site zinc ion is disrupted (for example, by proteolytic removal of the pro-peptide or by the action of organomercurials and chaotropic agents on the thiol of the cysteine residue), the active site becomes accessible, and the MMP has been ‘activated’. The pro-domain does not need to be removed for a proMMP to acquire activity; only disruption of the zinc–thiol interaction is absolutely required.

or GPI

Fr

Catalytic

Cys

Hemopexin-like or C5 Hemopexin-like

IgG-like

Figure 1 | Domain structure of the mammalian MMP family. The important features of matrix metalloproteinases (MMPs) are illustrated, showing the minimal domain structures. Although MMPs are often subdivided into groups on the basis of differences in domain composition (shown here), there is little consensus in the field about how such subdivisions should be assigned. Domain structure alone does not predict function. One clear division is between MMPs that are secreted and those that are anchored to the cell surface by an intrinsic motif: namely, a transmembrane (TM) domain (MMP14, -15, -16 and -24), a glycosylphosphatidylinositol (GPI) anchor (MMP17 and MMP25) or an amino (N)-terminal signal anchor (SA) (MMP23). Both the TM domains and GPI anchors are attached to the hemopexin-like domain by a short linker. As discussed in the text, the secreted MMPs might still be confined to the cell surface through interactions with specific accessory macromolecules. Because the mechanisms that control activation (that is, conversion of proMMP to active MMP) are key steps in the regulation of proteolysis, another grouping of the MMPs can be made on the basis of intracellular activation by furin proteinases. Nine MMPs, including all of the membrane-anchored enzymes, have a furin-recognition domain. C5, type-V-collagen-like domain; Col; collagenase-like protein; Cs, cytosolic; Cys, cysteine array; Fn, fibronectin repeat; Fr, furin-cleavage site; Pro, pro-domain; SH, thiol group; SP, signal peptide; Zn, zinc.

(MMP2 and MMP9) have GELATIN-BINDING DOMAINS that resemble a similar motif in fibronectin. This motif is involved in the binding of fibronectin to denatured collagen, and in MMP2 and MMP9, it probably enhances the interaction with gelatin or gelatin-like substrates. In addition to a common three-dimensional structure3, MMPs have a similar gene arrangement, indicating that they probably arose by duplications of an ancestor gene. At least eight of the known human MMP genes (MMP1, -3, -7, -8, -10, -12, -13 and -20) are clustered on chromosome 11 at 11q21–23, whereas other MMP genes are ‘scattered’ along chromosomes 1, 8, 12, 14, 16, 20 and 22 (REF. 4). Regulation of MMP activity

Similar to all secreted proteinases, the catalytic activity of MMPs is regulated at four points — gene expression, compartmentalization (that is, the pericellular accumulation of enzymes), pro-enzyme (or zymogen) activation and enzyme inactivation — and is further controlled by substrate availability and affinity. Typically, MMPs are not expressed in normal healthy tissues. By contrast, MMP expression can be detected in all repair or remodelling processes, in all diseased or inflamed tissues and in all cell types grown in culture. Although the qualitative patterns and quantitative levels of MMP expression vary among tissues, diseases, tumours, inflammatory conditions and cell types, a reasonable generalization is that activated cells, whether in


a tissue or in culture, express MMPs. For the most part, the production of MMPs is regulated at the level of transcription by specific signals that are temporally limited and spatially confined. ProMMPs are kept in a catalytically inactive state by the interaction between the thiol group of a pro-domain cysteine residue and the zinc ion of the catalytic site. They are converted to active proteinases by disruption of this interaction (a process known as the CYSTEINE-SWITCH 5 MECHANISM ), which can be achieved by proteolysis of the pro-domain or by modification of the cysteine thiol group. Several MMPs contain an RXKR or RRKR sequence between the pro- and catalytic domains, which functions as a target sequence for pro-protein convertases or furins; this is known as a furin cleavage site (FIG. 1). For the other MMPs, the mode of activation is more presumed than proven. The best described non-furin proMMP activation mechanism is probably the cellsurface activation of proMMP2 by active MMP14 (REFS 6–9). After the pro-domain has been cleaved, the active MMPs can be inhibited by natural inhibitors (BOX 1) and internalization10,11. Furthermore, and of relevance to inflammation, oxidants that are generated by leukocytes or other cells can both activate MMPs (through oxidation of the pro-domain thiol group) and subsequently inactivate MMPs (through modification of amino acids that are crucial for catalysis), providing a mechanism to control quantum bursts of proteolytic activity12–16.

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Box 1 | Blocking MMP activity

Natural inhibitors Tissue inhibitors of metalloproteinases (TIMPs). Four TIMPs (TIMP1, -2, -3 and -4) inhibit matrix-metalloproteinase (MMP) activity by binding to the catalytic site of MMPs91. However, the TIMPs differ in their affinity for specific MMPs, and their interaction does not always lead to inhibition. Indeed, binding of TIMP2 to the hemopexin-like domain of MMP2 is required for activation of the enzyme (in a complex with active MMP14). Some TIMPs also have activities that are independent of MMPs92,93. RECK (reversion including cysteine-rich protein with kazal motifs). This membranebound glycoprotein inhibits MMP2, -9 and -14. RECK probably has other functions, because a null mutation in the gene is embryonic lethal in mice94. α2-Macroglobulin. This broad-spectrum inhibitor traps several classes of active enzymes in the circulation, thereby mediating their uptake by scavenger receptors.

Synthetic inhibitors (For a discussion of the use and promise of MMP inhibitors in clinical trials for cancer see Coussens et al.95 and Overall and López-Otin96.) Peptidomimetics. These small hydroxamic-acid-based molecules mimic the substraterecognition site of collagen, chelate zinc ions and effectively inhibit MMP activity. Examples are batimastat and marimastat. Similar to the other synthetic inhibitors, the hydroxamic-acid-based inhibitors lack specificity. Not only do they block the activity of MMPs, but they can act on most metalloenzymes. Non-peptidomimetics. These are based on the conformation of the MMP active site and include tanomastat and prinomastat. Tanomastat is particularly potent against MMP2, -3 and -9. Tetracycline and derivatives. Tetracycline and non-antibiotic derivatives reduce both the synthesis and activity of MMPs. The only MMP inhibitor that has been approved by the United States Food and Drug Administration is the doxycycline known as periostat, which is used for treatment of periodontal and skin diseases. Others (natural and synthetic). These include the biphosphonates, neovastat (an extract of shark cartilage), green tea catechins, aspirin and CT-PCPE (a carboxy (C)-terminal fragment of the pro-collagen C-terminal proteinase enhancer97).

TIMPs

Tissue inhibitors of metalloproteinases. A family of four (TIMP1, -2, -3 and -4) endogenous matrixmetalloproteinase (MMP) inhibitors that bind the catalytic site in activated enzymes. TIMP1 and TIMP3 also bind the hemopexin-like domain of the MMP9 and MMP13 zymogens, whereas TIMP2, -3 and -4 can bind this domain in the MMP2 zymogen.

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In vitro studies have shown considerable overlap in the substrates that MMPs can cleave, particularly among the extracellular-matrix (ECM) substrates17. For example, fibronectin, laminins, elastin and type IV collagen can be degraded by various MMPs in vitro. In a setting such as inflammation, in which essentially all MMPs are present, the shared substrate potential would seemingly allow biochemical redundancy. However, substrate selectivity can be honed by two processes: enzyme affinity and compartmentalization. Kinetic studies using model substrates have shown that specific enzymes degrade some substrates more efficiently than others. For example, both MMP2 and MMP9 degrade cleaved collagen more efficiently than other gelatinolytic MMPs18, and MMP7 is a more potent proteoglycanase than MMP3 or MMP9 (REF. 19). So, in tissues, which contain many potential substrates, the selectivity of MMP catalysis, in addition to being regulated by the concentration of active enzyme, might be partly directed by the concentration of a preferred substrate relative to that of other potential substrates that are in proximity to a secreted MMP. Compartmentalization (that is, where and how in the pericellular environment an MMP is released and held) is equally, if not more, important for regulating


the specificity of proteolysis than the affinity of the enzyme–substrate interaction. An important concept is that cells do not indiscriminately release proteinases. Instead, proteinases, such as MMPs, are typically anchored to the cell membrane, thereby maintaining a high enzyme concentration locally and targeting their catalytic activity to specific substrates in the pericellular space. In addition to the membrane-bound MMPs, several examples of specific cell–MMP interactions have been reported, such as the binding of MMP2 to the αvβ3-integrin20, MMP1 to the α2β1-integrin21,22, MMP9 to CD44 (REF. 23) and MMP7 to surface proteoglycans24,25. As has been suggested for CD44 (REF. 23) and the α2β1integrin21, these membrane anchors might function as accessory factors that mediate both the activation of the pro-enzyme and the binding of both substrate and proteinase, thereby increasing the probability of proteolysis (FIG. 2). So, as for most protein–protein interactions, MMP specificity might be driven by an additional component. It is probable that other MMPs are also attached to cells or matrix through similar specific interactions, and determining these anchors will be a key advance towards identifying activation mechanisms and substrates. Do MMPs do more than degrade the ECM?

MMPs are thought to be responsible for the turnover and degradation of connective-tissue proteins. In fact, the first MMP, discovered by Gross and Lapiere in 1962 (REF. 26), was found in the regressing tadpole tail during their search for an endogenous collagenase that functions at neutral pH — the pH at which collagens turn over in most tissues. Following this lead, essentially all MMPs that have since been isolated have been shown to be capable of degrading various protein components of the ECM17. Consequently, as a family, MMPs are often assigned the role of being the enzymes responsible for the turnover, degradation, catabolism and destruction of the ECM. This assumption has led to some unexpected results in clinical trials using MMP inhibitors and, eventually, led to a reconsideration of MMP function. The role in ECM degradation attributed to MMPs has mostly been based on findings in defined systems — typically, using a purified MMP incubated under optimal conditions with a purified ECM protein — showing what a proteinase is capable of and not what it actually does in tissues. Despite this caveat, a large body of compelling biochemical and observational data, together with recent genetic findings, indicate that some MMPs do act on ECM proteins in vivo (TABLE 1). An important concept is that in a complex setting, such as an inflamed tissue or tumour, many diverse cell types are present and carry out various processes, including angiogenesis, remodelling and phagocytosis. As discussed earlier, cells produce a spectrum of MMPs, and as a result of the distinct localization of MMPs, a specific MMP secreted by one cell type (for example, a macrophage) would probably carry out a different function than the same enzyme produced by another cell type (for example, an epithelial cell). Furthermore, a particular MMP that is produced by one cell type probably has more than one function.


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CHEMOKINES

A family of structurally related, small glycoproteins (70–90 amino acids) that have potent leukocyte activation and/or chemotactic activity. They have pivotal roles in innate and acquired immunity. These molecules, of which there are more than 50, are classified into four subfamilies depending on the arrangement of the aminoterminal conserved cysteine residues: CC-, CXC-, C- and CX3C-chemokines (where X denotes any amino acid). In general, CC-chemokines attract monocytes, lymphocytes, basophils and eosinophils, whereas CXC-chemokines are chemotactic for neutrophils.

That is, particular MMPs can act on several protein substrates in a given tissue, and recent evidence indicates that these multiple functions are not always related to ECM degradation. The key to understanding MMP function is to identify the physiological substrates. Finding physiological substrates

Many proteinases, particularly MMPs, are nonspecific in vitro, so biologically relevant approaches have been required to identify enzyme function: that is, to identify the natural physiological substrates. Because MMPs are secreted or anchored to the cell surface, their potential substrates include all membrane proteins and proteins in the secretory pathway and extracellular space, but it is probable that only a small proportion of these are actual substrates. A key question is how we find and, importantly, verify substrates. The identification of substrates is not straightforward, but several approaches have been used, including affinity-based approaches using substrate-binding motifs27, proteomics28,29 and deduction (BOX 2). Undoubtedly, the development of protein-expression technology and genetically defined mice has provided extremely powerful investigative tools for modern biologists. The combination of a lack of protein cleavage in a specific knockout mouse, together with the acquisition of proteolysis upon ectopic expression of a specific MMP, is a great tool for verifying substrates. Recently, we have found evidence that MMPs can act on as many non-matrix substrates as ECM substrates30. In TABLE 1, we provide a list of MMP substrates that is limited to those that have been identified and verified (at least in part) by targeted mutagenesis and/or overexpression. Of the MMPs that have been genetically targeted so far, all show no phenotype or only a minor phenotype in unchallenged mice, except MMP14-deficient mice, which have severe bone deformations31,32. Although these negative observations could be interpreted to indicate that many MMPs do not have a direct role in the turnover of ECM proteins, this function is known to be carried out by some family members. For example, Reaction product (gain or loss of substrate function)

Substrate

Accessory/ adaptor protein

ProMMP (inactive enzyme)

Active MMP

Figure 2 | Minimal components of the proteolytic process. In vitro, proteolysis requires only a proteinase and a substrate. By contrast, in vivo, at least one additional component is typically included to augment, if not define, specificity, as well as perhaps catalytic rate. Various types of these accessory factors or adaptors are used: they can be either proteins or glycosaminoglycans, and either membrane-associated or extracellular-matrix components. As indicated in this figure, a transmembrane accessory factor would simultaneously interact with the proteinase and substrate, bringing both together at an effective concentration. In addition, the activation of some pro-matrix metalloproteinases (proMMPs), whether autolytic or non-autolytic, might be mediated by interaction with these factors. These factors could also have other functions.


MMP14 is a key physiological collagenase31,33,34, and gain-of-function studies indicate that MMP7 expressed by human macrophages is the relevant elastase produced by these cells35. However, matrix degradation is neither the sole nor the main function of these enzymes. An emerging view of MMPs is that they act primarily on non-matrix proteins — such as cytokines, CHEMOKINES, receptors and antimicrobial peptides — often potentiating the activity of these proteins (TABLE 1). After challenge, such as by injury, cancer, inflammation or infection, MMP-deficient mice reveal various phenotypes (TABLE 2), indicating that these enzymes have specific, and at times, essential roles in tissue repair, angiogenesis, host defence, tumour progression and, in particular, inflammation. So, it seems that MMPs (at least those that have been studied using knockout mice) have evolved to respond to the insults and pressures of the extra-uterine environment. If any generalization can be made about MMP function, it is that these proteinases have a role in inflammation. This has been recognized for MMP9 for several years. However, as this topic has been reviewed previously36, and because recent data have provided new clues to the mechanisms by which MMP7 influences inflammation, here we often use MMP7 as an example. MMPs in inflammation

Before discussing the role of MMPs as modulators of inflammatory processes, we briefly describe the process of inflammation and the possible roles MMPs might have in the response of host tissues to environmental insults. The inflammatory process (FIG. 3) comprises a series of cellular responses that depend on integrating information associated with the following: detection of an injury and/or the presence of microorganisms, the accumulation and intervention of cells that eliminate invading microorganisms and infected host cells, and the repair of tissues that are damaged by the initial insult, trauma or the response of the host. In a recent review of inflammation37, Nathan classified inflammatory disorders according to their possible origin and the particular role of inflammation. In each category there are diseases in which members of the MMP family are upregulated. In fact, increased or misregulated levels of many MMPs are observed in any disease that is characterized by or associated with inflammation. If only because matrix proteolysis is a hallmark of the inflammatory process that is associated with many conditions, the role of MMPs as matrixdegrading proteinases justifies their inclusion as important components of the host response to traumatic, infectious, toxic or autoimmune insults, which we have broadly defined as inflammation. Although MMP inhibitors (BOX 1) are used as anti-inflammatory drugs in periodontal disease38 and have been suggested as a therapy to halt tissue destruction in inflammatory conditions such as arthritis39 and vascular disease40, the exact role of most MMPs in inflammatory conditions has not yet been elucidated, even to the extent of understanding whether they function to improve or worsen inflammation. Indeed, using arthritis as an example, mouse studies


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Box 2 | Identifying and verifying substrates

Identifying substrates Proteomics. The basic strategy is to compare the pattern of pericellular proteins that is produced when a specific proteinase is expressed with the pattern in the absence of the proteinase28,29. Affinity approaches. Proteinases bind substrates using regions outside the catalytic domain (exosites), and these interactions can be exploited to design approaches to find binding partners, such as direct binding in a yeast two-hybrid assay97. Deduction. By far the most common approach to finding candidate substrates. Close examination of the phenotypes of knockout mice might indicate candidates, such as excess type I collagen deposition in matrix metalloproteinase 14 (MMP14)-deficient mice31 and reduced apoptosis in the prostate glands of castrated MMP7-deficient mice53.

Verifying substrates Location. A proteinase and substrate need to be in the same microenvironment during proteolysis. This can be assessed by methods such as immunostaining, fractionation, co-immunoprecipitation or ‘pull-down’. A caveat for immunoprecipitation and pulldown approaches is that a proteinase might only interact transiently with its substrate. However, a catalytically dead proteinase should bind stably to its substrate. The activity of MMPs can be blocked by mutating the nucleophile-providing glutamatic acid residue that is present in the conserved motif (HEXXHXXGXXH, where X denotes any amino acid) of the catalytic domain. Cleavage sites. If an MMP cleaves a protein in vivo, then it should make the same cut(s) and only that cut (those cuts) in vitro using purified reagents. Loss-of-function. At present, the favoured method is showing that ablation or mutation of an MMP can prevent a specific proteolytic process. This can be carried out using targeted mutagenesis, RNA interference, dominant-negative proteins or blocking antibodies. Gain-of-function. Overexpression or ectopic expression of an MMP or substrate can be easily manipulated to examine questions about cleavage-site specificity, accessory factors and more. As discussed in TABLE 1, overexpression studies need to be used with caution in the identification of candidate substrates.

indicate that MMPs long thought to contribute to joint destruction, such as MMP2 and MMP3, might actually provide protection41,42 (TABLE 2). As we discuss later, there are now several examples that show that these proteinases are important effectors in various processes controlling repair and leukocyte recruitment — processes that are central to inflammation (FIG. 4). MMPs in innate immunity and repair

INNATE IMMUNITY

The term generally refers to innate pathogen-recognition systems, as well as to antimicrobial peptides. Innate immunity comprises immediate responses that are generated without the requirement for memory of, or prior exposure to, the pathogen. It is mostly mediated by receptors that have broad specificity (such as Toll-like receptors): that is, receptors that recognize many related pathogen-associated molecular patterns.

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Epithelial repair. INNATE IMMUNITY comprises various ready-to-go mechanisms that defend against invading microorganisms, contribute to tissue repair, and regulate the activity and influx of cells that are involved in acquired immunity. By maintaining a tight barrier against the external environment, secreting antimicrobial peptides, and producing chemokines and homing receptors, the epithelium is the first line of innate defence. Injury disrupts the barrier function of the epithelium, creating an entry point for microorganisms and toxins. However, injured epithelial cells respond rapidly to close wounds, a process that involves cell proliferation, spreading and migration. Injury also induces the expression of several MMPs43, some of which are crucial for wound closure. For example, the repair of skin wounds requires the catalytic activity of MMP1. MMP1 alters the migratory substratum, which consists


of type I collagen, from a high-affinity ligand for the α2β1-integrin to one of lower affinity. In this way, it functions as the enzymatic machinery that drives the forward movement of the repairing cells by allowing them to attach, dislodge, then reattach to the woundbed matrix44. In mucosal tissues, such as lung and gut, MMP7 is expressed by wound-edge epithelial cells and is required for RE-EPITHELIALIZATION45. This presumably occurs by the shedding of epithelial (E)-cadherin ectodomains, which would loosen cell–cell contacts, thereby facilitating cell migration46. Although MMP9 has been shown to be required for the migration of airway epithelial cells over a collagen matrix in cell culture47, no defect in epithelial closure is observed in MMP9-deficient mice48. Killing bacteria. Unlike the many MMPs that are expressed in response to injury, inflammation or other overt stimuli, MMP7 is expressed by non-injured, noninflamed mucosal epithelium in most, if not all, adult tissues49. The expression of MMP7 in healthy epithelium indicates that it functions in common homeostatic processes, which seem to provide defence against microorganisms and enable apoptosis. In mice, MMP7 activates intestinal pro-α-DEFENSINS (also known as cryptdins)50, and because of the lack of mature, active α-defensins, MMP7-deficient mice have an impaired ability to battle enteric pathogens, such as pathogenic Escherichia coli and Salmonella typhimurium51. In addition to killing bacteria through the action of defensins and other antimicrobials, intestinal epithelial cells that are infected with invasive bacteria participate in defence by suicide: that is, by undergoing apoptosis and sloughing into the lumenal space (from which they are excreted by the host), and thereby decreasing the bacterial load52. Among its verified substrates (TABLE 1), MMP7 also sheds membrane-bound FAS ligand (FASL, also known as CD95L). The binding of FASL to the death receptor FAS controls programmed cell death53. Although MMP7-mediated effects on apoptosis have not yet been shown to be involved in defence mechanisms, these effects might be another way in which MMP7 is involved in innate immune responses (FIG. 4). Whereas MMP7 has an indirect role in killing bacteria, preliminary observations indicate that the hemopexinlike domain of MMP12, which is readily released from the intact enzyme, has bactericidal activity54. The induction and activation of MMP7 in mucosal epithelium is highly sensitive to the presence of virulent bacteria, further extending a role for this MMP in innate immunity55,56. All epithelial tissues in which MMP7 is expressed are exposed to the external environment, so the widespread production of MMP7 in epithelium might be sustained by continual, low-level bacterial exposure. Consistent with this idea, MMP7 is not detected in fetal or perinatal tissues57 that have little exposure to microorganisms. In the small intestine of germ-free, adult mice, MMP7 is present at almost undetectable levels, but its production is induced in ex-germ-free mice that are colonized with a single species of commensal bacteria55. So, bacterial exposure seems to be the physiological signal


REVIEWS that regulates MMP7 expression in intact epithelium. In accordance with the suggested role of epithelial cells as ‘sensors’ for microbial infection58, MMP7 might be part of a more general activation response to bacteria. MMPs and chemokines

Chemokines are a group of chemotactic molecules that specifically attract and recruit populations of immune effector cells (including neutrophils, monocytes and eosinophils) to sites of injury or infection and thereby shape the evolution and outcome of the inflammatory response59. Several studies have shown that specific MMPs control chemokine activity. This control can be direct, by MMP-mediated cleavage of these molecules27,60–63, which results in enhancement, inactivation or antagonism of chemokine activities, or it can be indirect, by the proteinases acting on other substrates that bind, retain or concentrate the chemotactic molecules in particular locations64–66.

MMPs directly modulate chemokine activity. One potential action of MMPs is to convert chemokines from their true (or initial) nature as chemotactic molecules into antagonistic derivatives, thereby disrupting the further recruitment of cellular components and contributors to sites of inflammation. This repressive processing has been examined in detail by Overall and colleagues27,60,61, who studied the monocyte-chemotactic protein (MCP) group of CC-chemokines. Using the C-terminal hemopexin-like domain of MMP2 as a bait in a yeast two-hybrid system, they discovered, unexpectedly, that CC-chemokine ligand 7 (CCL7; also known as MCP3) is a substrate of MMP2, which cleaves the four amino (N)-terminal amino acids of the chemokine. Truncated CCL7 still binds its cognate CC-chemokine receptors (CCRs), but it no longer promotes chemotaxis; instead, it functions as a chemokine antagonist. Similarly, MMP1, -3, -13 and -14 cleave the N-terminus of CCL2 (also known as MCP1), CCL8 (also known as

Table 2 | Inflammatory and immune phenotypes of Mmp-null mice Mouse

Phenotypes that indicate a function in innate or acquired immunity, including repair-related processes

Mmp2–/–

Decreased allergic inflammation More-severe immune-mediated arthritis Reduced tumour, corneal, retinal and choroidal neovascularization

Mmp3–/–

More-severe immune-mediated arthritis Reduced contact hypersensitivity Reduced neutrophil influx in immune-mediated lung injury Reduced number of macrophages in atherosclerotic plaques Reduced cartilage-derived macrophage-chemotactic activity

Mmp7–/–

Lack of activation of cell-surface TNF displayed on macrophages Lack of active intestinal α-defensins and impaired ability to kill enteric pathogens Spatially constrained chemokines and reduced neutrophil influx in injured lung Markedly impaired re-epithelialization and reduced shedding of E-cadherin in injured lung Reduced shedding of active FAS ligand (CD95L) and epithelial-cell apoptosis Increased neovascularization in corneal wounds

Mmp8–/–

Marked increased in chemically induced skin tumours*

RE-EPITHELIALIZATION

A mechanism of repair that involves epithelial-cell proliferation and migration across a denuded surface to re-establish cell contact and close a wound. During reepithelialization, cells receive and process cues from a new microenvironment (that is, the exposed wound) and coordinate various responses, including the induction of matrix metalloproteinases and pro-inflammatory mediators, and the activation and expression of integrins.

–/–

Mmp9

DEFENSINS

A class of antimicrobial peptides that have activity against Grampositive and Gram-negative bacteria, fungi and viruses. Defensins are classified into two main categories on the basis of the position of conserved cysteine and hydrophobic residues and the linkages of disulphide bonds: α-defensins are produced by intestinal Paneth cells and neutrophils, and β-defensins are expressed by most epithelial cells. A third category, the θ-defensins, arises from the splicing of two αdefensin-related peptides into a circular molecule; at present, these defensins have been detected only in the neutrophils of rhesus macaques.

64 41 119–122 42 123 124 125 72 106 51 66 45,46 53 126 68

Prolonged contact hypersensitivity Reduced experimental autoimmune encephalomyelitis Protection against endotoxin-mediated shock Reduced dendritic (Langerhans)-cell migration Reduced antigen-mediated blister formation Reduced angiogenesis in developing bone, tumours* and ischaemic tissues Impaired bronchiolization after acute lung injury Altered chemokine gradients in models of allergen-induced airway inflammation Altered leukocyte influx in models of allergen-induced airway inflammation Modulation of IL-13-induced lung inflammation Less-severe experimental arthritis Impaired macrophage infiltration in atherosclerosis Protection against macrophage-induced aneurysm formation* Reduced ischaemia-induced cerebral injury

Mmp2–/–Mmp9–/– Reduced choroidal neovascularization –/–

References

123 127 128 129 130 131–133 48 71 71,134,135 136 41 137 138,139 140 141

Mmp11

ND

Mmp12–/–

Reduced macrophage migration and influx in smoke-induced emphysema Reduced spinal cord injury Reduced release of active TNF from smoke-exposed macrophages Reduced neutrophil influx and epithelial permeability in immune-mediated lung injury Modulation of IL-13-induced lung inflammation

Mmp14–/–

ND

–/–

Mmp20

ND

Mmp28–/–

Markedly reduced inflammation in models of lung injury‡

73,142 143 106 144 136

*These phenotypes were reversed following transplantation of wild-type bone marrow, indicating that the effect observed in knockout mice was caused by the lack of the matrix metalloproteinase (MMP) in an inflammatory cell or group of inflammatory cells. ‡W.C.P., unpublished observations. E-cadherin, epithelial cadherin; IL, interleukin; ND, not determined; TNF, tumour-necrosis factor.



REVIEWS

Lumen

a

β-defensins

Cathelicidins

Epithelial cell Glycocalyx

Desmosomes and adherens junctions Blood vessel

Neutrophil

Injury and infection Macrophage

Pathogen

c b

ICAM1

b

Chemokines, cytokines and lipid mediators

Neutrophil

Figure 3 | Mucosal immunity. The mechanisms that epithelia use to defend against microorganisms encompass at least four processes. a | Barrier function. Epithelia form specialized macromolecular structures and polymers that are diplayed on their lumenal face, such as the cornified layer of the epidermis (which is mostly crosslinked intermediate filaments), the highly ordered uroplakin layer that lines the bladder and the glycocalyx of mucosal epithelia (shown). In addition to barring or partly restricting the passage of solutes and water, these lumenal structures provide a physical barrier to invasive microorganisms. The barrier function of epithelia is augmented by various junctional complexes, such as desmosomes and adherens junctions, that weld epithelial cells together. Also, intact epithelia release a variety of antimicrobial peptides and proteins, such as β-defensins, cathelicidins and lysozyme, that directly kill microorganisms. b | Re-epithelialization. Epithelia are constantly subjected to injury and trauma, which ranges from the denudation of a few cells to large wounds. A breached epithelium provides an entry point for infection. In response to injury, the wound-edge epithelium of all tissues responds rapidly and in a similar manner to close the tissue gap. That is, the cells at the edge of the wound change from a stationary to a migratory phenotype, spread out and cover the wound. This process of re-epithelialization uses the daughters of hyperproliferative cells that are present just behind the wound front. c | Bacterial clearance. There are several means that epithelia use to remove invading bacteria at a wound site: first, enhanced production and release of antimicrobial compounds; second, (potentially) apoptosis (not shown),which might be selective for cells that have been infected with invasive pathogens; and third, various physical processes that drive bacteria from the body, such as the mucociliary apparatus in the airways, fluid flow in the sweat glands and excretion. These mechanisms would also be active in the defence of intact epithelium. d | Inflammation. By their production of chemotactic molecules, such as chemokines and lipid mediators, and their expression of adhesion receptors, such as intercellular adhesion molecule 1 (ICAM1), epithelia provide the initial host-derived signals that mediate and direct the influx of inflammatory cells, such as neutrophils, to sites of injury or infection.

624


MCP2) and CCL13 (also known as MCP4) to produce antagonist factors60. Although MMPs have long been considered to augment inflammation and the associated tissue damage, these examples indicate that they can also dampen inflammatory processes (FIG. 4). In addition to MCPs, the function of other CC- and CXC-chemokines is altered by direct MMP proteolysis. CXC-chemokine ligand 11 (CXCL11; also known as SDF1) is a substrate of several MMPs (MMP1, -2, -3, -9, -13 and -14) both in vitro using purified proteins and in cell-culture models, and the processing of CXCL11 by MMP2 yields a product that has potent neurotoxic activity67. Similarly, processing by MMP9 leads to a loss of chemotactic activity of various chemokines, such as CXCL5 (also known as ENA78), CXCL6 (also known as GCP2) and mouse CXCL5 (also known as GCP2 or LIX). By contrast, the processing of CXCL8 (also known as interleukin-8, IL-8) by MMP9 markedly increases its chemotactic activity62,63. In vitro, MMP8 can also cleave mouse CXCL5, generating peptides with enhanced neutrophil-chemotactic activity; however, in vivo, high concentrations of processed mouse CXCL5 are recovered from the inflamed lungs of MMP8-deficient mice68, indicating that other proteinases can function to regulate the activity of this CXC-chemokine. In addition, MMPs might regulate the expression of chemokine receptors and signalling through these receptors, providing an indirect mechanism by which these proteinases can regulate leukocyte influx69. Although the studies discussed here indicate that MMPs can act on chemokines and alter their activity, a complete deficiency in the proteolytic processing of a specific chemokine has not yet been shown in any model of cellular recruitment in MMP-deficient mice. MMPs establish chemokine gradients. The bioavailability of chemokines is regulated by the level of biosynthesis, the expression of the cognate receptor(s) and the modification of chemokines by proteinases. In addition, it is also regulated by immobilization of chemokines to the ECM or to cell surfaces. In vivo, chemokines form chemotactic gradients by binding to accessory macromolecules (typically the glycosaminoglycan side chains of proteoglycans), thereby providing directional cues to migrating leukocytes. By acting on these accessory molecules, MMPs can indirectly regulate chemokine activity and, in turn, the influx of inflammatory cells. A good demonstration of this mechanism is provided by the ability of epithelial-derived MMP7 to shed the ectodomain of syndecan-1, a transmembrane heparan sulphate proteoglycan70. In this situation, three epithelial components — a secreted proteinase (MMP7), a cellbound proteoglycan (syndecan-1), and a chemokine (mouse CXCL1; also known as KC) — function coordinately to control and confine acute inflammation, specifically NEUTROPHIL TRANSEPITHELIAL MIGRATION, to sites of injury (FIG. 4). In response to mucosal injury, epithelial cells synthesize, secrete and deposit CXCL1 (or CXCL8 in humans) onto the heparan sulphate chains of preexisting syndecan-1 molecules. MMP7, which is also induced by injury, cleaves the syndecan-1 core protein at


REVIEWS a juxtamembrane site to release the ectodomain– CXCL1 complex. The shed complex either actively or passively creates a chemotactic gradient that guides neutrophils into the alveolar space. In bleomycin-injured lungs of MMP7-deficient mice, neutrophils extravasate easily from the vasculature, but they are either held at the epithelial–interstitial interface or in markedly

Lumen

expanded perivascular spaces, and do not enter the lumen of the lung. Interestingly, the interaction between MMP7 and heparan-sulphate-containing molecules increases the catalytic activity of MMP7 (REF. 24). So, the glycosaminoglycan chains on syndecan-1 might function as an accessory factor (FIG. 2) that links MMP7 to its substrate, the core protein of the proteoglycan.

MMP9

Soluble FASL FASL

c

d

Neutrophil elastase

Pathogen Adherens junctions Epithelial cell

a Syndecan-1 MMP7

CXCL8/ CXCL1

e

MMP12

b TNF-α

ADAM17

Macrophage CCR3/CCR1

CXCR2

CCL7

f Fibroblast MMP2

Blood vessel

Neutrophil

NEUTROPHIL TRANSEPITHELIAL MIGRATION

During bacterial infections at mucosal sites, neutrophils migrate from the vasculature through the interstitial compartment and across the epithelial barrier. The activation and migration of neutrophils into lungs also contributes to inflammatory tissue injury and remodelling of tissue architecture

Figure 4 | MMPs in inflammation in response to tissue injury. Injury initiates a programmed, coordinated series of responses to both repair the damaged tissue and to defend against infection. Almost all resident cells, particularly epithelial cells, endothelial cells and fibroblasts, participate in these processes and contribute to the regulation of inflammation. This occurs partly through the specific activity of a variety of matrix metalloproteinases (MMPs) that are produced by these cells. a | Soon after injury, epithelial cells at the wound edge produce a chemokine (in humans, CXC-chemokine ligand 8, also known as interleukin-8, and in mice CXCL1, also known as KC) that accumulates on the heparan sulphate chains of syndecan-1, a transmembrane proteoglycan. At the same time, these cells release MMP7, which sheds the ectodomains of syndecan-1, thereby establishing a local chemokine gradient that controls the influx and activation of neutrophils. b | Later on in the repair process, epithelial-derived MMP7 cleaves the ectodomains of epithelial (E)-cadherin, thereby disrupting adherens junctions and, in turn, facilitating cell migration. Re-epithelialization is also facilitated by the action of other MMPs, such as MMP1 in skin and MMP9 in lung cells. c | MMP7 also sheds and activates FAS ligand (FASL, also known as CD95L) that is produced by epithelial cells, thereby mediating apoptosis, which is a potential innate defence mechanism (discussed in text). d | After activation, neutrophils release several proteases. Among them, neutrophil elastase, a serine protease that is exclusively produced by neutrophils, which has direct antimicrobial activity. Mice deficient in this enzyme have an impaired ability to defend against Gram-negative bacteria98. Activated neutrophils also release MMP9, which degrades and neutralizes the serine protease inhibitor α1-antiproteinase99, a potent inhibitor of neutrophil elastase. In this setting, MMP9 provides cover for the antimicrobial activity of neutrophil elastase, thereby assigning it an indirect role in innate immunity. e | The activation of the latent form of tumour-necrosis factor (TNF) on the surface of cells such as macrophages is due to metalloproteinase-mediated proteolysis. In addition to ADAM17 (a disintegrin and metalloproteinase; also known as TNF-converting enzyme, TACE), MMP7 and MMP12 can activate latent TNF (TABLE 1). f | The influx of inflammatory cells is mainly directed by specific chemokines that are released by resident cells. In addition to indirect effects on chemokine activity, as discussed in a, MMPs also directly act on chemokines, either enhancing or abrogating their activity. For example, MMP2, which is typically produced by mesenchymal cells, can cleave and inactivate CC-chemokine ligand 7 (also known as macrophage-chemotactic protein 3, MCP3). CCR, CC-chemokine receptor; CXCR, CXC-chemokine receptor.



REVIEWS This inhibition of neutrophil recruitment in MMP7deficient mice that have been subjected to acute lung injury results in reduced lethality compared with wildtype animals66. However, if the neutrophils are forced into the alveolar space, by administration of a bacterial chemotactic peptide, nFNLP, then the MMP7-deficient mice are more susceptible to the lethal effects of acute lung injury than wild-type mice. These observations indicate that neutrophil activation and its oxidative burst, which probably cause the lethality, are held in check until these leukocytes reach the lumenal space, where they would first encounter any microorganism attempting to enter the tissue through the breached epithelium. Therefore, MMP7-mediated proteolysis would be high in the hierarchy of events that control neutrophil activation. The observation that MMP7-deficient mice are protected against acute lung injury and the subsequent lethal effects of a pro-inflammatory insult does not indicate that in all cases MMP7 is a detrimental proteinase. Although in the experimental model discussed, massive neutrophil influx and oxidative burst cause indiscriminate, severe and potentially mortal damage, neutrophils comprise an essential cellular component of innate host defence; and in alternative situations, MMP7 has a beneficial function in neutrophil influx and activation, as well as in mucosal immunity and epithelial migration66. Although MMP7 might not directly process chemokines at these sites60, it has a leading role in regulating the formation of a chemotactic gradient that controls neutrophil influx and activation. Other MMPs also regulate the formation of chemokine gradients. Allergen-induced airway inflammation is dampened in MMP2-deficient mice, an observation associated with reduced levels of CCL11 (also known as eotaxin) in lavage fluid64. How MMP2 controls the bioavailability of CCL11 is not known, but it is interesting that this pro-inflammatory function is distinct from the anti-inflammatory action of MMP2 on MCP chemokines (CCL2, -7, -8 and -13). In the same allergen model, MMP9 was also recently shown to be required for formation of transepithelial gradients of CCL11, as well as CCL7 and CCL17 (also known as TARC). But again, the mechanism by which MMP9 facilitates the movement of these chemokines from one tissue compartment to another is not known71. MMP9 also affects the ability of CXCL8 to stimulate the release of leukocytes from bone marrow, but similar to the allergen model, the mechanism by which this occurs (that is, the identity of the target substrate) has not been determined. In addition, MMP3 is known to mediate the release of a macrophage-chemotactic activity from chondrocytes72, and MMP12 is required for the influx of macrophages into smoke-exposed lungs73. But the nature of these activities, and whether they involve chemokines, is not known. Collectively, these findings show that several MMPs can regulate an inflammatory response by controlling the activity and mobilization of chemokines, and further work to identify the key substrates in these processes will provide insight into fundamental mechanisms of inflammation.

626


MMPs and cytokines

Several studies have indicated that MMPs can directly or indirectly affect the activity of various cytokines that function in inflammation and repair processes, including interferon-β74, vascular endothelial growth factor75, epidermal growth factors76, fibroblast growth factors77 and transforming growth factor-β1 (TGF-β1). As shown using TGF-β1-deficient mice, this cytokine functions to restrain mononuclear inflammation78–80. TGF-β1 is released by cells with its cleaved pro-domain still latently associated, and several mechanisms, including MMP proteolysis, have been proposed to release the active cytokine from this complex. In both cells and tissueexplant models, MMP3 (REF. 81), MMP9 (REF. 23) and MMP14 (REF. 82) have been shown or suggested to activate a proportion of total TGF-β1. If these or other MMPs can activate TGF-β1 in vivo, then this would be another mechanism by which MMPs restrain, rather than augment, inflammation. Similarly, IL-1β, a potent pro-inflammatory cytokine, requires proteolytic processing for activation, a process attributed to the IL-1β-converting enzyme (ICE, also known as caspase-1). Although the function of ICE had been well established in vitro, studies using ICE-deficient mice provided evidence of other mechanisms of IL-1β activation83. At least three members of the MMP family, MMP2, -3 and -9, can cleave and activate the IL-1β precursor84. Furthermore, after activating IL-1β, MMP3 degrades the biologically active cytokine84, which can also be inactivated in vitro by MMP1, -2 and -9 (REF. 85). These data indicate a dual role for MMPs in biphasic modulation of inflammatorymediator activity: they are involved in both activation (MMP3 and MMP9, and more weakly MMP2) and inactivation (MMP3). Another essential pro-inflammatory mediator that is regulated by metalloproteinase activity is tumournecrosis factor (TNF), which is produced as a 26-kDa membrane-associated protein (proTNF) and is cleaved by TNF-converting enzyme (TACE) into a soluble 17.5-kDa cytokine. Because synthetic metalloproteinase inhibitors block this cleavage, it was suggested that TACE was an MMP86. However, when the convertase activity was purified and cloned, TACE was found to be identical to ADAM17 (REFS 87,88), a member of the disintegrin family of metalloproteinases (ADAMs). The cleavage of proTNF by ADAM17 is specific89, and because the release of active TNF is reduced by 90% in cells derived from ADAM17-deficient mice, ADAM17 does seem to be the principal physiological TNF-converting enzyme in vivo. Even if ADAM17 is the main modulator of the generation of TNF activity, several MMPs (including MMP1, -2, -3, -9 and -17) can process proTNF to its active form in vitro89,90. Furthermore, as shown using cells from knockout mice, MMP7 and MMP12 also activate proTNF on macrophages (TABLE 1). So, whereas ADAM17 is seemingly involved in the inducible, high-level release of TNF in response to bacteria and toxic shock, MMP7 and MMP12 might elicit the constitutive release of TNF from macrophages that is required for common


REVIEWS functions, such as tissue resorption and resolution in response to injury (FIG. 4). The future

We have discussed many examples of how MMPs function in innate immunity and inflammation, but we have not mentioned acquired immunity. Because many MMPs influence macrophage behaviour (TABLE 2), this might indicate that they also affect the antigenpresentation function of these cells. In addition, functions associated with lymphocyte influx and T helper 2

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cell cytokines, such as IL-13 (TABLE 2), support the contention that MMPs have diverse functions in immunity. One challenge for the MMP field is to further uncover the function of MMPs: that is, to identify authentic substrates using physiologically relevant approaches and systems, and to do this with an open mind. Importantly, determining precise MMP–substrate interactions might provide an alternative and more precise strategy to block specific and potentially detrimental processes that are associated with inflammation and immune-mediated disease.

Although these mechanisms have not yet been shown in vivo, they are probably important for the regulation of MMPs in inflammation. Sternlicht, M. D. & Werb, Z. How matrix metalloproteinases regulate cell behavior. Annu. Rev. Cell Dev. Biol. 17, 463–516 (2001). Mackay, A. R., Hartzler, J. L., Pelina, M. D. & Thorgeirsson, U. P. Studies on the ability of 65-kDa and 92kDa tumor cell gelatinases to degrade type IV collagen. J. Biol. Chem. 265, 21929–21934 (1990). Halpert, I. et al. Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme. Proc. Natl Acad. Sci. USA 93, 9748–9753 (1996). Brooks, P. C. et al. Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin αvβ3. Cell 85, 683–693 (1996). Dumin, J. A. et al. Procollagenase-1 (matrix metalloproteinase-1) binds the integrin α2β1 upon release from keratinocytes migrating on type I collagen. J. Biol. Chem. 276, 29368–29374 (2001). Stricker, T. P. et al. Structural analysis of the α2 integrin I domain/procollagenase-1 (matrix metalloproteinase-1) interaction. J. Biol. Chem. 276, 29375–29381 (2001). Yu, Q. & Stamenkovic, I. Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-β and promotes tumor invasion and angiogenesis. Genes Dev. 14, 163–176 (2000). Yu, W. H. & Woessner, J. F. Jr. Heparan sulfate proteoglycans as extracellular docking molecules for matrilysin (matrix metalloproteinase 7). J. Biol. Chem. 275, 4183–4191 (2000). Yu, W. H., Woessner, J. F. Jr, McNeish, J. D. & Stamenkovic, I. CD44 anchors the assembly of matrilysin/MMP-7 with heparin-binding epidermal growth factor precursor and ErbB4 and regulates female reproductive organ remodeling. Genes Dev. 16, 307–323 (2002). This paper provides a good example of how a ‘secreted’ MMP is bound to and compartmentalized by a cell-surface molecule. Gross, J. & Lapiere, C. M. Collagenolytic activity in amphipian tissues: a tissue culture assay. Proc. Natl Acad. Sci. USA 48, 1014–1022 (1962). McQuibban, G. A. et al. Inflammation dampened by gelatinase A cleavage of monocyte chemoattractant protein-3. Science 289, 1202–1206 (2000). This paper shows how exosite scanning and yeast two-hybrid techniques can be used to identify novel MMP substrates: in this case, chemokines. Together with other studies by these investigators (references 54 and 55), this study provides evidence that MMP proteolysis directly regulates chemokine activity. Guo, L. et al. A proteomic approach for the identification of cell-surface proteins shed by metalloproteases. Mol. Cell. Proteomics 1, 30–36 (2002). Tam, E. M., Morrison, C. J., Wu, Y. I., Stack, M. S. & Overall, C. M. Membrane protease proteomics: isotopecoded affinity tag MS identification of undescribed MT1matrix metalloproteinase substrates. Proc. Natl Acad. Sci. USA 101, 6917–6922 (2004). This paper describes a proteomics study using stateof-the-art technology to identify MMP substrates. This knowledge is essential for understanding the function of these enzymes in vivo. McCawley, L. J. & Matrisian, L. M. Matrix metalloproteinases: they’re not just for matrix anymore! Curr. Opin. Cell Biol. 13, 534–540 (2001). Holmbeck, K. et al. MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue

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Competing interests statement The authors declare no competing financial interests.

Online links DATABASES The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene ADAM17 | CCL7 | CXCL1 | CXCL8 | FAS | FASL | IL-1β | MMPs | syndecan-1 | TGF-β1 | TNF Access to this interactive links box is free online.
