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phil chemotaxis was found [47], whereas in other investiga- tions, direct [41] ..... Rudd, P. M., Mattu, T. S., Masure, S., Bratt, T., Van den Steen, P. E.,. Wormald, M.
Gelatinase B functions as regulator and effector in leukocyte biology Ghislain Opdenakker, Philippe E. Van den Steen, Be´ne´dicte Dubois, Inge Nelissen, Els Van Coillie, Stefan Masure, Paul Proost, and Jo Van Damme Laboratory of Molecular Immunology, Rega Institute, University of Leuven, Leuven, Belgium

Abstract: Matrix metalloproteinases (MMPs) form a family of enzymes with major actions in the remodeling of extracellular matrix (ECM) components. Gelatinase B (MMP-9) is the most complex family member in terms of domain structure and regulation of its activity. Gelatinase B activity is under strict control at various levels: transcription of the gene by cytokines and cellular interactions; activation of the pro-enzyme by a cascade of enzymes comprising serine proteases and other MMPs; and regulation by specific tissue inhibitors of MMPs (TIMPs) or by unspecific inhibitors, such as ␣2-macroglobulin. Thus, remodeling ECM is the result of the local protease load, i.e., the net balance between enzymes and inhibitors. Glycosylation has a limited effect on the net activity of gelatinase B, and in contrast to the all-or-none effect of enzyme activation or inhibition, it results in a higher-level, fine-tuning effect on the ECM catalysis by proteases in mammalian species. Fast degranulation of considerable amounts of intracellularly stored gelatinase B from neutrophils, induced by various types of chemotactic factors, is another level of control of activity. Neutrophils are firstline defense leukocytes and do not produce gelatinase A or TIMP. Thus, neutrophils contrast sharply with mononuclear leukocytes, which produce gelatinase A constitutively, synthesize gelatinase B de novo after adequate triggering, and overproduce TIMP-1. Gelatinase B is also endowed with functions other than cleaving the ECM. It has been shown to generate autoimmune neo-epitopes and to activate pro-IL-1␤ into active IL-1␤. Gelatinase B ablation in the mouse leads to altered bone remodeling and subfertility, results in resistance to several induced inflammatory or autoimmune pathologies, and indicates that the enzyme plays a crucial role in development and angiogenesis. The major human neutrophil chemoattractant, IL-8, stimulates fast degranulation of gelatinase B from neutrophils. Gelatinase B is also found to function as a regulator of neutrophil biology and to truncate IL-8 at the aminoterminus into a tenfold more potent chemokine, resulting in an important positive feedback loop for neutrophil activation and chemotaxis. The CXC chemokines GRO-␣, CTAPIII, and PF-4 are degraded by gelatinase B,

whereas the CC chemokines MCP-2 and RANTES are not cleaved. J. Leukoc. Biol. 69: 851– 859; 2001. Key Words: matrix metalloproteinases TIMP 䡠 neutrophils

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extracellular matrix

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INTRODUCTION The orchestration of leukocyte biology at the molecular level has been linked primarily to cytokine functions and is much less associated with protease activities. Proteases, secreted by leukocytes, have been associated mainly with degrading extracellular matrix (ECM) components, rather than with regulatory functions. Nevertheless, many examples of interaction, complementation, and control exist among proteases, cytokines, and chemokines. This interconnection was emphasized recently at the International Meeting on Inflammatory Cytokines and Chemokines in the Context of Extracellular Matrix (Sept. 10 –14, 2000, Ma’ale Hachamisha, Israel), and particular aspects are evident from recent reviews [1–3]. We will discuss here part of this context and exemplify the interrelations with recent studies. Although the ECM is crucial for migration, recirculation, and homing of any leukocyte type, we will focus mainly on neutrophil biology.

GELATINASE B IS A COMPLEX MATRIX METALLOPROTEINASE (MMP) In a comprehensive overview of proteases [4], gelatinase B (MMP-9, MIM #120361, EC 3.4.24.35) is described as a multifunctional member of the ECM-degrading MMPs [5]. Figure 1 depicts the domain structure of gelatinase B in comparison with that of other MMPs. It shares with gelatinase A (MMP-2) a fibronectin-like domain, which functions in the binding of gelatin, and is unique in having a serine/proline/ threonine-rich collagen type V-like domain, which probably provides the attachment sites of multiple O-linked oligosac-

Correspondence: Ghislain Opdenakker, Laboratory of Molecular Immunology, Rega Institute, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail: [email protected] Received November 27, 2000; revised January 16, 2001; accepted January 17, 2001.

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Fig. 1. Comparison of the MMP family. Three nomenclatures of the MMPs are compared. At the left, the functional names of the enzymes are given, in which MT-MMP stands for membrane-type MMP. The MMP numbering is compared further with that of the IUPAC EC. At the right, the human chromosomal location of the various MMP genes is indicated. The latter shows gene clustering on human chromosome 11, subband q22. Furthermore, the protein domain structures are indicated by a color code and named at the bottom. On top, the zinc ion coordination by three histidines (H) in the zinc-binding domain is indicated. For the pro-enzyme forms, the fourth coordination is with the sulfhydryl group (-SH) of the unique cysteine (C) in the propeptide. By the activation process, the propeptide is clipped, and a water molecule (H2O) takes place in the active enzyme domain and ensures hydrolysis. Dashed lines indicate domains that are not present in particular MMPs.

charides. Gelatinase B possesses, like the other MMPs, a signal peptide, which guides the molecules to the extracellular milieu [6], and a propeptide, which imposes the latency state on these molecules [7, 8]. This propeptide of 80 –90 residues contains a unique cysteine of which the sulfhydryl group coordinates with and covers the zinc ion in the active site to keep the enzyme inactive. Activation in vivo occurs by proteolysis of the prodomain, which enables the “hydrolytic” water molecule to take place in the active site. This forms the basis of the cysteine switch model [7]. The interactions between the cysteine sulfhydryl group in the latent state, the water molecule in the active state, and the unique zinc ion were proven recently for intact, natural neutrophil gelatinase B by Kleifeld et al. [9]. The central core shared by all MMPs is the active site and the zinc-binding part in which three histidines form coordinations with the metal ion. Presumably, the conserved domain structure of the MMPs is the evolutionary consequence of the fact that many MMPs are relatived at the genomic level and are syntenic by gene duplication. For instance, it has been found that most collagenase 852

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and stromelysin genes cluster on human chromosome 11 (Fig. 1). Obviously, gelatinases and membrane-type MMPs are more distantly related because their genes are more dispersed across the human genome. Gelatinase B is a glycoprotein, and although this was already recognized when the human cDNA was cloned [10], it is not yet well-appreciated that glycosylation, as a major posttranslational modification, contributes 15% of the mass of the glycoprotein. Recently, the oligosaccharides of natural, human neutrophil gelatinase B have been sequenced [11], and functional studies of the oligosaccharides have been initiated.

CONTRASTS BETWEEN GELATINASE B FROM NEUTROPHILS AND OTHER CELLS Some 25 years ago, gelatinase B was discovered as a neutrophil product [12] and was recovered from monocyte supernatants a decade later [13]. The cDNA of human gelatinase B was cloned from tumor cells (HT1080), and the aminoterminus was idenhttp://www.jleukbio.org

tified as Ala-Pro-Arg-Glu-Arg-Glu-Ser-Thr-Leu-Val-Leu-PhePro-Gly [10]. However, when the first samples of gelatinase B from normal human cells, i.e., neutrophils and monocytes, were purified to homogeneity and sequenced at the protein level, another aminoterminal sequence was identified [14, 15]. Indeed, the latter two cell types produce truncation variants of gelatinase B that lack 8 or 10 aminoterminal residues. The aminoterminus in neutrophil gelatinase B is thus the same as in monocytes but differs from the one in tumor cells. At that time, there was still debate and confusion about the natural substrate of the enzyme (reflected by multiple names: type IV collagenase, type V collagenase, or 92-kDa collagenase). Following purification and identification of the natural enzyme from neutrophils, the name gelatinase B (EC 3.4.24.35) was assigned by the Enzyme Commission (EC) of the International Union of Pure and Applied Chemistry, because gelatinase A (EC 3.4.24.24) had already been accepted as a name, and gelatin (denatured collagen) was obviously a good substrate [14]. Human leukemic tumor cells, e.g., myelomonocytic THP-1 cells, produce gelatinase B in its proform [16] with the same aminoterminus as the above-mentioned tumor cells [10]. When considering all the collected data on the identified aminoterminal forms of progelatinase B from different cell sources, it can be concluded that neutrophil and peripheral blood monocyte gelatinases B originate from the same gene as the one in tumor cells, but their processing is different [10, 14 –16]. Neutrophils do not produce gelatinase A, whereas most other cell types do. Monocytes, lymphocytes, dendritic cells, fibroblasts, and tumor cell lines produce gelatinase A constitutively, albeit sometimes in small quantities (e.g., in fibroblasts). The two gelatinases A and B co-purify on gelatin-Sepharose affinity chromatography [17]. Remarkably, gelatinase B is an inducible enzyme in these cell types. Depending on the cell type used for gelatinase B induction in vitro, the soluble inducer may be bacterial [e.g., lipopolysaccharides (LPS)], viral (e.g., double-stranded RNA), plant products (e.g., lectins, phorbol esters), or cytokines, such as IL-1␤. Infection of cells with viruses also induces gelatinase B [17]. Alternatively, gelatinase B may be induced by cell-cell contacts (vide infra). Neutrophils do not produce tissue inhibitor-1 of MMP (TIMP-1) and consequently, do not produce gelatinase B/TIMP-1 complexes [14, 17]. This is in sharp contrast with monocytes and tumor cells that produce, after adequate triggering, gelatinase B and TIMP-1. For instance, when gelatinase B from normal, human monocytes [15] or THP-1 cells [16] was purified by gelatin-Sepharose affinity chromatography, an excess of TIMP-1 was co-purified as a complex with gelatinase B. Because this complex is not linked covalently, the addition of sodium dodecyl sulfate (SDS) to the samples results in the dissociation of gelatinase/TIMP complexes. This is illustrated in Figure 2. Both components of the complex were identified experimentally by amino acid sequencing [15, 16]. Neutrophils produce specific covalent complexes: homodimers of 200 kDa and heterodimers with neutrophil gelatinase B-associated lipocalin (NGAL) of 120 kDa (Fig. 2). Both these types of dimers are linked covalently by disulfide bridges and do not dissociate by the addition of SDS. The dimers may be dissociated by the addition of ␤-mercaptoethanol [18]. NGAL was identified by protein sequence analysis [19] and

Fig. 2. Comparison of gelatinase B from mononuclear cells and neutrophils. Gelatinases were purified by affinity chromatography on gelatin-Sepharose, and the proteins in the eluates were visualized by staining with Coomassie brilliant blue after SDS-polyacrylamide gel electrophoresis (PAGE). The proteins shown in the left panel are from THP-1 cells. This part illustrates that mononuclear cells produce gelatinases A and B and TIMP-1. Gelatinase A is barely visible but was visualized readily by zymography analysis. In this particular case, the THP-1 cells were stimulated with 100 ng/ml phorbolmyristate-acetate for 24 h, which results in the production of gelatinase B [16]. At the right, the gelatinases from formyl-Met-Leu-Phe (fMLP)-activated neutrophils are visualized. The latter cells produce monomers, dimers, and a heterodimer of gelatinase B with NGAL but do not produce TIMP-1. The schedule, used to obtain electrophoretically pure, natural gelatinase B from neutrophils, was a combination of biological and biochemical purification steps. Erythrocytes were removed by suspension in hydroxyethyl starch and sedimentation for 30 min at 37°C. Neutrophils were separated from mononuclear leukocytes by density-gradient centrifugation on Ficoll-sodium metrizoate. Then, neutrophils were degranulated under the pressure of 0.5 ␮M formylpeptide, and the supernatants were filtered to remove cell debris. The biochemical purification steps consisted of substrate-affinity chromatography and elution with 1.5 M NaCl plus 10% dimethyl sulfoxide (DMSO) [14], which results in a preparation as shown in the right panel. To remove the gelatinase B-NGAL complexes from the mixture, NGAL-specific mAb affinity chromatography is used [18].

may also occur as monomer or homodimer. Its exact physiological role remains elusive so far. Neutrophil gelatinase B and NGAL are distinctive in that their oligosaccharide structures are defined completely [11]. Because neutrophils do not produce TIMP-1 or gelatinase A, they have been shown to be an excellent source of natural gelatinase B for biochemical and biological studies. In fact, we ascribe our relative success in generating highly specific inhibitory monoclonal antibodies (mAbs) against human gelatinase B (without cross-reaction with gelatinase A) at least in part to the use of neutrophil gelatinase B as both antigen and selection reagent [20]. The latter reagent was completely devoid of gelatinase A (Fig. 2). Because the human gelatinase B cDNA was cloned already in 1989 [10], it may come as a surprise that electrophoretically homogeneous, natural gelatinase B was only generated recently [18]. Neutrophils mature from bone-marrow stem cells under the influence of growth and differentiation factors. During this developmental program, various markers are made within a specific frame of time and space. As reviewed recently, gelatinase B turns out to be a rather late and specific marker of final Opdenakker et al. Gelatinase B in leukocyte biology

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neutrophil maturation [21]. Thus, it is logical that such an enzyme may also assist in the regulation of leukocytosis and stem-cell mobilization [2, 22]. It needs to be stressed that other factors are involved in the mechanisms leading to stem-cell mobilization or that these mechanisms may be different according to animal species, because gelatinase B-deficient mice show normal mobilization. As illustrated in Figure 3, gelatinase B is stored in granules of mature neutrophils. Degranulation of pre-stored gelatinase B from neutrophils versus de novo synthesis and secretion by other leukocyte types is another characteristic (vide infra) implying that neutrophils immunostain optimally for gelatinase B in resting conditions. Conversely, to visualize gelatinase B in mononuclear leukocytes, the enzyme needs to be induced with appropriate cytokines, and usually the intracellular amount is smaller and more dispersed in the cytoplasm than in neutrophils (Fig. 3). Lymphocytes may be induced with phorbol esters [23], interleukin (IL)-2 [24], or by cell-cell contacts to produce gelatinase B [25], whereas IL-1, lectins, LPS, and viruses or viral products are good stimuli with which to generate gelatinase B in monocytes [15, 17].

FUNCTIONS OF GELATINASE B Gelatinolysis, i.e., the degradation of denatured collagens, is suggested to be a main function of gelatinase A and gelatinase B but remains to be proved in vivo. Generally, it is thought to be more important to consider basement membrane collagens of type IV as possible substrates. This function is executed in normal processes, such as the regulation of leukocytosis [2], and may be used for peripheral stem-cell mobilization by

Fig. 3. Production of gelatinase B by human leukocytes. Gelatinase B was visualized by immunocytochemistry in various leukocyte types. (a) Unstimulated neutrophils contain granules with gelatinase B and consequently stain brightly. (b and c) Mononuclear cells do not produce gelatinase B spontaneously but need stimulation by soluble factors or cellular interactions. In this case, mononuclear cells were purified by gradient centrifugation and then stimulated in vitro for 24 h with 100 units/ml IL-1␤ and immunostained. Fine and dispersed immunostaining of monocytes is shown (b), whereas the small rim of cytoplasmic gelatinase B is visualized in a lymphocyte and some immunoreactivity in the perinuclear area in a monocyte (c). The absence of staining of unstimulated monocytes is illustrated (d and e), whereas the same result is shown (f) on a stimulated monocyte when the primary antibody preparation was omitted. The primary antiserum [14] was used in combination with a second biotinylated goat anti-rabbit antiserum and an antibiotin/alkaline phosphatase conjugate. Controls with omission of the primary or secondary antiserum or the enzyme substrate showed no immunoreactivity, and a May-Gru¨nwald-Giemsa staining indicated that the mononuclear cell preparations were 99% pure.

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induction of gelatinase B release [22]. In most cell types—the neutrophil is the exception— gelatinase A is produced constitutively, whereas gelatinase B is inducible by many agonists. Therefore, the ratio of gelatinase B over gelatinase A has been used as a marker of the induction state in inflammation, including auto-immune diseases such as rheumatoid arthritis [17], multiple sclerosis (MS) [26, 27], and animal models thereof [28]. The abundant presence of gelatinase B (and granulocytes) in the synovium and synovial fluid of arthritic patients [17], where the enzyme will find ample substrate to digest, is the logical consequence of neutrophils being attracted and activated by synovial IL-8. It was much more intriguing to detect gelatinase B in cerebrospinal fluids in central nervous system (CNS) inflammations such as MS. In the latter case, the function of gelatinase B may be to assist in the degradation of the bloodbrain barrier collagen IV or other substrates. In addition, gelatinase B is also able to clip myelin compounds, such as myelin basic protein (MBP), within the human species [29] and even across species [28]. Remarkably, the resulting MBP fragments are potent encephalitogens (in animal models) and constitute immunodominant epitopes for T cells [1, 29]. We coined this the “remnant epitopes generate autoimmunity” or REGA model. This model summarizes qualitative and quantitative aspects of peptide neo-antigen formation in autoimmunity and the role of (extracellular) proteases in generating substrates for antigen presentation and T-cell activation. Qualitative elements are, for instance, the regulation of extracellular enzyme activity by cytokines and chemokines, the control by inhibition and activation, and the production of extracellular neoepitopes. Quantitative aspects include the fact that a gelatinase B molecule will cleave one substrate molecule, e.g., MBP, into several peptides, resulting in several-fold molar excesses of immunogenic peptides for processing and presentation, and that recruited T-cells can enter the CNS more easily through a damaged blood-brain barrier [1]. Is this REGA model useful for extending our understanding of the mechanisms and defining novel targets for the treatment of autoimmune diseases? Definitely yes, because cytokines or cytokine anatgonists with an effect on the protease load as well as aselective protease inhibitors have been shown to be efficient in the treatment of experimental animal models of autoimmune diseases (reveiwed in [1, 30, 31]). Recently, another proof of concept was provided with knock-out experiments. Gelatinase B gene knock-out in the mouse was made possible by homologous recombination [32] and by the public availability of the complete gene sequence as early as 1993 [33]. It has been shown that young gelatinase-B knock-out mice are resistant to development of experimental autoimmune encephalomyelitis and that young and adult animals are resistant to osteocartilaginous lesions, resembling chondrodermatitis nodularis helicis in humans [34]. These data indicate that selective gelatinase B inhibition may become an efficient treatment of acute and chronic inflammations including autoimmune diseases. Application of gene knock-out technology is an efficient way to define the functions of gelatinase B in vivo, to define the importance of the many in vitro properties of gelatinase B, and to complement phenomenological studies (e.g., the correlation http://www.jleukbio.org

of increased gelatinase B levels with the severity of particular diseases) with biological proof of concept. Table 1 shows the phenotypes demonstrated in gelatinase-B knock-out studies [34 – 47]. Two spontaneous phenotypes underline the role of gelatinase B in the remodeling of cartilage and bone and the reproductive tract [35, 36]. All other in vivo data were from studies in which the induction of gelatinase B was crucial. In many instances, this induction is in leukocytes, which are supposed to play a role in the pathology model. It should be noticed that detailed analysis and comparison of these studies are important. For instance, in one study, no defect in neutrophil chemotaxis was found [47], whereas in other investigations, direct [41] or indirect evidence [37] for reduced (neutrophil) chemotaxis was obtained. It may be that, depending on the animal model, the used gene knock-out strategy, or the involvement of particular (white blood) cell types, the phenotype of the deficient mouse is different (or not) from the wild type. Certainly, the list of phenotypes by gelatinase B ablation will increase considerably in the future, but already now, Table 1 illustrates the importance of the gelatinase B molecule in vivo. Several polymorphisms in the human gelatinase B gene [48] were shown to have a transcriptional effect. A cytidylateadenylate (CA) microsatellite, influencing transcriptional activity [49], has been detected in the promoter-enhancer region and used to study the genetics of aneurysma [50] and MS [51]. No associations were found between the occurrence of these diseases and specific alleles. In a similar approach, a single nucleotide polymorphism (SNP) with transcriptional effect was studied in atherosclerosis [52] and MS [51]. This SNP was associated with the severity of coronary heart disease but not with MS. Extracellular protease expression has been associated with invasive cancer development. By analogy with other proteases (urokinase on urokinase receptors, membrane-type MMPs anchored by glycosylphosphatidylinositol anchors, or hydrophobic-transmembrane domains), attempts to discover gelati-

TABLE 1.

Spontaneous and Induced Phenotypes by Gelatinase B Deficiency

Phenotypes of gelatinase B ablations in the mouse Transient delay in ossification of long bones Spontaneous infertility/subfertility Resistance to induced bullous pemphigoid Resistance to experimental autoimmune encephalomyelitis and tail necrosis Resistance to cancer cell invasion Resistance to post-ischemic cardiac rupture Diminished alveolar bronchiolization in the lung Diminished neutrophil chemotaxis toward mouse GCP-2 Prolonged, delayed hypersensitivity Serpin cleavage Angiogenic shift Resistance to development of abdominal aortic aneurysm Altered, post-myocardial infarction cardiac remodeling Normal migration of neutrophils in adult mice

Reference [35] [36] [37] [34] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47]

nase-B receptors have been made. The hyaluronan receptor, CD44, was found to be a gelatinase-B receptor [53, 54]. The localization of gelatinase B to the cell surface, by CD44, leads to activation of latent transforming growth factor (TGF)-␤ and constitutes a mechanism that may operate in normal tissue remodeling as well as in tumor growth and invasion [55].

THE REGULATION OF GELATINASE B IS COMPLEX The regulation of the cellular production and activity of gelatinase B has been the subject of recent reviews [56, 57]. However, the emphasis has been laid mainly on regulation by transcription, activation, and specific inhibition. Transcription of the gelatinase-B gene is stimulated in leukocytes by cytokines, viruses, bacterial products [16, 17], and plant lectins [17, 58]. In addition to soluble factors, cellular interactions activate MMP-9 gene transcription [59, 60]. Subsequent activation of secreted, latent progelatinase B into active gelatinase B occurs by proteolysis. Stromelysin-1 [61] and gelatinase A [62] catalyze this conversion and form part of a whole cascade that comprises plasmin and plasminogen activators [31]. It is important to realize that activation of pro-enzymes and inactivation of gelatinase B by TIMP are all-or-none phenomena. Although leukocytes are key players in the regulation of gelatinase B activity, the contribution of other cell types to the enzyme activation cascade or the production of inhibitors is obvious and equally important. Less well-recognized levels of control are by degranulation and glycosylation. To understand the latter two activity checkpoints, kinetic studies or multiple assays and extensive titrations are necessary. Release of gelatinase B by degranulation is a fast event in neutrophils and occurs within ⬍1 h when these cells are stimulated with chemotactic factors (Fig. 4), including the major neutrophil chemokine IL-8. This contrasts with the de novo synthesis of gelatinase B by monocytes, which is at least tenfold slower. Gelatinase B activity control by glycosylation is a fine-tuning effect, which developed in eukaryotes. It is much more difficult to assess because differential glycosylation of enzymes may influence the catalytic activity only two- to threefold, as has been found for plasminogen, tissue-type plasminogen activator [63], and ribonuclease [64]. Gelatinase B has three potential N-linked glycosylation sites, one of which is located in the propeptide [11]. This site and at least one of the two other glycosylation sequons (Asn-Xaa-Ser/Thr; Xaa is any amino acid except proline) in the active domain are occupied, but it has been impossible to deglycosylate the latter two sites enzymatically under native conditions. Such experiments are necessary to compare the specific activities of the aglycosyl with the fully N-glycosylated gelatinase B. Complete, native desialylation of the N- and O-linked sugars has been successful, as evidenced by lectin-blot analysis. Although desialylation does not affect the catalytic activity toward gelatin and synthetic peptides and, similarly, does not change the activation rate by stromelysin-1 and gelatinase A, it alters the interaction of gelatinase B with TIMP-1. After desialylation, the net activity of gelatinase B is increased significantly in the presence of equimolar or excess amounts of TIMP-1. Opdenakker et al. Gelatinase B in leukocyte biology

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Fig. 4. Gelatinase degranulation from human neutrophils. Peripheral blood neutrophils were treated with 3 U/ml pure, natural IL-8, with 100 U/ml pure, natural IL-1␤ at various time intervals, or were left untreated. The supernatants were analyzed by zymography, followed by scanning densitometry. Gelatinase B activities are expressed as percent activity versus control. Means ⫾ SE of six different experiments (except for IL-8 stimulation at 45 and 60 min, n⫽2) are shown. The decline of gelatinase B (as percent of control) activity after 1 h exposure to IL-8 is mainly because the untreated cells are releasing gelatinase B spontaneously from their granular content with time. Because this granular content is limited and not replaced by de novo synthesis of gelatinase B, the endpoint of the experiment is at 100 percent. This also implies that the degranulation effect can only be measured at early time intervals, although the gelatinase B enzyme is rather stable in cell culture media.

with TIMP-1 and with a highly specific inhibitory mAb [20]. Finally, the proform of gelatinase B did not catalyze the conversion of IL-8. The effects of the truncation by gelatinase B on the functions of IL-8 were documented at several levels (Table 2). These ranged between ten- and 27-fold, depending on the biological assay system used (receptor binding, increase of intracellular calcium concentration, release of gelatinase B from neutrophils, and neutrophil chemotaxis; Fig. 5B). With the use of chemokine receptor-transfectant cell lines, it was clarified further that the potentiating effect was mediated mainly by CXCR1 and less by CXCR2 [18]. Some relevant questions may be raised as a consequence of these observations. Are other CXC or CC chemokines also potentiated by gelatinase B? Is gelatinase A also mediating this clipping? What is the relevance for inflammatory and neoplastic diseases? Meanwhile, several answers have become available in the literature. We showed that the CXC chemokines connective tissue-activated peptides (CTAP-III), platelet factor 4 (PF-4), and growth-related open reading frame (GRO-␣) are degraded slowly, whereas the CC chemokines—regulated on activation, normal T expressed and secreted (RANTES), and

GELATINASE B AS A REGULATOR OF CYTOKINE AND CHEMOKINE FUNCTION Gelatinase B has been shown to clip many cytokines and chemokines. Although it is not surprising that a protease degrades cytokines and chemokines, it was a remarkable observation that specific cytokines are activated by MMPs. In particular, it has been shown that the proform of IL-1␤ is converted into the active cytokine [65]. After the purification of neutrophil gelatinase B to homogeneity, the activity on chemokine substrates was tested, and a positive feedback loop between IL-8 and gelatinase B (Fig. 5A) was discovered [18]. The major neutrophil chemoattractant, IL-8, is processed by gelatinase B at the aminoterminus at one particular site (P1⬘– P1⫽Ser-Ala). This results in a truncation variant with increased activity [IL-8(7-77)]. Because the intact IL-8 molecule possesses chemotactic activity, the clipping by gelatinase B into a more active form is an example of potentiation rather than activation. The major problem with proteolysis experiments is the purity of the enzyme and substrate. The clipping of IL-8 was observed with purified natural and recombinant IL-8. Furthermore, five types of control experiments were done to exclude the possibility that some minor contaminant (in the electrophoretical, pure, natural gelatinase B) might catalyze the conversion. Aspecific MMP inhibitors such as ethylenediaminetetraacetate (EDTA) and 1,10-phenanthroline blocked the conversion, whereas inhibitors of other protease classes had no effect on the clipping. The truncation was blocked completely 856

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Fig. 5. Positive feedback between IL-8 and gelatinase B. (A) IL-8, induced by infection or cytokines or produced by tumor cells, triggers neutrophil chemotaxis (⫽cell recruitment) and activation (⫽degranulation). This results in the release of gelatinase B, which converts IL-8(1-77) into IL-8(7-77). The latter is at least tenfold more potent and will result in further chemotaxis. Because tumor cells may have the capacity to produce IL-8 and gelatinase B, this clipping of IL-8 may occur initially, even in the absence of the neutrophil but will then lead to maximum neutrophil chemotaxis and activation immediately. (B) Effector levels of IL-8 potentiation by gelatinase B on neutrophils. At the left, a CXCR represents the IL-8 binding site. Triggering CXCRs by IL-8 binding results in the increase in intracellular calcium levels and the association and interaction of motor molecules. This leads to chemotaxis and release of granules with gelatinase B. At the right, the potentiation effect of the conversion of IL-8(1-77) into IL-8(7-77) by gelatinase B is indicated as the ratios IL-8(7-77)/IL-8(1-77) for the four indicated parameters of neutrophil functions [18].

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TABLE 2.

Chemokine Modification by Gelatinases

Gelatinase A (MMP-2) [66] Truncation of MCP-3(1-76) into MCP-3(5-76) Effects: Loss of activation of CCR1 and CCR2 and calcium release in THP-1 cells Antagonist of MCP-1 and MCP-3 Loss of chemotactic activity Dampening of inflammation No truncation of CC chemokines MCP-1, -2, or -4 Gelatinase B (MMP-9) [18] Truncation of IL-8(1-77) into IL-8(7-77) Effects: Increased binding on neutrophil IL-8 receptors Increase in [Ca2⫹]i signaling in neutrophils Increased gelatinase B release from neutrophils More effective neutrophil chemotaxis Increased binding to and activation of CXCR1 and CXCR2 transfectants Degradation of CTAP-III, GRO-␣, and PF-4 No truncation of CC chemokines MCP-1, MCP-2, MCP-3, MCP4, or RANTES [18, 66]

monocyte chemoattractant proten (MCP)-2—are not digested [18]. In another study [66], it was shown that recombinant gelatinase A, but not gelatinase B, cleaves MCP-3 selectively at the aminoterminus into an antagonist. In addition, gelatinase A did not cleave the other CC chemokines of the MCP subfamily, including MCP-1, MCP-2, and MCP-4 (Table 2). Degradation of chemokines by gelatinases A and B may thus lead to negative-feedback mechanisms. In the case of MCP-3, which is a chemoattractant for various types of mononuclear leukocytes [67, 68], the aminoterminus is crucial for biological activity [69, 70], and clipping leads to dampening the inflammatory response [66]. In neutrophil biology, a completely different context exists, which depends on positive feedback. Human neutrophils are chemoattracted and activated by IL-8, and this results in gelatinase-B release (Fig. 4). Gelatinase B then truncates specific IL-8 variants into the IL-8(7-77) variant, which is at least tenfold more active (Fig. 5). This results in an efficient amplification of neutrophil influx to combat infections. Gelatinase B is thus not only an effector but also a regulator of leukocyte function. In addition, gelatinase B degrades serine protease inhibitors [43] and has a regulatory effect on other members of the protease cascade. All these functions of gelatinase B result in pro-inflammatory effects. For inflammatory diseases, the role of the interaction between IL-8 and gelatinase B is clear, but this interaction also has consequences for neoplastic diseases [57]. We postulated the countercurrent principle of cancer-cell invasion [71] on the basis of studies of chemokine expression by tumor cells. In this model, chemokine-attracted inflammatory cells—the so-called tumorassociated leukocytes—assist in invasion and metastasis by the production of matrix-degrading enzymes [72]. A recent study [73] is in line with this concept and indicates that chemokines (e.g., IL-8) and inflammatory cells, including neutrophils and proteases such as gelatinase B, are also key players in tumor biology. Thus, our finding that gelatinase B potentiates IL-8 activity [18] is also important for tumor biology and suggests that anti-inflammatory drugs that target neutro-

phils or other gelatinase B-producing cells may be beneficial in the therapy of invasive cancers.

ACKNOWLEDGMENTS The present study was supported by the Fund for Scientific Research (FWO-Vlaanderen), Charcot Foundation, Belgian Federation against Cancer, and Cancer Research Foundation of Fortis Insurances AB, Belgium. P. E. V. d. S., B. D., and P. P. hold fellowships from FWO-Vlaanderen, and I. N. is a doctoral fellow of the Foundation for Research on Multiple Sclerosis. The authors thank Dr. Pauline Rudd and Prof. Raymond Dwek (University of Oxford) and Dr. Bernd Arnold (Deutsches Krebsforschungszentrum, Heidelberg) for many years of outstanding collaborations and two reviewers for constructive criticisms. This work is dedicated to Prof. A. Billiau (Leuven, Belgium), Prof. H. Teuchy, and Prof. M. Van Poucke (Diepenbeek, Belgium).

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