Brain Advance Access originally published online on April 8, 2003
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Brain, Vol. 126, No. 6, 1371-1381,
June 2003
© 2003 Guarantors of Brain
doi: 10.1093/brain/awg129
Gelatinase B/matrix metalloproteinase-9 cleaves interferon-ß and is a target for immunotherapy
Rega Institute for Medical Research, Laboratories of Molecular Immunology and Immunobiology, University of Leuven, Leuven, Belgium
Correspondence to: Professor Ghislain Opdenakker, MD, PhD, Rega Institute for Medical Research, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium E-mail: ghislain.opdenakker{at}rega.kuleuven.ac.be
Received October 9, 2002. Revised January 16, 2003. Accepted January 16, 2003.
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Parenteral administration of interferon (IFN)-ß is one of the currently approved therapies for multiple sclerosis. One characteristic of this disease is the increased production of gelatinase B, also called matrix metalloproteinase (MMP) 9. Gelatinase B is capable of destroying the blood–brain barrier, and of cleaving myelin basic protein into immunodominant and encephalitogenic fragments, thus playing a functional role and being a therapeutic target in multiple sclerosis. Here we demonstrate that gelatinase B proteolytically cleaves IFN-ß, kills its activity, and hence counteracts this cytokine as an antiviral and immunotherapeutic agent. This proteolysis is more pronounced with IFN-ß-1b than with IFN-ß-1a. Furthermore, the tetracycline minocycline, which has a known blocking effect in experimental autoimmune encephalomyelitis, an in vivo model of acute inflammation in multiple sclerosis, and other MMP inhibitors prevent the in vitro degradation of IFN-ß by gelatinase B. These data provide a novel mechanism and rationale for the inhibition of gelatinase B in diseases in which IFN-ß has a beneficial effect. The combination of gelatinase B inhibitors with better and lower pharmacological formulations of IFN-ß may reduce the side-effects of treatment with IFN-ß, and is therefore proposed for multiple sclerosis therapy and the immunotherapy of viral infections.
Keywords: gelatinase B; interferon; multiple sclerosis; inflammation; viral infection
Abbreviations: CHO = Chinese hamster ovary; CPE = cytopathogenic effect; EAE = experimental autoimmune encephalomyelitis; EDTA = ethylenediaminetetraacetic acid; HSA = human serum albumin; IFN = interferon; MMP = matrix metalloproteinase; SDS–PAGE = sodium dodecyl sulphate–polyacrylamide gel electrophoresis
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Introduction |
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Currently, recombinant interferon (IFN)-ß is an approved treatment for multiple sclerosis (IFNB Multiple Sclerosis Study Group, 1993
; Jacobs et al., 1996; Durelli et al., 2002), but its mechanism of action in vivo is not understood. Natural human IFN-ß is an antiviral cytokine that was purified for clinical use from human diploid fibroblasts and osteosarcoma cells at the Rega Institute (Billiau et al., 1977). Early studies were directed to the determination of its efficacy as a broad-spectrum antiviral agent, but the list of diseases treated with IFN-ß is rather limited and less extensive in comparison with that for IFN-
(Baron et al., 1991; Gutterman, 1994). In contrast to the -IFNs, which are encoded by a multigene family, human IFN-ß is a glycoprotein derived from a single-copy gene. Hence, any recombinant preparation of human IFN-ß is biochemically different from the ensemble of natural glycoforms (Opdenakker et al., 1995). As soon as the human cDNA of IFN-ß was cloned and expressed (Derynck et al., 1980), large-scale production of recombinant IFN-ß was started and it became possible to perform more clinical studies. Many clinical trials have studied the successful treatment of relapsing-remitting multiple sclerosis with recombinant IFN-ß. The frequency of relapses and disease activity, as measured by MRI, has been shown to be reduced after parenteral administration of rather high doses of either the recombinant aglycosyl IFN-ß from Escherichia coli or the glycosylated variant expressed in Chinese hamster ovary (CHO) cells (IFNB Multiple Sclerosis Study Group, 1993; Jacobs et al., 1996; Durelli et al., 2002). However, these treatment regimens result in side-effects such as fever, fatigue, nausea and flu-like symptoms, and with the present pharmacological formulations patients may develop neutralizing antibodies (Durelli et al., 2002; Scagnolari et al., 2002).
The expression of gelatinase B, also called matrix metalloproteinase (MMP)-9, has been associated with infections and autoimmune diseases, including multiple sclerosis and rheumatoid arthritis (Opdenakker and Van Damme, 1994; Opdenakker et al., 2001; Van den Steen et al., 2002). In multiple sclerosis and its animal models, experimental autoimmune encephalomyelitis (EAE), biochemical, biological and in vivo evidence has been found for the claim that gelatinase B is disease-promoting (Opdenakker and Van Damme, 1994; Dubois et al., 1999; Yong et al., 2001). Gelatinase B is increased in the serum and CSF of multiple sclerosis patients (Gijbels et al., 1992; Paemen et al., 1994; Lee et al., 1999), and cleaves human myelin basic protein into encephalitogenic and immunodominant peptides (Proost et al., 1993). In multiple sclerosis, gelatinase B contributes to the destruction of the blood–brain barrier (Mun-Bryce and Rosenberg, 1998; Lukes et al., 1999), and further regulates the inflammatory response by activating or destroying chemokines and cytokines (Schönbeck et al., 1998; Van den Steen et al., 2000), and by assisting the in vivo migration of leucocytes to sites of inflammation under the influence of chemotactic gradients (D’Haese et al., 2000; Opdenakker et al., 2001). The net activity of the ensemble of MMPs and their inhibitors always results from a subtle balance (Yong et al., 2001), but accumulating evidence favours the notion that MMP-9 may rather function as a molecular target in multiple sclerosis (Opdenakker and Van Damme, 1994; Opdenakker et al., 2001).
IFN-ß has been shown to downregulate the expression of gelatinase B protein (Leppert et al., 1996; Stüve et al., 1996; Nelissen et al., 2002; Bauvois et al., 2002) and mRNA (Galboiz et al., 2001), and to upregulate the levels of its physiological inhibitor, tissue inhibitor of metalloproteinase-1 (Özenci et al., 2000; Waubant et al., 2001). It seems logical that such changes in expression levels of enzymes and enzyme inhibitors should lead to alterations in net activity, but this suggested mechanism of action has not yet been proven in vivo. In fact, little is known about the tissue distribution of administered versus endogenous IFN-ß, and it is not yet clear whether protease inhibition affects multiple sclerosis in the CNS or in other tissues, such as the bone marrow. Only recently, it was demonstrated in vitro with the use of human dendritic cell cultures that IFN-ß is indeed capable of downregulating the net activity of gelatinase A and B that results from the balance between proteases and inhibitors (Bartholomé et al., 2001). It remains difficult to demonstrate whether such a net effect is also evident in the IFN-ß-treated multiple sclerosis patient. Equally unknown and relevant is the effect of gelatinase B on IFN-ß itself. Here we prove that gelatinase B cleaves IFN-ß, and show how this affects its biological activity and how such destruction can be prevented in vitro. As gelatinase B inhibitors we used the classical MMP inhibitor ethylenediaminetetraacetic acid (EDTA) and the therapeutically applicable minocycline (Brundula et al., 2002; Popovic et al., 2002). Out of a series of more than 20 chemically modified tetracyclines, minocycline has previously been found to be a potent MMP-9 inhibitor at high doses (Paemen et al., 1996).
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Interferon-ß and gelatinase B preparations
IFN-ß preparations were purchased from commercial sources. Thirty-microgram amounts (6 MIU) of IFN-ß-1a (Avonex®; Biogen, Hoofddorp, The Netherlands), expressed in CHO cells, were reconstituted in the supplied solvent [human serum albumin (HSA), NaH2PO4, Na2HPO4, NaCl], according to the instructions of the manufacturer. Recombinant bacterial IFN-ß-1b (Betaferon®; Schering, Berlin, Germany) was reconstituted in doses of 250 µg (8 MIU) in the supplied 1.25% (w/v) glucose solution, containing 1.25% (w/v) HSA. These IFN-ß preparations were brought to electrophoretic purity by gel filtration chromatography on Superdex 200 HR 16/60 (Amersham Biosciences, Uppsala, Sweden) in chromatography buffer (10 mM CaCl2, 100 mM NaCl, 100 mM Tris, pH 7.4) at 4°C. Fractions of 1 ml were collected at a flow rate of 0.5 ml/min. The column had been calibrated previously with a mixture of protein standards [apoferritin (443 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), desoxyribonuclease I (31 kDa), cytochrome c (12.4 kDa), aprotinin (6.5 kDa), vitamin B12 (1.35 kDa)]. The total protein concentration of the different IFN-ß fractions was determined by the Bradford Coomassie Brilliant Blue method (Bio-Rad, Hercules, CA, USA) using bovine serum albumin as a standard. The gelatinase B preparation that was used for the digestion of human IFN-ß was purified to homogeneity from human neutrophils and activated with 4-aminophenylmercuric acetate-activated stromelysin-1 (molar stromelysin-1 : gelatinase B ratio, 1 : 100), as described previously (Van den Steen et al., 2000
).
Digestion of IFN-ß by gelatinase B
IFN-ß was incubated with the stromelysin-1-activated gelatinase B or with an equivalent amount of stromelysin-1 in digestion buffer (10 mM CaCl2, 100 mM NaCl, 100 mM Tris–HCl, pH 7.4) as a control, as detailed previously for other substrates (Van den Steen et al., 2000). The incubations were performed at 37°C for various time intervals. Control experiments with gelatinase B inhibitors were done in the presence of activated gelatinase B and an inhibitor (EDTA or minocycline).
Analysis of IFN-ß cleavage
To assess the extent of proteolysis of IFN-ß by gelatinase B and the ability of different inhibitors of gelatinase activity to prevent this fragmentation, sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) was used. After digestion, 
200 ng of IFN-ß was diluted in 2x loading buffer [125 mM Tris–HCl, pH 6.8, 4% (w/v) sodium dodecyl sulphate, 20% (v/v) glycerol, bromophenol blue] containing 10% (v/v) ß-mercaptoethanol, and incubated at 95°C for 5 min to destroy the disulphide bonds by chemical reduction. When non-reducing conditions were required, samples were diluted in 2x loading buffer without ß-mercaptoethanol. Proteins were resolved on 15% Tris–glycine mini-gels under denaturing conditions with the use of Tris–glycine buffer (25 mM Tris, 192 mM glycine, pH 8.3), and protein bands on the gels were detected by silver staining (SilverQuestTM Silver Staining Kit; Invitrogen, Groningen, The Netherlands). For molecular weight calibration, a wide-range polypeptide standard mixture was included in the gels (Mark 12TM; Invitrogen). The proportion of degradation products relative to intact IFN-ß was measured semiquantitatively using scanning densitometry (Masure et al., 1990).
To detect the IFN-ß fragments in the digestion mixture more specifically, western blot analysis was performed. After reduction and denaturation, 20 ng of IFN-ß was electrophoresed on a 15% Tris–glycine gel and transferred onto a polyvinylidene difluoride membrane (ProBlottTM; Applied Biosystems, Foster City, CA, USA) at room temperature in a semi-dry blotting apparatus (Bio-Rad) using transfer buffer [50 mM Tris, pH 9.2, 40 mM glycine, 0.038% (w/v) sodium dodecyl sulphate and 20% (v/v) methanol]. Protein bands were detected by standard procedures on the blots using a goat anti-human IFN-ß antibody (R&D Systems, Minneapolis, MN, USA) and a secondary donkey anti-goat horseradish peroxidase-conjugated immunoglobulin G (Santa Cruz Biotechnologies, Santa Cruz, CA, USA). The blots were developed by the enhanced chemiluminescence method according to the manufacturer’s protocol (ECL Plus Western Blotting Detection Reagents; Amersham Bio sciences) and exposed to autoradiographic film (Kodak BioMax MR; Eastman Kodak Company, Rochester, NY, USA). To control the transfer of the proteins from the gel to the membrane and to standardize the molecular weight of the observed protein signals on the film, a prestained multicoloured protein marker was included in each gel (ProSieve® Color Protein Marker; Cambrex, East Rutherford, NJ, USA).
Site-specificity of IFN-ß cleavage
For the identification of cleavage sites in IFN-ß-1b, a purified monomer fraction of the cytokine was digested with activated gelatinase B. The resulting mixture was reduced with 1% ß-mercaptoethanol, desalted using C18 ZIPTIPs (Millipore, Bedford, MA, USA), and subsequently analysed by mass spectrometry on a quadrupole time-of-flight apparatus (QTOF-II; Micromass, Manchester, UK). Specific peptides were identified by comparison of the measured masses with the theoretical masses of all possible peptides from IFN-ß using the Biolynx software (Micromass). The identity of these peptides was confirmed by peptide sequencing using fragmentation analysis (tandem mass spectrometry) on the same mass spectrometer. Confirmation of the intact IFN-ß sequence was done using Edman degradation on a Procise-cLC sequencer (Applied Biosystems). Furthermore, the intact protein and large fragments resulting from digestion with gelatinase B were identified using the combination of reversed-phase high-pressure liquid chromatography and mass spectrometry. The samples were loaded on a PepMapTM C18 column (300 µm inside diameter x 15 cm; FusicaTM II; LC Packings, Amsterdam, The Netherlands) with precolumn flow splitter (Acurate; LC Packings) in 0.1% trifluoroacetic acid. Subsequently, they were eluted in a gradient of 0.1% acetic acid and 80% acetonitrile in 0.1% acetic acid, monitored online and analysed on an electrospray Ion Trap mass spectrometer at a flow rate of 3 µl per minute (Esquire-LC; Brüker, Bremen, Germany).
Antiviral activity assays
The biological activities of the recombinant IFNs were titrated in classical antiviral cytopathogenic effect (CPE) inhibition assays (Armstrong, 1981). Epidermoid larynx carcinoma CCL23 (Hep-2) cells were plated into 96-well plates and serial dilutions of the IFN-ß test samples were added. Approximately 24 h later, the cells were challenged with vesicular stomatitis virus. The IFN-ß concentration conferring 50% protection from viral killing was assessed after 1 day by staining the cells with crystal violet solution (0.5% w/v crystal violet, 1.75% v/v formaldehyde, 50% v/v denatured ethanol, 0.85% w/v NaCl), followed by macroscopic evaluation of viable cells. Titres in different tests were determined by side-by-side titration with a stable laboratory standard, which had previously been calibrated by comparison with the international human fibroblast IFN-ß standard. During the course of this study, vesicular stomatitis virus was banned because of biosafety regulations. Subsequently, a number of antiviral IFN-ß assays were therefore done with Mengo virus (Picornaviridae) as a challenge.
Molecular modelling
To assign the cleavage sites on the IFN-ß molecule, the crystal structure data for the dimer (Karpusas et al., 1997) were used to generate a monomer, which was rotated so as to maximize the view of positions where gelatinase B cleaves. The P1' positions, before which the cleavages occurred, were indicated manually in green using the Vector NTI Suite v. 6.0 software (InforMax, Oxford, UK).
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Human gelatinase B cleaves human IFN-ß
Two commercial preparations of recombinant human IFN-ß, which are currently used for the treatment of multiple sclerosis patients, were employed in this study. Recombinant IFN-ß-1a (Avonex) is expressed in CHO cells, possesses the same amino acid sequence as natural human IFN-ß and is glycosylated at the asparagine residue on position 80. The IFN-ß-1b preparation (Betaferon) is produced by expression in E. coli, and hence possesses no asparagine-linked sugar, contains a cysteine-to-serine mutation at residue 17, and lacks the amino-terminal methionine. Both IFN-ß preparations were purified by gel filtration chromatography from reconstituted samples. This purification step was performed in the absence of detergent to mimic physiological conditions. A considerable amount of HSA (eluting between 70 and 80 ml), which was added as a stabilizing protein, was observed in the elution profile of both IFN-ß preparations (Figs 1A and 2A). As noticed previously (Runkel et al., 1998
), the IFN-ß-1b preparation was confirmed to contain a considerable fraction of large, soluble aggregates (at 45 ml; Fig. 1A). These appeared to be mainly non-covalently bound, since this fraction, which eluted as high molecular weight material from the gel filtration column, migrated mainly as monomeric IFN-ß protein under non-reducing conditions, as observed by SDS–PAGE (Fig. 1B) and western blot (Fig. 1C) analysis. Only a minor proportion of IFN-ß-1b monomers (at 91 and 93 ml) was observed, alongside a large fraction (at 101 and 107 ml) that eluted in the chromatography at low molecular mass (<10 kDa), but was confirmed to consist of monomeric IFN-ß-1b by western blot analysis. This fraction was therefore assumed to be retarded on the chromatography column through interactions with the column matrix, which were probably more likely to occur after removal of HSA, and to contain mainly monomers. The latter supposition was confirmed by a second gel filtration analysis of the aggregated IFN-ß-1b fraction (at 45 ml) that was collected after the first purification step. This fraction eluted again almost entirely at 45 ml, and only small amounts of HSA (at 72 ml) and retarded monomers (at 108 ml) were observed (data not shown). In contrast to the IFN-ß-1b preparation, the IFN-ß-1a product contained a much smaller proportion of aggregated protein (at 44 ml; Fig. 2A, B and C). Moreover, a relatively larger fraction of monomeric IFN-ß-1a (at 91 and 93 ml) was present, which was observed under non-reducing conditions to be in equilibrium with its dimeric form and a minority of oligomers (Fig. 2B and C). In this preparation too, a large fraction of the mixture of monomers and dimers showed retarded elution (at 107 ml). Analysis by SDS–PAGE after reduction with ß-mercaptoethanol showed that monomeric IFN-ß produced by CHO cells exists as different glycosylated isoforms (glycoforms) and has an apparent molecular weight of 22 kDa (Fig. 2B), whereas the non-glycosylated form of the protein from E. coli migrates as a single 18.5 kDa protein band (Fig. 1B; see also Fig. 4). Based on the purity and abundance of IFN-ß in the gel filtration fractions described above, we selectively chose fractions between 100 and 110 ml of the IFN-ß-1b preparation, and between 90 and 95 ml of the IFN-ß-1a product to perform digestion experiments with gelatinase B.
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We then evaluated whether IFN-ß is proteolysed by gelatinase B and whether, depending on the source of recombinant cytokine, the fragmentation pattern is different. Indeed, the amino acid sequences of Avonex and Betaferon are not identical, and these IFN-ß forms also differ in glycosylation. Therefore, the most abundant species of chromatographically pure IFN-ß from both commercial preparations were compared. IFN-ß-1b and IFN-ß-1a fractions were digested with stromelysin-1-activated human neutrophil gelatinase B and analysed by reducing SDS–PAGE. Both forms were cleaved by gelatinase B, lending support to the view that the endogenous human IFN-ß glycoforms are also substrates. Time-kinetic studies (Fig. 3A and B) showed that IFN-ß was degraded gradually, but incompletely. We noticed a stepwise digestion of IFN-ß-1b. A fast first-step clipping resulted in a truncated molecule, whereas after longer incubation intervals IFN-ß-1b was further degraded (Fig. 3A). Full-length IFN-ß-1a was observed to disappear almost completely when fresh enzyme was added to the digestion reaction after a first 24 h incubation period, and the reaction was prolonged for another 24 h (Fig. 3C). However, whereas purified IFN-ß-1b monomers were already fragmented at a molar substrate : enzyme ratio of 10 : 1, cleavage of IFN-ß-1a monomer and dimer molecules required a 10-fold greater amount of gelatinase B (molar substrate : enzyme ratio, 1 : 1) to obtain a fragmentation pattern comparable to that of IFN-ß-1b (Fig. 3D). Furthermore, at any enzyme concentration tested, IFN-ß-1a appeared to be more resistant to degradation than bacterial IFN-ß-1b, since a larger proportion of IFN-ß-1a remained intact and fewer fragments were generated (Fig. 3A–D). For further comparison, IFN-ß-1b and IFN-ß-1a were cleaved by gelatinase B and analysed by western blot. These analyses (Fig. 4A and B) confirmed the generation of the cleavage products from both IFN-ß preparations and the fact that the cleavage was much more efficient with IFN-ß-1b than with IFN-ß-1a. From Fig. 4B, which shows the western blot analysis of the reaction mixture that is presented as stained proteins after SDS–PAGE in Fig. 3C, it becomes clear that the major digestion product of IFN-ß-1a gave rise to only a small shift to a lower molecular mass than that of the intact protein band. Only minor proportions of lower molecular weight fragments were generated. In addition, the clipping of IFN-ß was inhibited by the MMP inhibitors EDTA (20 mM) and minocycline at high concentrations (200 and 400 µg/ml; Fig. 5), substantiating the specificity of the enzymatic reaction. Inhibition by minocycline was dose-dependent, since concentrations of
100 µg/ml were not effective. In conclusion, human IFN-ß is cleaved by gelatinase B, and this enzyme destroys IFN-ß-1b much faster than IFN-ß-1a.
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Assignment of the cleavage sites in IFN-ß by gelatinase B
For the identification of cleavage sites in human IFN-ß, a sample of 4.5 µg of purified recombinant IFN-ß-1b was digested by activated gelatinase B at a molar sub strate : enzyme ratio of 10 : 1 for 24 h and the peptide fragments were determined by mass spectrometry analysis. Identification of specific IFN-ß peptides using tandem mass spectrometry revealed five gelatinase B fragmentation sites (Fig. 6A). In comparison with the consensus clipping sites of collagen II, a well-studied substrate of gelatinase B with efficient cleavages before hydrophobic residues (Van den Steen et al., 2002
), leucine was also most frequently detected at the P1' cleavage position in IFN-ß.
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As can be deduced from the previous SDS–PAGE results, the first clipping of IFN-ß by gelatinase B happens fast and yields the first-step cleavage product of the observed doublet in Figs 3A, B and 5. This cleavage product represents amino- or carboxy-terminal truncation of the intact IFN-ß by only a few amino acids. Based on our tandem mass spectrometry data, we suggest that this favoured truncation corresponds to cleavage after the amino-terminal (Met)-Ser-Tyr-Asn sequence, since the peptide Leu-Leu-Gly-Phe-Leu was identified most abundantly (Fig. 6B). Also, the complementary intact 162 amino acid carboxy-terminal fragment Leu-Leu-Gly-Phe-Leu-...-Gly-Tyr-Leu-Arg-Asn of IFN-ß-1b, resulting from the first-step cleavage, was the only one that was identified using a combination of liquid chromatography and mass spectrometry (Fig. 6C). A sample of non-digested IFN-ß-1b monomers was sequenced by automated Edman degradation to corroborate the intactness of the amino-terminus, which would confirm the possible occurrence of the observed cleavages close to the amino-terminus. The sequence obtained was Ser-Tyr-Asn-Leu-Leu-Gly-Phe-... (data not shown), and thus we excluded the possibility that the used expression system resulted in amino-terminal variants.
As a summary of our observations, Fig. 7 shows the 3D crystal structure of an IFN-ß monomer (Karpusas et al., 1997) with the indication where and how gelatinase B cleaves the IFN-ß-1b protein backbone (blue arrows). All cleavage sites are present in the helices A and C. Attempts to assign cleavage sites of gelatinase B in the glycosylated IFN-ß-1a were unsuccessful. Because of the high levels of enzyme that were required to obtain degradation (see above), no IFN-ß-1a fragments could be identified by mass spectrometry or Edman degradation analysis (data not shown). This is in line with the resistance of the glycosylated IFN-ß-1a against proteolysis and the glycobiological observation that sugars protect against proteolysis. To visualize this protective effect of the highly mobile oligosaccharides, Fig. 7 shows that the asparagine-linked major sugar of CHO-derived IFN-ß-1a has such a size and location that it can cover all accessible cleavage sites.
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Interferon-ß activity is destroyed by gelatinase B
The biological effect of the cleavage of IFN-ß by gelatinase B on IFN-responsive cells was studied in a classical IFN assay, i.e. the viral CPE reduction test. Compared with intact IFN-ß-1b with high specific antiviral activity, loss of IFN activity was observed in samples of gelatinase B-digested IFN-ß-1b monomers (Fig. 8A). Proteolytic clipping resulted in complete loss of biological activity, which was evidenced by a >30-fold reduction in bioactivity. Together with the protein sequencing data, this indicates that gelatinase B destroys IFN-ß structurally and kills its biological activity. We also evaluated the aggregates of IFN-ß that are abundantly present in the commercially available and clinically used IFN-ß-1b preparation. They constituted up to 60% of the IFN protein (Fig. 1A), in agreement with a previous study (Runkel et al., 1998
). The antiviral-specific activity of the aggregates was also abolished by treatment with active human gelatinase B (Fig. 8B). When the biological activity of IFN-ß-1a monomers and dimers from CHO cells (consequently glycosylated) was analysed in a CPE reduction assay after digestion with gelatinase B, only a three-fold reduction in antiviral potency compared with the intact fraction was detected. This is in accordance with the limited fragmentation observed (Fig. 8C).
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In multiple sclerosis (Gijbels et al., 1992
; Paemen et al., 1994; Lee et al., 1999) and in EAE animal models (Gijbels et al., 1993, 1994), gelatinase B levels are found to be increased in CSF and in plasma. Our present study shows that the effect of endogenous IFN-ß production and exogenous IFN-ß treatment may fade out in acute disease unless gelatinase B activity is inhibited. Such inhibition may be obtained with minocycline (Paemen et al., 1996). Recently, EAE development was indeed reversed by intraperitoneal administration of minocycline (Brundula et al., 2002; Popovic et al., 2002).
Our study was executed with exclusively human molecules to prevent eventual species-specific artefacts. It contains various elements with important consequences for the IFN-ß therapy of multiple sclerosis and other diseases with increased gelatinase B production. It contributes at a novel level to understanding of the interactions between cytokines and metalloproteinases. We show that gelatinase B, a key regulator and effector in autoimmune diseases, destroys bioactive IFN-ß. This cytokine is currently an approved treatment of multiple sclerosis. Our studies imply that in vivo activity of gelatinase B will influence the bioavailability of administered IFN-ß, and consequently its activity. We observed that the glycosylated IFN-ß-1a expressed in CHO cells was less susceptible to degradation by gelatinase B than the aglycosyl IFN-ß-1b from E. coli. This difference may be explained by enhanced stabilization of the IFN-ß-1a structure and possibly by less accessible cleavage sites, due to glycosylation at a single site, as shown previously for other cytokines (Opdenakker et al., 1995; Runkel et al., 1998) (Fig. 7). Alternatively, since IFN-ß-1a (in contrast to IFN-ß-1b) was observed by us and by others (Karpusas et al., 1998) to associate preferentially as a dimer, this dimerization may shield the potential cleavage sites from gelatinase B and make them less accessible.
Along with the HSA that is added as a stabilizer to the recombinant proteins, we also observed considerable amounts of protein aggregates in the clinically used preparation of IFN-ß-1b. These aggregates may be assembled predominantly through non-covalent interactions, as the presence of a large amount of monomeric IFN-ß in this gel-filtration chromatography fraction was observed. The tendency to aggregate was much lower in the IFN-ß-1a product, which is a consequence of shielding of solvent-exposed hydrophobic residues near the glycosylation site by the sugar (Karpusas et al., 1998). It was a side-observation that the preparation with fewer aggregates [IFN-ß-1a (Avonex) versus IFN-ß-1b (Betaferon)] was also recently shown to be less immunogenic (Durelli et al., 2002; Scagnolari et al., 2002). Therefore, the discussion about immunogenicity by differences in protein structure (absence of amino-terminal methionine, mutation of cysteine to serine) or in glycosylation needs to be extended to differences in aggregated forms and in altered susceptibility to proteolysis.
We demonstrated that the biochemical cleavage of IFN-ß by gelatinase B destroys the bioactivity of IFN-ß. Since gelatinase B levels have been found to be increased in CSF and plasma in multiple sclerosis (Gijbels et al., 1992; Paemen et al., 1994; Lee et al., 1999) and EAE animal models (Gijbels et al., 1993, 1994), the effect of endogenous IFN-ß production and exogenous IFN-ß treatment may thus fade out in acute disease, unless gelatinase B activity is inhibited. As we observed that IFN-ß inactivation by gelatinase B was prevented in vitro with minocycline, such effects may be obtained with gelatinase B inhibitors in vivo. Recently, EAE development has indeed been found to be reversed by intraperitoneal administration of minocycline (Brundula et al., 2002; Popovic et al., 2002). Minocycline is one of the most potent gelatinase B-inhibitory tetracyclines (Paemen et al., 1996) and can penetrate into the CNS (Yrjanheikki et al., 1999; Brundula et al., 2002). Whereas currently used therapies of multiple sclerosis (copolymer or IFN-ß) and most experimental drugs that inhibit EAE are administered parenterally, minocycline may be effective by the peroral route and has a long history of safety. These results are a stimulus to controlled studies of IFN-ß versus IFN-ß plus gelatinase B inhibitors in patients with multiple sclerosis, and other diseases. The rationales for this suggestion are now multiple. Increases in gelatinase B levels have been found to be associated with multiple sclerosis and other inflammatory or infectious diseases, and with EAE. Inhibitors of MMPs have been shown to possess beneficial effects in animal models of inflammatory CNS diseases (Gijbels et al., 1994; Hewson et al., 1995; Clements et al., 1997; for review see Cuzner and Opdenakker, 1999). Previously, we documented the mechanism of action of tetracyclines on gelatinase B activity (Paemen et al., 1996). As reported earlier, the combination of metacycline with D-penicillamine for the treatment of secondary progressive multiple sclerosis failed to result in clinical improvement, but it should be noted that patients under treatment with IFN-ß were excluded in this study (Dubois et al., 1998).
The novel direct link between IFN-ß and gelatinase B is superimposed on the findings that IFN-ß downregulates the production (Leppert et al., 1996; Stüve et al., 1996; Nelissen et al., 2002; Bauvois et al., 2002) and net bioactivity (Bartholomé et al., 2001) of gelatinase B. It is clear that protein substitution therapy (e.g. with IFN-ß) has to be evaluated within the context of all pathological changes and side-effects. In inflammatory diseases, various proteases are induced, for example CD26 (De Meester et al., 1999) and gelatinase B (Opdenakker et al., 2001), and these may modify the biological effects of cytokines and chemokines (Van den Steen et al., 2000). If such mechanisms are established, protease inhibition may be a logical and safe way to enhance the bioavailability of the therapeutic cytokine in specific diseases. Our study illustrates a novel physiopathological feedback mechanism and how metalloproteinase inhibition may be exploited in a beneficial way to enhance the treatment of multiple sclerosis by IFN-ß. Furthermore, cost-effective and peroral treatment with minocycline or other gelatinase B inhibitors, alone or in combination with lower doses of IFN-ß, deserves further evaluation.
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Acknowledgements |
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The authors thank Jean-Pierre Lenaerts for his excellent help with cell culture. The present study was supported by the Fund for Scientific Research (FWO-Vlaanderen), the Charcot Foundation of Belgium, the Geconcentreerde OnderzoeksActies (GOA) and Fortis AB, Belgium. I.N. is a doctoral fellow of the Foundation for Research on Multiple Sclerosis (WOMS vzw) and P.P. is a postdoctoral fellow of the Fund for Scientific Research (FWO-Vlaanderen). G.O. is a recipient of an International Grant from the British–Dutch consortium for research on multiple sclerosis and presents this work to Professor A. Billiau on the occasion of his retirement.
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References |
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