Journal of Neuroimmunology, 41 (1992)29-34
29
© 1992 Elsevier SciencePublishers B.V. All rights reserved 0165-5728/92/$05.00 JNI 02238
Gelatinase in the cerebrospinal fluid of patients with multiple sclerosis and other inflammatory neurological disorders K o e n r a a d Gijbels a, Stefan Masure a Herwig Carton b and Ghislain O p d e n a k k e r a Rega Institute, Leuven, Belgium, and h Department of Brain and Behavior Research, University of Leuven Medical School, Leuven, Belgium
(Received2 January 1992) (Revision received 13 April 1992) (Accepted 13 April 1992)
Key words: Multiplesclerosis; Demyelination;Gelatinase;Cerebrospinal fluid; Blood-brain barrier
Summary A substrate conversion assay was used to detect gelatinase activity in the cerebrospinal fluid (CSF) of patients with various neurological disorders. Two main forms of gelatinase with an apparent molecular mass of 65 and 85 kDa, respectively, could be discerned. The high molecular mass gelatinase was detectable only in samples of patients with multiple sclerosis or other inflammatory neurological disorders. A statistically significant correlation was found between the level of the 85-kDa gelatinase and the CSF cytosis. This protease could play a role in the process of demyelination and breakdown of the blood-brain barrier in certain neurological disorders, such as multiple sclerosis.
Introduction Multiple sclerosis (MS) is a chronic neurological disorder. In its active stages MS is characterized pathologically by breakdown of the blood-brain barrier (BBB), infiltration of inflammatory cells into the central nervous system (CNS) and demyelination. T ceils, specific for certain CNS antigens, have been implicated in the etiopathogenesis of these events (Khoury et al., 1990). However, T cells are probably not the only cells responsible for demyelination or for breakdown of the BBB. Evidence points to the macrophages present in the MS lesion as important effector cells, since they can be seen in close contact with the degenerating myelin sheet. Furthermore, macrophages can secrete lytic enzymes of diverse substrate specificity and have also been found to be able to engulf myelin fragments by phagocytosis (Raine, 1984). Proteases have since long been implicated in myelin degradation (Hallpike and Adams, 1969) with indirect evidence pointing to neutral proteases (Cammer et al., 1978; Cuzner et al., 1978; Alvord et al., 1979; Inuzuka et al,, 1987). Correspondence to." K. Gijbels, Rega Institute, Minderbroedersstraat
10, B-3000 Leuven,Belgium.
Gelatinases are neutral proteases, belonging to the group of the matrix metalloproteases. They are known to degrade denatured collagen, gelatin and some other extracellular matrix components (Matrisian, 1990). To investigate the possible role of gelatinases in the pathogenesis of demyelinating disease, the levels of gelatinases were measured in the cerebrospinal fluid (CSF) of MS patients and patients with other inflammatory neurological disorders, and the physicochemicai properties of the CSF enzymes were studied. Materials and Methods Patients and C S F sampling
In this study, CSF samples of 14 patients with definite or probable MS (clinically or laboratory-supported) were included (Poser et al., 1983). Inflammatory neurological disorders other than MS (OIND) included Guillain-Barr6 syndrome (two patients), herpes encephalitis (three patients), viral menigoencephalitis (two patients), viral meningitis (one patient) and bacterial meningitis (three patients), Samples from eight patients with disc herniations or spinal canal stenosis were grouped as non-inflammatory neurological disorder (NIND) controls. Further clinical and lab-
30 TABLE 1
and lgG concentrations were determined on the corresponding serum samples. The IgG index was determined using the formula of Tibbling and Link (Link and Tibbling, 1977; Tibbling et al., 1977).
Patient data Group
n
Mean age
(range)
M/F
MS Definite Probable OIND NIND
10 4 11 8
37.4 47 44.2 42.9
(23-55) (37-52) (21-60) (26-74)
4/6 1/3 7/4 4/4
Detection of gelatinase activity
Fourteen patients with MS and 19 with non-MS neurological disorders were grouped into MS, other inflammatory neurological disorders (OIND) and non-inflammatory neurological diseases (NIND). Range and m e a n of the ages in years and sex are indicated (M = male; F = female). T h e MS patients were grouped as 'definite' and 'probable' MS according to Poser et al. (1983).
oratory data of the patients in this study are shown in Tables 1 and 2. None of the patients received corticosteroids, ACTH or any other immunosuppressive therapy in the 6 months preceding CSF sampling. CSF samples were obtained by atraumatic lumbar puncture as a part of normal diagnostic procedures. Samples in the first part of the study were immediately aliquoted and frozen at -20°C. During the second part of the study, samples were first centrifuged to remove the cells, then aliquoted and stored at -70°C. These different sample treatments did not influence the results of the gelatinase assays. Furthermore, at the time of sampling, the following parameters were also determined on all the CSF samples studied: cytosis, formula, total protein, lgG and albumin concentrations. The presence of oligoclonal bands was detected using polyacrylamide gel isoelectric focusing and subsequent protein staining. Oligoclonal bands present in CSF were only reported as positive if they were absent in the corresponding serum samples. Serum albumin
Gelatinase activity in the CSF samples was determined by SDS/PAGE zymography according to a modification of the method of Heussen and Dowdle (Heussen and Dowdle, 1980; Masure et ai., 1990). Briefly, 25/xl of CSF was applied without prior denaturation on 7.5% (w/v) polyacrylamide gels to which 0.1% (w/v) gelatin (tissue culture grade, Sigma Chemical Company, St Louis, MO) was added and co-polymerised. Stacking gels were 5% (w/v) polyacrylamide and did not contain gelatin. The gels (20 cm x 20 cm x 0.15 cm) were run at 4°C for approximately 16 h at 100 V. After electrophoresis the gels were washed for 2 x 20 min (washing buffer: 50 mM Tris" HCi pH 7.5, 10 mM CaCI 2, 0.02% (w/v) NAN3, 2.5% (v/v) Triton X-100) to remove the SDS, then incubated at 37°C for 24 h (buffer as for the washing, but containing only 1% (v/v) Triton X-100) for development of the enzyme activity, stained with Coomassie brilliant blue R-250 and destained in methanol/acetic acid. Gelatinase activity was detected as unstained bands on a blue background. Quantitative determination of gelatinase activity was done by computerised image analysis through two-dimensional scanning densitometry. Gelatinase activity was expressed in arbitrary scanning units, representing the scanning area under the curves, which is an integration ratio that takes into account both brightness and width of the substrate lysis zone. A sample containing tumor cell-derived gelatinase was run in parallel as a positive control (Masure et al., 1990).
TABLE 2 Clinical data and CSF characteristics of the MS patient group Number
Class
Age
Sex
Protein (mg%)
IgG index
OCB
Cytosis (cells/ram 3)
Gelatinase ( h i g h / l o w tool. mass)
1.13 0.37 2.53 0.46 1.24 2.82 1.39 0.69 0.86
+
3
0.00
+ + + + + +
1 8 2 6 54 6 1 2
0.00 0.09 0.03 0.06 0.79 0.16 0.04 0.00
1
D
51
M
2 3 4 5 6 7 8 9
D D D D D D D D
26 49 55 38 23 31 29 40
F F M M F M F F
40 35 50 41 33 62 70 33 39
10
D
32
F
47
1.82
+
6
0.12
11 12 13 14
P P P P
52 52 47 37
M F F F
50 37 36 35
0.77 0.61 0.80 0.64
+ + + +
4 2 1 2
0.05 0.00 0.00 0.03
The CSF of ten patients with definitive (D) and four with probable (P) MS were analysed for biochemical and biological parameters, Protein content (rag%), the lgG index, the presence ( + ) or absence ( - ) of oligoclonal bands (OCB), the cytosis ( c e l l s / m m 3) and the ratio of high versus low molecular mass gelatinase are shown.
31
Inhibition of gelatinase-activity To assess the effect of proteinase inhibitors on the gelatinase activity present in the CSF, 5 /xl of CSF (showing the highest activity in the gelatinase assay) from an MS patient was run in multiplicate on an S D S / P A G E gel as described. After electrophoresis, the gel was sliced into the individual lanes and these slices were washed in the washing buffer. The individual slices were then kept for 24 h at 37°C in the incubation buffer containing the different proteinase inhibitors. The following inhibitors were tested at the indicated final concentrations: 1,10-phenanthroline, 5 mM; disodium-EDTA, 10 mM; soybean trypsin inhibitor, 100 ~ g / m l ; N-ethylmaleimide, 10 mM; aminomethylcyclohexane carboxylic acid, 500 / z g / m l (Sigma Chemical Company, St Louis, MO); phosphoramidon, 1 0 0 / z g / m l ; aprotinin, 2 / x g / m l ; pepstatin, 10 lzg/ml (Boehringer Mannheim, Mannheim, Germany). After incubation, the gel slices were stained and destained as described for the zymography procedure.
Statistical analysis Mann-Whitney U-test and linear regression analysis were used when appropriate for statistical analysis.
Results Gelatinase activity could be detected zymographically in all CSF samples tested. Two main forms of gelatinase were detectable: one with an apparent molecular mass of approximately 65 kDa and one with a molecular mass of approximately 85 kDa. The 65-kDa form was present in all samples. In contrast, the 85-kDa
form was only found in two of the eight N I N D samples, in nine of the 14 MS samples and in eight of the 11 O I N D samples tested. Since there existed differences in the final staining intensity of the zymographies, samples of the three groups of neurological disorders were run on the same gel to compare the values obtained for the different disease categories: six N I N D samples, nine definite MS samples and a selection of samples from several other inflammatory neurological disorders were included (see legend to Fig. 1). Most MS and O I N D samples scored positive for the presence of the higher molecular mass form of gelatinase, the bacterial meningitis CSF scoring highest. The CSF samples that did not contain detectable 85-kDa gelatinase were further analysed after concentration. All previously negatively scoring samples remained negative for the 85-kDa gelatinase when the equivalent of 50/zl of CSF was analysed (data not shown). As can be seen in Fig. 2, the levels of the 65-kDa form were in the same range for the three categories of diseases. However, for the 85-kDa form, there was a marked difference. Levels in the N I N D group did not reach higher than the lowest scoring detectable levels in the MS group, the highest level in the latter group being almost 20 times as high as the lowest. Values for the 85-kDa form in the MS group were also significantly higher than in the N I N D control group ( P < 0.05). The lowest detectable level in the O I N D group was three times as high as its counterpart in the MS group and the highest level two times as high as in the MS group. The ratio between the levels of activity of the 85-kDa form and the 65-kDa form was used to allow for comparison of results between different zymographies. The relationship between the gelatinase activity in the
Fig. 1. Gelatinases in the CSF of patients with various neurological disorders. Samples containing 25/~1 of CSF (obtained by lumbar puncture) were taken from patients with non-inflammatory neurological disorders (lanes 1-6), multiple sclerosis (lanes 7-9 and 11-16, control sample in lane 10) and other inflammatory neurological diseases (lanes 17-23). The latter group included samples from patients with Guillain-Barr6 syndrome (lanes 17 and 18), viral meningitis (lane 19), viral meningoencephalitis (lanes 20 and 21) and bacterial meningitis (lanes 22 and 23). After electrophoretic separation of the proteins, gelatinase activitywas developed by substrate zymography.Lane mw contains a molecular mass size marker and three proteins of this marker are indicated in kDa. The arrowheads locate the 85- and 65-kDa gelatinase species.
32
.~
104
8
[
88
.E
1000
85~
o
o o
65~
10o c m Q
0
...T
T
oooo
NIND
MS
OIND
Fig. 2. Quantification of gelatinases in CSF. Gelatinase activities were enzymatically determined (gel shown in Fig. 1) and expressed as arbitrary scanning units. For the different groups of neurological disorders, the 65-kDa (filled circles) and the 85-kDa (open circles) gelatinases are quantitated and averaged (cross-bar) where appropriate. The level of the 85-kDa gelatinase in the CSF from MS patients differed significantly from the level in the N1ND group (Mann-Whitney U-test; P < 0.05). (As the samples in the OIND group in this experiment were selected to have different diseases represented, these results were not included in the statistical analysis.)
CSF and the CSF-cytosis for each individual patient (all 14 MS patients and all 11 OIND patients) is shown in Fig. 3. A statistically significant correlation was found between CSF cytosis and ratios of 85-kDa/65kDa gelatinase levels (P < 0.05). The same relationship between CSF gelatinase levels and cytosis existed in samples that were centrifuged immediately after collection of the CSF as well as in those that were frozen before centrifugation. Table 2 reviews data on the MS patients in this study. The 85-kDa/65-kDa gelatinase ratio in the CSF was also correlated with CSF IgG index and with the CSF protein level.
Fig. 4. Inhibition of CSF gelatinase from an MS patient by various proteinase inhibitors. A sample of CSF, containing high 85-kDa gelatinase activity, from an MS patient (Fig. 1, lane 9; patient 6 in Table 2) was run in multiplicate in a zymography gel. Gelatinase activity was developed in the presence of inhibitors (at the indicated concentrations) of most classes of secreted proteinases. Residual activity was visualised by staining of the gelatin substrate. The arrowheads indicate the locations of the 85-kDa and 65-kDa gelatinase bands. E D T A and 1,10-phenathroline inhibited the gelatinase activity in the CSF sample, whereas N-ethylmaleimide caused only a partial inhibition. None of the other protease inhibitors caused any effect. NEM, N-ethylmaleimide (10 mM); PHEN, 1,10-phenanthroline (5 mM); EDTA, ethylene diamine tetraacetate disodium salt (10 raM); STI, soybean trypsin inhibitor (100 mg/ml); APROT, aprotinin (2 /xg/ml); PEPST, pepstatin (10 /zg/ml); PHOS, phosphoramidon (100 /xg/ml); AMCA, aminomethylcyclohexane carboxylic acid (500/~g/ml).
Figure 4 shows the effect of various proteinase inhibitors on the gelatinolytic activity in the CSF of one of the MS patients (no. 6 in Table 2). 1,10Phenanthroline and EDTA totally inhibited the gelatinase activity present in the CSF, whereas N-ethylmaleimide caused a partial inhibition. Both the 85-kDa and the 65-kDa form were equally inhibited. Soybean trypsin inhibitor, aprotinin, pepstatin, phosphoramidon and tranexamic acid did not exert a clear inhibitory effect.
1.5
Discussion
.Jo
1.2
,~
o.9
~
o.6
,~
0,3
o 0.0
!
,..!* ? ~- ......
oo ,
•
10
CSF
,
. ,,,,,,
........
100
~
1000
........
, 104
cytosi$ (cell$/ld)
Fig. 3. Relationship between gelatinase levels and cytosis in the CSF of patients with MS and other inflammatory neurological disorders. Gelatinase levels in the CSF of 14 patients with MS (stars) and 11 patients with other inflammatory neurological diseases (circles) were determined. To compare the results taken from different gels, the ratio of high/low molecular mass gelatinase levels was used. This ratio correlated with the CSF cytosis (linear regression analysis; P < 0.05). (It should be noticed that several datapoints represented in this graph are superimposed on each other.)
Gelatinolytic enzymes were detected in the CSF using a convenient substrate conversion assay. Two forms were observed: a 65-kDa gelatinase and an 85kDa gelatinase. The latter was only present in the CSF from patients with MS or other inflammatory neurological disorders. The 85-kDa gelatinase levels in the CSF of the MS group were significantly elevated when compared to the levels in the NIND group. The highest levels of high molecular mass gelatinase were attained in the CSF of patients with bacterial meningitis. In contrast, the 65-kDa gelatinase was detected in comparable levels in all CSF samples tested, including those of patients from the NIND control group. Hence, the 85-kDa gelatinase level in CSF may be a useful marker for inflammatory neurological disorders. These results are in accordance with a recent study on gelatinase activity in synovial fluids from arthritis patients (Opdenakker et al., 1991a).
33 The low (65 kDa) and high (85-91 kDa) molecular mass gelatinases are the products of two different genes. In most cell types examined, the 65-kDa gelatinase is constitutively produced, whereas production of the 85-kDa gelatinase is regulated by cytokines. Interleukin-1 (IL-1) induces the 85-kDa gelatinase in monocytes (Opdenakker et al., 1991a,b) and interleukin-8 stimulates neutrophils to release the enzyme (Masure et al., 1991), whereas interleukin-6 (IL-6) is a potent inducer of the natural inhibitor of metalloproteases, TIMP (tissue inhibitor of metalloproteinases) (Sato et al., 1990; Lotz and Guerne, 1991). It is worthwile to notice that both IL-6 and gelatinase are induced by the 'primary' cytokine IL-1 (Van Damme et al., 1987; Opdenakker et al., 1991a,b). IL-6 has been detected in the CSF during various inflammatory disorders of the CNS (Frei et al., 1988; Houssiau et al., 1988; Gijbels et al., 1990); however, not in the CSF of MS-patients (Hauser et al., 1990; Maimone et al., 1991), but production of IL-6 within the MS lesion is not excluded. In arthritic joint fluids, IL-6 and gelatinase levels were also correlated (Opdenakker et al., 1991a). From these observations, i.e. simultaneous production of both enzyme and the inducer of its inhibitor, one can infer a regulatory loop of possible importance in tissue remodelling (Masure and Opdenakker, 1989). It is as yet unclear which cells produce the variable high molecular mass gelatinase present in the CSF. Since the ratio of 85-kDa/65-kDa gelatinase levels in the CSF correlates with the CSF cytosis (Fig. 3), the enzyme is likely to be produced by cells within the CSF. For example, in the two samples, originating from patients with Guillain-Barr6 syndrome (lanes 17 and 18 in Fig. 1) there was no elevated cell count and the CSF was also devoid of 85-kDa gelatinase activity. In synovial fluids from arthritis patients, the high molecular mass form of the enzyme is presumably of neutrophilic origin (as judged by its molecular mass of 91 kDa, its molecular heterogeneity and by immunoprecipitation with specific antibodies) (Opdenakker et al., 1991a). The 85-kDa gelatinase in the CSF of MS patients probably has its origin in monocytes, as suggested by the observation that (i) all CSF samples tested contained monocytes and lymphocytes but no neutrophils, and (ii) the physicochemical properties of the enzyme (molecular mass of 85 kDa, co-migration with monocytic gelatinase and little molecular heterogeneity) were found to differ from the gelatinase of neutrophilic origin. These differences in molecular mass between neutrophilic and monocytic gelatinase were also apparent from the analysis of CSF samples of patients with bacterial meningitis (lanes 22 and 23 in Fig. 1), which contained predominantly neutrophils and yielded a 91-kDa gelatinase instead of the 85-kDa enzyme observed in CSF of MS patients. Direct proof, however, for the monocytic origin of the 85-kDa gelati-
nase in CSF of MS patients will require comparative demonstration of the presence of the enzyme in different CSF cells by means of immunohistochemistry. Unfortunately, antibodies that selectively recognize the 85-kDa gelatinase have sofar not been available. Detection of gelatinase mRNA by in situ hybridisation - albeit an indirect proof - - might be an alternative to define the gelatinase-producing cells. In the present study, the presence of gelatinases was only studied in the CSF. Although CSF is the most accessible way of sampling in the CNS, it may only represent 'the overflow' of the active lesions or altel-natively it may not represent what is going on inside the MS lesions. Therefore, further studies on post-mortem samples need to be done. We identified a specific neutral metalloproteinase, gelatinase, in the cell-free fraction of the CSF. This is in accordance with the observations that CSF from patients with inflammatory neurological disorders contains proteolytic activity for myelin basic protein (Cuzner et al., 1978) or whole myelin (Alvord et al., 1979; Inuzuka et al., 1987). Myelin, and especially the MBP fraction, is also degraded by neutral proteases secreted by stimulated macrophages (Cammer et al., 1978). This activity can be inhibited by EDTA, implying the activity of metalloproteases in this process. In our study, the gelatinase activity could be totally inhibited in the CSF from an MS patient by EDTA and 1,10-phenanthroline. Both products are chelators and inhibitors of metalloproteases. The partial activity of N-ethylmaleimide (a serine protease inhibitor) might indicate that the gelatinase in the CSF needs activation (by a serine protease) for full activity as described previously (Matrisian, 1990). The observation that EAE, an animal model of MS, can be suppressed by administration of inhibitors of serine proteinases, tranexamic acid (Brosnan et al., 1980) or camostat mesilate (Inuzuka et al., 1988) does not exclude a role for gelatinase in inflammatory demyelinating disorders, since both serine- and metalloproteases are interconnected in an enzyme cascade (Vaes, 1988). Gelatinase may contribute to the breakdown of the BBB seen in MS and in various other inflammatory neurological disorders. It is not certain whether gelatinase/type IV collagenase can digest native type IV collagen (Mackay et al., 1990), but the enzyme is certainly catalytically active on denatured basement membrane collagens (Matrisian, 1990). Gelatinase, purified to homogeneity (Masure et al., 1991), also cleaves intact MBP (manuscript in preparation). This activity might be of fundamental importance in the pathogenesis of MS as it has both structural (the desintegration of the myelin sheet) and immunological (the release of possible immunogenic MBP-fragments) implications. The in vivo inhibition of protease activity has been shown to suppress EAE (Brosnan et al., 1980; Inuzuka
34
et al., 1988). Hence the in vitro demonstration of gelatinase activity as well as the suppression of gelatinase activity in MS-CSF with specific inhibitors in the present study opens new perspectives for the elucidation of the pathogenesis and hopefully also for the design of an effective treatment of CNS inflammatory diseases and of MS in particular.
Acknowledgements Work in the authors' laboratory was supported by the Belgian National Fund for Scientific Research (NFWO) and by a special grant from the Lions Club (Belgium). The authors wish to thank Professor A. Billiau for support and critically reviewing the manuscript and the numerous assistants in the Department of Neurology for CSF collection. K.G. is a research assistant and G.O. is a research associate of the NFWO.
References Alvord, E.C., Hruby, S. and Sires, L.R. (1979) Degradation of myelin basic protein by cerebrospinal fluid: preservation of antigenic determinants under physiological conditions. Ann. Neurol. 6, 474-482. Brosnan, C.F., Cammer, W., Norton, W.T. and Bloom, B.R. (1980) Proteinase inhibitors suppress the development of experimental allergic encephalomyelitis. Nature 285, 235-237. Cammer, W., Bloom, B.R., Norton, W.T. and Gordon, S. (1978) Degradation of basic protein in myelin by neutral proteases secreted by stimulated macrophages: A possible mechanism of inflammatory demyelination. Proc. Natl. Acad. Sci. USA 75, 1554-1558. Cuzner, M.L., Davison, A.N. and Rudge, P. (1978) Proteolytic enzyme activity of blood leukocytes and cerebrospinal fluid in multiple sclerosis. Ann. Neurol. 4, 337-344. Frei, K., Leist, T.P., Meager, A., Gallo, P., Leppert, D., Zinkernagel, R.M. and Fontana, A. (1988) Production of B cell stimulatory factor-2 and interferon-gamma in the central nervous system during viral meningitis and encephalitis. J. Exp. Med. 168, 449453. Gijbels, K., Van Damme, J., Proost, P., Put, W., Carton, H. and Billiau, A. (1990) lnterleukin 6 production in the central nervous system during experimental autoimmune encephalomyelitis. Eur. J. Immunol. 20, 233-235. Hallpike, J.F. and Adams, C.W.M. (1969) Proteolysis and myelin breakdown: a review of recent histochemical and biochemical studies. Histochem. J. 1,559-578. Hauser, S.L., Doolittle, T.H., Lincoln, R., Brown, R.H. and Dinarello, C.A. (1990) Cytokine accumulations in CSF of multiple sclerosis patients: frequent detection of interleukin-1 and tumor necrosis factor but not interleukin-6. Neurology 40, 1735-1739. Heussen, C. and Dowdle, E.B. (1980) Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal. Biochem. 102, 196-202. Houssiau, F.A., Bukasa, K., Sindic, C.J.M., Van Damme, J. and Van Snick, J. (1988) Elevated levels of the 26K human hybridoma growth factor (interleukin-6) in cerebrospinal fluid of patients
with acute infection of the central nervous system. Clin. Exp. Immunol. 71,320-323. lnuzuka, T., Sato, S., Baba, H. and Miyatake, T. (1987) Neutral protease in cerebrospinal fluid from patients with multiple sclerosis and other neurological diseases. Acta Neurol. Scand. 76, 18-23. lnuzuka, T., Sato, S., Baba, H. and Miyatake, T. (1988) Suppressive effect of camostat mesilate (FOY 305) on acute experimental allergic encephalomyelitis (EAE). Neurochem. Res. 13, 225-228. Khoury, S.J., Weiner H.L. and Hailer, D.A. (1990) Immunologic basis of multiple sclerosis. In: S.D. Cook (Ed.), Handbook of Multiple Sclerosis, Marcel Dekker, New York, NY, pp. 129-149. Link, H. and Tibbling, G. (1977) Principles of albumin and lgG analysis in neurological disorders. II. Evaluation of lgG synthesis within the central nervous system in multiple sclerosis. Scand. J. Clin. Lab. Invest. 37, 397-401. Lotz, M. and Guerne, P.-A. (1991) Interleukin-6 induces the synthesis of tissue inhibitor of metalloproteinases-1/erythroid potentiating activity (TIMP-1/EPA). J. Biol. Chem. 266, 2017-2020. Mackay, A.R., Hartzler, J.L., Pelina, M.D. and Thorgeirsson, U.P. (1990) Studies on the ability of 65-kDa and 92-kDa tumor cell gelatinases to degrade type IV collagen. J. Biol. Chem. 265, 21929-21934. Maimone, D., Gregory, S., Arnason, B.G.W. and Reder, A.T. (1991) Cytokine levels in the cerebrospinal fluid and serum of patients with multiple sclerosis. J. Neuroimmunol. 32, 67-74. Masure, S. and Opdenakker, G. (1989) Cytokine-mediated proteolysis in tissue remodelling. Experientia 45, 542-549. Masure, S., Billiau, A., Van Damme, J. and Opdenakker, G. (1990) Human hepatoma cells produce an 85 kDa gelatinase regulated by phorbol 12-myristate 13-acetate. Biochim. Biophys. Acta 1054, 317-325. Masure, S., Proost, P., Van Damme, J. and Opdenakker, G. (1991) Purification and identification of 91-kDa neutrophil gelatinase. Release by the activating peptide interleukin-8. Eur. J. Biochem. 198, 391-398. Matrisian, L.M. (1990) Metalloproteinases and their inhibitors in matrix remodeling. Trends Genet. 6, 121-125. Opdenakker, G., Masure, S., Grillet, B. and Van Damme, J. (1991a) Cytokine-mediated regulation of human leukocyte gelatinases and role in arthritis. Lymphokine Cytokine Res. 10, 317-324. Opdenakker, G., Masure, S., Proost, P., Billiau, A. and Van Damme, J. (1991b) Natural human monocyte gelatinase and its inhibitor. FEBS Lett. 284, 73-78. Poser, C.M., Paty, D.W., Scheinberg, L., McDonald, W.I., Davis, F.A., Ebers, G.C., Johnson, K.P., Sibley, W.A., Silberberg, D.H. and Tourtellotte, W.W. (1983) New diagnostic criteria for multiple sclerosis: Guidelines for research protocols. Ann. Neurol. 13, 227-231. Raine, C.S. (1984) Analysis of autoimmune demyelination: Its impact upon multiple sclerosis. Lab. Invest. 50, 608-635. Sato, T., Ito, A. and Mori, Y. (1990) Interleukin 6 enhances the production of tissue inhibitor of metalloproteinases (TIMP) but not that of matrix metalloproteinases by human fibroblasts. Biochem. Biophys. Res. Commun. 170, 824-829. Tibbling, G., Link, H. and Ohman, S. (1977) Principles of albumin and lgG analysis in neurological disorders. I. Establishment of reference values. Scand. J. Clin. Lab. Invest. 37, 385-390. Vaes, G. (1988) Cellular biology and biochemical mechanism of bone resorption. A review of recent developments on the formation, activation and mode of action of osteoclasts. Clin. Ortop. 231, 239-271. Van Damme, J., Opdenakker, G., Simpson, R.J., Rubira, M.R., Cayphas, S., Vink, A., Billiau, A. and Van Snick, J. (1987) Identification of the human 26-kD protein, Interferon /32 (IFN/32), as a B cell hybridoma/plasmacytoma growth factor induced by interleukin 1 and tumor necrosis factor. J. Exp. Med. 165, 914-919.
29
© 1992 Elsevier SciencePublishers B.V. All rights reserved 0165-5728/92/$05.00 JNI 02238
Gelatinase in the cerebrospinal fluid of patients with multiple sclerosis and other inflammatory neurological disorders K o e n r a a d Gijbels a, Stefan Masure a Herwig Carton b and Ghislain O p d e n a k k e r a Rega Institute, Leuven, Belgium, and h Department of Brain and Behavior Research, University of Leuven Medical School, Leuven, Belgium
(Received2 January 1992) (Revision received 13 April 1992) (Accepted 13 April 1992)
Key words: Multiplesclerosis; Demyelination;Gelatinase;Cerebrospinal fluid; Blood-brain barrier
Summary A substrate conversion assay was used to detect gelatinase activity in the cerebrospinal fluid (CSF) of patients with various neurological disorders. Two main forms of gelatinase with an apparent molecular mass of 65 and 85 kDa, respectively, could be discerned. The high molecular mass gelatinase was detectable only in samples of patients with multiple sclerosis or other inflammatory neurological disorders. A statistically significant correlation was found between the level of the 85-kDa gelatinase and the CSF cytosis. This protease could play a role in the process of demyelination and breakdown of the blood-brain barrier in certain neurological disorders, such as multiple sclerosis.
Introduction Multiple sclerosis (MS) is a chronic neurological disorder. In its active stages MS is characterized pathologically by breakdown of the blood-brain barrier (BBB), infiltration of inflammatory cells into the central nervous system (CNS) and demyelination. T ceils, specific for certain CNS antigens, have been implicated in the etiopathogenesis of these events (Khoury et al., 1990). However, T cells are probably not the only cells responsible for demyelination or for breakdown of the BBB. Evidence points to the macrophages present in the MS lesion as important effector cells, since they can be seen in close contact with the degenerating myelin sheet. Furthermore, macrophages can secrete lytic enzymes of diverse substrate specificity and have also been found to be able to engulf myelin fragments by phagocytosis (Raine, 1984). Proteases have since long been implicated in myelin degradation (Hallpike and Adams, 1969) with indirect evidence pointing to neutral proteases (Cammer et al., 1978; Cuzner et al., 1978; Alvord et al., 1979; Inuzuka et al,, 1987). Correspondence to." K. Gijbels, Rega Institute, Minderbroedersstraat
10, B-3000 Leuven,Belgium.
Gelatinases are neutral proteases, belonging to the group of the matrix metalloproteases. They are known to degrade denatured collagen, gelatin and some other extracellular matrix components (Matrisian, 1990). To investigate the possible role of gelatinases in the pathogenesis of demyelinating disease, the levels of gelatinases were measured in the cerebrospinal fluid (CSF) of MS patients and patients with other inflammatory neurological disorders, and the physicochemicai properties of the CSF enzymes were studied. Materials and Methods Patients and C S F sampling
In this study, CSF samples of 14 patients with definite or probable MS (clinically or laboratory-supported) were included (Poser et al., 1983). Inflammatory neurological disorders other than MS (OIND) included Guillain-Barr6 syndrome (two patients), herpes encephalitis (three patients), viral menigoencephalitis (two patients), viral meningitis (one patient) and bacterial meningitis (three patients), Samples from eight patients with disc herniations or spinal canal stenosis were grouped as non-inflammatory neurological disorder (NIND) controls. Further clinical and lab-
30 TABLE 1
and lgG concentrations were determined on the corresponding serum samples. The IgG index was determined using the formula of Tibbling and Link (Link and Tibbling, 1977; Tibbling et al., 1977).
Patient data Group
n
Mean age
(range)
M/F
MS Definite Probable OIND NIND
10 4 11 8
37.4 47 44.2 42.9
(23-55) (37-52) (21-60) (26-74)
4/6 1/3 7/4 4/4
Detection of gelatinase activity
Fourteen patients with MS and 19 with non-MS neurological disorders were grouped into MS, other inflammatory neurological disorders (OIND) and non-inflammatory neurological diseases (NIND). Range and m e a n of the ages in years and sex are indicated (M = male; F = female). T h e MS patients were grouped as 'definite' and 'probable' MS according to Poser et al. (1983).
oratory data of the patients in this study are shown in Tables 1 and 2. None of the patients received corticosteroids, ACTH or any other immunosuppressive therapy in the 6 months preceding CSF sampling. CSF samples were obtained by atraumatic lumbar puncture as a part of normal diagnostic procedures. Samples in the first part of the study were immediately aliquoted and frozen at -20°C. During the second part of the study, samples were first centrifuged to remove the cells, then aliquoted and stored at -70°C. These different sample treatments did not influence the results of the gelatinase assays. Furthermore, at the time of sampling, the following parameters were also determined on all the CSF samples studied: cytosis, formula, total protein, lgG and albumin concentrations. The presence of oligoclonal bands was detected using polyacrylamide gel isoelectric focusing and subsequent protein staining. Oligoclonal bands present in CSF were only reported as positive if they were absent in the corresponding serum samples. Serum albumin
Gelatinase activity in the CSF samples was determined by SDS/PAGE zymography according to a modification of the method of Heussen and Dowdle (Heussen and Dowdle, 1980; Masure et ai., 1990). Briefly, 25/xl of CSF was applied without prior denaturation on 7.5% (w/v) polyacrylamide gels to which 0.1% (w/v) gelatin (tissue culture grade, Sigma Chemical Company, St Louis, MO) was added and co-polymerised. Stacking gels were 5% (w/v) polyacrylamide and did not contain gelatin. The gels (20 cm x 20 cm x 0.15 cm) were run at 4°C for approximately 16 h at 100 V. After electrophoresis the gels were washed for 2 x 20 min (washing buffer: 50 mM Tris" HCi pH 7.5, 10 mM CaCI 2, 0.02% (w/v) NAN3, 2.5% (v/v) Triton X-100) to remove the SDS, then incubated at 37°C for 24 h (buffer as for the washing, but containing only 1% (v/v) Triton X-100) for development of the enzyme activity, stained with Coomassie brilliant blue R-250 and destained in methanol/acetic acid. Gelatinase activity was detected as unstained bands on a blue background. Quantitative determination of gelatinase activity was done by computerised image analysis through two-dimensional scanning densitometry. Gelatinase activity was expressed in arbitrary scanning units, representing the scanning area under the curves, which is an integration ratio that takes into account both brightness and width of the substrate lysis zone. A sample containing tumor cell-derived gelatinase was run in parallel as a positive control (Masure et al., 1990).
TABLE 2 Clinical data and CSF characteristics of the MS patient group Number
Class
Age
Sex
Protein (mg%)
IgG index
OCB
Cytosis (cells/ram 3)
Gelatinase ( h i g h / l o w tool. mass)
1.13 0.37 2.53 0.46 1.24 2.82 1.39 0.69 0.86
+
3
0.00
+ + + + + +
1 8 2 6 54 6 1 2
0.00 0.09 0.03 0.06 0.79 0.16 0.04 0.00
1
D
51
M
2 3 4 5 6 7 8 9
D D D D D D D D
26 49 55 38 23 31 29 40
F F M M F M F F
40 35 50 41 33 62 70 33 39
10
D
32
F
47
1.82
+
6
0.12
11 12 13 14
P P P P
52 52 47 37
M F F F
50 37 36 35
0.77 0.61 0.80 0.64
+ + + +
4 2 1 2
0.05 0.00 0.00 0.03
The CSF of ten patients with definitive (D) and four with probable (P) MS were analysed for biochemical and biological parameters, Protein content (rag%), the lgG index, the presence ( + ) or absence ( - ) of oligoclonal bands (OCB), the cytosis ( c e l l s / m m 3) and the ratio of high versus low molecular mass gelatinase are shown.
31
Inhibition of gelatinase-activity To assess the effect of proteinase inhibitors on the gelatinase activity present in the CSF, 5 /xl of CSF (showing the highest activity in the gelatinase assay) from an MS patient was run in multiplicate on an S D S / P A G E gel as described. After electrophoresis, the gel was sliced into the individual lanes and these slices were washed in the washing buffer. The individual slices were then kept for 24 h at 37°C in the incubation buffer containing the different proteinase inhibitors. The following inhibitors were tested at the indicated final concentrations: 1,10-phenanthroline, 5 mM; disodium-EDTA, 10 mM; soybean trypsin inhibitor, 100 ~ g / m l ; N-ethylmaleimide, 10 mM; aminomethylcyclohexane carboxylic acid, 500 / z g / m l (Sigma Chemical Company, St Louis, MO); phosphoramidon, 1 0 0 / z g / m l ; aprotinin, 2 / x g / m l ; pepstatin, 10 lzg/ml (Boehringer Mannheim, Mannheim, Germany). After incubation, the gel slices were stained and destained as described for the zymography procedure.
Statistical analysis Mann-Whitney U-test and linear regression analysis were used when appropriate for statistical analysis.
Results Gelatinase activity could be detected zymographically in all CSF samples tested. Two main forms of gelatinase were detectable: one with an apparent molecular mass of approximately 65 kDa and one with a molecular mass of approximately 85 kDa. The 65-kDa form was present in all samples. In contrast, the 85-kDa
form was only found in two of the eight N I N D samples, in nine of the 14 MS samples and in eight of the 11 O I N D samples tested. Since there existed differences in the final staining intensity of the zymographies, samples of the three groups of neurological disorders were run on the same gel to compare the values obtained for the different disease categories: six N I N D samples, nine definite MS samples and a selection of samples from several other inflammatory neurological disorders were included (see legend to Fig. 1). Most MS and O I N D samples scored positive for the presence of the higher molecular mass form of gelatinase, the bacterial meningitis CSF scoring highest. The CSF samples that did not contain detectable 85-kDa gelatinase were further analysed after concentration. All previously negatively scoring samples remained negative for the 85-kDa gelatinase when the equivalent of 50/zl of CSF was analysed (data not shown). As can be seen in Fig. 2, the levels of the 65-kDa form were in the same range for the three categories of diseases. However, for the 85-kDa form, there was a marked difference. Levels in the N I N D group did not reach higher than the lowest scoring detectable levels in the MS group, the highest level in the latter group being almost 20 times as high as the lowest. Values for the 85-kDa form in the MS group were also significantly higher than in the N I N D control group ( P < 0.05). The lowest detectable level in the O I N D group was three times as high as its counterpart in the MS group and the highest level two times as high as in the MS group. The ratio between the levels of activity of the 85-kDa form and the 65-kDa form was used to allow for comparison of results between different zymographies. The relationship between the gelatinase activity in the
Fig. 1. Gelatinases in the CSF of patients with various neurological disorders. Samples containing 25/~1 of CSF (obtained by lumbar puncture) were taken from patients with non-inflammatory neurological disorders (lanes 1-6), multiple sclerosis (lanes 7-9 and 11-16, control sample in lane 10) and other inflammatory neurological diseases (lanes 17-23). The latter group included samples from patients with Guillain-Barr6 syndrome (lanes 17 and 18), viral meningitis (lane 19), viral meningoencephalitis (lanes 20 and 21) and bacterial meningitis (lanes 22 and 23). After electrophoretic separation of the proteins, gelatinase activitywas developed by substrate zymography.Lane mw contains a molecular mass size marker and three proteins of this marker are indicated in kDa. The arrowheads locate the 85- and 65-kDa gelatinase species.
32
.~
104
8
[
88
.E
1000
85~
o
o o
65~
10o c m Q
0
...T
T
oooo
NIND
MS
OIND
Fig. 2. Quantification of gelatinases in CSF. Gelatinase activities were enzymatically determined (gel shown in Fig. 1) and expressed as arbitrary scanning units. For the different groups of neurological disorders, the 65-kDa (filled circles) and the 85-kDa (open circles) gelatinases are quantitated and averaged (cross-bar) where appropriate. The level of the 85-kDa gelatinase in the CSF from MS patients differed significantly from the level in the N1ND group (Mann-Whitney U-test; P < 0.05). (As the samples in the OIND group in this experiment were selected to have different diseases represented, these results were not included in the statistical analysis.)
CSF and the CSF-cytosis for each individual patient (all 14 MS patients and all 11 OIND patients) is shown in Fig. 3. A statistically significant correlation was found between CSF cytosis and ratios of 85-kDa/65kDa gelatinase levels (P < 0.05). The same relationship between CSF gelatinase levels and cytosis existed in samples that were centrifuged immediately after collection of the CSF as well as in those that were frozen before centrifugation. Table 2 reviews data on the MS patients in this study. The 85-kDa/65-kDa gelatinase ratio in the CSF was also correlated with CSF IgG index and with the CSF protein level.
Fig. 4. Inhibition of CSF gelatinase from an MS patient by various proteinase inhibitors. A sample of CSF, containing high 85-kDa gelatinase activity, from an MS patient (Fig. 1, lane 9; patient 6 in Table 2) was run in multiplicate in a zymography gel. Gelatinase activity was developed in the presence of inhibitors (at the indicated concentrations) of most classes of secreted proteinases. Residual activity was visualised by staining of the gelatin substrate. The arrowheads indicate the locations of the 85-kDa and 65-kDa gelatinase bands. E D T A and 1,10-phenathroline inhibited the gelatinase activity in the CSF sample, whereas N-ethylmaleimide caused only a partial inhibition. None of the other protease inhibitors caused any effect. NEM, N-ethylmaleimide (10 mM); PHEN, 1,10-phenanthroline (5 mM); EDTA, ethylene diamine tetraacetate disodium salt (10 raM); STI, soybean trypsin inhibitor (100 mg/ml); APROT, aprotinin (2 /xg/ml); PEPST, pepstatin (10 /zg/ml); PHOS, phosphoramidon (100 /xg/ml); AMCA, aminomethylcyclohexane carboxylic acid (500/~g/ml).
Figure 4 shows the effect of various proteinase inhibitors on the gelatinolytic activity in the CSF of one of the MS patients (no. 6 in Table 2). 1,10Phenanthroline and EDTA totally inhibited the gelatinase activity present in the CSF, whereas N-ethylmaleimide caused a partial inhibition. Both the 85-kDa and the 65-kDa form were equally inhibited. Soybean trypsin inhibitor, aprotinin, pepstatin, phosphoramidon and tranexamic acid did not exert a clear inhibitory effect.
1.5
Discussion
.Jo
1.2
,~
o.9
~
o.6
,~
0,3
o 0.0
!
,..!* ? ~- ......
oo ,
•
10
CSF
,
. ,,,,,,
........
100
~
1000
........
, 104
cytosi$ (cell$/ld)
Fig. 3. Relationship between gelatinase levels and cytosis in the CSF of patients with MS and other inflammatory neurological disorders. Gelatinase levels in the CSF of 14 patients with MS (stars) and 11 patients with other inflammatory neurological diseases (circles) were determined. To compare the results taken from different gels, the ratio of high/low molecular mass gelatinase levels was used. This ratio correlated with the CSF cytosis (linear regression analysis; P < 0.05). (It should be noticed that several datapoints represented in this graph are superimposed on each other.)
Gelatinolytic enzymes were detected in the CSF using a convenient substrate conversion assay. Two forms were observed: a 65-kDa gelatinase and an 85kDa gelatinase. The latter was only present in the CSF from patients with MS or other inflammatory neurological disorders. The 85-kDa gelatinase levels in the CSF of the MS group were significantly elevated when compared to the levels in the NIND group. The highest levels of high molecular mass gelatinase were attained in the CSF of patients with bacterial meningitis. In contrast, the 65-kDa gelatinase was detected in comparable levels in all CSF samples tested, including those of patients from the NIND control group. Hence, the 85-kDa gelatinase level in CSF may be a useful marker for inflammatory neurological disorders. These results are in accordance with a recent study on gelatinase activity in synovial fluids from arthritis patients (Opdenakker et al., 1991a).
33 The low (65 kDa) and high (85-91 kDa) molecular mass gelatinases are the products of two different genes. In most cell types examined, the 65-kDa gelatinase is constitutively produced, whereas production of the 85-kDa gelatinase is regulated by cytokines. Interleukin-1 (IL-1) induces the 85-kDa gelatinase in monocytes (Opdenakker et al., 1991a,b) and interleukin-8 stimulates neutrophils to release the enzyme (Masure et al., 1991), whereas interleukin-6 (IL-6) is a potent inducer of the natural inhibitor of metalloproteases, TIMP (tissue inhibitor of metalloproteinases) (Sato et al., 1990; Lotz and Guerne, 1991). It is worthwile to notice that both IL-6 and gelatinase are induced by the 'primary' cytokine IL-1 (Van Damme et al., 1987; Opdenakker et al., 1991a,b). IL-6 has been detected in the CSF during various inflammatory disorders of the CNS (Frei et al., 1988; Houssiau et al., 1988; Gijbels et al., 1990); however, not in the CSF of MS-patients (Hauser et al., 1990; Maimone et al., 1991), but production of IL-6 within the MS lesion is not excluded. In arthritic joint fluids, IL-6 and gelatinase levels were also correlated (Opdenakker et al., 1991a). From these observations, i.e. simultaneous production of both enzyme and the inducer of its inhibitor, one can infer a regulatory loop of possible importance in tissue remodelling (Masure and Opdenakker, 1989). It is as yet unclear which cells produce the variable high molecular mass gelatinase present in the CSF. Since the ratio of 85-kDa/65-kDa gelatinase levels in the CSF correlates with the CSF cytosis (Fig. 3), the enzyme is likely to be produced by cells within the CSF. For example, in the two samples, originating from patients with Guillain-Barr6 syndrome (lanes 17 and 18 in Fig. 1) there was no elevated cell count and the CSF was also devoid of 85-kDa gelatinase activity. In synovial fluids from arthritis patients, the high molecular mass form of the enzyme is presumably of neutrophilic origin (as judged by its molecular mass of 91 kDa, its molecular heterogeneity and by immunoprecipitation with specific antibodies) (Opdenakker et al., 1991a). The 85-kDa gelatinase in the CSF of MS patients probably has its origin in monocytes, as suggested by the observation that (i) all CSF samples tested contained monocytes and lymphocytes but no neutrophils, and (ii) the physicochemical properties of the enzyme (molecular mass of 85 kDa, co-migration with monocytic gelatinase and little molecular heterogeneity) were found to differ from the gelatinase of neutrophilic origin. These differences in molecular mass between neutrophilic and monocytic gelatinase were also apparent from the analysis of CSF samples of patients with bacterial meningitis (lanes 22 and 23 in Fig. 1), which contained predominantly neutrophils and yielded a 91-kDa gelatinase instead of the 85-kDa enzyme observed in CSF of MS patients. Direct proof, however, for the monocytic origin of the 85-kDa gelati-
nase in CSF of MS patients will require comparative demonstration of the presence of the enzyme in different CSF cells by means of immunohistochemistry. Unfortunately, antibodies that selectively recognize the 85-kDa gelatinase have sofar not been available. Detection of gelatinase mRNA by in situ hybridisation - albeit an indirect proof - - might be an alternative to define the gelatinase-producing cells. In the present study, the presence of gelatinases was only studied in the CSF. Although CSF is the most accessible way of sampling in the CNS, it may only represent 'the overflow' of the active lesions or altel-natively it may not represent what is going on inside the MS lesions. Therefore, further studies on post-mortem samples need to be done. We identified a specific neutral metalloproteinase, gelatinase, in the cell-free fraction of the CSF. This is in accordance with the observations that CSF from patients with inflammatory neurological disorders contains proteolytic activity for myelin basic protein (Cuzner et al., 1978) or whole myelin (Alvord et al., 1979; Inuzuka et al., 1987). Myelin, and especially the MBP fraction, is also degraded by neutral proteases secreted by stimulated macrophages (Cammer et al., 1978). This activity can be inhibited by EDTA, implying the activity of metalloproteases in this process. In our study, the gelatinase activity could be totally inhibited in the CSF from an MS patient by EDTA and 1,10-phenanthroline. Both products are chelators and inhibitors of metalloproteases. The partial activity of N-ethylmaleimide (a serine protease inhibitor) might indicate that the gelatinase in the CSF needs activation (by a serine protease) for full activity as described previously (Matrisian, 1990). The observation that EAE, an animal model of MS, can be suppressed by administration of inhibitors of serine proteinases, tranexamic acid (Brosnan et al., 1980) or camostat mesilate (Inuzuka et al., 1988) does not exclude a role for gelatinase in inflammatory demyelinating disorders, since both serine- and metalloproteases are interconnected in an enzyme cascade (Vaes, 1988). Gelatinase may contribute to the breakdown of the BBB seen in MS and in various other inflammatory neurological disorders. It is not certain whether gelatinase/type IV collagenase can digest native type IV collagen (Mackay et al., 1990), but the enzyme is certainly catalytically active on denatured basement membrane collagens (Matrisian, 1990). Gelatinase, purified to homogeneity (Masure et al., 1991), also cleaves intact MBP (manuscript in preparation). This activity might be of fundamental importance in the pathogenesis of MS as it has both structural (the desintegration of the myelin sheet) and immunological (the release of possible immunogenic MBP-fragments) implications. The in vivo inhibition of protease activity has been shown to suppress EAE (Brosnan et al., 1980; Inuzuka
34
et al., 1988). Hence the in vitro demonstration of gelatinase activity as well as the suppression of gelatinase activity in MS-CSF with specific inhibitors in the present study opens new perspectives for the elucidation of the pathogenesis and hopefully also for the design of an effective treatment of CNS inflammatory diseases and of MS in particular.
Acknowledgements Work in the authors' laboratory was supported by the Belgian National Fund for Scientific Research (NFWO) and by a special grant from the Lions Club (Belgium). The authors wish to thank Professor A. Billiau for support and critically reviewing the manuscript and the numerous assistants in the Department of Neurology for CSF collection. K.G. is a research assistant and G.O. is a research associate of the NFWO.
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with acute infection of the central nervous system. Clin. Exp. Immunol. 71,320-323. lnuzuka, T., Sato, S., Baba, H. and Miyatake, T. (1987) Neutral protease in cerebrospinal fluid from patients with multiple sclerosis and other neurological diseases. Acta Neurol. Scand. 76, 18-23. lnuzuka, T., Sato, S., Baba, H. and Miyatake, T. (1988) Suppressive effect of camostat mesilate (FOY 305) on acute experimental allergic encephalomyelitis (EAE). Neurochem. Res. 13, 225-228. Khoury, S.J., Weiner H.L. and Hailer, D.A. (1990) Immunologic basis of multiple sclerosis. In: S.D. Cook (Ed.), Handbook of Multiple Sclerosis, Marcel Dekker, New York, NY, pp. 129-149. Link, H. and Tibbling, G. (1977) Principles of albumin and lgG analysis in neurological disorders. II. Evaluation of lgG synthesis within the central nervous system in multiple sclerosis. Scand. J. Clin. Lab. Invest. 37, 397-401. Lotz, M. and Guerne, P.-A. (1991) Interleukin-6 induces the synthesis of tissue inhibitor of metalloproteinases-1/erythroid potentiating activity (TIMP-1/EPA). J. Biol. Chem. 266, 2017-2020. Mackay, A.R., Hartzler, J.L., Pelina, M.D. and Thorgeirsson, U.P. (1990) Studies on the ability of 65-kDa and 92-kDa tumor cell gelatinases to degrade type IV collagen. J. Biol. Chem. 265, 21929-21934. Maimone, D., Gregory, S., Arnason, B.G.W. and Reder, A.T. (1991) Cytokine levels in the cerebrospinal fluid and serum of patients with multiple sclerosis. J. Neuroimmunol. 32, 67-74. Masure, S. and Opdenakker, G. (1989) Cytokine-mediated proteolysis in tissue remodelling. Experientia 45, 542-549. Masure, S., Billiau, A., Van Damme, J. and Opdenakker, G. (1990) Human hepatoma cells produce an 85 kDa gelatinase regulated by phorbol 12-myristate 13-acetate. Biochim. Biophys. Acta 1054, 317-325. Masure, S., Proost, P., Van Damme, J. and Opdenakker, G. (1991) Purification and identification of 91-kDa neutrophil gelatinase. Release by the activating peptide interleukin-8. Eur. J. Biochem. 198, 391-398. Matrisian, L.M. (1990) Metalloproteinases and their inhibitors in matrix remodeling. Trends Genet. 6, 121-125. Opdenakker, G., Masure, S., Grillet, B. and Van Damme, J. (1991a) Cytokine-mediated regulation of human leukocyte gelatinases and role in arthritis. Lymphokine Cytokine Res. 10, 317-324. Opdenakker, G., Masure, S., Proost, P., Billiau, A. and Van Damme, J. (1991b) Natural human monocyte gelatinase and its inhibitor. FEBS Lett. 284, 73-78. Poser, C.M., Paty, D.W., Scheinberg, L., McDonald, W.I., Davis, F.A., Ebers, G.C., Johnson, K.P., Sibley, W.A., Silberberg, D.H. and Tourtellotte, W.W. (1983) New diagnostic criteria for multiple sclerosis: Guidelines for research protocols. Ann. Neurol. 13, 227-231. Raine, C.S. (1984) Analysis of autoimmune demyelination: Its impact upon multiple sclerosis. Lab. Invest. 50, 608-635. Sato, T., Ito, A. and Mori, Y. (1990) Interleukin 6 enhances the production of tissue inhibitor of metalloproteinases (TIMP) but not that of matrix metalloproteinases by human fibroblasts. Biochem. Biophys. Res. Commun. 170, 824-829. Tibbling, G., Link, H. and Ohman, S. (1977) Principles of albumin and lgG analysis in neurological disorders. I. Establishment of reference values. Scand. J. Clin. Lab. Invest. 37, 385-390. Vaes, G. (1988) Cellular biology and biochemical mechanism of bone resorption. A review of recent developments on the formation, activation and mode of action of osteoclasts. Clin. Ortop. 231, 239-271. Van Damme, J., Opdenakker, G., Simpson, R.J., Rubira, M.R., Cayphas, S., Vink, A., Billiau, A. and Van Snick, J. (1987) Identification of the human 26-kD protein, Interferon /32 (IFN/32), as a B cell hybridoma/plasmacytoma growth factor induced by interleukin 1 and tumor necrosis factor. J. Exp. Med. 165, 914-919.
