Resistance of young gelatinase B–deficient mice to experimental autoimmune encephalomyelitis and necrotizing tail lesions

Regulated expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) plays a role in various physiological processes. To determine in vivo how unbalanced expression of these factors can promote or affect the course of pathologies, we knocked out the mouse gelatinase B gene by replacing the catalytic and zinc-binding domains with an antisense-oriented neomycin resistance gene. Adult gelatinase B–deficient mice and wild-type controls could be induced to develop experimental autoimmune encephalomyelitis (EAE) with similar scores for neurologic disease, blood-brain barrier permeability, and central nervous system histopathology. However, whereas diseased control animals showed necrotizing tail lesions with hyperplasia of osteocartilaginous tissue, adult gelatinase B–deficient mice were resistant to this tail pathology. Gelatinase B–deficient mice younger than 4 weeks of age were significantly less susceptible to the development of EAE than were age matched controls and, even as they aged, they remained resistant to tail lesions. These data illustrate that gelatinase B expression plays a role in the development of the immune system and that, in ontogenesis, the propensity to develop autoimmunity is altered by the absence of this MMP.

J. Clin. Invest.104:1507–1515 (1999).

Matrix metalloproteinases (MMPs) are involved in various physiological (1–5) as well as pathological (6–17) processes. Gelatinases belong to the secreted matrix metalloproteinases and constitute a terminal step in the enzyme cascade that processes extracellular matrices including basement membranes (18–20). Two types are distinguished: gelatinase A (MMP-2) and gelatinase B (MMP-9) (Figure 1a). The former has a molecular weight of 65–75 kDa, lacks a collagen-like domain and is, in general, constitutively present in all body fluids. The latter is inducible and has a molecular weight of more than 85 kDa. The MMPs are synthesized as zymogen-like latent precursors and are converted subsequently to the active form of the zinc proteases by the cysteine switch mechanism (21). In terms of protein and domain structure, gelatinase B is the largest and most complex member identified so far. From in vitro studies, it has been deduced that gelatinase B expression is characterized by a complex regulation with tight control at several levels, e.g., gene transcription and protein secretion by cytokines and chemokines (8, 19, 22–24), action of tissue inhibitors of matrix metalloproteinases (TIMPs) (18, 25, 26), and proenzyme activation by components of the plasminogen activation system (19, 20, 22, 27) or other MMPs (28).

Figure 1

Generation of gelatinase B–deficient mice. (a) Gelatinase A and B protein domain structure and gene organization of gelatinase B. Gelatinase B contains a type V collagen-like domain, not present in gelatinase A. Each domain is named, and a color code is used to indicate within the mouse genomic structure the exon parts that encode the respective protein domains. (b) Construction of targeting vector pPNT/mGEL-B. The sequence of the mouse gelatinase B gene (accession number X72794) was extended at the 5′- and 3′-ends, and fragments from the wild-type gene were used to generate the targeting vector pPNT/mGEL-B. Representative restriction sites for subcloning and analysis of wild-type or homologously recombined knockout genes are indicated. The scale bar indicates the fragment sizes in kbp. The orientation of the HSV-TK and NEO genes are antisense to the gelatinase B gene segments as indicated by the arrows. The localization of the 5′-end probes, used for Southern blot analysis, is indicated. (c) Genotyping by Southern blot analysis. Hybridization of EcoRV-digested tail DNA with a 5′-end probe resulted in bands of approximately 20 kb or 7.5 kb. Lanes 1 show the hybridization patterns of genomic DNA from wild-type mice, whereas lanes 2 are from heterozygote, and lanes 3 from gelatinase B–deficient, mice. (d) Screening by PCR analysis. Lanes 1 indicate the results of PCR, performed with a primer set to view the wild-type allele. Lanes 2 show the reaction products obtained with a knockout-specific primer set. The resulting genotype is indicated by heterozygous (HZ), knockout (KO), and wild-type (WT). The sizes of the amplified DNA-fragments are indicated in basepairs. (e) Functional analysis by zymography. Gelatinase activity was determined by SDS/PAGE gelatin substrate conversion. Lanes 1–4 show the analysis of leukocyte supernatants of 4 individual wild-type mice; lanes 5–8, those of 4 gelatinase B–deficient mice. The arrows indicate the gelatinase B zymolysis. Similar to control preparations of recombinant mouse gelatinase B, this zymolysis occurs mainly at >200 kDa (dimer, solid arrow), but also at the monomer position (110 kDa, arrowhead). M, molecular weight standardization.

The possible functions of this enzyme cascade, including gelatinase B, in central nervous system (CNS) inflammation may be manifold (19, 22, 29): cleavage of myelin proteins (30–32), degradation of collagen IV in the basement membranes (33, 34), activation or degradation of disease-modifying cytokines (35), and direct damage of CNS cells.

Gelatinase B has been detected in the cerebrospinal fluid of patients with optic neuritis, multiple sclerosis (MS), and other inflammatory neurologic diseases, but not in normal control patients, whereas gelatinase A was constitutively present in all samples (10, 11). Immunohistochemical reaction with gelatinase B–specific mAb’s (36) was documented within human MS plaques in concert with the expression of other metallo- and serine proteases and specific inhibitors (13).

Experimental autoimmune encephalomyelitis (EAE) is used as an animal model for MS. However, there exist many variants of EAE depending on the animal species (rat, mouse, primates), the inducing agent (microbial or myelin components, spinal cord homogenates, adoptive transfer of specific T-cell clones), and the clinical course (acute, chronic with relapses) (37, 38). In mouse EAE, gelatinase B was increased in the cerebrospinal fluid of diseased mice (31). Recently, it was demonstrated that levels of MMP-9 mRNA were increased in adoptive transfer EAE at times of maximum disease severity. Positive immunochemical staining with an MMP-9–specific mAb was observed along the meninges, around blood vessels, and within the parenchyma in diseased but not in normal animals (39). Biochemical (30, 31), histological, and immunochemical (13) studies, as well as EAE experiments in which protease inhibitors were used (40–45), illustrate that CNS inflammation is mediated by proteases. The disease-limiting effects of protease inhibitors are indicative of a role for serine proteases or MMPs in EAE. Finally, the beneficial effect of IFN-β on MS may be mediated by a control of the protease balance, the net activity of proteases and protease inhibitors (19), e.g., by an inhibition of the gelatinase B expression resulting in a reduction of T-lymphocyte infiltration into the CNS (46, 47).

All of these elements constitute indirect evidence for a role for gelatinase B in the damage caused by inflammation and demyelination in the CNS. In this study, gelatinase B–deficient mice were generated, and the clinical and histopathological effects of gelatinase B action were compared in wild-type versus knockout mice in a specific model of EAE.

Generation of gelatinase B–deficient mice. A targeting vector pPNT/mGEL-B was constructed in which the essential segments of the mouse gene (48), coding for the enzymatic activity of gelatinase B, were deleted and replaced by an antisense-oriented neomycin resistance gene (Figure 1b). The vector pPNT/mGELB was injected into ES cells, and the blastocysts were implanted in pseudo-pregnant mice. Ten chimeras derived from 3 independent ES cell lines transmitted the disrupted MMP-9 gene through the germline as shown by Southern blot analysis. Matings of heterozygous mice resulted in the expected mendelian frequency of MMP-9^–/– (81/359, 22.6%), MMP-9^+/–(177/359, 49.3%), and MMP-9^+/+ (101/359, 28.1%) mice, suggesting that embryonic and fetal development of homozygous mutant mice were not impaired. Further inbreeding and genotyping (Figure 1, c and d) yielded the gelatinase B–deficient animals and controls used in this study. During a follow-up period of more than 3 years, gelatinase B–deficient mice did not show gross phenotypic abnormalities, nor histopathological changes in parenchymatous organs when compared with wild-type animals. Both wild-type and gelatinase B–deficient mice appeared normal, and there was no difference in viability.

Expression of gelatinase B. To verify the gelatinase status in control and knockout mice, gelatinases were analyzed by zymography. Peripheral blood leukocytes were collected from individual wild-type and gelatinase B–deficient mice, purified, and stimulated with phorbol ester. As is clear from the zymography, shown in Figure 1e, leukocytes from gelatinase B–deficient mice did not express gelatinase B, in contrast to their wild-type controls, in which substantial levels of gelatinase B activity are present. As an internal control for sample preparation and handling, all samples contained constitutive gelatinases. From the zymography analysis, we could deduce that the deficiency in gelatinase B was not quantitatively compensated for by increased levels of other gelatinases.

Resistance to development of EAE in young gelatinase B–deficient mice. Because we did not observe a “spontaneous phenotype” in the gelatinase B–deficient mice, and as gelatinase B had previously been shown to be an inducible enzyme, we induced EAE and compared its clinical course in wild-type and gelatinase B–deficient mice. Clinical symptoms progressed in a caudal to rostral direction. The signs started with tail paralysis, which subsequently spread to the back limbs. The symptoms usually peaked with paralysis of the forelimbs. This was often followed by a remission that was, however, not always complete.

When EAE experiments were performed in 3- to 4-week-old mice, the disease course differed between gelatinase B–deficient and control mice. Three independent experiments (pooled data in Figure 2a) confirmed a lower susceptibility of these young gelatinase B–deficient mice to EAE when compared with age-matched wild-types. Statistical analysis yielded significant differences for the mean disease score (P = 0.02), mortality (P = 0.007), incidence of disease (P = 0.02), and mean day of onset (P = 0.02).

Figure 2

Induction of acute EAE in gelatinase B–deficient and wild-type mice. (a) Groups of 3- to 4-week-old mice were immunized with SJL-spinal cord homogenates to develop EAE. Mean disease score, mean day of onset, incidence, and mortality (insets) were calculated on the pooled data of 3 independent experiments. Dead animals were scored 6 from the day of death until the end of the study. (b) EAE was induced in adult (7- to 8-week-old) gelatinase B–deficient and wild-type mice. The insets show the disease parameters that were calculated on the pooled data of 2 experiments.

Susceptibility to EAE in adult gelatinase B–deficient mice. EAE was also induced in adult (7- to 8-week-old) gelatinase B–deficient and wild-type mice (Figure 2b). Statistical analysis on the pooled data from Figure 2b yielded no differences between gelatinase B–deficient and wild-type mice. Mean disease score (P = 0.44), mortality (P = 0.58), and incidence of disease (P = 0.31) did not differ significantly between both groups. However, disease tended to start later in the knockout mice (P = 0.07).

Histopathology: inflammation and demyelination assessment. An extensive histopathological comparison of the CNS lesions was made between young as well as adult gelatinase B–deficient and control mice and scored as outlined in Methods (Figure 3a). In young gelatinase B–deficient mice the inflammatory score (mean value = 1.29) of each spinal cord part (n = 35) was significantly lower (P = 0.0015) than that of the wild-type controls (mean value = 2.44; n = 18) (Figure 3b). In the knockout mice, the inflammatory infiltrates consisted of less polymorphonuclear cells (P = 0.003), whereas the presence of mononuclear cells was equivalent in both groups (P = 0.94). In the adult animals (Figure 3b), similar analyses did not yield significant differences between knockout and wild-type mice. For instance, the inflammatory score was not different in gelatinase B–deficient mice (mean value = 1.69; n = 26) compared with wild-type animals (mean value = 1.58; n = 24). Mononuclear cells and neutrophils were equally distributed in both groups of adult mice (mononuclear cells: P = 0.91; polymorphonuclear cells: P = 0.31). No signs of demyelination were observed within the experimental period of 26 days (Klüver-Barrera stains in Figure 3b).

Figure 3

Neuropathological findings in gelatinase B–deficient mice with EAE. (a) Shows different inflammation stages (stage 1, left; stage 3, right) on the basis of which the inflammatory score was calculated. ×42. (b) Shows hematoxylin-eosin (HE) and Klüver-Barrera (KB) stains of the spinal cord of young and adult gelatinase B–deficient (KO) or wild-type (WT) mice in a diseased (EAE) or in a normal state. ×110. The histology in normal wild-type mice was similar to that of gelatinase B–deficient animals.

Blood-brain barrier permeability. Table 1 summarizes the results of experiments that were performed to assess the blood-brain barrier permeability. Intravenous injection of Evans blue resulted in equivalent staining of the CNS in diseased knockout and wild-type mice, both in the young and in the adult animals. In contrast, the blood-brain barrier permeability increased consistently in mice that developed EAE symptoms compared with healthy controls. Because the overall permeability of the entire CNS was lower than that of the spinal cord, the results are in line with an ascending damage of the blood-brain barrier in EAE. This observation is in accordance with the neurological symptoms of an ascending myelitis. Although the A₆₅₀ ratios observed in the young gelatinase B–deficient mice (1.85 and 1.44) do not reach the level of significance, they appear to be very close to the values in adult mice (2.00 and 1.39). These small differences were strengthened by observations in a further experiment in which the blood-brain barrier permeability in young knockout animals that were resistant to the development of EAE was compared with that of normal (noninduced) gelatinase B–deficient mice. In this case, a mean value of A₆₅₀ ratio (EAE-resistant KO/control KO) was found to be 1.33 (P = 0.02) for the total CNS. This implies that EAE-induction per se damages the blood-brain barrier, even without the development of clinical signs. As expected, when overt clinical disease develops, these ratios increase up to a value of about 2.00.

Table 1

Blood-brain barrier permeability in gelatinase B–deficient mice

Necrotizing tail lesions. In young animals, 28 of 33 wild-type mice developed necrotizing lesions of the tail (Figure 4). In the gelatinase B–deficient mice, much less severe tail lesions were seen in only 13 of 33 animals. Statistical analysis between wild-type and gelatinase B–deficient mice, performed on the pooled data of 3 independent experiments, revealed a significant difference (P = 0.0005) for the incidence of tail lesions.

Figure 4

Tail necrosis in wild-type animals. Various forms of tail necrosis were observed in wild-type animals: loss of necrotic tail end (upper panel) and ulceration and subsequent necrosis (middle) leading to amputation with blunt end (lower panel).

Similar results (P = 0.0002) were obtained for adult mice. Of 21 wild-type mice, 17 developed tail lesions. Only 3 knockout mice of 20 developed such lesions, which again were much less severe and usually healed.

In both age groups, the neurological disease score was not directly related to the occurrence of tail lesions, but neurologically nonaffected animals retained a healthy tail and the lesions appeared usually when the disease process was at its height.

The histopathology (Figure 5) of these tail lesions consisted in the earliest phase of an accumulation of eosinophilic fibrinoid material underneath the epidermis. This was accompanied by dilatation of small blood vessels, some edema, and a sparse inflammatory infiltrate in the superficial stroma of the dermis. Subsequently, the epidermis became increasingly thinner, ultimately resulting in complete ulceration and covering by a fibrinopurulent exudate. The adjacent, uninvolved epidermis showed reactive irregular hyperplasia. After ulceration, the osteocartilaginous tissue in the center of the tail showed pronounced hyperplasia, with many irregular outgrowths of osseous tissue, in particular, underneath the ulceration. In some cases, this hyperplastic reaction resulted in extrusion of the osseous tissue through the ulceration (Figure 5). In addition, areas of chondroid tissue with high cellularity appeared within the osseous tissue. The tonguelike osseous outgrowths were lined by numerous osteoblasts and smaller numbers of osteoclasts and were surrounded by a fibroblastic reaction of the stroma. Vasculitis was not observed in any of the sections. The mild and infrequent tail lesions in the gelatinase B–deficient mice represented the initial stage of the histopathological evolution and can macroscopically be best compared with an exfoliation or an erosion. In some instances, these lesions were exudative or mildly bleeding. These never resulted in complete skin ulceration or tail amputation.

Figure 5

Histopathological findings on the tail lesions in wild-type animals suffering from EAE. (a) In the earliest stage fibrinoid material (→) appears immediately underneath the thinned epithelium. Inflammatory cells are present in the overlying horny layer. (b) Next, the adjacent epithelium shows irregular hyperplasia (→). (c) At the periphery of the lesion a fibroblastic reaction (*) occurs in the dermis. (d) Irregular outgrowth of osteocartilaginous tissue (*) occurs when the epithelium is completely ulcerated. Hematoxylin and eosin stain; all panels, ×110.

Gelatinase B has been associated with the pathogenesis and/or development of MS, as well as EAE (19). However, the pathogenic role of gelatinase B has never been proved. Indeed, specific inhibitory mAb’s against (mouse) gelatinase B are not available for testing in EAE. Knockout technology offers an elegant alternative to study the effect of gelatinase B. In this study, a phenotype of resistance to EAE development was observed to be limited to young gelatinase B–deficient mice of less than 4 weeks of age. The incidence of disease and the mean disease score were significantly lower in young gelatinase B–deficient animals versus controls. In 2 other studies with gelatinase B–deficient mice, the phenotypic changes were also observed in young mice. Abnormal bone formation was seen in 2- to 4-week-old mice (4), whereas a model of bullous pemphigoid was used in neonatal mice (50). However, in the latter study, no parametric comparisons were done with adult animals. The phenotypic changes in 3- to 4-week-old mice, observed in our experiments, illustrate the importance of gelatinase B function in the postnatal period. It was therefore crucial to know whether lack of gelatinase B was compensated for in adult life. In our mouse EAE model, no differences in mortality, incidence, mean day of onset, and mean disease score were observed in adult gelatinase B–deficient versus control mice. The histopathological findings corroborated the clinical data and suggest the action of alternative or compensatory pathways by other enzymes, as was also observed in the remodeling of the postpartum uterus (51). For instance, matrilysin (MMP-7) or metallo-elastase (MMP-12) might be important enzymes in damaging the CNS in MS (14) and EAE (45).

We also quantified the influx of polymorphonuclear cells in young and adult mice. When the EAE disease was at its height, young gelatinase B–deficient mice showed a significant reduction in infiltrated neutrophils in comparison to controls. This observation was not made in the adult animals. Significant differences in polymorphonuclear myeloperoxidase activity after 12 hours in neonatal knockout versus wild-type mice were previously also noticed in a model of bullous pemphigoid by Liu et al. (50). In our experiments, mononuclear cells were equally distributed in knockout and control animals, both in the young and adult ones. Finally, our study shows that absence of gelatinase B does not preclude destruction of the blood-brain barrier. However, the barrier permeability in young gelatinase B–deficient mice is less affected than in adult, as could be deduced from the absence of significant differences for the ratio diseased KO/control KO in Table 1. This finding is consistent with our clinical data of EAE disease.

Of the 5 MMPs that have been knocked out, namely MMP-12 (52), MMP-2 (53–54), MMP-7 (55), MMP-9 (4, 50, and this study), MMP-3 (56), none have been lethal to embryos, despite their potentially important roles in development, and several explanations have been proposed to explain lack of expected phenotypes (57). We were able to document the development of compensatory mechanisms during ontogenesis by comparison of EAE between adult and 3- to 4-week-old mice.

Another remarkable observation in this study was the appearance of necrotizing tail lesions with bone hyperplasia in the wild-type mice, whereas the gelatinase B–deficient mice were resistant to this pathology. Our observations extend those of Vu et al. (4), who demonstrated differences in osteocartilaginous tissue between wild-type animals and gelatinase B–deficient animals at young age. As anticipated by its role in remodeling of extracellular matrices, and in particular of collagen types, gelatinase B is a crucial enzyme in bone formation and metabolism. In view of the fact that gelatinase B has been supposed to function in blood vessel remodeling (15), it is tempting to associate the necrotizing lesions to vascular pathology. However, and because there were no clear signs of vasculitis, there exist alternative explanations for this pathology. For instance, the susceptibility for EAE and tail lesion development may be indirectly associated. Indeed, exclusively neurologically affected mice developed this necrosis. Which element or combination of agents of the immunization protocol might play a role in the development of the tail lesions will be the subject of further studies. In acute EAE protocols in SJL/J mice (n = 103) and in 129SvEv (n = 47), the aforementioned tail lesions were not observed.

Because the expression levels of 2 induced phenotypes (EAE and tail necrosis) were clinically followed, we were able to show that in gelatinase B–deficient mice, 1 phenotype (EAE) was compensated for, but the resistance to develop tail lesions remained through ontogenesis. This does not necessarily mean that gelatinase B is not important in adult EAE, rather, it means that other mediators may subserve its function when it is deleted for the life of the mouse (58). In addition, our study shows that gelatinase B is a causative, but replaceable, link in CNS inflammation and therefore that the development of gelatinase B or general MMP inhibitors might be important in the fight against diseases such as MS or those with pathological bone remodeling. Finally, the concept that regulation of extracellular protease–inhibitor balances is involved in autoimmunity (19) is now proved in vivo for 1 specific anticipated MMP family member, gelatinase B.

The authors appreciate the excellent technical help of S. Schmitt, J. Weik, G. Küblbeck, C. Dillen, P. Fiten, and M. Groenen and thank P.M. Rudd (Oxford University), A. Billiau, and J. Van Damme (University of Leuven) for critical reading of the manuscript. Helpful discussions with Z. Werb (University of California–San Francisco, San Francisco, California, USA) and V.W. Yong (University of Calgary, Alberta, Canada) are appreciated. This work was supported by the Cancer Foundation of the General Savings and Retirement Fund, the Geconcerteerde Onderzoeksacties, the Scientific Foundation for Multiple Sclerosis Research (Belgium, program 1998–2002), the Fund for Scientific Research (Vlaanderen), the Deutsche Forschungsgemeinschaft (SFB 405, EU BIO4-CT-96007) and the VW-Foundation (I/72-422). Bénédicte Dubois is a research assistant of the Fund for Scientific Research.

Bénédicte Dubois and Stefan Masure contributed equally to this work.

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