Collagenase-2 Deficiency or Inhibition Impairs Experimental Autoimmune Encephalomyelitis in Mice

Artigo Acesso aberto Revisado por pares

Collagenase-2 Deficiency or Inhibition Impairs Experimental Autoimmune Encephalomyelitis in Mice

2008; Elsevier BV; Volume: 283; Issue: 14 Linguagem: Inglês

10.1074/jbc.m709522200

ISSN

1083-351X

Autores

Alicia R. Folgueras, Antonio Fueyo, Olivia García‐Suárez, Jennifer H. Cox, Aurora Astudillo, Paolo Tortorella, Cristina Campestre, Ana Gutiérrez‐Fernández, Miriam Fanjul‐Fernández, Caroline J. Pennington, Dylan R. Edwards, Christopher M. Overall, Carlos López‐Otín,

Tópico(s)

Blood Coagulation and Thrombosis Mechanisms

Resumo

Matrix metalloproteinases (MMPs) have been implicated in a variety of human diseases, including neuroimmunological disorders such as multiple sclerosis. However, the recent finding that some MMPs play paradoxical protective roles in these diseases has made necessary the detailed study of the specific function of each family member in their pathogenesis. To determine the relevance of collagenase-2 (MMP-8) in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, we have performed two different analyses involving genetic and biochemical approaches. First, we have analyzed the development of EAE in mutant mouse deficient in MMP-8, with the finding that the absence of this proteolytic enzyme is associated with a marked reduction in the clinical symptoms of EAE. We have also found that MMP-8-/- mice exhibit a marked reduction in central nervous system-infiltrating cells and demyelinating lesions. As a second approach, we have carried out a pharmacological inhibition of MMP-8 with a selective inhibitor against this protease (IC50 = 0.4 nm). These studies have revealed that the administration of the MMP-8 selective inhibitor to mice with EAE also reduces the severity of the disease. Based on these findings, we conclude that MMP-8 plays an important role in EAE development and propose that this enzyme may be a novel therapeutic target in human neuro-inflammatory diseases such as multiple sclerosis. Matrix metalloproteinases (MMPs) have been implicated in a variety of human diseases, including neuroimmunological disorders such as multiple sclerosis. However, the recent finding that some MMPs play paradoxical protective roles in these diseases has made necessary the detailed study of the specific function of each family member in their pathogenesis. To determine the relevance of collagenase-2 (MMP-8) in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, we have performed two different analyses involving genetic and biochemical approaches. First, we have analyzed the development of EAE in mutant mouse deficient in MMP-8, with the finding that the absence of this proteolytic enzyme is associated with a marked reduction in the clinical symptoms of EAE. We have also found that MMP-8-/- mice exhibit a marked reduction in central nervous system-infiltrating cells and demyelinating lesions. As a second approach, we have carried out a pharmacological inhibition of MMP-8 with a selective inhibitor against this protease (IC50 = 0.4 nm). These studies have revealed that the administration of the MMP-8 selective inhibitor to mice with EAE also reduces the severity of the disease. Based on these findings, we conclude that MMP-8 plays an important role in EAE development and propose that this enzyme may be a novel therapeutic target in human neuro-inflammatory diseases such as multiple sclerosis. Collagenase-2 deficiency or inhibition impairs experimental autoimmune encephalomyelitis in mice.Journal of Biological ChemistryVol. 293Issue 30PreviewVOLUME 283 (2008) PAGES 9465–9474 Full-Text PDF Open Access Multiple sclerosis (MS) 2The abbreviations used are: MSmultiple sclerosisCNScentral nervous systemEAEexperimental autoimmune encephalomyelitisMMPMatrix metalloproteinaseMOGmyelin oligodendrocyte glycoproteinPBSphosphate-buffered salineTNFtumor necrosis factorIFNinterferonILinterleukinTGFtransforming growth factorWTwild type. is an inflammatory disease of the central nervous system (CNS) characterized by autoreactive T-cell infiltration that causes myelin sheath destruction and axonal loss (1Steinman L. Martin R. Bernard C. Conlon P. Oksenberg J.R. Annu. Rev. Neurosci. 2002; 25: 491-505Crossref PubMed Scopus (206) Google Scholar, 2Hafler D.A. Slavik J.M. Anderson D.E. O'Connor K.C. De Jager P. Baecher-Allan C. Immunol. Rev. 2005; 204: 208-231Crossref PubMed Scopus (259) Google Scholar). Although the origin of MS remains unclear, CD4+ Th1 cells are believed to be the main mediators of the autoimmune reaction (3Sospedra M. Martin R. Annu. Rev. Immunol. 2005; 23: 683-747Crossref PubMed Scopus (1792) Google Scholar, 4Hemmer B. Archelos J.J. Hartung H.P. Nat. Rev. Neurosci. 2002; 3: 291-301Crossref PubMed Scopus (495) Google Scholar). According to this, the injection of myelin peptides into susceptible mice generates experimental autoimmune encephalomyelitis (EAE), a murine model of human multiple sclerosis, and leads to myelin-specific recruitment of T cells that subsequently differentiate into Th1-type effector cells (5Bernard C.C. Johns T.G. Slavin A. Ichikawa M. Ewing C. Liu J. Bettadapura J. J. Mol. Med. 1997; 75: 77-88Crossref PubMed Scopus (234) Google Scholar). The extravasation of reactive leukocytes into the CNS parenchyma across the blood-brain barrier correlates with the appearance of clinical symptoms (6Muller D.M. Pender M.P. Greer J.M. J. Neuroimmunol. 2005; 160: 162-169Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). multiple sclerosis central nervous system experimental autoimmune encephalomyelitis Matrix metalloproteinase myelin oligodendrocyte glycoprotein phosphate-buffered saline tumor necrosis factor interferon interleukin transforming growth factor wild type. Matrix metalloproteinases (MMPs) have been largely implicated in MS and EAE progression due to their ability to degrade the main extracellular matrix components that maintain the integrity of the blood-brain barrier (7Yong V.W. Power C. Forsyth P. Edwards D.R. Nat. Rev. Neurosci. 2001; 2: 502-511Crossref PubMed Scopus (889) Google Scholar, 8Rosenberg G.A. Glia. 2002; 39: 279-291Crossref PubMed Scopus (730) Google Scholar). Consistent with this putative role of MMPs in the development or progression of MS, several members of this metalloproteinase family have been shown to be up-regulated in serum, cerebrospinal fluid, and brain samples of MS patients (9Lindberg R.L. De Groot C.J. Montagne L. Freitag P. van der Valk P. Kappos L. Leppert D. Brain. 2001; 124: 1743-1753Crossref PubMed Scopus (153) Google Scholar, 10Yong V.W. Zabad R.K. Agrawal S. Goncalves Dasilva A. Metz L.M. J. Neurol. Sci. 2007; 259: 79-84Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) as well as in murine models of demyelinating lesions (11D'Souza C.A. Mak B. Moscarello M.A. J. Biol. Chem. 2002; 277: 13589-13596Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 12Nygardas P.T. Hinkkanen A.E. Clin. Exp. Immunol. 2002; 128: 245-254Crossref PubMed Scopus (64) Google Scholar). Likewise, expression of MMPs has been detected in diverse cell types involved in the pathogenesis of the disease (13Bar-Or A. Nuttall R.K. Duddy M. Alter A. Kim H.J. Ifergan I. Pennington C.J. Bourgoin P. Edwards D.R. Yong V.W. Brain. 2003; 126: 2738-2749Crossref PubMed Scopus (292) Google Scholar, 14Toft-Hansen H. Nuttall R.K. Edwards D.R. Owens T. J. Immunol. 2004; 173: 5209-5218Crossref PubMed Scopus (130) Google Scholar). Interestingly, the administration of MMP inhibitors has reduced the severity of the disease in different EAE murine models (15Brundula V. Rewcastle N.B. Metz L.M. Bernard C.C. Yong V.W. Brain. 2002; 125: 1297-1308Crossref PubMed Scopus (387) Google Scholar, 16Giuliani F. Fu S.A. Metz L.M. Yong V.W. J. Neuroimmunol. 2005; 165: 83-91Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 17Opdenakker G. Nelissen I. Van Damme J. Lancet Neurol. 2003; 2: 747-756Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 18Liedtke W. Cannella B. Mazzaccaro R.J. Clements J.M. Miller K.M. Wucherpfennig K.W. Gearing A.J. Raine C.S. Ann. Neurol. 1998; 44: 35-46Crossref PubMed Scopus (111) Google Scholar). Furthermore, young MMP-9-deficient mice and MMP-2/MMP-9 double knock-out mice are more resistant to the development of the disease than wild-type animals (19Dubois B. Masure S. Hurtenbach U. Paemen L. Heremans H. van den Oord J. Sciot R. Meinhardt T. Hammerling G. Opdenakker G. Arnold B. J. Clin. Invest. 1999; 104: 1507-1515Crossref PubMed Scopus (243) Google Scholar, 20Agrawal S. Anderson P. Durbeej M. van Rooijen N. Ivars F. Opdenakker G. Sorokin L.M. J. Exp. Med. 2006; 203: 1007-1019Crossref PubMed Scopus (379) Google Scholar). However, and somewhat unexpectedly, MMP-2-/- and MMP-12-/- mice are more susceptible to EAE induction than their wild-type counterparts suggesting that these metalloproteinases could play protective roles in the course of MS (21Esparza J. Kruse M. Lee J. Michaud M. Madri J.A. FASEB J. 2004; 18: 1682-1691Crossref PubMed Scopus (95) Google Scholar, 22Weaver A. Goncalves da Silva A. Nuttall R.K. Edwards D.R. Shapiro S.D. Rivest S. Yong V.W. FASEB J. 2005; 19: 1668-1670Crossref PubMed Scopus (128) Google Scholar). These findings, together with the variety of processes in which MMPs are involved (23Folgueras A.R. Pendás A.M. Sánchez L.M. López-Otín C. Int. J. Dev. Biol. 2004; 48: 411-424Crossref PubMed Scopus (484) Google Scholar, 24Brinckerhoff C.E. Matrisian L.M. Nat. Rev. Mol. Cell. Biol. 2002; 3: 207-214Crossref PubMed Scopus (969) Google Scholar), suggest that the contribution of these enzymes to the progression of neuro-inflammatory diseases is much more complex than originally anticipated. Thus, and besides the degradative action of MMPs during blood-brain barrier disruption, these enzymes may also contribute to the progression of MS through their ability to degrade myelin components releasing encephalitogenic peptides (25Gijbels K. Proost P. Masure S. Carton H. Billiau A. Opdenakker G. J. Neurosci. Res. 1993; 36: 432-440Crossref PubMed Scopus (185) Google Scholar, 26Proost P. Van Damme J. Opdenakker G. Biochem. Biophys. Res. Commun. 1993; 192: 1175-1181Crossref PubMed Scopus (178) Google Scholar). Likewise, MMPs may play important roles in the regulation of the inflammatory stimuli responsible for autoreactive T cell recruitment and infiltration (27Parks W.C. Wilson C.L. López-Boado Y.S. Nat. Rev. Immunol. 2004; 4: 617-629Crossref PubMed Scopus (1464) Google Scholar). In fact, MMPs have been demonstrated to modulate inflammation through the proteolytic cleavage of a number of cytokines and chemokines that have been implicated in the pathobiology of MS (27Parks W.C. Wilson C.L. López-Boado Y.S. Nat. Rev. Immunol. 2004; 4: 617-629Crossref PubMed Scopus (1464) Google Scholar, 28Zhang K. McQuibban G.A. Silva C. Butler G.S. Johnston J.B. Holden J. Clark-Lewis I. Overall C.M. Power C. Nat. Neurosci. 2003; 6: 1064-1071Crossref PubMed Scopus (283) Google Scholar). This functional diversity of MMPs makes necessary the detailed analysis of the specific role that each individual member of this complex family of metallo-proteinases might play in the pathogenesis of MS. Our study in this regard has focused on the analysis of the putative implication of collagenase-2 (MMP-8) in the development and progression of neuro-inflammatory diseases such as MS. MMP-8 is a potent collagenolytic enzyme frequently associated with inflammatory conditions, including asthma, hepatitis, ulcerative colitis, atherosclerosis, periodontitis, and rhinosinusitis (29Balbín M. Fueyo A. Knauper V. Pendás A.M. López J.M. Jiménez M.G. Murphy G. López-Otín C. J. Biol. Chem. 1998; 273: 23959-23968Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 30Owen C.A. Hu Z. López-Otín C. Shapiro S.D. J. Immunol. 2004; 172: 7791-7803Crossref PubMed Scopus (201) Google Scholar, 31Gueders M.M. Balbín M. Rocks N. Foidart J.M. Gosset P. Louis R. Shapiro S. López-Otín C. Noël A. Cataldo D.D. J. Immunol. 2005; 175: 2589-2597Crossref PubMed Scopus (130) Google Scholar, 32Sorsa T. Tjaderhane L. Salo T. Oral Dis. 2004; 10: 311-318Crossref PubMed Scopus (450) Google Scholar, 33Van Lint P. Wielockx B. Puimege L. Noël A. López-Otín C. Libert C. J. Immunol. 2005; 175: 7642-7649Crossref PubMed Scopus (92) Google Scholar, 34Pirila E. Ramamurthy N.S. Sorsa T. Salo T. Hietanen J. Maisi P. Dig. Dis. Sci. 2003; 48: 93-98Crossref PubMed Scopus (52) Google Scholar). Interestingly, several works have also evidenced the up-regulation of MMP-8 in EAE, and its expression has been correlated with disease severity (12Nygardas P.T. Hinkkanen A.E. Clin. Exp. Immunol. 2002; 128: 245-254Crossref PubMed Scopus (64) Google Scholar, 14Toft-Hansen H. Nuttall R.K. Edwards D.R. Owens T. J. Immunol. 2004; 173: 5209-5218Crossref PubMed Scopus (130) Google Scholar). To further explore the possibility that this metalloproteinase could play a role in MS pathogenesis, we have used mutant mice deficient in MMP-8 and analyzed their susceptibility to EAE. In this work, we report that MMP-8-/- mice are more resistant to EAE than their wild-type counterparts, and show a marked reduction in CNS-infiltrating cells and demyelinating lesions. On this basis, we propose that MMP-8 plays an important role in EAE development and can be a therapeutic target in human neuro-inflammatory diseases such as MS. EAE Induction and Clinical Evaluation—Wild-type (MMP-8+/+) and MMP-8-null mice (MMP-8-/-) were generated in a C57BL6/129Sv background, as previously described (35Balbín M. Fueyo A. Tester A.M. Pendás A.M. Pitiot A.S. Astudillo A. Overall C.M. Shapiro S.D. López-Otín C. Nat. Genet. 2003; 35: 252-257Crossref PubMed Scopus (403) Google Scholar). Control and mutant mice used in all experiments were littermates derived from interbreeding of MMP-8+/- heterozygotes. For EAE induction, 8- to 10-week-old female mice were injected subcutaneously in the flank on days 0 and 7 with 300 μg of myelin oligodendrocyte glycoprotein (MOG35-55) peptide. The peptide was thoroughly emulsified in 100 μl of complete Freund's adjuvant containing 500 μg of heat-inactivated Myco-bacterium tuberculosis H37Ra (Difco Laboratories). Mice were also injected intraperitoneally on days 0 and 2 with 200 μl of PBS containing 500 ng of Pertussis toxin (List Biologicals Laboratories). After immunization with MOG, mice were observed daily, and the disease severity was scored on a scale of 0-5 with graduations of 0.5 for intermediate clinical signs. The score was defined as follows: 0, no detectable clinical signs; 1, weakness of the tail; 2, hind limb weakness or abnormal gait; 3, complete paralysis of the hind limbs; 4, complete hind limb paralysis with forelimb weakness or paralysis; 5, moribund or death. Paralyzed mice were given easy access to food and water. Mouse experimentation was done according to the guidelines of the Universidad de Oviedo, Oviedo-Spain. In Vivo MMP-8 Inhibition Studies—The cyclohexylamine salt of (R)-1-(3′-methylbiphenyl-4-sulfonylamino)methylpropyl phosphonic acid, a new phosphonate inhibitor with potent (IC50 = 0.4 nm) and selective action against MMP-8 (36Biasone A. Tortorella P. Campestre C. Agamennone M. Preziuso S. Chiappini M. Nuti E. Carelli P. Rossello A. Mazza F. Gallina C. Bioorg. Med. Chem. 2007; 15: 791-799Crossref PubMed Scopus (40) Google Scholar), was dissolved in PBS with 5% Me2SO at 2.5 mg/ml. Treated mice were injected intraperitoneally daily with a dose of 25 mg/kg body weight of the inhibitor, starting at the time of MOG immunization. All mice were monitored daily until the time of sacrifice. Analysis of Expression of MMPs, TIMPs, ADAMs, and ADAMTSs—Spinal cords from immunized wild-type and MMP-8-/- mice were isolated during the chronic phase of the disease. Tissues were homogenized and total RNA was extracted by using a commercial kit (RNeasy MiniKit, Qiagen). One microgram of RNA was reverse transcribed to make cDNA. TaqMan PCR was used to profile mRNA levels of all members of the MMP family and the four TIMPs and several members of the ADAM and ADAMTS families, as previously described (14Toft-Hansen H. Nuttall R.K. Edwards D.R. Owens T. J. Immunol. 2004; 173: 5209-5218Crossref PubMed Scopus (130) Google Scholar). The 18 S rRNA gene was used as an endogenous internal control. Isolation of Splenocytes and Cytokine Assays—Spleens from immunized wild-type and MMP-8-/- mice were isolated and dispersed into a single cell suspension. After lysis of red blood cells, the splenocytes were washed and resuspended in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, l-glutamine, sodium pyruvate, 2-mercaptoethanol, and streptomycin/penicillin. Cells were plated at 4 × 106 cells/well in 24-well plates containing 1 ml of culture medium with 0, 5, or 50 μg/ml MOG35-55. Supernatants were collected at 48 h for cytokine analysis. Quantitative enzyme-linked immunosorbent assay was performed for TNF-α, IFN-γ (R&D), IL-4, IL-10 (BD Biosciences), and TGF-β (Promega) according to the protocol supplied by the manufacturer. Splenocyte Proliferation Assay—Spleens from wild-type and MMP-8-/- mice were removed 21 days after immunization and processed as described above. Cells were plated in triplicates at 7 × 105 cells/well in 96-well microtiter plates containing 200 μl of culture medium with 0, 5, or 50 μg/ml MOG35-55. They were cultured for 48 h and then pulsed with 1 μCi/well [methyl-3H]thymidine for the last 16 h. Cells were collected and precipitated with 5% trichloroacetic acid for 4 h at 4 °C. The precipitated DNA was filtered using G/C glass fiber filters and radioactivity was determined in a scintillation counter. Histological Analysis and Immunofluorescence—Wild-type and MMP-8-/- immunized mice with representative clinical scores were selected from each group. Brains and spinal cords were isolated, fixed in 4% paraformaldehyde, and embedded in paraffin. Each sample was serially sectioned 5-μm thick at 100-μm intervals and stained with hematoxylin and eosin. To evaluate the degree of inflammation, the “depth” of inflammatory infiltrates was measured in serial sections of spinal cord samples and quantified using Image Tool HUCA software. To assess the degree of demyelinization, an immunohistochemical analysis was performed using a primary antibody against myelin basic protein. To perform immunohistochemistry, deparaffined, and rehydrated sections were rinsed in PBS (pH 7.5). Endogenous peroxidase activity and nonspecific binding were blocked with peroxidase block buffer (DakoCytomation) and 1% bovine serum albumin, respectively. Sections were incubated overnight at 4 °C with a monoclonal antibody anti-myelin basic protein (a gift from Dr. Sternberger), diluted 1:1500. Then, sections were incubated with an anti-mouse EnVision system-labeled polymer (Dako-Cytomation) for 30 min, washed in buffer solution, and visualized with diaminobenzidine. Sections were counterstained with Mayer's hematoxylin, dehydrated, and mounted in Entellan®. Inflammatory and demyelinating lesions were evaluated by a neuropathologist. To quantify the different cellular profiles, sampling was systematically randomized, and two sections of the spinal cord from wild-type and MMP-8-/- immunized mice, with representative clinical scores, were selected to perform the immunohistochemical analysis. To detect T lymphocytes, deparaffined and rehydrated sections were heated in 10 mm citrate buffer solution (pH 6.5) in a pressure cooker for 7 min. Antibody nonspecific binding was blocked using 1% bovine serum albumin in PBS. Samples were incubated overnight at 4 °C with a rabbit anti-human CD3 antibody (Aton Pharma Inc.), diluted 1:100. Then, slides were incubated with an anti-rabbit EnVision system-labeled polymer for 30 min, washed in buffer solution, and visualized with diaminobenzidine. To perform macrophage and polymorphonuclear (PHN) immunohistochemistry, sections were incubated for 2 h at 37 °C and overnight at 4 °C with a rat anti-mouse neutrophils (Serotec) or a rat anti-mouse F4/80 (Serotec), diluted 1:50. After that, slides were incubated with a goat anti-rat secondary antibody diluted 1:50. Sections were counterstained with Mayer's hematoxylin, dehydrated, and mounted in Entellan®. The total number of each cellular profile was referred to the area of white matter analyzed. This area was calculated in each section using Image Tool HUCA software. For double immunofluorescence, spinal cords were isolated, fixed in 4% paraformaldehyde, and embedded in OCT. Cryosections were blocked with 20% serum in PBS and 0.2% Triton X-100 for 30 min. Then, slides were incubated overnight at 4 °C with a rat monoclonal antibody to mouse Ly6G (BD Pharmingen) diluted 1:25. After washing with PBS, samples were incubated for 1 h at room temperature with Alexa Fluor 488-goat anti-rat IgG secondary antibody diluted 1:100. A primary antibody against MMP-8 (35Balbín M. Fueyo A. Tester A.M. Pendás A.M. Pitiot A.S. Astudillo A. Overall C.M. Shapiro S.D. López-Otín C. Nat. Genet. 2003; 35: 252-257Crossref PubMed Scopus (403) Google Scholar) was added to the slides diluted 1:3000 in blocking buffer and incubated for 1 h. Finally, samples were incubated for 1 h at room temperature with Alexa Fluor 594-goat anti-rabbit IgG secondary antibody diluted 1:2000. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole. Sections were examined using a Confocal-Ultra Espectral Leica TCS-SP2-AOBS microscope. MMP and ADAM Inhibition Assays—MMP inhibition assays have been previously described (36Biasone A. Tortorella P. Campestre C. Agamennone M. Preziuso S. Chiappini M. Nuti E. Carelli P. Rossello A. Mazza F. Gallina C. Bioorg. Med. Chem. 2007; 15: 791-799Crossref PubMed Scopus (40) Google Scholar). Recombinant ADAM-10 and ADAM-17 were purchased from R&D Systems. For assaying ADAMs, the inhibitor (R)-1-(3′-methylbiphenyl-4-sulfonylamino)methylpropylphosphonic acid stock solution (100 mm) was further diluted at six different concentrations (0.01 nm to 10 μm) in the fluorometric assay buffer (25 mm Tris, pH 8.0, 25 μm ZnCl2, 0.005% Brij-35). The enzyme (final concentration of 19 nm for ADAM-10 and 1.4 nm for ADAM-17) and inhibitor solutions were incubated in the assay buffer for 4 h at 25 °C. After addition of 5 μm (final concentration) of the fluorogenic substrate Mca-Pro-Leu-Ala-Gln-Ala-Val-Dap (Dpn)-Arg-Ser-Ser-Ser-Arg-NH2 (Bachem), the hydrolysis was monitored recording the increase of fluorescence (λex = 320 nm, λem = 405 nm) using a LS55 spectrofluorometer from PerkinElmer Life Sciences. The assays were performed in duplicate in a total volume of 100 μl per well in 96-well microtiter plates (Nunc). The percentage of inhibition was calculated from control reactions without the inhibitor. IC50 was determined using the formula: Vi/V0 = 1/(1 + [I]/IC50), where Vi is the initial velocity of substrate cleavage in the presence of the inhibitor at concentration [I] and V0 is the initial velocity in the absence of the inhibitor. Results were analyzed using Graph-Pad Software. Statistical Analysis—Values shown are mean ± S.E. Comparison of clinical scores, cytokine production levels, and cellular profiles between the various treatment groups were analyzed by using two-tailed Student's t test. A value of p ≤ 0.05 was considered significant. Statistically significant differences are shown with asterisks. MMP-8-/- Mice Are More Resistant to EAE—To investigate the possible contribution of MMP-8 to the initiation and progression of EAE, wild-type and MMP-8-deficient mice were immunized with the encephalitogenic peptide MOG35-55 and scored according to 8-/-, n = 34) were immunized in three independent experiments, and the results of a representative experiment are shown in Fig. 1A. Although the disease onset is similar in both groups, after day 20 from the immunization, the clinical scores observed in MMP-8-/- mice were significantly reduced during the chronic phase of the disease. Also, the maximal disease score was diminished in MMP-8-/- mice compared with wild type (wild type = 3.4 versus knock-out = 2.1, p = 0.09). Considering that the lack of MMP-8 could be compensated in the adult life by other members of the MMP family or by alternative proteolytic pathways, we also induced EAE in 4-week-old wild-type (n = 7) and MMP-8-/- mice (n = 8). As shown in Fig. 1B, the severity and the onset of the disease were significantly reduced in young MMP-8-/- compared with young wild-type mice. As expected, the maximal disease severity score of young MMP-8-/- mice (wild type = 2.5 versus knock-out = 0.8, p ≤ 0.05) was also diminished in comparison to that obtained with MMP-8+/+ young mice. The greatest differences between wild-type and MMP-8-/- mice EAE susceptibility obtained in this experiment suggest the existence of a compensatory mechanism in the adult life. To evaluate this possibility, we analyzed the expression levels of all murine MMPs and their endogenous inhibitors (TIMPs), as well as selected members of the structurally related families of ADAMs and ADAMTSs, in the spinal cords of 8-week-old (n = 4 mice per group) and 4-week-old (n = 4 mice per group) wild-type and MMP-8-/- immunized mice, sacrificed during the chronic phase of the disease. Interestingly, the relative expression level of MMP-8 was one of the highest in the wild-type diseased mice (Figs. 2A and 3A). This analysis revealed that there are six genes, MMP-9, MMP-12, MMP-15, MMP-17, MMP-24, and MMP-28, whose expression levels were significantly different between MMP-8+/+ andFIGURE 2Analysis of MMP, ADAM, ADAMTS, and TIMP expression levels in 8-week-old wild-type and MMP-8-/- immunized mice. TaqMan real-time PCR analysis of MMPs (A), TIMPs (B), and ADAMs and ADAMTSs (C) expressed in spinal cord samples from wild-type (black bar) and MMP-8-/- immunized mice (white bar). mRNA levels on the y-axis are expressed relative to 18 S rRNA levels. Both forms of murine MMP-1 (43Balbín M. Fueyo A. Knauper V. López J.M. Álvarez J. Sánchez L.M. Quesada V. Bordallo J. Murphy G. López-Otín C. J. Biol. Chem. 2001; 276: 10253-10262Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) were undetectable. Values are means ± S.E. *, p ≤ 0.05; **, p ≤ 0.01. n = 4 mice per group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Analysis of MMP, ADAM, ADAMTS, and TIMP expression levels in 4-week-old wild-type and MMP-8-/- immunized mice. TaqMan real time PCR analysis of MMPs (A), TIMPs (B), and ADAMs and ADAMTSs (C) expressed in spinal cord samples from wild-type (black bar) and MMP-8-/- immunized mice (white bar). mRNA levels on the y-axis are expressed relative to 18 S rRNA levels. Both forms of murine MMP-1 were undetectable. Values are means ± S.E. *, p ≤ 0.05; **, p ≤ 0.01. n = 4 mice per group.View Large Image Figure ViewerDownload Hi-res image Download (PPT) MMP-8-/- 8-week-old mice. However, three of them, MMP-9, MMP-12, and MMP-17, also showed different expression patterns in young mice (Figs. 2A and 3A). These results suggest that the occurrence of compensatory events, elicited by the lack of MMP-8, may have appeared during earlier stages of knockout mice development. By contrast, there are certain genes whose expression pattern is markedly different between 8- and 4-week-old MMP-8-/- mice. Thus, MMP-15, MMP-24, and MMP-28 were significantly up-regulated in adult MMP-8-/- mice compared with wild type, whereas there were not significant differences between their expression levels in MMP-8+/+ and MMP-8-/- 4-week-old mice (Figs. 2A and 3A). In an opposite way, ADAMTS1 expression levels were significantly up-regulated in MMP-8-/- 4-week-old mice compared with wild-type, whereas no difference was observed between MMP-8+/+ and MMP-8-/- adult mice. The relative expression levels of the four TIMPs and the analyzed ADAMs remained unaltered in both groups (Figs. 2B, 2C, 3B, and 3C). Altogether, these findings confirm the existence of different expression patterns between 8- and 4-week-old MMP-8-/- mice that may contribute to explain the differences observed between the EAE susceptibility of adult and young MMP-8-/- mice. Reduced Inflammation and Demyelination in MMP-8-/- EAE Mice—To evaluate whether MMP-8 may contribute to activated cell recruitment and CNS inflammation, histopathological examination of spinal cord and brain samples from 8-week-old wild-type and MMP-8-/- immunized mice was performed at different times in the course of the disease. We first analyzed samples extracted 10 days after the initiation of the experiment, before any clinical symptoms were observed. As expected, no detectable lesions were present at this time point in both wild-type and MMP-8-/- mice (Fig. 4, A-D). In contrast, samples extracted during the acute phase of the disease (21 days) showed a marked increase in the number of infiltrating cells, although the demyelinating lesions were still very limited. Despite the fact that this time marks the point of divergence in EAE progression between wild-type and MMP-8-/- mice, as the severity of EAE symptoms continues to increase in wild-type animals while is maintained in MMP-8-/- mice, no histological differences were observed between both groups (Fig. 4, E-H). Finally, we analyzed CNS tissues from wild-type and MMP-8-/- mice sacrificed at the chronic phase of the disease. Interestingly, although animals were chosen with similar clinical symptoms in both genotypes, the extent of the inflammatory infiltrates were significantly reduced in knock-out tissues compared with wild-type counterparts (wild type = 334.1 ± 57.2 μm, n = 3, versus knock-out = 125.0 ± 39.5 μm, n = 3, p ≤ 0.05) (Fig. 4, I, K, M, and O). In addition, myelin basic protein immunohistochemistry revealed extensive demyelinating lesions in the wild-type tissues, while demyelination was less severe in MMP-8-/- mice (Fig. 4, J, L, N, and P). A detailed analysis of the cellular types present in the inflammat

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Collagenase-2 Deficiency or Inhibition Impairs Experimental Autoimmune Encephalomyelitis in Mice