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Matrix Metalloproteinase-12 Deficiency Worsens Relapsing-Remitting Experimental Autoimmune Encephalomyelitis in Association with Cytokine and Chemokine Dysregulation

2009; Elsevier BV; Volume: 174; Issue: 3 Linguagem: Inglês

10.2353/ajpath.2009.080952

1525-2191

Angelika Goncalves DaSilva, V. Wee Yong,

Multiple Myeloma Research and Treatments

The elevation of several members of the matrix metalloproteinase (MMP) family promotes the pathophysiology of both multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Nonetheless, given the multiple activities of MMPs, it remains possible that increased levels of a particular MMP may have beneficial functions during disease progression. We reported previously that MMP-12−/− mice of the 129/SvEv strain had a poorer EAE outcome than wild-type controls. However, we did not determine further differences in disease profiles between these groups. Using the EAE model in 129/SvEv mice, we report that disease in both wild-type and MMP-12−/− mice follows a relapsing-remitting course. Although both mouse groups had similar clinical onsets, subsequent relapses were more severe in MMP-12−/− mice; their residual disability at remission was also higher compared with wild-type controls. The worsened relapses and remissions in MMP-12−/− mice occurred despite a deficiency of the antigen recall capacity of lymph node-derived cells as well as a reduction in the proportion of macrophages in the spinal cord during the chronic phase of EAE. Significantly, large increases of levels of chemokines and cytokines were found in the spinal cords of MMP-12−/− mice during chronic EAE. These results highlight MMP-12 as a beneficial enzyme in EAE and suggest that therapeutic interventions in multiple sclerosis should avoid targeting MMP-12. The elevation of several members of the matrix metalloproteinase (MMP) family promotes the pathophysiology of both multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Nonetheless, given the multiple activities of MMPs, it remains possible that increased levels of a particular MMP may have beneficial functions during disease progression. We reported previously that MMP-12−/− mice of the 129/SvEv strain had a poorer EAE outcome than wild-type controls. However, we did not determine further differences in disease profiles between these groups. Using the EAE model in 129/SvEv mice, we report that disease in both wild-type and MMP-12−/− mice follows a relapsing-remitting course. Although both mouse groups had similar clinical onsets, subsequent relapses were more severe in MMP-12−/− mice; their residual disability at remission was also higher compared with wild-type controls. The worsened relapses and remissions in MMP-12−/− mice occurred despite a deficiency of the antigen recall capacity of lymph node-derived cells as well as a reduction in the proportion of macrophages in the spinal cord during the chronic phase of EAE. Significantly, large increases of levels of chemokines and cytokines were found in the spinal cords of MMP-12−/− mice during chronic EAE. These results highlight MMP-12 as a beneficial enzyme in EAE and suggest that therapeutic interventions in multiple sclerosis should avoid targeting MMP-12. Multiple sclerosis (MS) is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS). There have been significant efforts in elucidating the mechanisms that regulate the entry of leukocytes into the CNS in MS, including in the expression of members of the matrix metalloproteinase (MMP) family. The production of MMPs by leukocytes helps degrade the basement membrane surrounding cerebral vessels,1Agrawal S Anderson P Durbeej M van Rooijen N Ivars F Opdenakker G Sorokin LM Dystroglycan is selectively cleaved at the parenchymal basement membrane at sites of leukocyte extravasation in experimental autoimmune encephalomyelitis.J Exp Med. 2006; 203: 1007-1019Crossref PubMed Scopus (379) Google Scholar, 2Toft-Hansen H Buist R Sun XJ Schellenberg A Peeling J Owens T Metalloproteinases control brain inflammation induced by pertussis toxin in mice overexpressing the chemokine CCL2 in the central nervous system.J Immunol. 2006; 177: 7242-7249PubMed Google Scholar and, within the CNS, the expression of MMPs may produce demyelination and axonal injury, and promote neuroinflammation.3Kieseier BC Seifert T Giovannoni G Hartung HP Matrix metalloproteinases in inflammatory demyelination: targets for treatment.Neurology. 1999; 53: 20-25Crossref PubMed Google Scholar, 4Yong VW Power C Forsyth P Edwards DR Metalloproteinases in biology and pathology of the nervous system.Nat Rev. 2001; 2: 502-511Crossref Scopus (889) Google Scholar Several members of the 25-MMP family are elevated in MS and experimental autoimmune encephalomyelitis (EAE)5Anthony DCC Ferguson B Matyzak MK Millert KM Esiri MM Perry VH Differential matrix metalloproteinase expression in cases of multiple sclerosis and stroke.Neuropathol Appl Neurobiol. 1997; 23: 406-415Crossref PubMed Scopus (240) Google Scholar, 6Weaver A Goncalves da Silva A Nuttall RK Edwards DR Shapiro SD Rivest S Yong VW An elevated matrix metalloproteinase (MMP) in an animal model of multiple sclerosis is protective by affecting Th1/Th2 polarization.FASEB J. 2005; 19: 1668-1670Crossref PubMed Scopus (128) Google Scholar and, in support of their negative roles, pharmacological inhibitors of metalloproteinase activity reduce severity of EAE.7Yong VW Giuliani F Xue M Bar-Or A Metz LM Experimental models of neuroprotection relevant to multiple sclerosis.Neurology. 2007; 68: S32-S54Crossref PubMed Scopus (36) Google Scholar Compared with wild-type animals, EAE is attenuated in mice deficient for MMP-88Folgueras AR Fueyo A Garcia-Suarez O Cox J Astudillo A Tortorella P Campestre C Gutierrez-Fernandez A Fanjul-Fernandez M Pennington CJ Edwards DR Overall CM Lopez-Otin C Collagenase-2 deficiency or inhibition impair experimental autoimmune encephalomyelitis in mice.J Biol Chem. 2008; 283: 9465-9474Crossref PubMed Scopus (60) Google Scholar and -99Dubois B Masure S Hurtenbach U Paemen L Heremans H van den Oord J Sciot R Meinhardt T Hammerling G Opdenakker G Arnold B Resistance of young gelatinase B-deficient mice to experimental autoimmune encephalomyelitis and necrotizing tail lesions.J Clin Invest. 1999; 104: 1507-1515Crossref PubMed Scopus (243) Google Scholar and in MMP-2 and -9 double-null mice.1Agrawal S Anderson P Durbeej M van Rooijen N Ivars F Opdenakker G Sorokin LM Dystroglycan is selectively cleaved at the parenchymal basement membrane at sites of leukocyte extravasation in experimental autoimmune encephalomyelitis.J Exp Med. 2006; 203: 1007-1019Crossref PubMed Scopus (379) Google Scholar Although the majority of MMP members are thought to promote the EAE and MS disease process, it is possible that particular MMPs may have contrasting beneficial roles to help attenuate disease, given the wide range of substrates of MMPs.10Sternlicht MD Werb Z How matrix metalloproteinases regulate cell behavior.Annu Rev Cell Dev Biol. 2001; 17: 463-516Crossref PubMed Scopus (3242) Google Scholar, 11Van Lint P Libert C Chemokine and cytokine processing by matrix metalloproteinases and its effect on leukocyte migration and inflammation.J Leukoc Biol. 2007; 82: 1375-1381Crossref PubMed Scopus (446) Google Scholar In particular, we have found that MMP-12−/− mice on the 129/SvEv strain had worse EAE clinical disease compared with wild-type controls when daily disease scores were summed (sum of scores) throughout 73 days of evaluation.6Weaver A Goncalves da Silva A Nuttall RK Edwards DR Shapiro SD Rivest S Yong VW An elevated matrix metalloproteinase (MMP) in an animal model of multiple sclerosis is protective by affecting Th1/Th2 polarization.FASEB J. 2005; 19: 1668-1670Crossref PubMed Scopus (128) Google Scholar However, we were unable to differentiate the long-term disease course in the two groups given that the day of onset of initial clinical signs was highly variable among animals in that study. The potential role of MMP-12 in alleviating disease requires further attention because MMP-12 is highly expressed in MS lesions.12Vos CMP van Haastert ES De Groot CJA Van der Walk P de Vries HE Matrix metalloproteinase-12 is expressed in phagocytotic macrophages in active multiple sclerosis lesions.J Neuroimmunol. 2003; 138: 106-114Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar Moreover, the use of nonselective MMP inhibitors to reduce inflammation may prove counterproductive if MMP-12 inhibition is not spared pharmacologically. Indeed, an inhibitor that targets MMP-12 has been contemplated for clinical trials in MS.13Hu J Van den Steen PE Sang QX Opdenakker G Matrix metalloproteinase inhibitors as therapy for inflammatory and vascular diseases.Nat Rev Drug Discov. 2007; 6: 480-498Crossref PubMed Scopus (660) Google Scholar Thus, with increasing experience in producing more consistent EAE disease onset in 129/SvEv mice, we have examined EAE disease activity in MMP-12−/− mice and sought to understand its roles in secondary lymphoid organs and within the spinal cord. We report that EAE in 129/SvEv wild-type and MMP-12−/− mice follows a relapsing-remitting course. Although MMP-12−/− and wild-type mice have similar onset of disease, subsequent relapses were more severe in MMP-12−/− mice and their residual disability at remission was also higher compared with wild-type controls. The worsened relapses and remission in MMP-12−/− mice was accompanied by significant alterations within the spinal cord of CD4CD25+ regulatory T cells and macrophages in conjunction with an increase in the expression of pro- and anti-inflammatory cytokines during chronic EAE. These results provide new insights into the regulation of inflammation during relapsing-remitting EAE and MS and point to a role for MMP-12 in resolving disease. MMP-12−/− mice on the 129/SvEv strain were a kind gift from Dr. Steve Shapiro (Harvard Medical School, Boston, Massachusetts).6Weaver A Goncalves da Silva A Nuttall RK Edwards DR Shapiro SD Rivest S Yong VW An elevated matrix metalloproteinase (MMP) in an animal model of multiple sclerosis is protective by affecting Th1/Th2 polarization.FASEB J. 2005; 19: 1668-1670Crossref PubMed Scopus (128) Google Scholar, 14Shipley JM Wesselschmidt RL Kobayashi DK Ley TJ Shapiro SD Metalloelastase is required for macrophage-mediated proteolysis and matrix invasion in mice.Proc Natl Acad Sci USA. 1996; 93: 3942-3946Crossref PubMed Scopus (410) Google Scholar Wild-type 129/SvEv mice were purchased from Taconics (Germantown, NY). These two lines were expanded in-house and used in the nonlittermate experiments. The lines were then crossed to produce heterozygote (MMP-12+/−) breeders and the offspring (≥F2 generation) were used as littermate mice. Genotyping (Supplemental Figure S1, available at http://ajp.amjpathol.org) confirmed the identity of the MMP-12+/+, MMP-12+/−, and MMP-12−/− mice. EAE was induced in female mice when they were 8 to 9 weeks of age by injecting subcutaneously two injections (day 0 and day 7) of 100 μg myelin oligodendrocyte glycoprotein (MOG)35–55 in complete Freund's adjuvant (Fisher, Pittsburgh, PA) supplemented with 4 mg/ml of Mycobacterium tuberculosis (H37Ra) (Difco Laboratory, Detroit, MI). No pertussis toxin was used because the 129/SvEv strain did not require this reagent for development of EAE. Animals were assessed daily using a 15-point disease score scale6Weaver A Goncalves da Silva A Nuttall RK Edwards DR Shapiro SD Rivest S Yong VW An elevated matrix metalloproteinase (MMP) in an animal model of multiple sclerosis is protective by affecting Th1/Th2 polarization.FASEB J. 2005; 19: 1668-1670Crossref PubMed Scopus (128) Google Scholar, 15Giuliani F Metz LM Wilson T Fan Y Bar-Or A Yong VW Additive effect of the combination of glatiramer acetate and minocycline in a model of MS.J Neuroimmunol. 2005; 158: 213-221Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 16Dasilva AG Yong VW Expression and regulation of matrix metalloproteinase-12 in experimental autoimmune encephalomyelitis and by bone marrow derived macrophages in vitro.J Neuroimmunol. 2008; 199: 24-34Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar that replaced the more commonly used 5-point scale because the 15-point scale differentiates individual limb disability, rather than lumping both fore- or hindlimbs together, thus allowing for better characterization of disease progression. The 15-point scale ranges from 0 to 15 and is the sum of the disease state for the tail (0 to 2) and all four limbs (scored 0 to 3). Based on this scoring system a fully quadriplegic mouse would attain a score of 14 and mortality would be given a score of 15. The observer was blinded to genotype in all EAE experiments during behavioral and histological (see below) assessments. All animals were handled in accordance with the policies outlined by the Canadian Council for Animal Care and the University of Calgary. All mice were genotyped to determine their genetic background. Pups from heterozygous breeders were genotyped by isolating DNA from ear samples. A total of 5 ng of DNA in 2 μl was added to 12 μl of polymerase chain reaction (PCR) master mix (10× PCR buffer, 10 mmol/L dNTP, 50 mmol/L MgCl2, 20 μmol/L oIMR0297 5′-CACGAGACTAGTGAGACGTG-3′, 20 μmol/L oIMR3207 5′-GCTAGAAGCAACTGGGCAAC-3′, 20 μmol/L oIMR3208 5′-5′-ACATCCTCACGCTTCATGTC-3′, ddH2O, and 1.25 U Taq polymerase). The PCR was run using the following conditions: 94°C for 3 minutes, 35 cycles of 94°C for 30 seconds, 65°C for 1 minute, and 72°C for 1 minute, followed by 72°C for 2 minutes. The PCR products were resolved on a 1% agarose gel with the following molecular weights: MMP-12+/+, 1064 bp fragments; MMP-12+/−, 1064 and 1500 bp fragments; and MMP-12−/−, 1500-bp fragments (Supplemental Figure S1, available at http://ajp.amjpathol.org). Animals were sacrificed via an overdose of ketamine/xylazine (Bimeda-MTC Animal Health Inc., Cambridge, Canada) and bled by aortic puncture for 5 minutes while the heart was pumping. Whole spinal cords were removed and fixed by submersion in 10% buffered formalin (Surgipath Canada Inc., Winnipeg, Canada) for 24 hours. Spinal cords were divided into thoracic and lumbar sacral portion and embedded in paraffin blocks. Thoracic cords were cut longitudinally on a microtome and four adjacent 6-μm-thick sections were placed on glass slides (Fisher) for a total of four series of 48 sections from each animal. One series was then processed for hematoxylin, eosin, and Luxol fast blue (LFB). Our experience with these tissue-processing methods allowed us to adequately differentiate between cells trapped in blood vessels and those that are in the parenchyma, and perfusion before tissue collections did not affect our ability to differentiate between intravascular and parenchymal leukocytes (data not shown). For histology, slides were warmed at 60°C for 1 hour, deparafinized through a series of ethanol steps, and then placed in LFB (solvent 38; Sigma, St. Louis, MO) at 60°C for 3 hours. Slides were then placed in 95% ethanol, followed by water, and then 0.05% lithium carbonate before being placed in 70% ethanol. Once the appropriate myelin color differentiation was achieved slides were placed in hematoxylin (EMD, San Diego, CA) for 4 minutes. Slides were then sequentially dipped in 1% acid alcohol and ammonia water, with water washes in between, and then rinsed in water again, followed by 70% ethanol and 95% ethanol before being dipped in alcoholic eosin (EMD). Slides were then passed through a dehydrating step before being coverslipped using acrytol (Surgipath Canada Inc.). Images were captured using an Olympus BH-2 microscope (Olympus, Port Moody, Canada) and QCapture Pro software (version 5.1.1.14; Media Cybernetics Inc., Bethesda, MD). For semiquantitative assessment of the extent of inflammation in the spinal cord, all stained longitudinal sections from the same coded mouse were first evaluated using the scoring system outlined in the Results (below). In this regard, the location (pial versus parenchymal) and number of inflammatory aggregates per section were documented, with parenchymal inflammation and larger and higher number of aggregates being ascribed greater inflammation scores. Finally, the number of inflammatory lesions/aggregates was counted per longitudinal section. Six different sections per animal were examined by a blinded observer and the average number of lesions per section was documented. Mice were sacrificed on day 10 after MOG immunization, just before clinical disease signs were expected, and LNs were removed. LNs were homogenized into a single cell suspension and washed in phosphate-buffered saline (PBS) two times at 1200 rpm. LN cells were plated in round-bottom plates at a density of 250,000 cells/100 μl in RPMI containing 1% mouse serum (Invitrogen, Carlsbad, CA). The LN populations served to provide T cells for subsequent antigen-recall proliferation. Cells for antigen-presenting cell (APC) function were harvested from spleens taken from nonimmunized animals. Spleens were homogenized and then layered onto Ficoll (GE Healthcare Bioscience, Uppsala, Sweden). The buffy layer containing cells was removed and washed two times in PBS before being suspended in RPMI containing 1% mouse serum. A tube containing the APC cells was then γ-irradiated at room temperature with a Gamma Cell 1000 (Nordion International Inc., Vancouver, Canada) using Cs-137 for 14 minutes at 300 rad, which was then followed by incubation of the APC cells with MOG35–55 for 30 minutes at 4°C. The APCs (250,000 APCs/100 μl) were then added to the LN cells described above for a total of 500,000 cells/well and incubated for 3 days at 37°C in 5% CO2. During the last 18 hours of incubation cells were pulsed with 1 μCi of [H3] thymidine (Perkin Elmer, Waltham, MA) to determine the proliferative state of the cultures. After incubation, cells were harvested using a PHD cell harvester (Brandel Inc., Gaithersburg, MD) and [H3] thymidine incorporation was determined by using a Beckman LS3801 scintillation counter (Beckman Coulter, Missassauga, Canada). In one set of experiments T cells and APCs were plated together based on matching their genotype whereas in other experiments we plated T cells with nonmatching APCs. Mice induced for EAE were sacrificed via an overdose of ketamine/xylazine and perfused with PBS. Draining LNs and lumbar sacral spinals cords were harvested at specific disease time points: Normal (no disease induction), presymptomatic (day 10 after MOG injection, just before signs of EAE symptoms), remission (2 days after the first signs of a decrease in peak EAE score), and chronic disease (day 35 after MOG injection) and placed in Hanks' balanced salt solution (Invitrogen). The tissue was pushed through a sieve with nylon mesh (pore size, 70 μmol/L; BD Bioscience, Mississauga, Canada) and single cell suspensions were collected in RPMI containing 10% fetal bovine serum (BD Bioscience). The cells were washed twice with RPMI for 10 minutes at 1200 rpm. Cells were counted and plated at 0.5 to 1 × 106/well before being stained for analysis. Cells were then washed in fluorescence-activated cell sorting (FACS) buffer [PBS, 2% fetal calf serum (v/v), 1% bovine serum albumin (w/v), 0.1% NaN3 (v/v); BD Bioscience] and then blocked with mouse BD Fc block (BD Bioscience) for 20 minutes at 4°C. Cells were washed twice with FACS buffer and incubated with primary antibodies or isotype controls for 30 minutes at 4°C Antibodies were CD3 PE-Cy5 (and isotype control: Armenian hamster IgG1 κ; dilution range per 106 cells/well, 0.2 μg/μl), CD4 R-PE (rat IgG2a κ, 0.2 μg/μl), CD8a FITC (rat IgG2a κ, 0.5 μg/μl), CD45 PerCP (rat IgG2b κ, 0.2 μg/μl), CD11b FITC (rat IgG2b κ, 0.5 μg/μl), CD4 PerCP (rat IgG2a κ, 0.2 μg/μl), and CD25 R-PE (rat IgG1 λ, 0.2 μg/μl). These antibodies were from BD Bioscience. Cells were then washed twice with FACS buffer before being fixed with 1% formalin for 30 minutes; finally cells were suspended in 500 μl of FACS buffer. For FoxP3 (FoxP3 FITC, rat IgG2a κ, 0.5 μg/μl) staining cells were suspended in 1× cytofix/cytoperm (BD Bioscience) for 20 minutes at 4°C, washed twice with permeabilization/wash buffer (BD Bioscience), and incubated with FoxP3 for 30 minutes at 4°C. Cells were then washed twice more and suspended in a final volume of 500 μl of FACS buffer. All cells were captured and analyzed using a Becton Dickinson LSR 3 laser flow cytometer (BD Bioscience) and data analysis was done using Cell Quest Pro V4.0.2 software (BD Bioscience). Two separate experiments were analyzed and the results of the total percentage of gated events (n = 4 to 13 animals per genotype per time point) are then pooled. The percentage of gated events was evaluated using flow cytometry plots (Supplemental Figure S2, available at http://ajp.amjpathol.org) in which the region of interest was determined using side (x axis) and forward (y axis) scatter plots. Cells were then sorted based on their expression of specific markers and the dot plots show the region of interest used to determine the final gated events, which is shown as a percentage of gated events on the y axis of the summary line graph. EAE was induced in MMP-12+/+ and MMP-12−/− mice as outlined above and thoracic spinal cords from each genotype were removed during the following phases: normal (no disease induction), presymptomatic (day 10 after MOG injection, no signs of EAE symptoms), remission (2 days after the first signs of a decrease in peak EAE score), and chronic disease (day 35 after MOG injection) (n = 3 mice/genotype in each group). Spinal cords were homogenized in lysis buffer and supernatant used after centrifugation at 1300 rpm. The protein expression of 40 immune-related molecules was assessed using the mouse RayBio inflammatory antibody array from RayBiotech Inc. (Norcross, GA) (Supplemental Figure S3, available at http://ajp.amjpathol.org). All samples were processed together as follows; glass chip slides coated with the antibody arrays were first treated with blocking buffer before being incubated for 1 hour with various protein samples (100 μg protein per slide) from the different genotypes during EAE. Slides were then washed and incubated sequentially with a biotin-conjugated secondary antibody solution, horseradish peroxidase-conjugated streptavidin, and horseradish peroxidase detection buffer. The signals were detected using chemiluminescence and densitometric analysis performed to determine the difference between the various samples. Slides were processed using a Gene Pix array 4000B scanner (Molecular Dynamics Inc., Sunnyvale, CA) and Gene Pix pro software (Molecular Dynamics Inc.). Positive control signals (a known amount of biotinylated antibody) were used to normalize the level of expression between samples being examined. For all arrays, each protein intensity value is divided by the average intensity value of the positive control on the same array; this value is then multiplied by the average intensity value of the positive control on the reference array giving the normalized level of expression for each protein examined. Normalization is essential for the identification and removal of the effects of systematic variation in the measured intensities, which are not attributable to differential expression. Statistical analysis was performed using Prism 5 (version 5.0) (GraphPad Software Inc., San Diego, CA). Statistical differences between groups with respect to disease onset, mean peak score, mean average disease score, mean cumulative disease, and histopathology were evaluated using a nonparametric analysis Mann-Whitney U-test. Statistical differences between groups with respect to daily clinical disease were evaluated using repeated measures two-way analysis of variance and Bonferroni post-hoc analyses. Statistical differences between groups with respect to T-cell cultures and antibody arrays were evaluated using one-way analysis of variance and Tukey's multiple comparison test or two-way analysis of variance and Bonferroni post-hoc analyses. Nonlittermate MMP-12+/+ and MMP-12−/− mice were induced for EAE using MOG35–55 and animals were examined daily for their EAE progression for 58 days. There was no significant difference in onset of clinical signs between MMP-12+/+ and MMP-12−/− mice, and both genotypes followed a relapsing-remitting course of disease (Figure 1A). Significantly, MMP-12−/− mice displayed a higher severity during a relapse, and they failed to remit to the low baseline disability of MMP-12+/+ mice (Figure 1A). At the termination of the experiment on day 58, the residual deficit in MMP-12−/− mice was clearly higher than that of MMP-12+/+ mice. To further differentiate the severity of disease burden between individual MMP-12+/+ and MMP-12−/− mice, the daily clinical disease for each mouse was summed throughout the 58 days of observation to provide the cumulative disease score per mouse. Figure 1B shows that MMP-12−/− mice had a significantly higher cumulative score per animal (163.1 ± 16.3 versus 70.8 ± 7.9) in comparison with MMP-12+/+ mice, emphasizing the higher overall disease burden in the former. Moreover, when the percentage of time in which mice spent with mild (percent time at score 0 to 3) and severe disability (percent time at score ≥4) was documented, we found that MMP-12−/− mice spent significantly less time with mild disability in comparison with MMP-12+/+(Figure 1C). In this regard, the MMP-12−/− mice spent a significant amount of time with tail involvement, hindlimb paralysis, and forelimb paresis whereas the MMP-12+/+ mice spent greater periods of time suffering only from tail limpness and mild hindlimb paresis. Overall, these results show that MMP-12−/− nonlittermate mice succumb to a worse relapsing-remitting disease than their MMP-12+/+ counterparts. We examined the thoracic spinal cords from MMP-12+/+ and MMP-12−/− using LFB and H&E to determine whether differences in EAE severity translated to worsened neuropathology. Using a semiquantitative classification system (Figure 2), we were able to document general inflammation score. Normal spinal cords from both MMP-12+/+ and MMP-12−/− mice were absent of demyelination and infiltrates, and we did not observe any gross anatomical or myelin structure abnormalities between the genotypes in nondisease condition. In EAE-afflicted mice, both MMP-12+/+ and MMP-12−/− mice displayed inflammatory infiltrates and we found that MMP-12−/− mice trended toward a higher inflammation score in comparison with MMP-12+/+ although statistical significance was not achieved (Figure 3A). We next counted the number of inflammatory lesions per longitudinal section of the thoracic cord, sampling six sections per mouse. Figure 3B shows that the average number of lesions per section was higher in MMP-12−/− mice compared with that in MMP-12+/+ mice (P = 0.03). We did not observe any qualitative differences in the lesions between the genotypes in terms of the size, distribution, or location relative to blood vessels.Figure 3The degree of neuropathology in the spinal cord trended toward higher severity in the MMP-12−/− animals. A: Inflammation score of the spinal cords of mice, assessed in sections stained with LFB/H&E, is similar between MMP-12+/+ and MMP-12−/− animals, although there were eight null mice with an inflammation score of 5 or higher, compared with four in the wild-type group. Individual values are plotted and the bar represents the mean for the group. B: The lesion number, referring to the average number of inflammatory lesions that are encountered within each of six longitudinal spinal cord sections of a given mouse, was significantly higher in MMP-12−/− animals compared with wild-type (P = 0.03). Nonparametric Mann-Whitney tests were significant when *P < 0.05 (MMP-12+/+, n = 10; and MMP-12−/−, n = 12).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The above work consisted of the use of wild-type or MMP-12−/− mice generated from separate breeding colonies. To preclude the possibility that EAE phenotype was confounded by epigenetic or environmental factors rather than the deletion of MMP-12, we sought to produce littermates for further analyses. MMP-12+/+ and MMP-12−/− breeders were crossed to generate heterozygote breeders, which were further cross-bred to produce littermates of MMP-12+/+, MMP-12+/−, and MMP-12−/− mice, which were genotyped (Supplemental Figure S1, available at http://ajp.amjpathol.org). Examination of the genotype of >200 pups showed that the distribution of MMP-12+/+, MMP-12+/−, and MMP-12−/− mice was similar to what would be expected for Mendelian inheritance of this gene (Supplemental Figure S1, available at http://ajp.amjpathol.org). We deemed it important to use littermate mice (Figure 4) to confirm the data from nonlittermate experiments (Figure 1) because differential breeding, nursing, or housing properties, besides unknown genetic influences, may adversely affect outcomes. EAE was induced in littermate MMP-12+/+, MMP-12+/−, and MMP-12−/− using MOG35–55 and animals were observed daily for 60 days. As with nonlittermates, there was no difference in disease onset between the genotypes and the course of disease in all groups was relapsing-remitting (Figure 4A). Significantly, MMP-12−/− mice did not remit to the same level as MMP-12+/+, and their relapse was more severe. Heterozygote MMP-12+/− mice behaved in the same manner as wild-type controls, so the loss of both MMP-12 alleles was necessary to confer a worsened EAE outcome. These results were confirmed by examination of average cumulative score throughout 60 days (Figure 4B) and the percentage of time mice spent with severe disability (Figure 4C), both of which were higher in the MMP-12−/− mice. Overall, in littermate or nonlittermate experiments, the presence of MMP-12 plays a role in conferring a greater degree of