Expression and Regulation of Matrix Metalloproteinase-12 in Experimental Autoimmune Encephalomyelitis and by Bone Marrow Derived
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Journal of Neuroimmunology 199 (2008) 24–34 www.elsevier.com/locate/jneuroim Expression and regulation of matrix metalloproteinase-12 in experimental autoimmune encephalomyelitis and by bone marrow derived macrophages in vitro ⁎ Angelika Goncalves DaSilva, V. Wee Yong Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada Department of Clinical Neurosciences, University of Calgary, Calgary, Alberta, Canada Department of Oncology, University of Calgary, Calgary, Alberta, Canada Received 3 November 2007; received in revised form 2 April 2008; accepted 21 April 2008 Abstract Several matrix metalloproteinase (MMP) members contribute to pathology in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). As the role of MMP-12 in EAE has been understudied, we examined its expression in EAE and in vitro. MMP-12 transcripts increased with EAE, and protein was localized to a subset of macrophages/microglia. The temporal expression of MMP-12 largely corresponded to that of cytokines, and we demonstrate that IL-1β and TNF-α promoted MMP-12 expression in cultured macrophages. We postulate that cytokine — MMP-12 interactions are important in the disease process of EAE. © 2008 Elsevier B.V. All rights reserved. Keywords: Cytokines; Metalloproteinases; Experimental autoimmune encephalomyelitis; Neuroinflammation; Proteases 1. Introduction cerebral vascular vessels (Cossins et al., 1997; Kouwenhoven et al., 2002; Agrawal et al., 2006; Toft-Hansen et al., 2006). Multiple sclerosis (MS) is an autoimmune neurological Besides participating in BBB breakdown, MMPs can also disease primarily affecting young adults. Experimental auto- produce demyelination and axonal injury, promote neuroin- immune encephalomyelitis (EAE), a commonly used animal flammation via the proteolysis of zymogens, and participate in model of MS, has provided many clues to the general mediating cell death (Kieseier et al., 1999; Yong et al., 2001; understanding of MS, but the etiology of the disease remains Opdenakker et al., 2003). Thus, inhibiting the activity of MMPs uncertain. Whatever the initial trigger(s) of MS, a major feature has been a goal of providing for novel therapeutics in MS of the disease is the infiltration of activated leukocytes through (Kieseier et al., 1999; Rosenberg, 2001; Hu et al., 2007; Yong the blood-brain barrier (BBB) into the central nervous system et al., 2007). (CNS) to produce neuropathology (Sospedra and Martin, 2005; The MMPs constitute a family of 25 members, and several of Frohman et al., 2006). these are found elevated in serum, leukocytes, cerebrospinal Matrix metalloproteinases (MMPs) are a subgroup of the fluid and the CNS tissues of patients with MS, or in animals metzincin proteases (Parks et al., 2004). It is now well afflicted with EAE. These include MMP-2, -3, -8, -9, -10, -11, established that infiltrating leukocytes secrete MMPs and that -12, -13, -14 and -25 (Anthony et al., 1997; Cossins et al., 1997; these MMPs help degrade the basement membrane surrounding Pagenstecher et al., 1998; Kouwenhoven et al., 2001; Lindberg et al., 2001; Weaver et al., 2005). The contribution of MMPs to ⁎ Corresponding author. University of Calgary, 3330 Hospital Drive, Calgary, disease evolution and progression has been supported by EAE Alberta, Canada T2N 4N1. Tel.: +1 403 220 3544; fax: +1 403 210 8840. studies where a variety of inhibitors of metalloproteinase E-mail address: [email protected] (V.W. Yong). activity, including GM6001 (Gijbels et al., 1994), BB1101 0165-5728/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2008.04.034 A.G. DaSilva, V.W. Yong / Journal of Neuroimmunology 199 (2008) 24–34 25 (Chandler et al., 1997; Liedtke et al., 1998), Ro31-9790 peak when symptoms were stabilized at a score for 5 days. We (Hewson et al., 1995) and minocycline (Brundula et al., 2002) also used control animals that received saline injections in CFA can either prevent disease manifestation or suppress ongoing instead of MOG and mice were killed 12 days after the saline disease activity. The MS medication, interferon-β, reduces injection; these control mice received pertussis toxin at days 0 MMP activity and levels in leukocytes and in the serum of and 2. patients (Leppert et al., 1996; Stuve et al., 1996; Galboiz et al., In our experience with female C57BL/6 mice aged 8– 2001; Avolio et al., 2005). 9 weeks, MOG-induced EAE causes inflammation which is It is uncertain if all the MMP members found elevated in MS distributed along the thoracic, lumbar and sacral spinal cord. or EAE contribute to disease activity. Conversely, it is also Thus, we have used (see below) the lumbar sacral cord for possible that an MMP member is elevated to help suppress protein analysis while the thoracic region was allocated for disease activity (Parks et al., 2004; Hu et al., 2007). In this RNA analysis. The use of multiple regions from the same regard, we have previously determined that MMP-12 null mice mouse thus allows for better control for disease variability have a more severe EAE course than wildtype controls (Weaver amongst mice; also, fewer mice were killed to perform these et al., 2005). Thus, while most MMP members may serve experiments as outlined in our animal care directives. detrimental roles in MS and EAE, as supported by the data that metalloproteinase inhibitors reduce EAE disease activity, there 2.2. RNA extraction from thoracic spinal cords of EAE is at least one MMP member, MMP-12, which may function to C57BL/6 mice dampen inflammation. Since the profile of MMP-12 expression during EAE has not been described previously, it would be Animals were killed via an overdose of ketamine/xylazine important to address its temporal expression and cellular (Bimeda-MTC animal Health Inc., Cambridge, ON) and bled by sources during EAE, and to determine how its levels are an incision to the atrium of the heart (N of 5 per time point). regulated during neuroinflammation. In this manuscript, we Immediately after bleeding the thoracic spinal cord was describe the expression and regulation of MMP-12 in EAE and removed and placed into 1 ml of TRIzol® (GibcoBRL), in tissue culture studies. homogenized via syringe, flashed frozen in liquid nitrogen and stored at −80 °C until further RNA processing. 2. Materials and methods Total RNA was isolated from thoracic spinal cords of mice using a standard extraction protocol for TRIzol® as described by 2.1. Disease induction and EAE analysis the manufacturer (Invitrogen, Missisauga, ON). Only samples with an A260/280 ratio N1.60 were used for subsequent analysis. EAE was induced in female C57BL/6 mice (Charles River, Montreal, QC), aged 8–9 weeks, by injecting subcutaneously 2.3. Generation of cDNA from RNA isolated from thoracic (s.c.) 50 μg MOG35–55 in Complete Freund's Adjuvant (CFA) spinal cords of EAE C57BL/6 mice (Fisher, Michigan USA). Intraperitoneal (i.p.) pertussis toxin (0.3 μg/200 μl, List Biological labs, Hornby, ON) was A total of 1 μg of RNA was used from each sample evaluated administered on days 0 and 2. and cDNA was made. RNA was treated with RQ1 RNAse-Free Animals were assessed daily using a 15-point disease score DNAse at 37 °C for 45 min and 65 °C for 10 min. Following scale (Giuliani et al., 2005; Weaver et al., 2005) that replaced DNAse treatment the cDNA master mix (5× First strand buffer, the more commonly used 5-point scale since the 15-point scale 0.1 M DTT, 5 mM dNTPs, Random primers (d(N)6), 40 U/ml differentiates individual limb disability, rather than grouping RNAsin/RNAse out and 200 U/ml Superscript reverse tran- both fore- or hind-limbs together, thus allowing for better scriptase) was added to each sample. Samples were incubated characterization of disease progression. The 15-point scale using the following protocol: 37 °C for 1 1/2 h, 70 °C for 5 min ranges from 0 to 15 and is the sum of the disease state for the tail and then 4 °C. cDNAwas diluted to a final volume of 100 μl and (which is scored from 0–2) and all 4 limbs (each limb is scored stored at −20 °C until further use. from 0–3). Based on this scoring system a fully quadriplegic mouse would attain a score of 14 and mortality would be given a 2.4. Real-time PCR of cDNA from thoracic spinal cords of EAE score of 15. All animals were handled in accordance with the C57BL/6 mice policies outlined by the Canadian Council for Animal Care and the University of Calgary. Real-time PCRs were done using SYBR green and relative Animals were grouped and killed at the following time fold expression values were calculated based on a housekeeping points post immunization with MOG: (1) Pre-symptomatic gene and using the comparative CT method for relative − ΔΔC animals (disease score of 0) were killed 5 days after MOG quantization using 2 T. For each sample 5 μl of cDNA immunization and before clinical signs of disease, (2) Disease was added to 20 μl of the PCR master mix (40 mM Tris–HCl onset animals (disease score 1–3) were killed during the first pH 8.4, 100 mM KCl, 6 mM MgCl2, 400 μM dGTP, 400 μM clinical signs of disease, (3) Disease progression (score 4–6) dTTP, 400 μM dCTP, 400 μM dATP, 10% Glycerol, 0.1% mice were killed during the rise of clinical disability, (4) Disease Tween 20, SYBER Green I (1/50,000), Fluroscein (1/10,000), peak (score 7 or more) mice were killed during peak clinical 5 μM primer mix, and Taq polymerase) and was incubated disease, and (5) Disease stabilization animals were killed post- using the following conditions: 95 °C for 5 min, 45 cycles of 26 A.G.