Metalloproteinases Are Enriched in Microglia Compared with Leukocytes and They Regulate Cytokine Levels in Activated Microglia
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GLIA 55:516–526 (2007) Metalloproteinases Are Enriched in Microglia Compared with Leukocytes and They Regulate Cytokine Levels in Activated Microglia ROBERT K. NUTTALL,1 CLAUDIA SILVA,2 WALTER HADER,2 AMIT BAR-OR,3 KAMALA D. PATEL,4,5 1 2 DYLAN R. EDWARDS, AND V. WEE YONG * 1School of Biological Sciences, University of East Anglia, Norwich, United Kingdom 2Department of Clinical Neurosciences, University of Calgary, Calgary, Alberta, Canada 3Montreal Neurological Institute, Montreal, Quebec, Canada 4Department f Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada 5Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada KEY WORDS ries to the CNS by becoming activated (Kreutzberg, 1996; microglia; cytokines; metalloproteinases; cell activation Streit et al., 1999). In this regard, they change their mor- phology from ramified to large round cells resembling macrophages. They proliferate and become more motile, ABSTRACT they phagocytose cellular debris, and they produce a num- Microglia are resident immune cells within the central ner- ber of cytokines including tumor necrosis factor-a (TNF-a), vous system (CNS). They become activated following neuro- b b logical insults and increase their expression of cytokines. interleukin-1 (IL-1 ), and IL-10 (Becher et al., 2000; Also elevated in CNS injuries are proteases, including matrix Buttini et al., 1994; Chabot and Yong, 2000; Kreutzberg, metalloproteinases (MMPs) and A disintegrin and metallo- 1996; Streit et al., 1999). The full spectrum of microglia proteinases (ADAMs). The spectrum of metalloproteinase activity imparts complex outcomes on the integrity of the members expressed by microglia and by the systemic leuko- CNS. While the persistent activation of microglia and the cytes that infiltrate the injured CNS is unknown, as are their elaboration of a range of molecules are thought to be detri- functions. We determined the levels of transcripts encoding mental, some microglial responses such as the production all 24 MMPs, nine ADAMs, and their four physiological of neurotrophic factors and removal of toxic products antagonists, tissue inhibitor of metalloproteinases (TIMPs), (Batchelor et al., 1999; Simard et al., 2006) may serve to in human microglia, B and T cells, monocytes, and neutro- limit injury and enhance repair. Understanding microglial phils. We found a distinct pattern for each immune subset and an enrichment of metalloproteinases in microglia com- activity and controlling their actions can have an impor- pared with leukocytes. When microglia were activated, there tant consequence on the recovery of the nervous system. was an upregulation of transcripts for nine metalloprotei- The metalloproteinase subfamilies of matrix metallopro- nases, and reduction of TIMP3. Activation of microglia also teinases (MMPs) and A disintegrin and metalloprotei- resulted in increased levels of tumor necrosis factor (TNF)-a, nases (ADAMs) are proteolytic enzymes that are promptly interleukin (IL)-1b, and IL-10 protein in the conditioned expressed and elevated following all types of insults to the media of cells. The amount of secreted TNF-a,butnotIL-1b CNS (Cunningham et al., 2005; Yong, 2005; Yong et al., or IL-10, was suppressed by BB94, a broad spectrum metal- 2001; Zhao et al., 2006). There are 24 MMP members that loproteinase inhibitor, and by TIMP3 but not TIMP1 or are secreted or membrane-bound, and all have metallopro- TIMP2. This inhibitory profile suggests the involvement of an ADAM member in TNF-a secretion. We conclude that teinase activity. Of the 32 human membrane-associated microglia bear a metalloproteinase signature distinct from ADAMs, many are restricted to the testes and only about systemic cells, and that following activation, microglia half the family have the HExxHxxGxxH catalytic core upregulate TNF-a protein levels through a combination of required for metalloproteinase activity. There are only elevated cytokine transcripts, increased metalloproteinase nine ADAMs that have both a broad tissue distribution level and activity, and through the decrease of TIMP3. The and the capacity to be proteolytically active (Blobel, 2005). results have implications for the regulation of neuroinflam- The activity of metalloproteinases is inhibited principally mation and its outcomes following CNS injuries. by the four physiological tissue inhibitors of metalloprotei- VC 2007 Wiley-Liss, Inc. This article contains supplementary material available via the Internet at http:// www.interscience.wiley.com/jpages/0894-1491/suppmat. INTRODUCTION Grant sponsor: Canadian Institutes of Health Research. *Correspondence to: V. Wee Yong, PhD, Hotchkiss Brain Institute and the Depart- Microglia are resident immune cells within the central ments of Clinical Neurosciences and Oncology, University of Calgary, 3330 Hospital Drive, Calgary, Alberta T2N 4N1, Canada. E-mail: [email protected] nervous system (CNS). In the uninjured adult brain, they Received 19 July 2006; Revised 26 November 2006; Accepted 28 November 2006 are highly dynamic surveyors that continually send and DOI 10.1002/glia.20478 retract processes into the local environment (Nimmerjahn Published online 10 January 2007 in Wiley InterScience (www.interscience. et al., 2005). Microglia respond rapidly to all types of inju- wiley.com). VC 2007 Wiley-Liss, Inc. MMPs AND ADAMs IN MICROGLIA 517 nases (TIMPs). For the most part, each of the TIMPs can the hippocampus, and they comprise material that had to inhibit MMPs, although TIMP1 cannot block the mem- be resected enroute to the epileptic focus. In previous brane-type MMPs effectively (Baker et al., 2002). Selectiv- work, we had described that microglia so obtained were ity for ADAMs is more pronounced: TIMP3 is the only in- not different from microglia derived from other neural hibitor of ADAM17 (Amour et al., 1998), but cannot inhibit pathologies or from normal brain (Williams et al., 1992). ADAM8 and ADAM9 (Amour et al., 2002); while TIMP1 We have also not found any obvious differences in the and TIMP3 can also inhibit ADAM10 (Amour et al., 2000). microglia characteristics in culture when these are As with microglia actions, the functions of MMPs and obtained from different areas of the CNS. In addition, ADAMs are complex and they not only have proinflamma- adult microglia are comparable to microglia obtained tory and neurotoxic properties but also reparative ones from fetal human brain with respect to their production (Cunningham et al., 2005; Yong, 2005; Zhao et al., 2006). of inflammatory cytokines (Chabot et al., 2002; Giuliani These differential functions are likely due to their clea- et al., 2005). vage of extracellular matrix proteins, growth factors, cyto- Microglia from human adult brain were derived using kines, chemokines, adhesion molecules, and cell surface methods that were previously published (Chabot and Yong, receptors (McCawley and Matrisian, 2001). For example, 2000; Williams et al., 1992). In brief, fragmented brain tis- TNF-a is actually synthesized as pro-TNF-a (26 kDa) that sue was incubated with 0.25% trypsin and 100 lg/mL is converted by metalloproteinase activity to the mature DNase for 1 h at 37°C, and dissociated into single cells by 17 kDa cytokine TNF-a (Black et al., 1997; Moss et al., forcing them through a mesh of 130 lm pore size. Follow- 1997). ing centrifugation through 30% Percoll, which allowed In the adult CNS, most MMPs are expressed at low or myelin, debris, and red blood cells to be separated from undetectable levels, but several become upregulated in viable neural cells, the latter cell layer was removed, cen- neurological diseases, such as multiple sclerosis, malig- trifuged, washed, and plated into T-25 uncoated flasks. nant glioma, and stroke (reviewed in Yong et al., 2001). Feeding medium was 10% fetal bovine serum in minimum The sources of the increase in MMP and ADAM members essential medium supplemented with 0.5% dextrose. Micro- in response to an insult within the CNS are likely multi- glia from adult human brain are very adherent cells, as ple, and they include neural cells as well as leukocytes opposed to other cell types (e.g. oligodendrocyte), on un- that have infiltrated into the CNS. A systematic compari- coated flasks. Thus, one day after plating, floating cells son of the relative capacity of different cell types to ex- were removed to leave behind adherent microglia. The lat- press the full spectrum of MMP and ADAM family mem- ter was removed by retrypsinization 2–3 days after, re- bers is nonexistent. In particular, a comparison between plated, and experiments (see below) were then initiated microglia and their systemic counterparts in immunity, 1–2 days following replating. The purity of microglia was leukocytes, is lacking. This is important since all these typically in excess of 95%, as evaluated using markers cell types mediate inflammatory responses within the such as CD11b and Iba1 (Fig. 1). CNS in response to an insult, and the roles of MMPs pro- In experiments to obtain microglia RNA, 500,000 cells duced by these different cell types in modulating immune were seeded per well of a six-well plate. In experiments responses need clarification. to collect conditioned media for cytokine protein analyses, In this study, we have profiled all 24 MMP members 5,000 microglia cells were seeded per well of a 96-well and nine ADAMs expressed by microglia and leukocyte plate. The initial seeding medium consisted of minimum subsets. We found distinct profiles of metalloproteinases