Differential Regulation and Function of Fas Expression on Glial Cells Sung Joong Lee, Tong Zhou, Chulhee Choi, Zheng Wang and Etty N. Benveniste This information is current as of September 28, 2021. J Immunol 2000; 164:1277-1285; ; doi: 10.4049/jimmunol.164.3.1277 http://www.jimmunol.org/content/164/3/1277 Downloaded from References This article cites 70 articles, 33 of which you can access for free at: http://www.jimmunol.org/content/164/3/1277.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 28, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Differential Regulation and Function of Fas Expression on Glial Cells1 Sung Joong Lee,* Tong Zhou,† Chulhee Choi,* Zheng Wang,† and Etty N. Benveniste2* Fas/Apo-1 is a member of the TNF receptor superfamily that signals apoptotic cell death in susceptible target cells. Fas or Fas ligand (FasL)-deficient mice are relatively resistant to the induction of experimental allergic encephalomyelitis, implying the involvement of Fas/FasL in this disease process. We have examined the regulation and function of Fas expression in glial cells (astrocytes and microglia). Fas is constitutively expressed by primary murine microglia at a low level and significantly up- regulated by TNF-␣ or IFN-␥ stimulation. Primary astrocytes express high constitutive levels of Fas, which are not further affected by cytokine treatment. In microglia, Fas expression is regulated at the level of mRNA expression; TNF-␣ and IFN-␥ induced Fas mRNA by ϳ20-fold. STAT-1␣ and NF-␬B activation are involved in IFN-␥- or TNF-␣-mediated Fas up-regulation in microglia, ␤ respectively. The cytokine TGF- inhibits basal expression of Fas as well as cytokine-mediated Fas expression by microglia. Upon Downloaded from incubation of microglial cells with FasL-expressing cells, ϳ20% of cells underwent Fas-mediated cell death, which increased to ϳ60% when cells were pretreated with either TNF-␣ or IFN-␥. TGF-␤ treatment inhibited Fas-mediated cell death of TNF-␣-or IFN-␥-stimulated microglial cells. In contrast, astrocytes are resistant to Fas-mediated cell death, however, ligation of Fas induces expression of the chemokines macrophage inflammatory protein-1␤ (MIP-1␤), MIP-1␣, and MIP-2. These data demonstrate that Fas transmits different signals in the two glial cell populations: a cytotoxic signal in microglia and an inflammatory signal in the astrocyte. The Journal of Immunology, 2000, 164: 1277–1285. http://www.jimmunol.org/ as (CD95) is a type I transmembrane glycoprotein belong- phoadenopathy and autoimmunity reminiscent of systemic lu- ing to the TNF receptor superfamily (1). It is expressed on pus erythematosus (13). F activated T and B cells, thymocytes, malignant T and B In experimental allergic encephalomyelitis (EAE), an autoim- cells, and in a variety of tissues outside the immune system, in- mune inflammatory disease of the CNS, the involvement of Fas/ cluding liver and lung (2). Upon ligation with agonistic Ab or the FasL was clearly demonstrated by observations that lpr or gld mice 3 natural Fas ligand (FasL), Fas trimerizes and recruits a number of are relatively resistant to the clinical development of EAE (14, 15). proteins sharing a death domain that leads to the formation of a These data suggested that the Fas/FasL pathway plays a significant specific death-inducing signaling complex (DISC) at the intracel- role in the development of EAE, possibly by mediating apoptosis by guest on September 28, 2021 lular region of the Fas receptor (3). The recruitment of caspase-8 of potential target cells. However, in two separate studies, oligo- to DISC results in proteolytic activation of the enzyme, which, in dendrocytes, the myelin-producing cells in the CNS that are con- turn, activates a series of other caspase members (4). FasL is a type sidered a major target in the disease of EAE, were resistant to II transmembrane glycoprotein that induces apoptosis in target FasL-mediated apoptosis and did not undergo apoptosis during the cells in both the membrane-bound form and the soluble form pathogenesis of EAE (16, 17). To the contrary, infiltrating CD4ϩ (sFasL) (5, 6). In the immune system, cell death through the Fas/ T cells and parenchymal microglia were killed by apoptosis, sug- FasL system is involved in B and T cell homeostasis and is also gesting that the effector cells, rather than the target cells, are elim- critical for CTL-mediated cytotoxicity (7–10). Recently, it was inated by apoptosis in the EAE disease process (16). More re- reported that the Fas/FasL system is responsible for immune cently, the involvement of Fas/FasL in EAE progression has been down-regulation in previously known immune-privileged sites dissected using the adoptive transfer model of EAE (18, 19). Ac- such as cornea and testis (11, 12). Mouse strains carrying cording to these studies, Fas expressed in the recipient animal as mutations in the Fas (lpr) or FasL (gld) genes exhibit lym- well as FasL expressed on donor cells are both important for the development of EAE. These data suggest that Fas expression on Departments of *Cell Biology and †Medicine, University of Alabama at Birmingham, CNS-resident glial cells contributes to EAE induction. Birmingham, AL 35294 In an attempt to elucidate the involvement of Fas/FasL in EAE Received for publication May 3, 1999. Accepted for publication November 22, 1999. as well as MS pathogenesis, Fas expression and function on glial The costs of publication of this article were defrayed in part by the payment of page cells has been investigated in several in vitro studies. In a study charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. using human oligodendrocytes, Fas ligation with anti-Fas Ab in- 1 This work was supported in part by National Institutes of Health Grants MH55795, duced rapid lysis of oligodendrocytes, supporting the idea of Fas- NS36765, and NS29719 (to E.N.B.). We acknowledge the support of the University dependent oligodendrocyte elimination in MS (20). However, this of Alabama at Birmingham Flow Cytometry Core Facility (Grant AM20614). Fas-dependent cell lysis did not exhibit characteristics of typical 2 Address correspondence and reprint requests to Dr. Etty N. Benveniste, Department apoptosis. High constitutive expression of Fas on mouse astrocytes of Cell Biology, 350 MCLM, University of Alabama at Birmingham, Birmingham, AL 35294-0005. E-mail address: [email protected] has been reported as well, but astrocyte susceptibility to FasL- 3 Abbreviations used in this paper: FasL, Fas ligand; DISC, death-inducing signaling mediated apoptosis is still controversial (21, 22). In a study using complex; EAE, experimental allergic encephalomyelitis; MIP, macrophage inflam- astrocyte cultures from fetal CNS, although Fas was constitutively matory protein; MS, multiple sclerosis; MAPK, mitogen-activated protein kinase; RPA, RNase protection assay; ERK, extracellular signal-related kinase; MEK, MAP/ expressed on astrocytes, Fas ligation failed to induce astrocyte cell ERK kinase; FAP-1, Fas-associated phosphatase-1. death (23). In another report, Fas was constitutively expressed by Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 1278 FAS EXPRESSION AND FUNCTION ON GLIAL CELLS human fetal astrocytes as well as adult astrocytes and was up- Analysis of Fas protein expression by immunofluorescence flow regulated by several proinflammatory cytokines such as IL-1, IL-6, cytometry ␥ ␣ IFN- , and TNF- (24). In addition, the fetal astrocytes underwent Primary astrocytes were plated at 5 ϫ 105 cells/well into six-well plates apoptosis when treated with agonistic anti-Fas Ab. It has been (Costar, Cambridge, MA) and treated with IFN-␥ or TNF-␣ in the absence suggested that the susceptibility of human astrocytes to Fas-me- or the presence of TGF-␤1 for various time periods. The cells were diated apoptosis is dependent on cell passage and other microen- trypsinized, washed with PBS, incubated with 20 ␮g/ml of PE-conjugated anti-Fas Ab (Jo2) for1hat4°C, washed, then fixed in a final volume of vironmental factors; only late passage astrocytes are sensitive to ␮ ␥ 100 l of 1% paraformaldehyde. The cells were then analyzed on the Fas-induced apoptosis and the presence of IFN- enhances apo- FACScan (Becton Dickinson, Mountain View, CA). Total fluorescence ptosis (25). Apoptosis in microglia has not been well studied com- intensity was calculated as the mean fluorescence intensity ϫ percentage of pared with that in astrocytes. In several studies using immunohis- positive cells. Negative controls were incubated with isotype-matched Ab. tochemistry in EAE brain, microglial cells were positively stained For the analysis of primary microglia or EOC13 cells, cells were treated as described above, then scraped to detach from the well and incubated with by the TUNEL assay more frequently than astrocytes (16). Re- 50 ␮l of 2.4G2 supernatant (which contains rat anti-mouse Fc␥R Ab) sup- cently, Fas-mediated apoptosis was reported in cultured microglia plemented with 10% normal mouse serum for 30 min at 4°C before addi- upon TNF-␣ or IFN-␥ stimulation (26). tion of PE-conjugated anti-Fas Ab.