The Glycosyl-Inositol-Phosphate and Dimyristoylglycerol Moieties of the Glycosylphosphatidylinositol Anchor of the Trypanosome Variant-Specific Surface This information is current as Glycoprotein Are Distinct of September 27, 2021. Macrophage-Activating Factors Stefan Magez, Benoi?t Stijlemans, Magdalena Radwanska, Etienne Pays, Michael A. J. Ferguson and Patrick De Baetselier J Immunol 1998; 160:1949-1956; ; Downloaded from http://www.jimmunol.org/content/160/4/1949 References This article cites 40 articles, 21 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/160/4/1949.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 27, 2021 • Fast Publication! 4 weeks from acceptance to publication *average 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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Glycosyl-Inositol-Phosphate and Dimyristoylglycerol Moieties of the Glycosylphosphatidylinositol Anchor of the Trypanosome Variant-Specific Surface Glycoprotein Are Distinct Macrophage-Activating Factors1 Stefan Magez,2* Benoıˆt Stijlemans,* Magdalena Radwanska,† Etienne Pays,† Michael A. J. Ferguson,‡ and Patrick De Baetselier* The TNF-a-inducing capacity of different trypanosome components was analyzed in vitro, using as indicator cells a macrophage cell line (2C11/12) or peritoneal exudate cells from LPS-resistant C3H/HeJ mice and LPS-sensitive C3H/HeN mice. The variant- specific surface glycoprotein (VSG) was identified as the major TNF-a-inducing component present in trypanosome-soluble extracts. Both soluble (sVSG) and membrane-bound VSG (mfVSG) were shown to manifest similar TNF-a-inducing capacities, Downloaded from indicating that the dimyristoylglycerol (DMG) compound of the mfVSG anchor was not required for TNF-a triggering. Detailed analysis indicated that the glycosyl-inositol-phosphate (GIP) moiety was responsible for the TNF-a-inducing activity of VSG and that the presence of the GIP-associated galactose side chain was essential for optimal TNF-a production. Furthermore, the results showed that the responsiveness of macrophages toward the TNF-a-inducing activity of VSG was strictly dependent on the activation state of the macrophages, since resident macrophages required IFN-g preactivation to become responsive. Comparative analysis of the ability of both forms of VSG to activate macrophages revealed that mfVSG but not sVSG stimulates macrophages http://www.jimmunol.org/ toward IL-1a secretion and acquisition of LPS responsiveness. The priming activity of mfVSG toward LPS responsiveness was also demonstrated in vivo and may be relevant during trypanosome infections, since Trypanosoma brucei-infected mice became gradually LPS-hypersensitive during the course of infection. Collectively, the VSG of trypanosomes encompasses two distinct macrophage-activating components: while the GIP moiety of sVSG mediates TNF-a induction, the DMG compound of the mfVSG anchor contributes to IL-1a induction and LPS sensitization. The Journal of Immunology, 1998, 160: 1949–1956. umor necrosis factor a is a pleiotropic cytokine, produced In several of these infections, TNF-a plays a bidirectional role, mainly by activated macrophages (1). Although its name influencing both parasite and host (8). In the case of trypanosome by guest on September 27, 2021 T is derived from its capacity to cause necrosis of certain infections, TNF-a seems to be involved in the neuropathology of parenchymal organs and tumors, the molecule was initially also sleeping sickness, and a correlation has been described between isolated as a factor named “cachectin,” responsible for systemic the disease severity in human African trypanosomiasis and high suppression of lipoprotein lipase activity in trypanosome-induced serum levels of the cytokine (9). Furthermore, a possible associ- cachexia (2, 3). As TNF-a is a main mediator in inflammatory ation between TNF-a production by monocytes from trypano- responses, its influence in a number of infectious disease has been some-infected cattle and the severity of disease-associated anemia studied. Among other effects, TNF-a was found to be involved in has been documented (10). Using mouse models, the immunopa- the pathology of several parasitic diseases, including trypanoso- thology of experimental African sleeping sickness has been linked miasis (4), Chagas’ disease (5), leishmaniasis (6), and malaria (7). to TNF-a-mRNA detection in the brain of Trypanosoma brucei- infected mice (11), and a role for TNF-a involvement in trypano- somiasis-associated immunosuppression has been put forward a *Laboratory of Cellular Immunology, Flanders Interuniversity Institute for Biotech- (12). Finally, a direct involvement of TNF- in trypanosomiasis nology, Free University of Brussels (Vrije Universiteit Brussel), and †Department of control and parasite growth was documented in vivo (4, 13), and a Molecular Biology, Free University of Brussels (Universite´Libre de Bruxelles), Brus- trypanolytic effect of TNF-a on T. brucei was confirmed in vitro sels, Belgium; and ‡Department of Biochemistry, The University Dundee, Scotland, United Kingdom (14–16). Information concerning the trypanosome-derived fac- a Received for publication April 17, 1997. Accepted for publication October 31, 1997. tor(s) that may be responsible for the induction of TNF- The costs of publication of this article were defrayed in part by the payment of page production by host cells is scarce. In analogy with plasmodia-as- charges. This article must therefore be hereby marked advertisement in accordance sociated TNF-a induction, trypanosomiasis-associated TNF-a in- with 18 U.S.C. Section 1734 solely to indicate this fact. duction has been suggested to be mediated by the glycosylphos- 1 This project has been funded by the UNDP/World Bank/WHO Special Programme phatidylinositol (GPI)3 anchor on the variant-specific surface for Research and Training in Tropical Diseases, The Belgian National Fund for Sci- entific Research (FWO-No. 6.0325.95 and FNRS-No. 3.4502.96), the Flemish Gov- glycoprotein (VSG) of the trypanosome (17). VSG is the crucial ernment (Vlaams Actieprogramma Biotechnologie-VLAB), the European Commu- molecule involved in the escape of the host immune response, as nity (Cost contract No. TS3-CT94-0293), and the Communaute´Franc¸aise de Belgique 107 identical VSGs cover each trypanosome and form a dense coat (ARC 94/99-189). This project was performed within the framework of an Interuni- versity Attraction Pole Programme, financed by the Belgian state government (Dien- around each cell (18, 19). The mechanism of immune escape is sten van de Eerste Minister-Federale diensten voor wetenschappelijke, technische en culturele aangelegenheden). S.M. is a Postdoctoral Research Fellow of the Foundation of Scientific Research-Flanders (FWO). 3 Abbreviations used in this paper: GPI, glycosylphosphatidylinositol; DMG, dimyr- 2 Address correspondence and reprint requests to Stefan Magez, Eenheid CIMM istoylglycerol; GIP, glycosyl-inositol-phosphate; VSG, variant-specific surface gly- (IMOL 2), Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit coprotein; sVSG, soluble VSG; mfVSG, membrane form VSG; PEC, peritoneal ex- Brussel, Paardenstraat 65, 1640 Sint Genesius Rode, Belgium. udate cell; TLCK, Na-( p-tosyl)lysine chloromethyl ketone. Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 1950 VSG-MEDIATED INDUCTION OF CYTOKINE PRODUCTION based on a regular switch in the expression of VSG variants, jeop- recombinant N-glycosidase F from Flavobacterium meningosepticum, ardizing the induction of an effective Ab response against the par- a-galactosidase from green coffee beans, Pronase from Streptomyces gri- asite. Interestingly, in response to environmental stress, trypano- seus, Proteinase K from Tritirachium album, and the protease inhibitors Pefabloc SC, PMSF, and TLCK. All enzymes and inhibitors were used somes are capable of liberating their VSG through a VSG lipase according to the manufacturer’s instructions. (20). This enzyme cleaves the GPI anchor, leaving the dimyris- N-Glycosidase F digestion was performed in PBS using 5 U/ml enzyme toylglycerol (DMG) compound of the GPI anchored in the mem- (24 h/37°C). brane, and releasing the glycosyl-inositol-phosphate (GIP)-VSG a-Galactosidase digestion of VSG was performed in 0.1 M sodium acetate buffer, pH 5.0, using 50 U/ml enzyme (2 3 18 h/37°C). part (21). In this report, we compare the macrophage-activating Proteinase K digestion was performed in PBS, pH 8.0, using 5 U/ml capacity, analyzed in terms of cytokine secretion (TNF-a/IL-1a, enzyme (24 h/37°C). IL-6, IL-10, and IL-12) and induction of LPS hyper-responsive- Pefabloc SC was used when indicated (1 mM final concentration) to ness, of the released soluble VSG (sVSG) and the membrane- prevent proteolysis during lysate preparation