Cutting Edge: Chemotactic Activity of Soluble Fas Ligand Against Phagocytes Ken-ichiro Seino, Kazuhisa Iwabuchi, Nobuhiko Kayagaki, Ryukou Miyata, Isao Nagaoka, Akio Matsuzawa, Katashi This information is current as Fukao, Hideo Yagita and Ko Okumura of September 27, 2021. J Immunol 1998; 161:4484-4488; ; http://www.jimmunol.org/content/161/9/4484 Downloaded from References This article cites 36 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/161/9/4484.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 27, 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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. c Cutting Edge: Chemotactic Activity of Soluble Fas Ligand Against Phagocytes1 Ken-ichiro Seino,2,3*† Kazuhisa Iwabuchi,3‡ Nobuhiko Kayagaki,* Ryukou Miyata,‡ Isao Nagaoka,‡ Akio Matsuzawa,§ Katashi Fukao,† Hideo Yagita,*¶ and Ko Okumura*¶ physiologic roles of the shedding of TNF family members have not A recombinant soluble form of human Fas ligand (sFasL) was been well characterized. Downloaded from tested for its chemotactic activity against human and mouse FasL induces apoptotic cell death by binding to its receptor, Fas polymorphonuclear neutrophils (PMN) by the Boyden cham- (also called APO-1 or CD95), which is a member of the TNF ber method. sFasL exhibited a potent chemotactic activity receptor family (4). FasL is predominantly expressed in activated against both human and mouse PMN and HL-60 cells when T cells and NK cells, while Fas is ubiquitously expressed on var- differentiatedintoneutrophilsormonocytes.Aneutralizinganti- ious cells. FasL-mediated cell death is involved in the T or NK FasL mAb abolished the chemotactic activity, while control cell-mediated cytotoxicity, some pathologic tissue damages, and http://www.jimmunol.org/ mAb did not. Ligation of Fas by either IgM- or IgG-type anti- the regulation of lymphocyte homeostasis (4). FasL is also ex- Fas mAb also induced PMN migration. PMN derived from lpr pressed in the testis (5), eye (6), and some malignant tumor cells mice that express few Fas molecules did not respond to sFasL. (7, 8), which has been proposed to contribute to their immune- In contrast, those derived from lprcg mice that express Fas privileged status. FasL expressed in such immune-privileged tis- molecules with a mutated death domain normally responded to sues may eliminate infiltrating immune cells. By applying this con- sFasL chemotaxis. These results directly indicated a chemo- cept, Lau et al. reported that syngeneic myoblasts expressing FasL tactic activity of sFasL against PMN and suggest a novel sig- protected allogeneic pancreatic islets from immune rejection when naling function of Fas, which appears to be independent of the cotransplanted under the kidney capsule (9). death domain-mediated apoptosis. The Journal of Immunol- In contrast, we and others have found that enforced FasL ex- by guest on September 27, 2021 ogy, 1998, 161: 4484–4488. pression in tumor cells or islets elicited neutrophilic inflammation and destruction of the graft. When implanted s.c. or i.p., various tumor cells expressing FasL induced neutrophil infiltration and he Fas ligand (FasL)4 belongs to the TNF family, which rapid rejection (10). Similarly, expression of functional FasL in the includes TNF, lymphotoxin, TNF-related apopto- pancreatic islets of FasL-transgeneic mice induced granulocytic T sis-inducing ligand (TRAIL), CD40 ligand, CD27 ligand, infiltrates and damage of the islets (11, 12). In this context, we here CD30 ligand, and OX40 ligand (1). Most members of the TNF verified the chemotactic activity of FasL against polymorphonu- family, except for lymphotoxin-a, are type II membrane proteins. clear neutrophils (PMN). A novel signaling function of Fas, which However, a soluble form of FasL (sFasL) is naturally produced by appears to be independent of the functional death domain, was metalloproteinase-mediated processing such as TNF-a (2, 3). The noted. *Department of Immunology, Juntendo University School of Medicine, Tokyo, Ja- Materials and Methods pan; †Department of Surgery, University of Tsukuba School of Medicine, Ibaraki, Preparation of sFasL Japan; ‡Department of Biochemistry, Juntendo University School of Medicine, To- kyo, Japan; §Laboratory Animal Research Center, Institute of Medical Science, Uni- A recombinant soluble form of human FasL (sFasL) was produced by versity of Tokyo, Tokyo, Japan; and ¶Core Research for Evolutional Science and using the baculovirus expression system as described previously (13). Technology, Japan Science and Technology Corporation, Tokyo, Japan Briefly, the full-length FasL cDNA was subcloned into PVL1393 Received for publication June 1, 1998. Accepted for publication August 24, 1998. (PharMingen, San Diego, CA) and transfected into Spodoptera frugiperda (SF9) cells according to the manufacturer’s instruction. The culture super- The costs of publication of this article were defrayed in part by the payment of page natant was applied to an affinity column of protein Sepharose A conjugated charges. This article must therefore be hereby marked advertisement in accordance with an anti-FasL (NOK-1) mAb (2). The bound sFasL was eluted with 0.1 with 18 U.S.C. Section 1734 solely to indicate this fact. M glycine-HCl buffer (pH 4.0). The concentration of sFasL was deter- 1 This work was supported by grants from the Science and Technology Agency, the mined by sandwich ELISA as described previously (2). Ministry of Education, Science and Culture, and the Ministry of Health, Japan. K.S. is a research fellow of the Japan Society for the Promotion of Science. 2 Address correspondence and reprint requests to Dr. Ken-ichiro Seino, Department of Preparation of PMN Surgery, University of Tsukuba School of Medicine, 1-1-1 Tennodai, Tsukuba C3H/He wild-type and lpr mice were purchased from SLC (Shizuoka, Ja- Science-City, Ibaraki 305-8575, Japan. E-mail address: [email protected] pan). CBA lprcg mice, which have a loss-of-function mutation in the death 3 The first two authors contributed equally to this work. domain of the Fas molecule (14), had been backcrossed 12 times to C3H cg 4 Abbreviations used in this paper: FasL, Fas ligand; sFasL, soluble FasL; PMN, mice, and the C3H lpr mice were maintained in the animal facilities of polymorphonuclear neutrophils. Tokyo University and used for the experiments. Mouse PMN were isolated Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 c The Journal of Immunology 4485 from peritoneal exudates 4–5 h after an i.p. injection of 3 ml 4% thiogly- shown), indicating that the cells undergoing chemotaxis were collate (Difco, Detroit, MI) as described previously (15). Mouse PMN neutrophils. 1 preparations contained 87% Gr-1 cells as estimated by flow cytometry. Next, we examined whether ligation of Fas by anti-Fas mAb Human PMN were isolated from citrate-anticoagulated peripheral blood of healthy donors by Polymorphoprep (Nycomed Pharma, Oslo, Norway) might also induce PMN migration. We used IgM (CH-11)- or IgG centrifugation techniques as described previously (16). The purity of hu- (DX-2)-type anti-Fas mAbs, which are cytotoxic or noncytotoxic man PMN was .95% as estimated by Wright-Giemsa stain. PMN were in solution, respectively. As indicated in Fig. 1B, not only cyto- suspended in PBS containing 1 mM CaCl2 and 1 mM MgSO4. toxic CH-11 but also noncytotoxic DX-2 induced PMN migration, Cell culture suggesting that the signal transduction for the chemotaxis may be different from that for apoptosis. Human promyelocytic leukemia HL-60 cells were maintained in RPMI It has been well characterized that a human promyelocytic leu- 1640 medium (Nissui, Tokyo, Japan) containing 10% FBS, 2 mM glu- tamine, 100 mg/ml streptomycin, and 100 U/ml penicillin. To differentiate kemia cell line HL-60 can be differentiated into neutrophils or HL-60 cells into neutrophilic or monocytic lineage, the cells were cultured monocytes in the presence of DMSO or PMA, respectively (17, with 1.3% DMSO or 5 nM PMA for 4 days, respectively (17, 18), and then 18). Untreated, DMSO-treated, and PMA-treated HL-60 cells ex- used for the chemotaxis assay. The differentiation was confirmed by pressed Fas at a comparable level as estimated by FACS analysis Wright-Giemsa stain of cytospin preparations and expression of cell sur- (data not shown). Then, we examined the ability of sFasL to in- face CD11b (17, 18). duce migration of the HL-60 cells that were differentiated into Chemotaxis assay neutrophilic or monocytic lineage. As shown in Fig. 2, A and B, both untreated and DMSO-treated HL-60 cells significantly re- Cell migration was assayed by employing a modified Boyden chamber Downloaded from with cellulose nitrate filter, as described previously (19). We used a 3-mm sponded to sFasL. Similarly, PMA-treated HL-60 cells also re- pore size filter for PMN and untreated or DMSO-treated HL-60 cells and sponded to sFasL (Fig. 2C). These results suggest that sFasL has a5-mm pore size filter for PMA-treated HL-60 cells. Varying concentra- a chemotactic activity against not only neutrophils but also tions of purified sFasL, anti-Fas mAb (CH-11 (IgM, Medical Biologic monocytes.