Cutting Edge: Chemotactic Activity of Soluble Fas 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

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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. ●

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 , 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 (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 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 . 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, , 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-␣, are type II membrane . 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-␣ (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 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

● 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. Ͼ ϩ 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 Ͼ95% 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 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 ␮g/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-␮m 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-␮m 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. Laboratories, Nagoya, Japan) or DX-2 (IgG1, PharMingen)), or control mAb (mouse IgM or IgG1, PharMingen) in PBS supplemented with 1 mM Further, we evaluated the ability of sFasL to induce mouse PMN Ca2ϩ,Mg2ϩ, and 1 mg/ml BSA (Fraction V, Sigma, St. Louis, MO) were migration. As shown in Fig. 3A, sFasL ranging from 0.1 to 10 nM added to the upper or lower wells of the chamber. One million cells in 250 induced as potent a migration as did IL-8 when PMN were ob- http://www.jimmunol.org/ ϩ ϩ ␮l PBS supplemented with 1 mM Ca2 ,Mg2 , and 1 mg/ml BSA were tained from wild-type C3H mice. The chemokinetic movement of placed into the upper wells. The entire apparatus was incubated at 37°C for neutrophils was not enhanced as in Fig. 1A. The anti-FasL mAb, 45 min for PMN and untreated or DMSO-treated HL-60 cells, and 90 min for PMA-treated HL-60 cells. The membrane was then fixed with neutral but not control mAb (data not shown), abolished the FasL-induced buffered formalin for 30 min and stained with hematoxylin, hematoxylin migration. The chemotactic activity of sFasL was not observed and eosin, or Wright-Giemsa. With a ϫ40 objective, the distance (␮m) against PMN obtained from the lpr mice, which lack Fas (Fig. 3B), from the top of the filter to the furthest two cells at the same focal plane indicating that the FasL-induced PMN migration is mediated was measured microscopically. This measure was taken for 10 fields across through Fas on PMN. In contrast, as shown in Fig. 3C, PMN the filter. In each assay, human IL-8 (Genzyme, Cambridge, MA) or FMLP cg was used as a positive control. For a blocking experiment, anti-FasL mAb obtained from the lpr mice responded to sFasL almost compa- (NOK-1) (2) or control mAb (mouse IgG1, PharMingen) at a concentration rably to those from wild-type mice, suggesting that the signal by guest on September 27, 2021 of 10 ␮g/ml was added to both upper and lower chambers. transduction for cell migration may not need a functional death domain in the cytoplasmic region of Fas. Statistical analysis The statistical significance of differences from control was evaluated by Student’s t test. Values of p Ͻ 0.05 were considered significant. Discussion In the present study, we demonstrated that sFasL acts as a che- Results moattractant against human and mouse PMN in vitro. This con- We first evaluated the ability of sFasL to induce human PMN clusion is consistent with our in vivo observation that FasL trans- migration across the cellulose nitrate filters. The mean data from fectants injected in syngeneic mice induced neutrophil infiltration three individual experiments is shown in Fig. 1A. Significant PMN and rejection of the cells (10). The FasL-induced rejection ap- migration for sFasL ranging from 0.1 to 10 nM was observed, peared to be dependent upon the expression of Fas on the host although the positive chemoattractant IL-8 was more active. sFasL leukocytes, because wild-type3lpr, but not lpr3wild-type, bone as well as IL-8 showed a bell-shaped chemotactic activity. When marrow chimeras rejected the FasL transfectants with neutrophilic the same concentration of sFasL was added to both upper and infiltrates (10). Therefore, it has been speculated that FasL might lower chambers, the PMN migration was not observed, indicating directly act on infiltrating cells that were mainly composed of neu- that chemokinetic movement of PMN was not enhanced. A neu- trophils. Although the present study does not exclude a possible tralizing mAb to FasL, but not control mAb (data not shown), contribution of other indirect mechanisms, it is strongly suggested abolished the FasL-induced migration, indicating that the PMN that the chemotactic action of sFasL is directly involved in the migration was indeed induced by FasL but not by some contam- FasL-induced inflammation in vivo. inant in the rsFasL preparation. We used human sFasL in this study. We and other groups dem- Because it was possible that Fas/FasL interaction might trigger onstrated that mouse FasL also induced a similar inflammation the production of some chemotactic factor responsible for the che- (10–12). Although mouse sFasL has been shown to have little motaxis, we obtained supernatant of human PMN that had under- cytotoxic activity (3), it may have another function such as che- gone chemotaxis to sFasL and tested its chemotactic activity motaxis, which remains to be determined in further studies. Re- against PMN in the presence of anti-FasL mAb. However, the cently, several groups demonstrated a greatly reduced cytotoxic supernatant failed to induce chemotaxis (data not shown), exclud- activity of sFasL as compared with membrane-bound FasL (20– ing this possibility. 22), arguing against a systemic cytotoxic role of sFasL. Consis- To characterize the cells undergoing chemotaxis to sFasL, we tently, the sFasL preparation used in this study did not enhance the stained the filters with hematoxylin and eosin or Wright-Giemsa. apoptosis of Jurkat cells (13) and PMN (data not shown) after The migrating cells had no eosinophilic or basophilic granules (not several hours of incubation. Our present results suggest that sFasL 4486 CUTTING EDGE

FIGURE 1. sFasL and anti-Fas mAbs induce human PMN migration. A, Effect of sFasL on human PMN migra- tion. IL-8 and sFasL (0.001–1000 nM) were assessed for chemotactic activity against human peripheral blood PMN by the Boyden chamber method. The column designated as 1/1 represents the presence of sFasL (1 nM) in both upper and lower chambers. For blocking, anti-FasL mAb or control mAb (10 ␮g/ml) was added to both upper and lower cham- bers. B, Effect of anti-Fas mAbs on human PMN migration. ␮ Control mouse IgM and IgG1 mAbs (1 g/ml) and anti-Fas Downloaded from mAbs (CH-11 (IgM) and DX-2 (IgG1) 0.01–10 ␮g/ml) were ,ء .assessed for chemotactic activity against human PMN p Ͻ 0.01 compared with negative control ,ءء ;p Ͻ 0.05 (cont.). ns, Not significant. Data are shown as mean Ϯ SD of three independent experiments using different donors. http://www.jimmunol.org/ by guest on September 27, 2021

can act proinflammatory by recruiting PMN rather than proapop- it has been demonstrated that crosslinking of Fas by Ab could totically in certain pathophysiologic conditions. induce activation of the transcription factor NF-␬B (26, 27) and Recently, signaling pathways for Fas-mediated apoptosis have secretion of IL-8 (28) in certain cell lines. In this study, both IgM- been extensively characterized. Recruitment of FADD/Mort-1 and and IgG-type anti-Fas mAbs induced PMN migration (Fig. 1B). activation of -8 mediate apoptosis through the activation of Although it is generally believed that trimerization of Fas is down-stream (23). Fas can also induce apoptosis through needed for the induction of apoptosis, these results suggest that RIP and RAIDD followed by caspase-2 activation (24), or through trimerization may not be necessary for the induction of chemo- Daxx followed by jun kinase activation (25). In contrast, signaling taxis, but dimerization of Fas may be sufficient. Furthermore, in for Fas-mediated inflammation has not been characterized. So far, the present study the chemotaxis was normally induced in PMN

FIGURE 2. Effect of sFasL on migration of undifferentiated or differentiated HL-60. FMLP (10 nM) and sFasL (0.1–10 nM) were assessed for p Ͻ 0.01 compared with negative control ,ءء ;p Ͻ 0.05 ,ء .chemotactic activity against untreated (A), DMSO-treated (B), and PMA-treated (C) HL-60 cells (cont.). ns, Not significant. Data are shown as mean Ϯ SD of three independent experiments. The Journal of Immunology 4487

FIGURE 3. Effect of sFasL on mouse PMN migration. IL-8 (20 nM) and sFasL (0.1–10 nM) were assessed for chemo- tactic activity against thioglycollate-elic- ited PMN obtained from wild-type (A), lpr (B), or lprcg (C) mice. The column designated as 1/1 represents the presence of sFasL (1 nM) in both upper and lower chambers. For blocking, anti-FasL mAb or control mAb (10 ␮g/ml) was added to p Ͻ ,ء .both upper and lower chambers -p Ͻ 0.01 compared with neg ,ءء ;0.05 ative control (cont.). ns, Not significant. Data are shown as mean Ϯ SD of three independent experiments. Downloaded from obtained from lprcg mice (Fig. 3C) whose Fas molecules have a 8. Strand, S., W. J. Hofmann, H. Hug, M. Muller, G. Otto, D. Strand, S. M. Mariani, point mutation in the death domain and cannot induce apoptosis W. Stremel, P. H. Krammer, and P. R. Galle. 1996. Lymphocyte apoptosis in- duced by CD95 (APO-1/Fas) ligand-expressing tumor cells: a mechanism of due to the inability to recruit FADD or RIP (14). These data sug- immune evasion? Nature Med. 2:1361. gest that some other signaling components may be recruited to the 9. Lau, H. T., M. Yu, A. Fontana, and C. J. Stoeckert, Jr. 1996. Prevention of islet Fas molecules independently of the known death domain-interact- allograft rejection with engineered myoblasts expressing FasL in mice. Science 273:109. ing molecules for the chemotactic activity, which remained to be 10. Seino, K., N. Kayagaki, K. Okumura, and H. Yagita. 1997. Antitumor effect of http://www.jimmunol.org/ identified in further studies. locally produced CD95 ligand. Nature Med. 3:165. It has been shown that soluble human TNF-␣ has a chemotactic 11. Allison, J., H. M. Georgiou, A. Strasser, and D. L. Vaux. 1997. Transgenic expression of CD95 ligand on islet ␤ cells induces a granulocytic infiltration, but activity against human PMN in vitro (29, 30). The chemotactic does not confer immune privilege upon islet allografts. Proc. Natl. Acad. Sci. activity of sFasL demonstrated in this study appears to be compa- USA 94:3943. rable to that of TNF-␣ described in these studies. Thus, it will be 12. Kang, S.-M., D. B. Schneider, Z. Lin, D. Hanahan, D. A. Dichek, P. G. Strock, and S. Baekkeskov. 1997. Fas ligand expression in islets of Langerhans does not interesting to see whether other members in the TNF family have confer immune privilege and instead targets them for rapid destruction. Nature similar function. It has been reported that low concentrations of Med. 3:738. TNF-␣ act synergistically with IL-1 to induce neutrophil infiltra- 13. Oyaizu, N., N. Kayagaki, H. Yagita, S. Pahwa, and Y. Ikawa. 1997. Requirement

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