A recombinant soluble form of human Fas ligand (sFasL) was tested for its chemotactic activity against human and mouse polymorphonuclear neutrophils (PMN) by the Boyden chamber method. sFasL exhibited a potent chemotactic activity against both human and mouse PMN and HL-60 cells when differentiated into neutrophils or monocytes. A neutralizing anti-FasL mAb abolished the chemotactic activity, while control mAb did not. Ligation of Fas by either IgM- or IgG-type anti-Fas mAb also induced PMN migration. PMN derived from lpr mice that express few Fas molecules did not respond to sFasL. In contrast, those derived from lprcg mice that express Fas molecules with a mutated death domain normally responded to sFasL chemotaxis. These results directly indicated a chemotactic activity of sFasL against PMN and suggest a novel signaling function of Fas, which appears to be independent of the death domain-mediated apoptosis.
The Fas ligand (FasL)4 belongs to the TNF family, which includes TNF, lymphotoxin, TNF-related apoptosis-inducing ligand (TRAIL), CD40 ligand, CD27 ligand, CD30 ligand, and OX40 ligand (1). Most members of the TNF family, except for lymphotoxin-α, are type II membrane proteins. However, a soluble form of FasL (sFasL) is naturally produced by metalloproteinase-mediated processing such as TNF-α (2, 3). The physiologic roles of the shedding of TNF family members have not been well characterized.
FasL induces apoptotic cell death by binding to its receptor, Fas (also called APO-1 or CD95), which is a member of the TNF receptor family (4). FasL is predominantly expressed in activated T cells and NK cells, while Fas is ubiquitously expressed on various cells. FasL-mediated cell death is involved in the T or NK cell-mediated cytotoxicity, some pathologic tissue damages, and the regulation of lymphocyte homeostasis (4). FasL is also expressed in the testis (5), eye (6), and some malignant tumor cells (7, 8), which has been proposed to contribute to their immune-privileged status. FasL expressed in such immune-privileged tissues may eliminate infiltrating immune cells. By applying this concept, Lau et al. reported that syngeneic myoblasts expressing FasL protected allogeneic pancreatic islets from immune rejection when cotransplanted under the kidney capsule (9).
In contrast, we and others have found that enforced FasL expression 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 rapid rejection (10). Similarly, expression of functional FasL in the pancreatic islets of FasL-transgeneic mice induced granulocytic infiltrates and damage of the islets (11, 12). In this context, we here verified the chemotactic activity of FasL against polymorphonuclear neutrophils (PMN). A novel signaling function of Fas, which appears to be independent of the functional death domain, was noted.
Materials and Methods
Preparation of sFasL
A recombinant soluble form of human FasL (sFasL) was produced by using the baculovirus expression system as described previously (13). Briefly, the full-length FasL cDNA was subcloned into PVL1393 (PharMingen, San Diego, CA) and transfected into Spodoptera frugiperda (SF9) cells according to the manufacturer’s instruction. The culture supernatant was applied to an affinity column of protein Sepharose A conjugated with an anti-FasL (NOK-1) mAb (2). The bound sFasL was eluted with 0.1 M glycine-HCl buffer (pH 4.0). The concentration of sFasL was determined by sandwich ELISA as described previously (2).
Preparation of PMN
C3H/He wild-type and lpr mice were purchased from SLC (Shizuoka, Japan). CBA lprcg mice, which have a loss-of-function mutation in the death domain of the Fas molecule (14), had been backcrossed 12 times to C3H mice, and the C3H lprcg mice were maintained in the animal facilities of Tokyo University and used for the experiments. Mouse PMN were isolated from peritoneal exudates 4–5 h after an i.p. injection of 3 ml 4% thioglycollate (Difco, Detroit, MI) as described previously (15). Mouse PMN preparations contained >87% Gr-1+ cells as estimated by flow cytometry. Human PMN were isolated from citrate-anticoagulated peripheral blood of healthy donors by Polymorphoprep (Nycomed Pharma, Oslo, Norway) centrifugation techniques as described previously (16). The purity of human PMN was >95% as estimated by Wright-Giemsa stain. PMN were suspended in PBS containing 1 mM CaCl2 and 1 mM MgSO4.
Human promyelocytic leukemia HL-60 cells were maintained in RPMI 1640 medium (Nissui, Tokyo, Japan) containing 10% FBS, 2 mM glutamine, 100 μg/ml streptomycin, and 100 U/ml penicillin. To differentiate HL-60 cells into neutrophilic or monocytic lineage, the cells were cultured with 1.3% DMSO or 5 nM PMA for 4 days, respectively (17, 18), and then used for the chemotaxis assay. The differentiation was confirmed by Wright-Giemsa stain of cytospin preparations and expression of cell surface CD11b (17, 18).
Cell migration was assayed by employing a modified Boyden chamber with cellulose nitrate filter, as described previously (19). We used a 3-μm pore size filter for PMN and untreated or DMSO-treated HL-60 cells and a 5-μm pore size filter for PMA-treated HL-60 cells. Varying concentrations of purified sFasL, anti-Fas mAb (CH-11 (IgM, Medical Biologic Laboratories, Nagoya, Japan) or DX-2 (IgG1, PharMingen)), or control mAb (mouse IgM or IgG1, PharMingen) in PBS supplemented with 1 mM Ca2+, Mg2+, and 1 mg/ml BSA (Fraction V, Sigma, St. Louis, MO) were added to the upper or lower wells of the chamber. One million cells in 250 μl PBS supplemented with 1 mM Ca2+, Mg2+, and 1 mg/ml BSA were placed into the upper wells. The entire apparatus was incubated at 37°C for 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 buffered formalin for 30 min and stained with hematoxylin, hematoxylin and eosin, or Wright-Giemsa. With a ×40 objective, the distance (μm) from the top of the filter to the furthest two cells at the same focal plane was measured microscopically. This measure was taken for 10 fields across the filter. In each assay, human IL-8 (Genzyme, Cambridge, MA) or FMLP was used as a positive control. For a blocking experiment, anti-FasL mAb (NOK-1) (2) or control mAb (mouse IgG1, PharMingen) at a concentration of 10 μg/ml was added to both upper and lower chambers.
The statistical significance of differences from control was evaluated by Student’s t test. Values of p < 0.05 were considered significant.
We first evaluated the ability of sFasL to induce human PMN migration across the cellulose nitrate filters. The mean data from three individual experiments is shown in Fig. 1 A. Significant PMN migration for sFasL ranging from 0.1 to 10 nM was observed, although the positive chemoattractant IL-8 was more active. sFasL as well as IL-8 showed a bell-shaped chemotactic activity. When the same concentration of sFasL was added to both upper and lower chambers, the PMN migration was not observed, indicating that chemokinetic movement of PMN was not enhanced. A neutralizing mAb to FasL, but not control mAb (data not shown), abolished the FasL-induced migration, indicating that the PMN migration was indeed induced by FasL but not by some contaminant in the rsFasL preparation.
Because it was possible that Fas/FasL interaction might trigger the production of some chemotactic factor responsible for the chemotaxis, we obtained supernatant of human PMN that had undergone chemotaxis to sFasL and tested its chemotactic activity against PMN in the presence of anti-FasL mAb. However, the supernatant failed to induce chemotaxis (data not shown), excluding this possibility.
To characterize the cells undergoing chemotaxis to sFasL, we stained the filters with hematoxylin and eosin or Wright-Giemsa. The migrating cells had no eosinophilic or basophilic granules (not shown), indicating that the cells undergoing chemotaxis were neutrophils.
Next, we examined whether ligation of Fas by anti-Fas mAb might also induce PMN migration. We used IgM (CH-11)- or IgG (DX-2)-type anti-Fas mAbs, which are cytotoxic or noncytotoxic in solution, respectively. As indicated in Fig. 1 B, not only cytotoxic CH-11 but also noncytotoxic DX-2 induced PMN migration, suggesting that the signal transduction for the chemotaxis may be different from that for apoptosis.
It has been well characterized that a human promyelocytic leukemia cell line HL-60 can be differentiated into neutrophils or monocytes in the presence of DMSO or PMA, respectively (17, 18). Untreated, DMSO-treated, and PMA-treated HL-60 cells expressed Fas at a comparable level as estimated by FACS analysis (data not shown). Then, we examined the ability of sFasL to induce migration of the HL-60 cells that were differentiated into neutrophilic or monocytic lineage. As shown in Fig. 2, A and B, both untreated and DMSO-treated HL-60 cells significantly responded to sFasL. Similarly, PMA-treated HL-60 cells also responded to sFasL (Fig. 2 C). These results suggest that sFasL has a chemotactic activity against not only neutrophils but also monocytes.
Further, we evaluated the ability of sFasL to induce mouse PMN migration. As shown in Fig. 3,A, sFasL ranging from 0.1 to 10 nM induced as potent a migration as did IL-8 when PMN were obtained from wild-type C3H mice. The chemokinetic movement of neutrophils was not enhanced as in Fig. 1,A. The anti-FasL mAb, but not control mAb (data not shown), abolished the FasL-induced migration. The chemotactic activity of sFasL was not observed against PMN obtained from the lpr mice, which lack Fas (Fig. 3,B), indicating that the FasL-induced PMN migration is mediated through Fas on PMN. In contrast, as shown in Fig. 3 C, PMN obtained from the lprcg mice responded to sFasL almost comparably to those from wild-type mice, suggesting that the signal transduction for cell migration may not need a functional death domain in the cytoplasmic region of Fas.
In the present study, we demonstrated that sFasL acts as a chemoattractant against human and mouse PMN in vitro. This conclusion is consistent with our in vivo observation that FasL transfectants injected in syngeneic mice induced neutrophil infiltration and rejection of the cells (10). The FasL-induced rejection appeared to be dependent upon the expression of Fas on the host leukocytes, because wild-type→lpr, but not lpr→wild-type, bone marrow chimeras rejected the FasL transfectants with neutrophilic infiltrates (10). Therefore, it has been speculated that FasL might directly act on infiltrating cells that were mainly composed of neutrophils. Although the present study does not exclude a possible contribution of other indirect mechanisms, it is strongly suggested that the chemotactic action of sFasL is directly involved in the FasL-induced inflammation in vivo.
We used human sFasL in this study. We and other groups demonstrated that mouse FasL also induced a similar inflammation (10, 11, 12). Although mouse sFasL has been shown to have little cytotoxic activity (3), it may have another function such as chemotaxis, which remains to be determined in further studies. Recently, several groups demonstrated a greatly reduced cytotoxic activity of sFasL as compared with membrane-bound FasL (20, 21, 22), arguing against a systemic cytotoxic role of sFasL. Consistently, the sFasL preparation used in this study did not enhance the apoptosis of Jurkat cells (13) and PMN (data not shown) after several hours of incubation. Our present results suggest that sFasL can act proinflammatory by recruiting PMN rather than proapoptotically in certain pathophysiologic conditions.
Recently, signaling pathways for Fas-mediated apoptosis have been extensively characterized. Recruitment of FADD/Mort-1 and activation of caspase-8 mediate apoptosis through the activation of down-stream caspases (23). Fas can also induce apoptosis through RIP and RAIDD followed by caspase-2 activation (24), or through Daxx followed by jun kinase activation (25). In contrast, signaling for Fas-mediated inflammation has not been characterized. So far, it has been demonstrated that crosslinking of Fas by Ab could induce activation of the transcription factor NF-κB (26, 27) and secretion of IL-8 (28) in certain cell lines. In this study, both IgM- and IgG-type anti-Fas mAbs induced PMN migration (Fig. 1,B). Although it is generally believed that trimerization of Fas is needed for the induction of apoptosis, these results suggest that trimerization may not be necessary for the induction of chemotaxis, but dimerization of Fas may be sufficient. Furthermore, in the present study the chemotaxis was normally induced in PMN obtained from lprcg mice (Fig. 3 C) whose Fas molecules have a point mutation in the death domain and cannot induce apoptosis due to the inability to recruit FADD or RIP (14). These data suggest that some other signaling components may be recruited to the Fas molecules independently of the known death domain-interacting molecules for the chemotactic activity, which remained to be identified in further studies.
It has been shown that soluble human TNF-α has a chemotactic activity against human PMN in vitro (29, 30). The chemotactic activity of sFasL demonstrated in this study appears to be comparable to that of TNF-α described in these studies. Thus, it will be interesting to see whether other members in the TNF family have similar function. It has been reported that low concentrations of TNF-α act synergistically with IL-1 to induce neutrophil infiltration in vivo (31). Similarly, it is possible that low concentrations of sFasL may enhance proinflammatory function of other cytokines. Actually, sFasL has been detected in the sera of patients suffering from some inflammatory diseases such as systemic lupus erythematosus (32), rheumatoid arthritis (32), Sjogren’s syndrome (32), leukemia (33), lymphohistiocytosis (34), myocarditis (35), and alcoholic liver disease (36). Recently, Hashimoto et al. (37) reported that sFasL was detected in the joints of patients with rheumatoid arthritis, where neutrophil influx is often observed. The sFasL was produced by lymphocytes in the synovial fluid, and cleaved sFasL accumulated in the inflamed joints. They also reported that the concentration of sFasL was remarkably higher in patients with severe rheumatoid arthritis than in patients with its mild form. Further clinical studies may elucidate the pathophysiologic role of sFasL at a local site of inflammation in other diseases.
This work was supported by grants from the Science and Technology Agency, the 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.
Abbreviations used in this paper: FasL, Fas ligand; sFasL, soluble FasL; PMN, polymorphonuclear neutrophils.