Involvement of IL-17 in Fas ligand-induced inflammation
Masayuki Umemura1,4,
Takaya Kawabe1,
Koyo Shudo1,
Hiroyasu Kidoya1,
Masayuki Fukui1,
Masahide Asano2,
Yoichiro Iwakura3,
Goro Matsuzaki4,
Ryu Imamura1 and
Takashi Suda1
1 Center for the Development of Molecular Target Drugs, Cancer Research Institute and 2 Department of Transgenic Animal Science, Graduate School of Medical Science, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan
3 Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shiroganedai, Minato-ku, Tokyo 108-8639, Japan
4 Center of Molecular Biosciences, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan
Correspondence to: T. Suda; E-mail: sudat{at}kenroku.kanazawa-u.ac.jp
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Abstract
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Fas ligand (FasL) has been well characterized as a death factor. However, recent studies revealed that ectopic expression of FasL induces inflammation associated with massive neutrophil infiltration. We previously demonstrated that the neutrophil infiltration-inducing activity of FasL is partly dependent on, but partly independent of, IL-1ß. Here we investigated the cytokine profile of peritoneal lavage fluid obtained from mice that received i.p. injections of FFL, a FasL-expressing tumor cell line. We found that FFL injection caused a marked increase of not only IL-1ß but also IL-6, IL-17, IL-18, KC/chemokine CXC ligand 1 and macrophage inflammatory protein (MIP)-2, but not of IL-1
, IFN-
, TGF-ß or TNF-
. The FFL-induced cytokine production was not observed in Fas-deficient lpr mice. Among cells transfected to express individually IL-1ß, IL-6, IL-17, or IL-18, only those expressing IL-1ß and IL-17 induced neutrophil infiltration. In these analyses, as little as 20 pg of peritoneal IL-17 induced neutrophil infiltration. The peritoneal IL-17 levels after FFL-injection were greatly diminished in IL-1-deficient mice. However, the IL-17 level was still above the threshold for neutrophil infiltration. Consistent with this, co-administration of the anti-IL-17 antibody with FFL diminished the peritoneal KC levels and neutrophil infiltration in IL-1-deficient mice. In addition, the expression of IL-17 by the tumor cells inhibited tumor growth in wild-type and nude mice. These results indicate that FasL is an upstream inflammatory factor that induces a variety of other inflammatory cytokines in vivo, and suggest that IL-17 is involved in FasL-induced inflammation in the absence of IL-1ß.
Keywords: cytokines, inflammation, rodent, T lymphocytes, tumor immunity
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Introduction
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Fas ligand (FasL, also called CD95L) is a cell membrane-associated factor that induces apoptotic cell death and plays important roles in various aspects of the immune system, including cell-mediated cytotoxicity and self-tolerance (1). FasL seems to play an immunosuppressive role against not only self-antigens but also exogenous antigens. FasL expression in the eyes and testes has been correlated with the immune-privileged property of these organs (2,3), and its expression in tumors has been implicated in tumor immune escape (4). However, it has not been easy to reproduce the immune-privileged status by FasL gene-transfer, because artificial expression of FasL in tissues or tumors induces inflammation accompanied by massive neutrophil infiltration, resulting in the destruction of the tissues or rejection of the tumors (58). In parallel with these findings, FasL plays a pathogenic role in a variety of inflammatory diseases in mice, including hepatitis, graft-versus-host diseases and pulmonary fibrosis (913).
We previously found that IL-1ß plays an important role in FasL-induced neutrophil infiltration. FasL induces apoptosis and, simultaneously, the conversion of inactive pro-IL-1ß into active IL-1ß in neutrophils; both of these processes are mediated by caspases (8). However, our results also suggested that a significant amount of the FasL-induced neutrophil infiltration is IL-1-independent. Other cytokines induced by FasL stimulation may be involved in the IL-1-independent infiltration. Consistent with this idea, Fas triggering induces IL-6 production in human fibroblast SV80 cells (14), IL-8 in the human colon adenocarcinoma cell line, HT-29 and rheumatoid arthritis synoviocytes (15,16), IL-18 in Propionibacterium acnes-primed mouse Kupffer cells and splenic macrophages (17), and macrophage inflammatory protein (MIP)-1, MIP-2 and MCP-1 in peritoneal resident macrophages (18). To elucidate the mechanism of inflammatory and immunosuppressive functions of FasL and to manipulate them, it is important to list the inflammatory cytokines.
IL-17 (also called CTLA8) is a recently described cytokine produced by activated CD4+ T cells in humans and
ßTCR+CD4CD8 thymocytes in mice (1921). IL-17 stimulates the production of IL-6, IL-8, G-CSF and PGE2 in epithelial, endothelial and/or fibroblastic cells (22) and IL-1ß and TNF-
in macrophages (23). IL-17 also induces the production of PGE2 and osteoclast differentiation factor in osteoblast cells, and has been implicated in bone resorption in rheumatoid arthritis (24). More recently, a role for IL-17 in neutrophil recruitment has emerged. Neutrophil infiltration in response to Klebsiella pneumoniae lung infection and trinitrochlorobenzene-induced contact hypersensitivity are impaired, respectively, in IL-17R and IL-17 knockout (KO) mice (25,26). Consistent with this, IL-17 stimulates the production of neutrophil chemokines such as IL-8 in bronchial epithelial cells and venous endothelial cells (27) and GRO
/KC in mesothelial cells (28). Furthermore, intratracheal and intraperitoneal administration of IL-17 induces neutrophil infiltration (27,28). In these activities, IL-17 resembles IL-1.
It is not well understood why FasL induces immune privilege in some organs but elicits inflammation when expressed ectopically. To explain these apparently conflicting phenomena, it is important to investigate the mechanism of FasL-induced inflammation in detail. Such study will also be useful to develop a novel remedy for inflammatory diseases such as hepatitis. For these purposes, here we investigated the peritoneal levels of various cytokines after inoculation with FasL-expressing tumor cells. We found that the i.p. injection of FasL-expressing cells induces the production of various inflammatory cytokines, including IL-17. Then we investigated the possible involvement of these cytokines in FasL-induced neutrophil infiltration and tumor growth inhibition.
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Methods
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Mice
Female wild-type and lpr/lpr C57BL/6 and BALB/c nu/nu mice were purchased from SLC Inc. (Shizuoka, Japan), and female wild-type and scid/scid CB-17 mice were purchased from Clea Japan Inc. (Tokyo, Japan) at 8 weeks old and used between 812 weeks of age. C57BL/6 mice with both their IL-1
and ß genes homozygously disrupted (IL-1 KO) were described previously (29). The Kanazawa University Committee on Animal Welfare approved all animal protocols.
Cell lines
All the stable transfectants used in this study were derived from the FBL-3 erythroleukemia cell line. The FFL cell line, bearing a human FasL mutation lacking most of its cytoplasmic region, and the FBH cell line, a control vector transfectant, were described previously (30). Stable transfectant lines expressing an artificial secretory form of mouse IL-1ß or IL-18, or natural mouse IL-6 or IL-17 were generated as follows. FBL-3 cells were co-transfected with a pEF-B0S-EX mammalian expression vector carrying the cDNA for secretory IL-1ß [a fusion cDNA encoding the signal sequence of mouse Fas (amino acids 124) and an active form of mouse IL-1ß (amino acids 118269)], for secretory IL-18 [a fusion cDNA encoding the signal sequence of mouse Fas (amino acids 124) and an active form of mouse IL-18 (amino acids 36192)], or for full-length mouse IL-6 or IL-17 with pBL-hygB, carrying the hygromycin B-resistance gene. Hygromycin B-resistant clones producing the corresponding cytokine were selected by ELISA as described below.
Isolation of peritoneal exudate cells (PEC) and peritoneal lavage fluid (PLF)
Mice were inoculated i.p. with various transfectant cell lines (4 x 106 cells). In some experiments, 10 µg of rat anti-mouse IL-17 neutralizing mAb (Genzyme, Cambridge, MA) or normal rat IgG were injected along with the cells. Twenty hours later, the peritoneal cavities of the mice were washed with 1.5 ml PBS. The peritoneal wash was separated into the cell fraction (PEC) and the supernatant (PLF) by centrifugation at 275 g for 5 min. PLF was then filtrated through 0.22 µm membrane filter.
Cytokine ELISA
The concentration of IL-1
, IL-1ß, IL-6, IL-17, IL-18, IFN-
, TNF-
, TGF-ß, KC and MIP-2
in the PLF or culture supernatant was determined using the ANALYZA ELISA Kit (Genzyme) or OptEIATM Set (PharMingen) according to the manufacturers' protocols. The amount of each cytokine in the peritoneal cavity was determined by multiplying the concentration by the volume of PLF (1.5 ml).
Flow cytometry analysis
PEC were pretreated with Fc-Blocker (PharMingen, San Diego, CA) and stained with FITC-conjugated anti-Gr-1 mAb and PE-conjugated anti-B220 mAb (PharMingen). In some experiments, PEC pretreated with Fc-Blocker were stained with FITC-conjugated anti-B220, CD3,
ßTCR, 
TCR, CD4, or CD8 mAb and biotin-conjugated anti-CD3 or Thy1.2 mAb followed by allophycocyanin (APC)-conjugated streptavidin (PharMingen). Cells were then fixed with 1% paraformaldehyde, permeabilized with 0.1% saponin, and stained with PE-conjugated anti-mouse IL-17 mAb (PharMingen). Cells were then analyzed using a FACSCalibur (Becton Dickinson, Mountain View, CA). Tumor cells and dead cells were excluded from analyses using the 2-dimensional profile of forward vs side scatter.
Assay for in vivo tumor growth
Female C57BL/6 or BALB/c nu/nu mice, 812 weeks old, were inoculated intradermally with 2.5 x 106 tumor cells; the cells were injected into the right and left sides of the dorsal skin. The long and short diameters of the resulting tumors were measured using calipers at intervals of 3 days. Tumor size was calculated as follows: tumor size (mm2) = long diameter (mm) x short diameter (mm).
Statistical analysis
The statistical significance of data was evaluated by a two-tailed Student's t-test. A P-value <0.05 was considered significant.
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Results
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FasL induces the production of a variety of cytokines in vivo
We previously reported that MethA tumor cells transfected with FasL induce massive neutrophil infiltration, when injected into the peritoneal cavity of syngeneic BALB/c mice (8). We demonstrated that IL-1ß plays an important role in the FasL-induced neutrophil infiltration. However, at that time, we also noticed a reduced but significant neutrophil infiltration in IL-1 KO mice. These observations were confirmed using other FasL-transfected tumor cell lines derived from FBL-3 erythroleukemia and syngeneic wild-type and IL-1KO C57BL/6 mice (data not shown). To investigate the possible involvement of cytokines other than IL-1ß in FasL-induced neutrophil infiltration, we determined by ELISA the amounts of various cytokines in the PLF collected from mice 20 h after inoculation with the human FasL-expressing cell line FFL or the control cell line FBH. As shown in Fig. 1(A), in addition to IL-1ß, large amounts of IL-6, IL-17, IL-18, KC and MIP-2 were detected. In contrast, only small amounts of IL-1
, IFN-
and TNF-
were produced in response to the FFL injection. There was no significant difference between the amounts of TGF-ß produced after FFL or FBH injection. FFL injection in lpr mice did not induce production of IL-1ß, IL-6, KC or IL-17 (Fig. 1B), confirming that the cytokine production in wild-type mice was a result of the interaction between Fas and FasL.

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Fig. 1. FasL induces various cytokines in vivo. Wild-type (A, B) or Fas-deficient lpr/lpr C57BL/6 mice (B) received i.p. injections of FBH or FFL cells. PLF was collected from untreated mice (0 h) or FBH- or FFL-injected mice 20 h after the injection, and the amount of the indicated cytokines was determined by ELISA. In (A), each data point represents an individual mouse. In (B), the vertical lines indicate SD (n = 3).
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Physiologically relevant amount of IL-17 induces neutrophil infiltration in vivo
IL-1 and IL-17 induce production of neutrophil chemokines such as KC and MIP-2 (27,28,31,32). Consistent with this, i.p. injection of 1 ng recombinant IL-1ß induces a strong neutrophil infiltration in mice (30). In contrast, injection of recombinant IL-6, IL-17 or IL-18 within the ranges of the amounts we found in the peritoneal cavity of FFL-injected mice induced little or no neutrophil infiltration, although in agreement with a previous report (33), more than 10 ng of IL-18 induced moderate levels of neutrophil infiltration (data not shown). Despite the lack of an effect at the tested levels, it was possible that these cytokines might induce neutrophil infiltration when they were continuously produced in vivo. To this end, we established transfectants expressing IL-6 (F6), IL-17 (F17), and a secretory form of IL-1ß (FF1b) and IL-18 (F18s) and injected them i.p. Among these cell lines, F17 and the FF1b lines induced strong neutrophil infiltration, although F17 was less potent than FF1b (Fig. 2A, upper panels). The amounts of IL-17 in the PLF of the mice that received the F17 injection were much higher than in the FFL-injected mice (Fig. 2A, second to the right in lower panels). To determine the threshold amount of IL-17 in the peritoneal cavity to induce neutrophil infiltration, we titrated the number of F17.5 cells, while the total number of injected cells was kept constant by adding FBH cells. The amount of IL-17 in the PLF and the proportion of neutrophils in the PEC of individual mice 20 h after the injection were plotted in Fig. 2(B). The result indicates that as little as 20 pg of IL-17 in the peritoneal cavity induced significant neutrophil infiltration.

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Fig. 2. Neutrophil infiltration-inducing activity of cytokines produced in vivo. (A) C57BL/6 mice received injections of the indicated transfectants. Twenty hours later, the peritoneal lavage was separated into PEC and PLF. The proportion of Gr-1 positive cells (i.e. neutrophils) in the PEC was determined by flow cytometry (upper panel). The amount of the indicated cytokines in the PLF was determined by ELISA (lower panel). Vertical lines indicate SD (n = 3). Two or three independent clones of FF1b, F6, F17 and F18s were used. (B) F17.5 and FBH cells were mixed at the indicated ratios and a total of 4 x 106 cells were injected i.p. Twenty hours later, the proportion of Gr-1+ cells in the PEC and amount of IL-17 in the PLF were determined as described above. Each data point represents an individual mouse.
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ß or 
TCR+CD4CD8 T cells are the major producers of IL-17 upon FasL stimulation
T cells produce IL-17 in both humans and mice (20,21). To investigate whether T cells produce IL-17 upon FasL stimulation, we injected FFL into wild-type and scid/scid mice (Fig. 3A). The level of peritoneal IL-17 after the FFL injection in scid/scid mice was approximately one-third of that in wild-type mice, indicating that mainly T and/or B cells and partly non-T/B cells are responsible for the observed IL-17 production. To determine which cells produce IL-17 upon FFL-injection, PEC from mice that received injections of FBH or FFL were stained for cell-surface markers and cytoplasmic IL-17 using fluorescent antibodies, and analyzed by flow cytometry. IL-17-producing cells were detected among the CD3+B220 T cells but not among the CD3B220+ B cells from FFL-injected mice (Fig. 3B, lower panels). Consistent with the ELISA results shown in Fig. 1(A), neither the B nor T cells from FBH-injected mice produced IL-17 (Fig. 3B, upper panels). We could not determine whether there were some IL-17+CD3B220 cells, because of the presence of cells with high auto-fluorescence in this subset. Among the Thy-1.2+ cells, the
ß and 
TCR+ cells produced IL-17 (Fig. 3C). The ratio of IL-17+ cells to 
TCR+ cells was higher than to
ßTCR+ cells. Among the CD4/CD8 subsets, CD4CD8 cells were the major producers of IL-17, although some CD4+CD8 cells also produced it. A few CD4CD8+ cells also appeared to produce a small amount of IL-17.

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Fig. 3. CD4CD8 T cells are the major producers of IL-17 upon FFL injection. (A) Wild-type and scid/scid CB-17 mice received injections of FBH or FFL, and the amount of IL-17 in the PLF 20 h after the injection was determined by ELISA. Vertical lines indicate SD (n = 3). Representative results of three independent experiments are shown. (B) C57BL/6 mice received injections of FBH or FFL. Twenty hours later, PEC were collected and stained with FITCanti-B220, biotinanti-CD3 plus APCstreptavidin, and PEanti-IL-17 mAb. CD3B220+ B cells (R1) and CD3+B220 T cells (R2) were gated and analyzed for IL-17 expression. The numbers in the right panels are the frequency of IL-17+ cells per 10 000 cells of each subset of cells. (C) C57BL/6 mice received injections of FBH or FFL. Twenty hours later, PEC were collected and stained with FITC-conjugated mAb for CD3, ßTCR,  TCR, CD4, CD8, or both CD4 and CD8, together with biotinanti-Thy1.2 mAb plus APCstreptavidin and PEanti-IL-17 mAb. Thy-1.2+ cells were gated and analyzed for TCR or CD4/CD8 expression and IL-17 expression.
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IL-17 is involved in the IL-1-independent FasL-induced KC production and neutrophil infiltration
A recent report showed that IL-1ß has the potential to induce IL-17 production in human peritoneal blood mononuclear cells (34). To investigate the possible involvement of IL-1ß in the FasL-induced IL-17 production, we compared the peritoneal levels of IL-17 in wild-type and IL-1ß KO mice after FFL injection. The IL-17 level in the IL-1 KO mice was markedly diminished compared with that in wild-type mice (Fig. 4A, left panel). However, it was not completely eliminated (Fig. 4A, right panel). Thus, the FasL-induced IL-17 production was largely dependent on IL-1, but there must be an IL-1-independent mechanism for this response. The amount of peritoneal IL-17 in the FFL-injected IL-1 KO mice was
40 pg on average. Taking the results shown in Fig. 2(B) into account, we could not rule out the possibility that the small amount of IL-17 played a role in the FFL-induced neutrophil infiltration in IL-1 KO mice. To investigate whether IL-17 is involved in FasL-induced inflammation, an anti-IL-17 neutralizing mAb was injected i.p. together with FFL into wild-type or IL-1KO mice. The antibody used in these experiments significantly inhibited neutrophil infiltration induced by up to 800 pg of IL-17, and completely inhibited that induced by up to 250 pg of IL-17 produced by F17 cells in the peritoneal cavity (see Supplementary fig. 1, available at International Immunology Online). In the wild-type mice, the anti-IL-17 mAb treatment did not affect the neutrophil infiltration and KC production induced by FFL (Fig. 4B and C). However, surprisingly, it significantly inhibited both of these responses in the IL-1 KO mice. These results indicate that IL-17 is not essentially involved in the FasL-induced KC production and neutrophil infiltration in wild-type mice, but it is at least in part responsible for these responses in IL-1 KO mice.

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Fig. 4. IL-17 is involved in the IL-1-independent mechanism of FasL-induced neutrophil infiltration and KC production. (A) Wild-type and IL-1-deficient C57BL/6 mice received injections of FBH or FFL. Twenty hours later, the amount of peritoneal IL-17 was determined by ELISA. Vertical lines indicate SD (n = 3). Representative results of three independent experiments are shown. (B and C) Wild-type and IL-1-deficient C57BL/6 mice received injections of FBH or FFL with or without 10 µg rat anti-mouse IL-17 mAb or normal rat IgG. Twenty hours later, the proportion of Gr-1+ cells in the PEC (B) and the amount of KC in the PLF (C) were determined. Vertical lines indicate SD (n = 6 or 3). The asterisks indicate P < 0.05. Representative results of three independent experiments are shown.
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Expression of IL-17 in tumor cells inhibits tumor growth
Artificial expression of FasL in tumor cells suppresses tumor growth (30,35). Therefore, we next investigated the effect of IL-17 on in vivo tumor growth. First, we confirmed that all of the FBH, FFL and F17 clones used here showed a similar growth rate in vitro (Fig. 5A). We inoculated syngeneic C57BL/6 mice or nude mice intradermally with FBH, FFL or F17 cells (Fig. 5B and C). As reported previously (30), control FBH transfectants spontaneously regressed within about 3 weeks when 2.5 x 106 cells were transplanted intradermally in syngeneic mice (Fig. 5B). T cells were responsible for this rejection, so the same tumor cells grew progressively in nude mice [(36) and Fig. 5C]. Consistent with our previous results (30), the FFL tumor was rejected more rapidly than the FBH tumor. The rejection of FFL was T cell independent, because the tumors were rejected with similar kinetics in both wild-type and nude mice. This finding is consistent with a previous report demonstrating that neutrophils are responsible for the tumor growth suppression caused by FasL expression (5). Tumors from all the F17 clones were also rejected earlier in syngeneic mice and grew slower in nude mice than tumors from FBH cells, although the suppression of tumor growth was not as prominent as with the FFL cells. Massive neutrophil infiltration was observed around the FFL but not FBH tumors in wild-type mice (Fig. 6). Importantly, a large number of neutrophils were also observed around F17 tumors. These results indicate that IL-17 inhibited tumor growth in a T cell-independent manner, probably by inducing neutrophil infiltration in our system, and supports the idea that IL-17 partly mediates the tumor growth inhibition by FasL.

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Fig. 5. Expression of IL-17 in tumor cells inhibits tumor growth in vivo but not in vitro. (A) 1 x 104 cells of the indicated transfectants derived from the FBL-3 tumor cell line were cultured in 10 ml culture medium, and the cell number/ml was determined daily using a hemocytometer. Vertical lines indicate SD (n = 3). (B and C) The indicated tumor cells (2.5 x 106 cells) were injected into the dorsal skin of syngeneic C57BL/6 mice or BALB/c nu/nu mice, and the tumor size was measured at intervals of 3 days. Vertical lines indicate SD (n = 6). Three independent clones of F17 were tested. Symbols represent the same cell lines as those shown in the top panel.
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Fig. 6. Histological analyses of tumors. FBH, FFL and F17 tumor cells were transplanted into C57BL/6 mice as described in Fig. 5B, and the tumors were excised on day 3. Sections of tumor tissues fixed with 10% formalinPBS were stained with hematoxylin and eosin. Arrowheads indicate neutrophils. The bars correspond to 100 µm. Original magnification, upper panels, x200; lower panels, x400.
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Discussion
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In this study, we demonstrated that i.p. injection of FasL-expressing cells into mice induces, in addition to IL-1ß, various cytokines including IL-6, IL-17, IL-18, KC, and MIP-2, but not much IL-1
, IFN-
, TNF-
or TGF-ß (Fig. 1A). Nelson et al. (37) reported that transgenic expression of FasL in the heart resulted in the production of not only IL-1ß and IL-6 but also TNF-
and TGF-ß. Therefore, the repertoire of cytokines produced by FasL stimulation may depend on the cell type or tissue environment. They also reported that FasL expression in the heart caused mild leukocyte infiltration but not the severe tissue damage that has been observed in islets of Langerhans that expressed FasL via genetic engineering (6,7). In this context, it is interesting to note that TGF-ß and TNF have been suggested to be involved in modulating the consequences of FasL expression to immune suppression in the immune-privileged organs (38,39). Thus, it is possible that this heterogeneity of the cytokine repertoire explains the different consequences of FasL expression, i.e. immune privilege or inflammation, in different tissues.
Among the cytokines that were found at high levels in the PLF of FFL-injected mice, only IL-1ß and IL-17 induced neutrophil infiltration within the range of the amounts that we found in the PLF (Fig. 2A). It has been demonstrated that administration of a large amount of Escherichia coli-expressed IL-17 induces neutrophil infiltration in vivo. For example, 1 µg E. coli-expressed hIL-17 injected intratracheally or 500 ng E. coli-expressed mIL-17 injected i.p. led to neutrophil infiltration (27,28). In our hands, up to 50 ng of E. coli-expressed mIL-17 did not induce neutrophil infiltration (data not shown). On the other hand, as little as 20 pg of mIL-17 produced i.p. by F17 cells induced significant neutrophil infiltration (Fig. 2B). This may be because the IL-17 produced in mammalian cells that is glycosylated (40) is more stable in vivo than the E. coli-expressed IL-17 protein that is unglycosylated. Alternatively, both forms of IL-17 may have a very short half-life in vivo, and thus its continuous production in situ may be essential for the highly sensitive neutrophil infiltration. It has been reported that IL-18 induces neutrophil infiltration in vivo. Consistent with a previous report (33), injection of 10 ng or more IL-18 induced significant neutrophil infiltration, although IL-18 up to 1 ng failed to induce significant neutrophil infiltration (data not shown). Thus, IL-18 may play a role in neutrophil infiltration, when a large amount of IL-18 is produced in some tissues upon FasL stimulation.
A novel finding of this study is that FasL induces IL-17 production in mice. Activated CD4+ T cells have been generally described as the major producers of IL-17, based on human data. However, in mice,
ßTCR+CD4CD8 (but not CD4+CD8 or CD4CD8+) thymocytes, activated by an anti-CD3 mAb, were shown to produce IL-17 (21). Production of IL-17 by other cell types has not been well investigated. Here we found that
ß or 
TCR+CD4CD8 peritoneal T cells were the major producers of IL-17 in our system (Fig. 3BD). It is also worth noting that the proportion of IL-17-producing 
-T cells to the total 
-T cells was higher than that of IL-17-producing
ß-T cells to the total
ß-T cells. Furthermore, FasL induced IL-17 production better in peritoneal T cells than in spleen T cells, while PMA plus ionomycin did the opposite (T. Kawabe, M. Umemura and T. Suda, unpublished observation). Thus, different stimuli might induce IL-17 production in different subsets of T cells. We also found that a small amount of IL-17 was produced in scid/scid mice upon FFL injection (Fig. 3A). Recently, it was reported that neutrophils produce IL-17 in a model of LPS-induced lung inflammation (41). Although we failed to demonstrate the presence of IL-17 in a non-T/B cell population because of a technical reason, it is possible that neutrophils produced a small amount of IL-17 in our system also.
It was previously reported that human IL-1 and IL-15 induce IL-17 production in human peritoneal blood mononuclear cells (34). IL-15 also induces IL-17 production in mouse CD4+ T cells (41). We found here that the peritoneal levels of IL-17 after FFL injection were severely diminished in IL-1 KO mice (Fig. 4A), indicating that FasL-induced IL-17 production is mainly mediated by IL-1ß in wild-type mice. However, a small amount of IL-17 was reproducibly detected after FFL injection in the PLF of IL-1 KO mice, suggesting that FFL injection led to the production of mediators other than IL-1 for IL-17 production. IL-15 is one such candidate mediator. Alternatively, FasL may have the potential to induce IL-17 production directly in certain cells. Interestingly, in our hands, recombinant IL-1 alone induced only weak IL-17 production in peritoneal cells (M. Umemura, T. Kawabe and T. Suda, unpublished observation) Thus, it seems that IL-1ß induced IL-17 in synergy with some other cytokines in our system. The details of the mechanism for FasL-induced IL-17 production are currently under investigation.
The peritoneal level of KC and the neutrophil infiltration after FFL injection in IL-1 KO mice were significantly diminished by the co-injection of the anti-IL-17 mAb (Fig. 4B and C). Thus, IL-17 is involved in the IL-1-independent mechanism of FasL-induced inflammation. On the other hand, IL-17 seems to be dispensable for FasL-induced neutrophil infiltration in wild-type mice where IL-1ß is produced. However, it was reported that IL-17 but not IL-1ß increases elastase and myeloperoxidase activity in neutrophils in vivo (42). Thus, IL-17 may play an important role in the activation of neutrophils in FasL-induced inflammation.
Expression of IL-17 in cells derived from the FBL-3 mouse erythroleukemia cell line inhibited the in vivo tumor growth, suggested that IL-17 is partly involved in the FasL-induced tumor growth inhibition. Further investigations using IL-1ß and IL-17 double knockout mice will provide conclusive answer to the question of whether this notion is correct. The effect of IL-17 expression in tumor cells on in vivo tumor growth has been controversial. Initially, Tartour et al. (43) reported that the expression of hIL-17 cDNA into two transfected human cervical tumor cell lines results in the enhancement of tumor growth in nude mice. In contrast, Benchetrit et al. (44) reported that IL-17 inhibits tumor growth by a T cell-dependent mechanism, because the growth of mIL-17-transfected P815 mouse mastocytoma and J558L plasmacytoma in wild-type syngeneic mice, but not in nude mice, was inhibited compared with the growth of mock transfectants. More recently, Numasaki et al. (45) reported that transfection of MCA205 mouse fibrosarcoma and MC38 mouse colon adenocarcinoma, which are poorly immunogenic tumor cell lines, with mIL-17 cDNA, enhanced the tumor growth in syngeneic mice. They also showed that IL-17 promoted neovascularization, and suggested that IL-17 promotes tumor growth when tumor cells are weakly or poorly immunogenic. Here, we found that F17 tumors exhibited reduced growth in both wild-type and nude mice (Fig. 5). Although, consistent with the Numasaki's rule, FBL-3 is a highly immunogenic cell line, the fact that the growth inhibition was also observed in nude mice indicates that it is T cell independent. Because F17 induced significant neutrophil infiltration in vivo, and neutrophils are responsible for FasL-induced tumor growth inhibition (5), our results are more consistent with the idea that neutrophils play an important role in tumor growth inhibition (46). The differences between our results and previous reports may depend on the sensitivity of tumor cells to neutrophils.
Although we mainly focused on IL-17 and neutrophil infiltration in this study, other cytokines may play important roles in other aspects of FasL-induced inflammation. For example, IL-18 has been reported to be a positive regulator of FasL expression (47). Thus, it is possible that FasL and IL-18 create a positive-feedback loop to enhance their expression. IL-6 is a pleiotropic cytokine with a wide range of biological activities in immune regulation, hematopoiesis and inflammation. IL-6 is also an important mediator to induce acute phase proteins that are thought to protect tissues from the detrimental effects of inflammation. The roles of these cytokines in FasL-induced inflammation remain to be investigated.
Previous studies demonstrated that FasL plays an important role in various murine models of inflammatory diseases (913). We initially thought that FasL directly killed cells in inflammatory tissues. However, our results shown here suggest that FasL is an upstream inflammatory factor that induces various other inflammatory cytokines. It is likely that this inflammatory activity of FasL is an important part of its pathogenic activity. Thus, understanding in detail the mechanism of FasL-induced inflammation may lead us to a novel remedy for FasL-related inflammatory diseases.
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Supplementary data
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Supplementary data are available at International Immunology Online.
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Acknowledgements
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We thank Ms I. Hashitani for secretarial and technical assistance. We also thank Dr A. Matsuzaki-Mukasa for assistance in histological analyses. This work was supported in part by Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government.
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Abbreviations
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FasL | Fas ligand |
MIP | macrophage inflammatory protein |
PEC | peritoneal exudate cells |
PLF | peritoneal lavage fluid |
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Notes
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M. Umemura and T. Kawabe contributed equally to this work
Transmitting editor: N. Shigekazu
Received 19 November 2003,
accepted 10 May 2004.
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References
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