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Fas Ligand on Tumor Cells Mediates Inactivation of Neutrophils ¹

Yi-Ling Chen^*, Shun-Hua Chen, Jiu-Yao Wang and Bei-Chang Yang^2,

^* Institute of Basic Medical Sciences, and Departments of Microbiology and Immunology and Pediatrics, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The expression of Fas ligand (FasL) on tumor cells (tumor FasL) has been implicated in their evasion of immune surveillance. In this study, we investigated the cellular mechanism for FasL-associated immune escape using melanoma B16F10-derived cells as a model. Transfectants carrying FasL-specific ribozymes expressed low levels of FasL (FasL^low tumor cells) as compared with those carrying enhanced green fluorescent protein-N1 plasmids (FasL^high tumor cells). When injected s.c. into C57BL/6 mice, FasL^low tumor cells grew more slowly than did FasL^high melanoma cells. FasL^high tumor cells showed more intensive neutrophilic infiltration accompanied by multiple necrotizing areas than did FasL^low tumor cells. The average size of FasL^low tumors, but not of FasL^high tumors, was significantly enhanced in mice depleted of neutrophils. Consistently, a local injection of LPS to recruit/activate neutrophils significantly delayed tumor formation by FasL^low tumor cells, and slightly retarded that of FasL^high tumor cells in both C57BL/6 and nonobese diabetic/SCID mice. Neutrophils killed FasL^low melanoma cells more effectively than FasL^high melanoma cells in vitro. The resistance of FasL^high melanoma cells to being killed by neutrophils was correlated with impaired neutrophil activation, as demonstrated by reductions in gelatinase B secretion, reactive oxygen species production, and the surface expression of CD11b and the transcription of FasL. Local transfer of casein-enriched or PMA-treated neutrophils delayed tumor formation by melanoma cells. Taken together, inactivation of neutrophils by tumor FasL is an important mechanism by which tumor cells escape immune attack.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fas ligand (FasL)³ (CD95L, APO-1L) is a member of the TNFR superfamily and triggers a death signal in Fas-bearing cells after engagement with the Fas molecule (1, 2). In contrast to a tightly regulated manner in lymphocytes, FasL is constitutively expressed in several nonlymphoid tissues as well as in tumors of various origins (3, 4, 5, 6, 7, 8, 9, 10). FasL is required to maintain an immune-privileged status in organs such as eyes and testes (11). During the progress of melanomas, tumor FasL gradually and predominantly increases in metastatic melanomas (12, 13). The expression of FasL on tumor cell surfaces led to the assumption that tumor FasL also contributes to immune escape by tumors (14, 15, 16). However, contradictory reports exist. Ectopic expression of FasL in transgenic animals or tumors revealed that FasL might enhance neutrophil-mediated tissue destruction of certain FasL-positive cells (17, 18, 19, 20, 21, 22). In addition, the findings that some malignant human melanoma cells do not express FasL (23), that Fas/FasL interaction might suppress melanoma lung metastasis (24, 25, 26, 27), and that there is no correlation between tumor FasL and tumor-infiltrating cells argue against a major contribution of FasL to the immune escape mechanism by melanomas (14, 15, 16).

The fact that neutrophils are recruited into a FasL-positive tumor region without causing growth retardation in tumors suggests a mechanism by which tumor cells can attenuate the antitumor processes of neutrophils in situ during these encounters. Another point that remains to be answered is whether the effect of FasL on immunity observed in ectopic expression systems still holds true in other systems. This argument is implied by the finding that some FasL-positive tumors do induce a mild degree of inflammation, but which, however, fails to cause tumor rejection (28). In this study, these questions were further addressed using a functional knockdown strategy with a hammerhead ribozyme to specifically suppress the FasL gene (27). The important role of neutrophils in antitumor immunity was demonstrated by depletion experiments using Abs for CD4⁺ cells, CD8⁺ T cells, and neutrophils. Coculture experiments were used to evaluate the alterations in functional activities of neutrophils in contact with tumor cells expressing different levels of FasL. A possible antitumor therapy that attempts to activate neutrophils was also tested. The results obtained not only complement previous findings using FasL overexpression systems, but also clearly demonstrate a crucial role for neutrophils in determining tumor immunity.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents and Abs

H&E, glycerol gelatin, propidium iodide (PI), LPS from Escherichia coli (055:b5 strain), acetylcysteine, and PMA were purchased from Sigma-Aldrich (St. Louis, MO). An aminoethyl carbazole substrate kit was purchased from Zymed (San Francisco, CA). The 2',7'-dichlorofluorescein diacetate (H₂DCFDA) was purchased from Molecular Probes (Eugene, OR). The following Abs were used in this study: rat IgG Ab (ICN Pharmaceuticals, Cappel, OH); recombinant mouse FasFc chimeric protein (mFasFc; R&D Systems, Minneapolis, MN); rat anti-NK mAb (DX5), rat anti-CD4 mAb (H129.19), rat anti-CD8 mAb (53-6.7), rat anti-Ly-6G (RB6-8C5) mAb, anti-CD4 FITC (H129.19), anti-CD8 PE (53-6.7), anti-Ly-6G FITC (RB6-8C5), and anti-CD11b (M1/70, PE conjugated) purchased from BD PharMingen (San Diego, CA); rabbit anti-FasL Ab (N-20) (Santa Cruz Biotechnology, Santa Cruz, CA); goat anti-rat IgG peroxidase conjugate (Chemica, San Diego, CA); and goat anti-rabbit IgG peroxidase conjugate (Calbiochem, San Diego, CA).

Transfectants and cell culture

The B16F10-derived melanoma cells used in this study were established, as previously reported (27). After DNA transfection followed by antibiotic selection, stable transfectants were established. Applying FasL^Ribozyme suppressed the expression of FasL in melanoma cells in vitro. B16F10 transfectants carrying the pEGFP (enhanced green fluorescent protein)-N1 plasmid were named Vn for FasL^high tumor clones; B16F10 transfectants carrying the FasL^Ribozyme plasmid were named Rn for FasL^low tumor clones. The transcripts of Fas and FasL were measured by RT-PCR, as described previously (27). R2, R4, R5, R6, and R7 showed various extents of reduced expression of FasL at both the transcriptional and translational levels (27). Cells were regularly maintained in DMEM, supplemented with 10% FCS and 2 mM L-glutamine at 5% CO₂/37°C in a humidified atmosphere. All cell culture medium was purchased from Life Technologies (Grand Island, NY).

Mice

Eight-week-old C57BL/6 (H₂^b) mice and nonobese diabetic (NOD)/SCID (lymphocyte- and NK cell-deficient) mice were purchased from the Laboratory Animal Center of National Cheng Kung University and were maintained under pathogen-free conditions. All animal experiments were conducted with the approval of the ethics committee of our institution.

Tumor formation

Tumor cells cultured for 3 days were harvested with 0.05% trypsin-EDTA and washed twice with sterile PBS. Mice were given an s.c. injection of tumor cells (5 x 10⁵) in the left flank. A tumor mass appeared 10 days after cell inoculation. Tumor volumes were calculated with a caliper using the formula: /6 x length x width² (29). To evaluate the effect of LPS administration on tumor formation, LPS from E. coli (055:b5 strain) with 100 µg in 0.1 ml of saline was administered s.c. after 24 h and on the 7th and 14th days. Local transfer of neutrophils was done as follows. On approximately the 10th day following tumor cell injection, tumor-bearing mice were instilled s.c. with saline, casein-elicited neutrophils, or neutrophils pretreated with 100 ng/ml PMA (5 x 10⁶ cells/animal in 0.3 ml PBS). Two days after neutrophil transfer, animals were once again instilled with saline, neutrophils, or PMA-treated neutrophils.

Histological examination and immunohistochemistry

Tissues were surgically obtained and immersed in a buffered 10% Formalin solution for at least 24 h. Sections 4 µm thick were dehydrated, embedded, and stained with H&E. The tissues for immunohistochemical staining were embedded in OCT compound (Miles, Elkhart, IN), frozen in liquid nitrogen, and stored at -20°C. Five-micrometer-thick cryosections were placed on poly(L-lysine)-coated glass slides and fixed with 3.7% formaldehyde in either PBS for 15 min plus acetone for 3 min or 3.7% paraformaldehyde for 5 min plus acetone for 1 min. The endogenous peroxidase activity in tissue sections was depleted by incubation in PBS containing 3% H₂O₂ for 3 min. The primary Ab was diluted with Ab diluent (DAKO, Carpenteria, CA), and included rat anti-NK mAb (DX5), rat anti-CD4 mAb (H129.19), rat anti-CD8 mAb (53-6.7), rat anti-Ly-6G (RB6-8C5) mAb, or rabbit anti-FasL (N-20). The secondary Abs were sheep anti-rat IgG conjugate or goat anti-rabbit IgG peroxidase. Reddish-brown peroxidase staining was developed with an aminoethyl carbazole substrate kit. Sections were counterstained with hematoxylin and mounted in glycerol gelatin.

Apoptosis

Tumor cells or neutrophils were analyzed for apoptosis by a PI analysis that detected hypodiploid nuclei. Cells that appeared in sub-G₀/G₁ peaks were apoptotic. In brief, cells were harvested, suspended in ice-cold 70% ethanol overnight at 4°C, and stained with a solution containing 5 µg/ml of PI (30). In neutrophil and tumor cell cocultures, viable tumor cells were distinguished from neutrophils or dead tumor cells by side and forward scatter parameters. In situ detection of apoptosis was performed with TUNEL staining. Cytospin cells were stained using a commercially available kit (ApoTag in situ apoptosis detection kit-peroxidase; Intergen, Purchase, NY), following the manufacturer’s instructions. The apoptotic cells were examined with conventional light microscopy at x400 to evaluate tumor cells, and 100 cells were counted per sample. Results shown are from individual mice, and data were reported as the percentage of cells with apoptotic morphology.

CD4⁺ T cell, CD8⁺ T cell, or neutrophil depletion

CD4⁺ T cell-, CD8⁺ T cell-, and neutrophil-specific Abs produced by hybridomas GK1.5 (31) and 2.43 (31), respectively, were purified by affinity chromatography on a protein G-Sepharose column (Pharmacia, LKB Biotechnology, Piscataway, NJ) and adjusted to a final concentration of 3 mg/ml. To deplete CD4⁺ or CD8⁺ T cells, 100 µg of anti-CD4 or anti-CD8 Ab was given i.p. to mice 2 days before tumor injection. After inoculation, boosters of Abs were given on the 7th and 14th days. To deplete neutrophils, mice were injected (i.p.) with 100 µl ascitic fluid generated from RB6-8C5 (32) 5 h before tumor injection. On days 3 and 5, mice received 150 µl ascitic fluids (i.p.). On days 7 and 9, they received 200 µl of ascitic fluids (i.p.). On days 12 and 15, they received 250 µl of ascitic fluids (i.p.). Combined depletions of CD4⁺/CD8⁺, CD4⁺/neutrophils, and CD8⁺/neutrophils followed same protocols as those for single depletion. Control mice received the same treatment of purified rat IgG Ab. To evaluate the effectiveness of depletion, splenocytes or neutrophils from the blood were incubated with anti-CD4 FITC (H129.19), anti-CD8 PE (53-6.7), or anti-Ly-6G FITC (RB6-8C5) at 4°C for 40 min in the dark. The mixture was washed twice with ice-cold HBSS, and the cells were resuspended in HBSS containing 2% FCS/0.1% NaN₃. Stained lymphocytes or neutrophils were analyzed by flow cytometry (FACScan; BD Biosciences, San Jose, CA). The depressed number of CD4⁺ T cells or CD8⁺ T cells in CD4- or CD8-depleted mice, or neutrophils in peripheral blood in neutrophil-depleted mice was confirmed on the 6th and 18th days. Flow cytometry analyses showed that CD4⁺ and CD8⁺ T cells were reduced by >90% in the spleen (27). After depletion with RB6-8C5 ascitic fluids, the number of neutrophils in peripheral blood was reduced by >75% (27).

Isolation of mouse neutrophils from peritoneal fluid

Mouse neutrophils in peritoneal fluid were isolated according to the procedures described previously (33). In brief, about 35 x 10⁷ peritoneal exudate cells were separated with a 9-ml Percoll gradient solution at room temperature in a 10-ml Beckman (Fullerton, CA) ultracentrifuge tube, and then the mixture was centrifuged for 20 min at 60,650 x g at 4°C. Neutrophils distributed in the second opaque layer were washed once with 10 ml of 5% FCS/RPMI 1640 medium, stained with Liu’s staining solution, and observed under conventional light microscopy to evaluate their purity. Cell viability was determined by an eosin Y exclusion assay. Usually, the neutrophil preparation obtained had a purity of >90% and a viability of 99%. CD11b, an activation marker of neutrophils, was stained by an Ab specific for mouse CD11b (M1/70, PE conjugated).

Direct coculture of tumor cells with neutrophils and Transwell experiments

Freshly isolated neutrophils or those fixed with 1% paraformaldehyde for 1 h at 4°C and tumor cells were cocultured in RPMI 1640–10% FCS at 37°C in a humidified CO₂ incubator (5% CO₂/95% air) for 20 h in six-well tissue culture plates (20, 34). To avoid cell-to-cell contact, neutrophils were separated from B16F10 transfectants by a 0.4-µM membrane in a well of Transwell six-well plates (Costar, Cambridge, MA). Neutrophils were placed in the upper chambers of the Transwell culture inserts. After 20 h, tumor cells from the lower chambers were harvested, washed, and analyzed for apoptosis, as described above. In some experiments, the interaction of Fas and FasL was interrupted by a 1-h preincubation using 500 ng mFasFc protein.

SDS-PAGE gelatin zymography

Release of gelatinase B was analyzed to indicate neutrophil activation. Tumor cells and neutrophils were cocultured for 20 h. Supernatants were collected. The gelatinase activity was determined by zymography, as described previously (35). Briefly, supernatants were subjected to electrophoresis on 7.5% SDS acrylamide gels containing 0.1% gelatin under nonreducing conditions. After electrophoresis, gels were washed with 2.5% Triton X-100, rinsed with water, and incubated at 37°C overnight in 50 mM Tris and 5 mM CaCl₂, pH 7.5. Gels were stained with Coomassie brilliant blue R-250 and destained in a solution of methanol/acetate. Quantitative determination of gelatinase activity was achieved by a densitometric image analysis of unstained bands.

Assay for reactive oxygen species (ROS) detection

The oxidative burst of neutrophils can be monitored quantitatively using H₂DCFDA. Intracellular H₂DCFDA, a nonfluorescent fluorescein analog, can be oxidized to highly fluorescent DCF by activated neutrophils (36). Briefly, after 20 h of coincubation with tumor cells, neutrophils were harvested and incubated with serum-free medium containing 10 µM H₂DCFDA at 37°C for 20 min. Fluorescence was monitored by FACScan with excitation at 488 nm and emission at 530 nm. This generation of ROS was completely suppressed by the addition of the antioxidant acetylcysteine.

Statistical analysis

Results are expressed as the mean ± SE. Values were compared using Student’s t test for independent experiments. For multiple factors comparison between different groups, data were analyzed by a multiway ANOVA (FasL, LPS, host, and time) using SAS statistical software package version 8.2 (SAS Institute, Cary, NC). Statistical significance was set at p 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
FasL^high tumor cells formed larger tumors with massive neutrophil infiltration than did FasL^low tumor cells

C57BL/6 mice were given an injection of 5 x 10⁵ FasL^high (Vn) or FasL^low (Rn) tumor cells in the left flank, and tumor nodules were palpable 10 days after inoculation. FasL^low tumor cells (R4, R5, R6, or R7) formed smaller tumors in mice as compared with FasL^high tumor cells (V1, V2, V5, or V13) measured on days 12, 15, or 18 postinoculation (Table I). The average incidence of tumor formation was much lower in mice inoculated with FasL^low tumor cells (44% by day 12; 61% by day 15) than with FasL^high tumor cells (100% by day 12; 100% by day 15).

View larger version (146K): [in this window] [in a new window]   FIGURE 1. Immunohistochemical analysis of FasL expression and neutrophil infiltration of tumor nodules on day 18. Tumors from mice inoculated with FasLhigh (A) and FasLlow (D) were immunostained for FasL, also showing FasL on vessels. Neutrophils were stained with the anti-Ly-6G Ab (B, C, E, and F). A–C, FasLhigh tumor cells; D–F, FasLlow tumor cells. A, C, D, and F, Magnification at x400; B and E, magnification at x100.

To determine which immune cells were required for controlling tumor formation, mice were depleted of CD4⁺ T cells, CD8⁺ T cells, or neutrophils by specific Abs (Fig. 2). Although FasL^high tumors had a greater amount of neutrophilic infiltration, to our surprise, the development of FasL^high tumors was not significantly enhanced in neutrophil-depleted mice (Fig. 2A). The average size of FasL^high tumors (V3, V4, or V5) in mice depleted of CD4⁺ T cells was similar to that in mice that received control IgG on day 18 postinoculation. CD8⁺ T cell depletion did not affect the tumor size of V3 and V4, but reduced the size of V5. The size of V13 tumors was reduced by combined depletion of CD4⁺/CD8⁺ T cells, but not significantly affected by CD4⁺/neutrophils and CD8⁺/neutrophil cell depletion (Fig. 2B). In contrast, depletion of immune cells affected the size of FasL^low tumors to various extents. The average tumor volume of R4 was increased by CD4⁺ or CD8⁺ T cell depletion. The size of R6 tumors in mice was significantly enhanced by CD8⁺ T cell depletion, but not by CD4⁺ T cell depletion. With neutrophil depletion, the size of FasL^low tumors (R4 and R6) increased by 2- to 4-fold on day 18 postinoculation (Fig. 2A). Multiple depletions of CD4⁺/CD8⁺ T cells, CD4⁺/neutrophils, and CD8⁺/neutrophils further enhanced the size of R4 (Fig. 2B).

View larger version (42K): [in this window] [in a new window]   FIGURE 2. Effect of CD4+ T cell-, CD8+ T cell-, and neutrophil-depleted mice on tumor formation. FasLlow or FasLhigh tumor cells at 5 x 105 were injected into the left flank of mice. Abs were used to deplete immune cells of mice. Student’s t test compared tumor size on day 18 in control mice to determine their immune suppression (*, p < 0.05; **, p < 0.01). A, Single depletion; B, multiple depletion.

We further investigated whether neutrophils could directly lyse tumor cells in vitro. Apoptotic cells were stained with PI and appeared as a sub-G₀/G₁ population in flow cytometric analysis. Murine neutrophils killed tumor cells at an E:T ratio of above 25:1 under coculture conditions (Fig. 3A). FasL^high tumor cells were more resistant to neutrophil-mediated apoptosis than were FasL^low tumor cells (compare V13 with R7 in Fig. 3A; V8, V11, and V13 with R4, R6, and R7 in Table II). In accordance with the results of PI staining, the TUNEL assay detected a higher percentage of apoptotic cells in FasL^low tumor cells cultured with neutrophils than in FasL^high tumor cells (p < 0.001, Fig. 3B). These results suggest that tumor FasL may inhibit neutrophil-mediated apoptosis in tumor cells.

View larger version (34K): [in this window] [in a new window]   FIGURE 3. In vitro cytotoxicity assay. Peritoneal exudate neutrophils were coincubated with V13 (open bars) or R7 (hatched bars) for 20 h. Apoptosis in tumor cells was determined with PI staining at the indicated E:T ratios (A). Apoptotic cells were also stained by TUNEL to detect DNA strain breaks (B). Data represent the mean ± SE of triplicate samples. Student’s t test was used to compare FasLhigh and FasLlow tumors (*, p < 0.005; **, p < 0.001).

Tumor FasL impaired the activation of neutrophils

The above results showed that FasL^high tumor cells were more resistant to neutrophil-mediated apoptosis than were FasL^low tumor cells in vitro. To elucidate how tumor FasL affected the function of neutrophils, we analyzed gelatinase B release, ROS production, and CD11b expression of neutrophils. Neutrophils coincubated with FasL^low tumor cells (R4, R6, or R7) released more gelatinase B to the culture supernatants than did those with FasL^high tumor cells (V7, V8, or V13) (p < 0.001, Fig. 4, A and C). In contrast, tumor cells did not release gelatinase B (Fig. 4B), indicating that the gelatinase activities detected in the culture medium were mainly produced by neutrophils. In coculture with FasL^low tumor cells (R4 or R7), the expression of CD11b on the surface of neutrophils was up-regulated compared with those with FasL^high tumor cells (V8 or V13) (p < 0.001, Fig. 4D). In accordance with the suppressive effect of tumor FasL on gelatinase B release and CD11b expression, neutrophils cocultured with FasL^low tumor cells produced more ROS than did those with FasL^high tumor cells (compare V8/V11 with R4/R7; p < 0.001, Fig. 4E). Neutrophils expressed Fas and FasL (Fig. 4F). Levels of Fas in neutrophils did not change under the culture conditions used in this study. Neutrophils cultured in vitro for 20 h expressed low level of FasL (Fig. 4F, 20 h). Activation of neutrophils by LPS or PMA enhanced the expression of FasL. Interestingly, the expression of FasL in neutrophils was significantly augmented by a 20-h coculture with FasL^low tumor cells (R4 or R7), but not with FasL^high tumor cells (V5, V11, or V13). We further used recombinant mFasFc chimera proteins to interfere with the interaction of Fas and FasL. Treatment with mFasFc did not significantly affect the spontaneous apoptosis of tumor cells cultured alone in vitro. In addition, adding mFasFc to the culture medium did not alter the basal ROS production of casein-enriched neutrophils. The ROS production of neutrophils upon contact with FasL^high tumor cells (V11 or V13) was enhanced when tumor cells were pretreated with mFasFc for 1 h before coculture (Fig. 5A). In contrast, mFasFc did not affect the ROS production of neutrophils cocultured with FasL^low tumor cells. Accordingly, mFasFc pretreatment elevated the susceptibility of FasL^high (V11 or V13) tumor cells to neutrophil killing, but not that of FasL^low cells (R4 or R7) (Fig. 5B). These results indicate thus that tumor FasL can impair the activation and oxidative bursts in neutrophils.

View larger version (63K): [in this window] [in a new window]   FIGURE 4. FasL on tumor cells impaired the activation of neutrophils. A, Release of gelatinase B in cell supernatants from coculture of neutrophils and tumor cells coincubated for 20 h. Gelatinase B was detected by zymography. P, Positive control; neutrophils were incubated with LPS (10 µg/ml) for 6 h. B, Gelatinase B activity in culture supernatants of tumor cells alone. C, Summary of gelatinase activity. D, Effects of tumor FasL on CD11b surface expression of neutrophils. The expression of the CD11b on neutrophils was up-regulated by coculture for 20 h with FasLlow tumor cells (E:T ratio, 50:1). N, Neutrophils cultured alone in vitro. E, PMA-induced generation of ROS of neutrophils was measured. C, Neutrophils treated by PMA served as a control. F, Fas and FasL transcripts in neutrophils. Neutrophils and B16F10 derivatives (FasLhigh tumor cells, V5, V11, or V13; Faslow tumor cells, R4 or R7) were cultured together for 20 h. Neutrophils were harvested in separate, and the transcripts of Fas and FasL in those cells were detected by RT-PCR. F, Fresh neutrophils; 20 h, neutrophils cultured for 20 h in vitro; LPS, neutrophils treated with LPS 10 µg/ml for 6 h; PMA, neutrophils treated with 100 ng/ml PMA for 30 min.

View larger version (31K): [in this window] [in a new window]   FIGURE 5. Blockage of Fas and FasL interaction by mFasFc. Tumor cells were preincubated with mFasFc protein for 1 h and then were subjected to coculture with neutrophils (E:T ratio, 50:1) for 20 h. A, ROS production in neutrophils and B, tumor cell apoptosis detected by PI staining. Neutrophils cultured alone for 20 h served as a control. Values analyzed by Student’s t test differed between FasLhigh and FasLlow tumor cells, which either received treatment with mFasFc protein or did not (*, p < 0.005; **, p < 0.001).

As the functions of neutrophils were impaired upon contact with FasL^high tumor cells, we tested whether activating neutrophils by LPS inhibits tumor growth in vivo. An LPS injection slightly inhibited the growth of FasL^high tumors (V5 or V13), while it significantly inhibited that of FasL^low tumors (R6 or R7) (Fig. 6). By day 12, the tumor incidence of C57BL/6 mice implanted with FasL^low tumor cells was lower in LPS-treated groups (4 of 8; average incidence, 50%) than in saline-treated groups (6 of 8; average incidence, 75%) (Fig. 6A). LPS can activate both neutrophils and B cells (37). To answer the question of whether LPS, in the absence of an adaptive immune response, is still capable of activating neutrophils to kill tumors, we grew tumors in NOD/SCID mice. Then LPS was serially injected into the tumor area. The melanoma transfectants showed the same tumorigenicity in NOD/SCID mice as those in C57BL/6 mice in terms of tumor size and tumor incidence. FasL^low tumors developed in NOD/SCID mice were more sensitive to LPS treatment by significant reduction in tumor volumes (Fig. 6, B and D). With an LPS injection, the incidence of tumor formation in NOD/SCID mice decreased (average incidence, 66.7% for FasL^high tumors and average incidence, 0% for FasL^low tumors in LPS-treated groups compared with average incidence, 100% for FasL^high tumors and average incidence, 62.5% for FasL^low tumors in saline-treated groups) (Fig. 6B).

View larger version (55K): [in this window] [in a new window]   FIGURE 6. Tumor volumes of FasLhigh and FasLlow derivatives in LPS-treated mice. FasLlow or FasLhigh tumor cells at 5 x 105 were injected into the left flank of mice. A multiple-way ANOVA analysis on factors of FasL, LPS, host, and time compared tumor size on days 12 and 18 to determine the LPS effect (*, p < 0.05; **, p < 0.001).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

B16F10 transfectants formed tumors in NOD/SCID mice as quickly as in immune-intact C57BL/6 mice. We barely detected T, B, and NK cells in tumor regions. Unexpectedly, FasL^low tumors grew faster in CD4⁺ T cell-, CD8⁺ T cell-, or neutrophil-depleted mice, although those cells did not intensively infiltrate into tumor sites as detected by immunohistochemical staining. In agreement with the established inflammation-inducing effect of FasL, down-regulation of FasL of melanoma cells reduced neutrophil infiltration. Interestingly, although FasL^high tumors showed heavy neutrophilic infiltration, the size of tumor nodules progressively increased and eventually killed the mice. Moreover, neither depletion of neutrophils nor depletion of CD4⁺ T cells significantly affected the development of FasL^high tumors, indicating a FasL-associated suppressive mechanism operated in situ during the encounter between the tumor and immune cells. That CD8 depletion reduced the size of V5 tumor is a surprise finding. Similarly, double depletion of CD4⁺ and CD8⁺ cells reduced the size of V13. Previous studies showed that T cells could help the formation of some tumors (43, 44, 45). A distinct profile of cytokines produced by those tumor-infiltrating T cells is believed to cause immune dysfunction and thus stimulate tumor formation (46, 47). However, this supposition cannot be applied as a general rule to all FasL^high clones. Not all tumor clones were affected by CD8/CD4 depletion, suggesting that other unknown factor contributed to the tumor surveillance mechanism in host animals.

Results of the in vitro cytotoxicity assay showed that tumor cells became more susceptible to neutrophils when their FasL was down-regulated by ribozymes. In addition, the findings that paraformaldehyde-fixed neutrophils were less cytotoxic to tumor cells, that the addition of supernatants from coculture of tumor cells plus neutrophils failed to induce tumor cell apoptosis, and that the separation of neutrophils and tumor cells by the Transwell culture system prevented apoptosis in tumor cells indicate that tumor cell apoptosis induced by neutrophils involves surface proteins and requires cell-to-cell contact. We found no evidence that FasL^high tumor cells could directly kill neutrophils. As a matter of fact, tumor cells reduced the spontaneous apoptosis in neutrophils cultured with tumor cells regardless of their levels of tumor FasL. Recently, we found that when in contact with human glioma cells, the spontaneous apoptosis in peripheral circulating neutrophils was reduced due to the elevated levels of IL-6 and IL-8 in culture medium. Furthermore, glioma cells produce these survival factors upon the activation of Fas (48). We believe that the prolonged survival of the murine neutrophils upon incubation with melanoma cells is operated through a similar mechanism by human neutrophils. Thus, direct cell cytotoxicity to deplete neutrophils cannot explain why FasL^high tumor cells were less sensitive to neutrophils and suggests a functional deterioration in neutrophils induced by tumor FasL. To test this hypothesis, we examined the activities of neutrophils in detail, focusing on the productions of secretary proteases and ROS, which are major forces in tumor destruction (49, 50, 51). We observed that the resistance of FasL^high tumor cells to killing by neutrophils was correlated with impaired neutrophil activation, as demonstrated by reductions in gelatinase B secretion, ROS production, and the surface expression of CD11b. Neutrophils express Fas and FasL. Activating neutrophils by LPS, PMA, or coculture with FasL^low tumor cells can enhance the expression of FasL in neutrophils. Interestingly, coculture with FasL^high tumor cells did not augment the expression of FasL in neutrophils. As Fas/FasL system is one of the major cytotoxic mechanisms used by neutrophils, the loss of FasL on neutrophils upon cell contact may contribute to the resistance of FasL^high tumor cells against neutrophil killing. Moreover, the application of mFasFc to block tumor FasL resulted in a greater amount of ROS production and higher tumor cytotoxicity of neutrophils. These data support tumor FasL inhibiting the function of neutrophils.

To test whether the restoration of neutrophil activity could inhibit tumor formation, we applied LPS and PMA to stimulate neutrophils. The tumor formation of FasL^low transfectants in C57BL/6 mice was delayed by serial injections of LPS or by adoptive transfer of casein-elicited or PMA-treated neutrophils. Similarly, in NOD/SCID mice, injection of LPS was also effective in slowing down the growth of tumors with low FasL, suggesting that adaptive immunity is not essential for LPS-induced neutrophil activation in antitumor immunity against FasL^low tumors. However, FasL^high tumor cells showed different responses to LPS treatment. Injection of LPS alone did not significantly affect the growth of FasL^high tumors in either NOD/SCID or C57BL/6 mice. But when the neutrophils were activated in vitro and reinjected into tumor-bearing mice, they were effective in delaying the growth of FasL^high tumors. These results suggest that tumor FasL causes local suppression of neutrophil activities very quickly, and the treatment with LPS alone cannot fully reactivate those neutrophils in tumors. Instead, preactivated neutrophils, which have not been affected by tumor FasL before cell contact, can suppress tumor growth in vivo. The pivotal role of innate immune cells in tumor combat has previously been recognized in several tumors, but combined therapy with drugs and LPS to improve innate immunity has exhibited various degrees of effectiveness against tumors (52, 53, 54, 55, 56, 57, 58, 59, 60). In this study, our results show that tumor FasL determines the effectiveness of innate immunity. Cell therapy with neutrophils, particularly those preactivated in vitro, may achieve a better antitumor effect than LPS treatment for tumors expressing high levels of FasL.

In conclusion, we demonstrate in this work that the expression of FasL on melanoma cells leads to neutrophil inactivation and helps tumor development in s.c. regions. An understanding of how tumor cells defeat neutrophils through the Fas/FasL pathway will facilitate the development of effective strategies that will be beneficial to antitumor therapy.

	Footnotes

 
¹ This work was supported by grants from the National Science Council (NSC89-2320-B006-020) and MOE Program for Promoting Academic Excellence of Universities from Minister of Education (91-FA09-1-4).

² Address correspondence and reprint requests to Dr. Bei-Chang Yang, Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, 70101, Republic of China. E-mail address: y1357{at}mail.ncku.edu.tw

³ Abbreviations used in this paper: FasL, Fas ligand; H₂DCFDA, 2',7'-dichlorofluorescein diacetate; mFasFc, mouse FasFc; NOD, nonobese diabetic; PI, propidium iodide; ROS, reactive oxygen species.

Received for publication December 13, 2002. Accepted for publication June 2, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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