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From the MRC Centre for Immune Regulation at University of Birmingham Liver Research Labóratories, Queen Elizabeth Hospital, Birmingham, United Kingdom
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is substantial evidence that lymphocyte-mediated mechanisms are important in controlling tumor growth in some cancers.4-6 However, despite being infiltrated by lymphocytes, the growth of colorectal hepatic metastases is relentless, suggesting that the local antitumor immune response is impaired.7,8 Most mononuclear cells infiltrating colorectal hepatic metastases are confined to the peritumoral margin between the advancing edge of the tumor and the adjacent uninvolved liver tissue.8 Although the infiltrate contains activated T cells, the cells are unable to lyse autologous tumor targets, even following in vitro activation with rIL-2,8,9 suggesting that the tumor deletes antitumor effector lymphocytes. It has been proposed recently that one mechanism responsible for this is Fas-mediated killing of lymphocytes by Fas ligand (Fas-L)-expressing tumor cells.10 Activated cytolytic lymphocytes (CTL) express Fas-L, a member of the tumor necrosis superfamily, which can trigger apoptosis in target cells that bear its cell surface receptor Fas (CD95/Apo-1).11 Fas-L/Fas interactions are involved in CTL killing of target cells, including virally infected hepatocytes in viral hepatitis.12 However, in addition to Fas-L, activated CTL also express Fas. Lymphocyte-lymphocyte interactions involving Fas and Fas-L trigger apoptosis and provide a mechanism for down regulating immune responses in vivo.11,13 The potent ability of Fas-L-bearing cells to kill Fas-expressing lymphocytes means that Fas-L expression is tightly controlled in vivo and tissues in which Fas-L is constitutively expressed, such as the eye and testis, are immune-privileged sites.14,15 Several tumor types, including primary colorectal cancer and hepatocellular carcinoma, express Fas-L and exploit its ability to kill Fas-expressing lymphocytes to evade immune recognition.10,16 In the present studies we have extended these observations to colorectal hepatic metastases and propose that the Fas pathway not only is responsible for killing infiltrating lymphocytes, but also facilitates local tumor growth by inducing apoptotic cell death in normal hepatocytes at the tumor margin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Surgically resected colorectal hepatic metastases were obtained from 15 patients (6 males and 9 females; median age 59 years, range, 48–79 years) treated at the Liver Unit, Queen Elizabeth Hospital (Birmingham, UK). Six were histologically well differentiated, 5 moderately differentiated, and 4 poorly differentiated adenocarcinoma. None of the patients had received immunosuppressive drugs, radiotherapy, or chemotherapy prior to surgery. Tissue containing both tumor and adjacent, macroscopically normal liver and a sample of noninvolved liver tissue distant from the tumor were snap-frozen in liquid nitrogen and stored at -70°C until analyzed. A separate sample of fresh tumor tissue was used for isolation of lymphocytes. Peripheral blood lymphocytes (PBL) were isolated from venous blood obtained from the same patients immediately before surgery.
Lymphocyte Isolation
Tumor-infiltrating lymphocytes (TIL) were isolated from fresh tumor tissues removed at surgery as described previously.8 Tumor tissues were immediately cut into small pieces, washed, and digested using RPMI-1640 (Gibco/Life Technologies, Paisley, UK) supplemented with 0.2% (w/v) collagenase type IV (Sigma, Poole, UK) and 20% fetal calf serum (Gibco) for 2–3 hours with continuous stirring at room temperature. The tumor digest was then passed through a nylon mesh to obtain a single cell suspension that was washed with phosphate-buffered saline (PBS) until the supernatant became clear. The single cell suspension was layered onto Ficoll Hypaque (Lymphoprep, Nycomed, Oslo, Norway) and centrifuged at 1600 rpm for 30 minutes at room temperature. TIL and tumor cells were recovered from the interface. Autologous PBL were isolated from heparinized venous blood obtained from the same patients immediately prior to surgery. PBL were separated by Ficoll Hypaque centrifugation at 1600 rpm for 30 minutes at room temperature and then washed twice with PBS.
Isolation and Culture of Primary Human Hepatocytes
Primary human hepatocytes were isolated from surplus liver tissue obtained from adult donor livers that had been reduced in size for transplantation into pediatric recipients as described previously.17 Approximately 150 g of human liver tissue were treated with segmental perfusion with 5 mmol/L calcium chloride, 0.05% Collagenase H (Life Technologies), 0.025% dispase (Boehringer-Mannheim, Mannheim, Germany), 0.0125% type 1-S hyaluronidase (Sigma), and 0.005% DNase (Boehringer Mannheim) in 10% Hanks' balanced salt solution. The resulting cell suspension was filtered and centrifuged at 28 x g to pellet hepatocytes. The hepatocytes were further purified by Percoll density gradient centrifugation and their viability determined by trypan blue dye exclusion. Immediately after isolation, hepatocytes were resuspended in Williams E medium containing hydrocortisone, insulin, and glutamine and seeded onto collagen-coated eight-well tissue culture plates (Costar Corp., Cambridge, MA), which were then incubated at 37°C in humidified air and 5% CO2. The hepatocytes were allowed to adhere for 2 hours, following which the medium was replaced with fresh culture medium and the cells allowed to recover for 24 hours before further studies began.
Culture of SW620
The human colon cancer cell line SW620 was obtained from American Type Culture Collection (Manassas, VA) and grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS) in a humidified 5% CO2 at 37°C.
Immunohistochemistry
Localization of Fas and Fas-L expression in peritumoral tissue was carried out on 6-µm cryostats that contained both tumor and adjacent normal hepatocytes, using standard immunohistochemistry as described previously.18,19 Tissue sections were fixed in acetone for 10 minutes at room temperature and then incubated with rabbit polyclonal anti-human Fas- or Fas-L-specific IgG (Santa Cruz Biotechnology, Santa Cruz, CA) at 2 µg/ml. The primary antibody binding was localized by mouse anti-rabbit (Dako, High Wycombe, UK) followed by rabbit anti-mouse (Dako) secondary antibodies, developed by the indirect alkaline phosphatase-anti-alkaline phosphatase (Sigma) technique, and the resulting enzyme complex visualized with napthol-AX (Sigma) and Fast Red TR (Sigma). Sections were counterstained with Meyer's haematoxylin. Incubations were done at room temperature for 45 minutes and sections were washed for 5 minutes with 2 changes of Tris buffer between incubations. Normal tonsil sections were used as positive controls. Negative controls consisted of sections in which the primary antibody was omitted. In addition, the specificity of anti-Fas or anti-Fas-L antibody was confirmed by the addition of a competitive inhibitor, either a Fas or Fas-L peptide (20 µg/ml, Santa Cruz Biotechnology), during the primary antibody incubation. The results of immunohistochemical studies for Fas and Fas-L expression were assessed by scoring the intensity of immunoreactivity for a given antigenic determinant relative to control staining, using a scale of 0 = negative, 1 = weak, 2 = moderate, and 3 = strong. The mean (± SE) was then calculated for all individual scores. We have previously validated this quantitative scoring system using confocal microscopy.20
Cellular proliferation was determined by immunostaining of paraffin-embedded sections with mouse anti-human Ki-67 (nuclear proliferation antigen; mib-1 clone of IgG1 isotype from Coulter Electronics, Luton, UK) at 1 µg/ml. Sections were dewaxed with xylene/alcohol and treated with 3% hydrogen peroxide in methanol for 10 minutes to block endogenous peroxidase, followed by microwaving for 30 minutes in citric acid (2.1 g/L adjusted to pH 6.0 with NaOH) to enhance antigen retrieval. Anti-Ki-67 was detected by biotinylated goat anti-mouse (Dako) and streptavidin-avidin-biotin complex/horseradish (Dako) and developed using diaminobenzidine tetrahydrochloride (Sigma). The sections were counterstained with Mayer's haematoxylin. Sections stained with an irrelevant mouse mAb were used as negative control. Normal tonsil sections stained with anti-Ki-67 were employed as positive controls. Approximately 100 tumor cells, mononuclear cells, and hepatocytes were enumerated separately in each high power field (x400) and 10 random high-power fields were counted on each section using light microscopy. The results were expressed as mean ± SE of 10 samples.
The proliferative index was calculated as follows:
 | (1) |
Determination of Apoptosis
Apoptosis was assessed by three methods: cellular morphology according to Kerr's criteria (cytoplasmic shrinkage, pyknosis, and/or nuclear fragmentation),21 in situ end-labeling (ISEL) to detect DNA fragmentation on paraffin-embedded tumor sections, and immunofluorescent staining of cryostats using mouse anti-human APO 2.7-PE (2.7A6A3 clone, IgG1 isotype, Coulter Electronics) at 4 µg/ml. ISEL was performed on paraffin-embedded tissue sections as described elsewhere.22 Sections 6 µm in width were dewaxed, dehydrated, and air-dried for 15 minutes, followed by incubation for 30 minutes at 37°C with 5 µg/ml proteinase K (Sigma) diluted in 50 mmol/L TBS, pH 8.2, containing 1 mmol/L EDTA. The sections were then washed 3x in distilled water, dehydrated with alcohol, and air-dried. One hundred microliters of the following labeling mixture were applied to each section: 0.01 mmol/L aATP, dCTP, dGTP, and digoxigenin-labeled dUTP (Boehringer Mannheim, Lewes, UK) made up in 50 mmol/L TBS, pH 7.4, containing 5 mmol/L MgCl2, 10 mmol/L beta-mercaptoethanol, 5 mg/ml BSA, and 20 units/ml Klenow DNA polymerase fragment (Boehringer Mannheim). Siliconized coverslips were placed on each section to minimize drying and spread of nucleotide mixture and sections were further incubated for 1 hour at 37°C. The reaction was terminated by 3 washes in distilled water. DNA polymerase was omitted in negative control sections. Digoxigenin-labeled cells were identified using alkaline phosphatase-conjugated F(ab)2 fragments of a polyclonal sheep anti-digoxigenin antibody (Boehringer Mannheim) diluted 1:200 in TBS, pH 7.4, and incubated for 1 hour at room temperature. Sections were then washed 3x in TBS, pH 8.2, and developed with alkaline phosphatase/Fast Red working solution. Approximately 100 tumor cells, mononuclear cells, and hepatocytes were enumerated separately in each high-power field (x400) and 10 random high-power fields were counted on each section using light microscopy. Results were expressed as mean ± SE of ten samples.
The apoptotic index was calculated as follows:
 | (2) |
Flow Cytometry
The expression of Fas and Fas-L on TIL, primary human hepatocytes, and tumor cells was assessed by immunofluorescent staining with rabbit polyclonal anti-human Fas- or Fas-L-specific antibodies (Santa Cruz Biotechnology) at 2 µg/ml followed by 1:20 dilution of FITC-labeled goat anti-rabbit immunoglobulin (Dako). The phenotypic composition of freshly isolated TIL and autologous PBL was analyzed by two-color flow cytometry using standard techniques.23 Single-cell suspensions (106 cells/ml) were incubated with 5 µl of primary unconjugated mouse mAb followed by 1:20 dilution of FITC-conjugated F(ab)2 fragments of rabbit anti-mouse immunoglobulin (Dako). Thereafter, the cell suspension was incubated with normal mouse serum to saturate the free binding sites on the F(ab)2 fragments before a final incubation with PE-conjugated anti-CD3 (Dako). All incubations were carried out at 4°C for 30 minutes and cells were washed twice with PBS (0.02% w/v sodium azide and 2% v/v FCS) between incubations. The cell suspension was fixed with 1% paraformaldehyde and analyzed using the FACS 440 (Becton Dickinson). A lymphocyte gate was set to exclude dead cells and debris and at least 104 cells were analyzed in each sample. Irrelevant isotype-matched mAb or nonimmune serum was used as a control. In addition, the specificity of the anti-Fas or anti-Fas-L antibody was confirmed by the addition of a competitive inhibitor, either a Fas or Fas-L peptide (20 µg/ml, Santa Cruz Biotechnology), during the primary antibody incubation.
Induction of Fas-Mediated Apoptosis of Primary Human Hepatocytes
Cell surface Fas receptor expression on isolated primary human
hepatocytes was studied by flow cytometric analysis of single-cell
suspensions of confluent hepatocyte monolayers before and after
treatment with varying doses of IL-1 for 24 hours at 37°C. The effect
of Fas ligation on human hepatocytes was evaluated by treating the
cells with 20 ng/ml anti-Fas antibody for 12, 24, and 48 hours at
37°C, following which apoptosis was determined by ISEL staining of
cytospin sections. The ability of TIL derived from colorectal liver
metastases and SW620 human colon cancer cells to induce apoptosis of
human hepatocytes was investigated by a cell coculture cytotoxicity
assay. Effector TIL or SW620 tumor cells were cocultured with confluent
monolayers of target human hepatocytes for 24 hours at an
effector:target ratio of 5:1 (TIL) or 2:1 (SW620). In some experiments,
40 ng/ml of antibody to human Fas-L (mAb NOK-1, Pharmingen, San Diego,
CA) were added to the hepatocyte culture medium before coculturing the
hepatocytes with TIL or SW620. This mAb inhibits Fas-mediated apoptosis
by specifically binding to human Fas-L on effector cells.24
Each experiment was carried out in triplicate. Hepatocytes were
harvested by trypsinization for 5 minutes with 0.25% v/v bovine
pancreatic trypsin and then neutralized by 10% FCS. Apoptosis of
hepatocytes was determined by ISEL of DNA fragmentation and quantified
on cytospin sections by calculating approximately 200 hepatocytes on
ten random high-power fields (x400) on each cytospin section using
light microscopy. The enumeration of apoptotic hepatocytes is
calculated as follows:
 | (3) |
Statistical Analysis
Kruskal-Wallis analysis of variance was used to determine variation between groups of samples. Differences between unpaired samples were analyzed by the Mann-Whitney U test for nonparametric data. Differences between paired samples were analyzed by the nonparametric Wilcoxon's rank sum test. The flow cytometric data of Fas expression on primary human hepatocytes were analyzed by the unpaired Student's t-test assuming unequal variance. The level of significance was set at P < 0.05.
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Results |
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. A large subset of
these T cells also expressed both Fas and Fas-L (Figure 1)
. Despite
their activated phenotype, these tumor-derived lymphocytes are unable
to lyse autologous or allogeneic tumor cell targets in
vitro.8
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Immunohistochemistry was used to analyze the pattern and
distribution of Fas and Fas-L protein expression in the different
cellular compartments at the margin of the tumor. Ten of the cases we
studied were moderately or well differentiated colorectal hepatic
metastases consisting of glandular structures with two distinct
domains: the basolateral membrane, which is in contact with the
extracellular matrix and the peritumoral stroma, and a luminal (apical)
domain, which is not. Fas expression was restricted to the luminal
surface of carcinoma cells (Figure 2a)
,
whereas Fas-L protein was detected on the membrane throughout the tumor
cells (Figure 2b)
. Fas and Fas-L proteins were also detected on
lymphocytes infiltrating the stroma between the tumor cells and
adjacent uninvolved hepatocytes (Figure 2, a and b)
. The strongest
expression of Fas was on the uninvolved hepatocytes, which showed
intense membranous and cytoplasmic staining for Fas protein extending
for several cell layers from the interface with the peritumoral stroma
(Figure 2a)
. Fas-L protein was not detected in either the peritumoral
or more distant hepatocytes (Figure 2b)
. The specificity of the
anti-Fas or anti-Fas-L antibody was confirmed by absent immunostaining
in the presence of a competitive inhibitor, Fas or Fas-L peptide, in
the primary antibody incubation.
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To determine which of the Fas-expressing cells were targets
of Fas-L attack, we looked for apoptosis in the different cellular
compartments at the tumor margin, ie, tumor cells, inflammatory cells,
and hepatocytes. Many Fashigh hepatocytes at the tumor
margin as well as many infiltrating lymphocytes were undergoing
apoptosis as defined by morphology, Apo 2.7 immunostaining, and ISEL of
DNA fragmentation (Figures 2, c and d
, 3,
and 4). Hepatocytes farther from the
tumor margin were Faslow and very few (<3%) were
apoptotic. Apoptotic lymphocytes were detected in lymphoid aggregates
as well as among mononuclear cells in direct contact with tumor cells,
suggesting that lymphocytes were being killed by neighboring
Fas-L-expressing lymphocytes via a bystander effect as well as by Fas-L
on tumor cells (Figure 2, c and d)
. In contrast, very few tumor cells
were apoptotic (Figure 2, c and d)
and many were in active cell cycle
as determined by staining for the nuclear proliferation antigen Ki-67
(Figures 2e and 4)
. Thus, both the infiltrating lymphocytes and the
surrounding hepatocytes were targets for Fas-mediated apoptosis.
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Increased Fas expression has been reported on hepatocytes during
liver inflammation in vivo.12,25
Thus, the
increased Fas expression detected on hepatocytes at the tumor margin in
the present study could be a consequence of the inflammatory response
at this site. In support of this we detected the proinflammatory
cytokines IL-1 and TNF-
in the peritumoral infiltrate by
immuno-histochemistry (data not shown). Furthermore, although the
level of surface Fas on human hepatocytes was high after 24 hours in
culture (85% of cells positive) it could be increased further by
treatment with exogenous IL-1 (Figure 5)
.
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).
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Flow cytometric and immunohistochemical studies show that
lymphocytes infiltrating colorectal hepatic metastases express cell
surface Fas-L (Figures 1 and 2)
. In order to determine if the cell
surface Fas-L was functional, TIL isolated from colorectal hepatic
metastases were cocultured for 24 hours with cell surface Fas-positive
primary human hepatocytes at a TIL:hepatocyte ratio of 5:1. At the end
of the coculture 47 ± 9% of hepatocytes were apoptotic as
determined by ISEL of DNA fragmentation when cocultured with TIL
compared to 4.3 ± 1.0% apoptosis of hepatocytes in control wells
treated with medium alone (P = 0.009, Figure 7
).
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). The NOK-1 monoclonal
antibody alone did not effect apoptosis of hepatocytes, as we
have reported similar observations previously.24
Thus the Fas receptor and Fas-L on human hepatocytes and TIL,
respectively, are functional in vitro. Tumor Cells from Colon Cancer Metastases Can Induce Fas-Mediated Apoptosis of Human Hepatocytes in Vitro
Because tumor cells express Fas-L it is possible that these cells
also induce apoptosis of normal hepatocytes. In order to test this we
used SW620, a human colonic tumor cell line derived from a lymph node
metastasis of a colon cancer and previously shown to express Fas-L at
both the mRNA and protein levels.10
The expression of
surface Fas-L on SW620 was confirmed by flow cytometric analysis, which
showed that 87% of the tumor cells used for coculture experiments were
positive for cell surface Fas-L (Figure 8)
. In order to determine the ability
of metastatic colon cancer cells to kill hepatocytes, SW620 cells
were cocultured with primary human hepatocytes for 24 hours at 37°C
using a ratio of 2 SW620 cells to 1 hepatocyte. Coculture resulted in
apoptosis of 26 ± 2% of hepatocytes as determined by ISEL
compared with only 5.6 ± 1.7% apoptosis in control wells
in which the hepatocytes were treated with culture medium alone
(P = 0.004) (Figure 9)
.
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. There was no significant difference between the
apoptosis in hepatocytes cocultured with NOK-1-treated SW620 (11.2
± 2%) and in those treated with NOK-1 alone (9.6 ± 1.6%,
Figure 9
). Thus, SW620 colon cancer cells can induce apoptosis of human
hepatocytes in vitro in a Fas-dependent manner. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One reason for the failure of the immune response against colorectal
metastases is the lack of lymphocyte entry into tumor tissue. The
inflammatory infiltrate is confined to the peritumoral margin, with
very few lymphocytes detected in the tumor parenchyma (Figure 2)
.
Moreover, <5% of the lymphocytes at the tumor margin were
proliferating, as determined by expression of the nuclear proliferation
antigen Ki-62, whereas up to 50% of them were dying by apoptosis
(Figures 3 and 4)
.
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The outcome of these strategies is that activated lymphocytes accumulate in the stroma at the tumor periphery but cannot penetrate the tumor itself. In the absence of Fas-bearing tumor targets, the Fas-Lhigh lymphocytes might interact with other Fas-bearing cells including neighboring lymphocytes or adjacent hepatocytes (see below). Thus, lymphocyte death occurs either by suicide, mediated by interactions between Fas and Fas-L within the same cell, or by fratricide, in which Fas-L-positive lymphocytes kill neighboring Fas-positive lymphocytes. Similar mechanisms of fratricidal lymphocyte killing are believed to be responsible for resolving inflammation and maintaining lymphocyte homeostasis at sites of inflammation after the offending antigen has been removed.13,28,29
We propose not only that colorectal metastases protect themselves by
eliminating activated CTLs but also that they utilize the frustrated
immune response at the tumor margin to enhance local invasion into the
adjacent uninvolved liver. The peritumoral infiltrate, which includes
large numbers of activated monocytes as well as CTLs,8
is
associated with the expression of proinflammatory cytokines, including
IL-1, at the tumor margin. Proinflammatory cytokines can increase the
expression of Fas on hepatocytes in vitro (Figure 5)
and the
marked up-regulation of Fas we detected on hepatocytes at the tumor
margin is thus likely to be a consequence of the adjacent inflammatory
infiltrate (Figure 2A)
. In the absence of Fas-expressing tumor targets
it is possible that Fas-Lhigh CTLs at the tumor margin will
engage Fas on adjacent hepatocytes and thereby induce them to undergo
apoptosis, allowing the tumor to expand into the surrounding hepatic
tissue. A similar mechanism of bystander hepatocyte killing has been
proposed to explain hepatocyte destruction in alcoholic
hepatitis.25
We provide evidence that TIL derived from
human colorectal liver metastases can kill hepatocytes because they
were able to induce apoptosis in primary human hepatocytes when
cocultured with these cells in vitro. Much of the apoptosis
could be prevented by blocking Fas-L with the NOK-1 monoclonal
antibody, confirming a role for Fas-mediated killing of hepatocytes by
TIL. However, the failure of NOK-1 to completely inhibit apoptosis of
hepatocytes by TIL suggests that other apoptotic pathways might
also be involved.
We used SW620, a colon cancer cell line, as a model for metastatic colon cancer cells because this cell line was derived from a lymph mode metastasis of a human colon cancer. Our flow cytometric analysis show that approximately 87% of SW620 cells express surface Fas-L and this finding is consistent with a previous study by O'Connell et al, who reported that Fas-L is expressed by SW620 at both the mRNA and protein levels.10 The fact that tumor cells also express Fas-L suggests the possibility that the Fas-L-positive tumor cells can also kill Fas-bearing hepatocytes directly. Further evidence for this mechanism is provided by the experiments in which we have shown that SW620 can also kill primary human hepatocytes in vitro in a Fas-dependent manner. In these experiments the Fas-mediated pathway could account for all of the hepatocyte apoptosis because anti-human Fas-L mAb could completely inhibit the killing of hepatocytes by tumor cells. Despite these intriguing observations, tumor killing of hepatocytes is likely to be less important than TIL-mediated killing because the peritumoral stroma separates the tumor from the surrounding hepatocytes, whereas the CTL are in direct contact with surrounding normal tissue. An alternative source for Fas-L would be soluble ligand secreted or shed from the tumor or the activated lymphocytes, although recent evidence suggests that soluble Fas-L may actually inhibit apoptosis induced by cell-bound Fas-L.30
These studies suggest that an inflammatory response does not always protect the host against an infiltrating tumor and may help to explain the paradoxical observation that some tumors occur less frequently and are less aggressive in patients who are immunosuppressed.31 To our knowledge this is the first time a metastatic tumor has been shown to promote Fas-dependent apoptosis of surrounding uninvolved tissue. These findings may also explain why the liver is a common site for the development of metastatic spread from colon cancers. It is noteworthy that not all primary colon cancers express Fas-L, but all of the colorectal liver metastases we have studied so far are positive for Fas-L (Yoong and Adams, unpublished observation). The Fas pathway would allow Fas-L-expressing cancer cells to establish at the secondary site and grow into adjacent normal liver tissue in an orderly manner. Thus we suggest that the Fas system is pivotal in facilitating local tumor growth of colon cancer cells in the liver. Whether these observations are unique to the liver or are more widely applicable mechanism of tumor invasion in other tissues remains to be determined.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Endowment Fund of the United Birmingham Hospitals and a research fellowship to KFY from the Royal College of Surgeons (England).
Accepted for publication December 10, 1998.
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