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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Department of General Surgical Science, Gunma University, Graduate School of Medicine, Maebashi, Japan
Received July 26, 2003; accepted September 18, 2003.
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Abstract |
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; Berenbaum, 1979); however, other reports indicate that the natural killer (NK) cytotoxicity in the spleen is reinforced by 5-FU (Blomgren et al., 1965; Goto et al., 1981, Okamoto et al., 1998), which is one of the most frequently used anticancer drugs. Heretofore, there have only been a few reports about the immunological effect of 5-FU on cancer.
5-FU is a potent inducer of several cytokines, such as interferon-
, tumor necrosis factor-
and -
, and interleukin-12 (IL-12) (Okamoto et al., 1998). Foremost, IL-12 has been described as a growth factor for activated NK cells and NK1.1+ (NKT) cells (Kobayashi et al., 1989; Perussia et al., 1992; Brunda et al., 1993; Trinchieri, 1994; Hashimoto et al., 1995; Smyth et al., 2000), and these cells have been known to be cytotoxic effector cells and to comprise the main antimetastatic lymphocyte population in the liver (Watanabe et al., 1995; Kobayashi et al., 2002). Meanwhile, systemic administration of the IL-12 protein was reported to augment the number of hepatic mononuclear cells (hMNCs) in mice, suppress the growth of a variety of established tumors in mice, and prolong the survival of tumor-bearing mice (Brunda et al., 1993; Hashimoto et al., 1995; Zou et al., 1995).
The objective of this study is to determine the immunological effect and mechanisms of the intrasplenic effect of 5-FU against liver metastases in mice and to establish whether the administration routes of 5-FU have some relationship with its effects. We investigated the cytotoxicity of splenic cells or hMNCs and the level of IL-12 in the spleen or liver. A subset of hMNCs having an anti-tumor effect was also identified using monoclonal antibodies.
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Materials and Methods |
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Mice. Normal inbred BALB/c mice aged 7 weeks were routinely used (Charles River Japan, Tokyo, Japan). All mice were housed in the Institute of Experimental Animal Research, Gunma University, Graduate School of Medicine, under conditions with a laminar air-flow. They were maintained on standard laboratory feed and in 12-h light/dark cycles.
Preparation and Administration of Colon Tumor Cells. The mice were anesthetized using diethyl ether and were laparotomized using a median incision on day 0. One hundred thousand colon 26 cells, a mouse colon carcinoma cell line capable of liver metastasis of BALB/c mice origin, were obtained by the in vivo selection method (Fidler, 1973) and injected into the portal vein. The wound was closed with continuous sutures of 3-0 silk in layers. Liver metastasis of colon tumor cells was demonstrated on the surface of the liver macroscopically 2 weeks after inoculation.
Administration Routes of 5-FU. The subjects were divided into four groups. On day 14, all mice were anesthetized, and laparotomy was performed again. In the first group, 25 mg/kg 5-FU (a kind gift from Kyowa Hakko Co., Tokyo, Japan) was injected directly into the spleen. The second group took the agent injected via the portal vein, and the third group, via the common iliac vein. Normal saline was injected into the spleen in the last group as a control. Each group consisted of 40 mice.
Survival Rates. Survival rates were calculated using 10 mice from each group from the day administered 5-FU or normal saline.
Collection of Samples and Measurement of Hepatic Metastases. The mice were sacrificed by decapitation under adequate anesthesia with pentobarbital according to the guidelines of Gunma University, Graduate School of Medicine, on day 18. The removed spleen and the liver of 10 mice from each group were weighed. Two days after fixation in 10% formalin, the number of metastatic foci on the surface of each fixed liver was counted. The very small nodes were determined by microscopic analysis.
Cytotoxic T Lymphocyte (CTL) and NK Cytotoxicity Assay of hMNCs and Splenic Cells. Hepatic MNCs were isolated using the method of Wiltrout et al. (1984), with some modifications. Four days after injection of 5-FU (on day 18), 10 mice from each group were sacrificed, and the liver and the spleen were extracted. The liver was minced into small pieces and centrifuged, and the supernatant was removed. A prewarmed enzyme solution, containing 0.005% collagenase type 4 (Sigma-Aldrich, St. Louis, MO) and 500 U/ml DNase type 1 (Biozyme Laboratories, Ltd., Gwent, South Wales, UK) in PBS, was added to this extract. The enzyme extract mixture was incubated at 37°C for 30 min, and the enzymatic digest was washed in a cold medium and centrifuged. The cell suspension was placed through a nylon mesh (50 µm; Tokyo Screen Co., Tokyo, Japan). For liver lymphocyte isolation, Percoll (Amersham Biosciences Inc., Piscataway, NJ) density gradient was used. Cells at the top band were collected and washed in a cold medium. These cells were considered to be hMNCs, and their total number was recorded. The spleens were also finely minced and placed in a 50-ml polypropylene tube in a cold medium. This extract was placed through a nylon mesh, and the total number of collected splenic cells was counted.
Antitumor cellular immunity was determined in terms of the NK and CTL cytotoxicity using51Cr-release assay. One million colon 26 cells incubated with 100 µg/ml Mitomycin-C (Kyowa Hakko Co., Tokyo, Japan) before use and isolated 1 x 106 hMNCs or splenic cells were mix-cultured at 37°C for 6 days as described previously (Mosley et al., 1989; Rodolfo et al., 1996). For delivered CTL isolation, Ficoll-Paque (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK) was used as the medium. After centrifugation, the lymphocyte layer was collected because it contains the effector cells for the CTL cytotoxicity assay. In this study, the NK-sensitive Moloney virus-induced YAC-1 lymphoma cell line of A/Sn mice was used as the target for NK cytotoxicity, and colon 26, as the target for CTL cytotoxicity. These cells were labeled with 100 µCi (per 1 x 106 cells) of Na251CrO4 (1 mCi = 37 MBq/ml; Amersham Biosciences Inc.) for 30 min at 37°C in a complete medium and washed with the medium. Effector cells were splenic cells or hMNCs from each group. The 51Cr-release assay was performed in 96-well round-bottom tissue culture plates. A graded number of effector cells in 100 µl were mixed with 5 x 105 labeled target cells in 100 µl, with an target-to-effector (T:E) ratio of 1:2.5, 1:5, and 1:10. For the determination of maximum release, five wells of target cells in 100 µl of the medium were mixed with 100 µl of Triton-X (Calbiochem-Novabiochem, San Diego, CA). After 4 h of incubation, the supernatant was harvested and counted using a gamma counter. Cytotoxicity was calculated as the percentage of releasable counts after subtraction of the spontaneous release.
Enzyme-Linked Immunosorbent Assay for IL-12 Using Liver and Spleen Homogenates. Liver and spleen homogenates from 10 mice from each group were collected to measure the IL-12 level using the method of Borovikova et al. (2000) with some modifications. In brief, the spleen was rapidly excised, rinsed of blood, and homogenized by Polytron (Brinkmann Instruments, Westbury, NY) instrumentally in an amount of a homogenization buffer (PBS containing 0.5% Triton-X and a protease inhibitor; pH 7.2; 4°C) that was equal to the liver or spleen weight. Homogenates were centrifuged at 12,000g for 10 min, and supernatants were collected.
To measure the concentration of IL-12 in the serum and homogenates, the enzyme-linked immunosorbent assay kit (BioSource International, Camarillo, CA) was used. The optical density was examined at 450 nm by a microplate reader. The concentration of IL-12 had been calibrated from a dose-response curve based on reference standards. We multiplied the values obtained for samples from the homogenate by 2 to correct for the 1:2 dilutions.
In Vivo Cell Depletion. To investigate the principal cells that have high NK cytotoxicity, starting 2 days before the tumor inoculation, other tumor-implanted mice received an intraperitoneal injection of 50 µg/mouse anti-asialoGM1 (ASGM1) Ab (Wako Pure Chemicals, Tokyo, Japan) or 200 µg/mouse anti-NK1.1 Ab (BD PharMingen, San Diego, CA) every 6 days (Seki et al., 1997; Kawamura et al., 1999). Each antibody was administered by 60 mice. Twenty mice from each group were injected with 5-FU on day 14 by one of three methods: intrasplenic, intraportal, or intravenous injection. These mice were sacrificed on day 18. The liver weight and the number of metastatic foci were measured using half of these mice, and the NK cytotoxicity of hMNCs was calculated using remaining half.
rmIL-12 Injection as a Substitute for 5-FU. To investigate the effect of the IL-12 to the hepatic metastases, recombinant murine IL-12 (rmIL-12) (R&D Systems, Minneapolis, MN) was injected into other 90 tumor-implanted mice in a volume of 0.2 ml on day 14. Three methods were used: directly into the spleen, via the portal vein, and via the common iliac vein. Each group consisted of 30 mice. The liver weight and the number of metastatic foci were measured using 10 mice from each group, the NK cytotoxicity of hMNCs was calculated using another 10 mice, and the concentration of IL-12 in the spleen and the liver was measured using the remaining 10 mice.
Statistical Analyses. To determine the significance of the difference between the experimental groups concerning total cell number (TCN), organ weight, the number of metastatic foci, and cytotoxicity, analysis of variance and post hoc test (Bonferroni/Dunn test) were used. Survival rates were calculated by the method of Kaplan-Meier. p values of <0.05 were judged to be statistically significant.
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Results |
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To investigate the condition of cell proliferation in the spleen and the liver, the number of splenic cells and hMNCs was counted. The total number of splenic cells in the group with an intrasplenic injection of 5-FU was significantly more abundant in the other groups that received 5-FU (p < 0.001) (Table 2). But there was not a significant difference between the group with an intrasplenic injection of 5-FU and normal saline. Although the number of cells per spleen weight was significantly smaller in the group that received intravenous injections of 5-FU compared with the other groups (p < 0.001), that in the group injected with 5-FU into the spleen was not significantly larger than that in the group with an intraportal injection of 5-FU or with an intrasplenic injection of normal saline. With respect to the total number of hMNCs, there was no significant difference among all groups. To expresses the relative increase or the capacity of hMNCs that attack metastatic foci, the number of hMNCs per liver weight was calculated. The number in the group injected with 5-FU into the spleen was significantly higher than that in the other groups (p < 0.01) (Table 3). These results suggest that the intrasplenic injection of 5-FU with a single 25-mg/kg dose plays a critical role in the involution of liver metastases and prolongs the survival period through the augmentation of hMNCs in comparison with the other groups. They also suggest that the injection to the spleen restrains the reduction of splenic cells as the systemic administration of 5-FU.
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Cytotoxic Activities of hMNCs and Splenic Cells. We examined the cytotoxicity of splenic cells and hMNCs isolated from mice in all groups using Yac-1 lymphoma cells and colon 26 as targets. There was no significant difference in the CTL and NK cytotoxicity of splenic cells among the groups (data not shown). The same result was obtained with regard to the CTL cytotoxicity of hMNCs in each group (data not shown). On the other hand, the NK cytotoxicity of hMNCs in the group injected with 5-FU into the spleen was significantly increased in comparison with that in the other groups in any point of the T:E ratio (p < 0.001) (Fig. 2). In particular, the difference was nearly twice as high as that in the group with an intrasplenic injection of normal saline. These results suggest that the injection of 5-FU into the spleen augments the NK cytotoxicity of hMNCs but not that of splenic cells. Furthermore, they demonstrate that the intrasplenic injection of 5-FU does not augment the CTL cytotoxicity of both spleen and liver.
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Up-Regulation of IL-12 by Treatment with 5-FU into the Spleen. Because IL-12 reportedly acts as a growth factor for activated NK cells, we examined the IL-12 level in spleen and liver homogenates of mice after 5-FU administration. As shown in Fig. 3, the IL-12 level in spleen or liver in the group injected 5-FU into the spleen was significantly higher than the levels in the other groups (p < 0.001). Particularly, the IL-12 level in the liver was nearly twice as high in the group with intrasplenic injection with 5-FU as it was in the other groups. These facts support the findings that the splenic injection of 5-FU stimulates the splenic immune system to produce IL-12 and up-regulate the IL-12 level in the liver.
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Involvement of NK Cells and NKT Cells. To investigate the principal cells that have high NK cytotoxicity, we used two antibodies, which depleted NK cells or both NK and NKT cells. Anti-ASGM1 Ab depletes only NK cells, and anti-NK1.1 Ab depletes NK and NKT cells. The liver weight and number of metastatic foci of the liver in the group pretreated with anti-ASGM1 Ab and injected with 5-FU into the spleen were significantly less than those in the other groups (p < 0.01) (Table 4). The NK cytotoxicity of hMNCs in the group injected with 5-FU into the spleen with ASGM1 Ab was significantly higher than that in other groups with Ab in any point of the T:E ratio (p < 0.001). The results described above suggest that, even in the context that NK cells are depleted and only NKT cells exist, an antitumor effect persists.
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Antitumor Effect of rmIL-12 after rmIL-12 Administration. The spleen and liver levels of IL-12 in the group injected with rmIL-12 into the spleen were significantly higher (p < 0.001) than those in the other groups (Fig. 4, A and B). The weight of the liver and the number of metastatic foci of liver in the group with an intrasplenic injection of rmIL-12 decreased significantly more than those in the other groups (p < 0.01) (Table 5). There was no difference between the group with an intraportal and intravenous injection of rmIL-12. Moreover, the NK cytotoxicity of hMNCs in the group injected with rmIL-12 into the spleen was significantly higher than that in other groups in any point of the T:E ratio (p < 0.01) (Fig. 5). The results described above suggest that rmIL-12 injected into the spleen activates the hMNCs and, secondarily, reduces the volume of metastases.
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On the other hand, the IL-12 level in the liver (Fig. 4B) and the NK cytotoxicity of hMNCs (Fig. 5) in the group injected with rmIL-12 via the portal vein were higher than those in the groups that received an intravenous injection of rmIL-12 and an intrasplenic injection of normal saline (p < 0.05). These facts show that, in spite of the augmentation of the NK cytotoxicity by injection of IL-12 through the portal vein, the liver weight and the number of metastases did not diminish as they did when IL-12 was administered into the spleen.
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Discussion |
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The spleen is a large lymphoid organ that produces various kinds of cytokines (Hood et al., 1978), which are known to stream into the liver via the splenic and portal veins and to enhance NK cytotoxicity in the liver (Shiratori et al., 1995). NK cells have been known to mediate spontaneous cytotoxicity against tumor cells and their metastases (Gorelik et al., 1982). It has also been reported that hepatic NK cells act as the first line of defense in the hepatic metastasis of colon cancer (Shiratori et al., 1992; Bouwens et al., 1998).
The splenic cells from mice given 5-FU intraperitoneally exhibited higher NK activities (Okamoto et al., 1998). The present study was carried out to determine whether the immune system in the spleen is up-regulated and whether it has an anticancer effect when 5-FU is administered into the spleen. As a result, in the mice that received 5-FU into the spleen, the weight of the liver, and the number of metastases were diminished. Furthermore, hMNCs increased, and their NK cytotoxicity was significantly augmented. These results demonstrate that the splenic injection of 5-FU plays a critical role in the involution of liver metastases. Furthermore, the survival in mice with an intrasplenic injection of 5-FU was significantly longer than that in mice injected with normal saline, and at least, no less than that in mice with an intraportal or intravenous injection of 5-FU. These facts reveal the possibility that an intrasplenic injection of 5-FU can prolong the survival period in combination with other treatments if it reduces the number and the volume of metastases.
Although 5-FU-based chemotherapy is widely considered to be associated with the suppression of immune function (at least, transiently) (Mitchell and DeConti, 1970; Berenbaum, 1979), other reports have described the increase in the immune function to result from the inhibition of suppressor T cell cytotoxicity associated with this drug, inversely (Blomgren et al., 1965; Goto et al., 1981, Okamoto et al., 1998). Thus, the effect of 5-FU on the immune system is controversial. In this study, the total number of cells per spleen weight was significantly diminished only in mice that were injected with 5-FU via the common iliac vein. And that in mice injected with 5-FU into the spleen is not diminished in comparison with the control. These results suggest that splenic cells are involved in the effect on the involution of liver metastases.
Until a certain size, a metastatic tumor in the liver grows by securing nutrients through the hepatic artery (Izumi et al., 1986). Archer and Gray (1990) reported that branches of the portal vein at the tumor periphery become invaded and compressed during the growth of metastases, which further diminishes drug delivery to the tumor site. In addition, 5-FU is an agent with a short half-life in serum of several tens of minutes. Therefore, it is conceivable that there is a new effect other than the original anticancer effect with this method. These findings suggest that the splenic administration of 5-FU reduces the metastatic lesion by augmenting the NK cytotoxicity of hMNCs rather than by the original effect of 5-FU, which is the so-called inhibition of DNA synthesis.
5-FU is a potent inducer of several T helper 1-type cytokines, such as interferon-, tumor necrosis factor- and -, and IL-12, and of effector cells carrying anticancer cytotoxicity mediated by NK cells as well as T cells; in addition, these abilities are closely associated with the in vivo anticancer effect of this agent (Okamoto et al., 1998). Especially, IL-12 was originally described as an NK stimulatory factor (Kobayashi et al., 1989) or a cytotoxic lymphocyte maturation factor (Trinchieri, 1994) produced principally by antigen-presenting cells. In this study, a 5-FU injection into the spleen was found to augment the level of IL-12 in the spleen or liver more than in the other groups. Furthermore, the fact that the increase of IL-12 is not followed by the augmentation of splenic cytotoxicity substantiates the view that most of the IL-12 produced in the spleen flows into the liver via the splenic vein and induces an increase in the number of hMNCs and augmentation of the cytotoxicity of hMNCs. Eberl and MacDonald (2000) reported that the proportion of NK and NKT cells made up about 8% of the total splenic cells and about 35% of the hMNCs. Their findings tend to substantiate this view. Actually, the cytotoxicity of hMNCs and the IL-12 level in the liver of mice that received injections of 5-FU into the spleen were significantly higher than those in mice that received 5-FU via the other administration routes.
Recent studies on IL-12 show that it acts as a growth factor for activated NK cells and NKT cells (Perussia et al., 1992; Smyth et al., 2000). Watanabe et al. (1995) reported that NKT cells, which proliferate in the liver after the administration of IL-12, are cytotoxic effector cells and comprise the main antimetastatic lymphocyte population in the liver. The systemic administration of IL-12 markedly activates hepatic NKT cells and induces the cytotoxic activities of hMNC against targets (Brunda et al., 1993; Hashimoto et al., 1995; Smyth et al., 2000). Wiltrout et al. (1984) reported that metastatic lesions became enlarged in mice that were treated by anti-ASGM1 Ab. Seki and colleagues reported that in vivo anti-ASGM1 Ab treatment only depletes NK cells in mice, whereas anti-NK1.1 Ab treatment depletes NK and NKT cells (Seki et al., 1997; Kawamura et al., 1999). This study revealed that antitumor effects persist and NKT cells play a major role in preventing metastases, even when NK cells are depleted and only NKT cells exist.
Systemic administration of IL-12 protein significantly suppressed the growth of a variety of established mouse tumors and prolonged the survival of tumor-implanted mice (Brunda et al., 1993; Zou et al., 1995). The number of hMNCs in mice that received rmIL-12 increased more significantly than that in mice that did not receive it (Hashimoto et al., 1995). In this study, to determine whether IL-12 influences the activation of hepatic NKT cells, rmIL-12 was injected into the spleen in tumor-implanted mice. The results revealed that the rmIL-12 injected into the spleen activated the hepatic NKT cells and reduced the volume of metastases. However, in spite of the augmentation of the NK cytotoxicity by rmIL-12 flowing through the portal vein, the effect did not directly aid in tumor involution. These data imply that some other factor, which is produced in the spleen by IL-12, plays a supplementary role.
We believe that the use of the spleen as a potential reservoir for the activation of cell-mediated immunity in vivo should be taken into account when considering the development of clinical anticancer immunotherapeutic strategies.
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Footnotes |
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ABBREVIATIONS: 5-FU, 5-fluorouracil; NK, natural killer; IL-12, interleukin-12; hMNC, hepatic mononuclear cell; CTL, cytotoxic T lymphocyte; PBS, phosphate-buffered saline; Ab, antibody; T:E ratio, target-to-effector ratio; ASGM1, asialoGM1; TCN, total cell number; rmIL-12, recombinant murine interleukin-12.
Address correspondence to: Dr. Kenichi Kanoh, Department of General Surgical Science, Gunma University, Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi 371-8511, Japan. E-mail: kenkanoh{at}showa.gunma-u.ac.jp
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) represent the cytotoxicity in the group under intrasplenic injection of 5-FU; closed squares (
), that under intraportal injection of 5-FU; closed circles (
), that under intravenous injection of 5-FU; and crosses (x), that under intrasplenic injection of normal saline. The NK cytotoxicity of hMNCs in the group injected with 5-FU into the spleen was markedly increased in comparison with that in the other group injected with 5-FU in any point of the T:E ratio (p < 0.01). Moreover, that in the group injected 5-FU via the portal vein was significantly higher than that in the group injected with normal saline into the spleen in any point of the T:E ratio (p < 0.001). Error bars represent S.D. Statistical differences are shown as ***, p < 0.001; **, p < 0.01.
, intrasplenic injection of rmIL-12;