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Mitochondrial membrane potential is reduced in peripheral natural killer cells following partial hepatectomy
Immunology Letters 82 (2002) 225 /233 www.elsevier.com/locate/
Mitochondrial membrane potential is reduced in peripheral natural killer cells following partial hepatectomy Shingo Iwata *, Yoshiro Taki, Yasuhiro Kawai, Michiyuki Kanai, Arimichi Takabayashi Department of Surgery, Tazuke-Kufukai Medical Research Institute, Kitano Hospital, 2-4-20 Ohgimachi, Kita-ku, Osaka 530-8480, Japan Received 9 January 2002; received in revised form 24 January 2002; accepted 27 February 2002
Abstract The mechanism underlying immunosuppression after partial hepatectomy remains unclear. Hepatectomy induces lymphopenia, which is related to immunomodulation. The aim of this study was to determine whether peripheral blood lymphocytes (PBL) are susceptible to mitochondria-mediated apoptosis after hepatic resection. We compared the changes in mitochondrial membrane potential in lymphocytes from hepatectomized patients with metastatic liver tumor with the corresponding changes in lymphocytes from cholechystectomized patients, because changes in mitochondrial membrane potential have been reported to frequently occur during the early stages of apoptosis. Mitochondrial membrane potential, subpopulation, and apoptosis of lymphocytes were estimated with flow cytometry. Hepatectomy significantly (P B/0.001) reduced postoperative mitochondrial membrane potential, while cholecystectomy slightly decreased it. Apoptosis of lymphocytes was increased on post-hepatectomy day, and this increase was correlated with the extent of mitochondrial membrane potential reduction. The major subset of lymphocytes with low mitochondrial membrane potential consisted of CD56 natural killer (NK) cells, and NK cell activity and cell counts significantly decreased after hepatectomy. Mitochondrial membrane potential of PBL was reduced after hepatectomy, and some lymphocytes underwent apoptosis through the mitochondrial pathway, which was one of the causes for lymphopenia. NK cells were more responsible for the decrease of mitochondrial membrane potential after hepatectomy than other lymphocytes, and the reduction in mitochondrial membrane potential in NK cells appeared to reflect modulation of the innate immune system. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Apoptosis; Surgical stress; Innate immunity; Lymphocyte; Mitochondrial membrane potential; Hepatectomy; Natural killer cell
1. Introduction Partial hepatectomy may lead to changes in local or systemic immunological responses, since the liver functions as a hepatolymphoid organ closely associated with the systemic immune network [1,2]. Deterioration of the immune system after hepatectomy is thought to contribute to increased morbidity and mortality from infections and tumor growth and dissemination in cancer patients [3 /6]. Potential effects of the immune system on growth and regeneration in the liver after hepatic resection have been reported [7], and it has also
* Corressponding author. Present address: Department of Surgery, Kyoto City Hospital, 1-2 Higashitakada Mibu, Nakagyo-ku, Kyoto 602-8845, Japan. Tel.: 81-75-311-5311; fax: 81-75-321-6025. E-mail address: [email protected] (S. Iwata).
been demonstrated that surgical trauma reduces the total circulating lymphocyte count and that alterations in this count reflect immunosuppression [8]. Reduction in lymphocyte count may indicate that circulating cells are being mobilized from the blood to the tissues important for postoperative antigen processing, such as the reticuloendothelial system and the operative wound. Recent findings indicate that circulating lymphocytes in the early postoperative period are susceptible to Fas-mediated apoptosis, which may cause depletion of circulating lymphocytes after surgery [9,10]. However, it is difficult to detect end-stage apoptosis in peripheral lymphocytes freshly isolated from the circulation because of phagocytic removal of cells undergoing apoptosis in vivo. Mitochondria appear to play an important role in the early events of apoptosis [11 /13]. Several biochemical
0165-2478/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 7 8 ( 0 2 ) 0 0 0 5 1 - 2
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events, including reduced mitochondrial membrane potential (DC m), hyperproduction of reactive oxygen species, calcium ion influx, caspase activation, and apoptosis-inducing factor and cytochrome c release from mitochondria, have been proposed to be necessary for commitment of a cell to apoptosis [14]. These biochemical changes can result from alterations in the function of mitochondria [15]. Many studies have demonstrated that mitochondrial membrane depolarization frequently occurs during the early stages of apoptosis and is often an early warning sign of apoptosis[14,16 /19]. Measurement of DC m may thus provide a useful index of mitochondrial disruption and indicate whether a cell is involved in apoptosis. We therefore determined the changes in DC m in peripheral blood lymphocytes (PBL) from hepatectomized patients with metastatic liver tumor and compared our findings with those for patients undergoing laparoscopic cholecystectomy. We found that the DC m of PBL was reduced after hepatic resection and that the DC m of natural killer (NK) cells was affected more than that of other PBL associated with the functional and numerical changes in NK cells.
2. Patients and methods 2.1. Patients and isolation of lymphocytes This study involved ten patients who underwent hepatic resection for liver metastasis of colon cancer, seven patients who underwent laparoscopic cholecystectomy for cholecystolithiasis at Kitano Hospital between June 1999 and October 2000, and 20 normal volunteers as a control. Patient characteristics are listed in Table 1. The 20 normal volunteers included 12 men and eight women from 23 to 56 years of age. None of the subjects had any viral or bacterial infection, or had received immunosuppressive agents such as corticosteroids, antibiotics or anticancer agents during the 1-month period preceding the study. Informed consent was obtained from all of the patients and volunteers according to the Helsinki Declaration and the Ethics Table 1 Patient charasteristics and surgical data
Age (years) Sex ratio (M:F) Duration of operation (h) Blood loss (ml)
Partial hepatectomy (n 10)
Laparoscopic chelecystectomy (n 7)
61.992.5 6:4 6.390.7***
53.995.0 3:4 2.090.3
10529666*
1092
Values are expressed as means9SE. *P B 0.05, ***P B 0.001 compared with the value for the laparoscopic chelecystectomy group.
Committee of our hospital approved this study. Heparinized blood samples were obtained preoperatively at 09:00 h on the day of operation as well as on postoperative days (POD) 1, 3 and 7 from the patients undergoing partial hepatectomy, and on POD 1 and 3 from the patients undergoing laparoscopic cholecytectomy. Lymphocytes were prepared with Ficoll Hypaque (Pharmacia, Uppsala, Sweden) as described by Bo¨yun [20]. 2.2. Flowcytometric analysis of DC m Flow cytometry was performed with a FACSCalibur (Becton Dickinson, San Jose, CA). To measure DC m cells were incubated with 1 nM of 3,3-dihexyloxacarbocyanine iodide (DiOC6(3); Molecular Probes, Inc., Eugene, OR) for 20 min at 37 8C [14]. Some of the cells were then incubated for 30 min at 37 8C with 40 mM carbonyl cyanide m -chlorophenyl hydrazone (CCCP; Sigma Chemical Co., St Louis, MO), an uncoupling agent that abolishes DC m. DiOC6(3)high cells were defined as those with fluorescence higher than that of cells treated with CCCP (Fig. 1A). For the simultaneous assessment of DC m and surface markers, cells were stained with DiOC6(3) and then with phycoerythrin-labeled anti-human CD3, CD20 or CD56 antibodies (Becton Dickinson) for 30 min on ice. Apoptosis was measured by flow cytometry after staining with fluorescein-conjugated annexin V (AV-FITC; R&D Systems, Minneapolis, MN) and propidium iodide (PI; R&D Systems) as previously described [21,22]. We used dual staining with PI to distinguish membrane-disrupted cells, since cells that have lost their membrane integrity can show positive AV staining. Briefly, PBL in a 24-h culture were dual-stained with AV and PI diluted in binding buffer for 15 min at room temperature. Isolated lymphocytes were cultured in RPMI with 10% FCS at 37 8C in a 5% CO2 humidified incubator. All culture reagents were purchased from GIBCO, Grand Island, NY. For one determination, 10 000 cells per sample were counted. 2.3. Measurement of natural killer cell activity NK cell activity was determined by measuring radioactivity released from chromium 51-labeled K562 target cells as previously described [23]. Briefly, target cells and effector cells were plated in triplicate in 96-well microtiter plates. The effector-to-target ratios studied ranged from 5:1 to 40:1. The plates were incubated for 4 h at 37 8C in a 5% CO2 humidified atmosphere. Released radioactivity was measured with a gamma scintillation counter. The percentage of NK cell cytotoxicity was calculated as (experimental cpm /spontaneous cpm)/ (total cpm /spontaneous cpm) /100. Lytic units were calculated with a non-linear least-square fit equation
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Fig. 1. Serial changes in DCm of PBL before and after surgery. (A) Definition of DiOC6(3) high cell. DiOC6(3) high cells were defined as cells with a fluorescence higher than that of cells treated with carbonyl cyanide m-chlorophenyl hydrazone (CCCP). (B) Representative flow cytometry profiles using DiOC6(3) staining. Numbers in the figure represent the percentages of DiOC6(3) high cells. (C) Changes in DCm of PBL before and after surgery. Data are expressed as mean9SE. Pre, preoperative day; POD, postoperative day; Hx, hepatectomy; Ch, cholecystectomy; DiOC6(3), 3,3dihexyloxacarbocyanine iodide. **P B 0.01 compared with the preoperative value. ***P B 0.001 compared with the preoperative value.
described by Pross et al. [23]. One lytic unit was defined as the number of effector cells needed to lyse 20% of 104 target cells, and the number of units per 106 effector cells was calculated.
3. Results
3.1. Patient background and serial changes in postoperative biochemical data and catecholamine and cortisol levels
2.4. Statistical analysis All data were expressed as the mean9/SE, and ANOVA, Student’s t -test and thex 2-test were used for statistical analysis. Non-parametric data were analyzed with the Mann/Whitney rank-sum test. Probability values less than 0.05 were considered significant. Correlations were calculated by regression analysis using the least-squares method.
There was no significant difference between the two groups in either age or gender. However, operating time and operative blood loss were significantly longer and larger for the hepatectomy (Hx) than for the cholecystectomy (Ch) group, as shown in Table 1. There was no correlation between blood loss and DCm reduction on POD 1 (r /0.08, P /0.827, n /10). The changes in DC m did not appear to be related to blood loss. Three of
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the ten hepatectomized patients were transfused with 1200, 2000, and 4200 ml of red blood cells. There was no correlation between blood transfusion and DC m reduction on POD 1 (r /0.017, P /0.9633, n /10). Blood transfusion did not appear to affect DC m reduction. As expected, postoperative values of biochemical tests were significantly higher in the Hx group than in the Ch group (Table 2). Postoperative changes in plasma adrenaline and cortisol levels were not remarkable and within the normal range for both groups (Table 3). Plasma noradrenaline levels significantly increased on POD 1 and 3 to above normal and returned to within the normal range on POD 7 in the Hx group (Table 3). In the Ch group, plasma noradrenaline levels tended to increase postoperatively but were within normal range. None of the patients died or suffered from postoperative complications.
3.3. Hepatectomy induced lymphocyte apoptosis
3.2. Serial changes in DC m of PBL before and after operation
3.4. Prominent reduction in DC m in natural killer cells
The first study was carried out to assess the preoperative DC m of PBL in the two groups and to compare it with DC m of PBL in normal volunteers. There was no significant difference among the patients and normal volunteers (86.19/3.8% for the Hx group, 90.19/2.1% for the Ch group, and 89.29/2.0% for normal volunteers). The aim of the next study was to assess postoperative changes in the DC m of PBL. Fig. 1B shows a representative example of flow cytometric analysis of PBL stained with DiOC6(3) from a hepatectomized and a cholecystectomized patient. In the Hx group, the percentage of DiOC6(3)high cells was reduced on POD 1 compared with the preoperative value (from 94 to 74%), increased to 86% on POD 3 and had become normal by POD 7. As shown in Fig. 1C, DiOC6(3)high cells were reduced significantly to 61.09/4.6% on POD 1 and returned to 71.59/3.3% on POD 3 in the Hx group. On the other hand, DiOC6(3)high cells decreased slightly to 88.49/2.5% of the preoperative value on POD 1 and to 87.99/3.7% on POD 3 in the Ch group. Thus the reduction in DC m was far smaller in the Ch than in the Hx group.
Flow cytometry analysis using AV-FITC and PI dual staining showed that spontaneous apoptosis of PBL isolated from patients on POD 1 after Hx was enhanced after 24-h incubation compared with that of PBL isolated from the same patients before surgery and patients in the Ch group (Fig. 2A). The percentage of cells that underwent spontaneous apoptosis after 24-h in vitro culture was positively correlated with the percentage of low DC m cells determined ex vivo (r /0.848, P B/0.0001) (Fig. 2B). The frequency of DiOC6(3)low cells was assessed for PBL isolated from patients on POD 1 in both groups (n /17 points). In addition, cells were cultured for 24 h at 37 8C without any external stimulation, followed by determination of apoptosis using the AV staining method.
After determining that the DC m of PBL was reduced after Hx, we investigated which lymphocyte subpopulation was mainly responsible for this reduction by using two-color staining with DiOC6(3) and lymphocyte surface markers (CD3, CD56 and CD20). Fig. 3A and B shows representative data from patients who underwent Hx or Ch. Before operation, the percentage (97%) of DiOC6(3)high cells to total CD56 cells, the equivalent of DC m of NK cells, was higher in the hepatectomized patient than in the cholecystectomized patient (87%). The preoperative DC m of NK cells in the Ch group was as almost the same as that for normal volunteers. Preoperative DC m of NK cells from the patients with metastatic liver tumor was higher than that of NK cells from the patients with gall-stone disease and normal volunteers. On POD 1 after Hx, two-color staining of PBL with DiOC6(3) and CD3 or CD56 clearly demonstrated that 42% of the CD56 cells exhibited low levels of DiOC6(3) while a high percentage (93%) of CD3 cells were DiOC6(3)high cells. The DC m in CD20 B cells indicated high levels of DiOC6(3) during the perioperative period (data not shown). As summarized in Fig. 3C, DC m of NK cells significantly decreased on POD 1 and
Table 2 Biochemical data and white blood cell counts in before and after operation in groups of partial hepatectomy and laparoscopic chelecystectomy Partial hepatectomy (n 10)
T-Bil (mg/dl) AST (IU/l) ALT (IU/l) LDH (IU/l) CRP (mg/dl)
Laparoscopic cholecystectomy (n 7)
Before
POD 1
POD 3
POD 7
Before
POD 1
0.790.1 3096 3699 343916 0.390.0
1.990.3*** 266951*** 330998*** 773967*** 3.090.5
1.590.2 89917 203958* 460932* 8.492.1***
0.990.1 41910 79922 383917 2.390.6
0.890.1 1993 2094 ND 0.390.0
1.290.2 4397 4295 ND 1.690.4
Note. Normal range: T-Bil B 1.0 mg/dl; ASTB 40 IU/l; LDHB 460 IU/l; CRPB 0.5mg/dl; ND, not detected. Values are expressed as means9SE.*P B 0.05, **P B 0.01, ***P B 0.001 compared with the preoperative value.
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Table 3 Plasma cortisol, adrenaline and noradrenaline levels in before and after operation in groups of partial hepatectomy and laparoscopic chelecystectomy Partial hepatectomy (n 10)
Cortisol (ng/ml) Adrenaline (pg/ml) Noradrenaline (pg/ml)
Laparoscopic cholecystectomy (n 7)
Before
POD 1
POD 3
POD 7
Before
POD 1
POD 3
118910 4592 286940
119912 50914 743998***
9697 47914 464978*
10397 50910 383968
110910 2797 258915
119912 3899 289917
876910 3496 323956
Plasma adrenaline (normal rangeB 100 pg/ml) and noradrenaline (normal range: 100 /450 pg/ml) levels were assessed by high-performance liquid chromatography in conjunction with electrochemical detection. Plasma cortisol levels (normal range: 45 /211 ng/ml) were determined with a chemiluminoscent immunoassay. The sensitivity of the cortisol assay was 2 ng/ml and that of the catecholamines assay 5 pg/ml. Values are expressed as means9SE. *P B 0.05, ***P B 0.001 compared with the preoperative value.
3 after Hx, DC m of NK cells from the Ch group tended to be decreased on POD 1, while DCm of T3 lymphocytes from the Hx group exhibited the same tendency in the postoperative period.
postoperative patients in both groups (n /85 points). A significant correlation was observed between NK cell activity and the percentage of NK cells with high DC m to total PBL (r /0.763, P B/0.0001), as shown in Fig. 4B.
3.5. NK cell activity was correlated with the fraction of CD56-positive cells with high DC m
3.6. Postoperative changes in absolute lymphocyte counts
Fig. 4A shows the changes in NK cell activity before and after operation. The preoperative values for the Hx group (24.09/6.7) tended to be higher than those for the Ch group (13.69/3.6) and normal volunteers (14.39/3.0). NK cell activity was significantly (P /0.0301) reduced in the Hx group (10.99/2.1) on POD 1 compared with the preoperative value. In the Ch group, a tendency toward decrease from the preoperative value was observed on POD 1 (12.39/4.6). We examined the relationship between NK cell activity and the ratio of DiOC6(3)high and CD56 cells to total lymphocytes for
The changes in total and T3 lymphocytes and NK cells in peripheral blood before and after surgery are shown in Fig. 5. In the Hx group, the number of total lymphocytes was significantly decreased on POD 1 (11749/169/ml) and POD 3 (10829/136/ml) when compared with the preoperative value (16869/165/ml), but had increased to 14699/137/ml on POD 7. The number of T3 lymphocytes in the Hx group exhibited no significant change during either the preoperative or the perioperative period. NK cells were significantly reduced in number on POD 1 (1959/41/ml) and POD 3
Fig. 2. Spontaneous lymphocyte apoptosis in patients undergoing hepatectomy or cholecystectomy. (A) Representative flow cytometry profiles obtained with 24-h culture of annexine V-FITC and propidium iodide staining. (B) The percentage of cells spontaneously undergoing apoptosis after 24-h in vitro culture correlated positively with the percentage of low DCm cells determined ex vivo. Hx, hepatectomy; Ch, cholecystectomy.
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Fig. 3. Serial changes in DCm in subpopulation of lymphocytes. (A) Representative flow cytometry profiles produced by double color staining with DiOC6(3) and CD3. (B) Representative flow cytometry profiles obtained with double color staining with DiOC6(3) and CD56. (C) Variation in proportions of cells which have CD3 positive or CD56 positive and high DiOC6(3) staining, that is, perioperative changes in DCm of T3 lymphocytes and DC m of NK cells. Data are expressed as mean9SE. Pre, preoperative day; POD, postoperative day; Hx, hepatectomy; Ch, cholecystectomy. *P B 0.05 compared with the preoperative value for the Ch group. **P B 0.01 compared with the preoperative value and the value for the Ch group on POD 3. ***P B 0.001 compared with the preoperative value and the value for the Ch group on POD 1.
(2249/32/ml) compared with the preoperative value (4889/70/ml).
4. Discussion This study demonstrated that partial hepatectomy reduced postoperative DC m of lymphocytes from pa-
tients with metastatic liver tumors and that some PBL could undergo apoptosis through the mitochondrial pathway. In addition, DC m of NK cells was more strongly affected by hepatectomy than that of other PBL and the magnitude of decrease in DC m in NK cells was closely associated with reduction in their function. Recent reports on clinical applications of DCm have suggested that changes in DC m of cells reflect their
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Fig. 4. (A) Serial changes in NK cell activity (lytic unit 20%) before and after surgery. Data are expressed as mean9SE. (B) Significant correlation between NK cell activity and the ratio of high DCm in NK cells (CD56 positive cells) to total PBL was observed (r 0.763, P B 0.0001). Pre, preoperative day; POD, postoperative day; Hx, hepatectomy; Ch, cholecystectomy.*P B 0.05 compared with the preoperative value.
function [17,19]. Thus, measurement of DC m could provide useful information not only on cell death but also cellular biological activity. To clarify whether the reduction in DC m was a consequence of hepatectomy or major surgery unrelated to the fact that the selected procedure was hepatectomy, we measured DC m of lymphocytes in patients after gastrectomy, colectomy, or pancreatico-duodenectomy. However, these procedures did not significantly affect DC m of lymphocytes like cholecystectomy (data not shown). Therefore, it is suggested that our results be linked to the hepatectomy but not to the major surgical trauma which is much more in patients with gastrectomy, colectomy, or pancreaticoduodenectomy than in cholecystectomized patients. Hepatectomy impaired NK cell cytotoxicity and this impairment was related to the reduction in NK cell count associated with decrease in NK cell activity. The mechanism underlying the reduction in circulating NK cells may be related to cell death or sequestration. Plasma catecholamine concentration, which appears to play an important role in liver regeneration [24],
increased after hepatic resection, similar to findings reported by other authors [25]. Catecholamines induced a rapid redistribution of NK cells from reservoirs such as the spleen and marginal pool where NK cells might differ in their DC m [26,27]. Since the liver also functions as a reservoir of NK cells, Hx may reduce this function and contribute to lymphopenia. In animal experiments, extrathymic T cells or NKT cells expanded in the liver in the early phase of hepatocyte regeneration after partial hepatectomy, while the change in the proportion of NK cells was minimal in the liver of mice subjected to partial hepatectomy [28]. Decrease in circulating NK cells may thus result mainly from cell death rather than from sequestration to the liver in this clinical setting. Shakhar et al. reported that catecholamine suppressed NK cell activity and compromised resistance to tumor metastasis [29]. Noradrenaline induces apoptosis of cardiac myocytes [30] and lymphocytes [31], and is thus one of the many candidates for molecules that commit NK cells to undergo apoptosis. Noradrenaline increases c-AMP and calcium influx via b-adrenergic receptor stimulation, and is thought to increase calcium levels in mitochon-
Fig. 5. Serial changes in total, T3 lymphocytes, and NK cells in peripheral blood before and after surgery. Data are expressed as mean9SE. Pre, preoperative day; POD, postoperative day, *P B 0.05 compared with the preoperative value. **P B 0.01 compared with the preoperative value. ***P B 0.001 compared with the preoperative value.
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dria as well as induce DC m perturbation [32]. To clarify the causal relationship between NK cells and catecholamines, further in vitro studies must be performed in which the effect of exogenous catecholamine on NK cells is evaluated. The major mechanism of target cell apoptosis by NK cells involves not only exocytosis of cytoplasmic granules from the NK cells towards the target cells, but also engagement of the Fas ligand or tumor necrosis factor on NK cells with the Fas or tumor necrosis factor receptor on the target cell [33]. NK cells also can undergo apoptosis following target cell and NK cell receptor engagement. It was observed that DC m of NK cells in the Hx group before surgery was higher than that for normal control. Elevation of DCm has been shown to have implications for a variety of pathophysiological conditions, particularly apoptosis [34,35]. The NK cells with high DC m we found were thought to be unstable and to be about to change to low DC m or undergo apoptosis after hepatectomy. DC m elevation and DC m reduction may thus take place at two different stages of the apoptotic process. NK cells are central in the innate immune response against parasites, infected cells, and tumors. Transgenic mice with defective natural killing and selective deficiency in NK cells displayed impaired in vivo tumor rejection [36]. This study using NK cell-deficient mice revealed that NK cells play an important role in suppressing tumor metastasis and growth. On the other hand, NK cells are involved in liver regeneration after partial hepatectomy [7,28] and are capable of killing regenerating liver hepatocytes [37], which express Fas constitutively and are susceptible to Fas-mediated apoptosis [38], since infiltration of NK cells into the liver can occur under pathological conditions such as hepatectomy [37]. Our study suggests that the cytotoxicity of NK cells is temporarily suppressed after partial hepatectomy and that they are noncytotoxic for regenerating liver. The reduction in DC m observed after partial hepatectomy is restricted to NK cells and may affect on liver regeneration. The assay currently used for the analysis of NK cell cytotoxicity is based on the measurement of radioactive 51 Cr release by target cells. Although this assay is the most commonly used to determine NK cell activity, it has a number of important drawbacks including the use of radioactive compounds, high cost, and the need for special laboratory equipment [39]. A relationship was observed between NK cell activity and the ratio of high DC m in NK cells to total PBL. Our results indicate that our novel method to measure DC m of CD56-positive cells can serve as a rapid assay useful for analysis of NKmediated cytotoxicity. Measurement of DC m of PBL may thus provide a useful method for examination of the effects of hepatectomy on the immune system.
Our study indicates that DC m of PBL is reduced after hepatectomy and that measurement of DCm can determine whether a cell will undergo apoptosis, which is one of the causes of lymphopenia. Decrease of DC m in NK cells is principally responsible for that of DC m in PBL after partial hepatectomy, and is associated with a reduction in the count and activity of NK cells. Reduction of DC m in NK cells may thus be important for liver regeneration after hepatectomy.
Acknowledgements This research was supported in part by Grants-in-Aid (11470251) for Scientific Research from the Ministry of Education, Science and Culture of Japan.
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Mitochondrial membrane potential is reduced in peripheral natural killer cells following partial hepatectomy Shingo Iwata *, Yoshiro Taki, Yasuhiro Kawai, Michiyuki Kanai, Arimichi Takabayashi Department of Surgery, Tazuke-Kufukai Medical Research Institute, Kitano Hospital, 2-4-20 Ohgimachi, Kita-ku, Osaka 530-8480, Japan Received 9 January 2002; received in revised form 24 January 2002; accepted 27 February 2002
Abstract The mechanism underlying immunosuppression after partial hepatectomy remains unclear. Hepatectomy induces lymphopenia, which is related to immunomodulation. The aim of this study was to determine whether peripheral blood lymphocytes (PBL) are susceptible to mitochondria-mediated apoptosis after hepatic resection. We compared the changes in mitochondrial membrane potential in lymphocytes from hepatectomized patients with metastatic liver tumor with the corresponding changes in lymphocytes from cholechystectomized patients, because changes in mitochondrial membrane potential have been reported to frequently occur during the early stages of apoptosis. Mitochondrial membrane potential, subpopulation, and apoptosis of lymphocytes were estimated with flow cytometry. Hepatectomy significantly (P B/0.001) reduced postoperative mitochondrial membrane potential, while cholecystectomy slightly decreased it. Apoptosis of lymphocytes was increased on post-hepatectomy day, and this increase was correlated with the extent of mitochondrial membrane potential reduction. The major subset of lymphocytes with low mitochondrial membrane potential consisted of CD56 natural killer (NK) cells, and NK cell activity and cell counts significantly decreased after hepatectomy. Mitochondrial membrane potential of PBL was reduced after hepatectomy, and some lymphocytes underwent apoptosis through the mitochondrial pathway, which was one of the causes for lymphopenia. NK cells were more responsible for the decrease of mitochondrial membrane potential after hepatectomy than other lymphocytes, and the reduction in mitochondrial membrane potential in NK cells appeared to reflect modulation of the innate immune system. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Apoptosis; Surgical stress; Innate immunity; Lymphocyte; Mitochondrial membrane potential; Hepatectomy; Natural killer cell
1. Introduction Partial hepatectomy may lead to changes in local or systemic immunological responses, since the liver functions as a hepatolymphoid organ closely associated with the systemic immune network [1,2]. Deterioration of the immune system after hepatectomy is thought to contribute to increased morbidity and mortality from infections and tumor growth and dissemination in cancer patients [3 /6]. Potential effects of the immune system on growth and regeneration in the liver after hepatic resection have been reported [7], and it has also
* Corressponding author. Present address: Department of Surgery, Kyoto City Hospital, 1-2 Higashitakada Mibu, Nakagyo-ku, Kyoto 602-8845, Japan. Tel.: 81-75-311-5311; fax: 81-75-321-6025. E-mail address: [email protected] (S. Iwata).
been demonstrated that surgical trauma reduces the total circulating lymphocyte count and that alterations in this count reflect immunosuppression [8]. Reduction in lymphocyte count may indicate that circulating cells are being mobilized from the blood to the tissues important for postoperative antigen processing, such as the reticuloendothelial system and the operative wound. Recent findings indicate that circulating lymphocytes in the early postoperative period are susceptible to Fas-mediated apoptosis, which may cause depletion of circulating lymphocytes after surgery [9,10]. However, it is difficult to detect end-stage apoptosis in peripheral lymphocytes freshly isolated from the circulation because of phagocytic removal of cells undergoing apoptosis in vivo. Mitochondria appear to play an important role in the early events of apoptosis [11 /13]. Several biochemical
0165-2478/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 7 8 ( 0 2 ) 0 0 0 5 1 - 2
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events, including reduced mitochondrial membrane potential (DC m), hyperproduction of reactive oxygen species, calcium ion influx, caspase activation, and apoptosis-inducing factor and cytochrome c release from mitochondria, have been proposed to be necessary for commitment of a cell to apoptosis [14]. These biochemical changes can result from alterations in the function of mitochondria [15]. Many studies have demonstrated that mitochondrial membrane depolarization frequently occurs during the early stages of apoptosis and is often an early warning sign of apoptosis[14,16 /19]. Measurement of DC m may thus provide a useful index of mitochondrial disruption and indicate whether a cell is involved in apoptosis. We therefore determined the changes in DC m in peripheral blood lymphocytes (PBL) from hepatectomized patients with metastatic liver tumor and compared our findings with those for patients undergoing laparoscopic cholecystectomy. We found that the DC m of PBL was reduced after hepatic resection and that the DC m of natural killer (NK) cells was affected more than that of other PBL associated with the functional and numerical changes in NK cells.
2. Patients and methods 2.1. Patients and isolation of lymphocytes This study involved ten patients who underwent hepatic resection for liver metastasis of colon cancer, seven patients who underwent laparoscopic cholecystectomy for cholecystolithiasis at Kitano Hospital between June 1999 and October 2000, and 20 normal volunteers as a control. Patient characteristics are listed in Table 1. The 20 normal volunteers included 12 men and eight women from 23 to 56 years of age. None of the subjects had any viral or bacterial infection, or had received immunosuppressive agents such as corticosteroids, antibiotics or anticancer agents during the 1-month period preceding the study. Informed consent was obtained from all of the patients and volunteers according to the Helsinki Declaration and the Ethics Table 1 Patient charasteristics and surgical data
Age (years) Sex ratio (M:F) Duration of operation (h) Blood loss (ml)
Partial hepatectomy (n 10)
Laparoscopic chelecystectomy (n 7)
61.992.5 6:4 6.390.7***
53.995.0 3:4 2.090.3
10529666*
1092
Values are expressed as means9SE. *P B 0.05, ***P B 0.001 compared with the value for the laparoscopic chelecystectomy group.
Committee of our hospital approved this study. Heparinized blood samples were obtained preoperatively at 09:00 h on the day of operation as well as on postoperative days (POD) 1, 3 and 7 from the patients undergoing partial hepatectomy, and on POD 1 and 3 from the patients undergoing laparoscopic cholecytectomy. Lymphocytes were prepared with Ficoll Hypaque (Pharmacia, Uppsala, Sweden) as described by Bo¨yun [20]. 2.2. Flowcytometric analysis of DC m Flow cytometry was performed with a FACSCalibur (Becton Dickinson, San Jose, CA). To measure DC m cells were incubated with 1 nM of 3,3-dihexyloxacarbocyanine iodide (DiOC6(3); Molecular Probes, Inc., Eugene, OR) for 20 min at 37 8C [14]. Some of the cells were then incubated for 30 min at 37 8C with 40 mM carbonyl cyanide m -chlorophenyl hydrazone (CCCP; Sigma Chemical Co., St Louis, MO), an uncoupling agent that abolishes DC m. DiOC6(3)high cells were defined as those with fluorescence higher than that of cells treated with CCCP (Fig. 1A). For the simultaneous assessment of DC m and surface markers, cells were stained with DiOC6(3) and then with phycoerythrin-labeled anti-human CD3, CD20 or CD56 antibodies (Becton Dickinson) for 30 min on ice. Apoptosis was measured by flow cytometry after staining with fluorescein-conjugated annexin V (AV-FITC; R&D Systems, Minneapolis, MN) and propidium iodide (PI; R&D Systems) as previously described [21,22]. We used dual staining with PI to distinguish membrane-disrupted cells, since cells that have lost their membrane integrity can show positive AV staining. Briefly, PBL in a 24-h culture were dual-stained with AV and PI diluted in binding buffer for 15 min at room temperature. Isolated lymphocytes were cultured in RPMI with 10% FCS at 37 8C in a 5% CO2 humidified incubator. All culture reagents were purchased from GIBCO, Grand Island, NY. For one determination, 10 000 cells per sample were counted. 2.3. Measurement of natural killer cell activity NK cell activity was determined by measuring radioactivity released from chromium 51-labeled K562 target cells as previously described [23]. Briefly, target cells and effector cells were plated in triplicate in 96-well microtiter plates. The effector-to-target ratios studied ranged from 5:1 to 40:1. The plates were incubated for 4 h at 37 8C in a 5% CO2 humidified atmosphere. Released radioactivity was measured with a gamma scintillation counter. The percentage of NK cell cytotoxicity was calculated as (experimental cpm /spontaneous cpm)/ (total cpm /spontaneous cpm) /100. Lytic units were calculated with a non-linear least-square fit equation
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Fig. 1. Serial changes in DCm of PBL before and after surgery. (A) Definition of DiOC6(3) high cell. DiOC6(3) high cells were defined as cells with a fluorescence higher than that of cells treated with carbonyl cyanide m-chlorophenyl hydrazone (CCCP). (B) Representative flow cytometry profiles using DiOC6(3) staining. Numbers in the figure represent the percentages of DiOC6(3) high cells. (C) Changes in DCm of PBL before and after surgery. Data are expressed as mean9SE. Pre, preoperative day; POD, postoperative day; Hx, hepatectomy; Ch, cholecystectomy; DiOC6(3), 3,3dihexyloxacarbocyanine iodide. **P B 0.01 compared with the preoperative value. ***P B 0.001 compared with the preoperative value.
described by Pross et al. [23]. One lytic unit was defined as the number of effector cells needed to lyse 20% of 104 target cells, and the number of units per 106 effector cells was calculated.
3. Results
3.1. Patient background and serial changes in postoperative biochemical data and catecholamine and cortisol levels
2.4. Statistical analysis All data were expressed as the mean9/SE, and ANOVA, Student’s t -test and thex 2-test were used for statistical analysis. Non-parametric data were analyzed with the Mann/Whitney rank-sum test. Probability values less than 0.05 were considered significant. Correlations were calculated by regression analysis using the least-squares method.
There was no significant difference between the two groups in either age or gender. However, operating time and operative blood loss were significantly longer and larger for the hepatectomy (Hx) than for the cholecystectomy (Ch) group, as shown in Table 1. There was no correlation between blood loss and DCm reduction on POD 1 (r /0.08, P /0.827, n /10). The changes in DC m did not appear to be related to blood loss. Three of
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the ten hepatectomized patients were transfused with 1200, 2000, and 4200 ml of red blood cells. There was no correlation between blood transfusion and DC m reduction on POD 1 (r /0.017, P /0.9633, n /10). Blood transfusion did not appear to affect DC m reduction. As expected, postoperative values of biochemical tests were significantly higher in the Hx group than in the Ch group (Table 2). Postoperative changes in plasma adrenaline and cortisol levels were not remarkable and within the normal range for both groups (Table 3). Plasma noradrenaline levels significantly increased on POD 1 and 3 to above normal and returned to within the normal range on POD 7 in the Hx group (Table 3). In the Ch group, plasma noradrenaline levels tended to increase postoperatively but were within normal range. None of the patients died or suffered from postoperative complications.
3.3. Hepatectomy induced lymphocyte apoptosis
3.2. Serial changes in DC m of PBL before and after operation
3.4. Prominent reduction in DC m in natural killer cells
The first study was carried out to assess the preoperative DC m of PBL in the two groups and to compare it with DC m of PBL in normal volunteers. There was no significant difference among the patients and normal volunteers (86.19/3.8% for the Hx group, 90.19/2.1% for the Ch group, and 89.29/2.0% for normal volunteers). The aim of the next study was to assess postoperative changes in the DC m of PBL. Fig. 1B shows a representative example of flow cytometric analysis of PBL stained with DiOC6(3) from a hepatectomized and a cholecystectomized patient. In the Hx group, the percentage of DiOC6(3)high cells was reduced on POD 1 compared with the preoperative value (from 94 to 74%), increased to 86% on POD 3 and had become normal by POD 7. As shown in Fig. 1C, DiOC6(3)high cells were reduced significantly to 61.09/4.6% on POD 1 and returned to 71.59/3.3% on POD 3 in the Hx group. On the other hand, DiOC6(3)high cells decreased slightly to 88.49/2.5% of the preoperative value on POD 1 and to 87.99/3.7% on POD 3 in the Ch group. Thus the reduction in DC m was far smaller in the Ch than in the Hx group.
Flow cytometry analysis using AV-FITC and PI dual staining showed that spontaneous apoptosis of PBL isolated from patients on POD 1 after Hx was enhanced after 24-h incubation compared with that of PBL isolated from the same patients before surgery and patients in the Ch group (Fig. 2A). The percentage of cells that underwent spontaneous apoptosis after 24-h in vitro culture was positively correlated with the percentage of low DC m cells determined ex vivo (r /0.848, P B/0.0001) (Fig. 2B). The frequency of DiOC6(3)low cells was assessed for PBL isolated from patients on POD 1 in both groups (n /17 points). In addition, cells were cultured for 24 h at 37 8C without any external stimulation, followed by determination of apoptosis using the AV staining method.
After determining that the DC m of PBL was reduced after Hx, we investigated which lymphocyte subpopulation was mainly responsible for this reduction by using two-color staining with DiOC6(3) and lymphocyte surface markers (CD3, CD56 and CD20). Fig. 3A and B shows representative data from patients who underwent Hx or Ch. Before operation, the percentage (97%) of DiOC6(3)high cells to total CD56 cells, the equivalent of DC m of NK cells, was higher in the hepatectomized patient than in the cholecystectomized patient (87%). The preoperative DC m of NK cells in the Ch group was as almost the same as that for normal volunteers. Preoperative DC m of NK cells from the patients with metastatic liver tumor was higher than that of NK cells from the patients with gall-stone disease and normal volunteers. On POD 1 after Hx, two-color staining of PBL with DiOC6(3) and CD3 or CD56 clearly demonstrated that 42% of the CD56 cells exhibited low levels of DiOC6(3) while a high percentage (93%) of CD3 cells were DiOC6(3)high cells. The DC m in CD20 B cells indicated high levels of DiOC6(3) during the perioperative period (data not shown). As summarized in Fig. 3C, DC m of NK cells significantly decreased on POD 1 and
Table 2 Biochemical data and white blood cell counts in before and after operation in groups of partial hepatectomy and laparoscopic chelecystectomy Partial hepatectomy (n 10)
T-Bil (mg/dl) AST (IU/l) ALT (IU/l) LDH (IU/l) CRP (mg/dl)
Laparoscopic cholecystectomy (n 7)
Before
POD 1
POD 3
POD 7
Before
POD 1
0.790.1 3096 3699 343916 0.390.0
1.990.3*** 266951*** 330998*** 773967*** 3.090.5
1.590.2 89917 203958* 460932* 8.492.1***
0.990.1 41910 79922 383917 2.390.6
0.890.1 1993 2094 ND 0.390.0
1.290.2 4397 4295 ND 1.690.4
Note. Normal range: T-Bil B 1.0 mg/dl; ASTB 40 IU/l; LDHB 460 IU/l; CRPB 0.5mg/dl; ND, not detected. Values are expressed as means9SE.*P B 0.05, **P B 0.01, ***P B 0.001 compared with the preoperative value.
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Table 3 Plasma cortisol, adrenaline and noradrenaline levels in before and after operation in groups of partial hepatectomy and laparoscopic chelecystectomy Partial hepatectomy (n 10)
Cortisol (ng/ml) Adrenaline (pg/ml) Noradrenaline (pg/ml)
Laparoscopic cholecystectomy (n 7)
Before
POD 1
POD 3
POD 7
Before
POD 1
POD 3
118910 4592 286940
119912 50914 743998***
9697 47914 464978*
10397 50910 383968
110910 2797 258915
119912 3899 289917
876910 3496 323956
Plasma adrenaline (normal rangeB 100 pg/ml) and noradrenaline (normal range: 100 /450 pg/ml) levels were assessed by high-performance liquid chromatography in conjunction with electrochemical detection. Plasma cortisol levels (normal range: 45 /211 ng/ml) were determined with a chemiluminoscent immunoassay. The sensitivity of the cortisol assay was 2 ng/ml and that of the catecholamines assay 5 pg/ml. Values are expressed as means9SE. *P B 0.05, ***P B 0.001 compared with the preoperative value.
3 after Hx, DC m of NK cells from the Ch group tended to be decreased on POD 1, while DCm of T3 lymphocytes from the Hx group exhibited the same tendency in the postoperative period.
postoperative patients in both groups (n /85 points). A significant correlation was observed between NK cell activity and the percentage of NK cells with high DC m to total PBL (r /0.763, P B/0.0001), as shown in Fig. 4B.
3.5. NK cell activity was correlated with the fraction of CD56-positive cells with high DC m
3.6. Postoperative changes in absolute lymphocyte counts
Fig. 4A shows the changes in NK cell activity before and after operation. The preoperative values for the Hx group (24.09/6.7) tended to be higher than those for the Ch group (13.69/3.6) and normal volunteers (14.39/3.0). NK cell activity was significantly (P /0.0301) reduced in the Hx group (10.99/2.1) on POD 1 compared with the preoperative value. In the Ch group, a tendency toward decrease from the preoperative value was observed on POD 1 (12.39/4.6). We examined the relationship between NK cell activity and the ratio of DiOC6(3)high and CD56 cells to total lymphocytes for
The changes in total and T3 lymphocytes and NK cells in peripheral blood before and after surgery are shown in Fig. 5. In the Hx group, the number of total lymphocytes was significantly decreased on POD 1 (11749/169/ml) and POD 3 (10829/136/ml) when compared with the preoperative value (16869/165/ml), but had increased to 14699/137/ml on POD 7. The number of T3 lymphocytes in the Hx group exhibited no significant change during either the preoperative or the perioperative period. NK cells were significantly reduced in number on POD 1 (1959/41/ml) and POD 3
Fig. 2. Spontaneous lymphocyte apoptosis in patients undergoing hepatectomy or cholecystectomy. (A) Representative flow cytometry profiles obtained with 24-h culture of annexine V-FITC and propidium iodide staining. (B) The percentage of cells spontaneously undergoing apoptosis after 24-h in vitro culture correlated positively with the percentage of low DCm cells determined ex vivo. Hx, hepatectomy; Ch, cholecystectomy.
230
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Fig. 3. Serial changes in DCm in subpopulation of lymphocytes. (A) Representative flow cytometry profiles produced by double color staining with DiOC6(3) and CD3. (B) Representative flow cytometry profiles obtained with double color staining with DiOC6(3) and CD56. (C) Variation in proportions of cells which have CD3 positive or CD56 positive and high DiOC6(3) staining, that is, perioperative changes in DCm of T3 lymphocytes and DC m of NK cells. Data are expressed as mean9SE. Pre, preoperative day; POD, postoperative day; Hx, hepatectomy; Ch, cholecystectomy. *P B 0.05 compared with the preoperative value for the Ch group. **P B 0.01 compared with the preoperative value and the value for the Ch group on POD 3. ***P B 0.001 compared with the preoperative value and the value for the Ch group on POD 1.
(2249/32/ml) compared with the preoperative value (4889/70/ml).
4. Discussion This study demonstrated that partial hepatectomy reduced postoperative DC m of lymphocytes from pa-
tients with metastatic liver tumors and that some PBL could undergo apoptosis through the mitochondrial pathway. In addition, DC m of NK cells was more strongly affected by hepatectomy than that of other PBL and the magnitude of decrease in DC m in NK cells was closely associated with reduction in their function. Recent reports on clinical applications of DCm have suggested that changes in DC m of cells reflect their
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Fig. 4. (A) Serial changes in NK cell activity (lytic unit 20%) before and after surgery. Data are expressed as mean9SE. (B) Significant correlation between NK cell activity and the ratio of high DCm in NK cells (CD56 positive cells) to total PBL was observed (r 0.763, P B 0.0001). Pre, preoperative day; POD, postoperative day; Hx, hepatectomy; Ch, cholecystectomy.*P B 0.05 compared with the preoperative value.
function [17,19]. Thus, measurement of DC m could provide useful information not only on cell death but also cellular biological activity. To clarify whether the reduction in DC m was a consequence of hepatectomy or major surgery unrelated to the fact that the selected procedure was hepatectomy, we measured DC m of lymphocytes in patients after gastrectomy, colectomy, or pancreatico-duodenectomy. However, these procedures did not significantly affect DC m of lymphocytes like cholecystectomy (data not shown). Therefore, it is suggested that our results be linked to the hepatectomy but not to the major surgical trauma which is much more in patients with gastrectomy, colectomy, or pancreaticoduodenectomy than in cholecystectomized patients. Hepatectomy impaired NK cell cytotoxicity and this impairment was related to the reduction in NK cell count associated with decrease in NK cell activity. The mechanism underlying the reduction in circulating NK cells may be related to cell death or sequestration. Plasma catecholamine concentration, which appears to play an important role in liver regeneration [24],
increased after hepatic resection, similar to findings reported by other authors [25]. Catecholamines induced a rapid redistribution of NK cells from reservoirs such as the spleen and marginal pool where NK cells might differ in their DC m [26,27]. Since the liver also functions as a reservoir of NK cells, Hx may reduce this function and contribute to lymphopenia. In animal experiments, extrathymic T cells or NKT cells expanded in the liver in the early phase of hepatocyte regeneration after partial hepatectomy, while the change in the proportion of NK cells was minimal in the liver of mice subjected to partial hepatectomy [28]. Decrease in circulating NK cells may thus result mainly from cell death rather than from sequestration to the liver in this clinical setting. Shakhar et al. reported that catecholamine suppressed NK cell activity and compromised resistance to tumor metastasis [29]. Noradrenaline induces apoptosis of cardiac myocytes [30] and lymphocytes [31], and is thus one of the many candidates for molecules that commit NK cells to undergo apoptosis. Noradrenaline increases c-AMP and calcium influx via b-adrenergic receptor stimulation, and is thought to increase calcium levels in mitochon-
Fig. 5. Serial changes in total, T3 lymphocytes, and NK cells in peripheral blood before and after surgery. Data are expressed as mean9SE. Pre, preoperative day; POD, postoperative day, *P B 0.05 compared with the preoperative value. **P B 0.01 compared with the preoperative value. ***P B 0.001 compared with the preoperative value.
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dria as well as induce DC m perturbation [32]. To clarify the causal relationship between NK cells and catecholamines, further in vitro studies must be performed in which the effect of exogenous catecholamine on NK cells is evaluated. The major mechanism of target cell apoptosis by NK cells involves not only exocytosis of cytoplasmic granules from the NK cells towards the target cells, but also engagement of the Fas ligand or tumor necrosis factor on NK cells with the Fas or tumor necrosis factor receptor on the target cell [33]. NK cells also can undergo apoptosis following target cell and NK cell receptor engagement. It was observed that DC m of NK cells in the Hx group before surgery was higher than that for normal control. Elevation of DCm has been shown to have implications for a variety of pathophysiological conditions, particularly apoptosis [34,35]. The NK cells with high DC m we found were thought to be unstable and to be about to change to low DC m or undergo apoptosis after hepatectomy. DC m elevation and DC m reduction may thus take place at two different stages of the apoptotic process. NK cells are central in the innate immune response against parasites, infected cells, and tumors. Transgenic mice with defective natural killing and selective deficiency in NK cells displayed impaired in vivo tumor rejection [36]. This study using NK cell-deficient mice revealed that NK cells play an important role in suppressing tumor metastasis and growth. On the other hand, NK cells are involved in liver regeneration after partial hepatectomy [7,28] and are capable of killing regenerating liver hepatocytes [37], which express Fas constitutively and are susceptible to Fas-mediated apoptosis [38], since infiltration of NK cells into the liver can occur under pathological conditions such as hepatectomy [37]. Our study suggests that the cytotoxicity of NK cells is temporarily suppressed after partial hepatectomy and that they are noncytotoxic for regenerating liver. The reduction in DC m observed after partial hepatectomy is restricted to NK cells and may affect on liver regeneration. The assay currently used for the analysis of NK cell cytotoxicity is based on the measurement of radioactive 51 Cr release by target cells. Although this assay is the most commonly used to determine NK cell activity, it has a number of important drawbacks including the use of radioactive compounds, high cost, and the need for special laboratory equipment [39]. A relationship was observed between NK cell activity and the ratio of high DC m in NK cells to total PBL. Our results indicate that our novel method to measure DC m of CD56-positive cells can serve as a rapid assay useful for analysis of NKmediated cytotoxicity. Measurement of DC m of PBL may thus provide a useful method for examination of the effects of hepatectomy on the immune system.
Our study indicates that DC m of PBL is reduced after hepatectomy and that measurement of DCm can determine whether a cell will undergo apoptosis, which is one of the causes of lymphopenia. Decrease of DC m in NK cells is principally responsible for that of DC m in PBL after partial hepatectomy, and is associated with a reduction in the count and activity of NK cells. Reduction of DC m in NK cells may thus be important for liver regeneration after hepatectomy.
Acknowledgements This research was supported in part by Grants-in-Aid (11470251) for Scientific Research from the Ministry of Education, Science and Culture of Japan.
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