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Impaired Kupffer Cell Function and Effect of Immunotherapy in Obstructive Jaundice
Journal of Surgical Research 92, 276 –282 (2000) doi:10.1006/jsre.2000.5868, available online at http://www.idealibrary.com on
Impaired Kupffer Cell Function and Effect of Immunotherapy in Obstructive Jaundice Minetoshi Tomioka, M.D., 1 Hisae Iinuma, Ph.D., 2 and Kota Okinaga, M.D., Ph.D. Second Department of Surgery, Teikyo University School of Medicine, 2-11-1, Kaga, Itabashi-ku, Tokyo, 173-0003, Japan Submitted for publication December 3, 1999
Background. Obstructive jaundice is frequently associated with septic complications. This study examined the influence of biliary obstruction on bacterial clearance and translocation. The study focused on the phagocytic and killing activities of Kupffer cells and the preventive effect on bacterial translocation of OK432, which is a hemolytic streptococcal preparation developed as a biological response modifier. Methods. To study the mechanism of sepsis in obstructive jaundice, two groups of Wistar rats were examined: rats subjected to common bile duct ligation (CBDL) and rats subjected to a sham operation. Bacterial clearance, organ distribution, hepatic blood flow, and phagocytic function of Kupffer cells were examined. To evaluate the effect of OK-432 on bacterial translocation, rats were divided into three groups: sham operation ⴙ phosphate-buffered saline (PBS), CBDL ⴙ PBS, and CBDL ⴙ OK-432. Results. In this study, clearance of Escherichia coli. from the peripheral blood in CBDL rats was decreased significantly compared with that in sham-operated rats. Significant decreases in E.coli trapped in the liver and in hepatic blood flow were observed in CBDL rats compared with sham-operated rats. Phagocytic activity and superoxide production of Kupffer cells isolated from CBDL rats were significantly lower than in sham-operated rats. The incidence of bacterial translocation in CBDL rats was increased significantly, and oral administration of OK-432 prevented it. Conclusion. The results suggest that susceptibility to infection in obstructive jaundice is due to impaired phagocytic function of Kupffer cells. Furthermore, obstructive jaundice promotes bacterial translocation,
1 Present address: Department of Surgery, Tokiwadai Surgical Hospital, Tokyo, Japan. 2 To whom reprint requests and correspondence should be addressed. E-mail: [email protected].
0022-4804/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
and OK-432 may be useful in preventing this translocation. © 2000 Academic Press Key Words: obstructive jaundice; Kupffer cells; bacterial translocation; immunotherapy. INTRODUCTION
Sepsis and renal insufficiency are major complications leading to increased postoperative morbidity and mortality in patients with obstructive jaundice [1, 2]. Although clinical and experimental studies have demonstrated a correlation between obstructive jaundice and the development of sepsis, the mechanism of these complications has not been fully elucidated. It is postulated that at least two factors contribute: one is impaired reticuloendothelial system (RES) function, the other is increased bacterial translocation from the gastrointestinal tract across the gut mucosal barrier into the portal circulation, which allows “spillover” of bacteria or endotoxin into the systemic circulation [3– 6]. The liver, which the largest reticuloendothelial organ, is affected by obstruction of bile ducts. When biliary obstruction occurs the stationary macrophages in the sinusoids of the liver, the Kupffer cells, do not function normally [3, 4]. Recent experimental studies have shown that bacterial translocation occurs in obstructive jaundice [5, 6]. Factors that contribute to this process include disrupted gut microecology, impaired host immunity, and injury of the gut mucosa. A hemolytic streptococcus preparation, OK-432, has been developed as a biological response modifier (BRM) and widely used for immunotherapy of cancer patients in Japan, Korea, and the Republic of China [7]. Studies demonstrated that OK-432 activates lymphocytes, macrophages, and neutrophils through the induction of many cytokines, such as interleukin (IL)-1, IL-2, interferon-␥, and colony-stimulating factor [8, 9]. OK432 was shown to be effective when administered to
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TABLE 1 Liver Function Tests a Group
Bilirubin (mol/liter)
ALAT (Kat/liter)
ASAT (Kat/liter)
ALP (Kat/liter)
Sham operation CBDL
1.92 ⫾ 0.08 106.33 ⫾ 4.11 b
0.82 ⫾ 0.04 3.09 ⫾ 0.17 c
1.18 ⫾ 0.14 4.56 ⫾ 0.29 d
7.54 ⫾ 0.42 24.97 ⫾ 0.49 e
Note. Six animals in each group. Means ⫾ SD. a CBDL, common bile duct ligation; ALAT, alanine aminotransferase; ASAT, aspargine aminotransferase; ALP, alkaline phosphatase. b– e P ⬍ 0.01 versus sham operation.
patients by various routes, such as subcutaneously, intramuscularly, intraperitoneally, intratumorally, and orally [10, 11]. The aim of this study was to investigate the influence of biliary obstruction on bacterial clearance and translocation. The study also investigated the preventive effect of oral administration of OK-432 on bacterial translocation in rats with obstructive jaundice. MATERIAL AND METHODS Animals and operative procedures. Specific pathogen-free (SPF) 8-week-old male Wistar rats (Charles River Inc., Japan) were used. Animals were maintained in the animal care facilities of Teikyo University (Tokyo, Japan) at 22 ⫾ 2°C with a time-regulated light period and provided water and dry food ad libitum. Double ligation and division of the common bile duct prepared the model of obstructive jaundice in rats. Sham-operated rats had bile ducts dissected but not ligated. Two weeks later, these animals were used to study bacterial clearance, organ distribution, bacterial translocation, hepatic blood flow, and Kupffer cell function. To evaluate bacterial translocation, we prepared antibiotic-decontaminated rats according to the method described by Berg [12]. Briefly, rats were given drinking water containing 1500 U/ml penicillin-G (Sigma Chemical Co., St. Louis, MO) and 2 mg/ml streptomycin sulfate (Sigma) from 4 days before operation until sacrifice of the animals. Bacterial clearance from the blood and survival. Rats were divided into two groups: common bile duct ligation (CBDL) and sham operation. Two weeks after the operation, rats were challenged with 10 8 CFU of Escherichia coli (Serotype O3; kindly provided by Dr.S. Murayama, Department of Microbiology, Teikyo University, Japan) intravenously (iv) or intraperitoneally (ip). To examine bacterial clearance, blood samples were obtained from tail vessels 1, 3, and 6 h after the challenge, and survival rates of rats were recorded. Organ distribution of 35S-labeled E. coli. E. coli (Serotype O3) was cultured at 37°C for 18 h in a shaker bath in 10 ml of chemically defined liquid medium (NH 4Cl, Na 2PO 4, KH 2PO 4, NaCl, MgCl 2, glucose, Na 2SO 4) containing 5 Ci/ml Na 2 35SO 4 (NEN Life Science Products, Boston, MA). Two weeks after the operation, rats were challenged with 10 8 CFU 35S-labeled E. coli iv. At 10 min after challenge, the rats were killed and the liver, spleen, and lung removed. Small tissue samples (100 mg) were dissolved by a tissue solubilizer NCS II (Amersham Pharmacia Biotech, Sweden) at 50°C overnight. The solubilized tissues were decolorized by treating with benzoyl peroxide (Sigma), and then 10 ml of OCS scintillation fluid (Amersham Pharmacia Biotech) was added. After 3 days, radioactivity was determined using a liquid scintillation system (Aloka Co., Japan). Bacterial translocation. Antibiotic-decontaminated SPF rats were divided into three groups as follows: phosphate-buffered saline (PBS)-administered sham operation group, OK-432-administered CBDL group, and PBS-administered CBDL group. One KE (0.1 mg)
of OK-432 (kindly provided by Chugai Pharm.Co., Japan) was administered directly into the stomach of rats using a plastic catheter (Nihon Clea Co., Japan) on the 7th, 10th, and 13th days after operation. Two weeks after the operation, rats were inoculated orally with 10 8 CFU of streptomycin-resistant E. coli C25 (kindly provided by Dr. K. Itoh, Department of Veterinary Public Health,Tokyo University, Japan). Twenty-four hours after inoculation, rats were sacrificed and the mesenteric lymph nodes (MLNs), liver, and spleen were removed. The tissues were weighed, placed in a glass homogenizer containing 5 ml of sterile PBS, and homogenized. One-tenthmilliliter aliquots of the homogenate samples diluted serially in PBS were placed on DHL agar plates (Nissui Pharmaceutical Co., Japan) containing 1 mg/ml streptomycin. The plates were incubated at 37°C for 24 h, and the E. coli were counted. Isolation of Kupffer cells. Kupffer cells were isolated using the method of Page and Garvey [13]. Briefly, the liver was perfused with Hanks’ balanced salt solution (HBSS, Nissui), then with HBSS solution containing 0.2% Pronase E (Pronase solution) (Merck, Rahway, NJ) and sliced with scissors. Sliced tissues were shaken at 37°C for 1 h in Pronase solution. Kupffer cells were then separated using Percoll gradients (Amersham Pharmacia Biotech). After 45 min of centrifugation at 2200 rpm, the interface (1.04 –1.06 g/ml) was collected, washed with HBSS, and resuspended in RPMI 1640 (Nissui) containing 10% fetal calf serum. The purity of the Kupffer cells was determined to be approximately 92% by nonspecific esterase staining and viability was found to be 98% using the trypan blue exclusion test. Kupffer cells were incubated at 37°C for 18 h under 5% CO2 in tissue culture chambers (Nunc Inc. Denmark) for phagocytosis assay or plastic tubes for chemiluminescence assays. Phagocytosis of Kupffer cells. E. coli opsonized with normal rat serum at 37°C for 1 h were added to adherent Kupffer cells (cells: bacteria ⫽ 1:25). After incubation at 37°C for 30 min under 5% CO 2, cells were washed three times, fixed with methanol, and stained with Giemsa solution (Sigma). Phagocytic activity was determined using two different methods: (1) The phagocytic ratio was the percentage of cells that ingested E. coli of all Kupffer cells. (2) The phagocytic index was the mean number of E. coli ingested per cell. In these assays, at least 200 cells in 20 fields were counted for each sample. Chemiluminescence assay of Kupffer cells. The release of reactive oxygen species from Kupffer cells was assessed by luminol-enhanced chemiluminescence using a modification of the technique of Lee et al. [14]. Briefly, 5 ⫻ 10 6 Kupffer cells were incubated at 37°C for 5 min in Hepes–MEM buffer, and luminol (5-amino-2,3-dihydro-1,4phthalazinedione; Tokyokasei, Japan) was added. To stimulate the release of reactive oxygen species, opsonized zymosan or E. coli was added to the vial, and the titer was recorded after 30 min using a Biolumat LB9500 (Berthold, Germany). Blood flow of liver tissue. Hepatic blood flow was measured using laser Doppler flowmeter (ALF21; Advance Co., Japan). The standard probe was applied to the liver surface, and blood flow of the left and right lobe was measured separately. Statistical analysis. Values are presented as means ⫾ SD. Differences between the two groups were analyzed with the Student’s t
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FIG. 1. Clearance of E. coli from peripheral blood. One hundred million E. coli were inoculated intravenously (A) or intraperitoneally (B) into sham-operated (E) or common bile duct-ligated (F) rats. The number of viable E. coli was measured 1, 3, and 6 h after challenge with E. coli. There were six animals in each group. Mean ⫾ SD. *P ⬍ 0.05.
test. Multiple groups were evaluated by analysis of variance (ANOVA) and the post hoc Bonferroni multiple-range test. The incidence of bacterial translocation was analyzed by 2 test. Survival rates were evaluated by the generalized Wilcoxon test. Probabilities less than 0.05 were considered significant.
RESULTS
Liver function tests. Ligation and division of the common bile duct in rats resulted in cystic dilation of the proximal bile duct and jaundice. Two weeks after the surgical procedure, the serum bilirubin level in the CBDL group increased significantly, about 55 times higher than that of the sham-operated control group (Table 1). Serum alanine aminotransferase (ALAT), asparagine aminotransferase (ASAT), and alkaline phosphatase (ALP) levels were also elevated significantly in the CBDL group compared with the shamoperated group. Blood clearance and survival after E. coli challenge. Blood clearances of ip or iv injected viable E. coli were examined in the CBDL and sham-operated rats (Fig.
1). After the intraperitoneal challenge with E. coli, CBDL rats showed a significant decrease in clearance of E. coli from the peripheral blood compared with sham-operated rats. After the intravenous challenge, a significant decrease in bacterial clearance from the blood was shown in CBDL rats compared with shamoperated rats. The survival rates after intraperitoneal or intravenous challenge with E. coli were 100% in sham-operated rats and 0% in CBDL rats (Fig. 2). All bile duct-ligated rats died of sepsis within 6 days of the bacterial challenge. These results demonstrate the increased susceptibility to sepsis in obstructive jaundice. Organ localization of 35S-labeled E. coli. The uptake of 35S-labeled E. coli was higher in the liver than in either the spleen or the lung. In the CBDL group, a significant decrease in bacterial trapping in the liver was found compared with sham-operated rats (Fig. 3). The percentages of radiolabeled E.coli trapped in the liver were 76% in sham-operated rats and 44% in CBDL rats. In contrast, there were no significant dif-
FIG. 2. Survival rates after E. coli administration. One hundred million E. coli were inoculated intravenously (A) or intraperitoneally (B) into sham-operated (…) or common bile duct-ligated (—) rats. There were six animals in each group. *P ⬍ 0.01 versus sham operation.
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TABLE 2 Phagocytic Activity of Cultured Kupffer Cells a Group
Phagocytic ratio b
Phagocytic index c
Sham operation CBDL
95.0 ⫾ 3.0% 84.0 ⫾ 4.0% d
12.9 ⫾ 3.1 10.0 ⫾ 2.2
Note. Six animals in each group. Means ⫾ SD. a CBDL, common bile duct ligation. b Percentage of cells injecting E. coli. c Mean number of E. coli ingested per cell. d P ⬍ 0.05 versus sham operation.
FIG. 3. Organ distribution of intravenously injected 35S-labeled E. coli. Sham-operated (䊐) and common bile duct-ligated (■) rats were inoculated with 1 ⫻ 10 8 radiolabeled E. coli intravenously, and bacterial localization in liver, spleen, and lung was determined 10 min after inoculation. There were six animals in each group. Values repesent means ⫾ SD expressed as percentage of injected E. coli per total organ. *P ⬍ 0.01.
ferences in the uptake of radiolabeled E. coli in the spleen and lung between these two groups. Number of Kupffer cells. Kupffer cells isolated from the liver were counted by nonspecific esterase staining. The number tended to be higher in CBDL rats, than in sham-operated rats; the difference was not statistically significant (Fig. 4). Phagocytic function of isolated Kupffer cells. Uptake of E. coli by Kupffer cells was evaluated as the phagocytic ratio and phagocytic index (Table 2). The phagocytic ratio of Kupffer cells isolated from CBDL rats was 84 ⫾ 4%, which was significantly lower than that of sham-operated rats (95 ⫾ 3%). The phagocytic index of Kupffer cells was 10.0 ⫾ 2.2 in CBDL rats and 12.9 ⫾ 3.1 in sham-operated rats; there was no significant difference. These results suggest that the phagocytic function of Kupffer cells decreases in obstructive jaundice.
FIG. 4. Number of Kupffer cells. The number of Kupffer cells per gram of liver of sham-operated (䊐) and common bile duct-ligated (■) rats was calculated by nonspecific esterase staining. There were six animals in each group. Mean ⫾ SD.
Chemiluminescence of isolated Kupffer cells. Superoxide production of Kupffer cells, which represents their bacteria-killing activity, was studied (Fig. 5). Kupffer cells were stimulated with zymosan or E. coli opsonized with normal rat serum. When stimulated with E. coli, the maximal count was observed at 10 min in the sham-operated group and 12 min in the CBDL group. This count decreased immediately. In contrast, the superoxide levels of Kupffer cells stimulated with zymosan increased gradually. The maximal count was at 15 min in the sham-operated group and at 20 min in the CBDL group. These counts remained elevated throughout the measurement period. Although the stimulation differed, the maximal count and total count for 30 min of superoxide in CBDL rats were significantly lower than those of sham-operated rats. These results suggest that the bacteria-killing activity of Kupffer cells decreases significantly with bile duct obstruction. Blood flow in the liver. In the left lobe of the liver, the mean level of hepatic blood flow was 13.7 ⫾ 1.4 in CBDL rats, which was significantly lower than 24.0 ⫾ 1.5 in sham-operated rats (Fig. 6). In the right lobe of the liver, the mean level of hepatic blood flow was 13.3 ⫾ 1.0 in CBDL rats, which was significantly lower than 27.6 ⫾ 3.8 in sham-operated rats. Bacterial translocation. In the sham-operated group receiving PBS, the incidence of bacterial translocation both in the MLNs and the liver was 16.7%, and no bacteria were found in the spleen (Table 3). In contrast, all rats in the CBDL group showed translocation of bacteria to the MLNs, liver, and spleen. The numbers of streptomycin-resistant E. coli in the MLNs, liver, and spleen of PBS-administered CBDL rats were increased significantly compared with PBSadministered sham-operated rats (Fig. 7). Oral administration of OK-432 significantly reduced the incidence of bacterial translocation to the liver and spleen in CBDL rats (Table 3). The numbers of E. coli in the liver and spleen of OK-432-administered CBDL rats were decreased significantly compared with PBS-administered CBDL rats (Fig. 7). These results suggest that obstructive jaundice increases the translocation of bacteria, and
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FIG. 5. Chemiluminescence of Kupffer cells. Isolated Kupffer cells (5 ⫻ 10 6) from sham-operated (E) or common bile duct-ligated (CBDL, F) rats were cultured for 24 h in plastic tubes, then stimulated with zymosan opsonized with normal rat serum (A) or opsonized E. coli (B) for 30 min. Data are expressed as maximal counts (a) and total counts for 30 min (b). There were six animals in each group. Mean ⫾ SD. *P ⬍ 0.01 versus sham operation. †P ⬍ 0.01 versus sham operation.
oral administration of OK-432 could prevent bacterial translocation. DISCUSSION
Several studies have reported that the increased incidence of sepsis in obstructive jaundice might be due to depressed reticuloendothelial system (RES) function [3– 6]. The present study also showed a significant decrease in bacterial clearance from the circulating blood and decrease in E. coli uptake into the liver in jaundiced animals. Despite many reports suggesting RES dysfunction in obstructive jaundice, more specific information has been sparse regarding phagocytosis in jaundice. Ding et al. [4] examined in vivo phagocytic index, which is the slope of the semilogarithmic plot of radioactivity in
blood after intravenous injection of radiolabeled E. coli. They demonstrated that RES phagocytic function gradually decreases during the course of obstructive jaundice, and that longstanding biliary obstruction (3 weeks) causes a more marked depression of RES function. In this study, we focused on in vitro phagocytic function of Kupffer cells isolated from the liver. It is known that Kupffer cells account for about 80% of the cells in the RES [15]. Only a few studies have reported on the phagocytic function of isolated Kupffer cells. Sheen-Chen et al. suggested that the phagocytic activity of yeast in isolated Kupffer cells increased significantly 1 week after bile duct ligation [16]. In contrast, our data showed a significant decrease in the phagocytic ratio of E. coli by Kupffer cells 2 weeks after bile duct ligation. Although the differences in methods may influence the results, the exact reason remains to be determined. Furthermore, we examined the superoxide production of isolated Kupffer cells. It is known that superoxide production can be used to assess bacteria-killing activity. Sung et al. [17] reported that superoxide production by Kupffer cells isolated from biliary-obstructed rats was significantly lower than TABLE 3 Incidence of Bacterial Translocation a
FIG. 6. Hepatic blood flow in left and right lobes of liver. Hepatic tissue blood flow in sham-operated (䊐) and common bile duct-ligated (■) rats 2 weeks after operation was assessed by laser Doppler flowmetry. There were six animals in each group. Mean ⫾ SD. *P ⬍ 0.01.
Group
MLNs
Liver
Spleen
Sham operation ⫹ PBS CBDL ⫹ PBS CBDL ⫹ OK-432
1 (16.7%) 6 (100.0%) b 6 (100.0%) c
1 (16.7%) 6 (100.0%) b 3 (50.0%) c,d
0 (0.0%) 6 (100.0%) b 3 (50.0%) c,d
a Six animals in each group. Mean (%) MLNs, mesenteric lymph nodes; CBDL, common bile duct ligation. b P ⬍ 0.01 versus sham operation ⫹ PBS. c P ⬍ 0.01 versus sham operation ⫹ PBS. d P ⬍ 0.01 versus CBDL ⫹ PBS.
TOMIOKA, IINUMA, AND OKINAGA: KUPFFER CELL FUNCTION IN OBSTRUCTIVE JAUNDICE
FIG. 7. Number of E. coli translocated into the mesenteric lymph nodes (MLNs), liver, and spleen. The numbers of E. coli in the MLNs, liver, and spleen were compared in the PBS-administered shamoperated group (䊐), PBS administered common bile duct ligated (CBDL) group (■), and OK-432-administered CBDL group (o). There were six animals in each group. Mean ⫾ SD. *P ⬍ 0.05. †P ⬍ 0.01.
that in sham-operated rats, and that after resting for 2 h, a significant recovery in superoxide production occurred. In contrast, the present study showed that production of superoxide by Kupffer cells isolated from CBDL rats was significantly depressed compared with the sham-operated rats, even after resting for 24 h. Differences in the methods of isolating Kupffer cells may affect the recovery of function. These results suggest that RES dysfunction in biliary obstruction is due to impaired phagocytosis and killing activity of Kupffer cells. The role of hepatic blood flow in obstructive jaundice has not been fully investigated. Several studies showed that liver blood flow is reduced in obstructive jaundice using colloidal radiogold ( 108Au) or 133Xe [18, 19] to measure. Other studies reported a significant decrease in blood flow in the hepatic artery and the portal vein in obstructive jaundice using the electromagnetic flowmeter [20]. The present study used laser Doppler flowmetry to measure liver blood flow. Arvidsson et al. [21] reported that this method is a reliable, reproducible, and continuous method to estimate hepatic blood flow. Our results showed a significant decrease in hepatic blood flow in CBDL rats, suggesting that the decreased bacterial elimination from circulating blood may be partly due to decreased blood flow. It is important to clarify the source of the bacteria that cause systemic infection in obstructive jaundice. It is known that translocation of intestinal bacteria increases after bile duct ligation [5, 6]. The present study also demonstrated that bile duct ligation promotes translocation of E. coli to the MLNs, liver, and spleen. This finding supports the hypothesis that the source of bacterial infection in obstructive jaundice can originate in the gastrointestinal tract even without other in-
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fected sites. Bacterial translocation is defined as the passage of viable indigenous bacteria from the gastrointestinal tract to normally sterile extraintestinal sites [22]. Although this process involves disrupted gut microecology, bacterial overgrowth, impaired host immunity, and physical injury of the gut mucosa, the factors that promote translocation are not fully understood. It was reported that bile salts inhibit the growth of intestinal bacteria and contribute to the regulation of gut microflora [23]. Therefore, removal of intraluminal bile salts by ligation of the bile duct may cause a change in the endogenous bacterial flora, loss of mucosal integrity, and increased bacterial translocation to the MLNs and liver. Furthermore, inadequate RES control of portal bacteremia by impaired phgocytic function of Kupffer cells may result in bacterial “spillover” followed by subsequent systemic bacteremia. Various agents have been proposed for preventing bacterial translocation. In patients with total parenteral nutrition, the use of oral neomycin, fluoroquinolone, polymyxin B, and tobramycin was found effective in preventing bacterial translocation by regulating the intestinal flora [24]. Experimental trials to prevent bacterial translocation by enhancing the host immunological function have been studied. Hagiwara et al. [25] reported that the transfer of a macrophage-enriched fraction of spleen cells collected from mice vaccinated with formalin-killed Propionibacterium acnes prevented translocation of bacteria. Suzuki et al. [26] demonstrated that the oral administration of a culture condensate of Bifidobacterium longum prevents bacterial translocation from the gastrointestinal tract of mice. In this study, we demonstrated the preventive effect of oral administration of OK-432 on bacterial translocation. OK-432 is a heat- and penicillin G-treated lyophilized powder of the Su strain of Streptococcus pyogenes A which has been used clinically as an immunomodulator for treating patients with gastric cancer [7]. It is known that OK-432 activates lymphocytes and macrophages by inducing various cytokines such as IL-1, IL-2, and IL-12 [8, 9]. Therefore OK-432 may activate the lymphocytes and macrophages of gutassociated lymphoid tissue that regulate bacterial translocation. However, the exact mechanism must be determined in future studies. CONCLUSION
Increased susceptibility to infection in rats with obstructive jaundice is associated with impaired phagocytic function, the killing activity of Kupffer cells, and decreased hepatic blood flow. Furthermore, promoted bacterial translocation in obstructive jaundice may influence gut-associated systemic infection. Oral administration of OK-432 seems to reduce the incidence of bacterial translocation.
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ACKNOWLEDGMENTS We thank Dr. R. Fukushima for his helpful advice on bacterial translocation and Ms. J. Tamura and Ms. H. Kumagai for their excellent technical support.
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from the gastrointestinal tracts of mice by oral treatment with penicillin, clindamycin, or metronidazole. Infect. Immun. 33: 854, 1981. Page, D., and Garvey, J. S. Isolation and characterization of hepatocytes and Kupffer cells. J. Immunol. Methods 27: 159, 1979. Lee, P., Walker, E. R., Miles, P. R., and Castranova, V. Differential generation of chemiluminescence from various fraction obtained by dog lung gavage. In K. Van Dyke and V. Castranova (Eds.), Cellular Chemiluminescence. Boca Raton: CRC Press, 1987. Vol. 3, p. 53. Saba, T. Physiology and pathophysiology of the reticuloendothelial system. Arch. Intern. Med. 126: 1031, 1970. Sheen-Chen, S-M., Chau, P., and Harris, H. W. Obstructive jaundice alters Kupffer cell function independent of bacterial translocation. J. Surg. Res. 80: 205, 1998. Sung, J. J. Y., and Go, Y. Y. M. Reversible Kupffer cell suppression in biliary obstruction is caused by hydrophobic bile acids. J. Hepatol. 30: 413, 1999. Aronsen, K. F., Nylander, G., and Ohlsson, G. Liver blood flow studies during and after various periods of total biliary obstruction in the dog. Acta Chir. Scand. 135: 55, 1969. Baer, H. U., Guastella, T., Wheatley, A. M., Zimmermann, A., and Blumgart, L. H. Acute effects partial hepatectomy on liver blood flow in the jaundiced rat. J. Hepatol. 19: 377, 1993. Mathie, R. T., Nagorney, D. M., Lewis, M. H., and Blumgart, L. H. Hepatic hemodynamics after chronic obstruction of the biliary tract in the dog. Surg. Gynecol. Obstet. 166: 125, 1988. Arvidsson, D., Svensson, H., and Haglund, U. Laser Doppler flowmetry for estimating liver blood flow. Am. J. Physiol. 254: G471, 1988. Berg, R. D., and Garlington, A. W. Translocation of certain indigeneous bacteria from the gastrointestinal tract to the mesenteric lymph nodes and other organs in a gnotobiotic mouse model. Infect. Immun. 23: 403, 1979. Williams, R. C., Showalter, R., and Kern, F. In vivo effect of bile salts and cholestyramine on intestinal anaerobic bacteria. Gastroenterology 69: 483, 1975. Batt, R. M., and Mclean, L. Comparison of the biochemical changes in the jejunal mucosa of dogs with aerobic and anaerobic bacterial overgrowth. Gastroenterology 93: 986, 1987. Hagiwara, T., Itoh, K., Takahashi, E., Honjo, K., and Hirota, Y. Effects of Propionibacterium acnes injection on inhibition of bacterial translocation and change of lymphocyte subsets. J. Germfree Life Gnotobiol. 24: 56, 1994. Suzuki, T., Itoh, K., Kaneko, T., and Suzuki, H. Inhibition of bacterial translocation from the gastrointestinal tract of mice by oral administration of a culture condensate of Bifidobacterium longum. J. Vet. Med. Sci. 59: 665, 1997.
Impaired Kupffer Cell Function and Effect of Immunotherapy in Obstructive Jaundice Minetoshi Tomioka, M.D., 1 Hisae Iinuma, Ph.D., 2 and Kota Okinaga, M.D., Ph.D. Second Department of Surgery, Teikyo University School of Medicine, 2-11-1, Kaga, Itabashi-ku, Tokyo, 173-0003, Japan Submitted for publication December 3, 1999
Background. Obstructive jaundice is frequently associated with septic complications. This study examined the influence of biliary obstruction on bacterial clearance and translocation. The study focused on the phagocytic and killing activities of Kupffer cells and the preventive effect on bacterial translocation of OK432, which is a hemolytic streptococcal preparation developed as a biological response modifier. Methods. To study the mechanism of sepsis in obstructive jaundice, two groups of Wistar rats were examined: rats subjected to common bile duct ligation (CBDL) and rats subjected to a sham operation. Bacterial clearance, organ distribution, hepatic blood flow, and phagocytic function of Kupffer cells were examined. To evaluate the effect of OK-432 on bacterial translocation, rats were divided into three groups: sham operation ⴙ phosphate-buffered saline (PBS), CBDL ⴙ PBS, and CBDL ⴙ OK-432. Results. In this study, clearance of Escherichia coli. from the peripheral blood in CBDL rats was decreased significantly compared with that in sham-operated rats. Significant decreases in E.coli trapped in the liver and in hepatic blood flow were observed in CBDL rats compared with sham-operated rats. Phagocytic activity and superoxide production of Kupffer cells isolated from CBDL rats were significantly lower than in sham-operated rats. The incidence of bacterial translocation in CBDL rats was increased significantly, and oral administration of OK-432 prevented it. Conclusion. The results suggest that susceptibility to infection in obstructive jaundice is due to impaired phagocytic function of Kupffer cells. Furthermore, obstructive jaundice promotes bacterial translocation,
1 Present address: Department of Surgery, Tokiwadai Surgical Hospital, Tokyo, Japan. 2 To whom reprint requests and correspondence should be addressed. E-mail: [email protected].
0022-4804/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
and OK-432 may be useful in preventing this translocation. © 2000 Academic Press Key Words: obstructive jaundice; Kupffer cells; bacterial translocation; immunotherapy. INTRODUCTION
Sepsis and renal insufficiency are major complications leading to increased postoperative morbidity and mortality in patients with obstructive jaundice [1, 2]. Although clinical and experimental studies have demonstrated a correlation between obstructive jaundice and the development of sepsis, the mechanism of these complications has not been fully elucidated. It is postulated that at least two factors contribute: one is impaired reticuloendothelial system (RES) function, the other is increased bacterial translocation from the gastrointestinal tract across the gut mucosal barrier into the portal circulation, which allows “spillover” of bacteria or endotoxin into the systemic circulation [3– 6]. The liver, which the largest reticuloendothelial organ, is affected by obstruction of bile ducts. When biliary obstruction occurs the stationary macrophages in the sinusoids of the liver, the Kupffer cells, do not function normally [3, 4]. Recent experimental studies have shown that bacterial translocation occurs in obstructive jaundice [5, 6]. Factors that contribute to this process include disrupted gut microecology, impaired host immunity, and injury of the gut mucosa. A hemolytic streptococcus preparation, OK-432, has been developed as a biological response modifier (BRM) and widely used for immunotherapy of cancer patients in Japan, Korea, and the Republic of China [7]. Studies demonstrated that OK-432 activates lymphocytes, macrophages, and neutrophils through the induction of many cytokines, such as interleukin (IL)-1, IL-2, interferon-␥, and colony-stimulating factor [8, 9]. OK432 was shown to be effective when administered to
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TABLE 1 Liver Function Tests a Group
Bilirubin (mol/liter)
ALAT (Kat/liter)
ASAT (Kat/liter)
ALP (Kat/liter)
Sham operation CBDL
1.92 ⫾ 0.08 106.33 ⫾ 4.11 b
0.82 ⫾ 0.04 3.09 ⫾ 0.17 c
1.18 ⫾ 0.14 4.56 ⫾ 0.29 d
7.54 ⫾ 0.42 24.97 ⫾ 0.49 e
Note. Six animals in each group. Means ⫾ SD. a CBDL, common bile duct ligation; ALAT, alanine aminotransferase; ASAT, aspargine aminotransferase; ALP, alkaline phosphatase. b– e P ⬍ 0.01 versus sham operation.
patients by various routes, such as subcutaneously, intramuscularly, intraperitoneally, intratumorally, and orally [10, 11]. The aim of this study was to investigate the influence of biliary obstruction on bacterial clearance and translocation. The study also investigated the preventive effect of oral administration of OK-432 on bacterial translocation in rats with obstructive jaundice. MATERIAL AND METHODS Animals and operative procedures. Specific pathogen-free (SPF) 8-week-old male Wistar rats (Charles River Inc., Japan) were used. Animals were maintained in the animal care facilities of Teikyo University (Tokyo, Japan) at 22 ⫾ 2°C with a time-regulated light period and provided water and dry food ad libitum. Double ligation and division of the common bile duct prepared the model of obstructive jaundice in rats. Sham-operated rats had bile ducts dissected but not ligated. Two weeks later, these animals were used to study bacterial clearance, organ distribution, bacterial translocation, hepatic blood flow, and Kupffer cell function. To evaluate bacterial translocation, we prepared antibiotic-decontaminated rats according to the method described by Berg [12]. Briefly, rats were given drinking water containing 1500 U/ml penicillin-G (Sigma Chemical Co., St. Louis, MO) and 2 mg/ml streptomycin sulfate (Sigma) from 4 days before operation until sacrifice of the animals. Bacterial clearance from the blood and survival. Rats were divided into two groups: common bile duct ligation (CBDL) and sham operation. Two weeks after the operation, rats were challenged with 10 8 CFU of Escherichia coli (Serotype O3; kindly provided by Dr.S. Murayama, Department of Microbiology, Teikyo University, Japan) intravenously (iv) or intraperitoneally (ip). To examine bacterial clearance, blood samples were obtained from tail vessels 1, 3, and 6 h after the challenge, and survival rates of rats were recorded. Organ distribution of 35S-labeled E. coli. E. coli (Serotype O3) was cultured at 37°C for 18 h in a shaker bath in 10 ml of chemically defined liquid medium (NH 4Cl, Na 2PO 4, KH 2PO 4, NaCl, MgCl 2, glucose, Na 2SO 4) containing 5 Ci/ml Na 2 35SO 4 (NEN Life Science Products, Boston, MA). Two weeks after the operation, rats were challenged with 10 8 CFU 35S-labeled E. coli iv. At 10 min after challenge, the rats were killed and the liver, spleen, and lung removed. Small tissue samples (100 mg) were dissolved by a tissue solubilizer NCS II (Amersham Pharmacia Biotech, Sweden) at 50°C overnight. The solubilized tissues were decolorized by treating with benzoyl peroxide (Sigma), and then 10 ml of OCS scintillation fluid (Amersham Pharmacia Biotech) was added. After 3 days, radioactivity was determined using a liquid scintillation system (Aloka Co., Japan). Bacterial translocation. Antibiotic-decontaminated SPF rats were divided into three groups as follows: phosphate-buffered saline (PBS)-administered sham operation group, OK-432-administered CBDL group, and PBS-administered CBDL group. One KE (0.1 mg)
of OK-432 (kindly provided by Chugai Pharm.Co., Japan) was administered directly into the stomach of rats using a plastic catheter (Nihon Clea Co., Japan) on the 7th, 10th, and 13th days after operation. Two weeks after the operation, rats were inoculated orally with 10 8 CFU of streptomycin-resistant E. coli C25 (kindly provided by Dr. K. Itoh, Department of Veterinary Public Health,Tokyo University, Japan). Twenty-four hours after inoculation, rats were sacrificed and the mesenteric lymph nodes (MLNs), liver, and spleen were removed. The tissues were weighed, placed in a glass homogenizer containing 5 ml of sterile PBS, and homogenized. One-tenthmilliliter aliquots of the homogenate samples diluted serially in PBS were placed on DHL agar plates (Nissui Pharmaceutical Co., Japan) containing 1 mg/ml streptomycin. The plates were incubated at 37°C for 24 h, and the E. coli were counted. Isolation of Kupffer cells. Kupffer cells were isolated using the method of Page and Garvey [13]. Briefly, the liver was perfused with Hanks’ balanced salt solution (HBSS, Nissui), then with HBSS solution containing 0.2% Pronase E (Pronase solution) (Merck, Rahway, NJ) and sliced with scissors. Sliced tissues were shaken at 37°C for 1 h in Pronase solution. Kupffer cells were then separated using Percoll gradients (Amersham Pharmacia Biotech). After 45 min of centrifugation at 2200 rpm, the interface (1.04 –1.06 g/ml) was collected, washed with HBSS, and resuspended in RPMI 1640 (Nissui) containing 10% fetal calf serum. The purity of the Kupffer cells was determined to be approximately 92% by nonspecific esterase staining and viability was found to be 98% using the trypan blue exclusion test. Kupffer cells were incubated at 37°C for 18 h under 5% CO2 in tissue culture chambers (Nunc Inc. Denmark) for phagocytosis assay or plastic tubes for chemiluminescence assays. Phagocytosis of Kupffer cells. E. coli opsonized with normal rat serum at 37°C for 1 h were added to adherent Kupffer cells (cells: bacteria ⫽ 1:25). After incubation at 37°C for 30 min under 5% CO 2, cells were washed three times, fixed with methanol, and stained with Giemsa solution (Sigma). Phagocytic activity was determined using two different methods: (1) The phagocytic ratio was the percentage of cells that ingested E. coli of all Kupffer cells. (2) The phagocytic index was the mean number of E. coli ingested per cell. In these assays, at least 200 cells in 20 fields were counted for each sample. Chemiluminescence assay of Kupffer cells. The release of reactive oxygen species from Kupffer cells was assessed by luminol-enhanced chemiluminescence using a modification of the technique of Lee et al. [14]. Briefly, 5 ⫻ 10 6 Kupffer cells were incubated at 37°C for 5 min in Hepes–MEM buffer, and luminol (5-amino-2,3-dihydro-1,4phthalazinedione; Tokyokasei, Japan) was added. To stimulate the release of reactive oxygen species, opsonized zymosan or E. coli was added to the vial, and the titer was recorded after 30 min using a Biolumat LB9500 (Berthold, Germany). Blood flow of liver tissue. Hepatic blood flow was measured using laser Doppler flowmeter (ALF21; Advance Co., Japan). The standard probe was applied to the liver surface, and blood flow of the left and right lobe was measured separately. Statistical analysis. Values are presented as means ⫾ SD. Differences between the two groups were analyzed with the Student’s t
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FIG. 1. Clearance of E. coli from peripheral blood. One hundred million E. coli were inoculated intravenously (A) or intraperitoneally (B) into sham-operated (E) or common bile duct-ligated (F) rats. The number of viable E. coli was measured 1, 3, and 6 h after challenge with E. coli. There were six animals in each group. Mean ⫾ SD. *P ⬍ 0.05.
test. Multiple groups were evaluated by analysis of variance (ANOVA) and the post hoc Bonferroni multiple-range test. The incidence of bacterial translocation was analyzed by 2 test. Survival rates were evaluated by the generalized Wilcoxon test. Probabilities less than 0.05 were considered significant.
RESULTS
Liver function tests. Ligation and division of the common bile duct in rats resulted in cystic dilation of the proximal bile duct and jaundice. Two weeks after the surgical procedure, the serum bilirubin level in the CBDL group increased significantly, about 55 times higher than that of the sham-operated control group (Table 1). Serum alanine aminotransferase (ALAT), asparagine aminotransferase (ASAT), and alkaline phosphatase (ALP) levels were also elevated significantly in the CBDL group compared with the shamoperated group. Blood clearance and survival after E. coli challenge. Blood clearances of ip or iv injected viable E. coli were examined in the CBDL and sham-operated rats (Fig.
1). After the intraperitoneal challenge with E. coli, CBDL rats showed a significant decrease in clearance of E. coli from the peripheral blood compared with sham-operated rats. After the intravenous challenge, a significant decrease in bacterial clearance from the blood was shown in CBDL rats compared with shamoperated rats. The survival rates after intraperitoneal or intravenous challenge with E. coli were 100% in sham-operated rats and 0% in CBDL rats (Fig. 2). All bile duct-ligated rats died of sepsis within 6 days of the bacterial challenge. These results demonstrate the increased susceptibility to sepsis in obstructive jaundice. Organ localization of 35S-labeled E. coli. The uptake of 35S-labeled E. coli was higher in the liver than in either the spleen or the lung. In the CBDL group, a significant decrease in bacterial trapping in the liver was found compared with sham-operated rats (Fig. 3). The percentages of radiolabeled E.coli trapped in the liver were 76% in sham-operated rats and 44% in CBDL rats. In contrast, there were no significant dif-
FIG. 2. Survival rates after E. coli administration. One hundred million E. coli were inoculated intravenously (A) or intraperitoneally (B) into sham-operated (…) or common bile duct-ligated (—) rats. There were six animals in each group. *P ⬍ 0.01 versus sham operation.
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TABLE 2 Phagocytic Activity of Cultured Kupffer Cells a Group
Phagocytic ratio b
Phagocytic index c
Sham operation CBDL
95.0 ⫾ 3.0% 84.0 ⫾ 4.0% d
12.9 ⫾ 3.1 10.0 ⫾ 2.2
Note. Six animals in each group. Means ⫾ SD. a CBDL, common bile duct ligation. b Percentage of cells injecting E. coli. c Mean number of E. coli ingested per cell. d P ⬍ 0.05 versus sham operation.
FIG. 3. Organ distribution of intravenously injected 35S-labeled E. coli. Sham-operated (䊐) and common bile duct-ligated (■) rats were inoculated with 1 ⫻ 10 8 radiolabeled E. coli intravenously, and bacterial localization in liver, spleen, and lung was determined 10 min after inoculation. There were six animals in each group. Values repesent means ⫾ SD expressed as percentage of injected E. coli per total organ. *P ⬍ 0.01.
ferences in the uptake of radiolabeled E. coli in the spleen and lung between these two groups. Number of Kupffer cells. Kupffer cells isolated from the liver were counted by nonspecific esterase staining. The number tended to be higher in CBDL rats, than in sham-operated rats; the difference was not statistically significant (Fig. 4). Phagocytic function of isolated Kupffer cells. Uptake of E. coli by Kupffer cells was evaluated as the phagocytic ratio and phagocytic index (Table 2). The phagocytic ratio of Kupffer cells isolated from CBDL rats was 84 ⫾ 4%, which was significantly lower than that of sham-operated rats (95 ⫾ 3%). The phagocytic index of Kupffer cells was 10.0 ⫾ 2.2 in CBDL rats and 12.9 ⫾ 3.1 in sham-operated rats; there was no significant difference. These results suggest that the phagocytic function of Kupffer cells decreases in obstructive jaundice.
FIG. 4. Number of Kupffer cells. The number of Kupffer cells per gram of liver of sham-operated (䊐) and common bile duct-ligated (■) rats was calculated by nonspecific esterase staining. There were six animals in each group. Mean ⫾ SD.
Chemiluminescence of isolated Kupffer cells. Superoxide production of Kupffer cells, which represents their bacteria-killing activity, was studied (Fig. 5). Kupffer cells were stimulated with zymosan or E. coli opsonized with normal rat serum. When stimulated with E. coli, the maximal count was observed at 10 min in the sham-operated group and 12 min in the CBDL group. This count decreased immediately. In contrast, the superoxide levels of Kupffer cells stimulated with zymosan increased gradually. The maximal count was at 15 min in the sham-operated group and at 20 min in the CBDL group. These counts remained elevated throughout the measurement period. Although the stimulation differed, the maximal count and total count for 30 min of superoxide in CBDL rats were significantly lower than those of sham-operated rats. These results suggest that the bacteria-killing activity of Kupffer cells decreases significantly with bile duct obstruction. Blood flow in the liver. In the left lobe of the liver, the mean level of hepatic blood flow was 13.7 ⫾ 1.4 in CBDL rats, which was significantly lower than 24.0 ⫾ 1.5 in sham-operated rats (Fig. 6). In the right lobe of the liver, the mean level of hepatic blood flow was 13.3 ⫾ 1.0 in CBDL rats, which was significantly lower than 27.6 ⫾ 3.8 in sham-operated rats. Bacterial translocation. In the sham-operated group receiving PBS, the incidence of bacterial translocation both in the MLNs and the liver was 16.7%, and no bacteria were found in the spleen (Table 3). In contrast, all rats in the CBDL group showed translocation of bacteria to the MLNs, liver, and spleen. The numbers of streptomycin-resistant E. coli in the MLNs, liver, and spleen of PBS-administered CBDL rats were increased significantly compared with PBSadministered sham-operated rats (Fig. 7). Oral administration of OK-432 significantly reduced the incidence of bacterial translocation to the liver and spleen in CBDL rats (Table 3). The numbers of E. coli in the liver and spleen of OK-432-administered CBDL rats were decreased significantly compared with PBS-administered CBDL rats (Fig. 7). These results suggest that obstructive jaundice increases the translocation of bacteria, and
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FIG. 5. Chemiluminescence of Kupffer cells. Isolated Kupffer cells (5 ⫻ 10 6) from sham-operated (E) or common bile duct-ligated (CBDL, F) rats were cultured for 24 h in plastic tubes, then stimulated with zymosan opsonized with normal rat serum (A) or opsonized E. coli (B) for 30 min. Data are expressed as maximal counts (a) and total counts for 30 min (b). There were six animals in each group. Mean ⫾ SD. *P ⬍ 0.01 versus sham operation. †P ⬍ 0.01 versus sham operation.
oral administration of OK-432 could prevent bacterial translocation. DISCUSSION
Several studies have reported that the increased incidence of sepsis in obstructive jaundice might be due to depressed reticuloendothelial system (RES) function [3– 6]. The present study also showed a significant decrease in bacterial clearance from the circulating blood and decrease in E. coli uptake into the liver in jaundiced animals. Despite many reports suggesting RES dysfunction in obstructive jaundice, more specific information has been sparse regarding phagocytosis in jaundice. Ding et al. [4] examined in vivo phagocytic index, which is the slope of the semilogarithmic plot of radioactivity in
blood after intravenous injection of radiolabeled E. coli. They demonstrated that RES phagocytic function gradually decreases during the course of obstructive jaundice, and that longstanding biliary obstruction (3 weeks) causes a more marked depression of RES function. In this study, we focused on in vitro phagocytic function of Kupffer cells isolated from the liver. It is known that Kupffer cells account for about 80% of the cells in the RES [15]. Only a few studies have reported on the phagocytic function of isolated Kupffer cells. Sheen-Chen et al. suggested that the phagocytic activity of yeast in isolated Kupffer cells increased significantly 1 week after bile duct ligation [16]. In contrast, our data showed a significant decrease in the phagocytic ratio of E. coli by Kupffer cells 2 weeks after bile duct ligation. Although the differences in methods may influence the results, the exact reason remains to be determined. Furthermore, we examined the superoxide production of isolated Kupffer cells. It is known that superoxide production can be used to assess bacteria-killing activity. Sung et al. [17] reported that superoxide production by Kupffer cells isolated from biliary-obstructed rats was significantly lower than TABLE 3 Incidence of Bacterial Translocation a
FIG. 6. Hepatic blood flow in left and right lobes of liver. Hepatic tissue blood flow in sham-operated (䊐) and common bile duct-ligated (■) rats 2 weeks after operation was assessed by laser Doppler flowmetry. There were six animals in each group. Mean ⫾ SD. *P ⬍ 0.01.
Group
MLNs
Liver
Spleen
Sham operation ⫹ PBS CBDL ⫹ PBS CBDL ⫹ OK-432
1 (16.7%) 6 (100.0%) b 6 (100.0%) c
1 (16.7%) 6 (100.0%) b 3 (50.0%) c,d
0 (0.0%) 6 (100.0%) b 3 (50.0%) c,d
a Six animals in each group. Mean (%) MLNs, mesenteric lymph nodes; CBDL, common bile duct ligation. b P ⬍ 0.01 versus sham operation ⫹ PBS. c P ⬍ 0.01 versus sham operation ⫹ PBS. d P ⬍ 0.01 versus CBDL ⫹ PBS.
TOMIOKA, IINUMA, AND OKINAGA: KUPFFER CELL FUNCTION IN OBSTRUCTIVE JAUNDICE
FIG. 7. Number of E. coli translocated into the mesenteric lymph nodes (MLNs), liver, and spleen. The numbers of E. coli in the MLNs, liver, and spleen were compared in the PBS-administered shamoperated group (䊐), PBS administered common bile duct ligated (CBDL) group (■), and OK-432-administered CBDL group (o). There were six animals in each group. Mean ⫾ SD. *P ⬍ 0.05. †P ⬍ 0.01.
that in sham-operated rats, and that after resting for 2 h, a significant recovery in superoxide production occurred. In contrast, the present study showed that production of superoxide by Kupffer cells isolated from CBDL rats was significantly depressed compared with the sham-operated rats, even after resting for 24 h. Differences in the methods of isolating Kupffer cells may affect the recovery of function. These results suggest that RES dysfunction in biliary obstruction is due to impaired phagocytosis and killing activity of Kupffer cells. The role of hepatic blood flow in obstructive jaundice has not been fully investigated. Several studies showed that liver blood flow is reduced in obstructive jaundice using colloidal radiogold ( 108Au) or 133Xe [18, 19] to measure. Other studies reported a significant decrease in blood flow in the hepatic artery and the portal vein in obstructive jaundice using the electromagnetic flowmeter [20]. The present study used laser Doppler flowmetry to measure liver blood flow. Arvidsson et al. [21] reported that this method is a reliable, reproducible, and continuous method to estimate hepatic blood flow. Our results showed a significant decrease in hepatic blood flow in CBDL rats, suggesting that the decreased bacterial elimination from circulating blood may be partly due to decreased blood flow. It is important to clarify the source of the bacteria that cause systemic infection in obstructive jaundice. It is known that translocation of intestinal bacteria increases after bile duct ligation [5, 6]. The present study also demonstrated that bile duct ligation promotes translocation of E. coli to the MLNs, liver, and spleen. This finding supports the hypothesis that the source of bacterial infection in obstructive jaundice can originate in the gastrointestinal tract even without other in-
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fected sites. Bacterial translocation is defined as the passage of viable indigenous bacteria from the gastrointestinal tract to normally sterile extraintestinal sites [22]. Although this process involves disrupted gut microecology, bacterial overgrowth, impaired host immunity, and physical injury of the gut mucosa, the factors that promote translocation are not fully understood. It was reported that bile salts inhibit the growth of intestinal bacteria and contribute to the regulation of gut microflora [23]. Therefore, removal of intraluminal bile salts by ligation of the bile duct may cause a change in the endogenous bacterial flora, loss of mucosal integrity, and increased bacterial translocation to the MLNs and liver. Furthermore, inadequate RES control of portal bacteremia by impaired phgocytic function of Kupffer cells may result in bacterial “spillover” followed by subsequent systemic bacteremia. Various agents have been proposed for preventing bacterial translocation. In patients with total parenteral nutrition, the use of oral neomycin, fluoroquinolone, polymyxin B, and tobramycin was found effective in preventing bacterial translocation by regulating the intestinal flora [24]. Experimental trials to prevent bacterial translocation by enhancing the host immunological function have been studied. Hagiwara et al. [25] reported that the transfer of a macrophage-enriched fraction of spleen cells collected from mice vaccinated with formalin-killed Propionibacterium acnes prevented translocation of bacteria. Suzuki et al. [26] demonstrated that the oral administration of a culture condensate of Bifidobacterium longum prevents bacterial translocation from the gastrointestinal tract of mice. In this study, we demonstrated the preventive effect of oral administration of OK-432 on bacterial translocation. OK-432 is a heat- and penicillin G-treated lyophilized powder of the Su strain of Streptococcus pyogenes A which has been used clinically as an immunomodulator for treating patients with gastric cancer [7]. It is known that OK-432 activates lymphocytes and macrophages by inducing various cytokines such as IL-1, IL-2, and IL-12 [8, 9]. Therefore OK-432 may activate the lymphocytes and macrophages of gutassociated lymphoid tissue that regulate bacterial translocation. However, the exact mechanism must be determined in future studies. CONCLUSION
Increased susceptibility to infection in rats with obstructive jaundice is associated with impaired phagocytic function, the killing activity of Kupffer cells, and decreased hepatic blood flow. Furthermore, promoted bacterial translocation in obstructive jaundice may influence gut-associated systemic infection. Oral administration of OK-432 seems to reduce the incidence of bacterial translocation.
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ACKNOWLEDGMENTS We thank Dr. R. Fukushima for his helpful advice on bacterial translocation and Ms. J. Tamura and Ms. H. Kumagai for their excellent technical support.
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