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Anti-rat IL-8 (CINC) monoclonal antibody administration reduces ischemia-reperfusion injury in small intestine
Anti-Rat IL-8 (CINC) Monoclonal Antibody Administration Reduces Ischemia-Reperfusion Injury in Small Intestine T. Tsuruma, A. Yagihashi, K. Tarumi, and K. Hirata
I
SCHEMIA-reperfusion injury in transplantation can cause primary nonfunction and is a matter similar in importance to rejection. Recent studies indicate that neutrophil infiltration plays an important role in ischemiareperfusion injury. We previously reported that there was a correlation between the degrees of mucosal damage and the peak level of IL-8 (CINC), known as a leukocyte chemoattractant,1 after the reperfusion,2 and that IL-8 (CINC) is a potential mediator of the ischemia-reperfusion injury in rat small intestine.2 Thus, we investigated whether anti-rat IL-8 (CINC) monoclonal antibody (mAb IL-8) administration reduces the ischemia-reperfusion injury. MATERIALS AND METHODS Male Lewis (LEW) rats weighing approximately 200 to 300 g were purchased from CLEA Japan Inc (Shizuoka, Japan). In each of the following experiments, five rats were used. All animals were anesthetized with an intraperitoneal injection of 35 mg/kg body weight of sodium pentobarbital. Then through a midline neck incision, using aseptic technique, the right common carotid artery was exposed and cannulated with polyethylene catheters (PE 50) for taking blood samples. The ischemia of the small intestine was induced by occlusion of the anterior mesenteric artery for 3 hours, and then it was subjected to the reperfusion. To investigate the protective effect of mAb IL-8 (CINC) (Rabbit anti-rat GRO/CINC-1 antibody, Immuno Biological Laboratories, Japan) on the ischemia-reperfusion injury, the rats were divided into two experimental groups. Group 1 was injected intraperitoneally with mAb IL-8 (200 mg/body), and group 2 was injected intraperitoneally with phosphate buffer saline (PBS) just before the reperfusion. Plasma tumor necrosis factor-alpha (TNF-a) and IL-8 (CINC) were measured by a two-step sandwich enzyme immunoassay (ELISA) method using a commercially available ELISA kit (IL-8, Immuno Biological Laboratories, Japan., TNF-a, Bio-source, USA) according to the manufacturer’s instruction. The detail was described previously.3 To assess the mucosal damages of small intestine, samples were collected and processed Table 1. The Mean Peak Levels of Plasma IL-8 (CINC) and TNF-a After Ischemia-Reperfusion
Group 1 Group 2
IL-8 (CINC) P , .05*
TNF-a P , .05*
440.2 6 49.4 pg/mL 588.6 6 46.1 pg/mL
8.3 6 7.68 pg/mL 44.4 6 28.02 pg/mL
Data were expressed as the mean 6 SE. *Mann Whitney’s U test.
Table 2. 24-Hour Survival Rates After Lethal Ischemia for 3 Hours Survival Rate
Group 1 Group 2
60% 40%
by routine techniques before embedding in paraffin. Sections were stained with hematoxilin and eosin.
RESULTS
In group 2, which was injected with PBS, the mean peak levels of plasma TNF-a and IL-8 (CINC) were 588.6 pg/mL and 44.4 pg/mL, respectively (Table 1). In group 1, which was injected with mAb IL-8 (CINC), the mean peak levels of these mediators (IL-8: 440.2 pg/mL, TNF-a: 8.3 pg/mL) were lower than those in group 2 (Table 2). The mucosal damage in group 2 was very severe : villous necrosis, inflammatory cell infiltration to mucosal layer, edema in proper muscle layer (Fig 1B), and crypt depth and villous height in group 2 were decreased compared with that in group 1 (Fig 1A, 1B). In addition, the 24-hour survival rate after lethal ischemia was also increased in group 1 (60%) in comparison with group 2 (40%). DISCUSSION
Ischemia-reperfusion injury is an inflammatory reaction, and its pathogenesis is complex and multifactorial.4 It is considered that reactive oxygen species are the major mediators of this injury.5,6 However, inflammatory cytokines, such as TNF-a, IL-1, IL-6, and IL-8, and neutrophils also have an important role in this injury. IL-8 is produced from many types of cells, including monocytes, macrophages, and endothelial cells, along with TNF-a and IL-1. In the organ subjected to ischemia-reperfusion, IL-8 (CINC) may attract neutrophils from the bloodstream into From the Departments of Surgery and Laboratory Diagnosis, Sapporo Medical University School of Medicine, Sapporo, Japan. Supported by a grant-in-aid for scientific research from the Ministry of Education, Culture, and Science of Japan. Address reprint requests to Tetsuhiro Tsuruma MD, Department of Surgery, Sapporo Medical University School of Medicine, S1, W16, Chuo-ku, Sapporo, 060, Japan.
0041-1345/98/$19.00 PII S0041-1345(98)00765-9
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2644
Transplantation Proceedings, 30, 2644–2645 (1998)
ANTI-RAT IL-8
2645
the injury sites, or locally produced IL-8 (CINC) may bind to the surface of endothelium and promote the migration of adherent neutrophils.7 The primed neutrophils may induce tissue damage and be associated with the development of ischemia-reperfusion injury. Moreover, this injury may induce lung injury as distant organ damage. Our study demonstrated that mAb IL-8 (CINC) administration decreased the peak levels of plasma IL-8 (CINC) and TNF-a. Myeloperoxidase activity, which represents neutrophil sequestration, in small intestine tissue in group 1 was also reduced in correlation with the peak level of IL-8 (CINC) (data not shown). Thus the administration of mAb IL-8 (CINC) reduced the infiltration of neutrophil and protected small intestine from ischemia-reperfusion injury. IL-8 (CINC) promotes tissue damage by inducing neutrophil infiltration as well as releasing lysosomal enzymes and superoxide anions by neutrophils. Therefore, blocking the action of IL-8 (CINC) by mAb IL-8 may be a beneficial treatment for ischemia-reperfusion injury in organ transplantation. REFERENCES
Fig 1. A: Group 1: Histologic findings in rat injected with mAb IL-8 (CINC) 12 hours after reperfusion. B: Group 2: Histologic findings in rat injected with PBS 12 hours after reperfusion.
1. Watanabe K, Konishi K, Fujioka M, et al: J Bio Chem 264:19559, 1989 2. Tsuruma T, Yagihashi A, Hirata K, et al: Transplant Proc 28:1917, 1996 3. Yagihashi A, Zou XM, Hirata K, et al: Transplant Proc 27:1632, 1995 4. Serizawa A, Nakamura S, Suzuki S, et al: Hepatology 23:1656, 1996 5. McCord JM: N Engl J Med 312:159, 1985 6. Granger DN: Am J Physiol 255:H1269, 1988 7. Sekido K, Mukaida N, Harada A, et al: Nature 365:654, 1993
I
SCHEMIA-reperfusion injury in transplantation can cause primary nonfunction and is a matter similar in importance to rejection. Recent studies indicate that neutrophil infiltration plays an important role in ischemiareperfusion injury. We previously reported that there was a correlation between the degrees of mucosal damage and the peak level of IL-8 (CINC), known as a leukocyte chemoattractant,1 after the reperfusion,2 and that IL-8 (CINC) is a potential mediator of the ischemia-reperfusion injury in rat small intestine.2 Thus, we investigated whether anti-rat IL-8 (CINC) monoclonal antibody (mAb IL-8) administration reduces the ischemia-reperfusion injury. MATERIALS AND METHODS Male Lewis (LEW) rats weighing approximately 200 to 300 g were purchased from CLEA Japan Inc (Shizuoka, Japan). In each of the following experiments, five rats were used. All animals were anesthetized with an intraperitoneal injection of 35 mg/kg body weight of sodium pentobarbital. Then through a midline neck incision, using aseptic technique, the right common carotid artery was exposed and cannulated with polyethylene catheters (PE 50) for taking blood samples. The ischemia of the small intestine was induced by occlusion of the anterior mesenteric artery for 3 hours, and then it was subjected to the reperfusion. To investigate the protective effect of mAb IL-8 (CINC) (Rabbit anti-rat GRO/CINC-1 antibody, Immuno Biological Laboratories, Japan) on the ischemia-reperfusion injury, the rats were divided into two experimental groups. Group 1 was injected intraperitoneally with mAb IL-8 (200 mg/body), and group 2 was injected intraperitoneally with phosphate buffer saline (PBS) just before the reperfusion. Plasma tumor necrosis factor-alpha (TNF-a) and IL-8 (CINC) were measured by a two-step sandwich enzyme immunoassay (ELISA) method using a commercially available ELISA kit (IL-8, Immuno Biological Laboratories, Japan., TNF-a, Bio-source, USA) according to the manufacturer’s instruction. The detail was described previously.3 To assess the mucosal damages of small intestine, samples were collected and processed Table 1. The Mean Peak Levels of Plasma IL-8 (CINC) and TNF-a After Ischemia-Reperfusion
Group 1 Group 2
IL-8 (CINC) P , .05*
TNF-a P , .05*
440.2 6 49.4 pg/mL 588.6 6 46.1 pg/mL
8.3 6 7.68 pg/mL 44.4 6 28.02 pg/mL
Data were expressed as the mean 6 SE. *Mann Whitney’s U test.
Table 2. 24-Hour Survival Rates After Lethal Ischemia for 3 Hours Survival Rate
Group 1 Group 2
60% 40%
by routine techniques before embedding in paraffin. Sections were stained with hematoxilin and eosin.
RESULTS
In group 2, which was injected with PBS, the mean peak levels of plasma TNF-a and IL-8 (CINC) were 588.6 pg/mL and 44.4 pg/mL, respectively (Table 1). In group 1, which was injected with mAb IL-8 (CINC), the mean peak levels of these mediators (IL-8: 440.2 pg/mL, TNF-a: 8.3 pg/mL) were lower than those in group 2 (Table 2). The mucosal damage in group 2 was very severe : villous necrosis, inflammatory cell infiltration to mucosal layer, edema in proper muscle layer (Fig 1B), and crypt depth and villous height in group 2 were decreased compared with that in group 1 (Fig 1A, 1B). In addition, the 24-hour survival rate after lethal ischemia was also increased in group 1 (60%) in comparison with group 2 (40%). DISCUSSION
Ischemia-reperfusion injury is an inflammatory reaction, and its pathogenesis is complex and multifactorial.4 It is considered that reactive oxygen species are the major mediators of this injury.5,6 However, inflammatory cytokines, such as TNF-a, IL-1, IL-6, and IL-8, and neutrophils also have an important role in this injury. IL-8 is produced from many types of cells, including monocytes, macrophages, and endothelial cells, along with TNF-a and IL-1. In the organ subjected to ischemia-reperfusion, IL-8 (CINC) may attract neutrophils from the bloodstream into From the Departments of Surgery and Laboratory Diagnosis, Sapporo Medical University School of Medicine, Sapporo, Japan. Supported by a grant-in-aid for scientific research from the Ministry of Education, Culture, and Science of Japan. Address reprint requests to Tetsuhiro Tsuruma MD, Department of Surgery, Sapporo Medical University School of Medicine, S1, W16, Chuo-ku, Sapporo, 060, Japan.
0041-1345/98/$19.00 PII S0041-1345(98)00765-9
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2644
Transplantation Proceedings, 30, 2644–2645 (1998)
ANTI-RAT IL-8
2645
the injury sites, or locally produced IL-8 (CINC) may bind to the surface of endothelium and promote the migration of adherent neutrophils.7 The primed neutrophils may induce tissue damage and be associated with the development of ischemia-reperfusion injury. Moreover, this injury may induce lung injury as distant organ damage. Our study demonstrated that mAb IL-8 (CINC) administration decreased the peak levels of plasma IL-8 (CINC) and TNF-a. Myeloperoxidase activity, which represents neutrophil sequestration, in small intestine tissue in group 1 was also reduced in correlation with the peak level of IL-8 (CINC) (data not shown). Thus the administration of mAb IL-8 (CINC) reduced the infiltration of neutrophil and protected small intestine from ischemia-reperfusion injury. IL-8 (CINC) promotes tissue damage by inducing neutrophil infiltration as well as releasing lysosomal enzymes and superoxide anions by neutrophils. Therefore, blocking the action of IL-8 (CINC) by mAb IL-8 may be a beneficial treatment for ischemia-reperfusion injury in organ transplantation. REFERENCES
Fig 1. A: Group 1: Histologic findings in rat injected with mAb IL-8 (CINC) 12 hours after reperfusion. B: Group 2: Histologic findings in rat injected with PBS 12 hours after reperfusion.
1. Watanabe K, Konishi K, Fujioka M, et al: J Bio Chem 264:19559, 1989 2. Tsuruma T, Yagihashi A, Hirata K, et al: Transplant Proc 28:1917, 1996 3. Yagihashi A, Zou XM, Hirata K, et al: Transplant Proc 27:1632, 1995 4. Serizawa A, Nakamura S, Suzuki S, et al: Hepatology 23:1656, 1996 5. McCord JM: N Engl J Med 312:159, 1985 6. Granger DN: Am J Physiol 255:H1269, 1988 7. Sekido K, Mukaida N, Harada A, et al: Nature 365:654, 1993