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Inhibition of CD95 ligand-mediated inflammation
Inhibition of CD95 Ligand-Mediated Inflammation K. Seino, T. Tun, N. Ohshima, H. Hamada, K. Yoshino, S. Ikeda, K. Fukunaga, H. Taniguchi, Y. Takada, K. Yuzawa, M. Otsuka, T. Todoroki, and K. Fukao
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D95 (Fas/APO-1) is a type I integral membrane protein that transduces an apoptotic death signal.1 CD95 is expressed in hematopoietic cells and various tissues. CD95 ligand (CD95L/FasL/APO-1L) is a type II integral membrane protein that can induce apoptosis in CD95bearing cells.1 CD95/CD95L interaction has been described to regulate immune responses and induce some tissue damage.1 Recent studies have suggested a possibility that a graft expressing CD95L could evade the rejection by inducing apoptotic death of CD95-expressing leukocytes.2,3 In contrast, we have demonstrated that CD95L-transfected cells were rejected with severe neutrophil infiltration when subcutaneously transplanted into syngenic mice, due to proinflammatory function of CD95L.4 Furthermore, we have demonstrated that a soluble form of CD95L has a chemotactic activity against neutrophils.5 We investigated the way to inhibit the CD95L-induced inflammation by using an antiinflammatory cytokine and a matrix metalloproteinase (MMP) inhibitor.
MATERIALS AND METHODS Animals Six week-old male BALB/c, C3H/HeN (C3H) and DBA/2 mice, and 8 week-old male Sprague-Dawley (SD) rats were purchased from Japan Clea (Tokyo, Japan). BALB/c mice were made diabetic by a single intraperitoneal injection of 250 mg/kg of streptozotocin 2 weeks before the experiments. Diabetic mice were defined as
From the Department of Surgery, Institute of Clinical Medicine (K.S., K.F., H.T., Y.T., K.Y., M.O., T.T., K.F.) and the Department of Biomedical Engineering, Institute of Basic Medical Sciences (T.T., N.O.), University of Tsukuba; the Department of Molecular Biotherapy Research, Cancer Chemotherapy Center, Cancer Institute (H.H.); and Nippon Organon K.K. (K.Y., S.I.), Ibaraki, Japan. This work was supported by a Grant-in-Aid for Highly Advanced Medical Technology from the Ministry of Education, Science and Culture of Japan. Address reprint requests to K. Seino, MD, PhD, Department of Surgery, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba-City, Ibaraki 305-8575, Japan.
Fig 1. MH134 and CD95L/ MH134 cells (2 ⫻ 106) were subcutaneously injected into syngenic C3H and allogenic DBA/2 mice. Some mice were injected with cells coexpressed with IL10. Tumor growth was assessed twice per week.
0041-1345/00/$–see front matter PII S0041-1345(00)01548-7
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2038
Transplantation Proceedings, 32, 2038–2039 (2000)
INHIBITION OF CD95 LIGAND-MEDIATED INFLAMMATION
Fig 2. Survival of SD rat islets transplanted into BALB/c recipients. Some islets were transfected with CD95L and/or IL-10. those with two consecutive nonfasting blood glucose levels of more than 400 mg/dL.
Xenogenic Islet Transplantation Pancreatic islets were obtained from SD rats by the collagenase digestion method and were isolated by dextran gradient separation as described previously.6 In the control group, 500 free islets were implanted under the kidney capsule of diabetic BALB/c mice. In the study group, islets were transfected with CD95L and/or IL-10 by an adenovirus vector (provided from H.H.) (MOI ⫽ 25) and then transplanted under the kidney capsule. Blood samples were taken everyday to measure blood glucose levels. A graft was considered to have failed if two consecutive determinations of blood glucose levels were greater than 250 mg/dL.
Cells CD95-negative MH134 hepatoma derived from a C3H mouse was obtained from the Japanese Cancer Research Resources Bank (Setagaya, Tokyo). Mouse CD95L cDNA was transfected into MH134 cells as described previously.4 Some MH134 and CD95L/ MH134 cells were transfected with IL-10 cDNA by using an adenoviral vector (MOI ⫽ 10). These cells were maintained in RPMI 1640 (Nissui, Tokyo) supplemented with 10% FCS, 2 mmol/L of glutamine, 100 g/mL streptomycin, and 100 U/mL penicillin.
In Vivo Evaluation of Tumor Growth Cells (2 ⫻ 106) in 200 L phosphate buffered saline (PBS) were subcutaneously injected into mice (five per group). Tumor growth was assessed twice per week. Some mice were injected with 10 mg/kg of a MMP inhibitor (KB7785, provided from Nippon Organon K.K., Osaka).
2039
Fig 3. MH134 and CD95L/MH134 cells (2 ⫻ 106) were subcutaneously injected into syngenic C3H mice. Some mice were treated with 10 mg/kg of a matrix metalloproteinase inhibitor (KB7785) every day. Tumor growth was assessed twice per week.
ment was decreased by IL-10 coexpression (data not shown). We next examined the effect of IL-10 in CD95Linduced inflammation using a xenogenic islet transplantation model. As indicated in Fig 2, coexpression of IL-10 in CD95L-expressing rat pancreatic islets induced a slightly prolonged graft survival compared with no IL-10 coexpression, but the grafts were finally rejected. These results suggest that coexpression of antiinflammatory cytokines such as IL-10 may suppress CD95L-induced inflammation. Consistently, Nabel’s group recently reported that tumor growth factor- can inhibit neutrophil activation induced by CD95L.8 Thus, it is possible that expression of CD95L and some other cytokines together generate a microenvironment that promotes immunologic tolerance. However, the grafts were finally rejected in the allogenic and xenogenic models in this study. It may imply an uncertainty of this strategy. It has been reported that CD95L is processed by matrix metalloproteinases and becomes a soluble form9 that can be chemotactic against neutrophils.5 Then, we verified whether an inhibitor of MMP (KB7785) could prevent the FasL-mediated inflammation. In vitro, the same kind of MMP inhibitor accumulated membrane-bound CD95L on CD95L/MH134 cells (data not shown). In vivo, growth of CD95L/MH134 cells in the mice treated with KB7785 was faster than in the untreated controls. Histologic studies indicated that neutrophil recruitment was decreased by KB7785 treatment (data not shown). In this study, we demonstrated that coexpression of IL-10 as well as MMP inhibitor treatment may be effective in inhibiting CD95L-induced inflammation. REFERENCES
RESULTS AND DISCUSSION
We examined whether IL-10, one of the antiinflammatory cytokines, can inhibit the CD95L-induced inflammation because IL-10 has been reported to downregulate neutrophil functions.7 IL-10 was transfected into MH134 and CD95L/MH134 cells as described in Materials and Methods. After confirmation of the IL-10 expression by FACS analysis, the cells were subcutaneously injected into syngenic C3H and allogenic DBA/2 mice. When IL-10 was coexpressed, the CD95L/MH134 cells exhibited more progressive growth than IL-10-negative CD95L/MH134 cells (Fig 1). Histologic studies indicated that neutrophil recruit-
1. Nagata S, Golstein P: Science 267:1449, 1995 2. Bellgrau D, Gold D, Selawry H, et al: Nature 377:630, 1995 3. Lau HT, Yu M, Fontana A, et al: Science 273:109, 1996 4. Seino K, Kayagaki N, Okumura K, et al: Nature Med 3:165, 1997 5. Seino K, Iwabuchi K, Kayagaki N, et al: J Immunol 161:4484, 1998 6. Tun T, Inoue K, Hayashi H, et al: Cell Transplant 5:559, 1996 7. Zuany-Amorim C, Haile S, Leduc D, et al: J Clin Invest 95:2644, 1995 8. Chen JJ, Sun Y, Nabel GJ: Science 282:1714, 1998 9. Kayagaki N, Kawasaki A, Ebata T, et al: J Exp Med 182:1777, 1995
C
D95 (Fas/APO-1) is a type I integral membrane protein that transduces an apoptotic death signal.1 CD95 is expressed in hematopoietic cells and various tissues. CD95 ligand (CD95L/FasL/APO-1L) is a type II integral membrane protein that can induce apoptosis in CD95bearing cells.1 CD95/CD95L interaction has been described to regulate immune responses and induce some tissue damage.1 Recent studies have suggested a possibility that a graft expressing CD95L could evade the rejection by inducing apoptotic death of CD95-expressing leukocytes.2,3 In contrast, we have demonstrated that CD95L-transfected cells were rejected with severe neutrophil infiltration when subcutaneously transplanted into syngenic mice, due to proinflammatory function of CD95L.4 Furthermore, we have demonstrated that a soluble form of CD95L has a chemotactic activity against neutrophils.5 We investigated the way to inhibit the CD95L-induced inflammation by using an antiinflammatory cytokine and a matrix metalloproteinase (MMP) inhibitor.
MATERIALS AND METHODS Animals Six week-old male BALB/c, C3H/HeN (C3H) and DBA/2 mice, and 8 week-old male Sprague-Dawley (SD) rats were purchased from Japan Clea (Tokyo, Japan). BALB/c mice were made diabetic by a single intraperitoneal injection of 250 mg/kg of streptozotocin 2 weeks before the experiments. Diabetic mice were defined as
From the Department of Surgery, Institute of Clinical Medicine (K.S., K.F., H.T., Y.T., K.Y., M.O., T.T., K.F.) and the Department of Biomedical Engineering, Institute of Basic Medical Sciences (T.T., N.O.), University of Tsukuba; the Department of Molecular Biotherapy Research, Cancer Chemotherapy Center, Cancer Institute (H.H.); and Nippon Organon K.K. (K.Y., S.I.), Ibaraki, Japan. This work was supported by a Grant-in-Aid for Highly Advanced Medical Technology from the Ministry of Education, Science and Culture of Japan. Address reprint requests to K. Seino, MD, PhD, Department of Surgery, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba-City, Ibaraki 305-8575, Japan.
Fig 1. MH134 and CD95L/ MH134 cells (2 ⫻ 106) were subcutaneously injected into syngenic C3H and allogenic DBA/2 mice. Some mice were injected with cells coexpressed with IL10. Tumor growth was assessed twice per week.
0041-1345/00/$–see front matter PII S0041-1345(00)01548-7
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2038
Transplantation Proceedings, 32, 2038–2039 (2000)
INHIBITION OF CD95 LIGAND-MEDIATED INFLAMMATION
Fig 2. Survival of SD rat islets transplanted into BALB/c recipients. Some islets were transfected with CD95L and/or IL-10. those with two consecutive nonfasting blood glucose levels of more than 400 mg/dL.
Xenogenic Islet Transplantation Pancreatic islets were obtained from SD rats by the collagenase digestion method and were isolated by dextran gradient separation as described previously.6 In the control group, 500 free islets were implanted under the kidney capsule of diabetic BALB/c mice. In the study group, islets were transfected with CD95L and/or IL-10 by an adenovirus vector (provided from H.H.) (MOI ⫽ 25) and then transplanted under the kidney capsule. Blood samples were taken everyday to measure blood glucose levels. A graft was considered to have failed if two consecutive determinations of blood glucose levels were greater than 250 mg/dL.
Cells CD95-negative MH134 hepatoma derived from a C3H mouse was obtained from the Japanese Cancer Research Resources Bank (Setagaya, Tokyo). Mouse CD95L cDNA was transfected into MH134 cells as described previously.4 Some MH134 and CD95L/ MH134 cells were transfected with IL-10 cDNA by using an adenoviral vector (MOI ⫽ 10). These cells were maintained in RPMI 1640 (Nissui, Tokyo) supplemented with 10% FCS, 2 mmol/L of glutamine, 100 g/mL streptomycin, and 100 U/mL penicillin.
In Vivo Evaluation of Tumor Growth Cells (2 ⫻ 106) in 200 L phosphate buffered saline (PBS) were subcutaneously injected into mice (five per group). Tumor growth was assessed twice per week. Some mice were injected with 10 mg/kg of a MMP inhibitor (KB7785, provided from Nippon Organon K.K., Osaka).
2039
Fig 3. MH134 and CD95L/MH134 cells (2 ⫻ 106) were subcutaneously injected into syngenic C3H mice. Some mice were treated with 10 mg/kg of a matrix metalloproteinase inhibitor (KB7785) every day. Tumor growth was assessed twice per week.
ment was decreased by IL-10 coexpression (data not shown). We next examined the effect of IL-10 in CD95Linduced inflammation using a xenogenic islet transplantation model. As indicated in Fig 2, coexpression of IL-10 in CD95L-expressing rat pancreatic islets induced a slightly prolonged graft survival compared with no IL-10 coexpression, but the grafts were finally rejected. These results suggest that coexpression of antiinflammatory cytokines such as IL-10 may suppress CD95L-induced inflammation. Consistently, Nabel’s group recently reported that tumor growth factor- can inhibit neutrophil activation induced by CD95L.8 Thus, it is possible that expression of CD95L and some other cytokines together generate a microenvironment that promotes immunologic tolerance. However, the grafts were finally rejected in the allogenic and xenogenic models in this study. It may imply an uncertainty of this strategy. It has been reported that CD95L is processed by matrix metalloproteinases and becomes a soluble form9 that can be chemotactic against neutrophils.5 Then, we verified whether an inhibitor of MMP (KB7785) could prevent the FasL-mediated inflammation. In vitro, the same kind of MMP inhibitor accumulated membrane-bound CD95L on CD95L/MH134 cells (data not shown). In vivo, growth of CD95L/MH134 cells in the mice treated with KB7785 was faster than in the untreated controls. Histologic studies indicated that neutrophil recruitment was decreased by KB7785 treatment (data not shown). In this study, we demonstrated that coexpression of IL-10 as well as MMP inhibitor treatment may be effective in inhibiting CD95L-induced inflammation. REFERENCES
RESULTS AND DISCUSSION
We examined whether IL-10, one of the antiinflammatory cytokines, can inhibit the CD95L-induced inflammation because IL-10 has been reported to downregulate neutrophil functions.7 IL-10 was transfected into MH134 and CD95L/MH134 cells as described in Materials and Methods. After confirmation of the IL-10 expression by FACS analysis, the cells were subcutaneously injected into syngenic C3H and allogenic DBA/2 mice. When IL-10 was coexpressed, the CD95L/MH134 cells exhibited more progressive growth than IL-10-negative CD95L/MH134 cells (Fig 1). Histologic studies indicated that neutrophil recruit-
1. Nagata S, Golstein P: Science 267:1449, 1995 2. Bellgrau D, Gold D, Selawry H, et al: Nature 377:630, 1995 3. Lau HT, Yu M, Fontana A, et al: Science 273:109, 1996 4. Seino K, Kayagaki N, Okumura K, et al: Nature Med 3:165, 1997 5. Seino K, Iwabuchi K, Kayagaki N, et al: J Immunol 161:4484, 1998 6. Tun T, Inoue K, Hayashi H, et al: Cell Transplant 5:559, 1996 7. Zuany-Amorim C, Haile S, Leduc D, et al: J Clin Invest 95:2644, 1995 8. Chen JJ, Sun Y, Nabel GJ: Science 282:1714, 1998 9. Kayagaki N, Kawasaki A, Ebata T, et al: J Exp Med 182:1777, 1995