- Email: [email protected]
Attempts to reveal the mechanism of CD95-ligand-mediated inflammation
Attempts to Reveal the Mechanism of CD95-LigandMediated Inflammation K. Seino, T. Ogino, K. Fukunaga, H. Taniguchi, Y. Takada, K. Yuzawa, M. Otsuka, H. Yagita, K. Okumura, and K. Fukao
C
D95 and the ligand for CD95 (CD95L) belong to the tumor necrosis factor (TNF)-receptor family and TNF family, respectively. CD95/CD95L interaction can induce apoptosis and has been described to regulate immune response.1 Recent studies have suggested that CD95L expression at the site of transplantation might be useful to help avoid rejection by inducing apoptotic death of CD95-expressing leukocytes.2,3 In contrast, we have demonstrated that CD95L expression can induce severe neutrophilic inflammation.4,5 In this series, we investigate the mechanism of CD95L-mediated inflammation and attempt to manipulate it.
subcutaneously into the mice. Tumor growth was assessed twice per week. For histologic studies, sections of the sites of tumor growth were stained with hematoxylin-eosin.
MATERIALS AND METHODS Animals
DISCUSSION
Six-week-old male AKR/J and BALB/c nude mice were purchased from SLC (Shizuoka, Japan).
RESULTS
Two million 5D5 cells were injected subcutaneously into syngeneic AKR/J and BALB/c nude mice. As Fig 1 shows, 2C11-treated 5D5 cells, which express CD95L, grew slower than untreated cells with neutrophil infiltration. The results indicate that CD95L, which was expressed through an endogeneous process but not by transfection, can induce neutrophilic inflammation when injected subcutaneously.
We found that tumor cells that were reinforced to express CD95L by transfection induced neutrophilic inflammation and rejection.5 However, other observations indicated that CD95L expression on cell surfaces, such as the testis and
Cells and In Vivo Evaluation of Tumor Growth T-cell hybridoma, 5D5, was kindly provided by Dr Shyr-Te Ju (Department of Medicine, Boston University School of Medicine). 5D5 cells were derived from lymph nodes of the lpr mouse and fused with BW5174 (AKR/J origin) cells as described previously.6 Some 5D5 cells were treated with plate-coated 2C11 (anti-CD3; Pharminogen, San Diego, Calif) monoclonal antibodies (MAb; 5 g/mL) for 48 hours, and then expressing CD95L.6 The cells were maintained in RPMI-1640 (Nissui, Tokyo) supplemented with 10% FCS, 2 mmol/L glutamine, 100 g/mL streptomycin, and 100 U/mL penicillin. Cells (2 ⫻ 106 cells) in 200 L of PBS were injected
From the Department of Surgery, University of Tsukuba School of Medicine, Tsukuba-City, Japan (K.S., K.F., H.T., Y.T., K.Y., M.O., K.F.); and Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan (T.O., H.Y., K.O.). 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 Dr K. Seino, Department of Surgery, University of Tsukuba School of Medicine, 1-1-1 Tennodai, Tsukuba-City, Ibaraki 305-8575, Japan.
Fig 1. Two million 5D5 cells with or without 2C11 treatment were injected subcutaneously into syngeneic AKR/J and BALB/c nude mice (five per group). Tumor growth was assessed twice per week. Data represent mean ⫾ standard error. Open circles: untreated; shaded circles: 2C11 treated. 0041-1345/99/$–see front matter PII S0041-1345(99)00219-5
© 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1942
Transplantation Proceedings, 31, 1942–1943 (1999)
LIGAND-MEDIATED INFLAMMATION
the eye, induced immune privilege. To exclude the possibility that the expression enforced by gene transfer changed the property of CD95L, we verified the CD95L-mediated inflammation by using 5D5 cells, which can endogeneously express CD95L upon stimulation. The present results indicate that not only transfected CD95L, but also endogenous CD95L, can act as a proinflammatory factor when injected subcutaneously. Recently, we found that the soluble form of CD95L shows a chemotactic activity toward neutrophils.7 CD95L can be released from the cell surface by matrix metalloproteinase and can assume a soluble form. In the context that soluble CD95L may induce inflammation, we examined the effect of a matrix metalloproteinase inhibitor upon CD95Lmediated inflammation (not shown). Furthermore, we tried to suppress the inflammation by coexpression of other cytokines, such as IL-10 (not shown). Data from these experiments will provide useful information in the study of
1943
clinical organ and cellular transplantation, including Langerhans islet transplantation. ACKNOWLEDGMENTS The authors thank Dr Shyr-Te Ju (Boston University School of Medicine) for providing the 5D5 cells.
REFERENCES 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. Yagita H, Seino K, Kayagaki N, et al: Nature 379:682, 1996 5. Seino K, Kayagaki N, Okumura K, et al: Nature Med 3:165, 1997 6. Cui H, El-Khatib M, Sherr DH, et al: Cell Immunol 167:302, 1996 7. Seino K, Iwabuchi K, Kayagaki N, et al: J Immunol 161:4484, 1998
C
D95 and the ligand for CD95 (CD95L) belong to the tumor necrosis factor (TNF)-receptor family and TNF family, respectively. CD95/CD95L interaction can induce apoptosis and has been described to regulate immune response.1 Recent studies have suggested that CD95L expression at the site of transplantation might be useful to help avoid rejection by inducing apoptotic death of CD95-expressing leukocytes.2,3 In contrast, we have demonstrated that CD95L expression can induce severe neutrophilic inflammation.4,5 In this series, we investigate the mechanism of CD95L-mediated inflammation and attempt to manipulate it.
subcutaneously into the mice. Tumor growth was assessed twice per week. For histologic studies, sections of the sites of tumor growth were stained with hematoxylin-eosin.
MATERIALS AND METHODS Animals
DISCUSSION
Six-week-old male AKR/J and BALB/c nude mice were purchased from SLC (Shizuoka, Japan).
RESULTS
Two million 5D5 cells were injected subcutaneously into syngeneic AKR/J and BALB/c nude mice. As Fig 1 shows, 2C11-treated 5D5 cells, which express CD95L, grew slower than untreated cells with neutrophil infiltration. The results indicate that CD95L, which was expressed through an endogeneous process but not by transfection, can induce neutrophilic inflammation when injected subcutaneously.
We found that tumor cells that were reinforced to express CD95L by transfection induced neutrophilic inflammation and rejection.5 However, other observations indicated that CD95L expression on cell surfaces, such as the testis and
Cells and In Vivo Evaluation of Tumor Growth T-cell hybridoma, 5D5, was kindly provided by Dr Shyr-Te Ju (Department of Medicine, Boston University School of Medicine). 5D5 cells were derived from lymph nodes of the lpr mouse and fused with BW5174 (AKR/J origin) cells as described previously.6 Some 5D5 cells were treated with plate-coated 2C11 (anti-CD3; Pharminogen, San Diego, Calif) monoclonal antibodies (MAb; 5 g/mL) for 48 hours, and then expressing CD95L.6 The cells were maintained in RPMI-1640 (Nissui, Tokyo) supplemented with 10% FCS, 2 mmol/L glutamine, 100 g/mL streptomycin, and 100 U/mL penicillin. Cells (2 ⫻ 106 cells) in 200 L of PBS were injected
From the Department of Surgery, University of Tsukuba School of Medicine, Tsukuba-City, Japan (K.S., K.F., H.T., Y.T., K.Y., M.O., K.F.); and Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan (T.O., H.Y., K.O.). 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 Dr K. Seino, Department of Surgery, University of Tsukuba School of Medicine, 1-1-1 Tennodai, Tsukuba-City, Ibaraki 305-8575, Japan.
Fig 1. Two million 5D5 cells with or without 2C11 treatment were injected subcutaneously into syngeneic AKR/J and BALB/c nude mice (five per group). Tumor growth was assessed twice per week. Data represent mean ⫾ standard error. Open circles: untreated; shaded circles: 2C11 treated. 0041-1345/99/$–see front matter PII S0041-1345(99)00219-5
© 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1942
Transplantation Proceedings, 31, 1942–1943 (1999)
LIGAND-MEDIATED INFLAMMATION
the eye, induced immune privilege. To exclude the possibility that the expression enforced by gene transfer changed the property of CD95L, we verified the CD95L-mediated inflammation by using 5D5 cells, which can endogeneously express CD95L upon stimulation. The present results indicate that not only transfected CD95L, but also endogenous CD95L, can act as a proinflammatory factor when injected subcutaneously. Recently, we found that the soluble form of CD95L shows a chemotactic activity toward neutrophils.7 CD95L can be released from the cell surface by matrix metalloproteinase and can assume a soluble form. In the context that soluble CD95L may induce inflammation, we examined the effect of a matrix metalloproteinase inhibitor upon CD95Lmediated inflammation (not shown). Furthermore, we tried to suppress the inflammation by coexpression of other cytokines, such as IL-10 (not shown). Data from these experiments will provide useful information in the study of
1943
clinical organ and cellular transplantation, including Langerhans islet transplantation. ACKNOWLEDGMENTS The authors thank Dr Shyr-Te Ju (Boston University School of Medicine) for providing the 5D5 cells.
REFERENCES 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. Yagita H, Seino K, Kayagaki N, et al: Nature 379:682, 1996 5. Seino K, Kayagaki N, Okumura K, et al: Nature Med 3:165, 1997 6. Cui H, El-Khatib M, Sherr DH, et al: Cell Immunol 167:302, 1996 7. Seino K, Iwabuchi K, Kayagaki N, et al: J Immunol 161:4484, 1998