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Lack of human endothelial cell activation by adult porcine islets
Lack of Human Endothelial Cell Activation by Adult Porcine Islets C. Ehrnfelt and J. Holgersson
O
NLY transplantation of pancreatic tissue can cure type 1 diabetes. Due to the scarcity of human donor tissue, the use of animal, such as pig, donor tissue is considered a valid alternative. Nonvascularized xenografts, such as skin and pancreatic islets, are lost through a cell-mediated rejection shortly after transplantation.1 The events triggering the rejection is not yet fully elucidated, but T cells and macrophages are important effector cells in some models.2,3 Xenotransplanted islets have been shown to be revascularized with host-derived endothelial cells (ECs).4 This means that leukocytes recruited into the grafted islets have to transverse syngeneic ECs before they reach the graft. Recently, it was reported that resident macrophages within human islets produce IL-1, which inhibit -cell function and may contribute to the development of autoimmune diabetes.5 We have investigated whether inflammatory cytokines such as IL-1, IL-6, or TNF-␣ are produced in adult porcine islets (APIs) and, if so, can they activate human ECs? Such an activation would result in a potentiated recruitment of the recipient’s immune cells and subsequent graft rejection. In addition, we have performed cocultivation experiments to investigate if the APIs, through a direct contact or via other soluble factors, are able to activate human ECs in vitro.
(Transwell) preventing cell to cell contact but allowing free passage of soluble molecules. The cells were cocultivated for 6, 16, and 24 hours. mRNA was prepared from HAECs followed by a single-tube RT-PCR reaction, where E-selectin and IL-8 mRNA expression served as markers for EC activation.
RESULTS AND CONCLUSIONS
We detected mRNAs for IL-1 and TNF-␣ but not IL-6 in the APIs cultured for 2, 4, 8, and 11 days in vitro. These results indicate that cells within the islets, such as islet resident macrophages, are able to produce inflammatory cytokines, which, if secreted, may have an activating effect on the recipient’s endothelium. When culturing HAECs with porcine IL-1, IL-6, and TNF-␣, only porcine TNF-␣ caused an activation of HAECs as indicated by an upregulation of E-selectin and VCAM. However, when cocultivating APIs and HAECs, we could not detect an upregulation of E-selectin or IL-8. Thus, the levels of TNF-␣ secreted by the APIs or the number of APIs used in the cocultivation experiment may not be sufficient to cause an EC activation.
REFERENCES 1. Korsgren O: Xenotransplantation 4:11, 1997
MATERIALS AND METHODS Detection of Porcine Inflammatory Cytokines Using RT-PCR APIs were collected on days 2, 4, 8, and 11 after isolation. mRNA was prepared from the APIs followed by a single-tube RT-PCR to detect expression of porcine IL-1, IL-6, and TNF-␣.
Activation of Human Aortic Endothelial Cells (HAECs) by Porcine Inflammatory Cytokines HAECs were incubated with 40 ng/mL porcine IL-1, 8 ng/mL porcine IL-6, and 1000 units/mL porcine TNF-␣ for 22 hours at 37°C. The cells were stained with mouse anti-E-selectin and anti-VCAM antibodies and analysed by flow cytometry.
Activation of HAECs by Cocultivation APIs (2700 IEQ/cm2) and HAECs were cocultivated both in direct contact with each other and separated by a polycarbonate filter
0041-1345/01/$–see front matter PII S0041-1345(00)02170-9
2. Benda B, Sandberg J-O, Holstad M, et al: Transplantation 66:435, 1998 3. Wallgren A, Karlsson-Parra A, Korsgren O: Transplantation 60:594, 1995 4. Vajkoczy P, Olofsson AM, Lehr HA, et al: Am J Pathol 146:1397, 1995 5. Arnush M, Heitmeier MR, Scarim AL, et al: J Clin Invest 102:516, 1998
From the Division of Clinical Immunology F-79, Karolinska Institutet, Huddinge University Hospital Stockholm, Sweden. Supported by Juvenile Diabetes Foundation/Knut & Alice Wallenberg’s foundation and Swedish Medical Research Council, no. K99-06X-13031-01A. Address reprint requests to Cecilia Ehrnfelt, Division of Clinical Immunology F-79, Karolinska Institutet, Huddinge University Hospital AB, S-141 86 Stockholm, Sweden.
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
620
Transplantation Proceedings, 33, 620 (2001)
O
NLY transplantation of pancreatic tissue can cure type 1 diabetes. Due to the scarcity of human donor tissue, the use of animal, such as pig, donor tissue is considered a valid alternative. Nonvascularized xenografts, such as skin and pancreatic islets, are lost through a cell-mediated rejection shortly after transplantation.1 The events triggering the rejection is not yet fully elucidated, but T cells and macrophages are important effector cells in some models.2,3 Xenotransplanted islets have been shown to be revascularized with host-derived endothelial cells (ECs).4 This means that leukocytes recruited into the grafted islets have to transverse syngeneic ECs before they reach the graft. Recently, it was reported that resident macrophages within human islets produce IL-1, which inhibit -cell function and may contribute to the development of autoimmune diabetes.5 We have investigated whether inflammatory cytokines such as IL-1, IL-6, or TNF-␣ are produced in adult porcine islets (APIs) and, if so, can they activate human ECs? Such an activation would result in a potentiated recruitment of the recipient’s immune cells and subsequent graft rejection. In addition, we have performed cocultivation experiments to investigate if the APIs, through a direct contact or via other soluble factors, are able to activate human ECs in vitro.
(Transwell) preventing cell to cell contact but allowing free passage of soluble molecules. The cells were cocultivated for 6, 16, and 24 hours. mRNA was prepared from HAECs followed by a single-tube RT-PCR reaction, where E-selectin and IL-8 mRNA expression served as markers for EC activation.
RESULTS AND CONCLUSIONS
We detected mRNAs for IL-1 and TNF-␣ but not IL-6 in the APIs cultured for 2, 4, 8, and 11 days in vitro. These results indicate that cells within the islets, such as islet resident macrophages, are able to produce inflammatory cytokines, which, if secreted, may have an activating effect on the recipient’s endothelium. When culturing HAECs with porcine IL-1, IL-6, and TNF-␣, only porcine TNF-␣ caused an activation of HAECs as indicated by an upregulation of E-selectin and VCAM. However, when cocultivating APIs and HAECs, we could not detect an upregulation of E-selectin or IL-8. Thus, the levels of TNF-␣ secreted by the APIs or the number of APIs used in the cocultivation experiment may not be sufficient to cause an EC activation.
REFERENCES 1. Korsgren O: Xenotransplantation 4:11, 1997
MATERIALS AND METHODS Detection of Porcine Inflammatory Cytokines Using RT-PCR APIs were collected on days 2, 4, 8, and 11 after isolation. mRNA was prepared from the APIs followed by a single-tube RT-PCR to detect expression of porcine IL-1, IL-6, and TNF-␣.
Activation of Human Aortic Endothelial Cells (HAECs) by Porcine Inflammatory Cytokines HAECs were incubated with 40 ng/mL porcine IL-1, 8 ng/mL porcine IL-6, and 1000 units/mL porcine TNF-␣ for 22 hours at 37°C. The cells were stained with mouse anti-E-selectin and anti-VCAM antibodies and analysed by flow cytometry.
Activation of HAECs by Cocultivation APIs (2700 IEQ/cm2) and HAECs were cocultivated both in direct contact with each other and separated by a polycarbonate filter
0041-1345/01/$–see front matter PII S0041-1345(00)02170-9
2. Benda B, Sandberg J-O, Holstad M, et al: Transplantation 66:435, 1998 3. Wallgren A, Karlsson-Parra A, Korsgren O: Transplantation 60:594, 1995 4. Vajkoczy P, Olofsson AM, Lehr HA, et al: Am J Pathol 146:1397, 1995 5. Arnush M, Heitmeier MR, Scarim AL, et al: J Clin Invest 102:516, 1998
From the Division of Clinical Immunology F-79, Karolinska Institutet, Huddinge University Hospital Stockholm, Sweden. Supported by Juvenile Diabetes Foundation/Knut & Alice Wallenberg’s foundation and Swedish Medical Research Council, no. K99-06X-13031-01A. Address reprint requests to Cecilia Ehrnfelt, Division of Clinical Immunology F-79, Karolinska Institutet, Huddinge University Hospital AB, S-141 86 Stockholm, Sweden.
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
620
Transplantation Proceedings, 33, 620 (2001)