- Email: [email protected]
Effect of Cyclosporine on Expression of MIC in Human Hepatocytes
CELLULAR EFFECTS OF IMMUNOSUPPRESSANTS
Effect of Cyclosporine on Expression of MIC in Human Hepatocytes Y. He, S. Li, L. Feng, F. Cheng, Z. Ye, and Y. Li ABSTRACT Objectives. The human major histocompatibility complex class I chain-related antigen A, B (MICA, B) are believed to be “cell stress sensors,” which encode stress-inducible surface proteins that bind to NKG2D, an activating receptor of NK, alpha beta and gamma delta T cells. While cyclosporine (CsA) has improved patient and graft survival rates following solid organ transplantation, its clinical use is often limited by acute and chronic toxicity. Besides ischemia-reperfusion injury, which could up-regulate the expression of MIC, the toxicity of CsA may also be another violent stress factor. The purpose of this study was to investigate whether CsA can affect the expression of MIC. Methods. Various doses of CsA (5 or 20 g/mL) was added to cultured human hepatocytes for 1, 3, 5, 8, or 24 hours. The expression of MIC was measured by immunofluorescence using a laser scanning confocal microscope. Results. CsA (5 or 20 g/mL) upregulated MIC protein expression in hepatocytes at 1 hour, peaking at 3 hours, and persisting until 5 hours (P ⬍ .05), returning to normal levels after 8 to 24 hours (P ⬎ .05). Conclusion. This study reported that CsA up-regulated MIC expression on human hepatocytes. MIC, as a “cellular stress biomarker,” may play a significant role in activating NK, T, and B lymphocyte responses associated with transplantation. This study suggested that immunosuppressants produce deterioration in organ function in transplantation.
C
YCLOSPORINE (CsA) has been the cornerstone of immunosuppression for organ transplantation since its introduction into clinical practice. CsA has significantly improved patient and allograft survivals. However, the clinical use of CsA is often limited by its acute and chronic toxicity, which remain major problems. In addition to ischemia-reperfusion injury (IRI) and the immune response, the toxicity of CsA is another violent stress factor during transplantation. Previous studies have observed that hepatic IRI increased RAE-I and H60 (MICA, B homologues) mRNA levels in mouse liver. Furthermore, hypoxia/reoxygenation may upregulate expression of MICA, B in human hepatocytes. The human major histocompability complex class I chain-related genes (MICA and MICB) are located within the HLA class I region of chromosome 6. Their organiza-
tion, expression, and products differ considerably from classical HLA class I genes.1 The MIC have been deemed as “cell stress sensors,” which encode cellular stress-inducible surface proteins that bind to NKG2D, an activating receptor of NK, alpha beta, and gamma delta T cells. They could From the Lab of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, People’s Republic of China. Grant support: National Basic Research Program of China No. 2003CB515504, Natural Science Fund of China (NSFC) No. 30500486, Innovative Research Team in University, Ministry of Education. Address reprint requests to Youping Li, Lab of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, 610041, People’s Republic of China. E-mail: [email protected]
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.06.080
Transplantation Proceedings, 38, 2231–2233 (2006)
2231
2232
function as a bridge between the natural immune response and the acquired immune response.2 Based on the previous results, the present study explored whether CsA affected the expression of MIC, which could be a mechanism underlying CsA toxicity.
MATERIALS AND METHODS Cell and Cell Culture Normal human liver L-02 cells were cultured in RPMI1640 media (Gibco) supplemented with 10% fetal calf serum and incubated under standard air conditions. None of the cell cultures contained any antibiotics. Cells were studied under experimental conditions when they were approximately 80% confluent. CsA (Sandoz, Basel) was dissolved in absolute ethanol before being added to the cultured media. The final ethanol concentration for all concentrations of CsA and vehicle controls was 0.1%. The final CsA concentration in the culture media were 5 g/mL and 20 g/mL. Hepatocytes were incubated with CsA for 1, 3, 5, 8, or 24 hours. The absolute ethanol was the control.
Immunofluorescence Following incubation with CsA, the expressions of MIC proteins were detected by immunofluorescence with a rabbit-anti-human polyclonal antibody (diluted, 1:100; Santa Cruz Biotech Co; Santa Cruz, Calif, USA). A goat-anti-rabbit antibody conjugated with FITC was used as a second antibody. Finally, we observed the fluorescence and acquired pictures through laser scanning confocal microscopy (Nikon, TE300; Bio-Rad, Eclipse). The average fluorescence intensity was measured by an image analysis system (LaserPix, Bio-Rad).
Statistics Experimental variables were tested in triplicate cultures. One-way analysis of variance was used for statistical evaluation of data. The significance of differences in mean values ⫾ standard deviations of MIC protein levels in human hepatocyte-conditioned media from the CsA versus ethanol treatment groups was assessed by P ⬍ .05 considered to be significant.
HE, LI, FENG ET AL
RESULTS
In all experiments there were no significances between cell growth in the CsA treatment and the ethanol vehicle groups. All results were compared with the ethanol vehicle controls. CsA (5 or 20 g/mL) upregulated MIC protein expression in hepatocytes at 1 hour, peaking at 3 hours, and persisting until 5 hours (P ⬍ .05), when they returned to normal levels between 8 and 24 hours (P ⬎ .05). At the same time, ethanol alone did not change MIC protein on the surface of hepatocytes (Fig 1). DISCUSSION
Various stress reactions are involved in organ transplantation. The transplantation process is a violent and persistent stress, including IRI and toxicity of immunosuppressants. Many studies have shown that solid organ transplantation induces heat-shock protein expression. Reactivity to these stress proteins has been implicated in acute and chronic allograft rejection.3–5 Our previous studies demonstrated the up-regulated expression of another stress-inducible protein—MIC in relation to IRI. Furthermore, the present data showed that CsA induced a strong increase in MIC protein on the surface of human hepatocytes. The introduction of CsA into clinical practice in the early 1980s ushered in a new era in organ transplantation. CsA is a highly insoluble cyclic polypeptide consisting of 11 amino acids. For over two decades, CsA was adopted as the main immunosuppressive agent not only for renal but for all types of solid organ transplantation. However, CsA may be responsible for a number of side effects, such as nephrotoxicity, hypertension, dyslipidemia, hypertrichosis, and increased risk of cardiovascular events, of which the most relevant is nephrotoxicity.6,7 The activation of RAS and reactive oxygen metabolites may play a role in the nephrotoxicity of CsA,8,9 but the mechanisms underlying CsA toxicity in transplantation are not fully understood. Our data showed clues about this, reporting that CsA upregulated expression of MIC in human hepatocytes. MIC shows a close relationship with allograft rejection.10,11 MIC encodes a surface protein bound to NKG2D, which activates NK, alpha beta, and gamma delta T cells, functioning as a stress-inducible activator of the immune response. These immune reactions may aggravated cell and tissue injury. So, MIC, a “cellular stress biomarker,” may be another reason for CsA toxicity. REFERENCES
Fig 1. Intensity of immunofluorescence during different incubation times in two CsA treatment groups (5 and 20 g/mL) and the ethanol vehicle group. ⽧, CsA 5 g/mL; , CsA 20 g/mL; , ethanol control.
‘
1. Bahram S, Bresnahan M, Geraghty DE, et al: A second lineage of mammalian major histocompatibility complex class I genes. Proc Natl Acad Sci USA 91:6259, 1994 2. Bauer S, Groh V, Wu J, et al: Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285:727, 1999 3. Ogita K, Hopkinson K, Nakao M, et al: Stress responses in graft and native intestine after rat heterotopic small bowel transplantation. Transplantation 69:2273, 2000
CYCLOSPORINE AND EXPRESSION OF MIC 4. Flohe S, Speidel N, Flach R, et al: Expression of HSP 70 as a potential prognostic marker for acute rejection in human liver transplantation. Transpl Int 11:89, 1998 5. Granja C, Moliterno RA, Ferreira MS, et al: T-cell autoreactivity to Hsp in human transplantation may involve both proinflammatory and regulatory functions. Hum Immunol 65:124, 2004 6. Bennett WM, De Mattos A, Meyer MM, et al: Chronic cyclosporine nephropathy: the Achilles’ heel of immunosuppressive therapy. Kidney Int 50:1089, 1996 7. Shihab FS: Cyclosporine nephropathy. Pathophysiology and clinical impact. Semin Nephrol 16:536, 1996 8. Bantle JP, Nath KA, Sutherland DER, et al: Effects of cyclosporine on renin-angiotensin-aldosterone system and potas-
2233 sium excretion in renal transplant recepients. Arch Intern Med 145:505, 1985 9. Walker PD, Das C, Shnh SV: Cyclosporin A induced lipid peroxidation in renal cortical mitochondria. Kidney Int 29:311, 1986 10. Mizutani K, Terasaki P, Rosen A, et al: Serial ten-year follow-up of HLA and MICA antibody production prior to kidney graft failure. Am J Transplant 5:2265, 2005 11. Sumitran-Holgersson S, Wilczek HE, Holgersson J, et al: Identification of the nonclassical HLA molecules, mica, as targets for humoral immunity associated with irreversible rejection of kidney allografts. Transplantation 74:268, 2002
Effect of Cyclosporine on Expression of MIC in Human Hepatocytes Y. He, S. Li, L. Feng, F. Cheng, Z. Ye, and Y. Li ABSTRACT Objectives. The human major histocompatibility complex class I chain-related antigen A, B (MICA, B) are believed to be “cell stress sensors,” which encode stress-inducible surface proteins that bind to NKG2D, an activating receptor of NK, alpha beta and gamma delta T cells. While cyclosporine (CsA) has improved patient and graft survival rates following solid organ transplantation, its clinical use is often limited by acute and chronic toxicity. Besides ischemia-reperfusion injury, which could up-regulate the expression of MIC, the toxicity of CsA may also be another violent stress factor. The purpose of this study was to investigate whether CsA can affect the expression of MIC. Methods. Various doses of CsA (5 or 20 g/mL) was added to cultured human hepatocytes for 1, 3, 5, 8, or 24 hours. The expression of MIC was measured by immunofluorescence using a laser scanning confocal microscope. Results. CsA (5 or 20 g/mL) upregulated MIC protein expression in hepatocytes at 1 hour, peaking at 3 hours, and persisting until 5 hours (P ⬍ .05), returning to normal levels after 8 to 24 hours (P ⬎ .05). Conclusion. This study reported that CsA up-regulated MIC expression on human hepatocytes. MIC, as a “cellular stress biomarker,” may play a significant role in activating NK, T, and B lymphocyte responses associated with transplantation. This study suggested that immunosuppressants produce deterioration in organ function in transplantation.
C
YCLOSPORINE (CsA) has been the cornerstone of immunosuppression for organ transplantation since its introduction into clinical practice. CsA has significantly improved patient and allograft survivals. However, the clinical use of CsA is often limited by its acute and chronic toxicity, which remain major problems. In addition to ischemia-reperfusion injury (IRI) and the immune response, the toxicity of CsA is another violent stress factor during transplantation. Previous studies have observed that hepatic IRI increased RAE-I and H60 (MICA, B homologues) mRNA levels in mouse liver. Furthermore, hypoxia/reoxygenation may upregulate expression of MICA, B in human hepatocytes. The human major histocompability complex class I chain-related genes (MICA and MICB) are located within the HLA class I region of chromosome 6. Their organiza-
tion, expression, and products differ considerably from classical HLA class I genes.1 The MIC have been deemed as “cell stress sensors,” which encode cellular stress-inducible surface proteins that bind to NKG2D, an activating receptor of NK, alpha beta, and gamma delta T cells. They could From the Lab of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, People’s Republic of China. Grant support: National Basic Research Program of China No. 2003CB515504, Natural Science Fund of China (NSFC) No. 30500486, Innovative Research Team in University, Ministry of Education. Address reprint requests to Youping Li, Lab of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, 610041, People’s Republic of China. E-mail: [email protected]
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.06.080
Transplantation Proceedings, 38, 2231–2233 (2006)
2231
2232
function as a bridge between the natural immune response and the acquired immune response.2 Based on the previous results, the present study explored whether CsA affected the expression of MIC, which could be a mechanism underlying CsA toxicity.
MATERIALS AND METHODS Cell and Cell Culture Normal human liver L-02 cells were cultured in RPMI1640 media (Gibco) supplemented with 10% fetal calf serum and incubated under standard air conditions. None of the cell cultures contained any antibiotics. Cells were studied under experimental conditions when they were approximately 80% confluent. CsA (Sandoz, Basel) was dissolved in absolute ethanol before being added to the cultured media. The final ethanol concentration for all concentrations of CsA and vehicle controls was 0.1%. The final CsA concentration in the culture media were 5 g/mL and 20 g/mL. Hepatocytes were incubated with CsA for 1, 3, 5, 8, or 24 hours. The absolute ethanol was the control.
Immunofluorescence Following incubation with CsA, the expressions of MIC proteins were detected by immunofluorescence with a rabbit-anti-human polyclonal antibody (diluted, 1:100; Santa Cruz Biotech Co; Santa Cruz, Calif, USA). A goat-anti-rabbit antibody conjugated with FITC was used as a second antibody. Finally, we observed the fluorescence and acquired pictures through laser scanning confocal microscopy (Nikon, TE300; Bio-Rad, Eclipse). The average fluorescence intensity was measured by an image analysis system (LaserPix, Bio-Rad).
Statistics Experimental variables were tested in triplicate cultures. One-way analysis of variance was used for statistical evaluation of data. The significance of differences in mean values ⫾ standard deviations of MIC protein levels in human hepatocyte-conditioned media from the CsA versus ethanol treatment groups was assessed by P ⬍ .05 considered to be significant.
HE, LI, FENG ET AL
RESULTS
In all experiments there were no significances between cell growth in the CsA treatment and the ethanol vehicle groups. All results were compared with the ethanol vehicle controls. CsA (5 or 20 g/mL) upregulated MIC protein expression in hepatocytes at 1 hour, peaking at 3 hours, and persisting until 5 hours (P ⬍ .05), when they returned to normal levels between 8 and 24 hours (P ⬎ .05). At the same time, ethanol alone did not change MIC protein on the surface of hepatocytes (Fig 1). DISCUSSION
Various stress reactions are involved in organ transplantation. The transplantation process is a violent and persistent stress, including IRI and toxicity of immunosuppressants. Many studies have shown that solid organ transplantation induces heat-shock protein expression. Reactivity to these stress proteins has been implicated in acute and chronic allograft rejection.3–5 Our previous studies demonstrated the up-regulated expression of another stress-inducible protein—MIC in relation to IRI. Furthermore, the present data showed that CsA induced a strong increase in MIC protein on the surface of human hepatocytes. The introduction of CsA into clinical practice in the early 1980s ushered in a new era in organ transplantation. CsA is a highly insoluble cyclic polypeptide consisting of 11 amino acids. For over two decades, CsA was adopted as the main immunosuppressive agent not only for renal but for all types of solid organ transplantation. However, CsA may be responsible for a number of side effects, such as nephrotoxicity, hypertension, dyslipidemia, hypertrichosis, and increased risk of cardiovascular events, of which the most relevant is nephrotoxicity.6,7 The activation of RAS and reactive oxygen metabolites may play a role in the nephrotoxicity of CsA,8,9 but the mechanisms underlying CsA toxicity in transplantation are not fully understood. Our data showed clues about this, reporting that CsA upregulated expression of MIC in human hepatocytes. MIC shows a close relationship with allograft rejection.10,11 MIC encodes a surface protein bound to NKG2D, which activates NK, alpha beta, and gamma delta T cells, functioning as a stress-inducible activator of the immune response. These immune reactions may aggravated cell and tissue injury. So, MIC, a “cellular stress biomarker,” may be another reason for CsA toxicity. REFERENCES
Fig 1. Intensity of immunofluorescence during different incubation times in two CsA treatment groups (5 and 20 g/mL) and the ethanol vehicle group. ⽧, CsA 5 g/mL; , CsA 20 g/mL; , ethanol control.
‘
1. Bahram S, Bresnahan M, Geraghty DE, et al: A second lineage of mammalian major histocompatibility complex class I genes. Proc Natl Acad Sci USA 91:6259, 1994 2. Bauer S, Groh V, Wu J, et al: Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285:727, 1999 3. Ogita K, Hopkinson K, Nakao M, et al: Stress responses in graft and native intestine after rat heterotopic small bowel transplantation. Transplantation 69:2273, 2000
CYCLOSPORINE AND EXPRESSION OF MIC 4. Flohe S, Speidel N, Flach R, et al: Expression of HSP 70 as a potential prognostic marker for acute rejection in human liver transplantation. Transpl Int 11:89, 1998 5. Granja C, Moliterno RA, Ferreira MS, et al: T-cell autoreactivity to Hsp in human transplantation may involve both proinflammatory and regulatory functions. Hum Immunol 65:124, 2004 6. Bennett WM, De Mattos A, Meyer MM, et al: Chronic cyclosporine nephropathy: the Achilles’ heel of immunosuppressive therapy. Kidney Int 50:1089, 1996 7. Shihab FS: Cyclosporine nephropathy. Pathophysiology and clinical impact. Semin Nephrol 16:536, 1996 8. Bantle JP, Nath KA, Sutherland DER, et al: Effects of cyclosporine on renin-angiotensin-aldosterone system and potas-
2233 sium excretion in renal transplant recepients. Arch Intern Med 145:505, 1985 9. Walker PD, Das C, Shnh SV: Cyclosporin A induced lipid peroxidation in renal cortical mitochondria. Kidney Int 29:311, 1986 10. Mizutani K, Terasaki P, Rosen A, et al: Serial ten-year follow-up of HLA and MICA antibody production prior to kidney graft failure. Am J Transplant 5:2265, 2005 11. Sumitran-Holgersson S, Wilczek HE, Holgersson J, et al: Identification of the nonclassical HLA molecules, mica, as targets for humoral immunity associated with irreversible rejection of kidney allografts. Transplantation 74:268, 2002