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Transplantation of hepatocytes: elimination of recipient natural killer cells with irradiation and bone marrow reconstitution prevent early graft dysfunction
Transplantation of Hepatocytes: Elimination of Recipient Natural Killer Cells With Irradiation and Bone Marrow Reconstitution Prevent Early Graft Dysfunction A. Wesolowska, W.L. Olszewski, and M. Durlik ABSTRACT Transplanted isolated syngeneic and allogeneic hepatocytes rapidly disintegrate, irrespective of the origin or the site of engraftment namely spleen, liver, portal vein, peritoneum, or subcutaneous tissues. Although various methods have been applied to attenuate this reaction, none have been found effective. We applied a combined protocol consisting of administration of anti-asialoGM1 antiserum (eliminating NK cells), sublethal whole-body irradiation, and reconstitution with syngeneic bone marrow cells to intrasplenic hepatocyte transplantation and 3 consecutive partial hepatectomies. This method overcame the early disintegration of grafted hepatocytes. Ninety days after transplantation numerous hepatocyte clusters and dilated bile canaliculae, occupying two thirds of the spleen, were observed, with some hepatocytes adhering to the bile ducts forming Hering’s canals. Mitotic figures were noticed. There were no recipient mononuclear infiltrates around the hepatocyte clusters.
T
RANSPLANTS of isolated syngeneic and allogeneic hepatocytes (HC) are rapidly disintegrated irrespective of origin or site of engraftment, namely, spleen, liver, portal vein, peritoneum, or subcutaneous tissue.1–10 Although various methods have been applied to attenuate this reaction,8,11,12 none has been found effective. In this study, we transplanted HC into the spleen of a recipient pretreated with intravenous administration of anti-asialoGM1 antiserum (AAGM1), sublethal irradiation (Whole-body irradiation [WBI]), reconstitution with syngeneic bone marrow cells (BMCtx), and partially hepatectomy (PH). The AAGM1 antiserum eliminates the natural killer (NK) cells. Irradiation decreases the number of circulating blood granulocytes and monocytes. BMCtx reconstitutes the depleted bone marrow as a source of stem cells, capable of repopulating spleen and PH stimulates HC proliferation.
against OX6 (MHC DR), ED1 (macrophages), and CD 161a (NKR-P1A) NK antigens were used, according to the manufacturers instruction.
RESULTS
MATERIAL AND METHODS
Fourteen days after transplantation clusters of 3 to 5 HC were seen in the spleen that had been largely depleted of its own mononuclear cells. Some HC formed short longitudinal lobules. In some areas they formed circular structures resembling bile canaliculae with a narrow lumen. Glycogen granules and CK 10 were easily identified in HC. At the site of HC engraftment, no OX6, ED1, or CD 161a cells were observed. These cells were equally distributed in the spleen parenchyma. Thirty days after transplantation more clusters of HC were observed; however, no glycogen was present in their cytoplasm. The spleen was repopulated with splenocytes, but no infiltrates were observed around the HC clusters. Numerous bile canaliculae were noticed.
Lewis (LEW) rats that were irradiated with 8 Gy on day 0 received 0.1 mg of AAGM1 intravenous on day 2. The animals were reconstituted with 107 syngeneic BMC on day 3 and grafted on the same day with 107 syngeneic HC into the spleen, which was exposed at laparotomy. At 14, 30, and 90 days after transplantation specimens were obtained for immunohistochemical staining with PAS staining for HC glycogen granules, monoclonal antibody OCH1E5 (HC), and cytokeratin 10 (CK 10). To identify mononuclear cells infiltrating the site of HC transplantation, monoclonal antibodies
From the Department of Surgical Research and Transplantology, Medical Research Center, Polish Academy of Sciences and Central Clinical Hospital, Ministry of Internal Affaires, Warsaw, Poland. Address reprint requests to Anna Wesolowska, Department of Surgical Research and Transplantology, Medical Research Center, Polish Academy of Sciences, 5 Pawinski Street, 02106 Warsaw, Poland. E-mail: [email protected]
0041-1345/03/$–see front matter doi:10.1016/S0041-1345(03)00780-2
© 2003 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2358
Transplantation Proceedings, 35, 2358 –2360 (2003)
TRANSPLANTATION OF HEPATOCYTES
Fig 1.
2359
Multiple islands of HC and bile canaliculae in spleen. Spleen deprived of lymphocytes, HE ⫻400.
At 90 days after transplantation, the spleen was pale-red colored and hard at the site of engraftment. Multiple, dilated bile canaliculae filled with proteinaceous content dominated the histopathological picture. There were many HC adjacent to the canaliculae (Fig 1). HC clusters contained 15 to 20 HC with a few mitotic figures. The HC did not store glycogen. DISCUSSION
Transplanted autologous and allogeneic HC (HCtx) are rapidly destroyed by recipient mechanisms of innate immunity. The cell implantation procedure creates a wound which leads to local mobilization and activation of granulocytes and macrophages, as well as to deposition of complement and coagulation factors. The scavenging phase of
wound healing is initiated. Transplanted cells become the victims of the wound healing process, being removed with wound debris. Only a few cells survive among the large number of HC that had been transplanted in suspension into tissues and body cavities.1–10 We postulated that isolated HC, with uncovered surface intercellular molecules, are recognized as “alien” by granulocytes and macrophages, and subsequently lysed. Elimination of these cells by sublethal irradiation or Endoxan treatment increases the survival of HCtx.11,12 However, 7 days after engraftment, no HC can be detected. On histological examination, we noticed accumulation of 3.2.3-positive NK cells around foci of subcutaneously implanted syngeneic HC.12 This finding prompted us to pretreat the recipient with AAGM1. It did not, however, significantly increase nor prolong HC survival
2360
rate. A lethal dose of irradiation and BMC transfusion prior to intrasplenic HCtx resulted in the formation of HC islands at 14 days after grafting. In the present study, we applied a combined protocol consisting of administration of AAGM1, sublethal WBI, reconstitution with syngeneic BMCtx, intrasplenic HCtx, and 3 consecutive PH. This method seemed to overcome the early disintegration of engrafted HC. Moreover, the transplanted cells proliferated and formed bile canaliculae. The AAGM1 and irradization eliminated the cytotoxic granulocytes, macrophages, and NK cells. Thus, there were no recipient mononuclear infiltrates around the HC clusters. Numerous HC clusters and dilated bile canaliculae as well as mitotic figures were observed. The surviving HC and presumably oval cells began to slowly differentiate and proliferate, forming liver unit structures. The effect of the transplanted BMC could be dual. BMC stem cells could lodge in the cell-depleted spleen, giving rise to oval cells, further differentiating to HC and bile epithelial cells,13 and the hematopoietic cytokines accompanying BM reconstitution in addition to other growth factors produced after PH may stimulate HC proliferation.
WESOLOWSKA, OLSZEWSKI, AND DURLIK
REFERENCES 1. Ando K, Guidotti LG, Wirth S, et al: J Immunol 152:3245, 1994 2. Ganey PE, Bailie MB, Van Cise S, et al: Lab Invest 70:53, 1994 3. Henne-Bomas D, Kruger V, Sumplemann D, et al: Virchows Arch 419:45, 1991 4. Jiang B, Sawa M, Yamamoto T, et al: Transplantation 63:131, 1997 5. Kocken JM, Bouwman E, Sinaasappel M, et al: Cell Transplant 5:32, 1996 6. Mito M, Kusano M: Cell Transplant 2:65, 1993 7. Olszewski WL, Jasklowska-Englisz M, Interewicz B: Transplant Proc 26:3369, 1994 8. Olszewski WL: Transplant Proc 29:1113, 1997 9. Onodera K, Ebata H, Sawa M: Transplant Proc 26:3006, 1992 10. Ross WK, von Geusau BA, Bouwman E, et al: Transplantation 63:513, 1997 11. Olszewski WL, Interewicz B, Durlik M, et al: Transplant Proc 33:651, 2001 12. Olszewski WL, Rudowska A, Mecner B, et al: Transplant Proc 34:705, 2002 13. Wanhg X, Moritini E, Al-Dhalimy M, et al: Am J Pathol 161:565, 2002
T
RANSPLANTS of isolated syngeneic and allogeneic hepatocytes (HC) are rapidly disintegrated irrespective of origin or site of engraftment, namely, spleen, liver, portal vein, peritoneum, or subcutaneous tissue.1–10 Although various methods have been applied to attenuate this reaction,8,11,12 none has been found effective. In this study, we transplanted HC into the spleen of a recipient pretreated with intravenous administration of anti-asialoGM1 antiserum (AAGM1), sublethal irradiation (Whole-body irradiation [WBI]), reconstitution with syngeneic bone marrow cells (BMCtx), and partially hepatectomy (PH). The AAGM1 antiserum eliminates the natural killer (NK) cells. Irradiation decreases the number of circulating blood granulocytes and monocytes. BMCtx reconstitutes the depleted bone marrow as a source of stem cells, capable of repopulating spleen and PH stimulates HC proliferation.
against OX6 (MHC DR), ED1 (macrophages), and CD 161a (NKR-P1A) NK antigens were used, according to the manufacturers instruction.
RESULTS
MATERIAL AND METHODS
Fourteen days after transplantation clusters of 3 to 5 HC were seen in the spleen that had been largely depleted of its own mononuclear cells. Some HC formed short longitudinal lobules. In some areas they formed circular structures resembling bile canaliculae with a narrow lumen. Glycogen granules and CK 10 were easily identified in HC. At the site of HC engraftment, no OX6, ED1, or CD 161a cells were observed. These cells were equally distributed in the spleen parenchyma. Thirty days after transplantation more clusters of HC were observed; however, no glycogen was present in their cytoplasm. The spleen was repopulated with splenocytes, but no infiltrates were observed around the HC clusters. Numerous bile canaliculae were noticed.
Lewis (LEW) rats that were irradiated with 8 Gy on day 0 received 0.1 mg of AAGM1 intravenous on day 2. The animals were reconstituted with 107 syngeneic BMC on day 3 and grafted on the same day with 107 syngeneic HC into the spleen, which was exposed at laparotomy. At 14, 30, and 90 days after transplantation specimens were obtained for immunohistochemical staining with PAS staining for HC glycogen granules, monoclonal antibody OCH1E5 (HC), and cytokeratin 10 (CK 10). To identify mononuclear cells infiltrating the site of HC transplantation, monoclonal antibodies
From the Department of Surgical Research and Transplantology, Medical Research Center, Polish Academy of Sciences and Central Clinical Hospital, Ministry of Internal Affaires, Warsaw, Poland. Address reprint requests to Anna Wesolowska, Department of Surgical Research and Transplantology, Medical Research Center, Polish Academy of Sciences, 5 Pawinski Street, 02106 Warsaw, Poland. E-mail: [email protected]
0041-1345/03/$–see front matter doi:10.1016/S0041-1345(03)00780-2
© 2003 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2358
Transplantation Proceedings, 35, 2358 –2360 (2003)
TRANSPLANTATION OF HEPATOCYTES
Fig 1.
2359
Multiple islands of HC and bile canaliculae in spleen. Spleen deprived of lymphocytes, HE ⫻400.
At 90 days after transplantation, the spleen was pale-red colored and hard at the site of engraftment. Multiple, dilated bile canaliculae filled with proteinaceous content dominated the histopathological picture. There were many HC adjacent to the canaliculae (Fig 1). HC clusters contained 15 to 20 HC with a few mitotic figures. The HC did not store glycogen. DISCUSSION
Transplanted autologous and allogeneic HC (HCtx) are rapidly destroyed by recipient mechanisms of innate immunity. The cell implantation procedure creates a wound which leads to local mobilization and activation of granulocytes and macrophages, as well as to deposition of complement and coagulation factors. The scavenging phase of
wound healing is initiated. Transplanted cells become the victims of the wound healing process, being removed with wound debris. Only a few cells survive among the large number of HC that had been transplanted in suspension into tissues and body cavities.1–10 We postulated that isolated HC, with uncovered surface intercellular molecules, are recognized as “alien” by granulocytes and macrophages, and subsequently lysed. Elimination of these cells by sublethal irradiation or Endoxan treatment increases the survival of HCtx.11,12 However, 7 days after engraftment, no HC can be detected. On histological examination, we noticed accumulation of 3.2.3-positive NK cells around foci of subcutaneously implanted syngeneic HC.12 This finding prompted us to pretreat the recipient with AAGM1. It did not, however, significantly increase nor prolong HC survival
2360
rate. A lethal dose of irradiation and BMC transfusion prior to intrasplenic HCtx resulted in the formation of HC islands at 14 days after grafting. In the present study, we applied a combined protocol consisting of administration of AAGM1, sublethal WBI, reconstitution with syngeneic BMCtx, intrasplenic HCtx, and 3 consecutive PH. This method seemed to overcome the early disintegration of engrafted HC. Moreover, the transplanted cells proliferated and formed bile canaliculae. The AAGM1 and irradization eliminated the cytotoxic granulocytes, macrophages, and NK cells. Thus, there were no recipient mononuclear infiltrates around the HC clusters. Numerous HC clusters and dilated bile canaliculae as well as mitotic figures were observed. The surviving HC and presumably oval cells began to slowly differentiate and proliferate, forming liver unit structures. The effect of the transplanted BMC could be dual. BMC stem cells could lodge in the cell-depleted spleen, giving rise to oval cells, further differentiating to HC and bile epithelial cells,13 and the hematopoietic cytokines accompanying BM reconstitution in addition to other growth factors produced after PH may stimulate HC proliferation.
WESOLOWSKA, OLSZEWSKI, AND DURLIK
REFERENCES 1. Ando K, Guidotti LG, Wirth S, et al: J Immunol 152:3245, 1994 2. Ganey PE, Bailie MB, Van Cise S, et al: Lab Invest 70:53, 1994 3. Henne-Bomas D, Kruger V, Sumplemann D, et al: Virchows Arch 419:45, 1991 4. Jiang B, Sawa M, Yamamoto T, et al: Transplantation 63:131, 1997 5. Kocken JM, Bouwman E, Sinaasappel M, et al: Cell Transplant 5:32, 1996 6. Mito M, Kusano M: Cell Transplant 2:65, 1993 7. Olszewski WL, Jasklowska-Englisz M, Interewicz B: Transplant Proc 26:3369, 1994 8. Olszewski WL: Transplant Proc 29:1113, 1997 9. Onodera K, Ebata H, Sawa M: Transplant Proc 26:3006, 1992 10. Ross WK, von Geusau BA, Bouwman E, et al: Transplantation 63:513, 1997 11. Olszewski WL, Interewicz B, Durlik M, et al: Transplant Proc 33:651, 2001 12. Olszewski WL, Rudowska A, Mecner B, et al: Transplant Proc 34:705, 2002 13. Wanhg X, Moritini E, Al-Dhalimy M, et al: Am J Pathol 161:565, 2002