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Transplantation of hepatocytes: Temporary elimination of scavenger cells prevents early loss of grafted cells
Transplantation of Hepatocytes: Temporary Elimination of Scavenger Cells Prevents Early Loss of Grafted Cells A. Wesolowska, W.L. Olszewski, and M. Durlik ABSTRACT Transplants of isolated syngeneic and allogeneic hepatocytes are rapidly disintegrated, irrespective of the site of engraftment; be it spleen, liver, portal vein, peritoneum, or subcutaneous tissue. Host scavenger cells are responsible for this reaction. We designed a method overcoming early disintegration of the grafted hepatocytes. It consisted of administration of anti-asialoGM1 antiserum eliminating natural killer cells; sublethal whole body irradiation; and reconstitution with syngeneic bone marrow cells, ligation of host bile duct, intrasplenic hepatocyte transplantation, and three consecutive partial hepatectomies. Six months after transplantation a glycogen-rich, trabeculae-forming, dividing hepatocytes, situated along strands of newly-formed fibrous tissue and numerous dilated blind bile cannaliculae were observed. There was evidently more bile canaliculae in hosts with ligated bile duct than nonligated controls. This is the first study showing fibrous tissue formed at the site of hepatocyte implantation, and stellate cells are presumably involved in this process.
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AT SYNGENEIC and allogeneic hepatocytes (HC) are disintegrated within hours after transplantation irrespective of the site of engraftment, be it spleen, liver, portal vein, peritoneum, or subcutaneous tissue.1–10 They are lysed and eliminated by ED1⫹ scavenger cells.9 We used various experimental methods to attenuate this reaction.7–10 In the previous study, we transplanted HC into spleen of a recipient pretreated with intravenous administration of anti-asialoGM1 antiserum to eliminate natural killer cells, sublethally irradiated, reconstituted with syngeneic bone marrow cells (BMCtx), and partially hepatectomized (PH).11 Irradiation decreased the number of circulating blood granulocytes and monocytes. BMCtx reconstituted the depleted bone marrow cavities and PH stimulated hepatocyte proliferation. This method overcame the early disintegration of grafted HC and 90 days after transplantation numerous HC and bile cannaliculae were observed. In this study, the protocol was enriched by ligation of recipient bile duct to stimulate formation of bile cannaliculae and the follow-up was prolonged to 6 months. MATERIALS AND METHODS Lewis (LEW) rats irradiated with 8Gy on day 0 received 0.1 mg IV of anti-asialoGM1 antiserum on day 2 and were reconstituted with 107 syngeneic BMC on day 3 and grafted on the same day 107 syngeneic HC into the spleen, which was exposed at laparotomy. Forty percent hepatectomy was repeated twice after 2 and 4 months. Six months 0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2004.12.160 260
after transplantation specimens were obtained for gross evaluation and immunohistochemical staining, with PAS staining method for HC glycogen granules, monoclonal antibody OCH1E5 (hepatocyte) and cytokeratin 10 (CK 10). To identify collagen deposition at the site of implanted HC, the trichrome staining method was used. To count the total number of HC in spleen, the entire spleen was transversely cut into 10-m-thick sequential sections and number of HC was counted on each section.
RESULTS
Six months after transplantation, the total number of HC in the spleen ranged between 5 to 7⫻105 cells. The spleen was pale-red colored and hard at the site of engraftment. On histology, multiple, short longitudinal HC lobules and dilated bile canaliculae filled with proteinaceous content dominated the picture. Two to four canaliculi were seen per 50 hepatocytes. There were many HC adjacent to the canaliculae. HC clusters contained 15 to 20 HC with a few mitotic figures. HC From the Department of Surgical Research and Transplantology, Medical Research Center, Polish Academy of Sciences (A.W., W.L.O., M.D.) and Central Clinical Hospital, Ministry of Internal Affaires (W.L.O., M.D.), Warsaw, Poland. Address reprint requests to Waldemar L. Olszewski, Department of Surgical Research and Transplantology, Medical Research Center, Polish Academy of Sciences, 5 Pawinski Street, 02106 Warsaw, Poland. E-mail: [email protected] © 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 37, 260 –261 (2005)
TRANSPLANTATION OF HEPATOCYTES AND SCAVENGER CELLS
contained glycogen granules. Many HC lost their rectangular shape, became disfigured, and squeezed between fibroblasts and deposits of acellular substance. Staining with trichrome revealed collagen deposits. There were no mononuclear infiltrates close to HC. DISCUSSION
Our previous studies have shown that transplanted autologous and allogeneic hepatocytes are rapidly destructed by the recipient in the mechanism of innate immunity.7–10 We postulated that the isolated HC, with uncovered surface intercellular molecules are recognized as “nonself” by granulocytes and macrophages and subsequently lysed. To prevent rapid elimination of HC, we applied a combined protocol consisting of administration of anti-asialoGM1 antiserum, sublethal whole body irradiation, reconstitution with syngeneic bone marrow cells, intrasplenic HCtx, and three consecutive partial hepatectomies.11 This method allowed us to overcome the early disintegration of engrafted HC. Moreover, the transplanted cells proliferated, and bile canaliculi were formed. The surviving HC and presumably oval cells began to slowly differentiate and proliferate, forming liver unit structures. Apperance of bile canaliculae prompted us to investigate whether there are humoral signals from the host liver regulating differentiation of cholangiocyte precursors. We added to our protocol ligation of the bile duct and observation of the late events at the site of HC implantation. The follow-up was extended to 6 months. Specimens harvested 6 months after transplantation revealed high numbers of glycogen-containing HC, regular HC trabeculae, and large numbers of bile canaliculi. The density of canaliculi was evidently higher than observed in rats without bile duct ligation. Interestingly, the diameter of their lumen was twice that of a hepatocyte. The canaliculae formed blind cyst-like structures, which is not the case in a normal liver. The questions that remains to be answered are whether the canaliculae were dilated by the accumulating acellular material and what was the chemistry of that material. Fibroblast and collagen accumulated along the HC trabeculae but not in other parts of spleen. The stellate
261
cells are responsible for proliferation of fibrous tissue in cirrhotic liver. We did not identify these cells in the present study. Taken together, glycogen-rich, trabeculae-forming, dividing HC, located along strands of fibrous tissue and numerous dilated blind bile cannaliculae were observed 6 months after HC transplantation. Ligation of host bile duct intensified formation of bile canaliculae. This is the first study showing that fibrous tissue formed at the site of HC implantation, and stellate cells are presumably involved in this process. REFERENCES 1. Ando K, Guidotti LG, Wirth S, et al: Class I-restricted cytotoxic T lymphocytes are directly cytopathic for their target cells in vivo. J Immunol 152:3245, 1994 2. Ganey PE, Bailie MB, Van Cise S, et al: Activated neutrophils from rat injured isolated hepatocytes. Lab Invest 70:53, 1994 3. Henne-Bomas D, Kruger V, Sumplemann D, et al: Peritoneal macrophages destroy transplanted hepatocytes. Virch Arch A Pathol Anat Histopatol 419:45, 1991 4. Jiang B, Sawa M, Yamamoto T, et al: Enhancement of proliferation of intrasplenically transplanted hepatocytes in cirrhotic rats by hepatic stimulatory substance. Transplantation 63: 131, 1997 5. Kocken JM, Bouwman E, Sinaasappel M, et al: Acute death after intraportal hepatocyte transplantation in an allogeneic rat strain combination: a possible role for complement activation. Cell Transplant 5:32, 1996 6. Michio M, Mitsuo K: Hepatocyte transplantation in man. Cell Transplant 2:65, 1993 7. Olszewski WL, Jasklowska - Englisz M, Interewicz B: Hepatocytes transplanted intravenously are rapidly destroyed by granulocytes. Transplant Proc 26:3369, 1994 8. Olszewski WL: Hepatocyte transplantation-granulocytes recognize surface of isolated autologous hepatocytes as non-self and destroy them. Transplant Proc 29:1113, 1997 9. Olszewski WL, Interewicz B, Durlik M, et al: Early loss of transplanted autologous hepatocytes-lysis by leukocytes in vivo and in vitro. Transplant Proc 33:651, 2001 10. Olszewski WL, Rudowska A, Mecner B: Autologous transplanted hepatocytes: Depletion of recipient leukocytes prevents in vivo lysis. Transplant Proc 34:705, 2002 11. Wesolowska A, Olszewski WL, Durlik M: Transplantation of hepatocytes: Elimination of recipient natural killer cells with irradiation and bone marrow reconstitution prevent early graft dysfunction. Transplant Proc 35:2358, 2003
R
AT SYNGENEIC and allogeneic hepatocytes (HC) are disintegrated within hours after transplantation irrespective of the site of engraftment, be it spleen, liver, portal vein, peritoneum, or subcutaneous tissue.1–10 They are lysed and eliminated by ED1⫹ scavenger cells.9 We used various experimental methods to attenuate this reaction.7–10 In the previous study, we transplanted HC into spleen of a recipient pretreated with intravenous administration of anti-asialoGM1 antiserum to eliminate natural killer cells, sublethally irradiated, reconstituted with syngeneic bone marrow cells (BMCtx), and partially hepatectomized (PH).11 Irradiation decreased the number of circulating blood granulocytes and monocytes. BMCtx reconstituted the depleted bone marrow cavities and PH stimulated hepatocyte proliferation. This method overcame the early disintegration of grafted HC and 90 days after transplantation numerous HC and bile cannaliculae were observed. In this study, the protocol was enriched by ligation of recipient bile duct to stimulate formation of bile cannaliculae and the follow-up was prolonged to 6 months. MATERIALS AND METHODS Lewis (LEW) rats irradiated with 8Gy on day 0 received 0.1 mg IV of anti-asialoGM1 antiserum on day 2 and were reconstituted with 107 syngeneic BMC on day 3 and grafted on the same day 107 syngeneic HC into the spleen, which was exposed at laparotomy. Forty percent hepatectomy was repeated twice after 2 and 4 months. Six months 0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2004.12.160 260
after transplantation specimens were obtained for gross evaluation and immunohistochemical staining, with PAS staining method for HC glycogen granules, monoclonal antibody OCH1E5 (hepatocyte) and cytokeratin 10 (CK 10). To identify collagen deposition at the site of implanted HC, the trichrome staining method was used. To count the total number of HC in spleen, the entire spleen was transversely cut into 10-m-thick sequential sections and number of HC was counted on each section.
RESULTS
Six months after transplantation, the total number of HC in the spleen ranged between 5 to 7⫻105 cells. The spleen was pale-red colored and hard at the site of engraftment. On histology, multiple, short longitudinal HC lobules and dilated bile canaliculae filled with proteinaceous content dominated the picture. Two to four canaliculi were seen per 50 hepatocytes. There were many HC adjacent to the canaliculae. HC clusters contained 15 to 20 HC with a few mitotic figures. HC From the Department of Surgical Research and Transplantology, Medical Research Center, Polish Academy of Sciences (A.W., W.L.O., M.D.) and Central Clinical Hospital, Ministry of Internal Affaires (W.L.O., M.D.), Warsaw, Poland. Address reprint requests to Waldemar L. Olszewski, Department of Surgical Research and Transplantology, Medical Research Center, Polish Academy of Sciences, 5 Pawinski Street, 02106 Warsaw, Poland. E-mail: [email protected] © 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 37, 260 –261 (2005)
TRANSPLANTATION OF HEPATOCYTES AND SCAVENGER CELLS
contained glycogen granules. Many HC lost their rectangular shape, became disfigured, and squeezed between fibroblasts and deposits of acellular substance. Staining with trichrome revealed collagen deposits. There were no mononuclear infiltrates close to HC. DISCUSSION
Our previous studies have shown that transplanted autologous and allogeneic hepatocytes are rapidly destructed by the recipient in the mechanism of innate immunity.7–10 We postulated that the isolated HC, with uncovered surface intercellular molecules are recognized as “nonself” by granulocytes and macrophages and subsequently lysed. To prevent rapid elimination of HC, we applied a combined protocol consisting of administration of anti-asialoGM1 antiserum, sublethal whole body irradiation, reconstitution with syngeneic bone marrow cells, intrasplenic HCtx, and three consecutive partial hepatectomies.11 This method allowed us to overcome the early disintegration of engrafted HC. Moreover, the transplanted cells proliferated, and bile canaliculi were formed. The surviving HC and presumably oval cells began to slowly differentiate and proliferate, forming liver unit structures. Apperance of bile canaliculae prompted us to investigate whether there are humoral signals from the host liver regulating differentiation of cholangiocyte precursors. We added to our protocol ligation of the bile duct and observation of the late events at the site of HC implantation. The follow-up was extended to 6 months. Specimens harvested 6 months after transplantation revealed high numbers of glycogen-containing HC, regular HC trabeculae, and large numbers of bile canaliculi. The density of canaliculi was evidently higher than observed in rats without bile duct ligation. Interestingly, the diameter of their lumen was twice that of a hepatocyte. The canaliculae formed blind cyst-like structures, which is not the case in a normal liver. The questions that remains to be answered are whether the canaliculae were dilated by the accumulating acellular material and what was the chemistry of that material. Fibroblast and collagen accumulated along the HC trabeculae but not in other parts of spleen. The stellate
261
cells are responsible for proliferation of fibrous tissue in cirrhotic liver. We did not identify these cells in the present study. Taken together, glycogen-rich, trabeculae-forming, dividing HC, located along strands of fibrous tissue and numerous dilated blind bile cannaliculae were observed 6 months after HC transplantation. Ligation of host bile duct intensified formation of bile canaliculae. This is the first study showing that fibrous tissue formed at the site of HC implantation, and stellate cells are presumably involved in this process. REFERENCES 1. Ando K, Guidotti LG, Wirth S, et al: Class I-restricted cytotoxic T lymphocytes are directly cytopathic for their target cells in vivo. J Immunol 152:3245, 1994 2. Ganey PE, Bailie MB, Van Cise S, et al: Activated neutrophils from rat injured isolated hepatocytes. Lab Invest 70:53, 1994 3. Henne-Bomas D, Kruger V, Sumplemann D, et al: Peritoneal macrophages destroy transplanted hepatocytes. Virch Arch A Pathol Anat Histopatol 419:45, 1991 4. Jiang B, Sawa M, Yamamoto T, et al: Enhancement of proliferation of intrasplenically transplanted hepatocytes in cirrhotic rats by hepatic stimulatory substance. Transplantation 63: 131, 1997 5. Kocken JM, Bouwman E, Sinaasappel M, et al: Acute death after intraportal hepatocyte transplantation in an allogeneic rat strain combination: a possible role for complement activation. Cell Transplant 5:32, 1996 6. Michio M, Mitsuo K: Hepatocyte transplantation in man. Cell Transplant 2:65, 1993 7. Olszewski WL, Jasklowska - Englisz M, Interewicz B: Hepatocytes transplanted intravenously are rapidly destroyed by granulocytes. Transplant Proc 26:3369, 1994 8. Olszewski WL: Hepatocyte transplantation-granulocytes recognize surface of isolated autologous hepatocytes as non-self and destroy them. Transplant Proc 29:1113, 1997 9. Olszewski WL, Interewicz B, Durlik M, et al: Early loss of transplanted autologous hepatocytes-lysis by leukocytes in vivo and in vitro. Transplant Proc 33:651, 2001 10. Olszewski WL, Rudowska A, Mecner B: Autologous transplanted hepatocytes: Depletion of recipient leukocytes prevents in vivo lysis. Transplant Proc 34:705, 2002 11. Wesolowska A, Olszewski WL, Durlik M: Transplantation of hepatocytes: Elimination of recipient natural killer cells with irradiation and bone marrow reconstitution prevent early graft dysfunction. Transplant Proc 35:2358, 2003