- Email: [email protected]
Hepatic Stellate Cells in Biopsies From Liver Allografts With Acute Rejection
Hepatic Stellate Cells in Biopsies From Liver Allografts With Acute Rejection B. Bilezikçi, B. Demirhan, A. S¸ar, Z. Arat, H. Karakayalı, and M. Haberal ABSTRACT Backround. Hepatic stellate cells (HSCs) are nonparenchymal elements that play a major role in fibrogenesis due to various pathologies. HSCs are easily activated by certain injuries, which produce contraction and relaxation of HSCs, resulting in hepatic microcirculatory disturbances. The present study sought to analyze the expression of alpha-smooth muscle actin (alpha-SMA) positive HSCs in liver allografts during acute rejection episodes (ARE), determining whether it was related to the pathogenesis of this immune response. Materials and Methods. Using immunohistochemistry and a semiquantitative scoring system, the expression of alpha-SMA in HSCs was analyzed in liver allografts with ARE (group 1, n ⫽ 64) or without ARE (group 2, n ⫽ 20). Normal liver tissue from transplant donors (group 3, n ⫽ 53) served as the control materials. Results. Significantly more alpha-SMA positive HSCs were found in group 2 than in the other two groups (P ⬍ .05). The minimal difference observed between groups 1 and 3 was not statistically significant. As well, no statistical association was found between expression of alpha-SMA and the clinical parameters of age, gender, etiology of liver failure, donor type (partial or whole), posttransplantation period, and liver function tests. Conclusions. While these results represent preliminary findings, it may be possible that HSC expression is a protective mechanism during ARE in hepatic allograft patients. If this is true, enhanced expression of this protein may mitigate ARE in liver allograft patients.
A
CUTE REJECTION EPISODES (ARE) are clinically diagnosed based on a combination of clinical, immunological, and histological parameters, and usually confirmed by biopsy.1 Hepatic stellate cells (HSCs) are the principal collagen-producing elements in the liver; they are responsible for the fibrosis that characterizes chronic liver disease.2– 4 In normal liver tissue quiescent HSCs are inconspicuous, but, when activated, they are transformed into myofibroblast-like cells that produce alpha-smooth muscle actin (alpha-SMA). As a result, these cells are readily visualized with immunohistochemical stains.5 HSC activation has been studied in a number of animal models of liver injury, and in various human liver diseases.2,5 However, the process has not been studied extensively in biopsies of hepatic allografts with ARE. The purpose of this study was to examine the relation between HSC activation and the morphological components of ARE, including the portal tract, bile ducts, and venous endothelial inflammation. © 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 38, 589 –593 (2006)
MATERIALS AND METHODS Patients We studied 84 needle liver biopsy specimens from patients who had undergone orthotopic liver transplantation, including 64 specimens from allografts with ARE (group 1) and 20 without ARE (group 2). The 53 needle biopsy specimens obtained from liver donor candidates served as controls (group 3). Each of the 64 patient records was reviewed for age, sex, etiology of liver failure, liver enzyme levels when biopsies were performed, From the Departments of Pathology (B.B., B.D., A.S¸.), Nephrology (Z.A.), and General Surgery, Division of Transplantation (H.K., M.H.), Bas¸kent University, Faculty of Medicine, Ankara, Turkey. This study was supported by the research fund of Bas¸kent University, Faculty of Medicine, Ankara, Turkey. Address reprint requests to Banu Bilezikçi, MD, Department of Pathology, Bas¸kent University, Faculty of Medicine, 12. Sokak, 7/3, 06490 Ankara, Turkey. E-mail: [email protected] 0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2005.12.096 589
590 and general parameters related to transplantation, including type of surgery, findings on postoperative monitoring, and type of immunosuppressant therapy.
Histological Study Each liver biopsy specimen fixed in formalin and embedded in paraffin was serially sectioned at 3 to 4 m and mounted on slides. Sections were prepared with hematoxylin as well as eosin; Masson’s trichrome; Gomori’s reticulin stain to identify collagen fibers; as well as Perl’s method to identify iron. ARE was graded as absent, mild, moderate, or severe using the Banff classification for liver allograft rejection.6 This system involves an initial global assessment followed by three specific features scored semiquantitatively on a scale of 0 –3 (mild, moderate, severe): portal inflammation, bile duct inflammation/damage, and venular inflammation. The three scores were added together to arrive at a final value, the rejection activity index (RAI).
Immunohistochemical Evaluation Mounted sections from each specimen were also reacted with monoclonal mouse antibody for alpha-SMA (smooth muscle Ab-1, clone 1A4, NeoMarkers, Fremont, Calif, USA), and processed using the avidin-biotin-peroxidase method.7 We assessed alphaSMA positive cells in perisinusoidal spaces but not in portal tracts or fibrotic areas. The staining results were graded semiquantitatively as described by Schmitt-Graff et al1: 0 ⫽ no staining or occasional cells stained; 1 ⫽ minimal staining (stained HSCs occupying approximately 1% to 10% of the sinusoidal liver cell surface); 2 ⫽ more cells stained (stained HSCs occupying approximately 10% to 30% of the sinusoidal liver cell surface); 3 ⫽ extensive staining (stained HSCs occupying approximately 30% to 70% of the sinusoidal liver cell surface); and 4 ⫽ highly extensive staining (stained HSCs occupying more than 70% of the sinusoidal liver cell surface). Vascular smooth muscle cell immunoreactivity was used as an internal positive control.
BILEZIKÇI, DEMIRHAN, S¸AR ET AL
Histological Findings
The sections from the 53 biopsies obtained from donor candidates prior to transplantation (control group) showed either no abnormalities or mild, nonspecific lesions that featured mild evidences of sinusoidal cell hyperplasia, balloon cell changes, or lipofuscin pigment deposition. The biopsies from the 20 allografts without ARE (group 2) showed minimal cholestasis, minimal hepatocellular injury with regenerative changes. Among the sections from the 64 transplants with ARE (group 1), the RAI values ranged from 2 to 8; 37 cases were classified as mild, 26 as moderate, and 1 as severe ARE.
Immunohistochemical Findings
Normal livers (groups 2 and 3) showed few alpha-SMA positive cells in the perisinusoidal spaces as well as in the vascular walls of the portal tract and the central vein (Fig 1). The stained cells in the perisinusoidal spaces were starshaped with dendritic cytoplasmic processes (Fig 2). In the ARE cases, the number of alpha-SMA positive cells was increased in the perisinusoidal spaces (Fig 3). The reaction was significantly greater in group 2 versus group 1 (P ⬍ .05; Table 1).
Statistical Analysis The chi-square test and the Kruskal-Wallis test were used to analyze statistical relations among the groups with P values ⬍.05 considered significant.
RESULTS
The 60 male and 24 female patients had a mean age of 25.1 years. Their causes of liver failure were Wilson’s disease (n ⫽ 37), hepatitis B virus (n ⫽ 16), cryptogenic cirrhosis (n ⫽ 7), hepatitis C virus (n ⫽ 5), progressive familial intrahepatic cholestasis (n ⫽ 4), alcoholic cirrhosis (n ⫽ 3), Budd-Chiari syndrome (n ⫽ 2), biliary atresia (n ⫽ 2), Byler’s syndrome (n ⫽ 2), Crigler-Najjar syndrome type 1 (n ⫽ 2), Alagille syndrome (n ⫽ 1), primary sclerosing cholangitis (n ⫽ 1), toxic fulminant hepatic failure (n ⫽ 1), and congenital hepatic fibrosis (n ⫽ 1). Groups 1 and 2 showed similar results for all of the general transplantationrelated parameters: type of surgery, postoperative monitoring, and immunosuppressant regimens. No statistical differences were observed between the expression of alpha-SMA and the clinical parameters that were studied including age, sex, etiology, interval from transplantation to biopsy, and liver function tests.
Fig 1. In the normal livers, there were alpha-SMA positive cells in the vascular walls of the portal tract and the central vein.
HEPATIC STELLATE CELLS IN ARE OF ALLOGRAFTS
591
Fig 2. Alpha-SMA positive cells in the perisinusoidal spaces were star-shaped with dendritic cytoplasmic processes.
Fig 3. In the ARE cases, the number of alpha-SMA positive cells was increased in the perisinusoidal spaces.
DISCUSSION
actions relevant for tissue repair9 and have a proliferative capacity and regenerative potential for hepatocytes in patients with fulminant hepatic failure.10 Our results of increased activation in both group 1 and group 2 might be related to hepatic regeneration and extracellular matrix production in the posttransplantation period. One of our hypotheses was that this elevation might be influenced by transplantation type, either partial or whole. The present study did not show any statistical difference between groups 1 and 2 regarding transplantation type (Table 2).
HSCs, whose long cytoplasmic processes surround the sinusoids, are located within the space of Disse. Their main functions include reproduction of various extracellular matrix components, vitamin A storage, control of microvascular tone, and hepatic regeneration.7 A previous histopathological follow-up study in the liver allografts of 17 patients with recurrent viral hepatitis investigated HSC activation and cirrhotic evolution in biopsies performed 3 to 6 months and 10 to 15 months posttransplantation. This study showed that expansion of alpha-SMA positive HSCs was an early event in human viral hepatitis, occurring during the acute phase and related to a cirrhotic evolution. In the same study, no statistical differences were noted in the number of episodes of rejection among the groups.8 This description did not appear to match the abstract for this study, which described a study with 44 patients. Unlike the above study by Guido et al,8 in our study we were unable to show a relation between increased HSC activation and time of posttransplantation biopsy. It was expected that HSC activation was increased in transplantation cases compared with controls regardless of the presence or absence of ARE. Nevertheless, we found HSC activation to be increased in cases without ARE. We know that HSCs also respond to chemokines with biological
Table 1. Relation Between Grading of Actin Staining and Groups* Grading of Actin Staining
Group 1 (ARE)
Group 2 (Non-ARE)
Group 3 (Control)
0 1 2 3 4
26 (40.6%) 22 (34.4%) 6 (9.4%) 4 (6.3%) 6 (9.4%)
— 8 (40.0%) 5 (25.0%) 2 (10.0%) 5 (25.0%)
29 (54.7%) 18 (34.0%) 6 (11.3%) — —
Total
64 (100%)
20 (100%)
53 (100%)
*High-grade staining in the non-ARE group was more than in the ARE group. There were significantly more alpha-SMA positive HSCs in group 2 than in the other two groups (P ⬍ .05).
592
BILEZIKÇI, DEMIRHAN, S¸AR ET AL Table 2. Distribution of Donor Type, Partial or Whole According to Groups* Partial
Whole
Group 1 (ARE) Group 2 (non-ARE)
40 (72.7%) 15 (27.3%)
24 (82.8%) 5 (17.2%)
Total
55 (100%)
29 (100%)
*There was no association between stellate cell density and donor type (P ⬎ .05).
Allograft rejection occurs as a result of the genetic difference between the donor organ and the recipient immune system: an immunological response is produced as a result of the presence of foreign antigens. The major histocompatibility complex (MHC) and the major ABO blood group antigens are the most widely studied donor antigen-recipient antigen systems. Expression of MHC antigens on cell surfaces is a dynamic process influenced by many factors including infections, drug therapy, and lymphokines.11 Chemokine expression by stellate cells is regulated by soluble mediators, in particular pro-inflammatory cytokines, as well as growth factors, proteases, and products of oxidative stress. In addition, stellate cells also respond to chemokines with biological actions relevant for tissue repair, such as cell migration or induction of other chemokines.12 Our other hypothesis was that statistical differences between groups 1 and 2 regarding the density of HSCs may be related to a mediator which suppresses HSCs and is expressed during ARE. It is well known that the liver contains four different vasculatures: the portal vein, hepatic artery, liver sinusoid, and hepatic vein. The hepatic sinusoid is a specific capillary network system wherein a variety of metabolic substances are exchanged between the hepatic blood flow and hepatic parenchymal cells. The liver sinusoid includes four cell types: sinusoidal endothelial cells (SECs), Kupffer cells, stellate cells,13,14 and pit cells.13 SECs play several important roles in the physiology and pathology of the liver. Their structural characteristics, such as the membrane sieve and lack of basement membrane, facilitate direct contact of soluble and insoluble serum substances with hepatic parenchymal cells, resulting in enhanced hepatic metabolic activity. SECs are now regarded as a member of the scavenger endothelial cell family, which has the potential to eliminate a variety of macromolecules from the blood by receptor-mediated endocytosis.13,15 SECs have been shown to have an antigenpresenting function similar to dendritic cells. In cooperation with Kupffer cells and hepatic dendritic cells, SECs may participate in immunoregulatory functions in the liver. HSCs that lie in the spaces between parenchymal cells play pivotal roles in the regulation of homeostasis of retinoids in the whole body. As well, HSCs play key roles in liver regeneration. Extracellular matrix components can reversibly regulate morphology, proliferation, and functions of HSCs.13 As shown in previous studies, activated Kupffer cells secrete various cytokines, stimulating proliferation of HSCs.16,17 In
the absence of activated Kupffer cells and SECs, most HSC-stimulating factors are not available.16 Both activated Kupffer cells and HSCs represent an important blood clearance system for removal of macromolecules from the circulation in pigs.14 We hypothesized that the release of soluble stimulants by Kupffer cells, and the release of reactive oxygen intermediates, might have increased the HSCs17 in liver allograft biopsies. We also speculated that HSCs may play a role, shared with SECs and Kupffer cells, in the clearance of foreign antigens that occurs during allograft rejection. Further, they might express antigenpresenting cells which probably contribute to hepatic immune surveillance18 in the posttransplantation period. We know that activation and/or proliferation of HSCs does not necessarily coincide topographically with necroinflammatory lesions, an observation that supports the existence of soluble factors that are capable of modulating the function or phenotype of HSCs.8 The absence of statistical differences between severe portal inflammation and increased expression of HSCs seen in this study might be related to autocrine and paracrine effects of activated HSCs.5,8 In summary, the preliminary results of the present study on the role of HSCs in ARE of hepatic allografts showed increased activation in non-ARE patients. If further research proves that HSCs are a protective factor in ARE, activation of HSCs may be a valuable method to treat ARE in hepatic allograft patients. ACKNOWLEDGMENT The authors would like to thank Mrs. Emel Peker for her technical assistance.
REFERENCES 1. Snover DC: Pathology of liver transplantation. In Solez K, Racusen LC, Billingham ME (eds): Solid Organ Transplant Rejection. New York: Marcel Dekker; 1996, p 187 2. Graff AS, Krüger S, Bochard F, et al: Modulation of alpha smooth muscle actin and desmin expression in perisinusoidal cells of normal and diseased human livers. Am J Pathol 138:1233, 1991 3. Yamaoka K, Nouchi T, Marumo F, et al: ␣-Smooth muscle actin expression in normal and fibrotic human livers. Digest Dis Sci 38:1473, 1993 4. Gressner AM, Lotfi S, Gressner G, et al: Identification and partial characterization of a hepatocyte-derived factor promoting proliferation of cultured fat-storing cells (parasinusoidal lipocytes). Hepatology 16:1250, 1992 5. Washington K, Wright K, Shyr Y, et al: Hepatic stellate cell activation in nonalcoholic steatohepatitis and fatty liver. Hum Pathol 31:822, 2000 6. Demetris AJ, Batts KP, Dhillon AP, et al: Banff schema for grading liver allograft rejection: an international consensus document. Hepatology 25:658, 1997 7. MacSween RNM, Desmet VJ, Roskams T, et al: Developmental anatomy and normal structure. In MacSween RNM, Burt AD, Portmann BC, et al (eds): Pathology of the Liver. China: Elsevier Science; 2003, p 45 8. Guido M, Rugge M, Leandro G, et al: Hepatic stellate cell immunodetection and cirrhotic evolution of viral hepatitis in liver allografts. Hepatology 26:310, 1997
HEPATIC STELLATE CELLS IN ARE OF ALLOGRAFTS 9. Skalli O, Ropraz P, Trzeciak A, et al: A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol 103:2787, 1986 10. Theuerkauf I, Zhou H, Fischer HP: Immunohistochemical patterns of human liver sinusoids under different conditions of pathologic perfusion. Virchows Arch 348:498, 2001 11. Markin RS, Wisecarver JL, Radio SJ: Hepatic allograft pathology. In Kolbeck PC, Markin RS, McManus BM (eds): Transplant Pathology. Hong Kong: ASCP Press; 1994, p 241 12. Marra F: Chemokines in liver inflammation and fibrosis. Frontiers Biosci 7:1899, 2002 13. Enomoto K, Nishikawa Y, Omori Y, et al: Cell biology and pathology of liver sinusoidal endothelial cells. Med Electron Microsc 37:208, 2004
593 14. Senoo H: Structure and function of hepatic stellate cells. Med Electron Microsc 37:3, 2004 15. Nedredal GI, Elvevold KH, Ytrebo LM, et al: Liver sinusoidal endothelial cells represent an important blood clearance system in pigs. Comp Hepatol 2:1, 2003 16. Guido M, Rugge M, Chemello L, et al: Liver stellate cells in chronic viral hepatitis: the effect of interferon therapy. J Hepatol 24:301, 1996 17. Petrovic LM, Arkadopoulos N, Demetriou AA: Activation of hepatic stellate cells in liver tissue of patients with fulminant liver failure with bioartificial liver. Hum Pathol 32:1371, 2001 18. Knolle PA, Gerken G: Local control of the immune response in the liver. Immunol Rev 174:21, 2000
A
CUTE REJECTION EPISODES (ARE) are clinically diagnosed based on a combination of clinical, immunological, and histological parameters, and usually confirmed by biopsy.1 Hepatic stellate cells (HSCs) are the principal collagen-producing elements in the liver; they are responsible for the fibrosis that characterizes chronic liver disease.2– 4 In normal liver tissue quiescent HSCs are inconspicuous, but, when activated, they are transformed into myofibroblast-like cells that produce alpha-smooth muscle actin (alpha-SMA). As a result, these cells are readily visualized with immunohistochemical stains.5 HSC activation has been studied in a number of animal models of liver injury, and in various human liver diseases.2,5 However, the process has not been studied extensively in biopsies of hepatic allografts with ARE. The purpose of this study was to examine the relation between HSC activation and the morphological components of ARE, including the portal tract, bile ducts, and venous endothelial inflammation. © 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 38, 589 –593 (2006)
MATERIALS AND METHODS Patients We studied 84 needle liver biopsy specimens from patients who had undergone orthotopic liver transplantation, including 64 specimens from allografts with ARE (group 1) and 20 without ARE (group 2). The 53 needle biopsy specimens obtained from liver donor candidates served as controls (group 3). Each of the 64 patient records was reviewed for age, sex, etiology of liver failure, liver enzyme levels when biopsies were performed, From the Departments of Pathology (B.B., B.D., A.S¸.), Nephrology (Z.A.), and General Surgery, Division of Transplantation (H.K., M.H.), Bas¸kent University, Faculty of Medicine, Ankara, Turkey. This study was supported by the research fund of Bas¸kent University, Faculty of Medicine, Ankara, Turkey. Address reprint requests to Banu Bilezikçi, MD, Department of Pathology, Bas¸kent University, Faculty of Medicine, 12. Sokak, 7/3, 06490 Ankara, Turkey. E-mail: [email protected] 0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2005.12.096 589
590 and general parameters related to transplantation, including type of surgery, findings on postoperative monitoring, and type of immunosuppressant therapy.
Histological Study Each liver biopsy specimen fixed in formalin and embedded in paraffin was serially sectioned at 3 to 4 m and mounted on slides. Sections were prepared with hematoxylin as well as eosin; Masson’s trichrome; Gomori’s reticulin stain to identify collagen fibers; as well as Perl’s method to identify iron. ARE was graded as absent, mild, moderate, or severe using the Banff classification for liver allograft rejection.6 This system involves an initial global assessment followed by three specific features scored semiquantitatively on a scale of 0 –3 (mild, moderate, severe): portal inflammation, bile duct inflammation/damage, and venular inflammation. The three scores were added together to arrive at a final value, the rejection activity index (RAI).
Immunohistochemical Evaluation Mounted sections from each specimen were also reacted with monoclonal mouse antibody for alpha-SMA (smooth muscle Ab-1, clone 1A4, NeoMarkers, Fremont, Calif, USA), and processed using the avidin-biotin-peroxidase method.7 We assessed alphaSMA positive cells in perisinusoidal spaces but not in portal tracts or fibrotic areas. The staining results were graded semiquantitatively as described by Schmitt-Graff et al1: 0 ⫽ no staining or occasional cells stained; 1 ⫽ minimal staining (stained HSCs occupying approximately 1% to 10% of the sinusoidal liver cell surface); 2 ⫽ more cells stained (stained HSCs occupying approximately 10% to 30% of the sinusoidal liver cell surface); 3 ⫽ extensive staining (stained HSCs occupying approximately 30% to 70% of the sinusoidal liver cell surface); and 4 ⫽ highly extensive staining (stained HSCs occupying more than 70% of the sinusoidal liver cell surface). Vascular smooth muscle cell immunoreactivity was used as an internal positive control.
BILEZIKÇI, DEMIRHAN, S¸AR ET AL
Histological Findings
The sections from the 53 biopsies obtained from donor candidates prior to transplantation (control group) showed either no abnormalities or mild, nonspecific lesions that featured mild evidences of sinusoidal cell hyperplasia, balloon cell changes, or lipofuscin pigment deposition. The biopsies from the 20 allografts without ARE (group 2) showed minimal cholestasis, minimal hepatocellular injury with regenerative changes. Among the sections from the 64 transplants with ARE (group 1), the RAI values ranged from 2 to 8; 37 cases were classified as mild, 26 as moderate, and 1 as severe ARE.
Immunohistochemical Findings
Normal livers (groups 2 and 3) showed few alpha-SMA positive cells in the perisinusoidal spaces as well as in the vascular walls of the portal tract and the central vein (Fig 1). The stained cells in the perisinusoidal spaces were starshaped with dendritic cytoplasmic processes (Fig 2). In the ARE cases, the number of alpha-SMA positive cells was increased in the perisinusoidal spaces (Fig 3). The reaction was significantly greater in group 2 versus group 1 (P ⬍ .05; Table 1).
Statistical Analysis The chi-square test and the Kruskal-Wallis test were used to analyze statistical relations among the groups with P values ⬍.05 considered significant.
RESULTS
The 60 male and 24 female patients had a mean age of 25.1 years. Their causes of liver failure were Wilson’s disease (n ⫽ 37), hepatitis B virus (n ⫽ 16), cryptogenic cirrhosis (n ⫽ 7), hepatitis C virus (n ⫽ 5), progressive familial intrahepatic cholestasis (n ⫽ 4), alcoholic cirrhosis (n ⫽ 3), Budd-Chiari syndrome (n ⫽ 2), biliary atresia (n ⫽ 2), Byler’s syndrome (n ⫽ 2), Crigler-Najjar syndrome type 1 (n ⫽ 2), Alagille syndrome (n ⫽ 1), primary sclerosing cholangitis (n ⫽ 1), toxic fulminant hepatic failure (n ⫽ 1), and congenital hepatic fibrosis (n ⫽ 1). Groups 1 and 2 showed similar results for all of the general transplantationrelated parameters: type of surgery, postoperative monitoring, and immunosuppressant regimens. No statistical differences were observed between the expression of alpha-SMA and the clinical parameters that were studied including age, sex, etiology, interval from transplantation to biopsy, and liver function tests.
Fig 1. In the normal livers, there were alpha-SMA positive cells in the vascular walls of the portal tract and the central vein.
HEPATIC STELLATE CELLS IN ARE OF ALLOGRAFTS
591
Fig 2. Alpha-SMA positive cells in the perisinusoidal spaces were star-shaped with dendritic cytoplasmic processes.
Fig 3. In the ARE cases, the number of alpha-SMA positive cells was increased in the perisinusoidal spaces.
DISCUSSION
actions relevant for tissue repair9 and have a proliferative capacity and regenerative potential for hepatocytes in patients with fulminant hepatic failure.10 Our results of increased activation in both group 1 and group 2 might be related to hepatic regeneration and extracellular matrix production in the posttransplantation period. One of our hypotheses was that this elevation might be influenced by transplantation type, either partial or whole. The present study did not show any statistical difference between groups 1 and 2 regarding transplantation type (Table 2).
HSCs, whose long cytoplasmic processes surround the sinusoids, are located within the space of Disse. Their main functions include reproduction of various extracellular matrix components, vitamin A storage, control of microvascular tone, and hepatic regeneration.7 A previous histopathological follow-up study in the liver allografts of 17 patients with recurrent viral hepatitis investigated HSC activation and cirrhotic evolution in biopsies performed 3 to 6 months and 10 to 15 months posttransplantation. This study showed that expansion of alpha-SMA positive HSCs was an early event in human viral hepatitis, occurring during the acute phase and related to a cirrhotic evolution. In the same study, no statistical differences were noted in the number of episodes of rejection among the groups.8 This description did not appear to match the abstract for this study, which described a study with 44 patients. Unlike the above study by Guido et al,8 in our study we were unable to show a relation between increased HSC activation and time of posttransplantation biopsy. It was expected that HSC activation was increased in transplantation cases compared with controls regardless of the presence or absence of ARE. Nevertheless, we found HSC activation to be increased in cases without ARE. We know that HSCs also respond to chemokines with biological
Table 1. Relation Between Grading of Actin Staining and Groups* Grading of Actin Staining
Group 1 (ARE)
Group 2 (Non-ARE)
Group 3 (Control)
0 1 2 3 4
26 (40.6%) 22 (34.4%) 6 (9.4%) 4 (6.3%) 6 (9.4%)
— 8 (40.0%) 5 (25.0%) 2 (10.0%) 5 (25.0%)
29 (54.7%) 18 (34.0%) 6 (11.3%) — —
Total
64 (100%)
20 (100%)
53 (100%)
*High-grade staining in the non-ARE group was more than in the ARE group. There were significantly more alpha-SMA positive HSCs in group 2 than in the other two groups (P ⬍ .05).
592
BILEZIKÇI, DEMIRHAN, S¸AR ET AL Table 2. Distribution of Donor Type, Partial or Whole According to Groups* Partial
Whole
Group 1 (ARE) Group 2 (non-ARE)
40 (72.7%) 15 (27.3%)
24 (82.8%) 5 (17.2%)
Total
55 (100%)
29 (100%)
*There was no association between stellate cell density and donor type (P ⬎ .05).
Allograft rejection occurs as a result of the genetic difference between the donor organ and the recipient immune system: an immunological response is produced as a result of the presence of foreign antigens. The major histocompatibility complex (MHC) and the major ABO blood group antigens are the most widely studied donor antigen-recipient antigen systems. Expression of MHC antigens on cell surfaces is a dynamic process influenced by many factors including infections, drug therapy, and lymphokines.11 Chemokine expression by stellate cells is regulated by soluble mediators, in particular pro-inflammatory cytokines, as well as growth factors, proteases, and products of oxidative stress. In addition, stellate cells also respond to chemokines with biological actions relevant for tissue repair, such as cell migration or induction of other chemokines.12 Our other hypothesis was that statistical differences between groups 1 and 2 regarding the density of HSCs may be related to a mediator which suppresses HSCs and is expressed during ARE. It is well known that the liver contains four different vasculatures: the portal vein, hepatic artery, liver sinusoid, and hepatic vein. The hepatic sinusoid is a specific capillary network system wherein a variety of metabolic substances are exchanged between the hepatic blood flow and hepatic parenchymal cells. The liver sinusoid includes four cell types: sinusoidal endothelial cells (SECs), Kupffer cells, stellate cells,13,14 and pit cells.13 SECs play several important roles in the physiology and pathology of the liver. Their structural characteristics, such as the membrane sieve and lack of basement membrane, facilitate direct contact of soluble and insoluble serum substances with hepatic parenchymal cells, resulting in enhanced hepatic metabolic activity. SECs are now regarded as a member of the scavenger endothelial cell family, which has the potential to eliminate a variety of macromolecules from the blood by receptor-mediated endocytosis.13,15 SECs have been shown to have an antigenpresenting function similar to dendritic cells. In cooperation with Kupffer cells and hepatic dendritic cells, SECs may participate in immunoregulatory functions in the liver. HSCs that lie in the spaces between parenchymal cells play pivotal roles in the regulation of homeostasis of retinoids in the whole body. As well, HSCs play key roles in liver regeneration. Extracellular matrix components can reversibly regulate morphology, proliferation, and functions of HSCs.13 As shown in previous studies, activated Kupffer cells secrete various cytokines, stimulating proliferation of HSCs.16,17 In
the absence of activated Kupffer cells and SECs, most HSC-stimulating factors are not available.16 Both activated Kupffer cells and HSCs represent an important blood clearance system for removal of macromolecules from the circulation in pigs.14 We hypothesized that the release of soluble stimulants by Kupffer cells, and the release of reactive oxygen intermediates, might have increased the HSCs17 in liver allograft biopsies. We also speculated that HSCs may play a role, shared with SECs and Kupffer cells, in the clearance of foreign antigens that occurs during allograft rejection. Further, they might express antigenpresenting cells which probably contribute to hepatic immune surveillance18 in the posttransplantation period. We know that activation and/or proliferation of HSCs does not necessarily coincide topographically with necroinflammatory lesions, an observation that supports the existence of soluble factors that are capable of modulating the function or phenotype of HSCs.8 The absence of statistical differences between severe portal inflammation and increased expression of HSCs seen in this study might be related to autocrine and paracrine effects of activated HSCs.5,8 In summary, the preliminary results of the present study on the role of HSCs in ARE of hepatic allografts showed increased activation in non-ARE patients. If further research proves that HSCs are a protective factor in ARE, activation of HSCs may be a valuable method to treat ARE in hepatic allograft patients. ACKNOWLEDGMENT The authors would like to thank Mrs. Emel Peker for her technical assistance.
REFERENCES 1. Snover DC: Pathology of liver transplantation. In Solez K, Racusen LC, Billingham ME (eds): Solid Organ Transplant Rejection. New York: Marcel Dekker; 1996, p 187 2. Graff AS, Krüger S, Bochard F, et al: Modulation of alpha smooth muscle actin and desmin expression in perisinusoidal cells of normal and diseased human livers. Am J Pathol 138:1233, 1991 3. Yamaoka K, Nouchi T, Marumo F, et al: ␣-Smooth muscle actin expression in normal and fibrotic human livers. Digest Dis Sci 38:1473, 1993 4. Gressner AM, Lotfi S, Gressner G, et al: Identification and partial characterization of a hepatocyte-derived factor promoting proliferation of cultured fat-storing cells (parasinusoidal lipocytes). Hepatology 16:1250, 1992 5. Washington K, Wright K, Shyr Y, et al: Hepatic stellate cell activation in nonalcoholic steatohepatitis and fatty liver. Hum Pathol 31:822, 2000 6. Demetris AJ, Batts KP, Dhillon AP, et al: Banff schema for grading liver allograft rejection: an international consensus document. Hepatology 25:658, 1997 7. MacSween RNM, Desmet VJ, Roskams T, et al: Developmental anatomy and normal structure. In MacSween RNM, Burt AD, Portmann BC, et al (eds): Pathology of the Liver. China: Elsevier Science; 2003, p 45 8. Guido M, Rugge M, Leandro G, et al: Hepatic stellate cell immunodetection and cirrhotic evolution of viral hepatitis in liver allografts. Hepatology 26:310, 1997
HEPATIC STELLATE CELLS IN ARE OF ALLOGRAFTS 9. Skalli O, Ropraz P, Trzeciak A, et al: A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol 103:2787, 1986 10. Theuerkauf I, Zhou H, Fischer HP: Immunohistochemical patterns of human liver sinusoids under different conditions of pathologic perfusion. Virchows Arch 348:498, 2001 11. Markin RS, Wisecarver JL, Radio SJ: Hepatic allograft pathology. In Kolbeck PC, Markin RS, McManus BM (eds): Transplant Pathology. Hong Kong: ASCP Press; 1994, p 241 12. Marra F: Chemokines in liver inflammation and fibrosis. Frontiers Biosci 7:1899, 2002 13. Enomoto K, Nishikawa Y, Omori Y, et al: Cell biology and pathology of liver sinusoidal endothelial cells. Med Electron Microsc 37:208, 2004
593 14. Senoo H: Structure and function of hepatic stellate cells. Med Electron Microsc 37:3, 2004 15. Nedredal GI, Elvevold KH, Ytrebo LM, et al: Liver sinusoidal endothelial cells represent an important blood clearance system in pigs. Comp Hepatol 2:1, 2003 16. Guido M, Rugge M, Chemello L, et al: Liver stellate cells in chronic viral hepatitis: the effect of interferon therapy. J Hepatol 24:301, 1996 17. Petrovic LM, Arkadopoulos N, Demetriou AA: Activation of hepatic stellate cells in liver tissue of patients with fulminant liver failure with bioartificial liver. Hum Pathol 32:1371, 2001 18. Knolle PA, Gerken G: Local control of the immune response in the liver. Immunol Rev 174:21, 2000