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Plasma osteopontin levels in patients with fulminant hepatitis
Hepatology Research 29 (2004) 202–206
Short communication
Plasma osteopontin levels in patients with fulminant hepatitis Atsushi Matsui a , Satoshi Mochida a , Akihiko Ohno a , Sumiko Nagoshi a , Takanori Hirose b , Kenji Fujiwara a,∗ a
b
Division of Gastroenterology and Hepatology, Department of Internal Medicine, Saitama Medical School, 38 Morohongo, Moroyama-cho, Iruma-gun, Saitama 350-0495, Japan Department of Pathology, Saitama Medical School, 38 Morohongo, Moroyama-cho, Iruma-gun, Saitama 350-0495, Japan Received 9 March 2004; received in revised form 24 March 2004; accepted 25 March 2004 Available online 28 May 2004
Abstract Fulminant hepatitis is characterized by massive or submassive liver necrosis. Massive liver necrosis can be induced by activated macrophages infiltrating into the liver. Osteopontin, an extracellular matrix, is a secretory glycoprotein as well essential for Th1 immune response, contributing to macrophage activation and infiltration. To know the significance of osteopontin in the development of fulminant hepatitis, plasma osteopontin levels were measured in patients with fulminant hepatitis. The levels were significantly greater in patients with fulminant hepatitis than in those with acute or chronic hepatitis as well as healthy adults. Among patients with fulminant hepatitis except one in whom bacterial infection was complicated, plasma osteopontin levels were elevated especially in the patients who developed hepatic encephalopathy of grade II or more within 10 days of the disease onset, a clinical type characteristic of massive liver necrosis. Immunohistochemical examination revealed that osteopontin was stained in macrophages positive for CD68, a marker for macrophages, in necrotic areas of the liver in a patient with fulminant hepatitis. In conclusion, plasma osteopontin levels were elevated in patients with fulminant hepatitis, probably reflecting production of osteopontin in Kupffer cells and hepatic macrophages, which might be involved in the development of massive liver necrosis in fulminant hepatitis. © 2004 Elsevier B.V. All rights reserved. Keywords: Osteopontin; Fulminant hepatitis; Macrophage; Massive liver necrosis; Th1 immune response
1. Introduction Fulminant hepatic failure is the clinical syndrome presented by acute liver dysfunction developing within 4–8 weeks of the disease onset [1,2]. In Japan, fulminant hepatitis has been used in terminology for such acute liver failure, because its causes are mostly due to hepatitis viral infection. Thus, fulminant hepatitis is defined as acute liver failure in which prothrombin time shows less than 40% of normal value with hepatic encephalopathy of grade II or more appearing within 8 weeks of the disease onset, and classified into acute and subacute types depending on this time period to such hepatic encephalopathy, within 10 days and 11 days later, respectively, since the survival rate is remarkably different in both types [3]. Osteopontin is a secretory extracellular glycoprotein originally isolated from the bone, which contains an ∗ Corresponding author. Tel.: +81-49-276-1198; fax: +81-49-294-8404. E-mail address: [email protected] (K. Fujiwara).
1386-6346/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.hepres.2004.03.009
Arg-Gly-Asp amino acid sequence (RGD motif) [4]. This protein is expressed physiologically in the kidney and bone, and contributes to extracellular matrix formation and calcium deposition in these organs [5]. Also, it is reported that osteopontin can bind to ␣v3 integrin expressed on monocytes and macrophages through RGD motif [6], and promote migration of these cells both in vitro and in vivo [6,7]. In addition, osteopontin is now considered to act as a cytokine essential for Th1 immune response [8], suggesting that osteopontin may play a central role in activation and infiltration of macrophages in various organs. It has long been believed that osteopontin is not expressed in the liver of normal rats and mice [5]. We demonstrated that both mRNA and protein expressions of osteopontin were found abundantly in Kupffer cells, macrophages and stellate cells activated in the liver of rats given carbon tetrachloride (CCl4) or heat-killed Propionibacterium acnes (P. acnes) [9,10]. Recently, we have made the transgenic mice expressing abundant osteopontin in hepatocytes using a vector containing human serum amyloid P component (SAP) promoter, and found that cytotoxic T lymphocyte (CTL) infil-
A. Matsui et al. / Hepatology Research 29 (2004) 202–206
tration and Kupffer cell activation developed spontaneously in the liver of these mice [11]. From these observations, it seems that osteopontin expressed in activated Kupffer cells, macrophages and stellate cells as well as in hepatocytes may be responsible for activation and infiltration of macrophages in the liver in both autocrine and paracrine manners. Pathological features of fulminant hepatitis are characterized by massive or submassive necrosis with inflammatory cell infiltration in the liver [12]. Fulminant hepatitis is suggested to develop as a consequence of the imbalance between Th1 and Th2 immune responses, since the expression of IFN-␥, a representative Th1 cytokine, is up-regulated in hepatic as well as circulating lymphocytes in patients with fulminant hepatitis [13,14]. Stimulation of Th1 immune response can induce activation of Kupffer cells and macrophages to provoke massive liver necrosis through microcirculatory disturbance due to endothelial cell destruction and fibrin deposition in the hepatic sinusoids in rats [15,16]. Thus, it is possible that osteopontin can be involved in the development of massive liver necrosis in patients with fulminant hepatitis. In the present paper, plasma levels and hepatic production of osteopontin in patients with fulminant hepatitis were evaluated to know the significance of osteopontin in the pathogenesis of fulminant hepatitis.
203
2.3. Immunohistochemical examinations of osteopontin and CD68 Blocks of liver specimens were fixed in 20% formalin neutral buffer solution (Muto Pure Chemicals Ltd., Tokyo, Japan), and embedded in paraffin. Three-micrometer sequential sections of the liver block were deparaffinized, and the endogenous peroxidase activity was blocked in a solution containing 0.03% H2 O2 . The sections were treated with microwave heating for 5 min at 90 ◦ C in sodium citrate buffer, 10 mM at pH 6.0. Immunohistochemical staining was performed by indirect immunoperoxidase methods with the DAKO EnVision + System (Dako Corporation, Carpinteria, CA) using monoclonal antibodies against human osteopontin (Immuno-Biological Laboratories) and CD68 (Dako Corporation) as primary antibodies. Non-immune mouse IgG at the same concentrations was used as controls for the primary antibodies. The reaction product was developed by incubation in a solution of 0.25% diaminobenzidine tetrahydrochroride containing H2 O2 (Dako Corporation). Counter staining was done by hematoxylin.
3. Results 3.1. Demographic and clinical features of patients
2. Patients and methods 2.1. Patients and experiments Subjects were patients with fulminant hepatitis fulfilling the Japanese criteria and patients with acute liver diseases who admitted to Saitama Medical School Hospital between January 1995 and March 2000. Acute hepatitis was defined as acute liver disease with no encephalopathy or encephalopathy of grade I, and included acute exacerbation of hepatitis B virus carriers as well in the present study. In all the patients, blood was collected in a plastic tube containing EDTA before receiving therapies including blood transfusion, blood-products infusion, artificial liver support and administration of glucocorticoid and interferon. Plasma was separated from the blood, and stored at −80 ◦ C for measurement of osteopontin levels. Blood was also collected from healthy adults and chronic hepatitis patients positive for serum HCV-RNA, and the plasma was stored similarly as controls. Autopsy samples obtained from a patient with fulminant hepatitis of subacute type were subjected to immunohistochemical examination of osteopontin and CD68, a marker for macrophages including Kupffer cells, in the liver. 2.2. Measurement of plasma osteopontin levels Plasma osteopontin levels were measured using an ELISA kit (Immuno-Biological Laboratories, Gunma, Japan) according to the manufacturer’s instructions.
Nine patients with fulminant hepatitis and six patients with acute hepatitis were enrolled in the study. Among nine patients with fulminant hepatitis, three were of the acute type, and six of the subacute type. Demographic and clinical features of these patients were shown in Table 1. Male patients were more frequent in acute hepatitis than in fulminant hepatitis (P = 0.15), and patients were older in fulminant hepatitis than in acute hepatitis. The etiology of fulminant hepatitis was hepatitis A virus (HAV) infection in one, hepatitis B virus (HBV) infection in one, co-infection of hepatitis C virus (HCV) and TT virus (TTV) in one, and unknown in six, while the etiology of acute hepatitis was HAV infection in one, HBV infection in one, exacerbation of HBV carriers in two, and unknown in two. There were no differences in serum alanine aminotranferase (ALT) levels and total bilirubin concentrations between the patients with fulminant hepatitis and acute hepatitis. Although three of six patients with acute hepatitis showed prothrombin time less than 40% of normal value, the extent of its reduction was smaller in those patients than in the patients with fulminant hepatitis. Serum C-reactive protein (CRP) levels were less than 1.5 mg/dL in all the patients except one patient with fulminant hepatitis of subacute type showing the level at 7.66 mg/mL. Eleven healthy adults (eight males, three females) and 10 patients with chronic hepatitis (seven males, three females) positive for serum HCV-RNA were enrolled as controls. Ages of both groups (years, mean ± S.D.) were 49 ± 12 and 47 ± 14, respectively. Serum ALT levels were within
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Table 1 Demographic and clinical features of patients with fulminant hepatitis, and acute hepatitis Age
Sex
Diagnosis
Etiology
PT (%)
ALT (IU/L)
T.bil (mg/dL)
Alb (g/dL)
Plt (×104 /L)
WBC (/L)
CRP (mg/dL)
60 37 65 78 18 55 64 64 79 25 36 27 50 28 54
F F M F F F M F F M F M F M M
FH-A FH-A FH-A FH-S FH-S FH-S FH-S FH-S FH-S AH AH AH AH AH AH
HAV HBV Unknown HCV, TTV Unknown Unknown Unknown Unknown Unknown HAV HBV HBV carrier HBV carrier Unknown Unknown
22 9 14 35 11 33 33 15 7 42 50 35 24 53 31
2,035 5,310 2,276 88 395 490 2,418 447 1,854 6,790 939 2,619 9,400 572 348
9.2 6.3 13.8 14.4 20.2 14.8 11.4 29.0 9.7 3.6 9.3 8.8 3.6 23.8 17.8
3.2 3.4 3.4 2.3 3.2 3.4 3.2 2.3 3.4 3.6 3.2 4.3 4.1 3.4 3.3
27.6 10.2 11.1 5.5 43.2 24.0 8.7 18.7 24.6 7.8 11.9 13.0 11.0 17.0 15.0
10,720 6,160 4,960 5,560 1,150 7,100 6,170 9,800 11,500 2,970 7,560 5,760 4,260 6,770 9,240
0.46 1.48 1.40 7.66 0.31 0.52 1.42 1.21 0.78 1.48 1.02 1.16 1.48 0.92 0.14
F, female; M, male; FH-A, fulminant hepatitis of acute type; FH-S, fulminant hepatitis of subacute type; AH, acute hepatitis; HAV, hepatitis A virus; HBV, hepatitis B virus; HCV, hepatitis C virus; TTV, TT virus; PT, prothrombin time; ALT, alanine aminotransferase; T.bil, total bilirubin; Alb, albumin; Plt, platelet count; WBC, white blood cell count; CRP, C-reactive protein.
normal range in healthy adults, and 148 ± 100 (IU/L, mean ± S.D.) in patients with chronic hepatitis. Serum ALT levels were significantly lower in patients with chronic hepatitis than in those with fulminant hepatitis and acute hepatitis. 3.2. Plasma osteopontin levels As shown in Fig. 1, plasma osteopontin levels (ng/mL, mean ± S.D.) in patients with fulminant hepatitis (3260 ± 3030) and acute hepatitis (690±256) were higher than those in patients with chronic hepatitis (436 ± 385) as well as healthy adults (239 ± 76). The levels in all the patients with
fulminant hepatitis were higher than those in patients with acute hepatitis. There was a significant correlation between plasma osteopontin levels and prothrombin time in patients with fulminant hepatitis and acute hepatitis. However, such correlations were not seen regarding serum levels of ALT, total bilirubin, albumin and CRP, and peripheral platelet or white blood cell counts (Table 1, Fig. 1). The levels were especially elevated in patients with fulminant hepatitis of acute type showing the levels greater than 3,000 ng/mL, while the levels in patients with fulminant hepatitis of subacute type were less than 2000 ng/mL except one patient in whom serum CRP level was increased to 7.66 mg/mL due to bacterial infection (Fig. 1). The mean level of plasma osteopontin in the other five patients with fulminant hepatitis of subacute type was higher than that of patients with acute hepatitis, but the difference was not statistically significant (Fig. 1). There was no difference in plasma osteopontin levels between patients with chronic hepatitis and healthy adults. The mean (±S.D.) of plasma osteopontin levels was 505 (±435) ng/mL in patients with chronic hepatitis whose serum ALT levels were higher than 100 IU/L, but 275 (±213) ng/mL in the other chronic hepatitis patients (P = 0.42). 3.3. Immunohistochemical examinations of osteopontin and CD68
Fig. 1. Plasma osteopontin levels in patients with fulminant hepatitis (FH), acute hepatitis (AH), chronic hepatitis (CH), and in healthy adults. FH-S, FH of subacute type: FH-A, FH of acute type. Bars are mean levels of each disease group. In FH-S, the mean level of five patients in the column is presented. # P < 0.05 vs. AH, $ P < 0.05 vs. FH, P < 0.05 by Mann–Whitney U-test. & The patient in whom serum C-reactive protein level was 7.66 mg/mL.
Immunohistochemical examination of the liver was done with autopsy samples obtained from a patient with fulminant hepatitis of subacute type whose plasma osteopontin level was 1,050 ng/mL. Marked infiltration of mononuclear cells positive for CD68 was found in necrotic areas of the liver (Fig. 2a). Immunohistochemical examination using sequential sections revealed that osteopontin was stained markedly in the cytoplasm of these cells (Fig. 2b).
A. Matsui et al. / Hepatology Research 29 (2004) 202–206
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Fig. 2. Immunostaining of CD68 and osteopontin in the liver of a patient with fulminant hepatitis of subacute type, (a) CD68 staining. (b) Osteopontin staining. Marked infiltration of mononuclear cells positive for CD68, a marker for hepatic macrophages and Kupffer cells, are found in necrotic areas of the liver (a). Osteopontin is stained markedly in the cytoplasm of these cells on a sequential section (b). Original magnification at 40×.
4. Discussion The present study was designed to know the significance of osteopontin in the development of massive liver necrosis in patients with fulminant hepatitis. Hepatic expression of osteopontin can be rarely evaluated by liver biopsy in patients with fulminant hepatitis, because most patients have marked bleeding tendency. Osteopontin can act as a secretory protein as well as an extracellular matrix, and so plasma osteopontin levels were measured as markers reflecting the hepatic expression in the present study. Also, plasma osteopontin levels were measured in patients with acute hepatitis and chronic hepatitis as well as healthy adults. Plasma osteopontin levels were increased both in patients with fulminant hepatitis and acute hepatitis than in those with chronic hepatitis and healthy adults, but the levels were especially high in patients with fulminant hepatitis of acute type. As shown in Fig. 1, plasma osteopontin levels were greater than 3000 ng/mL in all the patients with fulminant hepatitis of acute type, while the levels were less than 2000 ng/mL in the patients with acute hepatitis and fulminant hepatitis of subacute type except for one patient in whom bacterial infection was complicated with elevated serum CRP level at 7.66 mg/mL. Other clinical and demographic features in this patient were not different from those of other patients. Considering that osteopontin is reported to be expressed abundantly in lymph nodes of patients with regional adenitis [17], and plasma osteopontin levels are significantly higher in patients with tuberculosis than in control subjects and patients with sarcoidosis [18], plasma osteopontin levels elevated in
this patient might originate from other organs than the liver. Hepatic production of osteopontin was evaluated in a patient with fulminant hepatitis of subacute type in whom plasma osteopontin levels increased to 1050 ng/mL, but serum CRP level was less than 1.0 mg/dL. In this patient, osteopontin was stained markedly in infiltrating mononuclear cells in necrotic areas of the liver. Immunohistochemical examination using sequential sections revealed that these cells were identical to Kupffer cells and hepatic macrophages because of their positivity for CD68. The similar observations were not obtained with the liver of patients with fulminant hepatitis of acute type. However, it would be reasonable to assume that plasma osteopontin levels markedly increasing in these patients may reflect osteopontin produced by macrophages in the liver, since the similar mechanisms of development can underlie massive and submassive liver necrosis. The moderate increase of plasma osteopontin levels in patients with acute hepatitis might be attributable to the minor necrosis in the liver. Massive liver necrosis is a characteristic pathological feature in patients with fulminant hepatitis of acute type [3]. We demonstrated that massive liver necrosis developed after endotoxin administration in rats pretreated with heat-killed P. acnes as a result of microcirculatory disturbance due to endothelial cell injury and fibrin deposition in the hepatic sinusoids [19]. In this model, Kupffer cells and hepatic macrophages were activated [20], and contributed to the development of microcirculatory disturbance in the liver [15,21]. Kupffer cells and hepatic macrophages with marked expression of osteopontin became activated through Th1 cy-
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A. Matsui et al. / Hepatology Research 29 (2004) 202–206
tokine network of IL-18 and IFN-␥ in rats given heat-killed P. acnes [9,22], suggesting that this activation may occur through action of osteopontin in an autocrine manner. Thus, it seems likely that osteopontin is responsible for the development of massive liver necrosis in patients with fulminant hepatitis through initiation of Th1 immune response similarly as is in rats pretreated with heat-killed P. acnes. These issues should be investigated in future. In general, the prognosis of fulminant hepatitis is worse in the subacute type than in the acute type [3]. However, plasma osteopontin levels were higher in the acute type than in the subacute type. The mortality of fulminant hepatitis patients depends on various complications such as DIC, renal failure, brain edema, gastrointestinal bleeding and cardiac failure as well as liver dysfunction [3]. Also, the impairment of liver regeneration seems different in both types. Even if plasma osteopontin levels can be a marker reflecting the production of osteopontin in the liver, it would be further necessary to study whether this marker can be useful for predicting the prognosis of fulminant hepatitis. In conclusion, plasma osteopontin levels were elevated in patients with fulminant hepatitis, probably reflecting production of osteopontin in Kupffer cells and hepatic macrophages. Such osteopontin might be involved in the development of massive liver necrosis in fulminant hepatitis.
Acknowledgements We thank Ms. Kayoko Naiki (Gastroenterology and Hepatology, Department of Internal Medicine, Saitama Medical School) for her technical help. References [1] Trey C, Davidson CS. The management of fulminant hepatic failure. In: Popper H, Schaffer F, editors. Progress in liver diseases, New York; Grunn & Stratton, 1970: 282–92. [2] Tandon BN, Bernauau J, O Grady J, et al. Recommendation of the International Association for the Study of the Liver Subcommittee on the nomenclature of acute and subacute liver failure. J Gastroenterol Hepatol 1999;14:403–4. [3] Mochida S, Fujiwara K. Symposium on clinical aspects in hepatitis virus infection. 2. Recent advances in acute and fulminant hepatitis in Japan. Intern Med 2001;40:175–7. [4] Butler WT. The nature and significance of osteopontin. Connective Tissue Res 1989;23:123–36. [5] Uede T, Katagiri Y, Iizuka J, Murakami M. Osteopontin, a coordinator of host defense system: a cytokine or an extracellular adhesive protein? Microbiol Immunol 1997;41:641–8.
[6] Singh RP, Patarca R, Schwartz J, Singh P, Cantor H. Definition of a specific interaction between the early T lymphocyte activation 1 (Eta-1) protein and murine macrophages in vitro and its effect upon macrophages in vivo. J Exp Med 1990;71:1931–42. [7] Murry CE, Giachelli CM, Schwartz SM, Vracko R, Schwartz SM. Macrophages express osteopontin during repair of myocardial necrosis. Am J Pathol 1994;145:1450–62. [8] Ashkar S, Weber GF, Panoutsakopoulou V, et al. Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity. Science 2000;287:860–4. [9] Kawashima R, Mochida S, Matsui A, et al. Expression of osteopontin in Kupffer cells and hepatic macrophages and stellate cells in rat liver after carbon tetrachloride intoxication: a possible factor for macrophage migration into hepatic necrotic areas. Biochem Biophys Res Commun 1999;256:527–31. [10] Wang Y, Mochida S, Kawashima R, et al. Increased expression of osteopontin in activated Kupffer cells and hepatic macrophages during macrophage migration in Propionibacterium acnes-treated rat liver. J Gastroenterol 2000;35:696–701. [11] Mochida S, Yoshimoto T, Mimura S et al. Transgenic mice expressing osteopontin in hepatocytes as a model of autoimmune hepatitis. Biochem Biophys Res Commun 2004;317:114–20. [12] Horney JT, Galambos JT. The liver during and after fulminant hepatitis. Gastroenterology 1977;73:639–45. [13] Kimura K, Ando K, Tomita E, et al. Elevated intracellular IFN-(levels in circulating CD8+ lymphocytes in patients with fulminant hepatitis. J Hepatol 1999;31:579–83. [14] Leifeld L, Cheng S, Ramakers J. Imbalanced intrahepatic expression of interleukin 12, interferon gamma, and interleukin 10 in fulminant Hepatitis B. Hepatology 2002;36:1001–8. [15] Arai M, Mochida S, Ohno A, Ogata I, Fujiwara K. Sinusoidal endothelial cell damage by activated hepatic macrophages in rat liver necrosis. Gastroenterology 1993;104:1466–71. [16] Mochida S, Arai M, Ohno A, Fujiwara K. Deranged blood coagulation equilibrium as a factor of massive liver necrosis following endotoxin administration in partially hepatectomized rats. Hepatology 1999;29:1532–40. [17] Nau GJ, Chupp GL, Emile J-F, et al. Chupp GL., Osteopontin expression correlates with clinical outcome in patients with mycobacterial infection. Am J Pathol 2000;157:37–42. [18] Koguchi Y, Kawakami K, Uezu K, et al. High plasma osteopontin level and its relationship with interleukin-12-mediated type 1 T helper cell response in tuberculosis. Am J Respir Crit Care Med 2003;167:1355–9. [19] Yamada S, Ogata I, Hirata K, Mochida S, Tomiya T, Fujiwara K. Intravascular coagulation in the development of massive hepatic necrosis induced by Corynebacterium parvum and endotoxin in rats. Scand J Gastroenterol 1989;24:293–8. [20] Mochida S, Ogata I, Ohta Y, Yamada S, Fujiwara K. In situ evaluation of the stimulatory state of hepatic macrophages based on their ability to produce superoxide anions in rats. J Pathol 1989;158:67–71. [21] Mochida S, Ohno A, Arai M, Tamatani T, Miyasaka M, Fujiwara K. Role of adhesion molecules in the development of massive hepatic necrosis in rats. Hepatology 1996;23:320–8. [22] Toshima K, Mochida S, Ishikawa K, et al. Contribution of CD14 to endotoxin-induced liver injury may depend on types of macrophage activation in rats. Biochem Biophys Res Commun 1998;246: 731–5.
Short communication
Plasma osteopontin levels in patients with fulminant hepatitis Atsushi Matsui a , Satoshi Mochida a , Akihiko Ohno a , Sumiko Nagoshi a , Takanori Hirose b , Kenji Fujiwara a,∗ a
b
Division of Gastroenterology and Hepatology, Department of Internal Medicine, Saitama Medical School, 38 Morohongo, Moroyama-cho, Iruma-gun, Saitama 350-0495, Japan Department of Pathology, Saitama Medical School, 38 Morohongo, Moroyama-cho, Iruma-gun, Saitama 350-0495, Japan Received 9 March 2004; received in revised form 24 March 2004; accepted 25 March 2004 Available online 28 May 2004
Abstract Fulminant hepatitis is characterized by massive or submassive liver necrosis. Massive liver necrosis can be induced by activated macrophages infiltrating into the liver. Osteopontin, an extracellular matrix, is a secretory glycoprotein as well essential for Th1 immune response, contributing to macrophage activation and infiltration. To know the significance of osteopontin in the development of fulminant hepatitis, plasma osteopontin levels were measured in patients with fulminant hepatitis. The levels were significantly greater in patients with fulminant hepatitis than in those with acute or chronic hepatitis as well as healthy adults. Among patients with fulminant hepatitis except one in whom bacterial infection was complicated, plasma osteopontin levels were elevated especially in the patients who developed hepatic encephalopathy of grade II or more within 10 days of the disease onset, a clinical type characteristic of massive liver necrosis. Immunohistochemical examination revealed that osteopontin was stained in macrophages positive for CD68, a marker for macrophages, in necrotic areas of the liver in a patient with fulminant hepatitis. In conclusion, plasma osteopontin levels were elevated in patients with fulminant hepatitis, probably reflecting production of osteopontin in Kupffer cells and hepatic macrophages, which might be involved in the development of massive liver necrosis in fulminant hepatitis. © 2004 Elsevier B.V. All rights reserved. Keywords: Osteopontin; Fulminant hepatitis; Macrophage; Massive liver necrosis; Th1 immune response
1. Introduction Fulminant hepatic failure is the clinical syndrome presented by acute liver dysfunction developing within 4–8 weeks of the disease onset [1,2]. In Japan, fulminant hepatitis has been used in terminology for such acute liver failure, because its causes are mostly due to hepatitis viral infection. Thus, fulminant hepatitis is defined as acute liver failure in which prothrombin time shows less than 40% of normal value with hepatic encephalopathy of grade II or more appearing within 8 weeks of the disease onset, and classified into acute and subacute types depending on this time period to such hepatic encephalopathy, within 10 days and 11 days later, respectively, since the survival rate is remarkably different in both types [3]. Osteopontin is a secretory extracellular glycoprotein originally isolated from the bone, which contains an ∗ Corresponding author. Tel.: +81-49-276-1198; fax: +81-49-294-8404. E-mail address: [email protected] (K. Fujiwara).
1386-6346/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.hepres.2004.03.009
Arg-Gly-Asp amino acid sequence (RGD motif) [4]. This protein is expressed physiologically in the kidney and bone, and contributes to extracellular matrix formation and calcium deposition in these organs [5]. Also, it is reported that osteopontin can bind to ␣v3 integrin expressed on monocytes and macrophages through RGD motif [6], and promote migration of these cells both in vitro and in vivo [6,7]. In addition, osteopontin is now considered to act as a cytokine essential for Th1 immune response [8], suggesting that osteopontin may play a central role in activation and infiltration of macrophages in various organs. It has long been believed that osteopontin is not expressed in the liver of normal rats and mice [5]. We demonstrated that both mRNA and protein expressions of osteopontin were found abundantly in Kupffer cells, macrophages and stellate cells activated in the liver of rats given carbon tetrachloride (CCl4) or heat-killed Propionibacterium acnes (P. acnes) [9,10]. Recently, we have made the transgenic mice expressing abundant osteopontin in hepatocytes using a vector containing human serum amyloid P component (SAP) promoter, and found that cytotoxic T lymphocyte (CTL) infil-
A. Matsui et al. / Hepatology Research 29 (2004) 202–206
tration and Kupffer cell activation developed spontaneously in the liver of these mice [11]. From these observations, it seems that osteopontin expressed in activated Kupffer cells, macrophages and stellate cells as well as in hepatocytes may be responsible for activation and infiltration of macrophages in the liver in both autocrine and paracrine manners. Pathological features of fulminant hepatitis are characterized by massive or submassive necrosis with inflammatory cell infiltration in the liver [12]. Fulminant hepatitis is suggested to develop as a consequence of the imbalance between Th1 and Th2 immune responses, since the expression of IFN-␥, a representative Th1 cytokine, is up-regulated in hepatic as well as circulating lymphocytes in patients with fulminant hepatitis [13,14]. Stimulation of Th1 immune response can induce activation of Kupffer cells and macrophages to provoke massive liver necrosis through microcirculatory disturbance due to endothelial cell destruction and fibrin deposition in the hepatic sinusoids in rats [15,16]. Thus, it is possible that osteopontin can be involved in the development of massive liver necrosis in patients with fulminant hepatitis. In the present paper, plasma levels and hepatic production of osteopontin in patients with fulminant hepatitis were evaluated to know the significance of osteopontin in the pathogenesis of fulminant hepatitis.
203
2.3. Immunohistochemical examinations of osteopontin and CD68 Blocks of liver specimens were fixed in 20% formalin neutral buffer solution (Muto Pure Chemicals Ltd., Tokyo, Japan), and embedded in paraffin. Three-micrometer sequential sections of the liver block were deparaffinized, and the endogenous peroxidase activity was blocked in a solution containing 0.03% H2 O2 . The sections were treated with microwave heating for 5 min at 90 ◦ C in sodium citrate buffer, 10 mM at pH 6.0. Immunohistochemical staining was performed by indirect immunoperoxidase methods with the DAKO EnVision + System (Dako Corporation, Carpinteria, CA) using monoclonal antibodies against human osteopontin (Immuno-Biological Laboratories) and CD68 (Dako Corporation) as primary antibodies. Non-immune mouse IgG at the same concentrations was used as controls for the primary antibodies. The reaction product was developed by incubation in a solution of 0.25% diaminobenzidine tetrahydrochroride containing H2 O2 (Dako Corporation). Counter staining was done by hematoxylin.
3. Results 3.1. Demographic and clinical features of patients
2. Patients and methods 2.1. Patients and experiments Subjects were patients with fulminant hepatitis fulfilling the Japanese criteria and patients with acute liver diseases who admitted to Saitama Medical School Hospital between January 1995 and March 2000. Acute hepatitis was defined as acute liver disease with no encephalopathy or encephalopathy of grade I, and included acute exacerbation of hepatitis B virus carriers as well in the present study. In all the patients, blood was collected in a plastic tube containing EDTA before receiving therapies including blood transfusion, blood-products infusion, artificial liver support and administration of glucocorticoid and interferon. Plasma was separated from the blood, and stored at −80 ◦ C for measurement of osteopontin levels. Blood was also collected from healthy adults and chronic hepatitis patients positive for serum HCV-RNA, and the plasma was stored similarly as controls. Autopsy samples obtained from a patient with fulminant hepatitis of subacute type were subjected to immunohistochemical examination of osteopontin and CD68, a marker for macrophages including Kupffer cells, in the liver. 2.2. Measurement of plasma osteopontin levels Plasma osteopontin levels were measured using an ELISA kit (Immuno-Biological Laboratories, Gunma, Japan) according to the manufacturer’s instructions.
Nine patients with fulminant hepatitis and six patients with acute hepatitis were enrolled in the study. Among nine patients with fulminant hepatitis, three were of the acute type, and six of the subacute type. Demographic and clinical features of these patients were shown in Table 1. Male patients were more frequent in acute hepatitis than in fulminant hepatitis (P = 0.15), and patients were older in fulminant hepatitis than in acute hepatitis. The etiology of fulminant hepatitis was hepatitis A virus (HAV) infection in one, hepatitis B virus (HBV) infection in one, co-infection of hepatitis C virus (HCV) and TT virus (TTV) in one, and unknown in six, while the etiology of acute hepatitis was HAV infection in one, HBV infection in one, exacerbation of HBV carriers in two, and unknown in two. There were no differences in serum alanine aminotranferase (ALT) levels and total bilirubin concentrations between the patients with fulminant hepatitis and acute hepatitis. Although three of six patients with acute hepatitis showed prothrombin time less than 40% of normal value, the extent of its reduction was smaller in those patients than in the patients with fulminant hepatitis. Serum C-reactive protein (CRP) levels were less than 1.5 mg/dL in all the patients except one patient with fulminant hepatitis of subacute type showing the level at 7.66 mg/mL. Eleven healthy adults (eight males, three females) and 10 patients with chronic hepatitis (seven males, three females) positive for serum HCV-RNA were enrolled as controls. Ages of both groups (years, mean ± S.D.) were 49 ± 12 and 47 ± 14, respectively. Serum ALT levels were within
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Table 1 Demographic and clinical features of patients with fulminant hepatitis, and acute hepatitis Age
Sex
Diagnosis
Etiology
PT (%)
ALT (IU/L)
T.bil (mg/dL)
Alb (g/dL)
Plt (×104 /L)
WBC (/L)
CRP (mg/dL)
60 37 65 78 18 55 64 64 79 25 36 27 50 28 54
F F M F F F M F F M F M F M M
FH-A FH-A FH-A FH-S FH-S FH-S FH-S FH-S FH-S AH AH AH AH AH AH
HAV HBV Unknown HCV, TTV Unknown Unknown Unknown Unknown Unknown HAV HBV HBV carrier HBV carrier Unknown Unknown
22 9 14 35 11 33 33 15 7 42 50 35 24 53 31
2,035 5,310 2,276 88 395 490 2,418 447 1,854 6,790 939 2,619 9,400 572 348
9.2 6.3 13.8 14.4 20.2 14.8 11.4 29.0 9.7 3.6 9.3 8.8 3.6 23.8 17.8
3.2 3.4 3.4 2.3 3.2 3.4 3.2 2.3 3.4 3.6 3.2 4.3 4.1 3.4 3.3
27.6 10.2 11.1 5.5 43.2 24.0 8.7 18.7 24.6 7.8 11.9 13.0 11.0 17.0 15.0
10,720 6,160 4,960 5,560 1,150 7,100 6,170 9,800 11,500 2,970 7,560 5,760 4,260 6,770 9,240
0.46 1.48 1.40 7.66 0.31 0.52 1.42 1.21 0.78 1.48 1.02 1.16 1.48 0.92 0.14
F, female; M, male; FH-A, fulminant hepatitis of acute type; FH-S, fulminant hepatitis of subacute type; AH, acute hepatitis; HAV, hepatitis A virus; HBV, hepatitis B virus; HCV, hepatitis C virus; TTV, TT virus; PT, prothrombin time; ALT, alanine aminotransferase; T.bil, total bilirubin; Alb, albumin; Plt, platelet count; WBC, white blood cell count; CRP, C-reactive protein.
normal range in healthy adults, and 148 ± 100 (IU/L, mean ± S.D.) in patients with chronic hepatitis. Serum ALT levels were significantly lower in patients with chronic hepatitis than in those with fulminant hepatitis and acute hepatitis. 3.2. Plasma osteopontin levels As shown in Fig. 1, plasma osteopontin levels (ng/mL, mean ± S.D.) in patients with fulminant hepatitis (3260 ± 3030) and acute hepatitis (690±256) were higher than those in patients with chronic hepatitis (436 ± 385) as well as healthy adults (239 ± 76). The levels in all the patients with
fulminant hepatitis were higher than those in patients with acute hepatitis. There was a significant correlation between plasma osteopontin levels and prothrombin time in patients with fulminant hepatitis and acute hepatitis. However, such correlations were not seen regarding serum levels of ALT, total bilirubin, albumin and CRP, and peripheral platelet or white blood cell counts (Table 1, Fig. 1). The levels were especially elevated in patients with fulminant hepatitis of acute type showing the levels greater than 3,000 ng/mL, while the levels in patients with fulminant hepatitis of subacute type were less than 2000 ng/mL except one patient in whom serum CRP level was increased to 7.66 mg/mL due to bacterial infection (Fig. 1). The mean level of plasma osteopontin in the other five patients with fulminant hepatitis of subacute type was higher than that of patients with acute hepatitis, but the difference was not statistically significant (Fig. 1). There was no difference in plasma osteopontin levels between patients with chronic hepatitis and healthy adults. The mean (±S.D.) of plasma osteopontin levels was 505 (±435) ng/mL in patients with chronic hepatitis whose serum ALT levels were higher than 100 IU/L, but 275 (±213) ng/mL in the other chronic hepatitis patients (P = 0.42). 3.3. Immunohistochemical examinations of osteopontin and CD68
Fig. 1. Plasma osteopontin levels in patients with fulminant hepatitis (FH), acute hepatitis (AH), chronic hepatitis (CH), and in healthy adults. FH-S, FH of subacute type: FH-A, FH of acute type. Bars are mean levels of each disease group. In FH-S, the mean level of five patients in the column is presented. # P < 0.05 vs. AH, $ P < 0.05 vs. FH, P < 0.05 by Mann–Whitney U-test. & The patient in whom serum C-reactive protein level was 7.66 mg/mL.
Immunohistochemical examination of the liver was done with autopsy samples obtained from a patient with fulminant hepatitis of subacute type whose plasma osteopontin level was 1,050 ng/mL. Marked infiltration of mononuclear cells positive for CD68 was found in necrotic areas of the liver (Fig. 2a). Immunohistochemical examination using sequential sections revealed that osteopontin was stained markedly in the cytoplasm of these cells (Fig. 2b).
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Fig. 2. Immunostaining of CD68 and osteopontin in the liver of a patient with fulminant hepatitis of subacute type, (a) CD68 staining. (b) Osteopontin staining. Marked infiltration of mononuclear cells positive for CD68, a marker for hepatic macrophages and Kupffer cells, are found in necrotic areas of the liver (a). Osteopontin is stained markedly in the cytoplasm of these cells on a sequential section (b). Original magnification at 40×.
4. Discussion The present study was designed to know the significance of osteopontin in the development of massive liver necrosis in patients with fulminant hepatitis. Hepatic expression of osteopontin can be rarely evaluated by liver biopsy in patients with fulminant hepatitis, because most patients have marked bleeding tendency. Osteopontin can act as a secretory protein as well as an extracellular matrix, and so plasma osteopontin levels were measured as markers reflecting the hepatic expression in the present study. Also, plasma osteopontin levels were measured in patients with acute hepatitis and chronic hepatitis as well as healthy adults. Plasma osteopontin levels were increased both in patients with fulminant hepatitis and acute hepatitis than in those with chronic hepatitis and healthy adults, but the levels were especially high in patients with fulminant hepatitis of acute type. As shown in Fig. 1, plasma osteopontin levels were greater than 3000 ng/mL in all the patients with fulminant hepatitis of acute type, while the levels were less than 2000 ng/mL in the patients with acute hepatitis and fulminant hepatitis of subacute type except for one patient in whom bacterial infection was complicated with elevated serum CRP level at 7.66 mg/mL. Other clinical and demographic features in this patient were not different from those of other patients. Considering that osteopontin is reported to be expressed abundantly in lymph nodes of patients with regional adenitis [17], and plasma osteopontin levels are significantly higher in patients with tuberculosis than in control subjects and patients with sarcoidosis [18], plasma osteopontin levels elevated in
this patient might originate from other organs than the liver. Hepatic production of osteopontin was evaluated in a patient with fulminant hepatitis of subacute type in whom plasma osteopontin levels increased to 1050 ng/mL, but serum CRP level was less than 1.0 mg/dL. In this patient, osteopontin was stained markedly in infiltrating mononuclear cells in necrotic areas of the liver. Immunohistochemical examination using sequential sections revealed that these cells were identical to Kupffer cells and hepatic macrophages because of their positivity for CD68. The similar observations were not obtained with the liver of patients with fulminant hepatitis of acute type. However, it would be reasonable to assume that plasma osteopontin levels markedly increasing in these patients may reflect osteopontin produced by macrophages in the liver, since the similar mechanisms of development can underlie massive and submassive liver necrosis. The moderate increase of plasma osteopontin levels in patients with acute hepatitis might be attributable to the minor necrosis in the liver. Massive liver necrosis is a characteristic pathological feature in patients with fulminant hepatitis of acute type [3]. We demonstrated that massive liver necrosis developed after endotoxin administration in rats pretreated with heat-killed P. acnes as a result of microcirculatory disturbance due to endothelial cell injury and fibrin deposition in the hepatic sinusoids [19]. In this model, Kupffer cells and hepatic macrophages were activated [20], and contributed to the development of microcirculatory disturbance in the liver [15,21]. Kupffer cells and hepatic macrophages with marked expression of osteopontin became activated through Th1 cy-
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tokine network of IL-18 and IFN-␥ in rats given heat-killed P. acnes [9,22], suggesting that this activation may occur through action of osteopontin in an autocrine manner. Thus, it seems likely that osteopontin is responsible for the development of massive liver necrosis in patients with fulminant hepatitis through initiation of Th1 immune response similarly as is in rats pretreated with heat-killed P. acnes. These issues should be investigated in future. In general, the prognosis of fulminant hepatitis is worse in the subacute type than in the acute type [3]. However, plasma osteopontin levels were higher in the acute type than in the subacute type. The mortality of fulminant hepatitis patients depends on various complications such as DIC, renal failure, brain edema, gastrointestinal bleeding and cardiac failure as well as liver dysfunction [3]. Also, the impairment of liver regeneration seems different in both types. Even if plasma osteopontin levels can be a marker reflecting the production of osteopontin in the liver, it would be further necessary to study whether this marker can be useful for predicting the prognosis of fulminant hepatitis. In conclusion, plasma osteopontin levels were elevated in patients with fulminant hepatitis, probably reflecting production of osteopontin in Kupffer cells and hepatic macrophages. Such osteopontin might be involved in the development of massive liver necrosis in fulminant hepatitis.
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