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Immunohistochemical study of tumor necrosis factor-alpha in acute liver injury induced by Propionibacterium acnes and lipopolysaccharide in rats
International
Hepatology
ELSEVIER
6 (1997)
Communications 179-190
Immunohistochemical study of tumor necrosis factor-alpha in acute liver injury induced by Propionibacterium acnes and lipopolysaccharide in rats Shin-ichiro SatoaTb, Tomoyuki Masuda”**, Takashi Satoh”, Kazuyuki Suzukib “Department bDepartment
Received
of Pathology,
of Internal
School
Medicine,
2 September
of Medicine, Iwute Medical Universsity, llchimanc 19-1, Morioka 020, Japan School of Medicine, Iwate Medical Universiry, Uchimaru 19-1, Morioka 020, Japan
1996: revised
18 November
1996; accepted
22 November
1996
Abstract
The effect of intravenous injection of Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS) on the distribution of tumor necrosis factor-cc (TNF-cr) in different organs have not previously been investigated. Immunohistochemistry and histological examination were employed in evaluating the distribution of TNF-c( in the liver, spleen, lungs and bone marrow in rats injected intravenously with P. acnes followed by LPS 7 days later. Granulomas containing EDl-positive macrophages were observed in the liver 7 days after P. acnes injection. Subsequent LPS injection resulted in proliferation of EDI-positive macrophages in the sinusoids and coagulation necrosis of hepatocytes after 6 h. TNF-(r was detected in ED2-positive macrophages (Kupffer cells) 1 day after P. acnes injection and in macrophages constituting the granulomas 7 days later, but prior to LPS injection. TNF-a was also detected in EDI-positive macrophages in the spleen, predominantly in the marginal zone. When granulomas were formed 7 days after P. acnes injection, TNF-x was observed in macrophages of the granulomas. TNF-c( was also detected in macrophages of the granulomas found in the lung 1 day after P. acnes injection. No macrophages expressing TNF-cc * Corresponding
author.
0928-4346/97/$17.00 PIZ SO928-4346(96)00347-7
Tel.:
+ 81 91 6515111
0 1997 Elsevier
Science
Ireland
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Hepatology
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6 (1997)
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were found in the granulomas of bone marrow. The highest expression was in the liver at any time interval and in macrophages constituting granulomas. Our results suggest that the high expression of TNF-a in the liver results in selective hepatic necrosis. The expression of TNF-a in macrophages of the liver after P. acnes injection and the subsequent development of hepatic necrosis after LPS injection suggest that P. acnes acts as an inducer of TNF-GI production in macrophages while LPS acts as a trigger for the release of TNF-a from macrophages. 0 1997 Elsevier Science Ireland Ltd. TNF-c(; Liver injury; histochemistry
Keywords:
P. acnes;
Endotoxin; Macrophages; Granulomas; Immuno-
1. Introduction Tumor necrosis factor-alpha (TNF-a) was originally described as a mediator of endotoxin-induced hemorrhagic necrosis of murine sarcoma [l]. TNF-o( is a pleiotropic cytokine, produced mainly by macrophages and monocytes [2,3]. It is the principal mediator of the lethal effect of endotoxins or gram-negative bacteria, and passive immunization against TNF-a protects the animal during lethal endotoxemia or bacteremia [4,5]. Recombinant TNF-a induces many of the deleterious effects of endotoxin [6]. Similarly, TNF-ol is also involved in the pathogenesis of experimental liver injury induced by endotoxin [7,8]. In patients with fulminant hepatic failure, in whom endotoxemia is common, there is a marked increase in the production of TNF-a [9]. Severe hepatitis can be induced by injection of a small dose of lipopolysaccharide (LPS) in mice primed with Propionibacterium acnes (P. acnes) [lo]. Macrophages accumulated in the liver of these animals is presumed to produce a variety of hepatocytotoxic factors that cause liver injury [lo]. The T cell-mediated immune responses and interferon-y produced by T cells also contribute to the development of hepatic injury by P. acnes and LPS [11,12]. In this regard, a new cytokine of T cells, interferon-y-inducing factor, was purified recently from the liver of mice treated with P. acnes and LPS [13]. TNF-cx is also thought to play a role in the pathogenesis of liver injury under these circumstances [7]. Thus, LPS activates macrophages within several days after injection of P. acnes to produce TNF-a in vitro [14]. Furthermore, LPS injection after P. acltes injection increases the level of serum TNF-(x [15]. Accordingly, P. acnes and LPS are considered as the priming and eliciting agents, respectively, in the production of TNF-a. While P. acnes is a powerful inducer of TNF-a in vitro [16], to our knowledge no study has provided a clear evidence that P. acnes acts as an inducer of TNF-a in vivo. LPS stimulates the synthesis of TNF-a in several organs, thus potentially causing multiple organ injury [17- 191. In experimental endotoxemia, TNF-cr and its mRNA are expressed in Kupffer cells, sinusoidal endothelial cells and hepatocytes [20-221. In the present study, we investigated the serial changes in the expression of TNF-a in the liver, spleen, lung and bone marrow of rats treated with P. acnes and LPS.
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2. Materials and methods 2.1, Animals Twenty-eight 14-week-old male Wistar rats, weighing 260-310 g, were used. The animals were provided with food and water ad libitum. Heat-killed P. acnes (Ribi Immunochem Research, Hamilton, MT) was injected into the tail vein at a dose of 1.5 mg per 100 g body weight. After 7 days, 3 yg of LPS (Sigma, St. Louis, MO) per 100 g body weight was also administered intravenously. P. OcneSand LPS were suspended in physiological saline to 20 mg/ml and 100 pg/ml, respectively. Four control rats received intravenous injection of 0.2 ml of physiological saline only. The rats were sacrificed 1 and 7 days after injection of P. acnes, and 1, 3, 6, and 12 h after LPS injection. 2.2. Preparation
of tissue sanzples for microscopical
The liver, spleen, removed. The organs hyde solution for 8 embedded in paraffin. eosin and were used
exawtination
lung and bone marrow of the femur were dissected and were fixed immediately in 4% periodate-lysine-paraformaldeh, dehydrated in a series of graded ethanol solution, and Serial 3 pm thick sections were stained with hematoxylin and for immunohistochemistry.
2.3. Immunohistochemistry Rabbit anti-TNF-cc polyclonal (Genzyme, Boston, MA), and ED1 and ED2 mouse monoclonal antibodies (Serotec, Oxford) were used. The antigen used for preparing TNF-a antibody was a purified recombinant human TNF-a and those of ED1 and ED2 were rat spleen cells. The antibody used against TNF-c( was confirmed to bind rat TNF-ix. ED1 recognizes rat monocytes and macrophages, while ED2 recognizes tissue macrophages including Kupffer cells [23]. Anti-TNF-cc antibody was diluted 1:500 with 0.01 M phosphate-buffered saline (PBS, pH 7.2) containing 1% bovine serum albumin. ED1 and ED2 were diluted 1:200 and 1:50 with PBS containing 0.1% bovine serum albumin, respectively. Immunohistochemical staining was performed by the streptavidin-biotin (SAB) method using commercially available kits (Histofine SAB-PO, R or M kit, Nichirei, Tokyo). The activity of endogenous peroxidase was abolished with methanol solution containing 1.2% hydrogen peroxide for 30 min. Non-specific binding of antibodies was blocked by incubation with normal goat or rabbit serum for 12 min at room temperature. The sections were incubated overnight with the primary antibodies at 4°C then incubated for 20 min with biotinylated goat anti-rabbit IgG or rabbit anti-mouse IgG . A. M antibodies containing 10% normal rat serum. Each step was followed by repeated washing in PBS. Coloration was developed in a 3,3’-diaminobenzidine tetrahydrochloride (DAB, Wako, Osaka) solution containing hydrogen peroxide. The sections were then counterstained with hematoxylin. For negative controls, the same steps were performed, except replacing the primary antibodies with PBS, and normal mouse or normal rabbit serum.
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Double immunohistochemical staining was performed using the following procedure. At first, TNF-a was stained using the SAB method and coloration was developed in a DAB solution. After washing three times in 0.1 M glycine-HCl buffer for 40 min, secondary ED2 staining was performed using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method (Dakopatts kit, Glostrup, Denmark). Non-specific binding of antibodies was blocked by incubation with normal rabbit serum for 12 min at room temperature. The sections were incubated overnight with the primary antibodies at 4°C then reincubated with rabbit antimouse Ig antibody for 30 min. Coloration was developed in a naphthol AS phosphate (Sigma) solution with fast blue BB salt (Sigma). Counterstaining was not performed. 2.4. Morphometry
TNF-a-positive cells were counted in 60 microscopic fields (magnification; 40 x objective and 10 x ocular) randomly selected in each section, The results were expressed as the number of cells per 1 mm*. 2.5. Analysis
Data were expressed as mean + S.D. Differences between groups were tested for statistical significance using the Scheffe multiple comps 1,on test. P < 0.05 was considered significant.
3. Results 3.1. Light microscopy 3.1.1. Liver
No pathognomonic changes were observed 1 day after the injection of P. acnes. After the injection, 7 days, granulomas were scattered throughout the lobules, consisting predominantly of mononuclear cells and measuring approximately 67 hum in diameter (range, 20-223 pm; n = 90) (Fig. la). Mononuclear cell infiltration was observed in the portal areas. No significant change was found in the number and size of these granulomas after the day 7. On the other hand, neutrophils and mononuclear cells began to infiltrate the hepatic sinusoids 1 h after LPS injection, and pyknotic cells were occasionally observed in the sinusoids. After the injection, 6 h, more severe changes were observed in the lobules in two of the five rats examined consisting of focal coagulation necrosis of hepatocytes with inflammatory cell infiltration. These changes were observed in all rats examined 12 h after injection (Fig. lb). There was no relationship between the distribution of focal necrosis and granuloma.
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3.1.2. Spleen
The white pulp was enlarged with narrow marginal zone and fewer cells were noted 1 day after injection of P. acmes. After the injection, 7 days, the white pulp diminished in size and the marginal zones became indistinct. Furthermore, granulo-
Fig. 1. Microscopic appearances of the liver and spleen. Hematoxylin and eosin staining. (a) Liver: 7 days after P. acne3 injection. Note the presence of granulomas (*). x 250. (b) Liver: 12 h after LPS injection. Note the presence of a necrotic focus (N). x 250. (c) Spleen: 7 days after injection of P. awes. Note the large number of granulomas (arrowheads) around the white pulp (W). x 100.
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mas, measuring 45 pm in diameter (range, 23-70 pm; n = 90), were observed throughout the red pulp but were predominantly around the white pulp. Granulomas were also present in the white pulp. In addition, marked hyperplasia was noted in the hematopoietic foci (Fig. lc). On the other hand, the white pulp increased in size after LPS injection. Granulomas increased in size around the white pulp, which coalesced 12 h after LPS injection. Pyknotic cells were occasionally observed 1 h after the injection and gradually increased in number, although coagulation necrosis similar to that observed in the liver was not present. 3.1.3. Lung
A small number of granulomas, measuring 27 pm in diameter (range, 15-58 pm; n = 30), were scattered in the lung 1 day after injection of P. acnes. After injection,
7 days, examination of the lung showed accumulation of mononuclear cells in and around the pulmonary arteries and an increase in number and size of granulomas. No further changes in the distribution, size and number of granulomas were observed at later stages. On the other hand, infiltration of mononuclear cells and neutrophils was observed throughout the lung 1 h after LPS injection and became more severe at the later stage. 3.1.4. Bone marrow One day after P. acnes injection,
no pathognomonic changes were evident. A small number of granulomas, measuring 30 pm in diameter (range, 15-75 pm; n = 60), were observed on the seventh post-injection day, together with marked hypercellularity. A small number of pyknotic hematopoietic cells were observed 1 h after LPS injection, increasing gradually in number in subsequent samples. No significant changes occurred thereafter in granulomas. 3.2. Immunohistochemical
TNF-a-positive
findings
cells were not detected in all organs in control rats.
3.2.1. Liver
One day after injection of P. acnes, TNF-m-positive cells were found scattered within the sinusoids (Fig. 2a). Double immunohistochemical staining showed that these cells were also EDZpositive (Fig. 2b). Macrophages in the sinusoids became almost negative for TNF-cr 7 days after injection. On the same day, TNF-cr was positive in approximately 20% of macrophages present in granulomas. Granulomas consisted of macrophages which were positive for ED1 (Fig. 3a). On the other hand, injection of LPS increased the number of EDl-positive macrophages in the sinusoids, reaching a maximum number at 6 h. TNF-ar was persistently positive in those macrophages within the granulomas (Fig. 3b). Moreover, double staining revealed that approximately 70% of these TNF-a-positive macrophages expressed ED2 (Fig. 3~). As shown in Fig. 4, the maximum number of TNF-a-positive macrophages was observed 1 day after injection of P. acnes (39.86 + 8.61 cells/mn?, mean f S.D.), but decreased thereafter although TNF-a-positive macrophages were
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Fig. 2. Immunohistochemistry of the liver and spleen I day after injection of P. ucnes. (a) Liver: note the TNF-cl-positive cells (arrowheads) present in the sinusoids. x 500. (b) Liver: double staining. TNF-rpositive cells (brown) also express ED2 (blue) as indicated by arrowheads. No counterstaining. x 500. (c) Spleen: note the large number of TNF-r-positive cells (macrophages. arrowheads) in the marginal zone. R, red pulp: W, white pulp. x 500.
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Fig. 3. Immunohistochemistry of granulomas. (a) Liver: ED1 staining. 6 h after LPS injection, note granulomas (*) consisting mainly of EDl-positive cells. Note also the proliferation of EDl-positive cells in the sinusoids (arrowheads). x 180. (b) Liver: TNF-a is expressed in macrophages in a granuloma 3 h after LPS injection. x 700. (c) Liver: double staining. 6 h after LPS injection. Arrowheads point to double-stained macrophages in granulomas (asterisks). TNF-c( (brown) is also expressed in an EDZnegative cell (arrow). No counterstaining. x 350. (d) Spleen: TNF-c( is observed in a granuloma in the white pulp 3 h after LPS injection. x 700.
present in the granulomas. After LPS injection, TNF-a-positive macrophages increased to reach a maximum 3 h later (31.10 ) 6.15 cells/mm*), but then decreased. The number of these macrophages at each time interval, except for 1 h after LPS injection, was significantly different from control. However, the number of these cells was not different at each time interval. TNF-a-positive macrophages in the sinusoids accounted for less than 5% of the total TNF-cc-positive macrophages 7 days after P. acne.s injection. Most TNF-a-positive macrophages were present in the granulomas. No TNF-a-positive macrophages were detected in the necrotic foci 6 h after LPS injection. 3.2.2. Spleen
TNF-cc-positive macrophages were observed throughout the spleen 1 day after injection of P. acnes, predominantly in the marginal zone (Fig. 2~). These macrophages were also positive for EDl. After the injection, 7 days, when
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granulomas had formed, TNF-a was observed in some of the macrophages present in the granulomas (Fig. 3d). TNF-u-positive macrophages, not within the granulomas, accounted for less than 20% at each experimental period. TNF-cl-positive macrophages significantly increased in number 1 day after the injection (12.12 + 7.83 cells/mm2), but significantly decreased after 7 days (2.34 + 2.16 cells /mm2) and remained at that level after LPS injection (Fig. 4). The number of these macrophages in the spleen was, in general, less than that present in the liver throughout the experiment.
cells/mm*
cells/mm*
control 1 day 7 days after P. acnes injection
lhr
3hrs 6hrs after LPS injection
12hrs
(2) (4) (6) (3) (5) (5) (3) Fig. 4. Comparison of the number of TNF-a-positive macrophages detected in the liver, spleen and lung. Column and bar represent the mean and S.D., respectively. The solid part of each column represents the number of TNF-cl-positive macrophages in areas other than granulomas, while the open part of the column represents the number of macrophages in granulomas. Numbers in parentheses represent the numbers of rats used in each experiment.
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3.2.3. Lung
TNF-cr was detected in a small number of macrophages within lung granulomas 1 day after injection of P. acnes (1.59 f 0.38 cells/mm2) and 3 h after LPS injection (1.41 f 1.21 cells/mm2). TNF-a was also detected in a few macrophages scattered in other areas of the lung tissue at these periods. At other time intervals, TNF-ar was expressed only in granulomas (Fig. 4). The positive cells detected in the lung were less than those in the liver and spleen at each time interval. 3.2.4. Bone marrow
No positive reaction for TNF-a was detected throughout period, although granuloma formation was present.
the experimental
4. Discussion Severe hepatitis can be induced by injection of a small dose of LPS in mice primed with P. acnes or BCG [10,24]. The single injection of P. acnes or BCG causes focal and diffuse infiltration of mononuclear cells in the liver [10,24]. According to Ferluga and Allison [lo], mononuclear cells or macrophages are recruited into the liver through priming with P. acne8 while the release of a hepatocytic toxic factor following exposure to LPS. Nagakawa et al. [7] described a massive hepatic necrosis associated with marked increase of plasma TNF-a following injection of LPS. They also showed that passive immunization against TNF-a prevented liver injury. In addition, they described infiltration of activated macrophages expressing Mac-2 in the liver [7]. On the other hand, previous studies showed that the superoxide, produced by sinusoidal macrophages, and adhesion molecules, produced by sinusoidal endothelial cells, also contribute in the development of liver injury [25-271. TNF-a activates sinusoidal endothelial cells to express intercellular adhesion molecule-l [28]. On the other hand, TNF-a also enables hepatic macrophages to cause injury of hepatocytes in vitro, although TNF-a itself has no hepatocytotoxic effect [29]. Our results, however, showed a lack of relationship between the distributions of focal necrosis and granulomas expressing TNF-a. Accordingly, we believe that TNF-a was released into the sinusoids from macrophages present in granulomas, and that TNF-a is involved in the development of liver injury via expression of adhesion molecule on sinusoidal endothelial cells and/or activation of sinusoidal macrophages. The major finding of the present study was the presence of TNF-a in the cytoplasm of ED2-positive macrophages in the sinusoids of the liver 1 day after intravenous injection of P. acnes, as evident on double immunohistochemical staining. A marked expression of TNF-a was present in EDl-positive macrophages in granulomas. However, TNF-a expression became negative in the sinusoidal macrophages 7 days after injection. These results indicate that macrophages constituting granulomas are the major source of TNF-a production. Previous studies showed that TNF-a was not present in the blood of P. acnes-primed mice without an LPS challenge [7,15,30]. The present experiment, however, demonstrated TNF-a
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expression in macrophages constituting granulomas following injection of P. acnes prior to administration of LPS. Our results showed that the maximum expression of TNF-a in the liver, spleen and lung occurred 1 day after injection of P. acnes. Thus, P. acne~ acts as an inducer in the process leading to the production of TNF-a while LPS acts as a trigger releasing TNF-a from the cells. Previous studies using immunoelectron microscopy, in situ hybridization and polymerase chain reaction showed that TNF-c( or its mRNA are expressed in various organs such as the liver, spleen, lung and bone marrow in endotoxemia [17- 19,211. The present study also demonstrated different levels of expression of TNF-a in macrophages within granulomas in the liver, spleen and lungs. Furthermore, the expression of TNF-cr was higher in the liver than in the spleen and lungs but was not expressed in the bone marrow, which contained a few granulomas. The different levels of the expression in these organs are thought to be due to differences in the basal absolute population of macrophages in each organ, as well as differences in the sensitivity of macrophages to P. acne~ in each organ. This argument is based on results of several studies demonstrating the presence of functional differences among ‘different populations of macrophages [31,32]. Moreover, this may explain the confinement of the main tissue injury to the liver despite the systemic exposure to P. acne.r and LPS.
References [l] Carswell EA, Old LJ, Kassel RL, Green S, Fiore N, Williamson B. An endotoxin-induced serum factor that causes necrosis of tumors. Proc Nat1 Acad Sci USA 1975; 72: 3666-3670. [2] Le J, VilEek J. Biology of disease. Tumor necrosis factor and interleukin 1: cytokines with multiple overlapping biological activities. Lab Invest 1987; 56: 2344248. [3] Beutler B, Cerami A. Cachectin: more than a tumour necrosis factor. N Engl J Med 1987; 316: 379-385. [4] Beutler B, Milsark IW, Cerami AC. Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin. Science 1985; 229: 8699871. [5] Tracey KJ, Fong Y, Hesse DG, et al. Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature 1987; 330: 662~-664. [6] Tracey KJ, Beutler B, Lowry SF, et al. Shock and tissue injury induced by recombinant human cachectin. Science 1986; 234: 470-474. [7] Nagakawa J, Hishinuma I, Hirota K, et al. Involvement of tumor necrosis factor-a in the pathogenesis of activated macrophage-mediated hepatitis in mice. Gastroenterology 1990; 99: 758-765. [8] Hishinuma I, Nagakawa J, Hirota K, et al. Involvement of tumor necrosis factor-cc in development of hepatic injury in galactosamine-sensitized mice. Hepatology 1990; 12: 1187- 1191. [9] Muto Y, Nouri-Aria KT, Meager A, Alexander GJM, Eddleston ALWF, Williams R. Enhanced tumor necrosis factor and interleukin-1 in fulminant hepatic failure. Lancet 1988; ii: 72-74. [lo] Ferluga J, Allison AC. Role of mononuclear infiltrating cells in pathogenesis of hepatitis. Lancet 1978; ii: 610-611. [ll] Katschinski T, Galanos C, Coumbos A, Freudenberg MA. Gamma interferon mediates Propionibacterium acnes-induced hypersensitivity to lipopolysaccharide in mice. Infect Immun 1992; 60: 1994-2001. [12] Tanaka Y, Takahashi A, Kobayashi K, et al. Establishment of a T cell-dependent nude mouse liver injury model induced by Propionibacterium acne~ and LPS. J Immunol Methods 1995; 182: 21-28.
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[13] Okamura H, Nagata K, Komatsu T, et al. A novel costimulatory factor for gamma interferon induction found in the livers of mice causes endotoxic shock. Infect Immun 1995; 63: 3966-3972. [14] Liu P, Ohnishi H, Moriwaki H, Muto Y. Enhanced tumor necrosis factor and interleukin-1 in an experimental model of massive liver cell necrosis/fatal hepatitis in mice. Gastroenterol Jpn 1990; 25: 339-342. [15] Green S, Dobrjansky A, Chiasson MA, Carswell E, Schwartz MK, Old LJ. Corynebacterium paruum as the priming agent in the production of tumor necrosis factor in the mouse. J Nat1 Cancer Inst 1977; 59: 1519-1522. [16] Ross01 S, Voth R, Brunner S, et al. Corynebacterium parvum (Propionibacterium acnes): an inducer of tumor necrosis factor-cc in human peripheral blood mononuclear cells and monocytes in vitro. Eur J Immunol 1990; 20: 1761-1765. [17] Giroir BP, Johnson JH, Brown T, Allen GL, Beutler B. The tissue distribution of tumor necrosis factor biosynthesis during endotoxemia. J Clin Invest 1992; 90: 693-698. [18] Hadjiminas DJ, McMasters KM, Peyton JC, Cheadle WG. Tissue tumor necrosis factor mRNA expression following cecal ligation and puncture or intraperitoneal injection of endotoxin. J Surg Res 1994; 56: 549-555. [19] Schmauder-Chock EA, Chock SP, Patchen ML. Ultrastructural localization of tumour necrosis factor-alpha. Histochem J 1994; 26: 142-151. [20] Chensue SW, Terebuh PD, Remick DG, Scales WE, Kunkel SL. In vivo biologic and immunohistochemical analysis of interleukin-1 alpha, beta and tumor necrosis factor during experimental endotoxemia. Kinetics, Kupffer cell expression, and glucocorticoid effects. Am J Path01 1991; 138: 395-402. [21] Hoffmann R, Grewe M, Estler HC, Schulze-Specking A, Decker K. Regulation of tumor necrosis factor-a-mRNA synthesis and distribution of tumor necrosis factor-cr-mRNA synthesizing cells in rat liver during experimental endotoxemia. J Hepatoi 1994; 20: 122-128. [22] Nagano T, Kita T, Tanaka N. The immunocytochemical localization of tumour necrosis factor and leukotriene in the rat liver after treatment with lipopolysaccharide. Int J Exp Path01 1992; 73: 675-683. [23] Dijkstra CD, Dopp EA, Joling P, Kraal G. The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in the rat recognized by monoclonal antibodies EDl, ED2 and ED3. Immunology 1985; 54: 589-599. [24] Shands JW, Jr, Senterhtt VC. Endotoxin-induced hepatic damage in BCG-infected mice. Am J Path01 1972; 67: 23-40. [25] Yamada S, Ogata I, Hirata K, Mochida S, Tomiya T. Fujiwara K. Intravascular coagulation in the development of massive hepatic necrosis induced by Corynebacterium paruum and endotoxin in rats. Stand J Gastroenterol 1989; 24: 293-298. [26] 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 Path01 1989; 158: 67-71. [27] 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: 320-328. [28] Ohira H, Ueno T, Shakado S, et al. Cultured rat hepatic sinusoidal endothelial cells express intercellular adhesion molecule-l (ICAM-1) by tumor necrosis factor-cc or interleukin-la stimulation. J Hepatol 1994; 20: 729-734. [29] Tsutsui H, Mizoguchi Y, Morisawa S. Importance of direct hepatocytolysis by liver macrophages in experimental fulminant hepatitis. Hepatogastroenterology 1992; 39: 553-559. [30] Smith SR, Calzetta A, Bankowski J, Kenworthy-Bott L, Terminelli C. Lipopolysaccharide-induced cytokine production and mortality in mice treated with Corynebacterium parvum. J Leukoc Biol 1993: 54; 23-29. [31] Sun D, Lohmann-Matthes ML. Functionally different subpopulations of mouse macrophages recognized by monoclonal antibodies. Eur J Immunol 1982; 12: 134- 140. [32] Shibata Y, Bautista AP, Pennington SN, Humes JL, Volkman A. Eicosanoid production by peritoneal and splenic macrophages in mice depleted of bone marrow by s9Sr. Am J Path01 1987; 127: 75-82.
Hepatology
ELSEVIER
6 (1997)
Communications 179-190
Immunohistochemical study of tumor necrosis factor-alpha in acute liver injury induced by Propionibacterium acnes and lipopolysaccharide in rats Shin-ichiro SatoaTb, Tomoyuki Masuda”**, Takashi Satoh”, Kazuyuki Suzukib “Department bDepartment
Received
of Pathology,
of Internal
School
Medicine,
2 September
of Medicine, Iwute Medical Universsity, llchimanc 19-1, Morioka 020, Japan School of Medicine, Iwate Medical Universiry, Uchimaru 19-1, Morioka 020, Japan
1996: revised
18 November
1996; accepted
22 November
1996
Abstract
The effect of intravenous injection of Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS) on the distribution of tumor necrosis factor-cc (TNF-cr) in different organs have not previously been investigated. Immunohistochemistry and histological examination were employed in evaluating the distribution of TNF-c( in the liver, spleen, lungs and bone marrow in rats injected intravenously with P. acnes followed by LPS 7 days later. Granulomas containing EDl-positive macrophages were observed in the liver 7 days after P. acnes injection. Subsequent LPS injection resulted in proliferation of EDI-positive macrophages in the sinusoids and coagulation necrosis of hepatocytes after 6 h. TNF-(r was detected in ED2-positive macrophages (Kupffer cells) 1 day after P. acnes injection and in macrophages constituting the granulomas 7 days later, but prior to LPS injection. TNF-a was also detected in EDI-positive macrophages in the spleen, predominantly in the marginal zone. When granulomas were formed 7 days after P. acnes injection, TNF-x was observed in macrophages of the granulomas. TNF-c( was also detected in macrophages of the granulomas found in the lung 1 day after P. acnes injection. No macrophages expressing TNF-cc * Corresponding
author.
0928-4346/97/$17.00 PIZ SO928-4346(96)00347-7
Tel.:
+ 81 91 6515111
0 1997 Elsevier
Science
Ireland
(ext. Ltd.
3513); All
fax: rights
+ 81 19 6299340. reserved.
180
S.-i.
Sato et al. 1 International
Hepatology
Communications
6 (1997)
179-190
were found in the granulomas of bone marrow. The highest expression was in the liver at any time interval and in macrophages constituting granulomas. Our results suggest that the high expression of TNF-a in the liver results in selective hepatic necrosis. The expression of TNF-a in macrophages of the liver after P. acnes injection and the subsequent development of hepatic necrosis after LPS injection suggest that P. acnes acts as an inducer of TNF-GI production in macrophages while LPS acts as a trigger for the release of TNF-a from macrophages. 0 1997 Elsevier Science Ireland Ltd. TNF-c(; Liver injury; histochemistry
Keywords:
P. acnes;
Endotoxin; Macrophages; Granulomas; Immuno-
1. Introduction Tumor necrosis factor-alpha (TNF-a) was originally described as a mediator of endotoxin-induced hemorrhagic necrosis of murine sarcoma [l]. TNF-o( is a pleiotropic cytokine, produced mainly by macrophages and monocytes [2,3]. It is the principal mediator of the lethal effect of endotoxins or gram-negative bacteria, and passive immunization against TNF-a protects the animal during lethal endotoxemia or bacteremia [4,5]. Recombinant TNF-a induces many of the deleterious effects of endotoxin [6]. Similarly, TNF-ol is also involved in the pathogenesis of experimental liver injury induced by endotoxin [7,8]. In patients with fulminant hepatic failure, in whom endotoxemia is common, there is a marked increase in the production of TNF-a [9]. Severe hepatitis can be induced by injection of a small dose of lipopolysaccharide (LPS) in mice primed with Propionibacterium acnes (P. acnes) [lo]. Macrophages accumulated in the liver of these animals is presumed to produce a variety of hepatocytotoxic factors that cause liver injury [lo]. The T cell-mediated immune responses and interferon-y produced by T cells also contribute to the development of hepatic injury by P. acnes and LPS [11,12]. In this regard, a new cytokine of T cells, interferon-y-inducing factor, was purified recently from the liver of mice treated with P. acnes and LPS [13]. TNF-cx is also thought to play a role in the pathogenesis of liver injury under these circumstances [7]. Thus, LPS activates macrophages within several days after injection of P. acnes to produce TNF-a in vitro [14]. Furthermore, LPS injection after P. acltes injection increases the level of serum TNF-(x [15]. Accordingly, P. acnes and LPS are considered as the priming and eliciting agents, respectively, in the production of TNF-a. While P. acnes is a powerful inducer of TNF-a in vitro [16], to our knowledge no study has provided a clear evidence that P. acnes acts as an inducer of TNF-a in vivo. LPS stimulates the synthesis of TNF-a in several organs, thus potentially causing multiple organ injury [17- 191. In experimental endotoxemia, TNF-cr and its mRNA are expressed in Kupffer cells, sinusoidal endothelial cells and hepatocytes [20-221. In the present study, we investigated the serial changes in the expression of TNF-a in the liver, spleen, lung and bone marrow of rats treated with P. acnes and LPS.
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2. Materials and methods 2.1, Animals Twenty-eight 14-week-old male Wistar rats, weighing 260-310 g, were used. The animals were provided with food and water ad libitum. Heat-killed P. acnes (Ribi Immunochem Research, Hamilton, MT) was injected into the tail vein at a dose of 1.5 mg per 100 g body weight. After 7 days, 3 yg of LPS (Sigma, St. Louis, MO) per 100 g body weight was also administered intravenously. P. OcneSand LPS were suspended in physiological saline to 20 mg/ml and 100 pg/ml, respectively. Four control rats received intravenous injection of 0.2 ml of physiological saline only. The rats were sacrificed 1 and 7 days after injection of P. acnes, and 1, 3, 6, and 12 h after LPS injection. 2.2. Preparation
of tissue sanzples for microscopical
The liver, spleen, removed. The organs hyde solution for 8 embedded in paraffin. eosin and were used
exawtination
lung and bone marrow of the femur were dissected and were fixed immediately in 4% periodate-lysine-paraformaldeh, dehydrated in a series of graded ethanol solution, and Serial 3 pm thick sections were stained with hematoxylin and for immunohistochemistry.
2.3. Immunohistochemistry Rabbit anti-TNF-cc polyclonal (Genzyme, Boston, MA), and ED1 and ED2 mouse monoclonal antibodies (Serotec, Oxford) were used. The antigen used for preparing TNF-a antibody was a purified recombinant human TNF-a and those of ED1 and ED2 were rat spleen cells. The antibody used against TNF-c( was confirmed to bind rat TNF-ix. ED1 recognizes rat monocytes and macrophages, while ED2 recognizes tissue macrophages including Kupffer cells [23]. Anti-TNF-cc antibody was diluted 1:500 with 0.01 M phosphate-buffered saline (PBS, pH 7.2) containing 1% bovine serum albumin. ED1 and ED2 were diluted 1:200 and 1:50 with PBS containing 0.1% bovine serum albumin, respectively. Immunohistochemical staining was performed by the streptavidin-biotin (SAB) method using commercially available kits (Histofine SAB-PO, R or M kit, Nichirei, Tokyo). The activity of endogenous peroxidase was abolished with methanol solution containing 1.2% hydrogen peroxide for 30 min. Non-specific binding of antibodies was blocked by incubation with normal goat or rabbit serum for 12 min at room temperature. The sections were incubated overnight with the primary antibodies at 4°C then incubated for 20 min with biotinylated goat anti-rabbit IgG or rabbit anti-mouse IgG . A. M antibodies containing 10% normal rat serum. Each step was followed by repeated washing in PBS. Coloration was developed in a 3,3’-diaminobenzidine tetrahydrochloride (DAB, Wako, Osaka) solution containing hydrogen peroxide. The sections were then counterstained with hematoxylin. For negative controls, the same steps were performed, except replacing the primary antibodies with PBS, and normal mouse or normal rabbit serum.
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Double immunohistochemical staining was performed using the following procedure. At first, TNF-a was stained using the SAB method and coloration was developed in a DAB solution. After washing three times in 0.1 M glycine-HCl buffer for 40 min, secondary ED2 staining was performed using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method (Dakopatts kit, Glostrup, Denmark). Non-specific binding of antibodies was blocked by incubation with normal rabbit serum for 12 min at room temperature. The sections were incubated overnight with the primary antibodies at 4°C then reincubated with rabbit antimouse Ig antibody for 30 min. Coloration was developed in a naphthol AS phosphate (Sigma) solution with fast blue BB salt (Sigma). Counterstaining was not performed. 2.4. Morphometry
TNF-a-positive cells were counted in 60 microscopic fields (magnification; 40 x objective and 10 x ocular) randomly selected in each section, The results were expressed as the number of cells per 1 mm*. 2.5. Analysis
Data were expressed as mean + S.D. Differences between groups were tested for statistical significance using the Scheffe multiple comps 1,on test. P < 0.05 was considered significant.
3. Results 3.1. Light microscopy 3.1.1. Liver
No pathognomonic changes were observed 1 day after the injection of P. acnes. After the injection, 7 days, granulomas were scattered throughout the lobules, consisting predominantly of mononuclear cells and measuring approximately 67 hum in diameter (range, 20-223 pm; n = 90) (Fig. la). Mononuclear cell infiltration was observed in the portal areas. No significant change was found in the number and size of these granulomas after the day 7. On the other hand, neutrophils and mononuclear cells began to infiltrate the hepatic sinusoids 1 h after LPS injection, and pyknotic cells were occasionally observed in the sinusoids. After the injection, 6 h, more severe changes were observed in the lobules in two of the five rats examined consisting of focal coagulation necrosis of hepatocytes with inflammatory cell infiltration. These changes were observed in all rats examined 12 h after injection (Fig. lb). There was no relationship between the distribution of focal necrosis and granuloma.
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3.1.2. Spleen
The white pulp was enlarged with narrow marginal zone and fewer cells were noted 1 day after injection of P. acmes. After the injection, 7 days, the white pulp diminished in size and the marginal zones became indistinct. Furthermore, granulo-
Fig. 1. Microscopic appearances of the liver and spleen. Hematoxylin and eosin staining. (a) Liver: 7 days after P. acne3 injection. Note the presence of granulomas (*). x 250. (b) Liver: 12 h after LPS injection. Note the presence of a necrotic focus (N). x 250. (c) Spleen: 7 days after injection of P. awes. Note the large number of granulomas (arrowheads) around the white pulp (W). x 100.
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mas, measuring 45 pm in diameter (range, 23-70 pm; n = 90), were observed throughout the red pulp but were predominantly around the white pulp. Granulomas were also present in the white pulp. In addition, marked hyperplasia was noted in the hematopoietic foci (Fig. lc). On the other hand, the white pulp increased in size after LPS injection. Granulomas increased in size around the white pulp, which coalesced 12 h after LPS injection. Pyknotic cells were occasionally observed 1 h after the injection and gradually increased in number, although coagulation necrosis similar to that observed in the liver was not present. 3.1.3. Lung
A small number of granulomas, measuring 27 pm in diameter (range, 15-58 pm; n = 30), were scattered in the lung 1 day after injection of P. acnes. After injection,
7 days, examination of the lung showed accumulation of mononuclear cells in and around the pulmonary arteries and an increase in number and size of granulomas. No further changes in the distribution, size and number of granulomas were observed at later stages. On the other hand, infiltration of mononuclear cells and neutrophils was observed throughout the lung 1 h after LPS injection and became more severe at the later stage. 3.1.4. Bone marrow One day after P. acnes injection,
no pathognomonic changes were evident. A small number of granulomas, measuring 30 pm in diameter (range, 15-75 pm; n = 60), were observed on the seventh post-injection day, together with marked hypercellularity. A small number of pyknotic hematopoietic cells were observed 1 h after LPS injection, increasing gradually in number in subsequent samples. No significant changes occurred thereafter in granulomas. 3.2. Immunohistochemical
TNF-a-positive
findings
cells were not detected in all organs in control rats.
3.2.1. Liver
One day after injection of P. acnes, TNF-m-positive cells were found scattered within the sinusoids (Fig. 2a). Double immunohistochemical staining showed that these cells were also EDZpositive (Fig. 2b). Macrophages in the sinusoids became almost negative for TNF-cr 7 days after injection. On the same day, TNF-cr was positive in approximately 20% of macrophages present in granulomas. Granulomas consisted of macrophages which were positive for ED1 (Fig. 3a). On the other hand, injection of LPS increased the number of EDl-positive macrophages in the sinusoids, reaching a maximum number at 6 h. TNF-ar was persistently positive in those macrophages within the granulomas (Fig. 3b). Moreover, double staining revealed that approximately 70% of these TNF-a-positive macrophages expressed ED2 (Fig. 3~). As shown in Fig. 4, the maximum number of TNF-a-positive macrophages was observed 1 day after injection of P. acnes (39.86 + 8.61 cells/mn?, mean f S.D.), but decreased thereafter although TNF-a-positive macrophages were
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Fig. 2. Immunohistochemistry of the liver and spleen I day after injection of P. ucnes. (a) Liver: note the TNF-cl-positive cells (arrowheads) present in the sinusoids. x 500. (b) Liver: double staining. TNF-rpositive cells (brown) also express ED2 (blue) as indicated by arrowheads. No counterstaining. x 500. (c) Spleen: note the large number of TNF-r-positive cells (macrophages. arrowheads) in the marginal zone. R, red pulp: W, white pulp. x 500.
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Fig. 3. Immunohistochemistry of granulomas. (a) Liver: ED1 staining. 6 h after LPS injection, note granulomas (*) consisting mainly of EDl-positive cells. Note also the proliferation of EDl-positive cells in the sinusoids (arrowheads). x 180. (b) Liver: TNF-a is expressed in macrophages in a granuloma 3 h after LPS injection. x 700. (c) Liver: double staining. 6 h after LPS injection. Arrowheads point to double-stained macrophages in granulomas (asterisks). TNF-c( (brown) is also expressed in an EDZnegative cell (arrow). No counterstaining. x 350. (d) Spleen: TNF-c( is observed in a granuloma in the white pulp 3 h after LPS injection. x 700.
present in the granulomas. After LPS injection, TNF-a-positive macrophages increased to reach a maximum 3 h later (31.10 ) 6.15 cells/mm*), but then decreased. The number of these macrophages at each time interval, except for 1 h after LPS injection, was significantly different from control. However, the number of these cells was not different at each time interval. TNF-a-positive macrophages in the sinusoids accounted for less than 5% of the total TNF-cc-positive macrophages 7 days after P. acne.s injection. Most TNF-a-positive macrophages were present in the granulomas. No TNF-a-positive macrophages were detected in the necrotic foci 6 h after LPS injection. 3.2.2. Spleen
TNF-cc-positive macrophages were observed throughout the spleen 1 day after injection of P. acnes, predominantly in the marginal zone (Fig. 2~). These macrophages were also positive for EDl. After the injection, 7 days, when
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granulomas had formed, TNF-a was observed in some of the macrophages present in the granulomas (Fig. 3d). TNF-u-positive macrophages, not within the granulomas, accounted for less than 20% at each experimental period. TNF-cl-positive macrophages significantly increased in number 1 day after the injection (12.12 + 7.83 cells/mm2), but significantly decreased after 7 days (2.34 + 2.16 cells /mm2) and remained at that level after LPS injection (Fig. 4). The number of these macrophages in the spleen was, in general, less than that present in the liver throughout the experiment.
cells/mm*
cells/mm*
control 1 day 7 days after P. acnes injection
lhr
3hrs 6hrs after LPS injection
12hrs
(2) (4) (6) (3) (5) (5) (3) Fig. 4. Comparison of the number of TNF-a-positive macrophages detected in the liver, spleen and lung. Column and bar represent the mean and S.D., respectively. The solid part of each column represents the number of TNF-cl-positive macrophages in areas other than granulomas, while the open part of the column represents the number of macrophages in granulomas. Numbers in parentheses represent the numbers of rats used in each experiment.
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3.2.3. Lung
TNF-cr was detected in a small number of macrophages within lung granulomas 1 day after injection of P. acnes (1.59 f 0.38 cells/mm2) and 3 h after LPS injection (1.41 f 1.21 cells/mm2). TNF-a was also detected in a few macrophages scattered in other areas of the lung tissue at these periods. At other time intervals, TNF-ar was expressed only in granulomas (Fig. 4). The positive cells detected in the lung were less than those in the liver and spleen at each time interval. 3.2.4. Bone marrow
No positive reaction for TNF-a was detected throughout period, although granuloma formation was present.
the experimental
4. Discussion Severe hepatitis can be induced by injection of a small dose of LPS in mice primed with P. acnes or BCG [10,24]. The single injection of P. acnes or BCG causes focal and diffuse infiltration of mononuclear cells in the liver [10,24]. According to Ferluga and Allison [lo], mononuclear cells or macrophages are recruited into the liver through priming with P. acne8 while the release of a hepatocytic toxic factor following exposure to LPS. Nagakawa et al. [7] described a massive hepatic necrosis associated with marked increase of plasma TNF-a following injection of LPS. They also showed that passive immunization against TNF-a prevented liver injury. In addition, they described infiltration of activated macrophages expressing Mac-2 in the liver [7]. On the other hand, previous studies showed that the superoxide, produced by sinusoidal macrophages, and adhesion molecules, produced by sinusoidal endothelial cells, also contribute in the development of liver injury [25-271. TNF-a activates sinusoidal endothelial cells to express intercellular adhesion molecule-l [28]. On the other hand, TNF-a also enables hepatic macrophages to cause injury of hepatocytes in vitro, although TNF-a itself has no hepatocytotoxic effect [29]. Our results, however, showed a lack of relationship between the distributions of focal necrosis and granulomas expressing TNF-a. Accordingly, we believe that TNF-a was released into the sinusoids from macrophages present in granulomas, and that TNF-a is involved in the development of liver injury via expression of adhesion molecule on sinusoidal endothelial cells and/or activation of sinusoidal macrophages. The major finding of the present study was the presence of TNF-a in the cytoplasm of ED2-positive macrophages in the sinusoids of the liver 1 day after intravenous injection of P. acnes, as evident on double immunohistochemical staining. A marked expression of TNF-a was present in EDl-positive macrophages in granulomas. However, TNF-a expression became negative in the sinusoidal macrophages 7 days after injection. These results indicate that macrophages constituting granulomas are the major source of TNF-a production. Previous studies showed that TNF-a was not present in the blood of P. acnes-primed mice without an LPS challenge [7,15,30]. The present experiment, however, demonstrated TNF-a
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expression in macrophages constituting granulomas following injection of P. acnes prior to administration of LPS. Our results showed that the maximum expression of TNF-a in the liver, spleen and lung occurred 1 day after injection of P. acnes. Thus, P. acne~ acts as an inducer in the process leading to the production of TNF-a while LPS acts as a trigger releasing TNF-a from the cells. Previous studies using immunoelectron microscopy, in situ hybridization and polymerase chain reaction showed that TNF-c( or its mRNA are expressed in various organs such as the liver, spleen, lung and bone marrow in endotoxemia [17- 19,211. The present study also demonstrated different levels of expression of TNF-a in macrophages within granulomas in the liver, spleen and lungs. Furthermore, the expression of TNF-cr was higher in the liver than in the spleen and lungs but was not expressed in the bone marrow, which contained a few granulomas. The different levels of the expression in these organs are thought to be due to differences in the basal absolute population of macrophages in each organ, as well as differences in the sensitivity of macrophages to P. acne~ in each organ. This argument is based on results of several studies demonstrating the presence of functional differences among ‘different populations of macrophages [31,32]. Moreover, this may explain the confinement of the main tissue injury to the liver despite the systemic exposure to P. acne.r and LPS.
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