Tumor necrosis facior (TNF) has been shown to mediate lipopolysaccharide-induced neutrophil adhesion to liver sinusoidal endothelium in vivo. Female NMRI ...
JournalofHepatology, 1988; 7:239-249
239
Elsevier HEP 00455
Involvement of tumor necrosis factor in endotoxin-triggered neutrophil adherence to sinusoidal endothelial cells of mouse liver and its modulation in acute phase*
H.-J. Schlayer I , H. L a a f f z, T. P e t e r s I , M. W o o r t - M e n k e r 2, H . C . Estler 1, U. K a r c k 1, H . E . S c h a e f e r 2 and K. D e c k e r l I Biochemisches Institut and 2Pathologisches lnstitut der Albert-Ludwigs.Universitiit, Freiburg i. Br. (F.R.G.)
(Received 25 January 1988) (Accepted 17 May 1988)
Summary T u m o r necrosis facior (TNF) has been shown to mediate lipopolysaccharide-induced neutrophil adhesion to liver sinusoidal endothelium in vivo. Female N M R I mice received either 5 Bg lipopolysaccharide (R595) per animal alone (model A) or together with l16~mol o-galactosamine (model B). One hour after injection, TNF activity in the serum was detectable to an equal extent in both models. Neutrophils in the liver, which had been identified by chloroacetate esterase staining of liver sections and quantitated by light microscopy, started to increase at 1 h and were elevated 10-fold above baseline at 6 h after application in (A) and (B). If 0.5/~g TNF instead of lipopolysaccharide was injected alone (model C) or together with o-galactosamine (model D), neutrophil influx into the liver was comparable to that observed in (A) or (B). Alanine aminotransferase activity in the serum was nearly normal in (A) and (C) 6 h after injection, while it reached levels up to 50-fold above baseline in models (B) and (D). This reflects the well-known o-galactosamine sensitization against lipopolysaccharide or TNF. Furthermore, degranulation of a large number of intrasinusoidal neutrophils could be observed 9 h after lipopolysaccharide-galactosamine injection. The administration of ll6/~tmol D-galactosamine per animal alone led neither to a measurable TNF activity in the serum nor to an increase in alanine aminotransferase activity or number of liver neutrophils. If the animals had received 50/,~1 turpentine subcutaneously 24 h prior to lipopolysaccharide, T N F or D-galactosamine injection, the induced acute-phase reaction suppressed the increase of liver neutrophils in all models. Acute-phase reaction also prevented neutrophil degranulation and the rise of alanine aminotransferase in (B) to a great extent, while serum TNF activity was only minimally affected. It is concluded that T N F mediates neutrophil adhesion to the sinusoidal endothelium in vivo and that acute-phase reactants prevent lipopolysaccharide- or TNF-induced neutrophil influx into the liver.
* Dedicated to Prof. Dr. Friedhelm Schneider on the occasion of his sixtieth birthday. This work was supported by grants from the Deutsche Forschungsgemeinschaft. Bonn-Bad Godesberg, through SFB 154 and from Fonds der Cbemischen lndustrie, Frankfurt. Correspondence. Prof. Dr. Karl Decker, Biochemisches Institut, Albert-Ludwigs-Universit~it, Hermann-Herder-Str. 7, D-7800 Freiburg i. Br., F.R.G. Tel. (0761) 203 3190. 0168-8278/88/$03.50 I~) 1988 Elsevier Science Publishers B.V. (Biomedical Division)
240 Introduction
Tumor necrosis factor (TNF) was first reported by Carswell et al_ in 1975 [1] as an antitumor substance present in the serum of endotoxin-treated animals such as mice, rats and rabbits that previously had been given injections of BCG, Corynebacterium parrum or zymosan. Several types of macrophages including monocytes [2] and Kupffer cells (to be published) have been identified as sources of TNF in invitro experiments. This factor is known to induce hemorrhagic necrosis, growth inhibition or regression of certain transplanted murine or human tumors
[3]. TNF/cachectin has also been reported to play a prominent role in mediating toxic effects in the endotoxic shock syndrome [4]: when administered intravenously to rats in quantities similar to those produced endogenously in endotoxic shock, human recombinant TNF was able to cause hypotension, metabolic acidosis, ischemic and hemorrhagic lesions of the lung, of the gastrointestinal tract and of the kidney. Passive immunization against TNF substantially mitigated the lethal effect of endotoxin [5]. In vitro neutrophil adhesion to human umbilical vein endothelial cells [6], neutrophil degranulation and phagocytosis [7] were shown to be triggered by TNF. The production of procoagulant activity by vascular endothelial cells was also stimulated [8], which may relate to the clotting abnormalities associated with endotoxinemia. It was shown in a previous study that supernatants of monocytes stimulated with lipopolysaccharide were able to increase neutrophil adherence to isolated sinusoidal endothelial cells of the liver in monolayer culture [9]. TNF was shown to be responsible for this effect in vitro. In vivo, however, the significance of TNF in mediating endotoxin-triggered neutrophil adhesion to liver sinusoidal endothelial cells remained to be established_ Endotoxin sensitivity is known to be increased in galactosamine-treated animals by several orders of magnitude [10]. Galactosamine has also been shown to cause hepatitis in rats [11]. The combined administration of endotoxin and galactosamine induces ful-
H.-J. SCHLAYER et al. minant hepatitis within 6 h with widespread zonal necroses, formation of single cell necrosis and an intense inflammatory reaction [12]. Inflammatory infiltration by neutrophils and monocytes was also seen in the lung. The present communication deals with the neutrophil influx into the mouse liver induced by treatment of the mice either with endotoxin alone or in combination with galactosamine. The main objectives are to investigate (a) whether TNF can be detected in the serum after lipopolysaccharide or lipopolysaccharide/galactosamine administration, (b) whether T N F can substitute for lipopolysaccharide in its effects on neutrophil influx into the liver, (c) whether acute-phase reactants can modify these effects and (d) whether neutrophils are pathogenetically involved in mediating lipopolysaccharide-induced damage of the liver.
Materials and Methods
Animals and chemicals Female N M R I mice weighing 25-30 g were fed with Altromin a standard diet and water ad libitum and were kept on an artificial light cycle with a daytime period of 12 h (7.0 a . m . - 7 . 0 p.m.). For acutephase induction a single subcutaneous injection of 50 ~tl turpentine (Balsamterpentin61 D A B 7 from Otto Fischer, Scheidt, F.R_G.) into the neck skin was performed 24 h before starting the experiment_ DGalactosamine.HCl (Hepasamin, Roth, Karlsruhe, F.R.G_) was dissolved in distilled water at a concentration of 100 mg/ml and neutralized to pH 7.0-7.2 with 1 M sodium hydroxide. Lipopolysaccharide from Sahnonella minnesota R595, which was kindly provided by Dr. C. Galanos, Max-Planck-Institut ffir Immunbiologie, Freiburg, was dissolved in distilled water and diluted to 100ug per ml. Human recombinant TNF-ct was generously provided by Ernst Boehringer Institut for Arzneimittelforschung, Vienna, Austria. Its specific activity was 6 x 10 7 U TNF/mg and its contamination with lipopolysaccharide was indicated as
