Co.) and control mice. The mice treated with PAF antagonist received indicated doses of TCV-309 ..... Fry, D. E., L. Pearlstein, R. L. Filton, and H. C. Polk. 1980.
INFECrION AND IMMUNITY, Feb. 1993, p. 699-704 0019-9567/93/020699-06$02.00/0 Copyright ©) 1993, American Society for Microbiology
Vol. 61, No. 2
An Antagonist of Platelet-Activating Factor Suppresses Endotoxin-Induced Tumor Necrosis Factor and Mortality in Mice Pretreated with Carrageenan MASANORI OGATA,1* TAKAHIRO MATSUMOTO,' KAZUNORI KOGA,1 ICHIRO TAKENAKA,' MASAYUKI KAMOCHI,1 TAKEYOSHI SATA,1 SHIN-ICHI YOSHIDA,2 ANDAKO SHIGEMATSU' Department of Anesthesiology' and Department of Microbiology,2 School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807, Japan Received 26 June 1992/Accepted 23 November 1992
We found that carrageenan (CAR), that is, sulfated polygalactose, can enhance both lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production and the rate of lethality in mice (M. Ogata, S. Yoshida, M. Kamochi, A. Shigematsu, and Y. Mizuguchi, Infect. Immun. 59:679-683, 1991). It has been reported that platelet-activating factor (PAF) antagonists reduce the rate of mortality from endotoxin shock. However, there are few reports regarding the effect of PAF antagonists on TNF production. The aim of the present study is to examine the effect of TCV-309, a new PAF antagonist, on LPS-induced TNF production and mortality in mice pretreated with CAR. ddY mice (6 to 7 weeks old) were injected intraperitoneally with CAR (5 mg per mouse) and were then divided into two groups: mice treated with a PAF antagonist (TCV-309; Takeda Pharmaceutical Co.) and control mice. The mice treated with PAF antagonist received indicated doses of TCV-309 subcutaneously (s.c.) at 30 min before LPS injection, while the control mice received 1 ml of saline s.c. at the same time. All mice were stimulated by intravenous in|jection of LPS (50 ,Ig per mouse) at 24 h after pretreatment with CAR. At intervals after injection of LPS, serum samples were obtained for a TNF assay in which cytotoxicity to L929 cells was measured. TCV-309 both significantly suppressed LPS-induced TNF production and reduced mortality in a dose-dependent manner. When TCV-309 was administered at 30 min before injection of LPS, the effect of TCV-309 on the suppression of TNF activity was at its peak. Treatment with TCV-309 (990 ,ug per mouse) s.c. significantly improved the survival rate after challenge with LPS compared with the survival rate of control mice. Although the 50% lethal dose of LPS was 15 ,ig per mouse for control mice, it increased to 102 lug per mouse for mice that were treated s.c. with TCV-309 (990 ,ug per mouse). Even in vitro, TCV-309 also inhibited LPS-induced TNF production in thioglycolate-elicited macrophages. It was concluded that PAF plays an important role in endotoxin-induced TNF production and
mortality. that mice pretreated with CAR provide a useful model to analyze the pathophysiological mechanism of development of endotoxin-induced lethality and MOF. There is increasing evidence that inflammatory mediators such as platelet-activating factor (PAF) (7, 10, 35), leukotrienes (LTs) (8, 9, 12, 32), and cyclooxygenase products (21) are involved in the pathophysiology of endotoxemia. PAF is now recognized as a potent mediator of inflammation because it is produced by and activates a variety of cells involved in inflammatory reactions, including platelets, neutrophils, eosinophils, endothelial cells, and macrophages (3). It has been reported elsewhere that many PAF antagonists prevent endotoxin-induced hypotension and lethality (10, 26, 35). However, there are few reports regarding the effect of PAF antagonists on LPS-induced TNF production both in vivo and in vitro. TCV-309, a new PAF antagonist, was synthesized from nicotinamide derivatives. This compound exerted a morepotent PAF antagonist activity than both CV-6209 and the diazepam derivative WEB-2086, without the undesirable side effects associated with the lipid-type analogs, such as hemolytic action and vascular damage at the site of action (34). The inhibitory effect of TCV-309 on PAF-induced hypotension in rats continued for more than 8 h at an intravenous (i.v.) dose of 10 ,ug kg of body weight-' or an oral dose of 1 mg- kg-' (34). We investigated the effect of TCV-309 on LPS-induced TNF production and lethality in
Tumor necrosis factor (TNF) is implicated as a major mediator of endotoxin shock (1) and causes adult respiratory distress syndrome (13), hemodynamic and cardiovascular dysfunction (22), intravascular coagulation (2, 36), and multiple-organ damage (6). We previously reported that when mice were pretreated intraperitoneally (i.p.) with carrageenan (CAR), a sulfated polygalactose, even a low dose of endotoxin (50 ,ug per mouse) caused an enormous production of TNF in sera and even death (24). Mice pretreated with CAR as well as mice treated with galactosamine (17) are useful for the study of the pathophysiological mechanism of the low-dose-endotoxin lethality model. Galactosamine enhanced only the sensitivity to TNF, but CAR as well as muramyl dipeptide (AcMur-L-Ala-D-iGln) and adrenalectomy enhanced lipopolysaccharide (LPS)-induced TNF production (25). It was assumed that inflammation and suppression of the reticuloendothelial system (RES) played an important role in the enhancement of LPS-induced TNF production and lethality by pretreatment with CAR, because CAR is used as an inflammatory agent and as a RES blocker. Even in the case of human patients, inflammation (6, 15, 18, 19) and depression of the RES (29, 30) are assumed to be important factors in developing endotoxin-induced mortality and multiple-organ failure (MOF). These findings suggest *
Corresponding author. 699
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INFECT. IMMUN.
OGATA ET AL.
TCV-309
FIG. 1. The structure of TCV-309 (molecular formula and weight, C30H34BrN5O7 and 656.5, respectively).
mice pretreated with CAR. The present study showed that TCV-309 significantly suppressed endotoxin-induced TNF production both in vivo and in vitro and reduced lethality in a dose-dependent manner.
MATERUILS AND METHODS Protocol. The protocol for this study was approved by the Institutional Animal Care Committee. Animals. Male ddY mice were obtained from the Seiwa Experimental Animal Co., Oita, Japan. All mice used were 7 to 8 weeks of age. The mice were housed in groups of 10 and were allowed food and water ad libitum. Reagents. Phenol-extracted Escherichia coli LPS (0127: B8) was purchased from Difco Laboratories, Detroit, Mich. Iota-CAR (lot no. 59C-0328) was purchased from Sigma, St. Louis, Mo. TCV-309, a selective PAF receptor antagonist [3-bromo-5-(N-phenyl-N-(2-((2-(1,2,3,4-tetrahydro-2-isoqui-
nolylcarbonyloxy)ethyl)carbamoyl)ethyl)carbamoyl)-1-propylpyridinium nitrate], was a kind gift from Takeda Pharmaceutical Co., Osaka, Japan (Fig. 1). LPS, TCV-309, and CAR were dissolved in pyrogen-free physiological saline (Otsuka Pharmaceutical Co., Naruto, Japan). CAR solution was autoclaved at 121°C for 15 min before use. Induction of endotoxin shock and treatments by TCV-309. Endotoxin shock was induced in mice as previously reported (24). In brief, CAR (5 mg in 0.5 ml of physiological saline) was injected intraperitoneally (i.p.) as a priming agent 24 h prior to challenge with LPS. LPS (indicated doses in 0.5 ml of physiological saline) was injected i.v. into the tail vein as an inducing agent. The indicated doses of TCV-309 and physiological saline (control) were both administered subcutaneously (s.c.) at a volume of 1 ml into the backs of mice 30 min before provocation with LPS. At intervals after injection of LPS, blood samples were collected by cardiac puncture under ether anesthetic and then kept at room temperature for clotting. The serum samples were stored at -80°C until they were used for the TNF assay. In vitro study of the effect of TCV-309 on LPS-induced TNF production. ddY mice (6 to 7 weeks old) were injected with 2 ml of 3% thioglycolate medium (Eiken Chemical Co., Ltd., Tokyo, Japan). Four days later, peritoneal exudate cells
were lavaged, washed twice in phosphate-buffered saline (PBS), and resuspended in RPMI medium. After the concentration of macrophages was adjusted to 2 x 106 cells per ml, 0.5 ml of the cell suspension was transferred to flat-bottom, 24-well tissue culture plates, and the plates were then incubated at 37°C for 2 h. After nonadherent cells were removed by being washed in PBS, indicated doses of TCV309 were added in 1 ml of RPMI medium and 5% fetal calf serum. Fifteen minutes later, the macrophages were stimulated by LPS (1 ,ug/ml) and then incubated for 4 h at 37°C in a CO2 incubator. Culture supernatants of macrophages were stored at -80°C until TNF activities were measured. TNF assay. TNF activity was assayed by the colorimetric determination of cytotoxicity to L929 cells as previously reported (28). TNF activity was expressed in units per milliliter, which is the reciprocal of the dilution necessary to cause lysis of 50% of the cells. TNF blood samples were usually collected 2 h after challenge with LPS. In our assay, 1 U/ml is equivalent to 0.63 pg of recombinant murine TNF-a (Genzyme, Cambridge, Mass.) per ml. Mortality. The mortality rate was determined by using 10 mice from each group and by counting the number of dead mice at 6, 18, 24, 36, 48, and 72 h after injection of LPS. The cumulative percentages of mortality in mice receiving each dose of LPS were determined by counting the cumulative numbers of dead mice at 72 h after injection of LPS. Statistics. The physiological variables between groups were compared by a two-factor, repeated-measure analysis of variance. When necessary, further analysis was performed by the unpaired t test. The effects of various treatments on endotoxin-induced lethality were analyzed by the chi-square test. A significant difference was presumed when P was
