European Journal of Pharrnacology, 210 (1992) 107-113
107
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52212
Cloricromene, a coumarine derivative, protects against lethal endotoxin shock in rats F r a n c e s c o S q u a d r i t o , D o m e n i c a AltaviUa, G i u s e p p e M. C a m p o , G i o a c c h i n o Calapai, M a r i a p a t r i z i a I o c u l a n o , Basilia Zingarelli, A n t o n i n o Saitta 1, M a r c o P r o s d o c i m i 2 a n d Achille P. C a p u t i Institutes of Pharmacology and 1 Chair of Medical Therapeutics, School of Medicine, University of Messina, Messina, Italy and e Fidia Research Laboratories, Abano Terme, Italy Received 10 May 1991, revised MS received 6 August 1991, accepted 15 October 1991
Endotoxin shock was induced in male rats by an intravenous (i.v.) injection of Salmonella enteriditis lipopolysaccharide (LPS; 20 mg/kg i.v.). Survival rate, macrophage and serum tumor necrosis factor (TNF-a), mean arterial blood pressure (MAP) and white blood cell count were then evaluated. Furthermore the in vitro effect of cloricromene on peritoneal macrophage phagocytosis and TNF-a release by primed peritoneal macrophages was investigated. LPS admininistration caused animal death (0% survival 24 h after endotoxin challenge), hypotension, marked leukopenia and increased the levels of TNF-a in both serum and macrophage supernatants. Cloricromene administration (0.5, 1 and 2 mg/kg i.v. 15 rain after endotoxin) protected against LPS-induced lethality (100% survival rate 24 h after endotoxin challenge), reverted LPS-induced hypotension and leukopenia, and decreased TNF-a in both serum and macrophage supernatants. Finally, cloricromene, added in vitro to peritoneal macrophages collected from endotoxin-treated rats increased macrophage phagocytosis and reduced TNF-a formation by activated mononuclear phagocytes. Our data suggest that cloricromene increases survival rate in endotoxin shock through an inhibition of TNF-a production. Cloricromene; Tumor necrosis factor (TNF-c~); Endotoxin shock
1. Introduction
Tumor necrosis factor (TNF-a) is a protein secreted by primed macrophages upon the activation by endotoxin or other stimuli (Carswell et al., 1985). This cytokine has pleiotropic activities both in vitro and in vivo, including cytotoxic effects against tumors and virus-infected cells, stimulation of interleukin-1 secretion, prostaglandin E 2 and collagen production, inhibition of lipogenic gene expression in adipocites, and stimulation of various immune effector cells (Beutler and Cerami, 1987). Moreover TNF-a appears to be a common mediator of inflammation and endotoxin-induced shock (Beutler et al., 1985a; Mannel et al., 1975). As a matter of fact, elevated serum levels of TNF-a are present in endotoxin shock (Bagby et al., 1989) and the administration of specific antibodies raised against T N F - a protects animals from the lethal
Correspondence to: F. Squadrito, Institute of Pharmacology, School of Medicine, Piazza XX Settembre 4, 98122 Messina, Italy. Tel. 39.90.675 833 or 712 533, fax 39.90.661 029.
effects of endotoxic shock (Tracey et al., 1987). In addition, infusion of recombinant human TNF-a in experimental animals has been shown to reproduce many of the deleterious effects of endotoxin (Tracey et al, 1986). Clinical evidence also suggests that plasma T N F - a concentrations are elevated in septic patients and the concentrations are significantly correlated with the severity of illness (Waage et al., 1987; Cannon et al., 1990). In partial disagreement with these reports, it has been shown that there is no clear relationship between the levels of TNF-a produced by endotoxin and the endotoxin-induced mortality (Feuerstein et al., 1990). However this finding does not negate the possibility that T N F - a has a role in endotoxin shock. It is possible that TNF-c~ plays a permissive role in inducing the release of other factors (i.e. interleukin 1 and 6, lymphotoxin), that are relevant to shock. Therefore these findings, taken together, indicate that TNF-a has a key role in endotoxin shock and suggest that drugs able to reduce T N F - a production might have beneficial effects in septic shock. In keeping with this hypothesis, it has been recently
108
shown that pentoxifylline exerts salutary effects in endotoxin shock by inhibiting T N F - a production (Schade, 1990). Moreover the toxic effects of T N F - a in vivo are partially prevented by cyclooxygenase inhibitors such as indomethacin or ibuprofen (Kettelhut et al., 1987). These findings justify the search for therapeutic approaches addressed to the aim of reducing the production of this cytokine. Cloricromene, originally called AD6, is a coumarine derivative which produces coronary dilation (Aporti et al., 1978), stimulates prostacyclin production from human cultured endothelial cells (Dejana et al., 1982) and lowers thromboxane B 2 synthesis from platelets (Galli et al., 1980). Furthermore cloricromene improves survival rate, decreases plasma myocardial depressant factor accumulation and reduces macrophage TxB 2 synthesis in splanchnic artery occlusion shock (Sturniolo et al., 1989; Squadrito et al., 1988). In the present study, the activity of cloricromene was studied in a rat model of endotoxin shock and was found to provide beneficial therapeutic effects and to reduce TNF-cr production.
2. Materials and methods
2.1. Endotoxin shock procedure Male S p r a g u e - D a w l e y rats (200-220 g), fed on a standard diet and with water ad libitum, were used. Environmental conditions were standardized, including a room temperature of 22+_ 2°C and 12 h artificial lighting. Endotoxin shock was induced by administering a single i.v. dose of 2(1 m g / k g of Salmonella enteriditis endotoxin (Boivin preparation, Difco Lab.). Control rats received an equal volume of vehicle (NaC1).
2.2. Surt,it,al ecaluation Fifteen minutes after endotoxin injection, control rats received a 1 m l / k g 0.9% NaCI i.v. bolus and treated rats received cloricromene (0.5, 1 and 2 m g / k g ) as an i.v. bolus. Survival rate was evaluated for 48 h after endotoxin administration.
arterial pressure (MAP) in mm Hg. Rats were subjected to the same experimental protocol described above.
2.4. Leukocyte count A group of rats (n = 18) were used to study leukopenia. Tail vein blood samples for leukocyte count (Brechner and Cronkite, 1950) were taken before endotoxin injection and 1 and 2 h after endotoxin challenge. Leukocyte count (WBC × 103/mm ~) is reported as mean +_ S.D.
2.5. Peritoneal macrophage actit,ity Animals subjected to endotoxin shock were killed by decapitation 2 h after endotoxin challenge. Macrophages, obtained by peritoneal lavage with RPMI 1640, were counted and diluted to 106/ml. The cells were then incubated in Petri dishes at 37°C in a humidified 5% CO 2 atmosphere for 1 h. Phagocytic activity was evaluated by using a micromethod based on vital staining of ingested yeasts with acridine orange (Altavilla et al., 1989). After adhesion of the cells to coverslips in Petri dishes for 1 h and removal of non-adherent cells by washing, peritoneal cells were incubated for 1 h at 37°C in a humidified 5% CO 2 atmosphere with an opsonized Candida albicans suspension containing 5 x 10 ~' y e a s t s / m l . Coverslips were washed with R P M I 1640 and stained with acridine orange (1.44 rag/100 ml). Under ultraviolet light, the nuclei of viable C. albicans and of peritoneal cells stained green, whereas dead C. albicans stained reddish yellow. A differential count of cells containing yeast was performed. At [east 200 macrophages were scored on each slide, and all experimental procedures were performed in triplicate. For the in vitro study, shocked and sham-shocked rats were not treated, and peritoneal macrophages, collected from these animals, were first incubated in Petri dishes at 37°C, as described above, and then incubated with cloricromene at different concentrations (25, 50 and 100 IxM) or with RPMI 1640 (control) for 2 h. Phagocytosis is reported in percentage.
2.6. Biological assay for TNF-a acticity 2.3. Arterial blood pressure A second group of rats (n = 16) was used to monitor blood pressure as described elsewhere (Caputi et al., 1980). Briefly, animals were anesthetized with urethane (1.3 g / k g ) and a cannula (PE 50) was inserted into the left common carotid artery. The arterial catheter was connected to a pressure transducer. The pressure pulse triggered a cardiotachometer, and arterial blood pressure, monitored for 6 h, was displayed on channels of a polygraph. Arterial blood pressure is reported as mean
Killing of L929 mouse tumor cells was used to measure T N F levels in plasma and in the peritoneal macrophage supernatants on the basis of a standard assay (Ruff and Gifford, 1980). L929 cells in R P M I 1640 medium containing 5% fetal calf serum were seeded at 3 x 10 4 cells per well in 96-well microdilution plates and incubated overnight at 37°C in an atmosphere of 5% CO 2 in air. Serial 1 : 2 dilutions of serum (drawn 2 h after endotoxin) and supernatants of peritoneal macrophage (collected 2 h after endotoxin
109
as previously described by Altavilla et al., 1989), were made in the above-described medium containing 1.0 p.g of actinomycin D per ml and 100 p.l volumes of each dilution were added to the wells. On the next day, cell survival was assessed by fixing and staining the cells with crystal violet (0.2% in 20% methanol), and 0.1 ml of 1% sodium dodecyl sulphate was added to each well to solubilize the stained cells. The absorbance of each well was read at 490 nm with a model BT-100 Microelisa Autoreader. Percentage of cytotoxicity was calculated as 1 - ( [ A 4 9 0 of sample/A490 of control)] × 100. One TNF-c~ unit was defined as the amount of T N F - a giving 50% cell cytotoxicity. TNF-c~ content in the samples was calculated by comparison to a calibration curve performed with recombinant murine TNF-a (Nuclear Laser Medicine, Milan, Italy). To verify if the cytotoxicity tested was due to the presence of TNF-a or to other factor(s), the assay was repeated after the positive samples were neutralized with a polyclonal rabbit anti-murine T N F antiserum which cross-reacts with rat TNF (Remick et al., 1990; Warren et al., 1989). A negative assay after this treatment confirmed that the cytotoxic activity was due to TNF. 2. 7. Drug Cloricromene (Fidia Laboratories, Abano Terme, Italy) was dissolved in 0.9% NaCI solution and injected into the tail vein in a volume of 0.1 m l / 1 0 0 g body weight 15 min after endotoxin injection. For all experiments, cloricromene was dissolved in 0.9% NaCI solution just before injection. 2.8. Statistics The difference between the means of the two groups was evaluated using A N O V A followed by Bonferoni's test and was considered significant when P < 0.05. For survival data, statistical analysis was performed using the Fisher's exact probability test.
3. Results
3.1. Survit,al rate Table 1 shows the ratio of animals surviving in each group to the total number of animals throughout the experimental period. In the control groups, all animals survived. The vehicle-treated endotoxin group had 1 and 0 survivors out of 8 within 24 and 48 h, respectively. Cloricromene, administered curative[y, significantly protected against endotoxin shock. The effect was dose-dependent and the endotoxin group given with the highest dose of cloricromene (2 mg/kg) had 9 survivors and 8 survivors out of 9 after 24 and 48 h, respectively. Therefore, we chose the 2 m g / k g dose as the most effective and we employed it in the functional studies. 3.2. Arterial blood pressure Rats (n = 8) injected with endotoxin experienced a sharp and long-lasting decrease in MAP (fig. 1). Cloricromene (2 mg/kg; n = 8), given 15 rain after endotoxin, significantly inhibited the sustained decrease in MAP. 3.3. Peritoneal macrophage activity in vitro and leukocyte count This experiment was carried out to investigate the effect of cloricromene on macrophage functions. The phagocytotic activity of macrophages from sham rats treated with vehicle averaged 54.7_+ 2.3%. Macrophages from shocked rats showed reduced phagocytotic activity (table 2). Cloricromene (25, 50 and 100 p.M) added in vitro to peritoneal macrophages from shocked rats, significantly increased phagocytosis. Endotoxin administration in rats (n = 9) significantly impaired the white cell count (fig. 2). This marked
TABLE 1 Effect of cloricromene (CRM) on survival rate in endotoxin shock. The statistical analysis was performed using the Fisher's probability test. Animals received cloricromene 15 rain after endotoxin injection (20 m g / k g i.v.). Control animals received the vehicle (1 m l / k g i.v. of a 0.9% of an NaCI solution) instead of cloricromene. Hours after endotoxin
Control + vehicle Control + CRM 2 m g / k g Endotoxin + vehicle Endotoxin + CRM 0.5 m g / k g Endotoxin + CRM 1 m g / k g Endotoxin + CRM 2 m g / k g
3
6
12
24
48
7/7 7/7
7/7 7/7
7/7 7/7
7/7 7/7
7/7 7/7
8/8
3/8
1/8
l/s
8/8
4/8
1/8
I/8
s/8
8/8"
6/8 ~'
3/8
I/8 I/8
9/9
9/9 b
9/9 c
9/9 c
8/9 ~
~' P < 0.05 vs. endotoxin +vehicle; b p < 0.01 vs. endotoxin +vehicle; c p < 0.001 vs. endotoxin + vehicle.
ll0 20,0 2O 17,5
10 {3..
,< z~ 15,(
i -10°
12,~
O ~lO,(
20
7,5
-30
40
~)
30
dO
;0
180
3;0
MINUTES Fig. 1. Effects of vehicle (el or cloricromene (11) (2 m g / k g , 15 min after endotoxin challenge) on mean arterial blood pressure in rats subjected to endotoxin shock. Each value represents the mean _+S.D. of eight experiments. * P < 0.005 vs. vehicle-treated rats.
5,0
2,5 0,0 HOURS Fig. 3. Time course of TNF-c~ appearance in the serum of vehicle (o) or cloricromene ( D ) ( 2 mg/kg, 15 min after endotoxin challenge)treated endotoxin-shocked rats. All values are the means_+ S.D. of 10 experiments. * P < 0.01 vs. vehicle treated rats.
TABLE 2 In vitro effects of cloricromene (CRM) on endotoxic-shocked rat peritoneal macrophages a. Drug
phagocytosis in
leukopenia was significantly inhibited by the injection of cloricromene (n = 9).
Phagocytosis
(%) Controls Endotoxin Endotoxin Endotoxin Endotoxin
CRM 100/zM Vehicle CRM 25 tzM CRM 50/xM CRM 100 ~ M
54.7+ 2.3 19.2 + 2.0 31.5 _+2.7 35.6 _+3.7 41.1 _+2.1
3.4. Serum TNF-a and macrophage TNF-a ex t'ieo b c d d
a Macrophages were collected 2 h after endotoxin challenge and then incubated with CRM (/xM) for 2 h. Each value represents the mean + S.D. of six experiments, b p < 0.01 vs. control + CRM; c p < 0.005 vs. endotoxin +vehicle; d p < 0.001 vs. endotoxin +vehicle.
18
TABLE 3
16
Effect of cloricromene (CRM) on peritoneal macrophage TNF-a release.
14
Peritoneal macrophages were collected 2 h after endotoxin administration. Each point represents the mean_+S.D, of 10 experiments. Animals received cloricromene 15 min after endotoxin injection (20 m g / k g i.v.). Control animals received the vehicle (1 m l / k g i.v. of a 0.9% NaCI solution) instead of cloricromene. N.D. = below 1 U / m l of TNF-ot.
tO r~
~
T N F - a was undetectable in the serum and in the supernatants of peritoneal macrophages of control rats treated with vehicle (n = 10) or cloricromene (n = 10) (fig. 3; table 3). In endotoxin-shocked rats (n = 10) serum T N F - a progressively rises upon endotoxin administration and reaches the maximum increase 120 min after endotoxin challenge (fig. 3). At this time T N F - a was significantly increased in the supernatants of peritoneal macro-
8
Macrophage TNF-a (U/ml) lh
2h
Time
Fig. 2. Effects of vehicle (o) or cloricromene ( D ) (2 m g / k g , 15 min after endotoxin challenge) on white blood cells (WBC) in rats subjected to endotoxin shock. All values are the means + S.D. of nine experiments. * P < 0.001 vs. vehicle treated rats.
Controls + vehicle Controls + CRM 2 m g / k g Endotoxin + vehicle Endotoxin + CRM 2 m g / k g P < 0.001 vs. endotoxin +vehicle.
N.D. N.D. 150 + 7 18 + 3 a
111
15C
IOC E a.
z[..,
Buffer
25 50 CLORICROMENE
100pM
Fig. 4. In vitro effect of c l o r i c r o m e n e (25, 50 and 1 0 0 / x M ) on T N F - a r e l e a s e by m a c r o p h a g e s c o l l e c t e d from e n d o t o x i n - s h o c k e d rats. * P < 0.05 vs. buffer; * * P < 0.001 vs. buffer. E a c h point r e p r e s e n t s the m e a n + S.D. of six e x p e r i m e n t s .
phages collected from endotoxin-shocked rats (n = 10) (table 3). The administration of cloricromene, given curatively to endotoxin-shocked rats (n = 10), decreased TNF-a appearance in serum and reduced the cytokine levels in macrophage supernatants (fig. 3; table 3). 3.5. Macrophage TNF-a in uitro
In order to investigate whether cloricromene interferes with TNF-a release by macrophages, we primed macrophages with an in vivo endotoxin stimulus (20 mg/kg). TNF-a was significantly enhanced in the macrophage supernatants of endotoxin-treated rats (fig. 4). Cloricromene (25, 50 and 100 /xM) was added in vitro to peritoneal macrophages collected two hours after the endotoxin challenge. Under these experimental conditions cloricromene inhibited, in a dose-dependent manner, TNF-a release by macrophages (fig. 4).
4. Discussion
It has been suggested that endotoxin acts via endogenous mediators, mainly produced by mononuclear phagocytes (Nathan, 1987). Among these mediators, TNF-a plays a key role in the endotoxin effect. In fact many of the pathophysiological effects of endotoxemia may be induced in experimental animals by TNF-a adminis'tration (Beutler et al., 1985a). Systemic administration of recombinant human TNF-a to experimental animals produces hypotension, alterations in glucose homeostasis, hemoconcentration, metabolic acidosis, intravascular thrombosis, intestinal hypoperfusion, generation of other vasoactive substances and death (Beutler et al., 1985b; Tracey et al., 1987). TNF-a induces other cells to produce interleukin-1, platelet-
activating factor, eicosanoids and thromboplastin (Bachwich et al., 1986; Nawroth et al., 1986); enhances the adherence of polymorphonuclear leukocytes to vascular endothelium (Pohlman et al., 1986); and specific antisera raised against this cytokine reduce the morbidity and the mortality of endotoxin shock (Tracey et al., 1987). Furthermore the involvement of TNF-a in other models of experimental shock, such as splanchnic artery occlusion shock, has been recently shown (Squadrito et al., 1991). Cloricromene is a drug with proved efficacy in several models of experimental shock (Sturniolo et al., 1989; Sturniolo et al., 1991), but the effect of cloricromene in endotoxin shock had not yet been investigated. Our results indicate that cloricromene exerts protective and curative properties in experimental endotoxemia in the rat: as a matter of fact cloricromene, given curatively, prevented endotoxin-induced lethality. The effect was quite dramatic and 8 animals out of 9 were still alive 48 h after endotoxin challenge. Endotoxin administration produced an increase in the serum level of TNF-a. The time course of TNF-a appearance in the serum showed that the highest concentration of the cytokine was achieved within 2 h. TNF-a was also significantly enhanced in supernatants of peritoneal macrophages collected 2 h after endotoxin administration and cloricromene, given curatively, blunted TNF-a in both serum and macrophage supernatants. Since TNF-a has been shown to be involved in the lethality of endotoxin shock (Tracey et al., 1987), cloricromene's protective effect on survival rate in endotoxin-shocked rats may be ascribed, at least in part, to a reduction in the production a n d / o r release of this inflammatory cytokine. Indeed, the maximum levels of TNF-a in the present study did not correlate in time with succumbing of the animals. This discrepancy would negate a role of TNF-a in septic shock. However, as suggested by others (Mozes et al., 1991; Feuerstein et al., 1990), TNF-a might have a permissive role in stimulating the release of other mediators (i.e. platelet-activating factor; eicosanoids) relevant to endotoxic shock. Endotoxin shock is also characterized by a severe leukopenia and marked hypotension (Matera et al., 1988). In keeping with these findings our endotoxin-shocked rats had decreased white blood cell counts and reduced levels of arterial blood pressure. Both these changes were significantly improved by cloricromene treatment. Recombinant human TNF-a administration in conscious rats produces hypotension and leukopenia (Turner et al., 1989): therefore, cloricromene's salutary activity against the pathological sequelae of endotoxin shock might be due to its inhibitory effect on the inflammatory cytokine. However the main finding of the present paper is the evidence that cloricromene acts as an inhibitor of TNF-a production a n d / o r release.
112 C l o r i c r o m e n e , in fact, possesses i n t e r e s t i n g p r o p e r ties which might be relevant to T N F - a inhibition. As a m a t t e r of fact, this c o u m a r i n e derivative increases m a c r o p h a g e phagocytosis i m p a i r e d by an e x p e r i m e n t a l m o d e l of shock ( S q u a d r i t o et al., 1989), a n d r e d u c e s t h r o m b o x a n e B 2 in m a c r o p h a g e s u p e r n a t a n t s of s p l a n c h n i c artery occlusion-shocked rats ( S t u r n i o l o et al., 1989), thus i n d i c a t i n g that the drug may interfere with p r i m e d m o n o n u c l e a r phagocytes. In k e e p i n g with these previous results, c l o r i c r o m e n e in vitro significantly i n c r e a s e d p e r i t o n e a l m a c r o p h a g e f u n c t i o n depressed by e n d o t o x i n shock. T h e s e findings p r o m p t e d us to investigate w h e t h e r c l o r i c r o m e n e may affect the p r o d u c t i o n of T N F - a by p r i m e d p e r i t o n e a l macrophages. T N F - a was significantly e n h a n c e d in perit o n e a l m a c r o p h a g e s collected from e n d o t o x i n - t r e a t e d rats a n d c l o r i c r o m e n e , a d d e d in vitro, significantly b l u n t e d T N F - a in m a c r o p h a g e s u p e r n a t a n t s , thus suggesting that the c o u m a r i n e derivative may be an inhibitor to T N F - a release. I n d e e d the possibility that a toxic effect of c l o r i c r o m e n e on m a c r o p h a g e s may kill the cells in t u r n m a k i n g the m o n o c y t e s u n a b l e to release T N F - a is easily r u l e d out since c l o r i c r o m e n e increases m a c r o p h a g e f u n c t i o n i m p a i r e d by s p l a n c h n i c artery occlusion shock ( S t u r n i o l o et al., 1989; S q u a d r i t o et al., 1989) and, m o r e specifically, e n d o t o x i n shock (see Results). Previous findings have suggested that s u b s t a n c e s c a p a b l e of i n c r e a s i n g cyclic A M P i n t e r f e r e with the synthesis of T N F - a in m a c r o p h a g e s ( K a t a k a m i et al., 1988). C l o r i c r o m e n e has b e e n previously r e p o r t e d to inhibit cyclic n u c l e o t i d e p h o s p h o d i e s t e r a s e s in h u m a n platelets, b u t the effect o n cyclic A M P - s p e c i f i c phosp h o d i e s t e r a s e was p r e s e n t at doses which were higher t h a n those used in the p r e s e n t study ( H a k i m et al., 1988). F u r t h e r m o r e , prostacyclin has b e e n also shown to r e d u c e T N F - a p r o d u c t i o n : in fact it has b e e n shown that the synthesis of p r o s t a g l a n d i n E 2 by m a c r o p h a g e s d o w n r e g u l a t e s the f o r m a t i o n of TNF-c~ ( K u n k e l et al., 1986). C l o r i c r o m e n e is able to i n d u c e the synthesis of prostacyclin in e n d o t h e l i a l cells ( D e j a n a et al., 1982). W h e t h e r c l o r i c r o m e n e activates this way in macrophages as well, thus i n d u c i n g the synthesis of an end o g e n o u s m o d u l a t o r of T N F - a p r o d u c t i o n is still u n clear. Finally, a c i o r i c r o m e n e i n h i b i t o r effect o n the f o r m a t i o n of m R N A for T N F - a , c a n n o t at this m o m e n t be excluded. I n c o n c l u s i o n our results clearly indicate that cloric r o m e n e exerts a curative effect in e n d o t o x i n shock. A l t h o u g h several questions r e g a r d i n g the m e c h a n i s m of c l o r i c r o m e n e ' s effect r e m a i n u n c l e a r , these data e n h a n c e the e x p e r i m e n t a l evidence suggesting that this c o u m a r i n e derivative might be a useful drug in shock conditions.
References Altavilla, D., M.C. Berlinghieri, S. Seminara, D. Ianello, A. Foca' and P. Mastroeni, 1989, Different effects of bacterial lipopolysaccharide on superoxide anion production by macrophages from normal and tumor bearing rats, Immunopharmacology, 17, 99. Aporti, F., M. Finesso and L. Granata, 1978, Effects of 8-monochloro-3-beta-diethylaminoethyl-4-methyl-7-ethoxycarboxyl-methoxy coumarine (AD6) on coronary circulation in the dog, Pharmacol. Res. Commun. 10, 469. Bachwich, P.R., S.W. Chensue and J.W. Laurick, 1986, Tumor necrosis factor stimulates interleukin-1 and prostaglandin E 2 production in resting macrophages, Biochem. Biophys. Res. Commun. 136, 94. Bagby, G.J., J.J. Thompson, L.A. Wilson and R.M. Hazel, 1989, Temporal response of lipoprotein lipase-suppressing mediator and tumor necrosis factor in lipopolysaccharide-injected and infused rats, Circ. Shock 28, 385. Beutler, B. and A. Cerami, 1987, Cachectin: more than a tumor necrosis factor, New Engl. J. Med. 316, 379. Beutler, B., I.W. Milsark and A. Cerami, 1985a, Cachectin/tumor necrosis factor: production, distribution, and metabolic fate in vivo, J. lmmunol. 135, 3972. Beutler, B., W. Milsark and A. Cerami, 1985b, Passive immunization against cachectin tumor necrosis factor (TNF) protects mice from the lethal effect of endotoxin, Science 229, 868. Brechner, G. and E.P. Cronkite, 1950, Morphology and enumeration of human blood platelets, J. Appl. Physiol. 3, 365. Cannon, J.G., R.G. Tompkins, J.A. Gelfand, H.R. Michie, G.G. Stanford, J.W.M. Van der Meer, S. Endres, G. Lonneman, J. Corsetti, B. Chernow, D.W. Wilmore, S.M. Wolff, J.F. Burke and C.A. Dinarello, 199(/, Circulating interleukin-1 and tumor necrosis factor in septic shock and experimental endotoxin fever, J. Infect. Dis. 161, 79. Caputi, A.P., F. Rossi, K. Karney and H.E. Brezenoff, 1980, Modulatory effect of brain acetylcholine on reflex-induced bradycardia and tachycardia in conscious rats, J. Pharmacol. Exp. Ther. 215, 309. Carswell, E.A., L.J. Old, R.L. Kassel, S. Green, N. Fiore, and B.D. Williamson, 1985, An endotoxin induced serum factor that causes necrosis of tumor, Proc. Natl. Acad. Sci. U.S.A. 72, 3666. Dejana, E., C. De Castellarnau, G. Balconi, D. Rotilio, A. Pietra and G. De Gaetano, 1982, AD6, a coronary dilating agent stimulates PGI2 production in rat aorta ex vivo and in human endothelial cells in culture, Pharmacol. Res. Commun. 14, 719. Feuerstein, G., J.M. Hallenbeck, B. Vanatta, R. Rabinovici, P.Y. Perera and S.T. Vogel, 1990, Effect of Gram-negative endotoxin on levels of serum corticosterone, TNF, circulating blood cells and survival of rats, Circ. Shock 30, 265. Galli, C., E. Agradi, A. Petroni and A. Socini, 1980, Effects 8-monochloro-3-beta-diethylamino-ethyl-4-methyl-7-ethoxycarbonylmethoxycoumarin (AD6) on aggregation, arachidonic acid metabolism and thromboxane B2 formation in human platelets, Pharmacol. Res. Commun. 12, 329. Hakim, G., D. Fiorentini, A. Falasca, M. Prosdocimi and C.A. Rossi, 1988, Effect of AD6 (8-monochloro-3-beta-diethylamino-ethyl-4methyl-7-ethoxycarbonylmethoxycoumarin) on cyclic nucleotide phosphodiesterases in human platelets, Experientia 44, 226. Katakami, Y., Y. Nakao, T. Koizumi, N. Katakami, R. Ogawa and T. Fujita, 1988, Regulation of tumor necrosis factor production by mouse peritoneal macrophages: The role of cellular cyclic AMP, Immunology 64, 719. Kettelhut, I.C., W. Fiers and A.L. Goldberg, 1987, The toxic effects of tumor necrosis factor in vivo and their prevention by cyclooxygenase inhibitors, Proc. Natl. Acad. Sci. U.S.A. 84, 4273.
113 Kunkel, S.L., R.C. Wiggins, S.W. Chensue and J. Larrick, 1986, Regulation of macrophage tumor necrosis factor production by prostaglandin E 2, Biochem. Biophys. Res. Commun. 137, 404. Mannel, D.N., H. Northoff, F. Bauss and W. Falk, 1987, Tumor necrosis Factor: A cytokine involved in toxic effects of endotoxin, Rev. Inf. Dis. 9 (Suppl. 5), 5602. Matera, G., J.A. Cook, R.A. Hennigar, G.E. Tempel, W.C. Wise, T.D. Oglesby and P.V. Halushka, 1988, Beneficial effects of a 5-1ipoxygenase inhibitor in endotoxic shock in the rat, J. Pharmacol. Exp. Ther. 247, 363. Mozes, T., S. Ben-Efraim, C.J.A.M. Tak, J.P.C. Heiligers, P.R. Saxena and I.E. Bonta, 1991, Serum levels of tumor necrosis factor determine the fatal or non-fatal course of endotoxic shock, lmmunol. Lett. 27, 157. Nathan, C.F., 1987, Secretory products of macrophages, J. Clin. Invest. 79, 319. Nawroth, P., I. Bunk and D. Handley, 1986, Tumor necrosis factor/cachectin interacts with endothelial cell receptors to induce release of interleukin-1, J. Exp. Med. 163, 1363. Pohlman, T.N., K.A. Stanness, P.G. Beatty, H.D. Ochs and J.M. Harlan, 1986, An endothelial cell surface factor(s) induced in vitro by lipopolysaccharide, interleukin-1 and tumor necrosis factor-a increases neutrophil adherence by a CDw 18-dependent mechanism, J. lmmunol. 136, 4548. Remick, D.G., R.M. Strieter, M.K. Eskandari, D.T. Nguyen, M.A. Genord, C.L. Raiford and S.L. Kunkel, 1990, Role of tumor necrosis factor alpha in lipopolysaccharide induced pathologic alterations, Am. J. Pathol. 136, 49. Ruff, M.R. and G.E. Gifford, 1980, Purification and physicochemical characterization of rabbit tumor necrosis factor, J. Immunol. 125, 671. Schade, U.S., 1990, Pentoxifylline increases survival in murine endotoxin shock and decreases formation of tumor necrosis factor, Circ. Shock 31, 171. Squadrito, F., R. Sturniolo, D. Altavilla and A.P. Caputi, 1988, AD6,
a coumarine derivative, and other pharmacological approaches to splanchnic artery occlusion (SAO) shock therapy, J. Chemother. 4, 473. Squadrito, F., R. Sturniolo, D. Altavilla, G. Santoro, G.M. Campo, A. Arena and A.P. Caputi, 1991, Platelet activating factor involvement in splanchnic artery occlusion shock in the rats, Eur. J. Pharmacol. 192, 47. Sturniolo, R., F. Squadrito, D. Altavilla, G.R. Trimarchi, M. Prosdocimi and A.P. Caputi, 1989, Cloricromene improves survival rate and peritoneal macrophage function in splanchnic artery occlusion shock in rats, Circ. Shock 28, 267. Sturniolo, R., F. Squadrito, G.M. Campo, R. Vinci, A. Calatroni, M. Prosdocimi and A.P. Caputi, 1991, Protective effect of cloricromene, a coumarine derivative, in hypovolemic haemorrhagic shock in the rat, J. Cardiovasc. Pharmacol. 17, 261. Tracey, K,J., B. Beutler, S.F. Lowry, J. Merryweather, S. Wolpe, I.W. Milsark, R.J. Hariri, T.J. Fahey III, A. Zentella, J.D. Albert, G.T. Shires and A. Cerami, 1986, Shock and tissue injury induced by recombinant human cachectin, Science (Washington DC) 234, 470. Tracey, K.J., Y. Fong, D.G. Hesse, K.R. Manogue, A.T. Lee, G.C. Kuo, S.F. Lowry and A. Cerami, 1987, Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia, Nature 330, 662. Turner, C., K. Esser, E.B. Wheeldon, M.J. Slivjak and E.F. Smith, III, 1989, The cardiovascular and pulmonary effects of human recombinant tumor necrosis factor in the conscious rat, Circ. Shock 28, 369. Waage, A,, A. Halstensen and T. Espevik, 1987, Association between tumor necrosis factor in serum and fetal outcome in patients with meningococcal disease, Lancet 1,355. Warren, J.S., K.R. Yabroff, D.G. Remick, S.W. Chenuse, S.L. Kunkel, R.G. Kunkel, K.J. Johnson and P.A. Ward, 1989, Tumor necrosis factor partecipates in the pathogenesis of acute immune complex alveolitis in the rat, J. Clin. Invest. 84, 1873.
107
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52212
Cloricromene, a coumarine derivative, protects against lethal endotoxin shock in rats F r a n c e s c o S q u a d r i t o , D o m e n i c a AltaviUa, G i u s e p p e M. C a m p o , G i o a c c h i n o Calapai, M a r i a p a t r i z i a I o c u l a n o , Basilia Zingarelli, A n t o n i n o Saitta 1, M a r c o P r o s d o c i m i 2 a n d Achille P. C a p u t i Institutes of Pharmacology and 1 Chair of Medical Therapeutics, School of Medicine, University of Messina, Messina, Italy and e Fidia Research Laboratories, Abano Terme, Italy Received 10 May 1991, revised MS received 6 August 1991, accepted 15 October 1991
Endotoxin shock was induced in male rats by an intravenous (i.v.) injection of Salmonella enteriditis lipopolysaccharide (LPS; 20 mg/kg i.v.). Survival rate, macrophage and serum tumor necrosis factor (TNF-a), mean arterial blood pressure (MAP) and white blood cell count were then evaluated. Furthermore the in vitro effect of cloricromene on peritoneal macrophage phagocytosis and TNF-a release by primed peritoneal macrophages was investigated. LPS admininistration caused animal death (0% survival 24 h after endotoxin challenge), hypotension, marked leukopenia and increased the levels of TNF-a in both serum and macrophage supernatants. Cloricromene administration (0.5, 1 and 2 mg/kg i.v. 15 rain after endotoxin) protected against LPS-induced lethality (100% survival rate 24 h after endotoxin challenge), reverted LPS-induced hypotension and leukopenia, and decreased TNF-a in both serum and macrophage supernatants. Finally, cloricromene, added in vitro to peritoneal macrophages collected from endotoxin-treated rats increased macrophage phagocytosis and reduced TNF-a formation by activated mononuclear phagocytes. Our data suggest that cloricromene increases survival rate in endotoxin shock through an inhibition of TNF-a production. Cloricromene; Tumor necrosis factor (TNF-c~); Endotoxin shock
1. Introduction
Tumor necrosis factor (TNF-a) is a protein secreted by primed macrophages upon the activation by endotoxin or other stimuli (Carswell et al., 1985). This cytokine has pleiotropic activities both in vitro and in vivo, including cytotoxic effects against tumors and virus-infected cells, stimulation of interleukin-1 secretion, prostaglandin E 2 and collagen production, inhibition of lipogenic gene expression in adipocites, and stimulation of various immune effector cells (Beutler and Cerami, 1987). Moreover TNF-a appears to be a common mediator of inflammation and endotoxin-induced shock (Beutler et al., 1985a; Mannel et al., 1975). As a matter of fact, elevated serum levels of TNF-a are present in endotoxin shock (Bagby et al., 1989) and the administration of specific antibodies raised against T N F - a protects animals from the lethal
Correspondence to: F. Squadrito, Institute of Pharmacology, School of Medicine, Piazza XX Settembre 4, 98122 Messina, Italy. Tel. 39.90.675 833 or 712 533, fax 39.90.661 029.
effects of endotoxic shock (Tracey et al., 1987). In addition, infusion of recombinant human TNF-a in experimental animals has been shown to reproduce many of the deleterious effects of endotoxin (Tracey et al, 1986). Clinical evidence also suggests that plasma T N F - a concentrations are elevated in septic patients and the concentrations are significantly correlated with the severity of illness (Waage et al., 1987; Cannon et al., 1990). In partial disagreement with these reports, it has been shown that there is no clear relationship between the levels of TNF-a produced by endotoxin and the endotoxin-induced mortality (Feuerstein et al., 1990). However this finding does not negate the possibility that T N F - a has a role in endotoxin shock. It is possible that TNF-c~ plays a permissive role in inducing the release of other factors (i.e. interleukin 1 and 6, lymphotoxin), that are relevant to shock. Therefore these findings, taken together, indicate that TNF-a has a key role in endotoxin shock and suggest that drugs able to reduce T N F - a production might have beneficial effects in septic shock. In keeping with this hypothesis, it has been recently
108
shown that pentoxifylline exerts salutary effects in endotoxin shock by inhibiting T N F - a production (Schade, 1990). Moreover the toxic effects of T N F - a in vivo are partially prevented by cyclooxygenase inhibitors such as indomethacin or ibuprofen (Kettelhut et al., 1987). These findings justify the search for therapeutic approaches addressed to the aim of reducing the production of this cytokine. Cloricromene, originally called AD6, is a coumarine derivative which produces coronary dilation (Aporti et al., 1978), stimulates prostacyclin production from human cultured endothelial cells (Dejana et al., 1982) and lowers thromboxane B 2 synthesis from platelets (Galli et al., 1980). Furthermore cloricromene improves survival rate, decreases plasma myocardial depressant factor accumulation and reduces macrophage TxB 2 synthesis in splanchnic artery occlusion shock (Sturniolo et al., 1989; Squadrito et al., 1988). In the present study, the activity of cloricromene was studied in a rat model of endotoxin shock and was found to provide beneficial therapeutic effects and to reduce TNF-cr production.
2. Materials and methods
2.1. Endotoxin shock procedure Male S p r a g u e - D a w l e y rats (200-220 g), fed on a standard diet and with water ad libitum, were used. Environmental conditions were standardized, including a room temperature of 22+_ 2°C and 12 h artificial lighting. Endotoxin shock was induced by administering a single i.v. dose of 2(1 m g / k g of Salmonella enteriditis endotoxin (Boivin preparation, Difco Lab.). Control rats received an equal volume of vehicle (NaC1).
2.2. Surt,it,al ecaluation Fifteen minutes after endotoxin injection, control rats received a 1 m l / k g 0.9% NaCI i.v. bolus and treated rats received cloricromene (0.5, 1 and 2 m g / k g ) as an i.v. bolus. Survival rate was evaluated for 48 h after endotoxin administration.
arterial pressure (MAP) in mm Hg. Rats were subjected to the same experimental protocol described above.
2.4. Leukocyte count A group of rats (n = 18) were used to study leukopenia. Tail vein blood samples for leukocyte count (Brechner and Cronkite, 1950) were taken before endotoxin injection and 1 and 2 h after endotoxin challenge. Leukocyte count (WBC × 103/mm ~) is reported as mean +_ S.D.
2.5. Peritoneal macrophage actit,ity Animals subjected to endotoxin shock were killed by decapitation 2 h after endotoxin challenge. Macrophages, obtained by peritoneal lavage with RPMI 1640, were counted and diluted to 106/ml. The cells were then incubated in Petri dishes at 37°C in a humidified 5% CO 2 atmosphere for 1 h. Phagocytic activity was evaluated by using a micromethod based on vital staining of ingested yeasts with acridine orange (Altavilla et al., 1989). After adhesion of the cells to coverslips in Petri dishes for 1 h and removal of non-adherent cells by washing, peritoneal cells were incubated for 1 h at 37°C in a humidified 5% CO 2 atmosphere with an opsonized Candida albicans suspension containing 5 x 10 ~' y e a s t s / m l . Coverslips were washed with R P M I 1640 and stained with acridine orange (1.44 rag/100 ml). Under ultraviolet light, the nuclei of viable C. albicans and of peritoneal cells stained green, whereas dead C. albicans stained reddish yellow. A differential count of cells containing yeast was performed. At [east 200 macrophages were scored on each slide, and all experimental procedures were performed in triplicate. For the in vitro study, shocked and sham-shocked rats were not treated, and peritoneal macrophages, collected from these animals, were first incubated in Petri dishes at 37°C, as described above, and then incubated with cloricromene at different concentrations (25, 50 and 100 IxM) or with RPMI 1640 (control) for 2 h. Phagocytosis is reported in percentage.
2.6. Biological assay for TNF-a acticity 2.3. Arterial blood pressure A second group of rats (n = 16) was used to monitor blood pressure as described elsewhere (Caputi et al., 1980). Briefly, animals were anesthetized with urethane (1.3 g / k g ) and a cannula (PE 50) was inserted into the left common carotid artery. The arterial catheter was connected to a pressure transducer. The pressure pulse triggered a cardiotachometer, and arterial blood pressure, monitored for 6 h, was displayed on channels of a polygraph. Arterial blood pressure is reported as mean
Killing of L929 mouse tumor cells was used to measure T N F levels in plasma and in the peritoneal macrophage supernatants on the basis of a standard assay (Ruff and Gifford, 1980). L929 cells in R P M I 1640 medium containing 5% fetal calf serum were seeded at 3 x 10 4 cells per well in 96-well microdilution plates and incubated overnight at 37°C in an atmosphere of 5% CO 2 in air. Serial 1 : 2 dilutions of serum (drawn 2 h after endotoxin) and supernatants of peritoneal macrophage (collected 2 h after endotoxin
109
as previously described by Altavilla et al., 1989), were made in the above-described medium containing 1.0 p.g of actinomycin D per ml and 100 p.l volumes of each dilution were added to the wells. On the next day, cell survival was assessed by fixing and staining the cells with crystal violet (0.2% in 20% methanol), and 0.1 ml of 1% sodium dodecyl sulphate was added to each well to solubilize the stained cells. The absorbance of each well was read at 490 nm with a model BT-100 Microelisa Autoreader. Percentage of cytotoxicity was calculated as 1 - ( [ A 4 9 0 of sample/A490 of control)] × 100. One TNF-c~ unit was defined as the amount of T N F - a giving 50% cell cytotoxicity. TNF-c~ content in the samples was calculated by comparison to a calibration curve performed with recombinant murine TNF-a (Nuclear Laser Medicine, Milan, Italy). To verify if the cytotoxicity tested was due to the presence of TNF-a or to other factor(s), the assay was repeated after the positive samples were neutralized with a polyclonal rabbit anti-murine T N F antiserum which cross-reacts with rat TNF (Remick et al., 1990; Warren et al., 1989). A negative assay after this treatment confirmed that the cytotoxic activity was due to TNF. 2. 7. Drug Cloricromene (Fidia Laboratories, Abano Terme, Italy) was dissolved in 0.9% NaCI solution and injected into the tail vein in a volume of 0.1 m l / 1 0 0 g body weight 15 min after endotoxin injection. For all experiments, cloricromene was dissolved in 0.9% NaCI solution just before injection. 2.8. Statistics The difference between the means of the two groups was evaluated using A N O V A followed by Bonferoni's test and was considered significant when P < 0.05. For survival data, statistical analysis was performed using the Fisher's exact probability test.
3. Results
3.1. Survit,al rate Table 1 shows the ratio of animals surviving in each group to the total number of animals throughout the experimental period. In the control groups, all animals survived. The vehicle-treated endotoxin group had 1 and 0 survivors out of 8 within 24 and 48 h, respectively. Cloricromene, administered curative[y, significantly protected against endotoxin shock. The effect was dose-dependent and the endotoxin group given with the highest dose of cloricromene (2 mg/kg) had 9 survivors and 8 survivors out of 9 after 24 and 48 h, respectively. Therefore, we chose the 2 m g / k g dose as the most effective and we employed it in the functional studies. 3.2. Arterial blood pressure Rats (n = 8) injected with endotoxin experienced a sharp and long-lasting decrease in MAP (fig. 1). Cloricromene (2 mg/kg; n = 8), given 15 rain after endotoxin, significantly inhibited the sustained decrease in MAP. 3.3. Peritoneal macrophage activity in vitro and leukocyte count This experiment was carried out to investigate the effect of cloricromene on macrophage functions. The phagocytotic activity of macrophages from sham rats treated with vehicle averaged 54.7_+ 2.3%. Macrophages from shocked rats showed reduced phagocytotic activity (table 2). Cloricromene (25, 50 and 100 p.M) added in vitro to peritoneal macrophages from shocked rats, significantly increased phagocytosis. Endotoxin administration in rats (n = 9) significantly impaired the white cell count (fig. 2). This marked
TABLE 1 Effect of cloricromene (CRM) on survival rate in endotoxin shock. The statistical analysis was performed using the Fisher's probability test. Animals received cloricromene 15 rain after endotoxin injection (20 m g / k g i.v.). Control animals received the vehicle (1 m l / k g i.v. of a 0.9% of an NaCI solution) instead of cloricromene. Hours after endotoxin
Control + vehicle Control + CRM 2 m g / k g Endotoxin + vehicle Endotoxin + CRM 0.5 m g / k g Endotoxin + CRM 1 m g / k g Endotoxin + CRM 2 m g / k g
3
6
12
24
48
7/7 7/7
7/7 7/7
7/7 7/7
7/7 7/7
7/7 7/7
8/8
3/8
1/8
l/s
8/8
4/8
1/8
I/8
s/8
8/8"
6/8 ~'
3/8
I/8 I/8
9/9
9/9 b
9/9 c
9/9 c
8/9 ~
~' P < 0.05 vs. endotoxin +vehicle; b p < 0.01 vs. endotoxin +vehicle; c p < 0.001 vs. endotoxin + vehicle.
ll0 20,0 2O 17,5
10 {3..
,< z~ 15,(
i -10°
12,~
O ~lO,(
20
7,5
-30
40
~)
30
dO
;0
180
3;0
MINUTES Fig. 1. Effects of vehicle (el or cloricromene (11) (2 m g / k g , 15 min after endotoxin challenge) on mean arterial blood pressure in rats subjected to endotoxin shock. Each value represents the mean _+S.D. of eight experiments. * P < 0.005 vs. vehicle-treated rats.
5,0
2,5 0,0 HOURS Fig. 3. Time course of TNF-c~ appearance in the serum of vehicle (o) or cloricromene ( D ) ( 2 mg/kg, 15 min after endotoxin challenge)treated endotoxin-shocked rats. All values are the means_+ S.D. of 10 experiments. * P < 0.01 vs. vehicle treated rats.
TABLE 2 In vitro effects of cloricromene (CRM) on endotoxic-shocked rat peritoneal macrophages a. Drug
phagocytosis in
leukopenia was significantly inhibited by the injection of cloricromene (n = 9).
Phagocytosis
(%) Controls Endotoxin Endotoxin Endotoxin Endotoxin
CRM 100/zM Vehicle CRM 25 tzM CRM 50/xM CRM 100 ~ M
54.7+ 2.3 19.2 + 2.0 31.5 _+2.7 35.6 _+3.7 41.1 _+2.1
3.4. Serum TNF-a and macrophage TNF-a ex t'ieo b c d d
a Macrophages were collected 2 h after endotoxin challenge and then incubated with CRM (/xM) for 2 h. Each value represents the mean + S.D. of six experiments, b p < 0.01 vs. control + CRM; c p < 0.005 vs. endotoxin +vehicle; d p < 0.001 vs. endotoxin +vehicle.
18
TABLE 3
16
Effect of cloricromene (CRM) on peritoneal macrophage TNF-a release.
14
Peritoneal macrophages were collected 2 h after endotoxin administration. Each point represents the mean_+S.D, of 10 experiments. Animals received cloricromene 15 min after endotoxin injection (20 m g / k g i.v.). Control animals received the vehicle (1 m l / k g i.v. of a 0.9% NaCI solution) instead of cloricromene. N.D. = below 1 U / m l of TNF-ot.
tO r~
~
T N F - a was undetectable in the serum and in the supernatants of peritoneal macrophages of control rats treated with vehicle (n = 10) or cloricromene (n = 10) (fig. 3; table 3). In endotoxin-shocked rats (n = 10) serum T N F - a progressively rises upon endotoxin administration and reaches the maximum increase 120 min after endotoxin challenge (fig. 3). At this time T N F - a was significantly increased in the supernatants of peritoneal macro-
8
Macrophage TNF-a (U/ml) lh
2h
Time
Fig. 2. Effects of vehicle (o) or cloricromene ( D ) (2 m g / k g , 15 min after endotoxin challenge) on white blood cells (WBC) in rats subjected to endotoxin shock. All values are the means + S.D. of nine experiments. * P < 0.001 vs. vehicle treated rats.
Controls + vehicle Controls + CRM 2 m g / k g Endotoxin + vehicle Endotoxin + CRM 2 m g / k g P < 0.001 vs. endotoxin +vehicle.
N.D. N.D. 150 + 7 18 + 3 a
111
15C
IOC E a.
z[..,
Buffer
25 50 CLORICROMENE
100pM
Fig. 4. In vitro effect of c l o r i c r o m e n e (25, 50 and 1 0 0 / x M ) on T N F - a r e l e a s e by m a c r o p h a g e s c o l l e c t e d from e n d o t o x i n - s h o c k e d rats. * P < 0.05 vs. buffer; * * P < 0.001 vs. buffer. E a c h point r e p r e s e n t s the m e a n + S.D. of six e x p e r i m e n t s .
phages collected from endotoxin-shocked rats (n = 10) (table 3). The administration of cloricromene, given curatively to endotoxin-shocked rats (n = 10), decreased TNF-a appearance in serum and reduced the cytokine levels in macrophage supernatants (fig. 3; table 3). 3.5. Macrophage TNF-a in uitro
In order to investigate whether cloricromene interferes with TNF-a release by macrophages, we primed macrophages with an in vivo endotoxin stimulus (20 mg/kg). TNF-a was significantly enhanced in the macrophage supernatants of endotoxin-treated rats (fig. 4). Cloricromene (25, 50 and 100 /xM) was added in vitro to peritoneal macrophages collected two hours after the endotoxin challenge. Under these experimental conditions cloricromene inhibited, in a dose-dependent manner, TNF-a release by macrophages (fig. 4).
4. Discussion
It has been suggested that endotoxin acts via endogenous mediators, mainly produced by mononuclear phagocytes (Nathan, 1987). Among these mediators, TNF-a plays a key role in the endotoxin effect. In fact many of the pathophysiological effects of endotoxemia may be induced in experimental animals by TNF-a adminis'tration (Beutler et al., 1985a). Systemic administration of recombinant human TNF-a to experimental animals produces hypotension, alterations in glucose homeostasis, hemoconcentration, metabolic acidosis, intravascular thrombosis, intestinal hypoperfusion, generation of other vasoactive substances and death (Beutler et al., 1985b; Tracey et al., 1987). TNF-a induces other cells to produce interleukin-1, platelet-
activating factor, eicosanoids and thromboplastin (Bachwich et al., 1986; Nawroth et al., 1986); enhances the adherence of polymorphonuclear leukocytes to vascular endothelium (Pohlman et al., 1986); and specific antisera raised against this cytokine reduce the morbidity and the mortality of endotoxin shock (Tracey et al., 1987). Furthermore the involvement of TNF-a in other models of experimental shock, such as splanchnic artery occlusion shock, has been recently shown (Squadrito et al., 1991). Cloricromene is a drug with proved efficacy in several models of experimental shock (Sturniolo et al., 1989; Sturniolo et al., 1991), but the effect of cloricromene in endotoxin shock had not yet been investigated. Our results indicate that cloricromene exerts protective and curative properties in experimental endotoxemia in the rat: as a matter of fact cloricromene, given curatively, prevented endotoxin-induced lethality. The effect was quite dramatic and 8 animals out of 9 were still alive 48 h after endotoxin challenge. Endotoxin administration produced an increase in the serum level of TNF-a. The time course of TNF-a appearance in the serum showed that the highest concentration of the cytokine was achieved within 2 h. TNF-a was also significantly enhanced in supernatants of peritoneal macrophages collected 2 h after endotoxin administration and cloricromene, given curatively, blunted TNF-a in both serum and macrophage supernatants. Since TNF-a has been shown to be involved in the lethality of endotoxin shock (Tracey et al., 1987), cloricromene's protective effect on survival rate in endotoxin-shocked rats may be ascribed, at least in part, to a reduction in the production a n d / o r release of this inflammatory cytokine. Indeed, the maximum levels of TNF-a in the present study did not correlate in time with succumbing of the animals. This discrepancy would negate a role of TNF-a in septic shock. However, as suggested by others (Mozes et al., 1991; Feuerstein et al., 1990), TNF-a might have a permissive role in stimulating the release of other mediators (i.e. platelet-activating factor; eicosanoids) relevant to endotoxic shock. Endotoxin shock is also characterized by a severe leukopenia and marked hypotension (Matera et al., 1988). In keeping with these findings our endotoxin-shocked rats had decreased white blood cell counts and reduced levels of arterial blood pressure. Both these changes were significantly improved by cloricromene treatment. Recombinant human TNF-a administration in conscious rats produces hypotension and leukopenia (Turner et al., 1989): therefore, cloricromene's salutary activity against the pathological sequelae of endotoxin shock might be due to its inhibitory effect on the inflammatory cytokine. However the main finding of the present paper is the evidence that cloricromene acts as an inhibitor of TNF-a production a n d / o r release.
112 C l o r i c r o m e n e , in fact, possesses i n t e r e s t i n g p r o p e r ties which might be relevant to T N F - a inhibition. As a m a t t e r of fact, this c o u m a r i n e derivative increases m a c r o p h a g e phagocytosis i m p a i r e d by an e x p e r i m e n t a l m o d e l of shock ( S q u a d r i t o et al., 1989), a n d r e d u c e s t h r o m b o x a n e B 2 in m a c r o p h a g e s u p e r n a t a n t s of s p l a n c h n i c artery occlusion-shocked rats ( S t u r n i o l o et al., 1989), thus i n d i c a t i n g that the drug may interfere with p r i m e d m o n o n u c l e a r phagocytes. In k e e p i n g with these previous results, c l o r i c r o m e n e in vitro significantly i n c r e a s e d p e r i t o n e a l m a c r o p h a g e f u n c t i o n depressed by e n d o t o x i n shock. T h e s e findings p r o m p t e d us to investigate w h e t h e r c l o r i c r o m e n e may affect the p r o d u c t i o n of T N F - a by p r i m e d p e r i t o n e a l macrophages. T N F - a was significantly e n h a n c e d in perit o n e a l m a c r o p h a g e s collected from e n d o t o x i n - t r e a t e d rats a n d c l o r i c r o m e n e , a d d e d in vitro, significantly b l u n t e d T N F - a in m a c r o p h a g e s u p e r n a t a n t s , thus suggesting that the c o u m a r i n e derivative may be an inhibitor to T N F - a release. I n d e e d the possibility that a toxic effect of c l o r i c r o m e n e on m a c r o p h a g e s may kill the cells in t u r n m a k i n g the m o n o c y t e s u n a b l e to release T N F - a is easily r u l e d out since c l o r i c r o m e n e increases m a c r o p h a g e f u n c t i o n i m p a i r e d by s p l a n c h n i c artery occlusion shock ( S t u r n i o l o et al., 1989; S q u a d r i t o et al., 1989) and, m o r e specifically, e n d o t o x i n shock (see Results). Previous findings have suggested that s u b s t a n c e s c a p a b l e of i n c r e a s i n g cyclic A M P i n t e r f e r e with the synthesis of T N F - a in m a c r o p h a g e s ( K a t a k a m i et al., 1988). C l o r i c r o m e n e has b e e n previously r e p o r t e d to inhibit cyclic n u c l e o t i d e p h o s p h o d i e s t e r a s e s in h u m a n platelets, b u t the effect o n cyclic A M P - s p e c i f i c phosp h o d i e s t e r a s e was p r e s e n t at doses which were higher t h a n those used in the p r e s e n t study ( H a k i m et al., 1988). F u r t h e r m o r e , prostacyclin has b e e n also shown to r e d u c e T N F - a p r o d u c t i o n : in fact it has b e e n shown that the synthesis of p r o s t a g l a n d i n E 2 by m a c r o p h a g e s d o w n r e g u l a t e s the f o r m a t i o n of TNF-c~ ( K u n k e l et al., 1986). C l o r i c r o m e n e is able to i n d u c e the synthesis of prostacyclin in e n d o t h e l i a l cells ( D e j a n a et al., 1982). W h e t h e r c l o r i c r o m e n e activates this way in macrophages as well, thus i n d u c i n g the synthesis of an end o g e n o u s m o d u l a t o r of T N F - a p r o d u c t i o n is still u n clear. Finally, a c i o r i c r o m e n e i n h i b i t o r effect o n the f o r m a t i o n of m R N A for T N F - a , c a n n o t at this m o m e n t be excluded. I n c o n c l u s i o n our results clearly indicate that cloric r o m e n e exerts a curative effect in e n d o t o x i n shock. A l t h o u g h several questions r e g a r d i n g the m e c h a n i s m of c l o r i c r o m e n e ' s effect r e m a i n u n c l e a r , these data e n h a n c e the e x p e r i m e n t a l evidence suggesting that this c o u m a r i n e derivative might be a useful drug in shock conditions.
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