PDF《Hepatitis, not for Septic Shock》

1997 and in revised form August 5, 1998) This study was presented as an oral communication at the American Thoracic So- ciety Annual Meeting

1997 in San Francisco (A263). Supported by grants from l'

Institut National de la Santé et de la Recherche Médi- cale, La ligue contre le Cancer, and l'

Association pour la Recherche contre le Cancer. A.M. is supported by Le Fonds D'

Etudes de l'

Assistance Publique, H?pi- taux de Paris. N.R. is a recipient of a fellowship from IFSBM. J.C.P. is supported by Assistance Publique, H?pitaux de Paris CANAM. Correspondence and requests for reprints should be addressed to Alexandre Mi- gnon, INSERM U129, 24, Rue du Faubourg Saint Jacques,

75014 Paris, France. E-mail: mignon@cochin.inserm.fr Mignon, Rouquet, Fabre, et al.: Caspase Inhibitors in Septic Shock

1309 the complement system, the caspases are arranged in a pro- teolytic cascade that serves to transmit and amplify numerous death signals. Among the terminator caspases has been clearly underlined the role of caspase-3 (CPP32)-like proteases, espe- cially in the liver (12). Direct inhibition of ICE and ICE- related proteases is now available with the use of synthetic peptides, such as YVAD-aldehyde (YVAD-CHO) or YVAD- chloromethylketone (YVAD-CMK), which bind to and block the catalytic site of ICE proteases (12). Using ICE inhibitor YVAD-CMK, we and others recently demonstrated its pro- tective effects on Fas (CD95)-mediated liver apoptosis in vitro as in vivo (13, 14). Characterization of this ICE protease in- hibitor prompted us to hypothesize whether YVAD-CMK would prevent caspases activation and lethality induced by low-dose LPS in D-GalN sensitized mice, thus confirming that this experimental setting accounts for a caspase-dependent fulminant apoptotic hepatitis induced by TNF-?, and not for septic shock. We also addressed the question of whether YVAD-CMK could also protect from endotoxic shock induced by high-dose LPS in mice unsensitized with D-GalN. Besides its determi- nant role during apoptosis, ICE is also involved in the patho- physiology of sepsis (15). Excessive production of the proin- flammatory cytokine IL-1? might constitute an important harmful factor during sepsis. This point has been underlined by: (1) the protective effect of a strategy aimed at inhibiting IL-1? signaling by IL-1Ra (16);

and (2) the observation that ICE-deficient mice challenged with lipopolysaccharide do not release any IL-1? and are resistant to lethal endotoxic shock (17). Moreover, YVAD-CHO was recently demonstrated to suppress, but only transiently, the release of IL-1? in vivo in a murine endotoxic shock without improving survival (18). We addressed the question of whether pharmacologic inhi- bition of ICE might be beneficial during sepsis, not only by blocking IL-1? release but also by inhibiting ICE and ICE- related pro-apoptotic proteolytic activities. In distinguishing between these two effects of YVAD-CMK on these two mod- els of sepsis, we wondered whether we could definitely estab- lish that LPS challenge combined with D-GalN does not ac- count for septic shock, but for acute TNF-?-related liver apoptotic destruction. METHODS Animals and Reagents Six- to eight-week-old female C57Bl/6 mice (18C20 g) obtained from IFA-CREDO (Paris, France) were used. All experimental procedures were carried out in compliance with French government regulations (Service Vétérinaires de la Santé et de la Production Animale, Min- istère de l'

Agriculture). Escherichia coli endotoxin (LPS, serotype 0127:B8) and D-galactosamine hydrochloride (D-GalN) were pur- chased from Sigma Chemical Co. (St. Louis, MO) and reconstituted in pyrogen-free sterile saline. Highly purified recombinant murine TNF-? was purchased from Genzyme (Paris, France) and reconsti- tuted in pyrogen-free sterile phosphate-buffered saline (PBS). Tet- rapeptide YVAD-CMK (Ac-Tyr-Val-Ala-Asp-chloromethylketone) was purchased from Bachem Biochimie SARL (Basel, Switzerland) and reconstituted in pyrogen-free sterile PBS (4 ?moles in