Endogenous IL-10 Protects Mice from Death During Septic Peritonitis’ Tom van der Arnaud Marchant,* Wim A. Buurman,’ Lisa Berman,* Christopher V. Keogh,* Douglas D. Lazarus,* Lan Nguyen,* Michel Goldman,* Lyle 1. M~ldawer,~* and Stephen F. Lowry* IL-10 production during endotoxic shock is part of a protective mechanism that involves IL-10-induced inhibition of TNF synthesis. We sought to determine the role of IL-10 in septic peritonitis induced by cecal ligation and puncture (CLP). CLP led to a rapid induction of IL-10 mRNA in various organs of C57B1/6 mice. In liver, IL-10 mRNA was detectable within 1 h following CLP, while in spleen and lungs, IL-10 mRNA was detected from 2 to 4 h and onward. IL-10 protein became detectable in plasma 2 h after CLP, reaching peak concentrations after 12 h (12.7 2 5.7 ng/ml). Pretreatment (-2 h) with anti-IL-10 mAb resulted in higher plasma TNF levels following CLP when comparedwith mice treated with control mAb. Plasma IL-1 activity and IFN-.)I remained undetectable in virtually all mice. Anti-IL-10 enhanced mortality after CLP (p < 0.05 by log-rank test). Addition of anti-TNF mAb did not influence the increased mortality associated with anti-IL-10 treatment. Septic peritonitis is associated with sustained production of IL-10 in various organs, which serves to protect the host against lethality. The journal of Immunology, 1995, 155: 5397-5401. S epsis is associated with the induction of several cytokine species, including proinflammatory and anti-inflammatory mediators that interact in a highly complex network (I). In animals administered live bacteria or bacterial products i.v., the proinflammatory cytokine TNF plays a pivotal role in the devel- opment of tissue injury and death (2, 3). In contrast, the role of TNF during experimental sepsis in which systemic infection is produced from a localized source, such as peritonitis induced by i.p. administration of bacteria or by cecal ligation and puncture (CLP),4 is markedly different from its detrimental role after intra- venous challenges. Indeed, anti-TNF does not protect against mor- tality in lethal peritonitis models (4-7), and has been reported to convert a nonlethal model of CLP into a lethal model (8). IL-IO is an 18-kDa cytokine produced under different conditions of immune activation by a variety of cell types, including T cells, B cells, monocytes, and macrophages (9). Elevated levels of IL-10 have been detected in plasma of patients with sepsis (10, 1 I), and ‘Cornel1 University Medical College, Laboratoryof Surgical Metabolism,De- partment of Surgery, New York, NY 10021 ; ‘Academic Medical Center, Depart- ment of Internal Medicine, University of Amsterdam, Amsterdam, The Nether- lands; *UniversiteLibrede Bruxelles, Department of Immunology, Brussels, Belgium; and ‘University of Limburg, Department of Surgery, Maastricht, The Netherlands Received for publication May 19, 1995. Accepted for publication September 14, 1995. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertkement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Sciences to T.v.d.P., from the National Fund for Scientific Research, Belgium to ’ This work was supported by grants from the Royal Dutch Academy of Arts and A.M., and from the Biotech Program in lmmunotoxlcology (Grant B102-CT92- 0316)ofthe European CommissiontoA.M.,W.A.B.,andM.G.,Grant CM- 34695 to S.F.L., and GM-40586 to L.L.M. ’ Address correspondence and reprint requests to Dr. Tom van der Poll, Cornell University Medical College, Laboratory of Surgical Metabolism, Room LC-705, 1300 York Avenue, New York, NY 10021. pery, Cainesville, FL 32605. ’ Present address: University of Florida College of Medicine, Department of Sur- Abbreviation used in this paper: CLP, cecal ligation and puncture. Copyright 0 1995 by The American Association of Immunologists after administration of LPS to animals (12-14). It has been pro- posed that endogenously produced IL- IO serves a protective func- tion in models of endotoxic shock. Several lines of evidence in- dicate that this IL-10-induced protection during endotoxemia involves inhibition of TNF synthesis. First, recombinant IL-10 po- tently suppresses LPS-induced production of TNF by mononuclear cells in vitro (15, 16) and in mice in vivo (17, 18). Second, neu- tralization of endogenous IL-10 enhances mortality in endotox- emic mice and is associated with enhanced production of TNF (I 3, 19). Finally, blockade of this increased TNF activity in mice treated with anti-IL-10 mAb, prevents the increased LPS-induced mortality observed in mice treated with anti-IL-IO only (19). At present, the role of IL-10 in sepsis evolving from peritonitis is unknown. Therefore, in the present study we sought insight into the expression and role of endogenous IL-IO in septic peritonitis produced by CLP. Materials and Methods Antibody preparations JES5-2A5 is a neutralizing rat anti-mouse IL-10IgGl mAb (20). In a previous study, JES5-2A5 prevented the effects of exogenous IL-10 in mice (17). LO-DNP (a gift from HervC Bazin, Leuven, Belgium), a rat IgGl mAb, was used as isotype-matched control Ab (13, 19). TN3 is a neutralizing hamster anti-mouse TNF mAb (kindly provided by Celltech, Slough, UK) (21). L2 was used as isotype matched control Ab. Cecal ligation and puncture Female C57BL/6 mice (Charles River Labs., Wilmington, MA) weighing 16 to 20 g were used in all studies. They were housed (five per cage) in a temperature-controlled room with alternating 12 h light-dark cycles and were allowed to equilibrate for at least 5 days before the study. The animals were anesthesized by an i.p. injection of sodium pentobarbital (I mg per mouse; Fort Dodge Labs., Fort Dodge, WI). A small abdominal midline incision was made and the cecum identified and ligated below the ileocecal valve. The cecum was then punctured with a 20-gauge needle (through and through, to obtain two holes), and gentle pressure was applied on the li- gated cecum to exteriorize a small amount of feces. The cecum was then returned into the peritoneal cavity and the abdomen closed. Sham-operated mice underwent the same procedure without ligation and puncture of the cecum. After CLP, blood and organs were collected after 1, 2,4, 6, 12, and 0022-1 767/95/$02.00