IL-12 Suppression During Experimental Endotoxin Tolerance: Dendritic Cell Loss and Macrophage Hyporesponsiveness 1 Maria Wysocka, 2 * Susan Robertson, 3 * Helge Riemann, 4 * Jorge Caamano, ² Christopher Hunter, ² Agnieszka Mackiewicz,* Luis J. Montaner,* Giorgio Trinchieri, 5 * and Christopher L. Karp 6‡ Endotoxin tolerance, the transient, secondary down-regulation of a subset of endotoxin-driven responses after exposure to bac- terial products, is thought to be an adaptive response providing protection from pathological hyperactivation of the innate immune system during bacterial infection. However, although protecting from the development of sepsis, endotoxin tolerance also can lead to fatal blunting of immunological responses to subsequent infections in survivors of septic shock. Despite considerable experi- mental effort aimed at characterizing the molecular mechanisms responsible for a variety of endotoxin tolerance-related phe- nomena, no consensus has been achieved yet. IL-12 is a macrophage- and dendritic cell (DC)-derived cytokine that plays a key role in pathological responses to endotoxin as well as in the induction of protective responses to pathogens. It recently has been shown that IL-12 production is suppressed in endotoxin tolerance, providing a likely partial mechanism for the increased risk of sec- ondary infections in sepsis survivors. We examined the development of IL-12 suppression during endotoxin tolerance in mice. Decreased IL-12 production in vivo is clearly multifactorial, involving both loss of CD11c high DCs as well as alterations in the responsiveness of macrophages and remaining splenic DCs. We find no demonstrable mechanistic role for B or T lymphocytes, the soluble mediators IL-10, TNF-a, IFN-ab, or nitric oxide, or the NF-kB family members p50, p52, or RelB. The Journal of Immunology, 2001, 166: 7504 –7513. L ipopolysaccharide-containing endotoxins, major compo- nents of the outer membrane of Gram-negative bacteria, are potent activators of the innate immune system. As such, endotoxins are central to the pathogenesis of Gram-negative sepsis, a state marked by pathological release of proinflammatory mediators, lymphocyte and endothelial cell apoptosis, and dissem- inated intravascular coagulation (1– 4). Despite the fact that Gram- negative sepsis is a major cause of death throughout the world, such deleterious hyperactivation of the innate immune system is an uncommon result of exposure to endotoxins. In part, this is thought to be attributable to the phenomenon of endotoxin tolerance, the transient down-regulation of a subset of endotoxin-driven re- sponses after initial exposure to endotoxin (5, 6). In turn, it re- cently has become clear that endotoxin tolerance can itself be harmful. A significant percentage of survivors of sepsis have per- sistent endotoxin tolerance-related alterations in monocyte func- tion. Patients exhibiting this hypoinflammatory state, termed im- munological paralysis, have an elevated risk of succumbing to bacterial superinfection before discharge from the hospital (7–9). Endotoxin tolerance is demonstrable both in vivo and in vitro, at the level of whole organisms as well as in isolated cells. This apparent tachyphylaxis to LPS has been shown to correlate with suppression of proinflammatory cytokine production (5, 6, 10, 11). Monocyte/macrophages have been the prime targets of research in endotoxin tolerance. Tolerance in such cells clearly involves a dis- tinct functional state of activation or differentiation, not a global inhibition of function. Although the production of several proin- flammatory cytokines (e.g., TNF-a, IL-1, and IL-6) and antiin- flammatory cytokines (e.g., IL-10) is suppressed, the production of other mediators (e.g., IL-1RA) remains unaltered (6, 12). Such observations have led to the concept that endotoxin tolerance rep- resents a reprogramming of macrophages as an adaptive response to bacterial infection (13). It also has become clear that LPS-driven tolerance is but a particular instance of a more general phenome- non of activation-induced reprogramming of monocyte/ macrophages: similar effects are seen with other bacterial prod- ucts (e.g., macrophage-activating lipopeptides, 2 kDa (MALP-2) 7 ) and endogenous proinflammatory mediators (e.g., IL-1 plus TNF-a) (14 –16). In addition to monocyte/macrophages, other endotoxin- responsive cells also exhibit endotoxin tolerance. In this regard, the likely relevance of microbial product-induced changes in dendritic cell (DC) function and localization to endotoxin toler- ance in vivo recently has become clear (17–19) IL-12 is an immunoregulatory cytokine that is critical to the orchestration of cell-mediated immune responses in both the innate and adaptive immune systems. Produced largely by monocyte/ *The Wistar Institute, Philadelphia, PA 19104; ² University of Pennsylvania, Veter- inary School, Philadelphia, PA 19104; and ‡ Johns Hopkins University School of Medicine, Baltimore, MD 21205 Received for publication January 16, 2001. Accepted for publication April 2, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 These studies were supported by National Institutes of Health Grants AI34412 and CA20833 (to G.T.) and AI40507 and DE12167 (to C.L.K.) 2 Address correspondence to Dr. Maria Wysocka, University of Pennsylvania, 238 CRB, 415 Curie Boulevard, Philadelphia, PA 19104. E-mail address: Mwysocka@mail.med.upenn.edu 3 Current address: Center for Rheumatic Diseases, University Department of Medi- cine, Glasgow Royal Infirmary 10, Alexandra Parade, Glasgow, G 312ER U.K. 4 Current address: University of Munster, 56 Von-Esmarch strasse, 48149 Munster, Germany. 5 Current address: Schering-Plough, 27 Chemin des Peupliers-B.P.-11, 69571 Dard- illy Cedex, France. 6 Current address: Children’s Hospital Research Foundation, University of Cincinnati, TCHRF 1566, 3333 Burnet Avenue, Cincinnati, OH 45229 7 Abbreviations used in this paper: MALP-2, macrophage-activating lipopeptides, 2 kDa; DC, dendritic cell; TLR, Toll-like receptor; iNOS, inducible NO synthase. Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00