INCREASED EXPRESSION OF TOLL-LIKE RECEPTOR-2 AND -4 ON LEUKOCYTES FROM PATIENTS WITH SEPSIS Luc Härter,* Ladislav Mica,* Reto Stocker, † Otmar Trentz,* and Marius Keel* *Division of Trauma Surgery, University Hospital Zurich, Zurich, Switzerland; and † Division of Surgical Intensive Care, University Hospital Zurich, Zurich, Switzerland Received 13 Sep 2003; first review completed 6 Nov 2003; accepted in final form 29 Jul 2004 ABSTRACT—The reduced responsiveness of monocytes or granulocytes toward endotoxin (endotoxin tolerance) during sepsis may depend on Toll-like receptors (TLR). The expression of TLR-2 and TLR-4 was measured on neutrophils (PMN) and monocytes from patients with sepsis (n = 21) or healthy controls (n = 12). Leukocytes (1 × 10 6 /mL) were incubated at 37°C with or without a TLR-4 (LPS 1 μg/mL) or a TLR-2 ligand (MALP-2 2 nM). Surface expression of TLR-2 and TLR-4 at 0, 4, and 16 h was determined in FACS after staining with specific antibodies. The release of IL-8 and TNF- was measured by ELISA. Freshly isolated PMN from patients with sepsis exhibited significantly (P < 0.05) higher mean fluorescence for TLR-2 (78.0 ± 18.6) and TLR-4 (11.4 ± 2.3) than controls (12.8 ± 2.2 and 2.3 ± 0.4). Similarly, monocytes from patients exhibited higher TLR-2 and TLR-4 expression (300.8 ± 40.6 and 92.7 ± 12.1) than cells from controls (149.5 ± 27.1 and 52.2 ± 7.6). In patients with sepsis, expression of TLR-2 and TLR-4 on PMN increased during 16 h of incubation (106.2 ± 22.1 and 34.5 ± 5.3), whereas it remained unchanged in controls (19.3 ± 6.1 and 5.4 ± 1.9). Incubation with LPS or MALP-2 had no effect on TLR-4 or TLR-2 expression in cells from either controls or patients. Despite increased TLR expression in cells from patients with sepsis, the endotoxin-induced release of TNF- and IL-8 was indistinguishable from that in controls. Therefore, the endotoxin tolerance seen in patients with sepsis does not depend solely on TLR-2 or TLR-4 expression, and other mechanisms must be involved. KEYWORDS—Endotoxin, TLR, monocytes, neutrophils, sepsis INTRODUCTION Patients with severe trauma are at high risk to develop a systemic inflammatory response syndrome (SIRS), which may lead to multiple organ dysfunction syndrome (MODS) and organ failure (MOF). Additionally, bacterial infection can lead to sepsis in these patients, significantly increasing mortality in the late posttraumatic period (1, 2). The reaction of immune cells toward pathogens is initiated and controlled by receptors that specifically recognize bacterial endotoxins such as lipo- polysaccharide (LPS). The mammalian homologues of the Drosophila gene Toll were discovered as the signaling recep- tors for bacterial endotoxins and were named Toll-like recep- tors (TLR) (3–5). To date up to 10 different TLRs have been described, each with different ligand specificities (6, 7). Most prominent, the TLR-4 has been found to signal for the endo- toxin of gram-negative bacteria (LPS), whereas the TLR-2 is activated by different toxins from gram-positive bacteria such as the macrophage-activating lipopeptide-2 (MALP-2) from Mycoplasma fermentans (8, 9). A reduced cytokine response has been shown in whole blood from patients with sepsis or SIRS after trauma (10, 11), and a decreased cytokine production on restimulation of the cells with endotoxins was also shown (12–14). However, other cyto- kines, such as IL-18 plasma levels in septic patients (15) or early IL-8 plasma levels in patients with severe trauma (16), were significantly increased. The reduced responsiveness of isolated leukocytes or whole blood toward repeated endotoxin (LPS) stimulation has been known for a long time and was termed endotoxin tolerance (17). However, the exact mecha- nisms that regulate LPS-induced endotoxin tolerance in human monocytic cells are still not fully elucidated (18). In mice endotoxin tolerance can be induced by a preexposure to a sublethal dose of LPS before restimulation of the cells with an otherwise lethal dose of endotoxin (19, 20). The hypothesis that endotoxin tolerance is a result of Toll-like receptor down- regulation was strengthened by the finding that murine macro- phages showed down-regulated TLR-4 expression on endotox- in tolerance (21). In contrast, Medvedev and co-workers (22) reported an increase in TLR-2 expression in LPS-tolerant human mono- cytes, and just recently, an increase of tissue TLR-2 and TLR-4 mRNA and protein expression was reported in endotoxin- tolerant mice (23). However, despite multiple studies, the mechanisms regulating the reduced leukocyte response toward endotoxin remains unclear. The response of leukocytes toward endotoxin is of major importance and determines severity and development of the sepsis syndrome. Therefore, we investi- gated whether alterations of the TLR-2 and TLR-4 receptors can be found in leukocytes from patients with sepsis compared with healthy controls. Here we show that polymorph nuclear granulocytes (PMN) and monocytes from patients with sepsis show a significant up-regulation of the TLR-2 and TLR-4 receptors despite reduced or unchanged cytokine release. MATERIALS AND METHODS Patients The study population consisted of 21 patients admitted to the Surgical Intensive Care Unit of the University Hospital Zürich. The Acute Physiology and Chronic Address reprint requests to Luc Härter, PhD, Division of Trauma Surgery, University Hospital Zurich, Raemistrasse 100, CH-8091 Zurich, Switzerland. E-mail: luc.haerter@usz.ch. Supported by grant 32-52932.97 from Swiss Science Foundation, United Banks of Switzerland (UBS), Swiss Life, and Heuberg Stiftung. DOI: 10.1097/01.shk.0000142256.23382.5d SHOCK, Vol. 22, No. 5, pp. 403–409, 2004 403