Anesthesiology 2005; 102:768 –73 © 2005 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Endotoxemia-induced Lymphocyte Apoptosis Is Augmented by a Hyperinsulinemic–Euglycemic Clamp Jeppe Sylvest Nielsen, M.B.,* Anders Larsson, D.M.Sc.,† Vibeke Brix-Christensen, M.D., Ph.D.,‡ Jens Randel Nyengaard, D.M.Sc.,§ Thomas Ledet, D.M.Sc., Else Tønnesen, D.M.Sc.# Background: Sepsis and endotoxemia are associated with lymphocyte apoptosis. This has been regarded as harmful, con- tributing to further immune suppression in already immune- compromised patients. Because normalization of blood glucose improves outcome in critically ill patients, the authors hypoth- esized that one of the effects of insulin and normoglycemia would be inhibition of lymphocyte apoptosis. Therefore, in this experimental study in pigs, the authors examined the separate and combined effects of acute endotoxemia and a hyperinsu- linemic– euglycemic clamp (HEC) on lymphocyte apoptosis. Methods: After 60 min of stabilization, 38 anesthetized and mechanically ventilated pigs (weight, 35– 40 kg) were divided (by randomization performed before the experiment) into four groups and were then studied for 570 min. Group 1 received no intervention. Group 2 received a HEC (5 mM p-glucose, insulin infusion rate of 0.6 mU · kg 1 · min 1 ) for 570 min. Group 3 received a lipopolysaccharide infusion for 180 min. Group 4 was given a combination of a HEC and a lipopolysaccharide infusion. After the 570-min study period, the pigs were killed, and tissue was sampled from the spleen and frozen. In four sections of each sample, the apoptosis of B and T lymphocytes were analyzed using stereologic methods: The number of apo- ptotic B and T cells was estimated by fluorescence immunohis- tochemistry with anti–active caspase-3 and either anti-CD21 (B lymphocytes) or anti-CD3 (T lymphocytes). The number of apoptotic B and T lymphocytes was then compared using two- way analysis of variance, and the interaction between endotox- emia and the clamp (hyperinsulinemia and euglycemia) was investigated. Results: Endotoxemia induced apoptosis of B (P < 0.001) and T lymphocytes (P  0.016) in the spleen, and this effect was independent of the clamp. The ratios of apoptotic cells in the spleen tissue of pigs with and without endotoxemia were 2.4 (confidence interval, 1.7–3.4) and 1.6 (confidence interval, 1.1– 2.2) for B and T lymphocytes, respectively. Independent of endotoxin infusion, HEC increased the number of apoptotic lymphocytes (P  0.029 and P  0.038 for B and T lymphocytes, respectively). The ratios of the number of apoptotic spleen cells in pigs treated and not treated with HEC were 1.5 (confidence interval, 1.0 –2.1) and 1.5 (confidence interval, 1.0 –2.1) for B and T lymphocytes, respectively. Conclusion: In this porcine model, both endotoxemia and a HEC increased the number of apoptotic B and T lymphocytes in the spleen. Contrary to our hypothesis, lymphocyte apoptosis during acute endotoxemia was augmented by a HEC. INCREASED lymphocyte apoptosis has been proposed to be an important cause of immunodepression and mor- tality in critical illness. 1 An increased number of apopto- tic lymphocytes has been observed in experimental en- dotoxemia and was later also found in autopsy studies of septic patients. 2–4 A reversal of lymphocyte apoptosis with caspase-3 inhibitors showed increased survival among septic mice. 5 Furthermore, apoptotic cells have shown excessive immune-modulating mechanisms. Fadok et al. 6 revealed that macrophages digesting apo- ptotic cells inhibited the production of proinflammatory cytokines, and Sauter et al. 7 demonstrated that dendritic cells consuming apoptotic cells did not exhibit costimu- latory factors and were unable to activate T cells. We have previously found that a hyperinsulinemic– euglycemic clamp (HEC) attenuates plasma concentra- tions of tumor necrosis factor , glucagon, and free fatty acids in an acute endotoxemic porcine model. 8 It has also been shown that the use of insulin for normalization of blood glucose concentrations decreases mortality and morbidity in critically ill patients. 9 Although it has been suggested that the metabolic control, as reflected by normoglycemia, rather than the infused insulin dose per se, was related to the beneficial effects of intensive insulin therapy, 10,11 we hypothesized that the positive effects of a HEC could in part be ascribed to an insulin- induced inhibition of lymphocyte apoptosis. We tried to elicit this question by studying the effects of a HEC on splenic lymphocyte apoptosis in a porcine model of acute endotoxemia. Materials and Methods The model has been presented previously. 8 The Na- tional Committee on Animal Research Ethics (Copenha- gen, Denmark) approved the protocol, and the work was conducted according to the guidelines in the Guide for the Care and Use of Laboratory Animals. 12 Thirty-eight female Landrace pigs (weight, 35– 40 kg) were fasted overnight but were allowed free access to water. They were premedicated with 10 mg/kg ket- amine and 0.25 mg/kg midazolam intramuscularly. Fur- ther ketamine (10 mg/kg) was given intravenously be- fore intubation and mechanical ventilation. Anesthesia was maintained with a continuous intravenous infusion of 60 g · kg -1 ·h -1 fentanyl and 6 mg · kg -1 ·h -1 * Department of Anesthesiology and Intensive Care, Clinical Institute, and Research Laboratory for Biochemical Pathology, ‡ Department of Anesthesiology and Intensive Care and Clinical Institute, # Professor, Department of Anesthesiology and Intensive Care and Clinical Institute, § Associate Professor, Stereological Re- search and Electron Microscopy Laboratory,  Professor, Research Laboratory for Biochemical Pathology, Aarhus University Hospital. † Professor, Clinical Institute, Aarhus University Hospital, and Aalborg University Hospital, Aalborg, Denmark. Received from the Research Laboratory for Biochemical Pathology and the Clinical Institute, Aarhus University Hospital, Aarhus, Denmark. Submitted for publication July 20, 2004. Accepted for publication November 24, 2004. Supported by the Danish Research Council, grant Nos. 22-2-0010 and 22-02-0174, Aarhus University Research Foundation, Aarhus, Denmark; The A. P. Møller Foundation for the Advancement of Medical Science, Copenhagen, Denmark; and Police Inspector J.P.N. Colind and Wife Asmine Colinds Memorial Founda- tion, Aarhus, Denmark. Address reprint requests to Dr. Nielsen: Department of Anesthesiology and Inten- sive Care Medicine, Aarhus University Hospital, Nørrebrogade 44, DK-8000 Århus C, Denmark. Address electronic mail to: sylvest@ki.au.dk. Individual article reprints may be purchased through the Journal Web site, www.anesthesiology.org. Anesthesiology, V 102, No 4, Apr 2005 768