To test whether generation of oxygen radicals during postischemic reperfusion might promote peroxidation of cardiac membrane lipids, four groups of Langendorff-perfused rabbit hearts were processed at the end of (a) control perfusion, (b) 30 min of total global ischemia at 370C without reperfusion, (c) 30 min of ischemia followed by reperfusion with standard perfusate, (d) 30 min of ischemia followed by reperfusion with the oxygen radical scavenger human recombinant superoxide dismutase (h-SOD). The left ventricle was homogenized and tissue content of malonyldialdehyde (MDA), an end product of lipid peroxidation, was measured on the whole homogenate as well as on various subcellular fractions. Reperfusion was accompanied by a significant increase in MDA content of the whole homogenate and of the fraction enriched in mitochondria and lysosomes. This phenomenon was not observed in hearts subjected to ischemia but not reperfused, and was similarly absent in those hearts which received h-SOD at reflow. Reperfused hearts also had significantly greater levels of conjugated dienes (another marker of lipid peroxidation) in the mitochondrial-lysosomal fraction. Again, this phenomenon did not occur in ischemic hearts or in reperfused hearts treated with h-SOD. Unlike the effect on tissue MDA and conjugated dienes, reperfusion did not significantly stimulate release of MDA in the cardiac effluent. Treatment with h-SOD was also associated with significant improvement in the recovery ofcardiac function. In conclusion, these data directly demonstrate that postischemic reperfusion results in enhanced lipid peroxidation of cardiac membranes, which can be blocked by h-SOD, and therefore is most likely secondary to oxygen radical generation at reflow. (J. Clin.

Trafficking of dendritic cells (DCs) to peripheral tissues and to secondary lymphoid organs depends on chemokines and lipid mediators. Here, we show that bone marrow-derived DCs (BM-DCs) express functional leukotriene B 4 (LTB 4 ) receptors as observed in dose-dependent chemotaxis and calcium mobilization responses. LTB 4 , at low concentrations, promoted the migration of immature and mature DCs to CCL19 and CCL21, which was associated with a rapid (30-minute) increase of CCR7 expression at the mem- IntroductionDendritic cells (DCs) play a unique role in the activation of antigen-specific naive T lymphocytes. 1,2 To perform this function, antigen-loaded DCs travel from peripheral tissues to lymph nodes. This migration is dependent on the expression of CCR7 by DCs and the production of CCR7 ligands, namely CCL19, by stromal cells and mature DCs in the lymph node and CCL21 by afferent lymphatic cells. [3][4][5][6][7] However, the expression of CCR7 is not sufficient to ensure the migration of mature DCs. [8][9][10] Experimental evidence generated in vitro and in vivo has shown that CCR7 function is dependent on the presence of costimulatory signals, including cysteinyl-leukotrienes and their membrane transporter (MRP1), as well as prostaglandin E 2 . 9,10 These results indicate that in vivo, the local inflammatory microenvironment acts critically in regulating the migration of maturing DCs. Inflammatory cytokines and microbial agents are known to induce phospholipid metabolism and the activation of arachidonic acid cascade. 11 Leukotriene B 4 (LTB 4 ) is a potent chemoattractant generated by sequential actions of cytosolic phospholipase A 2 , 5-lipoxygenase and leukotriene A 4 hydrolase on membrane phospholipids. 11,12 Previous studies have shown that LTB 4 is a mediator of innate immunity, based on its chemotactic effect for phagocytic leukocytes. 13,14 Two distinct G protein-coupled receptors, the high affinity BLT1 15 and the low affinity BLT2, 16 have been identified as LTB 4 receptors. In this study, we show that LTB 4 is also a key mediator of adaptive immunity through the regulation of DC migration to secondary lymphoid organs. Materials and methodsAll animal studies and procedures were approved by the Animal Care and Use Committee of University of Louisville Research Resources Center. ReagentsMurine CCL3, CCL19, and CCL21 were from PeproTech (Rocky Hill, NJ). Recombinant murine granulocyte macrophage-colony-stimulating factor (rmGM-CSF), human Fms-like kinase-3 (Flt-3) ligand (hFlt3L), and recombinant murine tumor necrosis factor ␣ (rmTNF-␣) were from R&D Systems (Minneapolis, MN). Cytokines were endotoxin free as assessed by Limulus amebocyte assay (BioWhittaker, Walkersville. MD). LTB 4 was purchased from Cayman Chemical (Ann Arbor, MI). Dendritic cell cultureBLT1 Ϫ/Ϫ and BLT1/2 Ϫ/Ϫ17 mice backcrossed onto B6 background for 7 generations and C57/B6 mice from the National Cancer Institute (NCI) (wild-type [WT]) were used at 8 to 12 weeks of age. CD34 ϩ -derived myeloid DCs were generated by positive i...

Dendritic cells (DC) are potent antigen-presenting cells capable of inducing T and B responses and immune tolerance. We have characterized some aspects of energy metabolism accompanying the differentiation process of human monocytes into DC. Compared to precursor monocytes, DC exhibited a much larger number of mitochondria and consistently (i) a higher endogenous respiratory activity and (ii) a more than sixfold increase in ATP content and an even larger increase in the activity of the mitochondrial marker enzyme citrate synthase. The presence in the culture medium of rotenone, an inhibitor of the respiratory chain Complex I, prevented the increase in mitochondrial number and ATP level, without affecting cell viability. Rotenone inhibited DC differentiation, as revealed by the observation that the expression of CD1a, which is a specific surface marker of DC differentiation, was strongly reduced. Cells cultured in the presence of rotenone displayed a lower content of growth factor-induced, mitochondrially generated, hydrogen peroxide. A similar drop in ROS was observed upon addition of catalase, which caused functional effects similar to those produced by rotenone treatment. These results suggest that ROS play a crucial role in DC differentiation and that mitochondria are an important source of ROS in this process. © 2008 Elsevier Inc. All rights reserved.Keywords: Dendritic cells; Mitochondria; Reactive oxygen species; Oxidative phosphorylation; Free radicals Effective immune responses require correct localization and functioning of dendritic cells (DC). Dendritic cells are the most potent and versatile antigen-presenting cells, with a unique ability to induce specific immune responses as well as tolerance [1,2]. In peripheral tissues they reside in an immature state waiting for incoming antigens. After capturing and processing the antigens, DC undergo a maturation process which culminates in dramatic changes in functions and migratory properties [3,4]. The localization of mature DC to the draining lymph nodes coincides with the presentation of processed antigens to naïve T cells, triggering the initiation of specific immune responses [2,5]. An in vitro method to differentiate immature DC from CD14 + monocyte precursors cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) is very well established [6,7].Reactive oxygen species (ROS) have been identified as important second messengers involved in the transduction of several signaling pathways [8,9], gene expression, and cell proliferation [10]. Furthermore, recent studies have shown that growth factors, through the actions of their specific receptors, are able to increase

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Carbon monoxide releasing molecules (CORMs) are an emerging class of pharmaceutical compounds currently evaluated in several preclinical disease models. There is general consensus that the therapeutic effects elicited by the molecules may be directly ascribed to the biological function of the released CO. It remains unclear, however, if cellular internalization of CORMs is a critical event in their therapeutic action. To address the problem of cellular delivery, we have devised a general strategy which entails conjugation of a CO-releasing molecule (here a photoactivated CORM) to the 5'-OH ribose group of vitamin B12. Cyanocobalamin (B12) functions as the biocompatible water-soluble scaffold which actively transports the CORM against a concentration gradient into the cells. The uptake and cellular distribution of this B12-photoCORM conjugate is demonstrated via synchrotron FTIR spectromicroscopy measurements on living cells. Intracellular photoinduced CO release prevents fibroblasts from dying under conditions of hypoxia and metabolic depletion, conditions that may occur in vivo during insufficient blood supply to oxygen-sensitive tissues such as the heart or brain.