SummaryLipoxin A4 (LXA4) triggers selective responses with human neutrophils that are pertussis toxin sensitive and binds to high affinity receptors (Kd = 0.5 + 0.3 nM) that are modulated by stable analogues of guanosine 5'-triphosphate (GTP). Here, we characterized [11,12-3H]LXA4 specific binding with neutrophil granule and plasma membranes, which each display high affinity binding sites (Kd = 0.7 _+ 0.1 riM) that were regulated by GTP3,S. Since functional LXAa receptors are inducible in HL-60 cells, we tested orphan cDNAs encoding 7-transmembrane region receptors cloned from these cells for their ability to bind and signal with LXA4. Chinese hamster ovary (CHO) cells transfected with the orphan receptor eDNA (plNF114) displayed specific 3H-LXA4 high affinity binding (1.7 nM). When displacement of LXA4 binding with plNFl14-transfected CHO cells was tested with other eicosanoids, including LXB4, leukotriene D4 (LTD4), LTB4, or prostaglandin E2, only LTD4 competed with LXA4, giving a Ki of 80 nM. In transfected CHO cells, LXA4 also stimulated GTPase activity and provoked the release of esterified arachidonate, which proved to be pertussis toxin sensitive. These results indicate that plNF114 eDNA encodes a 7-transmembrane region-containing protein that displays high affinity for 3H-LXA4 and transmits LXA4-induced signals. Together, they suggest that the encoded protein is a candidate for a LXA4 receptor in myeloid cells.poxygenase (LO)l-derived eicosanoids are important lipid mediators (1). The 5-LO-derived products include leukotriene B4 (LTB4), a potent stimulus for phagocytic cells, and peptido-leukotrienes C4, D4, and E4, which are potent bronchoconstrictors and are associated with the pathogenesis of asthma (1). Lipoxins (LX) are a newer class of bioactive LO-derived products that are generated by the interactions of either 5-and 12-LO and/or 15-and 5-LO followed by subsequent reactions (for a review see reference 2). The LXs are functionally distinct from leukotrienes and other eicosanoids and are primarily generated in human tissues during cell-cell interactions that are exemplified by leukocyte-platelet interactions (2). LXA4 displays intriguing biological re-1 Abbreuiations used in this paper: AMP-PNP, 5'-adenylylimidodiphosphate; CHO, Chinese hamster ovary cells; DPBS/PBS, Dulbecco's PBS; PBS 2-without divalent cations; FPR, formyl peptide receptor; GTPyS, guanosine 5'-O-(3-thiotriphosphate); leukotriene B4 (LTB4), 5S,12R-dihydroxy-6,14-cis-8,10-trans-dihydroxyeicosatetraenoic acid; leukotriene D4 (LTD4), 5S-hydroxy-6R- lipoxin A4 (LXA4), 5S,6R,9, lipoxin B4 (LXB4), 5S,14R,10,-ciseicosatetraenoic acid; LO, lipoxygenase; PGE2, 9-oxy-11o~,15S-dihydroxy-5-cis-13-trans-prostadienoic acid; PT, Pertussis toxin holotoxin.sponses in several tissues (2), and with neutrophils they involve G protein-mediated signal transduction events (3-5). A LXA4 receptor is induced in HL-60 cells upon differentiation, and it activates phospholipase D (5). LXA4-induced lipid remodeling events are similar to those of other leuko...

The CC chemokine receptor-1 (CCR1) is a prime therapeutic target for treating autoimmune diseases. Through high capacity screening followed by chemical optimization, we identified a novel non-peptide CCR1 antagonist, R-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl]-2-oxoethoxy]phenyl]urea hydrochloric acid salt (BX 471). Competition binding studies revealed that BX 471 was able to displace the CCR1 ligands macrophage inflammatory protein-1␣ (MIP-1␣), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity (K i ranged from 1 nM to 5.5 nM). BX 471 was a potent functional antagonist based on its ability to inhibit a number of CCR1-mediated effects including Ca 2؉ mobilization, increase in extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated a greater than 10,000-fold selectivity for CCR1 compared with 28 G-protein-coupled receptors. Pharmacokinetic studies demonstrated that BX 471 was orally active with a bioavailability of 60% in dogs. Furthermore, BX 471 effectively reduces disease in a rat experimental allergic encephalomyelitis model of multiple sclerosis. This study is the first to demonstrate that a non-peptide chemokine receptor antagonist is efficacious in an animal model of an autoimmune disease. In summary, we have identified a potent, selective, and orally available CCR1 antagonist that may be useful in the treatment of chronic inflammatory diseases.It is clear that the inappropriate interaction of immune cells, such as T lymphocytes and monocytes, can lead to extensive inflammation and tissue destruction, which is a hallmark of several autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. Immune cells are sent on their destructive journey by chemoattractant molecules known as chemokines, which interact with and signal through specific cell surface chemokine receptors. Chemokine receptors belong to the GPCR 1 superfamily and have been viewed as attractive therapeutic targets by the pharmaceutical industry mainly because of their central role in regulating leukocyte trafficking. The premise that drugs that can inhibit the directed migration and activation of immune cells could be useful therapeutically has prompted the search for specific and highly potent chemokine receptor antagonists.Autoimmune diseases like multiple sclerosis and rheumatoid arthritis are characterized by interactions between invading T lymphocytes and tissue macrophages that result in extensive inflammation, tissue damage, and chronic disease pathologies. Numerous studies have demonstrated CCR1 expression in these cell types, and a variety of evidence provides strong in vivo concept validation for a role of this receptor in animal models of these diseases. For example, Karpus et al. (1, 2) were able to show in a mouse EAE model of multiple sclerosis that antibodies to MIP-1␣ prevented the development of both initial and relapsing paralytic disease as well as infiltration of mononuclear cells into the central nervous system. Treatment wit...

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There is potential for a variety of stem cell populations to mediate repair in the diseased or injured CNS; in some cases, this theoretical possibility has already transitioned to clinical safety testing. However, careful consideration of preclinical animal models is essential to provide an appropriate assessment of stem cell safety and efficacy, as well as the basic biological mechanisms of stem cell action. This article examines the lessons learned from early tissue, organ and hematopoietic grafting, the early assumptions of the stem cell and CNS fields with regard to immunoprivilege, and the history of success in stem cell transplantation into the CNS. Finally, we discuss strategies in the selection of animal models to maximize the predictive validity of preclinical safety and efficacy studies.