Because of its requirement for signaling by multiple cytokines, Janus kinase 3 (JAK3) is an excellent target for clinical immunosuppression. We report the development of a specific, orally active inhibitor of JAK3, CP-690,550, that significantly prolonged survival in a murine model of heart transplantation and in cynomolgus monkeys receiving kidney transplants. CP-690,550 treatment was not associated with hypertension, hyperlipidemia, or lymphoproliferative disease. On the basis of these preclinical results, we believe JAK3 blockade by CP-690,550 has potential for therapeutically desirable immunosuppression in human organ transplantation and in other clinical settings.

SummaryThe Duffy antigen/receptor for chemokines (DARC), first identified on erythrocytes, functions not only as a promiscuous chemokine receptor but also as a receptor for the malarial parasite, Plasmodium vivax. The recent finding that DARC is ubiquitously expressed by endothelial cells lining postcapillary venules provides a possible insight into the function of this receptor because this anatomic site is an active interface for leukocyte trafficking. However, the biological significance of DARC is questionable since it has not yet been determined whether individuals lacking the expression of this protein on their erythrocytes (Duffy negative individuals), who are apparently immunologicaUy normal, express the receptor on endothelial cells. However, we report here that DARC is indeed expressed in endothelial cells lining postcapillary venules and splenic sinusoids in individuals who lack the erythrocyte receptor. These findings are based on immunohistochemical, biochemical, and molecular biological analysis of tissues from Duff-y negative individuals. We also present data showing that, in contrast to erythrocyte DAILC, cells transfected with DARC internalize radiolabeled ligand. We conclude that the DARC may play a critical role in mediating the effects of proinflammatory chemokines on the interactions between leukocyte and endothelial cells since the molecular pathology of the Duffy negative genotype maintains expression on the latter cell type.

Cultured monocytes and macrophages stimulated with LPS produce large quantities of proIL-1β, but release little mature cytokine to the medium. The efficiency at which the procytokine is converted to its active 17-kDa species and released extracellularly is enhanced by treating cytokine-producing cells with a secretion stimulus such as ATP or nigericin. To determine whether this need for a secretion stimulus extends to blood, individual donors were bled twice daily for 4 consecutive days, and the collected blood samples were subjected to a two-step IL-1 production assay. LPS-activated blood samples generated cell-free IL-1β, but levels of the extracellular cytokine were greatly increased by subsequent treatment with ATP or nigericin. Specificity and concentration requirements of the nucleotide triphosphate effect suggests a P2X7 receptor involvement. Quantities of IL-1β generated by an individual donor’s blood in response to the LPS-only and LPS/ATP stimuli were relatively consistent over the 4-day period. Between donors, consistent differences in cytokine production capacity were observed. Blood samples treated with ATP also demonstrated enhanced IL-18 production, but TNF-α levels decreased. Among leukocytes, monocytes appeared to be the most affected cellular targets of the ATP stimulus. These studies indicate that an exogenous stimulus is required by blood for the efficient production of IL-1β and IL-18, and suggest that circulating blood monocytes constitutively express a P2X7-like receptor.

Leukotriene B4 (LTB4) is a product of the 5-lipoxygenase pathway of arachidonic acid metabolism. LTB4 is a potent chemotactic factor for neutrophils and has been postulated to play an important role in a variety of pathological conditions including rheumatoid arthritis (RA), psoriasis, and inflammatory bowel disease. Rheumatoid arthritis (RA) is a chronic inflammatory polyarthritis that is inadequately treated with currently available drugs (1). One potential strategy to better treat this disease is to reduce the influx of leukocytes into the joint, since recent studies have shown that the extent of neutrophil infiltration into the joints of RA patients precedes clinical signs of inflammation and is predictive of pain (2). There are a number of mediators of the inflammatory response that could potentially be responsible for neutrophil accumulation, but leukotriene B4 (LTB4) is an attractive target since it is a potent chemotactic agent for human neutrophils (3), is produced in large amounts by these cells, and is found in the synovial fluid of patients with RA (4).In this paper, we describe experiments to assess the role of LTB4 in a murine model of RA, collagen-induced arthritis. The immunological and histological features of this model resemble those seen in RA patients. The strategy we employed was to use a LTB4 receptor antagonist to block the biological effects of endogenously produced LTB4. Several potent and selective LTB4 receptor antagonists, with a variety of structural types, have been reported (reviewed in ref. 5). However, there are no data on the efficacy of these agents in models of arthritis. CP-105,696, (+)-1-(3S,4R)-[3-(4-phenylbenzyl)-4-hydroxychroman-7-yl]cyclopentane carboxylic acid, is a newly discovered LTB4 receptor antagonist that has a high affinity OH for human and mouse LTB4 receptors and has a long plasma half-life in the mouse, which allows the maintenance of pharmacologically relevant concentrations of the drug with a once daily dosing protocol. Here we report the use of CP-105,696 to demonstrate the importance of LTB4 as a critical mediator in the pathogenesis of murine collagen-induced arthritis. MATERIALS AND METHODSIn Vitro LTB4 Receptor Ligand Binding Assays. The procedure for [3H]LTB4 binding was adapted from the method of Cheng and co-workers (6). Binding was performed in 150 ,ul in a buffer containing 50 mM Tris HCl (pH 7.3), 10 mM MgCl2, 9% methanol, 0.7 nM [3H]LTB4 (5920-7400 GBq/ mmol; New England Nuclear), and either 0.83 mg (murine spleen) or 0.13 mg (human neutrophil) of membrane per ml. Unlabeled LTB4 was added at a concentration of 5 ,uM to determine nonspecific binding. Incubations were carried out in microtiter plates at 4°C for 30 min and the bound ligand was separated from the free ligand with a Betaplate apparatus (Pharmacia LKB) with double-thickness glass fiber filter mats.In Vitro Chemotaxis Assay. Chemotaxis assays were performed as described by Harvath and co-workers (7). Neutrophils were isolated according to the procedure of Ferrante and Thong (8...