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...
To test the hypothesis that leukotriene (LT) B 4 antagonists may be clinically useful in the treatment of asthma, CP-105,696 was evaluated in vitro, using chemotaxis and flow cytometry assays, and in vivo, using a primate asthma model. CP
The interaction between leukotriene B4 (LTB4) and its metabolite, 20‐hydroxy LTB4 in the control of neutrophil emigration was examined in guinea‐pig skin.
Leukotriene B4 (10–300 ng) elicited a dose‐dependent increase in neutrophil infiltration (as measured by myeloperoxidase activity) 4 h after injection into guinea‐pig skin. In contrast, 20‐hydroxy LTB4 (30–1000 ng) displayed only weak inflammatory activity in this assay.
Although 20‐hydroxy LTB4 had low agonist activity, this metabolite caused a potent dose‐dependent inhibition of responses to LTB4 (100 ng), when administered systemically (ED50= 1.3 μg kg−1, s.c.) without significantly affecting neutrophil infiltration in response to C5a (2 μg). Systemic administration of 20‐carboxy LTB4 (10 μg) did not affect neutrophil accumulation in response to LTB4 or C5a. In addition, neither 15(S)‐hydroxy 5(S)‐HPETE(10 μg) nor lipoxin A4 (10 μg) inhibited responses to LTB4.
Addition of 20‐hydroxy LTB4 (10−11–10−8 m) to human blood prior to isolation of the neutrophils led to concentration‐dependent decrease in the number of LTB4 receptors and decreased chemotactic responsiveness to LTB4 without affecting responses to C5a. Incubation of blood with 20‐carboxy LTB4 (10−8 m) did not reduce LTB4 receptor number of chemotactic responsivness to LTB4.
These data indicate that although 20‐hydroxy LTB4 is a weak agonist at LTB4 receptors, it can desensitize neutrophils to the effects of LTB4 via down‐regulation of the high affinity receptor and thus provides evidence for a mechanism whereby inflammatory responses may be regulated.
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