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, 153 (3), 528-35

Cysteinyl Leukotrienes Mediate the Enhancing Effects of Indomethacin and Aspirin on Eosinophil Production in Murine Bone Marrow Cultures

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Cysteinyl Leukotrienes Mediate the Enhancing Effects of Indomethacin and Aspirin on Eosinophil Production in Murine Bone Marrow Cultures

P X Elsas et al. Br J Pharmacol.

Abstract

Background: Prostaglandin E(2) (PGE(2)) suppresses, while indomethacin and aspirin enhance, eosinophil production in murine liquid bone-marrow cultures. Because cysteinyl leukotrienes (cys-LTs) enhance human eosinophil colony formation, we investigated whether the effects of indomethacin and aspirin on murine bone-marrow were due to blockade of PGE(2) production alone, or involved further promotion of cys-LTs production/signalling.

Experimental approach: BALB/c liquid bone-marrow cultures were established with IL-5, alone or associated with indomethacin, aspirin, or cys-LTs. The effects of preventing cys-LT production or signalling were assessed.

Key results: Indomethacin and aspirin counteracted the suppression of eosinophil production by exogenous PGE(2). LTD(4), LTC(4) and LTE(4) enhanced IL-5-dependent eosinophil production and further counteracted the effect of exogenous PGE(2). The 5-lipoxygenase activating protein (FLAP) inhibitor, MK886, a leukotriene synthesis inhibitor, zileuton, the CysLT(1) receptor antagonists, MK571 and montelukast, or inactivation of the LTC(4) synthase gene, abolished effects of indomethacin and aspirin. MK886 and zileuton were ineffective but MK571 and montelukast were effective, against LTD(4). Indomethacin, aspirin and LTD(4) failed to enhance eosinophil production in bone-marrow from CysLT1 receptor-deficient mice. Indomethacin, aspirin and LTD(4) no longer counteracted the effects of exogenous PGE(2) in the presence of MK571 and montelukast. MK886, MK571 and montelukast had no effect by themselves, or in association with PGE(2).

Conclusions and implications: Dependence on the FLAP/5-lipoxygenase/LTC(4) synthase pathway and receptor signalling shows that cyclo-oxygenase inhibitors act here through endogenous cys-LTs. While PGE(2) does not act by suppressing cys-LT production, cys-LTs override PGE(2) signalling. Eosinophil production is therefore coordinately regulated by both pathways.

Figures

Figure 1
Figure 1
Effect of PGE2 and NSAIDs on eosinophil production. (a and b) The data are mean±s.e.mean of the number of EPO+ cells in liquid cultures established from BALB/c bone marrow, in the presence of IL-5 alone, or associated with the following: PGE2, indomethacin (Indo) or aspirin (Asp) (a), or the indicated combinations of these agents (b). Data are derived from 4 to 5 experiments. *P<0.05; significant differences relative to the indicated controls. (c) Morphological features of EPO+ cells (bearing brown cytoplasmic granules, and donut-, circle- or C-shaped nuclei) generated in the various conditions described above counterstained with Harris' haematoxylin and photographed under immersion (× 1000). Counterstaining was kept to a minimum to allow easier visualization of individual EPO+ debris inside macrophages. EPO, eosinophil peroxidase; IL, interleukin; NSAID, nonsteroidal anti-inflammatory drug; PGE2, prostaglandin E2.
Figure 2
Figure 2
Effects of cys-LTs and MK886 on murine eosinophil production. The data are mean±s.e.mean of the number of EPO+ cells in liquid cultures established from BALB/c bone marrow, in the presence of IL-5 alone, or associated with the following: LTD4, LTC4 or LTE4 (10−7 M) (a); LTD4 (10−8 to 10−12M) (b); FLAP inhibitor MK886 (c) and 5-lipoxygenase inhibitor zileuton (d), with or without LTD4, indomethacin or aspirin at the indicated concentrations. Data are derived from 3 to 5 experiments. *P<0.05 relative to IL-5 controls. **P<0.05 relative to positive controls without MK886 or zileuton. cys-LT, cysteinyl-leukotriene; IL, interleukin.
Figure 3
Figure 3
Effects of cys-LT1 receptor antagonists on the responses to indomethacin, aspirin and LTD4. The data are mean±s.e.mean of the number of EPO+ cells in liquid cultures established from BALB/c bone marrow, in the presence of IL-5 alone, or associated with the following: LTD4 (10−8M), indomethacin (10−7M) or aspirin (10−8M), in the absence or in the presence of MK571 (a), 10−7M, or montelukast (Mkt; b and c), at the indicated concentrations. Data are derived from 3 to 5 experiments. *P<0.05 relative to IL-5 controls. **P<0.05 relative to positive controls without MK571 or Mkt. cys-LT, cysteinyl-leukotriene; EPO, eosinophil peroxidase; IL, interleukin.
Figure 4
Figure 4
Inability of indomethacin and aspirin to enhance eosinophil production in bone marrow from cys-LT1 receptor- or LTC4 synthase-deficient mice. The data are mean±s.e.mean of the number of EPO+ cells in liquid cultures established from bone marrow of control (Wild Type) or mutant (cys-LT1R−/− (a and b); LTC4s−/− (c)) mice from the BALB/c (a and c) or the C57BL/6 strains (b), in the presence of IL-5 alone, or associated with the following: LTD4, indomethacin or aspirin, at the indicated molar concentrations (10−7M in (c)). Data are derived from 3 to 4 experiments. *P<0.01 relative to the IL-5 control. cys-LT, cysteinyl-leukotriene; EPO, eosinophil peroxidase; IL, interleukin.
Figure 5
Figure 5
The cys-LTs and NSAIDs depend on cys-LT1 receptors to override the PGE2 suppressive signalling. The data are mean±s.e.mean of the number of EPO+ cells in liquid cultures established from BALB/c bone marrow, in the indicated conditions, which include the presence of an agonist (PGE2, 10−7M), of modulators of its activity (LTD4, indomethacin or aspirin) and of inhibitors of cys-LT-dependent pathways (MK886, montelukast) as well as the appropriate combinations of these substances. Asterisks indicate significant differences between the indicated groups. cys-LT, cysteinyl-leukotriene; NSAID, nonsteroidal anti-inflammatory drug; PGE2, prostaglandin E2.

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