Platelet-driven leukotriene C4-mediated airway inflammation in mice is aspirin-sensitive and depends on T prostanoid receptors

Abstract

Cysteinyl leukotrienes (cysLTs) are bronchoconstricting lipid mediators that amplify eosinophilic airway inflammation by incompletely understood mechanisms. We recently found that LTC4, the parent cysLT, potently activates platelets in vitro and induces airway eosinophilia in allergen-sensitized and -challenged mice by a platelet- and type 2 cysLT receptor-dependent pathway. We now demonstrate that this pathway requires production of thromboxane A2 and signaling through both hematopoietic and lung tissue-associated T prostanoid (TP) receptors. Intranasal administration of LTC4 to OVA-sensitized C57BL/6 mice markedly increased the numbers of eosinophils in the bronchoalveolar lavage fluid, while simultaneously decreasing the percentages of eosinophils in the blood by a TP receptor-dependent mechanism. LTC4 upregulated the expressions of ICAM-1 and VCAM-1 in an aspirin-sensitive and TP receptor-dependent manner. Both hematopoietic and nonhematopoietic TP receptors were essential for LTC4 to induce eosinophil recruitment. Thus, the autocrine and paracrine functions of thromboxane A2 act downstream of LTC4/type 2 cysLT receptor signaling on platelets to markedly amplify eosinophil recruitment through pulmonary vascular adhesion pathways. The findings suggest applications for TP receptor antagonists in cases of asthma with high levels of cysLT production.

Figure 1

Effect of LTC₄ on eosinophilic pulmonary inflammation and platelet activation. OVA-sensitized C57BL/6 were challenged with 0.1% OVA on three successive days, with or without inhalation of LTC₄ (2.2 nmol) 12 h before each challenge. BAL fluid was collected 24 h after the last OVA challenge. A. Total cell counts, total eosinophils, and percentage of eosinophils and in BAL fluids. B. Levels of TXB₂ in BAL fluid lipid fractions. C. Levels of BAL fluid CXCL7. Results in A-C are from ten mice/group from two separate experiments. * p<0.05, *** p<0.001.

Figure 2

Effect of aspirin on LTC₄-induced pulmonary inflammation. Sensitized and challenged mice were treated with Lysine aspirin (1 mg/ml in drinking water) or water alone for 5 d before collection of BAL fluid. A. Numbers of total cells, eosinophils, neutrophils and lymphocytes in BAL fluids. B. Percentages of neutrophils and eosinophils in BAL fluid. C. Prostaglandins in BAL fluid lipid fractions and BAL fluid levels of CXCL7. Results in A-C are from ten mice/group from two separate experiments. * p<0.05, ** p<0.01, *** p<0.001.

Figure 3

Effect of TP receptor deletion on LTC₄-mediated eosinophil recruitment to the lung. A. Cytofluorographic detection of eosinophils and neutrophils (based on light scatter characteristics within the CD45+ gate) in the blood of representative C57BL/6 WT and tbxar^-/- mice challenged with OVA alone or OVA + LTC₄. B. Percentages of blood granulocytes identified as eosinophils from the same groups of mice. C. Total BAL fluid cells, eosinophils, and lymphocytes from sensitized WT and tbxar^-/- mice challenged with OVA with or without LTC₄. BAL fluid levels of TXB₂ and CXCL7 are shown from the same mice. Results in A-C are from 10 mice/group from two separate experiments. * p<0.05, ** p<0.01.

Figure 4

LTC₄-mediated upregulation of pulmonary ICAM-1 and VCAM-1 proteins. Lysates were prepared from whole lung of the indicated strains of sensitized mice obtained 24 h after challenge with OVA with or without LTC₄. Some mice received aspirin in their drinking water. A. Western blots showing ICAM-1, VCAM-1, and GAPDH from the lungs of representative mice. B. Quantitative densitometry from the lungs of 5 mice/group. C. Immunohistochemical stains of lungs of representative OVA-sensitized mice challenged with low dose OVA either with vehicle or with LTC4. * p<0.05, ** p<0.01.

Figure 5

Contributions from hematopoietic and non-hematopoietic TP receptors to LTC₄-induced pulmonary inflammation. Lethally irradiated mice from the indicated recipient strains received bone marrow cells from the indicated donors. Ten weeks later, the mice were sensitized and challenged with OVA with or without LTC₄. A. Total BAL fluid cell counts and B. BAL eosinophil counts and percentages are displayed for 5 mice in each group. * p<0.05, ** p<0.01, *** p<0.001.