The cysteinyl leukotrienes cysLTs LTC LTD and LTE

The cysteinyl leukotrienes (cysLTs) LTC, LTD, and LTE are increased in the airways of asthmatic subjects, with the ability to induce bronchoconstriction, vascular permeability, and mucus secretion., , , , CysLTs are well established as mediators of allergic rhinitis and asthma on the basis of the efficacy of antileukotriene therapy,, , and they might play a role in the accumulation of inflammatory cells to allergic tissue. Eosinophils and CD34 cells, which express cysLT receptors, are found at increased numbers in allergic tissue and have been shown to migrate in response to LTD., , Furthermore, cysLTs have been shown to play a role in survival of eosinophils and proliferation of eosinophil-basophil progenitor cells. Basophil numbers have been shown to increase in the airway after inhalation of LTE, supporting the hypothesis that cysLTs might have a direct effect on basophil accumulation in the airways. CysLT receptors are expressed on a variety of peripheral blood leukocytes, but of interest, they are also expressed on cells that synthesize LTC, such as eosinophils and mast cells. The expression of cysLT receptors has not been evaluated on basophils, and efforts to do this have been hampered by the limited number of basophils that can be purified from peripheral blood. Because cysLT1 receptors are expressed on the shared eosinophil and basophil progenitor cells, CD34 cells, and both cysLT1 and cysLT2 receptors are present on mature eosinophils, we speculated that basophils might also express cysLT receptors. Basophils were therefore evaluated for cysLT1 and cysLT2 message, protein, and functional responses to LTD. Methods
Results
Discussion We have shown that basophils express mRNA for cysLT1 and cysLT2 and cell-surface receptor by means of flow cytometry. Our data obtained from Western blotting experiments are not as clear. Probing with the available polyclonal SP600125 mg revealed bands at approximately 52 and 58 kd in eosinophil lysates, which was not the expected molecular weight. Different glycosylation or dimerization of receptors by mature leukocytes might explain our observation of higher-molecular-weight leukotriene receptors than previously reported. The calculated molecular mass based on the amino acid sequence of cysLT1 is reported to be 38.5 kd,30, 31 and the monomeric recombinant protein is reported to be 42 kd, although these authors also reported larger amounts of dimerized and oligomerized receptors in the presence of denaturing gel detergents. An earlier study with a different polyclonal antibody to cysLT1 has reported a single band at approximately 45 kd in CD34+ hematopoietic progenitors and a number of cell lines. Using the polyclonal antibodies in the current study, we would have expected to find a band for cysLT1 near 50 kd and a band for cysLT2 between 53 and 55 kd (Cayman Chemical, personal communication). However, in both basophil and eosinophil lysates, we observed a 58-kd band when probing for cysLT1 and cysLT2. That we observed a reduction in the intensity of the band at 58 kd with cysLT2 blocking peptide suggests this band is indeed the cysLT2 receptor. There was no reduction in the intensity of the 58-kd band when a cysLT1 blocking peptide was applied, suggesting this band is not the cysLT1 receptor. These Western blotting results for cysLT2 are consistent with the flow cytometric data in basophils, together demonstrating that cysLT2 protein is detectable on the cell surface and in cell lysates. However, protein for cysLT1 is only detectable by means of flow cytometry with the available cysLT1 antibody. Functional studies suggested the presence of a cysLT1 receptor. We observed an LTD4-induced cytosolic calcium response, albeit one that was very weak by most standards. The magnitude of the average response was partially explained by the low percentage of cells showing a very transient response. Because the level of receptor expression would regulate calcium flux, the nature of the observed response could have been caused by low receptor levels in the blood donors (undergoing leukaphoresis) whose atopic status was unknown. The response was ablated by zafirlukast, indicating that this was a response mediated by a cysLT1 receptor. Studies in other cells have found that cysLT1 and cysLT2 act through heterotrimeric guanosine triphosphate–binding proteins that mediate signaling through an increase of intracellular calcium.