A surprising second functional effect

A surprising second functional effect was found in culture experiments. Inclusion of LTD4 reduced the frequency of Fas receptor (CD95) expression. Once again, this effect, although modest, was reversed by a cysLT1 receptor antagonist. The biologic significance of this effect or its mechanism remained unclear. LTD4 alone did not induce histamine release in basophils, but given the very weak cytosolic calcium response, this is perhaps not surprising. It remains possible that histamine release was not induced by exogenous LTD4 because the concentrations tested were too low and that local concentrations in situ might exceed those tested. We tested whether released LTC4 could induce a feedback effect (positive or negative) by inclusion of a cysLT1 antagonist but did not observe a significant difference in histamine release. Because the selective cysLT1 antagonist did not have an effect, we then tested an antagonist acting on both cysLT1 and cysLT2 receptors, which also did not have an effect on histamine release. On this basis, we could tentatively conclude that the cysLT2 receptor did not mediate a feedback effect of released LTC4. Given the considerable length of time required for basophil purification before MK-5172 synthesis were tested for functional responses, it is possible that the receptors were no longer present at optimal levels. Basophils have been shown to accumulate in the airways of asthmatic subjects after inhalation of LTE4, suggesting that cysLTs might play a role in the accumulation of basophils in allergic tissues. This could be a result of higher levels of cysLTs in the airways of asthmatic subjects, higher expression of cysLT receptors on basophils of asthmatic subjects (as we have demonstrated by means of flow cytometry), or both. Furthermore, zafirlukast has been shown to inhibit the infiltration of Alcian blue–positive cells (presumed to be basophils) in bronchoalveolar lavage fluid after segmental antigen challenge, confirming involvement of cysLT1. We hypothesized that this accumulation of basophils might be a result of increased viability, and our data showing decreased Fas expression after incubation with LTD4 supports this possibility. Chemotaxis would be another potential mechanism resulting in accumulation of basophils in allergic tissues. Eosinophils and basophils share a common CD34+ hematopoietic progenitor. When CD34+ cells are stimulated with LTD4, there is intracellular calcium mobilization and chemotaxis, and mature eosinophils have also been shown to be weakly chemotactic to LTD4. Although the chemotaxis assay was problematic for these basophil preparations, we did find a modest and transient change in forward-scatter characteristics by means of flow cytometry, and this behavior has been related to the ability of leukocytes to move in response to stimulation. Our experiments have demonstrated that peripheral blood basophils express mRNA for cysLT1 and cysLT2, detectable levels of cysLT1 and cysLT2 protein, and functional responses to LTD4. We have shown that despite the variable level of cysLT1 on basophils, there is indeed a functionally active receptor. Because 2 identified functional effects, increase of cytosolic Ca++ and decreased expression of CD95, were fully reversed by the presence of a cysLT1 antagonist, we conclude that we have not identified a functional role for the cysLT2 receptor, despite its equivalent level of protein expression, as shown by means of flow cytometry. Whether the observed level of response can account for the accumulation of basophils in allergic tissue remains to be seen.
Introduction Cysteinyl–leukotrienes (Cys-LTs), namely leukotriene C4, D4 and E4, are synthesized de novo by transformation of arachidonic acid via the activity of the 5-lipoxygenase enzyme (Samuelsson, 1983). Experimental research characterized Cys-LTs as potent stimulators of smooth muscle contraction, their effects being of particular relevance in the respiratory system, where they can cause severe bronchoconstriction (Hanna et al., 1981, Griffin et al., 1983). In normal subjects, inhalation of leukotriene D4 (LTD4) produces the same degree of airway obstruction as 10,000 times more concentrated inhaled solution of histamine. Moreover, increased synthesis of leukotrienes has been demonstrated in vivo following antigen challenge of allergic subjects (Creticos et al., 1984). Early studies pertaining to Cys-LTs effects have been centered on their contractile activity whereas, more recently, several investigators demonstrated their contribution to airway hyperresponsiveness, mucus plug formation, epithelial cell damage, and airway remodeling (reviewed in Holgate et al., 2003). It is now clear that cysteinyl–leukotrienes are endowed with a variety of proinflammatory actions, and it has been therefore speculated that these mediators might play a pivotal role in the pathogenesis of asthma.