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A1310 - LTA4 Hydrolase Activation and LTB4 Production by Eosinophils in Asthma
Author Block: K. Pal1, J. W. Steinke2, Y. M. Shim1, S. J. Sung3, L. Borish2; 1Pulmonary and Critical Care, University of Virginia, Charlottesville, VA, United States, 2Allergy and Immunology, University of Virginia, Charlottesville, VA, United States, 3Medicine, University of Virginia, Charlottesville, VA, United States.
Rationale Asthma and eosinophilic nasal polyposis (NP) are associated with the over-production and over responsiveness to leukotrienes (LTs). Leukotriene B4 (LTB4) is produced from the sequential metabolism of arachidonic acid by 5-lipoxygenase (5-LO) and LTA4 Hydrolase (LTA4H). It acts via the 2 LTB4 receptors (BLT1 and BLT2) as a neutrophil chemoattractant and has been thought to play a role in severe asthma. However, the source of LTB4 is unclear, especially in eosinophilic asthma. Some severe asthma patients are highly responsive to 5-LO inhibition, which blocks production of both cysteinyl LTs and LTB4. We speculated that the benefit of 5-LO inhibition could be mediated, at least in part, by inhibition of eosinophil-derived LTB4. Aim We hypothesized that asthma and NP disease is associated with increased expression of LTA4H activity by eosinophils which leads to increased production of LTB4. Methods Eosinophils from peripheral blood and sinus/NP tissue from control and asthmatic subjects were enriched using density gradient centrifugation followed by dextran sedimentation. Eosinophils were then separated from neutrophils (PMNs) via anti-CD16 magnetic affinity. Eosinophils were activated with aspirin, platelet activating factor (PAF), trypsin and phorbol myristate acetate (PMA) + ionomycin (I). Supernatants were collected and LTB4 was quantified by EIA. In addition, mRNA was extracted and LTA4H determined by qPCR. Immunohistochemistry (IHC) was performed on cytospins of the peripheral blood eosinophils and stained for LTA4H and 5-LO. Results No differences in LTA4H transcript expression was observed amongst peripheral blood eosinophils, however, LTA4H transcripts were increased in NP eosinophils derived from the asthmatic compared to healthy sinus/NP tissue. Significant LTB4 production by blood eosinophils was observed in response to aspirin, PAF, and PMA/I, however, these were not significantly different in control versus asthma patients. Consistent with these observations, IHC showed similar expression of LTA4H in blood eosinophils from control and asthmatic subjects. Conclusions While normally ascribed to PMNs and mononuclear phagocytes, we demonstrated that eosinophils also robustly express LTA4H transcripts and protein. Eosinophils in peripheral blood were found to have no difference in their LTA4H expression or production of LTB4 in control versus asthma patients although higher levels of transcripts were observed in inflammatory airway tissue. We speculate that in many severe asthmatic patients, eosinophil-derived LTB4 production would thereby be markedly increased. It is possible that these patients are more likely to respond to 5-LO inhibition.