We investigated whether cysteinyl leukotrienes (cysLT) are intracrine signal transducers that regulate human eosinophil degranulation mechanisms. Interleukin (IL)-16, eotaxin, and RANTES stimulate vesicular transport–mediated release of preformed, granule-derived IL-4 and RANTES from eosinophils and the synthesis at intracellular lipid bodies of LTC4, the dominant 5-lipoxygenase–derived eicosanoid in eosinophils. 5-Lipoxygenase inhibitors blocked IL-16–, eotaxin-, and RANTES-induced IL-4 release; but neither exogenous LTC4, LTD4, nor LTE4 elicited IL-4 release. Only after membrane permeabilization enabled cysLTs to enter eosinophils did LTC4 and LTD4 stimulate IL-4, but not RANTES, release. LTC4-elicited IL-4 release was pertussis toxin inhibitable, but inhibitors of the two known G protein–coupled cysLT receptors (cysLTRs) (CysLT1 and CysLT2) did not block LTC4-elicited IL-4 release. LTC4 was 10-fold more potent than LTD4 and at low concentrations (0.3–3 nM) elicited, and at higher concentrations (>3 nM) inhibited, IL-4 release from permeabilized eosinophils. Likewise with intact eosinophils, LTC4 export inhibitors, which increased intracellular LTC4, inhibited eotaxin-elicited IL-4 release. Thus, LTC4 acts, via an intracellular cysLTR distinct from CysLT1 or CysLT2, as a signal transducer to selectively regulate IL-4 release. These results demonstrate that LTC4, well recognized as a paracrine mediator, may also dynamically govern inflammatory and immune responses as an intracrine mediator of eosinophil cytokine secretion.

IL-4 release is important in promoting Th2-mediated allergic and parasitic immune responses. Although human eosinophils are potential sources of IL-4, physiologic mechanisms to elicit its release have not been established. By flow cytometry and microscopy, eosinophils from normal donors uniformly contained preformed IL-4. In contrast to cytolytic IL-4 release from calcium ionophore-activated eosinophils, eotaxin and RANTES, but not IFN-γ, elicited IL-4 release by noncytotoxic mechanisms. With a dual Ab capture and detection immunofluorescent microscopic assay, IL-4 was released at discrete cell surface sites. IL-5 enhanced eotaxin-induced IL-4 release, which was mediated by G protein-coupled CCR3 receptors, detectable as early as 5 min and maximum within 1 h. IL-4 release was not diminished by transcription or protein synthesis inhibitors, but was suppressed by brefeldin A, an inhibitor of vesicle formation. Thus, CCR3-mediated signaling can rapidly mobilize IL-4 stored preformed in human eosinophils for release by vesicular transport to contribute to immune responses.

Human eosinophils are potential sources of inflammatory and immunomodulatory mediators, including cysteinyl leukotrienes, chemokines, and cytokines, which are pertinent to allergic inflammation. We evaluated the means by which IL-16, a recognized eosinophil chemoattractant, might act on eosinophils to affect their capacity to release leukotriene C4 (LTC4) or their preformed stores of chemokines (eotaxin, RANTES) or Th1 (IL-12) or Th2 (IL-4) cytokines. IL-16 dose dependently (0.01–100 nM) elicited new lipid body formation, intracellular LTC4 formation at lipid bodies, and priming for enhanced calcium ionophore-activated LTC4 release. IL-16 also elicited brefeldin A-inhibitable, vesicular transport-mediated release of preformed IL-4, but not IL-12, from eosinophils. CD4 is a recognized IL-16R, and accordingly anti-CD4 Fab, soluble CD4, and a CD4 domain 4-based IL-16 blocking peptide inhibited the actions of IL-16 on eosinophils. Although CD4 is not G-protein coupled, pertussis toxin inhibited IL-16-induced eosinophil activation. IL-16 actions were found to be mediated by the autocrine activity, not of platelet-activating factor, but rather of endogenous CCR3-acting chemokines. IL-16 induced the rapid vesicular transport-mediated release of RANTES. The effects of IL-16 were blocked by CCR3 inhibitors (met-RANTES, anti-CCR3 mAb) and by neutralizing anti-eotaxin and anti-RANTES mAbs, but not by platelet-activating factor receptor antagonists (CV6209, BN52021). RANTES and eotaxin each enhanced LTC4 and IL-4 (but not IL-12) release. Therefore, IL-16 activation of eosinophils is CD4-mediated to elicit the extracellular release of preformed RANTES and eotaxin, which then in an autocrine fashion act on plasma membrane CCR3 receptors to stimulate both enhanced LTC4 production and the preferential release of IL-4, but not IL-12, from within eosinophils.

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Some allergic rhinitis medications can be sedating; therefore, it is important to treat allergic rhinitis with medications that improve symptoms while producing few adverse effects. Medications such as the second-generation antihistamines and anticholinergic drugs are well tolerated, but have little effect on congestion. Intranasal corticosteroids reduce congestion, improve sleep and sleep problems, and reduce daytime sleepiness, fatigue, and inflammation. Recently, montelukast, a leukotriene receptor antagonist, has been added to the therapies approved for allergic rhinitis. Montelukast significantly improves both daytime and nighttime symptoms.

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3560 Introduction: Acute myeloid leukemia (AML) originates from rare leukemia stem cells (LSCs). LSCs are chemotherapy resistant and responsible for disease recurrence. AML containing a high percentage of LSCs displays aggressive biology in animal models. Using humanized mice Saito et al (Sci Transl Med 2010; 2: 1–11) have recently shown that xenografted CD25+ LSCs initiate AML and are chemotherapy resistant. Confirmation with clinical data from human AML is needed. Methods: In order to determine the prognostic impact of CD25 expression on AML outcome we have retrospectively investigated CD25 expression in 56 patients (pts) diagnosed and treated for AML at our institution between 02/2008 to 05/2011. 46 pts who had non-APL morphology, were treated with induction chemotherapy and had an adequate specimen for CD25 assessment were included in further analysis. CD25 expression was assessed in each specimen by flow cytometry and immunohistochemistry and correlated with clinical outcome. Patients: Median age was 61 years (22–84), 18 (39%) pts were older than 65; F:M ratio was 19:27, 3 (7%) patients had good risk (core binding factor leukemias), 26 intermediate (diploid karyotype and no good or high risk; 57%) and 17 (37%) high risk cytogenetics (complex, anbormality of 3q26, monosomy 7 and 5). 6 (13%) pts had NPM1mut/FLT3-ITDwt, 6 (13%) pts had NPM1wt/FLT3-ITDmut and 9 (13%) pts had NPM1mut/FLT3-ITDmut. As induction high dose cytarabine/anthracycline based regimen was used in 36 (78%) pts, 7 pts received 7+3 (15%) and 3 (7%) pts received hypomethylating agent. 24 (53%) pts received stem cell transplantation (SCT; 16 [35%] allogeneic and 8 [17%] autologous). The median follow up of the surviving pts was 11.2 months (1.1–38.7). Results: CD25 was detected in 17 pts (37%; 16 at diagnosis and 1 at relapse). Six CD25+ pts experienced relapse (3 pts with 3 or more relapses) heralded by increase in the percentage of CD25+ blasts. 65% of pts with CD25+ AML also had FLT3-ITDmut (p=0,0012). When comparing CD25+ and CD25- pts there was no statistical difference in distribution of the following characteristics: sex, age (65+), cytogenetics risk, presence of NPM1mut, type of induction, SCT. Fifteen (88%) of CD25+ pts experienced relapse compared to 8 (28%) of CD25- pts (p= 0.00007), 8 (47%) CD25+ pts died and 9 (31%) CD25- pts died (p=ns). The median relapse free survival (RFS) of all pts was 10.8 months with the median overall survival (OS) 12.2 months. The estimated 6-month RFS was significantly decreased in CD25+ pts compared to CD25- pts (26% vs 79%, p= 0.0003). This did not translate into a difference in OS between both groups (1-year OS: CD25+ 43% vs CD25- 65%, p=ns). In univariate analysis CD25 positivity was a stronger predictor for relapse (HR 5.28 [2.21–12.62], p=0.0002) than FLT3-ITDmut (HR 4.72 [2.04–10.92]; p= 0.0003). In multivariate analysis CD25 positivity was also a stronger predictor for relapse (HR 6.54 [1.34–9.15], p=0.01) than FLT3-ITDmut (HR 4.72 [2.04–10.92], p= 0.03). Pts undergoing SCT had significantly longer 1-year OS (66%) compared to pts without SCT (21%; p=0.0004). In multivariate analysis SCT was a predictor for improved OS (HR 0.2 [0.07–0.57], p=0.0002). CD25+ pts who received SCT had also significantly longer 1-year OS (63%) compared to CD25+ pts who did not receive SCT (0%; p=0.0098). SCT did not impact RFS in either group. Conclusion: CD25 represents a novel prognostic factor in AML. The increase in CD25+ blasts at relapse is associated with increased relapsed rate and refractory AML supporting the LSCs hypothesis. The detection of CD25 serves not only as a prognostic marker, but may be valuable for minimal residual disease assessment in patients who lack a molecular marker. In our experience treatment inclusive of stem cell transplantation abrogated the negative impact of CD25 expression on OS. Exploration of CD25 as a therapeutic target in AML is warranted. Disclosures: No relevant conflicts of interest to declare.