Correlation between CCL26 production by human bronchial epithelial cells and airway eosinophils: Involvement in patients with severe eosinophilic asthma.

BACKGROUND: High pulmonary eosinophil counts are associated with asthma symptoms and severity. Bronchial epithelial cells (BECs) produce CC chemokines, notably CCL26 (eotaxin-3), which recruits and activates eosinophils from asthmatic patients. This suggests that CCL26 production by BECs might be involved in persistent eosinophilia in patients with severe asthma despite treatment with high corticosteroid doses. OBJECTIVE: We sought to determine whether CCL26 levels correlate with eosinophilia and asthma severity. METHODS: Human CC chemokine expression was assessed by means of quantitative PCR or a quantitative PCR array in vehicle- or IL-13-treated BECs. CCL26 was quantitated by means of ELISA. Immunohistochemistry analyses of CCL26 and major basic protein were done on bronchial biopsy specimens. RESULTS: IL-13 selectively induced CCL26 expression by BECs. This increase was time-dependent and more prominent in BECs from patients with severe eosinophilic asthma. CCL26 levels measured in supernatants of IL-13-stimulated BECs also increased with asthma severity as follows: patients with severe eosinophilic asthma > patients with mild asthma ≈ healthy subjects. Immunohistochemistry analyses of bronchial biopsy specimens confirmed increased levels of CCL26 in the epithelium of patients with mild and those with severe eosinophilic asthma. Tissue eosinophil counts did not correlate with CCL26 staining. However, sputum CCL26 levels significantly correlated with sputum eosinophil counts (P < .0001), suggesting that CCL26 participates in the movement of eosinophils from the tissues to the airway lumen. CONCLUSIONS: These results show a relation between CCL26 production by IL-13-stimulated BECs, sputum eosinophil counts, and asthma severity. They also suggest a role for CCL26 in the sustained inflammation observed in patients with severe eosinophilic asthma and reveal CCL26 as a potential target for treating patients with eosinophilic asthma that are refractory to classic therapies.

20-Hydroxy- and 20-carboxy-leukotriene (LT) B4 downregulate LTB4 -mediated responses of human neutrophils and eosinophils.

Leukotriene B4 (LTB4 ) plays a prominent role in innate immunity as it induces phagocyte recruitment, the release of antimicrobial effectors, and as it potentiates the ingestion and killing of pathogens. In humans, LTB4 has a short half-life and is rapidly metabolized by leukocytes, notably into 20-OH- and 20-COOH-LTB4 by neutrophils. Although these LTB4 metabolites bind to the BLT1 receptor with high affinity, they activate neutrophils to a much lower extent than LTB4 . We thus postulated that LTB4 metabolites could dampen BLT1 -mediated responses, therefore limiting the impact of LTB4 on human neutrophil functions. We found that 20-OH-LTB4 and 20-COOH-LTB4 inhibited all of the LTB4 -mediated neutrophil responses we tested (migration, degranulation, leukotriene biosynthesis). The potencies of the different compounds at inhibiting LTB4 -mediated responses were 20-OH-LTB4  = CP 105,696 (BLT1 antagonist) > > 20-COOH-LTB4 ≥ resolvin E1 (RVE1 ). In contrast, the fMLP- and IL-8-mediated responses we tested were not affected by the LTB4 metabolites or RVE1 . 20-OH-LTB4 and 20-COOH-LTB4 also inhibited the LTB4 -mediated migration of human eosinophils but not that induced by 5-KETE. Moreover, using 20-COOH-LTB4 , LTB4 , and LTB4 -alkyne, we show that LTB4 is a chemotactic, rather than a chemokinetic factor for both human neutrophils and eosinophils. In conclusion, our data indicate that LTB4 metabolites and RVE1 act as natural inhibitors of LTB4 -mediated responses. Thus, preventing LTB4 ω-oxidation might result in increased innate immunity and granulocyte functions.

Comparison of eight 15-lipoxygenase (LO) inhibitors on the biosynthesis of 15-LO metabolites by human neutrophils and eosinophils.

Neutrophils and eosinophils are important sources of bioactive lipids from the 5- and the 15-lipoxygenase (LO) pathways. Herein, we compared the effectiveness of humans eosinophils and eosinophil-depleted neutrophils to synthesize 15-LO metabolites using a cocktail of different 15-LO substrates as well as their sensitivities to eight documented 15-lipoxygenase inhibitors. The treatment of neutrophils and eosinophils with linoleic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and arachidonyl-ethanolamide, led to the synthesis of 13-HODE, 15-HETrE, 15-HETE, 15-HEPE, 14-HDHA/17-HDHA, and 15-hydroxy-AEA. Neutrophils and eosinophils also metabolized the endocannabinoid 2-arachidonoyl-glycerol into 15-HETE-glycerol, although this required 2-arachidonoyl-glycerol hydrolysis inhibition. Neutrophils and eosinophils differed in regard to dihomo-γ-linolenic acid and linoleic acid utilization with 15-HETrE/13-HODE ratios of 0.014 ± 0.0008 and 0.474 ± 0.114 for neutrophils and eosinophils respectively. 15-LO metabolite synthesis by neutrophils and eosinophils also differed in regard to their relative production of 17-HDHA and 14-HDHA.The synthesis of 15-LO metabolites by neutrophils was concentration-dependent and rapid, reaching a plateau after one minute. While investigating the biosynthetic routes involved, we found that eosinophil-depleted neutrophils express the 15-lipoxygenase-2 but not the 15-LO-1, in contrast to eosinophils which express the 15-LO-1 but not the 15-LO-2. Moreover, 15-LO metabolite synthesis by neutrophils was not inhibited by the 15-LO-1 inhibitors BLX769, BLX3887, and ML351. However, 15-LO product synthesis was partially inhibited by 100 µM NDGA. Altogether, our data indicate that the best 15-LO-1 inhibitors in eosinophils are BLX3887, BLX769, NDGA and ML351 and that the synthesis of 15-LO metabolites by neutrophils does not involve the 15-LO-1 nor the phosphorylation of 5-LO on Ser-663 but is rather the consequence of 15-LO-2 or another unidentified 15-LO.

Regulation of Eosinophil and Group 2 Innate Lymphoid Cell Trafficking in Asthma.

Asthma is an inflammatory disease usually characterized by increased Type 2 cytokines and by an infiltration of eosinophils to the airways. While the production of Type 2 cytokines has been associated with TH2 lymphocytes, increasing evidence indicates that group 2 innate lymphoid cells (ILC2) play an important role in the production of the Type 2 cytokines interleukin (IL)-5 and IL-13, which likely amplifies the recruitment of eosinophils from the blood to the airways. In that regard, recent asthma treatments have been focusing on blocking Type 2 cytokines, notably IL-4, IL-5, and IL-13. These treatments mainly result in decreased blood or sputum eosinophil counts as well as decreased asthma symptoms. This supports that therapies blocking eosinophil recruitment and activation are valuable tools in the management of asthma and its severity. Herein, we review the mechanisms involved in eosinophil and ILC2 recruitment to the airways, with an emphasis on eotaxins, other chemokines as well as their receptors. We also discuss the involvement of other chemoattractants, notably the bioactive lipids 5-oxo-eicosatetraenoic acid, prostaglandin D2, and 2-arachidonoyl-glycerol. Given that eosinophil biology differs between human and mice, we also highlight and discuss their responsiveness toward the different eosinophil chemoattractants.

Leukotriene B4 Metabolism and p70S6 Kinase 1 Inhibitors: PF-4708671 but Not LY2584702 Inhibits CYP4F3A and the ω-Oxidation of Leukotriene B4 In Vitro and In Cellulo.

LTB4 is an inflammatory lipid mediator mainly biosynthesized by leukocytes. Since its implication in inflammatory diseases is well recognized, many tools to regulate its biosynthesis have been developed and showed promising results in vitro and in vivo, but mixed results in clinical trials. Recently, the mTOR pathway component p70S6 kinase 1 (p70S6K1) has been linked to LTC4 synthase and the biosynthesis of cysteinyl-leukotrienes. In this respect, we investigated if p70S6K1 could also play a role in LTB4 biosynthesis. We thus evaluated the impact of the p70S6K1 inhibitors PF-4708671 and LY2584702 on LTB4 biosynthesis in human neutrophils. At a concentration of 10 µM, both compounds inhibited S6 phosphorylation, although neither one inhibited the thapsigargin-induced LTB4 biosynthesis, as assessed by the sum of LTB4, 20-OH-LTB4, and 20-COOH-LTB4. However, PF-4708671, but not LY2584702, inhibited the ω-oxidation of LTB4 into 20-OH-LTB4 by intact neutrophils and by recombinant CYP4F3A, leading to increased LTB4 levels. This was true for both endogenously biosynthesized and exogenously added LTB4. In contrast to that of 17-octadecynoic acid, the inhibitory effect of PF-4708671 was easily removed by washing the neutrophils, indicating that PF-4708671 was a reversible CYP4F3A inhibitor. At optimal concentration, PF-4708671 increased the half-life of LTB4 in our neutrophil suspensions by 7.5 fold, compared to 5 fold for 17-octadecynoic acid. Finally, Michaelis-Menten and Lineweaver-Burk plots indicate that PF-4708671 is a mixed inhibitor of CYP4F3A. In conclusion, we show that PF-4708671 inhibits CYP4F3A and prevents the ω-oxidation of LTB4 in cellulo, which might result in increased LTB4 levels in vivo.

CCL26/eotaxin-3 is more effective to induce the migration of eosinophils of asthmatics than CCL11/eotaxin-1 and CCL24/eotaxin-2.

CCL11, CCL24, and CCL26 are chemokines involved in the recruitment of eosinophils into tissues and mainly activate CCR3. Whereas the genomic or pharmacological inhibition of CCR3 prevents the development of experimental asthma in rodents, it only impairs the recruitment of eosinophils by ∼40% in humans. As humans, but not rodents, express CCL26, we investigated the impact of CCL11, CCL24, and CCL26 on human eosinophils recruitment and evaluated the involvement of CCR3. The migration of eosinophils of healthy volunteers was similar for the three eotaxins. Eosinophils of mild asthmatics had a greater response to CCL11 and a much greater response to CCL26. Whereas all eotaxins induced the migration of eosinophil of asthmatics from 0 to 6 h, CCL26 triggered a second phase of migration between 12 and 18 h. Given that the CCR3 antagonists SB 328437 and SB 297006 inhibited the 5-oxo-eicosatetraenoate-induced migration of eosinophils and that the CCR3 antagonist UCB 35625 was not specific for CCR3, CCR3 blockade was performed with the CCR3 mAb. This antibody completely blocked the effect of all eotaxins on eosinophils of healthy subjects and the effect of CCL24 on the eosinophils of asthmatics. Interestingly, CCR3 blockade did not affect the second migration phase induced by CCL26 on eosinophils of asthmatics. In conclusion, CCL26 is a more effective chemoattractant than CCL11 and CCL24 for eosinophils of asthmatics. The mechanism of this greater efficiency is not yet defined. However, these results suggest that CCL26 may play a unique and important role in the recruitment of eosinophils in persistent asthma.

2-Arachidonoyl-glycerol- and arachidonic acid-stimulated neutrophils release antimicrobial effectors against E. coli, S. aureus, HSV-1, and RSV.

The endocannabinoid 2-AG is highly susceptible to its hydrolysis into AA, which activates neutrophils through de novo LTB(4) biosynthesis, independently of CB activation. In this study, we show that 2-AG and AA stimulate neutrophils to release antimicrobial effectors. Supernatants of neutrophils activated with nanomolar concentrations of 2-AG and AA indeed inhibited the infectivity of HSV-1 and RSV. Additionally, the supernatants of 2-AG- and AA-stimulated neutrophils strongly impaired the growth of Escherichia coli and Staphylococcus aureus. This correlated with the release of a large amount (micrograms) of α-defensins, as well as a limited amount (nanograms) of LL-37. All the effects of AA and 2-AG mentioned above were prevented by inhibiting LTB(4) biosynthesis or by blocking BLT(1). Importantly, neither CB(2) receptor agonists nor antagonists could mimic nor prevent the effects of 2-AG, respectively. In fact, qPCR data show that contaminating eosinophils express ∼100-fold more CB(2) receptor mRNA than purified neutrophils, suggesting that CB(2) receptor expression by human neutrophils is limited and that contaminating eosinophils are likely responsible for the previously documented CB(2) expression by freshly isolated human neutrophils. The rapid conversion of 2-AG to AA and their subsequent metabolism into LTB(4) promote 2-AG and AA as multifunctional activators of neutrophils, mainly exerting their effects by activating the BLT(1). Considering that nanomolar concentrations of AA or 2-AG were sufficient to impair viral infectivity, this suggests potential physiological roles for 2-AG and AA as regulators of host defense in vivo.

Leukotriene D4 and interleukin-13 cooperate to increase the release of eotaxin-3 by airway epithelial cells.

INTRODUCTION: Airway epithelial cells play a central role in the physiopathology of asthma. They release eotaxins when treated with T(H)2 cytokines such as interleukin (IL)-4 or IL-13, and these chemokines attract eosinophils and potentiate the biosynthesis of cysteinyl leukotrienes (cysLTs), which in turn induce bronchoconstriction and mucus secretion. These effects of cysLTs mainly mediated by CysLT(1) and CysLT(2) receptors on epithelial cell functions remain largely undefined. Because the release of inflammatory cytokines, eotaxins, and cysLTs occur relatively at the same time and location in the lung tissue, we hypothesized that they regulate inflammation cooperatively rather than redundantly. We therefore investigated whether cysLTs and the T(H)2 cytokines would act in concert to augment the release of eotaxins by airway epithelial cells. METHODS: A549 cells or human primary bronchial epithelial cells were incubated with or without IL-4, IL-13, and/or LTD(4). The release of eotaxin-3 and the expression of cysLT receptors were assessed by ELISA, RT-PCR, and flow cytometry, respectively. RESULTS: IL-4 and IL-13 induced the release of eotaxin-3 by airway epithelial cells. LTD(4) weakly induced the release of eotaxin-3 but clearly potentiated the IL-13-induced eotaxin-3 release. LTD(4) had no effect on IL-4-stimulated cells. Epithelial cells expressed CysLT(1) but not CysLT(2). CysLT(1) expression was increased by IL-13 but not by IL-4 and/or LTD(4). Importantly, the upregulation of CysLT(1) by IL-13 preceded eotaxin-3 release. CONCLUSIONS: These results demonstrate a stepwise cooperation between IL-13 and LTD(4). IL-13 upregulates CysLT(1) expression and consequently the response to cysLTs This results in an increased release of eotaxin-3 by epithelial cells which at its turn increases the recruitment of leukocytes and their biosynthesis of cysLTs. This positive amplification loop involving epithelial cells and leukocytes could be implicated in the recruitment of eosinophils observed in asthmatics.