Increased IL-6 and Potential IL-6 trans-signalling in the airways after an allergen challenge.

BACKGROUND: In asthma, IL-6 is a potential cause of enhanced inflammation, tissue damage and airway dysfunction. IL-6 signalling is regulated by its receptor, which is composed of two proteins, IL-6R and GP130. In addition to their membrane form, these two proteins may be found as extracellular soluble forms. The interaction of IL-6 with soluble IL-6R (sIL-6R) can trigger IL-6 trans-signalling in cells lacking IL-6R. Conversely, the soluble form of GP130 (sGP130) competes with its membrane form to inhibit IL-6 trans-signalling. OBJECTIVES: We aimed to analyse IL-6 trans-signalling proteins in the airways of subjects after an allergen challenge. METHODS: We used a model of segmental bronchoprovocation with an allergen (SBP-Ag) in human subjects with allergy. Before and 48 h after SBP-Ag, bronchoalveolar lavages (BALs) allowed for the analysis of proteins in BAL fluids (BALFs) by ELISA, and membrane proteins on the surface of BAL cells by flow cytometry. In addition, we performed RNA sequencing (RNA-seq) and used proteomic data to further inform on the expression of the IL-6R subunits by eosinophils, bronchial epithelial cells and lung fibroblasts. Finally, we measured the effect of IL-6 trans-signalling on bronchial fibroblasts, in vitro. RESULTS: IL-6, sIL-6R, sGP130 and the molar ratio of sIL-6R/sGP130 increased in the airways after SBP-Ag, suggesting the potential for enhanced IL-6 trans-signalling activity. BAL lymphocytes, monocytes and eosinophils displayed IL-6R on their surface and were all possible providers of sIL-6R, whereas GP130 was highly expressed in bronchial epithelial cells and lung fibroblasts. Finally, bronchial fibroblasts activated by IL-6 trans-signalling produced enhanced amounts of the chemokine, MCP-1 (CCL2). CONCLUSION AND CLINICAL RELEVANCE: After a bronchial allergen challenge, we found augmentation of the elements of IL-6 trans-signalling. Allergen-induced IL-6 trans-signalling activity can activate fibroblasts to produce chemokines that can further enhance inflammation and lung dysfunction.

Mepolizumab Attenuates Airway Eosinophil Numbers, but Not Their Functional Phenotype, in Asthma.

RATIONALE: Mepolizumab, an IL-5-blocking antibody, reduces exacerbations in patients with severe eosinophilic asthma. Mepolizumab arrests eosinophil maturation; however, the functional phenotype of eosinophils that persist in the blood and airway after administration of IL-5 neutralizing antibodies has not been reported. OBJECTIVES: To determine the effect of anti-IL-5 antibody on the numbers and phenotypes of allergen-induced circulating and airway eosinophils. METHODS: Airway inflammation was elicited in participants with mild allergic asthma by segmental allergen challenge before and 1 month after a single intravenous 750-mg dose of mepolizumab. Eosinophils were examined in blood, bronchoalveolar lavage, and endobronchial biopsies 48 hours after challenge. MEASUREMENTS AND MAIN RESULTS: Segmental challenge without mepolizumab induced a rise in circulating eosinophils, bronchoalveolar lavage eosinophilia, and eosinophil peroxidase deposition in bronchial mucosa. IL-5 neutralization before allergen challenge abolished the allergen-induced rise in circulating eosinophils and expression of IL-3 receptors, whereas airway eosinophilia and eosinophil peroxidase deposition were blunted but not eliminated. Before mepolizumab treatment, bronchoalveolar lavage eosinophils had more surface IL-3 and granulocyte-monocyte colony-stimulating factor receptors, CD69, CD44, and CD23 and decreased IL-5 and eotaxin receptors than blood eosinophils. This activation phenotype indicated by bronchoalveolar lavage eosinophil surface markers, as well as the release of eosinophil peroxidase by eosinophils in the bronchial mucosa, was maintained after mepolizumab. CONCLUSIONS: Mepolizumab reduced airway eosinophil numbers but had a limited effect on airway eosinophil activation markers, suggesting that these cells retain functionality. This observation may explain why IL-5 neutralization reduces but does not completely eradicate asthma exacerbations. Clinical trial registered with www.clinicaltrials.gov (NCT00802438).

Human eosinophil activin A synthesis and mRNA stabilization are induced by the combination of IL-3 plus TNF.

Eosinophils contribute to immune regulation and wound healing/fibrosis in various diseases, including asthma. Growing appreciation for the role of activin A in such processes led us to hypothesize that eosinophils are a source of this transforming growth factor-ß superfamily member. Tumor necrosis factor-α (TNF) induces activin A by other cell types and is often present at the site of allergic inflammation along with the eosinophil-activating common ß (ßc) chain-signaling cytokines (interleukin (IL)-5, IL-3, granulocyte-macrophages colony-stimulating factor (GM-CSF)). Previously, we established that the combination of TNF plus a ßc chain-signaling cytokine synergistically induces eosinophil synthesis of the remodeling enzyme matrix metalloproteinase-9. Therefore, eosinophils were stimulated ex vivo by these cytokines and in vivo through an allergen-induced airway inflammatory response. In contrast to IL-5+TNF or GM-CSF+TNF, the combination of IL-3+TNF synergistically induced activin A synthesis and release by human blood eosinophils. IL-3+TNF enhanced activin A mRNA stability, which required sustained signaling of pathways downstream of p38 and extracellular signal-regulated kinase mitogen-activated protein kinases. In vivo, following segmental airway allergen challenge of subjects with mild allergic asthma, activin A mRNA was upregulated in airway eosinophils compared with circulating eosinophils, and ex vivo, circulating eosinophils tended to release more activin A in response to IL-3+TNF. These data provide evidence that eosinophils release activin A and that this function is enhanced when eosinophils are present in an allergen-induced inflammatory environment. Moreover, these data provide the first evidence for posttranscriptional control of activin A mRNA. We propose that an environment rich in IL-3+TNF will lead to eosinophil-derived activin A, which has an important role in regulating inflammation and/or fibrosis.

Potent synergistic effect of IL-3 and TNF on matrix metalloproteinase 9 generation by human eosinophils.

TNF (designated as TNF-α under previous nomenclature) is the preeminent activator of MMP-9 generation from a variety of cells including eosinophils. We have previously established that TNF strongly synergizes with IFN-γ and IL-4 for eosinophil synthesis of Th1- and Th2-type chemokines respectively. Thus, we sought to determine if TNF-induced synthesis of MMP-9 would be enhanced by the presence of Th1, Th2, or the eosinophil-associated common beta chain (ßc) cytokines. Human blood eosinophils were cultured with TNF alone or in combination with either IFN-γ, IL-4, IL-3, IL-5, or GM-CSF. Concentrations and activities of MMP-9 in eosinophil culture supernates were measured by ELISA and gelatin zymography, mRNA transcription and stabilization by quantitative real-time PCR, and signaling events by immunoblotting and intracellular flow cytometric analysis. Individually, TNF, GM-CSF, or IL-3, but not IL-4 or IFN-γ, induced relatively small (<0.2 ng/ml) but statistically significant quantities of MMP-9. Remarkable synergistic synthesis of MMP-9 (ng/ml levels) occurred in response to TNF plus IL-3, GM-CSF or IL-5, in the order of IL-3>GM-CSF>IL-5. Zymography revealed that eosinophils release MMP-9 in its pro-form. Eosinophil stimulation with the combination of IL-3 plus TNF led to increased steady-state levels of MMP-9 mRNA, prolonged mRNA stabilization, and enhanced activation of ERK1/2 phosphorylation. Inhibition of NF-κB, MEK kinase, or p38 MAP kinase, but not JNK signaling pathways, diminished IL-3/TNF-induced MMP-9 mRNA and protein production. Thus, the synergistic regulation of eosinophil MMP-9 by IL-3 plus TNF likely involves cooperative interaction of multiple transcription factors downstream from ERK, p38, and NF-κB activation as well as post-transcriptional regulation of MMP-9 mRNA stabilization. Our data indicate that within microenvironments rich in ßc-family cytokines and TNF, eosinophils are an important source of proMMP-9 and highlight a previously unrecognized role for synergistic interaction between TNF and ßc-family cytokines, particularly IL-3, for proMMP-9 synthesis.

Human airway eosinophils respond to chemoattractants with greater eosinophil-derived neurotoxin release, adherence to fibronectin, and activation of the Ras-ERK pathway when compared with blood eosinophils.

Human blood eosinophils exposed ex vivo to hematopoietic cytokines (e.g., IL-5 or GM-CSF) subsequently display enhanced responsiveness to numerous chemoattractants, such as chemokines, platelet-activating factor, or FMLP, through a process known as priming. Airway eosinophils, obtained by bronchoalveolar lavage after segmental Ag challenge, also exhibit enhanced responsiveness to selected chemoattractants, suggesting that they are primed during cell trafficking from the blood to the airway. Earlier work has shown that chemoattractants stimulate greater activation of ERK1 and ERK2 following IL-5 priming in vitro, thus revealing that ERK1/ERK2 activity can be a molecular readout of priming under these circumstances. Because few studies have examined the intracellular mechanisms regulating priming as it relates to human airway eosinophils, we evaluated the responsiveness of blood and airway eosinophils to chemoattractants (FMLP, platelet-activating factor, CCL11, CCL5, CXCL8) with respect to degranulation, adherence to fibronectin, or Ras-ERK signaling cascade activation. When compared with blood eosinophils, airway eosinophils exhibited greater FMLP-stimulated eosinophil-derived neurotoxin release as well as augmented FMLP- and CCL11-stimulated adherence to fibronectin. In airway eosinophils, FMLP, CCL11, and CCL5 stimulated greater activation of Ras or ERK1/ERK2 when compared with baseline. Ras activation by FMLP in blood eosinophils was also enhanced following IL-5 priming. These studies are consistent with a model of in vivo priming of eosinophils by IL-5 or related cytokines following allergen challenge, and further demonstrate the key role of priming in the chemoattractant-stimulated responses of eosinophils. These data also demonstrate the importance of the Ras-ERK signaling pathway in the regulation of eosinophil responses to chemoattractants in the airway. Human airway eosinophils respond to several chemoattractants with increased activation of the Ras-ERK cascade, eosinophil-derived neurotoxin release, and adherence to fibronectin relative to blood eosinophils.

Potential contribution of IL-7 to allergen-induced eosinophilic airway inflammation in asthma.

The primary function of IL-7 is to promote maturation and survival of T cells. Through microarray expression analysis, we previously observed that human blood eosinophils express mRNA for IL-7R alpha (CD127) and its common gamma chain (CD132). The purpose of this study was to determine whether eosinophils have functional IL-7 receptors and to assess the potential contribution of IL-7 to eosinophilic airway inflammation by evaluating its presence in bronchoalveolar lavage (BAL) fluid of subjects with atopic asthma before and after segmental bronchoprovocation with allergen. Immunoblot analysis revealed that CD127 is present in highly purified human blood eosinophils. Furthermore, eosinophils responded to IL-7 with phosphorylation of STAT5, up-regulation of the activation marker CD69, and prolonged survival. Neutralization of GM-CSF but not IL-5 significantly blunted these functional responses, suggesting that IL-7 mediates its effects by promoting eosinophil release of autologous GM-CSF. Notably, the suppressive effect of anti-GM-CSF on STAT5 phosphorylation occurred within 10 min of eosinophil exposure to IL-7. Thus, IL-7 likely activates eosinophil release of preformed rather than newly synthesized GM-CSF. The biological relevance of IL-7 to eosinophilia in vivo was implicated in a study of airway allergen challenge in patients with allergic asthma. IL-7 concentrations in BAL fluid increased significantly 48 h after segmental allergen challenge and were highly correlated with BAL eosinophils (r = 0.7, p < 0.001). In conclusion, the airway response to allergen is associated with the generation of IL-7, which may contribute to airway inflammation by promoting enhanced eosinophil activation and survival. Activation of eosinophils is a novel function for IL-7.

Generation of Th1 and Th2 chemokines by human eosinophils: evidence for a critical role of TNF-alpha.

Emerging evidence suggests a role for eosinophils in immune regulation of T cells. Thus, we sought to determine whether human eosinophils may exert their effect via differential generation of Th1 and Th2 chemokines depending on cytokines in their microenvironment and, if so, to establish the conditions under which these chemokines are produced. Eosinophils cultured with TNF-alpha plus IL-4 had increased mRNA expression and protein secretion of the Th2-type chemokines, CCL17 (thymus and activation-regulated chemokine) and CCL22 (macrophage-derived chemokine). Conversely, the Th1-type chemokines, CXCL9 (monokine induced by IFN-gamma) and CXCL10 (IFN-gamma-inducible protein-10), were expressed after stimulation with TNF-alpha plus IFN-gamma. Addition of TNF-alpha appeared to be essential for IFN-gamma-induced release of Th1-type chemokines and significantly enhanced IL-4-induced Th2-type chemokines. Inhibition of NF-kappaB completely blocked the production of both Th1 and Th2 chemokines. Activation of NF-kappaB, STAT6, and STAT1 was induced in eosinophils by TNF-alpha, IL-4, and IFN-gamma, respectively. However, there was no evidence for enhancement of these signaling events when eosinophils were stimulated with the combination of TNF-alpha plus IL-4 or TNF-alpha plus IFN-gamma. Thus, independently activated signaling cascades appear to lead to activation of NF-kappaB, STAT1, and STAT6, which may then cooperate at the promoter level to increase gene transcription. Our data demonstrate that TNF-alpha is a vital component for eosinophil chemokine generation and that, depending on the cytokines present in their microenvironment, eosinophils can promote either a Th2 or a Th1 immune response, supporting an immunoregulatory role for eosinophils.

IL-5 and granulocyte-macrophage colony-stimulating factor activate STAT3 and STAT5 and promote Pim-1 and cyclin D3 protein expression in human eosinophils.

Allergic inflammation is characterized by elevated eosinophil numbers and by the increased production of the cytokines IL-5 and GM-CSF, which control several eosinophil functions, including the suppression of apoptosis. The JAK/STAT pathway is important for several functions in hemopoietic cells, including the suppression of apoptosis. We report in this study that STAT3, STAT5a, and STAT5b are expressed in human eosinophils and that their signaling pathways are active following IL-5 or GM-CSF treatment. However, in airway eosinophils, the phosphorylation of STAT5 by IL-5 is reduced, an event that may be related to the reduced expression of the IL-5Ralpha on airway eosinophils. Furthermore, IL-5 and GM-CSF induced the protein expression of cyclin D3 and the kinase Pim-1, both of which are regulated by STAT-dependent processes in some cell systems. Pim-1 is more abundantly expressed in airway eosinophils than in blood eosinophils. Because Pim-1 reportedly has a role in the modulation of apoptosis, these results suggest that Pim-1 action is linked to the suppression of eosinophil apoptosis by these cytokines. Although cyclin D3 is known to be critical for cell cycle progression, eosinophils are terminally differentiated cells that do not proceed through the cell cycle. Thus, this apparent cytokine regulation of cyclin D3 suggests that there is an alternative role(s) for cyclin D3 in eosinophil biology.

Expression of interleukin-5- and granulocyte macrophage-colony-stimulating factor-responsive genes in blood and airway eosinophils.

Because interleukin (IL)-5 family cytokines are critical regulators of eosinophil development, recruitment, and activation, this study was initiated to identify proteins induced by these cytokines in eosinophils. Using oligonucleotide microarrays, numerous transcripts were identified as responsive to both IL-5 and granulocyte macrophage-colony-stimulating factor (GM-CSF), but no transcripts were markedly affected by one cytokine and not the other. Expression of several gene products were seen to be increased following in vitro stimulation of human blood eosinophils, including the IL-3 receptor alpha subunit, lymphotoxin beta, Pim-1, and cyclin D3. Given that eosinophils recovered from the bronchoalveolar lavage fluid of allergic patients after antigen challenge are exposed to IL-5 or GM-CSF in the airway prior to isolation, the hypothesis was tested that selected IL-5- and GM-CSF-responsive genes are upregulated in airway eosinophils relative to the expression in blood cells. Airway eosinophils displayed greater cell surface expression of the IL-3 receptor alpha subunit, CD44, CD25, and CD66e, suggesting that these proteins may be markers of eosinophil activation by IL-5 family cytokines in airway eosinophils. Other genes that were induced by both IL-5 and GM-CSF showed protein expression at similar or decreased levels in airway eosinophils relative to their circulating counterparts (i.e., lymphotoxin beta and CD24). These studies have identified several transcriptional targets of IL-5 and GM-CSF in human eosinophils and suggest that a number of protein products are critical to the responsiveness of airway eosinophils.

Decreased expression of membrane IL-5 receptor alpha on human eosinophils: I. Loss of membrane IL-5 receptor alpha on airway eosinophils and increased soluble IL-5 receptor alpha in the airway after allergen challenge.

IL-5 is a key cytokine for eosinophil maturation, recruitment, activation, and possibly the development of inflammation in asthma. High concentrations of IL-5 are present in the airway after Ag challenge, but the responsiveness of airway eosinophils to IL-5 is not well characterized. The objectives of this study were to establish, following airway Ag challenge: 1) the expression of membrane (m)IL-5Ralpha on bronchoalveolar lavage (BAL) eosinophils; 2) the responsiveness of these cells to exogenous IL-5; and 3) the presence of soluble (s)IL-5Ralpha in BAL fluid. To accomplish these goals, blood and BAL eosinophils were obtained from atopic subjects 48 h after segmental bronchoprovocation with Ag. There was a striking reduction in mIL-5Ralpha on airway eosinophils compared with circulating cells. Furthermore, sIL-5Ralpha concentrations were elevated in BAL fluid, but steady state levels of sIL-5Ralpha mRNA were not increased in BAL compared with blood eosinophils. Finally, BAL eosinophils were refractory to IL-5 for ex vivo degranulation, suggesting that the reduction in mIL-5Ralpha on BAL eosinophils may regulate IL-5-mediated eosinophil functions. Together, the loss of mIL-5Ralpha, the presence of sIL-5Ralpha, and the blunted functional response (degranulation) of eosinophils to IL-5 suggest that when eosinophils are recruited to the airway, regulation of their functions becomes IL-5 independent. These observations provide a potential explanation for the inability of anti-IL-5 therapy to suppress airway hyperresponsiveness to inhaled Ag, despite a reduction in eosinophil recruitment.

Decreased expression of membrane IL-5 receptor alpha on human eosinophils: II. IL-5 down-modulates its receptor via a proteinase-mediated process.

In the accompanying study, we demonstrated that following Ag challenge, membrane (m)IL-5Ralpha expression is attenuated on bronchoalveolar lavage eosinophils, soluble (s)IL-5Ralpha is detectable in BAL fluid in the absence of increased steady state levels of sIL-5Ralpha mRNA, and BAL eosinophils become refractory to IL-5 for ex vivo degranulation. We hypothesized that IL-5 regulates its receptor through proteolytic release of mIL-5Ralpha, which in turn contributes to the presence of sIL-5Ralpha. Purified human peripheral blood eosinophils were incubated with IL-5 under various conditions and in the presence of different pharmacological agents. A dose-dependent decrease in mIL-5Ralpha was accompanied by an increase in sIL-5Ralpha in the supernatant. IL-5 had no ligand-specific effect on mIL-5Ralpha or sIL-5Ralpha mRNA levels. The matrix metalloproteinase-specific inhibitors BB-94 and GM6001 and tissue inhibitor of metalloproteinase-3 partially inhibited IL-5-mediated loss of mIL-5Ralpha, suggesting that sIL-5Ralpha may be produced by proteolytic cleavage of mIL-5Ralpha. IL-5 transiently reduced surface expression of beta-chain, but had no effect on the expression of GM-CSFRalpha. Pretreatment of eosinophils with a dose of IL-5 that down-modulated mIL-5Ralpha rendered these cells unable to degranulate in response to further IL-5 stimulation, but they were fully responsive to GM-CSF. These findings suggest that IL-5-activated eosinophils may lose mIL-5Ralpha and release sIL-5Ralpha in vivo, which may limit IL-5-dependent inflammatory events in diseases such as asthma.