Kita, H

Objective To evaluate eosinophil peroxidase (EPX) as a biomarker for tissue levels of eosinophilia, cytokines, and chemokines within chronic rhinosinusitis (CRS). Methods Twenty‐eight subjects undergoing sinonasal surgery were prospectively enrolled. Ethmoid tissue was analyzed with an in‐house EPX immunoassay and a 48‐plex cytokine‐chemokine array. Clinical severity was assessed using SNOT‐22 and Lund‐Mackay scores. Subjects were grouped as follows: controls, polyp status (CRS with [CRSwNP] and without nasal polyps [CRSsNP]), tissue eosinophilia (eosinophilic CRS [eCRS], non‐eosinophilic CRS [neCRS]), or combinations thereof (eCRSwNP, eCRSsNP, neCRSsNP). eCRS was defined as >10 eosinophils per high power field (HPF). Subjects without CRS or asthma were enrolled as controls. Results EPX was elevated in CRSwNP compared to control ( p = 0.007), in eCRS compared to neCRS ( p = 0.002), and in eCRSwNP along with eCRSsNP compared to neCRSsNP ( p = 0.023, p = 0.015, respectively). eCRS displayed elevated IL‐5 compared to neCRS ( p = 0.005). No significant differences in EPX or IL‐5 were observed between eCRSwNP and eCRSsNP. IL‐5 was elevated in eCRSwNP ( p = 0.019) compared neCRSsNP. Area under the receiver operator characteristic curve was 0.938 (95% CI, 0.835–1.00) for EPX and tissue eosinophilia, with an optimal cut‐point of 470 ng/mL being 100% specific and 81.25% sensitive for tissue eosinophilia. Linear regression revealed a strong correlation between EPX and IL‐5 (R 2 = 0.64, p < 0.001). Comparing EPX and IL‐5, only EPX displayed significant correlation with SNOT‐22 ( p = 0.04) and Lund‐Mackay score ( p = 0.004). Conclusion EPX is associated with tissue eosinophilia in CRS patients regardless of polyp status. EPX correlates with IL‐5 and could be potentially considered a biomarker for anti‐IL‐5 therapies. Level of Evidence 3 Laryngoscope , 134:69–78, 2024

Abstract CD9 is a cell surface glycoprotein belonging to the transmembrane 4 superfamily. In this report, we demonstrate that cross-linking CD9 with different forms of mAb activates distinct functions of human eosinophils. Anti-CD9 mAb (clone ALB6) immobilized onto tissue culture plates induced eosinophil degranulation. This effect of anti-CD9 mAb (clone ALB6) was unique because other immobilized clones of anti-CD9 mAb (clones FMC56 and ML13) or anti-HLA Class I mAb failed to induce degranulation. In addition, neutrophil degranulation was not provoked by the immobilized clone ALB6, consistent with a lack of expression of CD9 on neutrophils. Eosinophil degranulation induced by anti-CD9 mAb (clone ALB6) was abolished by pretreatment of eosinophils with anti-CD18 mAb, suggesting that beta2 integrins are involved in the reaction. In contrast to the results with immobilized clones, all clones of anti-CD9 mAb in soluble form enhanced eosinophil survival. This enhanced survival was inhibited by anti-granulocyte-macrophage-CSF (GM-CSF) mAb, suggesting that autocrine production of GM-CSF by eosinophils mediated the enhanced survival. In fact, by ELISA and RT-PCR, GM-CSF protein and mRNA were detected in supernatants and cell lysates of eosinophils stimulated by soluble anti-CD9 mAb, but not by immobilized mAb. These results indicate that CD9 serves as a molecule that delivers stimulation signals on eosinophils. The eosinophil's cellular responses may differ and be dependent on the manner of receptor ligation.

Abstract Human eosinophils have been reported to express both the mRNA and protein for the high affinity IgE receptor (FcεRI); it is speculated that this receptor plays a role in eosinophil mediator release in allergic diseases. However, questions still remain. How much of the FcεRI protein is actually expressed on the cell surface of the eosinophil? If they are present, are these IgE receptors associated with effector functions of eosinophils? To address these issues, we studied blood eosinophils from patients with ragweed hay fever. A high level of low affinity IgG receptor (FcγRII, CD32), but no expression of FcεRI, was detectable on the eosinophil surface by standard FACS analysis. However, after in vitro sensitization with biotinylated chimeric IgE (cIgE), cell-bound cIgE was detected by PE-conjugated streptavidin. This cIgE binding was partially inhibited by anti-FcεRI mAb, suggesting that eosinophils do express minimal amounts of FcεRI detectable only by a sensitive method. Indeed, FACS analysis of whole blood showed that eosinophils express ∼0.5% of the FcεRI that basophils express. When stimulated with human IgE or anti-human IgE, these eosinophils did not exert effector functions; there was neither production of leukotriene C4 or superoxide anion nor any detectable degranulation response. In contrast, eosinophils possessed membrane-bound human IgG and showed functional responses when stimulated with human IgG or anti-human IgG. Thus, IgG and/or cytokines, such as IL-5, appear to be more important for eosinophil activation in allergic diseases than IgE.

Abstract The low-affinity IgG Fc receptor, FcγRII (CD32), mediates various effector functions of lymphoid and myeloid cells and is the major IgG Fc receptor expressed by human eosinophils. We investigated whether FcγRII regulates both cell survival and death of human eosinophils. When cultured in vitro without growth factors, most eosinophils undergo apoptosis within 96 h. Ligation of FcγRII by anti-CD32 mAb in solution inhibited eosinophil apoptosis and prolonged survival in the absence of growth factors. Cross-linking of human IgG bound to FcγRII by anti-human IgG Ab or of unoccupied FcγRII by aggregated human IgG also prolonged eosinophil survival. The enhanced survival with anti-CD32 mAb was inhibited by anti-granulocyte-macrophage-CSF (GM-CSF) mAb, suggesting that autocrine production of GM-CSF by eosinophils mediated survival. In fact, mRNA for GM-CSF was detected in eosinophils cultured with anti-CD32 mAb. In contrast to mAb or ligands in solution, anti-CD32 mAb or human IgG, when immobilized onto tissue culture plates, facilitated eosinophil cell death even in the presence of IL-5. Cell death induced by these immobilized ligands was accompanied by DNA fragmentation and was inhibited when eosinophil β2 integrin was blocked by anti-CD18 mAb, suggesting that β2 integrins play a key role in initiating eosinophil apoptosis. Thus, FcγRII may pivotally regulate both survival and death of eosinophils, depending on the manner of receptor ligation and β2 integrin involvement. Moreover, the FcγRII could provide a novel mechanism to control the number of eosinophils at inflammation sites in human diseases.

Abstract Eosinophils play a key role in the pathogenesis of asthma and other allergic inflammatory diseases. We have previously shown that treatment of eosinophils with lidocaine preferentially inhibits IL-5-induced survival. This inhibition cannot be overcome by increasing concentrations of IL-5 and is not due to the blocking of Na+ channels by lidocaine. Here we report that one class of K+ channel blockers, the sulfonylureas, inhibits eosinophil survival in a manner similar to lidocaine. The sulfonylurea glyburide inhibits eosinophil survival even at high concentrations of IL-5. In contrast, increasing concentrations of IL-3 or granulocyte-macrophage CSF overcome glyburide inhibition. Glyburide also blocks cytokine-induced eosinophil superoxide production. Similar results were seen with the sulfonylureas tolbutamide and glipizide. Interestingly, the effects of glyburide are not antagonized by the ATP-sensitive K+ channel openers cromakalim, pinacidil, or diazoxide. Although Scatchard analysis of [3H]glyburide binding to eosinophil membranes indicated that the high affinity sulfonylurea receptor (SUR1) is not present on eosinophils, human eosinophils do express mRNA homologous to the sulfonylurea receptor family, in keeping with the presence of a sulfonylurea receptor. Finally, coculture of eosinophils with combinations of glyburide, lidocaine, and dexamethasone resulted in synergistic inhibition of cytokine-mediated eosinophil survival and superoxide production. These results have intriguing clinical implications for the treatment of eosinophil-associated diseases.

Abstract Eosinophils and cytokines active on eosinophils, especially IL-5, are believed to be critically involved in chronic allergic diseases. IL-5 activates eosinophils and enhances their survival in vitro by delaying apoptosis. In this study, we found that lidocaine and six analogues blunt responses of eosinophils to IL-5. Lidocaine and its derivatives inhibit IL-5-mediated eosinophil survival in a concentration-dependent manner (IC50 = 110 μM for 30 pg/ml IL-5). At suboptimal lidocaine concentrations, the eosinophil survival response to IL-5 shifts and more IL-5 is required to maintain survival. The inhibitory effect requires at least 24-h exposure of eosinophils to lidocaine, and the protein kinase C activator, PMA, completely reverses the inhibition. A multiparameter flow-cytometric analysis shows that lidocaine hastens the apoptosis of eosinophils normally delayed by IL-5. Lidocaine does not affect IL-5R expression or IL-5-induced protein tyrosine phosphorylation. Lidocaine also inhibits eosinophil survival mediated by IL-3 or granulocyte-macrophage CSF, although less potently than that mediated by IL-5. Furthermore, lidocaine inhibits eosinophil superoxide production stimulated by IL-5, granulocyte-macrophage CSF, or IL-3, but not that stimulated by platelet-activating factor, immobilized IgG, or PMA. Lidocaine and its derivatives show novel immunomodulatory properties and are able to blunt eosinophil responses to cytokines in addition to their local anesthetic or antiarrhythmic properties. Thus, lidocaine and its derivatives may represent a new class of therapeutic agents to treat patients with allergic diseases.

Abstract Eosinophil activation and subsequent release of inflammatory mediators are implicated in the pathophysiology of allergic diseases. Eosinophils are activated by various classes of secretagogues, such as cytokines (e.g., IL-5), lipid mediators (e.g., platelet-activating factor (PAF)), and Ig (e.g., immobilized IgG). However, do these agonists act directly on eosinophils or indirectly through the generation of intermediate active metabolites? We now report that endogenous PAF produced by activated eosinophils plays a critical role in eosinophil functions. Human eosinophils produced superoxide when stimulated with immobilized IgG, soluble IL-5, or PAF. Pretreating eosinophils with pertussis toxin abolished their responses to these stimuli, suggesting involvement of a metabolite(s) that acts on G proteins. Indeed, PAF was detected in supernatants from eosinophils stimulated with IgG or IL-5. Furthermore, structurally distinct PAF antagonists, including CV6209, hexanolamine PAF, and Y-24180 (israpafant), inhibited IgG- or IL-5-induced superoxide production and degranulation. Previous reports indicated that exogenous PAF stimulates eosinophil eicosanoid production through formation of lipid bodies. We found in this study that IgG or IL-5 also induces lipid body formation and subsequent leukotriene C4 production mediated by endogenous PAF. Finally, inhibition of cytosolic phospholipase A2, one of the key enzymes involved in PAF synthesis, attenuated both PAF production and effector functions of eosinophils. These findings suggest that endogenous PAF plays important roles in eosinophil functional responses to various exogenous stimuli, such as cytokines and Igs. Therefore, inhibition of PAF synthesis or action may be beneficial for the treatment of eosinophilic inflammation.

Abstract To study the capacity and regulation of cytokine production by normal peripheral blood eosinophils, we isolated eosinophils from healthy individuals and stimulated them with immobilized Ig or TNF-α, with or without exogenous IL-5. By reverse transcription-PCR, uncultured, freshly isolated eosinophils constitutively expressed mRNA for IL-4, IL-10, and TGF-β1. Eosinophils stimulated by immobilized secretory IgA, immobilized IgA, immobilized IgG, or TNF-α for 3 h expressed mRNA encoding IL-3, IL-4, IL-8, IL-10, granulocyte-macrophage CSF, TNF-α, TGF-β, and RANTES. The mRNA for IL-2, IL-5, or IFN-γ was not detected. IL-4 and IL-10 protein, but not IL-8, were measurable in lysates of fresh eosinophils or eosinophils cultured with medium alone for 24 h. Eosinophils incubated with immobilized Ig or TNF-α released IL-8 protein into the supernatants. In contrast, IL-4 and IL-10 proteins were not detectable. Soluble secretory IgA immune complexes also induced degranulation, as measured by eosinophil-derived neurotoxin, and IL-8 release, but not IL-4 or IL-10 release, from eosinophils. Release of IL-8 protein and storage of IL-4 and IL-10 proteins were enhanced by exogenous IL-5 and inhibited by a transcription inhibitor, actinomycin D. Degranulation of stored granule proteins was not affected by actinomycin D. Therefore, normal peripheral blood eosinophils can transcribe and synthesize several cytokines, including IL-4, IL-8, and IL-10; some are stored, and some are released. These cytokines may play important roles in modulating immune responses in diseases associated with eosinophils.

Abstract Eosinophils and their products are important in the pathophysiology of allergic inflammation in mucosal tissues. Secretory component bound to IgA mediates transepithelial transport of IgA and confers increased stability on the resultant secretory IgA; however, the effect of secretory component on the biologic activity of IgA is unknown. Here, we report that secretory IgA and secretory component preferentially activate human eosinophils. When eosinophils were stimulated with immobilized secretory IgA, degranulation and superoxide production were two- to threefold greater than when stimulated with serum IgA. In contrast, neutrophils responded similarly to secretory IgA and serum IgA. Flow cytometric analysis showed that eosinophils bound to purified secretory component. The binding of 125I-labeled secretory component was inhibited by unlabeled secretory component or secretory IgA but not by serum IgA. Superoxide production by eosinophils stimulated with cytokines or IgG was enhanced synergistically by immobilized secretory component; secretory component showed no effect on neutrophil activation. Finally, anti-CD18 mAb abolished eosinophil superoxide production stimulated with secretory IgA or secretory component but not with serum IgA, suggesting a crucial role for β2 integrins in eosinophil interactions with secretory IgA or secretory component. Thus, secretory component plays important roles in activating eosinophil functions but not neutrophil functions. This preferential interaction between secretory component and eosinophils may provide a novel mechanism to regulate mucosal tissue inflammation.