Katô, Masahiko

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 An IgG-coated surface, such as found on parasites, is one of the most effective physiologic stimuli for eosinophil activation. Recent evidence suggests that cellular adhesion, especially that through the β 2 integrin, is an important step in cellular activation and accumulation. Therefore, we investigated the role of adhesion molecules in IgG-stimulated eosinophil functions. Cross-linking of eosinophil cytophilic IgG by anti-IgG immobilized to tissue culture plates induced degranulation, whereas soluble anti-IgG did not. Similarly, eosinophils exposed to human IgG immobilized to plates adhered and degranulated; in addition, adherence and subsequent degranulation were inhibited by mAbs to CD18 and CD11b, but not by mAb to CD29, suggesting an important role of β 2 integrin for these responses. Eosinophil degranulation induced by IgG covalently coupled to Sepharose 4B beads was also inhibited by mAb to CD18. Furthermore, fibrinogen, a ligand for CD11b/18, showed synergistic enhancement of IgG-induced degranulation when it was co-immobilized with IgG to plates. A morphologic study showed that eosinophils, stimulated by immobilized IgG, protrude numerous pseudopods; this morphologic change was inhibited by mAb to CD18. This cellular adhesion seems to affect the early signaling events in eosinophils because the production of inositol phosphates was abolished by mAb to CD18. Interestingly, although superoxide production by eosinophils triggered by immobilized IgG was inhibited by mAb to CD18, superoxide production and morphologic change of neutrophils were not. These results suggest that cell adhesion through CD11b/18 is a crucial step for the activation, signaling, and effector function of eosinophils stimulated by IgG.

Abstract Degranulation of eosinophils and release of toxic granule proteins play key roles in allergic diseases such as bronchial asthma. However, the intracellular signaling mechanisms that trigger eosinophil degranulation remain unclear. In this study, we investigated protein tyrosine kinase (PTK) involvement in the degranulation of human blood eosinophils induced by immobilized Ig. Eosinophils stimulated with Sepharose beads coated with secretory IgA (slgA) or IgG showed rapid increases in the tyrosine phosphorylation of intracellular proteins with molecular masses of 50 to 56, 73, 78, 100, and 105 kDa. The Ig-induced phosphorylation response was not affected by pertussis toxin, a known inhibitor of Ig-dependent eosinophil activation. The tyrosine kinase inhibitors genistein and herbimycin A inhibited both the tyrosine phosphorylation and degranulation responses of eosinophils induced by sIgA- or IgG-coated beads. In contrast, eosinophil degranulation induced by PMA was not affected by genistein. Treatment of eosinophils with the protein phosphatase inhibitor pervanadate induced the phosphorylation of a similar set of intracellular proteins as well as cellular degranulation. Pervanadate also stimulated an increase in phosphoinositide hydrolysis, which was consistent with the activation of a phospholipase C-γ isoform by this stimulus. Genistein pretreatment blocked the Ig-induced phospholipase C activation, providing evidence for PTK involvement in this reaction. These findings indicate that a PTK-dependent signaling pathway plays an important role in triggering the degranulation responses of human eosinophils to immobilized sIgA and IgG.

Abstract In granulocytes, platelet-activating factor (PAF) shares many of its biological effects with other chemotactic factors, such as FMLP, complement fragments, and lipid mediators. Two unique effects are that PAF is relatively resistant to pertussis toxin (PTX) and that PAF activates the inflammatory functions of eosinophils more strongly than it activates those of neutrophils. To investigate the molecular mechanisms of the responses of eosinophils to PAF, we analyzed superoxide anion production by a chemiluminescence method that provides real-time kinetic data for the cellular responses. We found that PAF induced bimodal superoxide anion production in human eosinophils, consisting of an intense, but transient, first phase and a larger and sustained second phase. In contrast, PAF induced essentially a transient unimodal response in human neutrophils. The two phases of eosinophil response were mediated by distinct cellular mechanisms: the second phase was highly dependent on cellular adhesion and β2 integrins, but the first phase was independent of both adhesion and β2 integrins. The upstream signaling mechanisms were also different: the second phase was mediated by PTX-resistant G-protein(s) and through activation of phosphatidylinositol 3-kinase, while the first phase was mediated by PTX-sensitive G-protein(s). Furthermore, the second-phase response was ∼100-fold more resistant to inhibition by a competitive PAF receptor antagonist than the first phase. Thus, eosinophils and neutrophils react differently to PAF, and PAF activates two separate and distinct effector pathways in human eosinophils. These two activation pathways may explain the eosinophils’ strong and diverse biological responses to PAF.

Abstract Evidence suggests that cellular adhesion is critical for eosinophil effector functions. Here, we tested the hypothesis that an adhesion molecule, specifically β2 integrin, participates in intracellular signaling events of eosinophils. Eosinophils stimulated with interleukin (IL)-5 and adherent to protein-coated tissue culture plates via β2 integrin (CD18) showed tyrosine phosphorylation of a number of proteins. Among these proteins, tyrosine phosphorylation of the 105 kD and 115 kD proteins and the product of the c-cbl protooncogene, Cbl, was specifically inhibited using soluble anti-CD18 monoclonal antibody (mAb) to block eosinophil cell adhesion. Furthermore, phosphoinositide turnover of IL-5-stimulated adherent eosinophils was also inhibited by anti-CD18 mAb, suggesting that cellular adhesion plays important roles in eosinophil signal transduction. αMβ2 (Mac-1, CD11b/18) was one of the β2 integrins involved in eosinophil adhesion to protein-coated plates. We found that direct ligation of eosinophil αMβ2 with anti-CD11b mAb coupled to polystyrene microbeads induced tyrosine phosphorylation of a 115 kD protein and Cbl. Furthermore, anti-CD11b mAb microbeads induced increases in both phosphoinositide hydrolysis and the eosinophil degranulation response. Control antibodies, such as mouse myeloma IgG1 and anti-HLA class I antigen mAb, did not induce these cellular responses. These results suggest that engagement of β2 integrin either by cell adhesion or by anti-CD11b mAb triggers activation of an intracellular signaling cascade, including protein tyrosine phosphorylation and phosphoinositide turnover, and subsequent cellular degranulation in human eosinophils. Tyrosine phosphorylation of a 115 kD protein and Cbl may play important roles in adhesion-dependent cellular functions of eosinophils.