Hirokazu Kimura

Platelet-Activating Factor Activates Two Distinct Effector Pathways in Human Eosinophils

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.

Inhibition of human eosinophil activation by a cysteinyl leukotriene receptor antagonist (pranlukast; ONO-1078).

Eosinophils produce cysteinyl leukotrienes such as leukotriene C4 and D4 upon stimulation by platelet-activating factor or other mediators, and these cells themselves express cysteinyl leukotriene receptors. Pranlukast, a compound developed in Japan, antagonizes cysteinyl leukotriene receptors and inhibits contraction of airway smooth muscle, microvascular leakage into airways, and eosinophil infiltration. This agent can decrease symptoms of bronchial asthma, but its specific influences on effector functions of eosinophils important to the pathogenesis and exacerbation of asthma remain unknown. In the present study, we investigated the effect of pranlukast on human eosinophil functions. Eosinophils obtained from peripheral blood of normal volunteers were stimulated by platelet-activating factor, leukotriene D4, or phorbol ester. Superoxide anion generation was measured by reduction of cytochrome c. Expression of αMβ2 was analyzed by flow cytometry. To evaluate eosinophil degranulation, eosinophil protein X, a toxic granule constituent, was measured by radioimmunoassay in sample supernatants. Pranlukast partially inhibited major eosinophil effector functions of superoxide anion generation and degranulation induced by platelet-activating factor, although at concentrations tested pranlukast failed to significantly reduce platelet-activating factor- induced αMβ2 expression. Pranlukast completely inhibited leukotriene D4-induced superoxide generation and αMβ2 expression. In contrast, pranlukast at 10−6 M did not affect phorbol ester-induced superoxide generation at 120 minutes, degranulation, or αMβ2 expression. The results suggested that inhibition by pranlukast of platelet-activating, factor-induced eosinophil effector functions such as superoxide generation and degranulation might result at least partly from antagonism of autocrine mechanisms involving cysteinyl leukotrienes produced in response to platelet-activating factor.

Dual Signaling and Effector Pathways Mediate Human Eosinophil Activation by Platelet-Activating Factor

Platelet-activating factor (PAF) induces various cellular functions in eosinophils including chemotaxis, adhesion, superoxide anion (O2–) production, and degranulation. While PAF shares many biological effects with other chemotactic factors such as N-formyl-methionyl-leucyl-phenylalanine, complement fragments, and lipid mediators, PAF is unique in that its action is relatively resistant to pertussis toxin (PTX), and in activating eosinophils more strongly than neutrophils. In this review we consider how PAF might activate human eosinophils in preference to neutrophils, and discuss possible mechanisms of PAF-induced activation of human eosinophils via two distinct signaling and effector pathways. Recently we analyzed O2– production by eosinophils using a sensitive, real-time chemiluminescence method. Our results showed that in human eosinophils PAF activates two distinct signaling and effector pathways coupled to the PAF receptor: one linked to PTX-sensitive G protein(s) and another to PTX-resistant G protein(s), phosphatidylinositol 3-kinase, and cellular adhesion. This activation of two different G proteins by the eosinophil PAF receptor may explain the strong and diverse biological responses of human eosinophils to PAF.

Cellular adhesion is required for effector functions of human eosinophils via G-protein coupled receptors

BACKGROUNDnEosinophils play an important role in the pathogenesis of allergic diseases. Chemoattractants, including platelet-activating factor (PAF) and complement component 5a (C5a), induce eosinophil infiltration and promote eosinophil effector functions.nnnOBJECTIVEnTo compare eosinophil degranulation and superoxide anion (O2-) generation induced by various chemoattractants, and to elucidate the role of cellular adhesion on these effector functions.nnnMETHODSnHuman eosinophils were stimulated with PAF, C5a, eotaxin, or leukotriene B4 (LTB4). O2- generation was assayed by a chemiluminescence method using a Cypridina luciferin analog as the amplifier. Degranulation and adhesion were measured by quantitating eosinophil protein X by radioimmunoassay. Expression of CD11b on eosinophils was measured by flow cytometry.nnnRESULTSnPAF and C5a induced significant degranulation and O2- generation from eosinophils. In contrast, the potency of eotaxin or LTB4 for these functions was much less. PAF and C5a also significantly enhanced eosinophil adhesion, whereas eotaxin and LTB4 did not. CD11b expression on eosinophils was enhanced by all four stimulants, and the order of potency to induce CD11b expression was C5a > PAF > eotaxin > LTB4.nnnCONCLUSIONSnThe potency of PAF and C5a for inducing effector function in eosinophils was greater than that of eotaxin or LTB4. The magnitude of the effector function was consistent with the degree of eosinophil adherence induced by each stimulant. These results suggest that effector functions of eosinophils which are mediated through G-protein coupled receptors are dependent on cellular adhesion.

Distinct isoforms of protein kinase C are involved in human eosinophil functions induced by platelet-activating factor

Background: Platelet-activating factor (PAF) is a potent stimulator of eosinophils. Recently, treatment with a protein kinase C (PKC) inhibitor which generally inhibits PKC isoforms has been shown to modulate several eosinophil functions in distinct manners, in that PKC inhibition enhanced CD11b expression and cellular adhesion, but inhibited superoxide generation and degranulation in PAF-stimulated human eosinophils. These results suggested that distinct PKC isoforms were likely to be involved in each eosinophil function induced by PAF. We have therefore investigated whether or not the PKC isoforms involved in PAF-induced CD11b expression and superoxide generation were different. Methods: Human eosinophils prepared from healthy volunteers were treated with PKC inhibitors, bis-indolylmaleimide I (BisI; a general PKC inhibitor), myristoylated PKC inhibitor peptide (myr-ψPKC; a PKCα, β and δ inhibitor) and rottlerin (a PKCδ inhibitor), followed by stimulation with PAF. CD11b expression was determined using flow cytometry and superoxide generation was evaluated using a cytochrome c reduction assay. Results: BisI treatment led to enhancement of PAF-induced CD11b expression, while myr-ψPKC and rottlerin did not. In contrast, PAF-induced superoxide generation was inhibited by treatment with BisI, myr-ψPKC and rottlerin. Conclusions: PKCα, β and δ are not involved in PAF-induced CD11b expression, but PKCδ is involved in the PAF-induced activation of superoxide anion generation.

Respiratory Syncytial Virus Enhances the Expression of CD11b Molecules and the Generation of Superoxide Anion by Human Eosinophils Primed with Platelet-Activating Factor

Background: Human respiratory syncytial virus (RSV) infection in infancy and early childhood causes acute bronchiolitis and exacerbates bronchial asthma. Eosinophil infiltration may contribute to airway obstruction in RSV infection. Objective: We hypothesized that RSV affects eosinophil function. Methods: Eosinophil activation was evaluated by chemiluminescent detection of superoxide anion (O–2) generation. Expression of CD11b on eosinophils was determined by flow cytometry. Results: Although RSV did not induce O–2 generation by resting eosinophils, RSV enhanced O–2 generation of eosinophils primed with platelet-activating factor (PAF). Enhancement was significantly inhibited by either continuous agitation to prevent eosinophil adhesion to test tube surfaces or by pretreating cells with anti-CD18 antibody, suggesting that the stimulatory effects of RSV on eosinophils depend on cell adhesion via β2-integrins. In fact, RSV enhanced PAF-induced CD11b expression by eosinophils. Conclusion: These findings suggest that RSV enhances eosinophil CD11b expression and O–2 generation induced by PAF. Thus, RSV infection may exacerbate airway inflammation by enhancing mediator release from eosinophils.

INCREASED SUPEROXIDE RADICALS GENERATION FROM ALVEOLAR MACROPHAGES IN IMMATURE GUINEA‐PIGS

To study the effect of maturation on abilities of superoxide radicals (O−2) generation in the airways, we compared stimuli‐induced O−2 generation by alveolar macrophages in immature (aged 10±2 days) and adult (aged 90±2 days) guinea‐pigs. The production of O−2 was assayed by chemiluminescence method, using aCypridina luciferin analog as a highly sensitive and specific probe for O−2. Whereas no significant difference in cell components of bronchoalveolar lavage fluid was observed between immature and adult animals, O−2 generation induced by stimulation of alveolar macrophages was greater in immature than in adult animals, with significant differences observed after platelet‐activating factor (100nM) or phorbol myristate acetate (0.5μg/ml). The results suggest that alveolar macrophages from immature animals are far more potent O−2 generators than the same cells of adult animals.

Sulfonated human immunoglobulin enhances CD16-linked CD11b expression on human neutrophils

Intravenous human immunoglobulin therapy infrequently results in excessive inflammatory responses in vivo; these effects are not fully understood. We assessed whether sulfonated human immunoglobulin (SHIG) or polyethylene glycol‐treated human immunoglobulin (PHIG) enhanced expression of inflammatory receptors on peripheral blood neutrophils in vitro, such as αMβ2 (CD11b/CD18) and Fc gamma receptor type III (FcγRIII). CD11b and CD16 expression on neutrophils was measured by fluorescence flow cytometry. Various cytokines were assessed using a highly sensitive fluorescence microsphere system. SHIG enhanced/induced CD11b expression and partial aggregations on neutrophils, but PHIG did not. No detection of aggregation IgG was observed in SHIG and PHIG. SHIG‐induced CD11b expression was inhibited by treatment of corticosteroid (dexamethasone) and by anti‐CD16 monoclonal antibody. Concentrations of various cytokines such as interleukin (IL)‐1β, IL‐2, IL‐4, IL‐5, IL‐6, IL‐8, IL‐10, RANTES, tumor necrosis factor (TNF)‐α, and interferon (INF)‐γ in culture supernatant were not significantly changed by SHIG or PHIG. SHIG and PHIG did not enhance CD16 on neutrophils. SHIG enhanced CD16‐linked CD11b expression on neutrophils in vitro. CD11b induction was inhibited by dexamethasone and by anti‐CD16 antibody. These in vitro results suggest that aggregations and enhancement of CD11b on neutrophils by SHIG may induce excessive inflammatory responses in vivo.