Actin assembly is a crucial factor for superoxide anion generation from adherent human eosinophils

BACKGROUND: Cellular adhesion is crucial for eosinophil effector functions. OBJECTIVE: We sought to elucidate the role of the actin cytoskeleton in cellular adhesion and superoxide anion generation by human eosinophils. METHODS: Eosinophils were stimulated with platelet-activating factor (PAF) or complement component 5a on human serum albumin-coated plates with or without an actin-polymerization inhibitor, cytochalasin B (CB), or cytochalasin D (CD). Superoxide anion generation was measured on the basis of reduction of absorbance associated with cytochrome c.2 Eosinophil adhesion was assessed on the basis of eosinophil protein X content in adherent cells. Transient stimulus-induced increase of intracellular calcium and translocation of protein kinase C (PKC) betaII, PKC delta, PKC zeta, and p47 phagocyte oxidase (a component of nicotinamide adenine dinucleotide phosphate oxidase) were also investigated. RESULTS: CB, CD, or antibodies against CD18 (the beta2 chain of integrin, alphaMbeta2) inhibited stimulus-induced eosinophil superoxide anion generation. Stimulus-induced eosinophil adhesion was unaltered by CB, whereas it was significantly suppressed by CD or anti-CD18 antibodies. Transient PAF-induced intracellular calcium increase was also unaffected by CB or CD, but stimulus-induced eosinophil shape changes and translocation of PKCs and p47 phagocyte oxidase to the cell membrane region were completely inhibited by CB. PAF-induced eosinophil degranulation was inhibited by CB, CD, or anti-CD18 antibodies, whereas complement component 5-induced degranulation was not suppressed by CB. CONCLUSION: By itself, beta2 integrin-dependent cellular adhesion is not sufficient for promoting eosinophil effector function. Adequate actin assembly is required for eosinophil adhesion and also for full superoxide anion generation in eosinophils.     

Inhibition of protein kinases A and C demonstrates dual modes of response in human eosinophils stimulated with platelet-activating factor

BACKGROUND: Platelet-activating factor (PAF) is a potent stimulator of human eosinophils involved in the pathogenesis of allergic diseases. However, intracellular signaling mechanisms in eosinophils involving the PAF receptor are incompletely understood. OBJECTIVE: We sought to determine the roles of protein kinase C (PKC) and cyclic AMP-dependent protein kinase (protein kinase A [PKA]) in signaling pathways of human eosinophils stimulated with PAF. METHODS: After pretreatment with a PKC inhibitor, bisindolylmaleimide I, or a PKA inhibitor, H89, we investigated PAF-evoked functions, such as CD11b expression, cellular adhesion, superoxide anion generation, and degranulation in human eosinophils. RESULTS: Preincubation of eosinophils with bisindolylmaleimide I resulted in enhancement of upregulated CD11b expression and adhesion induced by PAF. H89 pretreatment also enhanced PAF-induced cellular adhesion. Superoxide anion generation and degranulation were suppressed by means of inhibition of either PKC or PKA. CONCLUSION: PKC and PKA negatively regulate PAF-induced CD11b upregulation and cellular adhesion but promote eosinophil effector functions, such as superoxide anion generation and degranulation. PKC and PKA modulate PAF-evoked intracellular signaling of the eosinophil function in distinct ways.     

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

BACKGROUND: Eosinophils 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. OBJECTIVE: To compare eosinophil degranulation and superoxide anion (O2-) generation induced by various chemoattractants, and to elucidate the role of cellular adhesion on these effector functions. METHODS: Human 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. RESULTS: PAF 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. CONCLUSIONS: The 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.     

Differential inhibition of inflammatory effector functions by petasin, isopetasin and neopetasin in human eosinophils

BACKGROUND: Priming of eosinophils with granulocyte-macrophage colony-stimulating factor (GM-CSF) and subsequent stimulation with platelet-activating factor (PAF) or the anaphylatoxin C5a is associated with a rapid production of leukotrienes (LTs) and release of eosinophil cationic protein (ECP). OBJECTIVE: This study was designed to determine the effects of the sesquiterpene esters petasin, isopetasin and neopetasin on LT generation and ECP release in eosinophils in vitro. METHODS: The model of eosinophil activation described above was used to induce LT production and ECP release. Cells were incubated with petasins and control inhibitors prior to priming and stimulation. To analyse intracellular steps of eosinophil activation and determine potential drug targets, some key signalling events were studied. Activity of cytosolic phospholipase A2 (cPLA(2)) was measured by analysing the generation of arachidonic acid (AA). Translocation of 5-lipoxygenase (5-LO) was observed using immunofluorescence microscopy. Intracellular calcium concentrations [Ca2+]i were measured by a bulk spectrofluorometric assay. RESULTS: Whereas all three compounds inhibited LT synthesis, ECP release from eosinophils was blocked by petasin only, but not isopetasin or neopetasin. Similarly, PAF- or C5a-induced increases in [Ca2+]i were completely abrogated by petasin only, whereas isopetasin and neopetasin had significant lower blocking efficacy. Moreover, only petasin, but not isopetasin or neopetasin, prevented increases in cPLA(2) activity and 5-LO translocation from the cytosolic compartment to the nucleus envelope in calcium ionophore-stimulated eosinophils. CONCLUSION: These data suggest that different petasins may at least partially block different intracellular signalling molecules. To reduce LT synthesis, isopetasin and neopetasin may act at the level of or distal to 5-LO. In contrast, petasin may inhibit inflammatory effector functions in human eosinophils by disrupting signalling events at the level of or proximal to phospholipase Cbeta (PLCbeta), besides its potential inhibitory activity within mitogen-activated protein kinase (MAPK) and LT pathways.     

IL-5 enhances the in vitro adhesion of human eosinophils, but not neutrophils, in a leucocyte integrin (CD11/18)-dependent manner.

In an attempt to explain the preferential accumulation of eosinophils at sites of allergic tissue reactions, we have studied the effects of interleukin-5 (IL-5) on the adherence of human eosinophils and neutrophils to plasma-coated glass (PCG) or human microvascular endothelial cells (HMVEC). IL-5 was compared with IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and platelet-activating factor (PAF), since all these agents have biological properties associated with eosinophil activation and/or survival in vitro. IL-5, IL-3 and GM-CSF induced a time-dependent increase in adherence of normal density eosinophils to PCG optimal at 60 min, whereas the effect of PAF was greater at 15 min. Similar results were obtained with neutrophils, with the exception that IL-5 had minimal and non-significant effects on this cell type. Unstimulated eosinophils and neutrophils also adhered to PCG or HMVEC, but in low numbers. Preincubation of eosinophils with IL-5, GM-CSF or PAF resulted in dose-dependent increases in the numbers of adherent cells to PCG. IL-3 had a smaller but significant effect on enhanced eosinophil adhesion to PCG, while IL-2 and lyso-PAF were ineffective. Neutrophils gave similar levels of baseline and stimulated adhesion to PCG as eosinophils, IL-5 again had no significant stimulatory effect. IL-5 also increased eosinophil, but not neutrophil, adherence to HMVEC in a concentration-dependent manner. Preincubation with the protein synthesis inhibitor cycloheximide had no effect on IL-5-, GM-CSF- or PAF-stimulated eosinophil adhesion. The contribution of the CD11/18 leucocyte integrins to IL-5- and PAF-induced eosinophil hyperadherence was investigated by inhibition experiments utilizing monoclonal antibodies (mAb). Enhanced adhesion to PCG (by PAF) or HMVEC (by IL-5) was inhibited by (ranked in order of potency) anti-CR3 alpha = common beta-chain greater than LFA-1 alpha. Anti-p150,95 alpha had no measurable effect. Baseline adhesion by unstimulated eosinophils was not significantly influenced by prior incubation with these mAb. Using flow cytometry, IL-5 and IL-3 were found to up-regulate cosinophil but not neutrophil CR3 expression. These findings demonstrate that IL-5 enhances cosinophil, but not neutrophil, adherence reactions, by a mechanism dependent, at least in part, on the CD11/18 family of adhesion glycoproteins.     

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.     

Detection of C5a receptors on human eosinophils and inhibition of eosinophil effector functions by anti-C5a receptor (CD88) antibodies

Eosinophils and complement activation are reported to play a crucial role in the pathogenesis of connective tissue diseases. Depositions of antigens and antigen-antibody complexes lead to complement activation with the generation of anaphylatoxins, particularly C5a, which is thought to be responsible for the infiltration and activation of eosinophils in the tissue. Previous studies suggested that the eosinophil C5a receptor differs structurally from the receptor expressed on neutrophils. In this study, we investigated the expression and functional properties of C5a receptors on human eosinophils using the C5a receptor monoclonal antibody S5/1 (anti-CD88 mAb). Flow cytometric analysis demonstrated that the anti-CD88 mAb bound homogeneously on the surface of human eosinophils from nonatopic healthy donors. In addition, no subpopulations with respect to C5a receptor expression were identified in normodense or hypodense eosinophils of patients with hypereosinophilia. Pre-incubation of eosinophils with anti-CD88 specifically inhibited C5a-induced intracellular calcium concentration transients. C5a-induced chemotactic activity of eosinophils was significantly inhibited after pre-incubation of cells with anti-CD88 mAb in a dose-dependent manner. Furthermore, anti-CD88 mAb inhibited dose-dependently the release of reactive oxygen species by eosinophils following stimulation with C5a. Thus, the human eosinophil C5a receptor is homogeneously expressed on normal eosinophils from healthy donors as well as on hypodense and normodense eosinophil subpopulations from patients with hypereosinophilia. Based on the inhibitory effect of the S5/1 mAb on C5a-stimulated eosinophil effector functions, we conclude that a single C5a receptor type exists on human eosinophils. In addition, the inhibitory effect of the S5/1 mAb on C5a functions may enable a new experimental approach to the treatment of diseases that have been associated with C5a-mediated activation.     

Regulation of interleukin-5-induced beta2-integrin adhesion of human eosinophils by phosphoinositide 3-kinase

We examined the role of phosphoinositide 3-kinase (PI3K) in integrin-mediated eosinophil adhesion. Deltap85, a dominant-negative form of the class IA PI3K adaptor subunit, was fused to an HIV-TAT protein transduction domain (TAT-Deltap85). Recombinant TAT-Deltap85 inhibited interleukin (IL)-5-stimulated phosphorylation of protein kinase B, a downstream target of PI3K. beta(2)-Integrin-dependent adhesion caused by IL-5 to the plated intracellular adhesion molecule-1 surrogate, bovine serum albumin, was inhibited by TAT-Deltap85 in a concentration-dependent manner. Similarly, two PI3K inhibitors, wortmannin and LY294002, blocked eosinophil adhesion to plated bovine serum albumin. By contrast, beta(1)-integrin-mediated eosinophil adhesion to vascular cell adhesion moelcule-1 was not blocked by TAT-Deltap85, wortmannin, or LY294002. Rottlerin, a protein kinase C (PKC)-delta inhibitor, also blocked beta(2)-integrin adhesion of eosinophils caused by IL-5, whereas beta(1) adhesion to vascular cell adhesion molecule-1 was not affected. IL-5 caused translocation of PKCdelta from the cytosol to cell membrane; inhibition of PI3K by wortmannin blocked translocation of PKCdelta. Western blot analysis demonstrated that extracellular signal-regulated kinase phosphorylation, a critical intermediary in adhesion elicited by IL-5, was blocked by inhibition of either PI3K or PKC-delta. These data suggest that extracellular signal-regulated kinase-mediated adhesion of beta(2)-integrin caused by IL-5 is mediated in human eosinophils by a class IA PI3K through activation of a PKCdelta pathway.     

Interleukin-5 enhances the in vitro adhesion of human eosinophils, but not neutrophils, in a leucocyte integrin (CD11/18)-dependent manner.

Interleukin (IL-5) was found to enhance the adhesion of eosinophils, but not neutrophils, to both microvascular and large vein endothelial cells in a dose-dependent manner. Granulocyte/macrophage-colony-stimulating factor (GM-CSF) and platelet-activating factor (PAF) enhanced both eosinophil and neutrophil adhesion. Significant increases in eosinophil CR3 expression, but not LFA-1, were observed following pre-incubation with PAF, IL-3, IL-5 or GM-CSF. Neutrophil CR3 expression was increased significantly by pre-incubation with PAF or GM-CSF, but not IL-3 or IL-5. Enhanced adhesion to human microvascular endothelial cells (HMVEC) or human umbilical vein endothelial cells (HUVEC) was inhibited by (ranked in order of potency) anti-CR3 alpha = common beta-chain greater than LFA-1 alpha. Anti-p150,95 alpha had no measurable effect. Basal expression of eosinophil CR3 with monoclonal antibody inhibited IL-5-induced eosinophil hyperadherence to HUVEC in a manner almost identical to inhibition in the presence of excess anti-CR3. Thus, a conformational or affinity change in adhesion receptors following activation seems more important than a simple increase in numbers. No inhibition of unstimulated eosinophil adhesion to HMVEC or HUVEC by CD11/18 monoclonal antibody was observed. These findings demonstrate that IL-5 enhances eosinophil, but not neutrophil, adherence reactions, by a mechanism dependent, at least in part, on the CD11/18 family of adhesion glycoproteins.     

Proline-rich tyrosine kinase 2 regulates spreading and migration of eosinophils after beta2-integrin adhesion

We examined the role of proline-rich tyrosine kinase (Pyk) 2 in the spreading and migration of human blood eosinophils after beta(2)-integrin ligation. Western blot analysis showed that Pyk2 was activated by phosphorylation at Y402 after eosinophil adhesion to BSA-coated plates after activation with IL-5, platelet-activating factor (PAF), formyl-met-leu-phe (fMLP), or Mn(2)(+). To determine the role of Pyk2 in regulating eosinophil migration, we used a transducable dominant-negative inhibitor of Pyk2, TAT-mediated protein transduction of dominant-negative C-terminal Pyk2 (TAT-Pyk2-CT), a fusion protein in which TAT peptide was fused to the C-terminal Pyk2. TAT-Pyk2-CT blocked tyrosine phosphorylation of Pyk2 caused by beta(2)-integrin adhesion, but did not block adhesion of eosinophils to plated BSA. TAT-Pyk2-CT also blocked subsequent spreading and migration of eosinophils caused by IL-5, PAF, or fMLP. Spreading eosinophils stained with FITC-conjugated phalloidin showed elongation and formation of multiple fillopodia and lamellipodia, whereas nonspreading eosinophils were smaller and round. Treatment of eosinophils with TAT-Pyk2-CT had no effect on the initial cell polarization, but blocked the formation of fillopodia and lamellipodia in adherent cells. Migration of eosinophils through Transwell plates caused by IL-5, PAF, or fMLP was blocked significantly after inhibition of Pyk2. These data indicate that Pyk2, although not involved in beta(2)-integrin adhesion, causes eosinophil spreading and regulates subsequent chemotactic migration after beta(2)-integrin ligation to endothelial counter ligands. We conclude that Pyk2 is activated by beta(2)-integrin adhesion and is a required signal for eosinophil spreading and subsequent chemotactic migration.     

Interleukin-3, -5, and granulocyte macrophage colony-stimulating factor induce adhesion and chemotaxis of human eosinophils via p38 mitogen-activated protein kinase and nuclear factor kappaB

Hematopoietic cytokines such as interleukin (IL)-3, IL-5, and granulocyte macrophage colony-stimulating factor (GM-CSF) play a fundamental role in eosinophil functions in allergic asthma. The intracellular signal transduction mechanisms of these cytokines regulating the activation of eosinophils have been potential therapeutic targets. We investigated the roles of p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-kappaB) in IL-3, IL-5, and GM-CSF-induced adhesion, morphological changes, and subsequence transmigration of human eosinophils. IL-3, IL-5, and GM-CSF could augment the phosphorylation of p38 MAPK and nucleus translocation of NF-kappaB in eosinophils. cDNA expression arrays demonstrated that the gene expression levels of several adhesion molecules including intercellular adhesion molecule-1 (ICAM-1), alpha6, beta2 integrin (CD18), and CD44 were upregulated by these cytokines. Results from functional assays showed that adhesion of eosinophils onto airway epithelial cells was enhanced after IL-3 and IL-5 but not GM-CSF stimulation. These cytokines could markedly induce shape change and augment the transmigration of eosinophils. Moreover, administration of either p38 MAPK inhibitor, SB 203580, or proteasome inhibitor, N-cbz-Leu-Leu-leucinal (MG-132), could inhibit the cytokine-induced adhesion, shape change, and transmigration of eosinophils. Together, our findings suggest that IL-3, IL-5, and GM-CSF regulated the adhesion and chemotaxis of human eosinophils through shared signaling pathways involving both p38 MAPK and NF-kappaB. Our results therefore shed light on the further development of more effective agents for allergic and inflammatory diseases.     

Eosinophilopoietic factors prime eosinophils for increased interleukin-8 generation

Recent studies have identified eosinophils as a cellular source of various cytokines, indicating that eosinophils play not only an effector role but also a regulatory role within the allergic inflammatory cell network. Because eosinophilopoietic factors are known to stimulate various functions of eosinophils, we examined the effect of interleukin (IL)-5 on chemoattractant-induced IL-8 generation from eosinophils. Although IL-5 alone induced little or no IL-8 production from eosinophils, short-term preincubation with IL-5 markedly enhanced the eosinophil IL-8 generation caused by C5a plus cytochalasin B (CB). IL-3 also potentiated C5a-induced IL-8 generation. Both factors were active at picomolar concentrations. Furthermore, competitive polymerase chain reaction (PCR) experiments revealed that the enhancement occurred at the pretranslational level. Since eosinophils in allergic inflammation are believed to be activated by these eosinophilopoietic factors, eosinophil-derived cytokines may play more important roles in the allergic inflammatory cell network than has been previously supposed.     

A surrogate method for assessment of beta(2)-integrin-dependent adhesion of human eosinophils to ICAM-1

We have developed and validated an inexpensive and equivalent method for measuring eosinophil adhesion by β₂-integrin to endothelial ICAM-1 using bovine serum albumin (BSA) as a surrogate for the immunoglobulin supergene. The number of adherent eosinophils on BSA or ICAM-1 coated microplates was quantified by residual eosinophil peroxidase activity. Non-stimulated eosinophils did not adhere to either BSA or ICAM-1. However, after IL-5 stimulation, either BSA or ICAM-1 caused comparable and concentration-dependent adhesion of eosinophils. Eosinophil adhesion was rapid and occurred within 15 to 30 min of incubation for either BSA or ICAM-1. Preincubation of cells with CD11b or CD18 antibody specifically decreased adhesion to either BSA or ICAM-1. IL-5, PAF and fMLP all induced adhesion of eosinophils to either BSA or ICAM-1 in a concentration-dependent manner, and the optimal IL-5, fMLP and PAF concentrations for adhesion to BSA were the same as for adhesion to ICAM-1. BSA-binding was specific for β₂-integrin; neither -CD49d mAb directed against the ₄-chain or -CD29 directed against the common β₁-chain of VLA-4 blocked adhesion to BSA or ICAM-1 controls. The protein tyrosine kinase inhibitor, genistein, the phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor, wortmanin, and mitogen-activated protein kinase kinase (MEK) inhibitor, U0126, all inhibited IL-5-induced eosinophil adhesion to either BSA or ICAM-1 comparably. These results indicate that BSA is a reliable and economical surrogate ligand for ICAM-1 adhesion to β₂-integrin-dependent adhesion to ICAM-1. Ligation characteristics of BSA are identical to those for soluble ICAM-1, and the assay is suitable for assessment of signal transduction pathways mediating adhesion.     

The effect of salmeterol on human eosinophils is both stimulus- and response-dependent

Salmeterol, a long-acting β₂-adrenoceptor agonist, also possesses some anti-inflammatory properties, but whether eosinophils are the target of such action has been equivocal. To clarify the direct effect of salmeterol on eosinophil functions, we have studied the effect of the drug on the various responses of purified human eosinophils. Superoxide anions (O₂⁻) release and adherence to fibronectin-coated plastic plates induced by platelet-activating factor (PAF), interleukin-5 (IL-5), leukotriene B₄ (LTB₄) and phorbol myristate acetate (PMA), as well as degranulation induced by C5a and formyl methionyl leucyl phenylalanine (FMLP), in the presence of cytochalasin B (CB) were studied. In the concentration range 10⁻⁸-10⁻⁵ M, the drug inhibited PAF- and IL-5-induced O₂ release, with an IC₅₀ values of 3.2±1.2×10⁻⁷, M and 2.2±0.4 × 10⁻⁶, M, respectively, Superoxide anion release by LTB₄ was only modestly inhibited while that due to PMA was completely unaffected. On the other hand, eosinophil adherence induced by all the 4 stimuli were significantly inhibited within the same concentration range. On eosinophil degranulation, the drug failed to significantly inhibit the release of eosinophil peroxidase (EPO) induced by either C5a or FMLP. In contrast, β-hexoseaminidase (β-HA) release by the same agents was significantly inhibited, the inhibition being more pronounced for FMLP-induced, than C5a-induced release. None of the effects of the drug was reversed by the selective β₂-adrenoceptor antagonist ICI 118551 at a concentration of 10⁻⁷ M. These results show that salmeterol may have some direct inhibitory effects on human eosinophil functions but that these effects are both stimulus- and response-dependent, and are unlikely to be mediated via β₂ adrenoceptors.     

Inhibition by fenoterol of human eosinophil functions including beta2-adrenoceptor-independent actions

Agonists at beta2 adrenoceptors are used widely as bronchodilators in treating bronchial asthma. These agents also may have important anti-inflammatory effects on eosinophils in asthma. We examined whether widely prescribed beta2-adrenoceptor agonists differ in ability to suppress stimulus-induced eosinophil effector functions such as superoxide anion (O2-) generation and degranulation. To examine involvement of cellular adhesion in such responses, we also investigated effects of beta2 agonists on cellular adhesion and on CD11b expression by human eosinophils. O2- was measured using chemiluminescence. Eosinophil degranulation and adhesion were assessed by a radioimmunoassay for eosinophil protein X (EPX). CD11b expression was measured by flow cytometry. Fenoterol inhibited platelet-activating factor (PAF)-induced O2- generation by eosinophils significantly more than salbutamol or procaterol. Fenoterol partially inhibited PAF-induced degranulation by eosinophils similarly to salbutamol or procaterol. Fenoterol inhibited phorbol myristate acetate (PMA)-induced O2- generation and degranulation by eosinophils, while salbutamol or procaterol did not. Fenoterol inhibition of PMA-induced O2- generation was not reversed by ICI-118551, a selective beta2-adrenoceptor antagonist. Fenoterol, but not salbutamol or procaterol, significantly inhibited PAF-induced eosinophil adhesion. Fenoterol inhibited O2- generation and degranulation more effectively than salbutamol or procaterol; these effects may include a component involving cellular adhesion. Inhibition also might include a component not mediated via beta2 adrenoceptors.     

A surrogate method for assessment of β2-integrin-dependent adhesion of human eosinophils to ICAM-1

We have developed and validated an inexpensive and equivalent method for measuring eosinophil adhesion by β₂-integrin to endothelial ICAM-1 using bovine serum albumin (BSA) as a surrogate for the immunoglobulin supergene. The number of adherent eosinophils on BSA or ICAM-1 coated microplates was quantified by residual eosinophil peroxidase activity. Non-stimulated eosinophils did not adhere to either BSA or ICAM-1. However, after IL-5 stimulation, either BSA or ICAM-1 caused comparable and concentration-dependent adhesion of eosinophils. Eosinophil adhesion was rapid and occurred within 15 to 30 min of incubation for either BSA or ICAM-1. Preincubation of cells with CD11b or CD18 antibody specifically decreased adhesion to either BSA or ICAM-1. IL-5, PAF and fMLP all induced adhesion of eosinophils to either BSA or ICAM-1 in a concentration-dependent manner, and the optimal IL-5, fMLP and PAF concentrations for adhesion to BSA were the same as for adhesion to ICAM-1. BSA-binding was specific for β₂-integrin; neither α-CD49d mAb directed against the α₄-chain or α-CD29 directed against the common β₁-chain of VLA-4 blocked adhesion to BSA or ICAM-1 controls. The protein tyrosine kinase inhibitor, genistein, the phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor, wortmanin, and mitogen-activated protein kinase kinase (MEK) inhibitor, U0126, all inhibited IL-5-induced eosinophil adhesion to either BSA or ICAM-1 comparably. These results indicate that BSA is a reliable and economical surrogate ligand for ICAM-1 adhesion to β₂-integrin-dependent adhesion to ICAM-1. Ligation characteristics of BSA are identical to those for soluble ICAM-1, and the assay is suitable for assessment of signal transduction pathways mediating adhesion.     

Leukotriene D4 and eosinophil transendothelial migration, superoxide generation, and degranulation via beta2 integrin.

BACKGROUND: Evidence indicates that cysteinyl leukotriene (cysLT) 1 receptor antagonists possess anti-inflammatory properties in asthmatic patients in vivo. Although the exact mechanisms of these actions remain unknown, cysLTs regulate the locomotion and functions of eosinophils. We previously reported that leukotriene D4 augments the expression of eosinophil beta2 integrin and the adhesion of eosinophils to rh intercellular adhesion molecule 1 via beta2 integrin. OBJECTIVE: To examine whether leukotriene D4 modifies the transendothelial migration (TEM) and effector functions of eosinophils. METHODS: We evaluated the effects of leukotriene D4 on (1) eosinophil TEM across human umbilical vein endothelial cells, (2) superoxide anion (O2-) generation, and (3) eosinophil-derived neurotoxin release in eosinophils isolated from the blood of healthy individuals. RESULTS: Leukotriene D4 (0.1-1 microM) significantly induced eosinophil TEM, O2- generation, and eosinophil-derived neurotoxin release. Pranlukast, a cysLT1 receptor antagonist, significantly inhibited all of these parameters, although the inhibitory effect on O2- generation was partial. All of these responses were significantly inhibited by anti-beta2 integrin but not by anti-alpha4 integrin antibodies. CONCLUSIONS: Leukotriene D4 directly up-regulates the TEM and effector functions of eosinophils mainly via the cysLT1 receptor and beta2 integrin. These effects of leukotriene D4 probably contribute to the manifestation of eosinophil inflammation in asthmatic airways.     

The role of protein kinase C in regulating equine eosinophil adherence and superoxide production

Objective: To determine if protein kinase C (PKC) regulates equine eosinophil function.Material or subjects: Blood eosinophils were obtained from healthy ponies.Methods: IL-5- and histamine-induced adherence to serum-coated plastic was measured as the eosinophil peroxidase content of adherent cells and serum treated zymosan (STZ)-and IL-5-induced superoxide production by the reduction of cytochrome C. Eosinophil PKC activity was quantitated as the rate of transfer of ³²P from ATP to substrate. The effects of Ro31-8220 (isotype non-selective PKC inhibitor), Gö6976 (conventional PKC inhibitor), and rottlerin (PKC inhibitor) were determined by ANOVA and Bonferronis or Dunnetts test.Results: Ro31-8220 and Gö6976 reduced superoxide production whereas only Gö6976 inhibited adherence. Rottlerin inhibited histamine-induced adherence and increased STZ-induced superoxide production. Ro31-8220 and Gö6976, but not rottlerin, inhibited PKC activity.Conclusions: PKC is involved in regulating equine eosinophil adherence and superoxide production. The role of PKC appears to depend upon the stimulus used and response measured.Received 13 September 2004; returned for revision 19 October 2004; accepted by N. Boughton-Smith 4 November 2004     

Asbestos-induced apoptosis is protein kinase C delta-dependent

Alveolar epithelial and mesothelial cells undergo apoptosis in response to asbestos, a phenomenon that may be important in injury and/or initiation of compensatory proliferation. Here, we report a functional role of protein kinase (PKC)delta in apoptosis by crocidolite asbestos. We first show that asbestos increases the kinase activity of PKC delta in alveolar type II epithelial cells (C10 line) and causes its translocation to mitochondria, events associated with caspase-9 cleavage and apoptosis as detected by the Apostain technique. Pretreatment of C10 cells with rottlerin (Rot), a PKC delta-selective inhibitor, before addition of asbestos prevented cleavage of caspase-9 and blocked the appearance of apoptotic cells. Asbestos-induced apoptosis also was inhibited in cells stably expressing a dominant-negative kinase-deficient mutant of PKC delta (dnPKC delta), but not dnPKC alpha. Activities of PKC alpha and PKC zeta increased after exposure to asbestos, but neither isoform migrated to mitochondria. A general inhibitor of PKCs, bisindolylmaleimide I, had no effect on asbestos-induced apoptosis. Hydrogen peroxide (H2O2) induced activation of PKCs delta, alpha, zeta, and theta, translocation of PKC delta to mitochondria, and caspase-9 cleavage. However, H2O2-induced apoptosis was not inhibited by cell lines stably expressing either dnPKC delta or dnPKC alpha, suggesting that activation of PKC delta has a distinct role in the development of asbestos-induced apoptosis.