Human Eosinophil Effector Functions Research Articles

Interferon-γ enhances human eosinophil effector functions induced by granulocyte-macrophage colony-stimulating factor or interleukin-5

T helper (Th) 2-type cytokines play a dominant role in allergic inflammation. Accumulating evidence suggests that Th1-type cytokines antagonize Th2-type cytokine responses; however, recent studies demonstrate that Th1 cytokines might enhance Th2 immune responses. We examined whether interferon (IFN)-γ, a representative Th1 cytokine, modifies the effector functions of human eosinophils stimulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-5. GM-CSF and IL-5 have significant functional homology, and contribute to the regulation of Th2 immunity. After the pretreatment of eosinophils with IFN-γ, GM-CSF- or IL-5-induced eosinophil functions were examined, including superoxide anion generation, degranulation, adhesion, expression of GM-CSF receptor (R), IL-5R, or CD11b, and phosphorylation of intracellular signaling molecules. Superoxide anion generation was measured using the cytochrome c reduction method. Degranulation and cell adhesion were evaluated based on eosinophil-derived neurotoxin (EDN) contents in supernatants or adherent cells. Phosphorylation of signaling molecules was analyzed using a multiplex beads array system. Preincubation with IFN-γ resulted in enhanced GM-CSF- or IL-5-induced superoxide anion generation and degranulation of human eosinophils, whereas stimulus-induced eosinophil adhesion was unaffected. In addition, IFN-γ did not influence the expression of GM-CSFR, IL-5R, and CD11b. Furthermore, IFN-γ upregulated GM-CSF- or IL-5-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and activating transcription factor (ATF)-2. Finally, we confirmed that MAPK inhibitors blocked the enhancement of stimuli-induced superoxide anion generation of IFN-γ treated eosinophils. In conclusion, IFN-γ might upregulate ERK, p38, or JNK/ATF-2 phosphorylation induced by GM-CSF or IL-5, leading to enhanced cytokine-induced eosinophil superoxide generation and degranulation.

Interferon-γ Enhances Human Eosinophil Effector Functions Induced by Th2-type Cytokines

Interferon-γ Enhances Human Eosinophil Effector Functions Induced by Th2-type Cytokines

An atypical protein kinase C, PKC ζ, regulates human eosinophil effector functions

Protein kinase (PK) C comprises a family of isoenzymes that play key roles in downstream signalling and cell functions. We studied PKC zeta participation in the effector functions of human eosinophils stimulated with platelet-activating factor (PAF) or complement (C) 5a. After pretreating eosinophils with a myristoylated specific PKC zeta inhibitor; bisindlolylmaleimide I (BisI), an inhibitor of conventional and novel PKCs; or rottlerin, a PKC delta inhibitor, we examined PAF- and C5a-evoked functions. Induced PKC translocation was characterized by confocal laser scanning microscopy. The PKC zeta inhibitor blocked PAF- or C5a-induced eosinophil superoxide anion generation as effectively as BisI or rottlerin. The PKC zeta inhibitor also attenuated PAF- or C5a-induced eosinophil degranulation and adhesion. In contrast, the PKC zeta inhibitor did not affect PAF- or C5a-induced CD11b expression. Finally, both eosinophil shape changes and the translocation of PKC zeta and p47phox, a component of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, to the plasma membrane induced by PAF or C5a were completely inhibited by the PKC inhibitor. Thus, the atypical PKC zeta regulates human eosinophil adhesion and effector functions.

An atypical protein kinase C, PKC ζ, regulates human eosinophil effector functions

Rationale Protein kinase C (PKC) comprises a family of isoenzymes playing key roles in downstream signaling and cell functions. PKCs are grouped according to molecular structure and mode of activation: “conventional” PKCs (α, βI, βII, γ) activated by calcium, phosphatidylserine (PS), and diacylglycerol (DAG); “novel” PKCs (δ, ϵ, μ, θ, η) requiring PS and DAG but not calcium; and “atypical” PKCs (ζ, τ/λ) activated by PS alone. We studied PKC ζ participation in effector functions of human eosinophils stimulated with platelet-activating factor (PAF) or complement component (C) 5a. Methods After pretreating eosinophils with a myristoylated PKC ζ specific inhibitor; a myristoylated PKC η specific inhibitor; bisindolylmaleimide I (Bis I), an inhibitor of conventional and novel PKCs; or rottlerin, a PKC δ inhibitor, we examined PAF- or C5a-evoked functions. Induced PKC translocation was characterized by confocal laser scanning microscopy. Results The PKC ζ inhibitor blocked PAF- or C5a-induced eosinophil superoxide anion generation, degranulation, and adhesion as effectively as Bis I or rottlerin. The PKC η inhibitor had no effect since human eosinophils lack PKC η; this confirmed specificity of PKC ζ inhibitor effects. Neither the PKC ζ inhibitor nor rottlerin affected PAF- or C5a-induced αMβ2 expression, while Bis I enhanced PAF-induced αMβ2 expression. Finally, PAF or C5a induced eosinophil shape changes and translocation of PKC ζ, βII, and δ to the cell membrane. Cell shape and translocation responses were completely inhibited by an actin-polymerization inhibitor, cytochalasin B. Conclusions An atypical PKC, PKC ζ, regulates human eosinophil adhesion and effector functions.

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

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. 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. 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. 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. 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.

Enhanced survival and multiple gene expression in human eosinphils exposed to soluble secretory IgA

6 O A Enhanced Survival and Multiple Gene Expression in Human ~,F-Ir Eosinphils Exposed to Soluble Secretory IgA Kathleen R Bartemes, Kate Cooper, Hirohito Kita Mayo Clinic, Rochester, MN In humans, eosinophils are tissue-resident cells with >99% residing in tissues, especially in the mucosal tissues; many eosinophils are found in the intestine of normal individuals. Secretory IgA (S-IgA) is the main antibody secreted by the mucosa of the airways and gastrointestinal tract and is considered to provide protective mucosal immunity. Because of this common spatial distribution of eosinophils and S-IgA in mucosa, we speculated that S-IgA may have more profound effects than previously appreciated on homeostasis, gene expression, or effector functions of human eosinophils. In this study, we addressed this question by using two different forms of SIgA, namely S-IgA immobilized onto polystyrene beads (immobilized SIgA) and S-IgA kept in solution (soluble S-IgA) to mimic in vivo environment. We found that eosinophils incubated with immobilized S-IgA showed marked degranulation, as detected by the release of eosinophilderived neurotoxin (EDN), and superoxide production, compared to unstimulated eosinophils or to eosinophils incubated with uncoated beads. Immobilized S-IgA did not affect eosinophil viability as examined by exclusion of propidium iodide after 48 hours of incubation. In contrast, soluble S-IgA showed minimal effects on eosinophil degranulation and superoxide production. Furthermore, soluble S-IgA did inhibit eosinophil apoptosis and markedly enhanced 48-hour survival, suggesting that soluble SIgA does not affect release of mediators by eosinophils, but does affect cell survival and perhaps gene expression. Indeed, messenger RNA microarray analyses using Affymetrix Human Genome U95A Array showed that more than 120 genes are upregulated >2-fold in human eosinophils incubated with soluble S-IgA (7 ktg/ml) for 6 hours compared to those incubated with soluble IgG (7 Bg/ml) or medium alone. An example of upregulated genes includes those for signal transduction molecules (e.g. Act-2, pim-l, NF~b), cytokines/chemokines (e.g. IL113, IL-8, TNF-tx), and cell surface molecules (e.g. CD44, [32 integrin otD). Furthermore, the production of several representative proteins (e.g. IL-8) was also confirmed by immunoassays. These findings suggest that S-IgA may have pivotal effects on human eosinophils; an immobilized form is associated with effector functions and a soluble form is associated with survival and gene expression. Furthermore, eosinophils interacting with S-IgA in mucosal tissues or the lumen may express sets of genes which may be unique compared to those expressed by peripheral blood eosinophils.

Trypsin induces activation and inflammatory mediator release from human eosinophils through protease-activated receptor-2.

Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors, which are activated by proteolytic cleavage of the amino terminus of the receptor itself. PARs are most likely involved in various biological responses, such as hemostasis and regulation of muscle tone; however, the roles of PARs in the functions of inflammatory and immune cells are poorly understood. Because eosinophils are most likely involved in allergic inflammation and are exposed to a variety of proteases derived from allergens and other inflammatory cells, we investigated whether PARs regulate effector functions of eosinophils. Human eosinophils constitutively transcribe mRNA for PAR2 and PAR3, but not those for PAR1 and PAR4. The expression of PAR2 protein was confirmed by flow cytometry. When trypsin, an agonist for PAR2, was incubated with eosinophils, it potently induced superoxide anion production and degranulation; 5 nM trypsin induced responses that were 50-70% of those induced by 100 nM platelet-activating factor, a positive control. In contrast, thrombin, an activator for PAR1, PAR3, and PAR4, showed minimal effects. The stimulatory effect of trypsin was dependent on its serine protease activity and was blocked 59% by anti-PAR2 Ab. Furthermore, a specific tethered peptide ligand for PAR2 potently induced superoxide production and degranulation; the effects of peptide ligands for PAR1, PAR3, and PAR4 were negligible. These findings suggest that human eosinophils express functional PAR2, and serine proteases at the inflammation site may play important roles in regulating effector functions of human eosinophils. The expression and functional relevance of other PARs still need to be determined.

Cysteine protease secreted by Paragonimus westermani attenuates effector functions of human eosinophils stimulated with immunoglobulin G.

An immunoglobulin G (IgG)-coated surface, such as that found on helminth parasites, is one of the most effective physiologic stimuli for eosinophil activation. The cysteine proteases secreted by tissue-invasive helminth larvae play an important role in evasion of the immune response by degrading the host immunoglobulins. In this study, we investigated whether cysteine proteases in the excretory-secretory product (ESP) produced by Paragonimus westermani newly excysted metacercariae (PwNEM), which cause pulmonary or extrapulmonary paragonimiasis in human beings, could modify effector functions of human eosinophils stimulated with IgG. We coated 96-well plates with human IgG in the absence or presence of the ESP produced by PwNEM. When eosinophils were incubated in the wells coated with IgG in the presence of the ESP, eosinophil degranulation and superoxide production were significantly reduced compared with results for cells incubated in wells coated with IgG alone. This inhibitory effect of the ESP on IgG-induced superoxide production was dose dependent and was significantly abolished by pretreatment of the ESP with heat. These findings suggest that the cysteine proteases secreted by PwNEM attenuate both activation and degranulation of eosinophils stimulated with IgG. Thus, the cysteine proteases produced by tissue-invasive helminth larvae play crucial roles in evasion of IgG-dependent eosinophil helminthotoxicity and in reduction of eosinophil-associated tissue inflammation during the migratory period.

Modulation of eosinophil effector functions: the potential role of monoclonal antibodies and chemokine receptor antagonists.

Increased numbers of eosinophils in the peripheral blood and inflammatory tissue are characteristic features of allergic diseases such as allergic asthma, rhinoconjunctivitis, and atopic dermatitis. Tissue damage and propagation of inflammation is thought to be mediated by the interaction among Th2-like T cells, antigen-presenting cells, and eosinophils. In this process eosinophils are activated by several inflammatory mediators, leading to the influx of eosinophils at sites of inflammation and to tissue damage by the release of reactive oxygen species and toxic granule proteins. Therefore, agents that would be able to inhibit or antagonize mediator-induced eosinophil activation seem to be possibilities as new therapeutic strategies. In this review we will focus on the modulation of human eosinophil effector functions by monoclonal antibodies and chemokine receptor antagonists. We will discuss whether modulation of eosinophil effector functions might be successful as a possible future strategy of diseases that are accompanied by activated eosinophils. Even when these compounds show antagonistic effects on human eosinophils, in vitro future studies will be necessary to investigate whether chemokine receptor antagonists and monoclonal antibodies are suitable in vivo in an animal model prior to studies in humans.

Activation of human eosinophil effector functions by CC chemokines.

Different kinds of mediators, particularly chemokines, are responsible for the attraction and activation of eosinophils to the site of inflammation. In the last decade, this new family of chemotactic cytokines have become interesting because of their restricted target cell specificity. This article focuses on the activation of eosinophil effector functions in response to different CC chemokines.

The CC Chemokine Receptor Antagonist Met–RANTES Inhibits Eosinophil Effector Functions

Eosinophils play an important role in allergic diseases such as allergic asthma, rhinoconjunctivitis and atopic dermatitis. Recruitement of eosinophils to the side of inflammation, the release of reactive oxygen species, leading to tissue damage, and the propagation of the inflammatory response are mediated by chemokines. Thus, the applicability of agents able to inhibit or antagonize chemokine–induced eosinophil activation seems to be of interest in the treatment of allergic diseases. Therefore, the effect of the CC chemokine antagonist, Met–RANTES, on its effect on human eosinophil effector functions in response to RANTES, MCP–3 and eotaxin was investigated. Met–RANTES had no intrinsic activity on [Ca²⁺]_i transients in eosinophils and was able to dose–dependently inhibit [Ca²⁺]_itransients in eosinophils following stimulation with RANTES, MCP–3 and eotaxin. Besides its effect on [Ca²⁺]_i transients, Met–RANTES dose–dependently inhibited actin polymerization in eosinophils and the release of reactive oxygen species following stimulation with RANTES, MCP–3 and eotaxin. The results of this study lead to the conclusion that Met–RANTES is an effective and powerful compound to antagonize effector functions of human eosinophils following stimulation with RANTES, MCP–3 and eotaxin and is therefore a promising therapeutic approach to prevent the invasion and destructive power of eosinophils in allergic diseases.

Interaction with Secretory Component Stimulates Effector Functions of Human Eosinophils But Not of Neutrophils

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 beta2 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.

Sulochrin inhibits eosinophil activation and chemotaxis.

Because eosinophils likely play important roles in the pathophysiology of allergic diseases, specific inhibitors of eosinophils may be desirable to treat such diseases. To evaluate the capacity of a novel compound, sulochrin, as an inhibitor of eosinophilic inflammation, we examined the effects of this compound on various effector functions of eosinophils. We examined the effects of sulochrin on degranulation of human eosinophils stimulated with platelet-activating factor (PAF) or Sepharose 4B beads coated with secretory IgA (sIgA) or IgG. The effects of sulochrin on other effector functions of human eosinophils, including superoxide anion (O2-) production, leukotriene (LT) C4 release, and interleukin (IL)-8 production induced by sIgA-beads were also studied. Finally, using PAF and LTB4 as chemoattractants, we evaluated the potency of sulochrin to inhibit eosinophil migration in vitro and in vivo. Sulochrin inhibited EDN release by eosinophils stimulated with sIgA-beads. IgG-beads and PAF in a concentration-dependent manner; IC50 values were 0.75 microM, 0.30 microM and 0.03 microM. Eosinophil O2- production, LTC4 release, and IL-8 production were also inhibited by sulochrin. Furthermore, PAF-induced chemotaxis of human eosinophils and LTB4-induced chemotaxis of guinea pig eosinophils were abolished by 1 microM of sulochrin. Finally, sulochrin potently inhibited LTB4-induced infiltration of eosinophils into the skin of guinea-pig in vivo. These results suggest that sulochrin is a potent inhibitor of various effector functions of eosinophils. Sulochrin and its derivatives may be useful in the development of therapeutic approaches for patients with allergic diseases.

The CC chemokine antagonist Met-RANTES inhibits eosinophil effector functions through the chemokine receptors CCR1 and CCR3.

Eosinophils are predominant effector cells not only in allergic diseases but also in connective tissue diseases. The recruitment of eosinophils to the site of inflammation and release of reactive oxygen species leading to tissue damage and propagation of the inflammatory response are mediated by chemokines. Thus, agents that would be able to inhibit or antagonize chemokine-induced eosinophil activation are interesting as therapeutical agents. We describe the effect of a chemokine receptor antagonist, Met-RANTES, on human eosinophil effector functions in response to RANTES, monocyte chemoattractant protein (MCP)-3 and eotaxin. Met-RANTES was able to inhibit dose-dependently [Ca2+]i transients in eosinophils following stimulation with RANTES, MCP-3 and eotaxin. Whereas maximal and half-maximal inhibitory effect of Met-RANTES following stimulation with RANTES and MCP-3 were observed at 2 micrograms/ml and 1 microgram/ml, respectively, maximal and half-maximal inhibitory effects of Met-RANTES in response to eotaxin were detected at 10 micrograms/ml and 3 micrograms/ml. Moreover, eotaxin-induced [Ca2+]i transients were only half reduced at a Met-RANTES concentration at which RANTES and MCP-3 were completely blocked. Besides its effect on [Ca2+]i transients, Met-RANTES dose-dependently inhibited actin polymerization in eosinophils following chemokine stimulation. Whereas Met-RANTES totally inhibited RANTES- and MCP-3-induced actin polymerization at 5 micrograms/ml, the eotaxin-induced response was only reduced by 50%. However, Met-RANTES inhibited dose-dependently the release of reactive oxygen species in response to RANTES, MCP-3 and eotaxin. Again, eotaxin-induced release of reactive oxygen species, however, was only half reduced at a Met-RANTES concentration (10 micrograms/ml) at which RANTES and MCP-3 were completely blocked. The results of this study show that (1) Met-RANTES is an effective and powerful antagonist of effector functions of human eosinophils following stimulation with RANTES, MCP-3 and eotaxin; (2) Met-RANTES seems to be able to antagonize the response of eosinophils through chemokine receptor 1 (CCR1) preferentially to CCR3; (3) Met-RANTES antagonizes eosinophil but not neutrophil effector functions and might be therefore of interest for a new therapeutical approach to prevent the invasion and destructive power of eosinophils in diseases that are accompanied by eosinophil infiltration such as allergic asthma and connective tissue diseases.