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.     

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.     

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.     

IL-5-induced integrin adhesion of human eosinophils caused by ERK1/2-mediated activation of cPLA2

We examined the mechanism by which interleukin (IL)-5 causes beta(2)-integrin adhesion of human eosinophils. IL-5 caused time-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38alpha in eosinophils as detected by their phosphorylation. Preincubation of eosinophils with U0126, a mitogen-activated protein kinase/ERK kinase inhibitor, suppressed IL-5-induced activation of cytosolic phospholipase A(2) (cPLA(2)) and eosinophil adhesion, and p38 inhibition by SB203580 had neither effect. ERK1/2 phosphorylation and eosinophil adhesion were blocked by inhibition of the src-family tyrosine kinase, Janus tyrosine kinase (JAK)2, or phosphoinositide-3 kinase (PI3K). Coimmunoprecipitation assay demonstrated that Lyn, a src-family tyrosine kinase, was constitutively associated with PI3K. Inhibition of src-tyrosine kinase but not JAK2 suppressed PI3K activation. Our data suggest that IL-5 induces beta(2)-integrin adhesion of human eosinophils by regulation of cPLA(2) activation caused by ERK1/2 phosphorylation. This phosphorylation results from activation of PI3K and protein tyrosine kinases. We also find that src-family tyrosine kinase, possibly Lyn, is the upstream kinase causing PI3K activation.     

Intracellular signaling mechanisms regulating the activation of human?eosinophils by the novel Th2 cytokine IL-33: implications for allergic?inflammation

The novel interleukin (IL)-1 family cytokine IL-33 has been shown to activate T helper 2 (Th2) lymphocytes, mast cells and basophils to produce an array of proinflammatory cytokines, as well as to mediate blood eosinophilia, IgE secretion and hypertrophy of airway epithelium in mice. In the present study, we characterized the activation of human eosinophils by IL-33, and investigated the underlying intracellular signaling mechanisms. IL-33 markedly enhanced eosinophil survival and upregulated cell surface expression of the adhesion molecule intercellular adhesion molecule (ICAM)-1 on eosinophils, but it suppressed that of ICAM-3 and L-selectin. In addition, IL-33 mediates significant release of the proinflammatory cytokine IL-6 and the chemokines CXCL8 and CCL2. We found that IL-33-mediated enhancement of survival, induction of adhesion molecules, and release of cytokines and chemokines were differentially regulated by activation of the nuclear factor (NF)-κB, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathways. Furthermore, we compared the above IL-33 activities with two structurally and functionally related cytokines, IL-1β and IL-18. IL-1β, but not IL-18, markedly upregulated cell surface expression of ICAM-1. IL-1β and IL-18 also significantly enhanced eosinophil survival, and induced the release of IL-6 and chemokines CXCL8 and CCL2 via the activation of the NF-κB, p38 MAPK and ERK pathways. Synergistic effects on the release of IL-6 were also observed in combined treatment with IL-1β, IL-18 and IL-33. Taken together, our findings provide insight into IL-33-mediated activation of eosinophils via differential intracellular signaling cascades in the immunopathogenesis of allergic inflammation.     

Interleukin-18 Primes the Oxidative Burst of Neutrophils in Response to Formyl-Peptides: Role of Cytochrome b558 Translocation and N-Formyl Peptide Receptor Endocytosis

Using flow cytometry, we observed that interleukin-18 (IL-18) primed human neutrophils (PMNs) in whole blood to produce superoxide anion (O₂°⁻) in response to N-formyl peptide (fMLP) stimulation, whereas IL-18 alone had no significant effect. In contrast to tumor necrosis factor alpha (TNF-α), which is a cytokine known to strongly prime O₂°⁻ production, IL-18 did not induce either p47^phox phosphorylation or its translocation from the cytosol to the plasma membrane. However, IL-18 increased PMN degranulation, as shown by increased levels of cytochrome b558 and CD11b expression at the PMN surface. Moreover, addition of IL-18 to whole blood for 45 min reduced the ability of PMNs to bind to fMLP, suggesting endocytosis of fMLP receptors, as visualized by confocal microscopy. 2,3-Butanedione 2-monoxime, which inhibits endosomal recycling of plasma membrane components back to the cell surface, concomitantly accentuated the diminution of fMLP binding at the PMN surface and increased IL-18 priming of O₂°⁻ production by PMNs in response to fMLP. This suggests that fMLP receptor endocytosis could account, at least in part, for the priming of O₂°⁻ production. In addition, genistein, a tyrosine kinase inhibitor, and SB203580, a p38 mitogen-activated protein kinase (p38MAPK) inhibitor, completely reversed the decreased level of fMLP binding and increased the level of CD11b expression after IL-18 treatment. Flow cytometric analysis of intact PMNs in whole blood showed that IL-18 increased p38MAPK phosphorylation and tyrosine phosphorylation. In particular, IL-18 induced phosphorylation of focal adhesion kinase (p125^FAK), which has been implicated in cytoskeleton reorganization. Taken together, our findings suggest several mechanisms that are likely to regulate cytokine-induced priming of the oxidative burst in PMNs in their blood environment.     

A microtiter assay for activation of eosinophils

A simple microtiter assay for eosinophil activation is described. The assay used 1000–4000 eosinophils/microtiter well, and the design allows for a separate or simultaneous quantitative assessment of eosinophil adhension to protein-coated microtiter wells and degranulation after stimulation with eosinophil-activating factors. The number of adherent eosinophils is quantified indirectly by expressing the amount of eosinophil cationic protein (ECP) extracted from the adherent fraction of cells in percentage of the total amount of ECP extracted from the adherent fraction of cells in percentage of the total amount of ECP extracted from the cells added to the wells. Degranulation is quantified in the same way by expressing the amount of ECP or eosinophil protein X (EPX) released to the supernatant in percentage of the total amount of ECP or EPX. Known eosinophil-activating agents such as PMA, interleukin (IL)-3, IL-5, GM-CSF, and platelet-activating factor (PAF) all induced a time- and does-dependent adhension to albumin-coated wells, whereas L_PAF did not. KInetic experiments showed that most adhesion occurred within the first 15–30 min, reaching a plateau around 60 min. After prolonged incubation, a decline in adhesion was detected. GM-CSF-induced adhesion was completely inhibited by incubation with monoclonal antibodies directed against the common β₂-chain (CD18) of the LFA-1, Mac-1, p150,95 complexes. Monoclonal antibodies against CD11a, CD11b, CD11c, VLA-4 ALFA, ICAM-1, VCAM-1, Sialyl-Le^m, ELAM-1, and LECAM had no inhibitory effect. Simultaneous monitoring of adhesion and degranulation after stimulation of eosinophils in albumincoated wells with either PMA or GM-CSF showed that adhesion always preceded degranulation. Replacing the albumin coating of the microtiter wells with IgG or secretory IgA augmented both the spontaneous and the PMA- or GM-CSF-stimulated responses. In conclusion, the assay allows dynamic evaluation of eosinophil activationa dn can be used to assess soluble eosinophil-activating factors as well as to study eosinophil activation by solidphase-bound proteins.     

ICAM-1-dependent pathways regulate colonic eosinophilic inflammation

Eosinophilic inflammation is a common feature of numerous eosinophil-associated gastrointestinal (EGID) diseases. Central to eosinophil migration into the gastrointestinal tract are the integrin-mediated interactions with adhesion molecules. Although the mechanisms regulating eosinophil homing into the small intestine have begun to be elucidated, the adhesion pathways responsible for eosinophil trafficking into the large intestine are unknown. We investigated the role of adhesion pathways in eosinophil recruitment into the large intestine during homeostasis and disease. First, using a hapten-induced colonic injury model, we demonstrate that in contrast to the small intestine, eosinophil recruitment into the colon is regulated by a beta7 -integrin addressin cell adhesion molecule-1-independent pathway. Characterization of integrin expression on colonic eosinophils by flow cytometry analysis revealed that colonic CC chemokine receptor 3+ eosinophils express the intercellular adhesion molecule-1 (ICAM-1) counter-receptor integrins alphaL, alphaM, and beta2. Using ICAM-1-deficient mice and anti-ICAM-1 neutralizing antibodies, we show that hapten-induced colonic eosinophilic inflammation is critically dependent on ICAM-1. These studies demonstrate that beta2 -integrin/ICAM-1-dependent pathways are integral to eosinophil recruitment into the colon during GI inflammation associated with colonic injury.     

Protopine Inhibits Heterotypic Cell Adhesion in MDA-MB-231 Cells Through Down-Regulation of Multi-Adhesive Factors

Background

A Chinese herb Corydalis yanhusuo W.T. Wang that showed anticancer and anti-angiogenesis effects in our previous studies was presented for further studies. In the present study, we studied the anticancer proliferation and adhesion effects of five alkaloids which were isolated from Corydalis yanhusuo.

Materials and Methods

MTT dose response curves, cell migration assay, cell invasion assay, as well as three types of cell adhesive assay were performed on MDA-MB-231 human breast cancer cells. The mechanism of the compounds on inhibiting heterotypic cell adhesion were further explored by determining the expression of epidermal growth factor receptor (EGFR), Intercellular adhesion molecule 1 (ICAM-1), αv-integrin, β₁-integrin and β₅-integrin by western blotting assay.

Results

In five tested alkaloids, only protopine exhibited anti-adhesive and anti-invasion effects in MDA-MB-231 cells, which contributed to the anti-metastasis effect of Corydalis yanhusuo. The results showed that after treatment with protopine for 90 min, the expression of EGFR, ICAM-1, αv-integrin, β₁-integrin and β₅-integrin were remarkably reduced.

Conclusion

The present results suggest that protopine seems to inhibit the heterotypic cell adhesion between MDA-MB-231 cells, and human umbilical vein endothelial cells by changing the expression of adhesive factors.     

Attenuated translocation of group IVa phospholipase A2 and up-regulated annexin-1 synthesis by glucocorticoid blocks beta 2-integrin adhesion in neutrophils

We examined the effect of glucocorticoid stimulation in blocking beta 2-integrin adhesion of polymorphonuclear leukocytes (PMNs) isolated from human subjects. Surface expression of CD11b and ERK-1/2-mediated gIVaPLA2 phosphorylation, which are required for beta 2-integrin adhesion, were not affected by treatment with < or =10(-6) M fluticasone propionate (FP) for PMNs activated by either 10(-7) M LTB4 or 30 ng/ml TNF-alpha and caused no significant blockade of beta 2-integrin adhesion in vitro. Baseline expression of annexin-1 (ANXA1) synthesis was increased only after 10(-6) M FP for PMNs; by contrast, comparable increase in ANXA1 expression was demonstrated in human eosinophils from the same subjects with 10(-8) M FP. Viability of PMNs was verified by propidium iodide and by the persistence of beta 2-integrin adhesion in treated groups. Exogenous administration of ANXA1 mimetic peptide fragment blocked significantly and comparably the beta 2-integrin adhesion in PMNs activated by LTB4 and TNF-alpha and in eosinophils activated by IL-5. Translocation of gIVaPLA2 from the cytosol to the nucleus also was refractory for activated PMNs treated with > or =10(-7) M FP; by contrast, complete blockade of nuclear translocation of cytosolic gIVaPLA2 was effected by 10(-9) M FP in eosinophils. Our data indicate that the cell surface ANXA1 synthesis is capable of blocking beta 2-integrin adhesion in both PMNs and eosinophils. However, in contrast to eosinophils, FP does not cause either substantial ANXA1 synthesis or nuclear transport of cytosolic gIVaPLA2 in PMNs and thus does not block beta2-integrin adhesion, a necessary step for granulocyte cell migration in vivo.     

The Impact of Eotaxin- and IL-5-Induced Adhesion and Transmigration on Eosinophil Activity Markers

Eosinophils accumulate at sites of allergic inflammation, and play important roles in asthma/allergic disorders. The mechanism of eosinophil recruitment into tissues is not fully understood. In this study, we evaluated whether adhesion and/or transmigration, in the presence of IL-5 and eotaxin, alter the expression of CD9, CD11b, the ₁₄-integrin, and the EG2-epitope on intracellular ECP. We also investigated whether CD9 is involved in the adhesion process. With flow cytometry the surface expression of CD9, CD11b and the ₁₄-integrin, and the intracellular expression of EG2, were analyzed before, and after transmigration/adhesion to fibronectin. To evaluate the eventual role of CD9 in adhesion, eosinophils were preincubated with monoclonal antibodies to CD9. We observed decreased expression of CD9, and increased expression of CD11b on eosinophils, after adhesion and transmigration. The transmigration did not change the expression of the ₁₄-integrin or EG2, whereas the adhesion resulted in a decreased EG2 expression. Antibodies to CD9 decreased the adhesion property of eosinophils. The eosinophils are activated after both adhesion and transmigration by means of decreased CD9 and increased CD11b expression. The expression of the EG2-epitope on intracellular ECP was decreased when eosinophils adhered to fibronectin, probably due to degranulation. Our results also indicate that CD9 is involved in the adhesion of eosinophils to fibronectin.     

Monocytes augment inflammatory responses in human aortic valve interstitial cells via β2-integrin/ICAM-1-mediated signaling

Objective

Inflammatory infiltration in aortic valves promotes calcific aortic valve disease (CAVD) progression. While soluble extracellular matrix (ECM) proteins induce inflammatory responses in aortic valve interstitial cells (AVICs), the impact of monocytes on AVIC inflammatory responses is unknown. We tested the hypothesis that monocytes enhance AVIC inflammatory responses to soluble ECM protein in this study.

Methods

Human AVICs isolated from normal aortic valves were cocultured with monocytes and stimulated with soluble ECM protein (matrilin-2). ICAM-1 and IL-6 productions were assessed. YAP and NF-κB phosphorylation were analyzed. Recombinant CD18, neutralizing antibodies against β₂-integrin or ICAM-1, and inhibitor of YAP or NF-κB were applied.

Results

AVIC expression of ICAM-1 and IL-6 was markedly enhanced by the presence of monocytes, although matrilin-2 did not affect monocyte production of ICAM-1 or IL-6. Matrilin-2 up-regulated the expression of monocyte β₂-integrin and AVIC ICAM-1, leading to monocyte-AVIC adhesion. Neutralizing β₂-integrin or ICAM-1 in coculture suppressed monocyte adhesion to AVICs and the expression of ICAM-1 and IL-6. Recombinant CD18 enhanced the matrilin-2-induced ICAM-1 and IL-6 expression in AVIC monoculture. Further, stimulation of coculture with matrilin-2 induced greater YAP and NF-κB phosphorylation. Inhibiting either YAP or NF-κB markedly suppressed the inflammatory response to matrilin-2 in coculture.

Conclusion

Monocyte β₂-integrin interacts with AVIC ICAM-1 to augment AVIC inflammatory responses to soluble matrilin-2 through enhancing the activation of YAP and NF-κB signaling pathways. Infiltrated monocytes may promote valvular inflammation through cell–cell interaction with AVICs to enhance their sensitivity to damage-associated molecular patterns.

     

Eotaxin-2 alters eosinophil integrin function via mitogen-activated protein kinases

Adhesion molecules and chemokines contribute to selective eosinophil recruitment in allergic inflammation. In this study, we examined the effects of eotaxin-2, a CCR3-specific chemokine, on integrin-mediated eosinophil adhesion to vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), or both using a parallel plate flow system. Tissue culture plates were coated with various combinations of VCAM-1, ICAM-1, and/or eotaxin-2. Human eosinophils were infused at physiologic shear stress (0.5 dyn/cm(2)) for 10 min, and the numbers of attached eosinophils were monitored using video microscopy. Cells accumulated efficiently on VCAM-1 and even better on surfaces co-coated with VCAM-1 and ICAM-1, but poorly on surfaces coated with ICAM-1 or bovine serum albumin alone. When eotaxin-2 was co-immobilized with adhesion proteins, fewer cells adhered to VCAM-1 and more adhered to ICAM-1, whereas levels of attachment to VCAM-1 plus ICAM-1 showed no net change. However, experiments with adhesion molecule blocking monoclonal antibody showed that the contribution of ICAM-1-mediated adhesion was always greater if eotaxin-2 was present. Pretreatment of cells with a CCR3-blocking mAb, or PD98059, a MAP-kinase inhibitor, prevented the eotaxin-2-induced changes in eosinophil attachment. These data suggest that eotaxin-2, acting via MAP kinases, may facilitate eosinophil recruitment at sites of allergic inflammation by shifting their adhesion molecule usage away from VCAM-1-dominated to ICAM-1-dominated pathways.     

Role of the β<Subscript>1</Subscript>-integrin subunit in the adhesion,extravasation and migration of T24 human bladder carcinoma cells

The abilities of tumor cells to extravasate from the blood vessel system and to migrate through the connective tissue are prerequisites in metastasis formation. Both processes are chiefly mediated by integrins, which mediate both cell–cell and cell–matrix interactions. We investigated the role of integrin subunits in the adhesion, extravasation and migration of the highly invasive human bladder carcinoma cell line T24. Here we show that inhibition of the β₁-integrin subunit using the specific β₁-integrin blocking antibody 4B4 significantly reduces the adhesion to HUVEC and transmigratory activity of T24 cells. The blockade of the β₁-integrin subunit also resulted in a significantly reduced locomotory activity of T24 cells. A detailed cell migration analysis on a single cell level revealed that blockade of the β₁-integrin subunit leads to an altered migration pattern of single cells but does not influence migration per se. Migration parameters such as time active, velocity and distance migrated were significantly reduced as compared to untreated control cells. Our observations strongly suggest a central role for the β₁-integrin subunit in forming the cell–cell and cell–matrix bonds necessary for adhesion, extravasation and 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.     

Regulation of eosinophil adhesion by lysophosphatidylcholine via a non-store-operated Ca2+ channel

We examined the mechanism by which lysophosphatidylcholine (LPC) regulates beta2-integrin-mediated adhesion of eosiniophils. Eosinophils were isolated from blood of mildly atopic volunteers by negative immunomagnetic selection. beta2-integrin-dependent adhesion of eosinophils to plated bovine serum albumin (BSA) was measured by residual eosinophil peroxidase activity. LPC caused maximal adhesion of eosinophils to plated BSA at 4 microM. Lysophosphatidylinositol, which has a similar molecular shape, mimicked the effect of LPC on eosinophil adhesion, while neither lysophosphatidylserine nor lysophosphatidylethanolamine had any effect. Phosphatidylethanolamine, a lipid that has a molecular orientation that is the inverse of LPC, blocked eosinophil adhesion caused by LPC. Unlike platelet-activating factor, a G-protein-coupled receptor agonist, LPC did not cause Ca2+-store depletion, but caused increased Ca2+ influx upon addition of Ca2+ to extracellular medium. This influx was not inhibited by U73122, a phospholipase C inhibitor, demonstrating independence from the G protein-activated phospholipase C pathway. Ca2+ influx was inhibited by either preincubation of phosphotidylethanolamine or La3+, a broad spectrum blocker of cation channels. LPC induced up-regulation of the active conformation of CD11b, which was blocked by preincubation with phosphatidylethanolamine. These data suggest that LPC causes a non-store-operated Ca2+ influx into eosinophils, which subsequently activates CD11b/CD18 to promote eosinophil adhesion.     

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 κB

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-κB) 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-κB in eosinophils. cDNA expression arrays demonstrated that the gene expression levels of several adhesion molecules including intercellular adhesion molecule-1 (ICAM-1), α6, β2 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-κB. Our results therefore shed light on the further development of more effective agents for allergic and inflammatory diseases.     

Interleukin-5 enhances eosinophil adhesion to bronchial epithelial cells

BACKGROUND: Eosinophil-bronchial epithelial cell interactions are thought to be central to the pathogenesis of asthma, both in terms of the epithelium as a source of pro-inflammatory mediators and as a target for eosinophil-mediated damage. We have therefore investigated adhesion interactions between these two cell types. OBJECTIVES: To determine the role of eosinophil and epithelial activation on eosinophil adhesion to bronchial epithelium and to characterize the adhesion receptors mediating eosinophil adhesion. METHODS: Eosinophils were purified from human peripheral blood by immunomagnetic selection and adhesion to confluent cultures of the airway epithelial cell lines A549 and BEAS-2B was studied. RESULTS: Stimulation of A549 cells with TNFalpha, IFNgamma or a combination of 50 ng/mL of TNFalpha, IFNgamma and IL-1 (cytomix) did not effect eosinophil binding despite an increase in ICAM-1 expression. Similarly stimulation of eosinophils with PAF or IL-5 had no effect on eosinophil binding to medium- or cytokine-treated A549 cells. In contrast stimulation of BEAS-2B cells with cytomix caused a significant increase in eosinophil adhesion. This was associated with an increase in expression of ICAM-1 and induced expression of VCAM-1. Treatment of eosinophils with Mn2+ and IL-5 but not eotaxin, RANTES or PAF also significantly enhanced eosinophil adhesion to medium-treated BEAS-2B cells. Using blocking mAbs we were able to demonstrate that the increased adhesion resulting from stimulation of eosinophils or BEAS-2B cells was in both cases mediated by a combination of CD18 and alpha4 integrins. CONCLUSIONS: This study demonstrates a selective role for IL-5 in mediating integrin-dependent eosinophil adhesion to airway epithelium and once again emphasizes the importance of this cytokine in controlling eosinophil activation in diseases such as asthma.     

Human peripheral eosinophils express functional interferon-gamma receptors (IFN-γR)

In order to determine whether or not IFN-γR is associated with regulatory mechanisms on human eosinophil function, we examined the expression of functional IFN-γR on human peripheral eosinophils. In this study, peripheral blood eosinophils were obtained from seven normal controls and 12 patients (bronchial asthma, n = 9, and hypereosinophilic syndrome (HES), n = 3), and the purity of eosinophils was 97.11 ± 2. 31%, n = 19. We first showed that anti-IFN-γR α-chain MoAb reacted with all tested eosinophils of both normal controls and patients by flow cytometry analysis. We also showed expression of mRNA for the α-chain of IFN-γR in all purified eosinophils of six individuals. Further, to characterize IFN-γR on eosinophils, we did binding experiments with ¹²⁵I-IFN-γ on purified peripheral eosinophils. The linear Scatchard plot indicated a single type of high-affinity binding sites (dissociation constant (Kd) = 3.89–4.95 × 10⁻¹⁰ m, numbers of binding sites = 183–233/cell, n = 3). To determine whether IFN-γR on eosinophils is functional, we examined surface eosinophilic cationic protein (ECP) and CD69 induction after IFN-γR ligation with recombinant human IFN-γ (rhIFN-γ) on eosinophils by flow cytometry. rhIFN-γ stimulation significantly induced both ECP and CD69 expression on the 2–18 h-cultured eosinophils in a dose-dependent manner. Further, the effects of rhIFN-γ stimulation were significantly blocked by both a neutralizing anti-IFN-γ MoAb and a blocking anti-IFN-γR MoAb. These results suggest that human peripheral eosinophils express functional IFN-γR.     

IL-4–induced apoptosis in peripheral blood eosinophils

Background: The regulation of eosinophil survival and apoptosis may play a major role in diseases demonstrating increased numbers of circulating and tissue eosinophils such as allergic reactions. Because few promoters of eosinophil apoptosis have been described so far, the objective of this study was to elucidate the role of endogenous factors on eosinophil survival and apoptosis. Methods and Results: Highly purified peripheral blood eosinophils were analyzed in the time course from 24 up to 144 hours in culture. Eosinophil survival was assessed with trypan blue dye exclusion and apoptosis was determined by DNA fragmentation gel analysis and ELISA technique with anti-histone antibodies. We confirmed previous results demonstrating prolonged eosinophil survival and inhibited apoptosis by IL-3, IL-5, and GM-CSF. In contrast, eosinophil apoptosis was significantly enhanced by corticosteroids, particularly dexamethasone and hydrocortisone. However, IL-1β, IL-8, IL-12, platelet-activating factor, TNF-α, and eotaxin had no effect on eosinophil survival or apoptosis when compared with culture medium alone. In contrast, IL-4 at concentrations of 100 U/mL or more inhibited eosinophil survival and induced apoptosis. This effect was time dependent and abrogated by preincubation with neutralizing anti–IL-4 antibodies. However, after 96 hours in coincubation, IL-4 did overcome the survival-prolonging effect of IL-3, IL-5, and GM-CSF. IL-4 did not enhance eosinophil surface expression of APO-1/Fas antigen (CD95). In assessing IL-4–mediated effects on eosinophil function, we found no response by means of the release of eosinophil cationic protein or reactive oxygen species. Conclusions: Taken together, our data present direct evidence for the presence of functional IL-4 receptors on human eosinophils and indicate that IL-4 may lead to resolution of chronic inflammation by induction of eosinophil apoptosis. (J Allergy Clin Immunol 1998;102:1013-20)