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PubMed1999

Simultaneous analysis of eosinophil and neutrophil adhesion to plasma and tissue fibronectin, fibrinogen, and albumin.

A simple and convenient assay for the simultaneous measurement of eosinophil and neutrophil adhesion is described. Incubations were performed in microtitre plates coated with different proteins. Adhesion of eosinophils and neutrophils was determined by the use of specific radioimmunoassays for eosinophil cationic protein (ECP) and myeloperoxidase (MPO). Using this assay, Mn2+ induced a significant increase of the adhesion of eosinophils to plasma fibronectin and fibrinogen in a time-dependent fashion, while a small increase of the adhesion of neutrophils to these two proteins was observed. In contrast, a time-dependent potent increment of the adhesion of both eosinophils and neutrophils to tissue fibronectin and albumin was found. Tissue fibronectin preferentially supported eosinophil adhesion compared with that of neutrophils in the presence of Mn2+. PMA (10(-9) mol/l) induced a significant increase in the adhesion of eosinophils and neutrophils of the same pattern to all four proteins. However, when granulocytes were stimulated by Mn2+ in combination with PMA, eosinophils and neutrophils showed different patterns of response to plasma fibronectin and fibrinogen, respectively, but the same pattern of response to tissue fibronectin. f-MLP stimulated an early increase of the adhesion of neutrophils to fibrinogen, while a weak stimulation of the adhesion of eosinophils to plasma fibronectin and fibrinogen and of neutrophils to plasma fibronectin was observed. Co-stimulation with f-MLP and Mn2+ did not induce any additive effects on granulocyte adhesion. In conclusion, the assay allows rapid quantification of eosinophil and neutrophil adhesion and can be used to directly compare the response of neutrophils and eosinophils. The assay is thus suitable for studies aimed at identifying agents with a selective effect on either of the cells.

Cell Adhesion↗
PubMed2000

Blood eosinophils from atopic donors express messenger RNA for the alpha, beta, and gamma subunits of the high-affinity IgE receptor (Fc epsilon RI) and intracellular, but not cell surface, alpha subunit protein.

BACKGROUND: Blood eosinophils from hypereosinophilic donors were previously reported to possess the functional high-affinity IgE receptor (Fc epsilon RI), so providing a potential mechanism to account for eosinophil degranulation in atopic allergic disease. Furthermore, tissue eosinophils from allergic tissue reactions were shown to be mRNA(+) for the alpha, beta, and gamma subunits of Fc epsilon RI and gave positive immunostaining with an anti-Fc epsilon RI-alpha antibody. Recent studies, however, revealed negative surface staining on peripheral blood eosinophils, but intracellular Fc epsilon RI-alpha protein was identified by Western blot analysis. OBJECTIVE: Our purpose was to examine on peripheral blood eosinophils from atopic subjects (1) surface expression and mRNA for Fc epsilon RI-alpha, (2) up-regulation of Fc epsilon RI-alpha by allergy-associated tissue factors, and (3) Fc epsilon RI-alpha-dependent release of eosinophil peroxidase (EPO). METHODS: We measured (1) Fc epsilon RI mRNA expression by in situ hybridization, (2) Fc epsilon RI-alpha by flow cytometry and immunocytochemistry (with use of nonpermeabilized and permeabilized cells), and (3) Fc epsilon RI-alpha-dependent release of EPO. RESULTS: Eosinophils from atopic donors had negligible surface expression of Fc epsilon RI-alpha, which was not enhanced by culture with IgE, IL-3, IL-4, IL-5, GM-CSF, or fibronectin or coculture with fibroblasts. Permeabilization, however, revealed appreciable intracellular staining for Fc epsilon RI-alpha. The majority of eosinophils were mRNA(+) for the alpha, beta, and gamma subunits of Fc epsilon RI. Small but significant (P =.03) increases in alpha chain mRNA expression were observed after coculture of eosinophils with fibroblasts but not with IgE, IL-4, or fibronectin. Cross-linking of Fc epsilon RI on the surface of eosinophils from atopic donors did not lead to detectable EPO release. CONCLUSION: Human blood eosinophils express negligible, nonfunctional membrane Fc epsilon RI-alpha but have intracellular Fc epsilon RI-alpha protein and mRNA expression for the alpha, beta, and gamma subunits.

Cell Membrane↗
PubMed1997

Regulation of alpha 4 integrin-mediated adhesion of human eosinophils to fibronectin and vascular cell adhesion molecule-1.

BACKGROUND: Eosinophils selectively accumulate at sites of allergic inflammation. Their recruitment is dependent on both the expression and functional activity of cell adhesion molecules. How the functional activity of cell adhesion molecules on eosinophils is regulated is poorly understood. OBJECTIVE: Our objective was to examine the functional activity of alpha 4 integrins on human eosinophils and its regulation by various agents. METHODS: Function of alpha 4 integrins on human eosinophils was examined by testing adhesion to immobilized fibronection and vascular cell adhesion molecule-1 (VCAM-1) in the presence or absence of a monoclonal antibody (mAb) (8A2) that activates beta 1 integrin function. RESULTS: Spontaneous eosinophil adhesion to VCAM-1 was enhanced by 8A2, but adhesion to fibronectin could only be detected in the presence of 8A2. Concentrations of 8A2 that were approximately 100-fold less than saturating induced maximal eosinophil adhesion. Adhesion to VCAM-1 in the presence of 8A2 was effectively inhibited by alpha 4 and beta 1 integrin mAbs: beta 7 mAb had partial inhibitory activity. Connecting segment-1 peptide and alpha 4 mAb blocked 8A2-dependent fibronectin binding: beta 1, beta 2, and beta 7 integrin mAbs had partial inhibitory activity. Eosinophils obtained from bronchoalveolar lavage fluids and blood eosinophils stimulated with IL-5, platelet-activating factor, or RANTES displayed increased beta 2 integrin-dependent, not alpha 4 integrin-dependent, attachment. Spontaneous adhesion of eosinophils to VCAM-1 was significantly reduced by the tyrosine kinase inhibitor tyrphostin B46 (inhibitory concentration of 50% approximately equal to 20 mumol/L); this effect was reversed by 8A2. CONCLUSIONS: The functional activity of integrins on eosinophils can be positively and negatively regulated. Altered integrin avidity may influence eosinophil recruitment in vivo.

Antibodies, Monoclonal↗
PubMed1997

Dexamethasone inhibits prolonged survival and autocrine granulocyte-macrophage colony-stimulating factor production by human eosinophils cultured on laminin or tissue fibronectin.

BACKGROUND: Eosinophil autocrine generation of viability-promoting cytokines resulting from interaction with extracellular matrix (ECM) proteins may be important in promoting their persistence in allergic inflammation. OBJECTIVE: We examined eosinophil interaction with fibronectin, laminin, collagen, fibrinogen, hyaluronate, or vitronectin in terms of adhesion and for their ability to promote prolonged eosinophil survival by autocrine generation of IL-3, IL-5, or granulocyte-macrophage colony-stimulating factor (GM-CSF). The effect of dexamethasone was examined because it may inhibit integrin-dependent autocrine generation of viability-enhancing cytokines. METHODS: Microtiter wells were coated with different ECM proteins, and assays were performed to determine their levels of eosinophil adhesion, and tests were performed to determine their prolonged survival in culture. The receptors involved in these processes, the cytokines generated, and the effect of a dose range of dexamethasone were all examined. RESULTS: Eosinophils cultured for 3 days on laminin or tissue fibronectin exhibited dose-dependent prolonged survival, which was not seen with any other ECM protein. Unstimulated eosinophils adhered to wells coated with laminin or fibronectin but not to wells coated with any of the other ECM proteins that were tested. Laminin-dependent eosinophil adhesion and prolonged survival were inhibited by specific monoclonal antibodies to beta1 and alpha6beta1. Laminin-dependent survival was inhibited by a neutralizing antibody to GM-CSF, an anti-IL-5 antibody achieved a measurable but insignificant inhibition of survival, and an antibody to IL-3 had no effect. Increasing concentrations of dexamethasone (maximal at 10(-7) mol/L) inhibited both laminin- and fibronectin-dependent enhanced eosinophil viability at 3 days. Eosinophils cultured on either laminin or fibronectin for 24 or 72 hours generated picogram quantities of GM-CSF, which were inhibited by the addition of dexamethasone (10(-7) mol/L). CONCLUSION: We have confirmed that eosinophils adhere to laminin in an alpha6beta1-dependent manner, and this interaction results in the autocrine generation of GM-CSF, which enhances eosinophil survival in culture. Both laminin and tissue fibronectin-dependent autocrine GM-CSF generation were inhibited by physiologically relevant concentrations of dexamethasone. This may represent an important mechanism by which glucocorticoids reduce the tissue eosinophilia in allergic disease.

Anti-Inflammatory Agents↗
PubMed1999

PGE 2 and dibutyryl cyclic adenosine monophosphate prolong eosinophil survival in vitro.

BACKGROUND: Apoptosis represents a mechanism by which the accumulation and inflammatory potential of eosinophils in asthma might be limited. Mediators derived from the airway epithelium may influence the rate of eosinophil apoptosis. OBJECTIVE: We have investigated the effects on eosinophil apoptosis of 3 mediators that are likely to be produced by the airway epithelium, namely PGE2, TNF-alpha, and nitric oxide. METHODS: Peripheral blood eosinophils from healthy adult volunteers were purified by density gradient centrifugation and negative immunomagnetic selection. Eosinophils were cultured for 16 or 40 hours with PGE2 (10 nmol/L), dibutyryl cyclic adenosine monophosphate (AMP; 100 micromol/L), TNF-alpha (500 U/mL), the nitric oxide donors, S-nitroso-N-acetylpenicillamine (100 micromol/L), and 2,2;-(hydroxynitrosohydrazono)bis-ethanamine (1 mmol/L), or dibutyryl cyclic guanosine monophosphate (100 micromol/L). Control cultures consisted of untreated, IL5-treated (100 U/mL), and anti-Fas-treated (400 ng/mL) cells. Eosinophil apoptosis was assessed by flow cytometric analysis of annexin V-FITC binding to externalized phosphatidylserine, by electrophoresis of phosphorus 32 end-labeled DNA fragments, and by flow cytometric assessment of hypodiploid DNA with propidium iodide. RESULTS: PGE2 and cyclic AMP inhibited spontaneous eosinophil apoptosis at both 16 and 40 hours as did the PGEP2 receptor agonist, 11-deoxy PGE1, at 40 hours, but these effects were not inhibited by a protein kinase A antagonist. TNF-alpha delayed apoptosis in eosinophil cultures at 16 hours, whereas S-nitroso- N-acetylpenicillamine, 2, 2;-(hydroxynitrosohydrazono)bis-ethanamine, and cyclic guanosine monophosphate had little effect. Anti-Fas had little effect on spontaneous eosinophil apoptosis but significantly reduced the inhibitory effects of PGE2, cyclic AMP, and TNF-alpha. Assessments of apoptosis by DNA fragmentation gave similar but quantitatively less sensitive results. CONCLUSION: Inhibition of spontaneous eosinophil apoptosis by PGE2 appears to be mediated by EP2 receptors but is not protein kinase A dependent. By enhancing eosinophil survival, PGE2 may increase the proinflammatory potential of these cells in chronic asthma.

Adult↗
PubMed1999

Degranulation of eosinophils mediated by intercellular adhesion molecule-1 and its ligands is involved in adhesion molecule expression on endothelial cells-selective induction of VCAM-1.

BACKGROUND: Adhesion molecules and eosinophils may play an important role in the pathogenesis of allergic inflammatory reactions. OBJECTIVE: We attempted to clarify eosinophil activation, such as degranulation, by signaling through adhesion molecule and to determine whether degranulation is involved in adhesion molecule expression on endothelial cells. METHODS: Eosinophils were cultured with or without recombinant soluble intercellular adhesion molecule-1 (ICAM-1), and the levels of eosinophil cationic protein and eosinophil-derived neurotoxin were determined. The influence of these eosinophil granule proteins or supernatant from eosinophil cultured with ICAM-1 on the expression of ICAM-1 or vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells was also examined by flow-cytometric analysis. RESULTS: Supernatant levels of eosinophil granule protein were significantly increased by culture for 4 hourss or 16 hours with recombinant soluble ICAM-1, suggesting degranulation by adherence to ICAM-1. Both granule proteins and the supernatants of eosinophils cultured with recombinant soluble ICAM-1 induced expression of ICAM-1 and VCAM-1 on endothelial cells, with the latter showing a more prominant increase. CONCLUSION: Degranulation mediated through adherence to endothelial cells by ICAM-1 and its ligands may be involved in the expression of adhesion molecules, such as ICAM-1 or VCAM-1, on these cells. Our finding of the selective induction of VCAM-1 expression suggests that eosinophil adherence to endothelial cells, even if it is because of ICAM-1, may be involved in selective eosinophil recruitment and accumulation at sites of allergic inflammation.

Cell Adhesion Molecules↗
PubMed1999

Macrophage-derived chemokine induces human eosinophil chemotaxis in a CC chemokine receptor 3- and CC chemokine receptor 4-independent manner.

BACKGROUND: Chemokines are believed to contribute to selective cell recruitment. Macrophage-derived chemokine (MDC) is a CC chemokine that causes chemotaxis of dendritic cells, monocytes, and activated natural killer cells. MDC binds to CC chemokine receptor 4 (CCR4) but not to CCR1, CCR2, CCR3, CCR5, CCR6, or CCR7. OBJECTIVE: Our aim was to determine the in vitro activity of MDC on human eosinophils by using chemotaxis and calcium flux assays. METHODS: Eosinophils were purified from peripheral blood of allergic donors, and chemotactic activity of MDC and other CC chemokines was compared in microchemotaxis chamber assays. The role of CCR3 in these assays was determined by using a CCR3-blocking antibody. Measurements of cytosolic Ca++ mobilization were performed by using fura-2AM labeling, with eosinophils and cell lines transfected with CCR3 or CCR4. Eosinophil expression of CCR3 and CCR4 mRNA was determined by using RT-PCR. RESULTS: MDC (0.1 to 100 nmol/L) caused dose-dependent chemotaxis of purified human eosinophils (maximum approximately 3-fold control). Compared with other CC chemokines, the potency and efficacy for eosinophil chemotaxis were similar for MDC and eotaxin but were less than that observed for RANTES, monocyte chemoattractant protein (MCP)-4, and eotaxin-2. Although MDC can act by means of CCR4, RT-PCR analysis failed to reveal CCR4 mRNA in eosinophils. Effects of MDC on eosinophils was also independent of CCR3, as a blocking mAb to CCR3 failed to inhibit MDC-induced chemotaxis. Furthermore, CCR3-transfected human embryonic kidney cells labeled with Fura-2AM exhibited a rapid rise in intracellular free calcium after stimulation with eotaxin, eotaxin-2, or MCP-4, but not with MDC. Eosinophils cultured for 72 hours in 10 ng/mL IL-5 also demonstrated increased intracellular free calcium after stimulation with eotaxin-2 or MCP-4, but not with up to 100 nmol/L MDC. CONCLUSION: MDC is a CCR3- and CCR4-independent activator of eosinophil chemotaxis, but it does not appear to elicit measurable cytosolic calcium elevations during these responses. MDC appears to act by means of another receptor in addition to CCR4 and may therefore contribute to eosinophil accumulation without working through CCR1 to CCR7.

Calcium Signaling↗
PubMed2001

Eotaxin expression and eosinophil infiltrate in the liver of patients with drug-induced liver disease.

BACKGROUND: Drug-induced liver disease is due to intrinsic or idiosyncratic hepatotoxins. Liver parenchyma is then infiltrated by immunocompetent cells. Eosinophils are primarily tissue leukocytes which are attracted into tissues by various chemoattractants, including chemokines. The aim of this study was to study eosinophils in the livers of patients with drug-induced liver disease. METHODS: Immunohistochemical studies with antibodies against eosinophil cationic proteins (major basic protein, eosinophil-derived neurotoxin and eosinophil cationic protein), cytokines (interleukin 5 (IL-5), interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF)) and chemokines (eotaxin and RANTES (Regulated upon Activation Normal T cell Expressed and Secreted)) were performed to assess the in situ activation of the liver-infiltrating eosinophils of 14 patients with drug-induced liver disease and 19 controls. RESULTS: Eosinophils were only observed in patients with drug-induced liver disease. Eosinophils were morphologically normal when hepatitis was due to paracetamol whereas eosinophils had granular changes when hepatitis was due to an idiosyncratic hepatotoxin. Eotaxin was detected in all patients with drug-induced liver disease, whereas RANTES was detected in three of them. IL-5, IL-3 or GM-CSF were not detected. CONCLUSIONS: In patients with drug-induced liver disease, the recruitment of eosinophils in the liver may depend on eotaxin expression. Eosinophil changes may vary according to the type of drug.

Acetaminophen↗
PubMed1999

Eosinophils isolated by magnetic cell sorting respond poorly to lipid chemoattractants.

BACKGROUND: Eosinophils play an important role in allergic inflammation. In vitro methods to isolate human eosinophils for the study of chemotactic responses are essential in understanding the mechanisms involved in tissue eosinophilia. OBJECTIVE: We compared LTB4 and PAF-induced chemotactic responses of eosinophils isolated by the standard Percoll (positive selection) versus the magnetic cell separation systems (MACS) (negative selection) technique. METHODS: Discontinuous Percoll gradients were preceded by dextran and Ficoll-Paque steps, and followed by gelatin wash and red blood cell (RBC) lysis. MACS isolation included Percoll 1.090 g/mL layering and RBC lysis; incubation with CD16 antibody conjugated to magnetic beads (to bind neutrophils); and isolation of eluate from column positioned in magnet. RESULTS: Percoll-isolated eosinophils migrated to the lipid mediators, LTB4 and PAF, in a dose-responsive fashion. Although MACS isolation provided a greater number and higher purity of eosinophils, these eosinophils migrated less to LTB4 and PAF. Neither dextran sedimentation, dextran and Ficoll-Paque, nor dextran Ficoll-Paque and Percoll prior to MACS isolation reversed the decreased chemotactic responses observed with MACS isolated eosinophils. Further, Percoll-isolated eosinophils further purified with CD16 MicroBeads did not respond as well to LTB4 or PAF. CONCLUSIONS: The technique used to isolate eosinophils clearly affects the chemotactic responsiveness of this cell to LTB4 and PAF. Since several in vivo studies suggest that LTB4 and PAF are eosinophil chemoattractants, Percoll isolation of these cells might be more appropriate for studies involving eosinophil chemotactic responses to these lipid mediators.

Blood Donors↗
PubMed1999

Bradykinin down-regulates LPS-induced eosinophil accumulation in the pleural cavity of mice through type 2-kinin receptor activation: a role for prostaglandins.

1. The role of both exogenously administered and endogenously generated bradykinin (BK) on LPS-induced eosinophil accumulation in the mice pleural cavity was investigated by means of treatment with BK selective receptor agonists/antagonists and captopril. 2. Intrathoracic (i.t.) injection of LPS (250 ng cavity(-1)) induced eosinophil influx at 24 h as previously described (Bozza et al., 1993). Pretreatment with the B1 receptor antagonist des-Arg9-[leu-8]BK (0.025 and 0.25 nmol cavity(-1)) showed no effect on this phenomenon, whereas pretreatment with the B2 receptor antagonists, NPC 17731 (0.025 and 0.25 nmol cavity(-1)) or HOE 140 (2.5 nmol cavity(-1)), increased LPS-induced eosinophil influx. Accordingly, pretreatment with captopril at 10 mg kg(-1) i.p., inhibited eosinophil infiltration induced by LPS in the pleural cavity, suggesting that endogenous BK is down-regulating LPS-induced eosinophil accumulation. 3. BK administered at 15 and 25 nmol cavity(-1), i.t. or i.p. also inhibited LPS-induced eosinophil accumulation. BK alone had no effect on the basal number of leucocytes in the pleural or peritoneal cavity in doses up to 25 nmol cavity(-1). Nevertheless, when injected at doses of 50 and 100 nmol cavity(-1) BK induced leucocyte influx characterized by neutrophil and eosinophil accumulation at 24 h. 4. Similarly to what was observed with BK, a specific B2 receptor agonist, Tyr8BK, administered at 0.25 nmol cavity(-1) i.p., significantly inhibited the eosinophil influx induced by LPS. 5. The mechanism by which B2 receptor agonists inhibit LPS-induced eosinophil accumulation was investigated by pretreating the animals with indomethacin or a selective cyclooxygenase-2 inhibitor, NS-398. Pretreatment with either indomethacin or NS-398 had no effect on eosinophil influx induced by LPS alone, but those drugs were able to restore the LPS-induced eosinophil influx in Tyr8BK (0.25 nmol cavity(-1)) injected mice. 6. In conclusion, endogenously generated bradykinin seems to modulate, through activation of B2 receptors, eosinphil accumulation induced by LPS via a mechanism dependent on prostanoid synthesis.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2001

alpha(4) integrin-dependent eosinophil recruitment in allergic but not non-allergic inflammation.

1. Although anti-alpha(4) integrin mAbs reduce eosinophil accumulation in several models of allergic inflammation, it is not clear whether this occurs via a direct action to block eosinophil alpha(4) integrins or indirectly on another cell type. The role of alpha(4) integrins on the accumulation of (111)In-labelled eosinophils in allergic and non-allergic inflammation in guinea-pig skin was therefore investigated. 2. Intradermal injection of antigen in sensitized skin sites induced accumulation of (111)In-eosinophils that was reduced up to 70% by two anti-alpha(4) integrin mAbs. In contrast, accumulation of (111)In-eosinophils to intradermal chemoattractants was unaffected by the same mAbs. 3. Accumulation of (111)In-eosinophils in allergic and non-allergic conditions was partly inhibited by a low dose of an anti-beta(2) integrin mAb. In combination with anti-alpha(4) integrin mAb, responses were not further reduced suggesting that these adhesion pathways are not additive or synergic. 4. Pretreating skin sites with antiserum or contaminating LPS did not reveal an alpha(4) integrin dependent pathway for chemoattractant-induced (111)In-eosinophil accumulation. These data suggest that alpha(4) integrins are involved in the response to antigen in sensitized skin sites. 5. Pretreating (111)In-eosinophil with alpha(4) integrin mAb blocked their adhesion to fibronectin in vitro but did not inhibit their accumulation in allergic inflammation suggesting that the blocking effect in vivo was eosinophil independent. 6. These data support the concept that targeting alpha(4) integrins on cells other than eosinophils could control eosinophil accumulation and have therapeutic potential in allergic diseases such as asthma and atopic dermatitis.

Animals↗
PubMed1998

Inhibition of interleukin-5 mediated eosinophil viability by fluticasone 17-propionate: comparison with other glucocorticoids.

BACKGROUND: Inhaled glucocorticoids are commonly employed to treat patients with asthma. Eosinophils are important effector cells in the pathogenesis of asthma, and, in vitro, glucocorticoids modulate eosinophil viability. OBJECTIVE: Using this glucocorticoid inhibition of eosinophil viability, we compared the in vitro potencies of several inhaled glucocorticoids with particular attention to fluticasone 17-propionate. METHODS: Eosinophils from normal or mildly atopic donors were purified, cultured with cytokines and glucocorticoids, and on day 4, after staining with propidium iodide, analysed by flow cytometry. RESULTS: Eosinophil viability was prolonged by interleukin (IL)-5 in a concentration-dependent manner; in contrast, dexamethasone inhibited the IL-5 effect. Fluticasone 17-propionate, 1.0-1000 nM, also inhibited the IL-5 effect in a concentration-dependent manner; interestingly, at 0.1 nM fluticasone 17-propionate modestly, but significantly, enhanced eosinophil survival. High concentrations of IL-5 and granulocyte-macrophage colony-stimulating factor essentially completely overcame the inhibitory effect of 1000 nM fluticasone 17-propionate on eosinophil survival. In contrast, although interferon-gamma-mediated eosinophil viability was inhibited by 1.0-1000 nM fluticasone 17-propionate, this inhibition was not overcome by increased concentrations of interferon-gamma. Comparison of the glucocorticoid inhibition of eosinophil viability in the presence of 10 pg/mL IL-5 resulted in these drug IC50 values (in nM): fluticasone 17-propionate, 1.3; budesonide, 8.5; triamcinolone acetonide, 25; flunisolide, 32; dexamethasone, 94; beclomethasone 17-monopropionate, 210; beclomethasone 17,21-dipropionate, 290; and hydrocortisone, >1000. CONCLUSION: Fluticasone 17-propionate's effect on cytokine-mediated eosinophil viability is similar qualitatively to other glucocorticoid preparations. However, quantitatively, fluticasone 17-propionate has the most potent suppressive effects on IL-5 mediated eosinophil viability among the currently available inhaled glucocorticoids in the United States.

Administration, Inhalation↗
PubMed2003

Localization and content of nerve growth factor in peripheral blood eosinophils of atopic dermatitis patients.

BACKGROUND: Atopic dermatitis (AD) is a chronic and relapsing eczematous skin disorder characterized by eosinophilia. Nerve growth factor (NGF) modulates the allergic response through interactions with immune-inflammatory cells. Eosinophils have been reported to store NGF as a preformed mediator. OBJECTIVE: To gain further insight into the significance of eosinophils in association with NGF in the pathogenesis of AD, the localization of NGF within eosinophils and the difference of the eosinophil-derived NGF content in the peripheral blood of normal volunteers vs. AD patients were investigated. METHODS: We examined the localization of NGF within human eosinophils using the post-embedding immunoelectron microscopy and compared NGF content in freshly isolated eosinophil sonicates from the peripheral blood of 31 normal volunteers vs. 42 AD patients by immunoenzymatic assay. A possible correlation between the levels of NGF and major basic protein was also examined. RESULTS: Immunoelectron microscopic studies revealed that NGF was localized in the central core of normal eosinophil granules, where major basic protein is also present as a preformed mediator, in homogeneous granules and in intergranular ductal or vesicular structures adjacent to specific granules of eosinophils. NGF content in eosinophils was significantly increased in AD patients. Furthermore, there was a significant correlation between levels of NGF and major basic protein in eosinophils of AD patients. CONCLUSIONS: Increased levels of NGF contained in eosinophils of the peripheral blood from AD patients, when released with other mediators such as basic proteins, could promote inflammation and local tissue damage.

Adult↗
PubMed2000

Pathogenic roles of eosinophils in guinea-pig contact sensitivity: regulation of dermal eosinophilia with remotely administered IL-5.

Eosinophils have a variety of functions. Although increasing evidence links the presence of eosinophils to airway damage, studies have not examined in detail if, and how, eosinophils affect skin inflammation. The purpose of this study was to determine whether eosinophil infiltration augments the contact sensitivity reaction in vivo. Guinea-pigs were sensitized with 2, 4-dinitrochlorobenzene and challenged on the dorsal skin or on the right ear lobe. The number of eosinophils and macroscopic changes of the skin lesion in the presence or absence of human recombinant IL-5 (rIL-5) administered at the remote site was assessed. The reaction on the dorsal skin was acutely eczematous with considerable basophil infiltration. In contrast, eosinophils had extensively infiltrated the right ear lobe and major basic protein was deposited in the dermis. A subcutaneous injection of rIL-5 (10 pmol/kg) at the remote site (left ear lobe) 12 h after challenge induced transient blood eosinophilia and enhanced eosinophil accumulation in the challenged ear lobe. These changes were accompanied by increased ear swelling and severe erythema. In contrast, eosinophil infiltration was significantly inhibited by rIL-5 administered at the time of challenge. Ear thickness, as well as the erythema and oedema, were also reduced. These data suggest that marked eosinophil infiltration enhances skin inflammation in allergic contact dermatitis. Moreover, locally administered IL-5 functions remotely by controlling eosinophil recruitment into the skin. The guinea-pig model of contact sensitivity may be useful for evaluating therapies and pharmaceuticals targeted at eosinophil infiltration.

Animals↗
PubMed2002

Activation of the specific neurotrophin receptors TrkA, TrkB and TrkC influences the function of eosinophils.

BACKGROUND: Recent studies have shown that nerve growth factor (NGF) can act on several immune cells as well as residential cells. But little is known about their role in modulating eosinophil function via activation of high-affinity receptors. OBJECTIVES: The aim of this study was to assess whether eosinophils express functional receptors and if their function is influenced by NGF, brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). METHODS: Eosinophils were purified by negative immunoselection (purity > 96%). High-affinity neurotrophin receptors were demonstrated by reverse transcription polymerase chain reaction, western blotting and flow-cytometry analysis. Functionality of receptors was demonstrated by receptor phosphorylation after ligand binding. Eosinophils were incubated with NGF, BDNF and NT-3, and cells and supernatants were collected for measurement of the mediators IL-4, IL-5, IL-8, transforming growth factor (TGF)-beta1, eosinophil cationic protein (ECP), eosinophil protein X (EPX) as well as eosinophil viability. RESULTS: Eosinophils expressed mRNA for neurotrophin receptors. Proteins were detectable by western blot and fluorescent-activated cell sorter analysis. The receptors were phosphorylated after stimulation with neurotrophins. After NGF stimulation, a significant increase in IL-4 was detectable. BDNF and NT-3 stimulation led to a significant increase in EPX. Eosinophil viability was not influenced. CONCLUSIONS: Eosinophils express the functionally active receptors TrkA, TrkB and TrkC. Receptor activation stimulates eosinophils. This might be an additional pathway regulating inflammatory responses in allergic reactions.

Blotting, Western↗
PubMed1998

Effects of TGF-beta on eosinophil chemotaxis.

Transforming growth factor-beta (TGF-beta), which can decrease the effects of interleukin (IL)-3, IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) on eosinophil viability, has been shown to be chemotactic for neutrophils. However, there is little information on its effects on eosinophil chemotaxis. Because TGF-beta has recently been found in increased concentrations in asthmatic sputum, we investigated whether TGF-beta could influence eosinophil migration and eosinophil viability. Purified eosinophils from normal donors were incubated with increasing concentrations of TGF-beta. Chemotaxis was measured with a modified Boyden chamber technique. In addition, eosinophils were incubated for 96 h with either IL-3, IL-5 or GM-CSF (1 ng/ml) together with increasing concentrations of TGF-beta. Eosinophil viability was then determined with propidium jodide and flow cytometry. Eosinophil chemotaxis was significantly increased in the presence of TGF-beta in concentrations between 10(-9) and 10(-4) microg/ml. The optimal concentration of TGF-beta in this assay was between 10(-9) and 10(-8) microg/ml. The chemotactic effect of TGF-beta diminished when higher as well as lower concentrations (between 10[-12] and 10[-3] microg/ml) were employed. In contrast, inhibition of eosinophil survival induced by IL-3, IL-5 and GM-CSF reached its maximum at concentrations of TGF-beta between 10(-4) and 10(-3) microg/ml. From these data we conclude that TGF-beta in low concentrations can induce eosinophil chemotaxis whereas higher concentrations reduce eosinophil survival mediated by IL-3, IL-5 and GM-CSF.

Cell Survival↗
PubMed2000

IL-9 expression by human eosinophils: regulation by IL-1beta and TNF-alpha.

BACKGROUND: IL-9 is a pleiotropic cytokine that exhibits biologic activity on cells of diverse hemopoietic lineage. IL-9 stimulates the proliferation of activated T cells, enhances the production of IgE from B cells, and promotes the proliferation and differentiation of mast cells and hematopoietic progenitors. OBJECTIVE: In this study we evaluated the expression of IL-9 messenger (m)RNA and protein by human peripheral blood eosinophils. We also investigated the role of IL-1beta and TNF-alpha in the release of IL-9 from human peripheral blood eosinophils. METHODS: RT-PCR, in situ hybridization, and immunocytochemistry were used to investigate the presence of IL-9 mRNA and protein in human peripheral blood eosinophils from asthmatic patients and normal control subjects. Furthermore, biologic assay was used to investigate the release of IL-9 protein from IL-1beta- or TNF-alpha-stimulated eosinophils in vitro. RESULTS: RT-PCR analysis showed the presence of IL-9 mRNA in human peripheral blood eosinophil RNA preparations from subjects with atopic asthma, as well as in the eosinophil-differentiated HL-60 cell line. By using in situ hybridization, a significant difference (P <.01) in IL-9 mRNA expression was detected in human peripheral blood eosinophils freshly isolated from asthmatic subjects compared with those isolated from normal control subjects. Furthermore, the percentage of IL-9 immunoreactive eosinophils from asthmatic patients was increased compared with that found in normal control subjects (P <.01). We also demonstrate that cultured human peripheral blood eosinophils from asthmatic subjects synthesize and release IL-9 protein, which is upregulated on stimulation with TNF-alpha and IL-1beta. CONCLUSION: Human eosinophils express biologically active IL-9, which suggests that these cells may influence the recruitment and activation of effector cells linked to the pathogenesis of allergic disease. These observations provide further evidence for the role of eosinophils in regulating airway immune responses.

Eosinophils↗
PubMed2000

Glucocorticoids preferentially upregulate functional CXCR4 expression in eosinophils.

BACKGROUND: Chemokines play an important role in accumulation of eosinophils at allergic inflammatory sites. Systemic administration of glucocorticoids (GCCs) attenuates tissue eosinophilia. In vivo chemokine actions are regulated at levels of both ligand production and receptor expression. The inhibitory effects of GCCs on the production of eosinophil-active chemokines, such as eotaxin, have been well established. However, no data exist regarding the effects of GCCs on expression of chemokine receptors in eosinophils per se. OBJECTIVE: The objective of this study was to investigate the regulation of chemokine receptor expression in eosinophils by GCCs. METHODS: Chemokine receptor expression was analyzed by using flow cytometry and reverse transcriptase PCR. Intracellular Ca(2+) influx and chemotaxis were also analyzed. RESULTS: Eosinophil CCR3 expression was slightly downregulated by 24-hour treatment with dexamethasone (DEX). On the other hand, DEX-treated eosinophils showed markedly increased CXCR4 expression ( approximately 6 fold) in a time- and dose-dependent fashion. In contrast to eosinophils, CXCR4 expression in neutrophils was only marginally affected by DEX. In DEX-treated eosinophils, stromal cell-derived factor 1alpha, a natural ligand for CXCR4, induced a higher level of Ca(2+) influx and chemotaxis compared with untreated cells. CONCLUSION: GCCs upregulate the expression of CXCR4 in eosinophils but not in neutrophils. Because stromal cell-derived factor 1alpha may play a role in baseline trafficking of eosinophils into extravascular tissues rather than recruiting them directly to inflammatory sites, upregulation of CXCR4 by GCCs may mediate the antiallergic property of these drugs by sequestering eosinophils from the circulation to extravascular tissues.

Apoptosis↗