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PubMed1985

Rapid method for isolation of normal human peripheral blood eosinophils on discontinuous Percoll gradients and comparison with neutrophils.

Previous studies on human eosinophils often have used cells from patients with hypereosinophilia syndrome or parasitosis owing to the difficulty in isolating pure populations of eosinophils from normal individuals. In the present study, human eosinophils were isolated with a purity of 97%, with 70% recovery from normal individuals with blood eosinophil counts of less than 3%. Human eosinophils are denser than neutrophils, but the range of densities of the two cell types overlap, making purification of eosinophils by density-gradient centrifugation difficult. However, if neutrophils were exposed to the chemotactic peptide (f-Met-Leu-Phe), which did not stimulate eosinophils, the neutrophils' density decreased, shifting them away from the density of eosinophils. Whole normal blood anticoagulated with EDTA was incubated at 37 degrees C for 15 minutes with 10(-6) mol/L f-Met-Leu-Phe and then layered over a discontinuous Percoll gradient (65% and 75% in diluted phosphate-buffered saline) and centrifuged at 400 g for 25 minutes at 22 degrees C. The cell layer between the 65% and 75% Percoll was collected and washed, and hypotonic lysis was used to remove erythrocytes. This cell layer contained 97.3 +/- 0.7% eosinophils (N = 8) with a yield of 4.9 X 10(4) eosinophils per milliliter of whole blood, or 70% of the total eosinophil count. The isolated eosinophils were in a quiescent state but responded to Escherichia coli endotoxin-activated serum with shape change and chemotaxis, membrane depolarization, and reduced nitroblue tetrazolium (96.0 +/- 1.0%), when stimulated with phorbol myristate acetate. In phagocytic assays, 89.3 +/- 1.3% of the eosinophils ingested Candida albicans v 96.0% +/- 1.0% of neutrophils. In contrast, the eosinophils did not respond chemotactically, alter membrane potential, or reduce nitroblue tetrazolium when treated with f-Met-Leu-Phe, and studies with f-Met-Leu-[3H]Phe showed that normal eosinophils lacked expression of receptors for f-Met-Leu-Phe. In control studies, normal eosinophils that were not exposed to f-Met-Leu-Phe during purification also failed to respond to f-Met-Leu-Phe, indicating intrinsic differences between normal eosinophils and neutrophils. Thus, exposure of whole blood to f-Met-Leu-Phe, followed by separation on Percoll is a simple method for rapid isolation of normal human eosinophils.

Binding Sites↗

PubMed1992

Release of granule proteins from eosinophils cultured with IL-5.

Eosinophils isolated from normal individuals were cultured in the presence of human rIL-5 (hrIL-5) for up to 14 days, and the effects of this exposure were determined. First, the hrIL-5-cultured eosinophils were activated and degranulated more readily than freshly isolated eosinophils. For example, eosinophils cultured for 7 days with hrIL-5 released 30 and 10% of granule eosinophil-derived neurotoxin (EDN) when exposed to Sepharose 4B beads coupled to secretory IgA and IgG, respectively, whereas freshly isolated eosinophils released only 19 and 4%, respectively, of their EDN in response to the same stimuli. Degranulation of hrIL-5-cultured eosinophils was not augmented by further exposure to hrIL-5, whereas degranulation of freshly isolated cells to secretory IgA and IgG beads was increased by exposure to hrIL-5. Second, eosinophils cultured with hrIL-5 had prolonged viability in vitro. For example, after four days of culture with 50 U/ml of hrIL-5, 86% of eosinophils were viable compared to 12% in medium alone. Third, hrIL-5-cultured eosinophils became hypodense, and electron microscopy showed that they contained granules with core and matrix lucency and with evidence of granule fusion. Fourth, hrIL-5-cultured eosinophils spontaneously lost 30 to 60% of their EDN, eosinophil cationic protein, and eosinophil peroxidase and about 50% of their eosinophil granule major basic protein content compared to freshly isolated eosinophils, and all four of the granule proteins were released into the culture medium. Fifth, detailed studies of eosinophils cultured in hrIL-5 showed that 89 +/- 10% of the starting quantity of EDN could be recovered at 7 days. Whereas 99 +/- 1% of the EDN at day 0 was cell associated, by 7 days 60 +/- 9% was in the cell supernatants. Thus, hrIL-5 activates eosinophils, increases their viability, decreases their density, and their content of granule proteins and causes release of the granule proteins into culture fluids. The striking loss of granule proteins during culture with hrIL-5 may be an important mechanism for deposition of these cationic toxins in various diseases where IL-5 plays a role.

Blood Proteins↗

PubMed2000

Conjunctival fibroblasts enhance the survival and functional activity of peripheral blood eosinophils in vitro.

PURPOSE: To examine the effect of human conjunctival fibroblasts on the survival and functional activity of human peripheral blood eosinophils. METHODS: Eosinophils were purified by negative immunoselection [magnetic activated cell sorter (MACS), purity > 97%] from volunteers with mild atopia. Fibroblasts were cultured from conjunctival specimens of healthy donors. Eosinophils were cultured on confluent monolayers of conjunctival fibroblasts or in culture medium alone. Eosinophil survival was evaluated by the trypan blue exclusion test. Eosinophil adherence was assessed by counting the attached cells after washing the cultures. Eosinophil viability and adherence in coculture were also assessed in the presence of anti-granulocyte-macrophage colony-stimulating factor (GM-CSF), anti-interleukin (IL)-3, and anti-IL-5 neutralizing antibodies. Cocultured eosinophils were activated by lipopolysaccharide (LPS) after 4 days in culture, and eosinophil peroxidase (EPO) release was determined as a marker of their activation. RESULTS: Eosinophils cocultured with conjunctival fibroblasts had a significantly increased viability of 35.9% (P = 0.004) and 12.8% (P = 0.003) on days 4 and 8, respectively. Fibroblast-conditioned medium did not enhance the survival of eosinophils. The increase in eosinophil survival in coculture was partially inhibited by anti-GM-CSF (P = 0.019), anti-IL-3 (P = 0.033), or anti-IL-5 (P = 0.011), whereas eosinophil adherence was reduced by anti-GM-CSF alone (P = 0.034). LPS activation of eosinophils cultured for 4 days with conjunctival fibroblasts induced higher EPO release than in freshly isolated eosinophils (P = 0.01). CONCLUSIONS: Human conjunctival fibroblasts induced prolonged survival and increased secretory function of human peripheral blood eosinophils. Increased survival is partially mediated by IL-3, IL-5, and GM-CSF. The coculture of conjunctival fibroblasts with eosinophils can serve as an in vitro system for the study of eosinophil behavior in the ocular surface and of cellular interactions in allergic eye diseases.

Adolescent↗

PubMed1994

Interferon-gamma induces interleukin-3 release from peripheral blood eosinophils.

Interferon-gamma (IFN-gamma) upregulates eosinophil effector functions and prolongs the in vitro survival of eosinophils. We examined the possible capacity of IFN-gamma to stimulate eosinophils to produce eosinophil-activating cytokines. Eosinophils purified from mild atopic volunteers were cultured with 100 U/ml IFN-gamma. Viability of eosinophils was counted and supernatants were tested for the presence of cytokines by neutralization of eosinophil viability-enhancing activity with specific antibodies to IFN-gamma, interleukin-5 (IL-5), IL-3, or granulocyte-macrophage colony-stimulating factor (GM-CSF). IFN-gamma-enhanced eosinophil viability was up to 95% on the 4th day of culture. Pretreatment with anti-IL-3 antibody partially blocked the IFN-gamma-enhanced eosinophil survival. IFN-gamma-stimulated eosinophil supernatants had eosinophil survival. IFN-gamma-stimulated eosinophil supernatants had eosinophil viability-enhancing activity that was blocked by pretreatment not only with anti-IFN-gamma but also with anti-IL-3. Antibodies to IL-5 or GM-CSF did not have the blocking effect. To further confirm the production of IL-3 by eosinophils, we performed reverse transcription polymerase chain reaction (RT-PCR) for IL-3 messenger RNA (mRNA) in IFN-gamma-stimulated eosinophils. Significant IL-3 mRNA expression in eosinophils was observed at 6 h of incubation with IFN-gamma. These results suggest that IFN-gamma stimulates the autocrine function of eosinophils and may play an important role in allergic inflammation.

Cell Survival↗

PubMed2000

The effect of transendothelial migration on eosinophil function.

In bronchial asthma, eosinophils found in the airways have an enhanced inflammatory capacity. We hypothesized that, at least in part, changes in functional phenotype are due to the effect of transendothelial migration. To model in vivo eosinophil trafficking to the lung, we cultured human pulmonary microvascular endothelial cell (HPMEC) monolayers on Transwell filters. The HPMECs were activated with interleukin (IL)-1beta to increase cell expression of intercellular adhesion molecule (ICAM)-1 and, hence, eosinophil transmigration. Peripheral blood eosinophils from allergic patients were added to HPMEC-covered Transwell filters and incubated for 3 h at 37 degrees C. The eosinophils were collected from below (migrated cells) and above (nonmigrated cells) the HPMEC monolayer to determine surface receptor expression, in vitro survival, and oxidative burst. Eosinophils never exposed to HPMECs were used as controls. Eosinophil cell surface expression of CD69, human leukocyte-associated antigen-DR (HLA-DR), and CD54 (ICAM-1) was significantly increased after transendothelial migration through IL-1beta-treated HPMECs compared with control cells (CD69: P<0.0005; HLA-DR and CD54: P<0.05) and nonmigrated eosinophils (CD69 and HLA-DR: P<0.05). Moreover, the percent in vitro survival (48 h) of migrated eosinophils was also significantly greater (P<0.0001 by trypan blue exclusion, P< 0.05 by flow cytometry) than that of control or nonmigrated eosinophils. Prolonged survival of migrated eosinophils was inhibited by addition of anti-granulocyte macrophage colony-stimulating factor (GM-CSF) antibodies (P<0.05) to the 48-h survival culture, suggesting that autocrine production of GM-CSF was, at least partially, responsible for increased eosinophil survival. Although GM-CSF protein was not measurable in survival culture supernates, GM-CSF messenger RNA (mRNA) was expressed in both nonmigrated and migrated eosinophils but not in control cells. Similarly, the eosinophils' oxidative burst induced by platelet-activating factor, formylmethionyl leucylphenylalanine, or phorbol myristate acetate was equally, and significantly, increased in both nonmigrated and migrated eosinophils (P<0.05 versus control). Therefore, whereas exposure of eosinophils to cytokine-activated HPMECs can increase surface receptor expression, in vitro survival, GM-CSF mRNA, and the respiratory burst, transendothelial migration can further potentiate receptor expression and survival in migrated cells. These results suggest that the process of transendothelial migration selectively participates in determining the eventual phenotype of airway eosinophils.

Antibodies↗

PubMed1992

[Eosinophilic granulocytes and their significance in allergic diseases].

Eosinophils are found in the blood and tissues of patients with allergic diseases such as asthma, allergic rhinitis and also atopic dermatitis. The number of eosinophils in the diseased tissue generally correlates with the expression of clinical symptoms. Originally, the eosinophil was regarded as having an exclusively protective role, for example in host defense against parasites. More recently, the eosinophil is recognized as being a pro-inflammatory cell that can mediate allergic disease. The eosinophil is active in inflammation through the release of granule proteins and the synthesis and release of inflammatory mediators. The important eosinophil granule proteins are major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO). These proteins have toxic effects on the surrounding tissue. Of additional importance for the allergic inflammatory reactions are membrane-derived mediators such as leukotriene C4 (LTC4), platelet activating factor (PAF) and Charcot-Leyden crystals. These mediators are synthesized and released after eosinophil activation, and act toxic on surrounding cells. Eosinophils are active in asthma, and increased numbers and eosinophil-derived mediator concentrations have been documented in bronchial biopsies, BAL and sputum. In addition, eosinophil granule proteins and inflammatory mediators are found in nasal secretions in allergic rhinitis. In atopic dermatitis one finds activated eosinophils and depositions of eosinophil granule proteins in skin biopsies. Eosinophils are not only active in mediating allergic inflammation, but interact in cellular networks with antigen presenting cells (APC), mast cells, and T lymphocytes. These other cells influence eosinophil maturation, mobilization, tissue localization and activation.

Asthma↗

PubMed1990

Eosinophil degranulation. An immunologic determinant in the pathogenesis of the Mazzotti reaction in human onchocerciasis.

Onchocerciasis patients treated with diethylcarbamazine often undergo a severe inflammatory response, the Mazzotti reaction. To assess the eosinophil's role in the pathogenesis of the Mazzotti reaction, we obtained serial blood, plasma, and skin biopsy specimens from 21 heavily infected patients and 3 endemic controls, both before and during therapy with diethylcarbamazine. Samples were analyzed for blood eosinophils, plasma levels of eosinophil granule major basic protein (MBP) and eosinophil-derived neurotoxin, eosinophil infiltration and eosinophil and mast cell degranulation in the skin. After the first dose of diethylcarbamazine, blood eosinophils fell from a pre-treatment level of 888 +/- 111 to 203 +/- 42 cells/mm3 at 8 h. This decrease was followed by a marked eosinophilia developing over the remaining 7 days of treatment and 14 days of follow-up. Plasma eosinophil-derived neurotoxin levels increased from 56 +/- 4 ng/ml pretreatment to a peak of 82 +/- 9 ng/ml at 8 h and returned to pretreatment levels by 48 h. Beginning at 12 h, plasma MBP levels increased from 730 +/- 74 ng/ml pretreatment to a peak of 1140 +/- 74 ng/ml after 5 days. Pretreatment skin biopsies stained for MBP by immunofluorescence showed a bright fibrillar pattern in the dermis consistent with chronic eosinophil degranulation; the MBP was localized on elastic tissue fibers. After treatment, skin biopsy specimens showed both the pretreatment fibrillar MBP staining pattern as well as focal eosinophil degranulation. Deposition of MBP around microfilariae in the papillary dermis was visible as early as 1.5 h. The lowest blood eosinophil levels and peak plasma eosinophil-derived neurotoxin levels coincided with the infiltration and degranulation of eosinophils in the skin. Mast cell degranulation in the skin was maximal by the first posttreatment biopsy (1.5 h) coincident with the beginning of eosinophil degranulation. Although the pathogenesis of the Mazzotti reaction is clearly complex, our results indicate that eosinophil degranulation is characteristic of the response and that it occurs with a time course suggestive of a role for the eosinophil in determining the clinical and pathologic manifestations of the reaction.

Adult↗

PubMed2004

Mast cell tryptase activates peripheral blood eosinophils to release granule-associated enzymes.

BACKGROUND: Mast cells and eosinophils are important effector cells in asthma. Understanding their interactions is essential for studying asthma pathophysiology. Inflammatory mediators released from mast cells, such as arachidonic acid metabolites, TNF and IL-5, are important in eosinophil biology. However, little is known about the effects of mast cell-specific mediators, such as tryptase, on eosinophils. Our objective was to investigate the effects of mast cell tryptase on human peripheral blood eosinophils. METHODS: Peripheral blood eosinophils isolated from asthmatic individuals were activated using various concentrations of tryptase- and protease-activated receptor-2 (PAR-2)-activating peptides (PAR-2 AP). Eosinophil activation was evaluated by the release of granule mediators, superoxide release, estimation of eosinophil survival, changes in intracellular Ca2+ concentration and mitogen-activated protein kinase activation. RESULTS: Tryptase induced the release of eosinophil peroxidase and beta-hexosaminidase from peripheral blood eosinophils but had no effect on RANTES release. Eosinophils isolated from two thirds of our donors responded to tryptase, while the remainder appeared not to respond. Release of granule mediators was dependent on tryptase enzymatic activity. To identify the mechanism of eosinophil activation by tryptase, we studied the expression of PAR-2 by eosinophils and its function. Using RT-PCR, we amplified PAR-2 from eosinophils. However, flow cytometry failed to detect significant PAR-2 expression on the surface of eosinophils. The PAR-2 AP SLIGRL-NH2 did not induce eosinophil activation by any of the methods we employed. CONCLUSION: Our data indicate that mast cell tryptase may affect eosinophil activation status independently of PAR-2.

Asthma↗

PubMed1992

Eosinophil granule cationic proteins regulate complement. I. Activity on the alternative pathway.

Eosinophil granules contain several cationic proteins that mediate tissue damage in allergic disease. The present study examined the capacity and mechanisms by which these cationic proteins regulate activity of the alternative pathway of C. Eosinophil peroxidase and eosinophil cationic protein inhibited formation of cell-bound alternative pathway C3 convertase, causing 50% inhibition of lysis at about 0.19 and 0.75 microgram/10(7) cellular intermediates, respectively. Major basic protein inhibited alternative pathway C3 activity by only 19% at 1.5 micrograms/10(7) cellular intermediates. Eosinophil-derived neurotoxin had no activity on the alternative pathway. The eosinophil granule proteins were examined for the mechanism by which they inhibited alternative pathway activity. Eosinophil peroxidase and major basic protein inhibited fluid phase factor B consumption in a reaction mixture that also contained factors D and C3b, eosinophil-derived neurotoxin had no activity on factor B consumption, and eosinophil cationic protein consumed factor B in the absence of C3b and factor D. Both eosinophil cationic protein and eosinophil peroxidase enhanced the decay of preformed alternative pathway convertase. Lysis of EAC4b,3b cellular intermediates formed to contain a low surface amount of C3b was more inhibited than was lysis of cells formed with a standard amount of C3b on the surface. This suggests that these eosinophil proteins acted predominantly on C3b to regulate alternative pathway activity. We also found that none of the eosinophil granule cationic proteins had any effect on later events after the formation of the C3 convertase. We conclude that although eosinophil-derived neurotoxin (isoelectric pH value (pI) = 8.9) does not regulate alternative pathway activity, the more highly charged eosinophil granule cationic proteins--major basic protein (pI = 10.9), eosinophil cationic protein (pI = 10.8), and eosinophil peroxidase (pI = 10.8)--do share the capacity to regulate C activity and may exert this activity in vivo.

Blood Proteins↗

PubMed1998

Pathways for eosinophil lipid body induction: differing signal transduction in cells from normal and hypereosinophilic subjects.

Although lipid bodies, inducible cytoplasmic inclusions active in arachidonic acid metabolism, are abundant in activated leukocytes, including eosinophils, mechanisms for eosinophil lipid body formation are not certain. Eosinophils from hypereosinophilic syndrome (HES) donors contained about twice (approximately 18/cell) as many lipid bodies as eosinophils froin normal donors (approximately 10/cell). By immunocytochemistry both 5- and 15-lipoxygenases were localized at lipid bodies in HES eosinophils. Platelet-activating factor (PAF) induced rapid, receptor-mediated increases in lipid bodies in normal and HES eosinophils. Protein kinase C (PKC) inhibitors, chelerythrine and calphostin C, inhibited PAF-induced lipid body formation partially in normal and HES eosinophils. In HES, but not normal, eosinophils, PAF-induced lipid body formation was completely blocked by two tyrosine kinase inhibitors, herbimycin A and genistein, which were not acting on 5-lipoxygenase because they also blocked 5-HETE-induced lipid body formation in HES, and not normal, eosinophils. After 24 h culture with eosinophil growth factor cytokines [interleukin (IL)-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) or GM-CSF alone but not IL-5 or IL-3 alone], normal eosinophils were induced to exhibit an HES-like phenotype, including increased lipid body numbers and tyrosine kinase-dependent signaling for PAF-induced lipid body formation. Thus, signal transduction mechanisms involved in PAF-induced lipid body formation in eosinophils can be differentially recruited. Tyrosine kinase-dependent signaling is not involved in normal eosinophils, but is active in HES eosinophils and in normal eosinophils cultured with GM-CSF. PKC- and tyrosine kinase-dependent pathways are involved in the formation of eosinophil lipid bodies, which may facilitate enhanced synthesis of lipoxygenase-derived eicosanoids.

Adult↗

PubMed1988

In vivo activation of equine eosinophils and neutrophils by experimental Strongylus vulgaris infections.

Eosinophils and neutrophils from ponies with Strongylus vulgaris-induced eosinophilia (eosinophilic ponies; activated eosinophils and neutrophils) were assayed in vitro for chemotactic and chemokinetic responses to zymosan-activated serum (ZAS) using the filter system in Boyden chambers, for Fc and complement (C) receptors using the EA and EAC-rosette assays, respectively, and for phagocytic and bactericidal activities using opsonized Escherichia coli and the acridine orange method. The responses of activated eosinophils and neutrophils in the above assays were compared with those of eosinophils and neutrophils from S. vulgaris-naive ponies without eosinophilia (noneosinophilic ponies; nonactivated eosinophils and neutrophils). Differences in cell density following centrifugation in a continuous Percoll gradient were used to further characterize the heterogeneity of activated eosinophils and neutrophils. Activated and nonactivated eosinophils demonstrated similar chemotactic responses to ZAS while activated and nonactivated neutrophils demonstrated similar chemokinetic responses to ZAS. A higher percentage of activated eosinophils and neutrophils expressed Fc and C receptors compared with nonactivated cells (P less than 0.05). Generally, higher percentages of eosinophils and neutrophils expressed C than Fc receptors. However, the percentage of neutrophils with both receptors was higher than that of eosinophils. Phagocytosis and killing of E. coli by either type of eosinophil were not consistently observed. Both activated and nonactivated neutrophils phagocytized E. coli and significant differences between the two cell types were not observed. The bacterial activity, however, of activated neutrophils was significantly greater than that obtained using nonactivated neutrophils (P less than 0.05). Activated eosinophils and neutrophils were both separated into two distinct fractions based on differences in cell densities. A higher percentage of band 2 eosinophils (density of 1.106) expressed C receptors than did band 1 eosinophils (density of 1.049) (P less than 0.05). A higher percentage of band 1 neutrophils (density of 1.072) expressed both Fc and C receptors and these neutrophils were more phagocytic and bactericidal than were band 2 neutrophils (density of 1.082) (P less than 0.05). These data suggest that equine eosinophils and neutrophils are activated by chronic S. vulgaris infections.

Animals↗

PubMed2001

Gastrointestinal eosinophils.

The gut-associated lymphoid tissue (GALT) is composed of lymphocytes residing in Peyer's patches, lamina propria, and intraepithelial compartments. In addition to these features which distinguish GALT from other peripheral sites of the immune system, the gastrointestinal immune system is also composed of resident eosinophils. Eosinophils are generally considered to be peripheral blood leukocytes that have an important pro-inflammatory role in various immune disorders. Although most research concerning this cell has focused on understanding its trafficking and function in the blood and lung, recent studies have also started to elucidate its regulation and function in the gastrointestinal tract. Interestingly, eosinophil numbers in the gastrointestinal tract are substantially higher than in other tissues. At baseline (healthy conditions), most eosinophils reside in the lamina propria in the stomach and intestine. Eosinophil homing to these sites occurs during embryonic development and their levels in perinatal mice are comparable to those in adults, indicating that their homing is not dependent upon the presence of intestinal flora. Furthermore, eosinophil localization to the lamina propria at baseline is critically regulated by eotaxin, a chemokine constitutively expressed throughout the gastrointestinal tract. Although eotaxin is required for eosinophil homing, its expression in the esophagus is not sufficient for eosinophil accumulation, since this organ is devoid of eosinophils at baseline. During Th2-associated inflammatory conditions (e.g. interleukin (IL)-5 overexpression or oral allergen challenge), marked increases of eosinophils occur not only in the lamina propria but also in Peyer's patches. The accumulation of Peyer's patch eosinophils, which mainly occurs in the outer cortex and interfollicular regions, is critically regulated by IL-5 and less significantly by eotaxin, suggesting the involvement of other eosinophil chemokines in this lymphoid compartment. Preliminary investigations have shown that gastrointestinal eosinophils express the alpha4beta7 integrin and that this molecule is responsible, in part, for eosinophil homing. In summary, eosinophils are resident cells of the gastrointestinal immune system whose levels can be induced by antigen exposure under Th2 conditions, in a manner that is critically regulated by eotaxin and IL-5. We propose that eosinophils are integral members of the gastrointestinal immune system and are likely to be important in innate, regulatory and inflammatory immune responses.

Administration, Oral↗

PubMed1994

Role of interleukin-5 in enhanced migration of eosinophils from airways of immunized guinea-pigs.

1. Platelet activating factor (PAF), leukotriene B4 (LTB4) and interleukin-5 (IL-5) are potent chemoattractants for guinea-pig eosinophils, which may be involved in eosinophil recruitment and up-regulation in allergic diseases. Eosinophils from the bronchoalveolar lavage fluid (BALF) of ovalbumin-sensitized guinea-pigs were collected 24 h after antigen provocation and migration induced by PAF, LTB4 and rhIL-5 was studied. 2. Total BALF content and distribution of eosinophils were greater in immunized, ovalbumin-challenged guinea-pigs (5.0 +/- 0.8 x 10(6)/guinea-pig; 12 +/- 1%) than in immunized, saline-challenged animals (3.0 +/- 0.7 x 10(6)/guinea-pig; 7 +/- 1%). 3. The chemoattraction of eosinophils isolated on a metrizamide gradient was studied in micro-Boyden chambers, results being expressed as the number of migrating cells (mean +/- s.e. mean). PAF and LTB4-induced migration of eosinophils from immunized and OA-challenged guinea-pigs were significantly enhanced, as compared to immunized and saline-challenged animals (170 +/- 36 vs 35 +/- 9 migrating eosinophils for 10 nM PAF; 271 +/- 60 vs 110 +/- 19 for 1 nM LTB4). 4. The IL-5 antibody TRFK-5, in vivo, reduced eosinophil recruitment in BALF of antigen-challenged immunized animals as well as the enhanced responsiveness of eosinophils from the challenged animals, suggesting a role for IL-5 in the priming of eosinophils in vivo. 5. In contrast to TRFK-5, nedocromil sodium reduced to a similar extent eosinophil, macrophage and lymphocyte recruitment into the BALF of antigen-challenged, but failed to down-regulate the enhanced responsiveness of eosinophils from the challenged animals. 6. The increased eosinophil content in lungs from antigen-challenged guinea-pigs is thus selectively reduced by the anti-IL-5 antibody, which also attenuates the concomitant enhancement of the eosinophil responsiveness, supporting the concept that IL-5 is essential for recruitment and priming of eosinophils in vivo. In contrast, nedocromil sodium reduced non-selectively the total cell recruitment to the airways,but failed to attenuate the enhanced responsiveness of those eosinophils which migrated, indicating that its effects involve a different target.

Animals↗

PubMed2006

Differential effects of salbutamol and montelukast on eosinophil adhesion and superoxide anion generation.

BACKGROUND: Beta2-agonists, a representative class of bronchodilators used for asthma, have been shown to modulate some functions of eosinophils, including cell adhesion. Similarly, a leukotriene receptor antagonist (LTRA) may be beneficial in controlling inflammation in asthma, as cysteinyl leukotrienes (cysLTs) can cause accumulation or activation of eosinophils. Recent evidence suggests that the addition of an LTRA, but not a long-acting beta2-agonist, to inhaled corticosteroid additionally reduces the number of eosinophils in sputum and blood from patients with asthma. The present study examined whether a beta2-agonist and an LTRA differentially modify eosinophil adhesion and activation induced by cysLTs and other activators. METHODS: Eosinophils were isolated from blood of healthy donors and then incubated in the presence or absence of salbutamol (albuterol) or montelukast. Eosinophils were then exposed to leukotriene D4 (LTD4) or another activator, and the generation of superoxide anion (O2-) was evaluated by cytochrome C reduction assay. Eosinophil adhesion was examined by an eosinophil peroxidase assay. RESULTS: Montelukast, but not salbutamol (both at 1 microM), inhibited LTD4-induced (100 nM) eosinophil adhesion to recombinant human intercellular adhesion molecule 1. Both drugs similarly and partially inhibited the 100 pM interleukin-5-induced adhesive response of eosinophils to recombinant human intercellular adhesion molecule 1. Montelukast, but not salbutamol, blocked LTD4-induced eosinophil O2- generation of eosinophils. Finally, neither salbutamol nor montelukast modified phorbol myristate acetate (1 ng/ml)-induced O2- generation from eosinophils. CONCLUSION: These results confirm that LTD4 directly induces activation of eosinophils via the cysLT1 receptor. Furthermore, the results suggest that a beta2-agonist has no effect on eosinophil adhesion and activation induced by cysLTs. These results explain the differential effects of an LTRA and a beta2-agonist in the treatment of eosinophilic inflammation in asthma.

Acetates↗

PubMed1996

Comparison of surface antigens on eosinophils from patients with eosinophilia.

Recent studies have suggested that there may be heterogeneity among human eosinophils. To study this further, surface antigens on blood eosinophils from patients with eosinophilia (23 bronchial asthma, 6 eosinophilic pneumonia, 1 Kimura's disease and 1 adult T-cell leukemia) and from 8 control subjects were examined using a new direct method for fluorescence detection of eosinophils. HLA-DR+ and CD4+ eosinophil counts were higher in patients with bronchial asthma and adult T-cell leukemia (ATL) than in patients from other groups and in control subjects. CD11b+ eosinophil counts in Kimura's disease and ATL were smaller than those in the other groups. CD45RO+ eosinophil counts in bronchial asthma and eosinophilic pneumonia were significantly higher (p < 0.05) compared with Kimura's disease, ATL and control subjects. CD44+ eosinophil counts in eosinophilic pneumonia were significantly higher (p < 0.05) compared with the other groups and control subjects. These results suggest the existence of functional heterogeneity in the different eosinophilic diseases, with eosinophils in bronchial asthma and eosinophilic pneumonia being more highly activated in migration, activation and immunoregulation. On the other hand, eosinophils in Kimura's disease and ATL might be functionally down-regulated. This heterogeneity of eosinophils may reflect differences in the pathogenesis of various eosinophilic diseases.

Adolescent↗

PubMed1997

CD44 expression on blood eosinophils is a novel marker of bronchial asthma.

Bronchial asthma is characterized by infiltration of the respiratory tracts by eosinophils. A wide variety of adhesion molecules expressed by eosinophils have been proposed to be involved in binding of eosinophils to the vascular endothelium and subsequent transmigration from the circulation to the airways, while little is known about CD44 expression on eosinophils. We introduced a novel staining combination with which surface markers on eosinophils could be analyzed without purification prior to staining, and examined the expression of CD44 on eosinophils. Staining of eosinophils with anti-CD 16 and anti-VLA-4 mAbs enabled us to delineate eosinophils as VLA-4high CD 16- cells from any other leukocyte populations in the whole blood. CD44 was found to be constitutively expressed on resting eosinophils, and expression increased upon cytokine-mediated activation. In all bronchial asthma patients examined, CD44high eosinophils were enriched in sputum relative to peripheral blood, indicating that eosinophils in sputum were more activated than those in blood. By comparing the extent of CD44 expression on blood eosinophils from poorly controlled and well-controlled asthma patients, we unexpectedly found that the density of CD44 expression is higher on blood eosinophils from the well-controlled group. Thus, the extent of CD44 expression on blood eosinophils is a novel marker indicative of the mangement of bronchial asthma. Deterioration of the asthma management with a concomitant decrease in CD44 expression on peripheral eosinophils implies that CD44 may play an important role in facilitating the transmigration of activated, CD44high eosinophils from the circulation to the respiratory tracts.

Asthma↗

PubMed1995

Infection, apoptosis, and killing of mature human eosinophils by human immunodeficiency virus-1.

Although human eosinophils express low concentrations of CD4, the capacity of mature, non-replicating eosinophils to be infected with human immunodeficiency virus-1 (HIV-1) has not been established. Using peripheral blood eosinophils isolated free of contaminating lymphocytes and mononuclear leukocytes, we evaluated eosinophil infection with HIV-1. Eosinophils could be infected with strains of HIV-1 as evidenced by HIV-induced cytolytic effects, progressive release of p24 antigen in cultures of infected eosinophils, recovery of HIV from infected eosinophils by co-cultivation, and detection of HIV-1 gag viral DNA from infected eosinophils by polymerase chain reaction (PCR) amplification. Greater p24 antigen release from infected eosinophils was elicited by the phorbol ester, PMA; and eosinophil killing by HIV-1 was enhanced by the cytokine GM-CSF. By light and electron microscopy, HIV-infected eosinophils demonstrated apoptosis and necrosis. Apoptotic subdiploid nuclear staining was detected by flow cytometric analyses of propidium iodide-stained nuclei from HIV-infected eosinophils, and DNA isolated from HIV-infected eosinophils showed both nucleosomal fragmentation and diffuse degradation. Thus, mature eosinophils, non-replicating terminally differentiated leukocytes, can be infected with HIV-1. HIV-1 expression in eosinophils is promoted by increased granulocyte-macrophage colony-stimulating factor (GM-CSF) and can cause eosinophils to undergo death due to apoptosis and necrosis.

Apoptosis↗

PubMed1996

Cell cultures from bronchial subepithelial myofibroblasts enhance eosinophil survival in vitro.

Mechanisms of eosinophil accumulation and activation in the bronchial mucosa are crucial for the pathogenesis of asthma. The location of specialized fibroblasts, myofibroblasts, beneath the bronchial basement membrane and their proximity to infiltrating eosinophils potentially enable the myofibroblasts to modulate eosinophil survival and function in asthma. The aim of this study was to investigate the effects of bronchial myofibroblasts on eosinophil survival in vitro. Eosinophils from human peripheral blood were exposed to cell cultures from bronchial myofibroblasts and to myofibroblast-conditioned media. Eosinophil viability was assessed and granulocyte/macrophage colony-stimulating factor (GM-CSF) production was examined in co-culture supernatants and as messenger ribonucleic acid (mRNA) in myofibroblasts. Eosinophil survival was significantly increased and eosinophil apoptosis was inhibited by co-culture with myofibroblasts. Conditioned medium from tumour necrosis factor-alpha (TNF-alpha)-stimulated myofibroblasts also prolonged eosinophil survival. This effect could be blocked by GM-CSF antibody. GM-CSF mRNA and secretion from myofibroblasts were increased in co-cultures and by eosinophil-conditioned medium. Addition of antibodies to TNF-alpha and interleukin-1 alpha (IL-1 alpha) to co-cultures resulted in significant reduction both in eosinophil survival and GM-CSF levels. Blocking of fibronectin in the co-cultures did not affect the eosinophil survival enhancing activity. Prednisolone inhibited the eosinophil survival enhancing activity of the co-cultures by suppression of GM-CSF production. Soluble eosinophil-derived cytokines are involved in the interaction of eosinophils with myofibroblasts, which results in a tumour necrosis factor-alpha/interleukin-1 alpha mediated release of granulocyte/macrophage colony-stimulating factor from myofibroblasts. Bronchial myofibroblasts can, thereby, contribute to allergic inflammation by granulocyte/macrophage colony-stimulating factor-mediated inhibition of eosinophil apoptosis.

Anti-Inflammatory Agents↗