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PubMed1992

Migration of primed human eosinophils across cytokine-activated endothelial cell monolayers.

Eosinophils are known to adhere to cytokine-activated endothelium. Whereas transendothelial migration for neutrophils is an inevitable consequence of this endothelial-dependent adherence, this has not yet been shown for eosinophils. By means of human umbilical vein endothelial cells (HUVE) grown to confluence on microporous filters as an in vitro model of leukocytic migration across postcapillary venules, we have characterized the conditions leading to endothelium-driven transmigration of blood eosinophils from normals and from patients with allergic asthma. Freshly isolated eosinophils from nonallergic donors adhered to interleukin-1 (IL-1) and tumor necrosis factor-activated HUVE, but did not penetrate these monolayers. In contrast, eosinophils from allergic asthma patients showed an increased adherence and transmigration capacity. This increased functional competence was not caused by a difference in density phenotype, because the eosinophils from both groups showed a comparable density distribution over discontinuous Percoll gradients. Moreover, no difference existed within one group among eosinophils harvested from the Percoll density bands 1.080, 1.085, and 1.090 g/mL in terms of transendothelial migration. In vitro cultivation of freshly isolated eosinophils from nonallergic individuals in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 induced a stepwise decrease of the density distribution over such gradients. In contrast, eosinophils from patients with allergic asthma directly shifted to a final density of 1.075 g/mL within 24 hours of culture. Notwithstanding the kinetics of density changes, eosinophils from nonallergic donors already expressed the capacity to transmigrate IL-1-activated HUVE monolayers 20 hours after cultivation with different combinations of GM-CSF, IL-3, and IL-5. Inhibition studies with monoclonal antibodies showed that endothelium-driven transmigration of eosinophils predominantly implicates CD11/CD18 structures on the eosinophil surface, whereas no significant inhibition was found with the anti-VLA-4 monoclonal antibody HP2/1. From cytofluorometric studies, we conclude that spontaneous transmigration of eosinophils from allergic asthma patients is not accompanied by quantitative upregulation of these antigens. Taken together, these results allow the conclusion that blood eosinophils from allergic asthma patients have undergone in vivo priming, mimicked in vitro by cytokines such as GM-CSF, IL-3, and IL-5, leading to induction of the capacity to migrate across cytokine-activated HUVE monolayers.

Asthma↗

PubMed1991

Mature eosinophils stimulated to develop in human cord blood mononuclear cell cultures supplemented with recombinant human interleukin-5. Part I. Piecemeal degranulation of specific granules and distribution of Charcot-Leyden crystal protein.

Human cord blood mononuclear cells were cultured for 35 days in media containing recombinant human interleukin 5 (rhIL-5) supplemented with a fraction of the culture supernatant of phytohemagglutinin (PHA)-stimulated human T lymphocytes from which interleukin 2 (IL-2) was eliminated. Cultured cells were studied by electron microscopy and an immunogold procedure to detect subcellular site(s) of Charcot-Leyden crystal (CLC) protein. The majority of cells (greater than 70%) developing in this system were mature eosinophils, with descending frequency of other cells, including macrophages, mature basophils, eosinophilic myelocytes, and mature neutrophils. Mature eosinophils were characterized by increased numbers of primary granules, small granules, tubulovesicular structures, and decreased secondary granules. These eosinophils showed extensive piecemeal degranulation (PMD) characterized by partially empty and empty secondary granule chambers in the cytoplasm. Small, smooth vesicles were evident within empty granule chambers as well as adjacent to them. Eosinophils formed close associations with phagocytic macrophages that contained both standard-shaped and irregularly shaped CLC within phagolysosomes. Subcellular sites of CLC protein were demonstrated by immunogold in eosinophils and macrophages arising in these cultures. Charcot-Leyden crystal protein was present in the nuclear matrix and extraorganellar cytoplasm of eosinophils. Primary granules and some cytoplasmic vesicles were labeled for CLC protein, but full and empty secondary granules and the extensive network of tubulovesicles were not. Charcot-Leyden crystals were absent from eosinophils, nor were they present in the extracellular space. Charcot-Leyden crystals were absent from eosinophils, nor were they present in the extracellular space. Charcot-Leyden crystals within phagosomes of macrophages were labeled by the immunogold procedure for CLC protein. These results demonstrate that rhIL-5-supplemented, PHA-stimulated, T-cell-conditioned media induced the development of mature human eosinophils from cord blood cells. These eosinophils underwent PMD of secondary granule contents. Immunogold analysis showed eosinophil CLC protein in the cytoplasm, nucleus, and primary granules of eosinophils. Macrophages also were present in these cultures. They contained CLC protein-containing crystals in their phagosomes, suggesting active sequestration of eosinophil CLC protein by macrophages in vitro.

Cell Degranulation↗

PubMed1990

IgG-dependent generation of platelet-activating factor by normal and low density human eosinophils.

We have compared normal and low density human eosinophils for their ability to generate platelet activating factor (PAF) in response to IgG-dependent and nonimmunologic stimulation. After 45 min incubation with IgG-coated Sepharose beads the concentrations of cell-associated PAF recovered from normal density eosinophils were significantly greater than from low-density eosinophils or neutrophils. Moreover, eosinophils stimulated with calcium ionophore A23187 had a considerably greater capacity to generate PAF than had previously been described. Although the quantities of cell-associated PAF recovered from normal and low density eosinophils and neutrophils after A23187 stimulation were similar, the amounts of extracellular PAF recovered from both eosinophil populations were significantly greater than from neutrophils. The amounts of PAF recovered from the low density eosinophils may not reflect the full synthetic capacity of these cells, because PAF-turnover was found to be more rapid than that observed with normal density eosinophils. When exogenous [3H]PAF was added to the two stimulated eosinophil populations subsequent analysis of the [3H]PAF metabolites by DIOL-HPLC revealed that low density eosinophils incorporated PAF into the phosphatidylcholine (PC) pool more rapidly than did normal density eosinophils or neutrophils. Alkaline hydrolysis of the PC fraction from whole cell extracts followed by treatment with acetic anhydride resulted in all the PC-associated radioactivity being converted to [3H]PAF, confirming PAF incorporation to PC via this pathway. These findings suggest that the contribution of eosinophils to inflammatory processes through the generation of PAF may be greater than previously appreciated, and that Ig-mediated stimulation may be important in initiating generation of the mediator. Low density eosinophils, that are presumed to be similar to tissue eosinophils, may have a role in regulating PAF concentrations in tissues through their enhanced rate of metabolism.

Calcimycin↗

PubMed1994

Differences between human eosinophils and neutrophils in the function and expression of sialic acid-containing counterligands for E-selectin.

Both neutrophils and eosinophils have been shown to bind to the inducible endothelial cell adhesion molecule E-selectin. For neutrophils, one of the reported ligands for E-selectin is the sialylated Lewis X Ag (sLe(x)). To analyze the counterligands on eosinophils for E-selectin, adhesion assays were performed in which purified leukocytes were allowed to adhere to a soluble recombinant form of the molecule immobilized on plastic plates. Eosinophils, like neutrophils, bound to immobilized E-selectin, but significantly more neutrophils than eosinophils adhered in this assay. Consistent with the greater ability of neutrophils to bind E-selectin was the observation by flow cytometry that neutrophils expressed significant levels of sLe(x) and a sialylated dimeric form of the Le(x) Ag (sialyl-dimeric Le(x), or sialyl-stage-specific embryonic Ag-1, recognized by mAb FH6), whereas the expression of these epitopes on eosinophils was extremely low or undetectable. Expression was similar on eosinophils from allergic and nonallergic donors, and was not altered on eosinophils after induction of L-selectin shedding in vitro by treatment with platelet-activating factor. For both eosinophils and neutrophils, treatment with sialidase was associated with the complete elimination of sLe(x) and sialyl-dimeric Le(x) surface expression, and abolished leukocyte adhesion to E-selectin. Another glycosidase, endo-beta-galactosidase, which specifically cleaves the beta 1-4 galactose linkage to N-acetyl-glucosamine when it exists in an extended chain form such as that found in sialyl-dimeric Le(x), significantly inhibited eosinophil and neutrophil adhesion and expression of sialyl-dimeric Le(x). Such treatment also reduced sLe(x) expression on eosinophils, while having little effect on total neutrophil sLe(x) expression. For both eosinophils and neutrophils the sialylated ligand did not appear to be a glycoprotein because pretreatment of leukocytes with several proteases had no effect on adhesion to E-selectin or on expression of sLe(x) and sialyl-dimeric Le(x). These data suggest that eosinophils, like neutrophils, use sialylated, protease-resistant structures to bind to E-selectin, although the eosinophil expresses much lower levels of these structures on its surface. A major proportion of the sLe(x)-containing E-selectin ligand on the surface of eosinophils appears to be in the form of sialyl-dimeric Le(x), whereas this represents a minor proportion on the surface of neutrophils. Based on results using endo-beta-galactosidase, it appears that these cells may rely disproportionately upon the cell surface sialyl-dimeric Le(x) to bind to E-selectin.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗

PubMed1994

Human eosinophils in vitro. An ultrastructural morphology primer.

An ultrastructural morphological primer of human eosinophils is presented. Mature and immature eosinophils, obtained from peripheral blood and bone marrow, as well as activated tissue eosinophils are all used to illustrate the various morphologies assumed by eosinophils in vivo. The various ultrastructural changes expressed by this cell lineage in vivo reflect the impact of differentiation, maturation, activation, secretion, and cell injury on morphology. Nearly all of the changes described in vivo are also evident in eosinophils arising in in vitro systems. We review published studies of these culture systems, which have been supplemented with various conditioned media containing naturally occurring growth factor(s) that are permissive (or not permissive) for eosinophils or with the recombinant growth factors, IL-5 or IL-3. These studies were helpful in the recognition of eosinophil-promoting, -sustaining and -activating properties of human IL-3 and IL-5. Moreover, mature and immature eosinophils were shown to release a granule matrix protein--eosinophil peroxidase (EPO)--by its transport in small cytoplasmic vesicles, a process termed piecemeal degranulation (PMD), accounting for the gradual emptying of granule contents in the absence of granule fusions to the plasma membrane. Also presented are eosinophil morphologies that occur in vitro in suspension cultures of human cord blood supplemented with the c-kit ligand from various sources. The wide variety of eosinophil subcellular changes in the c-kit ligand-supplemented cultures, like the changes of which eosinophils are capable in vivo, reflects the processes of differentiation, maturation, activation, secretion and cell injury. Presentation of this ultrastructural morphological primer of human eosinophils in vitro should enable investigators to recognize eosinophils in all of their diverse morphologic forms in cultures that contain differentiating and functioning members of other lineages, also present in c-kit ligand-supplemented cultures. These lineages include mast cells, basophils, neutrophils, monocytes, macrophages, megakaryocytes, and endothelial cells.

Eosinophils↗

PubMed1993

Expression of a functional laminin receptor (alpha 6 beta 1, very late activation antigen-6) on human eosinophils.

The mechanisms responsible for the accumulation of eosinophils at sites of allergic and other inflammatory reactions are unknown, but recent studies have implicated both eosinophil and endothelial adhesion molecules in this process. However, less well studied have been the adhesive interactions between eosinophils and the subendothelial basement membrane and interstitial connective tissues. To test the hypothesis that eosinophils might interact with extracellular matrix proteins, we analyzed purified human eosinophils for the expression and function of various beta 1 integrins. Using indirect immunofluorescence and flow cytometry, purified eosinophils from mildly allergic donors were found to consistently express the integrin subunits beta 1 (CD29), alpha 4 (CD49d, very late activation antigen [VLA]-4 alpha), and alpha 6 (CD49f, VLA-6 alpha). No significant expression of the alpha 1, alpha 2, alpha 3, alpha 5, or beta 4 subunits was detected. Platelet contamination of the eosinophil preparations was excluded by light microscopy and by the inability to detect expression of platelet glycoproteins alpha v, CD41b, and CD42b. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of eosinophils confirmed the expression of cell-surface beta 1, alpha 4, and alpha 6. Furthermore, eosinophils purified from allergic donors were shown to adhere to plate-bound laminin, but not to type 1 or type 4 collagen. Adhesion to laminin was concentration-dependent, required divalent cations, and was completely and specifically inhibited by the anti-alpha 6 monoclonal antibody (MoAb) GoH3 and by the anti-beta 1 MoAb 33B6. Interestingly, the anti-beta 1 MoAb 18D3 (which like 33B6 blocks eosinophil binding to VCAM-1) did not inhibit eosinophil adhesion to laminin, suggesting that there are functionally distinct epitopes on the beta 1 subunit. Eosinophils purified from 4 healthy, nonallergic donors also showed alpha 6-dependent adhesion to laminin, although these cells adhered less well. These studies establish the expression of alpha 6 beta 1 on human eosinophils and document its function as a laminin receptor. Interaction of eosinophil alpha 6 beta 1 with laminin, eg, in basement membranes, may contribute to the localization of these cells at inflammatory sites in vivo.

Animals↗

PubMed1995

Blood eosinophil leukotriene C4 production in asthma of different severities.

In asthma, activation and recruitment of eosinophils to the bronchial mucosa amplifies many cellular functions. The blood eosinophil count and the number of hypodense eosinophils increase with asthma severity. Eosinophils produce numerous proinflammatory mediators in response to a variety of agonists, notably the peptido-leukotriene (LT) C4, a potent bronchoconstrictor. In this study, we have evaluated blood eosinophil LTC4 release and its modulation by cytokines in normal individuals and in subjects with asthma of various severities: mild (beta 2-agonist on demand); moderate (inhaled steroids on a regular basis); and severe (inhaled and oral steroids on a regular basis). Eosinophils were isolated using a modified Percoll gradient technique, which recovers both hypodense and normodense eosinophils in a global cell population. Eosinophils released detectable amounts of LTC4 only in the presence of the stimulus (calcium ionophore A23187, 2 microM). The ionophore-induced LTC4 release was greater in moderate asthmatics (mean +/- SEM 5.7 +/- 1.3 pg x 10(3)/250,000 eosinophils) than in normal individuals (1.6 +/- 0.4 pg x 10(3)/250,000 eosinophils), mild asthmatics (1.8 +/- 0.3 pg x 10(3)/250,000 eosinophils) and severe asthmatics (2.0 +/- 0.3 pg x 10(3)/250,000 eosinophils). Granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5) amplified the ionophore-induced LTC4 release in the four groups from 1.9 to 2.6 and 1.9 to 2.8 fold, respectively. Interleukin-3 (IL-3) did not increase LTC4 production except by the eosinophils of the severe asthmatics whose ionophore-induced LTC4 production was enhanced by 1.9 fold. These data demonstrate that the asthmatic bronchial inflammatory process may modify blood eosinophil LTC4 release and its modulation by cytokines according to asthma severity and treatment.

Adult↗

PubMed1996

Functional and structural characterization of the eosinophil P-selectin ligand.

Our recent studies have indicated an important role for P-selectin in eosinophil adhesion. We have therefore compared eosinophil and neutrophil binding with nasal polyp endothelium as well as purified P-selectin. We have also compared the structure and expression of the eosinophil and neutrophil P-selectin ligands. Using the frozen section assay, eosinophils bound to 2-fold more blood vessels within the nasal polyp tissue than neutrophils. Up to 10-fold more eosinophils than neutrophils bound per unit length of endothelium. Neutrophil and eosinophil binding was inhibited by a mAb against P-selectin and a P-selectin chimera which binds to the P-selectin ligand. Eosinophils bound with approximately 2-fold greater avidity to purified P-selectin under flow conditions. Using SDS-PAGE we characterized the eosinophil P-selectin ligand as a sialylated, homodimeric glycoprotein consistent with the known structure of PSGL-1. However, expression of PSGL-1 by eosinophils was significantly greater than on neutrophils. The eosinophil ligand had a calculated molecular mass by SDS-PAGE of approximately 10 kDa greater than the neutrophil ligand, which was not due to differences in N-glycosylation. Eosinophils expressed the 15-decapeptide repeat form of PSGL-1 compared with neutrophils that have the 16-decapeptide repeat form. The increased binding of eosinophils, compared with neutrophils, to P-selectin in both an ex-vivo and in vitro assay suggests that P-selectin may have a role directing the specific migration of eosinophils in diseases such as asthma. The increased avidity may be due to increased expression of PSGL-1 by eosinophils, differences in the peptide backbone, or post-translational modifications.

Antibodies, Monoclonal↗

PubMed1996

Surface and mRNA expression of the CD52 antigen by human eosinophils but not by neutrophils.

Eosinophilic and neutrophilic granulocytes represent major effector cells in the inflammatory response. Whereas neutrophils are predominantly involved in bacterial infections, eosinophils are of essential importance in the allergic inflammation. Surface markers have been used to distinguish neutrophils (CD16+) from eosinophils (CD16-) and might indicate different functional properties of these cells. In this study, expression and functional activity of CD52 on human eosinophils and neutrophils was investigated in nonatopic healthy donors and from patients with hypereosinophilia. Flow cytometric analysis using different anti-CD52 monoclonal antibodies (MoAbs) (mouse IgG3, humanized IgG1, and rat IgM) showed significant and homogeneous expression of CD52 on human eosinophils, but not on neutrophils. In addition, reverse transcription-polymerase chain reaction and Northern blot analysis showed that CD52 mRNA was constitutively expressed in eosinophils but not in neutrophils. Furthermore, expression of CD52 could be diminished in a dose-dependent manner by preincubation of eosinophils with phosphatidylinositol-specific phospholipase C, suggesting that CD52 on eosinophils is anchored to the membrane through a glycosylphosphatidylinositol (GPI) molecule. Whereas the phorbolester phorbol myristate acetate was able to downregulate the expression of CD52 on eosinophils in a dose-dependent manner, different eosinophil activating cytokines and chemotaxins had no effect. Cross-linking of CD52 by mouse anti-CD52 MoAb (IgG3) and humanized anti-CD52 MoAb (IgG1) with goat antimouse antibody and mouse antihuman antibody, respectively, dose-dependently resulted in an inhibition of reactive oxygen species production of eosinophils after stimulation with C5a, platelet-activating factor, and granulocyte-macrophage colony-stimulating factor. In summary, this study shows that the GPI-anchored antigen CD52 is not only a useful marker to distinguish eosinophils from neutrophils. The data point out a novel role of the CD52 antigen on human eosinophils that might be of clinical relevance, because cross-linking of this molecule will stop the destructive power of human eosinophils in the inflammatory tissue.

Antibodies, Monoclonal↗

PubMed1997

Eosinophil adhesion regulates RANTES production in nasal epithelial cells.

Among the many known chemotactic factors for eosinophils, the proinflammatory chemokine RANTES is particularly important, because it is potently and selectively chemotactic for eosinophils. Throughout the process of the migration of eosinophils from the blood vessels into the nasal cavity, eosinophil functions are assumed to be regulated by surface adhesion molecules. Conversely, the messages conferred by the eosinophils to the endothelial and epithelial cells are also of great interest. In the present study, we showed that eosinophil adhesion to human nasal epithelial cells (HNECs) inhibits RANTES production in HNECs. Eosinophils were isolated from peripheral blood obtained from patients with allergic rhinitis. Human mucosal microvascular endothelial cells and HNECs were isolated from human nasal mucosa specimens. After stimulation of the HNECs in the presence of eosinophils, the secretion of RANTES, induced by a combination of TNF-alpha and IFN-gamma, appeared to have decreased. The amount of the decrease was a function of the number of involved eosinophils. On the other hand, the presence of eosinophils did not affect RANTES production by the endothelial cells. After pretreatment of the eosinophils with anti-CD18 mAb or coculture with HNECs in Transwell culture inserts, these cells did not inhibit the TNF-alpha- and IFN-gamma-induced RANTES production. These results were virtually identical with those observed on RANTES mRNA expression. The adhesion of eosinophils to HNECs plays a key role in the inhibition of RANTES production. Our data indicate that a certain established system causes the signal transfer from eosinophils to HNECs to inhibit RANTES production, thus decreasing the eosinophil infiltration.

Adult↗

PubMed1996

[The roles of adhesion molecules, cytokines, and chemokines in eosinophil activation during allergic inflammation].

Patients with hypereosinophila have at least two subpopulation of eosinophils: "normodense" and "hypodense". Hypodense eosinophils can be distinguished by their increased expression of various membrane receptors including IL-5 receptors (J Exp Med 172: 1347) and by the expression of particular proteins (J Immunol 142: 4416). Recently, adhesion molecules have also been found to play an important role in the inflammatory processes in allergic disease. 1) Adhesion molecules were found to be strongly expressed on eosinophils from patients with asthma. 2) Platelet activating factor and induced the expression of adhesion molecules as did the supernatant of mononuclear cells from mite-allergic patients with asthma stimulated either with mite allergen or with a combination of recombinant IL-3, GM-CSF, and IL-5 (Immunol, Lett. 42: 25, '94, & 46: 241, '95). 3) Patients with bronchial asthma had a high level of soluble ICAM-1) (Lancet. 343: 1108, '94). Moreover, the presence of a large variety of membrane receptors and the identification of cytotoxic molecules (mainly granule basic proteins) indicate that eosinophils should be considered effector cells. Therefore the release of granule proteins in response to ICAM-1 and its ligands was studied. The concentrations of eosinophil cationic protein and eosinophil-derived neurotoxin in supernatants of eosinophils were significantly greater in the presence of recombinant soluble ICAM-1 than in its absence (p < 0.05). These results suggest that signals from ICAM-1 and its ligands induce eosinophil activation and are involved in degranulation of eosinophil granule proteins. In addition, reactive oxygen species generated by eosinophils have also been considered capable of causing airway injury at sites of inflammation. The effect of recombinant soluble ICAM-1 and its ligands on eosinophil-induced radical oxygen products was studied. Recombinant soluble ICAM-1 augmented eosinophil oxidative metabolism. Therefore, signaling via adhesion molecules might play an important role in the pathogenesis of allergic inflammation. Specifically, it may activate eosinophils and increase oxidative metabolism or cause degranulation of eosinophil granule proteins.

Cell Degranulation↗

PubMed1998

Immunoelectron microscopic evidence for release of eosinophil granule matrix protein onto microfilariae of Onchocerca volvulus in the skin after exposure to amocarzine.

The involvement of eosinophils in the host reaction to microfilariae (mf) of Onchocerca volvulus was studied by immunohistochemistry and immunoelectron microscopy. Skin biopsies were obtained from patients after transepidermal administration of the microfilaricide amocarzine. At 20-28 h after the application of amocarzine, mf were degenerated or dead and a marked eosinophil-parasite adherence (EPA) reaction was seen, with intense staining for intra- and extracellular eosinophil granule proteins such as eosinophil cationic protein (ECP) surrounding the mf. Immunoelectron microscopically the eosinophil granule matrix in intact and necrotic eosinophils was specifically labeled, whereas granules whose matrix had dissolved showed no specific gold particle binding. As specific labeling was seen on lowly electron-dense material adjacent to matrix-depleted granules, the material was regarded as released eosinophil granule matrix material. Intact and necrotic eosinophils, matrix-containing as well as matrix-depleted eosinophil granules, and released eosinophil granule matrix material were observed on the surface of damaged mf and between collagen fibers. The coincidence of mf degeneration, EPA reaction, and release of eosinophil granule matrix material on damaged mf and collagen fibers indicated a role of eosinophils and eosinophil granule matrix protein in the host reaction to mf after amocarzine application.

Adult↗

PubMed1989

Injurious effect of the eosinophil peroxide-hydrogen peroxide-halide system and major basic protein on human nasal epithelium in vitro.

Tissue injury is observed in allergic and nonallergic eosinophilic rhinitis, but the mechanism of this injury is unclear. Because eosinophils are prominent in biopsy specimens in these conditions, we hypothesized that they may participate in the injury process. Initially, we developed techniques to isolate and purify human nasal epithelial cells from turbinate biopsies to use as target cells for eosinophil granule products. Primary cultures from explants were characterized by electron microscopy and indirect immunofluorescence with a panel of primary monoclonal and polyclonal antibodies. These studies revealed the homogeneity of the cells and confirmed their epithelial nature. Cultured nasal epithelial cells were then exposed to either purified human eosinophil peroxidase, bromide, and glucose plus glucose oxidase, as a continuous source of hydrogen peroxide, or eosinophil major basic protein. Neither eosinophil peroxidase alone nor glucose plus glucose oxidase in the absence of eosinophil peroxidase were injurious, but the combined addition of eosinophil peroxidase, glucose/glucose oxidase, and bromide produced marked target cell lysis. This effect was time- and eosinophil peroxidase dose-dependent. Catalase and azide significantly inhibited the lysis of these cells, suggesting the eosinophil peroxidase-catalyzed products of halide oxidation mediated this form of injury. The addition of purified human eosinophil major basic protein also caused dose- and time-dependent lysis of the nasal epithelial cells but required longer incubation periods to effect injury. We hypothesize that the eosinophil peroxidase-hydrogen peroxide-halide system and major basic protein may injure the nasal epithelium in inflammatory conditions such as allergic and nonallergic eosinophilic rhinitis.

Blood Proteins↗

PubMed1997

Diesel exhaust particulates enhance eosinophil adhesion to nasal epithelial cells and cause degranulation.

Diesel exhaust particulates (DEP) are a common air pollutant from diesel-engine-powered car exhaust and are thought to cause chronic airway diseases. On the other hand, eosinophils are major components of allergic inflammatory disorders such as asthma, nasal allergy and atopic dermatitis. We examined the effects of DEP and DEP extract (extract of polyaromatic hydrocarbons) on eosinophil adhesion, survival rate and degranulation. Eosinophils, human mucosal microvascular endothelial cells (HMMECs) and human nasal epithelial cells (HNECs) were preincubated in the presence or absence of DEP and DEP extract. 35S-labeled eosinophils were allowed to adhere to monolayers of HMMECs and HNECs. After washing, 35S radioactivity was determined and numbers of adherent eosinophils were calculated using each standard curve. The effects of DEP and DEP extract on eosinophil survival rate and degranulation were also determined. Although neither DEP nor DEP extract affected the adhesiveness of HMMECs and HNECs to eosinophils, 5 ng/ml of DEP extract and 50 ng/ml of DEP extract each significancy increased eosinophil adhesiveness to HNECs (134+/-9 and 143+/-8%, respectively; p<0.01 vs. control), but neither effected eosinophil adhesiveness to HMMECs. DEP extract also induced eosinophil degranulation without changing the eosinophil survival rate. Given that eosinophil-derived lipid mediators and toxic proteins play important roles in the development of nasal allergy, the above findings strongly suggest that DEP plays an important role in promoting the nasal hypersensitivity induced by enhanced eosinophil infiltration of epithelium and eosinophil degranulation.

Adult↗

PubMed1985

Electron-microscopic and immunohistochemical studies on endomyocardial biopsies from a patient with eosinophilic endomyocardial disease.

Light- and electron-microscopic studies and immunohistochemical procedures were carried out on blood eosinophils and left ventricular endomyocardial biopsies from a 68-year-old man with an eosinophilia of 8.2 X 10(9)/l and congestive cardiac failure due to eosinophilic endomyocardial disease. Some blood eosinophils were vacuolated and degranulated, and reversal of the normal staining pattern of eosinophil granules was seen by means of electron microscopy. The biopsies showed degenerative changes in the cardiac myocytes, with interstitial fibrosis and infiltration by numerous eosinophils, mast cells, and macrophages. Eosinophils infiltrating the myocardium showed a decrease in the number of granules, many of which were indistinct or contained dissolving crystalloids, which occasionally were seen to be discharged onto the surface of adjacent cardiac myocytes. Immunohistochemical studies of the endomyocardial biopsies with a monoclonal antibody, which is specific for activated eosinophils and binds to the secreted forms of eosinophil cationic protein (ECP) and eosinophil protein-X (EP-X), demonstrated that the lesions contained numerous activated eosinophils and secreted ECP and EP-X. These findings support the concept that in eosinophilic endomyocardial disease, activated eosinophils infiltrate and degranulate in the myocardium, releasing eosinophil cationic proteins which then damage adjacent myocardial cells.

Aged↗

PubMed1998

Nerve growth factor is preformed in and activates human peripheral blood eosinophils.

BACKGROUND: Recent studies have shown that nerve growth factor (NGF) is produced by and can act on several immune-inflammatory cells. OBJECTIVES: The objective of this study was to study the effects of NGF on human peripheral blood eosinophils and assess whether these cells produce and store NGF. METHODS: Eosinophils were purified by negative immunoselection (magnetic cell sorting systems, purity 98% to 100%) from 13 subjects (9 to 26 years old) with mild blood eosinophilia, mainly of allergic origin. Eosinophils were incubated with NGF (50 to 1000 ng/mL), and supernatants were collected for measurement of eosinophil peroxidase (EPO, 20 minutes, colorimetric enzymatic assay) and IL-6 (12 hours, ELISA). Eosinophil viability was evaluated by Trypan blue test (days 2, 3, and 4). NGF content in freshly isolated eosinophils, after ultrasound disruption, was determined with a 2-site immunoenzymatic assay. The presence of mRNA for NGF was evaluated by reverse transcription PCR. RESULTS: NGF caused EPO release (highly significant at 1000 ng/mL NGF). IL-6 release from eosinophils was not higher than IL-6 spontaneously released into culture medium alone. NGF did not significantly affect the number of viable eosinophils. NGF was found in the eosinophil sonicates (1.5 to 17.8 pg/mL per 106 cells). Similarly, mRNA for NGF was detected by reverse transcription PCR in the freshly isolated eosinophils. CONCLUSIONS: NGF activates human peripheral blood eosinophils from subjects with mild eosinophilia to selectively release inflammatory mediators. Eosinophils store and produce NGF. Therefore the capability of NGF to induce a secretory response and its production and storage by circulating human eosinophils suggest a possible role for NGF in conditions associated with eosinophilia, including allergic disease.

Adolescent↗

PubMedDate not supplied

Airway hyperresponsiveness: first eosinophils and then neuropeptides.

Airway hyperreactivity to bronchoconstrictor mediators is a main characteristic in the majority of asthmatic patients and correlates well with the severity of the disease. The airways of asthmatic patients are characterized by an inflammatory state resulting in activation of lung tissue cells and attraction and infiltration of leukocytes from the blood. The accumulation of eosinophilic leukocytes is a prominent feature of inflammatory reactions that occurs in allergic asthma. The increase in number of eosinophils is important since it correlates in time with an increase in bronchial hyperresponsiveness. Viral respiratory infections can also induce eosinophilia and airway hyperresponsiveness in humans and animals and can worsen asthmatic reactions. This report reviews current opinions on the relationship between inflammation-induced eosinophil accumulation/activation and the development of airway hyperresponsiveness and the possible role for sensory neuropeptides in this process. Firstly, CC chemokines play an important role in allergic airway inflammation and respiratory viral infections leading to eosinophil recruitment. Secondly, it can be concluded that IL5 is involved in the development in airway hyperresponsiveness. IL5 has profound effects on eosinophils as promoter of growth, differentiation and proliferation, chemoattractant, activator and primer. However, it is conceivable that in animal models for allergic asthma besides IL5 other regulatory mediators may be involved in eosinophil migration and activation in the lung, which in turn will lead to airway hyperresponsiveness. Recent data support the possible role of eotaxin and its eosinophil-specific receptor CCR-3 in eosinophil chemotaxis and activation in allergic asthma. Moreover, it is suggested that the development of airway eosinophilia in vivo involves a two-step mechanism, elicited by eotaxin and IL5. The precise mechanism by which eosinophils induce bronchial hyperresponsiveness is at present unknown. Sensory neuropeptides could be important mediators in this process, since it has been demonstrated that airway nerves are surrounded by and infiltrated with eosinophils after antigen challenge. Sensory neuropeptides could be the final, more downstream, common pathway after eosinophil infiltration and activation in inducing airway hyperresponsiveness due to allergen inhalation or respiratory viral infections. In conclusion, in the process of the development of airway hyperresponsiveness observed during viral infections or in allergic asthma, the IL5/eotaxin-induced infiltration and activation of eosinophils in the airways is evident. Following this step, eosinophil-derived inflammatory mediators will induce the release of sensory neuropeptides (possibly NK2-receptor activating tachykinins) which in turn will lead to airway hyperresponsiveness.

Animals↗

PubMed1997

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

In order to determine whether or not IFN-gammaR is associated with regulatory mechanisms on human eosinophil function, we examined the expression of functional IFN-gammaR 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-gammaR alpha-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 alpha-chain of IFN-gammaR in all purified eosinophils of six individuals. Further, to characterize IFN-gammaR on eosinophils, we did binding experiments with 125I-IFN-gamma on purified peripheral eosinophils. The linear Scatchard plot indicated a single type of high-affinity binding sites (dissociation constant (Kd) = 3.89-4.95 x 10(-10) M, numbers of binding sites = 183-233/cell, n = 3). To determine whether IFN-gammaR on eosinophils is functional, we examined surface eosinophilic cationic protein (ECP) and CD69 induction after IFN-gammaR ligation with recombinant human IFN-gamma (rhIFN-gamma) on eosinophils by flow cytometry. rhIFN-gamma 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-gamma stimulation were significantly blocked by both a neutralizing anti-IFN-gamma MoAb and a blocking anti-IFN-gammaR MoAb. These results suggest that human peripheral eosinophils express functional IFN-gammaR.

Adolescent↗