Hypodense Eosinophils Research Articles

Interleukin-5, interleukin-3 and granulocyte-macrophage colony-stimulating factor prime actin-polymerization in human eosinophils: A study with hypodense and normodense eosinophils from patients with atopic dermatitis

Characteristic features of atopic diseases (AD) are immigration and local activation of eosinophils. Reorganization of the cytoskeleton modulates the function of leukocytes and is a prerequisite for the motility response. In this work, the regulation of actin polymerization has been investigated by flow cytometry using NBD-phallacidin and right angle light scatter measurements in purified eosinophils isolated from patients with atopic dermatitis and normal individuals. Stimulation of eosinophils with chemotaxins such as complement fragment C5a (C5a), CC chemokine RANTES/ CCL5 and platelet activating factor (PAF) induced a reversible polymerization of actin. Normodense eosinophils purified from patients with AD showed a decreased chemotaxin-induced actin response as compared to normodense eosinophils from healthy subjects and hypodense eosinophils from patients. Stimulation of eosinophils with Th2-cytokines such as interleukin-3 (IL-3), interleukin-5 (IL-5), granulocyte-macrophage colony-stimulating factor (GM-CSF) did not exert a significant effect on actin polymerization. However, pretreatment with IL-3, IL-5 or GM-CSF potentiated the chemotaxin-induced actin polymerization and graded the differential responsiveness between normodense and hypodense eosinophils. We demonstrate a different actin responsiveness in eosinophils from atopic patients and healthy subjects which could be overcome by modulating effects of Th2-cytokines.

Murine hypodense eosinophils induce tumour cell apoptosis by a granzyme B-dependent mechanism.

Eosinophils have been shown to potentiate anti-tumour cytotoxicity in both clinical and animal studies. The mechanism by which eosinophils induce tumour cell damage, however, has largely been speculative. The purpose of this study was to identify the mechanisms involved in eosinophil-induced tumour cell cytotoxicity. To investigate eosinophil cytotoxicity, eosinophils were isolated from the peritoneal cavity of Mesocestoides corti-infected BALB/c mice, and were separated into normodense (ND) and hypodense (HD) populations using discontinuous Percoll density gradient centrifugation. The tumoricidal activity of ND and HD eosinophils was assessed using the [51Cr]-release cytotoxicity assay (a measure of cytolytic activity) and the JAM assay (a measure of apoptotic activity). Investigation of apoptosis-inducing molecules in HD eosinophils was undertaken by RT-PCR. The calcium chelator EGTA, serine protease inhibitor aprotinin and a competitive substrate for granzyme B were used to assess the role of perforin and granzyme B in HD eosinophil killing. Cytotoxic activity induced by HD eosinophils was significantly greater than that of ND eosinophils, and apoptosis was the principal killing mechanism. RT-PCR analysis revealed that HD eosinophils express mRNA for perforin, granzyme B and Fas ligand. Furthermore, HD eosinophil killing was markedly inhibited by EGTA, intracellular aprotinin and the granzyme B competitive substrate. These data are consistent with a hypothesis that murine HD eosinophils elicit tumoricidal activity via a granzyme B-dependent mechanism.

Distinct amplification of the C5a-receptor pathways in normodense and hypodense eosinophils of patients with atopic dermatitis.

In patients with atopic dermatitis two different types of blood eosinophils with distinct density can be isolated. The normodense cells represent the huge majority in count, whereas the hypodense eosinophils are characterized by higher effector activity. To understand the altered functional responsiveness of these two cell subtypes, the expression of C5a receptors as well as C5a-induced signal pathways and the production of reactive oxygen metabolites have been analyzed. Chemiluminescence measurements revealed significant higher production of reactive oxygen metabolites in hypodense eosinophils in comparison to normodense cells. However, no difference in the expression level of C5a receptors as well as in the C5a-induced Ca2+-transients between normodense and hypodense eosinophils were found. In contrast, hypodense eosinophils showed a significantly higher actin polymerization response and phosphatidylinositol 4,5 bisphosphate 3-kinase activation after stimulation with C5a than normodense eosinophils. Therefore, normodense and hypodense eosinophils from the blood of patients with atopic dermatitis are characterized by differential amplification of C5a-receptor signal pathways, which might explain the differences in their proinflammatory activity.

CD44 and CD69 represent different types of cell-surface activation markers for human eosinophils.

Eosinophils (EOS) purified from peripheral blood or late-phase bronchoalveolar lavage (BAL) were analyzed with 473 monoclonal antibodies (mAbs) from the Fifth International Workshop on Human Leukocyte Antigens in an attempt to identify markers of EOS activation. Two strategies were used: (1) to look for surface markers absent on fresh EOS but present after in vivo activation (e. g., in late-phase BAL fluid [BALF]) or after in vitro culture for up to 72 h with cytokines (<= 10 ng/ml of interleukin-3 [IL-3], IL-5, or granulocyte-macrophage colony-stimulating factor [GM-CSF]); and (2) to look for markers constitutively expressed on fresh EOS that were increased after activation in vivo or after culture in vitro. With indirect immunofluorescence and flow cytometry, the first approach revealed that among approximately 350 mAbs tested, only those recognizing CD69 became bound to late-phase BALF EOS or cytokine-cultured EOS, but not to fresh EOS. Using the second approach, we observed statistically significant concentration- and time-dependent increases in CD44 expression in EOS cultured with IL-3, IL-5, or GM-CSF (approximately 2-fold increase in fluorescence intensity, P < 0.05), but not with interferon-gamma (IFN-gamma) (up to 100 ng/ml), whereas levels of 15 other constitutively expressed markers were unchanged. Despite increased expression, neither fresh nor cytokine-cultured EOS adhered to immobilized hyaluronate, a ligand for CD44. Additionally, simultaneous comparison of hypodense (specific gravity < 1.085 g/liter) and normodense (specific gravity > 1.085 g/liter) EOS from allergic donors consistently revealed higher levels of CD44 expression (approximately 3- to 8-fold) but not CD69 expression on hypodense EOS. We conclude that CD69 and CD44 represent different types of activation markers for human EOS. These findings may be useful in assessing the state of EOS activation in vitro and in vivo.

Hypodense eosinophils in blood of pediatric patients with asthma

Determine the number of circulating hypodense eosinophils in the blood of atopic asthmatics. The hypothesis was that hypodense eosinophils, the activated cells, were higher in asthmatics than in non-atopic children. Granulocytes were separated from mononuclear cells by Ficoll-hypaque. Gradients of density were generated through dilutions of Percoll for fractionations of cells. Asthmatics had blood eosinophilia (768 +/- 75/mcl) when compared to normal control (200 +/- 28/mcl), and higher mean hypodense eosinophils (39%) as compared to normal children (11.7%). Eosinophils accumulate in asthmatic, especially the low density activated variant, suggesting its active role in the allergic inflammatory process.

Differentiation of eosinophils from cord blood cell precursors: kinetics of Fc epsilon RI and Fc epsilon RII expression.

Expression of Fc epsilonRI and Fc epsilonRII/CD23 was examined by immunocytochemistry and flow cytometry on eosinophils differentiated from human cord blood cells in the presence of human interleukin-3 (rhIL-3), granulocyte/macrophage colony stimulating factor (rhGM-CSF) and interleukin-5 (rhIL-5) and on blood eosinophils purified from normal donors or patients with idiopathic hypereosinophilic syndrome (HES). On cord blood derived eosinophils, Fc epsilonRI expression started at 1 week of culture and increased to reach a plateau at 3 weeks of culture. Fc epsilonRII/CD23 appeared slightly later, after 2 weeks of culture, and the percentage of Fc epsilonRII/CD23-positive eosinophilic cells increased and stayed in plateau. Fc epsilonRI expression on cord blood derived eosinophils was downregulated after culture with interleukin-2 (rhIL-2), interleukin-4 (rhIL-4), rhIL-5, interferon-alpha (rhIFN-alpha), interferon-gamma (rhIFN-gamma). In contrast, the expression of Fc epsilonRII/CD23 on cord blood derived eosinophilic cells was upregulated after culture with rhIL-4, rhIL-5 and rhIFN-gamma, and downregulated with rhIL-2 and rhIFN-alpha. Fc epsilonRI was expressed on about 30% normal donor eosinophils as well as on normodense eosinophils from HES patients but significantly decreased on hypodense eosinophils. In contrast, Fc epsilonRII/CD23, expressed on a very small proportion of normal donor eosinophils, increased from normodense to hypodense eosinophils. These results suggest that Fc epsilonRI on eosinophils might represent one differentiation antigen expressed relatively early, with decreased expression through maturation or activation, whereas Fc epsilonRII/CD23 might rather be considered as a marker of eosinophil activation.

Oxygen radical production by blood eosinophils is reduced during birch pollen season in allergic patients

The eosinophil granulocyte takes part in allergic inflammatory diseases, like asthma and rhinitis. The aim of this study was to investigate the oxidative metabolism of the blood eosinophils and neutrophils from birch pollen allergic patients, during and after exposure to their allergen. Thirteen birch pollen allergic patients, with seasonal symptoms of rhinitis, with or without asthma, were followed. The cells were purified using a percoll gradient and the MACS system. The eosinophil purity in all samples was > 95%. The oxidative metabolism of both the PMN and the eosinophils was measured by means of a chemiluminescence (CL) assay, with luminol or lucigenin as the amplifier, and using PMA (16 nM) as the activator. In relative terms, the lucigenin CL by the eosinophils was 5-10-fold higher than that of the PMN, P = 0.002. The eosinophils of the birch pollen allergic patients produced less oxygen radicals during the season, compared to the reference group, measured both with luminol CL and lucigenin CL, P = 0.01 and P = 0.02 respectively. Out of season, there was no difference. There was also no difference, during either period, between patients and references in PMN CL. Separating the eosinophils into hypodense and normodense fractions, showed a decreased oxidative metabolism by the hydrodense eosinophils. We conclude that the eosinophils of birch pollen allergic patients have reduced production of oxygen radicals when the patients are exposed to their allergen, which could depend on higher amounts of hypodense eosinophils in the blood during season.

MOLECULAR BIOLOGY OF CYTOKINES IN ALLERGIC DISEASES AND ASTHMA

Cytokines that are important in the pathophysiology of allergic disorders are summarized in Table 4. The IgE isotype switch results from the activities of IL-4 and IL-13 and is inhibited by IFN-? and TGF13. IL-2, IL-5, and IL-6 synergize with IL-4 and IL-13 to enhance IgE secretion. IL-4 is responsible for the differentiation of IL-4-producing lymphocytes, whereas IL-12 inhibits the differentiation of IL-4producing T cells. IL-5 is the most important eosinophilopoietin that together with GM-CSF and IL-3 prolongs the survival of and activates mature eosinophils. These three cytokines are responsible for the generation of the hypodense eosinophils that characterize the asthmatic state. Eosinophilia may also result from selective recruitment by the eosinophil chemotaxins RANTES, MIP-1a, and eotaxin. Mast cell proliferation results from the activities of IL-3, IL-9, IL-10, nerve growth factor, and stem cell factor. Finally, several cytokines contribute to the inflammatory state of allergic inflammatory disorders. IL-1, TNF, and IFN- ? increase expression of endothelial cell adhesion molecules and support the egress of mononuclear cells, neutrophils, and eosinophils into the lungs. Induction of VCAM-1 by IL-4 may promote the selective recruitment of eosinophils, basophils, and lymphocytes. Many cytokines may then contribute to the activation of these leukocytes once they have reached the airway.

Phenotypic analysis of airway eosinophils and lymphocytes in a Th-2-driven murine model of pulmonary inflammation.

In order to investigate whether the pulmonary response to helminth antigens mimics that seen in allergic inflammation of the airways, we have examined the phenotypic characteristics of lymphocytes and eosinophils recruited to the airways following Nippostrongylus brasiliensis (N.b.) infection. Specifically, the cellular response was divided into an early and a late phase. During the early response there was a small but significant increase in neutrophil numbers recovered by bronchoalveolar lavage (BAL). Phenotypic analysis of BAL leukocytes revealed an early rise in the percentage of BAL lymphocytes expressing the naive T cell markers CD45RB and L-selectin, and the activation marker IL-2R. In addition, during the early response, there was an increased percentage of lymphocytes expressing the gamma delta TCR, but not the alpha beta TCR. In contrast, the late response was marked by a much larger accumulation, in the lungs and BAL, of memory CD4+ T lymphocytes and an influx of small, hypodense eosinophils which produced LTB4 and LTC4 on stimulation with calcium ionophore. At this time there was a substantial increase in the number of T lymphocytes and eosinophils expressing ICAM-1 and the integrins VLA-4 and LFA-1, implicating these adhesion molecules in inflammatory cell recruitment to the airways. We conclude that the pattern and phenotypic characteristics of the cellular recruitment seen following N.b. infection resemble those seen in early- and late-phase allergic inflammation of the airways in asthma, and therefore N.b. may be used to model these aspects of the disease.

Detection of C5a receptors on human eosinophils and inhibition of eosinophil effector functions by anti-C5a receptor (CD88) antibodies.

Eosinophils and complement activation are reported to play a crucial role in the pathogenesis of connective tissue diseases. Depositions of antigens and antigen-antibody complexes lead to complement activation with the generation of anaphylatoxins, particularly C5a, which is thought to be responsible for the infiltration and activation of eosinophils in the tissue. Previous studies suggested that the eosinophil C5a receptor differs structurally from the receptor expressed on neutrophils. In this study, we investigated the expression and functional properties of C5a receptors on human eosinophils using the C5a receptor monoclonal antibody S5/1 (anti-CD88 mAb). Flow cytometric analysis demonstrated that the anti-CD88 mAb bound homogeneously on the surface of human eosinophils from nonatopic healthy donors. In addition, no subpopulations with respect to C5a receptor expression were identified in normodense or hypodense eosinophils of patients with hypereosinophilia. Pre-incubation of eosinophils with anti-CD88 specifically inhibited C5a-induced intracellular calcium concentration transients. C5a-induced chemotactic activity of eosinophils was significantly inhibited after pre-incubation of cells with anti-CD88 mAb in a dose-dependent manner. Furthermore, anti-CD88 mAb inhibited dose-dependently the release of reactive oxygen species by eosinophils following stimulation with C5a. Thus, the human eosinophil C5a receptor is homogeneously expressed on normal eosinophils from healthy donors as well as on hypodense and normodense eosinophil subpopulations from patients with hypereosinophilia. Based on the inhibitory effect of the S5/1 mAb on C5a-stimulated eosinophil effector functions, we conclude that a single C5a receptor type exists on human eosinophils. In addition, the inhibitory effect of the S5/1 mAb on C5a functions may enable a new experimental approach to the treatment of diseases that have been associated with C5a-mediated activation.

Serum levels of eosinophil cationic protein reflect the state of in vitro degranulation of blood hypodense eosinophils in atopic dermatitis.

In patients with atopic dermatitis (AD), serum levels of eosinophil cationic protein (ECP) have been shown to be a good reflector of disease severity. To elucidate what serum levels of ECP actually reflect, ECP levels in serum and plasma and cytological aspects of blood eosinophils were examined in AD patients (n = 27) and compared to healthy subjects (n = 12). Significantly elevated levels of serum ECP were noted in AD patients, while plasma ECP were uniformly recorded at nadir levels in both AD patients and normal subjects. In addition to blood eosinophilia, AD patients had significantly increased numbers of hypodense eosinophils (HEo) with morphological characteristics consistent with an activated state. Serum ECP levels strongly correlated with HEo numbers rather than with total eosinophil counts. These results indicate that elevated levels of serum ECP may be a consequence of in vitro degranulation of "activated" HEo, not of ECP supplementation from lesional skin. In addition, the dynamic correlations of eosinophil-associated parameters (total eosinophil counts, HEo numbers, and serum ECP levels) with AD severity suggest that inflammatory events in lesional skin may be involved in causing not only eosinophilopoiesis in bone marrow, but also development of HEo in the periphery, whose degree in turn may be mirrored in the levels of serum ECP in vitro.

Update on cytokines

Summary Cytokines that are important in the pathophysiology of allergic disorders are summarized on Table V. The IgE isotype switch results from the activities of IL-4 and IL-13 and is inhibited by IFN-ggg and TGF-β. IL-2, IL-5, and IL-6 synergize with IL-4 and IL-13 to enhance IgE secretion. IL-4 is responsible for the differentiation of IL-4-producing lymphocytes, whereas IL-12 inhibits the differentiation of IL-4-producing T cells. IL-5 is the most important eosinophilopoietin, and together with GM-CSF and IL-3, prolongs the survival of and activates mature eosinophils. These three cytokines are responsible for the generation of the hypodense eosinophils which characterize the asthmatic state. Eosinophilia may also result from selective recruitment by the eosinophil chemotactic factors RANTES, MIP-1α, and eotaxin. Mast cell proliferation results from the activities of IL-3, IL-9, IL-10, nerve growth factor, and stem cell factor. Finally, several cytokines contribute to the inflammatory state of allergic disorders. IL-1, TNF, and IFN-γ increase expression of endothelial cell adhesion molecules and support the egress of mononuclear cells, neutrophils, and eosinophils into the lungs. Induction of vascular cell adhesion molecule-1 by IL-4 may promote the selective recruitment of eosinophils, basophils, and lymphocytes. Many cytokines may then contribute to the activation of these leukocytes once they have reached the airways.

Hypereosinophilic syndrome with generalized myasthenia gravis

A patient with hypereosinophilic syndrome simultaneously had generalized myasthenia gravis. Ultrastructural and functional studies demonstrated an increase of the activated hypodense eosinophils. The eosinophilic cationic protein level in plasma and whole blood paralleled the severity of congestive heart failure. A high level of interleukin-5 was detected in both serum and in conditioned medium of T cells by interleukin-2 stimulation before therapy. The findings indicate that both diseases may be associated with a common T-cell abnormality. (J P EDIATR 1996; 128:369-72)

Cell surface antigen expression by peripheral blood monocytes in allergic asthma: results of 2.5 years therapy with inhaled beclomethasone dipropionate

At present, inhaled glucocorticoids are widely accepted as the therapy of choice in chronic asthma. Treatment with inhaled glucocorticoids significantly suppresses local airway inflammation in asthmatics, but may also have systemic effects, e.g. a reduction of the number of circulating hypodense eosinophils or a down-modulation of HLA-DR antigen (Ag) expression by T lymphocytes in peripheral blood. However, the effect of long-term therapy with inhaled glucocorticoids on peripheral blood monocytes (PBM), which are the precursors of the most numerous cell type in the lung, the alveolar macrophage, have not yet been evaluated. We therefore investigated the expression of various cell surface Ag on PBM from non-smoking patients with allergic asthma who were treated for 2.5 years with a β2-receptor agonist plus either an inhaled glucocorticoid (beclomethasone dipropionate, BDP) (n = 4) or an anticholinergic or placebo (n = 8). We compared the results with healthy volunteers (n = 7). Long-term treatment of allergic asthmatics with inhaled BDP, but not anticholinergic or placebo therapy, was associated with a significantly lower CDllb Ag expression (p < 0.04) and higher expression of CD13, CD14 and CD18 Ag (p < 0.05, p < 0.02 and p < 0.04, respectively) when compared with the healthy control subjects (n = 7). Most interestingly, PBM of asthmatics treated with inhaled BDP expressed an almost two-fold higher level of CD14 Ag on their cell surface than PBM of patients treated with anticholinergic or placebo (p < 0.03). No significant differences in the expression of CD16, CD23, CD25, CD32 and CD64 Ag or HLA-DR were observed between PBM from the different patient groups or healthy controls. Taken together, this study shows that long-term local therapy with inhaled BDP coincides with an altered expression of at least one cell surface Ag on PBM from allergic asthmatics.

Increased hypodense eosinophils after activation with PAF-acether and calcium ionophore in asthmatic subjects.

Eosinophils play a major role in the pathogenesis of bronchial asthma. In this study, we examined the density characteristics of blood eosinophils from 9 normal healthy individuals and 9 allergic asthmatic patients. Furthermore, the effect of platelet-activating factor, a potent mediator of inflammation, and calcium ionophore, A23187, on the density of normodense eosinophils (density > 1.085 g/ml) has also been examined. Initially, asthmatic patients had 27.0 +/- 1.1% eosinophils of lighter density (density < or = 1.081 g/ml), significantly greater than that in the normal individuals (7.5 +/- 0.5%). After exposure to platelet-activating factor (1 microM) or calcium ionophore (A23187, 1 microgram/ml), the normodense eosinophils switched to hypodense in both groups: 16.7 +/- 2.1% and 54.2 +/- 3.7%, respectively, in normal individuals, and 30.6 +/- 5.7% and 77.4 +/- 2.3%, respectively, in asthmatic patients. These data demonstrated that a certain percentage of normodense eosinophils from asthmatics and normal subjects switched to hypodense after activation with platelet-activating factor or calcium ionophore. Furthermore, eosinophils from asthmatics switched to a greater degree than in normal subjects, suggesting that the normodense eosinophils in asthmatics become primed probably by endogenously released mediators.

Purification of human blood eosinophils by a combination method using anti-CD16 monoclonal antibody, immunobeads, and Nycodenz density gradient

A simple method is described for the procurement of human blood eosinophil phenotypes by combining an anti-CD16 monoclonal antibody, immunobeads, and a non-toxic and non-ionic density gradient medium, Nycodenz. The purification depends on the removal of mononuclear cells using a 1.076/1.102 g/ml Nycodenz density gradient, partial removal of neutrophils based on different binding to plastic dishes, interaction of residual neutrophils with immunobeads via an anti-CD16 monoclonal antibody and, finally, extraction of eosinophil phenotypes by sifting the immunobeads-loaded neutrophils through an 1.080/1.102 g/ml Nycodenz density gradient. This method permits simultaneous preparation of highly purified normodense (>1.080 g/ml) and hypodense eosinophils (<1.080 g/ml) with reasonable chemiluminescence responses to opsonized zymosans and helminthotoxic activity to opsonized schistosomula corresponding to their own immunocytological properties.

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.

Potential role of interleukin-1 in allergen-induced late asthmatic reactions in guinea pigs: Suppressive effect of interleukin-1 receptor antagonist on late asthmatic reaction

Interleukin (IL)-1 is a pluripotential proinflammatory cytokine and is thought to be involved in the pathogenesis of bronchial asthma and late asthmatic reactions (LARs). To determine whether IL-1 plays a role in LAR, guinea pigs sensitized with Ascaris antigen were used. We evaluated IL-1 production by immunostaining with anti-IL-1β antibody and elucidated the action of IL-1 in LAR with recombinant IL-1 receptor antagonist. Immunostaining revealed that IL-1β–like immunoreactivity-positive cells increased in the airway walls and in bronchoalveolar lavage fluid after the antigen challenge. IL-1 receptor antagonist protein pretreatment reduced the generation of LAR in terms of pulmonary resistance. IL-1 receptor antagonist protein pretreatment did not change cellular components but reduced the percentage of hypodense eosinophils in bronchoalveolar lavage fluid. We also studied the direct effect of recombinant human IL-1β on pulmonary resistance and eosinophil activity measured as released eosinophil peroxidase activity. Recombinant human IL-1β did not change pulmonary resistance but primed eosinophils to release eosinophil peroxidase activity in response to platelet activating factor. Therefore these results suggest that IL-1 was produced in sensitized pulmonary tissue of guinea pigs by allergen exposure and played a role in the generation of LAR, at least partially by modulating the activation of eosinophils. (J A LLERGY C LIN I MMUNOL 1995;95:1236-45.)

Mechanisms of human eosinophil activation by complement protein C5a and platelet-activating factor: similar functional responses are accompanied by different morphologic alterations.

The complement system is an important amplification system for the propagation of allergic as well as pseudoallergic inflammatory reactions. In the present study, the effect of the major anaphylatoxin C5a was compared with that of platelet-activating factor (PAF) on highly purified eosinophils (> or = 95%) by functional as well as morphologic criteria. Upon stimulation with C5a, eosinophils maintained their spheric structure, developing short, pseudopodia-like protrusions, whereas PAF induced the generation of a number of digitating protrusions. As shown by functional and ultrastructural assay systems, both stimuli provoked significant extracellular and intracellular H2O2 production in eosinophils, which was inhibited by cytochalasin B. With C5a, a pronounced H2O2 production was detected within the small cytoplasmic vesicles, whereas PAF-induced H2O2 production was observed on the outer surface of the plasma membrane in the contact zones between adjacent cells. Morphologic signs of degranulation induced by C5a and PAF were accompanied by the significantly increased release of eosinophil cationic protein and eosinophil peroxidase in the presence of cytochalasin B. Like PAF, C5a induced a significant production of reactive oxygen species in eosinophils, as measured by lucigenin-dependent chemiluminescence (CL) responses in eosinophils. Maximal responses, comparable with those of interleukin-5 (100 U/ml), were observed at concentrations of 10(-5)-10(-6) and 10(-7)-10(-8) M for PAF and C5a, respectively. Separation of eosinophils by discontinuous density gradients revealed the existence of two hypodense eosinophil populations, one of them showing significantly reduced CL responses upon stimulation with C5a and PAF. In addition, CL responses upon stimulation with C5a and PAF were abrogated by cytochalasin B, staurosporine, and wortmannin, and were almost completely blocked by pertussis toxin. In conclusion, these data indicate that C5a induces events in human eosinophils comparable to those induced by PAF in the assay systems tested. Thus, C5a, generated after activation of the complement system, may be of major importance for the eosinophil activation observed in eosinophil-related disease.

The skin as a model to study the pathogenesis of IgE-mediated acute and late-phase responses

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