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PubMed1998

Comparison between eosinophilic markers in induced sputum and blood in asthmatic patients.

BACKGROUND: The usefulness and safety of the analysis of blood inflammatory markers in asthma are widely recognized. Recently, the analysis of induced sputum has been proposed as a safe, non-invasive tool in the study of airway inflammation in asthma. OBJECTIVE: Our aim was to test whether sputum analysis is more useful than blood analysis in the evaluation of airway inflammation in untreated and treated asthmatic patients. METHODS: Twelve untreated patients with mild to moderate asthma underwent a methacholine challenge test, sputum induction and blood sampling. A group of 14 normal subjects was also evaluated for baseline comparison. The same evaluation was repeated after 3 months of budesonide treatment. Before and after treatment, we tested the relationship of eosinophilic markers in induced sputum and blood with clinical and functional data. We also compared eosinophilic markers in induced sputum with the same markers in blood. RESULTS: Untreated patients showed a significant relationship between sputum eosinophils and symptom score, and between sputum eosinophilic cationic protein and symptom score, FEV1 and PD20FEV1. No relationship between blood eosinophilic markers and clinical or functional data was observed. In budesonide-treated patients, both sputum and blood eosinophils were significantly lower than in untreated patients, but eosinophil decrease was greater in sputum than in blood. Sputum eosinophilic proteins were also significantly lower in treated patients, whereas serum eosinophilic proteins were low at baseline and remained unchanged after treatment. Sputum eosinophilic markers were lower in normal subjects than in both untreated and treated patients, while blood eosinophils, but not serum eosinophilic cationic protein, were lower in normals than in untreated patients. CONCLUSIONS: The analysis of induced sputum is more useful than the analysis of blood in the evaluation of asthma severity and of the effect of glucocorticoid treatment in patients with mild to moderate asthma.

Adolescent↗

PubMed2005

Circulating eosinophils in asthma, allergic rhinitis, and atopic dermatitis lack morphological signs of degranulation.

BACKGROUND: In allergic diseases, eosinophils in affected tissues release granule proteins with cytotoxic, immunoregulatory, and remodelling-promoting properties. From recent observations, it may be assumed that eosinophils degranulate already in circulating blood. If degranulation occurs in the circulation, this could contribute to widespread systemic effects and provide an important marker of disease. OBJECTIVE: To determine the degranulation status of circulating eosinophils in common allergic diseases. METHODS: Using a novel approach of whole blood fixation and leucocyte preparation, the granule morphology of blood eosinophils from healthy subjects, non-symptomatic patients, symptomatic patients with asthma, asthma and Churg-Strauss syndrome, allergic rhinitis, and atopic dermatitis was evaluated by transmission electron microscopy (TEM) and eosinophil peroxidase (TEM) histochemistry. Plasma and serum levels of eosinophil cationic protein were measured by fluoroenzymeimmunoassay. Selected tissue biopsies were examined by TEM. RESULTS: Regardless of symptoms, circulating eosinophils from allergic patients showed the same granule morphology as cells from healthy subjects. The majority of eosinophil-specific granules had preserved intact electron-density (96%; range: 89-98%), while the remaining granules typically exhibited marginal coarsening or mild lucency of the matrix structure. Abnormalities of the crystalline granule core were rarely detected. Furthermore, granule matrix alterations were not associated with any re-localization of intracellular EPO or increase in plasma eosinophil cationic protein. By contrast, eosinophils in diseased tissues exhibited cytolysis (granule release through membrane rupture) and piecemeal degranulation (loss of granule matrix and core structures). CONCLUSION: In symptomatic eosinophilic diseases, circulating blood eosinophils retain their granule contents until they have reached their target organ.

Adolescent↗

PubMed2004

Danger signals derived from stressed and necrotic epithelial cells activate human eosinophils.

Eosinophilic granulocytes are found in tissues with an interface with the external environment, such as the gastrointestinal, genitourinary and respiratory tracts. These leucocytes have been associated with tissue damage in a variety of diseases. The aim of this study was to evaluate whether necrotic epithelial cells can activate eosinophils. The danger theory postulates that cells of the innate immune system primarily recognize substances that signal danger to the host. We damaged epithelial cell lines derived from the genital (HeLa cells), respiratory (HEp-2 cells) and intestinal tracts (HT29 cells) and assessed their capacity to cause eosinophilic migration, release of putative tissue-damaging factors, such as eosinophil peroxidase (EPO) and eosinophil cationic protein (ECP), as well as secretion of tissue-healing factors, e.g. fibroblast growth factors (FGF)-1 and -2 and transforming growth factor (TGF)-beta1. We found that necrotic intestinal cells induced chemotaxis in human eosinophils. EPO release was elicited in eosinophils stimulated with necrotic cells derived from all cell lines, as well as from viable HEp-2 and HT29 cells. Release of ECP was only seen in eosinophils incubated with necrotic intestinal or genital cells, not viable ones. Both necrotic intestinal and genital cells elicited FGF-2 secretion from eosinophils. TGF-beta1 was released from eosinophils exposed to viable and necrotic HT29 cells. These findings indicate that eosinophils are able to recognize and be activated by danger signals released from damaged epithelial cells, which may be of importance in understanding the role of eosinophils in the various inflammatory conditions in which they are involved.

Blood Proteins↗

PubMed1998

Release of granule proteins from human eosinophils stimulated with mast-cell mediators.

BACKGROUND: It has been suggested that mast cells and eosinophils are major effector cells in the pathogenesis of allergic diseases. However, the interaction of these cells has not been thoroughly elucidated. We examined eosinophil cationic protein (ECP) release and cytosolic free calcium concentration ([Ca2+]i) in human eosinophils induced by the major mast-cell mediators including cytokines. METHODS: Eosinophils from healthy donors were stimulated with the major mast-cell mediators for 20 min after preincubation with cytochalasin B for 10 min. ECP in supernatants was measured by radioimmunoassay. Moreover, to examine changes of [Ca2+]i in eosinophils, Fura-2-loaded eosinophils were monitored for fluorescence changes after stimulus addition. RESULTS: Of the tested mediators (prostaglandin [PG]D2, leukotriene (LT)B4, platelet-activating factor (PAF), histamine, LTC4, and eosinophil chemotactic factor of anaphylaxis [ECF-A]), LTB4 and PAF induced ECP release from eosinophils. Any cytokines produced by human mast cells, i.e., interleukin (IL)-4, IL-5, IL-8, tumor necrosis factor (TNF), or granulocyte-macrophage colony-stimulating factor (GM-CSF), did not induce ECP release in our system. ECP release triggered with LTB4 and PAF occurred at concentrations of 10(-8)-10(-6) M concentration-dependently. LTB4 and PAF also elicited a rise in [Ca2+]i in eosinophils. Neither PGD2, histamine, nor LTC4 induced ECP release, although they increased cytosolic calcium in eosinophils. CONCLUSIONS: Of mast-cell mediators, LTB4 and PAF induced eosinophil degranulation. The contribution of LTB4 and PAF from mast cells to eosinophil degranulation may be important in the pathogenesis of allergic inflammatory diseases.

Blood Proteins↗

PubMed1994

TNF alpha-induced activation of eosinophil oxidative metabolism and morphology--comparison with IL-5.

Human dermal mast cells are capable of releasing cytokines, particularly preformed TNF alpha, upon appropriate stimulation. Mast cell activation in vivo was shown to be associated with an influx and activation of inflammatory cells, initially PMN (polymorphonuclear neutrophilic granulocytes) then eosinophils. In order to learn more about the mechanisms by which TNF alpha is capable of activating eosinophils, in the present study the effect of TNF alpha on morphology and function of highly purified normal eosinophils (> or = 95%) was examined. As estimated by transmission and scanning electron microscopy, TNF alpha-stimulated eosinophils appeared to be strictly adherent and flattened exhibiting a characteristic "hemispheric" shape. TNF alpha induced a dose-dependent, long-lasting production of reactive oxygen species as measured by lucigenin-dependent chemiluminescence (CL), even at a concentration of 0.001 U/ml. The maximal response upon stimulation with TNF alpha, however, was significantly lower than optimal effects induced by IL-5. TNF alpha-induced responses were completely inhibited by cytochalasin B and staurosporin, and partially blocked by pertussis toxin. Separation of eosinophils by discontinuous density gradients revealed the existence of at least two hypodense eosinophil populations with a distinct susceptibility to stimulation with TNF alpha. Based on functional assay systems, in contrast to a significant extracellular, only a small intracellular H2O2 production was detected. Accordingly, H2O2 production, detected by an ultrastructural technique, was observed only on the outer surface of the plasma membrane in the contact zones in between adjacent cells. Extracellular as well as intracellular production of H2O2 was completely inhibited by cytochalasin B. TNF alpha-induced activation of eosinophils is most probably mediated by binding to the 55 kD and the 75 kD TNF-receptor since both receptor molecules could be detected by FACS analysis and immune electron microscopy using receptor-specific antibodies. However, in contrast to its effect on eosinophil oxidative response, TNF alpha did not induce the release of significant concentrations of eosinophil cationic protein or eosinophil peroxidase in supernatants of cytokine-stimulated eosinophils, as detected by functional as well as immunological assay systems. These results clearly indicate that TNF alpha represents a potent eosinophil-activating cytokine which may be of relevance in the allergic inflammatory response.

Blood Proteins↗

PubMed1999

Effect of interleukin-3, interleukin 5 and hyaluronic acid on cultured eosinophils derived from human umbilical cord blood mononuclear cells.

BACKGROUND: Several studies have shown that cultured eosinophils can be generated from human umbilical cord blood mononuclear cells (UCMC) in the presence of interleukin (IL)-3 and IL-5 in vitro. Other reports have indicated that cellular adhesion to hyaluronic acid (HA) enhances the proliferation of cultured eosinophils derived from CD34+ cells purified from UCMC. The aim of this study was to obtain large numbers of mature eosinophils from UCMC using IL-3, IL-5 and HA, and to investigate their functions. METHODS: We examined several combinations of IL-3 and IL-5 and their effect on eosinophil development from UCMC in HA-coated on non-coated flasks. We also examined whether cultured eosinophils degranulated eosinophil-derived neurotoxin (EDN) induced by secretory immunoglobulin A conjugated to sepharose beads (sIgA-beads) and responded to eotaxin. RESULTS: Culture with HA-coated flasks for 35 days (in the presence of IL-3 and IL-5, with IL-3 omitted after day 14 of culture) caused a 11.2-fold augmentation in the proliferation of UCMC. On day 35 of the culture, 98% of cultured cells were eosinophils judging from May-Grünwald and Giemsa staining and transmission electron micrographs. The EDN content of the cultured eosinophils on day 35 was 156 ng/105 cells. Cultured eosinophils degranulated EDN induced by sIgA-beads and responded to eotaxin by chemotaxis and intracellular Ca2+ mobilization. CONCLUSION: We found a useful culture system to obtain large numbers of eosinophils derived from UCMC, which may facilitate the investigation of eosinophil function, since there was no significant difference in response to sIgA-beads and eotaxin between cultured and peripheral eosinophils.

Cell Degranulation↗

PubMed1995

Eosinophil granule cationic proteins regulate the classical pathway of complement.

Major basic protein, the primary constituent of eosinophil granules, regulates the alternative and classical pathways of complement. Major basic protein and other eosinophil granule cationic proteins, which are important in mediating tissue damage in allergic disease, regulate the alternative pathway by interfering with C3b interaction with factor B to assemble an alternative pathway C3 convertase. In the present study, eosinophil peroxidase, eosinophil cationic protein and eosinophil-derived neurotoxin, as well as major basic protein, were examined for capacity to regulate the classical pathway. Eosinophil peroxidase, eosinophil cationic protein and major basic protein inhibited formation of cell-bound classical pathway C3 convertase (EAC1,4b,2a), causing 50% inhibition of complement-mediated lysis at about 0.19, 0.75 and 0.5 micrograms/10(7) cellular intermediates, respectively. Eosinophil-derived neurotoxin had no activity on this pathway of complement. The eosinophil granule proteins were examined for activity on the formation of the membrane attack complex. Major basic protein and eosinophil cationic protein had no activity on terminal lysis. In contrast, eosinophil peroxidase inhibited lysis of EAC1,4b,2a,3b,5b, but had only minimal activity on later events in complement lysis. These polycations were then examined to determine the site(s) at which they regulated the early classical pathway. Eosinophil granule polycationic proteins: (1) reduced the Zmax at all time points but had only minimal effect on the Tmax during the formation of the classical pathway C3 convertase (EAC1,4b,2a); (2) inhibited formation of EAC1,4b,2a proportional to C4 but independent of C2 concentration; (3) inhibited fluid phase formation of C1,4b,2a, as reflected by a decrease in C1-induced consumption of C2 over time; and (4) inhibited C1 activity over time without a direct effect on either C4 or C2. These observations suggest that polycations regulate the early classical pathway by interfering with C1 and may exert this activity in vivo.

Blood Proteins↗

PubMed2002

The eosinophilic pneumonias.

Acute and chronic eosinophilic pneumonia can be distinguished by their clinical, laboratory, and radiographic features. Patients with both acute and chronic eosinophilic pneumonia present with cough, dyspnea, and fever. Patients with chronic eosinophilic pneumonia present subacutely over weeks to months but patients with acute eosinophilic pneumonia present within 5 days of symptom onset. Chest radiographs in chronic eosinophilic pneumonia show peripheral alveolar infiltrates. In contrast, radiographs in acute eosinophilic pneumonia show mixed interstitial and alveolar infiltrates, Kerley B lines, and pleural effusions. Both disorders are characterized by high percentages of bronchoalveolar lavage eosinophils, but high numbers of blood eosinophils accompanies only chronic eosinophilic pneumonia. The diagnosis of both disorders can usually be made based on clinical and radiographic findings; however, lung biopsy is occasionally necessary to distinguish the eosinophilic pneumonias from other eosinophilic lung diseases. In both conditions, patients will respond rapidly and completely to corticosteroids but patients with chronic eosinophilic pneumonia usually relapse if less than 6 months of treatment is given, whereas patients with acute eosinophilic pneumonia do not relapse after a brief course of treatment.

Journal Article↗

PubMed2006

The glucocorticoid RU24858 does not distinguish between transrepression and transactivation in primary human eosinophils.

BACKGROUND: Glucocorticoids are used to treat chronic inflammatory diseases such as asthma. Induction of eosinophil apoptosis is considered to be one of the main mechanisms behind the anti-asthmatic effect of glucocorticoids. Glucocorticoid binding to its receptor (GR) can have a dual effect on gene transcription. Activated GR can activate transcription (transactivation), or by interacting with other transcription factors such as NF-kappaB suppress transcription (transrepression). RU24858 has been reported to transrepress but to have little or no transactivation capability in other cell types. The dissociated properties of RU24858 have not been previously studied in non-malignant human cells. As the eosinophils have a very short lifetime and many of the modern molecular biological methods cannot be used, a "dissociated steroid" would be a valuable tool to evaluate the mechanism of action of glucocorticoids in human eosinophils. The aim of this study was to elucidate the ability of RU24858 to activate and repress gene expression in human eosinophils in order to see whether it is a dissociated steroid in human eosinophils. METHODS: Human peripheral blood eosinophils were isolated under sterile conditions and cultured in the presence and/or absence RU24858. For comparison, dexamethasone and mometasone were used. We measured chemokine receptor-4 (CXCR4) and Annexin 1 expression by flow cytometry and cytokine production by ELISA. Apoptosis was measured by DNA fragmentation and confirmed by morphological analysis. RESULTS: RU24858 (1 microM) increased CXCR4 and Annexin 1 expression on eosinophils to a similar extent as mometasone (1 microM) and dexamethasone (1 microM). Like dexamethasone and mometasone, RU24858 did suppress IL-8 and MCP-1 production in eosinophils. RU24858 also increased spontaneous eosinophil apoptosis to a similar degree as dexamethasone and mometasone, but unlike dexamethasone and mometasone it did not reverse IL-5- or GM-CSF-induced eosinophil survival. CONCLUSION: Our results suggest that in human eosinophils RU24858 acts as transactivator and transrepressor like classical glucocorticoids. Thus, RU24858 seems not to be a "dissociated steroid" in primary human eosinophils in contrast to that reported in animal cells. In addition, functionally RU24858 seems to be a less potent glucocorticoid as it did not reverse IL-5- and GM-CSF-afforded eosinophil survival similarly to dexamethasone and mometasone.

Journal Article↗

PubMed2002

Eosinophilic gastroenteritis: epidemiology, diagnosis and management.

Eosinophilic gastroenteritis is a heterogeneous and uncommon disorder characterized by eosinophilic inflammation of the gastrointestinal tissues. The location and depth of infiltration determine its varied manifestations, and the latter is also the basis for the proposed classification into mucosal, muscular and serosal eosinophilic gastroenteritis. Abdominal pain, vomiting, and diarrhea are each present in nearly 50% of the patients, with some overlap. Peripheral eosinophilia is seen in approximately two-thirds of patients with eosinophilic gastroenteritis. It is now clear that eotaxin, a specific eosinophil chemoattractant, plays a pivotal role in the process of eosinophil production. The differential diagnosis of eosinophilic gastroenteritis in children includes parasitic infections, inflammatory bowel disease, connective tissue diseases, some malignancies and adverse effects of drugs. Eosinophilic gastroenteritis itself has been strongly associated with food allergies, and concomitant atopic diseases or a family history of allergies is elicited in about 70% of cases. The pediatric experience is unique with respect to recognition of distinctive entities such as allergic procto-colitis, almost exclusively seen in infants, and eosinophilic esophagitis being increasingly reported among children and young adults. The gold standard for diagnosis, usually demonstrated on endoscopic biopsies, is prominent tissue eosinophilia. However, the diagnosis may be obscured by the patchy nature of the disease, and muscular and serosal eosinophilic gastroenteritis subtypes. In the latter cases, full thickness biopsies would be indicated for a definitive diagnosis. There are many reports of successful treatment of eosinophilic gastroenteritis in children, using a variety of treatment regimens including elimination diets. Corticosteroids remain the most effective agents for controlling symptoms, but unfortunately the relapsing nature of the disease would mandate prolonged corticosteroid use. Reports of favorable responses to new leukotriene inhibitors in patients with eosinophilic gastroenteritis are encouraging; these responses should stimulate future research on the pathophysiology and management of eosinophilic gastroenteritis.

Adrenal Cortex Hormones↗

PubMed2002

Clinical features of eosinophilic bronchitis.

BACKGROUND: Eosinophilic inflammation of the airway is usually associated with airway hyper-responsiveness in bronchial asthma. However, there is a small group of patients which has the eosinophilic inflammation in the bronchial tree with normal spirometry and no evidence of airway hyper-responsiveness, which was named eosinophilic bronchitis. The objectives of this study are 1) to investigate the incidence of eosinophilic bronchitis in the chronic cough syndrome and 2) to evaluate the clinical features and course of eosinophilic bronchitis. METHODS: We evaluated 92 patients who had persistent cough for 3 weeks or longer. In addition to routine diagnostic protocol, we performed differential cell count of sputum. Eosinophilic bronchitis was diagnosed when the patient had normal spirometric values, normal peak expiratory flow variability, no airway hyper-responsiveness and sputum eosinophilia (> 3%). RESULTS: The causes of chronic cough were post-nasal drip in 33%, cough variant asthma in 16%, chronic bronchitis in 15% and eosinophilic bronchitis in 12% of the study subjects. Initial eosinophil percentage in the sputum of patients with eosinophilic bronchitis was 26.8 +/- 6.1% (3.8-63.7%). Treatment with inhaled steroid is related with a subjective improvement of cough severity and a significant decrease of sputum eosinophil percentage (from 29.1 +/- 8.3% to 7.4 +/- 3.3%). During the follow-up period, increase in sputum eosinophil percentage with aggravation of symptoms were found. CONCLUSION: Eosinophilic bronchitis is one of the important cause of chronic cough. Assessment of airway inflammation by sputum examination is important in investigating the cause of chronic cough. Cough in eosinophilic bronchitis is effectively controlled by inhaled corticosteroid, but may follow a chronic course.

Adult↗

PubMed2007

Comparative study of eosinophil chemotaxis, adhesion, and degranulation in vitro in ulcerative colitis and Crohn's disease.

BACKGROUND: Eosinophils have been identified in tissues from patients with Crohn's disease (CD) and ulcerative colitis (UC) but whether they contribute to IBD pathogenesis is unknown. This study aimed to investigate the functional activity and morphological aspects of peripheral-blood eosinophils from IBD patients compared to those from healthy volunteers (HVs). METHODS: Eosinophils from HVs and CD and UC patients were purified using a Percoll gradient and then a immunomagnetic cell separator. Functional activity in inactivated and previously activated cells was investigated by measuring adhesion to fibronectin and chemotaxis to fMLP, and degranulation was measured by release of eosinophil peroxidase (EPO). Cell morphology was investigated using electron microscopy. RESULTS: Eosinophil adhesion to human fibronectin in both inactivated and PAF-stimulated and PMA-stimulated eosinophils was markedly higher in patients with CD than in either patients with UC or HVs. Similarly, the chemotactic response was markedly higher in eosinophils isolated from CD patients than in those isolated from UC patients or HVs. Baseline EPO release was higher in eosinophils isolated from UC patients than in those isolated from HVs or CD patients. Stimulation with fMLP or PMA did not further increase EPO release in cells from UC or CD patients. Comparable expression of MAC- 1 and VLA-4 adhesion molecules was observed on the surfaces of eosinophils from all groups, and an greater number of granules was noted in the eosinophils from UC patients than in those from CD patients. CONCLUSIONS: Our results indicate that peripheral-blood eosinophils are potentially primed and activated in IBD patients. Whether the differences in the morphology and functional responses of eosinophil from UC and CD patients reflect differences in disease phenotype remains to be elucidated.

Cell Adhesion↗

PubMed1996

Monoclonal antibodies against eosinophils in liver allograft rejection.

There has been recent interest in eosinophils as a histological diagnostic marker of liver allograft rejection. However, the reliability of counting eosinophils in sections stained with hematoxylin and eosin (H&E) has not been evaluated previously. We quantified eosinophils in 10 day-5 protocol liver biopsy specimens in 10 patients. The control groups were 5 patients with cytomegalovirus infection, 5 patients with obscure liver dysfunction, and 6 patients with HCV infection. Eosinophil count was assessed using H&E and by specific monoclonal antibody staining using (1) an anti-ECP antibody (EG2) and (2) a monoclonal antibody against human eosinophil major basic protein (MBP) (BMK-13). The average percentage of the total inflammatory infiltrate of eosinophils in portal tracts was 9% in the moderate to severe rejection group as compared with 0.25% in the mild rejection group (P < .001) and 0% in the control group (P < .001). The eosinophil count decreased markedly after successful treatment of rejection. The H&E staining correlated with MBP+ the (BMK-13 immunoreactive) cells but were more numerous with BMK-13. BMK-13 also stained significantly more cells when compared with EG2 (P < .01). This difference may be because EG2 staining only activated eosinophils, whereas BMK-13 is a pan-eosinophilic maker, regardless of activation. This study confirms that eosinophils are a specific feature of acute cellular rejection and are an aid to its diagnosis. BMK-13 is a useful pan-eosinophilic marker that is more efficient in obtaining eosinophil count when compared with H&E.

Antibodies, Monoclonal↗

PubMed1995

Modulation of growth and differentiation of eosinophils from human peripheral blood CD34+ cells by IL5 and other growth factors.

Small numbers of CD34+ primitive hematopoietic progenitors are found in normal human peripheral blood. These cells differentiate to myeloid or lymphoid lineage under the influence of different growth factors. We investigated the effects of IL5 and other growth factors on the production of eosinophils from peripheral blood CD34+ cells. CD34+ cells were enriched from normal donors by apheresis and positive selection using an affinity column and plated in agarose with different combinations of cytokines. At 14 days of growth a triple stain technique was used to identify eosinophil, monocyte, and neutrophil colonies. IL5 alone did not support colony growth from CD34+ cells. In contrast, GM-CSF and IL3 alone or together without added IL5 supported the generation of more than 50% pure eosinophil colonies. Addition of IL5 did not change the total number of colonies, but increased the fraction of pure eosinophil colonies to over 70%. Addition of G-CSF reduced the percentage of eosinophil colonies and increased the percentage of neutrophil colonies. Under the best conditions for eosinophil colony growth (IL3+GM-CSF+IL5), the addition of interferon-alpha or bacterial lipopolysaccharide inhibited colony growth by 51 and 58%, respectively. Addition of interferon-gamma, tumor necrosis factor-alpha, or dexamethasone had no effect on eosinophil colonies. Since IL5 alone did not support colony growth from CD34+ cells, we determined when IL5-responsive cells appeared in culture. Cells were grown initially with IL3 + GM-CSF in suspension, washed, and plated in agarose with IL5 alone. Only when progenitors were grown at least 3 days could IL5 serve as the single growth factor supporting pure eosinophil colony growth (47 colonies/10(4) cells plated at Day 3 and 134 colonies/10(4) cells at Day 7). We used neutralizing anti-IL5 antibodies to demonstrate that this late acting IL5 growth effect was specific, and that differentiation of eosinophils in the presence of IL3 + GM-CSF was IL5 independent. Using reverse-transcriptase polymerase chain reaction, the mRNA encoding the eosinophil-specific protein eosinophil peroxidase (EPO) was not detected in Day 0 CD34+ cells, but was demonstrated by Day 3 of culture. We conclude that within 3 days of culture, peripheral blood CD34+ cells can become committed to the eosinophil lineage as demonstrated by responsiveness to IL5 and production of EPO transcripts.

Antigens, CD↗

PubMed2000

Degranulating eosinophils in human endometriosis.

Degranulating eosinophils have been described in most endometrial cancers. We hypothesized that endometriosis (ectopic, nonneoplastic endometrial tissue) would be an appropriate model system for determining whether eosinophil degranulation is part of a specific immune response to endometrial cancer or if it is related to the more general phenomenon of tissue remodeling (wound healing) that is common to both disorders. To test this hypothesis, we performed immunohistochemistry and Western blotting to evaluate the presence of eosinophil peroxidase (a marker of eosinophil degranulation) in normal endometrium (n = 20) and endometriosis samples (n = 24) and to define the coexpression of three eosinophil chemoattractants: interleukin-5 (IL-5), eotaxin, and regulated on activator-normal T cell expressed and secreted (RANTES). There was focally intense deposition of eosinophil peroxidase in the fibrotic connective tissue and blood vessels of 21 of 24 human endometriosis specimens; two samples showed weak staining, and only one tissue was negative for eosinophil degranulation. None of the 10 normal proliferative endometrial specimens had evidence of eosinophil degranulation, and four of 10 secretory tissues stained only weakly for eosinophil peroxidase. The presence of degranulating eosinophils was also associated with the presence of eotaxin and IL-5 in some samples and with RANTES in others. We conclude that the abundant presence of degranulating eosinophils in the fibrous regions of endometriosis supports the interpretation that eosinophils are involved in general tissue remodeling and wound healing rather than a tissue-directed immune response.

Blotting, Western↗

PubMed1999

Inhibition of eosinophil activation in bronchoalveolar lavage fluid from atopic asthmatics by Y-24180, an antagonist to platelet-activating factor.

We examined the effects of Y-24180, a potent and long-acting antagonist to platelet-activating factor (PAF) receptor, on the expression of adhesion molecules in peripheral blood and bronchoalveolar lavage fluid (BALF) eosinophils from atopic asthmatics. Y-24180 (20 mg/day) was administered to 4 atopic asthmatics for 8 weeks. The number of eosinophils, the level of eosinophil cationic protein (ECP), the bindings of soluble intercellular adhesion molecule-1 (sICAM-1) and fibronectin (FN), and the expressions of CD11b (alpha chain of Mac-1) and CD49d (alpha chain of VLA-4) on eosinophils were evaluated in peripheral blood (n=4) and BALF (n=3) before and after the administration of Y-24180. The infiltration of eosinophils into the bronchial wall was also examined by taking biopsies. Eosinophil count, sICAM-1 and FN binding to eosinophils in BALF significantly decreased after the administration of Y-24180 (p<0.05). The level of CD11b expression also decreased remarkably after the administration (n=2). In peripheral blood, eosinophil count and ECP level did not change. The binding of sICAM-1 and FN, and expression of CD11b on eosinophils in peripheral blood showed a tendency to decrease after the administration. The level of CD49d expression on eosinophils changed neither in BALF nor in blood. Eosinophil infiltration into the bronchial wall markedly decreased in one out of 3 cases after the administration. These results suggest that Y-24180 inhibits the activation of eosinophils by antagonizing the actions of PAF in atopic asthmatics.

Adult↗

PubMed1996

Eosinophilic vasculitis in connective tissue disease.

BACKGROUND: Neutrophilic and lymphocytic vascular inflammation is common in vasculitis associated with connective tissue disease (CTD). We recently identified eight patients with CTD and eosinophilic vasculitis. OBJECTIVE: The purpose of this study was to characterize a variant form of vasculitis in CTD with eosinophilic infiltration. METHODS: Of 98 CTD patients with cutaneous necrotizing vasculitis, eight were found with predominantly eosinophilic vascular infiltration. Nine CTD patients with cutaneous neutrophilic vasculitis were identified for comparison. Clinical and laboratory findings were reviewed and compared. Indirect immunofluorescence for eosinophil granule major basic protein (MBP), neutrophil elastase, and mast cell tryptase was performed on lesional tissue. MBP levels and eosinophil survival enhancing activity were assayed in sera from three patients. RESULTS: The patients with eosinophilic vasculitis had depressed serum complement levels and peripheral blood eosinophilia; MBP levels were elevated in serum and eosinophil survival was prolonged. Immunofluorescence of tissue showed marked angiocentric eosinophil MBP staining with peripheral neutrophil elastase staining; mast cell tryptase staining was notably absent. The patients with neutrophilic vasculitis were variably hypocomplementemic and did not have peripheral blood eosinophilia. Immunofluorescence showed marked angiocentric neutrophil elastase staining with scattered eosinophil MBP staining; mast cell tryptase staining showed normal mast cell numbers. CONCLUSION: Patients with eosinophilic vasculitis, CTD, and hypocomplementemia show vessel wall destruction in association with vessel wall deposition of cytotoxic eosinophil granule MBP, which suggests that eosinophils mediate vascular damage in this disease process. In addition, perivascular mast cells appear diminished, thereby suggesting that mast cell degranulation occurs.

Adult↗

PubMed2000

Effects of human lung fibroblasts on eosinophil degranulation.

BACKGROUND: Although eosinophils and fibroblasts are thought to contribute to the mechanisms of chronic asthmatic inflammation, the interaction between eosinophils and fibroblasts has not been thoroughly clarified. We examined eosinophil cationic protein (ECP) release from human eosinophils cultured in the presence of human lung fibroblast HFL-1. METHODS: Eosinophils from healthy donors were cultured with or without C5a for 16 h in the presence of human fetal lung fibroblasts which had previously been incubated with or without TNF for 4 h. ECP in supernatants was measured by RIA. RESULTS: ECP release was potentiated only when both eosinophils and fibroblasts were activated by C5a and TNF, respectively, while it was not significantly potentiated when either eosinophils or fibroblasts were activated. Paraformaldehyde fixation of fibroblasts had some suppressive effect on ECP enhancement, and mAb against GM-CSF partly inhibited ECP enhancement. Coculture of eosinophils and fibroblasts with stimulus treatment resulted in the enhancement of both eosinophil adhesion and ECP release. The potentiation of ECP release was partially inhibited by anti-ICAM-1 mAb, anti-CD18 mAb, and anti-CD29 mAb, which caused partial and comparable inhibition of the enhancement of eosinophil adhesion. CONCLUSIONS: This study suggests that the activation of fibroblasts may have some role in the potentiation of ECP release from cocultured eosinophils, and that adhesion of eosinophils to fibroblasts may partly be involved in the mechanism of ECP potentiation.

Antibodies, Monoclonal↗