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PubMed1999

Regulation of human eosinophil migration across lung epithelial monolayers by distinct calcium signaling mechanisms in the two cell types.

In asthmatic patients, eosinophils massively infiltrate the lung tissues and migrate through lung epithelium into the airways. The regulatory mechanisms involved are obscure. We studied the role of calcium in the migration of human eosinophils across monolayers of human lung epithelial H292 cell line cells induced by combined chemotactic solutions of platelet-activating factor and C5a. The transepithelial migration of eosinophils was attenuated by depletion of the external Ca2+ in the migration system, whereas the eosinophil migration itself was unaffected as evidenced by measuring eosinophil chemotaxis in the Boyden chamber in the absence of epithelial cells. Buffering of intracellular Ca2+ in eosinophils with 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM) inhibited both eosinophil transepithelial migration and eosinophil chemotaxis in the Boyden chamber, suggesting the importance of intracellular Ca2+ in eosinophil transmigration. Although loading of BAPTA/AM or addition of thapsigargin to the epithelial cells effectively changed their cytoplasmic free Ca2+ concentrations, neither of these treatments affected transepithelial migration of eosinophils. Interestingly, addition of La3+ (0.2 mM) to epithelial cells suppressed eosinophil transmigration whereas addition of La3+ to eosinophils did not. Taken together, these results show the importance of Ca2+ in eosinophil migration across lung epithelium and support a distinctive regulatory role of intracellular and extracellular Ca2+ for the two cell types involved in this process; i.e., the transmigration of human eosinophils across a monolayer of lung epithelial cells is regulated by the intracellular Ca2+ in eosinophils, whereas the ability of the lung epithelial cell monolayer to allow eosinophil passage is dependent on the extracellular Ca2+.

Buffers↗

PubMed1992

Induction and function of eosinophil intercellular adhesion molecule-1 and HLA-DR.

We have previously established that eosinophils studied ex vivo from the sputum of asthmatics express intercellular adhesion molecule-1 (ICAM-1) and HLA-DR, whereas peripheral blood eosinophils do not express these surface proteins. On incubation of highly purified (greater than 99.5% pure) blood eosinophils from normal subjects with T cell supernatants, eosinophil ICAM-1 was induced in 24 h, whereas HLA-DR was maximally induced within 48 h. Recombinant cytokines that enable eosinophil survival (IL-5, IL-3, and granulocyte macrophage-CSF) were found to be unable to induce ICAM-1 or HLA-DR, even when pooled at concentrations individually required for eosinophil survival. However, synergy between these eosinophil survival factors and TNF (-alpha and -beta) was found mainly responsible for ICAM-1 induction, whereas synergy between IL-3 and IFN-gamma occurred for HLA-DR induction. Culture of eosinophils in the presence of cytokines and cycloheximide prevented expression of ICAM-1 and HLA-DR, showing that de novo eosinophil protein synthesis is occurring. At a functional level we demonstrate that ICAM-1-bearing eosinophils have increased adhesion capacity for autologous T cells. In contrast, HLA-DR-expressing eosinophils mediated Ag-specific proliferation of an autologous HLA-DR-restricted T cell clone that was inhibitable by anti-HLA-DR and anti-ICAM-1 mAb. Since eosinophil-mediated Ag presentation was inhibitable by treatment of eosinophils with glutaraldehyde or chloroquine, this suggests that eosinophils participate in Ag uptake, processing, and presentation and have accessory functions. Thus, through the induction of ICAM-1 and HLA-DR on tissue eosinophils, eosinophils have the capacity to interact with leukocytes and present Ag to T cells.

Antigen-Presenting Cells↗

PubMed1991

The dynamics of intestinal eosinophil depletion in rats treated with dexamethasone.

We examined the effects of corticosteroid treatment on eosinophils in the jejunal mucosa of rats previously infected with Nippostrongylus brasiliensis. Rats received a single intraperitoneal injection of 1 mg of dexamethasone. At a light microscopic level, the number of eosinophils with typical nuclei and granules was significantly decreased as early as 3 hours after injection, and had diminished to 17% of starting values at 24 hours. Pyknotic cells containing eosinophilic granules or fragments were observed scattered in the subepithelial interstitial space 3 and 7 hours after injection. By electron microscopy, more than 20% of eosinophils demonstrated nuclear abnormalities. Degenerating eosinophils without granular changes (27 of 127, 14.1%) or with or with granular changes (9 of 127, 7.1%) increased at 3 hours compared with untreated rats (8 of 233, 3.4%; 4 of 233, 1.7%). At 7 hours, 47 of 96 (49.0%) eosinophils were located inside phagocytic vacuoles of macrophages. Single macrophages occasionally engulfed two or more eosinophils. Only a few degeneration eosinophils (7 of 96) were observed outside macrophages. At 13 hours, the percentage of degenerating eosinophils and eosinophils inside macrophages was decreased; at 24 hours, few eosinophils were seen and eosinophil structures could not be identified inside macrophages. The epithelium and lamina propria did not show structural damage typical of an inflammatory reaction at any time. Eosinophil numbers in mesenteric lymph nodes, spleens, and peripheral blood were also reduced by dexamethasone. Similar observations were made in the jejunal mucosa of noninfected rats. We observed the slow restoration of eosinophil numbers in the intestinal wall, finally reaching preinjection numbers after 14 days. We conclude that dexamethasone has important effects on eosinophils causing (a) nuclear degeneration and (b) changes in the granular matrix. Subsequently, these damaged eosinophils are engulfed by macrophages and swiftly disappear from the intestinal mucosa. These effects appear to be due to the induction of apoptosis. Our findings offer an explanation for one of the significant antiinflammatory effects observed with the use of corticosteroids.

Animals↗

PubMed1979

Immunopathology of Toxocara canis and Ascaris suum infections of the eye: the role of the eosinophil.

Intravitreal infection of guinea pigs with second-stage larvae of Toxocara canis and second- and fourth-stage larvae of Ascaris suum induced intraocular IgE antibodies and a dense eosinophil infiltration in the anterior chamber and throughout the uveal tract. The eosinophil infiltrate began within 1 day after infection and persisted for as long as 51 days. By day 12 after intravitreal infection, the injected ascarid larvae were surrounded by granulomas which consisted almost entirely of eosinophils. Firm adherence of eosinophils to the parasite cuticle, morphologic alterations and degranulation of eosinophils with the deposition of free eosinophil granules on the parasite surfaces, and ingestion of eosinophils and eosinophil granules by the parasite larvae also were observed. Intravitreal injectionof a soluble antigen derived from third-stage A. suum molting to the fourth-stage in defined media in vitro also induced intraocular IgE antibody and a diffuse ocular eosinophil infiltrate. Dense eosinophil infiltration of the choroid, not immediately adjacent to a parasite larva, was accompanied by destruction of the lverlying outer retina, with cystic changes in the retina and between the retina and the choroid. Few eosinophils were observed within the retina, and the retinal destruction may be the result of direct toxic action of constituents of the choroidal eosinophils. Evidence which indicates that the eosinophil is the principal effector cell in immunity to helminth infections and is cytotoxic for the parasites, possible mechanisms for the induction of the ocular eosinophil infiltrates, and evidence for autotoxicity by eosinophils are briefly reviewed, and the potential roles of the eosinophil in the ocular response to helminth parasites are discussed.

Animals↗

PubMed1983

Eosinophil activation by colony-stimulating factor in man: metabolic effects and analysis by flow cytometry.

Substantial increases in the killing capacity of human eosinophils after in vitro incubation with human placental conditioned medium (HPCM), a standard source of colony-stimulating factor (CSF), have recently been described. In this article, the interaction between HPCM and purified human eosinophils is analyzed by flow cytometry and by effects on iodination, superoxide production, and protein synthesis. HPCM increased the intensity of natural eosinophil autofluorescence (aFlu) (460 nm) after the absorption of ultraviolet light (360 nm) in a manner that was both time and dose dependent. Measured in arbitrary units, eosinophil aFlu was 72 +/- 7.3 (arithmetic mean +/- SEM) and 121 +/- 3.2 after 18-hr incubations in the absence or presence of HPCM, respectively. The activity in HPCM responsible for these changes cochromatographed on Ultrogel AcA44 columns with CSF and with the less hydrophobic variant of CSF (CSF-alpha) on phenyl Sepharose. Mouse spleen, but not mouse lung, conditioned medium was also active on human eosinophils in this assay. Both CSF-alpha and mouse spleen conditioned medium also contain eosinophil colony-stimulating activity (CSA), whereas inactive CSFs with no effect on mature eosinophils, CSF-beta, and mouse lung conditioned medium also lack eosinophil CSA. CSF-alpha stimulated superoxide production of resting eosinophils (from 0.03 +/- 0.03 to 0.47 +/- 0.08 nmole cytochrome-c reduced/10(5) eosinophils) and of eosinophils incubated with preopsonized zymosan (from 0.15 +/- 0.06 to 0.73 +/- 0.07). It also stimulated iodination by resting eosinophils (from 0.76 +/- 0.16 to 2.60 +/- 0.72 nmoles l/10(7) eosinophils/hr) and of eosinophils incubated with preopsonized zymosan (from 7.52 +/- 2.08 to 29.8 +/- 1.32). In contrast, CSF-beta was inactive in these assays. CSF-alpha also stimulated, between 2- and 15-fold, the new protein synthesis of eosinophils. Thus, substances that stimulate the differentiation of progenitor cells into eosinophils also interact with peripheral mature eosinophils, and the activation of postmitotic cells may be a physiologic role of CSF-like molecules.

Cell Separation↗

PubMed1980

Enzymes altering the binding capacity of human blood eosinophils for IgG antibody-coated erythrocytes (EA).

Blood eosinophils from some patients with an eosinophilia have a higher capacity to bind to IgC antibody-coated red cells (EA) than blood eosinophils from normal people. Twenty per cent of eosinophils from normal blood bound EA, whereas eight of nine patients with hypereosinophilic syndromes and all nine patients with filariasis who were studied had blood eosinophils with EA rosette-forming capacities of between 42 and 89%. High EA binding capacity was reduced in culture, and prednisolone and cytochalasins A and B inhibited normal blood eosinophil EA binding. Normal blood eosinophils developed small increases in EA binding capacity in culture, but marked increases occurred after stimulation with soluble immune complexes, endotoxins and lipid A. Supernatants from granulocytes cultured with zymosan-C3b caused rapid increases in eosinophils EA binding capacity which also occurred with neuraminidase, pronase and trypsin. In vitro alterations in EA rosetting did not require protein synthesis and did not affect eosinophil phagocytic capacity for EA. Substances in culture which did not affect eosinophil EA rosetting capacity included sera from patients with eosinophils with high EA binding capacity and chemotactic substances. Cultured eosinophils also developed an increased capacity to form rosettes with EAC3b, and soluble immune complexes stimulated this further. Conversely, blood eosinophils formed less C3b rosettes when separated from heparinized blood in which C3 activation had occurred. CytochalasinA (but not B) irreversibly inhibited eosinophils EAC3b rosette formation. Trypsin also inhibited, but this effect was reversed within 30 min after washing. It was concluded that eosinophils from normal blood have an intrinsically lwo capacity to bind EA, but that in vivo and in response to stimulation in vitro their ability to bind complexed IgG can approach that seen with blood neutrophils. It is suggested that enzymes in granulocyte secretion products may cause the membrane changes which lead to high eosinophils EA binding capacity. This increase, which can occur separately from alterations in EAC binding or phagocytic capacity, may enable eosinophils to take part more effectively in inflammatory reactions in tissues.

Adolescent↗

PubMed1987

Eosinophilia in children. Cytochemical and immunological analysis of increased eosinophils.

Cytochemical and immunological studies of increased eosinophils were performed in three children with marked eosinophilia. These children were diagnosed as having non-Hodgkin's lymphoma, transient eosinophilia, and hyper-eosinophilic syndrome. All showed marked eosinophilia, with eosinophil counts of more than 5,000/mm3 in the peripheral blood and with eosinophils in all maturational stages in the bone marrow. In contrast to normal eosinophils, the increased eosinophils in three patients on cytochemical analysis showed moderately to strongly positive reactions for staining with alpha-naphthyl-butyrate esterase. Also, the mature eosinophils in the patient with lymphoma showed a strongly positive reaction for periodic acid-Schiff staining. The positive reaction for alpha-naphthyl-butyrate esterase and periodic acid-Schiff stain indicates the presence of eosinophilic proliferative disorders in these cases. Surface marker analysis of peripheral eosinophils demonstrated that an increased proportion of eosinophils expressed My7, a marker of granulocytes, in two patients with transient eosinophilia or hyper-eosinophilic syndrome, but not in a patient with lymphoma. In addition, 30-40% of eosinophils expressed Ia antigen, a differentiation marker of eosinophils, in two patients with lymphoma or hypereosinophilic syndrome. Thus, eosinophils in transient eosinophilia showed mature features, and those in hypereosinophilic syndrome showed immature features immunologically. Whereas, eosinophils in lymphoma may have a dysmaturity on the expression of Ia and My7. Cytochemical and immunological analyses of increased eosinophils may play an important role in clarifying the etiology of eosinophilia, especially its association with malignancy or other proliferative disorders.

Antigens, Surface↗

PubMed1994

The eosinophil as an effector cell of the immune response during hepatic allograft rejection.

OBJECTIVE: To evaluate the role of the eosinophil granulocyte during hepatic allograft rejection. DESIGN: (a) A retrospective case-control study and (b) a prospective study of consecutive liver transplant recipients. PATIENTS: In the retrospective study, eight patients with severe rejection in the first month after liver transplantation were compared with six patients without rejection. In the prospective study, 20 consecutive patients were studied for the presence of liver allograft rejection between March 1989 and October 1989. MEASUREMENTS: Absolute eosinophil counts were determined whenever blood was drawn. Serum was analyzed for the presence of two eosinophil granule proteins, major basic protein and eosinophil-derived neurotoxin, on days 7, 14 and 21 after transplantation. Liver biopsy specimens were stained for the presence of major basic protein by means of immunofluorescence using a double-antibody staining technique. The degree of eosinophil infiltration and degranulation was graded using a panel of representative slides. RESULTS: Blood eosinophilia was increased in patients with hepatic allograft rejection (p < 0.05). Serum major basic protein and eosinophil-derived neurotoxin concentrations were similar in patients with and without rejection. Many portal tracts of patients with rejection contained an abundance of eosinophils, and staining for major basic protein revealed the presence of intact eosinophils. In addition, extracellular major basic protein was seen, sometimes in the absence of intact eosinophils or an extensive infiltrate. In patients with severe allograft rejection, major basic protein staining was present in littoral cells lining the sinusoids. CONCLUSIONS: Patients with severe rejection in the first month after liver transplantation often have blood eosinophilia and marked infiltration of portal tracts with eosinophils or evidence of eosinophil degranulation. The presence of major basic protein likely is direct evidence of tissue destruction and may indicate active rejection (major basic protein in eosinophils and extracellular major basic protein, presence of portal infiltrate) or the immediate postinflammatory rejection state (extracellular major basic protein and major basic protein inside littoral cells, absence of portal infiltrate and eosinophils, bile ducts damaged or vanished). These findings underline the importance of the eosinophil as an integral part of the rejection process. We conclude that the presence of eosinophils or their secretion products in the first month after liver transplantation is an indicator of ongoing or recent allograft rejection.

Antibody Formation↗

PubMed1998

IL-4-induced apoptosis in peripheral blood eosinophils.

BACKGROUND: The regulation of eosinophil survival and apoptosis may play a major role in diseases demonstrating increased numbers of circulating and tissue eosinophils such as allergic reactions. Because few promoters of eosinophil apoptosis have been described so far, the objective of this study was to elucidate the role of endogenous factors on eosinophil survival and apoptosis. METHODS AND RESULTS: Highly purified peripheral blood eosinophils were analyzed in the time course from 24 up to 144 hours in culture. Eosinophil survival was assessed with trypan blue dye exclusion and apoptosis was determined by DNA fragmentation gel analysis and ELISA technique with anti-histone antibodies. We confirmed previous results demonstrating prolonged eosinophil survival and inhibited apoptosis by IL-3, IL-5, and GM-CSF. In contrast, eosinophil apoptosis was significantly enhanced by corticosteroids, particularly dexamethasone and hydrocortisone. However, IL-1beta, IL-8, IL-12, platelet-activating factor, TNF-alpha, and eotaxin had no effect on eosinophil survival or apoptosis when compared with culture medium alone. In contrast, IL-4 at concentrations of 100 U/mL or more inhibited eosinophil survival and induced apoptosis. This effect was time dependent and abrogated by preincubation with neutralizing anti-IL-4 antibodies. However, after 96 hours in coincubation, IL-4 did overcome the survival-prolonging effect of IL-3, IL-5, and GM-CSF. IL-4 did not enhance eosinophil surface expression of APO-1/Fas antigen (CD95). In assessing IL-4-mediated effects on eosinophil function, we found no response by means of the release of eosinophil cationic protein or reactive oxygen species. CONCLUSIONS: Taken together, our data present direct evidence for the presence of functional IL-4 receptors on human eosinophils and indicate that IL-4 may lead to resolution of chronic inflammation by induction of eosinophil apoptosis.

Apoptosis↗

PubMed1991

Eosinophilic cationic protein (ECP) in skin disorders.

Eosinophil cationic protein (ECP) is exclusively secreted only by the eosinophilic leukocyte. In this study the ECP concentration in the serum was measured in patients (n = 155) with various skin disorders and compared with the number of circulating eosinophils. The presence of activated eosinophils in the skin was also studied immunohistochemically using the monoclonal antibody EG-2, which recognizes both the eosinophil protein X (EPX/EDN) and ECP. EG-2 distinctly revealed these proteins in the eosinophils and their granules. Non-activated eosinophils were studied with the monoclonal antibody EG-1. In most cases this did not disclose any more eosinophils and often it was located more diffusely and not seldom on collagen fibers. Elevated serum ECP but normal numbers of circulating eosinophils were found in half of the patients with progressive plaque psoriasis and long-standing daily chronic urticaria. In patients with prurigo nodularis, papular erythematous eruptions, vasculitis, purpura and toxic drug reactions, Wells' syndrome, porphyria cutanea tarda and persistent light reaction the serum ECP was increased, although in some cases the number of circulating eosinophils was normal. In these disorders an increased number of activated eosinophils was found in the skin. Both serum ECP and the number of activated eosinophils normalized when the patients' condition improved. In atopic dermatitis the serum ECP and the number of activated eosinophils in the skin were increased only during exacerbation of the disease. High serum levels of ECP and activated eosinophils in the skin are frequent findings in many skin disorders in spite of often normal blood eosinophil counts.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

PubMed1991

Eosinophil cationic protein's role in human hepatic allograft rejection.

Although it is known that eosinophils consistently infiltrate rejecting human liver allografts, their function is unknown. Infiltrating eosinophils can release a cytotoxic substance, eosinophil cationic protein. Furthermore, eosinophil cationic protein may be identified in biopsy specimens using immunoperoxidase staining of an eosinophil cationic protein-specific monoclonal antibody. To study a possible effector role of eosinophils in rejecting liver allografts, 38 serial allograft biopsy specimens from 12 patients with acute rejection, 54 biopsy specimens from 11 patients with allograft dysfunction caused by other causes and 22 biopsy specimens from 8 patients without allograft dysfunction were stained for extracellular eosinophil cationic protein. In addition, the absolute blood eosinophil counts and the portal tract eosinophil percent of the total number of portal tract inflammatory cells were tabulated in these patients until 30 days after transplantation. The blood absolute eosinophil count, portal tract eosinophil percent and incidence of positive extracellular eosinophil cationic protein staining were significantly increased in patients with rejection compared with patients with dysfunction from other causes (p less than 0.05 to 0.005, t test). Furthermore, each of these parameters predicted rejection with excellent sensitivity (75% to 92%) and specificity (91% to 100%). Of patients with rejection, 59% had significant elevation of all three parameters before or during rejection, and 92% had at least two parameters elevated. Conversely, of the patients with dysfunction from other causes, 0% had elevations of any parameter. The recognized cytotoxic properties of eosinophil cationic protein and its almost exclusive presence, along with eosinophils in the blood and allograft biopsy specimens of patients during acute rejection, support the role of the eosinophil as an effector cell in tissue injury during acute liver allograft rejection.

Blood Proteins↗

PubMed1993

FK506 and rapamycin modulate the functional activities of human peripheral blood eosinophils.

Numerous studies suggest that eosinophils may play a role in the inflammatory process of various diseases. Eosinophils are potent effectors of a variety of end-stage inflammatory activities. In addition, eosinophils are highly capable of synthesizing several cytokines which can stimulate the inflammatory responses of other cell types. Eosinophils are also responsive to a number of cytokines in that their in vitro survival as well as their effector functions can be enhanced by various cytokines. Finally, it is well established that the immunosuppressive drugs, FK506 and rapamycin, can inhibit the activation of T cells and mast cells. In the present study, the effects of FK506 and rapamycin on the activities of human peripheral blood eosinophils were investigated. It was first shown that human peripheral blood eosinophils upon stimulation with the calcium ionophore A23187 produced significant amounts of the cytokines, GM-CSF and IL3. FK506 was found to inhibit the synthesis of IL3 and GM-CSF, whereas rapamycin failed to suppress the cytokine production of eosinophils. Since FK506 and rapamycin are structurally related, the ability of rapamycin to reverse the FK506-mediated inhibition on cytokine production of eosinophils was next examined. It was observed that a 250-fold excess of rapamycin was required to antagonize the suppressive effects of FK506 on the cytokine secretion mediated by eosinophils. The capacity of rapamycin to alter the in vitro IL5-mediated survival of eosinophils was also studied. The results of this study demonstrated that rapamycin was effective in reducing the ability of IL5 to maintain the survival of eosinophils in culture; by contrast, FK506 had minimal effects on the IL5-mediated survival of eosinophils. In conclusion, the effects of FK506 and rapamycin on the activities of eosinophils appear to parallel those of mast cells and T cells. Hence, it is possible that FK506 and rapamycin might be acting on signal pathways or molecules that are shared by eosinophils, mast cells as well as T cells. Moreover, these findings suggest that both FK506 and rapamycin can potentially interfere with the capacity of eosinophils to engage in interactive activities that may contribute to the chronicity of inflammatory diseases such as asthma.

Calcimycin↗

PubMed1993

Eosinophils and cytokines.

Cytokines act on eosinophils to regulate eosinophil function, with IL-5 recognised to be especially important in control of eosinopoiesis, eosinophil survival and eosinophil priming. In addition, eosinophils have the capacity to produce cytokines involved in acute and chronic inflammatory and repair processes, as well as to produce cytokines that stimulate eosinophils within an autocrine loop. This paper describes (A) an immunomagnetic selection technique for the purification of human blood eosinophils, and (B) a method that employs immunofluorescence with flow cytometry for measurement of blood and sputum eosinophil surface markers. Having demonstrated that sputum eosinophils express HLA-DR, highly purified blood eosinophils were used to analyse (C) the induction and function of eosinophil HLA-DR. Cytokines have the capacity to induce eosinophil HLA-DR, and are produced by eosinophils as an accessory function during antigen presentation. Finally, preliminary data on (D) eosinophil production of IL-8 is presented. Hence, eosinophils have the capacity to act as immunomodulatory cells within cells networks in allergic and asthmatic inflammation.

Antigens, Surface↗

PubMed1996

Early commitment to the eosinophil lineage by cultured human peripheral blood CD34+ cells: messenger RNA analysis.

Early hematopoietic progenitors expressing the CD34+ phenotype can be harvested from the peripheral blood of normal individuals. We have optimized the liquid culture of human CD34+ peripheral blood progenitors (PBPs) to achieve differentiation into a population of cells consisting almost entirely of eosinophil progenitors and maturing eosinophils. Growth of CD34+ PBPs for 28 days in the presence of the combination of IL-3, granulocyte-macrophage colony-stimulating factor, and IL-5 resulted in an almost 250-fold increase in cell number, yielding a population that contained 83% maturing eosinophils. The residual population consisted of basophils and mast cells (3% by acidic toluidine blue staining, 15.2% by flow cytometric assay for binding to high-affinity IgE receptor) and immature cells. This provides an opportunity to examine the kinetics of the acquisition of specialized mature eosinophil characteristics during eosinophil differentiation. Several host-defense and bioactive proteins are found almost exclusively in eosinophil granules. In addition, stimulated eosinophils, like neutrophils, produce copious amounts of toxic oxygen radicals. We used our culture system and the sensitive technique of reverse-transcriptase polymerase chain reaction to analyze the kinetics of production of messenger RNA transcripts encoding several eosinophil proteins, including five eosinophil granule proteins and four subunit peptides of the superoxide-generating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in small numbers of differentiating eosinophils from peripheral blood CD34+ cells. Freshly isolated CD34+ PBPs contained transcripts for the ubiquitously present housekeeping protein phosphoglucokinase but contained no eosinophil granule protein transcripts and barely detectable amounts of some oxidase protein transcripts. On day 3 of culture, no cells recognizable by histochemical staining as eosinophils could be detected, but transcripts for all five eosinophil granule proteins were present. These transcripts increased several fold during the entire culture period. Similar kinetics were seen for all but one of the NADPH oxidase protein transcripts. However, transcripts for the p67phox NADPH oxidase protein were not detected until day 7, and functional oxidase activity did not appear until day 12. From that point, oxidase activity increased dramatically over the culture period. These studies demonstrate that commitment of CD34+ PBPs to the eosinophil lineage occurs very early, by day 3, but that further events in differentiation must take place before the appearance of histologically staining eosinophil granules and acquisition of functional oxidase capacity.

Adult↗

PubMed1997

A novel murine model of allergic inflammation to study the effect of dexamethasone on eosinophil recruitment.

1. We have developed a novel model of allergen-induced eosinophil into mouse air-pouches following sensitization and challenge with ovalbumin (Ova). This model was used to investigate the mechanism(s) underlying the anti-inflammatory action of the glucocorticoid hormone dexamethasone (Dex). 2. Injection of 10 micrograms Ova into 6-day-old dorsal air-pouches of mice sensitized to the same antigen provoked an intense cell accumulation as early as 6 h post-challenge (0.08 +/- 0.03 and 4.0 +/- 1.0 x 10(5) leucocytes in saline and Ova-treated air-pouches, respectively), maximal at 24 h (0.02 +/- 0.01 and 6.0 +/- 0.8 x 10(5) leucocytes in saline and Ova-treated air-pouches, respectively) and persisted up to 48 h. At the 24 h time-point, the cellular infiltrate consisted of 37% eosinophils, 18% neutrophils and 45% mononuclear cells, as assessed by histological examination. The same ratio of eosinophil/neutrophil was obtained by fluorescence-activated cell sorting (FACS) analysis, since 72% of the polymorphonuclear (PMN) population was positive for very-late antigen-4 (VLA-4) expression. 3. Subcutaneous (s.c.) administration of Dex (50 or 100 micrograms per mouse, -1 h) inhibited eosinophil accumulation into Ova challenged air-pouches by about 70% (P < 0.05) and 75% (P < 0.05), respectively, when compared to controls. Cell accumulation measured at 48 h after Ova injection was also significantly reduced (-75%) by Dex administration at the 24 h time-point (n = 12, P < 0.05). 4. Eosinophil numbers in the bone marrow and blood were quantitated. We found that the sensitization protocol induced a 3 fold increase in eosinophil numbers in the bone marrow (naive mice: 2.7 +/- 0.3 x 10(5); sensitized mice: 8.7 +/- 1.7 x 10(5), P < 0.05) and blood (naive mice: 0.5 +/- 0.2 x 10(5); sensitized mice: 1.5 +/- 0.3 x 10(5), P < 0.01). However, 24 h following Ova challenge, the eosinophil numbers in the bone marrow had dropped (3.7 +/- 0.8 x 10(5) with no change in the circulating pool, suggesting an equilibrium within the eosinophil pools had been reached. 5. Dex administration provoked a profound eosinopaenia in the blood of naive (5.2 +/- 1.5 to 0.9 +/- 0.6 x 10(4)) and sensitized mice (1.5 +/- 0.3 to 0.08 +/- 0.02 x 10(5)) at 4 h. This effect was reversed within 24 h. Dex also inhibited the release of eosinophils from the bone marrow in response to Ova challenge. 6. We show for the first time that express the steroid-inducible protein lipocortin 1 (LC1). FACS analysis of eosinophils emigrated into the Ova challenged air-pouches revealed detectable LC1-like immunoreactivity (373 x 10(3)). These data were also substantiated by LC1 detection in circulating eosinophils of interleukin-5 transgenic mice (strain: CBA/Ca). However, s.c. injection of Dex (50 micrograms) did not alter LC1 levels in blood eosinophils, such that 235 +/- 21 x 10(3) LC1-like molecules per cell were measured after vehicle treatment (n = 5), and 224 +/- 8 x 10(3) LC1-like molecules per cell were associated with this cell type 1 h after steroid treatment (n = 5, not significant). Finally, resident eosinophils (in the pleural cavity) were found to have much higher LC1 levels than that found in the blood circulation (2 fold increase, P < 0.05). 7. Passive immunization of mice against LC1 with a validated antiserum (termed LCS3) and protocol failed to modify the anti-migratory activity exerted by Dex towards eosinophil extravasation into Ova-challenged air-pouches. The steroid (50 micrograms s.c., -1 h) produced a similar degree of inhibition of eosinophil accumulation both in control animals (treated with a non-immune sheep serum) the LCS3-treated mice (-56% and 59%, respectively, n = 15-21, not significant). 8. In conclusion, the air-pouch provides a novel and convenient cavity to study allergen-induced cell recruitment which is sensitive to glucocorticoid hormone treatment. The effect of Dex on eosinophil distribution in these experimental conditions has been studied in detail and

Animals↗

PubMed2000

Interleukin-5 enhances eosinophil adhesion to bronchial epithelial cells.

BACKGROUND: Eosinophil-bronchial epithelial cell interactions are thought to be central to the pathogenesis of asthma, both in terms of the epithelium as a source of pro-inflammatory mediators and as a target for eosinophil-mediated damage. We have therefore investigated adhesion interactions between these two cell types. OBJECTIVES: To determine the role of eosinophil and epithelial activation on eosinophil adhesion to bronchial epithelium and to characterize the adhesion receptors mediating eosinophil adhesion. METHODS: Eosinophils were purified from human peripheral blood by immunomagnetic selection and adhesion to confluent cultures of the airway epithelial cell lines A549 and BEAS-2B was studied. RESULTS: Stimulation of A549 cells with TNFalpha, IFNgamma or a combination of 50 ng/mL of TNFalpha, IFNgamma and IL-1 (cytomix) did not effect eosinophil binding despite an increase in ICAM-1 expression. Similarly stimulation of eosinophils with PAF or IL-5 had no effect on eosinophil binding to medium- or cytokine-treated A549 cells. In contrast stimulation of BEAS-2B cells with cytomix caused a significant increase in eosinophil adhesion. This was associated with an increase in expression of ICAM-1 and induced expression of VCAM-1. Treatment of eosinophils with Mn2+ and IL-5 but not eotaxin, RANTES or PAF also significantly enhanced eosinophil adhesion to medium-treated BEAS-2B cells. Using blocking mAbs we were able to demonstrate that the increased adhesion resulting from stimulation of eosinophils or BEAS-2B cells was in both cases mediated by a combination of CD18 and alpha4 integrins. CONCLUSIONS: This study demonstrates a selective role for IL-5 in mediating integrin-dependent eosinophil adhesion to airway epithelium and once again emphasizes the importance of this cytokine in controlling eosinophil activation in diseases such as asthma.

Antigens, CD↗

PubMed1996

Integrin alpha 4 beta 7 mediates human eosinophil interaction with MAdCAM-1, VCAM-1 and fibronectin.

We have investigated the contribution of integrin alpha 4 beta 7 to human peripheral blood eosinophil adhesive interactions. Immunofluorescence and flow cytometry demonstrated constitutive expression of alpha 4 beta 7 by eosinophils. Expression of alpha 4 beta 7 or alpha 4 beta 7 was not enhanced by eosinophil activation with platelet-activating factor (PAF). Expression of alpha 4 beta 7 was confirmed by immuno-precipitation of 125I-labeled lysates analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE). Approximately 20% of unstimulated eosinophils were adherent to L1-2 cells transfected with vascular cell adhesion molecule-1 (VCAM-1) cDNA, while very few resting eosinophils adhered to mouse mucosal adressin cell adhesion molecule-1 (MAdCAM-1) transfectants. Binding of unstimulated eosinophils to VCAM-1 transfectant was inhibited by HPI 2 (an antibody that blocks both alpha 4 beta 1 and alpha 4 beta 7 functions), but not Act-1, and alpha 4 beta 1 monoclonal antibody (mAb). PAF stimulation resulted in increased binding of eosinophils to MAdCAM-1 transfectants, which was inhibited by both HPI 2 and Act-1. In contrast, PAF did not enhance binding to VCAM 1 transfectants, although binding of PAE-stimulated eosinophils to VCAM-1 could be partially inhibited by Act-1. Stimulation of eosinophils with the beta 7-activating mAb TS2 16 resulted in enhanced binding of eosinophils to both VCAM-1 and MAdCAM-1 transfectants. The increased binding was largely alpha 4 beta 7-dependent. Unstimulated eosinophils bound to soluble recombinant human (rh) VCAM-1 and fibronectin (Fn), coated on 96-well plates in dose-dependent manner. Binding was inhibited by HPI-2 and 4b4, an anti-beta 1 mAb, but not by Act-1. TS2 16 treatment increased adherent cell numbers and this enhanced binding was inhibited by Act-1. We have therefore confirmed that alpha 4 beta 7 is functionally active on unstimulated eosinophils. In contrast, PAF-induced enhancement of eosinophils binding to VCAM-1 or MAdCAM-1 was alpha 4 beta 7-dependent. In addition treatment with TS2 16 resulted in a alpha 4 beta 7-dependent enhancement of eosinophil binding to VCAM-1, MAdCAM-1 and Fn. We therefore hypothesize that alpha 4 beta 7 may have an important role in eosinophil localization in diseases such as asthma and inflammatory bowel disease.

Cell Adhesion↗

PubMed1995

Density of eosinophils reflects activity of disease in allergic asthmatic children.

BACKGROUND: Low density eosinophils are more prominent in asthmatic patients compared with healthy subjects. LDE are metabolically more active and produce more tissue-injuring and spasmogenic proteins than normal eosinophils. OBJECTIVE AND METHODS: With a method providing information about eosinophils of 12 different densities we were able to study eosinophil density characteristics in 24 young patients in detail with allergic asthma in a stable phase, and in 21 patients after a bronchial allergen challenge. RESULTS: Study of the eosinophil density profile of patients and healthy controls revealed two density populations. Patients had more low density eosinophils than controls. In the patients eosinophil density characteristics and in particular the number of low density eosinophils correlated strongly with both FEV1% predicted (p = -0.66, P < 0.001) and FEV1/FVC (p = -0.47, P < 0.01) as well as with bronchial responsiveness to histamine (p = -0.68, P < 0.001) and house dust mite (p = -0.37, P < 0.05). Allergen induced bronchial reactions were associated with an increase in the number (P < 0.001) and percentage (P < 0.05) of low density eosinophils. A selective rise in the number of eosinophils collected from fractions with a low density accounted for the observed rise in the total number of eosinophils. Density changes did not differ between patients with an isolated early reaction and patients with both an early and a late reaction, nor was there a relation between the severity of the late reaction and the shift in eosinophil density. CONCLUSION: In conclusion, peripheral blood eosinophil density characteristics and in particular numbers of low density eosinophils are closely related with indicators of the asthma severity under stable conditions. Allergen inhalation induces a further shift towards lower density suggesting additional activation of the eosinophils.

Adolescent↗