Surface Of Eosinophils Research Articles

Increased adhesive properties of eosinophils in sickle cell disease

Objective A role for leukocytes in vasoocclusion is becoming increasingly recognized. Here we investigate a possible role for the eosinophil in sickle cell anemia (SCA). Patients and methods Eosinophils were isolated from whole blood samples of 59 steady-state SCA homozygous SS and control individuals using Percoll gradient separation, followed by immunomagnetic sorting. Adhesion of cells to FN was evaluated using static adhesion assays (60 min, 37°C, 5% CO 2) and eosinophil adhesion molecular expression was observed by flow cytometry. Results SCA patients presented significantly elevated absolute eosinophil numbers. Furthermore, eosinophils isolated from these individuals demonstrated a significantly greater adhesion (∼70% increased) to fibronectin (FN) than normal eosinophils in static adhesion assays. Coincubation of eosinophils with integrin-blocking monoclonal antibodies in adhesion assays showed that an association of the VLA-4, LFA-1, and Mac-1 integrins mediate the adhesion of SCA eosinophils to FN. Flow cytometry demonstrated that the expression of these integrins, however, was unaltered on the surface of SCA eosinophils, suggesting that the increased SCA eosinophil adhesion is a consequence of increased integrin affinity or avidity. SCA eosinophil adhesion to FN was further increased by the cytokine, GM-CSF, indicating that inflammation processes may further stimulate eosinophil adhesion in these patients. Conclusion We report that eosinophil numbers may be significantly increased in SCA individuals and that these cells appear to exist in an activated state. Such alterations may indicate a role for the eosinophil in the vasooclusive process.

Histamine H4 antagonism: a therapy for chronic allergy?

Chronic inflammatory diseases such as bronchial asthma, allergic gastrointestinal disease, and atopic dermatitis are characterized by the selective recruitment and activation of distinct subtypes of leukocytes, particularly eosinophils, into the tissue from peripheral blood (Rothenberg, 1998). Eosinophils, along with mast cells, are postulated to play a key role in the pathophysiology of these chronic diseases. The striking accumulation of eosinophils in allergic disease has stimulated intense scientific examination leading to the discovery of numerous molecular mechanisms responsible for this process. Eosinophils, like all leukocytes, migrate from the vascular lumen, across the endothelial cell surface into the appropriate tissue sites. This elaborate mechanism, known as transendothelial migration, has been shown to be a highly orchestrated process (Springer, 1994). The migratory pathway, or chemotaxis, of the leukocyte is directly influenced by soluble molecules known as chemoattractants. These molecules reside along a tissue gradient, and bind to and activate G-protein coupled receptors (GPCRs) on the leukocyte cell surface. Several eosinophil chemoattractants have been extensively studied. Some of these molecules, the classical chemoattractants, include the complement cleavage fragments, leukotrienes and platelet-activating factor (PAF). These chemoattractants have been shown to stimulate the recruitment of a wide variety of leukocyte subtypes, and are therefore nonselective. In contrast, chemotactic cytokines (or chemokines) such as eotaxin-1, -2, -3, and monocyte chemoattractant protein (MCP)-4 are more selective for the recruitment of leukocytes associated with allergic inflammation, such as eosinophils, mast cells, and basophils.

Caspase-3-Mediated Apoptosis of Human Eosinophils by the Tissue-Invading Helminth Paragonimus westermani

Background: Eosinophils are important cells in host immunity to infections by parasitic worms. Tissue-invasive helminthic parasites have been known to induce apoptosis of immune cells for their successful establishment in vivo. We have previously found that high doses of excretory-secreted products (ESP) secreted by lung fluke Paragonimus westermani newly excysted metacercariae (PwNEM), which cause pulmonary or extrapulmonary paragonimiasis in human beings, accelerate eosinophil cell death. However, little is known about the mechanism of eosinophil apoptosis induced by the Paragonimus-derived products. Objective: We examined involvement of caspase 3 activation in eosinophil cell death induced by ESP produced by PwNEM. Methods: Eosinophils were isolated from the peripheral blood of healthy donors by CD16-negative immunomagnetic selection. We examined the inhibitory effect of pan-caspase inhibitor on ESP-triggered phosphatidylserine (PS) externalization on the outer surface of eosinophils and intracellular activation of caspase 3 in cell lysates treated with the ESP. Results: When ESP secreted by PwNEM were incubated for up to 3 h with eosinophils, they increased surface exposure of PS on eosinophils in a time- and dose-dependent manner. This proapoptotic effect of the ESP on eosinophils was significantly inhibited by pretreatment of cells with pan-caspase inhibitor z-VAD-fmk, and was completely abolished by heat inactivation of ESP at 56°C. The activated forms of caspase 3 were also clearly detected in eosinophils incubated with ESP. Moreover, ESP potently inhibited prolonged survival of eosinophils induced by cytokines such as IL-5, IL-3, and GM-CSF. Conclusion: These results suggest that ESP secreted by PwNEM contain biological active factors causing caspase-3-mediated apoptosis of human eosinophils, thereby enabling the larvae to evade and to subvert the tackle by eosinophils during the early phase of the infection.

Donor to donor variation of the chemokine receptor CCR1 on human eosinophils and its prolonged internalization by RANTES

Rationale Whereas recent studies underlies the fundamental importance of the CC chemokine receptor CCR3 for the recruitment of eosinophils in allergic diseases controversial data about the expression of CCR1 on eosinophils exist. Therefore, the purpose of this study was to investigate the expression and regulation of CCR1 on eosinophils. Methods Flow cytometric analysis of whole blood eosinophils or CD16-negative selected freshly isolated eosinophils from atopic and non-atopic donors using an anti-CCR1 mAb. Results We found that 32% of the donors (n=24) were positive and 68% (n=49) were negative to express CCR1. Pre-incubation of isolated CCR1-positive eosinophils for 24 h with IL-3 (p=0.021), RANTES (p<0.001), MCP-3 (p=0.004), MCP-4 (p=0.006) and MIP-1α (p=0.005) induced a significant down-regulation of CCR1 when compared to medium-incubated eosinophils. Internalization experiments using CCR1-positive donors revealed that RANTES (2 min: p=0.003; 60 min: p=0.038) is more effective to induce CCR1 internalization than MIP-1α. Whereas CCR1 re-expression after stimulation with MIP-1α reached pre-stimulation levels, RANTES showed a prolonged CCR1 internalization (120 min: p<0.001; 24 h: p=0.01). Conclusions A subpopulation of human donors are able to express CCR1 on the surface of eosinophils which helps to explain the different response of eosinophils to MIP-1α in previous studies. It is tempting to speculate whether the higher positive charge of RANTES in comparison to its overall negative charge of MIP-1α is responsible for the prolonged CCR1 internalization in response to RANTES.

The effect of fexofenadine on expression of intercellular adhesion molecule 1 and induction of apoptosis on peripheral eosinophils

Rationale The role of eosinophils in the pathogenesis of allergic disorders has been demonstrated by several studies. Recently, it has been suggested that II generation antihistamines, widely used to relieve allergic symptoms, may have anti-inflammatory effects. To assess the possible anti-inflammatory in vitro activity of fexofenadine, a selective H1 receptor antagonist, we evaluated its capacity to modulate the expression of adhesion molecules LFA-1 and ICAM-1 on eosinophil surface and to induce apoptosis of eosinophils. Methods To analyze the expression of adhesion molecules, eosinophils from healthy donors were cultured in the presence of INF-γ and TNF-α with various concentrations of fexofenadine, incubated with monoclonal antibodies anti-ICAM-1 and LFA-1 and then analyzed by flow-cytometry. In order to evaluate apoptosis of eosinophils, cells stimulated with IL-5, in the presence of different concentrations of fexofenadine, have been incubated with a phosphatidylserine (PS)-binding protein (annexin V) FITC conjugated, and then analyzed by flow-cytometry. Apoptosis was evaluated as a percentage of annexin V positive cells. Results In the present study fexofenadine did not cause any significant changes in the expression of LFA-1 but it was demonstrated to be able to inhibit the expression of ICAM-1 at concentrations between 10−3 M and 10−4 M. Moreover, concentrations of fexofenadine from 10−3 M to 6×10−4 M induced a significative increment in the percentage of apoptotic cells. Conclusions The data reported in this article indicate the possibility of obtaining relevant anti-inflammatory pharmacological effects, other than antihistamine activity, by fexofenadine, such as inhibition of ICAM-1 expression and induction of eosinophil apoptosis.

A novel metalloproteinase, leukolysin, and development of a standardized immunoassay

Rationale Leukolysin is a matrix metalloproteinase that is expressed on the cell surface of eosinophils and mast cells. Leukolysin degrades extracellular matrix components and allows eosinophils and mast cells to infiltrate into bronchial airways and mediate tissue damage. Leukolysin may be an ideal candidate target for the development of asthma drugs. The purpose of this experiment was to measure serum leukolysin and determine its relationship to airway inflammation. Methods A highly sensitive chemiluminescent enzyme immunoassay (ELISA) was developed utilizing guinea pig anti-leukolysin and rabbit anti-leukolysin reagents made in our laboratories. History, spirometry, exhaled breath condensate (EBC) and serum was collected on 31 asthmatic and 10 non-asthmatics volunteers and the serum and EBC leukolysin levels were determined by immunoassay. Results To validate the immunoassay blinded ELISAs were performed on serial standardized dilutions of leukolysin from 200 ng/ml to 0.08 ng/ml. Regression analysis of the standardized dilutions and relative light units (RLU) showed a linear relationship at 1:100 dilution R squared=0.9765. No definite trends in these small sample populations were found. Conclusions A standardized immnunoassay was developed that accurately measures leukolysin. This assay will prove to be valuable in future research for measuring leukolysin in serum and EBC and determining its role in bronchial asthma.

Endothelial cells modulate eosinophil surface markers and mediator release.

Migration from blood to tissue modulates eosinophil function, possibly through interactions with endothelial cells. The effects of contact with and migration through endothelial cells on eosinophil expression of surface markers and release of leukotriene C4 were evaluated. A small proportion (2.6%) of eosinophils spontaneously migrated through endothelial cell monolayers. Activation of endothelial cells by interleukin (IL)-4 or IL-1beta slightly increased this migration (to 12.4%), which became much greater when a chemoattractant was placed in the lower chamber (84.3%). However, the chemotactic effect was downregulated by pretreating endothelial cells with interferon gamma (IFN-gamma; 63.1%). At baseline, 5% of eosinophils expressed CD69; this increased to 30.7% in culture on untreated endothelial cells and to 50.9% on IL-1beta-pretreated endothelial cells. This effect was mediated through intercellular adhesion molecule-1/CD11b interaction. Eosinophil migration through endothelial cells further increased CD69 expression to 63.9% and also increased CD35 expression from 83.3 to 91.3%. Upon stimulation, eosinophils that had migrated through endothelial cells produced more leukotriene C4 than control cells (872.4 and 103.9 pg x mL(-1), respectively). Endothelial cell pretreatment with IL-4 or IL-1beta further increased leukotriene C4 release (1,789.1 and 2,895.1 pg x mL(-1), respectively), whereas pretreatment with IFN-gamma decreased it (293.7 pg x mL(-1)). These data show that in vitro interactions with endothelial cells upregulate eosinophil membrane receptor expression and mediator release and that these effects are differently modulated by T-helper cell type 1 and 2 cytokines. These eosinophil modulations may play an important role in asthma pathogenesis.

Ligation of intercellular adhesion molecule 3 inhibits GM-CSF production by human eosinophils

Intercellular adhesion molecule 3 (ICAM-3) has recently been identified on the surface of eosinophils. The purpose of this study was to characterize ICAM-3 expression on eosinophils in response to cytokines and to determine whether ligand binding of ICAM-3 modulates inflammatory responses of eosinophils, as it does in other leukocytes. To determine effects of ICAM-3 on eosinophil function, we isolated human eosinophils and used a monoclonal antibody directed against the epitope of ICAM-3 that binds to leukocyte-function antigen-1 to mimic binding of ICAM-3 and this natural ligand. We measured granulocyte-macrophage colony stimulating factor (GM-CSF) production by unstimulated eosinophils and eosinophils stimulated with ionomycin (1 micromol/L), both in the presence and absence of this anti-ICAM-3 antibody. We found that 99% of eosinophils expressed ICAM-3, regardless of whether allergic symptoms were present or absent. Expression of ICAM-3 was not enhanced by proinflammatory cytokines. Expression of ICAM-3 was reduced in apoptotic cells and in cells incubated with the combination of GM-CSF and tumor necrosis factor-alpha (n = 3). Antibody binding of ICAM-3, which mimics leukocyte-function antigen-1 binding, had no effect on baseline GM-CSF production but reduced by 80% the production of GM-CSF stimulated by ionomycin (control 1969 pg/mL +/- 1259 SD versus anti-ICAM-3 396 pg/mL +/- 207 SD, n = 8) and reduced GM-CSF mRNA content. ICAM-3 is highly expressed on the surface of human eosinophils, and downregulation of GM-CSF production by anti-ICAM-3 mAb suggests that ICAM-3 ligation may inhibit eosinophil inflammatory responses and survival.

The eosinophilias, including the idiopathic hypereosinophilic syndrome.

The eosinophilias, including the idiopathic hypereosinophilic syndrome.

CC chemokine receptor 1 enhances susceptibility to Leishmania major during early phase of infection.

CC chemokine receptor 1 (CCR1) is expressed on the surfaces of monocytes, lymphocytes, neutrophils and eosinophils. CC chemokine receptor 1 not only regulates leucocyte chemotaxis, but also plays a role in the regulation of Th1/Th2 cytokine responses. To determine the role of CCR1 in regulation of immune response during Leishmania major infection, we analysed the course of cutaneous L. major infection in CCR1-deficient C57BL/6 mice (CCR1-/-) and compared with similarly infected wild-type mice (CCR1+/+). Following L. major infection, CCR1-/- mice developed significantly smaller lesions containing fewer parasites than CCR1+/+ mice. Furthermore, the severity of the inflammation as assessed by the degree of leucocyte infiltration at the site of infection was similar in CCR1+/+ and CCR1-/- mice. Although both groups developed significant antibody responses following L. major infection, CCR1-/- mice produced significantly lower IgE. On day 20 postinfection, LmAg-stimulated lymph node cells from L. major-infected CCR1+/+ and CCR1-/- mice produced comparable levels of IL-12 and IFN-gamma, but those from CCR1-/- mice produced significantly less IL-4 and IL-10. By day 70, lymph node cells from both CCR1+/+ and CCR1-/- mice produced significant amounts of IL-12 and IFN-gamma but low IL-4. At both time points, the draining lymph nodes from CCR1+/+ and CCR1-/- mice contained similar number of leucocytes. These results demonstrate that CCR1 plays a role in pathogenesis of cutaneous L. major infection. Moreover, they also indicate that CCR1 exacerbates L. major infection in C57BL/6 mice by up-regulating Th2-like response rather than inhibiting Th1 development or/and influencing leucocyte chemotaxis.

Interleukin‐2 primes eosinophil degranulation in hypereosinophilia and Wells' syndrome

Patients with hypereosinophilia frequently suffer from eosinophil-mediated damages of the heart, lungs, skin, and other organs, while some do not. The reason(s) for this difference is not known. We observed that eosinophils from most patients with hypereosinophilia express the alpha-chain of the IL-2 receptor (CD25), and that IL-2 enhances platelet-activating factor-stimulated release of eosinophil cationic protein from CD25-expressing but not from CD25-negative eosinophils. Such a "priming" effect has previously been described for eosinophil hematopoietins. These data suggest that patients with increased eosinophil surface CD25 expression are at higher risk of eosinophil degranulation and subsequent tissue damage when IL-2 is present at inflammatory sites.

Blockade of focal clustering and active conformation in beta 2-integrin-mediated adhesion of eosinophils to intercellular adhesion molecule-1 caused by transduction of HIV TAT-dominant negative Ras.

We transduced dominant negative (dn) HIV TAT-Ras protein into mature human eosinophils to determine the signaling pathways and mechanism involved in integrin-mediated adhesion caused by cytokine, chemokine, and chemoattractant stimulation. Transduction of TAT-dnRas into nondividing eosinophils inhibited endogenous Ras activation and extracellular signal-regulated kinase (ERK) phosphorylation caused by IL-5, eotaxin-1, and fMLP. IL-5, eotaxin-1, or fMLP caused 1) change of Mac-1 to its active conformation and 2) focal clustering of Mac-1 on the eosinophil surface. TAT-dnRas or PD98059, a pharmacological mitogen-activated protein/ERK kinase inhibitor, blocked both focal surface clustering of Mac-1 and the change to active conformational structure of this integrin assessed by the mAb CBRM1/5, which binds the activation epitope. Eosinophil adhesion to the endothelial ligand ICAM-1 was correspondingly blocked by TAT-dnRas and PD98059. As a further control, we used PMA, which activates ERK phosphorylation by postmembrane receptor induction of protein kinase C, a mechanism which bypasses Ras. Neither TAT-dnRas nor PD98059 blocked eosinophil adhesion to ICAM-1, up-regulation of CBRM1/5, or focal surface clustering of Mac-1 caused by PMA. In contrast to beta(2)-integrin adhesion, neither TAT-dnRas nor PD98059 blocked the eosinophil adhesion to VCAM-1. Thus, a substantially different signaling mechanism was identified for beta(1)-integrin adhesion. We conclude that H-Ras-mediated activation of ERK is critical for beta(2)-integrin adhesion and that Ras-protein functions as the common regulator for cytokine-, chemokine-, and G-protein-coupled receptors in human eosinophils.

Acute radiation colitis in patients treated with short-term preoperative radiotherapy for rectal cancer.

The histopathologic features of acute radiation-induced colitis in humans have been described in occasional, >20-year-old studies, but they have not been analyzed in detail. We characterize such findings in 34 patients with rectal cancer who underwent surgery a few days after preoperative irradiation with 25 Gy given over 5-7 days, and we compare the results to the histopathologic features detected in 18 patients treated by a conventional preoperative irradiation protocol consisting of 45 Gy during 5 weeks followed by surgery after a time interval of at least 3 weeks. Short-term preoperative irradiation therapy generally induced severe mucosal inflammation characterized by increased cellularity of the lamina propria, prominent eosinophilic infiltrates, crypt disarray, surface and crypt epithelial damage, nuclear abnormalities, and presence of apoptotic bodies in the crypt epithelium. These histopathologic features were absent or detected only occasionally in the patient group treated according to the long-term preoperative irradiation protocol. Despite acute severe inflammation, none of the patients treated by short-term irradiation developed perioperative complications. These observations indicate that acute radiation colitis may remain clinically silent and resolve spontaneously within a few weeks after irradiation. Given the widening acceptance of short-term preoperative irradiation protocols for rectal cancer, pathologists should be aware of the rather characteristic histologic findings of acute radiation colitis and avoid unnecessary concern of clinicians. The differential diagnosis includes infectious colitis, collagenous and ischemic colitis, nonsteroidal anti-inflammatory drug-associated colitis, and chronic idiopathic inflammatory bowel disease.

Staphylococcal exotoxins exert proinflammatory effects through inhibition of eosinophil apoptosis, increased surface antigen expression (CD11b, CD45, CD54, and CD69), and enhanced cytokine-activated oxidative burst, thereby triggering allergic inflammatory reactions

Background: Staphylococcus aureus colonization of the skin represents a potent trigger factor of atopic dermatitis. Our previous studies demonstrated that in atopic dermatitis eosinophil apoptosis is significantly delayed. Objective: We sought to investigate the effect of staphylococcal exotoxins (SETs) on eosinophil apoptosis and functional activities. Methods: Apoptotic eosinophils were investigated by determining their hypodiploid DNA peak. Eosinophil surface-antigen expression and intracellular production of hydrogen peroxide were assessed by means of flow cytometric analysis and respiratory burst by lucigenin-dependent chemiluminescence. Results: The SETs SEA, SEB, SEC, and toxic shock syndrome toxin 1 significantly inhibited eosinophil apoptosis in a manner comparable with that of high concentrations of IL-3. The LPS inhibitor polymyxin B was found to significantly inhibit LPS-mediated, but not SET-mediated, inhibition of apoptosis. Neither SETs nor LPS was able to modulate eosinophil surface expression of CD9, CD11a, CD16, CD40, CD44, or CD63. However, 24- and 48-hour incubation with all SETs, but not with LPS, significantly upregulated expression of CD11b and CD45 in a manner similar to that of IL-3, whereas dexamethasone induced a downregulation. Moreover, all SETs resulted in a significant upregulation of CD54 after 24 hours but not after 48 hours. Interestingly, CD69 was upregulated by means of IL-3 and SEB only. Neither direct stimulation of eosinophils nor 24-hour incubation with SETs or stimulation with SETs after a 24-hour prestimulation with IL-3, IL-5, or GM-CSF resulted in a significant extracellular production of reactive oxygen species or in a significant production of intracellular hydrogen peroxide. However, SEB and toxic shock syndrome toxin 1 were able to enhance cytokine-induced respiratory burst. Conclusion: Taken together, our data demonstrate that SETs may modulate the course of the allergic inflammatory response through modulation of potent eosinophil effector functions. This may be of particular importance in atopic dermatitis, in which the skin is regularly colonized with SET-producing S aureus. (J Allergy Clin Immunol 2002;109:477-84.)

Effects of the cysteinyl leukotriene receptor antagonist, montelukast, on eosinophil differentiation in an experimental mouse model of allergic rhinitis

The cysteinyl leukotriene (CysLT) LTD 4 is a potent lipid mediator which is synthesized and released from various inflammatory cells, and contributes to the pathophysiology associated with allergic disease. The cloning and characterization of the LTD 4 receptor (CysLTtR), has provided a useful target for therapy of rhinitis and asthma; in particular, montelukast sodium is a selective antagonist that binds with high affinity to CysLT]R on the surface of eosinophils and their progenitors. In the current study, we investigated the effects of montelukast on bone marrow eosinophil progenitor responses, using our recently developed experimental murine model of allergic rhinitis. BALB/c mice were sensitized and challenged with ovalbumin (OVA), and montelukast (5 mg/kg) or placebo was administered by gavage, for 1 or 2 wk, 1 hr before daily intra-nasal OVA challenge. Bone marrow colony assays in methylcellulose were performed, and day 6 eosinophil/basophil colony forming units (Eo/Baso-CFU) were enumerated. Colony cells (day 10) were isolated and histologically stained to allow morphologic assessment of eosinophil differentiation. Bone marrow Eo/Baso-CFU numbers, grown in the presence of interleukin 5 (IL-5) in vitro, were significantly lower than that those from controls in 2 wk (p<0.002), but not 1 wk, montelukast-treated mice. Highly significant suppression of eosinophil maturation compared to control (p<0.001) was observed in colony cells from both 1 wk and 2 wk montelukast-treated groups. These results indicate that montelukast, presumably via CysLT] R antagonism, limits the number of progenitors that differentiate into mature eosinophils; the combination of reduced CFU, as well as lower numbers of mature eosinophils per CFU, suggests that these effects may be occurring at more than one stage of eosinophil differentiation. The anti-allergic effects of montelukast may include targeting the recruitment of eosinophil progenitors from the bone marrow.

The molecular biology of nasal polyposis.

The molecular biologic events in the development of nasal polyps are now becoming unraveled. It appears that eosinophils are the dominant inflammatory cell present in this tissue. The events leading up to the extravasation of eosinophils into the lamina propria nasal polyps are regulated by the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1 beta. These cytokines upregulate very late antigen-4 on the surface of eosinophils and vascular cell adhesion molecule-1 on the surface of the endothelial blood vessel. Chemokines such as RANTES (regulated upon activation, normal T-cell expressed and secreted) and eotaxin are responsible for the movement of eosinophils into the lamina propria of the nasal polyp. The release of major basic protein has an effect on alteration of the epithelial architecture and on the sodium and chloride flux into and out of the apical epithelial cell of the tissue. Finally, the alteration of the surface epithelium results in a defect in the migration of the cystic fibrosis transmembrane regulator protein to the apical surface. These two events, the release of major basic protein from the eosinophil and the alteration of the architecture of the surface epithelium, lead to an increase in sodium absorption and resultant edema: the hallmark of the pathology of the nasal polyp.

Regulation of Surface FcεRI Expression on Human Eosinophils by IL-4 and IgE

Background: Recent studies have demonstrated that eosinophils from allergic patients express low levels of FcΕRI on their surface, but the regulatory mechanisms of eosinophil surface FcΕRI expression are not fully understood. We investigated whether IL-4 and IgE, which are reported to regulate surface FcΕRI expression on human mast cells, are able to affect surface FcΕRI expression in normal human eosinophils. Methods: Eosinophils purified from peripheral blood were cultured with IL-5 and with or without IL-4 and/or IgE, and surface FcΕRI expression was analyzed by flow cytometry using an anti-FcΕRI mAb, CRA-1. Results: Apparent FcΕRI expression (∼1% of mast cell FcΕRI levels) was observed in eosinophils cultured with both IL-4 and IgE. A combination of IL-4 (≧1 ng/ml) and IgE (≧ 0.5 µg/ml) was necessary for the maximal induction of surface FcΕRI expression. In the presence of IL-4 and IgE, eosinophils cultured for 2 days demonstrated low but statistically significant levels of surface FcΕRI, which reached a plateau after 7 days of culture. However, cross-linkage of surface FcΕRI molecules by CRA-1 or anti-IgE did not induce any eosinophil activation. Conclusions: IL-4 and IgE can affect the levels of surface FcΕRI on normal human eosinophils. FcΕRI expression on eosinophils may be regulated by a mechanism similar to that in mast cells.

Aminooxypentane-RANTES Induces CCR3 Activation and Internalization of CCR3 from the Surface of Human Eosinophils

Eosinophils are predominant effector cells in allergic diseases attracted by several CC chemokines into the inflammatory tissue. According to their important role in attracting leukocytes, several kinds of chemokine receptor antagonists have been developed. Therefore, the aim of this study was to investigate the effect of aminooxypentane (AOP)-RANTES on the activation of the CC chemokine receptor 3, CCR3, exemplary on human eosinophils, because they represent the dominant CCR3+ cell type. AOP-RANTES dose-dependently induced an increase of intracellular calcium concentration ([Ca²⁺]_i) and a release of reactive oxygen species, which could be inhibited by pertussis toxin, in human eosinophils from normal nonatopic donors. AOP-RANTES was as effective as RANTES but less effective than eotaxin and eotaxin-2 in the activation of the respiratory burst. Flow-cytometric analyses revealed that eosinophils constitutively expressed the CC chemokine receptors CCR1 and CCR3, whereas CCR5 was not expressed. AOP-RANTES, RANTES, eotaxin and eotaxin-2, but not Met-RANTES, induced a downregulation of CCR3 at 37°C. Reexpression of CCR3 on eosinophils was observed within 120 min. Whereas no differences of CCR3 downregulation and recycling after stimulation with AOP-RANTES, RANTES, eotaxin and eotaxin-2 were found there exists a distinct profile of activity with respect to the activation of the respiratory burst in human eosinophils.

Stimulation of eosinophil IgE low-affinity receptor leads to increased adhesion molecule expression and cell migration.

Immunoglobulin binding on eosinophil surface receptors results in activation of these cells. Evaluating blood eosinophils from atopic subjects, it was investigated whether ligation of immunoglobulin E low-affinity receptor (FcepsilonRII/ CD23) with specific monoclonal antibodies (Mabs) resulted in enhanced eosinophil migration and adhesion molecule expression. Eosinophils from 20 subjects with allergic asthma (atopic individuals) and nine nonatopic normal individuals (controls) were purified using Percoll gradients. The effect of antihuman CD23 Mabs on: 1) eosinophil migration through human umbilical vein endothelial cells (HUVECs); and 2) eosinophil expression of the adhesion molecules leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18), macrophage antigen-1 (Mac-1, CD11b/CD18) and very late activation antigen-1 (VLA-4, CD49d/CD29) was evaluated by specific Mab staining and flow cytometric analysis. As compared to controls, freshly isolated eosinophils from atopic individuals showed enhanced migration through HUVECs (p<0.05) and increased LFA-1 expression (p<0.01), but similar Mac-1 and VLA-4 expression (p>0.1 for both). In both controls and atopic individuals, eosinophil incubation with antihuman CD23 Mabs induced a dose-dependent increase in cell migration through HUVECs, significant at antihuman CD23 Mab concentrations of 5 microg x mL(-1) (p>0.05 for all). Similarly, incubation of the cells with antihuman CD23 Mabs induced dose-dependent upregulation of LFA-1 and Mac-1 expression, whereas no changes in VLA-4 expression were observed (p>0.1). Finally, the enhanced eosinophil migration induced by antihuman CD23 Mab stimulation was significantly inhibited by antihuman LFA-1 (84+/-14% (mean+/-SEM); p<0.01) and VLA-4 Mabs (47+/-15%; p<0.05) but not by antihuman Mac-1 Mabs (p>0.1). In both atopic and control subjects, immunoglobulin E, low-affinity receptor stimulation induces functional changes in eosinophils characterized by increased eosinophil migration associated with enhanced late function antigen-1 and Mac-1 expression.

Differential Activation of CC Chemokine Receptors by AOP-RANTES

RANTES (regulated on activation normal T cell expressed) has been found at elevated levels in biological fluids from patients with a wide range of allergic and autoimmune diseases and is able to attract several subtypes of leukocytes including eosinophils and monocytes into inflamed tissue. Amino-terminal modifications of RANTES produce receptor antagonists which are candidates for blocking this cellular recruitment. Met-RANTES has been shown to modulate inflammation in vivo, while AOP-RANTES is a potent inhibitor of R5 human immunodeficiency virus type 1 (HIV-1) strains and has been shown to down-modulate CCR5 and prevent recycling of the receptor. We have studied the effect of AOP-RANTES in eosinophil activation and have found that it is able to efficiently elicit eosinophil effector functions through CCR3, as measured by the release of reactive oxygen species and calcium mobilization, whereas Met-RANTES is inactive in these assays. AOP-RANTES is found to inhibit CCR3-mediated HIV-1 infection with moderate potency, in contrast to its potent inhibition of CCR5-mediated HIV-1 infection. Furthermore, we have investigated the abilities of these modified proteins to down-modulate CCR1 and CCR3 from the surface of monocytes and eosinophils. We show here that AOP-RANTES is much less effective than RANTES in down-modulation of CCR1. Surprisingly, recycling of CCR1 was minimal after incubation with RANTES while there was complete recycling with AOP-RANTES. In the case of CCR3, no significant difference was found between RANTES and AOP-RANTES in down-modulation and recycling. It therefore appears that trafficking of RANTES receptors follows different patterns, which opens up potential new targets for therapeutic intervention.