Eosinophil activation by eotaxin--eotaxin primes the production of reactive oxygen species from eosinophils

BACKGROUND: The CC chemokine eotaxin has been shown to possess selective chemotactic activity for eosinophils, the major effector cells in allergic inflammation. Reactive oxygen species (ROS) from eosinophils may damage cells or tissue, such as the mucosal epithelium. In this study, we examined the effect of eotaxin on ROS from eosinophils and compared its activity with RANTES and interleukin (IL)-5. Moreover, we examined the signal transduction of eotaxin and the effect of dexamethasone on ROS from eosinophils. METHODS: Eosinophils were isolated by modified CD16-negative selection. ROS in luminol-dependent or lucigenin-dependent chemiluminescence reaction were examined. Calcium ionophore A23187 was added to the mixture of eosinophils with luminol or lucigenin, and then ROS were determined. RESULTS: Eotaxin primed the production of ROS in a dose-dependent manner. ROS from untreated eosinophils evoked with calcium ionophore A23187 in luminol-dependent chemiluminescence gave a maximal value of 4957+/-1035 intensity counts (IC) (mean+/-SE, n=7) and an integral value of 15.75+/-3.14 IC (x10(-4)), while eosinophils that were treated with eotaxin gave maximal values of 11 142+/-2300 IC (10 nM) and 29165+/-3718 IC (100 nM) and integral values of 41.07+5.44 IC (x10(-4)) (10 nM) and 152.90+/-22.38 IC (x10(-4))(100 nM). Moreover, eotaxin was less effective as a priming agent with lucigenin-sensitive pathways than luminol-sensitive pathways. Among several kinds of eosinophils activating cytokines and chemokines, the priming effect of eotaxin on RO5 was the most potent. Eotaxin-primed ROS were inhibited by pertussis toxin, which ADP-ribolysates G proteins; wortmannin, a phosphatidylinositol-3-kinase inhibitor; and genistein, a tyrosine kinase inhibitor, suggesting the involvement of pertussis toxin-sensitive G proteins, phosphatidylinositol-3-kinase, and tyrosine kinase in the signal transduction of eotaxin. Moreover, dexamethasone inhibited ROS from not only untreated eosinophils but also eosinophils treated with eotaxin. CONCLUSION: Eotaxin may play an important role in the pathogenesis of allergic inflammation through eosinophil activation by priming of eosinophil oxidative metabolism, as well as by involvement in selective eosinophil chemotaxis.     

Thioredoxin reduces C-C chemokine-induced chemotaxis of human eosinophils

Background:  Human thioredoxin (TRX) is one of redox-active proteins that regulate reactive oxidative metabolisms. In recent study, we found that serum levels of TRX were elevated in asthmatic patients with exacerbation; however, few details are known about the physiological role of TRX in allergic inflammation, involving eosinophil infiltration.
Objective:  In the present study, we examined whether TRX modulated C-C chemokine-induced chemotaxis of human eosinophils.
Methods:  Eosinophils were isolated from subjects with mild eosinophilia by modified CD16 negative selection. After incubation with or without recombinant TRX, chemotaxis of human eosinophils was measured using Boyden chamber.
Results:  Preincubation with TRX suppressed eotaxin- and regulated on activation, normal T-cell expressed and secreted (RANTES)-induced chemotaxis of eosinophils. Although, TRX had no effect on the expression of C-C chemokine receptor 3, which is a receptor of eotaxin and RANTES, we demonstrated that the activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinases, which play an important role in eosinophil migration, was attenuated by the treatment with TRX.
Conclusion:  Our results suggest that the elicited TRX is beneficial to reduce allergic inflammation through negative regulation of eosinophil functions and has potential in the treatment of allergic diseases, such as asthma.     

The CC chemokine receptor antagonist met-RANTES inhibits eosinophil effector functions

Eosinophils play an important role in allergic diseases such as allergic asthma, rhinoconjunctivitis and atopic dermatitis. Recruitement of eosinophils to the side of inflammation, the release of reactive oxygen species, leading to tissue damage, and the propagation of the inflammatory response are mediated by chemokines. Thus, the applicability of agents able to inhibit or antagonize chemokine-induced eosinophil activation seems to be of interest in the treatment of allergic diseases. Therefore, the effect of the CC chemokine antagonist, Met-RANTES, on its effect on human eosinophil effector functions in response to RANTES, MCP-3 and eotaxin was investigated. Met-RANTES had no intrinsic activity on [Ca2+]i transients in eosinophils and was able to dose-dependently inhibit [Ca2+]i transients in eosinophils following stimulation with RANTES, MCP-3 and eotaxin. Besides its effect on [Ca2+]i transients, Met-RANTES dose-dependently inhibited actin polymerization in eosinophils and the release of reactive oxygen species following stimulation with RANTES, MCP-3 and eotaxin. The results of this study lead to the conclusion that Met-RANTES is an effective and powerful compound to antagonize effector functions of human eosinophils following stimulation with RANTES, MCP-3 and eotaxin and is therefore a promising therapeutic approach to prevent the invasion and destructive power of eosinophils in allergic diseases.     

The CC chemokine antagonist Met-RANTES inhibits eosinophil effector functions through the chemokine receptors CCR1 and CCR3

Eosinophils are predominant effector cells not only in allergic diseases but also in connective tissue diseases. The recruitment of eosinophils to the site of inflammation and release of reactive oxygen species leading to tissue damage and propagation of the inflammatory response are mediated by chemokines. Thus, agents that would be able to inhibit or antagonize chemokine-induced eosinophil activation are interesting as therapeutical agents. We describe the effect of a chemokine receptor antagonist, Met-RANTES, on human eosinophil effector functions in response to RANTES, monocyte chemoattractant protein (MCP)-3 and eotaxin. Met-RANTES was able to inhibit dose-dependently [Ca²⁺]_i transients in eosinophils following stimulation with RANTES, MCP-3 and eotaxin. Whereas maximal and half-maximal inhibitory effect of Met-RANTES following stimulation with RANTES and MCP-3 were observed at 2 μg/ml and 1 μg/ml, respectively, maximal and half-maximal inhibitory effects of Met-RANTES in response to eotaxin were detected at 10 μg/ml and 3 μg/ml. Moreover, eotaxin-induced [Ca²⁺]_i transients were only half reduced at a Met-RANTES concentration at which RANTES and MCP-3 were completely blocked. Besides its effect on [Ca²⁺]_i transients, Met-RANTES dose-dependently inhibited actin polymerization in eosinophils following chemokine stimulation. Whereas Met-RANTES totally inhibited RANTES- and MCP-3-induced actin polymerization at 5 μg/ml, the eotaxin-induced response was only reduced by 50%. However, Met-RANTES inhibited dose-dependently the release of reactive oxygen species in response to RANTES, MCP-3 and eotaxin. Again, eotaxin-induced release of reactive oxygen species, however, was only half reduced at a Met-RANTES concentration (10 μg/ml) at which RANTES and MCP-3 were completely blocked. The results of this study show that (1) Met-RANTES is an effective and powerful antagonist of effector functions of human eosinophils following stimulation with RANTES, MCP-3 and eotaxin; (2) Met-RANTES seems to be able to antagonize the response of eosinophils through chemokine receptor 1 (CCR1) preferentially to CCR3; (3) Met-RANTES antagonizes eosinophil but not neutrophil effector functions and might be therefore of interest for a new therapeutical approach to prevent the invasion and destructive power of eosinophils in diseases that are accompanied by eosinophil infiltration such as allergic asthma and connective tissue diseases.     

Allergic challenge-elicited lipid bodies compartmentalize in vivo leukotriene C4 synthesis within eosinophils

Eosinophils are an important source of leukotriene (LT)C(4), which can be synthesized within lipid bodies-cytoplasmic organelles where eicosanoid formation may take place. Allergy-driven lipid body formation and function have never been investigated. Here, we studied the in vivo induction and role of lipid bodies within eosinophils recruited to sites of allergic inflammation. Using two murine models of allergic inflammation (asthma and pleurisy), we verified that parallel to the eosinophil influx, allergic challenge also induced lipid body formation within recruited eosinophils. Neutralizing antibodies to eotaxin/CCL11, RANTES/CCL5, or CCR3 partially inhibited lipid body formation within recruited eosinophils in the allergic pleurisy model. Likewise, intrapleural administration of RANTES or eotaxin also induced significant influx of eosinophils loaded with lipid bodies. By immunolabeling, we detected the presence of a key enzyme involved in the leukotriene metabolism-5-lipoxygenase-within eosinophil lipid bodies formed in vivo after allergen challenge. Furthermore, specific immunolocalization of newly formed LTC(4) demonstrated that lipid bodies were the sites of formation of this eicosanoid within infiltrating eosinophils. Therefore, allergic inflammation triggers in vivo formation of new lipid bodies within infiltrating eosinophils, a phenomenon largely mediated by eotaxin/RANTES acting via CCR3 receptors. Such in vivo allergen-driven lipid bodies function as intracellular compartments of LTC(4) synthesis.     

Blood eosinophils from asymptomatic allergies have a reduced capacity to produce oxygen-free radicals

Background The eosinophil granulocyte is an inflammatory cell that plays an active part in diseases such as asthma and rhinitis. This study aimed to investigate oxidative metabolism by blood eosinophils taken from allergic rhinitis patients, asthmatics, and nonallergic controls before and during the birch-pollen season.
Methods Twenty patients with allergy to birch pollen and seasonal symptoms of rhinitis, some of whom were also asthmatic, were followed before and during the birch-pollen season in Sweden. The cells were purified using a Percoll gradient and the MACS system. Eosinophil purity in all samples was >95%. Oxidative metabolism was measured by a chemiluminescence (CL) assay, with luminol and lucigenin acting as enhancers, and PMA, serum-treated zymosan (STZ), interleukin (IL)-5. or RANTES as stimuli. Results The allergic subjects showed reduced luminol CL when activated before the season with PMA (P = 0.40) or STZ (P = 0.0055). This was not seen during pollen exposure. STZ-activated lucigenin CL was also reduced before the season (P = 0.0027). The reduction was most evident in the group with asymptomatic rhinitis. In terms of eosinophil stimulation. IL-5 and RANTES were equally effective in allergic and nonallergic subjects, both before and during the pollen season.
Conclusions Blood eosinophils from asymptomatic allergies may have a lower capacity to produce oxygen-free radicals than eosinophils from nonallergics.     

The inhibitory effect of oxatomide on oxygen radical products from human eosinophils and an eosinophilic cell line]

Recently, much attention has been paid to the role played by the allergic inflammatory reaction in the role of asthma. Eosinophils are considered to be major inflammatory cells in bronchial asthma. Therefore, in this study, eosinophil-mediated oxygen radicals were examined by means of luminol-dependent chemiluminescence. Also, the effect of oxatomide, an anti-allergic agent, which has an inhibitory effect on eosinophil-mediated natural cytotoxicity against bronchial epithelial cells, on the production of oxygen radicals from eosinophils was studied. The results revealed the inhibitory effects of oxatomide on eosinophil-mediated oxygen radicals products. Furthermore, the inhibitory effect of this agent on oxygen radical products from eosinophilic cell-line named EoL-3, which has been established recently, was observed. We concluded from these results that oxatomide not only has anti-allergic activity but also anti-inflammatory properties for eosinophils.     

Human eotaxin represents a potent activator of the respiratory burst of human eosinophils

Increased numbers of eosinophils are found in parasitic infections, autoimmune diseases and allergic diseases such as allergic asthma. They are activated by distinct cytokines and chemokines leading to the immigration in the inflamed tissue and mediate tissue damage by releasing reactive oxygen species. Here, the effect of the recently cloned CC chemokine human eotaxin was investigated for its ability to affect different eosinophil effector functions and compared to the CC chemokines MCP-3 and RANTES. Human eotaxin induced chemotaxis of human eosinophils in a dose-dependent manner. The range of efficacy of the CC chemokines compared to the well-known chemotaxin C5a was eotaxin = RANTES > MCP-3 = C5a. In addition, eotaxin induced rapid and transient actin polymerization, a prerequisite for cell migration, in eosinophils in the same range of efficacy as observed for chemotaxis. To investigate whether eotaxin was able to activate the respiratory burst of eosinophils, release of reactive oxygen species was measured by lucigenin-dependent chemiluminescence. Eotaxin induced production of significantly high amounts of reactive oxygen species at a concentration between 10 ng/ml and 500 ng/ml. Surprisingly, the effect of eotaxin was comparable to the well-known eosinophil activator C5a. The range of efficacy of the CC chemokines compared to C5a in the activation of the respiratory burst was eotaxin = C5a > MCP-3 > RANTES. Production of reactive oxygen species was inhibited by pertussis toxin, staurosporin, genestein and wortmannin. Furthermore, eotaxin induced transient increases in intracellular calcium concentration ([Ca²⁺]i) in human eosinophils. Therefore, pertussis toxinsensitive Gi-proteins, protein kinase C, tyrosine kinase, phosphatidylinositol-3-kinase and transient increases in [Ca²⁺]i are involved in the signal transduction of eosinophils following stimulation with eotaxin. In summary, this study reveals the importance of the CC chemokine eotaxin as a potent activator of the respiratory burst, actin polymerization and chemotaxis. Eotaxin, therefore, plays an important role not only by attracting eosinophils to the site of inflammation but also by damaging tissue by its capacity to induce the release of reactive oxygen species.     

Procaterol upregulates peroxisome proliferator-activated receptor-gamma expression in human eosinophils

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear receptor that regulates immune reaction. We have previously demonstrated that human eosinophils express PPARgamma and that stimulation with a synthetic agonist for PPARgamma attenuated the factor-induced eosinophil activations. However, the modulator of PPARgamma expression in eosinophils has not yet been studied. In this study, we investigated the effect of procaterol, the synthetic beta2-adrenoceptor agonist widely used as bronchodilators in asthma, on the PPARgamma expression in eosinophils. Purified human peripheral blood eosinophil and the eosinophilic cell line EoL-1 were cultured with procaterol. This was followed by PPARgamma measurement using flow cytometer and quantitative real-time RT-PCR. We observed that PPARgamma was constitutively expressed by EoL-1 and the purified eosinophils and that the therapeutic concentration (10(-9)M) of procaterol markedly enhanced PPARgamma protein expression, which was reversed by the selective beta2-adrenoceptor antagonist ICI-118551. The PPARgamma mRNA expression in EoL-1 and eosinophils was also induced by procaterol. These findings suggest that procaterol could modulate the eosinophil function by increasing the expression of PPARgamma.     

Tests for bronchial asthma

One characteristic feature of bronchial asthma is an allergic inflammation of the airways involving eosinophil activation. Since adhesion molecules, cytokines, and chemokines play a critical role in eosinophil infiltration into the tissues, it is of paramount importance to utilize these inflammation-related factors as clinical parameters to assess the status of asthma. For this reason, we measured the level of RANTES and soluble ICAM-1 in patients with asthma. The concentration of plasma RANTES was significantly elevated in asthmatic patients as compared with normal subjects. Patients with asthma attacks exhibited higher RANTES levels than those in remission. sICAM-1 concentration was also higher in serum and sputum in patients with asthma than in healthy subjects. In order to detect eosinophil activation directly, we studied intracellular EG2 expression in eosinophils using whole-blood flow-cytometric analysis. The number of EG2-positive eosinophils was significantly greater in patients with attacks than in asymptomatic subjects. We measured the temperature of expiratory flow and temperature flux as an alternative approach to assess airway inflammation. This study was based on a concept that inflammation would produce heat resulting in the higher temperature. The coefficient of temperature flux was significantly greater in asthmatic patients than in normal controls. Therefore, these new tests may be useful for the evaluation and treatment of allergic inflammation in asthma.     

The potential role of interleukin-13 in eosinophilic inflammation in nasal mucosa

Background Recent studies have revealed that interleukin (IL)-13, as well as IL-4, causes de novo surface expression of vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells of the umbilical vein and accelerates selective eosinophil migration. However, its role in allergic rhinitis remains to be clarified. Of particular interest is whether IL-13 upregulates VCAM-1 expression in human mucosal microvascular endothelial cells (HMMECs), to which eosinophils adhere in nasal mucosa.
Methods To understand the potential role of IL-13 in eosinophilic inflammation in nasal mucosa, we examined the effects of IL-13 on the adhesiveness between HMMECs and eosinophils.
Results IL-13 increased VCAM-1 expression in HMMECs, the adhesiveness of endothelial cells to eosinophils, and the transendothelial migration. On the other hand, IL-13 decreased the adhesiveness of eosinophils to HMMECs, and, as a result, accelerated eosinophil infiltration. Those effects are more potent than was those of IL-4. In addition, we also report that the amount of IL-13 in nasal mucosa was higher than that of IL-4.
Conclusions These results strongly indicate that IL-13. as well as IL-4, may be important in eosinophilic inflammation in the nasal mucosa.     

Increased expression of RANTES, CCR3 and CCR5 in the lesional skin of patients with atopic eczema

BACKGROUND: Atopic eczema (AE) is a relapsing inflammatory disease based on IgE sensitization and characterized by peripheral blood eosinophilia and eosinophil infiltration into the lesional skin. In the patch test reaction of AE by allergens, an increased infiltration of activated eosinophils has been demonstrated peaking at 24-48 h. Regulated on activation normal T cell expressed and secreted (RANTES/CCL5) is a chemokine that induces eosinophil migration, and CCR3 and CCR5 are the receptors of RANTES. OBJECTIVE: In order to further clarify the pathomechanisms of eosinophil infiltration in ongoing chronic inflammation in the skin of patients with AE and its relation to disease severity, we examined the expression of RANTES and its receptors CCR3 and CCR5 in challenged and unchallenged lesional skin of AE. METHODS: We examined the number of RANTES+ cells, CCR3+ cells, CCR5+cells, activated (EG2+) eosinophils and CD3+ T cells in normal skin of healthy volunteers, and in challenged lesional skin (24 h after mite patch test) as well as unchallenged lesional skin of AE patients by immunohistochemistry. The cellular source of RANTES, CCR3 and CCR5 was analyzed by double immunohistochemistry using specific antibodies to RANTES, CCR3 or CCR5, and antibodies to ECP (EG2) or CD3. RESULTS: The numbers of RANTES+ cells, CCR3+ cells, CCR5+ cells, EG2+ cells and CD3+ cells were all significantly increased in challenged (mite patch-tested) lesional skin of AE patients as compared to those in unchallenged lesional skin and normal skin. The numbers of these cells in unchallenged lesional skin were greater than those in normal skin. The number of EG2+ cells in the unchallenged lesional skin correlated with both the peripheral blood eosinophil count and the SCORAD index. The number of EG2+ cells in challenged lesional skin correlated with the number of CCR5+ cells. Activated eosinophils and T cells expressed RANTES and various proportions of these cells were CCR3+ and CCR5+ in both challenged and unchallenged lesional skin. CONCLUSION: Taken together, these results suggest that RANTES as well as its receptors CCR3 and CCR5 may play important roles in the orchestration of eosinophil infiltration in ongoing chronic inflammation in AE, and also reflect the severity of disease.     

Chemokines induce eosinophil degranulation through CCR-3

BACKGROUND: Such CC chemokines as eotaxin and RANTES induce preferential eosinophil recruitment in allergic inflammation. They also elicit proinflammatory effector functions of eosinophils, such as enhanced adhesion and superoxide generation. Eosinophil degranulation by chemokines, however, has not been studied in detail. OBJECTIVE: The purpose of this study was to identify chemokines and their corresponding receptors that induce eosinophil degranulation by using a panel of chemokines and blocking antibodies to candidate receptors. METHODS: Highly purified eosinophils were preloaded with Fura-2 and stimulated with a panel of chemokine ligands for 14 known chemokine receptors: CCR1 to CCR8, CXCR1 to CXCR4, CX3CR1, and XCR1. Calcium influx was measured with fluorescence spectrometry. Eosinophils were also stimulated with the chemokines in the presence or absence of IL-5, and levels of eosinophil-derived neurotoxin were measured in the supernatant with RIA. Specific antibodies to chemokine receptors were used to block degranulation. RESULTS: Calcium influx was induced by monocyte chemotactic protein (MCP) 1, MCP-3, MCP-4, RANTES, eotaxin, IL-8, and stromal cell-derived factor 1alpha, which are chemokines that bind several chemokine receptors. However, degranulation was induced only by CCR3 ligands, including MCP-3, MCP-4, RANTES, and eotaxin. Priming of eosinophils with IL-5 enhanced CCR3 ligand-induced degranulation but did not cause non-CCR3 ligands to induce eosinophil-derived neurotoxin release. An antibody against CCR3 significantly inhibited degranulation induced by CCR3 ligands, eotaxin, or RANTES. CONCLUSION: These results suggest that chemokine-induced eosinophil degranulation, a major effector of eosinophil functions, is mediated through only CCR3, although some non-CCR3 ligands induce calcium influx in eosinophils. CCR3 may be an important target in the treatment of eosinophilic inflammation.     

Cyclophosphamide-induced blood and tissue eosinophilia in contact sensitivity: Mechanism of hapten-induced eosinophil recruitment into the skin

The mechanism leading to selective production and accumulation of eosinophils in certain allergic skin diseases is unknown. Cyclophosphamide treatment (150 mg/kg) of BALB/c mice 48 h before sensitization with picryl chloride (PCI) resulted in striking blood and tissue eosinophilia, maximal at 13 days. Blood eosinophilia was not induced by the sensitization with oxazolone and 2, 4-dinitrofluorobenzene. Challenge with 1% PCI, but not croton oil caused preferential eosinophil accumulation into the dermis, which was associated with the enhanced expression of vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells. Intraveneous administration of anti-VCAM-1 monoclonal antibody abrogated eosinophil infiltration. In this murine model, we examined the role of several cytokines, including chemokines in inducing selective tissue eosinophilia in vivo. Local administration of antibodies against interleukin (IL)-1β, IL-4, tumor necrosis factor (TNF)-α, and RANTES, but not against IL-5 before challenge inhibited hapten-induced eosinophil recruitment. Intradermal injection of recombinant (r)IL-1β, rIL-4, rTNF-α, rRANTES, and rMIP-1α induced marked eosinophil accumulation. Nonetheless, intradermal rIL-5 was not a chemoattractant for eosinophils in vivo. Our findings suggest that IL-1β, IL-4, TNF-α, and RANTES contribute to the selective accumulation of eosinophils in contact sensitivity reaction. Although circulating IL-5 can activate eosinophils and prolong their survival, locally secreted IL-5 is not crucial for inducing eosinophil recruitment into the skin.     

Tumour necrosis factor-α-induced expression of intercellular adhesion molecule-1 on human eosinophilic leukaemia EoL-1 cells is mediated by the activation of nuclear factor-κB pathway

BACKGROUND: Intercellular adhesion molecule-1 (ICAM-1) has been shown to mediate the adhesion and migration of eosinophils to the site of allergic inflammation. However, molecular mechanisms regulating the expression of ICAM-1 in eosinophils are still being elucidated. We investigated the effect of tumour necrosis factor-alpha (TNF-alpha) on ICAM-1 expression of eosinophils. METHODS: The surface expression of ICAM-1 on a human eosinophilic leukaemic cell line, EoL-1, was assessed by immunocytochemical staining. The phosphorylation of inhibitor kappa B-alpha (IkappaB-alpha) and p38 mitogen-activated protein kinase (MAPK) was detected by Western blot. Nuclear factor kappa-B (NF-kappaB) pathway-related genes were evaluated by the cDNA expression array system, whereas the activity of NF-kappaB was measured by electrophoretic mobility shift assay (EMSA). RESULTS: TNF-alpha was found to induce the cell surface expression of ICAM-1. A specific proteasome inhibitor N-cbz-Leu-Leu-leucinal (MG-132), but not a p38 MAPK inhibitor (SB 203580), was found to suppress the TNF-alpha-induced expression of ICAM-1 on EoL-1 cells. The gene expressions of ICAM-1, NF-kappaB and IkappaBalpha were up-regulated after the stimulation with TNF-alpha. Further, TNF-alpha was shown to induce IkappaB-alpha phosphorylation and degradation, thereby indicating the activation of NF-kappaB. In EMSA, there was a shifted NF-kappaB band on TNF-alpha-treated cells with or without SB 203580, but no shifted band was observed on MG-132-treated cells. CONCLUSION: In vitro studies of EoL-1 cells, an eosinophilic leukaemic cell line, confirmed that NF-kappaB plays an important role in the expression of ICAM-1 and recruitment of eosinophils in allergic inflammation.     

Red blood cells regulate eosinophil chemotaxis by scavenging RANTES secreted from endothelial cells

BACKGROUND: Eosinophils play a critical role in the pathogenesis of allergic diseases. CC chemokines, such as regulated on activation, normal, T cell expressed, and secreted (RANTES), are key regulators of eosinophil locomotion. Although eosinophils migrate from the bloodstream into tissues, mechanisms that generate a chemogradient across the endothelium remain to be fully elucidated. OBJECTIVE: We first examined the polar secretion of RANTES by endothelial cells. We also studied the functional scavenging effect of red blood cells (RBCs) on RANTES secreted into the intravascular side. METHODS and RESULTS: Endothelial cells were cultured in a transwell chamber with a membrane pore size of 0.45, 3.0, and 8.0 microm and stimulated with TNF-alpha, IL-1beta, or IFN-gamma from the apical or basolateral side for 16 h. The measurement of RANTES in the supernatant was performed by ELISA. We did not see any difference in the amount of RANTES secreted from the cytokine-stimulated endothelium between inner (intravascular side) and outer (extravascular side) wells separated by the 8.0-microm membrane, although apical polarization was observed with the 0.45-microm membrane. The addition of RBCs (hemoglobin (Hb): 0.5-15 g/dL) to the apical supernatant of TNF-alpha-stimulated endothelial cells reduced the RANTES level in a concentration-dependent manner. The treatment of supernatant on the intravascular side with RBCs significantly enhanced the migration of eosinophils. CONCLUSION: RBCs possess a scavenging effect on intravascular RANTES, and thereby regulate transendothelial migration of eosinophils. Our findings suggest a new role of RBCs in allergic inflammation.     

The role of interleukin-5, interleukin-8 and RANTES in the chemotactic attraction of eosinophils to the allergic lung

BACKGROUND: Bronchoalveolar lavage (BAL) fluid from patients with birch-pollen allergy lavaged during the season showed an elevated chemotactic activity for eosinophils compared with BAL fluid from the same patients before the start of the season. AIM: The aim of this study was to identify the eosinophil chemotactic agents in the BAL fluid, to compare these findings with in vitro studies on selected cytokines, and to investigate the interactions between these cytokines. METHODS: Neutralizing antibodies for interleukins (IL) -2, -5 and -8, RANTES and leukaemia inhibitory factor (LIF) were added to the BAL fluid, and the chemotactic activity was tested with eosinophils from allergic donors. Eosinophils from healthy donors were preincubated with IL-5 in order to mimic the primed state of eosinophils from allergics, and the migration towards recombinant IL-5, IL-8, and RANTES in different combinations was measured. Eosinophils from allergic donors were also used. RESULTS: Anti-IL-5, anti-IL-8 and anti-RANTES inhibited the chemotactic activity in the BAL fluid. Recombinant RANTES induced migration, which was enhanced by preincubation of the cells with IL-5. Only eosinophils from symptomatic allergics responded to IL-8, and IL-5 was not sufficient to prime normal eosinophils in vitro to an IL-8 response. A negative correlation was found between the level of in vivo activation of the cells and their response to IL-5, and a positive correlation with the response to RANTES. CONCLUSION: IL-8 and RANTES are important for eosinophil accumulation to the lung of pollen-allergic asthmatics. IL-5 alone may not be responsible for the priming of eosinophils in vivo, but is an essential cofactor for the other chemoattractants.     

CC chemokines and transmigration of eosinophils in the presence of vascular cell adhesion molecule 1.

BACKGROUND: Interaction between eosinophil alpha4 integrin and vascular cell adhesion molecule 1 (VCAM-1) expressed on activated endothelial cells may be a key step in the selective recruitment of eosinophils from the circulation to sites of inflammation. OBJECTIVE: To investigate the factor(s) that induces transmigration of eosinophils after firm adhesion via the alpha4 integrin/VCAM-1 pathway. METHODS: We examined the effects of a variety of inflammatory mediators on the migration of eosinophils across recombinant human (rh) intracellular adhesion molecule 1- or rhVCAM-1-coated Transwell filters or VCAM-1-expressing human pulmonary microvascular endothelial cells (HPMECs) that had been stimulated with interleukin 4 (IL-4) and tumor necrosis factor alpha. The number of eosinophils that had transmigrated was evaluated by measuring eosinophil peroxidase activity. RESULTS: The CC chemokines RANTES (regulated on activation, normal T-cell expressed, and secreted), eotaxin, eotaxin 2, monocyte chemotactic protein 3 (MCP-3), and MCP-4 each increased eosinophil transmigration across rhVCAM-1-coated filters compared with fetal calf serum-blocked or rh intracellular adhesion molecule 1-coated filters (P < .01). On the other hand, platelet-activating factor, C5a, formyl-methionyl-leucil-phenylalanine, granulocyte-macrophage colony-stimulating factor, IL-5, and IL-8 did not enhance migration across rhVCAM-1. The enhancement of migration by RANTES in the presence of rhVCAM-1 was blocked by an anti-alpha4 integrin monoclonal antibody. CC chemokines augmented eosinophil transmigration across VCAM-1-expressing HPMECs compared with resting HPMECs (P < .01). Conversely, the transmigration induced by platelet-activating factor, C5a, formyl-methionyl-leucil-phenylalanine, or IL-8 was not modified by the expression of VCAM-1 on HPMECs. CONCLUSIONS: CC chemokines induce transendothelial migration of eosinophils after interaction between eosinophil alpha4 integrin and endothelial VCAM-1.     

Human platelets produce granulocyte-macrophage colony-stimulating factor and delay eosinophil apoptosis

An association between eosinophils and platelets has been described in several diseases, most notably asthma. Although the mechanisms through which platelets influence eosinophil behavior are not well defined, platelets seem to contribute to the selective accumulation of eosinophils at sites of allergic inflammation by virtue of their ability to produce eosinophil chemotactic factors. We report here for the first time that platelets delay apoptosis, thus enhancing eosinophil survival. A marked inhibition of spontaneous apoptosis was observed using eosinophil:platelet ratios of 1:50, 1:25, 1:10, and 1:5. Moreover, promotion of eosinophil apoptosis by either pronase or dexamethasone was also inhibited greatly in the presence of platelets. The antiapoptotic effect mediated by platelets was dependent on the release of soluble products and was significantly inhibited by neutralizing antibodies directed to GM-CSF. Studies performed by flow cytometry, directed to analyze the cellular source of this cytokine, demonstrated that intracytoplasmic GM-CSF is present in resting platelets. Moreover, GM-CSF was found in platelet supernatants, at concentrations able to prevent eosinophil apoptosis. Our findings support a novel mechanism through which platelets may contribute to eosinophil accumulation at allergic inflammatory sites.