Degranulation of eosinophils mediated by intercellular adhesion molecule-1 and its ligands is involved in adhesion molecule expression on endothelial cells-selective induction of VCAM-1.

BACKGROUND: Adhesion molecules and eosinophils may play an important role in the pathogenesis of allergic inflammatory reactions. OBJECTIVE: We attempted to clarify eosinophil activation, such as degranulation, by signaling through adhesion molecule and to determine whether degranulation is involved in adhesion molecule expression on endothelial cells. METHODS: Eosinophils were cultured with or without recombinant soluble intercellular adhesion molecule-1 (ICAM-1), and the levels of eosinophil cationic protein and eosinophil-derived neurotoxin were determined. The influence of these eosinophil granule proteins or supernatant from eosinophil cultured with ICAM-1 on the expression of ICAM-1 or vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells was also examined by flow-cytometric analysis. RESULTS: Supernatant levels of eosinophil granule protein were significantly increased by culture for 4 hourss or 16 hours with recombinant soluble ICAM-1, suggesting degranulation by adherence to ICAM-1. Both granule proteins and the supernatants of eosinophils cultured with recombinant soluble ICAM-1 induced expression of ICAM-1 and VCAM-1 on endothelial cells, with the latter showing a more prominant increase. CONCLUSION: Degranulation mediated through adherence to endothelial cells by ICAM-1 and its ligands may be involved in the expression of adhesion molecules, such as ICAM-1 or VCAM-1, on these cells. Our finding of the selective induction of VCAM-1 expression suggests that eosinophil adherence to endothelial cells, even if it is because of ICAM-1, may be involved in selective eosinophil recruitment and accumulation at sites of allergic inflammation.

Inhibitory effect of ibudilast (KC-404) on the expression of the beta2 integrin family on an eosinophilic cell line (EoL-1).

BACKGROUND: Adhesion molecules are thought to play a key role in inflammatory processes in bronchial asthma. We previously observed an increased expression of the beta2-integrin family on an eosinophilic cell line (EoL-1) by platelet-activating factor (PAF). OBJECTIVE: In the present study, we examined the effect of ibudilast (KC-404), a novel anti-asthma agent, on beta2 integrin expression induced by PAF. MATERIAL AND METHODS: EoL-1 cells (1x10(6)/mL) were incubated in the presence or absence of 10(-6) M ibudilast (KC-404), then cells were cultured in the presence or absence of PAF (10(-7) M) for 45 minutes. Flow cytometric analysis for CD11a, CD11b, and CD18 expression was examined. RESULTS: Ibudilast had an inhibitory effect on beta2 integrin expression induced by PAF [CD11a: 84.8% versus 73.1% (preincubation with ibudilast), CD11b: 35.8% versus 26.2%, CD18 74.9% versus 65.6%]. CONCLUSIONS: Ibudilast (KC-404) has anti-inflammatory activities through its inhibitory effect on the expression of adhesion molecules on eosinophils.

Priming effect of RANTES on eosinophil oxidative metabolism.

BACKGROUND: RANTES has been shown to possess chemotactic activity for eosinophils, which have also been considered to play a role in allergic inflammation through reactive oxygen species. Thus, in this study, we examined the effect of RANTES on radical oxygen products from eosinophils. METHODS: Purified eosinophils by CD16-negative selection or an eosinophilic cell line (EoL-1) were incubated with or without RANTES (2.5 x 10(-6) M). To the mixture of eosinophils and luminol, calcium ionophore (A23187) or opsonized zymosan (OZ) was added, and radical oxygen products were determined by luminol-dependent chemiluminescence for 600 s. RESULTS: Eosinophil-mediated radical oxygen products of untreated eosinophils produced with A23187 gave a peak value of 14.09 +/- 2.40 (mean +/- SE, n = 12) relative light units (RLU) and an integrated value of 3232.20 +/- 513.09 RLU. However, with treatment with RANTES, a peak value of 18.66 +/- 2.40 RLU and an integrated value of 5301.05 +/- 561.02 RLU were obtained. Eosinophil oxidative metabolism-induced A23187 or OZ was apparently augmented by the preincubation with RANTES. In addition, the radical oxygen products of EoL-1 showed similar results. CONCLUSIONS: Thus, we concluded that RANTES may play an important role in the pathogenesis of allergic inflammation through its involvement in eosinophil activation, as evidenced by oxygen products, as well as in selective eosinophil infiltration as selective eosinophil chemoattractant.

Elevated local production of neopterin from alveolar macrophages in patients with internal lung diseases.

1. We measured the neopterin level in the supernatant of cultured alveolar macrophages from patients with interstitial lung diseases (ILD patients) as a marker for the activation of alveolar macrophage. 2. In ILD patients, the supernatant neopterin level (40.1 +/- 7.8 pmol/ml) was significantly higher (P < 0.01) than that in control subjects (10.0 +/- 1.6 pmol/ml). 3. We also found that macrophage-colony stimulating factor (M-CSF) and interleukin-2 (IL-2) augmented neopterin production from alveolar macrophage in both ILD patients (51.6 +/- 10.4 and 60.1 +/- 10.8 pmol/ml, respectively, P < 0.01) and control subjects (28.1 +/- 6.0 and 25.7 +/- 4.9 pmol/ml, respectively). 4. These findings suggest that alveolar macrophages produce neopterin by M-CSF or IL-2.

Possible involvement of mononuclear cells stimulated with specific allergen from asthmatic patients in ICAM-1 expression on the eosinophilic cell line.

Adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1), recently have been considered to play a key role in inflammatory processes in asthma. Thus, from the point of view of cell interactions between mononuclear cells and eosinophils, we examined whether the supernatant of mononuclear cells (MNC) obtained from mite-allergic asthmatic patients cultured with specific allergen is involved in ICAM-1 expression using an eosinophilic cell line (EoL). ICAM-1 expression was induced by the supernatant of MNC from mite-allergic asthmatic patients stimulated with mite allergen as well as by a combination of IL-3, GM-CSF, and IL-5. Thus, we could conclude that some cytokines produced by specific allergen-stimulated MNC in asthmatics might be involved in allergic inflammation through the induction of adhesion molecule expression such as ICAM-1 on eosinophils in asthma or allergic disorders.

Possible low response of C-reactive protein production in eosinophilic pneumonia.

C-reactive protein (CRP) in the patients with eosinophilic pneumonia was examined compared with that of bacterial pneumonia. While no difference between erythrocyte sedimentation rate (ESR) in eosinophilic pneumonia (mean +/- SE: 74.5 +/- 10.6 mm/hr) and that in bacterial pneumonia (86.2 + 7.7 mm/hr) was observed, serum CRP level (3.87 +/- 1.24 mg/dL) and alpha 2-macroglobulin level (182.53 +/- 13.00 mg/dL) in eosinophilic pneumonia were lower in comparison with those in bacterial pneumonia (14.89 +/- 1.34 mg/dL, 315.65 +/- 11.54 mg/dL, respectively), suggesting that the pathogenesis of eosinophilic pneumonia might involve defective secretion of certain cytokine related to the production of acute-phase reactant proteins such as interleukin-6.

Effect of RANTES on intracellular expression of EG2 antigen in eosinophils.

RANTES, which is released from thrombin-stimulated platelets, is a member of the 8-kDa cytokine family that has been shown to possess chemotactic activity for eosinophils. The effect of RANTES on the intracellular expression of EG2 antigen in eosinophils was examined in this study. RANTES augmented the intracellular expression of EG2 antigen in eosinophils. These findings suggest that RANTES not only modulates the chemotactic activity of eosinophils but also intensifies the function of eosinophil activation.

Adhesion to fibronectin augments eosinophil radical oxygen products.

Fibronectin, an extracellular matrix component, is a ligand for very late activation antigen (VLA)-4, which is one of the beta 1-integrin family of molecules expressed by eosinophils. This study examined the effect of adherence to fibronectin on radical oxygen products from eosinophils. Adhesion of eosinophils to fibronectin resulted in enhancement of eosinophil production of radical oxygen species, as determined by luminol-dependent chemiluminescence of eosinophils stimulated with calcium ionophore. It was concluded that eosinophil adhesion to extracellular matrix via adhesion molecules may be important in the pathogenesis of allergic inflammation through eosinophil activation.

Increased eosinophil oxidative metabolism by treatment with soluble intercellular adhesion molecule-1.

Adhesion molecules may play an important role not only in adherence of inflammatory cells (particularly eosinophils) to an inflamed focus but also in activation of these cells. It is therefore of interest to evaluate eosinophil activation via intercellular adhesion molecule-1 (ICAM-1) and the beta 2-integrin family, namely CR3 (Mac-1), lymphocyte function-associated antigen (LFA)-1 alpha and LFA-1 beta, which are ligands for ICAM-1. Reactive oxygen species generated by eosinophils have also been considered capable of causing airway injury at the inflamed focus. This study examined the effect of recombinant soluble ICAM-1 and its ligands on eosinophil-induced radical oxygen products in terms of luminol-dependent chemiluminescence. Recombinant soluble ICAM-1 augmented eosinophil oxidative metabolism. It was concluded that signaling via adhesion molecules might play an important role in the pathogenesis of allergic inflammation through activation of eosinophils, e.g. an increase in oxidative metabolism.

Possible release of eosinophil granule proteins in response to signaling from intercellular adhesion molecule-1 and its ligands.

The presence of a large variety of membrane receptors and the identification of cytotoxic molecules (mainly granule basic proteins) have indicated that eosinophils should br considered as effector cells. It has recently been suggested that adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1), play an important role in allergic inflammation, for example in bronchial asthma. This study therefore investigated the possible release of granule protein in response to signaling from ICAM-1 and its ligands. The concentrations of eosinophil cationic protein and eosinophil-derived neurotoxin in supernatants of eosinophils were significantly greater (p < 0.05) in the presence of recombinant soluble ICAM-1 than without it. These results suggest that signaling from ICAM-1 and its ligands might induce eosinophil activation and might be involved in degranulation of eosinophil granule proteins, e.g. eosinophil cationic protein and eosinophil-derived neurotoxin.

A comparative study of eosinophil isolation by different procedures of CD16-negative depletion.

Eosinophils were isolated by the three methods of CD16-negative depletion: 1) magnetic beads, 2) fluorescence-activated cell sorter (FACS), and 3) complement reaction. Their purity, yield, and viability were compared. The second procedure produced well purity and viability (94.65 +/- 1.51% and 94.98 +/- 1.40%, respectively) but low yield of eosinophils (65.47 +/- 2.47%). The viability of cells obtained by the third procedure was not efficient (80.83 +/- 2.85%), while the purity and the yield were efficient (96.23 +/- 1.09% and 90.75 +/- 1.72%, respectively). In conclusion, the magnetic beads method (purity: 98.02 +/- 0.45%, yield: 91.05 +/- 2.43%, viability: 97.57 +/- 0.37%) was the most advantageous of these three procedures. Moreover, in the functional assay, radical oxygen products from eosinophils isolated by the procedure with complement reaction were less than with the magnetic beads or FACS procedures.

Induction of beta 2 integrin expression on an eosinophilic cell line (EoL-1) by the supernatant of mononuclear cells stimulated with specific allergen from asthmatic patients.

Adhesion molecules recently have been considered to play an important role in inflammatory processes in bronchial asthma. Our previous study revealed high expression of beta 2-integrin family (CR3, LFA-1 alpha, CD18) on hypodense eosinophils. Thus, from the point of view of cell-to-cell interaction between mononuclear cells and eosinophils, we examined whether the supernatant of mononuclear cells obtained from mite-allergic asthmatic patients cultured with specific allergen mite-allergen is involved in adhesion molecule expression using an eosinophilic cell line (EoL-1). These characteristics of beta 2-integrin family expression (high expression of beta 2 integrin) were induced by the supernatant of mononuclear cells from mite-allergic asthmatic patients stimulated with mite-allergen as well as with a combination of the recombinant eosinophilopoietic growth cytokines (IL-3, GM-CSF and IL-5). Thus, we could conclude that some cytokines produced by specific allergen stimulated mononuclear cells in asthmatics might be involved in allergic inflammation through the induction of adhesion molecule expression on eosinophils in asthma or allergic disorders.

[Effect of platelet-activating factor (PAF) on eosinophil adhesion to plasma-coated glass using eosinophilic cell lines (EoL-1, EoL-3)].

Adhesion molecules on various inflammatory cells, especially on eosinophils are now considered to play an important role in allergic inflammations such as bronchial asthma. In this study, we examined the effect of platelet-activating factor (PAF) on eosinophil adhesion to plasma-coated glass using eosinophilic cell lines (EoL-1, EoL-3), since PAF might be an important mediator as an eosinophil chemotactic agent in bronchial asthma. PAF markedly augmented adherence to plasma-coated plates. This finding suggests that PAF might up-regulate the inflammatory reaction in bronchial asthma through its action as an augmentative agent for eosinophil adhesion as well as its action as an eosinophil chemotactic agent.

Characteristics of bone marrow cells of patients with eosinophilic pneumonia.

Bone marrow features were studied to clarify the pathogenesis of peripheral blood eosinophilia in eosinophilic pneumonia. We observed (1) increases in the number of nucleated cells in patients with this disease as compared with that of control subjects (205,944 +/- 104,253 versus 118,154 +/- 76,306) (P < .05); (2) increases in the myelocyte to erythrocyte ratio (6.95 +/- 4.06 versus 4.02 +/- 1.36) (P < .05) mainly due to a marked increase in the eosinophilic series; and (3) an increase in the eosinophilic series with advance in the maturation process, the degree of increase becoming greater with the increase in eosinophilic maturation. These findings suggest that differentiation and maturation of the precursor cells into mature eosinophils were accelerated in the bone marrow in eosinophilic pneumonia by some lymphokines as eosinophilopoietic growth factors.

RANTES augments radical oxygen products from eosinophils.

RANTES, which is released from thrombin-stimulated platelets, is a member of the 8-kD cytokine family that has been shown to possess chemotactic activity for eosinophils. Thus, in this study, we examined the effect of RANTES on radical oxygen products from eosinophils. RANTES treatment resulted in the enhancement of peak value and integrated value of productivity of eosinophil-mediated radical oxygen products determined by luminol-dependent chemiluminescence of EoL-3 cells stimulated with A23187. In addition, the radical oxygen products of EoL-1 or eosinophils showed similar results. Thus, we could conclude that platelets might play an important role in the pathogenesis of allergic inflammation through the involvement in the selective eosinophil infiltration and eosinophil activation by releasing RANTES.

Induction of expression of adhesion molecules on an eosinophilic cell line (EoL-1) by the supernatant of lymphocytes stimulated with specific allergen from asthmatic patients.

Recently, adhesion molecules have been considered to play an important role in inflammatory processes in bronchial asthma. To extend our understanding of the high-intensity expression of adhesion molecules (CR3, LFA-1 alpha, LFA-1 beta, ICAM-1) on hypodense eosinophils, which was observed in our previous study, we examined whether the supernatant of lymphocytes from mite-allergic asthmatic patients is involved in adhesion molecule expression using an eosinophilic cell line (EoL-1). These characteristics of adhesion molecule expression were induced by the supernatant of lymphocytes obtained from mite-allergic asthmatic patients cultured with specific allergen as well as a combination of recombinant cytokines (IL-3, GM-CSF, IL-5). Thus, we could conclude that some lymphokines produced by specific allergen in asthma might be involved in the high-intensity expression of adhesion molecules on hypodense eosinophils in asthma or allergic disorders.

Effect of platelet-activating factor and platelet factor 4 on eosinophil adhesion.

The effect of platelet-activating factor (PAF) and platelet factor 4 (PF4) on the adhesion of isolated human eosinophils or eosinophilic cell lines (EoL-1, EoL-3) was examined. Both PAF and PF4 augmented eosinophil adhesion to plates coated with AB plasma or recombinant soluble intercellular adhesion molecule-1 (r-sICAM-1). These findings suggest that PAF and PF4 not only modulate chemotactic activity of eosinophils but also intensify the function of eosinophil adhesion. Since PAF and PF4 induce the expression of adhesion molecules (LFA-1 alpha, LFA-1 beta, CR3) on eosinophils, we could conclude that PAF or PF4 are closely related to eosinophil accumulation not only as chemotactic agents but also as augmentative agents for eosinophil adhesion through involvement in functional eosinophil adherence as well as surface expression of adhesion molecules on eosinophils.

Possible induction of intercellular adhesion molecule-1 (ICAM-1) expression on endothelial cells by platelet-activating factor (PAF).

We demonstrated that PAF and IL-1 markedly induce ICAM-1 expression on endothelial cells. These findings suggest that PAF not only modulates inflammation by the attraction and activation of eosinophils or by platelet activation, but also intensifies such phenomena by induction of ICAM-1 expression on endothelial cells.

Induction of IgE-Fc receptor (Fc epsilon R2/CD23) expression on lymphocytes from patients with mite-allergic bronchial asthma by mite allergen.

The present study was designed to clarify whether Fc epsilon R2 can be induced on lymphocytes of patients with bronchial asthma by stimulation with specific antigen. Expression of Fc epsilon R2 on freshly isolated lymphocytes (at 0 hour) was significantly higher in both patients with mite-allergic asthma and those with nonmite-atopic asthma than in healthy individuals. In addition, expression of Fc epsilon R2 on lymphocytes was still higher in patients during acute asthma. Marked induction of Fc epsilon R2 on lymphocytes was observed 48 hours or more after addition of mite-allergen in patients with mite-allergic asthma while no Fc epsilon R2 expression was induced in patients with nonmite-atopic asthma or healthy individuals. Induction of Fc epsilon R2 expression was not observed at 30 minutes, two hours, and five hours, which are equivalent in kinetics to immediate asthmatic response and late asthmatic response in allergen inhalational challenges.