Dai Kurachi

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.

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.

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.

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.

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.

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.

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.

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.

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.

Induction of β2 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.