Inhibition Of Ccl11, Ccl24, And Ccl26-induced Degranulation In Hl-60 Eosinophilic Cells By Specific Inhibitors Of Mek1/mek2, P38 Map Kinase, And Pi 3-Kinase

Abstract

Eosinophilic leukocytes are the cellular hallmark of allergic inflammation. Apart from being potent eosinophils chemoattractants, the eotaxins CCL11, CCL24 and CCL26 are capable of activating eosinophils to generate reactive oxygen species, lipid mediators of inflammation and degranulation of toxic granule proteins. Due to their central role in eosinophil trafficking and activation, understanding the signal transduction mechanism of the eotaxin-induced eosinophil effector functions may provide an innovative therapeutic strategy for eosinophil-associated diseases. Thus, these investigations were conducted to delineate signal transduction mechanisms of CCL11, CCL24 and CCL26-induced eosinophil peroxidase (EPO) degranulation following pretreatment of cells with or without a specific inhibitor of MEK1/MEK2 (U0126), inhibitor of p38 MAP kinase (SB203580) or a specific inhibitor of PI 3-kinase (LY294002). Results have shown that CCR3-mediated eotaxin-induced eosinophilic degranulation was concentration-dependently reduced by specific inhibitors of ERK1/ERK2, p38 MAP kinase and PI 3-kinase. However, the rank order of U0126 with respect to inhibition of chemokine-induced degranulation was CCL11 = CCL24 > CCL26. Potentiation of eotaxin-induced EPO degranulation by IL-5 was also seen. These investigations have not only confirmed the reported co-operativity between IL-5 and the eotaxins but also showed that the eosinophil-degranulating capabilities of the eotaxin CCL11, CCL24 and CCL26 is a consequence of activation of ERK1/ERK2, p38 MAP kinase and PI 3-kinase. Thus, these signaling molecules may provide the biochemical basis for mechanism-based therapy of allergic inflammatory diseases.

Autoregulation of CCL26 synthesis and secretion in A549 cells: a possible mechanism by which alveolar epithelial cells modulate airway inflammation

“…In indicated experiments, aliquots of eosinophils were pretreated with 55 nM of the CCR3 antagonist (S)-methyl-2-naphthoylamino-3-(4-nitrophenyl)-propionate (SB-328437) (EMD Biosciences, La Jolla, CA) for 2 min (5). Microtiter plates were incubated at 35°C in an atmosphere of 5% CO 2 for 2 h, and absorbances were read at 550 nm to determine superoxide dismutase-inhibitable reduction of ferricytochrome C (2,40).…”

“…Clone 15 HL-60 eosinophils (ATCC CRL-1964) were purchased from American Type Culture Collection. Final differentiation and culturing were carried out in RPMI-1640 (Cellgro by Mediatech, Herndon, VA) supplemented with 10% fetal calf serum, 10 ng/ml IL-5 (Atlanta Biologicals), penicillin (100 U/ml), and streptomycin (100 g/ml) in a humidified atmosphere of 5% carbon dioxide at 35°C as previously described (2). Viability of cells harvested for experiments was assessed by trypan blue exclusion, and populations of cells with viability Ͼ95% were used for generation of superoxide.…”

“…Asthma attacks and experimental antigen challenge are both associated with immediate formation of O 2 Ϫ ⅐ (7), which persists throughout late asthmatic response (40a). Using clone 15 human eosinophilic HL-60 cells, we have recently reported that each of the three eotaxins stimulate a concentrationdependent O 2 Ϫ ⅐ generation, and their bioactivity occurs through occupation of the eosinophil CCR3 receptor.…”

“…To demonstrate that chemokine stimulation via the CCR3 receptor was responsible for O 2 Ϫ ⅐ generation, eosinophils were pretreated with 55 nM SB-328437. These data confirmed that the medium obtained from stimulated A549 cells contained proinflammatory mediators, CC chemokines included, that act through eosinophil CCR3 receptors to generate O 2 Ϫ ⅐ anion.…”

CCL26-Targeted siRNA Treatment of Alveolar Type II Cells Decreases Expression of CCR3-Binding Chemokines and Reduces Eosinophil Migration: Implications in Asthma Therapy

“…Clone 15 HL-60 EOSs (ATCC CRL-1964) were cultured in RPMI 1640 (Cellgro by Mediatech, Inc., Herndon, VA, USA) supplemented with 10% fetal calf serum, 10 ng/mL IL-5 (Atlanta Biologicals, Atlanta, GA, USA), penicillin (100 U/mL) and streptomycin (100 μg/ mL) in a humidifi ed atmosphere as described above. Final differentiation was carried out for 7 days in supplemented RPMI-1640 containing 0.5 mM n-butyric acid (Badewa and Heiman 2003). Viability of cells harvested for experiments was assessed by trypan blue exclusion, and only populations of cells with viability >95% were used for experiments.…”