Role of p38 MAPK and NF-kB for chemokine release in coculture of human eosinophils and bronchial epithelial cells

Abstract

Eosinophils are principal effector cells of inflammation in allergic asthma, characterized by their accumulation and infiltration at inflammatory sites mediated by the chemokine eotaxin and their interaction with adhesion molecules expressed on bronchial epithelial cells. We investigated the modulation of nuclear factor-kappaB (NF-kappaB) and the mitogen-activated protein kinase (MAPK) pathway on the in vitro release of chemokines including regulated upon activation normal T cell expressed and secreted (RANTES), monokine induced by interferon-gamma (MIG), monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-8, and interferon-inducible protein-10 (IP-10) upon the interaction of human bronchial epithelial BEAS-2B cells and eosinophils. Gene expression of chemokines was evaluated by RT-PCR and the induction amount of chemokines quantified by cytometric bead array. NF-kappaB and p38 MAPK activities were assessed by electrophoretic mobility shift assay and Western blot, respectively. The interaction of eosinophils and BEAS-2B cells was found to up-regulate the gene expression of the chemokines IL-8, MCP-1, MIG, RANTES and IP-10 expression in BEAS-2B cells, and to significantly elevate the release of the aforementioned chemokines except RANTES in a coculture of BEAS-2B cells and eosinophils. IkappaB-alpha phosphorylation inhibitor, BAY 11-7082, and p38 MAPK inhibitor, SB 203580 could decrease the release of IL-8, IP-10 and MCP-1 in the coculture. Together, the above results show that the induction of the release of chemokines in a coculture of epithelial cells and eosinophils are regulated by p38 MAPK and NF-kappaB activities of BEAS-2B cells, at least partly, through intercellular contact. Our findings therefore shed light on the future development of more effective agents for allergic and inflammatory diseases.

Fig. 1

Representative RT-PCR analysis of β-actin, IL-8, IP-10, MCP-1, MIG and RANTES mRNA expression in (a) BEAS-2B cells and (b) eosinophils. Total RNA was extracted from confluent BEAS-2B cells (6 × 10⁵/well) or eosinophils (3 × 10⁶/well) after coculture for 12 h in a 6-well plate, and then reverse transcribed and analysed by PCR. The β-actin housekeeping gene was used as the control. BE: BEAS-2B cells, EOS: eosinophils.

Fig. 2

Induction of the release of (a) IL-8 (b) IP-10 (c) MCP-1 (d) MIG and (e) RANTES upon the interaction of eosinophils and BEAS-2B cells. Confluent BEAS-2B cells (1·5 × 10⁵/well) were cultured with or without eosinophils (5 × 10⁵/well) for 2–18 h in a 24-well plate. Chemokine release in culture supernatant was determined using the human chemokine CBA kit and flow cytometry. Results are expressed as the arithmetic mean ± SEM from three independent experiments with different blood donors per single well on the tissue culture plate for each treatment. **P < 0·01, ***P < 0·001 when compared with the BEAS-2B and eosinophils control. BE: BEAS-2B cells, EOS: eosinophils.

Fig. 3

Induction of the release of (a) IL-8 and (b) MCP-1 upon the coculture of eosinophils and BEAS-2B cells using transwell inserts. Confluent BEAS-2B cells (1·5 × 10⁵/well) were cultured with or without eosinophils (5 × 10⁵/well) for 18 h in 24-well plate using transwell inserts (pore size 0·4 µm) to separate the two cell types in two compartments. Chemokines released in culture supernatant was determined by human chemokine CBA kit using flow cytometry. Results are expressed as the arithmetic mean ± SEM from three independent experiments with different blood donors with single well on the tissue culture plate for each treatment. ***P < 0·001 when compared with the BEAS-2B and eosinophils control. BE: BEAS-2B cells, EOS: eosinophils.

Fig. 4

Induction of the release of (a) IL-8 and (b) MCP-1 upon the coculture of fixed or not fixed eosinophils and BEAS-2B cells. Confluent normal or paraformaldhyde (1%) fixed BEAS-2B cells (1·5 × 10⁵/well) were cultured with or without normal or paraformaldhyde (1%) fixed eosinophils (5 × 10⁵/well) for 18 h in 24-well plate. Chemokines released in culture supernatant was determined by human chemokine CBA kit using flow cytometry. Results are expressed as the arithmetic mean ± SEM from three independent experiments with different blood donors with single well on the tissue culture plate for each treatment. **P < 0·01, ***P < 0·001 when compared with the BEAS-2B and eosinophils control. BE: normal BEAS-2B cells, EOS: normal eosinophils, BE*: fixed BEAS-2B cells, EOS*: fixed eosinophils.

Fig. 5

Activation of p38 MAPK activity in BEAS-2B cells and eosinophils. Confluent BEAS-2B cells (6 × 10⁵/well) were cultured with or without eosinophils (3 × 10⁶/well) in a 6-well plate for 30 min in the presence or absence of SB 203580 (20 µm). Total cellular proteins were extracted from BEAS-2B cells or eosinophils for the detection of (A) phosphorylated p38 MAPK protein level by Western blot analysis and (B) p38 MAPK activity by the detection of phosphorylated ATF-2 using a p38 MAP Kinase assay kit. Experiments were performed in three independent experiments with essentially identical results, and representative results are shown. BE: BEAS-2B cells, EOS: eosinophils, SB: SB 203580.

Fig. 6

Effect of interaction between eosinophils and BEAS-2B cells on NF-κB activity in BEAS-2B cells and eosinophils. (a) Confluent BEAS-2B cells (6 × 10⁵/well) were cultured with or without eosinophils (3 × 10⁶/well) in a 6-well plate for 30 min. Total protein was then extracted from BEAS-2B or eosinophils. Phosporylated IκB was then analysed by Western blot. (b, c) Confluent BEAS-2B cells (6 × 10⁵/well) were cultured with eosinophils (3 × 10⁶/well) in a 6-well plate for 2 h with or without 1 h pretreatment and subsequent 2 h incubation of BAY 11–7082 (1 µm). Nuclear proteins were then extracted from (b) BEAS-2B cells or (c) eosinophils, and 5 µg protein was then subjected to EMSA. Experiments were performed in three independent experiments with essentially identical results, and representative results are shown. BE: BEAS-2B cells, EOS: eosinophils, BAY: BAY 11–7082.

Fig. 7

Effect of BAY 11–7082 and SB 203580 on the release of IL-8, IP-10, MCP-1, MIG and RANTES upon the interaction between eosinophils and BEAS-2B cells. Confluent BEAS-2B cells (1·5 × 10⁵/well) were pretreated with BAY 11–7082 (1 µm) or SB 203580 (20 µm) for 1 h followed by culture with eosinophils (3 × 10⁵/well) in a 24-well plate for a further 15 h in the presence of BAY 11–7082 or SB 203580. Chemokines released in the culture supernatant were determined using the human chemokine CBA kit and flow cytometry. Results are expressed as the arithmetic mean ± SEM from three independent experiments with different blood donors per single well on the tissue culture plate for each treatment. The eosinophil and BEAS-2B single culture, and coculture of eosinophils and BEAS-2B cells contained 0·1% DMSO as a DMSO control. *P < 0·05, **P < 0·01, ***P < 0·001 when compared with the control. BE: BEAS-2B cells, EOS: eosinophils, SB: SB203580, BAY: BAY 11–7082.