Tissue remodeling induced by hypersecreted epidermal growth factor and amphiregulin in the airway after an acute asthma attack

Concentrations of EGF (A) and amphiregulin (AREG) (B) in the sputa of patients with asthma after an asthma attack (acute), during the recovery phase (recovery), and during the stable phase (stable); sputa of normal healthy controls; and sputa of patients with respiratory tract infection (^∗∗P < .01; ^∗∗∗∗P < .001). The number of donors is indicated under the figure. Because of the small volume of the sputum samples, the experiments could not be conducted in some cases.

Time-course of changes in the sputum concentrations of EGF (A), amphiregulin (AREG) (B), and tryptase (C) in children with asthma hospitalized for treatment of an acute asthma attack during the acute phase and during the recovery phase after treatment (n = 8 donors). Each symbol indicates an individual donor. Correlation between the sputum concentrations of EGF and AREG (D), between those of AREG and tryptase (E), and between those of EGF and tryptase (F). Even if the asterisked point was omitted, there was a significant correlation between the sputum concentrations of AREG and tryptase (E, P < .01, r = 0.416). NS, Not significant.

EGF and amphiregulin (AREG) induced distinct patterns of cell proliferation. Bromodeoxyuridine uptake in NHBE (A), BSMC (B), and NHLF (C) induced by EGF (filled circles) or AREG (open circles). Data are expressed as means ± SEMs of 3 independent experiments using different donors. ^∗P < .05, ^∗∗P < .01, ^∗∗∗P < .005, and ^∗∗∗∗P < .001 compared with cells not treated with recombinant human EGF or recombinant human AREG. D, EGFR tyrosine phosphorylation in NHBE and BSMC by EGF and AREG. This experiment was repeated 3 times. E, Phosphorylation of ^44/42ERK in NHBE and BSMC by EGF or AREG. Fold induction of phosphorylation of p42 ERK was determined by densitometry and normalized to the respective protein. One representative of 3 individual experiments is shown. Abs, Absorbances.

Long-term exposure of NHBEs to EGF and amphiregulin (AREG) results in mucous cell metaplasia in the ALI culture. After NHBE were cultured in the ALI culture for 2 weeks in the presence of recombinant human EGF or recombinant human AREG, the medium was replenished, and the cells were incubated in the presence or absence of recombinant human EGF or recombinant human AREG for 24 hours (hatched bars) and 48 hours culture (filled or open bars) for analysis of MUC5AC gene expression (A) and for 72 hours for analysis of MUC5 AC protein production (B) and immunocytochemistry (C). Data are expressed as means ± SEMs (n = 3). C, Mucins were measured in the cell lysate and in the supernatants. i, Without second antibody. ii, Non–AREG-treated or EGF-treated cells. iii, 100 ng/mL recombinant human EGF–treated cells. iv, 100 ng/mL recombinant human AREG–treated cells. The data in A, B, and C are representative of similar results obtained from 3 independent experiments using different donors. Green and red staining indicates MUC5AC and nuclei, respectively.

Comparison of the sputum levels of EGF and amphiregulin (AREG) between patient groups with atopic and nonatopic asthma (A), between patient groups showing and not showing elevation of the serum C-reactive protein (CRP) during an exacerbation as a marker of infection triggering the asthma attack (B), and between patient groups treated and not treated with leukotriene receptor antagonists (LTRAs) (C).

Effect of EGF and amphiregulin (AREG) on the MUC5AC expression in NCI-H292 cells. NCI-H292 cells were incubated with 1 to 100 ng/mL recombinant human EGF or AREG for 24 hours (A) or with 10 ng/mL recombinant human EGF or AREG for 0 to 24 hours (B). The results were expressed as fold-change (mean ± SEM) in the MUC5AC mRNA expression levels in the EGF-treated or AREG-treated cells compared with those in non–EGF-treated or AREG-treated cells (n = 4). ^∗P < .05, ^∗∗P < .01, and ^∗∗∗P < .005 compared with cells not treated with recombinant human EGF or AREG.