Dual effect of neutrophils on pIgR/secretory component in human bronchial epithelial cells: role of TGF-beta

Abstract

Neutrophils have a dual affect on epithelial pIgR/SC, the critical receptor for transcellular routing of mucosal IgA, but mechanisms of pIgR/SC upregulation remain elusive. Requirements of cytokine, redox, and signalling pathways for pIgR/SC production were assessed in human bronchial epithelial (Calu-3) cells cocultured with increasing numbers of blood neutrophils. Increased SC production was observed after incubation for 48 hrs with intermediate neutrophil numbers (1.25 to 2.5 x 10(6)), was favoured by the elastase inhibitor SLPI, and correlated with increased TGF-beta production. Exogenous TGF-beta stimulated SC production with a maximal effect at 48 hrs and both TGF-beta- and neutrophil-driven SC upregulation were dependent on redox balance and p38 MAP-kinase activation. This paper shows that activated neutrophils could upregulate epithelial pIgR/SC production through TGF-beta-mediated activation of a redox-sensitive and p38 MAPK-dependent pathway. An imbalance between the two neutrophil-driven opposite mechanisms (SC upregulation and SC degradation) could lead to downregulation of pIgR/SC, as observed in severe COPD.

Figure 1

Dual effect of neutrophils on SC secretion. Confluent monolayers of Calu-3 cells were incubated in triplicates for 48 hrs with increasing numbers of neutrophils, from 0.3 × 10⁶ to 15 × 10⁶ cells. (a) SC concentration and (b) elastase activity were measured in supernatants, as described in Methods. Data are means ± SEM (n = 6 independent experiments). ^#P = .08, *P ≤ .05 and **P ≤ .01 as compared with control.

Figure 2

Effect of Secretory Leucoproteinase Inhibitor (SLPI) on pIgR/SC production. Confluent Calu-3 monolayers were cultured with increasing numbers of neutrophils (0.6 × 10⁶ to 10 × 10⁶ cells) in the presence (dashed line) or in the absence (full line) of SLPI. After 48 hrs, SC production was measured in supernatants (black lines) and in cell lysates (grey lines). Data are means ± SD of one experiment (out of 2) in triplicates. *P ≤ .05 as compared with control.

Figure 3

TGF-β1 production in epithelial/neutrophil cocultures. (a) TGF-β1 production was measured by ELISA in supernatants of Calu-3 cells incubated for 48 hrs with increasing numbers of neutrophils (1.25 × 10⁶ to 5 × 10⁶ cells). (b) Kinetics (from 2 to 72 hrs) of TGF-β1 production by Calu-3 cells alone (open bars), neutrophils alone (5 × 10⁶ cells, stripped bars) or epithelial cell/neutrophil cocultures (5 × 10⁶ neutrophils, solid bars). The horizontal bar represents TGF-β1 concentration in complete medium, containing fetal calf serum (201 ± 40 pg/ml, mean ± SD, n = 3). Data are means ± SEM (n = 4 independent experiments, both for a and b). (a) *P ≤ .05 as compared with control; (b) *P ≤ .05 as compared with Calu-3 alone, ^#P ≤ .05 as compared with Calu-3 at 2 hrs.

Figure 4

Effect of TGF-β1 on pIgR/SC production. (a) Calu-3 cells were cultured for 48 hrs with or without TGF-β1 (20 ng/ml). SC production was evaluated in supernatants (solid bars) and in cell lysates (open bars). (b) Calu-3 cells were exposed to increasing concentration of TGF-β1 (0.2 to 80 ng/ml) for 48 hrs, and SC was measured in supernatants. (c) SC production was assessed in supernatants from resting Calu-3 cells as control (open bars), Calu-3 cells incubated with TGF-β1 (20 ng/ml, solid bars) and Calu-3 cells/neutrophil cocultures (stripped bars) for 1 hr to 48 hrs. Results are expressed as means ± SEM for (a) (n = 7 independent experiments with triplicates), (b) (n = 3) and (c) (n = 3). *P < .05 as compared with control (a, b, c); ^#P < .05 as compared with control at 1 hr (c).

Figure 5

Effect of redox changes on TGF-β and neutrophil-induced SC production. (a) Calu-3 cells were incubated (or not, as control) for 48 hrs with TFG-β1 (20 ng/ml) or neutrophils (5 × 10⁶ cells) in the presence or not of the antioxidants N-acetylcysteine (NAC,10 mM, grey bars) or glutathione (GSH, 10 mM, solid bars). Supernatants were assayed for SC by ELISA. Data are means ± SEM (n = 3). *P < .05 as compared with control. (b) Calu-3 cells were cocultured (in a 24-wells plate) with neutrophils (80 × 10⁶ cells) in the presence or not of NAC (10 mM) for 5 to 120 min, and phosphorylation of ERK1/2 and p38 was assayed by western blot, as compared to β-actin as loading control.

Figure 6

Effect of MAP kinase inhibitors on pIgR/SC production. (a) Calu-3 cells were cultured with TGF-β1 (20 ng/ml) for 5 to 120 min and phosphorylation of ERK1/2 and p38 was assayed by western blot, as compared to β-actin as loading control. (b) Calu-3 cells were cultured with TGF-β1 (20 ng/ml) or neutrophils (5 × 10⁶ cells) for 48 hrs in the presence of medium alone (control) or MAP Kinase inhibitors: PD98059 (grey bars), SB203580 (solid bars) or SP600125 (stripped bars) (all 50 μM) as selective inhibitors of ERK1/2 MAPK, p38 MAPK and JNK MAPK, respectively. SC production was assessed in supernatants and results were shown as means ± SEM (n = 4). *P < .05, **P < .01 as compared with control.