Airway epithelial cells produce B cell-activating factor of TNF family by an IFN-beta-dependent mechanism

Abstract

Activation of B cells in the airways is now believed to be of great importance in immunity to pathogens, and it participates in the pathogenesis of airway diseases. However, little is known about the mechanisms of local activation of B cells in airway mucosa. We investigated the expression of members of the B cell-activating TNF superfamily (B cell-activating factor of TNF family (BAFF) and a proliferation-inducing ligand (APRIL)) in resting and TLR ligand-treated BEAS-2B cells and primary human bronchial epithelial cells (PBEC). In unstimulated cells, expression of BAFF and APRIL was minimal. However, BAFF mRNA was significantly up-regulated by TLR3 ligand (dsRNA), but not by other TLR ligands, in both BEAS-2B cells (376-fold) and PBEC (224-fold). APRIL mRNA was up-regulated by dsRNA in PBEC (7-fold), but not in BEAS-2B cells. Membrane-bound BAFF protein was detectable after stimulation with dsRNA. Soluble BAFF protein was also induced by dsRNA (> 200 pg/ml). The biological activity of the epithelial cell-produced BAFF was verified using a B cell survival assay. BAFF was also strongly induced by IFN-beta, a cytokine induced by dsRNA. Induction of BAFF by dsRNA was dependent upon protein synthesis and IFN-alphabeta receptor-JAK-STAT signaling, as indicated by studies with cycloheximide, the JAK inhibitor I, and small interfering RNA against STAT1 and IFN-alphabeta receptor 2. These results suggest that BAFF is induced by dsRNA in airway epithelial cells and that the response results via an autocrine pathway involving IFN-beta. The production of BAFF and APRIL by epithelial cells may contribute to local accumulation, activation, class switch recombination, and Ig synthesis by B cells in the airways.

FIGURE 1

Effect of TLR ligands on the up-regulation of BAFF and APRIL in airway epithelial cells. BEAS-2B cells (A) or PBEC (B) were incubated for 18 h with 10 μg/ml PGN, 100 μg/ml zymosan, 25 μg/ml dsRNA, 1 μg/ml LPS, 10 ng/ml flagellin, 1 μg/ml R-848, or 3 μg/ml CpG-A, as indicated, and then mRNA was extracted and analyzed for BAFF and APRIL using real-time PCR. C, BEAS-2B cells or PBEC were incubated for 18 h with 25 μg/ml dsRNA, and then expression of mRNA for full-length BAFF (246 bp), ΔBAFF (189 bp), and GAPDH (226 bp) was analyzed by conventional RT-PCR. D, BEAS-2B cells were incubated for 6 h with 0.25–25 μg/ml dsRNA, and then expression of mRNA for BAFF was analyzed by real-time PCR. E, BEAS-2B cells were incubated with 25 μg/ml dsRNA (circle) or vehicle control (square) for 0.5–18 h, and then expression of mRNA for BAFF was analyzed by real-time PCR. The copy number is expressed as the number of transcripts/ng total RNA. The results are shown as the mean ± SEM of three to five independent experiments. *, p < 0.05.

FIGURE 2

Detection of BAFF protein in airway epithelial cells. A, Representative histograms from flow cytometry demonstrate evidence of mBAFF on BEAS-2B cells. Cells were stained with mAb specific for BAFF (dark line) or an isotope control mAb (shaded histogram) after incubation with or without (untreated) 25 μg/ml dsRNA for 24 h. Detection of sBAFF protein by ELISA in the culture supernatant of BEAS-2B (B) and PBEC (C, 72 h) stimulated with dsRNA for the indicated time. Results shown are mean ± SEM of three to four independent experiments. **, Not detectable. *, p < 0.05.

FIGURE 3

Effect of cytokines on the expression of BAFF in airway epithelial cells. BEAS-2B cells (A) and PBEC (B) were incubated for 6 h with 25 μg/ml dsRNA, 100 ng/ml IL-1β, 100 ng/ml IL-4, 100 ng/ml IL-6, 100 ng/ml IL-10, 100 ng/ml IL-12, 100 ng/ml IL-15, 100 ng/ml TNF-α, 1000 U/ml IFN-β, 100 ng/ml IFN-γ, 100 ng/ml TGF-β, 100 ng/ml G-CSF, or 100 ng/ml GM-CSF, as indicated. The level of BAFF mRNA was determined by real-time PCR. The results are shown as the mean ± SEM of three to four independent experiments. *, p < 0.05.

FIGURE 4

Effect of IFN-β on the up-regulation of BAFF by dsRNA in BEAS-2B cells. A, BEAS-2B cells were incubated for 6 h with 1–1000 U/ml IFN-β. B, BEAS-2B cells were incubated with 100 U/ml IFN-β for up to 6 h. C, BEAS-2B cells were incubated with 25 μg/ml dsRNA or control medium for up to 6 h. The levels of BAFF and IFN-β mRNA were determined by real-time PCR. The results are shown as the mean ± SEM of three independent experiments.

FIGURE 5

Effect of the IFN-STAT pathway on the expression of BAFF induced by dsRNA in BEAS-2B cells. A, BEAS-2B cells were pre-incubated with 0.01% DMSO or 0.1–10 mg/ml cycloheximide (CHX) for 1 h, and then stimulated with 25 μg/ml dsRNA for 3 h. B, BEAS-2B cells were preincubated with 0.1% DMSO or 0.1–10 μM JAKI for 1 h, and then stimulated with 25 μg/ml dsRNA for 6 h. C, BEAS-2B cells were transfected with siRNA against STAT1, IFNAR2, or control RNA at 5 or 20 nM for 72 h, and then stimulated with 1000 U/ml IFN-β and 25 μg/ml dsRNA for 6 h. The levels of BAFF and IFN-β mRNA were determined by real-time PCR. The results are shown as the mean ± SEM of four independent experiments. *, p < 0.05 vs dsRNA or IFN-β stimulated.

FIGURE 6

Effect of glucocorticoids on the up-regulation of BAFF by dsRNA in airway epithelial cells. BEAS-2B cells (A and C–E) and PBEC (B) were preincubated with 0.001% DMSO, 100 nM FP (A, B, D, and E), or 10⁻¹¹–10⁻⁶ M FP (C) for 2 h, and then stimulated with 25 μg/ml dsRNA for 18 h (A–C), 24 h (E), or 72 h (D). The level of BAFF mRNA was determined by real-time PCR (A–C). Concentrations of sBAFF protein in the culture supernatant were measured by ELISA (D). Cells were stained with mAb specific for BAFF (dark line) or an isotope control mAb (shaded histogram) and analyzed by flow cytometry (E). The results are shown as the mean ± SEM of three to four independent experiments. *, p < 0.05 vs dsRNA stimulated. **, Not detectable.

FIGURE 7

Effect of epithelial cell-derived BAFF on the cell survival of B cells. A, Purified human B cells were stimulated with 5 ng/ml BAFF or concentrated BEAS-2B supernatant for 5 days. B, Purified human B cells were stimulated with 5 ng/ml BAFF, 10 ng/ml IL-4, 10 μg/ml mouse anti-human CD40 mAb, concentrated supernatant, or the indicated combinations for 5 days in the presence or absence of 5 μg/ml control IgG1, 5 μg/ml BAFF-R:Fc, or 5 μg/ml TACI:Fc. MTT reagent was added for the last 4 h of incubation. The concentrated BEAS-2B supernatant was added at a final dilution of 1/20 to the B cell culture. Absorbance at 570 nm was measured using a microtiter plate reader. The results are shown as the mean ± SEM of five independent experiments.*, p < 0.05.