Figure 4

Quantification of soluble collagen content in the media and matrix. (A) Sircol soluble collagen assay was performed as described in the Materials and Methods, which quantifies the amount of soluble collagen in the cell culture supernatant and newly synthesized salt soluble collagen in the matrix. The amount of soluble collagen secreted in the media at day 25 in the IL-13 pre-treated NHBE at 1 and 10 ng/ml co-cultured with NHLF is augmented as compared to the untreated NHBE co-culture; * p < 0.01 and addition of TGFβ₂ neutralizing antibody (10 μg/ml) abolishes this increase (^§ p < 0.01 compared to respective condition without TGFβ₂ neutralizing antibody). (B) At day 25 there is an increase in newly synthesized salt soluble collagen content in the matrix in the IL-13 pre-treated NHBE at 1 and 10 ng/ml followed by co-culture with NHLF as compared to the untreated NHBE co-culture; * p < 0.01 and the IL-13 pre-treated NHBE at 10 ng/ml co-culture collagen levels are elevated as compared to the IL-13 pretreated NHBE at 1 ng/ml co-culture; # p < 0.01. Also, addition of the TGFβ₂ neutralizing antibody abolishes this increase (^§ p < 0.01 compared to respective condition without TGFβ₂ antibody). The media and matrix collagen levels are normalized to respective levels obtained from NHLF embedded in collagen gels ("NHLF only"). (C, D) Representative Second harmonic generated (SHG) images (scale bar = 50 μm) of collagen fibrils at day 25 are shown along with the quantification of signal intensities. The SHG signals from the collagen secreted by NHLF embedded in rat tail collagen gels which were co-cultured with the IL-13 pre-treated NHBE at 10 ng/ml are elevated compared to the untreated NHBE co-culture; * p < 0.01 and this increase is inhibited on incubation with TGFβ₂ neutralizing antibody in the 3 day co-culture period (^§ p < 0.01 compared to respective condition without TGFβ₂ antibody). Addition of goat IgG did not alter the increased levels of collagen in the matrix and media in the pre-treated NHBE-NHLF co-culture. (E) Exogenous active TGF-β₂ at 0.05, 0.1, 0.5, 1 and 10 ng/ml is added in 50:50 epithelial media to NHLF embedded in collagen gels for a period of 3 days. There is a significant increase in the newly synthesized salt soluble collagen content in the matrix with addition of increasing concentration of active TGF-β₂ (* p < 0.01 compared to only NHLF condition). All values are normalized to those obtained from "NHLF only" condition. All experiments were performed using 3 donors, grown in duplicate, with 3–6 wells for each condition.