Mast cells promote airway smooth muscle cell differentiation via autocrine up-regulation of TGF-beta 1

Abstract

Asthma is a major cause of morbidity and mortality worldwide. It is characterized by airway dysfunction and inflammation. A key determinant of the asthma phenotype is infiltration of airway smooth muscle bundles by activated mast cells. We hypothesized that interactions between these cells promotes airway smooth muscle differentiation into a more contractile phenotype. In vitro coculture of human airway smooth muscle cells with beta-tryptase, or mast cells with or without IgE/anti-IgE activation, increased airway smooth muscle-derived TGF-beta1 secretion, alpha-smooth muscle actin expression and agonist-provoked contraction. This promotion to a more contractile phenotype was inhibited by both the serine protease inhibitor leupeptin and TGF-beta1 neutralization, suggesting that the observed airway smooth muscle differentiation was driven by the autocrine release of TGF-beta1 in response to activation by mast cell beta-tryptase. Importantly, in vivo we found that in bronchial mucosal biopsies from asthmatics the intensity of alpha-smooth muscle actin expression was strongly related to the number of mast cells within or adjacent to an airway smooth muscle bundle. These findings suggest that mast cell localization in the airway smooth muscle bundle promotes airway smooth muscle cell differentiation into a more contractile phenotype, thus contributing to the disordered airway physiology that characterizes asthma.

FIGURE 1

Increased ASM α-SMA expression in vivo localized with mast cells. A, Photomicrographs of a bronchial biopsy from an asthmatic illustrating ASM bundles stained for α-SMA (left), sequential tryptase stained section revealing one mast cell within the ASM bundle and two in the 30-μm perimeter of the bundle (middle), and high intensity thresholded α-SMA-stained bundle covering 8.76% of the total ASM bundle area (right) (×400). B, Photomicrograph of another asthmatic subject again illustrating α-SMA-stained ASM bundle (left), sequential tryptase-stained section revealing one mast cell in the 30-μm perimeter and none within the ASM bundle (middle), and high intensity thresholded α-SMA-stained ASM bundle covering 0% of the total ASM bundle area (right) (×400). C, Correlation between the percentage of high intensity α-SMA staining and the number of mast cells in the ASM bundle alone (r = 0.87; p = 0.0003) and D, together with the number of mast cells in the 30 μm perimeter of the ASM bundle (r = 0.87; p = 0.0002).

FIGURE 2

Mast cells increase ASM α-SMA expression. A, Example flow cytometry histogram of ASM stained with isotype control or α-SMA mAb FITC direct conjugate cocultured with or without unstimulated HLMC (ratio 1:4, HLMC/ASM) for 7 days. Isotype controls for both conditions were overlapping, therefore only one is shown for illustration. B and C, Bar charts illustrating the fold change over control of α-SMA GMFI expression by ASM as assessed by flow cytometry after coculture with HLMC (ratio 1:4, HLMC/ASM) unstimulated or IgE sensitized (2.5 μg/ml) (n = 9 ASM donors, n = 9 HLMC) or sensitized and anti-IgE activated (1:1000) (n = 5 ASM donors, n = 7 HLMC) (B) or HLMC (n = 8 ASM donors, n = 7 HLMC) or HMC-1 lysates (n = 16 ASM donors) at a ratio of 1:4 mast cell lysate to ASM for 1, 3, or 7 days (C). D, Example immunofluorescence images from three experiments of ASM cocultured with unstimulated HLMC, HLMC, or HMC-1 lysates for 7 days (ratio 1:4, mast cell/ASM) bottom panels and the corresponding controls in the top panels. Cell nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI), ASM were labeled with α-SMA mAb FITC direct conjugate, ASM and HLMC cocultures were counterstained with tryptase biotin indirectly conjugated with streptavidin Texas red. Changes in ASM α-SMA expression are illustrated by an increase in the intensity of α-SMA mAb FITC fluorescence. *, p < 0.05 per time point; ~, p < 0.05 across all time points.

FIGURE 3

β-Tryptase increases ASM α-SMA expression. A, Bar chart illustrating the fold change over control of α-SMA GMFI expression by ASM as assessed by flow cytometry after culture with β-tryptase (0.5 μg/ml) with or without leupeptin (10.6 μg/ml) for 1, 3, and 7 days (n = 4 ASM donors). B, Example immunofluorescence images from three experiments of ASM cultured with β-tryptase with or without leupeptin for 7 days (bottom panels) and the corresponding controls (top panels). Changes in ASM α-SMA expression are illustrated by an increase in the intensity of α-SMA mAb FITC fluorescence. C, Changes in α-SMA mRNA expression by ASM cultured with β-tryptase with or without leupeptin for 7 days as assessed by quantitative PCR (n = 3 ASM donors).

FIGURE 4

Mast cells mediate increased ASM α-SMA expression via β-tryptase. Bar charts illustrating the fold change over control of α-SMA GMFI expression by ASM as assessed by flow cytometry after culture with HLMC (n = 3 ASM donors, n = 3 HLMC) (A), HLMC or HMC-1 lysates (ratio 1 mast cell to 4 ASM; n = 4 ASM donors) for 1 or 3 days, with or without leupeptin (10.6 μg/ml) (B).

FIGURE 5

Enhanced ASM contractility in response to β-tryptase. Example photographs (A) and time course of contraction of collagen gels impregnated with ASM with or without β-tryptase (0.32 μg/ml) with or without leupeptin (6.7 μg/ml) for 48 h (n = 5 ASM donors) (B), followed by subsequent histamine (100 μM) stimulation to the same gels compared with appropriate control analyzed using ImageJ (C). *, p < 0.05.

FIGURE 6

Mast cells mediate increased TGF-β1 expression via β-tryptase. Bar charts illustrating the increase in TGF-β1 production by ASM as assessed by ELISA after culture with HLMC, HLMC lysates or β-tryptase (ratio 1:4 mast cell/ASM or 0.5 μg/ml β-tryptase; n = 3) for 1 or 3 days (A), with or without leupeptin (10.6 μg/ml) or lactoferrin (80.6 ng/ml) (B). *, p < 0.05.

FIGURE 7

TGF-β1 and TGF-β2 increase ASM α-SMA expression and contractility. Bar chart illustrating the fold change over control of α-SMA GMFI expression by ASM assessed by flow cytometry after culture with TGF-β1 or TGF-β2 (0.25, 2, or 10 ng/ml) (n = 9 ASM donors) (A) or TGF-β1 (2 ng/ml) with or without TGF-β1 neutralizing Ab or appropriate isotype control for 1, 3, or 7 days (n = 3 ASM donors) (B). C, Time course after 100 μM/ml histamine stimulation of collagen gel contraction in gels previously impregnated with TGF-β1 (2 ng/ml) in the presence or absence of TGF-β1 neutralizing Ab or appropriate isotype control for 48 h (n = 3 ASM donors). *, p < 0.05.

FIGURE 8

The neutralization of TGF-β1 inhibited β-tryptase-mediated increased ASM contraction. Time course (A) and area under the curve of the time course (B), after histamine (100 μM/ml) stimulation of collagen gel contraction in gels previously impregnated with β-tryptase (0.32 μg/ml) in the presence or absence of TGF-β1-neutralizing Ab or appropriate isotype control for 48 h (n = 4 ASM donors). *, p < 0.05.