Tissue factor–bearing exosome secretion from human mechanically stimulated bronchial epithelial cells in vitro and in vivo

doi:10.1016/j.jaci.2012.05.031

Journal of Allergy and Clinical Immunology

Volume 130, Issue 6, December 2012, Pages 1375-1383

https://doi.org/10.1016/j.jaci.2012.05.031 Get rights and content

Background

Tissue factor (TF), a primary initiator of blood coagulation, also plays a pivotal role in angiogenesis. TF expression in the airways is associated with asthma, a disease characterized in part by subepithelial angiogenesis.

Objectives

To determine potential sources of TF and the mechanisms of its availability in the lung microenvironment.

Methods

Normal human bronchial epithelial cells grown in air-liquid interface culture were subjected to a compressive stress of 30 cm H₂O; this is comparable to that generated in the airway epithelium during bronchoconstriction in asthma. Conditioned media and cells were harvested to measure TF mRNA and TF protein. We also tested bronchoalveolar lavage fluid and airway biopsies from asthmatic patients and healthy controls for TF.

Results

TF mRNA was upregulated 2.2-fold after 3 hours of stress compared with unstressed cells. Intracellular and secreted TF proteins were enhanced 1.6-fold and more than 50-fold, respectively, compared with those of control cells after the onset of compression. The amount of TF in the bronchoalveolar lavage fluid from patients with asthma was found at mean concentrations that were 5 times greater than those of healthy controls. Immunohistochemical staining of endobronchial biopsies identified epithelial localization of TF with increased expression in asthma. Exosomes isolated from the conditioned media of normal human bronchial epithelial cells and the bronchoalveolar lavage fluid of asthmatic subjects by ultracentrifugation contained TF.

Conclusions

Our in vitro and in vivo studies show that mechanically stressed bronchial epithelial cells are a source of secreted TF and that exosomes are potentially a key carrier of the TF signal.

Section snippets

Culture of bronchial epithelial cells

Primary NHBECs were expanded and maintained in a humidified environment containing 95% air and 5% CO₂ as described previously.⁶ Passage-2 NHBECs were plated at a density of 2 × 10⁴ cells/cm² on 12-well Transwell plates with polyester membranes, 0.4-μm pores (Corning, Inc, Corning, NY) coated with 50 ng/mL of type 1 rat tail collagen (BD Biosciences, San Jose, Calif). Primary cells from 3 nonsmoker donors were used.

Exposure of NHBECs to compressive mechanical stress

To expose cells to compressive stress, nontoxic tapered silicon plugs were

Compressive mechanical stress induces TF mRNA expression and intracellular TF protein in well-differentiated NHBECs

Well-differentiated NHBECs grown in ALI conditions were exposed to 30 cm H₂O transcellular compressive stress for 3 hours. As shown in Fig 1, A, this duration of compressive stress resulted in an increase in TF mRNA expression by 2.2-fold compared with that in time-matched controls. By 8 hours, the magnitude of the induction had fallen to 1.8-fold; both differences were significant compared with their time-matched controls. Intracellular TF protein (Fig 1, B) was detected constitutively, and

Discussion

Our experiments show that NHBECs constitutively express TF protein expression in cell lysates, but there is no detectable secretion into its culture medium under basal ALI culture conditions. Compressive mechanical stress (30 cm H₂O), comparable in magnitude to that associated with asthmatic bronchoconstriction, led to modestly enhanced TF transcription and intracellular protein production and dramatically increased TF secretion. New RNA synthesis was required for the upregulated TF

References (45)

J.A. Park et al.
The chitinase-like protein YKL-40 is secreted by airway epithelial cells at base line and in response to compressive mechanical stress
J Biol Chem
(2010)
Y.W. van den Berg et al.
The relationship between tissue factor and cancer progression: insights from bench and bedside
Blood
(2012)
B. Button et al.
Role of mechanical stress in regulating airway surface hydration and mucus clearance rates
Respir Physiol Neurobiol
(2008)
K.J. Livak et al.
Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method
Methods
(2001)
E. Cocucci et al.
Shedding microvesicles: artefacts no more
Trends Cell Biol
(2009)
M. Record et al.
Exosomes as intercellular signalosomes and pharmacological effectors
Biochem Pharmacol
(2011)
S. Mathivanan et al.
Proteomics analysis of A33 immunoaffinity-purified exosomes released from the human colon tumor cell line LIM1215 reveals a tissue-specific protein signature
Mol Cell Proteomics
(2010)
S.T. Holgate
Epithelium dysfunction in asthma
J Allergy Clin Immunol
(2007)
I. Del Conde et al.
Tissue-factor-bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation
Blood
(2005)
A. Aharon et al.
Microparticles, thrombosis and cancer
Best Pract Res Clin Haematol
(2009)

B. Ressler et al.

Molecular responses of rat tracheal epithelial cells to transmembrane pressure

Am J Physiol Lung Cell Mol Physiol

(2000)

D.J. Tschumperlin et al.

Mechanotransduction through growth-factor shedding into the extracellular space

Nature

(2004)

M.A. Swartz et al.

Mechanical stress is communicated between different cell types to elicit matrix remodeling

Proc Natl Acad Sci U S A

(2001)

J.A. Park et al.

Chronic intermittent mechanical stress increases MUC5AC protein expression

Am J Respir Cell Mol Biol

(2009)

D.J. Tschumperlin et al.

Mechanical stress triggers selective release of fibrotic mediators from bronchial epithelium

Am J Respir Cell Mol Biol

(2003)

J. Chen et al.

Tissue factor–a receptor involved in the control of cellular properties, including angiogenesis

Thromb Haemost

(2001)

P. Carmeliet et al.

Role of tissue factor in embryonic blood vessel development

Nature

(1996)

M. Belting et al.

Regulation of angiogenesis by tissue factor cytoplasmic domain signaling

Nat Med

(2004)

E.C. Gabazza et al.

Thrombin in the airways of asthmatic patients

Lung

(1999)

B.R. Wiggs et al.

On the mechanism of mucosal folding in normal and asthmatic airways

J Appl Physiol

(1997)

C.L. Grainge et al.

The influence of bronchoconstriction on airway remodeling in asthma

N Engl J Med

(2011)

C. Thery et al.

Isolation and characterization of exosomes from cell culture supernatants and biological fluids

Curr Protoc Cell Biol

(2006)

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: This study was supported by National Institutes of Health grants HL88028 (to J.M.D.) and T32-HL007118 (to J.-A.P.) and Medical Research Council grant G0900453 (to P.H.).

: Disclosure of potential conflict of interest: J.-A. Park, A.S. Sharif, D.J. Tschumperlin, and J.M. Drazen have received grants from the National Institutes of Health. P. Howarth has received grants from the Medical Research Council. The rest of the authors declare that they have no relevant conflicts of interest.

View full text

Mechanisms of allergy and clinical immunologyTissue factor–bearing exosome secretion from human mechanically stimulated bronchial epithelial cells in vitro and in vivo

Background

Objectives

Methods

Results

Conclusions

Section snippets

Culture of bronchial epithelial cells

Exposure of NHBECs to compressive mechanical stress

Compressive mechanical stress induces TF mRNA expression and intracellular TF protein in well-differentiated NHBECs

Discussion

J Biol Chem

Blood

Respir Physiol Neurobiol

Methods

Trends Cell Biol

Biochem Pharmacol

Mol Cell Proteomics

J Allergy Clin Immunol

Blood

Best Pract Res Clin Haematol

Molecular responses of rat tracheal epithelial cells to transmembrane pressure

Am J Physiol Lung Cell Mol Physiol

Mechanotransduction through growth-factor shedding into the extracellular space

Nature

Mechanical stress is communicated between different cell types to elicit matrix remodeling

Proc Natl Acad Sci U S A

Chronic intermittent mechanical stress increases MUC5AC protein expression

Am J Respir Cell Mol Biol

Mechanical stress triggers selective release of fibrotic mediators from bronchial epithelium

Am J Respir Cell Mol Biol

Tissue factor–a receptor involved in the control of cellular properties, including angiogenesis

Thromb Haemost

Role of tissue factor in embryonic blood vessel development

Nature

Regulation of angiogenesis by tissue factor cytoplasmic domain signaling

Nat Med

Thrombin in the airways of asthmatic patients

Lung

On the mechanism of mucosal folding in normal and asthmatic airways

J Appl Physiol

The influence of bronchoconstriction on airway remodeling in asthma

N Engl J Med

Isolation and characterization of exosomes from cell culture supernatants and biological fluids

Curr Protoc Cell Biol

Mechanisms of allergy and clinical immunology
Tissue factor–bearing exosome secretion from human mechanically stimulated bronchial epithelial cells in vitro and in vivo