Use of a three-dimensional cell culture model to study airway smooth muscle-mast cell interactions in airway remodeling.
Increased airway smooth muscle (ASM) mass and infiltration by mast cells are key features of airway remodeling in asthma. We describe a model to investigate the relationship between ASM, the extracellular matrix, mast cells, and airway remodeling. ASM cells were cultured in a three-dimensional (3-D) collagen I gel (3-D culture) alone or with mast cells. Immunocytochemistry and Western blotting of ASM in 3-D cultures revealed a spindle-shaped morphology and significantly lower alpha-smooth muscle actin and vimentin expression than in ASM cultured in monolayers on collagen type I or plastic (2-D culture). In 3-D cultures, basal ASM proliferation, examined by Ki67 immunocytochemistry, was reduced to 33 +/- 7% (P < 0.05) of that in 2-D cultures. The presence of mast cells in cocultures increased ASM proliferation by 1.8-fold (P < 0.05). Gelatin zymography revealed more active matrix metalloproteinase (MMP)-2 in 3-D than in 2-D culture supernatants over 7 days. Functional MMP activity was examined by gel contraction. The spontaneous gel contraction over 7 days was significantly inhibited by the MMP inhibitor ilomastat. Mast cell coculture enhanced ASM gel contraction by 22 +/- 16% (not significant). Our model shows that ASM has different morphology, with lower contractile protein expression and basal proliferation in 3-D culture. Compared with standard techniques, ASM synthetic function, as shown by MMP production and activity, is sustained over longer periods. The presence of mast cells in the 3-D model enhanced ASM proliferation and MMP production. Airway remodeling in asthma may be more accurately modeled by our system than by standard culture systems.
Ceresa
Claudia C
CC
Divisions of Therapeutics and Molecular Medicine, University of Nottingham, Nottingham, United Kingdom.
Knox
Alan J
AJ
Johnson
Simon R
SR
D001249:Asthma / Q000473:pathology / Q000503:physiopathology; D001980:Bronchi / Q000166:cytology* / Q000502:physiology; D002450:Cell Communication / Q000502:physiology*; D002455:Cell Division / Q000502:physiology; D048430:Cell Shape / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D008407:Mast Cells / Q000166:cytology*; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D009130:Muscle, Smooth / Q000166:cytology* / Q000502:physiology; D011014:Pneumonia / Q000473:pathology* / Q000503:physiopathology
D020778:Matrix Metalloproteinase 2