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Airway smooth muscle proliferation and survival is not modulated by mast cells.

Kaur D, Hollins F, Saunders R, Woodman L, Sutcliffe A, Cruse G, Bradding P, Brightling C - Clin. Exp. Allergy (2009)

Bottom Line: Mast cell activation was confirmed by the measurement of histamine release.Using a number of techniques, we found that ASM proliferation and survival was not significantly different between cells derived from subjects with or without asthma.Our findings do not support a role for increased airway smooth proliferation and survival as the major mechanism driving ASM hyperplasia in asthma.

View Article: PubMed Central - PubMed

Affiliation: Department of Infection, Inflammation and Immunity, Institute for Lung Health, University of Leicester, Leicester, UK.

ABSTRACT

Background: Airway smooth muscle (ASM) hyperplasia and mast cell localization within the ASM bundle are important features of asthma. The cause of this increased ASM mass is uncertain and whether it is a consequence of ASM-mast cell interactions is unknown.

Objective: We sought to investigate ASM proliferation and survival in asthma and the effects of co-culture with mast cells.

Methods: Primary ASM cultures were derived from 11 subjects with asthma and 12 non-asthmatic controls. ASM cells were cultured for up to 10 days in the presence or absence of serum either alone or in co-culture with the human mast cell line-1, unstimulated human lung mast cells (HLMC) or IgE/anti-IgE-activated HLMC. Proliferation was assessed by cell counts, CFSE assay and thymidine incorporation. Apoptosis and necrosis were analysed by Annexin V/propidium iodide staining using flow cytometry and by assessment of nuclear morphology using immunofluorescence. Mast cell activation was confirmed by the measurement of histamine release.

Results: Using a number of techniques, we found that ASM proliferation and survival was not significantly different between cells derived from subjects with or without asthma. Co-culture with mast cells did not affect the rate of proliferation or survival of ASM cells.

Conclusion: Our findings do not support a role for increased airway smooth proliferation and survival as the major mechanism driving ASM hyperplasia in asthma.

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Related in: MedlinePlus

(a) Representative micrographs at day 3 showing 4′,6-diamidino-2-phenylindole (DAPI) staining of airway smooth muscle (ASM) cells in the presence of ITS media alone, (b) in co-culture with human mast cell line (HMC-1) cells [stained with DAPI (blue) and overlaid with anti-CD117 (red)]. (c) ASM cells in co-culture with human lung mast cells (HLMC) cells (stained with DAPI and overlaid with anti-CD117). (d) Percentage of apoptotic nuclei of non-asthmatic (n=9) and asthmatic ASM cells (n=7) identified by nuclear morphology over 10 days. (e) Percentage of apoptotic nuclei for ASM cells in ITS media ± HMC-1 cells over 3 days (n=7) and (f) ASM cells in ITS media ± HLMC over 10 days (n=9). Data presented as mean±SEM; P-values are as shown.
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fig05: (a) Representative micrographs at day 3 showing 4′,6-diamidino-2-phenylindole (DAPI) staining of airway smooth muscle (ASM) cells in the presence of ITS media alone, (b) in co-culture with human mast cell line (HMC-1) cells [stained with DAPI (blue) and overlaid with anti-CD117 (red)]. (c) ASM cells in co-culture with human lung mast cells (HLMC) cells (stained with DAPI and overlaid with anti-CD117). (d) Percentage of apoptotic nuclei of non-asthmatic (n=9) and asthmatic ASM cells (n=7) identified by nuclear morphology over 10 days. (e) Percentage of apoptotic nuclei for ASM cells in ITS media ± HMC-1 cells over 3 days (n=7) and (f) ASM cells in ITS media ± HLMC over 10 days (n=9). Data presented as mean±SEM; P-values are as shown.

Mentions: Example immunofluorescent photomicrographs of mast cells in co-culture with ASM are as shown (Figs 5a–e). Mast cells were identified as CD117+ and excluded from examination of nuclear morphology. ASM nuclear DAPI staining supported the Annexin V data as there was no difference between the percentage apoptotic cells from subjects with (n=7) and without asthma (n=9; Fig. 5d). ASM cells were unaffected by the presence of HMC-1 cells over 3 days (n=7; Fig. 5e) and 10 days for the HLMC (n=9; Fig. 5f).


Airway smooth muscle proliferation and survival is not modulated by mast cells.

Kaur D, Hollins F, Saunders R, Woodman L, Sutcliffe A, Cruse G, Bradding P, Brightling C - Clin. Exp. Allergy (2009)

(a) Representative micrographs at day 3 showing 4′,6-diamidino-2-phenylindole (DAPI) staining of airway smooth muscle (ASM) cells in the presence of ITS media alone, (b) in co-culture with human mast cell line (HMC-1) cells [stained with DAPI (blue) and overlaid with anti-CD117 (red)]. (c) ASM cells in co-culture with human lung mast cells (HLMC) cells (stained with DAPI and overlaid with anti-CD117). (d) Percentage of apoptotic nuclei of non-asthmatic (n=9) and asthmatic ASM cells (n=7) identified by nuclear morphology over 10 days. (e) Percentage of apoptotic nuclei for ASM cells in ITS media ± HMC-1 cells over 3 days (n=7) and (f) ASM cells in ITS media ± HLMC over 10 days (n=9). Data presented as mean±SEM; P-values are as shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2821816&req=5

fig05: (a) Representative micrographs at day 3 showing 4′,6-diamidino-2-phenylindole (DAPI) staining of airway smooth muscle (ASM) cells in the presence of ITS media alone, (b) in co-culture with human mast cell line (HMC-1) cells [stained with DAPI (blue) and overlaid with anti-CD117 (red)]. (c) ASM cells in co-culture with human lung mast cells (HLMC) cells (stained with DAPI and overlaid with anti-CD117). (d) Percentage of apoptotic nuclei of non-asthmatic (n=9) and asthmatic ASM cells (n=7) identified by nuclear morphology over 10 days. (e) Percentage of apoptotic nuclei for ASM cells in ITS media ± HMC-1 cells over 3 days (n=7) and (f) ASM cells in ITS media ± HLMC over 10 days (n=9). Data presented as mean±SEM; P-values are as shown.
Mentions: Example immunofluorescent photomicrographs of mast cells in co-culture with ASM are as shown (Figs 5a–e). Mast cells were identified as CD117+ and excluded from examination of nuclear morphology. ASM nuclear DAPI staining supported the Annexin V data as there was no difference between the percentage apoptotic cells from subjects with (n=7) and without asthma (n=9; Fig. 5d). ASM cells were unaffected by the presence of HMC-1 cells over 3 days (n=7; Fig. 5e) and 10 days for the HLMC (n=9; Fig. 5f).

Bottom Line: Mast cell activation was confirmed by the measurement of histamine release.Using a number of techniques, we found that ASM proliferation and survival was not significantly different between cells derived from subjects with or without asthma.Our findings do not support a role for increased airway smooth proliferation and survival as the major mechanism driving ASM hyperplasia in asthma.

View Article: PubMed Central - PubMed

Affiliation: Department of Infection, Inflammation and Immunity, Institute for Lung Health, University of Leicester, Leicester, UK.

ABSTRACT

Background: Airway smooth muscle (ASM) hyperplasia and mast cell localization within the ASM bundle are important features of asthma. The cause of this increased ASM mass is uncertain and whether it is a consequence of ASM-mast cell interactions is unknown.

Objective: We sought to investigate ASM proliferation and survival in asthma and the effects of co-culture with mast cells.

Methods: Primary ASM cultures were derived from 11 subjects with asthma and 12 non-asthmatic controls. ASM cells were cultured for up to 10 days in the presence or absence of serum either alone or in co-culture with the human mast cell line-1, unstimulated human lung mast cells (HLMC) or IgE/anti-IgE-activated HLMC. Proliferation was assessed by cell counts, CFSE assay and thymidine incorporation. Apoptosis and necrosis were analysed by Annexin V/propidium iodide staining using flow cytometry and by assessment of nuclear morphology using immunofluorescence. Mast cell activation was confirmed by the measurement of histamine release.

Results: Using a number of techniques, we found that ASM proliferation and survival was not significantly different between cells derived from subjects with or without asthma. Co-culture with mast cells did not affect the rate of proliferation or survival of ASM cells.

Conclusion: Our findings do not support a role for increased airway smooth proliferation and survival as the major mechanism driving ASM hyperplasia in asthma.

Show MeSH
Related in: MedlinePlus