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Research upregulation of CD23 (FcepsilonRII) expression in human airway smooth muscle cells (huASMC) in response to IL-4, GM-CSF, and IL-4/GM-CSF.

Belleau JT, Gandhi RK, McPherson HM, Lew DB - Clin Mol Allergy (2005)

Bottom Line: The protein content of IL-4/GMCSF stimulated cells was significantly elevated.Stimulation with IL-4 resulted in the phosphorylation of STAT-6 and an increase in the expression of the IL-2Rgammac.Upregulation of CD23 by IL-4/GM-CSF results in phenotypic changes in huASMC that could play a role in cell migration or a change in the synthetic function of the cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, Children's Foundation Research Center at the Le Bonheur Children's Medical Center, University of Tennessee Health Science Center, 50 North Dunlap Street, Rm401, WPT, Memphis, TN 38103, USA. jcbelleau@yahoo.com

ABSTRACT

Background: Airway smooth muscle cells play a key role in remodeling that contributes to airway hyperreactivity. Airway smooth muscle remodeling includes hypertrophy and hyperplasia. It has been previously shown that the expression of CD23 on ASMC in rabbits can be induced by the IgE component of the atopic serum. We examined if other components of atopic serum are capable of inducing CD23 expression independent of IgE.

Methods: Serum starved huASMC were stimulated with either IL-4, GM-CSF, IL-13, IL-5, PGD2, LTD4, tryptase or a combination of IL-4, IL-5, IL-13 each with GM-CSF for a period of 24 h. CD23 expression was analyzed by flow cytometry, western blot, and indirect immunofluorescence.

Results: The CD23 protein expression was upregulated in huASMC in response to IL-4, GM-CSF, and IL-4/GM-CSF. The percentage of cells with increased fluorescence intensity above the control was 25.1 +/- 4.2% (IL-4), 15.6 +/- 2.7% (GM-CSF) and 32.9 +/- 13.9% (IL-4/GMCSF combination)(n = 3). The protein content of IL-4/GMCSF stimulated cells was significantly elevated. Expression of CD23 in response to IL-4, GM-CSF, IL-4/GM-CSF was accompanied by changes in cell morphology including depolymerization of isoactin fibers, cell spreading, and membrane ruffling. Western blot revealed abundant expression of the IL-4Ralpha and a low level expression of IL-2Rgammac in huASMC. Stimulation with IL-4 resulted in the phosphorylation of STAT-6 and an increase in the expression of the IL-2Rgammac.

Conclusion: CD23 on huASMC is upregulated by IL-4, GM-CSF, and IL-4/GM-CSF. The expression of CD23 is accompanied by an increase in cell volume and an increase in protein content per cell, suggesting hypertrophy. Upregulation of CD23 by IL-4/GM-CSF results in phenotypic changes in huASMC that could play a role in cell migration or a change in the synthetic function of the cells. Upregulation of CD23 in huASMC by IL-4 and GM-CSF can contribute to changes in huASMC and may provide an avenue for new therapeutic options in asthma targeting ASMC.

No MeSH data available.


Related in: MedlinePlus

Phosphorylation of STAT-6 by IL-4 and IL-4/GM-CSF in huASMC. Starved huASMC were incubated with either BSA vehicle control (1 μg/ml), IL-4 (0.5 nM), GM-CSF (0.4 nM), or IL-4/GM-CSF (0.5 nM/0.4 nM) for 15 minutes. Standard Western blot analyses were performed to detect STAT-6 and phosphorylated-STAT-6 (p-STAT-6) using a anti-STAT-6 polyclonal rabbit antibody (Calbiochem) and anti-p-STAT-6 polyclonal rabbit antibody (Calbiochem). Anti-rabbit horseradish peroxidase linked antibody was used as the secondary antibody. Protein bands were detected by ECL. STAT-6 was abundantly expressed by all four groups, while p-STAT-6 was only expressed in the IL-4 and IL-4/GM-CSF groups. Fibronectin polyclonal rabbit antibody (Sigma) was used as an irrelevant isotype control and was abundantly expressed in all four groups.
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Figure 7: Phosphorylation of STAT-6 by IL-4 and IL-4/GM-CSF in huASMC. Starved huASMC were incubated with either BSA vehicle control (1 μg/ml), IL-4 (0.5 nM), GM-CSF (0.4 nM), or IL-4/GM-CSF (0.5 nM/0.4 nM) for 15 minutes. Standard Western blot analyses were performed to detect STAT-6 and phosphorylated-STAT-6 (p-STAT-6) using a anti-STAT-6 polyclonal rabbit antibody (Calbiochem) and anti-p-STAT-6 polyclonal rabbit antibody (Calbiochem). Anti-rabbit horseradish peroxidase linked antibody was used as the secondary antibody. Protein bands were detected by ECL. STAT-6 was abundantly expressed by all four groups, while p-STAT-6 was only expressed in the IL-4 and IL-4/GM-CSF groups. Fibronectin polyclonal rabbit antibody (Sigma) was used as an irrelevant isotype control and was abundantly expressed in all four groups.

Mentions: To confirm that IL-4 was activating the IL-4Rα during the stimulation of huASMC, we examined the phosphorylation of downstream STAT-6 by western blot. Human ASMC were starved for 24 h and then stimulated with IL-4 (0.4 nM) for fifteen minutes. Results of Western blot revealed an approximately a four fold increase in intensity of the band for phosphorylated-STAT-6 in IL-4 stimulated cells when compared to BSA control (Figure 7). This supports the role of an IL-4 mediated signal transduction pathway involvement in CD23 upregulation in huASMC.


Research upregulation of CD23 (FcepsilonRII) expression in human airway smooth muscle cells (huASMC) in response to IL-4, GM-CSF, and IL-4/GM-CSF.

Belleau JT, Gandhi RK, McPherson HM, Lew DB - Clin Mol Allergy (2005)

Phosphorylation of STAT-6 by IL-4 and IL-4/GM-CSF in huASMC. Starved huASMC were incubated with either BSA vehicle control (1 μg/ml), IL-4 (0.5 nM), GM-CSF (0.4 nM), or IL-4/GM-CSF (0.5 nM/0.4 nM) for 15 minutes. Standard Western blot analyses were performed to detect STAT-6 and phosphorylated-STAT-6 (p-STAT-6) using a anti-STAT-6 polyclonal rabbit antibody (Calbiochem) and anti-p-STAT-6 polyclonal rabbit antibody (Calbiochem). Anti-rabbit horseradish peroxidase linked antibody was used as the secondary antibody. Protein bands were detected by ECL. STAT-6 was abundantly expressed by all four groups, while p-STAT-6 was only expressed in the IL-4 and IL-4/GM-CSF groups. Fibronectin polyclonal rabbit antibody (Sigma) was used as an irrelevant isotype control and was abundantly expressed in all four groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 7: Phosphorylation of STAT-6 by IL-4 and IL-4/GM-CSF in huASMC. Starved huASMC were incubated with either BSA vehicle control (1 μg/ml), IL-4 (0.5 nM), GM-CSF (0.4 nM), or IL-4/GM-CSF (0.5 nM/0.4 nM) for 15 minutes. Standard Western blot analyses were performed to detect STAT-6 and phosphorylated-STAT-6 (p-STAT-6) using a anti-STAT-6 polyclonal rabbit antibody (Calbiochem) and anti-p-STAT-6 polyclonal rabbit antibody (Calbiochem). Anti-rabbit horseradish peroxidase linked antibody was used as the secondary antibody. Protein bands were detected by ECL. STAT-6 was abundantly expressed by all four groups, while p-STAT-6 was only expressed in the IL-4 and IL-4/GM-CSF groups. Fibronectin polyclonal rabbit antibody (Sigma) was used as an irrelevant isotype control and was abundantly expressed in all four groups.
Mentions: To confirm that IL-4 was activating the IL-4Rα during the stimulation of huASMC, we examined the phosphorylation of downstream STAT-6 by western blot. Human ASMC were starved for 24 h and then stimulated with IL-4 (0.4 nM) for fifteen minutes. Results of Western blot revealed an approximately a four fold increase in intensity of the band for phosphorylated-STAT-6 in IL-4 stimulated cells when compared to BSA control (Figure 7). This supports the role of an IL-4 mediated signal transduction pathway involvement in CD23 upregulation in huASMC.

Bottom Line: The protein content of IL-4/GMCSF stimulated cells was significantly elevated.Stimulation with IL-4 resulted in the phosphorylation of STAT-6 and an increase in the expression of the IL-2Rgammac.Upregulation of CD23 by IL-4/GM-CSF results in phenotypic changes in huASMC that could play a role in cell migration or a change in the synthetic function of the cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, Children's Foundation Research Center at the Le Bonheur Children's Medical Center, University of Tennessee Health Science Center, 50 North Dunlap Street, Rm401, WPT, Memphis, TN 38103, USA. jcbelleau@yahoo.com

ABSTRACT

Background: Airway smooth muscle cells play a key role in remodeling that contributes to airway hyperreactivity. Airway smooth muscle remodeling includes hypertrophy and hyperplasia. It has been previously shown that the expression of CD23 on ASMC in rabbits can be induced by the IgE component of the atopic serum. We examined if other components of atopic serum are capable of inducing CD23 expression independent of IgE.

Methods: Serum starved huASMC were stimulated with either IL-4, GM-CSF, IL-13, IL-5, PGD2, LTD4, tryptase or a combination of IL-4, IL-5, IL-13 each with GM-CSF for a period of 24 h. CD23 expression was analyzed by flow cytometry, western blot, and indirect immunofluorescence.

Results: The CD23 protein expression was upregulated in huASMC in response to IL-4, GM-CSF, and IL-4/GM-CSF. The percentage of cells with increased fluorescence intensity above the control was 25.1 +/- 4.2% (IL-4), 15.6 +/- 2.7% (GM-CSF) and 32.9 +/- 13.9% (IL-4/GMCSF combination)(n = 3). The protein content of IL-4/GMCSF stimulated cells was significantly elevated. Expression of CD23 in response to IL-4, GM-CSF, IL-4/GM-CSF was accompanied by changes in cell morphology including depolymerization of isoactin fibers, cell spreading, and membrane ruffling. Western blot revealed abundant expression of the IL-4Ralpha and a low level expression of IL-2Rgammac in huASMC. Stimulation with IL-4 resulted in the phosphorylation of STAT-6 and an increase in the expression of the IL-2Rgammac.

Conclusion: CD23 on huASMC is upregulated by IL-4, GM-CSF, and IL-4/GM-CSF. The expression of CD23 is accompanied by an increase in cell volume and an increase in protein content per cell, suggesting hypertrophy. Upregulation of CD23 by IL-4/GM-CSF results in phenotypic changes in huASMC that could play a role in cell migration or a change in the synthetic function of the cells. Upregulation of CD23 in huASMC by IL-4 and GM-CSF can contribute to changes in huASMC and may provide an avenue for new therapeutic options in asthma targeting ASMC.

No MeSH data available.


Related in: MedlinePlus