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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

Upregulation of CD23 by IL-4 and GM-CSF. Dose-ranging studies were performed to determine the optimum concentrations of IL-4 and GM-CSF. Alpha-smooth muscle isoactin positive Human ASMC (Clonetics) in T-75 flasks were starved for 24 h in 0.1% FBS containing medium M199. The cells were then stimulated with BSA (1 μg/ml), IL-4 (0.125. 0.25, 0.5, or 1.0 nM) or GM-CSF (0.1, 0.2, 0.4, or 0.8 nM) for 24 h. The cell lysates in RIPA buffer were subjected to western blot analysis for CD23. Mouse anti-human CD23 monoclonal antibody (clone M-L233, BD Biosciences, 1 μg/5 ml) was used as the primary antibody and anti-mouse horseradish peroxidase linked antibody as the secondary antibody (Amersham). The immunoreactive protein bands were detected by enhanced chemiluminescence light (ECL) (Amersham).
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Figure 1: Upregulation of CD23 by IL-4 and GM-CSF. Dose-ranging studies were performed to determine the optimum concentrations of IL-4 and GM-CSF. Alpha-smooth muscle isoactin positive Human ASMC (Clonetics) in T-75 flasks were starved for 24 h in 0.1% FBS containing medium M199. The cells were then stimulated with BSA (1 μg/ml), IL-4 (0.125. 0.25, 0.5, or 1.0 nM) or GM-CSF (0.1, 0.2, 0.4, or 0.8 nM) for 24 h. The cell lysates in RIPA buffer were subjected to western blot analysis for CD23. Mouse anti-human CD23 monoclonal antibody (clone M-L233, BD Biosciences, 1 μg/5 ml) was used as the primary antibody and anti-mouse horseradish peroxidase linked antibody as the secondary antibody (Amersham). The immunoreactive protein bands were detected by enhanced chemiluminescence light (ECL) (Amersham).

Mentions: Alpha-smooth muscle isoactin positive Human ASMC (Cambrex, Walkersville, MD) in T-75 flasks were starved for 24 hours in 0.1% (vol/vol) fetal bovine serum (FBS) containing medium M199 (Cellgro, Herndon, VA) supplemented with 1% (vol/vol) antibiotic/antimycotic solution (Sigma Chemical Co., St Louis, MO). The cells were then stimulated with either vehicle (bovine serum albumin, BSA, vehicle for cytokines (1 mg/ml), in M199; ethanol (EtOH), vehicle for LTD4 (6% final concentration) and PGD2 (0.001–0.01% final concentration); M199, vehicle for tryptase), an individual mediator, or a mediator in combination with GM-CSF at their optimum concentrations for 24 hours. The doses of cytokines used were up to four time ED50 including: IL-4 (0.04–1 nM), GM-CSF (0.07–0.8 nM), IL-13 (0.4 nM), IL-5 (0.01–0.07 nM), IL-13 (0.3–2.2 nM), PGD2 (1–10 μM), LTD4 (1–10 μM), tryptase (30 nM, a concentration sufficient to induce ASMC proliferation) (Sigma). Dose ranging studies were performed to determine the optimum concentration of IL-4 and GM-CSF on the expression of CD23, and the doses chosen were IL-4 (0.5 nM) and GM-CSF (0.4 nM) (Figure 1). All cytokines were obtained from R & D Systems Inc. Minneapolis, MN except GM-CSF which was obtained from Sigma. The cells were then harvested with a soft rubber edged scraper, centrifuged for 5 minutes at 1000 rpm (200 g), washed and resuspended in 1% BSA in phosphate buffered saline (PBS) and fixed with 70% ETOH. After washing twice more, the cells were resuspended in 1% BSA in PBS. Finally, they were filtered through a 40 μm nylon mesh to obtain single cell suspension and stained with (20 μl) of PE (phycoerythrin)-CD23 (EBVCS-5, BD Biosciences, San Jose, CA) or PE-mouse IgG1 for 15 minutes in the dark to facilitate staining for flow cytometry.


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)

Upregulation of CD23 by IL-4 and GM-CSF. Dose-ranging studies were performed to determine the optimum concentrations of IL-4 and GM-CSF. Alpha-smooth muscle isoactin positive Human ASMC (Clonetics) in T-75 flasks were starved for 24 h in 0.1% FBS containing medium M199. The cells were then stimulated with BSA (1 μg/ml), IL-4 (0.125. 0.25, 0.5, or 1.0 nM) or GM-CSF (0.1, 0.2, 0.4, or 0.8 nM) for 24 h. The cell lysates in RIPA buffer were subjected to western blot analysis for CD23. Mouse anti-human CD23 monoclonal antibody (clone M-L233, BD Biosciences, 1 μg/5 ml) was used as the primary antibody and anti-mouse horseradish peroxidase linked antibody as the secondary antibody (Amersham). The immunoreactive protein bands were detected by enhanced chemiluminescence light (ECL) (Amersham).
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Related In: Results  -  Collection

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Figure 1: Upregulation of CD23 by IL-4 and GM-CSF. Dose-ranging studies were performed to determine the optimum concentrations of IL-4 and GM-CSF. Alpha-smooth muscle isoactin positive Human ASMC (Clonetics) in T-75 flasks were starved for 24 h in 0.1% FBS containing medium M199. The cells were then stimulated with BSA (1 μg/ml), IL-4 (0.125. 0.25, 0.5, or 1.0 nM) or GM-CSF (0.1, 0.2, 0.4, or 0.8 nM) for 24 h. The cell lysates in RIPA buffer were subjected to western blot analysis for CD23. Mouse anti-human CD23 monoclonal antibody (clone M-L233, BD Biosciences, 1 μg/5 ml) was used as the primary antibody and anti-mouse horseradish peroxidase linked antibody as the secondary antibody (Amersham). The immunoreactive protein bands were detected by enhanced chemiluminescence light (ECL) (Amersham).
Mentions: Alpha-smooth muscle isoactin positive Human ASMC (Cambrex, Walkersville, MD) in T-75 flasks were starved for 24 hours in 0.1% (vol/vol) fetal bovine serum (FBS) containing medium M199 (Cellgro, Herndon, VA) supplemented with 1% (vol/vol) antibiotic/antimycotic solution (Sigma Chemical Co., St Louis, MO). The cells were then stimulated with either vehicle (bovine serum albumin, BSA, vehicle for cytokines (1 mg/ml), in M199; ethanol (EtOH), vehicle for LTD4 (6% final concentration) and PGD2 (0.001–0.01% final concentration); M199, vehicle for tryptase), an individual mediator, or a mediator in combination with GM-CSF at their optimum concentrations for 24 hours. The doses of cytokines used were up to four time ED50 including: IL-4 (0.04–1 nM), GM-CSF (0.07–0.8 nM), IL-13 (0.4 nM), IL-5 (0.01–0.07 nM), IL-13 (0.3–2.2 nM), PGD2 (1–10 μM), LTD4 (1–10 μM), tryptase (30 nM, a concentration sufficient to induce ASMC proliferation) (Sigma). Dose ranging studies were performed to determine the optimum concentration of IL-4 and GM-CSF on the expression of CD23, and the doses chosen were IL-4 (0.5 nM) and GM-CSF (0.4 nM) (Figure 1). All cytokines were obtained from R & D Systems Inc. Minneapolis, MN except GM-CSF which was obtained from Sigma. The cells were then harvested with a soft rubber edged scraper, centrifuged for 5 minutes at 1000 rpm (200 g), washed and resuspended in 1% BSA in phosphate buffered saline (PBS) and fixed with 70% ETOH. After washing twice more, the cells were resuspended in 1% BSA in PBS. Finally, they were filtered through a 40 μm nylon mesh to obtain single cell suspension and stained with (20 μl) of PE (phycoerythrin)-CD23 (EBVCS-5, BD Biosciences, San Jose, CA) or PE-mouse IgG1 for 15 minutes in the dark to facilitate staining for flow cytometry.

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