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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.


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Expression of CD23 in response to IL-4/GM-CSF is accompanied by changes in huASMC morphology. Alpha-smooth muscle isoactin positive huASMC (Clonetics) in T-75 flasks were starved for 24 h in 0.1% FBS containing medium M199. The cells were then stimulated with either BSA or IL-4 (0.5 nM)/GM-CSF (0.4 nM) for 24 h, and stained with either anti-smooth muscle-isoactin (A & B) or anti-CD23 antibody (C & D). Those cells stimulated with the combination of IL-4/GM-CSF demonstrated CD23 expression (D) and changes in cell morphology including depolymerization of isoactin fibers, cell spreading, and membrane ruffling (B). Cells stimulated with BSA (vehicle for IL-4/GM-CSF) alone did not increase the expression of CD23 (C) nor changes in phenotype (A). These findings were confirmed by three independent observers.
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Figure 4: Expression of CD23 in response to IL-4/GM-CSF is accompanied by changes in huASMC morphology. Alpha-smooth muscle isoactin positive huASMC (Clonetics) in T-75 flasks were starved for 24 h in 0.1% FBS containing medium M199. The cells were then stimulated with either BSA or IL-4 (0.5 nM)/GM-CSF (0.4 nM) for 24 h, and stained with either anti-smooth muscle-isoactin (A & B) or anti-CD23 antibody (C & D). Those cells stimulated with the combination of IL-4/GM-CSF demonstrated CD23 expression (D) and changes in cell morphology including depolymerization of isoactin fibers, cell spreading, and membrane ruffling (B). Cells stimulated with BSA (vehicle for IL-4/GM-CSF) alone did not increase the expression of CD23 (C) nor changes in phenotype (A). These findings were confirmed by three independent observers.

Mentions: Western blot analysis of huASMC stimulated with IL-4, GM-CSF, or Il-4/GM-CSF for 24 h showed an increase in CD23 expression compared to BSA vehicle control (Figure 3). Indirect immunofluorescence was used also to identify any morphological changes associated with the cytokine stimulation and upregulation of CD23 (Figure 4A–D). Those cells stimulated with the combination of IL-4/GM-CSF demonstrated CD23 expression along with changes in cell morphology including depolymerization of isoactin fibers, cell spreading, and membrane ruffling (Figure 4B). These changes in phenotype are consistent with flow cytometry results in that the larger cells expressed CD23 (Figure 4D). In contrast, the control BSA stimulated population showed no changes in cell cytoskeletal structure and morphology (Figure 4A) or specific staining for CD23 (Figure 4C).


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)

Expression of CD23 in response to IL-4/GM-CSF is accompanied by changes in huASMC morphology. Alpha-smooth muscle isoactin positive huASMC (Clonetics) in T-75 flasks were starved for 24 h in 0.1% FBS containing medium M199. The cells were then stimulated with either BSA or IL-4 (0.5 nM)/GM-CSF (0.4 nM) for 24 h, and stained with either anti-smooth muscle-isoactin (A & B) or anti-CD23 antibody (C & D). Those cells stimulated with the combination of IL-4/GM-CSF demonstrated CD23 expression (D) and changes in cell morphology including depolymerization of isoactin fibers, cell spreading, and membrane ruffling (B). Cells stimulated with BSA (vehicle for IL-4/GM-CSF) alone did not increase the expression of CD23 (C) nor changes in phenotype (A). These findings were confirmed by three independent observers.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Expression of CD23 in response to IL-4/GM-CSF is accompanied by changes in huASMC morphology. Alpha-smooth muscle isoactin positive huASMC (Clonetics) in T-75 flasks were starved for 24 h in 0.1% FBS containing medium M199. The cells were then stimulated with either BSA or IL-4 (0.5 nM)/GM-CSF (0.4 nM) for 24 h, and stained with either anti-smooth muscle-isoactin (A & B) or anti-CD23 antibody (C & D). Those cells stimulated with the combination of IL-4/GM-CSF demonstrated CD23 expression (D) and changes in cell morphology including depolymerization of isoactin fibers, cell spreading, and membrane ruffling (B). Cells stimulated with BSA (vehicle for IL-4/GM-CSF) alone did not increase the expression of CD23 (C) nor changes in phenotype (A). These findings were confirmed by three independent observers.
Mentions: Western blot analysis of huASMC stimulated with IL-4, GM-CSF, or Il-4/GM-CSF for 24 h showed an increase in CD23 expression compared to BSA vehicle control (Figure 3). Indirect immunofluorescence was used also to identify any morphological changes associated with the cytokine stimulation and upregulation of CD23 (Figure 4A–D). Those cells stimulated with the combination of IL-4/GM-CSF demonstrated CD23 expression along with changes in cell morphology including depolymerization of isoactin fibers, cell spreading, and membrane ruffling (Figure 4B). These changes in phenotype are consistent with flow cytometry results in that the larger cells expressed CD23 (Figure 4D). In contrast, the control BSA stimulated population showed no changes in cell cytoskeletal structure and morphology (Figure 4A) or specific staining for CD23 (Figure 4C).

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