Limits...
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, GM-CSF and IL-4/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 IL-4 (0.5 nM)/GM-CSF (0.4 nM) for 24 h. The FACS analysis showed that the smaller cells passed through Gate A and larger cells passed through Gate D. Background noise was eliminated using the BSA-stimulated control cells that were labeled with PE-anti-CD23 (EBVCS), represented by C in Gate A, and F in Gate D. The FACS results of a representative experiment showed 66% of the larger cells (Gate D) had an increase in cell expression of CD23 when compared to the controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1173127&req=5

Figure 2: Upregulation of CD23 by IL-4, GM-CSF and IL-4/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 IL-4 (0.5 nM)/GM-CSF (0.4 nM) for 24 h. The FACS analysis showed that the smaller cells passed through Gate A and larger cells passed through Gate D. Background noise was eliminated using the BSA-stimulated control cells that were labeled with PE-anti-CD23 (EBVCS), represented by C in Gate A, and F in Gate D. The FACS results of a representative experiment showed 66% of the larger cells (Gate D) had an increase in cell expression of CD23 when compared to the controls.

Mentions: Previous studies have shown that IgE immune complexes in atopic serum caused an increase in CD23 expression in ASMC [16]. To determine if other humoral factors in atopic serum effect CD23 expression in human ASMC, we have tested the effect of the relevant cytokines, arachidonic acid metabolites, and the mast cell enzyme tryptase. Flow cytometry was performed to evaluate differences in cell populations after stimulation of the huASMC for 24 hours with either individual mediators IL-4 (0.5 nM), GM-CSF (0.4 nM), IL-13 (0.4 nM), IL-5 (0.4 nM), PGD2 (10 μM), LTD4 (10 μM), tryptase (30 nM) or a combination of IL-4, IL-5, and IL-13 each with GM-CSF. Within the huASMC stimulated by IL-4, GM-CSF or the combination of IL-4/GM-CSF, two populations of cells were detected distinguishable by cell size. While the smaller cells did not show a significant expression of CD23, many of the larger cells showed increased expression of CD23. In the example in Figure 2, 66% of the larger cells (gate D) showed an increase in cell expression of CD23 when compared to the controls. As stated previously, the functions of ASMC are heterogeneous including proliferation and synthesis. Previous studies have shown, on flow cytometry of ASMC stimulated in vitro with IL-1β and TNF-α, only 20–60% of ASMC produce GM-CSF. The ASMC producing GM-CSF include some which also have increased proliferative properties. This suggests that considerable heterogeneity exists in the phenotypic expression of the ASMC in culture [25].


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, GM-CSF and IL-4/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 IL-4 (0.5 nM)/GM-CSF (0.4 nM) for 24 h. The FACS analysis showed that the smaller cells passed through Gate A and larger cells passed through Gate D. Background noise was eliminated using the BSA-stimulated control cells that were labeled with PE-anti-CD23 (EBVCS), represented by C in Gate A, and F in Gate D. The FACS results of a representative experiment showed 66% of the larger cells (Gate D) had an increase in cell expression of CD23 when compared to the controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1173127&req=5

Figure 2: Upregulation of CD23 by IL-4, GM-CSF and IL-4/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 IL-4 (0.5 nM)/GM-CSF (0.4 nM) for 24 h. The FACS analysis showed that the smaller cells passed through Gate A and larger cells passed through Gate D. Background noise was eliminated using the BSA-stimulated control cells that were labeled with PE-anti-CD23 (EBVCS), represented by C in Gate A, and F in Gate D. The FACS results of a representative experiment showed 66% of the larger cells (Gate D) had an increase in cell expression of CD23 when compared to the controls.
Mentions: Previous studies have shown that IgE immune complexes in atopic serum caused an increase in CD23 expression in ASMC [16]. To determine if other humoral factors in atopic serum effect CD23 expression in human ASMC, we have tested the effect of the relevant cytokines, arachidonic acid metabolites, and the mast cell enzyme tryptase. Flow cytometry was performed to evaluate differences in cell populations after stimulation of the huASMC for 24 hours with either individual mediators IL-4 (0.5 nM), GM-CSF (0.4 nM), IL-13 (0.4 nM), IL-5 (0.4 nM), PGD2 (10 μM), LTD4 (10 μM), tryptase (30 nM) or a combination of IL-4, IL-5, and IL-13 each with GM-CSF. Within the huASMC stimulated by IL-4, GM-CSF or the combination of IL-4/GM-CSF, two populations of cells were detected distinguishable by cell size. While the smaller cells did not show a significant expression of CD23, many of the larger cells showed increased expression of CD23. In the example in Figure 2, 66% of the larger cells (gate D) showed an increase in cell expression of CD23 when compared to the controls. As stated previously, the functions of ASMC are heterogeneous including proliferation and synthesis. Previous studies have shown, on flow cytometry of ASMC stimulated in vitro with IL-1β and TNF-α, only 20–60% of ASMC produce GM-CSF. The ASMC producing GM-CSF include some which also have increased proliferative properties. This suggests that considerable heterogeneity exists in the phenotypic expression of the ASMC in culture [25].

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