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Murine aortic smooth muscle cells acquire, though fail to present exogenous protein antigens on major histocompatibility complex class II molecules.

Maddaluno M, MacRitchie N, Grassia G, Ialenti A, Butcher JP, Garside P, Brewer JM, Maffia P - Biomed Res Int (2014)

Bottom Line: Treatment with Eα-GFP (100 μg/mL) for 48 h induced a significant (P < 0.05) increase in the percentage of GFP positive SMCs while it did not affect the percentage of Y-Ae positive cells, being indicative of antigen uptake without its presentation in the context of MHC class II.Finally, OVA-treated SMCs failed to induce DO11.10-GFP hybridoma activation, a process independent of costimulation.Our results demonstrate that while murine primary aortic SMCs express MHC class II and can acquire exogenous antigens, they fail to activate T cells through a failure in antigen presentation and a lack of costimulatory molecule expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University of Naples Federico II, 80131 Naples, Italy ; Centre for Immunobiology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, 120 University Place, Glasgow G12 8TA, UK ; Novartis Vaccines, Via Fiorentina 1, 53100 Siena, Italy.

ABSTRACT
In the present study aortic murine smooth muscle cell (SMC) antigen presentation capacity was evaluated using the Eα-GFP/Y-Ae system to visualize antigen uptake through a GFP tag and tracking of Eα peptide/MHCII presentation using the Y-Ae Ab. Stimulation with IFN-γ (100 ng/mL) for 72 h caused a significant (P < 0.01) increase in the percentage of MHC class II positive SMCs, compared with unstimulated cells. Treatment with Eα-GFP (100 μg/mL) for 48 h induced a significant (P < 0.05) increase in the percentage of GFP positive SMCs while it did not affect the percentage of Y-Ae positive cells, being indicative of antigen uptake without its presentation in the context of MHC class II. After IFN-γ-stimulation, ovalbumin- (OVA, 1 mg/mL) or OVA323-339 peptide-(0.5 μg/mL) treated SMCs failed to induce OT-II CD4(+) T cell activation/proliferation; this was also accompanied by a lack of expression of key costimulatory molecules (OX40L, CD40, CD70, and CD86) on SMCs. Finally, OVA-treated SMCs failed to induce DO11.10-GFP hybridoma activation, a process independent of costimulation. Our results demonstrate that while murine primary aortic SMCs express MHC class II and can acquire exogenous antigens, they fail to activate T cells through a failure in antigen presentation and a lack of costimulatory molecule expression.

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SMCs lack key costimulatory molecules. Representative flow cytometry histograms and relative graph showing the effect of IFN-γ (100 ng/mL) on costimulatory/adhesion molecules expression in murine SMCs. Red empty histograms: isotype control; gray filled histograms or white columns: unstimulated SMCs; black empty histograms or black columns: IFN-γ-stimulated SMCs. Results are expressed as mean ± SEM from three separate experiments run in triplicate. *P < 0.05, **P < 0.01, and ***P < 0.001 versus unstimulated cells.
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fig4: SMCs lack key costimulatory molecules. Representative flow cytometry histograms and relative graph showing the effect of IFN-γ (100 ng/mL) on costimulatory/adhesion molecules expression in murine SMCs. Red empty histograms: isotype control; gray filled histograms or white columns: unstimulated SMCs; black empty histograms or black columns: IFN-γ-stimulated SMCs. Results are expressed as mean ± SEM from three separate experiments run in triplicate. *P < 0.05, **P < 0.01, and ***P < 0.001 versus unstimulated cells.

Mentions: Previous studies have correlated the inability of human SMCs to activate memory T cells with the lack of costimulation [19]. Thus we examined whether murine SMCs express costimulatory/adhesion molecules at baseline and after IFN-γ (100 ng/mL) stimulation for 72 h. As shown in Figure 4, unstimulated SMCs expressed CD54 (ICAM-1), CD80, and CD44 (30%, 11%, and 87% positive cells, resp.). The stimulation with IFN-γ caused a 2-fold increase in the percentage of both ICAM-1 (P < 0.01) and CD80 (P < 0.001) positive cells while it did not affect the percentage of CD44 positive cells. In contrast, only low levels of OX40L, CD40, CD70, and CD86 expression were detectable in unstimulated SMCs. IFN-γ stimulation did not increase the percentage of SMCs positive to these molecules. The failure of SMCs to respond to IFN-γ, in this case, was selective for the costimulatory molecules because the percentage of MHC class II molecules was increased after IFN-γ stimulation under the same conditions (Figure 4).


Murine aortic smooth muscle cells acquire, though fail to present exogenous protein antigens on major histocompatibility complex class II molecules.

Maddaluno M, MacRitchie N, Grassia G, Ialenti A, Butcher JP, Garside P, Brewer JM, Maffia P - Biomed Res Int (2014)

SMCs lack key costimulatory molecules. Representative flow cytometry histograms and relative graph showing the effect of IFN-γ (100 ng/mL) on costimulatory/adhesion molecules expression in murine SMCs. Red empty histograms: isotype control; gray filled histograms or white columns: unstimulated SMCs; black empty histograms or black columns: IFN-γ-stimulated SMCs. Results are expressed as mean ± SEM from three separate experiments run in triplicate. *P < 0.05, **P < 0.01, and ***P < 0.001 versus unstimulated cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4127268&req=5

fig4: SMCs lack key costimulatory molecules. Representative flow cytometry histograms and relative graph showing the effect of IFN-γ (100 ng/mL) on costimulatory/adhesion molecules expression in murine SMCs. Red empty histograms: isotype control; gray filled histograms or white columns: unstimulated SMCs; black empty histograms or black columns: IFN-γ-stimulated SMCs. Results are expressed as mean ± SEM from three separate experiments run in triplicate. *P < 0.05, **P < 0.01, and ***P < 0.001 versus unstimulated cells.
Mentions: Previous studies have correlated the inability of human SMCs to activate memory T cells with the lack of costimulation [19]. Thus we examined whether murine SMCs express costimulatory/adhesion molecules at baseline and after IFN-γ (100 ng/mL) stimulation for 72 h. As shown in Figure 4, unstimulated SMCs expressed CD54 (ICAM-1), CD80, and CD44 (30%, 11%, and 87% positive cells, resp.). The stimulation with IFN-γ caused a 2-fold increase in the percentage of both ICAM-1 (P < 0.01) and CD80 (P < 0.001) positive cells while it did not affect the percentage of CD44 positive cells. In contrast, only low levels of OX40L, CD40, CD70, and CD86 expression were detectable in unstimulated SMCs. IFN-γ stimulation did not increase the percentage of SMCs positive to these molecules. The failure of SMCs to respond to IFN-γ, in this case, was selective for the costimulatory molecules because the percentage of MHC class II molecules was increased after IFN-γ stimulation under the same conditions (Figure 4).

Bottom Line: Treatment with Eα-GFP (100 μg/mL) for 48 h induced a significant (P < 0.05) increase in the percentage of GFP positive SMCs while it did not affect the percentage of Y-Ae positive cells, being indicative of antigen uptake without its presentation in the context of MHC class II.Finally, OVA-treated SMCs failed to induce DO11.10-GFP hybridoma activation, a process independent of costimulation.Our results demonstrate that while murine primary aortic SMCs express MHC class II and can acquire exogenous antigens, they fail to activate T cells through a failure in antigen presentation and a lack of costimulatory molecule expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University of Naples Federico II, 80131 Naples, Italy ; Centre for Immunobiology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, 120 University Place, Glasgow G12 8TA, UK ; Novartis Vaccines, Via Fiorentina 1, 53100 Siena, Italy.

ABSTRACT
In the present study aortic murine smooth muscle cell (SMC) antigen presentation capacity was evaluated using the Eα-GFP/Y-Ae system to visualize antigen uptake through a GFP tag and tracking of Eα peptide/MHCII presentation using the Y-Ae Ab. Stimulation with IFN-γ (100 ng/mL) for 72 h caused a significant (P < 0.01) increase in the percentage of MHC class II positive SMCs, compared with unstimulated cells. Treatment with Eα-GFP (100 μg/mL) for 48 h induced a significant (P < 0.05) increase in the percentage of GFP positive SMCs while it did not affect the percentage of Y-Ae positive cells, being indicative of antigen uptake without its presentation in the context of MHC class II. After IFN-γ-stimulation, ovalbumin- (OVA, 1 mg/mL) or OVA323-339 peptide-(0.5 μg/mL) treated SMCs failed to induce OT-II CD4(+) T cell activation/proliferation; this was also accompanied by a lack of expression of key costimulatory molecules (OX40L, CD40, CD70, and CD86) on SMCs. Finally, OVA-treated SMCs failed to induce DO11.10-GFP hybridoma activation, a process independent of costimulation. Our results demonstrate that while murine primary aortic SMCs express MHC class II and can acquire exogenous antigens, they fail to activate T cells through a failure in antigen presentation and a lack of costimulatory molecule expression.

Show MeSH
Related in: MedlinePlus