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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 acquire exogenous antigens but fail to present them in the context of MHC class II. Evaluation of antigen uptake/presentation by murine SMCs. SMCs were stimulated with IFN-γ (100 ng/mL) for 72 h and subsequently treated with Eα-GFP peptide (100 µg/mL) for the indicated time points. (a) MHC class II expression. (b) GFP expression. (c) Representative flow cytometry plots showing no positivity of SMCs to the Y-Ae Ab or (d) positivity of DCs, used as a positive control. Results are expressed as mean ± SEM from three separate experiments. *P < 0.05, **P < 0.01, versus unstimulated cells.
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fig1: SMCs acquire exogenous antigens but fail to present them in the context of MHC class II. Evaluation of antigen uptake/presentation by murine SMCs. SMCs were stimulated with IFN-γ (100 ng/mL) for 72 h and subsequently treated with Eα-GFP peptide (100 µg/mL) for the indicated time points. (a) MHC class II expression. (b) GFP expression. (c) Representative flow cytometry plots showing no positivity of SMCs to the Y-Ae Ab or (d) positivity of DCs, used as a positive control. Results are expressed as mean ± SEM from three separate experiments. *P < 0.05, **P < 0.01, versus unstimulated cells.

Mentions: Stimulation with IFN-γ (100 ng/mL) for 72 h resulted in a significant (P < 0.01) 5- to 6-fold increase in the percentage of MHC class II positive SMCs compared with unstimulated cells (Figure 1(a)). Similar results were observed in IFN-γ-stimulated SMCs subsequently treated with Eα peptide (100 µg/mL) for 1 and 24 h (P < 0.05), while no significant changes were observed after 48 h of treatment (Figure 1(a)). As shown in Figure 1(b), SMC treatment with Eα peptide induced an increase in the percentage of GFP positive cells, both in presence or absence of IFN-γ-stimulation, being indicative of antigen uptake. The increase in GFP positive cells observed was significant only at 48 h (P < 0.05). No significant changes were observed in the percentage of Y-Ae positive SMCs after IFN-γ-stimulation and/or treatment with Eα peptide (Figure 1(c)) suggesting that, although SMCs internalize the antigen, they are not able to present the Eα peptide in the context of MHC class II. Treatment of DCs with Eα peptide (100 µg/mL), used as positive control, caused an increase in the percentage of Y-Ae positive cells (Figure 1(d)).


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 acquire exogenous antigens but fail to present them in the context of MHC class II. Evaluation of antigen uptake/presentation by murine SMCs. SMCs were stimulated with IFN-γ (100 ng/mL) for 72 h and subsequently treated with Eα-GFP peptide (100 µg/mL) for the indicated time points. (a) MHC class II expression. (b) GFP expression. (c) Representative flow cytometry plots showing no positivity of SMCs to the Y-Ae Ab or (d) positivity of DCs, used as a positive control. Results are expressed as mean ± SEM from three separate experiments. *P < 0.05, **P < 0.01, versus unstimulated cells.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: SMCs acquire exogenous antigens but fail to present them in the context of MHC class II. Evaluation of antigen uptake/presentation by murine SMCs. SMCs were stimulated with IFN-γ (100 ng/mL) for 72 h and subsequently treated with Eα-GFP peptide (100 µg/mL) for the indicated time points. (a) MHC class II expression. (b) GFP expression. (c) Representative flow cytometry plots showing no positivity of SMCs to the Y-Ae Ab or (d) positivity of DCs, used as a positive control. Results are expressed as mean ± SEM from three separate experiments. *P < 0.05, **P < 0.01, versus unstimulated cells.
Mentions: Stimulation with IFN-γ (100 ng/mL) for 72 h resulted in a significant (P < 0.01) 5- to 6-fold increase in the percentage of MHC class II positive SMCs compared with unstimulated cells (Figure 1(a)). Similar results were observed in IFN-γ-stimulated SMCs subsequently treated with Eα peptide (100 µg/mL) for 1 and 24 h (P < 0.05), while no significant changes were observed after 48 h of treatment (Figure 1(a)). As shown in Figure 1(b), SMC treatment with Eα peptide induced an increase in the percentage of GFP positive cells, both in presence or absence of IFN-γ-stimulation, being indicative of antigen uptake. The increase in GFP positive cells observed was significant only at 48 h (P < 0.05). No significant changes were observed in the percentage of Y-Ae positive SMCs after IFN-γ-stimulation and/or treatment with Eα peptide (Figure 1(c)) suggesting that, although SMCs internalize the antigen, they are not able to present the Eα peptide in the context of MHC class II. Treatment of DCs with Eα peptide (100 µg/mL), used as positive control, caused an increase in the percentage of Y-Ae positive cells (Figure 1(d)).

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