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Dual nature of T cell–epithelium interaction in chronic rhinosinusitis

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Presentation on theme: "Dual nature of T cell–epithelium interaction in chronic rhinosinusitis"— Presentation transcript:

1 Dual nature of T cell–epithelium interaction in chronic rhinosinusitis
Tomasz M. Basinski, PhD, David Holzmann, MD, Thomas Eiwegger, MD, Maya Zimmermann, PhD, Sven Klunker, PhD, Norbert Meyer, MD, Peter Schmid-Grendelmeier, MD, Marek Jutel, MD, Cezmi A. Akdis, MD  Journal of Allergy and Clinical Immunology  Volume 124, Issue 1, Pages e8 (July 2009) DOI: /j.jaci Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 The expression of IP-10, Mig, iTac, HLA-DR, Fas, FasL, TNF-α, and TNFRs in HSECs. A, Basal expression of chemokines and HLA-DR mRNAs in unstimulated HSECs (n = 4; mean ± SD). B, Kinetics of chemokines and HLA-DR expression on HSECs in response to IFN-γ. C, Significantly increased TNFR2 expression decreased after 48 hours of stimulation with IFN-γ (B and C, n = 3; mean ± SD). Each experiment was carried out in triplicate. ∗P < .05. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Expression of TRAIL and TRAIL receptors on HSECs. A, Basal expression of TRAIL and TRAILRs in unstimulated HSECs (n = 4). B, Surface expression of TRAIL and its receptors on HSECs. C, IFN-γ upregulates TRAIL and downregulates TRAILR4 on HSECs. HSECs were incubated with IFN-γ (12.5 ng/mL). D, TRAIL (6 hours) and TRAILR4 (12 hours) mRNAs expression in HSEC after IFN-γ treatment (n = 3 for each group). Data are presented as means ± SDs; ∗P < .05. us, Unstimulated. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Sinus epithelial apoptosis in CRS. Immunofluorescent in situ TUNEL staining of frozen tissue samples. Bright nuclei indicate positive TUNEL staining. 4'-6-Diamidino-2-phenylindole, (DAPI) blue staining indicates nuclei. Note structural disruption because of erosion and shedding of epithelial cells in hematoxylin and eosin staining (H&E). Representative slides are shown (n = 3 for each group), original magnification ×400. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 IFN-γ and its combination with TRAIL induces HSEC death independent of the clinical status. A, Cell death determination by ethidium bromide staining followed by flow cytometry (n = 4, mean ±SD). B, Apoptosis in epithelial cells after 4 days of cytokine treatment (percentage of annexin-positive cells; n = 4, mean ± SD). C, Percentage of annexin and 7-AAD–negative cells after stimulation with IFN-γ and TRAIL in healthy subjects, patients with CRS, and patients with CRS and allergy (day 4, n = 3, mean ± SD). ∗P < .05. us, Unstimulated. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 A, Degradation of caspases in HSEC by IFN-γ ± TRAIL and sFasL. Cells were stimulated with cytokines (4 days), stained with fluorescein isothiocyanate (FITC)–labeled VAD-FMK (n = 3, mean ± SD). Degradation of caspases was assessed with flow cytometry. B, Influence of TRAILR1 and TRAILR2 blocking on the induction of HSEC apoptosis. HSECs were stimulated with IFN-γ for 24 hours, and then TRAIL was added for 2 days (n = 3, mean ± SD). ∗P < .05. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 Sinus T cells produce IFN-γ and TRAIL. A, IFN-γ and TRAIL in supernatants of anti-CD2, anti-CD3, anti-CD28 mAbs stimulated sinus T cells (24 hours, n = 12 for CRS, n = 7 for CRS + allergy, mean ±S D). B, T cells induce HSEC apoptosis. HSECs were cocultured with activated autologous T cells in the presence of α-IFN-γ, α-TRAIL, α-FasL, and α-TNF-α (n = 3, mean ± SD). C, Induction of HSEC apoptosis by supernatants of TH1 and TH2 cells stimulated with anti-CD2, anti-CD3, and anti-CD28 mAbs (72 hours, 1 of 2 independent experiments). IC, Isotype control; us, unstimulated. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Purity of established HSEC lines from sinus biopsies
Purity of established HSEC lines from sinus biopsies. Bronchial smooth muscle cells (BSMC) and A549 cells were used as positive controls for vimentin and cytokeratin expressions, respectively. Images were acquired with the ×40 oil objective of a confocal laser scanning microscope Leica TCS SP5. All HSECs expressed cytokeratin and were negative for vimentin expression. In vimentin images, green was digitally changed to red for ease of visualization. Original magnification ×400. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Kinetics of apoptosis induction by combined effect of IFN-γ (12
Kinetics of apoptosis induction by combined effect of IFN-γ (12.5 ng/mL) and TRAIL (100 ng/mL) in HSECs. Transition of cells from the viable stage through the early stage of apoptosis to cell death is visible. One of at least 3 experiments performed with different HSEC lines is shown. Numbers in the lower right quadrant correspond to early apoptosis, and numbers in the upper right quadrant represent late apoptosis. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Phase contrast morphology (×200) of HSECs 4 days after exposure to cytokines: TRAIL (100 ng/mL), IFN-γ (12.5 ng/mL), sFasL (100 ng/mL), TNF-α (25 ng/mL). Percentage of nonapoptotic (viable) cells determined by annexin and 7-AAD staining and flow cytometry is indicated in the upper right corner. IFN-γ and its combination with TRAIL and sFasL induce clearly visible changes in morphology of HSECs. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Demonstration of apoptosis of HSECs on cytospins by TUNEL staining
Demonstration of apoptosis of HSECs on cytospins by TUNEL staining. HSECs stimulated with TRAIL (100 ng/mL) and/or IFN-γ (12.5 ng/mL) for 4 days were subjected to TUNEL with DAPI counterstaining. Original magnification ×400. Acquisition of images was performed with a confocal laser scanning microscope Leica TCS SP5. Results are representative of 1 of 2 experiments performed. us, Unstimulated. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 IFN-γ mRNA expression in whole tissue from patients with CRS and controls.
Total RNA from whole tissue was extracted and relative IFN-γ mRNA expression was measured by real-time PCR. The lowest value within the dataset was defined as 1, and relative expression to this value is shown (n = 4 controls, n = 7 CRS–, n = 3 patients with CRS + allergy; mean ± SEM). Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

13 IFN-γ–producing cells are present in vivo in sinus epithelial tissue of patients with CRS. Frozen sections from patients with CRS were generated and stained for CD3+ IFN-γ–producing cells. Images were acquired with the ×63 oil objective of a confocal laser scanning microscope Leica TCS SP5. Sinus tissue from the control group did not show any CD3 and IFN-γ staining. Data represent 1 of 5 independent experiments. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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