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Fibronectin is a TH1-specific molecule in human subjects

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Presentation on theme: "Fibronectin is a TH1-specific molecule in human subjects"— Presentation transcript:

1 Fibronectin is a TH1-specific molecule in human subjects
Hilary Sandig, PhD, Joanne McDonald, BSc, Jane Gilmour, PhD, Matthew Arno, PhD, Tak H. Lee, MD, ScD, David J. Cousins, PhD  Journal of Allergy and Clinical Immunology  Volume 124, Issue 3, Pages e5 (September 2009) DOI: /j.jaci Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 TH1-specific expression of fibronectin. A, FN1 mRNA is selectively expressed by TH1 cells. R, Resting; A, activated. Data are shown as means ± SEMs (n = 4). B, FN1 mRNA is induced during TH1 differentiation. Data are representative of 2 experiments. C and D, Fibronectin is secreted by TH1 cells. Fibronectin ELISA of supernatants is shown (Fig 1, C, means ± SEMs, n = 4; Fig 1, D, data are representative of 2 experiments). E and F, Western blotting of cytoplasmic extracts and cell-culture supernatants. −, Resting; +, activated. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Ex vivo TH1 cells selectively express fibronectin. Ex vivo TH1 and TH2 cells were isolated based on the expression of CD3, CD4, and IFN-γ or IL-13. A, FACS plots show presort and postsort profiles. B, RT-PCR on sorted samples (means ± SEMs, n = 3). Gene expression is shown relative to 1 biologic replicate (TH1 for IFNG and FN1 and TH2 for IL13). Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Expression of fibronectin by immune cell types. The results of real-time RT-PCR for total FN1 (A) and EDA-FN1 (B) mRNA in a panel of immune cell types are shown. Cell types were isolated by sorting from PBMCs or PMNs, as described in the Methods section, and in vitro generated TH1 and TH2 cells are also shown. R, Resting cells; A, activated cells (PMA/ionomycin). Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 TH1 cell–derived fibronectin modulates monocyte gene expression. A, Monocytes were cultured overnight with or without IFN-γ as indicated. After overnight incubation, fibronectin (shaded bars) or PBS (solid bars) was added for 4 hours. Quantitative real-time RT-PCR was performed for IL-6 (B) and IL-10 (C). ∗P < .05 and ∗∗P < .01, 2-way ANOVA with the Tukey post test. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Model for the role of fibronectin in TH1 cell functions. FnBP, Fibronectin-binding protein; α4β1, integrin α4β1; MØ, macrophage. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 In vitro differentiation of human TH1 and TH2 cells
In vitro differentiation of human TH1 and TH2 cells. Naive CD4+ T cells were isolated from PBMCs and cultured for 28 days under TH1- or TH2-polarizing conditions. Where indicated, cells were activated for 4 hours with PMA (5 ng/mL) and ionomycin (Ion; 500 ng/mL), and intracellular cytokine staining was performed. Data are representative of 6 independent experiments from different healthy donors. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Variable splicing of TH1 cell fibronectin. A, Human fibronectin gene
Variable splicing of TH1 cell fibronectin. A, Human fibronectin gene. Blue boxes represent exons. Black boxes indicate regions of variable splicing. VAR, Variable region. B, RT-PCR for EDB, EDA, and variable regions using primers indicated in Fig E2, C and D. C and D, Variably spliced regions. Red boxes represent exons identified by sequencing. Frequency of exon splice variants (Fig E2, A-F) is shown. E, Coding sequence of TH1 cell fibronectin isoforms around the EDA and variable regions (black boxes). F, Alignments of the variable regions and LDV and REDV motifs are highlighted. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Variable splicing of TH1 cell fibronectin. A, Human fibronectin gene
Variable splicing of TH1 cell fibronectin. A, Human fibronectin gene. Blue boxes represent exons. Black boxes indicate regions of variable splicing. VAR, Variable region. B, RT-PCR for EDB, EDA, and variable regions using primers indicated in Fig E2, C and D. C and D, Variably spliced regions. Red boxes represent exons identified by sequencing. Frequency of exon splice variants (Fig E2, A-F) is shown. E, Coding sequence of TH1 cell fibronectin isoforms around the EDA and variable regions (black boxes). F, Alignments of the variable regions and LDV and REDV motifs are highlighted. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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