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Local administration of antisense phosphorothioate oligonucleotides to the c-kit ligand, stem cell factor, suppresses airway inflammation and IL-4 production.

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Presentation on theme: "Local administration of antisense phosphorothioate oligonucleotides to the c-kit ligand, stem cell factor, suppresses airway inflammation and IL-4 production."— Presentation transcript:

1 Local administration of antisense phosphorothioate oligonucleotides to the c-kit ligand, stem cell factor, suppresses airway inflammation and IL-4 production in a murine model of asthma  Susetta Finotto, PhD, Michael Buerke, MD, Karen Lingnau, PhD, Edgar Schmitt, PhD, Peter R. Galle, MD, Markus F. Neurath, MD  Journal of Allergy and Clinical Immunology  Volume 107, Issue 2, Pages (February 2001) DOI: /mai Copyright © 2001 Mosby, Inc. Terms and Conditions

2 Fig. 1 Efficient uptake of SCF antisense oligonucleotides in NIH 3T3 fibroblasts. Cells were coincubated with FITC-labeled antisense oligonucleotides for 24 hours followed by immunodetection of SCF (left panel) and FITC-labeled oligonucleotides (right panel) by immunofluorescence. Antisense oligonucleotides were mainly seen in the cytoplasm (C) and nucleus (N) of NIH 3T3 cells. One representative of 3 experiments performed in triplicate is shown. Similar data were obtained in SP1 keratinocytes (not shown). (Original magnification ×400.) Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2001 Mosby, Inc. Terms and Conditions

3 Fig. 2 Inhibition of SCF protein expression in NIH 3T3 fibroblasts by SCF antisense phosphorothioate oligonucleotides. Cells were coincubated with SCF antisense oligonucleotides (SCF-AS) or control oligonucleotides (mismatched DNA [SCF-MM], nonsense DNA [SCF-NS]; 10 μmol/L each) for 24 hours as indicated. Antisense but not control mismatched or nonsense oligonucleotides led to decreased SCF expression. Cells were stained with a polyclonal antibody against SCF. Cell viability in separate wells was assessed by trypan blue exclusion and was greater than 90% in all cases, suggesting that there was no direct toxic effect of the antisense DNA. Similar data were obtained in SP1 keratinocytes (not shown). (Original magnification ×400.) Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2001 Mosby, Inc. Terms and Conditions

4 Fig. 3 Inhibition of SCF protein expression in the lung after local administration of antisense oligonucleotides. Sections of OVA-immunized mice (B) and saline-treated control mice (A) were stained with an antibody against SCF. OVA immunization caused an upregulation of SCF expression. Intranasal administration of SCF antisense oligonucleotides suppressed SCF expression in the lung interstitium of OVA-immunized mice (C). In contrast, mismatched-treated mice showed high amounts of SCF protein (D). SCF expression in dexamethasone-treated lungs was similar to that in PBS-treated animals (E). a, Airways.(Original magnification ×400.) Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2001 Mosby, Inc. Terms and Conditions

5 Fig. 4 Localization of SCF antisense oligonucleotides in the lung of OVA-sensitized mice on local administration of DNA. There was a strong uptake of FITC-labeled antisense DNA by interstitial lung cells (arrows in A and C ) but not by epithelial cells. A weak staining for SCF after local antisense application was still observed in the airway epithelium (see bronchi No. 1 to 3 in B and D ), suggesting that SCF antisense treatment is more effective in downregulating SCF expression in the interstitium than in the epithelial areas (see Fig 3 for overall reduction of SCF expression on antisense administration). Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2001 Mosby, Inc. Terms and Conditions

6 Fig. 5 Western blot analysis of SCF protein expression in OVA-sensitized mice after treatment with corticosteroids (CS) or local administration of SCF antisense (AS) DNA. Lung tissue of OVA-sensitized mice was homogenized, and equal amounts of proteins were separated by SDS-PAGE and blotted to a nitrocellulose membrane followed by immunohybridization with SCF antibody (see Methods). Three representative mice per group are shown. There was a striking downregulation of lung SCF protein expression on corticosteroid and antisense DNA treatment. Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2001 Mosby, Inc. Terms and Conditions

7 Fig. 6 Histologic evidence of airway inflammation in OVA-sensitized and control mice: Effect of SCF antisense treatment. Paraffin-embedded lung sections were stained with toluidine blue. Note the presence of lung inflammation and mast cells (arrows) in OVA-sensitized mice (OVA) compared to antisense-treated mice (AS) and saline-treated control mice (PBS). Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2001 Mosby, Inc. Terms and Conditions

8 Fig. 7 Percentage of eosinophils in the BAL fluid of OVA-sensitized mice. Eosinophils on cytospins were detected by staining according to May Grünwald–Giemsa methods and quantified as specified in the Methods section. Data are reported as the mean percentage of eosinophils in the BAL fluid ± SEM. Significant differences compared with OVA treatment are indicated (*P < .05; **P < .01). SCF-AS, OVA-sensitized mice treated with SCF antisense DNA; SCF-MM, mice treated with mismatched DNA control oligonucleotides; Dex, OVA-sensitized mice treated with dexa-methasone. Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2001 Mosby, Inc. Terms and Conditions

9 Fig. 8 SCF antisense treatment selectively reduces IL-4 cytokine concentration in BAL fluid. Analysis of IFN-γ and IL-4 cytokine concentration in the BAL fluid from lungs of saline-treated mice (PBS; n = 5), OVA-sensitized mice (OVA; n = 5) and OVA-sensitized mice treated with SCF antisense DNA (SCF-AS; n = 4) or dexa-methasone (DEX; n = 4). Cytokine levels (IFN-γ, IL-4) were determined by specific ELISA and are reported as mean values ± SEM. IL-4 concentration in BAL fluid was significantly suppressed (P < .05) by SCF antisense treatment compared with levels in OVA-sensitized mice. Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2001 Mosby, Inc. Terms and Conditions

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