Blockade of transforming growth factor beta/Smad signaling in T cells by overexpression of Smad7 enhances antigen-induced airway inflammation and airway reactivity - PubMed (original) (raw)
Blockade of transforming growth factor beta/Smad signaling in T cells by overexpression of Smad7 enhances antigen-induced airway inflammation and airway reactivity
A Nakao et al. J Exp Med. 2000.
Abstract
Transforming growth factor (TGF)-beta has been implicated in immunosuppression. However, it remains obscure whether regulation of T cells by TGF-beta contributes to the immunosuppression in vivo. To address this issue, we developed transgenic mice expressing Smad7, an intracellular antagonist of TGF-beta/Smad signaling, selectively in mature T cells using a plasmid construct coding a promoter element (the distal lck promoter) that directs high expression in peripheral T cells. Peripheral T cells were not growth inhibited by TGF-beta in Smad7 transgenic mice. Although Smad7 transgenic mice did not spontaneously show a specific phenotype, antigen-induced airway inflammation and airway reactivity were enhanced in Smad7 transgenic mice associated with high production of both T helper cell type 1 (Th1) and Th2 cytokines. Thus, blockade of TGF-beta/Smad signaling in mature T cells by expression of Smad7 enhanced airway inflammation and airway reactivity, suggesting that regulation of T cells by TGF-beta was crucial for negative regulation of the inflammatory (immune) response. Our findings also implicated TGF-beta/Smad signaling in mature T cells as a regulatory component of allergic asthma.
Figures
Figure 1
Generation of Smad7 Tg mice. (A) Schematic diagram of constructs used for peripheral T cell–specific expression of the mouse Flag-tagged Smad7 transgene. (B) Expression of the Flag-tagged Smad7 transgene in Smad7 Tg mice. Whole cell lysates were prepared from thymus (thy.) or spleen (sp.) from Smad7 Tg mice or Wt littermates, and subjected to Western blot (WB) analysis using anti-Flag antibody to detect protein expression of the Flag-tagged Smad7 transgene. As a positive control, cell lysate from COS7 cells transfected with Flag-tagged Smad7 (F-Smad7) cDNA was used.
Figure 2
Altered responsiveness of splenic T cells from Smad7 Tg mice to TGF-β. (A) Lack of responsiveness to TGF-β in splenocytes from Smad7 Tg mice. Splenocytes from Smad7 Tg mice or Wt littermates were stimulated with anti-CD3 mAb in the presence or absence (control) of TGF-β (1 ng/ml). Aliquots of cells were pulsed with [3H]thymidine, and incorporated counts (in cpm) were determined. Shown are means ± SD of [3H]thymidine corporation from triplicate cultures. The results are from one representative experiment out of three. (B) Inhibition of TGF-β–mediated phosphorylation of Smad2 in purified T cells from Smad7 Tg mice. Cell lysates were prepared from purified CD3+ T cells from Smad7 Tg mice or Wt littermates, and then immunoblotted with antiphosphorylated Smad2 antibody (top, P-Smad2) or anti-Smad2 antibody (bottom). The results are from one representative experiment out of three.
Figure 3
Normal T cell and B cell development in Smad7 Tg mice. FACS® analysis of thymocytes (A) or splenocytes (B) using the indicated conjugated antibodies. All data were gated for viable cells. Percentages represent proportions of viable cells in each region or quadrant. The results are from one representative experiment out of three.
Figure 4
Antigen-induced airway inflammation in Smad7 Tg mice. OVA-sensitized mice were challenged with inhalation of OVA as described in Materials and Methods. Leukocyte infiltration in the BALF (A) and eosinophil infiltration into the trachea (B) of the sensitized mice were then examined at 48 h after OVA inhalation. Data are means ± SD for five mice in each group. The results are from one representative experiment out of three. Eos, eosinophils; Neut, neutrophils; Lymph, lymphocytes; Mac, macrophages. *P < 0.05, significantly different from the mean value of the corresponding control response (Wt).
Figure 5
Cytokine production in Smad7 Tg mice. OVA-sensitized mice were challenged with inhalation of OVA. At 48 h after the inhalation, BALF was collected as described in Materials and Methods. For detection of cytokines in the culture supernatant, spleen cells were isolated from the mice sensitized with OVA/alum and stimulated with OVA (100 μg/ml) in vitro. 72 h after the stimulation, the culture supernatants were collected. IL-5 and IFN-γ levels in the BALF (A), OVA-induced IL-4, IL-5, and IFN-γ production in the supernatant of spleen cells from the sensitized mice (B), and TGF-β1 levels in the BALF (C) were determined by ELISA. Data are means ± SD for five mice in each group. The results are from one representative experiment out of three. *P < 0.05, significantly different from the mean value of the corresponding control response (Wt).
Figure 6
Activated phenotype in T lymphocytes in the airways of Smad7 Tg mice. Representative FACS® profiles of CD4 versus CD8 staining (A), CD4 versus CD25 staining (B), and intracellular staining of IL-4 versus IFN-γ in CD4 T cells (C), on BALF cells using anti-CD4–FITC, anti-CD8–PE, anti-CD25–PE, anti–IFN-γ–FITC, and anti–IL-4–PE antibodies (n = 5 mice in each group). Percentages represent proportions of viable cells in each region or quadrant.
Figure 7
Airway responsiveness to acetylcholine of Smad7 Tg mice. OVA-sensitized mice were challenged with inhalation of OVA (black bars) or saline (white bars). At 24 h after the inhalation, airway responsiveness was assessed by the time-integrated change in peak airway pressure (APTI; cmH2O · s) after intravenous acetylcholine challenge. Data are means ± SD for five mice in each group. The results are from one representative experiment out of three. *P < 0.05, significantly different from the mean value of the corresponding control response (Wt).
References
- Roberts A.B., Sporn M.B. The transforming growth factors-β. In: Sporn M.B., Roberts A.B., editors. Handbook of Experimental Pharmacology. Springer-Verlag; Heidelberg: 1990. pp. 418–472.
- Wahl S.M. Transforming growth factor-β in inflammationa cause and a cure. J. Clin. Immunol. 1992;12:61–74. - PubMed
- Miyazono K., ten Dijke P., Heldin C.-H. Receptors for transforming growth factor-β. Adv. Immunol. 1994;55:181–220. - PubMed
- Letterio J.J., Roberts A.B. Regulation of immune responses by TGF-β. Annu. Rev. Immunol. 1998;16:137–161. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Medical
Molecular Biology Databases
Miscellaneous