Mechanisms of immune modulation by transforming growth factor-beta(1): A role for Smad3 as an intracellular mediator

TGF-β₁ (T&barbelow;ransforming g&barbelow;rowth f&barbelow;actor- β₁) produces a diverse range of physiological immune modulatory effects. Targeted deletion of the TGF-β₁ gene exemplifies the importance of TGF-β₁ on maintaining immune homeostasis. TGF-β₁−/− mice develop a multifocal inflammatory disease with a T cell-dependent autoimmune process. A role for TGF-β₁ in maintaining T cell tolerance and demonstration that TGF-β₁ signaling in T cells is critical for maintaining B cell tolerance is illustrated by spontaneous T cell differentiation and autoimmunity in transgenic mice expressing a dominant negative TGF-β receptor under the control of a T cell-specific promoter. Mice homozygous for the TGF-β₁ receptor-activated Smad3 (Smad3−/− ) succumb to a deficiency in mucosal immunity and defective T cell responsiveness to TGF-β₁. In light of these observations, the overall objective of this research was to test the hypothesis that TGF-β ₁ acts directly on T cells to regulate IL-2 (i&barbelow;nterleukin-2&barbelow;) expression through Smad-mediated signaling. RT-PCR and ELISA analyses demonstrated a concentration-dependent bifunctional augmentation and attenuation of IL-2 expression by TGF-β₁ in α-CD3 + α-CD28-activated T cells. In agreement, a concentration-dependent stimulation and suppression of immunoglobulin production by TGF-β₁ in antigen-stimulated B cells was also established. T cells obtained from Smad3−/− mice were refractory to inhibition of IL-2 mRNA and IL-2 protein secretion by TGF-β₁. Inhibition of α-CD3 + α-CD28-induced T cell growth by TGF-β₁ was also attenuated in Smad3 −/− T cells. Collectively, these results establish a role for Smad3 in regulating IL-2 production and T cell growth in response to TGF-β ₁. Inhibition of LPS (l&barbelow;ipopo&barbelow;lys&barbelow;accharide)-activated B cell growth was unaffected in Smad3−/− mice suggesting a cell-type specificity for Smad3 signaling. Demonstration of a reversal of TGF-β₁-induced T cell growth arrest by exogenous IL-2 suggested that Smad3 may modulate T cell growth through a direct effect on IL-2 production. These results provide a putative mechanism whereby TGF-β₁ may selectively and specifically target T cell growth. Smad3-null B cells were demonstrably less sensitive to the inhibitory effects of TGF-β ₁ on IgM production in vitro. Moreover, a role for Smad3 in mediating IgA production by TGF-β₁ in vitro was evident by an inability of TGF-β₁ to augment IgA secretion in LPS-activated Smad3−/− splenic B cells. Five putative CAGA Smad3 response elements were identified in the IL-2 promoter. EMSA analyses demonstrated that CAGA sequences were critical for constitutive and α-CD3 + α-CD28-induced DNA binding activity at the CD28RE and proximal AP-1 (a&barbelow;ctivating p&barbelow;rotein-1&barbelow;) site, but not the distal NFAT or NRE-A sites in the IL-2 promoter. Smad3 binding to the proximal AP-1 site was CAGA-independent suggesting that Smad3 may mediate its effects through protein•protein interactions. TGF-β ₁ stimulated ERK MAP (e&barbelow;xtracellular r&barbelow;egulated k&barbelow;inase m&barbelow;itogen a&barbelow;ctivated p&barbelow;rotein) kinase activity in splenocytes. Blockade of ERK MAP kinase activity augmented Smad3 nuclear expression and attenuated inhibition of IL-2 by TGF-β ₁