In these studies, using a regulatable TGF-β1–expressing plasmid (termed pTet-On-TGF-β1), we show that in vivo TGF-β1 secretion rapidly induces IL-10 secretion and that such induction occurs in Th1 cells via Smad4 binding to and activation of the IL-10 promoter. The close association of these two major “regulatory” cytokines thus revealed mirrors their association during suppressor function occurring in association with Th1-mediated inflammation (30
). Powrie et al. (31
) for instance, has shown that in the SCID-transfer model of colitis, the regulatory effect of the subpopulation of cells in the memory CD45RBlow
cells is abrogated by administration of anti–TGF-β1 or anti–IL-10R and that such cells either produce or induce TGF-β1 and/or IL-10 (8
). In addition, we have shown that the regulation of TNBS colitis by feeding TNP-substituted proteins requires both TGF-β1 and IL-10 (7
). Finally, we and others have also shown that both “self–MHC-reactive” and CD4+
regulatory T cells produce both TGF-β1 and IL-10 upon appropriate stimulation (32
The fact that TGF-β induces IL-10 as shown here whereas IL-10 does not induce TGF-β1 strongly suggests that IL-10 facilitates TGF-β1 regulatory function and not the other way around (4
). One way such facilitation could occur is through IL-10 enhancement of TGF-β1 signaling. This possibility is supported by Cottrez and Groux (35
) who showed that IL-10 maintains TGF-β1RII expression on activated cells that would otherwise down-regulate this receptor. In addition, it is known that Th1 cytokines such as IFN-γ and TNF-α up-regulate Smad7, a cytosolic intermediate that inhibits TGF-β1 signaling via other Smads (36
) and it is thus possible (but not yet proven) that IL-10 inhibits such Smad7 up-regulation.
Another way that IL-10 could facilitate TGF-β1 regulatory activity is via an ability to sustain TGF-β1 secretion or allow the expansion of TGF-β1–producing cells. Support for this possibility comes from our recent observation that TGF-β1–mediated regulatory function induced by feeding antigen is not only abrogated by anti–TGF-β1 administration, but also by anti–IL-10 administration and that in fact no TGF-β1 production occurs in such fed mice given anti–IL-10 (7
). Further work showed that the effect of anti–IL-10 does not occur during the induction of TGF-β1–producing cells (in the absence of Th1 cytokines), but rather during the expansion of these cells (in the presence of Th1 cytokines) when they might be expected to undergo antigen-induced proliferation. These findings suggest that either nascent TGF-β1–producing regulatory cells undergo a turn-off of TGF-β1 production or fail to expand in the face of Th1 cytokine production. Thus, it is reasonable to suggest that IL-10 sustains TGF-β1 secretion directly by counteracting the negative effect of Th1 cytokines on TGF-β1 production or indirectly by down-regulating Th1 cytokine secretion. Both of these possibilities would explain the observation that mice expressing large amount of IL-10 in epithelial cells as a result of an epithelial cell–specific IL-10 transgene exhibit an increase in TGF-β1 expression in the underlying LP (38
). In addition, it accords with the observation that T cell TGF-β1 secretion is not seen during the Th1 response observed when TNBS colitis is induced in SJL/J mice but is seen during the Th2 response observed in the oxazolone colitis occurring in the same strain (39
). Taken together, these considerations strongly suggest that TGF-β1 induction of IL-10 fulfills an important requirement of regulatory T cell activity.
Although TGF-β1 and IL-10 as discussed above might be interdependent during a regulatory response, it is possible that their cosecretion also occurs because these cytokines regulate different aspects of an inflammatory response and are thus synergistic in their activity. This is supported by the fact that in Th1-driven inflammation, although TGF-β1 regulates Th1 cells via its ability to down-regulate T-bet and the IL-12Rβ2 chain (4
), IL-10 regulates via down-regulation of IL-12 and TNF-α secretion (4
). Thus, the regulatory activity of TGF-β1 and IL-10 acting together is greater than either of these cytokines acting alone.
An important and interesting aspect of TGF-β1 induction of IL-10 relates to our observation that such induction affects Th1 cells rather than Th2 cells (which exhibit IL-10 secretion in the absence of TGF-β1 and which are not induced to produce additional IL-10 by the presence of TGF-β). In a previous study by Blokzijl et al. (43
) using the HT-2 cell line (a cell line that produces IL-10 but not IL-4 or IL-5), it was found that TGF-β1–induced Smad3 interacts directly with GATA-3 and the complex formed acts cooperatively to up-regulate IL-10 expression (by ~1.8-fold) in a Smad3- and GATA-3–dependent fashion. This result is somewhat at odds with our data showing that IL-10 production by freshly prepared Th2 cells is not enhanced by TGF-β1 and is best explained by differences in the cells being studied. Blokzijl et al. (43
) used an established cell line of uncertain lineage, whereas we used a freshly stimulated Th2 cell line. Finally, we observed that CD4+
T cells exposed to TGF-β1 during induction of T cell differentiation produced much lower amounts of IL-10 when stimulated under neutral priming conditions than under Th1 priming conditions (unpublished data). This indicates that activation of the IL-10 promoter is not solely dependent on TGF-β1, but also involves other unknown factors induced during the differentiation of Th1 cells.
In addition to its role as a suppressor of inflammation, TGF-β1 has a known capacity to induce collagen and fibronectin synthesis and thus to promote fibrosis. In contrast, IL-10 has been shown to inhibit collagen synthesis and prevent fibrosis (44
), not only that caused by TGF-β1, but also by TNF-α (46
) and IL-13 (14
). On this basis we explored the possibility that the fibrogenic properties of TGF-β1 can be brought under control by IL-10 cosecretion. In these studies we showed that intranasal administration of pTet-On-TGF-β1 did not induce fibrosis of the lung, the organ most exposed to TGF-β1 as a result of intranasal plasmid administration. In contrast, such administration prevented bleomycin-induced pulmonary fibrosis, a fibrosis that is at least in part mediated by endogenous TGF-β production (27
). That this seemingly paradoxical effect was due to the cosecretion of IL-10 was shown by the fact that TGF-β1 plasmid administration did not prevent bleomycin-induced fibrosis in IL-10–deficient mice (in whom IL-10 cosecretion cannot occur). These findings strongly suggest that the IL-10 antifibrotic effect is dominant over the TGF-β1 profibrotic effect, even in the face of endogenous (nonlymphoid origin) TGF-β production and that IL-10 protects the animal from TGF-β1–mediated fibrosis. By extension, they suggest that induction of TGF-β1–secreting cells by administration of a plasmid such as pTet-On-TGF-β1 that leads to superphysiologic IL-10 secretion may have an unexpected antifibrotic effect that bodes well for its use as a therapy for autoimmune diseases.