We have previously shown that following oral administration of myelin basic protein (MBP), regulatory T cells are generated from gut-associated lymphoid tissue and that these cells suppress experimental allergic encephalomyelitis (EAE). These regulatory T cells produce transforming growth factor-beta (TGF-beta) with various amounts of IL-4 and IL-10 and these TGF-beta-secreting T cells have been termed Th3 cells. T cells in lymphoid organs drained by mucosal sites secrete IL-4 as a primary T cell growth factor. In the present study, we examined the role of IL-4 on oral tolerance and in the generation of TGF-beta secreting cells. Treatment of (PLJ x SJL)F1 mice with intraperitoneal (i. p.) IL-4 and low-dose oral MBP (0.5 mg) given three times reduced the severity of EAE, whereas i.p. injection of IL-4 alone or oral MBP alone given in these suboptimal doses, showed no protection. Spleen cells from protected mice produced increased amounts of TGF-beta and reduced IFN-gamma upon stimulation with MBP in vitro. Mucosal MBP-specific IgA production was significantly increased in IL-4 plus MBP fed animals. Moreover, oral administration of IL-4 (1 microg per feeding) also enhanced the suppression of EAE by oral MBP and this protective effect was reversed by administration of anti-TGF-beta antibody in vivo. Reverse transcription-PCR showed enhanced suppression of IFN-gamma in Peyer's patch in animals fed MBP and IL-4 versus those fed MBP alone. We then investigated the role of IL-4 in the generation of TGF-beta-secreting cells using MBP Ac1-11 TCR transgenic animals. Cells were cultured with IL-2, IL-4, or IFN-gamma in the presence of MBP and limiting dilution analysis for cytokine-secreting cells performed. We found that IL-4, but not IL-2 or IFN-gamma, generated TGF-beta-secreting T cells from naive splenic T cells and that these cells provided help for IgA production. These findings demonstrate that IL-4 is a differentiation factor for TGF-beta-secreting Th3 cells and oral IL-4 has a synergistic effect on low-dose oral tolerance that is associated with increased TGF-beta secretion.