Allergy involves eosinophilia and Th2 polarization. Indoleamine 2,3-dioxygenase (IDO)-catalyzed conversion of tryptophan to kynurenines (KYN) regulates T cell function. We show that human eosinophils constitutively express IDO. Eosinophils treated with IFN-γ showed an 8-fold increase in IDO mRNA within 4 h; IL-3, IL-5, and GM-CSF had no effect on baseline IDO expression. IL-3 pretreatment of eosinophils reduced IFN-γ-induced IDO mRNA expression below baseline. Conversely, GM-CSF, but not IL-5, resulted in a 2-fold increase in IFN-γ-induced IDO. Treatment with IL-3, IL-5, GM-CSF, or IFN-γ alone expressed IDO enzymatic activity (the presence of KYN in supernatants 48 h postculture). CD28 cross-linking resulted in measurable KYN in culture supernatants, inhibitable by a neutralizing anti-IFN-γ. Coculture of eosinophils with an IFN-γ-producing T cell line, but not IL-4-producing T cell clone, led to apoptosis and inhibition of CD3 or CD3/CD28-induced proliferation. Eosinophils infiltrating asthmatic lung and associated lymphoid tissue exhibited intracellular IDO immunoreactivity. Eosinophils may, therefore, maintain Th2 bias through IDO.

Retinoic acid (RA) exerts diverse biological effects in the control of cell growth in embryogenesis and oncogenesis. These effects of RA are thought to be mediated by the nuclear retinoid receptors. Mannose-6-phosphate (M6P)/insulin-like growth factor-II (IGF-II) receptor is a multifunctional membrane glycoprotein that is known to bind both M6P and IGF-II and function primarily in the binding and trafficking of lysosomal enzymes, the activation of transforming growth factor-β, and the degradation of IGF-II. M6P/IGF-II receptor has recently been implicated in fetal development and carcinogenesis. Despite the functional similarities between RA and the M6P/IGF-II receptor, no direct biochemical link has been established. Here, we show that the M6P/IGF-II receptor also binds RA with high affinity at a site that is distinct from those for M6P and IGF-II, as identified by a photoaffinity labeling technique. We also show that the binding of RA to the M6P/IGF-II receptor enhances the primary functions of this receptor. The biological consequence of the interaction appears to be the suppression of cell proliferation and/or induction of apoptosis. These findings suggest that the M6P/IGF-II receptor mediates a RA response pathway that is important in cell growth regulation. This discovery of the interaction of RA with the M6P/IGF-II receptor may have important implications for our understanding of the roles of RA and the M6P/IGF-II receptor in development, carcinogenesis, and lysosomal enzyme-related diseases.

The expression of indoleamine 2,3-dioxygenase (IDO), which metabolizes tryptophan, an essential amino acid, into kynurenine, has been identified as having a key role in the prevention of the immune rejection of the semi-allogeneic fetus during pregnancy. We have previously demonstrated that IDO expressed in fibroblasts causes bystander CD4(+) T cell damage as well as THP-1 cell damage by apoptosis. As T cells are primarily responsible for graft rejection, here, we asked the question of whether engraftment of IDO-expressing xenogeneic fibroblasts populated in a collagen matrix can be immuno-protected in an animal model. The results show a significant reduction in the number of infiltrated CD3(+) T lymphocytes on days 14 and 28 post-transplantation in the wounds receiving IDO-expressing fibroblasts relative to controls. IDO-expressing human fibroblasts embedded in bovine collagen on wounds in a rat model accelerates wound healing by promoting neovascularization during the early stages and providing protection of the xenograft fibroblasts. Using a co-culture system, we further confirm that IDO can induce angiogenesis through the depletion of tryptophan. These findings suggest that IDO may have an application in promoting the engraftment of skin substitutes and other transplanted organs.

We previously demonstrated that the formation of hypertrophic scarring on the wounds of a rabbit ear fibrotic model was significantly reduced by grafting a bilayer skin substitute expressing indoleamine 2,3-dioxygenase (IDO). Here, we hypothesize that the improved healing quality is due to extracellular matrix modulatory effect of IDO-mediated tryptophan metabolites. To test this hypothesis, a series of in vitro and in vivo experiments were conducted and the findings revealed a significant increase in the expression of matrix metalloproteinase 1 (MMP-1) in fibroblasts either transduced with human IDO gene or cultured with conditioned media obtained from IDO-expressing cells. Consistent with this finding, kynurenine (Kyn) treatment markedly increased the levels of MMP-1 and MMP-3 expression through activation of the MEK (mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase)-ERK1/2 MAPK signaling pathway. On the other hand, Kyn significantly suppressed the expression of type I collagen in fibroblasts as compared with that of control. To test the anti-fibrogenic effect of Kyn in an in vivo model, rabbit ear fibrotic wounds were topically treated with cream containing 50 μg Kyn per l00 μl of cream per wound. The result showed a marked improvement in scar formation relative to the controls. These findings collectively suggest that Kyn can potentially be used as an anti-fibrogenic agent for treating hypertrophic scarring.

Previously, we have demonstrated that keratinocyte releasable stratifin, also known as 14-3-3 sigma protein, stimulates matrix metalloproteinase (MMP)-1 expression in dermal fibroblasts. In this study, we showed that stratifin induced fibroblast MMP-1 messenger ribonucleic acid (mRNA) and protein levels through p38 mitogen-activated protein kinase (MAPK). Our data indicated that treatment of dermal fibroblasts with stratifin resulted in rapid and transient upregulation of c-jun and c-fos mRNA levels. We also demonstrated that SB203580 (SB), a specific inhibitor of p38 MAPK activity, inhibited the activation of fibroblast MMP-1 mRNA expression by stratifin. Subsequently, western blot analysis revealed phosphorylation of p38 at 90 min after stratifin stimulation and this was decreased to approximately 50% of the maximum value by 120 min. Stratifin was demonstrated to increase MMP-1 protein levels starting at 4 h and reaching its peak at 12-24 h. Furthermore, SB significantly blocked the stratifin induction of MMP-1 protein levels (***p<0.005, n=3). Microarray analysis of stratifin-treated fibroblasts shows an increase in Elk4/Sap1 mRNA expression and this finding was confirmed by northern blot analysis. Our results indicate that stratifin markedly increase Elk4/Sap1 mRNA expression in a time-dependent fashion. In conclusion, stratifin stimulates fibroblast MMP-1 levels through the activation of c-fos and MAPK pathway.