Recent insights into the role of dipeptidyl aminopeptidase IV (DPIV) and aminopeptidase N (APN) families in immune functions.

BACKGROUND: In the past, different research groups could show that treatment of immune cells with inhibitors of post-proline splitting dipeptidyl aminopeptidases leads to functional changes in the immune system consistent with immunosuppression. This is due to the inhibition of proliferation of lymphocytes and the production of inflammatory cytokines of the TH1, TH2, and TH17, cells as well as the induction of immunosuppressive cytokines, such as transforming growth factor-beta1 (TGF-beta1) and interleukin (IL)-1RA. Until recently, most of the effects of these inhibitors on immune functions were attributed to the inhibition of dipeptidyl aminopeptidase IV (DPIV/CD26). With the identification of new peptidases of the DPIV family (DASH) with the same or similar substrate specificity [fibroblast activation protein (FAP), DP8/9], the question arose whether and to what extent the inhibition of intracellularly localized enzymes, DP8 and DP9, contribute to the observed immunosuppression. In addition, members of the aminopeptidase N (APN) family are also involved in the regulation of immune functions. Hence, the concept of a combined targeting of both families of peptidases for treatment of inflammatory diseases is a promising strategy. RESULTS/CONCLUSIONS: Summarizing data obtained from the usage of different non-selective and selective inhibitors of DPIV, DP8/9, FAP, and DPII, this review provides evidence that in addition to DPIV, DP8/9 also regulate the immune response via modulation of cell cycle progression and cytokine production. The strongest and most consistent effects in vitro were, however, observed with non-selective inhibitors for the suppression of DNA synthesis and cytokine production. Similar effects were provoked by APN inhibitors, which were also found to suppress DNA synthesis and the production of inflammatory cytokines in vitro. However, different mechanisms and signaling pathways appear to mediate the cellular effects resulting from the inhibition of either APN or DPIV family members. In particular, members of the APN family uniquely influence the function of CD4+CD25+ regulatory T-cells. Consequently, the concomitant inhibition of both APN and DPIV enzyme families by means of two separate inhibitors or by binary inhibitors with specificity for both enzyme families (PETIR, peptidase targeted immunoregulation) synergistically affects immune cells on the level of cell cycle regulation, suppression of TH1, TH2, and TH17 cytokines as well as the activation of regulatory T-cells. Besides leukocytes, dermal cells as sebocytes, keratinocytes, and fibroblasts are also targeted by these inhibitors. This strongly suggests a broad potential of the multiple anti-inflammatory effects of PETIR in treatment of chronic inflammatory diseases, such as autoimmune diseases, allergies, and transplant rejections, as well as of inflammatory skin diseases, such as acne, psoriasis, rosacea or atopic dermatitis. The first active dual inhibitor, IP10.C8, has been developed by IMTM for the treatment of inflammatory skin diseases and has just entered the first phase II study.

Inhibition of alanyl-aminopeptidase on CD4+CD25+ regulatory T-cells enhances expression of FoxP3 and TGF-beta1 and ameliorates acute colitis in mice.

Inhibitors of alanyl-aminopeptidase e.g. phebestin increase the expression of transforming growth factor (TGF)-beta1 in mononuclear cells. We investigated whether phebestin also produced this effect in CD4+CD25+ T-cells and whether phebestin-treated CD4+CD25+ T-cells were capable of ameliorating acute colitis in mice. The suppressive activity of mouse CD4+CD25+ T-cells was assessed in vitro by co-culture with splenocytes. mRNA expression associated with the suppressive phenotype was determined in vitro and in vivo. The in vivo role of phebestin-exposed CD4+CD25+ T-cells was studied in sodium dextran sulfate-induced acute colitis in mice. The proliferation of activated effector T-cells or splenocytes in vitro was inversely correlated with the number of CD4+CD25+ T-cells. Phebestin pre-treatment substantially enhanced the suppressive activity of these cells and increased expression levels of TGF-beta1 and FoxP3. Furthermore, transfer of CD4+CD25+ T-cells exposed to phebestin for a short time ex vivo significantly reduced the mouse colitis disease activity index. We conclude that aminopeptidase inhibitors support the suppressive activity as well as TGF-beta1 and FoxP3 expression of natural regulatory T-cells.

TGF-beta1-mediated control of central nervous system inflammation and autoimmunity through the inhibitory receptor CD26.

The T cell marker CD26/dipeptidyl peptidase (DP) IV is associated with an effector phenotype and markedly elevated in the human CNS disorder multiple sclerosis. However, little is known about the in vivo role of CD26/DP IV in health and disease, and the underlying mechanism of its function in CNS inflammation. To directly address the role of CD26/DP IV in vivo, we examined Th1 immune responses and susceptibility to experimental autoimmune encephalomyelitis in CD26(-/-) mice. We show that gene deletion of CD26 in mice leads to deregulation of Th1 immune responses. Although production of IFN-gamma and TNF-alpha by pathogenic T cells in response to myelin Ag was enhanced in CD26(-/-) mice, production of the immunosuppressive cytokine TGF-beta1 was diminished in vivo and in vitro. In contrast to the reduction in TGF-beta1 production, responsiveness to external TGF-beta1 was normal in T cells from CD26(-/-) mice, excluding alterations in TGF-beta1 sensitivity as a mechanism causing the loss of immune regulation. Natural ligands of CD26/DP IV induced TGF-beta1 production in T cells from wild-type mice. However, natural ligands of CD26/DP IV failed to elicit TGF-beta1 production in T cells from CD26(-/-) mice. The striking functional deregulation of Th1 immunity was also seen in vivo. Thus, clinical experimental autoimmune encephalomyelitis scores were significantly increased in CD26(-/-) mice immunized with peptide from myelin oligodendrocyte glycoprotein. These results identify CD26/DP IV as a nonredundant inhibitory receptor controlling T cell activation and Th1-mediated autoimmunity, and may have important therapeutic implications for the treatment of autoimmune CNS disease.

Assigning the phenotype of a natural regulatory T-cell to the human T-cell line, KARPAS-299.

The human CD30-positive anaplastic large (T-) cell lymphoma cell line, KARPAS-299 (DSM ACC31), was established from blast cells in the peripheral blood from a case of non-Hodgkin lymphoma in 1988. We describe the mRNA and surface expression in KARPAS-299 cells of a panel of markers highly restricted to human natural regulatory T-cells and associated with their suppressive activity, including FOXP3, CD25, IL-10, TGF-beta1, CD62L, and Lag-3. Results obtained from co-culturing human peripheral blood leukocytes with KARPAS-299 cells assigned a suppressive phenotype to the latter ones. In conclusion, KARPAS-299 cells show characteristics typical of natural regulatory T-cells and, thus, represent a valuable model for studying regulatory T-cell function, which may also facilitate drug development aimed at the modulation of regulatory T-cell activity for the pharmacological therapy of, for example, autoimmune diseases.

Transcriptional regulation of cytosol and membrane alanyl-aminopeptidase in human T cell subsets.

Aminopeptidase inhibitors strongly affect the proliferation and function of immune cells in man and animals and are promising agents for the pharmacological treatment of inflammatory or autoimmune diseases. Membrane alanyl-aminopeptidase (mAAP) has been considered as the major target of these anti-inflammatory aminopeptidase inhibitors. Recent evidence also points to a role of the cytosol alanyl-aminopeptidase (cAAP) in the immune response. In this study we used quantitative RT-PCR to determine the mRNA expression of both cAAP and mAAP in resting and activated peripheral T cells and also in CD4+, CD8+, Th1, Th2 and Treg (CD4+ CD25+) subpopulations. Both mAAP and cAAP mRNAs were expressed in all cell types investigated, and in response to activation their expression appeared to be upregulated in CD8+ cells, but downregulated in Treg cells. In CD4+ cells, mAAP and cAAP mRNAs were affected in opposite ways in response to activation. The cAAP-specific inhibitor, PAQ-22, did not affect either cAAP or mAAP expression in activated CD4+ or CD8+ cells, whereas in activated Treg cells it markedly upregulated the mRNA levels of both aminopeptidases. The non-discriminatory inhibitor, phebestin, significantly increased the amount of mAAP and cAAP mRNA in CD4+ and that of cAAP in Treg cells.

Synergistic action of DPIV and APN in the regulation of T cell function.

Inhibitors of the enzymatic activity of alanyl-aminopeptidases severely affect growth and typical functions of human peripheral T cells both in vitro and in vivo. The most prominent changes observed include the activation of cellular signal transduction pathways such as MAP kinases Erk1/2 or the Wnt-pathway, a decrease of production and release of "pro-inflammatory" cytokines (IL-2, IL-12) and, most importantly, an induction of expression and release of the immunosuppressive cytokine, TGF-beta1. Similar effects on T cell proliferation and function have been observed in response to inhibition of DPIV, which is strongly suggestive of a functional synergism of APN and DPIV. In support of this hypothesis evidence is provided showing that the simultaneous application of inhibitors of DPIV and APN further enhances the anti-inflammatory and immunosuppressive effects provoked by the inhibition of APN or DPIV alone. Therefore, the simultaneous inhibition of these enzymes represents a promising strategy for the pharmacological therapy of T cell mediated diseases such as autoimmune disease, inflammation, allergy, and allograft rejection.