Dipeptidyl peptidase IV, aminopeptidase N and DPIV/APN-like proteases in cerebral ischemia.

BACKGROUND: Cerebral inflammation is a hallmark of neuronal degeneration. Dipeptidyl peptidase IV, aminopeptidase N as well as the dipeptidyl peptidases II, 8 and 9 and cytosolic alanyl-aminopeptidase are involved in the regulation of autoimmunity and inflammation. We studied the expression, localisation and activity patterns of these proteases after endothelin-induced occlusion of the middle cerebral artery in rats, a model of transient and unilateral cerebral ischemia. METHODS: Male Sprague-Dawley rats were used. RT-PCR, immunohistochemistry and protease activity assays were performed at different time points, lasting from 2 h to 7 days after cerebral ischemia. The effect of protease inhibitors on ischemia-dependent infarct volumes was quantified 7 days post middle cerebral artery occlusion. Statistical analysis was conducted using the t-test. RESULTS: Qualitative RT-PCR revealed these proteases in ipsilateral and contralateral cortices. Dipeptidyl peptidase II and aminopeptidase N were up-regulated ipsilaterally from 6 h to 7 days post ischemia, whereas dipeptidyl peptidase 9 and cytosolic alanyl-aminopeptidase were transiently down-regulated at day 3. Dipeptidyl peptidase 8 and aminopeptidase N immunoreactivities were detected in cortical neurons of the contralateral hemisphere. At the same time point, dipeptidyl peptidase IV, 8 and aminopeptidase N were identified in activated microglia and macrophages in the ipsilateral cortex. Seven days post artery occlusion, dipeptidyl peptidase IV immunoreactivity was found in the perikarya of surviving cortical neurons of the ipsilateral hemisphere, whereas their nuclei were dipeptidyl peptidase 8- and amino peptidase N-positive. At the same time point, dipeptidyl peptidase IV, 8 and aminopeptidase N were targeted in astroglial cells. Total dipeptidyl peptidase IV, 8 and 9 activities remained constant in both hemispheres until day 3 post experimental ischemia, but were increased (+165%) in the ipsilateral cortex at day 7. In parallel, aminopeptidase N and cytosolic alanyl-aminopeptidase activities remained unchanged. CONCLUSIONS: Distinct expression, localization and activity patterns of proline- and alanine-specific proteases indicate their involvement in ischemia-triggered inflammation and neurodegeneration. Consistently, IPC1755, a non-selective protease inhibitor, revealed a significant reduction of cortical lesions after transient cerebral ischemia and may suggest dipeptidyl peptidase IV, aminopeptidase N and proteases with similar substrate specificity as potentially therapy-relevant targets.

Divergent actions by inhibitors of DP IV and APN family enzymes on CD4+ Teff cell motility and functions.

Dipeptidyl peptidase IV (DP IV)/CD26 and aminopeptidase N (APN)/CD13 family enzymes control T cell functions. We have previously defined these peptidases as targets to treat autoimmune disease, but the underlying mechanism is unclear. Here, we determined the effect of enzymatic inhibitors on chemotaxis by CD4+ effector T (Teff) cells. Exposure of Teff cells to the inhibitor of DP IV activity, Lys[Z(NO2)]-pyrrolidide (LZNP) and the inhibitor of APN activity, actinonin has no effect on chemotaxis or unstimulated cell migration, even at high inhibitor concentrations. LZNP and actinonin also fail to suppress migration of unfractionated lymph node cells, excluding paracrine action through other leukocyte subsets. In contrast, inhibition of DP IV and APN activities selectively suppresses lymphocyte functions including proliferation and production of the T helper type (Th)1 cytokine IFN-γ, the Th17 cytokine IL-17, as well as TNF-α, and ameliorates autoimmunity in vivo. The present results combined with previous studies suggest that LZNP and actinonin do not prevent migration of pathogenic Teff cells into target tissues, but rather suppress disease through inhibitor induced release of TGF-ß by T cells at the site of inflammation.

Novel aspects of cellular action of dipeptidyl peptidase IV/CD26.

The cellular dipeptidyl peptidase IV (DPIV, E.C.3.4.14.5, CD26) is a type II membrane peptidase with various physio-logical functions. Our main knowledge on DPIV comes from studies of soluble DPIV which plays a role in regulation of glucose homeostasis by inactivation of the incretins glucagon-like peptide-1 and glucose-dependent insulinotropic poly-peptide. It has been reported that membrane-bound DPIV plays a crucial role in the immune system and in other tissues and cells, but the knowledge on the action of cellular DPIV and its regulation is limited. In this study, we show particularly for immune cells that DPIV and not DP8 or DP9 is the most potent member of the DPIV family in regulating cellular immune functions. Moreover, we provide evidence that soluble and cellular DPIV differ in functions and hand-ling of substrates and inhibitors owing to the different accessibility of peptide substrates to the two access paths of DPIV. The different functions are based on the favored access path of the central pore of cellular DPIV and a special central pore binding site which assists substrate access to the active site of the enzyme. The newly discovered central pore binding site mediates an autosterical regulation of cellular DPIV and is its most crucial target site to regulate cellular functions such as growth and cytokine production. Neuropeptide Y (NPY) processing by cellular DPIV was found to be inhibited by ligands which interact with the central pore binding site. This finding suggests a crucial role of the immunosuppressive cytokine NPY in the function of DPIV in growth regulation.

PETIR-001, a dual inhibitor of dipeptidyl peptidase IV (DP IV) and aminopeptidase N (APN), ameliorates experimental autoimmune encephalomyelitis in SJL/J mice.

Cellular dipeptidyl peptidase IV (DP IV, CD26) and amino-peptidase N (APN, CD13) play regulatory roles in T cell activation and represent potential targets for treatment of inflammatory disorders. We have developed a novel therapeutic strategy, 'peptidase-targeted Immunoregulation' (PETIR™), which simultaneously targets both cellular DP IV and APN via selective binding sites different from the active sites with a single inhibitor. To prove the therapeutic concept of PETIR™ in autoimmunity of the central nervous system (CNS), we evaluated the effect of a single substance, PETIR-001, in an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) in SJL/J mice. Administration of PETIR-001 significantly delayed and decreased clinical signs of active EAE, when given in a therapeutic manner intraperitoneally from day 15 to day 24 after induction of EAE. Both the acute phase and the first relapse of EAE were markedly inhibited. Importantly, a similar therapeutic benefit was obtained after oral administration of PETIR-001 from day 12 to day 21 after disease induction. Our results demonstrate that PETIR-001 exhibits a therapeutic effect on EAE in SJL/J mice. Thus, PETIR™ represents a novel and efficient therapeutic approach for immunotherapy of CNS inflammation.

Outside or inside: role of the subcellular localization of DP4-like enzymes for substrate conversion and inhibitor effects.

The discovery of the DP4-related enzymes DP8 and DP9 raised controversial discussion regarding the physiological and pathophysiological function of distinct members of the DP4 family. Particularly with regard to their potential relevance in regulating immune functions, it is of interest to know which role the subcellular distribution of the enzymes play. Synthetic substrates as well as low molecular weight inhibitors are widely used as tools, but little is yet known regarding their features in cell experiments, such as their plasma membrane penetration capacity. The fluorogenic substrates Gly-Pro-AMC or (Ala-Pro)2-R110 predominantly detect plasma membrane-bound activities of viable cells (less than 0.1% of fluorochromes R110 or AMC inside viable cells after 1 h incubation). Additionally, the selective and non-selective DP8/9 inhibitors allo-Ile-isoindoline and Lys[Z(NO2)]-pyrrolidide were found to be incapable of passing the plasma membrane easily. This suggests that previously reported cellular effects are not due to inhibition of the cytosolic enzymes DP8 or DP9. Moreover, our enzymatic studies with viable cells provided evidence that DP8 and/or DP9 are also present on the surface of immune cells under certain circumstances and could gain relevance particularly in the absence of DP4 expression. In summary, in cells which do express DP4 on the surface, this archetypical member of the DP4 family is the most relevant peptidase in the regulation of cellular functions.

Role of dipeptidyl peptidase IV (DP IV)-like enzymes in T lymphocyte activation: investigations in DP IV/CD26-knockout mice.

BACKGROUND: Dipeptidyl peptidase IV (DP IV, CD26) and DP IV-like enzymes, such as dipeptidyl peptidase II (DP II), dipeptidyl peptidase 8 (DP8), and dipeptidyl peptidase 9 (DP9), have been recognized to regulate T lymphocyte activation. Lys[Z(NO2)]-thiazolidide (LZNT) and Lys[Z(NO2)]-pyrrolidide (LZNP), non-selective inhibitors of DP IV-like activity known to target DP IV as well as DP II, DP8, and DP9, suppress T lymphocyte proliferation in vitro. Moreover, these inhibitors are capable of attenuating the severity of autoimmune diseases, such as experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis, and experimental arthritis, a model of human rheumatoid arthritis, in vivo, particularly in combination with inhibitors of aminopeptidase N (APN, CD13) enzymatic activity. METHODS: Here, we studied the influence of non-selective and selective inhibitors of DP IV-like enzymes on DNA synthesis in mitogen-stimulated splenocytes from wild-type C57BL/6 mice and DP IV/CD26-knockout (DP IV/CD26-KO) mice. RESULTS: LZNT and LZNP, the non-selective inhibitors of DP IV-like activity, suppressed the DNA synthesis in stimulated splenocytes from wild-type and DP IV/ CD26-KO mice to a comparable extent. Further, a selective inhibitor of DP8/DP9 activity was capable of suppressing DNA synthesis in mitogen-stimulated splenocytes of both wild-type and knockout mice to the same extent. In contrast, selective inhibitors of DP IV and DP II lacked this suppressive activity. CONCLUSIONS: Our data support the hypothesis that DP8 and/or DP9 represent additional pharmacological targets for the suppression of T cell proliferation and for anti-inflammatory therapy.

Reduced neuronal expression of insulin-degrading enzyme in the dorsolateral prefrontal cortex of patients with haloperidol-treated, chronic schizophrenia.

Insulin-degrading enzyme (IDE) is a neutral thiol metalloprotease, which cleaves insulin with high specificity. Additionally, IDE hydrolyzes Abeta, glucagon, IGF I and II, and beta-endorphin. We studied the expression of IDE protein in postmortem brains of patients with schizophrenia and controls because: (1) the gene encoding IDE is located on chromosome 10q23-q25, a gene locus linked to schizophrenia; (2) insulin resistance with brain insulin receptor deficits/receptor dysfunction was reported in schizophrenia; (3) the enzyme cleaves IGF-I and IGF-II which are implicated in the pathophysiology of the disease; and (4) brain gamma-endorphin levels, liberated from beta-endorphin exclusively by IDE, have been reported to be altered in schizophrenia. We counted the number of IDE immunoreactive neurons in the dorsolateral prefrontal cortex, the hypothalamic paraventricular and supraoptic nuclei, and the basal nucleus of Meynert of 14 patients with schizophrenia and 14 matched control cases. Patients had long-term haloperidol treatment. In addition, relative concentrations of IDE protein in the dorsolateral prefrontal cortex were estimated by Western blot analysis. There was a significantly reduced number of IDE expressing neurons and IDE protein content in the left and right dorsolateral prefrontal cortex in schizophrenia compared with controls, but not in other brain areas investigated. Results of our studies on the influence of haloperidol on IDE mRNA expression in SHSY5Y neuroblastoma cells, as well as the effect of long-term treatment with haloperidol on the number of IDE immunoreactive neurons in rat brain, indicate that haloperidol per se, is not responsible for the decreased neuronal expression of the enzyme in schizophrenics. Haloperidol however, might exert some effect on IDE, through changes of the expression levels of its substrates IGF-I and II, insulin and beta-endorphin. Reduced cortical IDE expression might be part of the disturbed insulin signaling cascades found in schizophrenia. Furthermore, it might contribute to the altered metabolism of certain neuropeptides (IGF-I and IGF-II, beta-endorphin), in schizophrenia.

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.

Inflammatory bowel diseases: multiple benefits from therapy with dipeptidyl- and alanyl-aminopeptidase inhibitors.

Inflammatory bowel diseases (IBD) are driven by imbalances in innate and acquired immune response. In IBD two dysregulated T cell subsets are in the focus of interest: activated effector T cells and regulatory T cells. These T cell subsets are characterized by a strong expression of the ectopeptidases dipeptidyl peptidase IV (DPIV /CD26) and aminopeptidase N (APN/CD13), which are thought to a role in the control of immune activation and in regulating cellular communication by hydrolyzing bioactive polypeptides. Since inhibitors of both enzymes were shown to be effective in limiting immune activation processes in vitro as well as in vivo, they emerged as new drug candidates for the treatment of diseases associated with an imbalanced T cell response, such as IBD. In this review we intent to throw light on the putative role of DPIV, APN and related enzymes in the regulation of immune and non-immune processes in inflammatory bowel diseases, on possible benefits from peptidase inhibitor therapy in these diseases as well on the gaps of knowledge in this field.

Regional and cellular distribution patterns of insulin-degrading enzyme in the adult human brain and pituitary.

The regional distribution and cellular localization of insulin-degrading enzyme (IDE) was studied in adult human brain and pituitary by means of immunhistochemistry. We show that the enzyme is widely but unevenly distributed in human brain, with hypothalamic neurons showing the strongest immunoreaction. Strong to moderate immunostaining for the enzyme was observed in multiple cortical areas, hippocampus, cerebellum, and brain stem. Cellularly, IDE was mainly confined to neurons, but it was also present in oligodendrocytes, choroid plexus, and some blood vessel endothelial cells. A strong immunoreaction was seen in a subset of adenohypophysial cells. Some immunolabeling was also present in the neurohypophysis. The putative importance of the distribution of the enzyme in brain and pituitary is discussed in relation to its main known substrates, insulin, Abeta, and beta-endorphin.

Inhibitors of dipeptidyl peptidase IV-like activity mediate antifibrotic effects in normal and keloid-derived skin fibroblasts.

Suppression of collagen and matrix synthesis and inhibition of the fibrogenic cytokine transforming growth factor-beta(1) (TGF-beta(1)) is a major therapeutic goal in the treatment of fibrosis and keloids. Inhibitors of dipeptidyl peptidase IV (DP IV)-like activity affect cell growth and cytokine production and are currently under investigation for the treatment of metabolic, autoimmune and inflammatory diseases. We show here that the inhibitors of DP IV-like activity, Lys[Z(NO(2))]-thiazolidide and Lys[Z(NO(2))]-pyrrolidide, suppress proliferation in human skin fibroblasts and keloid-derived skin fibroblasts in vitro. They significantly decrease TGF-beta(1) expression and secretion of procollagen type I C-terminal peptide in supernatants of both cell types. Furthermore, they abrogate the TGF-beta(1)-induced stimulation of collagen synthesis, matrix deposition, and TGF-beta(1) and fibronectin expression. Both inhibitors lead to dephosphorylation of mitogen-activated protein kinases pp38 and pERK1/2, which are activated upon TGF-beta1 stimulation and have been implicated in fibrogenesis. In a mouse model of dermal fibrosis, induced by repetitive intracutaneous injections of TGF-beta(1), the profibrotic effect of TGF-beta(1) detected by dermal thickening, collagen I, and alpha-smooth muscle actin expression, is significantly suppressed in the presence of inhibitors. Inhibition of DP IV-like enzymatic activity may therefore represent a promising therapeutic approach for the treatment of fibrotic skin disorders and keloids.

DP IV/CD26, APN/CD13 and related enzymes as regulators of T cell immunity: implications for experimental encephalomyelitis and multiple sclerosis.

Multiple sclerosis (MS) is the most frequent demyelinating disease of the central nervous system. Peptidases like dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) play a regulatory role in T cell activation and represent potential targets for the treatment of inflammatory disorders. Synthetic inhibitors of DP IV and/or APN enzymatic activity induce production of the immunosuppressive cytokine TGF-beta1 and subsequently suppress DNA synthesis and Th1 cytokine production of activated human T cells. Compelling evidence has demonstrated that IL-17-producing CD4 cells (Th17) are a major contributor to the pathogenesis of autoimmune inflammation. Here, we report that inhibitors of DP IV-like activity as well as of APN activity inhibit IL-17 production in activated human and mouse T cells. Combining inhibitors of DP IV and APN increases the suppressive effect on T cell specific IL-17 production in vitro compared to a single peptidase inhibitor. In the following, we summarize the evidence for the role of both ectoenzymes in T cell activation in vitro and in vivo and provide a rationale for the use of combined or dual ectopeptidase inhibitors to treat autoimmune diseases like MS.

The ectopeptidases dipeptidyl peptidase IV (DP IV) and aminopeptidase N (APN) and their related enzymes as possible targets in the treatment of skin diseases.

Skin cells express dipeptidyl peptidase IV (DP IV) and aminopeptidase N (APN) and their related molecules of the DP IV-like family DP2, DP6, DP8, DP9 and fibroblast activation protein (FAP), as well as the cytoplasmic alanyl aminopeptidase (cAAP). The inhibitors of DP IV-like activity, Lys(Z(NO2))-thiazolidide (LZNT) and Lys(Z(NO2))-pyrrolidide (LZNP), and the APN inhibitors actinonin and bestatin affect proliferation, differentiation and cytokine production in sebocytes and keratinocytes, which are involved in the initiation of acne. Furthermore, they suppress proliferation of Propionibacterium acnes-stimulated T cells ex vivo and induce an anti-inflammatory cytokine profile. In the mouse tail model of psoriasis they have a pro-differentiative effect. In addition, these inhibitors suppress skin fibroblast proliferation, whereas only inhibition of DP IV-like activity decreases TGF-beta1 expression and abrogates the TGF-beta1 mediated stimulatory effects on TGF-beta1 and fibronectin production, collagen synthesis and matrix deposition in these cells. Targeting enzyme activity of DP IV and APN and their related molecules might be a novel approach for the treatment of acne, psoriasis or keloids.

Dual inhibition of dipeptidyl peptidase IV and aminopeptidase N suppresses inflammatory immune responses.

The ectopeptidases dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) are known to regulate T cell activation. Since selective inhibitors of DP IV and APN suppress DNA synthesis and cytokine production of stimulated T cells in a TGF-beta1-dependent manner, we tested whether combined application of DP IV and APN inhibitors enhances this immunomodulatory effect. The results show that simultaneous application of DP IV and APN inhibitors significantly suppressed DNA synthesis in mitogen- or anti-CD3-stimulated human T cells in vitro when compared to the use of a single DP IV or APN inhibitor. Moreover, the combined action of DP IV and APN inhibitors markedly increased TGF-beta1 production associated with the observed immunosuppressive effects. In vivo, targeting both DP IV and APN led to a potent treatment of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (MS). This review summarizes the evidence for the role of both enzymes in T cell activation in vitro and in vivo and provides a rationale for using combined and dual peptidase inhibitors to treat autoimmune diseases like MS.

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.

Inhibitors of dipeptidyl peptidase IV and aminopeptidase N target major pathogenetic steps in acne initiation.

Acne is a chronic disease hallmarked by sebaceous hyperplasia, follicular hyperkeratosis, and inflammation. Parallel targeting of these factors is required to treat acne effectively. Inhibitors of dipeptidyl peptidase IV (DP IV) and aminopeptidase N (APN) show strong anti-inflammatory effects on immune cells and therapeutic efficacy in autoimmune disorders. Our investigation focused on the expression and functional relevance of these ectopeptidases in three cell types which exhibit an altered phenotype in early acne lesions. We showed for the first time expression of DP IV and APN on human sebocytes. In the SZ95 sebocyte cell line, the DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrrolidide and the APN inhibitors actinonin and bestatin suppressed proliferation, enhanced terminal differentiation, and slightly decreased total neutral lipid production. The anti-inflammatory and differentiation-restoring cytokine IL-1 receptor antagonist was significantly upregulated in SZ95 sebocytes and the HaCaT keratinocyte cell line in the presence of inhibitors. Furthermore, the inhibitors suppressed proliferation and IL-2 production of Propionibacterium acnes-stimulated T cells ex vivo and enhanced the expression of the immunosuppressive cytokine transforming growth factor-beta1. Our data provide first evidence for a functional role of DP IV and APN in the sebaceous gland apparatus and for their inhibitors, used alone or in combination, as completely new substances possibly affecting acne pathogenesis in a therapeutic manner.

Triggering endogenous immunosuppressive mechanisms by combined targeting of Dipeptidyl peptidase IV (DPIV/CD26) and Aminopeptidase N (APN/ CD13)--a novel approach for the treatment of inflammatory bowel disease.

The ectopeptidases Dipeptidylpeptidase IV and Alanyl-Aminopeptidase N, strongly expressed by both, activated and regulatory T cells were shown to co-operate in T cell regulation. Based on the findings that DPIV and APN inhibitors induce the TGF-beta1 and IL-10 production and a suppression of T helper cell proliferation in parallel, and that particularly APN inhibitors amplify the suppressing activity of regulatory T cells, both peptidases represent a promising target complex for treatment of diseases associated with an imbalanced T cell response, such as inflammatory bowel diseases (IBD). The aim of the present study was to analyze the therapeutic potential of DPIV and APN inhibitors in vivo in a mouse model of colitis. Balb/c mice received 3% (w/v) dextran sulphate sodium with the drinking water for 7 days. After onset of colitis symptoms, inhibitor treatment started at day 3. Disease activity index (DAI) was assessed daily, supplemented by histological and immunological analysis. While the DPIV inhibitor Lys-[Z(NO])(2)]-pyrrolidide or the APN-inhibitor Actinonin alone had marked but no significant therapeutic effects, the simultaneous administration of both inhibitors reduced colitis activity in comparison to placebo treated mice, significantly (DAI 4.8 vs. 7.7, p<0.005). A newly developed compound IP12.C6 with inhibitory capacity toward both enzymes significantly attenuated the clinical manifestation of colitis (DAI 3.2 vs. 7.6, p<0.0001). TGF-beta mRNA was found to be up-regulated in colon tissue of inhibitor-treated animals. In summary our results strongly suggest that combined DPIV and APN inhibition by synthetic inhibitors represents a novel and efficient approach for the pharmacological therapy of IBD by triggering endogenous immunosuppressive mechanisms.

Dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) as regulators of T cell function and targets of immunotherapy in CNS inflammation.

The ectoenzymes dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) have been implicated in the regulation of T cell activation and function. Both DP IV and APN serve as targets of efficient enzymatic inhibitors which induce autocrine production of TGF-beta1 and subsequent suppression of T cell proliferation and cytokine release. Here, we tested the hypothesis that the simultaneous inhibition of DP IV and APN enzymatic activity on leukocytes potentiates the anti-inflammatory effect of single DP IV or APN inhibitors. Our data show that the combined application of DP IV and APN inhibitors increased suppression of DNA synthesis in human peripheral blood mononuclear cells and isolated T cells in vitro when compared to the use of a single ectopeptidase inhibitor. Moreover, the combined action of DP IV and APN inhibitors markedly increased TGF-beta1 production associated with the observed immunosuppressive effects. In vivo, targeting DP IV and APN provided a potent therapeutic approach for the treatment of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Taken together, our study suggests that combined DP IV and APN inhibition on pathogenic T cells represents a novel and efficient therapy for autoimmune disease of the central nervous system by a mechanism that involves an active TGF-beta1-mediated anti-inflammatory effect at the site of pathology.