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Cerebral infections are restrained by a complex interplay of tissue-resident and recruited peripheral immune cells. Whether innate lymphoid cells (ILCs) are involved in the initiation of an orchestrated neuroinflammatory response is not fully understood. Here we demonstrate that ILCs accumulate in the cerebral parenchyma, the choroid plexus, and the meninges in the early stage of cerebralToxoplasma gondii infection. Longitudinal analyses revealed that NKp46+NK1.1+Eomes-CD49a+ ILC1-like cells surpass NKp46+NK1.1+Eomes+CD49a- conventional natural killer (cNK) cells in the course of infection. Antibody-mediated depletion of cNK and ILC1s cells resulted in diminished cytokine expression and increased cerebral parasite burden. Using cNK- and ILC1-deficient mouse models, we demonstrate that only the lack of ILC1s affected cerebral immune responses. In summary, our results provide evidence that ILC1-like cells are an early source of IFN‑γ and TNF in response to cerebral T. gondii infection, thereby inducing host defence factors as well as the recruitment of protective Ly6Chi inflammatory monocytes.

The survival of peripheral T cells is dependent on their access to peripheral lymph nodes (pLNs) and stimulation by Interleukin-7 (IL-7). In pLNs fibroblastic reticular cells (FRCs) and lymphatic endothelial cells (LECs) produce IL-7 suggesting their contribution to the IL-7-dependent survival of T cells. However, IL-7 production is detectable in multiple organs and is not restricted to pLNs. This raises the question whether pLN-derived IL-7 is required for the maintenance of peripheral T cell homeostasis. Here, we show that numbers of naive T cells (TN ) remain unaffected in pLNs and spleen of mice lacking Il7 gene activity in pLN FRCs, LECs or both. In contrast, frequencies of central memory T cells (TCM ) are reduced in FRC-specific IL-7 knockout mice. Thus, steady state IL-7 production by pLN FRCs is critical for the maintenance of TCM , but not TN , indicating that both T cell subsets colonize different ecological niches in vivo. This article is protected by copyright. All rights reserved.

Cerebral toxoplasmosis is characterized by activation of brain resident cells and recruitment of specific immune cell subsets from the periphery to the central nervous system (CNS). Our studies revealed that the rapidly invaded Ly6G+ neutrophil granulocytes are an early non-lymphoid source of interferon-gamma (IFN-γ), the cytokine known to be the major mediator of host resistance to Toxoplasma gondii (T. gondii). Upon selective depletion of Ly6G+ neutrophils, we detected reduced IFN-γ production and increased parasite burden in the CNS. Ablation of Ly6G+ cells resulted in diminished recruitment of Ly6Chi monocytes into the CNS, indicating a pronounced interplay. Additionally, we identified infiltrated Ly6G+ neutrophils to be a heterogeneous population. The Ly6G+CD62-LhiCXCR4+ subset released cathelicidin-related antimicrobial peptide (CRAMP), which can promote monocyte dynamics. On the other hand, the Ly6G+CD62-LloCXCR4+ subset produced IFN-γ to establish early inflammatory response. Collectively, our findings revealed that the recruited Ly6G+CXCR4+ neutrophil granulocytes display a heterogeneity in the CNS with a repertoire of effector functions crucial in parasite control and immune regulation upon experimental cerebral toxoplasmosis.

The adoptive transfer of antigen-specific CD8+ T cells is a promising approach for the treatment of chronic viral and malignant diseases. In order to improve adoptive T cell therapy (ATT) of cancer, recent strategies aim at the antibody-based blockade of immunosuppressive signaling pathways in CD8+ T cells. Alternatively, adjuvant effects of immunostimulatory cytokines might be exploited to improve therapeutic CD8+ T cell responses. For example, Interleukin-7 (IL-7) is a potent growth, activation and survival factor for CD8+ T cells that can be used to improve virus- and tumor-specific CD8+ T cell responses. Although direct IL-7 effects on CD8+ T cells were studied extensively in numerous models, the contribution of IL-7 receptor-competent (IL-7R+) host cells remained unclear. In the current study we provide evidence that CD8+ T cell-mediated tumor rejection in response to recombinant IL-7 (rIL-7) therapy is strictly dependent on IL-7R+ host cells. On the contrary, CD8+ T cell expansion is independent of host IL-7R expression. If, however, rIL-7 therapy and peptide vaccination are combined, host IL-7R signaling is crucial for CD8+ T cell expansion. Unexpectedly, maximum CD8+ T cell expansion relies mainly on IL-7R signaling in non-hematopoietic host cells, similar to the massive accumulation of dendritic cells and granulocytes. In summary, we provide evidence that IL-7R+ host cells are major targets of rIL-7 that modulate therapeutic CD8+ T cell responses and the outcome of rIL-7-assisted ATT. This knowledge may have important implications for the design and optimization of clinical ATT protocols.

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.

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.

In addition to the mechanisms inducing the expression and secretion of cytokines under distinct pathophysiological conditions, the fate of cytokines after secretion at sites of inflammation is a field of growing interest. Proteolysis has been suggested to be a fundamental mechanism of regulating the activities of various components of the cytokine network. Evidence grows that besides highly specific cytokine converting proteases such as interleukin-1β-converting enzyme or tumor necrosis factor-converting enzyme, neutrophil-derived serine proteases are intimately involved in the modulation of the activities of cytokines and their receptors. Particularly at sites of inflammation, high amounts of the active serine proteases elastase, cathepsin G, and proteinase 3 are released from infiltrating polymorphonuclear cells in close temporal correlation to elevated levels of inflammatory cytokines, strongly indicating that these proteases are involved in the control of cytokine bioactivity and availability.

Various studies have shown that the ectoenzyme dipeptidyl peptidase IV (DP IV, CD26), expressed on T, NK and B cells in the human immune system, is involved in the regulation of DNA synthesis and cytokine production. The DP IV/CD26 was found also on mouse splenocytes and thymocytes. Here, we show that the specific DP IV inhibitors Lys[Z(NO2)]-thiazolidide, Lys[Z(NO2)]-pyrrolidide inhibit DNA synthesis as well as production of IL-2, IL-6 and IL-10 of PHA-stimulated mouse splenocytes and Con A-stimulated mouse thymocytes. Most importantly, these inhibitors induce a three to fourfold increased secretion of latent transforming growth factor beta 1 (TGF-beta 1) by mitogen-stimulated mouse immune cells, as measured with a specific TGF-beta 1 enzyme-linked immunosorbent assay (ELISA). These data demonstrate that CD26 plays a role also in regulation of DNA synthesis and cytokine production by murine immune cells, that the enzymatic activity is required for mediating these effects, and that TGF-beta 1 might have key functions in these processes.

The human immunodeficiency virus 1 (HIV-1) Tat protein is known to be capable of suppressing antigen- and CD3-induced activation of human T cells. Previously, it was shown that Tat can bind to the dipeptidyl peptidase IV (DP IV, CD26) and inhibit the degradation of the chromogenic substrate Gly-Pro-p-nitroanilide. Using the method of free zone capillary electrophoresis, here we have shown that the DP IV-catalyzed hydrolysis of the NH2-X-Pro-containing cytokine peptides IL-2(1-12), IL-1 beta(1-6), and IL-6(1-12) was also significantly inhibited by the Tat protein. Moreover, HIV-1 Tat at a concentration of 10 micrograms/ml was found to have a strong suppressive effect on DNA synthesis and IL-1 beta production, but stimulates secretion of IL-1 receptor antagonist (IL-1RA) and TNF-alpha of CD26-expressing U937-H cells. It did not impair neither DNA synthesis nor cytokine production of low CD26-expressing U937-L cells. Similar results have been found with synthetic DP IV/CD26 inhibitors (Immunobiol., 1994, vol. 192, pp. 121-136). These data strongly suggest that Tat protein is a potent "natural" inhibitor of DP IV/CD26, and they support the hypothesis that DPIV plays a role in Tat's immunosuppressive activity.

The multifunctional cytokine transforming growth factor-beta 1 (TGF-beta 1) is known to inhibit the proliferation of lymphocytes. However, the role of TGF-beta 1 in the production and secretion of various interleukins is not yet clear. In this study we have analysed in parallel the effects of TGF-beta 1 on both DNA synthesis and production of the cytokines IL-1, IL-2, IL-6, and TNF-alpha by pokeweed mitogen-stimulated peripheral blood mononuclear cells. With this stimulation system we show that TGF-beta 1 at a concentration of 15 ng/ml significantly suppresses IL-2 and IL-6 production. The release of IL-1 and TNF-alpha, however, was not influenced under these conditions. Under similar conditions DNA synthesis of PWM-stimulated PBMC was found to be inhibited by 50 +/- 10%. Using flow cytometric methods we could demonstrate that TGF-beta 1 arrested the cells in the G0/G1 phase of the cell cycle. Taken together, these results suggest that TGF-beta 1 may suppress immune responses by inhibiting the endogenous production of IL-2 and IL-6.

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.

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.

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-β by T cells at the site of inflammation.

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 TH 1 , TH 2 , and TH 17 cells as well as the induction of immunosuppressive cytokines, such as transforming growth factor-β1 (TGF-β1) 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 TH 1 , TH 2 , and TH 17 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.

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(NO 2 )]-thiazolidide (LZNT) and Lys[Z(NO 2 )]-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-knock-out (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.

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.

Inhibitors of alanyl-aminopeptidase e.g. phebestin increase the expression of transforming growth factor (TGF)-Bl 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-s1 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-s1 and FoxP3 expression of natural regulatory T-cells.

In conclusion, these findings from in vitro and in vivo studies demonstrate that DP IV activity associated with CD26 plays an important role in the activation of autoreactive T cells. Moreover, inhibition of both DP IV/CD26 and APN/CD13 activity in vitro and in vivo provides a new approach to modulate T cell functions and tissue-specific autoimmunity in the CNS. These results may have important implications for the treatment of human diseases with a putative autoimmune pathogenesis. At present, major research efforts are directed at the investigation of DP IV/CD26 and APN/CD13 as potentially powerful and safe pharmacological targets. Our preliminary data raise the possibility that simultaneous inhibition of DP IV/CD26 and APN/CD13 may be advantageous over targeting a single ectopeptidase, and support the development of inhibitors with dual specificities for both ectopeptidases.

In contrast to the excessively elevated immunochemically detectable concentrations of interleukin-6 (IL-6) in inflammatory exudates, the IL-6 bioactivities are significantly reduced, suggesting an inactivation of IL-6 at sites of inflammation. Since high amounts of proteases are released by invading neutrophils (PMN) in close temporal correlation to elevated IL-6 concentrations at site of inflammation, this study focused on effects of the PMN-derived proteases elastase (NE), proteinase 3 (PR 3) and cathepsin G (Cat G) on the bioactivity and molecular integrity of IL-6. Here, we demonstrate that these enzymes play a crucial role in the initiation of the degradation and subsequent inactivation of IL-6 at sites of inflammation. Soluble IL-6 receptor subunits elicit a protective effect against the IL-6 inactivation by Cat G, only. Possible consequences of the proteolytical IL-6 inactivation for local inflammatory processes will be discussed.

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.

With this review we wanted to present three aspects of proteinases in diseases, namely (i) the newly energing involvement of the ubiquitin mediated proteolysis system in a growing number of clinical manifestations; (ii) the importance of the fine tuning of the delicate balance of PMN proteases and their inhibitors in inflammatory diseases and (iii) the very special effects exerted by lysosomal cysteine proteinases on tumour metastasis and progression. In all three topics during the last few years have been published surprising new results leading to better understanding of the normal and pathologic functions influenced by proteolytic enzymes. It is easy to forecast the continuation of this process, and this makes one curious what might be the next steps of discovery in this field.

Human polymorphonuclear neutrophils (PMN) are regulated by soluble factors such as chemotactic peptides and cytokines. PMN themselves are capable of synthesizing and releasing various cytokines, thereby taking part in the afferent or inductive limb of the immune response. We report for the first time that TGFbeta2 can be synthesized and released by PMN obtained from peripheral blood. Reverse transcriptase polymerase chain reaction (RT-PCR) showed that preparations of PMN express mRNA for transforming growth factor (TGF)beta1 and TGFbeta2 but not for TGFbeta3. Only translation of TGFbeta2 into protein was observed applying different assays. Interestingly, TGFbeta2 in supernatants from stimulated PMN could be detected in the bioactive form.

Various studies have shown that the membrane ectoenzyme dipeptidyl peptidase IV (DP IV; CD26), expressed on T, natural killer (NK) and B cells in the immune system, is involved in the regulation of DNA synthesis and cytokine production. We show that the specific DP IV inhibitors Lys[Z(NO2)]-thiazolidide, Lys[Z(NO2)]-piperidide, and Lys[Z(NO2)]-pyrrolidide inhibit DNA synthesis as well as production of interleukin-2 (IL-2), IL-10, IL-12, and interferon-gamma (IFN-gamma) of pokeweed mitogen (PWM)-stimulated purified T cells. Most importantly, these inhibitors induce a three- to fourfold increased secretion of latent transforming growth factor-beta 1 (TGF-beta 1) by PWM-stimulated peripheral blood mononuclear cells (PBMC) and T cells, as measured with a specific TGF-beta 1 enzyme-linked immunosorbent assay and in the Mv1Lu bioassay. As we could demonstrate previously, TGF-beta 1 exhibits the same inhibitory effects as DP IV inhibitors on DNA synthesis and cytokine production (Cytokine 1994, 6, 382-8; J Interferon Cytokine Res 1995, 15, 685-90). A neutralizing chicken anti-TGF-beta 1 antibody was capable of abolishing the DP IV inhibitor-induced suppression of DNA synthesis of PWM-stimulated PBMC and T cells. These data suggest that TGF-beta 1 might have key functions in the molecular action of DP IV/CD26 in regulation of DNA synthesis and cytokine production.

Dipeptidyl peptidase IV (DP IV, EC 3.4.14.3) is a transmembrane type II glycoprotein present on most mammalian cells 1,2.

The proinflammatory cytokine interleukin-6 (IL-6) and its potential opponent, transforming growth factor-beta (TGF-beta 1), have been discussed as being involved in the regulation of inflammatory processes following trauma and infections. The aim of this study was to investigate the effect of these cytokines on the regulation of neutrophil degranulation. The posttraumatic time courses of the plasma concentrations of IL-6, and the elastase-alpha 1-proteinase-inhibitor complex as marker of degranulation in patients undergoing severe trauma were found to be highly correlated, whereas TGF-beta 1 levels were determined to be not significantly altered. The close temporal correlation of IL-6 and elastase levels could be confirmed by investigation of exudates derived from the surgical area. To prove these in vivo findings, the effect of IL-6 and TGF-beta 1 on the degranulation of isolated neutrophils of healthy donors was investigated in vitro. Pathological high IL-6 concentrations were found to be capable of inducing a significant release of lysosomal elastase in a concentration-dependent manner, whereas the degranulation was unaffected by TGF-beta 1. In conclusion, these data suggest an involvement of IL-6 in the regulation of neutrophil degranulation under pathological conditions. However, TGF-beta 1 seems to have no direct regulatory effects besides its described chemotactic function on neutrophils.

Various studies from different laboratories have shown that the membrane ectoenzyme dipeptidyl peptidase IV (DP IV, CD26) expressed in T and NK cells is involved in the regulation of DNA synthesis and cytokine production. In this paper, we performed a biochemical and functional characterization of dipeptidyl peptidase IV on the human histiocytic lymphoma cell line U937. Using U937 clones expressing low to high levels of membrane localized CD26, we found that the synthetic reversible inhibitors of DP IV, Lys-[Z(NO 2 )]-thiazolidide and Lys-[Z(NO 2 )]-piperidide, have different effects on all functions. In U937-H cells that strongly express high levels of CD26, DP IV inhibitors were shown to suppress DNA synthesis and production of IL-1β, but stimulate the secretion of the IL-1 receptor antagonist (IL-1RA) and of TNF-α. In contrast, both inhibitors did not influence the cytokine production and DNA synthesis in U937-L cells exhibiting low level CD26 expression. These data support the hypothesis that CD26 plays a crucial role in proliferation and cytokine production, not only in T cells, but also in other cell systems, and that enzymatic activity is essential for its function.

In the last few years recent studies implicating a deficiency of interleukins (ILs) in several diseases have underlined the importance of measuring in vitro both DNA synthesis and cytokine production (IL-2, IL-6) in the same cell system [1–3]. The production of ILs by peripheral blood mononuclear cells (PBMC) in vitro can be induced by mitogens, phorbol ester, or ionophores [4, 5]. Many different protocols exist that propose optimal conditions for IL-2 or IL-6 induction [1, 4–7].

The differential display reverse transcription polymerase chain reaction method was used to detect alterations in gene expression in the superior colliculus after optic nerve crush in adult rats. One of the most prominent changes observed was the selective induction of R-esp1, a homologue of the Drosophila enhancer of split locus (Groucho). Therefore, we studied the influence of R-esp1 on nerve growth factor (NGF)-induced cell survival of PC12 cells. Overexpression of R-esp1 promotes cell survival even in the absence of NGF and, conversely, it is reduced by antisense-mediated inhibition of R-esp1 expression. In conclusion, we propose a novel model in which R-esp1 protein mediates the NGF-signaling pathway.