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More than destructive: neutrophil‐derived serine proteases in cytokine bioactivity control

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.

Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor-beta 1 in PWM-stimulated PBMC and T cells.

Various studies have shown that the membrane ectoenzyme dipeptidyl peptidase IV (DPIV; 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‐γ (IFN‐γ) of pokeweed mitogen (PWM)‐stimulated purified T cells. Most importantly, these inhibitors induce a three‐ to fourfold increased secretion of latent transforming growth factor‐β1 (TGF‐β1) by PWM‐stimulated peripheral blood mononuclear cells (PBMC) and T cells, as measured with a specific TGF‐β1 enzyme‐linked immunosorbent assay and in the Mv1Lu bioassay. As we could demonstrate previously, TGF‐β1 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‐β1 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‐β1 might have key functions in the molecular action of DP IV/CD26 in regulation of DNA synthesis and cytokine production.

Effects of different anaesthetic agents on immune cell function in vitro

Background and objective: Anaesthesia may affect the regulatory balance of postoperative immune response. The aim of this study was to investigate the effects of different volatile and non‐volatile anaesthetic agents and particularly of clinically used agent combinations on the proliferation capacity and cytokine production of immune cells. Methods: Peripheral blood mononuclear cells from healthy donors were PHA‐activated in the presence or absence of various concentrations of thiopental, propofol, fentanyl, sufentanil, sevoflurane, nitrous oxide and combinations of these anaesthetics. Cell proliferation was assessed by tritiated thymidine uptake. Interleukin‐2 production and release of the soluble IL‐2 receptor were determined by enzyme immunoassays and used as measures of lymphocyte activation. Results: Thiopental inhibited cell proliferation in a dose dependent manner (P < 0.001) and reduced sIL‐2R release (2090‐970 pg mL−1; P < 0.05). Propofol reduced sIL‐2R release at the high concentration of 10 μg mL−1 (2220 pg mL−1‐1780 μg mL−1; P < 0.05). Fentanyl and sufentanil did not compensate for or enhance the inhibitory effects of thiopental. Nitrous oxide, but not sevoflurane, reduced the proliferation of human peripheral blood mononuclear cells (P < 0.05). In combinations with thiopental or nitrous oxide, sevoflurane compensated the inhibitory effects of these two agents. Fentanyl, sufentanil, sevoflurane and nitrous oxide did not affect PHA‐induced IL‐2 and sIL‐2 receptor release by human peripheral blood mononuclear cells. Conclusion: Thiopental and nitrous oxide have immunosuppressive activity. In contrast, sevoflurane may have a beneficial effect by alleviating the immunosuppressive effects of both substances.

Evidence for a crucial role of neutrophil‐derived serine proteases in the inactivation of interleukin‐6 at sites of inflammation

The bioactivity of interleukin‐6 (IL‐6) was found to be dramatically reduced in fluids from sites of inflammation. Here, we provide evidence that the neutrophil‐derived serine proteases elastase, proteinase 3 and cathepsin G are mainly involved in its degradation and subsequent inactivation. The initially hydrolyzed peptide bonds were detected to be Val11‐Ala12 and Leu19‐Thr20 (elastase), Phe78‐Asn79 (cathepsin G) and Ala145‐Ser146 (proteinase 3). The soluble IL‐6 receptor elicits a protective effect against the IL‐6 inactivation by cathepsin G only. The inactivation of IL‐6 by neutrophil‐derived serine proteases might act as a feedback mechanism terminating the IL‐6‐induced activation of neutrophils.

Dipeptidyl peptidase IV (CD26) on human lymphocytes. Synthetic inhibitors of and antibodies against dipeptidyl peptidase IV suppress the proliferation of pokeweed mitogen-stimulated peripheral blood mononuclear cells, and IL-2 and IL-6 production.

In the present report, we describe that synthetic inhibitors of and polyclonal and monoclonal antibodies against the membrane ectoenzyme dipeptidyl peptidase IV (DP IV, CD26) inhibit the production of IL-2 and IL-6 and, concomitantly, DNA synthesis of pokeweed mitogen-stimulated peripheral blood mononuclear cells (PBMC). The release of IL-1 and TNF-alpha, was not influenced under these conditions. The data support the hypothesis that DP IV, possibly in conjunction with other peptidase, is involved in the regulation of activation and proliferation of T lymphocytes.

Selective proteolytic cleavage of IL-2 receptor and IL-6 receptor ligand binding chains by neutrophil-derived serine proteases at foci of inflammation.

Traumatic brain injuries induce a strong, locally restricted inflammatory response. Here we demonstrate that activated neutrophils infiltrate the site of tissue destruction and release large amounts of enzymatically active elastase, cathepsin G, and proteinase 3. High intracerebral protease concentrations were found to be accompanied by a reduced inhibitory potential at foci of inflammation. In 39 neurotrauma patients, a temporal correlation between the protease release from neutrophils and the solubilization of interleukin-2 (IL-2) receptor and IL-6 receptor ectodomains at sites of tissue destruction was observed, suggesting that neutrophil-derived proteases may play a crucial role in the cytokine receptor shedding at foci of inflammation. Under in vitro conditions, the cleavage of membrane-bound IL-2Ralpha was found to be predominantly catalyzed by elastase and, to a lesser extent, by proteinase 3. Cathepsin G was found to be incapable of solubilizing this receptor. In contrast, the cleavage of the IL-6R 80 kDa chain was catalyzed by cathepsin G but not by elastase or proteinase 3. The receptor fragments released by the action of these enzymes were found to retain their ligand-binding capacity. These results strongly suggest a pathophysiologic role of neutrophil-derived serine proteases, particularly in regulation of the expression of functional IL-2 and IL-6 receptors at foci of inflammation.

Inhibitors of Dipeptidyl Peptidase IV (DP IV, CD26) Specifically Suppress Proliferation and Modulate Cytokine Production of Strongly CD26 Expressing U937 Cells

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(NO2)]-thiazolidide and Lys-[Z(NO2)]-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-1 beta, but stimulate the secretion of the IL-1 receptor antagonist (IL-1RA) and of TNF-alpha. 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.

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

Abstract 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-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 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. Clin Chem Lab Med 2009;47:253–61.

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

Abstract 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. Clin Chem Lab Med 2009;47:268–74.

Inhibitors of dipeptidyl peptidase IV (DP IV, CD26) induces secretion of transforming growth factor-β1 (TGF-β1) in stimulated mouse splenocytes and thymocytes

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 β1 (TGF-β1) by mitogen-stimulated mouse immune cells, as measured with a specific TGF-β1 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-β1 might have key functions in these processes.