Modulation of cytokine production and silica-induced lung fibrosis by inhibitors of aminopeptidase N and of dipeptidyl peptidase-IV-related proteases.

AIMS: Dipeptidyl peptidase IV (DP IV)-related proteases and aminopeptidase N (APN) are drug targets in various diseases. Here we investigated for the first time the effects of DP-IV-related protease inhibitors and APN inhibitors on chronic inflammatory lung diseases. MAIN METHODS: A murine model of silica (SiO2)-induced lung fibrosis and in vitro cultures of human lung epithelial cells and monocytes have been used and the influence of silica-treatment and inhibitors on inflammation and fibrosis has been measured. KEY FINDINGS: We found increased inflammation and secretion of the chemokines IL-6, MCP-1 and MIP-alpha 2 weeks after SiO2 application, and increased lung fibrosis after 3 months. Treatment with the APN inhibitor actinonin reduced chemokine secretion in the lung and bronchoalveolar lavage fluid, and in cell culture, and decreased the level of fibrosis after 3 months. Treatment with inhibitors of DP-IV-related proteases, or a combination of DP IV inhibitors and APN inhibitors, had no significant effect. We found no obvious side effects of long-term treatment with inhibitors of APN and DP IV. SIGNIFICANCE: Overall, our findings show that actinonin, an inhibitor of aminopeptidase N, might modulate chemokine secretion in the lung and thus attenuate the development of lung fibrosis. Additional targeting of DP-IV-related proteases had no significant effect on these processes.

Radiation-induced matrix production of lung fibroblasts is regulated by interleukin-8.

PURPOSE: Lung fibrosis can be caused by radiation therapy during cancer treatment and therefore can be the limiting factor of the treatment. The factors that cause the actual fibrosis and the interaction between different cell types were investigated. MATERIALS AND METHODS: Epithelial lung cells and fibroblasts were irradiated and different cytokines were measured in the supernatant. Also effects of radiation on the matrix production of fibroblasts were investigated. RESULTS: Irradiation of isolated lung fibroblasts did not cause increased extracellular matrix production; however, the co-culturing of fibroblasts and irradiated lung epithelial cells or the treatment of fibroblasts with supernatants of irradiated epithelial cells did result in an increase. We were able to show that increased interleukin-8 (IL-8) levels led to increased matrix production. CONCLUSIONS: IL-8 is not only a proinflammatory cytokine but it also stimulates collagen synthesis and matrix production and therefore could be a possible drug target in preventing radiation damage during cancer therapy.

Tiotropium suppresses acetylcholine-induced release of chemotactic mediators in vitro.

The driving force in the progression of COPD is the development of exacerbations which are mostly the result of excessive inflammation. Bronchodilatators play an important role in the treatment of COPD. The reported reduction in exacerbation rates in COPD is due to the inhibition of vagal-mediated bronchoconstriction and mucus secretion. However, recent studies have highlighted the existence of muscarinic receptors on inflammatory cells and we have explored the possibility that tiotropium bromide might also inhibit neutrophil migration. We analysed the influence of tiotropium on the release of neutrophil chemotactic activity in response to acetylcholine (ACh) and the expression of muscarinic receptors on human alveolar macrophages (AM), A549 cells, MonoMac6 cells, and human lung fibroblasts. We found significant levels of all muscarinic receptor subtypes on all analysed cells except the fibroblasts. Fibroblasts expressed predominantly M2, receptors and did not release chemotactic activity. AM, A549 cells, and MonoMac6 cells released chemotactic active mediators after incubation with ACh. The secretion could be suppressed by more than 70% after coincubation with tiotropium. Tiotropium alone did not influence the granulocyte migration. Most of the chemotactic activity could be attributed to leukotriene B4 (LTB4). The release of interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) was not induced by ACh. From this, we suggest that the suppression of the Ach-mediated release of chemotactic substances like LTB4 modulates the inflammatory reaction. This may contribute to the decreased rate of exacerbations in COPD, which was observed in clinical trials.

Structural aspects of the inhibition of DNase and rRNase colicins by their immunity proteins.

Nuclease E colicins that exert their cytotoxic activity through either non-specific DNase or specific rRNase action are inhibited by immunity proteins in a high affinity interaction that gives complete protection to the producing host cell from the deleterious effects of the toxin. Previous X-ray crystallographic analysis of these systems has revealed that in both cases, the immunity protein inhibitor forms its highly stable complex with the enzyme by binding as an exosite inhibitor-adjacent to, but not obscuring, the enzyme active site. The structures of the free E9 DNase domain and its complex with an ssDNA substrate now show that inhibition is achieved without deformation of the enzyme and by occlusion of a limited number of residues of the enzyme critical in recognition and binding of the substrate that are 3' to the cleaved scissile phosphodiester. No sequence or structural similarity is evident between the two classes of cytotoxic domain, and the heterodimer interfaces are also dissimilar. Thus, whilst these structures suggest the basis for specificity in each case, they give few indications as to the basis for the remarkably strong binding that is observed. Structural analyses of complexes bearing single site mutations in the immunity protein at the heterodimer interface reveal further differences. For the DNases, a largely plastic interface is suggested, where optimal binding may be achieved in part by rigid body adjustment in the relative positions of inhibitor and enzyme. For the rRNases, a large solvent-filled cavity is found at the immunity-enzyme interface, suggesting that other considerations, such as that arising from the entropy contribution from bound water molecules, may have greater significance in the determination of rRNase complex affinity than for the DNases.

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.