Annegret Gerber

Cathepsin L is involved in cathepsin D processing and regulation of apoptosis in A549 human lung epithelial cells

Abstract Cathepsins are implicated in a multitude of physiological and pathophysiological processes. The aim of the present study was to investigate the function of cathepsin L (catL) in the proteolytic network of human lung epithelial cells and its role in the regulation of apoptosis. We found that catL-deficient A549 cells as well as lung tissue extracts of catL[-/-] mice express increased amounts of single-chain cathepsin D (catD). Degradation experiments indicate that catL specifically degrades the singlechain isoform of catD. Furthermore, we found that catLdeficient cells showed increased sensitivity to apoptosis. Finally, we demonstrate that the inhibition of catD activity by pepstatin A decreased the number of apoptotic cells in catLdeficient A549 cells after anti-Fas treatment. In conclusion, catL is involved in catD processing and the accumulation of catD isoforms in catL-deficient cells is associated with increased rates of spontaneous and anti-Fas-induced apoptosis.

Regulation of in vitro and in vivo immune functions by the cytosolic adaptor protein SKAP-HOM.

ABSTRACT SKAP-HOM is a cytosolic adaptor protein representing a specific substrate for the Src family protein tyrosine kinase Fyn. Previously, several groups have provided experimental evidence that SKAP-HOM (most likely in cooperation with the cytosolic adaptor protein ADAP) is involved in regulating leukocyte adhesion. To further assess the physiological role of SKAP-HOM, we investigated the immune system of SKAP-HOM-deficient mice. Our data show that T-cell responses towards a variety of stimuli are unaffected in the absence of SKAP-HOM. Similarly, B-cell receptor (BCR)-mediated total tyrosine phosphorylation and phosphorylation of Erk, p38, and JNK, as well as immunoreceptor-mediated Ca2+ responses, are normal in SKAP-HOM−/− animals. However, despite apparently normal membrane-proximal signaling events, BCR-mediated proliferation is strongly attenuated in the absence of SKAP-HOM−/−. In addition, adhesion of activated B cells to fibronectin (a ligand for β1 integrins) as well as to ICAM-1 (a ligand for β2 integrins) is strongly reduced. In vivo, the loss of SKAP-HOM results in a less severe clinical course of experimental autoimmune encephalomyelitis following immunization of mice with the encephalitogenic peptide of MOG (myelin oligodendrocyte glycoprotein). This is accompanied by strongly reduced serum levels of MOG-specific antibodies and lower MOG-specific T-cell responses. In summary, our data suggest that SKAP-HOM is required for proper activation of the immune system, likely by regulating the cross-talk between immunoreceptors and integrins.

CATHEPSIN K EXPRESSION IN HUMAN LUNG

Tissue remodeling is crucial in different lung diseases, in the embryonal development as well as in bronchial carcinoma. Cathepsins were proposed to be involved in the degradation of matrix proteins. Cathepsin K is one of the most potent matrix-degrading cysteine proteinases known as yet. The elastinolytic and collagenolytic activity of this papain-like protease is comparable with that of neutrophil elastase. We have investigated the cathepsin K expression in normal adult lung tissues, in embryonal lung tissue and in bronchial carcinoma. With help of specific anti-cathepsin K antibodies it could be shown that cathepsin K was expressed in bronchial epithelial cells. These data could be confirmed at mRNA level using a quantitative RT-PCR as well as by visualisation of the specific enzymatic activity in epithelial cell lines. During the embryonal development cathepsin K was expressed in the epithelial cells of the developing bronchi. The expression seemed to be upregulated in parallel with the development of the bronchial and alveolar lumen. In the later phase of lung development the cathepsin K expression was restricted to bronchial epithelial cells. Furthermore, using quantitative RT-PCR it could be shown that cathepsin K-mRNA was upregulated in lung tumor tissues in comparison to normal tissues from the same patients. These data suggest that cathepsin K may play an important role in matrix remodeling of the lung under physiological and pathological conditions.

TGF-β1-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-γ and TNF-α by pathogenic T cells in response to myelin Ag was enhanced in CD26−/− mice, production of the immunosuppressive cytokine TGF-β1 was diminished in vivo and in vitro. In contrast to the reduction in TGF-β1 production, responsiveness to external TGF-β1 was normal in T cells from CD26−/− mice, excluding alterations in TGF-β1 sensitivity as a mechanism causing the loss of immune regulation. Natural ligands of CD26/DP IV induced TGF-β1 production in T cells from wild-type mice. However, natural ligands of CD26/DP IV failed to elicit TGF-β1 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.

Recombinant Treponema pallidum antigens in syphilis serology.

Treponema pallidum, the etiological agent of syphilis, is characterized by a paucity of surface exposed outer membrane proteins and a high content of cytoplasma membrane associated lipoproteins. At all stages of infection intense antibody responses against lipoproteins are detectable. In order to provide antigens for syphilis diagnosis the highly immunogenic lipoproteins TpN17, TpN29-35 (TpD), TpN44.5 (TmpA), TpN47, and TpN35 (TmpC) and the membrane protein TpN39 (BMP) were cloned. Insertion of PCR amplified DNA into an E. coli expression vector resulted in high level expression of antigens. N-terminal hexahistidine sequence allowed efficient purification of fusion proteins by metal chelate affinity chromatography. The recombinant antigens were tested in enzyme-linked immunosorbent assays. TpN17, TpN47, and TpN44.5 antigens showed high antibody titers. Assays with the three antigens combined resulted in a further improvement of diagnostic sensitivity in comparison with single antigens. Antibodies were found in 17 of 18 patients in all stages of syphilis, whereas 42 normal human sera were nonreactive. No cross-reactivity was detected in 24 sera of patients with Lyme borreliosis.

Expression of Cathepsins B and L in Human Lung Epithelial Cells is Regulated by Cytokines

The cathepsins B, L, and H are expressed ubiquitously and represent the major proportion of lysosomal enzymes. They are involved in bulk proteolysis in the lysosomes, processing of proteins and matrix degradation. Under pathological conditions the participation of cathepsins, especially their secreted forms, was observed in inflammation, tumor progression and metastasis. The enzymatic activity of cathepsins is regulated by posttranslational modification, localization, maturation, changes in pH, and their interaction with inhibitors. Regulation at the level of transcription is not well elucidated. The aim of this study was to investigate the effect of IL-1 beta, IL-6, IL-10, TGF-beta 1, and HGF on mRNA expression and protein level in human lung epithelial cell lines A-549 and BEAS-2B. IL-6 leads to a twofold increase in cathepsin L mRNA expression, whereas TGF-beta 1 decreases the amount of cathepsin L mRNA. At protein level, using enzyme immunoassay, it was shown that IL-6 induced increased amounts of cathepsin L but not cathepsin B. In contrast, after incubation of bronchial epithelial cells with TGF-beta 1 the cathepsin L concentration was decreased. In conclusion, gene expression of cathepsins B and L is variable. The cytokines IL-6 and TGF-beta 1 modulate cathepsin gene expression.

Effects of retinoids on the generation of neutrophil-derived reactive oxygen species studied by EPR spin trapping techniques

Abstract.Objective: To study the potential efficacy of retinoids on granulocytes with regard to their role in inflammatory dermatoses.¶Methods: We investigated the in vitro effect of acitretin and isotretinoin on the generation of reactive oxygen species (ROS) by stimulated human neutrophils using EPR spin trapping techniques.¶Results: The effects of the two retinoids on ROS generation by neutrophils were different. Acitretin increased the generation of hydroxyl radicals, whereas isotretinoin showed an antioxidant activity against the superoxide anion.¶Conclusions: Our study demonstrates retinoid type-dependent effects on ROS production by stimulated neutrophils.

Investigation of Annexin V Binding to Lymphocytes after Extracorporeal Photoimmunotherapy as an Early Marker of Apoptosis

Background: Induction of programmed cell death is assumed to be a possible effect of extracorporeal photoimmunotherapy (ECPI). Objective: In the present study lymphocytes of patients with cutaneous T cell lymphoma undergoing ECPI were investigated for early apoptotic events. Methods: Annexin V, known for its selective affinity to phospholipids, was used to detect early phases of apoptosis. Simultaneous staining with propidium iodide binding to DNA allowed detection of late apoptotic/necrotic cells. Results: At 1 h after ECPI, an increase in early apoptotic cells was found indicating a direct effect of ECPI. At 20 h after each ECPI session, a delayed increase in the number of apoptotic lymphocytes was observed in early apoptotic annexin-stained cells and in late apoptotic cells, whereas in nonirradiated cells no remarkable changes were found. Apoptosis was confirmed by altered light scattering properties and DNA fragmentation. Conclusion: The apoptotic cell death of reinfused lymphocytes is supposed to be a therapeutic effect of ECPI.

CD26/DP IV in T cell activation and autoimmunity

In summary, the present study demonstrates a critical role for CD26/DP IV in balancing Th1 immune responses. We observed a deregulation of the autoimmune process in EAE in vitro, the prototypic model of an immune-mediated CNS disorder. Our results support the notion of a critical regulatory role for the CD26/DP IV molecule within the immune system.

Functional consequences of cathepsin L deficiency in human lung epithelial cells.

Abstract Cathepsin L is a cysteine protease of the papain family. Lung epithelial cells play an important role in host defence. The aim of the present study was to investigate the functional role of cathepsin L in the human lung carcinoma cell line A549. Cathepsin Ldeficient A549 clones were generated. They showed a significant lower proliferation and secreted 5- to 8-fold more IL-8 than the control cells. The production of IL 6, IL-18, and TGFβ1/2 was not affected significantly. It was shown that the cells upregulate IL-8 transcription and that IL-8 in the culture supernatant is necessary for the containment of cellular proliferation. In conclusion, the data show that suppression of cathepsin L expression in A549 cells leads to a growth inhibition which is partially compensated by an upregulation of IL-8 production.