A proteomic approach for the identification of immunotoxic properties of Tulipalin A.

The immune system is permanently exposed to several environmental influences that can have adverse effects on immune cells or organs leading to immunosuppression or inappropriate immunostimulation, called direct immunotoxicity. The natural compound Tulipalin A (TUPA), a lactone with α-methylene-γ-butyrolactone moiety, can influence the immune system and lead to allergic contact dermatitis. This in vitro study focused on effects of TUPA using two immune cell lines (Jurkat T cells and THP-1 monocytes). To evaluate the immunotoxic potential of the compound, a proteomic approach applying 2D gel electrophoresis and MALDI-TOF/TOF-MS in combination with metabolomic analysis was used after exposure of the cells to IC10 of TUPA. THP-1 cells showed a strong robustness to TUPA treatment since only five proteins were altered. In contrast, in Jurkat T cells an increase in the abundance of 66 proteins and a decrease of six proteins was determined. These intracellular proteins were mapped to biological processes. Especially an accumulation of chaperones and an influence on the purine synthesis were observed. The changes in purine synthesis were confirmed by metabolomic analysis. In conclusion, the data indicate possible target processes of low doses of TUPA in Jurkat T cells and provides knowledge of how TUPA affects the functionality of immune cells.

Highly precise quantification of protein molecules per cell during stress and starvation responses in Bacillus subtilis.

Systems biology based on high quality absolute quantification data, which are mandatory for the simulation of biological processes, successively becomes important for life sciences. We provide protein concentrations on the level of molecules per cell for more than 700 cytosolic proteins of the Gram-positive model bacterium Bacillus subtilis during adaptation to changing growth conditions. As glucose starvation and heat stress are typical challenges in B. subtilis' natural environment and induce both, specific and general stress and starvation proteins, these conditions were selected as models for starvation and stress responses. Analyzing samples from numerous time points along the bacterial growth curve yielded reliable and physiologically relevant data suitable for modeling of cellular regulation under altered growth conditions. The analysis of the adaptational processes based on protein molecules per cell revealed stress-specific modulation of general adaptive responses in terms of protein amount and proteome composition. Furthermore, analysis of protein repartition during glucose starvation showed that biomass seems to be redistributed from proteins involved in amino acid biosynthesis to enzymes of the central carbon metabolism. In contrast, during heat stress most resources of the cell, namely those from amino acid synthetic pathways, are used to increase the amount of chaperones and proteases. Analysis of dynamical aspects of protein synthesis during heat stress adaptation revealed, that these proteins make up almost 30% of the protein mass accumulated during early phases of this stress.

Exoproteome Analysis of Human Pathogenic Dermatophyte Species and Identification of Immunoreactive Proteins.

PURPOSE: Increasing incidence of onychomycosis and tinea pedis in humans of industrialized countries together with deep tissue infections are a therapeutic challenge in clinical mycology. For a better understanding of the pathology and immunology of infection, the authors analyze the exoproteomes of three reference strains of the most common clinical dermatophyte species (Trichophyton rubrum, Trichophyton interdigitale, Arthroderma benhamiae) and of Trichophyton strains isolated from affected patients. EXPERIMENTAL DESIGN: Extracellular proteins of those in vitro grown strains are separated via 2D High Performance Electrophoresis and identified by mass spectrometry to find proteins with provoked host immune reactivity. RESULTS: More than 80 secreted proteins including virulence factors such as peptidases and other hydrolases are identified. By Western blotting with respective patient sera, up to 31 proteins with significant antigen-antibody reactions are detected in comparison with control sera, for example, peptidases as well as several oxidoreductases. One protein, beta-glucosidase F2SZI9 seems to be a commonly processed antigen in all Trichophyton infections. CONCLUSIONS AND CLINICAL RELEVANCE: These first global exoproteome data of three dermatophyte species can be a stepping stone on the way to further study the molecular mechanisms of Trichophyton pathogenicity-associated traits. Possible candidates for potential new diagnostic methods or vaccination have to be validated in further investigations.

Differential effects of Helenalin, an anti-inflammatory sesquiterpene lactone, on the proteome, metabolome and the oxidative stress response in several immune cell types.

Extracts of Arnica spp. are traditionally used due to their anti-inflammatory effects for the topical treatment of e.g. haematoma or muscle distortions. One of the main active compounds is Helenalin, a sesquiterpene lactone that can be found in various Asteraceae. However, immunotoxic effects of the compound are only poorly analysed. In this study, a 2D gel electrophoresis based proteomic approach together with a membrane based proteomic assay, metabolomics and the detection of intracellular reactive oxygen species (iROS) were used to investigate potential immunotoxic properties of Helenalin on the human immune cell lines Jurkat and THP-1 and on human peripheral blood mononuclear cells (PBMC). The study revealed a dose-dependent cytotoxicity towards both tested cell lines and the PBMC. However, the cell lines were less sensitive to the Helenalin treatment than the PBMC. The proteomic assays showed strong effects on the carbohydrate metabolism and the protein folding in THP-1 cells but only weak impact on Jurkat cells. Metabolomic studies as well as iROS detection in THP-1 cells verified the results of the proteomic analysis. In summary, the approaches used in this study were able to identify target pathways of Helenalin especially in THP-1 monocytes and thus enable a risk assessment of the substance.

Cytokine sensitivity of Langerhans' islets of diabetes-prone BB/OK rats under hypoglycemic conditions.

Islets of Langerhans isolated from diabetes-prone BB/OK rats were exposed to interleukin-1beta (IL-1beta) or to a combination of tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma) under hypoglycemia at glucose concentrations of 2.2 and 3.2 mmol/l or in the presence of stimulatory conditions at 6.0 and 11 mmol/l glucose. For estimating cytokine effects the islets were functionally assayed by measurement of glucose stimulated insulin secretion. Pancreatic islets exposed for 24 h to IL-1beta at a glucose concentration of 6.0 mmol/l exhibited a reduced insulin secretion following a 48h recovery period compared to islets which were cytokine treated at 2.2 or 3.2mmol/l glucose, respectively. Islets pre-exposed for 24h to TNF-alpha plus IFN-gamma at 2.2, 3.2 or 6.0 mmol/l glucose displayed no alterations of insulin secretion following a 48 h regeneration. A temporary (3 h) influence of IL-1beta under hyperglycemic conditions at 11 mmol/l glucose caused a reduction of the subsequent insulin secretion of Langerhans' islets prior incubated for 24 h at 6.0 mmol/l glucose without cytokines, but not of islets precultured at 2.2 mmol/l glucose. In contrast, a 3 h treatment with TNF-alpha plus IFN-gamma at 11 mmol/l glucose did not affect insulin secretion of islets prior held at 6.0 mmol/l glucose, whereas a transient exposure for 6h to IL-1beta as well as TNF-alpha plus IFN-gamma under similar conditions diminished insulin secretion of islets preincubated at 2.2 or 6.0 mmol/l glucose. In conclusion, hypoglycemia reduces the sensitivity of BB/OK rat islets to IL-1beta, whereas a slight elevation of glucose concentration to 6.0 mmol/l increases again their vulnerability. TNF-alpha plus IFN-gamma at concentrations capable to decrease insulin secretion of islets during hyperglycemia do not affect the insulin output in a range between 2.2 and 6.0 mmol/l glucose. During glucose stimulation at 11 mmol/l islets' insulin secretory machinery is protected from IL-1beta as well as TNF-alpha plus IFN-gamma for 3 h by a preceding 24 h hypoglycemia, but its vulnerability is restored within additional 3 h.

Stress response of pancreatic islets from diabetes prone BB rats of different age.

Protective and/or repair mechanisms are thought to be activated in pancreatic beta cells in response to injury during insulitis. Manifestation of type-1 diabetes may depend on an imbalance between beta cell damage and repair. To prove this hypothesis, the ability of collagenase-isolated islets to respond to heat stress depending on the age of BB rats was investigated. The islets were exposed either to 44 degrees C (HS) or 37 degrees C (control) for 30 min and then kept at 37 degrees C for 5 h. Immediately and 5 h after heat shock, insulin secretion in response to 20 mmol/l glucose and total protein synthesis of heat-exposed islets were significantly diminished as compared with controls. The islet proteins were separated by SDS-PAGE followed by immunoblotting. Islets from BB rats at an age of 6-90 days responded to heat shock with the expression of major heat shock protein 70 (HSP 70). Islets from 3-day old rats, however, did not respond with induction of HSP 70. In contrast we could detect inducible HSP 70 in islets from 3-day old diabetes-resistant LEW rats. In islets from 90-day old BB rats we observed a decreased amount of HSP 70 compared with islets from 9-, 12-, 30- and 60-day old animals. There was also a higher extent of HSP 70 to observe in islets from 90-day old LEW rats as compared with 90-day old BB rats. Differences in HSP 70 expression between islets of 3-day old BB and LEW rats and other age groups of BB rats might represent distinct stages of maturation of islets whereas diminished expression of HSP 70 in islets of 90-day old BB rats at the age of high probability of developing diabetes might result from reduced ability to induce protective mechanisms.

IL-1beta, IFN-gamma and TNF-alpha increase vulnerability of pancreatic beta cells to autoimmune destruction.

In the pathogenesis of type-1 diabetes insulin-producing beta-cells are destroyed by cellular autoimmune processes. The locality of beta-cell destruction is the inflamed pancreatic islet. During insulitis cytokines released from islet-infiltrating mononuclear cells affect beta-cells at several levels. We investigated whether cytokine-induced beta-cell destruction is associated with changes in the expression of the surface receptors intercellular adhesion molecule (ICAM)-1 and Fas. Islets from diabetes-prone and congenic diabetes-resistant BB rats were exposed to interleukin (IL)-1beta alone or in combination with interferon (IFN)-gamma plus tumour necrosis factor (TNF)-alpha. Cytokines decreased islet insulin content, suppressed glucose stimulated insulin secretion and generated enhanced amounts of nitric oxide and DNA-strand breaks. While no membrane alterations of IL-1beta treated islets cells were detectable, the cytokine combination caused damage of cell membranes. Independent of diabetes susceptibility IL-1beta treated islet beta-cells expressed a significantly increased amount of ICAM-1 on their surfaces which was not further increased by IFN-gamma+TNF-alpha. However, IL-1beta induced Fas expression was significantly enhanced only on beta-cells from diabetes-prone BB rats. From these results we suggest that IL-1beta mediates the major stimulus for ICAM-1 induction which is possibly a necessary but not sufficient step in the process of beta-cell destruction. Obviously, the additional enhancement of Fas expression on the surface of beta-cells is important for destruction. The combined action of all three cytokines induced the expression of Fas on the beta-cell surface independent of diabetes susceptibility, indicating that such a strong stimulus in vitro may induce processes different from the precise mechanisms of beta-cell destruction in vivo.

Prevention of autoimmune diabetes in NOD mice by troglitazone is associated with modulation of ICAM-1 expression on pancreatic islet cells and IFN-gamma expression in splenic T cells.

Thiazolidinediones acting as PPAR-gamma agonists are a new generation of oral antidiabetics addressing insulin resistance as a main feature of type-2 diabetes. In accordance to our results, pre-clinical studies have demonstrated that the thiazolinedione troglitazone prevents the development of insulin-dependent autoimmune type-1 diabetes. To investigate whether TGZ acts by affecting the ICAM-1/LFA-1 pathway and/or the Th1/Th2 cytokine balance in NOD mice, we analysed the IL-1beta-induced ICAM-1 expression on islet-cells and the LFA-1, CD25, IL-2, IFN-gamma, IL-4, and IL-10 expression on splenocytes. After 200 days of oral TGZ administration, islet cells from TGZ-treated NOD mice showed a reduced ICAM-1 expression in response to the pro-inflammatory cytokine IL-1beta. The expression of the ligand LFA-1 on CD4(+) and CD8(+) T-cells was comparable to that of placebo- and untreated controls. Also, the expression of Th1/Th2 cytokines was comparable in groups receiving TGZ or Placebo. Nevertheless, the investigated NOD mice segregated into IFN-gamma low- and IFN-gamma high producers as revealed by cluster analysis. Interestingly, the majority of TGZ-treated mice belonged to the cluster of IFN-gamma low producers. Thus, the prevention of autoimmune diabetes in NOD mice by TGZ seems to be associated with suppression of IL-1beta-induced ICAM-1 expression leading to a reduced vulnerability of pancreatic beta-cells during the effector stage of beta-cell destruction. In addition, IFN-gamma production was modulated, implicating that alteration of the Th1/Th2 cytokine balance might have contributed to diabetes prevention. The findings of this study suggest that TGZ exerts its effects by influencing both the beta-cells as the target of autoimmune beta-cell destruction and the T-cells as major effectors of the autoimmune process.

Cytokines activate caspase-3 in insulinoma cells of diabetes-prone NOD mice directly and via upregulation of Fas.

In type 1 diabetes, autoimmune inflammation of pancreatic islets of Langerhans ('insulitis') results in destruction of insulin-producing beta cells. Cytokines released from islet-infiltrating mononuclear cells are known to be cytotoxic both directly and by upregulating Fas for FasL-induced apoptosis. To investigate the role of caspase-3, a major effector of apoptosis in beta-cell death, we asked whether cytokine- and/or FasL-induced apoptosis was associated with increased activity of caspase-3 in NIT-1 insulinoma cells and islets of autoimmune diabetes-prone NOD mice. Measurement of caspase-3 activity using a fluorogenic cleavage assay was validated in NOD mouse thymocytes undergoing dexamethasone (Dex)-induced apoptosis. For cytokine-induced apoptosis, NIT-1 cells or islets were exposed to IL-1 beta and IFN-gamma for 24 h. Caspase-3-like activity was increased 2.1+/-0.7 and 2.4+/-0.9-fold in lysates of cytokine-treated NIT-1 cells and NOD mouse islets, respectively. However, NIT-1 cells exhibited 2.1% (4.7 pg active caspase-3/microg protein) and islets 0.8% (1.9 pg active caspase-3/microg protein) of the active caspase-3 content observed in Dex-treated thymocytes (225.1 pg active caspase-3/microg protein). After 24 h cytokine-exposure, the percentage of Fas-positive NIT-1 cells increased from 1.4+/-1.1 to 29.7+/-11.6%. Addition of FasL for a further 3 h increased caspase-3-like activity an additional 1.8-fold in cytokine-treated NIT-1 cells. In summary, exposure of NOD mouse insulinoma cells or islets to IL-1 beta and IFN-gamma for 24 h induced caspase-3-like activity that, in the case of insulinoma cells at least, can be further enhanced by interaction of cytokine-induced Fas receptor with FasL. Compared to thymocytes, insulinoma cells and islets from NOD mice were characterised by low basal and cytokine-induced caspase-3 activity.