A proteomic method for the analysis of changes in protein concentrations in response to systemic perturbations using metabolic incorporation of stable isotopes and mass spectrometry

While several techniques exist for assessing quantitative differences among proteomes representing different cell states, methods for assessing how these differences are mediated are largely missing. We present a method that allows one to differentiate between cellular processes, such as protein synthesis, degradation and PTMs which affect protein concentrations. An induced systemic perturbation of a cell culture was coupled to a replacement of the growth medium to one highly enriched in the stable isotope 15N. The relative abundance of the 15N- and 14N-enriched forms of proteins, isolated from cell cultures harvested at time points following the onset of the perturbation, were determined by MS. Alterations in protein synthesis and degradation were quantified by comparing proteins isolated from perturbed and unperturbed cultures, respectively. The method was evaluated by subjecting HeLa cells to heat stress. As expected, a number of known heat shock proteins (Hsp) increased in concentration during heat stress. For Hsp27, increased de novo synthesis accounted for the concentration increase, while for Hsp70, decreased degradation accounted for the increase. A protein that was detected only after prolonged heat stress, vimentin, was not primarily synthesized de novo, but appeared rather as a result of PTM.     

Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-6, IL-2, IFN-gamma and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation.

Production of interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) after stimulation by lipopolysaccharide (LPS) and phytohemagglutinin (PHA) was studied in 1/10 diluted whole blood (WB) culture and in peripheral blood mononuclear cell (PBMC) culture. Cytokines IL-1 beta, TNF-alpha and IL-6 are preferentially stimulated by LPS whereas IL-2, IFN-gamma and GM-CSF are stimulated by PHA. Combination of 5 micrograms/ml PHA and 25 micrograms/ml LPS gave the most reliable production of the six cytokines studied. IL-1 beta, TNF-alpha and IL-6 represent a homogeneous group of early-produced cytokines positively correlated among themselves and with the number of monocytes in the culture (LeuM3). Furthermore, IL-1 beta was negatively correlated with the number of T8 lymphocytes. IL-2, IFN-gamma and GM-CSF represent a group of late-produced cytokines. Kinetics and production levels of IL-6 and GM-CSF are similar in WB and PBMC cultures. In contrast, production levels of TNF-alpha and IFN-gamma are higher in WB than in PBMC whereas production levels of IL-6 and IL-2 are lower in WB than in PBMC. Individual variation in responses to PHA + LPS was always higher in PBMC cultures than in WB cultures. The capacity of cytokine production in relation to the number of mononuclear cells is higher in WB, or in PBMC having the same mononuclear cell concentration as WB, than in conventional cultures of concentrated PBMC (10(6)/ml). Because it mimics the natural environment, diluted WB culture may be the most appropriate milieu in which to study cytokine production in vitro.     

Detection of Hsp60 (GroEL)-like proteins in Vibrio parahaemolyticus and vibrio species by Western immunoblotting analysis

Detection of heat shock proteins in Vibrio parahaemolyticus was investigated by SDS-PAGE and Western immunoblotting procedure using an anti-Hsp 60 antibody. Results indicate that V. parahaemolyticus elicited at least one Hsp 60 (GroEL)-like protein with apparent molecular weight of about 58 000 (58 kDa) when submitted to a heat shift from 30 to 42C. Kanagawa phenomenon-positive and -negative strains of V. parahaemolyticus responded the same way. Six other Vibrio species also showed an increased synthesis of GroEL-like (58 kDa) protein after heat shock, while synthesis of 58 kDa protein of V. alginolyticus was at a similar level before and after heat shock. Vibrio nereis showed an increased synthesis of a 60 kDa GroEL-like protein.     

Differential responses of stress proteins, antioxidant enzymes, and photosynthetic efficiency to physiological stresses in the Florida red tide dinoflagellate, Karenia brevis

This study identifies stress proteins and antioxidant enzymes that may play a role in the survival strategies of the Florida red tide dinoflagellate, Karenia brevis. Heat shock protein 60 (Hsp 60), mitochondrial small heat shock protein (mitosHsp), chloroplastic small heat shock protein (chlsHsp), Mn superoxide dismutase (SOD), and Fe SOD were first identified by Western blotting. The induction of these proteins in laboratory cultures in response to elevated temperatures, hydrogen peroxide, lead, or elevated light intensities was next assessed. In parallel, F(V)/F(M), a measurement of photosynthetic efficiency and common proxy of cellular stress, was determined. Hsp 60, Fe SOD, and Mn SOD were induced following exposure to elevated temperatures, hydrogen peroxide, or lead. MitosHsp responded only to heat, whereas chlsHsp responded only to H(2)O(2)-induced stress. The expression of stress proteins and antioxidant enzymes appears to be a more sensitive indicator of heat or chemically induced stresses than F(V)/F(M). However, F(V)/F(M) decreased significantly in response to elevated light intensities that did not induce the expression of stress proteins. These results identify for the first time stress proteins and antioxidant enzymes in K. brevis, provide evidence for differential sensitivity of cellular organelles to various sources of stress, and confirm the presence of conserved stress responses observed across phyla in a dinoflagellate.     

Intrinsic capacity of monocytes to produce cytokines ex vivo in patients with acute lymphoblastic leukaemia

Monocytic cytokine profiles of fifteen children with acute lymphoblastic leukaemia (ALL) were included to determine whether malignancy per se contributes to impaired cytokine profiles in vivo and ex vivo. The ex vivo tumour necrosis factor-alpha (TNF-alpha) and interleukin 1beta (IL-1beta) production was positively correlated with the monocyte number and with the number of intracellular TNF-alpha or IL-1beta positive cells in lipopolysaccharide (LPS)-stimulated MNC cultures. The mean ex vivo TNF-alpha and IL-1beta production per 1x10(4)monocytes in these cultures was not significantly different in children at diagnosis of ALL, at remission or in controls. High IL-10 plasma levels at diagnosis of ALL had no effect on the ex vivo TNF-alpha and IL-1beta production of monocytes in LPS stimulated MNC cultures. These results show that monocytes of ALL patients have a normal intrinsic capacity to produce cytokines ex vivo. However, the decreased monocyte number is responsible for the lower TNF-alpha and IL-1beta concentrations ex vivo upon LPS stimulation.     

Evaluation of monensin and brefeldin A for flow cytometric determination of interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha in monocytes.

Flow cytometry has become a powerful technique to measure intracellular cytokine production in lymphocytes and monocytes. Appropriate inhibition of the secretion of the produced cytokines is required for studying intracellular cytokine expression. The aim of this study was to compare the capacity of cytokine secretion inhibitors, monensin and brefeldin A, in order to trap cytokine production (interleukin-1 beta [IL-1beta], IL-6, tumor necrosis factor-alpha [TNF-alpha]) within peripheral blood monocytes. A two-color flow cytometric technique was used to measure intracellular spontaneous and lipopolysaccharide (LPS)-stimulated IL-1beta, IL-6, and TNF-alpha production in monocytes (CD14+) of whole blood cultures. The viability of monensin-treated monocytes was slightly lower than that of brefeldin A-inhibited monocytes, as measured with propidium iodide (PI). The percentage of IL-6 and TNF-alpha-producing monocytes after 8 h of culture without stimulation revealed significant lower values for monensin-treated than for brefeldin A-treated monocytes. The percentages for stimulated cells did not differ. The spontaneous intracellular production in molecules of equivalent soluble fluorochrome units (MESF) of IL-1beta, IL-6, and TNF-alpha after 8 h of culture was higher in brefeldin A than in monensin-inhibited monocytes. The LPS-stimulated intracellular production of IL-1beta, IL-6, and TNF-alpha was increased in brefeldin A-inhibited monocytes. In conclusion, for flow cytometric determination of intracellular monocytic cytokines (IL-1beta, IL-6, and TNF-alpha), brefeldin A is a more potent, effective, and less toxic inhibitor of cytokine secretion than monensin.     

Comparison of cytokine production in cultures of whole human blood and purified mononuclear cells.

In order to examine inter- and intra-individual variations in cytokine production, blood was collected from 48 healthy subjects on each of 4 occasions separated by 4 weeks. Whole blood (diluted 1:10) and mononuclear cell (MNC) cultures were stimulated for 24 h with either concanavalin A (Con A) or bacterial lipopolysaccharide (LPS) and the concentrations of IL-1alpha, IL-1beta, IL-2, TNF-alpha, IL-10 and IFN-gamma in the culture medium measured. There were highly significant inter-individual variations in the production of each of the cytokines measured. However, the level of the production of each cytokine appeared to be characteristic of an individual. There were significant correlations between production of each cytokine in whole blood and MNC cultures. It is concluded that there is significant inter-individual variation in cytokine production which is unaffected by time or by the stimulus used to elicit cytokine production, and that whole blood cultures can be used instead of MNC cultures to measure cytokine production.     

Synergic activities of streptococcal pyrogenic exotoxin A and lipoteichoic acid in cytokine induction

The present study was carried out to gain insight into the mechanisms involved in the pathogenesis of streptococcal toxic shock syndrome (TSS) and other acute invasive diseases caused by Streptococcus pyogenes (GAS). Specifically, since both whole bacteria and their soluble products are often present in the blood in these conditions, we sought to detect possible synergic activities of somatic and extracellular products in inducing mediators release. For this purpose, whole blood cultures from healthy donors were incubated with different concentrations of streptococcal pyrogenic exotoxin A (SpeA), which is considered a major molecular effector of TSS, heat-killed GAS and cell-wall components such as lipoteichoic acid (LTA) and soluble peptidoglican (sPGN). Significant levels of TNF-alpha, IL-1 alpha and IFN-gamma were found in supernatants from cultures incubated with each of the four inducers alone. Whole GAS and both cell-wall components were more effective (p < 0.05) than SpeA in inducing cytokine release. Whole GAS, at weight basis, was a more potent inducer than LTA and sPGN and LTA, at weight basis, was a more potent inducer than sPGN. In order to verify possible additive or synergic effects of exotoxic and parietal compounds in inducing cytokine release, whole blood cells were incubated with mixtures of SpeA and LTA at different molecular ratio. TNF-alpha, IL-1 alpha and IFN-gamma levels in supernatants were significantly (p < 0.05) higher in supernatants of cultures stimulated simultaneously with the two components than those of cultures stimulated with a single agent. Moreover, these levels were significantly higher than the sum of cytokine levels induced by single components. This study shows that parietal compounds can act in synergy with exotoxins in inducing the release of cytokines, which appear to be the major mediators of TSS.     

Identification of human cell responses to benzene and benzene metabolites

Benzene is a common air pollutant and confirmed carcinogen, especially in reference to the hematopoietic system. In the present study we analyzed cytokine/chemokine production by, and gene expression induction in, human peripheral blood mononuclear cells upon their exposure to the benzene metabolites catechol, hydroquinone, 1,2,4-benzenetriol, and p-benzoquinone. Protein profiling showed that benzene metabolites can stimulate the production of chemokines, the proinflammatory cytokines TNF-alpha and IL-6, and the Th2 cytokines IL-4 and IL-5. Activated cells showed concurrent suppression of anti-inflammatory cytokine IL-10 expression. We also identified changes in global gene expression patterns in response to benzene metabolite challenges by using high-density oligonucleotide microarrays. Treatment with 1,2,4-benzenetriol resulted in the suppression of genes related to the regulation of protein expression and a concomitant activation of genes that encode heat shock proteins and cytochrome P450 family members. Protein and gene expression profiling identified unique human cellular responses upon exposure to benzene and benzene metabolites.     

Cell death mediated by Vibrio parahaemolyticus type III secretion system 1 is dependent on ERK1/2 MAPK, but independent of caspases

Vibrio parahaemolyticus, which causes gastroenteritis, wound infection, and septicemia, has two sets of type III secretion systems (TTSS), TTSS1 and TTSS2. A TTSS1- deficient vcrD1 mutant of V. parahaemolyticus showed an attenuated cytotoxicity against HEp-2 cells, and a significant reduction in mouse lethality, which were both restored by complementation with the intact vcrD1 gene. V. parahaemolyticus also triggered phosphorylation of mitogenactivated protein kinases (MAPKs) including p38 and ERK1/2 in HEp-2 cells. The ability to activate p38 and ERK1/2 was significantly affected in a TTSS1-deficient vcrD1 mutant. Experiments using MAPK inhibitors showed that p38 and ERK1/2 MAPKs are involved in V. parahaemolyticus-induced death of HEp-2 cells. In addition, caspase-3 and caspase-9 were processed into active forms in V. parahaemolyticus-exposed HEp-2 cells, but activation of caspases was not essential for V. parahaemolyticusinduced death of HEp-2 cells, as shown by both annexin V staining and lactate dehydrogenase release assays. We conclude that secreted protein(s) of TTSS1 play an important role in activation of p38 and ERK1/2 in HEp-2 cells that eventually leads to cell death via a caspaseindependent mechanism.     

The heat shock response and cytoprotection of the intestinal epithelium

Following heat stress, the mammalian intestinal epithelial cells respond by producing heat shock proteins that confer protection under stressful conditions, which would otherwise lead to cell damage or death. Some of the noxious processes against which the heat shock response protects cells include heat stress, infection, and inflammation. The mechanisms of heat shock response-induced cytoprotection involve inhibition of proinflammatory cytokine production and induction of cellular proliferation for restitution of the damaged epithelium. This can mean selective interference of pathways, such as nuclear factor kappa B (NF-kappaB) and mitogen-activated protein kinase (MAPK), that mediate cytokine production and growth responses. Insight into elucidating the exact protective mechanisms could have therapeutic significance in treating intestinal inflammations and in aiding maintenance of intestinal integrity. Herein we review findings on heat shock response-induced intestinal epithelial protection involving regulation of NF-kappaB and MAPK cytokine production.     

Relations between IL-3-induced proliferation and in vitro cytokine secretion of bone marrow cells from AML patients.

The influence of IL-3 on the bone marrow cells of 53 patients with acute myeloid leukaemia (AML) was investigated after 72 h suspension in cultures by analysing the proliferation of blasts and the secretion of cytokines. The titres of IL-1beta IL-6, TNF-alpha and IL-3 were measured in the supernatants of these cultures with ELISA tests. Comparing the percentage of cells in S-phases of control cultures and cultures with IL-3, the leukaemias were divided into two growth pattern groups: IL-3-insensitive (n=19) and IL-3-sensitive (n=34) leukaemias. The IL-3-insensitive AML cells show a greater ability for autonomous growth, first by the increase of S-phase in the control culture compared with the S-phase in vivo (P=0.0486) and second, by the higher constitutive secretion (control culture) of IL-1beta P =0.0004), IL-6 ( P =0.0395) and TNF-alpha P=0.0005). The IL-3-induced secondary cytokine secretion is also different in the two growth pattern groups. Whereas in the IL-3-insensitive AML cells a moderate increase of IL-1beta (1.48-fold increase) was present, in the IL-3-sensitive AML cells a 4.72-fold increase of IL-1beta 2.71-fold increase of IL-6 and 11.81-fold increase of the TNF-alpha titre could be detected. Overall, the data show an inverse correlation between the ability of AML cells to respond to IL-3 with increase of an S-phase and the constitutive secretion of IL-1beta, II-6 and TNF-alpha. A further effect of IL-3 is the induction of secondary cytokine secretion in the bone marrow of IL-3-sensitive growing AML cells.     

HMGB1 develops enhanced proinflammatory activity by binding to cytokines

High mobility group box 1 protein (HMGB1), originally characterized as a nuclear DNA-binding protein, has also been described to have an extracellular role when it is involved in cellular activation and proinflammatory responses. In this study, FLAG-tagged HMGB1 was inducibly expressed in the presence of culture media with or without added IL-1beta, IFN-gamma, or TNF-alpha. HMGB1 purified from cells grown in culture media alone only minimally increased cytokine production by MH-S macrophages and had no effect on murine neutrophils. In contrast, HMGB1 isolated from cells cultured in the presence of IL-1beta, IFN-gamma, and TNF-alpha had enhanced proinflammatory activity, resulting in increased production of MIP-2 and TNF-alpha by exposed cells. IL-1beta was bound to HMGB1 isolated from cells cultured with this cytokine, and purified HMGB1 incubated with recombinant IL-1beta acquired proinflammatory activity. Addition of anti-IL-1beta Abs or the IL-1 receptor antagonist to cell cultures blocked the proinflammatory activity of HMGB1 purified from IL-1beta-exposed cells, indicating that such activity was dependent on interaction with the IL-1 receptor. These results demonstrate that HMGB1 acquires proinflammatory activity through binding to proinflammatory mediators, such as IL-1beta.     

Silencing of mitochondrial NADP+-dependent isocitrate dehydrogenase by small interfering RNA enhances heat shock-induced apoptosis

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. Mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDPm) produces NADPH, an essential reducing equivalent for the antioxidant system. In this report, we demonstrate that silencing of IDPm expression in HeLa cells greatly enhances apoptosis induced by heat shock. Transfection of HeLa cells with an IDPm small interfering RNA (siRNA) markedly decreased activity of IDPm, enhancing the susceptibility of heat shock-induced apoptosis reflected by morphological evidence of apoptosis, DNA fragmentation, cellular redox status, mitochondria redox status and function, and the modulation of apoptotic marker proteins. These results indicate that IDPm may play an important role in regulating the apoptosis induced by heat shock and the sensitizing effect of IDPm siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer therapy.     

RNA from LPS-stirnulated macrophages induces the release of tumour necrosis factor-alpha and interleukin-1 by resident macrophages

The effect of exogenous RNA on many cellular functions has been studied in a variety of eukaryotic cells but there are few reports on macrophages. In the present study, it is demonstrated that cytoplasmatic RNA extracted from rat macrophages stimulated with Escherichia coli lipopolysaccharide (LPS), referred to as L-RNA, induced the release of TNF-alpha and IL-1 from monolayers of peritoneal resident macrophages. The activity of L-RNA was not altered by polymyxin B but was abolished by ribonuclease (RNase) pretreatment, indicating the absence of LPS contamination and that the integrity of the polynucleotide chain is essential for this activity. Both the poly A(-) and poly A(+) fractions obtained from L-RNA applied to oligo(dT)-cellulose chromatography induced TNF-alpha and IL-1 release. The L-RNA-induced cytokine release was inhibited by dexamethasone and seemed to be dependent on protein synthesis since this effect was abolished by cycloheximide or actinomycin-D. The LPS-stimulated macrophages, when pre-incubated with [5-(3)H]-uridine, secreted a trichloroacetic acid (TCA) precipitable material which was sensitive to RNase and KOH hydrolysis, suggesting that the material is RNA. This substance was also released from macrophage monolayers stimulated with IL-1beta but not with TNF-alpha, IL-6 or IL-8. The substance secreted ((3)H-RNA) sediments in the 4-5S region of a 5-20% sucrose gradient. These results show that L-RNA induces cytokine secretion by macrophage monolayers and support the idea that, during inflammation, stimulated macrophages could release RNA which may further induce the release of cytokines by the resident cell population.