Decreased levels of heat shock proteins in gut epithelial cells after exposure to plant lectins

BACKGROUND: The enterocytes of the intestinal epithelium are regularly exposed to potentially harmful substances of dietary origin, such as lectins. Expression of heat shock proteins (HSPs) by this epithelium may be part of a protective mechanism developed by intestinal epithelial cells to deal with noxious components in the intestinal lumen. AIM: To investigate if the lectins PHA, a lectin from kidney beans (Phaseolus vulgaris) and WGA, a lectin from wheat germ (Triticum aestivum) could modify the heat shock response in gut epithelial cells and to establish the extent of this effect. METHODS: Jejunal tissue sections from PHA and WGA fed rats were screened for expression of HSP70, HSP72, and HSP90 using monoclonal antibodies. Differentiated Caco-2 cells, the in vitro counterpart of villus enterocytes, were exposed to 100 microg/ml of PHA-E(4) or WGA for 48 hours and investigated for changes in DNA and protein synthesis by double labelling with [2-(14)C]thymidine and L-[methyl-(3)H]methionine. The relative concentrations of HSP60, HSP70, HSP72, and HSP90 and binding protein (BiP) in these cells exposed to lectins were analysed by polyacrylamide gel electrophoresis and immunoblotting. To establish if lectin exposed differentiated Caco-2 cells were still capable of producing a heat shock response, these cells received a heat shock (40 degrees C, 41 degrees C, and 42 degrees C) for one hour and were allowed to recover for six hours at 37 degrees C. During heat shock and recovery periods, lectin exposure was continued. RESULTS: Constitutive levels of HSPs were measured in the intestinal cells of lactalbumin fed (control) rats, as may be expected from the function of this tissue. However, in PHA and WGA fed rats a marked decline in the binding of antibodies against several HSPs to the intestinal epithelium was found. These results were confirmed by in vitro experiments using differentiated Caco-2 cells exposed to PHA-E(4) and WGA. However, after exposure to lectins, these cells were still capable of heat induced heat shock protein synthesis, and total protein synthesis was not impaired indicating specific inhibition of HSP synthesis in non-stressed cells. CONCLUSIONS: We conclude that PHA and WGA decrease levels of stress proteins in rat gut and enterocyte-like Caco-2 cells, leaving these cells less well protected against the potentially harmful content of the gut lumen.     

Expression of human HSP70 during the synthetic phase of the cell cycle.

Expression of the major heat shock and stress-induced protein, HSP70, is under complex regulatory control in human cells. In addition to being induced by physiological stress such as heat shock or transition metals, the HSP70 gene is induced by serum stimulation and immortalizing products of the adenovirus E1A 13S and polyoma large tumor antigen genes. Here we show that expression of the human HSP70 gene is tightly regulated during the cell cycle. Using selective mitotic detachment, a noninductive method to obtain synchronous populations of HeLa cells, we show that levels of HSP70 mRNA rapidly increase 10- to 15-fold upon entry into S phase and decline by late S and G2. A transient increase in HSP70 synthesis is detected during early S phase. The subcellular localization of HSP70 varies throughout the cell cycle; the protein is diffusely distributed in the nucleus and cytoplasm in G1, localized in the nucleus in S, and again diffusely distributed in G2 cells. We suggest that the temporal pattern of HSP70 expression during S phase, the nuclear localization, and activation by trans-acting immortalizing proteins indicate a role for HSP70 in the nucleus of replicating cells.     

Reactive oxygen species play an important role in the activation of heat shock factor 1 in ischemic-reperfused heart

BACKGROUND: The myocardial protective role of heat shock protein (HSP) has been demonstrated. Recently, we reported that ischemia/reperfusion induced a significant activation of heat shock factor (HSF) 1 and an accumulation of mRNA for HSP70 and HSP90. We examined the role of reactive oxygen species (ROSs) in the induction of stress response in the ischemic-reperfused heart. METHODS AND RESULTS: Rat hearts were isolated and perfused with Krebs-Henseleit buffer by the Langendorff method. Whole-cell extracts were prepared for gel mobility shift assay using oligonucleotides containing the heat shock element. Induction of mRNA for HSP70 and HSP90 was examined by Northern blot analysis. Repetitive ischemia/reperfusion, which causes recurrent bursts of free radical generation, resulted in burst activation of HSF1, and this burst activation was significantly reduced with either allopurinol 1 mmol/L (an inhibitor of xanthine oxidase) or catalase 2x10(5) U/L (a scavenger of H2O2). Significant activation of HSF1 was observed on perfusion with buffer containing H2O2 150 micromol/L or xanthine 1 mmol/L plus xanthine oxidase 5 U/L. The accumulation of mRNA for HSP70 or HSP90 after repetitive ischemia/reperfusion was reduced with either allopurinol or catalase. CONCLUSIONS: Our findings demonstrate that ROSs play an important role in the activation of HSF1 and the accumulation of mRNA for HSP70 and HSP90 in the ischemic-reperfused heart.     

Regulation of human heme oxygenase-1 gene expression under thermal stress

Heme oxygenase-1 is an essential enzyme in heme catabolism, and its human gene promoter contains a putative heat shock element (HHO-HSE). This study was designed to analyze the regulation of human heme oxygenase-1 gene expression under thermal stress. The amounts of heme oxygenase-1 protein were not increased by heat shock (incubation at 42 degrees C) in human alveolar macrophages and in a human erythroblastic cell line, YN-1-0-A, whereas heat shock protein 70 (HSP70) was noticeably induced. However, heat shock factor does bind in vitro to HHO-HSE and the synthetic HHO-HSE by itself is sufficient to confer the increase in the transient expression of a reporter gene upon heat shock. The deletion of the sequence, located downstream from HHO-HSE, resulted in the activation of a reporter gene by heat shock. These results suggest that HHO-HSE is potentially functional but is repressed in vivo. Interestingly, heat shock abolished the remarkable increase in the levels of heme oxygenase-1 mRNA in YN-1-0-A cells treated with hemin or cadmium, in which HSP70 mRNA was noticeably induced. Furthermore, transient expression assays showed that heat shock inhibits the cadmium-mediated activation of the heme oxygenase-1 promoter, whereas the HSP70 gene promoter was activated upon heat shock. Such regulation of heme oxygenase-1 under thermal stress may be of physiologic significance in erythroid cells.     

Expression of human interleukin-3 (multi-CSF) is restricted to human lymphocytes and T-cell tumor lines

While the cellular sources for granulocyte-macrophage colony- stimulating factor (GM-CSF) are known to be widely distributed among several cell types, interleukin-3 (IL-3) gene expression has been demonstrated in only certain T-cell clones and in blood mononuclear cells stimulated with phytohemagglutinin (PHA) and phorbol-myristate- acetate (PMA). To determine which blood cells were responsible for this expression, we fractionated PHA/PMA-stimulated mononuclear cells and identified T lymphocytes as the source of IL-3 mRNA. Low-level IL-3 expression was detected as well in several stimulated human T-cell lines. Hematopoietic stromal cells such as fibroblasts and endothelial cells could not be induced to express IL-3 mRNA. The kinetics of IL-3 mRNA induction in mononuclear cells and lymphocytes stimulated with PHA/PMA or anti-CD3 monoclonal antibody (MoAb) and interleukin-1 (IL-1) were similar to those observed for GM-CSF expression.     

A non-toxic heat shock protein 70 inducer, geranylgeranylacetone, suppresses apoptosis of cultured rat hepatocytes caused by hydrogen peroxide and ethanol

BACKGROUND/AIMS: A stress-inducible heat shock protein 70 (HSP70) is one of the best-known endogenous factors protecting cell injury under various pathological conditions. The aim of this study was to examine anti-apoptotic actions of a non-toxic HSP70 inducer, geranylgeranylacetone (GGA), on hepatocytes exposed to hydrogen peroxide (H2O2) or ethanol. METHODS: Primary cultures of rat hepatocytes were treated with different concentrations of GGA and exposed to 0.5 mM H202 or 100 mM ethanol. The heat shock response was assessed by measuring the activation of heat shock factor 1 (HSF1), HSP70 mRNA expression, and accumulations of HSP70, HSP90, and HSP27. Apoptosis was evaluated by DNA fragmentation. RESULTS: Pretreatment with 1 microM GGA for 2 h enhanced nuclear translocation and phosphorylation of HSF1, HSF1-DNA binding, HSP70 mRNA expression, and its accumulation, when the cells were exposed to H202 or ethanol. In association with this accelerated response, GGA suppressed the insult-induced activation of c-Jun N-terminal kinases, caspase 9, and caspase 3-like proteases, leading to significant inhibition of apoptosis. CONCLUSIONS: GGA exerted anti-apoptotic actions, at least in part, by priming hepatocytes for enhanced HSP70 induction. Our results suggest that GGA may have a potential benefit for the treatment of alcoholic and ischemia-reperfusion liver injuries.     

Contrasting effects of triiodothyronine on heat shock protein 27 induction and vascular endothelial growth factor synthesis stimulated by TGF-beta in osteoblasts

In osteoblast-like MC3T3-E1 cells, we recently reported that transforming growth factor-beta (TGF-beta) stimulates the induction of heat shock protein 27 (HSP27). In the present study, we investigated the effects of triiodothyronine (T(3)) on the TGF-beta-stimulated induction of HSP27 and synthesis of vascular endothelial growth factor (VEGF) in these cells. T(3) by itself had little effect on the level of HSP27, however, it significantly reduced the TGF-beta-stimulated HSP27 accumulation in a dose-dependent manner in the range between 1 pM and 100 nM. The TGF-beta-stimulated increase in the level of mRNA for HSP27 was also attenuated by T(3). On the other hand, T(3), which alone stimulated the release of VEGF, more than additively stimulated the TGF-beta-induced VEGF release. T(3) enhanced the TGF-beta-induced increase in the levels of mRNA for VEGF. These results strongly suggest that T(3) has contrasting effects on HSP27 induction and VEGF synthesis induced by TGF-beta in osteoblasts.     

Acetylsalicylic acid-induced release of HSP70 from mast cells results in cell activation through TLR pathway

OBJECTIVE: Mast cells are considered major players in IgE-mediated allergic responses, but have also recently been recognized as active participants in innate as well as specific immune responses. Heat stress can modulate innate immunity by inducing stress proteins such as heat shock proteins (HSPs). It has been reported that HSPs are capable of inducing the production of pro-inflammatory cytokines by the monocyte-macrophage system. In the current study, we explored whether the stress response induces HSPs and affects the signaling pathways of mast cells. METHODS: In mouse mast cells, derived from a culture of bone marrow cells of male BALB/cBy and null HSF-1(-/-) mice, responsiveness to exogenous and endogenous HSP70 was monitored by measuring cytokine release. RESULTS: Using BMMC, we show that treatment with heat shock or acetylsalicylic acid results in a selective induction of HSPs, and leads to release of HSP70 into the extracellular environment. The release of HSP70 from mast cells may be of functional importance. We found that after induction of HSP70, the production of TNF-alpha and IL-6 was increased. In a number of experiments, we demonstrated that exogenous/secreted HSP70 is most likely responsible for the activation of mast cells to produce cytokines. Extracellular HSP70 induced production of TNF-alpha and IL-6 through the activation of the TLR4 receptor pathway, which was evidenced by an abrogation of the response in mast cells cultured from TLR4(null) or HSF-1(-/-) mice. CONCLUSION: Our experiments suggest that stress conditions can induce pro-inflammatory cytokine production by mast cells through an autocrine or paracrine stimulation of TLR receptors after a heat shock response. The recognition that heat shock proteins induce mast cell activation suggests an involvement of these cells in the immunological processes induced by heat shock response.     

结核杆菌热休克蛋白质70作为乙型肝炎核心抗原细胞毒性T淋巴细胞表位肽载体的研究

目的探讨结核杆菌热休克蛋白质70（TB．HSP70）作为乙型肝炎病毒（HBV）核心抗原细胞毒性T淋巴细胞（CTL）表位肽载体，诱导HBV特异性免疫应答的可能性。方法体外观察重组TB．HSP70-CTL融合蛋白质和TB．HSP70／CTL复合物诱导慢性乙型肝炎患者外周血淋巴细胞增殖以及HBV特异性细胞毒活性；以Balb／c小鼠为体内研究对象，行流式细胞术分析免疫后小鼠外周血和脾细胞中CD4^＋与CD8^＋T淋巴细胞及自然杀伤（NK）细胞的比率，并观察能否诱导HBV特异性免疫保护作用。结果体内外研究表明TB．HSP70-CTL融合蛋白质和TB．HSP70／CTL复合物能有效地诱导HBV特异性细胞毒活性，体内能激活CD4^＋与CD8^＋T淋巴细胞及NK细胞增殖。在体内，TB．HSP70-CTL融合蛋白质较复合物能更有效地激活免疫应答，其杀伤率为28．9％。CD8^＋T淋巴细胞在脾细胞中比率为43．9％，NK细胞为13．6％。而单纯的TB．HSP70和CTL表位肽并不能有效地引起机体的免疫应答。结论TB．HSP70能够作为乙型肝炎核心抗原CTL表位肽的载体，提高小分子表位肽的免疫原性。     

Effects of idebenone on mitogen-induced proliferation of human lymphocytes

In vitro effect of idebenone on human lymphocytes isolated from old and young donors was determined. The effects of drug were the same with the old and young donors cells. At concentrations of 2 microg/ml (6 microM) or less in the culture medium, idebenone showed no effect on phytohemagglutinin (PHA)-induced proliferation and protein synthesis, or on cell viability measured by Trypan Blue exclusion. Concentrations of 25 and 50 microg/ml showed dose-dependent suppression of the proliferation and protein synthesis which was associated with significant cytotoxicity. At concentrations of 8-10 microg/ml the compound appears to have just detectable effect on lymphocyte viability or ability to respond to PHA stimulation. It seems clear that such in vivo concentrations which would be associated with lymphopenia and immunologic suppression are not achieved with therapeutic doses of idebenone. The pattern of protein bands observed on fluorograms of sodium dodecyl sulfate-polyacrylamide gels of cells incubated with [(3)H]leucine and [(35)S]methionine was similar in control and idebenone-treated samples, consistent with a slight, nonspecific inhibitory effect on protein synthesis in cultures with higher doses of the compound. At these concentrations, idebenone induced a slight, but detectable, enhancement of the intracellular stress proteins, HSP70 and HSP90.     

Detection of the genes induced in activated lymphocytes by modified differential display.

Activated lymphocytes induced by mitogens or antigens express various sets of genes, including those involved in the expression of cytokines, surface molecules, and nuclear proteins. To detect inducible genes in activated lymphocytes, we used the RNA arbitrarily primed polymerase chain reaction (RAP-PCR) method, which is modified by original differential display. By this method we identified eight clones; four contained sequences almost identical to that of the genes for heat shock 90Kd protein1 alpha, STAT2, Ig kappa constant region and interferon receptor 1, two had 70% homology to mitochondrial ATP synthase and bromodomain-containing 2 genes, and two had less than 40% homology to known DNA sequences. By RT-PCR, the heat shock 90 Kd protein1 alpha, STAT2 and interferon receptor 1 genes showed increased expression in phytohemagglutinin (PHA)-stimulated or antigen stimulated T cell line. Differential display is a useful method for detection of inducible genes from a small amount of material and at various time points, although the homology of PCR primer influences the displayed genes.     

Interferon-alpha stimulates production of interleukin-10 in activated CD4+ T cells and monocytes

In the present study, we investigated the effect of interferon-alpha (IFN-alpha) on the expression of interleukin-10 (IL-10) mRNA and protein synthesis in human monocytes and CD4+ T cells. In mononuclear cells, IFN-alpha induced expression of IL-10 mRNA and further enhanced lipopolysaccharide (LPS)-stimulated IL-10 expression. In purified monocytes, a strong expression of IL-10 mRNA induced by LPS was not further enhanced by IFN-alpha. In highly purified CD4+ T cells, IFN- alpha upregulated IL-10 mRNA upon activation with phytohemagglutinin and phorbol myristate acetate. In purified monocytes, an effect of IFN- alpha on IL-10 protein synthesis was dependent on costimulation with LPS. Maximal stimulation of IL-10 protein by IFN-alpha was seen after prolonged incubation periods of 48 to 96 hours, whereas IFN-gamma reduced IL-10 production in the early incubation period. Similar effects of IFN-alpha were observed in CD4+ T cells activated with CD3 and CD28 monoclonal antibodies. Addition of IFN-alpha caused an increase of IL-10 in culture supernatants of activated T-helper cells of more than 100% after 96 hours of incubation. In contrast, other cytokines, including IFN-gamma and IL-4, had no influence on IL-10 secretion stimulated by CD3 and CD28 in CD4+ T cells. In serum samples of IFN-alpha-treated individuals, we failed to detect an influence of cytokine treatment on IL-10 serum levels, confirming the requirement of additional activating signals for IFN-alpha-mediated effects on IL-10 synthesis. In conclusion, IFN-alpha enhances the late induction of IL- 10, which physiologically occurs upon stimulation of monocytes and T cells. Biologically, this effect might enhance the negative-feedback mechanism ascribed to IL-10, which limits inflammatory reactions.