Correlation of dendritic cell maturation and the formation of aggregates of poly-ubiquitinated proteins in the cytosol

Dendritic cells (DCs) are the most powerful antigen presenting cells (APCs) in the immune system. Therefore, they are able to take up antigen by phagocytosis, macropinocytosis or endocytosis, process it in the cytosol and present it to naive T cells. It is known that presentation of the immunodominant influenza virus nucleoprotein-derived CTL epitope is delayed in bone marrow-derived DCs (BMDCs) compared to non-professional APCs. This delay coincided with the formation of transient aggregations of ubiquitinated proteins (DALIS, dendritic cell aggresome-like induced structures), which contain probably defective ribosomal products (DRiPs). DRiPs appear in the cytosol of maturing DCs and macrophages. Normally, DRiPs are degraded rapidly by proteasomes. However, their storage in DALIS delays their degradation. So, it is hypothesized that DALIS can function as antigen depots allowing DCs to coordinate maturation and antigen presentation during their migration to the lymph nodes. Upon inhibition of several pathways among the in signal transduction pathways of DCs, like the phosphatidylinositol 3-kinase (PI3-K) or the mammalian target of Rapamycin (mTOR), the cells show a rendered maturation profile. The formation of DALIS is inhibited in these cells which can be expected to influence antigen processing and presentation. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 490, individual projects E6 to H.S. and E3 to R.H.).     

热休克蛋白对树突状细胞成熟的影响研究

目的:探讨热休克蛋白(HSP)对树突状细胞(DCs)成熟的影响,同时对其形态学动态变化进行研究。方法:从肝癌组织中提取HSP(gp96 )抗原肽复合物;肝癌细胞进行热休克处理诱导其表面表达HSP,再用DiI荧光标记;将两种HSP与DCs混合培养,动态观察DCs捕获抗原和成熟过程中的形态学变化。流式细胞仪检测不同情况下热休克蛋白对DCs成熟表型的影响。结果:使用DiI标记的方法良好地显示了DCs摄取抗原的形态学变化;DCs通过直接接触、包绕、形成囊泡和伸出伪足且末端形成囊泡4种方式摄取抗原;热休克蛋白可促进DCs成熟表型的表达。结论:DiI是适合DCs形态学研究的良好的荧光标记物;DCs捕获抗原有4种不同方式;不同热休克蛋白均可促进DCs成熟,但提取的热休克蛋白作用更显著。     

HSP110 induces "danger signals" upon interaction with antigen presenting cells and mouse mammary carcinoma

HSP110 is a large molecular weight heat shock protein highly capable of chaperoning large proteins. When chaperoning tumour antigens, HSP110 is capable of eliciting effective anti-tumour immune responses. In the present study, we have determined whether such immunoadjuvant properties of HSP110 stem from its ability to induce "danger signals" through interaction with antigen presenting cells (APCs) and with tumour cells. In the previous studies, endotoxin contamination of HSP preparations was always a matter of concern and controversy. Therefore, we prepared recombinant HSP110 with low endotoxin concentration at which LPS did not have any effect on dendritic cells (DCs). We then evaluated the ability of the HSP110 to induce "danger signals" while interacting with APCs or mouse mammary carcinoma cell line (MMC), as evaluated by modulation of cell surface receptors and cytokines involved in innate and adaptive immune responses. We also performed competition studies in order to rule out contribution of endotoxin in HSP110 preparations while interacting with DCs and MMC. We showed that low endotoxin HSP110 induced DCs to up-regulate the expression of MHC class II, CD40 and CD86 molecules, and to secrete pro-inflammatory cytokines IL-6, IL-12 and TNF-alpha. Importantly, HSP110 induced MMC to secrete IL-12 and elevate secretion of IL-6 and expression of CD40 molecule. These findings demonstrate that HSP110 acts as a "danger signal" through its interaction with DCs and tumour cells, regardless of its endotoxin component. These immunoadjuvant properties of HSP110 suggest that pre-existing immunity in tumour-bearing individuals,may be due to the release of HSPs from tumours upon necrosis alerting the immune system against the tumours.     

Enhanced susceptibility to cytotoxic T lymphocytes without increase of MHC class I antigen expression after conditional overexpression of heat shock protein 70 in target cells

Antigenic peptides have been found associated with heat shock proteins (HSP) including cytoplasmic HSP70 and heat shock cognate protein 70 as well as the endoplasmic reticulum-resident glucose-regulated protein 94. Recently, HSP70 transfection has been reported to increase MHC class I cell surface expression and antigen presentation on mouse melanoma B16 cells (Wells et al., Int. Immunol. 1998. 10: 609). To analyze the effect of HSP70 on MHC class I cell surface expression and lysability of target cells we transfected a human melanoma cell line with the rat Hsp70-1 gene using the Tet-On system for conditional overexpression of HSP70. Induction of HSP70 did not increase cell surface expression of HLA class I molecules in general or individual HLA-A and B antigens in particular. Nonetheless, induction of HSP70 enhanced susceptibility of these cells to lysis by allospecific CTL. The same effect was observed using an HLA-A2-restricted tyrosinase-specific CTL clone after pulsing the tyrosinase-negative target cells with the specific peptide. Thus, HSP70 induction can increase killing by CTL without affecting MHC class I cell surface expression or antigen processing. This effect of HSP70 appears to be different from the commonly found protection exerted by HSP70 against stress like heat shock, and might be mediated by improving CTL-induced apoptosis.     

Effect of endotoxin-induced cell injury on 70-kD heat shock proteins in bovine lung endothelial cells

Heat shock proteins (HSPs) have been remarkably conserved throughout evolution. It has been assumed that induction of HSPs remains a stereotypic response to injury, important for survival of eukaryotic cells during euthermic injury. However, there are few studies of this phenomenon in endothelial cells, and none in pulmonary endothelial cells. We studied the induction of synthesis of 70-kD proteins in bovine pulmonary artery endothelial cells (BPAECs) in response to heat shock and to euthermic injury induced by bacterial endotoxin. First, in response to heat, BPAECs showed rapid and reversible heat-induced synthesis of 70-kD proteins, readily detectable by one-dimensional SDS-PAGE of [35S]methionine-labeled BPAECs. Heat shock at 42 degrees C for 3 h or 43 degrees C for 2 h suppressed total protein synthesis by 30% (P less than 0.001) but an increased rate of synthesis of 70-kD protein continued, representing an increasing fraction of total protein synthesis. Heat-induced synthesis of 70-kD protein returned to baseline levels 8 h after heat shock. Northern analysis showed that mRNA for a protein homologous to a conserved amino acid sequence in the family of species-homologous 70-kD heat shock proteins (HSP 70) was induced by a 15-min incubation at 42 degrees C and remained detectably increased for 6 h. We next assessed whether euthermic injury by bacterial endotoxin (LPS) generated a similar response. LPS was cytotoxic by BPAECs as assessed morphologically, by release of 51Cr from prelabeled cells, and by a significant suppression of total protein synthesis (range, 35 to 70%; P less than 0.001). Despite cytotoxicity, LPS did not induce 70-kD protein at a level that could be detected by SDS-PAGE, and no increase in mRNA for HSP 70 was detected by Northern analysis. LPS-injured BPAECs remained "competent" to induce both 70-kD proteins and mRNA for HSP 70 in response to heat shock. We conclude that at least quantitatively, induction of HSP 70 by BPAECs is not a stereotypic response to injury but rather is at least relatively injury-specific. However, competence to induce HSP 70 appears to be extremely resilient: it is retained in dysfunctional BPAECs in the face of profound inhibition of global protein synthesis, suggesting an important homeostatic role.     

Human recombinant heat shock protein 70 affects the maturation pathways of dendritic cells in vitro and has an in vivo adjuvant activity

Heat shock proteins (HSPs) are potent inducers of an antigen-specific immunological response. A role of chaperon of immunogenic peptides and a direct effect on APC activation and function have been described. However, the signal transduction events involved in the activation of human APCs are poorly characterized. We investigated, using human monocyte-derived dendritic cells (DCs), the signal transduction pathways activated by a human recombinant HSP70 (r)HSP70 purified from eukaryotic cells. rHSP70 effectively induced a partial maturation of DCs in vitro and a significant increase in the titers of antigen-specific IgG when used as a vaccine adjuvant in vivo. rHSP70 did not desensitize human DCs to LPS stimulation and retained its adjuvant properties in C3H/HeJ mice, which are LPS-resistant as a result of a mutation in TLR-4, ruling out the potential interference of LPS contamination. Effects on DC maturation and in vivo functions correlate to the ability of rHSP70 to activate IkappaB-alpha/NF-kappaB and ERK1/2 pathways in human DCs. No activation of p38 was induced in the same experimental conditions. Our data suggest that the IkappaB-alpha/NF-kappaB pathway has a critical role in the partial maturation of DCs induced by rHSP70.     

Proteasome inhibitors differentially affect heat shock protein response in cancer cells

The heat shock proteins (HSPs) are molecular chaperones that are emerging as biochemical regulators of cell growth, apoptosis, protein homeostasis and intracellular targeting of peptides. The immunological function of the HSPs are imparted by tissue specific peptides associated with the HSPs and as such autologous cancer derived HSP-peptide complexes are unique therapeutic agents. Since a majority of the intracellular peptides are generated by the proteasome, we examined the consequence of abrogation of proteasome function by proteasome inhibitors (PIs) such as Lactacystin, MG-132 and LLM on the growth and induction profile of HSP70 and gp96 using hematopoietic, lymphoid, and epithelial derived cancer cell lines. The effect on growth was measured by the XTT assay and induction of the heat shock proteins by western blot analyses using HSP70 and gp96 specific antibodies. Of the PIs tested, cancer cells, were most sensitive to MG-132 and least sensitive to LLM. MG-132 also showed a 10-fold differential sensitivity between estrogen receptor positive, (ER+) MCF-7 cells and negative cells, (ER-) MDA-MB-231. Induction of heat shock proteins, gp96 and HSP70 was, however, noted in response to LLM. Since LLM exhibited minimal cytotoxic effect, metabolic stress that results in induction of HSPs may not be translated in cell growth inhibition and that there may exist a cell-type specific phenomenon in the HSP response to PI mediated metabolic stress.     

Induction of amyloid precursor protein mRNA after heat shock in cultured human lymphoblastoid cells.

In an attempt to examine a possible relationship between heat shock stress and an induction of amyloid precursor protein, cultured lymphoblastoid cells established from 12 human subjects were treated with heat shock at 42 degrees C for 30 min. The levels of mRNA for amyloid precursor protein (APP), heat shock protein (HSP) 70, and actin were examined by Northern blot at 1, 3, 8, 24, and 48 h after the heat shock treatment. HSP70 mRNA was induced at 1 and 3 h, and became undetectable again by 8 h. APP mRNA was also induced at 3 and 8 h, and recovered to the steady level by 48 h. No induction was observed in actin mRNA. These results indicate that APP mRNA is induced by heat shock treatment after the induction of HSP70 mRNA, suggesting a role of heat shock response in an induction of APP.     

Efficient induction of antitumor T cell immunity by exosomes derived from heat-shocked lymphoma cells

Exosomes secreted by tumor cells could serve as a promising immunotherapeutic tumor vaccine. Heat shock proteins (HSP) induced in tumor cells by heat shock are molecular chaperones with potent adjuvant activity in the induction of antigen-specific T cell responses. To improve exosome-based tumor vaccines, we have investigated the efficacy of exosomes derived from heat-shocked mouse B lymphoma cells (HS-Exo) in the induction of antitumor immune responses. We found that HS-Exo, compared with control exosomes derived from the same cells (Exo), contain more HSP60 and HSP90 and increased amounts of molecules involved in immunogenicity including MHC class I, MHC class II, CD40, CD86, RANTES and IL-1beta. Furthermore, HS-Exo induce both phenotypic and functional maturation of dendritic cells more efficiently. HS-Exo immunization activates T cell responses more potently. Importantly, HS-Exo induce dramatically increased antitumor immune responses compared to control exosomes from the same cells in prophylaxis and therapeutic in vivo lymphoma models. We further demonstrate that CD8(+) T cells are the predominant T cell subset responsible for the antitumor effect of HS-Exo and that CD4(+) T cells are necessary in the induction phase of tumor rejection in a prophylaxis model. These findings provide a novel strategy to improve the efficacy of exosome-based tumor vaccines.     

Heat shock proteins: linking danger and pathogen recognition

Besides their central function in protein folding and transport within the cell, heat shock proteins (HSP) have been shown to modulate innate and adaptive immune response: (1) HSP mediate uptake and MHC presentation of HSP-associated peptides by antigen-presenting cells (APC). (2) HSP function as endogenous danger signals indicating cell stress and tissue damage to the immune system. (3) HSP bind pathogen-associated molecular pattern (PAMP) molecules and modulate PAMP-induced Toll-like receptor (TLR) signaling. Thus, HSP contribute to both, recognition of “danger” defined as uncontrolled tissue destruction and recognition of dangerous “nonself”. In this review these different aspects of immune stimulation by HSP will be discussed. Anke Osterloh is funded by the Mildred-Scheel-Stiftung fuer Krebsforschung, Germany.     

Differential induction of brain heme oxygenase-1 and heat shock protein 70 mRNA in sepsis

We examined gene expression of heat shock protein 70 (HSP70) and heme oxygenase-1 (HO-1), which is the rate limiting enzyme in heme catabolism and is also known as heat shock protein 32 (HSP32), in the rat brain using a sepsis model induced by bacterial lipopolysaccharide (LPS). Intraperitoneal injection of LPS (10 mg/kg) to rats caused the elevation of body temperature and white blood cell (WBC) counts as well as marked elevation of serum interleukin-6 (IL-6) level, showing the typical pathological characteristics of sepsis. In this model, HO-1 mRNA increased at 6 h after LPS administration and continued to rise until 30 h. In contrast, HSP70 mRNA increased only between 3 h and 6 h after LPS administration, returning completely to the control level by 12 h. HO-1 mRNA was expressed predominantly in the cortex and the medulla oblongata, while HSP70 mRNA was expressed mainly in the striatum. HO-1 and HSP70 mRNA levels thus showed distinctive time courses and tissue distribution in the brain, suggesting that gene expression of these heat shock proteins (HSPs) is separately regulated.     

Antigen-presentation capacity of dendritic cells is impaired in ongoing enterovirus myocarditis

Coxsackievirus B3 (CVB3)-infection is a frequent cause of acute myocarditis, which may result in chronic myocarditis and virus persistence. Investigation of the initial immune responses to CVB3 may shed light on the mechanisms that contribute to ongoing disease. DCs, as key professional APCs, were investigated in two MHC-matched hosts: while C57BL/6 mice are resistant to chronic CVB3-myocarditis, the A.BY/SnJ mouse strain exhibits susceptibility. DC maturation and activation were critically impaired in A.BY/SnJ mice, as reflected by the failure of DCs to induce co-stimulatory molecules and cytokine/chemokine responses. MHC class I-restricted antigen presentation via the cross-presentation pathway was also affected in DCs from A.BY/SnJ mice. DC maturation involves the accumulation of DC aggresome-like induced structures (DALISs) and the transient storage of defective ribosomal products (DRiPs). DCs from A.BY/SnJ mice showed permanent DALIS accumulation; the detection of poly-ubiquitinylated CVB3 proteins in these DALISs suggested a limitation in the MHC class I antigenic supply in this host. In conclusion, ongoing chronic disease in A.BY/SnJ mice due to a failure to clear the virus may be attributed to defects in DC maturation/activation and DC MHC class I antigen processing.     

Heat shock proteins and scavenger receptors: role in adaptive immune responses

Tumor-derived heat shock proteins have shown promise as anti-cancer vaccines in clinical trials. Heat shock proteins (HSPs) can generate potent anti-tumor immunity and elicit antigen-specific CD8 + T cell responses in murine studies. Antigen presenting cells (APC), such as macrophages and dendritic cells (DCs), can elicit antigen-specific CD8 + T cell responses mediated by HSPs. CD91 was the first identified endocytic scavenger receptor for HSPs on APC that can facilitate the process of cross-presentation. Other scavenger receptors may also play a similar role in this process. The present review critically evaluates the identified HSP endocytic receptors on APCs that may generate adaptive immune responses. A better understanding of this interaction between HSPs and APCs may further unravel mechanisms of immunoadjuvant function of HSPs.     

Salmonella typhimurium stimulation combined with tumour-derived heat shock proteins induces potent dendritic cell anti-tumour responses in a murine model

Appropriate activation of the immune system and effective targeting of tumour cells are the primary hurdles to be overcome for cancer immunotherapy to be successful and applicable to a wide range of tumour types. Our studies have examined the ability of bacterial-stimulated dendritic cells (DCs), loaded with tumour-associated antigens, to inhibit tumour growth in a murine model. Immature murine bone marrow-derived DCs were stimulated in vitro with the cytoplasmic fraction (CM) of Salmonella typhimurium in combination with heat shock proteins (hsps) from 4T1 tumours, isolated using heparin affinity chromatography. Activated DCs were administered subcutaneously. Tumours were generated by orthotopic inoculation of 4T1 cells in Balb/c mice. Primary tumour growth was measured using Vernier calipers, while lung metastases were measured using the clonogenic assay. S. typhimurium CM induced potent tumour necrosis factor (TNF)-alpha responses from DCs accompanied by significant up-regulation of CD80 and CD86 expression. When injected into mice, bacterial-stimulated DCs loaded with 4T1 hsps inhibited the formation of new 4T1 tumours and reduced the growth rate of established tumours. In addition, the number of lung metastatic nodules was reduced significantly in the DC-treated mice (1.6 +/- 0.6 versus 245.9 +/- 55.6, P = 0.0015). DCs stimulated with CM alone, exposed to tumour hsps alone or exposed to tumour hsps from an unrelated tumour cell line did not induce a protective immune response. Dendritic cells primed with a proinflammatory bacterial stimulus and tumour-associated antigens induce a protective anti-tumour immune response in this murine model.     

HSP60通过Toll样受体对ApoE~(-/-)小鼠树突状细胞功能影响研究

目的:探讨HSP60通过Toll样受体(TLR)介导丝裂原活化蛋白激酶(MAPK)信号转导在体外对ApoE-/-小鼠树突状细胞(mDC)的功能影响。方法:将ApoE-/-小鼠构建动脉粥样硬化模型后,从骨髓提取DC,体外培养成熟后分三组:HSP组、LPS组及对照组,分别与HSP60、LPS及生理盐水孵育后,应用流式细胞仪检测细胞表面TLR及CD80水平,RT-PCR检测TLR的mRNA水平,免疫印迹法检测MAPK家族中p38及ERK水平;上清液用ELISA法检测IL-6及TNF-α水平。结果:与对照组比较,HSP组中DC表达TLR及MAPK家族水平、成熟标记物CD80水平、分泌IL-6及TNF-α水平均明显升高(P0.01);但与LPS组比较则无显著差异(P0.05)。结论:HSP60通过TLR介导MAPK信号转导参与动脉粥样硬化模型mDC的活化过程。     

Message in a bottle: role of the 70-kDa heat shock protein family in anti-tumor immunity

Extracellular heat shock protein 70 (HSP70) is a potent agent for tumor immunotherapy, which can break tolerance to tumor-associated antigens and cause specific tumor cell killing by cytotoxic CD8+ T cells. The pro-immune effects of extracellular HSP70 are, to some extent, extensions of its molecular properties as an intracellular stress protein. The HSP70 are characterized by massive inducibility after stress, preventing cell death by inhibiting aggregation of cell proteins and directly antagonizing multiple cell death pathways. HSP70 family members possess a domain in the C terminus that chaperones unfolded proteins and peptides, and a N-terminal ATPase domain that controls the opening and closing of the peptide binding domain. These properties not only enable intracellular HSP70 to inhibit tumor apoptosis, but also promote formation of stable complexes with cytoplasmic tumor antigens that can then escape intact from dying cells to interact with antigen-processing cells (APC) and stimulate anti-tumor immunity. HSP70 may be released from tumors undergoing therapy at high local extracellular concentrations, and send a danger signal to the host leading to APC activation. Extracellular HSP70 bind to high-affinity receptors on APC, leading to activation of maturation and re-presentation of the peptide antigen cargo of HSP70 by the APC. The ability of HSP70-peptide complexes (HSP70-PC) to break tolerance and cause tumor regression employs these dual properties as signaling ligand and antigen transporter. HSP70-PC thus coordinately activate innate immune responses and deliver antigens for re-presentation by MHC class I and II molecules on the APC cell surface, leading to specific anti-tumor immunity.     

应激相关的热休克信号转导通路的研究进展

随着社会的发展和医疗技术的不断进步与完善,人们的健康水平有了大幅度的提高。但急性梗塞(如脑梗、心梗)、感染性疾病(如败血症)等仍有较高的发病率和死亡率。特别是随着我国社会和经济的发展,老年人群的结构比例明显增高,心脑血管疾病和感染引起的疾病将进一步增加。而且随着     

Previous heat shock treatment attenuates bicuculline-induced convulsions in rats

Exposure to elevated temperature provokes a sequence of events (heat shock response) in all living organisms. Through this response, heat shock proteins (HSPs) are induced and protect the cells against subsequent injury. We investigated the effect of heat treatment on bicuculline-induced convulsions, and analyzed a possible role of HSPs. Screw electrodes were implanted in the brain of mature male Wistar rats for electroencephalogram (EEG) recording. Experimental rats were subjected to whole-body hyperthermia at 41–42° C for 15 min. Fifteen hours later, bicuculline was injected intra peritoneally to induce convulsions in both experimental and control groups. The heated rats showed a significant attenuation of the convulsive response, in terms of both spike discharges in EEG and clinical seizures. Furthermore, induction of HSP72 was detected in the brain of heat-treated rats by immunoblotting, appearing at 4 h and reaching a maximal level 16–24 h after the heat shock. We conclude that the previous heat treatment stabilized neuronal excitability, most probably through the induction of HSP72.