Myeloid differentiation factor 88 is required for cross-priming in vivo

We describe a role for myeloid differentiation factor 88 (MyD88) in the induction of functional CTLs in vivo, in response to exogenously administered Ag, using a heat shock fusion protein, hsp65-P1, as a model Ag. CD8 T cells transferred into MyD88-deficient animals produce normal numbers of CD8 effector cells that have normal activation marker profiles after immunization with hsp65-P1. However, these CD8 T cells produced significantly less IFN-gamma and showed reduced killing activity. This reduction in activation of functional CTLs appears to be unrelated to Toll-like receptor 4 function, because in vitro hsp65-P1-experienced Toll-like receptor 4-deficient dendritic cells (DCs), but not MyD88-deficient DCs, activated CD8 T cells to a similar extent to wild-type DCs. We identify a cross-presentation defect in MyD88-deficient DCs that, when treated with hsp65-P1 fusion protein, results in surface display of fewer SIYRYYGL/class I MHC complexes. Thus, MyD88 plays a role in the developmental maturation of DCs that allows them to prime CD8 T cells through cross-presentation.     

Saturation, competition, and specificity in interaction of heat shock proteins (hsp) gp96, hsp90, and hsp70 with CD11b+ cells

Heat shock proteins (hsp(s)) have been postulated to interact with APCs through specific receptors, although the receptors are yet to be identified. Specificity, saturation, and competition are the three defining attributes of a receptor-ligand interaction. We demonstrate here that the interaction of the heat shock proteins gp96 and hsp90 with CD11b+ cells is specific and saturable and that gp96 can compete with itself in gp96-macrophage interaction. Interestingly, the phylogenetically related hsp90 also competes quite effectively with gp96 for binding to macrophages, whereas the unrelated hsp70 does so relatively poorly, although it binds CD11b+ cells just as effectively. These data provide evidence that the heat shock proteins interact with APCs with specificity and for the existence of at least two distinct receptors, one for gp96 and hsp90 and the other for hsp70.     

Dendritic cells (DCs) in rheumatoid arthritis (RA): progenitor cells and soluble factors contained in RA synovial fluid yield a subset of myeloid DCs that preferentially activate Th1 inflammatory-type responses

There is evidence that mature dendritic cells (DCs) present in the rheumatoid arthritis (RA) joint mediate immunopathology in RA. In this study, we indicate that early myeloid progenitors for DCs and DC growth factors existing in RA synovial fluid (SF) are also likely participants in the RA disease process. A fraction of cells lacking markers associated with mature DCs or DC precursors and enriched in CD34(negative) myeloid progenitors was isolated from RA SF. These cells proliferated extensively when cultured in vitro with cytokines that promote the growth of myeloid DCs (GM-CSF/TNF/stem cell factor/IL-4) and, to a lesser degree, when cultured with monocyte/granulocyte-restricted growth factors (M-CSF/GM-CSF). Mature DCs derived from RA SF progenitors with CD14-DC cytokines known to be prevalent in the inflamed RA joint (GM-CSF/TNF/stem cell factor/IL-13) were potent stimulators of allogeneic T cells and inflammatory-type Th1 responses and included CD14-DC subtypes. Cell-free RA SF facilitated DC maturation from myeloid progenitors, providing direct evidence that the inflamed RA joint environment instructs DC growth. Enhanced development of CD14-derived DCs was correlated with the presence of soluble TNFR (p55), raising the possibility that soluble TNFR also regulate CD14-derived DC growth in vivo. SF from patients with osteoarthritis contained neither myeloid DC progenitors nor DC growth factors. The existence of DC progenitors and myeloid DC growth factors in RA SF supports the concept that RA SF may be a reservoir for joint-associated DCs and reveals a compelling mechanism for the amplification and perpetuation of DC-driven responses in the RA joint, including inflammatory-type Th1 responses.     

Rheumatoid arthritis synovium contains plasmacytoid dendritic cells

We have previously described enrichment of antigen-presenting HLA-DR⁺ nuclear RelB⁺ dendritic cells (DCs) in rheumatoid arthritis (RA) synovium. CD123⁺HLA-DR⁺ plasmacytoid DCs (pDCs) and their precursors have been identified in human peripheral blood (PB), lymphoid tissue, and some inflamed tissues. We hypothesized recruitment of pDCs into the inflamed RA synovial environment and their contribution as antigen-presenting cells (APCs) and inflammatory cells in RA. CD11c⁺ myeloid DCs and CD123⁺ pDCs were compared in normal and RA PB, synovial fluid (SF), and synovial tissue by flow cytometry, immunohistochemistry, and electron microscopy and were sorted for functional studies. Nuclear RelB^-CD123⁺ DCs were located in perivascular regions of RA, in a similar frequency to nuclear RelB⁺CD123^- DCs, but not normal synovial tissue sublining. Apart from higher expression of HLA-DR, the numbers and phenotypes of SF pDCs were similar to those of normal PB pDCs. While the APC function of PB pDCs was less efficient than that of PB myeloid DCs, RA SF pDCs efficiently activated resting allogeneic PB T cells, and high levels of IFN-γ, IL-10, and tumor necrosis factor α were produced in response to incubation of allogeneic T cells with either type of SF DCs. Thus, pDCs are recruited to RA synovial tissue and comprise an APC population distinct from the previously described nuclear RelB⁺ synovial DCs. pDCs may contribute significantly to the local inflammatory environment.     

热应激和吡虫啉对大豆蚜hsp70和hsc70基因mRNA表达的影响

【目的】昆虫在高温或农药的胁迫下,通过高效表达热休克蛋白(HSP)等建立应激自我保护机制。本研究为从转录组水平上认识大豆蚜Aphis glycines在热应激和吡虫啉胁迫下hsp70和hsc70 mRNA表达分子机制,进而寻找自我保护应激反应中的薄弱环节,为大豆蚜的生物防治提供理论基础。【方法】采用同源克隆、RACE技术和实时荧光定量PCR等方法研究不同热激时间和热激后不同恢复时间及不同吡虫啉浓度对大豆蚜4龄若虫hsp70和hsc70的表达影响。【结果】37℃热激后,大豆蚜4龄若虫中hsp70表达量先上调,1 h时升至对照组的10.36倍（P<0.05）,然后逐渐下降。同样热激后恢复时间的长短对大豆蚜若蚜中hsp70的表达具有显著影响。热激处理后,大豆蚜若蚜中hsp70立即大量表达,表达量为对照组的8.78倍（P<0.05）,随后表达量下降至对照组水平,而hsc70的表达量并没有显著变化（P>0.05）。大豆蚜若蚜受吡虫啉的胁迫时,其hsp70和hsc70的表达量受吡虫啉的浓度及胁迫的时间的影响,呈现先升高后下降的趋势,具有明显的短期效应。【结论】吡虫啉诱导大豆蚜hsp70和hsc70表达量的上调;而热胁迫对hsp70和hsc70 mRNA具有不同的表达模式,高温可以诱导hsp70的表达,但对hsc70没有明显的诱导作用。     

A novel hsp70-like protein (P70) is present in mouse spermatogenic cells.

Mouse spermatogenic cells contain relatively large amounts of a 70-kilodalton protein (P70) that is closely related to hsp70, the major inducible heat shock protein. When hsp70 from spermatogenic cells is heat induced, it migrates to the same location as does P70 on two-dimensional polyacrylamide gels, indicating that it has an apparently identical mass and isoelectric point. P70 reacts strongly and specifically with an anti-Drosophila hsp70 monoclonal antibody that is specific for products of the hsp70 gene family. Both P70 and hsp70 are also ATP-binding proteins and are purified by using ATP-affinity chromatography. However, P70 and hsp70 are unique proteins on the basis of peptide map analysis and are regulated differently in germ cells. P70 appears to be a novel heat shock protein of spermatogenic cells which is synthesized in association with germ cell differentiation.     

Heat shock protein (hsp70) expression and thermal tolerance in sublethally heat-shocked eastern oysters Crassostrea virginica infected with the parasite Perkinsus marinus

To investigate whether sublethal heat shock protects Perkinsus marinus (Dermo)-infected oysters Crassostrea virginica from lethal heat stress, and the effects of P. marinus infection on sublethal heat shock response, oysters were first experimentally challenged with P. marinus. Then, when infections in oysters progressed to moderate levels (parasite burden = 10(4) to 10(5) cells g(-1) wet tissue weight), oysters were treated with a sublethal heat shock at 40 degrees C for 1 h (heat shock + Dermo challenge). Other treatment groups included heat-shocked, unchallenged (non-P. marinus challenged) oysters and non-heat-shocked, P. marinus-challenged and -unchallenged oysters. Thermal tolerance was compared among these treatments by administering a lethal heat treatment at 44 degrees C for 1 h, 7 d after sublethal heat shock. Sublethal heat shock enhanced survival to lethal heat treatment in both P. marinus-challenged and -unchallenged oysters. Although levels of hsp70 isoforms (hsp69 and hsp72) did not vary significantly by heat shock or infection with P. marinus, responses due to these treatments were apparent when comparing hsp70 levels within infected and uninfected oysters. Infection enhanced expression of hsp69, regardless of whether oysters were heat shocked or not. In uninfected oysters, hsp72 increased due to heat shock 2 and 7 d post heat shock. Overall, this study demonstrates that heat shock can improve survival in oysters, even in oysters infected with P. marinus. Expression of hsp70 varied among isoforms after sublethal and lethal heat shocks and in infected and uninfected oysters. The heat shock response was not negatively affected by P. marinus infection.     

Heat induced expression of CD95 and its correlation with the activation of apoptosis upon heat shock in rat histiocytic tumor cells

The heat shock response is a universal phenomenon and is among the most highly conserved cellular responses. However, BC-8, a rat histiocytoma, fails to mount a heat shock response unlike all other eukaryotic cells. In the absence of induction of heat shock proteins, apoptotic cell death is activated in BC-8 tumor cells upon heat shock. We demonstrate here that stable transformants of BC-8 tumor cells transfected with hsp70 cDNA constitutively express hsp70 protein and are transiently protected from heat induced apoptosis for 6-8 h. In addition heat stress induces CD95 gene expression in these tumor cells. There is a delay in CD95 expression in hsp70 transfected cells suggesting a correlation between the cell surface expression of CD95 and the time of induction of apoptosis in this tumor cell line. Also expression of CD95 antigen appears to inhibit the interaction between heat shock factors and heat shock elements in these cells resulting in the lack of heat shock response.     

The constitutive and stress inducible forms of hsp 70 exhibit functional similarities and interact with one another in an ATP- dependent fashion

Mammalian cells constitutively express a cytosolic and nuclear form of heat shock protein (hsp) 70, referred to here as hsp 73. In response to heat shock or other metabolic insults, increased expression of another cytosolic and nuclear form of hsp 70, hsp 72, is observed. The constitutively expressed hsp 73, and stress-inducible hsp 72, are highly related proteins. Still unclear, however, is exactly why most eukaryotic cells, in contrast to prokaryotic cells, express a novel form of hsp 70 (i.e., hsp 72) after experiencing stress. To address this question, we prepared antibodies specific to either hsp 72 or hsp 73 and have compared a number of biological properties of the two proteins, both in vivo and in vitro. Using metabolic pulse-chase labeling and immunoprecipitation analysis, both the hsp 72 and hsp 73 specific antibodies were found to coprecipitate a significant number of newly synthesized proteins. Such interactions appeared transient and sensitive to ATP. Consequently, we suspect that both hsp 72 and hsp 73 function as molecular chaperones, interacting transiently with nascent polypeptides. During the course of these studies, we routinely observed that antibodies specific to hsp 73 resulted in the coprecipitation of hsp 72. Similarly, antibodies specific to hsp 72 were capable of coprecipitating hsp 73. Using a number of different approaches, we show that the constitutively expressed, pre-existing hsp 73 rapidly forms a stable complex with the newly synthesized stress inducible hsp 72. As is demonstrated by double-label indirect immunofluorescence, both proteins exhibit a coincident locale within the cell. Moreover, injection of antibodies specific to hsp 73 into living cells effectively blocks the ability of both hsp 73 and hsp 72 to redistribute from the cytoplasm into the nucleus and nucleolus after heat shock. These results are discussed as they relate to the possible structure and function of the constitutive (hsp 73) and highly stress inducible (hsp 72) forms of hsp 70, both within the normal cell as well as in the cell experiencing stress.     

Activation of Fas inhibits heat-induced activation of HSF1 and up-regulation of hsp70.

Activation of heat shock factor (HSF) 1-DNA binding and inducible heat shock protein (hsp) 70 (also called hsp72) expression enables cells to resist various forms of stress and survive. Fas, a membrane-bound protein, is a central proapoptotic factor; its activation leads to a cascade of events, resulting in programmed cell death. These two mechanisms with contradictory functions, promoting either cell survival or death, were examined for their potential to inhibit each other's activation. Induction of FAS-mediated signaling was followed by a rapid decrease in HSF1-DNA binding and inducible hsp70 expression. Inhibition of HSF1-DNA binding was demonstrated to be based on absent hyperphosphorylation of HSF1 during FAS signaling. These effects of FAS activation on the HSF1/hsp70 stress response were blocked by ICE (caspase 1) inhibitors, suggesting an ICE-mediated process. Furthermore, inhibition of HSF1/hsp70 was accompanied by an increase in apoptosis rates from 20% to 50% in response to heat stress. When analyzing the effects of HSF1/hsp70 activation on Fas-mediated apoptosis, protection from apoptosis was seen in cells with induced hsp70 protein levels, but not in cells that were just induced for HSF1-DNA binding. Thus, we conclude that inhibition of HSF1/hsp70 stress response during Fas-mediated apoptosis and vice versa may facilitate a cell to pass a previously chosen pathway, stress resistance or apoptosis, without the influence of inhibitory signals.     

The effects of heat shock and acclimation temperature on hsp70 and hsp30 mRNA expression in rainbow trout: in vivo and in vitro comparisons

Heat shock (25° C) of 10° C-acclimated rainbow trout Oncorhynchus mykiss led to increases in heat shock protein 70 (hsp70) mRNA in blood, brain, heart, liver, red and white muscle, with levels in blood being amongst the highest. Hsp30 mRNA also increased with heat shock in all tissues with the exception of blood. When rainbow trout blood was heat shocked in vitro , both hsp70 and hsp30 mRNA increased significantly. In addition, these in vitro experiments demonstrated that blood from fish acclimated to 17° C water had a lower hsp70 mRNA heat shock induction temperature than did 5° C acclimated fish (20 v. 25° C). The hsp30 mRNA induction temperature (25° C), however, was unaffected by thermal acclimation. While increases in hsp70 mRNA levels in blood may serve as an early indicator of temperature stress in fish, tissue type, thermal history and the particular family of hsp must be considered when evaluating stress by these molecular means.     

The relationship between hsp 70 localization and heat resistance

Using indirect immunofluorescence we have investigated the kinetics of nuclear accumulation and removal of hsp 70 in HA-1 Chinese hamster fibroblasts exposed to elevated temperatures. The kinetics of accumulation of hsp 70 in the nuclei were found to be time/temperature dependent at all temperatures tested (42-45 degrees C). At a given temperature, the fraction of cells manifesting nuclear localization of hsp 70 increased with exposure time. For a given duration of heating, the fraction of cells manifesting nuclear localization of hsp 70 increased with the temperature. The kinetics of the nuclear accumulation of hsp 70 were similar for normal HA-1 cells, their heat-resistant variants, and transiently thermotolerant cells (triggered by prior exposure to a brief heat shock or to sodium arsenite). Upon return to 37 degrees C after heat shock, the kinetics of removal of the hsp 70 associated with the nucleus was dependent on the severity of the initial heat challenge. However, for a given heat dose, the decay of nuclear localization of hsp 70 was more rapid in thermotolerant and heat-resistant cells than in their normal counterparts. These results suggest that the increased levels of hsp 70 associated with the transient or permanently heat-resistant state may play a direct role in restoring and/or repairing heat-induced nuclear and nucleolar alterations associated with heat-induced cell killing. Furthermore, they also suggest that the heat-resistant state may involve ameliorated repair of heat-induced cellular alterations.     

Characterization of native interaction of hsp110 with hsp25 and hsc70

The 110 kDa heat shock protein (HSP) (hsp110) has been shown to be a diverged subgroup of the hsp70 family and is one of the major HSPs in mammalian cells [1,2]. In examining the native interactions of hsp110, we observed that it is found to reside in a large molecular complex. Immunoblot analysis and co-immunoprecipitation studies identified two other HSPs as components of this complex, hsc70 and hsp25. When examined in vitro, purified hsp25, hsp70 and hsp110 were observed to spontaneously form a large complex and to directly interact with one another. When luciferase was added to this in vitro system, it was observed to migrate into this chaperone complex following heat shock. Examination of two deletion mutants of hsp110 demonstrated that its peptide-binding domain is required for interaction with hsp25, but not with hsc70. The potential function of the hsp110-hsc70-hsp25 complex is discussed.