• Heat shock proteins form part of a danger signal cascade in response to lipopolysaccharide and GroEL

      Davies, Emma L.; Bacelar, Maria M. F. V. G.; Marshall, Michael J.; Johnson, E.; Wardle, T. D.; Andrew, Sarah M.; Williams, John H. H.; University of Chester ; University of Chester ; Charles Salt Centre, The Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry ; Spinal Studies, The Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry ; Countess of Chester Hospital ; University of Chester ; University of Chester (Wiley, 2006-05-26)
      An increasing number of cell types, including peripheral blood mononuclear cells (PBMCs), have been demonstrated to release heat shock proteins (Hsps). This paper investigates further the hypothesis that Hsps are danger signals. PBMCs and Jurkat cells released Hsp70 (0·22 and 0·7 ng/106 cells, respectively) into medium over 24 h at 37°C. Release of Hsp70 was stimulated 10-fold by GroEL (P < 0·001) and more than threefold by lipopolysaccharide (LPS) (P < 0·001). Although Hsp60 could be detected in the medium of cells cultured at 37°C for 24 h, the low rates of release were due probably to cell damage. Significant release of Hsp60 was observed when Jurkat cells were exposed to GroEL (2·88 ng/106 cells) or LPS (1·40 ng/106 cells). The data are consistent with the hypothesis that Hsp70 and Hsp60 are part of a danger signalling cascade in response to bacterial infection.
    • Heat shock proteins: Interactions with bone and immune cells

      Williams, John H. H.; Davies, Emma L. (University of Liverpool (Chester College of Higher Education), 2004-09)
      Heat shock proteins (Hsps) are increasingly being seen as having roles other than those of intracellular molecular chaperones, particularly with regard to their potential to act as cytokines, and to stimulate the innate immune system. Hsps have also been found to promote bone resorption and osteoclast formation in vitro, although the mechanism has not been previously identified. The overall aims of this thesis were to determine whether Hsps could stimulate bone resorption by affecting the RANKL/OPG pathway, and to address the hypothesis that Hsps can act as a danger signal to the innate immune system. In order for Hsps to affect either the RANKL/OPG system of bone resorption or act as danger signals they would need to be actively released from cells, ideally in a controlled manner following exposure to the source of stress. Hsp60 and Hsp70 were found to be released from a range of immune cells including the cell lines Jurkat and U937, and also PBMCs, T-cells and B-cells. This release was not due to cell damage. The release of Hsp60 and Hsp70 were downregulated by inhibitors of protein secretion, in particular Hsp70 release was reduced by compounds that inhibited lysosomal pathways and Hsp60 release by classical secretion inhibitors. Hsp60, Hsp70, GroEL and LPS all affected the RANKL/OPG system of bone regulation; OPG production and release was down-regulated in the MG63 and GCT osteoblast-like cell lines following treatment with Hsp60, Hsp70 and LPS, and RANKL expression was upregulated following treatment with Hsp60, Hsp70, GroEL and LPS. This effect on the RANKL/OPG system was found to translate into an effect on osteoclast formation when conditioned media from treated osteoblasts was added to osteoclast precursors in the presence of M-CSF. A range of different factors that affected Hsp release were identified; PHA activation of PBMCs was found to upregulate Hsp60 release from PBMCs. GroEL and LPS caused an upregulation in Hsp70 release from PBMCs and GCT osteoblast like cells, and Hsp70 was found to stimulate Hsp60 release from PBMCs and GCT cells. These responses of Hsp release were used to form a theory of a cascade-like danger signal that may occur when cells are exposed to bacterial infection and which would result in activation of antigen presenting cells via previously identified receptors for Hsps such as CD14/TLR4 or by unidentified pathways. The elevated release of Hsps in response to GroEL and LPS was also identified as a mechanism that could stimulate bone loss during infection or autoimmuniry by affecting the RANKL/OPG system. hi conclusion, Hsp60 and Hsp70 can be released from immune cells under normal conditions, and from both immune and osteoblast-like cells following stimulation with LPS and other Hsps. The observed release responses provide a mechanism through which Hsps can act as danger signals to the innate immune system, and also as promoters of bone resorption via the RANKL/OPG system.
    • Hsp70 release from peripheral blood mononuclear cells

      Hunter-Lavin, Claire; Davies, Emma L.; Bacelar, Maria M. F. V. G.; Marshall, Michael J.; Andrew, Sarah M.; Williams, John H. H.; University of Chester ; University of Chester ; University of Chester ; The Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry ; University College Chester ; University College Chester (Elsevier, 2004-11-12)
      There are an increasing number of studies reporting the presence of Hsps in human serum. We have investigated the release of Hsp70 into blood and culture medium from peripheral blood mononuclear cells (PBMCs), and whether this release is due to cell damage or active secretion from the cells. Intact Hsp70 was released from cells within whole blood and from purified PBMCs under normal culture conditions. Hsp70 release was rapid (0.1 ng/106 cells/h) over the first 2 h of culture and continued at a reduced rate up to 24 h (<0.025 ng/106 cells/h). Using viable cell counts and lactate dehydrogenase release we were able to confirm that the release of Hsp70 was not due to cellular damage. Hsp70 release was inhibited by monensin, methyl-β-cyclodextrin, and methylamine, but not by brefeldin A. These data suggest that Hsp70 is released from cells via a non-classical pathway, possibly involving lysosomal lipid rafts.