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First published online 26 July 2005
doi: 10.1242/jcs.02494

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Induction of heat shock proteins in B-cell exosomes

Department of Clinical Oncology and Palliative Medicine, Wales College of Medicine, Cardiff University, Velindre Cancer Centre, Whitchurch, Cardiff, CF14 2TL, UK

View larger version (17K): [in a new window]   Fig. 1. Constitutive and induced expression of cellular hsps. Non-stressed B-cell lines were washed in PBS, fixed, permeabilised and stained with hsp-specific antibodies or isotype control (as indicated). Levels of hsps were examined by flow-cytometry. (a) The value for the mean fluorescence intensity (MFI) was taken (mean+s.e.m. of four B-cell lines). (b) Altered cellular hsp expression following heat stress. Four B-cell lines were incubated at 42°C for up to 3 hours, incubated for a total of 24 hours at 37°C before hsp expression was analysed by flow-cytometry. The graph is expressed in terms of fold-change relative to non-stressed expression (normalised to a value of 1, and indicated by the dotted line) (mean ± of four B-cell lines).

View larger version (32K): [in a new window]   Fig. 2. Hsps are specifically enriched in exosomes during stress. B-cell lines were subjected to heat stress (42°C for 0-3 hours), and subsequently incubated for 24 hours at 37°C before exosome and cell lysate preparation. (a) Western blot analysis of stress-dependent (time) hsp expression in exosomes, demonstrating selected elevations in stress proteins but not other exosome markers. (b) Western blot analysis of cell lysates and exosomes, showing the absence of gp96 and hsp60 in exosomes, even after heat stress. Flow-cytometric analysis of hsc70 and hsp70 levels in control IB4 cells or IB4 cells heat-stressed for 2 hours, demonstrating that heat stress reduces cellular hsc70, while hsp70 is elevated (mean fluorescence values of the histogram are shown). Western blots of cell lysates or exosomes from the same experiment were also performed, comparing cell-lysates with exosomes. Various exposure times are shown (10-180 seconds for hsc70). (c) Densitometric analysis (of 180 exposed blots), was performed to confirm decreased cellular hsc70 levels, and increased exosomal hsc70 levels upon heat stress. (d) TSG101, a component of the exosome formation machinery, is only slightly elevated in exosomes following heat stress. (e) Exosomes derived from Jurkat cells subjected to heat stress (42°C for 0-2 hours), also demonstrate elevated levels of stress proteins hsp90 and hsp70, although steady-state levels of hsps in Jurkat exosomes were lower than that in B-cell exosomes.

View larger version (46K): [in a new window]   Fig. 3. The quantity of exosome secretion changes following heat stress. Exosomes were purified from culture supernatants containing equal numbers of control or 3-hour heat-stressed cells [IB4, BLCL(LL), BLCL(PC) or Jurkat cell lines]. The exosome pellet of each purification was resuspended in 100 µl PBS, and total protein determined by BCA assay. The graph represents the ratio of control exosome-protein to heat-shock-exosome-protein, where a ratio of 1 (dotted line) would indicate no difference. (Mean ratio±s.e.m. of n purifications.) The overall mean ratio was 1.245±0.07 (n=20), indicating a small elevation (less than 1.3-fold) in exosome secretion due to stress.

View larger version (36K): [in a new window]   Fig. 4. Hsps are located within the exosome lumen. (a) Purified stock exosomes were analysed by western blot for induced hsp expression. Aliquots of these exosomes were subsequently immobilised onto latex beads and surface-hsp expression was analysed by (b) flow-cytometry; filled histograms represent exosome-bead complexes stained with irrelevant-isotype control antibody, unfilled histogram represent antibody staining as indicated. (c) Remaining exosome-bead complexes, not stained with antibodies, were boiled in SDS-sample buffer and analysed by western blot. (d) The remainder of the initial exosome stock (intact exosomes, not immobilized onto latex beads) was immunoprecipitated with antibodies as indicated and the relative quantity of precipitated exosomes determined by western blot, stained for MHC class I. Although exosomes were effectively precipitated with MHC class II antibodies, no bands were seen with hsp-specific antibodies. These data indicate that hsps are detectable following exosome disruption, but are not present on the exosome surface.

View larger version (34K): [in a new window]   Fig. 5. Stress-derived exosomes do not mature dendritic cells. MDDCs (day 6) were incubated alone, with control exosomes or with 3 hour heat-shocked exosomes (1-100 µg per 105 MDDCs), or with LPS (0.1 or 10 µg/ml). (a) After 48 hours, MDDCs were harvested, fixed and analysed for CD83 expression by flow-cytometry. (b) Culture supernatants were taken and cytokines (IL-6, IL-10, IL-12) were measured by ELISA. Graph shows mean+s.d. of triplicate measurements.