Chaperokine-induced signal transduction pathways. Schematic representation of the main steps involved in chaperokine-induced signal transduction. Exogenous HSP70 binds to either TLR2 or TLR4 (characterized by a cytoplasmic Toll-/IL-1 receptor (TIR) domain and extracellular leucine-rich repeats), or CD40 or CD36 or a yet unknown receptor (?) at the surface of APC. Upon engagement of HSP70 with TLR2 or TLR4, the TLRs activate a downstream MyD88/IRAK signaling cascade which bifurcates at TRAF6 and results in NF-κB and c-Jun/ATF2/TCF activation (solid lines). Engagement of both TLR2 and 4 by exogenous HSP70 synergistically activates a signaling cascade that results in the MyD88-independent activation of NF-κB and c-Jun/ATF2/TCF, via TRAF6 (dashed lines). Engagement of HSP70 to CD40 activates the MyD88-independent activation p38 (dashed lines). Key; MyD88, myeloid differentiation factor 88; IRAK, IL-1R-associated kinase; TRAF, TNF-associated factor, MAPK, mitogen activated protein kinase; ERK, extracellular-signal regulated kinase; JNK, Jun N-terminal kinase.

Chaperokine-induced IL-1β expression. Human monocytic cells THP1 were grown on Falcon CultreSlides (BD Labware, Franklin Lakes, NJ) overnight and co-transfected for 24 h with vector alone or dominant negative MyD88 (MyD88-DN) or dominant negative TLR2 (TLR2-DN) or dominant negative TLR4 (TLR4-DN) or a combination of TLR2-DN + TLR4-DN or TLR2-DN + TLR4-DN + MyD88-DN. Empty vector (Vector), was used as a control. Cells were then stimulated with 100 ng/ml HSP70 or control (not shown) at 37ºC for 4 h in the presence of 10 μM Brefeldin A (Sigma). Following this incubation period, cells were simultaneously fixed and permeabilized using Cytofix/Cytoperm™ kit (BD Biosciences, San Diego, CA) according to the manufacturer’s instructions and counter stained with anti-human IL-1β-FITC (BD Biosciences). One drop of mounting media containing DAPI stain to visualize nuclear staining (Oncogene, Boston, MA) was placed onto the glass slide before the cover slip was sealed with nail polish. Results show fluorescence microscope pictograms of an overlay of a phase contrast image (PhC) and DAPI stain showing nuclear morphology in blue (PhC + DAPI; upper panel), and an overlay of a FITC stain showing intracellular IL-1β expression in green (IL-1β), and nuclear morphology (IL-1β + DAPI; bottom panel). Results are a representative of two independently performed experiments with similar results.