HSP70 peptidembearing and peptide-negative preparations act as chaperokines

Abstract

We recently elucidated a novel function for the 70-kDa heat shock protein (HSP70) as a chaperone and a cytokine, a chaperokine in human monocytes. Here we show that peptide-bearing and peptide-negative HSP70 preparations isolated from EMT6 mammary adenocarcinoma cells (EMT6-HSP70) act as chaperokines when admixed with murine splenocytes. EMT6-HSP70 bound with high affinity to the surface of splenocytes recovered from naive BALB/c mice. The [Ca2+]i inhibitor BAPTA dose dependently inhibited HSP70- but not LPS-induced NF-kappaB activity and subsequent augmentation of proinflammatory cytokine TNF-alpha, IL-1beta, and IL-6 production. Taken together, these results suggest that presence of peptide in the HSP70 preparation is not required for spontaneous activation of cells of the innate immune system.

Fig 1.

A typical purification profile of tumor-derived HSP70-peptide complex using ADP affinity chromatography. Results show Coomassie Blue staining of different proteins fractions depicting the various stages of purification; lane 1, molecular weight marker; lane 2, wash following first PD10 column; lane 3, total protein following Dounce homogenization; lane 4, wash following high salt solution; lane 5, eluate following ADP-agarose column; lane 6, wash following second PD10 column; 7 final eluate (EMT6-HSP70)

Fig 2.

EMT6-HSP70 specifically binds to surface membrane of murine splenocytes. Laser confocal microscopy of a section through the center of a murine splenocyte incubated with (A) biotinylated-EMT6-HSP70, (B) biotinylated-OVA, or (C) streptavidin conjugated-FITC only. Bar = 10 μm. Results are representative of 2 independently performed experiments with similar results

Fig 3.

Exogenous EMT6-HSP70 augments the expression of TNF-α. Murine splenocytes treated at 37°C for 2 hours with 100 μg/mL OVA (top panel), 70 nM EMT6(+)HSP70 (second panel from top), or 70 nM EMT6(−)HSP70 (third panel from top) or at 37°C for 4 hours with 70 nM EMT6(+)HSP70 (fourth panel from top) or 70 nM EMT6(−)HSP70 (bottom panel). Cells were then simultaneously fixed and permeabilized using PermeaFix (OrthoDiagnostics), stained with TNF-α-PE (Pharmingen), and analyzed by flow cytometry. Results represent the relative number of murine splenocytes staining positive for TNF-α and are representative of 22 independently performed experiments with similar results

Fig 4.

Exogenous EMT6-HSP70-induces IL-1β production in murine splenocytes. Murine splenocytes treated with 100 μg/mL OVA (top panel), 70 nM EMT6(+)HSP70 for 2 hours (middle panel), or 70 nM EMT6(+)HSP70 for 4 hours (bottom panel). Cells were then simultaneously fixed and permeabilized using PermeaFix (OrthoDiagnostics), then stained with IL-1β-PE (Pharmingen) and analyzed by flow cytometry. Results represent the relative number of murine splenocytes staining for IL-1β and are representative of 2 independently performed experiments with similar results

Fig 5.

Role of [Ca²⁺]i in EMT6-HSP70-induced phosphorylation of I-κBα. Murine splenocytes were pretreated for 30 minutes at 37°C with various concentrations of intracelluar Ca²⁺ chelator, BAPTA-AM. Cells were then treated with either 70 nM HSP70 (filled circles) or 100 μg/mL LPS (filled squares). Thirty minutes posttreatment, cells were immediately fixed and permeabilized using PermeaFix (OrthoDiagnostics), then treated with phospho-specific I-κBα antibody (New England BioLabs, Beverly, MA, USA), stained with anti-rabbit conjugated-FITC, and analyzed by flow cytometry. Results are the mean inhibition of I-κBα (%) ± SD. *P < 0.01 vs respective controls (Student's t-test)