Role of the heat shock protein 90 in immune response stimulation by bacterial DNA and synthetic oligonucleotides

Abstract

To elucidate the mechanisms of immunostimulation by bacterial DNA and synthetic oligonucleotides, the effects of heat shock protein 90 (Hsp90) inhibitors on the activation of murine spleen cells and macrophages by these molecules were investigated. Murine spleen cells and J774 and RAW264.7 macrophages responded to a CpG-containing oligodeoxynucleotide (CpG ODN) and Escherichia coli DNA by increased production of interleukin 6 (IL-6), IL-12, tumor necrosis factor alpha, and nitric oxide (NO). Pretreatment with any of the three Hsp90 inhibitors geldanamycin, radicicol, and herbimycin A resulted in a dose-dependent suppression of cytokine production from the spleen cells and macrophages and of NO from macrophages stimulated with CpG ODN or E. coli DNA. These Hsp90 inhibitors, however, had no effect on Staphylococcus aureus Cowan strain 1-induced IL-12 production from either the murine spleen cells or macrophages. CpG ODN and E. coli DNA induced increased intracellular levels of phosphorylated extracellular signal-regulated kinases (ERK1 and -2), which are members of the mitogen-activated protein (MAP) kinase family, while geldanamycin and radicicol blocked the phosphorylation of ERK1 and -2 in J774 and RAW264.7 cells. These data indicate that DNA-induced activation of murine spleen cells and macrophages is mediated by Hsp90 and that Hsp90 inhibitor suppression of DNA-induced macrophage activation is associated with disruption of the MAP kinase signaling pathway. Our findings suggest that Hsp90 inhibitors may provide a useful means of elucidating the mechanisms of immunostimulation by bacterial DNA and CpG ODN as well as a strategy for preventing adverse effects of bacterial DNA as well as lipopolysaccharide.

FIG. 1

Effects of Hsp90 inhibitors on IL-12 production in DNA-stimulated murine spleen cells (upper and middle panels) and J774 macrophages (lower panel). Cells were pretreated with the indicated concentrations of geldanamycin (GA) or 0.1 μg of geldanamycin, radicicol, or herbimycin A per ml for 1 h and then stimulated with CpG ODN or E. coli DNA (10 μg/ml) in the presence or absence of the inhibitor for 12 h. The controls were incubated with medium alone or non-CpG ODN and CT DNA (10 μg/ml). IL-12 levels in the culture supernatants were determined by ELISA. Each value is the mean of triplicate results. Error bars indicate the standard deviations. ∗, P < 0.05 in a comparison of stimulated cells with and without the inhibitors.

FIG. 2

Time course for IL-6, IL-12, and TNF-α production induced by CpG ODN and inhibited by geldanamycin. J774 cells were pretreated with 0.1 μg of geldanamycin (GA) per ml for 1 h and then stimulated with 10 μg of CpG ODN per ml in the presence or absence of geldanamycin. Culture supernatants were collected at the indicated time intervals and examined for their levels of IL-6, IL-12, and TNF-α. Each value is the mean of triplicate results. Error bars indicate the standard deviations. ∗, P < 0.05 in a comparison of stimulated cells with and without the inhibitors.

FIG. 3

Inhibition of nitrite production from J774 cells by Hsp90 inhibitors. J774 cells were treated as described in the legend to Fig. 1. Culture supernatants were collected at 48 h and examined for their levels of nitrite by the Griess method. Geldanamycin (GA), radicicol, and herbimycin A (HA) inhibited nitrite production in J774 cells in a dose-dependent fashion. Each value is a mean of triplicate results. Error bars indicate the standard deviations. ∗, P < 0.05 in a comparison of stimulated cells with and without the inhibitors.

FIG. 4

Effects of geldanamycin and radicicol on IL-12 production in murine spleen cells treated with stimulatory DNA and S. aureus Cowan strain 1 (SAC). The spleen cells were pretreated with 0.1 μg of geldanamycin (GA) and radicicol per ml for 1 h, followed by stimulation with 10 μg of CpG DNA or E. coli DNA per ml or a 0.1% concentration of S. aureus Cowan strain 1 cells for 12 h. Each value is the mean of triplicate results. Error bars indicate the standard deviations. ∗, P < 0.05 in a comparison of stimulated cells with and without the inhibitors.

FIG. 5

Effects of geldanamycin on IL-12 and NO production by J774 cells. J774 cells were pretreated with 0.1 μg of geldanamycin (GA) per ml for 1 h and then stimulated with 10 μg of CpG ODN per ml in the presence or absence of the inhibitor for 12 h. Each value is the mean of triplicate results. ∗, P < 0.05 in a comparison of stimulated cells with and without the inhibitors. SAC, S. aureus Cowan strain 1.

FIG. 6

DNA-mediated activation of the MAP kinases ERK1 and -2 and their inhibition by geldanamycin in murine macrophages. J774 cells were pretreated with or without 1 μg of geldanamycin per ml for 1 h and then stimulated with 10 μg of CpG ODN (CpG) or E. coli DNA (EC) per ml or 1 μg of LPS per ml in the presence or absence of geldanamycin for 30 min. Cell lysates were analyzed for the presence of phosphorylated ERK1 and -2 by immunoblotting. Membranes were exposed using ECL, the images were scanned, and optical densities were determined. Values are presented as the fold increases in optical density against that of a medium control without geldanamycin treatment (Med). The data are representative of four separate experiments.

FIG. 7

Effects of geldanamycin on CpG ODN internalization. J774 cells were pretreated with or without 1 μg of geldanamycin per ml for 1 h, followed by incubation with 10 μg of FITC-labeled CpG ODN per ml at 37°C for 30 min. Cells were washed and examined under a confocal microscope. Note the similarity of uptake and peripheral distribution patterns of labeled CpG ODN in the cells with (B) or without (A) geldanamycin pretreatment. The data shown are from a representative experiment that was repeated with identical results.