Bacterial heat shock proteins (hsp) have been shown to be important immunogens stimulating both T cells and B cells. However, little is known concerning the direct interactions between hsp and macrophages. In this study, we demonstrated that treatment of macrophage cultures with purified bacterial hsp, including Legionelia pneumophila hsp6O, Escherichia coli GroEL, Mycobacterium tuberculosis hsp7O, Mycobacterium leprae hsp65, and Mycobacterium bovis BCG hsp65, increased the steady-state levels of cytokine mRNA for interleukin-la (IL-la), IL-10, IL-6, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor as well as supernatant IL-1 secretion. This effect was shown not to be due to contamination of the hsp preparations with bacterial lipopolysaccharide. However, not all hsp induced cytokines; M. tuberculosis hsplO showed minimal activity in our study. These results suggest that bacterial hsp might modulate immunity by rapidly and directly increasing cytokine production in macrophages.
SUMMARYHeat-shock proteins (hsp) are chaperon molecules important in protein folding and assembly. Furthermore, they may have functions in immunoregulatory processes, like T-cell stimulation and antigen presentation, which are not yet fully understood. It has been shown that several hsp of various species and family derivations modulate functions in macrophage immunity by directly increasing cytokine production. In the present study we showed that the 60 000 MW hsp of Legionella pneumophila (Lp-hsp 60) increased cellular steady-state levels of interleukin-1b (IL-1b) mRNA measured by quantitative reverse transcription-polymerase chain reaction and Northern blotting as well as IL-1 secretion, when added to cultures of thioglycollate-elicited mouse peritoneal macrophages in vitro. The level of mRNA increased in a dose-dependent manner with a minimum effective concentration of 0 . 5 m g/ml and peaked 3 hr after stimulation. Lp-hsp 60-coated latex beads also increased IL-1b mRNA levels in the presence of cytochalasin D, which inhibits bead uptake but permits binding, indicating that binding to the macrophage surface was sufficient for induction. Accumulation of IL-1b mRNA was completely blocked by pretreatment with the protein kinase C (PKC) inhibitor, H7, but not decreased by prior treatment with cycloheximide. The cell lysates of macrophages stimulated with Lp-hsp 60 showed an increased PKC activity measured by phosphorylation of PKC pseudosubstrate. The IL-1 bioactivity in culture supernatants after 24 hr of stimulation with Lp-hsp 60 was increased in a dose-dependent manner but at hsp concentrations in excess of those needed to increase mRNA. Thus, the present study demonstrates that Lp-hsp 60 rapidly increases the steady-state level of IL-1b mRNA, possibly through a cell surface receptor system involving a PKC-dependent signalling pathway.
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