Helicobacter pylori Heat Shock Protein 60 Mediates Interleukin-6 Production by Macrophages via a Toll-like Receptor (TLR)-2-, TLR-4-, and Myeloid Differentiation Factor 88-independent Mechanism

Abstract

Helicobacter pylori has been reported to induce interleukin-6 (IL-6) production in monocytes/macrophages and in chronically inflamed gastric tissues. The mechanism by which H. pylori induces IL-6 production in macrophages, however, has not been investigated. To identify the H. pylori factor responsible for this activity, we fractionated soluble proteins from H. pylori strain 26695 by ion exchange and size exclusion chromatography and screened the fractions for IL-6-inducing activity on RAW 264.7 macrophages. A single protein was purified and identified by mass spectrometry as H. pylori heat shock protein 60 (HSP60). Consistent with the observed IL-6-inducing activity of H. pylori HSP60, soluble protein extracts of H. pylori 26695 and SS1 strains that were depleted of this protein by affinity chromatography had dramatically reduced IL-6-inducing activities. The immunopurified HSP60 stimulated IL-6 production in macrophages. When stimulated with H. pylori HSP60 or intact bacteria, peritoneal macrophages from mice deficient in Toll-like receptor (TLR)-2, TLR-4, TLR-2/TLR-4, and myeloid differentiation factor 88 produced the same amount of IL-6 than macrophages from wildtype mice, demonstrating the independence of H. pylori HSP60 responses from these signaling molecules. H. pylori HSP60-induced IL-6 mRNA expression, and NF-B activation in RAW 264.7 cells was abrogated in the presence of MG-132, a proteasome inhibitor. In contrast, inhibitors of protein kinase A or C, mitogen-activated protein kinase kinase, and phosphoinositide 3-kinase had no effect on IL-6 mRNA levels. This study demonstrates the induction of innate immune responses by H. pylori HSP60, thereby implicating this highly conserved protein in the pathophysiology of chronic gastritis.

Hsp60C is required in follicle as well as germline cells during oogenesis in Drosophila melanogaster

“…In our earlier study (Sarkar and Lakhotia, 2005) on the effects of the Hsp60C 1 mutation on tracheal morphogenesis and spermatogenesis, we raised the possibility that the Hsp60C itself may also function as a signaling molecule, especially because such a function is known in other systems (Gobert et al, 2004;Sarkar et al, 2006). In addition to the possible signaling activity, the Hsp60C also appears to have essential roles in organization of the various Factin, tubulin, and other cytoskeletal elements during oogenesis.…”

“…In recent years, however, they have also been found to be involved in a variety of nonchaperonic functions such as cell signaling, epithelial cell remodeling/ motility, apoptosis, and so on (Maguire et al, 2002;Gobert et al, 2004;Zhang et al, 2004;Sarkar et al, 2006;Arya et al, 2007). Besides being present in the organelles (mitochondria or chloroplasts), the Hsp60 proteins are now known to be present in cytosol, on cell membrane or even outside the cell (Maguire et al, 2002;Sarkar et al, 2006).…”

Unfolding the relationship between secreted molecular chaperones and macrophage activation states

“…In contrast, the same proteins from Escherichia coli or Helicobacter pylori are not dependent on CD14/ TLR2/4 for monocyte activation (reviewed by Maguire et al 2002). Indeed, the H. pylori Hsp60 activates macrophages in a TLR2/4 and myeloid differentiation factor 88 (Myd-88)-independent manner (Gobert et al 2004). Furthermore, one of the three Hsp60 proteins of Rhizobium leguminosarum fails to activate human monocytes (Lewthwaite et al 2002a).…”

Endocytosis of Mycobacterium tuberculosis Heat Shock Protein 60 Is Required to Induce Interleukin-10 Production in Macrophages*

“…Several microbial heat shock proteins (HSPs) have been demonstrated to modulate the macrophage effector function by inducing various cytokines in both TLR-dependent and -independent ways in addition to their classical chaperonin functions (22)(23)(24)(25)(26)(27). Among the M. tuberculosis heat shock proteins, HSP65 and HSP70 were found to induce pro-inflammatory cytokines in human dermal endothelial cells in TLR2-and TLR4-dependent ways (28).…”