Heat shock protein 60: specific binding of lipopolysaccharide.
ABSTRACT Human heat shock protein 60 (HSP60) has been shown to bind to the surface of innate immune cells and to elicit a proinflammatory response. In this study we demonstrate that the macrophage stimulatory property of recombinant human HSP60 is tightly linked to the HSP60 molecule and is lost after protease treatment. However, inhibition of macrophage stimulation was reached by the LPS-binding peptide magainin II amide. Indeed, HSP60 specifically bound [(3)H]LPS. [(3)H]LPS binding to HSP60 was saturable and competable by the unlabeled ligand. To identify the epitope region of the HSP60 molecule responsible for specific LPS binding, we analyzed the effect of several anti-HSP60 mAbs on HSP60-induced production of inflammatory mediators from macrophages. We identified only one mAb, clone 4B9/89, which blocked the macrophage stimulatory activity of the chaperone. The epitope specificity of this mAb points to the region aa 335-366 of HSP60. Clone 4B9/89 also strongly inhibited [(3)H]LPS binding to HSP60. A more detailed analysis was performed by screening with selected overlapping 20-mer peptides of the HSP60 sequence, covering the region aa 331-380. Only one peptide blocked LPS binding to HSP60, thereby restricting the potential LPS-binding region to aa 351-370 of HSP60. Finally, analysis of selected 15-mer peptides and a 13-mer peptide of the HSP60 sequence revealed that most of the LPS-binding region was accounted for by aa 354-365 of HSP60, with the motif LKGK being critical for binding. Our studies identified a defined region of HSP60 involved in LPS binding, thereby implicating a physiological role of human HSP60 as LPS-binding protein.
Article: Pneumococcal capsular polysaccharide is immunogenic when present on the surface of macrophages and dendritic cells: TLR4 signaling induced by a conjugate vaccine or by lipopolysaccharide is conducive.[show abstract] [hide abstract]
ABSTRACT: Previously, we reported that a peptide, p458, from the sequence of the mammalian 60-kDa heat shock protein (hsp60) molecule can serve as a carrier in conjugate vaccines with capsular polysaccharide (CPS) molecules of various bacteria. These conjugate vaccines were effective injected in PBS without added adjuvants. We now report that p458 conjugated to pneumococcal CPS type 4 (PS4) manifests innate adjuvant effects: it stimulated mouse macrophages to secrete IL-12 and induced the late appearance of PS4 on the macrophage surface in a TLR4-dependent manner; PS4 alone or conjugated to other carriers did not stimulate macrophages in vitro. The injection of macrophages manifesting PS4 on the surface into mice induced long-term resistance to lethal Streptococcus pneumoniae challenge. The TLR4 ligand LPS could also induce the late appearance on the surface of unconjugated PS4 and resistance to challenge in injected mice. Resistance was not induced by macrophages containing only internalized PS4 or by pulsed macrophages that had been lysed. Glutaraldehyde-fixed macrophages pulsed with PS4 did induce resistance to lethal challenge. Moreover, bone marrow-derived dendritic cells activated by LPS and pulsed with unconjugated CPS were also effective in inducing resistance to lethal challenge. Resistance induced by the PS4-pulsed bone marrow-derived dendritic cell was specific for pneumococcal CPS serotypes (type 3 or type 4) and was associated with the induction of CPS-specific IgG and IgM Abs.The Journal of Immunology 03/2008; 180(4):2409-18. · 5.79 Impact Factor
Article: Polymyxin B inadequately quenches the effects of contaminating lipopolysaccharide on murine dendritic cells.[show abstract] [hide abstract]
ABSTRACT: Dendritic cell (DC) activation is commonly used as a measure of the immunomodulatory potential of candidate exogenous and endogenous molecules. Residual lipopolysaccharide (LPS) contamination is a recurring theme and the potency of LPS is not always fully appreciated. To address this, polymyxin B (PmB) is often used to neutralise contaminating LPS. However, the limited capacity of this antibiotic to successfully block these effects is neglected. Therefore, this study aimed to determine the minimum LPS concentration required to induce murine bone marrow-derived dendritic cell (BMDC) maturation and cytokine secretion and to assess the ability of PmB to inhibit these processes. LPS concentrations as low as 10 pg/ml and 20 pg/ml induced secretion of interleukin (IL)-6 and tumor necrosis factor (TNF)-α respectively, while a concentration of 50 pg/ml promoted secretion of IL-12p40. A much higher threshold exists for IL-12p70 as an LPS concentration of 500 pg/ml was required to induce secretion of this cytokine. The efficacy of PmB varied substantially for different cytokines but this antibiotic was particularly limited in its ability to inhibit LPS-induced secretion of IL-6 and TNF-α. Furthermore, an LPS concentration of 50 pg/ml was sufficient to promote DC expression of costimulatory molecules and PmB was limited in its capacity to reverse this process when LPS concentrations of greater than 20 ng/ml were used. There is a common perception that LPS is heat resistant. However, heat treatment attenuated the ability of low concentrations of LPS to induce secretion of IL-6 and IL-12p40 by BMDCs, thus suggesting that heat-inactivation of protein preparations is also an ineffective control for discounting potential LPS contamination. Finally, LPS concentrations of less than 10 pg/ml were incapable of promoting secretion of IL-6 independently but could synergise with heat-labile enterotoxin (LT) to promote IL-6, indicating that reducing contaminating endotoxin concentrations to low pg/ml concentrations is essential to avoid misleading conclusions regarding candidate immunomodulators.PLoS ONE 01/2012; 7(5):e37261. · 4.09 Impact Factor
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ABSTRACT: Damage-associated molecular patterns (DAMPs) include endogenous intracellular molecules released by activated or necrotic cells and extracellular matrix (ECM) molecules that are upregulated upon injury or degraded following tissue damage. DAMPs are vital danger signals that alert our immune system to tissue damage upon both infectious and sterile insult. DAMP activation of Toll-like receptors (TLRs) induces inflammatory gene expression to mediate tissue repair. However, DAMPs have also been implicated in diseases where excessive inflammation plays a key role in pathogenesis, including rheumatoid arthritis (RA), cancer, and atherosclerosis. TLR activation by DAMPs may initiate positive feedback loops where increasing tissue damage perpetuates pro-inflammatory responses leading to chronic inflammation. Here we explore the current knowledge about distinct signalling cascades resulting from self TLR activation. We also discuss the involvement of endogenous TLR activators in disease and highlight how specifically targeting DAMPs may yield therapies that do not globally suppress the immune system.Mediators of Inflammation 01/2010; 2010. · 3.26 Impact Factor