[Heat-shock protein 70 may be a putative endogenous ligand of Toll-like receptor-4 of human monocytes].
OBJECTIVE
To explore the relation between human heat shock protein 70 (HSP70) and Toll-like receptor-4 (TLR4) in human monocytes.


METHODS
Periphery blood mononuclear cells were isolated from the samples of healthy blood donors' whole blood and monocytes were prepared and cultured. HSP70 of the final concentrations of 2.5 microg/ml, 5.0 microg/ml, 7.5 microg/ml, and 10 microg/ml respectively was added; 6 hours later the concentration of TNF-alpha in the supernatant was detected. Another monocytes were cultured and HSP70 of the final concentration of 5.0 microg/ml was added and the concentrations of NF-kappaB were detected 0, 30, 60, and 120 minutes later respectively. TLR4 blocker of the final concentrations of 5 microg/ml, 20 microg/ml, and 30 microg/ml respectively was added into another culture for 30 minutes and 5.0 microg/ml HSP70 was added, then immunochemistry was used to detect the concentration of NF-kappaB 120 minutes after ELISA was used to detect the concentration of and TNF-alpha 8 hours later. In order to examine the influence of HSP70 on the TLR4 in the cytomembrane of monocytes, HSP70 of the final concentration of 5.0 microg/ml was added into the culture of monocytes for 0, 30, 60, and 120 minutes respectively then flow cytometry was used to detect the mean fluorescence intensity (MFI) of TLR4.


RESULTS
HSP70 stimulation increased the TNF-a concentration in the supernatant dose-dependently. The percentages of NF-kappaB positive monocytes were 38 +/- 6, 67 +/- 12, and 54 +/- 12 30 min, 60 min, 120 min after HSP70 stimulation, all significantly higher than that at the beginning of experiment (17 +/- 6, P < 0.05, P < 0.01, and P < 0.01). The percentages of NF-kappaB positive monocytes were 39% +/- 4%, 32% +/- 6%, and 28% +/- 6% 120 minutes after anti-TLR4 mAb stimulation, all significantly lower than that of the control group (67% +/- 12%, all P < 0.05). TLR4 blocker of different concentrations significantly inhibited the TNF-alpha secretion by the monocytes (all P < 0.05). The MFI of TLR4 in the cytomembrane of monocyte was significantly down-regulated 60 minutes, especially 120 minutes, after the HSP70 stimulation in comparison with that before the stimulation (P < 0.05 and P < 0.01).


CONCLUSIONS
TLR4 appears to be involved in HSP70-mediated activation of innate immunity.
Chen
Xin-Zhong
XZ
Department of Cardiovascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Sun
Zong-Quan
ZQ
DU
Xin-Ling
XL
Liu
Yi
Y
Wu
Long
L
Liu
Chao
C
Chen
Jia-Jun
JJ
D002478:Cells, Cultured; D018840:HSP70 Heat-Shock Proteins / Q000494:pharmacology / Q000502:physiology*; D006801:Humans; D009000:Monocytes / Q000378:metabolism*; D016328:NF-kappa B / Q000032:analysis; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis*
D018840:HSP70 Heat-Shock Proteins; D016328:NF-kappa B; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha