The effect of humic acid on the adhesibility of neutrophils.
Humic acid (HA), a fluorescent allomelanin, has been implicated as an etiological agent of Blackfoot disease (BFD), a peripheral vascular disease prevailing in the southwest of Taiwan. Clinical and pathological studies reveal that it is similar to atherosclerosis. In this report, the effect of HA on human neutrophils is studied because prolonged and enhanced activation of neutrophils adhered on endothelium may damage the endothelium and initiate the process of thrombosis and vasculitis.


METHODS
Neutrophils, treated with various concentrations of HA, were added to culture plates, cultured human umbilical vein endothelial cells (HUVECs), or human umbilical vein endothelium tissue culture for 15 or 30 min. The adhesion of neutrophils was measured qualitatively and quantitatively. The mechanism of neutrophil activation was studied with free radical production and various kinase measurements and their activities' assays.


RESULTS
HA was shown to enhance, in a dose-dependent manner, the adhesion of neutrophils on the culture plates, cultured human umbilical vein endothelial cells, and human umbilical vein endothelium tissue culture. The adhesion-enhancing ability of HA is elicited through activation of ERK, P38 mitogen-activated kinase (P38MAPK), and phosphoinositide 3 kinase (PI3K) in neutrophils. HA also induces the NF-kappaB activation in neutrophils.


CONCLUSIONS
HA treatment markedly enhanced adhesion and superoxide radical production of neutrophils, the characteristics of activated neutrophils; and all these stimulation effects were blocked by several kinase inhibitors, reflecting the involvement of the ERK, P38MAPK, and PI3K on the activation of neutrophils. The induction of NF-kappaB implied that the consequence of neutrophil activation by HA were similar to other stimulants. The prolonged neutrophil activation will further damage endothelium cell and cause thrombosis, vaculitis, as well as arteriosclerosis. This may partially explain why HA consumption will cause BFD.
Chen
Chong-Hua
CH
Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC.
Liu
Jun-Jen
JJ
Lu
Fung-Jou
FJ
Yang
Mei-Ling
ML
Lee
Yashang
Y
Huang
Tien-Shang
TS
D002448:Cell Adhesion / Q000187:drug effects; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006812:Humic Substances / Q000633:toxicity*; D066298:In Vitro Techniques; D020935:MAP Kinase Signaling System / Q000187:drug effects; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects* / Q000502:physiology; D016491:Peripheral Vascular Diseases / Q000139:chemically induced; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014874:Water Pollutants, Chemical / Q000633:toxicity; D048051:p38 Mitogen-Activated Protein Kinases
D004791:Enzyme Inhibitors; D006812:Humic Substances; D016328:NF-kappa B; D014874:Water Pollutants, Chemical; D013481:Superoxides; D019869:Phosphatidylinositol 3-Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases