[Role of cell-surface nucleolin in lipopolysaccharide-stimulated expression and secretion of TNF-alpha and IL-1beta].
OBJECTIVE
To explore the role of cell-surface nucleolin in lipopolysaccharide (LPS)-stimulated expression and secretion of TNF-alpha and IL-1beta in human THP-1 monocytes.


METHODS
Immuno-fluorescence assay and Western blot were used to identify the expression of nucleolin on the surface of THP-1 monocytes. Inactivation of nucleolin was induced by anti-nucleolin monoclonal antibody blockage, and the expression and secretion of TNF-alpha and IL-1beta were observed by using reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immuno-sorbent assay (ELISA)respectively in LPS-mediated human THP-1 monocyte inflammatory model.


RESULTS
Immuno-fluorescence showed that nucleolin was localized on the cell surface of THP-1 monocytes as indicated by dotted red fluorescence. Western blot assay indicated that nucleolin existed in the cell membrane fractions. RT-PCR assay showed that the expressions of TNF-alpha and IL-1beta mRNA significantly increased at 2 h and 3 h after the treatment with 1000 microg/L LPS. After 1 h pretreatment with anti-nucleolin antibody, the levels of TNF-alpha and IL-1beta mRNA decreased compared with an anti-nucleolin antibody untreated group and an irrelevant IgG+LPS group (P<0.05). ELISA assay showed that the pretreatment with anti-nucleolin antibody inhibited significantly the secretion of LPS-induced levels of TNF-alpha and IL-1beta after 4, 12 and 24 h treatment with 1000 microg/L LPS.


CONCLUSIONS
Nucleolin expresses on the cell surface of THP-1 monocytes and involves in the LPS-mediated expression and secretion of TNF-alpha and IL-1beta.
Fang
Li
L
Laboratory of Shock, Department of Pathophysiology, Xiangya School of Medicine, Central South University, and Department of Cardiology, Xiangya Hospital, Changsha 410008, China.
Wang
Kang-kai
KK
Jiang
Lei
L
Jiang
Bi-mei
BM
Wei
Xing
X
Song
Lan
L
Deng
Gong-hua
GH
Xiao
Xian-zhong
XZ
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D053583:Interleukin-1beta / Q000096:biosynthesis* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D009000:Monocytes / Q000166:cytology / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism / Q000502:physiology*; D016601:RNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000378:metabolism; D000097796:Nucleolin
D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D014409:Tumor Necrosis Factor-alpha; D000097796:Nucleolin