Lipopolysaccharide binding protein expression in primary human hepatocytes and HepG2 hepatoma cells.

B.J Grube,C.G Cochane,R.D Ye,C.E Green,M.E Mcphail,R.J Ulevitch,P.S Tobias

doi:10.1016/s0021-9258(17)37218-6

Abstract

Lipopolysaccharide (LPS)-binding protein (LBP) is a normal plasma protein and an acute phase reactant important for host responses to Gram-negative bacteria and LPS. LBP forms high affinity complexes with LPS which bind to CD14, a monocyte surface protein, to initiate the release of inflammatory mediators. We found that human primary hepatocytes synthesize LBP and that the synthesis is up-regulated by interleukin (IL)-6. To examine this phenomenon in more detail, we evaluated the capacity of IL-6, IL-1, and tumor necrosis factor to induce LBP synthesis in HepG2 cells in the presence or absence of dexamethasone. IL-6 induced LBP synthesis. Dexamethasone, IL-1, and tumor necrosis factor had a synergistic effect when combined with IL-6, but demonstrated minimal effect independently. LBP biosynthesis was evaluated by immunoprecipitation of 35S-labeled LBP from HepG2 supernatants, measurement of steady-state LBP mRNA levels, and analysis of LBP-dependent LPS binding to CD14 positive cells. An 35S-labeled, 60-kDa protein was immunoprecipitated with anti-LBP antibody from IL-6-stimulated HepG2 cell supernatants. Northern blot analysis of cellular RNA revealed an increase in LBP mRNA in IL-6-stimulated cells. CD14 expressing cells bound fluoresceinated LPS in the presence of supernatants from HepG2 cells treated with IL-6. These data provide the first information about specific cytokine and dexamethasone regulation of LBP expression in HepG2 cells. LBP behaves like a Type 1 acute phase protein.

Highlights

Lipopolysaccharide (LPS)-binding protein (LBP)is a [1].A variety of cytokines are released for host defense from normal plasma protein and an acute phase reactant imc-ells of monocytic origin in response to challengbey LPS [2]
Other mechanisms for LPS activation of monocytes are postulated, only LBP-dependent LPS binding to CD14 results in a lo3 greater production of TNF-a [11]
LBP Expression in Primary HumanHepatocytes and HepG2 Hepatoma Cells-In order to determine if LBP is synthesized were coated with gt-a-rhLBP (75 pg/ml) in carbonate-bicarbonate bubffyerhuman liver cells, the supernatants of primary cultured overnight a t 4 "C, washed with phosphate-buffered salin+e 0.1%Tween human hepatocytes were assayed for the secretion of LBP as a, blocked for 1h at 37 "C with 10% milk powder in phos- function of time after stimulation and compared with the sephate-buffered saline

Summary

Human LBP Expression

Primary human hepatocytes were cultuarseddescribed under "Experimental Procedures." Cells were traeat teimd e 0,12 h, and 39 h with fresh stimulatory media. The Scripps Research Institute (TSRI), La CJoAlIlaw,ere cultured (Calbiochem 112251) and HepG2 supernatants, followed by rb-a-hFIB to confluence in minimal essential media (MEM, Flow, 11-10022, Ir- (Calbiochem 341552) or rb-a-hAGP(AccurateChemical & Scientific vine, CA) with 10% FCS heat-inactivated at 56 "C for 30 min. The effect of single or combined mediators on LBP secretion i n HepG2 cells was evaluated inSM -c DEX plus 1)no cytokine, 2) IL-1,3)TNF, 4). Bovine serum albumin an0d.05%NaN3) were added tthoe concentrated cytokine-stimulated HepG2 supernatant(s5 pl) diluted ibnuffer (100pl of Hanks' as for cells), and incubated withFITC-Re595 LPS For metabolic labeling,the medium was changedt o SMCYS-meatt- supernatants diluted in buffer were incubated with either polyclonal.

RESULTS

LP LP LP LPLP

DISCUSSION

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