RECONSTITUTED HIGH-DENSITY-LIPOPROTEIN NEUTRALIZES GRAM-NEGATIVE BACTERIAL LIPOPOLYSACCHARIDES IN HUMAN WHOLE-BLOOD

We have tested hypotheses relating lipoprotein structure to function a s measured by the relative ability to neutralize endotoxin by comparin g natural human lipoproteins, a chemically defined form of reconstitut ed high-density lipoprotein (R-HDL), and a lipid emulsion (Intralipid) . The human whole-blood system was used as an in vitro model of lipopo lysaccharide (LPS) binding protein and CD14-dependent activation of cy tokine production. When lipoproteins were compared on the basis of pro tein content, R-HDL was most effective in reducing tumor necrosis fact or alpha (TNF-alpha) production followed in order by very low density lipoprotein, low-density lipoprotein, Intralipid, and natural HDL. How ever, when these particles were compared by protein, phospholipid chol esterol, or triglyceride content by stepwise linear regression analysi s, only phospholipid was correlated to effectiveness (r(2) = 0.873; P < 0.0001). Anti-CD14 monoclonal antibodies MY4 and 3C10 inhibited LPS binding protein and CD14-dependent activation of TNF-alpha production by LPS at LPS concentrations up to similar to-1.0 ng/ml. R-HDL (2 mg o f protein per ml) blocked TNF-alpha production by LPS From both smooth - and rough-type gram-negative bacteria at concentrations up to 100 ng of LPS per ml but had little effect on heat-killed gram-positive Stap hylococcus aureus and no effect on other LPS-independent stimuli teste d. These results support our hypothesis that LPS is neutralized by bin ding to phospholipid on the surface of R-HDL and demonstrate that R-HD L is a potent inhibitor of the induction of TNF-alpha by LPS from both rough- and smooth-form gram-negative bacteria in whole human blood.