In order to examine the ability of Limulus antilipopolysaccharide factor (LALF) to bind lipopolysaccharide (LPS), we purified LALF to homogeneity from Limulus amoebocyte lysate and coupled it covalently to agarose beads. LALF-coupled beads captured more tritiated LPS from rough and smooth strains of gram-negative bacteria than did control human serum albumin-coupled beads. Unlabeled homologous and heterologous LPS competed for the binding of 3H-LPS to LALF-coupled beads. LALF bound LPS in a dose-dependent manner as assessed by the precipitation of LPS-LALF complexes with 50% saturated ammonium sulfate. We also studied the ability of LALF to neutralize LPS. LPS preincubated with LALF was less mitogenic for murine splenocytes, was less pyrogenic in the rabbit fever assay, was less lethal in mice which had been sensitized to LPS with actinomycin D, and induced less fever, neutropenia, and pulmonary hypertension when infused into sheep. Our findings extend prior studies which suggested that LALF binds to and neutralizes LPS from multiple strains of gram-negative bacteria.

Lipopolysaccharides (LPSs) extracted from gram-negative bacteria are much less active when bound to serum lipoproteins. We present evidence here that the binding of radiolabeled LPS extracted from Escherichia coli 0113 and Salmonella typhimurium to lipoproteins in rabbit serum is increased 8 to 24 h after a single intravenous injection of homologous or heterologous LPS. Supernatants of activated macrophages containing interleukin-1 also stimulate increased binding. The isolated product of this binding does not induce the production of interleukin-1 by macrophages in vivo or in vitro and is unable itself to stimulate increased binding of LPS to lipoprotein. Normal rabbit sera spiked with lipoprotein fractions prepared from tolerant but not normal rabbit sera bind increased amounts of LPS. These data suggest that there may exist a self-regulated mechanism for decreasing the toxicity of LPS and the production of LPS-induced interleukin-1; this mechanism is controlled by a macrophage factor and functions through altering the binding of LPS to certain serum lipoproteins.The biological activities of bacterial lipopolysaccharide (LPS) have been studied for decades. Much of this information stems from experiments performed with extracted LPS dissolved in aqueous solutions. Recently, however, it has been shown that LPS binds to high-density lipoproteins in serum and plasma (39,40) and that the resulting LPSlipoprotein complex (LPS-LP) is markedly less active than unbound LPS in many systems studied including LPSinduced neutropenia (39), thrombocytopenia (40), complement activation (39), fever (24, 39), rate of development of hypotension (19), and death in adrenalectomized mice (39). As the LPS structure remains intact within these complexes (23,39), the loss of activity presumably occurs by a masking of the toxic lipid A moiety of the molecule by the lipoprotein particle.There have been several studies suggesting that the transformation of LPS to LPS-LP can be modulated by serum elements that are induced by injections of LPS. Tobias et al. have reported that the binding of radiolabeled LPS from rough mutant Salmonella minnesota Re 595 to lipoprotein is slowed in "acute-phase" rabbit (37) and human (34) sera.

We have previously described an assay to quantify the serum neutralization of bacterial lipopolysaccharide which is based on a spectrophotometric Limulus amoebocyte lysate test (T.

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