Stimulation of macrophages and neutrophils by complexes of lipopolysaccharide and soluble CD14.

Sensitive responses of monocytes, macrophages, and neutrophils to bacterial LPS require membrane-bound CD14 (mCD14) and a plasma protein called LPS-binding protein (LBP). Cells lacking mCD14 respond to complexes of LPS and soluble CD14 (sCD14); these responses do not require LBP. To determine whether LBP is necessary for responses of mCD14-bearing cells to LPS, we measured responses of macrophages and neutrophils to complexes of LPS and sCD14 formed in the absence of LBP. We found that the amount of LPS needed to induce adhesive responses of neutrophils or cytokine production by macrophages was the same whether LPS was added with LBP or as LPS-sCD14 complexes, and was >100-fold less than when LPS was added alone. This result supports the view that LBP transfers LPS to CD14, but is not directly involved in responses of CD14-bearing cells to LPS. Responses of neutrophils to LPS-sCD14 complexes could be inhibited partially by blocking mCD14, suggesting that LPS may move rapidly from sCD14 to mCD14. Additionally, we found that responses of neutrophils to LBP and smooth LPS were made 30 to 100 times more sensitive when sCD14 was added. Our findings show that LBP is not necessary for the activation of CD14-bearing cells with LPS, and suggest that LPS-sCD14 complexes are an important intermediate in the inflammatory responses of leukocytes to LPS.

Identification of a lipopolysaccharide binding domain in CD14 between amino acids 57 and 64.

CD14 is a 55-kDa glycoprotein which binds lipopolysaccharide (LPS) and enables LPS-dependent responses in a variety of cells. Recent limited proteolysis studies have implicated a region in CD14 between amino acids 57 and 64 as being involved in LPS interaction. To specifically assess the importance of this region with respect to LPS binding, we constructed a mutant sCD14 (sCD14 delta 57-64) lacking amino acids 57-64. sCD14 delta 57-64 was isolated from the serum-free conditioned medium of this cell line, and, in all assays, the purified protein failed to recognize LPS or enable LPS-dependent responses in cells. We also demonstrated that the region between amino acids 57 and 64 is required for binding of a neutralizing CD14 mAb, MEM-18. Native polyacrylamide gel electrophoresis assays were used to demonstrate that MEM-18 and LPS compete for the same binding site on CD14. These data strongly suggest that the region spanning amino acids 57-64 binds LPS and that formation of sCD14.LPS complex is required in order for sCD14-mediated responses to occur.

Soluble CD14 truncated at amino acid 152 binds lipopolysaccharide (LPS) and enables cellular response to LPS.

CD14 is a 55-kDa glycoprotein which binds lipopolysaccharide (LPS) and enables LPS-dependent responses in a variety of cells. In order to identify the domains in CD14 required for function, we deleted increasing amounts of CD14 from the C terminus. Truncated CD14 cDNA sequences were transfected into COS-7 cells and serum-free conditioned medium was analyzed for mutant CD14 expression and bioactivity. Mutant CD14s containing as few as 152 amino acids were found to have activity equivalent to full-length sCD14. To further characterize the mutant CD14, we constructed a stable Chinese hamster ovary cell line expressing sCD14(1-152) and purified the protein to homogeneity. sCD14(1-152) bound radioactive LPS, enabled U373 cells to synthesize interleukin 6 in response to LPS, and enabled human neutrophils to respond to smooth LPS. In all of these assays, the behavior of sCD14(1-152) was quantitatively similar to full-length sCD14. We also found that two neutralizing anti-CD14 antibodies (3C10 and MEM-18) bound and neutralized sCD14(1-152). We conclude from these experiments that the N-terminal 152 amino acids of CD14 are sufficient to bind LPS and confer essentially wild-type bioactivity in vitro.

Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14.

CD14 is a 55-kD protein found as a glycosylphosphatidylinositol (GPI)-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, and as a soluble protein in the blood. Both forms of CD14 participate in the serum-dependent responses of cells to bacterial lipopolysaccharide (LPS). While CD14 has been described as a receptor for complexes of LPS with LPS-binding protein (LBP), there has been no direct evidence showing whether a ternary complex of LPS, LBP, and CD14 is formed, or whether CD14 binds LPS directly. Using nondenaturing polyacrylamide gel electrophoresis (native PAGE), we show that recombinant soluble CD14 (rsCD14) binds LPS in the absence of LBP or other proteins. Binding of LPS to CD14 is stable and of low stoichiometry (one or two molecules of LPS per rsCD14). Recombinant LBP (rLBP) does not form detectable ternary complexes with rsCD14 and LPS, but it does accelerate the binding of LPS to rsCD14. rLBP facilitates the interaction of LPS with rsCD14 at substoichiometric concentrations, suggesting that LBP functions catalytically, as a lipid transfer protein. Complexes of LPS and rsCD14 formed in the absence of LBP or other serum proteins strongly stimulate integrin function on PMN and expression of E-selectin on endothelial cells, demonstrating that LBP is not necessary for CD14-dependent stimulation of cells. These results suggest that CD14 acts as a soluble and cell surface receptor for LPS, and that LBP may function primarily to accelerate the binding of LPS to CD14.

Cloning and characterization of a gene encoding extracellular metalloprotease from Streptomyces lividans.

The prt gene, encoding a protease (Prt) from Streptomyces lividans TK24, was cloned and sequenced. An S. lividans host with plasmid-borne prt secreted 200 micrograms/ml of a 22-kDa Prt into the culture medium. Prt is classified as a metalloprotease since its activity is significantly inhibited by 1,10-phenanthroline or EDTA. The region upstream from prt codes for an incomplete open reading frame (ORF) oriented opposite to prt. This ORF has a strong similarity to a gene family (lysR) whose members regulate the transcription of structural genes required for either biosynthesis or degradation.

Adult and child discrimination of CV syllables differing in voicing onset time.

The purpose of this experiment was to determine whether age-related differences would be observed for discrimination of synthesized, 5-formant consonant-vowel syllables that differed in voicing onset time (VOT) of the initial consonant. Just noticeable differences (JNDs) were measured relative to the end points of the stimulus continuum, using a "same"-"different," adaptive procedure with trial-by-trial feedback/reinforcement and "catch" trials, in which members of the stimulus pair were identical syllables. 6-8-year-old children required significantly longer VOTs for syllabic discrimination than did adults. Adults' JNDs relative to [pa] at the end of the stimulus continuum with longer VOTs were significantly larger than their JNDs relative to [ba], as would be expected on the basis of psychoacoustic considerations, but neither 6-8-nor 8-11-year-old children showed this trend. Performance on "catch" trials for both groups of children was significantly poorer than for adults. Outcomes corresponded to results of other investigations that have reported that children require more acoustic information than adults to achieve the same performance criterion.

Syllable identification by children and adults for two task conditions.

Both 6-year-old children and adults showed as good performance on a syllable identification task for a test condition having more trials per test run and a lower overall level of positive reinforcement as they did for a condition with fewer trials per test run and a higher, overall level of positive reinforcement. Differences between identification thresholds of children and adults are interpreted as reflecting age-related differences in speech processing rather than differences in responding to the test situation.