CD14 and innate recognition of bacteria.
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Animals↗
Biomedical subjects
S D Wright
Publications and source records attributed to S D Wright.
Energetics of leukocyte integrin activation.
Cell adhesion mediated by leukocyte integrin CR3 (CD11b/CD18, alpha m beta 2) may be rapidly modulated without changes in receptor number, and transient changes in adhesivity are thought to be driven by reversible alteration of the affinity of CR3 for ligand. Here we measure the binding affinity of CR3 using purified active and inactive receptor and the ligand, C3bi, coupled to alkaline phosphatase. Immobilized, active CR3 bound saturably and with high affinity (12.5 +/- 4.7 nM). In contrast, inactive CR3 exhibited no measurable binding. High affinity binding could be restored by the addition of the activating anti-CR3 monoclonal antibody KIM-127 to inactive CR3. Since the affinity of KIM-127 for active and inactive receptor was identical, it cannot contribute the energy to convert a low affinity receptor into a high affinity receptor. Rather, KIM-127 appears to facilitate binding of C3bi by lowering the activation energy for the shift from an inactive to an active state. These results suggest that CR3-mediated binding and detachment of cells is not driven by a reversible change in affinity but by two mechanistically distinct processes, an energetically neutral activation step for binding and an energy-dependent step that reverses binding of ligand.
Antibodies, Monoclonal↗
Soluble CD14 acts as a shuttle in the neutralization of lipopolysaccharide (LPS) by LPS-binding protein and reconstituted high density lipoprotein.
We have recently shown that lipopolysaccharide (LPS)-binding protein (LBP) is a lipid transfer protein that catalyzes two distinct reactions: movement of bacterial LPS (endotoxin) from LPS micelles to soluble CD14 (sCD14) and movement of LPS from micelles to reconstituted high density lipoprotein (R-HDL) particles. Here we show that LBP facilitates a third lipid transfer reaction: movement of LPS from LPS-sCD14 complexes to R-HDL particles. This action of LBP is catalytic, with one molecule of LBP enabling the movement of multiple LPS molecules into R-HDL. LBP-catalyzed movement of LPS from LPS-sCD14 complexes to R-HDL neutralizes the capacity of LPS to stimulate polymorphonuclear leukocytes. Our findings show that LPS may be transferred to R-HDL either by the direct action of LBP or by a two-step reaction in which LPS is first transferred to sCD14 and subsequently to R-HDL. We have observed that the two-step pathway of LPS transfer to R-HDL is strongly favored over direct transfer. Neutralization of LPS by LBP and R-HDL was accelerated more than 30-fold by addition of sCD14. Several observations suggest that sCD14 accelerates this reaction by serving as a shuttle for LPS: addition of LBP and sCD14 to LPS micelles resulted in LPS-sCD14 complexes that could diffuse through a 100-kD cutoff filter; LPS-sCD14 complexes appeared transiently during movement of LPS to R-HDL facilitated by purified LBP; and sCD14 could facilitate transfer of LPS to R-HDL without becoming part of the final LPS-R-HDL complex. Complexes of LPS and sCD14 were formed transiently when LPS was incubated in plasma, suggesting that these complexes may play a role as intermediates in the neutralization of LPS under physiological conditions. These findings detail a new activity for sCD14 and suggest a novel mechanism for lipid transfer by LBP.
Acute-Phase Proteins↗
CD14: physical properties and identification of an exposed site that is protected by lipopolysaccharide.
Soluble CD14 (sCD14) is a 55-kDa serum protein that binds lipopolysaccharide (LPS) and mediates LPS-dependent responses in a variety of cells. Using recombinant sCD14 expressed in Chinese hamster ovary (CHO) cells, we examined the structural characteristics of sCD14 and sCD14.LPS complexes. The circular dichroism and fluorescence spectra of the sCD14 indicate that it contains substantial beta-sheet (40%) and a well-defined tertiary structure with the tryptophan residues located in environments with different degrees of hydrophobicity and solvent exposure. The spectra of the sCD14.LPS complex are identical within experimental error to the uncomplexed sCD14. Changes in surface accessibility upon LPS binding were examined using limited proteolysis with endoproteinase Asp-N. This analysis revealed that aspartic acid residues at amino acids 57, 59, and 65 are susceptible to cleavage by Asp-N, while the same residues are protected from proteolytic cleavage in the sCD14.LPS complex. These results suggest that a region including amino acids 57 to 64 is involved in LPS binding by sCD14.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
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.
Amino Acids↗
Does endotoxin stimulate cells by mimicking ceramide?
Recent studies indicate that tumor necrosis factor (TNF) and interleukin 1 (IL-1) stimulate cells via the intracellular messenger ceramide. Bacterial endotoxin (lipopolysaccharide; LPS) shows strong structural and functional resemblance to ceramide. Here, Samuel Wright and Richard Kolesnick review data suggesting that LPS may provoke cellular responses by mimicking the action of ceramide.
Acute-Phase Proteins↗
Video Respite: an innovative resource for family, professional caregivers, and persons with dementia.
The development of a new and promising resource for family, professional caregivers, and persons with dementia is detailed in this article. The concept, Video Respite, refers to the initial goal of creating videotapes specifically for persons with dementia to capture and maintain their attention, enabling caregivers to have respite time. Ten 20-53-minute videotapes are described along with their potential benefits and limitations. Early research findings reveal that most persons with Alzheimer's disease (AD) watch and participate with the tapes, caregivers use the time for respite, and staff of SCU's report the tapes to be calming for the residents.
Aged↗
Reversible inactivation of purified leukocyte integrin CR3 (CD11b/CD18, alpha m beta 2) by removal of divalent cations from a cryptic site.
Integrins exhibit reversible changes in their ability to bind ligands and these changes enable transient cell adhesion. We recently showed that leukocyte integrin CR3 (complement receptor type three, CD11b/CD18, alpha m beta 2) may be purified in a form that is either capable or incapable of binding soluble, monomeric ligand and that "inactive" CR3 may be rendered capable of binding ligand by addition of an anti-CR3 mAb known as KIM-127 (Cai and Wright, JBC. 270: 14358, 1995). Here, we demonstrate that active CR3 may be rendered inactive by treatment of immobilized receptor with EDTA. EDTA-treated CR3 failed to bind ligand even in the presence of mM Ca2+ and Mg2+, suggesting that EDTA-treatment caused a change in the receptor that is not readily reversed. EDTA-treated receptor did, however, bind ligand upon addition of KIM-127 plus Mg2+ with an affinity (17.8 +/- 4.5 nM) similar to untreated, active receptor (12.5 +/- 4.7 nM). EDTA-treated CR3 thus exhibits the properties of inactive CR3, in which the ligand binding site is cryptic but subject to exposure by KIM-127. A candidate for the cryptic ligand binding site is the I-domain, a Mg2+-binding region in the alpha chain of CR3. We found that monomeric C3bi binds directly to recombinant I-domain in a Mg(2+)-dependent fashion with an affinity of 300 +/- 113 nM. These results thus suggest that CR3 may be inactivated by removing tightly bound divalent cation from a cryptic site in CR3.
Antibodies, Monoclonal↗
Selenium status of white-tailed deer in southern Florida.
Samples of serum, liver, kidney, and heart were collected for selenium analysis from 174 white-tailed deer (Odocoileus virginianus) in southern Florida (USA), 1984 to 1988, to determine the selenium status of these animals. Deer were obtained from eight sites and classified by five age-class groups. For serum and the three tissues analyzed, selenium concentrations varied significantly (P < 0.001) among sites. Differences between years (P < 0.0004) were found for heart and kidney, age-class (P < 0.004) for kidney and season (P < 0.02) for liver. Low selenium concentrations were evident, in that 75% of all serum samples analyzed contained less than the critical concentration (< 0.06 ppm) by livestock standards, with 50% of serum samples less than 0.03 ppm, evidence of a severe deficiency. Likewise, tissue selenium concentrations (dry basis) were below critical livestock concentrations in 13% of the liver samples (< 0.25 ppm), 36% in kidney (< 3.0 ppm) and 19% in heart (< 0.15 ppm). Based on serum and tissue data, selenium dietary intake was low and may have been deficient for white-tailed deer in southern Florida.
Age Factors↗
LPS-dependent interaction of Mac-2-binding protein with immobilized CD14.
CD14 is a glycosylphosphatidyl-inositol (GPI)-linked, 55 kDa protein that binds bacterial lipopolysaccharide (LPS, endotoxin) and plays a key role in mediating cellular responses to this potent inflammatory stimulus. Binding of LPS to CD14 is facilitated by serum proteins such as LPS binding protein (LBP). To determine if there are additional plasma proteins that bind to CD14, plasma was passed over immobilized CD14 in the presence or absence of LPS, and retained proteins were eluted. This procedure isolated not only LBP but also a serum protein known as Mac-2-binding protein (Mac-2-BP), a 97 kDa species without a known function. Binding of both LBP and Mac-2-BP to CD14 required the simultaneous presence of LPS. Experiments with purified Mac-2-BP showed that this protein alone neither enabled responses of CD14-bearing cells to LPS nor blocked the ability of plasma to enable responses of CD14-bearing cells to LPS. However, Mac-2-BP did slow the neutralization of LPS mediated by plasma lipoprotein. These studies describe the first potential function for Mac-2-BP, and suggest that neutralization of LPS in plasma may be controlled by proteins in addition to LBP and CD14.
Acute-Phase Proteins↗
Lipopolysaccharide (LPS) binding protein catalyzes binding of LPS to lipoproteins.
Lipoproteins isolated from normal human plasma can bind and neutralize bacterial lipopolysaccharide (LPS) and may represent an important mechanism in host defense against gram-negative septic shock. We sought to define the components of high-density lipoproteins (HDL) that are required for neutralization of LPS. To accomplish this we have studied the functional neutralization of LPS by native and reconstituted HDL using a rapid assay that measures the CD14-dependent activation of leukocyte integrins on human neutrophils. Reconstituted HDL particles (R-HDL), prepared from purified apolipoprotein A-I (apoA-I) combined with phospholipid and free cholesterol, were not sufficient to neutralize the biologic activity of LPS. However, addition of recombinant LPS binding protein (LBP), a protein known to transfer LPS to CD14 and enhance responses of cells to LPS, enabled prompt binding and neutralization of LPS by R-HDL. Thus, LBP appears capable of transferring LPS not only to CD14 but also to lipoprotein particles. These results suggest that in addition to its ability to transfer LPS to CD14, LBP may also transfer LPS to lipoproteins. Additional studies demonstrated that LBP copurifies with native apoA-I containing lipoprotein (Lp(A-I)) particles. Since LBP appears to be physically associated with lipoproteins in plasma, it is positioned to play an important role in the neutralization of LPS.
Acute-Phase Proteins↗
Lipopolysaccharide (LPS)-binding protein is carried on lipoproteins and acts as a cofactor in the neutralization of LPS.
Lipoproteins isolated from normal human plasma can bind and neutralize bacterial lipopolysaccharide (LPS) and may represent an important mechanism in host defense against gram-negative septic shock. Recent studies have shown that experimentally elevating the levels of circulating high-density lipoproteins (HDL) provides protection against death in animal models of endotoxic shock. We sought to define the components of HDL that are required for neutralization of LPS. To accomplish this we have studied the functional neutralization of LPS by native and reconstituted HDL using a rapid assay that measures the CD14-dependent activation of leukocyte integrins on human neutrophils. We report here that reconstituted HDL particles (R-HDL), prepared from purified apolipoprotein A-I (apoA-I) combined with phospholipid and free cholesterol, are not sufficient to neutralize the biologic activity of LPS. However, addition of recombinant LPS binding protein (LBP), a protein known to transfer LPS to CD14 and enhance responses of cells to LPS, enabled prompt binding and neutralization of LPS by R-HDL. Thus, LBP appears capable of transferring LPS not only to CD14 but also to lipoprotein particles. In contrast with R-HDL, apoA-I containing lipoproteins (LpA-I) isolated from plasma by selected affinity immunosorption (SAIS) on an anti-apoA-I column, neutralized LPS without addition of exogenous LBP. Several lines of evidence demonstrated that LBP is a constituent of LpA-I in plasma. Passage of plasma over an anti-apoA-I column removed more than 99% of the LBP detectable by ELISA, whereas 31% of the LBP was recovered by elution of the column. Similarly, the ability of plasma to enable activation of neutrophils by LPS (LBP/Septin activity) was depleted and recovered by the same process. Furthermore, an immobilized anti-LBP monoclonal antibody coprecipitated apoA-I. The results described here suggest that in addition to its ability to transfer LPS to CD14, LBP may also transfer LPS to lipoproteins. Since LBP appears to be physically associated with lipoproteins in plasma, it is positioned to play an important role in the neutralization of LPS.
Acute-Phase Proteins↗
Afamin is a new member of the albumin, alpha-fetoprotein, and vitamin D-binding protein gene family.
A novel human serum protein with a molecular mass of 87,000 daltons was purified to homogeneity and subjected to amino acid sequence analyses. These sequences were used to design oligonucleotide primers and to isolate a full-length cDNA. The amino acid sequence encoded by the cDNA shares strong similarity to albumin family members and shares the characteristic pattern of Cys residues observed in this family. In addition, the gene maps to chromosome 4 as do other members of the albumin gene family. Based upon these observations, we conclude that the 87,000-dalton protein, which we designate afamin (AFM), is the fourth member of the albumin family of proteins. Afamin cDNA was stably transfected into Chinese hamster ovary cells and recombinant protein (rAFM) was purified from conditioned medium. Both rAFM and AFM purified from human serum react with a polyclonal antibody that was raised against a synthetic peptide derived from the deduced amino acid sequence of AFM.
Albumins↗
Bacterial lipopolysaccharide has structural similarity to ceramide and stimulates ceramide-activated protein kinase in myeloid cells.
Bacterial lipopolysaccharide (LPS), tumor necrosis factor (TNF)-alpha and interleukin-1 beta (IL-1 beta) stimulate similar cellular responses. TNF-alpha and IL-1 beta are known to initiate signaling through a pathway involving hydrolysis of sphingomyelin to ceramide (Kolesnick, R. N., and Golde, D. W. (1994) Cell 77, 325-328). In this system, ceramide acts as a second messenger stimulating a ceramide-activated serine/threonine protein kinase. The present studies demonstrate that LPS, like TNF and IL-1, stimulates ceramide-activated protein kinase activity in human leukemia (HL-60) cells and in freshly isolated human neutrophils. Lipid A, the biologically active core of LPS, enhanced kinase activity in a time- and concentration-dependent manner. As little as 10 nM lipid A was effective, and a maximal effect occurred with 500 nM lipid A, increasing kinase activity 5-fold. Native LPS similarly induced kinase activation. This effect of LPS was markedly enhanced by LPS binding protein and required the LPS receptor CD14. In contrast to TNF and IL-1, LPS did not cause sphingomyelin hydrolysis and thus stimulates ceramide-activated protein kinase without generating ceramide. Molecular modeling showed strong structural similarity between ceramide and a region of lipid A. Based on these observations, we propose that LPS stimulates cells by mimicking the second messenger function of ceramide.
Amino Acid Sequence↗
A fluorescence microassay for the quantitation of integrin-mediated adhesion of neutrophil.
The avidity of leukocyte integrin CR3 (Mac-1, CD11b/CD18, alpha m beta 2) on neutrophils (PMN) may be rapidly modulated by several agonists. We describe a method for determining the avidity of these receptors by measuring the adhesion of PMN to fibrinogen-coated surfaces. Cells are loaded with a succinimidyl ester of carboxyfluorescein diacetate, which is deacetylated by intracellular esterases yielding a highly fluorescent carboxyfluorescein that remains trapped within the PMN. The number of cells adhering to fibrinogen-coated wells of Terasaki microplates is quantitated with a fluorescence plate reader. Stimulation of PMN with a number of agonists, including PMA, fNLLP, Ca-ionophore A23187, interleukin-8, tumor necrosis factor and lipopolysaccharide strongly increased adhesion to fibrinogen, which was CD11b/CD18 dependent. The extent of cell adhesion depended on stimulus concentration and incubation time. This assay requires little time, utilizes small numbers of cells and does not require hazardous reagents.
Cell Adhesion↗
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.
Acute-Phase Proteins↗
Soluble CD14 from urine copurifies with a potent inducer of cytokines.
Here we report that soluble CD14 isolated from the urine of nephrotic patients (uCD14) contains a potent cytokine inducing activity. CD14 derived from urine appeared to consist of two major polypeptides of about 54 and 48 kDa. In uCD14 isolated from three different nephrotic patients the cytokine-inducing activity appeared to co-migrate with the 48-kDa polypeptide which upon sequencing had the same N-terminal sequence as native CD14. Treatment of human monocytes and the human astrocytoma cell line U373 with uCD14 resulted in a strong secretion of tumor necrosis factor (TNF) and interleukin-6, respectively. The cytokine-inducing activity of the uCD14 preparations was unaffected by the absence of serum. This is in contrast to the activation of human monocytes and U373 cells by lipopolysaccharide (LPS) which is highly dependent on the presence of serum. The cytokine-inducing activity was not affected by LPS-binding protein (LBP) or polyclonal rabbit antibodies against LBP. The TNF-inducing activity of uCD14 was also heat labile in contrast to the cytokine-inducing activity of LPS, which was relatively heat resistant. The results suggest that CD14 may exist in at least two forms of which one is involved in cytokine induction.
Acute-Phase Proteins↗