Plasma CD14 decreases monocyte responses to LPS by transferring cell-bound LPS to plasma lipoproteins.

CD14, a myeloid cell-surface receptor and soluble plasma protein, binds LPS and other microbial molecules and initiates the innate immune response to bacterial invasion. The blood concentration of soluble CD14 (sCD14) increases during the systemic response to infection. Although high sCD14 blood levels have correlated with increased risk of dying from severe sepsis, sCD14 can diminish cell responses to LPS. We show here that in human serum, sCD14 increases the rate at which cell-bound LPS is released from the monocyte surface and binds to plasma lipoproteins. This enhanced rate of LPS efflux is associated with a significant reduction in the ability of monocytes to produce cytokines in response to LPS. Serum from septic patients reduced the LPS-monocyte interaction by as much as tenfold, and depletion of sCD14 from the serum restored LPS-monocyte binding and release kinetics to near normal levels. In serum from septic patients, monocyte-bound LPS also moved more rapidly into lipoproteins, which completely neutralized the biologic activity of the LPS that bound to them. In human plasma, sCD14 thus diminishes monocyte responses to LPS by transferring cell-bound LPS to lipoproteins. Stress-related increases in plasma sCD14 levels may help prevent inflammatory responses within the blood.

MD-2 binds to bacterial lipopolysaccharide.

The exact roles and abilities of the individual components of the lipopolysaccharide (LPS) receptor complex of proteins remain unclear. MD-2 is a molecule found in association with toll-like receptor 4. We produced recombinant human MD-2 to explore its LPS binding ability and role in the LPS receptor complex. MD-2 binds to highly purified rough LPS derived from Salmonella minnesota and Escherichia coli in five different assays; one assay yielded an apparent KD of 65 nm. MD-2 binding to LPS did not require LPS-binding proteins LBP and CD14; in fact LBP competed with MD-2 for LPS. MD-2 enhanced the biological activity of LPS in toll-like receptor 4-transfected Chinese hamster ovary cells but inhibited LPS activation of U373 astrocytoma cells and of monocytes in human whole blood. These data indicate that MD-2 is a genuine LPS-binding protein and strongly suggest that MD-2 could play a role in regulation of cellular activation by LPS depending on its local availability.

Lipopolysaccharide-binding protein and phospholipid transfer protein release lipopolysaccharides from gram-negative bacterial membranes.

Although animals mobilize their innate defenses against gram-negative bacteria when they sense the lipid A moiety of bacterial lipopolysaccharide (LPS), excessive responses to this conserved bacterial molecule can be harmful. Of the known ways for decreasing the stimulatory potency of LPS in blood, the binding and neutralization of LPS by plasma lipoproteins is most prominent. The mechanisms by which host lipoproteins take up the native LPS that is found in bacterial membranes are poorly understood, however, since almost all studies of host-LPS interactions have used purified LPS aggregates. Using native Salmonella enterica serovar Typhimurium outer membrane fragments (blebs) that contained (3)H-labeled lipopolysaccharide (LPS) and (35)S-labeled protein, we found that two human plasma proteins, LPS-binding protein (LBP) and phospholipid transfer protein (PLTP), can extract [(3)H]LPS from bacterial membranes and transfer it to human high-density lipoproteins (HDL). Soluble CD14 (sCD14) did not release LPS from blebs yet could facilitate LBP-mediated LPS transfer to HDL. LBP, but not PLTP, also promoted the activation of human monocytes by bleb-derived LPS. Whereas depleting or neutralizing LBP significantly reduced LPS transfer from blebs to lipoproteins in normal human serum, neutralizing serum PLTP had no demonstrable effect. Of the known lipid transfer proteins, LBP is thus most able to transfer LPS from bacterial membranes to the lipoproteins in normal human serum.

Plasma constituents regulate LPS binding to, and release from, the monocyte cell surface.

Innate immunity to Gram-negative bacteria involves regulated mechanisms that allow sensitive but limited responses to LPS. Two important pathways that lead to host cell activation and LPS deactivation involve: (i) LPS interactions with CD14 and Toll-like receptor 4 on cells (activation); and (ii) LPS sequestration by plasma lipoproteins (deactivation). Whereas these pathways were previously thought to be independent and essentially irreversible, we found that they are connected by a third pathway: (iii) the movement of LPS from host cells to plasma lipoproteins. Our data show that, in the presence of human plasma, LPS binds transiently to monocyte surfaces and then moves from the cell surface to plasma lipoproteins. Soluble CD14 enhances LPS release from cells in the presence of lipoproteins, whereas LPS binding protein and phospholipid transfer protein do not. The transfer of cell-bound LPS to lipoproteins is accompanied by reduced cell responses to the LPS, suggesting that the movement of LPS from leukocytes into lipoproteins may attenuate host responses to LPS in vivo. Preliminary data suggest that changes that occur in the plasma after trauma or during sepsis decrease LPS binding to leukocytes while greatly increasing the rate of LPS release from cells.

Plasma lipoproteins promote the release of bacterial lipopolysaccharide from the monocyte cell surface.

When bacterial lipopolysaccharide (LPS) enters the bloodstream, it is thought to have two general fates. If LPS binds to circulating leukocytes, it triggers innate host defense mechanisms and often elicits toxic reactions. If instead LPS binds to plasma lipoproteins, its bioactivity is largely neutralized. This study shows that lipoproteins can also take up LPS that has first bound to leukocytes. When monocytes were loaded with [(3)H]LPS and then incubated in plasma, they released over 70% of the cell-associated [(3)H]LPS into lipoproteins (predominantly high density lipoprotein), whereas in serum-free medium the [(3)H]LPS remained tightly associated with the cells. The transfer reaction could be reproduced in the presence of pure native lipoproteins or reconstituted high density lipoprotein. Plasma immunodepletion experiments and experiments using recombinant LPS transfer proteins revealed that soluble CD14 significantly enhances LPS release from the cells, high concentrations of LPS-binding protein have a modest effect, and phospholipid transfer protein is unable to facilitate LPS release. Essentially all of the LPS on the monocyte cell surface can be released. Lipoprotein-mediated LPS release was accompanied by a reduction in several cellular responses to the LPS, suggesting that the movement of LPS from leukocytes into lipoproteins may attenuate host responses to LPS in vivo.

Bacterial lipopolysaccharide can enter monocytes via two CD14-dependent pathways.

Host recognition and disposal of LPS, an important Gram-negative bacterial signal molecule, may involve intracellular processes. We have therefore analyzed the initial pathways by which LPS, a natural ligand of glycosylphosphatidylinositol (GPI)-anchored CD14 (CD14-GPI), enters CD14-expressing THP-1 cells and normal human monocytes. Exposure of the cells to hypertonic medium obliterated coated pits and blocked 125I-labeled transferrin internalization, but failed to inhibit CD14-mediated internalization of [3H]LPS monomers or aggregates. Immunogold electron microscope analysis found that CD14-bound LPS moved principally into noncoated structures (mostly tubular invaginations, intracellular tubules, and vacuoles), whereas relatively little moved into coated pits and vesicles. When studied using two-color laser confocal microscopy, internalized Texas Red-LPS and BODIPY-transferrin were found in different locations and failed to overlap completely even after extended incubation. In contrast, in THP-1 cells that expressed CD14 fused to the transmembrane and cytosolic domains of the low-density lipoprotein receptor, a much larger fraction of the cell-associated LPS moved into coated pits and colocalized with intracellular transferrin. These results suggest that CD14 (GPI)-dependent internalization of LPS occurs predominantly via noncoated plasma membrane invaginations that direct LPS into vesicles that are distinct from transferrin-containing early endosomes. A smaller fraction of the LPS enters via coated pits. Aggregation, which greatly increases LPS internalization, accelerates its entry into the nonclathrin-mediated pathway.

Phosphatidylinositides bind to plasma membrane CD14 and can prevent monocyte activation by bacterial lipopolysaccharide.

Although bacterial lipopolysaccharides (LPS) and several other microbial agonists can bind to mCD14 (membrane CD14), a cell-surface receptor found principally on monocytes and neutrophils, host-derived mCD14 ligands are poorly defined. We report here that phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate, and other phosphatidylinositides can bind to mCD14. Phosphatidylserine (PS), another anionic glycerophospholipid, binds to mCD14 with lower apparent affinity than does PtdIns. LPS-binding protein, a lipid transfer protein found in serum, facilitates both PS- and PtdIns-mCD14 binding. PtdIns binding to mCD14 can be blocked by anti-CD14 monoclonal antibodies that inhibit LPS-mCD14 binding, and PtdIns can inhibit both LPS-mCD14 binding and LPS-induced responses in monocytes. Serum-equilibrated PtdIns also binds to mCD14-expressing cells, raising the possibility that endogenous PtdIns may modulate cellular responses to LPS and other mCD14 ligands in vivo.

Treponema pallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptides activate monocytic cells via a CD14-dependent pathway distinct from that used by lipopolysaccharide.

Lipoproteins of Treponema pallidum and Borrelia burgdorferi possess potent proinflammatory properties and, thus, have been implicated as major proinflammatory agonists in syphilis and Lyme disease. Here we used purified B. burgdorferi outer surface protein A (OspA) and synthetic lipopeptides corresponding to the N-termini of OspA and the 47-kDa major lipoprotein immunogen of T. pallidum to clarify the contribution of CD14 to monocytic cell activation by spirochetal lipoproteins and lipopeptides. As with LPS, mouse anti-human CD14 Abs blocked the activation of 1,25-dihydroxyvitamin D3-matured human myelomonocytic THP-1 cells by OspA and the two lipopeptides. The existence of a CD14-dependent pathway was corroborated by using undifferentiated THP-1 cells transfected with CD14 and peritoneal macrophages from CD14-deficient BALB/c mice. Unlike LPS, cell activation by lipoproteins and lipopeptides was serum independent and was not augmented by exogenous LPS-binding protein. Two observations constituted evidence that LPS and lipoprotein/lipopeptide signaling proceed via distinct transducing elements downstream of CD14: 1) CHO cells transfected with CD14 were exquisitely sensitive to LPS but were lipoprotein/lipopeptide nonresponsive; and 2) substoichiometric amounts of deacylated LPS that block LPS signaling at a site distal to CD14 failed to antagonize activation by lipoproteins and lipopeptides. The combined results demonstrate that spirochetal lipoproteins and lipopeptides use a CD14-dependent pathway that differs in at least two fundamental respects from the well-characterized LPS recognition pathway.

CD14-dependent internalization of bacterial lipopolysaccharide (LPS) is strongly influenced by LPS aggregation but not by cellular responses to LPS.

We analyzed the impact of ligand aggregation and LPS-induced signaling on CD14-dependent LPS internalization kinetics in human monocytic THP-1 cells and murine macrophages. Using two independent methods, we found that the initial rate and extent of LPS internalization increased with LPS aggregate size. In the presence of LPS binding protein (LBP), large LPS aggregates were internalized extremely rapidly (70% of the cell-associated LPS was internalized in 1 min). Smaller LPS aggregates were internalized more slowly than the larger aggregates, and LPS monomers, complexed with soluble CD14 in the absence of LBP, were internalized very slowly after binding to membrane CD14 (5% of the cell-associated LPS was internalized in 1 min). In contrast, the initial aggregation state had little or no effect on the stimulatory potency of the LPS. Previous studies suggest that LPS-induced signal responses may influence the intracellular traffic and processing of LPS. We found that elicited peritoneal macrophages from LPS-responsive (C3H/HeN) and LPS-hyporesponsive (C3H/HeJ) mice internalized LPS with similar kinetics. In addition, pre-exposure of THP-1 cells to LPS had no effect on their ability to internalize subsequently added LPS, and pre-exposure of the cells to the LPS-specific inhibitor, LA-14-PP, inhibited stimulation of the cells without inhibiting LPS internalization. In these cells, LPS is thus internalized by a constitutive cellular mechanism(s) with kinetics that depend importantly upon the physical state in which the LPS is presented to the cell.

Enzymatically deacylated lipopolysaccharide (LPS) can antagonize LPS at multiple sites in the LPS recognition pathway.

Like other tetraacyl partial structures of lipopolysaccharide (LPS) and lipid A, LPS that has been partially deacylated by acyloxyacyl hydrolase can inhibit LPS-induced responses in human cells. To identify the site(s) of inhibition in the LPS recognition pathway, we analyzed the apparent binding affinities and interactions of 3H-labeled enzymatically deacylated LPS (dLPS) and [3H]LPS with CD14, the LPS receptor, on THP-1 cells. Using (i) incubation conditions that prevented ligand internalization and (ii) defined concentrations of LPS binding protein (LBP), which facilitates LPS and dLPS binding to CD14, we found that dLPS can antagonize LPS in at least three ways. 1) When the concentration of LBP in the medium was suboptimal for promoting LPS-CD14 binding, low concentrations of dLPS were able to compete with LPS for binding CD14, suggesting competition between LPS and dLPS for engaging LBP. 2) When LBP was present in excess, dLPS could compete with LPS for binding CD14, but only at dLPS concentrations that were at or above its KD for binding CD14 (100 ng/ml). 3) In contrast, substoichiometric concentrations of dLPS (1 ng/ml) inhibited LPS-induced (3 ng/ml) interleukin-8 release without blocking LPS binding to CD14. Functional antagonism was possible without competition for cell-surface binding because both LPS-induced interleukin-8 release and dLPS inhibition occurred at concentrations that were far below their respective CD14 binding KD values. In addition to its expected ability to compete with LPS for binding LBP and CD14, dLPS thus potently antagonizes LPS at an undiscovered site that is distal to LPS-CD14 binding in the LPS recognition pathway.

Modulation of protein tyrosine phosphorylation in human cells by LPS and enzymatically deacylated LPS.

Previous studies have shown that the antagonism of LPS-induced responses by lipid A analogs and the induction of tolerance (adaptation) to LPS can occur without decreasing LPS binding to CD14. To learn more about these inhibitory mechanisms, we studied the effects of LPS and dLPS on an early response in the LPS signal pathway, protein tyrosine phosphorylation. Using CD14-expressing THP-1 cells, we found that very low concentrations of LPS stimulated tyrosine phosphorylation of a 42 kDa protein (p42). dLPS did not stimulate detectable phosphorylation of a 42 or any other cellular protein, but it inhibited the ability of LPS to induce this response. dLPS was a potent inhibitor when added to the cells at the same time as LPS, but not when added one or two minutes after LPS. Exposing cells to LPS for 3 hours induced a state of tolerance (adaptation) in which the cells were refractory to restimulation of p42 phosphorylation by LPS, but not by other agonists. The duration of LPS-induced tolerance (8-16 hours) was much longer than the duration of the refractory state resulting from dLPS pre-treatment (under 2 hours). Cellular binding and uptake of LPS were not significantly reduced by preexposing the cells to LPS or dLPS. The data indicate that the mechanisms of dLPS antagonism and LPS-induced tolerance occur distal to CD14 binding and proximal to p42 tyrosine phosphorylation.

Bacterial lipopolysaccharide binds to CD14 in low-density domains of the monocyte-macrophage plasma membrane.

We report that gram-negative bacterial lipopolysaccharide (LPS) binds to CD14 on lipid-enriched, low-density domains of the human monocyte-macrophage (THP-1 cell) plasma membrane. After brief incubation with [3H]LPS under conditions that prevent its internalization, THP-1 cells were disrupted using a detergent-free method and plasma membrane fragments were separated on density gradients. The [3H]LPS-binding fragments had low bouyant densities and were enriched, when compared to high-density membrane fragments, in CD14 (a receptor for LPS and other microbial molecules), p53/56lyn, GTP-binding proteins, ouabain-inhibitable Na+/K+ ATPase, sphingomyelin, and GM1 ganglioside. Monoclonal anti-CD14 antibody 60bca blocked [3H]LPS binding to these membrane fragments. Immunoelectron microscopic analysis identified clusters of CD14 on both large (200-1,000 nm) and small (< or = 200 nm) low-density membrane fragments. GM1 and CD14 were usually found on the same fragments, yet their distributions on those fragments infrequently overlapped. These cells seem to lack arrays of caveolae, the ordered membrane structures that harbor glycosylphosphatidyl-anchored proteins and GM1 in many other cell types. Finding that LPS binds to CD14 predominantly in low-density plasma membrane domains suggests, however, that discrete regions of the monocyte-macrophage plasma membrane may be organized to facilitate rapid responses to, and internalization of, molecules that bind CD14.

Lipopolysaccharide (LPS) partial structures inhibit responses to LPS in a human macrophage cell line without inhibiting LPS uptake by a CD14-mediated pathway.

Lipopolysaccharides (LPS) that lack acyloxyacyl groups can antagonize responses to LPS in human cells. Although the site and mechanism of inhibition are not known, it has been proposed that these inhibitory molecules compete with LPS for a common cellular target such as a cell-surface binding receptor. In the present study, we used an in vitro model system to test this hypothesis and to evaluate the role of CD14 in cellular responses to LPS. Cells of the THP-1 human monocyte-macrophage cell line were exposed to 1,25 dihydroxyvitamin D3 to induce adherence to plastic and expression of CD14, a binding receptor for LPS complexed with LPS-binding protein (LBP). The uptake of picograms of [3H]LPS (agonist) and enzymatically deacylated LPS [3H]dLPS (antagonist) was measured by exposing the cells to the radiolabeled ligands for short incubation periods. The amounts of cell-associated LPS and dLPS were then correlated with cellular responses by measuring the induction of nuclear NF-kappa B binding activity and the production of cell-associated interleukin (IL)-1 beta. We found that similar amounts of [3H]LPS or [3H]dLPS were taken up by the cells. The rate of cellular accumulation of the ligands was greatly enhanced by LBP and blocked by a monoclonal antibody to CD14 (mAb 60b), yet no cellular responses were induced by dLPS or dLPS-LBP complexes. In contrast, LPS stimulated marked increases of NF-kappa B binding activity and IL-1 beta. These responses were enhanced by LBP and inhibited by mAb 60b. dLPS and its synthetic lipid A counterpart, LA-14-PP (also known as lipid Ia, lipid IVa, or compound 406) strongly inhibited LPS-induced NF-kappa B and IL-1 beta, yet neither antagonist inhibited the uptake of LPS via CD14. dLPS did not inhibit NF-kappa B responses to tumor necrosis factor (TNF) alpha or phorbol ester. Our results indicate that (a) both stimulatory and nonstimulatory ligands can bind to CD14 in the presence of LBP; (b) the mechanism of inhibition by dLPS is LPS-specific, yet does not involve blockade of LPS binding to CD14; and (c) in keeping with previous results of others, large concentrations of LPS can stimulate the cells in the absence of detectable binding to CD14. The findings indicate that the site of dLPS inhibition is distal to CD14 binding in the LPS signal pathway in THP-1 cells, and suggest that molecules other than CD14 are important in LPS signaling.

Rearrangements of the tal-1 locus as clonal markers for T cell acute lymphoblastic leukemia.

Normal and aberrant immune receptor gene assembly each produce site-specific DNA rearrangements in leukemic lymphoblasts. In either case, these rearrangements provide useful clonal markers for the leukemias in question. In the t(1;14)(p34;q11) translocation associated with T cell acute lymphoblastic leukemia (T-ALL), the breakpoints on chromosome 1 interrupt the tal-1 gene. A site-specific deletion interrupts the same gene in an additional 26% of T-ALL. Thus, nearly one-third of these leukemias contain clustered rearrangements of the tal-1 locus. To test whether these rearrangements can serve as markers for residual disease, we monitored four patients with T-ALL; three of the leukemias contained a deleted (tald) and one a translocated (talt) tal-1 allele. These alleles were recognized by a sensitive amplification/hybridization assay. tald alleles were found in the blood of one patient during the 4th mo of treatment but not thereafter. Using a quantitative assay to measure the fraction of tald alleles in DNA extracts, we estimated that this month 4 sample contained 150 tald copies per 10(6) genome copies. The patient with t(1;14)(p34;q11) (talt) leukemia developed a positive assay during the 20th mo of treatment. By standard criteria, all four patients remain in complete remission 11-20 mo into treatment. We conclude that tal-1 rearrangements provide useful clonal markers for approximately 30% of T-ALLs.

Detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin hypervariable region specific oligonucleotide probes.

To develop a sensitive and specific assay for minimal residual disease in acute lymphoblastic leukemia (ALL), we exploited the enormous diversity of genomic sequences created by immune receptor gene rearrangements. To isolate clone-specific sequences, we first synthesized oligonucleotides that match conserved variable (VH) and joining (JH) sequences flanking the third hypervariable region (HVR3) in the rearranged immunoglobulin heavy chain (IgH) locus. In polymerase chain reactions (PCR), these primers were then used to amplify the intervening HVR3 segments from leukemic DNA samples. Of 12 B-lineage ALLs studied, ten generated one or more fragments of the size expected for HVR3 gene segments. Thus, this single pair of amplimers was sufficient to isolate HVR3 sequences from a majority of acute lymphoblastic leukemias. To verify that the amplified fragments originated from HVR3 alleles and to assess their diversity, we sequenced 7 PCR products derived from 6 leukemias. In addition to elements of recognized D segments, each of the 7 fragments contained novel VH-D and D-JH junctional sequences, including N nucleotides, not known to be present in the germline. Each sequence was unique, and allele-specific oligonucleotide probes hybridized only to HVR3 segments from which the probes were derived. Therefore, as anticipated, these HVR3 segments appeared to possess the diversity required to serve as clonal markers for leukemic populations. To demonstrate that these amplified HVR3 alleles could serve as the basis for a sensitive and specific assay to detect rare leukemic cells, we analyzed in detail one pre-B leukemia that had rearranged 2 IgH alleles. The HVR3 sequences were shown to be linked to rearranged JH-containing restriction fragments in digests of genomic DNA, establishing their origin in the leukemic cells. We synthesized oligonucleotides corresponding to the unique junctional sequences in the HVR3 segments. Using these novel amplimers in an allele-specific amplification and hybridization procedure, we showed that this assay can detect 10 leukemic cells in a background of 10(6) normal blood mononuclear cells. In contrast, the leukemic HVR3 sequences were not detected in extracts of normal or unrelated remission leukemic leukocytes. We conclude that the assay for specific IgH HVR3 sequences is a realistic strategy for detection of minimal residual disease in B-lineage ALL.

Phenotypic heterogeneity of TDT+ cells in the blood and bone marrow: implications for surveillance of residual leukemia.

Terminal deoxynucleotidyl transferase (TdT) is a useful marker for normal lymphocyte precursors and acute lymphoblastic leukemia (ALL). Our previous studies, however, have shown that for monitoring minimal residual disease in the circulation, assay for TdT alone is not sufficiently specific to distinguish leukemia cells from the background of rare normal blood TdT+ cells. In an attempt to increase specificity for leukemic cells, we have used double and triple immunophenotypic analysis to characterize normal circulating and bone marrow TdT+ cells. Overall, normal TdT+ cells were about 1000-fold more frequent in the marrow than in the blood. More than 75% of TdT+ cells in both the blood and marrow expressed the CD34, CD22, and HLA-DR antigens. However, circulating TdT+ cells infrequently expressed CD19 (4.5%) and CD9 (2.3%), compared with their marrow counterparts (74% and 47%, respectively). The brightly staining CD10+ phenotype, frequently associated with ALL blasts, was significantly less common among normal blood (5.7%) than marrow (31%) TdT+ cells. Although T-lineage markers were rarely expressed on TdT+ cells in either site, CD7+ cells were far more prevalent within the circulating TdT+ subset (4%) than among the marrow population (less than 0.2%). The results suggest a selective release of lineage-uncommitted and/or thymus-destined TdT+ cells from the marrow into the circulation. Moreover, since CD19, CD9, and high-density CD10 are frequently found on ALL blasts, staining for these markers on TdT+ cells in the circulation should improve the specificity of assay for residual common ALL cells. Likewise, assay for CD5+ and possibly CD7+ TdT+ cells in either marrow or blood should provide a very sensitive method of detection of T-ALL blasts.

Extraction and quantification of daunomycin and doxorubicin in tissues.

The measurement of intracellular concentrations of the anti-cancer drug doxorubicin was performed by the application of a simple cell extraction technique combined with a rapid high-performance liquid chromatographic separation. Quantitation was done by fluorescence detection. The extraction procedure was non-degradative and the mean recovery of drug was 95%. A high drug extraction efficiency was confirmed with radiolabeled [3H] doxorubicin. The method is applicable to normal and neoplastic tissue.