Richard L. Kitchens

Modulatory effects of sCD14 and LBP on LPS-host cell interactions.

LPS binding protein (LBP) and CD14 play key roles in promoting innate immunity to Gram-negative bacteria by transferring LPS to the signaling receptor complex, MD-2/Toll-like receptor 4 (TLR4). LBP and soluble CD14 (sCD14) can also inhibit responses to LPS by mechanisms that depend on their concentration and environment; during acute inflammation and infection, their concentrations increase in plasma and extravascular fluids. Whereas low concentrations of LBP enhance responses to LPS, high LBP concentrations can inhibit LPS bioactivity in vitro and in vivo. sCD14 also inhibits cell responses by diverting LPS from membrane-bound CD14 (mCD14) and by promoting LPS efflux from cell-surface mCD14 and transferring it to plasma lipoproteins. In vivo studies support the hypothesis that sCD14 has systemic anti-inflammatory effects, whereas in the tissues it may have pro-inflammatory effects that increase resistance to bacteria. Likewise, LBP increases resistance to Gram-negative bacteria by rapidly triggering pro-inflammatory responses to LPS. Thus, the dual stimulatory and inhibitory mechanisms of sCD14 and LBP may benefit the infected host by promoting inflammation in local sites, where it is needed, while at the same time preventing potentially detrimental systemic responses to LPS.

High-Density Lipoprotein Promotes Endothelial Cell Migration and Reendothelialization via Scavenger Receptor-B Type I

Vascular disease risk is inversely related to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL provides vascular protection are unclear. The disruption of endothelial monolayer integrity is an important contributing factor in multiple vascular disorders, and vascular lesion severity is tempered by enhanced endothelial repair. Here, we show that HDL stimulates endothelial cell migration in vitro in a nitric oxide-independent manner via scavenger receptor B type I (SR-BI)-mediated activation of Rac GTPase. This process does not require HDL cargo molecules, and it is dependent on the activation of Src kinases, phosphatidylinositol 3-kinase, and p44/42 mitogen-activated protein kinases. Rapid initial stimulation of lamellipodia formation by HDL via SR-BI, Src kinases, and Rac is also demonstrable. Paralleling the in vitro findings, carotid artery reendothelialization after perivascular electric injury is blunted in apolipoprotein A-I−/− mice, and reconstitution of apolipoprotein A-I expression rescues normal reendothelialization. Furthermore, reendothelialization is impaired in SR-BI−/− mice. Thus, HDL stimulates endothelial cell migration via SR-BI-initiated signaling, and these mechanisms promote endothelial monolayer integrity in vivo.

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.

Lipopolysaccharide-Binding Protein and Phospholipid Transfer Protein Release Lipopolysaccharides from Gram-Negative Bacterial Membranes

ABSTRACT 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 nativeSalmonella enterica serovar Typhimurium outer membrane fragments (blebs) that contained 3H-labeled lipopolysaccharide (LPS) and 35S-labeled protein, we found that two human plasma proteins, LPS-binding protein (LBP) and phospholipid transfer protein (PLTP), can extract [3H]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 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 [3H]LPS and then incubated in plasma, they released over 70% of the cell-associated [3H]LPS into lipoproteins (predominantly high density lipoprotein), whereas in serum-free medium the [3H]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.

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.

Apolipoprotein CI stimulates the response to lipopolysaccharide and reduces mortality in Gram-negative sepsis

Gram‐negative sepsis is a major death cause in intensive care units. Accumulating evidence indicates the protective role of plasma lipoproteins such as high‐density lipoprotein (HDL) in sepsis. It has recently been shown that septic HDL is almost depleted from apolipoprotein CI (apoCI), suggesting that apoCI may be a protective factor in sepsis. Sequence analysis revealed that apoCI possesses a highly conserved consensus KVKEKLK binding motif for lipopolysaccharide (LPS), an outer‐membrane component of Gram‐negative bacteria. Through avid binding to LPS involving this motif, apoCI improved the presentation of LPS to macrophages in vitro and in mice, thereby stimulating the inflammatory response to LPS. Moreover, apoCI dose‐dependently increased the early inflammatory response to Klebsiella pneumoniae‐induced pneumonia, reduced the number of circulating bacteria, and protected mice against fatal sepsis. Our data support the hypothesis that apoCI is a physiological protector against infection by enhancing the early inflammatory response to LPS and suggest that timely increase of apoCI levels could be used to efficiently prevent and treat early sepsis.—Berbée, J. F. P., van der Hoogt, C. C., Kleemann, R., Schippers, E. F., Kitchens, R. L., van Dissel, J. T., Bakker‐Woudenberg, I. A. J. M., Havekes, L. M., Rensen, P. C. N. Apolipoprotein CI stimulates the response to lipopolysaccharide and reduces mortality in Gram‐negative sepsis. FASEB J. 20, E1560 –E1569 (2006)

Impact of sepsis-induced changes in plasma on LPS interactions with monocytes and plasma lipoproteins: roles of soluble CD14, LBP, and acute phase lipoproteins.

Sepsis-induced changes in human plasma decrease LPS association with monocytes by regulating dynamic interactions among LPS, monocytes, and plasma lipoproteins. In the physiological environment of undiluted human serum, we have found that: (i) LPS binds transiently to monocytes and is released into plasma lipoproteins; (ii) the release of LPS from monocytes is dependent upon lipoprotein acceptors and is enhanced by soluble CD14 (sCD14); and (iii) both lipoproteins and sCD14 can attenuate cytokine responses in monocytes that have already bound LPS. Whereas LPS binding protein (LBP) also inhibited LPS responses after LPS had bound to monocytes, this did not require extensive release of cell-bound LPS as was observed with sCD14. In the serum of septic patients, both free LPS and monocyte-bound LPS were usually transferred to lipoproteins at an accelerated rate. In spite of a sharp decline in HDL levels, HDL remained the dominant LPS acceptor in many severely septic patients, whereas in some cases LPS binding shifted largely to a non-HDL lipoprotein fraction that co-eluted according to size with very low-density lipoprotein (VLDL). Preliminary data suggest that these lipoproteins have a very low density, and they contain apolipoprotein E and higher than normal proportions of the total lipoprotein cholesterol, phospholipid, apolipoprotein B, and serum amyloid A. The data suggest that the VLDL fraction contains acute phase lipoproteins of significantly altered composition that can replace HDL as the dominant LPS acceptor during sepsis when HDL levels are low.

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.

Native high-density lipoprotein augments monocyte responses to lipopolysaccharide (LPS) by suppressing the inhibitory activity of LPS-binding protein.

High-density lipoprotein (HDL) is an abundant plasma lipoprotein that is generally thought to be anti-inflammatory in both health and infectious disease. It binds and neutralizes the bioactivity of the potent bacterial lipids, LPS and lipoteichoic acid, that stimulate host innate immune responses. LPS-binding protein (LBP) plays an important role in augmenting leukocyte responses to LPS, whereas high concentrations of LBP, in the range of those found in plasma, can be inhibitory. We found that native HDL (nHDL) augmented human monocyte responses to LPS in the presence of inhibitory concentrations of LBP as measured by production of TNF and other cytokines. HDL did not stimulate cells in the absence of LPS, and it did not augment responses that were stimulated by IL-1β or lipoteichoic acid. This activity of HDL was inhibited by trypsin treatment, suggesting that one or more protein constituents of HDL are required. In contrast to nHDL, low-density lipoprotein, and reconstituted HDL did not possess this activity. The total lipoprotein fraction of normal plasma had activity that was similar to that of nHDL, whereas lipoproteins from septic patients with reduced HDL levels had a reduced ability to augment responses to LPS; this activity was restored by adding normal HDL to the patient lipoproteins. Our results demonstrate a novel proinflammatory activity of HDL that may help maintain sensitive host responses to LPS by suppressing the inhibitory activity of LBP. Our findings also raise the possibility that the decline of HDL during sepsis may help control the response to LPS.