CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein.

Leukocytes respond to lipopolysaccharide (LPS) at nanogram per milliliter concentrations with secretion of cytokines such as tumor necrosis factor-alpha (TNF-alpha). Excess secretion of TNF-alpha causes endotoxic shock, an often fatal complication of infection. LPS in the bloodstream rapidly binds to the serum protein, lipopolysaccharide binding protein (LBP), and cellular responses to physiological levels of LPS are dependent on LBP. CD14, a differentiation antigen of monocytes, was found to bind complexes of LPS and LBP, and blockade of CD14 with monoclonal antibodies prevented synthesis of TNF-alpha by whole blood incubated with LPS. Thus, LPS may induce responses by interacting with a soluble binding protein in serum that then binds the cell surface protein CD14.

Structure and function of lipopolysaccharide binding protein.

The primary structure of lipopolysaccharide binding protein (LBP), a trace plasma protein that binds to the lipid A moiety of bacterial lipopolysaccharides (LPSs), was deduced by sequencing cloned complementary DNA. LBP shares sequence identity with another LPS binding protein found in granulocytes, bactericidal/permeability-increasing protein, and with cholesterol ester transport protein of the plasma. LBP may control the response to LPS under physiologic conditions by forming high-affinity complexes with LPS that bind to monocytes and macrophages, which then secrete tumor necrosis factor. The identification of this pathway for LPS-induced monocyte stimulation may aid in the development of treatments for diseases in which Gram-negative sepsis or endotoxemia are involved.

Participation of tumor necrosis factor in the mediation of gram negative bacterial lipopolysaccharide-induced injury in rabbits.

Macrophages are induced by LPS to release a number of products that determine the host response during gram negative sepsis. To examine the role of one such substance, tumor necrosis factor (TNF), in mediating LPS-induced injury, we employed a rabbit model of endotoxic shock to (a) determine the kinetics and extent of release of TNF into plasma after injection of LPS, and (b) to evaluate the protective effect of in vivo neutralization of LPS-induced TNF by prior infusion of anti-TNF antibody. TNF was maximally induced 45-100 min after injection of 10 micrograms i.v. parent Salmonella minnesota Re595 LPS or 250 micrograms Re595 LPS-HDL complexes. Maximal induction of TNF by LPS was associated with development of hypotension, focal hepatic necrosis, intravascular fibrin deposition and lethality. Based on (a) the peak levels of TNF observed in serum, 2.5 X 10(3) U/ml, (b) the specific activity of purified rabbit macrophage-derived TNF, 1 X 10(8) U/mg, and (c) the biphasic disappearance of intravenously injected purified TNF (t1/2 = 0.5 min, 11 min) we constructed a kinetic model showing that at least 130 micrograms of TNF (1.3 X 10(7) U) was released into plasma 30-200 min postinjection of LPS. Prior infusion of anti-TNF antibody (30-45 min before LPS injection) resulted in neutralization of the LPS-induced serum TNF activity and provided significant protection from the development of hypotension, fibrin deposition, and lethality. Thus, these results provide further evidence that TNF plays a central role mediating the pathophysiologic changes that occur during gram negative endotoxic shock.

Lipopolysaccharide (LPS) recognition in macrophages. Participation of LPS-binding protein and CD14 in LPS-induced adaptation in rabbit peritoneal exudate macrophages.

Exposure of rabbit peritoneal exudate macrophages (PEM) or whole blood to picomolar concentrations of LPS induces adaptation or hyporesponsiveness to LPS. Because of the importance of plasma LPS-binding protein (LBP) and the macrophage cell membrane protein CD14 in recognition of LPS, we examined the effect of LBP on LPS-induced adaptation in PEM. PEM exposed to LPS in the presence of LBP for 8 h were markedly less responsive to subsequent stimulation by LPS than monocytes/macrophages (M phi) adapted in the absence of LBP. LPS-induced expression of TNF was sharply reduced in LBP-LPS-adapted PEM, but in contrast these cells remained fully responsive to Staphylococcus aureus peptidoglycan. We considered that specific hyporesponsiveness in LPS-adapted M phi or in blood monocytes could be due to decreased expression of CD14 or diminished binding of LBP-LPS complexes to CD14. However, flow cytometry analysis revealed only minimal reduction of CD14 expression or CD14-dependent binding of a fluorescent LPS derivative when normo- and hyporesponsive cells were compared. These results show that complexes of LPS and LBP are more effective than LPS alone in inducing adaptation to LPS, and LPS-induced hyporesponsiveness probably results from changes in cellular elements distinct from CD14 that are involved in either LPS recognition or LPS-specific signal transduction.

Adaptation to bacterial lipopolysaccharide controls lipopolysaccharide-induced tumor necrosis factor production in rabbit macrophages.

These experiments provide an explanation for the observation that two intravenous injections of lipopolysaccharide (LPS) spaced 5 h apart in rabbits cause tumor necrosis factor/cachectin (TNF) levels to rise in the blood only after the first LPS injection. Herein we show that treatment of elicited peritoneal exudate rabbit macrophages (PEM) with two doses of LPS given 9 h apart results in a marked reduction in TNF production by the second LPS exposure. This state of hyporesponsiveness is a result of adaptation to LPS, is induced by LPS concentrations that are 1,000-fold less than required to induce TNF production (picograms vs. nanograms), is characterized by a decrease in LPS-induced TNF mRNA without any change in TNF mRNA half-life, is not changed by including indomethacin in cultures, and is specific for LPS since LPS-adapted cells display a TNF response to heat-killed Staphylococcus aureus that is at least as good as that observed in control PEM.

Lipopolysaccharide binding protein enhances the responsiveness of alveolar macrophages to bacterial lipopolysaccharide. Implications for cytokine production in normal and injured lungs.

A plasma lipopolysaccharide (LPS)-binding protein (LBP) has been shown to regulate the response of rabbit peritoneal macrophages and human blood monocytes to endotoxin (LPS). We investigated whether LBP is present in lung fluids and the effects of LBP on the response of lung macrophages to LPS. Immunoreactive LBP was detectable in the lavage fluids of patients with the adult respiratory distress syndrome by immunoprecipitation followed by Western blotting, and also by specific immunoassay. In rabbits, the LBP appeared to originate outside of the lungs, inasmuch as mRNA transcripts for LBP were identified in total cellular RNA from liver, but not from lung homogenates or alveolar macrophages. Purified LBP enhanced the response of human and rabbit alveolar macrophages to both smooth form LPS (Escherichia coli O111B:4) and rough form LPS (Salmonella minnesota Re595). In the presence of LBP and LPS, the onset of tumor necrosis factor-alpha (TNF alpha) production occurred earlier and at an LPS threshold dose that was as much as 1,000-fold lower for both types of LPS. In rabbit alveolar macrophages treated with LBP and LPS, TNF alpha mRNA appeared earlier, reached higher levels, and had a prolonged half-life as compared with LPS treatment alone. Neither LPS nor LPS and LBP affected pHi or [Cai++] in alveolar macrophages. Specific monoclonal antibodies to CD14, a receptor that binds LPS/LBP complexes, inhibited TNF alpha production by human alveolar macrophages stimulated with LPS alone or with LPS/LBP complexes, indicating the importance of CD14 in mediating the effects of LPS on alveolar macrophages. Thus, immunoreactive LBP accumulates in lung lavage fluids in patients with lung injury and enhances LPS-stimulated TNF alpha gene expression in alveolar macrophages by a pathway that depends on the CD14 receptor. LBP may play an important role in augmenting TNF alpha expression by alveolar macrophages within the lungs.

Manifestations and outcome of extra-pulmonary tuberculosis: impact of human immunodeficiency virus co-infection.

SETTING: Metropolitan New Orleans. OBJECTIVE: To determine the impact of human immunodeficiency virus (HIV) co-infection on the manifestations and outcome of extra-pulmonary tuberculosis (EPTB). DESIGN: Retrospective analysis of 136 patients diagnosed with EPTB between 1 January 1993 to 31 December 2001. Characteristics of EPTB were compared by HIV serostatus. RESULTS: Of those tested for HIV (n = 87), 42.5% were seropositive. Except for a higher frequency of disseminated TB among co-infected persons, the manifestations, laboratory diagnostic yield and outcome of EPTB were similar between HIV-infected and non-infected persons. The overall fatality rate was 20%; HIV-infected patients had a three-fold higher mortality compared to non-infected persons. In multivariate logistic regression analysis, factors associated with death were: HIV-seropositive (adjusted odds ratio [aOR] 5.2, 95% CI 1.1-24.65) compared to HIV-seronegative, disseminated and meningeal compared to lymphatic disease (aOR 16.87, 95% CI 12.31-123.34), and lack of TB treatment compared to receipt of TB treatment (aOR 29.23, 95% CI 14.47-191.23). CONCLUSION: Manifestations of EPTB were non-specific and did not differ between HIV-infected and non-infected persons. Severe disease, lack of TB treatment and HIV co-infection were associated withdeath. Approaches are needed to reduce EPTB morbidity and mortality, especially among HIV-infected persons.

Interactions of lipopolysaccharide with neutrophils in blood via CD14.

The functional characteristics of neutrophils are exceedingly sensitive to physiological conditions as well as the details of isolation. Exposure to lipopolysaccharide (LPS) or even contamination of the isolating media with traces of LPS is known to play an important role in regulating cell function and expression of receptors. Because of the suspected role of CD14 as a receptor for LPS, we used anti-CD14 monoclonal antibodies both to identify CD14 in the cell surface of polymorphonuclear leukocytes and to inhibit functional changes elicited by LPS. Cytometric techniques were used to investigate the regulation of CD14 and CR3 on the neutrophil cell surface in whole blood to minimize any effects of isolation. In whole blood neutrophil express low levels of formyl peptide receptor, CD14, and CR3, which increase substantially in response to formyl peptide and LPS. The increases in CR3 and CD14 occurred in parallel and were independent of protein synthesis and tumor necrosis factor (TNF) production. The increase in CR3 was inhibited by antibodies MY4, 3C10, and 28C5 against CD14. These findings are consistent with the notion that in blood the observed receptor up-regulation is in direct response to the action of LPS on neutrophils through CD14 and does not require products from macrophages such as TNF or the production of C5a from the plasma.

The CD14 differentiation antigen mediates the development of endotoxin responsiveness during differentiation of mononuclear phagocytes.

The CD14 antigen was originally described as a differentiation antigen on mononuclear cells. The purpose of this study was to investigate the relationship between the appearance of surface CD14 and the acquisition of lipopolysaccharide (LPS) responsiveness during maturation of mononuclear phagocytes. Immature THP-1 cells responded poorly to LPS in the absence or presence of serum. Treatment with the maturational agent calcitriol caused a dose- and time-dependent increase in CD14 mRNA and surface CD14 and enhanced the responsiveness of THP-1 cells to smooth and rough form LPS, complexes of LPS and lipopolysaccharide-binding protein (LBP), and LPS in low concentrations of serum. Monoclonal antibodies to CD14 blocked the responses of THP-1 to LPS, LPS-LBP complexes and LPS in serum. Immunodepletion of LBP from serum also inhibited the effect of LPS in serum. The data show that maturation of the response of THP-1 cells to LPS and LPS-LBP complexes depends on the appearance of CD14 on the cell surface. Maturation of the response to LPS in serum depends in large part on the appearance of CD14 on the cell surface and the presence of LBP in serum.

Cholera in Louisiana. Widening spectrum of seafood vehicles.

The largest cholera outbreak in the United States in over a century occurred in Louisiana from August through October 1986. Eighteen persons in 12 family clusters had stool culture or serologic evidence of infection with toxigenic Vibrio cholerae 0-group 1. Thirteen of these persons had severe diarrhea, and 4 required intensive care unit treatment. Although all 18 survived, 1 96-year-old woman with suspected cholera died shortly after hospital admission. A case-control study showed that case-patients were more likely than neighborhood control subjects to have eaten cooked crabs or cooked or raw shrimp during the week before illness. Case-patients who ate crabs were more likely than control subjects who ate crabs to have undercooked and mishandled the crabs after cooking. A third vehicle from the Gulf waters, raw oysters, caused V cholerae 01 infection in two persons residing in Florida and Georgia. All three seafood vehicles came from multiple sources. Stool isolates from the Louisiana case-patients were genetically identical to other North American strains isolated since 1973, but differ from African and Asian isolates. While crabs are the most important vehicle for V cholerae 01 infection in the United States, shrimp and oysters from the Gulf coast can also be vehicles of transmission. A persisting reservoir of V cholerae 01 along the Gulf coast may continue to cause sporadic cases and outbreaks of cholera in Gulf states and in states importing Gulf seafood.

Blockade of CD14 aggravates experimental shigellosis.

Shigella infections lead to severe inflammation associated with destruction of colonic mucosa. We assessed the effect of in vivo blockade of CD14 on the outcome of experimental Shigella infection in rabbits. A total of 17 rabbits were divided into two groups: 8 received a single i.v. dose of anti-rabbit CD14 monoclonal antibody prior to infection with an invasive Shigella flexneri strain; the remainder served as controls. The anti-CD14-treated rabbits exhibited more severe tissue destruction and a 50-fold increase in bacterial invasion of the intestinal mucosa when compared to controls. Similar numbers of polymorphonuclear leukocytes were recruited to the intestinal mucosa in both groups despite the massive bacterial invasion seen in the CD14-blocked group. No statistically significant differences were seen in levels of IL-1beta nor in the ratio of IL-1RA/IL-1beta for either group. In contrast, higher quantities of TNF-alpha were observed in the CD14-blocked group. To conclude, anti-CD14 treatment had a detrimental effect on the capacity of Shigella-infected animals to clear the infection.

Ophthalmic pain following cataract surgery: a comparison between local and general anaesthesia.

Ophthalmic pain following uncomplicated extracapsular cataract surgery was assessed postoperatively in 61 patients; 55% undergoing ophthalmic surgery had no pain or discomfort postoperatively, and 32% reported slight discomfort. Approximately 8% of patients reported mild pain and the remaining 5% experienced moderate to severe pain. Local anaesthesia was shown to be more comfortable postoperatively than general anaesthesia in the immediate postoperative period with both groups receiving similar amounts of postoperative analgesics.

Uptake and subcellular localization of bacterial lipopolysaccharide in the adrenal gland.

For determination of the kinetics of uptake and subcellular localization of lipopolysaccharide (LPS) from LPS-high density lipoprotein (LPS-HDL) complexes in the adrenal gland, LPS-HDL complexes were isolated by immunoaffinity chromatography of 125I-Salmonella minnesota Re595 LPS that had been incubated with 20 mM EDTA-rabbit plasma. After intravenous injection of LPS-HDL complexes in rabbits, preferential uptake of the LPS was observed in the adrenal, so that by 5 hours, adrenal-tissue-bound LPS concentrations (determined by use of 131I-BSA blood marker) exceeded all other tissues examined, including liver and spleen, by at least three-fold. For determination of the subcellular localization of LPS, cholesterol-rich (lipid droplet) fractions and cholesterol-depleted fractions were obtained by ultracentrifugation of homogenates of adrenal tissue from rabbits killed at various times after injection of LPS-HDL complexes. As much as 40% of the adrenal-tissue-bound LPS was recovered in the cholesterol-rich fraction 2.5-24 hours after injection of LPS-HDL complexes. Electron-microscopic autoradiographic and immunocytochemical analysis of adrenal cortex of animals killed 5 hours after injection of LPS-HDL complexes demonstrated specific localization of LPS in lipid droplets. These data thus provide direct evidence for the uptake of LPS into the adrenal cortex of animals with intravascular LPS-HDL complexes and indicate that further study of the effect of LPS on adrenocortical function is warranted.

A family of lipopolysaccharide binding proteins involved in responses to gram-negative sepsis.

The lipopolysaccharides (LPS) of Gram-negative bacteria initiate potentially fatal processes in many host organisms. Recently published amino acid sequence data suggest that there is a family of LPS binding proteins that may participate in the host response to Gram-negative bacteremia. The first two members of the family to be identified are an LPS binding protein present in serum after an acute phase response in humans, mice, rabbits, and rats and a bactericidal/permeability increasing protein present in the primary granules of human and rabbit neutrophils. LPS binding protein and bactericidal/permeability increasing protein share an ability to bind to LPS, have homologous NH2-terminal amino acid sequences, and are immunologically cross-reactive. Nevertheless, these two molecules differ in their effects on LPS and Gram-negative bacteria, in their sites of biosynthesis, and localization in vivo.

Regulation of the host response to bacterial lipopolysaccharides.

Bacterial endotoxins or lipopolysaccharides (LPS) are unique glycolipids present in the outer cell membrane of all gram-negative bacteria. It is now generally recognized that LPS is of primary importance in initiating the pathophysiological changes that often accompany gram-negative bacillary infections in humans including hypotensive shock, disseminated intravascular coagulation, and metabolic abnormalities. Although the biochemical mechanisms of these changes are not well understood, increasing emphasis has been placed on defining the biochemical response of the macrophage (M phi) to LPS. In this paper we describe two M phi-derived factors induced by LPS that may be important in the expression of endotoxic activity in the host. These are a procoagulant activity, which is present on the cell membrane of LPS-treated rabbit liver M phi and acts by directly activating coagulation factor X, and a factor released into the supernatant by LPS-treated peritoneal exudate M phi, which suppresses steroidogenesis in explanted adrenocortical cells. The potential role of the M phi in regulating the binding of LPS to high-density lipoproteins through the induction of acute phase proteins is also considered.

Interactions of bacterial lipopolysaccharides with tissue macrophages and plasma lipoproteins.

Several of the cellular and molecular interactions of LPS within the experimental host have been examined in an attempt to elucidate the potential role of these interactions in initiating the pathophysiologic events of endotoxemia. Using 125I-LPS, the clearance of LPS from the blood and its tissue, cellular and subcellular localization was established. The H-Mø was found to be the major host site of intravenous LPS localization and, subsequently, the effect of LPS on explanted H-Mø was demonstrated to be both directly cytotoxic and stimulatory of a selective increase in several cellular enzymes. Both the depression in H-Mø function and the stimulation of release of local and systemic mediators by LPS give the H-Mø a potential central role in initiating endotoxemic shock and DIC. Finally, the marked reduction in the clearance rate of LPS remaining in plasma after the initial rapid tissue localization was found to coincide with a density shift to less than 1.2 g/cm3 for the parent LPS. This density shift was found to be dependent upon binding of the LPS to HDL in the serum or plasma and, with the presence of cellular HDL receptors, accounts for a shift in tissue localization to the adrenals. A postulated effect of direct adrenal damage by LPS can thus be invoked as contributing to the endotoxemic syndrome.

Regulatory mechanisms of host responsiveness to endotoxin (lipopolysaccharide).

During Gram-negative endotoxemia, precise regulation of monocyte/macrophage (M phi) responsiveness to lipopolysaccharide (LPS) is critical to preserve host defense while avoiding complications such as organ failure and death. We will discuss regulation of LPS-M phi interactions by LPS-binding plasma proteins and by LPS-induced changes in M phi responsiveness. Upon exposure to plasma, LPS binds to either lipoproteins or LPS-binding protein (LBP; a 60-kilodalton glycoprotein with a high-affinity binding site for the lipid A moiety of rough and smooth LPS). The LPS-LBP complex stimulates the M phi by binding to its cellular receptor, CD14 (a monocyte/M phi-specific, phosphatidylinositol-anchored surface glycoprotein). Pretreatment of whole blood with anti-CD 14 monoclonal antibody reduces the responsiveness of monocytes to LPS [determined by tumor necrosis factor-alpha (TNF-alpha) release]at least 10-fold. Similarly, cellular responsiveness to LPS is diminished at least 100-fold by depletion of plasma LBP with anti-LBP antibody. Compared to LPS-LBP induction of TNF-alpha, LPS-lipoprotein complexes are as much as 10,000-fold less active. Thus, partitioning of LPS between LBP and lipoproteins markedly influences M phi responsiveness to LPS. LPS also directly induces M phi hyporesponsiveness to itself by a process known as adaptation; exposure of M phi to less than or equal to LPS/ml (subthreshold for TNF induction) for 6-9 reduces the sensitivity of the M phi to subsequent challenge up to 1,000-fold, so that 1 microgram/ml rather than 1 ng/ml of LPS is required for maximal induction of TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)