Felix Stelter

Lipopolysaccharide-binding protein is required to combat a murine gram-negative bacterial infection.

An invading pathogen must be held in check by the innate immune system until a specific immune response can be mounted. In the case of Gram-negative bacteria, the principal stimulator of the innate immune system is lipopolysaccharide (LPS), a component of the bacterial outer membrane. In vitro, LPS is bound by lipopolysaccharide-binding protein (LBP) and transferred to CD14—the LPS receptor on the macrophage surface,—or to high-density lipoprotein (HDL) particles,. Transfer to CD14 triggers an inflammatory response which is crucial for keeping an infection under control. Here we investigate how LBP functions in vivo by using LBP-deficient mice. Surprisingly, we find that LBP is not required in vivo for the clearance of LPS from thecirculation, but is essential for the rapid induction of an inflammatory response by small amounts of LPS or Gram-negative bacteria and for survival of an intraperitoneal Salmonella infection.

Lipopolysaccharide and ceramide docking to CD14 provokes ligand‐specific receptor clustering in rafts

The glycosylphosphatidylinositol‐anchored receptor CD14 plays a major role in the inflammatory response of monocytes to lipopolysaccharide. Here, we describe that ceramide, a constituent of atherogenic lipoproteins, binds to CD14 and induces clustering of CD14 to co‐receptors in rafts. In resting cells, CD14 was associated with CD55, the Fcγ‐receptors CD32 and CD64 and the pentaspan CD47. Ceramide further recruited the complement receptor 3 (CD11b/CD18) and CD36 into proximity of CD14. Lipopolysaccharide, in addition, induced co‐clustering with Toll‐like receptor 4, Fcγ‐RIIIa (CD16a) and the tetraspanin CD81 while CD47 was dissociated. The different receptor complexes may be linked to ligand‐specific cellular responses initiated by CD14.

Soluble CD14 acts as a negative regulator of human T cell activation and function

T cell activation is controlled by the coordination of stimulatory and negative regulatory signals which are not completely defined. In this study we tested for a possible direct effect of CD14 on the regulation of T cell activation and function. We show that soluble CD14 (sCD14) induces inhibition of antigen‐mediated peripheral blood mononuclear cells (PBMC) proliferation and anti‐CD3‐mediated proliferation of CD4+CD8−, CD4−CD8+ and CD4+CD8+ T cell clones. This effect is not due to cell death, but results from a marked inhibition of IL‐2 production. Proliferation of T cell clones due to exogenous IL‐2 is not affected by sCD14. We also found that sCD14 inhibits production of another Th1‐like cytokine, IFN‐γ and a Th2‐like cytokine, IL‐4. Importantly, sCD14 induces a progressive accumulation of the inhibitory protein IκB‐α. We show that sCD14 binds to activated T cells. Following cell activation, biotinylated sCD14 stains CD3+ PBMC, as well as human T cell clones with varying intensity. The binding is saturable, can be inhibited by excess of unlabeled sCD14 and, following binding, sCD14 is internalized. Collectively, these findings reveal a previously unrecognized function of sCD14, namely its capacity to negatively regulate T lymphocyte activation and function by interacting directly with activated T cells.

Cutting Edge: Human B Cell Function Is Regulated by Interaction with Soluble CD14: Opposite Effects on IgG1 and IgE Production

The mechanism(s) controlling activation of naive B cells, their proliferation, Ag receptor affinity maturation, isotype switching, and their fate as memory or plasma cells is not fully elucidated. Here we show that between 24 and 60% of CD19+ cells in PBMC bind soluble CD14 (sCD14). Tonsillar B cells also bind sCD14, but preferentially the CD38−ve/low cells. Interaction of sCD14 with B cells resulted in higher levels of IgG1 and marked inhibition of IgE production by activated tonsillar B cells and Ag-stimulated PBMC. We found that sCD14 interfered with CD40 signaling in B cells, inhibited IL-6 production by activated B cells, and increased the kinetics and magnitude of CD40 ligand expression on T cells. Together with the previously reported effects on T cells, these findings define sCD14 as a novel soluble regulatory factor capable of modulating cellular and humoral immune responses by interacting directly with T and B cells.

LBP, CD14, TLR4 and the murine innate immune response to a peritoneal Salmonella infection

In mice, defense against an intraperitoneal Salmonella infection depends on a vigorous innate immune response. Mutations which lead to an inadequate early response to the pathogen thus identify genes involved in innate immunity. The best studied host resistance factor, NRAMP-1, is an endosomal membrane protein whose loss leads to an inability of the animals to hold the infection in check. However, innate defense against Salmonella is not restricted to mechanisms which directly attack the pathogen within macrophages. Here we have examined the contribution of the LBP, CD14 and TLR4 gene products to innate defense against Salmonella. To this end, we have generated mice which carry a wild-type allele of NRAMP-1, but which are deficient for the LBP, CD14 or TLR4 genes. Loss of any of these genes leads to a susceptibility to Salmonella as dramatic as that seen in animals lacking functional NRAMP-1 protein. This indicates that LBP, CD14 and TLR4 are all critical elements required in the proper induction of this innate defense system.

Elevated Levels of Lipopolysaccharide-Binding Protein and Soluble CD14 in Plasma in Neonatal Early-Onset Sepsis

ABSTRACT No data on lipopolysaccharide-binding protein (LBP) in newborns with sepsis have been available up to now. We therefore determined levels of LBP and soluble CD14 (sCD14) in plasma of healthy and septic neonates in order to evaluate their potential diagnostic role. The study included prospectively collected patient samples of two recently published studies on cytokine expression in neonatal sepsis. Twenty-nine septic patients were enrolled in the present analysis. Samples—either cord blood or peripheral blood—from patients admitted within the first 24 h of life for suspicion of sepsis and cord blood samples of a control group of 40 healthy mature infants delivered spontaneously were analyzed. For seven patients of the septic group, a second sample collected between 24 and 48 h of life was available. Levels of sCD14 and LBP in plasma were determined by an enzyme immunoassay using recombinant CD14 and LBP as standards. LBP and sCD14 were correlated to cytokine plasma levels. In septic neonates, LBP (median, 36.6 versus 7.8 μg/ml; P < 0.001) and sCD14 (median, 0.42 versus 0.28 μg/ml; P < 0.001) levels were highly elevated when compared to those of healthy neonates and strongly correlated to granulocyte colony-stimulating factor (G-CSF), interleukin-1β (IL-1β), IL-6, and IL-8 levels. LBP levels in septic neonates analyzed between 24 and 48 h of life even increased when compared to samples obtained at or shortly after delivery (median, 36.6 versus 60 μg/ml; P = 0.038). In summary, levels of LBP in plasma of neonates with early-onset sepsis are significantly elevated; the elevated plasma levels seem to persist for more than 24 h, which could provide the clinician with a prolonged time period to identify the newborn with bacterial sepsis.

Different efficacy of soluble CD14 treatment in high- and low-dose LPS models

About 50% of septic shock cases are attributed to Gram‐negative bacteria or their cell wall compound lipopolysaccharide (LPS, endotoxin). An attractive therapeutic strategy could target the binding of LPS to its cellular receptors. In vitro the soluble form of the endotoxin receptor CD14 (sCD14) competitively prevents binding of LPS to membrane‐bound CD14 and inhibits LPS‐stimulated macrophage responses.

CHO transfectants produce large amounts of recombinant protein in suspension culture

Chinese hamster ovary (CHO) cells transfected with various genes are widely used as adherent cell monolayers to produce recombinant proteins. In this report we present a new culture technique for CHO cells transfected with the vector pPOL-DHFR-CD14 using a minifermenter (miniPERM, Heraeus) for the production of recombinant human endotoxin receptor CD14 (rCD14). The transfectants were cultured for 12-17 days under serum-free conditions and formed spheroids. From this system we harvested supernatants containing up to 3.1 mg/ml recombinant CD14 (rCD14). This represents a 200-fold increase of rCD14 yield compared to conventional adherent CHO cell culture.

Implications for a general role of LPS-binding proteins (CD14, LBP) in combating bacterial infections

An invading pathogen must be held in check by the innate immune system until a specific immune response can be mounted. In the case of Gram-negative bacteria, the principal stimulator is LPS, a component of the outer membrane of the bacteria. In vitro LPS is bound by LBP and transferred to the LPS receptor CD14 on the macrophage surface. Binding to CD14 triggers an inflammatory response which is crucial for keeping an infection under control. In vitro, LBP mediates a response not only to LPS but also to intact Gram-negative bacteria. We show that whole Escherichia coli bacteria are recognised by CD14 on human monocytes, and subsequently may become phagocytosed. Although neither LBP nor CD14 interact with the heat inactivated, intact Gram-positive bacterium Bacillus subtilis both proteins form stable complexes with lipoteichoic acid derived from the bacterial cell wall. A brief exposure of B. subtilis to serum or antibiotics converts them into a form which can be recognised by CD14 in an LBP-dependent manner followed by phagocytosis. In preliminary experiments, it is shown that LBP is essential in vitro for the oxidative burst response of mouse macrophages induced by living Salmonella typhimurium as well as Staphylococcus epidermidis. Our results indicate that, in addition to CD14, LBP is also a pattern recognition element and is required to induce a rapid inflammatory response to Gram-negative as well as to Gram-positive bacteria and to initiate their phagocytosis.