LBP complexes: a short review

Rex Immunol. 1992, 143, 11-15

0 INSTITUT PASTEUR/ELSEVIER

Paris 1992

Function of lipopolysaccharide (LPS)-binding protein (LBP) and CD14, the receptor for LPWLBP complexes: a short review R.R.

Schumann

Institute for Molecular and Cell Biology, Department of Internal Medicine Albert-Ludwigs-University of Freiburg, 7800 Freiburg (Germany)

I,

SUMMARY With the recent discovery and cloning of the lipopolysaccharide-binding protein (LBP), the “adapter-molecule” for LPS-binding to the cell surface receptor CD14 was found. The ligand-receptor pair LPS/LBP-CD14 seems to be one important element in LPSmediated activation of monocytic cells and possibly granulocytes and B cells. Here, some of the known functions of the proteins involved, LBP and CD14, are reviewed in the context of other endotoxin recognition studies, and the outlook for ongoing and future investigations is described.

Key-words: tics;

Short

LPS, CD14, review.

Monocyte;

Binding

Introduction

One of the cell wall products of Gram negative bacteria, namely, lipopolysaccharide (LPS), is responsible for a cascade of cellular responses in the host organism when it enters the bloodstream. Different cells appear to be the targets of LPS and react in a way that subsequently leads to endotoxic shock (Young, 1990). Monocytic target cells react to LPS with the release of a number of cytokines (Adams and Koerner, 1988), B cells start proliferation and surface IgG production (Dziarski, 1989) and neutrophils are primed by LPS (Vosbeck et al., 1990). Monocytes after LPS induction also start the release of complement components and tissue factor (Semeraro et al., 1983) and LPS induces the transcription of the HIV1 proviral genome in latently infected human monocytes (Pomerantz et a/. , 1990). An important early event in LPS-induced cell activation seems to be the tyrosine phosphorylation of a number of

Submitted November 8, 1991, accepted December 4, 1991.

protein,

Septic

shock,

TNF,

Therapeu-

proteins in murine and human macrophages (Weinstein et al., 1991a). In vivo as well as in vitro experiments have shown that, in Gram negative sepsis, one of the primary and most dramatic events for the host is the activation of monocytic cells and the induction of cytokine production in these cells (Mathison et al., 1988). While mild stimulation of the defence system via LPS-induced monocyte activation, resulting in an appropriate response, has a beneficial effect, flooding of the organism with cytokines like tumour necrosis factor (TNF) or interleukind (IL6), or disturbance of the coagulation balance, may be detrimental to the organism. Because of the broad range of effects of LPS on the organism, many studies have focussed on the regulatory mechanisms involved in LPSmediated cell responses. Several molecules have been found which appear to be candidates for cellular LPS receptors and may act synergistically or specifically for certain LPS subtypes (Couturier et al., 1991).

12

R.R. SCHUMANN

Studies have found that most of the circuiating LPS in serum is bound to a protein that was recently discovered, purified and cloned and that was termed lipopolysaccharide-binding protein (LBP) (Tobias el al., 1986 ; Schumann el al., 1990). Complexes of LPS and LBP now seem to be recognized and specifically bound by a cellular receptor on myeloid cells, the surface antigen CD14 (Wright et af., 1990). This newly discovered ligand-receptor pair has been studied in different experimental setups using different cell types, monoclonal antibodies (mAb) and activity assays to gain further insight in to LPS-mediated cell activation. In parallel, investigations of other possible LPS recognition systems in the host are currently in progress. Recent findings

24 h (Schumann et al., unpublished results); thus far, no clear interpretation of this phenomenon has been found. The rise in LBP synthesis could occur as a reaction to depletion of circulating LBP during shock; however, the (relatively late) rise in LBP does not explain the development of shock, since the first shock symptoms are usually seen much earlier (after 6-8 h). As far as we know, the LPS effects on B cells cannot been augmented by the addition of LBP; moreover, in experimental setups in which high levels of LPS were used, no LPS-enhancing LBP effect could be seen either. Blocking experiments revealed that pretreatment of serum with antibodies against LBP and subsequent depletion of LBP from the serum resulted in a much weaker response to LPS challenge compared to LBPcontaining serum (Schumann et al., 1990).

of CD14

Function LBP is produced in hepatocytes as a 50-kDa protein and is constitutively secreted into the bloodstream at a concentration of approximately 500 rig/ml (Ulevitch et al. 1990b). The protein concentration rises in the “acute phase” to 50 Kg/ml, and LBP, which does not have activity by itself, binds to LPS with high affinity. Upon binding to LPS, LBP does not suppress or block the effects of LPS, but enhances endotoxin effects and abrogates cellular responses at subthreshhold LPS levels. A brief summary of published LBP effects is given in table I. LPS-induced TNF production and TNF-mRNA expression in rabbit peritoneal macrophages, for exampie, is enhanced strongly when LPS is complexed to LBP (Schumann et al., 1990). Rabbit peritoneal macrophages that are rendered unresponsive to LPS stimulation by a process called adaptation (Mathison et al., 1990) can have their ability to produce TNF restored by the addition of LBP. Also, macrophages detect and bind LPS much faster when it is complexed with LBP ; it thus acts as an opsonin for Gram-negative bacteria (Wright el al., 1989). Furthermore, the LPS-induced response in neutrophilic granulocytes can be enhanced by the addition of LBP to the system (Vosbeck et al., 1990). The biological effects of LBP can thus be summarized as complexing to LPS and subsequently enabling the organism to detect small amounts of LPS better and to trigger the defence cascade in a stronger way as compared to LPS alone. During the “acute phase”, synthesis of LBP-RNA and protein rises dramatically within

LBP LPS mAb

= = =

LPS-binding protein. lipopolysaccharide. monoclonal antibody.

CD14 is a 53-kDa glycoprotein found on the cell surface of myeloid cells and was generally termed “differentiation antigen”, as no specific function of this antigen was known (Goyert et al., 1986). Several cell lines express CD14, and immature myeloid cell lines can be driven to express CD14 by reagents known to induce myeloid cell differentiation (Graziano er al., 1983). Cellular CD14 is anchored in the plasma membrane, and a soluble form of the protein also exists and can be found in human serum (Bazil et al., 1989). Several mAb are commercially available which apparently detect different subepitopes of the CD14 antigen. They differ in their ability to block certain cellular functions, as is shown briefly in table II. In experiments that gave first evidence for CD14 being the receptor for LBP/LPS complexes, anti-CD 14 mAb 3C IO and 6Ob were able to inhibit LBP/LPS-mediated effects, including TNF production and opsonin function (Wright et al., 1990). The 3C10 mAb was also recently shown to be able to block LPS-induced protein tyrosine phosphorylation at concentrations similar to those blocking TNF production (Weinstein et al., 1991b). Engagement of monocytes with anti-CD14 mAb P9, ICM2 and My4, but not 3C10, revealed certain additional effects : LFA-1 /ICAM-l-dependent homotypic adhesion (Lauener ef al., 1990) along with suppression of monocyte-dependent T-cell proIiferation induced by various agents (Lue et al., 1991) in reaction to binding of the antibody to monocytes was observed. Induction of oxidative burst activity

PI TNF

= =

phosphatidyi inositol. turnour necrosis factor.

FUNCTION

OF LPS-BINDING

PROTEIN

AND

Table I. Effects of LBP in different experimental

Experimental

system

Rough and smooth form of LPS Rabbit macrophages (PEM) : - subthreshhold LPS stimulation - low-dose LPS stimulation - “adapted” cells, LPS stimulation Monocyte LPS attachment Human neutrophilic granulocytes : - 0, production, LPS-induced - FMLP receptor expression

CD14

13

setups.

LBP effect Binding to lipid A TNF response Enhanced TNF response Restored TNF response Enhanced (opsonin) Enhanced Enhanced

PEM = peritoneal elicited macrophages. FMLP = N-formyl-methionyl-Ieucyl-phenylalanine.

Table II. Blocking and enhancing effects of different

anti-CD14

mAb in different systems.

Anti-CD 14

Effects

3ClO

Blocking of TNF production and opsonin function of LBP, blocking of protein tyrosine phosphorylation Blocking of TNF production and opsonin function of LBP LFA-1 /ICAM-I -dependent homotypic adhesion, blocking of monocyte-dependent T-cell proliferation Oxidative burst activity in human monocytes

60b P9, ICM2,

Romo 1

My4

References given in the text.

in human monocytes, as detected by luminoldependent chemiluminiscence, was seen when monocytes were incubated with the anti-CD14 mAb Romo 1 (Schutt ef al., 1988). Cellular receptors usually are transmembrane molecules, as they transduce signals into the cell. CD14, however, recently was found to be linked to the cell surface via a phosphatidyl isonitol anchor (PI) that leaves it mobile in the plane of the membrane (Haziot et al., 1988). Numerous proteins share the PI linkage, including : the adhesion molecules LFA-1, -3 and N-CAM, cell surface hydrolases, alkaline phosphatase and acetylcholinesterase, the lymphoid antigens Thy-l, -3, CD24 and -73, carcino-embryonic antigen and decay-accelerating factor. Although the mechanism of signal transduction via PI is not clear, several PI-anchored proteins exist in the organism

with signalling capacities, including CD14, Thy-l and LFA-3 (Robinson, 1991). Internalization of the receptor upon binding of the ligand or interaction with (“presentation” to) an additional membrane protein are possible mechanisms of signal transduction via the PI-linked CD14 molecule. The existence of a second, slightly smaller, soluble form of CD14 that can be found in normal serum, most likely due to .shedding of the surface CD14 (Bazil and Strominger, 1991), supports the theory that binding of the ligand followed by shedding of the receptor might be a defence mechanism for this type of protein (Huizinga et al., 1988). Interestingly, the gene for CD14 is located on the 5th chromosome in a region known to encode for several cytokines, including GM-CSF (granulocyte-macrophage colonystimulating factor), CSFl, IL3, and endothelial cell growth factor, as well as receptors like CSF-lr, platelet-derived growth factor receptor, human c-fms protooncogene and P-adrenergic receptor (Goyert et al., 1988). Loss of this region (“q23-31”), also referred to as the “critical region”, is associated with certain forms of leukaemia, like acute nonlymphoblastic leukaemia (ANLL), t-ANLL and myelodysplastic syndrome. Conclusion and outlook

Binding of LPS by the serum protein LBP and the direction of this complex to the CD14 receptor is an unusual mechanism that might reflect the importance and complexity of endotoxin recognition, but which is not yet thoroughly understood in detail. It appears most likely that, after LPS entry into the bloodstream, other soluble proteins and effector cell surface molecules also play a role. Known serum proteins that bind to the lipid A moiety of LPS include albumin, the complement proteins Cl and C3 and

the high density lipoproteins which seem to have a detoxifying as well as a down-modulating effect on LPS activities (Ulevitch ef al., 199Oa). Evidence exists that, on the cell surface, the CD1 l/CD18 Leu-CAM complex also binds directly to LPS (without involvement of LBP), at least non-specifically (Wright ef al., 1986). A 73-kDa protein that is present on many cell types (Lei el al., 1991), an I8-kDa protein in the membrane of 702/3 cells (Kirkland et al., 1990) and two proteins of 95 and 31 kDa in the membranes of RAW 263.1 cells (Hampton et al., 1988) have also recently been identified as binding to LPS. A lectinlike monocyte membrane molecule was shown to interact with the sugar part of LPS (HaeffnerCavaillon, 1985) and recently in the granules of neutrophilic granulocytes, a protein was found that binds to LPS, is bactericida1 and has 44@?0sequence homology with LBP (Weiss et al., 1978 ; Schumann et a/. , 1990). In addition, hybrophobic interactions, mediated by the lipid A moiety of LPS with the lipid bilayer of the cell membrane, have been postulated and must be taken into consideration. One explanation for this relatively large group of candidate and/or proven LPS receptors distinct from the CD14 receptor is that different LPS-recognizing molecules may be able to detect various subtypes of LPS (from different bacterial strains) or that distinct cellular LPS recognition and reaction pathways could be responsible for distinct cellular reactions (Couturier ef al., 1991; Raetz e? al., 1991). Also, an even more complex combination of LPS-recognizing entities appears possible, for example, the interaction of another membrane-bound receptor with the PIanchiored CD 1Ltreceptor . Recombinant and mutated proteins, synthetic LPS derivatives (Lam et al., 1991) and more advanced mAb will help in elucidating the LPS-recognition mechanisms. Measurements of the serum levels of proteins involved and of the surface expression of cellular proteins in normal and pathologic groups of patients have begun and will also aid in elucidating mechanisms that lead to endotoxic shock. In addition, a disease called paroxysmal nocturnal haemoglobinuria, in which patients lack all PI-anchored proteins, as well as CD14+ leukaemias, are interesting in viuo modeIs for studying CDI4-related endotoxin recognition events. One method currently under clinical investigation for interfering with processes that lead to endotoxic shock involves the application of anti-LPS mAb to patients in shock (Ziegler et al., 1991). In addition to this direct means of therapy by elimination of the primary cause of the disease, an understanding of the complex mechanisms of the host reaction may lead to additional therapeutic measures for interfering with the cascade of events involved in endotoxinaemia-mediated shock.

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