CD14 and other recognition molecules for lipopolysaccharide: A review

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Immunopharmacology

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Immunopharmacology 29 (1995) 187-205

Review Article

CD14 and other recognition molecules for lipopolysaccharide: a review Tammy L. Kielian, Frank Blecha* Department of Anatomy and Physiology. College oj Veterinary Medicine, Kansas State University, Manhattan. KS 66506. USA

Received 31 October 1994; accepted 4 January 1995

Abstract Lipopolysaccharide (LPS) or endotoxin elicits a broad, non-specific cascade of events in vivo, resulting in secretion of a variety of potent mediators and cytokines produced primarily by activated macrophages and monocytes. The overproduction of these effector molecules, such as interleukin-1 and tumor necrosis factor-~, contributes to the pathophysiology of endotoxic shock. Cellular recognition of LPS involves several different molecules, including cluster of differentiation antigen C D 14. A thorough understanding of the interaction of LPS with cells of the immune system is necessary before effective preventative or therapeutic measures can be designed to limit the host response to endotoxin. This review discusses the role of CD14 and other LPS-recognition molecules in LPS-mediated macrophage activation. KeA'words: CD14; Lipopolysaccharide receptor; LPS-binding protein; Macrophage

1. Introduction

Septic or endotoxic shock induced by Gramnegative bacteria is a serious medical problem associated with a high mortality rate. In the United States approximately 500,000 individuals suffer from sepsis annually, of which 175,000 die (Stone, 1994). It is

* Corresponding author. Tel: ( + 1-913) 532-4537; Fax: ( + 1-913) 532-4557. Abbreviations: CD, cluster of differentiation; EC, endothelial cell; GM-CSF, granulocyte macrophage-colony stimulating factor; HIV-I, human immunodeficiency virus-l; HUVEC, human umbilical vein endothelial cell: IL, interleukin; LPS, lipopolysaccharide; LBP, lipopolysaccharidc binding protein; mCDI4, men)brane CDI4; mAb, monoclonal antibody; PI-PLC, phosphatidylinositol-specific phospholipase C; PGE> prostaglandin E,; sCD14, soluble CD14; TNF-~, tumor necrosis factor-:~ 0162-3109/95/$9.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 2 - 3 1 0 9 ( 9 5 ) 0 0 0 0 3 - 8

o of septic cases originate from estimated that 4 0.5 Gram-negative infections (Stone, 1994), in which lipopolysaccharide (LPS), a constituent of the outer cell wall of Gram-negative bacteria, is responsible for the overwhelming immune response in the host. Lipopolysaccharide or endotoxin is composed of three major subunits; an O-specific antigen comprised of long repeating side chains; the central polysaccharide core linked by KDO regions to the final major subunit, lipid A, considered to represent the biologically active component of LPS. For several years, investigators have attempted to produce both preventative and therapeutic measures to attenuate the host response to endotoxin. The pathophysiology observed with cases of endotoxic shock, such as hypotension, disseminated intravascular coagulopathy, and multiorgan failure are not a consequence of direct effects of endotoxin, but rather

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originate from the overproduction of secretory mediators from LPS-activated monocytes and macrophages, the most potent of which include interleukin1-beta (IL-1/~) and tumor necrosis factor-alpha (TNF-~) (Morrison and Ryan, 1987). Some therapeutic regimens which have been attempted to counteract the overactive immune system include, systemic administration of antibody directed against the lipid A portion of LPS, administration of lipid A precursors that function as competitive antagonists of lipid A, or antibody directed against IL-1/~ and TNF-c~ (Stone, 1994). However, these efforts have met with limited success, due likely to the ability of endotoxin to induce a broad activation of nonspecific responses in the host. The redundancy of effects elicited by LPS provide a challenge for the development of therapy, since targeting one aspect of the endotoxin response will more than likely have no effect on the outcome of the patient. Therefore, the search for effective therapeutic regimens for individuals suffering from LPS-induced septic shock continues. Recently, a cell surface receptor has been identified on monocytes and macrophages which displays affinity for LPS in association with LPS-binding protein (LBP), an acute-phase reactant produced by the liver. In the presence of LBP, CD14 has been implicated as a high-affinity LPS receptor, facilitating LPS-induced macrophage activation in response to subnanogram concentrations ofendotoxin. In addition, several alternate LPS receptors have been described which may participate in the host response to endotoxin. Elucidation of the mechanisms by which LPS interacts with and activates effector cells to produce mediators such as TNF-:~ and IL-1/3 will lead to more efficient methods of interventive and preventative therapy for Gram-negative sepsis. This review addresses the functional role of CDI4 in LPS-mediated macrophage activation, LBP and soluble CD14 (sCD14), two proteins involved with LPS-induced cell activation, in addition to alternate LPS recognition molecules.

2. Overview of CD14

Cluster of differentiation antigen 14 (CD14) was originally described during the First Leukocyte

Typing Workshop in 1982 (Bernard et al., 1984), in which a panel of ten monoclonal antibodies (mAb) displayed high affinity for human peripheral blood monocytes (Todd et al., 1984; Griffin and Schlossman, 1984). At the Third Leukocyte Typing Conference in 1986, these CD14 mAb were shown to identify a broad range of epitopes (Hogg and Horton, 1986) and recognized purified CD14 antigen in Western blots (Bazil et al., 1986a) and immunoprecipitates (Goyert and Ferrero, 1986) of CD14positive cells. CD14 is a 55 kilodalton (kDa) glycoprotein expressed on the surface of monocytes, macrophages, and neutrophils (Todd et al., 1984; Griffin and Schlossman, 1984; Hogg and Horton, 1986). The number of CD14 molecules on peripheral blood monocytes has been estimated at 50,000 cell- 1 (Van Voorhis et al., 1983), whereas the level of expression on peripheral blood neutrophils is tenfold lower (Wright et al., 1991a). Its expression on B cells has been reported (Labeta et al., 1991; Morabito et al., 1987; Zeigler-Heitbrock et al., 1994a), however, it is generally accepted that normal B cells are CD14negative. CD14 was originally identified as a myeloid differentiation antigen, present on mature cells but absent on myeloid precursors. Its expression has also been correlated with various forms of malignant myeloid leukemias, and has been used in aiding diagnosis. Recently, CD14 has been assigned a functional role, serving as a receptor for LPS in association with LBP in which heparinized human blood cultured for 16 h with LPS resulted in TNF-:~ production, whose synthesis and release could be nearly eliminated by pre-treatment with a CD14 blocking mAb (Wright et al., 1990a). This suggested that CD14 plays an important role in LPS-mediated TNF-c~ production. Furthermore, binding of erythrocyte-LPS complexes to macrophages could be inhibited by preincubation of macrophages with anti-CD14 blocking mAb (Wright et al., 1990a). However, it has become apparent that the available CD 14 mAb recognize a substantial array of epitopes on the CD14 molecule (Schuman, 1992). The predicted amino acid composition of CD14 does not contain a traditional transmembrane sequence that would be involved in cell signaling (Haziot et al., 1988). Rather, CD14 is anchored to the plasma membrane by a phosphatidylinostiol linkage and is

T.L. Kielian, F. Blecha / lmmunopharmacology 29 (1995) 187-205

classified as a member of the PI-anchored family of proteins (Haziot et al., 1988; Simmons et al., 1989). Treatment of monocytes with phosphatidylinositolspecific phospholipase C (PI-PLC), specific for the cleavage of all PI-anchored molecules, resulted in incomplete removal of CD14 from the cell surface (Haziot et al., 1988). Additionally, in patients suffering from paroxysmal nocturnal hemoglobinuria, a condition characterized by a deficiency of all PIanchored molecules, monocytes are CD 14 deficient (Haziot et al., 1988; Simmons et al., 1989). Because CD 14 lacks a traditional transmembrane domain, its direct role in signal transduction in response to LPS has been seriously questioned, and remains somewhat controversial. Cell surface CD14 has been shown to co-precipitate with tyrosine kinase activity in monocyte whole-cell lysates, suggesting a potential mechanism of signal transduction by CD14 in response to LPS (Stefanova et al., 1991). Additionally, LPS-induced protein tyrosine phosphorylation has been shown to be specifically inhibited by antiCD14 mAb treatment in response to low concentrations of LPS (1 ng ml- 1) (Weinstein et al., 1993; Han et al., 1993). However, at higher concentrations of LPS (i.e. > 10 ng ml- L), anti-CD14 mAb do not abrogate protein tyrosine phosphorylation, suggesting a lower affinity CD14-independent pathway also participates in the tyrosine phosphorylation response. An alternative mechanism has been proposed for CD14-dependent signal transduction which involves intracellular G-protein translocation (Yasui et al., 1992). The serum-dependent binding of LPS to CD14 resulted in the redistribution of intracellular G-protein to the cell membrane and likewise, antibody directed against CD 14 reduced the amount of G-protein associated with the membrane in response to LPS and serum. Other lines of evidence suggesting that CD14 plays a pivotal role in signal transduction have included the ability of certain CD 14 mAb to induce superoxide anion production, Ca 2+ flux, and IL-1 synthesis in human peripheral blood monocytes (Lund-Johansen etal., 1993; Schutt et al., 1988; Macintyre et al., 1989). Additionally, electron microscopic analysis of human monocytes incubated with the gold-labelled RoMo- 1 anti-CD14 mAb, demonstrate that CD14 receptor internalization occurs through uncoated invaginations characteristic of receptor-mediated endocyto-

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sis, which may serve to transduce a signal (Schutt et al., 1988; Jonas et al., 1990). Alternative mechanisms have been proposed for CD14-mediated macrophage activation by LPS. Comparing responses of GPI-anchored versus integral membrane CD14 to LPS in terms of nuclear factor-~c/3 activation and protein tyrosine phosphorylation, it has been suggested that binding of endotoxin to cell-surface CD 14 is followed by subsequent interaction of LPS-CD14 with other membrane component(s) which actually serve to transduce a signal (Lee et al., 1993). This model implies that CD 14 functions as the principal binding unit for LPS and is not directly involved in cell signaling. It is also possible that CD14 may serve to transport LPS to the cell surface in such a manner that other proteins or receptor subunit(s) are stimulated (Raetz etal., 1991). Because CD14 is a GPI-anchored membrane glycoprotein, it should possess adequate mobility in the plane of the cell membrane to associate with other membrane receptors/components which may in turn initiate signal transduction. Additionally, it has been suggested that the CD14 receptor may be similar to the IL-2 high-affinity receptor where both c~and/3 subunits are required for high-affinity binding, whereas expression of only one subunit confers low affinity interactions with ligand (Lee et al., 1992). Alternatively, CD14 may resemble the IL-6 receptor, a heterodimer comprised of a ligand binding unit and a non-ligand binding glycoprotein. Whether CD14 directly participates in transmembrane signaling remains elusive, and a complete understanding of CD14 in LPS-induced cell signaling warrants further study. The CD14 cDNA sequences for human, mouse, and rabbit have been elucidated and reveal a considerable amount of conservation between species. Amino acid sequence comparisons deduced from cDNA revealed that the rabbit CD14 gene shares 73~o and 64~o homology with human and murine CD14, respectively (Lee et al., 1992). The murine CD14 gene has been cloned (Matsuura et al., 1989) and mapped (Ferrero et al., 1990) to chromosome 18 and the human CD14 gene has been mapped to chromosome 5 (Goyert et al., 1988). The human CD 14 gene clusters within a region that encodes for other growth factors and receptors such as IL-3, granulocyte-macrophage colony stimulating factor

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(GM-CSF), M-CSF, platelet-derived growth factor receptor, and the fl-adrenergic receptor and is located within the 'critical region' that is frequently deleted in certain myeloid leukemias (Goyert et al., 198S). The CD 14 protein sequence contains several features that are characteristic of glycoproteins. This includes 17 hydrophobic and neutral amino acids at the carboxyl terminus and five potential sites for N-linked glycosylation (Goyert et al., 1988). The molecule also contains a leucine-rich motif which has been proposed to interact with lipids by forming amphipathic fl-pleated sheets (Setoguchi etal., 1989). Properties of sCD14, which may represent a truncated form of membrane CD14 (mCD14), include a 42 kDa single polypeptide chain containing intrachaln disulfide bonds, and a sialyated carbohydrate of approximately 11 kDa (Bazil et al., 1986b).

3. Functional role and regulation of CD14 expression

A functional role for CD14 in LPS recognition was first described in 1990, where Wright and colleagues demonstrated that LPS-induced TNF-:~ production in whole blood was CD 14-dependent, as evident by the ability of an anti-CD 14 mAb to inhibit LPS-induced TNF-c~ production (Wright etal., 1990a). Others have reported that CD14 plays an important role in macrophage production of TNF-:~ (Martin et al., 1992; Huemann et al., 1992; Couturier et al., 1992) and IL-1 (Couturier et al., 1992) in response to LPS. A recent report described the importance of CD14 in LPS recognition by human monocytes and alveolar macrophages, leading to the synthesis and release of TNF-~, IL-6, and IL-8 (Dentener et al., 1993). The LPS-induced synthesis of these cytokines could be blocked with the antiCD14 mAb MEM-18, whereas cytokine secretion induced by stimulation with either zymosan, PMA, IL-lfl, or TNF-~ was not affected by anti-CD14 treatment, reflecting the importance of CD14 in LPS-mediated cell activation. The significance of CD14 in LPS recognition was demonstrated using the murine 70Z/3 pre-B cell line transfected with human CD14 cDNA (Lee et al., 1992). 70Z/3 cells are sensitive to LPS, however, activation requires

high concentrations of LPS. Transfection of human CD 14 cDNA reduced the amount of LPS needed to activate these B cells (as measured by surface IgM expression) by up to 10,000-fold as compared to the 70Z/3 parent cells, suggesting that CD 14 represents a high affinity receptor for LPS. Recently, a human CD14 transgenic mouse model has been described that may serve as an excellent model to study the role of CD14 in response to LPS in vivo (Ferrero et al., 1993). These mice express high levels of CD 14 on peripheral blood monocytes, macrophages, and Thy-1 positive lymphocytes. With this model, CD 14 transgenic mice were hypersensitive to intraperitoheal injection of LPS as compared to non-transgenic mouse controls, and exhibited symptoms of endotoxin shock and mortality at a threefold lower dose of LPS than the LD100 for non-transgenics. CD14 expression has been reported to be influenced by a variety of factors including in vitro monocyte to macrophage maturation, various cytokines, and lipopolysaccharide. Several studies have reported increases in CD 14 antigen expression during the in vitro differentiation of peripheral blood monocytes to macrophages (Landmann et al., 1990; Gessani et al., 1993; Kreutz et al., 1992). However, these findings may not be representative of a universal pathway of monocyte differentiation events in vivo, where one finds a heterogeneous expression of CD14 on mature tissue macrophages which is dependent on the tissue of origin. For example, human alveolar macrophages have been reported to express low levels of CD 14, whereas peritoneal macrophages possess much higher levels of membrane CD14 (Passlick et al., 1989; Biondi et al., 1984; Andreesen et al., 1990). Therefore, differences in tissue microenvironment, either in terms of cytokine exposure or cell-cell interactions may play a role in dictating the mature macrophage phenotype in vivo. A recent study has examined the relationship between the appearance of CD 14 expression and the acquisition of LPS responsiveness during monocyte differentiation (Martin et al., 1994). The addition of 1-25 dihydroxyvitamin D 3 to the premonocytic cell line THP-1 induced CD14 expression which correlated with LPS responsiveness. The addition of mAb to CD14 blocked the responses to both smooth and rough forms of LPS in the differentiated THP-1 cells. Therefore, these data suggest that the maturation of

T.L. Kielian, F. Blecha / Immunopharmacology 29 (1995) 187-205

the response to LPS in serum is partially dependent on CD14 expression and the presence of LBP in serum. lnterleukin-4 has been shown to induce a dosedependent decrease in CD14 expression on monocytes in vitro in several studies (Landmann et al., 1990; Ruppert et al., 1991; Lauener et al., 1990a; Landmann et al., 1992). The reason for the observed decrease in CD 14 expression when cells are cultured with IL-4 is not clear. A recent study demonstrated that IL-4 and IFN-v down-regulate sCD14 release in human monocytes and macrophages in vitro (Landmann et al., 1992), suggesting that decreased mCD14 expression resulting from IL-4 treatment may not be the result of receptor shedding into culture supernatant. In addition, IFN- 7 has been reported to down-regulate mCD14 expression in vitro (Landmann et al., 1990; Lauener et al., 1990a; Landmann etal., 1991), whereas other cytokines such as GM-CSF, IL-lfi, IL-2, IL-3, 1L-5, IL-6, and transforming growth factor-/3 do not appear to exert effects on CD14 expression in human macrophages (Landmann et al., 1990; Lauener et al., 1990a; Ruppert et al., 1991; Landmann et al., 1991). Tumor necrosis factor-~ has been reported in one study to enhance CD14 expression on 4-day-old human monocyte cultures (Wright, 1991b). However, other studies report that TNF-~ has no effect on CD14 expression (Landmann et al., 1990; Lauener et al., 1990a; Ruppert et al., 1991; Landmann et al., 1991). In human peripheral blood neutrophils, TNF-e, GM-CSF, G-CSF, and formyl peptide have been shown to induce a twofold increase in CD14 expression (Wright et al., 1991a). Due to the short period of time required to detect upregulation (20 min), receptors were determined to be derived from preformed cytoplasmic stores of protein. However, another report suggests that the in vivo infusion of rhG-CSF but not rhGM-CSF enhances CD14 expression on peripheral blood neutrophils (Hansen et al., 1993). Whether the variations observed in CD14 receptor regulation in response to cytokine treatment are a consequence of the cell type examined, species variation, the concentration of cytokines used in experimental treatments, or the time intervals used to evaluate CDI4 expression is currently unknown. The effects exerted by LPS on CD14 expression,

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appear controversial varying from no effect to either enhancement or reduction in CD 14 expression. Several reports have demonstrated that LPS stimulation of whole blood (Marchant et al., 1992), normal peripheral blood monocytes (Brugger et al., 1991; Birkenmaier et al., 1992), alveolar macrophages (Kielian et al., 1995), or monocyte-like cell lines (Ziegler-Heitbrock et al., 1994b; Ikewaki et al., 1993) induces an increase in cell-surface CD14 expression. However, LPS has also been reported to reduce CD14 expression on monocyte-derived macrophages (Wright, 1991b), monocytes (B azil and Strominger, 1991), and on monocytes isolated from human sepsis patients (Birkenmaier et al., 1992). In addition, intravenous infusion of endotoxin into healthy human volunteers had no effect on CD14 expression on alveolar macrophages 6 h postinfusion (Smith et al., 1994), as did the in vitro stimulation of alveolar macrophages 4 h following LPS exposure (Martin et al., 1992). A recent study using the human monocyte-like cell line Mono Mac 6, demonstrated that 757o of unstimulated cells stain with the anti-CD14 mAb My4 which could be enhanced more than twofold with LPS treatment (Zeigler-Heitbrock et al., 1994b). LPS-induced CD 14 upregulation coincided with elevated levels of CD14 mRNA, which achieved a maximal ninefold increase two days following LPS stimulation. Therefore, the presence of LPS appears to upregulate CD 14 transcripts, followed by increased expression of C D I 4 protein on the cell surface. These differences in the effects of LPS stimulation on CD14 expression suggest that the regulation of CD14 is dependent on both the stage of cell maturation and the length of time that the cell is exposed to LPS. It is not clear what has contributed to the discrepancy of effects observed with LPS treatment on CD14 expression, but some possibilities include experimental variations such as the LPS chemotype and amount used for stimulation, culture conditions, time intervals when CD14 expression is examined, or quite possibly the phenotype and stage of differentiation of the cell in question. Since CD14 is regarded as a sensitive receptor for LPS, it is logical to think that its expression can be either directly regulated by LPS or indirectly by the action of LPSinduced mediators (i.e., cytokines, prostaglandins, etc). Therefore, the effects of LPS on both neutro-

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phil and macrophage CD14 expression warrant further investigation. Aside from its involvement as a myeloid differentiation antigen and LPS receptor, other functions have been suggested for CD14. A role for CD14 in cell-cell interactions has been reported (Beekhuizen et al., 1991) in which anti-CD14 mAb treatment prevented the adhesion of monocytes to cytokineactivated endothelial cells. Another report has demonstrated that treatment of normal human monocytes with a murine anti-CD 14 mAb induced homotypic adhesion, mediated by Ag-1/ICAM-1 interactions (Lauener et al., 1990b). Cellular adhesion induced by anti-CD 14 mAb treatment was demonstrated to be temperature and Mg dependent, and did not involve FcR engagement or CD14 crosslinking since F(ab)~ fragments could reproduce effects observed with the intact murine mAb (Lauener et al., 1990b). In a separate study, adherence of human peripheral blood neutrophils to human umbilical vein endothelial cells (HUVEC) was shown to be mediated at least in part, by CD14 since anti-CD14 mAb was capable of blocking adhesion (Worthen et al., 1992). Furthermore, an antiCD14 mAb has been shown to abrogate neutrophil actin assembly and retention in response to LPS, a mechanism believed to increase neutrophil stiffness and retention in small capillaries primarily in the lung (Erzurum et al., 1992). Some anti-CD14 mAb have been demonstrated to inhibit T cell proliferation induced by antigen through a mechanism requiring cell-cell contact between T cells and monocytes (Lue et al., 1991). A recent report has suggested the importance of CD14 in controlling the expression of human immunodeficiency virus- 1 (H IV- 1) in response to LPS using a latently infected cell line (Bagasra et al., 1992). The response to LPS by the latently infected cell line U1 is only observed when G M - C S F is added during culture, which induces CD14 expression on a subpopulation of U1 cells. The appearance of CD14 expression correlated with HIV-1 production stimulated by LPS and LPSinduced viral replication could be inhibited by pretreating cells with an anti-CD 14 mAb. In summary, it appears that CD 14 may participate in diverse cellular responses in addition to LPS recognition.

4. Soluble CD14 A soluble form of CD14 (sCD14) was originally described in cell culture supernatants based on its ability to block staining of monocytes with antiCD14 mAb (Maliszewski et al., 1985). Estimates of the molecular weight of sCD14 by SDS-PAGE are reported to be 50-53 kDa (Bazil et al., 1986b) which is smaller than the membrane form since sCD14 is believed to originate from enzymatic cleavage of mCD 14 via the action of specific phospholipases or proteases (Bazil et al., 1989) which have not yet been identified. Molecular characterization of sCD14 isolated from the urine of patients with secondary amyloidosis revealed that sCD14 is a single-chain protein containing intrachain disulfide bonds. Concentrations of sCD14 in normal human plasma have been estimated at 6 ~tg ml- 1 and increase in hospitaiized patients, especially those suffering from autoimmune disease (Bazil et al., 1986b). A recent study demonstrated that stimulation of the monocyte-like cell line Mono Mac 6 with LPS or prostaglandin E 2 (PGE2) resulted in increased concentrations of sCD14 detected in culture supernatants (Ziegler-Heitbrock et al., 1994b). It appears that normal peripheral blood monocytes may respond similarly depending on the stimulus; LPS and TNF-c~ have been shown to produce a dose-dependent increase in sCD14 (Schutt et al., 1992), whereas IL-4 decreases sCD14 levels in tissue culture supernatants (Landmann et al., 1992; Schutt et ai., 1992). Additionally, during monocyte to macrophage differentiation in vitro, the cumulative release of sCD 14 has been shown to be linear from days 1 to 7 of culture and correlated with cell number. Because of the relatively high concentrations of sCD14 in normal human plasma (6/zg ml- 1), investigators have become intrigued at elucidating a functional role for sCD14. Soluble CD14 has been shown to prevent the oxidative burst in human monocytes in response to LPS in vitro, theoretically by competing with mCD14 for LPS (Schutt et al., 1992). This observation was extended to suggest that sCD14 may function as a 'sponge' for LPS in the bloodstream, and might prove to be a new therapeutic concept in the prevention of endotoxic shock. Additionally, recombinant sCD14 produced using a baculovirus expression system was shown to specifically bind LPS

T.L. Kielian, F. Blecha / Immunopharmacology 29 (1995) 187-205

and was capable of inhibiting LPS-induced TNF-~ release from whole human blood in addition to monocytes and mononuclear cells (Haziot et al., 1994). However, recent evidence has suggested that sCD14-LPS complexes can activate cells that are normally deficient in membrane CD14, assigning a functional role to sCD14-LPS. Soluble CD14 has been shown to directly interact with LPS by use of native P A G E analysis of sCD14-LPS complexes (Hailman etal., 1994). These sCD14-LPS complexes have been reported to activate HUVEC, which are CD14-deficient (Haziot et al., 1993; Frey etal., 1992; Read etal., 1993). Activation of HUVEC by low concentrations of LPS is serum dependent, and immunodepletion of sCD14 from serum has been shown to abrogate cell activation (Frey et al., 1992; Read et al., 1993). A recent study has demonstrated that anti-CD14 mAb treatment was able to inhibit the release of IL-6 and E-selectin expression in HUVEC in response to LPS under serum-containing conditions, which was accompanied with a reduction in mRNA for the products (Von Asmuth etal., 1993). Inhibition of LPSinduced IL-6 and E-selectin expression by anti° CD14 mAb was maximal at low concentrations of LPS; with higher doses of LPS the degree of inhibition was less dramatic. In a separate study measuring the changes in endothelial cell monolayer integrity induced by LPS, the flux of 14C-labelled bovine serum albumin was measured under different experimental conditions (Goldblum et al., 1994). LPS-induced changes in enodthelial cell (EC) monolayer integrity were only observed under serum-containing conditions, and LPS-induced albumin flux across the monolayer could be prevented with anti-LB P or anti-CD 14 mAb. The effect of LP S in the presence of both LBP and sCD14 exceeded the effect observed in the presence of one protein alone, suggesting that both sCD14 and LBP function independently to present LPS to the endothelial cell. However, a recent report has suggested that the activation of EC by LPS is indirectly mediated by monocytes and mCD14 through the secretion of soluble mediators and downplays the direct pathway of LPS-sCD14 in EC activation (Pugin et al., 1993). Therefore, it seems possible that the serum pool of sCD14 may indeed function as an enormous reser-

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voir for LPS in the circulation, but that its function may serve to activate LPS-sensitive cells deficient in mCD14, resulting in widespread damage via production of cytokines and upregulation of adhesion molecules. From the in vitro data, one could speculate that a possible role for sCD 14 in endotoxic shock would include the interaction of LPS-sCD 14 complexes with EC inducing cell activation and resulting in massive upregulation of adhesion molecules and the synthesis of cytokines such as IL-1 and IL-8, as well as enhanced procoagulant activity on the endothelial cell surface. These changes induced by LPS-sCD 14 would result in enhanced cell adhesion/ trafficking, chemotactic activity, and coagulation respectively, all of which are pro-inflammatory and when in excess can be detrimental to the host. Therefore, sCD14 may not function merely to bind LPS and prevent its subsequent interaction with cellular LPS receptors, but rather may serve to activate CD14-deficient cells in response to LPS.

5. Lipopolysaccharide binding protein (LBP) Lipopolysaccharide-binding protein is a 60 kDa glycoprotein found in normal serum at concentrations of 0.5-10/~g ml- 1, with levels in acute-phase sera varying widely to above 200 #g ml- 1 (Tobias et al., 1992). In hepatocytes, LBP is synthesized intracellularly as a 50 kDa precursor and secreted as the mature 60 kDa glycosylated protein found in plasma (Ramadori et al., 1990). Definitive studies investigating cells capable of synthesizing LBP are limited; alveolar macrophage mRNA does not hybridize with probes for LBP and at this time hepatocytes have been identified as a major source of LBP (Tobias and Ulevitc, 1993). LBP has been identified in the serum of rabbits (Tobias et al., 1986), humans (Tobias et al., 1985), rats, mice, pigs, nonhuman primates (Tobias and Ulevitc, 1993) and cattle (Khemlani et al., 1994). The specificity of LBP is directed towards the hydrophobic lipid A portion of LPS (Tobias et al., 1989) and the affinity of this interaction has been estimated to be 10 -9 M for both smooth and rough forms of LPS (Mathison et al., 1992). Using cloned cDNAs to deduce the primary structure of human and rabbit LBP, it was found that a hydrophobic signal sequence of 25 residues

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precedes the mature protein of 452 residues (Schumann et al., 1990). The mature protein contains four cystines and five potential glycosylation sites, and shares approximately 44% sequence homology with human bactericidal/permeability-increasing protein found in neutrophil granules. Lipopolysaccharide-binding protein appears to be highly conserved, with human and rabbit LBP sharing 69°,o sequence homology (Schumann etal., 1990). To better characterize the structure-function relationship ofLBP, a truncated form of human LBP was constructed which contained amino acid residues 1-197 of native LBP (Han et al., 1994). This truncated form of LBP retained its ability to bind LPS but could not transfer LPS to either mCD14 or sCD14. From this study it appears that the LPS binding activity is contained within the aminoterminal half of LBP and the CD14 interaction site is found at the carboxyl-terminal half of LBP. Lipopolysaccharide-binding protein facilitates the interaction of LPS with CD14 by two basic processes. First, LBP acts as an opsonin for LPSbearing particulates enhancing the interaction with CD14 (Wright et al., 1989a), and secondly, LBP enables cells to respond to extremely low concentrations of LPS via a CD14-dependent pathway (Schumann et al., 1990). Recent evidence suggests that the LPS-binding activity of LBP is located in a structurally distinct site from its CD14-stimulatory activity, represented by the N-terminal 197 amino acids and C-terminal amino acids respectively, of the mature protein (Theofan et al., 1994). Lipopolysaccharide-binding protein opsonized both erythrocytes with LPS inserted into the cell membrane and LPS on the surface of Gram-negative bacteria and dramatically enhanced the interaction and uptake with neutrophils and macrophages (Wright et al., 1991a; Wright et al., 1989a). These LBP-LPS complexes were found to interact directly with mCD14 on monocytes/macrophages by use of a novel technique (Wright et al., 1990a). Macrophages were allowed to adhere to surfaces coated with anti-CD14 mAb, and because CD14 is a relatively mobile membrane protein, it diffused to the substrate attached portion of the adherent macrophage and was trapped by interactions with the CD 14 mAb. This in effect, depleted CD 14 from the apical surface of the cell, which in turn, abrogated

the ability of macrophages to bind LBP-opsonized LPS-erythrocyte complexes. Additionally, pretreatment of LPS-erythrocyte complexes with LBP resulted in enhanced uptake by macrophages, whereas pretreatment of macrophages with LBP had no effect suggesting that LBP does not form a stable complex with CD14. Moreover, macrophages did not recognize erythrocytes coated with LBP unless LPS was added which suggested that LPS-LBP complexes undergo a conformational change, subsequently recognized by CD14. Lipopolysaccharide-binding protein has also been shown to dramatically increase the sen sitivity of leukocytes to subnanogram concentrations of LPS. Experiments have utilized either purified LBP from acute-phase serum or 10~o FBS which is reported to contain substantial quantities of LBP to substitute as a source for purified LBP (Lee et al., 1992). In rabbit peritoneal macrophages, addition of LBP enhanced TNF-~ production from 40 U ml-~ to 10,000 U ml ~ when cells were stimulated with 100 ng ml- ~ of LPS (Schumann et al., 1990). This increase in TNF-~ protein was accompanied by a concomitant increase in the rate and synthesis of TNF-z~ mRNA as visualized on Northern blots. Immunodepletion of LBP from plasma with antiLBP mAb inhibited Salmonella minnesota Re 595 LPS-induced TNF-:~ production from rabbit whole blood without disrupting the response to Staphylococcus aureus-dependent TNF-~ synthesis (Schumann et al., 1990). In another report examining effects of LBP on rabbit macrophages, when LBP was added during LPS stimulation, macrophages responded with a more rapid induction of TNF-:~ and IL-lfl mRNA, higher steady state mRNA levels, and increased mRNA stability which was shown to correlate with increased TNF-~ and IL-lfi protein (Mathison et al., 1992). These effects of LBP were demonstrated to be lipid A dependent and extended to either smooth or rough forms of LPS in which lipid A is highly conserved. In contrast, macrophage responses to substances that lack lipid A, such as heat-killed Staphylococcus aureus, peptidoglycan, or phorbol ester are not enhanced by LBP treatment (Mathison et al., 1992). It is reported that the increased sensitivity of macrophages to LPS can be observed at LBP concentrations of 1 ng ml ~ which are far less than the concentrations of

T.L. Kielian. F. Blecha / Immunopharrnacology 29 (1995) 187-205

LBP estimated in normal human serum (0.5 to 10 #g ml 1). Several investigators have demonstrated that the LBP-dependent enhancement of T N F - ~ induced by LPS can be inhibited by antiCD14 mAb, suggesting recognition of LBP-LPS complexes by CD 14 (Wright et al., 1990a; Mathison et al., 1991). A recent study has addressed the importance of LBP in vivo by injecting mice with an anti-LBP antibody prior to, or at the time of LPS challenge (Gallay et al., 1993). The anti-LBP treatment protected mice against a lethal dose of LPS, but only when Ab was administered concurrently with LPS challenge. Anti-LBP treatment given 15 min post-LPS challenge conferred no protection. The protective effect of anti-LBP mAb was correlated with decreased plasma TNF-:~ activity, however, anti-LBP treatment was only effective at LPS concentrations < 100 ng ml-1. This suggests that with higher concentrations of LPS, LBP-independent mechanisms are triggered culminating in cell activation. The role of LBP in LPS-dependent activation of macrophages from both CBA (LPS-responsive) and C 3 H / H e J (LPS-hyporesponsive) mice was examined in which macrophages from C 3 H / H e J mice exhibited enhanced binding of LPS in the presence of LBP, suggesting that hyporesponsiveness in this strain involves a step subsequent to LPS binding (Corradin et al., 1992). The addition of LBP with LPS resulted in increased concentrations of T N F - ~ and nitric oxide synthesis by CBA macrophages when compared to LPS stimulation alone. Lipopolysaccharide-binding protein has also been shown to enhance the LPS-dependent priming of neutrophils for phorbol ester-induced superoxide anion production (Vosbeck et al., 1990). An alternative role for LBP has been suggested in rabbit peritoneal macrophages, where cells exposed to picomolar concentrations of LPS in the presence of LBP were less responsive to subsequent LPS stimulation (Mathison et al., 1993), (a process known as adaptation or LPS hyporesponsiveness), than macrophages adapted in the absence of LBP. This suggested that LBP may play a role in LPS-induced adaptation by sensitizing cells to picomolar concentrations of LPS and making them refractory to normal stimulatory levels of LPS. Early studies examining CD14 and LBP in LPS-

195

mediated macrophage activation implied that cell activation was dependent on the physical interaction of LBP-LPS complexes with CD14. However, recent data have suggested that the primary role of LBP is to function as a lipid transfer protein, enhancing the rate o f L P S translocation to CD 14 rather than forming a stable complex with it (Hailman et al., 1994). Using native P A G E techniques it was demonstrated that LPS can bind directly to sCD14 in the absence of LBP at higher LPS concentrations (Hailman et al., 1994). The direct interaction of LPS with CD 14 is of low stoichiometry, where it is estimated that one molecule of CD14 binds one to two molecules of LPS. When LBP was added to a mixture of CD14 and LPS at substoichiometric concentrations, LBP increased both the rate of binding and the amount of LPS bound to CD14. This evidence suggested that LBP serves as a lipid transfer protein facilitating the rate of transfer of LPS to sCD 14 and is not exhausted in the reaction, since it was included at a lower concentration than LPS and CD 14. These studies estimated that one molecule of LBP can transfer at least ten molecules of LPS to sCD14 in 30 rain (Hailman et al., 1994). Other reports have demonstrated that LPS can interact directly with sCD14 (Haziot et al., 1993), again suggesting that LBP may not be essential for CD14-LPS interactions. The biological effects of LBP may thus be summarized as complexing/transferring LPS to CD 14 and subsequently enabling the cell to respond to extremely low levels of LPS, which in the absence of serum (LBP) are unable to elicit a physiological response (Schuman, 1992). A recent study has suggested a novel role for LBP (Wurfel et al., 1994). The addition of LBP to reconstituted high density lipoprotein particles containing purified apoprotein A-I (apo A-I), phospholipid, and free cholesterol enabled binding and neutralization of LPS. The reconstituted high density lipoprotein alone was not capable of binding LPS. In addition, an immobilized anti-LBP mAb co-precipitated apo A-I from plasma which further suggests that LBP interacts with lipoproteins in vivo. This study suggests that in addition to its ability to transfer LPS to CD14, LBP is associated with plasma lipoproteins that may play an important role in the neutralization of LPS in vivo. Septin is another serum 'factor' which has been

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T.L. Kielian, F. Blecha / lmmunopharmacology 29 (1995) 187-205

identified in mediating LPS recognition by CD14 (Wright et al., 1992). This activity observed in normal human plasma was described for its ability to mediate binding of opsonized LPS particles to phagocytes, which could not be blocked by antibodies directed against LBP. However, since its initial description, septin has received little attention and its significance in mediating LPS responses is unknown.

6. Other receptors which recognize endotoxin (LPS) Although CD 14 has been shown to be an important effector molecule in LPS-mediated macrophage activation, evidence exists for alternate receptors for LPS. Several observations made within experiments examining CD14 in LPS-mediated macrophage activation point indirectly to the presence of alternative functional LPS receptors. Using FITC-LPS to measure LPS binding sites on human monocytes, a CD14 blocking mAb could only partially inhibit FITC-LPS binding, demonstrating the presence of other LPS recognition molecules (Gessani et al., 1993). A recent article examined the importance of serum and CD14 in the monocyte-like cell line THP-1 adapted under serum-free conditions and stimulated with 1-25 dihydroxyvitamin D3 to induce CD 14 upregulation (Lynn et al., 1993). The addition of anti-CD14 mAb to cells stimulated with LPS under serum-free conditions did not inhibit cell activation as measured by TNF-c~ production suggesting CD 14-independent pathways of LPS activation. In a separate experiment using tritiated LPS to measure LPS-binding sites on neutrophils under serumfree conditions, LPS internalization occurred despite the presence of a CD 14 blocking antibody, suggesting that interaction with CD 14 is not the only means by which LPS can traffic beyond the cell membrane (Luchi and Munfor, 1993). Likewise, in experiments examining the role of CD 14 in LP S-mediated cytokine production, reports have demonstrated that anti-CD14 mAb were only partially effective in abrogating T N F - a production in human monocytes in response to higher concentrations of LPS (Couturier et al., 1992; Lynn et al., 1993). A separate study has demonstrated that an LPS antagonist, deacylated LPS, was able to inhibit

the IL-lfl and nuclear factor-~cfi responses in 1-25 dihydroxyvitamin D 3 differentiated THP-1 cells without affecting CD14-mediated uptake of LPS suggesting that molecules other than CD14 are important in LPS signaling (Kitchens et al., 1992). In addition, examination of leukocytes from patients with paroxysmal nocturnal hemoglobinufia, a condition where cells lack GPl-anchored proteins (thus little to no CD14), cells were still capable of responding normally to LPS as measured by LPSinduced TNF-c~ and IL-lfl production (Couturier et al., 1992). Several lines of evidence suggests that under serum-free conditions CD14 does not facilitate binding of LPS, in that treatment of monocytes with an anti-CD14 mAb as well as PI-PLC treatment had no effect on FITC-LPS binding to the cell surface (Corrales et al., 1993). Collectively, these data argue that other LPS receptors, in addition to CD14, are expressed on macrophages which have the ability to mediate cell activation. Considerable progress has been made in identifying alternate cell surface receptors for LPS. Those identified to date can be found in Table 1; a brief description of each follows. 6.1. CD18

One of the well-characterized receptors displaying affinity for LPS is the leukocyte integrin CD18 (Wright and Jon, 1986). All three members of the CD18 family ( C D l l a / C D 1 8 , C D l l b / C D 1 8 , and CD 1 I c/CD 18) are capable of binding LPS, and participate in the phagocytosis of bacteria. However, evidence has suggested that CD18 does not play a role in LPS-mediated cell activation. In a study of leukocytes from CD18-deficient patients, it was demonstrated that cells were able to produce normal levels of TNF-:¢, IL-1B, and superoxide anion in response to LPS, suggesting that CD18 is not essential for mediating cellular responses to LPS (Wright et al., 1990b). LPS treatment has been shown to induce a rapid upregulation of C D l l b / CD 18 expression on human neutrophils, believed to be mediated by CD14, since anti-CD14 mAb specifically inhibited LPS-induced C D l l b / C D 1 8 expression (Lynn et al., 1991). In contrast, a separate study reported that the binding of LPS to human monocytes was not mediated by C D l l / C D 1 8 be-

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Table 1 Characteristics and functions of membrane receptors for LPS LPS receptor (Mw)

Cell types~

Possible functions

References

CD14 (55 kDa)

monocyte macrophage neutrophil

Interacts with LPS _+LBP, leading to cell activation Adhesion molecule

Wright et al., 1990a Hailman et al., 1994 Beekhuizen et al., 1991 Launer et al., 1990b

CD18 CDlla/CD18 CDllb/CDI8 CDllc/CD18

leukocytes

Adhesion molecule Recognizes LPS and LPS in association with whole bacteria CD18-deficient cells still respond normally to LPS

Wright et al., 1986 Lynn et al., 1991 Wright et al., 1990b

p73 (73 kDa)

splenic B & T lymphocytes, PMN, macrophages, MO, platelets, rat trophoblast cells, 70Z/3, YAC-1, EL-4, J774A.1 L929, COS-7, and P815 cell lines

mAb 5D3 (anti-p73) activates MO for killing of tumor & VSV infected cells

Chen et al., 1990 Green et al., 1992 LeBlanc, 1994 Morrison et al., 1990

p38 (38 kDa)

lymphocytes, macrophages, splenocytes, J774.1, and 70Z/3 cell lines

Recognizes inner core oligosaccharide determinant(s) of LPS

Lei et al., 1993

p18 p25

70Z/3

None

Kirkland et al., 1990

p65 p55

J774.1

None-55 kDamolecule may represent CD14

Hara-Kuge et al., 1990

(95 kDa)

a MO, monocyte; PMN, polymorphonuclear leukocyte; VSV, vesicular stomatitis virus; LBP, LPS-binding protein.

cause m A b directed against the receptor complex did not inhibit specific L P S binding (Couturier et al., 1991). Moreover, L P S was not able to effectively c o m p e t e with labeled m A b directed against C D l l / CD18. The protein ligands for C D 1 8 ( I C A M , fibrinogen) are not structurally related to L P S , and evidence suggests that interactions between these ligands occur at distinct sites on the C D 18 molecule (Wright et al., 1989b). Thus, it seems that the role o f C D 1 8 is not directed t o w a r d s mediating secretory responses to L P S , but rather serves to internalize and eliminate L P S . 6.2. 73 k D a L P S receptor

P e r h a p s one o f the m o s t well-characterized L P S receptors in addition to CD18 and CD14, is the 73 k D a (80 k D a ) receptor (Lei and M o r r i s o n , 1988). Using a photoactivatable, disulfide-reducible, iodin a t e d L P S derivative (12SI-ASD-LPS), an 80 k D a

protein displaying affinity for L P S was identified on p o p u l a t i o n s o f m u r i n e splenic B and T lymphocytes, m a c r o p h a g e s and rat t r o p h o b l a s t cells (Lei and M o r r i s o n , 1988; H u n t et al., 1989). This protein was also found on the surface o f the pre-B cell line 70Z/3, and the YAC-1 and EL4 T cell lines. Interestingly, this L P S binding protein was also present on splenocytes o f both C 3 H e B / F e J ( L P S responsive) and H e J ( L P S hyporesponsive) mice at c o m p a r a b l e levels. Using one-dimensional S D S - P A G E , a more accurate molecular weight estimate was later assigned to this molecule o f 73 k D a , to which it is n o w referred (Lei et al., 1991). The ubiquitous nature of this L P S receptor is intriguing, its w i d e s p r e a d distribution is m a r k e d l y different than CD14, whose expression is limited to a small group of target cells, namely monocytes, m a c r o p h a g e s , and neutrophils. As a m e t h o d to more effectively examine the p73 receptor, m A b against the p73 receptor were produced by injection o f partially purified p73 antigen

198

T.L. Kielian, F. Blecha / Immunopharmacology 29 (1995) 187-205

into hamsters (Bright et al., 1990). Two mAb were obtained from a series of clones, designated mAb 3D7 and mAb 5D3 which were determined to recognize the carbohydrate and protein determinants on p73, respectively. Both mAb were able to inhibit the interaction of LPS with the 73 k D a LPS receptor in a dose-dependent manner, reflecting specificity. The mAb 5D3 directed against the 73 kDa LPS receptor was shown to serve as an agonist for macrophage activation in vitro, which suggested that p73 may be involved in LPS-induced signal transduction (Chen etal., 1990). Macrophages from C 3 H / H e N mice pre-treated with mAb 5D3 exhibited tumor cell cytotoxicity against P815 mastocytoma targets, a response which could be enhanced by the addition of exogenous murine IFN-7. In contrast, neither mAb 5D3 nor LPS induced tumor cell cytotoxicity in C3H/HeJ (LPS hyporesponsive) macrophages, which have previously been shown to express normal levels of the 73 k D a LPS receptor. The cytotoxic activity of mAb 5D3 was not attributable to contaminating endotoxin, because mAb treatment with polymyxin B did not abrogate its effects. In a separate study, pre-treatment of resident peritoneal macrophages with mAb 5D3 in combination with IFN-7 resulted in high levels of nitric oxide production which paralleled potent tumor cytotoxicity (Green et al., 1992). In addition, a recent article demonstrated that mAb 5D3 could activate bone marrow culture-derived macrophages for cytolysis of vesicular stomatitis virus-infected 3T3 cells in vitro (LeBlanc, 1994). It was suggested that mAb 5D3 and LPS trigger a common receptor on macrophages to achieve activation since mAb 5D3 mimicked effects observed with LPS alone, albeit at higher levels. These data further suggest a functional role for p73 in LPS-mediated macrophage activation. Galactosamine-induced sensitization to LPS was first described in rabbits, mice, and rats in 1979 (Galanos et al., 1979). Injection of D-galactosamine (300 mg/kg) concurrently with LPS challenge resulted in a 10,000 to 100,000-fold increase in LPS sensitivity which was shown to be species dependent. The mechanism of LPS sensitization induced by galactosamine is believed to involve direct action on hepatocytes (Galanos et al., 1979). Within 30 min following injection of galactosamine, there is a dras-

tic depletion of hepatic UTP, which in turn leads to cessation of biosynthesis ofmacromolecules such as RNA, glycoproteins, and proteins. These alterations eventually lead to cell death and necrosis. The importance of U T P depletion in the pathogenesis of galactosamine-induced LPS sensitization is reflected by protection from galactosamine-induced sensitization with uridine administration. In studies using the o-galactosamine sensitization model, it was found that administration of low concentrations of endotoxin intraperitoneally to mice prior to LPS + galactosamine challenge provided protection against the lethal effects of LPS (Galanos et al., 1979). A recent study has shown that administration of 7.5 #g of mAb 5D3 intraperitoneally, 80 min prior to LPS + galactosamine challenge also confers protection against LPS-mediated lethality (Morrison et al., 1990). Injection of mAb 5D3 conferred protection against a dose of endotoxin tenfold greater than that required to kill 100% of mice in an untreated control group. The mechanism believed to provide protection in these models involves macrophage activation. In support of this hypothesis, it has been shown that mAb 5D3 can induce macrophage activation in vitro, and evidence suggests that rapid internalization of T N F - ~ receptors occurs in response to LPS stimulation. Therefore, LPS-pretreatment may reduce the number of cell-surface associated T N F - ~ receptors and as a result, cells are no longer sensitive to the toxic effects of LPS, many which are believed to be mediated by TNF-:~. Likewise, this study demonstrated that mAb 5D3 pretreatment protected against the lethal effects of T N F - ~ in the galactosamine sensitization model (Morrison et al., 1990), where T N F - ~ has been shown to mimic the lethal effects of L P S + galacto samine. The binding of LPS to p73 is independent of the chemotype or the source of LPS; smooth LPS and rough mutants were both capable of binding which suggested that this receptor displays affinity for the lipid A portion of LPS (Lei et al., 1991). Additionally, the p73 receptor has been demonstrated to bind soluble peptidoglycan on mouse B lymphocytes with the use of a photoreactivc crosslinking soluble peptidoglycan (Dziarski, 1991). The p73 receptor has also been shown to be expressed on a variety of transformed cell lines including J774A.I (murine

T.L. Kielian, F. Blecha / lmmunopharnTacology 29 (1995) 187-205

monocyte/macrophage line), L929 (murine fibroblast), COS-7 (monkey kidney), and P815 (murine mastocytoma). In all of these cells, p73 has been described as the major detectable LPS-binding entity. Recently, the 73 kDa LPS receptor has been demonstrated to be expressed on human monocytes, lymphocytes, PMN, and platelets (Halling et al., 1992). Several other LPS-binding proteins were identified on both lymphocyte and monocyte subsets of approximately 50, 31, and 20 kDa, although no functional role has been described for these molecules to date. Therefore, it appears that p73 represents another functional LPS recognition molecule that may be capable of signal transduction in response to LPS-receptor interactions. The rather ubiquitous distribution of this receptor suggests that it may play an important role in LPS-mediated cell activation. 6.3. Other L P S receptors

Other potential LPS recognition molecules have been described including 18 and 25 kDa proteins on the 70Z/3 pre-B cell line (Kirkland et al., 1990), and 65 and 55 kDa molecules expressed on the J774.1 macrophage-like cell line (Hara-Kuge et al., 1990). Currently, the functional significance of these LPSbinding proteins is unknown, although the 55 kDa molecule mentioned above may represent CD14. A 38 kDa cell surface protein displaying affinity for LPS was identified on mouse lymphocytes, macrophages, splenocytes, J774.1 cells and 70Z/3 cells using 125I-ASD-LPS (Lei et al., 1993). Binding of LPS to p38 could not be inhibited by purified lipid A, even in fiftyfold excess. However, the interaction of 125I-ASD-LPS with the p38 protein could be competed with ReLPS (possessing only lipid A and two K D O regions), indicating that this protein (p38) may specifically recognize an inner core oligosaccharide determinant(s) on EPS. A separate report (Lebbar et al., 1986) has implicated regions in the core oligosaccharide (KDO) region that are capable of inducing IL-1 synthesis by human monocytes. Most effects of LPS have been shown to be mediated by the lipid A portion of LPS, but this evidence suggests that other EPS determinants have the ability to elicit cellular responses.

199

7. Mechanism for macrophage activation by LPS A theoretical mechanism for monocyte/ macrophage activation by LPS can now be proposed (Fig. 1). This mechanism also addresses differences that may occur in a mononuclear phagocyte's response to LPS depending on its microenvironment in vivo. This model is based on two assumptions; (1), monocytes/macrophages have a defined number of LPS receptors on the cell surface that must be occupied in order to achieve a threshold stimulus capable of inducing cell activation; and (2), in the presence of LBP (serum), CD 14 represents the highaffinity EPS recognition molecule. Several lines of evidence suggest that the latter assumption may indeed by accurate. First, recent evidence has shown that transfection of human CD 14 cDNA into 70Z/3 B cells (normally CD14 negative) increased sensitivity to LPS in the presence of LBP by 10,000-fold as compared to 70Z/3 parent cells (Lee et al., 1992). Secondly, several reports have shown CD14 to be critical in the response of macrophages to low concentrations of LPS ( < 1 ng ml- l) in the presence of LBP, since anti-CD14 mAb are able to abrogate TNF-~ secretion in response to stimulation (Wright et al., 1990a; Martin et al., 1992; Dentener et al., 1993). In this model, to introduce the macrophage response to LPS occurring extravascularly, we have used the alveolar macrophage as an example. It is proposed that in tissues (i.e., lung), three pathways are possible in the macrophages' response to LPS, all of them dependent upon the local concentration of EPS or LBP in the tissue. The healthy lung norreally contains low to undetectable levels of LBP, however, it has been demonstrated that the concentration of LBP in the lung can become elevated under certain pathophysiological conditions (Martin et al., 1992) such as exudation from the plasma or by direct damage to the blood-gas barrier. The first pathway available for macrophages in response to LPS is one of no activation (Fig. 1, arrow 1). This could occur when macrophages are exposed to subnanogram concentrations of LPS with concurrently low levels of LBP. In this scenario, LPS is eliminated via phagocytosis by macrophages without subsequent cell activation (defined as production of secretory mediators such as TNF-c~, IL-1, IL-6, etc.). With

T.L. Kielian, F. Blecha / Immunopharmacology 29 (1995) 187-205

200

I

2#1

Low LPS

"i'h'B" I /

~-~

~

t/ ..~///~

LUNG

(Tissue)

I

I

,,-8

,L_0

~"

CD18 ~ 3

LOW LPS L°wLBP /

I

CD18 V

\

TNFo

/

/

H,ghLPS Low LBP

IL-1

/4

Low LPS Hi0hL,P L

J

No Activation Fig. 1. Mechanism for macrophage activation by LPS. Upon exposure to LPS, resident tissue macrophages can respond by one of three basic pathways, each dependent on the local concentrations of LPS and/or LBP in the tissue. Extremely low levels of LPS with low LBP do not trigger a LPS-mediated secretory response (i.e., TNF-~, IL-1) since receptor threshold is not reached (arrow 1). In this scenario, endotoxin is eliminated via non-specific mechanisms (i.e., phagocytosis) that does not initiate cytokine release, and the final outcome is a quiescent cell. When the concentration of LPS increases locally, as in a Gram-negative pneumonia or enteritis, the threshold of LPS receptors is readily attained due to the locally saturating concentration of LPS, culminating in macrophage activation and release of secretory mediators (arrow 2). Another mechanism culminating in macrophage activation is proposed when concentrations of LBP become elevated in tissue as a result of exudation from the vascular compartment in inflammation (arrow 3). This LBP-rich exudate enables macrophages to respond to much lower concentrations of LPS via a CD14-dependent pathway, which in the absence of LBP are incapable of eliciting a response. This allows the receptor threshold stimulus to be reached, resulting in cell activation. Peripheral blood monocytes are continuously exposed to high concentrations of LBP in the bloodstream (0.5-10/zg ml - ~), and as a consequence are highly sensitive to low concentrations of LPS via a CD14-dependent pathway' (arrow 4). However, this sensitivity may lead to deleterious consequences under certain conditions. For example, during Gram-negative septicemia, chronic and widespread activation of monocytes leads to pathophysiological damage of host tissues mediated by the over-production of secretory products by activated monocytes, such as TNF-c~ and IL-1. In porcine alveolar macrophages, LPS induces upregulation of CD14 in vitro, which may' impart greater sensitivity ofmacrophages to subsequent LPS exposure (arrow 5). 'Low LPS' represents LPS concentrations < 1 ng m l - 1, and 'high LPS' reflects concentrations of LPS > 10 ng ml - i.

low concentrations of LPS and undetectable LBP, the threshold occupancy of receptors is not reached, therefore, the macrophage does not become activated. The second pathway may be initiated when concentrations of LBP are elevated due to inflammation in the lung. In this circumstance, LBP facilitates the transfer of LPS to CD14 at such a rate that the threshold for activation is attained (Fig. 1, arrow 2). Thirdly, an alternative mechanism exists for the macrophage response to LPS which may occur in a

focal area of Gram-negative infection (Fig. 1, arrow 3). In this situation, LPS concentrations become elevated due to the focal nature of the lesion, and despite low levels of LBP, macrophages become activated. With high concentrations of LPS, engagement of lower affinity LPS receptors can occur, along with LPS interacting directly with CD14. Recent evidence has demonstrated that sCD14 can bind LPS alone (Hailman et al., 1994; Haziot et al., 1993) which may extend to m C D 14 at high concentrations

T.L. Kielian. F. Blecha / lmmunopharmacology 29 (1995) 187-205

of LPS. The ultimate result of this interaction is cell activation achieved by reaching the receptor threshold. Recently, we have shown that LPS induces CD 14 upregulation on porcine alveolar macrophages after a 24 h stimulation period (Kielian et al., 1995). It is not clear what effect this might have on the macrophage response to LPS, but one could postulate that increased CD14 expression may in turn result in enhanced sensitivity to subsequent LPS exposure (Fig. 1, arrow 5). Collectively, these events lead to activation of the macrophage and the resultant release of secretory mediators such as TNF-e, IL-lfl, IL-6, IL-8, and prostaglandins, to mention a select few. Peripheral blood monocytes are continuously exposed to high concentrations of LBP in the bloodstream (0.5-10 #g ml 1). Because of their continuous access to LBP, monocytes are highly sensitive to low levels of endotoxin (Fig. 1, arrow 4). Normally, very little endotoxin is detectable in the bloodstream, however, when present it is rapidly eliminated. However, this enhanced sensitivity to LPS could lead to deleterious consequences if the secretory response to LPS is not tightly regulated. For example, during Gram-negative septicemia, chronic and widespread activation of monocytes could lead to pathophysiological damage of host tissues mediated by the overproduction of secretory products derived from activated monocytes, such as T N F - e and IL-1. In summary, this model provides a basis for compartmentalization of the mononuclear phagocyte response to LPS in vivo. Elucidation of mechanisms involved in LPS-induced macrophage activation may provide avenues for therapeutic management of septic shock. Evidence suggests that several cell surface molecules have the ability to recognize and bind LPS. The identification and function of LPS receptors will remain an area of intense research interest because the elucidation of the cellular response to LPS is necessary before effective strategies of attenuating a dysregulated response to LPS can be designed.

Acknowledgements The authors would like to thank David C. Morrison for his helpful discussions and comments

201

on this review. This is contribution number 95-305-J of the Kansas Agricultural Experiment Station.

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