Agonists and antagonists for lipopolysaccharide-induced cytokines

Immunobiol., vol. 187, pp. 303-316 (1993) Forschungsinstitut Borstel, Institut fiir Experimentelle Biologie und Medizin, Borstel, Germany

Agonists and Antagonists for Lipopolysaccharide-Induced Cytokines HANS-DIETER FLAD, HARALD LOPPNOW, ERNST THEODOR RIETSCHEL, and ARTURJ. ULMER

Abstract Agonistic and antagonistic properties of LPS and partial structures in the induction of cytokines are reviewed. Studies on structure-activity relationships of LPS and lipid A with human mononuclear cells reveal that S- and notably R-form LPS are very potent cytokine inducers. Synthetic E. coli lipid A is somewhat less active, whereas synthetic s. minnesota-type lipid A is significantly less active. Pentaacylated forms of lipid A are less potent than hexaacylated forms, and tetraacylated synthetic precursor Ia and bisacylated disaccharides and monosaccharides are completely inactive, indicating that a structure-dependent hierarchy of LPS and lipid A partial structures determines the monokine-inducing capacity in human mononuclear cells. Precursor Ia is a potent LPS antagonist. The mechanism of its inhibitory activity is shown to be due to competitive binding to cellular binding sites (receptors). Proinflammatory and antiinflammatory cytokines, receptor antagonists, and soluble cytokine receptors influence the cytokine-inducing activity of LPS, suggesting a complex regulatory network.

Introduction Lipopolysaccharide (LPS, endotoxin), a major constituent of the outer membrane of Gram-negative bacteria, causes a variety of pathophysiological effects in experimental animals and in humans. These effects are not direct but induced by the interaction of LPS with the membrane of target cells. Cells of the mononuclear phagocyte system, B lymphocytes, granulocytes, platelets, fibroblasts, and endothelial cells are primary target cells of LPS. As a consequence of the interaction with LPS the cells produce peptide and lipid mediators such as interleukin-1a (IL-1a), interleukin-1~ (IL-1~), tumor necrosis factor (TNF), interleukin-6 (IL-6), interleukin-8 (IL-8), and oxidative metabolites of fatty acids. The mediators produced and released by these cells are responsible for the endotoxic effects such as fever, leukopenia, hypotension, acute phase response, and septic shock. Mediators in the Acute Phase Response and in Sepsis Infections, injuries, malignant tumors, and a variety of immunological disorders trigger a complex reaction of the organism, known as the acute

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phase response. In infections with Gram-negative bacteria, the active principle inducing this response is LPS. The acute phase response is characterized by fever, leukocytosis, a negative nitrogen balance, increased vascular permeability, alterations in plasma metal and steroid concentrations, and an increase in the synthesis of hepatic acute phase proteins. These proteins include a1-acid glycoprotein, aI-antitrypsin, al-antichymotrypsin, haptoglobin, hemopexin, and fibrinogen, in most species also a2macroglobulin, and in humans C-reactive protein, complement components C3 and factor B, and serum amyloid A. Some proteins, like albumin and transferrin, show decreased plasma levels and are, therefore, called negative acute phase proteins. For many years, investigators tried to identify the mediators involved in the triggering of this hepatic acute phase reaction. IL-l and TNF were reported to stimulate liver protein synthesis, but none of these cytokines was capable of inducing the full spectrum of acute phase proteins that followed exposure to crude monocyte supernatants. This missing active protein was termed hepatocyte-stimulating factor (1) and later IL-6. The importance of IL-6 as an inducer of the acute phase response has since been confirmed by the observation that it induces acute phase proteins in vivo (2) and that it stimulates the full spectrum of acute phase proteins in adult human hepatocytes. Besides its role as an inducer of acute phase proteins, IL-6 contributes to the body's defense by inducing fever (3). After infection, levels of IL-6 rise after 30 min and peak at 2 h with a maximal concentration in the range of 1-10 nM (4). An early increase of IL-6 levels is also observed in the serum of patients with sepsis (5). In septic shock, IL-6 levels appear to correlate with disease activity and may be of prognostic value. The relative lack of toxicity of IL-6 in experimental animals would even argue against a causative role of IL-6 in septic shock. High titers of 1L6 associated with acute infection seem to result from complex interactions involving 1L-l and TNF, which both induce potent 1L-6 responses in vivo. Recently, it has been proposed that measurement of IL-6 levels in sera provided information on the preceding exposure of the host to either LPS or 1L-l and/or TNF and on the LPS/IL-lITNF susceptibility and shock status of the host (6). Thus, endotoxin-susceptible patients are characterized by high levels of serum IL-6 and relatively low levels of serum endotoxin, and non-susceptible patients with low or undetectable serum IL-6 and high levels of serum endotoxin (6), an observation of relevance for the rapid diagnosis of endotoxin shock. Further studies involving molecular biological approaches are needed to better define the endotoxin susceptibility status of a patient.

Structure-Activity Relationship of LPS and Lipid A Partial Structures Studies on the structure-activity relationships of LPS and lipid A could provide insight into the basic principles underlying the endotoxic properties of LPS. Previous studies have demonstrated that the lipid A moiety of LPS

Agonists and Antagonists for Lipopolysaccharide-Induced Cytokines . 305

is the structure responsible for endotoxic activity of the molecule (7). The availability of synthetic lipid A and partial structures enabled us to investigate the biological activity of a given structure in vitro. As a model system we have chosen the production and release of the cytokines IL-1, IL-6 and TNF from human peripheral blood mononuclear cells in vitro. These studies have shown that S- and notably R-form LPS are very potent inducers of monokines acting in a concentration range of 1-1000 pg/ml (Fig. 1) (8-13). Synthetic E. coli lipid A (compound 506) is active at a concentration range of 1-100 ng/ml, whereas synthetic s. minnesota-type

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Figure 1. Upper graph: IL-l-inducing capacity of various LPS, lipid A and partial structures. The bars represent the range of minimal concentration of agonists found with mononuclear cells of different donors (After 9). Lower graph: TNF-inducing capacity of various LPS, lipid A and partial structures. The bars represent the range of minimal concentrations found with mononuclear cells of different donors (After 10).

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Agonists and Antagonists for Lipopolysaccharide-Induced Cytokines . 307

heptaacyllipid A (compound 516) is significantly less active. Furthermore, pentaacylated forms of lipid A are less potent than hexaacylated forms (data not shown). Interestingly, the pattern of phosphorylation appears to influence the biological activity, since the monophosphoryl partial structure (compound 504) is less active than the bisphosphorylated partial structure (compound 506). The tetraacylated synthetic precursor Ia (compound 406), the bisacyl compound 606 (data not shown), and the bisacylated monosaccharides (compound 401 [lipid X] and 410) were completely inactive. This inability to stimulate IL-1 (8,9) or TNF-a (10, 13) in human monocytes or mononuclear cells also holds true for IL-6 (11) and pertains not only to the induction of cytokine release, but also to the induction of intracellular cytokine proteins and of mRNA (Figs. 2 and 3) (8-13). The data summarized here and elsewhere (14) indicate that the biological activity of lipid A and lipid A partial structures depends on the phosphorylation and acylation pattern of the hexosamine disaccharide. Maximal monokine activity appears to be associated with the bisphosphorylated lipid A possessing six acyl residues, which structurally corresponds to E. colllipid A. Partial structures lacking one of these components or containing different constituents are either less active or not active at all. Taken together, a structure-dependent hierarchy of LPS and lipid A partial structures seems to determine the monokine-inducing capacity in human mononuclear cells. Precursor la, a biosynthetic precursor of E. coli lipid A (15), is structurally identical to compound 406. This structure and its derivatives, such as KDOrprecursor la, are also biologically inactive in human cells, but are agonists for murine cells (16, 17), suggesting different requirements for structure-function relationships in different species.

LPS Antagonists Glucosamine-based precursors and analogs of E. coli lipid A have been demonstrated to antagonize the biological activity of LPS. While monosaccharide analogs are predominantly antagonistic, disaccharides are usually mixed antagonists-agonists (18). Lipid X, a monosaccharide lipid A precursor, protects animals from LPS lethality and Gram-negative bacterial infection (19, 20). In vitro, lipid X inhibits priming of human neutrophils by LPS. In platelets, lipid X has been claimed to inhibit the action of LPS by inhibition of protein kinase C rather than by specific antagonism of LPS (21). However, some synthetic preparations of lipid X may have become inadvertently contaminated with glucosamine-based disaccharides, i.e., the presence of lipid A-like molecules may explain some of the biological effects observed with lipid X (22). In our case, monosaccharide precursor lipid X is less potent than the synthetic diglucosamine-based analog of lipid A, precursor Ia (compound 406) in inhibiting LPS-induced IL-l~, TNF, and IL-6 production by human monocytes or mononuclear cells (9, 11-13) (Figs. 2 and 3). The

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inhibition of IL-l and TNF production by compound 406 was found not only to occur at the level of release, but also at the level of IL-l ~ or TNF synthesis (23, 24) (Fig. 3) and at the level of mRNA for IL-l~ and TNF (9, 12) (Fig. 4). The inhibition of LPS- or lipid A-induced IL-6 production by compound 406 does also occur in the presence of recombinant interferon-y, suggesting that compound 406 and recombinant interferon-y act via different and independent pathways (11). These findings may indicate 't hat the inhibition of IL-6 production by lipid A partial structures may modulate the acute phase response proteins in Gram-negative infection. More recently, compound 406 has also been shown to inhibit LPS-induced IL-6 production of human vascular endothelial cells or smooth muscle cells (25). The inhibition of cellular responses by synthetic precursor Ia (compound 406) is specific for LPS or lipid A, as the cytokine production induced by other stimuli such as Gram-positive bacteria, lectins, lipoproteins, or other cytokines is not affected by compound 406 (9, 13).

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Agonists and Antagonists for Lipopolysaccharide-Induced Cytokines . 309

Recently, new synthetic analogs of lipid A have been developed, which are more readily accessible to chemical synthesis and which contain an aoxyethyl-linked (-O-CH r CH2) phosphoryl group in position 1 of the reducing glucosaminyl residue (26). One of these compounds, termed PE-4, the a-oxyethyl analog of compound 406, is unable to induce IL-l, IL-6, and TNF in human monocytes and inhibits, like precursor la, LPS-induced monokine production (27). These data suggest that a replacement of the aglycosidic phosphoryl group by the a-glycosidic phosphonooxyethyl group does not influence the biological activity and the antagonistic action of tetraacylated partial structures. A number of natural LPS and partial structures have also been shown to be antagonists of the biological activity of LPS. Biosynthetic precursor Ia of E. coli lipid A and structurally identical to compound 406, inhibits LPSstimulated release of IL-l, IL-6, TNF and prostaglandin E2 (PGE 2) from human mononuclear cells in vitro (17), TNF release from diluted whole blood in vitro (28), and the activation of neutrophils (29). In contrast on other LPS antagonists, precursor la is an agonist for murine cells, as it stimulates the release of TNF and of arachidonic acid metabolites from cells of the murine macrophage tumor line RAW 264.7 (17). The granulocyte enzyme acyloxyacyl hydrolase partially de-O-acylates LPS (30). This enzyme liberates the secondary acyl groups linked to the 3hydroxy fatty acid at positions 2' and 3' to produce a tetraacyl molecule identical to precursor la. Deacylated LPS inhibits cellular functions like precursor la, and among those the production of plasminogen activator inhibitor-l, of PGlb and PGE 2 of human vascular endothelial cells (31), and the LPS-induced adherence of neutrophils to endothelial cells (32). The detoxification of LPS by this granulocyte enzyme may be an important mechanism antagonizing the endotoxic effects of Gram-negative bacteria. LPS of Bacteriodes fragilis, a LPS of low toxicity, reduces also the adherence of neutrophils to vascular endothelial cells in response to E. coli LPS (33). Another nontoxic LPS derived from Rhodobacter capsulatus inhibits IL-l, IL-6 and TNF production of LPS stimulated mononuclear cells comparable to precursor Ia (34). Moreover, lipid A of Rhodobacter sphaeroides is able to specifically inhibit biological activities of LPS in vitro and in vivo (35).

Mechanism of Inhibition: Competition of Binding to Cellular Binding Sites Various research groups have been involved in the study of binding of radiolabeled LPS to cells with the aim to examine the question whether LPS antagonists would exert their inhibitory effect by competing with radiolabeled LPS for LPS binding sites. Radiolabeled S. minnesota R595 LPS (Re LPS) was used in a recently established binding assay (14, 36). In these studies, synthetic precursor Ia (compound 406) was found to inhibit

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completely the binding of 125I_ReLPS to human monocytes as efficiently as the unlabeled ligand, i.e. at concentrations of 50-400 ng/ml (37) (Fig. 5). Similarly to compound 406, preparation PE-4, the synthetic analog of precursor Ia having an oxyethyl-linked phosphoryl group in position 1 of the reducing glucosaminyl residue, is also inhibitory for the binding of 125 1_ ReLPS (37). If one assumes that inhibition of binding is of competitive nature, then there should be a correlation between inhibition of binding and affinity to a given binding structure. With regard to compound 406 there is, however, a discrepancy between its strong competitive activity in the 1251_LPS binding assay and its lack of IL-l-, IL-6- and TNF-a-inducing capacity. Whether LPS and compound 406 bind indeed to the same receptor( s) or whether binding of compound 406 to separate receptor(s) leads to negative intracellular signalling, is presently unknown. This argument does also not take into account the possibility that LPS and compound 406 are amphiphilic in aqueous solutions and may form different supramolecular structures (38). Furthermore, the physical state of these molecules may be different in the cytokine-inducing and binding assays, respectively. In addition, several different structures for the binding of LPS have been described (reviewed in 39). The binding of LPS or lipid A to one of the receptors, the CD14 molecule, involves the binding of LPS or lipid A to LPS-binding protein (LBP), whereas the binding to CD18 and to 80 and 40 kDa receptor proteins does not involve LBP (36), but this binding structure may be in equilibrium with other binding proteins present in soluble form in the serum (40-42). While the contribution of the different LPS and lipid A binding proteins to cellular activation and binding competition is uncertain, the present knowledge should provide a basis for further studies aimed at elucidating the fine specificity in the interaction of the LPS/lipid A ligand

Agonists and Antagonists for Lipopolysaccharide-Induced Cytokines . 311

with cell-associated binding proteins at the molecular level. (For a more detailed discussion of this problem see chapter 1.) Influence of Cytokines on Biological Effects of LPS

Interferon-y The possibility that cytokines themselves may influence biological effects of LPS has been studied in LPS-resistant C3H/He] mice, whose macrophages are defective in the ability to produce TNF and IL-1 in response to LPS. Administration of IFN -y before LPS in He] mice has no effect on IL-1 mRNA, but partially restores LPS-induced increase in hepatic and splenic mRNA for TNF and serum TNF levels, which was also accompanied by the increase in hepatic lipogenesis (43). On the other hand, TNF confers antimicrobial activity on interferon-y-treated macrophages of mice by its macrophage-activating property (44). In cancer patients, IFN-y prevents the reduction of cytokine production (TNF, IL-6, IL-8, G-CSF) following a second challenging dose of LPS (45). The complex regulatory mechanisms which, after in vivo infection, down-regulate the production of IL-1a, IL-1~, IL-6 and TNF in patients with septicemia and with noninfectious shock, are not well understood (46).

Interleukin-4 IL-4 is a cytokine which counteracts IFN -y-mediated effects. A potential anti-inflammatory effect has been attributed to IL-4. This cytokine has been shown to suppress in monocytes the production of IL-1, TNF, PGE 2 (47), and IL-6 (48). This down-regulation relates to both mRNA and proteins of IL-1~ and TNF (49). IL-4 enhances programmed cell death (apoptosis) in LPS- or IL-1-stimulated monocytes, which suggests that the antiinflammatory effect of IL-4 in chronic lesions is at least partially due to reducing survival of activated monocytes (50). As expected, this enhancement of apoptosis by IL-4 is abolished by IFN-y. IL-4 and IFN-y have been reported to down-regulate CD14 antigen expression on human monocytes and blood monocyte-derived macrophages, whereas LPS is upregulating CD14 expression (51). IL-4 also inhibits LPS-induced gene activation of human macrophage inflammatory protein 1~ in human monocytes (52). A LPS-responsive element has been localized within 455 bp 5' to the start of transcription of this gene (52).

Interleukin-IO Recombinant murine IL-10 suppresses LPS-induced TNF release from mouse peritoneal macrophages (53). Thus, IL-10 might be permissive for the growth of microbial pathogens, whereas TNF, reactive oxygen or nitrogen intermediates are major antimicrobial macrophage products. During activation of human monocytes by LPS, IL-10 is generated which

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inhibits the synthesis of IL-la, IL-l~, IL-6, IL-8, TNF, GM-CSF, and GCSF (54). This autoregulatory circuit may play an important role in controlling macrophage function in inflammatory responses (55).

Lipoxygenase products LPS-induced TNF production by murine macrophages has been effectively blocked by inhibitors of lip oxygenase (56). Likewise, the presence of TNF in serum of D-galactosamine-sensitized mice after challenge with LPS can be suppressed by lipoxygenase inhibitors. A lip oxygenase product, 13hydroxylinoleic acid, was isolated from LPS-stimulated macrophages, which is covalently bound to cellular constituents and which, when exogenously added to macrophages, neutralized the lipoxygenase inhibitorinduced suppression of TNF synthesis (14, 56). Further details are reported in this volume.

IL-J receptor antagonist This protein, which inhibits the binding of IL-l to IL-l receptor type I and type II, has been isolated from culture supernatants of stimulated monocytes (57). The mature protein has 26 % sequence homology with ILIa and 19% with IL-l~. This protein has been shown to counteract the pyrogenic and proinflammatory and other cytokine-inducing capacity of IL-l and, thus, to prevent LPS- and IL-l-induced acute inflammation (58) and E. coli- induced septic shock in rabbits (59).

Soluble TNF receptors Soluble TNF receptors are potent antagonists for TNF by inhibiting its biological activity. Soluble TNF receptor proteins have been shown to partially protect mice from LPS-induced lethality (60). Besides the potential application in cerebral malaria, soluble TNF receptor proteins may represent a novel therapeutic strategy in the treatment of septic shock. Concluding Remarks The molecular and functional characterization of LPS and lipid A partial structures has led to a significant progress in the understanding of the pathophysiology of Gram-negative bacterial infections. In this context the study of structure-function relationships allowed to define agonistic and antagonistic properties of lipid A partial structures in the induction of cytokines. Evidence has been accumulated which suggests that LPS antagonists act at least in part by competing for cellular binding sites. Binding sites for LPS, lipid A, and partial structures have been defined, however, the functional consequences of ligand-receptor interaction, such as signal transduction pathways and/or internalization of the ligand-receptor complex, are largely unknown. Nevertheless, the competitive action of

Agonists and Antagonists for Lipopolysaccharide-Induced Cytokines . 313

lipid A partial structures such as compound 406 represents in our opinion one of the biological principles with the potential of therapeutic application in Gram-negative sepsis. Several cytokine genes contain in the 5' flanking region regulatory elements which have been demonstrated to be responsive to LPS stimulation. At the level of released cytokines, pro inflammatory (IL-l, IL-6, IL-8, TNF) and antiinflammatory (IL-4, IL-IO, IL-l receptor antagonist) cytokines can be discriminated and regarded as members of a complex regulatory network. It appears that the experimental basis is now being laid for a rational therapy of Gram-negative sepsis involving antibiotics, LPS-neutralizing antibodies, LPS antagonists, and cytokine inhibitors such as IL-l receptor antagonist, soluble TNF receptors, and pentoxifyllin inhibiting TNF synthesis. Acknowledgements The financial support of Fonds der Chemischen Industrie (HDF, EThR) is gratefully acknowledged. We thank Mrs. F. RICHTER for preparing drawings and Mrs. G. STEGELMANN and Mrs. B. KelHLER for preparing the photographs. Special thanks to Mrs. R. HINZ for expert secretarial assistance.

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