A Pseudomonas aeruginosa hepta-acylated lipid A variant associated with cystic fibrosis selectively activates human neutrophils.

Pseudomonas aeruginosa (PA) infection in cystic fibrosis (CF) lung disease causes airway neutrophilia and hyperinflammation without effective bacterial clearance. We evaluated the immunostimulatory activities of lipid A, the membrane anchor of LPS, isolated from mutants of PA that synthesize structural variants, present in the airways of patients with CF, to determine if they correlate with disease severity and progression. In a subset of patients with a severe late stage of CF disease, a unique hepta-acylated lipid A, hepta-1855, is synthesized. In primary human cell cultures, we found that hepta-1855 functioned as a potent TLR4 agonist by priming neutrophil respiratory burst and stimulating strong IL-8 from monocytes and neutrophils. hepta-1855 also had a potent survival effect on neutrophils. However, it was less efficient in stimulating neutrophil granule exocytosis and also less potent in triggering proinflammatory TNF-α response from monocytes. In PA isolates that do not synthesize hepta-1855, a distinct CF-specific adaptation favors synthesis of a penta-1447 and hexa-1685 LPS mixture. We found that penta-1447 lacked immunostimulatory activity but interfered with inflammatory IL-8 synthesis in response to hexa-1685. Together, these observations suggest a potential contribution of hepta-1855 to maintenance of the inflammatory burden in late-stage CF by recruiting neutrophils via IL-8 and promoting their survival, an effect presumably amplified by the absence of penta-1447. Moreover, the relative inefficiency of hepta-1855 in triggering neutrophil degranulation may partly explain the persistence of PA in CF disease, despite extensive airway neutrophilia.

Hexa-acylated lipid A is required for host inflammatory response to Neisseria gonorrhoeae in experimental gonorrhea.

Neisseria gonorrhoeae causes gonorrhea, a sexually transmitted infection characterized by inflammation of the cervix or urethra. However, a significant subset of patients with N. gonorrhoeae remain asymptomatic, without evidence of localized inflammation. Inflammatory responses to N. gonorrhoeae are generated by host innate immune recognition of N. gonorrhoeae by several innate immune signaling pathways, including lipooligosaccharide (LOS) and other pathogen-derived molecules through activation of innate immune signaling systems, including toll-like receptor 4 (TLR4) and the interleukin-1ß (IL-1ß) processing complex known as the inflammasome. The lipooligosaccharide of N. gonorrhoeae has a hexa-acylated lipid A. N. gonorrhoeae strains that carry an inactivated msbB (also known as lpxL1) gene produce a penta-acylated lipid A and exhibit reduced biofilm formation, survival in epithelial cells, and induction of epithelial cell inflammatory signaling. We now show that msbB-deficient N. gonorrhoeae induces less inflammatory signaling in human monocytic cell lines and murine macrophages than the parent organism. The penta-acylated LOS exhibits reduced toll-like receptor 4 signaling but does not affect N. gonorrhoeae-mediated activation of the inflammasome. We demonstrate that N. gonorrhoeae msbB is dispensable for initiating and maintaining infection in a murine model of gonorrhea. Interestingly, infection with msbB-deficient N. gonorrhoeae is associated with less localized inflammation. Combined, these data suggest that TLR4-mediated recognition of N. gonorrhoeae LOS plays an important role in the pathogenesis of symptomatic gonorrhea infection and that alterations in lipid A biosynthesis may play a role in determining symptomatic and asymptomatic infections.

MyD88-dependent SHIP1 regulates proinflammatory signaling pathways in dendritic cells after monophosphoryl lipid A stimulation of TLR4.

We previously showed that monophosphoryl lipid A (MLA) activates TLR4 in dendritic cells (DCs) in a Toll/IL-1R domain-containing adaptor inducing IFN-ß (TRIF)-biased manner: MLA produced from Salmonella minnesota Re595 induced signaling events and expression of gene products that were primarily TRIF dependent, whereas MyD88-dependent signaling was impaired. Moreover, when tested in TRIF-intact/MyD88-deficient DCs, synthetic MLA of the Escherichia coli chemotype (sMLA) showed the same activity as its diphosphoryl, inflammatory counterpart (synthetic diphosphoryl lipid A), indicating that TRIF-mediated signaling is fully induced by sMLA. Unexpectedly, we found that the transcript level of one proinflammatory cytokine was increased in sMLA-treated cells by MyD88 deficiency to the higher level induced by synthetic diphosphoryl lipid A, which suggested MyD88 may paradoxically help restrain proinflammatory signaling by TRIF-biased sMLA. In this article, we demonstrate that sMLA induces MyD88 recruitment to TLR4 and activates the anti-inflammatory lipid phosphatase SHIP1 in an MyD88-dependent manner. At the same time, MyD88-dependent signaling activity at the level of IL-1R-associated kinase 1 is markedly reduced. Increased SHIP1 activity is associated with reductions in sMLA-induced IκB kinase α/ß and IFN regulatory factor 3 activation and with restrained expression of their downstream targets, endothelin-1 and IFN-ß, respectively. Results of this study identify a pattern that is desirable in the context of vaccine adjuvant design: TRIF-biased sMLA can stimulate partial MyD88 activity, with MyD88-dependent SHIP1 helping to reduce proinflammatory signaling in DCs.

A unique role of the cholera toxin A1-DD adjuvant for long-term plasma and memory B cell development.

Adjuvants have traditionally been appreciated for their immunoenhancing effects, whereas their impact on immunological memory has largely been neglected. In this paper, we have compared three mechanistically distinct adjuvants: aluminum salts (Alum), Ribi (monophosphoryl lipid A), and the cholera toxin A1 fusion protein CTA1-DD. Their influence on long-term memory development was dramatically different. Whereas a single immunization i.p. with 4-hydroxy-3-nitrophenyl acetyl (NP)-chicken γ-globulin and adjuvant stimulated serum anti-NP IgG titers that were comparable at 5 wk, CTA1-DD-adjuvanted responses were maintained for >16 mo with a half-life of anti-NP IgG ∼36 wk, but <15 wk after Ribi or Alum. A CTA1-DD dose-dependent increase in germinal center (GC) size and numbers was found, with >60% of splenic B cell follicles hosting GC at an optimal CTA1-DD dose. Roughly 7% of these GC were NP specific. This GC-promoting effect correlated well with the persistence of long-term plasma cells in the bone marrow and memory B cells in the spleen. CTA1-DD also facilitated increased somatic hypermutation and affinity maturation of NP-specific IgG Abs in a dose-dependent fashion, hence arguing that large GC not only promotes higher Ab titers but also high-quality Ab production. Adoptive transfer of splenic CD80(+), but not CD80(-), B cells, at 1 y after immunization demonstrated functional long-term anti-NP IgG and IgM memory cells. To our knowledge, this is the first report to specifically compare and document that adjuvants can differ considerably in their support of long-term immune responses. Differential effects on the GC reaction appear to be the basis for these differences.

The influence of local and systemic preconditioning on oxygenation, metabolism and survival in critically ischaemic skin flaps in pigs.

Stress proteins represent a group of highly conserved intracellular proteins that provide adaptation against cellular stress. The present study aims to elucidate the stress protein-mediated effects of local hyperthermia and systemic administration of monophosphoryl lipid A (MPL) on oxygenation, metabolism and survival in bilateral porcine random pattern buttock flaps. Preconditioning was achieved 24h prior to surgery by applying a heating blanket on the operative site (n = 5), by intravenous administration of MPL at a dosage of 35 microg/kg body weight (n = 5) or by combining the two (n = 5). The flaps were monitored with laser Doppler flowmetry, polarographic microprobes and microdialysis until 5h postoperatively. Semiquantitative immunohistochemistry was performed for heat shock protein 70 (HSP70), heat shock protein 32 (also termed haem oxygenase-1, HO-1), and inducible nitrc oxide synthase (iNOS). The administration of MPL increased the impaired microcirculatory blood flow in the proximal part of the flap and partial oxygen tension in the the distal part by approximately 100% each (both P<0.05), whereas both variables remained virtually unaffected by local heat preconditioning. Lactate/pyruvate (L/P) ratio and glycerol concentration (representing cell membrane disintegration) in the distal part of the flap gradually increased to values of approximately 500 mmol/l and approximately 350 micromol/l, respectively (both P<0.01), which was substantially attenuated by heat application (P<0.01 for L/P ratio and P<0.05 for glycerol) and combined preconditioning (P<0.01 for both variables), whereas the effect of MPL was less marked (not significant). Flap survival was increased from 56% (untreated animals) to 65% after MPL (not significant), 71% after heat application (P<0.05) and 78% after both methods of preconditioning (P<0.01). iNOS and HO-1 were upregulated after each method of preconditioning (P<0.05), whereas augmented HSP70 staining was only observed after heat application (P<0.05). We conclude that local hyperthermia is more effective in preventing flap necrosis than systemic MPL administration because of enhancing the cellular tolerance to hypoxic stress, which is possibly mediated by HSP70, whereas some benefit may be obtained with MPL due to iNOS and HO-1-mediated improvement in tissue oxygenation.

Regulation of cementoblast function by P. gingivalis lipopolysaccharide via TLR2.

Although cementoblasts express Toll-like receptors (TLR)-2 and -4, little is known regarding the possible participation of cementoblasts in the inflammatory response. We investigated the effects of Porphyromonas gingivalis lipopolysaccharide (LPS), tetra- and penta-acylated lipid A species (designated PgLPS(1435/1449) and PgLPS(1690), respectively), on gene expression of osteoclastogenesis-associated molecules in murine cementoblasts. Real-time quantitative RT-PCR analysis revealed that receptor activator of NF-kappaB ligand (RANKL), interleukin-6, Regulated on activation, normal T-cell expressed, and secreted (RANTES), macrophage inflammatory protein-1alpha, and monocyte chemoattractant protein-1 were rapidly and dramatically induced upon stimulation with PgLPS(1690), but only slightly induced with PgLPS(1435/1449). Osteoprotegerin, which was expressed constitutively, was not altered significantly. ELISA demonstrated synthesis of corresponding proteins. PgLPS(1690) significantly induced transcripts for NF-kappaB, and this activation was inhibited by pre-treatment with anti-TLR-2 but not with TLR-4 antibodies. These results suggest that cementoblasts participate in the recruitment of osteoclastic precursor cells by up-regulation of chemokines/cytokines.

The Lipid A substructure of the Sinorhizobium meliloti lipopolysaccharides is sufficient to suppress the oxidative burst in host plants.

Medicago sativa (alfalfa), Medicago truncatula and Nicotiana tabacum cell suspension cultures, responding to elicitation with the production of reactive oxygen species (ROS), were used to analyse the suppressor (and elicitor) activity of lipopolysaccharides (LPS) of the symbiotic soil bacterium Sinorhizobium meliloti. In order to identify the epitopes of the LPS molecule recognized by the plant, S. meliloti mutants defective in LPS biosynthesis and hydrolytically obtained Lipid A were analysed for biological activity. Lipopolysaccharides isolated from Sinorhizobium meliloti mutants 6963 (altered core region) and L994 (no long-chain fatty acid) showed the same ability to suppress the oxidative burst in host plant cell cultures as the wild-type LPS. Lipid A also displayed the same suppressor activity. By contrast, rhizobial LPS, but not Lipid A, was active as an inducer of the oxidative burst reaction in cell cultures of the nonhost Nicotiana tabacum. In host plants of Sinorhizobium meliloti the Lipid A part is sufficient to suppress the oxidative burst, but in non-host plants at least some sugars of the LPS core region are required to induce defence reactions.

Synthesis and biological evaluation of a lipid A derivative that contains an aminogluconate moiety.

A highly convergent strategy for the synthesis of several derivatives of the lipid A of Rhizobium sin-1 has been developed. The synthetic derivatives are 2-aminogluconate 3 and 2-aminogluconolactone 4, both of which lack C-3 acylation. These derivatives were obtained by the preparation of disaccharides in which the two amino groups and the C-3' hydroxy group could be modified individually with acyl or beta-hydroxy fatty acyl groups. Detailed NMR spectroscopy and MS analysis of 3 and 4 revealed that, even under neutral conditions, the two compounds equilibrate. The synthetic compounds lack the proinflammatory effects of Escherichia coli lipopolysaccharide (LPS), as indicated by an absence of tumor necrosis factor production. Although 3 and 4 were able to antagonize E. coli LPS, they were significantly less potent than the synthetic compound 2, which is acylated at C-3, and R. sin-1 LPS; these results indicate that the beta-hydroxy fatty acyl group at C-3 contributes to the antagonistic properties of R. sin-1 LPS. Based on a comparison of the biological responses of the synthetic lipid A derivatives with those of the R. sin-1 LPS and lipid A, the 3-deoxy-D-manno-octulosonic moieties appear to be important for the optimal antagonization of enteric LPS-induced cytokine production.

Diphosphoryl lipid A from Rhodobacter sphaeroides blocks the binding and internalization of lipopolysaccharide in RAW 264.7 cells.

Diphosphoryl lipid A derived from the nontoxic LPS of Rhodobacter sphaeroides (RsDPLA) has been shown to be a powerful LPS antagonist in both human and murine cell lines. In addition, RsDPLA also can protect mice against the lethal effects of toxic LPS. In this study, we complexed both the deep rough LPS from Escherichia coli D31 m4 (ReLPS) and RsDPLA with 5- and 30-nm colloidal gold and compared their binding to the RAW 264.7 cell line by electron microscopy. Both ReLPS and RsDPLA bound to the cells with the following observations. First, binding studies revealed that pretreatment with RsDPLA completely blocked the binding and thus internalization of ReLPS-gold conjugates to these cells at both 37 degrees C and 4 degrees C. Second, ReLPS was internalized via micropinocytosis (noncoated plasma membrane invaginations) involving formation of caveolae-like structures and leading to the formation of micropinocytotic vesicles, macropinocytosis (or phagocytosis), formation of clathrin-coated pits (receptor mediated), and penetration through plasma membrane into cytoplasm. Third, in contrast, RsDPLA was internalized predominantly via macropinocytosis. These studies show for the first time that RsDPLA blocks the binding and thus internalization of LPS as observed by scanning and transmission electron microscopy.

CD11/CD18 and CD14 share a common lipid A signaling pathway.

The activation of phagocytes by the lipid A moiety of LPS has been implicated in the pathogenesis of Gram-negative sepsis. While two LPS receptors, CD14 and CD11/CD18, have been associated with cell signaling, details of the LPS signal transduction cascade remain obscure. CD14, which exists as a GPI-anchored and a soluble protein, lacks cytoplasmic-signaling domains, suggesting that an ancillary molecule is required to activate cells. The CD11/CD18 integrins are transmembrane proteins. Like CD14, they are capable of mediating LPS-induced cellular activation when expressed on the surface of hamster fibroblasts Chinese hamster ovary (CHO)-K1. The observation that a cytoplasmic deletion mutant is still capable of activating transfected CHO-K1 argues that CD11/CD18 also utilizes an associated signal transducer. We sought to identify further similarities between the signaling systems utilized by CD14 and CD11/CD18. LPS-binding protein, which transfers LPS to CD14, enhanced both LPS-induced cellular activation and binding of Gram-negative bacteria in CD11/CD18-transfected CHO-K1, thus implying that LPS-binding protein can also transfer LPS to CD11/CD18. When synthetic lipid A analogues were analyzed for their ability to function as LPS agonists, or antagonists, in the CHO transfectants, we found the effects were identical regardless of which LPS receptor was expressed. This supports the hypothesis that a receptor distinct from CD14 and CD11/CD18 is responsible for discriminating between the lipid A of LPS and the LPS antagonists. We propose that this receptor, which is the target of the LPS antagonists, functions as the true signal transducer in LPS-induced cellular activation for both CD14 and CD11/CD18.

A lethal role for lipid A in Salmonella infections.

Salmonella infections in naturally susceptible mice grow rapidly, with death occurring only after bacterial numbers in vivo have reached a high threshold level, commonly called the lethal load. Despite much speculation, no direct evidence has been available to substantiate a role for any candidate bacterial components in causing death. One of the most likely candidates for the lethal toxin in salmonellosis is endotoxin, specifically the lipid A domain of the lipopolysaccharide (LPS) molecule. Consequently, we have constructed a Salmonella mutant with a deletion-insertion in its waaN gene, which encodes the enzyme that catalyses one of the two secondary acylation reactions that complete lipid A biosynthesis. The mutant biosynthesizes a lipid A molecule lacking a single fatty acyl chain and is consequently less able to induce cytokine and inducible nitric oxide synthase (iNOS) responses both in vivo and in vitro. The mutant bacteria appear healthy, are not sensitive to increased growth temperature and synthesize a full-length O-antigen-containing LPS molecule lacking only the expected secondary acyl chain. On intravenous inoculation into susceptible BALB/c mice, wild-type salmonellae grew at the expected rate of approximately 10-fold per day in livers and spleens and caused the death of the infected mice when lethal loads of approximately 10(8) were attained in these organs. Somewhat unexpectedly, waaN mutant bacteria grew at exactly the same rate as wild-type bacteria in BALB/c mice but, when counts reached 10(8) per organ, mice infected with mutant bacteria survived. Bacterial growth continued until unprecedentedly high counts of 10(9) per organ were attained, when approximately 10% of the mice died. Most of the animals carrying these high bacterial loads survived, and the bacteria were slowly cleared from the organs. These experiments provide the first direct evidence that death in a mouse typhoid infection is directly dependent on the toxicity of lipid A and suggest that this may be mediated via pro-inflammatory cytokine and/or iNOS responses.

Vaccine adjuvants: A new hope for effective immunization

Two important issue regarding the use of immunization to control infections and malignancies in the futureare: 1) the need to render poorly immunogenic, often highly purified, antigens more effective; and 2) the desire to direct the immune response in specific ways to achieve the most relevant response for each disease. The first issue can be solved by a broad range of vaccine adjuvants. The second requires careful selection among the adjuvants to allow directing of the immune response in the most appropriate manner. For exemple, in different settings expansion of a B cell response, cytotoxic T cell response, or enhancement of either a Th1 or Th2 subset response may be desired. These goals are accomplished by the use of several newly developed non-cytokine adjuvants, or by direct injection of the relevant cytokines. Some non-cytokine molecular adjuvants and cytokines used as adjuvants have already been proven effective in animal models and/or in clinical trials. Here, we review the present state of art in the use of vaccine adjuvants for control of various infections diseases.

A novel Escherichia coli lipid A mutant that produces an antiinflammatory lipopolysaccharide.

A unique screen was used to identify mutations in Escherichia coli lipid A biosynthesis that result in a decreased ability to stimulate E-selectin expression by human endothelial cells. A mutation was identified in the msbB gene of E. coli that resulted in lipopolysaccharide (LPS) that lacks the myristoyl fatty acid moiety of the lipid A. Unlike all previously reported lipid A mutants, the msbB mutant was not conditionally lethal for growth. Viable cells or purified LPS from an msbB mutant had a 1000-10,000-fold reduction in the ability to stimulate E-selectin production by human endothelial cells and TNF alpha production by adherent monocytes. The cloned msbB gene was able to functionally complement the msbB mutant, restoring both the LPS to its native composition and the ability of the strain to stimulate immune cells. Nonmyristoylated LPS acted as an antagonist for E-selectin expression when mixed with LPS obtained from the parental strain. These studies demonstrate a significant role for the myristate component of LPS in immune cell activation and antagonism. In addition, the msbB mutant allowed us to directly examine the crucial role that the lipid A structure plays when viable bacteria are presented to host defense cells.

Influence of CD14, LBP and BPI in the monocyte response to LPS of different polysaccharide chain length.

In this study we examined the involvement of human serum, recombinant lipopolysaccharide binding protein (rLBP), recombinant (r)CD14, CD14 antibodies and recombinant bactericidal permeability-increasing factor (rBPI) in the induction of TNF by Salmonella minnesota LPS of different polysaccharide chain lengths. Soluble rCD14 and rLBP markedly enhanced LPS 6261 TNF production and to a lesser degree also enhanced TNF production from Re 595 LPS and lipid A DP. Addition of anti-CD14 antibodies resulted in nearly complete inhibition of LPS 6261-induced TNF production and partial inhibition of Re 595 LPS and lipid A DP-induced TNF release. The ability of lipid A MP to induce TNF production increased with addition of rCD14. Addition of rLBP or anti-CD14 antibodies had no detectable effect on lipid A MP-induced TNF production. The effect of rBPI was also tested and the results showed that only the TNF-inducing ability from smooth LPS was completely inhibited by rBPI. Recombinant BPI was considerably less effective in inhibiting Re 595 LPS-induced TNF production, and lipid A DP was not affected by rBPI. Our data suggest that the ability of rLBP, rCD14, CD14 antibodies and rBPI to modulate LPS induced TNF production is strongly dependent on the LPS polysaccharide chain length.

Inhibition of lipid A- and lipopolysaccharide-induced cytokine secretion, B cell mitogenesis, and lethal shock by lipid A-specific murine monoclonal antibodies.

Three murine hybridomas secreting IgM monoclonal antibodies (MAbs) to lipid A (LA) of Salmonella minnesota R595 were generated. These MAbs serologically cross-reacted with LA and lipopolysaccharide (LPS) of unrelated gram-negative bacterial species. All three MAbs significantly suppressed the ability of LA and LPS from various gram-negative bacteria to induce tumor necrosis factor (TNF)-alpha (36%-67%) and interleukin-1 (30%-98%) in murine peritoneal macrophages and to stimulate B lymphocytes (37%-78%). Lipid A-induced TNF alpha production was also suppressed in mice (86%-88%). All three antibodies protected adrenalectomized mice against lethal shock induced by LA of S. minnesota R595. Optimal protection was achieved with one of the antibodies (MLA-1), if it was administered 2 h before injection of lipid A, and full protection persisted < or = 24 h. Moreover, MLA-1 was able to protect adrenalized or D(+)-galactosamine-sensitized mice against lethal shock induced by LPS derived from various gram-negative bacteria. This cross-protection could be predicted on the basis of serologic cross-reactivity and cross-neutralization by MLA-1 of the bioactivity of the heterologous LA or LPS in vitro.

Pathophysiology of renal failure in obstructive jaundice.

This paper aims to consider the several factors which may participate in renal failure occurring in surgery in patients with obstructive jaundice. The role of bilirubin and bile acids and the endotoxin effects related to lipid A, the active fragment of bacterial lipopolysaccharides, have been investigated to analyze their responsibility in propensities to hypotension and to postoperative acute renal failure.

Macrophage activation by an ornithine-containing lipid or a serine-containing lipid.

alpha-N-(3-Acyloxyacyl)-ornithine (or -serine) is the structure of lipoamino acids obtained by us previously from some gram-negative bacteria (Y. Kawai and I. Yano, Eur. J. Biochem. 136:531-538, 1983; Y. Kawai, I. Yano, and K. Kaneda, Eur. J. Biochem. 171:73-80, 1988; Y. Kawai, I. Yano, K. Kaneda, and E. Yabuuchi, Eur. J. Biochem. 175:633-641, 1988). The 3-acyloxyacylamide structure is present in both the lipoamino acids and lipid A of lipopolysaccharide (endotoxin). The efficacy of lipoamino acids (an ornithine-containing lipid and a serine-containing lipid) in activating C3H/HeSlc mouse peritoneal exudate macrophages was compared with that of bacterial lipopolysaccharide, because the two types of substances were expected to exhibit similar biological activities and physiological functions on the basis of their structural similarities. Actually, the lipoamino acids, as well as lipopolysaccharide, strongly activated the macrophages to generate the immunoregulatory substances prostaglandin E2 and interleukin-1, but their effect on the induction of L929 cell cytolytic factor (a possible tumor necrosis factor), another immunoregulatory substance, was weaker than that of lipopolysaccharide. The effect of lipoamino acids on the cytotoxicity of macrophages for EL-4 leukemia cells was very weak. However, all of these activities, as far as tested, were strongly enhanced by synergistic action with gamma interferon. Only the serine-containing lipid killed both C3H/HeSlc and C3H/HeJ macrophages to almost the same degree as endotoxin killed C3H/HeSlc macrophages. On the other hand, lethal toxicity for mice was not found with either the ornithine-containing lipid or the serine-containing lipid, even when 7 mg of compound was injected into a mouse. These studies suggest that the lipoamino acids are nontoxic characteristic immunoactivators.

Diphosphoryl lipid A from Rhodopseudomonas sphaeroides ATCC 17023 blocks induction of cachectin in macrophages by lipopolysaccharide.

Purified diphosphoryl lipid A (DPLA) obtained from the nontoxic lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023 was shown to block the induction of cachectin (tumor necrosis factor) in the RAW 264.7 macrophage cell line by toxic deep-rough-chemotype lipopolysaccharide (ReLPS) of Escherichia coli in a concentration-dependent manner. The ReLPS-to-DPLA mass ratios of 1:10 and 1:100 (when 1.0 ng of ReLPS per ml was used) gave 55 and 95% inhibitions, respectively, of the induction of cachectin. Since the structure of the DPLA from R. sphaeroides is so similar to that of the lipid A moiety of the toxic ReLPS from E. coli, we suggest that this inhibition could have been due to competitive binding by DPLA to the active sites on the macrophage. This DPLA could become a useful reagent to study the nature of lipopolysaccharide/lipid A binding in macrophages and perhaps other responding cells.

Structure-activity relationship of lipid A: comparison of biological activities of natural and synthetic lipid A's with different fatty acid compositions.

To investigate the structure-activity relationships, various biological activities, including pyrogenicity, lethal toxicity, elicitation of Shwartzman reaction, mitogenicity and tumor necrosis factor (TNF)-inducing activity, were compared among natural and synthetic lipid A's differing in fatty acid composition. In all these tests, natural lipid A's from Escherichia coli and Salmonella minnesota and synthetic LA-15-PP, which carries 3-hydroxy- and 3-acyloxy-tetradecanoyl groups at the 2, 3 and 2', 3' positions, respectively, showed the strongest activities among the tested lipid A's. In contrast, LA-16-PP, in which the amide-bound 3-hydroxytetradecanoic acid at position 2 of LA-15-PP is replaced by 3-hexadecanoyloxytetradecanoic acid, exhibited lower activity than LA-15-PP and natural lipid A's. Although LA-16-PP has been assumed to have a typical Salmonella lipid A structure (and, in fact, it has a structure corresponding to one of the components of Salmonella lipid A), the activity of this synthetic compound was not comparable to that of natural Salmonella lipid A. LA-17-PP, in which tetradecanoic acid is the sole fatty acid component, exhibited relatively strong mitogenicity and TNF-inducing activity, but very low pyrogenicity. The activities of LA-18-PP, which has ester-bound tetradecanoic acid and amide-bound 3-hydroxytetradecanoic acid, were lower than those of LA-17-PP. The results indicate that the differences in fatty acid composition of lipid A's have important influences on the biological activities studied.

Mitogenic and polyclonal B cell activation activities of synthetic lipid A analogues.

The activation of murine B cells and macrophages by synthetic lipid A analogues, solubilized with triethylamine and complexed with bovine serum albumin, were investigated in vitro. The analogues used are nonphosphorylated, C-1 or C-4' monophosphorylated and C-1,4' diphosphorylated derivatives of beta-1,6-linked D-glucosamine disaccharide possessing both ester- and amide-bound fatty acid substituents. They were divided into 4 groups, A, B, C and D in terms of the fatty acid substitution. Ester- and amine-bound fatty acids of the analogues are both tetradecanoic acids (C14) in group A, C14 and (R)-3-hydroxytetradecanoic acids (C14-OH) in group B, both C14-OH in group C and C14-OH and (R)-3-tetradecanoyloxytetradecanoic acids in group D. Mitogenic activity was exhibited in spleen cells from C3H/HeN mice by the C-1 monophosphorylated analogues in groups A and B, and by the C-4' monophosphorylated analogues in groups B, C and D, but not in cells from C3H/HeJ mice. Nonphosphorylated analogues in groups A, B and C, and C-4' monophosphorylated analogue in group A showed negative mitogenic activity. Only the nonphosphorylated analogue in group D exhibited mitogenic activity in spleen cells from C3H/HeJ mice as well as those from C3H/HeN mice. None of the other analogues exhibited the activity in C3H/HeJ spleen cells. Polyclonal B cell activation activity was exhibited by the C-1 monophosphorylated analogues in groups A and B, and by the C-4' monophosphorylated analogues in groups C and D. Nonphosphorylated analogues in all groups and C-4' monophosphorylated analogues in groups A and B showed negative PBA activity. None of the analogues tested could induce any cytostatic macrophages from thioglycollate-elicited peritoneal macrophages.