In vitro spleen cell proliferation following in vivo treatment with a synthetic glycolipid or lipid A in three mouse strains

Synthetic glycolipid, maltose hexastearate (MHS), like LPS or lipid A is a B cell mitogen for outbred Swiss mice, inbred C₃H/HeN and C₃H/HeJ × C₃H/HeN F₁ hybrid but not for C₃H/HeJ mice. MHS administered i.p. (10 μg) to Swiss, C₃H/HeN, C₃H/HeJ and C₃H/HeJ × C₃H/HeN F₁ hybrid mice confers within 90 min an increased ability in the spleen cell populations such that they exhibit increased incorporation of ³H thymidine into cellular DNA in vitro. The spleen cells of MHS treated mice also respond in vitro more vigorously than controls to a T cell mitogen (Con A) but not at all differently from controls to a B cell mitogen (LPS). The stimulated cells are sensitive to anti θ serum + C′ (experiment done with Swiss mice) and the Lyt 1,1 monoclonal antibody + C′ (experiment done with C₃H/HeN mice), indicating them functionally to be of T-helper variety. Unlike MHS, lipid A administration i.p. resulted in MHS like response in C₃H/HeN but not in C₃H/HeJ mice.MHS action has been traced to an induction of IL1 release by macrophages of MHS treated animals by chromatographic analysis of macrophage derived supernatants. IL1, in turn, according to the prevalent concepts would stimulate immature T cells to become mature IL2 producer cells, allowing T helper cell proliferation through IL2 release.     

Structural features of lipid A of Rickettsia typhi

Lipid A isolated from the Rickettsia typhi lipopolysaccharide (LPS) was investigated for its composition and structure using chemical analyses, gas chromatography-mass spectrometry (GC-MS), and electrospray ionization (ESI) combined with the tandem mass spectrometry (MS/MS). Our studies revealed a noticeable compositional and structural heterogeneity of lipid A with respect to the content of phosphate groups and the degree of acylation. It appeared that at least two molecular species were present in lipid A. The major species represented the hexaacyl lipid A consisting of the β-(1--> 6)-linked D-glucosamine (GlcN) disaccharide backbone carrying two phosphate groups. One of them was linked to the glycosidic hydroxyl group of the reducing GlcN I and the other was ester linked to the O-4′ position of the non-reducing GlcN II. The primary fatty acids consisted of two 3-hydroxytetradecanoic [C14:0(3-OH)] and two 3-hydroxyhexadecanoic [C16:0(3-OH)] acids. The former were ester- and the latter amide-linked to both GlcN. Two secondary fatty acids were represented by the octadecanoic (C18:0) and hexadecanoic (C16:0) acids that were ester-linked at the N-2′ and O-3′ positions, respectively. In the minor lipid A species, ester-linked C18:0 was substituted by C16:0 at the C16:0(3-OH) of GlcN II. The R. typhi lipid A resembles structurally the classical forms of enterobacterial lipids A with high endotoxicity.     

Structural requirements for inducing in vitro B lymphocyte activation by chemically synthesized derivatives related to the nonreducing D-glucosamine subunit of lipid A

Mitogenic and polyclonal B cell activation (PBA) activities of 16 synthetic compounds related to the nonreducing D-glucosamine (GlcN-II) subunit of lipid A were investigated. Among compounds possessing the GlcN backbone, a 4-O-phosphorylated GlcN derivative carrying N-linked 3-tetradecanoyloxytetradecanoyl [C14-O-(C14)] and 3-O-linked tetradecanoyl (C14) groups, GLA-27, expressed the highest degree of both activities. Omission of the 3-O-linked C14 group from GLA-27 and transfer of the C14 group to the C-6 position induced critical changes in expression of activities. Both 4-O-phosphorylated compounds carrying an N-linked C14 or 3-hydroxytetradecanoyl (C14OH) group instead of the C14-O-(C14) group in GLA-27 showed no detectable activity. Substituting a 3-O-linked C14 group in GLA-27 for the C14-O-(C14) group also markedly decreased mitogenic and PBA activities. Change of phosphorylation site from the C-4 to the C-6 position and bisphosphorylation at the C-4 and C-6 positions induced somewhat weak depression. Much weaker activities were observed in a compound carrying N-linked 3-dodecanoyloxydodecanoyl [C12-O-(C12)] and 3-O-dodecanoyl (C12) as fatty acid substituents. No detectable activity was seen in a compound carrying N-linked 3-hexadecanoyloxyhexadecanoyl [C16-O-(C16)] and 3-O-hexadecanoyl (C16), indicating that the most suitable carbon chain length for expressing the activities is C14. Regarding structural change of the GlcN backbone, a 1-deoxy derivative of GLA-27 exhibited stronger activity than did GLA-27 itself. Mitogenic and PBA activity of GLA-27 were stronger than those of lipid X, which corresponds to the reducing D-GlcN (GlcN-I) subunit of Escherichia coli lipid A and is a 1-O-phosphorylated GlcN derivative carrying N- and 3-O-linked C14OH groups. These results indicate that N-linked acyloxyacyl and 3-O-linked acyl groups and phosphorylation are critical for expressing both mitogenic and PBA activities.     

The effect of anti-lymphocytic antibody on the humoral immune response in different strains of mice: I. The response to bovine serum albumin

The effect of a single batch of horse anti-mouse thymocyte globulin on the humoral immune response has been extensively investigated in 10–12-week-old male mice of six inbred strains. The preparation under test readily suppressed the primary immune response of A/HeJ, C57B1, DBA/1, CBA and C₃H mice to 0.1, 1.0 and 10.0 mg doses of alum precipitated bovine serum albumin (alum BSA). However, at the doses used it generally failed to significantly suppress the primary immune response of BALB/c mice to 0.1 and 1.0 mg doses of this antigen. Furthermore, the effectiveness of the suppression was somewhat diminished by the incorporation of bordetella pertussis organisms into the alum BSA inoculum. The product also suppressed the secondary immune response to alum BSA in C₃H mice and C57B1 mice but, at the same doses, was without significant effect on the secondary responses of A/HeJ and CBA mice.     

Biological properties of lipid A isolated from Flavobacterium meningosepticum

The biological properties of the lipid A from Flavobacterium meningosepticum, which we recently isolated and whose complete chemical structure has been determined (H. Kato, T. Iida, Y. Haishima, A. Tanaka, and K. Tanamoto. J. Bacteriol. 180:3891--3899, 1998), were studied. The lipid A exhibited generally moderate activity compared to Salmonella enterica subsp. enterica serovar abortus equi lipopolysaccharide (LPS) used as a control in the assay systems tested; lethal toxicity in galactosamine-sensitized mice, mitogenicity in mouse spleen cells, induction of tumor necrosis factor alpha (TNF-alpha) release from mouse peritoneal macrophages and J774-1 mouse macrophage-like and human THP-1 line cells, nitric oxide induction activity from J774-1 cells, and Limulus gelation activity. The moderate activity of the F. meningosepticum lipid A may be explained by its unique fatty acid composition and the lack of a phosphate group in position 4'. It is noteworthy that the lipid A apparently induced TNF-alpha release from peritoneal macrophages in LPS-unresponsive C3H/HeJ mice and that the activation was suppressed by the LPS-specific antagonist, succinylated lipid A precursor. Significant splenocyte mitogenicity in C3H/HeJ mice was also observed with the lipid A. Taken together with the previous results concerning Porphyromonas gingivalis lipid A, which has a high level of structural similarity to the lipid A of F. meningosepticum, and the induction of TNF-alpha release in macrophages from C3H/HeJ mice, the lipid A of F. meningosepticum, which has novel fatty acids, may possibly play an role for the activation of C3H/HeJ macrophages.     

Comparative phenotypic assessment of cardiac pathology, physiology, and gene expression in C3H/HeJ, C57BL/6J, and B6C3F1/J mice

Human cardiomyopathies often lead to heart failure, a major cause of morbidity and mortality in industrialized nations. Described here is a phenotypic characterization of cardiac function and genome-wide expression from C3H/HeJ, C57BL/6J, and B6C3F1/J male mice. Histopathologic analysis identified a low-grade background cardiomyopathy (murine progressive cardiomyopathy) in eight of nine male C3H/HeJ mice (age nine to ten weeks), but not in male C57BL/6J and in only of ten male B6C3F1/J mice. The C3H/HeJ mouse had an increased heart rate and a shorter RR interval compared to the B6C3F1/J and C57BL/6J mice. Cardiac genomic studies indicated the B6C3F1/J mice exhibited an intermediate gene expression phenotype relative to the 2 parental strains. Disease-centric enrichment analysis indicated a number of cardiomyopathy-associated genes were induced in B6C3F1/J and C3H/HeJ mice, including Myh7, My14, and Lmna and also indicated differential expression of genes associated with metabolic (e.g., Pdk2) and hypoxic stress (e.g. Hif1a). A novel coexpression and integrated pathway network analysis indicated Prkaa2, Pdk2, Rhoj, and Sgcb are likely to play a central role in the pathophysiology of murine progressive cardiomyopathy in C3H/HeJ mice. Our studies indicate that genetically determined baseline differences in cardiac phenotype have the potential to influence the results of cardiotoxicity studies.     

Biological activities of lipopolysaccharides of Proteus spp. and their interactions with polymyxin B and an 18-kDa cationic antimicrobial protein (CAP18)-derived peptide

The saccharide constituents of lipopolysaccharides (LPS) of Proteus spp. vary with the strain and contain unique components about which little is known. The biological activities of LPS and lipid A from S- and R-forms of 10 Proteus strains were examined. LPS from all S-form Proteus strains was lethal to D-(+)-galactosamine (GalN)-loaded, LPS-responsive, C3H/HeN mice, but not to LPS-hypo-responsive C3H/HeJ mice. P. vulgaris 025 LPS evoked strong anaphylactoid reactions in N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP)-primed C3H/HeJ mice. LPS from S- and R-form Proteus strains induced production of nitric oxide (NO) and tumour necrosis factor (TNF) by macrophages isolated from C3H/HeN but not C3H/HeJ mice. Lipid A from Proteus strains also induced NO and TNF production, although lipid A was less potent than LPS. The effects of LPS were mainly dependent on CD14; LPS-induced NO and TNF production in CD14+ J774.1 cells was significantly greater than in CD14-J7.DEF.3 cells. All LPS from Proteus strains, and especially from P. vulgaris 025, exhibited higher anti-complementary activity than LPS from Escherichia coli or Pseudomonas aeruginosa. Polymyxin B inactivated proteus LPS in a dose-dependent manner, but these LPS preparations were more resistant to polymyxin B than E. coli LPS. CAP18(109-135), a granulocyte-derived peptide, inhibited proteus LPS endotoxicity only when the LPS:CAP18(109-135) ratio was appropriate, which suggests that CAP18(109-135) acts through a different mechanism than polymyxin B. The results indicate that LPS from Proteus spp. are potently endotoxic, but that the toxicity is different from that of LPS from E. coli or Salmonella spp. and even varies among different Proteus strains. The variation in biological activities among proteus LPS may be due to unique components within the respective LPS.     

Enhancement of nonspecific resistance to bacterial infections and tumor regressions by treatment with synthetic lipid A-subunit analogs. Critical role of N- and 3-O-linked acyl groups in 4-O-phosphono-D-glucosamine derivatives

Enhancement of nonspecific resistance against Pseudomonas aeruginosa infection and regression of growth of Meth A fibrosarcoma by chemically synthesized lipid A-subunit analogs, 4-O-phosphono-D-glucosamine derivatives carrying 3-O- and N-linked acyl groups, were investigated. Compounds carrying an (R)-3-hydroxytetradecanoyl (C14-OH) group at the 2-N-position with (R)-3-tetradecanoyloxytetradecanoyl [C14-O-(C14)] or (R)-3-dodecanoyloxytetradecanoyl [C14-O-(C12)] groups at the 3-O-position, termed GLA-60 or GLA-63, respectively, showed strong activity about one-tenth that of natural lipid A. The protective activity of compounds carrying an (R)-3-hexadecanoyloxytetradecanyl group instead of a C14-O-(C14) or C14-O-(C12) group was very weak. GLA-59 carrying the same acyl components as those of GLA-60 but with reversed binding sites showed significant but not so strong protective activity. The activity of compounds possessing a tetradecanoyl group instead of a C14-OH group in GLA-60 or GLA-63 was weaker than that of GLA-60 or GLA-63. Intravenous or intratumoral administration of GLA-59, GLA-60 and GLA-63 induced significant regression of Meth A fibrosarcoma in terms of tumor size, tumor weight and number of cured mice. The activity of GLA-59 was almost equivalent to that of GLA-60. None of the tested compounds exhibited significant pyrogenicity at a dose of 10 micrograms/kg in rabbits.     

Biological Properties of Lipid A Isolated from Flavobacterium meningosepticum

The biological properties of the lipid A from Flavobacterium meningosepticum, which we recently isolated and whose complete chemical structure has been determined (H. Kato, T. Iida, Y. Haishima, A. Tanaka, and K. Tanamoto. J. Bacteriol. 180:3891–3899, 1998), were studied. The lipid A exhibited generally moderate activity compared to Salmonella enterica subsp. enterica serovar abortus equi lipopolysaccharide (LPS) used as a control in the assay systems tested; lethal toxicity in galactosamine-sensitized mice, mitogenicity in mouse spleen cells, induction of tumor necrosis factor alpha (TNF-α) release from mouse peritoneal macrophages and J774-1 mouse macrophage-like and human THP-1 line cells, nitric oxide induction activity from J774-1 cells, and Limulus gelation activity. The moderate activity of the F. meningosepticum lipid A may be explained by its unique fatty acid composition and the lack of a phosphate group in position 4′. It is noteworthy that the lipid A apparently induced TNF-α release from peritoneal macrophages in LPS-unresponsive C3H/HeJ mice and that the activation was suppressed by the LPS-specific antagonist, succinylated lipid A precursor. Significant splenocyte mitogenicity in C3H/HeJ mice was also observed with the lipid A. Taken together with the previous results concerning Porphyromonas gingivalis lipid A, which has a high level of structural similarity to the lipid A of F. meningosepticum, and the induction of TNF-α release in macrophages from C3H/HeJ mice, the lipid A of F. meningosepticum, which has novel fatty acids, may possibly play an role for the activation of C3H/HeJ macrophages.     

Adjuvant actions of linear mannan-possessing lipopolysaccharide (LPS) in LPS-resistant C3H/HeJ mice.

Immunopotentiation has been demonstrated when Klebsiella O3 lipopolysaccharide (KO3 LPS), which possesses a linear mannan as the O-specific side chain, was injected subcutaneously into endotoxin resistant C3H/HeJ mice together with soluble protein antigens. The LPS exhibited significantly positive adjuvant effects on antibody responses in vivo after secondary antigen challenge and on delayed-type hypersensitivity (DTH) reactions against protein antigens. However, KO3 LPS was not a polyclonal B cell activator (PBA) in C3H/HeJ mice nor mitogenic in cultures of spleen cells of C3H/HeJ. Thus, the activity of the LPS in C3H/HeJ mice is confined to the potentiation of T-dependent immune responses. The contribution of the mannan O side chain to the adjuvant action of KO3 LPS was suggested.     

Mitogenic response of C3H/HeJ mouse lymphocytes to polyanionic polysaccharides obtained from Bordetella pertussis endotoxin and from other bacterial species.

Lipopolysaccharide extracted from Bordetella pertussis was mitogenic for spleen cells of endotoxin-resistant C3H/HeJ mice. Although endotoxic lipid A was inactive, mitogenic activity of lipopolysaccharide was exhibited by purified preparations of polysaccharides I and II, which constitute the carbohydrate moiety of the macromolecule. These low-molecular-weight (2,800 and 3,600) polysaccharides, containing carboxyl groups, were not mitogenic for thymocytes and splenic T-cells of C3H/HeJ mice, but did show mitogenic activity for splenic B-cells of C3H/HeJ mice and for spleen cells of C57BL/6 athymic nude mice. The mitogenic activities of polysaccharides I and II were also compared with those of other polyanionic polysaccharides, and the results indicate that high molecular weight is not necessary, and negative charges are not sufficient, for mitogenicity.     

Structural features of lipid A of Piscirickettsia salmonis, the etiological agent of the salmonid rickettsial septicemia

The composition and structure of lipid A isolated from the lipopolysaccharide (LPS) of Piscirickettsia salmonis were investigated by chemical analyses, gas chromatography/mass spectrometry (GCMS), and electrospray ionization (ESI) combined with the tandem mass spectrometry (MS/MS). Our study revealed moderate compositional and structural heterogeneity of lipid A with respect to the content of phosphate groups and 4-amino-4-deoxy-L-arabinopyranose (Ara4N) residues and with regard to the degree of acylation. It appeared that at least two molecular species were present in lipid A. The major species represented the hexaacyl lipid A consisting of the ss-(1--> 6)-linked D-glucosamine (GlcN) disaccharide backbone carrying two phosphate groups. The first one at the glycosidic hydroxyl group of the reducing GlcN I and the second one at the O-4' position of the non-reducing GlcN II. The primary fatty acids consisted of three 3-hydroxytetradecanoic [C14:0(3-OH)] and one 3-hydroxyhexadecanoic [C16:0(3-OH)] acids. The latter was amide-linked to GlcN I and one C14:0(3-OH) was amide-linked to GlcN II. Two secondary fatty acids were represented by C14:0(3-OH) and were equally distributed between the O-2' and O-3' positions. The phosphate group at O-4' carried a non-stoichiometric substituent Ara4N. The minor lipid A species contained exclusively C14:0(3-OH) with an asymmetric distribution (4+2) at GlcN II and GlcN I, respectively. The P. salmonis lipid A resembles structurally strongly endotoxic enterobacterial lipid A. This could be one of the reasons for the observed high endotoxicity of P. salmonis.     

Opposite effects of lipopolysaccharide and dextran sulfate on membrane phospholipid metabolism of murine B lymphocytes

The metabolism of [3H]inositol- and [14C]arachidonate-labeled phospholipids of B lymphocytes from normal (C3H/HePAS) and endotoxin-hyporesponsive (C3H/HeJ) mice, after incubation with two B cell mitogens, lipopolysaccharide (LPS) and dextran sulfate (DxS) was examined. The early effects of the two mitogens on the biosynthesis of phosphoinositides were different. DxS enhanced the levels of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in C3H/HeJ and C3H/HePAS cells, whereas LPS did not modify the levels of these components. When mixed with DxS, LPS reduced the effects of this stimulant. Analysis of the metabolism of fatty acids gave opposite results. Incorporation of arachidonate in all phospholipids, and particularly in phosphatidic acid, was inhibited in the two cell types after incubation with DxS, but was enhanced in C3H/HePAS and remained unchanged in C3H/HeJ cells after incubation with LPS. This activation of acyltransferases by LPS in B lymphocytes from endotoxin-responsive mice was inhibited when DxS was added in the stimulating mixture. The outcome of these opposite biochemical effects of LPS and DxS on the mitogenic responses of B cells was also examined. Preincubation with DxS for a 15-min period blocked the mitogenic effect of LPS in C3H/HePAS cells, whereas preincubation with LPS blocked the mitogenic effect of DxS in C3H/HeJ cells. Early changes in phospholipid metabolism induced by the two stimulants are therefore correlated with their late mitogenic effect.     

Differentiation of Bordetella avium and related species by cellular fatty acid analysis.

The fatty acids of 18 strains of Bordetella avium, 3 strains of Alcaligenes faecalis, 5 strains of Bordetella bronchiseptica, and 12 strains of a B. avium-like organism were examined by gas chromatography-mass spectrometry. The presence of a significant amount of the acid 2-OH C14:0 characterized B. avium and the B. avium-like organism. B. avium and the B. avium-like organism differed in their relative concentrations of C16:1 and 3-OH C14:0 acids. B. bronchiseptica and A. faecalis were distinguishable by comparison of the relative concentrations of C18:0 and C18:1 acids.     

The lipid A region of lipopolysaccharides from Rhizobiaceae activates bone marrow granulocytes from lipopolysaccharide-hyporesponsive C3H/HeJ and C57BL/10ScCr mice

We established in previous studies that the binding of Salmonella lipopolysaccharide (LPS) to constitutive receptors of low affinity triggers the expression of the inducible LPS-binding molecule CD14 in bone marrow cells (BMC) of C3H/HeOU mice, but not in BMC from C3H/HeJ mice. We show in this study that BMC from C3H/HeJ and C57BL/10ScCr mice do not express CD14 after exposure to LPSs from Salmonella enterica and Bordetella pertussis, but do express this marker when treated with several LPSs from Rhizobiaceae, or their lipid A fragments. This shows that the constitutive LPS receptor in BMC from C3H/HeJ and C57BL/10ScCr mice is fully able to trigger a complete signalling cascade. Results of cross-inhibition of the binding of radiolabelled LPS indicated that active LPSs (from R. species Sin-1 and R. galegae) and inactive LPSs (from S. enterica and B. pertussis) bind to the same site of the constitutive LPS receptor of C3H/HeJ cells. Furthermore, binding of R. species Sin-1 LPS, and signalling induced by this LPS, were both inhibited by pre-exposure of C3H/HeJ cells to B. pertussis lipid A. This correlation between binding and signalling suggests that in C3H/HeJ cells, the constitutive receptor, which recognizes a large panel of LPSs from different origins, appears selectively unable to be activated by some particular LPSs, such as those of Enterobacteria and Bordetella.     

Evidence for B-lymphocyte mitogen activity in Borrelia burgdorferi-infected mice.

Borrelia burgdorferi produces a mitogen for murine B lymphocytes which can be measured in vitro by polyclonal stimulation of proliferation and immunoglobulin production (R. Schoenfeld, B. Araneo, Y. Ma, L. Yang, and J. J. Weis, Infect. Immun. 60:455-464, 1992). Sonicated B. burgdorferi cells also stimulated IL-6 production by splenocyte cultures. We have used the murine model for Lyme disease described by Barthold et al. (S. W. Barthold, D. S. Beck, G. M. Hansen, G. A. Terwilliger, and K. D. Moody, J. Infect. Dis. 162:133-138, 1990) to determine whether the B. burgdorferi B-cell mitogen is expressed during active infection. To correlate arthritic changes with immune events, we have studied two strains of mice injected with B. burgdorferi; one of them, C3H/HeJ, developed severe disease, and the other, BALB/c, developed only mild disease. C3H/HeJ mice displayed a persistent 10-fold increase in circulating immunoglobulin G (IgG) levels, a 2-fold increase in IgM levels, and a 15-fold increase in peripheral lymph node B-cell numbers, providing evidence of mitogenic activity. Infected BALB/c mice also had evidence for mitogen activity, since the IgG level in serum increased three- to fourfold. The bulk of the increase in circulating IgG levels was not directed against B. burgdorferi antigens, supporting the occurrence of polyclonal B-cell activation. Analysis of IgG isotypes pointed out a contrast between C3H/HeJ and BALB/c mice in that levels of all isotypes were elevated somewhat in both strains of infected mice but IgG2a levels were much more dramatically increased in the C3H/HeJ mice (28-fold) than in the BALB/c mice (4-fold). In this study, interleukin-6 levels were found to be persistently elevated in the serum of infected C3H/HeJ mice. Interestingly, interleukin-6 levels in serum were much lower in the infected BALB/c mice. These findings indicate that the B. burgdorferi mitogen is active in infected animals and may contribute to the inflammatory and immune response to infection.     

Immunomodulatory activity of monophosphoryl lipid A in C3H/HeJ and C3H/HeSnJ mice.

Treatment with nontoxic monophosphoryl lipid A (MPL) derived from a polysaccharide-deficient, heptoseless Re mutant of either Salmonella typhimurium or Salmonella minnesota R595 enhanced the immunoglobulin M (IgM) anti-type III pneumococcal polysaccharide (SSS-III) antibody response of C3H/HeSnJ mice. Such an adjuvant effect was not observed in lipopolysaccharide-nonresponder C3H/HeJ mice. Nevertheless, C3H/HeJ spleen cells produced a weak mitogenic response to both preparations of MPL in vitro, and C3H/HeJ mice showed a significant increase in serum IgM levels without an increase in numbers of splenic IgM-secreting plaque-forming cells after in vivo treatment with MPL. A significant increase in serum IgG3 levels was accompanied by a transient decrease in serum IgG1 levels in C3H/HeSnJ mice given MPL; such non-antigen-specific polyclonal effects were not observed in C3H/HeJ or in athymic nu/nu mice. Since the enhanced antibody response to SSS-III has been attributed to the inactivation of suppressor T cells by MPL and since suppressor-T-cell activity is demonstrable in both C3H/HeSnJ and C3H/HeJ mice, these findings imply that (i) the suppressor T cells of C3H/HeJ mice are refractory to inactivation by MPL and (ii) some of the polyclonal and mitogenic effects produced in C3H/HeJ mice are due to the direct action of MPL on B lymphocytes.     

Biological activities of Brucella abortus lipopolysaccharides.

Purified lipopolysaccharide (LPS) from smooth (s) and rough (R) strains of Brucella abortus and lipid A isolated from S-LPS by mild acid hydrolysis were examined in several assays of biological activity. Brucella S- and R-LPSs and Brucella lipid A activated the complement cascade. Previously reported mitogenic activation by Brucella LPSs of spleen cells from endotoxin-resistant C3H/HeJ mice was confirmed and also produced by isolated Brucella lipid A. Mitogenicity was not inhibited by polymyxin B, and amino acid analysis showed no binding of polymyxin B to Brucella LPS under conditions in which mitogenicity of phenol-water-extracted Escherichia coli LPS was inhibited. S and R Brucella LPSs and lipid A all produced equivalent polyclonal stimulation of C3H/HeJ and C3H/HeAU spleen cells. Crude and purified LPS from S but not from R B. abortus was toxic for outbred mice, with 50% lethal doses approximately six times greater than that for E. coli LPS. S- and R-LPSs were abortifacient in pregnant outbred mice. S Brucella LPS was lethal for carrageenen-pretreated C3H/HeJ and C3H/HeAU mice, whereas only C3H/HeAU mice were killed by E. coli LPS. The data are consistent with the hypothesis that the unique fatty acid composition of Brucella lipid A is responsible for its biological activity in endotoxin-resistant C3H/HeJ mice. The participation of the protein strongly bound to the lipid A cannot be excluded, but its mode of action, if any, is different from that of the lipid A-associated protein of enterobacterial LPS.     

B cell activation and adjuvant activities of chemically synthesized analogues of the nonreducing sugar moiety of lipid A

Immunological activities of synthetic lipid A analogues (derivatives of 3-O-tetradecanoyl D-glucosamine) were investigated. Compounds possessing a N-tetradecanoyloxytetradecanoyl group with or without a 4-O-phosphoryl group exhibited both mitogenic and polyclonal B cell activating activities. Among these, the phosphorylated compound exhibited strong activity. 4-O-phosphorylated analogues with a N-tetradecanoyl group or 6-O-tetradecanoyl group in addition to N-3-hydroxyacyl groups showed no activity. All the analogues tested showed adjuvant activity, which was determined by augmentation of IgM antibody response against sheep erythrocytes. Relationship between chemical structure and biological activities is also discussed.