Enzyme-linked immunosorbent assay for bovine immunoglobulin subclass-specific response to Brucella abortus lipopolysaccharides.

An enzyme-linked immunosorbent assay was developed to follow the bovine response, by immunoglobulin class and subclass, to defined smooth and rough lipopolysaccharides (LPS) of Brucella abortus. Binding to smooth LPS of immunoglobulin G1 (IgG1) and IgG2 in sera from Brucella-infected animals was significantly greater than binding in sera from normal uninfected animals. Competition or steric blocking among IgM, IgG1, and IgG2 for binding sites on smooth LPS was shown to occur. Binding of IgM to Brucella smooth LPS with sera from uninfected animals was elevated above the assay control levels, and attempts to eliminate this nonspecific IgM binding were not successful. The same levels of nonspecific IgM binding were also seen with Brucella rough LPS, Escherichia coli LPS, and Pseudomonas solanacearum LPS. Sera from some, but not all, Brucella-infected animals showed elevated binding of IgG1 and IgM to both E. coli LPS and Brucella rough LPS as well as to Brucella smooth LPS. This was interpreted as specific antibody. Cross-reactions between B. abortus smooth or rough LPS and E. coli LPS could not be shown by immunodiffusion.     

Brucella abortus and its closest phylogenetic relative, Ochrobactrum spp., differ in outer membrane permeability and cationic peptide resistance

The outer membrane (OM) of the intracellular parasite Brucella abortus is permeable to hydrophobic probes and resistant to destabilization by polycationic peptides and EDTA. The significance of these unusual properties was investigated in a comparative study with the opportunistic pathogens of the genus Ochrobactrum, the closest known Brucella relative. Ochrobactrum spp. OMs were impermeable to hydrophobic probes and sensitive to polymyxin B but resistant to EDTA. These properties were traced to lipopolysaccharide (LPS) because (i) insertion of B. abortus LPS, but not of Escherichia coli LPS, into Ochrobactrum OM increased its permeability; (ii) permeability and polymyxin B binding measured with LPS aggregates paralleled the results with live bacteria; and (iii) the predicted intermediate results were obtained with B. abortus-Ochrobactrum anthropi and E. coli-O. anthropi LPS hybrid aggregates. Although Ochrobactrum was sensitive to polymyxin, self-promoted uptake and bacterial lysis occurred without OM morphological changes, suggesting an unusual OM structural rigidity. Ochrobactrum and B. abortus LPSs showed no differences in phosphate, qualitative fatty acid composition, or acyl chain fluidity. However, Ochrobactrum LPS, but not B. abortus LPS, contained galacturonic acid. B. abortus and Ochrobactrum smooth LPS aggregates had similar size and zeta potential (-12 to -15 mV). Upon saturation with polymyxin, zeta potential became positive (1 mV) for Ochrobactrum smooth LPS while remaining negative (-5 mV) for B. abortus smooth LPS, suggesting hindered access to inner targets. These results show that although Ochrobactrum and Brucella share a basic OM pattern, subtle modifications in LPS core cause markedly different OM properties, possibly reflecting the adaptive evolution of B. abortus to pathogenicity.     

Immunochemical identification of Brucella abortus lipopolysaccharide epitopes.

Sera from Brucella abortus-infected and -vaccinated bovines recognized four lipopolysaccharide (LPS) determinants: two in the O-polysaccharide (A and C), one in the core oligosaccharide from rough Brucella LPS (R), and one in lipid A (LA). From 46 different hybridomas secreting monoclonal antibodies (MAbs) against various LPS moieties, 9 different specificities were identified. Two epitopes, A and C/Y, were present in the O-polysaccharide. Two epitopes were found in the core oligosaccharide (R1 and R2) of rough Brucella LPS. MAbs against R1 and R2 epitopes reacted against LPS from different rough Brucella species; however, MAbs directed to the R2 epitope also reacted against enterobacterial LPS from deep rough mutants. Three epitopes (LA1, LA2, and LA3) were located in the lipid A backbone. Different sets of MAbs recognized two epitopes in the lipid A-associated outer membrane protein (LAOmp3-1 and LAOmp3-2). LPS preparations from smooth brucellae had small amounts of rough-type LPS. Although LPS from rough brucellae did not show smooth-type LPS in western blots (immunoblots), two hybridomas generated from mice immunized with rough B. abortus produced antibodies against smooth B. abortus LPS. Results are discussed in relation to the structure and function of B. abortus LPS and to previous findings on the epitopic density of the molecule.     

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.     

Role of Toll-like receptor 4 in induction of cell-mediated immunity and resistance to Brucella abortus infection in mice

Initial host defense to bacterial infection is executed by innate immunity, and therefore the main goal of this study was to examine the contribution of Toll-like receptors (TLRs) during Brucella abortus infection. CHO reporter cell lines transfected with CD14 and TLRs showed that B. abortus triggers both TLR2 and TLR4. In contrast, lipopolysaccharide (LPS) and lipid A derived from Brucella rough (R) and smooth (S) strains activate CHO cells only through TLR4. Consistently, macrophages from C3H/HePas mice exposed to R and S strains and their LPS produced higher levels of tumor necrosis factor alpha (TNF-alpha) and interleukin-12 compared to C3H/HeJ, a TLR4 mutant mouse. The essential role of TLR4 for induction of proinflammatory cytokines was confirmed with diphosphoryl lipid A from Rhodobacter sphaeroides. Furthermore, to determine the contribution of TLR2 and TLR4 in bacterial clearance, numbers of Brucella were monitored in the spleen of C3H/HeJ, C3H/HePas, TLR2 knockout, and wild-type mice at 1, 3, and 6 weeks following B. abortus infection. Interestingly, murine brucellosis was markedly exacerbated at weeks 3 and 6 after infection in animals that lacked functional TLR4 (C3H/HeJ) compared to C3H/HePas that paralleled the reduced gamma interferon production by this mouse strain. Finally, by mass spectrometry analysis we found dramatic differences on the lipid A profiles of R and S strains. In fact, S lipid A was shown to be more active to trigger TLR4 than R lipid A in CHO cells and more effective in inducing dendritic cell maturation. In conclusion, these results indicate that TLR4 plays a role in resistance to B. abortus infection and that S lipid A has potent adjuvant activity.     

Immunochemical characterization of rough Brucella lipopolysaccharides

Lipopolysaccharides (LPS) were extracted from rough strains of Brucella abortus and Brucella melitensis and from strains of the naturally occurring rough species Brucella ovis and Brucella canis. Brucella rough lipopolysaccharides (R-LPS) were readily distinguished from Brucella smooth lipopolysaccharides (S-LPS) and enterobacterial R-LPS, by their chemical, physical, and serological characteristics. B. ovis R-LPS was differentiated from B. abortus, B. melitensis, and B. canis R-LPS by its reaction of partial identity in immunodiffusion. Monospecific mouse sera against B. ovis R-LPS agglutinated only the homologous bacteria but not R cells of other species of Brucella. B. ovis R-LPS contained more 2-keto, 3-deoxyoctonate, and glucosamine as a percentage of dry weight than any other R-LPS tested. B. abortus R-LPS was identified by the absence of an unidentified sugar present in the other R-LPS molecules, and B. melitensis R-LPS could be differentiated from B. canis R-LPS by its higher content of fatty acids. In contrast to S-LPS, all of the R-LPS studied lacked quinovosamine. In electron micrographs, Brucella R-LPS had a granular appearance, in contrast to typical lamellar structures formed by Brucella S-LPS and Escherichia coli R-LPS.     

Lipopolysaccharide (LPS) from Brucella abortus is less toxic than that from Escherichia coli, suggesting the possible use of B. abortus or LPS from B. abortus as a carrier in vaccines.

Brucella abortus may be useful as a component of vaccines. This is because it possesses several unique properties as a carrier that enable it to stimulate human B cells even in the relative absence of T cells. Human immunodeficiency virus type 1 proteins conjugated to B. abortus could induce neutralizing antibodies against human immunodeficiency virus type 1. Recently we showed that the characteristics of lipopolysaccharide (LPS) derived from B. abortus are similar to those of the whole bacterium in that the LPS acts as a T-independent type 1 carrier in mice. In this study we wanted to determine whether LPS derived from B. abortus is associated with the adverse effects seen with other bacterial endotoxins. LPS purified from B. abortus by butanol extraction was shown to have less than 2% (wt/wt) contamination by protein and less than 1% (wt/wt) contamination by nucleic acids and to contain 1% (wt/wt) ketodeoxyoctanic acid. Compared with LPS derived from Escherichia coli, B. abortus LPS was 10,000-fold less potent in eliciting fever in rabbits, 268-fold less potent in killing D-galactosamine-sensitized mice, and 1,400-fold and 400-fold less potent in inducing interleukin-1 beta and tumor necrosis factor alpha production, respectively. These results suggest that B. abortus LPS is much less likely than the LPS from E. coli to evoke endotoxic shock; therefore, it may be feasible to incorporate B. abortus as a component of vaccines.     

Effect of Brucella abortus lipopolysaccharide on oxidative metabolism and lysozyme release by human neutrophils.

Both Brucella abortus lipopolysaccharide (LPS) and lipid A were low activators of nitroblue tetrazolium reduction and lysozyme release in human neutrophils. The stimulation was dose dependent and was higher in the presence of autologous plasma than in its absence. The comparison between Brucella LPS and lipid A versus Salmonella LPS revealed that at least 100 times more LPS and 1,000 times more lipid A of the former genus were required to induce significant nitroblue tetrazolium reduction and a corresponding lysozyme release in neutrophils. Low Brucella LPS-mediated superoxide and lysozyme production might contribute to the survival of these facultative intracellular bacteria in phagocytic cells.     

Physical and morphological characteristics of eucaryotic ribosomes and lipopolysaccharide complexes.

Lipopolysaccharides (LPS) from Pasteurella multocida or Brucella abortus were complexed with Aspergillus fumigatus ribosomes by mixing and fixation for 3 days in 3.8% formaldehyde. To investigate the nature of their physical association, ribosomes, LPS, and ribosome-LPS complexes were (i) centrifuged in CsCl gradients to determine buoyant densities, (ii) examined by electron microscopy, and (iii) monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ribosomes were found to bind to LPS from either P. multocida or B. abortus, producing complexes with densities of 1.45 to 1.50 g/ml. The buoyant density of the fixed ribosomes was 1.54 g/ml, and the buoyant densities of the fixed P. multocida and B. abortus LPS were 1.41 and 1.35 g/ml, respectively. Electron microscopy showed that formaldehyde-fixed ribosomes were attached to the LPS. Complexing of ribosomes to LPS may be of importance as a potentiator or carrier for experimental subunit vaccines.     

Interaction of Brucella abortus lipopolysaccharide with major histocompatibility complex class II molecules in B lymphocytes.

Lipopolysaccharide (LPS), a major amphiphilic molecule located at the outer membrane of gram-negative bacteria, is a potent antigen known to induce specific humoral immune responses in infected mammals. LPS has been described as a polyclonal activator of B lymphocytes, triggering the secretion of antibodies directed against distinct sugar epitopes of the LPS chain. But, how LPS is handled by B cells remains to be fully understood. This task appears to be essential for a better knowledge of the anti-LPS humoral immune response. In this study, we examine the internalization of LPS and its interaction with antigen-presenting major histocompatibility complex (MHC) class II molecules in murine and human B-cell lines. By use of immunofluorescence, we observe that structurally different LPSs from Brucella and Shigella strains accumulate in an intracellular compartment enriched in MHC class II molecules. By use of immunoprecipitation, we illustrate that only Brucella abortus LPS associates with MHC class II molecules in a haplotype-independent manner. Taken together, these results raise the possibility that B. abortus LPS may play a role in T-cell activation.     

Humoral immune response against lipopolysaccharide and cytoplasmic proteins of Brucella abortus in cattle vaccinated with B. abortus S19 or experimentally infected with Yersinia enterocolitica serotype 0:9.

The humoral immune responses against three different antigens of Brucella abortus were monitored by enzyme-linked immunosorbent assay in cattle vaccinated with B. abortus S19 or experimentally infected with Yersinia enterocolitica serotype 0:9. Immunoglobulin G (IgG) and IgM responses against (i) B. abortus lipopolysaccharide (LPS), (ii) total cytoplasmic proteins depleted of LPS (LPS-free CYT), and (iii) B. abortus 18-kDa cytoplasmic protein were measured. Vaccinated animals and Yersinia-infected animals developed high anti-LPS IgM and IgG titers, which overlapped with those obtained with sera from B. abortus 544-infected animals used as positive controls. In contrast, only a slight or negative IgG and IgM response against LPS-free CYT and the 18-kDa protein was detected in vaccinated or Yersinia-infected cattle, although its levels were always significantly lower than those of B. abortus 544-infected animals. These data indicate that cytoplasmic proteins of B. abortus could be useful for the differential diagnosis of bovine brucellosis.     

The outer membrane of Brucella ovis shows increased permeability to hydrophobic probes and is more susceptible to cationic peptides than are the outer membranes of mutant rough Brucella abortus strains.

The permeability of the outer membrane (OM) to hydrophobic probes and its susceptibility to bactericidal cationic peptides were investigated for natural rough Brucella ovis and for mutant rough Brucella abortus strains. The OM of B. ovis displayed an abrupt and faster kinetic profile than rough B. abortus during the uptake of the hydrophobic probe N-phenyl-naphthylamine. B. ovis was more sensitive than rough B. abortus to the action of cationic peptides. Bactenecins 5 and 7 induced morphological alterations on the OMs of both rough Brucella strains. B. ovis lipopolysaccharide (LPS) captured considerably more polymyxin B than LPSs from both rough and smooth B. abortus strains. Polymyxin B, poly-L-lysine, and poly-L-ornithine produced a thick coating on the surfaces of both strains, which was more evident in B. ovis than in rough B. abortus. The distinct functional properties of the OMs of these two rough strains correlate with some structural differences of their OMs and with their different biological behaviors in animals and culture cells.     

Live Brucella spp. fail to induce tumor necrosis factor alpha excretion upon infection of U937-derived phagocytes.

Tumor necrosis factor alpha (TNF-alpha) plays a central role in activation of first-line defenses of a host against foreign organisms. To determine whether Brucella infection modulated TNF-alpha production, we measured the biological activity of this cytokine in supernatants of U937 cell-derived macrophages and of fresh human monocytes infected with Brucella spp. Neither the smooth nor rough Brucella strains used induced any measurable TNF-alpha excretion upon infection. On the contrary, as reported before for other gram-negative bacteria, phagocytosis of nonpathogenic Escherichia coli was followed by a rapid and transient induction of TNF-alpha release, suggesting an involvement of this cytokine in some autocrine process. As expected, the Brucella strains tested survived and/or multiplied within U937-derived macrophages, whereas E. coli was rapidly eliminated after phagocytosis. Immunoglobulin G opsonization of E. coli strains enhanced their intracellular killing and strongly potentiated TNF-alpha secretion. Immunoglobulin G opsonization of Brucella strains, in contrast, did not lead to TNF-alpha production, although their rate of intracellular multiplication was reduced. Killed brucellae, however, promoted a significant excretion of TNF-alpha from U937-derived macrophages into cell culture supernatants. We finally demonstrated that pretreatment of U937-derived macrophages with exogenous TNF-alpha significantly inhibited intracellular multiplication of Brucella spp. These results and experiments performed on fresh human monocytes or with isolated lipopolysaccharide (LPS) showed that (i) differences in TNF-alpha production observed during macrophage infection by Brucella spp. and E. coli were not due to differences in LPS structure but resulted from active inhibition of TNF-alpha production by a specific process linked to Brucella spp. and (ii) the capacity of Brucella spp. to use pathways avoiding TNF-alpha production during infection may be considered a major attribute of virulence.     

High levels of nitric oxide production decrease early but increase late survival of Brucella abortus in macrophages.

Nitric oxide (NO), produced by the iNOS protein, is known as a defense mechanism against various pathogens and an apoptotic inducer of cells. Apoptosis can also be a host protective mechanism against intracellular bacteria. The intracellular survival of Brucella abortus in RAW264.7 macrophages was examined under conditions of the apoptotic inducer, NO. Since B. abortus does not induce high output of NO, Escherichia coli LPS and IFN-gamma, as potential therapeutic modalities, were added to increase the expression of iNOS, and thus NO. Using 10 ng/ml E. coli LPS and 25 U/ml IFN-gamma, nitrite production was as high as 140 microM by 72 h. However, when macrophages were infected with B. abortus, the nitrite concentration was 60 microM after 72 h post infection, greater than a two-fold decrease. The number of surviving bacteria decreased, from 6 to 24 h, in the presence of nitrite accumulation. In the absence of B. abortus there was an increase in apoptotic cells at 72 h with high nitrite accumulation. In contrast, the number of macrophage apoptotic bodies decreased in the presence of B. abortus. The data suggest that: (i) NO accelerates the killing of intracellular B. abortus, but not to completion during the first 24 h of infection; (ii) B. abortus can prevent apoptosis as an advantage for bacterial survival inside macrophages and (iii) surviving intracellular bacteria then replicate steadily after 24 h. B. abortus probably expresses genes that counteract the effect of a high NO environment or activates genes to utilize NO as a nitrogen source, as the Brucella genome codes for nitric and nitrous oxide reductase genes.     

Analysis of Brucella lipopolysaccharide with specific and cross-reacting monoclonal antibodies.

Monoclonal antibodies which bind Brucella A lipopolysaccharide (LPS)-specific, M LPS-specific, or cross-reactive epitopes were used as reagents in quantitative dot blot, Western blot (immunoblot), and immunoprecipitation analysis of Brucella whole cells, whole-cell extracts, and purified LPS preparations. This set of monoclonal antibodies detected four unique epitopes on Brucella LPS. The specificity of monoclonal antibodies reactive with Brucella unique (A and M) and common (C and C/Y) LPS epitopes was demonstrated by blot analysis. The serotype specificity of monoclonal antibodies for A LPS of B. abortus 1119.3 or M LPS of Brucella melitensis 16M was confirmed. Type C monoclonal antibodies recognized epitopes on Brucella A and M LPS and did not cross-react with Yersinia enterocolitica O:9. In Western blots, type C monoclonal antibodies were bound by epitopes on Brucella A and M LPSs ranging in Mrs from 30,000 to 70,000, relative to marker proteins. Type C/Y monoclonal antibodies were cross-reactive with Y. enterocolitica O:9 and recognized Brucella A LPS epitopes with a restricted Mr ranging only from 40,000 to 50,000, relative to marker proteins. Type C/Y monoclonal antibodies also displayed a more restricted pattern of binding to Brucella M LPS. The monoclonal antibodies were able to detect 5 to 50 pg of a purified A LPS preparation in dot blots. The limits of detection by the monoclonal antibodies of a purified M LPS preparation ranged from 0.05 to 50 pg. Monoclonal antibody analysis of whole-cell preparations also demonstrated quantitative differences in the presence of the respective epitopes. The binding profiles of the monoclonal antibodies to Brucella whole cells varied between acetone- and chloroform-killed organisms as well as between species and strains. The lower limit of detection of any whole-cell preparation by the dot blot technique was 10(5) CFU. Binding profiles in Western blots and endotoxin activity of immunoprecipitates obtained with these monoclonal antibodies further defined the Brucella LPS antigens. These monoclonal antibodies and the techniques described may be useful in monitoring the antigenic content of Brucella vaccines and diagnostics.     

Liposome potentiation of humoral immune response to lipopolysaccharide and O-polysaccharide antigens of Brucella abortus.

Liposomes were evaluated for their effectiveness as vaccine carriers in the potentiation of the mouse humoral response to the lipopolysaccharide (LPS) and O-polysaccharide (OPS) antigens of Brucella abortus. LPS and OPS were extracted from a pathogenic strain of B. abortus and were encapsulated within multilamellar vesicles. Groups of mice, immunized with liposome-encapsulated and free LPS or OPS, were bled weekly and the specific IgM and IgG levels in the sera were determined by an indirect fluorogenic enzyme-linked immunosorbent assay. Humoral response to these antigens were found to be dose-dependent. Mice immunized with LPS and OPS encapsulated within liposomes were found to have significantly higher IgG levels than mice immunized with free LPS and OPS. In addition, the antibody levels in mice that were immunized with liposome-encapsulated LPS and OPS were more sustained and remained at elevated levels--even after 5 weeks post immunization. As expected, OPS was found to be less immunogenic than LPS, but multiple injections of OPS encapsulated within liposomes greatly improved the immunogenicity. These results indicate that the humoral response to LPS and OPS of B. abortus can be enhanced when these antigens are encapsulated within liposomes.     

Genetic Characterization of a Tn5-Disrupted Glycosyltransferase Gene Homolog in Brucella abortus and Its Effect on Lipopolysaccharide Composition and Virulence

We constructed a rough mutant of Brucella abortus 2308 by transposon (Tn5) mutagenesis. Neither whole cells nor extracted lipopolysaccharide (LPS) from this mutant, designated RA1, reacted with a Brucella O-side-chain-specific monoclonal antibody (MAb), Bru-38, indicating the absence of O-side-chain synthesis. Compositional analyses of LPS from strain RA1 showed reduced levels of quinovosamine and mannose relative to the levels in the parental, wild-type strain, 2308. We isolated DNA flanking the Tn5 insertion in strain RA1 by cloning a 25-kb XbaI genomic fragment into pGEM-3Z to create plasmid pJM6. Allelic exchange of genomic DNA in B. abortus 2308 mediated by electroporation of pJM6 produced kanamycin-resistant clones that were not reactive with MAb Bru-38. Southern blot analysis of genomic DNA from these rough clones revealed Tn5 in a 25-kb XbaI genomic fragment. A homology search with the deduced amino acid sequence of the open reading frame disrupted by Tn5 revealed limited homology with various glycosyltransferases. This B. abortus gene has been named wboA. Transformation of strain RA1 with a broad-host-range plasmid bearing the wild-type B. abortus wboA gene resulted in the restoration of O-side-chain synthesis and the smooth phenotype. B. abortus RA1 was attenuated for survival in mice. However, strain RA1 persisted in mice spleens for a longer time than the B. abortus vaccine strain RB51, but as expected, neither strain induced antibodies specific for the O side chain.     

Induction of immune and adjuvant immunoglobulin G responses in mice by Brucella lipopolysaccharide

The immunogenic and adjuvant properties of Brucella abortus and Escherichia coli lipopolysaccharides (LPSs) were studied in endotoxin-responsive, athymic, and euthymic BALB/c mice and in responsive C3H/HeAu mice and congenic nonresponsive C3H/HeJ mice. Consistent with previous reports, E. coli LPS did not stimulate significant primary or secondary antibody responses in C3H/HeJ mice and induced the production of immunoglobulin M (IgM) and low levels of IgG in C3H/HeAu mice. In contrast, B. abortus smooth and rough LPS stimulated primary and secondary antibody responses and induced the production of IgM and high levels of IgG in both responsive and nonresponsive strains of C3H/He mice and in nude mice. When used as adjuvant, B. abortus LPS augmented the IgG plaque-forming-cell response of C3H/HeAu and BALB/c euthymic mice to the T-dependent antigen sheep erythrocytes. E. coli LPS augmented only the IgM plaque-forming-cell response in the same mouse strains. Neither B. abortus nor E. coli LPS was adjuvant for C3H/HeJ or nude mice. The dichotomy between the antibody and adjuvant responses of both C3H/HeJ mice and athymic mice to B. abortus LPS may be a function of the true thymus independence and dependence of these responses. In addition, the refractiveness of C3H/HeJ and nude mice to B. abortus LPS as adjuvant, but not as mitogen or polyclonal B cell activator, clearly dissociates these phenomena.     

Induction and regulation of Th1-inducing cytokines by bacterial DNA, lipopolysaccharide, and heat-inactivated bacteria

Th1 immune responses, characterized by production of gamma interferon (IFN-gamma), are associated with protective immunity to viruses and intracellular bacteria. Heat-killed Brucella abortus promotes secretion of Th1-inducing cytokines such as interleukin-12 (IL-12) and IFN-gamma and has been used as a carrier to induce Th1 responses to vaccines. To explore which bacterial constituents could mediate this response and how it is regulated, murine spleen cells were cultured with B. abortus derived DNA, lipopolysaccharide (LPS), or whole killed organisms. Each constituent induced similar, substantial amounts of IL-10. However, only B. abortus and B. abortus DNA induced high levels of IFN-gamma and IL-12. B. abortus and B. abortus DNA-stimulated IL-12 production was maximal by 6 to 18 h, while IL-10 production steadily accumulated over this time period. These kinetics suggested that IL-10 may eventually downmodulate the Th1-like cytokine response to B. abortus and B. abortus DNA, which was confirmed by using neutralizing antibody. In the absence of IL-10, B. abortus LPS induced strong IFN-gamma responses, but IL-12 p70 levels were still undetectable from BALB/c spleen cells. LPS induced IL-12 if the spleen cells were primed with IFN-gamma and IL-10 was neutralized, indicating that LPS can stimulate IL-12 production under the most favorable conditions. Responses to Escherichia coli LPS and DNA mirrored the responses to B. abortus components, suggesting that immune effects observed with these constituents may be generalizable to many microbial species. In vivo experiments demonstrated the same hierarchy of responses for IL-12 production. These findings support the likelihood that microbial components, if used as carriers or adjuvants, can differ substantially in their ability to effect a Th1 response.     

Lipopolysaccharides of Brucella abortus and Brucella melitensis induce nitric oxide synthesis in rat peritoneal macrophages

Smooth lipopolysaccharide (S-LPS) and lipid A of Brucella abortus and Brucella melitensis induced the production of nitric oxide (NO) by rat adherent peritoneal cells, but they induced lower levels of production of NO than Escherichia coli LPS. The participation of the inducible isoform of NO synthase (iNOS) was confirmed by the finding of an increased expression of both iNOS mRNA and iNOS protein. These observations might help to explain (i) the acute outcome of Brucella infection in rodents, (ii) the low frequency of septic shock in human brucellosis, and (iii) the prolonged intracellular survival of Brucella in humans.