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Members of the genus Brucella are gram-negative bacteria that produce chronic infections in a large number of mammals, including humans (10). Brucella species are facultative intracellular pathogens which survive within a variety of cells, including macrophages, and the virulence of these species and the establishment of chronic infections by them are thought to be essentially due to their ability to avoid the killing mechanisms within macrophages (3, 36). The molecular mechanisms accounting for these properties are incompletely understood, and only some aspects of the processes involved have been identified as yet. Brucella does not evade phagocytosis by macrophages or neutrophils (6) but inhibits the degranulation of both primary and secondary neutrophil granules (30, 33, 34) and the myeloperoxidase-hydrogen peroxide-halide system (5, 7). The virulence of Brucella abortus, Brucella melitensis, and Brucella suis is associated with the smooth colony morphotype which contains the full lipopolysaccharide (LPS) (36, 38). The low biological activity induced by Brucella smooth LPS (S-LPS) compared with that produced by enterobacterial endotoxin might be one of the factors contributing to the survival of these pathogens in phagocytic cells (32). Further, a major proportion of the protective antibody response is directed against the O-chain component of the S-LPS (11) and Brucella LPS itself is a virulent factor because it is the main cause of the resistance of Brucella to lysosomal cationic proteins (15).

Nitric oxide (NO) has been shown to play an important role in diverse functions, including vasoregulation, neurotransmission, immune response regulation (23, 24, 37), and macrophage-mediated cytotoxic activity against tumor cells and a variety of pathogens, including bacteria, fungi, viruses, helminths, and protozoa (22). NO has been implicated in host defenses against intracellular pathogens and might play a role in persistent or latent infections (14). NO is derived from L-arginine in a reaction catalyzed by the enzyme NO synthase (NOS), of which three different isoforms have been identified (29). The inducible isoform of NOS (iNOS) is responsible for the high-output path of NO production involved in antimicrobial activity (28). iNOS expression is induced by proinflammatory cytokines such as gamma interferon (IFN-), tumor necrosis factor alpha, and interleukin 1 (IL-1), as well as by microbial products such as LPS and lipoteichoic acid (14). The purpose of this study was to determine whether Brucella S-LPS induces NO production in rat peritoneal macrophages and compare the effects of S-LPS from various Brucella species with that of Escherichia coli LPS. Since most biological effects of LPS have been associated with the lipid A moiety (25), we also investigated NO induction by Brucella lipid A. We report that B. abortus and B. melitensis S-LPS and lipid A induce NO production in rat peritoneal macrophages by a mechanism involving transcriptional up-regulation of the iNOS gene.

Pathogen-free Wistar rats (200 to 300 g) were used for all studies. B. melitensis 16M (biotype 1) and B. abortus 544 (biotype 1) smooth virulent strains were grown on Brucella broth (Difco Laboratories, Detroit, Mich.). Phenol-inactivated bacteria were harvested by centrifugation and washed twice with saline. S-LPS was extracted by the phenol-water method modified for Brucella organisms as described by Leong et al. (20), which allows the separation of Brucella S-LPS in the phenolic phase. Purification of crude S-LPS isolated in the phenolic phase was performed according to the procedure of Moreno et al. (26). The thiobarbituric method was used to measure 2-keto-3-deoxyoctonate (KDO) (2). Under these experimental conditions B. melitensis 16M purified S-LPS contained 0.79% KDO, B. abortus 544 purified S-LPS contained 0.70% KDO, and E. coli O:26,B:6 LPS (Sigma Chemical, St. Louis, Mo.) contained 1.6% KDO. Lipid A from E. coli, B. abortus, and B. melitensis was obtained by hydrolysis of purified S-LPS with 2% acetic acid at 100°C for 1 h (E. coli) or 5 h (Brucella) (25). Resident peritoneal cells were collected from the peritoneal cavity with cold phosphate-buffered saline as reported previously (4). Adherent macrophage monolayers were obtained by plating the cells in 24-well tissue culture plates at 1.5 × 10⁶ cells/well for 2 h at 37°C. The production of NO was determined by the accumulation of nitrite measured by the Griess reaction (16). Statistical analysis was carried out using the unpaired Student t test for comparison of two means and one-way analysis of variance with the Bonferroni test for multiple comparisons of means. Statistical significance was set at a P value of <0.05.

Production of NO by rat peritoneal cells stimulated Brucella S-LPS and lipid A. Peritoneal cells stimulated with E. coli, B. abortus, and B. melitensis LPS generated nitrite in a dose-dependent manner (Fig. 1A). Since E. coli LPS is a microbial product inducing NO production by murine macrophages (39), it was included as a control of our cellular system and for comparison with Brucella S-LPS induction. E. coli LPS induced a significant level of production of NO from a dose of 0.01 µg/ml, plateau values being reached for LPS doses of 1 µg/ml. B. abortus S-LPS showed significant levels of NO production at doses of >0.1 µg/ml and produced maximal levels at a dose of 10 µg/ml. B. melitensis S-LPS induced NO production at concentrations higher than 0.01 µg/ml, with maximal induction at doses of 1 µg/ml. These data indicate that E. coli LPS is the most potent inductor of NO, whereas there was no significant difference in the NO production levels induced by B. abortus and B. melitensis S-LPS. The generation of nitrite by E. coli, B. abortus, and B. melitensis LPS was time dependent (Fig. 1B), first showing a lag of 8 h before significant production could be assayed and then increasing up to 24 h and showing plateau levels at 48 and 72 h (data not shown). Because most of the biological effects of the LPS have been associated with the lipid A moiety, we studied the ability of these lipids to induce NO production. Lipid A from both Brucella stimulated the production of NO in a dose-dependent manner (Fig. 1C). E. coli lipid A was again a potent inductor, significant production of NO being detected with concentrations of lipid A as low as 0.001 µg/ml, whereas maximal production was observed at a concentration of 0.1 µg/ml. B. abortus and B. melitensis lipid A showed similar potencies to elicit NO induction, i.e., significant amounts of nitrite were obtained in the presence of a concentration of 0.1 µg/ml and maximal production was obtained with concentrations of 1 µg/ml. Evidence of the involvement of the L-arginine pathway was obtained with the NOS inhibitor N^G-methyl-L-arginine (L-NMA), which suppressed nitrite production induced by each LPS and lipid A (Fig. 2).

View larger version (18K): [in this window] [in a new window]   FIG. 1.   (A) Production of nitrite by rat peritoneal adherent cells stimulated with different concentrations of E. coli LPS (), B. abortus S-LPS (), and B. melitensis S-LPS (). Adherent rat peritoneal cells were stimulated with LPS at the concentrations indicated. NO2 production was assayed 24 h after stimulation. Data are the means ± the standard errors of the means of results of four experiments with duplicate samples. (B) Kinetics of nitrite synthesis by adherent peritoneal cells stimulated with 1 µg of E. coli LPS (), B. abortus S-LPS (), or B. melitensis S-LPS () per ml. Cells were allowed to adhere to plastic dishes and then were stimulated as indicated. Open circles indicate cells without any stimulus. Data are the means ± the standard errors of the means of results of three experiments performed in duplicate. (C) Production of nitrite by rat peritoneal adherent cells stimulated with different concentrations of E. coli (), B. abortus (), or B. melitensis () lipid A. Nitrite production was assayed after 24 h of incubation. Data are the means ± the standard errors of the means of results of four experiments with duplicate samples. The nitrite levels induced by Brucella S-LPS and lipid A were significantly different from those of E. coli. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

View larger version (33K): [in this window] [in a new window]   FIG. 2.   Effect of L-NMA on nitrite production induced by E. coli, B. abortus, and B. melitensis LPS and lipid A. Adherent cells were incubated with 1 µg of the indicated LPS (A) or lipid A (B) per ml, in the presence (solid bars) or absence (hatched bars) of 0.5 mM L-NMA. Nitrite production was assayed after 24 h. Open bars show the production by control cells. Data are the means ± the standard errors of the means of results of three experiments performed in duplicate. The nitrite levels induced in the presence of L-NMA were significantly different from those induced with only the LPS or lipid A. *, P < 0.001.

Induction of iNOS mRNA expression in adherent peritoneal cells stimulated with LPS and lipid A. To address the involvement of iNOS in response to Brucella S-LPS the expression of iNOS mRNA was studied by Northern blot analysis. For this purpose, total cellular RNA was prepared from cell cultures according to the guanidium isothiocyanate method (8). Aliquots of total RNA (10 µg) were denatured and then separated by electrophoresis (35). After capillary transference of the RNA to nylon membranes, the membranes were hybridized overnight (9) with a radiolabeled DNA probe specific for mouse macrophage iNOS. The membranes were rehybridized, with ³²P-labeled -actin probe used as an internal control to demonstrate the integrity of the RNA, the equivalence of loading, and the specificity of mRNA induction. As shown in Fig. 3, cells incubated with E. coli, B. abortus, and B. melitensis LPS showed enhanced expression of iNOS mRNA compared to cells incubated with vehicle. E. coli LPS induced an increased expression of iNOS mRNA that was already detected at 2 h and that showed a peak at 8 h and an elevation above resting levels at 24 h (Fig. 4A and B). This time course of iNOS mRNA induction closely parallels nitrite production. B. abortus S-LPS also showed a time-dependent induction of iNOS mRNA (Fig. 4C and D), with increased levels after 2 h of stimulation and a maximal expression at 4 h. Incubation with B. melitensis S-LPS also increased iNOS mRNA in a time-dependent manner (Fig. 4E and F). Nitrite production induced by lipid A also correlated with iNOS mRNA expression. As shown in Fig. 3, the addition of E. coli, B. abortus, and B. melitensis lipid A enhanced iNOS mRNA expression, thus confirming the involvement of the lipid A moiety in this biological effect. Induction of iNOS mRNA was accompanied by the expression of iNOS protein, as judged from Western blot analysis. For this purpose, cell lysates were subjected to electrophoresis on a sodium dodecyl sulfate-8% polyacrylamide gel, transferred to a nitrocellulose membrane, and then incubated with rabbit anti-mouse iNOS antibody (Calbiochem). The detection was performed using the enhanced chemiluminescence system (Amersham). Figure 5 shows the expression of iNOS protein in adherent peritoneal cells incubated with 10 µg of LPS from either class per ml.

View larger version (37K): [in this window] [in a new window]   FIG. 3.   Effect of E. coli, B. abortus, and B. melitensis LPS and lipid A on iNOS mRNA. Adherent cells were treated with 1 µg of LPS or lipid A per ml. (A) Total RNA was isolated after 24 h of stimulation and evaluated by Northern blot analysis with 32P-labeled iNOS and -actin DNA probes. The relative level of NOS mRNA expression was determined for each lane after normalization to the respective -actin signal. The iNOS signal obtained in the absence of LPS was assigned a value of 1 to allow calculation of a relative level of mRNA expression for all other treatments. (B) The histogram shows the densitometric analysis of the autoradiograph shown in panel A. This is a representative experiment of four similar ones.

View larger version (69K): [in this window] [in a new window]   FIG. 4.   Time course of iNOS mRNA induction by E. coli LPS (A and B), B. abortus S-LPS (C and D), and B. melitensis S-LPS (E and F). Total RNA was isolated at the indicated times and used for Northern blot analysis with 32P-labeled iNOS and -actin DNA probes. The relative level of iNOS mRNA expression was determined for each lane after normalization to the respective -actin signal. The iNOS signal at 0 h was assigned a value of 1 to allow calculation of a relative level of mRNA expression for all other treatments. Results of scanning densitometric analysis of the autoradiographs in panels A, C, and E are presented in the histograms of panels B, D, and E (hatched bars indicate cells incubated for 24 h in the absence of LPS). These results are representative of those from three similar experiments.

View larger version (37K): [in this window] [in a new window]   FIG. 5.   Analysis of iNOS protein in peritoneal macrophages treated with E. coli, B. abortus, and B. melitensis LPS. Adherent cells were incubated with 10 µg of LPS per ml for 24 h. Cell lysates containing 40 µg of protein were analyzed by Western blotting with a rabbit anti-mouse iNOS antibody and peroxidase-conjugated anti-rabbit immunoglobulin G. These results are representative of three experiments.