Previous studies showed that local macrophages phagocytose nonantigenic chitin particles (1–10 μm polymers of N-acetyl-d-glucosamine) through mannose receptors and produce IL-12, IL-18, and TNF-α. These cytokines lead to the production of IFN-γ by NK cells. To determine whether chitin could down-regulate Th2 responses, chitin was given orally (8 mg/day for 3 days before and 13 days during ragweed allergen immunization) in BALB/c and C57BL/6 mice. These ragweed-immunized mice were given ragweed intratracheally on day 11. Three days after the challenge, the immunized mice with saline (controls) showed increases in serum IgE levels and lung eosinophil numbers. The chitin treatment resulted in decreases of these events in both strains. To dissect the inhibitory mechanisms of Th2 responses, spleen cells (4 × 106 cells/ml) isolated from the ragweed-immunized mice (controls) were cultured in the presence of ragweed and/or chitin for 3 days (recall responses). Ragweed alone stimulated the production of IL-4 (0.6 ng/ml), IL-5 (20 U/ml), and IL-10 (3.2 ng/ml), but not IFN-γ. Ragweed/chitin stimulation resulted in significant decreases of IL-4, IL-5, and IL-10 levels and the production of IFN-γ (48 U/ml). Moreover, spleen cells isolated from the chitin-treated mice showed ragweed-stimulated IFN-γ production (15 U/ml) and significantly lower levels of the Th2 cytokines, suggesting that the immune responses were redirected toward a Th1 response. Collectively, these results indicate that chitin-induced innate immune responses down-regulate Th2-facilitated IgE production and lung eosinophilia in the allergic mouse.

The siderophores produced by iron-starved Bordetella pertussis and B. bronchiseptica were purified and were found to be identical. Using mass spectrometry and proton nuclear magnetic resonance, we determined that the siderophore produced by these organisms was identical to alcaligin, a siderophore produced by Alcaligenes denitrificans.Organisms of the genus Bordetella are obligate pathogens of the upper respiratory tract of a variety of animal hosts. Bordetella pertussis and Bordetella parapertussis are obligate human pathogens (15). Although occasionally seen in infected humans (15), Bordetella bronchiseptica is a common pathogen of several other mammalian hosts (7). Bordetella avium infects the upper respiratory tract of domestic fowl (10). These organisms cause similar syndromes characterized by infection of the respiratory mucosal surface, without subsequent bloodstream dissemination (15). Our studies (1, 6) and others (2, 8) suggested that B. pertussis and B. bronchiseptica removed Fe from lactoferrin (LF) and transferrin by producing a hydroxamate siderophore. We have now purified the siderophores produced by these organisms and found that these siderophores were identical. Although we previously suggested the term ''bordetellin'' to denote the Bordetella siderophore (1), the siderophores produced by B. pertussis and B. bronchiseptica were identical to alcaligin, the Fe chelator produced by Alcaligenes denitrificans (13).B. pertussis DBP2 (a streptomycin-resistant derivative of strain Tohama I) and B. bronchiseptica MBORD846 were used in these studies; growth conditions have been described previously (1, 6). To prepare desferri-siderophore, 2 g of lyophilized culture supernatant from a logarithmic-phase iron-starved culture was dissolved in 10 ml of water and passed through two C 18 Sep-Pak cartridges (Waters Associates), connected in series. The cartridges were washed with 20 ml of water, and the siderophore was eluted with 50% methanol. The methanol was removed by rotary evaporation, and the material was dissolved in 0.1% trifluoroacetic acid (TFA) and applied to a Vydac C 18 high-pressure liquid chromatography (HPLC) column (4.6 mm by 25 cm). The column was washed with 0.1% TFA (solvent A) for 10 min, and then a linear gradient of 0.08% TFA in 70% acetonitrile (solvent B) was applied at 1% B per min (flow rate, 1 ml/min). Purification of the ferri-siderophore complex was similar, except that this material required 80% methanol-1% acetic acid for elution from the C 18 Sep-Pak cartridges. Siderophores were purified to homogeneity by this protocol (Fig. 1). B. pertussis and B. bronchiseptica culture supernatants yielded desferri-and ferri-siderophores that behaved identically during purification, suggesting that these were the same iron chelator (data not shown).We previously showed that a Tn5lac insertion mutation in B. bronchiseptica DBB22 blocked siderophore production, while other Fe transport components were intact (6). Strain DBB22 was also unable to use LF as an iron source for growth (6). We were ...

Intravenous (i.v.) administration of phagocytosable chitin particles (1 to 10 m) in C57BL/6 mice and SCID mice primed alveolar macrophages (M) within 3 days to yield up to a 50-fold increase in their oxidative burst when elicited in vitro with phorbol myristate acetate (PMA). C57BL/6 mice pretreated with monoclonal antibodies (MAbs) against mouse gamma interferon (IFN-␥) or NK1.1 showed a markedly decreased level of alveolar M priming following injection of chitin particles. To confirm IFN-␥ production in vitro, spleen cells isolated from normal C57BL/6 mice and SCID mice were cultured with chitin particles. Significant IFN-␥ production was observed following stimulation with chitin but not with chitosan or latex beads. When spleen cells were treated with anti-NK1.1 MAb, IFN-␥ production was significantly inhibited. Another set of experiments showed that when C57BL/6 mice were pretreated i.v. with a small dose IFN-␥, a higher level of priming was induced with not only phagocytosable chitin particles but also phagocytosable chitosan and even latex beads. Likewise, the spleen cell cultures preconditioned with IFN-␥ provided an up-regulation of IFN-␥ production by these phagocytosable particles. Taken together, the in vivo and in vitro results suggest that (i) the alveolar M priming mechanism is due, at least in part, to direct activation of M by IFN-␥, which is produced by NK1.1 ؉ CD4 ؊ cells; (ii) IFN-␥ would have an autocrine-like effect on M and make them more responsive to particle priming; and (iii) phagocytosis of particulates, probably by a postmembrane event such as interiorization, appears to be important for the up-regulation of alveolar M priming and IFN-␥ production.

No abstract

Recent studies have shown that Bordetella bronchiseptica utilizes a siderophore-mediated transport system for acquisition of iron from the host iron-binding proteins lactoferrin and transferrin. We recently identified the B. bronchiseptica siderophore as alcaligin, which is also produced by B. pertussis. Alcaligin production by B. bronchiseptica is repressed by exogenous iron, a phenotype of other microbes that produce siderophores. In this study, we report that alcaligin production by B. bronchiseptica RB50 and GP1SN was repressed by the Bordetella global virulence regulator, bvg, in addition to being Fe repressed. Modulation of bvg locus expression with 50 mM MgSO 4 or inactivation of bvg by deletion allowed strain RB50 to produce alcaligin. In modulated organisms, siderophore production remained Fe repressed. These observations contrasted with our previous data indicating that alcaligin production by B. bronchiseptica MBORD846 and B. pertussis was repressed by Fe but bvg independent. Despite bvg repression of alcaligin production, strain RB50 was still able to acquire Fe from purified alcaligin, suggesting that expression of the bacterial alcaligin receptor was not repressed by bvg. We tested 114 B. bronchiseptica strains and found that bvg repression of alcaligin production was strongly associated with Bordetella phylogenetic lineage and with host species from which the organisms were isolated.