Induction and regulation of Th1-inducing cytokines by bacterial DNA, lipopolysaccharide, and heat-inactivated bacteria - PubMed (original) (raw)

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

L Huang et al. Infect Immun. 1999 Dec.

Abstract

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.

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Figures

FIG. 1

FIG. 1

IFN-γ induction by B. abortus, bDNA, and LPS. BALB/c spleen cells were cultured with the indicated stimuli; supernatants were harvested at 24, 48, and 72 h, and supernatants were assayed for IFN-γ by ELISA. LPS was derived from either E. coli (LPS-EC) or B. abortus (LPS-BA). Controls include cells stimulated with eukaryotic herring testis DNA (DNA-HT) and untreated cells.

FIG. 2

FIG. 2

IL-12 is induced by B. abortus and bDNA but not LPS. BALB/c spleen cells were cultured with LPS and bDNA (from_B. abortus_ and E. coli) and with B. abortus. Supernatants were removed at the indicated times and assayed for IL-12 p70 levels by ELISA. Cells cultured with LPS, and untreated cells produced similar constitutive amounts of IL-12 p70, at levels near the detection limits of the ELISA (5 pg/ml). In contrast, bDNA and B. abortus both elicited IL-12 production in excess of the control cultures at early time points. The results of one of three similar experiments are shown.

FIG. 3

FIG. 3

IFN-γ induction by B. abortus, LPS, and bDNA is IL-12 dependent. BALB/c spleen cells were cultured for 72 h with or without anti-IL-12 (10 μg/ml), and supernatants were assayed by ELISA. More than 90% of the IFN-γ induction by bDNA and_B. abortus_ was prevented by anti-IL-12. This experiment is representative of four separate experiments in two strains of mice (BALB/c and B10.D2), which all showed significant reduction of IFN-γ secretion by antibodies to IL-12.

FIG. 4

FIG. 4

IL-10 production is not enhanced in LPS cultures. BALB/c spleen cells were cultured with the indicated additives for 6 to 72 h. Supernatants were assayed for IL-10 by ELISA. IL-10 production, like IFN-γ, peaked at the latest time point and showed similar kinetics for all constituents.

FIG. 5

FIG. 5

IFN-γ produced when endogenous IL-10 is blocked is IL-12 dependent. BALB/c spleen cells were cultured with various stimuli in the presence of anti-IL-10, anti-IL-12, or both antibodies (10 μg/ml). Supernatants were collected at 72 h and analyzed for IFN-γ by ELISA. The results from one of two similar experiments are shown.

FIG. 6

FIG. 6

B. abortus and bDNA-induced IL-12 are enhanced by IFN-γ priming, especially in the presence of anti-IL-10. LPS only induced detectable IL-12 with the combination of IFN-γ addition and anti-IL-10. Spleen cells from IFN-γ KO mice (BALB/c background) were stimulated with B. abortus, LPS, or bDNA in the presence of IFN-γ (50 ng/ml) and/or anti-IL-10 (10 μg/ml). LPS, bDNA, or B. abortus was added to the culture 10 min after the addition of anti-IL-10 and IFN-γ. IL-12 p70 was measured by ELISA 18 h later. The results of one of three representative experiments are shown. The results in normal BALB/c mice were similar.

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