Bacterial induction of beta interferon in mice is a function of the lipopolysaccharide component

Abstract

We investigated the reason for the inability of lipopolysaccharide (LPS)-resistant (Lps-defective [Lps(d)]) C57BL/10ScCr mice to produce beta interferon (IFN-beta) when stimulated with bacteria. For this purpose, the IFN-beta and other macrophage cytokine responses induced by LPS and several killed gram-negative and gram-positive bacteria in LPS-sensitive (Lps-normal [Lps(n)]; C57BL/10ScSn and BALB/c) and Lps(d) (C57BL/10ScCr and BALB/c/l) mice in vitro and in vivo were investigated on the mRNA and protein levels. In addition, double-stranded RNA (dsRNA) was used as a nonbacterial stimulus. LPS and all gram-negative bacteria employed induced IFN-beta in the Lps(n) mice but not in the Lps(d) mice. All gram-positive bacteria tested failed to induce significant amounts of IFN-beta in all four of the mouse strains used. As expected, all other cytokines tested (tumor necrosis factor alpha, interleukin 1alpha [IL-1alpha], IL-6, and IL-10) were differentially induced by gram-negative and gram-positive bacteria. Stimulation with dsRNA induced IFN-beta and all other cytokines mentioned above in all mouse strains, regardless of their LPS sensitivities. The results suggest strongly that LPS is the only bacterial component capable of inducing IFN-beta in significant amounts that are readily detectable under the conditions used in this study. Consequently, in mice, IFN-beta is inducible only by gram-negative bacteria, but not in C57BL/10ScCr or other LPS-resistant mice.

FIG. 1

Kinetics of IFN-β and TNF-α mRNA induction in Sn macrophages stimulated with different agents. Macrophages (10⁶/ml) were stimulated with LPS (10 μg/ml), dsRNA (10 μg/ml), serovar Typhimurium (100 μg/ml), and S. aureus (100 μg/ml) for different times. IFN-β and TNF-α mRNAs were detected by Northern blot analysis of total macrophage RNA (pools of duplicates), as described in Materials and Methods. The exposure time for IFN-β was 2 weeks, and that for TNF-α was 12 h. RNA applied to the gel (approximately 2 μg/lane) was visualized for each sample by the intensity of the ethidium bromide-stained 18S rRNA bands. Stimulation times were as follows: lanes 1, 20 min; lanes 2, 1 h; lanes 3, 3 h; lanes 4, 6 h; lanes 5, 9 h; lanes 6, 12 h; lanes 7, 24 h; lane C, unstimulated macrophages.

FIG. 2

Induction of IFN-β and TNF-α mRNAs in Lpsⁿ and Lps^d macrophages stimulated with LPS and different bacteria. Untreated control macrophages (10⁶/ml [lanes 1]) or macrophages treated with LPS (10 μg/ml [lanes 2]), serovar Typhimurium (100 μg/ml [lanes 3] and 500 μg/ml [lanes 3a]), S. aureus (100 μg/ml [lanes 4] and 500 μg/ml [lanes 4a]), P. acnes (100 μg/ml [lanes 5] and 500 μg/ml [lanes 5a]), E. coli (100 μg/ml [lanes 6] and 500 μg/ml [lanes 6a]), S. thermophilus (100 μg/ml [lanes 7] and 500 μg/ml [lanes 7a]), and dsRNA (10 μg/ml [lanes 8]) were cultured for 3 h. Thereafter, IFN-β and TNF-α mRNAs were detected by Northern blot analysis of total macrophage RNA (pools of duplicates), as described in Materials and Methods. The exposure time for IFN-β was 2 weeks, and that for TNF-α was 3 h. RNAs applied to the gel (approximately 6 μg/lane) were visualized for each sample by the intensity of the ethidium bromide-stained 18S rRNA bands.

FIG. 3

Levels of cytokines in supernatants of Lpsⁿ and Lps^d macrophages stimulated with different agents. Macrophages (10⁶/ml) of Sn, BALB/c (Balb), Cr, and BALB/c/l (Balb/l) mice were cultured with LPS (0.1 μg/ml), serovar Typhimurium (Stm; 100 μg/ml), S. aureus (Sa; 100 μg/ml), and dsRNA (10 μg/ml) for 24 h. The cytokines in cell-free supernatants were measured as described in Materials and Methods. The values are means of duplicates. One representative experiment of four is shown.

FIG. 4

Expression of IFN-β, TNF-α, and IL-6 mRNAs in the spleens of Lpsⁿ and Lps^d mice injected with serovar Typhimurium and S. aureus. Lpsⁿ (Sn and BALB/c [Balb]) and Lps^d (Cr and BALB/c/l [Balb/l]) mice (three animals per strain) were injected intravenously with 15 μg of killed serovar Typhimurium (Stm) or S. aureus (Sa) per g of body weight. Untreated mice served as controls (C). One hour after treatment, the mice were sacrificed and their spleens were removed and homogenized, as described in Materials and Methods. Total spleen RNA was extracted separately for each animal and pooled for the three identically treated animals of each group. Ten micrograms of each RNA pool were used for detection of cytokine mRNA in an RPA with a cytokine template set. Two constitutive gene probes (L32 and GAPDH [glyceraldehyde-3-phosphate dehydrogenase]) for standardization of RNA amounts were also used according to the instructions of the manufacturer.