In mice, sensitivity to lipopolysaccharide (LPS) is determined by a locus on chromosome 4 which has been designated the Lps
). Mice with defective Lps
genes are highly resistant to the biological activity of LPS. The LPS-resistant phenotype has been described in three mouse strains, C57BL/10ScCr (Cr) (5
), C3H/HeJ (30
), and C57BL/ScN (37
). In addition to these naturally occurring mutants, a fourth LPS-resistant strain, BALB/c/l, was produced independently in two laboratories (32
) by backcrossing the defective Lps
gene from C3H/HeJ into the BALB/c background. The LPS-resistant strains are designatedLpsd
defective), and the LPS-sensitive strains are designated Lpsn
normal). Very recently, evidence has been presented that Lps
and Toll-like receptor-4 (Tlr4
) genes are identical. Cr mice were shown to be homozygous for a null mutation of the Lps/Tlr4
gene, while C3H/HeJ mice carry a missense mutation of the gene, predicted to replace a proline with a histidine at position 712 of the Tlr4 polypeptide chain (24
). Mice of all the Lpsd
strains mentioned above are, under normal conditions, highly resistant to all LPS effects. There is, however, an important difference between the Cr and the C3H/HeJ and BALB/c/l strains. This concerns their abilities to produce gamma interferon (IFN-γ) in response to microorganisms; the response is normal in C3H/HeJ and BALB/c/l but highly impaired in Cr mice (11
). As demonstrated in an earlier study, IFN-γ is a key mediator of the LPS hypersensitivity induced by infection (10
). Consequently, when C3H/HeJ or BALB/c/l mice are infected or treated with killed bacteria, they become partial LPS responders, while Cr mice retain their LPS-resistant phenotype (12
Recently, we observed that splenocytes of Cr mice, supplemented with macrophages of the related, IFN-γ-normal C57BL/10ScSn (Sn) mice, acquire the ability to produce IFN-γ in response to gram-negative bacteria. A similar effect was also achieved with supernatants of Sn macrophages that had been stimulated with killed gram-negative bacteria. Such supernatants by themselves do not directly induce IFN-γ in Cr splenocytes; however, they enable these cells to produce IFN-γ in the presence of a bacterial stimulus. The helper factor present in the active supernatants was identified as IFN-β. This provided evidence that IFN-β is a cofactor for IFN-γ production by gram-negative bacteria and that it is missing in Cr mice (40
). The reason for the absence of an IFN-β production in Cr mice, however, remained unclear.
One interesting biological activity of IFN-β that we described earlier (40
), and which has subsequently been confirmed by other studies (16
), is its property of acting as a cofactor in the induction of IFN-γ. It was also shown that gram-negative bacteria induce IFN-β in theLpsn
Sn mice but not in the relatedLpsd
Cr mice (40
). This finding was unexpected, because bacteria (gram negative and gram positive) generally induce different cytokines in bothLpsn
). We investigated the reason for the above-mentioned inability of Cr mice to produce IFN-β by comparing their IFN-β and other macrophage cytokine responses to those ofLpsd
BALB/c/l and Lpsn
Sn and BALB/c mice following stimulation with different gram-negative and gram-positive bacteria.
IFN-β mRNA and IFN-β activity were inducible by LPS and by a large number of gram-negative bacteria in macrophages ofLpsn mice (Sn and BALB/c) cultured in vitro. Further, IFN-β mRNA was detectable in bothLpsn strains of mice injected with LPS or serovar Typhimurium. In contrast, neither LPS nor gram-negative bacteria induced IFN-β in Lpsd mice that was in any way comparable to that seen in Lpsn mice. Thus, in BALB/c/l mice IFN-β mRNA was practically undetectable and in Cr mice only extremely weak bands were visible after long exposure. Gram-positive bacteria induced no IFN-β activity in any of the mice, regardless of their LPS sensitivity, and in only some cases a very weak IFN-β mRNA signal was recognizable after prolonged exposure of the autoradiography film. All bacteria, however, induced comparable amounts of TNF-α, IL-1α, IL-6, and IL-10 in vitro or in vivo inLpsn and Lpsd mice. Thus, while macrophage cytokines known to be induced by LPS are also inducible by all bacteria in Lpsn andLpsd mice, IFN-β is an exception, being inducible practically only by LPS and therefore generally by gram-negative microorganisms. The inability of Cr and BALB/c/l mice to produce significant amounts of IFN-β when stimulated by gram-negative bacteria is, therefore, related to their lack of LPS responsiveness. A similar absence of IFN-β response to bacteria is predicted for the C3H/HeJ and C57BL/10ScN Lpsd mice (not investigated here). Therefore, LPS emerges as the only notable bacterial component capable of inducing IFN-β, and gram-positive bacteria emerge as a class of microorganisms that are practically incapable of inducing this cytokine.
Recently, evidence has been accumulating that the induction of cytokines by bacterial components is effected via Toll-like receptors (Tlr) 2 and 4 acting as signaling molecules (2
). In the present study, the induction of IFN-β and other cytokines by LPS in normal mice and its complete absence inTlr4/Lps
-defective mice (Cr and BALB/c/l) is evidence that LPS signal transduction proceeded solely via Tlr4. Further, the cytokine responses to bacteria obtained in the absence of LPS signaling, i.e., those induced by gram-positive bacteria or by gram-negative bacteria in Cr and BALB/c/l mice, must proceed via Tlr2 (and/or by as-yet-unidentified signaling receptors). Since IFN-β is selectively absent from such responses, it may be concluded that in mice the induction of IFN-β is strictly Tlr4 dependent, while the induction of other cytokines by LPS or other bacterial components can proceed by the Tlr4 or Tlr2 signaling pathway, respectively.
It has been shown that IFN-β is the predominant type I interferon induced in mice by LPS (39
). IFN-α and IFN-β share the same cellular receptor, which explains their similar biological activities (23
). In this connection, we have shown that both type I IFNs act as cofactors of IFN-γ induction (40
). We also investigated the possibility of IFN-α being produced as an alternative to IFN-β but could obtain no evidence for its presence, either on Northern blots or in supernatants from stimulated macrophages, using a bioassay. Interestingly, synthetic oligodeoxynucleotides containing unmethylated CpG motifs from bacterial DNA and nucleic acid fraction from Mycobacterium bovis
BCG have been shown to induce IFN-α/β (31
). Therefore, the absence of detectable IFN-α/β activity inLpsn
mice stimulated with different gram-positive bacteria and also inLpsd
mice stimulated with gram-negative bacteria observed in our study was surprising. It raises the question of how much of the bacterial DNA is really available for bioactivity in bacterium-treated mice.
As mentioned above, IFN-β acts as a cofactor in the induction of IFN-γ. The present results also allow further conclusions to be made regarding IFN-γ induction by bacteria. Since LPS is the only bacterial component inducing significant amounts of IFN-β, it follows that generally only gram-negative bacteria induce IFN-γ via the IFN-β-dependent pathway. An IFN-β-dependent production of IFN-γ is therefore absent from the LPS-resistant Cr and BALB/c/l mice. BALB/c/l mice, however, produce IFN-γ in response to gram-negative bacteria, indicating that these bacteria induce IFN-γ by additional, IFN-β-independent mechanisms. The latter pathway is also utilized very efficiently by gram-positive bacteria, since many of these bacteria are known to be excellent inducers of IFN-γ. Therefore, the inability of Cr mice to produce IFN-γ in response to any bacteria is related not only to the absence of IFN-β but also to a general inability to respond to the array of IFN-γ-inducing components present in bacteria and, as shown in earlier studies, also in parasites, such as P. chabaudi chabaudi
) andL. major
). It is interesting that when exogenous IFN-β is administered to Cr mice, they acquire the ability to produce IFN-γ after stimulation with gram-negative (reference40
and this study) but not gram-positive (unpublished data) bacteria. This indicates that the IFN-β-dependent pathway of IFN-γ induction is intact in these mice and that this pathway is utilized only by gram-negative bacteria.
Type I interferons are strongly inducible by viruses and play an important role in antiviral defence (23
). Consequently, they have been investigated most extensively in this connection. The significance of IFN-α or IFN-β in bacterial infections has been much less studied. It has been shown that mice deficient for the IFN-α/β receptor are indistinguishable from wild-type mice in their susceptibility to L. monocytogenes
). In view of the present results, the above-mentioned investigation allows no conclusions to be made regarding the significance of IFN-α/β in this infection model, since L. monocytogenes
induces no IFN-α/β and it would make no difference if the mice are deficient for the IFN-α/β receptor or not. In our hands, administration of exogenous recombinant murine IFN-β to mice infected with serovar Typhimurium had no detectable protective effect (M. Matsuura and C. Galanos, unpublished data). This may be understandable, considering that IFN-β is already induced during serovar Typhimurium infection and additional exogenous IFN-β may have no further discernible effect. For this reason, any antibacterial effect of exogenously administered IFN-β might be best recognized during infections by gram-positive bacteria, since these do not induce this cytokine. In this connection, it is interesting that a protective effect of exogenously administered IFN-β has been reported in the case of infection with L. monocytogenes
). For future investigations of the role of IFN-α/β in infections by gram-negative bacteria, new models will have to be developed. Of special interest would be the use of IFN-α/β receptor-deficient mice.