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Allergies to nickel (Ni(2+)) are the most frequent cause of contact hypersensitivity (CHS) in industrialized countries. The efficient development of CHS requires both a T lymphocyte-specific signal and a proinflammatory signal. Here we show that Ni(2+) triggered an inflammatory response by directly activating human Toll-like receptor 4 (TLR4). Ni(2+)-induced TLR4 activation was species-specific, as mouse TLR4 could not generate this response. Studies with mutant TLR4 proteins revealed that the non-conserved histidines 456 and 458 of human TLR4 are required for activation by Ni(2+) but not by the natural ligand lipopolysaccharide. Accordingly, transgenic expression of human TLR4 in TLR4-deficient mice allowed efficient sensitization to Ni(2+) and elicitation of CHS. Our data implicate site-specific human TLR4 inhibition as a potential strategy for therapeutic intervention in CHS that would not affect vital immune responses.

The innate immune response to Gram-negative bacteria depends mainly on the ability of the host to respond to the LPS component. Consequently, the state of LPS sensitivity at the time of infection and the numbers of invading bacteria (i.e. the amounts of LPS) are primary factors determining the innate responses provoked by Gram-negative pathogens. LPS sensitivity increases following treatment of mice with live or killed micro-organisms. Two types of sensitization have been recognized, strong, IFN-gamma-dependent and moderate IFN-gamma-independent. IL-12 and IL-18 are intimately involved in the induction of IFN-gamma by bacteria. We showed that Gram-negative bacteria induce IFN-gamma in mice also by an IFN-beta-dependent pathway that requires IL-18 and is independent of IL-12 signaling. This pathway is STAT4 dependent, the activation of which is directly linked to IFN-beta. Further, IFN-beta can be replaced by IFN-alpha. While different components of Gram-negative bacteria induce IL-12 and IL-18, LPS seems to be the only component in these bacteria capable of inducing IFN-beta. Therefore, the IFN-beta pathway of IFN-gamma induction, unlike the IL-12 pathway, proceeds only in LPS responder mice. The IFN-alpha/beta-dependent pathway is expected to play a role whenever IFN-alpha or IFN-beta, and IL-18 are produced concomitantly during infection.

C57BL/10ScCr (Cr) mice carry a deletion of the Toll-like receptor 4 (tlr4) gene (i.e. they are tlr4(0/0)) and are thus refractory to LPS effects. Insertion of wild-type tlr4 transgene into the tlr4(0/0) Cr germ line endowed LPS susceptibility in the two transgenic lines created, indicating that TLR4 is the only limiting factor for LPS responsiveness in Cr mice. The absolute levels of tlr4 mRNA expressed by the heterozygous transgenic (tlr4(Tr/0)), wild-type C57BL/10ScSn (Sn) (tlr4(+/+)) and heterozygous F1 (Sn x Cr) (tlr4(+/0)) mice varied markedly. However, the pattern of distribution of expression in the different organs was the same in all strains. In different biological assays (B cell mitogenicity, cytokine induction and lethal toxicity) the degree of LPS response obtained in the different strains of mice correlated with the levels of tlr4 mRNA expression. In macrophages, investigation of the LPS-induced cytokine (IL-6) response revealed a linear relationship between the response and the logarithm of TLR4-MD-2 levels.

Lipopolysaccharide (endotoxin, LPS) is a major recognition marker for the detection of gram-negative bacteria by the host and a powerful initiator of the inflammatory response to infection. Using S- and R-form LPS from wild-type and R-mutants of Salmonella and E. coli, we show that R-form LPS readily activates mouse cells expressing the signaling receptor Toll-like receptor 4/myeloid differentiation protein 2 (TLR4/MD-2), while the S-form requires further the help of the LPS-binding proteins CD14 and LBP, which limits its activating capacity. Therefore, the R-form LPS under physiological conditions recruits a larger spectrum of cells in endotoxic reactions than S-form LPS. We also show that soluble CD14 at high concentrations enables CD14-negative cells to respond to S-form LPS. The presented in vitro data are corroborated by an in vivo study measuring TNF-alpha levels in response to injection of R- and S-form LPS in mice. Since the R-form LPS constitutes ubiquitously part of the total LPS present in all wild-type bacteria its contribution to the innate immune response and pathophysiology of infection is much higher than anticipated during the last half century.