Nagging Questions Take Toll on Researchers (1)- Mouse mutants pinpoint Gram-negative sepsis co-receptor

For this article Karen Young Kreeger interviewed Bruce Beutler, professor of immunology at the Scripps Research Institute in La Jolla, Calif. Data from the Web of Science (ISI, Philadelphia) show that this paper has been cited significantly more often than the average paper of the same type and age. A. Poltorak, X. He, I. Smirnova, M-Y. Liu, C. Van Huffel, X. Du, D. Birdwell, E. Alejos, M. Silva, C. Galanos, M. Freudenberg, P. Castagnoli, B. Layton, B. Beutler, "Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: Mutations in Tlr4 gene," Science, 282:2085-8, 1998. (Cited in 275 papers) Infection by Gram-negative bacteria is no small matter: In the United States an estimated 20,000 people die each year due to septic shock from Gram-negative bacteria. The deadliness of this condition is partially due to a bacterial cell component called lipopolysaccharide (LPS), or endotoxin. When the human immune system encounters bacterial LPS, it triggers the release of toxic cytokines that can lead to sepsis. On the other hand, immune cells still need to recognize LPS to effectively clear the bacteria from the body. For many years, the question remained, by what mechanism does the signal from LPS travel into the cell to engage the host immune system? Investigators have known since early last century that endotoxin is one of the most toxic components of Gram-negative bacteria; and since 1965, it's been known that certain strains of mice are highly resistant to endotoxin, notes Bruce Beutler, professor of immunology at the Scripps Research Institute in La Jolla, Calif. "This was a persistent mystery in the LPS field," he recalls. Researchers suspected that the mutation responsible for these mice was related to the receptor for LPS. And later when CD14 was shown to be an important binding protein for LPS on the surface of cells, they suspected that LPS was a co-receptor because CD14 doesn't have a cytoplasmic domain and can't signal across the membrane. Bruce Beutler "But no one could isolate such a co-receptor," says Beutler. After trying various cDNA and biochemical methods, Beutler's lab finally used positional cloning to locate the mutant receptor. "This took us five years, but it worked where all other approaches failed," he recounts. "We narrowed the location of the gene to a very small area. We looked at every candidate in that area and finally we found the mutation in C3H/HeJ mice and also in C57BL/10ScCr mice." In the case of C3H/HeJ mice there is a point mutation, and in C57BL/10ScCr mice there is a deletion of the entire toll-like receptor 4 (TLR4) gene locus. Pinpointing these mutations in the TLR4 gene led Alexander Poltarak, then a postdoc in Buetler's lab, and the group to conclude that in mammals this TLR4 membrane protein receptor recognizes, and hence, tranduces the LPS signal across the plasma membrane. A 1999 paper from a Japanese group on a TLR4 knockout mouse1 confirmed the role of TLR4 as the inducer of the Escherichia coli LPS signal transduction pathway. Beutler mentions a few implications of their work: "It seems to be the case that C3H/HeJ mice can only sustain an innoculum of one or two bacterium and five to ten is usually a lethal dose; whereas, in a normally sensitive mouse you can give many more-10,000 or more. The question then arises: 'Do people with gram-negative infections have a similar defect?'" Another important ramification is that TLR4 turns out to be part of a family of receptors that now includes 10 members in all. "Each of them seems to recognize a different component of bacteria or fungi, and collectively they act to perceive probably all microbes," concludes Beutler. "The TLRs are the basis of innate immune recognition, just as immunoglobulins and T-cell receptors are the basis of acquired immune recognition. Toll-like receptors are what 'see' the microbial world." 1. K. Hoshino et al., "Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: Evidence for TLR4 as the LPS gene product," Journal of Immunology," 162:3749-52, 1999.

Nagging Questions Take Toll on Researchers (1)- Mouse mutants pinpoint Gram-negative sepsis co-receptor

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