Physical contact between lipopolysaccharide and toll-like receptor 4 revealed by genetic complementation

Abstract

Some mammalian species show an ability to discriminate between different lipopolysaccharide (LPS) partial structures (for example, lipid A and its congener LA-14-PP, which lacks secondary acyl chains), whereas others do not. Using a novel genetic complementation system involving the transduction of immortalized macrophages from genetically unresponsive C3H/HeJ mice, we now have shown that the species-dependent discrimination between intact LPS and tetra-acyl LPS partial structures is fully attributable to the species origin of Toll-like receptor 4 (Tlr4), an essential membrane-spanning component of the mammalian LPS sensor. Because Tlr4 interprets the chemical structure of an LPS molecule, we conclude that LPS must achieve close physical proximity with Tlr4 in the course of signal transduction.

Figure 1

Phenotypic characterization of the myc-immortalized MTC3H/HeJ macrophage line by flow cytometric analysis. Fourteen antigenic determinants were surveyed by using a panel of monoclonal reagents that distinguish T, B, and macrophage cell lineages. Open histograms show binding of specific antibodies, whereas isotype-matched control antibodies or secondary reagents are represented by dotted histograms. Abscissa, for all graphs: fluorescence intensity (logarithmic scale). Ordinate, for all graphs: cell number.

Figure 2

Pure populations of retrovirally transduced MTC3H/HeJ cells expressing human or mouse Tlr4. Cells were enriched by two cycles of cell sorting, based on GFP expression originating from the bicistronic PINCO vector. More than 99% of the population carries the transducing retroviral vector as assessed by fluorescence microscopy. (×400.) Fluorescence intensity was adjusted so that only the most intensely fluorescent cells are visible. Note the difference in morphology between cells transduced to express the mouse protein (Center) as compared with the human protein (Right).

Figure 3

Expression of human and mouse Tlr4 mRNAs in MTC3H/HeJ cells transduced with each cDNA. Northern blot was probed with the mouse Tlr4 cDNA. RNA was obtained from cells transfected with the mouse Tlr4 cDNA, the human Tlr4 cDNA, or the PINCO vector as indicated and stained with ethidium bromide (Left). The blot was exposed to film for 5 hr to produce the autoradiogram (Right). Under these conditions, only the recombinant mRNA (which is present in great excess over the native Tlr4 mRNA) is visualized.

Figure 4

Responses of MTC3H/HeJ cells to LPS and LPS partial structures: effect of Tlr4 species origin. (A) MTC3H/HeJ cells transfected with vector control. (B) MTC3H/HeJ cells transfected to express mouse Tlr4. (C) MTC3H/HeJ cells transfected to express human Tlr4. Each point represents the mean of four TNF assays, and error bars reflect SD. Curves were drawn to fit a one-site hyperbola model by using prism 3.0. Note that the y axis in A is drawn to a different scale than in B and C to highlight the complete nonresponsiveness of the control-transfected cells. The dashed line parallel to the abscissa indicates the level of TNF produced without the addition of any ligand (No Stimulus).