Toll-like receptor (TLR) 2 has recently been associated with cellular responses to numerous microbial products, including LPS and bacterial lipoproteins. However, many preparations of LPS contain low concentrations of highly bioactive contaminants described previously as “endotoxin protein,” suggesting that these contaminants could be responsible for the TLR2-mediated signaling observed upon LPS stimulation. To test this hypothesis, commercial preparations of LPS were subjected to a modified phenol re-extraction protocol to eliminate endotoxin protein. While it did not influence the ability to stimulate cells from wild-type mice, repurification eliminated the ability of LPS to activate cells from C3H/HeJ (Lpsd) mice. Additionally, only cell lines transfected with human TLR4, but not human or murine TLR2, acquired responsiveness to both re-extracted LPS and to a protein-free, synthetic preparation of lipid A. These results suggest that neither human nor murine TLR2 plays a role in LPS signaling in the absence of contaminating endotoxin protein.

Lipopolysaccharide (LPS) derived from the periodontal pathogen Porphyromonas gingivalis has been reported to differ structurally and functionally from enterobacterial LPS. These studies demonstrate that in contrast to protein-free enterobacterial LPS, a similarly purified preparation of P. gingivalis LPS exhibited potent Toll-like receptor 2 (TLR2), rather than TLR4, agonist activity to elicit gene expression and cytokine secretion in murine macrophages and transfectants. More importantly, TLR2 stimulation by this P. gingivalis LPS preparation resulted in differential expression of a panel of genes that are normally induced in murine macrophages by Escherichia coli LPS. These data suggest that (i) P. gingivalis LPS does not signal through TLR4 and (ii) signaling through TLR2 and through TLR4 differs quantitatively and qualitatively. Our data support the hypothesis that the shared signaling pathways elicited by TLR2 and by TLR4 agonists must diverge in order to account for the distinct patterns of inflammatory gene expression.Lipopolysaccharides (LPS) are among the most potent inflammatory bacterial mediators and have been strongly implicated in the inflammatory response associated with gram-negative sepsis. Most LPS signaling studies have used LPS preparations derived from species within the Enterobacteriaceae, which possess relatively well-conserved lipid A structures (reviewed in reference 36). A convergence of data suggest that these prototypic LPS preparations, when highly purified, elicit LPS responses that are restricted in the use of TLR4 as the principal signal-transducing molecule (reviewed in reference 21), which is strongly supported by the finding that synthetic E. coli lipid A activated Toll-like receptor 4 (TLR4) and not TLR2 transfectants (8). However, the lipid A of nonenterobacterial species, e.g., Porphyromonas gingivalis, which has been implicated in the inflammation associated with chronic periodontitis (reviewed in reference 9), differs both structurally and functionally from enterobacterial lipid A. Specifically, the major species of P. gingivalis lipid A is composed of unique branched fatty acids, with longer carbon chains than in enterobacterial lipid A, the absence of a phosphoryl group at position 4Ј of the nonreducing glucosamine, as well as other modifications ( Fig. 1) (1). Consistent with these structural differences is the finding that P. gingivalis LPS activity is poorly inhibited by polymyxin B (12), which has been postulated to inactivate LPS by binding electrostatically to negatively charged phosphate groups, leading to a subsequent interaction of polymyxin B with the hydrophobic fatty acids (25, 33). Although P. gingivalis-induced signaling was shown some time ago to be CD14 dependent (34), site-specific mutagenesis of CD14 suggests that the substitution of certain charged amino acids differentially affects the abilities of Escherichia coli and P. gingivalis LPS to bind CD14 (4, 5). In addition, binding of P. gingivalis LPS to LPS binding protein has been reported to be 100-fo...

Identity of the Epstein-Barr virus (EBV) receptor with the complement receptor type 2 (CR2) was established in three sets of experiments using the monoclonal antibodies, HB-5 and anti-B2, which recognize a Mr 145,000 Blymphocyte membrane protein that is CR2. First, the rank order for binding of fluoresceinated EBV to four lymphoblastoid cell lines (SB, JY, Raji, and Molt-4) was identical to the rank order for binding of HB-5 and anti-B2 by analytical flow cytometry. Second, pretreatment of cells with HB-5 followed by treatment with goat F(ab')2 fragments to mouse IgG blocked binding of fluoresceinated EBV on SB, a B-lymphoblastoid cell line. Virus attachment was not inhibited by alone, second antibody alone, rabbit anti-C3b receptor, or UPC10 (an irrelevant monoclonal antibody). Third, transfer of CR2 from SB to protein A-bearing Staphylococcus aureus particles, to which HB-5 had been absorbed, conferred on them the specific ability to bind 1251-labeled EBV. We conclude that CR2 is the EBV receptor of human B lymphocytes.pressed on phagocytes and large granular lymphocytes having natural killer and antibody-dependent cytotoxic activities, but it is not expressed on B lymphocytes (18-21). It consists of two polypeptide chains of Mr 155,000-185,000 and Mr 95,000-105,000 (20,22,23). The C3d receptor or CR2 binds the C3d region of C3d,g, iC3b and, with less affinity, C3b. It is found on mature B lymphocytes and on certain Bcell lines (24-29). It has been characterized as a Mr 140,000-145,000 membrane protein (26,27) that is recognized by the monoclonal antibodies (mAbs) termed anti-B2 (28) and , respectively.In the present study, these mAbs have been used to show that the EBVR and CR2 are quantitatively coexpressed on four cell lines, that binding of antibody to CR2 can prevent attachment of EBV, and that CR2 that has been immunoabsorbed onto particles of Staphylococcus aureus Cowan I strain (SACI) specifically binds EBV.

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