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Recognition of microbial components by TLR2 requires cooperation with other TLRs. TLR6 has been shown to be required for the recognition of diacylated lipoproteins and lipopeptides derived from mycoplasma and to activate the NF-κB signaling cascade in conjunction with TLR2. Human TLR2 is expressed on the cell surface in a variety of cells, including monocytes, neutrophils, and monocyte-derived, immature dendritic cells (iDCs), whereas the expression profile of TLR6 in human cells remains obscure. In this study we produced a function-blocking mAb against human TLR6 and analyzed TLR6 expression in human blood cells and cell lines and its participation in ligand recognition. TLR6 was expressed, although at a lower level than TLR2, on the cell surface in monocytes, monocyte-derived iDCs, and neutrophils, but not on B, T, or NK cells. Confocal microscopic analysis revealed that TLR6 was colocalized with TLR2 at the plasma membrane of monocytes. Importantly, TLR2/6 signaling did not require endosomal maturation, and anti-TLR6 mAb inhibited cytokine production in monocytes and iDCs stimulated with synthetic macrophage-activating lipopeptide-2 or peptidoglycan, indicating that TLR6 recognized diacylated lipopeptide and peptidoglycan at the cell surface. In addition, TLR2 mutants C30S and C36S (Cys30 and Cys36 in TLR2 were substituted with Ser), which were expressed intracellularly in HEK293 cells, failed to induce NF-κB activation upon macrophage-activating lipopeptide-2 stimulation even in the presence of TLR6. Thus, coexpression of TLR2 and TLR6 at the cell surface is crucial for recognition of diacylated lipopeptide and peptidoglycan and subsequent cellular activation in human cells.

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We compared the structural and functional properties of three recombinant human interleukin-18 (rIL-18) preparations, commercially available (Pep rIL-18) and prepared in our laboratory (active and inactive, according to their ability to potentiate IL-12-mediated interferon-gamma [IFN-gamma] induction in lymphocytes). All three preparations showed multimer formation on SDS-PAGE/immunoblotting using monoclonal antibodies (mAb) against the inactive form of rIL-18. In contrast, only the 18-kDa bands were recognized in each sample by mAb against the active form of rIL-18. The amounts of multimers and the 18-kDa moiety of Pep rIL-18 resembled those of the inactive rather than the active form. Likewise, the reaction profile of Pep rIL-18 toward mAb was very similar to that of inactive but not active rIL-18 on sandwich ELISA. Pep rIL-18 potentiated IFN-gamma-inducing activity together with IL-12, but its potency was 100-fold less than that of the active rIL-18, and excess doses were required for its activity. The inactive rIL-18 showed virtually no IFN-gamma-inducing ability, but when reduced and reconstituted, it inhibited the IFN-gamma-inducing activity of active rIL-18. These results suggest that there are two categories of recombinant IL-18 that are structurally, functionally, and antigenically different, and the mAb 125-2H and 21 can discriminate these two IL-18 populations by recognizing the epitopes specifically expressed on active and inactive IL-18, respectively.

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