Cellular mediators of innate immunity do not express rearranged V regions, yet have the capacity to recognize and respond to target cells in the absence of antibody. Recently an ancient family of pathogen recognition receptors, the toll like receptors (TLRs) was discovered in humans and mice and several have been shown to trigger inflammatory responses to pathogen derived substances. We hypothesized that pathogen recognition by immature dendritic cells (iDC) is mediated by TLRs and showed that human iDC do express and signal through TLRs. We have also shown that histamine generates signals in iDC that interact with the LPS/TLR4 signaling pathway, profoundly altering the maturation process. Histamine is produced in the peripheral microenvironment by the binding of allergens to IgE on mast. We demonstrated that iDC express two active histamine receptors, H1 and H2. Although histamine failed to affect the LPS-driven maturation of iDC with regard to phenotypic changes or capacity to prime naive T cells, it dramatically altered the repertoire of cytokines and chemokines secreted by mature DC. In particular, histamine, acting upon the H2 receptor for a short period of time, increased IL-10 production and reduced IL-12 secretion. As a result, histamine-matured DC polarized naive CD4+ T cells toward a Th2 phenotype, as compared with DC that had matured in the absence of histamine. We propose that the Th2 cells favor IgE production, leading to increased histamine secretion by mast cells. Thus a positive feedback loop would be established that could contribute to the severity of atopic diseases such as allergy and asthma and explain the observation that Th2 cells and serum IgE antibodies are increased in atopic patients. The molecular mechanisms by which TLRs recognize pathogens and initiate inflammatory responses is poorly understood, and we have examined two aspects of the TLR signaling process.
The aim of one study was to determine a function for TLR1. The mammalian TLRs constitute a family of ten members, several of which initiate inflammatory responses upon recognition of pathogen derived substances. TLR4 in particular recognizes LPS, and is essential for LPS-induced septic shock. By contrast, TLR1, the most widely expressed of the TLRs, has no defined function. Human microvascular endothelial cells (HMEC) express TLR4 but not TLR1 and activate NF-kB through TLR4 in response to LPS. When HMEC cells were transfected with TLR1, they lost their capacity to respond to LPS. Inhibition was specific for TLR1 because a different paralog, TLR5, failed to block TLR4 function. Moreover, TLR1 had no effect upon the TNF-a induced activation of NF-kB, indicating that TLR1 inhibited early events in the TLR signaling pathway. Constructs encoding the transmembrane and extracellular domains of TLR1 blocked TLR4 function. In addition, TLR1 physically associated with TLR4 in co-precipitation experiments. Our results suggest that the function of TLR1 is to moderate the potentially dangerous innate response to LPS by binding to TLR4 and diminishing its capacity to activate the TLR signaling pathway. The TLR4 response to LPS requires the binding of MD-2 to its extracellular domain. MD-2 contains a leader sequence but lacks a transmembrane domain, and we asked whether it is secreted into the medium as an active protein. As a source of secreted MD-2 (sMD-2) we used culture supernatants from cells stably transduced with epitope-tagged human MD-2. We showed that sMD-2 exists as a heterogeneous collection of large disulfide linked oligomers formed from stable dimeric subunits, and that concentrations of sMD-2 as low as 50 pM enhanced the responsiveness of TLR4 reporter cells to LPS. An MD-2 like activity was also released by monocyte-derived iDC from normal donors. When co-expressed with MD-2, TLR4 indiscriminantly associated in the ER/cis Golgi with different sized oligomers of MD-2, and excess MD-2 was secreted into the medium. We concluded that normal and transfected cells secrete a soluble form of MD-2 that binds with high affinity to TLR4. Thus, sMD-2 might play a role in regulating responses to LPS and other pathogen derived substances in vivo.
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