Cellular mediators of innate immunity have the capacity to recognize and respond to pathogens in the absence of antibody or T cells. It is now known that the principal cellular receptors mediating pathogen recognition are the Toll like receptors (TLRs), a family of ten paralogs that respond to a wide variety of pathogen derived substances by initiating inflammatory reactions. In addition, TLRs on immature dendritic cells (iDC) drive maturation in response to pathogens, an essential component of acquired immunity. We have previously shown that the H2 histamine receptor on iDC can profoundly affect the maturation process induced by LPS through TLR4. Histamine is produced by mast cells, which are located in the same microenvironment as DC, and we demonstrated that human monocyte-derived 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 monocyte-derived DC histamine increased IL-10 and reduced IL-12 secretion, and in plasmacytoid DC histamine caused a decrease in type-I interferon production. As a result of its affects upon cytokine secretion, histamine-treated DC induced a Th2 phenotype during T cell priming. We are currently testing in mice the hypothesis that maturation of DC in a microenvironment that is rich in histamine will promote the generation of Th2 cells in the draining lymph nodes. To do this we have established a system in which T cells from TCR transgenic mice are adoptively transferred into syngeneic normal mice. The TCR is specific for ovalbumin, and we have shown that subcutaneous injection with the antigen together with either LPS or CpG ODNs (which stimulate TLR4 and TLR9 respectively) as adjuvant induce proliferation of nave transgenic T cells in the draining lymph nodes. Interestingly, we have found that stimulators of mast cell degranulation, administered with antigen and adjuvant modify the polarization of T cells in the draining lymph nodes, as seen by abrogation of IFN-g production. These data provide evidence that allergy can affect the generation of acquired immune responses, which could lead to the exacerbation of the disease.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 is secreted into the medium as an active protein or when co-expressed with TLR4, forms a TLR4/MD-2 complex in the ER/cis Golgi . By using culture supernatants from cells stably transfected 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. Secreted MD-2 binds to LPS and to TLR4 on cell surfaces, and MD-2 rapidly and irreversibly loses its capacity to bind to both LPS and TLR4 at physiological temeperature. LPS, but not lipid A, prevents this loss in activity by forming a stable complex with MD-2, in a CD14-dependent process. Once formed, the stable MD-2/LPS complex activates TLR4 in the absence of CD14 or free LPS indicating that the activating ligand of TLR4 is the MD-2/LPS complex. Finally we showed that the MD-2/LPS complex, but not LPS alone, induces epithelial cells, which express TLR4 but not MD-2, to secrete IL-6 and IL-8. We proposed that the soluble MD-2/LPS complex plays a crucial role in the LPS response by activating epithelial and other TLR4+/MD-2- cells in the inflammatory microenvironment.TLR9 recognizes bacterial DNA and oligodeoxynucleotides (ODN) containing unmethylated CpG motifs. TLR9 is not located on the cell surface, but rather interacts with bacterial DNA in an interior compartment as expected, since DNA is released from bacteria after phagocytosis. We have shown that both the cytoplasmic and ecto-domains of TLR9 contain localization signals, and have investigated these signals in the TLR9 cytoplasmic domain by mutational analysis. Interestingly, we found that prior to signaling, TLR9 is located, exclusively in the endoplasmic reticulum. Following exposure to CpG DNA, TLR9 transits to endocytic vesicles where it encounters the incoming CpG, but it does so without passing through the Golgi. We have identified at least two localization motifs in the cytoplasmic domain, one that directs ER localization, and a second that leads to vesicle trafficking. We are currently investigating how these motifs cooperate to direct TLR9 to the correct intracellular compartments.A major unresolved question concerns the molecular basis of TLR function. To determine how TLRs recognize PAMPs at the molecular level, we have cloned the extracellular domains of all ten TLRs and have expressed protein for several of them. Currently we are studying ligand binding and molecular structure using purified protein.
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