Increased concentrations of DNA-containing immune complexes in the serum are associated with systemic autoimmune diseases such as lupus. Stimulation of Toll-like receptor 9 (TLR9) by DNA is important in the activation of plasmacytoid dendritic cells and B cells. Here we show that HMGB1, a nuclear DNA-binding protein released from necrotic cells, was an essential component of DNA-containing immune complexes that stimulated cytokine production through a TLR9-MyD88 pathway involving the multivalent receptor RAGE. Moreover, binding of HMGB1 to class A CpG oligodeoxynucleotides considerably augmented cytokine production by means of TLR9 and RAGE. Our data demonstrate a mechanism by which HMGB1 and RAGE activate plasmacytoid dendritic cells and B cells in response to DNA and contribute to autoimmune pathogenesis.

Aggregation of the receptor with high affinity for IgE (FceRI) on the surface of mast cells and basophils stimulates phosphorylation of protein tyrosines, a process in which p53/56'yn kinase has been implicated. We measured the association between FceRI and the kinase, using chemical crosslinking to stabilize their interaction. In the rat basophilic leukemia mast cell line, 3-4%, and at most 20%, of FceRI appear to be associated with the kinase prior to aggregation, even though there is an excess of total cell lyn kinase. Aggregating the FcERI causes three to four times more of the kinase to associate with receptors, a process requiring a prior phosphorylation step. In an in vitro assay, the lyn associated with the aggregated receptors becomes disproportionately more phosphorylated than would be predicted from the amount of lyn associated with the receptors. These and other data are consistent with a model in which aggregation of the receptor leads to its transphosphorylation by constitutively associated lyn kinase. We propose that additional molecules of this kinase are thereby recruited and that this markedly enhances transphosphorylation of tyrosine on the receptor and associated proteins, thereby initiating a cascade of further biochemical changes.This model is also consistent with data on receptors such as the clonotypic receptors on B and T lymphocytes, which share structural and functional features with FcERI.

Many ligands stimulate cellular responses by aggregating the cell-surface receptors to which they are bound. We investigated several mechanistic questions related to aggregation of receptors by using the high-affinity receptor for IgE (Fc6RI) on mast cells as a model system. We briefly exposed cells to covalently cross-linked oligomers of IgE and then added excess monomeric IgE to prevent further aggregation. Early events were examined by monitoring the phosphorylation of protein tyrosines; later events were examined by monitoring secretion. We found that aggregated receptors continue to signal both late and early events in the absence of formation of new aggregates. Additional experiments suggested that the clustered receptors undergo a dynamic process of phosphorylation and dephosphorylation. Our frndings suggest that for these and related receptors that function by aggregation, the persistence of signal transduction is directly related to the intrinsic affinity of the ligand for the individual receptor.Many plasma membrane receptors require aggregationoften dimerization-in order to initiate signal transduction (1,2). The mechanism by which such clustering initiates a signal is still uncertain even for the most extensively studied receptors, but activation of tyrosine kinase(s) as one of the earliest consequences has been well documented in many systems (3, 4). We are investigating the high-affminty receptor for IgE (Fc6RI) on rat mast cells-a system closely related to the one for which the importance of aggregation was first recognized (5) and to cells bearing related "multisubunit immune response receptors" (6). Like the latter but unlike the receptors for growth factors, Fc6RI has no known intrinsic kinase activity. However, phosphorylation of tyrosines on the receptor's subunits and on several other cellular proteins has been demonstrated as a proximate consequence of aggregating the receptor (4,(7)(8)(9).In MATERIALS AND METHODSReagents and Cells. All the reagents we used have been described (11)(12)(13)(14)(15). Rat basophilic leukemia 2H3 (RBL) cells were cultured as described (16).Generation and Purification of Oligomeric IgE. IgE oligomers were prepared essentially as described (17). Monomeric, dimeric, and trimeric IgE were separated on a Superose 6 column (Pharmacia) by repetitive chromatography in borate-buffered saline or for use with permeabilized cells, in 119 mM NaCl/5 mM KCl/5.4 mM glucose/25 mM Pipes, pH 7.2 (assay buffer).Activation ofCells. Cells from 3-day cultures were detached with trypsin. The cells were washed once with medium and twice with warm assay buffer containing 1 mM CaCl2, 0.4mM MgCl2, and 0.1% bovine serum albumin (BSA). Routinely the cells were suspended in the wash buffer at 5 x 106 cells per ml and stimulated at 370C with periodic mixing.Preparation and Analysis of Samples. The total cell proteins or immunoprecipitates of the receptor were analyzed for phosphotyrosines as described (15). A standard sample was used to normalize the densitometric scans (Imagequant; ...

An early event that follows aggregation of the high affinity receptor for IgE (Fc⑀RI) is the phosphorylation of protein tyrosines, especially those on the ␤-and ␥-subunits of the receptor. Disaggregation of the receptors leads to their rapid dephosphorylation, but even stably aggregated receptors undergo continual rounds of phosphorylation and dephosphorylation. We developed assays to study dephosphorylation of the receptors and other cellular proteins. Whole cell extracts dephosphorylated both subunits of the receptors rapidly and were as active against aggregated as against disaggregated Fc⑀RI. Upon disaggregation, the in vivo dephosphorylation of the Fc⑀RI and several other proteins followed first-order kinetics with closely similar rate constants despite substantial differences in the extent of phosphorylation. These results suggest that the level of phosphorylation of Fc⑀RI is largely controlled by the aggregation-induced action of kinase(s) and not from changes in susceptibility to or activity of the phosphatases. Much of the total phosphatase is lost when the cells are permeabilized, but the rate of dephosphorylation of disaggregated Fc⑀RI was comparable in intact and permeabilized cells. Thus, much of the activity utilized by the cell to dephosphorylate the Fc⑀RI is likely to be associated with the plasma membrane.The high affinity IgE receptor on mast cells and basophils (Fc⑀RI) has a central role in mediating allergic responses (1, 2). Aggregation of the receptors results in phosphorylation of protein tyrosines as an early event that leads to a variety of later cellular phenomena (3, 4). The receptor itself lacks sequences typical for intrinsic protein-tyrosine kinase activity (5), and experimentally, receptors purified by affinity columns or well washed immunoprecipitates show virtually no kinase activity in vitro (6, 7). However, a variety of studies have implicated a kinase weakly associated with the receptor (6,8,9). In rats, the critical initial kinase appears to be Lyn (9). The receptorassociated Lyn from resting cells displays tyrosine kinase activity toward exogenous substrates, but little or no phosphorylation of the receptor itself is observed unless the receptors are aggregated (10 -13). These observations and those made on the effect of inhibitors of phosphatases (12,14) imply that proteintyrosine phosphatases (PTPs) 1 are continuously modulating the resting system. The studies with inhibitors and other experimental approaches (15) show that the PTPs also continuously act on aggregated receptors. Finally, when individual receptors dissociate from the aggregate, e.g. by addition of monomeric hapten after stimulation by multivalent antigen, rapid dephosphorylation of the receptor and of other cellular proteins is observed (7, 10 -13).The PTPs involved in these processes remain undefined, and the purpose of the current study was to investigate some of their characteristics. We first developed an in vitro assay to test the PTP activity in total cell lysate toward receptors that had been phosp...