Receptor cross-talk spatially restricts p-ERK during TLR4 stimulation of autoreactive B cells.

To maintain tolerance, autoreactive B cells must regulate signal transduction from the BCR and TLRs. We recently identified that dendritic cells and macrophages regulate autoreactive cells during TLR4 activation by releasing IL-6 and soluble CD40 ligand (sCD40L). These cytokines selectively repress Ab secretion from autoreactive, but not antigenically naive, B cells. How IL-6 and sCD40L repress autoantibody production is unknown. In this work, we show that IL-6 and sCD40L are required for low-affinity/avidity autoreactive B cells to maintain tolerance through a mechanism involving receptor cross-talk between the BCR, TLR4, and the IL-6R or CD40. We show that acute signaling through IL-6R or CD40 integrates with chronic BCR-mediated ERK activation to restrict p-ERK from the nucleus and represses TLR4-induced Blimp-1 and XBP-1 expression. Tolerance is disrupted in 2-12H/MRL/lpr mice where IL-6 and sCD40L fail to spatially restrict p-ERK and fail to repress TLR4-induced Ig secretion. In the case of CD40, acute signaling in B cells from 2-12H/MRL/lpr mice is intact, but the chronic activation of p-ERK emanating from the BCR is attenuated. Re-establishing chronically active ERK through retroviral expression of constitutively active MEK1 restores tolerance upon sCD40L, but not IL-6, stimulation, indicating that regulation by IL-6 requires another signaling effector. These data define the molecular basis for the regulation of low-affinity autoreactive B cells during TLR4 stimulation; they explain how autoreactive but not naive B cells are repressed by IL-6 and sCD40L; and they identify B cell defects in lupus-prone mice that lead to TLR4-induced autoantibody production.

Macrophages prevent the differentiation of autoreactive B cells by secreting CD40 ligand and interleukin-6.

Activation of the innate immune system promotes polyclonal antibody secretion to eliminate invading pathogens. Inherent in this process is the potential to activate autoreactive B cells and induce autoimmunity. We showed previously that TLR-stimulated dendritic cells and macrophages regulate B cell tolerance to Smith antigen, in part through the secretion of interleukin-6 (IL-6). In this manuscript, we show that neutralization of IL-6 fails to abrogate macrophage-mediated repression and identify soluble CD40 ligand (CD40L) as a second repressive factor secreted by macrophages. CD40L selectively repressed Ig secretion by chronically antigen-experienced (anergic) immunoglobulin transgenic and nontransgenic B cells but not by transiently stimulated B cells. The importance of macrophages in maintaining B cell tolerance was apparent in lupus-prone MRL/lpr mice. Compared with C57BL/6 mice, macrophages from MRL/lpr mice were significantly less efficient at repressing immunoglobulin secretion coincident with diminished IL-6 and CD40 ligand production. These data indicate that macrophages regulate autoreactive B cells by secreting repressive factors that prohibit terminal differentiation of B cells. The regulation of autoreactive B cells by macrophages is diminished in lupus-prone mice suggesting a role in autoimmunity.

In vivo murine CD23 destabilization enhances CD23 shedding and IgE synthesis.

To investigate the effects of in vivo CD23 destabilization on CD23 shedding and IgE production, an anti-CD23 stalk monoclonal (19G5), previously shown to enhance proteolysis of CD23 in vitro, was utilized. Compared to isotype control-treated mice, BALB/cJ mice injected with 19G5 displayed significantly enhanced serum soluble CD23 and IgE. Soluble CD23 and IgE levels were also increased in 19G5-treated C57BL/6J mice (intermediate IgE responders); however, the kinetics of the responses differed between the high (BALB/cJ) and intermediate responder mice, suggesting a potential role for CD23 in regulating IgE responder status. The 19G5-induced IgE response was dependent on IL-4 and independent of CD21 as demonstrated through use of IL-4Ralpha and CD21/35-deficient mice, respectively. Overall, the data provide a direct demonstration for CD23's role in regulating IgE production in vivo and suggest that therapies aimed at stabilizing cell surface CD23 would be beneficial in controlling allergic disease.

Low-affinity, Smith antigen-specific B cells are tolerized by dendritic cells and macrophages.

Polyclonal B cell activation promotes immunity without the loss of tolerance. Our data show that during activation of the innate immune system, B cell tolerance to Smith Ag Sm is maintained by dendritic cells (DCs) and macrophages (MPhi). TLR4-activated myeloid DCs and MPhi, but not plasmacytoid or lymphoid DCs, repressed autoreactive B cells through the secretion of soluble mediators, including IL-6. Although IL-6 promotes plasma cell differentiation of B cells acutely stimulated by Ag, we show that it repressed cells that were chronically exposed to self-Ag. This mechanism of tolerance was not limited to Smith Ag-specific B cells as hen egg lysozyme- and p-azophenylarsonate-specific B cells were similarly affected. Our data define a tolerogenic role for MPhi and DCs in regulating autoreactive B cells during activation of the innate immune system.

CD23 trimers are preassociated on the cell surface even in the absence of its ligand, IgE.

Allergic disease is mediated by high levels of allergen-specific IgE. IgE binding to CD23, the low affinity receptor for IgE, results in a negative feedback signal leading to a decrease in IgE production. Previous studies have shown that CD23 associates as an oligomer and that cooperative binding of at least two lectin domains is required for high affinity IgE binding to CD23. We have previously shown that cooperative binding is required for regulation of IgE production. This study describes the production of several mAbs that bind the stalk region of murine CD23. One of the Abs, 19G5, inhibited the IgE/CD23 interaction at 37 degrees C, but not at 4 degrees C. Analysis of the binding properties of these Abs revealed that CD23 dissociates at high temperatures, such as 37 degrees C; however, the N terminus is constitutively associated, indicating partial, rather than complete, dissociation. A novel finding was that the stalk region, previously thought to mediate trimer association, was not required for oligomerization. These data reveal important information about the structure of CD23 that may be useful in modulating IgE production.

Temperature effect on IgE binding to CD23 versus Fc epsilon RI.

A chimeric soluble CD23, consisting of the extracellular domain of mouse CD23 and a modified leucine zipper (lz-CD23), has been shown to inhibit IgE binding to the FcepsilonRI. A similar human CD23 construct was also shown to inhibit binding of human IgE to human FcepsilonRI. In both systems, the inhibition was found to be temperature dependent; a 10-fold molar excess of lz-CD23 gave 90-98% inhibition at 4 degrees C, dropping to 20-30% inhibition at 37 degrees C. Surface plasmon resonance analysis of lz-CD23 binding to an IgE-coated sensor chip suggested that the effective concentration of lz-CD23 was lower at the higher temperatures. Analysis of (125)I-IgE binding to CD23(+)-Chinese hamster ovary cells also indicated that increased temperature resulted in a lower percentage of IgE capable of interacting with CD23. In contrast, IgE interacts more effectively with FcepsilonRI(+)-rat basophilic leukemia cells at 37 degrees C compared with 4 degrees C. The results support the concept that the open and closed IgE structures found by crystallography interact differently with the two IgE receptors and suggest that temperature influences the relative percentage of IgE in the respective structural forms. Changes in CD23 oligomerization also plays a role in the decreased binding seen at physiological temperatures.