Impairment of PD-L2 positive B1a cells enhances susceptibility to sepsis in RasGRP1-deficient mice.

RasGRP1 is a key molecule that mediates antigen-initiated signaling for activation of the RAS-MAPK pathway in lymphocytes. Patients with aberrant RasGRP1 expression experience lymphocyte dysfunction and are afflicted with recurrent microbial infections. Yet, the underlying mechanism that accounts for microbial infection remains unknown. We previously reported that B1a cells are heterogeneous with respect to PD-L2 expression and that RasGRP1 deficiency preferentially impairs PD-L2+ B1a cell development. In the present study, we show that PD-L2+ B1a cells exhibit increased capacity for differentiation to CD138+ plasma cells that secrete natural IgM antibody, as well as IL-10 and GM-CSF, in response to TLR stimulation. In keeping with this, we show here that RasGRP1-deficent mice are much more susceptible to septic infection triggered by cecalligation and puncture than wild type mice, and that reconstitution of RasGRP1-deficient mice with wild type PD-L2+ B1a cells greatly rescues RasGRP1-deficient mice from sepsis. Thus, this study indicates a mechanism for the association of RasGRP1 deficiency with predispostion to infection in the loss of a particular B1a subpopulation.

IL-4 rescues surface IgM expression in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) cells express poor levels of surface immunoglobulin (sIg), and many are minimally activated or anergic in response to B-cell receptor (BCR) crosslinking in vitro. Paradoxically, CLL cells in patients are highly activated through BCR signaling and expand in proliferation centers, suggesting that the function of sIg signaling is rescued. Here, we find that, compared with normal naïve B cells, CLL cells express a low level of total CD79b protein but normal levels of CD79a and IgM protein. Association of both CD79a and CD79b to IgM is markedly reduced. We further find that interleukin-4 (IL-4) markedly rescues CD79b and sIgM protein in CLL samples. These changes significantly enhance signaling in response to BCR crosslinking. Furthermore, we find that these changes are more pronounced in immunoglobulin heavy chain variable (IGHV)-unmutated CLL cells than IGHV-mutated CLL cells. The results described herein reveal that reduced sIgM is due to low expression of total CD79b protein in CLL cells. IL-4 substantially restores CD79b protein expression, sIgM expression, and BCR signaling.

RasGRP1 Is an Essential Signaling Molecule for Development of B1a Cells with Autoantigen Receptors.

B1a cells, particularly the PD-L2(+) B1a cell subset, are enriched with autoantigen-specific receptors. However, the underlying molecular mechanism responsible for the skewed selection of autoreactive B1a cells remains unclear. In this study, we find that B1 cells express only Ras guanyl nucleotide-releasing protein (RasGRP) 1, whereas B2 cells express mostly RasGRP3 and little RasGRP1. RasGRP1 is indispensable for transduction of weak signals. RasGRP1 deficiency markedly impairs B1a cell development and reduces serum natural IgM production; in particular, B1a cells that express autoantigen receptors, such as anti-phosphatidylcholine B1a cells, are virtually eliminated. Thus, unlike Btk and other signalosome components, RasGRP1 deficiency selectively affects only the B1a cell population with autoantigen receptors rather than the entire pool of B1a cells.

The osteopontin transgenic mouse is a new model for Sjögren's syndrome.

Osteopontin (Opn) is a cytokine involved in both physiological and pathological processes, and is elevated in many autoimmune diseases. Sjögren's syndrome (SS) is an autoimmune disease with a strong female predilection characterized by lymphocytic infiltration of exocrine glands. We hypothesized that Opn contributes to SS pathogenesis. We examined an established SS model and found increased Opn locally and systemically. Next, we examined Opn transgenic (Opn Tg) mice for evidence of SS. Opn Tg animals exhibited lymphocytic infiltration of salivary and lacrimal glands, and Opn co-localized with the infiltrates. Moreover, saliva production was reduced, and SS autoantibodies were observed in the serum of these mice. Finally, female Opn Tg mice showed more severe disease compared to males. Taken together, these data support a role for Opn in SS pathogenesis. We identify a new model of spontaneous SS that recapitulates the human disease in terms of sex predilection, histopathology, salivary deficits, and autoantibodies.

IL-4 upregulates Igα and Igß protein, resulting in augmented IgM maturation and B cell receptor-triggered B cell activation.

IL-4 is critical for optimal B cell activation and germinal center B cell expansion in T-dependent immune responses; however, the underlying mechanism remains elusive. In the current study, we found that primary B cells express little Igα and Igß protein despite substantial levels of mRNA. IL-4 markedly upregulates Igα and Igß protein expression that requires STAT6. Elevated Igα and Igß protein form heterodimers that associate with IgM and significantly promote IgM maturation and surface IgM expression, resulting in amplified BCR-initiated signaling that is Lyn dependent. In vivo, we found that pregerminal center B cells express upregulated Igα, Igß, and surface IgM expression, in conjunction with elevated BCR-triggered phosphorylated ERK ex vivo, that are dependent on IL-4 and reversed by in vivo administration of neutralizing anti-IL-4 Ab. Thus, this study elucidates a novel mechanism for cross-talk between the IL-4 and BCRs that programs enhancement of subsequent BCR signaling.

A novel Lyn-protein kinase Cδ/ε-protein kinase D axis is activated in B cells by signalosome-independent alternate pathway BCR signaling.

BCR signaling initiates multiple activities critical for B-cell function. Recently, we identified an alternate BCR signaling pathway, induced by IL-4, that is signalosome-independent, unlike the classical signalosome-dependent pathway, and that leads to activation of the MAP kinase, ERK. Here we questioned whether alternate pathway signaling extends to other key downstream events, especially protein kinase D (PKD) activation. We found that in murine spleen-derived B cells the IL-4-induced alternate pathway for BCR signaling results in PKD and PKD substrate phosphorylation, and that alternate pathway phosphorylation of HDAC5/7 and other key substrates requires PKD. Furthermore, we found that tyrosine phosphorylation of PKCδ/ε occurs as a result of alternate but not classical pathway signaling and is required for phosphorylation of PKD and PKD substrates. This result identifies PKCδ/ε tyrosine phosphorylation as a unique outcome of the alternate pathway. The alternate pathway is mediated by Lyn that is not required for classical pathway signaling and we found that Lyn associates directly with PKCδ/ε and is required for phosphorylation of PKCδ/ε and of PKD. These findings indicate that IL-4 influences B-cell activation by inducing a novel signaling pathway from BCR to Lyn to PKCδ/ε to PKD.

B cell receptor crosstalk: B cells express osteopontin through the combined action of the alternate and classical BCR signaling pathways.

Classical BCR signaling requires a number of signalosome mediators that are bypassed when BCR signaling follows an alternate pathway produced by prior exposure of B cells to IL-4. The two pathways, classical and alternate, co-exist in IL-4-treated B cells. Here we report that operation of the IL-4-induced alternate pathway in combination with the classical pathway changes the nature of the B cell response to BCR engagement so that the cytokine, osteopontin (Opn), is produced and secreted. Although Opn expression by B cells has not previously been noted, anti-Ig-induced secretion by IL-4-treated B cells amounts to levels comparable to those secreted by activated T cells. However, unlike T cell Opn expression, B cell expression of Opn is not mediated by T-Bet. Because elevated levels of IL-4 occur in association with severe illness, and because Opn is strongly associated with autoimmunity, these results suggest that the IL-4-induced alternate BCR signaling pathway may participate in the pathophysiology of autoimmune dyscrasias.

B cell receptor cross-talk: exposure to lipopolysaccharide induces an alternate pathway for B cell receptor-induced ERK phosphorylation and NF-kappa B activation.

BCR signaling in naive B cells depends on the function of signalosome mediators; however, prior engagement of CD40 or of IL-4R produces an alternate signaling pathway in which Bruton's tyrosine kinase, PI3K, phospholipase Cgamma2, and protein kinase Cbeta are no longer required for BCR-induced downstream events. To explore the range of mediators capable of producing such an alternate pathway for BCR signaling, we examined the TLR4 agonist, LPS. B cell treatment with LPS at relatively low doses altered subsequent BCR signaling such that ERK phosphorylation and NF-kappaB activation occurred in a PI3K-independent manner. This effect of LPS extended to MEK phosphorylation and IkappaBalpha degradation, and it developed slowly over a period of 16-24 h. The involvement of TLRs is suggested by similar effects observed with a structurally distinct TLR agonist, PAM3CSK4 and by the need for MyD88 for induction of alternate BCR signaling by LPS. Thus, LPS-mediated TLR engagement produces an alternate pathway for BCR-triggered signal propagation that differs from the classical, signalosome-dependent pathway.

Cutting Edge: B cell receptor (BCR) cross-talk: the IL-4-induced alternate pathway for BCR signaling operates in parallel with the classical pathway, is sensitive to Rottlerin, and depends on Lyn.

B cell exposure to IL-4 alters subsequent BCR signaling such that ERK phosphorylation becomes signalosome-independent; however, the nature of this new, alternate signaling pathway and its relationship to the classical, signalosome-dependent signaling pathway are not known. In this study, we report that the alternate and classical pathways for BCR signaling are differentially affected by rottlerin, and by Go6976 or LY294002, respectively. Furthermore, in B cells lacking protein kinase C (PKC)beta, the classical pathway for BCR signaling is blocked, whereas the alternate pathway is little affected. Conversely, in B cells lacking Lyn, the alternate pathway for BCR signaling is blocked, whereas the classical pathway is little affected. The rottlerin-sensitive element is not PKCdelta, inasmuch as the alternate pathway is not blocked in PKCdelta-deficient B cells. These results indicate that the rottlerin-sensitive, Lyn-dependent alternate pathway, and the classical pathway, for BCR signaling operate in parallel when BCR engagement follows IL-4 exposure.

B cell receptor (BCR) cross-talk: IL-4 creates an alternate pathway for BCR-induced ERK activation that is phosphatidylinositol 3-kinase independent.

IL-4 has pleiotropic effects on B cells. These effects include alteration of subsequent BCR-triggered responses. To identify a molecular basis for this receptor cross-talk, we examined ERK activation and NF-kappaB induction. We found that treatment with IL-4, but not other cytokines, affected subsequent BCR signaling by creating a new pathway in which the need for PI3K in ERK activation was eliminated. In contrast, the need for PI3K in NF-kappaB induction was not altered. The new pathway for ERK required time to develop, depended on STAT6, and was blocked by inhibition of macromolecular synthesis. As in the classical pathway, BCR-induced ERK activation in the new, PI3K-independent pathway required MEK and was reflected in c-Raf. Thus, IL-4 promotes an alternate pathway through which BCR is coupled to Raf/MEK/ERK that may function to heighten the responsiveness of B cells during times of immunological stress.