Critical Regulators of B Cell Receptor Signaling

Cumulative evidence indicates that the CD19 coreceptor can induce positive signals that could enhance B cell responses. By regulating Src kinases and PI3K activity, it lowers the threshold of BcR-mediated signaling. Strikingly, study of CD19-/- mice revealed an apparent tight regulation of CD19 cell surface density during B cell development (Saito et al., 2002). In contrast to mice that overexpress CD19, CD19-/- mice have a markedly elevated BcR signaling threshold compared with wild-type mice. Reversibly, transgenic expression of low levels of human CD19 with normal levels of mouse CD19 resulted in hyperactive B cells and loss of tolerance to nuclear Ags (Sato etal., 2000). Since the product of PI3K, phosphatidyl inositol 3,4,5 trisphosphate, activates protein kinase B (PKB), which in turn promotes B cell survival, CD19 participates positively in B cell activation. Its positive effect on PI3K activity also may result in survival of immature B cells with low-affinity-binding BcRs, a potential source of the autoreac-tivity seen in mice that have only a 15-30% increase in CD19 expression. The CD19 transgenic mice exhibit a profoundly different phenotype from normal controls and develop SLE-like manifestations. Like mice that overexpress CD19, the tight skin (TSK) mouse, a genetic model for human systemic sclerosis, also contains spontaneously activated B cells and autoAbs against systemic sclerosis-specific target autoAgs. In TSK mice, CD19 deficiency results in quiescent B cells, with significantly reduced autoAb production and skin fibrosis (Saito et al., 2002). Thus, even subtle increases in CD19 density were sufficient to predispose mice to autoimmune manifestations, suggesting that modest alterations in CD19 expression could contribute to the development of autoAbs in humans.

The transmembrane CD45 PTP is expressed on all nucleated hemopoietic cells and constitutes up to 10% of all membrane proteins in T and B cells. It possesses an intracytoplasmic region bearing two domains, D1 and D2, and an extracellular domain with variable composition and structure, due to alternative splicing of several exons and differential glycosylation. The function of CD45 in immunore-ceptor signaling is to regulate the activity of Src family kinases by dephosphorylat-ing their regulatory C terminal tyrosine. It is thought that dimerization of CD45 inhibits PTP activity through symmetrical interactions between its inhibitory structural wedge and its catalytic site. Recently, the phenotype of a gain-of-function mutation in CD45 that may enhance activity of Src family PTKs has been reported (Majeti et al., 2000). Homozygous and heterozygous mice in which a single point mutation, glutamate 613 to arginine, that inactivates the inhibitory wedge of CD45 exhibited polyclonal lymphocyte activation, lymphoproliferation, autoAb production, and severe glomerulonephritis (GN), resulting in death. These findings demonstrate the in vivo importance of negative regulation of CD45 and suggest that the level of Src family PTK activity is an important determinant of immune tolerance.

Members of the novel serine/threonine protein kinase C (PKC) family, which includes PKC-8, -e, -0 and -n, can phosphorylate a multitude of cellular substrates. It is emerging that the different family members have diverse roles in the immune system and are implicated in various cellular processes, such as growth, differentiation, and death. Among the 12 known isoforms, several PKC

members are expressed in B lineage cells and activated by BcR stimulation, suggesting a contribution of PKCs in the transduction of B cell-mediated immune responses. One of them, PKC-8, is unique in that its overexpression can have a potent negative influence on cell behavior, inhibiting proliferation and growth and enhancing death. This isoform is known to be highly expressed in B cells and to be involved in BcR signaling. It has the unusual property of being tyro-sine-phosphorylated and this phosphorylation event takes place within a minute of BcR engagement. To understand its role in B cell immunity, mice deficient for PKC-8 were analyzed (Mecklenbrauker et al., 2002; Miyamoto et al., 2002). The most overt abnormalities were splenomegaly and lymphadenopathy, both attributed to an increase in the number of conventional B 2 cells. This augmentation was not observed in the bone marrow, indicating that it occurred in secondary lymphoid organs. In the absence of Ag stimulation, PKC-8-deficient mice exhibit B cell expansion, formation of unusually high numbers of numerous germinal centers (GCs), and defective B cell tolerance to self-Ag. With age, the B cell abnormalities had pathological consequences, including increased concentrations of serum IgG1 and IgA, autoAbs, immune-complex (IC)-type GN, and lymphocyte perivascular infiltration in many organs. Additionally, while acute exposure of PKC-8-deficient B cells to self-Ag elicits immunity, chronic stimulation with the same Ag failed to induce tolerance via anergy, as it does for wild-type B cells (Mecklenbrauker et al., 2002), further underscoring the importance of PKC-8 in negative regulation of B cell proliferation and in establishing B cell tolerance.

While Lyn is not required to initiate BcR signaling, it is an essential inhibitor of transduction pathways. B cells from mice homozygous for a disruption at the Lyn locus had a delayed, but increased, Ca2+ flux and an exaggerated negative selection response to Ag, and a spontaneous hyperactivity (Hibbs et al., 1995; Nishizumi et al., 1995). The deficient mice also had circulating autoreactive Abs and severe GN caused by the deposition of ICs in the kidney, a pathology reminiscent of SLE. The role of Lyn in BcR-mediated signaling and in establishing B cell tolerance to self-Ags also stems from the phenotype of Lyn deficiency on a C57BL/6 background. The mutant mice are more susceptible to myelin oligodendrocyte glycoprotein-induced experimental allergic encephalo-myelitis (Du and Sriram, 2002). This strain dependence suggests that Lyn may represent a genetic susceptibility factor for autoimmune disease.

Further analysis of signaling pathways in Lyn-/- mice demonstrated that regulation of BcR signaling is a complex quantitative trait in which the Lyn activating role is mediated by the phosphorylation of tyrosine residues within immunoreceptor-activating motifs (ITAMs) of Iga, IgP and CD19, and the subsequent recruitment of signaling enzymes, such as Syk, PLCy2, and PI-3Kinase (Cornall et al., 1998). These positive effects are balanced by the negative regulatory role of Lyn in B cells through at least two pathways. First, Lyn is responsible for phosphorylating tyrosine residues of the negative BcR coreceptors FcyRIIb, platelet endothelial cell adhesion molecule-1 (PECAM-1), and PD-1 (see below). This activity may account, at least in part, for the enhanced BcR signaling seen in Lyn-/- B cells and for the autoimmune phenotype observed in mice lacking these BcR coreceptors. Second, Lyn was shown to negatively regulate

B cells by opposing the effect of Syk on BcR-mediated activation of Akt/PKB. Deregulation of Akt/PKB correlates with the BcR-mediated hyperresponsiveness of Lyn-/- B cells and might contribute to the autoimmune syndrome that develops in Lyn-deficient animals.

The key role of Lyn in establishing and maintaining peripheral tolerance also comes from studies of the consequences of sustained activation of Lyn in vivo using a targeted gain-of-function mutation (Hibbs et al., 2002). The mice, designated Lynup/up, carry a single point mutation (Y508) in a sequence that negatively regulates Lyn activity and express a constitutively activated form of Lyn, allowing study of the consequences of constitutive engagement of both stimulatory and inhibitory signaling pathways. The mutant mice have reduced numbers of conventional B-2 lymphocytes, downregulated surface IgM and costimulatory molecules, and elevated numbers of B-1a cells. In vitro, there is a heightened Ca2+ flux in response to BcR stimulation and exaggerated positive signaling. While there is a constitutive phosphorylation of negative regulators of BcR signaling (SHP-1 and SHIP-1), Syk and phospholipase Cy2 are constitutively phosphorylated. Surprisingly, Lynup/up mice developed circulating autoreactive Abs and lethal autoimmune GN, suggesting that enhanced positive signaling eventually overrides constitutive negative signaling. The breakdown of self-tolerance seen in these mice may result from an imbalance between chronic negative signaling and enhanced positive signaling. It indicates that Lyn plays a central role in maintaining the equilibrium between positive and negative B cell signaling pathways, and in regulating B cell tolerance and development of autoimmunity. Moreover, the fact that both Lyn-/- and Lynup/up mice show a breakdown in self-tolerance and develop circulating autoreactive autoAbs and severe lupus-like nephritis (Hibbs et al., 1995, 2002; Nishizumi et al., 1995) clearly demonstrates that Lyn is a key regulator of B cell signaling and suggests that any imbalance in signaling, either by deletion on activation, may result in severe autoimmunity.

Also important in BcR signaling and in self-tolerance is SHP-1, a PTP with several targets in immunoreceptor signaling. SHP-1 is reportedly recruited to CD22 or CD72, even in the absence of BcR coaggregation. It also can dephosphorylate the ITAMs of Src kinases, Syk kinases, adapters such as SLP-76, effectors such as Vav and PI-3' kinase, as well as receptors such as CD19. There also is strong indication that SHP-1 can dephosphorylate immunoreceptor inhibitory motif (ITIM)-containing receptors (see below), thereby providing a potential mechanism of autoregulation (Reth and Wienands, 1997; Benschop and Cambier, 1999). A significant clue regarding the role of SHP-1 in immune cell homeostasis was provided by the finding that its gene is mutated in motheaten (me) and viable motheaten (mev) mice (Shultz et al., 1993; Tsui et al., 1993). The mutant mice exhibit a spontaneous point mutation in SHP-1 that results in a protein with decreased PTP activity (10-20% of normal activity) and immune defects, including expansion of B-1 cells, a low threshold of membrane Ig signaling, hypergammaglobulinemia, autoantibody production, and GN. Their B cells exhibit augmented BcR-induced proliferation, protein tyrosine phosphorylation, and mitogen-activated protein kinase (MAPK) activation. Similar defects were observed in a B cell line expressing a dominantnegative form of SHP-1. Thus, SHP-1 is a critical negative regulator of immunore-ceptor signaling in B cells.

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