CD19-CD21 complex regulates an intrinsic Src family kinase amplification loop that links innate immunity with B-lymphocyte intracellular calcium responses.

CD19 is a B-lymphocyte cell surface molecule that functions as a general response regulator or rheostat, which defines intrinsic and B-cell antigen receptor-induced signalling thresholds that are critical for humoral immunity and expansion of the peripheral B-cell pool. In addition, B-cell responses are influenced by signals transduced through a CD19-CD21 cell surface receptor complex, where the binding of complement C3d to CD21 links humoral immune responses with the innate immune system. This review outlines recent biochemical and genetic studies that characterize the signal transduction pathways utilized by this receptor complex to regulate B-cell intracellular calcium responses.

CD19 amplification of B lymphocyte Ca2+ responses: a role for Lyn sequestration in extinguishing negative regulation.

B lymphocyte antigen receptor (BCR) signals are regulated by CD19, with BCR-induced intracellular calcium ([Ca(2+)](i)) responses enhanced by CD19 co-ligation. In this study, CD19 engagement using a dimeric anti-CD19 antibody induced [Ca(2+)](i) mobilization and significantly enhanced BCR-induced [Ca(2+)](i) responses without a requirement for CD19/BCR co-ligation. Although simultaneous CD19 and BCR engagement significantly enhanced CD19/Lyn complex formation and [Ca(2+)](i) responses, downstream tyrosine phosphorylation of CD22 and multiple other cellular proteins was inhibited, as was SHP1 recruitment to phosphorylated CD22. CD19 overexpression also enhanced BCR-induced [Ca(2+)](i) responses, but down-regulated tyrosine phosphorylation of CD22 and multiple other cellular proteins following BCR ligation. Because CD19 and Lyn expression are genetically titrated in B cells, CD19 engagement may augment BCR-induced [Ca(2+)](i) responses by sequestering the available pool of functional Lyn away from downstream negative regulatory proteins such as CD22. Consistent with this, simultaneous CD19 engagement did not further enhance the BCR-induced [Ca(2+)](i) responses of Lyn- or CD22-deficient B cells. Thus, CD19 recruitment of Lyn may preferentially activate selective signaling pathways downstream of the CD19/Lyn complex to the exclusion of other downstream regulatory and effector pathways. Other receptors may also utilize a similar strategy to regulate kinase availability and downstream intermolecular signaling.

CD19 can regulate B lymphocyte signal transduction independent of complement activation.

B lymphocytes are critically regulated by signals transduced through the CD19-CD21 cell surface receptor complex, where complement C3d binding to CD21 supplies an already characterized ligand. To determine the extent that CD19 function is controlled by complement activation, CD19-deficient mice (that are hyporesponsive to transmembrane signals) and mice overexpressing CD19 (that are hyperresponsive) were crossed with CD21- and C3-deficient mice. Cell surface CD19 and CD21 expression were significantly affected by the loss of CD21 and C3 expression, respectively. Mature B cells from CD21-deficient littermates had approximately 36% higher cell surface CD19 expression, whereas CD21/35 expression was increased by approximately 45% on B cells from C3-deficient mice. Negative regulation of CD19 and CD21 expression by CD21 and C3, respectively, may be functionally significant because small increases in cell surface CD19 overexpression can predispose to autoimmunity. Otherwise, B cell development and function in CD19-deficient and -overexpressing mice were not significantly affected by a simultaneous loss of CD21 expression. Although CD21-deficient mice were found to express a hypomorphic cell surface CD21 protein at low levels that associated with mouse CD19, C3 deficiency did not significantly affect B cell development and function in CD19-deficient or -overexpressing mice. These results, and the severe phenotype exhibited by CD19-deficient mice compared with CD21- or C3-deficient mice, collectively demonstrate that CD19 can regulate B cell signaling thresholds independent of CD21 engagement and complement activation.

A CD19-dependent signaling pathway regulates autoimmunity in Lyn-deficient mice.

CD19 and the Src family protein tyrosine kinases (PTKs) are important regulators of intrinsic signaling thresholds in B cells. Regulation is achieved by cross-talk between Src family PTKs and CD19; Lyn is essential for CD19 phosphorylation, while CD19 establishes an Src family PTK activation loop that amplifies kinase activity. However, CD19-deficient (CD19(-/-)) B cells are hyporesponsive to transmembrane signals, while Lyn-deficient (Lyn(-/-)) B cells exhibit a hyper-responsive phenotype resulting in autoimmunity. To identify the outcome of interactions between CD19 and Src family PTKs in vivo, B cell function was examined in mice deficient for CD19 and Lyn (CD19/Lyn(-/-)). Remarkably, CD19 deficiency suppressed the hyper-responsive phenotype of Lyn(-/-) B cells and autoimmunity characterized by serum autoantibodies and immune complex-mediated glomerulonephritis in Lyn(-/-) mice. Consistent with Lyn and CD19 each regulating conventional B cell development, B1 cell development was markedly reduced by Lyn deficiency, with further reductions in the absence of CD19 expression. Tyrosine phosphorylation of Fyn and other cellular proteins induced following B cell Ag receptor ligation was dramatically reduced in CD19/Lyn(-/-) B cells relative to Lyn(-/-) B cells, while Syk phosphorylation was normal. In addition, the enhanced intracellular Ca(2+) responses following B cell Ag receptor ligation that typify Lyn deficiency were delayed by the loss of CD19 expression. BCR-induced proliferation and humoral immune responses were also markedly inhibited by CD19/Lyn deficiency. These findings demonstrate that while the CD19/Lyn amplification loop is a major regulator of signal transduction thresholds in B lymphocytes, CD19 regulation of other Src family PTKs also influences B cell function and the development of autoimmunity.

CD19, CD21, and CD22: multifaceted response regulators of B lymphocyte signal transduction.

B lymphocyte development and function depend upon the activity of intrinsic and B cell antigen receptor (BCR)-induced signals. These signals are interpreted, amplified, fine-tuned, or suppressed through the precise actions of specialized cell surface coreceptors, or "response regulators," that inform B cells of their extracellular environment. Important cell surface response regulators include the CD19/CD21 complex, CD22, and CD72. CD19 establishes a novel Src-family protein tyrosine kinase (PTK) amplification loop that regulates basal signaling thresholds and intensifies Src-family PTK activation following BCR ligation. In turn, CD22 limits the intensity of CD19-dependent, BCR-generated signals through the recruitment of potent phosphotyrosine and phosphoinositide phosphatases. Herein we discuss our current understanding of how CD19/CD21 and CD22 govern the emergence and intensity of BCR-mediated signals, and how alterations in these tightly controlled regulatory activities contribute to autoimmunity in mice and humans.

CD19 regulates Src family protein tyrosine kinase activation in B lymphocytes through processive amplification.

CD19 regulates constitutive and antigen receptor-induced signaling thresholds in B lymphocytes through its unique cytoplasmic domain. Herein, we demonstrate a novel molecular mechanism where interactions between CD19 and Lyn amplify basal and antigen receptor-induced Src family kinase activation. Lyn expression was required for CD19 tyrosine phosphorylation in primary B cells. Experiments with purified proteins demonstrated that CD19-Y513 was Lyn's initial phosphorylation and binding site. This led to processive phosphorylation of CD19-Y482, which recruited a second Lyn molecule, allowing for transphosphorylation and amplification of Lyn activation. In vivo, CD19 deficiency impaired, and CD19 overexpression enhanced, Lyn kinase activity. Thus, CD19 functions as a specialized adapter protein for the amplification of Src family kinases that is crucial for intrinsic and antigen receptor-induced signal transduction.

CD19 and CD22 regulate a B lymphocyte signal transduction pathway that contributes to autoimmunity.

The fate of B lymphocytes is dependent on intrinsic and B cell antigen receptor (BCR)-induced signals. These signals are modified and interpreted by other cell-surface molecules such as CD19 and CD22 that govern mature B cell activation. This review assesses our current understanding of how CD19 and CD22 regulate B lymphocyte signaling and how alterations in these response-regulators contribute to autoimmunity in mice and humans. We propose that CD19 functions as a specialized adapter protein that regulates B lymphocyte signaling and autoantibody production. Overexpression of CD19 by B cells in systemic sclerosis patients correlates with autoantibody production and transgenic mice that overexpress CD19 produce similar autoantibodies. CD19 establishes a novel Src-family kinase activation loop that regulates basal signal transduction thresholds in resting B cells and amplifies Src-family kinase activation following BCR ligation. Reciprocally, CD22 is a potent regulator of CD19 function. These observations provide insight into how CD19 and CD22 govern the molecular ordering and intensity of signals transduced in B cells that may contribute to autoimmunity.

CD22 forms a quaternary complex with SHIP, Grb2, and Shc. A pathway for regulation of B lymphocyte antigen receptor-induced calcium flux.

CD22 is a cell surface molecule that regulates signal transduction in B lymphocytes. Tyrosine-phosphorylated CD22 recruits numerous cytoplasmic effector molecules including SHP-1, a potent phosphotyrosine phosphatase that down-regulates B cell antigen receptor (BCR)- and CD19-generated signals. Paradoxically, B cells from CD22-deficient mice generate augmented intracellular calcium responses following BCR ligation, yet proliferation is decreased. To understand further the mechanisms through which CD22 regulates BCR-dependent calcium flux and proliferation, interactions between CD22 and effector molecules involved in these processes were assessed. The adapter proteins Grb2 and Shc were found to interact with distinct and specific regions of the CD22 cytoplasmic domain. Src homology-2 domain-containing inositol polyphosphate-5'-phosphatase (SHIP) also bound phosphorylated CD22, but binding required an intact CD22 cytoplasmic domain. All three molecules were bound to CD22 when isolated from BCR-stimulated splenic B cells, indicating the formation of a CD22.Grb2.Shc.SHIP quaternary complex. Therefore, SHIP associating with CD22 may be important for SHIP recruitment to the cell surface where it negatively regulates calcium influx. Although augmented calcium responses in CD22-deficient mice should facilitate enhanced c-Jun N-terminal kinase (JNK) activation, BCR ligation did not induce JNK activation in CD22-deficient B cells. These data demonstrate that CD22 functions as a molecular "scaffold" that specifically coordinates the docking of multiple effector molecules, in addition to SHP-1, in a context necessary for BCR-dependent SHIP activity and JNK stimulation.

CD19 regulates intrinsic B lymphocyte signal transduction and activation through a novel mechanism of processive amplification.

The fate of B lymphocytes is dependent on intrinsic and B cell antigen receptor (BCR)-induced signals. These signals are interpreted and modified by response regulators such as CD19 that govern mature B cell activation. The current understanding of how CD19 governs B lymphocyte signaling is outlined in this review. Primarily, CD19 establishes a novel Src-family kinase amplification loop that regulates basal signal transduction thresholds in resting B cells. Moreover, CD19 amplifies Src-family kinase activation following BCR ligation. CD19 amplification of Lyn activity leads to processive phosphorylation of CD19 and downstream substrates including CD22. Phosphorylated CD19 recruits other effector molecules including Vav, Grb2, phosphoinositide 3-kinase, phospholipase Cgamma2, and c-Abl, which may contribute to CD19 regulation of B cell function. CD19/Lyn complex formation also regulates phosphorylation of CD22 and FcgammaRIIB, which inhibit B cell signal transduction through the recruitment of the SHPI and SHIP phosphatases. These observations provide insight into how CD19 governs the molecular ordering and intensity of signals transduced in B cells, and how perturbations in CD19 expression or signaling function may contribute to autoimmunity.

Modulation of B lymphocyte antigen receptor signal transduction by a CD19/CD22 regulatory loop.

CD19 and CD22 are B lymphocyte cell-surface molecules that positively and negatively regulate antigen receptor signal transduction, respectively. Biochemical studies with B cells from CD19-deficient and CD22-deficient mice indicated that these two regulatory molecules influenced each other's functions: CD22 expression negatively regulated CD19 tyrosine phosphorylation, while optimal CD22 function was dependent on CD19 expression. Functional CD19 and CD22 interactions were also assessed in vivo by generating CD19/CD22 double-deficient mice. Remarkably, the CD19 mutation was dominant to the CD22 mutation in most instances. B lymphocytes from CD19/CD22-deficient and CD19-deficient mice were functionally equivalent despite the negative influence normally provided by CD22 expression. These data collectively suggest that CD19 activates the CD22/SHP1 inhibitory pathway that then acts primarily on CD19.

CD19 amplifies B lymphocyte signal transduction by regulating Src-family protein tyrosine kinase activation.

Ligation of the B cell Ag receptor (BCR) induces cellular activation by stimulating Src-family protein tyrosine kinases (PTKs) to phosphorylate members of the BCR complex. Subsequently, Src-family PTKs, particularly Lyn, are proposed to phosphorylate and bind CD19, a cell-surface costimulatory molecule that regulates mature B cell activation. Herein, we show that B cells from CD19-deficient mice have diminished Lyn kinase activity and BCR phosphorylation following BCR ligation. Tyrosine phosphorylation of other Src-family PTKs was also decreased in CD19-deficient B cells. In wild-type B cells, CD19 was constitutively complexed with Vav, Lyn, and other Src-family PTKs, with CD19 phosphorylation and its associations with Lyn and Vav increased after BCR ligation. Constitutive CD19/Lyn/Vav complex signaling may therefore be responsible for the establishment of baseline signaling thresholds in B cells before Ag receptor ligation, in addition to accelerating signaling following BCR engagement or other transmembrane signals. In vitro kinase assays using purified CD19 and purified Lyn revealed that the kinase activity of Lyn was significantly increased when coincubated with CD19. Thus, constitutive and induced CD19/Lyn complexes are likely to regulate basal signaling thresholds and BCR signaling by amplifying the kinase activity of Lyn and other Src-family PTKs. These in vivo and in vitro findings demonstrate a novel mechanism by which CD19 regulates signal transduction in B lymphocytes. The absence of this CD19/Src-family kinase amplification loop may account for the hyporesponsive phenotype of CD19-deficient B cells.

CD40 signaling of monocyte inflammatory cytokine synthesis through an ERK1/2-dependent pathway. A target of interleukin (il)-4 and il-10 anti-inflammatory action.

Ligation of CD40 on monocytes through its interaction with CD40 ligand (CD154) present on activated T helper cells, results in activation of monocyte inflammatory cytokine synthesis and rescue of monocytes from apoptosis induced through serum deprivation. Both of these consequences of CD40 stimulation have been shown to be dependent on the induction of protein tyrosine kinase activity. CD40-mediated activation of protein tyrosine kinase activity and subsequent inflammatory cytokine production are abrogated by treatment of monocytes with the T helper type 2 cytokines interleukin 4 (IL-4) and interleukin 10 (IL-10). In the current study we demonstrate that stimulation of monocytes through CD40 resulted in the phosphorylation and activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein kinases, whereas phosphorylation of mitogen-activated protein kinases family members p38 and c-Jun N-terminal kinase was not observed in response to this stimuli over the time course examined. PD98059, an inhibitor of the upstream activator of ERK1/2, the MAP/ERK kinase MEK1/2, suppressed IL-1beta and tumor necrosis factor-alpha production in a dose-dependent fashion. Pretreatment of monocytes with IL-4 and IL-10 inhibited CD40-mediated activation of ERK1/2 kinase activity when used individually, and are enhanced in effectiveness when used in combination. Together, the data demonstrate that CD40-mediated induction of IL-1beta and tumor necrosis factor-alpha synthesis is dependent on a MEK/ERK pathway which is obstructed by signals generated through the action of IL-4 and IL-10.

CD19 regulates B lymphocyte responses to transmembrane signals.

CD19 is a component of a cell surface receptor complex that regulates B lymphocyte responses to transmembrane signals including those generated through the B cell antigen receptor. Studies in mice which lack or overexpress CD19 show that changes in CD19 expression levels have significant effects on B cell development and function. Recent studies suggest that CD19 establishes a Src-family kinase activation loop that amplifies tyrosine phosphorylation of numerous downstream effector molecules including potentially positive and negative regulatory elements. These observations provide an understanding of how CD19 governs the molecular ordering and intensity of signals transduced through multiple B cell receptors.

IL-4 and IL-10 modulation of CD40-mediated signaling of monocyte IL-1beta synthesis and rescue from apoptosis.

Previous studies have demonstrated that the interaction of CD40 on monocytes with CD40 ligand, present on activated CD4+ T cells, induces monocyte inflammatory cytokine synthesis and rescues monocytes from apoptosis. These findings suggest a role for CD40 signaling of monocyte activation in the maintenance and/or exacerbation of nonseptic (e.g., autoimmune) inflammatory responses. In the present study the effects of the modulatory cytokines IL-4 and IL-10 on CD40-mediated signaling of monocyte IL-1beta synthesis and rescue from apoptosis were examined. Both IL-4 and IL-10 decreased CD40-dependent IL-1beta synthesis in a dose-dependent manner individually and synergized in this effect when used concurrently, with minimal effect on CD40 surface expression. CD40 signaling of IL-1beta synthesis was shown to be dependent on the induction of protein tyrosine kinase (PTK) activity, and both IL-4 and IL-10 diminished CD40-mediated tyrosine phosphorylation of monocyte cellular proteins. However, IL-4, but not IL-10, blocked CD40-mediated rescue from apoptosis, an event that we have demonstrated previously to be dependent on PTK activity as well. Together these results suggest that in monocytes 1) both IL-4 and IL-10 target CD40-induced PTK activity in the down-regulation of IL-1beta synthesis; and 2) IL-4 and IL-10 have divergent effects on the CD40 signaling pathway, in that these cytokines are synergistic with respect to their abilities to inhibit CD40-mediated IL-1beta synthesis and differ in their abilities to block CD40-mediated rescue from apoptosis.

T cell rescue of monocytes from apoptosis: role of the CD40-CD40L interaction and requirement for CD40-mediated induction of protein tyrosine kinase activity.

Circulating monocytes have a limited life span and will undergo apoptosis in the absence of specific stimuli. Recent studies have demonstrated that monocytes can be rescued from apoptosis via lipopolysaccharide (LPS) activation or stimulation with interleukin-1 or tumor necrosis factor-alpha. Based on previous studies from our laboratory, we hypothesized that, in nonseptic (e.g., autoimmune) inflammation, the presence of activated T cells may enhance monocyte longevity through T cell contact-dependent signaling. Plasma membranes prepared from 6 h activated (TmA) and resting (TmR) purified CD4+ T cells were added to resting elutriation-purified monocytes cultured in serum-free medium. Cells were assayed for degree of apoptosis occurring over a 72-h incubation using both agarose gel electrophoresis and flow cytometry. The addition of TmA (but not TmR) was capable of blocking monocyte apoptosis and the ability of TmA to rescue monocytes was abrogated by the addition of anti-CD40L antibodies. Rescue of monocytes from apoptosis could also be mediated by direct cross-linking of monocyte CD40. Inhibitors of tyrosine kinase activity blocked both TmA and anti-CD40-mediated rescue of monocytes from apoptosis, suggesting a primary role of a tyrosine kinase signaling pathway in the events controlling monocyte longevity.