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Altered B-cell signaling in lupus
Autoimmunity Reviews 8 (2009) 214–218
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Autoimmunity Reviews j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t r ev
Altered B-cell signaling in lupus Kui Liu ⁎, Chandra Mohan ⁎ Division of Rheumatology, and Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390-8884, USA
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Available online 21 August 2008 Keywords: B cell Lupus Signaling
a b s t r a c t Systemic lupus erythematosus (SLE) is an autoimmune disease of complex etiology primarily characterized by the presence of high titers of autoantibodies targeting many nuclear as well as cytoplasmic antigens, with resultant end-organ damage. Aberrant signaling events have been documented in various lymphocyte populations, and they have constituted attractive targets for therapeutic intervention. Murine models of lupus (conventional or engineered) have yielded interesting snapshots of the signaling status of lupus lymphocytes, and many of these alterations in cell signaling observed in murine models of lupus have also been documented in patient samples. Analyses of B-cell signaling in various murine lupus models have not only provided an in-depth perspective of the signaling status and possibly the underlying mechanisms leading to enhanced survival of autoimmune B cells, but have also presented us with potential strategies for treating lupus. © 2008 Published by Elsevier B.V.
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Altered B-cell signaling in SLE patients. . . . . . . . . . . . . . . . . . . . . . . 3. Altered B-cell signaling in conventional murine lupus models . . . . . . . . . . . . 4. Altered B-cell signaling in genetically simplified congenic models of lupus . . . . . . 5. Lessons from engineered mouse models . . . . . . . . . . . . . . . . . . . . . . 6. Inhibition of systemic autoimmunity by targeting hyperactivated signaling pathways . Take-home messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease of complex etiology primarily characterized by the presence of high titers of autoantibodies targeting many nuclear as well as cytoplasmic antigens, with resultant end-
⁎ Corresponding authors. Department of Internal Medicine/Rheumatology UT Southwestern Medical Center, Mail Code 8884, Y8.204, 5323 Harry Hines Boulevard Dallas, TX 75390-8884, USA. Tel.: +1 214 648 9675; fax: +1 214 648 7995. E-mail addresses: [email protected] (K. Liu), [email protected] (C. Mohan). 1568-9972/$ – see front matter © 2008 Published by Elsevier B.V. doi:10.1016/j.autrev.2008.07.048
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organ damage. Antibody and immune-complex-mediated inflammatory processes in this disease can lead to the development of glomerulonephritis, dermatitis, serositis, and vasculitis. It is estimated to affect about 1 in 2000 people with a strong gender bias (female to male ratio is about 9:1). Furthermore, African-Americans and Hispanics are approximately 2 to 4 times more likely to develop this disease than Caucasians [1–3]. Both genetic and environmental factors contribute to its pathogenesis [1–3]. Although deficiencies in some of the early components of the complement cascade, such as C1 and C4, can lead to the development of SLE, this disease is typically polygenic in origin [1–5]. Studies from both SLE patients and mouse models indicate that production of antinuclear antibodies (ANA) is a key feature
K. Liu, C. Mohan / Autoimmunity Reviews 8 (2009) 214–218
of the dysregulated immune system in lupus. The emergence of ANAs in these studies is typically associated with quantitative and qualitative alterations in B-cell and/or T cell populations and a breach of immune tolerance at some level. Aberrant signaling has been reported in both of these cell populations in lupus [6,7], and these changes undoubtedly constitute attractive targets for therapeutic intervention. In this regard, murine models of lupus, both conventional models as well as the more recently studied genetically simplified congenic models of lupus, have collectively yielded interesting snapshots of the signaling status of lupus lymphocytes. Importantly, many of these alterations in cell signaling observed in murine models of lupus have also been documented in patient samples. This review is aimed at summarizing recent progress in this area, with a specific focus on B cells. 2. Altered B-cell signaling in SLE patients Cross-linking of antigen receptors on lymphocytes is a critical trigger in initiating downstream intracellular events that involve an intricate cascade of signaling molecules. These events lead to various outcomes, including activation/proliferation, inhibition/apoptosis, and cytokine secretion. In B lymphocytes, clustering of the B-cell receptor (BCR) leads to activation of protein tyrosine kinases, such as Lyn, which phosphorylate crucial immunoreceptor tyrosine-based activation motif (ITAM) tyrosine residues on the BCR-associated Igα and Igβ signaling molecules, as reviewed elsewhere [8]. Phosphorylation of ITAM mediates the recruitment and activation of the tyrosine kinase Syk, which initiates several downstream signaling pathways, including protein kinase C, MAP kinases, or phospholipase Cγ2 (PLCγ2). Upon activation, PLCγ2 cleaves the membrane phosphoinositide, phosphatidyl inositol 4,5-biphosphate (PIP2), generating diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). DAG activates protein kinase C while IP3 binds to specific receptors on the endoplasmic reticulum inducing the release of calcium from intracellular stores, resulting in the activation of transcription factors and gene expression., BCR activation also initiates a series of mechanism for negative regulation mediated by the activation of FCγRIIB, CD22, and other ITIM-containing inhibitory coreceptors, presumably for “fine-tuning” the degree of signaling. Besides BCR-mediated activation, signaling events mediated by various cytokines also play important roles in deciding a lymphocyte's fate [9]. It is of interest that SLE patient B cells have been reported to exhibit ‘generalized B-cell hyperactivation’ [10]. Both enhanced B-cell activation and insufficient negative regulation can potentially lead to the elevated levels of B-cell activation seen in SLE. Documented findings include increased intracellular calcium flux, and phosphorylation of various signaling molecules [11,12]. Stimulation of freshly isolated peripheral blood B cells from patients with SLE led to unusually high calcium responses and increased tyrosine phosphorylation of proteins relative to peripheral blood B cells from healthy individuals and disease controls [11,12]. This aberrant signaling status in SLE B cells could be the consequence of various (yet to be defined) susceptibility genes, which may potentially impinge B-cell signaling directly, or indirectly influence the degree of positive and/or negative regulators of B-cell signaling. A well documented
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example of a positive regulator is BLyS. Significant elevation of B lymphocyte stimulator (BLyS) has been reported in patients with SLE [13]. BLyS is a member of the human TNF family and is known to induce B-cell proliferation and immunoglobulin secretion [14]. It has been demonstrated that BLyS stimulation activates at least two independent signaling pathways, AKT/ mTOR and Pim2, associated with cell growth and survival [14]. Hence an increase in BLyS may potentially promote the signaling cascades that lead to increased survival and proliferation of autoreactive B lymphocytes in SLE. While it has been documented that serum level of BLyS is elevated in SLE, more resounding evidence has been reported documenting reduced inhibition of B-cell signaling in SLE patients [12,15,16]. Activation of FcγRIIB is followed by signaling events that down-regulate antigen-induced B-cell activation, which include phosphoinositide hydrolysis, Ras activation and elevation of intracellular calcium [8]. This downregulation through FcγRIIB is mainly mediated by the activation of the SH2-containing inositol 5′-phosphatase (SHIP) which hydrolyzes PIP3, an essential element of BCR signaling. CD19-mediated PI3K activation and generation of PI (3,4,5)P3 can also be suppressed by FcγRIIB-mediated inhibition. Consequently, insufficient inhibition mediated by FcγRIIB can lead to enhanced downstream signaling and increased calcium flux, as noted in SLE B cells. It has been shown that intracellular calcium levels following BCR crosslinking in B cells of SLE patients with defective FcγRIIB signaling is increased compared to healthy controls [12,15,17]. It has been suggested that the defect in FcγRIIB signaling may be due to polymorphisms in coding sequence or in the promoter region of this gene [12,15,16]. Another example of reduced inhibitory BCR signaling in SLE B cells comes from the analysis of Lyn in SLE patients [18]. Lyn exerts inhibitory effects on BCR signaling through phosphorylating CD22, FcγRIIB, and other ITIM-containing inhibitory coreceptors [19]. It was reported that expression of Lyn is significantly decreased in both resting and BCR-stimulated peripheral blood B cells from two-thirds of SLE patients [20]. It was also shown by another group that altered Lyn expression was found to be associated with increased spontaneous proliferation and production of anti-dsDNA autoantibodies [18]. Recently, it has been demonstrated that altered translocation of CD45 correlated with reduced expression of Lyn in SLE patients [21]. More recent genome-wide association studies have indicated that B-cell signaling molecules such as BANK1 and BLK may also constitute disease genes in human SLE [22,23]. In addition to the above players, it is important to ascertain the involvement of other key surface molecules and intracellular signaling intermediates in contributing to the B-cell hyperactivity observed in human SLE. At present, it remains unclear if any specific intracellular signaling cascade in human SLE may serve as a suitable therapeutic target, based on the limited human literature available. In contrast, we currently know more about the status of various signaling axes in murine lupus B cells. 3. Altered B-cell signaling in conventional murine lupus models A few studies have analyzed alterations in B-cell signaling in spontaneous murine lupus. In NZB mice, it has been
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reported that the levels of tyrosine phosphorylated proteins in whole cell lysates of B cells were different from that in control non-autoimmune mice, with the differences being even more prominent in older mice [24]. It has also been shown that B cells from NZB mice produce a stronger response to BCRmediated activation [24]. Moreover, NZB B cells have a higher baseline level of tyrosine phosphorylation as compared to BALB/c B cells, possibly due to hyperactivity of tyrosine kinases and/or the relative inactivity of tyrosine phosphatases. Significantly lower phosphatase activity and abnormalities in SHIP-1 activity were also documented in NZB B cells [24]. More recently, various B-cell signaling axes have been examined more comprehensively in murine lupus. Interestingly, B cells from MRL-lpr/lpr and BXSB-Yaa lupus mice reveal increased levels of activation of AKT/mTOR, ERK1/2, NF-κB and STAT3 pathways [25]. Collectively, the above studies indicate that generalized B-cell hyperactivity, and hyperactivation of various signaling axes may constitute a common theme in murine lupus, just as they do in human SLE. Since the traditionally studied lupus models are polygenic in nature, it is difficult to establish gene to pathway causality. An important step towards unraveling this complex puzzle is to examine genetically simplified congenic mouse models of lupus. 4. Altered B-cell signaling in genetically simplified congenic models of lupus Congenic dissection is a strategy in which individual disease susceptibility loci that contribute to a polygenic disease (such as lupus) can be segregated as a collection of unique sub-strains, each bearing an individual locus, thus allowing one to study component phenotypes contributed by each locus individually. Using this strategy, various lupus susceptibility loci have been successfully introgressed onto the genome of lupus “resistant” (relatively speaking) strains, such as the C57BL/6 (B6). These newly generated congenic mouse strains constitute a unique and powerful tool for dissecting lupus pathogenesis [26]. Since most lupus congenics derived to date have been generated on the B6 background, one can easily breed them to existing mouse tools such as transgenics and knockouts (involving various molecules of immunological importance), many of which already exist on the B6 background. This would then allow the scientist to study the roles of specific cells or molecules in the context of different lupus susceptibility loci. Moreover, the same breeding approach used to create congenic strains can be repeatedly applied in order to further “narrow” the relevant disease-support intervals. A good illustration of how lupus pathogenesis can be “dissected” using congenic mouse strains stems from the genetic studies in the NZM2410 inbred mouse model of lupus. In this model, the development of lupus is contingent upon at least three non-MHC chromosomal intervals — Sle1, Sle2, and Sle3/5. Functional analyses of B6-based congenic strains bearing Sle1, Sle2, or Sle3/5 have demonstrated that each interval is responsible for very different component phenotypes. Among them, the breach of immune tolerance to nuclear antigens mediated by Sle1 appears to be essential for lupus development [27]. However, Sle1 by itself does not lead to the development of fatal lupus but only modest serological
autoreactivity [27]. In contrast, Sle1 mediates highly penetrant fatal glomerulonephritis in epistasis with Sle2, Sle3/5, Yaa or lpr [26,28]. Studies of the NZM2410-derived susceptibility intervals support a multi-step pathogenesis model, in which development of fatal lupus appears to be “initiated” by Sle1 and further exacerbated by additional susceptibility loci or genes [5,26,28]. These genetically simplified congenic mice have also served as ideal tools for studying the impact of altered B-cell signaling in initiating lupus. Comprehensive analysis of signaling pathways have been conducted in B cells isolated from B6, B6.Sle1 and B6.Sle1Sle3 congenic mice [25]. Several signaling axes were noted to be elevated either within Sle1/ Sle1ab B cells themselves, or only within Sle1Sle3 bicongenic B cells. It was shown that enhanced activation of the ERK pathway that is downstream of BCR signaling was a prominent feature in B6.Sle1 mice [25,29]. AKT is a serine/threonine kinase that regulates cell growth, survival, metabolism and cell-cycle progression. The level of phosphorylation of AKT in unstimulated B cells was significantly elevated in B6.Sle1and B6.Sle1Sle3 mice, even at the age of 2 mo, prior to disease, and became progressively more activated in step with the disease [25]. Interestingly, although neither NF-κB nor inhibitor of NFκB (IκB) was upregulated or more phosphorylated in unmanipulated B6.Sle1 B cells, this pathway appeared to be more active in B6.Sle1Sle3 B cells, indicating that epistatic interaction of Sle1with Sle3 was essential for the spontaneous upregulation of this axis [25]. This finding also supports the observation that significantly more activated B cells are present in B6.Sle1Sle3 mice then in B6.Sle1 mice [30]. It was also demonstrated that development of lupus is associated with the upregulation of the anti-apoptotic molecule Bcl-2, with a concomitant decrease in proapoptotic molecules, such as Bim, Bax, and Bad [25]. It appears that at least some of these alterations could occur as a consequence of hyperactivation of various upstream signaling axes. For instance, AKT that is known to control cell survival by inactivating Bad, and this relationship might explain the observed changes in lupus B cells [25]. These studies have also uncovered dysregulation of signaling pathways that are typically activated through other surface receptors, such as cytokine receptors and coreceptors [25,29]. It was shown that the STAT3 pathway was constitutively active in B cells of B6.Sle1ab mice, although its suppressor, SOCS3, was also upregulated [29]. Further analysis indicated that IL-6 was expressed at a higher level in B6.Sle1ab mice, where the level of STAT3 activation also correlated with autoantibody levels [29]. These findings resonate well with the presence of elevated IL-6 in systemic autoimmunity, as observed in human SLE [31]. These results suggest that elevations of certain cytokines may further modulate the signaling status of lupus B cells. The above studies have also revealed that multiple signaling cascades fire up in B cells as lupus evolves, in a gene-dose dependent fashion, as the activation of these pathways were more prominent in B cells from B6.Sle1Sle3 mice than that from age-matched B6.Sle1 mice [25]. Moreover, the enhanced signaling profiles exhibited by B6.Sle1 and B6. Sle1Sle3 B cells were also reproduced in B cells from other mouse models of lupus, BXSB-Yaa and MRL-lpr/lpr, that are genetically distinct from the B6.Sle1 and B6.Sle1Sle3 strains
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and the related NZB/NZW as well as NZM2410 strains [25]. The challenge ahead is to unravel how each disease gene impacts intracellular signaling in lymphocytes, and to ascertain if the upregulated axes may serve as suitable targets for therapy. 5. Lessons from engineered mouse models Engineered mouse models have also highlighted signaling molecules that can potentially promote or thwart systemic autoimmunity. These molecules fall into either B-cell signaling pathways that promote autoimmunity or those that suppress autoimmunity. Molecules in the first category include CD19 and BAFF, and those in the second category include CD22, CD45, FcγRIIB, Lyn, PD-1, PKCδ, and Pten [14,19,32–39]. (PI) 3-kinases (PI3K) activation is a critical control point at various stages of B-cell development, proliferation and differentiation. Activation of BCR results in activation of PI3K which in turn leads to the activation of AKT. Increased CD19 expression in CD19-transgenic mice leads to SLE-like phenotypes [34]. As CD19 is a co-receptor that regulates PI3K activity and lowers the threshold of BCR-mediated signaling, the consequence of its overexpression is increased activity of the PI3K/AKT/mTOR pathway [34]. Survival of B cells requires BAFF (also known as BLyS), produced by stromal cells and various cell types of myeloid origin [14]. BAFF promotes B-cell survival by engaging BAFF receptor (BAFF-R) on B cells (5, 6). Both BCR and BAFF-R signals are crucial for B-cell survival. BAFF-mediated B-cell survival involves activation and upregulation of Bim, promoting cytoplasmic retention of PKCδ, and activation of the NF-κB, PKCβ and Akt axes [40]. Importantly, the overexpression of BAFF in BAFF-transgenic mice extended B-cell survival and triggered the development of lupus-like autoimmune disease [14,39]. It is also of interest that the same signaling axes triggered by BAFF are also elevated within the B cells of lupus mice [25]. Most of the reported engineered lupus models were generated by disrupting molecules in the second category. The roles of these molecules in controlling cell signaling and preventing autoimmunity were revealed in the respective knockout mouse models. One of the first studies relating immune-receptor signaling to autoimmunity involved the PTK, Lyn. Lyn is an essential inhibitor of B-cell signaling, and Lyn-deficient mice have circulating autoreactive antibodies and severe glomerulonephritis caused by the deposition of immune complexes in the kidneys [19]. The transmembrane CD45 phosphatase regulates the activity of the Src-family kinases by dephosphorylating their regulatory C-terminal tyrosine. Mice with a mutation in this molecule that abolishes its inhibitory function also exhibit polyclonal lymphocyte activation, autoantibody production, and severe GN [32]. Mutations in SHP-1, a phosphotyrosine phosphatase (PTP) that down-regulates BCR signaling, also result in autoantibody production and GN [19]. Mice with deletion of other inhibitory receptors, such as FcγRIIB, CD22, and PD-1, also develop severe autoimmunity with lupus-like disease [19,35,36]. Although the classical PKCs (Ca2+-dependent PKCs) are mediators of positive BCR signaling, PKCδ, a member of the novel Ca2+-independent PKC, has been established as an important negative regulator of BCR signaling, and mice deficient in PKCδ develops B-cell abnormalities and lupuslike systemic autoimmunity [37,38]. In-keeping with the
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involvement of enhanced AKT activation in development of autoimmunity, reducing its suppressor, PTEN, in PTEN+/− mice leads to the development of systemic autoimmunity [33]. Collectively, the above studies using engineered mouse models demonstrate that hyperactivation of lymphocyte signaling or negation of inhibitors of lymphocyte signaling can lead to systemic autoimmunity with lupus-like phenotypes. The challenge ahead is to fathom the role of these diverse signaling molecules in various leukocyte subsets in the pathogenesis of spontaneous systemic autoimmunity. 6. Inhibition of systemic autoimmunity by targeting hyperactivated signaling pathways Analyses of B-cell signaling in various mouse models of lupus have not only provided an in-depth perspective of the signaling status in this disease, and possibly the mechanisms leading to enhanced survival of autoimmune B cells, but also present us with potential opportunities for treating lupus by targeting selected nodes. Understanding the complexity and possible interactions among these signaling pathways is critical for therapeutic intervention. An interesting example is provided by the recent attempt to target the PI3K/AKT/ mTOR axis in lupus [25]. As aforementioned, this axis is involved in signal transduction from various receptors, and it is critical in promoting the growth and survival of selfreactive lymphocytes. An inhibitor of this pathway, RAD001 (everolimus), has demonstrated efficacy in controlling the development of murine lupus [25]. Interestingly, the targeting of this axis also resulted in the dampening of several other hyperactivated signaling axes, indicating that different signaling cascades in lymphocytes may be intricately interconnected and inter-dependent [25]. On the other hand, strategies that aim to augment negative regulatory pathways may also potentially be therapeutic. By targeting critical signaling nodes that are altered in lupus leukocytes using safer and more specific agents, we believe that effective treatments for human SLE will soon become a reality. Take-home messages • Aberrant signaling in lymphocytes is a common feature of both human SLE and murine lupus. • Congenic and genetically-engineered murine models have provided unique and powerful tools for studying the contribution of aberrant signaling towards lupus development. • Targeting aberrant signaling events in lupus can potentially be used as therapeutics for lupus in the coming years. References [1] Nath SK, Kilpatrick J, Harley JB. Genetics of human systemic lupus erythematosus: the emerging picture. Curr Opin Immunol 2004;16(6): 794–800. [2] Tsao BP. The genetics of human systemic lupus erythematosus. Trends Immunol 2003;24(11):595–602. [3] Vyse TJ, Kotzin BL. Genetic susceptibility to systemic lupus erythematosus. Annu Rev Immunol 1998;16:261–92. [4] Pickering MC, Walport MJ. Links between complement abnormalities and systemic lupus erythematosus. Rheumatology 2000;39(2):133–41 (Oxford, England). [5] Wakeland EK, Liu K, Graham RR, Behrens TW. Delineating the genetic basis of systemic lupus erythematosus. Immunity 2001;15(3):397–408.
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Angiotensin receptor agonistic autoantibodies induce pre-eclampsia in pregnant mice The clinical hallmark of pre-eclampsia include hypertention, proteinuria, endothelial dysfunction and placental defects. Advanced stage clinical symptoms include cerebral hemorrhage, renal failure and HELLP (hemolysis, elevated liver enzymes and low platelets) syndrome. Numerous recent studies have shown that women with pre-eclampsia possess autoantibodies, termed AT (1)-AAs, that bind and activate the angiotensin II receptor type 1a (AT (1) receptor). Here, Zhou CC. et al. (Nat Med 2008; 14:810-2) show that key features of pre-eclampsia, including hypertension, proteinuria, glomerular endotheliosis (a classical renal lesion of pre-eclampsia), placental abnormalities and small fetus size appeared in pregnant mice after injection with either total IgG or affinity-purified AT (1)-AAs from women with pre-eclampsia. These features were prevented by coinjection with losartan, an AT (1) receptor antagonist, or by an antibody neutralizing seven-amino-acid epitope peptide. Thus, our studies indicate that pre-eclampsia may be a pregnancy-induced autoimmune disease in which key features of the disease result from autoantibody-induced angiotensin receptor activation. This hypothesis has obvious implications regarding preeclampsia screening, diagnosis and therapy.
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Autoimmunity Reviews j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t r ev
Altered B-cell signaling in lupus Kui Liu ⁎, Chandra Mohan ⁎ Division of Rheumatology, and Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390-8884, USA
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Available online 21 August 2008 Keywords: B cell Lupus Signaling
a b s t r a c t Systemic lupus erythematosus (SLE) is an autoimmune disease of complex etiology primarily characterized by the presence of high titers of autoantibodies targeting many nuclear as well as cytoplasmic antigens, with resultant end-organ damage. Aberrant signaling events have been documented in various lymphocyte populations, and they have constituted attractive targets for therapeutic intervention. Murine models of lupus (conventional or engineered) have yielded interesting snapshots of the signaling status of lupus lymphocytes, and many of these alterations in cell signaling observed in murine models of lupus have also been documented in patient samples. Analyses of B-cell signaling in various murine lupus models have not only provided an in-depth perspective of the signaling status and possibly the underlying mechanisms leading to enhanced survival of autoimmune B cells, but have also presented us with potential strategies for treating lupus. © 2008 Published by Elsevier B.V.
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Altered B-cell signaling in SLE patients. . . . . . . . . . . . . . . . . . . . . . . 3. Altered B-cell signaling in conventional murine lupus models . . . . . . . . . . . . 4. Altered B-cell signaling in genetically simplified congenic models of lupus . . . . . . 5. Lessons from engineered mouse models . . . . . . . . . . . . . . . . . . . . . . 6. Inhibition of systemic autoimmunity by targeting hyperactivated signaling pathways . Take-home messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease of complex etiology primarily characterized by the presence of high titers of autoantibodies targeting many nuclear as well as cytoplasmic antigens, with resultant end-
⁎ Corresponding authors. Department of Internal Medicine/Rheumatology UT Southwestern Medical Center, Mail Code 8884, Y8.204, 5323 Harry Hines Boulevard Dallas, TX 75390-8884, USA. Tel.: +1 214 648 9675; fax: +1 214 648 7995. E-mail addresses: [email protected] (K. Liu), [email protected] (C. Mohan). 1568-9972/$ – see front matter © 2008 Published by Elsevier B.V. doi:10.1016/j.autrev.2008.07.048
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organ damage. Antibody and immune-complex-mediated inflammatory processes in this disease can lead to the development of glomerulonephritis, dermatitis, serositis, and vasculitis. It is estimated to affect about 1 in 2000 people with a strong gender bias (female to male ratio is about 9:1). Furthermore, African-Americans and Hispanics are approximately 2 to 4 times more likely to develop this disease than Caucasians [1–3]. Both genetic and environmental factors contribute to its pathogenesis [1–3]. Although deficiencies in some of the early components of the complement cascade, such as C1 and C4, can lead to the development of SLE, this disease is typically polygenic in origin [1–5]. Studies from both SLE patients and mouse models indicate that production of antinuclear antibodies (ANA) is a key feature
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of the dysregulated immune system in lupus. The emergence of ANAs in these studies is typically associated with quantitative and qualitative alterations in B-cell and/or T cell populations and a breach of immune tolerance at some level. Aberrant signaling has been reported in both of these cell populations in lupus [6,7], and these changes undoubtedly constitute attractive targets for therapeutic intervention. In this regard, murine models of lupus, both conventional models as well as the more recently studied genetically simplified congenic models of lupus, have collectively yielded interesting snapshots of the signaling status of lupus lymphocytes. Importantly, many of these alterations in cell signaling observed in murine models of lupus have also been documented in patient samples. This review is aimed at summarizing recent progress in this area, with a specific focus on B cells. 2. Altered B-cell signaling in SLE patients Cross-linking of antigen receptors on lymphocytes is a critical trigger in initiating downstream intracellular events that involve an intricate cascade of signaling molecules. These events lead to various outcomes, including activation/proliferation, inhibition/apoptosis, and cytokine secretion. In B lymphocytes, clustering of the B-cell receptor (BCR) leads to activation of protein tyrosine kinases, such as Lyn, which phosphorylate crucial immunoreceptor tyrosine-based activation motif (ITAM) tyrosine residues on the BCR-associated Igα and Igβ signaling molecules, as reviewed elsewhere [8]. Phosphorylation of ITAM mediates the recruitment and activation of the tyrosine kinase Syk, which initiates several downstream signaling pathways, including protein kinase C, MAP kinases, or phospholipase Cγ2 (PLCγ2). Upon activation, PLCγ2 cleaves the membrane phosphoinositide, phosphatidyl inositol 4,5-biphosphate (PIP2), generating diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). DAG activates protein kinase C while IP3 binds to specific receptors on the endoplasmic reticulum inducing the release of calcium from intracellular stores, resulting in the activation of transcription factors and gene expression., BCR activation also initiates a series of mechanism for negative regulation mediated by the activation of FCγRIIB, CD22, and other ITIM-containing inhibitory coreceptors, presumably for “fine-tuning” the degree of signaling. Besides BCR-mediated activation, signaling events mediated by various cytokines also play important roles in deciding a lymphocyte's fate [9]. It is of interest that SLE patient B cells have been reported to exhibit ‘generalized B-cell hyperactivation’ [10]. Both enhanced B-cell activation and insufficient negative regulation can potentially lead to the elevated levels of B-cell activation seen in SLE. Documented findings include increased intracellular calcium flux, and phosphorylation of various signaling molecules [11,12]. Stimulation of freshly isolated peripheral blood B cells from patients with SLE led to unusually high calcium responses and increased tyrosine phosphorylation of proteins relative to peripheral blood B cells from healthy individuals and disease controls [11,12]. This aberrant signaling status in SLE B cells could be the consequence of various (yet to be defined) susceptibility genes, which may potentially impinge B-cell signaling directly, or indirectly influence the degree of positive and/or negative regulators of B-cell signaling. A well documented
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example of a positive regulator is BLyS. Significant elevation of B lymphocyte stimulator (BLyS) has been reported in patients with SLE [13]. BLyS is a member of the human TNF family and is known to induce B-cell proliferation and immunoglobulin secretion [14]. It has been demonstrated that BLyS stimulation activates at least two independent signaling pathways, AKT/ mTOR and Pim2, associated with cell growth and survival [14]. Hence an increase in BLyS may potentially promote the signaling cascades that lead to increased survival and proliferation of autoreactive B lymphocytes in SLE. While it has been documented that serum level of BLyS is elevated in SLE, more resounding evidence has been reported documenting reduced inhibition of B-cell signaling in SLE patients [12,15,16]. Activation of FcγRIIB is followed by signaling events that down-regulate antigen-induced B-cell activation, which include phosphoinositide hydrolysis, Ras activation and elevation of intracellular calcium [8]. This downregulation through FcγRIIB is mainly mediated by the activation of the SH2-containing inositol 5′-phosphatase (SHIP) which hydrolyzes PIP3, an essential element of BCR signaling. CD19-mediated PI3K activation and generation of PI (3,4,5)P3 can also be suppressed by FcγRIIB-mediated inhibition. Consequently, insufficient inhibition mediated by FcγRIIB can lead to enhanced downstream signaling and increased calcium flux, as noted in SLE B cells. It has been shown that intracellular calcium levels following BCR crosslinking in B cells of SLE patients with defective FcγRIIB signaling is increased compared to healthy controls [12,15,17]. It has been suggested that the defect in FcγRIIB signaling may be due to polymorphisms in coding sequence or in the promoter region of this gene [12,15,16]. Another example of reduced inhibitory BCR signaling in SLE B cells comes from the analysis of Lyn in SLE patients [18]. Lyn exerts inhibitory effects on BCR signaling through phosphorylating CD22, FcγRIIB, and other ITIM-containing inhibitory coreceptors [19]. It was reported that expression of Lyn is significantly decreased in both resting and BCR-stimulated peripheral blood B cells from two-thirds of SLE patients [20]. It was also shown by another group that altered Lyn expression was found to be associated with increased spontaneous proliferation and production of anti-dsDNA autoantibodies [18]. Recently, it has been demonstrated that altered translocation of CD45 correlated with reduced expression of Lyn in SLE patients [21]. More recent genome-wide association studies have indicated that B-cell signaling molecules such as BANK1 and BLK may also constitute disease genes in human SLE [22,23]. In addition to the above players, it is important to ascertain the involvement of other key surface molecules and intracellular signaling intermediates in contributing to the B-cell hyperactivity observed in human SLE. At present, it remains unclear if any specific intracellular signaling cascade in human SLE may serve as a suitable therapeutic target, based on the limited human literature available. In contrast, we currently know more about the status of various signaling axes in murine lupus B cells. 3. Altered B-cell signaling in conventional murine lupus models A few studies have analyzed alterations in B-cell signaling in spontaneous murine lupus. In NZB mice, it has been
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reported that the levels of tyrosine phosphorylated proteins in whole cell lysates of B cells were different from that in control non-autoimmune mice, with the differences being even more prominent in older mice [24]. It has also been shown that B cells from NZB mice produce a stronger response to BCRmediated activation [24]. Moreover, NZB B cells have a higher baseline level of tyrosine phosphorylation as compared to BALB/c B cells, possibly due to hyperactivity of tyrosine kinases and/or the relative inactivity of tyrosine phosphatases. Significantly lower phosphatase activity and abnormalities in SHIP-1 activity were also documented in NZB B cells [24]. More recently, various B-cell signaling axes have been examined more comprehensively in murine lupus. Interestingly, B cells from MRL-lpr/lpr and BXSB-Yaa lupus mice reveal increased levels of activation of AKT/mTOR, ERK1/2, NF-κB and STAT3 pathways [25]. Collectively, the above studies indicate that generalized B-cell hyperactivity, and hyperactivation of various signaling axes may constitute a common theme in murine lupus, just as they do in human SLE. Since the traditionally studied lupus models are polygenic in nature, it is difficult to establish gene to pathway causality. An important step towards unraveling this complex puzzle is to examine genetically simplified congenic mouse models of lupus. 4. Altered B-cell signaling in genetically simplified congenic models of lupus Congenic dissection is a strategy in which individual disease susceptibility loci that contribute to a polygenic disease (such as lupus) can be segregated as a collection of unique sub-strains, each bearing an individual locus, thus allowing one to study component phenotypes contributed by each locus individually. Using this strategy, various lupus susceptibility loci have been successfully introgressed onto the genome of lupus “resistant” (relatively speaking) strains, such as the C57BL/6 (B6). These newly generated congenic mouse strains constitute a unique and powerful tool for dissecting lupus pathogenesis [26]. Since most lupus congenics derived to date have been generated on the B6 background, one can easily breed them to existing mouse tools such as transgenics and knockouts (involving various molecules of immunological importance), many of which already exist on the B6 background. This would then allow the scientist to study the roles of specific cells or molecules in the context of different lupus susceptibility loci. Moreover, the same breeding approach used to create congenic strains can be repeatedly applied in order to further “narrow” the relevant disease-support intervals. A good illustration of how lupus pathogenesis can be “dissected” using congenic mouse strains stems from the genetic studies in the NZM2410 inbred mouse model of lupus. In this model, the development of lupus is contingent upon at least three non-MHC chromosomal intervals — Sle1, Sle2, and Sle3/5. Functional analyses of B6-based congenic strains bearing Sle1, Sle2, or Sle3/5 have demonstrated that each interval is responsible for very different component phenotypes. Among them, the breach of immune tolerance to nuclear antigens mediated by Sle1 appears to be essential for lupus development [27]. However, Sle1 by itself does not lead to the development of fatal lupus but only modest serological
autoreactivity [27]. In contrast, Sle1 mediates highly penetrant fatal glomerulonephritis in epistasis with Sle2, Sle3/5, Yaa or lpr [26,28]. Studies of the NZM2410-derived susceptibility intervals support a multi-step pathogenesis model, in which development of fatal lupus appears to be “initiated” by Sle1 and further exacerbated by additional susceptibility loci or genes [5,26,28]. These genetically simplified congenic mice have also served as ideal tools for studying the impact of altered B-cell signaling in initiating lupus. Comprehensive analysis of signaling pathways have been conducted in B cells isolated from B6, B6.Sle1 and B6.Sle1Sle3 congenic mice [25]. Several signaling axes were noted to be elevated either within Sle1/ Sle1ab B cells themselves, or only within Sle1Sle3 bicongenic B cells. It was shown that enhanced activation of the ERK pathway that is downstream of BCR signaling was a prominent feature in B6.Sle1 mice [25,29]. AKT is a serine/threonine kinase that regulates cell growth, survival, metabolism and cell-cycle progression. The level of phosphorylation of AKT in unstimulated B cells was significantly elevated in B6.Sle1and B6.Sle1Sle3 mice, even at the age of 2 mo, prior to disease, and became progressively more activated in step with the disease [25]. Interestingly, although neither NF-κB nor inhibitor of NFκB (IκB) was upregulated or more phosphorylated in unmanipulated B6.Sle1 B cells, this pathway appeared to be more active in B6.Sle1Sle3 B cells, indicating that epistatic interaction of Sle1with Sle3 was essential for the spontaneous upregulation of this axis [25]. This finding also supports the observation that significantly more activated B cells are present in B6.Sle1Sle3 mice then in B6.Sle1 mice [30]. It was also demonstrated that development of lupus is associated with the upregulation of the anti-apoptotic molecule Bcl-2, with a concomitant decrease in proapoptotic molecules, such as Bim, Bax, and Bad [25]. It appears that at least some of these alterations could occur as a consequence of hyperactivation of various upstream signaling axes. For instance, AKT that is known to control cell survival by inactivating Bad, and this relationship might explain the observed changes in lupus B cells [25]. These studies have also uncovered dysregulation of signaling pathways that are typically activated through other surface receptors, such as cytokine receptors and coreceptors [25,29]. It was shown that the STAT3 pathway was constitutively active in B cells of B6.Sle1ab mice, although its suppressor, SOCS3, was also upregulated [29]. Further analysis indicated that IL-6 was expressed at a higher level in B6.Sle1ab mice, where the level of STAT3 activation also correlated with autoantibody levels [29]. These findings resonate well with the presence of elevated IL-6 in systemic autoimmunity, as observed in human SLE [31]. These results suggest that elevations of certain cytokines may further modulate the signaling status of lupus B cells. The above studies have also revealed that multiple signaling cascades fire up in B cells as lupus evolves, in a gene-dose dependent fashion, as the activation of these pathways were more prominent in B cells from B6.Sle1Sle3 mice than that from age-matched B6.Sle1 mice [25]. Moreover, the enhanced signaling profiles exhibited by B6.Sle1 and B6. Sle1Sle3 B cells were also reproduced in B cells from other mouse models of lupus, BXSB-Yaa and MRL-lpr/lpr, that are genetically distinct from the B6.Sle1 and B6.Sle1Sle3 strains
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and the related NZB/NZW as well as NZM2410 strains [25]. The challenge ahead is to unravel how each disease gene impacts intracellular signaling in lymphocytes, and to ascertain if the upregulated axes may serve as suitable targets for therapy. 5. Lessons from engineered mouse models Engineered mouse models have also highlighted signaling molecules that can potentially promote or thwart systemic autoimmunity. These molecules fall into either B-cell signaling pathways that promote autoimmunity or those that suppress autoimmunity. Molecules in the first category include CD19 and BAFF, and those in the second category include CD22, CD45, FcγRIIB, Lyn, PD-1, PKCδ, and Pten [14,19,32–39]. (PI) 3-kinases (PI3K) activation is a critical control point at various stages of B-cell development, proliferation and differentiation. Activation of BCR results in activation of PI3K which in turn leads to the activation of AKT. Increased CD19 expression in CD19-transgenic mice leads to SLE-like phenotypes [34]. As CD19 is a co-receptor that regulates PI3K activity and lowers the threshold of BCR-mediated signaling, the consequence of its overexpression is increased activity of the PI3K/AKT/mTOR pathway [34]. Survival of B cells requires BAFF (also known as BLyS), produced by stromal cells and various cell types of myeloid origin [14]. BAFF promotes B-cell survival by engaging BAFF receptor (BAFF-R) on B cells (5, 6). Both BCR and BAFF-R signals are crucial for B-cell survival. BAFF-mediated B-cell survival involves activation and upregulation of Bim, promoting cytoplasmic retention of PKCδ, and activation of the NF-κB, PKCβ and Akt axes [40]. Importantly, the overexpression of BAFF in BAFF-transgenic mice extended B-cell survival and triggered the development of lupus-like autoimmune disease [14,39]. It is also of interest that the same signaling axes triggered by BAFF are also elevated within the B cells of lupus mice [25]. Most of the reported engineered lupus models were generated by disrupting molecules in the second category. The roles of these molecules in controlling cell signaling and preventing autoimmunity were revealed in the respective knockout mouse models. One of the first studies relating immune-receptor signaling to autoimmunity involved the PTK, Lyn. Lyn is an essential inhibitor of B-cell signaling, and Lyn-deficient mice have circulating autoreactive antibodies and severe glomerulonephritis caused by the deposition of immune complexes in the kidneys [19]. The transmembrane CD45 phosphatase regulates the activity of the Src-family kinases by dephosphorylating their regulatory C-terminal tyrosine. Mice with a mutation in this molecule that abolishes its inhibitory function also exhibit polyclonal lymphocyte activation, autoantibody production, and severe GN [32]. Mutations in SHP-1, a phosphotyrosine phosphatase (PTP) that down-regulates BCR signaling, also result in autoantibody production and GN [19]. Mice with deletion of other inhibitory receptors, such as FcγRIIB, CD22, and PD-1, also develop severe autoimmunity with lupus-like disease [19,35,36]. Although the classical PKCs (Ca2+-dependent PKCs) are mediators of positive BCR signaling, PKCδ, a member of the novel Ca2+-independent PKC, has been established as an important negative regulator of BCR signaling, and mice deficient in PKCδ develops B-cell abnormalities and lupuslike systemic autoimmunity [37,38]. In-keeping with the
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involvement of enhanced AKT activation in development of autoimmunity, reducing its suppressor, PTEN, in PTEN+/− mice leads to the development of systemic autoimmunity [33]. Collectively, the above studies using engineered mouse models demonstrate that hyperactivation of lymphocyte signaling or negation of inhibitors of lymphocyte signaling can lead to systemic autoimmunity with lupus-like phenotypes. The challenge ahead is to fathom the role of these diverse signaling molecules in various leukocyte subsets in the pathogenesis of spontaneous systemic autoimmunity. 6. Inhibition of systemic autoimmunity by targeting hyperactivated signaling pathways Analyses of B-cell signaling in various mouse models of lupus have not only provided an in-depth perspective of the signaling status in this disease, and possibly the mechanisms leading to enhanced survival of autoimmune B cells, but also present us with potential opportunities for treating lupus by targeting selected nodes. Understanding the complexity and possible interactions among these signaling pathways is critical for therapeutic intervention. An interesting example is provided by the recent attempt to target the PI3K/AKT/ mTOR axis in lupus [25]. As aforementioned, this axis is involved in signal transduction from various receptors, and it is critical in promoting the growth and survival of selfreactive lymphocytes. An inhibitor of this pathway, RAD001 (everolimus), has demonstrated efficacy in controlling the development of murine lupus [25]. Interestingly, the targeting of this axis also resulted in the dampening of several other hyperactivated signaling axes, indicating that different signaling cascades in lymphocytes may be intricately interconnected and inter-dependent [25]. On the other hand, strategies that aim to augment negative regulatory pathways may also potentially be therapeutic. By targeting critical signaling nodes that are altered in lupus leukocytes using safer and more specific agents, we believe that effective treatments for human SLE will soon become a reality. Take-home messages • Aberrant signaling in lymphocytes is a common feature of both human SLE and murine lupus. • Congenic and genetically-engineered murine models have provided unique and powerful tools for studying the contribution of aberrant signaling towards lupus development. • Targeting aberrant signaling events in lupus can potentially be used as therapeutics for lupus in the coming years. References [1] Nath SK, Kilpatrick J, Harley JB. Genetics of human systemic lupus erythematosus: the emerging picture. Curr Opin Immunol 2004;16(6): 794–800. [2] Tsao BP. The genetics of human systemic lupus erythematosus. Trends Immunol 2003;24(11):595–602. [3] Vyse TJ, Kotzin BL. Genetic susceptibility to systemic lupus erythematosus. Annu Rev Immunol 1998;16:261–92. [4] Pickering MC, Walport MJ. Links between complement abnormalities and systemic lupus erythematosus. Rheumatology 2000;39(2):133–41 (Oxford, England). [5] Wakeland EK, Liu K, Graham RR, Behrens TW. Delineating the genetic basis of systemic lupus erythematosus. Immunity 2001;15(3):397–408.
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Angiotensin receptor agonistic autoantibodies induce pre-eclampsia in pregnant mice The clinical hallmark of pre-eclampsia include hypertention, proteinuria, endothelial dysfunction and placental defects. Advanced stage clinical symptoms include cerebral hemorrhage, renal failure and HELLP (hemolysis, elevated liver enzymes and low platelets) syndrome. Numerous recent studies have shown that women with pre-eclampsia possess autoantibodies, termed AT (1)-AAs, that bind and activate the angiotensin II receptor type 1a (AT (1) receptor). Here, Zhou CC. et al. (Nat Med 2008; 14:810-2) show that key features of pre-eclampsia, including hypertension, proteinuria, glomerular endotheliosis (a classical renal lesion of pre-eclampsia), placental abnormalities and small fetus size appeared in pregnant mice after injection with either total IgG or affinity-purified AT (1)-AAs from women with pre-eclampsia. These features were prevented by coinjection with losartan, an AT (1) receptor antagonist, or by an antibody neutralizing seven-amino-acid epitope peptide. Thus, our studies indicate that pre-eclampsia may be a pregnancy-induced autoimmune disease in which key features of the disease result from autoantibody-induced angiotensin receptor activation. This hypothesis has obvious implications regarding preeclampsia screening, diagnosis and therapy.