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Signaling pathways regulating RAG expression in B lymphocytes
Autoimmunity Reviews 8 (2009) 599–604
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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
Signaling pathways regulating RAG expression in B lymphocytes Sophie Hillion, Caroline Rochas, Pierre Youinou ⁎, Christophe Jamin Université Européenne de Bretagne, France Université de Bretagne Occidentale, EA 2216 Immunology and Pathology, IFR 148 ScInBioS, Brest F-29609, France Brest Medical School Hospital, Morvan, Brest F-29609, France
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Article history: Received 2 January 2009 Accepted 4 February 2009 Available online 9 February 2009 Keywords: B lymphocytes RAG Cytokines Autoimmunity Up-regulation
a b s t r a c t Development of B-cell lymphopoiesis is dependent on the presence of recombination activating genes RAG1 and RAG2 enzymes. They control the rearrangements of immunoglobulin variable, diversity and joining region segments, and allow progression of the cellular maturation. RAG1 and RAG2 are successively up- and down-regulated at each B-cell stage to progressively generate a B-cell receptor for which unforeseeable antigenic specificity results from a stochastic process. Therefore, in autoreactive immature B cells, new round of RAG re-expression can be observed to eliminate self-reactivity. In some circumstances, RAG up-regulation can also be found in peripheral mature B lymphocytes, specifically in autoimmune diseases. It is therefore of utmost importance to unravel signaling pathways that trigger RAG induction in normal and pathological conditions. Therapeutic modulation of cytokines or intracellular contacts involved in RAG expression might restrict the development of inappropriate autoimmune repertoire. © 2009 Elsevier B.V. All rights reserved.
Contents 1. Introduction . . . . . . . . . . . . . . 2. Cell surface signals. . . . . . . . . . . 3. Intra-cellular pathways. . . . . . . . . 4. Nucleus factors . . . . . . . . . . . . 5. RAG up-regulation in disease conditions Take-home messages . . . . . . . . . . . . References . . . . . . . . . . . . . . . . .
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1. Introduction Maturation of B lymphocytes is closely associated with immunoglobulin (Ig) gene rearrangements. Beginning in the bone marrow by commitment of lymphoid progenitors to the B lineage, initial diversity of the heavy chain (DH) to joining (JH) gene segments rearrange in pro-B cells. This is followed ⁎ Corresponding author. Laboratory of Immunology, Brest University Medical School Hospital, BP824, F-29609, Brest, France. Tel.: +33 298 22 33 84; fax: +33 298 22 38 47. E-mail address: [email protected] (P. Youinou). 1568-9972/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2009.02.004
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by recombination of a variable (VH) gene with the DJH segment, thus creating a complete VDJH rearrangement. Pro-B cells then differentiate into pre-B cells subdivided into proliferating pre-BI cells bearing the pre-B cell receptor (BCR) that utilize the rearranged heavy chain, and resting pre-BII cells that initiate Ig κ or λ light (L) chain gene rearrangement between VL and JL segments. Pre-BII cells differentiate into immature B cells that express a complete BCR corresponding to the surface expression of IgM. All these Ig gene rearrangements require the action of the recombination activating genes (RAG)1 and RAG2 enzymes, of which the expression is strikingly related to the recombination process.
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RAG1 and RAG2 are highly expressed in pro-B and pre-BII cells but down-regulated in pre-BI and immature B cells. BCRs are assembled via stochastic VDJH and VJL rearrangements, and comprised randomly selected H and L chains. The resulting complexes have an unforecasted specificity that can include recognition of auto-antigens. To avoid the terminal differentiation of autoreactive B lymphocytes, tolerance mechanisms take place in the bone marrow, such as elimination (clonal deletion) or anergy (inactivation) of the self-reactive B cells. However, newly L chain gene rearrangement, called receptor editing [1], can be re-induced at the immature B-cell stage due to a secondary up-regulation of RAG1 and RAG2 proteins, which appears to be the main mechanism of B cell tolerance. When they have reach the periphery, immature B cells progress as transitional B lymphocytes that can differentiate into mature B cells to develop a germinal centre (GC) response following encounter with antigen in secondary lymphoid organs. Initially observed in vitro using murine B cells, it has been suggested that RAG proteins could be reinduced in mature B cells [2]. Later, the presence of RAGpositive mature B lymphocytes within human tonsil tissues [3], has supported the notion that RAG up-regulation could also occur in peripheral lymphoid organs. The ensuing peripheral Ig gene rearrangements in mature B cells have been referred as receptor revision and would appear after somatic hypermutations [1]. This mechanism seems to be independent of tolerance purpose, but rather is induced in B cells expressing low-affinity BCRs to generate high affinity antibodies during affinity maturation in developing GCs. Activation, proliferation, differentiation or tolerance outcomes depend not only on the developmental stage, but also on the cell surface stimulated molecules. Therefore, engagement of BCR, of positive and negative regulators of BCR signaling, as well as of cytokine receptors instigates activation of intra-cellular signaling pathways that involve adaptor proteins, enzymes and secondary messengers. The resulting events are the recruitment of transcription factors that translocate into the nucleus to regulate gene expression. The current review is aimed at deciphering the molecular processes regulating expression of RAGs at the different stage of B-cell development. 2. Cell surface signals Several studies have shown that BCR signaling plays an important role in regulating RAG expression. Immature IgM+ IgD− B cells, and specifically those expressing a self-reactive BCR, can re-induce expression of RAG proteins following binding of the self-Ag in the bone marrow [4]. Stimulation of autoreactive BCR with soluble auto-Ag until the late immature B cell stage [5], provides an alternative process for the tolerance. Modification of the Ag specificity of newly developing cells eliminates autoreactivity without B-cell deletion. However, other models suggest that BCR cross-linking can lead to a down-regulation of RAG2 mRNA [6]. Therefore, another hypothesis has been proposed by Tze et al. who demonstrated that engagement of the BCR with self-Ag down-regulates surface Ig receptors via endocytosis, and triggers RAG up-regulation and subsequent receptor editing [7]. It is likely that diminished surface Igs results in a loss of
basal signal that allows re-induction of RAG expression and edition of Ig L genes under self-Ag stimulation. In contrast, competent BCR basal signaling inhibits RAG induction and receptor editing [8], and thereby allows the B-cell development to carry on. It is thought that RAG up-regulation in immature B cells would occur within a window during B-cell development (Fig. 1). The size of this window may vary according to the type of Ag and its concentration, as well as the avidity and the duration of Ag encounter [9]. The presence of positive regulator of BCR signaling such as CD19 plays also an important role in the B-cell differentiation. Thus, autoreactive immature B cells that lack CD19 have elevated tonic signals. Elevation of tonic signals up-regulates RAGs genes and stimulates receptor editing [10]. By contrast, lowering tonic signals suppress RAG expression and receptor editing, due to strong signalling transmission following BCR stimulation [11]. Similarly, immature autoreactive B cells deficient in CD45, which is a tyrosine phosphatase that functions as a positive regulator of the BCR signaling, are developmentally arrested in the presence of self-Ag, and undergo receptor editing. All these data therefore suggest that BCR-independent tonic signals can control RAG expression to suppress receptor editing and promote positive selection of immature B cells. The presence of RAG-positive cells in the GC of secondary lymphoid organs [12], raises the question as to whether the same signalings, as those observed in immature lymphocytes, would produce the same effects in mature B cells. Although it has been initially suggested that BCR cross-linking increases RAG1 mRNA level in a human mature B cell line, BCR ligation turns off RAG expression in human GC B cells [13], as well as in murine splenic B lymphocytes [14]. RAG induction may be inversely associated with either surface Ig expression on mature B cells [15], or with antigenic avidity of the BCRs [14]. The ensuing receptor revision process would thus occur to raise the BCR affinity in order to trigger maturation of the GC response [16]. Stimulation of BCR co-receptors can modulate RAG upregulation. Induction of RAG requires the co-ligation of CD40 with the BCR [17] or with another signal [2]. Moreover, RAG up-regulation in human peripheral B cells depends on the presence of CD5, known as a negative regulator of the BCR [17], suggesting that BCR threshold may play a role in the regulation of RAG expression in mature B cells. Consistent with this hypothesis, co-ligation of CD21/CD35 with the BCR abrogates much more efficiently RAG2 expression in murine splenic B cells than BCR engagement alone [18]. Several observations strongly suggest that two separate signals are required for the induction of RAG. This is the case for progenitor B cells that lack BCR on their surface. Upregulation of RAG mRNA is observed when a signal from cell surface molecule on stromal cells is associated with a second from cytokines [19]. Among the cytokines, IL-3, IL-6 and IL-7, but not IL-2, IL-4 and GM-CSF induce RAG gene expression in human lymphoid progenitor cell line (Fig. 1). It is likely that progenitor B lymphocytes may be sensitive to RAG upregulation during a window-time. Thus, after two days of stimulation with bone marrow stromal cells, these latter secrete IL-7 that up-regulates CD19 on progenitor B cells, and one day later down-regulates RAG1 and RAG2 expression. Interestingly, cross-linking of CD19 has no direct effect on
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Fig. 1. Signaling molecules involved in the control of RAG expression in pre-B, immature and mature B cells. Extra-cellular signals from interleukins in association with cell-surface molecules induce (arrows) or inhibit (blunted arrows) intra-cellular pathways. Intra-cytoplasmic signalings implicate adaptor proteins (BLNK), kinases activation (PKA, PKC, PI3K, MAPK, ERK, BtK, Akt), phosphatase inhibition (PP1−), phospholipase activation (PLCγ2), and increased concentration of secondary messengers (cAMP, iCa). These pathways recruit transcription factors involved in either activation (IRF4, E2A, Foxp1, Foxo1, NF-κB, Pax5, c-Myb, LEF-1) or inhibition (NF-kB1) of RAG expression.
RAG expression but blocks the IL-7 down-regulation of RAG, indicating the important role of co-receptor molecules [20]. Up-regulation of RAG in mature B cells also largely depends on cytokines. When polyclonally stimulated with Staphylococcus aureus Cowan I, more than half of peripheral human circulating B cells express RAG1 and RAG2 proteins in the presence of IL-2 [21]. IL-4 is another effective co-factor for RAG expression when human peripheral B cells are stimulated with anti-CD40 antibody, or when murine splenic B cells are stimulated with either lipopolysaccharide (LPS), antiCD40 or anti-IgM antibody [2]. It should be noted that LPS associated with other cytokines such as IL-2, IL-3 or IL-5 is ineffective. When co-stimulated with CD40 and BCR, human peripheral B cells as well as tonsillar B cells up-regulate RAG1 and RAG2 [22], due to the induced-secretion of IL-6 [23]. Finally, IL-7 also contributes to the re-expression of RAG observed in IgD+ mouse GC B cells [24]. All these data indicate
that complex intra-cellular pathways, induced from different surface molecules, are required to up-regulate RAG expression in B lymphocytes (Fig. 1). 3. Intra-cellular pathways Identification of intra-cellular signals involved in the control of RAG expression is complicated due to the differential responses observed during B-cell lymphopoiesis. In pre-B lymphocytes, increased level of cAMP, activator of protein kinase (PK) A, induces RAG expression [25], which is also up-regulated by inhibition of serine/threonine protein phosphatase PP1. By contrast, activation of PKC or elevated level of intra-cellular calcium inhibits RAG1 and RAG2 mRNA transcription [26]. In immature B cells, increases in intracellular free calcium, i.e. as occurs after BCR aggregation, induces up-regulation of RAG [27]. Autoreactive immature B
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cells from BLNK-deficient mice fail to promote Ig L chain gene recombination under BCR engagement, suggesting that the adaptor protein BLNK plays a role in the regulation of RAG expression after BCR stimulation [28]. Among the downstream molecules activated following binding to BLNK, phospholipase γ2 (PLCγ2) signaling pathway is important in activation of receptor editing, and thereby likely in RAG upregulation. Thus, PLCγ2-deficient mice exhibit impaired Aginduced receptor editing in autoreactive immature B cells [29]. Therefore, it may be that PLCγ2 which produces diacyglycerol (DAG) and inositol triphosphate, favors activation of PKC by DAG and releases of intra-cellular calcium that promotes RAG up-regulation and secondary Ig gene rearrangements. This signaling cascade proceeds also with activation of mitogen-activated protein kinases (MAPKs). Among the MAPK, extracellular signal-regulated kinase (ERK) activation is involved in RAG2 gene up-regulation. Thus, inhibition of the ERK signaling pathway impairs RAG2 expression and receptor editing in autoreactive immature murine B cells following auto-Ag stimulation [30]. Regulation of RAG induction is the result of a complex balance between several intra-cellular signaling pathways. In immature murine B lymphocytes that carry a nonautoreactive BCR, phosphatidylinositol 3-kinase (PI3K) signaling is required to suppress RAG expression, otherwise inappropriate receptor editing undergoes, as occurs in mice deficient in the PI3K. This basal BCR-directed PI3K signaling activates the downstream effector PLCγ2, Akt and Btk. Of these, PLCγ2 seems to play a significant role in down-regulating RAG expression [31], though the serine/threonine kinase, Akt can also repress the transcription of RAG1 and RAG2 [32]. In mature B cells, regulation of RAG expression appears to be only partially similarly controlled. While inhibition of PP1 up-regulates the expression of RAG1 and RAG2 as in pre-B cells [25], increased level of cAMP has no effect. Furthermore, in human plasma cell lines expressing RAG1 and RAG2, inhibition of PP1 does not affect the expression level of the RAG proteins although Ig L chain shifting can be induced [32]. These observations suggest that phosphatase inhibition can not only regulate RAG expression in mature B lymphocytes, but also their activity in plasma cells. However, in contrast to the effects observed in pre-B cells [26], stimulation of mature B lymphocytes with a combination of phorbol myristic acetate and inomycin, that activates PKC and increases the level of intra-cellular free calcium, up-regulates the level of RAG mRNA [31]. Finally, splenic mature B cells from PLCγ2deficient mice have a reduced frequency of lambda-expressing B cell subpopulation, and their Ig L chain gene rearrangements are barely detectable following self-Ag stimulation [29]. These data indicates that receptor editing is altered in mature B lymphocytes when PLCγ2 is lacking, and thereby that PLCγ2 is required for the up-regulation of RAG1 and RAG2 at the mature B-cell stage. These regulatory pathways are summarized in Fig. 1. 4. Nucleus factors Activation of these intra-cellular pathways works towards the recruitment of nuclear factors to control RAG1 and RAG2 gene expression (Fig. 1). Most of available informations have been achieved from studies on pre-B and
immature B lymphocytes. The transcription factor Pax-5, essential for the differentiation into the B-cell specific lineage, cooperates with c-Myb, another transcription factor known to be predominantly expressed in immature hematopoietic cells, to bind to the RAG2 promoter. This cooperative binding, restricts expression of RAG2 in immature B cells, which requires LEF-1 a third transcription factor to activate in concert the RAG2 promoter [33]. However, additional transcription factors may be needed. Through its binding to the enhancer element Erag, E2A activates the RAG2 promoter in early B cell precursors [34]. This means that RAG transcription can be regulated by distinct elements likely required for optimal levels of RAG expression in the different B-cell stages. Regarding the BCR-induced RAG mRNA transcription, NFkappa B family members play important roles. Thus, studies involving transduction of a superrepressive I kappaB alpha protein indicate that increased level of RAG mRNA is dependent of NF-kappaB/Rel proteins [35]. Moreover, BCR-mediated upregulation of RAG is counter-balanced by NF-kappaB1, because immature B cells of NF-kappaB1-deficient mice overexpress RAGs and undergo exaggerated receptor editing response [35]. Among the transcription factors activated by the NF-kappaB canonical pathway, interferon regulatory factor 4 (IRF-4) has been recently identified as a nuclear effector of BCR signalings that promote receptor editing. IRF-4-deficient mice present defective secondary Ig gene rearrangements in response to membrane-bound Ag on immature B cells [29]. This suggests that IRF-4 is involved in the up-regulation of RAG at the immature Bcell stage, as it has already been demonstrated in pre-B cells. Recent studies on essential transcriptional regulator of Bcell development have demonstrated that forkhead protein Foxp1 binds to the Erag enhancer and controls Ig H chain gene recombinations. Furthermore, in Foxp1-deficient mice, RAG1 and RAG2 expression is diminished and associated with a blockade in the pro-B to pre-B cell transition [36]. Foxp1 appears as a key contributor in the transcriptional network that up-regulates RAG1 and RAG2, and thereby the B-cell lymphopoiesis. However, transcription of RAG1 and RAG2 can also be regulated in Erag-deficient mice, indicating that other transcription factors play important role. Foxo1, a member of the Fox family transcription factors, directly activates RAG1 and RAG2 expression in pro-B, pre-B, as well as autoreactive immature B cells [37]. It is suggested that in non-autoreactive immature B cells, basal tonic signal activates the PI3K/Akt pathway that phosphorylates Foxo1 inducing its cytoplasmic translocation and degradation. Consequently, RAG1 and RAG2 transcription is repressed [37]. Engagement of autoreactive BCR recruits BLNK that counteracts Akt activity and promotes Foxo1 function on the RAG1 and RAG2 up-regulation. Whether Foxo1 is activated in mature B cells to increase RAG1 and RAG2 transcription has to be demonstrated. However, the positive effect observed when serine/threonine phosphatases are inhibited suggests that RAG up-regulation in mature B cells may be independent of Foxo1. 5. RAG up-regulation in disease conditions The stochastic Ig gene recombination during B-cell ontogenesis generates autoreactive cells that are tolerized
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following receptor editing in the bone marrow. Re-induction of RAG1 and RAG2 expression at the immature B-cell stage is an important mechanism for the establishment of the B-cell repertoire. Deficiency in RAG up-regulation would contributes to the persistence of autoreactive B cells and their migration in the periphery would facilitate the development of autoimmune responses [38]. However, defective control of RAG expression would encourage uncontrolled Ig gene recombinations and the possibility that autoreactive BCR arise. Abnormal increased frequency of RAG-expressing B cells has thus been detected in the peripheral blood of systemic lupus erythematosus (SLE) patients [39], as well as within in the synovial tissues of rheumatoid arthritis (RA) patients [40]. The mechanism by which such aberrant RAG upregulation occurs is not well understood. Nevertheless, it seems in SLE B lymphocytes that inappropriate BCRmediated signals are triggered following their engagements in vitro, inducing the persistence of RAG mRNA and the inability to switch their expression off [39]. This effect is the result of an aberrant production of IL-6 that further raises the level of p27Kip1 expression, thereby maintaining RAG2 into the nucleus. Such uncontrolled secondary Ig gene rearrangements can contribute to the generation of pathogenic autoreactivity. In the synovial tissues of RA patients, two cooperative signals are responsible for the abnormal up-regulation of RAG1 and RAG2. One comes from the presence of the B cell activating factor, BAFF, on the membrane of synoviocytes that can interact with its receptor BR3 on the surface of the B lymphocytes. The second is IL-6 which is highly produced by RA synoviocytes and not dispensable for the induction of RAG mRNA (Rochas et al., in press). Both signals induce RAG upregulation and secondary Ig recombination that could lead to the local production of autoreactive B cells. Taken as a whole, molecular pathways contributing to the regulation of RAG expression provide valuable therapeutic targets to avoid the production of autoAbs in autoimmune diseases.
Take-home messages • RAG up-regulation in pre-B cells requires cell-to-cell contact with bone marrow stromal cells associated with cytokine stimulation. • RAG up-regulation in autoreactive immature B cells is induced following BCR engagement associated with BCRindependent incompetent signal or high tonic signal. • RAG up-regulation in mature B cells is triggered by cell surface molecule stimulations in association with cytokine signalings. • RAG up-regulation is controlled by complex intra-cellular pathways and transcription factor recruitments. • Deficiency in RAG up-regulation or aberrant RAG expression can potentially promote the development of autoreactive B cells. • Molecules involved in the control of RAG expression are therapeutic targets to circumvent the appearance of pathogenic autoreactive B cells.
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[33] Jin ZX, Kishi H, Wei XC, Matsuda T, Saito S, Muraguchi A. Lymphoid enhancer-binding factor-1 binds and activates the recombinationactivating gene-2 promoter together with c-Myb and Pax-5 in immature B cells. J Immunol 2002;169:3783–92. [34] Hsu LY, Lauring J, Liang HE, Greenbaum S, Cado D, Zhuang Y, et al. A conserved transcriptional enhancer regulates RAG gene expression in developing B cells. Immunity 2003;19:105–17. [35] Verkoczy L, Aït-Azzouzene D, Skog P, Märtensson A, Lang J, Duong B, et al. A role for nuclear factor kappa B/rel transcription factors in the regulation of the recombinase activator genes. Immunity 2005;22: 519–31. [36] Hu H, Wang B, Borde M, Nardone J, Maika S, Allred L, et al. Foxp1 is an essential transcriptional regulator of B cell development. Nat Immunol 2006;7:819–26. [37] Amin RH, Schlissel MS. Foxo1 directly regulates the transcription of recombination-activating genes during B cell development. Nat Immunol 2008;9:613–22. [38] Youinou P, Hillion S, Jamin C, Pers JO, Saraux A, Renaudineau Y. B lymphocytes on the front line of autoimmunity. Autoimmun Rev 2006;5:215–21. [39] Hillion S, Garaud S, Devauchelle V, Bordron A, Berthou C, Youinou P, et al. Interleukin-6 is responsible for aberrant B-cell receptor-mediated regulation of RAG expression in systemic lupus erythematosus. Immunology 2007;122:371–80. [40] Rochas C, Hillion S, Youinou P, Jamin C, Devauchelle-Pensec V. RAGmediated secondary rearrangements of B-cell antigen receptors in rheumatoid synovial tissue. Autoimmun Rev 2007;7:155–9.
Anti-elastin antibodies and elastin turnover in normal pregnancy and recurrent pregnancy loss The role of elastin turnover and autoimmunity in patients with a history either of recurrent pregnancy loss (RPL) or during normal pregnancy has not been studied before. Konova E. et al. (Am J Reprod Immunol 2009: 61: 167–74) have recently investigated the role of anti-alpha-elastin and anti-tropoelastin IgG and IgM antibodies in sera amples of 60 medically and obstetrically normal pregnant women, classified to three trimester groups, 18 female patients with RPL and 18 healthy non-pregnant women with a history of successful pregnancies. They were measured by a home-made ELISA. One way analyses of variance and Least Significant Difference method were used for a statistical analysis. As results they found that anti-alpha-elastin IgG autoantibodies were significantly decreased in the third trimester pregnant women. IgM anti-alpha-elastin autoantibodies were significantly decreased in all pregnancy groups compared with the controls. Synthesis/degradation ratio of elastin was significantly increased in the third trimester pregnancy group, suggesting decreased elastin degradation during this period of pregnancy. Comparing the RPL patients with the healthy non-pregnant controls showed a significantly increased anti-alpha-elastin IgG antibody and significantly decreased synthesis/degradation ratio in the patient's group, suggesting increased elastin degradation in RPL. They concluded that elastin degradation is decreased during normal pregnancy. Increased anti-elastin IgG antibodies may contribute to the pathogenesis of pregnancy losses.
Beta(2)-glycoprotein I is a cofactor for tissue plasminogen activator-mediated plasminogen activation The regulation of the conversion of plasminogen to plasmin by tissue plasminogen activator (tPA) is critical in the control of fibrin deposition. While several plasminogen activators have been described, soluble plasma cofactors that stimulate fibrinolysis have not been characterized. In their study Bu C, et al. (Arthritis Rheum. 2009; 60:559–568), intended to investigate the effects of beta (2)glycoprotein I (beta(2)GPI), an abundant plasma glycoprotein, on tPA-mediated plasminogen activation. The effect of beta (2)GPI on tPA-mediated activation of plasminogen was assessed using amidolytic assays, a fibrin gel, and plasma clots. Binding of beta (2)GPI to tPA and plasminogen was determined in parallel. The effects of IgG fractions and anti-beta (2)GPI antibodies from patients with antiphospholipid syndrome (APS) on tPA-mediated plasminogen activation were also measured. They found that beta (2)glycoprotein I stimulated tPA-dependent plasminogen activation in the fluid phase and within a fibrin gel. The beta(2)GPI region responsible for stimulating tPA activity was shown to be at least partly contained within beta(2)GPI domain V. In addition, beta (2) GPI bound tPA with high affinity (K(d) approximately 20 nM), stimulated tPA amidolytic activity, and caused an overall 20-fold increase in the catalytic efficiency (K(cat)/K(m)) of tPA-mediated conversion of Glu-plasminogen to plasmin. Moreover, depletion of beta(2)GPI from plasma led to diminished rates of clot lysis, with restoration of normal lysis rates following beta(2)GPI repletion. Stimulation of tPA-mediated plasminogen activity by beta (2)GPI was inhibited by monoclonal anti-beta (2)GPI antibodies as well as by anti-beta (2)GPI antibodies from patients with APS. Those findings suggest that beta (2)GPI may be an endogenous regulator of fibrinolysis. Impairment of beta (2)GPI-stimulated fibrinolysis by anti-beta(2)GPI antibodies may contribute to the development of thrombosis in patients with APS.
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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
Signaling pathways regulating RAG expression in B lymphocytes Sophie Hillion, Caroline Rochas, Pierre Youinou ⁎, Christophe Jamin Université Européenne de Bretagne, France Université de Bretagne Occidentale, EA 2216 Immunology and Pathology, IFR 148 ScInBioS, Brest F-29609, France Brest Medical School Hospital, Morvan, Brest F-29609, France
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Article history: Received 2 January 2009 Accepted 4 February 2009 Available online 9 February 2009 Keywords: B lymphocytes RAG Cytokines Autoimmunity Up-regulation
a b s t r a c t Development of B-cell lymphopoiesis is dependent on the presence of recombination activating genes RAG1 and RAG2 enzymes. They control the rearrangements of immunoglobulin variable, diversity and joining region segments, and allow progression of the cellular maturation. RAG1 and RAG2 are successively up- and down-regulated at each B-cell stage to progressively generate a B-cell receptor for which unforeseeable antigenic specificity results from a stochastic process. Therefore, in autoreactive immature B cells, new round of RAG re-expression can be observed to eliminate self-reactivity. In some circumstances, RAG up-regulation can also be found in peripheral mature B lymphocytes, specifically in autoimmune diseases. It is therefore of utmost importance to unravel signaling pathways that trigger RAG induction in normal and pathological conditions. Therapeutic modulation of cytokines or intracellular contacts involved in RAG expression might restrict the development of inappropriate autoimmune repertoire. © 2009 Elsevier B.V. All rights reserved.
Contents 1. Introduction . . . . . . . . . . . . . . 2. Cell surface signals. . . . . . . . . . . 3. Intra-cellular pathways. . . . . . . . . 4. Nucleus factors . . . . . . . . . . . . 5. RAG up-regulation in disease conditions Take-home messages . . . . . . . . . . . . References . . . . . . . . . . . . . . . . .
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1. Introduction Maturation of B lymphocytes is closely associated with immunoglobulin (Ig) gene rearrangements. Beginning in the bone marrow by commitment of lymphoid progenitors to the B lineage, initial diversity of the heavy chain (DH) to joining (JH) gene segments rearrange in pro-B cells. This is followed ⁎ Corresponding author. Laboratory of Immunology, Brest University Medical School Hospital, BP824, F-29609, Brest, France. Tel.: +33 298 22 33 84; fax: +33 298 22 38 47. E-mail address: [email protected] (P. Youinou). 1568-9972/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2009.02.004
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by recombination of a variable (VH) gene with the DJH segment, thus creating a complete VDJH rearrangement. Pro-B cells then differentiate into pre-B cells subdivided into proliferating pre-BI cells bearing the pre-B cell receptor (BCR) that utilize the rearranged heavy chain, and resting pre-BII cells that initiate Ig κ or λ light (L) chain gene rearrangement between VL and JL segments. Pre-BII cells differentiate into immature B cells that express a complete BCR corresponding to the surface expression of IgM. All these Ig gene rearrangements require the action of the recombination activating genes (RAG)1 and RAG2 enzymes, of which the expression is strikingly related to the recombination process.
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RAG1 and RAG2 are highly expressed in pro-B and pre-BII cells but down-regulated in pre-BI and immature B cells. BCRs are assembled via stochastic VDJH and VJL rearrangements, and comprised randomly selected H and L chains. The resulting complexes have an unforecasted specificity that can include recognition of auto-antigens. To avoid the terminal differentiation of autoreactive B lymphocytes, tolerance mechanisms take place in the bone marrow, such as elimination (clonal deletion) or anergy (inactivation) of the self-reactive B cells. However, newly L chain gene rearrangement, called receptor editing [1], can be re-induced at the immature B-cell stage due to a secondary up-regulation of RAG1 and RAG2 proteins, which appears to be the main mechanism of B cell tolerance. When they have reach the periphery, immature B cells progress as transitional B lymphocytes that can differentiate into mature B cells to develop a germinal centre (GC) response following encounter with antigen in secondary lymphoid organs. Initially observed in vitro using murine B cells, it has been suggested that RAG proteins could be reinduced in mature B cells [2]. Later, the presence of RAGpositive mature B lymphocytes within human tonsil tissues [3], has supported the notion that RAG up-regulation could also occur in peripheral lymphoid organs. The ensuing peripheral Ig gene rearrangements in mature B cells have been referred as receptor revision and would appear after somatic hypermutations [1]. This mechanism seems to be independent of tolerance purpose, but rather is induced in B cells expressing low-affinity BCRs to generate high affinity antibodies during affinity maturation in developing GCs. Activation, proliferation, differentiation or tolerance outcomes depend not only on the developmental stage, but also on the cell surface stimulated molecules. Therefore, engagement of BCR, of positive and negative regulators of BCR signaling, as well as of cytokine receptors instigates activation of intra-cellular signaling pathways that involve adaptor proteins, enzymes and secondary messengers. The resulting events are the recruitment of transcription factors that translocate into the nucleus to regulate gene expression. The current review is aimed at deciphering the molecular processes regulating expression of RAGs at the different stage of B-cell development. 2. Cell surface signals Several studies have shown that BCR signaling plays an important role in regulating RAG expression. Immature IgM+ IgD− B cells, and specifically those expressing a self-reactive BCR, can re-induce expression of RAG proteins following binding of the self-Ag in the bone marrow [4]. Stimulation of autoreactive BCR with soluble auto-Ag until the late immature B cell stage [5], provides an alternative process for the tolerance. Modification of the Ag specificity of newly developing cells eliminates autoreactivity without B-cell deletion. However, other models suggest that BCR cross-linking can lead to a down-regulation of RAG2 mRNA [6]. Therefore, another hypothesis has been proposed by Tze et al. who demonstrated that engagement of the BCR with self-Ag down-regulates surface Ig receptors via endocytosis, and triggers RAG up-regulation and subsequent receptor editing [7]. It is likely that diminished surface Igs results in a loss of
basal signal that allows re-induction of RAG expression and edition of Ig L genes under self-Ag stimulation. In contrast, competent BCR basal signaling inhibits RAG induction and receptor editing [8], and thereby allows the B-cell development to carry on. It is thought that RAG up-regulation in immature B cells would occur within a window during B-cell development (Fig. 1). The size of this window may vary according to the type of Ag and its concentration, as well as the avidity and the duration of Ag encounter [9]. The presence of positive regulator of BCR signaling such as CD19 plays also an important role in the B-cell differentiation. Thus, autoreactive immature B cells that lack CD19 have elevated tonic signals. Elevation of tonic signals up-regulates RAGs genes and stimulates receptor editing [10]. By contrast, lowering tonic signals suppress RAG expression and receptor editing, due to strong signalling transmission following BCR stimulation [11]. Similarly, immature autoreactive B cells deficient in CD45, which is a tyrosine phosphatase that functions as a positive regulator of the BCR signaling, are developmentally arrested in the presence of self-Ag, and undergo receptor editing. All these data therefore suggest that BCR-independent tonic signals can control RAG expression to suppress receptor editing and promote positive selection of immature B cells. The presence of RAG-positive cells in the GC of secondary lymphoid organs [12], raises the question as to whether the same signalings, as those observed in immature lymphocytes, would produce the same effects in mature B cells. Although it has been initially suggested that BCR cross-linking increases RAG1 mRNA level in a human mature B cell line, BCR ligation turns off RAG expression in human GC B cells [13], as well as in murine splenic B lymphocytes [14]. RAG induction may be inversely associated with either surface Ig expression on mature B cells [15], or with antigenic avidity of the BCRs [14]. The ensuing receptor revision process would thus occur to raise the BCR affinity in order to trigger maturation of the GC response [16]. Stimulation of BCR co-receptors can modulate RAG upregulation. Induction of RAG requires the co-ligation of CD40 with the BCR [17] or with another signal [2]. Moreover, RAG up-regulation in human peripheral B cells depends on the presence of CD5, known as a negative regulator of the BCR [17], suggesting that BCR threshold may play a role in the regulation of RAG expression in mature B cells. Consistent with this hypothesis, co-ligation of CD21/CD35 with the BCR abrogates much more efficiently RAG2 expression in murine splenic B cells than BCR engagement alone [18]. Several observations strongly suggest that two separate signals are required for the induction of RAG. This is the case for progenitor B cells that lack BCR on their surface. Upregulation of RAG mRNA is observed when a signal from cell surface molecule on stromal cells is associated with a second from cytokines [19]. Among the cytokines, IL-3, IL-6 and IL-7, but not IL-2, IL-4 and GM-CSF induce RAG gene expression in human lymphoid progenitor cell line (Fig. 1). It is likely that progenitor B lymphocytes may be sensitive to RAG upregulation during a window-time. Thus, after two days of stimulation with bone marrow stromal cells, these latter secrete IL-7 that up-regulates CD19 on progenitor B cells, and one day later down-regulates RAG1 and RAG2 expression. Interestingly, cross-linking of CD19 has no direct effect on
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Fig. 1. Signaling molecules involved in the control of RAG expression in pre-B, immature and mature B cells. Extra-cellular signals from interleukins in association with cell-surface molecules induce (arrows) or inhibit (blunted arrows) intra-cellular pathways. Intra-cytoplasmic signalings implicate adaptor proteins (BLNK), kinases activation (PKA, PKC, PI3K, MAPK, ERK, BtK, Akt), phosphatase inhibition (PP1−), phospholipase activation (PLCγ2), and increased concentration of secondary messengers (cAMP, iCa). These pathways recruit transcription factors involved in either activation (IRF4, E2A, Foxp1, Foxo1, NF-κB, Pax5, c-Myb, LEF-1) or inhibition (NF-kB1) of RAG expression.
RAG expression but blocks the IL-7 down-regulation of RAG, indicating the important role of co-receptor molecules [20]. Up-regulation of RAG in mature B cells also largely depends on cytokines. When polyclonally stimulated with Staphylococcus aureus Cowan I, more than half of peripheral human circulating B cells express RAG1 and RAG2 proteins in the presence of IL-2 [21]. IL-4 is another effective co-factor for RAG expression when human peripheral B cells are stimulated with anti-CD40 antibody, or when murine splenic B cells are stimulated with either lipopolysaccharide (LPS), antiCD40 or anti-IgM antibody [2]. It should be noted that LPS associated with other cytokines such as IL-2, IL-3 or IL-5 is ineffective. When co-stimulated with CD40 and BCR, human peripheral B cells as well as tonsillar B cells up-regulate RAG1 and RAG2 [22], due to the induced-secretion of IL-6 [23]. Finally, IL-7 also contributes to the re-expression of RAG observed in IgD+ mouse GC B cells [24]. All these data indicate
that complex intra-cellular pathways, induced from different surface molecules, are required to up-regulate RAG expression in B lymphocytes (Fig. 1). 3. Intra-cellular pathways Identification of intra-cellular signals involved in the control of RAG expression is complicated due to the differential responses observed during B-cell lymphopoiesis. In pre-B lymphocytes, increased level of cAMP, activator of protein kinase (PK) A, induces RAG expression [25], which is also up-regulated by inhibition of serine/threonine protein phosphatase PP1. By contrast, activation of PKC or elevated level of intra-cellular calcium inhibits RAG1 and RAG2 mRNA transcription [26]. In immature B cells, increases in intracellular free calcium, i.e. as occurs after BCR aggregation, induces up-regulation of RAG [27]. Autoreactive immature B
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cells from BLNK-deficient mice fail to promote Ig L chain gene recombination under BCR engagement, suggesting that the adaptor protein BLNK plays a role in the regulation of RAG expression after BCR stimulation [28]. Among the downstream molecules activated following binding to BLNK, phospholipase γ2 (PLCγ2) signaling pathway is important in activation of receptor editing, and thereby likely in RAG upregulation. Thus, PLCγ2-deficient mice exhibit impaired Aginduced receptor editing in autoreactive immature B cells [29]. Therefore, it may be that PLCγ2 which produces diacyglycerol (DAG) and inositol triphosphate, favors activation of PKC by DAG and releases of intra-cellular calcium that promotes RAG up-regulation and secondary Ig gene rearrangements. This signaling cascade proceeds also with activation of mitogen-activated protein kinases (MAPKs). Among the MAPK, extracellular signal-regulated kinase (ERK) activation is involved in RAG2 gene up-regulation. Thus, inhibition of the ERK signaling pathway impairs RAG2 expression and receptor editing in autoreactive immature murine B cells following auto-Ag stimulation [30]. Regulation of RAG induction is the result of a complex balance between several intra-cellular signaling pathways. In immature murine B lymphocytes that carry a nonautoreactive BCR, phosphatidylinositol 3-kinase (PI3K) signaling is required to suppress RAG expression, otherwise inappropriate receptor editing undergoes, as occurs in mice deficient in the PI3K. This basal BCR-directed PI3K signaling activates the downstream effector PLCγ2, Akt and Btk. Of these, PLCγ2 seems to play a significant role in down-regulating RAG expression [31], though the serine/threonine kinase, Akt can also repress the transcription of RAG1 and RAG2 [32]. In mature B cells, regulation of RAG expression appears to be only partially similarly controlled. While inhibition of PP1 up-regulates the expression of RAG1 and RAG2 as in pre-B cells [25], increased level of cAMP has no effect. Furthermore, in human plasma cell lines expressing RAG1 and RAG2, inhibition of PP1 does not affect the expression level of the RAG proteins although Ig L chain shifting can be induced [32]. These observations suggest that phosphatase inhibition can not only regulate RAG expression in mature B lymphocytes, but also their activity in plasma cells. However, in contrast to the effects observed in pre-B cells [26], stimulation of mature B lymphocytes with a combination of phorbol myristic acetate and inomycin, that activates PKC and increases the level of intra-cellular free calcium, up-regulates the level of RAG mRNA [31]. Finally, splenic mature B cells from PLCγ2deficient mice have a reduced frequency of lambda-expressing B cell subpopulation, and their Ig L chain gene rearrangements are barely detectable following self-Ag stimulation [29]. These data indicates that receptor editing is altered in mature B lymphocytes when PLCγ2 is lacking, and thereby that PLCγ2 is required for the up-regulation of RAG1 and RAG2 at the mature B-cell stage. These regulatory pathways are summarized in Fig. 1. 4. Nucleus factors Activation of these intra-cellular pathways works towards the recruitment of nuclear factors to control RAG1 and RAG2 gene expression (Fig. 1). Most of available informations have been achieved from studies on pre-B and
immature B lymphocytes. The transcription factor Pax-5, essential for the differentiation into the B-cell specific lineage, cooperates with c-Myb, another transcription factor known to be predominantly expressed in immature hematopoietic cells, to bind to the RAG2 promoter. This cooperative binding, restricts expression of RAG2 in immature B cells, which requires LEF-1 a third transcription factor to activate in concert the RAG2 promoter [33]. However, additional transcription factors may be needed. Through its binding to the enhancer element Erag, E2A activates the RAG2 promoter in early B cell precursors [34]. This means that RAG transcription can be regulated by distinct elements likely required for optimal levels of RAG expression in the different B-cell stages. Regarding the BCR-induced RAG mRNA transcription, NFkappa B family members play important roles. Thus, studies involving transduction of a superrepressive I kappaB alpha protein indicate that increased level of RAG mRNA is dependent of NF-kappaB/Rel proteins [35]. Moreover, BCR-mediated upregulation of RAG is counter-balanced by NF-kappaB1, because immature B cells of NF-kappaB1-deficient mice overexpress RAGs and undergo exaggerated receptor editing response [35]. Among the transcription factors activated by the NF-kappaB canonical pathway, interferon regulatory factor 4 (IRF-4) has been recently identified as a nuclear effector of BCR signalings that promote receptor editing. IRF-4-deficient mice present defective secondary Ig gene rearrangements in response to membrane-bound Ag on immature B cells [29]. This suggests that IRF-4 is involved in the up-regulation of RAG at the immature Bcell stage, as it has already been demonstrated in pre-B cells. Recent studies on essential transcriptional regulator of Bcell development have demonstrated that forkhead protein Foxp1 binds to the Erag enhancer and controls Ig H chain gene recombinations. Furthermore, in Foxp1-deficient mice, RAG1 and RAG2 expression is diminished and associated with a blockade in the pro-B to pre-B cell transition [36]. Foxp1 appears as a key contributor in the transcriptional network that up-regulates RAG1 and RAG2, and thereby the B-cell lymphopoiesis. However, transcription of RAG1 and RAG2 can also be regulated in Erag-deficient mice, indicating that other transcription factors play important role. Foxo1, a member of the Fox family transcription factors, directly activates RAG1 and RAG2 expression in pro-B, pre-B, as well as autoreactive immature B cells [37]. It is suggested that in non-autoreactive immature B cells, basal tonic signal activates the PI3K/Akt pathway that phosphorylates Foxo1 inducing its cytoplasmic translocation and degradation. Consequently, RAG1 and RAG2 transcription is repressed [37]. Engagement of autoreactive BCR recruits BLNK that counteracts Akt activity and promotes Foxo1 function on the RAG1 and RAG2 up-regulation. Whether Foxo1 is activated in mature B cells to increase RAG1 and RAG2 transcription has to be demonstrated. However, the positive effect observed when serine/threonine phosphatases are inhibited suggests that RAG up-regulation in mature B cells may be independent of Foxo1. 5. RAG up-regulation in disease conditions The stochastic Ig gene recombination during B-cell ontogenesis generates autoreactive cells that are tolerized
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following receptor editing in the bone marrow. Re-induction of RAG1 and RAG2 expression at the immature B-cell stage is an important mechanism for the establishment of the B-cell repertoire. Deficiency in RAG up-regulation would contributes to the persistence of autoreactive B cells and their migration in the periphery would facilitate the development of autoimmune responses [38]. However, defective control of RAG expression would encourage uncontrolled Ig gene recombinations and the possibility that autoreactive BCR arise. Abnormal increased frequency of RAG-expressing B cells has thus been detected in the peripheral blood of systemic lupus erythematosus (SLE) patients [39], as well as within in the synovial tissues of rheumatoid arthritis (RA) patients [40]. The mechanism by which such aberrant RAG upregulation occurs is not well understood. Nevertheless, it seems in SLE B lymphocytes that inappropriate BCRmediated signals are triggered following their engagements in vitro, inducing the persistence of RAG mRNA and the inability to switch their expression off [39]. This effect is the result of an aberrant production of IL-6 that further raises the level of p27Kip1 expression, thereby maintaining RAG2 into the nucleus. Such uncontrolled secondary Ig gene rearrangements can contribute to the generation of pathogenic autoreactivity. In the synovial tissues of RA patients, two cooperative signals are responsible for the abnormal up-regulation of RAG1 and RAG2. One comes from the presence of the B cell activating factor, BAFF, on the membrane of synoviocytes that can interact with its receptor BR3 on the surface of the B lymphocytes. The second is IL-6 which is highly produced by RA synoviocytes and not dispensable for the induction of RAG mRNA (Rochas et al., in press). Both signals induce RAG upregulation and secondary Ig recombination that could lead to the local production of autoreactive B cells. Taken as a whole, molecular pathways contributing to the regulation of RAG expression provide valuable therapeutic targets to avoid the production of autoAbs in autoimmune diseases.
Take-home messages • RAG up-regulation in pre-B cells requires cell-to-cell contact with bone marrow stromal cells associated with cytokine stimulation. • RAG up-regulation in autoreactive immature B cells is induced following BCR engagement associated with BCRindependent incompetent signal or high tonic signal. • RAG up-regulation in mature B cells is triggered by cell surface molecule stimulations in association with cytokine signalings. • RAG up-regulation is controlled by complex intra-cellular pathways and transcription factor recruitments. • Deficiency in RAG up-regulation or aberrant RAG expression can potentially promote the development of autoreactive B cells. • Molecules involved in the control of RAG expression are therapeutic targets to circumvent the appearance of pathogenic autoreactive B cells.
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Anti-elastin antibodies and elastin turnover in normal pregnancy and recurrent pregnancy loss The role of elastin turnover and autoimmunity in patients with a history either of recurrent pregnancy loss (RPL) or during normal pregnancy has not been studied before. Konova E. et al. (Am J Reprod Immunol 2009: 61: 167–74) have recently investigated the role of anti-alpha-elastin and anti-tropoelastin IgG and IgM antibodies in sera amples of 60 medically and obstetrically normal pregnant women, classified to three trimester groups, 18 female patients with RPL and 18 healthy non-pregnant women with a history of successful pregnancies. They were measured by a home-made ELISA. One way analyses of variance and Least Significant Difference method were used for a statistical analysis. As results they found that anti-alpha-elastin IgG autoantibodies were significantly decreased in the third trimester pregnant women. IgM anti-alpha-elastin autoantibodies were significantly decreased in all pregnancy groups compared with the controls. Synthesis/degradation ratio of elastin was significantly increased in the third trimester pregnancy group, suggesting decreased elastin degradation during this period of pregnancy. Comparing the RPL patients with the healthy non-pregnant controls showed a significantly increased anti-alpha-elastin IgG antibody and significantly decreased synthesis/degradation ratio in the patient's group, suggesting increased elastin degradation in RPL. They concluded that elastin degradation is decreased during normal pregnancy. Increased anti-elastin IgG antibodies may contribute to the pathogenesis of pregnancy losses.
Beta(2)-glycoprotein I is a cofactor for tissue plasminogen activator-mediated plasminogen activation The regulation of the conversion of plasminogen to plasmin by tissue plasminogen activator (tPA) is critical in the control of fibrin deposition. While several plasminogen activators have been described, soluble plasma cofactors that stimulate fibrinolysis have not been characterized. In their study Bu C, et al. (Arthritis Rheum. 2009; 60:559–568), intended to investigate the effects of beta (2)glycoprotein I (beta(2)GPI), an abundant plasma glycoprotein, on tPA-mediated plasminogen activation. The effect of beta (2)GPI on tPA-mediated activation of plasminogen was assessed using amidolytic assays, a fibrin gel, and plasma clots. Binding of beta (2)GPI to tPA and plasminogen was determined in parallel. The effects of IgG fractions and anti-beta (2)GPI antibodies from patients with antiphospholipid syndrome (APS) on tPA-mediated plasminogen activation were also measured. They found that beta (2)glycoprotein I stimulated tPA-dependent plasminogen activation in the fluid phase and within a fibrin gel. The beta(2)GPI region responsible for stimulating tPA activity was shown to be at least partly contained within beta(2)GPI domain V. In addition, beta (2) GPI bound tPA with high affinity (K(d) approximately 20 nM), stimulated tPA amidolytic activity, and caused an overall 20-fold increase in the catalytic efficiency (K(cat)/K(m)) of tPA-mediated conversion of Glu-plasminogen to plasmin. Moreover, depletion of beta(2)GPI from plasma led to diminished rates of clot lysis, with restoration of normal lysis rates following beta(2)GPI repletion. Stimulation of tPA-mediated plasminogen activity by beta (2)GPI was inhibited by monoclonal anti-beta (2)GPI antibodies as well as by anti-beta (2)GPI antibodies from patients with APS. Those findings suggest that beta (2)GPI may be an endogenous regulator of fibrinolysis. Impairment of beta (2)GPI-stimulated fibrinolysis by anti-beta(2)GPI antibodies may contribute to the development of thrombosis in patients with APS.