Cracking the BAFF code.

The tumour necrosis factor (TNF) family members B cell activating factor (BAFF) and APRIL (a proliferation-inducing ligand) are crucial survival factors for peripheral B cells. An excess of BAFF leads to the development of autoimmune disorders in animal models, and high levels of BAFF have been detected in the serum of patients with various autoimmune conditions. In this Review, we consider the possibility that in mice autoimmunity induced by BAFF is linked to T cell-independent B cell activation rather than to a severe breakdown of B cell tolerance. We also outline the mechanisms of BAFF signalling, the impact of ligand oligomerization on receptor activation and the progress of BAFF-depleting agents in the clinical setting.

Heterologous desensitization of the glucagon-like peptide-1 receptor by phorbol esters requires phosphorylation of the cytoplasmic tail at four different sites.

Glucagon-like peptide-1 stimulates glucose-induced insulin secretion by binding to a specific G protein-coupled receptor that activates the adenylyl cyclase pathway. We previously demonstrated that heterologous desensitization of the receptor by protein kinase C correlated with phosphorylation in a 33-amino acid-long segment of the receptor carboxyl-terminal cytoplasmic tail. Here, we determined that the in vivo sites of phosphorylation are four serine doublets present at positions 431/432, 441/442, 444/445, and 451/452. In vitro phosphorylation of fusion proteins containing mutant receptor C-tails, however, indicated that whereas serines at position 431/432 were good substrates for protein kinase C (PKC), serines 444/445 and 451/452 were poor substrates, and serines 441/442 were not substrates. In addition, serine 416 was phosphorylated on fusion protein but not in intact cells. This indicated that in vivo a different PKC isoform or a PKC-activated kinase may phosphorylate the receptor. The role of phosphorylation on receptor desensitization was assessed using receptor mutants expressed in COS cells or Chinese hamster lung fibroblasts. Mutation of any single serine doublet to alanines reduced the extent of phorbol 12-myristate 13-acetate-induced desensitization, whereas substitution of any combination of two serine doublets suppressed it. Our data thus show that the glucagon-like peptide-1 receptor can be phosphorylated in response to phorbol 12-myristate 13-acetate on four different sites within the cytoplasmic tail. Furthermore, phosphorylation of at least three sites was required for desensitization, although maximal desensitization was only achieved when all four sites were phosphorylated.

Evidence for multiple B- and T-cell epitopes in Plasmodium falciparum liver-stage antigen 3.

Liver-stage antigen 3 (LSA-3) is a new vaccine candidate that can induce protection against Plasmodium falciparum sporozoite challenge. Using a series of long synthetic peptides (LSP) encompassing most of the 210-kDa LSA-3 protein, a study of the antigenicity of this protein was carried out in 203 inhabitants from the villages of Dielmo (n = 143) and Ndiop (n = 60) in Senegal (the level of malaria transmission differs in these two villages). Lymphocyte responses to each individual LSA-3 peptide were recorded, some at high prevalences (up to 43%). Antibodies were also detected to each of the 20 peptides, many at high prevalence (up to 84% of responders), and were directed to both nonrepeat and repeat regions. Immune responses to LSA-3 were detectable even in individuals of less than 5 years of age and increased with age and hence exposure to malaria, although they were not directly related to the level of malaria transmission. Thus, several valuable T- and B-cell epitopes were characterized all along the LSA-3 protein, supporting the antigenicity of this P. falciparum vaccine candidate. Finally, antibodies specific for peptide LSP10 located in a nonrepeat region of LSA-3 were found significantly associated with a lower risk of malaria attack over 1 year of daily clinical follow-up in children between the ages of 7 and 15 years, but not in older individuals.

Human invariant V alpha 24-J alpha Q TCR supports the development of CD1d-dependent NK1.1+ and NK1.1- T cells in transgenic mice.

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR V alpha 24-J alpha Q and V alpha 14-J alpha 18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. V alpha 24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. V alpha 24-J alpha Q TCR chain in all T cells. The expression of the human inv. V alpha 24 TCR in TCR C alpha(-/-) mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand alpha-galactosylceramide (alpha-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in V alpha 24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR V beta 7 and a corresponding decrease in TCR V beta 8.2 use. Despite the forced expression of the human CD1d-restricted TCR in C alpha(-/-) mice, staining with mCD1d-alpha-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1(-) T cells that bind CD1d-alpha-GalCer tetramers. These findings indicate that human inv. V alpha 24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. V alpha 24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.

Identification of proteoglycans as the APRIL-specific binding partners.

B cell activating factor of the tumor necrosis factor (TNF) family (BAFF) and a proliferation-inducing ligand (APRIL) are closely related ligands within the TNF superfamily that play important roles in B lymphocyte biology. Both ligands share two receptors--transmembrane activator and calcium signal--modulating cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA)--that are predominantly expressed on B cells. In addition, BAFF specifically binds BAFF receptor, whereas the nature of a postulated APRIL-specific receptor remains elusive. We show that the TNF homology domain of APRIL binds BCMA and TACI, whereas a basic amino acid sequence (QKQKKQ) close to the NH2 terminus of the mature protein is required for binding to the APRIL-specific "receptor." This interactor was identified as negatively charged sulfated glycosaminoglycan side chains of proteoglycans. Although T cell lines bound little APRIL, the ectopic expression of glycosaminoglycan-rich syndecans or glypicans conferred on these cells a high binding capacity that was completely dependent on APRIL's basic sequence. Moreover, syndecan-1-positive plasma cells and proteoglycan-rich nonhematopoietic cells displayed high specific, heparin-sensitive binding to APRIL. Inhibition of BAFF and APRIL, but not BAFF alone, prevented the survival and/or the migration of newly formed plasma cells to the bone marrow. In addition, costimulation of B cell proliferation by APRIL was only effective upon APRIL oligomerization. Therefore, we propose a model whereby APRIL binding to the extracellular matrix or to proteoglycan-positive cells induces APRIL oligomerization, which is the prerequisite for the triggering of TACI- and/or BCMA-mediated activation, migration, or survival signals.

Cathepsin X cleavage of the beta2 integrin regulates talin-binding and LFA-1 affinity in T cells.

T cell migration, essential for immune surveillance and response, is mediated by the integrin LFA-1. CatX, a cysteine carboxypeptidase, is involved in the regulation of T cell migration by interaction with LFA-1. We show that sequential cleavage of C-terminal amino acids from the β(2) cytoplasmic tail of LFA-1, by CatX, enhances binding of the adaptor protein talin to LFA-1 and triggers formation of the latter's high-affinity form. As shown by SPR analysis of peptides constituting the truncated β(2) tail, the cleavage of three C-terminal amino acids by CatX resulted in a 1.6-fold increase of talin binding. Removal of one more amino acid resulted in a 2.5-fold increase over the intact tail. CatX cleavage increased talin-binding affinity to the MD but not the MP talin-binding site on the β(2) tail. This was shown by molecular modeling of the β(2) tail/talin F3 complex to be a result of conformational changes affecting primarily the distal-binding site. Analysis of LFA-1 by conformation-specific mAb showed that CatX modulates LFA-1 affinity, promoting formation of high-affinity from intermediate-affinity LFA-1 but not the initial activation of LFA-1 from a bent to extended form. CatX post-translational modifications may thus represent a mechanism of LFA-1 fine-tuning that enables the trafficking of T cells.

Antigen Receptor Signaling to NF-kappa B via CARMA1, BCL10, and MALT1

The signaling pathway controlling antigen receptor-induced regulation of the transcription factor NF-kappa B plays a key role in lymphocyte activation and development and the generation of lymphomas. Work of the past decade has led to dramatic progress in the identification and characterization of new players in the pathway. Moreover, novel enzymatic activities relevant for this pathway have been discovered, which represent interesting drug targets for immuno-suppression or lymphoma treatment. Here, we summarize these findings and give an outlook on interesting open issues that need to be addressed in the future.

BAFF mediates survival of peripheral immature B lymphocytes.

B cell maturation is a very selective process that requires finely tuned differentiation and survival signals. B cell activation factor from the TNF family (BAFF) is a TNF family member that binds to B cells and potentiates B cell receptor (BCR)-mediated proliferation. A role for BAFF in B cell survival was suggested by the observation of reduced peripheral B cell numbers in mice treated with reagents blocking BAFF, and high Bcl-2 levels detected in B cells from BAFF transgenic (Tg) mice. We tested in vitro the survival effect of BAFF on lymphocytes derived from primary and secondary lymphoid organs. BAFF induced survival of a subset of splenic immature B cells, referred to as transitional type 2 (T2) B cells. BAFF treatment allowed T2 B cells to survive and differentiate into mature B cells in response to signals through the BCR. The T2 and the marginal zone (MZ) B cell compartments were particularly enlarged in BAFF Tg mice. Immature transitional B cells are targets for negative selection, a feature thought to promote self-tolerance. These findings support a model in which excessive BAFF-mediated survival of peripheral immature B cells contributes to the emergence and maturation of autoreactive B cells, skewed towards the MZ compartment. This work provides new clues on mechanisms regulating B cell maturation and tolerance.

Association of BAFF/BLyS overexpression and altered B cell differentiation with Sjögren's syndrome.

BAFF (BLyS, TALL-1, THANK, zTNF4) is a member of the TNF superfamily that specifically regulates B lymphocyte proliferation and survival. Mice transgenic (Tg) for BAFF develop an autoimmune condition similar to systemic lupus erythematosus. We now demonstrate that BAFF Tg mice, as they age, develop a secondary pathology reminiscent of Sjögren's syndrome (SS), which is manifested by severe sialadenitis, decreased saliva production, and destruction of submaxillary glands. In humans, SS also correlates with elevated levels of circulating BAFF, as well as a dramatic upregulation of BAFF expression in inflamed salivary glands. A likely explanation for disease in BAFF Tg mice is excessive survival signals to autoreactive B cells, possibly as they pass through a critical tolerance checkpoint while maturing in the spleen. The marginal zone (MZ) B cell compartment, one of the enlarged B cell subsets in the spleen of BAFF Tg mice, is a potential reservoir of autoreactive B cells. Interestingly, B cells with an MZ-like phenotype infiltrate the salivary glands of BAFF Tg mice, suggesting that cells of this compartment potentially participate in tissue damage in SS and possibly other autoimmune diseases. We conclude that altered B cell differentiation and tolerance induced by excess BAFF may be central to SS pathogenesis.

The scaffold protein IB1/JIP-1 controls the activation of JNK in rat stressed urothelium.

The c-Jun N-terminal kinase (JNK) is critical for cell survival, differentiation, apoptosis and tumorigenesis. This signalling pathway requires the presence of the scaffold protein Islet-Brain1/c-Jun N-terminal kinase interacting protein-1 (IB1/JIP-1). Immunolabeling and in situ hybridisation of bladder sections showed that IB1/JIP-1 is expressed in urothelial cells. The functional role of IB1/JIP-1 in the urothelium was therefore studied in vivo in a model of complete rat bladder outlet obstruction. This parietal stress, which is due to urine retention, reduced the content of IB1/JIP-1 in urothelial cells and consequently induced a drastic increase in JNK activity and AP-1 binding activity. Using a viral gene transfer approach, the stress-induced activation of JNK was prevented by overexpressing IB1/JIP-1. Conversely, the JNK activity was increased in urothelial cells where the IB1/JIP-1 content was experimentally reduced using an antisense RNA strategy. Furthermore, JNK activation was found to be increased in non-stressed urothelial cells of heterozygous mice carrying a selective disruption of the IB1/JIP-1 gene. These data established that mechanical stress in urothelial cells in vivo induces a robust JNK activation as a consequence of regulated expression of the scaffold protein IB1/JIP-1. This result highlights a critical role for that scaffold protein in the homeostasis of the urothelium and unravels a new potential target to regulate the JNK pathway in this tissue.

Desensitization and phosphorylation of the glucagon-like peptide-1 (GLP-1) receptor by GLP-1 and 4-phorbol 12-myristate 13-acetate.

Glucagon-like peptide-1 (GLP-1) stimulates glucose-induced insulin secretion by binding to a specific G protein-coupled receptor linked to activation of the adenylyl cyclase pathway. Here, using insulinoma cell lines, we studied homologous and heterologous desensitization of GLP-1-induced cAMP production. Preexposure of the cells to GLP-1 induced a decrease in GLP-1-mediated cAMP production, as assessed by a 3- to 5-fold rightward shift of the dose-response curve and an approximately 20 percent decrease in the maximal production of cAMP. Activation of protein kinase C by the phorbol ester phorbol 12-myristate 13-acetate (PMA) also induced desensitization of the GLP-1-mediated response, leading to a 6- to 9-fold shift in the EC50 and a 30% decrease in the maximal production of cAMP. Both forms of desensitization were additive, and the protein kinase C inhibitor RO-318220 inhibited PMA-induced desensitization, but not agonist-induced desensitization. GLP-1- and PMA-dependent desensitization correlated with receptor phosphorylation, and the levels of phosphorylation induced by the two agents were additive. Furthermore, PMA-induced, but not GLP-1-induced, phosphorylation was totally inhibited by RO-318220. Internalization of the GLP-1 receptor did not participate in the desensitization induced by PMA, as a mutant GLP-1 receptor lacking the last 20 amino acids of the cytoplasmic tail was found to be totally resistant to the internalization process, but was still desensitized after PMA preexposure. PMA and GLP-1 were not able to induce the phosphorylation of a receptor deletion mutant lacking the last 33 amino acids of the cytoplasmic tail, indicating that the phosphorylation sites were located within the deleted region. The cAMP production mediated by this deletion mutant was not desensitized by PMA and was only poorly desensitized by GLP-1. Together, our results indicate that the production of cAMP and, hence, the stimulation of insulin secretion induced by GLP-1 can be negatively modulated by homologous and heterologous desensitization, mechanisms that involve receptor phosphorylation.

Connexin26 is regulated in rat urothelium by the scaffold protein IB1/JIP-1.

Proper function of the wall of bladder requires gap junctional communication for coordinating the responses of smooth muscle (SMC) and urothelial cells exposed to urine pressure. In the rat bladder, Cx43 is expressed by SMC and urothelial cells, whereas Cx26 expression is restricted to the epithelium. We used a model of bladder outlet obstruction, in which a ligature is placed around the urethra to increase voiding pressure. Increased fluid pressure was associated with increased Cx43 and Cx26 mRNA expression and with the activation of a signaling cascade including the transcription factor c-Jun, which is a component of the AP-1 complex. The signaling pathway of the c-Jun NH2 terminal kinase (JNK) requires the presence of the scaffold protein Islet-Brain1/c-Jun amino-terminal kinase Interacting Protein-1 (IB1/JIP-1). Under stress conditions resulting from urine retention, we have found a reduced content of IB1/JIP-1 in urothelial cells, which in turn induced a drastic increase of JNK and AP-1 binding activities. The stress-induced activation of JNK was prevented by overexpressing IB1/JIP-1, using a viral gene transfer approach, a condition which also resulted in a decrease in Cx26 mRNA. The data show that: 1) mechanical stress of urothelial cells activates in vivo JNK, as a consequence of a regulated expression of IB1/JIP-1 and 2) that urothelial Cx26 may be directly regulated by the AP-1 complex.

MALT1 and Caspase-10 : two cysteine proteases controlling lymphocyte activation

Τ cell activation via the Τ cell receptor (TCR) through antigen recognition is one of the key steps to initiate the adaptive immune response. The mechanisms controlling TCR-induced signaling pathways are the subject of intense research, since deregulated signaling in lymphocytes can lead to immunodeficiency, autoimmunity or lymphomas. In Τ lymphocytes a complex composed of CARMA1, BCL10 and MALT1 has been identified to receive signals from TCR proximal events and to induce further signals crucial for Τ cell activation. MALT1 is scaffold protein and a cysteine protease and both functions have been shown, among other effects, to be crucial to initiate the activation of the transcription factors of the nuclear factor κ 14; (NF-κ 14;) family after TCR-stimulation. Several proteolytic targets have been described recently and all of them play roles in modulating NF-κ 14; activation or other aspects of Τ cell activation. In this study, we describe a novel target of MALT1, Caspase-10. Two isoforms of Caspase-10 are cleaved by MALTI in Τ and 14; cells after antigen receptor stimulation. Caspases are a family of cysteine proteases that are known for their roles in cell death and certain immune functions. Caspase-10 has so far only been reported to be involved in the induction of apoptosis. However it is very closely related to the well-characterized Caspase-8 that has been reported to be involved in Τ cell activation. In the present study, we describe a crucial role for Caspase-10, but not Caspase-8, in Τ cell activation after TCR stimulation. Jurkat Τ cells silenced for Caspase-10 expression exhibit a dramatic reduction in IL-2 production following stimulation. The data obtained revealed that this is due to severely reduced activation of activator protein-1 (AP-1), another transcription factor family with key functions in the process of Τ cell activation. We observed strongly reduced expression levels of the AP-1 family member c-Fos after Τ cell stimulation. This transcription factor is expressed upon TCR stimulation and is a crucial component of AP-1 transcription factor dimers required for Τ cell activation. In further analysis, it was shown that this defect is not based on reduced transcription, as the c-Fos mRNA levels are not altered, but rather seems to be caused by a defect in translation or protein stability in the absence of Caspase-10. Furthermore, we report a potential interaction of the c-Fos protein and Caspsae-10. This role of Caspase-10 in AP-1 activation however is independent of its cleavage by MALT1, leaving the role of Caspase-10 cleavage in activated lymphocytes unclear. Taken together, these results give new insights into the complex matter of lymphocyte activation whose understanding is crucial for the development of new drugs modulating the immune response or inhibiting lymphoma progression.

Expression of an uncleavable N-terminal RasGAP fragment in insulin-secreting cells increases their resistance toward apoptotic stimuli without affecting their glucose-induced insulin secretion.

Apoptosis of pancreatic beta cells is implicated in the onset of type 1 and type 2 diabetes. Consequently, strategies aimed at increasing the resistance of beta cells toward apoptosis could be beneficial in the treatment of diabetes. RasGAP, a regulator of Ras and Rho GTPases, is an atypical caspase substrate, since it inhibits, rather than favors, apoptosis when it is partially cleaved by caspase-3 at position 455. The antiapoptotic signal generated by the partial processing of RasGAP is mediated by the N-terminal fragment (fragment N) in a Ras-phosphatidylinositol 3-kinase-Akt-dependent, but NF-kappaB-independent, manner. Further cleavage of fragment N at position 157 abrogates its antiapoptotic properties. Here we demonstrate that an uncleavable form of fragment N activates Akt, represses NF-kappaB activity, and protects the conditionally immortalized pancreatic insulinoma betaTC-tet cell line against various insults, including exposure to genotoxins, trophic support withdrawal, and incubation with inflammatory cytokines. Fragment N also induced Akt activity and protection against cytokine-induced apoptosis in primary pancreatic islet cells. Fragment N did not alter insulin cell content and insulin secretion in response to glucose. These data indicate that fragment N protects beta cells without affecting their function. The pathways regulated by fragment N are therefore promising targets for antidiabetogenic therapy.

Aggregation of integrins and RhoA activation are required for Thy-1-induced morphological changes in astrocytes.

Thy-1, a cell adhesion molecule abundantly expressed in mammalian neurons, binds to a beta(3)-containing integrin on astrocytes and thereby stimulates the assembly of focal adhesions and stress fibers. Such events lead to morphological changes in astrocytes that resemble those occurring upon injury in the brain. Extracellular matrix proteins, typical integrin ligands, bind to integrins and promote receptor clustering as well as signal transduction events that involve small G proteins and cytoskeletal changes. Here we investigated the possibility that the cell surface protein Thy-1, when interacting with a beta(3)-containing integrin on astrocytes, could trigger signaling events similar to those generated by extracellular matrix proteins. DI-TNC(1) astrocytes were stimulated with Thy-1-Fc immobilized on beads, and increased RhoA activity was confirmed using an affinity precipitation assay. The effect of various inhibitors on the cellular response was also studied. The presence of Y-27632, an inhibitor of Rho kinase (p160ROCK), a key downstream effector of RhoA, significantly reduced focal adhesion and stress fiber formation induced by Thy-1. Similar effects were obtained when astrocytes were treated with C3 transferase, an inhibitor of RhoA. Alternatively, astrocytes were transfected with an expression vector encoding fusion proteins of enhanced green fluorescent protein with either the Rho-binding domain of Rhotekin, which blocks RhoA function, or the dominant-negative N19RhoA mutant. In both cases, Thy-1-induced focal adhesion formation was inhibited. Furthermore, we observed that RhoA activity after stimulation with soluble Thy-1-Fc molecule was augmented upon further cross-linking using protein A-Sepharose beads. The same was shown by cross-linking beta(3)-containing integrin with anti-beta(3) antibodies. Together, these results indicate that Thy-1-mediated astrocyte stimulation depended on beta(3) integrin clustering and the resulting increase in RhoA activity.