Stimulation Of B-cell Receptor Research Articles

Activation of NF-κB in B cell receptor signaling through Bruton's tyrosine kinase-dependent phosphorylation of IκB-α.

The antigen-mediated triggering of B cell receptor (BCR) activates the transcription factor NF-κB that regulates the expression of genes involved in B cell differentiation, proliferation, and survival. The tyrosine kinase Btk is essentially required for the activation of NF-κB in BCR signaling through the canonical pathway of IKK-dependent phosphorylation and proteasomal degradation of IκB-α, the main repressor of NF-κB. Here, we provide the evidence of an additional mechanism of NF-κB activation in BCR signaling that is Btk-dependent and IKK-independent. In DeFew B lymphoma cells, the anti-IgM stimulation of BCR activated Btk and NF-κB p50/p65 within 0.5min in absence of IKK activation and IκB-α degradation. IKK silencing did not affect the rapid activation of NF-κB. Within this short time, Btk associated and phosphorylated IκB-α at Y289 and Y305, and, concomitantly, p65 translocated from cytosol to nucleus. The mutant IκB-α Y289/305A inhibited the NF-κB activation after BCR triggering, suggesting that the phosphorylation of IκB-α at tyrosines 289 and 305 was required for NF-κB activation. In primary chronic lymphocytic leukemia cells, Btk was constitutively active and associated with IκB-α, which correlated with Y305-phosphorylation of IκB-α and increased NF-κB activity compared with healthy B cells. Altogether, these results describe a novel mechanism of NF-κB activation in BCR signaling that could be relevant for Btk-targeted therapy in B-lymphoproliferative disorders. KEY MESSAGES: Anti-IgM stimulation of BCR activates NF-κB p50/p65 within 30s by a Btk-dependent and IKK-independent mechanism. Btk associates and phosphorylates IκB-α at Y289 and Y305, promoting NF-κB activation. In primary CLLs, the binding of Btk to IκB-α correlates with tyrosine phosphorylation of IκB-α and increased NF-κB activity.

The Regulation of STAT3 and Its Role in the Adhesion and Migration of Chronic Lymphocytic Leukaemia Cells

The bone marrow and lymph node microenvironments are important in promoting cell proliferation, survival and protection from drug induced apoptosis in chronic lymphocytic leukaemia (CLL). Chemokine networks, such as the CXCR4/CXCL12 axis, in combination with selectins, such as CD62L and integrins allow the migration of CLL cells to these protective niches. The B-cell receptor (BCR) signalling pathway is the most important pathway involved in micro-environmental crosstalk and CLL cell survival. Further, it has been shown to interact with the signal transducer and activator of transcription 3 (STAT3) signalling pathway. The role of the STAT3 in CLL pathogenesis is unclear; however, it is constitutively phosphorylated on serine residue 727 (serine pSTAT3) in CLL cells.Here, we investigate the role of STAT3 in CLL cell survival and migration, using pharmacological inhibition and siRNA knockdown. Phospho-tyrosine and phospho-serine STAT3 were assessed by flow cytometry and western blotting. Apoptosis was assessed by Annexin V/Propidium Iodide staining by flow cytometry. The expression of cell surface markers involved in cell adhesion and homing was determined by multicolour flow cytometry.Stimulation of the BCR using immunoglobulin F(ab´)2 fragments induced tyrosine phosphorylation of STAT3 in CLL cells with unmutated immunoglobulin (IgVH) genes (n=7) but not mutated IgVH genes (n=5). This induced tyrosine phosphorylation was abrogated by pre-treatment with the Janus kinase (JAK) inhibitor Ruxolitinib and the BCR inhibitors Ibrutinib and Idelalisib (p<0.05, n=5). Gene expression studies using Taqman Assays showed BCR stimulation resulted in an upregulation of STAT3 regulated genes in CLL cells with unmutated IgVH genes. Interestingly, stimulation of BCR resulted in a significant increase in CD62L expression, which was inhibited by pre-treatment with Ibrutinib and Ruxolitinib (p<0.05, n=5).STAT3 inhibition was shown to have a divergent effect on CLL cell survival: In patient samples with >70% positive serine pSTAT3 cells, the STAT3 inhibitor cucurbitacin I induced apoptosis with a concurrent downregulation in serine phosphorylation (n=3); while in patient samples with <70% positive serine pSTAT3 cells, treatment with cucurbitacin I resulted in a decrease in apoptosis and a concurrent increase in serine phosphorylation (n=3). The STAT3 inhibitor S3I-201 had a similar effect but the upstream JAK inhibitor Ruxolitinib had no effect on serine phosphorylation and no effect on the apoptosis of CLL cells. In addition, siRNA mediated STAT3 knockdown and treatment with cucurbitacin I and S31-201, resulted in a significant decrease in CD62L positive CLL cells (p<0.0001, n=29).The role of STAT3 in CLL cell adhesion under shear flow conditions was investigated using a microfluidics system including a neMESYS Low Pressure syringe pump system and Human Umbilical Vein Endothelial Cells (HUVEC) coated biochips. Treatment of CLL cells with cucurbitacin I resulted in a significant decrease in adhesion to endothelial cells (p<0.001, n=4). The effect of STAT3 inhibition on the chemotaxis of CLL cells was investigated using Neuroprobe 96-well ChemoTx plates. Treatment with cucurbitacin I resulted in a significant decrease in CLL cells migrated in response to the chemokine CXCL12 compared to control (p=0.0001, n=8). In addition, treatment of CLL cells with CXCL12 resulted in an increase in serine pSTAT3 that was downregulated by pretreatment with cucurbitacin I.This study has shown:1. Activation of STAT3 by BCR stimulation occurs in poor prognostic unmutated IgVH genes2. Serine pSTAT3 has a role in cell survival in response to STAT3 inhibition3. A role for STAT3 in CLL cell adhesion and migration, in particular in the regulation of the expression of CD62L.In conclusion, this study shows a role for the STAT3 pathway in cell survival and CLL cell-microenvironment crosstalk, suggesting therapeutic potential by interfering with the migration and homing of CLL cells to the lymph node and bone marrow microenvironments. DisclosuresNo relevant conflicts of interest to declare.

T-bet-Expressing B Cells Are Positively Associated with Crohn's Disease Activity and Support Th1 Inflammation.

Pathogenesis of Crohn's disease (CD) is thought to involve the chronic activation of T helper 1 (Th1)- and Th17-mediated inflammation, such as the production of interferon-gamma (IFN-γ) and interleukin 17 (IL-17). However, studies have also shown that although IFN-γ is required, IFN-γ-producing or T-bet-expressing Th1 cells are dispensable. We therefore examined T-bet-expressing B cells as another source of IFN-γ that potentially supported intestinal inflammation in CD patients. We found that the frequencies of T-bet-expressing B cells were significantly upregulated and abundantly present in the gut of active, but not quiescent, CD patients. The frequencies of T-bet-expressing B cells were also directly correlated with CD disease activity. These T-bet+ B cells were almost exclusively IgG expressing and produced significantly higher amounts of IFN-γ, IL-6, and IL-12 than IgA- and IgM-expressing T-bet- B cells. These B cells also supported IFN-γ production of CD4+ T cells. T-bet expression was induced in vitro in peripheral blood B cells through the stimulation of B-cell receptor (BCR), Toll-like receptor 7 (TLR7), and IFN-γ, which resembled gut T-bet+ B cells in terms of elevated IFN-γ. We found that these stimulated B cells, but not unstimulated B cells, supported the IFN-γ and IL-12 production from autologous CD4+ T cells. In addition, in patients with higher gut T-bet+ B-cell percentage, a higher frequency of gut-infiltrating IFN-γ+ and IL-12+ T cells was also observed. Together, our results suggested that T-bet-expressing B cells could contribute to the intestinal Th1 inflammation in CD patients.

PAX5 tyrosine phosphorylation by SYK co-operatively functions with its serine phosphorylation to cancel the PAX5-dependent repression of BLIMP1: A mechanism for antigen-triggered plasma cell differentiation

Plasma cell differentiation is initiated by antigen stimulation of the B cell receptor (BCR) and is regulated by BLIMP1. Prior to the stimulation of BCR, BLIMP1 is suppressed by PAX5, which is a key transcriptional repressor that maintains B cell identity. The upregulation of BLIMP1 and subsequent suppression of PAX5 by BLIMP1 are observed after the BCR stimulation. These events are considered to trigger plasma cell differentiation; however, the mechanisms responsible currently remain unclear. We herein demonstrated that the BCR signaling component, SYK, caused PAX5 tyrosine phosphorylation in vitro and in cells. Transcriptional repression on the BLIMP1 promoter by PAX5 was attenuated by this phosphorylation. The BCR stimulation induced the phosphorylation of SYK, tyrosine phosphorylation of PAX5, and up-regulation of BLIMP1 mRNA expression in B cells. The tyrosine phosphorylation of PAX5 co-operatively functioned with PAX5 serine phosphorylation by ERK1/2, which was our previous findings, to cancel the PAX5-dependent repression of BLIMP1. This co-operation may be a trigger for plasma cell differentiation. These results imply that PAX5 phosphorylation by a BCR signal is the initial event in plasma cell differentiation.

Could DNA-reactive B lymphocytes be activated through HIV-1 DNA-stimulation involving BCR/TLR-9 pathway to yield antibodies targeting viral DNA?

This paper discusses potential avenues to abate the last bulwark to the HIV-1 eradication i.e. viral reservoirs in the body, consisting mainly of viral DNA hidden, in latent form, into long-lived memory CD4+ T-cells, resulting unaffected by either drugs or immune system. Activation of the innate immune system is necessary for the induction of adaptive immune responses against invading pathogens. In part, this is achieved by recognition of molecules associated with infection by a plethora of pattern recognition receptors comprising Toll-like receptors (TLRs) which are express on numerous cells of immune system such as, dendritic cells and B cells, and are activated by some TLR ligands. TLR-9, localized in endosomal compartment, specifically recognizes unmethylated oligonucleotide sequences containing CpG motifs which are particularly abundant in microbial genome, including HIV-1 DNA. Naïve B cells are activated following antigen binding to the B cell receptor (BCR). The complex antigen/BCR, internalises and synergises with TLR leading to hyper activation of B cells and antibodies production. Cross-link of the BCR with TLR9, followed by upregulation of TLR-9, in response to self DNA-containing antigens, on DNA-specific B lymphocytes lead to the development of DNA-specific autoantibodies. This occur in systemic autoimmune disorders, such as, systemic lupus erythematosus (SLE). Although such DNA-specific B lymphocytes, are usually present in B cell repertoire at low frequency, they normally do not produce autoantibodies, they can be activated by antigen BCR ligation, with plasmacytoid dendritic cells (pDCs) providing helper signals. The hypothesis here derives by insight that stimulation of BCR, by exogenously added HIV-1 DNA, on potentially reactive HIV-1 DNA-specific B cells, from HIV-1 and/or HIV-1/SLE patients, co-cultured with autologous viral DNA-stimulated pDCs, could lead to desirable in vitro production of antibodies direct against HIV-1 DNA by involving BCR/TLR9 pathway. These antibodies, suitably delivered into infected or uninfected CD4+ T-cells, by a carrier, such as, peptide transduction domain of Tat, could selectively recognize viral nucleic acid, rich in CpG motifs, respect to host DNA, both in productively than latently infected T cells. Because HIV-1 DNA into latently infected cells is heavily methylated and deacetylated, co-treatment with a small molecule inhibitor of DNA methylation, such as 5-aza-2′-deoxycytidine (aza-Cdr), and histone deacetylase inhibitors (HDACi), they would favor higher accessibility by antibodies to viral DNA, which is harbored into human chromosomes. The in vitro production of anti-HIV-1 DNA antibodies and their in vivo reintroduction, could find future application as interesting strategy to pave the way to an eventual cure to decrease and/or eliminate viral reservoirs from the body, and providing possible therapeutic applications not only for HIV-1/AIDS, but also for other infectious diseases.

Defective B-cell proliferation and maintenance of long-term memory in patients with chronic granulomatous disease

Chronic granulomatous disease (CGD) is a primary immune deficiency characterized by a defect in reactive oxygen species production. Although the effect of CGD mainly reflects on the phagocytic compartment, B-cell responses are also impaired in patients with CGD. We sought to investigate how defective gp91(phox) expression in patients with CGD and CGD carriers might affect the B-cell compartment and maintenance of long-term memory. We studied the B-cell compartment of patients with CGD in terms of phenotype and ability to produce reactive oxygen species and proliferate on stimuli differently directed to the B-cell receptor and Toll-like receptor 9. We further studied their capacity to maintain long-term memory by measuring cellular and serologic responses to measles. We show that the memory B-cell compartment is impaired among patients with CGD, as indicated by reduced total (CD19(+)CD27(+)) and resting (CD19(+)CD27(+)CD21(+)) memory B cells in parallel to increased naive (CD19(+)CD27(-)IgD(+)) B-cell frequencies. Data on CGD carriers reveal that such alterations are related to gp91(phox) expression. Moreover, proliferative capabilities of B cells on selective invitro stimulation of B-cell receptor or Toll-like receptor 9 pathways were reduced in patients with CGD compared with those seen in age-matched healthy control subjects. Significantly lower measles-specific antibody levels and antibody-secreting cell numbers were also observed, indicating a poor ability to maintain long-term memory in these patients. Altogether, our data suggest that patients with CGD present a defective B-cell compartment in terms of frequencies of memory B cells, response to invitro stimulation, and maintenance of long-term antigen-specific memory.

The Target Genes and Prognostic Significance of Mir-150 in Chronic Lymphocytic Leukemia

AbstractAbstract 3859Chronic lymphocytic leukemia (CLL) is the first disease in which miRNAs (hsa-miR-15a-16–1) were directly linked to cancer pathogenesis (Calin et al. PNAS, 2002). We and others have also shown that expression of certain miRNAs associates with disease activity in patients with CLL (Calin et al. NEJM, 2005; Mraz et al. Blood, 2012; Mraz et al. Leukemia, 2009). Moreover, patients with more aggressive disease have CLL cells that generally express unmutated IGHV and/or ZAP-70 and have a miRNA expression profile that differs from that of CLL cells from patients with indolent disease (Calin et al. NEJM, 2005). However, we still have very limited understanding of how miRNAs affect CLL cell-biology and expression of genes that play a critical role in either promoting or arresting the disease. We used pooled samples from 10 CLL patients to screen (TaqMan miRNA Cards-ABI, 750 miRNAs) for abundantly expressed miRNAs that could hypothetically influence CLL B cell biology. We identified miR-150 as the most abundant miRNA in CLL cells and also as being strongly expressed when compared to CD19+ blood lymphocytes of normal adults (N=5, P=0.008). This miRNA already has been reported to influence the differentiation and gene expression of normal B cells (Xiao et al. Cell, 2007) suggesting its possible relevance for CLL B cell biology. We examined additional CLL cell samples (N=168) and confirmed high miR-150 levels and also noted heterogeneity in its expression between CLL cells of patients with aggressive versus indolent disease. In our cohort, CLL cells of patients that expressed ZAP-70 (20% cut-off, N=74) or had unmutated IGHV (N=72) expressed significantly lower median-levels of miR-150 (fold change −1.7 and −2.0 respectively, p<0.005). Moreover, the lower levels of miR-150 also directly associated with higher response to stimulation of B-cell receptor (BCR) on CLL cells with anti-IgM (P<0.05, N=36, quantified by flow cytometric measurement of calcium mobilization). To understand the gene network regulated by miR-150 in CLL we performed array-based transcriptome analyses (HG-U133 Plus 2.0, Affymetrix) of 110 patient samples, which identified differential expression of 215 genes between CLL cells expressing low versus high levels of miR-150 (SAM analysis of upper and lower terciles). Thirty-eight of these 215 genes (17%) are predicted targets of miR-150 (determined by TargetScan, www.targetscan.org). Two well annotated genes (GAB1 and FOXP1) have evolutionary conserved binding sides for miR-150 in their 3‘UTRs, suggesting the possible importance of miR-150 in their regulation. GAB1 is an adaptor molecule and plays a key role in variety of cell signaling pathways (PLCγ, Ras/Erk, PI3K/Akt, CrkL). Interestingly, GAB1 modulates PI3K/Akt-pathway through binding domain identical to Bruton's tyrosine kinase (Rameh et al. JBC, 1997) and is a key molecule involved in regulating BCR-signaling (Ingham et al. JBC, 1998, 2001), a process that factors prominently in the pathogenesis and progression of CLL. FOXP1 is an essential participant in the transcriptional regulatory network of B lymphopoiesis and has been identified as playing a role in disease progression of other B-cell lymphomas (Hu et al. Nat Immunol, 2006). The immunoblot analysis of GAB1 and FOXP1 in CLL cells confirmed their higher protein levels in cases with low miR-150 expression (P<0.005, fold change >10.0). Importantly, cells with higher expression of GAB1 or FOXP1 were more responsive to BCR stimulation in vitro (P<0.01, N=36) and higher expression of each associates with shorter overall survival (OS) (13.9 vs. 22.7 years, 13.9 vs. 21.1 years; N=168; P<0.05). Most notably, a reverse trend was observed for miR-150, where higher levels (>median) were associated with significantly longer OS (not-reached vs. 13.9 years, N=168, P=0.006). Additionally, the expression level of miR-150 was an independent predictor of OS and time to first treatment (TTFT) in multivariate analyses, which included IGHV status, ZAP-70, CD38, Rai stage, gender, and age (OS HR: 3.4 [CI 1.4–8.6], P=0.009; TTFT HR: 2.3 [CI 1.3–4.2], P=0.004). We conclude that there is an inverse association between high-risk disease and expression of miR-150, which may reflect its capacity to regulate the expression of genes encoding proteins that may contribute to BCR-signaling and/or survival of CLL B cells. Disclosures:No relevant conflicts of interest to declare.

CIN85 is required for Cbl-mediated regulation of antigen receptor signaling in human B cells

AbstractThe aberrant regulation of B-cell receptor (BCR) signaling allows unwanted B cells to persist, thereby potentially leading to autoimmunity and B-cell malignancies. Casitas B-lineage lymphoma (Cbl) proteins suppress BCR signaling; however, the molecular mechanisms that control Cbl function in human B cells remain unclear. Here, we demonstrate that CIN85 (c-Cbl interacting protein of 85 kDa) is constitutively associated with c-Cbl, Cbl-b, and B-cell linker in B cells. Experiments using CIN85-overexpressing and CIN85-knockdown B-cell lines revealed that CIN85 increased c-Cbl phosphorylation and inhibited BCR-induced calcium flux and phosphorylation of Syk and PLCγ2, whereas it did not affect BCR internalization. The Syk phosphorylation in CIN85-overexpressing and CIN85-knockdown cells was inversely correlated with the ubiquitination and degradation of Syk. Moreover, CIN85 knockdown in primary B cells enhanced BCR-induced survival and growth, and increased the expression of BcLxL, A1, cyclin D2, and myc. Following the stimulation of BCR and Toll-like receptor 9, B-cell differentiation- associated molecules were up-regulated in CIN85-knockdown cells. Together, these results suggest that CIN85 is required for Cbl-mediated regulation of BCR signaling and for downstream events such as survival, growth, and differentiation of human B cells.

A Stim1-dependent, noncapacitative Ca2+-entry pathway is activated by B-cell-receptor stimulation and depletion of Ca2+

The depletion of intracellular Ca(2+) stores activates capacitative Ca(2+) entry (CCE), which is a Ca(2+)-selective and La(3+)-sensitive entry pathway. Here, we report a novel mechanism of La(3+)-resistant Ca(2+) entry that is synergistically regulated by B-cell-receptor (BCR) stimulation and Ca(2+) store depletion. In DT40 cells, stimulation of BCRs with anti-IgM antibodies induced Ca(2+) release and subsequent Ca(2+) entry in the presence of 0.3 microM La(3+), a condition in which CCE is completely blocked. This phenomenon was not observed in inositol 1,4,5-trisphosphate receptor-deficient DT40 (IP3R-KO) cells. However, in response to thapsigargin pretreatment, BCR stimulation induced La(3+)-resistant Ca(2+) entry into both wild-type and IP3R-KO cells. These results indicate that BCR stimulation alone does not activate Ca(2+) entry, whereas BCR stimulation and depleted Ca(2+) stores (either due to IP3R-mediated Ca(2+) release or Ca(2+) uptake inhibition) work in concert to activate La(3+)-resistant Ca(2+) entry. This Ca(2+) entry was inhibited by genistein. In addition, BCR-mediated Ca(2+) entry was completely abolished in Stim1-deficient DT40 cells and was restored by overexpression of YFP-Stim1, but was unaffected by double knockdown of Orai1 and Orai2. These results demonstrate a unique non-CCE pathway, in which Ca(2+) entry depends on Stim1- and BCR-mediated activation of tyrosine kinases.

Targeting lipid metabolism by the lipoprotein lipase inhibitor orlistat results in apoptosis of B-cell chronic lymphocytic leukemia cells

Constitutively activated pathways contribute to apoptosis resistance in chronic lymphocytic leukemia (CLL). Little is known about the metabolism of lipids and function of lipases in CLL cells. Performing gene expression profiling including B-cell receptor (BCR) stimulation of CLL cells in comparison to healthy donor CD5+ B cells, we found significant overexpression of lipases and phospholipases in CLL cells. In addition, we observed that the recently defined prognostic factor lipoprotein lipase (LPL) is induced by stimulation of BCR in CLL cells but not in CD5+ normal B cells. CLL cellular lysates exhibited significantly higher lipase activity compared to healthy donor controls. Incubation of primary CLL cells (n=26) with the lipase inhibitor orlistat resulted in induction of apoptosis, with a half-maximal dose (IC(50)) of 2.35 microM. In healthy B cells a significantly higher mean IC(50) of 148.5 microM of orlistat was observed, while no apoptosis was induced in healthy peripheral blood mononuclear cells (PBMCs; P<0.001). Orlistat-mediated cytotoxicity was decreased by BCR stimulation. Finally, the cytotoxic effects of orlistat on primary CLL cells were enhanced by the simultaneous incubation with fludarabine (P=0.003). In summary, alterations of lipid metabolism are involved in CLL pathogenesis and might represent a novel therapeutic target in CLL.

Synergistic activation of PKD by the B cell antigen receptor and CD19 requires PI3K, Vav1 and PLCγ

Antigens coated with complement fragments coligate the B cell receptor (BCR) with the CD21/CD19 complex which results in synergistic activation of B cells. Previous studies identified PI3K, Vav proteins and PLCγ as important components of this synergy. We now show that protein kinase D (also known as PKCμ) is also a point of convergence of these signalling pathways. We found that PKD activation upon BCR engagement or coligation of the BCR with CD19 is entirely dependent on PI3K and PLCγ but differ in the requirement for Vav proteins. Whereas PKD activation is Vav1 and Vav2 dependent in response to BCR cross-linking, PKD activation is sensitive to the lack of Vav1 under synergistic stimulation of BCR and CD19. These findings show that Vav proteins and PI3K regulation of PLCγ contributes to the activation of PKD in response to BCR and or CD19 cross-linking.

Aberrant HS1 molecule in a patient with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the activation of autoreactive B lymphocytes, which are supposed to carry aberrant signal transduction after the stimulation of B-cell receptor (BCR). To investigate abnormalities in BCR-mediated signaling pathway in lupus B lymphocytes, we analyzed HS1, a molecule downstream of BCR, in 80 Japanese SLE patients. We identified 37 amino acid deletion of HS1 in a 25-year-old female patient, and the aberrant HS1 lacked a part of a functional motif. Analysis of genomic DNA revealed that the aberrant HS1 was caused by exon skipping. Family study showed that the patient as well as her father and sister are heterozygous for the abnormality. WEHI-231 cell, a mouse B cell line, transfected with the aberrant HS1 displayed a significantly increased cell death upon cross-linking of BCR. Additionally, peripheral B lymphocytes from the patient exerted increased apoptosis after BCR stimulation compared to those from control SLE patients. These data suggest that the aberrant HS1 molecule may transmit an accelerated signal after BCR stimulation and may play a role in the activation of autoreactive B lymphocytes.

Distribution of the Src-homology-2-domain-containing inositol 5-phosphatase SHIP-2 in both non-haemopoietic and haemopoietic cells and possible involvement of SHIP-2 in negative signalling of B-cells

The termination of activation signals is a critical step in the control of the immune response; perturbation of inhibitory feedback pathways results in profound immune defects culminating in autoimmunity and overwhelming inflammation. FcγRIIB receptor is a well described inhibitory receptor. The ligation of B-cell receptor (BCR) and FcγRIIB leads to the inhibition of B-cell activation. Numerous studies have demonstrated that the SH2-domain-containing inositol 5-phosphatase SHIP (referred hereto as SHIP-1) is essential in this process. The cDNA encoding a second SH2-domain-containing inositol 5-phosphatase, SHIP-2, has been cloned [Pesesse, Deleu, De Smedt, Drayer and Erneux (1997) Biochem. Biophys. Res. Commun. 239, 697-700]. Here we report the distribution of SHIP-2 in mouse tissues: a Western blot analysis of mouse tissues reveals that SHIP-2 is expressed in both haemopoietic and non-haemopoietic cells. In addition to T-cell and B-cell lines, spleen, thymus and lung are shown to coexpress SHIP-1 and SHIP-2. Moreover, SHIP-2 is detected in fibroblasts, heart and different brain areas. SHIP-2 shows a maximal tyrosine phosphorylation and association to Shc after ligation of BCR to FcγRIIB but not after stimulation of BCR alone. Our results therefore suggest a possible role for SHIP-2 in the negative regulation of immunocompetent cells.

Distribution of the Src-homology-2-domain-containing inositol 5-phosphatase SHIP-2 in both non-haemopoietic and haemopoietic cells and possible involvement of SHIP-2 in negative signalling of B-cells

The termination of activation signals is a critical step in the control of the immune response; perturbation of inhibitory feedback pathways results in profound immune defects culminating in autoimmunity and overwhelming inflammation. FcgammaRIIB receptor is a well described inhibitory receptor. The ligation of B-cell receptor (BCR) and FcgammaRIIB leads to the inhibition of B-cell activation. Numerous studies have demonstrated that the SH2-domain-containing inositol 5-phosphatase SHIP (referred hereto as SHIP-1) is essential in this process. The cDNA encoding a second SH2-domain-containing inositol 5-phosphatase, SHIP-2, has been cloned [Pesesse, Deleu, De Smedt, Drayer and Erneux (1997) Biochem. Biophys. Res. Commun. 239, 697-700]. Here we report the distribution of SHIP-2 in mouse tissues: a Western blot analysis of mouse tissues reveals that SHIP-2 is expressed in both haemopoietic and non-haemopoietic cells. In addition to T-cell and B-cell lines, spleen, thymus and lung are shown to coexpress SHIP-1 and SHIP-2. Moreover, SHIP-2 is detected in fibroblasts, heart and different brain areas. SHIP-2 shows a maximal tyrosine phosphorylation and association to Shc after ligation of BCR to FcgammaRIIB but not after stimulation of BCR alone. Our results therefore suggest a possible role for SHIP-2 in the negative regulation of immunocompetent cells.