Molecular Cloning of DOCK10/Zizimin3, a Novel Cdc42/Rac-Interacting Protein Specifically Induced by IL4 in B Lymphocytes.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 939-939
Author(s):  
Estefania Yelo ◽  
Lourdes Gimeno ◽  
Maria Victoria Bernardo ◽  
Maria Juliana Majado ◽  
Maria Rocio Alvarez ◽  
...  
Keyword(s):  
Amino Acids ◽  
T Lymphocytes ◽  
Lymph Nodes ◽  
B Cell ◽  
B Lymphocytes ◽  
Expression System ◽  
Interleukin 4 ◽  
B Cell Differentiation ◽  
Nucleotide Exchange

Abstract Interleukin-4 (IL4) induces proliferation, differentiation and survival of B lymphocytes. IL4 protects CLL B cells from death by apoptosis. Gene expression analysis suggest that IL4 pathways are activated in CLL cells. We have identified DOCK10/Zizimin3 as an IL4-induced gene in CLL cells, and have obtained its full length sequence after cloning 1960 bp at its 5′ terminus by RACE-PCR. The human DOCK10/ZIZ3 sequence coded for a protein with 2180 amino acids and a predicted Mr of 250K. DOCK10/ZIZ3 shared homology with the other two members of the Zizimin family, and is the largest among them: DOCK9/ZIZ1 (2069 amino acids) and DOCK11/ZIZ2 (2073 amino acids) are 52% and 50% identical, respectively, to DOCK10/ZIZ3, and 58% identical between them. DOCK10 was predominantly expressed in hematopoietic tissues, particularly in peripheral blood (PB), but also in lymph nodes, thymus and spleen. Among the PB subpopulations, DOCK10 was expressed in B and T lymphocytes and, at lower levels, in monocytes. DOCK10 was also expressed in several non-hematopoietic tissues, most significantly in brain and kidney. Its homologue DOCK9, compared to DOCK10, was predominantly expressed in placenta, and less significantly in hematopoietic tissues, particularly in B lymphocytes and monocytes. DOCK11, like DOCK10, was predominantly expressed in PB. Compared to DOCK10, DOCK11 was expressed more prominently in placenta, thyroid and PB monocytes, and less significantly in brain and lymph nodes. Therefore, each of the Zizimin family members had a specific tissue distribution. Among the three genes, only DOCK10 was induced by IL4 in CLL cells in vitro. Induction of DOCK10 by IL4 was a common event in CLL, since it was observed in 10 out of 10 cases. IL4 also induced DOCK10 expression in normal PB B lymphocytes, suggesting that DOCK10 induction by IL4 in CLL cells may be normal, rather than pathological. Western blot analysis using a polyclonal antibody raised against a peptide which mapped at the N terminus of DOCK10, detected a band of the expected size of 250K. Interestingly, IL4 did not induce DOCK10 expression in CD4 or CD8 T lymphocytes in vitro. Expression of DOCK10 was also studied in 4 B-ALL, 2 T-ALL, and 1 T-CLL. DOCK10 neither was expressed at significant levels nor induced by IL4 in vitro in these patients, except for a weak induction in a common B-ALL case, suggesting that expression of DOCK10, and its induction with IL4, may be restricted to certain stages of B cell differentiation, and/or certain B cell malignancies. DOCK10 was distributed both in cytosolic and nuclear extracts of CLL cells, and IL4 increased its expression in both compartments. K562 clones stably transfected with DOCK10 using the inducible tet-off expression system showed significantly higher levels of DOCK10 in cytoplasm than in nucleus. Immunofluoresce analysis of HA-tagged DOCK10 K562 clones showed preferent staining of the cytoplasm, and dotted structures were frequently observed. GST-pulldown assays showed that DOCK10 bound to nucleotide-free (nf) Cdc42, but not to GTP- or GDP-loaded Cdc42. In addition, DOCK10 bound to nf Rac1, albeit with less affinity than to Cdc42. DOCK10 did not bind to RhoA. These results suggest that, like DOCK9 and DOCK11, DOCK10 may act as a novel Cdc42 guanine-nucleotide exchange factor (GEF) and, in addition, as a Rac1 GEF.

The development of functional B lymphocytes in conditional PU.1 knock-out mice

Blood ◽  
2005 ◽  
Vol 106 (6) ◽  
pp. 2083-2090 ◽  
Author(s):  
Matthew Polli ◽  
Aleksandar Dakic ◽  
Amanda Light ◽  
Li Wu ◽  
David M. Tarlinton ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Fetal Liver ◽  
Cell Receptor ◽  
Interleukin 4 ◽  
Knock Out ◽  
B Lineage ◽  
And Function

Abstract An abundance of research has entrenched the view that the Ets domain containing transcription factor PU.1 is fundamental to the development and function of B lymphocytes. In this study, we have made use of a conditional PU.1 allele to test this notion. Complete deletion of PU.1 resulted in the loss of B cells and all other lineage-positive cells in the fetal liver and death between E18.5 and birth; however, specific deletion of PU.1 in the B lineage had no effect on B-cell development. Furthermore, deletion of PU.1 in B cells did not compromise their ability to establish and maintain an immune response. An increased level of apoptosis was observed in vitro upon B-cell receptor (BCR) cross-linking; however, this was partially rescued by interleukin-4 (IL-4). These findings suggest that PU.1 is not essential for the development of functional B lymphocytes beyond the pre-B stage. (Blood. 2005;106:2083-2090)

scholarly journals Expression of interleukin-4 receptors on early human B-lineage cells

Blood ◽  
1991 ◽  
Vol 78 (3) ◽  
pp. 703-710 ◽  
Author(s):  
CL Law ◽  
RJ Armitage ◽  
JG Villablanca ◽  
TW LeBien
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Constitutive Expression ◽  
Lymphoid Cells ◽  
Interleukin 4 ◽  
B Cell Differentiation ◽  
Fluorescent Intensity ◽  
B Cell Growth Factor ◽  
B Lineage

Interleukin-4 (IL-4) regulates multiple stages of the antigen-dependent phase of B-cell development. However, its precise role in regulating B lymphopoiesis in bone marrow is not as well defined. We examined whether surface IgM- normal and leukemic human B-cell precursors (BCP) expressed IL-4 receptors using biotinylated IL-4. Constitutive expression of IL-4 receptors was detected on both normal and leukemic BCP. A higher percentage of normal BCP (82% +/- 15%) expressed IL-4 receptors compared with leukemic BCP (44% +/- 8%). Using mean fluorescent intensity as an indicator of receptor level on the IL-4 receptor positive cells, normal (91 +/- 41) and leukemic (44 +/- 37) BCP expressed comparable numbers of receptors. IL-4 induced the expression of CD23 on 30% of the leukemic BCP cases examined. IL-4 induced CD23 on surface IgM+ fetal bone marrow lymphoid cells but not on the surface IgM- normal BCP, despite the presence of detectable receptors on the surface IgM- cells. IL-4 did not stimulate proliferation of normal BCP, nor could it enhance the effect of recombinant IL-7 or low molecular weight B-cell growth factor. However, IL-4 increased the expression of surface IgM and surface Ig kappa on in vitro differentiated pre-B cells. Our collective results identify no role for IL-4 in the proliferation of normal or leukemic BCP, but identify a role in the enhancement of surface Ig expression during pre- B to B-cell differentiation.

scholarly journals Arhgap25 deficiency leads to decreased numbers of peripheral blood B cells and defective germinal center reactions

2020 ◽  
Author(s):  
Silke E. Lindner ◽  
Colt A. Egelston ◽  
Stephanie M. Huard ◽  
Peter P. Lee ◽  
Leo D. Wang
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Peripheral Blood ◽  
Cell Development ◽  
B Cell Development ◽  
Dependent Manner ◽  
B Cell Differentiation ◽  
Nucleotide Exchange

ABSTRACTRho family GTPases are critical for normal B cell development and function and their activity is regulated by a large and complex network of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). However, the role of GAPs in B cell development is poorly understood. Here we show that the novel Rac-GAP ARHGAP25 is important for B cell development in mice in a CXCR4-dependent manner. We show that Arhgap25 deficiency leads to a significant decrease in peripheral blood B cell numbers, as well as defects in mature B cell differentiation. Arhgap25-/- B cells respond to antigen stimulation in vitro and in vivo but have impaired germinal center formation and decreased IgG1 class switching. Additionally, Arhgap25-/- B cells exhibit increased chemotaxis to CXCL12. Taken together, these studies demonstrate an important role for Arhgap25 in peripheral B cell development and antigen response.

scholarly journals Activation of mouse lymphocytes by anti-immunoglobulin. II. A thymus-independent response by a mature subset of B lymphocytes.

1978 ◽  
Vol 148 (6) ◽  
pp. 1628-1643 ◽  
Author(s):  
D G Sieckmann ◽  
I Scher ◽  
R Asofsky ◽  
D E Mosier ◽  
W E Paul
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
B Cell ◽  
B Lymphocytes ◽  
Cell Populations ◽  
Direct Role ◽  
Membrane Immunoglobulin ◽  
Independent Process ◽  
Nylon Wool

Mouse spleen cells can be stimulated to proliferate in vitro by purified anti-mu or anti-gamma,kappa antibodies. These responses can be obtained in cell populations bearing membrane immunoglobulin (Ig), purified by the fluorescence activated cell sorter (FACS), but they are not observed in FACS-purified Ig- cell populations. Furthermore, treatment of spleen cell populations with anti-Thy 1.2 and complement does not impair the response, nor does addition of nylon wool-purified T lymphocytes enhance it. These results indicate that B lymphocytes respond to anti-Ig and that their response does not require T cells. On the other hand, cells from athymic nude (nu/nu) mice respond slightly less well to anti-mu than do cells from heterozygous littermate (nu/+) controls; nu/nu cells are almost unresponsive to anti-gamm,kappa and addition of nylon wool-purified T cells from nu/+ controls does not restore the response. This suggests that T lymphocytes or the thymus may control the appearance of cells responsive to anti-gamma,kappa. Responsiveness of normal mice to anti-mu does not appear until 4 wk of age and does not reach maximum levels until 8 wk of age. Acquisition of full responsiveness to anti-gamma,kappa is even more delayed. This, together with the failure of mice with the CBA/N B-cell defect to respond to anti-Ig, suggests that cells stimulated to proliferate by anti-Ig are a mature subset of B cells. Depletion of adherent cells by Sephadex G-10 treatment or by treatment with carbonyl iron and exposure to a magnetic field does not diminish anti-mu or anti-gamma,kappa responses, suggesting that the responsiveness does not require the presence of macrophages. Thus, activation of B-cell proliferation by anti-Ig appears to be a T-cell independent, macrophage-independent process in which membrane Ig plays a direct role in signal generation.

scholarly journals Expression of interleukin-4 receptors on early human B-lineage cells

Blood ◽  
1991 ◽  
Vol 78 (3) ◽  
pp. 703-710 ◽  
Author(s):  
CL Law ◽  
RJ Armitage ◽  
JG Villablanca ◽  
TW LeBien
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Constitutive Expression ◽  
Lymphoid Cells ◽  
Interleukin 4 ◽  
B Cell Differentiation ◽  
Fluorescent Intensity ◽  
B Cell Growth Factor ◽  
B Lineage

Abstract Interleukin-4 (IL-4) regulates multiple stages of the antigen-dependent phase of B-cell development. However, its precise role in regulating B lymphopoiesis in bone marrow is not as well defined. We examined whether surface IgM- normal and leukemic human B-cell precursors (BCP) expressed IL-4 receptors using biotinylated IL-4. Constitutive expression of IL-4 receptors was detected on both normal and leukemic BCP. A higher percentage of normal BCP (82% +/- 15%) expressed IL-4 receptors compared with leukemic BCP (44% +/- 8%). Using mean fluorescent intensity as an indicator of receptor level on the IL-4 receptor positive cells, normal (91 +/- 41) and leukemic (44 +/- 37) BCP expressed comparable numbers of receptors. IL-4 induced the expression of CD23 on 30% of the leukemic BCP cases examined. IL-4 induced CD23 on surface IgM+ fetal bone marrow lymphoid cells but not on the surface IgM- normal BCP, despite the presence of detectable receptors on the surface IgM- cells. IL-4 did not stimulate proliferation of normal BCP, nor could it enhance the effect of recombinant IL-7 or low molecular weight B-cell growth factor. However, IL-4 increased the expression of surface IgM and surface Ig kappa on in vitro differentiated pre-B cells. Our collective results identify no role for IL-4 in the proliferation of normal or leukemic BCP, but identify a role in the enhancement of surface Ig expression during pre- B to B-cell differentiation.

Histological and Immunological Study of Patients with Lung and Digestive Tract Tumors. Analysis of T and B Lymphocytes from the Peripheral Blood and from the Lymph Nodes Draining the Tumor.

1975 ◽  
Vol 61 (6) ◽  
pp. 547-557
Author(s):  
F. Franchi ◽  
S. Colizza ◽  
R. D'Amelio ◽  
G. Corelli ◽  
S. Bigliocchi ◽  
...  
Keyword(s):  
Lymph Node ◽  
T Lymphocytes ◽  
Lymph Nodes ◽  
Digestive Tract ◽  
B Lymphocytes ◽  
Peripheral Blood ◽  
Lung Tumor ◽  
Tritiated Thymidine ◽  
Immunological Study

The nature of the immune response to the tumor cell is complex and has yet to be clearly defined. Although past research has focused on the cytotoxic effect of T lymphocytes versus tumor cells, it has been shown in animal studies that B lymphocytes may also be implicated. Lymphocytes from patients with respiratory and digestive tract tumors were studied. B and T lymphocytes of peripheral blood and of draining lymph node tumors were studied using the following techniques: E rosettes (marker for T cells); membrane Ig, EAC rosettes, aggregated-Ig (marker for B lymphocytes); PHA and PKW in vitro response of lymphocytes using tritiated thymidine incorporation. It was observed that both groups of patients had normal or depressed B and T populations. PHA response was depressed in the majority of the cases with lung tumor. No difference was observed between lymphocytes from peripheral blood and from lymph node suspensions. As in normal lymph nodes the EAC rosettes were constantly observed in the cortical area of lymph node draining tumors. The immune defect observed in part of these cases is discussed in relation to the local and general immunological factors probably responsible for this defect.

scholarly journals B Cell–attracting Chemokine 1, a Human CXC Chemokine Expressed in Lymphoid Tissues, Selectively Attracts B Lymphocytes via BLR1/CXCR5

1998 ◽  
Vol 187 (4) ◽  
pp. 655-660 ◽  
Author(s):  
Daniel F. Legler ◽  
Marcel Loetscher ◽  
Regula Stuber Roos ◽  
Ian Clark-Lewis ◽  
Marco Baggiolini ◽  
...  
Keyword(s):  
Amino Acids ◽  
T Lymphocytes ◽  
B Cell ◽  
B Lymphocytes ◽  
Human Blood ◽  
Dna Sequences ◽  
Chemokine Receptors ◽  
Expressed Sequence Tag ◽  
Lymphoid Tissues ◽  
Cxc Chemokine

Although most leukocytes, T lymphocytes in particular, respond to several different chemokines, there is virtually no information on chemokine activities and chemokine receptors in B lymphocytes. A putative chemokine receptor, BLR1, that is expressed in Burkitt's lymphoma cells and B lymphocytes was cloned a few years ago. Deletion of the gene for BLR1 yielded mice with abnormal primary follicles and germinal centers of the spleen and Peyer's patches, reflecting the inability of B lymphocytes to migrate into B cell areas. By screening expressed sequence tag DNA sequences, we have identified a CXC chemokine, termed B cell–attracting chemokine 1 (BCA-1), that is chemotactic for human B lymphocytes. BCA-1 cDNA encodes a protein of 109 amino acids with a leader sequence of 22 residues. The mature protein shares 23–34% identical amino acids with known CXC chemokines and is constitutively expressed in secondary lymphoid organs. BCA-1 was chemically synthesized and tested for activity on murine pre–B cells 300-19 transfected with BLR1 and on human blood B lymphocytes. In transfected cells, BCA-1 induced chemotaxis and Ca2+ mobilization demonstrating that it acts via BLR1. Under the same conditions, no activity was obtained with 10 CXC and 19 CC chemokines, lymphotactin, neurotactin/fractalkine and several other peptide ligands. BCA-1 was also a highly effective attractant for human blood B lymphocytes (which express BLR1), but was inactive on freshly isolated or IL-2–stimulated T lymphocytes, monocytes, and neutrophils. In agreement with the nomenclature rules for chemokine receptors, we propose the term CXCR5 for BLR1. Together with the observed disturbance of B cell colonization in BLR1/ CXCR5-deficient mice, the present results indicate that chemotactic recruitment by locally produced BCA-1 is important for the development of B cell areas of secondary lymphoid tissues.

scholarly journals Altered Erythrocytes and a Leaky Block in B-Cell Development in CD24/HSA-Deficient Mice

Blood ◽  
1997 ◽  
Vol 89 (3) ◽  
pp. 1058-1067 ◽  
Author(s):  
P.J. Nielsen ◽  
B. Lorenz ◽  
A.M. Müller ◽  
R.H. Wenger ◽  
F. Brombacher ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
B Cell ◽  
Embryonic Stem ◽  
Cell Development ◽  
B Cell Development ◽  
Surface Glycoprotein ◽  
Malarial Parasite ◽  
Heat Stable Antigen ◽  
Deficient Mice

Abstract The heat stable antigen (HSA, or murine CD24) is a glycosyl phosphatidylinositol-linked surface glycoprotein expressed on immature cells of most, if not all, major hematopoietic lineages, as well as in developing neural and epithelial cells. It has been widely used to stage the maturation of B and T lymphocytes because it is strongly induced and then repressed again during their maturation. Terminally differentiated lymphocytes, as well as most myeloid lineages, are negative for HSA. Erythrocytes are an exception in that they maintain high levels of HSA expression. HSA on naive B cells has been shown to mediate cell-cell adhesion, while HSA on antigen-presenting cells has been shown to mediate a costimulatory signal important for activating T lymphocytes during an immune response. Here, we characterize mice that lack a functional HSA gene, constructed by homologous recombination in embryonic stem cells. While T-cell and myeloid development appears normal, these mice show a leaky block in B-cell development with a reduction in late pre-B and immature B-cell populations in the bone marrow. Nevertheless, peripheral B-cell numbers are normal and no impairment of immune function could be detected in these mice in a variety of immunization and infection models. We also observed that erythrocytes are altered in HSA-deficient mice. They show a higher tendency to aggregate and are more susceptible to hypotonic lysis in vitro. In vivo, the mean half-life of HSA-deficient erythrocytes was reduced. When infected with the malarial parasite Plasmodium chabaudi chabaudi, the levels of parasite-bearing erythrocytes in HSA-deficient mice were also significantly elevated, but the mice were able to clear the infection with kinetics similar to wild-type mice and were immune to a second challenge. Thus, apart from alterations in erythrocytes and a mild block in B-cell development, the regulated expression of HSA appears to be dispensable for the maturation and functioning of those cell lineages that normally express it.

scholarly journals AB0024 IN VITRO CHARACTERIZATION OF CENERIMOD, A POTENT AND SELECTIVE SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 (S1P1) MODULATOR IN PREVENTING MIGRATION OF NON-ACTIVATED AND ACTIVATED PRIMARY HUMAN B CELLS IN THE PRESENCE OR ABSENCE OF GLUCOCORTICOIDS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 1046.1-1046
Author(s):  
L. Schlicher ◽  
P. Kulig ◽  
M. Murphy ◽  
M. Keller
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Cell Activation ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
B Cell Activation ◽  
Human B Cells ◽  
Circulating Lymphocytes

Background:Cenerimod is a potent, selective, and orally active sphingosine 1-phosphate receptor 1 (S1P1) modulator that is currently being evaluated in a Phase 2b study in patients with systemic lupus erythematosus (SLE) (NCT03742037). S1P1 receptor modulators sequester circulating lymphocytes within lymph nodes, thereby reducing pathogenic autoimmune cells (including B lymphocytes) in the blood stream and in inflamed tissues. Extensive clinical experience has become available for the nonselective S1P receptor modulator fingolimod in relapsing forms of multiple sclerosis, supporting this therapeutic concept for the treatment of autoimmune disorders.Objectives:Although the effect of S1P-receptor modulators in reducing peripheral B cells is well documented1,2, the role of the S1P1 receptor on this cell type is only incompletely understood. In this study, the mode of action of cenerimod on primary human B cells was investigated in a series of in vitro experiments, including S1P1 receptor cell surface expression and chemotaxis towards S1P. Moreover, S1P1 expression following B cell activation in vitro was studied. As glucocorticoids (GC) are frequently used in the treatment of patients with autoimmune disorders including SLE, the potential influence of GC on the mode of action of cenerimod was evaluated.Methods:Primary human B lymphocytes from healthy donors were isolated from whole blood. In one set of experiments, cells were treated with different concentrations of cenerimod to measure S1P1 receptor internalization by flow cytometry. In a second set of experiments, isolated B cells were activated using different stimuli or left untreated. Cells were then analysed for S1P1 and CD69 cell surface expression and tested in a novel real-time S1P-mediated migration assay. In addition, the effect of physiological concentrations of GCs (prednisolone and prednisone) on cenerimod activity in preventing S1P mediated migration was tested.Results:In vitro, cenerimod led to a dose-dependent internalization of the S1P1 receptor on primary human B lymphocytes. Cenerimod also blocked migration of nonactivated and activated B lymphocytes towards S1P in a concentration-dependent manner, which is in line with the retention of lymphocytes in the lymph node and the reduction of circulating lymphocytes observed in the clinical setting. Upon B cell activation, which was monitored by CD69 upregulation, a simultaneous downregulation of S1P1 expression was detected, leading to less efficient S1P-directed cell migration. Importantly, physiological concentrations of GC did not affect the inhibitory activity of cenerimod on B cell migration.Conclusion:These results show that cenerimod, by modulating S1P1, blocks B lymphocyte migration towards its natural chemoattractant S1P and demonstrate compatibility of cenerimod with GC. These results are consistent with results of comparable experiments done previously using primary human T lymphocytes.References:[1]Nakamura M et al., Mult Scler. 2014 Sep; 20(10):1371-80.[2]Strasser DS et al., RMD Open 2020;6:e001261.Disclosure of Interests:None declared

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