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David J Rawlings

Publications and source records attributed to David J Rawlings.

28 records · Page 2Linked to original sources
PubMed2004

Naked DNA transfer of Factor VIII induced transgene-specific, species-independent immune response in hemophilia A mice.

The development of antibodies to a previously unexpressed protein product may limit the success of human gene therapy approaches. We inserted B-domain-deleted factor VIII (FVIII) cDNA of human, canine, or murine origin into the multiple cloning site of a liver-specific vector, pBS-HCRHPI-A, to yield plasmids pBS-HCRHPI-FVIIIA, pBS-HCRHPI-cFVIIIA, and pBS-HCRHPI-mFVIIIA, respectively. Fifty micrograms of each plasmid in 2 ml of solution was rapidly injected into the tail vein of three groups of hemophilia A mice. Factor VIII levels ranging from 3 to 12 IU/ml were obtained from all three groups (normal is 1 IU/ml in human plasma) 3 days after treatment. These initial very high levels of functional human, canine, or murine factor VIII, however, fell gradually to undetectable levels within 2-3 weeks, and their disappearance correlated with the generation of high-titer, inhibitory anti-FVIII antibodies. Notably, this immune response occurred independent of the species of origin of the exogenous factor VIII. Antibody titers to factor VIII were detected beginning at 2 weeks, reached a plateau and remained at high levels for over 6 months. The majority of anti-hFVIII IgG was IgG1 isotype specific, suggesting a humoral response mediated by Th2-induced signals. Consistent with this idea, in a separate group of mice treated with pBS-HCRHPI-FVIIIA, transient immunosuppression by cyclophosphamide significantly delayed (5/6) or abolished (1/6) inhibitory antibody formation against the transgene.

Animals↗
PubMed2004

Signaling in transitional type 2 B cells is critical for peripheral B-cell development.

Splenic peripheral B-cell development and the events regulating this functionally significant but relatively poorly defined developmental process have become a major focus in recent studies in B-cell immunology. Following the exit from the bone marrow, peripheral B cells develop through transitional type 1 (T1) and transitional type 2 (T2) B-cell stages. Emerging data suggest that the T2 subset is the immediate precursor of the mature B-cell populations present in the spleen. In this review, we first elaborate on the evidence describing the unique properties of CD21hiCD24hiCD23hiIgMhiIgDhi T2 B cells. T2 cells uniquely activate a proliferative, pro-survival, and differentiation program in response to B-cell antigen receptor (BCR) engagement. The potential mechanisms leading to the differential BCR responsiveness of T1 versus T2 B cells are discussed. We also review evidence that distinguishes key BCR-dependent signaling pathways operative in T2 and mature B cells. These signaling cascades include a protein kinase Cbeta (PKCbeta)-dependent cell-survival pathway and a second PKCbeta-independent pathway essential for BCR-driven differentiation. Finally, we discuss recent intriguing results suggesting that the type of signal(s) encountered by T2 cells leads to their differential maturation toward the follicular mature versus marginal zone mature B-cell populations. These combined observations suggest important implications with regard to B-cell selection and tolerance, potential novel therapeutic targets for B-cell lymphomas, and how the intricate balance of commensal organisms and other microenvironmental signals interact to promote the generation of 'innate-like' versus adaptive effector B-cell populations.

Animals↗
PubMed2003

A Ras activation pathway dependent on Syk phosphorylation of protein kinase C.

Protein kinase C (PKC) and Syk protein tyrosine kinase play critical roles in immune cell activation including that through the high-affinity IgE receptor, FcepsilonRI. Mechanisms by which PKC activation leads to the activation of Ras, a family of GTPases essential for immune cell activation, have been elusive. We present evidence that Tyr-662 and Tyr-658 of PKCbetaI and PKCalpha, respectively, are phosphorylated by Syk in the membrane compartment of FcepsilonRI-stimulated mast cells. These phosphorylations require prior PKC autophosphorylation of the adjacent serine residues (Ser-661 and Ser-657, respectively) and generate a binding site for the SH2 domain of the adaptor protein Grb-2. By recruiting the Grb-2/Sos complex to the plasma membrane, these conventional PKC isoforms contribute to the full activation of the Ras/extracellular signal-regulated kinase signaling pathway in FcepsilonRI-stimulated mast cells.

Adaptor Proteins, Signal Transducing↗
PubMed2003

B cell developmental requirement for the G alpha i2 gene.

Null mutation of the Galphai2 trimeric G protein results in a discrete and profound mucosal disorder, including inflammatory bowel disease (IBD), attenuation of IL-10 expression, and immune function polarized to Th1 activity. Genetic and adoptive transfer experiments have established a role for B cells and IL-10 in mucosal immunologic homeostasis and IBD resistance. In this study, we addressed the hypothesis that Galphai2 is required for the development of IL-10-producing B cells. Galphai2(-/-) mice were reduced in the relative abundance of marginal zone (MZ), transitional type 2 (T2), and B-1a B cells and significantly increased in follicular mature and B-1b B cells. Reconstitution of RAG2(-/-) mice with Galphai2(-/-) bone marrow induced an IBD-like colitis and a deficiency in absolute numbers of MZ, T2, and B-1 B cells. Thus, the Galphai2(-/-) genotype in colitis susceptibility and B cell development involved a cis effect within the hemopoietic compartment. In vitro, the B cell population of Galphai2(-/-) mice was functionally deficient in LPS-induced proliferation and IL-10 production, consistent with the exclusive capacity of T2 and MZ cell subpopulations for LPS responsiveness. In vivo, Galphai2(-/-) mice were selectively impaired for the IgM response to T-independent type II, consistent with the relative depletion of MZ and peritoneal B-1 subpopulations. Collectively, these results reveal a selective role for Galphai2 in MZ and B-1 B cell development. Disorders of this Galphai2-dependent process in B cell development may represent a mechanism for IBD susceptibility.

Animals↗
PubMed2003

BTK regulates PtdIns-4,5-P2 synthesis: importance for calcium signaling and PI3K activity.

Intracellular signaling by most cell surface receptors requires the generation of two major second messengers, phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3) and inositol-1,4,5-trisphosphate (IP3). The enzymes that produce these second messengers, phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC), utilize a common substrate, phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2). Until now, it has not been clear whether de novo PtdIns-4,5-P2 synthesis is necessary for PtdIns-3,4,5-P3 and IP3 production. Here we show that BTK, a member of the Tec family of cytoplasmic protein tyrosine kinases, associates with phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks), the enzymes that synthesize PtdIns-4,5-P2. Upon B cell receptor activation, BTK brings PIP5K to the plasma membrane as a means of generating local PtdIns-4,5-P2 synthesis. This enzyme-enzyme interaction provides a shuttling mechanism that allows BTK to stimulate the production of the substrate required by both its upstream activator, PI3K, and its downstream target, PLC-gamma2.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2002

Dysregulated TCL1 promotes multiple classes of mature B cell lymphoma.

The TCL1 protooncogene is overexpressed in many mature B cell lymphomas, especially from AIDS patients. To determine whether aberrant expression promotes B cell transformation, we generated a murine model in which a TCL1 transgene was overexpressed at similar levels in both B and T cells. Strikingly, transgenic mice developed Burkitt-like lymphoma (BLL) and diffuse large B cell lymphoma (DLBCL) with attendant Bcl-6 expression and mutated J(H) gene segments at a very high penetrance beginning at 4 months of age. In contrast, only one mouse developed a T cell malignancy at 15 months, consistent with a longer latency for transformation of T cells by TCL1. Activation of premalignant splenic B cells by means of B cell antigen receptor (BCR) engagement resulted in significantly increased proliferation and augmented AKT-dependent signaling, including increased S6 ribosomal protein phosphorylation. Transgenic spleen cells also survived longer than wild-type spleen cells in long-term culture. Together these data demonstrate that TCL1 is a powerful oncogene that, when overexpressed in both B and T cells, predominantly yields mature B cell lymphomas.

Animals↗
PubMed2002

SHP-1 regulates Fcgamma receptor-mediated phagocytosis and the activation of RAC.

Fcgamma receptor-mediated phagocytosis is a complex process involving the activation of protein tyrosine kinases, events that are potentially down-regulated by protein tyrosine phosphatases. We used the J774A.1 macrophage cell line to examine the roles played by the protein tyrosine phosphatase SHP-1 in the negative regulation of Fcgamma receptor-mediated phagocytosis. Stimulation with sensitized sheep red blood cells (sRBCs) induced tyrosine phosphorylation of CBL and association of CBL with CRKL. These events were completely or partially abrogated by PP1 or the heterologous expression of dominant-negative SYK, respectively. Heterologous expression of wild-type but not catalytically inactive SHP-1 also completely abrogated the phagocytosis of IgG-sensitized sRBCs. Most notably, overexpressed SHP-1 associates with CBL and this association led to CBL dephosphorylation, loss of the CBL-CRKL interaction, and the suppression of Rac activation. These data represent the first direct evidence that SHP-1 is involved in the regulation of Fcgamma receptor-mediated phagocytosis and suggest that activating signals mediated by SRC family kinases SYK, CBL, phosphatidyl inositol-3 (PI-3) kinase, and Rac are directly opposed by inhibitory signals through SHP-1.

Animals↗
PubMed2002

PKC-beta controls I kappa B kinase lipid raft recruitment and activation in response to BCR signaling.

NF-kappa B signaling is required for the maintenance of normal B lymphocytes, whereas dysregulated NF-kappa B activation contributes to B cell lymphomas. The events that regulate NF-kappa B signaling in B lymphocytes are poorly defined. Here, we demonstrate that PKC-beta is specifically required for B cell receptor (BCR)-mediated NF-kappa B activation. B cells from protein kinase C-beta (PKC-beta)-deficient mice failed to recruit the I kappa B kinase (IKK) complex into lipid rafts, activate IKK, degrade I kappa B or up-regulate NF-kappa B-dependent survival signals. Inhibition of PKC-beta promoted cell death in B lymphomas characterized by exaggerated NF-kappa B activity. Together, these data define an essential role for PKC-beta in BCR survival signaling and highlight PKC-beta as a key therapeutic target for B-lineage malignancies.

Animals↗
PubMed2002

Transitional B lymphocyte subsets operate as distinct checkpoints in murine splenic B cell development.

Signaling through the Ag receptor is required for peripheral B lymphocyte maturation and maintenance. Defects in components of the B cell receptor (BCR) signalosome result in developmental blocks at the transition from immature (heat-stable Ag (HSA)(high)) to mature (HSA(low)) B cells. Recent studies have subdivided the immature, or transitional, splenic B cells into two subsets, transitional 1 (T1) and transitional 2 (T2) cells. T1 and T2 cells express distinct surface markers and are located in distinct anatomic locations. In this report, we evaluated the BCR signaling capacity of T1 and T2 B cell subsets. In response to BCR engagement, T2 cells rapidly entered cell cycle and resisted cell death. In contrast, T1 cells did not proliferate and instead died after BCR stimulation. Correlating with these results, T2 cells robustly induced expression of the cell cycle regulator cyclin D2 and the antiapoptotic factors A1/Bfl-1 and Bcl-x(L) and exhibited activation of Akt. In contrast, T1 cells failed to up-regulate these markers. BCR stimulation of T2 cells also led to down-regulation of CD21 and CD24 (HSA) expression, resulting in a mature B cell phenotype. In addition, T2 cells from Bruton's tyrosine kinase-deficient Xid mice failed to generate these proliferative and survival responses, suggesting a requirement for the BCR signalosome specifically at the T2 stage. Taken together, these data clearly demonstrate that T2 immature B cells comprise a discrete developmental subset that mediates BCR-dependent proliferative, prosurvival, and differentiation signals. Their distinct BCR-dependent responses suggest unique roles for T1 vs T2 cells in peripheral B cell selection.

Agammaglobulinaemia Tyrosine Kinase↗