Interleukin 7 receptor engagement stimulates tyrosine phosphorylation, inositol phospholipid turnover, proliferation, and selective differentiation to the CD4 lineage by human fetal thymocytes.

The purposes of this study were to elucidate the effects of recombinant human interleukin 7 (rhIL-7) on proliferation as well as differentiation of human fetal thymocytes and to analyze the biochemical nature of the IL-7 receptor-linked transmembrane signal. In the absence of costimulants, rhIL-7 stimulated the in vitro proliferation and colony formation of CD4+CD8+ double-positive immature fetal thymocytes. Furthermore, rhIL-7 promoted partial differentiation of immature thymocytes with a selective advantage for the development of CD4+CD8- single-positive thymocytes. Our observations suggest that IL-7 likely has an important regulatory role during the earliest stages of human T-cell ontogeny. Stimulation of fetal thymocytes with rhIL-7 resulted in enhanced tyrosine phosphorylation of three distinct phosphoproteins with molecular masses of 72, 98, 123, and 190 kDa and induced a rapid and biphasic increase in the production of inositol 1,4,5-trisphosphate, which was inhibitable by the tyrosine protein kinase inhibitor genistein. Thus, the transmembrane signal triggered by engagement of the IL-7 receptor is intimately linked to a functional tyrosine protein kinase pathway and stimulates the inositol phospholipid turnover and proliferation, as well as selective differentiation to the CD4 lineage, by human fetal thymocytes.     

Predominant expression and activation-induced tyrosine phosphorylation of phospholipase C-gamma 2 in B lymphocytes.

The triggering of T- or B-cell antigen-specific receptors is accompanied by rapid tyrosine phosphorylation of distinct cellular substrates, one of which is the gamma 1 isoform of inositol phospholipid-specific phospholipase C (PLC-gamma 1). This phosphorylation event, mediated by a putative protein tyrosine kinase coupled to the antigen receptor, probably stimulates the enzymatic activity of PLC-gamma 1, thereby promoting inositol phospholipid hydrolysis and other downstream signal transduction events. Recently, another ubiquitously expressed PLC isoform, PLC-gamma 2 (which shares 50.2% amino acid homology with PLC-gamma 1), has been identified. PLC-gamma 2-specific antibodies were used to evaluate the distribution and potential signaling role of this isoform in lymphocytes. Here, we report that, in contrast to T lymphocytes that express predominantly PLC-gamma 1, the major isoform expressed in murine and human resting B cells is PLC-gamma 2. Among B-cell tumor lines, all five murine B-lymphoma lines tested and one of six human B-lymphoblastoid cell lines also expressed predominantly PLC-gamma 2. However, three other human lines preferentially expressed PLC-gamma 1, and two others displayed similar levels of the two PLC-gamma isoforms. Furthermore, the triggering of B-cell surface immunoglobulin by anti-receptor antibodies was accompanied by a rapid tyrosine phosphorylation of PLC-gamma 2, which peaked after 5 min of stimulation. Conversely, and in agreement with recent reports, triggering of the T-cell antigen receptor complex led to the predominant phosphorylation of PLC-gamma 1 on tyrosine. These findings identify PLC-gamma 2 as a substrate for a B-cell putative protein tyrosine kinase coupled to the antigen receptor and suggest that its tyrosine phosphorylation constitutes a critical and early event in B-cell activation and, furthermore, that PLC-gamma 1 and PLC-gamma 2 may participate in similar but distinct signal transduction pathways in lymphocytes.     

Developmental hierarchy during early human B-cell ontogeny after autologous bone marrow transplantation using autografts depleted of CD19+ B-cell precursors by an anti-CD19 pan-B-cell immunotoxin containing pokeweed antiviral protein.

Sequential immunophenotypes of bone marrow (BM) and peripheral blood (PBL) lymphoid cells from 15 B-lineage acute lymphoblastic leukemia (ALL) patients who underwent autologous bone marrow transplantation (BMT) during complete remission were determined by dual-color immunofluorescence and multiparameter flow cytometry. Autografts were depleted of CD19+ B-cell precursors by an immunochemopurging protocol that combines B43-PAP, a potent anti-CD19 immunotoxin, and the cyclophosphamide congener 4-hydroperoxycyclophosphamide (4-HC). A marked interpatient variation was observed in the appearance and expansion of B-cell precursors repopulating the posttransplant marrow. The expression of CD10 and CD19 antigens during early B-cell ontogeny post-BMT preceded the expression of CD20, CD21, CD22, CD40, and sIgM. The surface antigen profiles of the emerging B-cell precursors were similar to those of fetal liver or fetal bone marrow B-cell precursors. Our comparisons of BM and PBL samples from patients in the early post-BMT period demonstrated that (1) PBL initially contains fewer B-lineage cells than does BM, and (2) circulating B-lineage lymphoid cells have a more mature immunophenotype than do BM B-lineage lymphoid cells. Comparison of the surface antigen profiles of day 30 versus day 100 or year 1 BM or PBL lymphoid cells showed an increase in the percentages of CD10+CD22- undifferentiated lymphocyte precursors, as well as CD19+sIgM- B-cell precursors (pre-pre-B), consistent with a time-dependent expansion of these B-cell precursor populations post-BMT. Importantly, the percentages of CD10+CD22+ and CD19+sIgM+ B-cell precursor (pre-B) populations also increased between 30 days and 1 year post-BMT, confirming the ability of emerging immature B-cell precursors to differentiate along the B-precursor pathway. The acquisition and expression of B-lineage differentiation antigens at different stages of the post-BMT B-cell ontogeny support the notion that the expression of these antigens is developmentally programmed. Similar to patients in previous autologous BMT studies, recipients of B-cell precursor-depleted autografts had normal or nearly normal serum immunoglobulin levels, suggesting that the maturing B-cell/plasma cell populations can produce and secrete immunoglobulins. The development of a functional CD19+ B-lineage lymphoid compartment in recipients of autografts which were depleted of CD19+ B-cell precursors corroborates the previously postulated existence of CD19- B-lineage lymphoid progenitor cells.     

Engagement of interleukin-7 receptor stimulates tyrosine phosphorylation, phosphoinositide turnover, and clonal proliferation of human T-lineage acute lymphoblastic leukemia cells.

The purposes of this study were to examine the biologic effects of the engagement of the interleukin-7 receptor (IL-7R) with recombinant human interleukin-7 (rhIL-7) in immunophenotypically distinct T-lineage acute lymphoblastic leukemia (ALL) blasts and to elucidate the biochemical nature of the IL-7R-linked transmembrane signal in rhIL-7-responsive T-lineage ALL blast populations. In the absence of costimulants, rhIL-7 stimulated the in vitro proliferation and colony formation of freshly isolated leukemic blasts from six to eight T-lineage ALL patients with a mean plating efficiency of 196 +/- 53 (background subtracted) colonies/10(5) blasts plated. Stimulation of T-lineage ALL blasts with rhIL-7 resulted in markedly enhanced tyrosine phosphorylation of six distinct phosphoproteins with molecular weights of 57, 72, 98, 123, 150, and 190 Kd, and induced a rapid increase in the production of inositol-1,4,5-trisphosphate (Ins-1,4,5-P3), which was inhibitable by the tyrosine-specific protein kinase inhibitor genistein, but not by the serine/threonine-specific protein kinase C inhibitor H7. Similarly, rhIL-7 stimulated Ins-1,4,5-P3 production in CEM-1.3 T-lineage ALL cells and this stimulation was inhibitable by the tyrosine-specific protein kinase inhibitors genistein and herbimycin A, but not by H-7. Thus, the transmembrane signal triggered by engagement of the IL-7R is intimately linked to a functional tyrosine-specific protein kinase pathway and stimulates the phosphoinositide (PI) turnover and proliferation of T-lineage ALL blasts. The presented data confirm and extend previous studies on the expression of functional IL-7R on T-lineage ALL blasts and support the hypothesis that IL-7 may play an important regulatory role in the biology of T-lineage ALL.     

Inhibitory effect of interleukin-4 on the in vitro growth of Ph1-positive acute lymphoblastic leukemia cells

We investigated the effect of recombinant human interleukin-4 (rhIL-4) on the in vitro growth of human leukemia cells in liquid culture and 3H-thymidine incorporation and found inhibitory effects on the growth of leukemic cells from patients with Ph1-positive acute lymphoblastic leukemia (Ph1 ALL) and three Ph1 ALL cell lines. However, no inhibitory effects were seen in Ph1-positive leukemic cell lines derived from patients with chronic myelogenous leukemia in blast crisis and various types of Ph1-negative leukemia cells, including B-lineage leukemia cells. In a flow cytometry assay of IL-4 receptor (IL-4R), all three Ph1-positive ALL cell lines showed the presence of IL-4R on their cell surfaces, and the IL-4-dependent inhibition on the growth of Ph1-positive ALL cells was abrogated by the addition of either monoclonal or polyclonal antibodies against rhIL-4. Other cytokines, including IL-2, IL-3, granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, and IL-6, showed no inhibitory effects on the growth of Ph1-ALL cells, but tumor necrosis factor-alpha (TNF-alpha) and interferon (IFN)-alpha, -beta, and -gamma displayed slight inhibitory effects in a high concentration. The growth inhibition induced by rhIL-4 in the Ph1-positive ALL cells was not abrogated by the addition of antibodies against either IFN-gamma or TNF-alpha. Furthermore, these cells showed no significant production of IFN-alpha, -beta, or -gamma or TNF-alpha after exposure to rhIL-4, thus indicating that the growth inhibition of Ph1-positive ALL cells by rhIL-4 is not associated with IL-4-stimulating production of these factors. rhIL-4 caused significant inhibition of the tyrosine kinase activity in these Ph1-positive ALL cells, similar to Herbimycin A, an inhibitor of tyrosine kinase that inhibited the tyrosine kinase activity in these cells. Our finding suggests that the clinical evaluation of rhIL-4 may offer promising therapeutic possibilities for patients with Ph1-positive ALL.     

A recombinant single-chain IL-7/HGFbeta hybrid cytokine induces juxtacrine interactions of the IL-7 and HGF (c-Met) receptors and stimulates the proliferation of CFU-S12, CLPs, and pre-pro-B cells

A novel recombinant interleukin-7/hepatocyte growth factor beta-chain (IL-7/HGFbeta) hybrid cytokine was constructed as a single chain (sc) composed of IL-7 and HGFbeta connected by a flexible linker. Unlike recombinant (r) IL-7, which stimulated pro-B cells and pre-B cells only, scIL-7/HGFbeta stimulated the proliferation of pre-pro-B cells, common lymphoid progenitors (CLPs), and colony-forming unit (CFU)-S12 in cultures of IL-7-/- mouse BM cells. When injected in vivo, 3- to 4-fold more splenic B-lineage cells appeared in recipients of bone marrow (BM) cells from the scIL-7/HGFbeta-stimulated cultures than from rIL-7-stimulated cultures. Moreover, on a per-cell basis, scIL-7/HGFbeta culture-generated cells produced 16- to 20-fold more BM and splenic B-lineage cells than did normal BM cells. Antibody blocking, receptor phosphorylation, and confocal microscopy demonstrated that scIL-7/HGFbeta signals though both the IL-7 and HGF (c-Met) receptors, which form IL-7R/c-Met complexes on the surface of CLPs and pre-pro-B cells. In addition, the IL-7Ralpha chain, gammac chain, and c-Met were coisolated from purified CLPs and pre-pro-B cells on scIL-7/HGFbeta affinity gels, indicating that they are major components of the IL-7/HGFbeta receptor. Hence, the present results demonstrate that the IL-7/HGFbeta hybrid cytokine efficiently and selectively stimulates the most primitive B-lineage precursors in BM by inducing juxtacrine interactions between the IL-7 and c-Met receptors.     

Activation of phosphatidylinositol-3 kinase by ligation of the interleukin-7 receptor is dependent on protein tyrosine kinase activity

Ligation of the interleukin-7 receptor (IL-7R) results in a rapid phosphorylation of tyrosine residues on multiple substrates. In addition, we have recently shown that the IL-7R mediates activation of phosphatidylinositol-3 (PI-3) kinase. Because PI-3 kinase activity can be immunoprecipitated with antiphosphotyrosine antibodies in most receptor systems studied, it has been examined that either PI-3 kinase or an associated protein become tyrosine-phosphorylated after ligand binding. We studied here the possibility that PI-3 kinase, which is directly linked to mitogenic responses in growth factor receptors, is tyrosine-phosphorylated after stimulation of the IL-7R. Using anti-p85 alpha or anti-p85 beta antibodies raised against the p85 subunit of PI- 3 kinase for immunoprecipitation and subsequent blotting with antiphosphotyrosine clearly shows that IL-7-stimulated human precursor cells contain both p85 alpha and p85 beta proteins phosphorylated on tyrosine residues. Specific protein tyrosine kinase inhibitors such as tyrphostin AG-490 block total cell lysate phosphorylation and tyrosine phosphorylation on p85. Similar concentrations of this inhibitor also block in vitro and in vivo PI-3 kinase activity suggesting that this enzyme activation is dependent on the phosphorylation event of p85. In addition, AG-490 blocks IL-7-mediated proliferation in a dose-dependent manner, suggesting a link between the early events of PI-3 kinase phosphorylation and activation with IL-7R-induced cell growth.     

Interleukin 7 independent development of human B cells.

Mammalian hematopoietic stem cell (HSC) commitment and differentiation into lymphoid lineage cells proceed through a series of developmentally restricted progenitor compartments. A complete understanding of this process, and how it differs from HSC commitment and differentiation into cells of the myeloid/erythroid lineages, requires the development of model systems that support HSC commitment to the lymphoid lineages. We now describe a human bone marrow stromal cell culture that preferentially supports commitment and differentiation of human HSC to CD19+ B-lineage cells. Fluorescence activated cell sorterpurified CD34++/lineage-cells were isolated from fetal bone marrow and cultured on human fetal bone marrow stromal cells in serum-free conditions containing no exogenous cytokines. Over a period of 3 weeks, CD34++/lineage- cells underwent commitment, differentiation, and expansion into the B lineage. Progressive changes included: loss of CD34, acquisition of and graded increases in the level of cell surface CD19, and appearance of immature B cells expressing mu/kappa or mu/lambda cell surface Ig receptors. The tempo and phenotype of B-cell development was not influenced by the addition of IL-7 (10 ng/ml), or by the addition of goat anti-IL-7 neutralizing antibody. These results indicate a profound difference between mouse and human in the requirement for IL-7 in normal B-cell development, and provide an experimental system to identify and characterize human bone marrow stromal cell-derived molecules crucial for human B lymphopoiesis.     

Evidence for a signaling role for the alpha chains of granulocyte- macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors: divergent signaling pathways between GM-CSF/IL-3 and IL-5

In the present study, we have used a human erythroleukemia cell line, TF-1, that proliferates in response to granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3), and interleukin-5 (IL-5) to investigate the role of receptors for these cytokines in signal transduction mechanisms involved in proliferative responses. The receptors for GM-CSF, IL-3, and IL-5 each possess a cytokine specific alpha subunit, but all three share a common beta chain. Using an immunoblotting system designed to detect phosphotyrosine containing proteins and a permeabilized cell system to detect rapid changes in phosphate turnover on proteins, we show that while GM-CSF and IL-3 use tyrosine phosphorylation to mediate mitogenic signal transduction, IL-5 uses tyrosine dephosphorylation in its signaling pathway. The use of different signaling pathways by these cytokines can be confirmed in a biologic system whereby the proliferation induced in culture by GM-CSF and IL-3 is inhibited by tyrosine kinase inhibitors, but that induced by IL-5 is enhanced. Conversely, GM-CSF- and IL-3-induced proliferation is stimulated by a tyrosine phosphatase inhibitor, yet IL-5-induced proliferation is inhibited. Inhibitors of protein kinase C inhibit IL-3- and GM-CSF-, but not IL-5-induced proliferation. We suggest that, because all these cytokines share the identical beta chain of their receptors, the cytokine specific alpha chain mediates the linkage of each receptor to the individual biochemical signal transduction pathways responsible for the different biologic activities of these cytokines.     

Phorbol esters promote alpha 1-adrenergic receptor phosphorylation and receptor uncoupling from inositol phospholipid metabolism.

DDT1 MF-2 cells, which are derived from hamster vas deferens smooth muscle, contain alpha 1-adrenergic receptors (54,800 +/- 2700 sites per cell) that are coupled to stimulation of inositol phospholipid metabolism. Incubation of these cells with tumor-promoting phorbol esters, which stimulate calcium- and phospholipid-dependent protein kinase, leads to a marked attenuation of the ability of alpha 1-receptor agonists such as norepinephrine to stimulate the turnover of inositol phospholipids. This turnover was measured by determining the 32P content of phosphatidylinositol and phosphatidic acid after prelabeling of the cellular ATP pool with 32Pi. These phorbol ester-treated cells also displayed a decrease in binding affinity of cellular alpha 1 receptors for agonists with no change in antagonist affinity. By using affinity chromatography on the affinity resin Affi-Gel-A55414, the alpha 1 receptors were purified approximately equal to 300-fold from control and phorbol ester-treated 32Pi-prelabeled cells. As assessed by NaDodSO4/polyacrylamide gel electrophoresis, the Mr 80,000 alpha 1-receptor ligand-binding subunit is a phosphopeptide containing 1.2 mol of phosphate per mol of alpha 1 receptor. After phorbol ester treatment this increased to 3.6 mol of phosphate per mol of alpha 1 receptor. The effect of phorbol esters on norepinephrine-stimulated inositol phospholipid turnover and alpha 1-receptor phosphorylation showed the same rapid time course with a t1/2 less than 2 min. These results indicate that calcium- and phospholipid-dependent protein kinase may play an important role in regulating the function of receptors that are coupled to the inositol phospholipid cycle by phosphorylating and deactivating them.     

HGAL, a lymphoma prognostic biomarker, interacts with the cytoskeleton and mediates the effects of IL-6 on cell migration

HGAL is a newly identified germinal center (GC)-specific gene whose expression by the tumor cells correlates with a favorable prognosis in patients with diffuse large B-cell and classical Hodgkin lymphomas. The function of HGAL is unknown. Previous studies demonstrated that HGAL is dispensable for GC formation, immunoglobulin gene class-switch recombination, and somatic hypermutation. Herein, we identify a role for HGAL in the regulation of cell motility. We demonstrate that IL-6 induces the phosphorylation of the C-terminal tyrosine residue of the HGAL protein via the Lyn kinase, and promotes its relocalization from the cytoplasm to podosome-like structures. Further, IL-6-induced HGAL phosphorylation increases its interaction with myosin II and is associated with inhibition of cell migration. Knockdown of endogenous HGAL ameliorates IL-6-induced inhibition of cell migration, whereas overexpression of HGAL imparts inhibitory effects of IL-6 on cell migration. Taken together, our results suggest that HGAL is involved in negative regulation of lymphocyte migration, thus constraining lymphocytes to the GC. Inhibition of lymphocyte migration might contribute to the less aggressive clinical behavior of HGAL-expressing lymphomas.     

Temporal association of CD40 antigen expression with discrete stages of human B-cell ontogeny and the efficacy of anti-CD40 immunotoxins against clonogenic B-lineage acute lymphoblastic leukemia as well as B-lineage non-Hodgkin's lymphoma cells.

Detailed immunophenotypic analyses of immunologically classified leukemias and lymphomas showed that CD40 displays an exquisite B-lineage specificity within the human lymphopoietic system. Notably, 82% of B-lineage chronic lymphocytic leukemias (CLLs), 82% of B-lineage hairy cell leukemias (HCLs), 86% of B-lineage non-Hodgkin's lymphomas (NHLs), and 29% of B-lineage acute lymphoblastic leukemias (ALLs) were CD40+. Quantitative analyses of the correlated expression of CD40 and other B-lineage differentiation antigens on fetal lymphoid precursor cells by multiparameter two-color/three-color flow cytometry, combined with analyses of sequential antigen expression on fluorescence-activated cell fluorescence activated cell sorter (FACS) isolated immunologically distinct fetal B-cell precursor subpopulations during in vitro proliferation and differentiation, provided evidence that the acquisition of CD40 antigen in human B-cell ontogeny occurs subsequent to the expression of CD10 and CD19 antigens but before the surface expression of CD20, CD21, CD22, CD24, and surface immunoglobulin M (sIgM). Some leukemic pro-B cells from ALL patients as well as normal pro-B cell clones from fetal livers displaying germline Ig heavy chain genes were CD40+, indicating that the acquisition of CD40 antigen likely precedes the rearrangement of Ig heavy chain genes. CD40+ FACS-sorted malignant cells from B-lineage ALL as well as B-lineage NHL patients were capable of in vitro clonogenic growth, indicating the CD40 antigen is expressed on clonogenic leukemia and lymphoma cells. This hypothesis was confirmed by the ability of an anti-CD40 immunotoxin that we used as an antigen-specific cytotoxic probe to effectively kill clonogenic B-lineage ALL and NHL cells.     

Sustained correction of B-cell development and function in a murine model of X-linked agammaglobulinemia (XLA) using retroviral-mediated gene transfer

X-linked agammaglobulinemia (XLA) is a human immunodeficiency caused by mutations in Bruton tyrosine kinase (Btk) and characterized by an arrest in early B-cell development, near absence of serum immunoglobulin, and recurrent bacterial infections. Using Btk- and Tec-deficient mice (BtkTec(-/-)) as a model for XLA, we determined if Btk gene therapy could correct this disorder. Bone marrow (BM) from 5-fluorouracil (5FU)-treated BtkTec(-/-) mice was transduced with a retroviral vector expressing human Btk and transplanted into BtkTec(-/-) recipients. Mice engrafted with transduced hematopoietic cells exhibited rescue of both primary and peripheral B-lineage development, recovery of peritoneal B1 B cells, and correction of serum immunoglobulin M (IgM) and IgG(3) levels. Gene transfer also restored T-independent type II immune responses, and B-cell antigen receptor (BCR) proliferative responses. B-cell progenitors derived from Btk-transduced stem cells exhibited higher levels of Btk expression than non-B cells; and marking studies demonstrated a selective advantage for Btk-transduced B-lineage cells. BM derived from primary recipients also rescued Btk-dependent function in secondary hosts that had received a transplant. Together, these data demonstrate that gene transfer into hematopoietic stem cells can reconstitute Btk-dependent B-cell development and function in vivo, and strongly support the feasibility of pursuing Btk gene transfer for XLA.     

Membrane-associated CD19-LYN complex is an endogenous p53-independent and Bc1-2-independent regulator of apoptosis in human B-lineage lymphoma cells.

CD19 receptor is expressed at high levels on human B-lineage lymphoid cells and is physically associated with the Src protooncogene family protein-tyrosine kinase Lyn. Recent studies indicate that the membrane-associated CD19-Lyn receptor-enzyme complex plays a pivotal role for survival and clonogenicity of immature B-cell precursors from acute lymphoblastic leukemia patients, but its significance for mature B-lineage lymphoid cells (e.g., B-lineage lymphoma cells) is unknown. CD19-associated Lyn kinase can be selectively targeted and inhibited with B43-Gen, a CD19 receptor-specific immunoconjugate containing the naturally occurring protein-tyrosine kinase inhibitor genistein (Gen). We now present experimental evidence that targeting the membrane-associated CD19-Lyn complex in vitro with B43-Gen triggers rapid apoptotic cell death in highly radiation-resistant p53-Bax- Ramos-BT B-lineage lymphoma cells expressing high levels of Bcl-2 protein without affecting the Bcl-2 expression level. The therapeutic potential of this membrane-directed apoptosis induction strategy was examined in a scid mouse xenograft model of radiation-resistant high-grade human B-lineage lymphoma. Remarkably, in vivo treatment of scid mice challenged with an invariably fatal number of Ramos-BT cells with B43-Gen at a dose level < 1/10 the maximum tolerated dose resulted in 70% long-term event-free survival. Taken together, these results provide unprecedented evidence that the membrane-associated anti-apoptotic CD19-Lyn complex may be at least as important as Bcl-2/Bax ratio for survival of lymphoma cells.     

Identification, cloning, and characterization of a novel human T-cell- specific tyrosine kinase located at the hematopoietin complex on chromosome 5q

Signal transduction through the T-cell receptor and cytokine receptors on the surface of T lymphocytes occurs largely via tyrosine phosphorylation of intracellular substrates. Because neither the T-cell receptor nor cytokine receptors contain intrinsic kinase domains, signal transduction is thought to occur via association of these receptors with intracellular protein tyrosine kinases. Although several members of the SRC and SYK families of tyrosine kinases have been implicated in signal transduction in lymphocytes, it seems likely that additional tyrosine kinases involved in signal transduction remain to be identified. To identify unique T-cell tyrosine kinases, we used polymerase chain reaction-based cloning with degenerate oligonucleotides directed at highly conserved motifs of tyrosine kinase domains. We have cloned the complete cDNA for a unique human tyrosine kinase that is expressed mainly in T lymphocytes (EMT) and natural killer (NK) cells. The cDNA of EMT predicts an open reading frame of 1866 bp encoding a protein with a predicted size of 72 Kd, which is in keeping with its size on Western blotting. A single 6.2-kb EMT mRNA and 72-Kd protein were detected in T lymphocytes and NK-like cell lines, but were not detected in other cell lineages. EMT contains both SH2 and SH3 domains, as do many other intracellular kinases. EMT does not contain the N-terminal myristylation site or the negative regulatory tyrosine phosphorylation site in its carboxyterminus that are found in the SRC family of tyrosine kinases. EMT is related to the B-cell progenitor kinase (BPK), which has recently been implicated in X-linked hypogammaglobulinemia, to the TECI mammalian kinase, which has been implicated in liver neoplasia, to the more widely expressed TECII mammalian kinase, and to the Drosophila melanogaster Dsrc28 kinase. Sequence comparison suggests that EMT is likely the human homologue of a recently identified murine interleukin-2 (IL-2)-inducible T cell kinase (ITK). However, unlike ITK, EMT message and protein levels do not vary markedly on stimulation of human IL-2-responsive T cells with IL-2. Taken together, it seems that EMT is a member of a new family of intracellular kinases that includes BPK, TECI, and TECII. EMT was localized to chromosome 5q31-32, a region that contains the genes for several growth factors and receptors as well as early activation genes, particularly those involved in the hematopoietic system. Furthermore, the 5q31-32 region is implicated in the genesis of the 5q- syndrome associated with myelodysplasia and development of leukemia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bivalent binding and signaling characteristics of Leridistim, a novel chimeric dual agonist of interleukin-3 and granulocyte colony-stimulating factor receptors

Leridistim is a member of a novel family of engineered chimeric cytokines, myelopoietins, that contain agonists of both interleukin-3 (IL-3) receptors (IL-3R) and granulocyte colony-stimulating factor (G-CSF) receptors (G-CSFR).To more clearly understand Leridistim's function at the molecular level, binding to both IL-3R and G-CSFR and subsequent signaling characteristics have been delineated.The affinity of Leridistim for the human G-CSFR was found to be comparable to that of native G-CSF (IC(50) = 0.96 nM and 1.0 nM, respectively). Both Leridistim and G-CSF induced receptor tyrosine phosphorylation to a similar maximal level. Compared with native recombinant human IL-3 (rhIL-3), Leridistim was found to possess higher affinity for the IL-3R alpha chain (IL-3Ralpha) (IC(50) = 85 nM and 162 nM, respectively). However, the increase in Leridistim binding affinity to the functional, high-affinity heterodimeric IL-3Ralphabeta(c) receptor is lower than that observed with rhIL-3 (85 nM and 14 nM vs 162 nM and 3.5 nM, respectively). Leridistim induced tyrosine phosphorylation of beta(c) to a level comparable to native IL-3, and the level of JAK2 tyrosine phosphorylation in cells expressing both IL-3R and G-CSFR was comparable to that observed with IL-3 or G-CSF alone. The ability of Leridistim to interact with IL-3R and G-CSFR simultaneously was demonstrated using surface plasmon resonance analysis. These studies were extended to demonstrate that Leridistim exhibited a higher affinity for the IL-3R on cells that express both the IL-3Ralphabeta(c) and the G-CSFR (IC(50) = 2 nM) compared with cells that contain the IL-3Ralphabeta(c) alone (IC(50) = 14 nM).Leridistim binds to both IL-3R and G-CSFR simultaneously and has been shown to activate both receptors. The bivalent avidity may explain the unique biologic effects and unexpected potency of Leridistim in hematopoietic cells compared with rhIL-3 or G-CSF alone or in combination.     

Geranylgeranylacetone, an anti-ulcer drug, stimulates hexosamine production in a rat gastric mucosal cell line through binding to a specific cytosolic protein

An anti-ulcer drug, geranylgeranylacetone (GGA), stimulates hexosamine production in a rat gastric mucosal cell line (RGM-1). The aim of this study was to elucidate the mechanism of this action. The role of protein kinase A, inositol phospholipid turnover and tyrosine kinase in the stimulatory action of GGA on hexosamine production in RGM-1 was determined by observing cAMP production, [³H]-inositol phosphate turnover and western blotting of tyrosine phosphorylation, respectively. Any trophic effect of GGA on RGM-1 was also checked by [³H]-thymidine incorporation. Our experiments showed that GGA has no effect on cAMP production, inositol phospholipid turnover, tyrosine phosphorylation or DNA synthesis in RGM-1. Finally, a [¹⁴C]-GGA competitive receptor binding assay was performed on RGM-1 and we found that [¹⁴C]-GGA specifically bound to RGM-1 cytosolic protein. Although retinoic acid (RA), another polyisoprenoid compound significantly stimulated hexosamine production in RGM-1, we confirmed that the [¹⁴C]-GGA binding site in RGM-1 is different from the RA binding site. In summary, GGA stimulates hexosamine production in RGM-1 and this action is probably mediated through its binding to a specific cytosolic protein in RGM-1.     

Recombinant human interleukin-9 induces protein tyrosine phosphorylation and synergizes with steel factor to stimulate proliferation of the human factor-dependent cell line, M07e.

Human interleukin-9 (IL-9) was originally identified and cloned based on its stimulatory effect on proliferation of human myeloid cell line, M07e. IL-9 synergized with Steel factor, the ligand for the c-kit product, to stimulate M07e cell proliferation. To investigate potential mechanisms for this, IL-9 was assessed for effects on protein tyrosine kinase activities in M07e cells by immunoblotting with anti-phosphotyrosine monoclonal antibody; results were compared with those of Steel factor alone and in combination with IL-9, and those of 12-0-tetradecanoyl phorbol-13-acetate (TPA). Recombinant human IL-9 (10 ng/mL) rapidly and transiently induced or enhanced at least four tyrosine phosphorylated protein bands with molecular weights of 105, 97, 85, and 81 Kd. This tyrosine phosphorylation pattern was different from that generated by recombinant murine Steel factor or TPA stimulation and the combination of IL-9 and Steel factor did not change the IL-9-induced pattern. IL-9-induced tyrosine phosphorylated bands were completely blocked by treatment of IL-9 with anti-IL-9 antibody under conditions that also neutralized the synergistic effect of IL-9 with Steel factor on M07e cell proliferation. Genistein, a tyrosine kinase inhibitor, blocked phosphorylation of IL-9 and Steel factor-induced bands. Unlike Steel factor or TPA, IL-9 did not appear to stimulate phosphorylation of 42-Kd mitogen-activated protein (MAP) kinase or Raf-1, or enhance MAP kinase activity. MAP kinase and Raf-1 are serine/threonine kinases that are phosphorylated and activated by many growth factors and by agonists for protein kinase C. While the combination of IL-9 plus SLF did not appear to induce phosphorylation of new bands not already seen with either IL-9 or SLF alone, or enhance the phosphorylation of those bands seen with either cytokine alone, the results suggest that IL-9 activates specific and unique signal transduction pathways.     

Expression and function of the interleukin 7 receptor in murine lymphocytes.

A monoclonal antibody, A7R34, that recognizes the high-affinity interleukin 7 receptor (IL-7Ra) and blocks the binding between IL-7 and IL-7Ra has been produced. Cell surface staining with A7R34 demonstrated that IL-7Ra is expressed in both B- and T-cell lineages. In the bone marrow, immature B-lineage cells that do not express surface IgM were IL-7Ra+. In the thymus, IL-7Ra was detected in CD4-8- T cells and also in CD4 or CD8 single-positive cells but not in CD4+8+ double-positive cells. In the peripheral lymphoid tissues, both CD4 and CD8 single-positive cells were the major cell types that express IL-7Ra. Addition of A7R34 to a long-term B-precursor-cell culture inhibited proliferation of the B-lineage cells, indicating that IL-7 is an absolute requirement for in vitro B-cell genesis. Consistent with this in vitro result, continuous injection of A7R34 into an adult mouse resulted in a decrease of B-precursor cells and also of thymocytes, whereas a considerable fraction of mature B and T cells in the peripheral tissues persisted over 2 weeks of the experiment. When A7R34 injection is started from day 14 of gestation, it is possible to produce mice that lack B cells. These results indicate that IL-7 is an essential molecule for generation of both B and T cells in murine bone marrow and thymus, respectively. Moreover, IL-7Ra would be the sole receptor system regulating these processes.