Biomedical subjects

H E Broxmeyer

Publications and source records attributed to H E Broxmeyer.

At least 19 recordsLinked to original sources

PubMed2002

Role of the CC chemokine receptor 9/TECK interaction in apoptosis.

Chemokine receptors are members of the G protein coupled receptor (GPCR) supergene family whose expression is highly restricted to hematopoietic cells. Although the primary role of chemokine and chemokine receptor interaction is believed to be regulation of chemotaxis of leukocytes, subsequent information clearly suggests that multiple immune regulatory functions are attributed to chemokine receptor signaling. We recently showed that activation of the CC chemokine 9 receptor (CCR9), a thymus-specific chemokine receptor, led to potent cFLIP(L)-independent resistance to cycloheximide-induced apoptosis and modest resistance to Fas-mediated apoptosis possibly via activation of multiple signaling components involving Akt and glycogen synthase kinase 3beta. The fact that these two apoptotic signals involve activation of similar arrays of death execution machinery such as caspase-8, caspase-9, or caspase-3, suggests that chemokine receptor signaling may provide a wide range of antiapoptotic activities to hematopoietic cells under certain biological conditions. GPCR is a large family of cell surface receptors, many of which are critically involved in hormonal and behavioral control. Recent observations also suggest that GPCR signaling plays a pivotal role in immune cell activation. Heterotrimeric G protein is an integral part of GPCR signaling. Thus, dissection of signaling components involved in the CCR9-mediated antiapoptosis could be a framework for cell survival mechanisms and may provide options for therapeutic interventions for neurdegenerative diseases or T cell malfunctioning.

Animals↗

PubMed2001

Cutting edge: IL-17F, a novel cytokine selectively expressed in activated T cells and monocytes, regulates angiogenesis and endothelial cell cytokine production.

A novel secreted cytokine, termed IL-17F, was cloned using nested RACE PCR. This cytokine bears homology to IL-17. IL-17F was expressed only in activated CD4(+) T cells and activated monocytes. Recombinant human IL-17F did not stimulate the proliferation of hematopoietic progenitors or the migration of mature leukocytes. However, it markedly inhibited the angiogenesis of human endothelial cells and induced endothelial cells to produce IL-2, TGF-beta, and monocyte chemoattractant protein-1.

Amino Acid Sequence↗

PubMed2001

Blocking of c-FLIP(L)--independent cycloheximide-induced apoptosis or Fas-mediated apoptosis by the CC chemokine receptor 9/TECK interaction.

Chemokines play a pivotal role in regulating leukocyte migration as well as other biological functions. CC chemokine receptor 9 (CCR9) is a specific receptor for thymus-expressed CC chemokine (TECK). It is shown here that engagement of CCR9 with TECK leads to phosphorylation of Akt (protein kinase B), mitogen-activated protein kinases (MAPKs), glycogen synthase kinase--3 beta (GSK-3 beta), and a forkhead transcription factor, FKHR, in a human T-cell line, MOLT4, that naturally expresses CCR9. By means of chemical inhibitors, it is shown that phosphoinositide-3 kinase (PI-3 kinase), but not MAPK, is required for CCR9-mediated chemotaxis. Akt, GSK-3 beta, FKHR, and MAPK have been previously implicated in cell survival signals in response to an array of death stimuli. When MOLT4 cells, which expressed Fas as well as CXCR4, were stimulated with cycloheximide (CHX), an agonistic anti-Fas antibody, or a combination of these, the cells rapidly underwent apoptosis. However, costimulation of MOLT4 cells with TECK or stromal derived factor--1 significantly blocked CHX-mediated apoptosis, whereas stimulation only with TECK partially blocked Fas-mediated apoptosis. Concomitant with this blocking, cleavage of poly (adenosine 5'-diphosphate--ribose) polymerase and activation of caspase 3 were significantly attenuated, but the expression level of FLICE inhibitory protein c-FLIP(L), which had been shown to be regulated by CHX, was unchanged. This demonstrates that activation of CCR9 leads to phosphorylation of GSK-3 beta and FKHR and provides a cell survival signal to the receptor expressing cells against CHX. It also suggests the existence of a novel pathway leading to CHX-induced apoptosis independently of c-FLIP(L). (Blood. 2001;98:925-933)

Apoptosis↗

PubMed2001

Mzf1 controls cell proliferation and tumorigenesis.

MZF1 is a transcription factor belonging to the Krüppel family of zinc finger proteins, expressed in totipotent hemopoietic cells as well as in myeloid progenitors. Here we have inactivated Mzfi1 by gene targeting. Mzf1(-/-) mice develop lethal neoplasias characterized by the infiltration and complete disruption of the liver architecture by a monomorphic population of cells of myeloid origin reminiscent of human chloromas. Mzf1 inactivation results in a striking increase of the autonomous cell proliferation and of the ability of Mzf1(-/-) hemopoietic progenitors to sustain long-term hemopoiesis. These findings demonstrate that Mzf1 can act as a tumor/growth suppressor in the hemopoietic compartment.

Animals↗

PubMed2001

Synergistic activation of RSK correlates with c-fos induction in MO7e cells stimulated with GM-CSF plus Steel factor.

Steel factor (SLF) plus GM-CSF induces proliferative synergy in factor-dependent cell line MO7e and hematopoietic progenitor cells. We previously reported ERK1/2 involvement in this synergy, but its downstream signaling molecules are not defined. Here, we investigated activation of the 90-kDa ribosomal S6 kinase (RSK) proteins by measuring the phosphorylation status and in vitro kinase activity in MO7e cells. Both GM-CSF and SLF induced activation of RSK, and the combined stimulation with these two cytokines induced synergistic and persistent activation of RSK. RSK activity was reduced by PI3 kinase inhibitor LY294002 or MEK1 inhibitor PD98059, suggesting that the ERK as well as the PI3 kinase pathways are involved in regulation of RSK activity. Sensitivities of RSK activity to inhibitory drugs correlated well with those of c-fos gene induction. Taken together, synergistic activation of RSK may contribute, at least in part, to the synergistic induction of c-fos after combined stimulation with GM-CSF plus SLF.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed2001

The interphase microtubule damage checkpoint defines an S-phase commitment point and does not require p21(waf-1).

Cell cycle checkpoints ensure orderly progression of events during cell division. A microtubule damage (MTD)-induced checkpoint has been described in G(1) phase of the cell cycle (G(1)MTC) for which little is known. The present study shows that the G(1)MTC is intact in activated T lymphocytes from mice with the p21(waf-1) gene deleted. However, p21(waf-1) gene deletion does affect the ratio of cells that arrest at the G(1)MTC and the spindle checkpoint after MTD. The G(1)MTC arrests T lymphocytes in G(1) prior to cdc2 up-regulation and prior to G(1) arrest by p21(waf-1). Once cells have progressed past the G(1)MTC, they are committed to chromosome replication and metaphase progression, even with extreme MTD. The G(1)MTC is also present in a human myeloid cell line deficient in p21(waf-1) gene expression. The p21-independent G(1)MTC may be important in cellular responses to MTD such as those induced by drugs used to treat cancer.

Animals↗

PubMed2001

Transcriptional and ERK1/2-dependent synergistic upregulation of p21(cip1/waf1) associated with steel factor synergy in MO7e.

Steel factor (SLF) plus GM-CSF induces proliferative synergy in myeloid progenitors and factor-dependent cell line MO7e. We previously reported that the protein level of cyclin-dependent kinase inhibitor p21(cip1/waf1) (p21) increased synergistically when MO7e cells were stimulated with SLF plus GM-CSF and that p21 induction was required for SLF synergistic responses. Here we show that this p21 induction is regulated at the transcriptional level. Based on use of a multiprobe RNase protection assay, the synergistic increase of p21 mRNA was unique among many cell cycle regulators. While STAT5A and 5B were activated after stimulation with GM-CSF alone or SLF plus GM-CSF, there was no difference in activation between the groups. p44/42 MAP kinase (ERK1/2) was synergistically activated by SLF plus GM-CSF, but SAPK/JNK and p38 MAP kinase were not. Synergistic induction of p21 was significantly decreased with a MEK1 inhibitor, suggesting that the ERK1/2 pathway is involved in the synergistic increase of p21 after GM-CSF plus SLF stimulation.

Cell Cycle↗

PubMed2001

Regulation of hematopoiesis by chemokine family members.

Chemokines, originally designated as chemoattractant cytokines, comprise a large family of molecules that have been implicated in a number of different functions mediated through chemokine receptors. Among these functions are regulatory roles in hematopoiesis that encompass effects on the proliferation, survival, and homing/migration of myeloid progenitor cells. This article reviews the field of chemokine regulation of hematopoiesis at the level of myeloid progenitor cells.

Animals↗

PubMed2001

Current status of cord blood banking and transplantation in the United States and Europe.

Cord blood (CB) transplantation has expanded the ability of the transplantation community to meet the growing needs of their patients. Clinical data over the last decade show promising results in CB transplantation using blood from related as well as unrelated donors. Basic science continues to look for ways to expand the quality and quantity of CB. CB banks are now established around the world, with major efforts to standardize banking to facilitate regulation, collection, processing, and distribution as a way of providing the highest-quality CB for patient use. This review article discusses the current status of CB transplantation and banking in the United States and Europe.

Blood Banks↗

PubMed2001

Therapeutic potential of 4-1BB (CD137) as a regulator for effector CD8(+) T cells.

A fundamental problem of antitumor immunity is tumor-induced immunosuppression. Tumor cells often down-regulate expression of co-stimulatory molecules, tumor antigens, and major histocompatibility complex (MHC) molecules on tumor cells, secrete immunosuppressive substance such as transforming growth factor-beta (TGF-beta) or interleukin-4 (IL-4), and induce apoptosis of effector T cells to escape surveillance. A major goal of antitumor or antivirus immunotherapy is to generate long-lived protective T cells that enable killing of target cells. In this review, we discuss the importance of 4-1BB for development or survival of functionally active effector CD8(+) T cells against tumors, virus infection, and allogeneic immune responses and for potential therapeutic application.

Antigens, CD↗

PubMed2001

Human recombinant interferon-inducible protein-10: intact disulfide bridges are not required for inhibition of hematopoietic progenitors and chemotaxis of T lymphocytes and monocytes.

Human recombinant interferon-inducible protein-10 (rIP-10), a C-X-C chemokine, inhibits proliferation of human hematopoietic progenitors responsive to co-stimulation by recombinant steel factor (rSLF), is chemotactic for human monocytes and T-lymphocytes, and promotes T-lymphocyte adhesion to endothelial cells. Because chemokines have four conserved cysteines forming two intramolecular disulfide bridges, we decided to investigate their contribution in the biological activity of rIP-10. Since amino acid residues 22-98 of the sequence predicted by the cDNA constitute the naturally occurring IP-10, they were cloned after an initiating methionine into expression vector pET-3d. Subsequently rIP-10 was purified by enzymatic cell lysis, solubilization of refractile bodies with guanidine hydrochloride, renaturation by dialysis against dilute acetic acid, and sequential ion-exchange and reverse-phase high-performance liquid chromatography. Purified rIP-10 was reduced with 20 mM dithiothreitol, and chemically modified with 100 mM iodoacetamide (IAA), or S-methyl-methanethiosulfonate (MMTS), or N-methylmaleimide (NMM). Radiolabeling experiments demonstrated that 95% of the rIP-10 thiols were modified, and this was confirmed with SDS-PAGE. The biological activity of modified rIP-10 was determined in vitro by inhibition of rSLF-responsive human bone marrow hematopoietic progenitor proliferation and by chemotaxis assays using human T-lymphocytes and monocytes. In both assay systems, the biological activity was evident at rIP-10 concentrations of 20-100 ng/ml. The activity was preserved after modification of rIP-10 by IAA or MMTS, but was abolished after modification by NMM. We conclude that disulfide bridges are not essential for the biological activity of rIP-10.

Cell Division↗

PubMed2001

Chemokine regulation of hematopoiesis and the involvement of pertussis toxin-sensitive G alpha i proteins.

Chemokines have been implicated in regulation of various aspects of hematopoiesis, including negative regulation of the proliferation of immature subsets of myeloid progenitor cells (MPCs), chemotaxis of MPCs, and survival enhancement of MPCs after delayed growth factor addition. Since chemokine receptors are seven-transmembrane-spanning G-protein-linked receptors and the chemotactic effect in vitro of the CXC chemokine SDF-1 is pertussis toxin (PT)-sensitive, implying the involvement of G alpha i proteins as mediators of SDF-1-induced chemotaxis, we evaluated the effects of PT on other chemokine actions influencing MPCs. While the in vitro survival-enhancing effects of SDF-1 on GM-CSF and steel factor-dependent mouse bone marrow granulocyte macrophage progenitors (CFU-GM) were pertussis toxin-sensitive, the suppressive effects of the CC chemokine MIP-1 alpha and the CXC chemokine IL-8 on colony formation by GM-CSF and steel factor-sensitive CFU-GM were insensitive to pertussis toxin. These results suggest that not all chemokine-mediated effects on MPCs are necessarily mediated through pertussis toxin-sensitive G alpha i proteins.

Animals↗

PubMed2001

Steel factor regulates cell cycle asymmetry.

Asymmetric segregation of cell-fate determinants during mitosis (spatial asymmetry) is an essential mechanism by which stem cells are maintained while simultaneously giving rise to differentiated progenitors that ultimately produce all the specialized cells in the hematopoietic system. Temporal cell cycle asymmetry and heterogeneity are attributes of cell proliferation that are also essential for maintaining tissue organization. Hematopoietic stem cells (HSCs) are regulated by a complex network of cytokines, some of which have very specific effects, while others have very broad ranging effects on HSCs. Some cytokines, like steel factor (SLF), are known to synergize with other cytokines to produce rapid expansion of progenitor cells. Using the human growth factor-dependent MO7e cell line as a model for synergistic proliferation, we present evidence that links proliferation asymmetry to SLF synergy with GM-CSF, and suggests that temporal asymmetry and cell cycle heterogeneity can be regulated by SLF in vitro. We also show that CDK-inhibitor and cell cycle regulator, p27kip-1, may be involved in this temporal asymmetry regulation. We propose that SLF/GM-CSF synergy is, in part, due to a shift in proliferation pattern from a heterogeneous and asymmetric one to a more synchronous and symmetric pattern, thus contributing dramatically to the rapid expansion that accompanies SLF synergy observed in MO7e cells. This kinetic model of asymmetry is consistent with recent evidence showing that even though SLF synergy results in a strong proliferative signal, it does not increase primary HSC self-renewal, which is believed to be highly dependent on asymmetric divisions. The factor-dependent MO7e/SCF- synergy/asymmetry model described here may therefore be useful for studies of the effects of various cytokines on cell cycle asymmetry.

Cell Cycle↗

PubMed2000

An expansion phase precedes terminal erythroid differentiation of hematopoietic progenitor cells from cord blood in vitro and is associated with up-regulation of cyclin E and cyclin-dependent kinase 2.

The dynamics of cell cycle regulation were investigated during in vitro erythroid proliferation and differentiation of CD34(+) cord blood cells. An unusual cell cycle profile with a majority of cells in S phase (70.2%) and minority of cells in G1 phase (27.4%) was observed in burst-forming unit-erythrocytes (BFU-E)-derived erythroblasts from a 7-day culture of CD34(+) cells stimulated with interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), Steel factor, and Epo. Terminal erythroid differentiation was accompanied by a rapid increase of G0/G1 phase cells. Expression of cyclin E and cyclin-dependent kinase 2 (cdk2) correlated with the proportion of S phase cells. Cyclin D3 was moderately up-regulated during the proliferation phase, and both cyclin E and D3 were rapidly down-regulated during terminal differentiation. This suggests that the high proliferation potential of erythroblasts is associated with temporal up-regulation of cyclin E and cdk2. (Blood. 2000;96:3985-3987)

CDC2-CDC28 Kinases↗

PubMed2000

Analysis of engraftment, graft-versus-host disease, and immune recovery following unrelated donor cord blood transplantation.

Unrelated cord blood (UCB) is being used as a source of alternative hematopoietic stem cells for transplantation with increasing frequency. From November 1994 to February 1999, 30 UCB transplant procedures were performed for both malignant and nonmalignant diseases in 27 children, aged 0.4 to 17.1 years. Patients received either HLA-matched (n = 3) or 1- or 2-antigen-mismatched (n = 27) UCB following 1 of 2 standardized preparative and graft-versus-host disease regimens (hyperfractionated total body irradiation, cyclophosphamide, and antithymocyte globulin [ATG] with cyclosporine A and methotrexate; or busulfan, melphalan, and ATG with cyclosporine A and prednisone). The median time to neutrophil and platelet engraftment was 27 days (12-60 days) and 75 days (33-158 days) posttransplantation, respectively. No correlation was noted between neutrophil and platelet engraftment and nucleated cells per kilogram, CD34(+) cells per kilogram infused, or cytomegalovirus status of recipient. The cumulative probability of acute grade 2 or greater graft-versus-host disease (GVHD) was 37.2%, and of grade 3 or greater GVHD was 8.8%. No patients developed chronic GVHD. CD4, CD19, and natural killer cell recovery was achieved at a median of 12, 6, and 2 months, respectively. CD8 recovery was delayed at a median of 9 months. Normal mitogen response was achieved at 6 to 9 months. The probability of survival, disease-free survival, and event-free survival at 1 year was 52.3% (34.1%-70.5%), 54.7% (34.5%-74.9 %) and 49.6% (29.9%-69.4%), respectively. This series of 30 UCB transplants suggests that although CD8 cell recovery is delayed, the pattern of immune reconstitution with UCB is similar to that reported for other stem cell sources. (Blood. 2000;96:2703-2711)

Adolescent↗

PubMed2000

Flt3 ligand induces tyrosine phosphorylation of gab1 and gab2 and their association with shp-2, grb2, and PI3 kinase.

The receptor tyrosine kinase Flt3 has been shown to play an important role in proliferation, differentiation, and survival of hematopoietic stem and progenitor cells. Although some postreceptor signaling events of Flt3 have been characterized, the involvement of Gab family proteins in Flt3 signaling is not known. In this study, we show that both Gab1 and Gab2 are rapidly tyrosine phosphorylated after Flt3 ligand stimulation of Flt3 ligand-responsive cells. They interact with tyrosine-phosphorylated Shp-2, p85, Grb2, and Shc. The results suggest that Gab proteins are engaged in Flt3 signaling to mediate downstream activation of Shp-2 and PI3 kinase pathways and possibly the Ras/Raf/MAPK pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Essential role of signal transducer and activator of transcription (Stat)5a but not Stat5b for Flt3-dependent signaling.

The receptor tyrosine kinase Flt3 plays an important role in proliferation and survival of hematopoietic stem and progenitor cells. Although some post-receptor signaling events of Flt3 have been characterized, the involvement of the Janus kinase/signal transducer and activator of transcription (Jak/Stat) pathway in Flt3 signaling has not been thoroughly evaluated. To this aim, we examined whether Flt3 activates the Jak/Stat pathway in Baf3/Flt3 cells, a line stably expressing human Flt3 receptor. Stat5a, but not Stats 1-4, 5b, or 6, was potently activated by Flt3 ligand (FL) stimulation. Interestingly, FL did not activate any Jaks. Activation of Stat5a required the kinase activity of Flt3. A selective role for Stat5a in the proliferative response of primary hematopoietic progenitor cells to FL was documented, as FL did not act on progenitors from marrows of Stat5a(-/-) mice, but did stimulate/costimulate proliferation of these cells from Stat5a(+/+), Stat5b(-/-), and Stat5b(+/+) mice. Thus, Stat5a is essential for at least certain effects of FL. Moreover, our data confirm that Stat5a and Stat5b are not redundant, but rather are at least partially distinctive in their function.

Animals↗

PubMed2000

The CC chemokine CK beta-11/MIP-3 beta/ELC/Exodus 3 mediates tumor rejection of murine breast cancer cells through NK cells.

CK beta-11 chemoattracts T cells, B cells, dendritic cells, macrophage progenitors, and NK cells and facilitates dendritic cell and T cell interactions in secondary lymphoid tissues. We hypothesized that expression of CK beta-11 in tumor cells may generate antitumor immunity through these interactions. After transduction with the retroviral vector L(CK beta 11)SN, the murine breast cancer cell line C3L5 (C3L5-CK beta 11) showed expression of retroviral mRNA by Northern analysis and production of functional CK beta-11 by chemotaxis of human NK cells to C3L5-CK beta 11 supernatant. Only 10% of mice injected with C3L5-CK beta 11 developed tumors, compared with 100% of mice injected with a transduced control C3L5 line (C3L5-G1N). Importantly, the in vitro growth characteristics of the CK beta-11-transduced cell line were unaffected, suggesting the difference in growth in vivo was a result of chemokine production. Vaccination with C3L5-CK beta 11 partially protected animals from parental C3L5 challenge. Immunodepletion with anti-asialo-GM1 or anti-CD4 during C3L5-CK beta 11 vaccination significantly reduced CK beta-11 antitumor activity compared with control and anti-CD8-treated groups. Splenocytes from NK-depleted animals transferred the acquired immunity generated with C3L5-CK beta 11 vaccination, while splenocytes from the CD4-depleted animals did not. These results indicate, for the first time, that expression of CK beta-11 in a breast cancer cell line mediates rejection of the transduced tumor through a mechanism involving NK and CD4+ cells. Furthermore, CK beta-11-transduced tumor cells generate long-term antitumor immunity that requires CD4+ cells. These studies demonstrate the potential role of CK beta-11 as an adjuvant in stimulating antitumor responses.

Animals↗