Myeloid Lineage Development Research Articles

Evidence for a Novel Blood Lineage Commitment Pathway from Lympho-Myeloid Hematopoietic Stem Cells.

We recently identified a novel Lin−Sca-1+c-kithiCD34+Flt3hi (LSKCD34+Flt3hi) lymphoid-primed multipotent progenitor (LMPP) in adult mouse bone marrow which, although possessing a combined lymphoid (B and T cell) and myeloid (granulocyte-monocyte; GM) differentiation potential, have little or no ability to adopt erythroid (E) and megakaryocyte (MK) lineage fates (Adolfsson et al, Cell 121:295, 2005). The identification of this lineage restricted lymphomyeloid progenitor implicates the existence of alternative roadmaps for lineage commitment of pluripotent hematopoietic stem cells (HSCs), distinct from the classical model suggesting that the first HSC commitment step results in a strict separation into common lymphoid and myeloid progenitors. Herein we provide further, genetic evidence for such a model. Affymetrix global gene profiling, quantitative PCR, and multiplex single cell PCR analysis of LSKCD34−Flt3− long-term (LT)-HSCs, LSKCD34+Flt3− short-term (ST)-HSCs and LSKCD34+Flt3hi LMPPs, demonstrate that LMPPs in contrast to LT-HSCs and ST-HSCs down-regulate or turn off a number of genes critically involved in MkE lineage development, including GATA-1 and the receptors for erythropoietin and thrombopoietin. In contrast, a number of genes specific for early lymphoid development, including Rag-1, sterile Ig and IL-7 receptor are upregulated in LMPPs but absent in LT-HSCs and ST-HSCs. Importantly, within the LMPP, these lymphoid genes are typically co-expressed with a number of GM associated genes such as G-CSF receptor and MPO, but virtually never co-expressed with MkE associated genes. Investigating fetal liver day 14.5 we also provide evidence for existence of the LSKCD34+Flt3hi LMPPs at this early stage of development, and using a single cell clonal assay promoting combined B, T and myeloid lineage development, we demonstrate that a large fraction of fetal LMPPs lacking MkE potential possess a combined GM, B and T cell potential. Thus, evaluation at the single cell level of combined lineage potentials and multilineage gene expression provide compelling evidence for lymphoid-priming within the HSC compartment being preceeded by a loss of MkE potential, but occurring prior to loss of GM potential.

PU.1 silencing leads to terminal differentiation of erythroleukemia cells

The transcription factor PU.1 plays a central role in development and differentiation of hematopoietic cells. Evidence from PU.1 knockout mice indicates a pivotal role for PU.1 in myeloid lineage and B-lymphocyte development. In addition, PU.1 is a key player in the development of Friend erythroleukemia disease, which is characterized by proliferation and differentiation arrest of proerythrocytes. To study the role of PU.1 in erythroleukemia, we have used murine erythroleukemia cells, isolated from Friend virus-infected mice. Expression of PU.1 small interfering RNA in these cells led to significant inhibition of PU.1 levels. This was accompanied by inhibition of proliferation and restoration in the ability of the proerythroblastic cells to produce hemoglobin, i.e., reversion of the leukemic phenotype. The data suggest that overexpression of PU.1 gene is the immediate cause for maintaining the leukemic phenotype of the disease by retaining the self-renewal capacity of transformed erythroblastic cells and by blocking the terminal differentiation program towards erythrocytes.

Persistence of GATA1+ Erythroid Progenitors In Vivo in Absence of Zebrafish SCL.

The stem cell leukemia (SCL) gene encodes a bHLH transcription factor essential to the primitive and definitive hematopoietic systems in vertebrates. In the zebrafish, studies of hematopoiesis have shown significant functional conservation with mammals. In order to assess the functions of scl in zebrafish primitive hematopoiesis, zebrafish embryos were analyzed after ablation of Scl protein by morpholino-modified antisense RNA injection. Embryos injected with morpholinos targeting the scl translation initiation site or two independent splice donor sites lacked circulating blood and exhibited cardiac edema by 28 hours post fertilization. RNA in situ hybridization analysis with a panel of hematopoietic-specific genes showed that myeloid lineage development in the anterior lateral mesoderm was absent upstream of draculin and pu.1 expression. In contrast, erythroid development in the posterior lateral mesoderm occurred with little or no functional Scl, as shown by expression of draculin, pu.1, and gata1 in this PLM region. Primitive erythroid cells failed to mature, as evidenced by the absence of circulating cells after 24 hpf. Only upon titration of scl morpholinos to near toxic levels was gata1 reduced or absent in embryos at 21 somites. These findings show that scl plays a vital role in development of all hematopoietic lineages in zebrafish. However, these data reveal differential effects of reduction or loss of Scl on hematopoietic development in the anterior lateral mesoderm versus the posterior lateral mesoderm. The results reveal that reduction or loss of Scl affects erythroid development in the posterior lateral mesoderm or ICM at a progenitor stage of erythroid development, after initiation of gata1 and embryonic globin expression. These data suggest that early erythroid development in the zebrafish requires little or no Scl, while maturation of erythroid progenitors cannot occur in the absence of Scl. Myeloid development in the anterior lateral mesoderm is dependent upon Scl. This embryonic analysis in the zebrafish is the first to demonstrate a transient, in vivo population of erythroid precursors in embryos with severely reduced or absent expression of Scl.

Functional Differences in GATA-1 Affinity for Double Versus Single GATA-1 Binding Sites Dictate Synergistic Versus Antagonistic Interactions of PU.1 and GATA-1 in Myeloid Gene Transcription.

Instructive roles for both GATA-1 and PU.1 have been demonstrated in hematopoiesis, and recent studies have identified both antagonistic and synergistic interactions between them in myeloid gene transcription and lineage development. In prior studies, we reported that PU.1 synergizes with rather than antagonizes GATA-1 for transactivation of a hallmark eosinophil gene, the major basic protein P2 promoter (MBP-P2), which possesses a novel dual (double) GATA-binding site, similar to the palindromic double site in the murine GATA-1 control locus that may specify eosinophil lineage-specific expression of GATA-1 and eosinophil development. To address the transcriptional mechanism for PU.1-GATA-1 synergy through the MBP-P2 dual GATA site, we investigated GATA-1 and PU.1 physical and functonal interactions via their binding sites in the MBP-P2 promoter. DNA binding affinities of GATA-1 and its C- versus N-terminal zinc fingers were assessed for single versus double GATA sites in the presence or absence of PU.1. Our results show that the dual GATA site strongly binds full length GATA-1 with higher affinity than either of the single sites, using both zinc fingers, but that mutant GATA-1 proteins with C-finger or N-finger deletions retain their ability to bind, albeit at lower affinity, to the dual site. DNA binding activities of the two zinc fingers with the dual GATA site were confirmed using peptides containing only the C-finger or N-finger region. Of note, formation of GATA-1 complexes with the dual GATA site was not inhibited by the addition of PU.1, whereas formation of binding complexes for mutants of GATA-1 containing only the C- or N-finger region could be completely inhibited in a dose-response fashion by PU.1. These unique features of PU.1/GATA-1 interactions on a dual versus single GATA-1 site were confirmed using peptides containing only the C- or N-finger regions of GATA-1. Our findings indicate that both zinc fingers of GATA-1 are involved in formation of the high-affinity GATA-1 complex with the dual site. Importantly, we show that the higher affinity dual GATA-1 site complex is not affected by the addition of PU.1, whereas formation of the binding complex with a single GATA-1 site is eliminated by PU.1, emphasizing the different mechanisms of GATA-1/PU.1 interactions on dual versus single GATA binding sites. Functional analyses by transactivation confirmed that synergistic activation of the MBP-P2 promoter by GATA-1 and PU.1 is mediated by their protein-protein interactions through this unique high affinity dual GATA-1 binding site. We suggest two possible mechanisms for PU.1/GATA-1 synergy on dual GATA sites: (1) PU.1 may change GATA-1 conformation and its high affinity for the dual site, enhancing its availability for interaction with the basal transcriptional machinery. Alternatively, (2) PU.1 could impede interactions of GATA-1 with a co-repressor, e.g. FOG-1, which we and others have shown represses GATA-1 function in the eosinophil lineage.

Physiological Concentrations of Retinoic Acid Favor Myeloid Dendritic Cell Development over Granulocyte Development in Cultures of Bone Marrow Cells from Mice

Differentiation of hematopoietic progenitors to dendritic cells (DCs) is a complex, poorly understood process regulated by cytokines, colony-stimulating factors, growth factor receptors, and transcription factors. However, nutritional factors may play an important role. Vitamin A is essential for proper immune function and is implicated in the development of myeloid lineage cells, especially granulocytes. We investigated the role of vitamin A in the differentiation of myeloid DCs. Cultures of bone marrow cells from mice stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) in medium with reduced serum retinol demonstrated significantly decreased DC development compared with control cultures containing retinol. Surprisingly, granulocytes predominated in cultures stimulated with GM-CSF when retinol was depleted. The addition of all-trans or 9-cis retinoic acid to cultures depleted of retinol significantly restored DCs and inhibited granulocyte development. The DC-promoting effect of vitamin A was specific to myeloid lineage development stimulated by GM-CSF because vitamin A significantly inhibited DC development stimulated by flt-3 ligand. Vitamin A also affected DC major histocompatibility complex (MHC) class II and costimulatory molecule expression. In response to increasing concentrations of vitamin A, the expression of MHC class II decreased on the DC, whereas the expression of costimulatory molecules increased, especially CD86. Our data suggest that vitamin A favors the differentiation of myeloid progenitors to immature myeloid DC instead of granulocytes when dietary vitamin A is adequate, and that vitamin A deficiency may compromise adaptive immune responses that depend on myeloid DC antigen presentation.

Transcriptional Regulation of Mouse μ Opioid Receptor Gene by PU.1

We previously reported that the 34-bp cis-acting element of the mouse micro opioid receptor (MOR) gene represses transcription of the MOR gene from the distal promoter. Using a yeast one-hybrid screen to identify potential transcription factors of the MOR promoter, we have identified PU.1 as one of the candidate genes. PU.1 is a member of the ets family of transcription factors, expressed predominantly in hematopoietic cells and microglia of brain. PU.1 plays an essential role in the development of both lymphoid and myeloid lineages. Opioids exert neuromodulatory as well as immunomodulatory effects, which are transduced by MOR. Moreover, MOR-deficient mice exhibit increased proliferation of hematopoietic cells, suggesting a possible link between the opioid system and hematopoietic development. The PU.1 protein binds to the 34-bp element of the MOR gene in a sequence-specific manner confirmed by electrophoretic mobility shift assay and supershift assays. We have also determined endogenous PU.1 interactions with the 34-bp element of MOR promoter by chromatin immunoprecipitation assays. In co-transfection studies PU.1 represses MOR promoter reporter constructs through its PU.1 binding site. When the PU.1 gene is disrupted as in PU.1 knock-out mice and using small interfering RNA-based strategy in RAW264.7 cells, the transcription of the endogenous target MOR gene is increased significantly. This increase is probably mediated through modification of the chromatin structure, as suggested by the reversal of the PU.1-mediated repression of MOR promoter activity after trichostatin A treatment in neuroblastoma NMB cells. Our results suggest that PU.1 may be an important regulator of the MOR gene, particularly in brain and immune cells.

Functional analysis of PU.1 domains in monocyte-specific gene regulation

The Ets family transcription factor PU.1 is required for the development of various lymphoid and myeloid cell lineages, and regulates the expression of several genes in a cell type-specific manner. Recently we found that overproduction of PU.1 in mouse bone marrow-derived mast cell progenitors induced the expression of monocyte-specific genes. This prompted us to analyze the functions of each domain of PU.1 in monocyte-specific gene expression, using transfection of mast cell progenitors with a series of retrovirus vectors for overexpression of various truncation mutants. Both the acidic region and the Ets domain of PU.1 were required for expression of monocyte-specific genes, and for enhanced interleukin-6 production in response to lipopolysaccharide. The Gln-rich region was suggested to be involved in expression of both MHC class II and F4/80. On the other hand, when PU.1 protein lacking the PEST domain was produced in the progenitor cells, expression of monocyte-specific genes was substantially enhanced, suggesting that the PEST domain plays a negative role in monocyte-specific gene expression.

NF-kB inhibitor blocks B cell development at two checkpoints

Members of the NF-kB transcription factor family are differentially expressed in the B cell lineage. Disruption of individual or two NF-kB subunits exhibits distinct defects in B lymphocyte development, activation, and survival. However, the role each NF-kB plays during B cell development has been obscured by molecular compensation. To address this issue, a trans-dominant form of IkBα was transduced into bone marrow cells to act as a pan-inhibitor of NF-kB using a retroviral system. While the development of T-lymphocytes and myeloid cell lineages was not grossly affected by the transduced IkBα gene, a significant reduction in the number and percentage of B lineage cells was apparent in IkBα transduced chimeric mice. IkBα expression decreased the percentage of pre-B and immature B cell subsets in the bone marrow and further impaired the development of follicular mature B cells and marginal zone B cells in the periphery. Introduction of the Bcl-X transgene completely restored the pre-B and immature B cell pool in the bone marrow. However, despite a significant improvement of overall viability of the B cell lineage, Bcl-X expression was insufficient to overcome the maturation block resulting from NF-kB inhibition. Together, our study suggests that NF-kB activity is required for two distinct checkpoints during B cell development: one is for pre-B/immature B cell viability, the other is to provide both survival and maturation signals to ensure the proper development of follicular mature B cells.

Overproduction of PU.1 in mast cell progenitors: its effect on monocyte- and mast cell-specific gene expression

The Ets family transcription factor PU.1 is required for development of various lymphoid and myeloid cell lineages, and regulates the expression of several genes in a cell type-specific manner. Mouse bone marrow-derived hematopoietic progenitor cells are programmed to differentiate into mast cells, when the cells are maintained in the presence of pokeweed mitogen-stimulated spleen-conditioned medium. However, by retroviral introduction of PU.1 cDNA, the progenitor cells expressed MHC class II, CD11b, CD11c, and F4/80, and acquired the ability to stimulate T cells. Furthermore, PU.1-overproducing cells exhibited the morphology, in part, similar to that of monocyte. These results indicate that the mast cell progenitors still have the ability to express monocyte-specific genes by increased expression of PU.1.

Pax5 determines B- versus T-cell fate and does not block early myeloid-lineage development

AbstractProgenitor B cells deficient in Pax5 are developmentally multipotent, suggesting that Pax5 is necessary to maintain commitment to the B-cell lineage. Commitment may be mediated, in part, by Pax5 repression of myeloid-specific genes. To determine whether Pax5 expression in multipotential cells is sufficient to restrict development to the B-cell lineage in vivo, we enforced expression of Pax5 in hematopoietic stem cells using a retroviral vector. Peripheral blood analysis of all animals reconstituted with Pax5-expressing cells indicated that more than 90% of Pax5-expressing cells were B220+ mature B cells that were not malignant. Further analysis showed that Pax5 completely blocked T-lineage development in the thymus but did not inhibit myelopoiesis or natural killer (NK) cell development in bone marrow. These results implicate Pax5 as a critical regulator of B- versus T-cell developmental fate and suggest that Pax5 may promote commitment to the B-cell lineage by mechanisms that are independent of myeloid gene repression.

Influence of lineage‐specific cytokines on commitment and asymmetric cell division of haematopoietic progenitor cells

We examined the influence of cytokines on erythroid- and myeloid-lineage development of AC133+ cells during primary and secondary cultures. Cells cultured for 14 d in liquid medium containing erythropoietin (EPO) were amplified 831-fold with 98.2% erythroid cells. A similar culture exposed to granulocyte colony-stimulating factor (G-CSF) grew 1350-fold with 97.4% myeloid cells. To assess whether the cells with EPO inducement could respond at this point to G-CSF signal, or vice versa, the EPO-stimulated population was re-grown with G-CSF, constituting 95.2% myeloid, of 5075-fold, cells after 14 d of re-culture. Conversely, reculture of the G-CSF-stimulated population with EPO resulted in a 4083-fold growth with 81.4% erythroid cells. Semisolid culture containing EPO orG-CSF showed that some individual colonies had self- renewal potential after 14 d culture and could be induced todevelop into a different lineage. Analysis of primitive markers, CD34 and Notch1, or lineage markers, EPO-R and CD13, by single-cell reverse transcription polymerase chain reaction showed that individual colonies of 2-16 cells contained at least one CD34-positive cell with expression ofNotch1 and co-expression of EPO-R and CD13 appeared on either CD34-positive or CD34-negative cells. In situ hybridization with the same cell surface markers in cell populations confirmed the asymmetric cell division and co-expression from single cell data. The study provides a useful model for the analysis of multipotential progenitor development, and indicates that progenitor cells co-express genes from different lineage pathways before commitment and that cytokines influence lineage commitment.

Granulocyte inducer C/EBPα inactivates the myeloid master regulator PU.1: possible role in lineage commitment decisions

AbstractSeveral transcription factors have been implicated as playing a role in myelopoiesis. PU.1, an ets-family transcription factor, is required for the development of myeloid and lymphoid lineages, whereas the transcription factor CCAAT–enhancer binding protein family member C/EBPα is essential for granulocyte development. We present here the first evidence that C/EBPα blocks the function of PU.1. PU.1 and C/EBPα interact physically and colocalize in myeloid cells. As a consequence of this interaction, C/EBPα can inhibit the function of PU.1 to activate a minimal promoter containing only PU.1 DNA-binding sites. We further demonstrate that the leucine zipper in the DNA-binding domain of C/EBPα interacts with the β3/β4 region in the DNA-binding domain of PU.1 and as a result displaces the PU.1 coactivator c-Jun. Finally, C/EBPα blocks PU.1-induced dendritic cell development from CD34+ human cord blood cells. The functional blocking of PU.1 by C/EBPα could be the mechanism by which C/EBPα inhibits cell fates specified by PU.1 and directs cell development to the granulocyte lineage.

Dok-related protein negatively regulates T cell development via its RasGTPase-activating protein and Nck docking sites.

Downstream of kinase (Dok)–related protein (DokR, also known as p56dok/FRIP/Dok-R) is implicated in cytokine and immunoreceptor signaling in myeloid and T cells. Tyrosine phosphorylation induces DokR to bind the signal relay molecules, RasGTPase-activating protein (RasGAP) and Nck. Here, we have examined the function of DokR during hematopoietic development and the requirement for RasGAP and Nck binding sites in its biological function. Retroviral-mediated expression of DokR in bone marrow cells dramatically inhibited their capacity to form colonies in vitro in response to the cytokines macrophage colony–stimulating factor and stem cell factor, whereas responses to interleukin-3 and granulocyte macrophage colony–stimulating factor were only weakly affected. When introduced into lethally irradiated mice, hematopoietic cells expressing DokR showed a drastically reduced capacity to repopulate lymphoid tissues. Most notably, DokR dramatically reduced repopulation of the thymus, in part by reducing the number of T cell precursors seeding in the thymus, but equally, through inhibiting the transition of CD4−CD8− to CD4+CD8+ T cells. Consequently, the number of mature peripheral T cells was markedly reduced. In contrast, a minimal effect on B cell and myeloid lineage development was observed. Importantly, functional RasGAP and Nck binding sites were found to be essential for the biological effects of DokR in vitro and in vivo.

Targeted disruption of c-myb in the chicken pre B-cell line DT40.

The c-myb proto-oncogene is highly expressed in a wide variety of immature hematopoietic cells and plays a key role in the development of the hematopoietic system. c-myb and its retroviral counterpart v-myb encode transcription factors which have been implicated in the regulation of certain target genes. Targeting of c-myb in mouse embryonic stem cells by homologous recombination has provided clear evidence that c-myb is necessary for the proper development of most myeloid lineages of the hematopoietic system as well as of T-lymphocytes. Here we have explored the function of c-myb in the B-lymphoid lineage. We have used the chicken DT40 cells, a pre B-cell line which shows extremely high efficiencies of homologous recombination, as a model system to disrupt c-myb. DT40 cells lacking a functional c-myb gene are viable and show only minor perturbations of their growth parameters, indicating that c-myb is not an essential gene in these cells. We have used the c-myb null DT40 cells to analyse the expression of genes which have been previously been identified as myb target genes. Neither c-myc nor bcl-2, two putative myb targets, showed altered expression in the cells lacking c-myb. However, expression of the Pdcd4 gene, a myb target gene originally identified in a myelomonocytic cell line expressing a conditional form of v-myb, was diminished in the absence of c-myb. The c-myb knock-out cells described here should provide a useful model system for the identification and characterization of c-myb target genes in B-lymphoid cells.

Redefinition of lymphoid progenitors.

Similarities between T and B lymphocytes might have led to the idea that these functionally distinct cells develop from a common lymphoid progenitor. However, investigations with a new clonal assay which allows for T-, B- and myeloid-lineage development indicate that commitment to T-cell and B-cell lineages occurs instead through myeloid/T and myeloid/B bipotential stages, respectively. These findings provide an opportunity to reconsider the ontogeny and phylogeny of T- and B-cell development.

Cloning and expression pattern of the lysozyme C gene in zebrafish

Here, we report isolation and developmental expression pattern of the zebrafish lysozyme C gene. Amino acid sequence analysis showed that the zebrafish lysozyme C protein shared ∼37–80% identities with the mouse, human, chicken, and carp counterparts. Whole-mount in situ hybridization showed that the lysozyme C gene was expressed in macrophages, as its expression was co-localized with the known myeloid lineage markers L-plastin and PU.1. At 20 hours postfertilization (hpf), most of the lysozyme C positive cells were localized in the yolksac and head mesenchyme but not in the intermediate cell mass, supporting the notion that the primitive macrophage originated from the yolksac (Development 126 (1999) 3735). At 36 hpf, the lysozyme C positive cells scattered within the head and yolksac, and began to appear in the caudal part of axial vein. By 6 days postfertilization (dpf), the lysozyme C positive cells accumulated in the kidney where hematopoiesis had been indicated to take place after 4 dpf (Dev. Dyn. 214 (1999) 323). Taken together, our results demonstrate that the lysozyme C gene is specifically expressed in myeloid lineage, suggesting that it could serve as an excellent marker for genetic screening of both primitive and definitive myeloid lineage development in zebrafish.

Ex Vivo Hematopoiesis and Lymphopoiesis

Bone marrow is the hematopoietic tissue and a primary lymphoid organ. Its intricate, three-dimensional architecture facilitates cell-cell and cell-matrix interactions and provides a microenvironment that supports self-renewal and multilineal differentiation. Traditional in vitro culture systems (flask cultures) are limited by the absence of normal marrow spatial organization and cellular interactions with extracellular matrices. They deviate from the three-dimensional marrow structure, induce artificial lipogenesis and cell flattening, and produce only limited cell lineages. Our hypotheses are: 1) the marrow’s function in promoting multilineal hemopoiesis depends on its three-dimensional structure and 2) this function can be (substantially) reproduced in vitro only if its three-dimensional structure is preserved. We have developed a novel human ex vivo bone marrow model that mimics bone marrow both structurally and functionally. By providing an artificial scaffolding, we were able to obtain a three- dimensional growth configuration with high cell density and intimate physical contact between hematopoietic and stromal cells. Characterization of the stroma developed demonstrated that an anatomical tissue-like structure was obtained. More importantly, the three-dimensional culture system supported multilineal hematopoiesis, including the development of myeloid, erythroid, and lymphoid lineages. The ability to engineer a structural and functional human bone marrow mimicry ex vivo provides the possibilities of generating lineage-specific cells from the hematopoietic stem cells for adoptive cellular therapies. In this presentation, the B-lymphocyte development in our three-dimensional bone marrow culture system will be compared with B-lymphopoiesis in the traditional two-dimensional culture system.

Identification and characterization of a novel member of olfactomedin-related protein family, hGC-1, expressed during myeloid lineage development

We have cloned a novel hematopoietic granulocyte colony-stimulating factor (G-CSF)-induced olfactomedin-related glycoprotein, termed hGC-1 (human G-CSF-stimulated clone-1). mRNA differential display was used in conjunction with a modified two-phase liquid culture system. Cultures were enriched for early precursors of erythroid, myeloid, and megakaryocytic lineages, which were isolated after induction with erythropoietin, G-CSF, and thrombopoietin, respectively. RNA from the enriched cells was subjected to differential display analysis to identify lineage-specific expressed genes. One clone specifically induced by G-CSF, hGC-1, was characterized. The 2861 bp cDNA clone of hGC-1 contained an open reading frame of 1530 nucleotides, translating into a protein of 510 amino acids with a signal peptide and six N-linked glycosylation motifs. The protein sequence of hGC-1 showed it to be a glycoprotein of the olfactomedin family, which includes olfactomedin, TIGR, Noelin-2 and latrophilin-1. Olfactomedin-like genes show characteristic tissue-restricted patterns of expression; the specific tissues expressing these genes differ among the family members. hGC-1 was strongly expressed in the prostate, small intestine, and colon, moderately expressed in the bone marrow and stomach, and not detectable in other tissues. In vitro translation and ex vivo expression showed hGC-1 to be an N-linked glycoprotein. The hGC-1 gene locus mapped to chromosome 13q14.3. Together, our findings indicate that hGC-1 is primarily expressed as an extracellular olfactomedin-related glycoprotein during normal myeloid-specific lineage differentiation, suggesting the possibility of a matrix-related function for hGC-1 in differentiation.

Differential requirement for the transcription factor PU.1 in the generation of natural killer cells versus B and T cells

AbstractPU.1 is a member of the Ets family of transcription factors required for the development of various lymphoid and myeloid cell lineages, but its role in natural killer (NK) cell development is not known. The study shows that PU.1 is expressed in NK cells and that, on cell transfer into alymphoid Rag2/γc−/−mice, hematopoietic progenitors of PU.1−/−fetal liver cells could generate functional NK cells but not B or T cells. Nevertheless, the numbers of bone marrow NK cell precursors and splenic mature NK cells were reduced compared to controls. Moreover,PU.1−/− NK cells displayed reduced expression of the receptors for stem cell factor and interleukin (IL)-7, suggesting a nonredundant role for PU.1 in regulating the expression of these cytokine receptor genes during NK cell development.PU.1−/− NK cells also showed defective expression of inhibitory and activating members of the Ly49 family and failed to proliferate in response to IL-2 and IL-12. Thus, despite the less stringent requirement for PU.1 in NK cell development compared to B and T cells, PU.1 regulates NK cell differentiation and homeostasis.

Myeloid specific human CD33 is an inhibitory receptor with differential ITIM function in recruiting the phosphatases SHP-1 and SHP-2

AbstractCD33 is a myeloid specific member of the sialic acid-binding receptor family and is expressed highly on myeloid progenitor cells but at much lower levels in differentiated cells. Human CD33 has two tyrosine residues in its cytoplasmic domain (Y340 and Y358). When phosphorylated, these tyrosines could function as docking sites for the phosphatases, SHP-1 and/or SHP-2, enabling CD33 to function as an inhibitory receptor. Here we demonstrate that CD33 is tyrosine phosphorylated in the presence of the phosphatase inhibitor, pervanadate, and recruits SHP-1 and SHP-2. Co-expression studies suggest that the Src-family kinase Lck is effective at phosphorylating Y340, but not Y358, suggesting that these residues may function in the selective recruitment of adapter molecules and have distinct functions. Further support for overlapping, but nonredundant, roles for Y340 and Y358 comes from peptide-binding studies that revealed the recruitment of both SHP-1 and SHP-2 to Y340 but only SHP-2 to Y358. Analysis using mutants of SHP-1 demonstrated that binding Y340 of CD33 was primarily to the amino Src homology-2 domain of SHP-1. The potential of CD33 to function as an inhibitory receptor was demonstrated by its ability to down-regulate CD64-induced calcium mobilization in U937. The dependence of this inhibition on SHP-1 was demonstrated by blocking CD33-mediated effects with dominant negative SHP-1. This result implies that CD33 is an inhibitory receptor and also that SHP-1 phosphatase has a significant role in mediating CD33 function. Further studies are essential to identify the receptor(s) that CD33 inhibits in vivo and its function in myeloid lineage development.