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HEMATOPOIESIS
From the Department of Molecular Preventive Medicine
and CREST, School of Medicine, and the Department of Hematopoietic
Factor, Institute of Medical Science, University of Tokyo,
Japan.
Both SDF-1 and CXCR4 disruption are lethal to mice at the
embryonic stage and cause abnormalities in B lymphopoiesis,
myelopoiesis, cardiogenesis, vasculogenesis, and cerebellar
development. To investigate the role of SDF-1 and CXCR4 in
hematopoiesis during the adult stage, mice reconstituted with bone
marrow-derived hematopoietic progenitor cells transduced with either
the SDF-1 or a genetically modified SDF-1-intrakine gene using a
retroviral expression vector were analyzed. Flow cytometric (FCM)
analysis showed a dramatic reduction of CXCR4 expression on the cells
of intrakine-transduced mice, whereas CCR7 and CCR1 expression was
unchanged or marginally decreased on splenocytes. Migration of
splenocytes and bone marrow cells to SDF-1 was markedly suppressed in
intrakine-transduced mice. FCM analysis of bone marrow cells of
intrakine-transduced mice exhibited decreased numbers of pro-B
(B220+ CD43+), pre-B (B220+
CD43 Hematopoiesis consists of developmental cascades in
which the hematopoietic stem cells (HSCs) generate lineage-committed
cells and repeat the process of self-renewal.1,2 HSCs are
defined as cells that have dual capability for self-renewal and
multilineage differentiation. During embryonic development,
hematopoietic progenitor cells (HPCs) sequentially appear in the yolk
sac, para-aortic splanchnopleura mesoderm, aorta-gonad-mesonephros,
fetal liver, and, finally, bone marrow.3,4 The
molecular mechanism for the migration of HSCs and HPCs during
development remains unknown.
Chemokines are a family of chemoattractive polypeptides that are
classified into 4 groups, depending on the position of conserved cysteine residues. Chemokines mediate their effect by binding to 7 transmembrane G protein-coupled receptors, and they attract leukocyte
subsets to inflammation sites.5,6 Recently, several chemokines were shown to be expressed constitutively in lymphoid tissues, indicating that these chemokines have homeostatic functions in
regulating lymphocyte trafficking between and within lymphoid organs.7-9
Stromal cell-derived factor-1 (SDF-1), a CXC chemokine, was originally
cloned from bone marrow stromal cells by using the signal sequence trap
method10 and was later found to be pre-B-cell stimulating
factor (PBSF).11 SDF-1 is chemoattractive for
hematopoietic progenitor cells, B lymphocytes, T lymphocytes, and
monocytes.12,13 CXCR4, a receptor for SDF-1, has been
identified as an orphan receptor by several groups, including
us,14-17 and is expressed constitutively in various
tissues. Mice with disrupted SDF-1 or CXCR4 genes die in utero and show
severe abnormalities in B lymphopoiesis, myelopoiesis in bone marrow,
cardiogenesis, vascular development, and cerebellar
development.18-20 CXCR4 also acts as a coreceptor for the
T-tropic human immunodeficiency virus (HIV)-1, and SDF-1 has been also
shown to competitively block the viral entry of T-tropic isolates of
HIV-1.21,22
Genetically modified intracellular chemokines, or intrakines, which
have been linked with an endoplasmic reticulum retention signal
sequence (KDEL) on their carboxy termini, have been shown to remain
inside cells, to bind intracellularly the newly synthesized cognate
chemokine receptors, and to prevent the transport of the receptors to
cell surfaces.23,24 Lymphocytes transduced with a gene
expressing SDF-1-intrakine were found to be resistant to ligand
stimulation and HIV-1 infection in vitro, indicating that intrakine can
be used as a therapeutic approach for acquired immunodeficiency syndrome (AIDS). It remains to be established, however, whether intrakine blocks CXCR4 expression in vivo and how safe the
down-regulation of CXCR4 by intrakine is in adults.
In this study, using mice reconstituted with bone marrow-derived
hematopoietic progenitor cells (BM-HPCs) transduced with either SDF-1
or intrakine genes by a bicistronic retroviral expression vector, we
demonstrated that the SDF-1-intrakine could successfully suppressed
the expression and function of CXCR4 in vivo and that intrakine-transduced adult mice exhibited impaired lymphopoiesis and myelopoiesis.
Cytokine and cell lines
Construction of expression vectors
A murine SDF-1 gene was amplified by PCR reaction with the primers 5'-GCGAATTCCACCATG TGGACGCCAAGGTCGTC-3' and 5'-GCGAATCCTTACTTGTTTAAAGCTTTCTG-3'). The murine SDF-1 gene was linked with an ER retention signal (SEKDEL) by a PCR reaction with the primers 5'-GCGAATTCCACCATGGACGCCAAGGTCGTC-3' and 5'-GCGAATTCTTACAGCTCGTCCTTCTCGCTCTTGTTTAAAGCT- TTCTG-3'). These DNA fragments were digested with EcoRI and inserted into the EcoRI site of pEGFPMY. These bicistronic vectors were designated pSDFMY and pSDFKMY, respectively. All constructs were sequenced using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Branchburg, NJ) and an Applied Biosystems model 377 DNA sequencer. Preparation of antibody The cDNA encoding the full length of mSDF-1 and NH2-terminal portion of mCXCR4 from mCXCR4-pBlue Script KS (+) was amplified by PCR reaction. PCR primers for SDF-1 were 5'-GCGGATCCATGGAGGCCAAGGTCGTC-3' and 5'-GCCTCGAGTTACTTGTTTAAAGCTTTCTC-3'. Primers for CXCR4 were 5'-GCGGATCCATGGAACCGATCAGTGTG-3' and 5'-GCCTCGAGTTAGGTGGGCAGGAAGATCCT-3'. Amplified DNA fragments were digested with BamHI/XhoI and subcloned into the GST-fusion protein expression vector, pGEX-4T-3 (Amersham Pharmacia Biotech, Bucks, UK). These expression vectors were introduced into Escherichia coli BL21 (Trx) cells, and preparations of fusion protein were made according to a previously published method.28 Immunization of fusion protein and preparation of polyclonal antibodies to GST-mSDF-1 and GST-mCXCR4 fusion protein were performed according to a previously published method.29 Rabbit polyclonal antibodies against a synthetic peptide corresponding to the N-terminal region of mCCR7 were prepared according to standard methods.30 Preparation of antibodies against mCCR1 was described previously.31The immunologic binding specificity of these prepared antibodies against a chemokine and chemokine receptors was confirmed using murine recombinant chemokines (mMIP-II and mSLC) and chemokine receptor transfectants (mCCR1, mCCR2, mCCR7, and mCXCR4), respectively. Retrovirus-mediated bone marrow transduction and transplantation Bone marrow (BM) mononuclear cells were obtained from the tibias and femurs of C57BL/6 mice and prepared by gradient centrifugation on LYMPHOPREP (Daiichi Pure Chemicals, Tokyo, Japan). For the purification of c-kit+ cells, BM cells were treated with a combination of anti-c-kit antibody and Microbeads in the MACS system (Miltenyi Biotech, Bergisch Gladbach, Germany). All isolated cells were confirmed to be highly purified (greater than 98%) by an immunofluorescence flow cytometry analysis using EPICS ELITE ESP cell sorter (Beckman Coulter, Fullerton, CA).The c-kit+ cells were precultured in serum-free medium, SF03 (Sankou-junyaku, Tokyo, Japan) with 10 ng/mL murine SCF, 10 ng/mL murine IL-6, and 10 ng/mL human Flt3L for 48 hours. The production of supernatants containing retroviruses was performed according to the method described previously.27 For infection of c-kit+ cells, precultured 1 × 106 cells were transduced by centrifugation with 1 mL retrovirus supernatants containing 10 ng/mL mSCF, 10 ng/mL mIL-6, 10 ng/mL hFlt3L, and 8 µg/mL polybrene (Sigma, St. Louis, MO) at 2500g for 2 hours at 28°C to 30°C on day 1 and then cultured with DMEM containing 10% FCS, 10 ng/mL mSCF, 10 ng/mL mIL-6, and 10 ng/mL hFlt3L for 22 hours. On day 2, cells were also transduced by the centrifugation method with retrovirus supernatants. On day 3, GFP+ cells were purified with a cell sorter. Purified 1 × 105 GFP+ c-kit+ cells were transplanted by tail-vein injection into lethally irradiated (11 Gy total body irradiation) C57BL/6 recipient mice. Mice were maintained in a specific pathogen-free environment and with acidic water. Reverse transcription-polymerase chain reaction Total RNAs were extracted from cells using RNA-zolB (Biotex Laboratories, Houston, TX), according to the manufacturer's instructions. First-strand cDNA was synthesized at 37°C for 1 hour from 2 µg total RNA in 20 µL reaction mixture using random primers (Promega, Madison, WI). Thereafter, cDNA was amplified for 30 cycles at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds, with a pair of primers corresponding to each gene. Primers for GFP and SDF-1 were described above. Primers for G3PDH were described previously.31 Primers for CXCR4 were 5'-GCGAATTCATGGAACCGATCAGTGTG-3' and 5'-GCGAATTCTTAGCTGGAGTGAAAACT-3'. Primers for intrakine were 5'-GCGAATCCACCATGTGGAC GCCAAGGTCGTC-3 and 5'-GCGAATTCTTACAGCTCGTCCTTCTCGCT-3'. PCR products were fractionated on 1.2% agarose gel and visualized by ethidium bromide staining.Chemotaxis assay Chemotaxis assays were performed using a 96-well chemotaxis chamber (Neuroprobe, Pleasanton, CA) with polycarbonate filter (5-µm pore size). Cells were suspended at a density of 1 × 106 /mL in RPMI 1640 medium containing 20 mmol/L HEPES, pH 7.2, and 0.5% bovine serum albumin (BSA; Sigma). Twenty-five-microliter cell suspensions were added to upper chambers, and diluted chemokines (final volume, 29 µL) were added to lower chambers. Chemotaxis chambers were incubated for 2 hours at 37°C in 5% CO2. Cells that migrated to lower chamber were transferred to polystyrene tubes. Numbers of migrated cells were determined by EPICS ELITE ESP cell sorter (Beckman Coulter).Flow cytometric analysis Single-cell suspensions were prepared from tissues and peripheral blood after red blood cell depletion. Cells were incubated with monoclonal antibodies against cell surface markers for 30 minutes at 4°C. Phycoerythrin (PE), cy-chrome (CYC), or biotin-conjugated antibodies specific for murine CD11b, Gr-1, CD4, CD8, B220, CD43, and IgM (PharMingen, San Diego, CA) were used for flow cytometric analysis. Biotinylated antibodies were developed with CYC and allophycocyanin-conjugated streptavidin. Cells were analyzed with EPICS XL/XL-MCL System II and EPICS ELITE ESP cell sorter (Beckman Coulter). Dead cells were excluded by propidium iodide staining.In vitro colony assay Bone marrow cells were incubated in IMDM with 1.2% methylcellulose, 20% FCS, 1% BSA (Sigma), 50 µmol/L 2-mercaptoethanol, and 10 ng/mL of SCF or IL-3 or human EPO or mouse IL-7. Colony forming units (CFU) were monitored at 4 to 6 days (BFU-E) or 7 to 10 days (CFU-mix, CFU-GM, and CFU-IL-7) after inoculation.ELISA for circulating immunoglobulin Serum immunoglobulin levels were measured using antibody pairs specific for different mouse immunoglobulin isotypes (Southern Biotechnology Associates, Birmingham, AL) on microtiter plates. P-nitrophenylphosphatase-conjugated secondary antibodies were added, and the absorbance at 405 nm was measured in a microplate reader (Molecular Devices, Sunnyvale, CA). Concentrations were calculated by using purified immunoglobulin standards (Bio Pure AG, Bubendorf, Switzerland).
Generation of GFP-, SDF-1-, and intrakine-transduced mice We used a bicistronic retroviral expression vector system to generate mice reconstituted with BM-HPC transduced with either SDF-1 or SDF-1-intrakine genes. The long terminal repeats (LTRs) derived from PCC-4, cell-passaged myeloproliferative sarcoma virus (PCMV) and myeloproliferative sarcoma virus (MPSV) with wide host range were used to express SDF-1 or intrakine in vivo. The SDF-1 or intrakine genes to be expressed were placed upstream of the IRES and the GFP gene (Figure 1A). The c-kit+ (BM-HPC) cells, which were precultured for 2 days in serum-free medium containing recombinant mouse SCF, IL-6, and human Flt3 ligand, were transduced by using the retroviruses expressing GFP, SDF-1-GFP, or intrakine-GFP by a centrifugal transduction method. After transduction, GFP+ cells were purified by cell sorting (Figure 1B), and 1 × 105 cells were injected into lethally irradiated recipient mice.
After transplantation, GFP expression in reconstituted mice was analyzed (Figure 1C). Fifty-six days after transplantation, reconstitution efficiency with transduced cells was up to 90.3% ± 7.8% and 95.5% ± 8.4% in the peripheral blood of the GFP- or SDF-1-transduced mice, respectively (Figure 1C). High-level reconstitution with GFP+ cells was also observed in the spleen, bone marrow, and thymus of these mice. On the other hand, reconstitution efficiency was 12% to 15% lower in intrakine-transduced mice than in others (Figure 1C). It has been reported that 56 to 84 days are required to establish full reconstitution in recipient mice; therefore,32 we used mice at 70 days after transplantation for further experiments. The absolute cell number in the peripheral blood, spleen, bone marrow, and thymus from GFP-transduced mice was 4.78 ± 0.47 × 106/mL, 1.56 ± 0.27 × 108 mL, 1.71 ± 0.29 × 107 mL, and 4.61 ± 0.46 × 107, respectively, 70 days after transplantation. In addition, the absolute cell number of those from SDF-1 and intrakine-transduced mice was 5.61 ± 0.49 × 106/mL, 2.06 ± 0.15 × 108 mL, 1.98 × 0.24 × 107, and 3.90 ± 0.72 × 107 mL versus 3.18 ± 0.77 × 106/mL, 1.18 ± 0.15 × 108, 1.15 ± 0.24 × 107, and 1.73 ± 0.42 × 107, respectively. Next, we confirmed the mRNA expression level of the transferred genes in the reconstituted mice. Total RNA was extracted from splenocytes of reconstituted mice, and reverse transcription (RT)-PCR was performed using the primers for specific genes. High expression of SDF-1 mRNA was detected in the splenocytes from SDF-1-transduced mice compared with those from GFP- and intrakine-transduced mice (Figure 1D). On the other hand, specific and high expression of intrakine mRNA was detected in the splenocytes from intrakine-transduced mice but not in the GFP- or SDF-1-transduced mice (Figure 1D). No differences were observed in the CXCR4 mRNA expression of these mice. Similar results were obtained from the peripheral blood, bone marrow, and thymus. These results suggested that genes transferred by retroviral vector were highly expressed in the reconstituted mice. In vivo effects of SDF-1-intrakine on the expression and function of CXCR4 in adult reconstituted mice FCM analysis revealed that cell surface CXCR4 expression was dramatically decreased in the splenocytes of intrakine-transduced mice than in those of GFP- or SDF-1-transduced mice (Figure 2Ai,iii,v). The suppression of CXCR4 expression was also observed in B220+ cells of peripheral blood (Figure 2A, panels ii, iv, vi), bone marrow, and thymus (data not shown). Similar expression levels of CXCR4 were observed in GFP- and SDF-1-transduced mice (Figure 2A, panels i-iv). In contrast, there were comparable expression levels of CCR7, a receptor for SLC/6Ckine and ELC/MIP-3 /CK-11, on the splenocytes of GFP-, SDF-1-, and
intrakine-transduced mice (Figure 2B). The expression level of CCR1, a
receptor for MIP-1 and RANTES, on the splenocytes of
intrakine-transduced mice was lower than that of GFP- and
SDF-1-transduced mice, though the decrease in CCR1 expression in
intrakine-transduced mice was less prominent than that in CXCR4
expression (Figure 2C). Chemotaxis assay showed that in
intrakine-transduced mice, splenocyte response to SDF-1 was
dramatically decreased, whereas the responses to SLC and MIP-1 were
slightly reduced (Figure 3A). Chemotaxis
assay using bone marrow cells from intrakine-transduced mice also
showed a dramatic decrease in response to SDF-1 (Figure 3B). Similar results were observed in the cells from peripheral blood, lymph node,
and thymus (data not shown). Splenocytes derived from GFP- or
SDF-1-transduced mice showed prominent responses to SDF-1, with
profiles characterized by a typical sharp bell curve (Figure 3A). These
results suggested that the dramatic inhibition of response to SDF-1 in
intrakine-transduced mice resulted from the specific down-regulation of
CXCR4 expression on the cell surfaces induced by the
intrakine.
B lymphopoiesis and myelopoiesis in reconstituted mice To investigate the role of SDF-1 in adult hematopoiesis, bone marrow cells from reconstituted mice were subjected to flow cytometric analysis. The numbers of pro-B (B220+ CD43+), pre-B (B220+ CD43 ), and immature-B
(B220+ IgM+) cells were significantly decreased
in intrakine-transduced mice than in those in GFP-transduced mice
(3.7%± 0.7% versus 8.8% ± 1.1%, 12.3% ± 1.7% versus
25.3% ± 2.2%, 1.2% ± 0.4% versus 6.6% ± 0.9%,
respectively) (Figure 4A). Similar
reductions in the B-cell number in the spleen and peripheral blood were
observed in intrakine-transduced mice (Figure 4C). The numbers of
granulocytes/myeloid cells (Gr-1+ CD11b+) in
the bone marrow and peripheral blood were severely reduced in
intrakine-transduced mice (16.7% ± 1.4% versus 31.5% ± 2.4%, respectively) (Figure 4A,C). The percentages of CD4+ and
CD8+ T cells in peripheral blood of intrakine-transduced
mice were 15.4% ± 1.3% and 10.7% ± 1.2%, respectively (data
not shown).
On the other hand, the numbers of pro-B (B220+
CD43+), pre-B (B220+ CD43 In vitro colony assay revealed that the relative CFU of CFU-mix,
CFU-GM, and CFU-IL-7 were reduced in intrakine-transduced mice
compared to GFP-transduced mice (0.70 ± 0.13 versus
1.0 ± 0.18, 0.62 ± 0.12 versus 1.0 ± 0.15, and 0.52 ± 0.10
versus 1.0 ± 0.14, respectively) (Figure 4B). In contrast, the
relative CFU of CFU-mix, CFU-GM, and CFU-IL-7 were increased in
SDF-1-transduced mice compared to those in GFP mice
(1.43 ± 0.20 versus 1.0 ± 0.18, 1.38 ± 0.11 versus
1.0 ± 0.15, and 1.52 ± 0.17 versus 1.0 ± 0.1, respectively)
(Figure 4B). Percentages of c-kit+ cells in the bone marrow
was increased in the SDF-1-transduced mice (9.4% ± 0.3%), whereas
it was decreased in intrakine-transduced mice (4.1% ± 0.1%)
compared to GFP-transduced mice (6.4% ± 0.3%). These results
suggested that the inhibition of CXCR4 expression on the cell surfaces
by intrakine results in impaired B lymphopoiesis and myelopoiesis in
adult mice and that the overexpression of SDF-1 in SDF-1-transduced
mice induces enhanced B lymphopoiesis and myelopoiesis in bone marrow.
As shown in Table 1, serum immunoglobulin concentrations in reconstituted mice were measured by isotype-specific enzyme-linked immunosorbent assay (ELISA). The mean concentrations of
IgG, IgM, and IgA in sera were decreased in intrakine-transduced mice
compared to those of GFP-transduced mice, whereas the mean concentrations of IgG, IgM, and IgA in sera were increased in SDF-1-transduced mice (Table 1).
T lymphopoiesis in reconstituted mice FCM analysis of the thymocytes revealed that the number of CD4+dull CD8+dull double-positive cells was significantly increased in SDF-1-transduced mice in comparison with GFP-transduced mice and that CD4+high and CD8+high single-positive cells were extremely few in these mice (Figure 5). In contrast, an aberrant increase in CD4/dull CD8+/ cells was
observed in the thymi of intrakine-transduced mice (Figure 5). FCM
analysis showed no difference in thymocyte size. The total number of
thymocytes in intrakine-transduced mice was decreased by approximately
30% to 40% compared with GFP-transduced mice. Histologic analysis of
the thymus revealed hyperplasia but no increase in total cell number in
the cortices of SDF-1-transduced mice compared with GFP-transduced
mice. Similar patterns were observed in the thymic medullas of
SDF-1-transduced mice. On the other hand, histologic studies revealed
thin cortical areas and hypoplasia in the medullas of
intrakine-transduced mice.
We have demonstrated in this study that the expression and
function of CXCR4 was blocked in mice reconstituted with BM-HPCs expressing SDF-1 intrakine and that hematopoiesis, including T lymphopoiesis, was markedly impaired in intrakine-transduced mice. This
is the first in vivo study that shows the successful use of an
intrakine to regulate the function of specific chemokines and chemokine
receptors. Ma et al33 reported that CXCR4 is required for
the retention of the B lineage and granulocytic precursors in the bone
marrow microenvironment. They detected a decrease in the number of
B-cell lineages (B220+) in bone marrow and an increase in
the number of immature B cells (B220+ CD43+
IgM Because an ELISA system for measuring serum levels of murine SDF-1 is unavailable, we could not confirm higher concentrations of SDF-1 in SDF-1-transduced mice than in GFP-transduced control mice. Although a higher expression of messenger RNA for SDF-1 was observed in SDF-1-transduced mice than in GFP-transduced mice by RT-PCR analysis, similar expression levels of CXCR4 on the cells by FACS analysis and migration activity in GFP- and SDF-1-transduced mice were observed. This is rather unexpected because the overexpression of SDF-1 could be expected to result in the down-regulation of CXCR4 expression on leukocytes in SDF-1-transduced mice. The reason for this phenotype is unclear, but it could be because of the accelerated turnover of CXCR4 in SDF-1-transduced mice. Interestingly, we found abnormalities in T-cell maturation in the
thymus, both in SDF-1- and intrakine-transduced mice. There was an
increase of CD4+dull CD8+dull cells in the
thymi of SDF-1-transduced mice in contrast to an increase of
CD4 Preliminary results from immunohistochemical staining showed that SDF-1 is highly expressed in the thymic medulla, whereas CXCR4 is expressed in the thymic cortex. This suggests that preferential localization of SDF-1 in the thymic cortex plays a role in positive selection and that down-regulation of CXCR4 during positive selection enables selected thymocytes to move toward the thymic medulla. Kim et al36 reported that high levels of mRNA of SDF-1 and CXCR4 were detected in the thymus and that SDF-1 had a chemotactic activity for immature thymocytes (subsets of triple-negative thymocytes and double-positive thymocytes) over mature single-positive thymocytes. They speculated that SDF-1 might be a chemoattractant regulating the migration of immature double-negative and double-positive thymocytes at premedullar stage (cortex) migration. Based on these results and our own, we speculate that the overexpression of SDF-1 may cause the retention of immature thymocytes in the cortex and inhibit intrathymic trafficking required for positive selection. On the other hand, the migration of thymocytes in intrakine-transduced mice is also interfered with by down-regulated CXCR4 expression on those by intrakine. FCM analysis on thymocytes in intrakine-transduced mice revealed the
accumulation of the CD4 The discovery that several chemokine receptors act as coreceptors for HIV-1 provides multiple opportunities for new therapeutic interventions into the process of viral pathogenesis in AIDS.37,38 T-tropic HIV uses the chemokine receptor CXCR4 as a coreceptor and emerges during the late stages of AIDS progression associated with a decline in CD4+ T lymphocytes.39,40 This suggests that CXCR4 may be related to AIDS pathogenesis and that it is a suitable target for AIDS therapeutic application. Our results may provide a new therapeutic approach to AIDS through the use of the intrakine gene. It should be noted, however, that the transduction of an intrakine into human hematopoietic stem cells from patients and the transplantation of those into AIDS patients might result in impaired hematopoiesis and lymphopoiesis. In summary, this study represents the first successful use of intrakine in vivo to down-regulate the expression and function of specific cognate receptors. By using this novel approach, we have demonstrated that SDF-1/CXCR4 has an important role in B lymphopoiesis, myelopoiesis, and T lymphopoiesis in adult hematopoiesis.
We thank Dr Masataka Osawa (Kirin Brewery, Tokyo, Japan) for valuable technical advice on bone marrow transplantation.
Submitted October 5, 1999; accepted May 10, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Kouji Matsushima, Department of Molecular Preventive Medicine, School of Medicine, University of Tokyo, 7-3-1 Hongo, Bonkyo-ku, Tokyo 113-0033, Japan; e-mail: koujim{at}m.u-tokyo.ac.jp.
1. Dexter TM, Allen TD, Lajtha LG. Conditions controlling the proliferation of hematopoietic stem cells in vitro. J Cell Physiol. 1977;91:335-344[Medline] [Order article via Infotrieve]. 2. Ikuta K, Uchida N, Friedman J, Weissman IL. Lymphocyte development from stem cells. Annu Rev Immunol. 1992;10:759-783[Medline] [Order article via Infotrieve]. 3. Muller AM, Medvinsky A, Strouboulis J, Grosveld F, Dzierzak E. Development of hematopoietic stem cell activity in the mouse embryo. Immunity. 1994;1:291-301[Medline] [Order article via Infotrieve].
4.
Godin I, Dieterlen-Lievre F, Cumano A.
Emergence of multipotent hematopoietic cells in the yolk sac and paraaortic splanchnopleura in mouse embryos, beginning at 8.5 days postcoitus [published correction appears in Proc Natl Acad Sci U S A 1995;92:10815].
Proc Natl Acad Sci U S A.
1995;92:773-777 5. Harada A, Mukaida N, Matsushima K. Interleukin 8 as a novel target for intervention therapy in acute inflammatory diseases. Mol Med Today. 1996;2:482-489[Medline] [Order article via Infotrieve]. 6. Baggiolini M. Chemokines and leukocyte traffic. Nature. 1998;392:565-568[Medline] [Order article via Infotrieve]. 7. Yoshie O, Imai T, Nomiyama H. Novel lymphocyte-specific CC chemokines and their receptors. J Leukoc Biol. 1997;62:634-644[Abstract]. 8. Sallusto F, Lanzavecchia A, Mackay CR. Chemokines and chemokine receptors in T-cell priming and Th1/Th2-mediated responses. Immunol Today. 1998;19:568-574[Medline] [Order article via Infotrieve]. 9. Kim CH, Broxmeyer HE. Chemokines: signal lamps for trafficking of T and B cells for development and effector function. J Leukoc Biol. 1999;65:6-15[Abstract].
10.
Tashiro K, Tada H, Heilker R, Shirozu M, Nakano T, Honjo T.
Signal sequence trap: a cloning strategy for secreted proteins and type I membrane proteins.
Science.
1993;261:600-603
11.
Nagasawa T, Kikutani H, Kishimoto T.
Molecular cloning and structure of a pre-B-cell growth-stimulating factor.
Proc Natl Acad Sci U S A.
1994;91:2305-2309
12.
Bleul CC, Fuhlbrigge RC, Casasnovas JM, Aiuti A, Springer TA.
A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1).
J Exp Med.
1996;184:1101-1109
13.
Aiuti A, Webb IJ, Bleul C, Springer T, Gutierrez-Ramos JC.
The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood.
J Exp Med.
1997;185:111-120
14.
Nomura H, Nielsen BW, Matsushima K.
Molecular cloning of cDNAs encoding a LD78 receptor and putative leukocyte chemotactic peptide receptors.
Int Immunol.
1993;5:1239-1249 15. Herzog H, Hort YJ, Shine J, Selbie LA. Molecular cloning, characterization, and localization of the human homolog to the reported bovine NPY Y3 receptor: lack of NPY binding and activation. DNA Cell Biol. 1993;12:465-471[Medline] [Order article via Infotrieve]. 16. Federsppiel B, Melhado IG, Duncan AM, et al. Molecular cloning of the cDNA and chromosomal localization of the gene for a putative seven-transmembrane segment (7-TMS) receptor isolated from human spleen. Genomics. 1993;16:707-712[Medline] [Order article via Infotrieve]. 17. Jazin EE, Yoo H, Blomqvist AG, et al. A proposed bovine neuropeptide Y (NPY) receptor cDNA clone, or its human homologue, confers neither NPY binding sites nor NPY responsiveness on transfected cells. Regul Pept. 1993;47:247-258[Medline] [Order article via Infotrieve]. 18. Nagasawa T, Hirota S, Tachibana K, et al. Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature. 1996;382:635-638[Medline] [Order article via Infotrieve]. 19. Tachibana K, Hirota S, Iizasa H, et al. The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract [see comments]. Nature. 1998;393:591-594[Medline] [Order article via Infotrieve]. 20. Zou YR, Kottmann AH, Kuroda M, Taniuchi I, Littman DR. Function of the chemokine receptor CXCR4 in hematopoiesis and in cerebellar development. Nature. 1998;393:595-599[Medline] [Order article via Infotrieve]. 21. Bleul CC, Farzan M, Choe H, et al. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature. 1996;382:829-833[Medline] [Order article via Infotrieve]. 22. Oberlin E, Amara A, Bachelerie F, et al. The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T-cell-line-adapted HIV-1 [published correction appears in Nature 1996;384:288]. Nature. 1996;382:833-835[Medline] [Order article via Infotrieve]. 23. Chen JD, Bai X, Yang AG, Cong Y, Chen SY. Inactivation of HIV-1 chemokine co-receptor CXCR-4 by a novel intrakine strategy. Nat Med. 1997;3:1110-1116[Medline] [Order article via Infotrieve].
24.
Yang AG, Bai X, Huang XF, Yao C, Chen S.
Phenotypic knockout of HIV type 1 chemokine coreceptor CCR-5 by intrakines as potential therapeutic approach for HIV-1 infection.
Proc Natl Acad Sci U S A.
1997;94:11567-11572 25. Wang J, Mukaida N, Zhang Y, Ito T, Nakao S, Matsushima K. Enhanced mobilization of hematopoietic progenitor cells by mouse MIP-2 and granulocyte colony-stimulating factor in mice. J Leukoc Biol. 1997;62:503-509[Abstract].
26.
Ogata M, Zhang Y, Wang Y, et al.
Chemotactic response toward chemokines and its regulation by transforming growth factor-beta1 of murine bone marrow hematopoietic progenitor cell-derived different subset of dendritic cells.
Blood.
1999;93:3225-3232
27.
Kitamura T, Onishi M, Kinoshita S, Shibuya A, Miyajima A, Nolan GP.
Efficient screening of retroviral cDNA expression libraries.
Proc Natl Acad Sci U S A.
1995;92:9146-9150
28.
Yasukawa T, Kanei-Ishii C, Maekawa T, Fujimoto J, Yamamoto T, Ishii S.
Increase of solubility of foreign proteins in Escherichia coli by coproduction of the bacterial thioredoxin.
J Biol Chem.
1995;270:25328-25331 29. Su SB, Mukaida N, Wang J, Nomura H, Matsushima K. Preparation of specific polyclonal antibodies to a C-C chemokine receptor, CCR1, and determination of CCR1 expression on various types of leukocytes. J Leukoc Biol. 1996;60:658-666[Abstract]. 30. Liu FT, Zinnecker M, Hamaoka T, Katz DH. New procedures for preparation and isolation of conjugates of proteins and a synthetic copolymer of D-amino acids and immunochemical characterization of such conjugates. Biochemistry. 1979;18:690-693[Medline] [Order article via Infotrieve].
31.
Tokuda A, Itakura M, Onai N, Kimura H, Kuriyama T, Matsushima K.
Pivotal role of CCR1 positive leukocytes in bleomycin induced lung fibrosis in mice.
J Immunol.
2000;164:2745-2751
32.
Osawa M, Hanada K, Hamada H, Nakauchi H.
Long-term lymphohematopoietic reconstitution by a single CD34 33. Ma Q, Jones D, Springer TA. The chemokine receptor CXCR4 is required for the retention of B lineage and granulocytic precursors within the bone marrow microenvironment. Immunity. 1999;10:463-471[Medline] [Order article via Infotrieve].
34.
Kawabata K, Ujikawa M, Egawa T, et al.
A cell-autonomous requirement for CXCR4 in long-term lymphoid and myeloid reconstitution.
Proc Natl Acad Sci U S A.
1999;96:5663-5667
35.
Okada S, Nakauchi H, Nagayoshi K, Nishikawa S, Miura Y, Suda T.
In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells.
Blood.
1992;80:3044-3050
36.
Kim CH, Pelus LM, White JR, Broxmeyer HE.
Differential chemotactic behavior of developing T cells in response to thymic chemokines.
Blood.
1998;91:4434-4443 37. Feng Y, Broder CC, Kennedy PE, Berger EA. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science. 1996;272:872-877[Abstract]. 38. Cairns JS, D'Souza MP. Chemokines and HIV-1 second receptors: the therapeutic connection. Nat Med. 1998;4:563-568[Medline] [Order article via Infotrieve]. 39. D'Souza MP, Harden VA. Chemokines and HIV-1 second receptors: confluence of two fields generates optimism in AIDS research. Nat Med. 1996;2:1293-1300[Medline] [Order article via Infotrieve].
40.
Unutmaz D, Littman DR.
Expression pattern of HIV-1 coreceptors on T cells: implications for viral transmission and lymphocyte homing [comment].
Proc Natl Acad Sci U S A.
1997;94:1615-1618
© 2000 by The American Society of Hematology.
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|
 |
|
  A. Banerjee, R. Grumont, R. Gugasyan, C. White, A. Strasser, and S. Gerondakis NF-{kappa}B1 and c-Rel cooperate to promote the survival of TLR4-activated B cells by neutralizing Bim via distinct mechanisms Blood, December 15, 2008; 112(13): 5063 - 5073. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Nazari, X. Z. Ma, and S. Joshi Inhibition of human immunodeficiency virus-1 entry using vectors expressing a multimeric hammerhead ribozyme targeting the CCR5 mRNA J. Gen. Virol., September 1, 2008; 89(9): 2252 - 2261. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Gotoh, Y. Tanaka, A. Nishikimi, A. Inayoshi, M. Enjoji, R. Takayanagi, T. Sasazuki, and Y. Fukui Differential requirement for DOCK2 in migration of plasmacytoid dendritic cells versus myeloid dendritic cells Blood, March 15, 2008; 111(6): 2973 - 2976. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. M. Fuhler, A. L. Drayer, S. G. M. Olthof, J. J. Schuringa, P. J. Coffer, and E. Vellenga Reduced activation of protein kinase B, Rac, and F-actin polymerization contributes to an impairment of stromal cell derived factor-1 induced migration of CD34+ cells from patients with myelodysplasia Blood, January 1, 2008; 111(1): 359 - 368. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Kim, H. H. Kim, J. H. Kim, Y. H. Choi, Y. H. Kim, and J. Cheong Chemokine stromal cell-derived factor-1 induction by C/EBP{beta} activation is associated with all-trans-retinoic acid-induced leukemic cell differentiation J. Leukoc. Biol., November 1, 2007; 82(5): 1332 - 1339. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. E. McCandless, Q. Wang, B. M. Woerner, J. M. Harper, and R. S. Klein CXCL12 Limits Inflammation by Localizing Mononuclear Infiltrates to the Perivascular Space during Experimental Autoimmune Encephalomyelitis J. Immunol., December 1, 2006; 177(11): 8053 - 8064. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Banerjee, R. Gugasyan, M. McMahon, and S. Gerondakis Diverse Toll-like receptors utilize Tpl2 to activate extracellular signal-regulated kinase (ERK) in hemopoietic cells PNAS, February 28, 2006; 103(9): 3274 - 3279. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Segal, R. Shah, A. Afzal, C. M. Perrault, K. Chang, A. Schuler, E. Beem, L. C. Shaw, S. Li Calzi, J. K. Harrison, et al. Nitric Oxide Cytoskeletal-Induced Alterations Reverse the Endothelial Progenitor Cell Migratory Defect Associated With Diabetes Diabetes, January 1, 2006; 55(1): 102 - 109. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Bonig, G. V. Priestley, and T. Papayannopoulou Hierarchy of molecular-pathway usage in bone marrow homing and its shift by cytokines Blood, January 1, 2006; 107(1): 79 - 86. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. J. Bendall, R. Baraz, J. Juarez, W. Shen, and K. F. Bradstock Defective p38 Mitogen-Activated Protein Kinase Signaling Impairs Chemotaxic but not Proliferative Responses to Stromal-Derived Factor-1{alpha} in Acute Lymphoblastic Leukemia Cancer Res., April 15, 2005; 65(8): 3290 - 3298. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. V. Gulino, D. Moratto, S. Sozzani, P. Cavadini, K. Otero, L. Tassone, L. Imberti, S. Pirovano, L. D. Notarangelo, R. Soresina, et al. Altered leukocyte response to CXCL12 in patients with warts hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome Blood, July 15, 2004; 104(2): 444 - 452. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Yoneyama, K. Matsuno, Y. Zhang, T. Nishiwaki, M. Kitabatake, S. Ueha, S. Narumi, S. Morikawa, T. Ezaki, B. Lu, et al. Evidence for recruitment of plasmacytoid dendritic cell precursors to inflamed lymph nodes through high endothelial venules Int. Immunol., July 1, 2004; 16(7): 915 - 928. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Papayannopoulou Current mechanistic scenarios in hematopoietic stem/progenitor cell mobilization Blood, March 1, 2004; 103(5): 1580 - 1585. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Plotkin, S. E. Prockop, A. Lepique, and H. T. Petrie Critical Role for CXCR4 Signaling in Progenitor Localization and T Cell Differentiation in the Postnatal Thymus J. Immunol., November 1, 2003; 171(9): 4521 - 4527. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. N. Kremer, T. D. Humphreys, A. Kumar, N.-X. Qian, and K. E. Hedin Distinct Role of ZAP-70 and Src Homology 2 Domain-Containing Leukocyte Protein of 76 kDa in the Prolonged Activation of Extracellular Signal-Regulated Protein Kinase by the Stromal Cell-Derived Factor-1{alpha}/CXCL12 Chemokine J. Immunol., July 1, 2003; 171(1): 360 - 367. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ara, M. Itoi, K. Kawabata, T. Egawa, K. Tokoyoda, T. Sugiyama, N. Fujii, T. Amagai, and T. Nagasawa A Role of CXC Chemokine Ligand 12/Stromal Cell-Derived Factor-1/Pre-B Cell Growth Stimulating Factor and Its Receptor CXCR4 in Fetal and Adult T Cell Development in Vivo J. Immunol., May 1, 2003; 170(9): 4649 - 4655. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Kollet, I. Petit, J. Kahn, S. Samira, A. Dar, A. Peled, V. Deutsch, M. Gunetti, W. Piacibello, A. Nagler, et al. Human CD34+CXCR4- sorted cells harbor intracellular CXCR4, which can be functionally expressed and provide NOD/SCID repopulation Blood, September 26, 2002; 100(8): 2778 - 2786. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Gugasyan, C. Quilici, S. T.T. I, D. Grail, A. M. Verhagen, A. Roberts, T. Kitamura, A. R. Dunn, and P. Lock Dok-related protein negatively regulates T cell development via its RasGTPase-activating protein and Nck docking sites J. Cell Biol., July 8, 2002; 158(1): 115 - 125. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Onai, M. Kitabatake, Y.-y. Zhang, H. Ishikawa, S. Ishikawa, and K. Matsushima Pivotal role of CCL25 (TECK)-CCR9 in the formation of gut cryptopatches and consequent appearance of intestinal intraepithelial T lymphocytes Int. Immunol., July 1, 2002; 14(7): 687 - 694. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Zaitseva, T. Kawamura, R. Loomis, H. Goldstein, A. Blauvelt, and H. Golding Stromal-Derived Factor 1 Expression in the Human Thymus J. Immunol., March 15, 2002; 168(6): 2609 - 2617. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Hernandez-Lopez, A. Varas, R. Sacedon, E. Jimenez, J. J. Munoz, A. G. Zapata, and A. Vicente Stromal cell-derived factor 1/CXCR4 signaling is critical for early human T-cell development Blood, January 15, 2002; 99(2): 546 - 554. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Suzuki, M. Rahman, and H. Mitsuya Diverse Transcriptional Response of CD4+ T Cells to Stromal Cell-Derived Factor (SDF)-1: Cell Survival Promotion and Priming Effects of SDF-1 on CD4+ T Cells J. Immunol., September 15, 2001; 167(6): 3064 - 3073. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
), bone marrow (
),
spleen (
), and thymus (
) of each reconstituted mouse transplanted
with 1 × 105 GFP+ cells per mouse.
Each point represents mean percentage ± SE from 4 separate
experiments. (D). The mRNA expression level of transferred
genes in the reconstituted mice. Total RNA was extracted from
splenocytes. Total RNA was reverse transcribed with random primer and
amplified using the specific primers for GFP, CXCR4, SDF-1, intrakine,
and G3PDH. These results represent 3 independent experiments.
Indicates the packaging symbol.
