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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1145-1156
Coreceptor/Chemokine Receptor Expression on Human Hematopoietic
Cells: Biological Implications for Human Immunodeficiency Virus-Type
1 Infection
By
Benhur Lee,
Janina Ratajczak,
Robert W. Doms,
Alan M. Gewirtz, and
Mariusz Z. Ratajczak
From the Department of Medicine and the Department of Pathology and
Laboratory Medicine, Hospital of the University of Pennsylvania,
Philadelphia, PA.
 |
ABSTRACT |
The recent discovery of chemokine receptors as coreceptors for human
immunodeficiency virus-type 1 (HIV-1) entry offers new avenues for investigating the pathogenesis of acquired immunodeficiency syndrome (AIDS)-related cytopenias. To this end, we sought to (1)
phenotype human hematopoietic cells for CD4 and the HIV-1 coreceptors
CXCR4, CCR5, CCR3, and CCR2b; (2) correlate CD4 and chemokine receptor
expression with their susceptibility to HIV-1 infection; and
(3) examine any potential interplay between inflammatory cytokines
released during HIV-1 infection and regulation of chemokine receptor
expression. Fluorescence-activated cell sorting (FACS) analysis of bone
marrow mononuclear cells (BMMNC), cells derived from serum-free
expanded hematopoietic lineages (colony-forming unit-granulocyte-macrophage [CFU-GM], colony-forming
unit-megakaryocyte [CFU-Meg], and burst-forming unit-erythroid
[BFU-E]), and CD34+ cells showed
differential expression of chemokine receptors and CD4 with some
lineage specificity. Significantly, FACS-sorted CXCR4+/CD34+ cells had the same clonogeneic
potential as CXCR4 /CD34+ cells. Reverse
transcriptase-polymerase chain reaction (RT-PCR) analysis of
FACS-sorted human candidate stem cells (HSC; CD34+,
c-kit+, Rho123low) showed the presence of
CXCR4 mRNA but not CD4 mRNA. Infection studies with HIV-1
Env-pseudotyped luciferase reporter viruses indicated that X4 Env
(CXCR4-using) pseudotypes infected megakaryocytic cells, whereas R5 Env
(CCR5-using) pseudotypes did not. Similarly, R5 but not X4
Env-pseudotyped viruses infected granulocyte-macrophage cells in a
CD4/CCR5-dependent manner. Erythroid cells were resistant to R5 or X4
viral infection. Finally, we found that  -interferon treatment
upregulated CXCR4 expression on primary hematopoietic cells. In
summary, the delineation of chemokine receptor expression on primary
hematopoietic cells is a first step towards dissecting the
chemokine-chemokine receptor axes that may play a role in hematopoietic
cell proliferation and homing. Furthermore, susceptibility of
hematopoietic cells to HIV-1 infection is likely to be more complicated
than the mere physical presence of CD4 and the cognate chemokine
receptor. Lastly, our results suggest a potential interplay between
-interferon secretion and CXCR4 expression.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PATIENTS INFECTED BY human
immunodeficiency virus-type 1 (HIV-1) frequently exhibit
a variety of different hematological abnormalities, including anemia,
neutropenia, and thrombocytopenia, in addition to the invariable loss
of CD4+ lymphocytes.1,2 The discovery of
chemokine receptors as coreceptors for HIV-1 entry offers new avenues
for increasing our understanding of the mechanisms underlying
HIV-1-associated bone marrow dysfunction.3 At this point,
there are 11 reported chemokine or orphan receptors that function as
HIV-1 coreceptors: CXCR-4, CCR5, CCR2b, CCR3, CCR8, STRL33, GPR1, V28,
ChemR23, GPR15, and APJ (reviewed previously4,5). All HIV-1
strains studied to date use CCR5 (R5 strains), CXCR4 (X4), or both
receptors (R5X4) to enter cells, and individuals who lack CCR5 are
highly resistant to virus infection (reviewed in McNicholl et
al6). The in vivo relevance of coreceptors other than CCR5
and CXCR4 has yet to be determined, although their ability to support
infection by more limited numbers of virus strains raises the
possibility that their use may be involved in the myriad pathologies
associated with HIV-1 infection, including the hematologic
abnormalities. As such, exploring the chemokine receptor expression
pattern on subsets of hematopoietic progenitors may shed light on the
susceptibility of various subsets to either direct infection by HIV-1
or other forms of modulation such as chemokine-induced
inhibition/proliferation or perhaps envelope (Env)-mediated toxicity.
With regard to the latter point, recent studies have shown that soluble
HIV-1 and SIV Env can induce G-protein-mediated signal transduction
through their cognate coreceptors.7,8 Therefore,
intracellular signaling cascades mediated through chemokine receptors
by HIV-1 Env may lead to hematopoietic derangements even in the absence
of productive infection of hematopoietic progenitor populations. This
is supported by studies showing an inhibitory effect of recombinant
viral envelope glycoprotein on CD34+ progenitor
cells.9-11
Studies to date have looked at HIV-1 coreceptor expression in bone
marrow progenitor cells only at the mRNA level.12,13 The
use of in vitro serum-free cultures for expanding relatively pure,
lineage-committed hematopoietic progenitors along with recently developed monoclonal antibodies (MoAbs) against the major HIV-1 coreceptors has allowed us to define coreceptor/chemokine receptor expression on erythroid, megakaryocytic, and granulo-macrophage lineages. Although the pathogenesis of acquired immunodeficiency syndrome (AIDS)-related cytopenias is likely to be multifactorial (reviewed in Moses et al14), the delineation of coreceptor
and CD4 antigen expression will allow a preliminary determination of
hematopoietic subsets that may be susceptible to either direct infection by HIV-1 or to HIV-1 Env-mediated cytotoxicity. In addition, it will now be possible to determine whether the many proinflammatory cytokines (tumor necrosis factor- [TNF-], -interferon
[-IFN], etc) secreted in excess during chronic HIV-1
infection15,16 have any influence on cognate coreceptor
expression. Because the chemokine-chemokine receptor axes may be
involved in hematopoietic proliferation and homing,17 any
pertubation of chemokine receptor expression may not only result in the
expansion or restriction of HIV-1 tropism, but also contribute to the
pathogenesis of the many cytopenias observed in HIV-1 disease.
We report here that HIV-1 coreceptor expression exhibited some lineage
specificity and that megakaryocytic cells were infectable by X4
viruses, whereas granulo-macrophage lineage cells were infectable by R5
viruses. Furthermore, we determined that CXCR4 was expressed even on
the earliest candidate human stem cells (HSC), although only about half
of clonogeneic hematopoietic progenitor cells (HPC) were
CXCR4+/CD34+ cells. We also found that -IFN
could upregulate the expression of CXCR4 on BMMNC, suggesting that
proinflammatory cytokines released during chronic HIV infection may
influence the dynamics of HIV-1 replication by altering chemokine
receptor expression levels.
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MATERIALS AND METHODS |
Selection of HSC candidates by fluorescence-activated cell sorting
(FACS).
Light-density bone marrow mononuclear cells (BMMNC) were obtained from
12 consenting healthy donors and depleted of adherent cells and T
lymphocytes (ATMNC) as
described.18 MNC (~3 to 6 × 107) were
simultaneously labeled with phycoerythrin (PE)-conjugated anti-CD34
MoAb (anti-HPCA-2PE; Becton Dickinson, Mountain View, CA), an antihuman
Kit receptor MoAb (SR-1; kind gift of Dr V. Broudy, University of
Washington, Seattle, WA) detected with a Cy 5-labeled conjugate, and
Rh123 at concentrations previously shown to be nontoxic to
hematopoietic cells. CD34+, Kit+,
Rh123low (defined as the dimmest 5% to 10% of
Rh123-labeled cells) were isolated by FACS as described
previously.19 We have also isolated by FACS a fraction of
CD34+, Kit+, Rh123bright cells
(defined as the brightest 50% of Rh123 labeled cells) that is enriched
in HPC.19
Isolation of CXCR4+ cells.
BMMNC were stained with CXCR4 MoAb and subsequently isolated by using
immunomagnetic beads (Dynal, Oslo, Norway) according to the
manufacturer's protocol and as described.20 In some
experiments, FACS-sorted CD34+/CXCR4+ and
CD34+/CXCR4 cells were isolated from
total bone marrow. Briefly, BMMNC were stained with CXCR4 MoAb (R&D
Systems, Minneapolis, MN) and detected with fluorescein isothiocyanate
(FITC)-conjugated goat antimouse polyclonal Abs (Sigma, St Louis, MO),
followed by staining with PE-conjugated CD34+ MoAb.
Subsequently, cells were washed twice (1× phosphate-buffered saline [PBS] with 2% calf serum) and FACS sorted for both
CD34+/CXCR4+ and
CD34+/CXCR cells using FACStarPlus
(Becton Dickinson).
In vitro clonogeneic assays for hematopoietic progenitors.
Immunomagnetically isolated CXCR4+ cells (as described
above) or FACS-sorted CXCR+/CD34+ or
CXCR4/CD34+ cells were plated in HCC-17
methylcellulose medium (StemCell Technologies, Vancouver, British
Columbia, Canada) as described.19 Colony-forming unit-mix
(CFU-Mix) colonies were stimulated with a cocktail of
recombinant human (rH) growth factors: kit ligand (KL; 10 ng/mL), interleukin-3 (IL-3; 20 U/mL), granulocyte-macrophage colony-stimulating factor (GM-CSF; 5 ng/mL), erythropoietin (Epo; 2 U/mL), and IL-6 (40 U/mL). Burst-forming unit-erythroid (BFU-E) growth
was stimulated with Epo (2 U/mL) and KL (10 ng/mL) and colony-forming
unit-granulocyte-macrophage (CFU-GM) growth was stimulated with IL-3
(20 U/mL) and GM-CSF (5 ng/mL), whereas colony-forming unit-megakaryocyte (CFU-Meg) growth was stimulated with
thrombopoietin (TPO; 50 ng/mL) and IL-3 (20 U/mL).
Cytokines were from R&D Systems. Cultures were incubated at 37°C in
a fully humidified atmosphere supplemented with 5% CO2.
Colonies were scored at day 15 (CFU-Mix) and day 11 (BFU-E, CFU-GM, and
CFU-Meg), respectively.
Ex vivo expansion of normal human hematopoietic cells.
CD34+ cells were expanded in serum-free liquid system as
described.19-22 Briefly, CD34+
AT BMNC were resuspended in
Iscove Dulbecco's modified Eagle's medium (DMEM; GIBCO BRL, Grand
Island, NJ; 104/mL) supplemented with 25% of artificial
serum containing 1% delipidated, deionized, and charcoal-treated
bovine serum albumin (BSA), 270 µg/mL iron-saturated transferrin,
insulin (20 µg/mL), and 2 mmol/l L-glutamine (all from Sigma). BFU-E
growth was stimulated with rH Epo (2 U/mL) and rH KL (10 ng/mL) and
CFU-GM growth was stimulated with rH IL-3 (20 U/mL) and rH GM-CSF (5 ng/mL), whereas CFU-Meg growth was stimulated with rH TPO (50 ng/mL)
and IL-3 (20 U/mL). Cytokines were from R&D Systems. Cultures were
incubated at 37°C in a fully humidified atmosphere supplemented
with 5% CO2. Under these conditions, approximately 100%
of BFU-E-derived cells were glycophorin A positive, 65% to 80% of
CFU-Meg cells were gpIIa/IIIb positive, and 100% of CFU-GM-derived
cells were glycophorin A and gpIIb/IIIa negative and expressed
CD33.21-23
Flow cytometry analysis.
The expression of CXCR4, CCR5, CCR2, CCR3, and CD4 on normal human
hematopoietic cells was evaluated by FACS. The following MoAbs were
used: 12G5 (J.A. Hoxie, University of Pennsylvania, Philadelphia, PA)
and clone #701 (R&D Systems) for CXCR4; clones #529, #531, and #549 for
CCR5 (R&D Systems); biotinylated clone #RO2 and #R05 for CCR2 (a
generous gift from Carlos Martinez-A., Universidad Autonoma de Madrid,
Madrid, Spain); 7B11 for CCR3 (NIH AIDS Reference Reagent Program); and
Leu3A for CD4 (Becton Dickinson). Flow cytometric staining and analysis
of the receptors were performed as described.23 Briefly,
the cells were stained in PBS (Ca and Mg free) supplemented with 5%
bovine calf serum (BCS). Primary MoAbs were detected with
secondary PE- or FITC-conjugated goat antimouse MoAbs (Sigma; 1:100) or
PE-conjugated streptavidin (Pharmingen, San Diego, CA) at 0.25 mg/mL
for biotinylated primary antibodies. After the final washes, cells were
fixed in 1% paraformaldehyde before FACS analysis using FACScan
(Becton Dickinson, San Jose, CA). BMMNC or cells isolated from in vitro
expanded liquid cultures of BFU-E, CFU-GM, and CFU-Meg cells were also
assayed for the binding of biotinylated macrophage inflammatory
protein-1 and monocyte chemotactic protein-1 (R&D Systems) according
to the manufacturer's protocols. Data analysis was performed using the Cell Quest (Becton Dickinson, San Jose, CA).
Reverse transcription-polymerase chain reaction
(RT-PCR) studies.
RNA was extracted from FACS-sorted CD34+, Kit+,
Rh123dull and CD34+, Kit+,
Rh123bright cells using a poly A-mRNA purification kit
(Pharmacia, Piscataway, NJ) according to the manufacturer's protocol.
The isolated RNA was dissolved in triple-distilled and autoclaved water
and stored at  20°C until used. For RT-PCR, mRNA (0.5 µg)
was reverse-transcribed with 500 U of Moloney murine leukemia virus
reverse transcriptase (MoMLV-RT) and 50 pmol of an ODN primer
complementary to the 3' end of the following sequence of CXCR4
(CAA GGA AGC TGT TGG CTG AAA) or CD4
(5'-TTGGCGCCTTCGGTGCCGGCA-3'),24 according to
reported cDNA sequences. The resulting cDNA fragments were amplified
using 5 U of Thermus aquaticus (Taq) polymerase with the addition of primers specific for the 5' end of CXCR4 (5'-CGA GGC AAG
TGA CGC CGA GGG CCT G-3') and CD4 (5'-GTGTGG
GGACCCACCTCCCCTAAG-3').24 Amplified products (10 µL) were electrophoresed on a 2% agarose gel and documented
photographically. Specificity of the amplified products was further
confirmed by Southern blotting. Electrophoresed gel fragments were
transferred to a nylon filter and filters were prehybridized and probed
with a 32P end-labeled ODN specific for the cDNA of CXCR4
or CD4. Hybridization was detected by autoradiography as
described.18
Viral infection assay.
Luciferase reporter viruses were prepared as previously
described25,26 by cotransfecting 293T cells with the
indicated Envs and the NL4-3 luciferase virus backbone
(pNL-luc-ER) plasmids.
Full-length gp160 env genes from R5 (ADA, JRFL) and X4 (HXB2, NL4-3)
viruses were cloned into pSV7d, where expression is driven off a
constitutive SV40 promoter. These plasmids were generously provided
by John Moore (Aaron Diamond AIDS Research Center, New York, NY). The
NL4-3 luciferase virus backbone
(pNL-luc-ER) was provided by Ned
Landau (Aaron Diamond AIDS Research Center). This backbone was
constructed with a frame-shift mutation in its env gene and a
luciferase gene inserted into the nef coding region. Forty-eight hours after CaPO4 transfection, the supernatant
was collected, filtered through a 0.2-µm filter, and stored at
80°C until further use. Infections were performed on the
indicated target cells in the presence of 8 µg/mL of diethyl
aminoethyl (DEAE)-dextran. Four days postinfection, cells were lysed
with 0.5% TX-100 in PBS and an appropriate aliquot was analyzed for luciferase activity. Chemiluminescence from substrate conversion by
luciferase was measured in a Wallac Microbeta Trilux luminometer and
data were presented in relative light units (RLU). For inhibition assays, the appropriate inhibitor (chemokine or antibody) at the indicated concentrations was added 30 minutes before the addition of
the reporter virus.
Statistics.
Arithmetic means and standard deviations were calculated on a MacIntosh
computer using Instat 1.14 (GraphPad, San Diego, CA) software. Data
were analyzed using the Student's t-test for unpaired samples.
Statistical significance was defined as P < .01.
| |
RESULTS |
Expression of CXCR4, CCR5, CCR2, and CCR3 on normal human BMMNC.
Because the expression of the major HIV-1 coreceptors in the various
bone marrow hematopoietic populations has not been systematically examined, we first evaluated the expression of chemokine receptors on
normal human BMMNC isolated by Ficoll-gradient centrifugation. As can
be seen in Fig 1, CXCR4 (58% ± 6%
positive), CCR5 (13% ± 2% positive), and CCR2 (51% ± 6%
positive) but not CCR3 were variously present in total BMMNC. Because
no detectable CCR3 was expressed on BMMNC, no further analysis of CCR3
was performed. We next determined the expression of these chemokine
receptors in different subpopulations of BMMNC (lymphocyte-R1,
monocyte-R2, and granulocyte-R3 gates; Fig
2) based on their forward versus side-scatter properties (Fig 1). As
summarized in Table 1, we found that CXCR4
was expressed predominantly on cells from the lymphocyte and monocyte
gates, CCR5 predominantly in the monocyte gate, and CCR2 mostly in the
monocyte and granulocyte progenitor gates. These results indicate that
chemokine receptor expression exhibits some degree of lineage
specificity.
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| Fig 1.
Expression of chemokine receptors on total BMMNC. BMMNC
were isolated from bone marrow aspirates of healthy donors by
Ficoll-gradient centrifugation; stained with MoAbs to CXCR4 (B), CCR5
(C), CCR2 (E), and CCR3 (F); and subjected to FACS analysis as
described in Materials and Methods. The histogram represents analysis
of 10,000 events acquired in the total ungated population (A and D).
The isotyped matched negative control is shown in the overlay. M1 gate
represents the positive populations. Data from at least 3 different
donors were analyzed with similar results. Data from a representative
donor are presented.
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| Fig 2.
FACS analysis of CXCR4, CCR5, and CCR2 on subpopulations
of BMMNC. Total BMMNC were stained with anti-CXCR4, anti-CCR5, and
anti-CCR2 antibodies as described and FACS analysis was performed on
the gated populations as indicated in Fig 1A and D. R1, R2, and R3
represent the lymphocyte, granulocyte precursor, and monocyte gates,
respectively. The isotype negative controls are overlaid (bold line),
and M1 represents the positive populations. Data from at least 3 different donors were analyzed. The mean percentage of positive cells
for each chemokine receptor plus or minus the standard deviation is
summarized in Table 1. Histograms from a representative donor are
presented.
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Chemokine receptor expression in in vitro expanded hematopoietic
lineages.
To further examine the apparent lineage specificity of chemokine
receptor expression in hematopoietic subsets and to control for
uncharacterized factors in serum that might unduly affect chemokine
receptor expression, we sought to determine chemokine receptor
expression in erythroid, megakaryocyte, and granulo-macrophage cells
expanded under serum-free conditions21,22 in liquid
culture. Table 2 summarizes the expression
of CCR5, CXCR4, and CCR2 on liquid cultured ex vivo expanded BFU-E-,
CFU-Meg-, and CFU-GM-derived cells, as well as on mature erythrocytes
and platelets. CCR5 and CXCR4 were both present on CFU-Meg- and
CFU-GM-derived cells but were absent on BFU-E-derived cells. In
contrast, CCR2 was predominantly present on erythroid cells. Thus, the
pattern of chemokine receptor expression reflects some lineage
specificity, with CCR2 restricted to the erythroid lineage cells and
CCR5 and CXCR4 restricted to megakaryocytic and granulo-macrophage
lineage cells.
Chemokine receptor expression on CD34+ BMMNC.
Because ligands to CXCR4, CCR5, and CCR2 have been reported to have
effects on hematopoiesis, we next tried to determine if they were
expressed on the surface of CD34+ BMMNC. Dual-color flow
cytometric analysis of CD34+ BMMNC showed that, whereas
greater than 50% of CD34+ cells were positive for CXCR4
(Fig 3B), less than 5% of
CD34+ cells were positive for CCR2 (Fig 3D). At the same
time, CCR5 was not present on CD34+ cells (Fig 3C).
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| Fig 3.
FACS analysis of chemokine receptor expression on
CD34+ cells using MoAbs or biotinylated ligands. BMMNC
were costained with an FITC-conjugated MoAb to CD34 and MoAbs to CXCR4
(B), CCR5 (C), and CCR2 (D), followed by PE-conjugated antimouse IgG or
streptavidin-PE as described. Alternatively, cells stained with
PE-conjugated anti-CD34 MoAb were also costained with biotinylated
MIP-1 (E) or MCP-1 (F), followed by avidin-FITC. The forward versus
side-scatter characteristics of the gated population, R1, is shown (A).
Negative gates were drawn according to the threshold seen with either
the isotype-matched negative controls or in the case of the
biotinylated ligands, after the addition of neutralizing antichemokine
antibodies provided by the manufacturer (R&D Systems).
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Although MoAbs to CCR2, CCR3, CCR5, and CXCR4 are available, antibodies
to many other chemokine receptors have not yet been developed.
Therefore, we used the biotinylated chemokines MIP-1 and MCP-1 as
probes to determine if additional chemokine receptors are expressed on
CD34+ cells. MIP-1 binds to CCR1 and CCR4 in addition to
CCR5, whereas MCP-1 also binds to CCR4 and CCR1 in addition to
CCR2.27,28 Because CCR5 was not detectable on
CD34+ cells and CCR2 was only detectable on less than 5%
of the cells, binding to MIP-1 or MCP-1 would indicate the presence
of either CCR1 or CCR4. Therefore, BMMNC were bound to biotinylated
MIP-1 and MCP-1 and costained with an anti-CD34 MoAb. Dual-color
FACS analysis showed that close to 50% of CD34+ cells were
also positive for MIP-1 and MCP-1 receptors (Fig 3E and F). This
binding was specific, because coincubation with neutralizing
antichemokine antibodies abolished all specific binding activity (data
not shown). There was also a distinct CD34
population of cells positive for MIP-1 and MCP-1 receptors, consistent with the CCR5+/CD34 and
CCR2+/CD34 populations seen in Fig 3C
and D. These binding data suggest that CCR1 and/or CCR4 must be
present in significant amounts on CD34+ cells, although it
is possible that as yet uncharacterized receptors to MIP-1 and MCP-1
may account for these data.
CXCR4+ cells are enriched in clonogeneic
hematopoietic progenitors.
Because greater than 50% of human CD34+ cells coexpress
CXCR4, we were interested if CXCR4 is expressed not only on
CD34+ cells, but also on the clonogeneic human HPC. This
issue is particularly germane, because mice lacking the SDF-1 gene, the
natural ligand for CXCR4, appear to have severe defects in B-cell
lymphopoiesis and bone marrow myelopoiesis.29 To address
this issue, the CXCR4+ cells were isolated by using
immunomagnetic beads as described in Materials and Methods. Immediately
after isolation, CXCR4+ cells were plated in serum-free
methylcellulose cloning medium and stimulated to grow CFU-Mix, BFU-E,
CFU-GM, and CFU-Meg colonies by adding the appropriate cytokine
cocktail. We found that human bone marrow CXCR4+ cells were
clonogeneic and contain hematopoietic progenitors belonging to all
major hematopoietic lineages (data not shown).
To further evaluate the distribution of clonogeneic HPC between
CD34+/CXCR4+ and
CD34+/CXCR4 cells, we FACS-sorted
CD34+/CXCR4+ and
CD34+/CXCR4 cells from nonadherent
T-cell-depleted BMMNC (Fig 3B). Both fractions of cells were
subsequently plated serum-free in methylcellulose cultures and
stimulated to grow CFU-Mix, BFU-E, CFU-GM, and CFU-Meg colonies. We
found that both fractions of CD34+ cells, positive or
negative for CXCR4, contained hematopoietic progenitors belonging to
the mixed, erythroid, myeloid, and megakaryocytic lineage
(Table 3). Therefore, we conclude that
human HPC were distributed equally in both
CXCR4+CD34+ and
CXCR4CD34+ cells and that lack of CXCR4
in amounts that will allow for their isolation does not restrict the
clonogeneic potential of HPC, at least not in the in vitro assays used.
Expression of CXCR4 mRNA in early human hematopoietic cells.
Our results clearly demonstrate that CXCR4 is expressed on the surface
of CD34+ cells from human BMMNC. Moreover,
CXCR4+ cells isolated from BMMNC have clonogeneic potential
as they grow in vitro colonies belonging to all hematopoietic lineages. Therefore, we tried to determine if CXCR4 is expressed on the earliest
human HSC. To address this issue, we isolated human
CD34+c-kitR+Rh123low cells, which
we have previously demonstrated to be highly enriched in
HSC19 and CD34+, c-kitR+,
Rh123bright cells that are enriched in HPC.19
The mRNA was extracted from both populations of cells, and CXCR4 mRNA
expression was analyzed by RT-PCR. As shown in
Fig 4, both populations of cells enriched in either HSC
(CD34+c-kitR+Rh123low) or in HPC
(CD34+c-kitR+Rh123bright) expressed
mRNA encoding for CXCR4. To determine if HSC also harbor CD4, the
primary receptor for HIV-1 entry, we also performed RT-PCR analysis for
CD4 mRNA. However, we were unable to demonstrate the expression of CD4
mRNA in HSC (data not shown).
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| Fig 4.
RT-PCR analysis of CXCR4 mRNA expression in candidate
HSC. CD34+, c-kit+, Rh123dull
(lanes 1 and 2) and CD34+, c-kit+,
Rh123bright (lanes 3 and 4) cells were FACS-sorted as
described and subjected to RT-PCR analysis for CXCR4 mRNA. Negative
control reaction (no template) is shown in lane 5. Specificity of the
PCR products shown was confirmed by Southern blotting (data not shown)
.
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Megakaryocytic cells are infectable by X4 and myeloid cells by R5
viruses.
Whereas BFU-E-, CFU-Meg-, and CFU-GM-derived cells all expressed one
or more HIV-1 coreceptors, virus infection would be expected to occur
only if CD4 were also expressed. Therefore, we also phenotyped these
cells for CD4 antigen expression. CD4 was barely present on
BFU-E-derived cells (Fig 5F) but was
substantively present on CFU-Meg-derived (64% ± 9%) and
CFU-GM-derived (47% ± 10%) cells (Fig 5D and B), respectively.
Because megakaryocytic cells and granulo-macrophage cells cloned under
serum-free conditions appear to have CD4 and both of the major HIV-1
coreceptors, we next tried to determine if these cells were indeed
infectable by either R5 (M-tropic) or X4 (T-tropic) viruses. Classical
viral infection assays rely on culturing virus-innoculated cells for up
to 2 weeks and measuring levels of viral p24 or RT activity in the
culture supernatant as evidence for a productive viral infection.
However, culturing in vitro expanded hematopoietic colony cells even
under serum-free conditions for such a long period may lead to changes in cellular phenotype unaccounted for their initial characterization, particularly if HIV-1 infection itself can lead to the secretion of
proinflammatory and hematopoietic cytokines.14 Therefore, to determine if these megakaryocytic cells were permissive for viral
replication at the time of our characterization of its cellular phenotype, we infected CFU-Meg-derived cells with pseudotyped luciferase reporter viruses. The luciferase reporter virus consists of
the NL4-3 provirus with a frame-shift mutation, its env gene rendering it replication incompetent, and a luciferase gene inserted into its nef coding region.25,26 Because this
provirus does not have a functional Env of its own, it can be
pseudotyped by cotransfecting the proviral backbone with a plasmid
coding for any viral Env of interest into the appropriate packaging
cells. Viruses thus produced will be capable of a single-cycle
infection and if infection proceeds to the point of viral integration
and LTR-transcription, luciferase will be produced and productively infected cells can be assayed for luciferase activity. This reporter virus system has been widely used to measure the ability of various cell types to support virus entry and integration.26,30
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| Fig 5.
Expression of CD4 on serum-free expanded hematopoietic
progenitor-derived cells. CFU-GM (A and B), CFU-Meg (C and D), and
BFU-E (E and F) progenitors were serum-free expanded from
CD34+ BMMNC as described and FACS analyzed for CD4
antigen expression. Histograms (B, D, and F) represent the gated
populations as indicated (A, C, and E). The isotype negative controls
are overlaid (bold line), and M1 represents the positive populations.
Data from at least 3 different donors were analyzed. The mean
percentage of positive cells for each chemokine receptor plus or minus
the standard deviation is summarized in the test. Histograms from a
representative donor are presented.
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Somewhat surprisingly, we found that megakaryocytic cells were
infectable by X4 (HxB, NL4-3) and not R5 (ADA, JRFL) viruses, whereas
granulo-macrophage cells were infectable by R5 (ADA, JRFL) but not X4
(HxB, NL4-3) viruses, despite the fact that cells from both lineages
express both of the HIV-1 coreceptors (Fig
6A). To show that viral entry into these cells was indeed mediated by
CD4 and the cognate coreceptor, reporter virus infection was performed
in the presence of Leu3A (an anti-CD4 antibody) or antibodies to either
CXCR4 (12G5, R&D701, 702,708) or CCR5 (R&D 531). As can be seen in Fig
6B and C, Leu3A was highly effective in neutralizing viral entry (as
measured by luciferase production), and both anti-CXCR4 and anti-CCR5
antibodies were variously effective in blocking cognate viral entry.
The differential susceptibility of the NL4-3 or HxB Env to
CXCR4-specific MoAb or SDF-1 inhibition is consistent with reports in
the literature showing that inhibition of CXCR4-mediated entry by
either anti-CXCR4 MoAb or SDF-1 is highly strain
specific.31,32 This indicates that viral entry into
megakaryocytic cells was CD4 and CXCR4-dependent and that entry into
granulo-macrophage cells was CD4 and CCR5-dependent. By contrast,
BFU-E-derived cells were not infectable by either R5 or X4 viruses,
consistent with our failure to detect CXCR4 or CCR5 in this cell
population. However, these erythroid cells were readily infectable by
viruses bearing the amphotorpic MLV Env protein, indicating that the
block to infection by R5 and X4 viruses was at the level of viral entry (Fig 6A). Infection with pseudotyped GFP reporter viruses confirmed that only CD41+ cells in CFU-Meg-derived cells are
infeactable by X4-env pseudotyped viruses (data not shown). These
results in toto indicate that megakaryocytic cells were infectable by
X4 viruses and that infection of these cells was mediated through CD4
and CXCR4.
View larger version (48K):
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[in a new window]
| Fig 6.
Infection of lineage-specific hematopoietic cells with
pseudotyped reporter viruses. (A) Approximately 2 × 105
megakaryocytic (Meg colony), erythroid (Ery colony), and
granulocyte-macrophage (GM colony) cells were infected with X4 or R5
Env pseudotyped viruses as indicated. Four days after infection, cells
were lysed and analyzed for luciferase activity (RLU). The amphotropic
MLV Env pseudotyped virus was used to control for cell viability.
Megakaryocytic (B) and granulo-macrophage cells (C) were infected with
either two different X4 Env pseudotyped viruses or a prototypic R5 Env
pseudotyped virus, respectively, in the presence or absence of blocking
agents. Leu3A is an MoAb against CD4 that recognizes the HIV Env
binding epitope on CD4; SDF-1 is the natural ligand for CXCR4; R&D 701, 702, 708, and 12G5 are MoAbs against CXCR4; and R&D 531 is an MoAb
against CCR5. The RLU obtained in the presence of blocking agents is
normalized to the RLU obtained without any blocking agents, and the
data for infection efficiency are presented as the percentage of
unblocked control. Note that none of the blocking agents had any effect
on the entry of the MLV pseudotyped virus, indicating the specificity
of any blocking effects. All infection and blocking experiments were
repeated 2 to 3 independent times with different donors with similar
results. Representative experiments are shown.
|
|
Upregulation of CXCR4 expression in human BMMNC after
-IFN treatment.
Because different proinflammatory cytokines (IL-1, TNF-, TNF- ,
-IFN, -IFN, and -IFN) secreted during chronic infections have
been reported to either induce or suppress HIV infection in various
cell types, we evaluated if these cytokines were able to modulate the
expression of HIV-1 coreceptors (CXCR4, CCR5, and CD4) on human BMMNC.
To address this issue, BMMNC were resuspended in serum-free medium and
stimulated for 36 hours with different proinflammatory cytokines.
Subsequently, we evaluated changes in CXCR4, CCR5, and CD4 expression
by FACS. As can be seen in Fig 7, of all
the proinflammatory cytokines tested, only -IFN increased expression
of CXCR4. In three independent experiments, -IFN increased the
number of CXCR4-expressing cells by approximately 20% to 30% of total
BMMNC. However, this upregulation was not a global effect on all CXCR4
expressing cells. When the BMMNC were gated based on their forward
versus side scatter characteristics, most of the CXCR4 upregulation
occurred in the granulocyte precursor and monocyte gates (data not
shown). In the parallel experiments, none of the proinflammatory
cytokines (IL-1, TNF-, TNF-, -IFN, -IFN, and -IFN)
evaluated had any effect on the expression of CCR5 or CD4 (not shown).
These results suggest that -IFN released during the course of a
chronic infection may affect the susceptibility of certain BMMNC to X4
virus infection.
View larger version (40K):
[in this window]
[in a new window]
| Fig 7.
Regulation of CXCR4 expression by -IFN. Freshly
isolated BMMNC in serum-free media were either left alone or treated
with a variety of proinflammatory cytokines as indicated for 36 hours.
Expression of CXCR4 was monitored by FACS analysis after the treatment
period. The negative isotype control is overlaid on each histogram. A
representative experiment is shown of three independent repeats with
similar results. M1 represents the positive population; the percentage
of positive cells is indicated within each histogram.
|
|
| |
DISCUSSION |
The pathogenesis of HIV-1-associated hematopoietic dysfunction has
been a subject of intense investigation and considerable debate. It is
likely that no one mechanism can account for the spectrum of
hematological abnormalities seen in AIDS. The confluence of
experimental results thus far seem to implicate the ability of virus
infection or viral gene products to disrupt the hematoregulatory function of bone marrow auxiliary cells (reviewed in Moses et al14). However, the recent discovery of certain chemokine
receptors such as HIV-1 coreceptors coupled with the reported ability
of cognate chemokine receptor ligands such as MIP-1 and SDF-1 to modulate hematopoietic development29,33,34 has opened a new arena of investigative opportunities regarding the pathogenesis of
AIDS-related cytopenias.
Cellular infection by HIV-1 requires the presence of CD4 and at least
one additional coreceptor. Accordingly, R5 viruses require CCR5 and X4
viruses require CXCR4 in addition to CD4 for cellular entry.35 Because chemokine receptors may mediate some of
the negative influences of the chemokines on the clonogeneic growth of
early hematopoietic cells3 and both HIV-1 Env and the
ligands to HIV-1 coreceptors can be secreted in excess during HIV-1
infection, deciphering the chemokine/chemokine receptor axes in
hematopoietic cells will allow a first approximation as to which
hematopoietic subsets might be susceptible to detrimental effects of
direct viral infection or Env-mediated cytotoxicity as well as
chemokine-mediated hematodysregulation.
In this report, we have examined cell surface expression of the major
HIV-1 coreceptors, CCR5 and CXCR4, on various subsets of hematopoietic
cells. Although there was pleiotropic expression of these receptors as
well as CCR2b to varying degrees on the cells from the lymphocyte,
monocyte, and granulocyte gates in total BMMNC, chemokine receptor
expression appeared more lineage restricted when examined on cells from
serum-free expanded hematopoietic progenitors. CCR5 and CXCR4 were both
expressed on cells expanded from CFU-GM and CFU-Meg, whereas CCR2 was
predominantly expressed on BFU-E-derived cells. Because we found that
CD4 is also expressed on both myeloid and megakaryocytic cells, it was
surprising that myeloid cells were only infectable by R5 Env
pseudotyped viruses and megakaryocytic cells were only infectable by X4
Env pseudotyped viruses. This finding implies that the physical
presence of the appropriate receptors and coreceptors on the cell
surface does not necessarily ensure a productive infection. The
infectability of megakaryocytic cells by X4 but not R5 viruses has been
reported recently.36 Our results confirm and extend these
findings by characterizing the coreceptors responsible for infection of
both CFU-Meg and CFU-GM derived cells. The apparent discrepancy between the expression of CD4 and the appropriate coreceptor and the
restrictive tropism of certain primary cells has precedence in the
HIV-1 infection of macrophages. It is becoming increasingly clear that,
although both CCR5 and CXCR4 are expressed on macrophages, only R5
viruses can productively replicate in these cells.37,38
However, certain R5/X4 viruses can productively infect CCR5-deficient
macrophages via CXCR4.37 Whether this restriction of
tropism is due to the affinity of the particular Env for the coreceptor
in question, the CD4/coreceptor ratio required for productive membrane
fusion,39,40 or postentry determinants in the cellular
milieu of the target cell remains to be determined.7,8,41
However, the restrictive tropism of CFU-Meg and CFU-GM cells offers an
additional model in which to sort out the effects that determine viral
entry and replication. Furthermore, the susceptibility of
CFU-Meg-derived cells to X4 virus infection supports the notion that
HIV-1-related thrombocytopenia may be partially explained by the
cytopathic effects resulting from direct infection of megakaryocytic
precursors. To our knowledge, this is also the first demonstration that
erythroid cells are resistant to infection with R5 and X4 viruses. This could be explained by our findings that, although erythroid precursor cells express low levels of CD4, they did not express CXCR4 or CCR5.
Therefore, direct infection of erythroid precursor cells probably does
not play a major role in the pathogenesis of HIV-related anemia.
It has also been reported recently that CD34+ cells express
mRNA for CXCR4 and, to a lesser degree, CCR5.13 In this
report, we characterized the expression of chemokine receptors on
CD34+ cells at the protein level and the expression of a
variety of other chemokine receptors on CD34+ BMMNC. We
found that CD34+ BMMNC express CXCR4 but not CCR5, CCR3, or
CCR2 proteins. It is also significant that we not only demonstrated
cell surface expression of CXCR4 on hematopoietic progenitor cells
(CD34+), but also that FACS-sorted
CD34+/CXCR4+ cells were clonogeneic and capable
of giving rise to all major hematopoietic lineages (CFU-mix, CFU-GM,
CFU-Meg, and BFU-E). Interestingly, the clonogeneic potential of
hematopoietic precursor cells did not appear to be limited to
CXCR4+ cells, because
CD34+/CXCR4 cells were also capable of
giving rise to multilineage colony formation. This finding implies that
CXCR4 may not be a sensitive selection marker for all hematopoietic
progenitors. However, in vitro colony-forming assays are only a
surrogate for true stem-like regenerative capacity. It remains to be
seen if CD34+/CXCR4+ and
CD34+/CXCR4 cells possess true stem-like
clonogeneic potential by a more stringent test such as SCID-mice
repopulation. The potential presence of CXCR4 on human HSC is supported
by the fact that we could detect CXCR4 mRNA by RT-PCR in
CD34+ Kit+ Rh123low cells that are
highly enriched in human hematopoietic stem cells.19 Interestingly, our failure to detect expression of CD4 mRNA in the same
population of cells could explain why human HSC are resistant to
infection by HIV.14,42,43 Nevertheless, the expresssion of
CXCR4 on candidate human stem cells as well as on a variety of
clonogeneic human progenitor cells has implications for lentiviral gene
therapy, because there are HIV-1 and HIV-2 viruses that can use CXCR4
for entry independent of CD4.44-46 Therefore, pseudotyping lentiviral vectors with these CXCR4-dependent, CD4-independent Envs may
provide a way of specifically targeting therapeutic genes to
hematopoietic stem and progenitor cells.
We also found that, although CD34+ cells were negative for
CCR5 and CCR2 by MoAb staining, they were clearly positive for other MIP-1 and MCP-1 receptors as shown by FACS analysis with
biotinylated ligands. Because MIP-1 and MCP-1 are also known ligands
for CCR1 and CCR4,28 these results imply that CCR1
and/or CCR4 are also present on CD34+ cells. CCR1
and CCR4 mRNA have recently been reported to be expressed in
CD34+ cells and the inhibitory effects of MIP-1 on
erythropoiesis has been shown to be mediated through
CCR1.47 This study shows that CCR1 and/or CCR4 on
human CD34+ cells can indeed bind to their respective
ligands. Considering that cognate ligands to many of the chemokine
receptors examined are secreted in excess during chronic HIV
infection,48-50 the delineation of chemokine receptor
expression on various subsets of hematopoietic progenitors represents a
first step towards teasing apart the intricate network of relationships
between chemokine receptors, HIV infection, and
hematopoiesis.3
We also tested the hypothesis that some of the proinflammatory
cytokines released during chronic infections may modulate the course of
HIV infection by augmenting the expression of particular chemokine
coreceptors on the surface of hematopoietic cells. Our finding that
-IFN can upregulate the expression of CXCR4 underscores the
interplay between cytokine release during chronic HIV infection and the
chemokine/chemokine receptor axes. -IFN is greatly increased in
lymphoid tissues during HIV-1 infection,51 and other
cytokines such as GM-CSF and IL-10 have been shown to decrease or
increase the expression of CCR5, respectively.52,53 Thus,
cytokine-mediated modulation of coreceptor expression may play a role
in the dynamics of HIV replication in vivo.
In conclusion, we have determined the pattern of CD4 and major HIV-1
coreceptor expression on a variety of HPC and correlated this with
their susceptibility to HIV-1 infection. We found that productive
infection of cells is likely more complicated than the mere physical
presence of CD4 and coreceptor on the cell surface. Finally, we also
determined that -IFN can upregulate the expression of CXCR4 on
BMMNC. The results presented represent a guide towards future
investigations into the biological consequences of chemokine receptor
expression on hematopoietic cells and offer an initial framework in
which to sort out the web-like complexity between the myriad cytokine
and chemokine networks that may impinge upon the dynamics of HIV-1 replication.
| |
ACKNOWLEDGMENT |
The authors thank Monica Tsang (R&D Systems) and Carlos Martines-A.
(Universidad Autonoma de Madrid) for generously providing the chemokine
receptor antibodies.
| |
FOOTNOTES |
Submitted June 9, 1998; accepted October 12, 1998.
B.L. was supported by the Measey Foundation Fellowship for Clinicians
(Wistar Institue). R.W.D. was supported by National Institutes of
Health (NIH) Grant No. AI-40880. A.M.G. and M.Z.R. were supported by a
program project grant in stem cell biology NIH PO1 DK52558-01A1, NIH
R01 HL 61796-01.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Presented in part at the American Society of Hematology Meeting, San
Diego, CA, 1997 and published in abstract form in Blood
90:2144, 1997 (abstr, suppl 1).
Address reprint requests to Mariusz Z. Ratajczak, MD, PhD, Department
of Medicine, Hospital of the University of Pennsylvania, 1007 Stellar-Chance Laboratories, 422 Curie Blvd, Philadelphia, PA 19104.
| |
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Top
Abstract
Introduction