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Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 907-915
Neither Human Immunodeficiency Virus-1 (HIV-1) nor HIV-2 Infects
Most-Primitive Human Hematopoietic Stem Cells as Assessed in
Long-Term Bone Marrow Cultures
By
Frank F. Weichold,
Davide Zella,
Oxana Barabitskaja,
Jaroslaw P. Maciejewski,
Daniel E. Dunn,
Elaine M. Sloand, and
Neal S. Young
From the Institute of Human Virology, University of Maryland,
Baltimore; the Department of Internal Medicine, University of Nevada,
School of Medicine, Reno; and the Hematology Branch, National Heart,
Lung and Blood Institute, Bethesda, MD.
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ABSTRACT |
Attempts to clarify the pathophysiology of human immunodeficiency
virus (HIV)-mediated bone marrow (BM) dysfunction have yielded inconsistent results regarding the susceptibility of BM progenitors to
the viral infection. To specifically address this question, we exposed
highly purified subpopulations of human BM progenitor cells to various
HIV-1 and HIV-2 strains and assessed (pro)viral gene presence and
expression in more-committed (CD34+CD38+)
as well as most-primitive (CD34+CD38 )
cells in long-term BM cultures. Quantitative analysis of long-term culture-initiating cells (LTCIC) failed to demonstrate adverse effects
of exposing hematopoietic stem cells to HIV. Our results show that
HIV-2, similar to HIV-1, does not infect hematopoietic stem cells in
vitro with any significant frequency and infected cells are not present
within LTCICs. Cytofluorometric analysis of CD34+ cells
for surface molecules that facilitate HIV entry was consistent with the
functional assay in that expression of virus receptors was
predominantly on the more-committed subsets of BM progenitors. The
failure to detect productive or latent HIV in the most-primitive human
BM progenitor and stem cells has important implications for future
therapeutic strategies, including those dealing with transduction of
these cells with protective genes as a treatment modality for AIDS.
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INTRODUCTION |
A;4 LARGE NUMBER of studies have been
conducted to identify and characterize the pathophysiologic mechanisms
leading to bone marrow (BM) dysfunction in patients with acquired
immunodeficiency syndrome (AIDS).1-3 Understanding of these
mechanisms is essential not only for the management of hematologic
complications in AIDS but also for successful disease intervention,
since T-cell depletion in AIDS is thought to be, at least in part, due
to the failure of T-cell development from lymphohematopoietic stem
cells. While the results of experiments performed with patient-derived
material can be difficult to interpret due to the multifactorial nature of the hematopoietic failure in AIDS,4-13 experiments using
ex vivo infected normal BM cells9,14-19 may not properly
reflect the pathophysiology operating during disease progression.
Generally, the results of all these experiments with regard to both
ability of human immunodeficiency virus (HIV) to directly inhibit
hematopoiesis7,9-16,19 as well as to the susceptibility of
hematopoietic progenitors to HIV infection,4-9,15,17,18
have not been uniform and are often contradicting.
Our previous in vitro studies have demonstrated that indirect
mechanisms involving release of tumor necrosis factor (TNF- ) from
accessory cells in response to HIV-1 envelope proteins could account
for hematopoietic suppression observed in vivo.20
This and other studies failed to show, however, a direct inhibitory effect of HIV-1 in vitro.1,8,9,21 Since there is no doubt that the therapy-related side-effects, as well as secondary
pathophysiologic mechanisms (eg, cytokine disturbances, cachexia, and
opportunistic infections) can impair BM function,22 the
direct infection of immature hematopoietic cells need not necessarily
be postulated to account for the hematopoietic suppression in
AIDS.20,23,24-27 In several studies, the infection of
phenotypically defined populations of hematopoietic progenitor cells
was assessed,4-9,16,18-21,28 and most of them suggest that
HIV does not infect a significant portion of progenitor
cells.4-5,7-9,28 However, the susceptibility of
hematopoietic stem cells to HIV infection is still unclear. Resolution
of this central issue could shed light on the pathophysiologic significance of direct virus-mediated cell depletion and the reservoir function of infected hematopoietic cells for the latent virus. Theoretically, integration of proviral DNA into stem cell genomes could
lead to the spread of HIV infection through the expansion of infected
clones, or conversely, it could interfere with normal stem cell
maturation and proliferation resulting in the interruption of normal
hematopoiesis.
Bone marrow long-term cultures (BMLTC) and long-term culture-initiating
cell (LTCIC) assays offer an opportunity to study human hematopoiesis
and quantify the most-immature human hematopoietic cells amenable to in
vitro assessment. In BMLTC, the immature hematopoietic progenitor cells
are selected over a period of 5 weeks. These cells may subsequently be
evaluated for their ability to initiate and form colonies in secondary
methylcellulose assays.29-32 The number of colony-forming
cells (CFC) can serve as a measure of LTCIC, the best estimate of
hematopoietic stem cells in vitro.29-31 Although some
strains of HIV-215 and to a lesser degree monocytotropic strains of HIV-1,15,19,33,34 were shown to affect the
function and number of LTCIC, the question about the susceptibility of LTCIC to HIV infection has not been systematically studied. Therefore, we investigated the effects of HIV-1 and HIV-2 on the growth of most-primitive human hematopoietic progenitor cells in BMLTC and determined under stringent conditions the ability of HIV to replicate or latently persist in these cells.
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MATERIALS AND METHODS |
Bone marrow cell preparation.
BM was obtained from healthy donors (age 26 to 42 years) by aspiration
from the posterior iliac crest into syringes containing media
supplemented 1:10 with heparin (O'Neill and Feldman, St Louis, MO).
Informed consent was obtained according to a protocol approved by the
Institutional Review Board of the National Heart, Lung and Blood
Institute. Mononuclear BM cells (BM MNC) were isolated by density
gradient centrifugation using lymphocyte separation medium (Organon,
Durham, NC). After washing in Hank's balanced salt solution (HBSS;
GIBCO-BRL/Life Technologies, Gaithersburg, MD), cells were resuspended
in Iscove's modified Dulbecco's medium (IMDM; GIBCO) supplemented
with 20% fetal calf serum (FCS; heat inactivated, Life Technologies).
Separation of CD34+ cells and flow cytometry.
After washing with phosphate buffered saline (PBS) supplemented with
2% human albumin and labeling with anti-CD34 monoclonal antibody
(MoAb), BM cells were passed through an affinity column (Cellpro,
Bothell, WA) for selection, and the CD34+ cell fraction was
eluted with PBS. An aliquot of eluted cells was stained with
phycoerythrin (PE)-conjugated anti-CD34 HPCA-2 MoAb (Becton Dickinson,
Mountain View, CA) to assess the purity, which usually ranged from 80%
to 94%. For higher purity preparations, cells were further
fractionated: column-purified cells were stained with fluorescein
isothiocyanate-labeled (FITC) anti-CD34+ MoAb, washed with
PBS, and sorted by microcytofluorometry (Epics V; Coulter, Hialeah,
FL). The purity of cells prepared by combining affinity chromatography
and flow cytometry was 97% to 99%. To obtain the most-immature BM
progenitor cells from the CD34+ population, cells were
stained with anti-CD38 MoAb (PE-labeled, Becton Dickinson) and the
negative gate was collected by cytofluorometry.
In several experiments, more detailed analysis of BM cells was
performed using FITC-conjugated HPCA-1 MoAb directed against human CD34
antigen (Becton Dickinson), PE-conjugated anti-CD38 MoAb, and
PerCP-conjugated anti-CD4 or anti-CD14 MoAb (Becton Dickinson). For
analysis of chemokine receptor molecules on
CD34+CD38 cells, mouse MoAb to CXCR-4 (12G5,
mIgG2a) and CCR-5 (12D1, mIgG2a; both MoAbs
obtained from the AIDS Research and Reference Reagent Program,
Rockville, MD) were detected with an antimouse-biotin/streptavidin-PE system (DAKO A/S, Denmark, and Molecular Probes). In all flow cytometry
experiments, appropriate isotype MoAb conjugate controls were used to
determine the levels of background fluorescence.
Long-term bone marrow cultures.
BMLTC and the determinations of the number of LTCIC were performed
according to a modification of described methods.29-32
Approximately 107 BM MNC were used to initiate stromal
cultures. Allogeneic BM cells were grown until they formed a confluent
stromal layer. Culture media consisted of stem cell media (MyeloCult
H5100, Stem Cell Technologies, Vancouver, BC, Canada) supplemented with
106 mol/L hydrocortisone succinate (Sigma, St Louis,
MO), 4 mmol/L L-glutamine, 50 U/mL penicillin, 50 µg/mL streptomycin
(all Mediatech Inc, Herndon, VA), and the media was renewed to 50%
twice a week. After approximately 3 weeks of culture, stromal cells
were trypsinized, washed, and placed in 48 well plates. After
reestablishment of a confluent cellular layer the plates were
irradiated (15 Gy of 250-KV x-rays) to prevent further proliferation of
the feeder cells.29 Subsequently to their viral exposure,
total BM MNC, isolated CD34+ cells, or sorted
CD34+CD38 cells were added to preestablished
irradiated stomal feeder layers in duplicate (for each treatment
regimen), and cultured for 5 weeks at 35°C in humidified atmosphere,
containing 5% CO2. Every 2 days, 50% of the stem cell
culture media (as described above, without hydrocortisone) was replaced
by fresh media containing stem cell factor (rhSCF, 50 ng/mL, Amgen,
Thousand Oaks, CA), rhlL-3 (20 ng/mL, Genzyme, Boston, MA), and rhLIF
(20 ng/mL, Biosource International, Camarillo, CA). Starting 1 week
after BM cell addition, the number of secondary colonies generated from
nonadherent cell fractions of BMLTC was analyzed weekly in triplicate
methylcellulose cultures (secondary cultures, performed as described
below). After 5 weeks, the remaining cells in BMLTC were harvested and
replated in methylcellulose so as to assess the numbers of LTCIC (cells able to form secondary colonies after 5 weeks of BMLTC). Cells and
supernatants derived from these cultures were used for determining contents of viral antigen (p24, p27) and proviral DNA (HIV-1 and HIV-2
gag).
Secondary hematopoietic methylcellulose cultures.
Hematopoietic CFC in BMLTC were measured in methylcellulose cultures
under standard conditions. BMLTC derived cells were plated in
methylcellulose (MethoCult H4100, Stem Cell Technologies) in the
presence of 50 ng/mL IL-3 (Genzyme), 20 ng/mL granulocyte-macrophage colony stimulating factor (GM-CSF; Boehringer, Indianapolis, IN), 50 ng/mL stem cell factor (SCF), and 2 U/mL erythropoetin (EPO, both
Amgen). After 2 weeks, colonies formed were microscopically evaluated
for quality and enumerated using a grid.
Tertiary suspension cultures.
In some experiments, 14-day colonies from methylcellulose cultures were
dispersed, washed twice in PBS, resuspended in IMDM supplemented with
FCS (both GIBCO-BRL) and growth factors (at concentrations used for
secondary methylcellulose cultures) and cultured for an additional 14 days. Cells and supernatants derived from these cultures were used for
proviral DNA analysis by PCR and viral antigen (p24, p27) measurement
by ELISA, respectively.
Virus and recombinant viral proteins.
Viral stocks of HIV-1 strains BAL and RF, or HIV-2 strains ST and ROD
were propagated in primary human cells20,35 and cell free
supernatants were used for BM infection experiments. Two primary
isolates of HIV-1 (passage 9 of isolate MN, passage 3 of isolate 571),
which were included in some experiments, were isolated in our
laboratory and propagated exclusively in PBMNC as
described.20,36 All virus stocks and defined lots were
individually titered for infectivity in PHA-P activated PBMNC cultures
under standardized conditions.35 Virus incubations were
carried out with the viral dose of 50 × tissue culture infectious
dose 50 (TCID50), corresponding approximately to 2 × 105
cpm/mL of reverse transcriptase activity, for 2 hours at 37°C. For
control experiments, mock-virus stocks were prepared by irradiation (20 Gy of 250-KV x-rays) or heat-inactivation for 30 minutes at 59°C. To
account for potential transfer of bio-active molecules such as
cytokines, chemokines, and growth factors in supernatants, additional
controls were generated by exposing peripheral blood (PB) derived MNC
fractions to a variety of stimuli. Cells were cultured in IMDM
(GIBCO-BRL) containing 10% human AB serum (ABI, Cambridge, MA), and
treated with LPS (2 µg/mL; Sigma) or with IL-4 (0.1 µg/mL) and
GM-CSF (50 mg/mL), or with rIFN- (0.1 µg/mL, all Boehringer) for 3 days. Culture media was changed on day 3 and cell free supernatants
were collected on day 6 and stored in aliquots at  80°C until used.
Incubations of BM-derived cell fractions with the differently
conditioned media were carried out, similar to viral exposure, for 2 hours at 37°C. Commercially available baculovirus derived HIV-1 gp120
(ABI, Cambridge, MA; 5 µg/mL) was added to some control cultures at
the concentration of 2 µg/mL. After exposure, cells were thoroughly
washed three times with PBS, enumerated by using the trypan blue
exclusion method, and placed into cultures.
Semiquantitative PCR.
To determine the amount of HIV-DNA in the infected cells, a
quantitative PCR36 was performed using primers
complementary to the gag region of HIV-1.37 Plasmid
pAC38/93 was used as the internal control.38
Both HIV-1 gag sequences were amplified by the same set of
primers (SK38 and SK39) and the products were differentiated by size
and hybridization to specific probes.37 For quantification,
serial dilutions of the positive standard were amplified in parallel
with test samples (previously normalized for total DNA using  -globin
gene amplification)36 in the presence of 200 copies of the
internal control in order to normalize the results. The amplification
products were transferred onto nitrocellulose filters, hybridized with
5 -end P32-labeled specific probes in a slot blot format
and densitometrically quantified. The limit of detection using this
method was at a range of 20 HIV-1 gene copies per 2 × 105
cells (1 µg of total DNA). To amplify HIV-2 gag proviral DNA primers SK145 and SK431 (Perkin Elmer) were used in a semiquantitative PCR assay, applying similar reaction conditions as described above. The
amplification products (139 bp) were detected with a labeled probe
(SK102, Perkin Elmer) and the number of gene copies in test samples was
estimated based on a standard curve. Detection limit of the
semiquantitative assay was 40 HIV-2 gene copies per 2 × 105 cells.
ELISA.
For determination of p24 (HIV-1) and p27 (HIV-2) Coulter ELISA kits
(Coulter, Hialeah, FL) were used according to the protocols supplied.
Samples were stored at 80°C until assayed in duplicate determinations using a Bio-Rad microplate reader (Model 3550 Bio-Rad Laboratories, Hercules, CA) and manufacturer's software. The limit of
viral core protein detection for the p24 assay was 8 pg/mL and 14 pg/mL
for the p27 assay.
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RESULTS |
Effects of HIV-1 and -2 exposure on total bone marrow as assessed by
number of CFC and LTCIC in BMLTC.
The effects of HIV on the colony formation of committed progenitor
cells has been previously studied using primary methylcellulose assays.9-13,16,20,28,38 We investigated the long-term
effects of a short-term exposure to HIV-1 and HIV-2 by CFC-assays in
BMLTC. To minimize the viral effects on stroma formation and function, BM MNC fractions were exposed to HIV-1 and HIV-2 before being placed on
preformed irradiated allogeneic stromal layers. Excess of inoculated
virus was removed by repeated washing steps. When total BM cells were
used in the experiments, differences in the number of secondary CFC
were observed in the first weeks of the BMLTC (Table
1). Exposure to infectious HIV-1 (strains
RF, BaL) as well as HIV-2 (strains ST, Rod) decreased colony formation to 76% ± 10%, 72% ± 8%, 75% ± 6%, and 78% ± 8%,
respectively, as compared with controls (paired t-test,
P < .05). Mock virus generated by irradiation or heat
inactivated viral stocks had similar effects (72% ± 6% and
68% ± 6% of controls, respectively). Consistent with
our previous results,20 recombinant viral envelope protein
(gp120) alone had a potent inhibitory effect on CFC in the first 2 weeks of BMLTC. Conditioned medium generated from activated PBMNC
derived cultures, representing a control for potential transfer of
cytokines, chemokines, and growth factors from cell cultures used for
virus stock propagation, failed to mediate the suppressive effects on
CFC. During the further course of culture, beginning at week 3, the
number of colonies derived from BMLTC exposed to the various
inactivated (noninfectious) HIV preparations, including gp120,
increased moderately (mean 120% ± 5% of controls at weeks 4, P < .05). The number of CFCs obtained in experiments where
total BM was exposed to the different infectious viral preparations was
lower than in controls throughout the entire period of assessment up to
4 weeks. However, no significant differences in the number of LTCICs
were determined (Table 1).
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Table 1.
Effects of HIV-1 and HIV-2 Exposure on BM Cells,
Measured as the Number of Secondary CFC Derived From BMLTC
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Effects of HIV-1 and -2 exposure on CD34+ cell subsets
assessed by CFC and LTCIC in BMLTC.
To exclude the possible interaction of monocytes and lymphocytes
(contained in total BM preparations, susceptible to infection with
HIV-1 and HIV-2) with BM progenitor and stem cells, purified populations of BM-derived CD34+ cells were used in
subsequent experiments. Exposure of CD34+ cells to HIV-1 or
HIV-2, followed by long-term culture on preformed stromal layers,
revealed no inhibitory effects on secondary colony formation. Instead,
a moderate increase in CFC was noted after the first week of BMLTC
initiation. This effect was not seen with controls exposed to
conditioned medium. The number of secondary CFCs for weeks 2 to 5 were
unaffected by infectious virus or mock virus controls (Fig
1). Similar results were obtained when
previously described primary isolates of HIV-1, MN and
571,35,56 were used instead of laboratory strains (data not
shown).
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| Fig 1.
Secondary colonies formation as derived from LTBMC
initiated with purified CD34+ cells exposed to HIV-1 and
HIV-2. CFC are expressed as percentage of control cultures performed
with heat inactivated virus for each time point. All cultures were
performed in duplicate. The control CFC per 105 cells were
2,890 ± 310, 128 ± 22, and 26 ± 6 for 1, 3, and 5 weeks, respectively.
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To further analyze the viral influence on immature progenitor and stem
cell maintenance and development in vitro, CD34+
cells were subdivided based on the expression of CD38 antigen (Fig 2A,
B, and C).29-31 While the measurable effects on the more committed CD34+CD38+ BM cells were comparable
with those seen in cultures of total CD34+ cells, secondary
colony formation by the most-primitive,
CD34+CD38 cells exposed to HIV-1 and
HIV-2 was unaffected for the entire period of 5 weeks of
BMLTC (Table 2). All HIV strains tested showed similar activity.
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| Fig 2.
Flow cytometric analysis of CD34+,
CD34+CD38+, and
CD34+CD38 cell subsets used to initiate
BMLTC. (A) Scatter diagram of CD34+ selected cells
stained with FITC-labeled CD34 MoAb and PE-labeled CD38 MoAb, in which
Log PE fluorescence intensity was displayed versus Log FITC
fluorescence intensity. (B) CD34+ cells were gated and
sorted. (C) A scatter diagram of gated CD34+ cells sorted
for absence of the CD38 activation marker (most-primitive BM stem
cells). (D) The log PE fluorescence intensity (CD38) of an affinity
column purified CD34+ cell population was displayed
versus Log PerCP-fluorescence intensity (CD4). Results of one
representative experiment illustrated.
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Table 2.
Secondary Colony Formation in the Course of BMLTC
Initiated With Purified CD34+ Cells Exposed to HIV-1 and
HIV-2
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Lack of HIV-1 and -2 propagation in immature hematopoietic cells
capable of secondary colony formation after BMLTC.
Previous results suggested that neither HIV-1 nor HIV-2 directly exert
significant adverse effects on immature BM progenitor and stem cells as
measured in BMLTC. Theoretically, it is possible that HIV-1 or HIV-2
infect these cell types and persist in them or in the progeny generated
from these cells relatively inactive, without notably affecting their
growth in vitro. To investigate the possibility of a limited
HIV replication, we measured the levels of p24 (HIV-1) and p27 (HIV-2)
viral protein in BMLTC of purified CD34+ cells as well as
in those initiated by CD34+CD38 + and
CD34+CD38 cells. While no p24 or p27
production was detected in culture supernatants of most-immature
CD34+CD38 cells up to 6 weeks after viral
exposure, a low level of HIV core production was detected in the
cultures of CD34+CD38+ cells in the first 2 weeks of culture (Table 3). In comparison, levels of viral core protein in BMLTC initiated with total BM cells
increased over a period of 3 weeks, reaching p24 and p27 levels of 3.7 ± 1.4 ng/mL and 7.4 ± 5.1 ng/mL, respectively.
Since infections may persist in a "silent," nonproductive state
and since ELISA assays only detect expressed viral gene products, we
used a highly sensitive PCR technique to detect HIV-1 and HIV-2 proviral DNA in BMLTC initiated with CD34+CD38+
and CD34+CD38 cells (Table 4). HIV-1 and
HIV-2 DNA was detected in cultures of
CD34+CD38+ cells up to 3 weeks after infection.
However, in cultures of CD34+CD38 cells,
proviral DNA could only be detected immediately after viral exposure
and during the first week in BM-LTCIC, representing most likely a
residual contamination by the viral inoculum. No proviral DNA was
detectable at any later time point when cultures initiated with
CD34+CD38 were analyzed (Table
4). In contrast, HIV proviral DNA detected in cultures initiated with total BM reached levels of more than 105 copies per 106 cells.
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Table 4.
HIV Proviral DNA PCR Analysis in BM Cells Derived From
BMLTC Initiated With CD34+CD38+ and
CD34+CD38 Cells
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BM stem cells, measured as LTCIC in our experimental system, are
present only in a low number even within enriched
CD34+CD38 cell populations. To achieve the
highest sensitivity of detection of the infection of stem cells in
vitro, secondary 5-week colonies derived from LTCIC were harvested and
used for further analysis. PCR performed on the viable cell populations
failed to demonstrate the presence of HIV-1 or HIV-2 DNA. In attempts
to detect low levels of HIV infection, cells derived from secondary
colony formation assays initiated after 5 weeks of BM-LTCIC were
further subcultured in suspension (tertiary suspension cultures) to
achieve their complete differentiation. Neither HIV gene products as
assayed in p24 and p27 ELISA nor proviral DNA was found in these
cultures (data not shown). This demonstrates that all LTCIC-derived
cell cultures, independent from the initially used subpopulation, were not infected by HIV-1 or HIV-2.
Lack of HIV receptor molecules on hematopoietic progenitor and stem
cell subpopulations.
To estimate the potential influence of contaminating terminally
differentiated cells (macrophages and lymphocytes) on the results of
our experiments, the number of these cells was quantified with anti-CD3
and anti-CD14 MoAb using flow cytometry. Preparations of
CD34+CD38 cells were virtually free of
monocytes and lymphocytes while a low number of CD14+ cells
were present in the preparations of CD34+CD38+
cells (data not shown). Since CD4, the high affinity receptor for HIV
infection, has been reported to be present on purified CD34+ cells free of lymphocytic
population,39-41 we studied whether a limited replication
of HIV in cultures of CD34+CD38+ cells could be
correlated with the presence of receptors required for infection. Using
3-color flow cytometric analysis, CD4, CXCR-4, and CCR-5 molecules were
assessed on CD34+ cells. In agreement with our infection
assays, CD4 expression could only be found within
CD34+CD38+ but not within
CD34+CD38 cell populations (Fig
2D). Expression of HIV coreceptors such as
CXCR-4 and CCR-5 was also not detectable on most-primitive hematopoietic progenitors (CD34+CD38) by
means of flow cytometry. The increase of CD38 molecules on CD34+ BM cells correlates with the presence of HIV receptor
molecules CD4, CXCR-4, and CCR-5. On cells purified on the basis of
CD34, approximately 6% (±2.6%) of the cell population expressed
CD4, 12% ± 5% CXCR-4 and 3.5% ± 2.2% CCR-5, as determined in
three independent experiments.
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DISCUSSION |
The effects of HIV-1 and HIV-2 on committed hematopoietic progenitor
cells assayed in methylcellulose cultures have been extensively studied.9-13,16,20,28,38 Fewer reports have addressed the question of whether or not the most-primitive hematopoietic progenitors and stem cells can be infected by the viridae and to what degree the
proliferative function of these cells might be affected. Unfortunately, the results of these studies are inconsistent, possibly due to variability of the different methods used and the technical demands of
such experiments. However, virus-mediated stem cell depletion or
functional impairment due to direct viral cytotoxicity and host immune
response may account for some of the hematologic manifestations of
AIDS. At least theoretically, the infection of dormant stem cells with
retroviridae such as HIV-1 and HIV-2 could be associated with long-term
viral persistence and production of a large number of infected progeny.
Using highly stringent conditions to select for the most-primitive
hematopoietic stem cells in vitro, we demonstrated that these cells are
not susceptible to either HIV-1 or HIV-2 infection.
We first investigated the viral effects on the survival and function of
the most-immature human hematopoietic progenitor and stem cells in
BMLTC. Using purified populations of
CD34+CD38 cells (enriched in hematopoietic
stem cells), we showed that the number of LTCICs, as assessed by
secondary colony formation, remains unchanged after in vitro exposure
to HIV-1, or to HIV-2. Because LTCIC assessment is considered the best
in vitro assay of human hematopoietic stem cells,29-31
these results suggest that the BM stem cells are not directly affected
by HIV. Compatible results were reported when the number of LTCIC or
cobblestone-area-forming cells (cells functionally comparable with
LTCIC) was quantified in the peripheral blood and BM of patients with
HIV-1 infection.8,22 These studies suggested that stem
cells (measured as LTCIC) are affected by the cytotoxic therapies,
opportunistic infections and immune dysregulation rather than by the
direct infection with HIV itself. This interpretation is supported by a
number of reports where unchanged proliferative capacity of committed
progenitor cells in methylcellulose cultures was observed after in
vitro challenge with HIV-1.9,18,20,21 However, it is
noteworthy that decreased progenitor cell growth was reported under
varying experimental conditions which selected for, and assessed, more mature hematopoietic progenitor cells.7,11,13-15,17,28
We have also demonstrated that, in contrast to LTCIC, proliferation of
committed progenitor cells (CFCs derived from total BM after 2 to 4 weeks of BMLTC) was decreased after exposure to HIV-1 or HIV-2. Similar
findings were reported in previous studies demonstrating the impairment
of hematopoietic function in BMLTC after exposure to a variety of HIV
strains.19,20,33,34 These results and our own observations
are compatible with the theory that the virus may alter the
proliferation of hematopoietic progenitor cells indirectly,
eg., by the induction of cytokine production such as TNF-,
IFN- or TGF-. A large body of literature provides substantial
evidence for cytokine dysregulation in HIV infections. In addition, it
was hypothesized that HIV infection may influence the function of BM
stromal cells, which support the hematopoietic system.42,43
Although our present study was not designed to investigate this issue,
several other reports as well as the most recent studies from our
laboratory do not sustain this theory, showing that stromal layers
derived from AIDS patients adequately support hematopoiesis initiated
by normal CD34+ cells.22,44
The second important element of this study is the demonstration that
only a limited propagation of HIV-1 and HIV-2 is possible in
CD34+ cells. Cells susceptible to HIV-infection were
contained within the more committed subpopulation of
CD34+CD38+ cells and not in
CD34+CD38 cells.
Viral infection and replication was found to be restricted to committed
progenitors since no HIV-1 or HIV-2 proviral DNA presence nor viral
core protein production could be demonstrated in secondary CFC derived
from LTCIC, which represent a readout for the most-primitive stem cell
subpopulation. Even continued culture of secondary CFCs in a medium
designed to activate HIV-replication (tertiary cultures, supplemented
with a cocktail of growth factors) failed to reveal a possible viral
presence by any means of detection. These findings are compatible with
the demonstration that HIV-1 could be found only in a small
subpopulation of CD34+ cells purified from AIDS patient
derived BM.5,6,28
In the third part of our study, we demonstrated that the most-primitive
hematopoietic stem cells lack the surface molecules CD4, CXCR-4 and
CCR-5, which are required for HIV-infection. It has been reported that
CD34+ cells can express some level of CD4
molecules.18,39-41 In our study, CD4+ cells
were found only within the maturer CD34+CD38+
cell populations, explaining their susceptibility to HIV-infection and
limited traces of viral replication at the beginning of BMLTC. In
contrast, no CD4 expression was detectable on less mature
CD34+CD38 cells and no evidence for
infection of these cells was found in BMLTC. Several reports using
phenotypic rather than functional criteria for the characterization of
stem cells support our findings.4,5,7-9,28 Other
investigators have described that LTCIC were found within a
CD4lowCD34+ cell population and that CD4
expression was higher on CD34+ cells with the
CD38 phenotype.41 Although we cannot explain
these differences, it is commonly recognized that CD4 expression
appears to be generally low and infrequent on CD34+ cells.
It is possible that different protocols for obtaining BM cells (marrow
aspiration versus leukapheresis) and their storage (fresh cells versus
cryo-preservation) may influence the results of phenotype analysis.
CXCR-4 or CCR-5 molecules, which represent in conjunction with CD4 a
functional unit for high-efficacy HIV entry into CD34+
cells, were found to be expressed on the more mature progenitors in our
study. This seems somewhat surprising and appears intriguing, since it
was suggested that chemokines and their receptor-mediated signals might
play an important role in hematopoiesis.45,46 However, the
inability to detect HIV receptors on most-immature hematopoietic cells
by means of flow cytometry supports our results obtained from stringent
long-term culture assessment. The tropism of the different HIV strains
used in our experiments should have allowed for an effective entry, if
the surface molecules were present on the cells in a sufficient
density. The HIV-1 strain Bal represents a non-syncytium inducing (NSI)
isolate, which readily infects monocytes/macrophages (monocytotropic).
Bal entry into cells is mainly supported by RANTES- receptors,
including CCR-5. The primary isolate HIV-1 571 has been found to have
very similar characteristics regarding cell tropism.20
HIV-1 RF and NM have been demonstrated to induce syncytium in culture
(SI) and are preferentially T cells tropic with the ability to use
CXCR-4 for entry.47 The HIV-2 strains ST and Rod were
reported to use a variety of receptors, including CCR-5 and
CXCR-4.48-50
It has been demonstrated that subsets of
CD34+CD4+ cells, selected from leukapheresis
products, contain mRNA specific for CXCR-4 and CCR-5 as detected by
RT-PCR. Sequences for both chemokine receptors were also amplified in
some subpopulations of CD34+CD4
cells.51 Theoretically, it is possible that the levels of
chemokine receptor mRNA detectable in primitive hematopoietic precursor cells may not necessarily correlate with receptor expression on the
cell surface. An alternative explanation for the insusceptibility of BM
stem cell subsets to HIV infection could be derived from the hypothesis
that the virus would enter, but the immaturity and/or
activational stage of the cell may not support the complex process of
HIV replication. Clarification of these aspects, however, remains an
issue of future investigations.
We have previously demonstrated that exposure of total BM cells to
HIV-1 or HIV-2 is associated with TNF-
production.20,52-54 In our experiments, a restricted
TNF- release was also observed in correlation with a temporary
modulation of the CFC function (data not shown). The number of LTCICs
derived from CD34+ cells was not significantly affected in
our experimental system, suggesting that LTCIC are either more
resistant to the action of TNF- (that may have been released in
culture) or that continuous or recurrent presence of HIV is required
for the sustained levels of TNF- capable of affecting the function
of hematopoietic stem cells. In support of this hypothesis, it was
recently demonstrated that continuous exposure of hematopoietic stem
cells to IFN- was required to induce a decrease of the LTCIC number
in BMLTC.55 Similar mechanisms may operate in vivo
in AIDS patients. Continuous presence of virus or of viral gene
products may cause elevated levels of TNF-, sufficient to modulate
the function of hematopoietic progenitor cells. The stimulus for
continuous TNF- release was not present in BMLTC cultures of
CD34+ cells challenged with HIV or gp120, since inoculum
was removed through extensive washing and no viral replication was
detectable. However, when total BM cells were used to initiate the
BMLTC, the decrease in CFC (1 to 3 weeks of BMLTC) correlated with the release of TNF-, as well as the subsequent detection of viral replication in these cultures.
When purified CD34+ cell preparations were exposed to HIV-1
or HIV-2 viral stocks, a moderate increase in the number of 1-week secondary CFC was noted. Similar findings were reported in other studies.33,34,42 It is possible that virus stocks contain certain growth factors with transient effects on the proliferation of
progenitor cells.23 To account for these factors in our
experiments, we included controls derived from cell cultures similar to
those that were used to propagate viral stocks. These controls showed that growth factors contained in the viral supernatant do
not appear to play a relevant role in the observed increase in colony formation seen in the first weeks of cultures. It has been demonstrated that viral gene products such as envelope (gp120, gp41) or Tat represent potent modulators of cell activation and could also trigger
the release of a variety of cytokines responsible for the observed
differences in colony formation.23,51,56,57 In addition to
the proposed indirect mechanisms, some studies suggested a direct
effect of viral proteins on CD34+ cells, resulting in
inhibition of proliferation and the onset of
apoptosis.26,38,58 In our experiments, however, we were unable to observe such a direct effect on LTCIC.
Our results show that HIV-2, similar to HIV-1, does not infect
hematopoietic stem cells in vitro with any significant frequency, and
that infected cells are not represented within LTCICs. This finding
supports the hypothesis that direct infection of hematopoietic stem
cells by HIV-1 and HIV-2 does not play a major role in the hematologic
pathophysiology of AIDS-associated BM failure, nor are hematopoietic
stem cells likely candidates to serve as a significant reservoir of
virus in vivo. The failure to detect productive or latent HIV in the
most-primitive human BM progenitor and stem cells has important
implications for future therapeutic strategies, including those dealing
with immune modulation and gene therapy.
| |
FOOTNOTES |
Submitted June 3, 1997;
accepted October 2, 1997.
Address reprint requests to Frank F. Weichold, MD, PhD, Institute of
Human Virology, University of Maryland, 725 West Lombard St, Baltimore,
MD 21201.
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.
| |
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Abstract
Introduction
Abstract