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Journal of Virology, July 1999, p. 5865-5874, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Expression and Use of Human Immunodeficiency Virus
Type 1 Coreceptors by Human Alveolar Macrophages
Stefan
Worgall,1
Ruth
Connor,2
Robert J.
Kaner,1
Elizabeth
Fenamore,2
Kristine
Sheridan,2
Ravi
Singh,1 and
Ronald G.
Crystal1,*
Division of Pulmonary and Critical Care
Medicine, The New York Hospital-Cornell Medical
Center,1 and Aaron Diamond AIDS
Research Center, The Rockefeller University,2
New York, New York
Received 27 July 1998/Accepted 26 March 1999
 |
ABSTRACT |
Human immunodeficiency virus type 1 (HIV-1) requires, in addition
to CD4, coreceptors of the CC or CXC chemokine families for productive
infection of T cells and cells of the monocyte-macrophage lineage.
Based on the hypothesis that coreceptor expression on alveolar
macrophages (AM) may influence HIV-1 infection of AM in the lung, this
study analyzes the expression and utilization of HIV-1 coreceptors on
AM of healthy individuals. AM were productively infected with five
different primary isolates of HIV-1. Levels of surface expression of
CCR5, CXCR4, and CD4 were low compared to those of blood monocytes, but
CCR3 was not detectable. mRNA for CCR5, CXCR4, CCR2, and CCR3 were all
detectable, but to varying degrees and with variability among donors.
Expression of CCR5, CXCR4, and CCR2 mRNA was downregulated following
stimulation with lipopolysaccharide (LPS). In contrast, secretion of
the chemokines RANTES, MIP-1
, and MIP-1
was upregulated with LPS
stimulation. Interestingly, HIV-1 replication was diminished following
LPS stimulation. Infection of AM with HIV-1 in the presence of the CC
chemokines demonstrated blocking of infection. Together, these studies
demonstrate that AM can be infected by a variety of primary HIV-1
isolates, AM express a variety of chemokine receptors, the dominant
coreceptor used for HIV entry into AM is CCR5, the expression of these
receptors is dependent on the state of activation of AM, and the
ability of HIV-1 to infect AM may be modulated by expression of the
chemokine receptors and by chemokines per se.
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INTRODUCTION |
The human immunodeficiency virus
type 1 (HIV-1) requires interaction of the viral envelope glycoprotein
gp120 with CD4 and a second coreceptor for productive infection of its
target cell (4, 5, 9, 19, 34). These recently identified
coreceptors include the -chemokine receptors (CCR5, CCR3, and CCR2b)
and the -chemokine receptor CXCR4 (2, 3, 11, 16, 20, 21, 23,
24, 46-48). HIV-1 tropism and entry cofactor utilization are
important determinants of pathogenesis (4, 5, 9, 19, 34).
During primary HIV-1 infection and throughout the asymptomatic phase of
infection, isolates from blood are predominantly macrophagetropic and
CCR5 dependent (7, 15, 52, 53). In contrast, strains that
emerge later in many infected individuals can use CXCR4, the main
coreceptor for HIV-1 infection of T cells (7, 15, 52, 53,
58).
The focus of the present study is to characterize the pattern and usage
of the HIV-1 coreceptors on healthy human alveolar macrophages
(AM), the pulmonary representative of the mononuclear phagocyte
system. Other than evidence of productive infection of AM in
HIV-1-positive individuals (1, 12, 30, 35, 38-40, 45, 50),
little is known about the interactions of HIV-1 with this cell type.
Pulmonary infections are a major cause of the morbidity and mortality
associated with infection with HIV-1, and a majority of individuals
with AIDS develop one or more episodes of pulmonary infection during
the course of their disease (29, 36). AM represent the major
cellular host defense against microorganisms on the respiratory
epithelial surface (6, 43). In this context, understanding
the mechanisms of HIV-1 infection of AM may be central to understanding
the loss of respiratory epithelial surface host defense associated with
HIV-1 infection.
Based on the knowledge that AM are differentiated from blood monocytes
and that HIV-1 mainly uses CCR5 as a coreceptor on blood monocytes and
in vitro monocyte-derived macrophages (6, 9, 34, 42, 61, 64)
but that the type and level of coreceptor expression on monocytes can
be influenced by differentiation and activation (8, 18, 37, 44,
45), it is reasonable to assume that the coreceptors are
expressed on AM. Interestingly, the data demonstrate that the
coreceptor expression on healthy human AM generally parallels
that of autologous blood monocytes. However, most coreceptor
expression on AM is markedly lower and is only mildly influenced by
activation. Concomitant production of chemokines such as RANTES,
MIP-1, and MIP-1 may also markedly influence the ability of HIV-1
to infect AM.
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MATERIALS AND METHODS |
Cells.
Human AM were obtained by bronchoalveolar lavage from
healthy volunteers as previously described (49). The lavage
fluid was filtered through gauze to remove debris and cells were
pelleted, washed with phosphate-buffered saline (PBS) (pH 7.4) and
resuspended in RPMI 1640 medium containing 10% fetal bovine serum, 2 mM glutamine, 100 U of penicillin/ml, and 10 µg of streptomycin
(GIBCO BRL, Gaithersburg, Md.)/ml. For most experiments, AM were
purified by adherence to plastic (2 h, 37°C). For flow cytometry
studies, the cells were cultured in Teflon-coated vials (Savillex
Corp., Minnetonka, Minn.) until evaluation. Peripheral blood monocytes (PBM) and peripheral blood lymphocytes (PBL) were obtained from the
blood of the AM donors and purified by Ficoll gradients. The monocytes
were separated from the lymphocytes by adherence and maintained in RPMI
1640 media containing 10% human serum, 100 U of penicillin/ml, and 10 µg of streptomycin/ml for 18 h. For RNA analysis, PBM and PBL
were isolated by using immunomagnetic beads (Dynal, Lake Success, N.Y.)
coated with anti-CD14 for the isolation of monocytes and with anti-CD3
for the isolation of lymphocytes.
Infection with HIV-1 primary isolates.
AM were cultured in
48-well plates and infected with five different primary HIV-1 isolates
with known coreceptor usage (AD2-3 [CCR5], AD2-6 [CCR5 and CXCR4],
AD3-3 [CCR5], AD3-7 [CCR5, CCR2b, CCR3, and CXCR4]
[15], and JRFL [CCR5]). Twenty-four hours following infection, the cells were washed, and fresh medium was added. Productive infection was determined by measuring HIV-1 p24 antigen in
the supernatant by enzyme-linked immunosorbent assay (ELISA) (Abbott
Laboratories, North Chicago, Ill.) 5, 8, and 14 days after infection.
Flow cytometry.
To analyze surface HIV-1 coreceptor
expression on AM, PBM, and PBL, the cells were incubated with PBS
containing 2% bovine serum albumin and 10% human serum (4°C for 15 min), followed by incubation with primary antibodies against CCR5
(2D7), CCR3 (7B11), CXCR4 (12G5) (all antibodies were obtained from the
AIDS Research and Reference Reagent Program, National Institutes of
Health, Bethesda, Md.). After washing with PBS, the cells were
incubated with fluorescein isothiocyanate (FITC)-conjugated goat
anti-mouse immunoglobulin G (IgG) [F(ab')2] fragments
(Boehringer Mannheim, Indianapolis, Ind.) or FITC-conjugated anti-CD4
(Pharmingen, San Diego, Calif.) for 30 min. The cells were then washed
and incubated with 10% mouse serum for 15 min, followed by incubation
with phycoerythricin (PE)-labeled antibodies against HLA-DR (AM), CD14
(PBM), or CD3 (PBL) (Pharmingen), washed, and then analyzed by flow
cytometry. Isotype-matched unlabeled and PE-labeled antibodies served
as negative controls. To analyze CCR5 surface expression using
antibodies other than clone 2D7, the FITC-conjugated primary antibodies
recognizing CCR5 (FAB180F, FAB181F, FAB182F, and FAB183F [all from R&D
Systems, Minneapolis, Minn.]) were used to stain AM, PBM, and PBL as
described above. FITC-labeled isotype-matched antibody was used as a control.
mRNA analysis.
Two strategies were used to evaluate
coreceptor mRNA in the AM in comparison to PBM and PBL, reverse
transcription (RT)-PCR and Northern analysis. For RT-PCR, total RNA was
extracted from AM, PBM (CD14 purified), or PBL (CD3 purified) by using
Trizol reagent (GIBCO BRL) and reverse transcribed (45 min, 48°C; 2 min, 94°C), and the resulting DNA was amplified by PCR (9600 Gene
Amp; Perkin-Elmer) by 40 cycles of 94°C for 30 s, 56°C for 1 min,
and 68°C for 2 min by using synthetic oligonucleotide primers
specific for CCR3 (sense primer, TCCACACTCGAGAATGACCATCT;
antisense primer, ACTGGAAGTTTGAAGGACTGTTTT; product
size, 578 bp), CCR5 (sense primer, CAGGGCTGTGAGGCTTATCTT;
antisense primer, CCCAGGCTGTGTATGAAAACT; product size,
437 bp), CXCR4 (sense primer, TTGTCTGAACCCCATCCTCTAT; antisense primer, ACTCCTGAAAACTGAAAAACCA; product
size, 626 bp), CCR2B (sense primer, CCAACGAGAGCGGTGAAGAAGT;
antisense primer, GGGAGTCCAGAAGAGAAAGTAAACA; product
size, 737 bp), and CD4 (sense primer, AGTTGCATCAGGAAGTGAACCT;
antisense primer, CTGAGACATCCGCTCTGCTTGG; product
size, 383 bp). Primers for glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) (sense primer, CCTTCATTGACCTCAACTACA; antisense primer, GGCAGTGATGGCATGGACTGT; product size, 443 bp) served
as an internal control. The PCR products were analyzed on a 1.5% agarose gel. DNA contamination was ruled out by pretreatment of the
samples with DNase (GIBCO BRL) for 15 min at 37°C and by omitting the
reverse transcriptase from the PCR as a control.
To analyze coreceptor expression by Northern analysis, total cellular
RNA (10 µg) was transferred to Duralon membranes (Stratagene, La
Jolla, Calif.) after electrophoretic separation through a 1% agarose
gel under denaturing conditions. Probes for CCR5, CCR3, CCR2B, and
CXCR4 (kindly provided by Ned Landau, Aaron Diamond AIDS Research
Center, New York, N.Y.) were gel purified and labeled with
[32P]dCTP by random priming (Stratagene). Hybridizations
were performed in hybridization solution (Quickhyb; Stratagene) for
2 h at 65°C, followed by sequential washes in 1× SSC (0.15 M
NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS)
for 30 min and 0.1% SSC-0.1% 2× SDS for 30 min. Following
hybridization, the membranes were analyzed by autoradiography or phosphorimaging.
Influence of AM stimulation on HIV-1 coreceptor expression.
To analyze if stimulation of AM influenced the expression of the HIV
coreceptors, AM were treated with 100 ng of lipopolysaccharide (LPS)/ml
for either 4 or 48 h. Total RNA was extracted and analyzed for
coreceptor expression by RT-PCR and Northern analysis as described above. To determine if LPS stimulation resulted in increased secretion of chemokines, the levels of MIP-1, MIP-1, RANTES, and eotaxin in
the supernatant were quantified by ELISA (R&D Systems).
To evaluate if stimulation with LPS influences HIV-1 infection and
replication, AM were inoculated with 200 50% tissue culture infective
doses (TCID50) of five different primary isolates as described above after overnight stimulation with 100 ng of LPS/ml. HIV-1 p24 levels were measured in the culture supernatant by ELISA at
days 5, 8, and 14 postinfection. To ensure that cell viability was not
affected by stimulation with LPS, AM were plated in 96-well plates, and
viability was assessed in the presence or absence of 100 ng of LPS/ml
after 7 and 14 days by using an MTT-based cytotoxicity assay (Sigma,
St. Louis, Mo.).
Chemokine blocking of HIV-1 infection in AM.
To assess the
ability of chemokines to block HIV-1 infection of AM, cells were
infected with 100 TCID50 of HIV-1 AD2-3, AD2-6, AD3-3,
AD3-6, or JRFL in the presence of 250 ng of RANTES, MIP-1, MIP-1,
or SDF-1/ml, either alone or in combination. After 48 h, the cells
were washed, and the appropriate chemokines were added back to the
wells. p24 levels in the supernatant were measured by ELISA on day 7 after infection and were compared to those of control cultures infected
in the absence of added chemokines. Percent inhibition was calculated
as (1 
the mean p24 concentration of duplicate wells with
chemokines/mean of control wells) × 100.
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RESULTS |
Infection of AM with primary HIV-1 isolates.
Inoculation of AM
from healthy individuals with primary isolates of HIV-1 demonstrated
virus replication in AM from all donors for all tested isolates (Fig.
1). Peak p24 levels among the different donors ranged from 313 to 778 pg/ml for AD2-3, 25 to 951 pg/ml for
AD2-6, 341 to 1,516 pg/ml for AD3-3, 86 to 4,408 pg/ml for AD3-7, and
604 to 20,000 pg/ml for JRFL.
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FIG. 1.
Replication of primary HIV-1 isolates in AM from
different donors. AM were obtained by bronchoalveolar lavage of healthy
individuals and infected in vitro with five different primary HIV-1
isolates. HIV-1 p24 antigen was measured by ELISA in the supernatant on
days 4, 8, and 14. Values are peak p24 levels measured on day 14. The
known coreceptor usage of each primary isolate is listed
(15), as is the clinical stage of the individual at the time
the virus was isolated. Note that AD2-3 and AD2-6 are from the same
individual at different stages, as are AD3-3 and AD3-7. Asx,
asymptomatic. Each symbol represents data for one individual.
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HIV-coreceptor expression on AM.
Flow cytometry analysis of
the HIV-1 coreceptors CCR5, CCR3, and CXCR4, as well as CD4, on the
surface of AM demonstrated very low levels of these receptors (Fig.
2 and 3).
In contrast, surface expression of HLA-DR, a marker for AM, was
detectable on 93 to 98% of the cells. Higher surface expression of
CCR5, CXCR4, and CD4 was detectable on PBM and PBL from the same
individuals stained in parallel, while the levels of CCR3 were very low
on PBM and PBL. On the average, CCR5 and CXCR4 levels on AM were significantly lower than on autologous PBM and PBL (P < 0.01, all comparisons), while CCR3 levels were similar
(P > 0.1, all comparisons). Using antibody clones
against CCR5 other than clone 2D7 (Fig.
4), AM demonstrated low-to-undetectable
levels of cells positive for FAB180F (1.2% ± 0.9%) and FAB181F
(0.2% ± 0.2%), whereas some cells stained positive for FAB182F
(8.1% ± 1.0%) and FAB183F (6.2% ± 0.8%). Further testing of these
antibodies on PBM and PBL showed staining similar to the CCR5 antibody
2D7 for clone FAB182F (PBM, 38.5% ± 6.1%; PBL, 5.9% ± 0.4%),
whereas using the other clones, positive cells were less frequently
observed or not detectable (clone FAB180F: PBM, 2.3% ± 0.9%, PBL,
2.0% ± 0.7%; clone FAB181F: PBM 6.5% ± 2.1%, PBL, 1.8% ± 0.5%;
clone FAB183F: PBM, 5.3% ± 1.5%, PBL, 2.2% ± 0.3%). These results
suggest that CCR5 surface expression levels on AM are low, although a small subpopulation stained positive with the CCR5 clones FAB182F and
FAB183F, suggesting that certain epitopes may be masked by using
different antibody clones.
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FIG. 2.
Flow cytometry evaluation of expression of chemokine
receptors on AM. AM were obtained by bronchoalveolar lavage and stained
with anti-CCR3, CCR5, or CXCR4 monoclonal antibodies (followed by
FITC-labeled anti-IgG) or FITC-conjugated anti-CD4. Blood monocytes and
lymphocytes obtained from the same donors were evaluated in parallel.
Shown are representative samples from one individual of AM (panels A to
D), PBM (panels E to H), and PBL (panels I to M). In addition to
coreceptor staining, AM were double stained with PE-labeled HLA-DR,
blood monocytes were double stained with PE-labeled CD14, and
lymphocytes were double stained with PE-labeled CD3. The histograms
shown represent the cells selected by these markers. IgG-irrelevant
controls for each antibody are depicted by the dotted lines. Surface
expression of CCR3 (panels A, E, and I); CCR5 (panels B, F, and J);
CXCR4 (panels C, G, and K); and CD4 (panels D, H, and L) is shown. The
solid horizontal line represents the region selected for
quantification.
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FIG. 3.
Quantitative analysis of chemokine receptor surface
expression on AM compared to that on autologous blood monocytes and
blood lymphocytes. AM, PBM, and PBL from healthy individuals were
labeled with antibodies against CCR5, CCR3, CXCR4, and CD4 and
evaluated by flow cytometry (as described in the legend for Fig. 2).
Shown are the means ± standard errors of the means for six
individuals.
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FIG. 4.
Flow cytometry analysis of expression of the chemokine
receptor CCR5 using different monoclonal antibodies. AM were obtained
by bronchoalveolar lavage and stained with the following
FITC-conjugated monoclonal antibodies against CCR5: FAB180F, FAB181F,
FAB182F, and FAB183F. AM were double stained with PE-labeled HLA-DR.
The histograms represent the cells selected by this marker. IgG-matched
control antibody is depicted by the dotted line. Shown is surface
expression of representative samples from one individual for FAB180F
(A), FAB181F (B), FAB182F (C), and FAB183F (D).
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Analysis of CCR5, CCR3, CXCR4, CCR2b, and CD4 expression at the mRNA
level using RT-PCR demonstrated detectable expression
of each receptor
on AM, PBM, and PBL (not shown). Northern analysis
demonstrated mRNA
transcripts of CCR5, CXCR4, and CCR2b in cells
from all AM donors
evaluated, although there was variability in
the mRNA levels from donor
to donor (Fig.
5). In contrast, mRNA
expression of the control GAPDH was similar among all individuals.
CCR3
transcripts were not detected in AM, PBM, or PBL by Northern
analysis
from any donor (not shown). The mRNA levels for CCR5
and CXCR4 tended
to be lower on the AM than on PBM and PBL, but
not with the more
variable expression of CCR2b. Interestingly,
while PBL (and to a lesser
extent PBM) showed two mRNA bands of
1.4 and 3.0 kb for CXCR4 as has
been previously reported (
25),
AM showed expression of the
1.4-kb band only.
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FIG. 5.
Chemokine receptor expression in AM assessed by Northern
analysis. RNA obtained from AM of three individuals (lanes A, B, and C)
and, as a control, from PBM and PBL from individual A. The PBM and PBL
were purified with immunomagnetic beads coated with antibodies against
CD14 (PBM) and CD3 (PBL), respectively. The cells were analyzed with
specific probes for CCR5, CXCR4, and CCR2b. From top to bottom are
shown expression of CCR5, CXCR4, CCR2b, and GAPDH. Lanes: 1 to 3, AM
for three healthy individuals; 4, PBM from individual A; 5, PBL from
individual A. The sizes of the mRNAs are indicated in kilobases (kb).
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Influence of AM activation on coreceptor expression, chemokine
expression, and HIV-1 replication.
To determine if activation of
AM influences the expression of CCR3, CXCR4, CCR5, and CCR2b, the cells
were cultured in the presence of LPS for either 4 or 48 h.
Northern analysis demonstrated markedly decreased mRNA levels of CXCR4
after 4 h of stimulation and mildly decreased levels of CCR5 mRNA
after 48 h of stimulation, (Fig.
6A). CCR3 was not detectable by Northern
analysis. GAPDH levels remained unchanged.
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FIG. 6.
Consequences of activation of AM on HIV-1 coreceptor
expression, chemokine expression, and the ability of AM to be infected
by primary isolates of HIV-1. (A) AM were stimulated with LPS (100 ng/ml) for either 4 or 48 h and then analyzed for HIV-1 coreceptor
expression by Northern analysis. CCR5, CXCR4, and CCR2b expression
analyzed by Northern analysis in unstimulated () and LPS-stimulated
(+) cells. GAPDH expression is used as a control. The sizes of the
mRNAs are indicated in kilobases (kb). This pattern is representative
of three different donors analyzed. (B) RANTES, MIP-1, MIP-1, and
eotaxin secretion in culture supernatant as measured by ELISA following
stimulation of AM with LPS ( ) or without LPS stimulation ( ).
Dashed line represents the limit of detection of the assay. (C)
Influence of LPS stimulation on HIV-1 replication in AM. AM were
stimulated with LPS for 12 h and then infected with five primary
HIV-1 isolates. After 14 days, HIV-1 p24 antigen levels in the
supernatant were measured by ELISA. Shown are data obtained with (+)
and without () LPS stimulation. The HIV-1 isolates are the same as in
Fig. 1, and the coreceptor use of these isolates is indicated. Data are
means ± standard errors of the means of triplicate
measurements.
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To determine whether activation of AM resulted in increased secretion
of chemokines, the levels of RANTES, MIP-1
, MIP-1
,
and eotaxin,
were measured in the culture supernatant. Increased
levels of RANTES,
MIP-1
, and MIP-1
, but not eotaxin, were found
following
stimulation with LPS (Fig.
6B). Although there was some
donor-to-donor
variability in the response to LPS, there was a
marked increase for
MIP-1
, MIP-1
, and RANTES following LPS stimulation
in all samples
(Table
1).
To analyze if LPS simulation of AM influences infection of AM with
HIV-1, LPS-stimulated cells were inoculated with five different
primary
isolates of HIV-1 in the presence of LPS, and the levels
of HIV-1
replication were determined. Strikingly, HIV-1 replication
of
LPS-stimulated AM was diminished for all of the isolates tested
compared to infection of unstimulated cells (Fig.
6C;
P < 0.05,
all comparisons). The viability of the cells was not
affected
by LPS
stimulation.
Chemokine blocking of HIV-1 replication in AM.
To determine if
the ligands for the coreceptors could block HIV-1 replication in AM,
cells were infected with the primary isolates AD2-3, AD2-6, AD3-3,
AD3-7, or JRFL in the presence of either RANTES, MIP-1, MIP-1,
SDF-1, or all four combined. HIV-1 replication was inhibited in the
presence of RANTES (44 to 84%), MIP-1 (20 to 62%), and MIP-1
(55 to 85%) for all the HIV-1 isolates (Fig.
7). All chemokines combined had a >80%
inhibitory effect on HIV-1 replication. Interestingly, for one HIV-1
isolate which, like CCR5, can use CXCR4 (AD2-6), SDF-1 blocked HIV-1
infection to 67%, whereas for all the other isolates SDF-1 did not
block HIV-1 infection. However, for AD2-6, the blocking effect of SDF-1 did not exceed the effect seen by MIP-1 (92%), MIP-1 (55%), RANTES (44%), or all chemokines combined (86%).
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FIG. 7.
Inhibition of HIV-1 replication in AM by chemokines. AM
were infected with primary HIV-1 isolates in the presence of 250 ng of
RANTES, MIP-1, MIP-1, or SDF-1/ml or all chemokines together.
HIV-1 p24 antigen was measured in the culture supernatants on day 10 after virus inoculation. The percent inhibition was calculated based on
control cultures infected without added chemokines. Data are means of
duplicate wells from one (of three) representative experiment. Shown
are results for isolates AD2-2 (coreceptor used, CCR5), AD2-6
(coreceptors used, CCR5 and CXCR4), AD3-3 (coreceptor used, CCR5),
AD3-7 (coreceptors used, CCR5, CXCR4, CCR2b, and CCR3), and JRFL
(coreceptor used, CCR5).
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DISCUSSION |
The present study analyzes the expression and utilization of the
major chemokine receptors for HIV-1 entry into normal human AM. AM were
productively infected with several primary isolates of HIV-1.
Expression of the major known HIV-1 coreceptors (CCR5 and CXCR4) was
detectable at the RNA level, whereas surface expression of these
receptors occurred at lower levels. However, CCR5-specific chemokines
were able to significantly inhibit HIV-1 replication in AM as did
stimulation of AM with LPS, which leads to increased expression of
CCR5-specific chemokines. These data suggest that CCR5 is the
predominant coreceptor used by HIV-1 to infect AM, but that coreceptor
expression levels are far lower on AM than on blood monocytes.
Importantly, the combined observations that activation of normal human
AM decreases replication of HIV-1, downregulates CCR5 expression, and
increases the release of RANTES, MIP-1, and MIP-1 together
suggest that the interplay of chemokine receptors and chemokine
production plays a major role in the susceptibility of AM to HIV-1 infection.
AM and HIV-1 infection.
AM play an important role in the
pulmonary host defense and are known targets for HIV-1 infection
(1, 6, 12, 30, 35, 38-40, 43, 45, 50). Although pulmonary
infections are common in HIV-1-infected individuals and in patients
with AIDS, the role of the AM in the progression of HIV-1-related lung
disease is not well defined. Importantly, it is not known if the
infection of AM takes place within the lung or is secondary to systemic infection of AM precursors or both (1). AM obtained by
bronchoalveolar lavage from HIV-1-infected individuals harbor HIV-1
(1, 12, 30, 35, 38-40, 45, 50). In general, the absolute
number of HIV-1-infected AM in individuals with AIDS is lower than that of blood monocytes (35, 38, 39), although a significant increase of HIV-1 in AM, but not monocytes, has been reported in some
patients as the disease progresses (56).
HIV-1 entry in AM.
From the results of the present study, it
is clear that healthy human AM can be productively infected with
primary isolates of HIV-1 in vitro. The entry mechanisms for HIV-1 into
AM have not been extensively studied. It is known that CD4 is critical for HIV infection of AM, and AM are known to express CD4 (26, 32). The discovery that HIV-1 also requires coreceptors of the CC
and CXC chemokine family for entry into lymphocytes and cells of the
monocyte-macrophage lineage has shed new light on the pathogenesis of
HIV-1 infection (4, 9, 19, 34). Based on a variety of
studies of the coreceptors for HIV-1 entry into blood monocytes and
monocyte-derived macrophages, it is likely that CCR5 is the main
receptor used for entry into these cells (18, 37, 42, 55, 61,
64), although recent data obtained by using CCR5-deficient monocytes demonstrate that CXCR4 can also be used (63). CCR5 seems to play a central role in the transmission of HIV-1 in vivo, as
individuals homozygous for a 32-bp deletion in CCR5 have increased resistance to HIV-1 infection (33, 51). HIV-1 strains that use CCR5 are present throughout the course of the disease, whereas in
some individuals, variants that use additional coreceptors emerge later
in the course of the disease (15, 52).
Current knowledge of the pattern of coreceptor expression on tissue
macrophages is limited. Studies with human microglial
cells of the
brain have demonstrated the expression of CCR3 and
CCR5, although
recent data suggest a dominant role for CCR5 in
infection (
28,
54). Human AM are known to express the two
orphan
seven-transmembrane receptors, GPR-1 and GPR-15, which
can be used for
simian immunodeficiency virus entry (
22). The
present study
demonstrates that primary HIV-1 isolates which solely
use CCR5 can
replicate in AM, in addition to isolates which use
more than one
coreceptor. The potential usage of additional coreceptors,
including
GPR-1 and/or GPR-15, has not been determined, and thus
we cannot rule
out the possibility that these receptors play a
role in HIV-1 entry
into
AM.
Coreceptor expression on AM.
While all of the evidence is
consistent with the concept that HIV-1 uses predominantly CCR5 to enter
AM, the surface expression of CCR5 and the other major chemokine
receptors is much lower on AM than on autologous blood monocytes,
despite the presence of mRNA. Although there was some variability with
different clones of anti-CCR5 antibodies, the overall detectable
surface expression of CCR5 was well below 10% of the cells. When using
antibodies against CCR5 other than clone 2D7, a small, distinct,
positive subpopulation could be seen with the clones FAB182F and
FAB183F, suggesting that certain CCR5 epitopes could be masked on
AM, but this may represent only a small percentage of the total
population. Likewise, as has been previously shown (32), the
expression of CD4 on normal AM is low, suggesting that low-level
surface expression of CD4 and coreceptors is sufficient for infection of HIV-1. It has been reported that, in retrovirus-modified HeLa cells,
CD4 and CCR5 interact in a concentration-dependent manner, i.e., in the
presence of low levels of CD4 expression, high levels of CCR5 are
sufficient for HIV infection and vice versa (41). In the
present study, however, the levels of surface expression of both CD4
and CCR5 were found to be similarly low on AM. Although expression
levels of all of the major chemokine receptors are low on healthy AM,
the level of expression of CCR3 is by far the lowest, detectable only
by RT-PCR. This is similar to that previously noted in blood monocytes
(18, 37).
Interestingly, the levels of CXCR4 and CCR2 mRNA in AM vary
considerably among individuals. As with blood monocyte-derived
macrophages (
8,
18,
37,
44,
45), the state of activation
of
AM influences the expression of some HIV-1 coreceptors. Although
dependent on time after activation, expression of CCR2, CXCR4,
and CCR5
mRNA is suppressed by activation of AM. CCR2 mRNA levels
in
monocyte-derived macrophages and monocytic cell lines are diminished
by
activation, with moderate decreases in CCR5 mRNA levels
(
55).
Stimulation of human endothelial cells with LPS also
leads to
a decrease in CXCR4 mRNA levels (
27).
In addition to receptor mRNA regulation, activation of AM results in
the secretion of the CCR5 ligands RANTES, MIP-1
, and
MIP-1
.
Chemokines, as the natural ligands of the HIV-1 coreceptors,
are able
to competitively block HIV-1 infection (
13,
59).
Consistent
with these observations, activation of blood-derived
monocytes results
in decreased replication of HIV-1 (
31) and,
as shown in the
present study, activation of AM results in a similar
decrease in
replication of HIV-1 isolates which primarily use
CCR5 as the
coreceptor for infection. This may be due in part
to the decreased
expression of this receptor following activation
and, in part, to the
secretion of CCR5 ligands RANTES, MIP-1
,
and MIP-1
, which can
compete with HIV-1 for the coreceptor. Furthermore
the decreased
coreceptor expression could have resulted from the
secretion of
endogenous beta chemokines, which themselves could
act to downregulate
receptor mRNA. RANTES, MIP-1
, and MIP-1
blocked infection of AM
with all the five primary HIV-1 isolates
tested in our study, a
phenomenon which has been recently described
for infection of AM with
the HIV-1 strain BAL (
14). SDF-1, the
ligand for CXCR4, was
able to block infection to some extent for
only one of two primary
isolates utilizing both CCR5 and CXCR4,
but the inhibitory effect was
less than that seen for the CCR5-specific
chemokines. Although it is
possible that CXCR4 may be used to
some degree for HIV-1 entry,
indirect effects of SDF-1 rather
than direct blocking may also be an
explanation for decreased
HIV-1 replication in the presence of SDF-1.
For blood monocyte-derived
macrophages, HIV-1 replication is inhibited
by activation via
the release of RANTES, MIP-1
, and MIP-1
(
60). Consistent with
this concept, MIP-1
has been shown
to be produced by AM in increased
amounts in HIV-1-infected individuals
(
17).
Taken as a whole, the present study demonstrates that AM express a
variety of chemokine receptors relevant for HIV-1 entry,
that HIV-1
likely enters AM mainly through CCR5, and that activation
of AM can
result in decreased infection of this cell type with
HIV-1. Strategies
to prevent infection via blockage of chemokine
receptors on
macrophages, including chemically modified chemokines
such as
AOP-RANTES (
57), are currently being developed.
Intracellular
blocking of CCR5 receptor expression via "intrakines"
may be equally
useful to prevent HIV-1 infection of AM (
10,
62).
| |
ACKNOWLEDGMENTS |
S.W. and R.C. participated equally in this study.
We thank Simon Monard for help with the flow cytometry studies, Barbara
Ferris for technical assistance, Philip L. Leopold and Neil R. Hackett
for helpful discussions, and N. Mohamed for help in preparing the manuscript.
These studies were supported, in part, by NIH grants P01 HL59312 and
R01 HL59861-01; the Will Rogers Memorial Fund, Los Angeles, Calif.; and
GenVec, Inc., Rockville, Md. R.C. was also supported, in part, by grant
AI 41373 from The Aaron Diamond Foundation, New York, N.Y.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Weill Medical
College of Cornell University-New York Presbyterian Hospital, Starr
505, New York, NY 10021. Phone: (212) 746-2258. Fax: (212) 746-8383. E-mail: geneticmedicine{at}mail.med.cornell.edu.
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Journal of Virology, July 1999, p. 5865-5874, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
