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Picower Institute for Medical Research, Manhasset, NY 11030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and -1ß. LPS did not act by down-regulating CCR5 mRNA
(mRNA levels actually increased slightly after LPS treatment) or by
enhancing the degradation of internalized receptor. Rather, the
observed failure of LPS-treated macrophages to rapidly restore CCR5
expression at the cell-surface appeared to result from altered
recycling of chemokine receptors. Taken together, our results suggest a
novel pathway of CCR5 recycling in LPS-stimulated human macrophages
that might be targeted to control HIV-1
infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The surface marker CD4, expressed in a wide variety of cell types, was the first cell-surface molecule found to bind HIV-1. Recently, several members of an unrelated family of surface proteins that specifically bind ligands of the chemokine super family have been shown to function as HIV-1 coreceptors; that is, HIV must engage CD4 in combination with one of several distinct chemokine receptors for efficient entry of the virus into cells. These HIV-1 coreceptors belong to a large family of seven transmembrane domain G-protein-coupled receptors. This family of receptors can be subdivided into two major groups designated CC and CXC chemokine receptors after the subclass of chemokine (CC or CXC) that binds the receptor (3, 4). Individual chemokines affect the proliferation, differentiation, trafficking, phenotype, and activation of particular populations of hematopoietic cells, and the differential expression of chemokine receptors by distinct cell types is an important determinant of the specificity of chemokine-induced biological effects (5). In parallel, the preferential tropism that some HIV-1 strains show for specific cell types can be explained, at least partially, by selective adaptation to specific subclasses of chemokine receptors. For example, lymphotropic strains of HIV-1 (such as NL4-3, NDK, and Bru) preferentially infect activated T cells through binding to CXCR4, whereas CCR5 has been demonstrated to be the main coreceptor for macrophage-tropic strains (such as ADA and JRFL) (6, 7). The requirement of CCR5 for productive infection of macrophages by HIV-1 correlates with the observation that an increase in CCR5 expression in monocyte-derived macrophages (MDMs)5 during the first week after isolation from the blood coincides with an increase in permissiveness for HIV-1 (8, 9, 10). It is of particular note that patients presenting a genetically mutated CCR5 have been shown to be resistant to HIV-1 infection despite multiple exposures to the virus (11, 12, 13). In addition to the key role played by chemokines and their receptors in HIV pathogenesis (14), they regulate hematopoiesis (15, 16), tumor cell growth (17), and angiogenesis (18, 19).
Bacterial LPS, a major constituent of the cell wall of Gram-negative bacteria (20), is a potent stimulus for macrophages. Exposure to low levels of LPS activates multiple macrophage effector functions that serve to coordinate host-protective immune and inflammatory responses. However, increased exposure to LPS drives macrophage activation, which leads to tissue injury and, in extreme cases of endotoxemia, to circulatory collapse, multiorgan failure, shock, and death (21, 22, 23). Several recent studies have demonstrated that LPS treatment protects primary macrophages from productive HIV-1 infection in vitro (24, 25, 26, 27). In agreement with these previous reports, we describe in this paper that preincubation of MDM with LPS markedly decreased HIV-1 infection via inhibition of viral entry. We further demonstrate that the inhibitory effect of LPS was secondary to a direct and rapid down-regulation of CCR5 receptors from the cell surface, which was independent of de novo protein synthesis. Low levels of cell-surface chemokine receptor expression were maintained for an extended period compared with down-regulation mediated by binding of natural ligands. These results suggest that receptor down-regulation is the main mechanism of LPS-mediated resistance of macrophages to HIV-1 infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HIV-1ADA was obtained from Dr. H. E.
Gendelman (Department of Pathology and Microbiology, University of
Nebraska Medical Center, Omaha, NE) (28). HIV-1 primary
isolates 92US660, 92US657, and 92UG021 were obtained from the AIDS
Research and Reference Reagent Program (Rockville, MD). LPS from
Escherichia coli 0127:B8 was purchased from Difco
(Detroit, MI). The cell culture medium DMEM was from Life Technologies
(Gaithersburg, MD). Normal human serum was from BioWhittaker
(Walkersville, MD), and FBS was obtained from Life Technologies. Human
recombinant M-CSF, the antibiotic cycloheximide, and the
Ca+2 ionophore ionomycin calcium salt
from Streptomyces conglobatus were obtained from Sigma (St.
Louis, MO). BSA, fraction V, was purchased from USB-Amersham
(Cleveland, OH). Human recombinant chemokines macrophage-inflammatory
protein (MIP)-1, MIP-1ß, and RANTES were from PeproTech (Rocky
Hill, NJ) and radiolabeled chemokines
[I125]MIP-1 and
[I125]MIP-1ß were from DuPont NEN (Boston,
MA). Fura 2-acetoxymethyl ester was from Molecular Probes
(Eugene, OR).
Isolation and differentiation of human MDMs
Human PBMCs were isolated from buffy coats of healthy
seronegative donors (Long Island Blood Services, Melville, NY) by
Ficoll density gradient centrifugation (Ficoll-Paque PLUS, Pharmacia
Biotech, Piscataway, NJ) (29). PBMCs were cultured in
Primaria flasks (Becton Dickinson, Franklin Lakes, NJ) under culture
medium (DMEM) supplemented with 10% heat-inactivated normal human
serum, 2 mM glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin
(all from Life Technologies) at 37°C in a humidified atmosphere of
5% CO2 in air and at a cell density of 8 x
106 cells/ml. Two hours after the PBMC fraction
was plated, nonadherent cells were aspirated, and the adherent cells
were washed three times with culture medium. Culture medium containing
M-CSF (at 2 ng/ml) was added to the cells, which were then cultured
overnight. At 
18 h after initial plating, adherent cells were
harvested by detachment with 10 mM EDTA/PBS and cultured in 24-well
Primaria plates (Becton Dickinson) or in Teflon flasks (Nalgene-VWR,
Rochester NY) for 7 days in the presence of recombinant human M-CSF (at
2 ng/ml) at a density of 106 cells/ml. After 7
days, final MDM cultures comprised about 98% macrophages as judged by
morphology and nonspecific esterase activity (28).
Infection with HIV
The macrophage-tropic strain HIV-1ADA was carried in MDM cultures. Immediately before experimental infections, harvested virus was pretreated with RNase-free DNase (Boehringer Mannheim, Indianapolis, IN) for 1 h at room temperature and then filtered through a 0.45 µm nitrocellulose membrane. MDM cultures were infected for 2 h at 37°C with an amount of virus corresponding to 1 x 105 cpm of reverse transcriptase activity per 1 x 106 cells. After infection, unabsorbed virus was washed away, and the cells were cultured without M-CSF.
DNA-PCR
Samples from HIV-1-infected macrophage cultures were prepared and subjected to PCR analysis as described previously (27). The following primer pairs were used: LTR/gag, 5'-CAGATATCCACTGACCTTTGG and 5'-GCTTAATACTGACGCTCTCGCA; 2LTR, 5'-TCCCAGCGTCAGATCTGGTCTAAC and 5'-GCCTCAATAAAGCTTGCCTTG; tubulin, 5'-GTTGGTCTGGAATTCTGTCAG and 5'-AAGAAGTCCAAGCTGGAGTTC. PCR products were analyzed by Southern blotting using 32P-labeled probes (LTR/gag, 5'-GAGGCTTAAGCAGTGGGTTC, or a 330-bp Mro1/Nar1 fragment of the plasmid MJ2 containing HIV-1NL4-3 genome) (30). The results were quantified in a phosphorimager (Direct Imager; Packard, Meriden, CT).
Radiolabeled chemokine binding studies
For chemokine binding experiments, cells were exposed to
different treatments and then washed extensively before carrying out
binding protocols. For each sample, 1 x 106
cells were incubated with 0.5 nM of
[I125]MIP-1 or
[I125]MIP-1ß (each having a specific activity
of 2200 Ci/mmol) in a binding buffer comprising DMEM and 1% BSA for
2 h at 4°C. Cells were washed twice with cold PBS, lysed in RIPA
buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40,
0.5% deoxycholic acid, and 1% SDS), and then counted in a gamma
counter (Gamma Track 1193; TM Analytic, Brandon, FL) (31).
Nonspecific background binding was determined by counting radioactivity
bound in the presence of a 100-fold molar excess of unlabeled MIP-1
or MIP-1ß. Specific binding of MIP-1ß was markedly lower than that
of MIP-1; therefore, MIP-1ß binding was presented as percent
binding relative to control. In receptor recovery experiments,
macrophage cultures were incubated with a mixture of chemokines
containing MIP-1, MIP-1ß, and RANTES at 10 nM each or with 100 ng
LPS/ml. After a 3-h incubation period, cells were washed extensively,
and duplicate samples were assayed for binding of radiolabeled MIP-1
(time 0) or returned to the incubator for later binding
experiments.
Calcium signaling studies
Macrophages cultured in suspension for 7 days in Teflon flasks were resuspended in a prewarmed (37°C) HBSS containing 2 mM Ca+2 and 10% FBS at a density of 1 x 107 cells/ml. Five microliters of fura 2 (from a 1-mM stock) was added and cells were incubated at 37°C for 20 min. The cell suspension was then diluted 3-fold in prewarmed HBSS containing 10% FBS for 5 min at 37°C. The cells were centrifuged for 5 min at 700 rpm and the pellet was resuspended in 2 ml of HBSS containing 2 mM Ca+2. Three aliquots of 600 µl each were dispensed in Eppendorf centrifuge tubes and spun for 5 s. Cell pellets were resuspended in fresh HBSS and transferred to luminescence spectrometer cuvets for readings. The data acquisition and analysis were performed using the software FL-winlab (Perkin-Elmer, Norwalk, CT).
Immunofluorescence microscopy
Human MDMs were obtained as described above and treated with LPS (100 ng/ml) or medium alone for 3 h. For visualization of cell-surface CCR5 expression, MDMs were subjected to the following immunostaining protocol at the end of either the 3-h incubation with LPS or the 24-h period after the removal of LPS. Cells were washed and then incubated with an unlabeled mouse monoclonal anti-CCR5 Ab (2D7) for 30 min. After extensive washing, a secondary FITC-labeled anti-mouse Ab was applied for 30 min, after which the cells were washed and resuspended in PBS. All the incubations were done with cells in suspension and carried at 4°C. Small aliquots of cells were submitted to cytocentrifugation and then analyzed using an immunofluorescence microscope (Olympus, Melville, NY) or a confocal system in an inverted microscope (PCM 2000; Nikon, Melville, NY). Acquired images were later assembled using the Microsoft PowerPoint program. In experiments analyzing intracellular as well as extracellular CCR5 expression, MDMs were first stained for cell-surface CCR5 as described above and then were incubated with a fixing and permeabilizing solution (Cytofix/Cytoperm; PharMingen, San Diego) for 20 min, washed twice with Perm/Wash (PharMingen) solution, and then incubated again with unlabeled 2D7 Ab for 30 min, always in the presence of the permeabilizing solution. The cells were washed and incubated with a rhodamine-labeled secondary Ab for 30 min. The cells were extensively washed and resuspended in PBS. Cells were processed and analyzed for fluorescence as described above.
RNase protection assay
Total RNA was isolated (Ultraspec RNA isolation system, Biotecx Laboratories, Houston, TX) from macrophages treated with LPS or medium alone at different time points after the treatment, and 2 µg of RNA was used in an RNase protection assay (Riboquant, PharMingen). Samples were separated by SDS-PAGE and quantified by electronic radiography in a Direct Imaging system (Packard).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A previous study demonstrated that LPS, when present both at the
time of infection of macrophages with HIV-1 and during subsequent
cultivation of infected cells, potently inhibited HIV-1 infection
(27). To determine whether continuous exposure to LPS was
required for this effect or whether brief treatment of macrophages with
LPS might be sufficient to induce an antiviral state, MDMs were exposed
to LPS for 2 h, then washed extensively, and cultured for various
periods in fresh medium without LPS until the time of infection (time
0) with HIV-1ADA. A 2-h preincubation with LPS
was sufficient to produce a profound and lasting inhibition of HIV-1
infection (Fig. 1
). HIV infection was
assessed by PCR with primers specific for linear forms of the viral DNA
(primers LTR/gag) because formation of 2 LTR-circles
(markers of intranuclear localization of the HIV-1 DNA (32, 33)) was inhibited below the levels of detection. A similar
inhibition was observed with primers specific for the early products of
reverse transcription (primers U3/R; data not shown), indicating that
pretreatment with LPS inhibited a very early step (entry or initiation
of reverse transcription) in macrophages. Thus, brief (2-h) exposure of
MDMs to LPS renders them resistant to HIV-1 infection for a period
lasting at least 24 h.
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and MIP-1ß
binding in a time- and dose-dependent manner
Because CCR5 expression is required for HIV-1 infection of MDMs,
ligand binding studies were undertaken to investigate whether
cell-surface CCR5 expression was regulated by LPS treatment. MDMs were
first treated with increasing concentrations of LPS and then analyzed
for [125I]MIP-1 binding. A dose-dependent
decrease of [125I]MIP-1 binding was observed
with increasing LPS concentration (Fig. 2a). There is some donor
variability in LPS sensitivity, therefore LPS at 100 ng/ml (a dose that
abrogated binding and calcium signaling in nearly every donor) was used
for all additional experiments. As shown in Fig. 2b,
LPS-induced down-regulation of MIP-1 binding also correlated with
time of exposure to LPS, and substantial inhibition of MIP-1 binding
occurred as early as 1.5 and 2 h after the addition of
LPS.
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binding receptor, CCR1, and the total
[I125]MIP-1 binding activity observed in
Fig. 2
, a and b, might be attributable to binding
to one or both of those receptors (34). To specifically
determine the fate of CCR5 after LPS stimulation, parallel binding
studies with [I125]MIP-1ß, which has been
reported to bind exclusively to CCR5 receptors, were performed
(4). As can be observed in Fig. 2c,
preincubation of macrophages with LPS resulted in a 90% reduction of
MIP-1ß binding, confirming that CCR5-mediated chemokine binding was
down-modulated as a consequence of exposure to LPS.
LPS-mediated inhibition of MIP-1 binding is maintained for an
extended period of time compared with chemokine-induced down-regulation
To study the kinetics of receptor down-regulation and reappearance
at the cell surface, MDM cultures were incubated with either LPS or a
mixture of chemokines, and receptor binding was analyzed as a function
of time (Fig. 3,
a–c). A 3-h incubation with LPS resulted in
complete inhibition of [125I]MIP-1 binding
for up to 24 h after LPS was removed from the cell culture. About
40% of [125I]MIP-1 binding activity was
recovered by 48 h, and by 96 h MIP-1 binding activity was
fully restored. When cells were rechallenged with LPS 5 days after
primary challenge, cell-surface CCR5 was again down-regulated (120-h
point; Fig. 3a). To rule out the possibility that these
LPS-induced changes in cell-surface CCR5 expression were a reflection
of LPS-induced cytotoxicity, the viability of untreated and LPS-treated
cultures were compared. No significant effect on cell viability was
observed up to 5 days after LPS stimulation (at 100 ng/ml) as measured
by MTT enzymatic assay (Thiazolyl blue) or neutral red viability assay
(data not shown).
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binding
induced by LPS, ligand-mediated down-regulation of MIP-1 binding,
though nearly 90% at time 0 (Fig. 3b), was rapidly reversed
after the removal of chemokines (Fig. 3c). The inhibition of
MIP-1 binding observed upon restimulation with a fresh mixture of
chemokines 5 days (120 h) after removal of the initial
chemokine-conditioning stimulus shows that the cultures had fully
regained their chemokine sensitivity (see Fig. 1). LPS abrogates calcium signaling responses to chemokines and prolongs cell desensitization to chemokines
To assess whether LPS-mediated down-regulation of the
ligand-binding activity of CCR5 chemokine receptor affected
intracellular calcium signaling induced by chemokine binding, calcium
concentrations were estimated in LPS-treated macrophages in response to
MIP-1 or MIP-1ß by a fluorometric method. Preincubation of cells
with LPS for 2 h completely abrogated the calcium response
elicited by MIP-1 (Fig. 4a)
or MIP-1ß (Fig. 4b). To evaluate the kinetics of this
effect, calcium signaling studies were performed at different times
after an LPS preincubation period. As illustrated in Fig. 4c, the cells exposed to LPS remained unresponsive to
exogenous chemokines 24 h after LPS exposure (at which time
chemokine binding is still undetectable (Fig. 3a)), and an
attenuated response was seen after 48 h. By 72 h after LPS
exposure, calcium signaling responses to exogenous chemokine (MIP-1ß)
were approaching normal. Therefore, LPS reduces the number or the
functional activity of CCR5 receptors on macrophages.
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To directly analyze the effect of LPS treatment on CCR5 expression
at the single-cell level, macrophages were stained with FITC-labeled
mAb (2D7) specific for CCR5 protein to identify cell-surface receptors.
The same cells were then permeabilized and stained with unlabeled 2D7
and then with secondary rhodamine-labeled anti-mouse Ig to detect
newly exposed intracellular CCR5. Samples were evaluated by
fluorescence microscopy and quantitatively analyzed using an imaging
system (Metamorph; Universal Imaging, West Chester, PA). Fig. 5 compares dual staining in untreated
(top panel) and LPS-treated cells (bottom
panel) and shows a substantial decrease in membrane staining
(green) in conjunction with stronger intracellular staining (red) in
LPS-treated cells. Therefore, we conclude that LPS induces
internalization of CCR5.
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a (compare left
panel and middle panel), a 3-h incubation with
chemokines results in down-regulation of cell-surface CCR5. Similar
results are observed after a 3-h incubation with LPS (compare
left panel and middle panel of Fig. 6b). But surprisingly, cell-surface expression of CCR5 was
still completely absent in the LPS-treated cells 24 h after
removal of LPS, whereas surface expression in ligand-treated cells was
recovered 24 h after chemokine was removed from the media. To
further characterize the prolonged LPS-induced down-modulation of CCR5
expression, MDM cultures were stimulated with LPS for 3 h, washed,
and then replenished with fresh media. Twenty-four hours after removal
of LPS, cells were permeabilized and stained for CCR5 by first
incubating with unlabeled primary Ab (2D7) and then incubating with a
rhodamine-labeled secondary anti-IgG Ab. The intensity of
intracellular staining for CCR5 observed in the cell population that
had LPS removed 24 h earlier was comparable to the group that was
stained immediately after the 3-h incubation with LPS (Fig. 6c). The control represents untreated cells that show a
stronger CCR5 staining at the membrane. These results demonstrate that
CCR5 surface receptors remain in the cytoplasmic compartment for a
prolonged period of time after LPS-induced internalization and suggest
that the receptors are not being targeted for degradation for at least
24 h after LPS stimuli is removed.
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To determine whether LPS-induced down-regulation of CCR5
correlated with susceptibility of macrophages to HIV-1 infection, we
inoculated LPS-stimulated MDM cultures with HIV-1 at various intervals
after pretreatment with LPS. Viral entry was evaluated by PCR using
primers specific for the early products of reverse transcription
(R/U5). As shown in Fig. 7,
susceptibility of cells to HIV-1 infection was inhibited during the
first 24 h after LPS stimulation but returned to control levels by
72 h, which is in good correlation with the kinetics of CCR5
recovery. In additional experiments in which the 48-h time point was
analyzed, viral entry was still 50% inhibited compared with control
(data not shown).
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LPS induces general stimulation of macrophages and promotes, among
many other activation-specific responses, secretion of the
proinflammatory cytokines TNF and IL-1 as well as of the chemokines
MIP-1 and MIP-1ß. These LPS-induced mediators could modulate
macrophage expression of CCR5 in an autocrine manner, as has been
suggested previously (35). Our observation that after a
1-h exposure to LPS (at a time when MIP-1 peptide levels were
undetectable by ELISA; data not shown) MIP-1 binding was inhibited
by 60% (Fig. 2b) suggested that the down-regulation
occurred too rapidly to be mediated by chemokine secretion, which
occurs over a 3- to 6-h time course. To further investigate possible
mechanisms by which LPS might affect CCR5 receptor expression and
activity, cells were incubated with either media alone or LPS in the
presence or absence of the protein synthesis inhibitor cycloheximide.
Binding activity for [I125]MIP-1ß was then
assessed as a measure of cell-surface CCR5 expression. Cells treated
with LPS showed significantly decreased binding of MIP-1ß, whether
stimulated with LPS in the presence or absence of cycloheximide (Fig. 8a). Control cells not exposed
to LPS exhibited the same MIP-1ß binding activity in the presence or
absence of cycloheximide. Further addition of a chemokine mixture
containing MIP-1, MIP-1ß, and RANTES at 10 nM each to cultures
exposed to LPS in the presence of cycloheximide did not further reduce
MIP-1ß binding (Fig. 8a). Therefore, the LPS-mediated CCR5
down-regulation in macrophages as a consequence of LPS exposure does
not require synthesis of new protein.
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binding
studies were performed on MDMs that were incubated with LPS for 3
h, washed free of LPS, and incubated for 24 h, at which time they
were again washed and replenished with medium alone or medium
containing cycloheximide for an additional 3 h. In case secreted
chemokines were involved in CCR5 down-regulation, it would be expected
that the last 3-h incubation without cycloheximide would result in
receptor down-regulation, whereas in the presence of cycloheximide no
CCR5 down-regulation would be observed. However, comparable levels of
inhibition of MIP-1 binding between the MDMs replenished with medium
in the presence or absence of cycloheximide was observed (Fig. 8b). This result demonstrates that new protein synthesis is
not required for the LPS-induced prolonged down-regulation of
CCR5. LPS mediates a slight early down-regulation and a more pronounced late up-regulation of the mRNA for CCR5
To determine the effect of LPS treatment on CCR5 mRNA
expression, the levels of CCR5 mRNA were analyzed in LPS-stimulated
macrophage cultures and compared with CCR5 mRNA levels in cells not
exposed to LPS. Levels of CCR5 mRNA were found to be slightly decreased
after LPS stimulation, showing a return to normal levels by 7 h
after the addition of the endotoxin stimulus (Fig. 9). From this time until 75 h after
LPS stimulation, message levels were higher than those in controls. In
all experiments, a decrease in CCR5 mRNA was also observed for the
control at the 3-h time point, returning to normal levels at later time
points. All results were normalized according to GAPDH mRNA signals.
Thus, we conclude that LPS-induced down-regulation of CCR5 is not
mediated by reduction of CCR5 mRNA expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Consistent with the above results, LPS added together with HIV-1 in macrophage cultures has been shown to increase viral entry into those cells, as assessed by accumulation of early products of reverse transcriptase (27). The observed increase in viral entry under these conditions might be explained by the overt LPS-induced internalization of CCR5, which would facilitate the transport of surface-bound HIV-1 to the intracellular compartment.
One possible explanation for the loss of cell-surface CCR5 seen after
LPS treatment is that chemokines, which are secreted by the macrophages
as a consequence of LPS stimulation, act in an autocrine fashion to
down-modulate their own receptors. And, in fact, a recent report
suggests that LPS-induced down-regulation of cell-surface CCR5 on
macrophages is mediated by CC chemokines released as a consequence of
activation (35). Our results argue against this
hypothesis. Indeed, a 50% reduction of MIP-1 binding was observed
as early as 1 h after the addition of LPS (Fig. 2b),
when no chemokines were detected in the culture medium (data not
shown). This suggests that de novo protein synthesis is not required
for promoting the inhibitory effect. Further proof that down-regulation
of CCR5 is chemokine release-independent was obtained in an experiment
in which macrophages were stimulated with LPS in the presence of
cycloheximide, a specific inhibitor of protein synthesis. Cycloheximide
completely blocked synthesis and secretion of chemokines, as measured
by ELISA (data not shown). Still, LPS-induced down-regulation of CCR5
(as judged by MIP-1ß binding) in cycloheximide-treated cells was
comparable to that in control cultures and was not increased by
exogeneously added CC chemokine mixture (Fig. 8a),
indicating that LPS is directly responsible for the down-regulation of
CCR5. Interestingly, exogeneously added chemokines reduced binding of
MIP-1 to LPS-stimulated, cycloheximide-treated macrophages by
50% (data not shown). Because MIP-1 binds to CCR1 as well as to
CCR5 on macrophages, this result suggests that although for some
receptors (e.g., CCR5) direct LPS-induced internalization constitutes
the main mechanism of receptor deactivation, other receptors (e.g.,
CCR1) might be deactivated through secondary mechanisms such as
LPS-stimulated chemokine secretion.
Surprisingly, recovery of CCR5 on the cell surface after LPS-mediated
down-regulation was markedly delayed compared with receptor recovery
after exposure to a natural ligand, MIP-1. Indeed, 100% inhibition
of MIP-1 binding was observed 24 h after LPS treatment (Fig. 3a), whereas 80% of the original binding was restored
within 30 min after removal of MIP-1 (Fig. 3c). ß
Chemokine-induced CCR5 down-regulation and recovery was similar to that
observed for CXCR4 in response to stromal cell-derived factor-1
(38). The delay in CCR5 surface reexpression after LPS
treatment did not appear to be due to continuous down-regulation of
chemokine receptors by LPS-induced release of chemokines in long term
culture. This was demonstrated by the sustained down-regulation of
MIP-1 binding in MDM cultures that had been stimulated with LPS
24 h in advance of receiving fresh media containing cycloheximide
for 3 h before carrying out the binding experiments. If chemokine
exposure were responsible for down-regulation of CCR5 surface
expression, cyclohexamide would be expected to block further chemokine
expression and 3 h should have sufficed to allow reappearance of
CCR5 at the cell surface. The failure of MIP-1 binding to be
restored after LPS-induced mediators were removed from the culture and
synthesis of new inflammatory mediators was blocked by cycloheximide
demonstrated that the observed prolonged down-regulation of CCR5 is not
chemokine-dependent (Fig. 8b). Although we have observed
donor variability in both cell-surface expression of CCR5 and magnitude
of calcium signaling response after chemokine stimulation, sustained
LPS-dependent down-regulation of surface CCR5 was observed in all
donors tested.
Immunofluorescence analysis of CCR5 receptor expression (Figs. 5 and 6)
immediately after and 24 h after chemokine or LPS treatment
indicate that CCR5 is rapidly internalized in response to both stimuli
but that in the case of ligand-induced internalization, cell-surface
receptor expression is quickly restored, whereas in the case of
LPS-induced internalization, the receptor remains undegraded and
sequestered within the cell for >24 h. These results suggest that LPS
treatment induces an alternate route of intracellular trafficking of
the receptors after internalization. It is important to note that the
striking difference in the recovery rate of cell-surface CCR5 after
LPS- vs chemokine-induced down-regulation might be even more
significant if we consider that the recovery of surface receptors after
chemokine treatment apparently occurs between 45 and 60 min, as
suggested by earlier binding studies (Fig. 3c). This is not
an unprecedented phenomenon because a similar inhibition of CCR5
recycling after internalization recently has been reported for a
modified form of RANTES (aminooxypentane-RANTES) (39).
These results suggest that chemokine receptors can undergo differential
intracellular pathways after the internalization process, depending on
the nature of the triggering stimuli.
Although the molecular mechanisms by which LPS down-regulates surface
expression of chemokine receptors are not yet defined,
immunofluorescence analysis (Fig. 5) suggests that they involve
internalization of receptors. It has been documented that LPS, via
activation of p38 mitogen-activated protein kinase, can induce
rearrangement of actin filaments and thus promote endocytosis
(40, 41). Experiments analyzing whether CCR5 colocalizes
with markers of endosomes and/or lysosomes are in progress and are
expected to reveal whether LPS-mediated down-regulation of this
receptor occurs via enhanced endocytosis or whether another mechanism
is involved. In addition, such experiments may elucidate the fate of
the receptor after its internalization.
Interestingly, in contrast to previous work by others
(42), there was no significant decrease in CCR5 mRNA
levels in LPS-treated compared with control macrophages (Fig. 9). On
the contrary, message levels for CCR5 progressively increased starting
7 h after LPS stimulation, returning to levels comparable with
controls in the subsequent 30 h. This temporary increase in CCR5
mRNA may be important for eventual replenishing of the receptor on the
cell membrane, which is observed around 96 h after the removal of
LPS. The absence of CCR5 mRNA down-regulation supports the hypothesis
that LPS-induced down-modulation of CCR5 is through inhibition of
recycling and/or trafficking of the receptors to the membrane.
The results presented herein suggest a model whereby CCR5 receptors on
macrophages are down-modulated in response to LPS exposure
independently of autocrine chemokine stimulation, leading to resistance
to HIV-1 infection. In addition, the data strongly suggest the use of a
pathway of chemokine receptor recycling that differs from recycling
initiated by ligand binding and delays the return of biologically
active CCR5 to the cell surface. In a recent publication, LPS was shown
to induce a prolonged down-modulation of CXCR1 and CXCR2 in macrophages
through a tyrosine kinase-dependent pathway (43). Whether
such a pathway is involved in LPS-dependent CCR5 down-modulation
remains to be evaluated. Preliminary experiments in our lab indicate
that LPS-mediated down-modulation of surface CCR5 is a specific effect
because the cell-surface expression of the FMLP receptor, another G
protein-coupled seven-transmembrane receptor, was not affected by LPS
treatment (data not shown). In addition, our results suggest that CCR1
and CCR5 (receptors for MIP-1) have different sensitivity to LPS. It
will be important to determine whether this effect is restricted to LPS
or whether other macrophage activators elicit the same response. Our
results raise the important clinical question of whether certain states
of tissue macrophage activation (e.g., concomitant Gram-negative
bacterial infection) render such cells more resistant to HIV infection,
thus slowing the spread of the virus. Further studies on the
elucidation of the unique intracellular pathway that CCR5 undergoes as
a consequence of LPS stimulation will provide a more complete
understanding of the intricacies of CCR5 receptor regulation and may
reveal new targets for anti-HIV therapies.
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Acknowledgments |
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Footnotes |
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2 Current address: Pfizer Central Research, Eastern Point Roard,Groton, CT 06340.
3 Current address: Molecular Infection Biology, Research Center Borstel, Parkallee 22, D-23845 Borstel, Germany.
4 Address correspondence and reprint requests to Dr. Barbara Sherry, Picower Institute for Medical Research, 350 Community Drive, Manhasset, NY 11030. E-mail address:
5 Abbreviations used in this paper: MDM, monocyte-derived macrophage; M-CSF, macrophage colony-stimulating factor; MIP, macrophage-inflammatory protein; LTR, long terminal repeat.
Received for publication August 17, 1999. Accepted for publication December 15, 1999.
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