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The 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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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bacterial LPS, the main constituent of the outer leaflet of the cell
wall of Gram-negative bacteria (10), is a major stimulus for
macrophages. LPS was reported to activate three different
MAPK3 families, i.e., ERK,
JNK/SAPK, and p38, in macrophages (11, 12, 13, 14, 15). Among these kinases, p38
plays an important role in transducing activating signals from LPS
receptor, CD14, to TNF-
promoter (16). This effect is probably
mediated by p38-induced activation of specific transcription factors,
such as ATF2 (17, 18, 19). Activated p38 phosphorylates MAPK-activated
protein kinase 2 and/or MAPK-activated protein kinase 3, which, in
turn, phosphorylate small heat shock protein, hsp27 (18, 20, 21). This
modulates dynamics of actin filaments (22, 23, 24), which may affect
various cellular activities, including phagocytosis and chemotaxis. The
macrophage response to LPS can protect the host from infection, but it
can also cause tissue injury, cachexia, circulatory collapse,
multiorgan failure, and death (25, 26, 27). The activation of macrophages
is only deleterious at the extremes and normally forms part of the
protective immune response. Activated macrophages recognize and kill
malignant tumor cells and microorganisms more effectively than
quiescent cells, a capacity that can be manipulated
therapeutically (28).
Consistent with this concept, LPS has been shown to protect primary macrophages from productive infection by HIV-1 in vitro (29, 30, 31, 32). In contrast, activation by LPS has also been reported to stimulate HIV-1 expression in monocytoid tumor cell lines (33). Most of these studies relied on the measurement of the release of virus particles into the culture supernatant for evaluation of LPS-mediated effects. However, the level of virus production is determined to a large extent by the efficiency of the first steps in virus replication, such as entry, RT, and nuclear import of the viral preintegration complex (PIC). Thus, we focused on the analysis of LPS-mediated effects at the early stages of HIV-1 infection of human macrophages. In this report we show that addition of LPS during infection resulted in an increase in virus entry, followed by a marked reduction of late RT products and inhibition of nuclear translocation of the viral PIC. This inhibitory effect was mediated via activation of the p38 MAPK and was reversed by compounds that block this pathway. These results suggest new targets for interference with HIV-1 infection of macrophages.
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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) (34). HIV-1 primary isolates 92US660, 92US657, and 92UG021 were obtained from the AIDS Research and Reference Reagent Program (Rockville, MD). LPS (Escherichia coli 0111:B4), lipid A (Salmonella minnesota RE595), and polymyxin B sulfate were obtained from Sigma (St. Louis, MO). Compound 406 was a gift from Dr. A. J. Ulmer, Department of Immunology and Cell Biology, Research Center Borstel (Borstel, Germany). Anti-CD14 (MEM18) was purchased from Sanbio (AM Uden, The Netherlands). SB203580 and SB202474 were obtained from Alexis and Calbiochem (both in San Diego, CA). CNI-1493 was provided by Dr. K. J. Tracey, The Picower Institute for Medical Research (Manhasset, NY).
Isolation and differentiation of human MDMs
Human PBMC 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). PBMC were cultured in DMEM supplemented with 10% heat-inactivated human serum (BioWhittaker, Walkersville, MD), 2 mM glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin (all from Life Technologies, Gaithersburg, MD) at 37°C in 5% CO2. After 18 h, adherent cells were detached with ice-cold 10 mM EDTA/PBS and cultured in 24-well Primaria plates (Becton Dickinson, Franklin Lakes, NJ) for 7 days in the presence of recombinant human macrophage CSF (2 ng/ml; Sigma) at a density of 106 cells/ml. Cells were >98% macrophages as judged by morphology and nonspecific esterase activity.
Infection with HIV-1
The macrophage-tropic strain HIV-1ADA was grown on PBMCs. Before infection, the 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. Seven days after isolation, adherent macrophages were infected for 2 h at 37°C with an amount of virus corresponding to 1 x 105 cpm reverse transcriptase activity/1 x 106 cells. After infection, unabsorbed virus was washed away, and cells were cultured in the medium without macrophage CSF.
DNA PCR
Infected macrophages (106 cells) were resuspended in 200 µl of 1x PCR buffer (50 mM KCl, 10 mM Tris-HCl (pH 8.3), 3.5 mM MgCl2, and 0.1 mg/ml gelatin) supplemented with 0.45% Nonidet P-40, 0.45% Tween 20, and proteinase K (500 µg/ml; Boehringer Mannheim). Samples were digested overnight at 56°C, and proteinase K was inactivated at 95°C for 10 min. For each PCR replicate, 25 µl of sample was added to 25 µl of PCR mix containing 2.5 µl of 10x PCR buffer, 1 µl each of sense and antisense primers (from a 10-µM stock), 1 µl of 10 mM deoxynucleotide triphosphates (Life Technologies), 19.25 µl of H2O, and 1.25 U of Taq polymerase (Perkin-Elmer, Norwalk, CT). The primers used in this study have been described recently (35).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several previous reports demonstrated that LPS inhibits
replication of HIV-1 in long term cultures of MDMs (29, 30, 31, 32). Consistent
with these results, we observed that virus replication, as measured by
the amount of viral reverse transcriptase activity released into the
culture medium, was blocked when LPS was added to the cells together
with the virus (Fig. 1
A). To
evaluate the effect of LPS on preintegration steps of HIV-1
replication, we used PCR analysis of virus-specific DNA. Monocytes from
healthy, HIV-seronegative blood donors were differentiated into
macrophages in vitro by adherence to plastic (34). After 7 days in
culture, MDMs were infected with HIV-1ADA in the absence or
the presence of increasing concentrations of LPS (0.01–10 ng/ml) for
2 h (Fig. 1B). MDMs were harvested 48 h
postinfection, which is the time required to complete RT and nuclear
import of the viral PICs in MDMs (36), and 2LTR circle forms of the
viral DNA were amplified by PCR. These DNA forms are produced
exclusively inside the nucleus after completion of RT and nuclear
import (37) and are convenient markers in the analysis of
preintegration steps of HIV-1 replication (38). As a control for the
amount of DNA in each sample, PCR using tubulin-specific primers was
performed in parallel on each sample (not shown). Results were scored
in the linear phase of amplification, as demonstrated by PCR performed
on serial dilutions of HIV-1-infected H9 cells (Fig. 1B, upper panel). After Southern blotting, the
products of the PCR reaction were revealed by autoradiography (Fig. 1B, upper panel) and quantified on an Instant
Imager (bar graph). As shown in Fig, 1B, LPS
inhibited the appearance of the 2LTR circle forms of HIV-1 DNA in a
dose-dependent manner. Maximal inhibition (80–90%) was typically
observed at LPS concentrations from 1–10 ng/ml, in good correlation
with the results obtained with long term cultures (Fig. 1A).
The results varied to some extent between different donors. Even at the
low concentration (10 pg/ml), LPS consistently reduced the amounts of
viral 2LTR circle DNA forms by >30%. This effect was not specific for
the strain HIV-1ADA, as the same inhibition was observed
with primary NSI isolates 92US660 and 92US657 (not shown). Because the
sensitivity to the inhibitory effect of LPS was donor dependent, a
concentration of 10 ng/ml was used in all additional experiments to
ensure maximal inhibition. To control for possible cytotoxic effects
due to simultaneous exposure of macrophages to both LPS and
HIV-1, we used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide assay. No difference in the viability of HIV-1-infected MDMs
with or without LPS was observed.
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To define the effect of LPS in more detail, we followed the fate
of the entering virus using PCR primers specific for the early,
intermediate, and late products of viral RT and for the intranuclear
forms of viral DNA, thus monitoring the stages from viral entry to
nuclear import. LPS was added to cells either together with the virus
or 2 h after infection. For the analysis of early RT products,
cells were harvested 6 h postinfection, and viral DNA was analyzed
with primers (U3/R) specific for the strong stop HIV-1 cDNA that is
produced early in infection cycle (35, 39). Regardless of whether LPS
was present only during infection or was added 2 h after
infection, a dramatic, more than threefold, increase in the amount of
early RT products was observed (Fig. 2A). LPS-enhanced HIV-1 entry
was still dependent on virus interaction with receptors, as Ab to CD4
blocked entry into both unactivated and activated macrophages (not
shown). In addition, treatments that down-regulate HIV receptors (e.g.,
pretreatment of macrophages with RANTES or LPS) also blocked HIV-1
entry (G. Franchin, B. Sherry, and M. B., manuscript in
preparation). These results suggest that LPS promotes entry of the
receptor-bound virus into human macrophages.
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70%) of
pol-specific PCR products in all LPS-treated samples (Fig. 2
B). An even stronger inhibition (up to 90%) was observed
at the level of nuclear HIV-1 DNA, detected 48 h after infection
using primers specific for the 2LTR circle DNA (Fig. 2C).
Taken together, these results suggest that addition of LPS during or
immediately after infection stimulates virus entry, but strongly
inhibits RT and nuclear import of the viral PIC. Disruption of the activation pathway restores HIV-1 infection of LPS-treated macrophages
The inhibitory effect of LPS on HIV-1 infection was probably
mediated by LPS-induced signaling events. LPS initiates intracellular
signaling through the CD14 molecule, a
glycosylphosphatidylinositol-anchored membrane protein of myeloid cells
(40). Although other surface proteins have been suggested to function
as LPS receptors, CD14 is the only protein of fully defined structure
that binds LPS and mediates LPS-induced cell activation. To investigate
the role of CD14-mediated LPS signaling in the inhibition of HIV-1
infection of MDMs, we used inhibitors of LPS-CD14 interaction. Compound
406, also called precursor Ia or lipid IVa, is a precursor in the
synthesis of lipid A and has been shown to bind to CD14. In contrast to
lipid A, compound 406 has no biologic activity, but effectively
inhibits the effects of LPS, such as induction of cytokine production
in human monocytes (41). Preincubation of MDMs with compound 406 for 30
min before the simultaneous addition of LPS (10 ng/ml) and
HIV-1ADA completely eliminated the inhibitory effect of LPS
on HIV-1 infection, as measured by 2LTR circle-specific PCR (Fig. 3A). As a control, compound
406 alone had no effect on infection. A similar result was obtained
with a mAb MEM-18 directed against CD14 (Fig. 3B). This Ab
has been shown to specifically block LPS binding and signaling
(42). Together, these results suggest that CD14-mediated signal
transduction events regulate the LPS-mediated effect on HIV-1
infection of human macrophages.
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A). Interestingly, the
inhibitor alone had a weak stimulatory effect on HIV-1 infection (see
below). Cell viability in the presence of the inhibitor (used at 1
µM) was measured by trypan blue exclusion and by a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and
was 95% of the viability of control (untreated) cultures. In addition,
the fact that cells treated with the compound supported higher levels
of virus replication than untreated cells attests to their high
metabolic activity and viability.
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B). Cell viability was 93% that
of control cells. Taken together, these results indicate that
activation of macrophages, and of the p38 MAPK pathway in
particular, is critical in mediating the inhibitory effect of LPS on
the preintegration steps of HIV-1 infection of human macrophages. Inhibition of p38 MAPK stimulates replication of HIV-1 in human macrophages
Since it has been reported that compound SB203580 blocks
transcriptional activation of the HIV-1 LTR by cytokines and stress
(46), while we observed a stimulatory effect on the preintegration
steps of viral infection (Fig. 2A), we investigated the
cumulative effect of this compound on HIV-1 replication in MDMs.
Macrophages were infected with HIV-1ADA or
HIV-192UG021 (a primary SI isolate) in the presence of
SB203580 or its inactive control, SB202474. The amount of the virus
released into the medium was determined by reverse transcriptase
activity in culture supernatants. Figure 5 shows that SB203580 stimulated
replication of both virus isolates (HIV-1ADA by more than
twofold and HIV-192UG021 by about fivefold). A similar
stimulation of virus replication was observed with the compound
CNI-1493 or when LPS and inhibitor were added together (data not
shown). These results suggest that, in contrast to infection of primary
T cells (45), inhibition of p38 MAPK (and of macrophage activation)
enhances virus replication, most likely through stimulation of virus
entry.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A recently published report (51) demonstrated that inhibition of p38 reduces IL-1ß-induced HIV-1 production by promonocytic cell line U1. This seeming conflict with our results is probably explained by the fact that the U1 cell line represents undifferentiated monocytes that contain two copies of integrated defective HIV-1 genome. Therefore, early (preintegration) steps of viral infection cannot be studied on these cells. As the inhibitory effect of LPS on HIV-1 replication (mediated, at least in part, by p38) occurs at the step of virus entry, it conceals any stimulatory effect of p38 activation at the later step. In addition, transcription of the wild-type HIV-1 genome in primary macrophages might be regulated differently from that of a Tat-defective virus (52) in the U1 cell line and might depend less on p38 activation. These differences are underscored by diametrically opposite effects of LPS on HIV-1 replication in primary macrophages (29, 30, 31, 32) and in U1 cells (33).
It has been shown recently (31) that preexposure of macrophages to LPS
suppresses HIV-1 infection through the release of C-C chemokines, which
can compete with the virus for the CCR5 receptor. This mechanism,
however, is unlikely to account for the effects described in this study
for several reasons. First, we added LPS to cells during or after
addition of the virus, thus allowing virus-receptor interaction to
occur. Second, in contrast to chemokine-mediated inhibition (53, 54),
we observed an enhancement, rather than an inhibition, of virus entry
in LPS-treated macrophages (Fig. 2A). Third, the low LPS
concentrations used in our experiments (100-fold lower than those used
by Verani et al. (31)) did not induce the high levels of C-C chemokines
required for inhibition of HIV-1 entry into macrophages (35) (results
not shown).
Restriction of infection in activated macrophages appears to occur at the postentry step, but before integration. To monitor these steps of HIV-1 replication, we used PCR analysis of HIV-1 DNA. The 2LTR circle forms of viral DNA were used as a marker for successful completion of RT and translocation of the PICs into the nucleus. Because these HIV-1 DNA forms are produced exclusively inside the nucleus (55), they are an excellent indicator for the completion of preintegration steps. Although previously this approach has been used to monitor specifically nuclear importation of the viral PIC under one-cycle conditions (38, 56), there is a good correlation between the efficiency of production of 2LTR circle DNA forms and the level of HIV-1 replication in a long term culture (57, 58, 59, 60, 61). With PCR-based analyses there is always a risk of contaminating material. In our case, macrophage populations contained 2% of contaminating cells, some of which might be HIV-1-susceptible CD4+ T lymphocytes. However, as we have demonstrated previously (35), the contribution of T cells to HIV-1 replication in our culture conditions is minimal.
Our results indicate that whereas HIV-1 entry into activated
macrophages was enhanced, subsequent steps of replication (RT and
nuclear import of the PIC) were greatly inhibited (Fig. 2). The most
likely explanation of this result is that LPS treatment switched
internalization of receptor-bound virus from a normally used fusion
between the viral and cellular membranes to endocytosis. This
hypothesis is consistent with the previously reported LPS-mediated
increase in phagocytic and endocytic activity of MDMs (62). In
addition, LPS has previously been reported to rapidly reduce the number
of binding sites for MCP-1, the ligand for CCR2 (63). LPS also
down-regulates CCR5 (J. Franchin, B. Sherry, and M. Bukrinsky,
unpublished result), most likely via internalization of surface
molecules.
While internalized virus was able to initiate RT (Fig. 2A),
subsequent steps of replication in activated macrophages were
suppressed (Fig. 2, B and C), suggesting that
virus particles did not gain access to the right subcellular
compartment that normally supports viral RT and nuclear import. As a
result, LPS-treated macrophages became resistant to productive
infection.
The effect of LPS on HIV-1 infection appears to be mediated by
signaling from the CD14 molecule (Fig. 3), the principal receptor
for LPS (40, 42). One of the best documented pathways of LPS-induced
intracellular signal transduction is the
ras/raf/p38 MAPK pathway, by which p38 MAPK is
phosphorylated rapidly during activation with LPS and is crucial in
mediating LPS-specific effects (14, 43). It was thus conceivable that
the LPS effect on HIV-1 infection was also mediated by p38. Consistent
with this hypothesis, preincubation of MDMs with a specific inhibitor
of p38 MAPK activation, compound SB203580, restored HIV-1 infection in
the presence of LPS (Fig. 4A), indicating the central role
of p38 MAPK in LPS-induced resistance to HIV-1 infection. Additional
experiments will be necessary to determine the link between activation
of the p38 MAPK by LPS and inhibition of virus replication, but two
major mechanisms may be envisioned. First, LPS, via activation of p38,
might induce rearrangement of actin filaments (22, 23, 24), thus
stimulating virus internalization via endocytosis. Virus internalized
via this mechanism would be targeted to lysosomes, where it would be
rapidly degraded. This mechanism would explain both the enhanced uptake
and the decreased RT/nuclear import observed after LPS treatment.
Second, p38 activation might directly affect viral RT and/or nuclear
import through phosphorylation of downstream targets of p38. For
instance, another member of the MAPK family, ERK, has been shown
recently to phosphorylate HIV-1 matrix protein (64), thus regulating
PIC formation and RT.
The idea of LPS-induced resistance is consistent with a recent report (65) demonstrating that macrophages isolated from mucosal surfaces of the gastrointestinal tract, where exposure to bacterial LPS is very likely, showed reduced susceptibility to HIV-1 infection compared with blood-derived monocytes. Of course, our results should not be interpreted as an indication that LPS or bacterial infections can help fight HIV-1 disease, since such treatments lead to the secretion of proinflammatory cytokines and give rise to activated T cells that are highly permissive for HIV-1. However, alternative, less radical, methods of macrophage stimulation, especially those that target the p38 MAPK pathway, might prove useful in future therapeutic and vaccine approaches.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael Bukrinsky, The Picower Institute for Medical Research, 350 Community Dr., Manhasset, NY 11030. E-mail address:
3 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; PIC, preintegration complex; MDM, monocyte-derived macrophage; LTR, long terminal repeat.
Received for publication May 14, 1998. Accepted for publication September 21, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, MIP-1ß receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272:1955.[Abstract]
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