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The Journal of Immunology, 2004, 173: 6735-6744.
Copyright © 2004 by The American Association of Immunologists

Mechanisms for Macrophage-Mediated HIV-1 Induction

Krishnakumar Devadas1,*, Neil J. Hardegen{ddagger}, Larry M. Wahl§, Indira K. Hewlett*, Kathleen A. Clouse{dagger}, Kenneth M. Yamada and Subhash Dhawan1,*

* Immunopathogenesis Section, Laboratory of Molecular Virology, Center for Biologics Evaluation and Research, and {dagger} Laboratory of Cell Biology, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892; and {ddagger} Cellular Immunology Section, Oral Infection and Immunity Branch, § Immunopathology Section, Developmental Mechanisms Section, Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Viral latency is a long-term pathogenic condition in patients infected with HIV-1. Low but sustained virus replication in chronically infected cells can be activated by stimulation with proinflammatory cytokines such as TNF-{alpha}, IL-1 {beta}, or other host factors. However, the precise mechanism by which cellular activation induces latently infected cells to produce virions has remained unclear. In the present report, we present evidence that activation of HIV-1 replication in latently infected U1 or ACH2 cells by human macrophages is mediated by a rapid nuclear localization of NF-{kappa}B p50/p65 dimer with concomitant increased expression of proinflammatory cytokines. Multiplexed RT-PCR amplification of mRNA isolated from cocultures of macrophages and U1 and ACH2 cells showed significant induction of IL-1{beta}, IL-6, IL-8, TNF-{alpha}, and TGF-{beta} expression within 3 h of coincubation. Fixation of macrophages, U-1, or ACH2 cells with paraformaldehyde before coculture completely abrogated the induction of NF-{kappa}B subunits and HIV-1 replication, suggesting that cooperative interaction between the two cell types is an essential process for cellular activation. Pretreatment of macrophage-U1 or macrophage-ACH2 cocultures with neutralizing anti-TNF-{alpha} Ab down-regulated the replication of HIV-1. In addition, pretreatment of macrophage-U1 or macrophage-ACH2 cocultures with the NF-{kappa}B inhibitor (E)3-[(4-methylphenyl)sulfonyl]-2-propenenitrile (BAY 11-7082) prevented the induction of cytokine expression, indicating a pivotal role of NF-{kappa}B-mediated signaling in the reactivation of HIV-1 in latently infected cells by macrophages. These results provide a mechanism by which macrophages induce HIV-1 replication in latently infected cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Infection with HIV-1, the causative agent of AIDS, is characterized clinically by a long asymptomatic period of latency preceding the development of AIDS. Even during this period of clinical latency, the virus is continuously replicating and causing new rounds of infection. Recent studies using combination antiviral therapy have revealed a population of latently infected cells that are refractory to antiviral therapy, which is believed to be the leading cause of the persistence of infection seen in HIV-1 disease (1). Furthermore, there is evidence of ongoing virus replication even in patients on highly active antiretroviral therapy in whom viremia is suppressed to undetectable levels (2). Thus, a detailed study of the mechanisms that modulate viral replication during this clinically latent stage would help to further understand the pathogenesis of this disease and to develop targeted therapies for eradicating latently infected cells or preventing the activation of viral replication in these cells.

CD4+ T cells are thought to be the major reservoirs of both actively replicating and latent HIV-1 throughout the course of disease. Several in vitro studies have identified potentially stable reservoirs of inducible latently infected CD4+ cells carrying an integrated form of the viral genome (3, 4, 5). Recently, it was shown that integrated proviral DNA is present at low frequency in resting, memory CD4+ (CD45RO+, HLA-DR–) cells (6). Because memory CD4+ T cells can persist for months to years, these resting memory CD4+ cells carrying replication-competent viral genomes may represent an important long-term viral reservoir in patients (7). This population of latently infected cells has been clearly identified even in patients who have been on triple therapy for long periods (>30 mo) of time (8, 9), suggesting that these resting T cells provide a stable reservoir of HIV-1 for extended periods during the course of HIV-1 disease.

In addition to CD4+ T cells, monocytes are a major reservoir for HIV-1 in vivo, since a number of blood monocytes are maintained in HIV-1-infected patients even during late-stage disease when T cells may be practically undetectable (10, 11, 12). Unlike CD4+ T cells, monocytes are nonlytically infected. These cells serve as viral reservoirs in infected tissues, vectors for virus transmission to target cells, and sources of potent cytokines that can affect cell function and virus replication, and they thereby play a key role in the pathogenesis of HIV-1 disease. Peripheral blood monocytes that are latently infected in vivo can be activated by a number of agents to produce large quantities of virus (13). These observations suggest that infected macrophages (M{phi})2 may serve as a major reservoir for HIV-1, causing the persistence and spread of the virus.

The chronically infected, promonocytic cell line U1 harbors two copies of the HIV-1 provirus and under unstimulated conditions expresses low levels of viral transcripts encoding Tat, Rev, and Nef, but little or no full-length viral RNA (14, 15, 16). In a similar manner, the chronically infected T cell line ACH2 contains a single copy of the integrated HIV-1 provirus (16). Viral replication is enhanced in U1 cells following treatment with exogenous Tat, Nef, LPS, GM-CSF, or a variety of other factors (16, 17, 18, 19). The progeny viruses from U1 cells are defective and cannot infect susceptible cells. In contrast, ACH2 cells can produce viral particles that can infect susceptible T cells. Several studies have reported the activation of HIV-1 replication in these cells when cocultured with several other cell types, including endothelial cells (19, 20, 21, 22, 23, 24, 25, 26). However, conditioned medium from endothelial cells failed to up-regulate HIV-1 replication. Thus, the activation of HIV-1 replication by coculture required cell–cell contact. The activation of HIV-1 replication was preceded by the activation of NF-{kappa}B and cytokine secretion (27). It is probable that the activation of NF-{kappa}B by coculture could involve the induction of transcription factors such as C/EBP and other cellular factors (28). NF-{kappa}B plays a critical role in the activation of HIV-1 gene expression by cytokines and other stimuli, but the pathways that regulate the switch from latency to the infectious phase have not been elucidated.

Immune activation as a result of the host response to HIV-1 infection itself or the presence of exogenous stimuli has a profound effect on the life cycle of HIV-1. Persistent immune activation caused by the release of endogenous cytokines can result in increased HIV-1 replication and lead to phenotypic and genotypic changes in the virus. HIV-1 replication is dependent on the host transcriptional machinery and is closely regulated by a complex pathway of proinflammatory and immunoregulatory cytokines (29, 30). Some of these cytokines are known to activate NF-{kappa}B, which in turn can further induce long terminal repeat (LTR)2-directed gene expression (31, 32). For example, TNF-{alpha} secretion induced by the virus can up-regulate HIV-1 replication in an autocrine and paracrine manner in chronically infected cells (33, 34).

In this report, we have examined the factors associated with activation of HIV-1 replication in latently infected cells by cultured M{phi}. The coculture of ACH2 and U1 cells with uninfected M{phi} resulted in activation of HIV-1 replication. Cell–cell contact caused the rapid nuclear localization of NF-{kappa}B p65/p50 dimers with a concomitant increase in the expression of proinflammatory cytokines, suggesting a crucial role for M{phi} in the reactivation of HIV-1 in latently infected cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
M-CSF, anti-TNF-{alpha} Ab, anti-TRAIL Ab, and goat anti-rabbit IgG were purchased from R&D Systems (Minneapolis, MN). CytoXpress multiplex PCR primer pairs and TNF-{alpha} and GAPDH PCR primer pairs were obtained from BioSource International (Camarillo, CA). NF-{kappa}B inhibitor (E)3-[4-methylphenyl)sulfonyl]-2-propenenitrile (BAY 11-7082) was purchased from Calbiochem (San Diego, CA). Proteinase K and Pronase E were purchased from Sigma-Aldrich (St. Louis, MO).

Isolation and culture of monocyte-derived M{phi}

Monocytes were isolated from PBMC of donors seronegative for HIV-1 and hepatitis B after leukophoresis and were purified by countercurrent centrifugal elutriation (35). Cell suspensions contained >95% monocytes by criteria of cell morphology on Wright-stained cytosmears, by granular peroxidase and by nonspecific esterase. The cells were cultured for 5 days in DMEM supplemented with 10% FBS, 20 µg/ml gentamicin, and 1000 U/ml M-CSF. After 5 days, culture medium was completely removed, and the cells were washed three times with PBS and transferred to RPMI 1640 medium supplemented with 10% FBS and 20 µg/ml gentamicin, and the resultant M{phi} were cultured for 2 additional days without M-CSF. All cell culture reagents were tested using the Limulus lysate assay (Associates of Cape Cod, Cape Cod, MA) for endotoxin contamination and the levels were found to be <0.06 endotoxin units/ml.

Culture of chronically infected U1 and ACH2 cells

U1 cells, a subclone of HIV-1-infected U937 cells (36), and ACH2 cells, a subclone of HIV-1 infected A301 cells (37), were cultured in RPMI 1640 supplemented with 10% FBS and 20 µg/ml gentamicin at 37°C in 5% CO2.

Coculture of M{phi} and U1 or ACH2 cells

M{phi} (1 x 106) and U1 or ACH2 cells (1 x 105) were cocultured in 24-well plates in 2 ml of RPMI 1640 medium supplemented with 10% FBS and 20 µg/ml gentamicin. For Transwell experiments, M{phi} were plated on the plastic substrate of 24-well plates, and U1 or ACH2 cells were plated in the Transwell insert. Supernatants were collected at 24 and 48 h, respectively.

Coculture of monocytes and U1 or ACH2 cells

Freshly isolated monocytes (1 x 106) and U1 or ACH2 cells (1 x 105) were cocultured in 24-well plates in 2 ml of RPMI 1640 medium supplemented with 10% FBS and 20 µg/ml gentamicin. Supernatants were collected at 24 and 48 h, respectively.

Coculture of PBMC and U1 or ACH2 cells

Freshly isolated PBMC (5 x 106) were washed once in RPMI 1640 medium and cocultured with U1 or ACH2 cells (5 x 105) in RPMI 1640 medium supplemented with 10% FBS and 20 µg/ml gentamicin. Supernatants were collected at 24 and 48 h, respectively.

Quantitation of HIV-1 replication

Culture supernatants were assayed for HIV-p24 using a NEN/DuPont ELISA analysis kit (PerkinElmer Life Sciences, Boston, MA) according to the manufacturer’s instructions. Assays were performed in triplicate.

Treatment of cells with paraformaldehyde

M{phi}, U1 cells, and ACH2 cells were washed three times in PBS (pH 7.2) and incubated at 4°C for 2 h with 10% paraformaldehyde in PBS. After 2 h the cells were washed three times with PBS and then twice with RPMI 1640 medium before placing them in coculture.

Treatment of cells with proteinase K and Pronase E

Adherent M{phi} were treated overnight with 100 µg/ml proteinase K in serum-free medium for 18 h at 37°C or with 200 µg/ml Pronase E for 4 h in serum-free medium at 37°C. After incubation the cells were washed six times with PBS and placed in coculture.

Isolation of RNA from cells

Cocultures of M{phi} and U1 or ACH2 cells, grown at a ratio of 2:1, were harvested and washed in ice-cold PBS three times. Total RNA was extracted using Qiagen RNAeasy columns (Qiagen, Valencia, CA) according to the manufacturer’s instructions. RNA concentration and purity were determined by spectrophotometry at A260 and A280. RNA samples were aliquoted and stored at –70°C until further use.

Isolation of nuclear extract

Cocultures of M{phi} and U1 or ACH2 cells, grown at a ratio of 2:1, were harvested and washed three times in ice-cold PBS. Nuclear extracts were prepared as previously reported (38). Briefly, 1 x 103 cells were allowed to swell in 400 µl of ice-cold buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF) for 15 min on ice, after which 25 µl of 0.5% Nonidet P-40 was added and the tubes were thoroughly mixed for 10 s. The homogenate was centrifuged for 30 s and the pellet was saved. The nuclear pellet was resuspended in 50 µl of ice-cold buffer C (20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM DTT, 1 mM EDTA, 1 mM EGTA, and 1 mM PMSF) and incubated on ice for 15 min. The nuclear extract was centrifuged at 1100 x g for 5 min, and the supernatant containing the nuclear proteins was transferred to a fresh tube. Protein concentration was determined using a Pierce BCA protein assay kit (Pierce, Rockford, IL), and the concentration was adjusted to 1 mg/ml protein. The nuclear extracts were aliquoted and stored at –70°C until use.

NF-{kappa}B analysis

NF-{kappa}B p50 and p65 activities were determined using the BD Mercury transfactor kit (BD Clontech, Palo Alto, CA) according to the manufacturer’s instructions. The assay was performed in triplicate using 15 µg of nuclear extract in each well.

RT-PCR

First-strand cDNA synthesis was conducted using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. The first-strand cDNA was synthesized using 1–2 µg of total cell RNA. The cDNA was subsequently analyzed by PCR using an AccuPrime TaqDNA Polymerase System (Invitrogen Life Technologies) and CytoXpress multiplex PCR human IFN cytokine set 1 primers according to the manufacturer’s instructions. The PCR products were separated on a 2% agarose gel using a Tris-borate buffer and visualized by ethidium bromide staining.

TNF-{alpha} immunoassay

Supernatants from cocultures of M{phi} and U1 or ACH2 cells, grown at a ratio of 2:1, were collected at 24 and 48 h, respectively, and TNF-{alpha} concentrations were determined using the Quantikine kit (R&D Systems) according to the manufacturer’s instructions. Assays were performed in triplicate.

FACS analysis

M{phi} (1 x 106), cocultured with U1 or ACH2 cells (5 x 105), were treated with 100 µg/ml anti-TNF-{alpha} Ab or 100 µg/ml goat anti-rabbit IgG for 48 h. After 48 h the cells were harvested and washed three times in PBS. After centrifugation, the cell pellets were permeabilized using 0.5% saponin, and intracellular p24 was labeled with FITC-labeled mAb to HIV-1 core protein p24 (Coulter Immunology, Hialeah, FL) or control IgG as described previously (39) and acquired for FACS analysis.

Treatment with anti-TNF-{alpha} Ab

U1 or ACH2 cells (5 x 105) and M{phi} (1 x 106) were treated for 2 h with 100 µg/ml anti-TNF-{alpha} Ab or 100 µg/ml anti-TRAIL Ab and placed in coculture. As a control, cells were treated with 100 µg/ml goat anti-rabbit IgG for 2 h and placed in coculture. Supernatants were collected at 24 and 48 h, respectively. Culture supernatants were assayed for HIV-p24. Viable cell numbers were determined using trypan blue dye and counting in a hemocytometer.

Treatment with NF-{kappa}B inhibitors

To study the possible down-regulation of cytokine expression and HIV-1 replication by the NF-{kappa}B inhibitor BAY 11-7082 (Calbiochem, San Diego, CA), ACH2, U1 and M{phi} were pretreated with 30 µM BAY 11-7082 for 30 min and placed in coculture along with the inhibitor for the specified durations. Culture supernatants were collected at specified times, and TNF-{alpha} levels and HIV-1 p24 levels were determined. No evidence of cell toxicity was detected at the concentrations of BAY 11-7082 used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of HIV-1 replication in U1 and ACH2 cells by coculture with M{phi}

To determine the effect of coculture on the induction of HIV-1 replication in chronically infected U1 cells, M{phi} and U1 cells were cultured together at a ratio of 10:1, and culture supernatants were collected after 24 and 48 h and assayed for HIV-1 p24 by ELISA. Coculture of M{phi} with U1 cells at a ratio of 10:1 induced HIV-1 replication within 24 h. HIV-1 p24 levels reached greater than 8000 pg/ml in culture supernatants after 48 h in coculture (Fig. 1). Neither M{phi} nor U1 cells alone showed any appreciable amounts of HIV-1 p24. In addition, the supernatants from coculture of U1 cells with proteinase K- and Pronase E-treated M{phi} showed no significant inhibition of HIV-1 induction (Fig. 1). However, supernatants from coculture of U1 cells with paraformaldehyde-fixed M{phi} showed no significant increase of HIV-1 p24 levels even after 48 h of culture (Fig. 1). In a similar manner, coculture of ACH2 and M{phi} at a ratio of 1:10 induced viral replication within 24 h and resulted in 3500 pg/ml HIV-1 p24 in culture supernatants after 48 h (Fig. 1). The M{phi} or ACH2 cells not in coculture produced a low amount of viral p24 even after 48 h in culture. In addition, proteinase K- and Pronase E-treated M{phi} did not inhibit HIV-1 induction in ACH2 cells. However, paraformaldehyde-fixed M{phi} failed to induce viral replication (Fig. 1). Furthermore, when a permeable membrane physically separated M{phi} and U1 cells, a 10-fold reduction of HIV-1 induction was seen (Fig. 2A). Likewise, when ACH2 cells and M{phi} were grown in Transwell inserts that prevented cell–cell contact, viral replication was significantly lower (Fig. 2B). In addition, induction of HIV-1 replication was also observed when latently infected U1 and ACH2 cells were cocultured with PBMC (Fig. 3) and freshly isolated monocytes (Fig. 4).



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  		FIGURE 1. Determination of HIV-1 p24 production in culture supernatants by ELISA. Briefly, 1 x 105 U1 and 1 x 106 M{phi} or 1 x 105 ACH2 and 1 x 106 M{phi} were cocultured at a ratio of 1:10, and culture supernatants were collected at 24 and 48 h. Culture supernatants were analyzed in triplicate. Results expressed as mean ± SEM are representative of three independent experiments.

 


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  		FIGURE 2. Comparison of HIV-1 production in supernatants derived from cells grown in a Transwell system and in coculture. A, One x 105 U1 cells or 1 x 106 M{phi} were plated in Transwell plates and in coculture in 24-well plates. B, One x 105 ACH2 cells or 1 x 106 M{phi} were plated in Transwell plates and in coculture in 24-well plates. After 24 and 48 h, culture supernatants were collected and the production of HIV-1 p24 was determined by ELISA. Culture supernatants were analyzed in triplicate. Results expressed as mean ± SEM are representative of two independent experiments.

 


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  		FIGURE 3. Induction of HIV-1 replication by PBMC. Briefly, 1 x 105 U1 and 1 x 106 PBMC or 1 x 105 ACH2 and 1 x 106 PBMC were cocultured at a ratio of 1:10, and culture supernatants were collected at 24 and 48 h. Culture supernatants were analyzed in triplicate. Results expressed as mean ± SEM are representative of three independent experiments.

 


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  		FIGURE 4. Induction of HIV-1 replication by freshly isolated monocytes. Briefly, 1 x 105 U1 and 1 x 106 monocytes or 1 x 105 ACH2 and 1 x 106 monocytes were cocultured at a ratio of 1:10, and culture supernatants were collected at 24 and 48 h. Culture supernatants were analyzed in triplicate. Results expressed as mean ± SEM are representative of three independent experiments.

 
Induction of NF-{kappa}B

Several studies have demonstrated that coculture of latently infected cells with M{phi} and other cell types induced the nuclear localization of NF-{kappa}B (20, 25, 26, 27, 28). To determine the kinetics of the activation of NF-{kappa}B by coculture, nuclear proteins were isolated from cocultures of U1 or ACH2 and M{phi} at 0, 3, 6, and 24 h, and the amounts of NF-{kappa}B p50 and p65 in the nuclear extracts were determined by ELISA. The results (Fig. 5A) demonstrated a rapid nuclear accumulation of NF-{kappa}B p50 among the nuclear proteins isolated from U1 and M{phi} cocultures within 3 h, followed by a continuous increase to the 24-h time point. The kinetics of nuclear localization of NF-{kappa}B p65 were slightly different from that of NF-{kappa}B p50. NF-{kappa}B p65 induction was slower than that of NF-{kappa}B p50 and showed maximum activity at 6 h followed by a decrease in activity after 24 h. Neither the M{phi} nor the U1 cells alone demonstrated nuclear localization of NF-{kappa}B p50 or p65 subunits at the different time points tested (Fig. 5B). In a similar manner, activation of the NF-{kappa}B p50 subunit was observed in nuclear proteins isolated from ACH2 and M{phi} cocultures (Fig. 5C). The kinetics of activation indicated a rapid nuclear accumulation at 3 h that continued to the 24-h time point. In nuclear proteins isolated from ACH2 and M{phi} coculture, no activation of the NF-{kappa}B p65 subunit was observed. The inclusion of a large excess of competitor oligomers or mutant oligomers greatly reduced the binding affinity, demonstrating that the binding assays were specific for NF-{kappa}B p50 and NF-{kappa}B p65, respectively. Treatment of U1 cells, ACH2 cells, and M{phi} with paraformaldehyde before coculture abolished activation of both NF-{kappa}B subunits (Fig. 6). Thus, the activation of NF-{kappa}B p50 and p65 subunits required the interaction of viable M{phi} and U1 cells.



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  		FIGURE 5. Determination of NF-{kappa}B in cocultures. NF-{kappa}B p50 and p65 activity was determined using the BD Mercury transfactor kit. Fifteen micrograms of nuclear proteins isolated from M{phi} (A) cocultured with U1 cells, U1 cells or M{phi} alone (B), or M{phi} (C) cocultured with ACH2 cells were used in each well. A 100-fold excess of competitor oligonucleotide was used where indicated. The assay was performed in triplicate. Results expressed as mean ± SEM are representative of three independent experiments.

 


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  		FIGURE 6. Determination of NF-{kappa}B in cocultures treated with paraformaldehyde. NF-{kappa}B p50 and p65 activities were determined using the BD Mercury transfactor kit. A, Fifteen micrograms of nuclear proteins isolated from M{phi} cocultured with U1 cells, M{phi} fixed with paraformaldehyde and cocultured with U1 cells, M{phi} cocultured with U1 cells fixed with paraformaldehyde, M{phi} fixed in paraformaldehyde alone, and U1 cells fixed in paraformaldehyde alone was used in each well. B, Fifteen micrograms of nuclear proteins isolated from M{phi} cocultured with ACH2 cells, M{phi} fixed with paraformaldehyde and cocultured with ACH2 cells, M{phi} cocultured with ACH2 cells fixed with paraformaldehyde, M{phi} fixed in paraformaldehyde alone, and ACH2 cells fixed in paraformaldehyde alone was used in each well. The assay was done in triplicate. Results expressed as mean ± SEM are representative of two independent experiments.

 
Induction of cytokines by coculture

The effect of coculture of U1 or ACH2 cells with M{phi} on the induction of cytokine mRNA was examined by RT-PCR. The U1 or ACH2 cells and M{phi} were cocultured, and total RNA was isolated at 0, 3, 6,, and 24 h and subjected to RT-PCR analysis. The RT-PCR results from U1 coculture clearly demonstrated that the mRNAs of the proinflammatory cytokines IL-1{beta}, IL-8, and TNF-{alpha} were up-regulated, in comparison to GAPDH mRNA (Fig. 7A). The mRNAs for IL-1{beta} and IL-8 were up-regulated at the 3-h time point and continued to persist even after the 24-h time point. In contrast, TNF-{alpha} transcripts showed up-regulation only at 3 h, after which the mRNA returned to basal levels (Fig. 7A). In contrast, the levels of IL-6, TGF-{beta}, and GM-CSF remained unchanged at all of the time points tested (Fig. 7A).



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  		FIGURE 7. Induction of proinflammatory cytokines. PCR products from amplification of cDNA derived from U1 cells alone, M{phi} alone, and U1 and macrophage coculture (A) and ACH2 cells alone and ACH2 and macrophage coculture (B)were separated on a 2% agarose gel and stained with ethidium bromide. M denotes molecular markers and Std denotes a positive control for the PCR. The data are representative of three independent experiments.

 
Likewise, the RT-PCR results from ACH2 and M{phi} coculture demonstrated up-regulation of TNF-{alpha}, IL-1{beta}, IL-6, and IL-8 (Fig. 7B). Analogous to the results obtained with U1 and M{phi} coculture, increased transcription of IL-1{beta} and IL-8 was also observed. In addition, increased IL-6 mRNA expression was detected as early as 3 h and remained constant up to 24 h. In contrast, only a 2-fold induction of TNF-{alpha} was seen at the 3-h time point. The levels of TGF-{beta} and GM-CSF mRNAs remained unchanged at all time points tested (Fig. 7B). Neither the M{phi} alone nor the U1 and ACH2 cells alone showed any up-regulation of mRNAs for these cytokines (Fig. 7).

The effect of coculture of U1 or ACH2 cells with M{phi} on the induction of TNF-{alpha} was examined by ELISA. The results demonstrated that there was an increase in TNF-{alpha} levels in supernatants collected at 24 h which declined after 48 h (Fig. 8). Neither M{phi} alone nor the U1 and ACH2 cells alone showed any elevation of TNF-{alpha} levels at 24 h (Fig. 8)



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  		FIGURE 8. Induction of TNF-{alpha}. Briefly, 5 x 105 U1 and 1 x 106 monocytes or 5 x 105 ACH2 and 1 x 106 monocytes were cocultured at a ratio of 1:2, and culture supernatants were collected at 24 and 48 h. TNF-{alpha} levels in the culture supernatants were determined by ELISA. Culture supernatants were analyzed in triplicate. Results expressed as mean ± SEM are representative of three independent experiments.

 
Role of TNF-{alpha} in induction of HIV-1 replication

The RT-PCR results from the cocultures of U1 or ACH2 and M{phi} clearly demonstrated an up-regulation of mRNAs of the proinflammatory cytokines TNF-{alpha}, IL-1{beta}, and IL-8. TNF-{alpha} is known to induce HIV-1 replication in chronically infected U1 cells (33). Thus, to study the role TNF-{alpha} in the induction of HIV-1 replication by coculture, U1 or ACH2 and M{phi} cocultures were treated with anti-TNF-{alpha} Abs or goat anti-rabbit IgG for 48 h, and the production of nascent cell-associated HIV-1 was determined by FACS analysis using Abs to HIV-1 p24. The FACS results confirmed that when U1 and ACH2 were cocultured, induction of HIV-1 replication occurred (Fig. 9, A and C). However, treatment with anti-TNF-{alpha} Ab considerably blocked the induction of HIV-1 replication as seen in Fig. 9 (B and D). Isotype-matched control IgG did not produce any shift in the relative fluorescence (Fig. 9) indicating the specificity of anti-p24 Ab.



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  		FIGURE 9. FACS analysis of nascent HIV-1 particles. A, Coculture of U1 and M{phi} treated with 100 µg/ml goat anti-rabbit IgG. B, Coculture of U1 cells and M{phi} treated with 100 µg/ml anti-TNF-{alpha} Ab. C, Coculture of ACH2 cells and M{phi} treated with 100 µg/ml goat anti-rabbit IgG. D, Coculture of ACH2 and M{phi} treated with 100 µg/ml anti-TNF-{alpha} Ab. The results are representative of three independent experiments.

 
In a similar manner, treatment of cocultures with anti-TNF-{alpha} Ab greatly reduced the amount of HIV-1 p24 detected in culture supernatants even after 48 h (Fig. 10A). However, treatment with anti-TRAIL Ab or normal goat anti-rabbit IgG did not have any effect on HIV-1 induction (Fig. 10A). Analysis of viable U1 and ACH2 cells from coculture with M{phi} revealed that the anti-TNF-{alpha} Ab treatment protected U1 and ACH2 cells from cytopathic effects caused by coculture (Fig. 10B). In contrast, considerable reduction in viable cells was detected in U1 or ACH2 cocultures treated with normal goat anti-rabbit IgG or anti-TRAIL Ab (Fig. 10B). Treatment of cocultures with neutralizing Abs against IL-1{beta}, IL-6, IL-8, and M-CSF had no significant effect on the induction of HIV-1 replication (data not shown).



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  		FIGURE 10. Effect of anti-TNF-{alpha} Ab treatment. U1 or ACH2 cells and M{phi} treated with 100 µg/ml goat anti-rabbit IgG, U1 or ACH2 cells and M{phi} treated with 100 µg/ml anti-TRAIL Ab, or U1 or ACH2 cells and M{phi} treated with 100 µg/ml anti-TNF-{alpha} Ab were cocultured at a ratio of 1:2, and culture supernatants were collected at 24 and 48 h. A, HIV-1 replication was determined by measuring HIV-1 p24 in culture supernatants by ELISA. Culture supernatants were analyzed in triplicate. B, The number of viable cells was determined by trypan blue staining and counting in a hemocytometer. Each sample was assayed in triplicate. Results expressed as mean ± SEM are representative of two independent experiments.

 
Effect of NF-{kappa}B inhibitors

Treatment of M{phi} and U1 or M{phi} and ACH2 cocultures with the NF-{kappa}B inhibitor BAY 11-0782 at 30 µM (40, 41) completely blocked the induction of NF-{kappa}B (Fig. 11). In addition, PCR analysis of cDNA derived from M{phi} and U1 or M{phi} and ACH2 cocultures treated with the inhibitor demonstrated a marked decrease in the induction of IL-1{beta} (Fig. 12). A modest decrease in the induction of TNF-{alpha} and TGF-{beta} was observed in U1 and M{phi} cocultures (Fig. 12A). However, only a marginal inhibition of TGF-{beta} was observed with no change in the ongoing expression of TNF-{alpha} in M{phi} and ACH2 cocultures (Fig. 12B). Nevertheless, the induction of IL-8 expression was unaffected in M{phi} and U1 or M{phi} and ACH2 coculture (Fig. 12). Although only a modest decrease in the induction of TNF-{alpha} mRNA was observed when M{phi} and U1 or ACH2 cocultures were treated with NF-{kappa}B inhibitor BAY 11-7082, ELISA results of coculture supernatants treated with BAY 11-7082 demonstrated a profound decrease in TNF-{alpha} (Fig. 13). In a similar manner, treatment of M{phi} and U1 or M{phi}-ACH2 cocultures with this inhibitor substantially inhibited viral induction (Fig. 14).



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  		FIGURE 11. Determination of NF-{kappa}B in cocultures treated with BAY 11-0782. NF-{kappa}B p50 activity was determined using the BD Mercury transfactor kit. Fifteen micrograms of nuclear proteins per well isolated from U1 and M{phi} cocultures pretreated with 30 µM BAY 11-0782 (A) and ACH2 and M{phi} cocultures pretreated with 30 µM BAY 11-0782 (B). The assay was performed in triplicate. Results expressed as mean ± SEM are representative of two independent experiments.

 


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  		FIGURE 12. Inhibition of cytokine induction by NF-{kappa}B inhibitors. PCR products from amplification of cDNA derived from U1 and M{phi} coculture pretreated with 30 µM BAY 11-0782 (A) and ACH2 and M{phi} coculture pretreated with 30 µM BAY 11-0782 (B) were separated on a 2% agarose gel and stained with ethidium bromide.

 


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  		FIGURE 13. TNF-{alpha} levels in coculture supernatants treated with BAY 11-0782. TNF-{alpha} concentration was determined in culture supernatants derived from U1 and M{phi} coculture pretreated with 30 µM BAY 11-0782 and ACH2 and M{phi} coculture pretreated with 30 µM BAY 11-0782 by ELISA. The data are representative of three independent experiments.

 


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  		FIGURE 14. Effect of NF-{kappa}B inhibitors on HIV-1 induction. ACH2, U1, and M{phi} were pretreated with 30 µM BAY 11-0782 before placing them in coculture as described above. Culture supernatants were collected at 24 and 48 h, respectively. HIV-1 p24 production in culture supernatants was determined by ELISA. Culture supernatants were analyzed in triplicate. Results expressed as mean ± SEM are representative of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that HIV-1 expression in U1 and ACH2 cells is markedly induced by coculture with PBMC, monocytes, and M{phi}. Several studies have documented the secretion of cytokines and other soluble factors by M{phi} in response to viral infection (42, 43, 44, 45, 46). To determine whether soluble factors were a source of the inductive stimuli, we cultured M{phi} and U1 or ACH2 cells in Transwell inserts separated by a permeable membrane. The results from these experiments clearly demonstrated a substantial but not complete reduction of HIV-1 replication when the two cell types were physically separated. Our data indicate that even though some interactions by soluble factors (e.g., cytokines) can occur, close proximity or contact of cells is required for full induction. These results are consistent with other investigations demonstrating that conditioned medium from M{phi} and other cell types did not induce viral replication in U1 and ACH2 cells to the same magnitude as that obtained by cell–cell contact (20, 27). The inductive effect required the presence of viable M{phi}, since we found that paraformaldehyde-fixed M{phi} did not induce viral replication. The failure of fixed M{phi} to induce HIV-1 replication may be due to the denaturation of membrane-bound TNF-{alpha} (47) or a ligand or cellular receptor interacting with a cognate receptor or ligand on the U1 or ACH2 cells involved in triggering signals for activating HIV-1 replication.

The life cycle of HIV-1 is closely associated with the activation state of its host cells. HIV-1 is dependent on a number of host factors for entry, transcription, and viral gene expression. The HIV-1 LTR contains at least six defined elements that bind cellular transcription factors (48). Among these, the best characterized is the NF-{kappa}B, which plays a central role in mediating inducible gene expression. Binding of the cellular transcription factor SP1 to three adjacent sites further enhances NF-{kappa}B activity (49). Apart from the activation of HIV-1, the family of NF-{kappa}B transcriptional activators is also involved in the transduction of immunological response, cellular differentiation, and cell growth.

The activation of NF-{kappa}B is a rapid immediate early event that occurs within minutes after exposure to relevant inducers, does not require de novo protein synthesis, and results in the strong transcriptional stimulation of several genes. Other factors, including cell type specificity, subcellular localization, differential regulation by I{kappa}B, and phosphorylation of Rel (50) play important roles in regulating gene expression. In most cell types, the NF-{kappa}B complex is sequestered in the cytoplasm by a family of inhibitory proteins (I{kappa}B). I{kappa}B binds to the NF-{kappa}B complex and masks the nuclear localization signal, thereby preventing the translocation of the NF-{kappa}B complex into the nucleus. Upon activation by various upstream signals, I{kappa}B is phosphorylated and degraded, releasing the NF-{kappa}B complex, which is then free to translocate to the nucleus where it can regulate gene expression. Cytokines such as IL-1 and TNF-{alpha}, phorbol esters, radical oxygen intermediates, and UV irradiation can rapidly stimulate NF-{kappa}B activation (50). In addition, NF-{kappa}B is also activated by viral infection. Many viral proteins like Tat and Tax are known to activate NF-{kappa}B. The activation of NF-{kappa}B mediates the immune response to the invading pathogens and in several instances promotes viral replication (51). The cell–cell contact of U1 or ACH2 cells with M{phi} in coculture may trigger transducing signals that result in the observed activation of the NF-{kappa}B p50/p65 subunits, which in turn can act on the NF-{kappa}B motif on the HIV-1 LTR for inducing viral replication. The persistent induction of NF-{kappa}B subunit p50 observed in this study indicates that there is continuous stimulation of the cells. Fixation of U1, ACH2, and M{phi} with paraformaldehyde disrupted this cooperative interaction and hence blocked activation of the NF-{kappa}B p50-p65 complex. Likewise, paraformaldehyde-fixed M{phi} failed to induce HIV-1 replication in U1 or ACH2 cells by coculture. Thus, taken together, our data suggest that cooperative interactions involving intimate cell association is essential for the activation of NF-{kappa}B and subsequent induction of HIV-1 replication.

Consistent with the activation of NF-{kappa}B, an induction of downstream proinflammatory cytokines was observed. RT-PCR results clearly demonstrated the induction of IL-1{beta}, IL-6, IL-8, and TNF-{alpha} expression within 3 h of coculture. However, the expression of other cytokines like GM-CSF and TGF-{beta} was not significantly induced. In addition, quantitation of TNF-{alpha} levels in culture supernatants clearly indicate high levels of TNF-{alpha} accumulation after 24 h. Several in vitro and in vivo studies have demonstrated that HIV-1 infection can induce the secretion and elevation of IL-1{beta}, IL-6, and TNF-{alpha} (42, 43, 44, 45, 46, 52). Activated M{phi} secrete IL-1 {beta}, IL-6, IL-8, and TNF-{alpha} during inflammatory reactions (9) that promote leukocyte recruitment and initiate the adaptive immune response. Consequently, the up-regulation of proinflammatory cytokines by both U1 and ACH2 when in contact with M{phi} may provide a mechanism for induction of HIV-1 replication in latently infected cells.

A direct correlation between elevated levels of these cytokines and viral load during the progression of HIV-1 infection indicate an important role of cytokines in the activation of NF-{kappa}B (53, 54). Up-regulated TNF-{alpha} can activate NF-{kappa}B within minutes and can induce apoptosis upon prolonged stimulation (55, 56). Soluble and the membrane-bound forms of TNF-{alpha} are known to enhance HIV-1 replication (57, 58) by the direct activation of NF-{kappa}B in T cells (18) and in M{phi} (43). Although IL-1{beta} and IL-6 are also known to induce HIV-1 replication, their inductive capacity is much lower than that of TNF-{alpha}. Unlike TNF-{alpha}, the mechanism of action of IL-1{beta} and IL-6 for the induction of HIV-1 replication is mediated by an alternative pathway independent of NF-{kappa}B activation (17). The role of IL-8 in HIV-1 pathogenesis is not clear. IL-8 does not induce HIV-1 replication in U1 cells (59), but primarily promotes the recruitment of leukocytes to areas of inflammation and chemotaxis.

The treatment of U1 or ACH2 and M{phi} cocultures with anti-TNF-{alpha} Ab had a profound effect on the production of nascent HIV-1 particles, indicating that TNF-{alpha} plays a major role in the induction of HIV-1 replication in U1 and ACH2 cells during coculture with M{phi}. Consistent with these findings, treatment of U1-M{phi} or ACH2-M{phi} cocultures with NF-{kappa}B inhibitor demonstrated a complete inhibition of the induction of NF-{kappa}B, with a concomitant down-regulation of the proinflammatory cytokines IL-1{beta} and TNF-{alpha}. The inhibition of NF-{kappa}B activation and the down-regulation of NF-{kappa}B-induced cytokines TNF-{alpha} and/or IL-{beta} resulted in complete inhibition of HIV-1 production (Fig. 14), indicating that NF-{kappa}B and cytokines play a pivotal role in the reactivation of HIV-1 in latently infected cells by M{phi}.

Some members of the TNFR superfamily are also known to play an important role in inducing the activation of HIV-1 replication. Recent studies have demonstrated that the activation of the TNFR superfamily of proteins, including TNFR1, TNFR11, CD30, 4-1BB, CD40, and OX40, activate signal transducers that are responsible for the activation of NF-{kappa}B (46, 60, 61, 62). The triggering of CD30 can also induce HIV-1 replication through the NF-{kappa}B pathway in chronically infected T cell lines (63). Cross-linking of 4-1BB with agonistic mAbs significantly enhances HIV-1 replication in CD4+ T cells (64). Similarly, the stimulation of CD40 in the presence of IL-4 and IL-2 up-regulates HIV-1 replication in B cells (65). Thus, TNF-{alpha} and its receptors play a central role in the induction of HIV-1 replication by the activation of NF-{kappa}B. Since the TNF-{alpha} promoter also contains NF-{kappa}B binding sites, TNF-{alpha} can positively up-regulate its own synthesis, leading to the persistence of infection (66).

In summary, our results indicate that in M{phi} grown in coculture, close cell interactions generate intracellular signals that activate the NF-{kappa}B p50-p65 complex. The active forms of the NF-{kappa}B subunits p50 and p65 are then translocated into the nucleus, where they can induce the expression of proinflammatory cytokines. When these cytokines are secreted into the culture medium, they can in turn act upon the U1 or ACH2 cells triggering the signal transduction cascade leading to the activation and translocation of NF-{kappa}B to the nucleus, where it can act upon the HIV-1 LTR inducing viral replication. In addition, the activated NF-{kappa}B can up-regulate the transcription of proinflammatory cytokines which, when secreted into the culture medium, can act as an autocrine inducer of cell signaling in U1 or ACH2 cells. The persistent activation of NF-{kappa}B supports continuous activation of the cells in the coculture system. The exact mechanism of the induction of HIV-1 replication in U1 or ACH2 cells by M{phi} is not known at present. Future studies aimed at understanding the molecular mechanisms involved in the reactivation of HIV-1 from latently infected cells would help us to understand further the pathogenesis of this disease.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Subhash Dhawan or Dr. Krishnakumar Devadas, Immunopathogenesis Section, Laboratory of Molecular Virology, Center for Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike (HFM-315), Rockville, MD 20852-1448. E-mail addresses, respectively: dhawan{at}cber.fda.gov; devadas{at}cber.fda.gov Back

2 Abbreviations used in this paper: M{phi}, macrophage; BAY 11-7082, (E)3-[(4-methylphenyl)sulfonyl]-2-propenenitrile; LTR, long terminal repeat. Back

Received for publication September 5, 2003. Accepted for publication September 21, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Chun, T. W., R. T. Davey, Jr, M. Ostrowski, J. S. Justemente, D. Engel, J. I. Mullins, A. S. Fauci. 2000. Relationship between pre-existing viral reservoirs and the re-emergence of plasma viremia after discontinuation of highly active anti-retroviral therapy. Nat. Med. 6:757.[Medline]
  2. Blankson, J. N., D. Persaud, R. Siliciano. 2002. The challenge of viral reservoirs in HIV-1 infection. Annu. Rev. Med. 53:557.[Medline]
  3. Butera, S. T., T. M. Folks. 1992. Application of latent HIV-1 infected cellular models to therapeutic interventions. AIDS Res. Hum. Retroviruses 8:991.[Medline]
  4. Bukrinsky, M. I., T. L. Stanwick, M. P. Dempsey, M. Stevenson. 1991. Quiescent T lymphocytes as an inducible virus reservoir in HIV-1 infection. Science 254:423.[Abstract/Free Full Text]
  5. Chun, T. W., D. Finzi, J. Margolick, K. Chadwick, D. Schwartz, R. F. Siliciano. 1995. In vivo fate of HIV-1-infected T cells: quantitative analysis of the transition to stable latency. Nat. Med. 1:1284.[Medline]
  6. Chun, T. W., L. Stuyver, S. B. Mizell, L. A. Ehler, J. A. Mican, M. Baseler, A. L. Lloyd, M. A. Novak, A. S. Fauci. 1997. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc. Natl. Acad. Sci. USA 94:13193.[Abstract/Free Full Text]
  7. Wong, J. K., M. Hezareh, H. F. Günthard, D. V. Havlin, C. C. Ignacio, C. A. Spiria, D. D. Richman. 1997. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science 278:1291.[Abstract/Free Full Text]
  8. Finzi, D., M. Hermankova, T. Pierson, L. M. Carruth, C. Buck, R. E. Chaisson, T. C. Quinn, K. Chadwick, J. Margolick, R. Brookmeyer, et al 1997. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 278:1295.[Abstract/Free Full Text]
  9. Aquaro, S., R. Calio, J. Balzarini, M. C. Bellocchi, E. Garaci, C. F. Perno. 2002. Macrophages and HIV infection: therapeutical approaches toward this strategic virus reservoir. Antiviral Res. 55:209.[Medline]
  10. Crowe, S. M.. 1995. Role of macrophages in the pathogenesis of human immunodeficiency virus (HIV) infection. Aust. N Z J. Med. 25:777.[Medline]
  11. Herbein, G.. 1997. Cytokines, viruses and macrophages: an interactive network. An immune dysregulation involving the members of the tumor necrosis factor (TNF) receptors superfamily could be critical in AIDS pathogenesis. Pathol. Biol. 45:115.[Medline]
  12. Orenstein, J. M., C. Fox, S. M. Wahl. 1997. Macrophages as a source of HIV during opportunistic infections. Science 276:1857.[Abstract/Free Full Text]
  13. Kutsch, O., E. N. Benveniste, G. M. Shaw, D. N. Levy. 2002. Direct and quantitative single-cell analysis of human immunodeficiency virus type 1 reactivation from latency. J. Virol. 76:8776.[Abstract/Free Full Text]
  14. Yang, X., Y. Chen, D. Gabzuda. 1999. ERK MAP kinase links cytokine signals to activation of latent HIV-1 infection by stimulating a cooperative interaction of AP-1 and NF-{kappa}B. J. Biol. Chem. 274:27981.[Abstract/Free Full Text]
  15. Grossman, Z., M. B. Feinberg, W. E. Paul. 1998. Multiple modes of cellular activation and virus transmission in HIV infection: a role for chronically and latently infected cells in sustaining viral replication. Proc. Natl. Acad. Sci. USA 95:6314.[Abstract/Free Full Text]
  16. Rieckmann, P., G. Poli, J. H. Kehrl, A. S. Fauci. 1991. Activated B lymphocytes from human immunodeficiency virus-infected individuals induce virus expression in infected T cells and a promonocytic cell line U1. J. Exp. Med. 173:1.[Abstract/Free Full Text]
  17. Poli, G., A. L. Kinter, A. S. Fauci. 1994. Interleukin 1 induces expression of the human immunodeficiency virus alone and in synergy with interleukin 6 in chronically infected U1 cells: inhibition of inductive effects by the interleukin 1 receptor antagonists. Proc. Natl. Acad. Sci. USA 91:108.[Abstract/Free Full Text]
  18. Duh, E. J., W. J. Maury, T. M. Folks, A. S. Fauci, A. B. Rabson. 1989. Tumor necrosis factor {alpha} activates human immunodeficiency virus type 1 through induction of nuclear factor binding to NF-{kappa}B sites in the long terminal repeat. Proc. Natl. Acad. Sci. USA 86:5974.[Abstract/Free Full Text]
  19. Owatari, S., J. Zhang, M. Gao, A. Tanabe-Tochikura, N. Wakamiya, H. Tsuchie, T. Kurimura. 1997. Contribution to the regulation of virus replication in cells latently infected with human immunodeficiency virus 1. Acta Virol. 41:21.[Medline]
  20. Faure, E., N. Yahi, A. Zider, C. Cavard, S. Champion, J. Fantini. 1994. Physical contact with lymphocytes is required for reactivation of dormant HIV-1 in colonic epithelial cells: involvement of the HIV-1 LTR. Virus Res. 34:1.[Medline]
  21. Gilles, P. N., J. L. Lathey, S. A. Spector. 1995. Replication of macrophage-tropic and T-cell-tropic strains of human immunodeficiency virus type 1 is augmented by macrophage-endothelial cell contact. J. Virol. 69:2133.[Abstract]
  22. Ho, W., Y. Li, X. Zhu, L. Song, S. D. Douglas. 1996. Induction of HIV-1 expression in chronically infected promonocytic cells cocultured with human lung fibroblasts. Clin. Immunol. 80:171.
  23. Bacsi, A., E. Cosma, Z. Beck, I. Andirko, J. Konya, L. Gergely, F. D. Toth. 2001. Induction of HIV-1 replication in latently infected synctiotrophoblast cells by contact with placental macrophages: role of interlukin-6 and tumor necrosis factor {alpha}. J. Interferon Cytokine Res. 21:1079.[Medline]
  24. Fan, S. T., K. Hsia, T. S. Eddington. 1994. Upregulation of human immunodeficiency virus-1 in chronically infected monocytic cell line by both contact with endothelial cells and cytokines. Blood 84:1567.[Abstract/Free Full Text]
  25. Borghi, M. O., P. Panzeri, R. Shattock, S. Sozzani, A. Dobrina, P. L. Meroni. 2000. Interaction between chronically HIV-infected promonocytic cells and human umbilical vein endothelial cells: role of proinflammatory cytokines and chemokines in viral expression modulation. Clin. Exp. Immunol. 120:93.[Medline]
  26. Hoshino, Y., K. Nakata, S. Hoshino, Y. Honda, D. B. Tse, T. Shioda, W. N. Rom, M. Weiden. 2002. Maximal HIV-1 replication in alveolar macrophages during tuberculosis requires both lymphocyte contact and cytokines. J. Exp. Med. 195:495.[Abstract/Free Full Text]
  27. Faure, E., P. Lecine, C. Lipcey, S. Champion, J. Imbert. 1997. Cell-to-cell contact activates the long terminal repeat of human immunodeficiency virus 1 through its NF-{kappa}B motif. Eur. J. Biochem. 244:568.[Medline]
  28. Lee, E., H. Zhou, A. J. Henderson. 2001. Endothelial cells enhance immunodeficiency virus type 1 replication in macrophages through a C/EBP-dependent mechanism. J. Virol. 75:9703.[Abstract/Free Full Text]
  29. Butera, S. T., B. D. Roberts, T. M. Folks. 1993. Regulation of HIV-1 expression by cytokine networks in a CD4+ model of chronic infection. J. Immunol. 150:625.[Abstract]
  30. Nagira, M., A. Sato, S. Miki, T. Imai, O. Yoshie. 1999. Enhanced HIV-1 replication by chemokines constitutively expressed in secondary lymphoid tissue. Virology 264:422.[Medline]
  31. Frankel, A. D., J. A. Young. 1998. HIV-1: fifteen proteins and an RNA. Annu. Rev. Biochem. 67:1.[Medline]
  32. Conant, K., M. Ma, A. Nath, E. O. Major. 1996. Extracellular human immunodeficiency virus type 1 Tat protein is associated with an increase in both NF-{kappa}B binding and protein kinase C activity in primary human astrocytes. J. Virol. 70:1384.[Abstract]
  33. Folks, T. M., K. A. Clouse, J. Justemente, A. Rabson, E. Duh, J. H. Kehrl, A. S. Fauci. 1989. Tumor necrosis factor {alpha} induces expression of human immunodeficiency virus in a chronically infected T-cell clone. Proc. Natl. Acad. Sci. USA 86:2365.[Abstract/Free Full Text]
  34. Poli, G., A. Kinter, J. S. Justemente, J. H. Kehrl, P. Bressler, S. Stanley, A. S. Fauci. 1990. Tumor necrosis factor functions in an autocrine manner in the induction of human immunodeficiency virus expression. Proc. Natl. Acad. Sci. USA 87:782.[Abstract/Free Full Text]
  35. Wahl, L., I. M. Katona, R. L. Wilder, C. C. Winter, B. Haraoui, I. Scher, S. M. Wahl. 1984. Isolation of human mononuclear cell subsets by counterflow centrifugal elutriation (CCE). I. Characterization of B-lymphocyte-, T-lymphocyte-, and monocyte-enriched fractions by flow cytometric analysis. Cell. Immunol. 85:373.[Medline]
  36. Folks, T. M., J. Justement, A. Kinter, C. A. Dinarello, A. S. Fauci. 1987. Cytokine-induced expression of HIV-1 in a chronically infected promonocytic cell line. Science 238:800.[Abstract/Free Full Text]
  37. Clouse, K. A., D. Powell, I. Washington, G. Poli, K. Strebel, W. Farrar, P. Barstad, J. Kovacs, A. S. Fauci, T. M. Folks. 1989. Monokine regulation of human immunodeficiency virus-1 expression in a chronically infected human T cell clone. J. Immunol. 142:431.[Abstract]
  38. Dhawan, S., S. Singh, B. B. Aggarwal. 1997. Induction of endothelial cell surface adhesion molecules by tumor necrosis factor is blocked by protein tyrosine phosphatase inhibitors: role of the nuclear factor NF-{kappa}B. Eur. J. Immunol. 27:2172.[Medline]
  39. Lafernie, R. M., L. M. Wahl, J. S. Epstein, I. K. Hewlett, K. M. Yamada, S. Dhawan. 1996. HIV-1 Tat modulates the function of monocytes and alters their interactions with microvessel endothelial cells. J. Immunol. 156:1638.[Abstract]
  40. Izban, K. F., M. Ergin, J. Qin, R. L. Martinez, R. J. Pooley, S. Saeed, S. Alkan. 2000. Constitutive expression of NF-{kappa}B is a characteristic feature of mycosis fungiodes: implications for apoptosis resistance and pathogenesis. Hum. Pathol. 31:1482.[Medline]
  41. Mori, N., Y. Yamada, S. Ikeda, Y. Yamasaki, K. Tsukasaki, Y. Tanaka, M. Tomonaga, N. Yamamoto, M. Fuji. 2002. BAY 11-7082 inhibits transcription factor NF-{kappa}B and induces apoptosis of HTLV-1-infected T-cell lines and primary adult T-cell leukemia cells. Blood 100:1828.[Abstract/Free Full Text]
  42. Longo, N., J. M. Zabay, J. M. Sempere, J. Navarro, E. Fernandez-Cruz. 1993. Altered production of PEG2, IL-1{beta} and TNF-{alpha} by peripheral blood monocytes from HIV-positive individuals at early stages of HIV infection. J. Acquired Immune Defic. Syndr. 6:1017.
  43. Biswas, P., G. Poli, J. M. Orenstein, A. S. Fauci. 1994. Cytokine-mediated induction of human immunodeficiency virus (HIV) expression and cell death in chronically infected U1 cells: do tumor necrosis factor {alpha} and {gamma} interferon selectively kill HIV-infected cells?. J. Virol. 68:2598.[Abstract/Free Full Text]
  44. Lafrenie, R. M., L. M. Wahl, J. S. Epstein, K. Yamada, S. Dhawan. 1997. Activation of monocytes by HIV-Tat treatment is mediated by cytokine expression. J. Immunol. 159:4077.[Abstract]
  45. Mogensen, T. H., S. R. Paludan. 2001. Molecular pathways in virus-induced cytokine production. Microbiol. Mol. Biol. Rev. 65:131.[Abstract/Free Full Text]
  46. Greenwell-Wild, T., N. Vasquez, D. Sim, M. Schito, D. Chatterjee, J. M. Orenstein, S. M. Wahl. Mycobacterium avium infection and modulation of human macrophage gene expression. J. Immunol. 169:6286.
  47. Kriegler, M., C. Perez, K. DeFay, I. Albert, S. D. Lu. 1988. A novel form of TNF/cachetin is a cell surface cytotoxic transmembrane protein: ramification for the complex physiology of TNF. Cell 53:45.[Medline]
  48. Pereira, L. A., K. Bentley, A. Peeters, M. J. Churchill, N. J. Deacon. 2000. A compilation of cellular transcription factor interactions with the HIV-1 LTR promoter. Nucleic Acids Res. 28:663.[Abstract/Free Full Text]
  49. Perkins, N. D., A. B. Agranoff, E. Pascal, G. Nabel. 1994. An interaction between the DNA-binding domains of RelA (p65) and Sp1 mediates human immunodeficiency virus gene activation. Mol. Cell. Biol. 14:6570.[Abstract/Free Full Text]
  50. Camano, J., C. A. Hunt. 2002. NF-{kappa}B family of transcription factors: central regulators of innate and adaptive immune functions. Clin. Microbiol. Rev. 15:414.[Abstract/Free Full Text]
  51. Baldwin, A. S.. 2001. NF-{kappa}B in defense and disease. J. Clin. Invest. 107:144.
  52. Chun, T., D. Engel, S. B. Mizell, L. A. Ehler, A. S. Fauci. 1998. Induction of HIV-1 replication in latently infected CD4+ T cells using a combination of cytokines. J. Exp. Med. 188:83.[Abstract/Free Full Text]
  53. Arany, I., M. Muldrow, S. K. Tyring. 2001. Correlation between mRNA levels of IL-6 and TNF {alpha} and progression rate in anal squamous epithelial lesions from HIV-positive men. Anticancer Res. 21:425.[Medline]
  54. Arditi, M., W. Kabat, R. Yogev. 1991. Serum tumor necrosis factor {alpha}, interleukin 1-{beta}, p24 antigen concentrations and CD4+ cells at various stages of human immunodeficiency virus 1 infection in children. Pediatr. Infect. Dis. J. 10:450.[Medline]
  55. Van der Meide, P. H., H. Schellekens. 1996. Cytokines and the immune response. Biotherapy 8:243.[Medline]
  56. Lee, E. G., D. L. Boone, S. Chai, S. L. Libby, M. Chien, J. P. Lodolce, A. Ma. 2000. Failure to regulate TNF-induced NF-{kappa}B and cell death responses in A20-deficient mice. Science 289:2350.[Abstract/Free Full Text]
  57. Lama, J., C. F. Ware. 2000. Human immunodeficiency virus type 1 nef mediates sustained membrane expression of tumor necrosis factor and the related cytokine LIGHT on activated T cells. J. Virol. 74:9396.[Abstract/Free Full Text]
  58. Marshall, W. L., B. M. N. Brinkman, C. M. Ambrose, P. A. Pesavento, A. M. Uglialoro, E. Teng, R. W. Finberg, J. L. Browning, A. Goldfeld. 1999. Signaling through the lymphotoxin-{beta} receptor stimulated HIV-1 replication alone and in cooperation with soluble or membrane-bound TNF-{alpha}. J. Immunol. 162:6016.[Abstract/Free Full Text]
  59. Tiemessen, C. T., B. Kilroe, D. J. Martin. 2000. Interlukin-8 fails to induce human immunodeficiency virus-1 expression in chronically infected promonocytic U1 cells but differentially modulates induction by proinflammatory cytokines. Immunology 101:140.[Medline]
  60. Edfjall, C., H. Jacobsen, H. Lotscher, J. Mous. 1996. Tumor necrosis factor receptor p55 mediates induction of HIV type 1 expression in chronically infected U1 cells. AIDS Res. Hum. Retroviruses 12:199.[Medline]
  61. Gruss, H. J., S. K. Dower. 1995. Tumor necrosis factor ligand superfamily: involvement of in the pathology of malignant lymphomas. Blood 85:3378.[Abstract/Free Full Text]
  62. Takahashi, Y., Y. Tanaka, A. Yamashita, Y. Koyonagi, M. Nakamura, N. Yamamoto. OX40 stimulation by gp34/OX40 ligand enhances productive human immunodeficiency virus type 1 infection. J. Virol. 75:6748.
  63. Biswas, P., C. A. Smith, D. Goletti, E. C. Hardy, R. W. Jackson, A. S. Fauci. 1995. Cross-linking of CD30 induces HIV expression in chronically infected T cells. Immunity 2:587.[Medline]
  64. Wang, S., Y. J. Kim, C. Bick, S. H. Kim, B. S. Kwon. 1998. The potential roles of 4-1BB costimulation in HIV type 1 infection. AIDS Res. Hum. Retroviruses 14:223.[Medline]
  65. Gras, G., C. Legendre, R. Krzysiek, D. dormant, P. Galanaud, Y. Richard. 1996. CD40/CD40L interactions and cytokines regulate HIV replication in B cells in vitro. Virology 220:309.[Medline]
  66. Udalova, I. A., J. C. Knight, V. Vidal, S. A. Nedospasov, D. Kwiatkowski. 1998. Complex NF-{kappa}B interactions at the distal tumor necrosis factor promoter region in human monocytes. J. Biol. Chem. 273:21178.[Abstract/Free Full Text]



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A. F. B. Victoriano, K. Asamitsu, Y. Hibi, K. Imai, N. G. Barzaga, and T. Okamoto
Inhibition of Human Immunodeficiency Virus Type 1 Replication in Latently Infected Cells by a Novel I{kappa}B Kinase Inhibitor
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