Follicular Dendritic Cells Activate HIV-1 Replication in Monocytes/Macrophages through a Juxtacrine Mechanism Mediated by P-Selectin Glycoprotein Ligand 1

Kenji Ohba, Akihide Ryo, Md. Zahidunnabi Dewan, Mayuko Nishi, Toshio Naito, Xiaohua Qi, Yoshio Inagaki, Yoji Nagashima, Yuetsu Tanaka, Takashi Okamoto, Kazuo Terashima and Naoki Yamamoto
J Immunol July 1, 2009, 183 (1) 524-532; DOI: https://doi.org/10.4049/jimmunol.0900371

Abstract

Follicular dendritic cells (FDCs) are located in the lymphoid follicles of secondary lymphoid tissues and play a pivotal role in the selection of memory B lymphocytes within the germinal center, a major site for HIV-1 infection. Germinal centers are composed of highly activated B cells, macrophages, CD4+T cells, and FDCs. However, the physiological role of FDCs in HIV-1 replication remains largely unknown. We demonstrate in our current study that FDCs can efficiently activate HIV-1 replication in latently infected monocytic cells via an intercellular communication network mediated by the P-selectin/P-selectin glycoprotein ligand 1 (PSGL-1) interaction. Upon coculture with FDCs, HIV-1 replication was significantly induced in infected monocytic cell lines, primary monocytes, or macrophages. These cocultures were found to synergistically induce the expression of P-selectin in FDCs via NF-κB activation and its cognate receptor PSGL-1 in HIV-1-infected cells. Consistent with this observation, we find that this response is significantly blocked by antagonistic Abs against PSGL-1 and almost completely inhibited by PSGL-1 small interfering RNA. Moreover, a selective inhibitor for Syk, which is a downstream effector of PSGL-1, blocked HIV-1 replication in our cultures. We have thus elucidated a novel regulatory mechanism in which FDCs are a potent positive bystander that facilitates HIV-1 replication in adjacent infected monocytic cells via a juxtacrine signaling mechanism.

The natural progression of HIV-1 infection consists of acute and chronic stages (1, 2). In the acute phase of viral infection, an initial peak level of plasma viremia appears within a couple of weeks of transmission. At this early time point in the course of infection, HIV-1 has disseminated to the lymphoid organs and viral reservoirs and latency have been established. The HIV-1 viral load stabilizes at a relatively low level after a period of acute viral infection, defined as the “set point,” during which an immunological activation against HIV-1 is initiated. However, in tandem with seroconversion, HIV-1 production in reservoir or latently infected cells will eventually resume upon specific immunological responses such as host cytokine secretion or cell-mediated immune reactions (3, 4, 5, 6).

Lymphoid organs have been proposed to function as a major reservoir for HIV-1 (7). During the course of HIV infection, T cells and macrophages in secondary lymphoid organs also become major reservoir cells for HIV-1 (8). Several in vitro studies have now identified potentially stable reservoirs of inducible latently infected CD4+ cells carrying an integrated form of the viral genome (7, 8, 9). In addition to CD4+ T cells, monocytes are thought to be major reservoirs for HIV-1 in vivo, since a number of blood monocytes are maintained in HIV-1-infected patients even during the late disease stages when T cells can be practically undetectable (10, 11). These observations suggest that infected CD4+ T cells and macrophages provide sites as a stable reservoir and producer of HIV-1, causing the persistent production of progeny virus in lymphoid organs. However, it has not been well investigated how these reservoir cells can maintain sufficient levels of viral replication that will retain a sufficient viral load during the long course of this disease.

It is generally believed that the central point in the immune system is the lymphoid organs and germinal centers (GCs)3 where several immune cell types are localized, although these circulate throughout the whole body (12, 13, 14). The GCs of secondary lymphoid tissues are composed of B cells, CD4+ T cells, macrophages, and follicular dendritic cells (FDCs) (15, 16, 17). FDCs are characterized by the expression of CD21, CD35, CD40, and specific cell surface adhesion molecules including ICAM-1, VCAM-1, and the surface dendritic cell (DC) markers DC-SIGN and DRC-1 (16, 18, 19, 20, 21). The FDCs play an important role in the immune response by interacting with CD4+ T or B cells and in organization of the follicular structure.

In HIV infection, human FDCs can capture and retain infectious HIV particles in a stable manner on their cell surfaces for several months or even years via Fc receptors or other molecules (22, 23, 24, 25). Unlike conventional DCs, FDCs are not themselves infected with HIV despite expression of chemokine receptors and DC-SIGN (24). Furthermore, active HIV infection is largely confined to sites surrounding the FDCs (24), suggesting that this microenvironment is highly conducive to infection with this virus. FDCs have also been shown to transmit signals to the GC microenvironment which also appears to increase HIV infection and replication (24, 25). Our previous study showed that FDCs stimulated virus production in MOLT-4 T cells preexposed to HIV-1(23). Very recently, Thacker et al. (26) also reported that FDCs contributed to virus replication in CD4+ T cells infected with HIV-1 obtained from peripheral blood and GCs by increasing viral transcription mediated by TNF-α upon coculture. However, the role of FDCs in HIV-infected monocytes/macrophages is largely unknown.

We here report that FDCs can activate HIV-1 production in surrounding infected monocytes or macrophages via a cell-cell interaction with a clear mechanistic distinction from CD4+ T cells reported by Thacker et al. (26). This enhancement in monocytic cells was found to be mediated mainly by an association between P-selectin on FDCs, acting as a ligand, and P-selectin glycoprotein ligand 1 (PSGL-1), the cognate receptor, on HIV-1-infected cells. Furthermore, we delineate the biological significance of the PSGL-1/spleen tyrosine kinase (Syk) pathway in the FDCs-mediated switch to induce HIV-1 replication. Our current findings thus shed new light on mechanisms involved in the HIV replication pathway that are mediated through intercellular communication and provide clues for the design of future novel therapeutic interventions against AIDS and related disorders.

Materials and Methods

Cell culture and reagents

Several FDC lines were established from fresh human palatine tonsils and maintained as described previously (23). Briefly, FDCs were isolated from fresh palatine tonsils surgically removed. Tonsils were cut into pieces in the thickness of 2–3 mm and then digested for 15 min at 37°C with collagenase (type I; Wako). Following rinsing with RPMI 1640 by centrifugation at 400 × g for 7 min, cells were filtered through at 70-μm nylon mesh and overlaid on a 1.25, 2.5, and 5% continuous BSA gradient at 1 × g for 2 h. The lowest fraction with a higher density fraction was resuspended and cultured in RPMI 1640 medium with 10% FCS. Cell clusters in the lowest fraction included cells positive for DRC-1, a specific maker of FDCs. One week after the culture, adherent spindle-shaped FDCs appeared from the cell clusters after having released lymphoid cells and spontaneously proliferated without additional cytokines or growth factors. The character of FDCs was checked with expression of FDCs makers such as CAN-42, S-100α, CD54, DC-SIGN, and CXCR4 on its surface. After culturing along more than 2 wk, FDCs were stocked in −80°C before use. PBMCs were separated from three healthy donors in accordance with the guidelines of the ethics committee of Tokyo Medical and Dental University. PBMCs were cultured in RPMI 1640 containing 10% FBS at 37°C in 5% CO2. Primary monocytes were obtained from three healthy donors with Rosette Sep (StemCell Technologies) according to the manufacturer’s instructions. Primary macrophages were differentiated from monocytes by culturing in RPMI 1640 containing 10% AB serum (Sigma-Aldrich) and 20 ng/ml M-CSF (R&D Systems) for 7 days. HIV-1 chronically infected monocytic cell line U1 cells (27) were cultured in RPMI 1640 supplemented with 10% FBS (Invitrogen Life Technologies). Coculturing and Transwell assays were performed using 1 × 105 HIV-infected cell lines or 2 × 105 primary cells with 1 × 104 FDCs. For the FDC supernatant assay, filtered (0.2 μm) supernatants from FDC cultures were collected and added to HIV-1-infected cells at a 1:4 volume supernatant:total volume of fluid ratio. In the cell fixation assay, FDCs incubated with 3% paraformaldehyde in PBS for 2 h were washed three times with PBS and then twice with RPMI 1640 before coculturing.

Virus preparation and infection

HIV-1JR-FL or HIV-1NL4-3 viruses were generated by transfection of the pJR-FL or pNL4-3 construct in 293T cells, respectively. Virus preparations were passed through a 0.4-μm filter and titrated using a conventional method as described previously (28). For the HIV-1 infection of primary cells, PBMCs were infected with HIV-1JR-FL for 24 h following stimulation with PHA-P (3 μg/ml) for 3 days. To adjust the culture condition for monocytes/macrophages with that for PBMCs, monocytes or macrophages were also infected with HIV-1JR-FL for 24 h following stimulation with PHA-P (3 μg/ml) for 3 days. All primary cell cultures were maintained in the absence of IL-2. Jurkat or FDCs were infected with HIV-1NL-4-3 (multiplicity of infection (MOI) = 0.05) for 1, 3, or 5 days.

Antibodies

Polyclonal Abs raised against phospho-p65 (Ser536), phospho-Syk (Tyr352), phospho-IκBα (Ser32), and unmodified Syk were purchased from Cell Signaling Technology. Anti-p65 polyclonal, actin, and PSGL-1 (KPL1) mAb were purchased from Santa Cruz Biotechnology. Anti-α-tubulin mAb was purchased from Sigma-Aldrich. Neutralizing Abs targeting PSGL-1 or ICAM-1 were purchased from R&D Systems. Anti-HIV-1 p24 mAb (2C2; mouse IgG1) was produced by Y. Tanaka (University of Ryukyus, Okinawa, Japan).

Isolation of total RNA from cells and quantitative RT-PCR

U1 cells and FDCs were harvested after coculturing and washed three times with PBS. Total RNA was then extracted using Isogen (Nippongene) according to the manufacturer’s instructions. RNA (1 μg) was reverse transcribed using Superscript III (Invitrogen) before semiquantitative RT-PCR, and quantitative RT-PCR was performed using a SYBER Green One-step Real-time PCR kit (Invitrogen) with mRNA-specific primer pairs. Analyzed genes and corresponding primers are listed in supplemental Table I.4

Neutralization assay

HIV-1-infected cells were pretreated with neutralizing Abs (anti-PSGL-1, anti-ICAM-1, or control mouse IgG) for 2 h before and during coculturing. Optimal concentrations were determined by the IC50 values in accordance with the manufacturer’s instructions. Culture supernatants were collected after 3 days and subjected to measurement of HIV-1 p24.

Chemicals and inhibitory assays

BAY11-7082 and JNK inhibitor II were purchased from Merck. The Syk-specific inhibitor ER-27319 (29, 30) was purchased from Sigma-Aldrich. Cells were pretreated with 30 μM ER-27319, 1 μM JNK inhibitor II, 1–2 μM BAY11-7082, or DMSO (Sigma-Aldrich) for 2 h. The inhibitor treated/untreated cells were then cocultured with FDCs in the presence of Syk or NF-κB inhibitor. In small interfering RNA (siRNA) experiments, U1 cells were transfected with control or PSGL-1 siRNA (Santa Cruz Biotechnology) by Nucleofector (Amaxa) and then cocultured with FDCs. Lysates and supernatants were collected from these cultures after 3 days for measurement of p24 and Western blotting analysis.

Flow cytometry

Cells were washed twice with staining buffer (3% FBS and 0.09% NaN3/PBS) and then stained with PSGL-1-RP-E (BD Biosciences) for 30 min on ice. Cells were then washed twice and processed for flow cytometry.

Measurement of HIV-1 p24

Cell culture supernatants were collected after centrifugation at 4000 rpm for 5 min at 4°C and then processed for measurement of HIV-1 p24 by using Lumipulse (Fujirebio) according to the manufacturer’s instructions. Assays were performed in triplicate.

Results

FDCs activate HIV-1 production in adjacent HIV-1-infected monocytic cells

To address whether FDCs can also activate HIV-1 replication in the surrounding infected monocytes/macrophages as an effective bystander or stimulator, several primary FDCs were established from fresh palatine tonsils of three healthy human donors (23). Since each of these established cell lines was very similar in nature, exhibiting typical properties of FDCs (positive for CAN-42, S-100α, CD54, DC-SIGN, and CXCR4; morphological character such as spine-like spiculae and intercellular gap junction), the FDC 1 line was mainly used in subsequent experiments. FDCs themselves were not productively infected with HIV-1 (Fig. 1⇓A), consistent with previous reports (22, 23, 24, 25).

FIGURE 1.
FIGURE 1.

FDCs activate HIV-1 production in adjacent HIV-1-infected cells. A, Jurkat or FDCs were infected with HIV-1NL-4-3 (MOI = 0.05) for 1, 3, or 5 days. Cells were collected and then lysed for the separation of total RNA. Total RNA were treated with DNase I followed by quantitative RT-PCR with specific primer sets for either HIV-1 Gag or G3PDH. The data shown are the fold inductions normalized by G3PDH. B, U1 cells (1 × 105 cells/well) were cultured alone or in coculture with either FDCs or 293T cells (1 × 104 cells/well) for 3 days. Cell supernatants were then collected and assayed for measurement of p24. C, p24 levels in culture supernatants were monitored at 3, 5, and 7 days. D, p24 levels in lysates were monitored at 1, 3, 5, and 7 days by Western blot. E, PBMCs separated from a healthy donor were cultured with 3 μg/ml PHA for 3 days followed by infection with HIV-1JR-FL (MOI = 0.05) for 24 h. The PBMCs (2 × 105 cells/well) were then cocultured with FDCs (1 × 104 cells/well) in the absence of IL-2 for 14 days. Culture supernatants were then assayed for measurement of p24. F, Monocytes separated from a healthy donor were cultured with 3 μg/ml PHA for 3 days followed by infection with HIV-1JR-FL (MOI = 0.05) for 24 h. The monocytes (1 × 105 cells/well) were then cocultured with FDCs (1 × 104 cells/well) for 14 days. G, Primary differentiated macrophages were cultured with 3 μg/ml PHA for 3 days followed by infection with HIV-1JR-FL (MOI = 0.05) for 24 h. The macrophages (1 × 105 cells/well) were then cocultured with FDCs (1 × 104 cells/well) for 7 days. Culture supernatants were then assayed for measurement of p24. The data shown in B are the average ± SD of at least three independent experiments. The data presented in E–G were obtained using samples of three donors (∗, p ≤ 0.05 and ∗∗, p ≤ 0.01 by the Student t test).

Initially, the FDCs were cocultured with chronically HIV-1-infected monocytic cell line U1 to examine whether they had the ability to activate HIV-1 replication. After 3 days of growth, HIV-1 production was analyzed for HIV-1 p24. The results showed that coculturing with FDCs significantly induced HIV-1 replication in the two infected cell types tested, whereas no such induction was observed when the U1 cells were cultured with 293T cells (Fig. 1⇑B). A parallel kinetic study further demonstrated that the p24 levels in supernatants and lysates were increased in a time-dependent manner in U1 cells grown under these coculture conditions (Fig. 1⇑, C and D). To address whether this trend occurred also in primary cells, FDCs were cocultured with PBMCs from healthy donors after infection with R5 (HIV-1JR-FL) virus. As shown in Fig. 1⇑E, the virus production was considerably augmented in coculture with FDCs. Furthermore, parallel experiments revealed that the virus production in monocytes or macrophages purified from PBMCs was also increased by coculturing with FDCs (Fig. 1⇑, F and G). These data thus strongly indicate that FDCs can indeed activate viral replication monocytes/macrophages infected with HIV-1.

The enhancement of HIV-1 production by FDCs requires direct cell-cell interactions

To investigate whether this stimulation by FDCs was achieved by direct cell-cell interaction or soluble factors, we used two different cell culture methods for FDCs and U1 cells as follows: 1) FDCs were separately cultured with U1 cells using Transwells and 2) U1 cells were grown in culture medium supplemented with FDC supernatant. Although both culture systems could partially induce HIV-1 replication in U1 cells, these effects were ∼20–30% of the full induction of those observed following coculture with FDCs (Fig. 2⇓). This suggested that direct cell-cell interactions might be required for the full induction of HIV-1 replication in monocytic cells, although certain soluble factors may also activate HIV-1 replication to a lesser degree. Furthermore, the fixation of FDCs with paraformaldehyde before coculture completely abrogated the induction of HIV-1 replication in U1 cells, suggesting a requirement for bioactive cell surface molecules in this response.

FIGURE 2.
FIGURE 2.

The enhancement of HIV-1 production by FDCs requires direct cell-cell interactions. U1 cells (1 × 105 cells/well) were cocultured with regular or paraformaldehyde-fixed FDCs (1 × 104 cells/well), cultured separately with FDCs on Transwell plates, or grown in medium supplemented with FDC culture supernatant at a 1:4 ratio of volume supernatant:total volume of fluid. Cell supernatants were collected after 3 days and assayed for measurement of p24. The data shown are the average ± SD of two independent experiments (∗, p ≤ 0.05 and ∗∗, p ≤ 0.01 by the Student t test).

Taken together, these data indicate that direct interactions via cell surface bioactive molecules are important to fully stimulate HIV-1 replication in monocytic U1 cells by FDCs.

Activation of NF-κB in both FDCs and HIV-1-infected cells following coculture

Our initial analysis demonstrated that FDCs can enhance HIV-1 replication in infected cells via cell-cell interaction. We thus examined whether this induction is initiated by the activation of the HIV-1 long-terminal repeat sequence (LTR). Quantitative and semiquantitative RT-PCR analyses revealed that the levels of HIV-1 mRNA were increased in U1 cells in tandem with increased supernatant p24 levels under coculture conditions with FDCs (Fig. 3⇓, A and B).

FIGURE 3.
FIGURE 3.

Activation of NF-κB in both FDCs and HIV-1-infected cells. A and B, U1 cells (1 × 105 cells/well) were cocultured with FDCs (1 × 104 cells/well) for 5 days and the mRNA levels for the indicated genes were measured by RT-PCR (A) or quantitative RT-PCR (B). C, Schematic representation of HIV-1 LTR-derived luciferase reporter constructs. D, U1 cells (1 × 105 cells/well) were initially transfected with the indicated reporter constructs and then cocultured with FDCs (1 × 104 cells/well) for 48 h, which was followed by a gene reporter assay. E, U1 cells (1 × 105 cells/well) were pretreated with the indicated concentrations of BAY 11-7082 for 2 h and then cocultured with FDCs (1 × 104 cells/well) for 3 days in the presence of the same concentration of BAY 11-7082. Cell supernatants were then collected and assayed for measurement of p24. F, U1 cells (1 × 105 cells/well) were cocultured with FDCs (1 × 104 cells/well) for 3 days and both cell types were collected and subjected to immunoblotting analysis for phospho-p65 (Ser536), phospho-IκBα (Ser32), p65, or actin. G, U1 cells (1 × 105 cells/well) were cocultured with FDCs (1 × 104 cells/well) and collected at the indicated time points. Cell lysates were subjected to immunoblot analysis using either phospho-p65 (Ser536) or α-tubulin Abs. The data shown are the average ± SD of three independent experiments (∗, p ≤ 0.05 and ∗∗, p ≤ 0.01 by the Student t test).

HIV-1 replication has been shown to be regulated by host transcription factors such as NF-κB, NF-AT, Sp1, and AP-1 that are recruited and bind directly to the HIV-1 LTR (31, 32, 33). To determine the identity of the cis-regulatory element(s) within the HIV-1 LTR that are the targets of FDC-mediated transcriptional activation, we examined various 5′-deletion mutants of these region as described in Fig. 3⇑C (34). Coculturing of U1 cells with FDCs resulted in the activation of CD12 and CD23 reporter constructs that harbor a NF-κB-binding sequence. However, the CD52 and CD54 constructs lacking this NF-κB consensus site were not activated, suggesting the involvement of NF-κB in the HIV-1 replication response (Fig. 3⇑D). Consistent with this notion, the reporter construct CD12 that contains a site-directed mutation within the NF-κB binding site, CD12mκB, was not responsive to FDC stimulation. These results together indicate that the stimulation of HIV-1 in infected cells by FDCs is mediated via the activation of the HIV-1 LTR via NF-κB.

To further address this in terms of biological function, cells were treated with the NF-κB inhibitor BAY 11-7082 to further delineate the role of NF-κB in FDC-mediated HIV-1 replication. Treatment with BAY 11-7082 significantly suppressed HIV-1 production from U1 cells, even when growing in coculture with FDCs (Fig. 3⇑E), although the viability of both cell types was not significantly affected by this exposure (data not shown). Taken together, our data thus indicate that intercellular communication pathways triggered by FDCs can promote and augment HIV-1 production in infected cells via NF-κB activation.

We next investigated whether NF-κB is in fact activated in FDCs as well as in U1 cells under coculture conditions. Consistent with our above gene reporter data, NF-κB activation was confirmed in U1 cells as revealed by the phosphorylation status of NF-κB p65 and IκBα (Fig. 3⇑F). Interestingly, parallel experiments showed NF-κB activation in FDCs also in our coculture system, as revealed by immunoblotting with phospho-specific Abs (Fig. 3⇑F). Furthermore, fractionation analysis demonstrated that the nuclear p65 (RelA) levels were significantly enhanced in both U1 and FDCs, indicating the nuclear accumulation of activated NF-κB (Fig. 3⇑F). Parallel kinetic analysis revealed that NF-κB activation in U1 cells was initiated at 12 h and persisted for at least 48 h (Fig. 3⇑G). These findings thus support our contention that cell-cell interactions between FDCs and U1 cells results in the constitutive activation of NF-κB in both cell types and that this is likely to be involved in the amplification of HIV-1 replication signals.

FDCs activate HIV-1 production via a P-selectin-PSGL-1 interaction

We were prompted to examine whether NF-κB up-regulates a specific cell surface ligand and its cognate receptor in FDCs and HIV-1-infected monocytic cells, eventually contributing to the amplification of HIV-1 replication signals via NF-κB activation. To this end, we examined the expression of different cell surface ligands and their cognate receptors which are known to be regulated by NF-κB. We chose three ligand/receptor combinations based upon a database search, ICAM-1/CD11b, VCAM-1/CD49d, and P-selectin/PSGL-1, and the expression of these molecules was analyzed by quantitative RT-PCR. Although the mRNA levels of ICAM-1 and VCAM-1 were not significantly altered upon stimulation, transcripts for P-selectin (CD62P/SLBP) were dramatically increased in FDCs (Fig. 4⇓A). Interestingly, transcripts for the cognate receptor for P-selectin, PSGL-1, were found to be significantly up-regulated in U1 cells grown in coculture with the FDCs (Fig. 4⇓B), but this was not the case for the CD11b and CD49d receptors. Quantitative RT-PCR and FACS analysis revealed that treatment with the NF-κB inhibitor BAY11-7082 significantly inhibited the increase of PSGL-1 mRNA expression and, consequently the cell surface expression of PSGL-1, in U1 cells cocultured with FDCs (Fig. 4⇓, C and D). This suggested a crucial role for NF-κB signaling in the induction of PSGL-1 during this coculture in HIV-1-infected cells. Likewise, we found that BAY11-7082 treatment also decreased the induction of P-selectin mRNA in FDCs, indicating that the NF-κB activation in FDCs could play a crucial role in the induction of P-selectin during the coculture with HIV-1-infected monocytic cells (Fig. 4⇓E).

FIGURE 4.
FIGURE 4.

Involvement of P-selectin/PSGL-1 in reactivation of HIV-1 replication by FDC. A and B, U1 cells (1 × 105 cells/well) were cocultured with FDCs (1 × 104 cells/well) for 3 days. The mRNA levels of the indicated genes were then measured by quantitative RT-PCR. Labels inside parentheses indicate counterpart ligand or receptor molecules. C–E, U1 cells (1 × 105 cells/well) were cocultured with FDCs (1 × 104 cells/well) in the presence of BAY 11-7082 (1 μM) for 3 days and the levels of PSGL-1 in these cells were then analyzed by quantitative RT-PCR (C). Cell surface PSGL-1 was analyzed by flow cytometry using an anti-PSGL-1 Ab (D). M1 denotes the range of positive cell populations. E, P-selectin expression in FDCs analyzed by quantitative RT-PCR. F, U1 cells (1 × 105 cells/well) were untreated or pretreated with either PSGL-1 or ICAM-1 Ab for 1 h. Cells were then cocultured with FDCs (1 × 104 cells/well) for 3 days followed by measurement of p24. G, U1 cells (1 × 105 cells/well) were transduced with either control or PSGL-1 siRNA (final 6 nM) by Nucleofector according to the manufacturer’s instructions. Cells were then cocultured with FDCs (1 × 104 cells/well) for 3 days followed by Western blot analysis with the indicated Abs. Numerical values below the blots indicate p24 signal intensities normalized by α-tubulin intensity derived by densitometry. The data shown are the average ± SD of three independent experiments (∗, p ≤ 0.05 and ∗∗, p ≤ 0.01 by the Student t test).

Next, to test the biological significance of a P-selectin-PSGL-1 interaction in terms of HIV-1 induction in our FDC coculture system, U1 cells were pretreated with blocking Ab against PSGL-1 before setting up these cultures. Treatment with PSGL-1 Ab, but not an ICAM-1 Ab, specifically suppressed HIV-1 production in a dose-dependent manner (Fig. 4⇑F). Consistent with this result, targeted disruption of PSGL-1 by specific siRNA significantly decreased HIV-1 production in U1 cells coculturing with FDCs (Fig. 4⇑G). These results together indicate that a juxtacrine signaling mechanism mediated by PSGL-1/P-selectin underlies the activation of HIV-1 replication in infected monocytic cells stimulated by FDCs.

Syk acts as a downstream effector of PSGL-1 during HIV-1 replication

Several previous reports have demonstrated that the cytoplasmic domain of PSGL-1 can directly interact with a Src family kinase, the Syk (35). Syk consists of two N-terminal Src homology 2 domains, which bind phosphorylated ITAM sequences, and a C-terminal tyrosine kinase domain (35, 36, 37). The phosphorylation of Syk at Tyr352 has been shown to be a hallmark of its activation. Indeed, phosphorylated Syk was found in our present analyses to be significantly increased in U1 cells during their cocultivation with FDCs (Fig. 5⇓A).

FIGURE 5.
FIGURE 5.

Syk is a mediator of P-selectin/PSGL-1 signaling for HIV-1 replication in U1 cells. A and B, U1 cells (1 × 105 cells/well) were untreated or pretreated with either ER-27319 (30 μM) or JNK inhibitor II (1 μM) for 1 h. Cells were then cocultured with FDCs (1 × 104 cells/well) for 3 days in the presence or absence of inhibitor. Cells were collected and subjected to immunoblotting analysis for phosphorylated Syk (Tyr352), unmodified Syk, and α-tubulin (A). The numbers below the blot indicate the band intensity ratios. Cell supernatants were assayed for measurement of p24 (B). The data shown are the average ± SD of two independent experiments (∗, p ≤ 0.05 and ∗∗, p ≤ 0.01 by the Student t test).

To next examine the possible biological functions of Syk during HIV-1 replication, we used a specific inhibitor of the molecule ER-27319 (29, 30) in our FDC cocultures. Treatment with ER-27319 significantly decreased HIV-1 production and this was accompanied by a reduction in the phosphorylated Syk levels in U1 cells (Fig. 5⇑), whereas JNK inhibitor II had no such effects. These results indicate that the juxtacrine signaling between FDCs and HIV-1-infected monocytic cells mediated by P-selectin/PSGL-1 results in the activation of Syk, which serves as a mediator of the function of NF-κB activation in the HIV-1 replication pathway.

PSGL-1 and Syk inhibition blocks FDC-induced HIV-1 replication in primary monocytes

Finally, we addressed whether FDCs can also activate HIV-1 production in infected primary cells via P-selectin/PSGL-1 pathway, in this case human primary monocytes from healthy donors that had subsequently been exposed to HIV-1JR-FL. At 24 h after viral infection, the primary monocytes were cocultured with FDCs in the presence or absence of either PSGL-1 Ab or the Syk inhibitor ER-27319. Both of these treatments significantly inhibited HIV-1 production in the primary monocytes in a manner similar to U1 cells (Fig. 6⇓). These results indicate that similar to U1 cells, the PSGL-1/Syk signaling is likely to be a major pathway mediating FDC-induced HIV-1 replication in primary monocytes.

FIGURE 6.
FIGURE 6.

Inhibition of the PSGL-1/Syk pathway abrogates FDC-induced HIV-1 replication in primary monocytes. A and B, Primary human monocytes were separated from three healthy donors as indicated in Materials and Methods and these cells were then treated with 3 μg/ml PHA for 3 days. After stimulation, the cells (1 × 105 cells/well) were infected with HIV-1JR-FL (MOI = 0.05) for 24 h and subsequently cocultured with FDCs (1 × 104 cells/well) in the presence of PSGL-1 Ab (25 μg/ml; A) or ER-27319 (30 μM; B) at 14 days, followed by measurement of p24 (∗, p ≤ 0.05 and ∗∗, p ≤ 0.01 by the Student t test).

Discussion

Previous studies have indicated that HIV-1 infection is largely confined to the GCs of secondary lymph nodes where FDCs commonly reside (15, 16, 17). This microenvironment could thus provide the site for highly productive HIV-1 infection whereby FDCs might execute “on-switch” signaling to increase HIV replication. Furthermore, cell-cell infection appears to be far more efficient for viral spread than cell-free virus infection (38, 39). We here report that FDCs can facilitate HIV-1 replication in adjacent infected monocytes/macrophages via a cell-cell interaction mechanism.

FDCs have been shown to interact with B or CD4+ T cells in the GCs of normal lymph nodes (16, 17, 20). It is also reported that in tonsils, CD150 (SLAM)+ monocytes were localized not only in T cell areas, but also within GCs, suggesting they play a role in B cell activation (40). Moreover, substantial numbers of HIV-infected macrophages were observed in GCs during the course of HIV infection (41). Thus, FDCs can interact with HIV-infected monocytes or macrophages under these conditions during HIV-1 infection. Furthermore, the dysfunctional FDC network is observed in secondary lymph nodes of lymphadenopathy, where the degeneration of the FDC network is usually seen following highly active antiretroviral therapy or administration of therapeutic vaccine in HIV or SIV infection (42, 43, 44, 45). One of the most common histological features of HIV-1-associated lymphadenopathy is hyperplastic lymphoid follicles that subsequently undergoes folliculolysis, in which FDCs can be scattered to the extra-GC within the lymph nodes such as cortical sinuses and mantle bodies (46, 47). Our results with immunohistochemical analysis indicate that FDCs reside with various types of HIV-1-infected cells including monocytes or macrophages in lymphoid organs of HIV-1-associated lymphadenopathy (supplemental Fig. 1). Therefore, our current proposed model for cell-cell interaction between FDCs and HIV-1-infected monocytic cells may reflect the biological or pathological aspects of the natural HIV infection in vivo. However, we could not determine the specific cell surface molecules for activating HIV-1 replication via the cell-cell interaction in vivo. Moreover, it is not well confirmed whether a multitude of other cells, cytokines, and other factors in vivo could influence the cell-cell interaction observed in our in vitro coculture system. Further careful analysis should be performed using human tissues as well as a humanized mouse model inoculated with HIV-1-infected human cells.

We clearly demonstrated here that FDCs, derived from human tonsils, can enhance HIV-1 production in infected monocytic cells in a coculture system. This enhancement requires direct cell-cell interactions via a juxtacrine signaling pathway that is mediated by P-selectin/PSGL-1. Our results are summarized as follows: 1) FDCs can activate HIV-1 replication in infected cells through cell-cell interactions; 2) HIV-1 replication is activated at the transcriptional level and is accompanied by the activation of the HIV-1 LTR through NF-κB; 3) P-selectin expression in FDCs and the up-regulation of its cognate receptor PSGL-1 in HIV-1-infected monocytes cells are facilitated via NF-κB activation; 4) the pathways leading to HIV-1 induction in cell lines also function in human primary monocytes and macrophages infected with HIV-1; and 5) selective inhibitors of PSGL-1 or Syk can efficiently block HIV-1 production in U1 and primary monocytes. These data together indicate for the first time that FDCs are a potent inducer of HIV-1 replication in surrounding infected monocytes and macrophages and that PSGL-1/Syk signaling plays a crucial role in this induction of HIV-1.

Very recently, Thacker et al. (26) reported a similar but distinct role of FDCs in the induction of HIV-1 replication in CD4+ T cells obtained from PBMCs and GCs. We also confirmed that FDCs could stimulate HIV-1 replication in MOLT-4 T cells (23) as well as in primary CD4+ T cells (data not shown). However, FDCs-induced HIV-1 replication in CD4+ T cells might be mediated by a distinct mechanism from HIV-1-infected monocytic cells since the involvement of the PSGL-1/Syk pathway in CD4+ T cells was found to be not prominent (K. Ohba, A. Ryo, and N. Yamamoto, unpublished observation). Therefore, the molecular mechanism for FDCs to stimulate HIV-1 replication in surrounding infected cells could be attributable to cell type specific.

Intercellular interactions via a ligand/receptor juxtacrine signaling system has been implicated in several virus infections. Tsukamoto et al. (48) reported that the juxtacrine function of the IL-15/IL-15 receptor system in human B cell lines might play a role in the infectivity of EBV (48). Pilotti et al. (49) have demonstrated a crucial protective role for CCL3L1/CCL3 (MIP-1αP/LD78α) signals in both HIV infection and subsequent disease progression. These intercellular communication processes may play an important role in the sustained infection of viruses in different microenvironments within lymphoid organs. Further careful analyses will be required in the future to elucidate the variety of intercellular communication systems that may operate during HIV-1 infection.

There is now some evidence for a role of PSGL-1 as a signal-transmitting receptor in neutrophils (50), monocytes (51), and T lymphocytes (52). This molecule has been reported to associate with Syk through its interaction with moesin and promotes the tyrosine phosphorylation and thus the activation of Syk (35). In addition, signals elicited through PSGL-1/Syk can induce the activation of downstream effectors such as ERK, c-Fos, and NF-κB (53). The activation of NF-κB via PSGL-1 has also been demonstrated in platelet-stimulated monocytes, although the details of the molecular pathways leading to NF-κB activation in this manner have not yet been elucidated (51). Consistent with this result also, we found from our current analyses that PSGL-1/Syk signaling can activate NF-κB. This observation suggests a linkage between PSGL-1 signaling and HIV-1 replication through the activation of NF-κB.

Recently, Gilbert et al. (54) have reported that Src and Syk tyrosine kinases play important roles in the spread of HIV-1 from immature monocyte-derived DCs to CD4+ T cells. They found that these kinases play a suppressive role in virus transfer in vitro probably by inhibiting the formation of the virological synapse. However, it has not been well characterized whether these signaling molecules contribute to the cell-cell interaction between HIV-1-infected cells and adjacent noninfected cells for virus replication. We showed in this current study that the activation of Syk through the PSGL-1 positively regulates HIV-1 replication in infected monocytic cells. Thus, Syk could be involved at multiple points in HIV-1 infection and its role could be dependent on each step of HIV-1 life cycle.

In summary, we demonstrate in our current study that FDCs are a potent activator of HIV-1 replication in surrounding infected monocytic cells. Furthermore, the PSGL-1/Syk pathway is important for this activation of HIV-1 replication. These results shed valuable new light on our understanding of the natural progression of HIV-1 infection over the long term and could provide a means for designing novel therapeutic interventions against AIDS and related disorders.

Acknowledgments

We thank S. Yamaoka and W. Sugiura for helpful discussions and H. Shimura, N. Sakamaki, C. Matsubara, M. Tanaka, and H. Soeda for technical assistance.

Disclosures

The authors have no financial conflict of interest.

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 This work was supported in part by grants from the Japanese Ministries of Education, Culture, Sports, Science and Technology (20390136, 13226027, 14406009, and 1941075) Health, Labour and Welfare (H18-005) and Human Health Science (H19-001) to N.Y. and A.R.

  • ↵2 Address correspondence and reprint requests to Dr. Akihide Ryo and Dr. Naoki Yamamoto, AIDS Research Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjyuku-ku, Tokyo 162-8640, Japan. E-mail addresses: aryo{at}nih.go.jp and nyama{at}nih.go.jp

  • ↵3 Abbreviations used in this paper: GC, germinal center; FDC, follicular dendritic cell; DC, dendritic cell; PSGL-1, P-selectin glycoprotein ligand 1; Syk, spleen tyrosine kinase; LTR, long terminal repeat; MOI, multiplicity of infection; siRNA, small interfering RNA.

  • ↵4 The online version of this article contains supplemental material.

  • Received February 3, 2009.
  • Accepted April 25, 2009.

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