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Originally published online as doi:10.1189/jlb.0603291 on January 23, 2004

Published online before print January 23, 2004
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(Journal of Leukocyte Biology. 2004;75:504-514.)
© 2004 by Society for Leukocyte Biology

Virally stimulated plasmacytoid dendritic cells produce chemokines and induce migration of T and NK cells

Nicholas J. Megjugorac*, Howard A. Young{dagger}, Sheela B. Amrute*, Stacey L. Olshalsky* and Patricia Fitzgerald-Bocarsly*,1

* University of Medicine and Dentistry of New Jersey, New Jersey Medical School, and The Graduate School of Biomedical Sciences, Newark; and
{dagger} Laboratory of Experimental Immunology, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, Maryland

1Correspondence: Department of Pathology and Laboratory Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 South Orange Ave., Newark, NJ 07103. E-mail: bocarsly{at}umdnj.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The natural interferon (IFN)-producing cell is now known to be identical to the plasmacytoid dendritic cell (PDC). These are Lin-, CD123+, CD11c-, and human leukocyte antigen-DR+ cells that secrete large amounts of IFN-{alpha} (1–2 IU/cell) when stimulated by enveloped viruses such as herpes simplex virus. In the current study, we have evaluated chemokine expression by virally stimulated PDC. Up-regulation of mRNA for CCL4, CCL3, CCL5, CCL2, and CXC chemokine ligand (CXCL)10 in herpes simplex virus-stimulated PDC was detected by RNAse protection assays. In contrast, PDC-depleted peripheral blood mononuclear cells did not up-regulate these mRNA species upon viral stimulation. Enzyme-linked immunosorbent assay and/or intracellular flow cytometry confirmed production of these proteins, and studies indicated overlapping production of IFN-{alpha} and the other cytokines/chemokines by PDC. Endocytosis plays a critical role in chemokine induction, as disruption of the pathway inhibits the response. However, transcription of viral genes is not required for chemokine induction. Autocrine IFN-{alpha} signaling in the PDC could account for a portion of the CXCL10 and CCL2 production in virally stimulated PDC but was not responsible for the induction of the other chemokines. To evaluate the functional role of the chemokines, chemotaxis assays were performed using supernatants from virally stimulated PDC. Activated T cells and natural killer cells, but not naïve T cells, were preferentially recruited by these PDC supernatants. Migration was subsequently inhibited by addition of neutralizing antibody to CCL4 and CXCL10. We hypothesize that virally induced chemokine production plays a pivotal role in the homing of leukocytes to PDC.

Key Words: cell trafficking • interferon • herpes simplex virus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) have received much attention as pivotal hinges in the machinery of a developing immune response. DC are a heterogeneous population of antigen presenting cells (APC) with the unique ability to take up exogenous antigen [1 2 3 4 5 ], process and present it via major histocompatibility complex classes I and II pathways [6 7 8 ], and go on to induce primary immune responses [9 10 11 12 ]. Our laboratory has been interested in a population of cells in peripheral blood, termed the natural interferon (IFN)-producing cells (NIPC) [13 , 14 ]. Although long thought to be within the DC lineage, these cells have only recently been identified as a lymphoid-derived DC subset known as precursors to type 2 DC (pDC2) or plasmacytoid DC (PDC) [15 16 17 ]. NIPC/PDC produce high levels of IFN-{alpha} (up to 3–10 pg/cell or 1–2 IU) in response to herpes simplex virus (HSV) as well as other enveloped viruses including influenza, Sendai virus, vesicular stomatitis virus (VSV), Newcastle disease virus, and human immunodeficiency virus (HIV) [13 , 18 19 20 21 ]. These light-density cells are phenotypically CD123 (IL-3 receptor {alpha}+), CD11c-, p T cell receptor (TCR){alpha}+, Lin-, and human leukocyte antigen (HLA)-DR+ and express two recently defined molecules, blood DC antigen 2 (BDCA-2) and BDCA-4 [15 , 22 ].

Although originally identified in the blood as an infrequent cell type representing 0.2–0.5% peripheral blood mononuclear cells (PBMC) [23 , 24 ], virus-responsive, IFN-{alpha}-producing PDC have also been found in the spleen, bone marrow (BM), tonsil, and lymph node [25 ] (P. Fitzgerald-Bocarsly, in preparation). There is also evidence of PDC localizing in skin in response to spirochete antigen inoculation [26 ], as well as localizing to the nasopharynx of allergic individuals [27 ].

A second subset of DC in the blood is known as DC1 or myeloid DC (MDC) [28 ]. These myeloid-derived cells are CD123 dim, CD11c+, pTCR{alpha}-, Lin-, and HLA-DR+ [29 , 30 ]. These subsets of DC are morphologically and phenotypically divergent and are believed to arise from distinct precursors derived from CD34+ hematopoietic stem cells in the BM [31 , 32 ]. Cells similar to DC1 can also be generated from CD14+ monocytes by culturing them in the presence of granulocyte macrophage-colony stimulating factor and interleukin (IL)-4 [30 , 33 , 34 ]. The immature DC1 then matures with the addition of tumor necrosis factor {alpha} (TNF-{alpha}), CD40L, and/or IL-1 [32 , 35 36 37 38 ] or can become Langerhans cells (LC) by culturing with transforming growth factor-ß [39 ]. The MDC have been well characterized with respect to cytokine and chemokine production. They produce large amounts of the T helper cell type 1 (Th1)-inducing cytokine, IL-12, upon stimulation with lipopolysaccharide [40 ]. DC1 also produce chemokines, members of a rapidly expanding family of chemoattractant proteins that signal through transmembrane receptors coupled to intracellular guanosine 5'-triphosphate-binding regions. Moreover, not only do immature DC1 produce chemokines, but different subsets of these DC are stimulated selectively through chemokine receptors. For example, LC migrate in response to macrophage-inflammatory protein (MIP)-3{alpha} via CCR6 [41 , 42 ] and immature DC1 respond to MIP-1{alpha}/ß and monocyte chemoattractant protein (MCP) chemokines via CCR1, -5, and -2. Mature DC lose responsiveness to these inflammatory chemokines but gain responsiveness to Epstein-Barr-induced 1 ligand chemokine/MIP-3ß and secondary lymphoid-tissue chemokine/6Ckine by up-regulating CCR7, which facilitates entry into the lymph node [43 ].

Somewhat less is known about cytokine production by PDC. Until recently, their only reported secreted products were IFN-{alpha}/ß [15 , 20 , 44 ], TNF-{alpha}, and IL-6 [45 ]. It has recently been reported that PDC produce the inflammatory chemokines MIP-1{alpha} and -ß, low levels of IFN-inducible protein 10 (IP-10) and MCP-1 in response to CpG, inactivated influenza virus, and CD40L stimulation [46 ]. PDC also produce IL-8 and IP-10 upon stimulation through Toll-like receptors (TLRs) [47 ]. It is interesting that the same percentage of PDC that produce IFN-{alpha} in response to TLR ligation by Imiquimod does so in response to HSV stimulation [48 ]. In each instance, a subpopulation of the PDC (roughly 35%) produces IFN-{alpha}, perhaps representing various levels of maturation or differentiation within the population. Their unique ability to produce massive amounts of IFN-{alpha} has led to the suggestion that the PDC are "professional" IFN-producing cells [15 ]. The IFN-{alpha}-producing PDC serve as excellent accessory cells to support natural killer (NK) cell-mediated lysis of virally infected targets [49 ]. Of particular interest is the decreased IFN-{alpha} production potential of PDC in HIV-seropositive patients [50 , 51 ]. The PDC not only lose their ability to produce IFN but also decrease in number as HIV progresses into AIDS [52 , 53 ]. With the ability to interact with cells in the periphery (such as NK cells) as well as cells in secondary lymphoid organs (T cells) [9 ], the PDC are uniquely equipped to act as a functional link between the innate and adaptive arms of the immune system. NK and activated T cells have been shown to respond to inflammatory chemokines such as MIP-1{alpha}/CCL3, MIP-1ß/CCL4, IP-10/CXCL10, and regulated on activation, normal T expressed and secreted (RANTES)/CCL5 [43 ]. The current studies seek to uncover the mechanisms involved in HSV-stimulated chemokine production in PDC. We report that PDC produce a subset of chemokines in response to viral stimulation that allows them to chemoattract NK and activated T cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Viruses
HSV-1 strain 2931 and VSV (originally obtained from Dr. Nicholas Ponzio, New Jersey Medical School, Newark) were grown and titrated by a plaque-forming assay in VERO cells [American Type Culture Collection (ATCC), Manassas, VA] as described previously [54 ]. Sendai virus (Sendai/Cantell strain) was also obtained from ATCC. All virus stocks were stored at –70°C until use. UV-irradiated HSV was obtained by exposing the above virus stock to 7800 J/cm2 in a UV Stratalinker 1800 (Stratagene, La Jolla, CA). This UV treatment inhibits viral replication in permissive VERO cells and enhanced green fluorescent protein (eGFP) expression by HSV strains containing an eGFP construct under immediate early promoters. Where noted, cells were preincubated in media containing 0.1 mM chloroquine (Sigma-Aldrich, St. Louis, MO) for 1 h, washed, resuspended, and subsequently stimulated with HSV.

Cell lines
GM-0459A (National Institute of General Medicine Sciences Human Genetic Mutant Cell Line Repository, Camden, NJ), a primary fibroblast cell line trisomic for chromosome 21, was grown in Dulbecco’s modified Eagle’s medium (DMEM; JHR Biosciences, Lenexa, KS) supplemented with 15% fetal calf serum (FCS; HyClone, Logan, UT), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (DMEM, 15%). VERO cells were grown in DMEM–10% FCS.

Preparation of PBMC
PBMC were isolated by Ficoll-Hypaque density centrifugation (Lymphoprep, Accurate Chemical and Scientific Co., Weatbury, NY) from heparinized peripheral blood obtained via venipuncture with informed consent from healthy volunteers. PBMC were washed twice with Hank’s balanced salt solution (Life Technologies, Grand Island, NY), resuspended in RPMI-1640 media (Life Technologies) containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 25 mM HEPES, and enumerated electronically with a Coulter counter series Z1 (Coulter Electronics, Hialeah, FL). Where noted, PBMC, PDC, and non-DC (1x106/ml) were stimulated with HSV [multiplicity of infection (MOI)=1] for 18 h, and supernatants and/or cells were collected and immediately stored at –20°C for later use.

Enrichment of PDC
PBMC were depleted of T, B, NK, and red blood cells and monocytes by using a modified version of the magnetic blood DC isolation kit (Miltenyi Biotec, Auburn, CA). PBMC were washed and resuspended in magnetic cell sorter (MACS) buffer [phosphate-buffered saline (PBS; Life Technologies)] with 0.5% bovine serum albumin (BSA) and 2 mM EDTA (Sigma-Aldrich) and were incubated at 4°C for 10 min with FcR-blocking reagent and hapten-conjugated murine antibodies against CD3, CD11b, and CD16. Cells were then washed twice and incubated with antihapten microbeads, CD19 microbeads, and glycophorin A microbeads at 4°C for 15 min. After washing, the labeled cells were applied to a LD-negative selection column pretreated with 2 ml magnetic activated cell sorter buffer and subsequently washed twice with 1 mL MACS buffer. Negatively selected DC were pelleted, counted, and evaluated for purity using flow cytometry (relative enrichment: 60% cells were HLA-DR+, Lin-, CD11c-, and CD123+ by flow cytometry). The PDC-depleted PBMC (non-DC) that remained inside the column were eluted by removing the cells from the magnetic field and flushing the column with 2 mL RPMI.

IFN bioassay
IFN bioassays were performed using a cytopathic effect reduction assay with GM-0459A cells infected with VSV as the challenging virus as described previously [54 ]. An IFN-{alpha} reference standard (National Institute of Allergy and Infectious Diseases, Bethesda, MD, standard G-023-901-527) was used at 100 IU/mL.

RNA purification
RNA purification was achieved using an RNeasy mini kit (Qiagen, Santa Clarita, CA) according to the manufacturer’s instructions. Briefly, cells were pelleted, lysed, and homogenized using a Qiashredder. Ethanol was added to adjust binding conditions of the sample, and the mixture was subsequently spun through an RNeasy spin column for adsorption of RNA to membrane. Contaminants were removed by washing with buffer RW1 and RPE, and mRNA was eluted in RNAse-free water.

RNAse protection assay
Multiprobe RNAse protection assays were performed according to the manufacturer’s (PharMingen, San Diego, CA) directions with the following modifications.

Hybridization
Probes were synthesized with 33P uridine 5'-triphosphate (70–80 µC/full reaction) using the PharMingen in vitro transcription kit. Following incubation, yeast tRNA and EDTA were added as described by the manufacturer, the reaction was placed on Amersham-Pharmacia G25 microspin columns, and the probe purified by centrifugation for 2 min at 3000 rpm. To each RNA, 0.5–1.0 x 106 cpm was added in a final hybridization volume of 10–20 µl (at least 50% PharMingen hybridization buffer).

RNAse inactivation
A master cocktail, containing 200 µl Ambion RNAse inactivation reagent (Ambion, Austin, TX), 50 µl ethanol, 5 µg yeast tRNA, and 1 µl Ambion GycoBlue coprecipitate per RNA sample, was used to precipitate the protected RNA. After adding the individual RNAse-treated samples to 250 µl of the inactivation/precipitation cocktail, the samples were mixed well, placed at –70°C for 30 min, and subjected to centrifugation at 14,000 rpm for 15 min in a room temperature (RT) microcentrifuge. The supernatants were decanted, a sterile cotton swab was used to remove excess liquid, and the pellet was resuspended in 3 µl PharMingen sample buffer.

Flow cytometry
After washing with PBS, cells were resuspended in PBS containing 5% heat-inactivated human serum and incubated with fluorochrome-conjugated antibodies at 4°C for 20 min. The cells were then washed twice with PBS and resuspended in 300 µl 1% paraformaldehyde. Antibodies used were as follows: anti-immunoglobulin G (IgG)2a, IgG1 (Dako D/S, Denmark), CD3, CD4, CD16, CD123, and HLA-DR (BD BioSciences, San Diego, CA).

Intracellular detection of IFN-{alpha} and chemokines
PBMC were prepared for intracellular detection of IFN-{alpha} and chemokines using a modification of the method described previously [55 ]. PBMC (2x106 cells/ml) were stimulated with 10,000 IU/ml recombinant (r) human IFN-{alpha}2b (Schering-Plough, Kenilworth, NJ) or HSV-1 strain 2931 (MOI=1) for 4 h at 37°C in 5% CO2. Brefeldin A (5 µg/ml; Sigma-Aldrich) was then added, and incubation continued for an additional 2 h. For IFN-{alpha}-blocking studies, cells were preincubated for 10 min at RT with a 1:700 dilution of sheep anti-IFN (G-026-501-568) or sheep control antibody (G-027-501-568) before stimulation (National Institute of Allergy and Infectious Diseases). Cells were washed with cold 0.1% BSA (Sigma-Aldrich) in PBS (Life Technologies), blocked with 5% heat-inactivated human serum, and stained with the fluorochrome-conjugated antibody sufficient to detect PDC; CD123 phycoerythrin (PE) and HLA-DR APC (BD Biosciences) for 20 min at 4°C were washed and fixed with 1% paraformaldehyde (Fisher, Pittsburgh, PA) in PBS at 4°C overnight. The following day, cells were washed twice with PBS, 2% FCS, and permeabilized with 0.5% saponin (Sigma-Aldrich) in PBS, 2% FCS, for 30 min at RT, and subsequently incubated with 50 ng biotinylated 293 monoclonal antibody (mAb) to IFN-{alpha} and/or anti-CCL3, CCL4, CCL5, CCL2 (R&D Systems, Minneapolis, MN), biotinylated anti-CXCL10, or TNF-{alpha} (BD PharMingen) for 30 min at RT. In the samples containing biotinylated primary antibodies (IFN-{alpha} and CXCL10), cells were then washed twice with saponin and incubated with Streptavidin-Quantum Red (Sigma-Aldrich) for 30 min at RT. Finally, the cells were washed and resuspended in 1% paraformaldehyde in PBS and analyzed using the FACSCalibur flow cytometer with CellQuest analysis software (BD Biosciences).

Enzyme-linked immunosorbent assays (ELISA)
Supernatants were generated as described above and used in ELISA according to the following manufacturer’s instructions: TNF-{alpha} and CXCL10 optEIA (PharMingen), CCL3 and CCL5 duoset (R&D Systems), and CCL4 Quantikine (R&D Systems). The assays were performed in Nunc-Immuno maxi-sorp plates (Nalge-Nunc International, Denmark), read immediately at 450 nm in a Tecan Genios microplate reader, and analyzed using Magellan 2.5 analysis software (Tecan, Research Triangle Park, NC).

Migration studies
NK and T cells were enriched from PBMC using Miltenyi NK and untouched T cell isolation kits according to the manufacturer’s instructions. Activated T cells were generated by culturing T cells for 1 week in RPMI–10% FCS supplemented with phytohemagglutinin (PHA; 1 µg/ml; Sigma-Aldrich) and rIL-2 (50 U/ml; R&D Systems). Migration experiments were performed by placing 100 µl purified NK or T cells (at 5x106 cell/ml) in the upper wells of 5 µm pore Transwell inserts (Corning, Somerset, NJ), which were then inserted into wells of a 24-well plate. Supernatants (400 µl) from unstimulated or HSV-1-stimulated PDC or media (prepared as above) were placed in the lower chambers of the wells. The plates were incubated for 4 h at 37°C with 5% CO2 in a humid environment and then for 10 min at 4°C to loosen cells stuck to the undersides of the membranes. The media in the lower chambers were then collected, and the number of migrated cells was counted using a Nuebauer hemacytometer and analyzed via fluorescein-activated cell sorter as described above. Where indicated, neutralizing antibodies to CCL4 (2 µg/mL) and CXCL10 (5 µg/mL; US Biological, Swampscott, MA) were incubated with PDC supernatants 10 min before the start of the assay. Migration inhibition data were determined by subtracting the amount of background migration from the experimental groups.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of chemokine mRNA by PDC
To determine if HSV stimulation induces chemokine mRNA, PBMC, PDC-depleted PBMC (non-DC), and PDC-enriched cell populations were mock- or HSV-stimulated. mRNA was isolated at 2 and 6 h following stimulation and analyzed via an RNAse protection assay, which included probes for CCL5, CCL3, CCL4, CXCL10 (Fig. 1A ), and CCL2 (6 h only, Fig. 1B ). Unstimulated PDC expressed little or no CCL3, CCL4, or CXCL10 mRNA (Fig. 1A , lane A). However, by 6 h (Fig. 1A , lane B, and B) and even as early as 2 h (Figure 1A , lane C), HSV-stimulated PDC showed prominent bands for CCL4 and CXCL10 and up-regulation of CCL5 and CCL3. However, DC-depleted fractions that were unstimulated (Fig. 1 , lane F) and HSV-stimulated (Fig. 1 , lane G) showed constitutive expression of chemokine mRNA but no evidence of virally induced up-regulation of mRNA. Unstimulated PBMC show constitutive expression of CCL3, CCL4, and CCL5 (Fig. 1A , lane D). The small up-regulation of virally induced chemokine mRNA seen in the HSV-stimulated PBMC samples (Fig. 1A , lane E) is most likely a result of the presence of naturally occurring PDC in the sample. The presence of chemokine mRNA in PDC within 2 h of viral stimulation is a strong indicator that induction is a result of viral stimulation and not autocrine stimulation by a cytokine such as IFN-{alpha}, as 2 h is insufficient time for the cells to produce IFN-{alpha} in response to HSV and then undergo autocrine stimulation [54 ].



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  		Figure 1. Expression of chemokine mRNA in virally stimulated PDC. Enriched PDC (lanes A–C), PBMC (lanes D and E), and PDC-depleted PBMC (lanes F and G) were stimulated with media (lanes A, D, F) or HSV for 2 or 6 h. mRNA was isolated and analyzed for the indicated chemokines via RNAse protection assay. Virally induced chemokine expression in PDC at 2 h (lane C) and 6 h (lane B). Chemokine mRNA expression in unstimulated PBMC (lane D) and after 6 h stimulation (lane E). Chemokine expression in PDC-depleted PBMC after 6 h stimulation with HSV (lane G). (B) CCL2 expression in purified PDC after 6 h HSV stimulation.

 
Production of chemokines, IFN-{alpha}, and TNF-{alpha} by PDC as detected by intracellular flow cytometry
Although PDC were shown to produce chemokine mRNA, it was important to demonstrate chemokine proteins in these cells. We used intracellular cytokine staining to detect cytokine and chemokine production in HSV-stimulated CD123+/HLA-DR+ PDC (Fig. 2A and 2B ). The PDC were gated and then analyzed for intracellular expression of the chemokines or cytokines. This procedure precludes the need for PDC isolation by enabling one to gate only on a specific population within PBMC and effectively analyzes a pure population. The solid lines represent cytokine and chemokine staining in unstimulated PDC, and the dotted lines indicate the expression in HSV-stimulated PDC of IFN-{alpha} (C, 25% of the cells), TNF-{alpha} (D, 29%), CCL4 (E, 55%), CXCL10 (F, 27%), CCL3 (G, 19%), CCL5 (H, 7%), and CCL2 (I, 13%) in PDC stimulated with HSV.



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  		Figure 2. Cytokine and chemokine protein expression in HSV-stimulated PDC. PBMC were stimulated with HSV for 6 h and analyzed via intracellular flow cytometry for production of the indicated chemokines and cytokines. The PDC were gated as CD123 bright, HLA-DR+ cells as shown in A and B. The bold histogram represents unstimulated populations, and the dotted lines denote up-regulation of the following chemokines in response to viral stimulation by HSV: IFN-{alpha} (C), TNF-{alpha} (D), CCL4 (E), CXCL10 (F), CCL3 (G), CCL5 (H) and CCL2 (I). Data are representative of eight different experiments. SSC, Side-scatter; FSC, forward scatter; BIO 293 + STR QR, biotinylated 293 (anti-IFN-{alpha}) + streptavidin QR; FITC, fluorescein isothiocyanate.

 
IFN-{alpha} producing PDC do not produce the entire panel of chemokines
The results in Figure 2 indicate that a portion of the PDC produces cytokines and chemokines in response to stimulation with HSV. To determine the overlap between IFN-{alpha} production and the TNF-{alpha} or chemokine expression, PBMC were stimulated with HSV, and the PDC fraction was analyzed for dual expression of the proteins by intracellular flow cytometry (Fig. 3 ). The dot plots show concurrent PDC expression of (A) IFN-{alpha} and CCL4; (B) IFN-{alpha} and TNF-{alpha}; (C) IFN-{alpha} and CCL3; and (D) IFN-{alpha} and CCL5. In the first group, 92% of the IFN-{alpha} producers were also producing CCL4. Similarly, 97% of the IFN-{alpha}-producing cells concurrently produced TNF-{alpha}. However, only 58% and 30% of the IFN-{alpha} producers coexpressed CCL3 and CCL5, respectively. We were unable to carry out coexpression studies for IFN-{alpha} and CXCL10 as a result of restrictions in antibody selections. These data suggest there may be partially nonoverlapping subsets of PDC responsible for differential responses to viral stimulation.



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  		Figure 3. Coexpression of IFN-{alpha} and TNF-{alpha} and chemokines by PDC. PBMC were stimulated as described in Figure 2 , and CD123 bright, HLA-DR+ PDC were analyzed via intracellular flow for simultaneous production of IFN-{alpha} (vertical axes) and CCL4 (A), TNF-{alpha} (B), CCL3 (C), and CCL5 (D). Statistics shown indicate the percentage of PDC in each quadrant. Data are representative of three different experiments.

 
Secretion of cytokines and chemokines by PDC
Having detected chemokines in PDC intracellularly, we next measured amounts of secreted chemokine or cytokine proteins in the supernatants of stimulated cells (Fig. 4 ). PBMC and enriched PDC populations were stimulated overnight with HSV, and the supernatants were assayed for the presence of chemokines and cytokines via ELISA assays or IFN-{alpha} bioassay. Results of these studies are illustrated in Figure 4 . Compared with PBMC, enriched PDC produced larger quantities of IFN-{alpha} (A), TNF-{alpha} (B), and CXCL10 (C). PDC also produced high levels of CCL4 (D) and CCL3 (E), which do not appear enriched above PBMC production. CCL5 (F) does not appear to be produced by either population in response to HSV stimulation.



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  		Figure 4. Secretion of cytokines and chemokines by PDC. PBMC and enriched PDC populations were stimulated overnight with HSV, and the supernatants were assayed for the presence of chemokines and cytokines via ELISA assays or IFN-{alpha} bioassay. The graphs show production of IFN-{alpha} (A), TNF-{alpha} (B), CXCL10 (C), CCL4 (D), CCL3 (E), and CCL5 (F). *, Statistical significance from the unstimulated population as determined by ANOVA analysis using Scheffe’s test with a 5% confidence interval (n=4).

 
Transcription of viral genes is not necessary for chemokine induction by PDC
In an effort to elucidate the mechanisms involved in chemokine production by PDC, we used UV-inactivated HSV (UV-HSV) to stimulate PBMC. The irradiated virus is unable to be transcribed and therefore does not produce an active viral infection. We have previously shown that UV-HSV retains the ability to induce IFN-{alpha} in PDC [56 ]. To determine whether transcription of viral genes is necessary for chemokine induction, PBMC were stimulated with HSV or UV-HSV for 6 h and analyzed via intracellular flow cytometry for PDC expression of the indicated chemokines (Fig. 5 ). UV-HSV was able to induce all of the measured chemokines; however, these levels are consistently lower than that of HSV stimulation.



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  		Figure 5. Transcription of viral genes is not necessary for chemokine induction by PDC. PBMC were mock- or UV-HSV-stimulated for 6 h, and the PDC was analyzed via intracellular flow cytometry for expression of IFN-{alpha}, CXCL10, CCL4, and CCL2.

 
Endosomal acidification is necessary for chemokine induction by HSV
Previous work by our laboratory and others demonstrates that endosomal acidification is necessary for virally induced IFN-{alpha} production by PDC. Chloroquine is a weak base that accumulates in endosomes and lysosomes and leads to elevation of vacuolar pH. To determine if increases in endosomal pH inhibit chemokine production by PDC, PBMC were pretreated with 0.1 mM chloroquine for 1 h and washed before addition of HSV and expression of IFN-{alpha}, CXCL10, CCL4, and CCL2 in PDC were determined. Expression of IFN-{alpha} and all three chemokines was inhibited by the chloroquine treatment (Fig. 6 ).



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  		Figure 6. Endosomal acidification is necessary for chemokine induction by HSV. PBMC were pretreated for 1 h with 0.1 mM choloroquine (Chlor), washed, and mock- or HSV-stimulated for 6 h, and the CD123+/HLA-DR+ PDC was analyzed via intracellular flow cytometry for expression of IFN-{alpha}, CXCL10, CCL4, and CCL2. Representative of four experiments. PERCP, Peridinin chlorophyll protein.

 
CXCL10 and CCL2 are induced by IFN-{alpha} and HSV
As PDC produce large amounts of IFN-{alpha} upon viral stimulation, it was necessary to determine whether this IFN-{alpha} was responsible for inducing chemokine production by PDC. PBMC were stimulated for 6 h with 10,000 IU/mL exogenous IFN-{alpha} and were analyzed via intracellular flow to detect cytokine and chemokine production by PDC. IFN-{alpha} pretreatment alone did not induce the expression of IFN-{alpha}, TNF-{alpha}, CCL4, CCL3, or CCL5 (data not shown). However, IFN-{alpha} did induce CXCL10 (Fig. 7A ) and CCL2 (Fig. 7E) production by PDC. To clarify whether HSV or autocrine IFN-{alpha} stimulation is responsible for CXCL10 and CCL2 induction, PBMC were preincubated with control (not shown) or anti-IFN-{alpha} antibody and then were stimulated with IFN-{alpha} or HSV. The IFN-{alpha}-induced CXCL10 and CCL2 production was completely ablated in the presence of the anti-IFN-{alpha} antibody (B and F). However, HSV-stimulated CXCL10 (C) and CCL2 (G) production was reduced only 30% by treatment with anti-IFN-{alpha} antibody (D and H, respectively). These data suggest that both HSV and IFN-{alpha} act to induce CXCL10 and CCL2 production by PDC.



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  		Figure 7. HSV and IFN-{alpha} each induce CXCL10 and CCL2 expression in PDC. PBMC were stimulated for 6 h with HSV or exogenous IFN-{alpha} in the presence or absence of anti-IFN antibodies. Histograms represent percentage of CD123+/HLA-DR+ PDC expressing CXCL10 or CCL2 in response to IFN-{alpha} (A and E), HSV (B and F), IFN-{alpha} plus anti-IFN-{alpha} antibody (C and G), or HSV plus anti-IFN-{alpha} antibody (D and H). Bold lines indicate mock treatment, and the dotted lines denote experimental conditions. Data are representative of three different experiments.

 
Chemokines secreted by PDC elicit chemotaxis of NK and activated CD4+ T cells
Having demonstrated chemokine production by PDC in response to HSV, it was important to determine which cell population(s) might be acted on during PDC stimulation in vivo. Two such populations, NK cells and T cells, had been shown to respond to some of these inflammatory chemokines [43 ]. To determine the migratory capacity of these cells in response to PDC-produced factors, NK cells (Fig. 8A ) and naïve T cells (Fig. 8B) were isolated as described, and a portion of the T cells was cultured with IL-2 and PHA to induce activation. Each of these three populations was then placed in the upper chambers of Transwell inserts. The lower chambers contained media, supernatant from unstimulated PDC, or supernatant from HSV-stimulated PDC (each after a 24-h incubation). The naïve T cells showed only slight preferential migration over media in response to supernatants from virally stimulated PDC but surprisingly, migrated threefold above the media control in response to supernatants from unstimulated PDC (C). Conversely, activated T cells (D) exposed to media or unstimulated PDC supernatant showed similar low levels of chemotaxis but were induced to migrate more than threefold greater than media controls in response to virally stimulated PDC supernatant. Migration of NK cells (E) exposed to unstimulated PDC supernatant was more than double that which was observed in media. However, in response to HSV-stimulated PDC supernatant, there were roughly four times the number of cells migrating over that of media. Graphs show the mean and standard deviation, and all samples have been found to be statistically significant via ANOVA analysis using Scheffe’s test with a 5% confidence interval (n=4).



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  		Figure 8. Supernatant from PDC stimulated with HSV induces chemotaxis of NK and activated CD4+ T cells. PBMC were sorted via magnetic separation to yield populations of NK cells (A) and naïve CD4+ T cells (B). Where noted, T cells were cultured in the presence of IL-2 and PHA to induce activation. The naïve T cells (C), activated T cells (D), and NK cells (E) were assayed for chemotaxis in response to media or supernatants from PDC or HSV-stimulated PDC. All samples have been found to be statistically significant via ANOVA analysis using Scheffe’s test with a 5% confidence interval (n=4). Y-axis of bar graph = number of migrating cells.

 
CCL4 and CXCL10 are integral for PDC-induced chemotaxis of activated T and NK cells
The observed migration of NK and activated T cells in response to supernatant from PDC + HSV could be attributed to several cytokines. To elucidate the principal modulators of chemotaxis, the supernatants were incubated for 10 min with neutralizing antibody to CCL4 or CXCL10 before the start of the assay. The antibodies had no effect on the number of cells migrating in response to media or PDC supernatants (data not shown) but did inhibit chemotaxis in response to supernatant from PDC + HSV. Neutralizing antibody to CCL4 inhibited NK cell migration by 78%, and activated T cell migration by 35% (Fig. 9 ) Conversely, neutralizing antibody to CXCL10 inhibited NK cell migration by 39% and activated T cell migration by 81%. Figure 9 is representative of several experiments.



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  		Figure 9. CCL4 and CXCL10 are integral for PDC-induced chemotaxis of activated T and NK cells. PDC were isolated and mock- or HSV-stimulated for 18 h, and the supernatants were used in a chemotaxis assay with NK and activated T cells (Act. T). Supernatants were preincubated for 10 min with neutralizing antibody to CCL4 (2 µg/mL) or CXCL10 (5 µg/mL). Migration was measured after 4 h by counting the number of cells in the bottom chamber of the Transwell plate. Data are representative of two experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Highly specific, adaptive-immune responses are the end result of a complicated set of cellular interactions. Only through the combined efforts of innate and acquired immunity can clearance of an invading pathogen be achieved. These interdependent processes are mediated predominantly through chemokines, cytokines, and cell-cell interactions. Critical to the initiation of immune responses are DC. This heterogeneous population of cells, classified into type 1 (MDC) and type 2 (PDC) DC [28 ], is among the first cells to respond to inflammatory stimuli. After antigen acquisition, mature MDC and PDC express CCR7, which enables them to traffic to secondary lymphoid organs [57 ]. Once inside the lymph node, DC interact with T cells and complete the processes necessary for maturation to fully competent APC. Throughout these events, cytokines and chemokines play pivotal roles in orchestrating the events necessary to achieve immune activation.

Aside from their role as APC, PDC are potent mediators of innate responses. TLR7, present on PDC, is reported to be involved in IFN-{alpha}, TNF-{alpha}, and CXCL10 induction by imidazoquinolins and may induce maturation/survival signals as well [58 ]. PDC also express TLR9, which contributes to nonspecific bacterial recognition through binding of bacterial CpG motifs [59 ]. Contrary to other reports, our laboratory has demonstrated that PDC express low levels of mannose receptor (MR) and that it plays a major role in IFN-{alpha} induction by HSV [55 ]. The IFN-{alpha} produced by PDC during viral stimulation is hypothesized to be a key player in host antiviral responses. In addition, virally stimulated PDC express IL-6 as well as TNF-{alpha}, the latter of which activates vascular endothelium and increases its permeability to allow entry of IgG, complement, and responding cells.

Subsequent to these numerous innate responses, MDC and PDC go on to activate naïve and memory T cells as part of the adaptive-immune response [60 ]. MDC are able to induce Th1 and Th2 responses, but IL-12 production by MDC influences T cell polarization to a Th1 phenotype. Recent work further substantiates the Th1 biasing nature of MDC by examining chemokine production [61 ]. MDC stimulated with IFN-{alpha} were found to produce the powerful chemoattractant CXCL10. The receptor for CXCL10, CXCR3, is present on Th1-polarized cells and may enable them to localize to and interact more efficiently with MDC [62 ]. PDC also react to CXCR3 ligation in a CXCR4-specific manner and use other chemokine signals, such as CCR7 ligation, to position themselves in the lymph node [63 ]. Although the role of PDC as potent IFN-{alpha}-producing cells is well documented, their ability to produce chemokines has only recently been reported. New data have shown that MDC and PDC produce different subsets of chemokines, including CXCL10, in response to bacterial antigens, CpG motifs, and influenza virus [46 , 63 ]. In our current study, we have investigated virus-induced chemokine expression by PDC and begin to address the mechanisms of inflammatory chemokine production by HSV-stimulated PDC and to determine their target populations.

Data at the mRNA level demonstrate that HSV stimulation leads to vigorous up-regulation of the inflammatory chemokines CXCL10, CCL4, CCL3, CCL2, and CCL5 by PDC in response to HSV. This up-regulation was hardly seen in PBMC, suggesting novel activity by the plasmacytoid DC. Protein expression of IFN-{alpha}, TNF-{alpha}, CXCL10, and CCL4 and to a lesser extent, CCL3 and CCL5 by virally stimulated PDC was confirmed using intracellular flow cytometry and/or ELISA.

Dual-staining studies indicated that the IFN-{alpha} and TNF-{alpha} or CCL4-producing PDC populations are largely overlapping. In contrast, however, CCL3 and CCL5 are produced in low levels by the non-IFN-{alpha}-producing PDC, perhaps suggesting heterogeneity within the PDC or representing a change in the maturation/activation state. Furthermore, CXCL10 production is up-regulated 20-fold in virally stimulated, enriched PDC versus PBMC. However, virally stimulated PDC do not produce CCL5 in any appreciable amounts over the unstimulated population. This divergent response again speaks to a possible change in maturity or activation state that may comprise a heterogeneous PDC population.

Induction of chemokines in PDC could be a direct effect of virus on the PDC or consequent to activation by a secreted product such as IFN-{alpha}. To investigate the possible role of PDC-produced IFN-{alpha} on chemokine induction, exogenous IFN-{alpha} was used to stimulate PDC. We used high levels of IFN-{alpha} (10,000 IU/ml) to mimic potentially high, local levels of IFN-{alpha} that might be produced. PDC produced only two of the chemokines, CXCL10 and CCL2, in response to this IFN-{alpha} stimulation. CXCL10 production has recently been reported in MDC upon stimulation with type I IFNs [61 ]. However, CXCL10 production by PDC is not solely a result of autocrine IFN-{alpha} stimulation, as CXCL10 mRNA is detectable by 2 h in HSV-stimulated PDC. This is not enough time for the virus to induce cytokines such as IFN-{alpha}, which would then act as autocrine-stimulatory signals. Further, anti-IFN-{alpha} mAb prevented IFN-{alpha}-induced CXCL10 and CCL2 production by PDC but only partially reduced the virally stimulated production of these chemokines. Although HSV-stimulated PDC produce other type I IFNs, such as IFN-ß, the relative amounts of these cytokines produced as compared with IFN-{alpha} are significantly lower. Additionally, previous experiments have shown complete loss of PDC-mediated antiviral activity after neutralization of IFN-{alpha}, indicating that IFN-{alpha} is the key mediator of this response [21 ]. Together, these data suggest that during HSV stimulation, a viral stimulus and then subsequent IFN-{alpha} signaling contribute to production of CXCL10 and CCL2.

In studies to elucidate the mechanisms of chemokine production by PDC, we demonstrated that PDC produce chemokines in response to UV-irradiated HSV. Although UV treatment inhibits viral replication, we have previously shown that PDC still respond to UV-HSV by producing IFN-{alpha} [56 ]. Similarly, we show here that UV-HSV is a good inducer of the chemokines CXCL10, CCL4, and CCL2. Although viral replication is not necessary to induce a response in PDC, our results indicate that the endocytic pathway is crucial for IFN-{alpha} and chemokine induction. Prior studies have shown that glycoprotein D (gD) is essential for IFN-{alpha} induction by HSV, as antibody to gD inhibits IFN-{alpha} production [64 ], and gD-negative mutants do not induce IFN-{alpha} [65 ]. This is most likely a result of decreased binding of the HSV to cell-surface receptors, resulting in loss of endocytosis. We have also identified the MR as an important mediator of PDC/HSV interactions, whereby blocking the MR results in decreased IFN-{alpha} production [54 ]. In addition, other inhibitors of endocytosis prevent IFN-{alpha} induction in response to virus (S. L. Olshalsky et al., in preparation). In this paper, we have shown that raising endosomal pH with chloroquine treatment inhibits PDC production of IFN-{alpha}, CXCL10, CCL4, and CCL2, as has been previously shown for IFN-{alpha} [21 , 64 ]. These data highlight the importance of the endolysosomal pathway in response to HSV and are further evidence that an intact, endocytic pathway is necessary for PDC-mediated responses to HSV.

Virally induced CCL4 and CXCL10 are critical mediators of activated T cells and NK cells, contributing to inflammation by attracting various leukocyte subsets to sites of infection. We hypothesized that large amounts of these chemokines produced by virally stimulated PDC may provide important chemotactic signals during viral responses in vivo. In support of this hypothesis, we observed that supernatants from HSV-stimulated PDC can indeed act as chemotactic agents for NK and activated T cells, which have been shown previously to migrate in response to numerous chemokines by virtue of their CCR5 and CXCR3 expression [43 , 66 , 67 ]. We hypothesized that CXCL10 would act as a potent inducer of migration in activated T cells as a result of high expression of CXCR3. Inhibition of NK and activated T cell chemotaxis was achieved using neutralizing antibodies to CCL4 and CXCL10. Not surprisingly, 78% inhibition of NK cell chemotaxis was seen after neutralizing CCL4, and only 35% neutralization was achieved by blocking CXCL10. Conversely, blocking CXCL10 (81% inhibition) mainly inhibited migration of activated T cells, and 39% inhibition was observed after neutralization of CCL4. As a result of CCR5 expression, T cells retain the ability to migrate even in the absence of CXCL10, as the data suggest. These experiments identify CCL4 and CXCL10 as important mediators in the chemotaxis of both cell types.

In contrast, naïve T cells were not preferentially attracted by supernatant derived from HSV-induced PDC. However, supernatant from unstimulated PDC caused a fourfold increase in numbers of migrating, naïve T cells as compared with media alone. This shift in migration patterns may be indicative of a change from a homeostatic state to one of antiviral activity. Normal lymphocyte trafficking to secondary lymphoid organs may be strongly influenced by resident PDC and changes during viral stimulation to induce chemotaxis of effector cells to sites of infection.

Once attracted by virally stimulated PDC, the NK and activated T cells can be functionally up-regulated by the cytokines and chemokines produced by PDC. We and others have previously shown that human peripheral blood NK cells require the participation of an accessory cell that is phenotypically identical to the PDC to be activated to kill HSV-infected fibroblast targets [49 , 68 , 69 ]. This accessory activity is partially a result of IFN-{alpha} secretion, leading to activation of the NK cells but also appears to be dependent on cell contact. Moreover, IFN-{alpha} is known to induce Th1 responses in humans [70 ]. Under these circumstances, PDC can respond to HSV with production of IFN-{alpha} and other cytokines/chemokines and augment some of these responses through autocrine IFN-{alpha} stimulation. We conclude that HSV stimulation of PDC leads to production of IFN-{alpha} as well as inflammatory chemokines that result in recruitment and subsequent activation of NK cells and activated T cells, providing effective antiviral immunity.


   ACKNOWLEDGEMENTS
 
We thank Dana Stein and Zenny Garcia of the UMDNJ-NJMS (Newark) Flow Cytometry Facility as well as Dr. Gunnar Alm of the Swedish University of Agricultural Sciences (Uppsala, Sweden) for providing the 293 anti-IFN-{alpha} antibody.

Received June 25, 2003; revised October 27, 2003; accepted October 29, 2003.


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 DISCUSSION
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