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Clinical and Diagnostic Laboratory Immunology, March 2005, p. 426-435, Vol. 12, No. 3
1071-412X/05/$08.00+0     doi:10.1128/CDLI.12.3.426-435.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Characterization of Virus-Responsive Plasmacytoid Dendritic Cells in the Rhesus Macaque

Eugene Chung,^1,2 Sheela B. Amrute,¹ Kristina Abel,^3,4 Gunjan Gupta,^1,2 Yichuan Wang,^3,4 Christopher J. Miller,^3,4,5,6 and Patricia Fitzgerald-Bocarsly^1,2*

Department of Pathology and Laboratory Medicine,¹ Graduate School for Biomedical Sciences, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey,² Center for Comparative Medicine,³ California National Primate Research Center,⁴ Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine,⁵ Department of Pathology and Laboratory Medicine, University of California at Davis, Davis, California⁶

Received 4 October 2004/ Returned for modification 23 November 2004/ Accepted 18 December 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Plasmacytoid dendritic cells (PDC) are potent producers of alpha interferon (IFN-) in response to enveloped viruses and provide a critical link between the innate and adaptive immune responses. Although the loss of peripheral blood PDC function and numbers has been linked to human immunodeficiency virus (HIV) progression in humans, a suitable animal model is needed to study the effects of immunodeficiency virus infection on PDC function. The rhesus macaque SIV model closely mimics human HIV infection, and recent studies have identified macaque PDC, potentially making the macaque a good model to study PDC regulation. In this study, we demonstrate that peripheral blood PDC from healthy macaques are both phenotypically and functionally similar to human PDC and that reagents used for human studies can be used to study macaque PDC. Both human and macaque PBMC expressed IFN- in response to herpes simplex virus (HSV), the prototypical activator of PDC, as measured by using an IFN bioassay and IFN--specific enzyme-linked immunospot assays. Similar to human PDC, macaque PDC were identified by using flow cytometry as CD123⁺ HLA-DR⁺ lineage^– cells. In addition, like human PDC, macaque PDC expressed intracellular IFN-, tumor necrosis factor alpha, macrophage inflammatory protein 1ß/CCL4, and IFN-inducible protein 10/CXCL10 upon stimulation with HSV, all as determined by intracellular flow cytometry. We found that IFN regulatory factor 7, which is required for the expression of IFN- genes, was, similar to human PDC, expressed at high levels in macaque PDC compared to monocytes and CD8⁺ T cells. These findings establish the phenotypic and functional similarity of human and macaque PDC and confirm the utility of tools developed for studying human PDC in this animal model.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dendritic cells (DC) are ubiquitous cells found in blood, lymphoid, and many other nonlymphoid tissues. These heterogeneous cells share the ability to take up (11, 23, 36, 38, 46) and process and present (31) exogenous antigens to CD4⁺ T cells (3, 5, 21, 44). Two distinct populations of DC have been identified in humans on the basis of their surface antigens: the myeloid DC (MDC), which are lineage^–, CD11c⁺, CD123^dim, and HLA-DR⁺ (32, 39), are phenotypically and functionally similar to monocyte-derived dendritic cells, which can be derived in vitro by culturing peripheral blood monocytes with granulocyte-macrophage colony-stimulating factor and interleukin-4 (37). These MDC produce little or no alpha interferon (IFN-) in response to herpes simplex virus (HSV) (41). The second peripheral blood subset of DC, the plasmacytoid DC (PDC), are lineage^–, CD11c^–, CD123^bright, and HLA-DR⁺ (32). Human PDC also express blood DC antigen 2 (BDCA-2) and BDCA-4, which are an endocytic C-type lectin receptor and neuropilin-1, respectively (10). PDC produce vast amounts of IFN- (3 to 10 pg/cell or 1 to 2 IU/cell) in response to enveloped viruses such as HSV and Sendai virus (SV), in addition to some bacteria and DNA-containing unmethylated CpG sequences (4, 7, 15, 16, 25, 41). In addition, PDC have been shown to produce inflammatory chemokines such as macrophage inflammatory protein 1 (MIP-1) and MIP-1ß, IFN-inducible protein 10 (IP-10) and MCP-1 in response to CpG, inactivated influenza virus, CD40L stimulation, and HSV stimulation (28, 34), and HSV-stimulated PDC express chemokines that attract both natural killer (NK) cells and activated T cells (28). Depending on the nature of the stimulus they receive, PDC can direct either Th1 or Th2 responses (6, 26).

Our lab and others have shown that the functional and numerical loss of PDC in peripheral blood is associated with disease progression and enhanced virus replication in human immunodeficiency virus type 1 (HIV-1) (12, 13, 33, 42, 43)-, dengue virus (35)-, and HCV (1)-infected patients. The PDC therefore play a critical role in the link between innate and adaptive immunity, and understanding PDC function and their role in antiviral immunity is important for both vaccine design and therapeutic interventions.

Although murine models have been established for PDC, murine PDC are not phenotypically identical to human PDC, making direct correlations from mouse to human difficult (2, 19, 30). In addition, it has been challenging to study the progression of certain diseases, such as HIV infection, in humans. The difficulty in controlling for duration of infection, coinfection with other agents, and drug treatment and the difficulty in obtaining tissue samples from human patients all demonstrate the need for a model to study HIV infection. Because the immune system of the rhesus macaque closely resembles the human, the macaque model provides a unique system for studying PDC. Particularly because, as seen with progressive HIV infection in humans, the macaque shows an absolute decrease in CD4⁺ T cells in SIV infection (17), the macaque provides an important animal model to study immunodeficiency virus pathogenesis.

Coates et al. have recently demonstrated that the PDC of fms-like tyrosine kinase 3-ligand (Flt3L)-treated rhesus macaques produce IFN- in response to HSV, as demonstrated by enzyme-linked immunosorbent assay (8). The goal of the present study was to characterize PDC in the peripheral blood of healthy, untreated rhesus macaques. Our aim was to not only to establish whether rhesus PDC are phenotypically similar to human PDC but, more importantly, also to establish whether rhesus PDC are functionally similar to human PDC. We demonstrate here that macaque PDC, similar to human PDC, respond to live virus stimulation with IFN- production. We show that many of the human reagents and techniques that we use to study human PDC in mixed preparations through intracellular flow cytometry, IFN bioassay, and enzyme-linked immunospot (ELISPOT) assay can also be applied to rhesus macaques. In addition, we demonstrate that macaque PDC, like their human counterparts (9, 22, 45), constitutively express high levels if IFN regulatory factor 7 (IRF-7) compared to monocytes and CD8⁺ T cells. Finally, we demonstrate that, similar to humans (28), macaque PDC produce tumor necrosis factor alpha (TNF-), IP-10/CXCL10, and MIP-1ß/CCL4 in response to viral stimulation.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals. Rhesus macaques (Macaca mulatta) were housed at the California National Primate Research Center in accordance with the regulations of the American Association for Accreditation of Laboratory Animal Care standards. All animals were negative for antibodies to HIV-2, simian immunodeficiency virus (SIV), type D retrovirus, and simian T-cell lymphotropic virus type 1.

Viruses. HSV type 1 (HSV-1) strain 2931 and vesicular stomatitis virus (originally obtained from Nicholas Ponzio, New Jersey Medical School) were grown, and titers were determined by plaque-forming assay in Vero cells (American Type Culture Collection, Manassas, Va.) as previously described (14). SV (Sendai/Cantell strain) was obtained from the Charles River SPAFAS, Inc. All virus stocks were stored at –70°C until use.

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

Preparation of PBMC. Human blood was obtained with informed consent from healthy human donors. Rhesus blood was drawn into heparinized tubes and either tested fresh at the University of California at Davis or shipped at room temperature overnight from the California National Primate Research Center to New Jersey. Human and macaque peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density centrifugation (Lymphoprep; Accurate Chemical and Scientific Co., Westbury, N.Y.). PBMC were washed twice with Hanks balanced salt solution (Life Technologies, Grand Island, N.Y.) and resuspended in RPMI 1640 (Life Technologies) containing 10% FCS, 2 mM L-glutamine, 100 U of penicillin/ml, 100 µg of streptomycin/ml, and 25 mM HEPES and then enumerated electronically with a Series Z1 Coulter Counter (Coulter Electronics, Inc., Hialeah, Fla.).

For some experiments, freshly isolated macaque PDC were immediately frozen in 10% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, Mo.)-90% fetal bovine serum, stored in liquid nitrogen, shipped from California to New Jersey on dry ice, and then stored again in liquid nitrogen until thawing.

Flow cytometry. For surface staining, cells were washed with cold 0.1% bovine serum albumin (BSA; Sigma-Aldrich) in phosphate-buffered saline (PBS) (Life Technologies), blocked with 5% heat-inactivated human serum, stained with fluorochrome-conjugated antibody for 20 min at 4°C, washed, and fixed with 300 µl of 1% paraformaldehyde in PBS (Fisher, Pittsburgh, Pa.) at 4°C overnight. The antibodies used for surface staining were as follows: CD8 (clone SK1), CD14 (clone7G3), CD123 (clone MP9), and HLA-DR (clone L243) (BD Biosciences, San Diego, Calif.).

Intracellular detection of IFN-, TNF-, IRF-7, and chemokines. PBMC were prepared for intracellular detection of IFN-, IRF-7, CXCL10/IP-10, and CCL4/MIP-1ß by using a modification of the method described previously (29). PBMC (2 x 10⁶ cells/ml)were either mock stimulated (placed in the incubator without any virus added) or stimulated with HSV-1 strain 2931 at a multiplicity of infection of 1 for 4 h at 37°C in 5% CO₂. Brefeldin A (5 µg/ml) (Sigma-Aldrich) was then added, and incubation was continued for an additional 2 h. Cells were surfaced stained, as described above, and fixed with 1% paraformaldehyde in PBS at 4°C overnight. The following day, cells were washed twice with PBS-2% FCS, permeabilized with 0.5% saponin (Sigma-Aldrich) in PBS-2% FCS for 30 min at room temperature, and then incubated with 50 ng of biotinylated 293 monoclonal antibody (MAb) to IFN- (obtained from G. V. Alm, Uppsala, Sweden) or a commercially available antibody to IFN- (clone MMHA-2, PBL Biomedical Laboratories, Piscataway, N.J.). Biotinylation was carried out by using the succinamide ester method. For intracellular staining of IRF-7, chemokines, and TNF-, polyclonal rabbit antibody to IRF-7 (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), anti-CCL4 (R&D Systems, Minneapolis, Minn.), or biotinylated anti-CXCL10 (U.S. Biologicals, Swampscott, Mass.), or anti-TNF- (BD Pharmingen) were incubated with the PBMC for 30 min at room temperature. Cells were subsequently washed twice with 0.5% saponin in PBS-2% FCS and incubated 30 min at room temperature with streptavidin-Quantum Red (Sigma-Aldrich) or fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G (IgG; BD Biosciences). Finally, the cells were washed and resuspended in 1% paraformaldehyde in PBS and analyzed by using a FACSCalibur flow cytometer with CellQuest analysis software (BD Biosciences).

ELISPOT assays. ELISPOT assays for detection of IFN--producing cells were carried out as previously described (13). Briefly, PBMC (10⁶/ml) were either mock stimulated (placed in the incubator with no virus) or stimulated for 6 h with HSV-1 at an multiplicity of infection of 1. We coated 96-well Multiscreen plates (Millipore) with AS94 (Glaxo SmithKline, Uxbridge, United Kingdom), a bovine polyclonal antibody to IFN-, for 5 h. Cell suspensions were added, and this was followed by incubation for 11 h at 37°C; the primary antibody to IFN-, MAb 293, was then added. After a 2-h incubation at room temperature, secondary antibody, horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson Immunoresearch Laboratories), was added for 1 h. Finally, diaminobenzidene with H₂O₂ was added for 5 min. After washing and drying steps, the spots were counted under a dissecting microscope to determine the number of IFN-producing cells (IPC).

IFN bioassays. IFN bioassays were performed by using a cytopathic effect reduction assay with GM cells infected with vesicular stomatitis virus as the challenging virus as previously described (14). An IFN- reference standard (G-023-901-527; National Institute of Allergy and Infectious Disease, Bethesda, Md.) was used at 100 IU/ml.

Purification of PDC from macaque blood. PBMC were depleted of T cells, B cells, NK cells, and monocytes by using a modified version of the magnetic blood dendritic cell isolation kit (Miltenyi Biotech, Inc., Auburn, Calif.). Briefly, PBMC were washed and resuspended in MACS buffer (PBS [Life Technologies] with 0.5% BSA and 2 mM EDTA [Sigma-Aldrich]) and then incubated at 4°C with anti-CD3, anti-CD16 beads, anti-CD14 beads, anti-IgG1 beads, and anti-CD20 beads. PDC were negatively selected for by using the MACS magnetic separation column (LD columns).

PDC were subsequently positively selected for by incubating the negatively selected cells at 4°C for 20 min with anti-CD123 PE. Resuspended cells were then incubated with anti-phycoerythrin beads (Miltenyi Biotech) for 15 min, and PDC were positively selected by using a MACS magnetic separation column (LS and MS columns).

Giemsa staining. PDC were enriched by using the magnetic bead separation method described above, cytopsin centrifuged onto slides, and allowed to air dry overnight. PDC were stained with Giemsa stain, mounted with Permount (Fisher), and observed under a microscope.

Identification of IFN--producing PDC by fluorescence microscopy. PDC were enriched by using the negative selection described above. Enriched PDC were either mock or HSV stimulated for 6 h, after which they were positively selected, as described above. The resulting cells were then subjected to cytospin centrifugation and allowed to air dry on slides overnight. Purified PDC were fixed with 1% paraformaldehyde in PBS for 15 min and then permeabilized with 0.2% Triton X-100 in PBS for 5 min. Slides were subsequently washed twice with PBS and blocked with 3% BSA in PBS and 10% normal goat serum for 30 min. Purified PDC were incubated for 30 min with mouse MAb to human IFN- (clone MMHA-2; PBL) that was labeled with Alexa Fluor-680 by using the Zenon labeling system (Molecular Probes, Eugene, Oreg.). Cells were washed twice with 0.2% Triton X-100 in PBS and washed twice with PBS. Purified PDC were mounted on slides with mounting medium (Vector) and then observed under a fluorescence microscope.

Statistical analysis. Data are expressed as mean values plus standard deviations. Statistical significance was determined by one-way analysis of variance with Scheffe's test. Differences were considered to be significant at P values of <0.05.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Phenotypic characterization of macaque PDC. Human PDC constitute <1% of PBMC, making them difficult to isolate and study in large numbers. However, human PDC can be identified by using flow cytometry, allowing us to gate in on the small population of cells for both phenotypic and functional analysis (13). Human PDC have been characterized as cells that are lineage marker negative, HLA-DR⁺, CD123^bright, and CD11c^– (Fig. 1A). Moreover, we have shown that the cells identified as HLA-DR⁺ and CD123^bright by four-color fluorescence-activated cell sorting (FACS) analysis are identical to the HLA-DR⁺ and CD123^bright population by using a simplified two-color analysis (Fig. 1A) (9). In addition, BDCA-2 and BDCA-4 have been used to identify human PDC in PBMC populations (8).

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FIG. 1. Two-color flow cytometry gating identifies macaque PDC that phenotypically resemble human PDC. Human (A) and macaque (B) PDC were initially defined by using four-color flow cytometry. PBMC were first selected based on forward and side scatter (R1, left panels) and then further defined as HLA-DR+ lineage– cells (R2, second panels) and CD123+ CD11c– cells (R3, third panels). The same cells from the four-color flow cytometry, represented by the blue dots, are also identified by two colors as CD123+ and HLA-DR+ cells (R4, right panels). The data are representative of typical flow cytometric analysis.

Using the same four- and two-color FACS analyses used to identify human PDC, we identified a population of cells in rhesus blood that is HLA-DR⁺ CD123^bright and thus phenotypically resemble human PDC (Fig. 1B). Interestingly, there was more variability in the mean fluorescence intensity (MFI) of expression of HLA-DR in macaque PDC than in the human PDC. However, antibodies to human PDC-specific surface markers BDCA-2 and -4 did not cross-react with macaque PDC (data not shown). Gating on the HLA-DR⁺ CD123^bright cells, there were, on average, 1,260 ± 411 PDC/3 x 10⁵ PBMC for humans (0.4%, n = 11 donors) and 264 ± 189 PDC/3 x 10⁵ PBMC for macaques (0.1%, n = 28 donors) (P < 0.05). Phenotypic analysis comparing PDC from matched donors in freshly isolated PBMC, in PBMC isolated in shipped peripheral blood, and in frozen PBMC yielded similar results (data not shown).

HSV-induced IFN- production by rhesus PBMC. In humans, the total IFN response can be tested by measuring IFN- release (as determined by IFN bioassay) after in vitro stimulation of PBMC with HSV (12). To determine whether this assay can also be used to assess IFN- production in rhesus PBMC, these cells were stimulated in vitro with HSV for 18 h, and the IFN- in supernatants was measured. Positive control cultures consisted of supernatants from human PBMC stimulated with HSV.

In both human and macaque cultures, unstimulated PBMC produced less IFN than the lower limits of detection of the assay (Fig. 2). In response to HSV stimulation, human PBMC produced a geometric mean of 2,220 IU of IFN/10⁶ cells (one standard deviation; range, 851 to 5,791), whereas macaque PBMC produced a geometric mean of 1,723 IU of IFN/10⁶ cells (one standard deviation; range, 867 to 3,424) (P = 0.66 [not significant]). In addition, in response to SV, which stimulates both monocytes and human PDC to produce IFN- (13), human PBMC produced a geometric mean of 6,049 IU of IFN/10⁶ cells (one standard deviation; range, 2,147 to 17,044), whereas macaque PBMC produced a geometric mean of 7,965 IU of IFN/10⁶ cells (one standard deviation; range, 3,250 to 19,518) (P = 0.62, NS). Thus, rhesus PBMC responded to HSV stimulation with a magnitude of IFN- secretion similar to that of human PBMC.

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FIG. 2. Bioactive IFN produced by macaque and human IPC. Bioassays were performed to measure the IFN produced by PBMC incubated for 18 h with medium, HSV, or SV. For both macaques and humans, the IPC produced less than the lower limits of detection (LD) in response to mock (medium) stimulation. Mean values for six human samples and eight macaque samples are shown. The error bars represent one standard deviation from the means. There were no significant differences in IFN production between humans and macaques.

To further determine whether the macaque model closely resembles the established human model, the frequency of HSV-responsive IPC was determined by using an IFN--specific ELISPOT assay. HSV and SV stimulation of human PBMC yielded average frequencies of 7.3 ± 2.7 and 45.1 ± 46.8 IPC/10⁴ PBMC, respectively, whereas averages of 2.1 ± 1.8 and 15.8 ± 9.4 IPC/10⁴ PBMC, respectively, were detected in rhesus samples after HSV and SV stimulation (Fig. 3). Both the HSV-induced (P = 0.0002) and SV-induced (P = 0.0473) ELISPOT frequencies were significantly lower in macaques than in humans. Moreover, in general, the sizes of the "spots" in the ELISPOT assays, as determined by visual observation, were smaller in macaque samples than in human samples.

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FIG. 3. Frequency of IFN--producing cells in macaque and human PBMC. Macaque and human PBMC were either mock stimulated (A and B), HSV stimulated (A), or SV stimulated (B) for 6 h. The frequency of IFN--producing cells was determined by ELISPOT assay. The mean values for 6 human samples and 12 macaque samples are shown. The error bars represent one standard error of the mean. The number of IPC detected in macaque PBMC in response to HSV and SV was significantly lower than in humans (P < 0.05).

Identification of PDC as the major IFN--producing cells after in vitro stimulation of rhesus PBMC with HSV. To determine whether rhesus PDC, like their human counterparts, are indeed the main IFN--producing cell type, PBMC from both macaques and humans were stimulated with HSV-1 for 6 h and then stained and analyzed by FACS for intracellular IFN-. As in humans, the majority of the cells staining positive for IFN- were CD123⁺ cells (Fig. 4). Using the same gating strategy as described above for enumeration of PDC, the percentages of PDC producing IFN- in response to 6 h of stimulation with HSV were determined for humans and macaques (representative results are shown in Fig. 5A and B, respectively). As we previously reported for human PDC (12), not all of the macaque PDC produced IFN- simultaneously upon viral stimulation. The percentages of PDC producing IFN- varied from subject to subject (Fig. 5C), ranging from 10 to 72% in human PDC and from 14 to 82% in macaque PDC. Although we only directly compared a limited number of fresh versus shipped blood samples for expression of IFN- after HSV stimulation, there was no statistical difference between these data (Fig. 5D). In contrast, although PDC in cryopreserved PBMC samples were similar in terms of phenotype and frequency to fresh PDC, there was variability in their ability to produce IFN-. Similar variability was obtained with cryopreserved human PBMC samples, indicating that our method of freezing yielded samples with inconsistent functional ability (data not shown).

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FIG. 4. PDC are the predominant IPC in HSV-stimulated macaque PBMC. PBMC were either mock (A) or HSV (B) stimulated and gated based on forward scatter and intracellular expression of IFN-. (C) Of the cells that are IFN-+, 90% are HLA-DR+ and CD123+. The data are shown for one representative experiment of two.

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FIG. 5. Intracellular flow cytometric detection of IFN- production by HSV-stimulated macaque and human PDC. (A) Human PBMC (left panel) were gated as CD123+ HLA-DR+ cells and then further gated for IFN- production to determine the percentage of PDC that produce IFN- in response to mock and HSV stimulation. The numbers in the upper right quadrants are the percentages of IFN-+ PDC. (B) Macaque PBMC were gated as described for human PDC in panel A to determine the percentage of PDC producing IFN- in response to mock and HSV stimulation. The data shown are representative experiments of 11 human samples and 28 macaque samples. (C) The percentage of PDC producing IFN- ranged from 12 to 80% in humans and 36 to 82% in macaques. (D) There was no significant difference in the percentages of PDC producing IFN- between freshly drawn macaque blood and overnight-shipped blood, where PBMC derived from fresh and shipped blood are shown with the same symbol for an individual animal. The boxes in panels C and D represent the upper and lower quartiles, with the medians shown inside the boxes. The lines extend out to either the upper quartiles plus 1.5 times the interquartile range or the lower quartiles minus 1.5 times the interquartile range.

Morphology of macaque PDC. PDC were enriched from PBMC by negative selection and subsequently Giemsa stained (Fig. 6A), revealing enrichment for large cells with lateralized reniform nuclei, a typical PDC morphology. For fluorescence microscopy, negatively enriched PDC were further purified by positive selection, stimulated with HSV for 6 h, and stained with anti-IFN-. Virtually no IFN- positive cells were seen in the mock-stimulated purified PDC (Fig. 6B), whereas the HSV-stimulated, purified PDC showed a bright fluorescence pattern in the cytoplasm of the cells (Fig. 6C).

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FIG. 6. Microscopic analysis of macaque PDC. (A) Macaque PDC were enriched from PBMC by negative selection and then Giemsa stained and analyzed for morphology, revealing cells with characteristic PDC morphology. Positively selected PDC were mock (B) or HSV (C) stimulated for 6 h and then stained with Alexa Fluor 680-conjugated anti-IFN- antibody.

IRF-7 expression in rhesus PDC. IRFs play an important role in the induction of IFN- and IFN-ß gene expression, with IRF-7 being specifically required for stimulation of the IFN- genes (27, 40, 47). We (9, 22) and others (45) have previously reported that IRF-7 is expressed at high constitutive levels in human PDC and at much lower levels in monocytes and T cells, thus making the PDC uniquely poised to rapidly produce high levels of IFN- in response to virus stimulation. Similar to human PDC, constitutive high levels of IRF-7 expression were observed in macaque PDC, with lower levels being observed in monocytes and CD8⁺ T cells (Fig. 7). Macaque PDC, however, had lower MFIs associated with IRF-7 than did human PDC (i.e., MFI = 127.9 [one standard deviation range from 85.6 to 191.0] versus MFI = 376.7 [one standard deviation range from 181.0 to 786.2], respectively; P = 0.0003) (9, 22).

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FIG. 7. Expression of IRF-7 in human and macaque PDC, monocytes, and CD8+ T cells. PBMC from humans (upper panels) and macaques (lower panels) were surface labeled for identification of PDC, monocytes, and CD8+ T cells. Cells were then permeabilized and stained with either control antiserum (dim lines) or anti-IRF-7 (bold lines), followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG. PDC (CD123+ HLA-DR+), monocytes (CD14+), and CD8+ T cells were gated, and intracellular expression of IRF-7 in the selected populations was determined as for the PDC. The data are representative of two experiments with similar results.

Macaque PDC produce CXCL10/IP-10, CCL4/MIP-1ß, and TNF- in response to HSV stimulation. IP-10 (CXCL10) and MIP-1ß (CCL4) are inflammatory chemokines that chemoattract Th1-polarized T cells and NK cells, respectively. We have previous demonstrated that human PDC produce CXCL10 in response to either IFN- or HSV, whereas HSV but not IFN- induces the expression of CCL4 (28). In the macaque model, utilizing intracellular flow cytometry, we detected significant IP-10/CXCL10 production in HSV-stimulated PDC from 7 of 11 monkeys tested (Fig. 8A and C). We also observed HSV-induced upregulation of MIP-1ß/CCL4 expression in macaque PDC (Fig. 8B and D), a finding similar to what we reported earlier for humans (28). Finally, similar to human PDC, macaque PDC expressed intracellular TNF- in response to HSV stimulation (Fig. 9).

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FIG. 8. CXCL10 and CCL4 production in macaque PDC. Macaque PBMC were either mock or HSV stimulated for 6 h. PDC were gated as CD123+ HLA-DR+ cells as described above. The intracellular expression of CXCL10 (A) and CCL4 (B) was measured by flow cytometry. CXCL10 and CCL4 production is compared between mock stimulation (dim lines) and HSV stimulation for 6 h (bold lines). Panels A and B are representative experiments of seven samples that showed an increase in CXCL10 in response to HSV and four samples for CCL4. The differences in MFI for CXCL10 (C) and CCL4 (D) are shown. Boxes are as described for Fig. 5. There was a significant difference in expression of both CXCL10 and CCL4 between mock and HSV stimulation.

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FIG. 9. TNF- expression in macaque PDC. Macaque PBMC were either mock or HSV stimulated for 6 h and then surface stained for identification of PDC, followed by permeabilization and staining for TNF- expression. The percentages of PDC that were positive for TNF- are shown for mock (A)- and HSV (B)-stimulated PBMC for one representative monkey out of nine. (C) The percentage of PDC positive for TNF- ranged from 0 to 4% in mock-stimulated PBMC and 2 to 27% in HSV-stimulated PBMC (P < 0.05).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although it is recognized that PDC play a critical role in the link between innate and adaptive immunity and that their numerical and functional dysfunction contributes to HIV pathogenesis (12, 42), study of the fate of PDC in HIV infection has been hampered by the difficulty of monitoring the PDC throughout the body. Likewise, it is difficult to study PDC in human hosts at the earliest periods after infection with HIV. Thus, an animal that allows study of PDC function in the context of immunodeficiency virus infection is very much needed. The present study was undertaken to determine the extent to which macaque PDC are similar to their human counterparts.

We used reagents that we routinely use in the study of human PDC to further describe the rhesus macaque PDC. Others have reported that macaque PDC, like their human counterparts, can be identified by using four markers: lineage, HLA-DR, CD123, and CD11c (8, 48). We demonstrate here that the macaque PDC, like their human counterparts, can be identified by using our two-color scheme (9), which utilizes CD123 and HLA-DR only. Using a four-color flow cytometer, defining the PDC by two colors, opens up two additional channels for additional studies, such as intracellular analysis of IFN-, chemokines, or IRF-7. Two existing antibodies frequently used to identify and/or isolate human PDC, namely, BDCA-2 and BDCA-4, however, failed to react with the macaque PDC PDC phenotype, and frequencies were found to be similar within macaque PBMC obtained from freshly isolated blood, PBMC separated from heparanized blood that had been shipped overnight, and in cryopreserved, thawed PBMC. The macaque, however, had a significantly lower percentage of PDC in the peripheral blood than human donors. In addition, we observed more variability in the expression of HLA-DR by the macaque than the human PDC, but this did not interfere with our ability to identify the PDC. By using Giemsa stain, isolated PDC were indistinguishable from human PDC.

In addition to their phenotypic similarity to human PDC, the macaque PDC within the PBMC vigorously produced IFN- in response to stimulation with HSV, as measured both by total IFN- activity in an IFN bioassay and by ELISPOT analysis with human IFN- specific reagents. Although the levels of IFN in supernatants of HSV and SV-stimulated samples were statistically indistinguishable, the ELISPOT frequencies of the IPC were lower in macaques than in humans. The lower frequency of HSV-responsive IPC, as measured by ELISPOT, is consistent with the observation that the monkeys had a lower percentage of PDC among PBMC than humans. The ability of the gated PDC to produce IFN-, as measured as the percent PDC positive for intracellular IFN-, was statistically equivalent between monkeys and humans, indicating that, as we previously demonstrated in humans (12, 28), not all PDC respond to HSV with IFN production, a finding that has also been seen with human PDC stimulated with the TLR7 agonist, imiquimod (18). The markedly lower frequency of SV-responsive IPC in monkeys compared to humans may reflect limitations to the ELISPOT assay. SV is known to induce both PDC and monocytes to produce IFN-, with the monocytes expressing 5- to 10-fold lower expression of IFN- on a per-cell basis than the PDC (13, 20). In the ELISPOT, this is seen by a mixture of small (monocyte-derived) and large (PDC-derived) spots. The number of smaller, monocyte-derived IFN- spots was noticeably lower in the macaque than in the human, perhaps reflecting spots that were too dim to detect, thus limiting the usefulness of the ELISPOT assay for detecting SV-induced IPC.

Also similar to the human PDC, macaque PDC produced both CXCL10/IP-10 and CCL4/MIP-1ß, as well as TNF-, in response to HSV. Thus, as in humans, the macaque PDC are uniquely poised to interact with other cell types such as NK cells and T cells (28) and to link innate and adaptive immune responses (24). Overall, the similarity of macaque PDC to human PDC in response to HSV demonstrates the usefulness of the macaque model for the study of PDC.

Coates et al. studied PDC in Flt3L-treated macaques (8). Although growth factors such as Flt3L may be useful in therapeutics, we have demonstrated that fresh, untreated PDC can be functionally studied in the macaque model. In addition, we were able, by using the two-color scheme to identify PDC, to demonstrate that macaque PDC, like their human counterparts, produce IFN-, IP-10, MIP-1ß, and TNF- in response to viral stimulation. The similarity in cytokine production of macaque to human PDC further establishes the macaque model as a good system for studying PDC.

In addition to the phenotypic and functional similarities between the macaque and human PDC, the macaque PDC, again similar to human PDC (9, 22), were found to express high levels of the transcription factor IRF-7 compared to other peripheral blood cell types. In humans, we have demonstrated that this IRF-7 can be rapidly translocated to the nucleus of PDC after stimulation with HSV. We postulate that this high constitutive IRF-7 is what makes PDC such exquisite "professional IFN-producing cells" (40).

In conclusion, the macaque PDC model provides a valuable system to study these important cells in a nonhuman primate setting. Furthermore, the similarity between SIV and HIV pathogenesis in rhesus macaques and humans, respectively, provides a useful model in the macaque for studying HIV pathogenesis. Studies are currently under way to evaluate the PDC system in the context of acute and chronic immunodeficiency virus infection. The demonstration of the macaque as a good model for PDC study will hopefully permit the elucidation of the role of PDC in viral pathogenesis, as well as in other human diseases.

 
This study was supported by grant 106449-34-RGIM from amfAR (P.F.-B.), Public Health Service Grants AI 26806 (P.F.-B.), AI44480 (C.J.M.), RR14555 (C.J.M.), RR01 69 (C.J.M.), AI055793 (C.J.M.), AI57264 (C.J.M.), and by the UMDNJ Graduate School for Biomedical Sciences (E.C. and G.G.).

 
* Corresponding author. Mailing address: UMDNJ-NJMS, Department of Pathology and Laboratory Medicine, 185 S. Orange Ave., Newark, NJ 07101. Phone: (973) 972-5233. Fax: (973) 972-3503. E-mail: Bocarsly{at}umdnj.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Clinical and Diagnostic Laboratory Immunology, March 2005, p. 426-435, Vol. 12, No. 3
1071-412X/05/$08.00+0     doi:10.1128/CDLI.12.3.426-435.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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