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The Journal of Immunology, 2008, 181, 4010 -4018
Copyright © 2008 by The American Association of Immunologists, Inc.
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A Human Dendritic Cell Subset Receptive to the Venezuelan Equine Encephalitis Virus-Derived Replicon Particle Constitutively Expresses IL-321

Kevin P. Nishimoto*,{ddagger}, Amanda K. Laust*,{ddagger} and Edward L. Nelson2,*,{dagger},{ddagger}

* Molecular Biology and Biochemistry, School of Biological Sciences, {dagger} Department of Medicine, Division of Hematology/Oncology, School of Medicine, and {ddagger} Center for Immunology, University of California at Irvine, Irvine, CA 92697


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DCs) are a diverse population with the capacity to respond to a variety of pathogens. Because of their critical role in pathogenesis and Ag-specific adaptive immune responses, DCs are the focus of extensive study and incorporation into a variety of immunotherapeutic strategies. The diversity of DC subsets imposes a substantial challenge to the successful development of DC-based therapies, requiring identification of the involved subset(s) and the potential roles each contributes to the immunologic responses. The recently developed and promising Venezuelan equine encephalitis replicon particle (VRP) vector system has conserved tropism for a subset of myeloid DCs. This immunotherapeutic vector permits in situ targeting of DCs; however, it targets a restricted subset of DCs, which are heretofore uncharacterized. Using a novel technique, we isolated VRP-receptive and -nonreceptive populations from human monocyte-derived DCs. Comparative gene expression analysis revealed significant differential gene expression, supporting the existence of two distinct DC populations. Further analysis identified constitutive expression of the proinflammatory cytokine IL-32 as a distinguishing characteristic of VRP-receptive DCs. IL-32 transcript was exclusively expressed (>50 fold) in the VRP-receptive DC population relative to the background level of expression in the nonreceptive population. The presence of IL-32 transcript was accompanied by protein expression. These data are the first to identify a subset of immature monocyte-derived DCs constitutively expressing IL-32 and they provide insights into both DC biology and potential mechanisms employed by this potent vector system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DCs),3 the most potent APCs, are a critical component of the immune system, based on their unique immunostimulatory and immunoregulatory roles. DCs were initially characterized as a population of cells that had predetermined properties to initiate and activate T cell responses, particularly naive Ag responses. However, it is becoming more apparent that DCs possess a high degree of functional plasticity with the capacity to elicit different classes of immune responses (1, 2). This plasticity may be due, in part, to the heterogeneous populations of DCs, which can be characterized in various ways, including by cell surface molecule profile, differential expression of pattern recognition receptors and developmental lineage, for example, from myeloid or lymphoid precursors (2, 3). This phenotypic and functional diversity of DCs, in addition to their fundamental potency as Ag-presenting and immune regulatory cells, has led to their increased study and application in strategies to modulate the immune system for the control of various diseases. Development of effective DC-based immunotherapy will be critically dependent on understanding the characteristics of the involved DC subset(s).

The uptake and/or expression of specific Ag(s) by DCs is a requirement for Ag processing, presentation, and, ultimately, elicitation of Ag-specific immune responses. Viral-derived vector systems are attractive options for the delivery of sequences encoding Ags of interest to DCs (4). Such vectors possess intrinsic immunogenicity, as well as the ability to generate high levels of heterologous protein to promote robust Ag-specific immune responses (5). The effectiveness of a given vector system depends on several factors, including the population of DCs susceptible to transduction by the vector, the efficiency of this transduction, and the functional capacity of the transduced DCs. There have been numerous efforts to identify viral vectors that have the ability to successfully target and deliver Ag sequences to DCs (4). The most common virus vectors that have been utilized to transduce DCs are derived from adenovirus, poxvirus, or Retroviridae families (6, 7, 8), but several issues, including preexisting anti-vector immunity (9, 10, 11), inhibition of normal DC function (12, 13), or genomic incorporation into the host DNA chromosome and potential insertional mutagenesis (14) have led to the exploration of alternative, virus-derived vectors to target DCs. Notably, among these is the Venezuelan equine encephalitis (VEE) virus-derived replicon particle (VRP) vector system. This alphavirus-derived vector system has in vivo and in vitro tropism for DCs (15, 16, 17), which along with its characteristics of being derived from a positive strand RNA virus with a life cycle entirely restricted to the cytoplasm (18, 19), make it a promising immunotherapeutic vector that is being evaluated in a number of model systems and recent human clinical studies (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30).

We and others have identified human immature DCs in the myeloid lineage as the target for VRP transduction (16, 17). However, not all immature monocyte-derived (myeloid) DCs are receptive to VRP transduction, a finding that is consistent with evidence that DCs generated from heterogeneous populations of monocytes have functional differences (31, 32). We sought to characterize the DC subset that is receptive to VRP, and likely to VEE, to gain insight into potential mechanisms employed by this potent vector system and into dendritic cell biology. FACS of VRP-receptive and -nonreceptive monocyte-derived DC subsets followed by microarray gene expression analysis revealed differential expression patterns, suggesting two distinct DC subsets within the monocyte-derived DC population. We further investigated the expression of NK transcript 4 (NK4), now commonly referred to as IL-32 (33, 34), as this transcript was highly expressed in the VRP-receptive DC subset. IL-32 is a recently described proinflammatory cytokine known to induce TNF-{alpha} (34), and it synergizes with nucleotide-binding oligomerization domains to induce the expression of IL-1β and IL-6 (35). We confirmed constitutive IL-32 expression at the mRNA and protein levels in the VRP-receptive human DC subset in contrast to the nonreceptive subset. This finding identifies another subset of myeloid DCs and provides potential mechanisms to account for the potency of this vector system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Replicon vector constructs

The construction and manufacturing of the VRP-enhanced green fluorescent reporter protein (EGFP) was performed using techniques previously described (20). Briefly, the split helper and VRP RNA constructs were subject to co-electroporation into Vero cells for the production and purification of the replicon particle (AlphaVax). Infectious unit titers (IU/ml) were obtained by plating serial dilutions on Vero cell monolayers with immunofluorescent evaluation of VEE nonstructural gene products or heterologous protein expression. VRPs were resuspended in an isotonic phosphate buffered solution with 1% human serum albumin (formulation buffer), frozen, and stored at –80°C until use.

Generation of human monocyte-derived DCs

All human blood samples were collected in accordance with Institutional Review Board reviewed and approved research protocols. The normal blood donor program administered and run by the University of California at Irvine General Clinical Research Center provided anonymous, fresh, single-unit normal donor samples from which human PBMCs were isolated by differential density centrifugation over lymphocyte separation media (ICN Biomedicals). PBMCs were subjected to continuous counterflow centrifugation to yield populations of resting lymphocytes and monocytes that were >95% pure. The monocytes were used for the generation of monocyte-derived DCs as previously described (36). Monocytes were cultured at 1 x 106 cells/ml in RPMI 1640 (Cellgro) supplemented with L-glutamine (2 mM), sodium pyruvate (1 mM), nonessential amino acid mixture (1x), penicillin (100 U/ml), streptomycin (100 µg/ml) (Invitrogen), 2-ME (50 µM) (Sigma-Aldrich), and 10% FCS (Summit Biotechnology) at 37°C and 10% CO2. GM-CSF (100 U/ml) (Berlex Oncology) and IL-4 (50 ng/ml) (PeproTech) were added at day 0. Cultures were fed every 2–3 days by 50% volume exchange with cytokine-containing media. Cells cultured under these conditions for 6–8 days were used for all of the following experiments.

VRP transduction of monocyte-derived DCs

Monocyte-derived DCs were harvested from tissue culture flasks as previously described (36), and the cell pellet was recovered after centrifugation. The cells were washed in 1x PBS and the cell pellet was exposed to VRPs or matched formulation buffer at indicated multiplicities of infection (MOIs) and incubated for 1 h at 37°C. The cells were then transferred to a tissue culture flask with media containing human GM-CSF and IL-4, as above, for 24 h. Fluorescent reporter protein expression was evaluated by flow cytometry.

Biotinylation of VRP, detection of cell surface binding, competition assay, and maintenance of transduction capacity

VRP was biotinylated using EZ-Link Sulfo-NHS-Biotin reagent (Pierce) at optimal concentrations following the manufacturer’s protocol. Formulation buffer was biotinylated and used as the negative control. DCs were harvested and kept at 4°C for 30 min before exposure to biotinylated VRP (bVRP) or biotinylated formulation buffer at a MOI of 100. For the competition assay, a mixture of bVRP at a MOI of 100 and nonbiotinylated VRP at various MOIs (100, 500, or 1000) was added to DCs. Bound bVRP was detected using streptavidin conjugated to PE (BioLegend), and standard flow cytometry was used to analyze DC preparations. All steps and conditions were done at 4°C to minimize internalization of the bVRP. To detect reporter protein expression, DCs exposed to bVRP-EGFP were incubated at 37°C and 10% CO2 for 24 h. EGFP expression was detected by flow cytometry or fluorescent microscopy.

FACS and microarray gene expression profiling of monocyte-derived DCs

The development of the FACS isolation procedure was described previously (37). Briefly, receptive and nonreceptive DC populations were sorted at 4°C directly into RNAlater (Ambion) or DC culture media. Cells were recovered from RNAlater after centrifugation, and Trizol reagent (Invitrogen) was added directly to the resultant cell pellet after removal of RNAlater. RNA was isolated as per the manufacturer’s instructions. Affymetrix U133 plus 2.0 chips (Affymetrix) were employed for microarray gene expression analysis. The microarray core facilities at the University of California at Irvine performed 1) first-strand cDNA synthesis, 2) biotin-labeled cRNA synthesis, 3) target hybridization, and 4) array scanning after confirmation of RNA integrity. The results were analyzed using GeneChip operating software (Affymetrix) and GeneSpring software (Agilent Technologies). The Cyber-T statistical analysis program (found at http://cybert.microarray.ics.uci.edu) was used to determine levels of significance and confidence of differentially expressed genes. Aliquots of sorted DC populations placed into culture were examined 24 h later for EGFP expression by fluorescent microscopy and flow cytometry.

IL-32 transcript detection using RT-PCR

RNA from FACS-isolated VRP-receptive and -nonreceptive DCs was used to make cDNA using reverse transcriptase SuperScript II (Invitrogen). Human PBMCs exposed with or without heat-killed (80°C at 20 min) Mycobacterium, bacillus Calmette-Guérin, at a MOI of 2 for 24 h were subjected to RNA isolation to generate cDNA. PCR was performed on a DNA Engine Peltier Thermal Cycler-200 (MJ Designs). The following primers were used to detect all four isoforms of IL-32 described previously (38): IL-32{alpha} and IL-32β, forward, 5'-CTG AAG GCC CGA ATG CAC CAG-3', reverse, 5'-GCA AAG GTG GTG TCA GTA TC-'3; IL-32{delta}, forward, 5'-TCT CTG GTG ACA TGA AGA AGC T-3', reverse, 5'-GCA AAG GTG GTG TCA GTA TC-3'; and IL-32{gamma}, forward, 5'-GTA ATG CTC CTC CCT ACT TC-3', reverse, 5'-GCA AAG GTG GTG TCA GTA TC-3'. As an internal control we used GAPDH, forward, 5'-CCC ATC ACC ATC TTC CAG GAG-3', reverse, 5'-GTT GTC ATG GAT GAC CTT GGC-3'. Several PCR conditions were used as previously described (38): 1) 2 min at 50°C and 10 min at 95°C, followed by 30 cycles of PCR reaction at 95°C for 45 s, 70°C for 2 min, and 59°C for 1 min, or 2) 2 min at 50°C and 10 min at 95°C, followed by 30 cycles of PCR reaction at 95°C for 45 s, 53°C for 30 s, and 72°C for 50 s. The PCR products were run on 1% agarose gels stained with ethidium bromide.

IL-32 protein detection

VRP-receptive and -nonreceptive DCs were sorted into RNAlater by FACS. Cells were recovered from RNAlater as described above. Radioimmunoprecipitation assay cell lysis buffer containing protease inhibitors PMSF (1 mM), aprotinin (5 µg/ml), pepstatin A (5 µg/ml), and leupeptin (5 µg/ml) was added to the cells and incubated for 30 min on ice. The cell lysate was clarified by centrifugation at 12,000 x g for 10 min at 4°C. The supernatants were collected and stored at –80°C. Tris-gylcine 4–20% gels (Cambrex) were used. Loading buffer (50 mM Tris-HCl, 30% glycerol, 10% SDS, 0.012% bromphenol blue, 2-ME) was added to cell lysates and heated at 95°C for 5 min. Human spleen lysate (ProSci) was used as a positive control for IL-32 protein. Protein from the gel was transferred onto Hybond ECL nitrocellulose membrane paper (GE Healthcare Bio-Sciences). Approximately 5 µg/ml of rabbit polyclonal anti-IL-32 (ProSci) was added to the membrane and incubated for 2 h at room temperature. For IL-32 peptide inhibitor (ProSci), a 50-fold molar excess was used and incubated with the IL-32 polyclonal Ab for 30 min at 37°C according to the manufacturer’s instructions. Goat anti-rabbit IgG (H+L) HRP was added to the membrane and detected using SuperSignal ECL detection reagent (Amersham). Membranes were exposed to HyBlot CL autoradiography film (Denville Scientific).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Evaluation of cell surface-bound VRP on monocyte-derived DCs

We generated DCs by culturing human monocytes in the presence of cytokines IL-4 and GM-CSF for 6–7 days. Flow cytometry was used to evaluate all preparations by detecting the expression of DC-associated cell surface markers (Fig. 1, A and B). We transduced monocyte-derived DCs with VRP-expressing EGFP and detected fluorescent protein expression 24 h later. An intriguing aspect of our study is that only a portion of human monocyte-derived DCs undergo VRP productive transduction (Fig. 1C). This observation has several possible etiologies: either poor VRP transduction efficiency or a specific subset of DCs is receptive to VRP. We think the latter is likely, based on Poisson analysis, that a MOI of 5 is sufficient to ensure that 99% of receptive cells are infected with a virus (39), and the observed plateau of the percentage of monocyte-derived DCs transduced with VRP at increasing MOIs, as previously shown (17). The results of the labeling methodology described below provide additional support for this view.


Figure 1
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  		FIGURE 1. A subset of monocyte-derived DCs are receptive to VRP transduction. Human monocytes were cultured in IL-4 and GM-CSF for 6–7 days. A, Forward scatter (FSC) and side scatter (SSC) characteristics of the generated DC culture along with the gates used to delineate the DC population. B, Flow cytometric histograms of cell-surface phenotype from the monocyte-derived DCs. Dashed line indicates isotype control; solid line, designated cell surface marker. C, Two-color flow cytometric dot plots of DCs transduced with VRP-EGFP, and fluorescent expression was analyzed 24 h later. DCs were stained with Abs against CD14, MHC class II, and the appropriate isotype control. The numbers in each quadrant depict the percentage of cells that are single- or double-positive for fluorescent protein expression and the designated cell-surface expression markers. These data are representative of five separate experiments.

 
Because VRP rapidly usurps host cell protein synthesis machinery and is postulated to activate transduced cells to some degree (16, 40), we developed a methodology to identify and isolate DCs that were receptive to VRP without requiring transduction and reporter protein expression. This was accomplished by bVRP and detection of bound VRP on the cell surface of monocyte-derived DCs, which were maintained at 4°C. Employing this method, we observed a distinct population of DCs with bVRP on the cell surface (Fig. 2A). Comparisons of transduction efficiency of DCs with bVRP-EGFP and nonbiotinylated VRP-EGFP demonstrated that at the level of biotinylation we used, there was no difference in percentage of EGFP expression 24 h after VRP exposure (Fig. 2B). To further support specific binding of the bVRP toward receptive DCs, a competition experiment using nonbiotinylated VRP at various MOIs showed an inhibition of bVRP cell surface binding in a dose-dependent manner (Fig. 3). These findings suggest that bVRP binding is specific and eliminates the possibility of enhanced nonspecific binding or inhibition of VRP binding due to biotinylation. These data also support the existence of a subset of monocyte-derived DCs that is receptive to VRP, and they provide a method to isolate VRP-receptive and -nonreceptive DCs before productive transduction.


Figure 2
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  		FIGURE 2. Biotinylated VRP is detected on the surface of a subset of human monocyte-derived dendritic cells. VRP-EGFP was biotinylated and used to detect cell surface-bound VRP on DCs. A, Flow cytometric dot plots of bVRP on the surface of DCs that were detected using streptavidin-PE. The numbers represent the percentage of DCs positive for cell surface-bound VRP. B, Flow cytometric dots plots of DCs that were transduced with bVRP or VRP; EGFP expression was analyzed 24 h later. Numbers represent the percentage of DCs positive for EGFP expression. These data are representative of five experiments.

 

Figure 3
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  		FIGURE 3. VRP competition of biotinylated VRP binding to DCs. Individual panels depict flow cytometric dot plots of bound bVRP on the cell surface of monocyte-derived DCs under the designated conditions. The gate identifies percentage of DCs with bound bVRP on the cell surface. A, DCs that were not exposed to VRP and definition of the background. B, DCs exposed to bVRP at MOI of 100. C, DCs exposed to an equal mixture of bVRP and unlabeled VRP. D, DCs exposed to bVRP in the presence of a 5-fold excess of unlabeled VRP. E, DCs exposed to bVRP in the presence of a 10-fold excess of unlabeled VRP. These data are representative of three experiments.

 
Isolation of VRP-receptive and -nonreceptive monocyte-derived DCs

The characterization of the VRP-receptive subset of DCs is complicated by the lack of a known marker to isolate these cells (17). Therefore, we implemented the use of our bVRP, as described above, to isolate the VRP-receptive population within monocyte-derived DCs. To provide proof of principle for the isolation of receptive and nonreceptive DC populations, we isolated these respective populations and evaluated the expression of the reporter protein. VRP-receptive and -nonreceptive DCs were gated based on cell surface-bound bVRP-EGFP (Fig. 4A). We observed high purity isolation of bVRP-EGFP binding and nonbinding DCs (Fig. 4B). These populations were cultured and evaluated for EGFP expression 24 h later by fluorescent microscopy. Fluorescent images showed enrichment of EGFP+ cells in the bVRP-EGFP binding relative to the nonbinding DC population (Fig. 4, C–F). This provided evidence that we can successfully enrich a population of VRP-receptive DCs for further characterization.


Figure 4
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  		FIGURE 4. FACS enriches VRP-receptive human monocyte-derived DCs. Cell-surface bVRP on monocyte-derived DCs was detected by flow cytometry. A, Flow cytometric dot plots that were used to gate VRP-receptive and -nonreceptive DC populations for FACS. Numbers represent the percentage of cells for each gate. B, Flow cytometric dot plots of isolated VRP-receptive and -nonreceptive DCs from FACS. The x-axis shows bound VRP y-axis, SSC. These cells were cultured for 24 h, and the expression of EGFP was analyzed by fluorescent microscopy. C, EGFP expression from isolated VRP-nonreceptive DCs. D, Phase contrast of isolated VRP non-receptive DCs. E, EGFP expression from isolated VRP-receptive DCs. F, Phase contrast of isolated VRP-receptive DCs. These data are representative of three experiments.

 
Gene expression profile of VRP-receptive and -nonreceptive monocyte-derived DCs

To characterize the differences between VRP-receptive and -nonreceptive DCs, we opted to use a global unbiased microarray analysis of gene expression profile. To provide an accurate profile of the monocyte-derived DC subset that interacts with VRP, it was important to avoid VRP-induced perturbation of host cellular RNAs. These potential modulations could occur as the result of 1) the transduction of these DCs with the associated potential activation, 2) the amplification of VRP subgenomic RNA, 3) the usurpation of normal protein expression machinery, and 4) the associated high-level expression of VRP-encoded heterologous protein. To minimize any of these effects and to maximize the isolation of unperturbed VRP-receptive and -nonreceptive DCs, we performed FACS of our cells at 4°C, as depicted in Fig. 3, and sorted them directly into RNAlater. RNAlater has been described to rapidly diffuse into cells and denature cellular proteins (manufacturer’s guidelines), thus minimizing any potential VRP-associated changes in transcript or protein levels.

Total RNA was isolated from VRP-receptive and -nonreceptive DCs and used for microarray analysis. A statistical analysis was performed using the Bayesian regularized paired t test between the two preparations of VRP-receptive and -nonreceptive DCs. From this analysis, we categorized the number of differentially expressed genes overlapping between DC preparations of two different donors based on p value (p ≤ 0.001) and the posterior probability of differential expression (PPDE). The PPDE is an important criterion to estimate the probability of false-positives when comparing differentially expressed genes (41, 42). The relative expression of 652 transcripts (p ≤ 0.001, PPDE ≥ 0.99) from VRP-receptive and -nonreceptive DCs obtained from two separate donors were compared and displayed as a heat map (Fig. 5). The observed differential gene expression profile supports the existence of two populations within bulk cultured monocyte-derived DCs.


Figure 5
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  		FIGURE 5. Microarray gene expression profile of differentially expressed genes between VRP-receptive and -nonreceptive human monocyte-derived DCs. A hierarchal cluster analysis of 652 genes from two isolated RNA preparations of VRP-receptive and -nonreceptive DCs was generated and displayed as a heat map. The y-axis consists of dendrograms of clustered genes. Gene expression levels are indicated by color (red indicates high; black, moderate; green, low) relative to the median signal strength. The Cyber-T statistical analysis program was used to determine levels of significance and confidence of differentially expressed genes. All genes from each preparation were compared using a paired regularized Bayesian t test. The 652 genes were chosen based on the posterior probability of differential gene expression (PPDE ≥ 0.99 and p values of ≤0.001) generated from the respective t test.

 
IL-32 transcript expression in VRP-receptive monocyte-derived DCs

A striking difference in transcript expression between VRP-receptive and -nonreceptive DCs from our microarray analysis was the NK4 mRNA. Statistical analysis of the NK4 transcript between the data from two separate donors showed a p value of 2.6 x 10–4 and an 88.7-fold difference in VRP-receptive compared with VRP-nonreceptive DCs, with background level expression detected in the nonreceptive DC population. NK4, now commonly referred to as IL-32, is a proinflammatory cytokine involved in the induction of cytokines such as TNF-{alpha}, IL-1β, and IL-6, and it has recently been shown to be associated with proinflammatory states and autoimmune diseases (34, 35, 43). The IL-32 gene encodes multiple isoforms (34, 44). We evaluated by RT-PCR each IL-32 isoform expressed in FACS-isolated VRP-receptive and -nonreceptive DCs. We observed high transcript levels of IL-32β and IL-32{delta}, but no detectable levels of IL-32{alpha} or IL32{gamma} in VRP-receptive DCs compared with VRP-nonreceptive DCs (Fig. 6). Our data are also in agreement with reports of constitutive and up-regulated transcript IL-32-β, -{delta}, and -{gamma} isoform expression from resting and Mycobacterium-stimulated human PBMCs (38). A single pair of primers was utilized to detect both IL-32{alpha} and IL-32β. If both are present, two bands should be observed, with IL-32{alpha} representing the smaller length transcript (38). Previous studies by Netea et al. showed inconsistent expression of IL-32{alpha} transcript levels from multiple experiments of human PMBCs exposed to M. tuberculosis (38). We were unable to observe the expression of IL-32{alpha} transcript in our PBMCs exposed to M. bovis (Fig. 6), and therefore, we are unable to draw any valid conclusions regarding IL-32{alpha} transcript levels in our VRP-receptive human DCs. Nevertheless, these data confirm and validate our initial exploratory microarray results and support the existence of specific IL-32 transcript isoforms constitutively expressed in VRP-receptive DCs, notably the β and {delta} isoforms.


Figure 6
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  		FIGURE 6. RT-PCR of IL-32 transcript level isoforms expressed in VRP-receptive and -nonreceptive monocyte-derived DCs. RNA isolated from VRP-receptive compared with nonreceptive monocyte-derived DCs was subjected to RT-PCR to identify the presence of IL-32{alpha}, IL-32β, IL-32{delta}, and IL-32{gamma}. PCR reactions were loaded onto a 1% agarose gel and detected with ethidium bromide. Lanes are represented as: 1, resting PBMCs; 2, PBMCs stimulated with bacillus Calmette-Guérin; 3, VRP-receptive DCs; and 4, VRP-nonreceptive DCs. RT-PCR of transcript GAPDH was used as an internal loading control.

 
IL-32 protein expression in VRP-receptive monocyte-derived DCs

Protein expression level was evaluated by Western blot using a polyclonal Ab directed against the cytoplasmic tail sequence common to all four IL-32 isoforms. We were able to detect a band representing the IL-32 protein (27 kDa) in VRP-receptive DC lysates, with no band having been observed from the nonreceptive DCs (Fig. 7A). The positive human spleen lysate was used as a control and demonstrated the reactive bands described by the manufacturer. We used the IL-32 peptide immunogen to block binding of the polyclonal Ab to confirm that the immunoreactive bands were specific to the IL-32 target Ag sequence. Co-incubation of the Ab with the peptide inhibitor/immunogen markedly decreased the IL-32 band compared with Ab without the blocking peptide, supporting IL-32 binding specificity (Fig. 7B). The slight discrepancy in apparent molecular weights of the immunoreactive bands seen in the human spleen lysates and DC cellular lysates (25 and 27 kDa, respectively) is observed in VRP-receptive DCs from three different donors. This is consistent with the literature (34, 44) and is likely attributed to the IL-32 isoforms and also potential N-glycosylation and/or N-myristolation sites, which have been described for IL-32 (34). Although we have no direct evidence of the identity of this particular isoform of IL-32 at the protein level, we think it may represent IL-32β and/or IL-32{delta} based on the presence of transcript levels of IL-32 β and {delta} in the VRP-receptive DC population. In conjunction with our microarray analysis and RT-PCR validation, these data further support the constitutive expression of both IL-32 transcript and protein in VRP-receptive DCs.


Figure 7
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  		FIGURE 7. IL-32 protein expression of VRP-receptive and -nonreceptive human monocyte-derived DCs. Protein lysates were isolated from VRP-receptive and -nonreceptive DCs. A, Western blot detecting the presence of IL-32 protein using a rabbit polyclonal anti-IL-32. B, Rabbit polyclonal anti-IL-32 Ab was incubated with IL-32 peptide immunogen before staining on Western blot. Lanes represent: 1, standard marker ladder; 2, human spleen; 3, VRP-receptive DCs; and 4, VRP-nonreceptive DCs. These data are representative of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have identified a subset of human immature myeloid DCs with constitutive expression of the recently identified proinflammatory cytokine IL-32 arising from our studies investigating the population of human DCs receptive to VRP transduction. This constitutive expression of IL-32 was not detected in the nonreceptive population of monocyte-derived DCs, supporting the identification of a heretofore-uncharacterized human DC subset. IL-32 is not thought to be expressed on the cell surface, and thus, it is unlikely that it is directly related to the restricted tropism and binding of VRPs to DCs. However, the constitutive expression of IL-32 may be related to the potency of the VRP vector system and potentially to the pathogenesis of VEE. Constitutive mRNA and protein expression of proinflammatory cytokines is not unprecedented. For example, the chemoattractant cytokine IL-8 is found to be present in resting human neutrophils and eosinophils (45). By analogy, this suggests that IL-32 may have an important role in the early phase of the immune response mediated by this subset of DCs. The absence of an identified murine homolog suggests that study of IL-32 biology will be primarily restricted to human systems until the murine homolog is identified.

IL-32 has been shown to consist of multiple different isoforms (34, 41), although the functional significance of these various IL-32 isoforms is not fully understood. The {alpha}, β, and {delta} IL-32 isoforms do not have an N-terminal signal sequence to support extracellular transport (38, 44). IL-32 protein has been reported to be primarily expressed within the cell, and thus those particular isoforms may have intracellular functions (38, 44). However, the functional significance of the various IL-32 isoforms remains to be determined. We observed the expression of IL-32β and IL-32{delta} transcript levels in VRP-receptive DCs, but we were unable to demonstrate the IL-32{alpha} isoform in control mycobacterial-stimulated PBMCs or either receptive or nonreceptive DCs. The source of this discrepancy between reports (38, 44) is unclear, but it should be kept in mind as the biology of IL-32 is more fully characterized. The expression of IL-32 has been evaluated from a number of different human cell types. Two groups have provided evidence for the induction of IL-32 transcript and protein expression from monocyte-derived DCs after exposure to mycobacterium, LPS, or TNF-{alpha} (38, 46). In contrast to our study, those groups did not detect IL-32 in DCs before the exposure to maturation or activating stimuli. It is likely that the relatively small size of this DC subset with constitutive IL-32 expression (10–15% of the total population) accounts for the observation that bulk DC preparations required stimulation for induction of IL-32 expression. Cell types described as having constitutive IL-32 mRNA transcript expression include resting human PBMCs, monocytes, and isolated lymphoid tissues (38, 46). Although the role of constitutively expressed IL-32 remains to be defined, it is reassuring that similar patterns of constitutive IL-32 isoform expression are seen in other primary cells (38), as in our VRP-receptive DC population.

Most functional studies for IL-32 have evaluated recombinant protein and its effects on specific cell types (34, 35, 43, 46). IL-32 is an intriguing cytokine because it may possess dual roles: acting as a proinflammatory cytokine to induce TNF-{alpha} (34, 35), and possibly being involved in the activation-induced cell death of human T cells (44). Activation-induced T cell death was correlated with IL-32 up-regulation (44), but whether this is causally related or similarly observed in other cell types remains to be investigated. If confirmed, this association of IL-32 with activation-induced cell death provides a potential mechanism for the profound lymphopenia associated with infection by the parent wild-type virus VEE in the absence of high viral loads and viral cytopathic effect (47). Netea et al. reported that the induction of IL-32 in human PBMCs through a caspase-1/IL-18/IFN-{gamma}-dependent pathway (38), but we found no evidence of detectable transcript levels of those particular proteins in our microarray analysis of the VRP-receptive DCs (data not shown). This is not surprising given our prediction that our method would limit the potential activation of the DC subset binding bVRP. Recombinant human IL-32 protein has been used in murine models with generally concordant findings, supporting the existence of an as yet unidentified murine homolog. However, findings in murine systems should be interpreted cautiously in the absence of an identified IL-32 homolog and an as yet undefined receptor (35, 43, 46). The role of IL-32 in our identified DC subset and the effects of VRP productive transduction on IL-32 expression will be important for a more complete understanding of this DC subset and the function of the VRP vector system.

Our method to biotinylate VRP provided a novel strategy to isolate VRP-receptive DCs before productive transduction. VRP transduction is thought to occur via an active energy-dependent, endosomal pathway that is also dependent upon endosomal acidification for conformational changes leading to internalization and uncoating of the nucleocapsid (19, 48). Thus, internalization of VRP into cells is minimized at 4°C, allowing us to detect biotinylated VRP on the cell surface of receptive DCs. The isolation of VRP-receptive and -nonreceptive DCs by FACS showed an enriched population of EGFP-expressing cells, although not an entirely pure population based on EGFP+ cells. Note that we observed decreases in the expression of EGFP in DC populations, either bulk or sorted populations, run through the cell sorter (data not shown). It is likely that DCs, as well as other cell types, are sensitive to external factors associated with FACS, such as pressure or droplet charge, and this is supported by our data that biotinylation of VRP does not affect binding or transduction efficiency of monocyte-derived DCs.

DCs consist of heterogeneous populations that have diverse phenotypes and functions (49, 50). The list of DC subsets continues to grow, with recent additions that have discrete capacities to stimulate immune responses (51). The in vitro generation of human DCs under different conditions has led to the identification of selected myeloid DC subsets that correlate with DC subsets identified in vivo, such as Langerhans cells and dermal DCs (52, 53). Human monocyte-derived DCs have been a mainstay for the study of the functional capacity of DCs. However, it is also clear that even freshly isolated peripheral blood monocytes have phenotypic heterogeneity (31). Thus, it is not unexpected that different culture conditions yield different DC bulk populations and that subsets exist within these bulk populations. It has been demonstrated that DCs derived from CD16+ and CD16– human monocytes display differential properties to elicit Th1 and Th2 responses (31). Furthermore, human CD2+ monocytes have also shown to result in functional differences when used to generate DCs (32). Our microarray data documents the existence of two distinct populations of DCs from in vitro-generated monocyte-derived DCs. Statistical analysis between these DCs populations, which we have defined as being receptive or nonreceptive to VRP, revealed significantly differentially expressed genes employing the strict criterion of PPDE of 0.99 or greater, which is equivalent to a global false-positive of 1% or less. The identification of new DC subsets is likely to continue, as the roles of individual DC subsets and their contribution to the diverse range of immune responses are just beginning to be understood. Although the relevance of monocyte-derived DCs compared with actual in vivo human DCs has been raised by Shortman and Naik (49), in the absence of more robust analytical tools to investigate in situ human DCs, the studies of ex vivo-generated DCs, with their acknowledged limitations, will have to suffice and provide direction for future investigations.

The capacity of VRP to target DCs in vitro and in vivo has made it an attractive immunotherapeutic vector system. VRP-based immunotherapy induces robust Ag-specific immune responses that have been observed in a variety of animal models (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30). We and others have demonstrated that VRP interacts with myeloid DCs, but the identity of the specific DC subset(s) involved and the mechanism of how VRP productive transduction modulates or changes receptive DC subsets are not fully understood. Nevertheless, the capacity of this recently developed vector system to elicit robust immune responses surpassing other viral-derived vector systems in multiple animal models in conjunction with its documented restricted cellular tropism argue strongly for receptive DCs having a critical role in eliciting these immune responses. Furthermore, the observation that transduced DCs can maintain their viability and their functional capacity to acquire the mature phenotype after VRP transduction suggests a distinct functional role for VRP-transduced DCs (17). Although there are proposed models suggesting the mechanisms involved in VRP-induced Ag-specific immune responses, most of these studies are from animal models, and thus, the translation to human systems may be quite different. Moran et al. demonstrated the induction of specific cytokines and Ag-specific T cell proliferation by human monocyte-derived DCs exposed to VRP (16), but these studies did not clearly define if these events were derived from DCs undergoing VRP productive transduction, from bystander DCs, or both. Our ability to isolate VRP-receptive from -nonreceptive DCs provides an opportunity to approach and resolve these issues.

In summary, our results of investigations into VRP tropism for human DCs has led to the identification of a previously unrecognized subset of monocyte-derived DCs that constitutively express IL-32. The association of VRP with IL-32-expressing DCs may provide a potential mechanism of how this contributes to induce robust immune responses, and it also offers insight into the high degree of lymphopenia observed with the parent wild-type virus VEE (48) by the nature of IL-32’s potential role in activation-induced cell death (44). Our data and methodology provide an opportunity to study and identify other potential downstream signaling events with this respective DC subset during interactions with VRP. The identification of this new human DC subset, along with the evolving characterization of IL-32 function, enhances our understanding of human DC biology, of potential critical mechanisms for VRP function, and may have a significant impact on our understanding of a diverse range of pathologic states.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts 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 by Pacific Southwest Regional Center of Excellence in Biodefense and Emerging Infectious Disease Research. Back

2 Address correspondence and reprint requests to Dr. Edward L. Nelson, University of California at Irvine, Hewitt Hall, Irvine, CA 92697. E-mail address: enelson{at}uci.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; bVRP, biotinylated VRP; MOI, multiplicity of infection; EGFP, enhanced green fluorescent reporter protein; NK4, NK transcript 4; PPDE, posterior probability of differential expression; VEE, Venezuelan equine encephalitis; VRP, VEE replicon particle. Back

Received for publication July 12, 2007. Accepted for publication July 16, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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