Porcine dendritic cells generated in vitro: morphological, phenotypic and functional properties

Abstract

Despite the central role that dendritic cells (DC) play in immune regulation and antigen presentation, little is known about porcine DC. In this study, two sources of DC were employed. Bone marrow haematopoietic cell-derived DC (BM-DC) were generated using granulocyte–macrophage colony-stimulating factor (GM-CSF) in the presence or absence of tumour necrosis factor-α (TNF-α). Monocyte-derived DC (Mο-DC) were generated with GM-CSF and interleukin-4 (IL-4). In both systems, non-adherent cells developed with dendritic morphology, expressing high levels of major histocompatibility complex (MHC) class II. The presence of TNF-α increased the BM-DC yield, and enhanced T-cell stimulatory capacity. Both BM-DC and Mο-DC expressed the pan-myeloid marker SWC3, as well as CD1 and CD80/86, but were also CD14⁺ and CD16⁺. The CD16 molecule was functional, acting as a low-affinity Fc receptor. In contrast, the CD14 on DC appeared to differ functionally from monocyte CD14: attempts to block CD14, in terms of lipopolysaccharide (LPS)-induced procoagulant activity (PCA), failed. The use of TNF-α or LPS for DC maturation induced up-regulation of MHC class II and/or CD80/86, but also CD14. Allogeneic mixed leucocyte reactions and staphylococcal enterotoxin B antigen presentation assays demonstrated that these DC possessed potent T-cell stimulatory capacity. No T helper cell polarization was noted. Both the BM-DC and the Mο-DC induced a strong interferon-γ and IL-4 response. Taken together, porcine DC generated in vitro possess certain characteristics relating them to DC from other species including humans, but the continued presence of CD14 and CD16 on mature and immature porcine DC was a notable difference.

Introduction

The characterization and understanding of the porcine immune system have progressed rapidly over the past 10–15 years, particularly in the area of lymphocyte and macrophage immunobiology.¹ Porcine immunology has recently been receiving additional attention, due to the potential of the pig as both a donor in xenotransplantation,^2,3 and a large animal model for immunological studies.^4,5 Despite these advances, knowledge of porcine dendritic cells (DC) remains poor, and has not yet evolved in a comparable manner to that of DC from other species. This is problematic for the advancement of porcine immunology, considering the important central role of DC in both the processing/presentation of antigen to T lymphocytes, and regulation of immune responses.^6–8 DC have also shown functional diversity, due to their capacity to act as both immunogenic and tolerogenic antigen-presenting cells (APC) within the immune system.⁹ Such characteristics are particularly interesting for transplantation immunology, and understanding the pathogenesis of immunocompromising viral diseases.

Due to the infrequency of DC in the circulation and lymphoid organs, methods to generate these in vitro were established to provide sufficient numbers for immunological analyses. Stimulation of bone marrow (BM) haematopoietic cells (BMHC) with granulocyte–macrophage colony-stimulating-factor (GM-CSF) has been particularly successful with mouse BMHC-derived DC.^10–13 Stimulation of DC development from BMHC taken from rat¹⁴ and cattle¹⁵ required not only GM-CSF, but also interleukin-4 (IL-4). With human DC, isolated CD34⁺ BMHC required stimulation by GM-CSF and tumour necrosis factor-α (TNF-α).¹⁶ Stem cell factor (SCF) and Flt-3L are two additional cytokines that have been employed as proliferative stimuli for DC expansion in BMHC-derived culture systems.^15,17,18 Overall, these reports illustrate the species-dependent differences and diversity in terms of the cytokine requirements for deriving DC from BMHC. This contrasts with the generation of DC from blood monocytes (Mο),¹⁹ wherein the use of GM-CSF and IL-4 has been consistent for all species to date.^6–8

The objective of the present study was to identify and characterize porcine DC generated in vitro from BMHC and blood Mο. An initial aim was to determine the cytokine requirements for this generation, and how this related to DC generation from other species. From this, the comparative immunobiology of porcine DC was investigated using morphological, phenotypic and functional characterization.

Materials and methods

Isolation/preparation of bone marrow and monocytic cells

Swiss White Landrace pigs were kept under specific pathogen-free (SPF) conditions at the institute. BMHC were isolated from the sternum of 3- to 6-month-old pigs as previously described.²⁰ Briefly, the bone was flushed with phosphate-buffered saline /0·03% ethylenediaminetetraacetic acid (w/v) at 37°, with the cell suspension obtained being depleted of erythrocytes and mature granulocytes by centrifugation over Ficoll-Paque (1·077 g/l; Amersham Pharmacia Biotech AG, Dübendorf, Switzerland) at 1000 g for 40 min at room temperature. Peripheral blood mononuclear cells (PBMC) were isolated using density centrifugation (1000 g, 25 min) over Ficoll-Paque (1·077 g/l; Pharmacia) as previously described.²¹ Mο were isolated by adherence to plastic for 16 h.²¹

Bone marrow and monocytic cultures

BMHC and Mο were cultured in phenol red-free Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Life Technologies AG, Basel, Switzerland), supplemented with 2 mm glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 50 µm 2-mercaptoethanol (all Gibco, BRL). Fetal calf serum (FCS) (10% v/v; Sigma, Buchs, Switzerland) or porcine serum (PS) (10% v/v; obtained from SPF pigs) was used as serum supplements for the medium. For BMHC cultures, 25 ng/ml recombinant porcine (rp) GM-CSF²² was also employed, either alone (BM-DC medium) or in combination with rpTNF-α²³ (30 U/ml). For the generation of Mο-DC, the medium was supplemented with 150 ng/ml rpGM-CSF, 100 U/ml rpIL-4 and PS (Mο-DC medium). The rpIL-4 was prepared in our laboratory by cloning the porcine IL-4 gene²⁴ in a pBAD-Thiofusion expression vector as described in the manufacturer's handbook (Invitrogen, Leiden, NL). A thioredoxin-IL-4-(His)₆ fusion protein was expressed in Escherichia coli and extracted from cell lysates as described in the handbook. Purification used affinity chromatography with HiTrap chelating columns and fast protein liquid chromatography (FPLC; Aekta, Amersham Pharmacia Biotech, Dübendorf, Switzerland). Alternatively, commercial rpIL-4 (Biosource, Lucernachem, Luzern, Switzerland) was used. The bioactivity of both sources of rpIL4 was determined using TF-1 cells, with the IL-4 concentration giving half-maximum proliferation being defined as 1 unit.

BMHC were cultured in 100-mm Petri dishes (Falcon, Becton Dickinson, Basel, Switzerland), with incubation at 39°. The culture method was based on protocols described for both human¹⁶ and murine BMHC-derived DC.^10,11,13 For initiation (day 0), BMHC were cultured at 4 × 10⁵ cells/ml in 10 ml of BM-DC medium. On day 3 of culture, another 10 ml of BM-DC medium was added. At days 6 and 8 of culture, half of the culture medium was replaced by fresh BM-DC medium. Mο-DC were generated by culture of monocytes (0·5 × 10⁶ cells/ml) in Mο-DC medium for 6 days. On days 2 and 4, half of the medium was replaced by fresh Mο-DC medium. For induction of DC maturation, half of the old medium was replaced by fresh complete medium with 250 U/ml rpTNF-α or 1 µg/ml lipopolysaccharide (LPS; Sigma) and the cells were cultured for a further 24 hr (LPS) or 48 hr (TNF-α).

Phenotyping

Non-adherent cells were phenotyped as previously described.²⁰ Briefly, after incubation with the antibody against the cell marker, fluorescein isothiocyanate (FITC), phycoerythrin (PE) or biotin-conjugated goat F(ab′)₂ anti-mouse isotype-specific immunoglobulins (Southern Biotechnology, Birmingham, AL) were added. For detection of the biotinylated conjugate a final incubation with Spectralred® (Southern Biotechnology) was added. The following monoclonal antibodies (mAbs) were used: anti-major histocompatibility complex class I (MHCI; 74-11-10),²⁵ anti-MHCII (MSA3),²⁶ anti-CD1 (76-4-2),²⁷ anti-CD3 (3E8, VMRD Inc., Pullmann, WA),²⁸ anti-CD4 (74-12-4),²⁸ anti-CD8α (76-2-11),²⁹ anti-CD14 (MIL2),³⁰ (MY-4,³¹ Beckman-Coulter, Nyon, Switzerland), anti-CD16 (G7),³² CD80/86 (hCTLA4-mouse immunoglobulin fusion protein, Alexis, Switzerland);³³ Swine Workshop Cluster (SWC) antibodies employed were against the pan-myeloid marker SWC3 (74-22-15),²⁸ and against SWC8 (MIL3).²⁸ Surface immunoglobulin (sIg) was detected using goat anti-pig IgG (heavy- and light-chain-specific, Jackson Immunoresearch Laboratories, Avondale, PA). The mAbs 74-12-4, 74-22-15, 74-11-10, 76-4-2, 76-2-11 and MSA3 were kindly provided by Dr J. Lunney (USDA, Beltsville, MD) and Dr A. Saalmüller (BFAV, Tübingen, Germany). The mAbs MIL2 and MIL3 were kindly provided by Dr K. Haverson (University of Bristol, UK), and G7 by Dr Y. B. Kim (Finch University of Health Sciences, North Chicago, IL).

T-cell stimulation assays

T cells were purified by magnetic cell sorting using the MACS system (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). To this end, a mAb against porcine CD6 (a38b2, kindly donated by Dr A. Saalmüller BFAV, Germany), which is expressed on all T helper (Th) and MHC-restricted cytolytic T lymphocytes,³⁴ was used. Purity of CD6⁺ T cells was 95–98%. Non-adherent cells were treated with mitomycin C (10 µg/ml, 1 hr, 39°; Sigma) and washed four times. For presentation assays of the superantigen staphylococcal enterotoxin B (SEB) (Toxin Technology, Sarasota, FL). APC were incubated simultaneously with SEB (100 ng/ml) and mitomycin C for 1 hr at 39°, followed by four washings. Quadruplicates of 1 × 10⁵ CD6⁺ T cells/well were seeded into a 96-well flat-bottom microtitre plate (Costar, Cambridge, UK) together with titrated numbers of APC. After 3 days for allogeneic responses or 2 days for SEB stimulation, co-cultures were pulsed with 1 µCi/well [³H]methyl-thymidine (Moravek Biochemicals Inc., Brea, CA) for 18 hr. The plates were harvested on to filter mats, and counted in a 1450 MicroBeta® TriLux radioactivity counter (Wallac, Turku, Finland).

Procoagulant activity (PCA)

PCA was measured by the determination of the clotting time of citrated human platelet-poor plasma (PPP), based on the method by Jungi.³⁵ Briefly, cells were untreated or stimulated with LPS (1 µg/ml), washed, and resuspended in 0·9% (w/v) NaCl. Then, 100 µl of the cell suspension was incubated with 100 µl PPP, and the clotting time was determined after recalcification (25 mm CaCl₂) by measuring the time to reach half-maximum turbidity at 370 nm in a VERSAmax™ microplate reader (Molecular Devices, Sunnyvale, CA). The clotting time was converted to arbitrary units by using a thromboplastin (Thromborel® S, Dade Behring, Marburg, Germany) calibration curve. For attempts to block LPS-induced PCA, cells were preincubated with anti-CD14 mAbs known to interfere with LPS binding to porcine Mο³⁰ (MEM-18, Sanbio®, Am Uden, NL; MIL2).

Measurement of low-affinity immunoglobulin receptors

Cells were preincubated with or without anti-CD16 mAb (G7, FcγRIII),³² followed by addition of heat-treated porcine serum (50% v/v). The serum had been heat-treated (60° for 1 hr) to induce immunoglobulin complexes. Immunofluorescence detection of the bound immunoglobulin used Protein A–FITC (Roche Biochemicals, Basel, Switzerland).

DC-induced T-cell polarization

SEB-pulsed DC were co-cultured with T cells at a 1 : 10 ratio for 3 days. At this time, half of the medium was replaced, and the cells were stimulated with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml) plus ionomycin (0·5 µg/ml) for 18 hr. Production of interferon-γ (IFN-γ) and IL-4 in the culture supernatants was analysed using enzyme-linked immunosorbent assay kits (Biosource).

Results

Generation of porcine BM-derived cells of DC morphology

Phase contrast microscopic analysis of BMHC stimulated with recombinant porcine cytokines generated cells with dendritic cell-like morphology. After 6–8 days of stimulation with GM-CSF, the in vitro BMHC culture consisted of heterogeneous adherent and non-adherent populations. The non-adherent population was dominated by cells, which possessed dendritic and veiled extensions, seen to be protruding from the cell surface (Fig. 1a–c). These cells, hereafter referred to as BM-DC, often formed clusters (Fig. 1a). In addition, the BMHC cultures contained smaller non-adherent cells, presumably immature precursors and granulocytic cells. The adherent cells within the BMHC cultures were dominated by cells of macrophage (Mφ) morphology (Fig. 1c, larger cell towards the upper right hand corner).

Figure 1.

Phase contrast photomicrographs of GM-CSF-stimulated BMHC cultures at day 6, showing the dendritic morphology of the non-adherent cells. (a) Cluster of non-adherent cells; (b) non-adherent cell with dendritic morphology and veils; (c) non-adherent cells with dendritic morphology, together with an adherent cell of Mφ morphology.

Phenotype of BM-DC

The majority (over 90%) of the non-adherent BMHC cultured in the presence of GM-CSF, as shown in Fig. 1, were SWC3⁺ (Fig. 2a), indicating their myeloid origin.^20,36 Using MHCII labelling, three overlapping populations were defined: MHCII⁻, MHCII^low and MHCII^high. Flow cytometric forward and side scatter (FSC/SSC) characteristics of these populations, after electronic gating, revealed two major populations. The MHCII^−/low cells had relatively low FSC/SSC (Fig. 2b, grey), while the MHCII^high population (defined in R1 of Fig. 2a) were FSC^high (depicted black in Fig. 2b). This MHCII^high population increased with time in culture (Fig. 2c), peaking at 8–10 days post-plating (Fig. 2c). There was variation between experiments with respect to the representation of these MHCII^high cells within the non-adherent population, ranging from 50 to 85%. Interestingly, the cell yield was higher when PS was employed in place of FCS (data not shown).

Figure 2.

MHCII and SWC3 expression on non-adherent BMHC cultured for 8 days with GM-CSF;

the cells with intermediate to negative MHCII expression in (a) (grey dots) were separated from the MHCII^high cells (black dots) by electronic gating, and their FSC/SSC characteristics analysed by multicolour dot plot.

The increase in the percentage of MHCII^high non-adherent BMHC with time in culture. The results shown in (a) to (c) are representative of five independent experiments.

Comparison of monocyte and granulocyte markers on BM-DC

Additional double- and triple-labelling experiments were performed using an electronic gate set on FSC^high cells, as defined in R2 of Fig. 2(b). As for the ungated cells above, over 95% of these cells expressed the myeloid differentiation antigen SWC3. Using SWC8/MHCII double labelling, three populations could be distinguished within the gated region (Fig. 3a,b). SWC8⁺ MHCII⁻ cells were probably immature precursors of the granulocytic lineage. SWC3⁺ SWC8⁺ MHCII⁻ cells were previously shown to belong to the granulocytic lineages of BMHC.²⁰ In the GM-CSF-stimulated cultures their representation within the total (ungated) non-adherent cells decreased from 80% at the initiation of the culture to less than 15% after 8 days (data not shown). The other two populations, both increasing with culture time, expressed high levels of MHCII and were distinguished by their SWC8 expression. The dominant population was SWC8⁻ and when PS instead of FCS was used as culture supplement, their representation was higher (Fig. 3a,b, upper and lower right quadrants). Nevertheless, the level of MHCII antigen expression was comparable in both PS- and FCS-supplemented cultures (Fig. 3a,b). The phenotype of the dominant SWC3⁺ SWC8⁻ MHCII⁺ non-adherent population would place these cells within the monocytic lineage.²⁰

Figure 3.

Phenotype of BM-DC generated by culture of BMHC for 8 days in the presence of GM-CSF. An electronic gate on FSC^high as shown in R2 of Figure 2(b) was used. (a) SWC8/MHCII, FCS-supplemented cultures; (b) SWC8/MHCII, PS-supplemented cultures; (c) CD14/MHCII, FCS-supplemented cultures; (d) CD16/MHCII, FCS-supplemented cultures; (e) CD14/CD1, FCS-supplemented cultures. The results shown in (a) to (e) are representative of five experiments.

BM-DC expression of markers related to maturation in other species

Interestingly, the MHCII^high cells also expressed CD14, with a fluorescence intensity correlating to the MHCII expression on a large proportion of the cells (Fig. 3c). CD16 expression was also noted on these cells (Fig. 3d). Although CD1⁻ CD14⁺ cells were found, the majority of the CD14⁺ BMHC-derived non-adherent cells were also CD1⁺ (Fig. 3e). These cells were confirmed as myeloid in origin, due to their lack of expression of the lymphoid antigens CD3, CD4, CD8α and sIg (data not shown).

It was of interest to determine if BM-DC could be characterized in terms of a ‘maturation status’,^6–8 as has been defined for DC from other species. Consequently, BM-DC derived by culture for 6 days in GM-CSF were incubated with either LPS or TNF-α for a further 48 hr. In the BM-DC culture, MHCI and MHCII expression was clearly enhanced only with TNF-α (Fig. 4a,b). Surprisingly, this was also noted with CD14 (Fig. 4d), while CD16 remained relatively constant (Fig. 4e). The LPS treatment had only a slight effect, if any, on these expressions. In contrast, the co-stimulatory molecules CD80/86 were up-regulated by both TNF-α and LPS (Fig. 4f), while CD1 expression was down-regulated, more so by LPS treatment (Fig. 4c).

Figure 4.

Influence of maturation signals on cell surface antigen expression of BM-DC. LPS and TNF-α were added to some cultures on day 6, and all cultures were harvested on day 8 of culture. Grey-filled histogram, conjugate control; dotted line histogram, untreated BM-DC; bold-line histogram, TNF-α-treated BM-DC; thin-line histogram, LPS-treated BM-DC. The results shown in

(a) to (e) are representative of three independent experiments.

Monocyte-derived DC

Comparing the porcine BM-DC described above with mouse and human BM-DC, the continued expression of both CD14 and CD16, especially after addition of maturation signals, was surprising. Consequently, it was of interest to know whether this expression pattern was also found on other in vitro generated porcine myeloid DC types. To this end, Mο-DC were generated using GM-CSF plus IL-4 stimulation. Similar to the reports on mouse and human Mο-DC, this cytokine combination induced the development of non-adherent cells with dendritic morphology and cluster formation. Their morphology was comparable to that for the BM-DC shown in Fig. 1. These cells first appeared after 2–3 days, further increasing up to 6 days of culture. At this time-point, to address the effect of maturation signals, either LPS or TNF-α was added for another 24 hr.

The Mο-DC were similar to BM-DC in that they expressed the pan-myeloid SWC3 (data not shown), as well as MHC molecules, CD1, CD14, CD16 and CD80/86 (Fig. 5a–f). Relative to the negative controls, the fluorescence intensity for these surface markers on the Mο-DC appeared low compared to the intensity seen on the BM-DC. This could be explained by the higher autofluorescence observed with Mο-DC than with BM-DC. Nevertheless, the MHCII expression on the Mο-DC did differ from that on BM-DC, being more heterogeneous and not significantly up-regulated by TNF-α (Fig. 5b). The possible heterogeneity of Mο-DC was also indicated by the CD1 staining profile, where the majority of cells were CD1^low, with only a minority having high CD1 expression (Fig. 5c).

Figure 5.

Influence of maturation signals on cell surface antigen expression of Mo-DC. LPS and TNF-α were added to some cultures on day 6, and all cultures were harvested on day 7 of culture. Grey-filled histogram, conjugate control; dotted line histogram, untreated Mο-DC; bold-line histogram, TNF-α-treated Mο-DC; thin-line histogram, LPS-treated Mο-DC. The results shown in (a) to (e) are representative of three independent experiments.

Interestingly, both CD14 and CD16 expression was a consistent feature of Mο-DC, similar to the BM-DC. TNF-α induced up-regulation of CD14 on the Mο-DC (Fig. 5d,e), as seen with the BM-DC, but the Mο-DC were different in that LPS stimulation resulted in a down-regulation of CD16 (Fig. 5e). The Mο-DC were comparable to BM-DC, in that maturation signals – particularly LPS – induced an up-regulation of CD80/86 (Fig. 5f).

T-cell stimulatory capacity

Considering the DC-like characteristics of BM-DC and Mο-DC, it was important to determine their T-cell stimulatory capacity (Fig. 6). BM-DC did act as potent stimulators of allogeneic T lymphocytes in a mixed lymphocyte reaction, especially in comparison with autologous monocytes. In Fig. 6(a), maximum proliferation of around 100 000 counts per minute (c.p.m.) was obtained. This depended on the MHC haplotypes of the stimulator and responder cells, whereby counts ranging from 20 000 to 200 000 c.p.m. could be obtained. Nevertheless, a strong stimulatory capacity of the BM-DC depended on the presence of at least 1 BM-DC per 10–40 T cells. It should also be noted that the non-adherent BMHC were comprised of a heterogeneous population of cells. Although the BM-DC dominated, they were not alone (see Fig. 2a,b). Consequently, a ratio of 10 BM-DC to 100 T cells quoted in Fig. 6 may well be more in the region of 5–9 BM-DC to 100 T cells.

Figure 6.

T-lymphocyte stimulatory capacity of in vitro generated DC. BM-DC were taken at day 8, while Mo-DC were harvested at day 7 of culture. The proliferative response of sorted CD6⁺ T cells (1 × 10⁵ cells/well) to different numbers of APC (x-axis) was determined by [³H]thymidine incorporation (c.p.m., y-axis). (a) Comparison of the allostimulatory activity of 24-hr adherent monocytes and BM-DC. (b) Comparison of the allostimulatory activity of BM-DC generated in FCS-supplemented medium and BM-DC generated in PS-supplemented medium. (c) Comparison of the SEB-presenting capacity of 24-hr adherent Mο and BM-DC. (d) Comparison of the SEB-presenting capacity of GM-CSF-derived BM-DC and GM-CSF-derived BM-DC matured with TNF-α (250 U/ml) for the final 48 hr of culture. (e) Comparison of the SEB-presenting capacity of GM-CSF/TNF-derived BM-DC and GM-CSF/TNF-derived BM-DC matured with TNF-α (250 U/ml) for the final 48 hr of culture. (f) Comparison of the SEB-presenting capacity of GM-CSF/IL-4-derived Mo-DC, the same Mo-DC matured with TNF-α (250 U/ml) for the final 24 hr of culture, and the same Mo-DC matured with LPS (1 µg/ml) for the final 24 hr of culture. The results shown in (a) to (f) are representative of three independent experiments.

The influence of serum supplements on the BM-DC was again noted in terms of their stimulatory capacity. BM-DC cultured in PS had a lower level of granulocytes (SWC8⁺ MHCII⁻) and the SWC3⁺ SWC8⁺ MHCII⁺ population (Fig. 3b compared to Fig. 3a). Yet it was the FCS culture, which provided BM-DC with the higher T-cell stimulatory capacity (Fig. 6b). Nevertheless, this was only noted at the 1 : 1 ratio of BM-DC : T cells. At other ratios, the allogeneic responses induced were comparable using cells from both the FCS and PS cultures.

Previous studies have shown that DC isolated from human blood are particularly potent presenters of microbial superantigens.³⁷ This is especially true of DC pulsed with SEB, in comparison with B cells and Mο,³⁸ or MHCII-transfected cells.³⁹ When SEB was employed in the T-cell proliferation assays with the porcine BM-DC, comparable values to the MLR data (Fig. 6a,b) were obtained (Fig. 6c). Once again, relatively high numbers of the BM-DC were required (> 1 DC: 80 T cells), but they were potent APC, especially when compared with 24-hr adherent blood Mο (Fig. 6c). High responses were obtained with a ratio of 1 DC per 10 T cells (Fig. 6c).

Influence of DC maturation on T-cell stimulatory capacity

As shown in Fig. 4, addition of TNF-α as maturation factor induced MHCII up-regulation. However, no significant influence of this TNF-α-induced maturation on the capacity of the BM-DC to induce SEB-dependent T-cell proliferation was observed (Fig. 6d). Similar results were obtained using LPS in place of the TNF-α as a maturation factor (data not shown).

Attempts to enhance the accessory capacity of the BM-DC culture system included addition of other cytokines at the onset of the BMHC culture. Although this early addition of TNF-α did not change the morphological/phenotypic characteristics of BM-DC (data not shown), a reproducible increase in T-cell stimulatory activity was noted (Fig. 6e).

The T-cell stimulatory capacity of Mο-DC was also tested in the SEB presentation assay. The representative experiment shown in Fig. 6(f) demonstrates that Mο-DC appeared to be superior APC compared to BM-DC. Although similar maximum c.p.m. were obtained, the titration curve of the Mο-DC did not drop as rapidly, and formed a ‘shoulder’. A clear T-cell stimulation was observed down to ratios of 1 DC per 100 T cells. Also contrasting with BM-DC, LPS-induced maturation enhanced the SEB presentation capacity of Mο-DC (Fig. 6f).

Function of CD14 on the DC as an LPS receptor

The continued expression of CD14 on BM-DC and Mο-DC, even after addition of maturation signals, is in contrast to the behaviour of CD14 on mouse and human DC.^6–8 This raised the question concerning the function of this CD14 on porcine DC. Based on the established role of CD14 as a major LPS receptor,⁴⁰ BM-DC and Mο were compared in terms of the ability of anti-CD14 mAbs to block LPS-induced PCA (Fig. 7). This assay was selected due to the reported CD14-dependent inducibility of PCA by LPS⁴⁰ and the reported PCA of DC.⁴¹ As shown in Fig. 7(a) and (b), LPS was capable of inducing PCA in both Mο and BM-DC. However, when the cells were preincubated with the anti-CD14 mAbs prior to LPS addition, this blocking of CD14 impaired the LPS-induced PCA only with the Mο, but not with the BM-DC. Such results would imply that the CD14 expressed on Mο and BM-DC clearly differed functionally in terms of being the major LPS receptor.

Figure 7.

CD14 and CD16 functionality of the CD14 and CD16 expressed on porcine APC. The Mο were 24-hr adherent blood Mο, while the BM-DC were generated after 8 days in culture in the presence of GM-CSF and TNF-α. The PCA of Mο (a) and BM-DC (b) is shown. The cells were stimulated with LPS (1 µg/ml) alone, treated with anti-CD14 mAb (MEM18 or MIL2) alone, or pretreated with anti-CD14 mAb followed by LPS stimulation as shown on the x-axis. Anti-porcine CD16 mAb G7 inhibition of heat aggregated porcine serum binding to: Mο (c) and BM-DC (d) The bound immunoglobulin was detected using Protein A–FITC, to reduce detection of bound monomeric immunoglobulin. Grey histogram, conjugate control; thin line, immunoglobulin binding, no mAb; bold line mAb G7 pretreatment before immunoglobulin staining. The results shown in (a) to (d) are representative of three independent experiments.

Function of CD16 as a low-affinity Fc receptor

The unexpected expression of CD16 on porcine BM-DC and Mο-DC also raised the question of its function. To this end, the binding of heat-aggregated immunoglobulin to BM-DC was quantitatively compared with binding to Mο (Fig. 7c,d). Although the signal of bound immunoglobulin was lower on BM-DC than in Mο, addition of anti-CD16 mAb reduced the binding of aggregated immunoglobulin to both cell types. This would indicate a related functionality of the CD16 on both BM-DC and Mο.

Analysis of the capacity of BM-DC and Mο-DC to induce Th cell polarization

T cells were stimulated with SEB-pulsed BM-DC or Mο-DC treated with different maturation signals (TNF-α and LPS), and the production of IFN-γ and IL-4 was measured (Fig. 8). In general, BM-DC and Mο-DC induced a strong IFN-γ and IL-4 response without polarizing the T cells towards a specific Th1 (IFN-γ) or Th2 (IL-4) pathway. Surprisingly, the maturation of BM-DC with TNF-α resulted in lower levels of both T-cell cytokines compared to untreated cultures (Fig. 8a,b), while LPS maturation induced only lower IFN-γ responses. This modulation was not evident with the Mο-DC. However, the Mο-DC were more effective at inducing IL-4 production compared to the BM-DC.

Figure 8.

DC-induced T-cell polarization. SEB-pulsed DC were co-cultured with CD6⁺ T cells at a 1 : 10 ratio. After 48 hr of co-culture, T cells were washed and restimulated with PMA (50 ng/ml) plus ionomycin (1 µg/ml). Supernatants were harvested after a further 18 hr of culture, and measured for the presence of IFN-γ (a) and IL-4 (b) (c) by enzyme-linked immunosorbent assay. The results shown in (a) to (b) are representative of three experiments.

Discussion

Knowledge on DC development from haematopoietic progenitors has been enhanced through models using in vitro cytokine stimulation of BMHC, particularly in terms of DC origin,^42,43 mechanism of antigen presentation,⁴⁴ and capacity to stimulate T-cell-dependent immunity.⁴⁵ Knowledge on both the haematopoietic^20,46 and monocytic^21,47,48 development of porcine leucocytes is being elucidated, yet the characterization of porcine DC is still poorly understood. For these reasons, the present work set out to identify porcine in vitro generated DC, and determine their immunobiological characteristics.

When porcine BMHC were stimulated with GM-CSF, a cytokine known to induce BMHC-derived DC development in the mouse,^10–13 three types of myeloid cells were generated. They were all SWC3⁺ and mostly CD14⁺, but could be divided into adherent and non-adherent cells. The adherent cells were morphologically, phenotypically and functionally like Mφ (data not shown), based on previous reports concerning porcine Mφ.^21,47,48 These Mφ were CD1⁻, and expressed lower levels of both MHCII and CD80/86 compared to the non-adherent BM-DC (data not shown).

As for the non-adherent GM-CSF-derived BMHC, granulocytic cells were identifiable as FSC^low CD1⁻ MHCII^−/low SWC3⁺ SWC8⁺,²⁰ and possessed a characteristic nuclear staining (data not shown). The dominant cells referred to as BM-DC, had a dendritic morphology typical of that reported for DC from other species, and a DC-like phenotype – MHCII^high CD1⁺ CD80/86⁺.^6–8 Surprisingly, the BM-DC were also positive for CD14 and CD16, and addition of maturation factors further increased CD14 expression. These phenotypic characteristics were also found on porcine Mο-DC. This would demonstrate a clear difference when compared to human blood DC⁴⁹ and both human and mouse haematopoietic progenitor-derived DC, all of which are negative for CD14 and CD16 in their final differentiation stage.^13,50 Yet, recent reports have described potent human monocytic APC with DC-like characteristics, which maintain CD14 and CD16 on their cell surface.^51,52 Bovine BMHC-derived DC were also reported to express CD14,¹⁵ relating to the closer phylogenetic relationship between cattle and swine, both being ungulates. A subset of DC-like cells in porcine intestinal lamina propria was found to be SWC3⁺ CD16⁺, but was negative for both CD1 and CD14.⁵³

With respect to porcine CD16, the cellular distribution of this molecule differs from the human counterpart.²⁰ Nevertheless, the CD16 on porcine DC was functionally similar to that on Mο, in terms of aggregated immunoglobulin binding. The same cannot be said for CD14. The molecule expressed on porcine DC is antigenically CD14. Like the Mο CD14, it is detected by the MY-4 mAb recognizing porcine CD14.³¹ The MIL2 mAb which competes with MY-4 for binding to Mο also recognizes the DC-expressed CD14. A recent report described in vitro derived porcine Mο-DC, which had apparently down-regulated CD14.⁵⁴ However, the authors did not employ MY-4 to identify CD14. Furthermore, their culture conditions differed from those employed for the present work. The identification of a subpopulation of CD14⁻ DC-like cells in porcine lamina propria⁵³ could be reflecting the existence of DC subpopulations which can be CD14⁻. Development of DC towards a CD14⁺ or CD14⁻ phenotype would be dependent on the cytokine and environmental signals received. However, certain anti-CD14 mAbs fail to recognize the molecule once Mο have differentiated towards Mφ (McCullough et al. unpublished data). Consequently, the definition of porcine cells as CD14⁻ should use the MY-4, which has a high affinity for and specifically reacts with the porcine CD14 molecule.³¹ This antibody identifies CD14 expression when other mAbs fail (McCullough et al. unpublished data). In addition, to identifying the antigenic expression of the CD14 molecule on porcine DC, it is also necessary to determine its function. CD14 on Mο is a functional LPS receptor.⁴⁰ However, with DC, in contrast to Mο, LPS-enhanced PCA was unaffected by anti-CD14 mAbs, indicating cell type-dependent functional differences of the CD14 receptor. Being a glycosyl-phosphatidylinositol-anchored protein, CD14 requires a bystander molecule to signal the cells.⁵⁵ Thus, the lack of CD14-dependent PCA-inducibility in porcine DC may be a consequence of the bystander molecule rather than of CD14 itself. PCA was still induced by the LPS, in an apparent CD14-independent manner. This would relate to LPS interacting possibly with one of its other receptors, such as Toll-like receptors (TLR), specifically TLR3.⁵⁶

The presence of the SWC8⁺ MHCII⁺ cells in the non-adherent GM-CSF-derived BMHC cultures was another interesting observation. SWC8 was shown to be a differentiation marker of the granulocytic lineage, acquired at an early stage during differentation.²⁰ This raised the question of the origin of this SWC8⁺ MHCII⁺ subset. A report with human polymorphonuclear leucocytes (PMNs) has shown that precursors of the neutrophilic lineage can be driven to acquire DC characteristics.⁵⁷

The in vitro generated DC possessed strong T-cell stimulatory capacity, in allogeneic mixed lymphocyte reactions and SEB-presentation assays. This stimulatory capacity was clearly superior to that of the PBMC alone, or when 24-hr adherent blood-derived Mο were employed as APC. Nevertheless, between 3 and 10 BM-DC were required to stimulate 100 T cells, maximum responses being obtained with approximately 30 DC/100 T cells. This did not appear to be a result of DC immaturity. Several agents reported to mature DC, including LPS and TNF-α, did not significantly enhance this stimulatory capacity. The rapid drop of T-cell stimulation with high DC : T-cell ratios might be explained by a heterogeneity in the DC population. It is possible that the efficient stimulators were only a subset within the heterogeneous non-adherent population. Phenotypic analysis did not identify any clear minor subset with higher MHCII or co-stimulatory molecule expression. Nevertheless, the T-cell stimulatory capacity observed was comparable to that of DC isolated from porcine Peyer's patch,⁵⁸ and also to that of mouse BMHC-derived DC.¹³

Taken together, the present report demonstrates the characteristics of porcine myeloid DC generated in vitro, and the species-related differences in comparative DC biology. This report and other recent publications on porcine DC subsets, including thymic DC,⁵⁹ dermal and epidermal DC,⁶⁰ Peyer's patch DC,⁵⁸ and lamina propria DC,⁵³ are the basis for further characterization within porcine DC immunology. The present porcine in vitro DC models will be particularly useful for studies on the role of DC in the immune response against infectious agents, allo-antigens and xeno-antigens.