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Macrophage-Derived Dendritic Cells Have Strong Th1-Polarizing Potential Mediated by ß-Chemokines Rather Than IL-12¹

Weiping Zou^*, Jozef Borvak^*, Florentina Marches^*, Shuang Wei^*, Pierre Galanaud, Dominique Emilie and Tyler J. Curiel^2,*

^* Baylor Institute for Immunology Research, Dallas, TX 75204; and Institut Paris-Sud Sur Les Cytokines, Institut National de la Santé et de la Recherche Médicale, Clamart, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Monocyte-derived dendritic cells (MDDCs) activate naive T lymphocytes to induce adaptive immunity, effecting Th1 polarization through IL-12. However, little is known about other potential DC Th1 polarizing mechanisms, or how T cell polarization may be affected by DCs differentiating in, or exposed to, a proinflammatory environment. Macrophages (Ms) are DC precursors abundant in inflamed tissues, lymph nodes, and tumors. Thus we studied the T cell-activating and -polarizing properties of M-derived DCs (DCs). Monocytes were cultured in M-CSF (M-CSF) to produce Ms, which were then differentiated into DCs following culture with GM-CSF plus IL-4. DCs activated a significant allogeneic MLR and were significantly better than MDDCs in activating T cells with superantigen. Most strikingly, DCs elicited up to 9-fold more IFN- from naive or Ag-specific T cells compared with MDDCs (with equivalent IL-4 secretion), despite producing up to 9-fold less IL-12. Neutralization of MDDC, but not DC IL-12 significantly inhibited T cell IFN- induction. DCs produced up to 12-fold more ß-chemokines (macrophage-inflammatory protein-1, -1ß, and RANTES) than MDDCs. Ab blockade of CCR5, but not CXC chemokine receptor 4, inhibited T cell IFN- induction by DCs significantly greater than by MDDCs. Thus DCs differentiating from Ms induce T cell IFN- through ß-chemokines with little or no requirement for IL-12. Myeloid DCs arising from distinct precursor cells may have differing properties, including different mechanisms of Th1 polarization. These data are the first reports of IFN- induction through chemokines by DCs.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Monocytes are peripherally circulating myeloid dendritic cell (DC)³ precursor cells. In vitro they differentiate into monocyte-derived dendritic cells (MDDCs) following culture with GM-CSF plus IL-4 (1). However, myeloid differentiation is complex, not fully understood, and subject to external influences (2, 3, 4). Monocytes cultured with macrophage colony-stimulating factor (M-CSF) differentiate into macrophages (Ms), and these Ms will differentiate into DCs upon withdrawal of M-CSF followed by further culture with GM-CSF plus IL-4 (5, 6). Monocytes reverse transmigrating across endothelial barriers differentiate into DCs in the absence of exogenous cytokines (7). Collectively, these myeloid DCs may be referred to as DC1s (8), although this designation generally relates to MDDCs.

Factors impinging upon DC1s during their differentiation may influence the functional phenotype of the differentiated DCs. For example, monocytes exposed to PGE₂ will differentiate into DCs in the presence of GM-CSF plus IL-4 and mature into CD1a⁺CD83⁺ DCs, but are impaired in IL-12 secretion. Exposure to IL-10 inhibits both maturation and IL-12 production (9). Addition of type I IFNs to monocytes differentiating in the presence of GM-CSF plus IL-4 significantly impairs their ability to differentiate into IL-12-secreting, IFN--inducing DCs (10, 11). In contrast, monocytes cultured in GM-CSF plus IFN- secrete more IL-12 compared with MDDCs generated with GM-CSF plus IL-4, resulting in significantly better T cell IFN- induction (12).

Thus, MDDCs are generally considered to be Th1 polarizing, and mediate this effect through secretion of IL-12. Although monocytes differentiating into MDDCs in the presence of PGE₂ induce Th2-polarized immunity (13), MDDC Th1 polarization in the absence of IL-12 has not previously been reported.

Plasmacytoid DCs (referred to as DC2s; Ref. 8) are distinct from myeloid DCs and may be Th0 or Th2 polarizing when freshly isolated from blood, stimulating T cell IL-4 and other cytokines, but little or no IFN- (8, 14). However, following viral infection of plasmacytoid DCs, they induce T cells to produce IFN- (simultaneous with IL-10 in some cases) with little or no IL-4 production (15).

Thus T cell activation by DCs varies according to the type of signals present at the time of activation. Furthermore, all prior work has examined the effects of different signals on the same precursor cell: monocytes in the case of DC1s and pDC2s in the case of DC2s. The effects on DC differentiation of different DC precursor cells have been little studied in the context of both DC1s and DC2s.

We considered that DC differentiation under local conditions of infection, inflammation, or tumor might differ from steady-state DC differentiation owing to different precursor cells of origin, in addition to local factors. Thus we studied Ms as the model local DC1 precursor cell, as they are abundant in inflamed tissues, tumors, and lymph nodes, and compared them to MDDCs as the model steady-state DC1.

We now show that compared with MDDCs, DCs are significantly more efficient in activating T cells, express a distinct pattern of T cell costimulatory molecules and Ag capture receptors, and are significantly more efficient at pinocytosis. Most strikingly, DCs induce T lymphocyte IFN- secretion through a mechanism that involves ß-chemokines with little or no dependence on IL-12. Induced T cell IL-4 secretion is equivalent to MDDCs, demonstrating a Th1-polarizing effect. Thus DCs may use distinct Th1-polarizing mechanisms depending on the precursor cell of origin, which in turn may be influenced by the local microenvironment.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human subjects

These studies were approved by the Institutional Review Board of Baylor University Medical Center. Subjects gave written informed consent for study. Toxoplasma gondii Ab testing of serum was performed in a commercial laboratory.

Generation of M-CSF-derived Ms, MDDCs, and DCs

Recombinant human IL-2 was the generous gift of Hoffman-LaRoche (Nutley, NJ). All other recombinant human cytokines were purchased from R&D Systems (Minneapolis, MN). PBMCs were obtained by Ficoll-Hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation of either heparinized peripheral blood obtained by phlebotomy, or from leukopheresis products obtained without cytokine-induced mobilization of precursor cells, and were frozen at -86°C until use. PBMCs were adhered to plastic plates (Costar, Corning, NY) for 2 h in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated FCS (Life Technologies), 10 mM HEPES buffer, 2 mM L-glutamine, and antibiotics. Nonadherent cells were gently rinsed away and frozen for later use as a source of responder T cells. Ms were produced by culture of adherent PBMCs in 25 ng/ml M-CSF for 4–7 days. M-CSF was replaced every 2 or 3 days. Immature MDDCs were produced by culturing adherent PBMCs in medium containing 25 ng/ml GM-CSF plus 5 ng/ml IL-4 and used on days 5–7 of culture. Fresh cytokines were replaced every 2 or 3 days.

To differentiate DCs from Ms, after 4–7 days of culture of monocytes in M-CSF, medium with M-CSF was removed, cells were washed, and medium with 25 ng/ml GM-CSF plus 5 ng/ml IL-4 was added. IL-13 (5 ng/ml) replaced IL-4 where indicated. Cells were cultured an additional 4–9 days as indicated before use. CD1a-expressing cells were positively selected using goat anti-mouse mAb-coated Miltenyi superparamagnetic microbeads (Miltenyi Biotec, Auburn, CA) coupled to murine anti-human CD1a mAb (BioSource International, Camarillo, CA) according to the manufacturer’s suggestions.

DCs and MDDCs were matured by 2 days of incubation with 1 µg/ml Escherichia coli LPS (Sigma, St. Louis, MO), 200 ng/ml recombinant soluble CD40 ligand (CD40L; Immunex, Seattle, WA), or a combination of 10 ng/ml TNF- plus 10 ng/ml IL-1ß.

Cells were counted by light microscopy in hemacytometer chambers using trypan blue dye. Cytospin slides were prepared by centrifuging 50,000 cells onto glass microscope slides. These were dried, fixed with methanol, stained with Giemsa stain, and visualized by light microscopy.

Responder T lymphocytes

Ag-specific polyclonal T cell lines were generated from T. gondii-seropositive subjects by repeated stimulation of PBMCs with Ag as we previously described (16, 17), except that autologous MDDCs were substituted for PBMCs as the APC. At the time of assays, these polyclonal cultures were comprised of 99% CD3⁺ T lymphocytes, which were a mixed population of both CD4⁺ and CD8⁺ cells by FACS analysis. The specific Ags used were heat inactivated (56°C for 2 h) T. gondii tachyzoites (maintained in human foreskin fibroblasts as described; Ref. 16) or tetanus toxoid (Massachusetts State Board of Health, Jamaica Plain, MA). The Ag specificity of these polyclonal cultures was confirmed by incubation with Ag-charged, autologous MDDCs. T cell cultures were maintained in recombinant human IL-2 at 5–10 U/ml and were used in functional assays 7–10 days following their last stimulation with Ag. Purified CD4⁺CD45RA⁺ naive T cells were obtained from adult PBMCs using Miltenyi paramagnetic microbeads.

Functional analyses

In all comparisons between DCs and MDDCs reported herein, cells were generated from the same donor and assayed at the same time under identical conditions, meaning that in some cases cultures were initiated at different times. T cell proliferation assays were performed in 200 µl total volume in 96-well round-bottom plastic tissue culture plates (Costar) using autologous or allogeneic CD4⁺CD45RA⁺ naive T lymphocytes or Ag-specific polyclonal T cell lines. Staphylococcus aureus enterotoxin B (SEB; Sigma) was used at a final concentration of 10 ng/ml, and tetanus toxoid was used at 4 U/ml. T. gondii tachyzoites were used at 10 per DC or MDDC. DCs or MDDCs were incubated with Ag overnight before coculture with responder T cells. [³H]methylthymidine (1 µCi; New England Nuclear, Boston, MA) was added during the final 8 h of incubation, which was 3 days for SEB and 5 days for other stimuli, and the mean ± SD of triplicate determinations is reported. Murine polyclonal neutralizing Abs to IL-12 (R&D Systems) were added at the time of coculture of DCs and T lymphocytes, at a final concentration of 10 µg/ml. Murine blocking Abs to CXC chemokine receptor (CXCR) 4 or CCR5 (PharMingen, San Diego, CA) were added to T lymphocytes 8 h before they were added to DCs, at a final concentration of 10 µg/ml.

CTL were elicited by incubating DCs with autologous, irradiated (6000 rads from a source) EBV-transformed B lymphocytes overnight, followed by addition to autologous PBMCs at a 1:100 ratio in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, HEPES buffer, glutamine, and antibiotics. Cytolytic activity was assessed 10 days later against autologous and allogeneic ⁵¹Cr-labeled EBV-transformed B lymphocytes as described (16, 18).

To assess pinocytosis, 1 x 10⁵ cells were suspended in 100 µl of medium and incubated 1 h with FITC-dextran (final concentration 200 µg/ml; Molecular Probes, Eugene, OR) at 37°C in a water bath, or at 4°C on ice. Cells were then washed in PBS without calcium or magnesium plus 5 mM EDTA and 2% FCS. FITC-dextran uptake was assessed by FACS. Dead cells were excluded from the analysis gate by the addition of propidium iodide (Sigma) at 4°C 2 min before data acquisition. Data were acquired on a FACScan (Becton Dickinson, Mountain View, CA) and analyzed with CellQuest software (Becton Dickinson). At least 5000 gated events were evaluated for each condition.

Cytokine and chemokine secretion and in situ cytokine production

To determine cytokine or chemokine production by MDDCs or DCs, supernatants were collected from immature DCs or MDDCs activated with CD40L, LPS, or IL-1ß plus TNF-, maintained at a uniform density of 200,000 cells/ml, and frozen at -86°C before analysis. Cytokine or chemokine concentration was determined by ELISA using commercial kits (R&D Systems) according to the manufacturer’s suggestions. The IL-12 ELISA kit we used detects biologically active p70.

To assess cytokine secretion by T cells following activation by MDDCs or DCs, T cells and DCs were cocultured at a 5–50:1 ratio, and supernatants were collected 3 days later, frozen until use, and analyzed by ELISA as described above. To detect in situ cytokine production, mature DCs or MDDCs were charged with 10 ng/ml SEB or specific Ag, and cocultured with autologous CD4⁺CD45RA⁺ naive autologous T cells or Ag-specific autologous polyclonal T cell lines, respectively, for 3 days. T cells were then stimulated with 50 ng/ml PMA (Sigma) plus 500 ng/ml ionomycin (Sigma) for 6 h. Monensin (2 µM; Sigma) was added during the final 2 h. Cells were then stained with murine mAbs against CD4 (FITC-labeled), CD8 (PE-labeled), and CD3 (peridinin chlorophyll protein-labeled), fixed with 1% paraformaldehyde in PBS, or permeabilized, and stained with murine mAbs detecting IL-2 (APC-labeled), IL-4 (FITC-labeled), or IFN- (PE-labeled). Stained cells were analyzed by FACS as described above, counting at least 8000 gated events per condition.

FACS analysis

mAbs were purchased from BioSource International (CD1a), Becton Dickinson (CD3, CD4, CD8, CD11c, CD19, CD25, CD45RO, CD45RA, CD80, MHC class I molecule (DR)), Caltag (Burlingame, CA) (CD14), PharMingen (CD36, CD40, CD51/61, CD86, CD95, HLA A,B,C, _vß₃, IL-2, IL-4, IFN-), Immunotech (Miami, FL) (CD54, CD83), and Dako (Glostrup, Denmark) (CD68). Cells were stained according to the manufacturer’s suggestions and then fixed in paraformaldehyde 1% in PBS. Data were acquired and analyzed as described above, counting at least 5000 cells per condition. Appropriate cell and mAb isotypic controls were included in each analysis, with acquisition and analysis gates set accordingly.

Statistics

Differences between groups were compared by Student’s two-tailed paired or unpaired t test as appropriate, assuming equal variances. Values of p < 0.05 were considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
M-CSF-derived Ms differentiated into DCs following culture in GM-CSF plus IL-4

Monocytes cultured with M-CSF differentiated into typical Ms (Fig. 1, A and D). Forty to fifty percent of these Ms became nonadherent cells morphologically similar to MDDCs in that dendrites were present by days 4–7 of culture in GM-CSF plus IL-4, in agreement with previous work (5). MDDCs comprised a uniform population of nonadherent cells, whereas DC cultures were comprised of an adherent monolayer interspersed with occasional adherent cell clumps and a detached, nonadherent population (Fig. 1, B and C). Ms in parallel cultures are shown for comparison (Fig. 1A). By days 7–9 of culture in GM-CSF plus IL-4, matured nonadherent DCs were larger, more spherical, and had more abundant cytoplasm by Giemsa staining and had longer, finer dendrites compared with MDDCs produced in parallel cultures (Fig. 1, E and F). Because these M-derived cells have DC morphologic characteristics, express CD1a, CD80, CD86, CD83 (upon maturation), and DR, and elicit a significant allogeneic MLR (see sections on activation of naive T cells), they are clearly DCs. We term these M-derived DCs "DCs" (pronounced "phi DCs") to denote their M origin.

View larger version (118K): [in this window] [in a new window]   FIGURE 1. Photomicrographs of Ms (A and D), DCs (B and E), and MDDCs (C and F) in culture. A–C, Light microscopic images of live cells in culture by inverted phase microscopy on day 6. D–F, Giemsa-stained cells from the same cultures following preparation on a cytospin slide. MDDCs and DCs for the photographs (D–F) were the nonadherent fraction following two additional days of culture (8 days total) with E. coli LPS to induce maturation. Adherent day 8 Ms were removed following treatment with trypsin. Original magnification x200 for inverted phase microscopy and x400 for Giemsa-stained cells.

DCs expressed typical myeloid DC surface molecules, but significantly more CD54, CD80, CD86, and MHC class I compared with MDDCs, and some continued to coexpress CD14

Nonadherent cells in cultures of Ms from days 6–9 in GM-CSF plus IL-4 were a heterogeneous population of cells, variably 40–70% CD1a⁺ and 30–60% CD1a^-. The nonadherent cells in the culture were referred to as immature DCs. For FACS analyses, data were collected on cells gated for CD1a expression. For MLR and other analyses, CD1a⁺ cells were purified (>90% purity) from the immature population using Miltenyi paramagnetic microbeads. These immature DCs expressed up to 9-fold more CD14 than immature MDDCs (Table I). Further culture in GM-CSF plus IL-4 beyond 9 days did not result in additional nonadherent DC formation, decreased CD14 expression, or increased CD1a expression. Thus immature DCs were studied between days 6 and 8 of culture in GM-CSF plus IL-4 (days 10–13 of total culture). Under these conditions, 40–70% of input Ms differentiated into nonadherent DCs. Substitution of IL-13 for IL-4 effected comparable differentiation of Ms into DCs, and culture of Ms in GM-CSF alone or with TNF-, but without IL-4, did not effect DC differentiation (data not shown). The adherent cells in these cultures morphologically resembled Ms, were CD1a^lo/^-CD14⁺DR^lo by FACS, and were significantly less efficient than nonadherent cells in eliciting an allogeneic MLR.

View this table: [in this window] [in a new window]   Table I. Ag/apoptotic body capture receptor expression on MDDCs and DCs

View larger version (56K): [in this window] [in a new window]   FIGURE 2. Comparison of the FACS profiles of LPS-matured MDDCs or DCs. Both MDDCs and DCs were cultured for 6 days in GM-CSF plus IL-4, and maturation was then induced by two additional days of culture following addition of 1 µg/ml E. coli LPS. Legends in bold represent molecules expressed at significantly different intensities (p < 0.05) by mature DCs compared with mature MDDCs. CD14, CD54, CD80, CD86, and MHC class I (HLA-ABC) were all expressed at significantly higher intensities by DCs compared with MDDCs. Although 95–100% of MDDCs and DCs expressed CD1a, DCs expressed a mean 0.5 log10 lower intensity of staining (p < 0.05), and 10–25% continued to coexpress CD14. Expression of CD4 and CD51/61 is not shown here, but was equivalent between MDDCs and DCs. Data shown were derived from cells obtained from a single donor and are representative of similar studies of five individuals with similar results.

The apoptotic body/Ag capture receptors CD14, CD36, and _vß₃ were expressed on a significantly higher proportion of DCs compared with MDDCs. _vß₅ expression on MDDCs and DCs was similar.

For these experiments, maturation was induced by incubation with LPS only. Results are shown in Table I. Immature DCs and MDDCs both down-regulated CD14, CD36, and _vß₅ following maturation with LPS, although baseline expression and degree of change in expression following maturation differed considerably between MDDCs and DCs. Immature DCs expressed almost twice as much CD36 as immature MDDCs, although both down-regulated this receptor to equivalent levels following maturation. _vß₅ expression by immature and mature MDDCs and DCs was similar and high. Mature DCs expressed significantly (8.5-fold) more _vß₃ than mature MDDCs. Ten to 25% (mean 14%) of DCs remained dually CD1a⁺CD14⁺ following maturation with LPS, which is 4- to 10-fold higher than MDDCs (Table I, and data not shown).

DCs efficiently activated allogeneic, naive CD4⁺CD45RA⁺ T cells (this activation was comparable to that effected by MDDCs)

DCs matured with LPS effected significant proliferation of naive CD4⁺CD45RA⁺ T cells in an allogeneic MLR, which was significantly stronger than control Ms as expected and comparable to that elicited by mature MDDCs (Fig. 3A). Similar results were obtained when DCs were matured with soluble, recombinant CD40L or with the combination of TNF- plus IL-1-ß (data not shown).

View larger version (47K): [in this window] [in a new window]   FIGURE 3. T cell activation by MDDCs compared with DCs. In all of the experiments shown, cultures were staggered so that MDDCs were compared with autologous DCs on the same day using the same responder cells. A, Induction of an allogeneic MLR was equivalent using MDDCs or DCs. LPS-matured MDDCs or DCs were incubated with 50,000 CD4+CD45RA+ naive, allogeneic T cells for 4 days, and 1 µCi [3H]methylthymidine was added during the final 8 h. Results are expressed as the mean ± SD of triplicate determinations. One representative experiment of >15 from five individuals is shown. B, DCs incubated with the superantigen SEB consistently induced significantly more naive T cell activation as measured by [3H]methylthymidine incorporation compared with autologous MDDCs. Experiments were set up as described above except that autologous T cells were used and MDDCs or DCs were added together with 10 ng/ml SEB. One representative experiment of >15 from five individuals is shown. Significantly greater T cell proliferation was effected by DCs compared with MDDCs at all three DC:T cell ratios studied in all five subjects studied. *, # Values of p are <0.05 for each of the determinations shown here. C, T. gondii-specific T cell line. D, TT-specific T cell line. C and D, DCs present specific Ag to T cells. Autologous DCs were charged with Ag, matured with LPS, and incubated with polyclonal T cell cultures for 5 days. T cell proliferation was determined by [3H]methylthymidine incorporation as described. One representative experiment of three is shown.

DCs were significantly more effective than MDDCs at autologous T cell activation by SEB

LPS-matured DCs induced superantigen-mediated autologous T cell proliferation significantly better than MDDCs as measured by T cell proliferation (Fig. 3B) and cytokine production (see below). Both DCs and MDDCs were significantly more potent than Ms in eliciting allogeneic and autologous T cell activation as expected (maximum M-induced proliferation was <2000 cpm in an allogeneic MLR and <4000 cpm for presentation of SEB).

DCs were potent at eliciting Ag-specific T cell proliferation

Immature day 6 DCs were incubated overnight with tetanus toxoid or with T. gondii tachyzoites, matured with LPS, and cultured with autologous, polyclonal Ag-specific T cells. Mature DCs were significant activators of Ag-specific T cells as measured by T cell proliferation (Fig. 3, C and D) and cytokine production (see below). These data also confirm the Ag specificity of these polyclonal T cell lines.

Following activation, DCs and MDDCs secreted comparable amounts of IL-10, but MDDCs secreted up to 9-fold more IL-12 p70

Baseline secretion of IL-10 or IL-12 p70 was undetectable by ELISA in MDDCs and DCs. MDDCs or DCs were matured by incubation for 2 days with recombinant, soluble CD40L, LPS, or IL-1ß plus TNF-. Following maturation, DCs produced levels of IL-10 that were comparable to those produced by MDDCs, but produced up to 9-fold less IL-12 p70, no matter which of the three activation signals was used (Table II).

View this table: [in this window] [in a new window]   Table II. IL-10 and IL-12 p70 production by MDDCs and DCs1

DCs elicited significantly more IFN- secretion from T cells in response to SEB or specific Ag compared with MDDCs despite producing up to 9-fold less IL-12 p70

LPS-matured DCs incubated with SEB induced significantly more IFN- secretion from autologous CD4⁺CD45RA⁺ T cells than did MDDCs (Fig. 4A). To determine whether this strong Th1-polarizing influence also occurred in an Ag-specific manner, we used DCs expressing either tetanus toxoid or T. gondii Ags. By in situ cytokine detection, a significantly greater proportion of T cells expressed IFN- in response to coculture with Ag-expressing DCs compared with Ag-expressing MDDCs (77 ± 15% vs 37 ± 8%; p < 0.001) (representative data in Fig. 4B). DCs also elicited significantly more T cell IL-2 than MDDCs (20 ± 5% vs 12 ± 3%; p < 0.05). Elicited T cell IL-4 was equivalent and <5% using either DCs or MDDCs. Data here are the mean ± SD of three independent experiments. Similar results were obtained using SEB stimulation of autologous naive CD4⁺CD45RA⁺ T cells, and in an allogeneic MLR (data not shown).

View larger version (39K): [in this window] [in a new window]   FIGURE 4. DCs elicit significantly more (up to 9-fold) T cell IFN- secretion and induce a significantly greater proportion of T cells to produce IFN- compared with MDDCs. A, Naive CD4+CD45RA+ T cells were induced to express cytokines using SEB presented either on autologous MDDCs or DCs. Supernatants were assayed for IFN- secretion by ELISA 4 days later. Each symbol connected by a line represents the same donor assayed at the same time, demonstrating the consistency of this observation. B, DCs or MDDCs were incubated with T. gondii Ags and matured for 2 days with LPS before culture with an autologous T. gondii-specific T cell line. In this experiment, the cell line contained 70% CD4+ T cells and 30% CD8+ T cells. A single experiment representative of three similar experiments with similar results is shown. C, DCs elicit IFN- secretion from CD8+ T lymphocytes in an Ag-specific manner. Autologous DCs were loaded with T. gondii Ags and used to elicit IFN- production from autologous, polyclonal T. gondii-specific CD8+ T lymphocytes. IFN- production using autologous DCs loaded with irrelevant TT Ag was <20%. One experiment representative of two is shown.

DCs activate Ag-specific CD8⁺ T lymphocytes

Although we were primarily interested in evaluating the effects of DCs on CD4⁺ T lymphocytes, we also asked whether DCs could activate Ag-specific CD8⁺ T lymphocytes. DCs elicited up to 26% specific cytotoxicity against autologous EBV-transformed B lymphocytes in polyclonal T cell cultures at an E:T ratio of 60:1, and induced IFN- secretion in autologous polyclonal cultures of T. gondii-specific CD8⁺ T lymphocytes in an Ag-specific manner (Fig. 4C).

Induction of T lymphocyte IFN- by DCs requires little or no IL-12

To assess for factors inducing T lymphocyte IFN- secretion, T. gondii Ag-charged DCs or MDDCs were incubated with autologous polyclonal cultures of T. gondii-specific T lymphocytes. Addition of anti-IL-12-neutralizing Ab significantly diminished T cell IFN- induction by MDDCs as expected, whereas there was no significant effect on induction of T cell IFN- by DCs (Fig. 5A). Anti-IL-12-neutralizing Ab also diminished T cell proliferation mediated by both DCs and MDDCs, although there was significantly more diminution with MDDCs (Fig. 5B).

View larger version (45K): [in this window] [in a new window]   FIGURE 5. Neutralization of IL-12 with a polyclonal Ab inhibits MDDC, but not DC-induced T cell IFN- production. A, Numbers within the quadrants are the percentages of cells in that quadrant. A single experiment representative of three is shown. B, Neutralizing anti-IL-12 Ab reduced T lymphocyte proliferation elicited by either DCs or MDDcs in an allogeneic MLR. However, suppression of MDDC-mediated T lymphocyte proliferation was significantly greater (p < 0.01 compared with DCs).

DCs secrete up to 12-fold more ß-chemokines following LPS activation than MDDCs

As ß-chemokines may be Th1 polarizing (19, 20, 21, 22, 23, 24) and may be produced by DCs (25, 26, 27), we determined the relative secretion of ß-chemokines by DCs and MDDCs by ELISA of cell culture supernatants following their maturation with LPS. DCs secreted a mean of up to 12-fold more macrophage-inflammatory protein (MIP)-1, -1ß, and RANTES compared with MDDCs (Fig. 6A). In addition to the higher mean production of ß-chemokines between DCs and MDDCs, DCs of each individual secreted more ß-chemokines compared with autologous MDDCs, demonstrating a consistent effect from subject to subject.

View larger version (44K): [in this window] [in a new window]   FIGURE 6. DCs secrete up to 12-fold more of the ß-chemokines MIP-1, MIP-1ß, and RANTES compared with MDDCs following maturation with E. coli LPS. A, ß-chemokine concentrations in culture supernatants after 2 days. Lines connect autologous MDDCs to DCs, demonstrating consistently higher ß-chemokine production in each individual subject studied using DCs (n = 7). B, Blockade of T lymphocyte CCR5, but not CXCR4, significantly reduced (p < 0.05) DC-mediated IFN- induction in an allogeneic MLR comparing anti-CCR5 to control (without Abs). One experiment representative of three is shown.

A CCR5 ligand mediates DC T cell IFN- induction

To confirm the role of ß-chemokines in T cell IFN- induction, we assessed the effect of a blocking Ab against the ß-chemokine receptor CCR5. CCR5 blockade inhibited DC-mediated T cell IFN- induction by up to 50% (p < 0.01), whereas blocking Ab against the CXCR4 had no significant effect (Fig. 6B). Anti-CCR5 (but not anti-CXCR4) blocking Ab slightly reduced MDDC-mediated T cell IFN- production, but to a significantly lesser extent than that observed with DCs (Fig. 6B, and data not shown).

Pinocytosis by DCs is significantly greater than that effected by MDDCs or Ms

Cells in cultures of immature DCs effected significantly more pinocytosis of FITC-dextran than MDDCs or Ms, displaying a 0.5 log₁₀ higher mean fluorescence intensity (n = 5; p < 0.05) following ingestion of FITC-dextran (representative data are shown in Fig. 7). Following maturation, DCs and MDDCs were equally poor in pinocytosing FITC-dextran, consistent with a mature DC functional phenotype.

View larger version (26K): [in this window] [in a new window]   FIGURE 7. FITC-dextran uptake mediated by immature DCs is significantly greater than that effected by MDDCs. One hundred thousand DCs or MDDCs were incubated with 200 µg/ml FITC-dextran for 1 h. Uptake at 37°C was compared with uptake at 4°C (control). M pinocytosis of FITC-dextran was significantly lower than MDDCs or DCs (data not shown). One experiment representative of 10 is shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We first demonstrated that DCs and MDDCs both expressed equivalent amounts of the myeloid differentiation marker CD11c as well as other molecules typically associated with DC1s including CD1a, CD4, CD83 (following maturation), and HLA DR. However, DCs expressed significantly higher levels of T cell activation molecules and apoptotic body/Ag capture receptors than MDDCs, suggesting that they are specialized to be efficient in Ag capture and subsequent T cell activation. This phenotype translated into significantly more efficient activation of T cells compared with MDDCs following presentation of superantigen to naive T lymphocytes, or specific Ag to Ag-specific autologous T lymphocytes. To what extent the enhanced expression of T cell costimulatory molecules contributes to efficient T cell activation is not completely defined. However, as activation of an allogeneic MLR was equivalent by either MDDCs or DCs, additional factors are likely to be involved.

Immature MDDCs express a variety of Ag capture receptors including CD14, CD36, _vß₃, and _vß₅, which are all involved in the capture of apoptotic bodies (30, 31, 32). These are typically down-regulated upon MDDC maturation at the same time as the immature MDDC loses its ability to acquire Ag and up-regulates its ability to present acquired Ag to T cells (28). Most of these well-characterized Ag capture receptors were expressed at significantly higher levels on DCs compared with MDDCs. However, the specific functional consequences have not yet been defined. The continued expression of CD14 on a subpopulation of matured DCs may reflect their lack of complete differentiation in these cultures.

As expected, immature DCs were avidly pinocytotic of FITC-dextran but lost this capacity following maturation, a functional DC hallmark. Unexpectedly, DCs pinocytosed significantly more FITC-dextran than MDDCs. Efficient pinocytosis by DCs may be due to their larger size, more efficient pinocytosis, or a combination of factors. Enhanced pinocytosis may make DCs highly proficient at capturing soluble Ags compared with MDDCs.

In addition to activation of naive CD4⁺ T lymphocytes, DCs also activate CD8⁺ T lymphocytes. DCs induced Ag-specific CTLs as assessed by Ag-specific target cell cytotoxicity, and induced IFN--producing CD8⁺ T lymphocytes following Ag-specific activation.

To investigate activation of T cells further, we studied biologically active IL-12 p70 secretion by DCs and MDDCs. Despite culture with optimal amounts of three different agents known to induce IL-12 secretion from DCs, DCs produced up to 9-fold less IL-12 p70 than MDDCs. Human Ms cultured in M-CSF are deficient in IL-12 production (33). Thus, the deficient IL-12 production of these DCs compared with MDDCs may be attributable in part to an M-CSF effect on the M precursor. If so, then IL-12 secretion by the mature DC may be dictated at the level of the DC precursor cell, which is earlier than at the level of the immature DC as previously reported (34).

IFN- is a potent Th1-inducing cytokine, which is produced by T cells in response to IL-12 secretion from DCs (35, 36, 37). DCs induced significant T cell proliferation and up to 9-fold more T cell IFN- secretion compared with MDDCs, despite producing up to 9-fold less IL-12. They also appear to be Th1 polarizing, as they do not induce significant T cell IL-4 production under these conditions. Neutralization of MDDC IL-12 significantly inhibited T cell IFN- induction, but had little effect on DC-induced T cell IFN-. Thus, it appeared that IL-12 was not primarily responsible for the potent IFN- inducing potential of DCs.

ß-chemokines may be Th1 polarizing or associated with Th1-polarized immunity in some instances (19, 20, 21, 22, 23, 24), and are DC products (25, 26, 27). Thus we investigated the production of the ß-chemokines MIP-1, -1ß, and RANTES, and determined that DCs secreted up to 12-fold more than MDDCs in response to LPS activation. Neutralization of these ß-chemokines did not significantly reduce DC-mediated T cell IFN-. However, blockade of the ß-chemokine receptor CCR5, but not the CXCR4, significantly reduced DC-mediated T cell IFN- induction. Anti-CCR5 Ab also reduced the mean fluorescence intensity of IFN- in DC-stimulated T cells by 59% compared with only 19% for MDDC-stimulated T lymphocytes (p < 0.01 for the comparison of DCs to MDDCs), demonstrating that IFN- production by individual cells was also diminished and suggesting that ß-chemokines may also play some role in MDDC-mediated T lymphocyte IFN- induction.

As MIP-1, -1ß, and RANTES are the only currently known ligands for CCR5 (21), these data strongly suggest that DCs induce T cell IFN- at least in part through secretion of ß-chemokines, with little or no dependence on IL-12. Inability to inhibit IFN- completely through neutralization of ß-chemokines likely relates to the enormous quantities of ß-chemokines produced in the cultures. These data also represent the first demonstration of IL-12-independent T cell IFN- induction by DC1s, and the first T cell IFN- induction by DC ß-chemokines to our knowledge. However, it is possible that an as yet unknown CCR5 ligand may mediate some of these effects. Furthermore, the specific roles of individual ß-chemokines and whether other factors cooperate with ß-chemokines in T cell IFN- induction by DCs require further investigation, as does determination of the relative efficiency of IL-12 compared with ß-chemokines in IFN- induction.

DC2s, which are not reported to secrete IL-12 (8, 15, 38), likewise induce T cell IFN- following viral infection (15). The mechanism of this IFN- induction has not yet been reported.

Regarding the T cell IFN- induced, it may be increased because more cells are producing it, because more is made on a per cell basis, or both. In our analyses of in situ production, 130–200% more individual T cells were positive for IFN- production when activated by DCs compared with MDDCs, whereas the supernatants collected at the same time contained 2- to 9-fold more IFN- protein. These data suggest that DCs induced more cells to produce IFN- and that more was produced per cell compared with MDDCs. Our data further demonstrate that IFN- production is induced by DCs from both CD4⁺ and CD8⁺ T lymphocytes.

DCs exhibit functional attributes (augmented Ag capture receptor expression; avid pinocytosis; high T cell costimulatory molecule expression; enhanced capacity to activate T cells and induce IFN- secretion, little or no IL-12 requirement for IFN- induction, and high-level ß chemokine secretion), which may be exploited in experimental therapeutics and may be important in vivo. We have confirmed that human in vivo Ms also differentiate into DCs and share some similarities with DCs derived from in vitro M-CSF-derived Ms (J. Borvak, W. Zov, A. Gordon, T. Pustilnik, T. Isaeva, and T. Curiel, manuscript in preparation). Their further study merits attention.

The functional capabilities and immune-mediating potential of DCs differentiated from distinct precursor cells may differ depending on the precursor cell in addition to the microenvironment. We now show that distinct DC1 precursor cells contribute to differences in DC1 function following their differentiation under identical conditions. This differential functional capacity includes reduced or absent requirements for IL-12 to induce T cell IFN- induction, which in the case of DCs appears to be mediated at least in part by ß-chemokines (or an as yet unidentified CCR5 ligand).

	Acknowledgments

 
We thank Elizabeth Kraus for excellent technical assistance with flow cytometry. Recombinant human IL-2 was the generous gift of Hoffman-LaRoche. Soluble, recombinant human CD40L was the generous gift of Immunex.

	Footnotes

 
¹ This work was supported by R01AI39379 and the Baylor Endowment (to T.J.C.).

² Address correspondence and reprint requests to Dr. Tyler J. Curiel, Baylor Institute for Immunology Research, 3434 Live Oak Street, Suite 205, Dallas, TX 75204.

³ Abbreviations used in this paper: DC, dendritic cell; M, macrophage; DC (pronounced "phi DC"), macrophage-derived dendritic cell; M-CSF, macrophage CSF; MDDC, monocyte-derived dendritic cell; SEB, Staphylococcus aureus enterotoxin B; CXCR, CXC chemokine receptor; DR, class II MHC molecule; CD40L, CD40 ligand; MIP, macrophage-inflammatory protein.

Received for publication February 25, 2000. Accepted for publication July 25, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Sallusto, F., A. Lanzavecchia. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor . J. Exp. Med. 179:1109.[Abstract/Free Full Text]
2. Oehler, L., O. Majdic, W. F. Pickl, J. Stockl, E. Riedl, J. Drach, K. Rappersberger, K. Geissler, W. Knapp. 1998. Neutrophil granulocyte-committed cells can be driven to acquire dendritic cell characteristics. J. Exp. Med. 187:1019.[Abstract/Free Full Text]
3. Geissmann, F., C. Prost, J. P. Monnet, M. Dy, N. Brousse, O. Hermine. 1998. Transforming growth factor ß1, in the presence of granulocyte/macrophage colony-stimulating factor and interleukin 4, induces differentiation of human peripheral blood monocytes into dendritic Langerhans cells. J. Exp. Med. 187:961.[Abstract/Free Full Text]
4. Akagawa, K. S., N. Takasuka, Y. Nozaki, I. Komuro, M. Azuma, M. Ueda, M. Naito, K. Takahashi. 1996. Generation of CD1⁺RelB⁺ dendritic cells and tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated giant cells from human monocytes. Blood 88:4029.[Abstract/Free Full Text]
5. Palucka, K. A., N. Taquet, F. Sanchez-Chapuis, J. C. Gluckman. 1998. Dendritic cells as the terminal stage of monocyte differentiation. J. Immunol. 160:4587.[Abstract/Free Full Text]
6. Zou, W., S. Wei, J. Borvak, F. Marches, T. Curiel. 1999. Properties of dendritic cells produced from in vitro or in vivo macrophages. Blood 94:208a.
7. Randolph, G. J., S. Beaulieu, S. Lebecque, R. M. Steinman, W. A. Muller. 1998. Differentiation of monocytes into dendritic cells in a model of transendothelial trafficking. Science 282:480.[Abstract/Free Full Text]
8. Rissoan, M. C., V. Soumelis, N. Kadowaki, G. Grouard, F. Briere, R. de Waal Malefyt, Y. J. Liu. 1999. Reciprocal control of T helper cell and dendritic cell differentiation. Science 283:1183.[Abstract/Free Full Text]
9. Kalinski, P., J. H. Schuitemaker, C. M. Hilkens, M. L. Kapsenberg. 1998. Prostaglandin E₂ induces the final maturation of IL-12-deficient CD1a⁺CD83⁺ dendritic cells: the levels of IL-12 are determined during the final dendritic cell maturation and are resistant to further modulation. J. Immunol. 161:2804.[Abstract/Free Full Text]
10. McRae, B. L., R. T. Semnani, M. P. Hayes, G. A. van Seventer. 1998. Type I IFNs inhibit human dendritic cell IL-12 production and Th1 cell development. J. Immunol. 160:4298.[Abstract/Free Full Text]
11. McRae, B. L., T. Nagai, R. T. Semnani, J. M. van Seventer, G. A. van Seventer. 2000. Interferon- and -ß inhibit the in vitro differentiation of immunocompetent human dendritic cells from CD14⁺ precursors. Blood 96:210.[Abstract/Free Full Text]
12. Santini, S. M., C. Lapenta, M. Logozzi, S. Parlato, M. Spada, T. Di Pucchio, F. Belardelli. 2000. Type I interferon as a powerful adjuvant for monocyte-derived dendritic cell development and activity in vitro and in Hu-PBL-SCID mice. J. Exp. Med. 191:1777.[Abstract/Free Full Text]
13. Kalinski, P., C. M. Hilkens, A. Snijders, F. G. Snijdewint, M. L. Kapsenberg. 1997. Dendritic cells, obtained from peripheral blood precursors in the presence of PGE₂, promote Th2 responses. Adv. Exp. Med. Biol. 417:363.[Medline]
14. Cella, M., D. Jarrossay, F. Facchetti, O. Alebardi, H. Nakajima, A. Lanzavecchia, M. Colonna. 1999. Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon. Nat. Med. 5:919.[Medline]
15. Kadowaki, N., S. Antonenko, J. Y.-N. Lau, Y.-J. Liu. 2000. Natural interferon /ß-producing cells link innate and adaptive immunity. J. Exp. Med. 192:219.[Abstract/Free Full Text]
16. Curiel, T. J., E. C. Krug, M. B. Purner, P. Poignard, R. L. Berens. 1993. Cloned human CD4⁺ cytotoxic T lymphocytes specific for Toxoplasma gondii lyse tachyzoite-infected target cells. J. Immunol. 151:2024.[Abstract]
17. Purner, M. B., E. C. Krug, P. Nash, D. R. Cook, R. L. Berens, T. J. Curiel. 1995. Cross-reactivity of human Toxoplasma-specific T cells: implications for development of a potential immunotherapeutic or vaccine. J. Infect. Dis. 171:984.[Medline]
18. Curiel, T. J., J. T. Wong, P. F. Gorczyca, R. T. Schooley, B. D. Walker. 1993. CD4⁺ human immunodeficiency virus type 1 (HIV-1) envelope-specific cytotoxic T lymphocytes derived from the peripheral blood cells of an HIV-1-infected individual. AIDS Res. Hum. Retroviruses 9:61.[Medline]
19. Lu, Y., K. Q. Xin, K. Hamajima, T. Tsuji, I. Aoki, J. Yang, S. Sasaki, J. Fukushima, T. Yoshimura, S. Toda, E. Okada, K. Okuda. 1999. Macrophage inflammatory protein-1 (MIP-1) expression plasmid enhances DNA vaccine-induced immune response against HIV-1. Clin. Exp. Immunol. 115:335.[Medline]
20. Kim, J. J., L. K. Nottingham, J. I. Sin, A. Tsai, L. Morrison, J. Oh, K. Dang, Y. Hu, K. Kazahaya, M. Bennett, T. Dentchev, D. M. Wilson, A. A. Chalian, J. D. Boyer, M. G. Agadjanyan, D. B. Weiner. 1998. CD8 positive T cells influence antigen-specific immune responses through the expression of chemokines. J. Clin. Invest. 102:1112.[Medline]
21. Rollins, B. J.. 1997. Chemokines. Blood 90:909.[Free Full Text]
22. Trumpfheller, C., K. Tenner-Racz, P. Racz, B. Fleischer, S. Frosch. 1998. Expression of macrophage inflammatory protein (MIP)-1, MIP-1ß, and RANTES genes in lymph nodes from HIV⁺ individuals: correlation with a Th1-type cytokine response. Clin. Exp. Immunol. 112:92.[Medline]
23. Schrum, S., P. Probst, B. Fleischer, P. F. Zipfel. 1996. Synthesis of the CC-chemokines MIP-1, MIP-1ß, and RANTES is associated with a type 1 immune response. J. Immunol. 157:3598.[Abstract]
24. Kennedy, K. J., W. J. Karpus. 1999. Role of chemokines in the regulation of Th1/Th2 and autoimmune encephalomyelitis. J Clin. Immunol. 19:273.[Medline]
25. Sallusto, F., B. Palermo, D. Lenig, M. Miettinen, S. Matikainen, I. Julkunen, R. Forster, R. Burgstahler, M. Lipp, A. Lanzavecchia. 1999. Distinct patterns and kinetics of chemokine production regulate dendritic cell function. Eur. J. Immunol. 29:1617.[Medline]
26. Rossi, D., A. Zlotnik. 2000. The biology of chemokines and their receptors. Annu. Rev. Immunol. 18:217.[Medline]
27. Zlotnik, A., O. Yoshie. 2000. Chemokines: a new classification system and their role in immunity. Immunity 12:121.[Medline]
28. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245.[Medline]
29. Gluckman, J. C., B. Canque, F. Chapuis, M. Rosenzwajg. 1997. In vitro generation of human dendritic cells and cell therapy. Cytokines Cell. Mol. Ther. 3:187.[Medline]
30. Albert, M. L., S. F. Pearce, L. M. Francisco, B. Sauter, P. Roy, R. L. Silverstein, N. Bhardwaj. 1998. Immature dendritic cells phagocytose apoptotic cells via _vß₅ and CD36, and cross-present antigens to cytotoxic T lymphocytes. J. Exp. Med. 188:1359.[Abstract/Free Full Text]
31. Devitt, A., O. D. Moffatt, C. Raykundalia, J. D. Capra, D. L. Simmons, C. D. Gregory. 1998. Human CD14 mediates recognition and phagocytosis of apoptotic cells. Nature 392:505.[Medline]
32. Fadok, V. A., M. L. Warner, D. L. Bratton, P. M. Henson. 1998. CD36 is required for phagocytosis of apoptotic cells by human macrophages that use either a phosphatidylserine receptor or the vitronectin receptor (_vß₃). J. Immunol. 161:6250.[Abstract/Free Full Text]
33. Smith, W., M. Feldmann, M. Londei. 1998. Human macrophages induced in vitro by macrophage colony-stimulating factor are deficient in IL-12 production. Eur. J. Immunol. 28:2498.[Medline]
34. Kalinski, P., C. M. Hilkens, A. Snijders, F. G. Snijdewint, M. L. Kapsenberg. 1997. IL-12-deficient dendritic cells, generated in the presence of prostaglandin E₂, promote type 2 cytokine production in maturing human naive T helper cells. J. Immunol. 159:28.[Abstract]
35. Wu, C. Y., C. Demeure, M. Kiniwa, M. Gately, G. Delespesse. 1993. IL-12 induces the production of IFN- by neonatal human CD4 T cells. J. Immunol. 151:1938.[Abstract]
36. Trinchieri, G.. 1998. Immunobiology of interleukin-12. Immunol. Res. 17:269.[Medline]
37. Sousa, C. R., S. Hieny, T. Scharton-Kersten, D. Jankovic, H. Charest, R. N. Germain, A. Sher. 1997. In vivo microbial stimulation induces rapid CD40 ligand-independent production of interleukin 12 by dendritic cells and their redistribution to T cell areas. J. Exp. Med. 186:1819.[Abstract/Free Full Text]
38. Kohrgruber, N., N. Halanek, M. Groger, D. Winter, K. Rappersberger, M. Schmitt-Egenolf, G. Stingl, D. Maurer. 1999. Survival, maturation, and function of CD11c^- and CD11c⁺ peripheral blood dendritic cells are differentially regulated by cytokines. J. Immunol. 163:3250.[Abstract/Free Full Text]

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