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IMMUNOBIOLOGY
From the Department of Surgery and the Harrison
Surgical Research Center, University of Pennsylvania, Philadelphia; the
Frederick Cancer Research and Development Center, National Cancer
Institute, MD; and the Center for Surgery Research, Cleveland Clinic
Foundation, OH.
Mature dendritic cells (DCs), in addition to providing
costimulation, can define the Th1, in contrast to the Th2, nature of a
T-cell response through the production of cytokines and chemokines. Because calcium signaling alone causes rapid DC maturation of both
normal and transformed myeloid cells, it was evaluated whether calcium-mobilized DCs polarize T cells toward a Th1 or a Th2 phenotype. After human monocytes were cultured for 24 hours in serum-free medium
and granulocyte-macrophage colony-stimulating factor to produce
immature DCs, additional overnight culture with either calcium
ionophore (CI) or interferon Dendritic cells (DCs) are responsible for priming
naive T lymphocytes.1-4 Immature DCs reside in peripheral
tissues and sample environmental antigens. After exposure to
"danger" signals or to various inflammatory signals, such as
interleukin-1 (IL-1), tumor necrosis factor- T-cell-dependent immune responses are often polarized in favor of T
cells that secrete high levels of interferon The induction of Th2 responses by DC2 may play a critical
role in several immunologically mediated disease states.
DC2 may activate Th2 cells that can prevent or decrease
graft-versus-host disease (GVHD),22,23 play a role in
regulating autoimmunity,24,25 and be involved in
preventing the rejection of solid organ transplants.26 Cytokines such as granulocyte colony-stimulating factor (G-CSF) appear
to increase DC2 precursor cells in the peripheral blood of
patients undergoing allogeneic transplantation, which may result in
decreased GVHD.27,28 It would be useful to explore the
ability to regulate DC2 activity for the treatment of
diseases thought to be mediated by excessive Th1 polarization, such as
GVHD and other autoimmune diseases.
We have previously shown that calcium signaling results in the
development of mature DCs29,30 that express high levels of
costimulatory molecules and CD83 and that present antigens to
naive T cells. Calcium signaling in myeloid precursor cells and chronic
myeloid leukemia cells also induces activation into mature
DCs.31,32 Many signal-transducing pathways, such as Fc
receptor or complement receptor binding, lead to calcium fluxes within monocytes and macrophages33 through the calcium
capacitance channel34-36 or from mobilization from
intracellular stores. Calcium signaling, therefore, appears to be
highly integrated into the regulation of DC activity. In the current
study we sought to determine whether calcium signaling in mature
myeloid-derived DCs differentially regulates a T-cell response toward
Th1 or Th2 development. We report here that calcium signaling can
promote mature DC2 that induces Th2/Tc2-type responses and
that selectively antagonizes the induction of IL-12 production by
DC1-promoting agents.
Preparation of human peripheral blood mononuclear cell
fractions
Reagents and antibodies
Preparation of T-lymphocyte subsets One hundred twenty to 140 lymphocyte-rich elutriation fractions were used to prepare CD4+ CD45RO T cells
using human T-cell subset columns (R&D Systems, Minneapolis, MN) by
quantitative negative-depletion techniques, as recommended by the
manufacturer. CD8+ T cells from HLA A2.1+
donors were isolated through the same technique using the
appropriate columns.
Monocyte cultures One hundred fifty to 190 monocyte-rich fractions were used fresh or thawed and were plated at 1.5 × 106 cells/mL in either 24- or 48-well Costar cluster plates (Corning, Corning, NY). The cells were cultured in macrophage-monocyte medium supplemented with 50 ng/mL GM-CSF. After overnight culture (14-24 hours) in GM-CSF, the cells were matured as follows: either CI (150 ng/mL) alone was added to the culture for an additional 20 hours or IFN- (1000 U/mL) and
TNF- (10 ng/mL) were added and maturation was completed 6 hours
later with the addition of CD40L at 1 µg/mL. Cells were cultured for
an additional 14 hours. Activated DCs from both groups were harvested
40 hours after their initial placement in culture. Cells were harvested
for coculture and fluorescence-activated cell sorter (FACS) analysis,
and supernatants were harvested for enzyme-linked immunosorbent assay
(ELISA) 14 to 24 hours after the addition of CI or CD40L. DCs were
washed 2 to 3 times before coculture with T cells. In experiments in
which IL-12 was added to DCs during activation with CI, the DCs were
washed 3 times before coculture with T cells.
Cell surface FACS analysis Elutriated fraction cells were analyzed by fluorescence multicolor flow cytometry (FACScan; Becton Dickinson, Mountain View, CA). Cells were stained at 4°C using Ca++/Mg++-free HBSS with 1% fetal bovine serum and 0.1% sodium azide as a diluent/wash buffer. Cells were preincubated with unconjugated goat-globulin (Sigma Chemical) for
10 minutes to block Fc receptor binding and, in most cases, were double
stained with PE- and FITC-conjugated antibodies for 30 minutes. After
washing with cold buffer, propidium iodide (PI) was added to
distinguish viable from nonviable cells, and 3-color analysis was
performed using a 5-W argon laser emitting 200 mW of 488-nm light.
Simultaneous measurement of fluorescein, PE, and PI emission was made
using 530, 575, and 650 filters with acquisition in logarithmic mode.
High PI-staining, nonviable cell events were acquired, but they were
eliminated from final analysis. For staining with anti-DC LAMP, the
cells were permeabilized in Cytofix/Cytoperm (Pharmingen) solution,
stained with anti-DC LAMP, and FITC-labeled with goat
anti-mouse antibody.
Antigen uptake in immature dendritic cells Cells from the monocyte-rich fractions of elutriation were cultured in SFM with GM-CSF as described above. At the time points of interest, the cells were pulsed with 1 mg/mL FITC-albumin (Sigma). After 2 hours of incubation at either 37°C or 4°C, the cells were washed 3 times with buffer and analyzed by flow cytometry. The difference in mean channel fluorescence between the groups at 37°C and 4°C was used as a measure of uptake by macropinocytosis. T and B cells were used as a negative control.Evaluation of RelB expression Dendritic cells were pelleted and lysed in a buffer containing 10 mM Tris (pH 7.4), 220 mM NaCl, 30 mM sodium phosphate, 50 mM NaF, 5 mM ZnCl2, 1% Triton X-100, and protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN) supplemented with additional protease inhibitor2-macroglobulin (1.25 U/mL; Roche
Molecular Biochemicals). The extract was incubated on ice for 10 minutes and centrifuged at 500g for 15 minutes at 4°C; the
supernatant constituted the whole cellular extract. Pelleted nuclei
were extracted with 30 to 50 µL solution containing 10 mM Tris (pH
7.0), 220 mM NaCl, 30 mM sodium phosphate, 50 mM NaF, 5 mM
ZnCl2, 0.05% Nonidet P40, and protease inhibitors at 4°C for 20 minutes with agitation. The extract was centrifuged at 15 000g for 15 minutes at 4°C; the supernatant
constituted the nuclear extract. Protein concentrations were measured
by using the Bio-Rad (Hercules, CA) protein assay using a
bovine serum albumin standard. Protein (5-10 mg) was resolved by 10%
tricine SDS-PAGE (Invitrogen, Carlsbad, CA) and transferred to
Immobilon-P (Millipore, Bedford, MA). The membrane was probed with
anti-relB antiserum 131937 and antiactin monoclonal
antibody (Chemicon, Temecula, CA). Immunoreactive proteins were
revealed with an enhanced chemiluminescence system (Amersham Pharmacia
Biotech, Arlington Heights, IL).
Allosensitization of CD4+ CD45RO T cells were cocultured
with CI or IFN--, TNF--, and CD40L-treated allogeneic
antigen-presenting cells in 48-well flat bottom tissue culture
plates at a ratio of 1 × 106 T cells to
1 × 105 antigen-presenting cells (ratio, 10:1) in the
absence of any added cytokines. After 6 days, T cells were harvested
and restimulated on anti-CD3, anti-CD28-coated 96-well plates at
1 × 105 cells/well. Supernatants were harvested 24 hours
later and analyzed by ELISA.
ELISA assays Capture and biotinylated detection antibodies and standards for IFN-, IL-4, IL-5, IL-6, IL-8, IL-10, and IL-12p70 ELISAs were
purchased from Pharmingen and used according to the manufacturer's recommended protocols. Reagents for MIP-1 ELISA were purchased from
R&D Systems and used according to the manufacturer's protocols.
Intracellular cytokine staining After primary allosensitization as described above, T cells were harvested and restimulated with phorbol 12-myristate-13-acetate (50 ng/mL) and calcium ionophore (1 µg/mL) for 6 hours in the presence of monensin (2 µM). Cells were then harvested, fixed, and permeabilized in Cytofix/Cytoperm (Pharmingen) solution and stained with FITC-anti-IFN- according to the manufacturer's
recommendations. Flow cytometric acquisition was performed as described
above with the omission of PI gating.
CD8+ T-cell cocultures Dendritic cells from healthy donors used in CD8+ T-cell cocultures were pulsed with MART-1 peptide 27-35 at 50 ng/mL (National Cancer Institute, Bethesda, MD) 2 hours before harvest. They were harvested, washed with fresh media, and replated with lymphocytes at a T cell-to-DC ratio of 20:1, with recombinant human IL-2 IU/mL was added once. After 1 week, the T cells were harvested and restimulated with relevant (Mel624 A2+) and irrelevant control (Mel624 A2 , MW115)
cell lines. Cell lines used were Mel 624 A2+, Mel 624 A2, and MW115
and were obtained from Dr Steven A. Rosenberg (National Cancer
Institute). Supernatants were harvested after 24 hours and analyzed
by ELISA.
Rapid development of immature dendritic cells from monocytes Before culture monocytes were initially CD14+, CD13+, CD11b+, and CD11c+, expressed MHC class 1 and class 2 molecules, and demonstrated some expression of CD86, CD54, and CD40, but they were CD80
and CD83 (Figure 1A). In
addition, the cells lacked CD3, CD56, and CD20, suggesting that they
were a relatively pure population of myeloid monocytes (Figure 1B).
When these cells were cultured for 20 to 24 hours in SFM with GM-CSF,
the cells had diminished expression of CD14 and increased expression of
CD54 and CD40 (Figure 1C). The cells also expressed some CD80 and CD86
but had minimal expression of DC-LAMP or CD83 (Figure 1C). These DCs
demonstrated increased uptake of antigen by macropinocytosis, as
demonstrated by the uptake of FITC-labeled human albumin, suggesting
they were immature DCs (Figure 1D).
Similar expression of CD83, RelB, and costimulatory molecules on mature dendritic cells Immature DCs (monocytes cultured overnight in SFM and GM-CSF) treated with 150 ng/mL CI resulted in rapid increased expression of CD80, CD86, CD83, DC-LAMP, CD40, and CD54, which was evident within 20 hours of the addition of CI or 40 hours from initiation of the cultures (Figure 2A). In addition, there was increased expression of MHC class 1 and class 2 molecules (Figure 2A). There was also high-level expression of CD4+ (Figure 2C) and continued uniform expression of CD11b, CD11c, and CD13 with low CD14 expression (Figure 2A). This phenotype is consistent with that of mature DCs. Typically, cell yields of CI-treated DCs were approximately half those of plated cells for cryopreserved cells and 75% to 80% of plated cells for fresh monocytes, similar to the yields of immature DCs, suggesting the entire population acquired a mature DC phenotype, as we had previously demonstrated.29
Immature DCs (monocytes cultured in SFM and GM-CSF overnight) cultured
with IFN- Transcription factors of the Rel/NF
Calcium signaling inhibits IL-12 production by dendritic cells Mature DCs activated with CI produced high levels of IL-8 and low levels of MIP-1, with no significant production of p70 IL-12 or IL-6
(Figure 4). Kinetic studies demonstrated
no production of IL-12 from CI-activated DC at any point from 4 hours
to 48 hours (not shown). In contrast, DCs activated with IFN-,
TNF-, and CD40L produced not only high quantities of IL-8 but high
levels of p70 IL-12 (1000-5000 pg/mL), IL-6 (2500-7000 pg/mL), and
MIP-1 (Figure 4). Calcium ionophore inhibited IL-12 production by
DCs treated with IFN-, TNF-, and CD40L (Figure
5) in a dose-dependent fashion to
concentrations as low as 9 ng/mL. CI added even as late as 6 hours
after the activation of DCs with IFN-, TNF-, and CD40L resulted
in more than 50% inhibition of IL-12 production (not shown). In
contrast, calcium signaling did not inhibit the production of MIP-1
by DCs treated with IFN-, TNF-, and CD40L (not shown).
Calcium-signaling-induced inhibition of IL-12 production is not mediated through calcineurin phosphatase We have previously reported that calcium-signaling induced activation of DCs is mediated through the activation of calcineurin phosphatase and is inhibited by CSA.30,39 Addition of CSA to DCs activated with IFN-, TNF-, and CD40L had no effect on the expression of CD83, CD80, or IL-12 secretion by the activated DCs
(Figure 6). In contrast, DCs activated
with calcium-signaling agents in the presence of CSA demonstrated
significantly decreased expression of CD80 and CD83 (Figure 6). CSA
also did not reverse the inhibition of IL-12 secretion by DCs activated
with IFN-, TNF-, and CD40L in the presence of CI (Figure 6),
suggesting that the inhibition of IL-12 secretion by calcium-signaling
agents is not mediated through calcineurin phosphatase, as is the
activation of DCs by calcium-signaling agents.30,39
Calcium-signaling-activated dendritic cells induce Th2 cells CI-activated DCs, when cocultured with naive CD45RA allogeneic CD4+ T cells, stimulated the T cells to proliferate significantly, as evidenced by a 3-fold expansion (Figure 7A). This was similar to DCs activated with IFN-, TNF-, and CD40L (Figure 7A). CI-activated DCs induced
the CD4+ T cells to secrete high levels of IL-4 (400-800 pg/mL) and IL-5 (900-3,000 pg/mL), relatively low levels of IFN-,
and no IL-10 (Figure 7B) when the T cells were restimulated. In
contrast, DCs activated with IFN-, TNF-, and CD40L stimulated
high levels of IFN- production (60 000-100 000 pg/mL) by the
CD4+ T cells, with minimal secretion of IL-4, IL-5, or
IL-10 at restimulation (Figure 7B). In addition, intracellular cytokine
analysis of IFN- demonstrated 52% (average, 51%; range, 47%-53%)
of the CD4+ T cells cultured with DCs treated with IFN-,
TNF-, and CD40L-secreted IFN-, whereas only 9% (average, 11%;
range, 6%-14%) of the CD4+ T cells induced by CI were
positive for intracellular IFN- (Figure 7C). When DCs were activated
with IFN-, TNF-, and CD40L in the presence of calcium-signaling
agents and then were cocultured with naive CD4+ T cells,
the T cells secreted high levels of IL-4 and IL-5 with lower level
production of IFN- (not shown).
Calcium-activated dendritic cells stimulate TC2 cells MART-1 27-35 is an immunodominant peptide derived from MART-1/MelanA melanoma tumor antigen that is presented on the HLA-A2.1 binding domain.40 When CI-activated DCs from healthy donors were pulsed with MART-1 and were cocultured with autologous CD8+ T cells at restimulation with tumor cells expressing MART-1, the CD8+ T cells secreted predominantly IL-5 (Figure 8). In contrast, CD8+ T cells sensitized with DCs activated with IFN-, TNF-, and CD40L
secreted high levels (6-10 ng/mL) of IFN- when the T cells were
restimulated with melanoma cell lines (Figure 8). The amount of IFN-
secreted by the MART-1-specific CD8+ T cells stimulated by
peptide-pulsed IFN--, TNF--, and CD40L-activated DCs was
usually on the order of 10- to 20-fold greater release than
CD8+ T cells generated by CI-pulsed DCs.
Calcium-activated dendritic cells retain DC2 activity in the presence of exogenous IL-12 When exogenous IL-12 was added with calcium ionophore to DC cultures during maturation and was washed out before coculture with T cells, the cytokine secretion profile of naive allogeneic CD4+ T cells was characterized by increased IFN- and
decreased IL-5 (Figure 9A). A
corresponding shift in chemokine secretion was not seen. Despite IL-12
treatment, these DCs produced only low levels of MIP-1 that was not
increased with the addition of IL-12 (Figure 9B).
Multiple studies have suggested that monocytes can serve as a reservoir for human DCs.41-43 Monocytes cultured in vitro in serum-containing medium (eg, fetal calf serum or human serum) develop characteristics of immature DCs after 6 or 7 days.4,43,44 In contrast to such lengthy culture activation, monocytes can also acquire characteristics of DCs more rapidly.45 Randolph et al46 have shown, using an endothelial reverse-transmigration model, that monocytes crossing an endothelial monolayer and contacting particulate collagen matrix can acquire characteristics of DCs in as few as 2 days. It appears that this system may model monocyte extravasation, entry into peripheral tissues, and phagocytosis of particulate matter that forms part of the normal DC maturation process. Similarly, in the current study, monocytes cultured in SFM down-regulate their CD14 expression and acquire characteristics of immature DCs, such as increased ability to take up antigen within 40 hours of culture initiation. Because interstitial fluids, in which postextravasation DC precursors would find themselves, differ from serum in protein and ionic content, it may be possible that removal from serum is one of many cues that sensitize these cells to additional signals that trigger the rapid acquisition of mature DC characteristics. This would explain why we see more rapid differentiation and maturation from CD14+ precursors cultured in SFM than from similar treatments in serum-containing medium. We have previously documented that calcium signaling in human monocytes
results in the rapid development of immunologically activated
DCs.29,30 These DCs express high levels of the
costimulatory molecules B7.1 and B7.2 and high levels of the DC
activation molecule CD83. The level of costimulatory molecule
expression on DCs activated with CI was similar to that on DCs treated
with IFN- Other signaling agents such as cholera toxin,20 a
nematode-derived phosphorylcholine glycoprotein,19 and
PGE Several known signal receptor complexes can trigger calcium fluxes in monocytes and DCs.33,49 For instance, it has been shown that Fc receptor binding or complement receptor binding induces calcium fluxes within the cells.33 Calcium signaling has been shown by Sutterwala et al33 to diminish IL-12 production in monocytes; in the current study, calcium signaling inhibits IL-12 production by mature DCs. Hence, antigen-antibody complexes, or immune complexes binding to Fc or complement receptors and taken up on DCs, could be expected to favor Dc2 activity through the mobilization of intracellular calcium. Calcium signaling induced by monocyte chemoattractant protein-1 (MCP-1) in macrophages and monocytes49 also provides a mechanism by which to explain the ability of MCP-1 to inhibit IL-12 production and to induce T cells that secrete IL-4 when it is used as a vaccine adjuvant in the gastrointestinal tract.50 Therefore, calcium signaling may provide a common pathway for environmental pathogens and regulatory molecules to interact with activated DCs to regulate Th1 and Th2 cell development. Plasmacytoid CD11b Th2 and DC2 cells are thought to be involved in preventing GVHD in allogeneic transplantation,22,23,27 preventing autoimmunity,24,25 and preventing rejection in solid organ transplantation.26 The ability to activate monocytes into DC2 by simple ex vivo manipulation with calcium-signaling agents may make it possible to explore treating some of these diseases with DC2 vaccines. Calcium-signaling agents such as calcium ionophore may be more desirable than other DC2-activating agents such as cholera toxin and PGE2 because of the unavoidable antigenicity of cholera toxin and the variable effects of PGE2 on DC activation at different stages of maturation.53,54 The DC2 phenotype induced by calcium signaling is
demonstrably stable because calcium-activated DCs exposed to exogenous
IL-12 during maturation induce an increase in IFN- Transcription factors of the Rel/NF- Signals other than IL-12 may be needed to induce a strong Th1 response.
Th1 cells express the chemokine receptor CCR5 on their surfaces,58,59 and MIP-1 Dendritic cells activated with INF- In summary, calcium signaling in immature DCs results in the development of mature DCs that express high levels of CD83 and the costimulatory molecules, CD80 and CD86. These cells, when used as antigen-presenting cells, appear to favor the development of Th2 cells. Calcium signaling can also inhibit the production of IL-12 by DC1. Calcium signaling provides a common pathway for multiple environmental signals to be translated within DCs to induce Th2 development. DCs activated with calcium-signaling agents may have clinical usefulness in treating disease states with excessive Th1 activity.
We thank Dr Dupont Guerry for critical review of this manuscript. We thank the dedicated staff of the apheresis unit and the Cell Processing Center at the University of Pennsylvania. We also thank Kathy Pichak and Immunex for supplying rhCD40L.
Submitted March 26, 2001; accepted June 18, 2001.
Supported by American Cancer Society RPG-99-029-01.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Brian J. Czerniecki, Department of Surgery, University of Pennsylvania, 4 Silverstein, 3400 Spruce St, Philadelphia, PA 19104; e-mail: czerniec{at}mail.med.upenn.edu.
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Top
Abstract
Introduction
resulting in further increases in costimulatory
molecule expression
B family, including p50, p52, and
RelB, are expressed by and play an important role in the biologic
function of DCs.
), 624mel MART-1+ A2
), and non-MART-1 cell line MW115 (
) for 24 hours. Supernatants
were then harvested and analyzed for IL-5 or IFN-
