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IMMUNOBIOLOGY
From the Laboratory of Immunology, Istituto Dermopatico
dell'Immacolata, IRCCS, Rome, Italy; the Department of Experimental
and Diagnostic Medicine, Section of General Pathology and Center for
the Study of Inflammatory Diseases, University of Ferrara, Italy; and
the Department of Experimental Dermatology, University of Freiburg,
Germany.
We previously reported that chronic stimulation with low,
noncytotoxic doses of extracellular adenosine triphosphate (ATP) induced a distorted maturation of dendritic cells (DCs) and impaired their capacity to initiate T-helper (Th) 1 responses in vitro. Here, we
examined the effects of ATP on chemokine-receptor expression and
chemokine production by DCs. ATP strongly induced expression of CXC
chemokine receptor 4 on both immature and lipopolysaccharide (LPS)-stimulated DCs and slightly up-regulated CC chemokine receptor (CCR) 7 on both DC types. In contrast, ATP reduced CCR5 expression on
immature DCs. These effects were confirmed at both the messenger RNA
and protein levels and were not produced by uridine triphosphate (UTP).
Consistent with the changed receptor expression, ATP increased migration and intracellular calcium of immature and mature DCs to stromal-derived factor 1 (CXC ligand [CXCL] 12) and macrophage inflammatory protein [MIP] 3 Migration of dendritic cells (DCs) from the site of
antigen capture to secondary lymphoid organs is a crucial event in the initiation and amplification of immune responses. DCs reside in an
immature state in peripheral tissues, where they are highly efficient
in capturing antigens. On exposure to danger signals such as pathogens,
dying cells, or inflammatory cytokines, DCs differentiate into mature
DCs. Maturing DCs migrate to draining lymph nodes, where they are very
effective at activating naive and central memory T
cells.1,2 In turn, inflammatory signals recruit
circulating DCs and their precursors from the blood to sites of
inflammation, replacing DCs that have migrated to lymph nodes.2 Trafficking of DCs through tissues is regulated by the pattern of chemokine receptors expressed on DCs and the local availability of chemokines. Immature DCs and monocytes express receptors for inflammatory chemokines (CXC chemokine receptor [CXCR]
1, CC chemokine receptor [CCR] 1, CCR2, and CCR5), which account for
the capacity of these cells to migrate to inflamed tissues where
cognate ligands are produced. Maturation of DCs is associated
with the coordinated down-regulation of receptors for inflammatory
chemokines and the induction of lymphoid chemokine receptors such as
CXCR4, CCR4, and CCR7.2-4 Thus, maturing DCs become
responsive to lymphoid chemokines such as CXC ligand (CXCL) 12 (stromal-derived factor [SDF] 1), CC ligand (CCL) 2 (macrophage-derived chemokine [MDC]), and CCL19 (macrophage
inflammatory protein [MIP] 3 DCs are also a relevant source of chemokines. Immature DCs
constitutively release MDC and CCL17 (thymus- and activation-regulated chemokine [TARC]).5 At early stages of maturation, DCs
produce high levels of inflammatory chemokines, such as CCL2 (monocyte chemoattractant protein 1 [MCP-1]), CCL3 (MIP-1 Extracellular nucleotides are important regulators of inflammatory and
immune responses.10 Nucleotides are present at high concentrations in the cytoplasm and can be released by regulated exocytosis or by passive leakage after cell damage. In the
extracellular compartment, nucleotides bind P2 purinergic receptors,
which are expressed by both human and murine DCs.11-14 We
previously reported that low, noncytotoxic doses of extracellular
adenosine triphosphate (ATP), but not uridine triphosphate (UTP),
suppressed IL-12 production by maturing DCs, thereby impairing their
ability to initiate T-helper (Th) 1 responses.15 In the
current study, we found that extracellular ATP, but not UTP, changes
chemokine-receptor expression and chemokine production by DCs. In
particular, ATP induced functional expression of CXCR4 and CCR7,
setting DCs for lymph node localization. Moreover, ATP up-regulated MDC
while inhibiting RANTES and IP-10 production by maturing DCs, thus
reducing their ability to attract type 1 T lymphocytes specifically.
Reagents and antibodies
Preparation and stimulation of DCs
Flow cytometry analysis of DCs DCs that were either untreated or stimulated for 24 hours with LPS in the presence or absence of nucleotides were washed and then incubated in phosphate-buffered saline (2% FCS and 0.01% sodium azide) with FITC-conjugated mAbs for 40 minutes at 4°C. When pure mAbs were used, cells were washed 3 times and a second incubation with an FITC-coupled goat (Fab')2 antimouse IgG (Southern Biotechnology Associates, Birmingham, AL) was done. For CCR7 staining, the second incubation was done with biotin-coupled rat antimouse IgM and was followed by extensive washing and additional incubation with FITC-conjugated streptavidin (BD PharMingen). Isotype-matched mouse Ig was used in control samples. Cells were analyzed with a fluorescence-activated cell-sorter scanner (FACScan; Becton Dickinson) and Cell Quest software. Data analysis used WinMDI software (http://facs.scripps.edu). Results are shown as the net mean fluorescence intensity (MFI), which is the mean fluorescence obtained with mAbs subtracted from the mean fluorescence measured with isotype-matched control Ig.Release of chemokines from DCs Cell-free supernatants from DCs were tested for chemokine content by enzyme-linked immunosorbent assay (ELISA). RANTES was assessed by using the Ab pair, rabbit polyclonal 20581D (for coating) and 20582D (for detection; BD PharMingen). IP-10 was assayed with the purified 4D5/A7/C5 and the biotinylated 6D4/D6/G2 antihuman IP-10 mAbs (BD PharMingen). MDC and TARC were measured by using ELISA kits from R&D Systems. SDF-1 was measured by using purified 79018.111 and
biotinylated antihuman SDF-1 goat IgG (AQL02; R&D Systems). The
plates were analyzed in an ELISA reader (3550 UV; Bio-Rad, Hercules,
CA). Samples for each condition were assayed in triplicate.
Ribonuclease protection assay Total RNA was extracted from purified DCs after 24 hours of incubation with the selected stimuli by using Trizol (Invitrogen Italia) according to the manufacturer's instructions. Two multiprobe template sets, hCR5 and hCR6 (RiboQuant; BD PharMingen), were used for in vitro transcription reactions in the presence of a GACU pool and a T7 RNA polymerase to synthesize phosphorus 32-UTP-labeled antisense probes. Ribonuclease (RNase) protection analysis of 5 µg total RNA was done after overnight hybridization at 60°C with 2.5 × 106 cpm hCR5 or hCR6, followed by digestion with RNase A and T1 according to standard protocols. Protected fragments were treated with proteinase K, extracted with phenol-chloroform and isoamyl alcohol (50:1), and precipitated in ethanol in the presence of ammonium acetate. The samples were electrophoresed on 5% denaturing sequencing gels and exposed to Kodak films.Calcium-flux analysis DCs (106 cells/mL) were loaded with 8 µM fluo-3-acetoxymethyl ester in the presence of 1 µM pluronic F-127 (Molecular Probes, Eugene, OR) in complete RPMI with 1% FCS for 40 minutes at 37°C and subjected to frequent gentle agitation. Cells were then washed twice; stimulated with SDF-1, MIP-1 , or MIP-3
(all at 100 ng/mL; R&D Systems); and analyzed on a FACScan device.
Cells and chemokines were kept at 37°C during the assay.
Migration assay Chemotaxis of DCs and T cells was evaluated by measuring their migration through 5-µm-pore polycarbonate filters in 24-well transwell chambers (Corning Costar, Cambridge, MA), as described previously.17 DC migration was studied by adding different concentrations of chemokines to the lower wells and 105 DCs suspended in complete RPMI with 0.5% bovine serum albumin (BSA) in the transwell insert. The chemotactic property of DC supernatants was evaluated by adding 105 T cells to the top chamber and various dilutions of the supernatants (0.6 mL) to the bottom chamber of the transwell. After 1 hour of incubation at 37°C with 5% carbon dioxide, cells that transmigrated into the lower chamber were recovered and acquired with a FACScan device for 60 seconds at a flow rate of 60 µL/min. Data acquisition and analysis were restricted to events with the forward and side scatter properties of cells and not cell debris. In some experiments, DC supernatants were used after 30 minutes of incubation with a neutralizing anti-IP-10 mAb, anti-MDC Ab, and anti-TARC Abs or mouse Ig. Results are shown as net migration, which is the number of cells in the lower chamber containing the agonistic chemokine or DC supernatant subtracted from the number of cells in chambers containing medium alone.T-cell lines and clones T-cell lines were generated by stimulating peripheral blood mononuclear cells (PBMCs) from healthy donors with 1 µg/mL phytohemagglutinin (PHA) in complete RPMI complemented with 5% human serum in the presence of 2 ng/mL rhu IL-12 and 10 µg/mL anti-IL-4 mAb (for type 1 cell polarization) or the presence of 250 U/mL rhu IL-4 and 10 µg/mL anti-IL-12 mAb (for type 2 cell polarization). After 10 to 14 days of culture in the presence of 30 U/mL IL-2, T cells were restimulated with plate-coated anti-CD3 and soluble anti-CD28 mAbs (both at 1 µg/mL; BD PharMingen) and examined for intracellular interferon (IFN- ) and IL-4 after 6 hours. For 2-color intracellular staining, monensin (10 µM; Sigma-Aldrich) and brefeldin A (10 µg/mL; Sigma-Aldrich) were added to the cultures before staining to
prevent cytokine secretion. T cells were then fixed with 2% paraformaldehyde, permeabilized with 0.5% saponin, stained with FITC-conjugated mouse anti-IFN- and phycoerythrin-conjugated rat
anti-IL-4 (BD PharMingen), and analyzed with a FACScan device. In
control samples, staining was done with isotype-matched control Ab. T
cells that were not restimulated did not show any lymphokine production
(data not shown).
T-cell clones were generated from CD4+ or CD8+
purified and polarized T-cell lines by limiting dilution (0.4 cells/well) in the presence of irradiated 2 × 105 PBMCs,
30 U/mL IL-2, and 1% PHA in complete RPMI plus 10% FCS. Clones were
grown in the presence of IL-2 and periodically stimulated with 1% PHA
and feeder cells or plate-coated anti-CD3 and soluble anti-CD28.
Phenotype was assessed by flow cytometry. Release of IL-4 and IFN- Statistical analysis The Mann-Whitney test was used to compare differences in chemokine release and cell migration. P values of .05 or less were considered to indicate significance.
Extracellular ATP, but not UTP, induces CCR7 and CXCR4 and down-regulates CCR5 expression RNase protection assays were done to study the pattern of expression of chemokine receptors in DCs stimulated with ATP or UTP (Figure 1). Immature DCs expressed high levels of CCR5 and low levels of CXCR4 and CCR7 messenger RNA (mRNA). Incubation with 250 µM ATP strongly up-regulated CXCR4 and, to a lesser extent, CCR7 and completely down-regulated CCR5 mRNA. In addition, ATP further increased CXCR4 and CCR7 mRNA levels induced by LPS. In contrast, UTP did not affect expression of receptors in either immature or LPS-stimulated DCs (Figure 1).
Expression of chemokine receptors on the membranes of immature or
mature DCs stimulated with ATP was also assessed by flow cytometry
(Figure 2). ATP, but not UTP, acted as a
maturation stimulus for DCs inducing up-regulation of CD83, as
described previously.15 DCs treated with ATP had high
membrane expression of CXCR4. In contrast, CXCR4 was not detected on
the surface of LPS-stimulated DCs despite the high mRNA expression, a
finding described previously.18,19 The presence of CCR7
and CCR5 molecules on the DC surface was consistent with the respective
mRNA expression: CCR7 was detected at low levels on ATP-treated DCs and
more abundantly on LPS-stimulated DCs, with DCs exposed to both
ATP and LPS expressing the highest amount. CCR5 was expressed by
immature and UTP-stimulated DCs but completely disappeared on DCs
treated with ATP, LPS, or both.
The functional activity of chemokine receptors was then examined by
measuring intracellular calcium flux (Figure
3) and cell migration (Figure
4) in response to agonistic chemokines.
Immature DCs showed intracellular calcium mobilization and migrated in response to the CCR5 ligand MIP-1
ATP up-regulates MDC and inhibits IP-10 and RANTES release by DCs and reduces the capacity of DCs to attract type 1 lymphocytes In the next series of experiments, we investigated the effect of ATP on chemokine production by DCs. Consistent with previous observations, immature DCs constitutively produced MDC and TARC but not IP-10 or RANTES.5,16 In these cells, treatment with ATP significantly increased release of MDC but not of TARC, IP-10, or RANTES (Table 1). Moreover, ATP increased the production of MDC and strongly inhibited secretion of IP-10 and RANTES induced by LPS (Table 1 and Figure 5). SDF-1 was not detected in the supernatants of immature or mature DCs, regardless of whether nucleotides were present (data not shown). Suppression of IP-10 and
enhancement of MDC were already evident at an ATP dose of 2.5 µM,
whereas the minimal ATP concentration inhibiting RANTES secretion was
100 µM. UTP did not affect chemokine production by
DCs.
Depending on their polarization, T lymphocytes differentially express
receptors for IP-10, RANTES, MDC, and TARC, with type 1 cells
preferentially expressing CXCR3 and CCR5 and type 2 cells expressing
CCR3, CCR4, and CCR8.18,20-22 We thus investigated whether
ATP could affect the capacity of DCs to attract type 1 and type 2 T
lymphocytes. First, polarized T-cell lines were generated by
stimulating PBMCs with PHA in the presence of IL-12 and neutralizing anti-IL-4 mAb or IL-4 and neutralizing anti-IL-12 mAb. T-cell lines
were about 60% CD8+ and 40% CD4+ (data not
shown). As expected, type 1 lymphocytes showed high levels of IFN-
Consistent with the high production of MDC and TARC by immature DCs, type 2 cells (Figure 6F) migrated more efficiently (P < .05) than type 1 cells (Figure 6E) to supernatants from immature DCs, regardless of the treatment with nucleotides. Type 1 and type 2 cells showed a similar, higher level of migration to supernatants from LPS-stimulated DCs compared with supernatants from immature DCs (Figure 6G-H). However, supernatants from DCs matured in the presence of ATP, but not UTP, showed a strongly reduced capacity to attract type 1 lymphocytes, whereas attraction of type 2 cells was not affected (Figure 6G-H). Preincubation of supernatants from immature or mature DCs with anti-MDC and anti-TARC Abs markedly reduced (70% inhibition) migration of type 2 cells but not type 1 cells. In contrast, IP-10 neutralization suppressed (60% inhibition) the migratory response of type 1 cells but only slightly inhibited (20% inhibition) the response of type 2 cells induced by supernatants from mature DCs (data not shown). We next assessed the migratory activity of DC supernatants on
type 1 and type 2 T-cell clones (Table
2). As shown in Figure 7, Th2 clones migrated more vigorously
than Th1 and T- cytotoxic (Tc) clones to supernatants from
immature DCs, a function that was not affected by treatment with ATP or
UTP. Supernatants from mature DCs were more potent than those from
immature DCs in inducing migration of Th1 and Tc1 clones. In contrast,
migration of Th2 and Tc2 clones was not influenced by the maturation
stage of donor DCs. Interestingly, supernatants from mature DCs that
had been treated with ATP attracted Th1 and Tc1 cells less efficiently, whereas migration of Th2 and Tc2 cells was not affected.
DCs express functional purinergic receptors of both the P2X and P2Y families, including P2X1, P2X2, P2X4, P2X5, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11.11-14 P2X receptors are membrane channels, whereas P2Y are G protein-coupled 7-membrane spanning receptors that can, on activation, mobilize calcium from the intracellular stores by means of generation of inositol triphosphate.10 Extracellular ATP can thus affect many biologic aspects of DCs through activation of distinct P2 purinergic receptors.10-14,23,24 In particular, extracellular ATP showed the ability to suppress production of proinflammatory cytokines and IL-12 by mature DCs.15 In addition, patch-clamp studies found that DCs reoriented their dendrites in the vicinity of a patch pipette containing ATP, thus suggesting sensitivity to an ATP-based chemotactic gradient.11 This preliminary finding was confirmed by a study showing that ATP can modulate DC chemotaxis by means of activation of pertussis toxin-sensitive P2Y receptors (M.I., S. Dichmann, D.F., et al, unpublished data, July 2001). These observations are consistent with data showing that extracellular nucleotides are powerful chemoattractants for inflammatory cells,25-27 and they support the hypothesis that these nucleotides have an important role in alerting the immune system after cell injury or tissue damage. The current study showed that ATP, but not UTP, modifies the pattern of
chemokine-receptor expression on DCs leading to up-regulation of the
lymphoid chemokine receptors CCR7 and CXCR4 and the coordinated down-regulation of CCR5. Functionally, DCs exposed to ATP migrated vigorously to MIP-3 ATP also affected the pattern of chemokine release from DCs by
up-regulating the constitutive production of MDC and inhibiting the
LPS-induced secretion of IP-10 and RANTES. ATP was found to block the
production of IP-10 by astrocytes stimulated with
IL-1 ATP and other nucleotides are present in the cell cytoplasm at
millimolar concentrations and can be released by exocytosis, transport
through unidentified plasma-membrane pathways, or (more easily),
passive leakage after cell damage.10 ATP is thus likely to
be present at high concentrations in the extracellular milieu at sites
of inflammation associated with tissue damage. ATP concentration in the
pericellular space after cell stimulation has been measured in a few
experimental systems and found to vary from the high nanomolar to the
low micromolar range.33,34 However, ATP acts as a
short-range extracellular messenger that can reach higher levels in the
space between adjacent cells. Extracellular ATP appears to have a dual
effect on maturing DCs. On the one hand, it can by itself stimulate
secretion of IL-1 In summary, we found that low doses of ATP change the chemokine-receptor aspect of DCs, conferring on them an enhanced capacity to localize to lymph nodes. We also observed an additional mechanism by which extracellular ATP can promote immune deviation toward type 2 responses, that is, by preventing DC recruitment of type 1 but not type 2 T lymphocytes. Our findings support the hypothesis that ATP released by damaged cells in inflamed tissues can act as a potent regulatory signal for DCs and for the polarization of the immune response.
We thank Brian Kelsall, Warren Strober, and Michael Braun for helpful discussions.
Submitted August 27, 2001; accepted October 19, 2001.
Supported by the Italian Ministry of Health, the Italian Ministry for Education, the Italian Association for Cancer Research, the Italian Space Agency, the National Research Council of Italy (Target Project on Biotechnology), and Telethon of Italy.
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: Giampiero Girolomoni, Istituto Dermopatico dell'Immacolata, IRCCS, Via Monti di Creta 104, 00167 Rome, Italy; e-mail: giro{at}idi.it.
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Top
Abstract
Introduction
DC
preparations. DCs were left untreated, incubated for 2 hours at 37°C
with nucleotides, or induced to mature with 10 µg/mL LPS in the
presence or absence of nucleotides. The cells were then washed and
incubated for 24 hours at 37°C.
) or UTP (
). After 24 hours of incubation,
IP-10 (A), RANTES (B), MDC (C), and TARC (D) were measured in culture
supernatants by ELISA. Results are mean ± SD nanograms per
milliliter/106 cells from triplicate cultures. Differences
in IP-10, RANTES, and MDC secretion of DCs stimulated with LPS alone
and those stimulated with both LPS and ATP were significant
(P < .05) when ATP amounts were at least 2.5 µM, at
least 100 µM, and at least 2.5 µM, respectively, for the 3 chemokine assessments.
)
or stimulated with 250 µM ATP (
) or 250 µM UTP (
) and results
with supernatants from DCs treated with 10 µg/mL LPS alone (
), LPS
and ATP (
