Abstract
Dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) is a monocyte-derived dendritic cell (MDDC)-specific lectin which participates in dendritic cell (DC) migration and DC-T lymphocyte interactions at the initiation of immune responses and enhances trans-infection of T cells through its HIV gp120-binding ability. The generation of a DC-SIGN-specific mAb has allowed us to determine that the acquisition of DC-SIGN expression during the monocyte-DC differentiation pathway is primarily induced by IL-4, and that GM-CSF cooperates with IL-4 to generate a high level of DC-SIGN mRNA and cell surface expression on immature MDDC. IL-4 was capable of inducing DC-SIGN expression on monocytes without affecting the expression of other MDDC differentiation markers. By contrast, IFN-α, IFN-γ, and TGF-β were identified as negative regulators of DC-SIGN expression, as they prevented the IL-4-dependent induction of DC-SIGN mRNA on monocytes, and a similar inhibitory effect was exerted by dexamethasone, an inhibitor of the monocyte-MDDC differentiation pathway. The relevance of the inhibitory action of dexamethasone, IFN, and TGF-β on DC-SIGN expression was emphasized by their ability to inhibit the DC-SIGN-dependent HIV-1 binding to differentiating MDDC. These results demonstrate that DC-SIGN, considered as a MDDC differentiation marker, is a molecule specifically expressed on IL-4-treated monocytes, and whose expression is subjected to a tight regulation by numerous cytokines and growth factors. This feature might help in the development of strategies to modulate the DC-SIGN-dependent cell surface attachment of HIV for therapeutic purposes.
Dendritic cells (DC)4 are professional APC critically involved in the initiation of T cell-dependent immune responses as a consequence of their high expression of MHC and costimulatory molecules (1). Myeloid DC are sparsely distributed throughout the body in an immature state, exhibiting a high capacity for Ag uptake and processing but weak T cell stimulatory activity (1, 2). Once activated by inflammatory stimuli or infectious agents, DC undergo a maturation process, migrate into lymphoid organs, and acquire the capacity to activate naive T lymphocytes (1, 2, 3, 4). Ontogenically, DC are heterogeneous and are derived from lymphoid or myeloid lineages (reviewed in Ref. 5). In human blood there are at least two types of DC precursors, myeloid monocytes (or pre-DC1) and plasmacytoid DC precursors (or pre-DC2) (5). In vitro, immature myeloid monocyte-derived DCs (MDDC) can be obtained from peripheral blood monocytes in the presence of GM-CSF and IL-4 (6, 7, 8, 9). The further addition of LPS or TNF-α leads to the appearance of MDDC with all the morphological, phenotypic, and functional characteristics of mature DC (6, 7).
DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN; CD209) is a type II membrane protein with a C-type lectin extracellular domain (10, 11). DC-SIGN plays an important role in establishing the initial contact between DC and resting T lymphocytes through its recognition of ICAM-3 (11), and it also mediates DC trafficking through interactions with endothelial ICAM-2 (12). Therefore, DC-SIGN appears to be a critical mediator of the migratory and T cell-interacting capabilities exhibited by maturing MDDC. In addition, DC-SIGN, originally described as a molecule with HIV gp120-binding ability (10), is now believed to capture HIV in the periphery and promote efficient infection in trans of cells expressing HIV receptors and coreceptors (13, 14, 15).
Given the relevance of the functional activities displayed by DC-SIGN (16), determination of the signaling pathways and factors controlling its expression might provide clues for modulating the effector functions of DC in the initiation of immune responses. Immunofluorescence analysis has revealed that DC-SIGN is only expressed on a small percentage of CD14+ blood cells, while it is highly expressed on immature DC in peripheral tissues and in vitro derived MDDC (11, 12). By contrast, DC-SIGN mRNA has been detected in PHA-activated PBL by means of RT-PCR analysis, an approach which has also demonstrated the existence of an extensive repertoire of DC-SIGN isoforms (17). To systematically analyze the regulation of DC-SIGN expression during MDDC differentiation, we have generated a DC-SIGN-specific mAb that allowed the identification of DC-SIGN as an IL-4-inducible molecule and its negative regulation by TGF-β and type I and II IFN.
Materials and Methods
Cytokines and reagents
−8–10−6 M. Escherichia coli 055:B5 LPS was purchased from Sigma (Barcelona, Spain) and used at 10 ng/ml. The Janus kinase (JAK)2/3 inhibitor AG-490 and the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)1/2 inhibitor U0126 were purchased from Calbiochem (San Diego, CA) and used at 30 μg/ml to 30 ng/ml (AG-490) and 2.5 μM (U0126).
Cells
Human PBMC were isolated from buffy coats from normal donors over a Lymphoprep (Nycomed, Oslo, Norway) gradient according to standard procedures. Monocytes were purified from PBMC by a 1-h adherence step at 37°C in complete medium. Nonadherent cells were washed off by extensive washing with PBS and the remaining adherent cells (>90% monocytes, as determined by flow cytometric analysis of forward scatter/side scatter, CD14 and CD11c staining) were immediately subjected to the DC differentiation protocol as previously described (6, 7, 18, 19). Briefly, monocytes were resuspended at 0.5–1 × 106 cells/ml and cultured in RPMI 1640 supplemented with 10% FCS, 25 mM HEPES, and 2 mM glutamine (complete medium) containing 1000 U/ml GM-CSF and 1000 U/ml IL-4. Cells were cultured for 5–7 days, with cytokine addition every second day, to obtain a population of immature MDDC. For maturation, immature MDDC were treated with LPS at 10 ng/ml.
Flow cytometry and Abs
Cellular phenotypic analysis was conducted by indirect immunofluorescence. mAbs used for cell surface staining included T3b (anti-CD3), TS1/2 (anti-MHC class II), HB1/5 (anti-CD83; Immunotech, Marseille, France), HC1/1 (anti-CD11c), UCH-M1 (anti-CD14; Santa Cruz Biotechnology, Santa Cruz, CA), and BL6 (anti-CD1a; Immunotech). All incubations were done in the presence of 50 μg/ml human IgG to prevent binding through the Fc portion of the Abs. The supernatant from the myeloma P3×63 (X63) was always included as a negative control. Flow cytometry analysis was performed with an EPICS-CS (Coulter Científica, Madrid, Spain) using log amplifiers. Where indicated, results are expressed as expression index: percentage of marker-positive cells multiplied by their mean fluorescence intensity (MFI).
Immunofluorescence staining
LPS-treated MDDC were resuspended in complete medium and allowed to adhere onto poly-l-lysine-coated coverslips (50 × 103 cells/coverslip) for 60 min. Cells were fixed in 3.7% formaldehyde in PBS (10 min at room temperature), permeabilized with 0.2% Triton X-100 (10 min at room temperature), and processed for immunofluorescence. Preparations were double-stained with the MR-1 anti-CD209 Ab and FITC-phalloidin (Sigma) for 45 min at room temperature, followed by an incubation with Cy3 goat anti-mouse Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1/500 in PBS. Additional Abs included TS1/2 (anti-MHC class II) and the anti-CD83 Ab HB1/5, which specifically reacts with mature MDDC. Coverslips were mounted in fluorescent mounting medium (DAKO, Glostrup, Denmark) and representative fields of cells were photographed through an oil immersion lens on a Nikon Eclipse E800 microscope (Nikon, Melville, NY) equipped for epifluorescence. Cells were also photographed using Nomarski optics.
Northern blot
After extensive washing in PBS, cells were harvested and total cellular RNA was isolated using RNeasy columns (Qiagen, Hilden, Germany) following the manufacturer’s recommendations. After confirming RNA integrity, denatured RNA (10 μg) was size-fractionated on formaldehyde-containing 1% agarose gels in the presence of ethidium bromide. Then, RNA was transferred overnight onto nitrocellulose membranes with 20× SSC. Prehybridization was conducted overnight at 42°C in 50% formamide, 5× SSC, 5× Denhardt’s, 50 mM sodium phosphate (pH 6.5), and 250 μg/ml denatured salmon sperm DNA. Membranes were hybridized for 16 h at 42°C in the same solution containing 106 cpm/ml oligo-labeled probe. Blots were sequentially washed in 2× SSC, 0.5% SDS at room temperature, and in 0.3× SSC, 0.5% SDS at 65°C, and exposed to x-ray film at −70°C. Detection of DC-SIGN mRNA was accomplished using the whole coding region of the DC-SIGN cDNA as probe (10).
Generation of stable DC-SIGN transfectants in K562 cells
The whole coding region of DC-SIGN was initially obtained by RT-PCR using oligonucleotides 5′-GGGAATTCAGAGTGGGGTGACATGAGTGAC-3′ and 5′-CCCCAAGCTTGTGAAGTTCTGCTACGCAGGAG-3′ as primers, where italicized letters mark the initiation and termination codons and underlined residues represent additional sequences containing EcoRI or HindIII restriction sites. Two micrograms of total RNA from immature MDDC was reverse transcribed in a total volume of 20 μl of amplification buffer (50 mM Tris-HCl (pH 8.2), 5 mM MgCl2, 10 mM DTT, 50 mM KCl, 1 mM of each deoxynucleotide, 0.5 μM random hexamers) including RNAsin and avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) at 1 U/μl. The mixture was incubated at 42°C for 60 min followed by a 30-min incubation at 52°C, and the final volume was taken to 100 μl with water. Amplification of the full-length DC-SIGN mRNA was conducted using 5 μl of the cDNA synthesis reaction in 50 μl of a solution containing 0.2 mM of each deoxynucleotide, 1 μM of each oligonucleotide primer, and 2.5 U of Pfu DNA polymerase (Stratagene, La Jolla, CA). The resulting fragment was digested with EcoRI and HindIII and gel purified and ligated into EcoRI- and HindIII-digested pCDNA3.1− to generate pCDNA3.1-DC-SIGN. Transfection of pCDNA3.1-DC-SIGN in K562 cells was accomplished using Superfect (Qiagen), and selection of transfected cells was done using G418 (300 μg/ml). Stable DC-SIGN expression of the selected population (K562-CD209) was verified using the anti-DC-SIGN Ab AZN-D1 (provided by Dr. Y. van Kooyk, Free University Medical Center, Amsterdam, The Netherlands) (11).
Generation of anti-DC-SIGN mAbs
BALB/c mice received three i.p. injections of 106 immature MDDC and a final i.v. boost with 4 × 106 immature MDDC. Three days later, spleen was removed and splenocytes were fused to SP2 cells at a 2:1 ratio using PEG 1500 (Sigma). Cells were split in eight 96-well plates and selection for hybridomas was accomplished with azaserine and hypoxanthine. Once hybridoma growth was evident, 100 μl of supernatant was removed from each well and subjected to screening for anti-DC-SIGN Abs. To that end, hybridoma supernatants were analyzed by indirect immunofluorescence against a 1/1 mixture of K562 and K562-CD209 cells, and those producing a bimodal distribution of cell fluorescence intensity were selected for further study. After two rounds of cloning, a single hybridoma (6G6) was obtained whose mAb (MR-1) specifically recognized K562-CD209 transfectants as well as immature MDDC.
HIV binding assays
Cells grown under distinct culture conditions (106/well) were pretreated or not with distinct dilutions (1/100, 1/500, 1/2500, 1/10000) of ascitic fluid of the anti-DC-SIGN MR-1 or the anti-CD3 T3b Ab, for 1 h at 4°C. Then HIV-1NL4.3 was added to the wells (multiplicity of infection = 1) and incubated for 3 h at 37°C. Cells were washed five times with 2% FCS in PBS and lysed with 0.5% Triton X-100, and HIV-1 binding was evaluated by an Ag p24 assay (Innogenetics, Barcelona, Spain). For each assay, trypsin was added to parallel wells to control for nonspecific binding.
Results
Production of anti-DC-SIGN mAbs
The full-length DC-SIGN cDNA was amplified from immature MDDC total RNA, and the resulting 1,239-bp fragment was inserted into the pCDNA3.1− expression plasmid and used to generate stable DC-SIGN transfectants in K562 cells (K562-CD209). Expression of DC-SIGN in K562-CD209 was demonstrated using the previously described AZN-D1 anti-DC-SIGN mAb (11) (Fig. 1⇓A). K562 and K562-CD209 cells were mixed (1/1) and the resulting population was used in the screening for hybridomas producing anti-DC-SIGN Abs. One hybridoma yielded a flow cytometry profile compatible with anti-DC-SIGN reactivity (Fig. 1⇓B), and it was selected and grown for further studies. After two rounds of cloning by limiting dilution, it became evident that the selected hybridoma produced an Ab (hereafter termed MR-1) that specifically recognized K562-CD209 cells while it was completely unreactive against untransfected K562 cells (Fig. 1⇓C). The DC-SIGN-specific reactivity of the MR-1 Ab was further demonstrated in flow cytometry and immunofluorescence analysis (see Fig. 1⇓D and below). MR-1 specifically recognized LPS-matured MDDC, which exhibited a high level of expression of MHC class II molecules and the mature MDDC marker CD83. By contrast, MR-1 did not recognize PBL, PHA-activated PBL, monocytes, granulocytes, or endothelial cells (data not shown).
Generation of hybridomas producing DC-SIGN-specific mAbs. A, DC-SIGN expression plasmid pCDNA3.1-CD209 was transfected in K562 cells and stable transfectants selected using G418. Stable expression of DC-SIGN in K562-CD209 was confirmed using the previously described anti-DC-SIGN Ab AZN-D1 (generously provided by Dr. Y. van Kooyk). The fluorescence produced by an irrelevant Ab (X63; open histogram) or AZN-D1 (filled histogram) is shown. B, A 1/1 mixture of K562 and K562-CD209 cells was used for the screening of DC-SIGN-specific hybridoma supernatants, as described in Materials and Methods, and supernatants yielding a bimodal reactivity were selected for further study. C, After two rounds of cloning by limiting dilution, a single hybridoma supernatant (MR-1) was selected which did not recognize untransfected K562 cells but recognized K562-CD209 cells. The fluorescence produced by an irrelevant Ab (X63; open histogram) or MR-1 (filled histogram) is shown. D, MDDC were matured with LPS and plated onto poly-l-lysine-coated coverslips, fixed with formaldehyde, permeabilized, and double-stained with FITC-phalloidin (actin) or MR-1 Ab plus Cy3-goat anti-mouse antiserum (MR-1). Cells were also stained with Abs against MHC class II or CD83. The upper left panel illustrates the cellular morphology using Nomarski optics.
DC-SIGN induction along DC differentiation is IL-4 dependent
The availability of the MR-1 Ab allowed us to dissect the changes in DC-SIGN expression that take place during DC differentiation. To that end, peripheral blood monocytes were isolated and differentiated along the macrophage, immature MDDC, or Langerhans cell-like (20) pathways in the presence of M-CSF, GM-CSF plus IL-4, or GM-CSF plus IL-4 plus TGF-β, respectively. As shown in Fig. 2⇓, M-CSF-treated monocytes retained CD14 on the cell surface and did not acquire CD1a or DC-SIGN expression after a 6-day treatment period. By contrast, immature MDDC and Langerhans-like cells lost expression of CD14 and acquired a high level of CD1a and DC-SIGN expression (Fig. 2⇓). In all cases, GM-CSF plus IL-4-treated monocytes (immature MDDC) showed a higher level of DC-SIGN expression than those cultured in the presence of GM-CSF plus IL-4 plus TGF-β (Fig. 2⇓), suggesting an inhibitory effect of TGF-β on the DC-SIGN expression (see below). Therefore, and in agreement with previous studies (11), DC-SIGN appears to be specifically induced along the DC differentiation pathway.
Acquisition of DC-SIGN expression during monocyte-MDDC differentiation. Peripheral blood monocytes were isolated and subjected to a 6-day treatment with either M-CSF (macrophages), GM-CSF plus IL-4 plus TGFβ (Langerhans cell-like cells), or GM-CSF plus IL-4 (immature MDDC). An aliquot of immature MDDC were additionally treated with LPS for 48 h (mature MDDC). Cell surface expression of CD1a, CD14, CD83, and CD209 (DC-SIGN) was determined on each cell type by indirect immunofluorescence and is represented by filled histograms. Open histograms indicate the fluorescence produced by an irrelevant Ab (X63). The percentage of positive cells (upper number) and the MFI (lower number) are indicated in each case. The experiment was performed twice on different donors and one of the experiments is shown.
To determine the factors responsible for the induction of DC-SIGN along MDDC differentiation, monocytes were treated with GM-CSF, IL-4, or GM-CSF plus IL-4, and the level of DC-SIGN expression was determined after 36–48 h. GM-CSF-treated monocytes retained CD14 expression, did not acquire CD1a, and showed a very weak reactivity with the MR-1 Ab (25% MR-1+ cells and 0.6 MFI) (Fig. 3⇓A). By contrast, IL-4-treated monocytes lost CD14 expression and, although they did not acquire CD1a, exhibited a moderate-to-high level of DC-SIGN expression (56% MR-1+ cells and 1.4 MFI) (Fig. 3⇓A), suggesting that DC-SIGN induction during MDDC differentiation is primarily IL-4 mediated. The combined addition of GM-CSF and IL-4 led to disappearance of CD14 and acquisition of CD1a and yielded the highest DC-SIGN expression after 48 h (78% MR-1+ cells and 2.7 MFI) (Fig. 3⇓A). The induction of DC-SIGN expression by IL-4 was found to be dose dependent, with IL-4 yielding maximum levels of DC-SIGN expression at 200 U/ml, although its effects were observed with concentrations as low as 10 U/ml (Fig. 3⇓B). Altogether these results indicate that the DC-SIGN induction during monocyte-MDDC differentiation is primarily triggered by IL-4 and that GM-CSF enhances the IL-4-dependent induction of DC-SIGN, suggesting that both factors exert a cooperative effect on DC-SIGN expression.
Effect of individual cytokines on DC-SIGN expression: dose-dependent effect of IL-4. A, Peripheral blood monocytes were isolated and either left untreated or treated with either GM-CSF, IL-4, or both factors. After 48 h, cell surface expression of CD1a, CD14, and CD209 (DC-SIGN) was determined on each cell type by indirect immunofluorescence and is illustrated by filled histograms. Open histograms indicate the fluorescence produced by an irrelevant Ab (X63). The percentage of positive cells (upper number) and the MFI (lower number) are indicated in each case. The experiment was performed four times on cells from different donors and a representative experiment is shown. B, Dose response analysis of IL-4-induced DC-SIGN expression. Monocytes were isolated and treated with increasing concentrations of IL-4. At the indicated time points, cell surface expression of CD209 was determined and indicated as expression index: percentage of marker-positive cells multiplied by their MFI. The experiment was performed on two distinct donors and one of the experiments is shown.
In vitro, the monocyte-MDDC differentiation protocol takes 6–7 days and requires the repeated addition of GM-CSF and IL-4 for MDDC to acquire their full repertoire of phenotypic markers and functional capabilities (6, 7, 8, 9). To determine whether a single exposure to the DC differentiation mixture (GM-CSF plus IL-4) sufficed for DC-SIGN expression, a kinetic analysis of DC-SIGN appearance on monocytes was conducted. As shown in Fig. 4⇓A, a single exposure to either IL-4 or GM-CSF plus IL-4 was enough to induce DC-SIGN expression after 24 h, and its expression reached the highest levels after 36–48 h (Fig. 4⇓A). This result was further confirmed upon detection of DC-SIGN mRNA during the initial stages of monocyte-MDDC differentiation. DC-SIGN mRNA was initially detected 6 h after GM-CSF plus IL-4 addition, peaked after 24–36 h, and started to decline thereafter: 48 h after GM-CSF plus IL-4 treatment, DC-SIGN mRNA level was 60% of that observed after 24 h of cytokine addition (Fig. 4⇓B). Therefore, DC-SIGN expression is primarily dependent on IL-4 and is acquired during the initial stages of the monocyte-MDDC differentiation pathway.
Kinetics of the induction of DC-SIGN mRNA and cell surface expression. A, Kinetics of DC-SIGN-inducible expression. Peripheral blood monocytes were isolated and treated with either GM-CSF, IL-4, or both factors. At the indicated time points, cell surface expression of CD209 (DC-SIGN) was determined by indirect immunofluorescence (filled histograms). Open histograms indicate the fluorescence produced by an irrelevant Ab (X63). The percentage of positive cells (upper number) and the MFI (lower number) are indicated in each case. The experiment was performed three times on cells from different donors and a representative experiment is shown. B, Inducibility of DC-SIGN mRNA during monocyte-MDDC differentiation. Peripheral blood monocytes were isolated and treated with GM-CSF plus IL-4. Upper panel, Total cellular RNA was obtained at the indicated time points and DC-SIGN mRNA levels were determined by Northern blot. Lower panel, The loading of each lane in the formaldehyde-containing gel before transfer onto the nitrocellulose membrane. The position of 28S and 18S rRNA is indicated.
Type I and type II IFN inhibit the IL-4-dependent inducibility of DC-SIGN
The IL-4-dependent induction of several genes is known to be primarily mediated through activation of the JAK-STAT pathway, with STAT6 being the key transcription factor mediating IL-4 inducibility (21). The involvement of the JAK-STAT signaling route in the IL-4-dependent induction of DC-SIGN in monocytes was initially evaluated by the use of tyrphostin AG490, an specific inhibitor of JAK2 and JAK3 activation (22, 23). Induction of DC-SIGN by IL-4 was completely abrogated in the presence of AG-490 concentrations known to specifically inhibit JAK2 and JAK3 (Fig. 5⇓A), and further dilution of the inhibitor still prevented a full induction of DC-SIGN (Fig. 5⇓A). Conversely, AG-490 did not affect the expression of CD14 (Fig. 5⇓A). Similarly, the GM-CSF plus IL-4-triggered induction of DC-SIGN was inhibited by AG-490 in a dose-dependent manner (Fig. 5⇓B). In this case, the expression of CD1a was affected in a similar fashion, while CD14 expression was not affected (Fig. 5⇓B). In parallel experiments, the MEK1/2 inhibitor U0126 also diminished the induction of DC-SIGN caused by either IL-4 or the combination of GM-CSF plus IL-4 (Fig. 5⇓), while it left unaltered the expression of CD14 and increased the expression of CD1a in response to GM-CSF plus IL-4 (Fig. 5⇓). Altogether, these results suggest a role for the MEK-extracellular signal-regulated kinase (ERK) and JAK-STAT pathways in the induction of DC-SIGN by IL-4.
Effect of AG-490 and U0126 on the inducible expression of DC-SIGN. Peripheral blood monocytes were isolated and treated with either IL-4 (A) or GM-CSF plus IL-4 (B) and in the absence or in the presence of the JAK2/3 inhibitor AG-490 at two different concentrations (30 μg/ml or 30 ng/ml), or the MEK1/2 inhibitor U0126 (2.5 μM). After 36 h, cell surface expression of CD209 (DC-SIGN), CD14, and CD1a was determined by indirect immunofluorescence. A, Results from IL-4-treated monocytes are shown as flow cytometry profiles, indicating the percentage of positive cells (upper number) and the MFI (lower number), and with open histograms indicating the fluorescence produced by an irrelevant Ab (X63). B, Results from GM-CSF plus IL-4-treated monocytes are given as expression index: percentage of marker-positive cells multiplied by their MFI. The experiment was performed twice on cells from different donors and a representative experiment is shown.
In contrast, the STAT6-mediated IL-4 inducibility of several genes has been found to be inhibited in the presence of either IFN-α or IFN-γ through transcriptional and posttranscriptional mechanisms (24). To find out whether DC-SIGN is subjected to a similar type of regulation, and because initial experiments revealed that IFN-α or IFN-γ did not induce DC-SIGN on monocytes (data not shown), DC differentiation was induced in the presence of either type of IFN. As shown in Fig. 6⇓A, IFN-α greatly reduced the IL-4-dependent induction of DC-SIGN (88% MR-1+ cells and 5.6 MFI vs 26% MR-1+ cells and 0.7 MFI). The inhibitory effect of IFN-α could be seen at concentrations as low as 50 U/ml (Fig. 6⇓B), demonstrating that the IL-4 inducibility of DC-SIGN expression is extremely sensitive to the presence of IFN-α. Although to a lower extent, the IFN-α inhibitory effect was also observed when peripheral blood monocytes were treated with GM-CSF plus IL-4 (94% MR-1+ cells and 13 MFI vs 60% MR-1+ cells and 1.9 MFI) (Fig. 6⇓A). IFN-γ also reduced the IL-4-inducible expression of DC-SIGN, but its inhibitory effect was weaker than that of IFN-α (88% MR-1+ cells and 5.6 MFI vs 64% MR-1+ cells and 2 MFI) (Fig. 6⇓A). Unlike IFN-α, IFN-γ was only capable of preventing the IL-4-mediated induction of DC-SIGN, while it had no effect on the DC-SIGN expression induced by IL-4 and GM-CSF (Fig. 6⇓A). These results confirm the cooperative effect of GM-CSF and IL-4 on DC-SIGN induction and indicate that type I and type II IFN differentially affect DC-SIGN induction during monocyte-MDDC differentiation. Therefore, like other IL-4-regulated genes, induction of DC-SIGN is negatively regulated by either type of IFN. Besides, because treatment of DC-SIGN-expressing immature MDDC with either IFN-α or IFN-γ had no major effect on DC-SIGN expression (Fig. 6⇓C), the negative regulatory role of IFNs appears to be exerted at an early time point during DC differentiation, possibly by inhibiting the STAT-mediated signaling cascades initiated upon IL-4 treatment.
Inhibitory effect of IFN-α and IFN-γ on DC-SIGN inducibility. A, Peripheral blood monocytes were isolated and treated with either IL-4 or GM-CSF plus IL-4 and in the absence or in the presence of IFN-α or IFN-γ. After 36 h, DC-SIGN cell surface expression was determined on each cell type by indirect immunofluorescence (filled histograms). Open histograms indicate the fluorescence produced by an irrelevant Ab (X63). The experiment was performed four times on cells from different donors and a representative experiment is shown. B, Dose response analysis of IFN-α on IL-4-induced DC-SIGN expression. DC-SIGN expression was determined on monocytes treated with IL-4 in the absence or presence of IFN-α at the indicated concentrations. DC-SIGN expression is given as expression index: percentage of marker-positive cells multiplied by their MFI. C, Immature MDDC were either left untreated or treated with IFN-α. After 48 h, DC-SIGN cell surface expression was determined on each cell type by indirect immunofluorescence (filled histograms). The experiment was performed twice with identical results and one of them is shown. In all cases, open histograms indicate the fluorescence produced by an irrelevant Ab (X63), and the percentage of positive cells (upper number) and the MFI (lower number) are indicated.
TGF-β and dexamethasone are negative regulatory factors for DC-SIGN expression
TGF-β is an essential factor for Langerhans cell generation (25), and TGF-β-based protocols have been established that generate Langerhans cell-like cells from monocytes (20). Given the lower DC-SIGN expression obtained upon Langerhans cell differentiation (Fig. 2⇑), we tested whether TGF-β had any effect on the inducible DC-SIGN expression. At a concentration of 10 ng/ml, TGF-β greatly reduced the IL-4-induced DC-SIGN expression (from 74% MR-1+ cells and 2.2 MFI to 33% and 0.7 MFI) (Fig. 7⇓A). To a lower extent, TGF-β also reduced the expression of DC-SIGN induced upon treatment with GM-CSF plus IL-4 (86% MR-1+ and 4.9 MFI vs 67% and 1.8 MFI) (Fig. 7⇓A). The TGF-β inhibitory effect could be observed as early as 24 h and was evident at all time points analyzed (Fig. 7⇓B). Therefore, TGF-β has a direct negative influence on the IL-4-dependent induction of DC-SIGN, which might explain the lower DC-SIGN expression observed when monocytes differentiate in vitro along the Langerhans cell differentiation pathway.
Inhibitory effect of TGF-β and dexamethasone on DC-SIGN inducibility. Peripheral blood monocytes were isolated and treated with either IL-4 or GM-CSF plus IL-4, and in the absence or in the presence of TGF-β (A and B) or dexamethasone (C and D). After 36 h (A, C, and D), or at the indicated time points (B), cell surface expression of CD209 (DC-SIGN) (A, B, and D), CD1a, and CD14 (C) was determined on each cell type by indirect immunofluorescence (filled histograms). In all cases, open histograms indicate the fluorescence produced by an irrelevant Ab (X63), and the percentage of positive cells (upper number) and the MFI (lower number) are indicated. B, Cell surface expression of CD209 is indicated as expression index: percentage of marker-positive cells multiplied by their MFI. Each experiment was repeated three times on cells from different donors with almost identical results, and a representative experiment is shown.
All the above results indicated that DC-SIGN expression is specifically induced during the monocyte-MDDC transition, in agreement with previous reports (11). Because GM-CSF plus IL-4-driven differentiation of monocytes into immature MDDC can be inhibited by glucocorticoids (26, 27), we also tested whether dexamethasone affected DC-SIGN induction. The presence of 10−6 M dexamethasone during the GM-CSF plus IL-4-driven differentiation prevented the acquisition of CD1a, the loss of CD14 expression, and considerably impaired DC-SIGN induction (from 95% MR-1+ cells and 12.6 MFI to 67% MR-1+ cells and 1.4 MFI) (Fig. 7⇑C). Moreover, the induction of DC-SIGN by IL-4 was completely prevented in the presence of 10−6 M dexamethasone (Fig. 7⇑D), and identical inhibitory effects were obtained with 10−7 and 10−8 M dexamethasone (data not shown), underscoring the physiological significance of this inhibitory effect. Therefore, dexamethasone, a well-known inhibitor of the monocyte-MDDC differentiation pathway, inhibits the induction of DC-SIGN that takes place during the process, and completely prevents the IL-4-triggered DC-SIGN expression.
IFN, TGF-β, and dexamethasone prevent the induction of DC-SIGN mRNA
To determine the mechanisms responsible for the effects of IFN-α, TGF-β, and dexamethasone on the DC-SIGN inducibility, the level of DC-SIGN mRNA was analyzed by Northern blot. As predicted from the cell surface expression results, DC-SIGN mRNA was absent in peripheral blood monocytes either untreated or exposed to IFN-α (Fig. 8⇓A). By contrast, monocyte treatment with IL-4 for 6 h led to induction of DC-SIGN mRNA (Fig. 8⇓A). Moreover, although GM-CSF alone had a minor impact on DC-SIGN mRNA, it further enhanced the level of DC-SIGN mRNA induced by IL-4 (Fig. 8⇓A), confirming the collaborative action of both factors on DC-SIGN expression. The inhibitory effect of IFN-α on DC-SIGN induction was also observed at the mRNA level, because IFN-α considerably reduced the DC-SIGN mRNA level yielded upon IL-4 treatment (Fig. 8⇓A). The negative effects of dexamethasone and TGF-β on DC-SIGN expression were also confirmed by analysis of the DC-SIGN mRNA level in monocytes from two independent donors. As shown in Fig. 8⇓B, the presence of either TGF-β or dexamethasone greatly prevented the induction of DC-SIGN mRNA produced by the combination of GM-CSF plus IL-4. All these results indicate that, in monocytes, DC-SIGN mRNA level is greatly up-regulated through the combined action of GM-CSF and IL-4, thus explaining the induction of DC-SIGN cell surface expression along the GM-CSF plus IL-4-driven monocyte-MDDC differentiation pathway, and that IFN-α, TGF-β, and dexamethasone inhibit the acquisition of DC-SIGN expression by decreasing the levels of DC-SIGN mRNA.
Inhibitory effect of IFN-α, TGF-β, and dexamethasone on the DC-SIGN mRNA inducibility during monocyte-MDDC differentiation. Peripheral blood monocytes were isolated and subjected to the indicated treatments. B, The influence of TGF-β and dexamethasone was analyzed on monocytes from two independent donors. After 6 h, total RNA from each culture condition was extracted and DC-SIGN mRNA levels were determined by Northern blot (upper panels). Lower panels, The loading of each lane in the formaldehyde-containing gels before transfer onto nitrocellulose membranes. The position of 28S and 18S rRNA is indicated.
Functional relevance of the regulators of DC-SIGN expression
To evaluate the functional relevance of the positive and negative regulators of DC-SIGN expression, we tested the DC-SIGN-dependent HIV-binding ability of differentiating MDDC in the presence of TGF-β, IFN-α, or dexamethasone. Treatment of monocytes with either IL-4 or GM-CSF plus IL-4 for 48 h, a time point at which DC-SIGN induction has taken place, considerably increased HIV-binding ability, as measured by HIV p24 Ag determination (Fig. 9⇓A). HIV binding was specifically and dose-dependently inhibited by the MR-1 Ab, thus confirming that the increased HIV binding was DC-SIGN dependent (Fig. 9⇓B). In addition, the presence of either TGF-β, IFN-α, or dexamethasone, all of which prevented DC-SIGN induction ( Figs. 6–8⇑⇑⇑), greatly inhibited the IL-4- or GM-CSGF plus IL-4-induced increase in HIV binding (Fig. 9⇓A). Therefore, TGF-β, IFN-α, and dexamethasone block the induction of DC-SIGN caused by IL-4 or during the monocyte-MDDC differentiation and, consequently, prevent the acquisition of DC-SIGN-dependent functions.
HIV binding to differentiating monocytes is mediated by DC-SIGN: inhibitory effect of IFN-α, TGF-β, and dexamethasone. A, Peripheral blood monocytes were left untreated or treated with either IL-4 or GM-CSF plus IL-4, and in the presence or in the absence of TGF-β, IFN-α, or dexamethasone. After 48 h, cells were incubated for 3 h with HIV-1NL4.3 and washed extensively, and bound HIV-1 was quantitated by a p24 detection assay. B, Peripheral blood monocytes were treated with GM-CSF plus IL-4 for 96 h, pretreated with the indicated dilutions of anti-CD3 (T3b) or anti-CD209 (MR-1) Abs (1 h at 4°C), and incubated for 3 h with HIV-1NL4.3. After extensive washing, bound HIV-1 was quantitated by a p24 detection assay. Each experiment was performed on two independent donors with identical results and one of the experiments is shown.
Discussion
DC-SIGN is a MDDC-specific lectin which participates in DC migration and in DC-T lymphocyte interactions at the initiation of immune responses. In addition, DC-SIGN enhances trans-infection of T cells through its HIV gp120-binding ability. The generation of a DC-SIGN-specific mAb has allowed us to determine that the acquisition of DC-SIGN expression during the monocyte-DC differentiation pathway is primarily induced by IL-4, and that GM-CSF cooperates with IL-4 to up-regulate DC-SIGN expression during MDDC differentiation. By contrast, TGF-β, IFNs, and inhibitors of the monocyte-MDDC transition have been identified as negative regulators of DC-SIGN expression and, as a consequence, inhibit the DC-SIGN-dependent binding of HIV-1 to differentiating MDDC. Altogether, these results indicate that DC-SIGN expression is regulated by numerous cytokines and growth factors and suggest that DC-SIGN, considered as a MDDC differentiation marker, is specifically expressed on IL-4-treated monocytes.
The generation of DCs from monocytes depends critically on IL-4 (6, 7, 8), which, based on its capacity to inhibit macrophage colony formation (28), is thought to function mainly by suppressing differentiation along the macrophage pathway. However, the IL-4 inducibility of DC-SIGN and the relevance of the DC-SIGN activities indicate that IL-4 not only suppresses the monocyte/macrophage lineage but actively promotes the differentiation of monocytes along the DC lineage. In this regard, and because of its DC-SIGN-inducing function, IL-4 appears to be required for MDDC to acquire their full repertoire of migratory and T cell-interacting capabilities, because DC-SIGN mediates both DC trafficking (through binding to ICAM-2) and the DC-T cell interactions during the generation of an immune response (through binding to ICAM-3) (29).
IL-4 signals through activation of STAT6 and, in fact, lymphocytes from STAT6−/− mice are unable to up-regulate IL-4-responsive genes (reviewed in Ref. 21). STAT6 mediates the IL-4 inducibility of several genes (30, 31) that exhibit STAT6-binding elements within their gene regulatory regions (32, 33). Accordingly, it is tempting to speculate that DC-SIGN induction is directly regulated by STAT6, a hypothesis supported by 1) the negative effect of the JAK2/3 inhibitor AG-490 on DC-SIGN expression; 2) the presence of consensus STAT-binding elements within the DC-SIGN gene proximal regulatory region (M. Relloso and A. L. Corbí, unpublished observations); and 3) the ability of IFN-α to inhibit DC-SIGN expression. The negative regulatory effect of IFN-α on the expression of several IL-4-regulated genes is now well established (reviewed in Ref. 24), and there are evidences that IFN-α and IFN-γ suppress IL-4-inducible gene expression by inhibiting tyrosine phosphorylation and nuclear translocation of STAT6 (34), possibly via suppressors of cytokine signaling expression (35). Therefore, it is conceivable that inhibition of IL-4-dependent STAT6 activation might contribute to the reduced DC-SIGN expression observed in the presence of IFN-α. By contrast, and despite the capacity of TGF-β to inhibit JAK-STAT activation in other systems (36), the inhibitory effect of TGF-β on DC-SIGN induction might be STAT6 independent because TGF-β fails to suppress STAT6 activation by IL-4 on monocytes (35). In any event, because IL-4 treatment does not lead to the appearance of DC-SIGN on PBL or endothelial cells (data not shown), it appears that cell type-specific factors distinct from STAT6 are also involved in the regulation of DC-SIGN expression.
In addition to IFN and TGF-β, dexamethasone also counteracts the positive effect of IL-4 on DC-SIGN expression. Dexamethasone, which inhibits monocyte-MDDC differentiation (26, 27), not only blocks the GM-CSF plus IL-4-dependent acquisition of CD1a but also that of DC-SIGN. Because dexamethasone is an NF-κB inhibitor (37, 38) and NF-κB activity greatly increases during MDDC differentiation (39), it is possible that DC-SIGN expression is positively regulated by NF-κB factors. This alternative is not supported by our findings that classical NF-κB activators such as LPS or TNF-α do not up-regulate, but reduce, DC-SIGN expression, and that the DC-SIGN proximal promoter is not transactivated by NF-κB proteins (A. L. Corbí, unpublished observations). An alternative possibility, which we favor, is that dexamethasone inhibits DC-SIGN expression by blocking IL-4-triggered intracellular signaling. The ability of dexamethasone to suppress JAK-STAT signaling has been recently reported (40) and indicates that dexamethasone is capable of inhibiting IL-4-induced STAT6 activation on T lymphocytes. Because dexamethasone abolishes the induction of DC-SIGN by IL-4, which by itself does not promote MDDC differentiation, it is reasonable to conclude that dexamethasone prevents DC-SIGN induction by impairing IL-4 signaling and independently of its inhibitory effect on the monocyte-MDDC transition.
Finally, it is worth noting that DC-SIGN expression is oppositely regulated by IL-4 and IFN-γ, whose production is mutually exclusive upon activation and polarization of naive T lymphocytes. IL-4 is the driving cytokine for Th2 polarization and a major product of Th2 lymphocytes, while IFN-γ is the most relevant product of Th1 cells (4, 5). Their differential production by Th1 and Th2 cells underlies the specific features of helper-dependent immune responses: IFN-γ (and other Th1 cytokines) promotes neutrophil recruitment and macrophage activation, thus leading to inflammatory responses, while IL-4 (and other Th2 cytokines) limits Th1-mediated inflammatory responses, thus preventing excessive tissue destruction and restraining inflammation. Consequently, while promoting macrophage differentiation and activation, Th1 lymphocytes would prevent the acquisition of DC-SIGN expression by monocytes within inflamed tissues, while DC-SIGN expression would be induced on monocytes during Th2 responses or once Th2 cytokines are produced to down-regulate Th1 immune responses. Thus, if DC-SIGN expression marks the differentiation of monocytes into DCs, a phenomenon known to occur in vivo (41), MDDC production would depend on the interplay between Th1- and Th2-derived cytokines within an inflamed tissue. According to this hypothesis, monocyte differentiation into DCs would be favored once inflammatory responses are down-regulated, thus allowing the inflamed tissue to regain their normal complement of DCs. Further studies on the mechanisms controlling DC-SIGN expression are required to determine whether its expression on monocytes correlates with DC differentiation and/or the distinct stages during inflammatory responses.
Acknowledgments
We are extremely grateful to Drs. Yvette van Kooyk, Antonio de la Hera, Eva Sanz, María Mittelbrunn, Francisco Sánchez-Madrid, and Carmelo Bernabéu for suggestions, reagents, and help during the course of the study.
Footnotes
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↵1 This work was supported by grants from Comisión Interministerial de Ciencia y Tecnología (SAF98/0068), Comunidad Autónoma de Madrid (08.3/0026/2000.1), and Fondo de Investigaciones Sanitarias (01/0063-01) to A.L.C.
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↵2 Current address: University of California, Davis, CA.
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↵3 Address correspondence and reprint requests to Dr. Angel L. Corbí, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Velázquez 144, 28006 Madrid, Spain. E-mail address: acorbi{at}cib.csic.es
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↵4 Abbreviations used in this paper: DC, dendritic cell; DC-SIGN, DC-specific ICAM-3 grabbing nonintegrin; MDDC, monocyte-derived DC; JAK, Janus kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein/ERK kinase; MFI, mean fluorescence intensity.
- Received September 10, 2001.
- Accepted January 14, 2002.
- Copyright © 2002 by The American Association of Immunologists
References
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