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* Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas, Madrid, Spain;
Servicio de Neumología, Hospital Universitario de la Princesa, Madrid, Spain; and
Servicio de Anatomía Patológica, Hospital General de Guadalajara, Guadalajara, Spain
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN, CD209) is a type II membrane C-type lectin (6, 7) whose IL-4-dependent expression is mostly restricted to interstitial DC, monocyte-derived DC, and a small subset of CD14+ peripheral blood DC (7, 8). Structurally, DC-SIGN contains a mannan-binding lectin domain, a "neck" region composed of seven repeats, and a transmembrane region followed by a cytoplasmic tail containing recycling and internalization motifs (7, 9, 10). DC-SIGN recognizes a large array of pathogens, including HIV (11), Ebola (12), hepatitis C (13, 14) and Dengue virus (15), Leishmania amastigotes (16), Mycobacterium tuberculosis (17, 18), and Candida albicans (19), in a mannan-dependent and Lewis oligosaccharide-dependent manner (20, 21). Moreover, DC-SIGN plays an important role in the establishment of the initial contact between DC and naive T lymphocytes through its recognition of ICAM-3 (7) and also mediates DC trafficking through interactions with endothelial ICAM-2 (22).
Nosocomial opportunistic fungal infections constitute a major life-threatening complication in immunocompromised hosts, particularly in AIDS patients and those subjected to cytotoxic chemotherapy and bone marrow transplantation (23). A large percentage of fungal infections worldwide are caused by Aspergillus species. Specifically, Aspergillus fumigatus appears as the most prevalent airborne fungal pathogen, being responsible for 90% of human cases of aspergillosis and causing fatal invasive infections (invasive aspergillosis) in immunologically impaired individuals (23, 24). In fact, invasive aspergillosis is currently a major direct cause of death in leukemia patients. The small size of the A. fumigatus conidia (2.5–3.5 µm) allow them to be continuously inhaled by humans and to reach all levels of the respiratory tract, where they are captured, ingested, and killed by resident alveolar macrophages in immunocompetent individuals (25). The finding that Th2-type cytokines increase the susceptibility to invasive aspergillosis (26) has prompted studies on the role of DC in Aspergillus infections. In this sense, murine pulmonary DC have been shown to bind and transport A. fumigatus conidia to the draining lymph nodes (27), and in vitro-generated human Langerhans cells were found to bind live A. fumigatus conidia via an unidentified lectin with galactomannan specificity (28). In the present article, we provide evidences that monocyte-derived DC and macrophages bind and internalize conidia from a clinical isolate of A. fumigatus through the DC-SIGN pathogen receptor, and that this binding can be inhibited by Aspergillus-derived galactomannans.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GM-CSF (Leucomax) was purchased from Schering-Plough (Kenilworth, NJ) and used at 1000 U/ml. IL-4 was obtained from PeproTech (Rocky Hill, NJ) and, unless otherwise indicated, used at 1000 U/ml.
Cells
Human PBMC were isolated from buffy coats from normal donors over a Lymphoprep (Nycomed, Norway) gradient according to standard procedures. Monocytes were purified from PBMC by a 1-h adherence step at 37°C in RPMI 1640 supplemented with 10% FCS, 25 mM HEPES, and 2 mM glutamine (complete medium). Nonadherent cells were removed 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 (29). Briefly, monocytes were resuspended at 0,5–1 x 106 cells/ml and cultured in 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 monocyte-derived DC (MDDC). For maturation, immature MDDC were treated with LPS from Escherichia coli 055:B5 at 100 ng/ml for 24–48 h. Evaluation of the maturation-inducing capacity of A. fumigatus was done by culturing 7 x 105 immature MDDC with live or heat-killed conidia at a 1:1 ratio and in the presence of amphotericin B (0.62 µg/ml) to prevent fungal overgrowth. Monocyte-derived macrophages (MDM) were generated by culture of isolated monocytes in complete medium containing 1000 U/ml GM-CSF for 5–7 days. Alternative activation of macrophages was accomplished by treating MDM with IL-4 for 48 h.
DC-SIGN-expressing K562 transfectants (K562-CD209) have been previously described (30) and were cultured in complete medium supplemented with 300 µg/ml G418 (Invitrogen Life Technologies, Grand Island, NY). Isolation of K562-CD209 cells expressing different levels of DC-SIGN was accomplished by cell sorting after staining with the anti-DC-SIGN MR-1 (30) mAb.
Alveolar macrophages were obtained from bronchoalveolar lavages (BAL) of healthy individuals or an acute myeloid leukemia-M2 patient subjected to chemotherapy and diagnosed with invasive pulmonary aspergillosis. BAL fluid was centrifuged and BAL cells were washed and processed for immunostaining or Western blot as indicated. Cytologic samples from lung tissue were obtained for conventional microscopic evaluation and immunocytochemistry. Tissue samples consisted of 5-mm sections of frozen pulmonary tissue obtained from the nonpathologic areas of different surgical lung specimens. Informed consent was obtained from all tissue donors.
Flow cytometry and Abs
Cellular phenotypic analysis was conducted by indirect immunofluorescence. mAbs used for cell surface staining included 9E10 (anti-c-Myc), HB1/5 (anti-CD83; Immunotech, Marseille, France), B-T7 (anti-CD86; Diaclone Research, Besan
on, France), HC1/1 (anti-CD11c), 2.1D10 (anti-CD206; mannose receptor, generously provided by Dr. S. J. Sung, Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, VA), and MR-1 (anti-DC-SIGN, CD209) (30). All incubations were done in the presence of 50 µg/ml human IgG to prevent binding through the Fc portion of the Abs. Flow cytometry analysis was performed with an EPICS-CS (Coulter Científica, Madrid, Spain) using log amplifiers.
Determination of cytokine levels
The level of IL-12 p70 and IL-10 was determined using the OptiEIA human IL-12 p70 set (BD Pharmingen, San Diego, CA) or the IL-10 ELISA set (Immunotools, Friesoythe, Germany), respectively, per the manufacturers’ recommendations. Based on preliminary experiments, supernatants from MDDC were assayed either undiluted or diluted 1/3 in complete medium.
MDDC-induced allogeneic T lymphocyte proliferation
Allogeneic T lymphocytes were obtained from peripheral blood of healthy adults after density centrifugation on Lymphoprep (Nycomed Pharma, Oslo, Norway) and monocyte removal by an adherence step. For T lymphocyte proliferation experiments, 1 x 105 T lymphocytes was stimulated in a flat-bottom 96-well plate with 0.5 x 103 or 2.5 x 103 allogeneic irradiated (150 rad/min for 10 min) MDDC matured with either LPS (100 ng/ml) or A. fumigatus conidia at a 1:1 ratio. Each stimulator:responder ratio was assayed in triplicate. After a 6-day incubation period, [3H]thymidine was added (1 µCi/well) during the last 16 h of coculture and thymidine incorporation was determined to evaluate the level of T cell proliferation.
Fungi: culture conditions and isolation of alkali-extractable water-soluble (F1SS) cell wall polysaccharides
For mycelium production, the fungi were grown as previously described (31). Conidia from A. fumigatus CBS 115.55, a strain derived from a clinical isolate, were obtained from 8-day-old cultures on petri dishes containing Bacto potato dextrose agar medium supplemented with 1 g/L Bacto yeast extract (Difco, Detroit, MI). Conidia were harvested by washing cultures with sterile distilled water and gently scraping the mycelium with a glass rod. Mycelium debris were removed by filtration through two layers of gauze and conidia were collected by centrifugation. Alkali-extractable water-soluble (F1SS) cell wall polysaccharides were prepared and their structures were determined as described by Ahrazem et al. (32).
A. fumigatus-binding assays
Conidia from A. fumigatus were washed twice, resuspended, and incubated in PBS containing 0.1 mg/ml FITC for 1 h at room temperature. Conidia were then extensively washed and either used immediately or stored at –20°C until use. Pathogen-binding assays were performed essentially as described previously (16). Briefly, cells grown under distinct culture conditions were washed, resuspended in complete medium (3 x 105/well), unless otherwise indicated, and pretreated for 20 min at room temperature with anti-DC-SIGN (MR-1), anti-CD206 (2.1D10), anti-CD11c (HC1/1), or anti-CD86 (B-T7) Abs or cell wall polysaccharides from different sources. Then cells were incubated with FITC-labeled A. fumigatus conidia at the indicated ratios, and the binding was allowed to proceed for 30 min at room temperature. After extensive washing to eliminate unbound conidia, cells were fixed with 1% paraformaldehyde for 1 h at 4°C, washed, and analyzed on a Coulter EPICS-CS (Coulter Científica, Madrid, Spain).
Immunofluorescence and immunocytochemistry
After FITC-labeled conidia binding, cells were resuspended in complete medium and allowed to adhere onto poly-L-lysine-coated coverslips (50 x 103 cells/coverslip) for 60 min. After a brief washing step with PBS, cells were fixed and permeabilized in a 1:1 solution of acethone:methanol for 10 min at –20°C, washed, and kept at 4°C until processed for immunofluorescence. Preparations were stained with a rabbit polyclonal antiserum against the neck region of DC-SIGN followed by an incubation with Cy3-labeled goat anti-rabbit Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1/500 in PBS. Coverslips were mounted in fluorescent mounting medium (DakoCytomation, Carpinteria, CA) 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. Alcohol-fixed material was used for immunocytochemical studies. Heat-induced epitope retrieval was performed using a microwave oven-heated citric acid solution (pH 6). For immunocytochemistry, a dextran polymer conjugate technique (EnVision+; Dakocytomed, Glostrup, Denmark) was used. For visualization, peroxidase was used as a chromogen. In frozen tissue sections, incubation of the primary Abs was done directly, without prior heat treatment.
DC-SIGN-dependent adhesion assays
DC-SIGN-dependent adhesion of K562-CD209 transfectants was evaluated using ICAM-3/Fc as specific ligand (kindly provided by Dr. D. Staunton, ICOS, Bothwell, WA). Ninety-six-well microtiter EIA II-Linbro plates were coated overnight with ICAM-3/Fc at 3 µg/ml in 100 mM NaHCO3 (pH 8.8) at 4°C, and the remaining sites were blocked with 0.4% BSA for 2 h at 37°C. Cells were labeled in complete medium with the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein acetoxymethyl ester (BCECF; Poortgebouw, Molecular Probes, The Netherlands) and then preincubated for 20 min at 37°C in RPMI 1640 medium containing 0.4% BSA with or without a function-blocking Ab against DC-SIGN (MR-1) or cell wall F1SS polysaccharides. Cells were then allowed to adhere to each well for 15 min at 37°C. Unbound cells were removed by three washes with 0.5% BSA in RPMI 1640 medium, and adherent cells were quantified using a fluorescence analyzer.
Western blot
Total cell lysates were obtained in 50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 2 mM Pefabloc, and 2 µg/ml aprotinin, antipain, leupeptin, and pepstatin. Ten micrograms of each lysate was subjected to SDS-PAGE under reducing conditions and transferred onto an Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA). After blocking of the unoccupied sites with 5% nonfat dry milk in 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween 20, protein detection was performed using the Supersignal West Pico Chemiluminescent system (Pierce, Rockford, IL). Detection of DC-SIGN was conducted using a polyclonal Ab against the C-terminal 20-residue peptide of DC-SIGN (C-20, sc-11038; Santa Cruz Biotechnology, Santa Cruz, CA) or a polyclonal antiserum raised against a peptide based on the 23-residue repeats that form the neck region of the DC-SIGN. For reprobing, membranes were incubated in stripping buffer (62.5 mM Tris-HCl (pH 6.7), 100 mM 2-ME, and 2% SDS) for 30 min at 50°C with occasional agitation.
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Results |
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Pathogen binding to DC-SIGN relies on the presence of internal mannose residues within oligosaccharide chains (20). To identify additional DC-SIGN-interacting pathogens, cell wall F1SS polysaccharides from fungi responsible for dermatomycosis, tinea (33), and pulmonary infections, and whose structure primarily consist of a mannan core with variable side chain substitutions (34), were screened for their ability to inhibit the adhesion of K562-CD209 cells to ICAM3-coated plates. The results of the screening assay revealed the capacity of fungal cell wall galactomannans to inhibit DC-SIGN-dependent functions (data not shown). Given the clinical relevance of A. fumigatus in pulmonary infections in immunosuppressed individuals (24), the ability of DC-SIGN to interact with A. fumigatus conidia was analyzed using K562-CD209 cells. FITC-labeled A. fumigatus conidia were specifically bound by K562-CD209 in a DC-SIGN-dependent manner, as the interaction was abrogated in the presence of a blocking mAb against DC-SIGN but was not affected by Abs against CD86 or other cell surface molecules (Fig. 1A and data not shown). The binding was evident at a conidia:cell ratio as low as 1:1 (data not shown) and reached maximum levels with a 10:1 ratio (Fig. 1, A and B). Furthermore, the binding was also observed when coincubation was done at 4°C (data not shown) and in the absence of serum (Fig. 1C), indicating that DC-SIGN interacts with conidial components in the absence of any opsonizing molecules. Taken together, these results clearly indicate that DC-SIGN binds A. fumigatus conidia.
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Previous reports have described the ability of murine pulmonary DC and human Langerhans cells to bind, internalize, and transport Aspergillus conidia via the macrophage mannose receptor (CD206) (27) or an unidentified lectin (28), respectively. Therefore, we tested whether the DC-SIGN-dependent recognition of A. fumigatus conidia also took place on the membrane of human MDDC (Fig. 2A). Both immature and mature MDDC bound A. fumigatus conidia at all cell:fungi ratios tested, and the binding was abrogated by a function-blocking Ab against DC-SIGN, but was left unaffected by a mAb against CD86 or against the mannose receptor (Fig. 2B). Immature MDDC exhibited a higher level of A. fumigatus conidia attachment than mature MDDC (Fig. 2B), in accordance with the lower DC-SIGN expression normally exhibited by mature MDDC (Fig. 2A) (7). Therefore, human MDDC binding of A. fumigatus conidia is primarily mediated by DC-SIGN.
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The differential ability of immature and mature MDDC to bind A. fumigatus conidia suggested that the level of expression of DC-SIGN might influence fungal attachment to DC. Since the level of DC-SIGN expression varies among individuals (Ref. 35 and our unpublished results), and low/moderate levels of DC-SIGN can be also found on alveolar macrophages (36, 37), the influence of the DC-SIGN expression level on Aspergillus conidia binding was evaluated. To that end, we initially compared conidial binding to K562-CD209 variants differing in DC-SIGN expression by more than one order of magnitude (Fig. 4A). All variants exhibited a high capacity to bind A. fumigatus conidia when assayed at a 10:1 ratio and, although to a variable extent, retained the DC-SIGN-dependent conidia-binding capacity at lower conidia:cell ratios (Fig. 4B). By contrast, variants with high DC-SIGN expression bound to ICAM-3-coated plates, but those with the lowest level of DC-SIGN weakly bound to surface-bound ICAM-3 (Fig. 4C). Therefore, whereas a low level of DC-SIGN is sufficient to confer the ability to bind A. fumigatus conidia, cells seem to require higher levels of DC-SIGN to display an optimal ICAM-3-binding ability. The differential dependence on the cell surface expression levels of both DC-SIGN-dependent functions is probably explained in terms of a higher avidity of DC-SIGN for multivalent ligands (such as galactomannans on A. fumigatus conidia) than for monovalent ligands (exemplified by individual ICAM-3-Fc molecules bound to a plastic surface).
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Galactomannans from seeds of Ceratonia siliqua have been previously shown to inhibit internalization of A. fumigatus conidia and hypha by murine pulmonary DC (27). To determine the structures involved in conidial recognition by DC-SIGN, A. fumigatus cell wall F1SS polysaccharides were isolated and used as competitors in binding assays. Chemical and nuclear magnetic resonance spectroscopy analyses revealed that the isolated polysaccharide is a galactomannan with the same structure as the polysaccharides from other Aspergillus species (data not shown and Ref. 34). For comparative purposes, Saccharomyces cerevisiae mannans were also included in the experiments, as they have been shown to compete most DC-SIGN-dependent pathogen-recognition functions (20). A. fumigatus cell wall galactomannans dose-dependently reduced conidia binding to either K562-CD209 transfectants or MDDC, with up to 50% inhibition at 50 µg/ml (Fig. 5A). At the same concentration, Aspergillus galactomannan was always slightly less inhibitory than S. cerevisiae mannans (43 vs 68% inhibition in immature MDDC; Fig. 5A). A similar inhibitory effect was observed when Aspergillus galactomannans were used to block the DC-SIGN-ICAM-3 interaction in plate adhesion assays. As shown in Fig. 5B, Aspergillus galactomannan and S. cerevisiae mannan reduced cell adhesion to ICAM-3 by >60%, whereas the blocking Ab against DC-SIGN exhibited the highest inhibitory effect. Altogether these results demonstrate that A. fumigatus cell wall polysaccharides inhibit DC-SIGN-dependent recognition capabilities and that A. fumigatus conidia attachment to immature MDDC is partly dependent on the recognition of outer cell wall galactomannans by DC-SIGN.
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To determine the functional consequences of A. fumigatus binding and internalization by DC, MDDC were incubated with fungal conidia at a 1:1 ratio. After a 24-h incubation, MDDC became adherent, formed homotypic aggregates (Fig. 6A), and exhibited phenotypic characteristics of mature DC, with increased CD86 and induced CD83 expression (Fig. 6B). Analysis of the cytokine profile in Aspergillus conidia-treated cells revealed that MDDC incubated with A. fumigatus conidia for 18 h released higher levels of IL-10 than LPS-matured MDDC (Fig. 6C). By contrast, no IL-12p70 was detected in the supernatant of conidia-treated MDDC, whereas LPS induced the release of low but detectable levels of IL-12p70 (data not shown). In addition, comparison with either immature or LPS-matured MDDC indicated that A. fumigatus conidia-treated MDDC exhibited an enhanced capacity to stimulate allogeneic T lymphocytes (Fig. 6D). Together, all of these morphologic, phenotypic, and functional changes demonstrate that binding and internalization of A. fumigatus conidia trigger MDDC maturation.
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A. fumigatus is responsible for pulmonary infections in immunocompromised patients (24). Since alveolar macrophages constitute the first line of defense against pathogens in the lung and to evaluate the potential pathologic significance of the DC-SIGN-Aspergillus interaction in pulmonary aspergillosis, the presence of DC-SIGN-expressing cells in the lung was analyzed. Immunochemical analysis of BAL cells from healthy individuals revealed the presence of DC-SIGN-positive cells, and immunohistochemical analysis of lung tissue sections revealed the expression of DC-SIGN on both alveolar macrophages and pulmonary DC (Fig. 7A and data not shown), confirming previous results (36, 37). Moreover, immunofluorescence analysis on BAL cells from a patient with invasive aspergillosis also revealed the presence of DC-SIGN-positive cells (Fig. 7A). DC-SIGN expression on alveolar macrophages was further confirmed by means of Western blot and immunofluorescence on cells obtained by BAL of a patient suffering from invasive aspergillosis. As shown in Fig. 7B, an anti-DC-SIGN-specific polyclonal Ab detected a 44-kDa band whose mobility was identical to that of DC-SIGN expressed on the K562-CD209 transfectants. Altogether, these results confirm the presence of DC-SIGN-expressing cells in the lumen of the alveolar cavities, where they are appropriately positioned for Aspergillus conidia capture.
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Discussion |
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The comparison of Aspergillus conidia attachment by distinct variants of K562-CD209 transfectants has revealed a correlation between DC-SIGN expression levels and fungal binding, and a similar result has been observed with other pathogen-recognition activities of DC-SIGN (D. Serrano and A. Corbí, unpublished observations). DC-SIGN expression is tightly regulated by pro- and anti-inflammatory cytokines: IL-4 and IL-13 enhance DC-SIGN expression on monocytes and macrophages (30, 36, 37), whereas IFN-
prevents DC-SIGN induction in response to IL-4 (30). Consequently, the involvement of DC-SIGN in the binding of fungal pathogens (including A. fumigatus conidia) by alveolar macrophages might be ultimately determined by the relative abundance of both cytokine types. Since IL-4 increases the susceptibility to invasive pulmonary aspergillosis by preventing the onset of protective Th1 responses (26, 39), it is tempting to speculate that an IL4-dependent augmented expression of DC-SIGN might increase fungal internalization and contribute to exacerbation of the infection.
Along the same line, DC-SIGN mediates the binding of numerous pathogens to DC, including virus (HIV, hepatitis C virus, Dengue, Ebola), protozoa (Leishmania), and bacteria (Mycobacterium, Helicobacter) (4, 5), and it is worth noting that most DC-SIGN-interacting pathogens modulate immune responses by skewing T lymphocyte polarization toward the Th2 route, what usually leads to disease progression (5). Previous studies have shown that a switch toward a Th2-predominant immune response contributes to the development and unfavorable outcome of invasive aspergillosis (26, 39). Our finding that A. fumigatus conidia trigger human MDDC maturation and IL-10 production, without detectable levels of IL-12p70, implies that fungal recognition and internalization would result in mature DC with a Th2/T regulatory-polarizing phenotype. Since A. fumigatus conidia binding and internalization by MDDC are DC-SIGN dependent, the lectin plays a necessary role in the acquisition of this skewed T cell polarizing capacity, which has been proposed to be ultimately determined by members of the TLR family of pathogen-recognition receptors (40, 41, 42, 43). Whether DC-SIGN-initiated intracellular signals also contribute to the pro-Th2/T regulatory-polarizing phenotype of A. fumigatus-treated DC is currently under investigation. In any event, and given the capacity of galactomannans to inhibit the DC-SIGN-dependent recognition of A. fumigatus conidia, it is tempting to speculate that, like in the case of Mycobacterium mannans (18), Aspergillus-derived galactomannans might contribute to IL-10 production by interacting with DC-SIGN, thus generating DC impaired in their ability to mount a Th1 response.
Galactomannans appear to be the main DC-SIGN ligand on the cell wall of A. fumigatus conidia (Fig. 5) and also reduce the T cell stimulatory ability of human MDDC in allogeneic MLR (Fig. 6). It has been known for a long time that dermatophyte fungi produce substances that diminish immune and inflammatory responses (33). Specifically, dermatophyte-derived mannans are capable of inhibiting lymphoproliferative responses of human mononuclear leukocytes to Ags, mitogens, and an anti-TCR Ab (anti-CD3) in vitro (44). Besides Aspergillus, we have observed that DC-SIGN mediates the binding of dermatophytic fungi containing a variety of mannans as constituents of their cell wall polysaccharides and that dermatophyte-derived mannans can abrogate or inhibit the DC-SIGN-dependent cell adhesion to ICAM-3 (data not shown). Therefore, mannans might prevent the initial interactions that take place between DC and naive T lymphocytes, which are prior to the establishment of the immunologic synapse and thought to depend on the DC-SIGN-ICAM-3 interaction. If this is the case, fungal mannans, through binding to DC-SIGN, could affect not only DC maturation but might also impair Ag presentation by inhibiting the establishment of DC-T lymphocyte contacts.
Like other pathogens, fungi can gain access to host cells via phagocytosis, macropinocytosis and receptor-mediated endocytosis. In the specific case of A. fumigatus, conidia internalization has been demonstrated not only in professional phagocytes but in lung epithelial and endothelial cells (45), with mannose receptor proposed as the major player in A. fumigatus binding and capture by murine phagocytic cells (46). Regarding DC, previous studies in the murine system have shown that pulmonary DC capture and transport A. fumigatus conidia to the lymph nodes and that conidia internalization is mainly mediated via the mannose receptor and DEC-205 lectins, with the CD11b/CD18 integrin playing a minor role (27). By contrast, the binding of live A. fumigatus conidia to in vitro-generated human Langerhans cells has been described to take place independently of the mannose receptor or Langerin (28). These latter results are in agreement with our data since we have observed that A. fumigatus conidia binding to MDDC is greatly reduced in the presence of blocking Abs against DC-SIGN and that IL-4-treated in vitro-generated human macrophages also bind A. fumigatus in a DC-SIGN-dependent manner. The distinct recognition of Aspergillus conidia by murine and human DC might be explained by a differential expression of lectin receptors on DC from distinct tissue locations or by the existence of species-specific receptors. In this regard, although five murine molecules have been described with homology to the DC-SIGN lectin domain (47), they all differ from DC-SIGN in either their domain structure or their tissue distribution.
In summary, the present report constitutes the first description of a cell surface receptor which confers human DC and macrophages the ability to bind and internalize A. fumigatus conidia and, given the ability of DC-SIGN to bind C. albicans (19), highlights a critical role for DC-SIGN in binding/uptake of the pathogenic agents responsible for the two most prevalent life-threatening fungal infections in immunocompromised patients.
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Footnotes |
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1 This work was supported by the Ministerio de Ciencia y Tecnología (Grant SAF2002-04615-C02–01), Fundación para la Investigación y Prevención del SIDA en España (FIPSE 36422/03), and Fondo de Investigaciones Sanitarias (Grant 01/0063–01) to A.L.C. This project has also been supported by a grant from Red Respiva (RTIC C03/011)-ISCIII-SEPAR. 
2 Address correspondence and reprint requests to Dr. Angel L. Corbí, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas, Ramiro de Maeztu, 9, Madrid 28040, Spain. E-mail address: acorbi{at}cib.csic.es
3 Abbreviations used in this paper: DC, dendritic cell; DC-SIGN, DC-specific ICAM-3-grabbing nonintegrin; MDDC, monocyte-derived DC; MDM, monocyte-derived macrophage; BAL, bronchoalveolar lavage; BCECF, 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein acetoxymethyl ester.
Received for publication February 9, 2004. Accepted for publication August 12, 2004.
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 J. B. Torrelles, A. K. Azad, and L. S. Schlesinger Fine Discrimination in the Recognition of Individual Species of Phosphatidyl-myo-Inositol Mannosides from Mycobacterium tuberculosis by C-Type Lectin Pattern Recognition Receptors J. Immunol., August 1, 2006; 177(3): 1805 - 1816. [Abstract] [Full Text] [PDF]
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 S. O. Dionne, A. B. Podany, Y. W. Ruiz, N. M. Ampel, J. N. Galgiani, and D. F. Lake Spherules Derived from Coccidioides posadasii Promote Human Dendritic Cell Maturation and Activation Infect. Immun., April 1, 2006; 74(4): 2415 - 2422. [Abstract] [Full Text] [PDF]
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G. M. Gersuk, D. M. Underhill, L. Zhu, and K. A. Marr Dectin-1 and TLRs Permit Macrophages to Distinguish between Different Aspergillus fumigatus Cellular States J. Immunol., March 15, 2006; 176(6): 3717 - 3724. [Abstract] [Full Text] [PDF]
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V. Gafa, R. Lande, M. C. Gagliardi, M. Severa, E. Giacomini, M. E. Remoli, R. Nisini, C. Ramoni, P. Di Francesco, D. Aldebert, et al. Human Dendritic Cells following Aspergillus fumigatus Infection Express the CCR7 Receptor and a Differential Pattern of Interleukin-12 (IL-12), IL-23, and IL-27 Cytokines, Which Lead to a Th1 Response Infect. Immun., March 1, 2006; 74(3): 1480 - 1489. [Abstract] [Full Text] [PDF]
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A. Vallon-Eberhard, L. Landsman, N. Yogev, B. Verrier, and S. Jung Transepithelial Pathogen Uptake into the Small Intestinal Lamina Propria J. Immunol., February 15, 2006; 176(4): 2465 - 2469. [Abstract] [Full Text] [PDF]
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 J. E. Crowther and L. S. Schlesinger Endocytic pathway for surfactant protein A in human macrophages: binding, clathrin-mediated uptake, and trafficking through the endolysosomal pathway Am J Physiol Lung Cell Mol Physiol, February 1, 2006; 290(2): L334 - L342. [Abstract] [Full Text] [PDF]
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K. Vermaelen and R. Pauwels Pulmonary Dendritic Cells Am. J. Respir. Crit. Care Med., September 1, 2005; 172(5): 530 - 551. [Abstract] [Full Text] [PDF]
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