| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Published online before print April 23, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* The Edward Jenner Institute for Vaccine Research, Compton, Berkshire, United Kingdom; and
Sir William Dunn School of Pathology, Oxford University, United Kingdom
1Correspondence: The Edward Jenner Institute for Vaccine Research, Compton, Berkshire, RG20 7NN, UK. E-mail: delyth.wong{at}jenner.ac.uk
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
s), neutrophils, and dendritic cells (DCs). On Ms and bone marrow-derived DCs, it has been shown to mediate the nonopsonic recognition of and response to soluble and particulate yeast ß-glucans. We have optimized the immunohistochemical detection of Dectin-1 and demonstrated its expression on neutrophils, subpopulations of Ms in splenic red and white pulp, alveolar Ms, Kupffer cells, and Ms and DCs in the lamina propria of gut villi. This is consistent with its role in pathogen surveillance. A significant proportion of CD11c+ splenic DCs expressed Dectin-1, but expression was not restricted to any one subset. Dectin-1 expression was low on resident Ms and DCs of skin and was not detected on resident Ms or DCs in kidney, heart, brain, or eye. The proposed, additional role of Dectin-1 as a coreceptor for T cell activation is supported by its expression on DCs in the T cell areas of the spleen and lymph nodes. Strong expression of Dectin-1 on subpopulations of Ms and DCs in the medullary and corticomedullary regions of the thymus suggests a role distinct from pathogen recognition. Tissue localization thus revealed potential roles of Dectin-1 in leukocyte interactions during innate immune responses and T cell development.
Key Words: innate immunity • macrophages • dendritic cells • spleen • thymus development
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
s), and it mediates their cellular responses to zymosan and live yeast pathogens [3
, 4
].
The mouse Dectin-1 gene was cloned independently by subtractive cDNA cloning for dendritic cell (DC)-specific genes [5
] and by screening a retroviral cDNA library derived from a M cell line for binding to zymosan particles [1
]. The cDNA of mouse Dectin-1 encodes a protein that consists of a C-type lectin-like domain [6
], a stalk, and a cytoplasmic tail with an immunoreceptor tyrosine-based activation motif (ITAM). The human homologue of Dectin-1 is 72% identical at the amino acid level and similar in structural organization to the mouse protein, but there are two major and several minor human Dectin-1 isoforms as a result of alternative mRNA splicing [7
]. Mouse and human Dectin-1 function as pathogen-recognition receptors that recognize particles such as zymosan, Saccharomyces cerevisiae, and heat-killed Candida albicans in a ß-glucan-dependent manner [1
]. Zymosan binding to Dectin-1-transfected RAW cells activates tyrosine phosphorylation of this receptor and results in the generation of reactive oxygen species (ROS) [8
]. The ITAM domain in the cytoplasmic tail of Dectin-1 is also required for the production of tumor necrosis factor 
(TNF-) by transfected cells in response to zymosan and live fungal pathogens [4
]. It is important that Toll-like receptor 2 acts cooperatively with Dectin-1 in response to zymosan and live fungal pathogens by enhancing the production of cytokines such as TNF- and interleukin (IL)-12 in Ms and bone marrow (BM)-derived DCs. The biological activities mediated by Dectin-1, as measured by in vitro assays, depend on the cell-surface expression of this receptor on M cell lines, transfected cells, and BM-derived DCs [4
, 8
].
Analysis of Dectin-1 expression on mouse spleen, primary Ms, BM, and peripheral blood by fluorescein-activated cell sorter (FACS) has demonstrated that its expression is not restricted to DCs as reported previously [5
] but also on cells of the monocyte/M and neutrophil lineages [9
]. Much lower levels of Dectin-1 expression were detected on minor lymphoid populations. Northern blot analysis revealed the presence of Dectin-1 transcripts in the thymus [9
], which suggests that Dectin-1 may also have a role distinct from pathogen recognition and generation of proinflammatory responses, but the identity and location of Dectin-1+ cells in the thymus and other tissues have not been reported. Previous findings that soluble Dectin-1 bound T cells and promoted T cell proliferation in the presence of suboptimal concentrations of anti-CD3 antibodies suggest that Dectin-1 is a coreceptor for T cell activation [5
]. It is interesting that T cell binding to NIH-3T3 cells transfected with mouse or human Dectin-1 is not inhibitable by soluble ß-glucans, which indicates the existence of a ß-glucan-independent binding site on Dectin-1 for an unknown T cell ligand [1
, 7
]. To gain further insights into the role of Dectin-1 in the thymus and in immune and proinflammatory responses, we have analyzed its tissue distribution in situ and its expression in newborn tissues.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Monoclonal antibodies (mAb)
mAb 2A11 [immunoglobulin G (IgG)2b] was developed from a Fischer rat immunized i.p. with NIH-3T3 cells transduced with full-length Dectin-1 [1
] and boosted with soluble, recombinant hemagglutinin-tagged Dectin-1 [3
]. mAb 2A11 was shown to specifically recognize the mouse ß-glucan receptor, Dectin-1. Commercially available, biotinylated mAb used in immunocytochemisty double-labeling experiments were from BD PharMingen (anti-mouse CD3, CD4, CD8, CD11c, and CD19; respective clones were 17A2, H129.19, 53-3.7, HL3, and 1D3) and Serotec (F4/80, FA11 for macrosialin/CD68 and 3D6 for sialoadhesin). The isotype control antibodies were purchased from BD PharMingen.
Immunohistochemistry
Single-labeling immunocytochemistry
Frozen sections of mouse tissues (5 µm) were cut (Leica cryostat) and collected on polylysine or electrostatically charged slides (VWR International, Ltd., Poole, UK), fixed in ethanol (4°C), air-dried, and stored frozen before use. Sections were rehydrated in phosphate-buffered saline (PBS) for 5 min, followed by blocking in 10% normal rabbit serum for 10 min. The slides were then drained and incubated with purified mAb 2A11 or the rat IgG2b isotype control at 10 µg/ml for 1 h. Antibodies used for the labeling of serial sections were used at dilutions recommended by suppliers. As the epitope of Dectin-1 recognized by the mAb 2A11 was sensitive to H2O2 treatment, the endogenous peroxidase quenching was delayed until after labeling with the primary antibodies. Endogenous peroxidase was quenched using 0.3% H2O2 (Sigma-Aldrich Co. Ltd., St. Louis, MO) in 5% normal rabbit serum for 4 min. After washing in PBS, sections were incubated with biotinylated mouse-adsorbed rabbit anti-rat IgG for 30 min. The slides were rinsed in PBS and treated with the avidin-biotin-complex (ABC)–horseradish peroxidase (HRP) reagent for 30 min. After rinsing in PBS, the slides were treated with the HRP substrate NovaRed. All reagents other than the primary antibodies were from Vector Laboratories (Burlingame, CA) unless otherwise stated. Slides were counterstained with Gill’s formula haematoxylin (Vector Laboratories), dehydrated with alcohol, cleared with Histoclear (Fisher Scientific, Loughborough, UK), and mounted with DPX (Fluka Chemical Corp., St. Louis, MO).
Double immunolabeling
Using HRP, color combinations were optimized for each double-labeling experiment. HRP was preferred over alkaline phosphatase, as it gave crisper, less diffuse reaction products. NovaRed and Vector SG gave the best contrast. Most immunocytochemistry was performed initially with mAb 2A11 and developed with NovaRed substrate as above. Thereafter, the slides were treated again with H2O2, as above, to quench the specifically bound HRP. The sections were then blocked for 30 min with 10% normal rat serum (Serotec, Oxford, UK), and exposed sites on the already-bound ABC were blocked using the avidin-biotin blocking kit from Vector Laboratories. Then, biotinylated, primary antibodies (to CD3, CD19, or CD11c and their isotype controls) were applied for 1 h. After rinsing, the sections were probed again with ABC–HRP, followed by development with Vector SG. In the case of double-labeling for Dectin-1 with CD4 or CD8 in the thymus, best results were obtained when biotinylated anti-CD4 or -CD8 was applied first and developed with NovaRed. After the appropriate blocking, as described above, thymus sections were incubated with biotinylated 2A11 and developed with Vector SG.
For analysis by fluorescence microscopy, when fluorochromes were used for antigen localization, the following modifications in the procedure were used: Biotinylated hamster anti-CD11c was used to study DCs and was detected with streptavidin AlexaFluor 568 (red) or 488 (green; Molecular Probes, Junction City, OR); 2A11 was detected with rabbit anti-rat Ig AlexaFluor 568 or 488. The marginal zone (MZ) metallophilic Ms were detected using cysteine-rich (CR)-Fc (a gift from Dr. Luisa Martinez-Pomares, Oxford University, UK). The CR-Fc is a chimeric molecule consisting of the CR region of the mouse M mannose receptor and human IgG1 Fc chain. The CR binds specifically to MZ metallophilic Ms [10
] and was detected with biotinylated anti-human IgG (Jackson Laboratory, Bar Harbor, ME), followed by streptavidin AlexaFluor 568. CR-Fc labeling was used together with mAb 2A11 and anti-rat AlexaFluor 488. Double labeling for Dectin-1 and macrosialin/CD68 was determined using biotinylated mAb 2A11 or its isotype control followed by streptavidin AlexaFluor 568 and fluorescein isothiocyanate (FITC)-FA11. No counterstain was performed, and the slides were mounted in Vectashield (Vector Laboratories). Results were recorded using software for the Leica (Knowlhill, UK) TCS NT confocal system.
DC isolation
Splenic DCs were obtained using a variation of the method described by Vremec et al. [11
]. Briefly, spleens from six to eight C57BL/6 mice were perfused with RPMI-1640 medium containing 10% heat-inactivated fetal calf serum (FCS), collagenase (1 mg/ml, type III, Worthington Biochemical Corp., Lorne Laboratories, Twyford, Reading, UK), DNase I (325 K units/ml, Sigma-Aldrich Co. Ltd.), 0.1 M EDTA, pH 7.2, and 100 units/ml polymyxin B. Spleens were digested for 30 min at 37°C, then the remaining tissue was disrupted mechanically, and the homogenate was passed through a 0.40-µm cell sieve. Cells were pelleted, resuspended in Nycodenz (1.077 g/ml, Life Technologies Ltd., Paisley, UK), layered on Nycodenz, and centrifuged in a Sorval RT7plus at 2000 g for 20 min. The low-density fraction was collected. This was stained directly or subjected to further purification of CD11c+ cells. In the latter case, cells were incubated on ice with anti-CD11c microbeads (Miltenyi Biotech Ltd., Bisley, Surrey, UK) for 20 min. The positive fraction was recovered by passing over a magnetic cell sorter separation column (Miltenyi Biotech Ltd.) and checked on a FACScalibur® (Becton Dickinson UK Ltd., Oxford) for purity; 98% purity was attained routinely.
Cell staining and flow cytometric analysis
Aliquots of 2–5 x 105 cells were treated with Serotec clone 2.4G2, an anti-Fc receptor for IgG types II/III (anti-CD16/32) antibody, to block Fc binding to FcR. Cells were then stained with mAb 2A11 conjugated to AlexaFluor 647 or a similarly conjugated IgG2b isotype control, together with BD PharMingen (San Jose, CA) antibody to CD11c (HL3, conjugated to FITC), CD4 [L3T4, conjugated to phycoerythrin (PE)], CD8 (Ly-2, conjugated to PE), and/or B220 (RA3-6B2, conjugated to PE) in PBS containing 2% FCS and 0.1% NaN3. The 2A11 mAb and the IgG2b isotype control were conjugated with AlexaFluor 647 (Molecular Probes) according to the manufacturer’s instructions. After staining, cells were fixed with 0.1–0.3% paraformaldehyde and analyzed on a FACScalibur using CellQuest software (Becton Dickinson UK Ltd.).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Ms and DCs in murine lymphoid tissues
We have previously studied the distribution of Dectin-1 surface expression in the blood and spleen by FACS analysis and reported that this receptor is broadly expressed with the highest levels found on monocytes, Ms, and neutrophils [9
]. However, there is considerable heterogeneity of Ms and DCs in any given tissue, especially in the spleen where M subpopulations are defined not only by phenotypic markers but also by their anatomical location. Both of these characteristics often reflect their functional capability and ability to interact with other cells in vivo [12
13
14
15
]. Identification of Dectin-1-expressing cells and their location within lymphoid tissues by immunohistochemistry would thus provide additional insights into the physiological role of Dectin-1. We focused our studies on Ms and DCs because of their important roles in linking innate and adaptive immune responses. In the spleen, mAb 2A11 labeled distinct populations of cells within the red and white pulp (RP and WP, respectively). mAb commonly used for identifying Ms and DCs are shown for comparison (Figs. 1
and 2
).
|
|
and DC markers (Fig. 1
and unpublished results). The mAb identified Dectin-1+ cells in M- and DC-occupied areas, which is consistent with its expression on subpopulations of both cell types. The 2A11 mAb recognized cells within the RP, the T cell areas of the WP (Fig. 1a
, small arrowhead), and also, a few within the MZ (Fig. 1a
, large arrowhead). Two mAb used for identifying Ms were compared with 2A11. First, the FA11 mAb, which identifies a lysosomal component (macrosialin/CD68) present in populations of Ms and DCs, strongly labeled RP Ms as well as some cells within the MZ and WP (Fig. 1b)
. The second mAb, F4/80, strongly labeled the RP Ms (Fig. 1c)
. In comparison with these two M markers, the mAb 2A11 labeling of RP was more heterogeneous. This might reflect the state of activation of these cells and the presence of a variable number of neutrophils and monocytes that are known to express Dectin-1 [9
]. Like F4/80, the 2A11 mAb did not recognize many MZ Ms or metallophilic Ms that were identified by a mAb to sialoadhesin/CD169 (Fig. 1d) . Dectin-1+ cells were occasionally detected in the outer MZ (Fig. 1a
, large arrowhead) but were more common at sites close to the central arteriole (Fig. 1a
, small arrowhead). From the anti-CD11c mAb labeling shown for comparison in Figure 1e , where many cells were CD11c+ at sites in the MZ, Dectin-1 was expressed on fewer cells than CD11c.
In lymph node, Dectin-1+ cells were most abundant within the paracortex adjacent to the B cell follicles and in the medullary regions. There were a number of Dectin-1+ cells within the follicles and around the germinal centers (Fig. 1f
, arrowhead), which were identified by biotinylated peanut agglutinin lectin staining of serial sections (unpublished results). In this location, some Dectin-1+ cells might be tingible body Ms involved in the phagocytosis of apoptotic centrocytes. The FA11 mAb, shown for comparison, labeled all areas of the lymph node, including several cells within the follicles (Fig. 1g)
. F4/80 labeled similar areas to that of FA11, but unlike 2A11 and FA11, it did not label within the follicles (Fig. 1h)
. There was little 2A11 labeling in the subcapsular sinus, an equivalent area to the MZ of the spleen where sialoadhesin labeling was strong (Fig. 1f and 1i
, respectively). The labeling for CD11c (Fig. 1j)
within the lymph node was much stronger than that for Dectin-1, although both labeled the paracortex.
Microanatomical location and identity of Dectin-1+ cells in spleen
Several Dectin-1+ cells were seen within the T cell areas of the WP, identified by labeling for CD3 and also by the presence of the central arteriole (Fig. 2a)
. The majority of Dectin-1+ cells was stellate in morphology, a distinct feature of DCs, and was in close contact with lymphocytes in the T cell area (Fig. 2b)
. This contact between Dectin-1+ cells and lymphocytes was not observed in the RP. Double labeling for CD19 and Dectin-1 revealed a few Dectin-1+ cells among the B cells but no same cell colocalization (Fig. 2c)
. Our previous FACS analysis of splenocytes has demonstrated that GR-1+ (a granulocyte marker) neutrophils highly express Dectin-1. It was possible to identify neutrophils by their nuclear morphology, and many of them in the splenic RP were Dectin-1+ (Fig. 2d
, arrowheads). Double-labeling immunofluorescence using mAb FA11 or anti-CD11c in conjunction with mAb 2A11 was performed to further analyze Dectin-1+ cells. These experiments showed that a small proportion of the CD11c+ cells were also positive for Dectin-1, suggesting that the double-labeled cells (yellow) were a subpopulation of DCs (Fig. 2e
, arrowheads). In contrast, double labeling with FA11 revealed that the majority of the FA11+ cells within the PALS was also Dectin-1+ (Fig. 2f) . Thus, the 2A11+ cells within the PALS likely represented a mixed population of DCs and Ms, and the M population represented the larger population of Dectin-1+ cells. Dectin-1-expressing cells were seen among the CD11c+ cells in the MZ area (Fig. 2g) , and a proportion of cells coexpressed these molecules (arrowheads). Although the CD11c–/Dectin-1+ cells within the MZ are likely to be Ms, clearly not all MZ Ms express Dectin-1. The labeling pattern of mAb 2A11 is very different from that of sialoadhesin (Fig. 1a
and 1d)
and in double immunofluorescence, mAb 2A11 did not recognize the MZ metallophilic Ms identified by CR-Fc labeling (Fig. 2h)
. Figure 2h
shows a region of MZ at a bridging zone [16
]. Here, the PALS is close to the MZ, and there are many CD11c+ DCs in these regions. mAb 2A11 labeling demonstrated the presence of Dectin-1+ cells within the bridging zone (Figs. 1a
and 2h)
, but inspection of many sections labeled for CD11c and Dectin-1 revealed only small numbers of cells expressing both molecules as shown in Figure 2g
.
Dectin-1 expression on splenic DC subpopulations
Subsets of DCs are defined by a combination of phenotypic markers, CD11c, CD4, CD8, and B220, and activation of different subsets helps to shape the subsequent adaptive immune response [17
]. The labeling of Dectin-1 on splenic DCs in the T cell zone (Figs. 1a and 2b
and 2e)
prompted us to investigate whether Dectin-1 is expressed preferentially by a particular DC subset. Splenic DCs were thus further purified from low buoyant density cells isolated from normal spleens using positive selection for CD11c and analyzed by FACS for 2A11 labeling. CD11c+ cells constitute 2–3% of the total splenocytes (unpublished results). A significant proportion (
20%) of CD11c+ cells were Dectin-1+. In contrast, the isotype control mAb stained less than 0.2% of the total CD11c+ cells (Fig. 3a
). Dectin-1 expression was consistently found on all subsets of splenic DCs (Fig. 3b)
, and the mean of fluorescence intensity for the individual subsets was very similar (unpublished results).
|
s and DCs, which are located in areas where the process of negative selection of thymocytes occurs.
|
areas of the Peyer’s patches, and along the lamina propria of the intestinal villi (Fig. 4g
and 4h)
. The labeling within the Peyer’s patches was similar to that seen with anti-CD11c on serial sections (unpublished results).
Ms and DCs in murine nonlymphoid tissues
Low numbers of Dectin-1+ cells were found within the dermal layer of BALB/c skin (Fig. 4i
and 4j)
. No Dectin-1+ cells were detected within the epidermal layer (Fig. 4j)
. Within the dermis, there were a few round cells weakly positive for Dectin-1 and major histocompatibility complex (MHC) class II molecule (unpublished results), which might represent plasmacytoid DCs, but the identity of these cells remains to be determined. Liver sinusoidal Kupffer cells were Dectin-1+, but significantly, endothelial cells were not (Fig. 4k
and 4l)
. Dectin-1+ cells were observed throughout the lung sections (Fig. 4m
and 4n)
, and double labeling for macrosialin/CD68 suggested that these were alveolar Ms (unpublished results) consistent with our previous FACS analysis [9
]. In sections of kidney, muscle or cardiac tissue mAb 2A11 weakly labeled a few small cells near blood vessels that were thought to be monocytes and neutrophils (unpublished results). Also, sections of whole mouse eye or brain were not labeled, which suggests that Dectin-1 is not normally expressed at detectable levels on cells in these tissues, including choroid Ms, retinal pigment epithelial cells of the eye and microglia, periarteriole, choroid plexus, and meningeal Ms of the brain.
Dectin-1 expression appears early in development
We have previously shown Dectin-1 to be strongly expressed in adult mouse thymus by Northern blot analysis [9
], and in this study, we have determined the location of Dectin-1+ cells in the thymus (Fig. 4a
4b
4c
4d
4e
4f)
. A possible role for Dectin-1 on thymic DCs in T cell development and its functions on other DCs and Ms prompted us to investigate Dectin-1 expression in newborn and young mice. We found Dectin-1 expression in all tissues examined by the time of birth in the mouse (Fig. 5
and unpublished results). In the developing thymus, clear differences could be seen in the distribution of Dectin-1-, CD11c-, and macrosialin/CD68-positive cells (Fig. 5
, top three panels). In the newborn thymus, although CD11c was largely restricted to the medulla, the mAb 2A11-labeled cells were more numerous in the cortex and might represent a population of Ms present at this time. By age 2 weeks, although cells in the cortex still expressed Dectin-1, there were greater numbers of Dectin-1+ cells in the medulla and around the corticomedullary junction (arrowhead). Macrosialin/CD68, as in the adult thymus (unpublished results), was distributed throughout the cortical and medullary regions.
|
lineage than DCs (Fig. 5
, middle two panels). Two weeks further into development, the localization of CD11c and Dectin-1 became more organized, and the spleen more closely resembled that of the adult. In newborn and 2-week-old mice, Dectin-1 expression in the lung (Fig. 5
, bottom panels), gut, and liver (unpublished results) was similar to that found in adult tissues (Fig. 4)
.
Regulation of splenic Dectin-1 expression in response to LPS in vivo
We have previously reported that cytokines and other agents affect Dectin-1 expression and function on resident and thioglycollate-elicited murine peritoneal Ms in vitro. In particular, Dectin-1 was highly up-regulated by T helper cell type 2 cytokines, IL-4 and IL-13, and down-regulated by IL-10 and LPS [18
]. We have investigated Dectin-1 expression in the spleen following a low dose (10 µg) of LPS injection. LPS clearly decreased Dectin-1 expression within the T cell areas around the central arterioles after 24 h (Fig. 6b
). In contrast, there appeared to be no dramatic changes with CD11c labeling at the MZ or Dectin-1 labeling in the RP (Fig. 6a
and 6b
, respectively). The effect was found to be relatively short-lived, and the mAb 2A11 immunohistochemistry on spleens taken after 48 h appeared similar to that of untreated, control spleens (Figs. 6c
and 1a
, respectively).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Dectin-1 expression was found in a variety of lymphoid and nonlymphoid tissues. Where the comparison can be made, the results are in agreement with our previous Northern analysis of Dectin-1 mRNA expression in various mouse tissues with the possible exceptions of kidney, heart, and skin [9 ]. It is possible that blood cells trapped within the heart and kidney tissues contributed to the detection of Dectin-1 mRNA by Northern analysis. In skin, the numbers of Dectin-1+ cells were low and were MHC class II-low or -negative, possibly representing migratory plasmacytoid DCs. The presence of Dectin-1 on this important type I interferon-producing DC subset requires confirmation from a more in-depth analysis.
The main cell types expressing Dectin-1 in all the tissues examined were Ms and DCs, although high expression was also detected in neutrophils in the splenic RP. This expression pattern is in good agreement with our previous FACS analysis of Dectin-1 surface expression on cells isolated from the spleen, blood, BM, lung, and the peritoneal cavity [9
]. Immunohistochemical labeling studies revealed considerable heterogeneity of Dectin-1 expression on M and DC populations. Part of the heterogeneity of Dectin-1 expression is likely to be a result of the different states of activation/maturation of Ms and DCs based on the following observations. High expression of Dectin-1 on Ms and DCs was found in tissues such as the lung, gut, and spleen, which are frequently exposed to microbes and microbial products, whereas Dectin-1 was not detected on quiescent Ms from immune-privileged sites such as the brain and eye or on Langerhans cells of normal skin. Dectin-1 is expressed on a significant and often variable proportion (20–40%) of splenic DCs but not restricted to any one DC subset as determined by FACS analysis. Our previous finding that resident peritoneal Ms had a much higher Dectin-1 expression following 1 day in culture also supports this conclusion [9
]. We have recently determined that Dectin-1 expression and function on primary Ms could be modulated by cytokines such as IL-4 and IL-13 and other agents including LPS [18
]. It is therefore likely that Dectin-1 expression on Ms and DCs in vivo is differentially regulated depending on the cell types and their local milieu. In the present study, we have found that there was a clear reduction of mAb 2A11 labeling in the splenic T cell areas after 24 h of LPS injection. Although this finding is consistent with our previous results showing that LPS down-modulates Dectin-1 expression on resident and thioglycollate-elicited Ms, we cannot exclude the possibility that Dectin-1-positive cells have emigrated or undergone apoptosis [21
]. In contrast, it was difficult to assess Dectin-1 expression changes in the RP following LPS treatment, as the mAb 2A11 labeling in this area was not strikingly different from the controls. It was not possible to examine the regulation of Dectin-1 expression in response to zymosan or ß-glucans, as binding of these pathogen-derived ligands to Dectin-1 blocked mAb 2A11 labeling of this receptor in vitro and in vivo (ref. [3
] and unpublished results, respectively). Studies are now in progress to determine the regulation of Dectin-1 expression on Ms and DCs during the course of microbial challenge to gain further insights into the role of Dectin-1 in innate immunity and adaptive immune responses.
It is known that Ms and DCs exhibit heterogeneity in their surface expression of phenotypic markers within the same tissue [22
, 23
]. A comparison of Dectin-1 with several M and DC markers revealed its expression on subpopulations of Ms and DCs within splenic RP, MZ, and WP, as Dectin-1 did not colocalize completely with F4/80+, FA11+, or CD11c+ cells. Few MZ Ms and DCs expressed Dectin-1, and they did not correspond to those expressing sialoadhesin or CR-Fc ligands, which include MZ Ms and metallophilic Ms [10
]. Expression patterns in the mouse spleen of three C-type lectin receptor family members are also different from that of Dectin-1. The M mannose receptor is expressed in the RP but not WP or MZ [24
], murine specific intercellular adhesion molecule 3-grabbing nonintegrin receptor 1 (mSIGNR1) is found on MZ Ms [25
, 26
], and DEC-205 is localized in the WP [27
]. It has been shown recently that Dectin-1 and the murine 7/4 antigen are useful markers to define murine myeloid cell heterogeneity ex vivo [28
]. Similarly, as more pathogen-recognition receptors and differentiation antigens on Ms and DCs are localized in various tissues, it may be possible to use a combination of markers to better define M and DC subsets operating in vivo during homeostasis, inflammation, and infection.
The functions of the mannose receptor and mSIGNR1 correlate well with their location in the spleen. The M mannose receptor mediates the clearance of reactive substances including tissue plasminogen activator and lysosomal hydrolases to maintain homeostasis [29
], and it recognizes pathogen-associated molecular patterns on a wide range of microorganisms triggering phagocytosis in the innate immune response [30
]. The localization of mannose receptor-positive Ms in splenic RP would be consistent with these two main functions. Similarly, the localization of mSIGNR1 on Ms in the MZ is ideally suited for a receptor involved in the trapping/clearing of blood-borne antigens such as polysaccharides from encapsulated bacteria [25
, 26
]. The currently known function of Dectin-1 in pathogen recognition also correlates well with the predominant expression of this receptor on neutrophils, monocytes, Ms, and DCs in the blood, gut, lung, and spleen. The lack of Dectin-1 expression on MZ Ms would suggest that it is unlikely for Dectin-1 to have an antigen trapping and clearing role similar to mSIGNR1. Dectin-1 is also different from the other known C-type lectin family members on DCs, as it contains an ITAM motif in its cytoplasmic domain [5
]. The ITAM motif is necessary for Dectin-1 to mediate responses to zymosan including TNF- and ROS production [4
, 8
]. Although the role of Dectin-1 in the phagocytosis and killing of fungal pathogens by neutrophils and Ms are clear, and the signaling mechanism mediated by Dectin-1 on Ms and BM-derived DCs is emerging [4
, 8
], the role of Dectin-1 in DC–T cell interactions is currently undefined.
DEC-205 is a mannose receptor family member, and DEC-205 and the mannose receptor have been shown to mediate the uptake of macromolecules by DCs [31 , 32 ]. Unlike the mannose receptor, DEC-205 is localized in the T cell areas of lymphoid organs. Anti-DEC-205 antibodies have been used to target antigens for processing and presentation by DCs in vivo because of the ability of DEC-205 to deliver antigens via an EDE sequence in its cytoplasmic domain to target MHC class II compartments following adsorptive endocytosis [33 ]. The localization of Dectin-1 on DCs in the T cell zones of spleen and lymph nodes and morphological evidence (e.g., stellate cells in close contact with T cells in the PALS region) from our studies provide support for a role of Dectin-1 on DCs similar to that of DEC-205. However, there are notable differences between Dectin-1 and DEC-205. One key difference is that Dectin-1 does not contain a tyrosine-based, coated pit localization sequence or an EDE sequence in its cytoplasmic domain. Whether Dectin-1 can enhance antigen processing and presentation following phagocytosis or adsorptive endocytosis warrants investigation. The potential of Dectin-1 on DCs to mediate T cell binding and activation has been suggested by recent studies using recombinant Dectin-1 and cells transfected with mouse or human Dectin-1 [5 , 7 , 20 ]. Dectin-1 on DCs during homeostasis may be able to provide a costimulatory signal to T cells directly by engaging T cell ligands or indirectly via the production of cytokines following Dectin-1 receptor triggering by non-ß-glucan ligands.
The localization of Dectin-1 to the corticomedullary and medullary regions of the thymus suggests a role of Dectin-1 in T cell development. It is known that thymic DCs are localized exclusively to the medulla [34
], a major site for deletion of autoreactive thymocytes [35
]. Dectin-1 on Ms and DCs in the thymus may be involved in antigen capture and processing and the clearance of apoptotic cells. Efficient antigen capture and processing by molecules such as Dectin-1 and DEC-205 would result in the production and presentation of MHC peptide complexes that are needed to delete self-reactive thymocytes. It is also possible that Dectin-1 has a role in delivering critical costimulatory signals to thymocytes expressing Dectin-1 ligand(s). Strong expression in neonatal and adult thymus suggests that Dectin-1 is required throughout thymus development, including the process of negative selection of thymocytes. Strong, early expression of Dectin-1 also occurs in other tissues such as the spleen and lung where Dectin-1 would likely be involved in immune surveillance as well as normal, developmental processes. In view of the receptor’s known function in pathogen recognition and its speculated role in T cell activation, the subpopulations of Ms and DCs, as defined by Dectin-1, are likely to have functional differences from their non-Dectin-1-expressing counterparts. It is therefore critically important to define soluble or T cell-associated ligands for Dectin-1 to understand how Dectin-1 mediates T cell activation in various tissues.
In summary, Dectin-1 is likely to be a versatile receptor in the immune system. It has an established pathogen-recognition role in Ms and DCs. Its wide distribution and expression patterns indicate that Dectin-1 has the potential to be involved in a number of leukocyte interactions in homeostasis, immune responses, and T cell development. Identification of the endogenous ligands recognized by Dectin-1 will provide further insights into Dectin-1-mediated events and the consequences of its regulation during T cell development and antimicrobial responses.
|
ACKNOWLEDGEMENTS |
|---|
Received January 21, 2004; revised March 5, 2004; accepted March 10, 2004.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  A. B. Tarabishy, B. Aldabagh, Y. Sun, Y. Imamura, P. K. Mukherjee, J. H. Lass, M. A. Ghannoum, and E. Pearlman MyD88 Regulation of Fusarium Keratitis Is Dependent on TLR4 and IL-1R1 but Not TLR2 J. Immunol., July 1, 2008; 181(1): 593 - 600. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Wells, J. A. Salvage-Jones, X. Li, K. Hitchens, S. Butcher, R. Z. Murray, A. G. Beckhouse, Y.-L.-S. Lo, S. Manzanero, C. Cobbold, et al. The Macrophage-Inducible C-Type Lectin, Mincle, Is an Essential Component of the Innate Immune Response to Candida albicans J. Immunol., June 1, 2008; 180(11): 7404 - 7413. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Koneti, M. J. Linke, E. Brummer, and D. A. Stevens Evasion of Innate Immune Responses: Evidence for Mannose Binding Lectin Inhibition of Tumor Necrosis Factor Alpha Production by Macrophages in Response to Blastomyces dermatitidis Infect. Immun., March 1, 2008; 76(3): 994 - 1002. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. G. Rothfuchs, A. Bafica, C. G. Feng, J. G. Egen, D. L. Williams, G. D. Brown, and A. Sher Dectin-1 Interaction with Mycobacterium tuberculosis Leads to Enhanced IL-12p40 Production by Splenic Dendritic Cells J. Immunol., September 15, 2007; 179(6): 3463 - 3471. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Vermijlen, P. Ellis, C. Langford, A. Klein, R. Engel, K. Willimann, H. Jomaa, A. C. Hayday, and M. Eberl Distinct Cytokine-Driven Responses of Activated Blood {gamma}{delta} T Cells: Insights into Unconventional T Cell Pleiotropy J. Immunol., April 1, 2007; 178(7): 4304 - 4314. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Gays, J. G. Aust, D. M. Reid, J. Falconer, N. Toyama-Sorimachi, P. R. Taylor, and C. G. Brooks Ly49B Is Expressed on Multiple Subpopulations of Myeloid Cells J. Immunol., November 1, 2006; 177(9): 5840 - 5851. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. W. Carter, C. Thompson, D. M. Reid, S. Y. C. Wong, and D. F. Tough Preferential Induction of CD4+ T Cell Responses through In Vivo Targeting of Antigen to Dendritic Cell-Associated C-Type Lectin-1 J. Immunol., August 15, 2006; 177(4): 2276 - 2284. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. S. Palma, T. Feizi, Y. Zhang, M. S. Stoll, A. M. Lawson, E. Diaz-Rodriguez, M. A. Campanero-Rhodes, J. Costa, S. Gordon, G. D. Brown, et al. Ligands for the beta-Glucan Receptor, Dectin-1, Assigned Using "Designer" Microarrays of Oligosaccharide Probes (Neoglycolipids) Generated from Glucan Polysaccharides J. Biol. Chem., March 3, 2006; 281(9): 5771 - 5779. [Abstract] [Full Text] [PDF]
|
|
|
