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From the Department of Molecular Cell Biology and Immunology,* Vrije Universiteit Medical Center, Amsterdam, The Netherlands; the Department of Pathology, University Medical Center Nijmegen, Nijmegen, The Netherlands; and the Medical Research Council Human Immunology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom
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Abstract |
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This immune reaction against peripheral antigens through active transport by DCs to lymph nodes holds true for part of the antigens only. Many antigens from lymph and interstitial tissue fluids reach lymph nodes in soluble form.3 Similar to peripheral tissues, lymph nodes contain immature DCs; recent studies even postulate that the majority of DCs in lymph nodes exist in an immature state, acting at these sites to capture soluble antigens.4-7 Accordingly, immature lymph node DCs require activation signals to efficiently present antigenic peptides to T lymphocytes.6,8,9 These studies were performed in mice; in humans, the existence of lymphoid immature DCs remains unclear. DC-specific molecules could be useful to address this issue.
We have recently described that dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN), a C-type lectin, is exclusively expressed by human DCs in peripheral tissues, such as skin and mucosa, and in lymphoid organs.10 In blood, we have characterized a subset of CD14+ DCs that express DC-SIGN.11 In the same study, plasmacytoid DCs did not express detectable levels of DC-SIGN, although lack of DC-SIGN expression on these interferon-producing DCs remains controversial.11-13 DC-SIGN has several properties contributing to the function of DCs. As has been described for other C-type lectins such as the mannose receptor,14 DC-SIGN can capture antigens for processing and subsequent presentation to T cells.15 A growing list of pathogens is bound by DC-SIGN,16 including human immunodeficiency virus,17 Mycobacterium tuberculosis,18 Leishmania amastigotes,19 and Dengue virus.20,21 Moreover, DC-SIGN regulates migration of DCs by binding its ligand ICAM-2 on endothelial cells and activation of resting T cells through ICAM-3 binding.22,23 In contrast to DC-SIGN, liver/lymph node-specific ICAM-3 grabbing nonintegrin (L-SIGN, also called DC-SIGNR), a functional homologue of DC-SIGN with similar binding activity, was not found to be expressed on DCs in peripheral tissues but on liver sinus endothelial cells, organ-resident antigen-presenting cells.24-26 On liver sinus endothelial cells, L-SIGN may function to facilitate interactions with lymphocytes as well as to bind antigens and pathogens. Interestingly, in lymph nodes, both SIGN family members are expressed, although the exact localization remains unclear.
In this study we have characterized the expression of DC-SIGN and L-SIGN in more detail in both normal and immunoreactive lymph nodes. We observed that in lymph nodes DC-SIGN is expressed by mature DCs present in the paracortex, where interactions with T lymphocytes take place, as well as on a large number of immature DCs in the outer zone of the paracortical areas, where antigen capture takes place. In the vicinity of these immature DC-SIGN+ DCs, L-SIGN+ cells are also detected. High-resolution staining demonstrated that cells expressing L-SIGN are specialized endothelial cells that co-express the recently described lymph endothelial markers LYVE-127 and CLEVER-1.28 During the induction of humoral and cellular responses, changes were observed in the number of mature DC-SIGN+ DCs in the paracortex and both immature DC-SIGN+ DCs and L-SIGN+ endothelial cells in the paracortical outer zones. Thus, DC-SIGN+ and L-SIGN+ cell populations are highly dynamic during immune activation and are potentially important players in lymph nodes. We propose that these immune cells use DC-SIGN and L-SIGN either for capturing antigens or for cellular interactions facilitating T-cell activation.
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Materials and Methods |
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The following antibodies were used: AZN-D1, AZN-D2, AZN-D3 (anti-DC-SIGN monoclonal antibodies23,24 ), CSRD (polyclonal antiserum obtained after immunization of rabbits with the following peptide from DC-SIGN coupled to KLH: CSRDEEQFLSPAPATPNPPPA), PTTS (polyclonal antiserum obtained after immunization of rabbits with the following peptide from L-SIGN coupled to KLH: PTTSGIRLFPRDFQFQQIH), anti-S100 (Z0311, DAKO, Glostrup, Denmark), KP-1 (anti-CD68, DAKO), anti-CD31 (B&D, Oxnard, CA), 3.29B1 (anti-mannose receptor, kind gift of Dr. M. Cella, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA), anti-von Willebrand factor (clone F8/86, DAKO), anti-LYVE-1,27 3-372 (anti-CLEVER, kind gift of Dr. S. Jalkanen28 , Medical Research Laboratory and Department of Medical Microbiology, Turku University, Turku, Finland), and anti-CD163 (described in van den Heuvel et al29 ).
Cells
K-562 cells were transfected with DC-SIGN or L-SIGN as described.23 Immature DCs were generated by culturing human blood monocytes in RPMI 1640/10% fetal calf serum containing interleukin-4 (500 U/ml, Schering-Plough) and GM-SCF (800 U/ml, Schering-Plough, Brussels, Belgium) for 5 to 8 days.
Human Tissues
Tissues were obtained from patients by surgical removal, following national ethical guidelines regarding the use of human tissues. One part was frozen for cryosections and one part was embedded in paraffin. Classification of immune reactive lymph nodes was performed on hematoxylin and eosin-stained paraffin sections. All lymph nodes showed mixed cellular and humoral responses using histology; in the classification the dominant response is indicated. The characteristics of the patients that underwent lymph node removal: patient 1: 9-year-old male, enlarged lymph node in neck because of skin allergy, classification: reactive lymph node, dermatopathic lymphadenopathy, and sinus histiocytosis, mild cellular response; patient 2: 25-year-old female, enlarged lymph node behind right ear because of eczema, histological classification: dermatopathic lymphadenopathy and sinus histiocytosis, humoral response; patient 3: 8-year-old male, enlarged lymph node in neck, histological classification: reactive paracortical hyperplasia and sinus histiocytosis, cellular response; patient 4: 85-year-old female, mammacarcinoma, enlarged lymph node in neck without tumor, histological classification: reactive paracortical hyperplasia, cellular response; patient 5: 28-year-old male, enlarged lymph node in neck without diagnosis, histological classification: reactive follicular hyperplasia, humoral response; patient 6: 33-year-old male, enlarged inguinal lymph node without diagnosis, histological classification: dermatopathic lymphadenopathy and sinus histiocytosis, cellular response.
Immunohistochemistry
Tissue cryosections (4 µm) were fixed in acetone for 10 minutes and incubated with primary and secondary antibodies (anti-mouse horseradish peroxidase and anti-rabbit biotin; Vector Laboratories, Burlingame, CA).23 Paraffin sections were rehydrated and subjected to antigen-retrieval by boiling in 0.01 mol/L citric acid (pH 6.0) for 10 minutes before incubation with antibodies. Staining was performed with the ABC-AP Vectastain kit (Vector Laboratories) or ABC-PO and diaminobenzidine tetrahydrochloride (0.5 mg/ml) and sections were counterstained with hematoxylin according to Pappanicolau. For immunofluorescence, Alexa 488 (Molecular Probes, Eugene, OR)- and Texas Red (Jackson, West Grove, PA)-conjugated secondary antibodies were used.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Expression of DC-SIGN, a DC-specific C-type lectin, can be detected using three different monoclonal antibodies that we have generated, AZN-D1, AZN-D2, and AZN-D3 (Figure 1A)
.22,23
Two of these antibodies, AZN-D2 and AZN-D3, cross-react with L-SIGN/DC-SIGNR, a structural and functional homologue of DC-SIGN (Figure 1A)
. To better distinguish between cells expressing DC-SIGN and/or L-SIGN, polyclonal antibodies were generated in rabbits by immunization with 
20-mer peptides coupled to keyhole limpet hemocyanin (KLH). Based on amino acid differences, a peptide corresponding to the C-terminal part of DC-SIGN (CSRD) was used and an amino acid stretch in the cytoplasmic tail of L-SIGN (PTTS). Using K562 cells stably transfected with either DC-SIGN or L-SIGN, the specificity of these antibodies was confirmed (Figure 1B)
; CSRD specifically recognizes DC-SIGN whereas PTTS reacts with L-SIGN only. Moreover, confirming our previous results of Northern blot analysis,24
staining with these antibodies clearly demonstrates that monocyte-derived DCs express DC-SIGN but not L-SIGN (Figure 1B)
.
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Using these antibodies, cryosections from various tissues were screened for the expression of DC-SIGN and L-SIGN; results are summarized in Table 1
. DC-SIGN expression was detected on DCs in peripheral tissues, such as placenta, skin, and mucosa and in T-cell areas of secondary lymphoid tissues, including lymph nodes and spleen, as described before.12,17,23
In contrast, L-SIGN expression was confined to endothelial cells in liver sinuses and in lymph nodes.24
Others have detected L-SIGN+ cells in placental villi;26
this discrepancy could be because of the developmental stage of the placenta. Thus, the SIGN family members have distinct expression patterns, except in lymph nodes, where both DC-SIGN and L-SIGN expression is found (see below).
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The distribution of DC-SIGN and L-SIGN was analyzed on cervical lymph nodes in more detail using immunohistochemistry. DC-SIGN is expressed on large cells with an irregular cell shape in the paracortex (Figure 2A)
. DC-SIGN-expressing cells are identified as DCs, based on co-expression of several markers on sequential sections, including MHC class II, the mannose receptor, CD11b and CD11c, and lack of surface expression of CD68 (Table 2)
. Moreover, DC-SIGN expression is also detected on cells in the outer zone of the paracortex, in proximity of paracortical and medullary sinuses (Figure 2A)
. In these areas, L-SIGN-expressing cells are also situated, although fewer in number as compared to DC-SIGN+ cells (Figure 2A)
. Similar expression patterns were observed in human mesenteric and inguinal lymph nodes (not shown). In comparison, the distribution of S100, an extensively used DC marker for paraffin-embedded lymphoid tissue,30
was studied. Expression of S100 was detected on a large population of DCs scattered throughout the paracortex and in B-cell follicles, whereas DC-SIGN expression was restricted to a subset of lymph node DCs (Figure 2B)
.
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. In the paracortical outer zone, most cells express either DC-SIGN or L-SIGN, and form a network of cells of which the cell extensions may overlap (Figure 3A)
. This is also demonstrated by overlapping of the extensions of DC-SIGN+ cells and adjacent L-SIGN+ cells because of close proximity. The fact that the adhesion molecules DC-SIGN and L-SIGN were expressed on adjacent cells prompted us to analyze interactions between these molecules. However, no binding of DC-SIGN to L-SIGN was observed (TBH Geijtenbeek, unpublished results). Because both ICAM-2 and ICAM-3 are present on both cell types (not shown), they may interact with each other using these cellular ligands. Because DC-SIGN and L-SIGN are expressed at the same site in lymph nodes but on distinct cell types, we set out to characterize these cells in more detail. Furthermore, we determined whether DC-SIGN- and L-SIGN-expressing cells are a subset of DC, macrophage, or endothelial cells.
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and Table 2
). These molecules are expressed by macrophages as well as DCs. No expression of the Langerhans’ cell-marker CD1a and a marker for mature DC, CD83, was found, but these DC-SIGN+ cells did express MHC class II molecules (Table 2)
. This prompted us to study expression of macrophage markers. Expression of CD68 was detected intracellularly in a spot in DC-SIGN+ cells (Figure 3B)
, a similar pattern as described before in immature DCs.31
This is in contrast to macrophages that exhibit an overall cytoplasmic and surface expression of CD68.31
A similar perinuclear staining pattern was observed for the lysosomal enzyme acid phosphatase in DC-SIGN-expressing cells (Table 2)
. In addition, neither CD163, a macrophage marker,29
nor CD14, a monocytic marker, were detected on DC-SIGN+ cells (Table 2)
. Taken together these results suggest that the DC-SIGN+ cells near the paracortical and medullary sinuses are not macrophages but represent DCs that have an immature phenotype.
In a previous study by Pohlmann and colleagues,26
it was postulated that lymph node endothelial cells express L-SIGN/DC-SIGNR. However, we observed L-SIGN expression in the proximity of the sinuses only and not on endothelium from small lymph vessels and high-endothelial venules. To further characterize L-SIGN+ cells, double stainings were performed using markers for DC, macrophage, and endothelial cells. The pan endothelial marker CD31 is highly expressed on the luminal side of the cell layer lining the sinuses, underneath which all cells express low amounts of CD31 (Figure 3C)
. L-SIGN expression is lacking on the most luminal part, but is expressed directly underneath on several layers of cells in a continuous lining. Another endothelial cell marker, von Willebrand factor, was expressed intracellularly in most L-SIGN+ cells (Table 2)
. In addition, expression of mannose receptor and low levels of CD11b and CD11c were detected on L-SIGN+ cells, but these cells lacked expression of the macrophage markers CD68, CD163, and acid phosphatase (Table 2)
. Because these results indicate that L-SIGN is expressed on certain cells of endothelial lineage, we analyzed expression of two markers for lymph endothelium, LYVE-127
and CLEVER-1.28
Whereas CLEVER-1 is expressed on both efferent and afferent lymph vessels and on high-endothelial venules,28
the hyaluronan receptor LYVE-1 is present on lymph endothelium in all tissues and on sinusoidal endothelium in liver and spleen.27,32
Interestingly, L-SIGN+ cells expressed both LYVE-1 and CLEVER-1 (Figure 3C
, arrows). However, L-SIGN was not expressed by CLEVER-1+ high-endothelial venules and not on LYVE-1+ lymph endothelium (Figure 3C
, arrowheads), but only on sinusoidal endothelium. Thus, L-SIGN expression is detected on a subset of endothelial cells near sinuses, that are present in several layers and co-express CD11b and CD11c and mannose receptor, that has been previously described on endothelial cells.33
Expression of DC-SIGN and L-SIGN in Immune Reactive Lymph Nodes
During an immune reaction, several changes occur in the various functional areas of lymph nodes in time, depending on the type and the intensity of the immune response. The tissue used for the detection of SIGN expression so far was, although taken from a patient with a dominant cellular reaction, relatively normal concerning cellular density and size (patient 1). We therefore performed immunohistochemical analyses using lymph nodes showing highly reactive humoral and cellular responses to assess the expression of DC-SIGN and L-SIGN in these circumstances.
During a dominant humoral response, hyperplasia of the cortex is observed in lymph nodes, with increased numbers of B-cell follicles and germinal centers (Figure 4A)
. None of the cells within the hyperplastic cortex were found to express either DC-SIGN or L-SIGN. The number of mature DC-SIGN+ DCs in the paracortex was normal in this lymph node (Figure 4A)
. However, high numbers of DC-SIGN+ immature DCs as well as L-SIGN+ endothelial cells were found in areas around the sinuses (compare Figure 4A
with Figure 2A
). Besides DCs and sinus endothelial cells, DC-SIGN and L-SIGN expression was not detected on other cells as determined by double stainings (not shown).
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. The size of the lymph node can increase dramatically, depending on the intensity of the immune reaction (compare top panels of Figure 4; A, B, and C
). In the paracortex, increased numbers of mature DC-SIGN+ DCs were present, in agreement with their T-cell stimulatory function (Figure 4B)
. A similar increase in the number of mature DC-SIGN+ DCs has been reported during acute Epstein-Barr virus infections.34
However, no changes in DC-SIGN- and L-SIGN-expressing cells in the paracortical outer zone were observed as determined by double stainings (not shown), although the amount of L-SIGN+ sinus endothelial cells increased dramatically in enlarged lymph nodes (Figure 4C
, right). |
Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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. Thus, similarly to rodents,4,6,7
in human lymph nodes a large and dynamic population of resident immature DCs might exist for capturing lymph-borne antigens. In rodents, it is not clear at which site these immature DCs are located. Our observation that human lymph nodes contain immature DCs that are situated in proximity of the sinuses would position these cells optimally for incoming tissue antigens. DC-SIGN and L-SIGN have distinct expression patterns, except in lymph nodes, where both are detected. Initial studies using reverse transcriptase-polymerase chain reaction indicated that co-expression of DC-SIGN and L-SIGN could occur on certain cell-types, including DCs.25 However, using highly specific antibodies, our studies have failed to detect simultaneous expression of L-SIGN on DC-SIGN+ DCs.24 In addition, using detailed immunohistochemical analysis, we here show that protein expression of DC-SIGN and L-SIGN does not coincide on the same cells in lymph nodes. Whereas DC-SIGN is restricted to lymph node DCs, L-SIGN is expressed on sinus endothelial cells. Thus, these two C-type lectins, that share binding to cellular ligands and pathogens, show a very restricted and distinct expression pattern.
L-SIGN+ endothelial cells are located exclusively to the outer zone of the paracortex and their number is increased during an immune response. Interestingly, L-SIGN+ cells co-express the lymph endothelial cell markers CLEVER-1 and LYVE-1. CLEVER-1 mediates binding of lymphocytes,28
indicating that these CLEVER-1+/L-SIGN+ endothelial cells may play a role in migration of lymphocytes leaving the paracortex. Remarkably, at certain sites multiple layers of L-SIGN+ sinus endothelial cells are detected forming a continuous lining that would have to be crossed by T lymphocytes leaving the lymph node (Figure 4)
. L-SIGN, by its ability to bind ICAM-3 on T cells,24
could assist in this process. The hyaluronan receptor LYVE-1 is co-expressed with L-SIGN on lymph sinuses as well as on LSEC.27,32
These specialized endothelial cells in liver can capture and present antigens locally to T cells, resulting not in activation of T cells, but in tolerance.40
The expression of both L-SIGN and mannose receptor on sinus endothelial cells would enable them to efficiently capture antigens.
Thus far, attempts to isolate L-SIGN+ lymph node endothelial cells for functional characterization have been unsuccessful. Recently, a murine homologue of DC- and L-SIGN, mSIGNR1, was cloned and was found to be expressed on specialized macrophages and liver sinusoidal endothelial cells.41,42 mSIGNR1 functions in efficient capture and internalization of soluble antigens,42 suggesting that L-SIGN in human lymph nodes might perform a similar function. In the murine system, more insight could be gained in the function of the dynamic population of L-SIGN-expressing cells in lymph nodes.
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Acknowledgements |
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Footnotes |
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Supported by the Heart Foundation (grant 97.078 to A.E.) and the AIDS Foundation (grant 5008 to T.G.).
Accepted for publication January 14, 2004.
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