Immature dendritic cells possess a sugar-sensitive receptor for human mannan-binding lectin

Abstract

Mannan-binding lectin (MBL) is a collectin synthesized by the liver and secreted into the bloodstream. It has a receptor for microbial structures in its C-type lectin domain and a separate receptor(s) located within its collagen-like region for autologous phagocytic cells. Here we demonstrate that human peripheral blood adherent cells (monocytes) and monocyte-derived dendritic cells are a source of MBL, and that a novel calcium-dependent and sugar-specific MBL receptor is up-regulated in immature (CD1a-positive) dendritic cells. These findings suggest a previously unsuspected autologous function for MBL, perhaps a regulatory role within the immune system.

Introduction

Mannan binding lectin (MBL) is a collectin (C-type lectin with a collagen-like region) synthesized by the liver and secreted into the bloodstream. It is thought to be an important component of the innate immune response as it can neutralize invading pathogens and interact with other components of the immune system by activating complement after recognizing and binding to sugar patterns typical of microbial surfaces. A substantial literature supports the belief that MBL deficiency or insufficiency is physiologically relevant, by increasing susceptibility to a broad spectrum of infectious agents or affecting the course of disease.^1,2

Dendritic cells (DC) also bridge the innate and adaptive immune systems and it is notable that dendritic cells express a variety of C-type lectins including the macrophage mannose receptor, dectin-1 and several asialoglycoprotein receptors like DC-SIGN/CD-209 and langerin/CD-207.^3,4 These lectins presumably mediate the recognition of non-self and the presentation of foreign antigens and there is evidence that DC-SIGN can regulate important adhesion processes.³ We therefore looked for the presence of mannan-binding lectin on the surface of human dendritic cells.

We report here that cell-surface MBL can indeed be detected in and on monocytes and monocyte-derived dendritic cells. An unexpected finding, however, was the presence of receptors sensitive to ethylenediaminetetra-acetic acid (EDTA) and specific monosaccharides, separate from the putative C1q/collectin receptor(s). This cation-sensitive receptor was markedly up-regulated specifically in immature dendritic cells. These findings indicate a previously unsuspected autologous function for human MBL.

Materials and methods

Reagents

Mouse monoclonal antibodies to human MBL (clones 131-1 and 131-11) were prepared in the State Serum Institute (Copenhagen). Clone 131-1 is specific for functional (oligomeric) MBL; clone 131-10 also appears to recognize functional MBL, but is directed against a separate epitope. MBL was isolated from Cohn fraction-III of pooled human plasma as previously described.⁵ MBL, anti-MBL antibodies, human transferrin (Sigma, Poole, UK) and human IgG (Miles Laboratories Ltd, Stoke Poges, UK) were biotinylated using N-hydroxysuccinimido-biotin.⁶ Interleukin (IL)-4 was purchased from R&D Systems (Abingdon, UK) and granulocye–macrophage colony-stimulating factor (GM-CSF) (Leucomax) from Sandoz/Schering-Plough Ltd (Welwyn Garden City, UK). d-Mannose, d-galactose, N-acetyl-d-glucosamine (GlcNAc), and N-acetyl-d-galactosamine (GalNAc) were purchased from Sigma-Aldrich, as were all other chemicals unless stated otherwise. The following antihuman antibody conjugates were used for flow cytometry: CD1a-PE (Pharmingen, Oxford, UK), CD14-PE (Pharmingen), CD14-FITC (Coulter, Hialeah, FL), CD80-FITC, CD83-PE (both Immunotech), and CD86-FITC (Pharmingen).

Human buccal epithelial cells were harvested from a mouthwash in phosphate buffered saline. Human foreskin fibroblasts and the murine myeloma cell line Sp 2/0 were the kind gifts of Drs Nick Hunter and Anne Mackie, respectively.

In vitro generation of dendritic cells

Buffy coats obtained from the local blood transfusion centre were used as a source of human leucocytes. Mononuclear cells were isolated by density gradient centifugation on Lymphoprep (Nycomed, Oslo, Norway), suspended in 1% human AB serum in RPMI-1640 medium, and allowed to adhere to the surface of plastic tissue culture flasks (Costar, Corning Inc., Corning, NY) for 1 hr at 37° in a 5%-CO₂ incubator. The resultant adherent cells were washed three times with medium before their incubation was continued in 1% serum-RPMI medium supplemented with IL-4 (15 ng/ml) and GM-CSF (50 ng/ml). After 3 days, the cells were either harvested as immature dendritic cells or incubated for a further 3 days in the presence of Poly (I:C) to obtain mature dendritic cells. On some occasions, the cells obtained by density gradient centrifugation were resuspended in X-Vivo 20 (BioWhitaker, Walkersville, MD). The adherence step and subsequent culture were then conducted in this medium without the addition of serum.

Cell labelling for flow cytometry

All cells were collected by centrifugation (after scraping the tissue culture flasks to remove any adherent cells) and washed twice in phosphate-buffered saline. In preliminary experiments, cells were fixed by incubation with 1% paraformaldehyde for 5 min at room temperature. Subsequently, unfixed cells were used in the presence of 0·1% sodium azide. The standard solution used was 1% bovine serum albumin (BSA) and 0·1% sodium azide in 20 mm–Tris/150 mm–NaCl/5–mM CaCl_2, adjusted to pH 7·4 (TBS-Ca), but when appropriate, 5 mm-EDTA was substituted for the CaCl₂ (TBS-EDTA). The cell suspensions (0·2 ml) were first incubated for 30 min at room temperature with either biotin–anti-MBL (10 µg/ml) or biotin–MBL (15 µg/ml) then washed twice with TBS-Ca (or TBS-EDTA). The cells were then incubated a further 30 min with Streptavidin–fluorescein isothiocyanate (FITC) (0·5 µg/ml) before analysis by flow cytometry. (For some confirmatory experiments, Streptavidin–FITC was replaced by streptavidin–Quantum Red conjugate (Sigma), at a dilution of 1 in 100.)

Flow cytometry

Cells were phenotyped by direct immunofluorescence for two-colour cytometry, using isotype-matched controls. The latter were murine myeloma immunoglobulins G (IgG) of the same subclasses as the test reagents and conjugated to the same fluorophors (BD Biosciences/Pharmingen). Data was collected on a FacScan and analysed using Cell Quest software (Becton Dickinson Ltd, San Jose, CA). Cells investigated for MBL and MBL receptors were first stained with anti-MBL/streptavidin–FITC or MBL–biotin/streptavidin–FITC prior to staining with phycoerythrin (PE)-conjugated anti-CD14, -CD1a or -CD83 antibodies.

Intracellular staining of MBL and MBL receptors was performed according to the manufacturer's instructions. Briefly, cells were incubated with PE-conjugated antibodies for surface staining for CD14, CD1a or CD83 as required followed by incubation with the Perm 2 reagent (Becton Dickinson Ltd). Cells were then stained internally with either anti-MBL (clone 131-1) or MBL–biotin (using either non-specific IgG or MBL, respectively, as controls) followed by streptavidin–FITC.

Statistics

Statistical analyses were performed using Prism for Windows software from Graph Pad (San Diego, CA) and Excel data analysis software from Microsoft (Redmond, WA).

Results

MBL is present on adherent cells and dendritic cells

Plastic-adherent cells obtained from buffy coats were used to prepare dendritic cells. The expected differentiation was confirmed by monitoring development by surface marker expression (Table 1).

Table 1.

Surface antigen expression during DC development

	Positive cells (%)
Antigen	Monocytes	D3-iDC	D6-iDC	D6-mDC
CD1a	0·5 + 0·1	48·3 + 6·9	76·2 + 7·0	78·6 + 3·3
CD80	2·3 + 0·3	12·6 + 3·7	46·4 + 6·6	89·0 + 2·6
CD83	0·5 + 0·1	4·5 + 4·6	10·3 + 2·3	45·2 + 12·9
CD86	8·0 + 0·7	85·0 + 3·4	52·8 + 6·7	85·9 + 4·5

Adherent cells (monocytes) were cultured with IL-4/G-CSF for 3 days (D3-iDC) then allowed to culture for a further 3 days with (D6-mDC) or without (D6-iDC) the addition of Poly (I:C) to induce maturation. Results are expressed as the proportion of positive cells (mean + SE of eight determinations) after selecting the cell population on the basis of light scattering properties.

A proportion of immature DC, mature DC and the original adherent cell population (Fig. 1a) bound biotinylated anti-MBL antibodies. This was true whether the monoclonal antibody used was clone 131-1 or clone 131-11, or whether the cells had been fixed with paraformaldehyde or used in the presence of sodium azide. Typically, 18% of the adherent cells expressed MBL, a significantly higher proportion than for immature DC (10%) or mature DC (10%) (Table 2). Immature DC allowed to culture for a further three days without a maturation agent were similar (12% MBL expression).

Figure 1.

MBL and MBL receptors detected by flow cytometry. Cells were labelled with biotinylated anti-MBL antibodies or biotinylated MBL followed by streptavidin–FITC as described in the Materials and Methods. (a) The profile for monocytes labelled with anti-MBL is shown in grey with the outline of the isotype control superimposed as a thick black line. (b) The profile for immature DC labelled with biotinylated MBL in the presence of EDTA is shown in grey with the negative control (unlabelled MBL) outline superimposed as a thick black line. (c) The profile for immature DC labelled with biotinylated MBL in the presence of calcium is shown in grey with the negative control (unlabelled MBL) outline superimposed as a thick black line.

Table 2.

Surface MBL during DC development

Stage	n	MBL-positive cells (%)
Monocytes (day 0)	21	18·1 + 2·4
Immature DC (day 3)	21	9·8 + 1·2	P < 0·004
Immature DC (day 6)	19	12·4 + 1·8	P < 0·05
Mature DC (day 6)	16	10·1 + 1·3	P < 0·01

Results are expressed as means + SE. The number of determinations (n) with each cell type is given. Significance values denote comparisons between monocytes and each developmental type of dendritic cell.

To eliminate the possibility that the MBL detected was derived from the human serum used for culture, labelled antibody binding was repeated on cells cultured for 3 days in serum-free medium, and which had had no contact with AB serum. Similar results were obtained.

When the cells were rendered permeable to permit intracellular labelling, MBL could be detected in over 60% of the adherent cells and over 80% of the dendritic cells (irrespective of age or maturation status). This too could be confirmed in cells cultured in serum-free medium.

No surface or intracellular staining was apparent when biotinylated anti-MBL (clone 131-1) was used in the same way with human buccal epithelial cells or human T lymphocytes.

No surface staining was apparent with intact human fibroblasts or murine myeloma cells either, but a substantial minority of both those cells displayed intracellular staining. Nevertheless, this apparently non-specific staining was not in any way comparable to the degree of staining obtained with human monocytes and dendritic cells.

Dual labelling experiments in which cells were reacted with biotinylated anti-MBL in combination with anti-CD1a or anti-CD83 or anti-CD14, indicated that the anti-MBL was costaining with the specific monocyte (CD14), immature dendritic cell (CD1a) or mature dendritic cell (CD83) surface markers.

To assess whether MBL was being secreted, culture media were tested for MBL. Buffy coat cells from 10 individuals were used (with plasma MBL concentrations ranging from <0·1–2·7 µg/ml, median 2·0). Adherent cells were either induced to form dendritic cells or simply allowed to culture for up to six days. The immature DC cultures after 3 days were either induced to mature with poly(IC) or simply allowed to culture for a further 3 days. In no instance could any MBL whatsoever be detected in any culture medium.

The results presented for MBL detection were obtained using fluorescein as the fluorochrome. These findings were confirmed for monocytes and immature dendritic cells after substituting streptavidin–Quantum Red for streptavidin–FITC on two occasions (data not given).

MBL receptors are present on adherent cells and dendritic cells

Biotinylated MBL was incubated with immature DC, mature DC and adherent cells. Statistically significant binding was detected in the presence of 5 mm EDTA compared with the negative control (Fig. 1b). This cation-independent binding was readily detected on approximately 20% of the cells although the highest proportion was for immature dendritic cells. However, MBL binding was always greatly enhanced in the presence of 5 mm calcium, irrespective of cell type; this was most pronounced for immature DC, with typically 70% of cells positive (Table 3).

Table 3.

MBL receptor expression during DC development

	MBL receptor-positive cells (%)
Stage	+ EDTA	+ Ca2+
Monocytes	13·4 + 3·3 [24]	26·3 + 2·8 [27]	P < 0·01
Immature DC (day 3)	25·9 + 5·5 [30]	70·7 + 4·1 [39]	P < 0·00001
Immature DC (day 6)	17·0 + 2·9 [25]	57·4 + 11·7 [31]	P < 0·004
Mature DC (day 6)	14·0 + 3·2 [24]	36·1 + 9·9 [26]	P < 0·03

The proportions of cells labelled with biotinylated MBL are expressed as means + SE of eight determinations. The geometric mean fluorescence intensity is also given in square brackets. The significance values shown are for comparisons between determinations in the presence (+Ca²⁺) and absence (+ EDTA) of calcium. In the presence of calcium, the proportion of positive cells was significantly higher in day 3 immature DC compared with monocytes (P < 0·00001) and day 6 mature DC (P < 0·003), but not compared with day 6 immature DC (P = 0·26).

The binding of biotinylated MBL to dendritic cell preparations in the presence of calcium displayed a strong and statistically significant inverse correlation with CD 83 coexpression (Spearman r = −0·75; P < 0·0001). The corresponding relationship for receptors detected in the presence of EDTA was much weaker (Spearman r = −0·41; P < 0·04).

When the cells were rendered permeable before exposure to biotinylated MBL, the outcome was radically different depending on the presence or absence of calcium (Table 4). Irrespective of cell type, in the presence of calcium, virtually all the cells (>90%) were positive. In the presence of EDTA, however, although most adherent cells and 3-day cultured immature dendritic cells were positive, 10% or less of dendritic cells (irrespective of maturation) had detectable MBL receptors after 6 days in culture.

Table 4.

MBL receptors in permeable cells

	MBL receptor positive cells (%)
Cell type	+ EDTA	+ Ca2+
Monocytes	67·4 + 23·7 (2)	91·6 + 0·1 (2)
Immature DC (day 3)	86·8 + 4·3 (3)	90·4 + 2·6 (3)
Immature DC (day 6)	10·3 + 1·3 (4)	89·6 + 1·4 (4)
Mature DC (day 6)	6·6 + 0·5 (2)	90·5 + 0·5 (2)

Results are expressed as means + SE with the number of determinations in parentheses.

Confirmation of binding of biotinylated MBL to immature dendritic cells with streptavidin–Quantum Red instead of streptavidin–FITC was carried out on two occasions (data not given). Both cell surface and intracellular staining was apparent in around 90% of the cells.

To check the specificity of the MBL–dendritic cell interaction, biotinylated human transferrin and biotinylated human IgG were each incubated with immature dendritic cells. There was no increase in surface binding compared to the negative control for either transferrin or IgG, despite a large majority of cells binding MBL under identical conditions. Similarly, when the cells were rendered permeable to permit intracellular staining, binding of biotinylated transferrin or IgG was not appreciably greater than the negative control while virtually all the cells bound MBL.

To investigate the possibility that an endogenous dendritic cell lectin could be interacting with the carbohydrate moiety of MBL, immature dendritic cells were incubated with mannan (20 mg/ml) for 20 min before unbound mannan was removed by harvesting the cells by centrifugation. These mannan-treated cells bound biotinylated MBL to an identical degree as their untreated counterparts.

MBL–MBL receptor interaction is inhibited by mannose and GlcNAc

The binding of MBL to its receptor on immature DC in the presence of calcium could be readily inhibited by mannose and GlcNAc. At 33 mm concentrations, the degree of binding approached the level detected in the presence of EDTA. In contrast, galactose and GalNAc had little effect on MBL binding, even at 100 mm (Fig. 2).

Figure 2.

Inhibition of MBL–MBL receptor interaction by specific monosaccharides. Biotinylated MBL was incubated with immature DC (day 3) and a tripling dilution series of each monosaccharide. (The positive control was obtained in the absence of added sugar). Most results are given by means ± SE of four determinations (but only one determination at 1·2 mm). d-mannose, N-acetyl-D-glucosamine, d-galactose and N-acetyl-D-galactosamine were tested in the presence of 5 mm Ca²⁺. The broken line represents the corresponding mean value obtained without calcium or sugars in the presence of EDTA.

Preincubation of immature dendritic cells with unlabelled MBL (20 µg/ml) or unlabelled anti-MBL antibodies (131-1; 20 µg/ml) for 20 min caused an inhibition of biotin–MBL binding similar to that obtained with mannose or GlcNAc. When the cells were rendered permeable to permit intracellular staining, however, excesses of MBL or anti-MBL produced only modest inhibition.

Discussion

We have found that most human monocytes and monocyte-derived dendritic cells contain intracellular MBL and that an appreciable minority express surface MBL. This appears to be the first report of extrahepatic MBL synthesis in human cells, although renal synthesis of MBL mRNA (and possibly MBL protein) has been described in the rat⁷ and MBL mRNA expression was found in various lymphoid and myeloid cell types from several murine tissues.^8,9 Extrahepatic synthesis of MBL is of considerable clinical significance as this observation could potentially explain the surprising finding of Mullighan et al.¹⁰ that low MBL in either donor or recipient was an independent risk factor for significant infections in allogeneic stem cell transplant recipients during the post-transplant period. The association with recipient MBL status is easily accounted for, but the relationship with the MBL status of the stem cell donor can only be explained if MBL can be synthesized by the donor's leucocytes in their new host. Unfortunately, we were unable to demonstrate any secretion from our cultured cells into the medium over a six-day period, but the leucocytes putatively responsible might well behave differently in a physiological environment.

The demonstration of cation-independent MBL receptors on some monocytes and dendritic cells was unsurprising. There is much evidence for a collectin/C1q receptor(s) on phagocytic cells, although its (their) nature(s) remains elusive. It appears that there may be two or more phagocytic cell receptors that can bind MBL by protein–protein interaction, and just how much overlap there is with similar receptors for other collectins and for C1q is unclear.^11,12 Our observed binding of MBL to monocytes and dendritic cells in the presence of EDTA was therefore expected.

However, the existence of a receptor, apparently synthesized for preferential expression on immature dendritic cells, was totally unexpected. Other reported MBL receptors on phagocytic cells bind to the collagen domain on MBL by protein–protein interaction, and although the activity of one such receptor, CR1/CD35, was enhanced by calcium¹³ the latter could not be inhibited with 100 mm GlcNAc.

MBL can bind to apoptotic cells¹⁴ and to certain phospholipids.¹⁵ MBL may recognize phospholipid structures present only on the surface of apoptotic cells, but which are accessible intracellularly when healthy cells are rendered permeable for intracellular staining. Therefore, intracellular staining with biotinylated MBL is impossible to interpret fully, as both MBL–carbohydrate and MBL–phospholipid interactions are inhibited to a similar degree by the same simple sugars.¹⁵ These considerations do not apply to the surface staining. The importance of the intracellular MBL binding is simply that it is consistent with the surface staining; any failure to demonstrate intracellular MBL binding would have cast suspicion on the validity of the surface binding observations.

The discovery of a new mannose/GlcNAc-sensitive, cation-dependent receptor for MBL present mainly on the surface of immature dendritic cells suggests a new direction for collectin research. MBL has hitherto been thought of as a pattern recognition molecule recognising carbohydrate structures on foreign (microbial) surfaces, and some apoptotic cells.¹⁴ However, MBL has not been suspected of binding to structures on healthy autologous cells. This observation suggests a new, unforeseen function for MBL, perhaps a regulatory role within the immune system.