IMMUNOLOGY
ORIGINAL ARTICLE
Variable antigen uptake due to different expression of the macrophage mannose receptor by dendritic cells in various inbred mouse strains Stella Eugenie Autenrieth1,2 and Ingo Birger Autenrieth1 1
Institut fu¨r Medizinische Mikrobiologie und Hygiene, Universita¨t Tu¨bingen, Tu¨bingen, and 2Interfakulta¨res Institut fu¨r Zellbiologie, Universita¨t Tu¨bingen, Tu¨bingen, Germany
doi:10.1111/j.1365-2567.2008.02960.x Received 22 July 2008; revised 3 September 2008; accepted 4 September 2008. Correspondence: S. E. Autenrieth, Institut fu¨r Medizinische Mikrobiologie und Hygiene, Universita¨t Tu¨bingen, ElfriedeAulhorn-Str. 6, 72076 Tu¨bingen, Germany. Email: Stella.Autenrieth@medizin. uni-tuebingen.de Senior author: Ingo Birger Autenrieth, email:
[email protected]
Summary Antigen uptake by dendritic cells is essential for the induction of antigenspecific T-cell responses. Here, we investigate the ability of dendritic cells from different mouse strains to endocytose antigens. The uptake of different fluorescently labelled soluble antigens by bone marrow-derived dendritic cells from BALB/c, C57BL/6 and C3H/HeN mice was analysed by flow cytometry. Using transferrin as a specific marker for clathrinmediated endocytosis, we observed no significant differences of transferrin uptake by dendritic cells from BALB/c, C57BL/6 and C3H/HeN mice. Similar results were obtained by analysing macropinocytosis with lucifer yellow. In contrast, analysing the uptake of ovalbumin, which is predominantly mediated by clathrin-mediated endocytosis via the macrophage mannose receptor, we found that dendritic cells from C3H/HeN mice take up three- to fivefold more ovalbumin than dendritic cells from BALB/c or C57BL/6 mice. Blocking the uptake of ovalbumin via the macrophage mannose receptor by using mannan led to a comparable uptake of ovalbumin by dendritic cells from all three mouse strains. Consistently, dendritic cells from C3H/HeN mice displayed significantly increased expression of the macrophage mannose receptor compared to dendritic cells from BALB/c or C57BL/6 mice. In conclusion, receptors involved in antigen uptake such as the macrophage mannose receptor may be differentially expressed and may explain variations of T-cell responses after vaccination in different individuals. Keywords: antigen uptake; dendritic cells; macrophage mannose receptor; mice
Introduction Endocytosis by dendritic cells (DCs) is essential for the presentation of antigens by major histocompatibility complex (MHC) molecules and subsequent T-cell activation.1,2 Dendritic cells take up soluble antigens via two different mechanisms, either by macropinocytosis3,4 or by clathrin-mediated endocytosis (previously referred to as receptor-mediated endocytosis).5 Macropinocytosis is a form of high-volume, non-specific endocytosis that involves extension of membrane ruffles. Clathrin-mediated endocytosis in DCs includes the endocytosis of receptors such as Fc receptors, transferrin receptor, macrophage mannose receptor (MMR, CD206), DEC 205 (CD205), and DC-SIGN.3,4,6,7 The MMR, a member of the C-type lectin family,8 has been shown to be involved in the recognition of several 2008 Blackwell Publishing Ltd, Immunology, 127, 523–529
microorganisms and the clearance of serum glycoproteins. In macrophages, the MMR functions as an endocytic receptor that binds mannosylated structures such as dextrans.9 The MMR is also expressed on DCs, Langerhans cells, monocytes and endothelial cells. In addition, it has been shown that in DCs, endocytosis of the model antigen ovalbumin (OVA) is predominantly mediated via MMR.10,11 The OVA is then transported to early endosomes, leading to cross-presentation via MHC class I molecules and subsequently to CD8+ T-cell activation.11,12 On the other hand, OVA uptake via macropinocytosis leads to classical presentation of OVA peptide on MHC class II molecules and CD4+ T-cell activation.12 These data demonstrate that the route of OVA uptake determines the presentation to CD4+ or CD8+ T cells (in bone marrow-derived DCs from C57BL/6 mice). As there are no studies comparing antigen uptake by DCs from 523
S. E. Autenrieth and I. B. Autenrieth distinct mouse strains, we investigated whether there are differences in the uptake of soluble antigens by bone marrow-derived DCs from BALB/c, C57BL/6 and C3H/HeN mice.
Materials and methods Mice BALB/c, C57BL/6 and C3H/HeN mice were purchased from Harlan Winkelmann (Borchen, Germany). The experiments were performed using 6- to 10-week-old female mice.
Generation of bone marrow-derived DCs Bone marrow-derived DCs were grown in RPMI-1640 medium (Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich, Hamburg, Germany), 2 mM glutamine (Invitrogen, Karlsruhe, Germany), 100 U/ml penicillin (Invitrogen), 100 lg/ml streptomycin (Invitrogen), 50 lM 2-mercaptoethanol (Sigma), 1% (v/v) non-essential amino acids (Biochrom) and 1 mM sodium pyruvate (Biochrom). The DCs were prepared using granulocyte–macrophage colony-stimulating factor (GM-CSF) as previously described.13,14 Briefly, 2 · 106 bone marrow cells, flushed from the femurs and tibias of BALB/c, C57BL/6 and C3H/HeN mice, were seeded in 100-mm dishes in 10 ml medium containing 200 U/ml GM-CSF. After 3 days, an additional 10 ml of fresh medium containing 200 U/ml GM-CSF was added to the cultures. On day 6, half of the culture supernatant was collected and centrifuged, and the resultant cell pellet was resuspended in 10 ml of fresh medium containing 200 U/ml GM-CSF and given back to the original plate. At day 8, the slightly attached cells were used for the experiments described in this report.
Splenic cell suspension Spleens were cut into small fragments and then digested for 30 min at 37 in 2 ml modified RPMI-1640 + 2% FCS medium containing collagenase (1 mg/ml; type IV; Sigma-Aldrich) and DNase I (Invitrogen). To disrupt DC–T-cell complexes, ethylenediaminetetraacetic acid (EDTA; 1 ml, 01 M, pH 72) was added, and mixing continued for 5 min. Undigested fibrous material was removed by filtration.
sciences, San Diego, CA), rat anti-mouse CD206 conjugated with fluorescein isothiocyanate (-FITC), rat anti-mouse CD206-bio (MR5D3; AbD Serotec, Du¨sseldorf, Germany) following streptavidin-phycoerythrin or streptavidin conjugated with peridinin chlorophyll protein, rat anti-mouse MHC II (M5/114.15.2), hamster antimouse CD80-FITC (16-10A1), rat anti-mouse CD86-FITC (GL-1), and hamster anti-mouse CD40-FITC (HM40-3), which were all obtained from BD Biosciences.
Measurement of antigen uptake by flow cytometry For antigen uptake, DCs (5 · 105) were incubated with OVA-AlexaFluor647 (10 lg/ml), dextran-FITC (1 mg/ml), Tf-AlexaFluor647 (50 lg/ml), or lucifer yellow (2 mg/ml; all from Molecular Probes, Invitrogen) at 37 for 30 min or 10 min in the case of lucifer yellow. In some cases, cells were pretreated with 3 lg/ml mannan (SigmaAldrich) for 30 min at 37. Unspecific binding was assessed by incubation of the DCs with the markers for 30 min on ice. Cells were washed three times in ice-cold phosphate-buffered saline containing 2% FCS. Subsequently, the DCs were labelled with fluorescent monoclonal antibodies according to the manufacturer’s instructions. For MMR expression an intracellular staining with anti-CD206 monoclonal antibody was performed after fixation and permeabilization of the cells with 1% paraformaldehyde and 0.1% Saponin plus 0.5% bovine serum albumin in phosphate-buffered saline, respectively. Dead cells were excluded by staining with 7-amino-actinomycin D (7-AAD; Sigma-Aldrich). In the case of intracellular staining fixation of the cells after 7-AAD staining was performed in the presence of 2 lg/ml actinomycin-D. The analysis of antigen uptake was performed on a fluorescence-activated cell sorter (FACS) Calibur or FACS Canto II flow cytometer (BD Biosciences) using Summit 4.3 (Dako Cytomation Dako, Hamburg, Germany) and DIVA software (Becton Dickinson). The uptake of antigens was quantified as mean fluorescence intensity of CD11c+/ 7-AAD) cells.
Statistics Data were analysed using the GRAPH PAD PRISM 4.0 software. Statistical analysis was performed using the unpaired two-tailed Student’s t-test. Differences were considered as statistically significant if P < 005.
Results Antibodies The following monoclonal antibodies were used for flow cytometry: allophycocyanin- or phycoerythrin-conjugated hamster anti-mouse CD11c (HL3; BD Biosciences, Heidelberg, Germany), rat anti-mouse CD8a (53/6.7; eBio524
Differences in antigen uptake by DCs from BALB/c, C57BL/6 and C3H/HeN mice To investigate whether endocytosis of different antigens is similar in DCs from several mouse strains, bone marrow 2008 Blackwell Publishing Ltd, Immunology, 127, 523–529
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Figure 1. Differences in antigen uptake by dendritic cells (DCs) from BALB/c, C57BL/6 and C3H/HeN mice. Bone marrow-derived DCs generated from BALB/c, C57BL/6 and C3H/HeN mice were incubated for 30 min with ovalbumin (OVA)-AlexaFluor647 (a, b), dextran-fluorescein isothiocyanate (FITC) (c), Tf-AlexaFluor647 (d) or lucifer yellow (e) for 10 min, washed three times and analysed by flow cytometry. Numbers in the upper right quadrant represent the percentage of CD11c+ 7-AAD) OVA+ cells (a). The mean fluorescence intensity (MFI) of OVA-AlexaFluor647 or dextran-FITC by CD11c+ 7-AAD) DCs is shown as mean + SD of triplicates. The data are representative of 6–10 individual experiments (b and c). (d) Tf-AlexaFluor647 data represent mean values + SD of three individual experiments. (e) Lucifer yellow uptake is shown as MFI values representative of three individual experiments. **P < 001, ***P < 0005 compared to DCs from BALB/c and C57BL/6 mice.
derived DCs from BALB/c, C57BL/6 and C3H/HeN mice were incubated with different fluorescently labelled soluble antigens and analysed for antigen uptake by flow cytometry. Analysing the uptake of OVA, which is mediated by macropinocytosis and clathrin-mediated endocytosis via the MMR (CD206),10,11 revealed that DCs from C3H/HeN mice took up threefold to fivefold more OVA-AlexaFluor647 than DCs from BALB/c or C57BL/6 mice (Fig. 1a,b). Furthermore, we analysed the endocytosis of another antigen, dextran, known to be taken up by DCs via macropinocytosis and clathrin-mediated endocytosis.9 For this purpose, we incubated DCs from BALB/ C, C57BL/6 and C3H/HeN mice with FITC-labelled dextran 40.000 and analysed the uptake of dextran by flow cytometry. As with OVA-AlexaFluor647, we observed significantly higher mean fluorescence intensity values of dextran-FITC in DCs from C3H/HeN mice compared to DCs from BALB/c and C57BL/6 mice (Fig. 1c). To find out whether the higher uptake of OVA and dextran was mediated by macropinocytosis or by clathrin-mediated endocytosis we analysed the uptake of transferrin, a specific marker for clathrin-mediated endocytosis via the transferrin receptor,15 and lucifer yellow as a specific marker for macropinocytosis.4 We observed comparable uptake of transferrin-AlexaFluor647 or lucifer yellow in DCs from the different inbred mouse strains (Fig. 1d,e). 2008 Blackwell Publishing Ltd, Immunology, 127, 523–529
During the maturation process of DCs macropinocytosis is down-regulated, whereas clathrin-mediated endocytosis is not affected.4 To exclude the possibility that the state of maturation is responsible for the difference in antigen uptake by the three mouse strains, we analysed the expression of maturation markers on the DCs at day 8 of culture and 24 hr post-stimulation with 1 lg/ml lipopolysaccharide (LPS). Figure 2 shows no differences in the expression of CD80, CD86 and CD40 by DCs from all mouse strains at day 8 of culture and at 24 hr after LPS stimulation, indicating similar maturation states. The expression of MHC class II with and without LPS stimulation by DCs from BALB/c and C57BL/6 mice was comparable, whereas DCs from C3H/HeN mice expressed lower levels of MHC class II in both cases. This effect has also been observed by others (personal communication T. Volz, University of Tu¨bingen). Nevertheless, the fold change of the mean fluorescence intensity of MHC class II from untreated to LPS-treated DCs was similar in all cases. In addition, spleen cells from C3H/HeN mice also exhibited a lower expression of MHC class II than spleen cells from C57BL/6 mice (personal communication Antje Lohmu¨ller, BD Biosciences Europe). Therefore, the different uptake of OVA by DCs from BALB/c, C57BL/6 and C3H/HeN mice was not the result of different maturation states. Altogether, these data indicate that macropinocytosis 525
S. E. Autenrieth and I. B. Autenrieth CD80
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Figure 2. Comparable maturation state of dendritic cells (DCs) from BALB/c, C57BL/6 and C3H/HeN mice. Bone marrow-derived DCs from BALB/c, C57BL/6 and C3H/HeN mice were stained for the expression of the maturation markers CD80, CD86, CD40 and major histocompatibility complex class II (MHC II) following stimulation for 24 hr with 1 lg/ml lipopolysaccharide (black thick line), no stimulation (dashed area) or isotype control (grey area).
by DCs from BALB/c, C57BL/6 and C3H/HeN is comparable but that there are differences in clathrin-mediated endocytosis of OVA and dextran.
Higher expression of MMR by DCs from C3H/HeN mice increase the amount of antigen uptake As the uptake of OVA by DCs from C3H/HeN mice is higher than that of DCs from BALB/c and C57BL/6 mice, we analysed whether the expression of MMR involved in antigen uptake was different in DCs from these mouse strains. By flow cytometry analysis, we observed a fourfold higher expression of the MMR by DCs from C3H/ HeN mice compared to DCs from BALB/c and C57BL/6 mice (Fig. 3a,b), whereas the expression of CD205, another member of the C-type lectin family involved in antigen uptake by DCs, was comparable by DCs from all three mouse strains (data not shown). Our group and that of Kurts have shown that bone marrow-derived DCs take up soluble OVA via macropinocytosis and clathrinmediated endocytosis via the MMR.10,11 By staining the DCs with a monoclonal antibody to MMR after incubation with dextran-FITC or OVA-AlexaFluor647, we could demonstrate that there is a correlation between the expression of MMR and the amount of dextran and OVA actually endocytosed (Fig. 3c), indicating that dextran and OVA uptake were mediated by MMR in DCs from BALB/c, C57BL/6 and C3H/HeN mice. Mannan has been shown to block MMR-mediated uptake of antigens by competitive binding to MMR.4 To investigate whether the correlation of the expression of MMR and the uptake of OVA is the result of a functional 526
role of MMR, DCs were preincubated with mannan, and then analysed for OVA uptake (Fig. 4a,b). The uptake of OVA was blocked in mannan-treated DCs from all three mouse strains compared to untreated DCs. The amounts of OVA taken up by DCs from BALB/c, C57BL/6 and C3H/HeN mice after pretreatment with mannan were similar, indicating that OVA uptake via MMR is completely blocked by mannan, while macropinocytosis of OVA is not affected. Consistently, the percentage of reduction of OVA uptake was higher in DCs from C3H/ HeN mice compared with DCs from BALB/c and C57BL/ 6 mice. Altogether our data show that the different expression of MMR by DCs of distinct mouse inbred strains leads to differences in the amount of antigen uptake by clathrin-mediated and MMR-mediated mechanisms.
Discussion Endocytosis of antigens by DCs is essential for the induction of T-cell responses during infection or vaccination. To determine whether the uptake of soluble antigens is similar in DCs from distinct mouse strains, we used bone marrow-derived DCs from the most common inbred mouse strains BALB/c, C57BL/6 and C3H/HeN. We could show that macropinocytosis, analysed by the use of lucifer yellow, is similar in DCs from all three mouse strains. Likewise, receptor-mediated endocytosis of transferrin via the transferrin receptor is also comparable in DCs from BALB/c, C57BL/6 and C3H/HeN mice. In contrast, we could demonstrate that the uptake of OVA and dextran by DCs from C3H/HeN mice was up to fivefold more 2008 Blackwell Publishing Ltd, Immunology, 127, 523–529
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Figure 3. Expression of macrophage mannose receptor (MMR) regulates differences in uptake of ovalbumin (OVA) and dextran by dendritic cells (DCs) from inbred mouse strains. (a) Bone marrow-derived DCs from BALB/c, C57BL/6 and C3H/HeN mice were stained with anti-CD206 antibody (black line) or isotype control antibody (grey area). (b) The diagram shows MMR expression as mean fluorescence intensity (MFI) + SD of triplicates representative of five independent experiments. ***P < 0005 compared to DCs from BALB/c and C57BL/6 mice. (c) Bone marrow-derived DCs generated from BALB/c, C57BL/6 and C3H/HeN mice were incubated either with fluorescein isothiocyanate-conjugated dextran or OVA-AlexaFluor647 for 30 min, stained for MMR expression, and analysed by flow cytometry. Numbers in the upper right quadrant represent the percentage of CD11c+ 7-AAD) OVA+ CD206+ or CD11c+ 7-AAD) dextran+ CD206+ cells.
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efficient than the uptake by DCs from BALB/c and C57BL/6 mice. The increased uptake of OVA and dextran by DCs from C3H/HeN mice compared to DCs from BALB/c, and C57BL/6 mice is a result of their higher expression of MMR mediating uptake of soluble antigens by DCs.10,11 The MHC class II restricted CD4 T-cell responses are crucial for immunity against many bacterial and fungal pathogens, e.g. Yersinia enterocolitica, Salmonella typhimurium and Candida albicans. It was shown for fungal 2008 Blackwell Publishing Ltd, Immunology, 127, 523–529
pathogens like Aspergillus fumigatus,16 Cryptococcus neoformans17 and C. albicans18 that they are phagocytosed by DCs via the MMR, leading to killing of C. albicans18 and the induction of T helper cell responses against A. fumigatus,16 C. neoformans17 and C. albicans.18 Our results suggest that as a result of the different expression of MMR by DCs the different inbred mouse strains should be more or less resistant to infection with these fungal pathogens, and should mount weaker or stronger pathogen-specific T-cell responses. As to whether MMR 527
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Figure 4. Blocking of macrophage mannose receptor (MMR) with mannan leads to comparable uptake of ovalbumin (OVA) by dendritic cells (DCs) from BALB/c, C57BL/6 and C3H/HeN mice. After 30 min pretreatment of bone marrow-derived DCs with 3 mg/ml mannan the cells were incubated for 30 min with OVA-AlexaFluor647 and the uptake of OVA was analysed by flow cytometry. (a) Histograms show the OVA-AlexaFluor uptake profile by untreated (black line), mannan-treated (dashed area), and ice control (grey area) CD11c+ 7-AAD) DCs from BALB/c, C57BL/6 and C3H/ HeN mice. (b) The diagram shows MFI of OVA uptake by untreated (black bars) and mannan-treated (white bars) CD11c+ 7-AAD) DCs from BALB/c, C57BL/6 and C3H/HeN mice. The data show triplicates representative of three independent experiments. ***P < 0005 compared to DCs from BALB/c and C57BL/6 mice.
is involved in resistance against Y. enterocolitica or S. typhimurium or whether antigen uptake via MMR is required for Y. enterocolitica-specific or S. typhimuriumspecific T-cell responses is unknown. Work by Burgdorf et al.12 demonstrated that the route of antigen uptake determines the presentation of soluble antigens to either CD4+ or CD8+ T cells. Using DCs from CD206)/) and C57BL/6 mice, it was demonstrated that OVA taken up by MMR is found in early endosomes, loaded on MHC class I molecules, and subsequently crosspresented to CD8+ T cells.11,12 Furthermore, OVA uptake via macropinocytosis leads to classical presentation of OVA peptide by MHC class II molecules, and to CD4+ T-cell activation.12 Therefore, targeting antigens to MMR would be one possibility of supporting vaccines in the induction of CD8+ T-cell-mediated immunity against viruses and tumours. Ramakrishna et al.19 used a human antibody against MMR genetically linked with the melanoma antigen pmel17 to target DCs. This resulted in the presentation of pmel17 in the context of HLA class I and class II molecules leading to the activation of pmel17-specific cytotoxic CD8 T cells as well as CD4 T helper cells.19 528
The data presented in this paper show that there are differences in the expression of MMR by DCs of different inbred strains of mice leading to greater or lesser antigen uptake by this receptor. In consequence, according to Kurts and colleagues,11,12 this should cause a weaker or stronger CD8+ T-cell activation. Unfortunately, this hypothesis could not be verified because of the lack of specific T cells or tetramers for the OVA epitope in the context of MHC class I from C3H/HeN mice. Nevertheless, ex vivo studies with splenic DCs revealed that MMR is expressed in all mouse strains by CD8a+ splenic DCs; however, expression of MMR by splenic CD8a+ DCs is limited in comparison with bone marrow-derived DCs. Moreover, while splenic CD8a+ DCs from C3H/HeN and BALB/c mice display moderate MMR expression, CD8a+ DCs from C57BL/6 mice display very limited expression only (Fig. S1). Nevertheless, the differences in antigen uptake via MMR in individuals should still be considered when targeting MMR for vaccination studies. In addition, further studies regarding the antigen uptake by DCs via MMR in humans should be instigated. Moreover, it cannot be excluded that the lower expression of MMR by DCs of individuals resulting in less antigen uptake might be compensated via increased macropinocytosis, leading to comparable antigen-specific CD4 and CD8 T-cell responses.
Acknowledgements We thank D. Gunst for excellent technical help. This work was supported by grants from the Deutsche Forschungsgemeinschaft.
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Supporting information Additional Supporting information may be found in the online version of this article: Figure S1. Expression of MMR by splenic DCs. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
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