Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.1204711 on February 22, 2005

Published online before print February 22, 2005
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(Journal of Leukocyte Biology. 2005;77:689-698.)
© 2005 by Society for Leukocyte Biology

Differential capability for phagocytosis of apoptotic and necrotic leukemia cells by human peripheral blood dendritic cell subsets

Jakob Dalgaard*,1, Karen J. Beckstrøm*, Frode L. Jahnsen{dagger} and Jan E. Brinchmann*

* Institute of Immunology and
{dagger} Laboratory for Immunohistochemistry and Immunopathology, Institute of Pathology, Rikshospitalet University Hospital and University of Oslo, Norway

1 Correspondence: Institute of Immunology, Rikshospitalet University Hospital, Sognsvannsveien 20, N-0027 Oslo, Norway. E-mail: jakob.dalgaard{at}labmed.uio.no


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD11c+ dendritic cells (DC) and plasmacytoid DC (PDC) are the two major DC subsets in human peripheral blood. For the purpose of immunotherapy with DC, it is important to investigate the phagocytosis of killed tumor cells by different DC subsets. Using immature monocyte-derived DC (iMoDC) as reference, we have compared the ability of CD11c+ DC and PDC to phagocytose apoptotic and necrotic K562 leukemia cells. Freshly isolated CD11c+ DC phagocytosed apoptotic and necrotic K562 cells, whereas PDC did not show any evidence of uptake of dead cells. Blocking studies showed that CD36 is importantly involved in uptake of apoptotic and necrotic material. CD91 and CD11c were also involved. In addition, we found that ß5 integrin was expressed on CD11c+ DC but not in its classical association with {alpha}V. Uptake of apoptotic K562 cells by CD11c+ DC was increased following incubation with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4, alone or in combination with transforming growth factor-ß1, to levels comparable with those observed for iMoDC. Phagocytosis of dead cellular material by the GM-CSF/IL-4-treated CD11c+ DC was largely restricted to a subset expressing low levels of human leukocyte antigen-DR and CD83. Thus, the relationship between phagocytosis of antigenic material and expression of maturation-related cell-surface molecules is similar for CD11c+ DC and MoDC. We conclude that CD11c+ DC in peripheral blood are precursor cells, which under the influence of cytokines, differentiate to cells with DC phenotype and function.

Key Words: CD11c+ • PDC • MoDC • uptake • CD36


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) are rare bone marrow-derived cells, specialized in antigen uptake, processing, and presentation to T cells. DC are unique antigen-presenting cells (APC) in the sense that they are able to prime immunologically naïve T cells [1 , 2 ]. Immature DC (iDC) are able to efficiently take up and process exogenous, antigenic material. Following maturation, antigenic peptides are presented in the context of major histocompatibility complex (MHC) class II molecules to CD4+ T cells [3 ]. It has been shown that exogenous antigens may also be presented by human DC in the context of MHC class I to CD8+ T cells, a phenomenon called cross-presentation [2 , 4 ], which is an important process and has been associated with the generation of CD8+ cytotoxic T lymphocytes against tumor cells [5 ]. The ability of mature DC to prime naïve as well as memory T cells results from the high level of expression of MHC complexes and costimulatory molecules compared with other APC, like B cells and monocytes. Other molecules unique to the DC may also be involved in T cell priming [1 ].

Peripheral blood contains two major populations of iDC: CD11c+ DC, which are also referred to as CD1c+ DC or myeloid DC, and the plasmacytoid DC (PDC), which are also called interleukin-3 receptor-positive (IL-3R{alpha}+) or CD123+ PDC [6 7 8 ]. Peripheral blood DC (PBDC) are defined by their lack of hematopoietic lineage-specific markers (CD3–, CD14–, CD16–, CD19–, CD56–) and high expression of MHC class II molecules and constitute less than 1% of peripheral blood mononuclear cells (PBMC) in healthy individuals [9 ]. PBDC can be isolated by negative selection, or they can be positively selected using monoclonal antibodies (mAb) specific for the DC subsets [10 ]. It is believed that the PBDC are en route to peripheral tissues. Here, they reside, and upon encounter with pathogen, they mature and migrate to secondary lymphoid organs [1 ].

Monocytes and CD11c+ DC are known to be of myeloid origin, and the ontogeny of PDC is still not fully characterized [1 , 8 , 11 ]. In vitro, monocytes differentiate to immature monocyte-derived DC (iMoDC) under the influence of granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-4 [12 ]. iMoDC have low expression of MHC classes I and II and low levels of costimulatory and adhesion molecules and do not express CD83 [1 , 2 ]. They capture antigens efficiently through phagocytosis and endocytosis. iMoDC can be matured with a variety of stimuli upon which they up-regulate expression of MHC classes I and II and costimulatory and adhesion molecules. Thus, they become potent APC. They also up-regulate CD83 and CC chemokine receptor 7, which is important for migration [12 13 14 15 ]. During the maturation process, the phagocytic and endocytic capabilities of iMoDC are reduced to a low level.

The in vitro differentiation and maturation potential of the PBDC subsets are less well-characterized. CD11c+ DC isolated from patients treated with FMS-like tyrosine kinase 3 ligand (Flt3L) have been shown to mature to cells expressing CD83 upon brief culture with GM-CSF and IL-4 [16 ]. Like iMoDC, CD11c+ DC can differentiate into cells resembling epidermal Langerhans cells (LC) in the presence of transforming growth factor-ß1 (TGF-ß1), which is an important factor for LC differentiation in mice and humans [1 , 17 , 18 ]. PDC require IL-3 for in vitro survival [6 ]. A recent study demonstrated that PDC may differentiate to myeloid DC upon virus infection in vitro [19 ]. This surprising observation underscores the need for more information on the ontogeny and differentiation potential of these DC subsets.

Certain functional aspects of CD11c+ DC and PDC, such as allostimulation of T cells, migration, secretion of cytokines, and uptake of particles, have been characterized extensively. In some of the studies, a comparison with monocytes, MoDC, or CD34+-derived DC has been done [10 , 16 , 18 , 20 21 22 23 24 ]. However, studies comparing the ability of the different DC subsets to take up and present antigenic material from tumor cells have, to the best of our knowledge, not been performed.

For the purpose of clinical cancer trials, DC subsets can be loaded with appropriate antigen by different strategies. One strategy is to load DC with material from apoptotic or necrotic tumor cells [2 ]. We have previously examined uptake of apoptotic and necrotic K562 leukemia cells by iMoDC [25 ]. Here, we have investigated the uptake of apoptotic and necrotic K562 leukemia cells by PBDC using the uptake by iMoDC as a reference. We show that CD11c+ DC but not PDC are able to take up apoptotic and necrotic K562 leukemia cells. Comparing CD11c+ DC with iMoDC, the uptake by iMoDC was significantly higher than that observed for freshly isolated CD11c+ DC. However, culture for 3 or 6 days in GM-CSF and IL-4 resulted in increased uptake by the CD11c+ DC to levels comparable with the iMoDC. Expression and blocking experiments indicated that CD36 may be one of the key molecules involved in the uptake of apoptotic and necrotic material from leukemia cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Media and cytokines
Macrophage serum-free medium (MSFM) with L-glutamine (Gibco, Invitrogen Corp., Paisley, Scotland) was supplemented with 2% autologous serum (AS), penicillin, 50 units/ml, and streptomycin, 50 µg/ml (Sigma Chemical Co., St. Louis, MO), referred to as MSFM 2% AS. RPMI 1640 with HEPES and L-glutamine (PAA Laboratories GmbH, Pasching, Austria) was supplemented with 10% heat-inactivated fetal calf serum (FCS; Gibco, Invitrogen Corp.), penicillin, and streptomycin, referred to as RPMI 10% FCS. The following recombinant human cytokines were used: GM-CSF (Leukomax, Sandoz, Basel, Switzerland), IL-4 and TGF-ß1 (both from R&D Systems, Minneapolis, MN), and IL-3 (Peprotech, Rocky Hill, NJ).

Antibodies
Conjugated mAb against the following proteins were used: CD3, CD14, CD16, CD19, CD20, CD56, CD80, CD86, human leukocyte antigen (HLA)-DR [conjugated to fluorescein isothiocyanate (FITC)], CD11c, CD83 [conjugated to phycoerythrin (PE)], HLA-DR [conjugated to peridinin chlorophyll protein (PerCP); all from Becton Dickinson Biosciences, San Jose, CA], and goat anti-mouse (GAM) immunoglobulin G (IgG; conjugated to allophycocyanin, Jackson Immunoresearch, West Grove, PA). Irrelevant IgG1 mAb conjugated to FITC and PE and irrelevant IgG2A mAb conjugated to PerCP (all from BD Biosciences) were used as isotype control mAb. The following unconjugated mAb were also used: CD11c (clone B-ly6), {alpha}5 (CD49e; clone IIA1), CD91 (clone A2MR-{alpha}2, BD Biosciences), isotype IgG1 (Diatec, Oslo, Norway), CD36 (clone L103, Ann Jackson, BD Biosciences, San Jose, CA), CD36 (clone SM{Phi}), {alpha}V (CD51; clone 13C2, Chemicon Europe Ltd., Chandlers Ford, UK), {alpha}Vß5 (clone P1F6), {alpha}V (clone AV1, Chemicon International, Temecula, CA), and ß5 (clone B5-IVF2, Upstate Biotechnology, Lake Placid, NY). All unconjugated mAb were isotype IgG1 except for anti-CD36 (clone SM&{Phi};), which was IgM. Unconjugated mAb were used for expression studies and blocking experiments. Anti-CD36 (clone SM&{Phi};) and anti-{alpha}V (clone 13C2) were only used in preliminary blocking experiments with iMoDC. NaN3 was removed from antibody solutions by centrifugation on a desalting column before use in blocking experiments.

Cells, cell cultures, and cell lines
Buffy coats and AS were obtained from healthy blood donors at the Blood Bank, Ullevaal University Hospital (Oslo, Norway). AS was not heat-inactivated. PBMC were isolated by density gradient centrifugation on Lymfoprep (Axis-Shield, Oslo, Norway). PBDC and CD14+ monocytes were isolated from PBMC from the same buffy coat in the following order: PDC, CD11c+ DC, CD14+ monocytes. The following kits were used according to the manufacturer’s recommendations: blood DC antigen (BDCA)-4 cell isolation kit, CD1c (BDCA-1) DC isolation kit, and CD14 microbeads (Miltenyi Biotec, Bergish Gladbach, Germany) for isolation of PDC, CD11c+ DC, and CD14+ monocytes, respectively. Positive selection of PDC and CD11c+ DC was repeated twice, yielding cells with purity greater than 98% and 99%, respectively (data not shown). The purity of CD14+ monocytes after one isolation procedure was greater than 96% (data not shown). After isolation, PDC and CD11c+ DC were resuspended in MSFM 2% AS with IL-3 (10 ng/mL) and GM-CSF (20 ng/mL), respectively, as survival factors and used for further experiments.

In differentiation studies, CD11c+ DC were cultured in MSFM 2% AS in Costar 96-well culture plates (Corning Inc., Corning, NY) in the presence of GM-CSF (100 ng/mL) and IL-4 (50 ng/mL) alone or with the addition of TGF-ß1 (1 ng/mL) for 3 or 6 (only with GM-CSF and IL-4) days. The cells were then used for studies of uptake of apoptotic material or expression of relevant molecules. When cultured for 6 days, half of the medium was removed on day 3 and replaced with an equal volume of fresh medium containing standard concentrations of cytokines. iMoDC were generated from CD14+ monocytes by culturing at 1 x 106/mL in Costar six-well plates (Corning Inc.) in 3 mL MSFM 2% AS with GM-CSF (100 ng/mL) and IL-4 (50 ng/mL) for 7 days. On days 2 and 4, 1 mL medium was removed, and an equivalent volume of fresh medium was added with full concentrations of the above-mentioned cytokines. For characterization of PBDC with flow cytometry, leukocytes were isolated from healthy laboratory personnel between 20 and 50 years of age. Venous blood was drawn into Vacutainers containing acid-citrate-dextrose solution A (BD Biosciences, PharMingen, San Diego, CA) and diluted 1:10 in a solution containing 150 mM NH4Cl, 10 mM NaHCO3, and 2 mM EDTA (pH 7.2, 20°C) to lyse red cells. The leukocytes were then pelleted by centrifugation at 180 g for 5 min, washed once in phosphate-buffered saline (PBS), supplemented with 1% FCS, and resuspended in PBS 1% FCS. The Institute of Clinical Pharmacology, Rikshospitalet University Hospital (Oslo, Norway), kindly provided the K562 cell line. Cells were maintained in RPMI 10% FCS.

Uptake of apoptotic K562 leukemia cells by iMoDC and PBDC
Prior to induction of apoptosis, K562 cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) at a concentration of 15 µM for 10 min. Apoptosis was induced as described previously [25 ]. Briefly, a combination of the specific BCR-ABL tyrosine kinase inhibitor CGP 57148 (STI 571; kindly provided by Dr. Elisabeth Buchdunger, Novartis Pharma AG, Basel, Switzerland) and {gamma} irradiation was used. K562 cells in the logarithmic phase of growth were exposed to 10 µM STI 571 on day 0 and 50 Gy {gamma} irradiation on day 2 and were kept in culture until day 6, when they were used for uptake studies. Day 6 apoptotic K562 cells were collected, washed, and resuspended in MSFM 2% AS and added to the different DC subsets at a ratio of 1:1 in Costar 96-well plates for 4 h at 37°C or 4°C (negative control). CFSE-labeled, living K562 cells were also added to the DC subsets as a control. Cells were then collected, washed, and labeled with anti-HLA-DR PerCP mAb and analyzed on a FACSCalibur flow cytometer using CellQuest Pro software (BD Biosciences). DC were identified as HLA-DR+ cells, and uptake was determined as the percentage of CFSE+HLA-DR+ double-positive DC (see Fig. 1 , upper-right quadrant) relative to all DC (see Fig. 1 , upper-right and upper-left quadrants). In the blocking experiments, DC were preincubated with various mAb at a concentration of 50 µg/mL for 30 min at 4°C before the cocultures were established.



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  		Figure 1. iMoDC and CD11c+ DC take up apoptotic K562 leukemia cells, but PDC do not. K562 cells were prelabeled with CFSE prior to induction of apoptosis. DC subsets were identified as HLA-DR (DR) PerCP+ by flow cytometry. Uptake of apoptotic K562 cells was determined as the percentage double-positive DC (upper-right quadrant) relative to all DC (upper-right+upper-left quadrants). One representative experiment out of four is shown.

 
Uptake of necrotic K562 leukemia cells by iMoDC and PBDC
Before induction of necrosis, K562 cells in the logarithmic phase of growth were labeled with CFSE at 10 µM for 10 min or PKH26 (Sigma Chemical Co.) at 15 µM for 10 min. Cells were washed and resuspended in MSFM 2% AS, and necrosis was induced by exposure to five cycles of freezing and rapid thawing in liquid nitrogen and 37°C water-bath, respectively. Necrotic cells were then added to the different DC subsets as described above. Necrotic K562 cells equivalent to a K562:DC ratio of 1:1 were used. The cultures were collected after 4 h, and uptake by the DC was measured as described above. Blocking experiments were also performed as described above.

Immunofluorescence stainings
CD11c+ DC were incubated with necrotic K562 cells prelabeled with PKH26 for 4 h at 4°C and 37°C, as described above. The cells were then stained with anti-HLA-DR FITC for 30 min at 4°C, washed, and fixed with 1% paraformaldehyde, and cytospins were prepared. Fluorescent images were obtained with a confocal laser-scanning microscope (Leica TCS SP, Heidelberg, Germany). The preparations were optically sectioned, and image stacks (Z-series) obtained for each wavelength were then merged and processed with Adobe PhotoShop CS (Version 8.0).

Flow cytometry
Four-color flow cytometry was used to analyze the expression of integrins and other relevant molecules potentially involved in uptake of apoptotic and necrotic cells in CD11c+ DC and PDC. A combined indirect and direct antibody staining was used. Leukocytes were preincubated with PBS with 5% human IgG for 5 min and then incubated with appropriate unconjugated mAb (10 µg/mL) for 20 min. Subsequently, the cells were washed, followed by incubation with 20% goat serum for 5 min. Cells were then labeled with GAM IgG-allophycocyanin for 20 min, washed, and incubated with 20% mouse serum for 5 min. Cells were then labeled with anti-CD3, -CD14, -CD16, -CD19, -CD20, and CD56 FITC (lineage cocktail), CD11c PE, and HLA-DR PerCP for 20 min. The entire staining procedure was performed on ice. Flow cytometry acquisition and analysis were performed on a FACSCalibur flow cytometer using CellQuest Pro software. Live PBMC were gated, and target DC were identified as lineage-negative, HLA-DR+, and CD11c±, the PDC being the CD11c– cells. Expression of relevant molecules could then be analyzed in fluorescence channel 4 (allophycocyanin). Two-color flow cytometry was used to analyze the expression of CD83 and the costimulatory molecules CD80 and CD86 on CD11c+ DC on day 0, 3, or 6. Cells were washed and incubated with 20% mouse serum for 5 min and then labeled with mAb for 30 min on ice. Again, flow cytometry acquisition and analysis were performed.

Statistical analysis
Nonparametric statistical analysis was performed using Prism® Version 4.0. Data are presented with median values. A two-tailed P value less than 0.05 was considered significant. Mann-Whitney test was used for comparison of unpaired data and Wilcoxon signed-rank test, for comparison of paired data.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Freshly isolated CD11c+ DC and iMoDC phagocytose apoptotic and necrotic K562 leukemia cells, but PDC do not
Uptake of apoptotic and necrotic cells is an important function of iDC, which has been well-examined using iMoDC. Uptake by PBDC has been less well-characterized. We therefore examined the uptake of apoptotic and necrotic K562 leukemia cells by PBDC and iMoDC from the same individuals. As PDC rapidly undergo apoptosis unless rescued by IL-3, we added IL-3 to the PDC as a survival factor, before examination of uptake [6 ]. We also added GM-CSF to the CD11c+ DC as a survival factor. Results from a single experiment with apoptotic K562 cells are shown in Figure 1 , and the accumulated results are shown in Figure 2A . Phagocytosis of apoptotic K562 cells was observed in 59% (median) of the iMoDC and in 16% (median) of the CD11c+ DC (P<0.01). Uptake of apoptotic K562 by the PDC was not different from the background level (4°C, controls; P<0.01 vs. CD11c+ DC). As necrotic cells are not one cellular unit, the K562 cells were labeled with dyes binding to membranous material (PKH26) or cytoplasmatic material (CFSE) [26 ]. Uptake of the two elements of necrotic cells was evaluated separately. Phagocytosis of necrotic K562 prelabeled with PKH26 was observed in 69% (median) of the iMoDC and in 54% (median) of the CD11c+ DC (Fig. 2C) . This difference did not reach statistical significance. Uptake of necrotic K562 prelabeled with CFSE by iMoDC was 32% (median), whereas 22% (median) of the CD11c+ DC showed uptake (P<0.01; Fig. 2D ). Again, the uptake of both types of necrotic material by the PDC was not different from the background level (4°C, controls; Fig. 2C and 2D ). Thus, CD11c+ DC and iMoDC phagocytose apoptotic and necrotic K562 leukemia cells, but PDC do not. Uptake of living K562 cells served as a control for apoptotic cells. We found a small uptake by the CD11c+ DC and the iMoDC (Fig. 2B) . However, for both subsets, the uptake of apoptotic cells was significantly higher (Fig. 2A and 2B ; P<0.05 in both cases). For iMoDC, uptake of necrotic K562 prelabeled with PKH26 was significantly higher than uptake of K562 prelabeled with CFSE (Fig. 2C and 2D ; P<0.01), whereas for the CD11c+ DC, no significant difference was found. A confocal laser-scanning microscope (Fig. 3 ) demonstrated phagocytosis by the CD11c+ DC of necrotic material from K562 cells stained with PKH26. Z-series of confocal images (0.7 µm increments) obtained at x1000 magnification confirmed that the fluorescent material was located intracellular (not shown).



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  		Figure 2. Summary of phagocytosis experiments. Uptake of apoptotic K562 cells (A), living K562 cells (B), necrotic K562 cells prelabeled with PKH26 (C), and necrotic K562 cells prelabeled with CFSE (D) by the different DC subsets. Results from all experiments performed at 37°C are shown with median values. Each point represents one experiment from one individual donor. Control experiments at 4°C were performed for all experiments. The percentage phagocytosing DC were always between 1% and 4% (data not shown). ", P < 0.05, compared with other DC subset; *, P < 0.01, compared with other DC subset.

 


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  		Figure 3. Demonstration of intracellular localization of phagocytosed material. Immunofluorescence staining of CD11c+ DC with HLA-DR FITC (green) and necrotic K562 cells prelabeled with PKH26 (red) after incubation for 4 h at 37°C (A, B) and 4°C (C, D), respectively. (B, D) The original images represent 0.7 µm optical sections.

 
Analysis of cell-surface molecules potentially involved in uptake of apoptotic and necrotic K562 leukemia cells by CD11c+ DC
Having shown that CD11c+ DC were capable of phagocytosis of apoptotic and necrotic K562 cells, we then examined molecules potentially involved in the uptake. It is known from previous studies that {alpha}Vß5 and CD36 are involved in the uptake of apoptotic cells by iMoDC [13 ]. It is also known that CD91 is involved in the uptake of necrotic cells by APC [27 ]. We therefore decided to determine the expression of these molecules and some other integrins on PBDC. PDC were included in the examination, although they did not show any uptake. We reasoned that molecules expressed by the CD11c+ DC and not by PDC might be responsible for the differential uptake of dead cells by these DC subsets. As shown in Table 1 , almost all of the CD11c+ DC expressed {alpha}4, {alpha}5, ß1, CD36, and CD91. Sixty percent (median) of these cells stained with a mAb specific for the {alpha}Vß5 heterodimer, and 29% (median) of the cells bound a ß5-specific mAb. Practically no cells stained with a mAb specific for {alpha}V (Fig. 4 and Table 1 ). All of the PDC expressed {alpha}4, ß1, and CD36. There was no expression of {alpha}5, {alpha}V, {alpha}Vß5, ß5, and CD91. We decided to perform blocking studies on the CD11c+ DC using mAb specific for {alpha}Vß5, ß5, {alpha}5, CD11c, CD91, and CD36. Antibodies specific for {alpha}4 and ß1 were not included, as these molecules were expressed by both DC subsets. A mAb specific for {alpha}V, which was not expressed by the CD11c+ DC, served as a control.


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  		Table 1. Expression of Integrins and Other Molecules Potentially Involved in Phagocytosis by PBDC

 


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  		Figure 4. CD11c+ DC express ß5 but not {alpha}V. Shown is PBDC (gated as lineage-FITC-negative and HLA-DR-PerCP-positive cells) with CD11c+ DC and PDC identified as CD11c-PE-positive or -negative cells. Expression of {alpha}V (A), {alpha}Vß5 (B), and ß5 (C) was analyzed using unconjugated mouse mAb in indirect staining techniques. Expression of the integrin chains on CD11c+ DC is shown as a percentage of the total population of CD11c+ DC. One representative experiment out of five is shown.

 
Figure 5 shows the results of the blocking studies with CD11c+ DC. We found a 50% inhibition of the uptake of apoptotic K562 cells using the anti-CD36 mAb. The anti-{alpha}Vß5 and anti-CD91 mAb inhibited uptake of apoptotic cells by ~20%, and the remaining mAb did not inhibit uptake. A "cocktail" of mAb specific for CD36, CD91, {alpha}Vß5, and CD11c did not inhibit the uptake more than the anti-CD36 mAb alone (Fig. 5A) . When examining uptake of membranous material from necrotic K562 cells prelabeled with PKH26 (Fig. 5B) , we observed a 15% inhibition with anti-{alpha}Vß5, anti-ß5, and anti-CD91 mAb, 20% inhibition with anti-CD11c mAb, and 30% inhibition with a mAb specific for CD36. Again, no further inhibition was observed using the mAb cocktail. There was no inhibition using the {alpha}5 or the control {alpha}V antibody. In fact, the uptake using the mAb specific for the nonexpressed {alpha}V molecule consistently showed values above those observed for untreated cells, suggesting the possibility of a small element of Fc receptor (FcR)-mediated uptake. If so, the values given for the blocking studies above may be too low.



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  		Figure 5. Summary of experiments using mAb to a number of receptors potentially involved in phagocytosis of apoptotic (A) and membranous-necrotic (B) K562 leukemia cells by CD11c+ DC. K562 cells were labeled with CFSE before induction of apoptosis and with PKH26 before induction of necrosis. Control experiments were performed with untreated CD11c+ DC, which were identified as HLA-DR PerCP+ by flow cytometry. The percent phagocytosing cells was determined as the percent double-positive DC relative to all DC. The degree of blockage was quantified as the percent phagocytosing DC using a receptor-specific mAb relative to the percent phagocytosing cells in the absence of mAb.

 
Cytokine induction of CD11c+ DC increases uptake of apoptotic K562 leukemia cells
PBDC are considered as precursors en route to tissue, where they are thought to differentiate to iDC under the influence of cytokines. Based on the hypothesis that cytokines might differentiate freshly isolated CD11c+ DC to iDC with improved capacity for phagocytosis in vitro, we examined uptake of apoptotic K562 cells by the CD11c+ DC after in vitro culture in mixtures of cytokines for 3 days. Two combinations of cytokines where chosen: GM-CSF and IL-4, which have been shown to induce differentiation to dermal/interstitial DC [1 , 28 ], and GM-CSF/IL-4 plus TGF-ß1, which have been shown to induce differentiation to LC-like cells [18 , 29 ]. We found, as shown in Figure 6 and in Table 2 , that culture for 3 days under both conditions resulted in increased uptake. Compared with the level of uptake on day 0, the uptake was enhanced almost twofold on day 3 and almost threefold on day 6. The x-axis mean fluorescence (indicating concentration of CFSE per cell) of the HLA-DR and CFSE double-positive CD11c+ DC was much higher on day 3 than on day 0, suggesting that the uptake of apoptotic material per DC was higher on day 3 than on day 0.



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  		Figure 6. Culture of CD11c+ DC with different combinations of cytokines for 3 days increases uptake of apoptotic K562 leukemia cells, which were labeled with CFSE before induction of apoptosis. CD11c+ DC were isolated and cultured for 3 days with GM-CSF and IL-4, without or with the addition of TGF-ß1. CD11c+ DC were identified as HLA-DR (DR) PerCP+ by flow cytometry. The percent phagocytosing DC were determined as the percentage of double-positive DC relative to all DC. Note the population of nonphagocytosing, HLA-DRhi cells (discussed in the text). One representative experiment out of four is shown.

 

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  		Table 2. Phagocytosis of Apoptotic K562 Leukemia Cells by CD11c+ DC after In Vitro Culture for 3 or 6 Days with Different Combinations of Cytokines

 
For both cytokine combinations, flow cytometric examination after 3 days of culture suggested that the CD11c+ DC consisted of two populations: one population with high forward-scatter (FSC) and low HLA-DR expression and another population with low FSC and high HLA-DR-expression (Fig. 7A and 7B ). Only the more abundant FSChi and HLA-DRlo population was able to take up apoptotic K562 cells (Figs. 6 and 7C) . This subpopulation was CD83-negative, and the FSClo HLA-DRhi-nonphagocytosing population contained CD83+ and CD83– cells (Fig. 7D and 7E) . We also examined the expression of CD83, CD80, and CD86 on CD11c+ DC on day 0 and on days 3 and 6 after culture with GM-CSF and IL-4. On day 0, all cells were CD86-positive, and there was no expression of CD83 or CD80. From days 0 to 3, there was a more than tenfold increase in the mean fluorescence of CD80 and CD86 with 76% and 80% positive cells (medians), respectively. However, only 39% (median) of the cells expressed CD83 on day 3 (Fig. 7D and 7E) . From days 3 to 6, there was a slight decrease in the number of positive cells and level of expression of the markers (data not shown).



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  		Figure 7. Uptake of apoptotic K562 cells by CD11c+ DC after 3 days of culture with GM-CSF and IL-4. Dot-plots of CD11c+ DC after 4 h of coincubation with apoptotic K562 cells at 37°C shows in (A) one population of smaller cells [FSClo and side-scatter (SSC)lo] and another population of larger cells (FSChi and SSChi). (B) The FSClo population has higher expression of HLA-DR than the FSChi population. (C) Only the FSChi population is able to phagocytose CFSE-stained apoptotic K562 cells. (D and E) The FSChi population is CD83– and HLA-DRlo. The FSClo population contains one CD83+ HLA-DRhi and one CD83– population.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Several DC subsets exist in vivo, such as CD11c+ DC and PDC in human blood, LC in the epidermis, and interstitial DC in the dermis. In vitro, MoDC can be generated from CD14+ monocytes, which resemble interstitial DC [28 ]. DC resembling LC can be generated from monocytes, CD34+ progenitor cells, or CD11c+ DC [17 , 18 , 29 ]. Indeed, it has been claimed that peripheral blood monocytes also represent a DC precursor in vivo [1 ]. It is important to understand differences in the biology between these subsets. This may contribute to our understanding of the immunobiology in vivo of these different subsets and how to use them in protocols for immunotherapy.

We show here using flow cytometry and confocal microscopy that freshly isolated CD11c+ DC are able to take up apoptotic and necrotic K562 leukemia cells, whereas PDC do not. As necrotic cells are not one cellular unit, the K562 cells were labeled with a membranous dye (PKH26) or a cytoplasmatic dye (CFSE) [26 ]. This was done to evaluate uptake separately. Membranous and cytoplasmatic material was taken up by CD11c+ DC. Compared with GM-CSF/IL-4-induced iMoDC, the uptake of apoptotic cells and necrotic cytoplasmatic material (CFSE) by the freshly isolated CD11c+ DC was significantly lower, suggesting that following GM-CSF/IL-4 induction, iMoDC perform phagocytosis of dead cellular material more efficiently than freshly isolated CD11c+ DC. The uptake by the iMoDC of necrotic-membranous (PKH26) material was significantly higher than the iMoDC uptake of cytoplasmatic (CFSE) material. A similar difference was found among the CD11c+ DC. This could reflect that for these phagocytosing DC subsets, different modes of uptake are used, depending on the type of material presented.

Several authors have examined uptake by freshly isolated CD11c+ DC and PDC of particles such as dextran, latex beads, and zymosan granules [6 , 10 , 18 , 23 ]. In some studies, a comparison with monocytes or iMoDC has been done. In all of the studies, the PDC showed no or very little uptake, whereas the CD11c+ DC took up particles. Monocytes and iMoDC showed higher uptake than the CD11c+ DC. The results of the present study, which is the first to examine the uptake of apoptotic and necrotic tumor cells by CD11c+ DC and PDC, are in accordance with these observations. One other study has examined cellular uptake of apoptotic and necrotic lymphocytes by tonsillar CD11c+ DC. However, in that study, the uptake of dead cells was no greater than the uptake of living cells, and no 4°C controls were included [24 ]. Another study demonstrated minimal uptake of apoptotic thymocytes by freshly isolated monocytes [30 ]. Thus, of the cells in peripheral blood with the potential for development to DC, only CD11c+ DC can take up dead, cellular material. PDC were previously thought to participate only in innate antiviral responses. However, recent studies have shown that the PDC are also involved in adaptive antiviral responses, suggesting that they could be capable of taking up viral material [31 ]. In vitro, PDC were shown to increase their antigen processing capacity following culture with IL-3 and tumor necrosis factor {alpha} (TNF-{alpha}) for 2 days [32 ]. In vivo, lung PDC were recently shown to be capable of taking up FITC-conjugated ovalbumin when administered as inhalation [33 ]. It is therefore possible that the PDC progenitor in blood, upon differentiation among cytokines in tissues, acquires the ability to take up and process antigen.

As mentioned above, CD11c+ DC have been considered to be en route to peripheral tissues, where they may function as immature APC. The CD11c+ DC in peripheral blood have been regarded as immature but may just as well be designated as precursors [1 ]. In vitro, exposure of CD11c+ DC to GM-CSF, IL-4, and TGF-ß1 was shown to generate cells that resemble LC in the epidermis, and culture with GM-CSF and IL-4 alone produced cells that were similar to dermal/interstitial DC [1 , 18 ]. Animal studies have shown that intestinal myeloid DC and CD11c+ splenic marginal zone DC are able to phagocytose apoptotic cells [34 , 35 ]. Thus, having demonstrated that human freshly isolated CD11c+ cells were able to phagocytose dead cellular material, we wanted to investigate if the uptake could be increased by cell-culture conditions, previously shown to induce DC differentiation. We found that uptake of apoptotic K562 cells was increased at least twofold upon 3 days of in vitro culture with GM-CSF and IL-4. Addition of TGF-ß1 did not change the level of uptake. There was an additional increase in uptake from days 3 to 6 when cultured with GM-CSF and IL-4. Uptake was now comparable with the uptake by the iMoDC. Robinson et al. [23 ], who demonstrated an increase in uptake of zymosan granules by CD11c+ DC after culture with GM-CSF and TNF-{alpha}, obtained similar results. These observations support the presumption that peripheral blood CD11c+ DC can be regarded as precursor cells and that some degree of differentiation is needed to acquire the properties of efficient APC.

It is generally accepted that iDC are CD83– and show a high degree of endocytic and phagocytic capacity. Upon maturation, when the DC becomes CD83+, they lose their endocytic and phagocytic capacity. However, this knowledge is based on experiments with MoDC [13 ]. In this system, monocytes differentiate to CD83– iMoDC after 5–6 days of exposure to GM-CSF and IL-4 [1 ]. Jefford et al. [16 ] have suggested that CD11c+ DC are different from MoDC, as they express the maturation marker CD83 after only 24 h of culture with GM-CSF and IL-4. However, the CD11c+ cells used in that study were isolated from the blood of patients treated with Flt3L [16 ]. In the present study, the freshly isolated CD11c+ DC did not express CD83, in accordance with previous findings [10 ]. After 3 days of culture with GM-CSF and IL-4, only a minor subset was CD83+. At 6 days, this subset was even smaller. At these time-points, phagocytosis of apoptotic K562 cells was identified only within the subset expressing the immaturity markers CD83– HLA-DRlo. We did not examine the expression of CD83 after 24 h of culture and so cannot exclude that all cells were CD83+ at that time. However, we would argue that the cells were unlikely to be mature at that time, as at later time-points, the majority of the cells demonstrated phenotypic and functional characteristics of immature cells. We also show here that upon culture for 3 or 6 days with GM-CSF and IL-4, CD11c+ DC increase expression of CD80 and CD86, and a large proportion of the cells are positive for these markers. Although costaining of all markers was not performed, the high percentage of CD80+ CD86hi cells suggests that some of the phagocytosing CD83– HLA DRlo immature cells express these maturity markers. Clearly, further studies are required to determine if these results point to the existence of two truly different subsets of CD11c+ DC.

Previously, it has been shown that iMoDC phagocytose apoptotic cells using the cell-surface molecules {alpha}Vß5 and CD36 [13 ]. We confirmed these observations on iMoDC (data not shown) and then investigated a number of molecules that could potentially be involved in uptake by the CD11c+ DC. CD36 was obviously involved in uptake of apoptotic and membranous elements of necrotic K562 cells. The inhibition of uptake of necrotic-membranous and apoptotic K562 was similar, suggesting that necrotic-membranous material is taken up by mechanisms similar to those used for apoptotic material. Apart from binding to oxidized low-density lipoprotein sites, CD36, in cooperation with {alpha}Vß5 and trombospondin, mediates phagocytosis by iMoDC [13 , 36 ]. The ligand on apoptotic cells is currently unknown. Our results show that CD91 is involved in the uptake of membranous-necrotic and apoptotic material from leukemia cells. It is well known that APC phagocytose necrotic material via CD91, recognizing the heat shock protein gp96 [27 ]. Macrophages phagocytose apoptotic cells via CD91 and calreticulin [37 , 38 ]. This again supports the hypothesis that necrotic-membranous material is phagocytosed using mechanisms similar to those used for apoptotic material. Material from apoptotic and necrotic tumor cells has been shown to be cross-presented by MoDC [2 , 5 , 13 ].

Functional integrins always exist as heterodimers of one {alpha} and one ß chain. To date, no other {alpha} chain than {alpha}V has been observed in combination with the ß5 integrin chain [39 ]. We observed expression of ß5 on a considerable proportion of the CD11c+ DC but surprisingly, never observed expression of {alpha}V. However, the failure of the CD11c+ DC to express {alpha}V has also been observed by de la Rosa et al. [20 ]. Using a mAb specific for the {alpha}Vß5 heterodimer, we observed expression on a large proportion of the CD11c+ DC. We take these results to suggest that CD11c+ DC do express ß5 but in combination with an {alpha} chain different from {alpha}V. There are 18 different {alpha} chains; the two {alpha} chains investigated in this study ({alpha}4 and {alpha}5) were expressed on CD11c+ DC. We expect that the staining with the anti-{alpha}Vß5 mAb is mediated by an epitope on the ß5 chain, which alone, is sufficient for binding of this mAb. These conclusions were supported by the phagocytosis-blocking experiments, which suggested some degree of blocking by the {alpha}Vß5 and ß5 mAb relative to the results using the {alpha}V mAb.

With the cocktail of mAb, we did not exceed the inhibition observed using only an anti-CD36 mAb alone. Albert et al. [13 ] observed the same phenomenon examining iMoDC. This suggests that a redundancy of receptors is involved in uptake of apoptotic and necrotic-membranous material. One possible additional candidate for uptake of apoptotic cells is the receptor for phosphatidylserine [40 ]. Complement receptor 3 (CR3; CD11b/CD18), CR4 (CD11c/CD18), and Fc{gamma}R type I (Fc{gamma}RI/CD64) and type II (Fc{gamma}RII/CD32) could also be involved [35 , 41 , 42 ]. The fact that the PDC did not show any uptake, despite high expression of CD36, suggests that other membrane proteins may cooperate with CD36 to provide intracellular uptake or that PDC do not have the intracellular components required for this type of phagocytosis.

In summary, we have shown that cultured CD11c+ DC take up apoptotic and necrotic tumor cells as efficiently as cultured iMoDC. The receptors involved may be the same for the two subsets. Many believe that monocytes in vivo represent a precursor DC [1 ]. Our findings suggest that CD11c+ DC and monocytes are precursor DC, which upon differentiation (using GM-CSF and IL-4 in vitro), become APC with the ability to phagocytose dead, cellular material efficiently.

Most clinical trials using DC for cancer immunotherapy have been using iMoDC or mature MoDC, as they are easy to generate in large numbers [43 , 44 ]. However, some trials have used PBDC with relatively good results compared with MoDC, suggesting that PBDC represent more physiologically normal APC than in vitro-generated MoDC [44 45 46 47 48 ]. The use of PBDC in clinical trials has been hampered by their limited number in peripheral blood. It is now possible to expand PBDC in vivo using Flt3L, thereby making it possible to expand these cells to numbers sufficient for clinical trials [16 , 45 , 49 ]. Several strategies can be used for loading DC with antigen for clinical purposes. The trials using PBDC as APC have all used peptides as antigens. However, by using whole, killed cells as antigen instead of peptides, one obviates the need of identifying tumor-specific peptides and the problems concerning MHC restriction [50 , 51 ]. As we show here, CD11c+ DC are fully capable of taking up apoptotic and necrotic tumor cells. Thus, CD11c+ DC represent an interesting alternative for investigators wishing to use autologous DC and dead cell antigenic material in clinical trials of cancer vaccines.


   ACKNOWLEDGEMENTS
 
We thank Fridtjof Lund-Johansen, M.D., Ph.D., for the generous gift of anti-CD36 and help performing four-color flow cytometry.

Received December 8, 2004; accepted January 21, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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