Rat monocyte-derived dendritic cells function and migrate ... - CiteSeerX

Rat monocyte-derived dendritic cells function and migrate in the same way as isolated tissue dendritic cells C. D. Richters,* I. Mayen,* C. E. G. Havenith,† R. H. J. Beelen,* and E. W. A. Kamperdijk* *Department of Molecular Cell Biology, Faculty of Medicine, VUMC, Amsterdam; and †Division of Immunological and Infectious diseases, TNO Prevention and Health, Leiden, The Netherlands

Abstract: Dendritic cells (DC) are the most potent antigen-presenting cells and are therefore useful to induce immune responses against tumor cells in patients. DC can be generated in vitro from monocytes using GM-CSF and IL-4, the so-called monocyte-derived DC (MoDC). To achieve antitumor responses, MoDC must be able to migrate to the draining lymph nodes after injection to induce cytotoxic T cells. Therefore, we studied migration of MoDC in a rat model. Functional rat MoDC were generated from PVG-RT7B rats and injected subcutaneously into PVG rats. These rat strains differ only at one epitope of the leukocyte-common antigen, which can be recognized by the antibody His 41. The advantage is that migrated cells can be detected in the draining lymph nodes by staining sections with His 41ⴙ; thus, migration is not influenced by labeling procedures. Rat MoDC migrated to the T-cell areas of the draining lymph nodes, just as isolated Langerhans cells or spleen DC do. In contrast, monocytes also migrated to the B-cell areas and the medulla. J. Leukoc. Biol. 71: 582–587; 2002. Key Words: antigen presentation 䡠 tumor cells 䡠 GM-CSF 䡠 Langerhans cells

INTRODUCTION Dendritic cells (DC) are the most important cells in the initiation of immune responses [1]. In vivo, precursor DC travel from the bone marrow via the blood to the tissues where they can take up antigens. Upon antigen capture and processing, they migrate to the T-cell areas of the draining lymph nodes where they activate T cells [1, 2]. Because of their superior antigen-presenting capacities, DC may be useful for the initiation of antitumor responses. They can be loaded with tumor antigens in vitro and then injected into patients. Several methods to generate DC in vitro have been developed from bone marrow [3, 4] or blood monocytes [5, 6]. The last method, giving rise to the monocyte-derived DC (MoDC), seems to be the most practical method for clinical trials to induce antitumor responses, because it is not difficult to obtain peripheral blood from the patient. In the first studies, MoDC were very effective when used as prevaccination against tumors in mice [7]. In patients, substantial positive results against 582

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melanoma have been demonstrated [8, 9]. To optimize results in patients, it is important to study the in vitro conditions resulting in MoDC with efficient capacity to activate cytotoxic T cells. In addition, it is crucial that MoDC are really able to migrate to T-cell areas of draining lymph nodes. However, little is known about the migratory capacities of MoDC so far. In earlier migration experiments with isolated Langerhans cells (LC) or spleen DC, we used a rat model using the PVG-RT7b rats as donors and the congeneic PVG rats as recipients [10 –12]. The leukocytes of the PVG-RT 7b rats bear an epitope of the leukocyte-common antigen that can be recognized by the antibody His 41. The congeneic PVG rats do not express this marker [13]. The advantage of this model is that injected cells can be visualized in the lymph node after migration. The process itself cannot be influenced by labeling procedures of the cells prior to injection. In the present study, we used this model to examine the migratory capacities of MoDC after subcutaneous (s.c.) injection in the hind footpad. Therefore, we generated MoDC from rat blood using rat recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4. Phenotypical and functional characterization showed that these rat MoDC were comparable with the described MoDC generated from human blood monocytes [5, 6]. We analyzed phenotype and function of the MoDC and the cells from where they were generated, i.e., monocytes. Furthermore, we quantified the numbers of migrated MoDC present in the lymph nodes to determine the efficiency of migration and compare this with the known capacities of LC [10, 11].

MATERIALS AND METHODS Animals Inbred male PVG-RT7b and PVG rats weighing 160 –180 g were obtained from Harlan CPB (Zeist, The Netherlands). They were kept under routine conditions until use. All experiments were performed at least three times.

Generation of MoDC Rats were placed in a mixture of O2/CO2 until they reached unconsciousness. Then, 6 –10 ml heparinized blood was obtained by heart puncture, resulting in

Correspondence: C. D. Richters, Department of Molecular Cell Biology, Faculty of Medicine, VUMC, Van der Boechorststraat 7, 1081 BT Amsterdam. E-mail: [email protected] Received March 5, 2001; revised December 7, 2001; accepted December 8, 2001.

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death of the animal. Peripheral blood mononuclear cells (PBMC) were separated from erythrocytes and granulocytes by centrifugation on a density gradient (Lymphoprep; Nycomed, Oslo, Norway). PBMC were placed into culture for 2 h in six-wells plates in ␣-RPMI [RPMI 1640, supplemented with 2 mmol/L glutamine, 100 ␮g/mL penicillin, 100 ␮g/mL streptomycin, and 10% heat-inactivated fetal calf serum (FCS), from Gibco Ltd., Paisley, washing gently using warm (37°C) medium. In some experiments, the nonadherent cells were used to obtain T cells for in vitro antigen-presentation assays (see below). Adherent cells were cultured for 7 days in medium containing rat recombinant GM-CSF and IL-4 at a concentration of 5 ng/ml (PharMingen, San Diego, CA). At day 7, nonadherent cells were isolated by collecting the medium. The cells were spun down and counted. Viability of the cells was ⬎95% as determined using trypan blue exclusion. In some additional experiments, we examined the effect of the maturation state of the MoDC on migration. We then added lipopolysaccharides (LPS) to the wells (1 ␮g/ml from Eschericia coli; Difco, Detroit, MI) at day 6 to induce maturation of the MoDC.

Isolation of monocytes Heparinized blood (6 –10 ml) was obtained by heart puncture. PBMC were separated from erythrocytes and granulocytes by centrifugation on a density gradient (Lymphoprep; Nycomed). PBMC were then incubated for 60 min in serum and 2% gelatin (Merck, Darmstadt, Germany)-coated culture flasks. Nonadherent cells were washed away, and adherent cells were isolated using trypsine (0.18%)/ethylenediaminetetraacetate (EDTA; 10 mM; Merck) solution. Purity of the isolated cells ranged from 80% to 95% monocytes; contaminating cells were T and B cells. The percentage of monocytes in cell population was identified with ED-9 [14] antibodies.

Characterization of MoDC

(OVA; type VII; Sigma Chemical Co.) for 60 min. After washing twice, 1 ⫻ 105 cells were injected s.c. into the hind footpads of syngeneic recipient rats. Six days after injection, the popliteal lymph nodes were removed. A cell suspension was prepared by mincing the node using scissors. The cells were separated further by passage through monodur gauze (Stokvis, Ijmuiden, The Netherlands). The cells (1⫻105 per well) were cultured in 96-well microtiter plates in triplicate with or without OVA (5 ␮g/ml) in the medium for 4 days. Six hours before cell harvesting, 3H-thymidine (1 ␮Ci per well, 25 Ci/mmol; Amersham) was added to the cells, and incorporation of the isotope was measured using a liquid scintillation counter.

Migration of cells To study the capacity of the isolated cells to migrate to the draining lymph node, we used the PVG-RT 7b rats as donors and the PVG rats as recipients. These animals differ only at one epitope on CD45, which can be recognized using the His 41 antibody [13]. MoDC or monocytes (2⫻105) in a volume of 0.1 ml PBS were s.c. injected in the hind footpad after the animals were anaesthetized with ether. After injection (1 or 4 days), animals were sacrificed, and the draining popliteal lymph nodes were taken out. To study localization of the His 41⫹ cells, the lymph nodes were frozen in liquid nitrogen. Cryostate sections were prepared and stained with the His 41 antibody to detect migrated cells. For examination of the efficieny of MoDC migration, we quantified the numbers of migrated His 41⫹ cells in the lymph node. After injection (1 or 4 days) of 2 ⫻ 105 MoDC, the lymph nodes were removed and minced thoroughly using scissors as described earlier [10, 11]. The cells were separated further by passage on nylon gauze (Monodur; Stokvis) and counted using a Bu¨ rker chamber. Cytocentrifuge preparations were made and stained with His 41 to count the numbers of positive cells.

Cytocentrifuge preparation of isolated cells was made to study their phenotype. Acid phosphatase activity was visualized according to M. S. Burnstone [15] with naphtol AS-BI phosphate (Sigma Chemical Co., St. Louis, MO) as substrate. The incubation was performed at 37°C for 60 min. Thereafter, the cytospins were counterstained using haematoxilin (Gurr; BDH Ltd., Poole, UK). The immunophenotype of the isolated cells was examined by flow cytometry. The following monoclonal antibodies (mAb) were used: OX-6 against major histocompatibility complex class II (MHC II), OX-62 recognizing spleen DC [16], anti-rat B7-2 (PharMingen), and anti-rat CD11c (Serotec, Oxford, UK). Cells were resuspended in phosphate-buffered saline (PBS) with 0.1% FCS and incubated with the first antibody for 45 min at 4°C. For isotype controls, mouse anti-human CD4 [immunoglobulin G (IgG)1] and CD58 (IgG2a) antibodies with no cross-reactivity with rat cells were used. Cells were washed twice with PBS and incubated for 45 min with rabbit anti-mouse phycoerythrin (PE) as secondary antibody (Dako, Denmark) and 2% normal rat serum to block nonspecific binding. Analysis was performed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) using CellQUest software.

Immunohistochemistry

In vitro antigen presentation

The generated rat MoDC showed features of dendritic cells such as cytoplasmic protrusions, but these were not so distinct as observed in earlier studies for isolated spleen DC or LC migrated in vitro from skin explants [10 –12]. Acid phosphatase activity was present as a spot near the nucleus (Fig. 1), which is characteristic for DC [18, 19]. Based on this expression and positivity for MHC II, the purity of the isolated MoDC population ranged from 90% to 95%. Fluorescein-activated cell sorter (FACS) analysis showed high expression of MHC II, CD11c, and B7-2 (Fig. 2) on the MoDC. In contrast, monocytes have low or no expression of these molecules (Fig. 2). OX-62 was not expressed on the MoDC or on monocytes. Addition of LPS (1 ␮g/ml) 24 h before harvesting the MoDC induced further up-regulation of MHC II (OX-6) and B7-2 molecules on these cells. OX-6 expression (mean fluorescence ratio) increased with 38% ⫾ 37.5 and B7-2 expression with 20% ⫾ 5.

In this assay, we compared the capacity of MoDC and monocytes to stimulate a secondary T-cell response. Autologous T cells were isolated from the nonadherent cells that were harvested during the procedure to generate the MoDC or during the isolation procedure of the monocytes. These nonadherent cells were incubated with beads coated with sheep anti-rat IgG to remove B cells (Dynal, Oslo, Norway). The remaining cells were 95–98% T cells, as determined by positive staining for OX-52. The different cell populations (MoDC, monocytes) were cultured in various concentrations with these autologous T cells in the presence of the antigen Candida albicans (5 ␮g/ml; ARTU Biologicals, Lelystad, The Netherlands). Cells (1⫻105 per well) were cocultured for 4 days in 96-well microtiter plates (Greiner, Alphen a/d, Rijn, The Netherlands) in triplo. Six hours before harvesting, 3H-thymidine (25 Ci/mmol; Amersham International, Amersham, UK) was added to each well. Incorporation of the isotope was measured using a liquid scintillation counter.

In vivo T-cell priming In this assay, the capacity of MoDC and monocytes to prime T cells in vivo was studied. Isolated MoDC and monocytes were pulsed with 5 mg/ml ovalbumin

To study migration of injected cells, His 41 was used as primary antibody. The cryostate sections or cytocentrifuge preparations of the lymph nodes were further stained with the indirect APAAP method described by Cordell et al. [17]. Briefly, the slides were fixed in acetone for 10 min. After incubation with His 41, the slides were washed and incubated with rabbit anti-mouse IgG (Dakopatts, Glostrup, Denmark). Normal rat serum was added at a final dilution of 1:50 to block aspecific binding. Thereafter, the slides were incubated with APAAP complexes (Dakopatts). Positive cells were stained with alkaline phosphatase substrate containing new fuchsine (Gurr) and Naphtol AS-BI phosphate (Sigma Chemical Co.). Levamisole (1 mM; Sigma Chemical Co.) was added to block endogenous alkaline phosphatase activity.

RESULTS Phenotypical characterization of MoDC

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Fig. 1. Cytocentrifuge preparation of MoDC stained for acid phosphatase. Reactivity is present as a red spot near the nucleus (original magnification, 600⫻).

During culture, we observed that not all cells changed into nonadherent MoDC. Up to 50% of the cells were still adherent to the plastic after 11 days of culture in the presence of GM-CSF and IL-4. When GM-CSF and IL-4 were not added during culture, the numbers of cells that could be harvested was more than tenfold lower. The few cells that could be obtained showed a more macrophage-like phenotype; acidphosphatase activity was not present in a spot near the nucleus but was throughout the whole cytoplasm of the cells.

Functional capacities of MoDC MoDC were capable of inducing higher secondary responses to C. albicans when compared with monocytes, as shown in Fig-

Fig. 2. FACS analysis of the MoDC and monocytes. MoDC show high expression of OX-6, CD11c, and B7-2 in contrast to monocytes. Bold lines, specific stainings; thin lines, isotype controls. Data of a representative experiment out of seven are shown.

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Fig. 3. Capacity of MoDC and monocytes to induce a secondary T-cell response with C. albicans as antigen. The ratio of MoDC or monocytes: responder T cells is expressed at the horizontal axis. Data are the means ⫾ SD of triplicate wells from a representative experiment out of four.

ure 3. MoDC were able to prime naı¨ve T cells in the popliteal lymph node when pulsed in vitro with OVA and injected s.c. into the hind footpad. They were more potent cells than pulsed monocytes, as can be seen in Figure 4.

In vivo migratory capacities of MoDC After s.c. injection into the hind footpad, the MoDC migrated to the paracortex of the draining popliteal lymph node (Figs. 5, A and B, and 6), the T-cell area. Monocytes migrated more randomly into the different areas of the lymph node. Monocytes could also be observed in the medulla and B-cell areas in between follicles (Fig. 5, C and D). The numbers of His 41⫹ cells in the sections of the draining lymph nodes were higher on day 4 after injection when compared with day 1. On the cryosections, the numbers of MoDC arriving in the T-cell areas of the draining lymph nodes were comparable with the observations in our earlier experiments with isolated rat LC and spleen DC [10–12]. In each section of the draining lymph node containing His 41⫹ cells, 4 –15 positive cells could be

Fig. 4. Capacity of MoDC and monocytes to prime T cells for OVA. Cells (1⫻105) were loaded in vitro with OVA and injected s.c. into the footpads of syngeneic-recipient rats. Six days later, popliteal lymph nodes were taken out. A cell suspension was prepared, which was restimulated with OVA in vitro. Shown are representative results of one experiment out of three experiments. Data are the means ⫾ SD of triplicate wells.

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Fig. 5. Representative pictures of draining lymph nodes from one experiment out of four. Shown are sections of lymph nodes from 1 day (A, C) and 4 days (B, D) after injection, containing migrated His 41⫹ cells (red cells). Original magnification, 90⫻ (A, C); 115⫻ (B); 130⫻ (C). MoDC are present in the T-cell areas (A, B); monocytes can also be observed in the medulla and B-cell areas (C, D). F, Follicle; PCA, paracortical area.

observed. After counting the numbers of His 41⫹ on the cytocentrifuge preparations of the minced, draining lymph nodes, we made an estimation of the total numbers of His 41⫹ cells present in the entire lymph nodes, taking into account the number of all cells in the nodes (Table 1). Using this method, the number of cells present in the lymph node at day 4 is higher (8.600⫾4.600 His 41⫹ cells) when compared with day 1 (3.000⫾360). In earlier experiments in which we injected LC obtained from cultured skin explants, we observed that the numbers of cells present in the lymph node at day 4 after injection were slightly higher (i.e., 9.500⫾1.080; data from ref. [11]). Of the initially injected MoDC, 4.3% (Table 1) has arrived in the lymph node at day 4. Using LC from explants, this was up to 5% [10, 11]. In part of the experiments, we compared migration of MoDC that were cultured the last 24 h in the presence of 1 ␮g LPS with MoDC cultured without LPS. On cryosections, we did not observe clear differences in numbers of migrated cells between MoDC cultured with or without LPS.

DISCUSSION In the present study, we show that it is possible to generate the monocyte-derived DC from rat peripheral blood. We compared

Fig. 6. Higher magnification (original, 250⫻) of a cyrosection, showing migrated MoDC (red cells) nearby high endothelial venules (arrows), characteristic of the T-cell area.

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TABLE 1. Estimation of the Numbers of MoDC Present in the Popliteal Lymph Node after Subcutaneous Injection of 2 ⫻ 105 of These Cells in the Hind Footpada

Time after injection

Number of His 41⫹ cells

Total number of cells in lymph node

Percentage of initially injected cells

Day 1 Day 4

3.000 ⫾ 360 8.600 ⫾ 4.600

3.6 ⫾ 0.6 ⫻ 106 10.4 ⫾ 1.6 ⫻ 106

1.5 ⫾ 0.15 4.3 ⫾ 2.3

a

The mean ⫾

SD

of three experiments is shown.

the phenotype, functional capacities, and migration pattern of the MoDC with that of the cells from where they were generated—monocytes. To compare the efficiency of migration of MoDC with the known capacities of LC isolated from rat skin explants [10, 11], we quantified the numbers of MoDC present in the lymph node at day 1 or 4 after s.c. injection. Rat MoDC resembled the described human MoDC [5, 6]. The cells express high levels of MHC II and B7-2 molecules and are able to induce T-cell responses. Monocytes were less potent, which can be explained at least partly by their lower expression of MHC II and costimulatory molecules. When compared with rat spleen DC, the MoDC differed in OX-62 expression, the latter cell type being negative. However, rat LC are also negative for OX-62 [10]. Because MoDC may be useful for tumor vaccination purposes, it is important that they can migrate to the draining lymph node after s.c. injection. The possibility to generate rat MoDC enabled us to investigate migration of these cells using PVG-RT7 rats as donors and PVGs as recipients. These rat strains differ only at one epitope of the leukocyte-common antigen (CD45), which can be recognized by the mAb His 41. The advantage of this model is that labeling migrated cells can be performed after migration without possible side effects of the labeling procedure on the process. Using this model, we have observed that rat LC or spleen DC [10 –12] migrate to the T-cell area of the popliteal lymph node after s.c. injection. In the present study, we show that rat MoDC are also able to migrate to the T-cell areas of the lymph node. In contrast, monocytes were observed in the medulla and B-cell areas. When compared with our earlier migration experiments using LC [10, 11], numbers of MoDC present in the lymph node at day 4 were slightly lower. We estimated that 4.3% of the initially injected cells had arrived in the draining lymph nodes, four days after s.c. injection; for the LC, this was up to 5% [11]. Thus, MoDC migration is almost as efficient as isolated tissue DC. Our percentages of MoDC present in the lymph nodes are about 10 times higher than data from one of the few published MoDC migration studies. Very low numbers were found (0.12% of the injected population, 36 h after injection) in this study, using rhesus macaques [20]. This may be a result of their labeling procedure prior to injection, which may change the migratory capacities of the cells or other differences in the experimental procedure to quantify migrated cells. In addition, that the state of maturation had no influence on the number of DC arriving in the lymph node was described in this study [20]. In our model, we also observed no differences in numbers of 586

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migrated cells on the cryosections between cells cultured with or without LPS. Once migrated into the T-cell area of the lymph node, it is at present not clear how long DC survive. Disappearance within 3 days has been demonstrated [21], probably depending on whether the DC get in contact with antigen-specific T cells [22]. In our experiments, the number of MoDC present in the lymph node is higher on day 4 than on day 1, suggesting that the MoDC stay alive for at least 3 days. However, we do not know the relative contribution of cells still migrating from the injection site to the lymph node after 1 day to the total number of cells present in the lymph node after 4 days. The freshly isolated monocytes seem to be a heterogeneic cell population. In contrast to the MoDC, monocytes, the precursor cells of the MoDC, migrated to various regions of the lymph node after s.c. injection. They could be observed in the medulla, the B-cell area, and sometimes in the T-cell area. Most probably, this migration to different areas of the lymph node is caused by expression of different chemokine receptors and chemokines [23, 24]. When placed into culture with GMCSF and IL-4, this heterogeneity remains, because only 50% of the cells became nonadherent with the typical features of DC. Further research is necessary to determine whether the adherent cells will finally develop into DC after longer culture periods or whether these cells remain adherent and develop characteristics of macrophages. In conclusion, we showed that functional MoDC can also be generated in the rat model. They can migrate to the T-cell areas of the draining lymph nodes after s.c. injection, with about the same efficiency when compared with isolated-tissue DC, such as LC. Whether this will be enough to initiate an antitumor response is a subject currently under study in a rat tumor model. Our present results show that the numbers of MoDC arriving in the lymph node are sufficient to induce a primary T-cell response, as shown by our experiments using OVA as antigen.

ACKNOWLEDGMENTS The work of C. D. Richters and I. Mayen was supported financially by the Dutch Burns Foundation, Beverwijk, The Netherlands.

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