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. 2002 Dec;107(4):500–506. doi: 10.1046/j.1365-2567.2002.01536.x

Phagocytosis of bacille Calmette–Guérin-infected necrotic macrophages induces a maturation phenotype and evokes antigen-presentation functions in dendritic cells

Oliver Goldmann 1, Manfred Rohde 1, Eva Medina 1
PMCID: PMC1782828  PMID: 12460195

Abstract

The interaction of pathogens with dendritic cells (DCs) seems to play a critical role in the initiation of the immune response. Tissue damage and induction of an inflammatory reaction are events frequently associated with the progression of the infection. Although DCs are very efficient at phagocytosing pathogens, the capacity of these cells to uptake microbes from a necrotic environment has not yet been proven. Here we have investigated the ability of murine bone marrow-derived DCs to maturate and acquire antigen-presentation functions when cocultured with bacille Calmette–Guérin (BCG)-infected necrotic macrophages. Immature DCs exhibited a prominent capacity to ingest necrotic material as demonstrated by flow cytometry analysis and confocal microscopy. Furthermore, after exposure to BCG-infected necrotic macrophages, DCs underwent phenotypic changes, including the up-regulation of maturation specific markers (major histocompatibility complex class II, CD40, CD80, and CD86) and the capacity to stimulate antigen-specific CD4+ T cells with higher efficiency than when they were directly infected with a similar number of bacteria. Antigen presentation following phagocytosis of BCG-infected necrotic macrophages was demonstrated by their ability to stimulate in vitro proliferation and interferon-γ production of antigen-specific CD4+ T cells. These results suggest that the functional changes occurring in DCs after interaction with a pathogen can be favoured when the encounter takes place in a necrotic environment and it may constitute an important mechanism for the amplification of class II-restricted immune responses induced during infection.

Introduction

The ingestion of microbes by specialized antigen-presenting cells (APCs) during infection is a critical event for the initiation of the host immune defence against pathogens. During the inflammatory reactions frequently induced by infection, microbes, dying cells and tissue debris can be removed from the site of inflammation by scavenger phagocytes such as neutrophils, which are devoid of the ability to initiate immune responses1 and also by professional APCs such as DCs and macrophages.2,3 DCs are the most potent APCs able to efficiently stimulate primary immune responses. Immature DCs located at body surfaces and mucosal sites are specialized for antigen uptake and processing.4 Immature DCs can capture antigens such as extracellular and intracellular bacteria, parasites, and dying cells5–8 and differentiate into mature DCs. This is followed by migration to the draining lymph nodes where they efficiently present antigens to lymphocytes and initiate T-cell mediated immune responses.9–12 The functional dichotomy of immature and mature DCs and their segregation may help to prevent chronic T-cell mediated inflammatory reactions of the mucosal surfaces which are continuously exposed to environmental antigens.

Because the immature DCs located in the periphery are responsible for the uptake of the infecting agents, it could be speculated that their efficiency might be determined, at least in part, by the availability and accessibility of the microbes in the infected tissue. Many in vitro studies have shown that DCs can efficiently internalize various micro-organisms and subsequently undergo maturation.5,6,13,14 These studies are generally performed in optimal experimental conditions which facilitate the interaction of the DC with the specific pathogen. However, the in vivo encounter between DCs and pathogens may take place in a more unfavourable environment. An example is provided by intracellular mycobacterial infections where protection in the host is a local event focused on granulomatous lesions and generally requires the lysis of infected cells. At the infection foci, mycobacteria are engulfed by macrophages, within which they subvert the normal hostile environment and multiply. Infected non-activated macrophages are destroyed by the dividing bacilli which are released and readily phagocytosed by additional phagocytes recruited to the site of infection. In addition, cell-mediated immunity can also lyse infected macrophages in an effort to eliminate the infecting pathogen. Therefore, DCs may encounter mycobacteria at the site of infection not as a single micro-organism but most probably in clumps of several and within a tangle of tissue debris. In this context, we have investigated here the ability of DCs to uptake bacille Calmette–Guérin (BCG) from necrotic tissue and initiate immunity following maturation. We found that exposure of DCs to BCG in a necrotic environment provided the requisite maturation signal(s) to DCs that resulted in the up-regulation of maturation-specific markers and the capacity to stimulate antigen-specific CD4+ T cells with much higher efficiency than when they were directly infected with a similar number of bacteria.

Materials and methods

Preparation of DCs

DCs were prepared from bone marrow suspensions obtained from femurs and tibias of BALB/c mice (Harlan-Winkelmann, Borchen, Germany). For DCs cultures, bone marrow cells were centrifuged at 259 g for 10 min, and red blood cells were lysed with NH4Cl–Tris solution. CD4+ and CD8+ T cells were removed using MiniMACS Magnetic microbeads according to the manufacturer's protocol (Mitenyi Biotech GmbH, Bergisch-Gladbach, Germany). The cell concentration was adjusted to 5 × 106 cells/ml, and cultured in six-well plates in Dulbecco's modified Eagle's minimal essential medium (DMEM; Gibco BRL, Paisley, UK) supplemented with 5% fetal calf serum (FCS), 2-mercaptoethanol (2-ME; 50 µm), l-glutamin (1 mm) and 10 ng/ml of recombinant murine granulocyte–macrophage colony-stimulating factor (GM-CSF; Sigma, Deisenhofen, Germany) and recombinant murine interleukin-4 (IL-4; Sigma). Cells were fed every 2 days. On day 4, non-attached cells (DCs) were resuspended in fresh medium and transferred to new wells. At day 6, cells were harvested and preparations containing approximately 80% DCs, as assessed by the high CD11c expression and the lack of CD14, were used in different experimental procedures.

Mycobacteria

Mycobacterium bovis BCG Pasteur strain was obatined from the American Type Culture Collection (Rockville, MD; TMC 1011). BCG was grown to mid-log phase in Middlebrook 7H9 medium (Middlebrook; Difco Laboratories, Inc., Detroit, MI), and frozen in aliquots at −70°. For infection purposes, bacteria were washed and resuspended in tissue culture medium, sonicated for 10 s, and then added to the cell cultures. Enumeration of viable bacteria to confirm the multiplicity of infection (MOI) was performed by plating for viable colony-forming units (CFU) on 7H10 Middlebrook medium and incubated for 20 days at 37° with 5% humified CO2.

Isolation and infection of peritoneal macrophages

Peritoneal macrophages were obtained by lavage of the peritoneal cavity with 5 ml of cold DMEM. Peritoneal lavage fluids were centrifuged (180 g) for 10 min at 4°. Cells were washed in cold DMEM and then suspended in DMEM supplemented with 10% FCS, 2 mm l-glutamine, 2-ME (50 µm), 100 U/ml penicillin, and 100 µg/ml streptomycin and plated in 24-well plates at 106 cells/well. After 2 hr of incubation at 37° to allow adherence, the cells were washed with warm complete medium and then incubated overnight. Before infection, cells were washed and resuspended in complete medium without antibiotics.

In vitro infection

For in vitro macrophage infection, BCG was suspended in complete medium without antibiotics at 37° and added to macrophage cultures at a MOI of 10 mycobacteria per macrophage. Phagocytosis was allowed for 6 hr, cultures were then washed and fresh medium was added. Infection was allowed to proceed for 48 hr.

For in vitro infection of DCs, DCs were seeded at 106 cells/well in 24-well plates and infected with BCG at a MOI of 10. After 12 hr, unincorporated bacteria were removed by pelleting the DCs at low speed (< 180 g) and further culturing them with fresh media.

CFU assay

For the determination of intracellular CFU, infected cells were lysed with 0·025% Triton-X-100 in dH2O to release intracellular bacilli. Bacterial suspensions were serially diluted in phosphate-buffered saline (PBS) plus 0·05% Tween and plated on 7H11 agar plates. The number of viable bacilli was evaluated by counting individual colonies after 3 weeks of growth at 37°.

Generation of necrotic macrophages and phagocytosis assay

Necrosis of BCG-infected and uninfected control macrophages was carried out by subjecting them to five cycles of freezing and thawing. Such treatment generally resulted in necrosis of 90% of the cells. For the phagocytosis assay, peritoneal macrophages were first dyed green using PKH67 cell linker according to the manufacturer's protocol (Sigma), infected with BCG and then induced to undergo necrosis. Immature DCs were dyed red with PKH26 (Sigma) and then cocultured with the necrotic macrophages at cell equivalent ratios of 1 : 1 for 48 hr at 37°. Phagocytosis of necrotic macrophages by immature DCs was defined by the percentage of green-fluorescent positive cells by fluorescence-activated cell sorting (FACS) analysis and visualized by confocal microscopy.

Flow cytometry analysis of cell surface markers

DCs were stained for surface markers using Ia-phycoerythrin (PE), CD11c–PE, CD40–PE, CD80–FITC, CD14–PE and CD86–fluoroscein isothiocyanate (FITC) antibodies and appropriate isotype controls. Cells were incubated for 30 min at 4°, washed and analysed by flow cytometry (FACScan) using CellQuest (analysis) software (Becton Dickinson Immunocytometry Systems, NJ). All antibodies were obtained from Pharmingen, San Diego, CA.

Scanning electron microscopy

Samples were fixed with a fixation solution containing 3% glutaraldehyde (w/v) and 5% formaldehyde (w/v) in cacodylate buffer (0·1 m cacodylate, 0·09 m sucrose, 0·01 m MgCl2, 0·01 m CaCl2, pH 6·9) for 1 hr on ice. After several washing steps with PBS, samples were dehydrated in a graded series of acetone and subjected to critical-point drying with liquid CO2. Samples were then fractured and sputter coated with an approximately 10 nm thick gold film and examined in a field emission-scanning electron microscope Zeiss DSM982 Gemini using the Everhart Thornley SE-detector and the inlens SE-detector in a 50 : 50 ratio and at an acceleration voltage of 5 kV.

T-cell proliferation assays

DCs were either infected with BCG or cocultured with BCG-loaded necrotic macrophages and assayed for their T-cell stimulatory capacity after 48 hr of culture. For this purpose, DCs were plated in triplicate in 96-well U-bottomed plates (Gibco) at 5 × 104 cells/well. As a source of sensitized T cells, lymphocytes were isolated from pulmonary lymph nodes of BALB/c mice at day 30 after intravenous infection with 107 CFU of BCG. CD8+ T and B cells were depleted using MiniMACS Magnetic microbeads according to the manufacturer's protocol (Mitenyi Biotech). CD4+ enriched cells were added at various concentrations to achieve 1 : 1–1 : 50 DCs : T-cell ratio. After 7 days in culture, T cells were harvested, and further cultured with DC as before in complete DMEM supplemented with murine IL-2 (20 U/ml; Sigma). After 3 days, 1 µCi/well [3H]thymidine was added for 16 hr and the incorporation of radioactivity was measured in a β-scintillation counter (WALLAC 1450, MICRO-b TRILUX). Immature DCs served as controls.

Cytokine production

Culture supernatants from DC-stimulated T cells were harvested during the second round of restimulation and stored at −70°. Interferon-γ (IFN-γ) production was measured by sandwich enzyme-linked immunosorbent assay (ELISA) using the antibodies (Pharmingen) according to the manufacturer's protocol. Recombinant murine IFN-γ (Pharmingen) was used to generate a standard curve.

Statistical analysis

Statistical analysis was performed by the Student's t-test.

Results

Visualization of uptake of BCG by immature DCs using electron microscopy

Previous studies have shown that immature DCs are capable of internalizing BCG with high efficiency.6,15,16 The process of uptake and internalization of BCG by DCs is shown in Fig. 1. Scanning electron microscopy analysis revealed that internalization started immediately after the attachment of BCG to the cell surface of the DCs. After attachment, emerging phagocytic appendages from the DCs can be observed engulfing the whole bacilli Fig. 1(a). The electron microphotograph shown in Fig. 1(b) shows the enclosed bacteria almost completely internalized.

Figure 1.

Figure 1

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Scanning electron microphotographs showing the uptake of BCG (a and b) and BCG-clumps containing numerous microorganisms (c and d) by DCs. Following attachment, BCG is enclosed by thin folds of plasma membrane protruding from the DCs (a) and efficiently internalize within the cell (b). (Bar size, 0·5 µm; bacteria are indicated by arrows). BCG was grown to form clumps and used for phagocytic assay with bone marrow-derived DCs. An initial contact between DC and bacteria (c) is followed by the total engulfment and ingestion of the bacterial cluster (d). (Bar size, 2·5 µm; bacterial clumps are indicated by arrows).

As DCs will encounter mycobacteria at the site of infection, not as a single micro-organism but most probably in clumps containing numerous bacilli, the ability of DCs to phagocyte clumps of BCG was determined by scanning electron microscopy (Figs 1c,d). BCG was grown in liquid medium and clumping bacteria were collected during the mid-log phase and used for infection assays. High-resolution scanning electron micrographs clearly show that after an initial contact with the clumping micro-organisms (Fig. 1c), DCs were capable of engulfing and very efficiently internalizing the entire cluster (Fig. 1d).

Phagocytosis of BCG-infected necrotic macrophages by immature DCs

The capacity of immature DCs to phagocyte necrotic macrophages was then determined. Peritoneal macrophages were first dyed green using PKH67 cell linker, infected with BCG and induced to undergo necrosis by subjecting them to five freeze and thawing cycles. The number of micro-organisms recovered after this treatment from the necrotic tissue was estimated to be approximately 1·5 × 105 CFU/well. Immature DCs were labelled red with PKH26 and cocultured with the necrotic cells at a ratio of 1 : 1. After 6 hr of incubation, phagocytic uptake of necrotic macrophages by DC was quantified by flow cytometric analysis. Results in Fig. 2a show that approximately 85% of the immature DCs engulfed PKH26-labelled necrotic material as demonstrated by an increase of green fluorescence compared with controls. The uptake of BCG-infected necrotic macrophages was further confirmed by visualizing the uptake with confocal microscopy (Fig. 2b).

Figure 2.

Figure 2

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Phagocytosis of BCG-infected necrotic macrophages by DCs. Peritoneal macrophages were labelled green with the PKH67 fluorescent cell linker, infected with BCG (MOI = 10) and induced to undergo necrosis via repeated freezing and thawing. Immature DCs were dyed red with PKH26 fluorescent cell linker and then cocultured with the necrotic cells at a ratio of 1 : 1. (a) Cells were analysed by FACScan where FL1-positive cells indicate uptake of the necrotic cells by the DCs. DCs cocultured with unlabelled BCG-infected necrotic macrophages were used as controls (dotted line). (b) Visualization by confocal microscopy of uptake of necrotic cell (green) by immature DCs.

DCs exposed to BCG-infected necrotic macrophages acquire a mature phenotype

Immature DCs are characterized by high phagocytic capacity and low levels of major histocompatibility complex (MHC) class II, CD40, and the costimulatory molecules CD86 and CD80.4 Upon receipt of a maturation signal, DCs down-regulate antigen-capture, and express higher levels of costimulatory and MHC molecules.4

The ability of BCG-loaded necrotic macrophages to induce maturation of DCs was then evaluated. DCs were cocultured for 48 hr with BCG-infected necrotic macrophages and with BCG in similar number than those recovered from the necrotic macrophages (0·2 MOI). Non-infected necrotic macrophages were included as control. DCs were then collected, stained for maturation markers, and analysed by flow cytometry. Results in Fig. 3a show that BCG-loaded necrotic macrophages were much more effective than BCG or non-infected necrotic macrophages at inducing up-regulation of MHC class II and CD40 in DCs.

Figure 3.

Figure 3

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Maturation of DCs after exposure to BCG-loaded necrotic macrophages. (a) DCs were either infected with BCG (MOI = 0·2) or cocultured with either uninfected (neMc) or BCG-infected (BCG-neMc) necrotic macrophages at a ratio of 1 : 1. After 48 hr, the DCs were stained for the maturation markers MHC class II and CD40 (unbroken line). Isotype-matched antibodies served as controls in all experiments (dotted line). (b) Up-regulation of the costimulatory molecules CD80 and CD86 by DCs in response to BCG-infected necrotic macrophages (BCG-neMc), BCG (MOI = 0·2), or uninfected necrotic macrophages (neMc) (thick line) respect to immature DCs (thin line). A representative of five different experiments is shown.

In addition, BCG-loaded necrotic macrophages induced increased surface expression of CD80 and CD86 costimulatory molecules in DCs (Fig. 3b). Little alteration in surface expression of these molecules was detected after DCs treatment with BCG or with non-infected necrotic macrophages.

Uptake of BCG-loaded necrotic macrophages by immature DCs results in efficient antigen presentation

To determine whether BCG-loaded necrotic macrophages could serve as a source of antigen for antigen presentation to CD4+ T cells by immature DCs, CD4+ T cells were isolated from lymph nodes of mice infected with BCG and cultured either with DCs, BCG-infected DCs (0·2 MOI) or with DCs pulsed with BCG-loaded necrotic macrophages at different APC : effector cells ratio (1 : 1, 1 : 10, 1 : 20 and 1 : 50). Only DCs pulsed with BCG-loaded necrotic macrophages induced substantial proliferation of T cells from infected mice (Fig. 4a). In contrast, uninfected immature DCs or BCG-infected DCs at MOI 0·2 were very poor CD4+ T-cell stimulators and the background proliferation observed may probably be because of the maturation of some DCs as a result of DC–T-cell interactions (Fig. 4a).

Figure 4.

Figure 4

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Stimulation of antigen-specific CD4+ T-cell proliferation and IFN-γ production by DCs infected with BCG (MOI = 0·2) or pulsed with BCG-infected necrotic macrophages (BCG-neMc). (a) Proliferation of antigen-specific CD4+ enriched cells was measured by [3H]thymidine incorporation. (b) Supernatants of the cultures containing 1 : 20 DC/effector cells ratio were tested for IFN-γ release by ELISA. Statistical analysis with Student's t-test shows that, compared with DC infected with BCG (MOI = 0·2) or DC alone, the increase in IFN-γ release in cultures of DCs stimulated with BCG-neMc is significant (**P < 0·005). A representative experiment (of three experiments) is shown.

A correlation was observed between the stimulation of CD4+ T-cell proliferation and IFN-γ production. DCs pulsed with BCG-loaded necrotic macrophages were capable to stimulate secretion of higher levels of IFN-γ by CD4+ T cells than uninfected DCs or DCs infected with BCG at MOI 0·2 (Fig. 4b).

Discussion

The interaction of DCs with infectious agents plays a critical role in the initiation of most T-cell mediated immune responses.4,12 In this regard, immature DCs are characterized by a high capability for antigen capture and processing, but low T-cell stimulatory capacity.3 Upon stimulation, immature DCs differentiate into mature DCs as they migrate to regional lymphoid organs where they efficiently present antigens to T cells.3,17,18 During the maturation process, DCs lose their ability to capture antigens and acquire the capacity to stimulate T cells.12 These effector T cells migrate back to the focus of infection and activate macrophages through the production of specific cytokines and also lyse target cells in an effort to eliminate the infectious agent. Therefore, induction of tissue damage and inflammation are events frequently associated with infection. In this context, the rapid acquisition of infecting pathogens as well as dying host cells by DCs present at the site of inflammation might contribute to the clearance of the infection as well as the amplification of the cellular immune responses. Although the capacity of DCs to internalize pathogens has been extensively demonstrated5,6,13,14,19 their ability to directly acquire microbes released from necrotic tissues has not yet been clarified. Here, we have investigated the ability of DCs to uptake BCG released from infected necrotic macrophages and initiate an immune response following maturation. After exposure to BCG-infected necrotic macrophages, immature DCs experienced phenotypic and functional changes such as up-regulation of maturation markers and acquire capacity to stimulate antigen-specific CD4+ T cells at a higher rate than when they were directly infected with a similar number of bacteria. The mechanisms underlying the superior efficiency of BCG-infected necrotic cells to stimulate DCs when compared with stimulation by direct infection with BCG remains to be clarified. However, we can suggest that, in this particular context, a combination of maturation signals provided by the pathogen and the necrotic tissue might be involved in activation and maturation of DCs. Supporting this view, it has been shown here and by others5,15,20 that DCs can efficiently internalize mycobacteria and subsequently undergo maturation. In addition, the capacity of DCs to maturate in response to endogenous signals derived from necrotic tissue such as heat-shock proteins, nucleotides, reactive oxygen intermediates, extracellular matrix breakdown products, and cytokines has been also demonstrated.21–23

On the other hand, the ability of DCs to present antigens from endocytosed particulate material as well as their capacity to extract peptides from phagocytosed cellular fragments has been previously reported.24 This could also be a feasible explanation for the observed efficiency of DCs pulsed with BCG-loaded necrotic macrophages to stimulated antigen-specific CD4+ T cells. Support for this notion was provided by the confocal microscopy and also by flow cytometric analysis showing the capacity of DCs to internalize fragments from either BCG-infected necrotic macrophages.

The results shown here could provide the basis for a better understanding of the role of DCs during infection with M. tuberculosis in the lungs. Upon inhalation, M. tuberculosis are engulfed by resident alveolar macrophages, within which they overcome the normal hostile environment and multiply. At this initial stage, resident alveolar macrophages might antagonize the activities of pulmonary interstitial DCs by sequestering antigen within the air spaces.25 The immunosuppressive effects of alveolar macrophages seems to be critical for limiting inflammation of the respiratory surface and for maintaining local homeostasis in response to a low dose of bacterial antigens.25 However, during the course of a tuberculosis infection, lysis of incapacitated infected macrophages allows the release of a high number of intracellular bacteria which are subsequently taken up by more efficient phagocytes. In this context, the DCs present at the site of infection can notably contribute to the stimulation and amplification of class II-restricted immune responses by ingesting, not only the released bacteria but also fragments of necrotic infected cells. Further evidence supporting the importance of the DCs for the local immune defence of the respiratory mucosa has been provided by the demonstration that a large number of DCs are recruited after a challenge of the airway mucosa with various antigens.26 Although necrosis of infected macrophages may contribute to spread of bacteria and severity of the infection, on the other hand, it may also constitute a host mechanism to counteract the well-known subverting influences of bacilli when they reside within macrophages.

In conclusion, the results shown here suggest that activation of immature DCs during infection can be enhanced when the encounter with the pathogen takes place in a necrotic environment and it may constitute an important mechanism for the amplification of class II-restricted immune responses induced during infection.

Acknowledgments

This work was supported by the European Community (grant QLK2-CT-1999–01093). We thank Dr Els Maas and Prof G.S. Chhatwal for carefully reading the manuscript and helpful discussions.

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