Am. J. Trop. Med. Hyg., 68(1), 2003, pp. 48–53 Copyright © 2003 by The American Society of Tropical Medicine and Hygiene
INCREASED APOPTOSIS AND EXPRESSION OF TUMOR NECROSIS FACTOR-␣ CAUSED BY INFECTION OF CULTURED HUMAN MONOCYTES WITH DENGUE VIRUS LUZ M. ESPINA, NEREIDA J. VALERO, JANETH M. HERNÁNDEZ, AND JESÚS A. MOSQUERA Sección de Virología y Sección de Inmunología y Biología Celular, Instituto de Investigaciones Clínicas Dr. Américo Negrette, Facultad de Medicina, Universidad del Zulia, Maracaibo, Venezuela
Abstract. Dengue (DEN) virus is responsible for one of the most significant viral diseases in tropical countries. Monocytes/macrophages (Mo/M) are the major target cells for DEN virus. To determine the effects of the interaction between DEN virus and Mo/M, human monocyte cultures were infected with DEN virus type 2. Apoptosis and production of tumor necrosis factor-␣ (TNF-␣) and nitric oxide were measured in control and infected cultures. Virus was taken up by phagocytosis, but no membrane-coated pits at the virus attachment sites were observed. Increased number of apoptotic cells and increased production of TNF-␣ were observed in infected monocyte cultures. No increase in production of nitric oxide was observed. These results may be related to early primary viral infection, in which virus could induce apoptosis in monocytes, but monocytes may contribute to host defense mechanisms against virus by viral phagocytosis, phagocytosis of infected apoptotic cells, and the release of proinflammatory cytokines. by centrifugation, and the virus supernatant was aliquoted and stored at −70°C until used. Virus was titrated by a plaque formation assay on Vero cells. Cells were seeded at a concentration of 1 × 106/well in 24-well plates, serial dilutions of virus were added, and the mixtures were incubated at 37°C for seven days. Plaques were visualized by staining with a solution of 1% crystal violet. Virus concentrations are given as plaque-forming units (PFU)/ml. The virus stock was free of endotoxin, as determined by a Limulus amebocyte lysate assay. Infection of monocyte cultures. Virus culture fluid was added to adhered monocytes at final concentrations ranging from 4 × 105 to 4 × 103 PFU/ml (multiplicity of infection [MOI] ⳱ 0.8−0.008) and incubated at 37°C in an atmosphere of 5% CO2 for six hours. In a second group of experiments, monocytes were infected with a virus concentration of 4 × 104 PFU/ml (MOI ⳱ 0.08) and incubated for one, two, four, and six hours. The controls used were monocytes cultured with supplemented medium without virus. In addition, monocyte cultures were incubated with heat-inactivated dengue virus (56°C for 30 min.) at a concentration of 4 × 104 PFU/ml for six hours. Culture media and infected cells were obtained for further studies. Direct immunofluorescence for DEN-2 antigens. Experiments were performed in eight-well plastic chamber slides. Monocytes were washed in phosphate-buffered saline and fixed with cold acetone for five minutes. Intracellular viral antigens were detected by a direct immunofluorescence assay using a fluorescein-conjugated DEN-2 virus-specific monoclonal antibody (Centers for Disease Control and Prevention, Fort Collins, CO). Electron microscopy. Monocytes seeded in 75-cm3 tissue culture flasks were incubated for six hours with DEN-2 virus (4 × 104 PFU/ml). Cells were then detached by incubation with 0.01% EDTA and removal with a cell scraper. After centrifugation, infected monocytes were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3. Cells were postfixed with 1% osmium tetraoxide, dehydrated in a graded ethanol series, and embedded in Epon 812. Samples were cut into ultrathin sections, stained with uranyl acetate, followed by lead citrate, and examined in an JEM 1010 (Jeol, Japan, Tokyo) electron microscope.
INTRODUCTION Monocytes/macrophages (Mo/M) are the major target cells of dengue (DEN) virus. These cells are responsible for the dissemination of the virus after its initial entry via the mosquito vector.1–3 It has been shown that soluble mediators released from DEN virus-infected Mo/M exerted prominent effects on the biologic properties of endothelial cells and the hematopoietic cell population,4–6 suggesting that the interaction of DEN virus with Mo/M could play a role in the pathogenesis of dengue. Since Mo/M are active phagocytic cells with cytoplasmic lysosomal components capable of eliminating microorganisms,7 and DEN virus may induce cell death by apoptotic mechanisms,8,9 the interaction of DEN virus with Mo/M may also result in deleterious effects on both virus and cells. To analyze the cellular damage induced by DEN virus, we conducted experiments to determine whether apoptosis and the release of tumor necrosis factor-␣ (TNF-␣) and nitric oxide occur in human monocyte cultures infected with DEN virus type 2.
MATERIALS AND METHODS Monocyte cultures. Monocytes were isolated from heparinized peripheral blood obtained from human healthy volunteers (n ⳱ 5) by density centrifugation over 1.077 Histopaque (Sigma Chemical Co., St. Louis, MO). Total mononuclear leukocytes recovered from the interface were washed and resuspended in RPMI 1640 medium, 10% fetal bovine serum, and penicillin (100 /ml)/streptomycin (100 g/ml). Samples (300 L/well) of a cellular suspension (4 × 106 cells/ml) were then layered on eight-well plastic chamber slides (Nunc, Roskilde, Denmark) and incubated for three hours at 37°C in an atmosphere of 5% CO2. Non-adherents cells were removed by washing with warm medium and adhered cells were used for the experiments. Preparation of virus stock and virus titration. The DEN-2 virus strain New Guinea C was propagated in C6/36HT mosquito cells that were cultured in Eagle’s minimal essential medium containing 10% fetal bovine serum prior to infection of monocytes with virus. The virus culture medium was harvested after incubation for five days, cell debris was removed
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Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. The method for nick endlabeling of apoptotic cells was adapted from that of Gavrieli and others10 with a commercial kit (Pharmingen, San Diego, CA). Adhered monocytes were treated according to the protocol provided with this kit. The assay is based on the preferential binding of the fluorescein isothiocyanate-dUTP by terminal deoxynucleotidyl transferase to the 3⬘ OH ends of the DNA. Positive apoptotic nuclei were assessed by fluorescence microscopy (Axioskop; Zeiss, Wetzlar, Germany). Quantitation of TNF-␣ and nitric oxide. Supernatants from DEN-2 virus-treated monocyte cultures (4 × 104 PFU/ml) were harvested at one, two, four, and six hours and tested for TNF-␣ using an enzyme-linked immunosorbent assay kit (Biosource International, Camarillo, CA). The assays were performed according to the instructions of the manufacturer. Total production of nitric oxide was determined by assaying for nitrite.11 To assay for nitrite, 50 L of supernatants from control or infected monocyte cultures were removed from individual wells at the indicated times and incubated with 50 L of Griess reagent (0.5% sulfanilamide, 0.05% N-(1naphthyl) ethylenediamine dihydrochloride in 2.5% H3PO4). The optical densities of the samples were then obtained on a microplate reader (Benchmark; Bio-Rad, Hercules, CA) at 540 nm. A standard curve using NaNO2 was used to calculate concentrations of nitric oxide. Statistical analysis. Quantification of the percentage of DEN+ cells and apoptotic cells was performed on at least 300 cells in duplicate samples. Results are shown as the mean ± SEM of at least three experiments. Statistical significance was assessed by analysis of variance or the paired t-test, as appropriate. Two-tailed P values < 0.05 were considered statistically significant.
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FIGURE 2. Vacuolar compartment containing partially digested cell material (large arrow) and virus-like particles inside the phagosome (small arrow). n ⳱ nucleus (magnification × 10,000).
taken up by phagocytosis, and many virus particles were engulfed by typical lamellipods and sequestered into intracytoplasmatic vacuoles (Figures 1, 2, and 3c). Many monocytes had huge vacuoles containing cellular material undergoing
RESULTS Infection of human monocytes by DEN-2 virus. As determined by electron microscopy studies, six hours after infection DEN-2 virus particles were observed on the plasma membrane of monocytes (Figure 1). Coated pits did not form at the virus attachment sites, and virus-containing vesicles were not found in the cytoplasm. In most cases, particles were
FIGURE 1. Monocyte culture six hours after infection with dengue type 2 virus, showing attachment of virus particles to the cell surface (large arrow) and several particles in an intracytoplasmic vacuole (small arrow) (magnification × 20,000).
FIGURE 3. Intravacuolar localization of dengue type 2 virus in infected monocytes six hours after infection. a, Intense cytoplasmic vacuolization (arrow) corresponds to immunofluorescent staining of viral antigens in b (arrow) (magnification × 1,000. c, Numerous viral particles are enclosed in several intracytoplasmic vacuoles (arrow) (magnification × 10,000).
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degradation and virus-like particles were observed in some of these phagosomes (Figure 2). The presence of viral antigen in cytoplasmic vacuoles was detected by immunofluorescence in approximately 29−63% of the cultured monocytes, depending on the virus concentration (Table 1and Figure 3b). This finding was confirmed by the presence of numerous cytoplasmic vacuoles containing viral particles as assessed by electron microcopy (Figure 3). Death of human monocytes after infection with DEN-2 virus. Light microscopy showed that DEN-2 virus-infected monocyte cultures displayed the typical features of apoptosis, such as nuclear fragmentation and shrinkage of the cytoplasm. Nicking of the DNA by endogenous endonucleases is another hallmark of apoptosis and the TUNEL method was used to detect intrachromosomal DNA strand breaks. Untreated monocytes showed low values of TUNEL-positive cells. However, six hours after infection, higher values were observed (Figure 4 and Table 1). We found a higher percentage of monocytes expressing DEN antigen at a viral dose of 4 × 103 PFU/ml in contrast to higher dose of 8 × 103 PFU/ml (Table 1). We have no clear explanation for this finding, but since apoptotic cells can detach from the surface of the culture plate, the lower apoptosis values found at 4 × 103 PFU/ml could be responsible for the higher DEN+ cells found at this dose. Apoptotic cells showed intense nuclear fluorescence (Figure 5a) and at the ultrastructural level, characteristic aspects of apoptosis were observed mainly in cells with scanty cytoplasm and cells with numerous intracytoplasmatic vacuoles (Figures 5b and c). Several apoptotic cells and apoptotic bodies were ingested by neighboring healthy monocytes (Figures 5a and 6). Few cells displayed ultrastructural features typical of lysis six hours after viral infection (Figure 7). Attempts to detect apoptotic cells positive for dengue virus antigen using double-staining techniques were unsuccessful. In addition, analysis by electron microscopy showed only scanty virus-like particles in cytoplasmic vacuoles of a very small number of apoptotic cells. Production of TNF-␣ and nitric oxide in DEN-2 virusinfected monocyte cultures. Since monocytes may be reactive to the entry of virus particles and transiently release various mediators, we attempted to detect the apoptosis mediators TNF-␣ and nitric oxide in the supernatants of controls and infected cultures. Increased concentrations of TNF-␣ were found in supernatants of infected cultures at different periods of time (Figure 8). However, undetectable reactivity for TNF-␣ was found in non-infected cultures. No differences were found in nitrite concentrations between controls and infected cultures (control ⳱ 119 ± 7.3, infected ⳱ 116 ± 7.4 nM after culturing for six hours). Monocytes cultured with heat-inactivated virus showed undetectable levels of TNF-␣ and low numbers of apoptotic cells
FIGURE 4. Frequency of TUNEL + cells (apoptosis) at various times after infection of monocyte cultures with dengue type 2 virus (4 × 104 plaque-forming units/ml). Noninfected cultures had 0.9 ± 0.15% (mean ± SEM) TUNEL+ cells. *P < 0.01; n ⳱ 5.
(1.5 ± 0.4); these values were similar to those in noninfected cultures. DISCUSSION Dengue viruses are important human pathogens; however, the pathogenesis of DEN virus is still poorly understood. Monocytes are one of the main targets of DEN virus,1–3 but the interactions between DEN-2 virus and monocytes regarding induction of apoptosis have not been studied. Our results showed that DEN-2 virus can be efficiently phagocytized by human monocytes, as assessed by electron microscopy and immunofluorescence assay (approximately 63% and 44% DEN-2+ cells at 4 × 105 and 4 × 104 PFU/ml, respectively). Similar data were reported in peripheral blood mononuclear cells (68.8%) from dengue-infected patients four days after onset of the disease.12 As shown by electron microscopy, most of the viral particles penetrated the monocytes by phagocytosis and were presumably degraded, suggesting that phagocytosis could be a mechanism of viral clearance. Indeed, phagocytosis leads to viral degradation; for example, it has been shown that human immunodeficiency virus entering human macrophages by phagocytosis is noninfectious.13 It has also been reported that only a small proportion of the monocyte population supports replication of DEN-2 virus.14 In addition, receptor-mediated endocytosis appears to be an effective mechanism for intracellular virus replication;15,16 however, uptake of viral particles via clathrin-coated pits was not observed. Since a small proportion of monocytes can support replication of dengue virus, infected monocytes can be a
TABLE 1 Effect of different concentrations of dengue type 2 (DEN-2) virus on apoptosis and intracellular viral antigen expression in cultured human monocytes* Virus concentration (PFU/ml)
%TUNEL+ %DEN-2+
0
4 × 105
4 × 104
8 × 103
4 × 103
1.1 ± 1 0
16.1 ± 5.6† 62.9 ± 2.4
15.4 ± 3.5† 43.8 ± 18.1
6.3 ± 1.3 28.7 ± 4.6
3.3 ± 0.5 35.4 ± 0.9
* Values are the mean ± SEM of positive cells from 6-hour monocyte cultures (n ⳱ 5). PFU ⳱ plaque-forming units. † P < 0.05.
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FIGURE 6. Apoptotic body (arrow) ingested by a neighboring monocyte (magnification × 8,000).
sis, as determined by the TUNEL assay and electron microscopy. This effect has previously shown in other DEN virusinfected mammalian cells.8,9,18,19 The percentage of apoptotic monocytes was related to the viral concentration and a very low percentage of apoptotic cells was observed in noninfected cultures, suggesting that the presence of virus is required to trigger cell death. The apoptotic event could prevent the release of infectious viral progeny.20 In addition, apoptotic cells and apoptotic bodies may be digested by neighboring monocytes. In this regard, macrophagic natural Kupffer cells have been shown to have a scavenger function for virus infectedapoptotic cells.8,21,22 Monocytes/macrophages can release various mediators in response to virus infection.23,24 These mediators released include interleukins, TNF, and nitric oxide that may interfere with the virus life cycle and/or initiate an inflammatory process during the course of dengue.24–26 Increased expression of TNF-␣ was observed in DEN-2 virus-infected monocytes at different periods of time. However, no differences in nitric oxide concentrations were observed at six hours between noninfected and infected monocytes. Tumor necrosis factor-␣ has been reported to be an apoptosis inducer in different systems.27,28 Since increased apoptosis was observed with in-
FIGURE 5. Apoptotic features of dengue type 2 virus-infected monocyte cultures. a, TUNEL staining of monocytes six hours after infection. Intense fluorescence was observed in apoptotic nuclei (small arrows) and intracytoplasmic apoptotic bodies were observed en several cells (large arrow) (magnification × 1,000. b, Apoptotic cell with condensed marginated chromatin (asterisk) and scarce cytoplasm (arrow) (magnification × 15,000. c, Apoptotic cell showing condensed marginated chromatin (asterisk), nuclear fragmentation, and striking accumulation of cytoplasmic vesicles (magnification × 10,000).
source of viral dissemination after cellular differentiation and release of virus and cytokines onto blood vessels.17 In addition to the ingestion of viral particles by monocytes, cells from DEN-2 virus-infected cultures underwent apopto-
FIGURE 7. Apoptotic (arrowhead) and lysed cells (arrow) observed six hours after infection of monocytes with dengue type 2 virus (magnification × 4,000).
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In conclusion, although monocytes could undergo apoptosis in contact with the DEN 2-virus, monocytes may protect against virus replication by eliminating the virus through phagocytosis, phagocytosis of infected apoptotic cells or apoptotic bodies, or cytokine autocrine or paracrine mechanisms. Received April 25, 2001. Accepted for publication March 21, 2002. Acknowledgment: We thank Dr. Duane Gubler (Centers for Disease Control and Prevention, Fort Collins, CO) for the monoclonal antibody to dengue virus type 2 used in the viral immunofluorescence studies. Authors’ addresses: Luz M. Espina, Nereida J. Valero, and Janeth M. Hernández: Seccion de Virologia. Instituto de Investigaciones Clinicas Dr. Americo Negrete, Facultad de Medicina, Universidad del Zulia, Apartado Postal 1151, Maracaibo 4001-A, Zulia, Venezuela, Jesús A. Mosquera, Seccion de Inmunologia y Biología Celular, Instituto de Investigaciones Clinicas Dr. Americo Negrete, Facultad de Medicina, Universidad del Zulia, Apartado Postal 1151, Maracaibo 4001-A, Zulia, Venezuela. FIGURE 8. Expression of tumor necrosis factor-␣ (TNF-␣) in dengue type 2 virus-infected monocyte cultures (4 × 104 plaque-forming units/ml). No detectable values were observed in untreated cultures at the different times and in heat-inactivated virus-treated cultures (n ⳱ 5).
Reprint requests: Jesus A. Mosquera, Apartado Postal 1151, Maracaibo 4001-A, Zulia, Venezuela, Telephone/Fax: 58-61-597-247, Email:
[email protected]
REFERENCES creased concentrations of TNF-␣, this cytokine could be involved in an autocrine or paracrine manner in the induction of apoptosis found in this study. Induction of apoptosis could represent a indirect effect of TNF-␣ to decrease viral progeny.8,20–22 In addition, TNF-␣ is involved in others anti-viral mechanisms.29 Nitric oxide is generated in macrophages if the inducible enzyme nitric oxide synthase is present. Previous reports have shown increased production of nitric oxide and increased expression of this enzyme in macrophage cultures (Kupffer cells) as early as one hour after infection with dengue virus.8 We did not observe increased amounts of nitrate/ nitrite after six hours after infection of dengue virus in monocyte cultures, suggesting that the initial virus-monocyte interaction is not involved in the generation of nitric oxide. No increased apoptotic cell number or production of TNF-␣ were detected when monocytes were exposed to heatinactivated virus. This suggests that viable viruses are required to trigger both processes. The source of increased levels of TNF-␣ found in patients infected with DEN virus30,31 could be partially related to the stimulation of monocytes by DEN virus. Our results demonstrated that the exposure of monocytes to DEN virus induced enhanced production of TNF-␣ in supernatants from infected cultures. In this regard, previous studies have also shown increased concentrations of TNF-␣ in the supernatants of monocytic-like cell cultures exposed to DEN-1 and DEN-3 viruses.32 Antibody-dependent enhancement is widely thought to be central to the development of the pathogenesis of the shock syndrome associated with dengue hemorrhagic fever. Nonneutralizing antibodies to DEN virus can enhance viral uptake and replication in monocytes.33,34 This study represents an in vitro and short-term interaction between DEN-2 virus and monocytes and could reflect the early primary viral infection. However, since the flavivirus status of the donors is not known, we can not rule out antibody-dependent enhancement mechanisms because small amounts of contaminating antibodies could be present upon isolation of monocytes.
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