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ARTICLE |
Early Intracellular Events During Internalization of Listeria monocytogenes by J774 Cells
Paul Websteraa House Ear Institute, Los Angeles, California
Correspondence to: Paul Webster, House Ear Institute, 2100 West Third Street, Los Angeles, CA 90057. E-mail: pwebster@hei.org
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Summary |
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The gram-positive bacillus Listeria monocytogenes gains entry into host cells through a phagosome membrane that forms around entering bacteria. During the early stages of internalization the invading bacteria appear to modify the protein composition of the forming phagosome membrane in J774 cells. MHC class II molecules on the cell surface and exposed surface molecules available for biotinylation are excluded from the bacteria–host cell membrane interface and from the forming phagosome. This exclusion of MHC class II molecules from the early phagosome may partially help to explain previous reports suggesting that L. monocytogenes is able to interfere with antigen presentation. Inside the host cell, MHC class II molecules are delivered to the phagosome membrane. This is followed by delivery of LAMP 1, a marker of late endocytic compartments, and fusion with low-pH compartments. The bacteria then escape into the cell cytoplasm, possibly assisted by rapid delivery of this low-pH environment. (J Histochem Cytochem 50:503–517, 2002)
Key Words: electron microscopy, phagocytosis, macrophages, MHC class II, immunocytochemistry, lysosomes
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Introduction |
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INTRACELLULAR PATHOGENS such as bacteria and protozoa enter mammalian cells using processes initiated by either the host cell or by the invading organism. Inside the cell, internalized microorganisms usually enter a membrane-bound phagosome (
The gram-positive bacterium Listeria monocytogenes enters and is able to survive in mammalian cells. This organism is a contaminant of the food we eat (
Experimental listeriosis in mice is a widely studied model for intracellular bacterial infections. With this model, it has been shown that L. monocytogenes is able to generate responses from both the MHC class I and class II pathways (
In contrast, viable L. monocytogenes exhibit a fine control over phagosome maturation. Newly formed phagosomes accumulate rab 5 and N-ethylmaleimide (NEM)-sensitive factor (NSF), which causes upregulation of fusion with early endosomes (
Although events occurring during the early stages of L. monocytogenes internalization are better understood than for most pathogenic bacteria, many questions remain unanswered. The interactions between viable bacteria and host cell plasma membranes have not yet been fully investigated. Similarly, intracellular events during the initial internalization of bacteria are only currently being elucidated. For this reason, a preliminary immunocytochemical study of the early phagosome formation process is reported here. In this study the processes of phagosome maturation are visualized during the critical early stages of infection when bacteria first associate with and enter host cells.
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Materials and Methods |
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Cells
All tissue culture reagents were obtained from Gibco Laboratories (Grand Island, NY) and were sterilized either by autoclave or filtration. The macrophage-like J774A.1 cultured cell line (ATCC TIB-67) was grown in Dulbecco's minimal essential medium (DMEM) containing 5% fetal calf serum (FCS) at 37C in a 5% CO2 atmosphere. The cells were maintained as monolayers in 75-cm2 flasks and passaged twice a week by washing once in PBS, scraping them into serum-free DMEM, and then diluting the resuspended cells in fresh DMEM with 5% FCS.
Bacteria
The wild-type L. monocytogenes strain (10403S), which belongs to serotype 1, was a gift from Dr. D. A. Portnoy and was previously described (
Western Blotting
SDS-PAGE separation of J774 cell lysates was performed as described previously (
Invasion Assays
J774 cells were seeded into sterile 9-mm culture dishes at a dilution of 1 x 104 per ml and incubated for 2 days at 37C before addition of L. monocytogenes suspensions. Electron microscopy was performed using these cultures. For light microscopy, J774 cells were grown on sterile glass coverslips at the bottom of culture dishes.
After 2 days of incubation at 37C, the J774 cells were counted (approximately 1 x 105 per dish) and infected with 20 mid-log phase bacteria (OD 0.6 at 660 nm) bacteria per cell (2 x 106). The cells and bacteria were left at 37C for 5 min, washed with DMEM, and then either fixed immediately or incubated in DMEM with 10% FCS for increasing time intervals. They were then fixed in buffered aldehyde and prepared for examination by either light or electron microscopy. Infection times were recorded from the time the bacteria were first added to the cells as time zero.
For some control experiments, cells were incubated for 10 min with 1-µm diameter latex beads coated with avidin (
Surface Biotinylation and Incubation with DAMP
Cells to be biotinylated were cooled on ice, washed with PBS, and then exposed to 2 mg/ml sulfo-NHS-biotin (Pierce; Rockford, IL) in PBS for 5 min on ice. They were then washed with cold PBS and then with cold DMEM containing 10% serum. Cold DMEM containing L. monocytogenes was then added to the cells, left for 5 min, and either fixed immediately on ice or warmed to 37C and incubated for increasing times before being fixed.
For some experiments, the J774 cells were incubated in DMEM containing 10% FCS and 20 µM 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP) at 37C (
Antibodies
Intracellular organelles of infected J774 cells were probed on thawed thin cryosections or on permeabilized whole-cell preparations with specific antibodies to LAMP 1 (Developmental Studies Hybridoma Bank; Iowa City, IA), biotin (Jackson Immunoresearch; West Grove, PA), and mouse Ia MHC class II (
BSA–Gold Incubation
Colloidal gold of 15-nm particle size, prepared as previously described (
Light and Electron Microscopy
For light microscopy, cells grown on glass coverslips were fixed in 4% phosphate-buffered formaldehyde, permeabilized with 0.01% Triton X-100 in PBS, and labeled with specific and fluorescent secondary antibodies. After labeling, the coverslips were examined by epifluorescent illumination with an Axiophot light microscope (Zeiss).
For immunocytochemistry, some cell monolayers were fixed for 1 hr in 0.5% glutaraldehyde buffered in 100 mM sodium phosphate (pH 7.4). Other cells were fixed in 4% phosphate buffered-formaldehyde for 1 hr and then left overnight in 8% formaldehyde in the same buffer. After fixation, the cells were prepared for cryosectioning and immunocytochemistry using previously described methods (
For routine morphological examination, cell monolayers were fixed in 2.5% glutaraldehyde buffered in 100 mM sodium cacodylate (pH 7.4), scraped from the culture dish as described, pelleted, postfixed in osmium tetroxide, en bloc-stained in 1% uranyl acetate in 50 mM sodium maleate (pH 5.2), dehydrated in ethanol, and embedded in epoxy resin. Sections were examined and photographed using a transmission electron microscope (FEI-Philips; Hillsboro, OR) operating at 80 kV.
Quantification
Co-localizations of L. monocytogenes with intracellular markers was examined after labeling with specific antibodies and fluorescent secondary antibodies. Samples were examined by epifluorescent illumination. Approximately 200–300 cells were sequentially sampled for each experiment.
Stereological estimates of anti-biotin labeling density over J774 cell membranes were performed on sequentially sampled electron micrographs using cross-lattice overlays. Gold particle numbers were combined with numbers of intersects between the membrane under investigation and a test line of known length (
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Results |
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Viability of L. monocytogenes
To assess the viability of the L. monocytogenes used in this work, growth and infection rates were examined. Although all incubation times used in this study were never longer than 20 min post infection (p.i.), longer incubation times were used to examine the normal internalization process of the bacteria being used. After 5-min exposure to bacteria, approximately 15% of J774 cells were observed with associated bacteria, with an average of 1 bacterium per cell (Table 1). The numbers of cells with associated bacteria increased with time (Table 1), with 35% of cells being affected after 2 hr and 74% of cells after 3 hr. The number of bacteria per cell also increased during this time, with average numbers increasing slowly for the first 2 hr but increasing to 10 per cell after 3 hr. Labeling of non-permeabilized cells showed only small numbers of extracellular bacteria associated with the J774 cells (data not shown).
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The numbers of actin tails associated with internalized L. monocytogenes, visualized with fluorescent phalloidin, were first detected 2 hr p.i. The mean number of actin tails was slightly less than the number of bacteria per cell but increased over time (Table 1).
Adding L. monocytogenes to J774 cell cultures had no obvious effect on the subcellular morphology of the cells. Intracellular bacteria were easily detected by light and electron microscopy and sufficient bacteria were found by electron microscopy to give unbiased overviews of early intracellular events.
Nonspecific Antibody Binding
Although monospecific antibody probes were used for labeling sections, controls to test for nonspecific binding were performed. Sections of L. monocytogenes-infected J774 cells were probed with non-relevant antibodies and PAG, PAG alone, or rabbit anti-mouse antibodies followed by PAG. No signal was detected with either the non-relevant antibodies or PAG (data not shown), and only small amounts of gold were detected on sections treated with anti-mouse antibodies (Fig 1A and Fig 1B). Small numbers of gold particles were seen over the nuclear envelope, throughout the cytoplasm, over the nucleus, and occasionally inside bacteria (data not shown). There was no signal over host cell membrane or over phagosome membranes (data not shown).
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Interaction of L. monocytogenes with Host Cell Surface
In the first few minutes of infection, L. monocytogenes bound to the host cell surface with a tight apposition of bacterial and host cell membranes (Fig 1A). This close interaction of host and bacterial membranes continued into early phagosomes (Fig 1B). No specific accumulation of DAMP-containing structures (Fig 1C) or BSA–gold-filled lysosomes (Fig 1D) was detected in the intracellular region close to extracellular surface-bound bacteria.
In cryosections, the MHC class II labeling of J774 cells could be easily detected on the cell surface and in intracellular structures. At the site where L. monocytogenes were attached to the plasma membrane of cells expressing surface MHC class II, an exclusion of labeling from the J774 cell surface was observed (Fig 2A). This suggested that L. monocytogenes was able to exclude some surface proteins from the plasma membrane during the binding phase before internalization. To confirm this, J774 cells were surface-biotinylated and then infected with L. monocytogenes. Biotinylation on the cell membrane can be detected by applying specific anti-biotin antibodies and PAG. In sections, this label was present on the whole length of the surface membrane. However, at the site where L. monocytogenes attached to the plasma membrane an exclusion of the specific biotin labeling from the interface between bacterium and cell surface was observed (Fig 2B and Fig 2C). The biotin signal was excluded when there was a tight apposition of bacterial and host cell membranes (Fig 2B) and when the association appeared to be a loose one (Fig 2C). Quantification of the labeling density of anti-biotin antibody at this site showed a significant reduction in the signal compared to plasma membrane where L. monocytogenes was not attached (Table 2).
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It is possible that the absence of labeling between the L. monocytogenes and the host cell was because the antigens at this site were inaccessible for antibody binding. For this reason, sections were probed with antibodies that recognized bacterial surface antigens. These antibodies did label the interface between bacteria and host cell membrane (Fig 2D). To confirm that antigens at this site were accessible for antibody binding, bacteria were biotinylated, bound to cells, sectioned, and probed with anti-biotin antibodies. The anti-biotin antibodies bound to the zone of contact between the biotinylated L. monocytogenes and the J774 cell membrane (Fig 2E). Both experiments clearly demonstrate that the site of bacterial and host cell membrane apposition was easily accessible to antibodies.
To further investigate the plasma membrane site where particles bind as a first step to internalization, J774 cells were incubated for 10 min with avidin-coated latex beads. Sections through aldehyde-fixed cells with attached latex beads were probed with anti-MHC class II antibody. Antibody binding was detected at the interface where the plasma membrane and latex bead surface were interacting (Fig 3). The method of internalization appeared to be very different from that occurring during L. monocytogenes internalization. For each bead examined associating with a cell membrane, there was a close apposition of membrane to the bead surface, with the cell membrane wrapping completely around the latex bead (Fig 3). In contrast, L. monocytogenes did not appear to stimulate the long phagocytic processes on the J774 cell membrane that engulfed latex beads.
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Expression of MHC Class II by J774 Cells
The J774 cell line is not usually considered to be an MHC class II-expressing cell line. However, class II-specific antibodies produced specific labeling patterns on cryosections of J774 cells. Light and electron microscopic quantification of the cells showed that approximately 70% of the cells in the population expressed MHC class II (data not shown).
To confirm the expression of MHC class II by the J774 cells, Western blots of homogenized cells were probed with antibodies to class II MHC antigens. A polypeptide pattern consistent with MHC class II expression in J774 cells was recorded (Fig 4). This pattern was similar to that seen in Western blots of A20 ß-lymphocytes, a known MHC class II-expressing cell line, probed with the same antibodies (Fig 4).
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and ß side chains are present in both cell types.Cells probed with antibodies to I-Ed epitopes specific to MHC class II molecules from Balb/c mice (the original source of the J774 cell line) and subjected to a fluorescently activated cell sorter (FACS) analysis demonstrated a positive signal on approximately 65% of the cells (Fig 5). In contrast, a less specific epitope, I-Ad, was seen on only 5% of the cells (Fig 5).
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The Early Phagosome and Intracellular Organelles
After incubation of cells and bacteria for 5 min at 37C, bacteria were found inside the cells in membrane-bound vacuoles. The membrane of early phagosomes showed reduced labeling with antibodies to class II MHC antigens (Fig 6A) and with anti-biotin antibodies in cells that had been surface-biotinylated (Fig 6B). With the biotin labeling, the mean number of gold particles associated with phagosome membranes was similar to the depleted signal estimated for the plasma membrane at the region of contact with L. monocytogenes (Table 2). Some phagosomes did not label with the anti-MHC class II antibodies (data not shown).
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After 10-min exposure to bacteria, some L. monocytogenes-containing phagosomes still did not label with the anti-MHC class II antibodies. Structures in close proximity to the early L. monocytogenes-containing phagosomes did show MHC class II labeling (Fig 7A). In other cells, it appeared that MHC class II-positive structures were fusing with the phagosome membrane (Fig 7B). Although many of the internalized bacteria had escaped into the cytoplasm at the 25-min time point (data not shown), phagosomes still containing bacteria were present and showed increased labeling with the MHC class II antibodies (Fig 7C). In multiple-label experiments the phagosome membranes showed variable labeling with antibodies to LAMP 1 and MHC class II. Although many phagosomes that labeled with anti-class II antibodies had little or no LAMP 1 label (Fig 7D and Fig 7E), some were present that did label with this marker (Fig 7E). In some cells, LAMP 1-positive structures were seen close to L. monocytogenes-containing phagosomes (Fig 8).
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The variable association of LAMP 1 and MHC class II labeling with L. monocytogenes-containing phagosomes observed by TEM was quantified by light microscopy. Co-localization of bacteria with either LAMP 1 or MHC class II antibodies was quantified on cells fixed at regular intervals after exposure to bacteria. At the first time point, when bacteria were first added to the cells, no co-localization of bacteria with MHC class II or LAMP 1 was observed (data not shown). However, after the 5-min p.i. time point, approximately 50% of the cell associated bacteria co-localized with MHC class II (Fig 8). After 10 min the number of bacteria co-localized with MHC class II had risen to 70%. This number dropped to just over 20% at 15 min p.i. and steadily dropped, until by 2 hr p.i. there was only approximately 10% co-localization (Fig 9).
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In comparison, co-localization with LAMP 1 was first observed 15 min p.i. with 
10% of the bacteria being associated with LAMP 1. This rose to 20% by 25 min and remained at this value until it rose to 50% at 3 hr p.i. (Fig 9).
Anti-MHC Class II Antibodies Do Not Bind to Surface Fc Receptors
To confirm that MHC class II labeling on cryosections was not an artifact due to crossreactivity with the Fc receptor, antibodies to mouse Fc receptor (24G2) were used as a blocking step before labeling with anti-class II antibodies. Sequential sections through cells infected for 5 min either were incubated with anti-MHC class II alone (Fig 10A) or were treated with 24G2 before labeling with anti-MHC class II (Fig 10B and Fig 10C). As expected, early phagosomes in untreated sections labeled with MHC class II (Fig 10A). Pretreatment of sections with 24G2 did not inhibit MHC class II labeling on the cell surface (Fig 10B) or on the L. monocytogenes-containing phagosome membrane (Fig 10C), suggesting that the MHC class II antibodies do not bind to Fc receptors in J774 cells. Similarly, sections labeled with the anti-Fc receptor antibody showed only a low labeling signal on the surface and over intracellular sites in J774 cells, which were not associated with intracellular L. monocytogenes (data not shown), also suggesting that the MHC class II antibody labeling was specific.
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Interaction of Phagosomes with Low-pH Compartments
To examine the process of acidification of L. monocytogenes-containing compartments, cells were incubated with DAMP before infection. Sections through aldehyde-fixed cells were probed with anti-DNP antibodies and PAG to identify intracellular sites of DAMP accumulation. Many membrane-bound organelles labeled with the anti-DNP antibodies, indicating sites of low pH. The cell nucleus (data not shown) and mitochondria labeled with anti-DNP antibodies, indicating an accumulation of DAMP (Fig 11A). Many other organelles showed accumulation of DAMP, some of which were observed fusing with L. monocytogenes-containing phagosomes (Fig 11A). The anti-DNP signal was found accumulating around the bacteria within the lumen of phagosomes (Fig 11B).
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Discussion |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Exposure of J774 cells to a large inoculum of bacteria for short incubation times has made it possible to examine early synchronous events of L. monocytogenes internalization using immunocytochemical methods. In this way it has been possible to demonstrate that, during the early stages of L. monocytogenes association with J774 cells, the initial binding to host plasma membrane is accompanied by a rearrangement of some surface proteins on the host cell surface. This results in the formation of an early phagosome membrane inside the cell with a peptide profile different from that of the plasma membrane. Surface molecules available for biotinylation, and MHC class II molecules at the site of attachment, are excluded from the phagosome membrane. The exclusion of biotin signal and MHC class II molecules from between the bacterium and the host cell membrane suggests that manipulation of the cell surface by the bacteria is occurring either before or during the binding event. This hypothesis is supported by the observation that MHC class II molecules are not excluded from the interface between cell membrane and phagocytosed latex beads and by an exclusion of surface molecules from the membranes of early phagosomes containing L. monocytogenes.
Once inside the cells the L. monocytogenes are again exposed to MHC class II molecules when organelles containing MHC class II are delivered to and fuse with the phagosome membrane just before the bacteria escape into the cell cytoplasm. The phagosome membrane also acquires lysosomal glycoproteins from organelles that fuse at this early internalization stage. Organelles with low luminal pH also fuse with the L. monocytogenes-containing phagosome during the first 25 min after exposure to bacteria.
The technical limitations of the labeling methods used in this study could lead to conclusions that some of the observations are the result of technical artifacts. For this reason it was important to perform extensive control experiments to demonstrate specificity. It is possible that the exclusion of label at sites where L. monocytogenes is bound to the cell membrane was a result of antigen inaccessibility for antibody binding. To exclude this possibility, sites with loosely bound bacteria on the cell membrane were examined. These sites showed exclusion of MHC class II and biotin label even though antibody accessibility appeared possible. In addition, antibodies that recognized antigens on the bacterial surface were used to label the site of binding between plasma membrane and attached bacteria. These antibodies were able to label this site. Biotin on the bacterial surface was also accessible to antibody binding. Finally, anti-MHC class II labeling was detected at the interface where latex beads bound to J774 cells before internalization. Together, these observations suggest that the loss of labeling on the host plasma membrane was probably a result of a bacterial effect and not the result of physical exclusion of labeling. This is supported by the modified peptide profiles observed on early phagosomes that appear similar to those observed on the cell membrane.
The use of antibodies that recognize mouse Igs could also be a source of incorrectly observed immunolabeling on mouse macrophages. However, the control experiments that show only a limited amount of signal on these cells effectively rule out this possibility. Similarly, control experiments rule out the possibility that nonspecific binding of antibody to Fc receptors is playing a role in the labeling experiments.
Although J774 cells have previously been considered to be a non-MHC class II-expressing cell, it appears that approximately 60–70% of the cells in culture do express MHC class II molecules on their cell surface and at intracellular sites. The peptide profile produced on blots and the presence of a Balb/c-specific MHC class II epitope associated with the J774 cells support the observation that these cells express MHC class II molecules. It remains to be determined whether infected J774 cells are good candidates for stimulation and presentation of L. monocytogenes antigens. However, presentation of antigen using the class II MHC pathway has been reported to occur in these cells (
When living L. monocytogenes are used to infect bone marrow macrophages, most of the intracellular bacteria are destroyed before escape from the phagosome has been accomplished (
Internalized L. monocytogenes appear to encounter MHC class II molecules only after they have entered the cells and before they have escaped into the cell cytoplasm. Once in the cytoplasm the L. monocytogenes pass into adjacent cells via LAMP 1-positive compartments (this study and (
In antigen-presenting cells, the exact site where peptide-MHC class II complexes are formed is still undetermined. Newly synthesized MHC class II molecules pass from the Golgi complex to specialized MHC class II-containing endosomal compartments (CIIVs) (
The ability of pathogens to modify phagosome membranes appears to be a common event (
It is commonly accepted that, during phagocytosis, newly formed phagosomes have a polypeptide profile similar to that of the plasma membrane (
In conclusion, intracellular pathogens must utilize host cell processes for the regulation of pathogenesis. The results of this work suggest that L. monocytogenes enters cells through a route that is different from the phagocytic pathway. In this way the organism can partially limit exposure to MHC class II by excluding this molecule from the phagocytic vacuole as it is formed. The subsequent proteolysis of the organism resulting from exposure to lysosomes is negated by a rapid escape into the cell cytoplasm, possibly assisted by rapid delivery of low pH organelles to the phagosome. Peritoneal macrophages are able to destroy most of the intracellular bacteria before they can escape from the phagosome (
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Acknowledgments |
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I thank Linda Chicoine for excellent technical assistance, Dan Portnoy for the L. monocytogenes cultures, Ira Mellman for immunoreagents, and Gareth Griffiths for advice on incubating latex beads with J774 cells.
Received for publication May 16, 2001; accepted November 21, 2001.
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