Lehrstuhl für Mikrobiologie, Theodor-Boveri-Institut für Biowissenschaften der Universität Würzburg, Würzburg, Germany,1 Pediatric Infectious Diseases, Johns Hopkins School of Medicine, Baltimore, Maryland2
Received 8 July 2002/ Returned for modification 6 September 2002/ Accepted 30 September 2002
ABSTRACT
Listeria monocytogenes causes meningitis and encephalitis in humans and crosses the blood-brain barrier by yet unknown mechanisms. The interaction of the bacteria with different types of endothelial cells was recently analyzed, and it was shown that invasion into, but not adhesion to, human brain microvascular endothelial cells (HBMEC) depends on the product of the inlB gene, the surface molecule InlB, which is a member of the internalin multigene family. In the present study we analyzed the role of the medium composition in the interaction of L. monocytogenes with HBMEC, and we show that invasion of HBMEC is strongly inhibited in the presence of adult human serum. The strong inhibitory activity, which is not present in fetal calf serum, does not inhibit uptake by macrophage-like J774 cells but does also inhibit invasion of Caco-2 epithelial cells. The inhibitory component of human serum was identified as being associated with L. monocytogenes-specific antibodies present in the human serum. Human newborn serum (cord serum) shows only a weak inhibitory activity on the invasion of HBMEC by L. monocytogenes.
INTRODUCTION
Listeria monocytogenes, a gram-positive, facultatively intracellular bacterium, is known to cause meningitis, encephalitis, and brain abscesses, mainly in immunocompromised individuals (21). Central nervous system (CNS) penetration by L. monocytogenes suggests that invasion of brain microvascular endothelial cells may be an important way of crossing the blood-brain barrier. During the last couple of years, several groups have reported on the capacity of L. monocytogenes to invade different types of human endothelial cells. However, the absolute values of invasion, as well as the dependency of invasion on the inlB gene product, differed markedly among the studies (5, 11, 12, 17, 22). It has previously been shown that invasion of, but not adhesion to, human brain microvascular endothelial cells (HBMEC) by L. monocytogenes is strictly dependent on the presence of the product of the inlB gene (2, 10, 11). InlB is a 630-amino-acid protein of the internalin family of leucine-rich repeat proteins which is found at the cell surface but is also secreted into the supernatant (3, 7, 9). Parida et al. (17) have reported a similar inlB-dependent invasion of human umbilical vein endothelial cells (HUVEC), which we could not detect in an earlier study (12). Furthermore, Drevets et al. (5) first reported an InlA-dependent invasion of HUVEC and later an inlA- and inlB-independent invasion of human microvascular endothelial cells (22). The differences in InlB dependency of endothelial cell invasion might be due, at least partially, to the different types of inlB mutants used in the studies as well as to differences in the target cells. On the other hand, differences in experimental conditions might also have influenced the outcomes of the experiments.
In the present study we analyzed the roles of normal human serum (HS) and fetal calf serum (FCS) in adhesion to and invasion of HBMEC by L. monocytogenes. We show that antibodies present in HS result in a dramatic decrease in HBMEC invasion. This finding may not only help to explain some of the discrepancies among recent publications on InlB-dependent invasion of endothelial cells by L. monocytogenes but also question the in vivo role of InlB-dependent invasion of endothelial cells in the course of human infections.
MATERIALS AND METHODS
Cell culture and infection. Culture of HBMEC, Caco-2 epithelial cells, and J774 macrophages and their infection with L. monocytogenes have been described in detail recently (2, 11). L. monocytogenes strain EGD was cultured aerobically in brain heart infusion (BHI) broth (Difco) at 37°C until it reached the mid-log phase of growth. After the bacteria were washed twice with phosphate-buffered saline (PBS), they were stored in aliquots in PBS with 20% (vol/vol) glycerol at -80°C until they were used for the infection experiments.
HBMEC were isolated from a brain biopsy specimen of an adult female with epilepsy and were cultured by methods described previously (19). HBMEC were subsequently immortalized by transfection with simian virus 40 large T antigen and maintained their morphological and functional characteristics for at least 30 passages (20). HBMEC were cultured in gelatin-coated flasks without the addition of antibiotics in complete HBMEC medium (RPMI 1640 medium [Gibco] supplemented with FCS [10%] [Gibco or Sigma], NuSerum IV [10%] [Becton Dickinson, Bedford, Mass.], nonessential amino acids [1%] and vitamins [1%], heparin [5 U/ml], sodium pyruvate [1 mM], L-glutamine [2 mM], and endothelial cell growth supplement [30 µg/ml] [all from Sigma]) and were incubated at 37°C under a humid atmosphere of 5% CO2. Caco-2 epithelial cells and J774 macrophages were cultured in RPMI 1640 medium supplemented with FCS (10%) according to standard procedures (2).
Forty-eight hours prior to infection, cells were split and seeded into normal (Caco-2 cells and J774 macrophages) or gelatin-treated 24-well tissue culture plates at a density of 105 cells per well. Immediately prior to the assay, each well was found to contain approximately 2 x 105 cells. Bacteria were diluted in RPMI 1640 medium, with or without serum, and 1 ml of the suspension was added to each monolayer in order to obtain the desired multiplicity of infection of 20 bacteria per cell.
To measure initial association, cultures were incubated at 37°C for 1 h in order to allow the bacteria to associate with the cells, which were then washed five times and lysed, and appropriate dilutions were plated on BHI agar. To measure invasion, cultures were incubated at 37°C for 1 h in order to allow the bacteria to invade the cells. One milliliter of RPMI medium containing 100 µg of gentamicin (Sigma)/ml was then added to the washed monolayers to kill extracellular bacteria, and the plates were further incubated for 1 h at 37°C. After the cells were washed twice with PBS, they were lysed and plated on BHI agar. All cellular association and invasion assays were performed in triplicate and repeated at least three times. The absolute numbers of intracellular bacteria were always around 105 bacteria per well, which means that about 5% of the bacteria added to the cells were taken up by the HBMEC.
For statistical analysis, the two-tailed, unpaired Student t test was applied, and P values of
(Tobias Hertzig Martin Weber Lars Greiffenberg Britta Schulte Holthausen Werner Goebel Kwang Sik Kim )
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