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The Journal of Immunology, 1998, 161: 3010-3018.
Copyright © 1998 by The American Association of Immunologists

Two Distinct Phospholipases C of Listeria monocytogenes Induce Ceramide Generation, Nuclear Factor-{kappa}B Activation, and E-Selectin Expression in Human Endothelial Cells1

Nicole Schwarzer*, Ralph Nöst*, Joachim Seybold*, Shreemanta K. Parida{dagger}, Oliver Fuhrmann*, Matthias Krüll*, Reinhold Schmidt*, Robert Newton{ddagger}, Stefan Hippenstiel*, Eugen Domann{dagger}, Trinad Chakraborty{dagger} and Norbert Suttorp2,*

* Department of Internal Medicine and {dagger} Institute of Medical Microbiology, Justus-Liebig University, Giessen, Germany; and {ddagger} Imperial College School of Medicine, National Heart and Lung Institute, Department of Thoracic Medicine, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Infection of endothelial cells by Listeria monocytogenes is an essential step in the pathogenesis of listeriosis. We recently reported that L. monocytogenes induces up-regulation of E-selectin and other endothelial adhesion molecules and subsequent polymorphonuclear leukocyte (PMN) adhesion into cultured human endothelial cells. In the present study, we characterized the mechanisms of enhanced E-selectin expression using L. monocytogenes wild type (EGD), the isogenic in-frame deletion mutants for phosphatidylcholine (PC)- and phosphatidylinositol (PI)-specific phospholipases EGD{Delta}plcA and EGD{Delta}plcB, as well as the nonvirulent control strain Listeria innocua. Infection of endothelial cells with EGD{Delta}plcA or EGD{Delta}plcB for 6 h induced, as compared with EGD wild type, intermediate levels of E-selectin mRNA and protein as well as PMN rolling and adhesion at a shear rate of 1 dyne/cm2, indicating that both bacterial phospholipases are required for a maximal effect. Similarly, ceramide content and NF-{kappa}B activity were increased in L. monocytogenes-exposed endothelial cells, but only to intermediate levels for PC- or PI-phospholipase C (PLC)-deficient listerial mutants. Phospholipase effects could be mimicked by exogenously added ceramides or bacterial sphingomyelinase. The data presented indicate that PI-PLC and PC-PLC are important virulence factors for L. monocytogenes infections that induce accumulation of ceramides that in turn may act as second messengers to control host cell signal-transduction pathways leading to persistent NF-{kappa}B activation, increased E-selectin expression, and enhanced PMN rolling/adhesion. The ability of L. monocytogenes to stimulate PMN adhesion to endothelial cells may be an important mechanism in the pathogenesis of severe listeriosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Listeria monocytogenes, a Gram-positive facultative intracellular bacteria, is an opportunistic pathogen for animals and humans (1). It causes food-borne septicemia and meningitis primarily in immunocompromised hosts, pregnant women, and neonates (2, 3, 4). During the course of systemic disease, many cell types are infected, including intestinal epithelial cells (5), macrophages (6), endothelial cells (7), hepatocytes (8), and fibroblasts (9), demonstrating the capability of Listeria to invade different professional and nonprofessional phagocytes. The infectious process of L. monocytogenes can be separated into the following steps: adhesion, invasion, escape from the phagosomal compartment, intracytosolic replication, and cell-to-cell spread. Recently, several genes encoding virulence determinants have been identified and found to be clustered on the chromosome. Among these are hly, plcA, and plcB, which encode the pore-forming listeriolysin (LLO),3 a phosphatidylinositol-specific phospholipase C (PI-PLC), and a phosphatidylcholine-specific phospholipase C (PC-PLC), respectively. These three virulence factors contribute, partly in a synergistic manner, to the lysis of the phagosome as well as to cell-to-cell spread (10, 11, 12).

Recent studies demonstrate that invasion and activation of endothelial cells by L. monocytogenes are critical events in the pathogenesis of listeriosis (13, 14, 15, 16). In major manifestations of this disease, such as meningitis and neonatal sepsis, bacteria must cross the endothelial barrier, thereby promoting the infection of different tissues and organs. Therefore, an understanding of the interaction between bacteria and endothelial cells is relevant to the pathophysiology of listeriosis.

While antilisterial immunity is accomplished primarily by T lymphocytes (17, 18), phagocytes such as macrophages and polymorphonuclear leukocytes (PMN) also contribute to the antilisterial resistance (19, 20, 21). Adhesion of circulating leukocytes to endothelial cells is an early step in an inflammatory reaction, and we have recently reported an increased expression of different adhesion molecules (ICAM-1 and VCAM-1 as well as E- and P-selectin) on cultured human endothelial cells after infection with L. monocytogenes, an effect that was accompanied by an enhanced adhesion of PMN (14).

A prerequisite for the expression of E-selectin and other proinflammatory mediators is activation and subsequent translocation of the transcription factor NF-{kappa}B from the cytoplasm to the nucleus, where it regulates the activity of different genes involved in the immune response (22, 23, 24). NF-{kappa}B is a member of the Rel family of transcriptional activator proteins (25). It is sequestered in the cytoplasm as a p50-p65 heterodimer, which is associated with two major inhibitory proteins, I-{kappa}B{alpha} and I-{kappa}Bß (26). Stimulation of cells leads to phosphorylation, polyubiquitination, and degradation of the inhibitory proteins, which results in a NF-{kappa}B activation (27, 28, 29, 30).

L. monocytogenes wild-type related NF-{kappa}B translocation into the nucleus of endothelial cells was demonstrated recently by fluorescence microscopy (13). In P388D1 macrophages, L. monocytogenes-induced NF-{kappa}B activation was biphasic (31). The initial transient translocation of NF-{kappa}B was induced by lipoteichoic acid, which was followed by a second persistent phase mediated by expression of listerial phospholipases and paralleled by I-{kappa}Bß degradation (31).

Ceramides are key mediators in coordinating cellular responses. They are generated via the sphingomyelinase pathway, which in turn is activated by the PLC product diacylglycerol (DAG) (32). NF-{kappa}B activation can occur in the presence of ceramides, suggesting that these sphingomyelinase products may represent the link between listerial PLC and endothelial E-selectin expression.

In the present study, besides the wild-type L. monocytogenes (EGD) and the nonpathogenetic Listeria innocua (INN), we made use of genetically engineered mutants that lack the genes required for the expression of PI- or PC-PLC. The data presented suggest that both PLCs of L. monocytogenes are necessary for maximal intracellular ceramide generation, NF-{kappa}B activation, and subsequent E-selectin expression. Moreover, addition of exogenous sphingomyelinase as well as ceramides to endothelial cells mimicked the effects of L. monocytogenes infection with respect to E-selectin expression. We therefore suggest that listerial PLC-induced ceramides act as potential second messengers in L. monocytogenes-infected endothelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Tissue culture plasticware was obtained from Becton Dickinson (Heidelberg, Germany) and Nunc (Wiesbaden, Germany). MCDB 131 medium, HBSS, PBS, trypsin-EDTA solution, HEPES, and FCS were from Life Technologies (Karlsruhe, Germany). Collagenase (CLS type II) was purchased from Worthington Biochemical (Freehold, NJ). Paraformaldehyde and Silica 60 high performance thin layer chromatography (HPTLC) plates were obtained from Merck (Darmstadt, Germany). Sodium chromate ([51Cr]Na2CrO4, 1 mCi/ml) was purchased from New England Nuclear (Dreieich, Germany). Ceramides C8 and C16 were obtained from Biomol (Hamburg, Germany). All other reagents were obtained from Sigma (Munich, Germany).

Monoclonal Abs

Purified freeze-dried mAb directed against E-selectin (1.2B6) was obtained from Immunotech (Marseille, France). Horseradish peroxidase-conjugated polyclonal sheep anti-mouse IgG Abs were from Amersham (Dreieich, Germany). All Abs used were azide free. To further characterize the adhesion system, endothelial cells were preincubated with 50 µg/ml inhibitory mAb for 45 min.

Preparation of HUVEC

Cells were isolated from umbilical cord veins and identified according to the method of Jaffe et al. (33). Briefly, cells obtained from collagenase digestion were washed, resuspended in MCDB 131/5% FCS, and seeded into well plates or flasks. Only confluent monolayers of primary cultures were used.

Isolation and labeling of human PMN

Heparinized human donor blood was centrifuged in a discontinuous Percoll gradient to yield a PMN fraction of >97% purity (34). Freshly isolated neutrophils were radiolabeled with 51Cr, according to Gallin et al. (35). Briefly, after isolation PMN were incubated with 100 µCi 51Cr at 37°C for 1 h in RPMI 1640 medium containing 10% FCS. Subsequently, cells were washed twice in HBSS (with calcium, without magnesium) to remove unincorporated 51Cr.

Bacterial strains and growth media

Bacteria were grown in brain heart infusion broth (Difco Laboratories, Detroit, MI) at 37°C and were used in the logarithmic phase of growth.

Generation of isogenic mutant: The wild-type L. monocytogenes serotype 1/2a strain EGD, the L. innocua serotype 6b strain ATCC 33090 (INN), and the in-frame deletion mutants L. monocytogenes {Delta}plcB have been described previously (16, 36, 37, 38). To generate the isogenic mutant strain {Delta}plcA, the flanking regions of the plcA gene (formerly pic gene, accession number X54618) had been amplified separately by PCR using chromosomal DNA from L. monocytogenes as template. The upstream region of the plcA gene was amplified with the oligonucleotide pair 238 (5'-TTCCCAAGTAGCAGGACATGC-3') and 3974 (5'-GTTCGAGGATTAGGCATACTAATCATG-3') and the downstream region with the oligonucleotide pair 3973 (5'-TGTAGTTACAGAGTTCTTTATTGGC-3') and 108 (5'-CATGGGTTTCACTCTCCTTCTAC-3'), resulting in products of 1055 and 378 bp, respectively. Both PCR products were ligated and again amplified by PCR with the flanking oligonucleotides 238 and 108 to obtain a DNA fragment of 1433 bp. Finally, plcA lacked the amino acids 46 to 290. The 1433-bp-long PCR product had been directly cloned into the SmaI restriction site of the multiple cloning site of the vector pUC18-generating plasmid pUC18-{Delta}plcA. The deletion allele of the plcA gene, located on a 1.5-kb large XbaI/SacI restriction fragment from plasmid pUC18-{Delta}plcA, was cloned into the restriction sites XbaI and SacI from suizid vector pAUL-A (39) to obtain plasmid pAUL-{Delta}plcA. After transformation of the wild-type strain EGD with pAUL-{Delta}plcA, the plcA deletion mutant was subsequently generated by procedures described previously (40, 41). Verification of the {Delta}plcA mutant came from corraborative data obtained by sequencing the chromosomal deletion generated, as well as by examining production with mAbs directed against PlcA (data not shown).

Listeria infection assay

Before infection with different Listeria strains, HUVEC were washed thrice with MCDB 131 medium without supplements or antibiotics. Bacterial concentrations from experimental cultures were adjusted by determining the OD at 600 nm, and appropriate dilutions were prepared. Then bacteria were centrifuged at 8000 rpm for 2 min, followed by two washes in PBS and one washing in plain MCDB 131 medium, and added in a bacteria to eukaryotic cell ratio of 10:1 directly to the HUVEC monolayer culture. After 2 h, the plates or flasks were washed extensively with plain medium, and subsequently cells were incubated in medium containing 50 µg/ml gentamicin to kill remaining extracellular bacteria. At times indicated in the figure legends, cells were processed for ceramide and NF-{kappa}B reporter gene assay, Northern blot for E-selectin, cell surface ELISA, neutrophil adhesion assay under stationary or flow conditions, and neutrophil rolling assay.

Assay for ceramide content

Ceramide content of endothelial cells preincubated with different Listeria strains or nonstimulated control cells was determined by HPTLC. Confluent, pretreated HUVEC monolayers in two T80 flasks were washed, scraped into ice-cold methanol, and disrupted by sonication (Branson Sonic Power Sonifier; Branson Instruments, Danburg, CT) three times for 15 s each on ice at a power output of 60 W. Lipids were extracted according to Bligh and Dyer (42). The organic phase was evaporated under a stream of N2, and inorganic phosphorus was measured by means of a colorimetric assay, as described (43). Total ceramides were separated from other lipids on HPTLC using Silica 60 plates, according to Squier et al. (44). This procedure was modified by the addition of a second HPTLC run to clearly separate ceramides and monoglycerides. Samples were applied to plates with a Linomat IV applicator (Camag, Muttenz, Switzerland). The first solvent system (44) contained chloroform:methanol:acetic acid (95:4.5:0.5, v/v/v). Ceramide spots identified via corresponding ceramide standard were scraped off, eluted with 10 ml chloroform, and dried under N2. These unknown samples as well as 6 C16 ceramide standard samples (ceramide concentrations increasing from 0.143–6.43 µg) were applied to a second HPTLC plate, which was developed in ethyl ether:hexane:acetic acid (70:29:1; v/v/v). Ceramide spots were visualized with primulin staining. Densitometric quantification of ceramide was done by scanning at 254 nm using a TLC scanner (Camag). Standard ceramide curves were obtained for each HPTLC plate, and integration and calibration were done by the use of Camag Cat software version 3.17.

HUVEC-derived ceramide spots were finally verified using gas-chromatographic analysis by comparison of fatty acid profile of stimulated vs nonstimulated cells. Upon stimulation, samples typically showed a ceramide characteristic increase in behenic acid (22:0), lignoceric acid (24:0), nervonic acid (24:1), palmitic acid (16:0), and stearic acid (18:0) content (45, 46). Ceramide recovery after lipid extraction and first HPTLC run was determined by gas-chromatographic analysis of 100 µg ceramide (C16) standard in relation to 20 µg 15:0 fatty acid methyl ester as internal standard, and amounted to 83 ± 7.4%.

NF-{kappa}B reporter gene assay

The minimal promoter vector, pGL3.BG, was created by inserting a XhoI/HindIII fragment, which contains the rabbit ß-globin TATA box and transcription start (bases -43/+3), from pADneo2BGluci (47) into XhoI/ HindIII-digested pGL3basic luciferase vector (Promega, Madison, WI). Subsequently, pGL3.BG was opened with SmaI, and after calf alkaline phosphatase treatment (Promega), ligated with phosphorylated double-stranded oligonucleotides containing either three copies of the consensus NF-{kappa}B recognition sequence (sense strand only underlined), 5'-GGG GAC TTT CCC TGG GGA CTT TCC CTG GGG ACT TTC CC-3', or three copies of the mutated sequence (mutated bases underlined), 5'-GGCCAC TTT CCC TGG CCA CTT TCC CTG GCC ACT TTC CC-3'. Recombinants containing two tandem repeats of the respective oligonucleotides were selected to create pGL3.BG.6{kappa}B and pGL3.BG.6{kappa}B-mut, and in each case identity was confirmed by double-stranded sequencing.

HUVEC were transiently transfected with 2 µg of the NF-{kappa}B plasmids pGL3.BG.6{kappa}B or pGL3.BG.6{kappa}B-mut (as negative control) using Tfx50 transfection reagent (Promega). Transfected HUVEC were stimulated for 6 h and harvested in reporter lysis buffer (Promega), and total protein was measured using the Bio-Rad reagent (Bio-Rad, München, Germany). NF-{kappa}B-luciferase assay was performed using a commercial kit (Promega). Luminescence was measured on a Lumat LB 9501 luminometer (Berthold, Bad Wildbad, Germany). Relative luminescence readings were normalized to total protein.

Northern blot analysis

RNA was extracted using the guanidinium isothiocyanate method, as described by Chomzcynski and Sacchi (48). Total RNA was quantified by measuring absorbance at 260 nm with a Uvikon 860 spectrophotometer (Kontron, Neufahrn, Germany). RNA samples (10 µg/lane) were electrophoresed on denaturing 1% formaldehyde-agarose gels, transferred onto Magna nylon membrane (MSI, Westborough, MA), and fixed by exposure to UV radiation using a Hoefer UVC 500 Crosslinker.

cDNA probes were labeled with [{alpha}-32P]dCTP (>3000 Ci/mmol) by random priming (Rediprime DNA labeling system; Amersham, Braunschweig, Germany) added to the prehybridization chambers at 1 x 106 cpm/ml and incubated for 12 to 16 h at 42°C. E-selectin cDNA probe was a kind gift from Dr. D. Simmons (Imperial Cancer Research Fund, Institute of Molecular Medicine, Oxford, U.K.). The 598-bp cDNA fragment of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was obtained as previously described (49). Following hybridization, the filters were washed to a stringency of 0.1x SSC/0.1% SDS for 30 min at 55°C. Membranes were autoradiographed overnight at -70°C by exposure to hyperfilm MP (Amersham). Bands were quantified by phosphoimaging (FUJIX-BAS 1000; Fuji, Düsseldorf, Germany). After exposure, blots were stripped in 50% formamide, 10 mM NaH2PO4 for 1 h at 65°C before subsequent rehybridization. To account for difference in loading or transfer of the RNA, hybridization was performed with 32P-labeled GAPDH-cDNA probe.

Cell surface ELISA for E-selectin expression

E-selectin expression on endothelial cells preincubated with different Listeria strains was determined by cell surface ELISA (14, 34). Confluent, pretreated HUVEC monolayers in 96-well flat-bottom microtiter plates were washed and finally fixed with 4% paraformaldehyde for 15 min. Human Ig was used to reduce nonspecific binding, and primary Abs were added for 30 min. Thereafter, cells were washed thrice and exposed to a horseradish peroxidase-conjugated rabbit anti-mouse Ig Ab for 30 min. After washing, o-phenylenediamine was added for 5 min. Data are indicated as OD at 492 nm.

Neutrophil adhesion assay

After the preincubation of the HUVEC with different Listeria strains, medium was aspirated and endothelial cells were washed twice with MCDB 131 medium. A total of 1 x 106 51Cr-labeled PMN in 1 ml medium was added to each well (24-well plate) and allowed to settle for 30 min at 37°C and 5% CO2. Subsequently, unbound PMN were removed by gentle aspiration, and each well was washed twice with HBSS. Adherent PMN and endothelial cells were lysed with 2 M H2SO4 for 30 min. Radioactivity of the lysate was quantitated with a gamma counter (Cobra Autogamma B5003; Canberra Packard, Frankfurt, Germany). Percentage of PMN adhesion was calculated as the 51Cr fraction in the lysate in relation to the total radioactivity added. Frequent counting of adhering PMN in several high power fields in the microscope confirmed that L. monocytogenes-related 51Cr retention in the culture wells accurately reflected neutrophil adhesion to endothelial cells.

Neutrophil rolling and adhesion assay under flow conditions

Leukocyte rolling and adhesion were determined using a parallel plate flow chamber, according to the method of Lawrence and Springer (50). Confluent endothelial monolayers grown on Themanox coverslips (22 x 60 mm; Nunc, Wiesbaden, Germany) were preincubated with wild-type and different deletion mutants of L. monocytogenes, as described above, and subsequently treated with culture medium alone or medium containing saturating concentration of mAb against E-selectin. A suspension of 3 x 106 leukocytes/ml was perfused through the chamber at a constant wall shear stress of 1 dyne/cm2 (syringe pump sp100i; WPI, Sarasota, FL). Interactions were visualized using a phase-contrast videomicroscope (with a KP-C551 CCD color camera; Hitachi, Rodgau, Germany) and videotaped (JVC HR-S7000EG; JVC Friedberg, Germany) the entire time period of leukocyte perfusion. Images were recorded at real time and played back at six- or ninefold slower speed. The tape was paused to mark the location of cells, and the displacement of the center of individual cells was measured 2 to 4 s later. Rolling was expressed as the number of rolling cells/high power field during a 3-min observation period (51). Leukocytes were considered to be adherent after 30 s of stable contact with the monolayer. Adhesion was determined after 5 min of perfusion by analysis of 10 to 12 high power (x40) fields from videotape (50).

Statistical methods

Depending on the number of groups and number of different time points studied, data of Figure 3Go were analyzed by a two-way ANOVA. A one-way ANOVA was used for data of Figures 1Go and 2Go and 4 through 7. Main effects were then compared by an F probability test. p < 0.05 was considered significant.



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  		FIGURE 3. Increased ceramide content in L. monocytogenes-infected HUVEC after 2 to 6 h. Endothelial cells in two T80 flasks were scraped off with ice-cold methanol. After lipid extraction, ceramide and phospholipid content were determined. Resting cells displayed 6.6 ± 0.24 ng ceramide/1 µg phospholipid. Percentage of ceramide increase in stimulated cells was calculated in relation to control cells. Data presented are mean ± SEM of five separate experiments.

 


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  		FIGURE 1. Left, Enhanced E-selectin expression in L. monocytogenes-infected HUVEC: L. monocytogenes wild-type (EGD), L. innocua (INN), and the deletion mutants EGD{Delta}plcA and EGD{Delta}plcB were incubated with HUVECs in antibiotic-free medium in 96-well plates in a bacteria to cell ratio of 10:1. After 2 h, wells were washed thrice with fresh medium supplemented with 50 µg/ml gentamicin to kill remaining extracellular bacteria. After incubation with medium containing gentamicin for another 4 h, cells were washed and processed for E-selectin cell surface ELISA. Data presented are mean ± SEM of six separate experiments. Right, Enhanced PMN adhesion to L. monocytogenes-infected HUVEC under stationary conditions: Listeria (10:1 bacteria:cell ratio) were added directly to medium of endothelial cells in 24-well plates. After 2 h, wells were washed thrice with fresh medium supplemented with 50 µg/ml gentamicin. After another 4 h, cells were washed again and processed for neutrophil adhesion assay. A total of 1 x 106 51Cr-labeled PMN in 1 ml medium was added to each well and allowed to adhere for 30 min. Subsequently, unbound PMN were removed by gentle aspiration. Adherent PMN and endothelial cells were lysed with H2SO4. Percentage of PMN adhesion was calculated as the 51Cr fraction in the lysate in relation to the total radioactivity added. Data presented are mean ± SEM of three separate experiments.

 


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  		FIGURE 2. Enhanced PMN rolling (left) and increased PMN adhesion (right) onto Listeria-stimulated HUVEC under flow conditions at a shear rate of 1 dyne/cm2: Different Listeria strains were added to endothelial cells in four-well plates containing rectangular Thermanox coverslips (for details, see Fig. 1Go). After 6 h, coverslips were processed for laminar flow adhesion assay. A total of 3 x 106 PMN/ml was injected into the flow system and perfused over endothelial cell monolayer for 5 min using a high precision syringe pump. Rolling PMNs (for definition and details, see Materials and Methods) were counted over a 3-min observation period (left). Adherent PMN were determined by counting 10 to 12 random high power fields (right). Data presented are mean ± SEM of five separate experiments. *Denotes experiments with endothelial cell monolayers pretreated with anti-E-selectin mAb.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PC-PLC and PI-PLC activity of L. monocytogenes were required for maximal E-selectin expression

We recently reported an increased expression of adhesion molecules and enhanced PMN adherence in cultured human endothelial cells infected with L. monocytogenes (14). Maximal effects had occurred with a bacteria to cell ratio of 10:1, and this bacterial concentration was therefore used throughout the present study. The expression of internalin B, a listerial cell wall-associated protein, is mandatory for invasion and infection of endothelial cells (HUVEC) with L. monocytogenes (16). Since all deletion mutants used in the present study were isogenic mutants, the level of internalin B expression was equal to wild-type Listeria. This aspect was also assessed by infection studies on endothelial cells (data not shown).

In HUVEC exposed, the L. monocytogenes in-frame deletion mutants {Delta}plcA and {Delta}plcB as compared with the Listeria wild-type EGD, an intermediate increase in E-selectin expression was demonstrated after 6 h (Fig. 1Go, left panel). There was no significant difference with respect to E-selectin expression between L. monocytogenes {Delta}plcA- and {Delta}plcB-infected HUVEC, indicating that both phospholipases PI-PLC and PC-PLC are required for a full response (Fig. 1Go, left). The nonpathogenic L. innocua strain (INN), also used as control, induced only a small, yet significant, up-regulation of E-selectin (INN vs non: OD 0.21 ± 0.018 vs 0.164 ± 0.007; n = 6) (Fig. 1Go, left panel).

The elevated E-selectin expression was accompanied by an increase of PMN adhesion in L. moncytogenes-exposed HUVEC 6 h postinfection, as demonstrated in the stationary adhesion assay (Fig. 1Go, right). Moreover, enhanced PMN rolling and adhesion was verified for infected endothelial cells under flow conditions at a shear rate of 1 dyne/cm2, as determined in the parallel plate flow chamber (Fig. 2Go, left and right). PMN rolling (left) and adhesion (right) in {Delta}plcA- or {Delta}plcB L. monocytogenes-infected HUVEC was clearly higher than in endothelial cells exposed to L. innocua, but significantly lower as compared with EGD-stimulated monolayers. PMN rolling and PMN adherence under physiologic flow conditions were reduced in EGD-infected HUVEC in the presence of an anti-E-selectin Ab by 51% ± 4.7 and 62% ± 5.27, respectively, indicating that E-selectin was the most important endothelial adhesion molecule 6 h after L. monocytogenes infection (for role of other adhesion molecules, see discussion below and also 14 .

L. monocytogenes increased intracellular ceramide level in HUVEC

Increased intracellular ceramide levels were noted in all virulent L. monocytogenes-infected HUVEC within 6 h (Fig. 3Go). L. monocytogenes wild-type (EGD) was most effective with respect to this ceramide increase, while L. innocua had a minimal effect. The phospholipase deletion mutants {Delta}plcA and {Delta}plcB induced half-maximal cellular ceramide levels without a significant difference between them. Taken together, these data suggest that both bacterial phospholipases are involved in intracellular ceramide generation.

L. monocytogenes infection induced persistent NF-{kappa}B activation in HUVEC

Infection of HUVEC with wild-type L. monocytogenes (EGD) for 6 h induced a 4.4-fold increase of the NF-{kappa}B reporter gene activity as compared with L. innocua-infected endothelial cells (Fig. 4Go). L. monocytogenes deletion mutants {Delta}plcA and {Delta}plcB, lacking either PI-PLC or PC-PLC, were not as effective as the wild-type Listeria strain (EGD), inducing an intermediate level of the NF-{kappa}B reporter gene activity, which was 2.3 ({Delta}plcA) and 2.1 ({Delta}plcB) times higher than the activity in L. innocua-infected cells (Fig. 4Go). EGD-stimulated HUVEC, which had been transiently transfected in parallel with the mutated NF-{kappa}B-luciferase plasmid (pGL3.BG.6{kappa}B-mut), revealed no detectable luciferase activity (data not shown).



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  		FIGURE 4. Enhanced levels of NF-{kappa}B-reporter gene activation in L. monocytogenes-infected HUVEC: Different Listeria strains were added to endothelial cells that had been transiently transfected with a NF-{kappa}B-directed luciferase reporter plasmid (for details, see Materials and Methods). After 6 h of Listeria infection, cells were washed and NF-{kappa}B activity was quantitated as chemoluminescence intensity of the reporter gene-luciferase assays/µg cell protein. Data presented are mean ± SEM of five separate experiments.

 
L. monocytogenes generated ceramides as possible second messengers for increased E-selectin expression in HUVEC

Cell membrane-permeable ceramides as well as exogeneous SMase (Staphylococcus aureus) were added to endothelial cells to elucidate the potential role of ceramides for NF-{kappa}B activation and E-selectin expression in L. monocytogenes-infected cells. Incubation of endothelial cells with SMase or ceramides (C8, C16) for 4 h increased E-selectin expression in a dose-dependent manner (Fig. 5Go) with maximal effects seen at 1 U/ml SMase and 1 µM C8 or C16 ceramide.



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  		FIGURE 5. Enhanced E-selectin expression in HUVEC stimulated with exogenously added sphingomyelinase (0.5 and 1 U/ml), C16 ceramide (0.1 and 1 µM), or purified LLO (500 ng/ml). White bars indicate addition of LLO or coaddition of LLO and SMase, as well as LLO and C16 ceramide. Ethanol in comparable amounts was used as control (E). After 4 h, 96-well plates were washed and processed for E-selectin cell surface ELISA. Data presented are mean ± SEM of four separate experiments.

 
These effects were accompanied by increased PMN rolling and PMN adhesion (Fig. 6Go, left and right), phenomena that were reduced by >70% in the presence of an anti-E-selectin mAb (Fig. 6Go). Exogenously applied SMase and ceramides also significantly increased NF-{kappa}B reporter gene activity (see Fig. 7Go). Again, SMase- or ceramide-stimulated HUVEC transiently transfected with the mutated NF-{kappa}B-luciferase plasmid (pGL3.BG.6{kappa}B-mut) showed no detectable luciferase activity (data not shown).



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  		FIGURE 6. Enhanced PMN rolling (left) and increased PMN adhesion (right) onto SMase (1 U/ml) or cell membrane-permeable ceramide (1 µM)-stimulated HUVEC under flow conditions with a shear rate of 1 dyne/cm2. Stimuli were added to endothelial cells for 4 h in four-well plates containing rectangular Thermanox coverslips (for details, see Fig. 2Go). Data presented are mean ± SEM of three separate experiments. *Denotes experiments with endothelial cell monolayers pretreated with anti-E-selectin mAb.

 


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  		FIGURE 7. Enhanced levels of NF-{kappa}B-reporter gene activation in SMase or C8 and C16 ceramide-stimulated HUVEC: 1 U/ml SMase or 1 µM ceramide was added to endothelial cells that had been transiently transfected with a NF-{kappa}B-directed luciferase reporter plasmid (for details, see Materials and Methods). After 6 h, cells were washed and NF-{kappa}B activity was quantitated as chemoluminescence intensity of the reporter gene-luciferase assays/µg cell protein. Data presented are mean ± SEM of four separate experiments.

 
Based on the published synergism between LLO as a pore-forming toxin and exogenously added PlcA (15) with respect to phosphoinositol metabolism, we examined whether there was also a synergistic interaction between LLO and SMase or between LLO and C16 ceramides. As demonstrated in Figure 5Go, coaddition of purified LLO and SMase or C16 ceramide resulted in a merely additive (not synergistic) effect regarding E-selectin expression.

L. monocytogenes infection resulted in up-regulation of E-selectin mRNA in HUVEC

Having demonstrated that both PI- and PC-PLC induce ceramide generation, NF-{kappa}B activation, and enhanced E-selectin expression on the endothelial cell surface, we finally characterized the effects of listerial PLC on E-selectin mRNA transcription by Northern blot hybridization using a human E-selectin cDNA probe. A maximal E-selectin mRNA content in human endothelial cells was observed after stimulation with wild-type L. monocytogenes (EGD) for 4 h (Fig. 8Go), whereas HUVEC exposed to L. monocytogenes {Delta}plcA and {Delta}plcB displayed intermediate E-selectin mRNA levels. Cells treated with the nonpathogenic strain L. innocua for the same incubation period did not show any detectable amount of E-selectin mRNA. These molecular data indicate, very similar to the above reported functional data, that both listerial PLC are required for maximal E-selectin mRNA expression in HUVEC.



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  		FIGURE 8. L. monocytogenes induced increase of E-selectin mRNA in HUVEC after 4 h: Listeria were added in a bacteria to cell ratio of 10:1. Total endothelial RNA was isolated, and E-selectin mRNA levels were determined by Northern blot hybridization utilizing an E-selectin cDNA probe. E-selectin expression was normalized to the constitutively expressed message of GAPDH. Total amount is demonstrated in relation to IL-1ß-induced mRNA expression after 4 h. Data presented are mean ± SEM of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In a previous report, we demonstrated up-regulation of endothelial adhesion molecules and subsequent increased PMN adhesion to L. monocytogenes-exposed HUVEC. These effects were characterized by a biphasic kinetic with an early peak at 15 to 30 min, which was P-selectin mediated and strictly dependent on the generation of the exotoxin listeriolysin (14, 34, 52, 53, 54, 55). To better characterize the later E-selectin-dependent phase, we analyzed the role of listerial PLC in the study presented using L. monocytogenes EGD{Delta}plcA or EGD{Delta}plcB in-frame deletion mutants. We present direct evidence for an involvement of both listerial phospholipases PI-PLC, as well as PC-PLC in the up-regulation of endothelial E-selectin. Both PLC are expressed, while L. monocytogenes resides inside the phagocytic vacuole and contributes to an efficient lysis of the phagosome, depending on the cell type (12, 56). Listerial PI-PLC is secreted in an active state, whereas PC-PLC is secreted as an inactive proenzyme whose activation is mediated by a listerial metalloprotease and by specific host cell factors (57). PC-PLC is a broad spectrum phospholipase that generates DAG from phosphoglycerides and ceramide from sphingomyelin (12, 58). PI-PLC is a PI-specific phospholipase that also produces DAG, which in turn can activate acidic SMase. This enzyme is located primarily in the lysosomal compartment and generates ceramides from sphingomyelin (11, 59, 60). Interestingly, each PLC induced intermediate ceramide levels, suggesting that both PC- and PI-PLC are essential for a maximal effect. This is in line with the observation that high levels of intracellular ceramide were observed only after infection with the Listeria wild type expressing both functional phospholipases. Indeed, Smith et al. could not detect an increase in ceramide levels in J774 cells infected with mutants lacking both phospholipases (56).

Our results suggest that ceramides generated by listerial PLCs act as second messengers in the signaling pathway that via NF-{kappa}B leads to enhanced E-selectin expression. Corroborative results were obtained using a reporter gene assay for NF-{kappa}B activity and Northern blot analysis for E-selectin. Thus, high level persistent NF-{kappa}B activity or maximal E-selectin gene transcription following infection with L. monocytogenes wild type was observed, which was clearly diminished in strains infected with the {Delta}plcA and {Delta}plcB phospholipase deletion mutants. There is strong evidence for ceramides as mediators of NF-{kappa}B activation (31, 61, 62, 63, 64, 65). The mechanisms of ceramide-induced NF-{kappa}B activation are not known in detail, but may be related to stimulation of ceramide-activated protein kinase or I-{kappa}B kinase (58, 66, 67).

The potential role of ceramides as second messengers is supported by studies involving exogenously added ceramide as well as sphingomyelinase. In these experiments, ceramides induced NF-{kappa}B activation/translocation, including all subsequent reactions, indicating that this lipid mediator can substitute for endogenously generated ceramide. This confirms earlier work by Modur, who demonstrated ceramide- and SMase-related activation of human endothelial cells (68). Interestingly, in our study, maximal effects were seen in the presence of L. monocytogenes wild type, while intermediate levels of NF-{kappa}B activation were noted with PLC deletion mutants or membrane-permeable ceramides. These data suggest that L. monocytogenes-associated phospholipases may have better access to their substrate pools, thereby generating more DAG and in turn ceramide as second messenger for NF-{kappa}B activation than the extracellularly applied ceramides or sphingomyelinase. This notion is supported by the recently described synergistic interaction between LLO, a pore-forming toxin, and exogeneously added PI-PLC (11, 15). In the study, present coaddition of LLO and exogenous SMase or ceramide resulted in a merely additive E-selectin expression. This observation, however, is consistent because sphingomyelin is distributed predominantly in the outer leaflet of the plasma membranes, whereas PIs are located exclusively in the inner leaflet (69, 15). Therefore, it is likely that LLO will enhance the accessibility of exogenously added PI-PLC, but not of added sphingomyelinase to its respective substrate.

The level of sphingomyelinase stimulation and ceramide accumulation in L. monocytogenes-infected endothelial cells is reminiscent of TNF-signaling mechanisms, thus placing sphingomyelin turnover as a central early event not only in TNF-, but also in Listeria-induced signal transduction (58, 59).

L. monocytogenes as well as ceramides and sphingomyelinase not only induce NF-{kappa}B activation in HUVEC, but also E-selectin gene expression, a process normally not detected in resting endothelium, but strongly and rapidly induced by inflammatory stimuli (70). The inducibility of the E-selectin gene requires NF-{kappa}B binding to at least three of the four positive regulatory domains in the E-selectin promotor region, a condition that is also similar for the ICAM-1 and VCAM-1 promoters (24, 71).

Up-regulation of endothelial adhesion molecules due to L. monocytogenes infection resulted in an increased PMN adhesion to the endothelium (14). At 6 h postinfection, this effect is dominated by E-selectin, as evidenced by experiments using anti-E-selectin mAb (Fig. 2Go). From previous studies, it is known that the other major endothelial adhesion molecule is ICAM-1, which contributes about 40% to PMN adhesion after 6 h and 85% after 18 h of Listeria infection (14). In this process, PMN adhesion is also promoted by ß2 integrins, namely CD11b and most importantly CD18 (14). While a direct Listeria-related PMN activation may occur, an indirect PMN stimulation by Listeria-exposed endothelial cells is also possible because interaction of neutrophil structures with E-selectin will activate ß2 integrins on the neutrophil surface, which in turn bind with high affinity to ICAM-1 on the endothelium (72).

As outlined before, up-regulation of endothelial adhesion molecules due to L. monocytogenes infection resulted in an increased PMN adhesion to the endothelium. Infection of endothelial cells with L. monocytogenes EGD{Delta}plcA or EGD{Delta}plcB in-frame deletion mutants resulted in intermediate E-selectin levels and showed correspondingly intermediate degrees of PMN-endothelial cell interactions. The nonproducing PLC Listeria strain INN did not induce PMN adhesion. To extend our previous observation on PMN adhesion under stationary conditions, we also analyzed PMN-endothelial cell interaction at a well-defined shear rate of 1 dyne/cm2 using a parallel plate flow chamber. This approach not only allowed the study of PMN adhesion under more physiologic conditions, but even more importantly allowed the quantitation of PMN rolling.

In a recent study, Drevets presented data suggesting that LLO is the most important virulence factor with respect to E-selectin expression in endothelial cells and PMN adhesion, while EGD{Delta}DplcA and EGD{Delta}DplcB mutants induced only a minor defect (73). The reasons for these quantitatively, but not qualitatively, different results are not clear. It is likely that a combination of differences in cell passage (we have used only primary HUVEC cultures) as well as culture conditions are responsible for the discrepancies observed.

Enhanced PMN adhesion to L. monocytogenes-exposed endothelial cells may be seen as a protective reaction in light of several studies that documented the importance of PMN in controlling early events following bacterial entry (19, 20). Hence, in the early phase of a listerial infection, PMN substantially contribute to the nonspecific antilisterial resistance, as PMN depletion within 24 h of Listeria inoculation rendered mice extremely sensitive to this bacteria (21). L. monocytogenes-exposed endothelial cells could also support the adhesion of other circulating leukocytes such as lymphocytes or monocytes, which contribute to the specific host defense (17, 18, 72, 74, 75). Infected monocytes, however, may play a double-edged role during listerial infection because they may act as a Trojan horse to facilitate bacterial spread to endothelial cells (7).

The interpretation of our study is limited to cultured human large vessel endothelial cells. For an exact analysis of Listeria-related alterations of endothelial function in clinical disorders, it would be desirable to also study human microvascular endothelial cells of different organs. The isolation and culture of these cells in sufficient quantities, however, is difficult, and therefore the applicability of the data presented to other important anatomical sites such as the blood brain barrier must be verified in further studies.

In conclusion, up-regulation of endothelial adhesion molecules and subsequent increased PMN adhesion to L. monocytogenes-exposed HUVEC occur in a biphasic manner with an early peak at 15 to 30 min, which was P-selectin mediated and strictly dependent on the generation of listeriolysin (14). For the second phase, which phenotypically results in E-selectin up-regulation, we now present evidence for a requirement of both (PC-PLC, PI-PLC) listerial phospholipases. A role for ceramide as a second messenger in the expression of E-selectin as an inflammatory gene product via NF-{kappa}B activation was inferred from the ability of listerial phospholipases to generate ceramide in endothelial cells. PI- and PC-phospholipase effects could be mimicked by exogenous addition of ceramides or sphingomyelinase. Therefore, we suggest ceramides to be a missing link between Listeria infection and cellular inflammatory responses.


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  		Table I. Listeria strains used in the present study

 

   Acknowledgments
 
We thank Heike Geisel for excellent technical assistance. Parts of this work will be included in the Ph.D. thesis of N. Schwarzer and in the M.D. thesis of R. Nöst. We thank the staff of the Delivery Services of the hospitals Lich and Ehringshausen, as well as the Department of Gynecology, University of Giessen, for their help in collecting umbilical cords.


   Footnotes
 
1 This work was supported by the Deutsche Forschungsgemeinschaft (SFB 547/B2 to N.S.), by the European Union through the BIOMED 2 Project (CT950659 to T.C.), and by a Hermann and Lilly Schilling professorship to N. Suttorp. Back

2 Address correspondence and reprint requests to Dr. Norbert Suttorp, Department of Internal Medicine, Justus-Liebig University, Klinikstrasse 36, 35392 Giessen, Germany. E-mail address: Back

3 Abbreviations used in this paper: LLO, listeriolysin; DAG, diacylglycerol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPTLC, high performance thin layer chromatography; I-{kappa}B, inhibitory-{kappa}B; PC, phosphatidylcholine; PI, phosphatidylinositol; PLC, phospholipase C; PMN, polymorphonuclear leukocyte; SMase, sphingomyelinase. Back

Received for publication January 5, 1998. Accepted for publication May 8, 1998.


   References
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 Introduction
 Materials and Methods
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 Discussion
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