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Infect Immun, January 1998, p. 232-238, Vol. 66, No. 1
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Listeria monocytogenes Virulence Factors
That Stimulate Endothelial Cells
Douglas A.
Drevets*
Departments of Medicine, Microbiology, and
Immunology, R. C. Byrd Health Sciences Center of West Virginia
University, Morgantown, West Virginia 26506-9163
Received 22 September 1997/Accepted 30 October 1997
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ABSTRACT |
Listeria monocytogenes infection of endothelial cells
upregulates surface expression of adhesion molecules and stimulates neutrophil adhesion to infected cell monolayers. The experiments presented here tested the roles of specific bacterial virulence factors
as triggers for this inflammatory phenotype and function. Human
umbilical vein endothelial cell (HUVEC) monolayers were infected with
wild-type L. monocytogenes or L. monocytogenes
mutants; then surface expression of E-selectin and neutrophil adhesion were measured. The results showed that 
hly and
prfA mutants were the most crippled, requiring 100-fold
more mutant bacteria than wild-type bacteria for analogous stimulation.
By comparison, L. monocytogenes mutants with deletions of
actA, inlA, inlB,
inlAB, plcA, and plcB resembled
their parent strains, and a plcA plcB mutant
displayed decreased intracellular growth rate but only a minor decrease
in stimulation of E-selectin or neutrophil adhesion. Other experiments
showed that cytochalasin D-treated HUVEC monolayers bound bacteria, but
internalization and increased surface E-selectin and intercellular
adhesion molecule-1 expression were profoundly inhibited. However,
cytochalasin D had no effect on the HUVEC response to stimulation with
lipopolysaccharide or tumor necrosis factor alpha. These data suggest
that listeriolysin O production by infecting L. monocytogenes contributes to increased expression of surface
E-selectin and intercellular adhesion molecule-1, but neither it nor
intracellular replication are directly responsible for this event.
Nonetheless it is possible that listeriolysin O potentiates the
effect(s) of an other molecule(s) that directly triggers this response.
Additionally, cellular invasion by L. monocytogenes appears
to be critical for initiating the HUVEC response, potentially by
providing a signal which results in upregulation of the necessary
bacterial genes.
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INTRODUCTION |
Interactions between vascular
endothelial cells and pathogenic bacteria are common events in many
infectious diseases and often result in endothelial cell stimulation
and enhance leukocyte adhesion to infected cells (1). Such
interactions are comprised of two components: endothelial cell
stimulation by bacterial products and direct microbial infection of the
endothelial cell. Bacterial products can stimulate endothelial cells in
the absence of cellular infection, or the two processes can act in
concert when bacteria invade endothelial cells. Bacterial products that
stimulate cells without infection include the gram-negative cell wall
component, lipopolysaccharide (LPS), the phospholipase C and
perfringolysin O of Clostridium perfringens, and
listeriolysin O (LLO) and the phosphoinositol-specific phospholipase C
of Listeria monocytogenes (4, 16, 27, 33, 40,
41). As mentioned above, several different pathogenic bacteria
have been shown to bind or invade endothelial cells and to stimulate
them in the process (9, 14, 15, 38, 39, 44, 50). Products
that could stimulate cells during binding and invasion include the
outer membrane protein A of Borrelia burgdorferi,
peptidoglycan from Leptospira icterohemorhagiae, and certain
bacterial heat shock proteins (9, 21, 48, 49). Endothelial
cell stimulation by either of these processes has profound effects on
expression of endothelial cell adhesion molecules as well as cytokine
and chemokine production and ultimately plays a critical role in the
inflammatory process and host defenses.
L. monocytogenes is a pathogenic facultative intracellular
bacterium able to invade and replicate within mammalian cells (14, 18, 35). Several L. monocytogenes genes involved in
cellular invasion and intracellular parasitism have been identified and their function and products studied in detail (reviewed in reference 36). These include the pleiotropic regulator of the
virulence gene cluster prfA, members of the gene cluster
(plcA, hly, mpl, actA, and
plcB), and the inl family of invasion genes
(5, 19). Products with roles in phagosomal lysis and escape
into the cytoplasm include LLO, a pore-forming toxin encoded by
hly, and two C-type phospholipases, a
phosphoinositol-specific phospholipase C encoded by plcA and
a broad-spectrum phospholipase C encoded by plcB that cleaves phosphatidylcholine (PC-PLC) (18, 30, 35, 42). These
enzymes act with LLO to facilitate phagosomal escape and cell-to-cell
spread and also may be involved in stimulating intracellular signaling
in the eukaryotic target. The mpl gene encodes an enzyme that processes the immature form of PC-PLC into a mature form (10,
30, 32). Intracellular motility and subsequent cell-to-cell spread is dependent upon the ActA protein, which is essential for
polymerization of host F-actin (11, 26). The recently described inl family of genes encode internalin A and
internalin B proteins that are involved in binding and invasion of
eukaryotic cells (13, 14, 20, 29).
As a pathogenic microbe, L. monocytogenes is a well-known
cause of bacteremia and central nervous system infections of
immunocompromised humans and of domesticated animals (22,
31). The predilection of L. monocytogenes to invade
the central nervous system from the bloodstream led to the hypothesis
that infection of vascular endothelial cells was an important event in
the pathophysiology of listeriosis (2, 14, 37). Previous
work from this laboratory showed that L. monocytogenes can
infect and replicate within human umbilical vein endothelial cells
(HUVEC) (14). In response to infection, there was
upregulated surface expression of the adhesion molecules E-selectin,
intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion
molecule-1 (VCAM-1) and stimulation of neutrophil (polymorphonuclear
leukocyte [PMN]) adhesion to infected monolayers (15).
Induction of this inflammatory phenotype and function did not occur
following infection with the nonpathogenic Listeria innocua
and Listeria welshimeri or following incubation of infected
HUVEC with uninfected cells separated by a permeable membrane or with
sterile-filtered supernatants from infected cells. These results
suggested that specific bacterial virulence factors and direct contact
of L. monocytogenes with HUVEC were required to trigger the
HUVEC response. The experiments presented here studied the roles of
specific virulence factors as stimuli for endothelial cell adhesion
molecule expression and PMN adhesion.
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MATERIALS AND METHODS |
Antibodies.
Mouse monoclonal antibodies directed against
human ICAM-1 (CD54, immunoglobulin G1 [IgG1]) and E-selectin (CD62E,
IgG1) were obtained from Serotec USA (Washington, D.C.). Horseradish
peroxidase-conjugated goat anti-mouse IgG was obtained from Bio-Rad
(Hercules, Calif.).
Bacteria.
L. monocytogenes EGD, originally obtained
from G. B. Mackaness, was a gift from Priscilla Campbell (National
Jewish Center, Denver, Colo.). This strain has been passed through mice
to maintain virulence and is post-10th passage. Mutants of L. monocytogenes containing in-frame deletions of inlA
(BUG 947), inlB (BUG 1047), and inlAB (BUG 949)
and the wild-type parent strain, EGD (designated EGD-Pasteur), were
gifts from P. Cossart (13). The nonhemolytic, avirulent
L. monocytogenes strain 43250, which harbors a mutation in
the prfA gene, was purchased from the American Type Culture Collection (ATCC) (Rockville, Md.) (28, 34). L. monocytogenes containing in-frame deletion mutants of
hly (DP-L2161), plcA (DP-L1552), plcB
(DP-L1935), actA (DP-L1942), and plcA plcB
(DP-L1936) and the wild-type parent strain, 10403s, were gifts from D. Portnoy (30, 42). The L. monocytogenes prfA
mutant EGD prfA1 was the gift of T. Chakraborty
(5).
Cultures containing 109 bacteria/ml were stored at 
70°C
in tryptose phosphate broth or brain heart infusion (BH1) (Difco, Detroit, Mich.) containing 15% glycerol. For each experiment, a fresh
culture of bacteria was prepared by inoculating 10 µl of stock
culture into 4 ml of broth. The prfA1 mutant was cultured in
broth containing erythromycin (5 µg/ml) (5). Cultures were incubated overnight at 37°C with shaking or, in the cases of
DP-L2161, DP-L1552, DP-L1942, and DP-L1936, at 30°C without shaking.
Bacteria were washed by centrifugation at 12,000 × g
for 3 min followed by resuspension and vortex mixing in Hanks'
balanced salt solution (HBSS).
Cells.
Cultures of primary normal HUVEC were purchased from
the ATCC or from Clonetics (San Diego, Calif.). Cells from ATCC were cultured in F-12K medium (Gibco BRL, Grand Island, N.Y.) containing 10% fetal calf serum (Hyclone Laboratories, Logan, Utah), 50 µg of
endothelial cell growth supplement (Collaborative Biomedical Products,
Bedford, Mass.)/ml, 100 µg of heparin/ml, and 0.05 mM ascorbic acid
(Gibco). The lyophilized growth supplement contained streptomycin at a
final concentration of 19.5 µg/ml in the complete medium. No
additional antibiotics were used. ATCC cells were used between passages
15 and 20. They were maintained in 100-mm-diameter dishes, fed twice
weekly, and split 1:2 or 1:3 weekly into 24- or 96-well plates as
needed for experiments. Cells purchased from Clonetics were cultured in
Clonetics EGM-2 medium in the absence of antibiotics. These cells were
used between passages 3 and 10. Cells from the two sources responded
similarly to L. monocytogenes infection with respect to
upregulation of surface expression of adhesion molecules, stimulation
of PMN adhesion, and responses to L. monocytogenes mutants.
The only differences were that Clonetics cells had a longer useful life
span and grew to a higher cell density than cells from the ATCC.
HUVEC infection.
Intracellular growth of Listeria
in HUVEC was measured by a standard gentamicin protection assay as
described previously and adapted for use with HUVEC (14,
19). Medium (0.25 ml) containing 105 bacteria/ml was
centrifuged for 10 min at 1,000 × g onto HUVEC cultured in 24-well plates, followed by a 60-min incubation at 37°C
in a CO2 incubator. Next, the cells were washed, covered with medium containing gentamicin (10 µg/ml) to kill extracellular bacteria, and then incubated for an additional 60 min. This time point
is designated time zero. The cells were washed twice, and bacteria
remaining with the monolayer, presumably intracellular, were collected
by the addition of 1.0 ml of sterile distilled H2O
containing 0.5% saponin plus 3 mM EDTA. The cell lysates were collected with a Pasteur pipette, and CFU from triplicate wells were
quantified by serial dilution in sterile distilled H2O and plating on tryptic soy agar (Difco). The remaining wells were incubated
for another 4 h (time plus 4 h) at 37°C in medium
containing 10 µg of gentamicin/ml, and then Listeria CFU
were measured as described above. Intracellular growth of bacteria
during the 4-h incubation was calculated as follows: log10
CFU at time plus 4 h log10 CFU at time zero.
In some experiments cytochalasin D (Sigma Chemical Co., St. Louis, Mo.)
was used to inhibit bacterial invasion of HUVEC. Endothelial
cells in
duplicate plates were incubated with twofold dilutions
of cytochalasin
D for 60 min and then infected with 10
5 bacteria as
described above. Cells and bacteria were cultured
for 60 min at 37°C
and washed, and CFU bound to the cells were
quantified by serial
dilution and plating. Next, gentamicin (final
concentration, 50 µg/ml) was added to the remaining plate, and
it was incubated for
another 60 min. The wells were washed again,
and intracellular
bacterial CFU were quantified by serial dilution
and plating.
Measurement of adhesion molecule expression.
HUVEC cultured
in 96-well plates were left uninfected or were infected with serial
dilutions of wild-type L. monocytogenes or L. monocytogenes mutants. Bacterial inocula were measured by serial
dilution and plating. HUVEC were cultured for 60 min, washed, and
covered with medium containing gentamicin (10 µg/ml). The cells were
cultured for another 4 h and washed, and surface E-selectin or
ICAM-1 expression was measured by whole-cell enzyme-linked immunosorbent assay (ELISA) as previously described (15).
Relative E-selectin expression for each bacterial strain or mutant was expressed as a percentage of the E-selectin absorbance elicited by the
indicated wild-type strain at an inoculum of 104 CFU/well,
calculated as follows: (E-selectin absorbance after infection with the
test bacterium/E-selectin absorbance after infection with the wild type
at 104 CFU per well) × 100.
Broth from overnight cultures was tested for E-selectin-stimulating
activity. Bacterial cultures in BHI broth were centrifuged,
sterile
filtered, and added to HUVEC in twofold increments to
achieve final
dilutions of 1:10 to 1:400. The cells were incubated
for 6 h, and
surface E-selectin expression was measured as before.
A 10% dilution
of sterile-filtered control BHI broth had no detectable
effect on
cellular viability or baseline surface E-selectin expression
compared
with those of control cells.
PMN adhesion to HUVEC.
Human peripheral blood PMN were
obtained by venipuncture from healthy, human immunodeficiency
virus-negative donors. Whole blood was drawn into EDTA-containing
Vacutainer tubes (Becton Dickinson, Lincoln Park, N.J.), and PMNs were
separated by centrifugation through Neutrophil Isolation Medium
(Cardinal Associates, Santa Fe, N.Mex.) as previously described
(14). HUVEC in 96-well plates were infected and incubated
for 5 h. Immediately prior to incubation with HUVEC, PMNs
(107/ml) were loaded with 5 µM calcein AM (Molecular
Probes, Eugene, Oreg.) in phosphate-buffered saline at room temperature
for 30 min and then were washed twice with 10 ml of HBSS without
Mg2+ or Ca2+ to remove excess label
(8). Infected HUVEC were washed twice with HBSS containing
Mg2+ and Ca2+, and then 105 calcein
AM-loaded PMNs in 100 µl of RPMI 1640 (Gibco) plus 5% fetal calf
serum were added to the wells. Plates containing HUVEC and PMNs were
incubated for 30 min at 37°C, and then HUVEC were washed five times
with divalent cation-containing HBSS to remove unbound PMNs.
Fluorescence from adherent PMNs was measured in a CytoFluor 4000 (PerSeptives Biosystems, Framingham, Mass.) fluorescence microplate
reader with excitation and emission wavelengths of 530 and 485 nm,
respectively. PMN adhesion is expressed as mean relative fluorescence
units ± standard deviation (SD) from quadruplicate wells.
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RESULTS |
L. monocytogenes strains with mutations in specific
virulence genes were used to study the roles of L. monocytogenes virulence factors in HUVEC stimulation. First, we
measured the ability of various wild-type bacteria and mutants to
replicate within HUVEC. Figure 1 shows
that wild-type bacteria replicated rapidly within HUVEC. By comparison,
hly and prfA mutants did not replicate but did
avoid being killed by gentamicin, suggesting an intracellular location.
Bacteria lacking either inlA or inlB or both
replicated as readily as their parent strain once they had entered the
cells. The actA mutant grew slightly less rapidly than
its parent strain, 10403s, whereas the plcA plcB
mutant demonstrated a more significant, slower net replication in the
monolayer, presumably due to defects in escape from the primary vacuole
and cell-to-cell spread (30, 42).
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FIG. 1.
Growth of wild-type L. monocytogenes and
L. monocytogenes mutants within HUVEC. Confluent HUVEC
monolayers were infected with 104 CFU. Cells and bacteria
were cocultured for 60 min, washed, and then incubated for another 60 min in medium containing gentamicin to kill extracellular bacteria.
Triplicate wells from one plate were lysed, and intracellular bacterial
CFU at time zero were quantified by serial dilution and plating. The
second plate was incubated for another 4 h (time plus 4 h),
and CFU were quantified as before. Intracellular growth during the 4-h
interval was calculated as follows: log10 CFU at time plus
4 h log10 CFU at time zero. Results are shown as
the mean (±SEM) log10 growth from three experiments.
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Next, HUVEC were infected with increasing numbers of wild-type L. monocytogenes or mutant bacteria, and relative surface E-selectin expression was measured by whole-cell ELISA. In these experiments the
response to infection with mutant bacteria was represented as a
percentage of the absorbance at 490 nm following infection with
104 wild-type L. monocytogenes organisms.
Dose-response curves for each type of bacterium were generated that
plotted percent E-selectin signal as a function of the inoculum for
that bacterium that was used to infect HUVEC. Each of the wild-type
bacteria stimulated E-selectin expression, and there were only minor
differences between them when the inoculum and the ELISA signal were
compared. By contrast, nonhemolytic prfA-negative or
hly L. monocytogenes mutants were clearly separated from
hemolytic bacteria (Fig. 2) in that
infection with 104 CFU/well did not increase surface
E-selectin expression over that of uninfected cells. However, when the
inoculum of infecting nonhemolytic mutants was increased to
106 CFU/well, these bacteria stimulated relative E-selectin
expression comparably to wild-type bacteria at 104
CFU/well. By comparison, the dose-response curve generated from three
separate experiments with the mutant DP-L1942 (actA) was superimposable upon that of its parent strain, 10403s, showing that
intracellular motility is not an essential factor for HUVEC stimulation
(data not shown). Individual deletions of the plcA and
plcB genes had no significant effect on relative E-selectin expression, whereas the plcA plcB (DP-L1936) mutant
was slightly less active than the parent strain, 10403s, but only at an
inoculum of 3.5 log10 CFU/well (Fig.
3). Figure
4 shows that the inlA, inlB, and inlAB mutants stimulated
E-selectin expression similarly to their parent strain (EGD-Pasteur).
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FIG. 2.
Nonhemolytic L. monocytogenes mutants are
crippled in their ability to stimulate increased surface expression of
E-selectin on endothelial cells. HUVEC were infected with increasing
numbers of wild-type L. monocytogenes ( ), prfA
mutants 43250 ( ) or EGD prfA1 ( ), or a
hly mutant ( ). Bacterial inocula were measured by
serial dilution and plating. After a 4-h incubation, surface E-selectin
expression was quantified by whole-cell ELISA. Relative percent
E-selectin expression for each bacterium was calculated as follows:
absorbance at 490 nm following infection with test bacterium/absorbance
at 490 nm following infection with 104 L. monocytogenes EGD organisms. The mean (±SEM) relative percent
E-selectin expression from three to seven experiments is shown.
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FIG. 3.
Stimulation of HUVEC surface E-selectin expression by
L. monocytogenes plcA, plcB, and
plcA plcB mutants. HUVEC were infected with increasing
numbers of the parent wild-type L. monocytogenes 10403s
( ) or L. monocytogenes plcA (),
plcB (), and plcA plcB ( ) mutants.
Bacterial inocula were measured by serial dilution and plating. After a
4-h incubation, surface E-selectin expression was quantified by
whole-cell ELISA. Relative percent E-selectin expression for each
bacterium was calculated as follows: absorbance at 490 nm following
infection with test bacterium/absorbance at 490 nm following infection
with 104 L. monocytogenes 10403s organisms. The
mean (±SEM) relative percent E-selectin expression from three
experiments is shown.
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FIG. 4.
Stimulation of HUVEC E-selectin expression by L. monocytogenes inlA, inlB, and
inlAB mutants. HUVEC were infected with increasing
numbers of the parent wild-type L. monocytogenes EGD-Pasteur
( ) or L. monocytogenes inlA (), inlB
(), or inlAB () mutants. Bacterial inocula were
measured by serial dilution and plating. After a 4-h incubation,
surface E-selectin expression was quantified by whole-cell ELISA.
Relative percent E-selectin expression for each bacterium was
calculated as follows: absorbance at 490 nm following infection with
test bacterium/absorbance at 490 nm following infection with
104 L. monocytogenes EGD-Pasteur organisms. The
mean (±SEM) relative percent E-selectin expression from three
experiments is shown.
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Subsequent experiments tested the relative abilities of these L. monocytogenes mutants to stimulate PMN adhesion to infected monolayers. Figure 5 shows that PMN
adhesion in response to bacterial infection generally paralleled the
expression of E-selectin. The nonhemolytic prfA and
hly mutants stimulated very little PMN adhesion at an
inoculum of 105 CFU/well. However, increasing the inocula
of nonhemolytic prfA and hly mutants to
107 CFU/well stimulated PMN adhesion in excess of that
caused by hemolytic bacteria at a 250- to 500-fold lower inoculum. The
plcA plcB mutant was similar to wild-type EGD and only
slightly less effective than 10403s. Similarly, the inlA,
inlB, and inlAB mutants were essentially
comparable to their parent strain in stimulating PMN adhesion to
infected monolayers (data not shown). These data show that loss of LLO
is the single most important mutation that cripples the bacterium's
ability to stimulate E-selectin expression and PMN adhesion. However,
the fact that at high inocula of nonhemolytic mutants HUVEC could
stimulate these features indicates that LLO production is not an
absolute requirement for endothelial cell stimulation.
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FIG. 5.
PMN adhesion to endothelial cells infected by wild-type
L. monocytogenes or L. monocytogenes mutants.
HUVEC were infected with increasing numbers of wild-type L. monocytogenes or L. monocytogenes mutants and then
cultured for another 4 h. Bacterial inocula were measured by
serial dilution and plating. Infected monolayers were washed and then
incubated for 30 min with 105 calcein-AM-loaded PMNs/well.
The monolayers were washed again, and fluorescence emission was
measured with a fluorescence microplate reader. PMN adhesion is
represented as mean (±SD) relative fluorescence units from
quadruplicate groups of wells from one of two experiments with
identical results.
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Next we tested whether bacterial products secreted into culture medium
during growth could elicit E-selectin expression. Concentrations of BHI
broth of >1% from cultures of the hemolytic L. monocytogenes 10403s and DP-L1936 (plcA plcB)
produced rapid cellular ballooning and death. In contrast, broth from
cultures of prfA or hly L. monocytogenes
mutants or L. innocua caused no obvious cytotoxicity at
concentrations up to 10%. Surface E-selectin expression was only
minimally increased, and there were no significant differences between
hemolytic or hly L. monocytogenes and L. innocua at concentrations of 
1% or between hly
L. monocytogenes and L. innocua at
concentrations up to 10% (data not shown). These data suggest that
secreted LLO acting alone or in combination with other secreted
bacterial products can cause cellular necrosis. However, these
experiments did not detect differences between broth from L. monocytogenes and broth from the nonpathogenic L. innocua in stimulation of surface E-selectin despite radical
differences in their abilities to stimulate HUVEC during cellular
infection (15).
Previous work showed that addition of bacteriostatic antibiotics to
HUVEC 30 min after infection did not affect subsequent surface E-selectin expression, suggesting that the cellular response was triggered early in the host-pathogen interaction
(15). Thus, in a separate series of experiments we used
cytochalasin D to separate the roles of binding and invasion to
test whether binding of wild-type bacteria was sufficient to
initiate upregulation of E-selectin. Untreated (control) HUVEC bound
4.66% ± 1.44% of the inoculum, and 34.66% ± 1.86% of
bound bacteria invaded the cells (mean ± standard error of the
mean (SEM); n = 3). Figure 6 shows that binding to HUVEC was
increased modestly but internalization was reduced to as low as 1.2%
of that of the control. Together with this, cytochalasin D caused a
dose-dependent inhibition of L. monocytogenes-elicited
E-selectin expression (Fig. 7). However, cytochalasin D did not alter increased surface expression of E-selectin in response to Salmonella typhimurium LPS (100 ng/ml) or
recombinant human tumor necrosis factor alpha (data not shown), showing
that HUVEC were still capable of responding to other inflammatory
stimuli.
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FIG. 6.
Cytochalasin D inhibits invasion of endothelial cells by
L. monocytogenes. HUVEC were cultured with increasing
amounts of cytochalasin D for 60 min and then were infected with
104 L. monocytogenes EGD organisms. Cells and
bacteria were incubated for 60 min and washed, and then one set of
triplicate wells were lysed and bound CFU were quantified by serial
dilution and plating. The remaining cells were incubated with medium
containing gentamicin for 60 min, and then intracellular bacterial CFU
were quantified as before. Percent bacterial binding (open bars) was
calculated as follows: CFU bound/CFU added. Percent invasion (hatched
bars) was calculated as follows: intracellular bacterial CFU/CFU bound.
Results presented are the mean (±SEM) percent control (no cytochalasin
D added) binding and invasion from three experiments.
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FIG. 7.
Cytochalasin D inhibits surface E-selectin and ICAM-1
upregulation in response to L. monocytogenes infection but
not to stimulation with LPS. HUVEC were incubated with increasing
concentrations of cytochalasin D for 60 min; then the cells were
infected with 104 L. monocytogenes EGD organisms
(A) or stimulated with 100 ng of LPS/ml (B). After 5 h E-selectin
(open bars) and ICAM-1 (hatched bars) surface expressions were measured
by whole-cell ELISA. Results presented are the mean (±SD) absorbance
at 490 nm from quadruplicate wells from one of two experiments with
similar results.
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Alternative interpretations of these results are that the critical
factor for HUVEC stimulation was either the event of invasion or the
bacterial load of infected cells when adhesion molecule expression was
measured. If the second interpretation is correct, increasing the
inoculum of bacteria should produce an incremental increase in surface
E-selectin expression on cytochalasin D-treated HUVEC. However, Fig.
8 shows that an eightfold increase in
added CFU stimulated surface E-selectin expression on control cells but
not on cytochalasin D-treated cells. However, there was no difference
in surface E-selectin or ICAM-1 (data not shown) between cytochalasin
D-treated cells infected with 1× or 8× inocula of bacteria,
presumably because a threshold number of invading bacteria was not
achieved. Taken together these data show that L. monocytogenes invasion of HUVEC is dependent upon microfilament
function and suggest that binding of L. monocytogenes to
HUVEC is not sufficient to trigger increased surface expression of
E-selectin or ICAM-1. They also indicate a potential role for bacterial
invasion as a critical step in HUVEC stimulation.
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FIG. 8.
Increasing the bacterial inoculum does not increase
surface E-selectin on cytochalasin D-treated cells. HUVEC were
incubated with 250 µg of cytochalasin D/ml for 60 min (black bars) or
left untreated (hatched bars) prior to infection with twofold
increments of L. monocytogenes EGD. The mean (±SD)
inoculum (1×) was 1.72 × 103 ± 0.23 × 103 CFU of bacteria/well. Cells were cultured for 60 min,
gentamicin was added, and the cells were incubated for another 4 h. E-selectin expression was measured by whole-cell ELISA. Results
presented are the mean (±SEM) absorbance at 490 nm from three
experiments.
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DISCUSSION |
PMNs occupy a critical role in the early host response to
infection by L. monocytogenes as well as to infection by
other pathogenic bacteria (6, 7, 23). Recruitment of PMNs to
foci of infection and inflammation is a multifacted process, in which
increased surface expression of adhesion molecules on vascular
endothelial cells occupies a critical role (reviewed in reference
45). Recent data from this laboratory and others
have shown that endothelial cell infection with L. monocytogenes or exposure to L. monocytogenes products
can stimulate PMN adhesion to HUVEC monolayers and that it
increases surface expression of the adhesion molecules E- and P-selectin, ICAM-1, and VCAM-1 (15, 27). Data from
this laboratory showed that PMN adhesion to infected HUVEC monolayers
paralleled the surface expression of E-selectin: monoclonal antibodies
against E-selectin and CD18 blocked approximately 75% and 35% of PMN
adhesion to infected monolayers, respectively (15). The
study presented here used mutants of L. monocytogenes
to test the roles of its virulence factors as stimuli for upregulating
HUVEC surface expression of E-selectin and stimulating PMN adhesion to
infected monolayers.
The most obvious distinction among the various bacteria tested was that
hly and prfA mutants were profoundly crippled
in their stimulatory capacity. L. monocytogenes hly
mutants are deficient only in the production of LLO, whereas
prfA mutants are defective for transcription of all the
prfA-dependent virulence genes, including hly;
however there can be low-level prfA-independent transcription, resulting in contact hemolysis (12). As a
result, these mutants are severely crippled in their capacity for
intracellular parasitism and were not able to replicate within
HUVEC. Nonetheless, HUVEC could be stimulated when the inocula of these
nonhemolytic mutants were increased at least 100-fold over that used
for wild-type bacteria. This is a clear contrast to the nonhemolytic,
avirulent Listeria species, L. innocua and
L. welshimeri, which did not stimulate HUVEC even when
108 CFU/well was used for infection (15).
None of the other L. monocytogenes mutants demonstrated
such an extreme loss of stimulatory capacity as that caused by the single loss of LLO production. Deletions of plcA and
plcB had no detectable inhibitory effect, whereas deletion
of both plcA and plcB led to a decreased
intracellular growth rate but caused little change in HUVEC activation.
The enzymes encoded by these genes, a phosphoinositol-specific
phospholipase C and a broad-spectrum phospholipase C,
participate with LLO in lysis of primary phagosomes and during
cell-to-cell spread, and they can mediate LLO-independent phagosomal
lysis in some cells (30, 42). The phosphoinositol-specific phospholipase C has also been shown to participate with LLO in stimulation of HUVEC phosphoinositide hydrolysis and diacylglycerol formation (41). Data presented here indicate that
L. monocytogenes phospholipases are not directly
involved in stimulating E-selectin expression or PMN adhesion. The lack
of intracellular growth by nonhemolytic mutants suggests that
LLO-independent phagosomal lysis does not occur in HUVEC, or at least
is infrequent compared with Henle 407 cells (30). If such an
event did occur, it is possible that plcA hly or
plcB hly mutants would be more crippled than single
hly mutants for stimulating E-selectin and PMN adhesion, suggesting a role for early phagosomal lysis in triggering these events. Another set of mutants, inlA, inlB,
and inlAB, were comparable to the wild type in
intracellular growth, stimulation of E-selectin expression, and
stimulation of PMN adhesion; however, invasion rates for these mutants
were not measured. Nevertheless, these results suggest that under
serum-containing conditions, which do not favor internalin-mediated
binding (14), inlA and inlB are not
required for HUVEC stimulation.
The data show that LLO production contributes significantly to optimal
stimulation of HUVEC surface E-selectin expression and PMN adhesion.
However, the fact that nonhemolytic mutants could stimulate the cells
shows that LLO production is not absolutely required for this event.
Similarly, the response of HUVEC to nonhemolytic L. monocytogenes suggests that intracellular replication is also not
a required trigger. This conclusion is consistent with our previous
data showing that bacteriostatic antibiotics applied to HUVEC 30 min
after infection did not inhibit the cells' E-selectin response and
with immunofluorescence microscopy showing the presence of infected
cells which were E-selectin negative (15). The lack of a
direct effect of LLO was also suggested by the finding that broth from
L. monocytogenes cultures did not stimulate increased surface expression of E-selectin compared with broth from L. innocua at a similar dilution and that bacteria bound to
cytochalasin D-treated cells did not stimulate them. Recent experiments
by Krüll et al. directly tested the role of LLO as a stimulus for HUVEC adhesion molecule surface expression (27). They showed that purified LLO and genetically engineered L. innocua
that overexpress LLO stimulated surface expression of
P-selectin but not E-selectin, ICAM-1, or VCAM-1, suggesting the
presence of an LLO-independent trigger for expression of these
molecules. Thus, a mechanism(s) that accounts for L. monocytogenes stimulation of HUVEC E-selectin, ICAM-1, and VCAM-1
surface expression is not known. However, it is possible that LLO
potentiates the effects of the L. monocytogenes virulence factor or factors that trigger these responses directly.
Other experiments used cytochalasin D to study the roles of wild-type
L. monocytogenes binding and invasion as stimuli for adhesion molecule expression. Increasing concentrations of cytochalasin D slightly enhanced bacterial binding to HUVEC but inhibited
internalization of bound bacteria profoundly. Along with this,
increased surface expression of E-selectin and ICAM-1 was also
prevented, suggesting that bacterial invasion, or at least
rearrangement of the cytoskeleton, is a critical event. By comparison,
cytochalasin D did not alter the effects of stimulation with LPS and
tumor necrosis factor alpha showing that not all signaling pathways
which upregulate E-selectin and ICAM-1, for example through CD14, were
interrupted (33). It is possible that cytochalasin D
interrupts cell signaling pathways triggered by bacterial binding that
are important for invasion as well as adhesion molecule expression.
Binding of L. monocytogenes to epithelial cells has
been shown to stimulate mitogen-activated protein kinase through LLO
and phosphoinositide 3-kinase activity through internalin B-mediated
binding (24, 46, 47). However, these events were not
influenced by cytochalasin D treatment of the target cells. This could
indicate that if cytochalasin D blocks pathways in the HUVEC system
which ultimately affect surface adhesion molecule expression, they are
distal to activation of phosphoinositide 3-kinase.
Bacterial invasion of mammalian cells is a complex process which
involves active participation by the microbe and the eukaryotic target
(17, 20). Current models indicate that pathogenic bacteria use several different mechanisms to sense their microenvironment and
adjust expression of their virulence factors accordingly (reviewed in
reference 17). Thus, it is possible that blockade of
invasion could prevent elaboration of bacterial products critical for
stimulating adhesion molecule expression. Recent data show that
expression of L. monocytogenes hly, plcB,
actA, and plcA and production of their respective
proteins is upregulated by incubation under stress conditions, such as
a shift from rich to minimal medium, heat shock, or growth within
mammalian cells (3, 25, 43). In the experiments presented
here, these virulence factors were not directly responsible for HUVEC
stimulation. Nevertheless, regulation of L. monocytogenes virulence genes in response to microenvironmental cues supports the concept that invasion could trigger expression of the
gene or genes necessary for stimulating HUVEC.
| |
ACKNOWLEDGMENTS |
I am grateful for the excellent technical assistance of Sandra
Wilson and to T. Chakraborty, P. Cossart, and D. Portnoy for providing
L. monocytogenes mutants.
This work was supported by a WVU School of Medicine research grant.
| |
FOOTNOTES |
*
Mailing address: Section of Infectious Diseases, P.O.
Box 9163, R. C. Byrd Health Sciences Center, Morgantown, WV
26506-9163. Phone: (304) 293-3357. Fax: (304) 293-8677. E-mail:
ddrevets{at}wvu.edu.
Editor: J. G. Cannon
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Abstract
Introduction
