1001
Penicillin G-Induced Microbicidal Activity of Endothelial Cells Cultured on
Gelfoam Blocks
Bin Zhang, Michelle Centra, Guan Liang Cao,
Robert M. Taylor, Roman E. Ratych,
and Gerald M. Rosen
Department of Pharmaceutical Sciences, Program in Pharmacology and
Toxicology, University of Maryland School of Pharmacy; Research
Service, VA Medical Center, and Department of Pathology and
Dermatology, Johns Hopkins Medical Institutions, Baltimore, Maryland
Neutrophils, monocytes, and macrophages are a group of
leukocytes that are recognized to have a pivotal role in the
control of bacterial and fungal infections by phagocytosing and
killing these organisms. In contrast, endothelial cells are viewed
as passive members of the immune system, acting as barriers
to the influx of microbes and secreting factors, which bring
professional phagocytes to sites of bacterial invasion. In recent
years, however, a body of evidence has surfaced documenting
the ability of endothelial cells to phagocytose bacteria [1-7].
However, unlike neutrophils, endothelial cells in these models
did not kill bacteria [l, 3, 4, 6, 7]. Given the fact that endothelial
cells may share a common genetic linkage with macrophages
[8, 9], we questioned whether endothelial cells may, under
certain conditions, have the ability to kill bacteria.
The endothelial cell is unique in that its luminal surface is
in contact with blood, while the other side is intimately associated with a subendothelial matrix containing collagen and laminin [10, 11]. This specific environment has spawned considerable efforts to develop in vitro matrices attempting to mimic
the in vivo situation [10, 12]. Of particular interest is the study
of Thompson et al. [13], in which the site-directed angiogenic
properties of the three-dimensional matrix, Ge1foam, was well
documented. The ability of Gelfoam to induce in vivo neovessel formation [13] suggested that this substrate might be an
Received 30 October 1995; revised 19 June 1996.
Grant support: NIH (HL-33550); Department of Veterans Affairs Research
Service; Council for Tobacco Research-USA.
Reprints or correspondence: Dr. Gerald M. Rosen, Dept. of Pharmaceutical
Sciences, Program in Pharmacology and Toxicology, University of Maryland
School ofPhannacy, 20 N. Pine St., Baltimore, MD 21201.
The Journal ofInfectious Diseases 1996;174:1001-9
© 1996 by The University of Chicago. All rights reserved.
02-189!6745~$O
1.00
ideal matrix for in vitro culturing of endothelial cells. With
this in mind, we demonstrated that endothelial cells grown on
Gelfoam simulated the morphology ofthe endothelium in small
vessels and capillaries [14], which was not observed when
these cells were cultured on monolayer matrices [3, 4].
Staphylococcus aureus is a virulent gram-positive organism,
causing infections in a variety of tissues [2]. Considering the
reported phagocytosis of S. aureus by endothelial cells [1, 6,
7] and similarities of endothelial cells cultured on Ge1foam
with their in vivo morphology [13, 14], we explored the possibility that endothelial cells might play a more active role in
host immune defense than previously reported [1-7].
Material and Methods
Chemicals. Medium Ml99, Hanks' balanced salt solution
(HBSS), penicillin-streptomycin, Fungizone, L-glutamine, sodium
hydrogen carbonate, and glucose were purchased from Life Technologies GIBCD BRL (Gaithersburg, MD). Collagenase type II
was obtained from Worthington Biochemical (Freehold, NJ). Fetal
calf serum was purchased from Hyclone (Logan, UT). Gelfoam
was obtained from Upjohn (Kalamazoo, MI). Cytochalasins Band
o and lysostaphin were obtained from Sigma (St. Louis). Benzyl
C4 C]penicilIin potassium (54 rnCi/rnmol) was purchased from Amersham (Amersham, UK). All other reagents were purchased from
commercial sources.
S. aureus strain and culturing. S. aureus ATCC 292I3 and
penicillin-resistant S. aureus ATCC 13301 (American Type Culture Collection, Rockville, MD) colonies were collected from 5%
sheep's blood agar plates and grown to mid-logarithmic phase in
10 mL of trypticase soy broth at 37°C for 2 h. The organisms were
harvested by centrifugation at 1000 g for 8 min and washed twice
with O. 1% gelatin-PBS at pH 7.4. Bacterial concentrations were
estimated spectrophotometrically and confirmed by plating on agar
plates and counting colony-forming units. The susceptibility of S.
aureus ATCC 29213 to penicillin G was tested during a 2-h incuba-
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A body of evidence has surfaced documenting the ability of endothelial cells cultured on monolayers to phagocytose but not kill bacteria. Several years ago, a new three-dimensional endothelial
cell culturing model was developed, which simulated the morphology of the endothelium in small
vessels and capillaries. Given that endothelial cells may be derived from the same pluripotent stem
cells as macrophages, the question of whether endothelial cells might phagocytose and kill bacteria
was explored. Endothelial cells grown on Gelfoam blocks exhibited bactericidal activity towards
Staphylococcus aureus, reaching maximal killing of >90% after 2 h. Evidence documents the
involvementof bacterial adherence to the plasma membrane of the endothelial cell. This is followed
by phagocytosisof S. aureus, leading to intracellular killing.Penicillin G, included in the endothelial
cell growth medium, was found to be a critical factor in the bactericidal activity demonstrated by
Gelfoam blocks laden with endothelial cells.
1002
Zhang et al.
extracellular and adherent S. aureus. In each case, there were no
colony-forming units of S. aureus from the lysostaphin-containing
supernatant. The Gelfoam endothelial cell cultures were washed
three times with 4 mL of HBSS per washing. Sterile 0.1% gelatin/
water was added to lyse the endothelial cells. The resultant bacterial suspension was vortexed, serially diluted, and plated on tryptic
soy agar. The plates were incubated for 18 h in a dry incubator at
37°C, and the total number of viable S. aureus was quantitated by
counting the colony-forming units.
To evaluate the role of actin microfilaments in the killing of S.
aureus by endothelial cells cultured on Gelfoam blocks, cytochalasin B (10 j.tg/mL) was added to endothelial cells 1 h before
addition of bacteria and continued during the times of incubation.
At specific time points, sterile 0.1 % gelatin/water was added to
lyse Gelfoam-contained endothelial cells. The sample was diluted
and plated on agar as described above. For some experiments with
endothelial cells cultured on Gelfoam in M199 without antibiotics,
either cytochalasin B (10 j.tg/mL) or cytochalasin D (10 j.tg/mL)
was included in the medium, 1 h before the addition of S. aureus.
Benzyl [14C}penicillin potassium-tracing study. Cultured M199,
containing penicillin G (200 U/mL, 340 j.tM), was spiked with benzyl
C4 C]penicillin potassium (0.06 j.tCi/rnL, 1.2 j.tM), and 2 mL was
added to either Ge1foam laden with endothelial cells or Ge1foam
blocks alone. Every 3 days, this medium was removed and replaced
with fresh medium (2 rnL). On day 10, medium was removed, and
Gelfoam blocks were washed as described above and dissolved with
collagenase (0.63 mg/mL). Samples (0.5 mL) of medium from day
10 and of each of the four washings and of supernatant of dissolved
Gelfoam blocks were transferred to glass vials. Endothelialcells were
collected by filtering through a cell harvester (Brandel M-24; Life
Technologies) and washed three times with buffer before filters were
transferred to glass vials. The counting solution containing toluenePPO-POPOP was added to each vial and then placed in a liquid
scintillation counter (LS 6800; Beckman, Brea, CA). Radioactive
counts were expressed as disintegrations per minute.
Transmission electron microscopy. Transmission electron micrographs of Gelfoam-contained endothelial cell cultures with S.
aureus were made as follows. At l-h intervals, the reaction medium
was decanted and the Gelfoam blocks containing the endothelial
cells were immediately placed in cold 2.5% glutaraldehyde/a. 1 M
Sorenson's phosphate buffer, pH 7.4, fixed for 3 h at 4°C, rinsed,
and sectioned into 1-mm blocks [16]. Specimens were then postfixed in 1% osmic acid for 1.5 h, embedded in Spurr's plastic
embedding compound, sectioned at 1 j.tm thickness, stained with
toluidine blue, and examined by light microscopy to determine
representative sites for ultrastructural analysis. Tissue blocks were
then ultrathin-sectioned (60-90 nm) on an ultramicrotome (MT
5000; DuPont, Wilmington, DE), placed on 200-mesh copper
grids, stained with uranyl acetate and lead citrate, and examined
with a transmission electron microscope (Zeiss EM lOA) [17].
Statistical handling of data. Data for the Gelfoam and monolayer growth curves are the internal controls of bacterial growth
taken from the experiments described above. Gelfoam and monolayer control incubations of S. aureus and medium were done
simultaneously with their respective bacteria, medium, and endothelial cell culture incubations. Data are expressed as the mean ::!::
SD of three to five separate experiments with three to five replicate
incubations per time interval.
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tion using a dose range from a to 0.5 U/mL in antibiotic-free and
serum-free M199. A 90% inhibition of bacterial growth at 2 h was
observed at 0.2 U/mL penicillin G.
Isolation and culturing of bovine pulmonary artery endothelial
cells. Endothelial cells were isolated from bovine pulmonary arteries by brief collagenase digestion, grown in T-75 polystyrene
flasks in the presence of antibiotics (200 U/mL penicillin G and
200 j.tg/mL streptomycin), and subcultured as described by Centra
et al. [14]. All cultures used in this study were second to fifth
passage. Confirmation of endothelial cell identity was obtained by
staining for factor VIn antigen, as described by Jaffe et al. [15],
and visual inspection of their typical morphology [3, 4]. For endothelial cells grown on monolayers, cell cultures (5 X 105 cells/
mL in 2 mL of M199 containing penicillin G, 200 U/mL, and
streptomycin, 200 j.tg/mL) were seeded onto 3.8-cm 2 petri dishes
until confluence was reached, usually within 3-4 days [14].
For endothelial cells grown on Gelfoam blocks, cells from T-75
polystyrene flasks (5 X 105 cells/rnL) were added to a precut 24 X
8 X 2 rnm autoclaved block of Gelfoam, previously placed in a 17
X 100 rnm polypropylene tube to a total volume of 2 rnL of M199.
Four separate culturing conditions were then used: M199 containing
penicillin G, 200 U/rnL, and streptomycin, 200 j.tg/mL; Ml99 without
antibiotics;M 199containing only penicillin G (200 U/mL); and M199
containing only streptomycin (200 j.tg/rnL). For each of these conditions, the endothelial cells were allowed to grow undisturbed for
several days, and thereafter appropriate media for each experimental
design were changed frequently for a total culturing period of 10 days
as described [14]. Cell-free controls were subjected to the identical
culturing conditions used with their respective Gelfoam or monolayer
cell cultures. At various time points throughout the different experiments, the viability of endothelial cells was closely monitored, using
trypan blue dye exclusion. For up to 2 h of incubation, no endothelial
cell death was observed.
Microbicidal assay. Before the introduction of bacteria to endothelial cells, these eukaryotic cells, grown either on Gelfoam
blocks (1 X 106 cells/block) or in monolayer cultures (5 X 105
cells/well), were extensively washed four times with 4 mL/washing
of HBSS to remove antibiotics. At this point, the endothelial cell
cultures were placed in 2 mL of an antibiotic-free and serum-free
modified M199 buffer, containing sodium hydrogen carbonate (24
roM) and HEPES buffer (20 mM) to maintain pH 7.4. Gelfoam
blocks, in the absence of endothelial cells, washed as for the Gelfoam laden with endothelial cells as described above, were used
as controls. Bacteria were then added to endothelial cell cultures
in a 10:1 endothelial cell-to-bacterium ratio. This dual cell system
was incubated at 37°C, with a gas mixture of 95% air/5% CO2 ,
At defined time intervals, 0.1% gelatin-Hjf) was added to the
cultures to lyse endothelial cells. The resultant bacterial suspension
was vortexed, serially diluted, and plated on tryptic soy agar. The
plates were incubated for 18 h in a dry incubator at 37°C, and the
total number of viable S. aureus was quantitated by counting the
colony-forming units.
To determine the number of viable internalized bacteria, lysostaphin (0.5 j.tg/mL) was added to the samples at various time
points after endothelial cell/bacteria incubation to kill extracellular
and adherent S. aureus, as described by Hamill et al. [1]. Lysostaphin-containing samples were incubated at room temperature
for 5 min. A portion ofthe medium from each sample was collected
and plated to determine the efficiency of lysostaphin killing of
JID 1996; 174 (November)
Microbicidal Activity of Endothelial Cells
JID 1996; 174 (November)
150
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Figure 1. No. of viable S. aureus after incubationwith bovine pulmonary artery endothelial cells cultured on Gelfoam blocks and
treated with antibiotic-containing medium 199 for 10 days. Bars reflect mean z SD of6 separateexperimentswith 3-5 replicateincubations per time interval. Ratio of endothelial cells to S. aureus in
endothelialcellcultureswas 10:I. G = Gelfoam blockswithoutendothelial cells; C = Gelfoam blocks laden with endothelial cells.
units of group B streptococci versus vortex treatment of these
Iysates.
Although our findings suggest that endothelial cells kill S.
aureus, experiments were designed to demonstrate that the
observed microbicidal activity was not due to antibiotics present in the growth medium. After M199 containing penicillin
G and streptomycin was removed from Gelfoam containing
endothelial cells, M 199 without antibiotics (4 mL) was added
to this matrix. The matrix was then gently shaken. After 30
min, the medium was removed and the washing procedure
was repeated three more times with 4 mL of M199 without
antibiotics. The efficiency of the washing procedure was documented by observing increased S. aureus growth in each sequential washing, achieving control bacterial growth by the
fourth wash (data not shown). This finding demonstrates that
the final medium wash no longer possessed the ability to kill
S. aureus.
Next, we explored the possibility that antibiotics were
trapped within this Gelfoam matrix or associated with the endo-
400
•
Results
When endothelial cells cultured on Gelfoam blocks for 10
days were incubated with nonopsonized S. aureus at an initial
ratio of 10: I (endothelial cells to bacteria), we observed bacterial killing compared with Gelfoam alone in the absence of
endothelial cells (figure 1). This is in line with similar microbicidal activity exhibited by neutrophils [18]. With time, this
microbicidal activity increased, such that >90% bacterial killing was noted at 2 h (figure I) . In concurrence with earlier
studies [1, 3, 4, 7], endothelial cells cultured on monolayers
did not exhibit bacterial killing, even after 4 h of incubation
(figure 2). In fact, a significantly faster S. aureus growth rate
was observed with the Gelfoam controls at 1-4 h compared
with growth curves for the monolayer controls in the absence
of endothelial cells in each model (data not shown). This increased rate of bacterial growth on Gelfoam obviates the
fact that this matrix may be contributing to aggregation (pseudokilling).
To ensure that bacterial counts after the gel/water dilution
represented single bacteria rather than clumps, fractions of the
gel/water dilution were sonicated for 20 s on ice and plated on
tryptic soy agar. We found no significant differences in counts
between the vortex and sonication treatments at 3 h of incuba tion (2.28 X 104 S. aureus cfu for vortexing vs. 1.73 X 104 S.
aureus cfu for sonication, n = 10). This demonstrates that
less traumatic vortexing alone is sufficient to prevent bacterial
clumping. This is consistent with the findings of Gibson et al.
[19], who showed that sonication of endothelial cell Iysates
did not increase the number of intracellular colony-forming
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Figure 2. No. of viable S. aureus after incubation with bovine pulmonary artery endothelial cells cultured on monolayers and treated
with antibiotic-containing medium 199 for 3 days. Bars reflect mean
:!: SD of 4 separate experimentswith 5 replicate incubationsper time
interval. Ratio of endothelial cells to S. aureus in endothelial cell
cultures was 10: I. M = monolayer without endothelial cells; Me =
monolayer with endothelial cells.
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Figure 3. No. of viable S. aureus after incubation with bovine pulmonary artery endothelial cells cultured on Gelfoam blocks and
treated with antibiotic-containing medium 199 for 10 days. G = Gelfoam blocks without endothelial cells; CS = endothelial cell suspension, initially cultured on Gelfoam blocks, then removed before introducing bacteria. Bar G is taken from figure 1. Other bars reflect mean
::t SD of 2 separate experiments with 3 replicate incubations per time
interval. Ratio of endothelial cells to S. aureus in endothelial cell
cultures was 10: I .
thelial cells. We designed studies to explore these possibilities.
In a first series of experiments, endothelial cells were separated
from Gelfoam and each component tested to determine their
role in the killing process. Extensively washed endothelial cell Gelfoam blocks were treated with collagenase (125 tlg /mL)
for 10 min at 37°C and centrifuged to separate the cells from
the dissolved matrix. Bacteria were then added to the dissolved
matrix and incubated for 2 h. Bacterial growth was not inhib ited. The remaining isolated endothelial cells were son icated
(model W-IO, power 3.5 and tune 4; Heat Systems Ultrasonics,
Plainview, NY) for 30 s on ice and incubated for 2 h with
bacteria. This cell lysate did not exhibit any bactericidal activity . To obviate the faint possibility that sonication destroyed
the activity of the antibiotics, independent experiments were
done in which penicillin G (0.1 U/mL) and streptomycin (0.1
tlg/mL) were sonicated for a comparable time and subsequently
incubated with S. aureus. There was no decrease in antibiotic
activity. To determine whether the endothelial cell lysate could
inactivate penicillin G, this ant ibiotic (0.5 U/mL) was added
to the endothelial cell lysate and incubated with S. aureus. The
degree of bacterial killing in these experiments was unchanged
compared with controls with penicillin G (0.5 U/mL) alone. In
contrast, intact endothelial cells, removed from their Gelfoam
matrix by coll agenase treatment, still retained their bactericidal
activity (figure 3).
A last series of experiments was designed to eliminate the
possibility that antibiotics were responsible for the observed
bacterial killing. This required the culturing of endothelial cells
on Gelfoam for 10 days in the absence of penicillin G and
streptomycin. To our surprise, the endothelial cells on Gelfoam
were no longer able to kill S. aureus (figure 4). This unexpected
finding suggested that the antibiotics may be playing a role in
the killing process that is not directly related to their specific
and well-described pharmacologic activity. This implies that
endothelial cells grown on this matrix were activated by treatment with antibiotics, leading to the observed microbicidal
activity.
Interestingly, we found that the bacterial growth in the presence of Gelfoam treated with 1\1 199 without antibiotics was
greater than when this matrix was placed in this medium with
the antibiotics. One possible explanation for this phenomenon
comes from the work of van den Broek et al. [20]. These
authors reported that very low concentrations of penicillin G
(0.0085-0.017 U/mL), levels below its bactericidal activity,
retarded growth of S. aureu s. In fact , penicillin G, at 0.01 UI
mL, inhibited S. aureus growth in a manner parallel to that
observed when this bacterium was added to Gelfoam blocks
pretreated with M 199 in the presence of antibiotics for 10 days,
followed by the washing treatments described above (data not
shown). Therefore, it is conceivable that our procedures did
not remove all of the antibiotic but merely lowered the concentration of penicillin G to levels that slowed the growth rate of
this bacterium. These experiments point to the fact that the
microbicidal activity exhibited by endothelial cells cultured on
Gelfoam is not due to contamination by the antibiotics added
to the growth medium, although residual levels retard growth
of S. aureus, but rather, bacterial killing requires viable, function ing endothelial cells .
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Figure 4. No. of viable S. aureus after incubation with bovine pulmonary artery endothelial cells cultured on Gelfoam blocks and
treated with antibiotic-containing or antibiotic-free (-) medium 199
for 10 days. G = Gelfoam blocks without endothelial cells; C =
Gclfoam blocks laden with endothelial cells. Bars G and C were
taken from figure I. Other bars reflect mean ::t SD of 3-6 separate
experiments with 3-5 replicate incubations per time interval. Ratio
of endothelial cells to S. aureus in endothelial cell cultures was 10: I.
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JID 1996; 174 (November)
Microbicidal Activity of Endothelial Cells
lID 1996 ; 174 (November)
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Figure 6. No. of viable penicillin-resistant S. aur eus after incubation with bovine pulmonary artery endoth elial cells cultured on Gelfoam blocks and treated with antibiotic-containing medium 199 for
10 days. G = Gelfoam blocks without endothelial cells ; C = Gelfoam
blocks laden with endothelial cells . Bars reflect mean :!: SD of 4
separate experiments with 3 replicate incubations per time interval.
Ratio of endothelial cell cultures to S. aureus in endoth elial cell
cultures was 10: I .
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time (hours)
No, of viable S aureus after incubation with bovine pulmonary artery endothelial cells cultured on Gelfoam blocks (C) and
treated with antibiotic-containing (P = penicillin, 200 U/mL ; S =
streptomycin, 200 ;.rg/mL) or antibiotic-free (-) medium 199 for 10
days. Bars reflect mean :!: SD of 3 separate experiments with 3
replicate incubations per time interval. Ratio of endothelial cells to
S aureus in endothelial cell cultures was 10:1.
Figure 5.
Since penicillin G and strep tom ycin were in M 199, we undertook experim ents to determine which of these antibiotics
was required for endotheli al cell - induced m icrobicidal activity. Endothelia l cells on Gelfoam were incubated with M199
in th e pre sence of either penicillin G (200 U/mL) or streptomycin (20 0 Ilg/mL). As shown in figure 5, only those endothelial
cell s on Gelfoam cultured in M 199 containing penicillin G
demonstrated bacterial killing. Therefore, penicillin G , and not
streptomycin, appears to be the putative antibiotic responsible
for the observed microb icidal act ivity.
If re sidual penicillin G we re solely responsible for the observed bact erial killing , as shown in figure I , then substituting
a penicillin-resistant strain of S. aureus should not alter the
bacterial growth curve in the pr esence of endothelial cells cultured on Gelfoam blocks. A s shown in figure 6 , after 2 h the
end othelial cell kill rate was > 90% compared with controls .
Using radiolabeled penicillin G , we discovered that even
after fou r washings, a sm all but sign ificant concentration of
radiolabeled material was adherent to Gelfoam blocks, independent of the presence of endothelial cells (table 1). However,
after separating endothelial cell s from the matrix, we were
unable to detect cell-associated radioactivity (table I). These
data further confirm tha t the penicillin G-dependent killing of
S. aureus was not the result of either local accumulation or
endothelial cell uptake of penicillin G.
Since we have now observed a poss ibl e inductive event,
it is important to focus on the endothelial cell to understand
the killing process. Sequential ultrastructure examination of
the interaction ofS. aureus with Gelfoam-contained endoth e lia l cell s is shown in figure 7. Adherence of the bacterium
Localizat ion of C4 C]penicillin G in Gelfoam blocks and
Gelfoam laden with endoth elial cells.
Table I.
Gelfoam control
Medium
Wash I
Wash 2
Wash 3
Wash 4
Supernate
Filter
299,728 :!:: 84 36
Gelfoam laden with
endothelial cells
300,481 :!:: 80 52
44,202 :!:: 10,41 5
44,623 :!:: 12,498
866 9 :!:: 32 02
9221 :!:: 24 73
205 0 :!:: 455
225 2 :!:: 685
784 :!:: 159
984 :!:: 327
4477 :!:: 728
4021 :!:: 754
27 :!:: 4
41 :!:: 10
NOTE. Gelfoam blocks and Gelfoam laden with endothelial cells were
cultured in 2 mL of medium 199 containing penicillin G (200 UlmL) and
[1 4C]penicillin G (0.06 pCi/m L ) for 10 days. Every 3 days medium was
changed with medium 199 of same composition. On day 10, Gelfoarn blocks,
with and without endothelial cells, were washed and radioactivity from 0 ,5
mL of each sample and washes along with dissolved Gelfoam matrix and
endothelial cells (collected on filters) were counted. Data are disintegrations
per min, and each value represents mean :': SO of 20 different determinations.
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degree of intracellul ar killing. Based on these data, > 90% of
the tota l bacterial killi ng takes place with in the endothelial cell .
We cannot overlook the po ssibility that the observe d bacterial killing takes pla ce within a phagocytic vacuole. To explore
the feasibility of this hypot hesis, we incubated Gelfoam-co ntained endo thelial cell s, cultured in M 199 withou t antibiotic for
10 days, with S. aureus in the pres ence of either cytoc halasi n
B (10 ttg /mL) or cytochalasin D ( 10 Jig/mL), folIowed by
lysos taphin (0.5 ttg/mL ) treatm en t. As shown in figure 9, both
cytochalasins demonstrated significant inhib ition of S. aureus
internaliza tion, with cytochal asin D exhibiting considerably
more pharmacologic activity.
Becau se of these findin gs and the fact that cytoch alasin B
interfere s with microfilament functi on in neutrophils [22, 23],
we investigated the po ssibility that cytochalasin B may inhibi t
the kilIing of S. aureus by our Ge lfoam-contained endothelial
celI model. This was not observed . Cyt ochalasin B was found
to only minimalIy, although nonsignifi cantly, affect the killing
of S. aur eus by these euk aryot ic cells (figure 10).
Discussion
This paper describes the ability of endothelial cells, cultured
on the three-dimensional mat rix , Ge lfoam, to kill S. aureus.
This bactericidal act ivity is dependent on a series of events.
As show n in figure 7, the adhere nce of S. aureus to the endothelial cells is the initial step . Then, phagocytosis interna lizes the
bacterium , which even tua lly results in the killing of S. aureus.
•
Figure 7. Transmission electron micrographs showing sequential phagocyt osis of S. aureus by endothelial cells cultured on Gelfoam. Lett,
Adherence of bacteria to endothelial cell plasma membrane: original magnification, X20,OOO. Middle, Pseudopodia! engulfment of bacteria by
endothelial cells attached to Gelfoam: original magnification, X 15,750. Right, Endothelial cell phagosomal vacuole containing bacteria; original
magnification, x 20,OOO.
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to th e endo thel ial ce ll is th e initial step of th e process (figure
7, le ft ). T his is foll o wed by the ex tension of pseudop odia,
whic h beg in to eng ulf th e bac te ria (figure 7, middle), si mi lar
to wha t is obse rv ed wi th phagocytic cells [2 1]. At 3 h of
inc u ba tio n, a representat ive electron ph ot o m icro gr ap h shows
multi ple bacter ia within an apparent ph agocyt ic vac uole
(figure 7, rig ht ). Alt houg h th e ult ras truc ture of the bacteria
wit hi n this vacuole doe s not a llow us to co ncl ude th at S.
aureus was killed, dat a from figure I un equi vocally de monstra te bacteri cid al activi ty by endo the lia l cells cultured on
Ge lfoa m blo cks . These micrographs suggest, howe ver , that
killing may be dep endent on the ph agocyto sis of S. a ureus.
T he refo re, biochemical studies we re desi gned to dis criminate betw een sites of bacterial killing.
Th e localization for endotheli al cell -mediated bactericidal
activity was investigated usin g endothe lial cells cultured on
Gelfoam blocks in the presence and absence of antibiotics. For
these experiments, lysostaphin (0 .5 tt g/mL), which ha s been
shown not to enter endothelial cells [I] , was added to quantify
the number of interna lized S. aureus. W ith the antibiotic-free
med ium - cultured endothelial ce lls, lysostaphin trea tment demonstrated an increased intracellular bact erial grow th with time
of incub ation (figure 8). ln co ntrast to this finding , endothelial
cells grown in the presen ce of antibiotics d id not perm it intracell ular bacterial grow th (figure 8) . Assuming that the pathway
for internalization is the same , independen t of whet her medium
co nta ins antibio tics, differences in viable S. aureus colonyformin g units in the two cell models a llow us to determine the
lID 1996; 174 (November)
Microbicidal Activity of Endothelial Cells
lID 1996; 174 (November)
thelial cells. A similar effect has been observed in the uptake of
20
A
Nocardia asteroides by a pulmonary artery endothelial cell line
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B
[25].
There are several possible explanations for why cytochal asin
B did not inhibit the ability of endothelial cells to kill S. aureus.
First, endotheli al cells are sensitive to cytochalasin B only
when these cells are cultured on monolayers. Second, alterna tive, non-cytochalasin B- sensitive phagocytic pathways are
responsible for the observed killing . Third, although cytochalasin B prevents phagosomal closure, microbic idal activity appears to takes place within an open phago cytic vacuole.
With regard to the initial hypothesis, a number of studies
have suggested that microv ascular endothelial cells in vivo are
morphologically different in their cytoskel etal structure than
when these cells are cultured on monolayer [26, 27]. Actin
microfilam ents, which are of importance in regulating structural
integrity of the endothelium, are located at central and periph-
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Figure 8. No . of viable S. aureus after incubation with bovine pulmonary artery endothel ial cells cultured on Ge1foam blocks (C) and
treated with antibiotic-free medium 199 (- ; A) or with antibioticcontaining medium 199 (B) for 10 days. At defined times, lysostaphin
(LS, 0.5 ,ug/mL) was added and intracellular viabl e bact erial counts
were determined. Bar s reflect mean ::+:: SD of 3 separate experiments
with 3 replicat e incub ations per time interv al. Ratio of endothelial
cell cultures to S. aureus in endothelial cell cultures was 10: 1.
Assuming that the uptake of bacteria by endothelial cells is
independent of whether these eukaryotic cells are cultured in
medium containing antibiotics, these data clearly demonstrate
that actin microfilament reorganization is an important component in the endothelial cell phagocytosis of S. aureus.
Cytochalasins have been shown to prevent phagocyte-mediated
uptake of particles, including microbes, when endothelial cells
are grown in monolayer cultures. For example, Gibson et al. [19]
have demonstrateda statistically significant cytochalasin D doseresponse inhibition of group B streptococcus invasion of human
endothelial cells. Likewise, Vora and Karasek [24] have shown
that cytochalasin B totally prevented the uptake of fluorescence
paramagnetic latex beads by human dermal microvascular endo-
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time (hours)
Figure 9. No. of viable S. aureus after incubation with bovine pulmonary art ery endothelial cell s cultured on Gelfoam blocks (C) in
antibiotic -free medium 199 for 10 days and pretreated with cyto chalasin B (CB) or cytochalasin D (CD) at 10 j.lg/mL for 1 h befo re
inoculation of bacteria or left untreated. At defined times , lysostaphin
(LS, 0.5 ,ug/mL) was added and intracellular viable bacterial counts
were determined. Bar C + LS is taken from figure 8A. Other bars
reflect me an::+:: SD of2 separate experiments with 3 replicate incubations per time interval. Ratio of endoth elial cells to S. aureus in
endothelial cell cultures was 10: 1.
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Zhang ct a!.
1008
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ill
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(hours)
Figure to. No. of viable S. aureus after incubation with bovine
pulmonary artery endothelial cells cultured on Gelfoam blocks in
antibiotic-containing medium 199 for 10 days and pretreated with
cytochalasin B (CB, 10 J.1.g/mL) for I h before inoculation of bacteria
or left untreated. G = Gelfoam blocks without endothelial cells; C
= Gclfoam blocks laden with endothelial cells. Bars G and C were
taken from figure I . Bar C+CB reflects mean ± SD of 3 separate
experiments with 3 replicate incubations per time interval. Ratio of
endothelial cells to S. aureu s in endothelial cell cultures was 10: I.
cral sites within the cell [26-28]. Peripheral microfilamcnts,
known as the dense peripheral band, participate in cell adhesion, whereas central microfilament bundles are important in
extracellular matrix adhesion. In vivo, however, the dense peripheral band is not as well developed as observed in vitro
[28]. Current knowledge suggests that the dense peripheral
bands are sensitive to the effects of cytochalasin B, whereas
central microfilament bundles appear to be nonresponsive towards cytochalasins [29].
The second hypothesis suggests that the observed bacterial
killing occurs by a non -cytochalasin B -sensitive mechanism.
Our data do not, however, discriminate between opposing
routes. The concept of an alternative bacterial phagocytic pathway has been reported by Oelschlaeger et a1. [30] , who demonstrated that cytochalasin-mediated effects were both bacterial
strain-dependent and target cell-dependent for Campylobact er jeju ni and Citroba cterfreundii. Similarly, Beaman and
Beaman [25], using bovine pulmonary artery endothelial cells,
showed that cytochalasin B increased the percentage of cellassociated N. asteroides. In fact , these authors termed this phe nomenon a " phagocytosis-inde p endent invasion factor. " In
addition, macrophages, to which endothelial cells are gcnetically related, are insensitive to the actions of cytochalasin B,
even though these cells continue to phagocytose microbes [31].
Our data support the third hypothesis, in that killing of S.
aureus is almost exclusively intracellular, within a vacuole.
Ev idence in support of this comes from the finding that in non penicillin G -treated endothelial cells, cytochalasins B and D,
by pr eventing elosure of the phagosome, allowed lysostaphin
to enter this cell and kill S. aureus. Since cytochalasin B did
not inhibit bacterial killing, an enclosed phagosomal vacuole
in the endothelial cells, unlike neutrophils, is not essential to
the bactericidal activity exhibited by these eukaryotie cells .
Penicillin G appears to pla y a dual role in the killing of S.
aureus. First, we believe that at residual levels, penicillin G retards
the growth of S. aureus. Support for this theory comes from our
findings that the Gelfoam matrix, in either the presence or absence
of endothelial cells , bound radiolabeled penicillin , even though
no penicill in was detected associated with these eukaryotic cells
(table I) . Since tracing studies cannot distinguish between the
presence of penicillin or its inactive hydrolyzed products, there
must undoubtedly still be some penicillin G remaining to retard
the growth of S. aureus. Additionally, Root et a!. [32] demonstrated that sub-MIC doses of penicillin G impede staphylococcal
growth by inhibiting crosswall form ation and causing the thickening of cell wall without subsequent separation. This pharmacologic activity enhances the susceptibility of S. aureus to be killed
by phagocytic cells. Second, our findings suggest that penicillin
G activates endothelial cells cultured on Gelfoam blocks to exhibit
bactericidal activity, rather than mediating its microbicidal properties through the direct killing of S. aureus. Evidence in support
of this hypothe sis comes from our data demonstrating a lack of
either the internalization of penicillin G or the binding of this
antibiotic to the surface of the endothelial cell (table I). Finally,
penicillin-resistant S. aureus is equally killed by endothelial cells
(figure 6).
Classically, endothelial cells are thought to be passive participants in host immune response; however, our findings suggest
a more active role for these cells , one in which they can be
activated to phagocytose and kill bacteria. In some situations,
this may provide an additional1ine of defense aga inst microbial
penetration. Our results clearly demonstrate that endothelial
cells cultured on Gelfoam blocks, which morphologically
mimic these cells in the microvasculature, are activated by
penicillin G to phagocytose and kill S. aureus. Future studies
are aimed at elucidating the mechanism by which Gelfoamcontained endothelial cells kill S. aureus.
Acknowledgments
We appreciate the helpful suggestions and numerous discussions
with Sovitj Pou, the continued technical assistance by Michael
Gentry (University of Maryland School of Pharmacy) , and the
photographic expertise of Milton D. Tudahl, Sr. (Director of Photographic Service s, Franc is Scott Key Medical Center, Baltimore).
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