Divergent Interactions of Ehrlichia chaffeensis - Infection and ...

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CONTENT ALERTS
Divergent Interactions of Ehrlichia
chaffeensis- and Anaplasma
phagocytophilum-Infected Leukocytes with
Endothelial Cell Barriers
Jinho Park, Kyoung-Seong Choi, Dennis J. Grab and J.
Stephen Dumler
Infect. Immun. 2003, 71(12):6728. DOI:
10.1128/IAI.71.12.6728-6733.2003.
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INFECTION AND IMMUNITY, Dec. 2003, p. 6728–6733 Vol. 71, No. 12
0019-9567/03/$08.000 DOI: 10.1128/IAI.71.12.6728–6733.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Divergent Interactions of Ehrlichia chaffeensis- and Anaplasma
phagocytophilum-Infected Leukocytes with Endothelial
Cell Barriers
Jinho Park, 1 Kyoung-Seong Choi, 1 Dennis J. Grab, 2 and J. Stephen Dumler 1 *
Division of Medical Microbiology, Department of Pathology, 1 and Division of Infectious Diseases, Department
of Pediatrics, 2 The Johns Hopkins University School of Medicine, Baltimore, Maryland
Received 27 May 2003/Returned for modification 26 July 2003/Accepted 20 August 2003
Human anaplasmosis (formerly human granulocytic ehrlichiosis) and human monocytic ehrlichiosis (HME)
are emerging tick-borne infections caused by obligate intracellular bacteria in the family Anaplasmataceae.
Clinical findings include fever, headache, myalgia, leukopenia, thrombocytopenia, and hepatic inflammatory
injury. Whereas Ehrlichia chaffeensis (HME) often causes meningoencephalitis, this is rare with Anaplasma
phagocytophilum infection. The abilities of infected primary host monocytes and neutrophils and of infected
HL-60 cells to cross human umbilical vein endothelial cell-derived EA.hy926 cell barriers and human brain
microvascular cells (BMEC), a human blood-brain barrier model, were studied. Uninfected monocyte/macrophages
crossed endothelial cell barriers six times more efficiently than neutrophils. More E. chaffeensis-infected
monocytes transmigrated than uninfected monocytes, whereas A. phagocytophilum suppressed neutrophil transmigration.
Differences were not due to barrier dysfunction, as transendothelial cell resistivities were the same
for uninfected cell controls. Similar results were obtained for HL-60 cells used as hosts for E. chaffeensis and
A. phagocytophilum. Differential transmigration of E. chaffeensis- and A. phagocytophilum-infected leukocytes
and HL-60 cells confirmed a role for the pathogen in modifying cell migratory capacity. These results support
the hypothesis that Anaplasmataceae intracellular infections lead to unique pathogen-specific host cell functional
alterations that are likely important for pathogen survival, pathogenesis, and disease induction.
Human infections caused by obligate intracellular bacteria
in the family Anaplasmataceae are on the rise in the United
States and around the world (4, 29). The predominant genera
that include human pathogens are Anaplasma and Ehrlichia,
and a unifying characteristic is that the bacteria infect peripheral
blood leukocytes (10). Similarly, a unifying theme of
diseases caused by these pathogens is the induction of a nonspecific
febrile illness characterized by leukopenia, thrombocytopenia,
and mild liver injury (4, 29). Both infections incite
inflammatory tissue injury and increase the potential for opportunistic
infections (25, 37). While Anaplasma phagocytophilum,
the causative agent of human anaplasmosis (formerly
human granulocytic ehrlichiosis, or HGE), infects neutrophils,
Ehrlichia chaffeensis infects monocytes and macrophages and
causes human monocytic ehrlichiosis. Despite the similar clinical
manifestations, one key difference between the two diseases
is that approximately 20% of E. chaffeensis infections
include central nervous system (CNS) infection (meningoencephalitis
or meningitis), whereas human CNS infection with
A. phagocytophilum has never been convincingly demonstrated
(1, 9, 29, 33). The mechanisms for induction of or resistance to
meningoencephalitis after infection with these different bacteria
are not understood. However, a limiting step in the development
of CNS infection is the ability of the pathogen, or in
the case of obligate intracellular pathogens the infected leu-
* Corresponding author. Mailing address: Division of Medical Microbiology,
Department of Pathology, The Johns Hopkins University
School of Medicine, Ross Research Building, Room 624, 720 Rutland
Ave., Baltimore, MD 21205. Phone: (410) 955-8654. Fax: (443) 287-
3665. E-mail: sdumler@jhmi.edu.
6728
kocyte, to transmigrate across the microvasculature that comprises
the blood-brain barrier (15, 19, 32, 36). In the studies
that follow, we present data which show that E. chaffeensisinfected
monocytic cells transmigrate across in vitro models of
systemic and brain microvascular endothelial cell barriers far
more efficiently than A. phagocytophilum-infected cells migrate
under similar circumstances. Moreover, these data indicate the
critical role of the pathogen in manipulating the infected cell
toward transmigration of endothelial cell barriers.
MATERIALS AND METHODS
Preparation of A. phagocytophilum- and E. chaffeensis-infected HL-60 cells. A.
phagocytophilum Webster strain was propagated in HL-60 cells in RPMI 1640
medium (GIBCO-BRL) supplemented with 5% fetal bovine serum (FBS;
GIBCO-BRL) and 2 mM L-glutamine (GIBCO-BRL) at 37°C in 5% CO 2.
Although we first sought to compare the transmigrating abilities of infected
primary cells, because of the potential for introducing a confounding factor with
the use of different cells we initially took advantage of the bifunctional capacity
of HL-60 cells to be used as models for the study of neutrophils and monocyte/
macrophages and the capacity of these cells to support the growth of both E.
chaffeensis and A. phagocytophilum (16). E. chaffeensis Arkansas strain (provided
courtesy of Jackie Dawson, Centers for Disease Control and Prevention, Atlanta,
Ga.) was initially propagated in the canine DH82 histiocytic cell line in minimal
essential medium (MEM) supplemented with 5% FBS and 2 mM L-glutamine.
Cell-free E. chaffeensis bacteria were prepared by lysis of heavily infected DH82
cells after serial passage through a 26-gauge syringe needle. Intact DH82 cells
were removed by centrifugation at 500 g. The supernatant was examined for
the presence of any residual infected cells and for cell-free bacteria by Romanowsky
staining (HEMA 3; Biochemical Science Inc., Swedesboro, N.J.).
Cell-free bacteria in the supernatant were then transferred to uninfected HL-60
cells cultivated in the same medium used for culture of A. phagocytophiluminfected
cells. The viability of cells was determined using trypan blue exclusion
and was always 95%. Infection of cells was monitored by Romanowsky staining.
Preparation of A. phagocytophilum-infected neutrophils and E. chaffeensis-
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VOL. 71, 2003 ENDOTHELIAL BARRIER TRANSMIGRATION BY ANAPLASMATACEAE 6729
infected monocytes. Human peripheral blood neutrophils and monocytes were
isolated from EDTA-anticoagulated blood of normal donors by dextran sedimentation
followed by Ficoll density gradient centrifugation (Histopaque 1077;
Sigma, St. Louis, Mo.). The contaminating residual erythrocytes present in the
neutrophil preparations were lysed by exposure to hypotonic solution (0.2%
NaCl) for 30 s and then adjusted back to isosmotic conditions with hypertonic
(1.8%) NaCl prior to washing in tissue culture medium. The purified cells were
suspended in MEM (monocytes) or RPMI 1640 (neutrophils) supplemented
with 5% FBS and then incubated at 37°C in5%CO 2 in a humidified environment.
The monocyte/macrophage cultures were allowed to become confluent by
growth for 3 to 4 days prior to use. Neutrophil cultures were used immediately.
For the infection of neutrophils or monocytes by A. phagocytophilum or E.
chaffeensis, cell-free bacteria were prepared by lysis of heavily infected HL-60
cells and DH82 cells after three to five serial passages through a 26-gauge syringe
needle. Intact cells were removed by centrifugation as above, and after confirmation
of purity by microscopic evaluation the cell-free bacteria were used to
infect the purified neutrophil and monocyte/macrophage cultures. After overnight
incubation at 37°C in5%CO 2, the viability of cells and the proportion of
infected cells were determined as above. Routinely, overnight infection of neutrophils
by A. phagocytophilum yielded a preparation with 95% viable neutrophils,
of which nearly 100% were infected. Similarly, overnight infection of
monocyte/macrophages by E. chaffeensis generally yielded 95% viable cells, of
which 50% were infected.
Endothelial cells. Human brain microvascular endothelial cells (BMEC) transformed
by the simian virus 40 large T antigen (35) were propagated in RPMI
1640 medium supplemented with 20% heat-inactivated FBS (Omega Scientific
Inc., Tarzana, Calif.), 2 mM L-glutamine, 1 mM MEM sodium pyruvate
(GIBCO), 1 MEM nonessential amino acid solution (Sigma), and 1 MEM
vitamin solution (Sigma). These cells, which have been shown to exhibit the
hallmark characteristics of the endothelium of the blood-brain barrier, have been
extensively used to examine how bacteria (Escherichia coli, group B Streptococcus
and Streptococcus pneumoniae, and Citrobacter spp.), monocytes, viruses (human
immunodeficiency virus), and fungi (Candida albicans) enter the brain (13, 14,
17–19, 21, 28, 32). EA.hy926 cells, which were derived as a fusion of A549 cells
with human umbilical vein endothelial cells (HUVEC) and are frequently used
as a model of systemic endothelial cells (5, 12), were grown in high-glucose (4.5
g/liter) Dulbecco’s modified Eagle medium (GIBCO) supplemented with 10%
heat-inactivated FBS and 1 hypoxanthine-thymine supplement (GIBCO). Both
endothelial cell cultures were plated and propagated in 25-cm 2 flasks (Sarstedt
Inc., Newton, N.C.).
Endothelial monolayer transmigration assay. The confluent cells were removed
from the flasks using trypsin-EDTA solution and adjusted to 10 5 /ml.
Human BMEC and EA.hy926 cells were seeded (200 l) on top of collagencoated
semipermeable Transwell polycarbonate tissue culture inserts (6.5-mm
diameter [0.33 cm 2 ], with a 3.0-m pore size; Corning Costar Corp.). This in vitro
model allows us separate access to the upper compartment (blood side) and
lower compartment (brain or tissue side). The cells were cultured for 5 to 7 days
in appropriate medium. Medium in both the top and bottom chambers was
changed every other day. One day before the experiments, the culture medium
was changed to experimental medium consisting of Ham’s F-12 nutrient medium
diluted 1:1 with medium M199 supplemented with 20% FBS and 2 mM Lglutamine
(experimental medium) and incubated overnight. Electrical resistance
measurements using an Endohm chamber with an EVOM voltometer (World
Precision Instruments, Sarasota, Fla.) were used to determine monolayer integrity
and are expressed as ohms times centimeters squared, as per the manufacturer’s
recommendation, after adjustment for the resistivity of the membrane
itself.
Some endothelial cell cultures were stimulated with 80 ng of recombinant
human tumor necrosis factor alpha (TNF-; R&D Systems, Inc., Minneapolis,
Minn.)/ml for 4hat37°C in5%CO 2 to induce expression of P-selectin and
E-selectin on endothelial cell surfaces. Expression of selectins on these cell lines
after exposure to TNF- was previously confirmed by flow cytometry (data not
shown).
Prior to the experiments, neutrophils, peripheral blood monocyte-derived
macrophages, or HL-60 cells that were uninfected or infected with A. phagocytophilum
or E. chaffeensis were labeled with PKH67 green fluorescent dye (a cell
tracker dye from Sigma Chemical Co.) according to the manufacturer’s instructions.
After labeling, the cells were washed and checked for adequate labeling by
fluorescence microscopy. The labeled cells were suspended to a concentration of
10 6 cells/ml in experimental medium, of which 200 l of the cell suspensions was
added to the upper chamber containing the appropriate Transwell inserts in
24-well plates. One milliliter of experimental medium was added into the lower
wells. Cultures were incubated overnight at 37°C in5%CO 2. After 4 to 5 h, the
Transwell inserts were transferred into new 24-well plates with wells containing
1 ml of fresh experimental medium and incubated overnight. Cells in the bottom
chambers were counted after 4 to 5 h and after overnight incubation using a
fluorescence microscope. Electrical resistance measurements of monolayer integrity
were done before and after the experiments. All experiments were conducted
in at least triplicate cultures. Experiments utilizing HL-60 cells were
conducted four times; those using primary cells were conducted in duplicate.
Pilot experiments using uninfected undifferentiated HL-60 cells, neutrophils,
and monocytes revealed cell transmigration of similar magnitude as observed in
previous reports (13, 14, 31, 34), with the exception of neutrophil transmigration
of human BMEC, which has not previously been assessed.
Statistical analysis. All statistical evaluations were conducted using paired or
unpaired one-tailed Student’s t tests; P values of 0.05 were considered significant.
Human subjects. Neutrophils and monocytes were obtained from human
volunteers with the approval of the Johns Hopkins University School of Medicine
Institutional Review Board, and all research conducted with these materials
was done so in accordance with the Declaration of Helsinki principles.
RESULTS
E. chaffeensis-infected monocytes/macrophages cross endothelial
cell barriers more efficiently than uninfected or A.
phagocytophilum-infected neutrophils. Resistivity measurements
for both cell lines were similar to the observations of
other investigators reported in the published literature (6, 14,
21, 28, 34). We first examined the ability of infected and uninfected
leukocytes to cross human BMEC and EA.hy926 barriers.
True to their blood-brain barrier function, human BMEC
consistently formed tighter cell barriers than did EA.hy926
cells; i.e., the resistivities of the BMEC monolayers were an
average of 2.3-fold higher than those for EA.hy926, but neither
cell line demonstrated significant alterations in resistivity after
4 h of TNF- stimulation (data not shown).
Thus, it was anticipated that leukocytes would cross
EA.hy926 cells more easily than human BMEC. Specifically,
normal monocytes/macrophages crossed both endothelial cell
barriers approximately six times more efficiently than did neutrophils
with or without recombinant TNF- (rTNF; P
0.001) (Fig. 1). While E. chaffeensis-infected monocyte/macrophages
crossed rTNF--activated endothelial cell barriers as
well as or more efficiently than uninfected monocyte/macrophages
(BMEC, P 0.014; EA.hy926, P 0.075), the ability of
A. phagocytophilum-infected neutrophils to cross both BMEC
and EA.hy926 cells was usually suppressed compared to the
abilities of normal neutrophils (BMEC, P 0.011; EA.hy926,
P 0.010) (Fig. 2). Similar results were obtained when unstimulated
endothelial cell cultures were used, although
TNF- treatment did allow modestly enhanced transmigration
of both monocyte/macrophages and neutrophils, whether infected
or uninfected (data not shown).
The kinetics of transmigration varied for E. chaffeensis-infected
monocyte/macrophages and uninfected cells. Normal
monocyte/macrophages crossed both the human BMEC and
EA.hy926 barriers to a higher degree during the first5h(P
0.017 and P 0.001, respectively); while somewhat delayed
during the initial 5 h, infected cells crossed to a higher degree
between 5 and 18 h (P 0.007 and P 0.030, respectively). In
contrast, A. phagocytophilum-infected neutrophils did not cross
human BMEC during the first 5 h, a time period during which
only 0.005% of uninfected neutrophils crossed (BMEC, P
0.010; EA.hy926, P 0.001). The slow transmigration persisted
so that by 18 h, 0.04 and 0.56% of uninfected neutrophils
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6730 PARK ET AL. INFECT. IMMUN.
FIG. 1. Total transmigration of infected and uninfected primary monocyte/macrophage cells and neutrophils through TNF--stimulated in vitro
models of systemic endothelial cells (EA.hy926) (A) and endothelium of the blood-brain barrier (BMEC) (B) after 18 h. The number of
transmigrating cells was counted after 5 to 8 h and again after 18 h. E. chaffeensis-infected (Echaff-monocytes) and uninfected monocytes
transmigrated through both endothelial cell lines to a greater degree than either uninfected or A. phagocytophilum-infected neutrophils (Aphagneutrophils)
(P 0.001). Although E. chaffeensis-infected monocytes migrated through both endothelial cell lines more rapidly than uninfected
monocytes, by 18 h the difference was significant only for BMEC (P 0.014).
had transmigrated BMEC and EA.hy926 cell monolayers, respectively.
During this same interval, considerably fewer A.
phagocytophilum-infected neutrophils had transmigrated compared
to uninfected neutrophils (0.01% for BMEC [P 0.010]
and 0.20% for EA.hy925 [P 0.007]). Although minor differences
in the magnitude of transmigrating cells were observed
between experiments, the overall differences between transmigration
of uninfected cells and cells infected with either bacterium
were not statistically different.
Increased transmigration of E. chaffeensis-infected monocyte/macrophages
could not be attributed to loss of endothelial
cell barrier function, as human BMEC resistivity continued to
increase over the entire 18 h of the experiment, and no differ-
ences in EA.hy926 resistivity change were noted regardless of
whether HL-60 cells were infected or whether endothelial cells
were TNF- activated (P 0.239 to 0.333).
E. chaffeensis-infected HL-60 cells transmigrate through endothelial
cell barriers more than uninfected or A. phagocytophilum-infected
HL-60 cells. The observation that E. chaffeensis-
and A. phagocytophilum-infected leukocytes differentially
crossed endothelial cell barriers prompted our investigation
into whether the effect was related only to the infected cell or
whether the infecting bacterium could influence transmigration.
Thus, undifferentiated HL-60 cells infected with either E.
chaffeensis or A. phagocytophilum were used in identical endothelial
cell transmigration assays. As for the assays conducted
FIG. 2. Compared to uninfected neutrophils, A. phagocytophilum infection of neutrophils (Aphag-neutrophils) suppresses transmigration
through both EA.hy926 systemic endothelial cell barrier (A and B) and BMEC blood-brain barrier (B) models (P 0.012) at 18 h. Panel B shows
the neutrophil data from panel A expanded to a different scale. Note the significant difference in migration through EA.hy926 cells compared with
that through BMEC cells regardless of infection status of neutrophils.
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VOL. 71, 2003 ENDOTHELIAL BARRIER TRANSMIGRATION BY ANAPLASMATACEAE 6731
FIG. 3. Transmigration of uninfected, A. phagocytophilum-infected (Ap-HL60), and E. chaffeensis-infected HL-60 (Ech-HL60) cells through
both EA.hy926 systemic endothelial cell barrier and BMEC blood-brain barrier models mimics the findings with infected primary leukocytes. Note
the significantly increased transmigration of E. chaffeensis-infected HL-60 cells through EA.hy926 endothelial cells after 5 h, compared with that
of either uninfected or A. phagocytophilum-infected cells (P 0.006) (A). Similarly, E. chaffeensis-infected HL-60 cells, but not A. phagocytophiluminfected
HL-60 cells, transmigrated by 18 h through the BMEC blood-brain barrier model more than uninfected HL-60 cells (P 0.04), regardless
of pretreatment of endothelial cells with TNF-. A. phagocytophilum-infected HL-60 cells migrated through both EA.hy926 and BMEC endothelial
cells to the same degree or less than uninfected cells (B). The results are averages of triplicate cultures and are representative of at least four
replicated experiments.
using primary monocyte/macrophages and neutrophils, many
more HL-60 cells, infected and uninfected, passed through
EA.hy926 cells than through human BMEC (Fig. 3A). In repeated
experiments, only a very small proportion of total cells
migrated through human BMEC during the first 5 h, ranging
from 0.003% of E. chaffeensis-infected cells to 0 to 0.003% of
A. phagocytophilum-infected and uninfected HL-60 cells, respectively.
The majority of human BMEC-transmigrating cells
were observed during the interval 5 to 18 h after inoculation,
when approximately 5- to 10-fold more (maximum, 0.02%) E.
chaffeensis-infected HL-60 cells and many fewer uninfected
HL-60 cells (maximum, 0.003%) and A. phagocytophilum-infected
HL-60 cells (maximum, 0.005%) transmigrated (Fig.
3B). Prior activation of human BMEC with TNF- did not
enhance overall transmigration of either infected or uninfected
cells.
In contrast, significant transmigration through the systemic
endothelial cell barrier model EA.hy926 cells occurred
throughout the entire 18-h period, including during the first
5 h. TNF- activation of EA.hy926 cells enhanced early migration
of infected and uninfected HL-60 cells, but only E.
chaffeensis-infected HL-60 cells transmigrated more through
TNF--activated EA.hy926 cells overall (P 0.001), although
the difference was small (0.25 versus 0.20%) (Fig. 3A).
E. chaffeensis-infected HL-60 cells transmigrated through
rTNF--activated (P 0.001) and unstimulated (P 0.006)
BMEC to a significantly higher degree than uninfected or A.
phagocytophilum-infected HL-60 cells after overnight incubation
(Fig. 3A), but not by 5 h (data not shown). In contrast, by
5hE. chaffeensis-infected HL-60 cell transmigration through
TNF--activated or unstimulated EA.hy926 endothelial cells
had occurred to a degree greater than that with either uninfected
(P 0.03) or A. phagocytophilum-infected HL-60 cells
(P 0.04) (Fig. 3B). A. phagocytophilum-infected HL-60 cells
migrated similarly to or in smaller numbers than uninfected
HL-60 cells at all time points examined, regardless of endothelial
cell type and TNF- stimulation (Fig. 3).
Transendothelial electrical resistivity of EA.hy926 cells and
human BMEC. Transendothelial resistance measurements
were conducted in order to estimate overall endothelial cell
barrier integrity after conclusion of the experiments. In general,
resistivity stayed high throughout all experiments, regardless
of manipulations by adding infected or uninfected HL-60
cells, or with TNF- activation. The average starting resistance
reading for the 6.5-mm inserts for human BMEC cultures was
25.9 1.35 cm 2 (range, 21.9 to 28.2), and no significant
differences were observed when the cells were TNF- activated.
The overall starting electrical resistance of the
EA.hy926 cells was lower, with a mean of 11.4 0.86 cm 2
(range, 9.6 to 12.9), and in only one of four experiments did
resistivity become significantly lower with TNF- pretreatment.
The observed differences in electrical resistance clearly
highlight the barrier function of BMEC as reflected in the
profound reduction in leukocyte migration across BMEC compared
to their migration across HUVEC. Inconsistently, E.
chaffeensis-infected HL-60 cells stimulated decreased resistivity
in either human BMEC or EA.hy926 cultures, but the
differences were generally very small and within the range of
variation observed at the beginning of experiments, except in
one instance for EA.hy926 cells (mean resistivity change, 3.3
cm 2 ). No differences in resistivity between cultures incubated
with uninfected and A. phagocytophilum-infected HL-60
cells were observed. In one experiment, the electrical resistance
continued to rise despite incubation with infected and
uninfected HL-60 cells, excluding a role for endothelial barrier
dysfunction in transmigration.
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6732 PARK ET AL. INFECT. IMMUN.
DISCUSSION
How infectious agents penetrate through the blood-brain
barrier as a prerequisite for entry into the CNS, a critical event
before meningoencephalitis, is an area of active study by many
investigators (13, 17–19, 21, 28). Similarly, the mechanisms by
which inflammatory cells respond to CNS and systemic infections
and pass through the blood-brain barrier or systemic
vasculature into tissues is increasingly the focus of study (5, 14,
15, 32). The entry of obligate intracellular bacteria into the
CNS and into tissues poses issues critical to both arenas of
study because of the strict requirement of host cell association.
Among the most intriguing of these situations is the passage
through systemic and brain endothelial cell barriers of leukocytes
infected with members of the Anaplasmataceae family,
especially including E. chaffeensis and A. phagocytophilum.
From a microbiological perspective, A. phagocytophilum and
E. chaffeensis are related but unique organisms. Although there
are significant differences between these two organisms, one
key aspect that differentiates the two is that the former infects
neutrophils and the latter infects mononuclear phagocytes
(29). From a clinical perspective, these two pathogens induce
similar systemic diseases, with the predominant exception that
meningoencephalitis is a frequent component of monocytic
ehrlichiosis (E. chaffeensis infection), whereas CNS infection
with anaplasmosis or HGE (A. phagocytophilum infection) is
exceedingly rare (1, 9, 29). Moreover, monocytic ehrlichiosis
has a higher rate of mortality and appears to be a more severe
infection, while anaplasmosis (HGE) is associated with moderate
inflammatory reactions sometimes accompanied by manifestations
of immune suppression and neutrophil dysfunction
(1, 25, 29, 30). The results of this study suggest that some of the
clinical observations recorded may relate to dramatic differences
in how leukocytes penetrate endothelial cell barriers,
including the blood-brain barrier, differences that can be modulated
in the host leukocyte by the bacterium. This novel observation
provides support for the emerging concept that interactions
of Anaplasmataceae bacteria with the host cells lead
to a dynamic alteration in leukocyte function modulated by the
bacterium to a great extent, an event that may substantially
contribute to human disease (29, 30).
E. chaffeensis-infected primary monocyte/macrophages rapidly
and more effectively penetrate endothelial cell barriers
than do either uninfected monocytes/macrophages, neutrophils,
or A. phagocytophilum-infected neutrophils. However,
this observation could potentially be explained by differences
in the nature of the host cells, their receptors, and other factors
unrelated to the bacterium itself. This is no moot point, as it is
well established that neutrophil migration occurs at a rate less
than, by about 14%, that of monocytes from the same donor
(27). By using undifferentiated HL-60 cell cultures that are
established as models of both neutrophil and macrophage biology
(14, 15, 20, 23, 31), we reasoned that it might be possible
to ascertain whether the differences in endothelial cell barrier
penetration result from cell lineage factors or from bacteriuminduced
cell changes. That E. chaffeensis-infected HL-60 cells
mimic the increased penetration and rate of transmigration
observed with primary cells and not observed with uninfected
or A. phagocytophilum-infected HL-60 cells suggests a highly
significant role for the pathogen in this process. Of additional
interest is the lack of any change or the reduction in migration
observed when neutrophils and HL-60 cells are infected with
A. phagocytophilum. Because transendothelial resistance measurements
were not significantly different among the endothelial
cell monolayer cultures with infected and uninfected leukocytes,
it is highly unlikely that injury to endothelial cell
barrier function accounts for the increased transmigration observed
with E. chaffeensis. This is consistent with the observation
that tight junctions are maintained after paracellular migration
of monocytes (14) and neutrophils (6).
The observations are consistent with other data regarding
the effects of these species on their respective host cells. E.
chaffeensis is known to enter into early endosomes that are
then directed into a salvage-recycling pathway to avoid lysosomal
fusion of the infected vacuole (3). However, infection in
the presence of antibody allows complexes to bind Fc receptors
that initiate signal transduction cascades, NF-B translocation
after degradation of IB, and significant induction of proinflammatory
cytokines (24). Empirically, E. chaffeensis infections
are more severely inflammatory than A. phagocytophilum
infections, and significant degrees of tissue inflammation and
cerebrospinal fluid pleocytosis, which may include infected
cells, are often observed (11, 30, 33, 37). In contrast, infection
of neutrophils with A. phagocytophilum leads to inhibition of
the respiratory burst via repression of rac-2 mRNA (7) and
downregulation of gp91 phox or proteolysis of p22 phox (2, 26),
secretion of chemokines but not proinflammatory cytokines
(22), inhibition of neutrophil phagocytosis and microbicidal
activity (38), and loss of surface selectin expression that mediates
interactions with endothelial cells (8). The loss of surface
selectin may explain the lack of transmigration through tight
endothelial cell barriers, such as that in the in vitro blood-brain
barrier used here, in spite of the unimpaired ability of infected
cells to migrate through Transwell filters without endothelial
cells or through porous endothelial cell barriers such as with
the HUVEC-derived EA.hy926 cells. Moreover, these data are
consistent with clinical observations that CNS infection by A.
phagocytophilum is a rare event.
In this study, we analyzed quantitative transmigration of E.
chaffeensis-infected, A. phagocytophilum-infected, and normal
cells. The data confirm the significant role of the bacterial
pathogens in modifying host cell behavior that potentially contributes
directly to human disease. It is not possible to exclude
whether the biological behavior observed resulted from direct
effects of infected cells, direct effects or bacterial components
in the medium, or both. However, the differential effects of the
two phylogenetically related organisms suggest that the main
effect is mediated at the level of the infected cell.
While increasing information about how A. phagocytophilum
affects normal leukocyte and neutrophil function is available,
detailed analyses of the mechanisms by which these bacteria
control cellular and molecular functions are still lacking, especially
for E. chaffeensis. The ability to transmigrate endothelial
cells, especially those of the blood-brain barrier, requires specific
cellular alterations, including those critical for binding and
initiating transient endothelial cell alterations that permit passage.
Thus, fruitful areas for continued study might examine
how E. chaffeensis effects surface adhesion molecule expression
on monocytes, how infected cells or products of infection in
monocytes affect endothelial cell actin architecture and integ-
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VOL. 71, 2003 ENDOTHELIAL BARRIER TRANSMIGRATION BY ANAPLASMATACEAE 6733
rity of junctional components, and whether bacterial components,
host cell components, or both directly influence transmigration.
Likewise, beneficial areas for study of A.
phagocytophilum infections may allow a greater focus on how
cells that are in part functionally paralyzed continue to manage
to induce continued inflammatory injury unrelated to the degree
of bacteria present. Regardless, such studies will continue
to emphasize the crucial differences in the biology among
members of the Anaplasmataceae rather than proposing broad
similarities in clinical disease by shared pathogenetic mechanisms.
ACKNOWLEDGMENTS
This work was supported by grant ROI AI44102 to J.S.D. D.J.G. was
supported in part by a grant from the Thomas Wilson Sanitarium for
Children of Baltimore. J. Park and K.-S. Choi were supported in part
by awards from the Korea Science and Engineering Foundation.
We thank Monique Stins and Kwang Sik Kim (Department of Pediatrics,
Johns Hopkins School of Medicine) for the transfected human
BMEC.
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