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Infection and Immunity, March 2000, p. 1125-1133, Vol. 68, No. 3
Department of Veterinary Biosciences, The
Ohio State University, Columbus, Ohio 43210-1093
Received 13 August 1999/Returned for modification 7 October
1999/Accepted 23 November 1999
In patients with human granulocytic ehrlichiosis (HGE), the HGE
agent has been seen only in the peripheral blood granulocytes, which
have a life span too short for ehrlichial proliferation. To determine
if the HGE agent delays the apoptosis of human peripheral blood
neutrophils for its advantage, peripheral blood granulocytes consisting
mostly of neutrophils were incubated with freshly freed host cell-free
HGE agent in vitro. The HGE agent induced a significant delay in
morphological apoptosis and the cytoplasmic appearance of
histone-associated DNA fragments in the granulocytes. This antiapoptotic effect was dose dependent. Although much weaker than the
HGE agent freshly freed from the host cells, noninfectious purified HGE
agent stored frozen and thawed also had antiapoptotic effect, which was
lost with proteinase K treatment but not with periodate treatment.
Treatment of neutrophils with a transglutaminase inhibitor,
monodansylcadaverine, blocked the antiapoptotic effect of the HGE
agent. Addition of oxytetracycline, however, did not prevent or reverse
the antiapoptotic effect of the HGE agent. These results suggest that
binding of a protein component(s) of the HGE agent to neutrophils and
subsequent cross-linking and/or internalization of the receptor and
ehrlichiae are required for antiapoptotic signaling, but ehrlichial
protein synthesis and/or proliferation is not required. MG-132, a
proteasome inhibitor, and cycloheximide accelerated the apoptosis of
neutrophils and overrode the antiapoptotic effect of the HGE agent.
Studies with specific inhibitors suggest that protein kinase A,
NF- Human granulocytic ehrlichiosis
(HGE) is a newly recognized tick-borne zoonosis in the United States
and Europe (3, 5, 6, 8, 9). HGE is an acute febrile systemic
disease associated with hematologic abnormalities, such as
thrombocytopenia and leukopenia, as well as increased serum
aminotransferase activity. HGE may be fatal when patients are
immunocompromised or antibiotic treatment is delayed. The etiologic
agent, called the HGE agent, is a small gram-negative coccus closely
related to previously known granulocytotropic Ehrlichia
spp. HGE agent and cell culture.
HGE isolate HZ (37)
was cultivated in HL-60 cells in RPMI 1640 medium (GIBCO, Grand Island,
N.Y.) supplemented with 5% fetal bovine serum (Atlanta Biologicals,
Norcross, Ga.), 2 mM L-glutamine (GIBCO), 0.1 mM minimal
essential medium-nonessential amino acid mixture (GIBCO), and 1 mM
minimal essential medium-sodium pyruvate (GIBCO). When >90% of the
cells were infected, as determined by examining cells stained with
Diff-Quik (Baxter Scientific Products, Obetz, Ohio), the infected cells
were sonicated and centrifuged at 500 × g for 5 min.
The supernatant was centrifuged at 10,000 × g for 10 min, and the pellet, containing the host cell-free, viable HGE agent,
was immediately used to infect human peripheral blood neutrophils or
peripheral blood leukocytes (PBL). Because the HGE agent is small and
multiplies as microcolonies, it is impractical to accurately count
individual organisms. Therefore, the number of host cell-free
ehrlichiae was estimated by using the following formula: number of
ehrlichial organisms = total infected cell number × average
number of morulae in an infected cell (typically five) × average
number of ehrlichial organisms in a morula (typically 19) × percentage of ehrlichiae recovered as host cell free (typically 50% as
determined by using metabolically [35S]methionine-labeled
ehrlichiae [36]).
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Intracellular Infection by the Human Granulocytic
Ehrlichiosis Agent Inhibits Human Neutrophil Apoptosis
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
B, and interleukin 1
are not involved in the antiapoptotic
mechanism of the HGE agent.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Ehrlichia phagocytophila, the agent of tick-borne fever in sheep and goats, and Ehrlichia equi, the agent of
equine ehrlichiosis (9). These bacteria are incapable of
extracellular survival and are seen to replicate in the cytoplasm of
peripheral blood granulocytes. A small number of individually dispersed
ehrlichiae are difficult to recognize under the light microscope.
However, microcolonies, called morulae, that result from ehrlichial
replication stain dark blue to purple with Romanowsky dye; they are
large and have a characteristic morphology and thus can be recognized by the trained eye (34). All 12 patients initially reported in Minnesota and Wisconsin had detectable morulae in 1 to 41% of their
peripheral blood granulocytes (5). In another study, intracytoplasmic morulae were seen in 3 of 12 patients in New York, and
the frequency of infected granulocytes ranged from 0.3 to 6%
(3). The majority of infected granulocytes seen were neutrophils. Mature neutrophils undergo rapid apoptosis (programmed cell death), with a half-life of several hours in vivo (11). Considering the low growth rate of cytoplasmic ehrlichiae and the
important roles neutrophils play in host defense and acute inflammation, the life span of neutrophils is a critical determinant for ehrlichial survival and HGE pathogenesis. Recently, the HGE agent
was reported to induce apoptosis in the human promyelocytic leukemia
cell line HL-60 (17). However, no information is available on whether the HGE agent modulates apoptosis of human neutrophils, its
natural host. In the present study, we examined whether the HGE agent
alters constitutive apoptosis of human peripheral blood neutrophils in
vitro. Furthermore, mechanisms by which apoptosis is delayed by the HGE
agent were examined by using an HGE agent treated with various reagents
and host cells inhibited in intracellular signaling pathways. This
study, therefore, is helpful in understanding not only the
intracellular survival strategy of the HGE agent but also the role of
neutrophils in the pathogenesis of HGE.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
80°C until it was used, as previously described (35).
Periodate-treated ehrlichiae were prepared by incubating purified HGE
agent with 20 mM sodium periodate (Sigma Chemical Co., St. Louis, Mo.)
in 50 mM sodium acetate buffer (pH 4.5) for 1 h at room
temperature in the dark followed by incubation with 50 mM sodium
borohydride (Sigma) in sterile phosphate-buffered saline (PBS; 2.7 mM
KCl-1.8 mM KH2PO4-137 mM NaCl-10 mM
NaH2PO4, pH 7.4) for 30 min at room temperature
(21, 22). For proteinase K treatment, the purified HGE agent
was incubated in 1 mg of proteinase K (GIBCO)/ml in distilled water at
60°C for 2 h. After incubation, 1 mM phenylmethylsulfonyl
fluoride (Sigma) was added, the mixture was incubated for 10 min at
60°C, and then the ehrlichiae were washed three times in RPMI 1640 medium (21, 22).
Treatment.
After being washed twice, purified neutrophils or
PBL were suspended at 106 cells/ml in RPMI 1640 medium.
Uninfected HL-60 cells, prepared by brief sonication of the cell
suspension at 106 cells/ml followed by differential
centrifugation as previously described (21), and the
purified HGE agent prepared as described above was added at a final
concentration of 100 µg of protein/ml. Host cell-free HGE agent was
added to purified neutrophils at a multiplicity of infection (MOI) of
100 immediately after the isolation of the HGE agent. Treated or
infected neutrophil suspensions were seeded in 96-well flat-bottom
plates (Becton Dickinson Co., Franklin Lakes, N.J.) at 200 µl/well in
triplicate wells per assay point, and these samples were incubated at
37°C in a 5% CO2 atmosphere for 
96 h. Cells were
harvested every 8 h. To consider individual human variations, all
experiments were independently repeated two or three times on different
days using neutrophils derived from different donors and the freshly
prepared host cell-free HGE agent each time. Donor cells were never
mixed, and each donor neutrophil assay included positive and negative
controls to ensure the quality of both neutrophil and HGE agent preparation.
(IL-1) (clone 8516.311; R & D Systems,
Minneapolis, Minn.)/ml or 100 µM acetyl-Tyr-Val-Ala-Asp-chloromethyl
ketone (YVAD-CMK) (Calbiochem, San Diego, Calif.) were added to
triplicate wells at 0 h or 8 h (10 µM H-89 only) after the addition
of HGE agent.
Microscopic determination of apoptosis.
Cell suspensions (80 µl) were centrifuged on a glass slide at 30 × g for
1 min with Cytospin 3 (Shandon Inc., Pittsburgh, Pa.). The cells were
stained with Diff-Quik and examined microscopically at ×1,000.
Apoptotic cells were determined based on their morphology, including
densely condensed and homogeneous nuclei, loss of connection between
the lobules of nuclei, and eosinophilic cytoplasm (Fig. 1). A total of 500 cells were scored from
each well. The time required for 50% of the neutrophils to show
morphological apoptosis (T50) was determined by
plotting the percentage of apoptotic cells observed at each incubation
time point.
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Detection of histone-associated DNA fragments. To detect the fragmentation of DNA in apoptotic neutrophils, histone-associated DNA fragments were examined by using a Cell Death Detection ELISA Plus kit (Boehringer Mannheim, Indianapolis, Ind.). This assay is based on the quantitative sandwich enzyme immunoassay principle, with mouse monoclonal antibodies directed against DNA and histones. Cell suspensions (80 µl) incubated in a 96-well plate for 8, 12, and 16 h were harvested and centrifuged at 200 × g for 10 min. The lysis buffer (200 µl) was added to the pellet and incubated at room temperature for 30 min. After centrifugation at 200 × g for 1 min, a sample of the supernatant was diluted 10-fold with lysis buffer, and 20 µl was applied in a well of the streptavidin-coated microtiter plate. An immunoreagent mixture (80 µl) containing anti-histone-biotin (4 µl), anti-DNA-peroxidase (4 µl), and incubation buffer (72 µl) was added to each well and incubated for 2 h at room temperature. After three washes with the incubation buffer, 100 µl of the substrate solution (2,2'-azino-di[3-ethylbenzthiazolin-sulfonate]) was added. The absorbances of samples at 405 nm and background at 490 nm were measured.
Statistical analysis. Statistical significance compared with the addition of the medium alone as a control was determined by Student's t test with Sigmaplot version 4.0. A P value of <0.05 was considered significant.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Morphology.
As a first approach, we investigated whether the
HGE agent could interfere with physiological apoptosis that occurs in
mature neutrophils isolated from blood and cultured. The results of
various treatment groups were compared using neutrophils prepared from a single donor for each experiment, and the experiments were repeated more than three times with neutrophils derived from different donors.
Neutrophil apoptosis can be assessed by various parameters, including
changes in cellular morphology. Apoptotic neutrophils have condensed
and homogenous nuclei whose lobules have lost their connection in
condensed eosinophilic cytoplasm (Fig. 1A). The percentage of apoptotic
neutrophils by these criteria rapidly increased in untreated
neutrophils after 8 h of incubation in vitro (Fig. 1A).
Neutrophils treated with HL-60 cell lysate as a negative control had a
rate of apoptosis similar to that without HL-60 cell lysate, indicating
that HL-60 cell lysate (a fraction of which was present in the host
cell-free HGE agent preparation because the HGE agent had been
cultivated in HL-60 cells) does not influence in vitro apoptosis (Fig.
2). Almost 100% of untreated as well as
HL-60 cell lysate-treated neutrophils were apoptotic after 24 h of
incubation in vitro (Fig. 1 and 2). In contrast, most neutrophils
incubated with freshly prepared host cell-free HGE agent for 24 h
showed normal cytoplasm and lobulation of nuclei (Fig. 1B and 2). Of
all neutrophils, >50% that were incubated with freshly freed HGE
agent were not apoptotic for up to 48 h, and 10 to 20% of
neutrophils were not apoptotic after 96 h of incubation. In these
nonapoptotic cells, formation of typical ehrlichial morulae was
observed after 24 h of incubation, although the neutrophils having
morulae were approximately 5% of all neutrophils (Fig. 1C). Morulae
were also observed in eosinophils present in the neutrophil preparation
after 24 h of incubation (Fig. 1D). Eosinophil numbers varied
widely among the blood donors, but they survived longer even after the
apoptosis of a majority of neutrophils at 96 h, and morulae became
larger in these cells.
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Detection of cytoplasmic histone-associated DNA fragments. Internucleosomal DNA fragmentation in apoptosis appears as detectable histone-associated DNA fragments in the cytoplasm of apoptotic cells, after enrichment of mono- and oligonucleosomes in the cytoplasm of the apoptotic cells caused by DNA degradation occurring several hours before plasma membrane breakdown (15). Because morphological apoptotic changes in uninfected neutrophils were observed in >50% of untreated neutrophils after ~12 h of incubation, we examined the numbers of these DNA fragments after 8 and 16 h of incubation (Fig. 3). Untreated cells revealed rapid increases in cytoplasmic histone-associated DNA fragments starting at 12 h of incubation, which were paralleled by an increase in morphologically apoptotic cells (Fig. 2). DNA fragments of neutrophils infected with freshly prepared host cell-free HGE agent did not increase up to a 16-h incubation period. The purified HGE agent had a weak but significant inhibitory effect on apoptosis at 16 h postincubation (Fig. 3). Chromatin fragmentation by internucleosomal endonuclease during apoptosis was also examined by the conventional agarose gel electrophoresis of DNA extracted from neutrophils. Although the typical "ladder patterns" were detectable in control neutrophils, but not in HGE agent-infected neutrophils, at more than 24 h of incubation, the assay was not quantitative and was less sensitive than the detection of cytoplasmic histone-associated DNA fragments (data not shown).
Dose response. The inhibitory effects on apoptosis of purified and freshly prepared host cell-free HGE agents were dose dependent (Fig. 4). The purified HGE agent at 1 µg of protein/ml significantly inhibited the morphological apoptosis of neutrophils, but at <0.1 µg/ml there was no effect. The level of inhibitory effect with freshly freed HGE agent at an MOI of 10 was comparable to that of 1 µg of protein/ml of the purified HGE agent.
Treatment of HGE agent with proteinase K or periodate. To examine the requirement for ehrlichial protein(s) or carbohydrate(s) in the antiapoptotic effect, the purified HGE agent was treated with 1 mg of proteinase K/ml or 20 mM sodium periodate. The HGE agent treated with proteinase K completely lost its inhibitory effect, whereas periodate treatment had no effect (Fig. 5). The results indicates that ehrlichial proteins are required for antiapoptotic effect.
Effects of MDC and oxytetracycline.
Previous results in our
laboratory have shown that internalization and infection of
Ehrlichia risticii in P388D1 cells and of
Ehrlichia chaffeensis and the HGE agent in THP-1 cells
(7, 26, 31, 36) was inhibited by the reversible
transglutaminase inhibitor MDC. Transglutaminase catalyzes the
formation of 
-(
-glutamyl)-lysine between protein molecules by
coupling amines and diamines to the -carboxyl residue of glutamine
(23). Receptor-mediated endocytosis of various ligands has
been shown to be inhibited by transglutaminase inhibitors
(23), suggesting an involvement of transglutaminase and
receptor-mediated endocytosis in ehrlichial uptake. Therefore, we
utilized MDC to determine whether internalization of the HGE agent is
required to prevent apoptosis. MDC blocked the antiapoptotic effect of
the host cell-free HGE agent: the means and standard deviations of the
percentage of apoptotic neutrophils were 49.9 ± 4.4 for the
medium control, 54.9 ± 1.8 for the medium-plus-MDC control,
25.5 ± 0.8 with the host cell-free HGE agent, and 45.1 ± 3.4 with the host cell-free HGE agent plus MDC (n = 3)
when MDC and the extracellular HGE agent were removed after 2 h of incubation and continuously incubated for 24 h. When MDC and the extracellular HGE agent were removed at 2 h, the means and
standard deviations of the percentage of infected cells at 24 h
were 0.9 ± 0.4 with MDC treatment and 6.2 ± 0.2 (n = 3) without MDC, indicating that MDC blocked
internalization of the HGE agent. Similar results were obtained when
MDC and the extracellular HGE agent were not removed at 2 h and
were kept throughout the incubation period. These results suggest that
clustering and/or internalization of the ehrlichiae and their receptors
is required for the inhibition of apoptosis.
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Effects of H-89.
The cyclic AMP (cAMP) analog 8-CTP-cAMP was
shown to delay the spontaneous and cycloheximide- or anti-FAS-induced
apoptosis of human neutrophils in vitro (32). cAMP-elevating
agentsprostaglandins and the phosphodiesterase type IV inhibitor RO 20-1724were shown to inhibit neutrophil apoptosis, and treatment of
human peripheral blood neutrophils with 1 µM H-89, a selective
inhibitor of protein kinase A (10), prevented prostaglandin
E2- and RO 20-1724-induced inhibition of cell apoptosis
(30). To investigate whether the host cell protein kinase A
is involved in the inhibition of apoptosis of neutrophils by the HGE
agent, H-89 (1 µM at 0 h or 10 µM at 8 h postinfection)
was added. The apoptosis of neutrophils in the presence or absence of
the viable HGE agent did not change with this concentration of H-89
(Fig. 7). A concentration of H-89 of >10
µM present for >48 h was toxic to neutrophils. The result suggests
that delayed in vitro apoptosis of neutrophils by the HGE agent is not
mediated by protein kinase A activation.
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Effects of genistein, MG-132, and SN-50.
Lipopolysaccharide
(LPS)- or granulocyte-macrophage colony-stimulating factor
(GM-CSF)-induced delay in the spontaneous apoptosis of human
neutrophils was reported to be blocked by a tyrosine kinase inhibitor,
herbimycin A, or by pyrrolidine dithiocarbamate (40). Both
herbimycin A and pyrrolidine dithiocarbamate inhibit NF-B activation
in human granulocytes in response to LPS (40). To
investigate whether NF-B activation was involved in the delayed apoptosis of neutrophils by the HGE agent, effects of 50 µM
genistein, a tyrosine kinase inhibitor (4); 100 mM MG-132, a
cell-permeable peptide-aldehyde protease inhibitor that blocks NF-B
activation via its effect on the proteasome (18); and 100 µg of SN-50/ml, a cell-permeable inhibitory peptide of nuclear
translocation of NF-B (24), were examined. Genistein had
no effect on apoptosis of neutrophils in the absence or presence of the
HGE agent (Fig. 8A). MG-132 accelerated
the apoptosis of neutrophils regardless of the presence of the HGE
agent. Of both infected and uninfected neutrophils, ~100% became
morphologically apoptotic after 16 h of incubation in vitro. This
suggests that degradation of some host proteins by proteasomes is
required for the inhibition of the apoptotic process in both normal and
HGE agent-infected neutrophils (Fig. 8B). On the other hand, 100 µg
of SN-50/ml slightly delayed apoptosis of uninfected neutrophils, but
the percentage of morphologically apoptotic neutrophils infected with
the HGE agent was the same whether SN-50 was present in the medium or
not (Fig. 8C). These results suggest that NF-B is not involved in
delaying apoptosis of neutrophils by the HGE agent.
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Effects of inhibitors of the IL-1 pathway.
Our results
showed that the HGE agent induces IL-1 expression in PBL and
neutrophils (H.-Y. Kim and Y. Rikihisa, Abstr. 99th Gen. Meet. Am. Soc.
Microbiol., abstr. D/B-129, p. 234, 1999). Two different
proinflammatory stimuli, LPS and GM-CSF, upregulate the expression of
IL-1-converting enzyme, also known as caspase-1, and delay the
apoptosis of neutrophils. The delay is blocked by blocking antibody to
IL-1 or a caspase-1 inhibitor (42). Therefore, we
examined whether endogenous IL-1 generated by neutrophils in
response to the HGE agent or caspase-1 is involved in inhibition of
neutrophil apoptosis by the HGE agent by adding blocking anti-human IL-1 antibody or an irreversible tetrapeptide inhibitor of
caspase-1, YVAD-CMK (39), to the assay system. There was no
change in the apoptosis of neutrophils regardless of the presence of
the HGE agent (Fig. 9A).
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B activation but blocks the antiapoptotic effect of LPS and GM-CSF by inhibiting IL-1 and caspase-1 upregulation (42). Cycloheximide treatment
accelerated apoptosis regardless of the presence of the HGE agent (Fig.
9B).
Effect of HGE agent on apoptosis of neutrophils in PBL.
Because other leukocytes, such as monocytes and lymphocytes, which
coexist in the blood influence neutrophil apoptosis through cytokines
and cell-cell interactions (11), we examined whether the
coexistence of other cell types in the blood influences the inhibition
of neutrophil apoptosis by the HGE agent in vitro. Freshly prepared
host cell-free HGE agent was more effective in inhibiting apoptosis of
neutrophils in PBL, which consisted of ~60% neutrophils, than in
purified neutrophils (Fig. 10). The
purified HGE agent was less effective than host cell-free HGE agent but was more effective in inhibiting apoptosis of neutrophils in PBL than
in purified neutrophils. The rate of apoptosis of uninfected neutrophils was, however, similar in PBL and purified neutrophil preparation.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The host of the HGE agent is the neutrophil, a suicidal effector cell equipped with the most powerful antibacterial armamentarium. Since ehrlichiae die if they remain extracellular, the HGE agent must enter neutrophils. Once it is intracellular, the HGE agent has solved the problem of lysosomal destruction by inducing the formation of a unique membrane-bound niche, a parasitophorus vacuole which does not fuse with lysosomes (28). However, normal neutrophils survive for a limited time in the peripheral blood. Unless the neutrophil's life span is extended, the intracellular HGE agent will die together with the host cell before having the chance to proliferate. The present study revealed that the HGE agent delays the apoptosis of human peripheral blood neutrophils sufficiently to allow intracellular proliferation of the HGE agent in vitro, resulting in a significant morula formation in 24 to 48 h of incubation at a level comparable to those seen in patients with HGE (3, 5). The actual percentage of infected cells may be greater, since by using immunolabeling and flow cytometry we previously found that almost 100% of P388D1 cells take up E. risticii at low levels at 3 h postincubation (26), although by Diff-Quik staining this low level of intracellular ehrlichiae is not apparent (31).
Although small, condensed, elementary-body-like and large, light, reticular body-like ehrlichiae have been seen (34), a chlamydia-like developmental cycle or eclipse stage has not been demonstrated in ehrlichiae. However, when we follow the time course of in vitro ehrlichia infection in a leukemia cell line, during the first day of culture we can seldom see infected cells (lag phase). After day 2 to 3 days of culture, logarithmic growth occurs, and 100% of the cells are infected with a large number of organisms by day 5 to 7 of culture, after which the cells are lysed (31). Since even with the delay the apoptosis of infected neutrophils takes place prior to complete ehrlichial proliferation and host cell lysis, the HGE agent in neutrophils must be horizontally transmitted to the next generation of neutrophils prior to host cell apoptosis in order to survive. This may be one reason why heavily infected neutrophils are rarely seen in patients. In the present study, nearly all neutrophils could be prevented from undergoing rapid apoptosis when stimulated with the host cell-free HGE agent at an MOI of 100. Delaying apoptosis of all neutrophils is advantageous for the HGE agent, because it gives more time for the HGE agent to survive and replicate inside neutrophils and to enhance the chance of its intercellular spreading. How the HGE agent spreads from infected to uninfected cells is unknown. Infected neutrophils were rarely seen filled with the HGE agent and/or lysed. Spreading of monocytic ehrlichiae can occur without lysis of the infected host cells (36), probably by ehrlichial exocytosis from the infected cells followed by endocytosis of freed ehrlichiae by other cells (36). The HGE agent may be transmitted by a similar mechanism from infected to uninfected neutrophils after brief intracellular proliferation.
Previous observations with other granulocytotropic ehrlichiae support our observation. In vitro incubation of peripheral blood granulocytes from dogs experimentally infected with Ehrlichia ewingii or heparinized whole blood from sheep experimentally infected with E. phagocytophila results in an increase in the proportion of infected neutrophils and the number of morulae in infected cells even after 2 to 4 days or 24 h, respectively (43, 44), suggesting that these infected granulocytes survive for a longer period in vitro to allow for the growth of ehrlichiae. Our result was the opposite of a previous report (17). This difference might be due to a difference in host cells, since the previous study used an immortalized leukemia cell line, HL-60.
As has been seen in patients' blood (3), we found that
eosinophils become infected with the HGE agent and survive much longer
than neutrophils in vitro. Eosinophils display a similar capacity to
undergo constitutive apoptosis when aged in vitro, but this process is
much slower than that observed for the neutrophil and is differentially
regulated; for example, it is stimulated rather than inhibited by
corticosteroids (25). Eosinophils, therefore, may
potentially serve as a reservoir for the increasing numbers of HGE
agents for longer periods of time than do neutrophils, in order to
infect circulating neutrophils. Several cytokines, such as granulocyte
CSF (1), GM-CSF, IL-1 (13, 42), IL-2 (11,
33), gamma interferon (IFN-) (13), and IL-8
(19), are reported to delay apoptosis of neutrophils in
vitro, but IL-6, tumor necrosis factor alpha (TNF)-
, and IL-10 are
reported to accelerate neutrophil apoptosis (2, 11, 40).
Although significant levels of TNF- and IL-6 were generated by PBL
in response to the HGE agent in vitro (H.-Y. Kim and Y. Rikihisa,
Abstr. 99th Gen. Meet. Am. Soc. Microbiol. 1999, abstr. D/B-129, p.
234, 1999), in the present study neutrophils in PBL, which may be
closer to the in vivo situation, survived as long as purified
neutrophils in vitro, and the effect of the HGE agent in delaying
apoptosis of neutrophils is greater in the presence of other
leukocytes, indicating that the influence of the HGE agent is
reproducible and the presence of other leukocyte populations does not
override or cancel the influence.
Unlike E. coli, which accelerates the apoptosis of
neutrophils (41), it is becoming clear that several
intracellular microorganisms delay apoptosis of host cells. Detailed
signaling pathways for the inhibition of apoptosis, however, are not
yet known for any of these agents. Rickettsia rickettsii
(12) activates NF-B to inhibit the apoptotic process of
the host endothelial cells. NF-B is expressed in human neutrophils,
and inhibition of an inducible form of NF-B is linked to the
induction of neutrophil apoptosis (40). Our present study,
however, suggests that NF-B activation is not involved in delaying
apoptosis of neutrophils by the HGE agent. Mycobacterium
tuberculosis, which has a longer doubling time than the HGE agent,
at low numbers delays apoptosis of human monocytes in vitro
(16). The inhibition is partially neutralized with
anti-TNF- antibodies, suggesting that TNF- partially mediates the
antiapoptotic effect of M. tuberculosis. Unlike monocytes,
TNF- is known to induce rapid apoptosis of human peripheral blood
neutrophils in vitro (11, 40). An intracellular infection by
the protozoan parasite Toxoplasma gondii counteracts apoptosis of murine lymphoma cell lines induced by several kinds of
stimuli, such as Fas ligation, granzyme B, - or UV irradiation, and
calcium ionophores (29), suggesting that a mechanism common to many apoptotic pathways is involved. In contrast to the HGE agent,
protection against apoptosis by T. gondii requires the continued presence of live organisms and ongoing protein synthesis (29). The intracellular protozoan parasite Leishmania
donovani or treatment with lipophosphoglycan, the major surface
molecule of the Leishmania promastigote, also inhibits mouse
bone marrow macrophage apoptosis induced by removal of macrophage CSF
in vitro (27). Although exogenous TNF- inhibits apoptosis
in this assay system and Leishmania infection induces
TNF- secretion, the inhibition was not restored by
anti-TNF--neutralizing antibodies. As far as we know, the HGE agent
is the first infectious agent known to delay apoptosis of neutrophils,
and the antiapoptotic mechanism of the HGE agent appears to be
different from the mechanisms of other intracellular microorganisms.
Therefore, the HGE agent may serve as a new tool for analysis of the
apoptotic mechanism of neutrophils.
Because the noninfectious purified HGE agent had an antiapoptotic effect and because inhibition of ehrlichial protein synthesis or proliferation did not prevent or turn off the antiapoptotic signal, infection per se is not essential for delaying apoptosis. However, the protein residue of the HGE agent, rather than carbohydrates, is required for the inhibition. When monocytic ehrlichiae are treated with trypsin, a milder proteolytic enzyme than proteinase K, ehrlichial binding and subsequent internalization in host cells is prevented (26). In addition, our MDC study showed that ehrlichial internalization and/or receptor cross-linking is required for apoptosis inhibition. The inhibitory effect of the HGE agent on apoptosis was dose dependent. These results suggest that initial occupation and cross-linking of host cell receptors by preformed proteins of the HGE agent may be sufficient to trigger the antiapoptotic signal. The reason the freshly prepared host cell-free HGE agent had stronger antiapoptotic activity than the purified HGE agent may be that the structural or conformational integrity present in the freshly prepared host cell-free HGE agent, which is lost in the purified HGE agent, is required for effective cross-linking of receptors or internalization.
E. chaffeensis, upon binding to THP-1 cells, increases
protein kinase A activity 25-fold within 30 min and inhibits tyrosine phosphorylation of Jak-1 and -2 and Stat1 in response to IFN-. This inhibition does not require the internalization of E. chaffeensis in THP-1 cells or the carbohydrate residue of the
organism, but binding of the protein of E. chaffeensis to
the host cells is required (22). It appears that these
conditions are similar to those required for delaying apoptosis by the
HGE agent. However, we did not find the involvement of protein kinase A
activation in apoptosis delay by the HGE agent. Similarly, inhibition
of basal protein kinase A activity by 25 µM H-89 has no influence on
(does not accelerate) spontaneous or cycloheximide- or anti-Fas-induced neutrophil apoptosis (32), although conditions that raise
intracellular cAMP are shown to delay spontaneous neutrophil apoptosis
and inhibit apoptosis induced by cycloheximide or anti-Fas (30,
32). Additionally, because host cell interactions with E. chaffeensis and the HGE agent differ in several respects, such as
the intracellular compartments they occupy (28) and
upregulation of host transferrin receptor mRNA (7) and
cytokine mRNA expression (21; Kim and Rikihisa, Abstr. 99th Gen. Meet. Am. Soc. Microbiol), host cell receptors and
intracellular signaling pathways may be different. The antiapoptotic mechanism may share the signaling pathway with dexamethasone-induced apoptosis, because suppression of apoptosis by both dexamethasone and
the HGE agent is abolished by cotreatment with cycloheximide (14).
The HGE agent might have LPS, since it belongs to the gram-negative
bacteria. E. coli LPS is reported to delay apoptosis of neutrophils in vitro (38, 42). Inhibition of apoptosis of neutrophils by bacterial LPS is mediated by induced pro-IL-1 and
caspase-1 through protein tyrosine phosphorylation-dependent activation
of NF-B (38, 42). IL-1 is known to delay apoptosis of
neutrophils (42), and IL-1 was also induced in
neutrophils exposed to the HGE agent (Kim and Rikihisa, Abstr. 99th
Gen. Meet. Am. Soc. Microbiol.). However, IL-1 and NF-B do not
appear to be involved in inhibition of apoptosis by the HGE agent. This suggests that the HGE agent either lacks LPS or the structure and
biological activity of ehrlichial LPS is distinct from those of
E. coli LPS.
Delayed apoptosis of neutrophils may help ehrlichial proliferation and prolong proinflammatory cytokine generation, making patients more ill and thus prone to hospitalization. In agreement with this speculation, Bakken et al. reported a higher percentage of neutrophils in the PBL of hospitalized than nonhospitalized HGE patients (6). Elucidation of an ehrlichial factor(s) and the signaling pathway in the neutrophils that inhibit apoptosis would be important in understanding the pathogenesis of HGE. The HGE agent or its protein components may also serve as a tool in analyzing the fundamental apoptotic mechanisms of neutrophils.
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ACKNOWLEDGMENTS |
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This research was supported by grant RO1AI30010 from the National Institutes of Health. K. Yoshiie was supported by a fellowship from The Japan Health Sciences Foundation.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, 1925 Coffey Rd., Columbus, OH 43210-1093. Phone: (614) 292-5661. Fax: (614) 292-6473. E-mail: rikihisa.1{at}osu.edu.
Present address: Department of Bacteriology, Faculty of Medicine,
Kagoshima University, Kagoshima 890-8520, Japan.
Editor: P. E. Orndorff
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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| 1. | Adachi, S., M. Kubota, Y. W. Lin, A. Okuda, K. Matsubara, Y. Wakazono, H. Hirota, K. Kuwakado, and Y. Akiyama. 1994. In vivo administration of granulocyte colony-stimulating factor promotes neutrophil survival in vitro. Eur. J. Haematol. 53:129-134[Medline]. |
| 2. |
Afford, S. C.,
J. Pongracz,
R. A. Stockley,
J. Crocker, and D. Burnett.
1992.
The induction by human interleukin-6 of apoptosis in the promonocytic cell line U937 and human neutrophils.
J. Biol. Chem.
267:21612-21616 |
| 3. |
Aguero-Rosenfeld, M. E.,
H. W. Horowitz,
G. P. Wormser,
D. F. McKenna,
J. Nowakowski,
J. Munoz, and J. S. Dumler.
1996.
Human granulocytic ehrlichiosis: a case series from a medical center in New York State.
Ann. Intern. Med.
125:904-908 |
| 4. | Akiyama, T., and H. Ogawara. 1991. Use and specificity of genistein as inhibitor of protein-tyrosine kinases. Methods Enzymol. 201:362-370[Medline]. |
| 5. |
Bakken, J. S.,
J. S. Dumler,
S. M. Chen,
M. R. Eckman,
L. L. van Etta, and D. H. Walker.
1994.
Human granulocytic ehrlichiosis in the upper Midwest United States. a new species emerging?
JAMA
272:212-218 |
| 6. |
Bakken, J. S.,
J. Krueth,
C. Wilson-Nordskog,
R. L. Tilden,
K. Asanovich, and J. S. Dumler.
1996.
Clinical and laboratory characteristics of human granulocytic ehrlichiosis.
JAMA
275:199-205 |
| 7. |
Barnewall, R.,
N. Ohashi, and Y. Rikihisa.
1999.
Ehrlichia chaffeensis and E. sennetsu, but not the Human granulocytic ehrlichiosis agent, colocalize with transferrin receptor and up-regulate transferrin receptor mRNA by activating iron-responsive protein 1.
Infect Immun.
67:2258-2265 |
| 8. | Brouqui, P. 1999. Ehrlichiosis in Europe, p. 220-232. In D. Raoult, and P. Brouqui (ed.), Rickettsiae and rickettsial diseases at the turn of the third millennium. Elsevier, Paris, France. |
| 9. |
Chen, S. M.,
J. S. Dumler,
J. S. Bakken, and D. H. Walker.
1994.
Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease.
J. Clin. Microbiol.
32:589-595 |
| 10. |
Chijiwa, T.,
A. Mishima,
M. Hagiwara,
M. Sano,
K. Hayashi,
T. Inoue,
K. Naito,
T. Toshioka, and H. Hidaka.
1990.
Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino) ethyl] 5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells.
J. Biol. Chem.
265:5267-5272 |
| 11. | Christa, H. E., B. Homburg, and D. Roos. 1996. Apoptosis of neutrophils. Curr. Opin. Hematol. 3:94-99[Medline]. |
| 12. |
Clifton, D. R.,
R. A. Goss,
S. K. Sahni,
D. V. Antwerp,
R. B. Baggs,
V. J. Marder,
D. J. Silverman, and L. A. Sporn.
1998.
NF-B-dependent inhibition of apoptosis is essential for host cell survival during Rickettsia rickettsii infection.
Proc. Natl. Acad. Sci. USA
95:4646-4651 |
| 13. |
Colotta, F.,
F. Re,
N. Polentarutti,
S. Sozzani, and A. Mantovani.
1992.
Modulation of granulocyte survival and programmed cell death by cytokines and bacterial products.
Blood
80:2012-2020 |
| 14. | Cox, G., J. Gauldie, and M. Jordana. 1992. Dexamethasone-induced suppression of apoptosis in human neutrophils requires continuous stimulation of new protein synthesis. J. Leukoc. Biol. 61:224-230[Abstract]. |
| 15. | Duke, R. C., and J. J. Cohen. 1986. IL-2 addiction: withdrawal of growth factor activates a suicide program in dependent T cells. Lymphokine Res. 5:289-299[Medline]. |
| 16. | Durrbaum-Landmann, I., J. Gercken, H.-D. Flad, and M. Ernst. 1996. Effect of in vitro infection of human monocytes with low numbers of Mycobacterium tuberculosis bacteria on monocyte apoptosis. Infect. Immun. 64:5384-5389[Abstract]. |
| 17. | Hsieh, T.-C., M. E. Aguero-Rosenfeld, J. M. Wu, C. Ng, N. A. Papanikolaou, S. A. Varde, I. Schwartz, J. G. Pizzolo, M. Melamed, H. W. Horowitz, R. B. Nadelman, and G. P. Wormser. 1997. Cellular changes and induction of apoptosis in human promyelocytic HL-60 cells infected with the agent of human granulocytic ehrlichiosis (HGE). Biochem. Biophys. Res. Commun. 232:298-303[CrossRef][Medline]. |
| 18. | Jensen, T. J., M. A. Loo, S. Pind, D. B. Williams, A. L. Goldberg, and J. R. Riordan. 1995. Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83:129-135[CrossRef][Medline]. |
| 19. |
Kettritz, R.,
M. L. Gaido,
H. Haller,
F. C. Luft,
C. J. Jennette, and R. J. Falk.
1998.
Interleukin-8 delays spontaneous and tumor necrosis factor--mediated apoptosis of human neutrophils.
Kidney Int.
53:84-91[CrossRef][Medline].
|
| 20. |
Kim, H.-Y., and Y. Rikihisa.
1998.
Characterization of monoclonal antibodies against the major outer membrane protein of human granulocytic ehrlichiosis agent.
J. Clin. Microbiol.
36:3278-3284 |
| 21. |
Lee, E. H., and Y. Rikihisa.
1996.
Absence of tumor necrosis factor-, interleukin-6 (IL-6), and granulocyte-macrophage colony-stimulating factor expression but presence of IL-1, IL-8, and IL-10 expression in human monocytes exposed to viable or killed Ehrlichia chaffeensis.
Infect. Immun.
64:4211-4219[Abstract].
|
| 22. |
Lee, E. H., and Y. Rikihisa.
1998.
Protein kinase A-mediated inhibition of gamma interferon-induced tyrosine phosphorylation of Janus kinases and latent cytoplasmic transcription factors in human monocytes by Ehrlichia chaffeensis.
Infect. Immun.
66:2514-2520 |
| 23. |
Levitzki, A.,
M. Willingham, and I. Pastan.
1980.
Evidence for participation of transglutaminase in receptor-mediated endocytosis.
Proc. Natl. Acad. Sci. USA
77:2706-2710 |
| 24. |
Lin, Y.,
S. Yao,
R. A. Veach,
T. R. Torgerson, and J. Hawiger.
1995.
Inhibition of nuclear translocation of transcription factor NF-B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence.
J. Biol. Chem.
270:14255-14258 |
| 25. | Meagher, L. C., J. M. Cousin, J. R. Seckl, and C. Haslett. 1996. Opposing effects of glucocorticoids on the rate of apoptosis in neutrophilic and eosinophilic granulocytes. J. Immunol. 156:4422-4428[Abstract]. |
| 26. |
Messick, J. B., and Y. Rikihisa.
1993.
Characterization of Ehrlichia risticii binding, internalization, and growth in host cells by flow cytometry.
Infect. Immun.
61:3803-3810 |
| 27. | Moore, K. J., and G. Matlashewski. 1994. Intracellular infection by Leishmania donovani inhibits macrophage apoptosis. J. Immunol. 152:2930-2937[Abstract]. |
| 28. |
Mott, J.,
R. Barnewall, and Y. Rikihisa.
1999.
Human granulocytic ehrlichiosis agent and Ehrlichia chaffeensis reside in different cytoplasmic compartments in HL-60 cells.
Infect. Immun.
67:1368-1378 |
| 29. |
Nash, P. B.,
M. B. Purner,
R. P. Leon,
P. Clarke,
R. C. Duke, and T. J. Curiel.
1998.
Toxoplasma gondii-infected cells are resistant to multiple inducers of apoptosis.
J. Immunol.
160:1824-1830 |
| 30. | Ottonello, L., R. Gonella, P. Dapino, C. Sacchetti, and F. Dallegri. 1998. Prostaglandin E2 inhibits apoptosis in human neutrophilic polymorphonuclear leukocytes: role of intracellular cyclic AMP levels. Exp. Hematol. 26:895-902[Medline]. |
| 31. |
Park, J., and Y. Rikihisa.
1991.
Inhibition of Ehrlichia risticii infection in murine peritoneal macrophages by gamma interferon, a calcium ionophore, and concanavalin A.
Infect. Immun.
59:3418-3423 |
| 32. |
Parvathenani, L. K.,
S. Buescher,
E. Chacon-Cruz, and S. J. Beebe.
1998.
Type I cAMP-dependent protein kinase delays apoptosis in human neutrophils at a site upstream of caspase-3.
J. Biol. Chem.
273:6736-6743 |
| 33. | Pericle, F., J. H. Liu, J. I. Diaz, D. K. Blanchard, S. Wei, G. Forni, and J. Y. Djeu. 1994. Interleukin-2 prevention of apoptosis in human neutrophils. Eur. J. Immunol. 24:440-444[Medline]. |
| 34. |
Rikihisa, Y.
1991.
The tribe Ehrlichieae and ehrlichial diseases.
Clin. Microbiol. Rev.
4:286-308 |
| 35. |
Rikihisa, Y.
1991.
Cross-reacting antigens between Neorickettsia helminthoeca and Ehrlichia spp. shown by immunofluorescence and Western immunoblotting.
J. Clin. Microbiol.
29:2024-2029 |
| 36. |
Rikihisa, Y.,
Y. Zhang, and J. Park.
1994.
Inhibition of infection of macrophages with Ehrlichia risticii by cytochalasins, monodansylcadaverine, and taxol.
Infect. Immun.
62:5126-5132 |
| 37. | Rikihisa, Y., N. Zhi, G. Wormser, B. Wen, H. W. Horowitz, and K. E. Hechemy. 1997. Direct isolation and cultivation of human granulocytic ehrlichia from a human patient. J. Infect. Dis. 175:210-213[Medline]. |
| 38. | Sweeney, J. F., P. K. Nguyen, G. M. Omann, and D. B. Hinshaw. 1998. Lipopolysaccharide protects polymorphonuclear leukocytes from apoptosis via tyrosine phosphorylation-dependent signal transduction pathways. J. Surg. Res. 74:64-70[CrossRef][Medline]. |
| 39. |
Thornberry, N.,
H. G. Bull,
J. R. Calaycay,
K. T. Chapman,
A. D. Howard,
M. J. Kostural,
D. K. Miller,
S. M. Molineaux,
J. R. Weidner,
J. Aununs,
K. O. Elliston,
J. M. Ayala,
F. J. Casano,
J. Chin,
G. J.-F. Ding,
L. A. Egger,
E. P. Gaffney,
G. Limjuco,
O. C. Palyha,
S. M. Raju,
A. M. Rolando,
J. P. Sally,
T.-T. Yamin,
T. D. Lee,
J. E. Shively,
M. MacCross,
R. A. Mumford,
J. A. Schmidt, and M. J. Tocci.
1992.
A novel heterodimeric cysteine protease is required for interleukin-1 processing in monocytes.
Nature
356:768-774[CrossRef][Medline].
|
| 40. |
Ward, C.,
E. R. Chilvers,
M. F. Lawson,
J. G. Pryde,
S. Fujihara,
S. N. Farrow,
C. Haslett, and A. G. Rossi.
1999.
NF-B activation is a critical regulator of human granulocyte apoptosis in vitro.
J. Biol. Chem.
274:4309-4318 |
| 41. | Watson, R. W. G., H. P. Redmond, J. H. Wang, C. Condron, and D. Bouchier-Hays. 1996. Neutrophils undergo apoptosis following ingestion of Escherichia coli. J. Immunol. 156:3986-3992[Abstract]. |
| 42. |
William, R.,
G. Watson,
O. D. Rotstein,
J. Parodo,
R. Bitar, and J. C. Marshall.
1998.
The IL-1-converting enzyme (caspase-1) inhibits apoptosis of inflammatory neutrophils through activation of IL-1.
J. Immunol.
161:957-962 |
| 43. | Winjum, N., and L. K. Riley. 1993. In vitro proliferation of a canine granulocytic Ehrlichia. Vet. Microbiol. 34:355-362[CrossRef][Medline]. |
| 44. | Woldehiwet, Z., and G. R. Scott. 1982. Stages in the development of Cytoectes phagocytophila, the causative agent of tick-borne fever. Comp. Pathol. 92:469-474. |
| 45. |
Zhi, N.,
N. Ohashi, and Y. Rikihisa.
1999.
Multiple p44 genes encoding major outer membrane proteins are expressed in the human granulocytic ehrlichiosis agent.
J. Biol. Chem.
274:17828-17836 |
| 46. | Zhi, N., Y. Rikihisa, H.-Y. Kim, G. P. Wormser, and H. W. Horowitz. 1997. Comparison of major antigenic proteins of six strains of human granulocytic ehrlichiosis agents by Western immunoblot analysis. J. Clin. Microbiol. 35:2606-2611[Abstract]. |
Infection and Immunity, March 2000, p. 1125-1133, Vol. 68, No. 3
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Martin, M. E., Caspersen, K., Dumler, J. S.
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158: 1881-1888
[Abstract] [Full Text] -
Sotomayor, E. A., Popov, V. L., Feng, H.-M., Walker, D. H., Olano, J. P.
(2001). Animal Model of Fatal Human Monocytotropic Ehrlichiosis. Am. J. Pathol.
158: 757-769
[Abstract] [Full Text] -
Mott, J., Rikihisa, Y.
(2000). Human Granulocytic Ehrlichiosis Agent Inhibits Superoxide Anion Generation by Human Neutrophils. Infect. Immun.
68: 6697-6703
[Abstract] [Full Text]
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