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Infection and Immunity, February 2000, p. 594-602, Vol. 68, No. 2
Department of Microbiology, Seoul National
University College of Medicine, Seoul 110-799,1
Department of Microbiology and Immunology, Sungkyunkwan University
School of Medicine, Suwon 440-746,2 and
Department of Microbiology, Cheju National University College of
Medicine, Cheju 690-756,3 Republic of
Korea
Received 9 August 1999/Returned for modification 10 September
1999/Accepted 4 November 1999
Scrub typhus, caused by Orientia tsutsugamushi
infection, is characterized by local as well as systemic inflammatory
manifestations. Inflammation is initiated by O. tsutsugamushi-infected macrophages and endothelial cells in the
dermis. We investigated the regulation of chemokine induction in
macrophage cell line J774A.1 in response to O. tsutsugamushi infection. The mRNAs for macrophage inflammatory proteins 1 Orientia tsutsugamushi,
an obligate intracellular bacterium, is the causative agent of scrub
typhus (20). The disease is characterized by fever, rash,
eschar, pneumonitis, menigitis, and disseminated intravascular
coagulation, which leads to severe multiorgan failure (1, 11,
62). O. tsutsugamushi causes local inflammations
accompanying eschars at the site of infection, which then spread
systemically (6). O. tsutsugamush infects a
variety of cells in vitro and in vivo, including macrophages, polymorphonuclear leukocytes (PMN), lymphocytes, and endothelial cells
(26, 38, 42, 47).
Analysis of early immunologic responses to O. tsutsugamushi
infection in mice showed that macrophage-mediated cellular immunity is
essential for resolution of this infection (8, 39).
Resistance to the lethal effects of acute rickettsia infection is under
unigenic dominant control by the Ric locus (21).
Macrophages infiltrate both susceptible (Rics)
and resistant (Ricr) mouse strains in response
to O. tsutsugamushi infection (25, 39). A slight
increase occurs in the number of infiltrating cells recovered from
resistant mice. Although susceptible mice experienced slower cellular
infiltration, the number of infiltrating macrophages was larger than
that in resistant mice (39). The resistant strain of mice
was reported to have less PMN response to O. tsutsugamushi
than a susceptible strain did (26). Induction of nonspecific
inflammation leading to the recruitment of PMN rendered resistant mice
susceptible to rickettsia infection (26). As a result,
susceptible mice died within 2 weeks of infection. By contrast,
Ricr strains showed a minimal level of infection
over 2 weeks and survived the infection (27, 39).
Mononuclear cells such as lymphocytes and macrophages as well as PMN
were observed in eschars and rashes caused by scrub typhus
(1). It is notable that in a patient who died after 2 days,
infiltration of considerable numbers of PMN was observed around some of
the blood vessels (1). Early host inflammatory responses
seem to play a key role in determining the fate of the host infected
with O. tsutsugamushi (39, 53). For these
reasons, the regulatory components that determine the quality and
magnitude of the cellular influx to the site of the rickettsia
infection should be analyzed. Proinflammatory mediators and chemokines
play an important role in these processes (4, 24). The
expression of chemokines and their kinetics, however, have not been
elucidated in the disease caused by O. tsutsugamushi.
Chemokines are the key players in the processes of leukocyte
recruitment into inflammatory tissues. The interaction of different chemokines with their receptors on leukocytes allows selective activation and chemotaxis of neutrophils, lymphocytes, or monocytes necessary for migration to the sites of evolving inflammation. It has
been shown that during infection, infected macrophages produce a subset
of chemokines (17, 46, 56). The cellular influx into
inflamed tissue is provoked by gradients of chemokines that contribute
to the adhesion of leukocytes to endothelium, transendothelial
migration, and movement through the extracellular matrix
(24).
Activated monocytes and macrophages are the major chemokine-secreting
cells (4). Various sets of chemokines are produced by
monocytes and macrophages infected with different pathogenic microorganisms (7, 17, 45, 46, 56). The kinetics of chemokine gene expression also varies according to the microorganism studied. The regulation of chemokine gene expression, as a defense mechanism against pathogenic microorganism, seems to be related to the
clinical courses of the infected host (33, 34, 37, 44).
The transcription factor NF- Cell culture.
J774A.1 cells were obtained from the American
Type Culture Collection, Rockville, Md., and cultured in Dulbecco's
modified Eagle's medium (Gibco BRL, Grand Island, N.Y.) containing
10% (vol/vol) heat-inactivated fetal bovine serum (FBS) (Gibco BRL), 100 µg of streptomycin per ml, 100 U of penicillin per ml, and 2 mM
L-glutamine (DMEM-10) in a humidified 5% CO2
atmosphere at 37°C. The cells were seeded onto six-well plates
(Becton Dickinson Labware, Franklin Lakes, N.J.) for the preparation of
mRNA or onto 100-mm dishes (Becton Dickinson Labware) for the
preparation of nuclear extract. The prototype strain, O. tsutsugamushi Karp (American Type Culture Collection) was
propagated in monolayers of L-929 cells as described previously
(32, 51). When more than 90% of the cells were infected, as
determined by an indirect immunofluorescent-antibody technique
(9), the cells were collected, homogenized with a glass
Dounce homogenizer (Wheaton Inc., Millville, N.J.), and centrifuged at
500 × g for 5 min. The supernatant was centrifuged at
10,000 × g for 10 min, and the rickettsia pellet was
resuspended in DMEM-10 and stored in liquid nitrogen until use. The
infectivity titer of the inoculum was determined as described previously with modification (31, 57). Briefly, fivefold
serially diluted rickettsia samples were inoculated onto L-929 cell
layers on 24-well tissue culture plates. After 3 days of incubation, the cells were collected, fixed, and stained as described previously (31). The ratio of infected cells to the counted number of
cells was determined microscopically, and infected-cell counting units (ICU) of the rickettsia sample were calculated as follows
(57): ICU = (total number of cells used in
infection) × (percentage of infected cells) × (dilution
rate of the rickettsiae suspension)/100.
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Expression of Chemokine Genes in Murine Macrophages
Infected with Orientia tsutsugamushi
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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(MIP-1/), MIP-2, and macrophage chemoattractant protein 1 were induced within 30 min, and their levels showed a
transitory peak for 3 to 12 h. However, the lymphotactin, eotaxin, gamma interferon-inducible protein 10, and T-cell activation gene 3 mRNAs were not detected by RNase protection assays. Heat-killed O. tsutsugamushi induced a similar extent of chemokine
responses. Induction of the chemokine genes was not blocked by the
eukaryotic protein synthesis inhibitor cycloheximide, suggesting that
de novo synthesis of host cell protein is not required for these transcriptional responses. The induction of chemokine mRNAs by O. tsutsugamushi was blocked by the inhibitors of NF-
B
activation. Furthermore, O. tsutsugamushi induced the
nuclear translocation and activation of NF-B. These results
demonstrate that heat-stable molecules of O. tsutsugamushi
induce a subset of chemokine genes and that induction involves
activation of the transcription factor NF-B.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
B is known to play an important role in
the regulation of inflammatory mediators, such as cytokines, acute-phase proteins, and adhesion molecules (5, 19). Since many of the chemokine genes are also regulated by NF-B
(61), it is possible that O. tsutsugamushi
induces the chemokine genes through activation of NF-B. In this
study, we analyzed the transcriptional activation of a subset of
chemokine genes in a murine macrophage cell line during O. tsutsugamushi infection. The activation of transcription factor
NF-B was also shown to be involved in the induction of chemokine
genes by O. tsutsugamushi.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
RNase protection assay.
Total RNA was prepared with RNeasy
kit (Qiagen GmbH, Hilden, Germany) as specified by the manufacturer and
was quantified spectrophotometrically. Detection and semiquantification
of various murine chemokine mRNAs were performed with the multiprobe
RNase protection assay system from Pharmingen (San Diego, Calif.). In brief, a mixture of [32P]CTP-labeled antisense riboprobes
was generated from chemokine template DNAs including lymphotactin
(Ltn), RANTES, eotaxin, macrophage inflammatory protein 1
(MIP-1), MIP-1, MIP-2, gamma interferon-inducible protein 10 (IP-10), macrophage chemoattractant protein-1 (MCP-1), and T-cell
activation gene 3 (TCA-3). The template DNAs for the murine
housekeeping genes encoding glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) and a murine ribosomal protein, L32, was also included to
ensure equal loading of total RNA onto the gels. Total RNAs from each
sample (10 µg each) were hybridized overnight at 56°C with 2 × 105 cpm of the 32P-labeled antisense
riboprobe mixture. After hybridization, the samples were digested with
a mixture of RNases A and T1. Nuclease-protected RNA
fragments were precipitated with ethanol. The samples were resolved on
a 5% polyacrylamide sequencing gel (52). The bands were
observed after autoradiography. The specific chemokine bands were
identified on the basis of their individual mobilities compared with
labeled standard probes. The band intensities shown in autoradiography were digitized by scanning the images and analyzed with TINA software (Raytest Isotopenmeßgeräte GmbH, Straubenhardt, Germany). The densitometric intensity was normalized with respect to the intensities of the band for the housekeeping genes, GAPDH and L32.
Semiquantitative RT-PCR.
Total RNA extracted from each
sample (2 to 5 µg per sample) was subjected to first-strand cDNA
synthesis at 42°C for 1 h in a 40-µl reaction mixture
containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2,
1 mM deoxynucleoside triphosphate mixture, 1 U of RNasin per µl, 2.5 µM oligo(dT) primer, and 100 U of murine leukemia virus reverse
transcriptase (RT) (all from Perkin-Elmer, Branchburg, N.J.). The cDNA
was heated at 94°C for 5 min and diluted with water. The cDNA amounts
equivalent to 100 ng of total RNA were subjected to PCR amplification
in a 20-µl reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM
KCl, 2 mM MgCl2, 0.5 mM deoxynucleoside triphosphate
mixture, 1 µM each primer, and 0.2 U of AmpliTaq DNA polymerase
(Perkin-Elmer) in a Gene Cycler (Bio-Rad Laboratories Inc., Hercules,
Calif.). The reaction mixture was prepared as a master mixture to
minimize reaction variation. One PCR cycle consisted of denaturation at
94°C for 30 s, annealing at 55°C for 30 s, and extension
at 72°C for 1 min. The PCR products (5-µl samples) were
electrophoresed in a 1.5% agarose gel containing 0.5 µg of ethidium
bromide per ml. If not otherwise specified, a 123-bp DNA ladder (Gibco
BRL) was used at 1 µg/lane as molecular size markers to provide bands
from 4,182 to 123 bp. The amplified DNA fragments in the gels were
identified according to their size predicted by cDNA sequences reported
previously (2, 15, 22, 30, 54, 58). The densities of the
bands were analyzed as described previously (52). The
densitometric intensity was normalized by comparing the ratio of
chemokine bands with that of -actin. PCR was performed for the
following number of cycles for each set of primers to ensure that the
assay was in the linear range according to the amount of template (data
not shown): RANTES, 30; MIP-1, 20; MIP-1, 25; MIP-2, 25; MCP-1,
25; -actin, 25. The 5' and 3' sequences of the primers and the size
of PCR products are as follows: RANTES (215 bp), 5'-CCT CAC CAT CAT CCT
CAC TGC A-3', 5'-TCT TCT CTG GGT TGG CAC ACA C-3'; MIP-1 (390 bp),
5'-AAC CCC GAG CAA CAC CAT GAA G-3', 5'-TGA ACG TGA GGA GCA AGG ACG
C-3'; MIP-1 (357 bp) 5'-GGT CTC CAC CAC TGC CCT TGC-3', 5'-GGT GGC AGG AAT GTT CGG CTC-3'; MIP-2 (536 bp), 5'-AGT TTG CCT TGA CCC TGA AGC
C-3', 5'-CCA TGA AAG CCA TCC GAC TGC A-3'; MCP-1 (582 bp), 5'-TCT CTT
CCT CCA CCA CCA TGC AG-3', 5'-GGA AAA ATG GAT CCA CAC CTT GC-3';
-actin (349 bp), 5'-TGG AAT CCT GTG GGA TCC ATG AAA C-3', 5'-TAA AAC
GCA GCT CAG TAA CAG TCC G-3'.
EMSA.
Nuclear extraction and electrophoretic mobility shift
assay (EMSA) were performed as described previously with some
modifications (10). Following infection with O. tsutsugamushi, J774A.1 cells were washed with cold
phosphate-buffered saline (PBS) and collected by centrifugation
(500 × g for 5 min). The cells were resuspended in 100 µl of buffer (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride), vigorously vortexed for 15 s, and allowed to stand in ice for 10 min. The nuclei were pelleted by centrifugation (400 × g for 2 min) and resuspended for 20 min on ice in 50 µl of cold
buffer containing 20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.2 mM
phenylmethylsulfonyl fluoride. Nuclear debris were removed by
centrifugation (13,000 × g for 5 min) at 4°C, and
nuclear extracts were collected. Protein concentrations were determined
with the bicinchoninic acid protein assay reagent (Pierce Chemical Co.
Rockford, Ill.). Aliquots of the supernatant were frozen in liquid
nitrogen and stored at 
70°C until use. Equal amounts of nuclear
extracts (10 µg of protein) from each sample were incubated for 30 min at 25°C in 30 µl of binding buffer (10 mM Tris-HCl [pH 7.5],
75 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 4% Ficoll) containing 2 µg of sonicated salmon sperm DNA and 30,000 cpm of an
NF-B-specific oligonucleotide probe that was radiolabeled with
[
-32P]ATP (Amersham Ltd., Little Chalfont, England).
The sequence of the NF-B-specific probe was 5'-AGT TGA GGG GAC TTT
CCC AGG C-3'. To ascertain the specific binding of nuclear extracts
with NF-B probe, a competition assay was performed with a 50-fold molar excess of unlabeled oligonucleotides. Nuclear translocation of
NF-B heterodimer was analyzed by a supershift assay with anti-p65 antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.). The nuclear extract proteins were mixed with 4 µg of anti-p65 antibody and were
hybridized with the NF-B-specific oligonucleotide probe. The
supershifted bands were analyzed after separation on 5% nondenaturing polyacrylamide gels and autoradiography.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Induction of chemokine gene expression.
Before and after
exposure of J774A.1 cells to O. tsutsugamushi, the levels of
chemokine transcripts were assayed at each time point by an RNase
protection assay and semiquantitative RT-PCR (Fig.
1). Although there were some variations
in the ratio of RNA transcripts for the control and the test groups
between the sets of RT-PCR experiments, the changes within a set of
experiments were reproducible throughout this study. The absence of
contamination of RNA with genomic DNA was monitored by the size of PCR
products from the pair of primers whose binding sites are located in
different exons. The mRNAs of the CC chemokines MIP-1, MIP-1, and
MCP-1 were constitutively expressed at low levels in noninfected
J774A.1 cells (Fig. 1). Basal levels of these chemokines are also
expressed constitutively in monocytes and macrophages (17,
56). The mRNAs for MIP-1, MIP-1, MCP-1, and MIP-2 were
up-regulated and detected as early as 30 min after infection, peaked at
6 h, and began to decrease from 6 to 12 h after infection.
While the MIP-1 and MIP-2 mRNAs persisted after incubation for
48 h, the levels of transcripts for MIP-1 and MCP-1 were
reduced to the levels in uninfected cells by 48 h. The transcript
for RANTES was also detectable as early as 3 h after infection.
However, the level of this transcript was significantly lower
than those of other induced chemokines. Expression of RANTES
mRNA was characterized by slower kinetics compared to those of
other induced chemokine mRNAs. The peak response for RANTES was
observed 12 h after infection and decreased to the level of
uninfected cells by 48 h. Similar kinetics of mRNA expression were
detected when the levels of chemokine mRNAs were analyzed by either the
RNase protection assay or semiquantitative RT-PCR (Fig. 1). No mRNA for
Ltn, eotaxin, IP-10, and TCA-3 was detected during infection by RNase
protection.
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X174 DNA digested with HaeIII; N,
negative control (reactions performed without cDNA).
and
MCP-1 showed an approximately fivefold increase in their optical
densities. Although the RANTES, MIP-1, and MCP-1 mRNAs were detected
in cells treated with medium or L-929 cell lysate, the levels in cells
incubated with O. tsutsugamushi increased by approximately
two- to fivefold as measured by their optical densities. Compared to
control groups, cells incubated for 6 h with O. tsutsugamushi resulted in higher levels of mRNAs of all the
chemokines tested (Fig. 2). To determine whether the chemokine
induction was a specific response to O. tsutsugamushi infection, we investigated whether the phagocytosis of polystyrene beads similar in size to O. tsutsugamushi would provide a
stimulus for chemokine gene expression. Although the levels of mRNA of RANTES, MIP-1, and MCP-1 were increased by incubation with
polystyrene beads, they were similar to those of the cells treated with
an L-929 cell lysate. The mRNA levels in the cells infected with O. tsutsugamushi were approximately 2- to 10-fold higher as
measured by their optical densities than were those in the cells
treated with polystyrene beads. The chemokine genes are induced by
proinflammatory cytokines such as interleukin-1 (IL-1) and tumor
necrosis factor alpha (61). To investigate whether the
chemokine induction was a consequence of the host cytokine expression,
cells were incubated for 1 h with CHX, a eukaryotic protein
synthesis inhibitor, and then infected with O. tsutsugamushi. Although the chemokine genes were induced when the
cells were treated only with CHX (16, 61), higher levels of
chemokine mRNAs were observed when CHX-treated cells were infected with
O. tsutsugamushi (Fig. 2).
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, and MCP-1, the levels of the mRNAs of MIP-1 and MIP-2 were
dramatically reduced to those similar to the levels in the control
group by polymyxin B treatment. In contrast, in cells treated with
polymyxin B and O. tsutsugamushi, mRNA levels for all the
chemokines tested did not differ significantly from those induced by
stimulation with O. tsutsugamushi. These results show that
possible exogenous sources of LPS are not responsible for the induction
of the chemokine genes.
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Chemokine expression by NF-B activation.
To examine whether
NF-B activation is involved in the chemokine induction of O. tsutsugamushi-exposed J774A.1 cells, we used two inhibitors of
NF-B activation, the antioxidant PDTC (50) and the
proteasome inhibitor TPCK (36). When the cells were incubated with O. tsutsugamushi in the presence of TPCK,
induction of RANTES, MIP-1, and MIP-2 was inhibited completely (Fig.
4). Although induction of MIP-1 and
MCP-1 was not completely blocked by the inhibitor, the levels of their
transcripts were reduced by one-half and one-third, respectively,
compared to those in the cells treated with O. tsutsugamushi
alone. Expression of MIP-1, MIP-2, and MCP-1 genes was also
inhibited in the presence of PDTC by approximately one-half to
one-third as judged by measurement of their optical densities, while
induction of RANTES and MIP-1 was also completely blocked in
O. tsutsugamushi-infected cells (Fig. 4).
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B activation by O. tsutsugamushi was directly
evaluated by nuclear translocation of NF-B and EMSA (Fig.
5). At 2 h after infection of
macrophages with O. tsutsugamushi, we could detect a
mobility-shifted complex which was competed off by an unlabeled probe
(50-fold excess of competitor) corresponding to the B binding domain
of the murine kappa light-chain gene enhancer. The bands at the front
of NF-B complex observed in all lanes might be probe binding to
nonspecific proteins (36). Although the basal levels of
NF-B complexes were detected in the cells treated with medium or
L-929 cell lysate, the activation and nuclear translocation of NF-B
were remarkably increased when stimulated with O. tsutsugamushi. When the levels of NF-B activation were
normalized to nonspecific bands, the level of activation in the
O. tsutsugamushi-infected cells was increased two- or
threefold compared to that in cells treated with L-929 cell lysate or
medium only. In the presence of NF-B activation inhibitors, however,
the levels of NF-B complex were decreased and comparable to those of
control groups. The proteasome inhibitor TPCK was more effective in
inhibiting NF-B activation than was PDTC. The p50/p65 heterodimeric
form of NF-B is the prototypical and transcriptionally active
complex, while the p50/p50 homodimeric form is constitutively
present and is thought to be an inactive or repressive complex (5,
19). The p50/p65 heterodimeric form of the NF-B complex was
confirmed by a supershift assay with a p65-specific antibody (Fig. 5B). The NF-B complexes shown in Fig. 5A were more widely separated, and,
furthermore, incubation with anti-p65 antibody resulted in the loss of
a band (Fig. 5B). The remaining lower complex of NF-B might
represent the p50/p50 homodimeric form of NF-B (16), although we did not identify the homodimeric complex by using a
p50-specific antibody. The basal level of the heterodimeric complex of
NF-B was also detected in the cells treated with medium only. The
level of the heterodimeric form in the O. tsutsugamushi-infected cells was approximately three times higher,
as measured by optical density, than in the control group after
nonspecific bands were normalized for. The supershifted complex was
detectable only in the O. tsutsugamushi-stimulated cells.
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Heat stability of the stimulating molecule.
To evaluate
whether active rickettsia replication was required for chemokine
induction, we exposed macrophages to heat-inactivated O. tsutsugamushi for 6 h. As shown in Fig.
6, mRNA levels of MIP-1, MCP-1, and
MIP-2 in cells treated with heat-inactivated O. tsutsugamushi were comparable to those in cells treated with the
live microorganism. The kinetics of chemokine expression in cells
stimulated with heat-inactivated O. tsutsugamushi were also
similar to those of expression in the cells treated with live O. tsutsugamushi (data not shown). However, in cells treated with
heat-inactivated O. tsutsugamushi, the levels of RANTES and
MIP-1 were reduced in optical density by 30 and 50%, respectively,
compared to those in cells treated with active O. tsutsugamushi.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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It has been well documented that macrophages play a pivotal role in early immune responses to O. tsutsugamushi infection (27, 39-41). Although the inactive tissue macrophages could support the growth of O. tsutsugamushi at the site of infection, subsequent cellular influxes, especially of activated macrophages and lymphocytes, have been suggested to be important in protection against O. tsutsugamushi infection (25, 26). Early PMN responses seem to provide a cellular population for rickettsia replication instead of providing antirickettsial activity in vivo (26). The cellular recruitment is controlled largely by chemokines which are secreted by stimulated cells such as macrophages at the site of primary infection (34, 61).
In this study, we showed that the murine macrophage cell line J774A.1
induced the expression of MIP-1, MIP-1, RANTES, MCP-1, and MIP-2
in response to O. tsutsugamushi infection. With the exception of RANTES, the induction of the chemokine genes occurred within 30 min and peaked transiently between 3 and 12 h. The
inducibility and the kinetics of these chemokines are different from
those of murine macrophages infected with other pathogenic
microorganisms (45, 46). The differences in the patterns of
early chemokine responses to various pathogens are likely to be related
to disease manifestations (49). Although we did not perform
assays to confirm the secretion of active chemokine protein following
gene induction, several recent studies have shown a correlation between
mRNA expression and chemokine protein secretion (7, 17, 46,
56).
The chemokine genes were induced specifically in response to O. tsutsugamushi infection. Ingestion of polystyrene beads by macrophages resulted in little or no induction of the chemokine genes tested. Phagocytosis of inert particles such as latex beads by murine macrophages did not affect the basal levels of cytokines and chemokines (45). Contamination by LPS during the preparation of O. tsutsugamushi was also examined. It has been previously reported that the cell wall component in O. tsutsugamushi is deficient in LPS (3). Blocking LPS with polymyxin B did not decrease chemokine responses in macrophages infected with O. tsutsugamushi. These results suggest that O. tsutsugamushi-mediated induction of chemokine genes requires certain signals which are not generated by nonspecific phagocytosis of macrophages and, in addition, are not mediated by LPS. This finding is intriguing in light of the fact that throughout the entire course of the O. tsutsugamushi infection, the macrophage is one of the main target cells for rickettsia parasitism. The ability of O. tsutsugamushi to selectively induce the expression of a subset of chemokines in vitro represents the earliest host response to infection and could play a role in early manifestations following skin infection in vivo.
Previous studies had already demonstrated that various cytokines stimulate chemokine expression in vitro. We therefore examined the role of newly synthesized proteins in chemokine gene expression (61). When CHX, the eukaryotic protein synthesis inhibitor, was included in macrophage cultures, the levels of the chemokine mRNAs were similar to those in cells infected with O. tsutsugamushi alone. This indicated that chemokine induction was not an indirect effect due to prior induction of tumor necrosis factor alpha or IL-1, which are known to induce chemokine production in macrophages (4).
The transcription factor NF-B/Rel family plays a central role in the
regulation of a variety of genes involved in host innate immunity,
including various chemokines (19). For all the chemokine genes tested in this study, regulation by NF-B either has been demonstrated or is suggested by the presence of the NF-B consensus motif in the promoter (14, 18, 61, 63). We have found that
O. tsutsugamushi induces an increase in the levels of
active NF-B in the nucleus, particularly of the p65/p50
heterodimer. The induction of the chemokine mRNAs by O. tsutsugamushi is completely or partially blocked by inhibitors of
NF-B activation. PDTC, an antioxidant, inhibits the phosphorylation
of IB (50), a prerequisite for its subsequent proteolytic
degradation. TPCK, an inhibitor of chymotryptic activity associated
with the proteasome, blocks activation of NF-B by inhibiting
proteasome-dependent degradation of the inhibitory peptides
(36). These chemically unrelated compounds reduce
O. tsutsugamushi-induced RANTES and MIP-1 mRNA
levels, implicating NF-B as the main transcription factor in the
expression of these chemokines. The mRNA expression of MIP-1 and
MCP-1 was partially blocked by treatment with PDTC and TPCK. These
findings strongly suggest that O. tsutsugamushi induces gene
expression of the chemokines in J774A.1 cells via proteasome-sensitive
and reactive oxygen intermediate-sensitive pathways that have been
implicated in the activation of NF-B (5, 19). Our data
indicates that TPCK is likely to be more effective in inhibiting the
expression of the chemokine genes, especially for MIP-2, and in
activating NF-B. Although NF-B is essential for the transcription
of the chemokine genes, a number of other transcription factors form
activating complexes capable of up-regulating chemokine gene
expression. Various transcriptional regulatory elements apart from
NF-B are required for the expression of MCP-1 (18, 60,
61). In addition, various potential cis-regulatory elements have been identified in the upstream region of the MIP-1 gene (63). For these reasons, it appears that O. tsutsugamushi activates signal transduction pathways leading to
activation of those transcription factors as well as to activation of
NF-B. Although direct evidence was not provided, this data suggests that the induction of chemokines in J774A.1 cells and the activation of
NF-B are physiologically relevant.
A recent report has suggested that NF-B activation by rickettsia
infection is related to the inhibition of apoptosis in endothelial cells and fibroblasts. This provides a possible mechanism to enable host cells to remain as a site for rickettsiae replication
(13). However, it has also been reported that apoptotic
death of macrophages and lymphocytes occurs in the spleen and lymph
nodes of O. tsutsugamushi-infected mice (29).
Furthermore, apoptosis was also observed in J774A.1 cells within
12 h of O. tsutsugamushi infection (12).
Further study of apoptosis modulation in rickettsia-infected cells by NF-B activation is needed.
In addition, we tried to investigate rickettsia molecules eliciting chemokine responses of macrophages. The physicochemical characteristic of the molecule was analyzed after O. tsutsugamushi was subjected to heat treatment. The expression of chemokine mRNAs was unchanged whether the cells were treated with heat-killed or living O. tsutsugamushi. This suggests that heat-stable rickettsia molecules may be involved in activating transcription factors and that proliferation of O. tsutsugamushi within infected macrophages is not a prerequisite for expression of those chemokines. Further studies on stimulatory components of O. tsutsugamushi and signal transduction pathways in host cells during rickettsia infection will provide valuable insights into the mechanisms controlling the inflammatory responses during O. tsutsugamushi infection.
Protective immunity against O. tsutsugamushi is largely due
to cell-mediated immune responses, particularly those provided by
macrophages and T cells (28, 40, 51). The explanation for a
susceptible/resistant mouse phenotype to O. tsutsugamushi infection was provided by the analysis of the early T-lymphocyte activation 1 (Eta-1)/osteopontine (Op) gene,
which maps to the Ric locus (21, 43). Eta-1/Op
has been thought to enhance resistance to rickettsia infection by
affecting the ability of macrophages to migrate to sites of infection
and/or to express bactericidal activity (43). However, the
infiltration of T lymphocytes and their secretion of Eta-1/Op in the
early stage of infection should be preceded by activation of
macrophages and their chemokine secretions, which recruit specific and
nonspecific immune cells. In other studies, genetic susceptibility to
infectious disease has been shown to be associated with the expression
of different cytokine profiles (23). Members of the CC
chemokine subfamily, which include RANTES, MIP-1, MIP-1, and
MCP-1, preferentially attract monocytes and lymphocytes. Those of the
CXC chemokine subfamily, such as IL-8 and MIP-2, are potent neutrophil
attractants (4). Furthermore, a correlation between
chemokines and a subset of T-cell responses has been described
(35, 48, 55). While the CC chemokines MIP-1, MIP-1,
and RANTES were found to be efficient chemoattractants for Th1 cells,
Th2 cells were not attracted by these chemokines (55).
Stimulation of T cells in the presence of MIP-1 enhanced gamma
interferon production by Th1 cells, while stimulation of T cells in the
presence of MCP-1 led to an increase IL-4 production (35).
Based on these studies, we hypothesize that a delicate balance of
chemokines exists between the induction of a resistant and a
susceptible immune response to rickettsia infection. Further study is
required to determine whether qualitative and quantitative differences
in the production of chemokines can be correlated with the resistant or
susceptible mouse phenotype.
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ACKNOWLEDGMENT |
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This work was supported by the Korea Research Foundation of the Republic of Korea (grant 97110814).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Republic of Korea. Phone: 82-2-740-8301. Fax: 82-2-743-0881. E-mail: seongsy{at}plaza.snu.ac.kr.
Editor: J. D. Clements
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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