INFECTION AND IMMUNITY, Aug. 2002, p. 4132–4141 Vol. 70, No.

8
0019-9567/02/$04.00⫹0 DOI: 10.1128/IAI.70.8.4132–4141.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Roles of p38 Mitogen-Activated Protein Kinase, NF-␬B, and Protein

Kinase C in Proinflammatory Cytokine mRNA Expression by
Human Peripheral Blood Leukocytes, Monocytes, and
Neutrophils in Response to Anaplasma phagocytophila
Hyung-Yong Kim and Yasuko Rikihisa*
Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio 43210-1093
Received 26 November 2001/Returned for modification 15 February 2002/Accepted 24 April 2002

Anaplasma phagocytophila, an obligately intracellular bacterium of granulocytes, causes human granulocytic

ehrlichiosis. Within 2 h after addition of A. phagocytophila, interleukin-1␤ (IL-1␤), tumor necrosis factor alpha
(TNF-␣), and IL-6 mRNAs are induced in human peripheral blood leukocytes (PBLs) or monocytes in vitro.
However, neutrophils generate only IL-1␤ mRNA. In the present study, signaling pathways for induction of
these three cytokines were examined. TNF-␣ and IL-6 mRNA expression by PBLs was inhibited with SB 203580
(a p38 mitogen-activated protein kinase [MAPK] inhibitor), MG-132 (a proteasome inhibitor), and SN-50 (an
NF-␬B inhibitor). Activation of p38 MAPK and NF-␬B mRNAs in monocytes was detectable within 15 to 30 min
after addition of A. phagocytophila. Expression of these two cytokine mRNAs in PBLs and monocytes was also
dependent on protein kinase C (PKC), protein kinase A (PKA), and protein tyrosine kinase (PTK). IL-1␤
mRNA expression by neutrophils was not dependent on p38 MAPK, and p38 MAPK was not activated in
neutrophils incubated with A. phagocytophila. IL-1␤ mRNA induction by PBLs, monocytes, and neutrophils was
dependent on PKC and PKA. Neutrophil expression of IL-1␤ mRNA was dependent on transglutaminase,
phospholipase C, and PTK, all of which are also required for internalization of A. phagocytophila. However,
monocyte expression of IL-1␤ mRNA was less dependent on these enzymes. These results suggest that A.
phagocytophila transduces different signals between its host neutrophils and monocytes for proinflammatory
cytokine generation.

Human granulocytic ehrlichiosis (HGE), an acute febrile
systemic disease first described in 1994, has been increasingly
recognized in the United States (5, 8, 33) and various parts of
Europe (23, 30, 38). HGE is characterized by fever, chills,
headache, myalgia, and hematological abnormalities such as
thrombocytopenia and leukopenia, as well as increased serum
aminotransferase activities (4, 25). HGE may be fatal in elderly
and/or immunocompromised patients or when antibiotic treat-
ment is delayed. The etiological agent of HGE is a strain of
Anaplasma phagocytophila (8, 11). A. phagocytophila, an obli-
gately intracellular gram-negative bacterium, propagates in the
membrane-bound inclusions of granulocytes. Because very few
organisms are usually detected in patients’ blood even at the
height of illness, we and other workers have speculated that the
illness is not caused directly by A. phagocytophila but rather is
caused by proinflammatory cytokines generated by the host in
response to A. phagocytophila infection (19).
Using human peripheral blood leukocytes (PBLs), we re-
cently found that A. phagocytophila HZ isolated from an acute-
stage HGE patient (42) and the recombinant 44-kDa major
outer membrane protein (rP44) cloned from this isolate (51)
induce rapid, strong proinflammatory cytokine (interleukin-1␤
[IL-1␤], tumor necrosis factor alpha [TNF-␣], and IL-6)
mRNA expression within 2 h and protein secretion within 24 h
in vitro (19). Within the 2-h period after incubation with A.
* Corresponding author. Mailing address: Department of Veteri-
nary 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@osu.edu.
phagocytophila, IL-8, IL-10, gamma interferon, IL-2, and trans-
forming growth factor ␤ mRNAs were not consistently upregu-
lated in PBLs, suggesting that expression of these cytokines
either is not induced or is induced at later time points in vitro.
Although human promyelocytic leukemia cell line HL-60 cells
are widely used for analysis of cytokine gene expression, IL-1␤,
TNF-␣, or IL-6 mRNA was not upregulated in HL-60 cells
within 2 h of incubation (Kim and Rikihisa, unpublished data).
Using HL-60 cells, other researchers have also reported an
absence of IL-1␤, TNF-␣, and IL-6 induction, and IL-8 mRNA
is not detectable at 12 h postinfection (2, 20). Since A. phago-
cytophila is granulocyte tropic, the monocyte responses to this
bacterium have been neglected. However, when we separated
neutrophils and monocytes in human PBLs prior to addition of
A. phagocytophila or 2 h after stimulation with A. phagocyto-
phila, we found that monocytes are the cells that express
mRNAs of IL-1␤, TNF-␣, and IL-6, whereas neutrophils ex-
press only IL-1␤ mRNA. Thus, collectively, PBLs produce all
three of these cytokines in response to A. phagocytophila. This
means that A. phagocytophila has the ability to selectively ac-
tivate monocytes to induce proinflammatory cytokine genera-
tion, but in neutrophils this activity may be suppressed (19).
We previously reported an analogous difference between hu-
man monocytes and neutrophils; A. phagocytophila inhibits
generation of superoxide in neutrophils but not in monocytes
in response to various stimuli (34). Differences in the signals
transduced by A. phagocytophila in these two types of primary
host defensive cells may be critical in understanding the mech-
anism of its selective survival in granulocytes and HGE patho-
genesis. For example, lesions found in the liver in HGE pa-

4132
VOL. 70, 2002 CYTOKINE SIGNALS BY A. PHAGOCYTOPHILA 4133

TABLE 1. Inhibitors used for investigation of signals of proinflammatory cytokine mRNA induction by A. phagocytophila

Length of Cellular target or mechanism

Concn Cells preincubation 50%
Inhibitor Refer- Source
useda pretreated at 37°C Cellular target or mechanism Inhibitory
(min)a ence
concnb

MG-132 50 ␮M PBLs, monocytes 60, 90 Proteasome inhibitor, inhibits NF-␬B acti- 3 ␮M 17 Biomol
vation by preventing I␬Bd degradation
c
SN-50 50 or 100 ␮g/ml PBLs, monocytes 30, 60, 90 NF-␬B nuclear translocation inhibitor 50 ␮g/ml 29 Biomol
SB 203508 10 ␮M PBLs, monocytes, 60, 90 p38 MAPK inhibitor 600 nM 24 Calbiochem-
neutrophils Novabiochem
PD 098059 20 ␮M PBLs, monocytes 60, 90 ERK/MEK inhibitor 2 ␮M 3 Biomol
H-7 25 ␮M PBLs, monocytes, 30 PKC inhibitor 6 ␮M 15 Biomol
neutrophils
H-89 50 ␮M PBLs, monocytes, 30 PKA inhibitor 48 nM 9 Biomol
neutrophils
Genistein 100 ␮M PBLs, monocytes, 30 PTK inhibitor 2.6 ␮M 1 Sigma
neutrophils
MDC 100 ␮M PBLs, monocytes, 30 Transglutaminase inhibitor, receptor- 15 ␮M 26 Sigma
neutrophils mediated endocytosis inhibitor
Verapamil 100 ␮M PBLs, monocytes, 30 Ca2⫹ channel blocker 60 ␮M 52 Sigma
neutrophils
2⫹
W-7 30 ␮M PBLs, monocytes, 30 Ca -calmodulin kinase antagonist 25 ␮M 16 Sigma
neutrophils
Neomycin sulfate 20 ␮M PBLs, monocytes, 30 PLC inhibitor 1–10 ␮M 48 Sigma
neutrophils
CHX 20 ␮g/ml PBLs, monocytes, 30 Eukaryote protein synthesis inhibitor ⬃0.1 ␮g/ml 6 Sigma
neutrophils
Oxytetracycline 10 ␮g/ml A. phagocytophila 30 Prokaryote protein synthesis inhibitor ⬃0.1 ␮g/ml 18 Sigma
a
When two or more concentrations or incubation times were used, the concentration or time in boldface type was the concentration or time used in the experiments
whose results are shown in the figures.
b
The values were obtained from the references and from the catalogs of Calbiochem-Novabiochem (La Jolla, Calif.) and Biomol Research Lab (Plymouth Meeting,
Pa.).
c
Concentration that resulted in ⬃85% inhibition.
d
I␬B, an inhibitor protein of NF-␬B.

tients are lymphohistiocytic rather than granulocytic infiltrates
(25).
Transcription of IL-1␤, TNF-␣, and IL-6 mRNAs is regu-
lated by at least two different mechanisms. One mechanism
involves nuclear translocation of cytoplasmic latent transcrip-
tion factors, such as activator protein 1 (AP-1)/c-Jun, c-Fos,
NF-␬B, or NF-IL-6, and binding of these factors to the appro-
priate enhancer elements present in the promoter regions of
IL-1␤, TNF-␣, and IL-6 genes (10, 27, 31, 37, 39, 40, 46).
Another mechanism involves activation of mitogen-activated
protein kinase (MAPK) family members that modulate the
activity of transcription factors by phosphorylation (10, 45). In
the present study, we examined the involvement of NF-␬B or
other transcription factors and the roles of MAPK and other
protein kinases in the rapid induction of proinflammatory cy-
tokines in human PBLs in order to understand responses by
the total mixed leukocyte population and in separated mono-
cytes and neutrophils in order to understand cell type-specific
responses to A. phagocytophila.
MATERIALS AND METHODS
Cultures. A. phagocytophila HZ isolated from an HGE patient (42) was prop-
agated in human promyelocytic leukemia cell line HL-60 (American Type Cul-
ture Collection, Manassas, Va.) as described elsewhere (51).
Preparation of host-cell-free A. phagocytophila. When ⬎90% of the HL-60 cells
were infected, as determined by Diff-Quik staining (Baxter Scientific Products,
Obetz, Ohio), a cell suspension (107 cells in 5 ml of RPMI 1640 medium) was
sonicated by using an ultrasonic processor (model W-380; Heat Systems, Farm-
ingdale, N.Y.) and the predetermined minimum damaging conditions for A.
phagocytophila (setting 2, 20 kHz for 7 s) and was centrifuged at 500 ⫻ g for 5
min. The supernatant containing host-cell-free A. phagocytophila was centrifuged
at 10,000 ⫻ g for 10 min at 4°C. Because A. phagocytophila is small and multiplies
as microcolonies (morulae) in the cytoplasm of granulocytes, it is impractical to
accurately count individual organisms. Therefore, the number of host-cell-free A.
phagocytophila cells was estimated as described previously (19).
Preparation of human PBLs, neutrophils, and monocytes. Human PBLs,
neutrophils, and monocytes were isolated from buffy coats from healthy human
immunodeficiency virus-negative donors as described previously (19). Briefly,
peripheral blood buffy coats were centrifuged at 500 ⫻ g for 5 min. Erythrocytes
were lysed in a sterile 0.83% NH4Cl solution for 5 min at room temperature, and
PBLs were washed twice in phosphate-buffered saline (PBS) (137 mM NaCl, 10
mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4; pH 7.2). To separate neutrophils,
buffy coats diluted 1:2 in PBS were layered on Ficoll-Paque Plus (Pharmacia,
Uppsala, Sweden) and centrifuged at 750 ⫻ g for 15 min at room temperature.
The pellet was washed twice in PBS with centrifugation at 400 ⫻ g for 5 min and
suspended in RPMI 1640 medium containing 10% fetal bovine serum (FBS).
The cell suspension was layered on top of a 62% Percoll (Pharmacia) solution
and centrifuged at 400 ⫻ g for 15 min at room temperature, and the band of
neutrophils was collected. The percentage of neutrophils in the preparation was
⬎95%, as assessed by morphological examination of Diff-Quik-stained cells. To
obtain adherent monocytes, the interface resulting from Ficoll-Paque Plus (Phar-
macia) gradient centrifugation was collected, washed twice in PBS, and incu-
bated in RPMI 1640 medium containing 10% FBS in 150-mm-diameter culture
dishes (Corning, Corning, N.Y.) at 37°C for 2 h; floating lymphocytes were
discarded, and adherent monocytes were used for the study. The viabilities of
PBLs, neutrophils, and monocytes were ⬎98%, as assessed by trypan blue dye
exclusion tests. All sets of experiments were independently repeated two or more
times by using human PBLs, monocytes, and neutrophils derived from different
blood donors. Donor cells were never mixed, and each donor leukocyte assay
included positive and negative controls to ensure the quality of both leukocyte
and A. phagocytophila preparations. Data obtained with blood specimens from 23
anonymous blood donors were included in this study.
Treatment of cells. Human PBLs, neutrophils, and monocytes (107 cells each
in 1 ml of RPMI 1640 medium in a well of a 24-well plate) or host-cell-free A.
phagocytophila cells were separately preincubated at 37°C with the inhibitors at
the concentrations and for the time periods shown in Table 1. After the prein-
cubation, cells were incubated for an additional 2 h with the host-cell-free A.
phagocytophila (100 bacteria/cell) in the presence of inhibitors. Host-cell-free A.
phagocytophila was preincubated with 10 ␮g of oxytetracycline (Sigma) per ml for
4134 KIM AND RIKIHISA
30 min and was then incubated with cells. As controls, cells in RPMI 1640
medium were incubated with 10 ␮l of the same medium supplemented with 10%
FBS containing either no lysate or the lysate derived from 107 uninfected HL-60
cells. The viabilities of PBLs, neutrophils, and monocytes were ⬎98%, as as-
sessed by trypan blue dye tests at the end of an experiment.
RNA isolation and RT-PCR. Total RNA was extracted from human PBLs,
monocytes, and neutrophils (107 cells each) by using TRIzol reagent (GIBCO-
BRL, Gaithersburg, Md.) as previously described (19). For reverse transcription
(RT)-PCR, total cellular RNA (2 ␮g) was reverse transcribed in a 30-␮l reaction
mixture containing 1⫻ reaction buffer (50 mM Tris-HCl [pH 8.3], 75 mM KCl,
3 mM MgCl2), deoxynucleoside triphosphates (0.5 mM each), 1 U of an RNase
inhibitor (GIBCO-BRL), 1.5 ␮M oligo(dT) primer, and 10 U of Moloney murine
leukemia virus reverse transcriptase (GIBCO-BRL) at 42°C for 1 h. The reaction
was terminated by heating the mixture at 94°C for 5 min, and the cDNA (2 ␮l)
was amplified in a 50-␮l reaction mixture containing 1⫻ PCR buffer (10 mM
Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl2), deoxynucleoside triphosphates
(0.2 mM each), and 0.4 ␮M (each) 3⬘ and 5⬘ primers (Clontech Laboratories,
Inc., Palo Alto, Calif.) in a DNA thermal cycler (model 9700; Perkin-Elmer
Corp., Norwalk, Conn.). Positive controls for three proinflammatory cytokines
were obtained from Clontech. To reduce nonspecific priming, all PCRs were
performed by the hot-start method. Taq DNA polymerase (2 U; GIBCO-BRL)
was added after incubation of the mixture at 94°C for 5 min. One PCR cycle
consisted of denaturation at 94°C for 45 s, annealing at 60°C for 45 s, and
extension at 72°C for 2 min. The PCR was conducted for 25 cycles in all
experiments. After a final extension for 7 min, 10 ␮l of PCR products was
electrophoresed in a 1.5% agarose gel containing ethidium bromide (final con-
centration, 0.5 ␮g/ml). DNA size markers (HaeIII fragments of ␾X174 replica-
tive-form DNA [GIBCO-BRL]) providing bands at 1,353 to 72 bp were run in
parallel. The amounts of the target PCR products were analyzed by using a gel
video system (Gel Print 2000i; BioPhotonics Corp., Ann Arbor, Mich.) and
image analysis software (ImageQuant; Molecular Dynamics, Sunnyvale, Calif.),
and values were normalized against glyceraldehyde-3-phosphate dehydrogenase
(G3PDH) mRNA in a corresponding sample.
p38 immunoblotting. Monocytes or neutrophils (2 ⫻ 107 cells) were incubated
for 15, 30, and 60 min with A. phagocytophila (100 bacteria/cell). Cells were lysed
by adding 100 ␮l of sodium dodecyl sulfate (SDS) sample buffer (62.5 mM
Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 50 mM dithiothreitol [DTT], 0.1%
bromophenol blue) and were transferred to a microcentrifuge tube. The extract
was sonicated on ice for 2 s and then boiled at 100°C for 5 min, cooled on ice, and
centrifuged for 5 min. Equal amounts (20 ␮l per lane) of supernatants were
subjected to SDS–12% polyacrylamide gel electrophoresis, in which 10 ␮l of
prestained broad-range molecular weight standards (Bio-Rad, Hercules, Calif.)
and 15-␮l portions of control p38 MAPK cell extracts (Cell Signaling Technol-
ogy, Beverly, Mass.) were included, and then transferred to nitrocellulose mem-
branes. Protein blots were incubated in blocking buffer (5% nonfat dry milk in
Tris-buffered saline–Tween 20 [TBST] buffer [50 mM Tris-HCl, 150 mM NaCl,
0.1% Tween 20; pH 7.6]) for 1 h at room temperature and washed three times
(5 min each) with 15 ml of TBST buffer. The membranes were incubated over-
night at 4°C with rabbit anti-p38 (pTpY180/182) MAPK antibody (Biosource
International, Inc., Camarillo, Calif.) or rabbit anti-p38 MAPK antibody (Cell
Signaling Technology) at a dilution of 1:1,000 in TBST buffer–5% bovine serum
albumin (Sigma). Horseradish peroxidase-conjugated goat anti-rabbit immuno-
globulin G (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.) was
added at a dilution of 1:2,000 in TBST buffer–5% bovine serum albumin and
incubated for 3 h at room temperature. The peroxidase-positive bands were
detected by immersing the blots in a developing solution (73 mM sodium acetate,
pH 6.2) containing 0.3% diaminobenzidine tetrahydrochloride (Nacalai Tesque,
Inc., Kyoto, Japan) and 0.04% H2O2 at room temperature for 5 min. The enzyme
reaction was terminated by washing the blots in 0.1 M H2SO4.
To investigate the dependence of activation of p38 MAPK on other kinases,
monocytes (107 cells) incubated for 30 min with genistein, H-89, and H-7 and
exposed to A. phagocytophila (100 bacteria/cell) were lysed for 30 min in 500 ␮l
of ice-cold radioimmunoprecipitation assay buffer (1% NP-40, 0.5% sodium
deoxycholate, and 0.1% SDS in 1⫻ PBS) containing several inhibitors (1 mM
phenylmethylsulfonyl fluoride [PMSF], 1 ␮M aprotinin, 14 ␮M leupeptin, 1 ␮M
pepstatin, 1 ␮M NaVO3, and 1 ␮M NaF). The peroxidase-positive bands were
detected by using an enhanced chemiluminesence Western blot detection system
according to the manufacturer’s protocol (Cell Signaling Technology).
Nuclear extract preparation. Human PBLs (108 cells/ml in each well) were
incubated for 2 h at 37°C, and adherent monocytes were used. Nuclear extract
was prepared at each time point (0, 30, 60, 120, and 240 min) after A. phagocy-
tophila infection. Briefly, cells were centrifuged and washed with Tris-buffered
saline (pH 7.9) and then were suspended in 400 ␮l of cold buffer A, which
INFECT. IMMUN.
contained 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA
[ethylene glycol-bis(␤-aminoethyl ether)-N,N,N⬘,N⬘-tetraacetic acid], 1 mM
DTT, 0.5 mM PMSF, 1 ␮M aprotinin, 14 ␮M leupeptin, 1 ␮M pepstatin, and 80
␮g of benzamidine per ml. After incubation on ice for 15 min, the cells were lysed
by adding 24 ␮l of 10% NP-40 (final concentration, 0.6%). Lysis was completed
by vigorous vortexing for 10 s. The homogenate was centrifuged at 13,000 ⫻ g for
30 s in a microcentrifuge, and the nuclear pellet was resuspended in 50 ␮l of cold
buffer B, which contained 20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA,
1 mM EGTA, 1 mM DTT, 1 mM PMSF, 1 ␮M aprotinin, 14 ␮M leupeptin, 1 ␮M
pepstatin, and 80 ␮g of benzamidine per ml. After centrifugation at 13,000 ⫻ g
for 30 s at 4°C, the resulting supernatant of nuclear extract was stored at ⫺85°C.
EMSA. Electrophoretic mobility shift assays (EMSA) were performed by using
a gel shift assay system (Promega Corp., Madison, Wis.) according to the man-
ufacturer’s protocol. NF-␬B (22 bp) and AP-1 (21 bp) consensus oligonucleo-
tides provided in the kit (Promega) were end labeled with [␥-32P]ATP by using
T4 nucleotide kinase, and 50,000 cpm of the probe per lane was used to assess
binding. The reaction mixture (final volume, 25 ␮l) for the DNA-protein inter-
action experiment contained 2 ␮l of the nuclear extract in 10 mM Tris-HCl (pH
7.5), 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, and 0.05 ␮g of
poly(dI-dC) per ␮l in 4% glycerol. The radioactive DNA probe was added to this
mixture and incubated at room temperature for 20 min. After incubation, equal
amounts of samples were loaded onto a 4% polyacrylamide gel that had been
prerun in 0.5⫻ TBE buffer (44 mM Tris, 44 mM borate, 1 mM EDTA; pH 8.3)
for 30 min and then were electrophoresed at 100 V for 3 h. After the gel was
exposed to X-ray Hyperfilm (Amersham-Pharmacia Biotech, Inc., Piscataway,
N.J.) for 16 h at ⫺85°C overnight, the film was developed. For the supershift
assay, 2 ␮l of rabbit polyclonal immunoglobulin G to p65 (C-20) (Santa Cruz
Biotechnology, Inc., Santa Cruz, Calif.) was added to the extract before incuba-
tion with the labeled NF-␬B oligonucleotide.
Effect of anti-PSGL-1 on IL-1␤ mRNA induction in human neutrophils. Neu-
trophils at a concentration of 107 cells per ml of RPMI 1640 medium were
preincubated with anti-PGSL-1 monoclonal antibody (MAb) (PL-1; 20 ␮g/ml;
Ancell Corp., Bayport, Minn.) for 40 min at 4°C and then incubated with host-
cell-free A. phagocytophila (100 organisms/cell) for 2 h at 37°C. After incubation,
total RNA was isolated and subjected to RT-PCR.
RESULTS
Involvement of p38 MAPK, NF-␬B, and protein kinase C
(PKC) in induction of IL-1␤, TNF-␣, and IL-6 mRNAs in hu-
man PBLs, neutrophils, or monocytes in response to A. phago-
cytophila. To determine whether p38 MAPK and/or extracel-
lular-signal-regulated kinase (ERK)/MAPK kinase (MEK)
and NF-␬B and/or AP-1 are required for proinflammatory
cytokine gene expression in response to A. phagocytophila, the
effects of several inhibitors on cytokine gene expression by
human PBLs were examined by RT-PCR at the linear range
determined previously based on our dose- and time-dependent
study in which competitive RT-PCR was used (19). Constitu-
tively expressed G3PDH mRNA levels were used to normalize
the amount of input RNA across the samples, and stimulation
with the medium or the HL-60 cell lysate was used as a control
for the basal and background cytokine gene expression by each
donor blood specimen. The contamination of RNA with
genomic DNA was negligible because the PCR products from
reverse transcriptase-minus controls were not detected in any
specimens. A densitometric analysis of the PCR products was
performed, and Table 2 shows the percentages of inhibition of
IL-1␤, TNF-␣, and IL-6 mRNA levels by each inhibitor in
PBLs, monocytes, and neutrophils from several different donor
blood specimens.
SB 203580, a selective inhibitor of p38 MAPK, binds with
high affinity to p38 MAPK near the ATP-binding site, thus
rendering p38 MAPK inactive (24). SB 203580 blocked induc-
tion of TNF-␣ and IL-6 mRNAs in PBLs in response to A.
phagocytophila, while IL-1␤ mRNA was partially blocked with
VOL. 70, 2002 CYTOKINE SIGNALS BY A. PHAGOCYTOPHILA 4135

TABLE 2. Inhibition of IL-1␤, TNF-␣, and IL-6 mRNAs in human

PBLs, monocytes, and neutrophils in response to A. phagocytophila

Cytokin % Inhibitiona
Inhibitor
mRNA PBLs Monocytes Neutrophils

IL-1␤ SN-50 50 ⫾ 12 8, 19 NDb

MG-132 44 ⫾ 7 23, 61 ND
SB 203580 60 ⫾ 34 26, 32 0, 0
PD 098059 17 ⫾ 10 9, 11 ND
H-7 95 ⴞ 4 84 ⴞ 8 75 ⴞ 20
H-89 41, 64 73 ⴞ 2 69 ⴞ 11
Genistein 49, 50 30, 52 90 ⴞ 7
MDC 19, 29 35, 54 64 ⴞ 3
Verapamil 0, 0 17, 27 12, 23
W-7 21, 33 5, 3 8, 16
Neomycin 0, 0 16, 24 68 ⴞ 12
TNF-␣c SN-50 74 ⫾ 34 25, 53
MG-132 91 ⴞ 25 32, 46
SB 203580 93 ⴞ 36 70 ⴞ 2
PD 098059 55 ⫾ 16 25, 44
H-7 94 ⴞ 4 82 ⴞ 5
H-89 44, 72 68 ⴞ 9
Genistein 79 ⴞ 6 75 ⴞ 13
MDC 32, 43 49, 57
Verapamil 67 ⴞ 9 81 ⴞ 7
W-7 46, 60 47, 66 FIG. 1. IL-1␤, TNF-␣, and IL-6 mRNA induction in human PBLs
Neomycin 63 ⴞ 7 0, 0 preincubated with several inhibitors and exposed to A. phagocytophila.
IL-6c SN-50 59 ⫾ 24 42, 78 PBLs (107 cells/ml) were preincubated for 1 h with SN-50 (100 ␮g/ml),
MG-132 95 ⴞ 1 34, 82 MG-132 (50 ␮M), SB 203580 (10 ␮M), PD 098059 (20 ␮M), and H-7
SB 203580 97 ⴞ 1 94 ⴞ 2 (25 ␮M) and exposed to A. phagocytophila (100 bacteria/cell) for 2 h.
PD 098059 44, 54 37, 54 Total RNA was extracted and subjected to RT-PCR. The amounts of
H-7 94 ⴞ 3 90 ⴞ 7 cDNAs were normalized against the amounts of G3PDH mRNA in
H-89 93 ⴞ 3 75 ⴞ 3 corresponding samples. The PCR products (10 ␮l each) were resolved
Genistein 91 ⴞ 1 39, 67 on agarose gels containing ethidium bromide. Molecular size markers
MDC 33, 34 11, 23 (HaeIII fragments of ␾X174 replicative-form DNA) were included.
Verapamil 39, 42 18, 19 The data (donor 3 data) are representative of the data from two
W-7 38, 40 0, 0 independent experiments (donors 1 and 3) that gave similar results.
Neomycin 37, 39 16, 17
a
Percentages of inhibition were calculated with the following formula: [1 ⫺
(band density in the presence of inhibitor/band density of G3PDH) ⫼ (band
density without inhibitor/band density of G3PDH)] ⫻ 100. The values are means involved in IL-1␤ mRNA expression than in TNF-␣ and IL-6
⫾ standard deviations from three independent experiments or actual values from
two independent experiments. Values that consistently were ⬎50% are in bold-
mRNA expression. In contrast, when the same donor PBLs
face type. were used, H-7, which selectively inhibits PKC activity via
b
ND, not determined. direct interaction with the catalytic site of the enzyme (15),
c
Neutrophils were not induced in response to A. phagocytophila.
showed significant inhibition of expression of all three proin-
flammatory cytokine mRNAs (Fig. 1; Table 2).
In a previous study (19), we found that A. phagocytophila
SB 203580 (Fig. 1; Table 2). PD 098059, a specific inhibitor induces all three cytokine mRNAs in human monocytes but
that binds inactive forms of MEK and prevents their activation only IL-1␤ mRNA in neutrophils within 2 h of incubation. To
and phosphorylation, resulting in inhibition of ERK/MEK (3), confirm that SB 203580 inhibits TNF-␣ and IL-6 mRNA in-
had consistently weaker inhibitory effects than SB 203580 upon duction by monocytes and to examine the effect of SB 203580
induction of any of the three proinflammatory cytokine on neutrophil generation of IL-1␤, cytokine mRNA expression
mRNAs (Fig. 1; Table 2). was examined separately in isolated monocytes and neutro-
At a concentration of 50 ␮M, MG-132, a cell-permeable phils. SB 203580 significantly blocked TNF-␣ and IL-6 mRNA
peptide-aldehyde protease inhibitor that blocks NF-␬B activa- induction and only weakly inhibited IL-1␤ mRNA expression
tion via its effect on the proteasome (17, 37), prevented induc- in monocytes (Fig. 2A; Table 2). However, in neutrophils, SB
tion of TNF-␣ and IL-6 mRNAs by PBLs, but IL-1␤ mRNA 230580 had no effect on IL-1␤ mRNA induction (Fig. 2B;
was less affected by this inhibitor than TNF-␣ and IL-6 Table 2). These results suggest that TNF-␣ and IL-6 mRNA
mRNAs (Fig. 1; Table 2). SN-50, a cell-permeable peptide that expression in monocytes in response to A. phagocytophila re-
inhibits nuclear translocation of NF-␬B (29), at a concentra- quires p38 MAPK activation, and IL-1␤ mRNA induction in
tion of 50 or 100 ␮g/ml inhibited cytokine mRNA expression both monocytes and neutrophils in response to A. phagocyto-
by PBLs, but it was less effective than MG-132 (Fig. 1; Table 2). phila is less dependent on p38 MAPK. In contrast, H-7 inhib-
These results suggest that activated p38 MAPK and a protea- ited induction of all three cytokine mRNAs in monocytes and
some–NF-␬B pathway for induction of TNF-␣ and IL-6 IL-1␤ mRNA induction in neutrophils from the same donors
mRNAs in PBLs are involved in the response to A. phagocy- (Fig. 2; Table 2).
tophila. p38 MAPK and a proteasome–NF-␬B pathway are less p38 MAPK is activated in monocytes but not in neutrophils
4136 KIM AND RIKIHISA
FIG. 2. Effects of SB 203580 and H-7 on proinflammatory cytokine
mRNA induction in human monocytes and neutrophils exposed to A.
phagocytophila. Monocytes or neutrophils (107 cells) were preincu-
bated for 30 min with SB 203580 (10 ␮M) or H-7 (25 ␮M) and then
incubated with host-cell-free A. phagocytophila (100 bacteria/cell) for
2 h. The total RNA was extracted and subjected to RT-PCR. The
amounts of cDNAs were normalized against the amounts of G3PDH
mRNA in corresponding samples. The PCR products were resolved on
agarose gels containing ethidium bromide. Molecular size markers
(HaeIII fragments of ␾X174 replicative-form DNA) were included.
The data (donor 8 data) are representative of the data from three
independent experiments (donors 6, 7, and 8) that gave similar results.
in response to A. phagocytophila. To verify the SB 230580
inhibition results (Fig. 1 and 2; Table 2), we examined p38
MAPK activation in both monocytes and neutrophils after A.
phagocytophila stimulation by immunoblotting. Activation of
p38 MAPK requires phosphorylation on Thr-180 and Tyr-182
(24) and can be assayed directly by using specific antibodies to
the phosphorylated form of p38 MAPK. The data showed that
phosphorylation of p38 MAPK in monocytes peaked at 15 min
and was maintained for up to 60 min after A. phagocytophila
stimulation (Fig. 3A). However, p38 MAPK was not activated
in neutrophils incubated with HL-60 cell lysate alone during
the same time period (Fig. 3B). These results confirmed the
results of experiments with SB 230580 which indicated that the
p38 MAPK activation at earlier time points is critical for
TNF-␣ and IL-6 generation in human monocytes within 2 h
after exposure to A. phagocytophila. These results also con-
firmed that IL-1␤ mRNA induction in neutrophils in response
to A. phagocytophila is not dependent on the p38 MAPK path-
way.
NF-␬B but not AP-1 was activated in human monocytes in
INFECT. IMMUN.
response to A. phagocytophila. Because induction of TNF-␣ and
IL-6 mRNAs in human PBLs in response to A. phagocytophila
was inhibited by MG-132 or SN-50 and monocytes are the
primary sources of cells generating TNF-␣ and IL-6 (19), ac-
tivation of transcription factors NF-␬B and AP-1 was examined
by EMSA. A time course study of monocytes responding to A.
phagocytophila showed that NF-␬B was activated 30 min after
stimulation (the earliest time point examined), but AP-1 was
not (Fig. 4). Thus, the EMSA data supported the results ob-
tained with MG-132 and SN-50 showing that A. phagocytophila
rapidly induces NF-␬B activation for expression of TNF-␣ and
IL-6 mRNAs in monocytes.
Effects of kinase inhibitors other than H-7 on induction of
IL-1␤, TNF-␣, and IL-6 mRNAs. Protein tyrosine kinase
(PTK) activities are required for proinflammatory cytokine
gene expression through NF-␬B in human peripheral blood
monocytes in response to lipopolysaccharide (LPS) (13).
Therefore, genistein, which inhibits the binding of ATP to PTK
(1), and H-89, a selective inhibitor of protein kinase A (PKA)
(9), were examined. Both genistein and H-89 inhibited IL-1␤,
TNF-␣, and IL-6 mRNA induction in PBLs (Fig. 5A; Table 2)
and in monocytes (Fig. 2 and 5B; Table 2). Both also inhibited
IL-1␤ mRNA induction in neutrophils (Fig. 5C; Table 2).
FIG. 3. Western blot analysis of activation and phosphorylation of
p38 MAPK in human monocytes (A) and neutrophils (B) in response
to A. phagocytophila. A total of 2 ⫻ 107 cells were incubated for
different times (0, 15, 30, and 60 min) with A. phagocytophila (100
bacteria/cell) or HL-60 cell lysate (negative control), lysed, blotted on
a nitrocellulose membrane, and probed with anti-phospho-p38 MAPK
antibody (pp 38) or with anti-p38 MAPK antibody (p 38). As controls,
nonphosphorylated (N) and phosphorylated (P) p38 MAPK extracts
from C-6 glioma cells prepared without and with anisomycin (Cell
Signaling Technology) treatment, respectively, were used. The positive
bands were detected by immersing the blots in a DAB developing
solution. The data (donor 12 data) are representative of the data from
two independent experiments (donors 9 and 12).
VOL. 70, 2002
FIG. 4. Time course of NF-␬B activation (A) and comparison of
NF-␬B activation and AP-1 activation (B) in human monocytes ex-
posed to A. phagocytophila, examined by EMSA. Human monocytes
(donor 5; 107 cells/ml in each well) were incubated with A. phagocyto-
phila (100 bacteria/cell) or E. coli LPS (1 ␮g/ml) (positive control) for
30 min. Nuclear extracts were prepared at different times (0, 0.5, 1, 2,
and 4 h) and used for EMSA. The data (donor 5 data) are represen-
tative of the data from three independent experiments (donors 5, 6,
and 7). N, uninfected monocytes.
However, H-7 most strongly inhibited induction of all three
cytokine mRNAs (Fig. 1 and 2; Table 2).
We have shown that internalization and infection, but not
binding of members of the family Anaplasmataceae (Neorick-
ettsia risticii in P388D1 cells [43] and Ehrlichia chaffeensis and
A. phagocytophila in THP-1 cells and neutrophils [7, 35]), are
inhibited by monodansylcadaverine (MDC), a transglutami-
nase inhibitor (26). Transglutaminase catalyzes the formation
of ε-(␥-glutamyl)-lysine between protein molecules by coupling
amines and diamines to the ␥-carboxyl residue of glutamine
(26). MDC significantly blocked IL-1␤ mRNA expression by
neutrophils in response to A. phagocytophila, suggesting that
clustering and/or internalization through binding to neutrophil
receptors of A. phagocytophila is required for IL-1␤ mRNA
induction (Fig. 5C; Table 2), whereas MDC had less effect on
CYTOKINE SIGNALS BY A. PHAGOCYTOPHILA 4137
cytokine mRNA expression in monocytes or PBLs. Oxytetra-
cycline, a prokaryotic protein synthesis inhibitor (18) known to
inhibit A. phagocytophila at the concentration used (49), had no
effect on expression of the three cytokine mRNAs in PBLs,
monocytes, or neutrophils (Fig. 5). Thus, a newly synthesized
protein of A. phagocytophila was not required, confirming our
previous observation made with recombinant major surface
protein rP44 of A. phagocytophila (19) that preformed proteins
of A. phagocytophila are sufficient for expression of these cy-
tokine genes.
An influx of Ca2⫹, calmodulin, and activation of phospho-
lipase C (PLC) are required for A. phagocytophila, N. risticii,
and E. chaffeensis infection (28, 35, 44). The involvement of
these signals in induction of IL-1␤, TNF-␣, and IL-6 mRNAs
in PBLs and monocytes or in induction of IL-1␤ mRNA in
neutrophils in response to A. phagocytophila was examined.
Verapamil (a phenyalkylamine Ca2⫹ channel blocker) (52) had
an inhibitory effect on TNF-␣ but not on IL-1␤ and had less
effect on IL-6 mRNA expression in PBLs or monocytes (Fig.
5A and B; Table 2). Neomycin (a PLC inhibitor) (48) had an
inhibitory effect on IL-1␤ mRNA expression by neutrophils
(Fig. 5C; Table 1). Cycloheximide (CHX) blocks eukaryotic
protein synthesis (6). CHX enhanced IL-1␤ and TNF-␣
mRNA expression but not IL-6 mRNA expression in PBLs,
TNF-␣ mRNA expression in monocytes, and IL-1␤ mRNA
expression in neutrophils (Fig. 5; Table 2), indicating that
preexisting host proteins are sufficient for expression of the
genes for IL-1␤ and TNF-␣ and newly synthesized host pro-
teins are rather suppressive (inflammation turn-off signals) for
induction of IL-1␤ and TNF-␣ mRNAs in monocytes and
neutrophils, respectively.
Effects of protein kinase and other kinase inhibitors on ac-
tivation of p38 MAPK and NF-␬B. To determine whether the
protein kinases are upstream signals required for p38 MAPK
and NF-␬B activation in monocytes, the effects of the inhibi-
tors on p38 MAPK and NF-␬B activation were examined. Be-
cause H-7 did not block p38 MAPK activation in monocytes in
response to A. phagocytophila (Fig. 6), PKC-independent p38
MAPK activation by monocytes in response to A. phagocyto-
phila is involved in induction of TNF-␣ and IL-6 mRNAs.
However, with genistein or H-89, p38 MAPK activation was re-
duced, suggesting that PTK and PKA may be partially involved
in p38 MAPK activation in monocytes in response to A. phago-
cytophila.
Because NF-␬B activation was not reduced by A. phagocy-
tophila after 45 min of preincubation of human monocytes with
H-7 and was significantly reduced with H-89, genistein, or SB
203580, the EMSA data obtained with these inhibitors sup-
ported PKC-independent NF-␬B activation and involvement
of other cytoplasmic kinase enzyme cascades, such as PTK,
PKA, and p38 MAPK, leading to NF-␬B activation for expres-
sion of TNF-␣ and IL-6 mRNAs in monocytes (Fig. 7).
Effect of anti-PSGL-1 MAb on the A. phagocytophila-induced
IL-1␤ mRNA expression by human neutrophils. Herron et al.
(14) showed that MAb PL-1 against PSGL-1 at a concentration
of ⬎8 ␮g/ml almost completely blocks binding of A. phagocy-
tophila to HL-60 cells. PL-1 at a concentration of 20 ␮g/ml did
not inhibit IL-1␤ mRNA induction in response to A. phagocy-
tophila in human neutrophils (data not shown), suggesting that
a receptor other than PSGL-1 is involved in this induction.
4138 KIM AND RIKIHISA INFECT. IMMUN.

FIG. 5. Effects of several inhibitors on induction of proinflammatory cytokine mRNAs in PBLs (A), monocytes (B), and neutrophils (C) in
response to A. phagocytophila. Cells (107 cells/ml) were preincubated for 30 min with CHX (20 ␮g/ml), MDC (100 ␮M), H-89 (50 ␮M), verapamil
(100 ␮M), W-7 (30 ␮M), neomycin (20 ␮M), and genistein (100 ␮M) and exposed to host-cell-free A. phagocytophila (100 bacteria/cell) for 2 h.
For the oxytetracycline (OTC) (10 ␮g/ml) treatment, host-cell-free A. phagocytophila (100 bacteria/cell) was preincubated for 30 min. Total RNA
was extracted and subjected to RT-PCR. The amounts of cDNAs were normalized against the amounts of G3PDH mRNA in corresponding
samples. The PCR products were resolved on agarose gels containing ethidium bromide. Molecular size markers (HaeIII fragments of ␾X174
replicative-form DNA) were included. (A) PBL data (donor 3 data) representative of the data from three independent experiments (donors 2, 3,
and 10) that gave similar results. (B) Monocyte data (donor 11 data) representative of the data from two independent experiments (donors 11 and
13). (C) Neutrophil data (donor 12 data) representative of the data from two independent experiments (donors 12 and 13).

DISCUSSION A. phagocytophila activated in human PBLs and monocytes

Using Escherichia coli LPS as a control, we previously dem-
onstrated that A. phagocytophila rapidly induces production of
major proinflammatory cytokines by human PBLs at a level
similar to the level induced by LPS (19). A. phagocytophila
cannot survive outside neutrophils or other granulocytes. Since
signals transduced by LPS activate the microbicidal activities of
neutrophils, A. phagocytophila may use signals different from
LPS to generate these cytokines. The present study revealed
that the signaling pathways for induction of these cytokines in
response to A. phagocytophila are indeed distinct from those
known for LPS and are also different in neutrophils and mono-
cytes.
some signals (p38 MAPK and NF-␬B) involved in TNF-␣ and
IL-6 production in response to LPS. LPS activates ERK1/2,
p38, c-Jun N-terminal kinase–stress-activated protein kinases,
and both AP-1 and NF-␬B (10). Because NF-␬B activation by
A. phagocytophila was partially blocked by SB 203580, p38
MAPK activation is upstream of NF-␬B activation in human
monocytes. There are four distinct isoforms of p38 MAPK,
termed p38␣, p38␤, p38␥/SAPK3, and p38␦/SAPK4. Because
SB 203580 inhibits p38␣ and p38␤ but not p38␥ or p38␦ (21),
it seems that p38␣ and/or p38␤ is responsible for induction of
TNF-␣ and IL-6 mRNAs in human monocytes in response to
A. phagocytophila. We found that both p38 MAPK activation
VOL. 70, 2002
FIG. 6. Western blot analysis of phosphorylated p38 MAPK. Hu-
man monocytes preincubated for 30 min with kinase inhibitors were
incubated with A. phagocytophila (100 bacteria/cell) for 30 min. The
same amount of protein was electrophoresed, blotted on a nitrocellu-
lose membrane, and probed with anti-phospho-p38 MAPK antibody
(pp38) and with anti-p38 MAPK antibody (p38). Horseradish peroxi-
dase-conjugated goat anti-rabbit immunoglobulin G was added, and
the positive bands were detected by using the enhanced chemilumi-
nescence method. The data (donor 16 data) are representative of the
data from five independent experiments (donors 15, 16, 20, 21, and 22)
that gave similar results.
and NF-␬B activation in human monocytes by A. phagocyto-
phila were PTK and PKA dependent. NF-␬B activation by LPS
has also been reported to be PTK dependent (13, 46, 50),
suggesting that A. phagocytophila may share with LPS the same
PTK-dependent pathway upstream of p38 MAPK activation.
PKA activation was reported to increase DNA binding of
NF-␬B in T cells (22), and PKA activates p38 MAPK in en-
docrine cells (41). The overall responses of PBLs and mono-
cytes were similar in the present study. However, a few differ-
ences were seen in sensitivities to some inhibitors. Whether
these differences are due to individual donor variation, the
monocyte isolation procedure, or a requirement of the mixed
or additional cell population for a PBL-type response remains
to be clarified.
In contrast to what happens in monocytes, A. phagocytophila
did not activate p38 MAPK in neutrophils. This does not seem
to be due to a universal lack of responsiveness of neutrophil
p38 MAPK, since p38 MAPK has been reported to be acti-
vated in human neutrophils by LPS (36). Whether NF-kB is
activated and required for IL-␤ mRNA induction in neutro-
phils in response to A. phagocytophila was not directly exam-
ined in the present study. However, since IL-␤ mRNA induc-
tion in PBLs in response to A. phagocytophila was less sensitive
to MG-132 or SN-50 treatment than TNF-␣ or IL-6 mRNA
induction, NF-kB may not play a major role in neutrophil IL-␤
mRNA induction in response to A. phagocytophila. This also
does not seem to be due to a universal inability of neutrophil
CYTOKINE SIGNALS BY A. PHAGOCYTOPHILA 4139
FIG. 7. NF-␬B activation in human monocytes preincubated with
several inhibitors and exposed to A. phagocytophila. Human monocytes
(107 cells/ml in each well) were preincubated with H-7, H-89, genistein,
and SB 203580 for 45 min and then exposed to A. phagocytophila (100
bacteria/cell) for 30 min. Nuclear extracts were prepared and used for
EMSA. The data (donor 19 data) are representative of the data from
five independent experiments (donors 15, 16, 17, 18, and 19) that gave
similar results. The nonspecific competitor AP-2 and the specific com-
petitor NF-␬B were included in this assay to verify the effects of these
competitors on binding of the NF-␬B complex.
NF-kB to be activated, since at least two reports have shown
NF-kB activation in neutrophils in response to LPS (32, 36).
Proinflammatory cytokine gene expression in response to A.
phagocytophila was sensitive to H-7 in PBLs, monocytes, and
neutrophils. The site of PKC action appeared to be either
independent from or downstream of p38 MAPK and NF-␬B
activation, because activation of either MAPK or NF-␬B in
monocytes in response to A. phagocytophila was not inhibited
by H-7. Of note, LPS-induced TNF-␣ release in human mono-
cytes has been reported to be PKC independent (47). Further-
more, neutrophil IL-1␤ mRNA expression was dependent on
transglutaminase and PLC, suggesting that cross-linking of
proteins, subsequent activation of PLC, and an increase in the
intracellular Ca2⫹ level are required (28). The signaling path-
way is somewhat similar to that described in a recent report
showing that IL-1␤ synthesis by human neutrophils in response
to granulocyte-macrophage colony-stimulating factor requires
PKC activation and an increase in the intracellular Ca2⫹ level
(12).
Leukocytes derived from 23 human immunodeficiency virus-
negative donors were included in the present study. The blood
donors were anonymous, and we could not obtain age, sex, or
other information from most of these donors. Elderly and/or
4140 KIM AND RIKIHISA
immunocompromised patients have more severe clinical signs
of HGE than other patients (4). It remains to be determined
whether and how the immune status of patients influences the
signaling induced in PBLs, monocytes, and neutrophils in re-
sponse to A. phagocytophila and thus the levels of parasitemia
and proinflammatory cytokines in the blood.
In summary, the present data suggest (i) a key role for p38
MAPK and NF-␬B in the induction of TNF-␣ and IL-6
mRNAs in human PBLs in response to A. phagocytophila, (ii)
the involvement of PKC either downstream of or independent
from p38 MAPK and NF-␬B activation in IL-1␤, TNF-␣, and
IL-6 mRNA expression in monocytes, (iii) the involvement of
PKA and PTK upstream of p38 MAPK and NF-␬B in TNF-␣
and IL-6 mRNA expression in human monocytes in response
to A. phagocytophila, and (iv) the involvement of PKC and
intracellular Ca2⫹ in IL-1␤ mRNA expression in neutrophils.
The cell type-specific signaling may be important for under-
standing the granulocyte-specific survival of A. phagocytophila
and pathological changes such as the liver damage and throm-
bocytopenia seen in HGE.
ACKNOWLEDGMENTS
This research was supported by grants R01AI30010 and R01AI40934
from the National Institutes of Health.
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