Heat shock protein 10 of Chlamydophila pneumoniae induces
proinflammatory cytokines through Toll-like receptor (TLR)
2 and TLR4 in human monocytes THP-1
Z. Zhou & Y. Wu & L. Chen & L. Liu & H. Chen & Z. Li &
C. Chen
Received: 13 April 2011 /Accepted: 29 June 2011 / Editor: T. Okamoto
# The Society for In Vitro Biology 2011
Abstract Inflammatory response is the first line of infec-
tion. Previous studies have suggested that Chlamydophila
pneumoniae heat shock protein (CHSP) 60 is present in
human atheromata, and it plays an important role on the
chronic infection elicited by C. pneumoniae. Here, we
demonstrated in vitro the impact of heat shock protein 10
(HSP10) of C. pneumoniae on THP-1 cells and the role of
Toll-like receptors (TLRs) in the procedures of inflamma-
tory response. The production of proinflammatory cyto-
kines, including tumor necrosis factor alpha (TNF-alpha),
interleukin (IL)-6, and IL-1beta were induced by recombi-
nant HSP10 dose-dependently, and the proinflammatory
activity of HSP10 was greatly reduced by heating and
deproteinization treatment. The expression of TLR4 and
TLR2 on the cultured cells were determined by reverse
transcriptase-polymerase chain reaction and immunofluo-
rescence. Peritoneal macrophages isolated from wild-type
(C3H/HeN) and TLR4-deficient mice (C3H/HeJ) were
respectively stimulated with endotoxin-free proteins. Cyto-
kine responses after stimulation were significantly different,
depending on the presence of TLR4. The effect on cytokine
expression was blocked by anti-TLR2 or anti-TLR4 MAb
partially or dramatically. Thus, HSP10 of C. pneumoniae
which could elicit inflammatory reactions in human mono-
cytes may contribute to the inflammatory processes in
Chlamydophila infection, and the effects were mediated by
TLR4 and, to a lesser extent, TLR2.
Keywords Chlamydophila pneumoniae . Heat shock
protein 10 . Proinflammatory cytokines . Toll-like
receptors . THP-1
Introduction
Chlamydophila pneumoniae is an obligate intracellular
pathogen which spread worldwide. The seroprevalence in
the adult population is about 50∼70%, and nearly every-
body has been infected at least once during his or her
lifetime (Miyashita et al. 2008; Jha et al. 2009). The
pathogen causes acute respiratory infections such as
pneumonia and bronchitis and has been implicated in
chronic processes including asthma and cardiovascular
disease (Grayston et al. 1993; Hahn et al. 1998; Cook
1999; Cosentini et al. 2008; Blasi et al. 2009). Although the
link between C. pneumoniae and atherosclerosis is well-
accepted, little has been known about the mechanisms of C.
pneumoniae-induced infections.
Inflammation seems to play a key role on the acute
infections and chronic inflammatory diseases, and the
cytokine response is one of its remarkable features. Several
studies have confirmed C. pneumoniae triggers an inflam-
matory response in human and animal cells (Blessing et al.
2000; Yamamoto et al. 2005; Didion 2008). The pathogen
could invade and replicate in the host cells and induce
activation of chronic inflammation with cytokines release
such as IL-1beta, IL-6, and TNF-alpha, but the specific
components of C. pneumoniae involved in these initiations
is not well identified.
Heat shock proteins (HSPs) are highly conserved
molecules. Because of molecular mimicry of human
Z. Zhou : Y. Wu (*) : L. Chen : L. Liu : H. Chen : Z. Li : C. Chen
Pathogenic Biology Institute, University of South China,
No. 28 Changsheng Western Road,
Hengyang 421001, People’s Republic of China
e-mail: yimouwu@sina.com
In Vitro Cell.Dev.Biol.—Animal
DOI 10.1007/s11626-011-9441-4

homologous HSP, some microbial HSPs seem to induce
humoral or cellular responses and involve in pathogenesis
such as inflammation and autoimmunity. It was proven that
HSP60 of C. pneumoniae can be detected in human
atheroma and may take part in the development of lesions
and directly activate monocytes/macrophages to secrete
cytokines from cells (Kol et al. 1998, LaVerda et al. 2000).
But very little is known about the role of HSP10 which is
genetically and physiologically linked to HSP60.
The present study aimed to determine whether HSP10 were
implicated in the inflammatory responses against infection and
whether the TLRs (TLR2 and TLR4) were required for C.
pneumoniae HSP10 (CHSP10)-induced cytokine expression
by monocyte cells. For this purpose, we incubated cultured
human monocytic cells with recombinant CHSP10 with or
without anti-TLR antibodies and measured the effects of
CHSP10 on IL-1beta, IL-6, and TNF-alpha production.
Materials and Methods
Cells. The human monocytic cell line THP-1 (American Type
Culture Collection) and human lung adenocarcinoma epithelial
cell line A549 (China Center for Type Culture Collection,
Wuhan University) were grown at 37°C with 5% CO2 in
RPMI 1640 medium (HyClone, Logan, UT). Human kidney
cell line HEK293T (China Center for Type Culture Collec-
tion, Wuhan University) was cultured in Dulbecco’s modified
Eagle’s medium (HyClone); all the cells were cultured in
mediums contained 10% heat-inactivated fatal bovine serum
(FBS; GIBCO; Langley, OK), 2 mM L-glutamine, 100 U/mL
penicillin, and 100 μg/mL streptomycin.
Reagents. Neutralizing anti-human TLR4 MAb (HTA125),
neutralizing anti-human TLR2 MAb (TL2.1), and isotype
control MAb (Mouse IgG2a, κ Isotype Ctrl) as a
blocking Ab were purchased from Biolegend (Clonetimes
Biotech Co., Ltd, Changsa, China). CyTM2-conjugated
goat anti-mouse IgG and CyTM3-conjugated goat anti-
mouse IgG were obtained from Jackson ImmunoResearch
Laboratories Inc. (Westgrove, PA). For reverse transcrip-
tase reaction, the RevertAidTM First-Strand cDNA Syn-
thesis Kit (Fermentas; Shenzhen, China) was applied. PCR
amplification was performed with Taq DNA polymerase
(Dongsheng Biotech; Zhejiang, China). Penicillin, ampi-
cillin, polymyxin B (PMB), and Escherichia coli lipo-
polysaccharide (LPS; 0111:B4) were purchased from
Sigma-Aldrich (Shanghai, China). All other chemicals
were obtained from commercial sources and were of
analytical or reagent grade.
Peritoneal macrophages from C3H/HeN and C3H/HeJ. A
4∼6-wk-ld C3H/HeN (Vital River Laboratory Animal
Technology Co. Ltd.) and C3H/HeJ female mice (Model
Animal Research Center of Nanjing University) were fed
with standard laboratory chow and water ad libitum.
Macrophages were induced by injecting 1 mL of 5% sterile
starch broth intraperitoneally, and peritoneal macrophages
were collected 4 or 5 d later by peritoneal lavage with
chilled serum-free culture medium. The macrophages were
then incubated in a 5% CO2 incubator at 37°C for 2∼4 h in
RPMI-1640 culture medium supplemented with 100 U/mL
penicillin G and 100 μg/mL streptomycin. After three times
vigorous washing, nonadherent cells were removed, and
macrophages were incubated in RPMI 1640 with 10% FBS
in 96-well plate (HyClone) at 1×104 cells per well for
cytokine assays.
Recombinant CHSP10 preparation. CHSP10 recombinant
plasmid was constructed with pQE30 vector (QIAGEN;
Shanghai, China) and transformed into E. coli M15,
induced, and purified as described elsewhere (Jiang et al.
2008). Briefly, cells were cultured in Luria-Bertani broth
containing 100 μg/mL ampicillin and 100 μg/mL kanamy-
cin, harvested by centrifugation, and disrupted by sonica-
tion on ice; soluble His-tagged protein CHSP10 was
purified by nickel-chelate affinity resin (QIAGEN) accord-
ing to the manufacturer’s protocol. Elution fractions were
then analyzed by sodium dodecyl polyacrilamide gel
electrophoresis using 15% running gel. The concentration
of the purified protein was determined by BCA Protein
Assay Kit (Pierce). To exclude possible LPS contamination,
proteins were collected and mixed with 50 μg/mL
polymyxin B (Sigma) or treated with Detoxi-GelTM
Endotoxin Removing Gel (Pierce; IL). The endotoxin
concentration of recombinant HSP10 was less than 60 pg/
mL, as determined by the Limulus amebocyte lysate assay
(Chinese Horseshoe Crab Reagent Manufactory, Ltd., Xiamen,
China). The protein was stored at −135°C until it was
used.
Stimulation experiments. For stimulation of THP-1 or
A549 cells with HSP10, approximately 3×105 cells per
well were seeded into 24-well plate. After incubation for
indicated conditions, cells were then stimulated with
HSP10 using different concentrations or in different times.
Cell-free supernatants were harvested after centrifugation
and was stored at −70°C until use.
Heat, deproteinized treatments of CHSP10. Heat experiment
was performed by heating CHSP10 at 56°C for 30 min or
100°C for 20 min, respectively. Deproteinized treatments
were performed by mixing purified proteins with 50%
nickel-chelate affinity resin and incubating at room tem-
perature for 1 h to bind the His-tagged CHSP10, and then
the mixture was loaded onto a column. Flow-through was
ZHOU ETAL.