Roles of toll-like receptors in C-C chemokine production by renal tubular epithelial cells.
ABSTRACT Pyelonephritis, in which renal tubular epithelial cells are directly exposed to bacterial component, is a major predisposing cause of renal insufficiency. Although previous studies have suggested C-C chemokines are involved in the pathogenesis, the exact source and mechanisms of the chemokine secretion remain ambiguous. In this study, we evaluated the involvement of Toll-like receptors (TLRs) in C-C chemokine production by mouse primary renal tubular epithelial cells (MTECs). MTECs constitutively expressed mRNA for TLR1, 2, 3, 4, and 6, but not for TLR5 or 9. MTECs also expressed MD-2, CD14, myeloid differentiation factor 88, and Toll receptor-IL-1R domain-containing adapter protein/myeloid differentiation factor 88-adapter-like. Synthetic lipid A and lipoprotein induced monocyte chemoattractant protein 1 (MCP-1) and RANTES production in MTECs, which strictly depend on TLR4 and TLR2, respectively. In contrast, MTECs were refractory to CpG-oligodeoxynucleotide in chemokine production, consistently with the absence of TLR9. LPS-mediated MCP-1 and RANTES production in MTECs was abolished by NF-kappaB inhibition, but unaffected by extracellular signal-regulated kinase inhibition. In LPS-stimulated MTECs, inhibition of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase significantly decreased RANTES, but did not affect MCP-1 mRNA induction. Thus, MTECs have a distinct expression pattern of TLR and secrete C-C chemokines in response to direct stimulation with a set of bacterial components.
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ABSTRACT: The most common cause of acute kidney injury (AKI) in hospitalized patients is sepsis. However, the molecular pathways and mechanisms that mediate septic AKI are not well defined. Experiments performed over the past 20 years suggest that there are profound differences in the pathogenesis between septic and ischemic AKI. Septic AKI often occurs independently of hypoperfusion, and is mediated by a concomitant pro- and anti-inflammatory state that is activated in response to various pathogen-associated molecular patterns, such as endotoxin, as well as damage-associated molecular patterns. These molecular patterns are recognized by Toll-like receptors (TLRs) found in the kidney, and effectuate downstream inflammatory pathways. Additionally, apoptosis has been proposed to play a role in the pathogenesis of septic AKI. However, targeted therapies designed to mitigate the above aspects of the inflammatory state, TLR-related pathways, and apoptosis have failed to show significant clinical benefit. This failure is likely due to the protean nature of septic AKI, whereby different patients present at different points along the immunologic spectrum. While one patient may benefit from targeted therapy at one end of the spectrum, another patient at the other end may be harmed by the same therapy. We propose that a next important step in septic AKI research will be to identify where patients lie on the immunologic spectrum in order to appropriately target therapies at the inflammatory cascade, TLRs, and possibly apoptosis.Critical care (London, England) 09/2014; 18(5):501. · 5.04 Impact Factor
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ABSTRACT: The innate immune system plays an important role as a first response to tissue injury. This first response is carried out via germline-encoded receptors. Toll-like receptors (TLRs) are the first identified and best studied family of pattern recognition receptors. TLRs are expressed on a variety of cell types, including epithelial cells, endothelia, dendritic cells, monocytes/macrophages, and B- and T-cells. TLRs initiate innate immune responses and concurrently shape the subsequent adaptive immune response. They are sensors of both pathogens, through the exogenous pathogen-associated molecular patterns (PAMPs), and tissue injury, through the endogenous danger-associated molecular patterns (DAMPs). TLR signaling is critical in defending against invading microorganisms; however, sustained receptor activation is also implicated in the pathogenesis of inflammatory diseases. Ischemic kidney injury involves early TLR-driven immunopathology, and the resolution of inflammation is needed for rapid regeneration of injured tubule cells. Notably, the activation of TLRs also has been implicated in epithelial repair. This review focuses on the role of TLRs and their endogenous ligands within the inflammatory response of acute kidney injury.International Journal of Nephrology and Renovascular Disease 01/2014; 7:241-51.
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ABSTRACT: Preconditioning is a preventative approach, whereby minimized insults generate protection against subsequent larger exposures to the same or even different insults. In immune cells, endotoxin preconditioning downregulates the inflammatory response and yet, preserves the ability to contain infections. However, the protective mechanisms of preconditioning at the tissue level in organs such as the kidney remain poorly understood. Here, we show that endotoxin preconditioning confers renal epithelial protection in various models of sepsis in vivo. We also tested the hypothesis that this protection results from direct interactions between the preconditioning dose of endotoxin and the renal tubules. This hypothesis is on the basis of our previous findings that endotoxin toxicity to nonpreconditioned renal tubules was direct and independent of immune cells. Notably, we found that tubular protection after preconditioning has an absolute requirement for CD14-expressing myeloid cells and particularly, macrophages. Additionally, an intact macrophage CD14-TRIF signaling pathway was essential for tubular protection. The preconditioned state was characterized by increased macrophage number and trafficking within the kidney as well as clustering of macrophages around S1 proximal tubules. These macrophages exhibited increased M2 polarization and upregulation of redox and iron-handling molecules. In renal tubules, preconditioning prevented peroxisomal damage and abolished oxidative stress and injury to S2 and S3 tubules. In summary, these data suggest that macrophages are essential mediators of endotoxin preconditioning and required for renal tissue protection. Preconditioning is, therefore, an attractive model to investigate novel protective pathways for the prevention and treatment of sepsis.Journal of the American Society of Nephrology 11/2014; · 9.47 Impact Factor
Roles of Toll-Like Receptors in C-C Chemokine Production by
Renal Tubular Epithelial Cells1
Naotake Tsuboi,* Yasunobu Yoshikai,†Seiichi Matsuo,* Takeshi Kikuchi,†Ken-Ichiro Iwami,†
Yoshiyuki Nagai,‡Osamu Takeuchi,§Shizuo Akira,§and Tetsuya Matsuguchi2†
Pyelonephritis, in which renal tubular epithelial cells are directly exposed to bacterial component, is a major predisposing cause
of renal insufficiency. Although previous studies have suggested C-C chemokines are involved in the pathogenesis, the exact source
and mechanisms of the chemokine secretion remain ambiguous. In this study, we evaluated the involvement of Toll-like receptors
(TLRs) in C-C chemokine production by mouse primary renal tubular epithelial cells (MTECs). MTECs constitutively expressed
mRNA for TLR1, 2, 3, 4, and 6, but not for TLR5 or 9. MTECs also expressed MD-2, CD14, myeloid differentiation factor 88, and
Toll receptor-IL-1R domain-containing adapter protein/myeloid differentiation factor 88-adapter-like. Synthetic lipid A and li-
poprotein induced monocyte chemoattractant protein 1 (MCP-1) and RANTES production in MTECs, which strictly depend on
TLR4 and TLR2, respectively. In contrast, MTECs were refractory to CpG-oligodeoxynucleotide in chemokine production,
consistently with the absence of TLR9. LPS-mediated MCP-1 and RANTES production in MTECs was abolished by NF-?B
inhibition, but unaffected by extracellular signal-regulated kinase inhibition. In LPS-stimulated MTECs, inhibition of c-Jun
N-terminal kinase and p38 mitogen-activated protein kinase significantly decreased RANTES, but did not affect MCP-1 mRNA
induction. Thus, MTECs have a distinct expression pattern of TLR and secrete C-C chemokines in response to direct stimulation
with a set of bacterial components. The Journal of Immunology, 2002, 169: 2026–2033.
ciency. For example, LPS, a principal constituent of Gram-nega-
tive bacteria, is an important factor in the development of acute
renal failure and acceleration of chronic nephritis, often leading to
renal insufficiency (1–5). In animal models, LPS induces experi-
mental glomerulonephritis or acute proximal tubule injury, such as
IgA nephropathy (2), lupus nephritis (3), antiglomerular basement
membrane disease (5), and acute tubular necrosis (4). In these
experimental models, LPS potently induces chemokines, which
play major roles in the pathogenesis by recruiting immune cells
into interstitium and glomerulus. Renal TECs have been proposed
yelonephritis, in which renal tubular epithelial cells
(TECs)3are directly exposed to bacterial component, is
one of the major predisposing causes of renal insuffi-
as an important source of C-C chemokines, such as monocyte che-
moattractant protein 1 (MCP-1), and RANTES (6). However, the
precise mechanisms whereby TECs produce chemokines in re-
sponse to bacterial infection remain ambiguous.
Toll-like receptors (TLRs) have been shown to play important
roles in the recognition of bacterial components (7). Ten members
of TLRs have been reported so far (7). Among TLRs, TLR4 me-
diates LPS signal transduction in collaboration with other mole-
cules, such as CD14, MD-2, myeloid differentiation factor 88
(MyD88), and Toll receptor-IL-1R domain-containing adapter pro-
tein (TIRAP)/MyD88-adapter-like (Mal) (7–10). On the other
hand, TLR2 is considered to be an essential receptor for other
bacterial components: lipoprotein, peptidoglycan (PGN), and lipo-
teichoic acid (7). It has recently been reported that TLR6 forms a
heterodimer with TLR2 to mediate the responsiveness to PGNs
and zymosan, but not lipoproteins (11). TLR3 (12), TLR5 (13),
and TLR9 (14) have recently been shown to mediate signals from
dsRNA, flagella, and bacterial DNA, respectively. Roles of TLRs,
especially TLR2 and TLR4, have been examined mainly in pro-
fessional immune cells such as monocytes (15), macrophages (16),
T cells (17, 18), and dendritic cells (19), but also in other cell
types, e.g., dermal endothelial cells (20), intestinal epithelial cells
(21), hepatocytes (22), and osteoblasts (23). These findings raise
the possibility that TLRs may be involved in C-C chemokine pro-
duction in renal cells after direct exposure to bacterial components.
Therefore, we examined the roles of TLRs in the C-C chemokine
production by mouse primary renal tubular epithelial cell (MTEC)
after stimulation with bacterial components including LPS/lipid A,
lipoprotein, and bacterial DNA. Our results indicate that MTECs
have a distinct expression pattern of TLR genes and directly
respond to a set of bacterial components by secreting C-C
chemokines. We have also shown that although both MCP-1
and RANTES are induced by LPS in MTECs, the molecular
mechanisms controlling their expression are different.
*Department of Internal Medicine, Division of Nephrology and†Laboratory of Host
Defense and Germfree Life, Research Institute for Disease Mechanism and Control,
Nagoya University Graduate School of Medicine, Nagoya, Japan;‡Toyama Institute
of Health, Toyama, Japan; and§Department of Host Defense, Research Institute for
Microbial Disease, Osaka University, Osaka, Japan
Received for publication February 2, 2002. Accepted for publication June 12, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by grants from the Ono Pharmaceutical Company;
the Yokoyama Research Foundation for Clinical Pharmacology; the Aichi Kidney
Foundation; and the Naito Foundation (to T.M.); Ministry of Education, Science and
Culture of the Japanese Government, JSPS-RFTF97L00703; and the Yakult Bio-
science Foundation (to Y.Y.).
2Address correspondence and reprint requests to Dr. Tetsuya Matsuguchi, Laboratory
of Host Defense and Germfree Life, Research Institute for Disease Mechanism and
Control, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku,
Nagoya 466-8550, Japan. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: TEC, tubular epithelial cell; MCP-1, monocyte
chemoattractant protein 1; TLR, Toll-like receptor; MyD88, myeloid differentiation
factor 88; TIRAP, Toll receptor-IL-1R domain-containing adapter protein; Mal,
MyD88-adapter-like; PGN, peptidoglycan; MTEC, mouse primary renal TEC; JNK,
c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; ODN, oligode-
oxynucleotide; ERK, extracellular signal-regulated kinase; MIP, macrophage inflam-
The Journal of Immunology
Copyright © 2002 by The American Association of Immunologists, Inc.0022-1767/02/$02.00
Materials and Methods
Reagents and Abs
LPS from Escherichia coli (serotype B6:026) and synthetic lipoprotein
(palmitoyl-Cys((RS)-2,3-di(palmitoyloxy)-propyl)-Ala-Gly-OH)) were ob-
tained from Sigma-Aldrich (St. Louis, MO) and Bachem (Bubendorf,
Switzerland), respectively. Recombinant mouse TNF-?, IFN-?, and IL-1?
were purchased from PeproTech (Seattle, WA). Synthetic lipid A analog
(ONO-4007) was kindly provided by Ono Pharmaceuticals (Osaka, Japan)
(24). DMEM and DMEM nutrient mixture F-12 Ham, cycloheximide, and
curcumin were obtained from Sigma-Aldrich. SP600125, PD98059, and
SB203580 were obtained from Calbiochem (San Diego, CA). Anti-mouse
CD14 mAb and anti-MyD88 polyclonal Ab were purchased from BD
PharMingen (San Diego, CA) and Alexis Biotechnology (San Diego, CA),
respectively. A polyclonal anti-c-Jun N-terminal kinase (JNK) 1 Ab and a
polyclonal anti-p38 Ab were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). A polyclonal anti-extracellular signal-regulated kinase
(ERK)1/2, a phosphospecific anti-ERK1/2 polyclonal Ab and a phospho-
specific anti-p38 mitogen-activated protein kinase (MAPK) polyclonal Ab
were purchased from New England Biolabs (Beverly, MA). Phosphoro-
thioate-stabilized CpG-oligodeoxynucleotide (ODN) (TCCATGACGTTC
CTGATGCT) (14) was purchased from Rikaken (Nagoya, Japan). For
immunofluorescence studies, anti-mouse cytokeratin mAb was obtained
from Enzo Diagnostics (New York, NY) and anti-rabbit brush border
vesicle polyclonal Ab was gift a from Dr. G. Andres (25).
C57BL/6, C3H/HeN, and C3H/HeJ mice were purchased from Japan SLC
(Shizuoka, Japan). These mice were bred in our institute under specific
pathogen-free conditions. Eight- to 10-wk-old female mice were used for
the experiments. The mutant mouse (F2interbred from 129/Ola ?
C57BL/6) strain deficient in TLR2 was generated by gene targeting, as
described previously (26).
Primary mouse renal TEC culture
TECs were harvested from murine renal cortex following microdissection
and brief collagenase digestion. Cells were grown in hormonally defined
K-1 medium (27) supplemented with epidermal growth factor (50 pg/ml),
insulin-transferrin-sodium selenite media supplement (0.12 IU/ml), PGE1
(1.25 ng/ml), T3 (34 pg/ml), hydrocortisone (18 ng/ml), 10% decomple-
mented FBS, and 1% penicillin/streptomycin at 37°C. Expression of epi-
thelial cell markers including cytokeratin and brush border vesicle was
verified by immunofluorescence studies of subconfluent monolayers.
SV40-transformed murine mesangial cell line, Mes13, purchased from
American Type Culture Collection (Manassas, VA) and a mouse macro-
phage cell line, RAW264.7, obtained from Riken Cell Bank (Tsukuba,
kines in MTECs after LPS or cytokine stimula-
tion. A, Immunofluorescence staining of MTECs
from C57BL/6 mice with mAb against mouse cy-
tokeratin. Original magnification, ?600. B, MCP-1
and RANTES gene transcription in MTECs and
Mes13 cells. MTECs were derived from C57BL/6
mice; Mes13 and RAW264.7 cells were cultured
under basal conditions or stimulated with 1 ?g/ml
LPS. Total RNAs were extracted at the indicated
time points after LPS exposure and examined for
the gene expressions of C-C chemokines (MCP-1,
RANTES, MIP-1?, and MIP-1?) by Northern
blot analysis. Ethidium bromide-stained gel is
shown as a control. C, MCP-1 and RANTES
mRNA after cytokine stimulation of MTECs.
MTECs were stimulated for 2 h by 10 ng/ml
TNF-?, 10 ng/ml IFN-?, or 10 ng/ml IL-1?, fol-
lowed by mRNAs extraction for Northern blots.
D, MTECs were pretreated with various concen-
trations of cycloheximide (CHX) for 30 min fol-
lowed by a 2-h stimulation with 1 ?g/ml LPS.
MCP-1 and RANTES mRNA were analyzed by
Northern blot as above. E, MTECs from C57BL/6
mice were cultured at 2.5 ? 104cells/0.5 ml for
24 h and treated with 10 ?g/ml neutralizing Abs
against TNF-? (anti-TNF-?) or control IgG for 30
min. Subsequently, MTECs were stimulated with
1 ?g/ml LPS for 12 h. MCP-1 (upper panel) and
RANTES (lower panel) concentrations were de-
termined by ELISA. These data are representative
of three experiments.
Gene expression of C-C chemo-
2027The Journal of Immunology
Japan), were maintained in DMEM supplemented with 10% FCS (Sigma-
Aldrich). Cells were cultured at 37°C in 5% CO2/95% air.
Northern blot analysis
cDNA was synthesized from 2 ?g of total RNA derived from RAW264.7
cells by RT-PCR. PCR of the synthesized cDNA was performed as previously
described (17). The synthesized PCR products were used for specific probes.
The primers were: mouse TLR1 sense, CTGAAGGCTTTGTCGATACA;
mouse TLR1 antisense, GGGAAACTGAGTTATGGTCG; mouse TLR3
sense, ATGTTTCAGTGCATCGGATT; mouse TLR3 antisense, AAACAT
TCCTCTTCGCAAAC; mouse TLR5 sense, GAATTCCTTAAGCGACGT
AA; mouse TLR5 antisense, GAGAAGATAAAGCCGTGCGA; mouse TLR6
sense, AGTGCTGCCAAGTTCCGACA; mouse TLR6 antisense, AGCAA
ACACCGAGTATAGCG; mouse TLR9 sense, CCAGACGCTCTTCGAG
AACC; mouse TLR9 antisense, GTTATAGAAGTGGCGGTTGT; mouse
MCP-1 sense, GCTGTTCACAGTTGCCGGCT; mouse MCP-1 antisense,
CATTAGCTTCAGATTTACGG; mouse RANTES sense, CCCTCTGCAC
CCCCGTACCT; mouse RANTES antisense, CCATTTTCCCAGGACCGA
GT; mouse macrophage inflammatory protein (MIP)-1? sense, CTCAACAT
CATGAAGGTCTC; mouse MIP-1? antisense, GGCATTCAGTTCCAGGT
CAG; mouse MIP-1? sense, CTCTCTCTCCTCTTGCTCGT; and mouse
MIP-1? antisense, CTCCATGGGAGACACGCGTC. cDNA fragments con-
taining the full coding regions of mouse TLR2, mouse TLR4 (16), mouse
MyD88 (28), mouse TIRAP/Mal (9, 10), and mouse MD-2 (29) were also
synthesized by RT-PCR and used for specific probes. Total cellular RNA
was prepared using TRIzol reagent (Life Technologies, Rockville, MD).
Total RNAs (5- to 15-?g aliquots) were used for Northern blot analysis as
previously described (17).
Cell extract preparation and immunoblotting
Cells were lysed in PLC buffer (50 mM HEPES (pH 7.0), 150 mM NaCl,
10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF,
10 mM NaPPi, 1 mM Na3VO4, 1 mM PMSF, 10 ?g/ml aprotinin, 10 ?g/ml
leupeptin) and analyzed by immunoblotting as previously described (17).
In vitro kinase assay
Cell lysates (107cells/sample) were immunoprecipitated with 0.4 ?g of
anti-JNK1 polyclonal Ab, anti-ERK1/2 polyclonal Ab, and anti-p38
MAPK polyclonal Ab for 2 h at 4°C, respectively, followed by incubation
with protein A-Sepharose beads for an additional 1 h. The beads were
extensively washed with PLC buffer three times followed by kinase buffer
(20 mM Tris-HCl (pH 7.4), 20 mM MgCl2, and 2 mM EGTA) once. The
kinase reaction was initiated by the addition of 30 ml of kinase buffer with
20 mM ATP, 5 ?Ci of [?-32P]ATP (New England Nuclear, Boston, MA)
and 0.5 ?g of GST-c-Jun for JNK (16), myelin basic protein (Sigma-
Aldrich) for ERK or GST-ATF2 for p38 MAPK (16), and was allowed to
proceed for 15 min at 30°C. The reaction was terminated by the addition
of 2? SDS sample buffer, resolved by SDS-PAGE, and the fixed gel was
exposed to an x-ray film.
MTECs from C57BL/6 mice were pretreated with various concentrations
of curcumin for 30 min followed by a 30-min stimulation with 1 ?g/ml
LPS. Subsequently, nuclear extracts were prepared from cells as previously
described (30). An oligonucleotide containing the NF-?B sense sequence
(5?-AGT TGA GGG GAC TTT CCC AGG C-3?) was used as a probe for
EMSA. Approximately 1 ? 104cpm of32P-labeled oligonucleotide, 10 ?g
of nuclear extract, and 1 ?g of poly(dI ? dC) were added to the binding
buffer (10 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 4% glycerol) and
incubated for 30 min at 4°C. The reaction mixtures were run in a 5%
nondenaturing polyacrylamide gel at 4°C in Tris-borate-EDTA buffer (90
mM Tris-borate, 2 mM EDTA).
Determination of chemokine production
Concentrations of MCP-1 and RANTES in the culture supernatants of
MTECs were measured by commercial ELISA kits (R&D Systems, Min-
neapolis, MN) according to the manufacture’s instructions. All samples
were assayed in triplicate and the data were presented as the mean ? SD.
Gene expression of C-C chemokines in MTECs and a mouse
mesangial cell line after LPS stimulation
MCP-1, RANTES, MIP-1?, and MIP-1? are major mononuclear
cell-directed chemokines expressed in the interstitium or glomeruli
of a variety of murine or human glomerulonephritis (6, 31–33). To
examine the expression of these four chemokines in LPS-treated
renal cells, MTECs were isolated and grown in primary culture as
described in Materials and Methods. The purity of the MTECs was
verified by the expression of epithelial cell markers such as cyto-
keratin (Fig. 1A) and brush border vesicle (data not shown) to be
95%. We also analyzed a moue mesangial cell line (Mes13) for a
comparison. These two cell types were stimulated with LPS for 2
mRNA induction is impaired by various inhibitors for
NF-?B or MAPKs. A and B, Upper panel, MTECs
from C57BL/6 mice were pretreated with indicated
concentrations of curcumin or SP600125 for 30 min
followed by a 2-h stimulation with 1 ?g/ml LPS. Total
RNAs (10 ?g/each) were analyzed by Northern blot. A
photograph of the ethidium bromide-stained gel is
shown as a control. Lower panel, JNK activation was
measured by the in vitro kinase assay using GST-c-Jun
as substrate. The nuclear extracts were also prepared
from MTECs, and EMSA was performed using a NF-
?B-specific consensus probe as described in Materials
and Methods. C and D, Upper panel, MTECs from
C57BL/6 mice were pretreated with various concentra-
tions of PD98059 or SB203580 for 30 min followed by
a 2-h stimulation with 1 ?g/ml LPS. After total RNA
extraction, Northern blot hybridization was performed
as above. Lower panel, Effective inhibition of LPS-me-
diated ERK and p38 MAPK by specific inhibitors was
shown. Cells were pretreated with PD98059 or
SB203580 as in the upper panel. After a 30-min stim-
ulation with LPS, LPS-mediated ERK or p38 MAPK
phosphorylation was measured by Western blot using
an anti-phospho-ERK or anti-phospho–p38 Ab. ERK
and p38 activation were measured by the in vitro kinase
assay using myelin basic protein and GST-ATF2 as
substrate, respectively. Data shown represent one of the
LPS-mediated MCP-1 and RANTES
2028ROLES OF TLRs IN CHEMOKINE PRODUCTION BY TECs
or 8 h, and the total RNA was extracted for the Northern blot
analysis (Fig. 1B). As a control, we used RAW264.7, a mouse
macrophage cell line, that produces various cytokines and chemo-
kines in response to LPS (34–36). Among all C-C chemokines
examined in the present study, MCP-1 mRNA was significantly
induced after LPS stimulation in both MTECs and Mes13 cells. On
the other hand, induction of RANTES mRNA was detected only in
MTECs. MIP-1? and MIP-1? were not expressed in either MTECs
or Mes13 cells even after LPS stimulation, whereas LPS signifi-
cantly induced these chemokine mRNAs in RAW264.7 cells.
MTECs were also stimulated for 2 h by several proinflammatory
cytokines, including TNF-?, IFN-?, and IL-1? and assessed for
their MCP-1 and RANTES gene transcripts. As shown in Fig. 1C,
these cytokines rapidly induced significant amounts of MCP-1 and
RANTES mRNA in MTECs. Together, these results suggest that
LPS, as well as inflammatory cytokines, directly affects MTECs to
express both MCP-1 and RANTES, but not MIP-1? or ?.
MTECs are able to produce various cytokines including TNF-?
by LPS or proinflammatory cytokines. To evaluate the possibility that
LPS increases chemokine mRNA indirectly by inducing cytokine
secretion, MTECs were pretreated with cycloheximide, a protein
synthesis inhibitor, before LPS stimulation. As shown in Fig. 1D,
cycloheximide rather enhanced the LPS-mediated increase of MCP-1
mRNA in MTECs, but partially inhibited RANTES mRNA induc-
tion. Furthermore, neutralization of TNF-? by a specific Ab did not
impair the LPS-induced MCP-1 and RANTES synthesis (Fig. 1E).
MAPKs do not regulate MCP-1 mRNA after LPS stimulation,
but JNK and p38 MAPK activation pathways are involved in
LPS-mediated RANTES mRNA expression in MTECs
To examine the molecular mechanisms for chemokine production
in MTECs, we pretreated the cells with inhibitors of various sig-
naling molecules. It has been reported that curcumin inhibits JNK
activation at a concentration of 5 or 10 ?M, whereas higher con-
centrations of curcumin (50 ?M or higher) inhibit NF-?B activa-
tion in various cell types (16). Consistent with the previous report,
LPS-mediated JNK activation was significantly inhibited at 10 ?M
curcumin, while inhibition of NF-?B activity needs 25 ?M cur-
cumin in MTECs (Fig. 2A). MCP-1 and RANTES mRNA induc-
tion by LPS was dramatically impaired by pretreatment with 25
?M curcumin (Fig. 2A). Pretreatment with 10 ?M curcumin did
not influence LPS-induced MCP-1 mRNA up-regulation, but in-
hibited RANTES gene transcription after LPS stimulation (Fig.
2A). SP600125 is a potent, cell-permeable, selective, and revers-
ible inhibitor of JNK (37, 38). In MTECs as well, SP600125 in-
hibited LPS-induced JNK activation in a dose-dependent manner,
but not NF-?B activity. When MTECs were preincubated with
SP600125, LPS-induced RANTES gene expression was signifi-
cantly decreased, whereas MCP-1 mRNA induction remained con-
stant (Fig. 2B). This is consistent with the result of curcumin,
clearly indicating that JNK activation is essential for LPS-medi-
ated RANTES mRNA induction. Next, MTECs were pretreated
with a specific inhibitor of ERK (PD98059) or p38 MAPK
(SB203580) pathway followed by LPS stimulation. As shown in
Fig. 2C, PD98059 treatment did not affect either chemokines
mRNA induction. On the other hand, pretreatment with SB203580
inhibited LPS-induced RANTES mRNA up-regulation in a dose-
dependent manner (Fig. 2D). Taken together, these findings sug-
gest that NF-?B activation seems essential for LPS-mediated up-
regulation of both MCP-1 and RANTES mRNA. Furthermore,
activation of p38 MAPK and JNK by LPS were also involved in
mRNA induction of RANTES but not MCP-1, indicating that the
induction mechanisms of these two chemokines are different.
Gene expression of TLRs in MTECs and Mes13 with or without
TLR4 has recently been revealed to work as a pattern recognition
receptor for LPS in various mammalian species including mouse.
MTECs and Mes13 cells were left untreated or stimulated with 1 ?g/ml
LPS for 2 or 8 h before total RNA extraction. Northern blot analysis for
TLR4 was performed. Left two lanes contain total RNAs isolated from
untreated or 1 ?g/ml LPS-treated RAW264.7 cells. The lowest panel
shows the ethidium bromide staining of the gel. B, Gene expression of
other TLRs in MTECs and Mes13 cells. RNAs were prepared as in A and
hybridized with TLR-specific cDNA probes. All blots are representative of
three independent experiments showing similar results.
Profile of TLR expressions in MTECs and Mes13. A,
2029 The Journal of Immunology
Thus, we attempted to disclose TLR4 expression in renal cells at
the transcriptional level in comparison with that of a macrophage
cell line, RAW264.7, which constitutively expresses a significant
amount of TLR4 mRNA (16). As shown in Fig. 3A, both MTECs
and Mes13 cells constitutively expressed TLR4 mRNA at compa-
rable levels to that of RAW264.7 cells. Similarly to RAW264.7
cells, LPS stimulation did not further increase TLR4 mRNA in
these renal cells.
We next characterized the mRNA expression of other TLRs in
renal cells (Fig. 3B). TLR2 mRNA was weakly expressed in both
renal cell types without stimulation, but significantly increased af-
ter LPS treatment. TLR3 gene expression was constitutively de-
tected in MTECs and further increased with LPS treatment. Mes13
cells, on the other hand, expressed barely detectable TLR3 mRNA,
which did not increase with LPS treatment. TLR5 gene expression
was constitutively identified in Mes13 cells, but almost undetect-
able in MTEC. TLR6 was constitutively expressed at lower levels
in both renal cell types. Furthermore, TLR1 mRNA expression
was constitutive but rather weak. A recent report revealed that
TLR9 recognizes bacterial DNA (14). However, TLR9 gene ex-
pression was undetectable in either renal cell type.
It is of note that multiple mRNA bands were always detected
using TLR6 and TLR9 probes. The nucleotide sequence of mam-
malian TLR6 is most similar to that of TLR1 (39). Especially,
nucleotide sequence bases 1333–2251 of the mouse TLR6 cDNA
has 98% identities to mouse TLR1 cDNA. To avoid cross-hybrid-
ization between TLR1 and TLR6 mRNA, we chose a mouse TLR6
cDNA region which is not homologous to mouse TLR1 as the
probe. Multiple transcripts were also detected using another partial
cDNA fragments encoding bases 1–450 and 883-1332 of mouse
TLR6 cDNA (data not shown). Human TLR9 was reported to be
expressed in at least two splice forms, one of which is monoexonic
and the other is biexonic. The latter encodes a protein with 57
additional amino acids at the N terminus (40). In addition, Hemmi
et al. (14) demonstrated two kinds of mouse TLR9 transcripts by
Northern blot analysis. In the present study, we could detect two
TLR9 transcripts. The same two TLR9 mRNA bands were de-
tected by other TLR9 probes containing bases 1–1356 of the
mouse TLR9 coding region (data not shown). These results indi-
cates that both mouse TLR6 and TLR9 genes produce multiple
mRNAs by alternative splicing.
Membranous CD14, MD-2, MyD88, and TIRAP/Mal expression
in MTECs and Mes13
At least four molecules, including CD14, MD-2, MyD88, and
TIRAP/Mal, have been shown to be essential for LPS signaling
(7–10). Thus, we examined their expression in renal cells. Western
blot analysis showed that MTECs expressed a significant amount
of membranous CD14 at the protein level. Mes13 cells, in contrast,
expressed much less CD14 protein compared with MTECs (Fig.
4A). As shown in Fig. 4, B and C, gene expression of MD-2,
MyD88, and TIRAP/Mal was detected both in MTECs and Mes13
and induced after LPS stimulation in a time-dependent manner.
The protein level of MyD88, however, did not increase in both
renal cell types as well as in RAW264.7 cells (Fig. 4D).
Synthetic lipid A induces C-C chemokine gene expression and
protein production in MTECs through TLR4
To investigate whether TLR4 expression in MTECs is responsible
for LPS-induced C-C chemokine production from MTECs, we iso-
lated MTECs from C3H/HeJ mice, which have a TLR4 mutation
that substitutes histidine for proline at position 712 (7). To elim-
inate the effects of the possible contamination of commercial grade
of LPS with cell stimulatory substances, we stimulated MTECs
with synthetic lipid A. Lipid A has long been established as the
bioactive component of LPS and this is the common structural
feature shared by every LPS (7). As shown in Fig. 5A, synthetic
lipid A induced MCP-1 and RANTES mRNA in MTECs from
C3H/HeN mice but not that from C3H/HeJ mice. We further mea-
sured the concentration of MCP-1 and RANTES protein in the
culture supernatants after synthetic lipid A stimulation and found
that the secretion of both chemokines was significantly induced in
MTECs from control C3H/HeN mice, but not in those from C3H/
HeJ mice (Fig. 5, B and C). These results clearly indicated that
TLR4 expressed on MTECs played a critical role in LPS-induced
Synthetic lipoprotein induces C-C chemokine gene expression
and protein production in MTECs through TLR2
Lipoprotein is one of cell wall components that exist in both Gram-
positive and Gram-negative bacteria. Bacterial lipoprotein acts
MD-2, MyD88, and TIRAP/Mal in renal cells. A,
Membranous CD14 expression on MTECs and Mes13
cells. Cells were untreated or treated with 1 ?g/ml
LPS for 2 h, followed by cell lysate preparation. West-
ern blot analysis was performed by using anti-CD14
Ab. Cell lysates from RAW264.7 cells and HEK293T
cells were used as a positive and negative control, re-
spectively. B and C, MTECs and Mes13 cells were
cultured with or without 1 ?g/ml LPS for the indicated
times. Total RNAs (10 ?g/each) were extracted for the
Northern blot analysis using mouse MD-2, MyD88,
and TIRAP/Mal cDNA probes. As a positive control,
total RNA was extracted from RAW264.7 cells. The
ethidium bromide-stained gel is also shown. D,
MyD88 protein expression. Cells were untreated and
treated with 1 ?g/ml LPS for 2 or 8 h, followed by
Western blot analysis using anti-MyD88 Ab. All blots
are representative of three independent experiments
showing similar results.
Protein or gene expression of CD14,
2030ROLES OF TLRs IN CHEMOKINE PRODUCTION BY TECs
synergistically with LPS to induce proinflammatory cytokine pro-
duction and lethal shock (41). Previous reports have suggested that
TLR2, but not TLR4, is the required receptor for the cellular re-
sponse to bacterial lipoproteins (42, 43). In our subsequent study,
we investigated the role of TLR2 in lipoprotein-mediated chemo-
kine production by MTECs using TLR2-deficient mouse. As
shown in Fig. 6A, MCP-1 and RANTES mRNA were not in-
creased in MTECs from TLR2-deficient mice after synthetic li-
poprotein treatment. In contrast, they were significantly induced in
MTECs from wild-type mice. ELISA revealed that the protein se-
cretion of both MCP-1 and RANTES was significantly induced
from MTECs of wild-type mice, but not from those of TLR2-
deficient mice after synthetic lipoprotein stimulation (Fig. 6, B and
C). These results indicate that TLR2 is essential for C-C chemo-
kine production induced by synthetic lipoprotein.
CpG-ODN does not induce MCP-1 and RANTES in MTECs
A recent report revealed that TLR9 recognizes bacterial DNA (14).
We next assessed MCP-1 and RANTES production by MTECs
after stimulation with CpG-ODN. RAW264.7 cells expressed
TLR9 at the transcriptional level (Fig. 3B) and secreted significant
MCP-1 and RANTES proteins after CpG-ODN stimulation. In
contrast, MTECs did not produce either chemokine in response to
CpG-ODN (Fig. 7). This is consistent with our finding that MTECs
are defective of TLR9 mRNA (Fig. 3B) and suggests that MTECs
are refractory to bacterial DNA for chemokine production.
Tubulointerstitial and glomerular accumulations of immune cells,
such as macrophages and T cells, are a prominent feature of a
variety of nephritis (6, 31–33). The C-C family of chemokines is
major mononuclear cell chemoattractants and may be central to the
MTECs through TLR4 at transcriptional and protein levels. A, MTECs
were isolated from C3H/HeN mice and C3H/HeJ mice and cultured with 1
?g/ml synthetic lipid A for the indicated times. Total RNAs were extracted
for Northern blot analysis for MCP-1 and RANTES. The ethidium bro-
mide-stained gel is also shown. The data are representative of three inde-
pendent stimulation experiments giving similar results. B and C, MTECs
from C3H/HeN mice and C3H/HeJ mice were cultured at 5 ? 104cells/0.5
ml for 24 h and then in fresh medium (0.5 ml) in the absence or presence
of synthetic lipid A. After 12 h, MCP-1 (B) and RANTES (C) protein
concentrations in the culture supernatant were determined by ELISA. All
samples were assayed in triplicate and the data were presented as the
mean ? SD.
Synthetic lipid A induces MCP-1 and RANTES from
tion in MTEC of TLR2-deficient mice. A, MTECs from TLR2?/?mice and
TLR2?/?mice were harvested and treated with 1 ?g/ml synthetic lipopro-
tein for the indicated times. Total RNAs were prepared and examined for
the expressions of MCP-1 and RANTES by Northern blot analysis. A
photograph of the ethidium bromide-stained gel is shown in the lowest
panel. The data are representative of three independent stimulation exper-
iments giving similar results. B and C, MTECs derived from TLR2?/?
mice and TLR2?/?mice were cultured as in Fig. 5 with or without syn-
thetic lipoprotein. MCP-1 (B) and RANTES (C) in the culture supernatants
were measured by ELISA after a 12-h stimulation. All samples were as-
sayed in triplicate and the data were presented as the mean ? SD.
The effects of synthetic lipoprotein on chemokines produc-
2031The Journal of Immunology
recruitment of these cells. As LPS is involved in the onset or pro-
gression of acute and chronic renal diseases (2–5), C-C chemo-
kines produced by renal cells after LPS exposure (44–46) may
play a predominant role in the pathogenesis. LPS initiates multiple
intracellular signaling events, including the activation of NF-?B,
AP-1, and three distinct MAPKs: p38 MAPK, ERK, and JNK (7).
Recently, it has been documented that LPS-induced MCP-1 gene
expression in rat tubular epithelial cells is NF-?B dependent (46). In
our studies performed by using various inhibitors, the LPS-mediated
MCP-1 mRNA increase was dependent on NF-?B activation, but not
on the three MAPK signaling pathways. In contrast, much less is
known about the regulation of RANTES mRNA in MTECs. In
LPS-stimulated macrophage cell line, RAW264.7 cells, it was shown
that JNK and NF-?B response elements were involved in RANTES
gene activation (47). Besides, both p38 MAPK and JNK signaling
pathways regulate RANTES production in influenza virus-infected
human bronchial epithelial cells (48). In our present study, we have
shown that LPS-mediated RANTES production in MTECs is at least
partially sensitive to JNK and p38 MAPK inhibition. Collectively, the
signal transduction pathway regulating RANTES gene transcription
may be cell and stimulus specific.
Transcription of MCP-1 and RANTES was induced by synthetic
lipid A stimulation in MTEC derived from C3H/HeN mice, but not
from C3H/HeJ mice, which lacks functional TLR4. On the contrary,
MTECs from TLR2?/?mouse failed to produce the two chemokines
in response to lipoprotein. These findings clearly demonstrated that
TLR4 and TLR2 are strictly required for chemokine production by
MTECs in response to lipid A and lipoprotein, respectively.
In Northern blot analyses, both MTECs and Mes13 express
TLR4 at a level comparable to that of a macrophage cell line,
RAW264.7 cells. Several proteins other than TLR4 are also in-
volved in LPS signaling. Both CD14 and MD-2 are helper mole-
cules for TLR4 and required for LPS recognition (7). We found
that CD14 was constitutively expressed on both MTECs and the
mesangial cell line, although the expression level of the latter was
significantly lower. MD-2 was also expressed on MTECs and Mes13
at the transcriptional level and, interestingly, the expression was
rapidly induced after LPS stimulation. MyD88, an adapter molecule,
is indicated as an essential component in the downstream signaling of
TLRs (49). In addition, TIRAP/Mal is recently reported as another
adapter protein that controls activation of MyD88-independent sig-
naling pathways downstream of TLR4 (9). In both MTECs and
Mes13, the expression of these adopter molecules was induced after
LPS stimulation at the transcriptional level, although the protein level
of MyD88 was not affected. Together, these data indicate that both
MTECs and Mes13 cells contain essential components of LPS re-
sponsiveness. The existence of the LPS signaling pathway through
TLR4 in renal interstitial and glomerular cells may be important as
these renal cells are directly exposed to both retrograde and blood-
derived Gram-negative bacteria.
We have also characterized gene expressions for TLR members
other than TLR4. TLR2 has previously been implicated in LPS
signaling as well as TLR4, but recent studies have suggested that
TLR2 recognizes other bacterial components such as PGN, li-
poarabinomannan, lipoteichoic acid, and mycobacterial lipoprotein
(7). As demonstrated in our previous report, mouse TLR2 gene
transcription is inducible after treatment of LPS in macrophages
(16). We also demonstrated that the two NF-?B binding sites in the
5? upstream region are essential for the responsiveness of TLR2 to
LPS (50). Because NF-?B was also activated in MTECs after LPS
stimulation, our current data that LPS rapidly induces TLR2 gene
expression at 2 h is compatible to our previous data in macro-
phages and T lymphocytes (16, 17, 50). In the process of nephritis,
renal TECs, which express excessive TLR2 in response to LPS,
may become more responsive to bacterial components.
TLR3 mRNA that recognizes dsRNA (12) were expressed in
MTECs, but scarcely in Mes13. TLR3 mRNA increased in
MTECs by LPS stimulation. In contrast, mRNA of TLR5, recently
identified as a receptor for bacterial flagella (13), was present in
Mes13 but undetectable in MTECs or in RAW264.7 cells. Also,
TLR6, that was shown to enhance the TLR2-dependent response to
phenol-soluble modulin (51) or zymosan (11), was expressed in all
renal cell lines examined, but their transcriptional levels were not
induced after LPS stimulation. TLR1 gene expression was induc-
ible in RAW264.7 cells after LPS stimulation, but was weakly
expressed in renal cells. TLR9, suggested as the receptor that rec-
ognizes bacterial DNA (14), was detected only in the macrophage
cell line, but not in either renal cell type. Consistent with these
findings, CpG-ODN did not induce MCP-1 or RANTES produc-
tion by MTECs. Taken together, TLR mRNA expression patterns
of renal cells are somewhat different from that of macrophages.
Based on these findings, we can speculate that renal cells respond
well to several bacterial components but not to the others. Recent
studies have revealed that renal tubular cells play important roles
in controlling immune responses by presenting Ags, expressing
costimulatory proteins, and secreting cytokines/chemokines (52).
As TLRs control these processes in professional APC, such as
dendritic cells, immunogenic actions of renal tubular cells may
also be affected by the presence of various bacterial components
through TLRs expressed on the cell surface.
In summary, we have demonstrated that TLR2 and TLR4 ex-
pressed in MTECs mediate their direct responses to bacterial com-
ponents. As excessive activation of TLRs in TECs may result in
further influx of inflammatory cells to the renal interstitium and in
subsequent development of renal dysfunction, the regulation of
TLR function may be one of the therapeutic targets for renal dis-
eases after bacterial infection. We have also found that MTECs,
unlike macrophages, are defective of TLR9 expression and do not
produce chemokines in response to CpG-ODN. This indicates that
CpG-ODN may be used as a therapeutic adjuvant for vaccination
without severe side effects of renal injury.
and MTECs after CpG-ODN stimulation. RAW264.7 cells and MTECs
from C57BL/6 mice were seeded at 5 ? 104cells/0.5 ml/well in 24-well
plates in triplicate for 24 h. Subsequently, the supernatant in each well
was removed and cultured by fresh DMEM (0.5 ml) in the absence or
presence of CpG-ODN at the indicated concentration. After 12 h, MCP-1
and RANTES protein concentrations in the culture supernatant were de-
termined by ELISA. All samples were assayed in triplicate and the data
were presented as the mean ? SD.
MCP-1 and RANTES production by macrophage cell line
2032ROLES OF TLRs IN CHEMOKINE PRODUCTION BY TECs
We thank K. Itano, A. Nishikawa, and N. Suzuki for their technical
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2033The Journal of Immunology