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and TNF-
Mediated Up-Regulation During Inflammation1
*
Nutrition and Toxicology Research Institute Maastricht, Department of General Surgery, University of Maastricht, Maastricht, The Netherlands; and
Department of Pulmonology, Leiden University Medical Center, Leiden, The Netherlands
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-
. These results indicate a potential
mechanism of increased immunosurveillance during inflammation at the
site in which ascending bacteria enter the kidney tissue, i.e., the
collecting ducts and the distal part of the
nephron. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B. The latter induces the
transcription of the genes encoding for cytokines, chemokines, and
adhesion molecules crucial to the inflammatory process aimed at
clearance of invading bacteria (1, 10).
Both TLR2 and TLR4 are predominantly expressed by monocytes/macrophages
and neutrophils (11). Lower expression of TLR is observed
in vitro by several other leukocytes, endothelial cells, epithelial
cells, and fibroblasts (12, 13, 14, 15, 16). In vitro experiments
indicate that TLR expression is modulated by bacterial products and
cytokines (11). Accordingly, an IFN--responsive element
is found in the promoter region of the gene encoding for TLR4
(17). Although the above indicates putative pathways that
regulate TLR expression, the factors that mediate TLR expression in
vivo remain to be elucidated.
Functional TLR4 expression is required for the resistance to
experimental pyelonephritis induced by a bladder inoculum with
Gram-negative bacteria. This is evidenced by the persistence of
Gram-negative bacteria in the kidney after pyelonephritis induction in
C3H/HeJ mice (18) that carry a malfunctioning TLR4
(3), while mice bearing intact TLR4 rapidly clear bacteria
from the urinary tract. The cellular origin of TLR expression in the
kidney responsible for resistance to bacteria is unknown. Considering
that the kidney is an important port of entry for bacteria, we set out
to localize TLR expression in the kidney and to study the role of
cytokines in the regulation of TLR expression in vivo. We used a renal
ischemia/reperfusion (I/R) model that leads to transient tissue damage
that is associated with an inflammatory process that develops rapidly
during reperfusion (19). The enhanced expression of
macrophage-inflammatory protein-2, KC, TNF-, IFN-, and MHC
class I and II molecules in this renal sterile inflammation model
(20, 21, 22) resembles the pattern of production of
immunological mediators after bacterial infections to a large extent.
This model enabled us to study the modulation of the expression of TLR2
and TLR4 mRNA by cytokines in vivo during inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following Abs were used: anti-murine IFN- mAb F3 and
anti-murine TNF- mAb TN3 were kindly provided by HBT (Uden, The
Netherlands) and Celltech (Slough, U.K.) respectively; polyclonal
rabbit anti-murine TLR4 serum was kindly provided by B. Beutler
(The Scripps Research Institute, La Jolla, CA); peroxidase-conjugated
goat anti-rat IgG and peroxidase-conjugated goat anti-rabbit
IgG were from Jackson ImmunoResearch Laboratories (West Grove, PA);
anti-Tamm Horsfall protein was from Cappel (Durham, NC); sheep
anti-digoxigenin/alkaline phosphatase was from Roche (Basel,
Switzerland); and biotinylated rabbit anti-goat IgG was from DAKO
(Glostrup, Denmark). Other reagents were proteinase K (Life
Technologies, Paisley, U.K.); dextran sulfate (Pharmacia, Uppsala,
Sweden); streptABComplex/AP (DAKO); polyvinylalcohol, formamide,
and 2-ME (Merck, Darmstadt, Germany); DTT and salmon sperm DNA
(Sigma-Aldrich, St. Louis, MO); and T7 and SP6 RNA polymerase, tRNA,
nitroblue tetrazolium, and bicholylindolyl phosphate (Boehringer
Mannheim, Mannheim, Germany).
Animal model and protocol
All experiments were approved by the Institutional Animal Care
Committee of the University of Maastricht (Maastricht, The
Netherlands). Renal ischemia was induced as described (20, 21). In brief, male Swiss mice (Charles River Breeding
Laboratories, Heidelberg, Germany) were anesthetized and body
temperature was maintained at 39°C. After laparotomy, ischemia was
induced by clamping the left renal pedicle for 45 min, during which the
wound was covered. Subsequent to removal of the clamp, the
contralateral kidney was removed and stored. After closing the abdomen,
mice were supplemented with prewarmed PBS to maintain fluid balance.
The animals were sacrificed at 1 and 6 h as well as 1, 3, and 5
days after ischemia. The experimental left kidney was harvested
immediately and divided into specimens for assays described below. Mice
subjected to ischemia were treated 10 min before reperfusion with 0.5
ml of PBS i.p. containing 300 µg of anti-IFN- mAb F3, 1
mg of anti-TNF- mAb TN3, or PBS only. Previously, we have shown
that mice receiving an isotype-matched control Ab did not differ in
inflammatory parameters or renal injury when compared with the
ischemia/PBS control group (20, 21), indicating the
specific effects of the respective Abs.
In situ hybridization
Oligonucleotide primers were designed for the specific PCR amplification of a fragment of murine TLR2 and TLR4. The TLR2 primers were 5'-TCT GGG CAG TCT TGA ACA TTT-3' (sense primer) and 5'-AGA GTC AGG TGA TGG ATG TCG-3' (antisense primer), yielding a 321-bp fragment. The TLR4 primers were 5'-GCA ATG TCT CTG GCA GGT GTA-3' (sense primer) and 5'-CAA GGG ATA AGA ACG CTG AGA-3' (antisense primer), yielding a product of 406 bp (all primers were synthesized by Eurogentec, Seraing, Belgium).
TLR2 and TLR4 cDNA fragments were amplified from murine kidney cDNA
prepared from total RNA by reverse transcription, as described below.
The fragments were both TA cloned into pGEM-Teasy (Promega, Madison,
WI). All clones obtained contained the correct TLR sequences that were
evaluated using the Big Dye termination cycle sequencing kit
(PerkinElmer/Cetus, Emeryville, CA), according to the manufacturer’s
instructions. DH5--competent cells were transformed by heat-shock
procedures. Subsequently, clones containing plasmids with the TLR
insert were selected, isolated, and purified using the Qiafilter
Plasmid midi kit (Qiagen, Hilden, Germany). Sense probes were prepared
after linearization of the plasmid with NcoI and
transcription with SP6 polymerase; antisense probes were prepared from
the plasmid after linearization with SpeI by transcription
with T7 polymerase. cRNA probes were labeled by incorporation of
digoxigenin-labeled UTP following the manufacturer’s protocol
(Boehringer Mannheim). In situ hybridization was performed as described
by de Boer et al. (23) using 3-µm paraffin sections
placed on coated slides (SuperFrost Plus; Menzel-Gläser,
Braunschweig, Germany). Briefly, after prewarming the sections and
subsequent rehydration with a decreasing xylene and ethanol gradient,
the sections were hybridized with 30 ng probe in 300 µl for 16
h. The hybridization was performed at 50°C for TLR2 and at 55°C for
TLR4 using a solution containing 50% formamide, 1 mg/ml tRNA, 10%
dextran sulfate, 10 mM DTT, 0.25 mg/ml salmon sperm DNA, and 4x SSC.
Subsequently, sections were first washed in 2x SSC with 50% formamide
at 37°C, second in 0.1x SSC with 20 mM 2-ME at 42°C, and, finally,
sections were treated with 100 U/ml RNase T1 in 2x SSC with 1 mM EDTA
at 37°C. Digoxigenin-labeled hybrids were detected with alkaline
phosphatase-conjugated sheep anti-digoxigenin, using nitroblue
tetrazolium as chromogen and bicholylindolyl phosphate as coupling
agent. Polyvinylalcohol was used to enhance the signal.
Evaluation of mRNA levels by RT-PCR
For RT-PCR, total RNA was extracted from kidneys using the SV
Total RNA isolation system (Promega) and treated with RQ1 RNase-Free
DNase (Promega). Total RNA was reverse transcribed using oligo(dT)
primer and Moloney murine leukemia virus reverse transcriptase (Life
Technologies) according to the supplier’s recommendations. cDNA
samples were standardized based on the content of 
-actin cDNA as
housekeeping gene. -actin cDNA was evaluated by performance of a
-actin PCR on multiple dilutions of each cDNA sample. The amount of
amplified product was estimated by densitometry of ethidium
bromide-stained 1.2% agarose gels using a CCD camera and Imagemaster
VDS software (Pharmacia). The TLR primers used for the generation of
the cRNA probes were also used for PCR amplification. Primers for
murine -actin were 5'-TAA AAC GCA GCT CAG TAA CAG TCG G-3' (sense
primer) and 5'-TGC AAT CCT GTG GCA TCC ATG AAA C-3' (antisense primer);
primers used for the amplification of murine TNF- mRNA, 5'-GGC AGG
TCT ACT TTG GAG TCA TTG C-3' and 5'-ACA TTC GAG GCT CCA GTG AAT TCG
G-3' (antisense primer); primers designed for amplification of murine
IFN- mRNA, 5'-AGC GGC TGA CTG AAC TCA GAT TGT AG-3' (sense primer)
and 5'-GTC ACA GTT TTC AGC TGT ATA GGG-3' (antisense primer). All
primers were used for PCR amplification of murine cDNA kidney samples
from mice exposed to the interventions. PCR with TLR2-, TLR4-,
-actin-, IFN--, or TNF--specific primers were performed using
appropriate dilutions of the cDNA. PCR were performed in a total volume
of 25 µl in PCR buffer (PerkinElmer/Cetus) in the presence of 0.2 mM
dNTP (Pharmacia), 1 µM of each primer, 0.3 mM
MgCl2, and 0.5 U of Taq polymerase
(PerkinElmer/Cetus). PCR conditions for each primer couple
were as follows: -actin, 95°C for 30 s, 60°C for 45 s,
and 72°C for 30 s during 21 cycles; TLR2, 95°C for 30 s,
57°C for 30 s, and 72°C for 45 s during 33 cycles;
TLR4, 95°C for 45 s, 61°C for 45 s, and 72°C for
45 s during 36 cycles; IFN-, 95°C for 30 s, 63°C
for 30 s, and 72°C for 30 s during 40 cycles; TNF-,
95°C for 30 s, 63°C for 45 s, and 72°C for 30 s
during 38 cycles. Levels of TLR2, TLR4, TNF-, and IFN- RNA
expression were evaluated by densitometric image analysis, as described
above. Relative TLR mRNA levels were calculated by comparison of band
intensities of the TLR RT-PCR products with standard curves prepared by
PCR amplifications on dilution series of a highly concentrated murine
kidney cDNA.
Immunohistochemistry
Staining with the anti-Tamm Horsfall protein on paraffin sections was performed to discriminate between distal tubules, proximal tubular epithelium, the loop of Henle, and collecting ducts. Immunohistochemistry was conducted on sections adjacent to those used for in situ hybridization. Paraffin sections treated as described above were incubated with appropriate dilutions of primary Ab, washed, developed using biotinylated rabbit anti-goat IgG and alkaline phosphatase-labeled strepABComplex, and visualized, as described above.
TLR4 Western blotting
Renal tissue samples for evaluation of TLR4 protein expression were obtained from mice of which the renal blood vessels were flushed, immediately after the mice were sacrificed, with ice-cold PBS containing nitroprusside and heparin by canulation of the left ventricle and opening of the vena cava. This was done to remove TLR4-expressing blood leukocytes. Western blotting on renal tissue was performed using 8% polyacrylamide SDS gels. Aliquots (50 µl) of kidney homogenates (50 mg/ml) in 2% SDS sample buffer were subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes (Immobilon P; Millipore, Bedford, MA). After transfer of the proteins, membranes were blocked with 5% nonfat dry milk in 50 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4 (TBST). Membranes were then incubated with polyclonal rabbit anti-murine TLR4 serum (24), kindly provided by B. Beutler (The Scripps Research Institute), at a 1/2500 dilution in TBST with 0.5% nonfat dry milk. Positive bands were visualized by chemiluminescence technology (Supersignal; Pierce, Rockford, IL) using peroxidase-conjugated goat anti-rabbit IgG at a 1/5000 dilution.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To gain insight into renal expression of TLR, we evaluated mRNA
expression of both TLR2 and TLR4 by RT-PCR in cDNA samples of healthy
kidneys. Constitutive expression of both renal TLR2 and TLR4 mRNA was
observed (Fig. 1
). To determine the
cellular origin of this constitutive expression, TLR mRNA
was localized by in situ hybridization.
As depicted in Figs. 2, A and B, and
3, A and B, basal
expression of TLR2 and TLR4 mRNA was observed predominantly in the
epithelial cells of the distal and proximal tubules and in Bowman’s
capsular epithelium. Expression in glomeruli and endothelial cells was
minor. While macrophages are reported to express relatively abundant
levels of TLR2 and TLR4 mRNA, it appeared that epithelial cells express
the majority of the TLR mRNA in the kidney. Only few resident
macrophage-like cell types were observed in the renal tissue, and these
cells stained to a similar extent as tubular epithelium. Proximal
epithelial cells in these healthy kidneys stained slightly more
intensely for TLR4 mRNA compared with the distal epithelium. Control
incubations using sense riboprobes were negative for healthy and
experimental kidneys (Fig. 3G).
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Mice were exposed to unilateral renal I/R, a process that leads to
tissue damage and a subsequent sterile inflammatory reaction. This
inflammatory process is characterized by initial tubular apoptosis,
followed by KC, macrophage-inflammatory protein-2, and TNF-
expression and subsequent neutrophil influx with apoptosis, necrosis,
and organ dysfunction peaking at day 1 (19, 20, 22).
Thereafter, a strongly enhanced IFN--mediated MHC-I and II
expression occurs from day 3 onward (21, 25). The effect
of this sterile inflammatory process on TLR2 and TLR4 mRNA levels was
assessed by RT-PCR analysis. Levels of renal TLR2 mRNA decreased to
undetectable levels in animals sacrificed at 1 and 6 h after
ischemia. TLR2 mRNA returned to basal level after 1 day (Fig. 1
). A
similar mRNA expression pattern was observed for TLR4 (Fig. 1): TLR4
mRNA expression was undetectable at 1 h; from 1 to 6 h, the
TLR4 synthesis returned to detectable levels. After 24 h of
reperfusion, TLR4 expression was enhanced when compared with controls.
TLR2 and TLR4 mRNA expression were similar in healthy and sham-operated
controls, indicating that the surgical procedure did not influence TLR
expression (data not shown). The results of densitometric
quantification of TLR2 and TLR4 mRNA levels at 1, 6, and 24 h
after ischemia are depicted in Fig. 4, A and B. At days 3 and 5 after renal ischemia, a
significant increase of both TLR2 and TLR4 mRNA was detected in the
kidney (Fig. 1). A 4- to 5-fold increase of TLR2 mRNA was observed when
compared with healthy controls (Fig. 5A). Similarly, a 4- and
5.5-fold enhancement of TLR4 mRNA was measured at 3 and 5 days
postischemia, respectively (Fig. 5B).
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D). In the medulla, TLR2 transcripts
appeared up-regulated in the thin limb of Henle’s loop (Fig. 2C). Moreover, TLR2 transcription was detected in
macrophage-like cells in the interstitial tissue (Fig. 2, C
and D). One day after ischemia, TLR4 expression was
predominantly observed in the epithelial cells of the distal tubules,
the thin limb of Henle’s loop, and collecting ducts, while low
expression was observed in the proximal tubules, glomeruli, and
capsular epithelium. A small number of macrophage-like cells were
present and stained positive. In concordance to the enhanced TLR mRNA
expression as measured by RT-PCR at 5 days after ischemia, intense
staining for both TLR2 and TLR4 mRNA was detected by in situ
hybridization at the same time point. The intense staining was
predominantly localized in the collecting ducts, the thin limb of
Henle’s loop, and distal tubules (Figs. 2, E and
F, and 3, E and F). Identification of
distal tubules in these experiments was performed by Tamm Horsfall
protein staining (Fig. 3H). At 5 days postischemia, a small
number of macrophage-like cells were present that stained positive for
TLR2 and TLR4. However, compared with the staining in collecting ducts,
the loop of Henle, and distal tubules, this TLR expression by
macrophage-like cells constituted only a minority of the total
expression (Figs. 2, E and F, and 3, E
and F). At day 5, only moderate TLR expression was detected
in the glomeruli and proximal tubules (Figs. 2, E and
F, and 3, E and F). Taken together,
renal I/R leads to marked enhancement of both TLR2 and TLR4 mRNA
levels, which appeared to be predominantly present in epithelial cells
of the distal tubules, the collecting ducts, and Henle’s thin
loop.
Role of IFN- and TNF- in regulation of renal TLR expression
Renal I/R results in TNF--modulated renal inflammation that is
followed by IFN--dependent up-regulation of MHC class I and II
molecules, indicating an active cytokine-mediated immune response in
the organ (20, 21, 25). The role of these cytokines in the
observed up-regulation of TLR mRNA expression in our I/R-induced renal
inflammation model was evaluated by blocking TNF- and IFN- with
inhibitory Abs. The observed enhancement of renal TLR2 mRNA expression
5 days after ischemia was completely prevented in animals treated with
anti-IFN- or anti-TNF- Abs (Fig. 6, A and B).
Moreover, blocking of either IFN- or TNF- reduced the TLR2 mRNA
expression at day 5 to below the constitutive level (Fig. 6B). In contrast to this major inhibitory effect of
anti-TNF- on TLR2 mRNA synthesis in our renal inflammation
model, blocking of TNF- resulted only in a partial reduction of TLR4
mRNA expression (Fig. 6, A and C). Similar to the
effect of anti-IFN- on TLR2 mRNA expression, blocking of IFN-
completely prevented the enhancement of TLR4 mRNA synthesis at 5 days
after ischemia, resulting in TLR4 mRNA expression below the
constitutive level. Our findings indicate that TLR2 and TLR4 expression
are regulated differentially by TNF- and IFN-. Because both
IFN- and TNF- are involved in the enhanced TLR mRNA expression,
we determined the effect of cytokine inhibition on cytokine mRNA
expression. Anti-IFN- treatment decreased IFN- mRNA translation
in the kidneys 5 days after the ischemic insult, whereas TNF- mRNA
was not decreased by inhibition of IFN- (Fig. 7A). Anti-TNF- Ab
administration did not decrease the renal TNF- and renal IFN-
mRNA levels when compared with PBS-treated animals (Fig. 7B). These findings show that IFN- does not enhance TLR
mRNA expression in our model by induction of TNF- and that TNF-
does not enhance TLR mRNA expression by induction of IFN-.
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Renal TLR4 expression was evaluated by Western blotting using
specific anti-murine TLR4 antiserum. Whole renal tissue samples,
and liver as positive control, were homogenized in SDS, and proteins
were separated by SDS-PAGE. Subsequent immunoblotting with the
anti-murine TLR4 antiserum revealed positive bands with an apparent
molecular mass of 
95 and 120 kDa in normal liver and in
kidney tissue 5 days after renal I/R (Fig. 8). The calculated molecular mass of
mature TLR4 based on its amino acid sequence is 93.5 kDa and is
consistent with the anti-TLR4-positive bands at 95 kDa in the
Western blot. The band at a molecular mass of 120 kDa in renal
tissue after I/R and in normal liver probably represents a glycosylated
form of the TLR4 protein. The observed molecular mass form of 120 kDa
of murine TLR4 is consistent with the reported apparent molecular mass
of human rTLR4 (26). Basal TLR4 protein expression was not
detectable with the used Western blotting technique in healthy
murine renal tissue (Fig. 8). These observations show that the
up-regulation of TLR4 mRNA expression during renal inflammation induced
by I/R is accompanied by a significant increased TLR4 protein
expression in the kidney.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF- and
is associated with a major increase of renal TLR4 protein expression.
While in vitro TLR4 mRNA expression and responsiveness had been
reported for several cell types such as leukocytes, endothelial cells,
epithelial cells, and fibroblasts, but most abundantly for macrophages
(11, 12, 13, 14, 15), the differential expression of TLR4 in vivo
remained to be elucidated. Our study indicates that murine renal
epithelial cells express TLR2 and TLR4 in vivo. The observation that
TLR2 and TLR4 mRNA expression in the kidney is predominantly located in
renal tubular epithelium appears to have some implications for the
understanding of the innate immune defense mechanism in the kidney
against pyelonephritis as a result of ascending urinary tract
infections (UTI). Gut-derived Gram-negative and Gram-positive bacteria
are the most prevalent microbial pathogens responsible for UTI, with
Escherichia coli being the most common (28).
Our data suggest that tubular epithelial cells can monitor the presence
of both types of bacteria. In this context, Hagberg et al.
(18) showed already in 1984 that C3H/HeJ mice, who lack
functional TLR4 (3), are highly susceptible to persistent
Gram-negative pyelonephritis. TLR-dependent cellular activation leads
to the translocation of NF-B to the nucleus, which leads to
transcription of genes encoding for cytokines, chemokines, adhesion
molecules, and antimicrobial peptides (1, 3, 10, 29).
Consistently, TLR4 signaling, induction of CXC chemokine expression and
CXCR signaling (18, 30), and subsequent neutrophil
recruitment are crucial for the clearance of Gram-negative bacteria
from the kidney (31, 32). Our results suggest that the
functional role of TLR4 in prevention of pyelonephritis as described
(18) could be the result of functional TLR4 expression by
the renal tubular epithelial cells. To monitor bacteria in the lumen of
tubules and collecting ducts, the TLRs should be expressed on the
apical membrane of the renal epithelial cells. It should be noted that
we located renal TLR expression at the mRNA level, and obviously these
results need to be confirmed by studies at the protein level by
immunohistochemistry and with functional studies. In regard of the
functionality of TLR4 in the kidney, it is noteworthy that the mRNA for
MD-2, the essential cofactor for TLR4, is abundantly present in the
murine kidney (3). We have confirmed the abundant renal
MD-2 mRNA expression by RT-PCR analysis, and also observed elevation of
MD-2 mRNA after I/R (data not shown). We applied a renal I/R model to elucidate the regulation of TLR expression during inflammation. This model allowed us to evaluate cytokine-mediated TLR mRNA expression in an inflammatory process that is not influenced by bacterial products. Most interestingly, a shift occurs from the observed diffuse proximal and distal staining for TLR2 and TLR4 mRNA in healthy kidneys to a predominant and enhanced expression 5 days after ischemia in the distal epithelial tubular cells, the thin limb of the loops of Henle, and collecting ducts. Following renal I/R injury, monocyte and T cell influx has been reported from day 3 onward (33). We observed a small number of macrophage-like cells that stained all positive for both TLR2 and TLR4; however, compared with the enhanced expression in collecting ducts, the thin limb of the loops of Henle, and distal tubules, this constituted only a minority of the total TLR expression. The increased TLR2 and TLR4 mRNA expression in our renal inflammation model by epithelial cells in the distal part of the nephron and the medulla suggests the mobilization of TLR-dependent antimicrobial potential during inflammation to the site in which ascending bacteria may enter the kidney.
The I/R-induced TLR mRNA expression in the later phase of reperfusion
(day 3–5) was dependent on the action of IFN- and TNF-.
Previously, we demonstrated that anti-TNF- reduces kidney
neutrophil influx and deterioration of renal function in our murine
model of renal I/R (20), whereas anti-IFN- did not
affect these parameters (21). The inhibition of TLR
up-regulation by anti-TNF- is not caused by a major effect on
the IFN- mRNA synthesis (Fig. 7). This observation is supported by
data showing that anti-TNF- does not block the up-regulation of
MHC molecules in this model, while this process is largely IFN-
dependent (20, 25). Because blocking of either TNF- or
IFN- results in inhibition of the majority of TLR2 expression, it
appears that the observed enhancement of TLR2 expression is elicited by
a synergistic action of these cytokines. TLR4 synthesis is only
partially blocked by anti-TNF- and almost completely by
anti-IFN-; therefore, it appears that the up-regulation of TLR4
in our inflammation model is primarily mediated by IFN-. Thus, the
regulation of TLR4 mRNA expression discerns with TLR2 in our in vivo
model in respect to the dependence on TNF-. Consistently, also in
vitro studies indicate that TLR2 and TLR4 expression are regulated via
distinct pathways (11). Our in vivo observation of
IFN--mediated TLR4 expression is in line with the presence of a
functional IFN response factor motif in the promoter region of the
human and mouse TLR4 gene (17). It should be mentioned
that we cannot exclude the possibility that TNF- or IFN- acts
indirectly by stimulation of a putative modulator of TLR
expression.
In general, IFN- is involved in bacterial clearance during the late
stages of infection with virulent Gram-negative or Gram-positive
bacteria (34, 35, 36). Furthermore, IFN- deficiency is
found to be associated with infection by poorly pathogenic
Mycobacterium strains and Salmonella
(37). Our observations suggest that IFN- augments
enhanced reactivity to bacteria in vivo by up-regulation of TLR2 and
TLR4 mRNA. Indeed, increased expression of TLR4 by IFN- may explain
the enhanced LPS-induced lethality of IFN--treated rabbits and the
enhanced LPS sensitivity of monocytes/macrophages induced by IFN-
(38, 39, 40). It appears that increased TLR2 and TLR4
expression may play a role in the mechanisms involved in
IFN--mediated resistance to virulent bacteria.
Although an obvious role for IFN- in UTI in humans is not reported,
it must be mentioned that the epithelial cells of the kidney stain
strongly positive with anti-IFN- receptor Abs in
immunohistochemistry (41), indicating the responsiveness
of these cells to IFN-. IFN- deficiency was observed to increase
the susceptibility of mice to experimental UTI with uropathogenic
E. coli (42). The latter is in line with the
IFN--mediated TLR4 expression (this study) and increased
susceptibility of TLR4-deficient mice for Gram-negative UTI
(18). Next to behavior and anatomical impairments of the
urinary tract, genetic factors also seem to be involved in human
ascending UTI (27). However, the genetic influence in
human UTI needs further investigation (see Ref. 30).
In conclusion, TLR2 and TLR4 are constitutively expressed in both
proximal and distal tubular renal epithelial cells in vivo. During
renal inflammation, TLR2 and TLR4 mRNA synthesis is enhanced by the
action of TNF- and IFN-, and this increased expression is mainly
localized in distal tubules, the thin limb of the loops of Henle, and
collecting ducts. The epithelial localization of TLR mRNA expression
suggests a role for epithelial-derived TLR signaling in the
inflammatory response observed during ascending UTI.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Cornelis van ’t Veer, Department of General Surgery, University of Maastricht, P.O. Box 616, 6200 MD Maastricht, The Netherlands. E-mail address: c.vantveer{at}ah.unimaas.nl
3 Abbreviations used in this paper: TLR, Toll-like receptor; I/R, ischemia/reperfusion; UTI, urinary tract infection.
Received for publication February 22, 2001. Accepted for publication November 26, 2001.
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 A. Rodrigues, D. B.C. Queiroz, L. Honda, E. J. R. Silva, S. H. Hall, and M. C. W. Avellar Activation of Toll-Like Receptor 4 (TLR4) by In Vivo and In Vitro Exposure of Rat Epididymis to Lipopolysaccharide from Escherichia Coli Biol Reprod, December 1, 2008; 79(6): 1135 - 1147. [Abstract] [Full Text] [PDF]
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 A. C. Shirali and D. R. Goldstein Tracking the Toll of Kidney Disease J. Am. Soc. Nephrol., August 1, 2008; 19(8): 1444 - 1450. [Abstract] [Full Text] [PDF]
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B. Zhang, G. Ramesh, S. Uematsu, S. Akira, and W. B. Reeves TLR4 Signaling Mediates Inflammation and Tissue Injury in Nephrotoxicity J. Am. Soc. Nephrol., May 1, 2008; 19(5): 923 - 932. [Abstract] [Full Text] [PDF]
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 H. Yao, I. Edirisinghe, S.-R. Yang, S. Rajendrasozhan, A. Kode, S. Caito, D. Adenuga, and I. Rahman Genetic Ablation of NADPH Oxidase Enhances Susceptibility to Cigarette Smoke-Induced Lung Inflammation and Emphysema in Mice Am. J. Pathol., May 1, 2008; 172(5): 1222 - 1237. [Abstract] [Full Text] [PDF]
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M. C. Banas, B. Banas, K. L. Hudkins, T. A. Wietecha, M. Iyoda, E. Bock, P. Hauser, J. W. Pippin, S. J. Shankland, K. D. Smith, et al. TLR4 Links Podocytes with the Innate Immune System to Mediate Glomerular Injury J. Am. Soc. Nephrol., April 1, 2008; 19(4): 704 - 713. [Abstract] [Full Text] [PDF]
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 R. A. Zager, A. C. M. Johnson, M. Naito, and K. Bomsztyk Maleate nephrotoxicity: mechanisms of injury and correlates with ischemic/hypoxic tubular cell death Am J Physiol Renal Physiol, January 1, 2008; 294(1): F187 - F197. [Abstract] [Full Text] [PDF]
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 A. Tsung, J. R. Klune, X. Zhang, G. Jeyabalan, Z. Cao, X. Peng, D. B. Stolz, D. A. Geller, M. R. Rosengart, and T. R. Billiar HMGB1 release induced by liver ischemia involves Toll-like receptor 4 dependent reactive oxygen species production and calcium-mediated signaling J. Exp. Med., November 26, 2007; 204(12): 2913 - 2923. [Abstract] [Full Text] [PDF]
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 V. Andreani, G. Gatti, L. Simonella, V. Rivero, and M. Maccioni Activation of Toll-like Receptor 4 on Tumor Cells In vitro Inhibits Subsequent Tumor Growth In vivo Cancer Res., November 1, 2007; 67(21): 10519 - 10527. [Abstract] [Full Text] [PDF]
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 K. Uno, K. Kato, T. Atsumi, T. Suzuki, J. Yoshitake, H. Morita, S. Ohara, Y. Kotake, T. Shimosegawa, and T. Yoshimura Toll-like receptor (TLR) 2 induced through TLR4 signaling initiated by Helicobacter pylori cooperatively amplifies iNOS induction in gastric epithelial cells Am J Physiol Gastrointest Liver Physiol, November 1, 2007; 293(5): G1004 - G1012. [Abstract] [Full Text] [PDF]
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 R. M. Levy, K. P. Mollen, J. M. Prince, D. J. Kaczorowski, R. Vallabhaneni, S. Liu, K. J. Tracey, M. T. Lotze, D. J. Hackam, M. P. Fink, et al. Systemic inflammation and remote organ injury following trauma require HMGB1 Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2007; 293(4): R1538 - R1544. [Abstract] [Full Text] [PDF]
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 C.-C. Yang, M.-C. Ma, C.-T. Chien, M.-S. Wu, W.-K. Sun, and C.-F. Chen Hypoxic preconditioning attenuates lipopolysaccharide-induced oxidative stress in rat kidneys J. Physiol., July 1, 2007; 582(1): 407 - 419. [Abstract] [Full Text] [PDF]
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H. J. Brown, H. R. Lock, T. G.A.M. Wolfs, W. A. Buurman, S. H. Sacks, and M. G. Robson Toll-Like Receptor 4 Ligation on Intrinsic Renal Cells Contributes to the Induction of Antibody-Mediated Glomerulonephritis via CXCL1 and CXCL2 J. Am. Soc. Nephrol., June 1, 2007; 18(6): 1732 - 1739. [Abstract] [Full Text] [PDF]
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A. A. Shigeoka, T. D. Holscher, A. J. King, F. W. Hall, W. B. Kiosses, P. S. Tobias, N. Mackman, and D. B. McKay TLR2 Is Constitutively Expressed within the Kidney and Participates in Ischemic Renal Injury through Both MyD88-Dependent and -Independent Pathways J. Immunol., May 15, 2007; 178(10): 6252 - 6258. [Abstract] [Full Text] [PDF]
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 S. Spiller, S. Dreher, G. Meng, A. Grabiec, W. Thomas, T. Hartung, K. Pfeffer, H. Hochrein, H. Brade, W. Bessler, et al. Cellular Recognition of Trimyristoylated Peptide or Enterobacterial Lipopolysaccharide via Both TLR2 and TLR4 J. Biol. Chem., May 4, 2007; 282(18): 13190 - 13198. [Abstract] [Full Text] [PDF]
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 C. I. Maratheftis, E. Andreakos, H. M. Moutsopoulos, and M. Voulgarelis Toll-like Receptor-4 Is Up-Regulated in Hematopoietic Progenitor Cells and Contributes to Increased Apoptosis in Myelodysplastic Syndromes Clin. Cancer Res., February 15, 2007; 13(4): 1154 - 1160. [Abstract] [Full Text] [PDF]
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 A. Tsung, N. Zheng, G. Jeyabalan, K. Izuishi, J. R. Klune, D. A. Geller, M. T. Lotze, L. Lu, and T. R. Billiar Increasing numbers of hepatic dendritic cells promote HMGB1-mediated ischemia-reperfusion injury J. Leukoc. Biol., January 1, 2007; 81(1): 119 - 128. [Abstract] [Full Text] [PDF]
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R. A. Zager, A. C. M. Johnson, S. Lund, and J. Randolph-Habecker Toll-like receptor (TLR4) shedding and depletion: acute proximal tubular cell responses to hypoxic and toxic injury Am J Physiol Renal Physiol, January 1, 2007; 292(1): F304 - F312. [Abstract] [Full Text] [PDF]
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C. Chassin, J.-M. Goujon, S. Darche, L. du Merle, M. Bens, F. Cluzeaud, C. Werts, E. Ogier-Denis, C. Le Bouguenec, D. Buzoni-Gatel, et al. Renal Collecting Duct Epithelial Cells React to Pyelonephritis-Associated Escherichia coli by Activating Distinct TLR4-Dependent and -Independent Inflammatory Pathways J. Immunol., October 1, 2006; 177(7): 4773 - 4784. [Abstract] [Full Text] [PDF]
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R. D. Pawar, P. S. Patole, M. Wornle, and H.-J. Anders Microbial nucleic acids pay a Toll in kidney disease Am J Physiol Renal Physiol, September 1, 2006; 291(3): F509 - F516. [Abstract] [Full Text] [PDF]
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R. A. Zager, A. C. M. Johnson, S. Lund, and S. Hanson Acute renal failure: determinants and characteristics of the injury-induced hyperinflammatory response Am J Physiol Renal Physiol, September 1, 2006; 291(3): F546 - F556. [Abstract] [Full Text] [PDF]
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H. J. Brown, H. R. Lock, S. H. Sacks, and M. G. Robson TLR2 Stimulation of Intrinsic Renal Cells in the Induction of Immune-Mediated Glomerulonephritis J. Immunol., August 1, 2006; 177(3): 1925 - 1931. [Abstract] [Full Text] [PDF]
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H. J. Brown, S. H. Sacks, and M. G. Robson Toll-Like Receptor 2 Agonists Exacerbate Accelerated Nephrotoxic Nephritis J. Am. Soc. Nephrol., July 1, 2006; 17(7): 1931 - 1939. [Abstract] [Full Text] [PDF]
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P. G. Tipping Toll-Like Receptors: The Interface between Innate and Adaptive Immunity J. Am. Soc. Nephrol., July 1, 2006; 17(7): 1769 - 1771. [Full Text] [PDF]
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G. Gatti, V. Rivero, R. D. Motrich, and M. Maccioni Prostate epithelial cells can act as early sensors of infection by up-regulating TLR4 expression and proinflammatory mediators upon LPS stimulation J. Leukoc. Biol., May 1, 2006; 79(5): 989 - 998. [Abstract] [Full Text] [PDF]
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T. M. El-Achkar, X. Huang, Z. Plotkin, R. M. Sandoval, G. J. Rhodes, and P. C. Dagher Sepsis induces changes in the expression and distribution of Toll-like receptor 4 in the rat kidney Am J Physiol Renal Physiol, May 1, 2006; 290(5): F1034 - F1043. [Abstract] [Full Text] [PDF]
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 C. F. Ortega-Cava, S. Ishihara, M. A. K. Rumi, M. M. Aziz, H. Kazumori, T. Yuki, Y. Mishima, I. Moriyama, C. Kadota, N. Oshima, et al. Epithelial Toll-Like Receptor 5 Is Constitutively Localized in the Mouse Cecum and Exhibits Distinctive Down-Regulation during Experimental Colitis Clin. Vaccine Immunol., January 1, 2006; 13(1): 132 - 138. [Abstract] [Full Text] [PDF]
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P Van der Meer, E Lipsic, R. Henning, K Boddeus, J van der Velden, A. Voors, D. van Veldhuisen, W. van Gilst, R. Schoemaker, J. Leemans, et al. Heart Failure after Myocardial Infarction--Benefit beyond Hemoglobin from Erythropoietin: Erythropoietin Induces Neovascularization and Improves Cardiac Function in Rats with Heart Failure after Myocardial Infarction J. Am. Soc. Nephrol., December 1, 2005; 16(12): 3449 - 3454. [Full Text] [PDF]
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J. C. Rice, T. Peng, J. S. Spence, H.-Q. Wang, R. M. Goldblum, B. Corthesy, and B. J. Nowicki Pyelonephritic Escherichia coli Expressing P Fimbriae Decrease Immune Response of the Mouse Kidney J. Am. Soc. Nephrol., December 1, 2005; 16(12): 3583 - 3591. [Abstract] [Full Text] [PDF]
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M. G. Netea, T. Azam, G. Ferwerda, S. E. Girardin, M. Walsh, J.-S. Park, E. Abraham, J.-M. Kim, D.-Y. Yoon, C. A. Dinarello, et al. IL-32 synergizes with nucleotide oligomerization domain (NOD) 1 and NOD2 ligands for IL-1{beta} and IL-6 production through a caspase 1-dependent mechanism PNAS, November 8, 2005; 102(45): 16309 - 16314. [Abstract] [Full Text] [PDF]
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R. P. Gomariz, A. Arranz, C. Abad, M. Torroba, C. Martinez, F. Rosignoli, M. Garcia-Gomez, J. Leceta, and Y. Juarranz Time-course expression of Toll-like receptors 2 and 4 in inflammatory bowel disease and homeostatic effect of VIP J. Leukoc. Biol., August 1, 2005; 78(2): 491 - 502. [Abstract] [Full Text] [PDF]
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L. S. Miller, O. E. Sorensen, P. T. Liu, H. R. Jalian, D. Eshtiaghpour, B. E. Behmanesh, W. Chung, T. D. Starner, J. Kim, P. A. Sieling, et al. TGF-{alpha} Regulates TLR Expression and Function on Epidermal Keratinocytes J. Immunol., May 15, 2005; 174(10): 6137 - 6143. [Abstract] [Full Text] [PDF]
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M. Ueta, T. Nochi, M.-H. Jang, E. J. Park, O. Igarashi, A. Hino, S. Kawasaki, T. Shikina, T. Hiroi, S. Kinoshita, et al. Intracellularly Expressed TLR2s and TLR4s Contribution to an Immunosilent Environment at the Ocular Mucosal Epithelium J. Immunol., September 1, 2004; 173(5): 3337 - 3347. [Abstract] [Full Text] [PDF]
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 L. Armstrong, A. R. L. Medford, K. M. Uppington, J. Robertson, I. R. Witherden, T. D. Tetley, and A. B. Millar Expression of Functional Toll-Like Receptor-2 and -4 on Alveolar Epithelial Cells Am. J. Respir. Cell Mol. Biol., August 1, 2004; 31(2): 241 - 245. [Abstract] [Full Text] [PDF]
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 R. Mahanonda, N. Sa-Ard-Iam, O. Charatkulangkun, A. Promsudthi, R.E. Schifferle, K. Yongvanichit, and S. Pichyangkul Monocyte Activation by Porphyromonas gingivalis LPS in Aggressive Periodontitis with the Use of Whole-blood Cultures Journal of Dental Research, July 1, 2004; 83(7): 540 - 545. [Abstract] [Full Text] [PDF]
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 J. A. Feulner, M. Lu, JohnM. Shelton, M. Zhang, J. A. Richardson, and R. S. Munford Identification of Acyloxyacyl Hydrolase, a Lipopolysaccharide- Detoxifying Enzyme, in the Murine Urinary Tract Infect. Immun., June 1, 2004; 72(6): 3171 - 3178. [Abstract] [Full Text] [PDF]
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P. Kropf, M. A. Freudenberg, M. Modolell, H. P. Price, S. Herath, S. Antoniazi, C. Galanos, D. F. Smith, and I. Muller Toll-Like Receptor 4 Contributes to Efficient Control of Infection with the Protozoan Parasite Leishmania major Infect. Immun., April 1, 2004; 72(4): 1920 - 1928. [Abstract] [Full Text] [PDF]
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H.-J. Anders, B. Banas, and D. Schlondorff Signaling Danger: Toll-Like Receptors and their Potential Roles in Kidney Disease J. Am. Soc. Nephrol., April 1, 2004; 15(4): 854 - 867. [Abstract] [Full Text] [PDF]
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S. Knapp, C. W. Wieland, C. van 't Veer, O. Takeuchi, S. Akira, S. Florquin, and T. van der Poll Toll-Like Receptor 2 Plays a Role in the Early Inflammatory Response to Murine Pneumococcal Pneumonia but Does Not Contribute to Antibacterial Defense J. Immunol., March 1, 2004; 172(5): 3132 - 3138. [Abstract] [Full Text] [PDF]
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P. N. Cunningham, Y. Wang, R. Guo, G. He, and R. J. Quigg Role of Toll-Like Receptor 4 in Endotoxin-Induced Acute Renal Failure J. Immunol., February 15, 2004; 172(4): 2629 - 2635. [Abstract] [Full Text] [PDF]
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 R. Bals and P.S. Hiemstra Innate immunity in the lung: how epithelial cells fight against respiratory pathogens Eur. Respir. J., February 1, 2004; 23(2): 327 - 333. [Abstract] [Full Text] [PDF]
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L. Guillot, S. Medjane, K. Le-Barillec, V. Balloy, C. Danel, M. Chignard, and M. Si-Tahar Response of Human Pulmonary Epithelial Cells to Lipopolysaccharide Involves Toll-like Receptor 4 (TLR4)-dependent Signaling Pathways: EVIDENCE FOR AN INTRACELLULAR COMPARTMENTALIZATION OF TLR4 J. Biol. Chem., January 23, 2004; 279(4): 2712 - 2718. [Abstract] [Full Text] [PDF]
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T. Goldammer, H. Zerbe, A. Molenaar, H.-J. Schuberth, R. M. Brunner, S. R. Kata, and H.-M. Seyfert Mastitis Increases Mammary mRNA Abundance of {beta}-Defensin 5, Toll-Like-Receptor 2 (TLR2), and TLR4 but Not TLR9 in Cattle Clin. Vaccine Immunol., January 1, 2004; 11(1): 174 - 185. [Abstract] [Full Text] [PDF]
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M. B. Drennan, D. Nicolle, V. J. F. Quesniaux, M. Jacobs, N. Allie, J. Mpagi, C. Fremond, H. Wagner, C. Kirschning, and B. Ryffel Toll-Like Receptor 2-Deficient Mice Succumb to Mycobacterium tuberculosis Infection Am. J. Pathol., January 1, 2004; 164(1): 49 - 57. [Abstract] [Full Text] [PDF]
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 R. Pawlinski, B. Pedersen, B. Kehrle, W. C. Aird, R. D. Frank, M. Guha, and N. Mackman Regulation of tissue factor and inflammatory mediators by Egr-1 in a mouse endotoxemia model Blood, May 15, 2003; 101(10): 3940 - 3947. [Abstract] [Full Text] [PDF]
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C. F. Ortega-Cava, S. Ishihara, M. A. K. Rumi, K. Kawashima, N. Ishimura, H. Kazumori, J. Udagawa, Y. Kadowaki, and Y. Kinoshita Strategic Compartmentalization of Toll-Like Receptor 4 in the Mouse Gut J. Immunol., April 15, 2003; 170(8): 3977 - 3985. [Abstract] [Full Text] [PDF]
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L. Liu, A. A. Roberts, and T. Ganz By IL-1 Signaling, Monocyte-Derived Cells Dramatically Enhance the Epidermal Antimicrobial Response to Lipopolysaccharide J. Immunol., January 1, 2003; 170(1): 575 - 580. [Abstract] [Full Text] [PDF]
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A. E. Medvedev, A. Lentschat, L. M. Wahl, D. T. Golenbock, and S. N. Vogel Dysregulation of LPS-Induced Toll-Like Receptor 4-MyD88 Complex Formation and IL-1 Receptor-Associated Kinase 1 Activation in Endotoxin-Tolerant Cells J. Immunol., November 1, 2002; 169(9): 5209 - 5216. [Abstract] [Full Text] [PDF]
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D. Wang, P. L. Pedraza, H. I. Abdullah, J. C. McGiff, and N. R. Ferreri Calcium-sensing receptor-mediated TNF production in medullary thick ascending limb cells Am J Physiol Renal Physiol, November 1, 2002; 283(5): F963 - F970. [Abstract] [Full Text] [PDF]
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