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1 Department of Clinical Pharmacology, Lund University Hospital, SE-221 85 Lund; and 2 Department of Chemistry and Biomedical Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Inducible nitric oxide
synthase (iNOS)-deficient mice were used to examine the role of
iNOS in Escherichia coli-induced urinary tract infection
(UTI). The toxicity of nitric oxide (NO)/peroxynitrite to bacteria and
host was also investigated. The nitrite levels in urine of
iNOS+/+ but not iNOS
/ mice increased after
infection. No differences in bacterial clearance or persistence were
noted between the genotypes. In vitro, the uropathogenic E. coli 1177 was sensitive to 3-morpholinosydnonimine, whereas the
avirulent E. coli HB101 was sensitive to both NO and 3-morpholinosydnonimine. E. coli HB101 was statistically
(P < 0.05) more sensitive to peroxynitrite than
E. coli 1177. Nitrotyrosine immunoreactivity was observed in
infected bladders of both genotypes and in infected kidneys of
iNOS+/+ mice. Myeloperoxidase, neuronal (n)NOS, and
endothelial (e)NOS immunoreactivity was observed in inflammatory cells
of both genotypes. Our results indicate that iNOS/ and
iNOS+/+ mice are equally susceptible to E. coli-induced UTI and that the toxicity of NO to E. coli
depends on bacterial virulence. Furthermore, myeloperoxidase and
nNOS/eNOS may contribute to nitrotyrosine formation in the absence of iNOS.
inducible nitric oxide synthase; myeloperoxidase; Escherichia coli; transgenic; nitrotyrosine
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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URINARY TRACT INFECTIONS (UTIs), including cystitis and pyelonephritis, are among the most common bacterial infections in man and Escherichia coli is the most causative agent of the infection. The bacteria are cleared from the urinary tract through the action of inflammatory cells, particularly by polymorphonuclear (PMN) cells (23). The production of antimicrobial factors such as nitric oxide (NO) may contribute to control UTIs. It has been demonstrated that patients with UTI have an elevated nitrite concentration in the urine compared with healthy controls (42). Increased gaseous NO concentrations in the urinary bladder in patients with lower UTI have also been reported (30). Previously, we have demonstrated increased urinary nitrite production in E. coli-infected mice (41). However, the actual role of NO in UTI has not been elucidated, and it is not clear whether NO has a bactericidal effect on UTI.
In inflammatory responses, NO is produced by an enzyme known as inducible nitric oxide synthase (iNOS). Genetically altered mice with a mutation of the iNOS gene have been used to define the role of NO production and iNOS expression in infection. Mice lacking iNOS were more susceptible to herpes simplex virus infection than their corresponding wild-type controls (31), and NO production seemed to be important in the host response to extracellular gram-positive bacteria (33). However, disruption of iNOS improved the clearance of Mycobacterium avium, and increased NO production seemed to exacerbate this infection rather than clear it up (19).
NO itself is neither highly reactive nor particularly toxic but forms
oxidants that are responsible for its toxicity. The chemical reactivity
and toxicity of NO can be increased by its diffusion-limited reaction
with superoxide (O
This study examined the significance of iNOS induction and NO production for the clearance of E. coli from the urinary tract of wild-type and iNOS-deficient mice. Furthermore, the toxicity of NO and peroxynitrite to different E. coli strains was examined in vitro and the toxicity of NO to the host tissue was assessed by investigation of nitrotyrosine formation.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Mice
C57BL/6J and C57BL/6-NOS2/ mice were obtained
from The Jackson Laboratory (Bar Harbor, ME). The mice were constructed
as previously described (29). Female mice were used at 9 wk of age. The mice were kept on a nitrate/nitrite-free pellet diet
(Altromin 1324N, Petersen, Ringsted, Denmark), starting 1 wk before the
experiment. A few days before infection, groups of five mice were
separated into individual cages, and urine samples were collected and
examined for bacterial growth and the leukocyte cell content was
determined microscopically using a hemocytometer chamber. Mice with
more than 5 × 105 leukocytes/ml in preinoculation
urine samples or with a positive bacterial culture were excluded from
the experiment. The experimental protocol was approved by the Animal
Ethics Committee, Lund University, Lund, Sweden.
Bacteria
In vivo experiments. E. coli 1177, of serotype O1:K1:H7, was isolated from a child with acute pyelonephritis (32). The strain is virulent in the mouse UTI model and evokes a strong inflammatory host response (10). It expresses type 1 and P fimbrial adhesins but is hemolysin negative. E. coli 1177 was maintained in deep agar stabs, passaged on tryptic soy agar (TSA; Difco Laboratories, Detroit, MI), grown overnight at 37°C in static Luria broth, and harvested by centrifugation at 3,200 rpm for 10 min. The pellet was resuspended in sterile PBS, pH 7.2, to a concentration of 109 colony-forming units (CFU)/ml.
In vitro experiments. When used for the in vitro experiments, E. coli 1177 was grown overnight at 37°C on TSA and harvested in sterile PBS by centrifugation at 3,200 rpm for 10 min. As an avirulent strain, a nonfimbriated K12 derivative, E. coli HB101, was used. HB101 was grown overnight at 37°C on TSA and harvested in sterile PBS by centrifugation at 3,200 rpm for 10 min. The pellets of both bacterial strains were resuspended and then diluted in sterile PBS to a final concentration of 109 CFU/ml.
Infection Procedure
The mouse bladder was emptied by gentle compression on the lower abdomen, and urine was saved for preinfection measurements of nitrite (see below). Experimental UTI was established in the mice by intravesical injection of E. coli 1177 as previously described (22). After anesthesia, 0.1 ml of bacterial suspension was slowly instilled into the bladder transurethrally, using a soft polyethylene catheter (outer diameter 0.61 mm; Kebolab, Malmö, Sweden). The catheter was immediately withdrawn after inoculation, and no further manipulations were carried out. The animals were placed in the cages after instillation and allowed food and water ad libitum. Infection was monitored at 6, 24, and 72 h and 7 days. Urine samples were stored on ice or at 4°C until further analysis.Infection was quantified by viable bacterial counts on tissue homogenates from mice killed by CO2 asphyxia at different times after inoculation. One-half of the bladder and one of the kidneys were aseptically harvested and homogenized in 5 ml of sterile PBS in sterile disposable plastic bags using a LAB Stomacher 80 Homogenizer (Seward Medical UAC House, London, UK). Serial dilutions of the tissue homogenates were plated on TSA. After overnight culture, plates with bacterial colonies were scored and bacterial numbers (CFU/ml of tissue homogenate) were determined after adjustment for the dilution factor. The other half of the bladder and the second kidney were processed for immunohistochemistry as described below.
Immunohistochemistry
Bladders and kidneys were immersion-fixed for 4 h in cold 4% formaldehyde in PBS (pH 7.4) and then rinsed for 3 days in PBS containing 15% sucrose. Both fixation and rinsing were performed at 4°C, after which the specimens were frozen in isopentane at
40°C
and stored at 70°C until sectioning. Sections were cut (10 µm) in
a cryostat (Leica CM3050; Leica Microsystems, Askik, Sweden) and
preincubated with PBS containing 0.2% Triton X-100 and 0.1% BSA for
2 h at room temperature. Sections were incubated with the
following primary antisera (diluted with PBS containing 0.2% Triton
X-100 and 0.1% BSA): a rabbit polyclonal antibody raised to murine
iNOS (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA); a rabbit
polyclonal antibody raised to nitrotyrosine (1:470; Upstate
Biotechnology, Lake Placid, NY); a sheep polyclonal antibody raised to
neuronal (n)NOS (1:1,000; a gift from Drs. I. Charles and P. C. Emson, Cambridge Univ., Cambridge, UK); or a rabbit polyclonal antibody
raised to endothelial (e)NOS (1:250; Santa Cruz Biotechnology)
overnight in a moisture chamber at room temperature. Sections were
incubated with a rabbit polyclonal antibody raised to MPO (1:200;
NeoMarkers, Fremont, CA) for 2 h at room temperature. The sections
were rinsed in PBS and incubated for 90 min with FITC-conjugated donkey
anti-rabbit IgG (1:80), Texas red (TR)-conjugated F(ab')2
fragment donkey anti-rabbit IgG (1:160; both from Jackson
Immunoresearch Laboratories, West Grove, PA), or FITC-conjugated donkey
anti-sheep IgG (1:80; Sigma) diluted in PBS. The sections were rinsed
in PBS and mounted in glycerol with p-phenylenediamine to
prevent fading.
To identify the inflammatory cells, a double-label immunofluorescence method was used. Sections were first incubated overnight with RB6-8C5, a rat IgG2b monoclonal antibody specific for murine neutrophils (a gift from Dr. A. Sjöstedt, Umeå University, Umeå, Sweden). After being rinsed in PBS, the iNOS antibody was added and the sections were incubated again overnight. RB6-8C5 staining was visualized by incubating the sections for 90 min with TR-conjugated F(ab')2 fragment donkey anti-rat IgG (1:160). After being rinsed, the sections were incubated for 90 min with FITC-conjugated donkey anti-rabbit IgG (1:80) to visualize iNOS. The sections were mounted as described above.
In control experiments, no immunoreactivity was detected in sections incubated with only the secondary antibodies. The specificity of the iNOS antibody was confirmed by incubating the antibody with excessive antigen. No iNOS immunoreactivity was detected in sections incubated with absorbed antibody. All micrographs of the immunolabeled sections were obtained using a digital camera system (Olympus BX60F-3 microscope and Olympus Digital camera DP-50; Olympus Optical, Tokyo, Japan), and the pictures were captured using appropriate filter settings for FITC and TR. Adobe Photoshop was used for image handling, and the three-color channels were handled separately. Only the background level, contrast, and brightness of the entire image were changed in the final picture.
Hematoxylin and Eosin Staining
The morphology of the inflammatory cells was examined after staining of the tissue sections with hematoxylin and eosin (Apoteksbolaget, Malmö, Sweden).DNA Isolation and Genotyping with PCR
PCR analyses for the intact and disrupted iNOS gene were performed to genotype the mice. Briefly, DNA was extracted from the tip of the tail by incubating the tail in lysis buffer (50 mM Tris · Cl, 5 mM EDTA, 100 mM NaCl, and 0.5% SDS; all from Sigma, St. Louis, MO) containing proteinase K (10 mg/ml; Sigma) overnight at 56°C. PCR was performed according to the Sigma PCR-Core kit with Taq DNA polymerase (Sigma), using 2 µg genomic DNA. The primers for mouse iNOS were obtained from MWG-Biotech (Ebersberg, Germany) and were as follows: sense, 5'-CAG ATC GAG CCC TGG AAG ACC-3', and antisense, 5'-CCT TGG TGT TGA AGG CGT AGC-3', amplifying a 533-bp product. PCR was performed in an automated thermal cycler (GeneAmp PCR System 2400, PerkinElmer, Foster City, CA), with one cycle at 94°C for 5 min, followed by 30 cycles at 94°C for 60 s, at 58°C for 60 s, at 72°C for 60 s, and a final extension at 72°C for 7 min. PCR products were separated by 2% agarose gel electrophoresis, and bands were visualized by ethidium bromide staining.Nitrite Assay
NO production was determined as urinary nitrite levels by the Griess reaction assay. Briefly, 10 µl of centrifuged urine were transferred to 96-well plates and mixed with 20 µl of water and 100 µl of Griess reagent [1 part 0.1% N-(1-naphtyl) ethylene-diamine dihydrochloride (Sigma) in water and 1 part 1% sulfanilamide (Sigma) in 5% concentrated H3PO4]. The mixture was incubated for 5 min at room temperature and read at 540 nm by spectrophotometry (Labsystems Multiscan PLUS; Labsystems, Lund, Sweden). Concentrations were determined compared with a standard curve of sodium nitrite. The detection limit of the assay was 1 µM nitrite.Bacterial Viability Experiments
Bacterial viability in response to exogenously applied NO and 3-morpholinosydnonimine (SIN-1), a generator of peroxynitrite in solution, was examined. One milliliter (109 CFU/ml) of E. coli 1177 or E. coli HB101 in PBS was transferred to a sterile test tube. The bacteria were exposed to either NO [2,2-(hydroxynitrosohydrazino)bis-ethanamine (DETA/NO); 500 µM, Alexis Biochemicals, Lausen, Switzerland] or SIN-1 (500 µM, Casella, Frankfurt am Main, Germany) for 24 h. Untreated bacteria were used as control. Serial dilutions were plated on TSA and, after overnight culture, plates with bacterial colonies were scored and bacterial numbers (CFU/ml) were determined after adjustment for the dilution factor.Analysis of Data
Data are expressed as means ± SE. Student's paired or unpaired t-test was used to compare two means, and ANOVA followed by the Bonferroni-Dunn post hoc test was used for multiple comparisons (GraphPad Prism 3.0). Differences were considered significant at P < 0.05.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The genotype of the mice was confirmed by PCR using primers
specific for the calmodulin-binding domain of the iNOS gene. A 533-bp
product, corresponding to the calmodulin-binding domain of iNOS, was
detected in all the investigated C57BL/6J mice (iNOS+/+)
but not in the investigated transgenic iNOS/ mice (Fig.
1).
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Nitrite Levels in Urine
The production of NO into the urine in response to infection was quantified as nitrite, a stable end product of NO (20). Most uropathogens are members of the family Enterobacteriaceae, known to reduce nitrate to nitrite. Urine contains nitrates from dietary sources, but the mice in our study were fed a nitrate/nitrite-free diet to ensure that we measured nitrite formed from endogenous NO and not from bacterially converted nitrate. The urinary nitrite levels increased in wild-type mice after infection with E. coli 1177 at 6 h postinfection (42 ± 12 µM, n = 12, P < 0.05) compared with at 0 h (12 ± 1 µM, n = 12) (Fig. 2A). The urinary nitrite levels were low at 24 and 72 h and 7 days after bacterial instillation. iNOS/ mice showed no urinary nitrite
response to infection (Fig. 2B).
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Bacterial Counts of Infected Tissue
Bladders and kidneys were harvested from the E. coli 1177-infected iNOS+/+ and iNOS/ mice, and
bacterial persistence was determined by viable counts on tissue
homogenates. There was no significant difference in bacterial
persistence or clearance in bladders obtained from iNOS wild-type or
iNOS-deficient mice (Fig. 3A).
The highest bacterial numbers were found 24 h after infection in
wild-type mice and 6 h after infection in iNOS-deficient mice.
Between 72 h and 7 days after infection, the bacterial numbers
were fairly constant and none of the mice strains managed to clear the
infection.
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There was no difference in bacterial numbers in kidneys from
iNOS/ and wild-type mice (Fig. 3B). After a
reduction between 24 and 72 h postinfection, the bacterial numbers
of wild-type mice remained stationary for up to 7 days. The bacterial
numbers in kidneys of iNOS/ mice increased up to
72 h and then decreased slightly between 72 h and 7 days to
reach the same numbers as observed in kidneys of iNOS+/+ mice.
Morphological Examination
Bladders and kidneys were examined for iNOS, nNOS, eNOS, nitrotyrosine, and MPO expression by immunohistochemistry at 6, 24, and 72 h and 7 days postinstillation.Identification of inflammatory cells. Hematoxylin and eosin staining showed that the majority of the inflammatory cells in bladders and kidneys were PMN cells. By double-label immunofluorescence using the neutrophil marker RB6-8C5, the majority of the iNOS-positive cells were identified as neutrophils (data not shown).
iNOS immunoreactivity.
Low levels of two abnormal iNOS transcripts have been detected in
macrophages isolated from iNOS/ mice (29).
These transcripts may produce low levels of immunoreactive peptides
that are enzymatically inactive but recognized by some iNOS antibodies
(The Jackson Laboratory, personal communication). The iNOS antibody
used in our study detected iNOS in inflammatory cells in bladders of
both wild-type and iNOS-deficient mice 6 h after infection (data
not shown). iNOS-positive uroepithelial cells were found in the
majority of bladders from iNOS+/+ mice from 6 h and up
to 7 days after infection (Fig.
4A). iNOS immunoreactivity
was, however, not observed in the bladder urothelium of
iNOS/ mice (Fig. 4B). Kidneys of infected
iNOS+/+ and iNOS/ mice were devoid of
iNOS-expressing inflammatory cells at all times. iNOS immunoreactivity
in transitional and columnar epithelial cells lining the
renal pelvis was found in the majority of wild-type mice at 6 and
24 h postinstillation, and this immunoreactivity was further
increased at 72 h and 7 days after infection (Fig. 4C).
The iNOS-deficient mice did not express iNOS immunoreactivity in
epithelial cells lining the renal pelvis (Fig. 4D).
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nNOS and eNOS immunoreactivity.
We next examined whether a compensatory increase in nNOS and eNOS
expression had occurred in iNOS-deficient mice. In the bladder, nNOS-immunoreactive neuronal structures were observed within the smooth
muscle and submucosa of E. coli-infected bladders of both wild-type and iNOS-deficient mice. In the kidney, nNOS immunoreactivity was sparse and only observed in the macula densa and in a few nerve
fibers associated with blood vessels. eNOS-positive immunoreactivity was observed in vascular endothelium. The inflammatory cells of both
genotypes were found to express nNOS and eNOS (Fig. 5, A and
B). As judged by visual
observation, no difference in nNOS or eNOS expression was observed when
infected iNOS-deficient mice were compared with infected wild-type
mice.
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Nitrotyrosine immunoreactivity.
Immunoreactivity to nitrotyrosine was not found in uninfected bladders
and kidneys. Nitrotyrosine immunoreactivity was, however, found in
bladders of infected iNOS+/+ and iNOS/ mice
at all times (Fig. 6A). Three
types of cells/structures were found to express nitrotyrosine. PMN
cells and large round cells, resembling inflammatory cells, showed
nitrotyrosine staining (Fig. 6B). In addition, small
nitrotyrosine-positive structures, with no visible nucleus, were found
close to the inflammatory cells in the submucosa (Fig. 6B).
In kidneys of wild-type mice, nitrotyrosine was observed in
inflammatory cells and in small structures in the renal pelvis
primarily at 72 h and 7 days postinfection (Fig. 6C).
Nitrotyrosine immunoreactivity was found in the glomeruli of one
iNOS+/+ mouse 72 h after bacterial infection (Fig.
6D). Nitrotyrosine was not detected in kidneys of
iNOS/ mice (Fig. 6E).
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MPO immunoreactivity.
Because nitrotyrosine immunoreactivity was observed in iNOS-deficient
mice, possible sources for protein nitration other than peroxynitrite
were investigated. MPO is an enzyme used by granulocytes during
phagocytosis. In the presence of nitrite, MPO can nitrate protein
tyrosines (13). No immunoreactivity to MPO was observed in
uninfected bladders and kidneys. Numerous MPO-immunoreactive inflammatory cells were observed in infected bladders of both iNOS+/+ (Fig. 7A)
and iNOS/ (Fig. 7B) mice at all times. The
MPO-immunoreactive cells were located within the bladder smooth muscle
layer, lamina propria, and close to or within the uroepithelium. In the
kidney, inflammatory cells expressing MPO were observed close to the
epithelium lining the renal pelvis mainly at 6 h after infection.
Some MPO-immunoreactive inflammatory cells were also observed in the
glomeruli (Fig. 7C) at all times. There was no difference in
the number or distribution of MPO-expressing cells between the
genotypes as judged by visual observation. However, MPO
immunoreactivity was by far more pronounced in the bladder than in the
kidney. The uroepithelial cells were not stained for MPO.
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Bacterial Viability
The effect of exogenously added NO and SIN-1, a peroxynitrite donor in solution, on bacterial viability was investigated. When E. coli 1177 was exposed to DETA/NO (500 µM), which spontaneously decomposes and provides a constant NO supply over hours (3), the number of colonies formed was only slightly (14 ± 8.4%, n = 3) decreased compared with untreated controls. However, when exposed to SIN-1 (500 µM), the number of viable colonies of E. coli 1177 was significantly decreased by 53 ± 12% (n = 3, P < 0.05) compared with untreated bacteria (Fig. 8A). HB101, the nonfimbriated E. coli strain, was sensitive to both NO and SIN-1. DETA/NO (500 µM) and SIN-1 (500 µM) caused a significant decrease in E. coli HB101 viability by 25 ± 7.8 (n = 3, P < 0.05) and 85 ± 3.5% (n = 3, P < 0.001), respectively (Fig. 8B). E. coli HB101 was statistically (P < 0.05) more sensitive to SIN-1 than E. coli 1177.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In this study, iNOS-deficient mice were used to examine the role
of iNOS expression and NO production in bacterial clearance after an
E. coli-induced UTI. Uropathogenic E. coli was
found to persist in bladders and kidneys of iNOS+/+ and
iNOS/ mice for up to 7 days after infection, and none
of the mice strains managed to clear the infection. Bacteria have
previously been reported to persist within the bladder of C57BL/6 mice
for days and weeks (26, 37), with the persistence being
attributed to the type 1 pili (10, 36). There was no
significant difference in bacterial clearance or persistence between
wild-type and iNOS/ mice, suggesting that the bacteria
persisted in bladders and kidneys irrespective of iNOS induction. These
findings are consistent with an earlier study, showing that
pharmacological inhibition of iNOS did not alter the sensitivity of
C3H/HeN and C3H/HeJ mice to renal infection caused by two different
E. coli uropathogens (39). Furthermore, in
experimental glomerulonephritis, no difference in the disease was
observed when iNOS-deficient mice were compared with wild-type mice
(9).
Immunoreactivity to iNOS was observed in bladder inflammatory cells of
iNOS+/+ mice and, surprisingly, also in the
iNOS/ mice. Low expression of two abnormal iNOS
transcripts, which may produce immunoreactive peptides, has been
described in macrophages isolated from the iNOS/ mice
used in this study (29). Some iNOS antibodies may
recognize the produced peptides and, indeed, several investigators have noticed iNOS-positive labeling in the iNOS/ mice (The
Jackson Laboratory, personal communication). Nevertheless, these
transcripts are enzymatically defective and do not produce NO. In our
study, no increase in urinary nitrite levels of infected iNOS/ mice was detected, confirming the absence of iNOS
activity in these animals. Genotypic studies further confirmed the
disruption of the iNOS gene in transgenic mice. The nitrite levels in
urine from E. coli-infected iNOS+/+ mice reached
a peak 6 h after instillation, which coincided in time with the
presence of numerous iNOS-positive inflammatory cells in the bladder.
iNOS immunoreactivity in uroepithelial cells was found up to 7 days
after infection, but urinary nitrite levels were not increased in
samples analyzed 1, 3, or 7 days postinfection. This suggests that
inflammatory cells, and not uroepithelial cells, are the main
contributors of NO/nitrite in urine samples of infected mice.
We have previously demonstrated iNOS immunoreactivity in uroepithelial
cells at 24 and 72 h after infection in C3H/HeN mice infected with
E. coli (41). In the present study, using
C57BL/6 wild-type mice and a different E. coli strain, iNOS
immunoreactivity in uroepithelial cells was seen as early as 6 h
postinfection, and the expression increased with time. Thus the genetic
background of the mice as well as the bacterial strain seem to affect
the rate and degree of the iNOS response in host uroepithelial cells. iNOS immunoreactivity was not found in uroepithelial cells in bladders
and kidneys of iNOS-deficient mice. This may suggest that uroepithelial
cells in iNOS/ mice, unlike inflammatory cells, do not
produce the abnormal iNOS transcripts.
It is generally believed that NO in inflammatory cells is antimicrobial and that it participates in the host defense against invading pathogens (15). However, the role of iNOS induction in uroepithelial cells is not clear. Induction of iNOS in uroepithelial cells may be involved in uroepithelial cell shedding by promoting deletion of infected and damaged cells (16, 34). Inhibition of iNOS was found to reduce intestinal permeability and to decrease bacterial translocation by limiting the damage to the gut mucosa (12, 43). This suggests that NO may favor bacterial translocation through the epithelium. Massive bladder uroepithelial cell shedding has been reported within 6 h after infection with type 1 piliated E. coli in the same mice strain, C57BL/6, used in our study (38). The degree of uroepithelial cell shedding was not specifically investigated in the present study, and it is unclear whether iNOS-deficient bladders and kidneys showed less shedding than tissue from wild-type mice.
Because the uropathogenic E. coli strain 1177 persisted in both genotypes, we examined the bactericidal effect of NO to E. coli in vitro. E. coli 1177 was not sensitive to NO exposure. The compound SIN-1, which in solution generates peroxynitrite, caused a significant decrease in bacterial viability. Consistent with our results, other studies have shown that exposure of E. coli to NO did not decrease the viability of the bacteria (8, 39), whereas exposure to peroxynitrite did (8, 49). However, when a nonfimbriated avirulent E. coli strain, HB101, was exposed to NO and SIN-1, a significant decrease in viability was observed with both agents. It may be speculated whether the effect of NO on E. coli HB101 is a direct effect of the exogenously applied NO or whether NO has reacted with bacterially derived oxygen and formed peroxynitrite. E. coli contains relatively high concentrations of catalases that could provide a source of oxygen (25). Added NO and bacterially derived oxygen may thus form low concentrations of peroxynitrite that is bactericidal to E. coli HB101 but not to E. coli 1177.
Microbial pathogens have multiple means of defending themselves against
the cytotoxic effects of NO. Recent evidence shows that most organisms,
including E. coli, are able to metabolize and detoxify NO.
Studies have shown that aerobic E. coli strains are
protected against NO toxicity by expressing an NO-inducible NO
deoxygenase (NOD) (17). NOD is a flavohemoglobin, which
oxidizes NO to NO
The cytotoxicity of NO/peroxynitrite is not only directed to invading
pathogens but may also affect NO-producing cells and surrounding tissue
(35). Increased iNOS expression and NO production have
been shown to coincide in time with maximal kidney damage in acute
pyelonephritis (27). Nitrotyrosine has become a useful marker of peroxynitrite formation in vivo (5).
Immunoreactivity to nitrotyrosine was found in bladder inflammatory
cells at all time points, demonstrating that urinary neutrophils
produce peroxynitrite. No difference in nitrotyrosine staining in the
bladder was detected between the genotypes, suggesting that
peroxynitrite formed in iNOS/ mice must depend on NO
sources other than iNOS. It has previously been demonstrated that the
lack of iNOS does not fully abolish tyrosine nitration
(50). Production of NO by other isoforms of NOS is a
possible source of NO in iNOS-deficient mice. In our study, nNOS and
eNOS immunoreactivity was observed in inflammatory cells in bladders of
E. coli-infected iNOS-deficient mice. It was recently
demonstrated that human neutrophils express eNOS (11) and
that human and rat neutrophils express nNOS mRNA (21). Also, rat neutrophils have been shown to express nNOS protein and
spontaneously release nitrite and nitrate anions (21).
Purified nNOS was found to produce both O/ mice,
at least not after immunohistochemical evaluation.
MPO, an enzyme implicated in various inflammatory diseases, has been
suggested as a potential pathway for nitrotyrosine formation. MPO is a
major neutrophil protein and is stored in granules and released during
phagocytosis (47). MPO may use either hydrogen peroxide
(H2O2) or hypochlorous acid (HOCl) to oxidize
nitrite and form reactive nitrogen intermediates that may result in
tissue nitration (24, 45). In our study, numerous
MPO-stained inflammatory cells were observed in infected bladders of
both wild-type and iNOS-deficient mice. This suggests that MPO may
contribute to the detected nitrotyrosine formation in bladders of
iNOS/ mice. Nitrotyrosine expression, but no iNOS
expression, was observed in inflammatory cells in kidneys of
iNOS+/+ mice. We have previously detected iNOS-positive
inflammatory cells in the kidney of infected C3H/HeN mice
(41). It is likely that iNOS expression in kidney
inflammatory cells in the present study, which used a different mouse
strain and a more virulent bacterial strain, peaked earlier than 6 h. Furthermore, MPO-positive cells were observed in the kidney and, as
discussed above, MPO may contribute to nitrotyrosine formation. Unlike
in the bladder, nitrotyrosine was not observed in kidneys of
iNOS/ mice, suggesting that no peroxynitrite- or
MPO-derived nitrotyrosine formation occurred in the kidney of
iNOS-deficient mice. Indeed, MPO and nNOS/eNOS expression, the possible
alternative sources for nitrotyrosine formation in the absence of iNOS,
was not as pronounced in the kidney as in the bladder.
Nitrotyrosine was also observed in the vicinity of inflammatory cells in small submucosal structures. The inflammatory cells may secret peroxynitrite and cause nitration in membrane or intracellular compartments of target cells (1). In our study, the small structures observed could be nitrated structural tissue proteins or nitrotyrosine formed on the bacteria (14). The modification of structural proteins may be a particularly important pathological target of nitration by causing disruption of the normal function of cellular structures (2).
In conclusion, our results indicate that wild-type and iNOS-deficient mice are equally susceptible to E. coli-induced UTI and that the outcome of NO toxicity to E. coli may depend on bacterial virulence. Furthermore, the lack of iNOS did not abolish nitrotyrosine formation. Myeloperoxidase and nNOS/eNOS may contribute to nitrotyrosine formation in the absence of iNOS.
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ACKNOWLEDGEMENTS |
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We thank Prof. Catharina Svanborg for generously providing the bacteria and valuable comments. Dr. Kristian Moller is acknowledged for help with the iNOS primers.
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FOOTNOTES |
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This project was supported by the Swedish Medical Research Council (12601, A0694), the Royal Physiographic Society, and the Foundations of Crafoord and Magnus Bergwall.
Address for reprint requests and other correspondence: K. Persson, Dept. of Clinical Pharmacology, Lund Univ. Hospital, SE-221 85 Lund, Sweden (E-mail: Katarina.Persson{at}klinfarm.lu.se).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
September 3, 2002;10.1152/ajprenal.00101.2002
Received 15 March 2002; accepted in final form 1 September 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Babior, BM.
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) and iNOS-deficient
(
) mice were inoculated with E. coli 1177, and the infection was monitored by viable bacterial counts of bladder
(A) and kidney homogenates (B) after 6, 24, and
72 h or 7 days. There was no significant difference in bacterial
clearance or persistence between the genotypes. Values are means ± SE expressed as colony-forming units (CFU)/ml (n = 3-6).
