1 Immunology and 3 Neuroscience Research Groups, Department of Physiology and Biophysics, and 2 Gastrointestinal Research Group, Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1
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ABSTRACT |
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Mice deficient in
both inducible nitric oxide synthase (iNOS) and interleukin (IL)-10
(iNOS/
/IL-10
/
) were
created to examine the role of iNOS in spontaneously developing intestinal inflammation.
IL-10
/
/iNOS
/
mice were compared with IL-10
/
(iNOS+/+) littermates over 6 mo. RT-PCR, Western blot
analysis, and immunohistochemistry were performed to measure iNOS
message and protein levels. Plasma nitrate/nitrite (NOx)
levels were assessed by HPLC. Damage scores (macroscopic and
microscopic) and granulocyte infiltration were assessed. At 3-4
wk, IL-10
/
and
IL-10
/
/iNOS
/
mice
had no signs of colonic inflammation or granulocyte infiltration. Plasma NOx levels were not different from controls. By
3-4 mo, IL-10
/
mice had increased damage
scores and granulocyte infiltration concurrent with increased mRNA and
protein synthesis (restricted to the epithelium) for iNOS in intestinal
tissues but not other tissues. Plasma NOx levels increased
fivefold. Interestingly, in the absence of iNOS induction or increased
plasma NOx,
iNOS
/
/IL-10
/
mice
had damage and granulocyte infiltration equivalent to those observed in
IL-10
/
littermates. These data suggest that
iNOS does not impact on the development or severity of spontaneous
chronic inflammation in IL-10-deficient mice.
inflammatory bowel disease; nitric oxide; intestine; inflammation; myeloperoxidase
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INTRODUCTION |
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IN 1993, KUHN ET AL. (20)
generated, using embryonic stem cell technology, interleukin
(IL)-10-deficient mice that spontaneously develop a chronic
enterocolitis within 3 mo of birth. The disease is characterized by
weight loss, splenomegaly, and mild to moderate anemia. If kept under
specific pathogen-free (SPF) conditions, the mice develop a limited
form of colitis predominantly affecting the colon. Histopathological
features of the inflammation include abnormal crypt formation, goblet
cell depletion, and a marked thickening of the intestinal wall. The
inflammatory infiltrate consists of lymphocytes, plasma cells,
macrophages, eosinophils, and neutrophils. The development of colitis
in IL-10-deficent mice appears to be mediated by CD4+ T
cells and an uncontrolled Th1 response (3). In addition to the
overproduction of numerous inflammatory mediators [IL-1, IL-6,
tumor necrosis factor (TNF)-] in colonic cultures (3), our own
preliminary work revealed an increase in inducible nitric oxide
synthase (iNOS) mRNA induction in colonic tissue from IL-10-deficient mice. The latter observation is most intriguing in light of the very
significant interest in iNOS in inflammatory bowel disease (IBD) (17,
30, 38) as well as other spontaneously developing autoimmune diseases
(1, 5, 9, 12).
Numerous laboratories have proposed that iNOS produces very significant
levels of nitric oxide (NO), resulting in the nitrosation of many
proteins, causing dysregulation of the inflammatory process and
inappropriate tissue injury (2, 16, 26, 41). However, inhibitors of NO
have provided both protection as well as exacerbation of experimentally
induced inflammation (14, 16, 25, 32, 33, 35). A criticism directed
against many of these studies has been the lack of specificity of the
inhibitors used; many of the inhibitors block all of the isoforms of
NOS (inducible, as well as neuronal and endothelial NOS). It is thought
that inhibition of the constitutive forms will cause tissue damage,
whereas selective inhibition of the inducible form will prevent tissue
injury. However, an NOS inhibitor that is absolutely selective for iNOS
and that can be used for many months without bioaccumulation and
potential nonselective effects remains unavailable. An alternative
approach to inhibitors is to use iNOS-deficient mice that have normal
production of the constitutive isoforms of NOS but have no capacity to
produce NO from the inducible isoform (22). In this study, we examined the effect of the production of high levels of NO from the iNOS isoform
during a spontaneously occurring chronic inflammatory response in
IL-10/
mice, a model that has some features
similar to IBD (20).
Acute inhibition of NO synthesis has been reported to increase the
production of cytokines in inflammatory conditions (10, 39), and iNOS
deficiency has been postulated to increase leukocyte recruitment (13)
and reduce tissue repair (23). In IL-10-deficient mice, it is possible
that there may be a compensatory overproduction of NO in an attempt to
reduce the development of inflammation in this model. If this was the
case, iNOS-deficient (iNOS/
) mice bred into
an IL-10-deficient (IL-10
/
) background would
have increased rate of onset or severity of IBD. Alternatively, iNOS,
and the overproduction of NO, may be detrimental in
IL-10
/
mice. Indeed, numerous investigators
have postulated a role for NO from iNOS in IBD based on levels of this
mediator and enzyme in colonic biopsies from patients with IBD (4, 34,
36). With this in mind, our aim was to study the role of iNOS over time
(6 mo) in the development of inflammation in
IL-10
/
mice. We first fully characterized the
profile of iNOS in IL-10
/
mice and then
monitored the development of disease from early in life (3 wk) to
adulthood (3 and 6 mo) in both iNOS-positive and iNOS-deficient
IL-10-deficient
(IL-10
/
/iNOS
/
) mice.
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MATERIALS AND METHODS |
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Mice deficient in IL-10 (20) and mice deficient in iNOS (22) were generated by gene targeting in embryonic stem cells as previously described and obtained from Dr. R. N. Fedorak (University of Alberta, Edmonton, AB, Canada) and Dr. J. Mudgett (Merck Research Laboratories, Rahway, NJ), respectively. The iNOS-deficient mice were on a mixed C57Bl6 × 129Sv/Ev background, and the IL-10-deficient mice were on a pure 129Sv/Ev background. All animals were generated in SPF facilities. All experimental procedures were approved by the Animal Care Committee of the University of Calgary and conform to the guidelines established by the Canadian Council for Animal Care.
Generation of IL-10/iNOS double-deficient mice.
IL-10/iNOS double-deficient
(IL-10/
/iNOS
/
) mice
were generated from IL-10-deficient mice heterozygous for iNOS
(IL-10
/
/iNOS+/
breeding
pairs). The double-deficient mice were compared directly with
IL-10-deficient wild-type mice for iNOS
(IL-10
/
iNOS+/+) that were
generated in the same litters. The development of spontaneous
intestinal inflammation was studied in male or female littermates over
a 6-mo period. Age-matched background mice were used for baseline
comparison. All mice were genotyped for IL-10 and iNOS by PCR analysis
of genomic DNA purified from tail biopsies. The PCR protocol used for
genotyping IL-10 was obtained from Jackson Laboratory (Bar Harbor, ME),
and the following primers were used: oIMR086 5'-GTG GGT GCA GTT
ATT GTC TTC CCG-3', oIMR087 5'-GCC TTC AGT ATA AAA GGG GGA
CC-3', and oIMR088 5'-CCT GCG TGC AAT CCA TCT TG-3'.
All three primers were used in a single PCR reaction. Genomic DNA from
an IL-10+/+ mouse resulted in a single 200-bp product,
amplified by primers oIMR086 and oIMR087 from the IL-10+
alleles. Genomic DNA from an IL-10+/
mouse resulted
in two products, 200 bp and 400 bp in length, amplified by primers
oIMR086 and oIMR087 from the IL-10+ allele and by primers
oIMR087 and oIMR088 from the IL-10
allele,
respectively. Finally, genomic DNA from an
IL-10
/
mouse resulted in a single 400-bp
product amplified by primers oIMR087 and oIMR088 from the
IL-10
alleles.
Analysis of iNOS mRNA expression by RT-PCR.
Colon (ascending and descending) tissue and terminal ileum samples were
obtained from mice at 3-4 wk, 3-4 mo, and 6 mo of age. In a
separate experiment, various tissues (colon, small intestine, stomach,
mesentery, liver, lung, and heart) were obtained from 3- to 4-mo-old
mice treated with or without lipopolysaccharide (LPS; 50 µg ip) for 4 h to ensure that all organs had the capacity to induce iNOS in
IL-10/
mice. Tissue samples were rinsed in
saline, weighed, and placed in guanidinium isothiocyanate to extract
total RNA (7). The final RNA concentrations were determined by
absorbance using a GeneQuant spectrophotometer (Pharmacia, Piscataway,
NJ). The RT and PCR steps were performed as described by Wong et al.
(42) and outlined previously (23).
Western blot analysis for iNOS protein.
Intestinal tissue was rinsed with saline, frozen in liquid nitrogen,
and stored at 80°C for no more than 1 wk before protein content was determined as follows. Tissue was cut into small pieces and
then homogenized in 250 µl of buffer [40 mM
Tris · HCl, pH 8.0, protease inhibitor cocktail
(Calbiochem), 1 mM phenylmethylsulfonyl fluoride, 1% Triton
X-100] with a hand-held motorized pestle (Kontes). Homogenates
were ultracentrifuged in a total volume of 1.25 ml (100,000 g, 4°C,
1 h). Total protein was determined using Bio-Rad Assay Reagent.
Homogenization buffer lacking Triton X-100 was added to 100 mg of
homogenate total protein to give a final volume of 25 µl. Samples
were boiled for 5 min in an equal volume of 2× sample buffer
(0.125 M Tris · HCl, pH 6.8, 4% SDS, 20% glycerol, 10%
-mercaptoethanol, 0.02% bromphenol blue). Samples were
resolved onto polyacrylamide gel (Bio-Rad mini gel system) and
transblotted to nitrocellulose membrane (Bio-Rad). The membrane
was blocked with buffer (PBS containing 0.05% Tween 20 and
5% skim milk powder, 1 h at room temperature) then incubated (2 h room temperature or overnight at 4°C) with a rabbit
polyclonal antibody raised against iNOS (1:500; N32030, Transduction
Laboratories, Lexington, KY) before bring washed. After incubation (2 h, room temperature) with horseradish peroxidase-conjugated donkey
anti-rabbit IgG (NA9340, Amersham) the membrane was washed again.
Finally, the membrane was incubated (5 min, room temperature) in
SuperSignal substrate (1:1 vol/vol) luminal substrate and stable
solution (Pierce). The membrane was exposed to Hyperfilm (Amersham Life Science) for 1, 2, 5, and 20 min (overexposure) to confirm the absence
of bands.
Nitrate/nitrite measurement by HPLC.
Blood samples were collected in heparin from mice by cardiac puncture.
The samples were centrifuged, and the plasma was frozen at
20°C until assayed. After centrifugation to remove
particulate matter (14,000 g, 2 min), plasma samples were subjected to
a fivefold dilution with HPLC-grade water and deproteinized by
ultracentrifugation through nitrocellulose filters (Centrisart C-4
ultracentrifugation tubes, mol wt cutoff 5,000; Sartorius) by
centrifugation (6,000 g, 60 min). One hundred microliters of the
ultrafiltrates were analyzed for their nitrite and nitrate content
according to the HPLC method previously described by Muscara and de
Nucci (29).
Assessment of severity of colitis.
Mice were killed by cervical dislocation at 3-4 wk, 3-4 mo,
and 6 mo of age. The colon was excised, and the severity of colonic damage was assessed (both the ascending and descending sections were
scored separately) using parameters outlined in Table
1. This scoring system includes features of
clinical colitis, the presence or absence of adhesions, strictures, and
diarrhea (diarrhea was defined as loose, watery stool), and bowel wall
thickness in millimeters. The terminal ileum was also assessed for
signs of inflammation. After gross macroscopic scoring, samples of
colonic tissue were fixed in neutral buffered formalin and processed
for subsequent histological examination. In addition, samples of colon were taken for estimation of myeloperoxidase (MPO) activity and immunohistochemical studies as described in Determination of tissue MPO activity and Immunohistochemistry for iNOS
protein.
|
Histological scoring. After overnight fixation in formalin, tissues were dehydrated (graded alcohols) and cleared (xylene) before being embedded in paraffin wax. Sections of tissue were cut and stained with hematoxylin and eosin (H and E) and scored in a blinded manner. Histological scoring was based on a semiquantitative scoring system in which the following features were graded: extent of destruction of normal mucosal architecture (0, normal; 1, 2, and 3, mild, moderate, and extensive damage, respectively), presence and degree of cellular infiltration (0, normal; 1, 2, and 3, mild, moderate, and transmural infiltration, respectively), extent of muscle thickening (0, normal; 1, 2, and 3, mild, moderate, and extensive thickening, respectively), presence or absence of crypt abscesses (0, absent or 1, present), and presence or absence of goblet cell depletion (0, absent or 1, present). The scores for each feature were summed with a maximum possible score of 11.
Determination of tissue MPO activity.
Samples of intestinal tissue were weighed, frozen on dry ice, and
processed for determination of MPO activity. MPO is an enzyme found in
cells of myeloid origin and has been used extensively as a biochemical
marker of granulocyte (mainly neutrophil) infiltration into
gastrointestinal tissues (18, 27). The samples were stored at
20°C for no more than 1 wk before the MPO assay was
performed. MPO activity was determined using an assay described
previously (18) but with the volumes of each reagent modified for use
in 96-well microtiter plates. The rate of change in absorbance at 450 nm over a 90-s period was determined using a kinetic microplate reader
(Molecular Devices). One unit of MPO activity was defined as that
degrading 1 µmol hydrogen peroxide/min at 25°C. Values are
expressed as units of MPO activity per milligram of tissue sampled.
Immunohistochemistry for iNOS protein. Samples of tissue were fixed overnight in Zamboni's fixative (pH 7.4) at 4°C, rinsed (3 × 10 min) in PBS, transferred to PBS containing 20% sucrose (pH 7.4), and stored at 4°C overnight. They were embedded in OCT embedding medium (Sakura Finetek, Torrance, CA), cryostat sectioned at 12 µm, and thaw-mounted onto poly-D-lysine-coated slides and dried. To assess the expression of iNOS protein in colonic tissue, frozen sections were rehydrated in PBS containing 0.1% Triton X-100 (PBS-T) and incubated with 2% normal goat serum in PBS for 30 min at room temperature to block nonspecific binding before being placed in primary antibody. Sections were then incubated for 48 h at 4°C with a rabbit polyclonal antibody raised against iNOS (1:500; N32030, Transduction Laboratories, Lexington, KY). The specificity of iNOS antibody was verified by substitution of the same concentration of normal rabbit IgG used for the anti-iNOS IgG. Antibodies were diluted in PBS-T containing 0.1% bovine serum albumin. Immunohistochemical controls routinely performed involved incubation with blocking solution and diluent in place of the primary antibody. Sections were rinsed (3 × 10 min) with PBS-T and incubated for 1 h at room temperature with sheep anti-rabbit IgG conjugated to CY3 (1:100). After a final wash (3 × 10 min) with PBS-T, sections were mounted in bicarbonate-buffered glycerol (pH 8.6) and examined using a Zeiss Axioplan fluorescence microscope. Sections were photographed using Kodak Ektachrome film.
Statistical analysis.
Data are expressed as means ± SE. Groups of data were compared using
nonparametric Mann-Whitney U-test or Kruskal-Wallis one-way ANOVA
followed by Dunn's multiple-comparison test. Probabilities (P)
of 5% were considered statistically significant.
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RESULTS |
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iNOS mRNA expression.
Levels of iNOS mRNA in descending colon samples were measured by RT-PCR
in IL-10/
/iNOS+/+,
IL-10
/
/iNOS
/
, and
wild-type control mice at 3-4 wk, 3-4 mo, and 6 mo of age. A
representative band for each group at each time point is shown in Fig.
1A. The RT-PCR product bands were
quantified using nonlinear densitometry, and the ratio of the iNOS to
internal standard (GAPDH) was determined. The mean optical density
ratios for each group of mice (n = 4) are illustrated in Fig.
1B. In IL-10
/
/iNOS+/+
mice there was a fivefold increase in message for iNOS (P < 0.05) as early as 3-4 wk of age. The level of iNOS message
increases at 3-4 mo of age and is still significantly elevated at
6 mo. Our data also reveal that an upregulation of iNOS message could be detected in the ascending colon and small intestine in a pattern similar to that observed for the descending colon, with the exception of small intestinal tissue from young mice, in which no increase in
iNOS mRNA was observed (data not shown). No message for iNOS was
detectable in double-deficient
(IL-10
/
/iNOS
/
) mice
at any time point (Fig. 1).
|
|
Measurement of iNOS protein.
To ensure that iNOS mRNA was translated into protein, Western blot
analysis was performed on tissues from wild-type control, IL-10/
/iNOS+/+, and
IL-10
/
/iNOS
/
mice.
Figure 2 shows a representative gel for
iNOS protein in ascending and descending colon and small intestinal
tissue from 3- to 4-mo-old mice. Our data illustrate that iNOS protein
is generated in the ascending and descending colon and small intestinal tissue from IL-10
/
/iNOS+/+.
However, no iNOS protein could be detected in
IL-10
/
/iNOS
/
or in
wild-type mice despite the presence of low levels of message in the
latter group. Figure 2 also illustrates that no protein for iNOS was
observed in lung tissue from any group. The presence of iNOS protein in
colonic tissue was confirmed by immunohistochemistry. Figure
3 illustrates fluorescence micrographs from
wild-type (Fig. 3A) and IL-10
/
(Fig.
3, B and C) mice stained for iNOS protein. Intense
fluorescent staining was observed in the epithelial cell border lining
the lumen of the gut in IL-10
/
mice (Fig. 3,
B and C), localizing iNOS protein to the epithelial cells. No specific staining was noted in tissues from wild-type (Fig.
3A) or
IL-10
/
/iNOS
/
(not
shown) mice.
|
|
Plasma nitrite levels.
To ensure that iNOS message was translated into a functional product,
plasma nitrates/nitrites were converted to nitrite; the levels are
reported in Fig. 4. At 3-4 wk of age
plasma nitrite levels in wild-type control,
IL-10/
/iNOS+/+, and
double-deficient mice were low (10-25 µM) and not significantly different among the groups, despite an increase in iNOS mRNA as measured semiquantitatively by RT-PCR in
IL-10
/
mice (Fig. 1). At this point, any iNOS
protein present does not increase NO output sufficiently to be detected
as an increase in plasma nitrite. However, by 3-4 mo of age plasma
nitrite levels had increased significantly in
IL-10
/
/iNOS+/+ mice (5-fold
higher than controls), and a profound increase was observed by 6 mo
(425 ± 75 µM). No increase in plasma nitrite levels from control
levels was observed in double-deficient mice at any time point. Because
only the intestinal tract had signs of iNOS message, the very high
plasma nitrite levels suggest very significant NO production within the
intestine.
|
Parameters of inflammation.
Figure 5 illustrates the macroscopic damage
observed in the descending colon of mice at different ages. At the
earliest time point examined (3 wk) there was very little notable
damage in any group studied (0.5-0.7). However, by 3-4 mo
profound macroscopic damage was observed in
IL-10/
/iNOS+/+ mice, with a mean
value of 4 ± 0.5 (P < 0.05), which represents hemorrhage
ulceration, diarrhea, and increased bowel wall thickness. At this age
mice were beginning to prolapse, and by 6 mo of age all mice had rectal
prolapse and were underweight. The level of macroscopic damage observed
in the double-deficient mice was very similar to that observed in the
iNOS-positive, IL-10-deficient mice at all time points. Furthermore,
each parameter measured to obtain the macroscopic damage score was very
similar for each group of animals, suggesting that the development and
progression of the disease was not different between the two groups of
animals. Signs of inflammation were also evident in the ascending colon of both the iNOS-positive and iNOS-deficient IL-10-deficient groups, with hyperplasia of the mucosa noted in most animals by 3-4 mo of
age. Small intestinal inflammation was rarely observed in any mice
studied.
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DISCUSSION |
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In this study we demonstrate for the first time that in IL-10-deficient animals there is a profound increase in iNOS mRNA as early as 3 wk of age that persists at least to 6 mo of age. Associated with the increase in iNOS mRNA is also a very substantial increase in iNOS protein at 3-4 mo, resulting in high levels of plasma nitrite/nitrate, the end product of NO metabolism. The increased nitrite levels are likely from iNOS because in the IL-10/iNOS double-deficient mice, no increase in plasma nitrite was noted. Nitrite levels rise in the circulation to >5-fold at 3 mo and 20-fold at 6 mo, remarkably reaching as much as 0.5 mM nitrite at the later stage of the disease. More impressive is the fact that iNOS is not produced by all organs but appears to be restricted to the gastrointestinal tract and more specifically to the colon (epithelium) and small intestine. This underscores the levels of NO that must be produced within the intestinal tract to achieve such profound levels of nitrite in the circulation. Despite the high level of iNOS activity within the gastrointestinal tract, this enzyme does not appear to be essential for the development or maintenance of colitis, because the IL-10-deficient mice rendered iNOS deficient developed a degree of inflammation, as assessed by microscopic and macroscopic damage, leukocytic infiltration, and intestinal dysfunction (diarrhea and rectal prolapse), commensurate with that of their iNOS-positive littermates.
The literature to date is equivocal with respect to the effect of IL-10
on iNOS production. Certainly, a number of studies suggest that IL-10
can directly inhibit iNOS production from macrophages stimulated with
bacteria or bacterial products (8, 11, 28). However, other studies have
proposed that IL-10 increases or has no effect on NOS activity caused
by LPS in macrophages (6, 15, 31). The results from our study reveal
that in the absence of endogenous IL-10, there is enhanced iNOS
production in the small and large intestine, suggesting that under
basal conditions IL-10 either directly or indirectly inhibits iNOS
synthesis and NO release. It remains unknown whether this observation
is caused by a direct regulation of IL-10 on iNOS production, perhaps
via nuclear factor-B as has been proposed for TNF-
and other
proinflammatory cytokines. Alternatively, it may be an indirect effect
caused by the absence of IL-10 increasing TNF-
and interferon-
,
which are known to induce iNOS production (3). The localized increase in iNOS caused by lack of IL-10 suggests that this cytokine is far more
important as a regulator of iNOS production in the intestine than in
other tissues, because only the intestinal tract revealed elevated
levels of the enzyme. This observation argues against the possibility
of a systemic effect such as enhanced levels of circulating
proinflammatory cytokines being entirely responsible for the increased
iNOS, because this would likely impact on all organs, not just the
gastrointestinal tract.
It is conceivable that luminal antigens are responsible for the enhanced iNOS production because the gastrointestinal tract is directly in contact with the external environment. Indeed, animals kept in SPF facilities develop a more limited intestinal inflammation (20). However, other organs in direct contact with the external milieu including the lung did not exhibit increases in iNOS or overt inflammation. Nevertheless, it is conceivable that the gastrointestinal tract is presented with a specific inducer of iNOS not found in other organs or that the load of antigen is only sufficient to induce iNOS in the absence of IL-10, in this and not in other organ systems. Interestingly, preliminary work from our laboratory suggests that the iNOS response to the same concentration of a bacterial product (LPS) is enhanced in IL-10-deficient mice relative to wild-type animals, raising the possibility that in the gastrointestinal tract, normal endogenous levels of antigen may be sufficient to induce iNOS production in IL-10-deficient but not wild-type mice.
In this study, our results reveal that IL-10-deficient mice have increased induction of iNOS mRNA and protein synthesis and very high levels of NO output (nitrite levels in plasma) concomitant with the development of intestinal inflammation. However, intestinal inflammation progressed at the same rate and with the same severity in IL-10-deficient mice deficient in iNOS and in the absence of elevated NO production. Clearly, in this spontaneously developing model of colitis, overproduction of iNOS, which appears to begin after weaning and continues throughout adult life, is neither the initiating factor nor a major contributor to the development of this disease. Although it may appear somewhat surprising that the iNOS-deficient mice are not spared from injury in this chronic autoimmune disease, this observation is not entirely inconsistent with observations from other models of autoimmune inflammation. For example, overproduction of iNOS was not a contributing factor to antigen-induced autoimmune myocarditis (1), Lyme arthritis (5), or bacterial septic arthritis (24). On the other hand, iNOS-deficient MRL-lpr/lpr mice have less vasculitis associated with glomerulonephritis (12), which is a hallmark of this autoimmune disease, whereas Fenyk-Melody et al. (9) reported a more than fourfold increase in incidence and severity of experimental autoimmune encephalomyelitis in iNOS-deficient mice relative to their wild-type littermates. Clearly, the role of iNOS cannot be predicted from these studies and appears to be dependent on the type of autoimmune disease or perhaps the afflicted organ. Our own data for the first time demonstrate that in the colon, the role of iNOS in a spontaneously developing model of inflammatory bowel disease is not a critical mediator.
This study has also revealed some new information regarding leukocyte adhesion and infiltration during chronic inflammation. We (19) and others (21) reported previously that constitutive NO synthesis inhibition caused an increase in leukocyte adhesion. Moreover, during an inflammatory response induced by LPS, leukocytes from iNOS-deficient mice adhered in response to concentrations of LPS that did not invoke an adhesive response in leukocytes of wild-type mice (13). In light of this work, it was reasonable to expect increased leukocyte recruitment in the IL-10/iNOS double-deficient mice relative to their IL-10-deficient counterparts that are producing a lot of iNOS. Indeed, in other models of intestinal inflammation including acute acetic acid-induced colitis (23), the amount of granulocytic infiltrate into the colon was enhanced in animals deficient in iNOS relative to wild-type mice. However, in our IL-10-deficient chronic model of IBD the amount of granulocyte infiltration was enhanced to similar degrees in both types of mutant mice. As a whole, these studies clearly suggest that although iNOS impacts on granulocyte recruitment and contributes to the resolution of an acute, rapidly resolving inflammation, in a persistent chronic inflammatory disease the mechanisms are sufficiently different that the presence of iNOS does not affect granulocyte infiltration. It should be noted that the absence of a role for iNOS in reducing the inflammatory response in this study may be specific to this chronic model and that iNOS may play a role in other models of chronic inflammation.
In summary, these are the first data documenting that there is a very significant increase in iNOS mRNA, protein, and product during the lifespan of IL-10-deficient mice and that this event does not contribute to the inflammatory process. This model of colitis allowed us to probe extensively the role of iNOS at early, middle, and late phases of the disease, and the results reveal that the progression of the disease is comparable with or without iNOS. Thus the relation between an autoimmune phenotype and increased systemic NO production is not necessarily correlated with injury. In this study we circumvented any issues of lack of specificity or incomplete inhibition of iNOS by making iNOS/IL-10 double-deficient mice. Although the argument could be levied that this IL-10-deficient model is not a suitable model of colitis, it has certain features that make it an attractive model. It is a spontaneously developing inflammation and therefore does not depend on the addition of toxic chemicals or other exogenous reagents. IL-10-deficient mice do not gain weight to the same extent as wild-type mice; they develop diarrhea and microscopically present with transmural cellular infiltration, increased muscle thickness, goblet cell depletion, and crypt abscess formation, all characteristic features of clinical disease. Interestingly, in this model of colitis we were also able to localize iNOS protein to the epithelial cells lining the gut (by immunohistochemistry), which is in accordance with what has been noted in many clinical studies (17, 30, 38). Most importantly, this mouse model has led to the use of IL-10 as a therapeutic molecule in human IBD with some very promising results (37, 40).
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ACKNOWLEDGEMENTS |
---|
The authors acknowledge the technical assistance of Lesley Marshall and Winnie Ho.
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FOOTNOTES |
---|
This study was supported by a grant from the Crohn's and Colitis Foundation of Canada (CCFC). P. Kubes is a Medical Research Council (MRC) scientist and Alberta Heritage Foundation for Medical Research (AHFMR) Senior Scholar. K. A. Sharkey is an AHFMR Senior Scholar. J. L. Wallace is a MRC scientist and an AHFMR scientist. M. Muscara is an AHFMR fellow.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. Kubes, Immunology Research Group, Univ. of Calgary, Health Sciences Center, 3330 Hospital Dr. N.W., Calgary, AB, Canada T2N 4N1 (E-mail: pkubes{at}ucalgary.ca).
Received 2 November 1999; accepted in final form 27 January 2000.
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