IL-2-deficient mice raised under germfree conditions develop
delayed mild focal intestinal inflammation
Michael
Schultz1,
Susan L.
Tonkonogy2,
Rance K.
Sellon2,
Claudia
Veltkamp1,
Virginia L.
Godfrey3,
Julie
Kwon2,
Wetonia B.
Grenther2,
Edward
Balish4,
Ivan
Horak5, and
R. Balfour
Sartor1
1 Center for Gastrointestinal
Biology and Disease, Department of Medicine, and
3 Department of Pathology and
Laboratory Medicine, University of North Carolina, Chapel Hill 27599;
and 2 College of Veterinary
Medicine, North Carolina State University, Raleigh, North Carolina
27606; 4 Departments of Surgery,
Medical Microbiology, and Immunology, University of Wisconsin, Madison,
Wisconsin 53706; and 5 Institute
of Molecular Pharmacology, Department of Molecular Genetics, 12207 Berlin, Germany
 |
ABSTRACT |
Interleukin-2
(IL-2) amplifies immune stimuli and influences B cell differentiation.
IL-2-deficient mice spontaneously develop intestinal inflammation if
raised under specific pathogen-free (SPF) conditions. We quantitatively
determined the aggressiveness and kinetics of gastrointestinal and
hepatic inflammation in the presence or absence of viable bacteria in
IL-2-deficient mice. Breeding colonies were maintained under SPF and
germfree (GF) conditions. Intestinal tissues, serum, and mesenteric
lymph nodes were obtained from mice at different ages for blind
histological scoring, immunoglobulin measurements, mucosal T cell
infiltration, and cytokine secretion. GF IL-2
/
mice
developed mild, focal, and nonlethal intestinal inflammation with
delayed onset, whereas the more aggressive inflammation in SPF IL-2
/
mice led to their death between 28 and 32 wk.
Periportal hepatic inflammation was equal in the presence or absence of
bacterial colonization. Intestinal immunoglobulin secretion decreased
significantly by 13 wk of age in IL-2
/
mice in
both GF and SPF environments. In contrast to other genetically
engineered rodents, IL-2
/
mice develop mild focal
gastrointestinal and active portal tract inflammation in the absence of
viable bacteria.
interleukin-2-deficient mice; luminal flora; colitis; animal models
 |
INTRODUCTION |
RECOMBINANT DNA TECHNOLOGY allows genes of interest to
be either introduced or deleted in rodents by targeted manipulation. To
determine the functions of cytokines in vivo, mice with selective deletion of various cytokine genes have been generated. The development of spontaneous colitis in a number of these "knockout" mice has led to new insights into the pathogenesis of chronic intestinal inflammation (38).
Since its introduction several years ago, the interleukin-2 knockout
mouse (IL-2
/
) has been regarded as a model for chronic, immune-mediated colitis in hosts with dysregulated T cell function (1,
35, 36). IL-2 is an important regulatory cytokine, produced by a subset
of activated CD4+ T cells that
amplifies immune stimuli by promoting T lymphocyte growth and
expansion, possibly contributing to B cell differentiation and
activating macrophages, LAK cells (lymphocyte-activated killer cells),
and NK cells (natural killer cells) (28, 46). However, conflicting data
exist regarding the role of IL-2 in vivo. In 3- to 6-wk-old
IL-2-deficient mice, immune responses are still relatively normal, and
no intestinal or hepatic inflammation is evident (20, 41); however, as
they age, IL-2-deficient mice raised in a conventional environment
progressively develop aggressive colitis, first detectable between 6 and 15 wk of age (36), that is mediated by T cells, especially
CD4+ T cells, and not B cells (25,
43). These T cells are thought to be thymus dependent and invade the
colon and the bone marrow to cause colitis, anemia, and loss of B
cells, respectively (18). Recent studies suggested that the
extraintestinal manifestations are independent of the microbial
environment (8).
In the original description of colitis in IL-2-deficient mice, Sadlack
et al. (36) demonstrated attenuated colitis in knockout mice raised in
a specific pathogen-free (SPF) environment and no clinical or
histological evidence of colitis in a limited number of germfree (GF,
sterile) IL-2
/
mice. The absence of histological evidence of colitis in young GF IL-2
/
mice was confirmed
recently by Contractor et al. (8), who also reported that anemia,
hepatic inflammation, and generalized lymphoid hyperplasia in these
mice was not dependent on bacterial colonization. These observations suggest that luminal microbial agents provide the persistent antigenic stimulus for this T cell-mediated colitis. A number of other rodent models support this hypothesis (37, 38). One of the best characterized models in this respect is the
HLA-B27/human
2-microglobulin transgenic rat.
Normal resident luminal bacteria play an important role in the
pathogenesis of inflammation in this model since these rats do not
develop colitis, gastritis, or arthritis if raised in a sterile
environment (30, 47). Furthermore,
Bacteroides species preferentially
induced colitis in reconstitution studies (30, 32), consistent with the
ability of metronidazole and broad-spectrum antibiotics to attenuate
disease (31). Similarly, IL-10-deficient mice, depending on their
genetic background, and T cell receptor (TCR) knockout mice develop
colitis, anemia, and growth retardation in the SPF environment and stay
healthy in the GF state (12, 19, 42). Luminal bacteria and bacterial
products also contribute to the inflammatory response in the
indomethacin-induced enterocolitis rat model. GF rats have attenuated
acute small intestinal ulceration, no chronic enteritis, and no cecal
ulcers (10, 34, 39), while monoassociation with
Escherichia coli accentuated small intestinal ulcers (34). Furthermore, chronic indomethacin-induced inflammation is attenuated by treatment with tetracycline and metronidazole (3, 51).
The ability of sterile bacterial components to induce intestinal
inflammation is documented by the observations that luminal peptidoglycan-polysaccharide polymers (PG-PS), the primary structural cell wall component of nearly all bacterial species, potentiates small
intestinal ulcers in indomethacin-treated rats (10) and acetic
acid-induced colitis (40) and that intramural injections of purified
PG-PS (26) or muramyl dipeptide in incomplete Freud's adjuvant (21)
induce chronic, immune-mediated granulomatous enterocolitis in
susceptible hosts. In vitro studies with colonic epithelial cell lines
further document the ability of bacteria and bacterial cell wall
components to stimulate inflammatory responses. Jung and co-workers
(16) demonstrated that bacterial invasion of epithelial cells stimulate
a characteristic array of proinflammatory cytokines, and we have shown
that a colonic epithelial cell line can be activated with PG-PS and
lipopolysaccharide (LPS) (15). This cumulative evidence strongly
suggests that products of the normal enteric gut flora can induce
colitis in the genetically susceptible host and are essential for
perpetuation of chronic disease. However, the recent observation that
GF mice fed dextran-sodium-sulfate (DSS) develop aggressive colitis
with increased mortality indicates that acute colonic injury can
develop in the absence of viable luminal bacteria (2).
The aim of this study was to investigate in a quantitative fashion the
relative time of onset, rate of progression, and degree of
aggressiveness of colitis, gastritis, and hepatitis in IL-2-deficient mice at various ages in the presence or absence of luminal bacteria. Previous studies of colitis in GF IL-2
/
mice have
investigated only early time points and have not quantified mucosal
inflammatory responses (8, 36). In contrast to previous reports in this and other rodent model systems, we found that mild colitis and active
gastritis and portal inflammation occur in IL-2-deficient mice in the
absence of viable luminal bacteria.
 |
MATERIALS AND METHODS |
Mice.
GF C57BL/6 × 129/Ola outbred IL-2-deficient mice were derived at
the University of Wisconsin by cesarian section as previously described
(30, 50). SPF and GF breeding colonies (heterozygous × heterozygous) were established at the Laboratory Animal
Facilities of the University of North Carolina at Chapel Hill and the
Gnotobiotic Facilities of the Center of Gastrointestinal Biology and
Disease at the North Carolina State University, College of Veterinary Medicine at Raleigh, respectively. Mice were genotyped by amplification of the IL-2 gene by PCR with IL-2-specific primers (Nucleic Acid Core
Facility, Lineberger Comprehensive Cancer Center, University of North
Carolina, Chapel Hill, NC) in tail digests as described elsewhere (41).
The GF colonies were housed in Trexler flexible film isolators with
autoclaved food and water ad libitum (50). Sterility was tested on a
monthly basis by fecal Gram stain and cultures of the feces and
bedding. In addition, cecal contents were documented to be sterile by
aerobic and anaerobic culture and Gram stain at the time of necropsy of
selected mice. PCR analysis of feces from SPF IL-2-deficient mice
excluded Helicobacter infection (4).
Animals were used between 4 and 46 wk of age.
Clinical assessment.
Clinical symptoms of intestinal inflammation, including diarrhea,
progressive wasting, and rectal prolapse, were assessed twice weekly.
Before necropsy, mice were weighed and then killed by
CO2 asphyxiation. Cardiac blood
was taken for serum immunoglobulin measurements. Rectum, colon, cecum,
small intestine, stomach, and liver were processed for histology,
immunohistochemistry, immunoglobulin, and cytokine measurements. In
addition, mesenteric lymph nodes were obtained for flow cytometric analysis.
Histological assessment of intestinal inflammation.
Intestinal tissues were fixed for 24 h in 10% buffered Formalin.
Tissues were embedded in paraffin and stained with hematoxylin and
eosin. A gastrointestinal histological inflammatory score ranging from
0 to 4 (Table 1), adapted from a scoring
system previously validated in
HLA-B27 transgenic rats (30), was
applied in a blinded fashion to quantitate intestinal inflammation. An overall large intestinal score was obtained by adding the individual scores from the rectum, transverse colon, and cecal tip and dividing by
three.
Immunohistochemical staining of colonic tissues.
Frozen sections (5 µM) of colons of GF IL-2
/
and
wild-type (WT) mice 14 wk of age were acetone fixed (10 min) and then washed in PBS. To block endogenous peroxidase the sections were then
incubated with 3%
H2O2
(10 min). After washing in PBS, nonspecific binding sites were blocked
with 10% normal rabbit serum (Vector; 20 min). Next, sections were
incubated with the primary antibody rat anti-mouse CD4 or rat
anti-mouse CD8 (both rat IgG2a
from Pharmingen) in a concentration of 2 µg/ml (30 min). Negative
controls were incubated only in PBS. After a wash in PBS the secondary antibody (Vector biotinylated) rabbit anti-rat biotinylated was added
at a concentration of 5 µg/ml (20 min). All incubations were done at
room temperature. After washing in PBS, sections were incubated with
horseradish peroxidase (HRP)-conjugated avidin-biotin complexes
(Vectastain ABC method, Vector Laboratories) followed by
3-amino-9-ethylcarbazole (AEC substrate from Vector) staining. Counterstaining was done with hematoxylin.
Intestinal fragment cultures.
Sections of the transverse colon of individual mice were prepared as
previously described (7). Briefly, colon sections were washed with
1× PBS (pH 7.5) to remove fecal contents, gently shaken at room
temperature for 30 min in PBS, and cut into small fragments. Tissue
fragments (100 mg) were incubated in 1.0 ml of RPMI 1640 (GIBCO, Grand
Island, NY), supplemented with 100 U/ml penicillin, 100 µg/ml
streptomycin, 0.25 µg/ml fungizone (all GIBCO), 5% heat-inactivated
FCS (Irvine Scientific, Santa Ana, CA), 2 mM
L-glutamine, 1 mM sodium
pyruvate, 5 × 10
5 M 2-mercaptoethanol,
and 50 µg/ml gentamicin (Sigma, St. Louis, MO) at 37°C for 24 h.
Supernatants were collected and stored at
20°C until further processing.
Isotype-specific ELISA to measure immunoglobulin.
Detection of immunoglobulin production in colon culture supernatants
and sera was performed as described in detail previously (48). The
following affinity-purified antibodies were used for capture: goat
anti-mouse IgA and goat anti-mouse IgM [Kirkegaard and Perry
Laboratories (KPL), Gaithersburg, MD], goat anti-mouse IgG1 and goat anti-mouse
IgG2a (Southern Biotechnology,
Birmingham, AL). HRP-labeled goat anti-mouse IgA, goat anti-mouse IgM
(KPL), and goat anti-mouse IgG1,
and IgG2a (Southern Biotechnology)
were used to detect the different isotypes. The
concentration of antibody was calculated by comparison with a
standard curve of purified mouse IgA, IgM,
IgG1, and
IgG2a (PharMingen, San
Diego, CA).
Cytokine measurements by ELISA.
IL-12 secretion in colon culture supernatants was measured by ELISA
using commercially available anti-IL-12 antibodies C15.6 and
biotinylated C17.8 (PharMingen) for capture and detection, respectively, of the IL-12 p40 subunit. The amount of binding of
biotinylated antibody was detected using HRP-labeled streptavidin (KPL). Concentrations of IL-12 were established by comparison of values
against a standard curve generated for each assay using recombinant
IL-12 p70 (PharMingen). IL-12 was measured in duplicate culture
supernatants in each separate experiment. Levels of interferon-
(IFN-
) and tumor necrosis factor-
(TNF-
) in the colon culture supernatants were measured by ELISA using commercially available antibiodies R-46A2 (Pharmingen) for capture of IFN-
and biotinylated XMG 1.2 (Pharmingen) for detection; MP6-XT22 for capture of TNF-
(Pharmingen) and rabbit anti-mouse TNF-
(Genzyme,
Cambridge MA) followed by HRP-labeled goat F(ab') 2 anti-rabbit
IgG (Zymed, So. San Francisco, CA) for detection.
Flow cytometric analysis.
Mesenteric lymph nodes were removed, and single cell suspensions were
prepared by gentle teasing. To identify T cell subpopulations, cells
were incubated overnight at 4°C in either culture medium alone, rat
monoclonal anti-mouse CD4 (GK1.5) (11) or anti-mouse CD8 (53-6.72)
(22) to detect CD4+ T cells and
CD8+ T cells, respectively, as
previously described (49). Hybridomas (GK1.5 and 53-6.72) were
obtained from American Type Culture Collection (ATCC, Rockville, MD).
Supernatants were prepared in this laboratory. Cells were then washed
twice and incubated with 25 µg/ml FITC-labeled goat anti-rat IgG
(Pharmingen) adsorbed with mouse serum (KPL) for 30 min at 4°C and
then washed twice and analyzed. For detection of B cells, cells were
incubated for 30 min at 4°C with either FITC-labeled goat
anti-mouse IgA-IgG-IgM, 12.5 µg/ml (KPL), and washed twice. All
analyses were done in a FACScan (Becton Dickinson, San Jose, CA).
Statistical analysis.
Values are expressed as means ± SE. For each measurement, one-way
ANOVA was carried out to compare groups.
P < 0.05 was regarded as significant.
 |
RESULTS |
Clinical parameters of mice with disrupted IL-2 gene in variable
microbial environments.
Offspring of both colonies (GF and SPF) appeared normal after birth,
but in the perinatal period IL-2
/
mice in both
environments were already generally smaller than WT animals and were
more difficult to maintain. At 8 wk of age there was a mortality rate
of 50% affecting only the SPF colony, following severe growth
retardation and progressive wasting. Sadlack et al. (36) provided
evidence that this early death is the result of severe autoimmune
anemia. By 16-18 wk of age, all of the surviving IL-2
/
mice in the SPF environment
(n = 17) developed diarrhea and gained
less weight than the WT controls (P < 0.0001, Fig. 1). Rectal prolapse was seen rarely (8% in SPF IL-2
/
). IL-2
/
mice in the SPF environment died usually between 28 and 32 wk of age
(n = 9). In contrast, IL-2
/
mice raised in the GF environment remained clinically healthy during the observation period of up to 46 wk
(n = 12) with no mortality, diarrhea,
or rectal prolapse, and gained weight normally in the first 8 wk of
life but then gradually lost weight and were generally smaller than
age-matched WTs (P < 0.05, Fig. 1).
However, GF IL-2
/
mice weighed more than SPF IL-2
/
mice in the first 13 wk
(P < 0.0001, Fig. 1).

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Fig. 1.
Body weight (g) of specific pathogen-free (SPF, ,
n = 26) and germfree (GF, ,
n = 12 ) interleukin-2
(IL-2)-deficient mice in comparison to wild-type (WT) SPF ( ,
n = 28) and GF ( ,
n = 14) at different ages. Values are
means ± SE. * P < 0.05 and
** P < 0.0001 SPF IL-2
/ vs. SPF WT. # P < 0.001 SPF IL-2 / vs. GF IL-2 / . + P < 0.05 GF IL-2
/ vs. GF WT.
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|
Gross and histological evaluation of gastrointestinal tract.
SPF IL-2-deficient animals (n = 6)
showed mild splenomegaly by 4-8 wk of age. Grossly, the intestines
of SPF IL-2
/
mice appeared normal at these time points,
although blinded histological examination at 8 wk revealed mild large
intestinal inflammation (Fig. 2),
predominantly affecting the rectum (rectal histological score: SPF IL-2
/
1.0 ± 0.2, SPF WT 0.1 ± 0.1;
P < 0.0001). By 13 wk of age,
intestinal inflammation in SPF IL-2
/
mice rapidly
progressed and became characterized by mucosal hyperplasia and massive
infiltration of the lamina propria with mononuclear cells, which in
some cases extended into the submucosa. By 28 wk of age, the entire
gastrointestinal tract, including liver, stomach, small intestine
(jejunum and ileum), and colon was involved in SPF IL-2
/
mice (Fig. 3). At this time point, advanced
disease was grossly manifested by a rigid, thickened colon, mesenteric lymphadenopathy, and marked splenomegaly. These later stages of the
disease were characterized histologically by a further loss of goblet
cells, frequent crypt abscesses, and widely separated crypts (Fig.
4B).
Ulcerations of the mucosa were rarely seen. The involvement of the
stomach was similar, with prominent lymphocytic infiltration and
hyperplasia of the mucosa. The glandular region (Fig.
4E) was more affected than the
squamous cell portion of the stomach, and inflammation extended into
the duodenum.

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Fig. 2.
Inflammation of large intestine in IL-2-deficient mice (SPF, ; GF,
) compared with GF WT ( ). Average blinded large intestinal
histological score was obtained by summing separate scores for rectum,
transverse colon, and the cecal tip and dividing by 3. * P < 0.001 and ** P < 0.0001 SPF
IL-2 / vs. GF WT. + P < 0.05 SPF IL-2
/ vs. GF IL-2 / mice.
# P < 0.05 GF IL-2
/ vs. GF WT.
## P < 0.001 GF IL-2
/ vs. GF WT.
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Fig. 3.
Gastrointestinal inflammation in older IL-2-deficient mice, comparing
SPF WT mice (solid bars, 28 wk, n = 20) with SPF (stippled bars, 28 wk, n = 17) and GF (hatched bars, 38-46 wk,
n = 6) IL-2 knockouts.
* P < 0.0001, ** P < 0.001, and + P < 0.05 vs. SPF WT mice.
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Fig. 4.
Histological evidence of colitis in GF and SPF mice.
A: stained normal colonic section from
healthy, 28-wk-old IL-2 WT mouse, raised under SPF conditions
[hematoxylin and eosin (H&E), ×100].
B: colon section from 26-wk-old SPF
IL-2-deficient mouse (×100). Characteristics of inflammatory
process are marked mucosal hyperplasia due to infiltrating mononuclear
cells, loss of goblet cells, and disrupted mucosal architecture.
C: section of colon of diseased GF
IL-2-deficient mouse, 36 wk of age (×100). Inflammation is focal
(arrowheads), manifested by mild infiltration of mononuclear cells and
the loss of goblet cells. Adjacent mucosa is entirely normal. In
contrast to SPF animals, there was no hyperplasia of mucosa.
D: low-power view of colon of
28-wk-old GF IL-2-deficient mouse demonstrating focal normal areas with
abundant goblet cells (open arrow) with more generalized mild
inflammation with mild infiltration of a small number of mononuclear
cells and loss of goblet cells, but no crypt hyperplasia. Focal areas
of moderate mononuclear cellular infiltration are present (solid arrow)
(×40). E: section of glandular
part of stomach from 26-wk-old SPF IL-2-deficient mouse (×100).
Inflammatory pattern is similar to that of colon with abundant
lymphocytic infiltration and hyperplasia of mucosa.
F: moderate mononuclear cell
infiltration of antrum of 28-wk-old GF IL-2-deficient mouse
(×100).
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|
Even though GF IL-2-deficient animals showed no clinical symptoms or
mortality up to 46 wk of age, they developed splenomegaly, lymphadenopathy, and histological features of mild gastrointestinal inflammation (Figs. 2 and 4C).
Sterility of the colony was confirmed by monthly culture and Gram
staining of feces and culture and Gram staining of cecal contents from
selected mice at necropsy. The disease in GF animals was delayed (onset
after 8 wk) but exhibited a 100% penetrance by 13 wk of age. Colonic
inflammation progressively increased between 8 and 28 wk, but then
reached a plateau. At each time point after 4 wk histological colonic
inflammation in GF IL-2
/
mice was significantly less
than in SPF IL-2
/
mice (P < 0.05; Fig. 2) and became
significantly greater than in WT controls by 13 wk of age
(P < 0.05; Fig. 2). Inflammation in
the large intestine in GF IL-2
/
mice was quite focal,
and mucosal hyperplasia was much less prominent than in the SPF
environment (compare Fig. 4B with Fig.
4, C and
D). Crypt abscesses were
occasionally present but no ulcerations or submucosal inflammation was
noted even at late time points in the colons of GF IL-2
/
mice. GF WTs showed no inflammation at any time point.
Immunohistochemical staining indicated infiltration of
CD4+ T lymphocytes in the colonic
mucosa of GF IL-2
/
mice (Fig. 5A);
CD4+ T cells were rare in the
mucosa of GF WT mice (Fig. 5B).
CD8+ T lymphocytes were less
evident in the mucosa of GF IL-2
/
mice (data not shown).
By 28-46 wk of age GF IL-2
/
mice exhibited increased histological inflammation in all gastrointestinal regions (Fig. 3), including focally aggressive mononuclear cell infiltration into basal regions of the antral mucosa (Fig.
4F).

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Fig. 5.
Infiltration of CD4+ T lymphocytes
in colonic mucosa of GF mice. Immunohistochemical staining (see
MATERIALS AND METHODS) with anti-CD4
antibody of mucosa of GF IL-2-deficient mouse 14 wk of age
(A) and GF WT mouse
(B).
CD4+ T cells are detected by brown
stain (×66).
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Colonic IL-12 production.
Mucosal secretion of IL-12 (p40 subunit) was measured as an independent
objective marker of immune-mediated colitis. The p40 subunit of IL-12
is upregulated with inflammation in a number of experimental mouse
models of chronic colitis (9, 33, 42). IL-2
/
mice of
both environments expressed increased colonic levels of IL-12 only with
advanced disease. Colonic IL-12 secretion corresponded to the
aggressiveness of colitis by histological criteria. Although colons of
GF IL-2
/
mice 38-46 wk of age secreted
approximately twofold more IL-12 than those of GF WT animals (443 ± 72 vs. 229 ± 68 pg/100 mg tissue weight,
P < 0.05), SPF IL-2
/
mice at 28 wk of age secreted as much as 20-fold more colonic IL-12
than SPF WT mice (4,211 ± 769 vs. 199 ± 32 pg/100 mg tissue,
P < 0.01). INF-
and TNF-
could
not be detected in cultured colonic supernatants.
Immunoglobulin production.
GF and SPF IL-2-deficient mice exhibited similar patterns of serum and
colonic mucosal immunoglobulin isotype production. After an initial
rise at 8 wk of age, which was less pronounced in GF IL-2
/
animals, colonic
IgG2a production decreased
dramatically with age, with ~100-fold lower values at 26 wk of age
(Fig. 6), compared with 8 wk of age.
Similar patterns were seen with serum and colonic IgM, IgA, and
IgG1 (data not shown). In SPF IL-2
/
mice, all of the analyzed isotypes (IgA, IgM,
IgG1,
IgG2a) were already increased at
the time of very mild histological inflammation (at 8 wk of age) and
before clinical signs of colitis became evident. At this time point,
SPF IL-2
/
mice showed 13-fold more secreted colonic
IgG2a compared with WT of the same
age and twofold more IgG2a
relative to GF IL-2
/
mice (Fig. 6). However, by 13 wk of
age, colonic production of immunoglobulins (Fig. 6) of IL-2
/
mice progressively decreased and was very low at 28 wk
of age in SPF and at 38 wk of age in GF IL-2
/
animals
compared with WT mice.

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Fig. 6.
IgG2a production by cultured
colonic mucosal fragments. Comparison between SPF (stippled bars,
n = 26) and GF (hatched bars,
n = 12) IL-2-deficient mice and SPF
(solid bars, n = 28) and GF (open
bars, n = 14) WT animals.
* P < 0.05 and
** P < 0.0001 vs. SPF WT. + P < 0.05 and
++ P < 0.0001 vs. GF WT. No
information was obtained from 13-wk-old GF IL-2 / mice.
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Flow cytometric analysis of mesenteric lymph node cells.
Macroscopically there was an increase in size and numbers of the
mesenteric lymph nodes in SPF and GF IL-2
/
mice with
colitis compared with their healthy IL-2
/
littermates.
We therefore examined the cellular composition of these lymphoid
organs. The loss of circulating and mucosal immunoglobulin production
was accompanied by a loss of mature surface
Ig+ B cells from mesenteric lymph
nodes in older IL-2-deficient mice, regardless of luminal bacterial
colonization (Fig.
7A).
Initially, at 4 wk of age, all mice had comparable proportions of
mesenteric lymph node cells expressing surface immunoglobulin, with a
slightly higher percentage in SPF IL-2
/
[SPF: IL-2
/
41.5 ± 7.0% vs. IL-2 WT 35.0 ± 2.2%, not
significant (NS) and GF: IL-2
/
33.5 ± 3% vs. IL-2
WT 35.2 ± 2.1%, NS]. However, during later stages of the
disease, the differences became very apparent, such that by 28 wk of
age, very few B cells were detected in SPF and GF IL-2
/
mice (SPF: IL-2
/
2.7 ± 0.3% vs. IL-2 WT 26.6 ± 3.4%, P < 0.0001 and GF: IL-2
/
1.6 ± 0.4% vs. IL-2 WT 30.6 ± 5.2%, P < 0.001). At the same time point,
we observed a reciprocal increase of
CD4+ and
CD8+ T cells in IL-2
/
mice with no significant differences between SPF and GF
mice (Fig. 7, B and
C). Proportions of
CD4+ and
CD8+ T cells remained increased in
GF IL-2
/
mice at 42 wk of age (Fig. 7,
B and
C).

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Fig. 7.
A: percentage of mesenteric lymph node
cells that express surface IgA-IgM-IgG, obtained by
fluorescence-activated cell sorting analysis at different time points.
(SPF IL-2 / , stippled bars,
n = 10; GF IL-2 / ,
hatched bars, n = 6; SPF WT, solid
bars, n = 19; GF WT, open bars,
n = 10).
B: percentage of mesenteric lymph node
cells that express CD4+.
C: percentage of mesenteric
lymph node cells that express
CD8+.
* P < 0.0001, ** P < 0.001, and + P < 0.05 vs. SPF WT.
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Inflammation of liver.
The liver of SPF IL-2-deficient mice showed early signs of multifocal
periportal infiltration, composed of macrophages and lymphocytes as
early as 4-8 wk of age (Fig. 8). By 13 wk of age there were diffuse periportal infiltrates with abundant
extramedullary hematopoiesis. The inflammation by 28 wk of
age was characterized by severe periportal infiltration of macrophages
and lymphocytes with penetration of the limiting plate, bile duct
proliferation, and single cell necrosis of hepatocytes adjacent to
lesions (Fig. 9B). In
contrast to the less severe gastrointestinal inflammation in GF IL-2
/
mice, portal inflammation was similar at all time points in SPF and GF IL-2
/
mice (Figs. 8 and 9,
B and
C). Interestingly, serum
transaminases were not elevated even with advanced histological inflammation (results not shown). WT mice showed minimal hepatic inflammation (Fig. 9A).

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Fig. 8.
Blinded liver histological scores at different ages. SPF ( ,
n = 26) and GF ( ,
n = 12) IL-2-deficient mice were
compared with GF ( , n = 14) WTs.
* P < 0.05 and
** P < 0.0001 vs. GF WT.
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Fig. 9.
Histological evidence of portal tract inflammation in GF and SPF.
A: hematoxylin and eosin (H&E)-stained
liver section of 28-wk-old SPF IL-2 WT (×400).
B: portal tract inflammation in
28-wk-old SPF IL-2-deficient animal with characteristic periportal
infiltrates and bile duct proliferation (×400).
C: H&E stained liver section of
28-wk-old GF IL-2-deficient mouse showing similar infiltration
(×400).
|
|
 |
DISCUSSION |
In this study we were able to confirm previous observations (8, 36)
that IL-2
/
mice raised under SPF conditions developed histological inflammation; however, in contrast to these previous reports of absent colitis in GF IL-2
/
mice, we
demonstrated mild, delayed, and focal gastrointestinal inflammation in
GF IL-2-deficient mice, which was first detected at 13 wk of age.
Colitis in GF IL-2
/
mice was mild, focal, delayed in
onset, and reached a plateau at 28 wk. Mononuclear infiltrates in the
lamina propria consisted predominantly of
CD4+ T lymphocytes with both
CD4+ and
CD8+ T cells increased in the
mesenteric lymph nodes of GF IL-2
/
mice. There were no
clinical symptoms or mortality in GF IL-2
/
mice up to 46 wk of age, and histological inflammation and colonic IL-12 production
were significantly less than in the SPF environment. The intestinal
inflammation in SPF IL-2
/
mice was accompanied by a
progressive loss of body weight and dramatically increased colonic
IL-12 production. In addition, we observed clinical and gross evidence
of progressive and lethal intestinal inflammation in our SPF IL-2
/
colony, in contrast to the absence of all but mild
histological evidence of colitis in the original report (36). The mild,
focal nature of the colitis in GF IL-2
/
mice older than
13 wk of age, our inclusion of older mice, and the different genetic
background of our animals may explain the absence of observed colonic
inflammation in other studies (8, 36). For example, the study by
Contractor et al. (8) concentrated on histological evidence of colonic
inflammation at early time points (4-8 wk of age), whereas our GF
IL-2
/
mice did not develop detectable colitis by
histological criteria until 13 wk of age and increased colonic IL-12
secretion was not evident until 36-38 wk of age. In addition, our
SPF and GF colonies of IL-2-deficient mice were C57BL/6 × 129/Ola
outbred populations derived from the same progenitors and were not
selectively backcrossed, whereas the previous studies were performed on
backcrossed C57BL/6 IL-2
/
mice (8, 36). In other models,
C57BL/6 mice are low responders. For example, Berg et al. (5) showed
that IL-10-deficient mice on an inbred 129/SvEv background developed
more aggressive colitis than those on an inbred C57BL/6 background and
that outbred 129/C57BL/6 mice had an intermediate phenotype (5). The
importance of the genetic background is further illustrated by
observations that IL-2 knockout mice are more sensitive to both SPF
bacterial and GF environments than IL-10 knockout mice and
HLA-B27 transgenic rats. In
previous studies, we demonstrated that no mortality or jejunoileal
inflammation occurred in IL-10
/
mice or
B27 transgenic rats housed in the
same SPF environment used in the current study and that neither of
these two rodent models developed inflammation if housed in identical
conditions as the GF IL-2
/
mice (30, 42). Additional
differences in the environmental conditions could account for disparate
results of the present and previous studies, since subtle alterations
in luminal bacteria (30), food (29), and temperature (45) can influence
experimental inflammation. We cannot exclude spontaneous mutations,
which enhance susceptibility to colitis, but we were careful to exclude
the presence of all detectable microbial pathogens, including
Helicobacter species, in our SPF colony.
The IL-2 knockout mouse model of chronic intestinal inflammation
described here is unique because it spontaneously develops mild
colonic, small bowel, and gastric lesions when raised under sterile
conditions. In other well-established genetically engineered models of
colitis, the involvement of the luminal bacterial flora on
gastrointestinal inflammation is more pronounced. IL-10 and TCR-
/
knockout mice, as well as HLA-B27
transgenic rats, do not develop colitis or gastritis if raised under GF
conditions (12, 30, 39, 42, 47), and IL-10
/
mice develop
even greater inflammation under conventional conditions where
environmental pathogens are common, compared with the SPF state (19).
Colitis in GF mice fed DSS established a precedent for mucosal
inflammation in the absence of luminal bacteria (2), but in this acute
situation luminal DSS could be considered the injurious agent.
Although our GF IL-2
/
mice were documented to have no
viable bacteria, they were still exposed to dead bacteria and bacterial constituents in the autoclaved food and bedding. We have previously postulated that nonviable bacterial products can provide the constant antigenic stimulus that drives chronic, relapsing colitis in
genetically susceptible hosts (30, 37) and demonstrated that sterile
bacterial components can induce or potentiate enterocolitis and
extraintestinal disease. For example, luminal sterile PG-PS can
potentiate toxin-induced intestinal inflammation in the indomethacin
and acetic acid models (10, 40). Similarly, chronic granulomatous
enterocolitis, hepatitis, and arthritis can be induced in susceptible
Lewis rats by the intramural injection of purified bacterial PG-PS
polymers (26), and heat-killed E. coli
causes hepatic inflammation in rabbits (17). Furthermore, mice with
experimental colitis display humoral and cellular immune responses to
luminal bacterial antigens, establishing immunologic mechanisms by
which nonviable bacterial antigens could cause inflammation (6, 13). It
is possible that nonviable bacterial cell wall components and protein
antigens present in the autoclaved food or bedding of GF animals
provide sufficient stimulation to generate the mild, focal
gastrointestinal inflammation seen in our GF IL-2
/
mice,
but that the less susceptible IL-10
/
mice and
HLA-B27 transgenic rats do not
respond to identical stimuli. The autoclaved food in our GF facility
contains dead bacteria by Gram stain and readily detectable LPS. In
keeping with our suggestion of heightened susceptibility to luminal
bacterial stimuli, SPF IL-2
/
mice have more aggressive
gastrointestinal disease with frequent crypt abscesses, higher
mortality by 28-32 wk of age, and increased extraintestinal
disease in the portal tracts and bone marrow than do SPF IL-10
/
mice (19, 42). Alternatively, dysregulated T cells may
react to self antigens for which bacterial constituents provide an
adjuvant effect. Although we stress the unique presence of spontaneous
gastrointestinal inflammation in sterile IL-2
/
mice, we
also need to emphasize the delayed onset of this mild, focal,
nonproliferative, and nonprogressive colitis and the lack of a lethal
inflammatory response, which is much more aggressive in the presence of
viable luminal bacteria.
Histological inflammation was present in the gastric antrum, small
intestine, colon, and hepatic portal tracts of GF IL-2
/
mice. However, not all organs responded equally to the bacterial environment. Although the gastrointestinal inflammation was delayed and
attenuated in GF animals, the periportal hepatic inflammation with
necrosis of adjacent hepatocytes in GF IL-2
/
mice was of
similar intensity and started at the same time as in SPF animals; however, liver enzymes remained relatively normal throughout. Similarly, Contractor et al. (8) documented increased numbers of
intrahepatic lymphocytes in IL-2
/
mice at 5 wk of age,
when mice did not have colitis, but did not compare hepatic
inflammation in GF vs. SPF IL-2
/
mice. Therefore, portal
tract inflammation in this model does not appear to be secondary to the
colonic inflammation and is independent of viable bacteria, in contrast
to studies by Lichtman et al. (23, 24) in the rat jejunal bacterial
overgrowth model. In addition, the degree of splenomegaly was similar
in GF and SPF IL-2
/
mice. A discrepancy between
gastrointestinal and certain systemic manifestations was also noted in
GF
HLA-B27/
2-microglobulin transgenic rats, where colitis, gastritis, and arthritis were absent,
but dermatitis and testicular inflammation were not influenced by the
absence of viable bacteria (30, 47).
The progressive and dramatic selective loss of B cells and
immunoglobulins in IL-2-deficient mice was not influenced by the bacterial environment, inasmuch as both GF and SPF animal colonies reacted similarly. Similar to the report by Sadlack et al. (36), we
demonstrated normal serum and secreted mucosal immunoglobulin levels at
4 wk in IL-2
/
mice, with increased levels at 8 wk of
age, followed by a rapid and progressive decrease in immunoglobulin levels and B lymphocytes in the mesenteric lymph nodes in older IL-2
/
mice with colitis. Low levels of immunoglobulins were observed in all subclasses examined (IgA, IgM,
IgG1,
IgG2a). At the same time, we
showed increasing proportions of T lymphocytes in the mesenteric lymph
nodes, which could either reflect preservation of T cells in the face
of the diminishing B cell population or an actual increase in the
number of T cells. The loss of B cells and immunoglobulin secretion
during progressive colitis provides further evidence that B cells are
not involved in the pathogenesis of the intestinal inflammation. Ma et
al. (25) demonstrated that IL-2
/
× RAG-2
/
double-mutant mice (T and B cell deficient) were
disease free, whereas IL-2
/
× JH
/
double-mutant mice (selective B cell deficient) developed equivalent
disease as IL-2
/
(25).
Colonic secretion of IL-12 was significantly increased in SPF IL-2
/
mice, and to a lesser extent in older GF IL-2-deficient mice. We measured IL-12 p40 because this subunit is upregulated in
antigen-presenting cells by microbial stimuli (33) and because IL-12 is
a key immunoregulatory molecule in experimental colitis (44) and
Crohn's disease (14). Most rodent models of chronic enterocolitis,
with the notable exception of TCR-
/
mice, display a
TH1 profile of cytokines (38),
despite the fact that human ulcerative colitis patients have more of a
TH2 pattern of lymphokines (14).
Increased colonic IFN-
expression has been reported in SPF
IL-2-deficient mice with colitis (1). IL-12 p40 is upregulated in
colonic tissue of SPF IL-10
/
mice (9, 42) and mice with
chronic trinitrobenzene sulfonic acid-induced colitis (27). In each of
these models, blockade of endogenous IL-12 with neutralizing antibody
dramatically reversed experimental colitis (9, 27).
In conclusion, we demonstrate that, in contrast with other genetically
engineered models, older GF IL-2-deficient mice develop mild, focal,
subclinical gastrointestinal inflammation in a sterile environment.
Although mild colitis, small intestinal inflammation, mucosal
infiltration of CD4+ T cells,
upregulation of colonic IL-12, and antral gastritis are present in the
absence of viable luminal bacteria, SPF mice have a much more rapidly
progressive and aggressive clinical course, confirming the importance
of resident luminal bacteria in the pathogenesis of chronic intestinal
inflammation in genetically susceptible hosts. The mild colitis in GF
IL-2-deficient mice is accompanied by moderate portal tract
inflammation and a progressive loss of mucosal B lymphocytes and
immunoglobulin secretion, which is not dependent on viable luminal
bacterial stimulation. The hypothesis that nonviable luminal bacterial
fragments play a role in the gastrointestinal inflammation seen in GF
IL-2
/
mice could be further investigated by eliminating
all bacterial antigens from the food and bedding of GF animals.
 |
ACKNOWLEDGEMENTS |
This work was supported by the National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-40249, DK-53347, and DK-34087,
the National Institute of Allergy and Infectious Diseases Grant
AI-01122, the Crohn's and Colitis Foundation of America, and the
Deutsche Forschungsgemeinschaft Schu 1131/1-2.
 |
FOOTNOTES |
Present address of M. Schultz: Klinik und Poliklinik fur Innere
Medizin, Klinikum der Universitat Regensburg, Franz-Josef-Straub-Allee 11, D-93042 Regensburg, Germany.
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: R. B. Sartor,
Div. of Digestive Diseases and Nutrition, School of Medicine, Dept. of
Medicine, CB 7080, Rm. 030, Glaxo Bldg., Univ. of North Carolina at
Chapel Hill, Chapel Hill, NC 27599-7080 (E-mail:
rbs{at}med.unc.edu).
Received 6 April 1998; accepted in final form 23 February 1999.
 |
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