Genes and Immunity (2001) 2, 309–316
 2001 Nature Publishing Group All rights reserved 1466-4879/01 $15.00
www.nature.com/gene
Dextran sulphate sodium-induced colitis is ameliorated
in interleukin 4 deficient mice
L Stevceva1, P Pavli1, A Husband2, A Ramsay3 and WF Doe1
1
Division of Molecular Medicine and 2Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research,
ANU, Canberra, ACT, 2601, Australia; 3Department of Veterinary Anatomy and Pathology, University of Sydney, NSW, 2006,
Australia
The importance of IL-4 and its effects in inflammatory bowel disease (IBD) was studied using the dextran sulphate
sodium-induced model of experimental colitis. The model resembles ulcerative colitis in humans. IL-4 deficient mice and
IL-4+/+ littermates were used to induce colitis. Activity of disease, extent of tissue damage, immunoglobulin isotypes, IFN␥
and IL-10 production was assessed. Both disease activity index (DAI) and histological scores were consistently lower in
the IL-4 deficient mice than in the IL-4+/+ littermates. Furthermore, the lower histological scores reflected the milder
inflammatory lesions and decreased ulceration found in the IL-4 deficient mice. Analysis of immunoglobulin subtypes
showed that IgG1 was almost absent in the sera of IL-4 deficient mice. IFN␥ contents was much higher in colonic tissues
from IL-4 deficient mice. Dextran sulphate sodium-induced colitis is ameliorated in IL-4 deficient mice. IL-4 either directly
or through its effects on T and B cells influences its severity. It is unclear if the higher immunoglobulin-producing cells in
the colonic tissues of IL-4 deficient mice before colitis was induced could have influenced the outcome of the disease.
The high IFN␥ contents in colonic tissues of IL-4 deficient mice argue against the role of this cytokine as a crucial
mediator of tissue damage during the acute phase of colitis. Genes and Immunity (2001) 2, 309–316.
Keywords: immunoglobulins; interferon-gamma; IL-10; knockout; ‘in situ’, colitis, IBD
Introduction
Immune responses to antigens are distinguished by a distinct pattern of cytokines produced by subsets of CD4+ T
helper (Th) cells that result in cell-mediated or humoral
immunity. Th1 cells produce IL-2, IFN-␥ and TNF-␣ and
promote delayed-type hypersensitivity reactions while
Th2 cells produce IL-4, IL-5, IL-6, IL-10 and IL-13 but not
IL-2 or IFN-␥ and promote allergic-type responses,1,2 by
stimulating B cells, mast cells and eosinophils.3 Differentiation of naive T cells into cells of the Th2 phenotype
requires the presence of IL-4.4–6 IL-4 also suppresses
development of IL-2 and IFN-␥ secreting effector cells.4
Even low levels of IL-4 are effective since blocking the
effects of IL-4 by adding neutralizing monoclonal antibodies has a dramatic effect even if the levels of IL-4 in
the supernatants of the cultures are low or undetectable.4,7
Several recent reports have confirmed the beneficial
effects of the absence of IL-4 on colitis in different models
of inflammatory bowel disease (IBD).8–12
Dextran sulphate sodium (DSS) given via drinking
water can induce a form of colitis in mice with features
similar to those found in human ulcerative colitis. Our
Correspondence: Liljana Stevceva, Basic Science Laboratory, National
Cancer Institute, Building 41/C303, National Institutes of Health,
Bethesda, MD 20892–5055, USA. E-mail: stevcevl얀mail.nih.gov
Part of this work has been presented at The Annual Gastroenterology Meeting in San Francisco, California and was published as an
abstract in Gastroenterology 1996; 110: 4:A1019.
Received 3 April 2001; revised 6 June 2001; accepted 26 June 2001
previous findings have confirmed that colitis is limited
to the large intestine and is more prominent in the distal
colon and rectum.13 In our hands, inflammatory changes
were diffuse and were restricted/most prominent in the
mucosa. Initially, an inflammatory infiltrate consisting of
macrophages, neutrophils and eosinophils appears, cryptitis and crypt abscesses follow and finally erosions and
broad-based mucosal ulceration develop.13,14 Cyclic
ingestion of DSS leads to chronic colitis characterized by
lymphocyte infiltration and distortion of crypt architecture while prolonged exposure leads to hyperplasia and
dysplasia.15–17
In this study DSS-induced experimental colitis was
induced in IL-4 deficient mice and the outcome of the
inflammation examined and compared to the IL-4+/+
mice. Interleukin 4-deficient mice have normal B and T
cell development, low serum levels of IgG1 and undetectable IgE,18 impaired peripheral eosinophilia and their
cells produce less IL-5, IL-9 and IL-10.19 They are characterized by an increase in IFN␥ production and an overall
Th1 type environment in the tissues.
Results
Activity of the disease is lower in IL-4 deficient than
in the IL-4 positive mice
The activity of the disease was assessed in six consecutive
experiments in which colitis was induced by ingestion
of either 2.5% or 5% DSS in the drinking water. IL-4+/+
littermates were used as a control group. In every one of
the experiments the severity of the disease was much
DSS-induced colitis is ameliorated in IL-4 deficient mice
L Stevceva et al
310
Figure 1 Activity of the disease. Disease activity index in IL-4−/−
and IL-4+/+ mice in DSS-induced colitis using 2.5% DSS (a) and
5% DSS (b). P values apply to day 6 of DSS ingestion.
lower in the IL-4 deficient mice than in the control group.
Shortly, IL-4 deficient mice lost less weight and even
though they also developed diarrhea, gross rectal bleeding and severe weight loss was rarely seen. Bloody
diarrhea was seen in 32.5% (13/40) of IL-4−/− and in
74.5% (29/39) of the IL-4+/+ mice in which colitis was
induced by 5% DSS. To demonstrate the delay in appearance of clinical signs, disease activity index (DAI) was
calculated on a daily basis in the first two experiments
(Figure 1a,b). The difference was statistically significant
starting from day 3 in the 2.5% DSS (P = 0.02) and from
day 2 in the 5% DSS (P = 0.0001).
As established by histopathological examination,
inflammation in IL-4 deficient mice was ameliorated
when compared to the control group (Figure 2). Both
mononuclear cell infiltrate and neutrophils infiltrate was
reduced in IL-4 deficient mice. Ulceration, when present,
was smaller and rarely extended through the whole
transverse section (Figure 3a). Mice from the control
group, on the other hand, had extensive infiltration with
mononuclear cells and neutrophils in the colon, severe
ulceration and often complete denudation of the epithelial layer (Figure 3b). Histopathological scoring of
Figure 3 Histopathological findings. Damage to the colon was
much less extensive in the IL-4 deficient mice. Ulceration was
smaller and never extended throughout the whole transverse section (a). On the other hand, ulceration in the IL-4+/+ littermates
was wide and epithelium was almost missing (b). Day 6. Original
magnification ×25.
samples revealed consistently lower scores in the IL-4
deficient group than in the control group (Figure 2).
However, when histological scores were analyzed by
Mann–Whitney U test for non-parametric data, P values
were close but not below 0.05 that was considered to be
a significance level. The reason for that could be that the
histological scoring system that we used did not account
for the extent of the damage but rather for the presence
or absence of it.
Figure 2 Histological scores. Histological scores after 6 days of DSS
ingestion were lower in the IL-4 deficient mice in every one of the
six experiments done.
Genes and Immunity
Immunoglobulin production in colons of healthy IL-4
deficient mice
The intestinal tract is a single, most important source of
immunoglobulin production20,21 and immunoglobulins
produced by intestinal plasma cells play an important
role in preventing intestinal injuries caused by enteric
pathogens. As IL-4 plays a significant role in immunoglobulin production and isotype switching, immunoglobulin levels were first assessed in sera of healthy IL-4
DSS-induced colitis is ameliorated in IL-4 deficient mice
L Stevceva et al
311
Figure 4 Immunoglobulin-producing cells in healthy colonic tissue. IL-4 deficient mice had twice as many IgG2- and IgG3-producing cells
in the colon compared to the IL-4+/+ littermates. Numbers of IgA, IgM- and IgG1-producing cells were similar in both strains.
deficient mice and the control littermates. Our findings
in the sera were very similar to what was initially
described by Kuhn et al,18 ie while IgG1 was almost
absent in IL-4 deficient mice, levels of other IgG isotypes
were similar to what was found in the IL-4+/+ littermates
(Table 1). Numbers of IgG1-producing cells in the colons
of these mice were similar to the control strain but there
was a two-fold higher number of IgG2-subclassesproducing cells in IL-4 deficient mice (Figure 4b).
Because of the importance of IgA in mucosal protection
and the postulated role that IL-4 may play in immunoglobulin switching from IgM to IgA, numbers of IgA and
IgM-producing cells in colonic mucosa was also assessed
and was similar in IL-4 deficient mice and in the IL-4+/+
littermates (Figure 4a).
Immunoglobulin production in IL-4 deficient mice
after induction of colitis
Numbers of IgA and IgM-producing cells decreased during the course of the colitis. IgA-producing cells
decreased to a lesser extent in the IL-4 deficient mice, so
that their numbers were two-fold higher in the inflamed
colons after 6 days of DSS ingestion (Figure 5a). There
were fewer and similar numbers of IgM-producing cells
in the inflamed colons of both mice strains (Figure 5a).
Induction of colitis resulted in a two-fold increase of
all IgG subtypes in IL-4 deficient mice. There was fourfold more IgG-producing cells in the inflamed colon of
the IL-4+/+ strain, so, when the two strains were compared, no significant difference could be seen (Figure 5b).
IgG2b was the dominant immunoglobulin that
increased in sera collected after 6 days of DSS exposure
in both mouse strains (Table 1).
IL-10 mRNA in colonic tissues
It has previously been shown by others that IL-10 is an
important cytokine that has the capacity to modify the
extent of colonic damage in several models of colitis.22,23
Similarly, it was found that IL-10 levels in DSS-induced
colitis increase24,25 and that treatment with IL-10 ameliorates the colitis while blocking endogenous IL-10 exacerbates it.24 Thus, the importance of looking into IL-10
expression in inflamed colonic tissue.
We did not attempt to quantify IL-10 mRNA
expression. However, in situ hybridization was done to
assess if IL-10 is expressed in colonic tissues of IL-4
Table 1 Immunoglobulin G levels in the serum
mg/ml
IgG1
IgG2a
IgG2b
untreated
mice
Day 6, 2.5%
DSS
Day 6, 5%
DSS
IL-4+/+
IL-4−/−
IL-4+/+
IL-4−/−
IL-4+/+
IL-4−/−
⬍to the limit
9.48–3.7
15.5–4.6
5.47–1.3
8.93–2.8
55.8–8.2
1.9–0.3
5.6–0.7
62.2–6.2
11.7–3.1
18.4–2.9
55.7–7.0
⬍to the limit
4.7–0.33
79.7–6.9
7.32–2.1
15.07–3.6
15.64–5.4
As shown by ELISA, IgG1 levels were significantly lower to absent in IL-4 deficient mice. IgG2b levels rose significantly during the course
of the disease but were not different in the IL-4 deficient mice when compared to the wild-type.
Genes and Immunity
DSS-induced colitis is ameliorated in IL-4 deficient mice
L Stevceva et al
312
Figure 5 Immunoglobulin-producing cells in inflamed colons. While numbers of IgA-producing cells change only slightly with induction
of inflammation in IL-4 deficient mice, it decreased significantly in the IL-4+/+ littermates. Numbers of IgG-producing cells increased in
both strains and were similar in the inflamed colons 6 days after 5% DSS ingestion. Findings were similar when colitis was induced with
2.5% DSS (data not shown).
deficient mice. We were able to demonstrate that cells containing mRNA for IL-10 were present in the inflamed
colonic tissue (Figure 6). Many of the IL-10-producing cells
were aggregated in the submucosa and the lymphoid follicles of lamina propria mucosae. IL-10-producing cells
could also be seen between crypts in the lamina propria.
It did not appear that there was a difference in the
expression between the IL-4 deficient mice and the control strain but actual quantification of the cells was not
attempted.
IFN␥ contents in colonic tissues was higher in the IL4 deficient mice regardless of the concentration of
DSS used to induce the colitis
Care was taken to standardize the assay for assessing
IFN␥ contents in the tissues by taking samples of colonic
tissue from the same location in each animal at 2 cm
proximal to the termination of rectum. IFN␥ content in
the inflamed colons was always higher in the IL-4
deficient mice regardless of the concentration of DSS
used to induce the colitis (Figure 7).
Figure 6 In situ hybridization for IL-10. IL-10 mRNA was detected
in aggregated cells of the submucosal layer but also in cells between
crypts and in the lymphoid follicles of the lamina propria mucosae.
IL-4 deficient mice. Day 6. Original magnification ×200.
Figure 7 IFN␥ contents in the colonic tissue. IFN␥ contents in the
inflamed colonic tissue were higher in the IL-4 deficient mice
regardless of the concentration of DSS used to induce the colitis.
Genes and Immunity
DSS-induced colitis is ameliorated in IL-4 deficient mice
L Stevceva et al
Discussion
Ingestion of DSS given in the drinking water in mice
causes colitis that closely resembles IBD. The pathogenetic events leading to colonic inflammation remain largely unknown. In this study, we looked at the effects of
IL-4 on the development and severity of the disease.
The results described were somewhat surprising and
suggest that IL-4 directly or via its effects on other proinflammatory mediators modifies the mechanisms of
inflammation in this model of colitis. In addition, a recent
study in this model of colitis has shown only a modest
increase in IL-4 mRNA that increased if higher concentration of DSS was used.25 There was a clear difference
in the severity of the disease as determined by both clinical and histopathological features. IL-4 deficient mice lost
less weight during the course of the colitis and the other
clinical signs (diarrhea and rectal bleeding) developed at
a later stage of the disease. The inflammatory infiltrate
and ulceration, when present, was much less extensive
in the IL-4 deficient mice; it never extended throughout
the entire transverse section.
In this study, for the first time, we examined changes
in immunoglobulin production during DSS-induced colitis. As it appears, there is a loss of IgA-producing cells
in the inflamed colonic mucosa. At the same time, colitis
is characterized with an increase of IgG2 immunoglobulins. In inflamed colonic tissues, there is an increase of
all of the IgG2 immunoglobulin-producing cells.
Assessment of IgG subclass levels in mice sera showed
that there was a three-fold increase in IgG2b levels during the course of the colitis. The capacity of DSS to induce
polyclonal expansion of B cells and to generate production of predominantly IgG2b and IgG3 immunoglobulins has been reported previously in vitro.26 Therefore,
the preferential increase in serum IgG2b during the
course of the colitis may be due to the direct effect of DSS
on B cells.
IL-4 has a well known array of effects on B cell proliferation and immunoglobulin production and isotype
switching. IL-4 deficient mice had a lower number of
IgM-producing-cells and similar numbers of IgA-producing cells indicating that there is no obligatory role for IL4 in isotype switching from IgM to IgA. While numbers
of IgA-producing cells decreased significantly during the
course of the colitis in the wild-type mice, in the IL-4
deficient mice they remained steady. It is unclear however, whether this effect is a cause or the consequence of
the ameliorated disease process in the colon.
One of the main activities of IL-4 is to act as a switch
factor for IgG1.27 Consequently, IL-4 deficient mice have
been described as having low levels of IgG1.18 Despite
significantly lower levels of IgG1 in the serum, the numbers of IgG1-producing cells in the colonic tissue were
similar in both mice strains and did not change significantly during the course of the colitis. Numbers of IgG2a,
IgG2b and IgG3-producing cells before the colitis was
induced were two-fold higher in the IL-4 deficient mice.
The higher numbers of IgG2a, IgG2b and IgG3-producing
cells in IL-4 deficient mice colonic mucosa before colitis
was induced may have had a beneficial effect sufficient
to ameliorate the disease process.
IL-4 has a profound effect on the ability of T cells to
differentiate into Th1 or Th2 type helper cells. IL-4
deficient mice lack Th2 type cytokines such as IL-5 and
IL-10.19 Our previous studies showed that IL-5 does not
influence the severity of the inflammation in this model
of colitis.28 In regard to IL-10, several studies have demonstrated that IL-10 has a beneficial effect in IBD.22,23 IL10 deficient mice bred in a normal environment as
opposed to a germ-free state develop IBD that is ameliorated by the administration of IL-10.22 Similar results were
achieved by treating mice with trinitrobenzenesulphonic
acid (TNBS)-induced colitis with IL-10.23 Treatment using
IL-10 restored tolerance to bacterial sonicates indicating
an important role for IL-10 in regulating tolerance
towards intestinal flora and opening exciting possibilities
of using IL-10 to treat patients with IBD.
It was shown by others that IL-10 levels in DSSinduced colitis increase.24 In chronic DSS-induced colitis,
treatment with IL-10 ameliorated the intestinal inflammation while blocking endogenous IL-10 caused exacerbation of the inflammation.24 We assessed IL-10 mRNA
expression by in situ hybridization and determined that
IL-10 was expressed in the inflamed tissues of IL-4
deficient mice and that there was no difference in the levels of expression of IL-10 between the IL-4 deficient mice
and the IL-4+/+ strain.
Our results that show higher levels of IFN␥ in the
inflamed colonic tissue of IL-4 deficient mice are in agreement with previous reports of the role of IL-4 has in T
cell differentiation. It has been shown that chronic DSSinduced colitis is characterized by both Th1- and Th2type response.29 Recent studies in chronic DSS-induced
colitis have shown that IFN␥ increases with development
of lesions and that treatment with neutralizing antibodies
to IFN␥ reduces histological scores.30 In another study, in
which DSS colitis was induced in CCR5 and CCR2deficient mice, IFN␥ has been implicated as a possible
contributing factor to tissue damage.31 The results of this
study indicate that, in the acute phase of DSS colitis, IFN␥
may not be a crucial mediator of tissue damage or that
its expression levels do not correlate with the histological
score at this stage of the colitis.
313
Materials and methods
Mice
IL-4 deficient mice used for induction of colitis were of
129SV × C57Bl/6 background and were kindly provided
by Manfred Kopf, Basel Institute of Immunology, Basel,
Switzerland.19 IL-4+/+ littermates were used as a control
group. Functional disruption of the IL-4 gene has been
regularly confirmed both by the absence of IL-4 mRNA
as measured by RT-PCR and by the absence of bioactivity
(as determined by the failure of the IL-4 sensitive cell line,
4S, to proliferate in response to supernatants from antiCD3 plus IL-2 stimulated IL-4−/− CD4+ T cells). Mice
were housed under standard laboratory conditions
according to the Australian National University guidelines and were fed sterile food and water ad libitum.
Females (total number 55 for 2.5% DSS-induced colitis
and 60 for 5% DSS-induced colitis) in a total number of
six experiments were used. The studies were approved
by The Animal Experimentation Ethics Committee of The
Australian National University.
Dextran sulphate sodium
The DSS used was a sodium salt of molecular weight of
41 K and with sulphur substitution of 16.3 (TdB
Genes and Immunity
DSS-induced colitis is ameliorated in IL-4 deficient mice
L Stevceva et al
314
Consultancy, Uppsala, Sweden). DSS was given as 2.5%
and 5% solutions in distilled water for 8 days.
Disease activity index (DAI)
DAI was derived from the three major clinical signs
(weight loss, diarrhea and rectal bleeding) as previously
described.13 The clinical signs were analyzed separately
and correlated with the histological score. The final formula for the DAI was evaluated by correlation with the
histological score as previously described.13 The DAI was
defined as:
DAI = (weight loss) + (diarrhea score)
+ (rectal bleeding score).
Body weight loss: Body weight loss was calculated as the
difference between the predicted body weight and the
actual body weight on a particular day. The formula for
predicted body weight was derived by simple regression
using the body weight data for the control group. The
following formula was derived: Y = a + kx, where
Y = body weight change (loss or gain), k = daily increase
in body weight, x = day, a = starting body weight. The
daily increase in body weight (k) was 0.251 for the IL4+/+ strain and 0.242 for the IL-4 deficient mouse strain.
Diarrhea: The appearance of diarrhea was defined as
mucus/fecal material adherent to anal fur. The presence
or absence of diarrhea was scored as either 1 or 0 respectively. The cumulative score for diarrhea was calculated
by adding the score for each day and dividing this number by the number of days of exposure.
Rectal bleeding: The appearance of rectal bleeding was
defined as diarrhea containing blood or mucus or frank
rectal bleeding. The final score for rectal bleeding was
calculated as described for diarrhea.
Histopathological examination and scoring
Tissue specimens were fixed in 10% neutral formalin and
embedded in paraffin. Colons were divided into right,
transverse and left and specimens stained with haematoxylin and eosin. Histological examination was performed on serial sections. Three serial sections of five to six
different sites of the colon (accounting for up to 18 sections per mouse) were examined and the most affected
part scored blindly using a standard 12-point histological
scoring system.32
Immunofluorescent staining
The specimens were fixed using cold ethanol,33 embedded in paraffin and 5-␮ sections cut on to gelatin-coated
slides. Immunoglobulin-producing cells were detected by
immunofluorescence as previously described.34 Briefly,
tissues were dewaxed, rehydrated and then incubated
with goat anti-mouse IgA (alpha heavy chain specific)
(Seralab, Crawley Down, Sussex, UK), 1/60; fluoresceinconjugated rabbit affinity-purified antibody to mouse
IgG1 (Cappel-Organon Teknika Corporation, Durham,
NC, USA) used in dilution, 1/100; fluorescein-conjugated
rabbit affinity-purified antibody to mouse IgG2a (CappelOrganon Teknika Corporation), 1/25; fluorescein-conjugated rabbit affinity-purified antibody to mouse IgG2b
(Cappel-Organon Teknika Corporation) used at dilution,
1/25 and fluorescein-conjugated rabbit affinity-purified
Genes and Immunity
antibody to mouse IgG3 (Cappel-Organon Teknika
Corporation), 1/40. Anti-goat immunoglobulin TRITC
conjugated (ATAB Atlantic Antibodies) was used at a
dilution of 1/100 as a secondary antibody.
Following each 20-min incubation, excess reagent was
removed and the slides rinsed in phosphate-buffered
saline (PBS). Finally slides were rinsed for 1 h in PBS and
mounted using 90% glycerol in PBS. The number of Igcontaining cells in the intestine were counted using a 40×
objective and 10× eyepieces using a Zeiss Axioskop 20
microscope with incident light illumination. Each section
of the intestine was scanned from the tips of the villi to
the base of the mucosa, and cells were counted in a total
of 30 such scans. The average number of cells per scan
was expressed as cells per linear cm of intestine by multiplying by 30.3, a correction factor determined from the
field width of 330 ␮m, as previously described.35
Enzyme-linked immunoabsorbent assay (ELISA) for
detection of immunoglobulin levels in the serum
Activated polyvinyl chloride immuno-assay plates were
coated with 1:2000 dilution of goat anti-mouse IgG (H+E)
(Caltag Immunodiagnostics, Burlingame, USA) in carbonate buffer for detection of immunoglobulin subtypes
in the serum and with 1:5000 dilution of goat anti-mouse
unlabelled antibodies in carbonate buffer as a standard.
Plates were stored overnight at 4°C, were washed five
times with PBS in 0.04% Tween 20. Skim milk powder
(4% in PBS, 100 ␮l per well) (Carnation, Australia) was
used for blocking for 1 h at 37°C. After washing the plates
five times with PBS in 0.04% Tween 20, two-fold serial
dilutions of the samples were performed starting from
1:1000, added at 50 ␮l per well and the wells were incubated at 37°C for 2 h. Goat anti-mouse biotinylated antibodies were diluted in PBS in 0.04% Tween 20 at 1:2000
for IgG1, 1:500 for IgG2a and 1:250 for IgG2b and were
added to the plates at 50 ␮l per well for 1 h. After washing with PBS in 0.04% Tween 20 five times, streptavidin
alkaline phosphatase (Amersham International, Buckinghamshire, UK) 1:1000 in PBS was added at 50 ␮l per
well and the plates were incubated for 1 h. Plates were
washed with PBS in 0.04% Tween 20, five times and substrate solution was added at 50 ␮l per well. Absorbance
was read at 405–490 nm. Samples were blinded for determination of immunoglobulin levels.
In situ hybridization for mouse IL-10
In situ hybridization was carried out according to the
method described by Pyke et al.36
IL-4+/+ and IL-4−/− mice were exposed to 2.5% and
5% DSS for 8 days. Colons were removed, washed in normal saline and then fixed in periodate-lysine-paraformaldehyde fixative for 3 h. Prior to embedding, tissue was
transferred to 20% sucrose for 3–4 h and to 7% sucrose
overnight. Tissues were embedded in paraffin. 3-␮ sections were cut on gelatin-coated, RNAase-free slides,
under RNAase-free conditions and were heated to 60°C
for 60 min.
Pre-hybridization treatment of tissue: Paraffin was removed
by immersing the slides twice for 5 min in fresh xylene.
The tissue was rehydrated by passing it through a series
of graded ethanol concentrations: 100, 100, 95, 95, 70 followed by PBS twice for 5 min. After treating the sections
with 0.2 M HCL for 20 min and washing them twice for
DSS-induced colitis is ameliorated in IL-4 deficient mice
L Stevceva et al
5 min in PBS, tissue was treated with proteinase K
(50 ␮g/ml) at 37°C for 30 min to improve the penetration
of the probe. Following washing in PBS (twice for 5 min),
sections were placed in 4% paraformaldehyde for 20 min
to stop residual proteinase K and RNAse activity. Tissues
were washed twice for 5 min in PBS and acetylated in
acetic anhydride (0.2% in 100 mm triethanolamine) in
order to reduce the electrostatic binding of the probe to
the section by acetylating positively charged amino
groups. Sections were dehydrated through a series of
graded ethanol steps (PBS, 70% ethanol, 95%, 95%, 100%
and 100%) and air-dried before applying the probe.
Hybridization: One ␮g of the IL-10 sense and antisense
riboprobes were diluted in 50% formamide, 1× Denhart
solution, 0.3 m NaCl, 5 × 10−4 EDTA, 0.01 m Tris HCL,
5 × 10−3m NaH2PO4, 10% dextran sulphate and transfer
RNA 1 ␮g/ml (Sigma Chemical Co, St Louis, USA). To
ensure a uniform solution, the mixture was heated at
80°C for 3 mins and cooled on ice. The probe was
applied, and the slides were incubated at 42°C overnight.
High-stringency washes were performed in a hybridization solution at 50 C twice for 1 h. The sections were
then washed in NTE (NaCl 0.5 m, Tris pH7.2 10 mm,
EDTA 1 mm) at 37°C for 30 min before treating them with
RNAse A, 20 ␮g/ml (Pharmacia, Uppsala, Sweden) in
NTE at 37°C to remove the unbound probe. To remove
the RNAse, two subsequent washes in NTE at 37°C were
performed and the sections were washed in 0.1 × SSc for
30 min.
Detection of digoxigenin-labelled probes: After a brief wash
in buffer 1 (0.1 mol/l maleic acid in 0.15 mol/l NaCl, pH
7.5), sections were incubated at room temperature for 30
min in buffer 2 (blocking stock solution 1:10 in buffer 1).
Anti-digoxigenin antibody conjugate (anti-digoxigeninAP Fab fragments: Boehringer, Mannheim Gmbh,
Mannheim, Germany) 1:5000 in buffer 2 was applied for
15 min at room temperature. Sections were equilibrated
in buffer 3 (Tris-HCL 100 mmol/l, NaCl 100 mmol/l and
MgCl2 50 mmol/l, pH 9.5) for 2 min before applying the
colour substrate: 33.8 ␮l 4-nitro-blue tetrazolium chloride
(NTE) (Boehringer Meinheim) and 35 ␮l 5-bromo-4chhloro-3-indolyl-phosphate (x-phosphate) (Boehringer
Mannheim) in 10 ml buffer 3. Slides were incubated overnight in the dark and at room temperature. Excess colour
substrate was removed by washing in buffer 4 (Tris-HCL,
10 mMol; EDTA 1 mMol, pH 8.0) and rinsing in tap
water. Sections were counterstained in Kernechtrof solution (0.2 g nuclear fast red and 10 g aluminium sulphate
in 200 ml distilled water)37 overnight, then washed in tap
water and air-dried. After immersion in xylene, the slides
were mounted using Histoclad (Clay Adams, Parsippany, NJ, USA).
IFN␥ quantification
IFN␥ contents were measured in tissue sonicates38 from
the inflamed colons after 6 days of DSS ingestion. Two
centimeters of the distal end of the rectum were taken,
thoroughly washed and placed in 600 ␮l of serum-free
RPMI 1640. Samples were stored at −70°C until assay.
Tissues were thawed, minced and sonicated for 60 sec
and centrifuged for 10 min at 3200 rpm. Supernatants
were collected, blinded and assayed for IFN␥ contents by
ELISA. Briefly, IFN␥ was measured on commercial
ELISA plates (R&D Systems, USA) pre-coated with
monoclonal anti-mIFN␥ according to manufacturer’s
instructions. Sensitivity of the assay was less than
2 pg/ml.
315
Statistical analysis
Disease activity index was derived by using correlation
coefficient to correlate clinical signs with the histological
score as previously described in detail.13 Disease activity
index and IFN␥ levels in tissues of the IL-4 deficient mice
and the wild-type strain were compared by using
unpaired, two-tailed Student’s t-test. P values of below
0.05 were considered to be significant. Histological scores
were compared by using Mann–Whitney U test for nonparametric data.
Acknowledgements
We would like to thank Dr Andrzej Wozniak for his
tremendous support and Aulikki Koskinnen from the
Division of Biochemistry and Molecular Biology, John
Curtin School of Medical Research that performed ELISA
for immunoglobulins in the serum. We are very grateful
to Prof Owen Dent from The Australian National University for his advice on the statistical analysis of the data.
References
1 Mosmann TR, Coffman RL. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional
properties. Annu Rev Immunol 1989; 7: 145–173.
2 Parish CR. The relationship between humoral and cell-mediated
immunity. Transplant Rev 1972; 13: 35–66.
3 Sher A, Coffman RL. Regulation of immunity to parasites by T
cells and T cell-derived cytokines. Annu Rev Immunol 1992; 10:
385–409.
4 Swain SL, Weinberg AD, English M, Huston G. IL-4 directs the
development of Th2-like helper effectors. J Immunol 1990; 145:
3796–3806.
5 Le Gros G, Ben-Sasson SZ, Seder R, Finkelman FD, Paul WE.
Generation of interleukin 4 (IL-4)-producing cells in vivo and
in vitro: IL-2 and IL-4 are required for in vitro generation of IL4- producing cells. J Exp Med 1990; 172: 921–929.
6 Hsieh CS, Heimberger AB, Gold JS, O’Garra A, Murphy KM.
Differential regulation of T helper phenotype development by
interleukins 4 and 10 in an alpha beta T-cell-receptor transgenic
system. Proc Natl Acad Sci USA 1992; 89: 6065–6069.
7 Seder RA, Paul WE, Davis MM, Fazekas de St Groth B. The
presence of interleukin 4 during in vitro priming determines
the lymphokine-producing potential of CD4+ T cells from T cell
receptor transgenic mice. J Exp Med 1992; 176: 1091–1098.
8 Stevceva L, Pavli P, Doe WF, Husband A, Ramsay A. The severity of the dextran sulphate sodium-induced colitis is significantly reduced in interleukin 4 deficient mice. Gastroenterology
1996; 10: A1019.
9 Mizoguchi A, Mizoguchi E, Bhan AK. The critical role of
interleukin 4 but not interferon gamma in the pathogenesis of
colitis in T-cell receptor alpha mutant mice. Gastroenterology
1999; 116: 320–326.
10 Iijima H, Takahashi I, Kishi D et al. Alteration of interleukin 4
production results in the inhibition of T helper type 2 cell-dominated inflammatory bowel disease in T cell receptor alpha chaindeficient mice. J Exp Med 1999; 190: 607–615.
11 Dohi T, Fujihashi K, Rannert PD et al. Hapten-induced colitis is
associated with colonic patch hypertrophy and T helper cell 2type responses. J Exp Med 1999; 189: 1169–1180.
12 Boirivant M, Fuss IJ, Chu A, Strober W. Oxazolone colitis: a
Genes and Immunity
DSS-induced colitis is ameliorated in IL-4 deficient mice
L Stevceva et al
316
13
14
15
16
17
18
19
20
21
22
23
24
25
murine model of T helper cell type 2 colitis treatable with antibodies to interleukin 4. J Exp Med 1998; 188: 1929–1939.
Stevceva L, Pavli P, Buffinton G, Wozniak A, Doe WF. Dextran
sodium sulphate-induced colitis activity varies with mouse
strain but develops in lipopolysaccharide-unresponsive mice. J
Gastroenterol Hepatol 1999; 14: 54–60.
Okayasu I, Hatakeyama S, Yamada M et al. A novel method in
the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology 1990; 98: 694–702.
Yamada M, Ohkusa T, Okayasu I. Occurrence of dysplasia and
adenocarcinoma after experimental chronic ulcerative colitis in
hamsters induced by dextran sulphate sodium. Gut 1992; 33:
1521–1527.
Tamaru T, Kobayashi H, Kishimoto S et al. Histochemical study
of colonic cancer in experimental colitis of rats. Dig Dis Sci 1993;
38: 529–537.
Stevceva L, Buffinton GD, Wozniak A, Doe WF. Disease activity
and histopathological changes in the chronic dextran sulphate
sodium (DSS)-induced colitis resemble those in chronic human
inflammatory bowel disease (IBD). J Gastroenterol Hepatol 1994;
9: 5, A:105.
Kuhn R, Rajewsky K, Muller W. Generation and analysis of
interleukin-4 deficient mice. Science 1991; 254: 707–710.
Kopf M, Le Gros G, Bachmann M et al. Disruption of the murine
IL-4 gene blocks Th2 cytokine responses. Nature 1993; 362:
245–248.
Mestecky, J. The common mucosal immune system and current
strategies for induction of immune responses in external
secretions. J Clin Immunol 1987; 7: 265–276.
van der Heijden PJ, Stok W, Bianchi AT. Contribution of
immunoglobulin-secreting cells in the murine small intestine to
the total ‘background’ immunoglobulin production. Immunology
1987; 62: 551–555.
Kuhn R, Lohler J, Rennick D, Rajewsky K, Muller W. Interleukin-10-deficient mice develop chronic enterocolitis. Cell 1993; 75:
263–274.
Duchmann R, Schmitt E, Knolle P, Meyer zum Buschenfelde
KH, Neurath M. Tolerance towards resident intestinal flora in
mice is abrogated in experimental colitis and restored by treatment with interleukin-10 or antibodies to interleukin-12. Eur J
Immunol 1996; 26: 934–938.
Tomoyose M, Mitsuyama K, Ishida H, Toyonaga A, Tanikawa
K. Role of interleukin-10 in a murine model of dextran sulfate
sodium- induced colitis. Scand J Gastroenterol 1998; 33: 435–440.
Egger B, Bajaj-Elliott M, MacDonald TT et al. Characterisation
of acute murine dextran sodium sulphate colitis: cytokine profile
and dose dependency. Digestion 2000; 62: 240–248.
Genes and Immunity
26 Bergstedt-Lindqvist S, Fernandez C, Severinson E. A synergistic
polyclonal response to dextran sulphate and lipopolysaccharide:
immunoglobulin secretion and cell requirements. Scand J Immunol 1981; 15: 439–448.
27 Isakson PC, Pure E, Vitetta ES, Krammer PH. T cell-derived B
cell differentiation factor(s). Effect on the isotype switch of
murine B cells. J Exp Med 1982; 155: 734–748.
28 Stevceva L, Pauli P, Husband A et al. Eosinophilia is attenuated
in experimental colitis induced in IL-5 deficient mice. Genes
Immun 2000; 1: 213–218.
29 Dieleman LA, Palmen MJ, Akol H et al. Chronic experimental
colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines. Clin Exp Immunol 1998; 114:
385–391.
30 Obermeier F, Kojouharoff G, Hans W et al. Interferon-gamma
(IFN-gamma)- and tumour necrosis factor (TNF)-induced nitric
oxide as toxic effector molecule in chronic dextran sulphate
sodium (DSS)-induced colitis in mice. Clin Exp Immunol 1999;
116: 238–245.
31 Andres PG, Beck PL, Mizoguchi E et al. Mice with a selective
deletion of the CC chemokine receptors 5 or 2 are protected
from dextran sodium sulfate-mediated colitis: lack of CC
chemokine receptor 5 expression results in a NK1.1+ lymphocyte-associated Th2-type immune response in the intestine. J
Immunol 2000; 164: 6303–6312.
32 Saverymuttu SH, Camilleri M, Rees H et al. Indium 111-granulocyte scanning in the assessment of disease extent and disease
activity in inflammatory bowel disease. A comparison with
colonoscopy, histology, and fecal indium 111-granulocyte
excretion. Gastroenterology 1986; 90: 1121–1128.
33 Sainte-Marie G. A parrafin embedding technique for studies
employing immunofluorescence. J Histochem Cytochem 1962; 10:
250–256.
34 Sheldrake RF, Husband AJ, Watson DL, Cripps AW. Selective
transport of serum-derived IgA into mucosal secretions. J Immunol 1984; 132: 363–368.
35 Husband AJ, Gowans JL. The origin and antigen-dependent distribution of IgA-containing cells in the intestine. J Exp Med 1978;
148: 1146–1160.
36 Pyke C, Kristensen P, Ralfkiaer E et al. Urokinase-type plasminogen activator is expressed in stromal cells and its receptor in
cancer cells at invasive foci in human colon adenocarcinomas.
Am J Pathol 1991; 138: 1059–1067.
37 Luna L. Manual of Histologic Staining Methods of the Armed Forces
Institute of Pathology. McGraw-Hill Book Company, 1968, p 89.
38 Staats HF, Lausch RN. Cytokine expression in vivo during
murine herpetic stromal keratitis. Effect of protective antibody
therapy. J Immunol 1993; 151: 277–283.