IL-6 Is Required For The Development of Th1 Cell-Mediated Murine Colitis

IL-6 Is Required for the Development of Th1 Cell-Mediated Murine Colitis -- Yamamoto et al.
164 (9): 4878 -- The Journal of Immunology
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The Journal of Immunology, 2000, 164: 4878-4882. This Article

Copyright © 2000 by The American Association of Immunologists
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Mitsunari Yamamoto*, Kazuyuki Yoshizaki , Tadamitsu Kishimoto and Hiroaki
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Abstract
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Proinflammatory cytokines have been demonstrated to play a crucial role in the
Abstract
pathogenesis of Crohn’s disease. Among those cytokines, strong expression of IL-6 has Introduction
been repeatedly demonstrated. To examine the role for IL-6 in the pathogenesis of Crohn’s Materials and Methods
disease, we introduced anti-IL-6R mAb to a murine model of colitis. Colitis was induced in Results
C.B-17-scid mice transferred with CD45RBhigh CD4+ T cells from BALB/c mice. Anti-IL- Discussion
6R mAb or rat IgG was administered weekly after T cell transfer. ICAM-1 and VCAM-1 References
expression were analyzed by immunohistochemistry. Colonic cytokine expression was
determined by RT-PCR. Mice treated with mAb showed normal growth, whereas controls lost weight. The average colitis
score was 0.64 for mAb-treated mice and 1.80 for controls. T cell expansion in treated mice was less remarkable than in
the controls. Colonic ICAM-1 and VCAM-1 expression were markedly suppressed by mAb. IFN- , TNF- , and IL-1ß
mRNA were reduced by the treatment. The results presented here show a crucial role for IL-6 in the pathogenesis of
murine colitis and suggest a therapeutic potential of anti-IL-6R mAb for treatment of human Crohn’s disease.
http://www.jimmunol.org/cgi/content/full/164/9/4878 (1 of 16)7/11/2006 7:47:55 AM

IL-6 Is Required for the Development of Th1 Cell-Mediated Murine Colitis -- Yamamoto et al. 164 (9): 4878 -- The Journal of Immunology
Introduction
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3
Efforts have been made to elucidate the pathogenesis of Crohn’s disease (CD), but the Abstract
mystery remains unsolved. The problem is that CD affects young adults, and there is no Introduction
satisfactory therapy. Whatever may trigger the initial event, there is substantial evidence Materials and Methods
that the chronic intestinal inflammation may be shaped by Th1 cell activation and Results
subsequent overproduction of proinflammatory cytokines such as IL-1ß, TNF- , and IL-6 Discussion
(1). References
Various immunosuppressive therapies have been shown to benefit patients with CD. Corticosteroids are the most
commonly used, but they only relieve the symptoms, without improvement of endoscopically visible lesions (2).
Azathioprine and other immunosuppressive medications are also used, but their effectiveness in inducing remission
remains controversial, and their utility is limited because of their side effects (3). Recently, anti-TNF- mAb has been
shown to be useful in patients with steroid-unresponsive CD (4). This might prove a great boon to patients, but its safety
and efficacy in long-term treatment have not yet been established.
It has been shown that IL-6 is present at very high levels in both serum and intestinal tissue from patients with CD (5).
The soluble form of IL-6R (sIL-6R) is also elevated (6), and this is known to potentiate IL-6 activity by forming IL-6-sIL-
6R complexes that bind the signaling-competent transmembrane receptor gp130 (7). IL-6 is known as a major modulator
of inflammation (8). Therefore, IL-6 might play a crucial role in the pathogenesis of CD, and we considered that blocking
IL-6 signaling should bring great benefits to patients with CD.
Congenic scid mice given CD45RBhigh CD4+ T cells from normal mice develop Th1 cell-mediated colitis called wasting
disease which resembles human CD (9). In this report, we utilized this model to examine the therapeutic potential of anti-
IL-6R mAb (MR16-1), which blocks both the transmembrane and soluble forms of IL-6R.
Materials and Methods

Top
Mice
Abstract
Introduction
BALB/c and C.B-17 scid mice were purchased from Charles River Breeding Laboratories Materials and Methods
(Shiga, Japan). Before use, the mice were maintained in the specific pathogen-free animal Results
facility at the Institute of Experimental Animal Sciences, Osaka University Medical School. Discussion
BALB/c mice were used at 7–9 wk, C.B-17 scid mice at 8–12 wk of age. References
Anti-IL-6R mAb
Anti-IL-6R mAb, which is rat IgG1 mAb against murine IL-6R, was provided by Chugai Pharmaceutical Co. (Gotemba,
Japan).
Purification of splenocyte subsets
All procedures were conducted under aseptic conditions. CD4+ T cells were enriched from single-cell suspensions of

BALB/c splenocytes in RPMI 1640 containing 2% FCS using an anti-L3T4 (anti-CD4) MACS magnetic separation
system (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instruction. Briefly, splenocytes were incubated
with anti-CD4 magnetic microbeads for 20 min at 40°C, washed, and then enriched by passage through magnetic flow
columns. Enriched CD4+ T cells (96–97% purity, confirmed by FACS) were then labeled with PE-conjugated anti-mouse
CD4 (L3T4) (RM4-5, rat IgG2a; PharMingen, San Diego, CA) and FITC -conjugated anti-CD45RB (16A, rat IgG2a;
PharMingen) and sorted into CD45RBhigh (brightest staining 40–50%) and CD45RBlow (dullest staining 13–17%)
fractions on a FACS Vantage (Becton Dickinson, Sunnyvale, CA).
Cell transfer and administration of anti-IL-6R mAb
Each C.B-17 scid mouse was injected i.p. with 100 µl of PBS containing 4 x 105 CD45RBhigh CD4+ T cells from normal
BALB/c mice. Mice were also given either rat IgG or anti-IL-6R mAb by i.p. injection (2 mg at the time of T cell transfer
and 1 mg weekly up to 8 wk).
Histological and immunohistological analyses
Colons were removed from recipient mice 8 wk after T cell reconstitution and were frozen in OCT compound at -80°C.
For histological analyses, 6-µm sections were fixed in 10% Formalin and stained with hematoxylin and eosin. Colitis was
graded on a scale of 0–3 as follows: 0, minimal, indistinguishable from normal BALB/c mice; 1, mild; 2, moderate, low to
intermediate degree of leukocytic infiltration and epithelial hyperplasia; and 3, severe, extensive leukocytic infiltration,
loss of goblet cells, and marked epithelial hyperplasia. Histological evaluation was conducted in a blinded fashion. For
immunohistochemistry, staining of the sections was performed using an immunofluorescence staining method. Six-
micrometer sections were incubated with PE-conjugated anti-ICAM-1 (BE29G1, rat IgG2a; Leico Technologies, St.
Louis, MO) and PE-conjugated anti-CD4. For staining of VCAM-1, anti-VCAM-1 (MK2, rat IgG1; Immunotech,
Marseille, France) and FITC-conjugated anti-rat IgG (Vector Laboratories, Burlingame, CA) were used. Fluorescence
micrographs were taken by using a LSM 310 microscope (Zeiss, Oberkochen, Germany).
RT-PCR for cytokine expression
The expression of IFN- , TNF- , IL-1ß, IL-4, IL-6, IL-10, and TGF-ß were determined by RT of total RNA, followed by
PCR. Total RNA was isolated from the colonic tissue by the acid guanidium thiocyanate-phenol chloroform extraction
method. cDNAs were synthesized by incubating 5 µg of total RNA with 600 U of Moloney murine leukemia virus reverse
transcriptase (Life Technologies, Gaithersburg, MD) in the presence of 0.05 U of oligo(dT) primers (Pharmacia,
Piscataway, NJ), 3 mg of acetylated BSA (Life Technologies), and 40 U of RNase inhibitor (Promega, Madison, WI) in a
volume of 30 ml for 1 h at 37°C and then for 5 min at 96°C to stop the reaction. PCR of the cDNA was performed in a
final volume of 50 µl containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001% (w/v) gelatin, 0.2 mM
of dNTP (Pharmacia), 1.25 U of AmpliTaq DNA polymerase (Perkin-Elmer, Branchburg, NJ), and each primer at 0.5 µM,
using the geneAmp 2400 PCR system (Perkin-Elmer). The amplification cycles were 94°C for 30 s, 60°C for 1 min, and
72°C for 1 min. The PCR products were separated by electrophoresis on 1.5% agarose gel after 25–33 cycles for IFN- ,
TNF- , IL-1ß, IL-4, IL-6, and IL-10, or 20–22 cycles for gylceraldehyde-3-phosphate dehydrogenase (G3PDH), and were
visualized by ethidium bromide staining. Amplification of G3PDH served as a control for sample loading and integrity.
The following primers were used: IFN- sense, 5'-GAA-AGC-CTA-GAA-AGT-CTG-AAT-AAC-T-3'; IFN- antisense,
5'-ATC-AGC-AGC-GAC-TCC-TTT-TCC-GCT-T-3'; TNF- sense, 5'-ATG-AGC-ACA-GAA-AGC-ATG-ATC-CGC-3';
TNF- antisense, 5'-CCA-AAG-TAG-ACC-TGC-CCG-GAC-TC-3'; IL-1ß sense, 5'-ATG-GCA-ACT-GTT-CCT-GAA-
CTC-AAC-T-3'; IL-1ß antisense, 5'-CAG-GAC-AGG-TAT-AGA-TTC-TTT-CCT-TT-3'; IL-4 sense, 5'-ATG-GGT-CTC-
AAC-CCC-CAG-CTA-GT-3'; IL-4 antisense, 5'-GCT-CTT-TAG-GCT-TTC-CAG-GAA-GTC-3'; IL-6 sense, 5'-GAT-

GCT-ACC-AAA-CTG-GAT-ATA-ATC-3'; IL-6 antisense, 5'-GGT-CCT-TAG-CCA-CTC-CTT-CTG-TG-3'; IL-10

sense, 5'-ACC-TGG-TAG-AAG-TGA-TGC-CCC-AGG-CA-3'; IL-10 antisense, 5'-CTA-TGC-AGT-TGA-TGA-AGA-
TGT-CAA-A-3'; G3PDH sense, 5'-CGG-TGC-TGA-GTA-TGT-CGT-GGA-GTC-T-3'; and G3PDH antisense; 5'-GTT-
ATT-ATG-GGG-GTC-TGG-GAT-GGA-A-3'. TGF-ß was analyzed using quantitative competitive PCR mouse TGF-ß
(Maxim Biotech, South San Francisco, CA).
Isolation of lamina propria lymphocytes (LPLs) and analysis by FACS
LPLs were isolated from the colon as described previously (10) and incubated with PE-conjugated anti-ICAM-1, FITC-
conjugated anti-CD4 (RM4-5, rat IgG2a; PharMingen), anti-Mac-1 (M1/70, rat IgG2b; PharMingen), and FITC-
conjugated anti-rat IgG. Flow cytometric analysis was performed on a FACScan (Becton Dickinson).
Statistical analysis
Data are expressed as means ± SEM. Results were analyzed using the t test. A p value of <0.05 was considered to be
statistically significant.
Results
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Anti-IL-6R mAb prevents wasting disease
Abstract
Introduction
To test whether blocking IL-6 signaling could prevent wasting disease, scid mice restored Materials and Methods
with CD45RBhigh CD4+ T cells were treated with anti-IL-6R mAb, control rat IgG, or PBS Results
alone. As shown in Fig. 1 , the mice restored with CD45RBhigh CD4+ T cells and given Discussion
PBS alone started to lose body weight by 3 wk after T cell transfer (wasting disease), References
whereas mice restored with CD45RBhigh CD4+ T cells and treated with anti-IL-6R mAb
experienced weight gain similar to that seen in normal scid mice. The final average weight of the mice given control rat
IgG was indistinguishable from that of the mice given PBS alone. Although it seemed that rat IgG had partial protection,
it was not statistically significant. The average body weight at the end of observation was: normal scid; 110.5 ± 0.9%, anti-
IL-6R mAb-treated mice; 108.9 ± 1.9%, mice given PBS alone; and 88.7 ± 3.0%, control rat IgG treatment; 93.3 ± 5.0%
of their initial weight. We also examined the mice restored with both CD45RBhigh and CD45RBlow CD4+ T cells which
were protected from wasting disease. The change of body weight was very similar to the normal scid mice and the mice
treated with anti-IL-6R mAb (data not shown).

FIGURE 1. Anti-IL-6R mAb prevents wasting disease. C.B-17 scid

mice were reconstituted with 4 x 105 CD45RBhigh CD4+ T cells and
treated weekly with anti-IL-6R mAb (•, n = 11), PBS ( , n = 16), or
control rat IgG ( , n = 5) for up to 8 wk. Body weights of these mice
were compared with normal unreconstituted scid mice ( , n = 8). Data
represent the average ± SEM.
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Anti-IL-6R mAb treatment prevents the development of colitis
The mice were sacrificed 8 wk after T cell transfer, and the colons were removed for histological evaluation. In gross
examination, colons of the mice restored with CD45RBhigh CD4+ T cells and given PBS alone were obviously enlarged,
and the walls were diffusely thickened, whereas colons from the mice treated with anti-IL-6R mAb were almost
indistinguishable from normal unreconstituted scid mice. Further observation under the microscope clearly demonstrated
the therapeutic effect of anti-IL-6R mAb on the development of colitis. Thirteen of 16 mice restored with CD45RBhigh
CD4+ T cells and given PBS alone showed moderate to severe colitis, with an average score of 2.3 ± 0.2 (Fig. 2 ),
characterized by marked epithelial hyperplasia, extensive leukocyte infiltration, and goblet cell depletion. Inflammatory
infiltrates, which consisted mainly of mononuclear cells, were observed in the lamina propria. The mice restored with
CD45RBhigh CD4+ T cells and given control rat IgG showed similar results with an average colitis score of 1.8 ± 0.6 (Fig.
3 B). In contrast, treatment with anti-IL-6R mAb provided significant protection from colitis: 7 of 11 had mild colitis and
4 of 11 had minimal pathological changes, yielding an average colitis score of 0.6 ± 0.2 (Fig. 3 C).
FIGURE 2. Anti-IL-6R mAb prevents colitis in scid mice restored with

CD4+ CD45RBhigh T cells. Colons were removed from scid mice 8–9 wk
after T cell transfer and stained with hematoxylin and eosin. Pathology
was graded on a scale of 0–3. Data represent the average ± SEM for the
group. The average score for the mice treated with anti-IL-6R mAb was
significantly different from the mice given PBS alone (p < 0.0001) or rat
IgG-treated mice (p < 0.02). Statistical analysis was performed using the
t test.

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FIGURE 3. Prevention of colitis in mice treated with anti-IL-6R mAb.

A, Normal unreconstituted scid mice. B, Severe colitis (grade 3) in mice
reconstituted with 4 x 105 CD45RBhigh T cells and given control rat IgG.
C, Prevention of colitis in mice restored with 4 x 105 CD45RBhigh T cells
View larger version (57K): and treated with anti-IL-6R mAb. Colonic sections were stained with
[in this window] hematoxylin and eosin and photographed at x40 magnification.
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Anti-IL-6R mAb suppressed the expression of ICAM-1 and VCAM-1
Cell adhesion between vascular endothelial cells and leukocytes is an important process for inflammatory changes. We
examined the expression of ICAM-1 and VCAM-1 in the colons by means of immunohistochemistry. In the mice restored
with CD45RBhigh CD4+ T cells and given control rat IgG, there was a massive accumulation of ICAM-1-positive cells in
the lamina propria. Vascular endothelial cells were also positive for ICAM-1 (Fig. 4 b). In contrast, the immunoreactivity
for ICAM-1 was less intense and no vascular staining was seen in the mice restored with CD45RBhigh CD4+ T cells and
treated with anti-IL-6R mAb (Fig. 4 c). VCAM-1 was positive in vascular endothelial cells in the mice given control rat
IgG (Fig. 4 e). Again, the expression of VCAM-1 was markedly suppressed in the mice treated with anti-IL-6R mAb
(Fig. 4 f). Analysis of isolated LPLs by FACS revealed that most of the ICAM-1+ cells were Mac-1+ and CD4- and were
most likely macrophages (data not shown).
FIGURE 4. ICAM-1 and VCAM-1 expression was suppressed by anti-

IL-6R mAb treatment. Frozen tissue sections of colons from T cell-
restored mice given control rat IgG (b and e), mice treated with anti-IL-
6R mAb (c and f), and normal scid mice (a and d) were stained by
immunofluorescence for the expression of ICAM-1 (a–c, x200
magnification) and VCAM-1 (c–e, x200 magnification).

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Anti-IL-6R mAb reduced the expansion of CD4+ T cells in the recipient scid mice
Increase of transferred T cells in the recipient scid mice was discussed in a previous report, with the scid mice that
received CD45RBhigh CD4+ T cells having more CD4+ T cells than originally injected and the majority of CD4+ T cells
being recovered from the spleens (9). To examine the effects of anti-IL-6R mAb on the expansion of T cells in the
recipients, we compared colonic infiltration of CD4+ cells by immunohistochemistry and determined the number of CD4+
T cells in the spleen. Fig. 5 clearly demonstrates marked reduction of CD4+ cells in the colon of mAb-treated mice

compared with those with colitis. As shown in Table I , the average number of splenic CD4+ T cells recovered from mice
that received CD45RBhigh CD4+ T cells and given PBS alone was 2.05 ± 0.37 x 106 and that from mice given control rat
IgG was 2.22 ± 0.54 x 106. In contrast, treatment with anti-IL-6R mAb significantly suppressed the expansion of CD4+ T
cells. The cell number in mice given anti-IL-6R mAb was 0.83 ± 0.19 x 106, which was significantly lower than in the
other groups. Therefore, anti-IL-6R mAb suppressed the expansion of both colonic and splenic CD4+ T cells.
FIGURE 5. Anti-IL-6R mAb reduced the expansion of CD4+ T cells in

the recipient scid mice. Colons from mice restored with CD45RBhigh T
cells and given control rat IgG (a) and the mice treated with anti-IL-6R
mAb (b) were stained with PE-conjugated anti-CD4. Original
magnification, x200.

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View this table: Table I. Expansion of CD4+ T cells in C.B-17 scid

[in this window] mice1
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Colonic expression of IFN- and proinflammatory cytokines were also reduced by treatment with anti-IL-6R mAb
Wasting disease is a Th1-mediated colitis, and Th1 cytokines, especially IFN- and TNF- , have been demonstrated to
play an important role in its pathogenesis (9). Therefore, by using RT-PCR, we compared cytokine expression in the
colons of the mice that developed wasting disease with the mice successfully treated with anti-IL-6R mAb. As shown in
Fig. 6 , the mice into which CD45RBhigh CD4+ T cells were transferred and given control rat IgG, which developed
wasting disease and colitis, showed strong expression of IFN- , TNF- , IL-1ß, and IL-6 mRNA, whereas the normal
unreconstituted scid mice exhibited only slight expression of those cytokines. The mice restored with both CD45RBhigh
and CD45RBlow CD4+ T cells, which were protected from the development of colitis, showed similar patterns of cytokine
expression to normal scid mice. In the colons of the mice restored with CD45RBhigh and treated with anti-IL-6R mAb,
mRNA for IFN- was almost undetectable, IL-1ß was remarkably reduced, and the level of TNF- expression was as low
as in normal scid mice and mice cotransferred with CD45RBlow CD4+ T cells. As compared with the other cytokines, the
amount of IL-6 mRNA in mAb-treated mice was considerable but slightly less than in colitic mice. Recently, colitis-
suppressing potential of CD45RBlowCD4+ T cells, T regulatory-1 cells, and their effector cytokines have been discussed
(11, 12). Therefore, we examined IL-4, IL-10, and TGF-ß. As shown in Fig. 6 , IL-10 and TGF-ß mRNA were detectable
almost equally in all experimental groups, whereas IL-4 was undetectable in any mice.

FIGURE 6. Anti-IL-6R mAb inhibits colonic expression of IFN- , TNF- , and IL-
1ß mRNA. The expression of IFN- , TNF- , IL-1ß, IL-4, IL-6, IL-10, and TGF-ß
mRNA in the colons was analyzed by RT-PCR. 1, normal scid mice; 2, scid mice
reconstituted with both CD45RBhigh and CD45RBlow CD4+ T cells; 3, scid mice
reconstituted with CD45RBhigh CD4+ T cells and treated with rat IgG; and 4, scid
mice reconstituted with CD45RBhigh CD4+ T cells and treated with anti-IL-6R mAb.
Amplification of G3PDH serves as a control for sample loading and integrity.

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Discussion
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The data in this report provide evidence that IL-6 plays a crucial role for the pathogenesis
Abstract
of murine wasting disease, and hold out hope that anti-IL-6 strategy will be effective in the Introduction
treatment of human CD. Materials and Methods
Results
Murine wasting disease is considered to be a Th1-mediated pathogenesis, as is human CD. Discussion
Treatment of wasting disease by blocking Th1 cytokines was discussed in a previous report, References
with only two doses of anti-IFN- mAb given to mice soon after T cell transfer preventing
the development of colitis for up to 12 wk, whereas continual neutralization of TNF with anti-TNF- and -ß mAb only
reduced the incidence of severe disease and anti-TNF mAb only for the first 3–4 wk had no effect, which suggested that
anti-TNF might act primarily to block TNF effector function (9). Th1 cytokine IFN- is the major physiologic activator of
macrophages and activated macrophages produce proinflammatory cytokines, including IL-1, TNF- , and IL-6.
Furthermore, IL-6 is produced by various kinds of cells following IL-1 and TNF- stimulation (13). IL-6 is considered to
play a central role in the regulation of inflammatory and immune reactions, and the requirement for IL-6 has been
demonstrated for murine models of rheumatoid arthritis and multiple sclerosis (14, 15). CD shares characteristics with
these diseases in terms of oligoclonal expansion, activation of CD4+ T cells, and participation of proinflammatory
cytokines. Therefore, it is not surprising that anti-IL-6R mAb prevented the development of wasting disease and colitis.
So far, we have not determined whether continual administration of anti-IL-6R mAb is indispensable or not. Treatment
starting the following day after T cell transfer was equally effective (data not shown). Questions such as how many doses
are required, and how much dosage is necessary remain to be answered.
Proinflammatory cytokines are known to up-regulate a spectrum of cell adhesion molecules, including ICAM-1, VCAM-
1, selectins, and integrins. In the study of human inflammatory bowel disease, ICAM-1+ inflammatory infiltrates and
venules were demonstrated to increase in parallel to the degree of inflammation, whereas VCAM-1 was not significantly
enhanced in the inflamed mucosa (16, 17). It has been shown that IL-6 in the presence of sIL-6R augmented ICAM-1
expression of endothelial cells, but did not modify the expression of VCAM-1 (8), which may explain the difference
between ICAM-1 and VCAM-1 expression in human CD. Stimulation with concomitant IL-6 and sIL-6R was required

because endothelial cells express only the gp130 signal-transducing chain and not the subunit-specific IL-6R (8).
Adhesion molecules in murine colitis appeared somewhat different from those in human CD. There was enhanced
expression of both ICAM-1 and VCAM-1 in wasting disease mice, and treatment with anti-IL-6R mAb remarkably
reduced the expression of both adhesion molecules. Reduction of expression was notable in small vessel walls as well as
in lamina propria. From the result of FACS analysis, ICAM-1+ lamina propria cells were Mac-1+ CD4- and were
considered to be macrophages. Suppression of VCAM-1 by anti-IL-6R mAb treatment was more than expected. It might
be attributable to indirect effects of the IL-6 blockade or to the difference between mice and humans.
CD40-CD40 ligand (CD40L) interactions play multifunctional roles in the immune system and are crucial for production
of IL-12 by APCs, which biases CD4+ T cell responses toward Th1 (18). Endothelial cell activation by CD40L may play
an important role not only in leukocyte recruitment through enhancement of adhesion molecule expression, but also in the
maintenance of an inflammatory loop through the increase in secretion of proinflammatory cytokines such as IL-6 (19).
Furthermore, the in vivo relevance of CD40-CD40L interaction was described in the hapten-induced experimental colitis
model (20). Effects of anti-IL-6R mAb may be exerted through blocking such an inflammatory loop, and this could
explain why anti-IL-6R mAb induced suppression of Th1 cytokine IFN- and proinflammatory cytokines, including TNF-
, IL-1ß, and IL-6.
Recently, it has been shown that chronic activation of CD4+ T cells in the presence of IL-10 gives rise to CD4+ T cell
clones with low proliferative capacity, producing high levels of IL-10, low levels of IL-2, and no IL-4 (T regulatory cells),
which prevent wasting disease (12). In our study, the levels of IL-10 seemed similar in both IL-6R mAb-treated mice and
nontreated controls. So far we cannot conclude that IL-6R mAb induced tolerance through induction of IL-10, although its
protein levels remain to be tested. In addition, TGF-ß has been demonstrated to be a key cytokine that mediates the colitis-
suppressive effect of CD45RBlow CD4+ T cells (11). Our data showed no significant increase of TGF-ß in mAb-treated
mice. Therefore, it did not appear that anti-IL-6R mAb suppressed wasting disease and colitis through the induction of
TGF-ß.
In summary, the results show the requirement of IL-6 in the development of wasting disease and colitis. Experiments
addressing the mechanism for the abrogation of colitis indicate the effects of IL-6 blockade on ICAM-1 and VCAM-1
expression, T cell proliferation, and IFN- and proinflammatory cytokine production. This study will provide a basis for
the practical application of anti-IL-6 mAb to the treatment of human CD.
Acknowledgments
We thank Chugai Pharmaceutical Company Ltd. for generously supplying rat anti-mouse IL-6R mAb (MR16-1).
Footnotes
1This work was supported by a grant-in-aid for scientific research from the Japanese Ministry of Education, Science and
Culture of Japan.
2 Address correspondence and reprint requests to Dr. Hiroaki Ito, Department of Molecular Medicine, Osaka University

Medical School, 2-2 Yamada-oka, Suita 565-0871, Japan.
3Abbreviations used in this paper: CD, Crohn’s disease; sIL-6R, soluble IL-6R; G3PDH, glyceraldehyde-3-phosphate
dehydrogenase; LPL, lamina propria lymphocyte; CD40L, CD40 ligand.
Received for publication August 23, 1999. Accepted for publication February 22, 2000.
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IL-6 Is Required For The Development of Th1 Cell-Mediated Murine Colitis

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IL-6 Is Required for the Development of Th1 Cell-Mediated Murine Colitis -- Yamamoto et al.

164 (9): 4878 -- The Journal of Immunology

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The Journal of Immunology, 2000, 164: 4878-4882. This Article

1 Alert me if a correction is posted

http://www.jimmunol.org/cgi/content/full/164/9/4878 (1 of 16)7/11/2006 7:47:55 AM

Materials and Methods

Purification of splenocyte subsets

http://www.jimmunol.org/cgi/content/full/164/9/4878 (2 of 16)7/11/2006 7:47:55 AM

Cell transfer and administration of anti-IL-6R mAb

Histological and immunohistological analyses

RT-PCR for cytokine expression

http://www.jimmunol.org/cgi/content/full/164/9/4878 (3 of 16)7/11/2006 7:47:55 AM

GCT-ACC-AAA-CTG-GAT-ATA-ATC-3'; IL-6 antisense, 5'-GGT-CCT-TAG-CCA-CTC-CTT-CTG-TG-3'; IL-10

Isolation of lamina propria lymphocytes (LPLs) and analysis by FACS

http://www.jimmunol.org/cgi/content/full/164/9/4878 (4 of 16)7/11/2006 7:47:55 AM

FIGURE 1. Anti-IL-6R mAb prevents wasting disease. C.B-17 scid

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Anti-IL-6R mAb treatment prevents the development of colitis

FIGURE 2. Anti-IL-6R mAb prevents colitis in scid mice restored with

View larger version (35K):

http://www.jimmunol.org/cgi/content/full/164/9/4878 (5 of 16)7/11/2006 7:47:55 AM

FIGURE 3. Prevention of colitis in mice treated with anti-IL-6R mAb.

Anti-IL-6R mAb suppressed the expression of ICAM-1 and VCAM-1

FIGURE 4. ICAM-1 and VCAM-1 expression was suppressed by anti-

View larger version (19K):

http://www.jimmunol.org/cgi/content/full/164/9/4878 (6 of 16)7/11/2006 7:47:55 AM

FIGURE 5. Anti-IL-6R mAb reduced the expansion of CD4+ T cells in

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View this table: Table I. Expansion of CD4+ T cells in C.B-17 scid

http://www.jimmunol.org/cgi/content/full/164/9/4878 (7 of 16)7/11/2006 7:47:55 AM

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http://www.jimmunol.org/cgi/content/full/164/9/4878 (9 of 16)7/11/2006 7:47:55 AM

Medical School, 2-2 Yamada-oka, Suita 565-0871, Japan.

http://www.jimmunol.org/cgi/content/full/164/9/4878 (10 of 16)7/11/2006 7:47:55 AM

D. Yen, J. Cheung, H. Scheerens, F. Poulet, T. McClanahan, B. Mckenzie, M.

M. Carol, N. Borruel, M. Antolin, M. Llopis, F. Casellas, F. Guarner, and J.-R.

M. L. Drakes, T. G. Blanchard, and S. J. Czinn

http://www.jimmunol.org/cgi/content/full/164/9/4878 (11 of 16)7/11/2006 7:47:55 AM

S Dogan, Y Beyazit, A Shorbagi, S Koklu, S Ustunel, N Guler, and A Uner

I. Rochman, W. E. Paul, and S. Z. Ben-Sasson

K. Kitamura, Y. Nakamoto, S. Kaneko, and N. Mukaida

M. Delgado, D. Pozo, and D. Ganea

L. Wang, B. Walia, J. Evans, A. T. Gewirtz, D. Merlin, and S. V. Sitaraman

http://www.jimmunol.org/cgi/content/full/164/9/4878 (12 of 16)7/11/2006 7:47:55 AM

B. Salh, K. Assi, V. Templeman, K. Parhar, D. Owen, A. Gomez-Munoz, and

D. Philippe, L. Dubuquoy, H. Groux, V. Brun, M. T. V. Chuoi-Mariot, C.

L. Boisse, M. D. V. Sickle, K. A. Sharkey, and Q. J. Pittman

P. Salmenpera, S. Hamalainen, M. Hukkanen, and E. Kankuri

T. Totsuka, T. Kanai, K. Uraushihara, R. Iiyama, M. Yamazaki, H. Akiba, H.

C. Pasare and R. Medzhitov

http://www.jimmunol.org/cgi/content/full/164/9/4878 (13 of 16)7/11/2006 7:47:55 AM

J. M. H. VAN DEN BRANDE, M. P. PEPPELENBOSCH, and S. J. H. VAN

I Monteleone, P Vavassori, L Biancone, G Monteleone, and F Pallone

R. R. Fenton, S. Molesworth-Kenyon, J. E. Oakes, and R. N. Lausch

R. G. Titus, G. K. DeKrey, R. V. Morris, and M. B. P. Soares

A. Rühl, S. Franzke, S. M. Collins, and W. Stremmel

S. V. Sitaraman, D. Merlin, L. Wang, M. Wong, A. T. Gewirtz, M. Si-Tahar,

http://www.jimmunol.org/cgi/content/full/164/9/4878 (14 of 16)7/11/2006 7:47:55 AM

A. Suzuki, T. Hanada, K. Mitsuyama, T. Yoshida, S. Kamizono, T. Hoshino,