J Korean Surg Soc 2010;79:317-325 DOI: 10.4174/jkss.2010.79.5.317
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Immunomodulatory Effects of Mesenchymal Stem Cells in a Murine Model of TNBS-Induced Colitis Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 1 University of Melbourne, Victoria, Australia, 2Center for Clinical Research, Samsung Biomedical Research Institute, 3Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 1
2
Yong Beom Cho, M.D., Min Shik Kim, M.D. , Min Jeong Kang, M.D. , 2 3 Hee Jung Shin, M.D. , Seok-Hyung Kim, M.D. , Hee Cheol Kim, M.D., Seong Hyeon Yun, M.D., Woo Yong Lee, M.D., Ho-Kyung Chun, M.D. Purpose: Mesenchymal stem cells (MSCs) are surfacing as a new method of treatment for various diseases that have poor outcome with drug treatments. In this study, we investigated the effects of MSCs in a murine intestinal inflammation model mimicking human Crohn’s disease (CD) using 2,4,5-trinitrobenzene sulfonic acid (TNBS). Methods: Colitis was induced by rectal administration of 2 mg of TNBS in 35% ethanol as experimental group compared to control group. Histological changes, surface molecules of T and B cells of the spleen and blood, and cytokine production (IFN-γ, TNF-α, IL-4, IL-6, IL-10, and IL-12) were determined among 3 groups comprised of control group, TNBS group and TNBS/MSC group. Results: In the mice treated with MSCs, there was a decrease in the wasting disease process and inflammatory histopathological changes. There was also a decrease in pro-inflammatory T-helper 1 (Th1) cytokines IFN-γ and IL-12 and T-helper 2 (Th2) cytokine IL-4. Anti-inflammatory cytokine IL-10 increased in mice treated with MSCs compared to colitic mice. The blood CD4＋CD25＋ T-regulatory cells also increased and splenic CD19 B-cells decreased. Conclusion: The results of this study suggest that MSCs may have a therapeutic effect in controlling the Th1 and Th2 mediated immune response in patients with CD and aid in tissue regeneration. (J Korean Surg Soc 2010;79:317-325) Key Words: Mesenchymal stem cell, Inflammatory bowel disease, Crohn’s disease especially activated T-lymphocytes.(1,2) T-lymphocytes secrete cytokines which regulate immune response and
INTRODUCTION
according to their function, are divided into T-helper 1 Inflammatory bowel disease (IBD) is defined as chronic
(Th1) (IL-2, interferon-γ; regulation of cell-mediated
inflammation of the bowel of unknown etiology and
immunity) and T-helper 2 (Th2) (IL-4, IL-5, IL-10, IL-13;
normally refers to Crohn’s disease (CD) and ulcerative
regulation of humoral immunity) cells.(3,4) Dysregulation
colitis. While specific causes are unknown, it is thought to
of T-lymphocytes in IBD leads to imbalance of these cyto-
be associated with the bowel’s immunological function,
kines, causing an influx of inflammatory cells such as T-lymphocytes and macrophages and subsequent tissue
Correspondence to: Ho-Kyung Chun, Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50, Irwon-dong, Gangnam-gu, Seoul 135-710, Korea. Tel: 02-34103465, Fax: 02-3410-0040, E-mail:
[email protected] Received March 10, 2010, Accepted August 24, 2010 This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2007-313-E00005).
damage.(5) Also, genetic and psychological susceptibility, altered bowel microflora and various other factors are thought to be involved in the inflammatory process.(6,7) IBD progresses either gradually over a long period or
317
318 J Korean Surg Soc. Vol. 79, No. 5 acutely over several days.(8) With lack of a definitive
to the recipient’s immune system.(15,16) Also, by forming
treatment option due to an ill-defined etiology, most pa-
chimeras, they have led to donor-specific immune tole-
tients enter a chronic disease state.(2)
rance.(17)
Current medical treatment for IBD mainly consists of
Cytotherapy using stem cells is receiving attention as the
pharmacological intervention. Rather than achieving a
next generation treatment method as it can be used in
cure, the current aim of treatment is to improve quality
rejection or incurable illnesses that current medicines
of life by inducing remission, preventing recurrence and
cannot solve.(12) Cytotherapy also can be applied to
controlling the symptoms.(9) Drugs used include amino-
various fields of medicine including cardiovascular, neuro-
salicylates (sulfasalazine, mesalamine), corticosteroid-based
logical, hematological, and immunological systems, and also
anti-inflammatories, antibiotics (metronidazole), immune
hereditary, hepatic and endocrine diseases. Utilization of
boosters (levamisole, Bacillus Calmette Guerin (BCG)), a
MSCs in the treatment of IBD requires more in-depth
mast cell stabilizer (cromolyn sodium), and allopathic medi-
research to clarify its mechanism of action. Therefore, in
cations including antidiarrheals, antispasmodics and
the present study, we examined the effect of MSCs in the
cholestyramine.(9,10) An immunosuppressant (azathioprine,
murine model of TNBS-induced colitis.
mercaptopurine, methotrexate, cyclosporine) can be used when other drug treatments are ineffective.(10) However, many of these drugs face limited use because of ineffectiveness and adverse side effects.
METHODS 1) Induction of colitis and treatments
Due to issues facing drug treatments, treatment using
Female 8-week-old BALB/c mice (weight range 20∼25
mesenchymal stem cells (MSC) is surfacing as a new method
g) were purchased from the Jackson Laboratory (Bar
of treatment. MSCs are multipotent non-hematopoietic
Harbor, ME, USA). All mice were housed in specific
progenitor cells from adult bone marrow which can
pathogen-free conditions and in accordance with institu-
differentiate into various cells that make up fat, cartilage,
tional guidelines. Colitis was induced by rectal admini-
bone, muscle, stroma, nerves, etc.(11) Currently, clinical
stration of 2 mg of 2,4,6-trinitrobenzene sulfonic acid (TNBS,
research is aimed towards achieving tissue regeneration
Sigma Chemical Co., St. Louis, MO, USA) in 35% ethanol
with MSCs.
(Merck, Germany) using a vinyl catheter positioned 4 cm
MSCs have a low expression of antigens such as HLA-
from the anus (20 mice per group). After instillation they
DR, and therefore produce less adverse immune responses
were kept vertically for 60 seconds with mice faced down
when injected in vivo.(12,13) MSCs can be genetically
for effective absorption and prevention of regurgitation of
modified, and they can produce various growth factors or
administered TNBS or ethanol. The MSC treatment group
cytokines continuously or for certain periods of time when
was treated with 5×105 cells/150μl in Dulbecco’s Phos-
injected.(14) They cannot present antigen due to absent
phate Buffered Saline (DPBS, GIBCO, NY, USA) isolated
MHC-II and co-stimulatory molecules, but they play an
from the mice inguinal fat pad, via tail vein 24 hrs after
important role in proliferation and growth of hemato-
colitis induction. The control group was treated with 35%
poietic stem cells and produce various cytokines and
ethanol (Et-OH).
regulatory molecules that regulate granulocyte, macrophage and dendritic cell production, hemopoiesis and immune response.(11,12)
2) Assessment of inflammation and histology Mice were weighed daily. Body weight loss was deter-
MSCs with low immunogenicity, immunomodulatory
mined by percentage of weight loss from baseline body
function, and ability to differentiate into various cell lineages
weight. Mice were sacrificed on each 3 (n=10) and 10 days
have not shown graft versus host disease or caused damage
(n=10) after the TNBS administration. The longitudinally
Yong Beom Cho, et al：Mesenchymal Stem Cells in TNBS-Induced Colitis
divided colons were rolled up and fixed in 4% buffered formalin. Fixed tissues were embedded in paraffin, and 4∼ 6μm sections were stained with H&E for histologic grad-
319
the authors.
3) Flow cytometric analysis and cytokine assay
ing. An experienced pathologist blinded to treatment allo-
The surface molecules of T cells were observed in the
cation scored all sections: (1) percentage of colon involved,
peripheral blood and spleen by the use of mAb directed
(2) infiltration of mononuclear cells, and (3) alteration &
to CD4, CD8a, CD19, CD25 and a control isotype conju-
erosion. The severity was scored on a scale of 0∼4 as
gated with fluorochromes (FITC, PE). All Abs were
follows: 0, absent; 1, weak; 2, moderate; 3, severe; 4, most
purchased from BD PharMingen (San Diego, CA, USA)
severe. Grading was done in a blinded fashion by one of
and analysis of the samples was carried out on a FACS Vantage flow cytometer (BD Biosciences, USA). Splenocytes were cultured in 200μl of RPMI 1640 medium supplemented with L-glutamine, antibiotics, 10% FBS and 2μg/ml anti-CD28 mAb in 96-well plates (Costar, USA) precoated with 5μg/ml anti-CD3 mAb for 72 hrs. Cytokine concentrations in the culture supernatants were determined by specific ELISA according to the manufacturer’s instructions (R&D system, USA).
4) Statistical analysis Statistical analysis was done using Graphpad Prism (ver. 4). Results were expressed as the mean±standard deviation Fig. 1. Survival rate and body weight after induction of TNBS colitis. Survival rate after induction of TNBS colitis in mice treated with MSC. Application of MSC caused a significant decrease in mortality rates in TNBS colitis. The graph represents data from six independent experiments with at least ten mice in each group.
(SD). Groups of data were compared using Student’s t-test. Statistical significance was established at P＜0.05.
RESULTS 1) Measurement of survival rate and weight loss To investigate the role of MSC in experimental colitis
Table 1. Histological score of colitic mice treated with MSC*
Fig. 2. TNBS colitis and influence of MSC on body weight. Body weight was recorded daily from days 1 to 10. The change in weight is expressed as the percentage of body weight from day 1. Administration of MSC reduced weight loss in TNBS colitis. Results are representative of six experiments.
3 days Involved region (%) Mononuclear cell Alteration & erosion 10 days Involved region (%) Mononuclear cell Alteration & erosion
Control/ Et-OH
TNBS
TNBS/ MSC
0 0.67±0.41 0.17±0.20
1.17±0.20 1.50±0.61 1.50±0.69
0.67±0.61 1.00±0.55 0.50±0.27
0 0.67±0.26 0.33±0.26
1.83±0.38 1.50±0.61 1.17±0.20
0.33±0.26 0.67±0.26 ‡ 0.33±0.26
†
‡
Data are presented as mean±SD on a scale of 0∼4. The results are representative of six experiments. *MSC = mesenchymal stem † ‡ cell; TNBS = trinitrobenzene sulfonic acid; P＜0.05.
320 J Korean Surg Soc. Vol. 79, No. 5 and the therapeutic effect of MSC, colitis was induced in
increased survival rate of 90% compared to TNBS colitic
BALB/c mice. Colitis was induced by intrarectal admini-
mice of 40% (Fig. 1).
stration of TNBS, which haptenates autologous colonic pro-
Similar decrease in body weight was observed in both
teins with trinitrophenol. Administration of TNBS resulted
TNBS colitic mice and MSC treated colitic mice at day 1
in diarrhea and wasting disease with an increased mortality
(Fig. 2). Decrease in body weight (maximum of more than
rate of approximately 60% after 10 days (Fig. 1). TNBS
10%) was observed until day 3 followed by gradual recovery
colitic mice treated with MSCs showed a markedly
in TNBS colitic mice (Fig. 2). However, MSC treated colitic
Fig. 3. Histological analysis of colonic specimens on day 3 and 10 after induction of TNBS colitis. (A) Control/Et-OH mice post-op day 3 (A-1) and day 10 (A-2), respectively. (B) TNBS colitis induced mice post-op day 3 (B-1) and day 10 (B-2), respectively. (C) TNBS colitis induced mice treated with MSCs post-op day 3 (C-1) and day 10 (C-2). Treatment with MSCs led to reduction in TNBS-induced inflammation. Significantly less inflammation, fewer cell infiltration and crypt destruction were present in MSC treated colitic mice (C) compared with TNBS colitic mice (B).
Yong Beom Cho, et al：Mesenchymal Stem Cells in TNBS-Induced Colitis
321
mice showed no further weight loss after day 1 and showed
control/Et-OH group, but showed a dramatic decrease
rapid weight recovery (Fig. 2).
after MSC treatment, and CD4＋CD25＋ T cells showed a
Thus, MSC treatment in colitis-induced mice resulted in increased survival rate and decreased weight loss.
2) Assessment of H&E staining After H&E staining and scores were compiled for each
marked increase in TNBS/MSC group compared to TNBS group in the blood on day 3 (Table 2).
4) Measurement of cytokine production in culture medium
mouse, statistically significant differences were observed in
In order to determine the treatment effect of MSC in
the subject in the involved region and alteration & erosion
TNBS colitis, splenocytes producing various cytokines were
on day 10 in MSC treated mice (Table 1, Fig. 3).
cultured and ELISA was performed. Concentrations of IFN-γ and IL-12 from the Th1
3) Flow cytometric analysis
group (IFN-γ, TNF-α, IL-12) were significantly lower in
Induction of TNBS colitis resulted in a slight decrease ＋
the MSC treated group compared to the control (Et-OH)
in CD4 T cells in the blood and spleen (except day 10
and TNBS groups, and concentrations of TNF-α were
in spleen), whereas MSC treatment showed increase in it.
significantly higher in the MSC treated group compared to
＋
CD8a
T cells showed a slight decrease after TNBS
the TNBS group on day 3 but markedly decreased by day
treatment in both blood and spleen samples, but increased
10 which showed that there is a decreasing effect on
＋
after MSC treatment. CD19 T cells in the spleen showed a 30∼40% decrease in the TNBS group compared to
pro-inflammatory markers (Fig. 4). Concentration of IL-4 in the Th2 group (IL-4, IL-6,
Table 2. Analysis of surface expression in T cells in the blood (A) and spleen (B)
(A) Blood 3 day CD4＋ ＋ CD8a CD19＋ ＋ ＋ CD4 CD25 10 day ＋ CD4 CD8a＋ ＋ CD19 ＋ CD4 CD25＋ (B) Spleen 3 day ＋ CD4 CD8a＋ ＋ CD19 ＋ CD4 CD25＋ 10 day ＋ CD4 CD8a＋ CD19＋ ＋ ＋ CD4 CD25
†
Control/Et-OH
TNBS*
TNBS/MSC
36.44±2.49 15.94±1.57 38.05±1.43 5.19±1.09
33.52±3.87 13.55±1.14 22.02±5.62 4.43±0.44
42.66±2.87 17.62±1.21‡ 20.79±0.70 6.21±0.44‡
36.53±2.86 12.44±1.93 7.7±1.20 3.56±0.34
23.37±1.44 11.87±1.00 16.43±2.59 3.24±0.29
30.57±1.77‡ 12.06±0.27 17.69±1.93 3.31±0.18
38.36±1.21 20.20±0.93 26.32±3.49 6.51±0.96
32.04±0.62 16.43±0.31 19.01±0.69 6.02±0.28
36.97±3.73 19.42±2.31 18.05±3.12 6.13±0.65
18.23±2.64 20.39±6.75 30.63±6.84 1.99±1.13
20.86±1.91 13.86±1.14 19.06±1.01 3.72±0.44
33.06±1.45‡ 15.01±1.37 15.36±0.56‡ 3.74±0.18
The surface molecules of T cells were observed in spleens by the use of mAb directed to CD4, CD8a, CD19, CD25 and control isotype † conjugated with fluorochromes (FITC, PE). Mean values from six independent experiments. *TNBS = trinitrobenzene sulfonic acid; MSC ‡ = mesenchymal stem cell; P＜0.05.
322 J Korean Surg Soc. Vol. 79, No. 5
Fig. 4. Effect of MSC treatment on cytokine production. The results are representative of six experiments. *P＜0.05.
IL-10) was significantly lower in the MSC treated group
10 compared to the TNBS group (Fig. 4).
compared to the TNBS group on day 10. In contrast, the concentration of IL-6 was significantly higher in the MSC
DISCUSSION
treated group on day 3. The concentration of IL-10 on day 3 showed no noticeable differences in all 3 groups, but the
In this study, we examined the effects of MSC on
MSC treated group showed a significant increase on day
TNBS-induced colitis, which is a CD4＋ T-cell mediated
Yong Beom Cho, et al：Mesenchymal Stem Cells in TNBS-Induced Colitis
autoimmune disease.
323
in both Th1 and 2 inflammatory processes by MSC. In
Clinical results from H&E staining showed a significant
addition, the significant increase in IL-6 level in day 3
decrease in both the microscopic inflammatory process and
TNBS/MSC group is explainable as IL-6 causes increased
in damage in the TNBS/MSC group compared to the
migration and activation of MSC.(23)
TNBS group. On day 10, all of the involved regions
Cell surface markers from the TNBS/MSC group also
showed a significant decrease in alteration and erosion.
showed the anti-inflammatory effect of MSC. A significant
Mononuclear cell infiltration was also decreased, but was
increase in CD4＋CD25＋ T cells in the blood sample of
not statistically significant. These are all signs of a decrease
the TNBS/MSC group showed that T-regulatory cells that
in the inflammatory process, which proves that MSC has
attenuate the inflammatory process increased.(24,25) This
an anti-inflammatory effect on microscopic inflammation.
is in line with the study by Veltkamp et al.(26), which had
IFN-γ and IL-12 decreased in the TNBS/MSC group
shown that CD4＋CD25＋ T-regulatory cells prevent
compared to the TNBS group, which showed a decrease
experimental colitis in the bone marrow transplanted
in Th1 response. IFN-γ and IL-12 both significantly
murine model. Also, a significant decrease in CD19 in the
decreased on both days 3 and 10. This is possibly due to
spleen sample of the TNBS/MSC group on day 10 showed
termination of the IFN-γ - IL-12 - IFN-γ - IL-12 loop
effective attenuation of the Th2 response as CD19＋ B cell
by MSC. In this loop, increase in IFN-γ leads to an
subset population blocks T regulatory cells and exacerbates
increase in IL-12, which itself causes the IFN-γ to in-
ileitis in murine model of CD.(27,28) However, the general
crease again in a loop.(18) This illustrates the Th1 polari-
decrease in CD19 level in the TNBS group compared to
zation (decrease in Th1 response that is responsible for
the control group requires further research.
cellular immunity) and inhibition of inflammation that is triggered by the inflammatory IFN-γ cytokine.
However, cell surface markers from the TNBS group yielded different results. CD8a (blood, day 4) and CD4
IL-4 decreased and IL-10 increased in the TNBS/MSC
(blood and spleen, day 10) in the TNBS/MSC group all
group compared to the TNBS group, which showed an
showed a significant increase compared to the TNBS group,
anti-inflammatory response in the Th2-mediated immune
which shows that there is still inflammation and a Th1
response. IL-4 acts on the T-helper 0 (Th0) cells to
response. This could have been due to a remaining inflam-
differentiate into Th2 cells.(19) Subsequently, IL-4 acts on
matory process that was not attenuated by MSCs.
Th2 cells to produce Th2 cytokines such as IL-5, IL-6,
Also, TNF-α levels did not correlate with other cytokine
IL-13 and even IL-4 (paracrine effect).(20,21). Therefore,
levels. TNF-α levels significantly increased on day 3 in the
a significant decrease in IL-4 cells attenuates the Th2
TNBS/MSC group compared to the TNBS group, which
immune response, leading to decreased humoral immunity.
shows that there is a significant inflammatory process
Also, IL-4 works with IFN-γ, where an increase in IFN-γ
present. This result did not correlate with other cytokine
also leads to a subsequent increase in IL-4.(19) IL-10 also
levels which all showed a decrease, signifying a decreased
antagonizes the production of Th1 cytokines IFN-γ and
inflammatory process. Whereas this study showed no
IL-12.(22) Therefore, the significant decrease in IFN-γ
significant decrease in the TNF-α levels by MSCs, Lee et
correlates with the significant decrease in IL-4 and the
al.(29,30) showed that DSG and anti-4-1 BB mAb leads
significant increase in IL-10, showing that the TNBS/MSC
to a decrease in TNF-α levels. However, TNF-α
group is effective in attenuating the inflammatory process.
significantly increased only on day 3 but not on day 10
Aggarwal and Pittenger(14) also reported a decrease in IFN-
in this study, which can be interpreted as MSCs working
γ and an increase in IL-10, but also an increase in IL-4
more effectively in the later part of the inflammation
in their study using human MSC, which showed a Th2
process and not in the earlier part. Also, results from H&E
shift. Our current results might suggest a general decrease
staining showed a significant decrease in the microscopic
324 J Korean Surg Soc. Vol. 79, No. 5 inflammatory process on day 10 which correlates to the lack of a significant rise in these cytokine levels by then. There is much research being done on the use of MSCs in treating intestinal diseases. It has been shown that adipose-derived MSCs can be used to treat rectovaginal fistulas in perianal CD without any adverse effects.(31) Research has suggested a great potential for the application of MSC. However, the mechanism of action still requires more in depth clarification, which will provide the direction and focus for future study. The current study would have been able to yield more reliable results if the sample sizes were larger. The addition of an apoptosis detection assay to monitor the decrease in specific cell populations would also have given more accurate findings. In addition, monitoring other cytokines such as CD11 (leukocyte adhesion, macrophage levels) would have given more insight into the anti-inflammatory process. Research comparing the effect of using MSC treatment along with conventional treatment versus conventional treatment alone could be meaningful. Because of MSC’s immunomodulatory effect, synergy with conventional treatment to increase treatment effectiveness and decrease side effects can be expected. In conclusion, MSC’s immunomodulatory effect in itself was shown to have a possible therapeutic effect in the murine IBD model, especially in CD. With further research, it could be possible to utilize MSC in the clinical treatment of IBD if subsequent experiments can prove the safety of MSC therapy.
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