Expression and secretion of interleukin-1b, tumour
necrosis factor-aand interleukin-10 by hypoxia- and
serum-deprivation-stimulated mesenchymal stem cells
Implications for their paracrine roles
Zongwei Li, Hua Wei, Linzi Deng, Xiangfeng Cong and Xi Chen
Research Center for Cardiac Regenerative Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
Introduction
Ischaemic heart disease is a life-threatening condition
that may cause sudden cardiac failure and death.
Many researchers have investigated cell transplantation
as an alternative treatment for heart disease. Bone
marrow-derived mesenchymal stem cells (MSCs) are
easily obtainable and expandable, multipotent progeni-
tor cells [1] that have emerged as attractive candidates
for cellular therapies for heart and other organ-system
disorders [2]. Although several mechanisms have been
proposed for the cardioprotective effects of MSCs,
including cardiomyocyte regeneration, spontaneous cell
fusion and paracrine action [3], there is a growing
Keywords
IL-10; IL-1b; mesenchymal stem cell;
paracrine; TNF-a
Correspondence
X. Chen; X. Cong, Research Center for
Cardiac Regenerative Medicine, The
Ministry of Health, Cardiovascular Institute
& Fu Wai Hospital, Chinese Academy of
Medical Sciences & Peking Union Medical
College, 167 Beilishilu, Beijing 100037,
China
Fax ⁄Tel: +86 10 88398584
E-mail: chenxifw@yahoo.com.cn;
xiangfeng_cong@yahoo.com.cn
(Received 26 April 2010, revised 27 June
2010, accepted 10 July 2010)
doi:10.1111/j.1742-4658.2010.07770.x
To understand the potential paracrine roles of interleukin-1b(IL-1b),
tumour necrosis factor-a(TNF-a) and interleukin-10 (IL-10), the expres-
sion and secretion of these factors by rat bone marrow-derived mesenchy-
mal cells stimulated by hypoxia (4% oxygen) and serum deprivation
(hypoxia ⁄SD) were investigated. We found that hypoxia ⁄SD induced
nuclear factor kappa Bp65-dependent IL-1band TNF-atranscription. Fur-
thermore, hypoxia ⁄SD stimulated the translation of pro-IL-1band its
processing to mature IL-1b, although the translation of TNF-awas
unchanged. Unexpectedly, the release of IL-1band TNF-afrom hypox-
ia ⁄SD-stimulated mesenchymal cells was undetectable unless ATP or lipo-
polysaccharide was present. This result suggests that IL-1band TNF-aare
not responsible for the paracrine effects of mesenchymal cells under ischae-
mic conditions. We also found that hypoxia ⁄SD induced the transcription
and secretion of IL-10, which were significantly enhanced by lipopolysac-
charide and the proteasomal inhibitor MG132. Moreover, both the condi-
tioned medium from hypoxia ⁄SD-stimulated mesenchymal cells (MSC-CM)
and IL-10 efficiently inhibited cardiac fibroblast proliferation and collagen
expression in vitro, suggesting that mesenchymal cell-secreted IL-10 pre-
vents cardiac fibrosis in a paracrine manner under ischaemic conditions.
Taken together, these findings may improve understanding of the cellu-
lar and molecular basis of the anti-inflammatory and paracrine effects of
mesenchymal cells.
Abbreviations
BrdU, 5-bromodeoxyuridine; DMEM, Dulbecco’s modified Eagle’s medium; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular
signal-regulated kinase; hypoxia ⁄SD, hypoxia and serum deprivation; IL, interleukin; IMDM, Iscove’s modified Dulbecco’s medium;
LPS, lipopolysaccharide; MSCs, mesenchymal stem cells; NF-jBp65, nuclear factor kappa Bp65; p38, p38 mitogen-activated protein kinase;
TNF-a, tumour necrosis factor-a.
3688 FEBS Journal 277 (2010) 3688–3698 ª2010 The Authors Journal compilation ª2010 FEBS
body of evidence supporting the hypothesis that para-
crine mechanisms mediated by MSC-secreted factors
play an essential role in the reparative process [4,5].
It has been reported that MSC-conditioned medium
under normoxic conditions significantly attenuates car-
diac fibroblast proliferation and type I and III collagen
expression, exerting paracrine anti-fibrotic effects.
However, researchers did not analyse the active compo-
nents of the conditioned medium [6]. Other researchers
have suggested that adrenomedullin and hepatocyte
growth factor are paracrine factors secreted by trans-
planted MSCs, decreasing myocardial fibrosis [7–9].
Whether other paracrine factors released by MSCs
mediate these cells’ anti-fibrotic effects remains largely
unknown.
Interleukin-1b(IL-1b) and tumour necrosis factor-a
(TNF-a) are present in the tissues or systemic circula-
tion in many inflammatory conditions. It has also been
reported that the expression of IL-1band TNF-ain
MSCs can be augmented by exposure to hypoxia [5].
Furthermore, IL-1bcan induce cardiomyocyte growth
but inhibits cardiac fibroblast proliferation in culture
[10]. By contrast, MSC transplantation in rat models
of myocardial infarction has anti-inflammatory effects,
decreasing protein production and gene expression for
IL-1band TNF-a[11]. To address these paradoxes of
both pro- and anti-inflammatory effects, the secretion
of IL-1band TNF-afrom MSCs under ischaemic con-
ditions must be further characterized.
IL-10 is an anti-inflammatory cytokine that has been
reported to be involved in the immunomodulation
mediated by transplanted MSCs [12,13]. IL-10 is also a
potential anti-fibrotic factor in the liver and kidney
[14–16]. In addition, the protective effect of MSCs
against sepsis is dependent on IL-10, which is not
directly produced by the injected MSCs but rather by
endogenous macrophages [17]. However, it is not
known whether MSCs can secrete IL-10 under ischae-
mic conditions, resulting in a paracrine anti-fibrotic
effect in the heart.
To assess the paracrine effects of IL-1b, TNF-aand
IL-10 released by MSCs on cardiac remodelling under
ischaemic conditions, conditioned medium from MSCs
(MSCs-CM) was collected during hypoxia and serum
deprivation (hypoxia ⁄SD). This medium was used to
treat cardiac fibroblasts, enabling observation of the
paracrine effects of MSCs. The expression and secre-
tion of IL-1b, TNF-aand IL-10 by hypoxia ⁄SD-stimu-
lated MSCs were also investigated. Our data
demonstrate that MSCs-CM can inhibit cardiac fibro-
blast proliferation and collagen synthesis, with
< 30 kDa molecules as its major active components.
MSCs did not secrete IL-1band TNF-aunder
hypoxia ⁄SD conditions, although MSC-secreted IL-10
hindered cardiac fibroblast proliferation and collagen
expression. These findings suggest that IL-10 may be
an important paracrine, anti-fibrotic mediator secreted
by MSCs.
Results
MSCs-CM inhibits cardiac fibroblast proliferation
and collagen synthesis
The effects of MSCs-CM on cardiac fibroblast prolifer-
ation and collagen synthesis were detected by [
3
H]-thy-
midine and [
3
H]-proline incorporation. As shown in
Fig. 1A, MSC-CM treatment significantly inhibited
[
3
H]-thymidine and [
3
H]-proline incorporation under
normoxic or hypoxic culture conditions. To further
clarify the molecular mass range of important active
factors in the MSCs-CM, the medium was divided into
AB
Fig. 1. MSCs-CM inhibits cardiac fibroblast proliferation and collagen synthesis. (A) The effects of MSCs-CM on the incorporation of [
3
H]-thy-
midine and [
3
H]-proline by cardiac fibroblasts under normoxic or hypoxic conditions. Each data point represents the mean ± SEM of at least
three independent experiments. ***P< 0.001 versus normoxic control (Cont) group; ###P< 0.001 and ##P< 0.01 versus hypoxic control
(Cont + h) group. (B) The effects of the > 30 kDa and < 30 kDa components of MSCs-CM on the incorporation of [
3
H]-thymidine and
[
3
H]-proline by cardiac fibroblasts under normoxic or hypoxic conditions. ***P< 0.001 versus Cont group; ###P< 0.001 versus Cont + h group.
Z. Li et al. Paracrine anti-fibrotic effects of MSCs in vitro
FEBS Journal 277 (2010) 3688–3698 ª2010 The Authors Journal compilation ª2010 FEBS 3689
> 30 and < 30 kDa components using a 30 kDa
molecular mass cut-off ultrafiltration membrane. Frac-
tionation revealed that the < 30 kDa components, but
not the > 30 kDa components, of the MSCs-CM
inhibited cardiac fibroblast proliferation and collagen
synthesis (Fig. 1B).
Hypoxia ⁄SD induces NF-jB-dependent IL-1band
TNF-atranscription
Because transcription of IL-1band TNF-acan be aug-
mented in MSCs by hypoxia [5], and because the
molecular masss of IL-1band TNF-aare both 17 kDa
(< 30 kDa), changes in IL-1band TNF-agene tran-
scription in hypoxia ⁄SD-stimulated MSCs were exam-
ined. As shown in Fig. 2A, the increased transcription
of IL-1band TNF-aoccurred after 3 h of hypoxia ⁄SD
with a gradual increase up to 6 h, after which tran-
scription decreased. We also found that transcription
of IL-1band TNF-awas mainly induced by SD,
whereas hypoxia simply augmented this effect
(Fig. 2B).
It has been reported that the nuclear factor-jB (NF-
jB) signalling pathway plays an important role in reg-
ulating IL-1band TNF-atranscription [18,19]. To
investigate the role of this pathway in hypoxia ⁄SD-
induced transcription, MSCs were exposed to BAY
11-7082, an NF-jB pathway inhibitor, followed by
hypoxia ⁄SD for 6 h. As shown in Fig. 2C, the tran-
scription of IL-1band TNF-awas significantly attenu-
ated by BAY 11-7082. Interestingly, the proteasomal
inhibitor MG132 also abrogated hypoxia ⁄SD-induced
IL-1band TNF-atranscription.
Next, to clarify the mechanism by which the NF-jB
pathway induces IL-1band TNF-atranscription, the
nuclear translocation of NF-jBp65 was assessed by
immunocytochemical staining. As shown in Fig. 2D,
NF-jBp65 was mainly distributed in the cytoplasm
of control cells. By contrast, hypoxia ⁄SD treatment
significantly stimulated the nuclear translocation of
AB
CD
Fig. 2. Hypoxia ⁄SD induces NF-jB-dependent IL-1band TNF-atranscription. (A) MSCs were incubated under hypoxia ⁄SD conditions for the
indicated number of hours, and the relative mRNA levels of IL-1band TNF-awere determined by real-time PCR. The data are the mean ±
SEM of at least three independent experiments. *P< 0.05 and **P< 0.01 versus control group (0 h). (B) The relative mRNA levels for IL-1b
and TNF-ain MSCs after hypoxia, SD or hypoxia ⁄SD for 6 h by real-time PCR. **P< 0.01 versus Cont group; #P< 0.05 versus SD group.
(C) MSCs were exposed to BAY 11-7082 or MG132, followed by hypoxia ⁄SD for 6 h and detection of relative mRNA levels of IL-1band
TNF-aby real-time PCR. *P< 0.05 and **P< 0.01 versus hypoxia ⁄SD treatment group. (D) A representative pattern of the nuclear translo-
cation of NF-jBp65, as assessed by immunocytochemical staining of MSCs using anti-(NF-jBp65 primary Ig) (red) and nuclear labelling with
4¢,6-diamidino-2-phenylindone (blue).
Paracrine anti-fibrotic effects of MSCs in vitro Z. Li et al.
3690 FEBS Journal 277 (2010) 3688–3698 ª2010 The Authors Journal compilation ª2010 FEBS
NF-jBp65, indicated by strong immunostaining in the
nucleus. Pretreatment with BAY 11-7082 inhibited
hypoxia ⁄SD-induced NF-jBp65 translocation, with
substantial levels of NF-jBp65 staining remaining in
the cytoplasm of most cells. These results demonstrate
that hypoxia ⁄SD induces IL-1band TNF-atranscrip-
tion, which are dependent on activation of the NF-jB
pathway.
Hypoxia ⁄SD-induced IL-1band TNF-atranscription
depend on the extracellular signal-regulated
kinase pathway
The extracellular signal-regulated kinase 1 ⁄2 (ERK1 ⁄2)
and p38 mitogen-activated protein kinase (p38) signal-
ling pathways play important roles in hypoxia ⁄
SD-induced apoptosis of MSCs [20,21] and may also
affect IL-1band TNF-atranscriptional regulation [22].
To confirm this, 20 lmU0126 (Fig. S1A) was used to
inhibit the ERK1 ⁄2 pathway in MSCs, followed by
measurement of IL-1band TNF-amRNA levels by
real-time PCR. As shown in Fig. 3A, U0126 completely
abolished hypoxia ⁄SD-induced IL-1band TNF-a
transcriptional upregulation. When the MSCs were
exposed to 15 lmSB202190 (Fig. S1B), a p38-specific
inhibitor, hypoxia ⁄SD-induced IL-1btranscription
was inhibited by 60%, although TNF-atranscription
was not affected (Fig. 3B). Like BAY 11-7082, U0126
could also inhibit NF-jBp65 nuclear translocation
(Fig. 3C), suggesting that hypoxia ⁄SD-induced activa-
tion of the NF-jB signalling pathway depends on the
ERK1 ⁄2 signalling pathway.
Hypoxia ⁄SD increases the translation of pro-IL-1b
but not TNF-a
Having demonstrated significant transcriptional upreg-
ulation, we next examined protein levels of IL-1band
TNF-ain MSCs-CM. Unexpectedly, neither IL-1bnor
TNF-awas detectable in MSCs-CM using enzyme-
linked immunosorbent assay (ELISA) analysis. To
determine the reason for this lack of IL-1band TNF-a
secretion by MSCs, changes in these factors’ transla-
tion in hypoxia ⁄SD-stimulated MSCs were investi-
gated. As shown in Fig. 4A, hypoxia ⁄SD increased
pro-IL-1btranslation in a time-dependent manner,
whereas TNF-aprotein expression remained
unchanged at each time point. Furthermore, MG132,
BAY 11-7082 and U0126, all of which abrogated
hypoxia ⁄SD-induced IL-1band TNF-atranscription,
also abolished pro-IL-1btranslational upregulation
(Fig. 4B,C) but failed to affect TNF-atranslation
A
C
B
Fig. 3. IL-1band TNF-atranscriptional
induction depends on the ERK1 ⁄2 pathway.
MSCs were exposed to the ERK1 ⁄2
inhibitor U0126 or the p38 inhibitor
SB202190, followed by hypoxia ⁄SD for 6 h.
(A,B) Relative mRNA levels for IL-1band
TNF-a, as determined by real-time PCR.
*P< 0.05 versus hypoxia ⁄SD group.
(C) A representative pattern of the nuclear
translocation of NF-jBp65, as assessed by
immunocytochemical staining of MSCs
using an anti-(NF-jBp65 primary Ig) (red)
and nuclear labelling with 4¢,6-diamidino-2-
phenylindone (blue).
Z. Li et al. Paracrine anti-fibrotic effects of MSCs in vitro
FEBS Journal 277 (2010) 3688–3698 ª2010 The Authors Journal compilation ª2010 FEBS 3691
(Fig. 4B). These results demonstrate that in hypox-
ia ⁄SD-stimulated MSCs, IL-1bmRNA can be effi-
ciently translated into pro-IL-1bprotein, whereas the
translation of TNF-amRNA is severely repressed.
Hypoxia ⁄SD induces cleavage of pro-IL-1binto
mature IL-1b
Given that hypoxia ⁄SD induced significant transla-
tional upregulation of pro-IL-1band that processing
of pro-IL-1binto mature IL-1brequires activating
cleavage of pro-caspase 1[23], the cleavage of both
pro-IL-1band pro-caspase 1was examined in hypox-
ia ⁄SD-stimulated MSCs. As shown in Fig. 5A, hypox-
ia ⁄SD promoted the processing of pro-IL-1binto
mature IL-1b, with a stronger induction effect in the
presence of the endotoxin LPS. Consistent with these
data, hypoxia ⁄SD also induced the cleavage of pro-
caspase 1, with stronger activation in the presence of
LPS (Fig. 5B).
Hypoxia ⁄SD-stimulated MSCs require a second
signal for IL-1band TNF-arelease
Although significant cleavage of pro-IL-1band pro-
caspase 1occurred intracellularly in hypoxia ⁄SD-stim-
ulated MSCs, mature IL-1bwas undetectable in
MSCs-CM (Fig. 6A). However, significant release of
IL-1bby hypoxia ⁄SD-stimulated MSCs in the presence
of ATP was detected. Furthermore, when both LPS
and ATP were present, hypoxia ⁄SD-stimulated MSCs
released a larger amount of IL-1b(Fig. 6A). We also
examined TNF-aexpression in hypoxia ⁄SD-stimulated
MSCs in the presence of LPS. As shown in Fig. 6B,
LPS relieved the translational inhibition of TNF-a.
Moreover, TNF-arelease by MSCs was detectable
after hypoxia ⁄SD treatment for 6 h in the presence of
LPS (Fig. 6C). These findings demonstrate that hypoxia ⁄
SD-stimulated MSCs require a second stimulatory
signal in order to secrete IL-1band TNF-a.
Hypoxia ⁄SD induces the transcription and
secretion of IL-10
Because of the lack of secretion of the inflammatory
cytokines IL-1band TNF-afrom hypoxia ⁄SD-stimu-
lated MSCs, as well as the significant anti-inflamma-
tory effects of MSCs, expression and secretion of the
anti-inflammatory cytokine IL-10 by these cells was
investigated. As shown in Fig. 7A, hypoxia ⁄SD
A
C
B
Fig. 4. Hypoxia ⁄SD increases translation of
pro-IL-1bbut not TNF-a. (A) Representative
western blots for pro-IL-1band TNF-a
expression in MSCs stimulated by
hypoxia ⁄SD for the indicated number of
hours. (B) Representative western blots
for pro-IL-1band TNF-aexpression in MSCs
in the presence and absence of
BAY 11-7082 or MG132. *, nonspecific
band. (C) Representative western blots for
pro-IL-1bexpression in MSCs in the pres-
ence and absence of U0126. *, nonspecific
band.
A
B
Fig. 5. Hypoxia ⁄SD induces cleavages of pro-IL-1band pro-cas-
pase 1. MSCs were stimulated by hypoxia ⁄SD in the presence or
absence of LPS for the indicated number of hours. (A) Representa-
tive western blots for pro-IL-1band mature IL-1bin MSCs. (B) Rep-
resentative western blots for pro-caspase 1and cleaved caspase 1
in MSCs.
Paracrine anti-fibrotic effects of MSCs in vitro Z. Li et al.
3692 FEBS Journal 277 (2010) 3688–3698 ª2010 The Authors Journal compilation ª2010 FEBS