Carbon Monoxide Produced by Heme

Oxygenase-1 Suppresses T Cell Proliferation

This information is current as
of December 27, 2016.
via Inhibition of IL-2 Production
Hyun-Ock Pae, Gi-Su Oh, Byung-Min Choi, Soo-Cheon
Chae, Young-Myeong Kim, Khee-Rhin Chung and
Hun-Taeg Chung
J Immunol 2004; 172:4744-4751; ;
doi: 10.4049/jimmunol.172.8.4744
http://www.jimmunol.org/content/172/8/4744

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 The Journal of Immunology

Carbon Monoxide Produced by Heme Oxygenase-1 Suppresses

T Cell Proliferation via Inhibition of IL-2 Production1

Hyun-Ock Pae,* Gi-Su Oh,* Byung-Min Choi, Soo-Cheon Chae, Young-Myeong Kim,
Khee-Rhin Chung, and Hun-Taeg Chung2*
Heme oxygenase-1 (HO-1) catabolizes heme into CO, biliverdin, and free iron and serves as a protective enzyme by virtue of its
anti-inflammatory, antiapoptotic, and antiproliferative actions. Previously, we have demonstrated that human CD4 T cells
express HO-1 and that HO-1-overexpressing Jurkat T cells tend to display lower proliferative response. The aim of this study is
to elucidate the mechanism(s) by which HO-1 can mediate its antiproliferative effect on CD4 T cells. Among the three HO-1
byproducts, only CO showed suppressive effect on T cell proliferation in response to anti-CD3 plus anti-CD28 Abs, mimicking the
antiproliferative action of HO-1. CO blocked the cell cycle entry of T cells, which was independent of the guanylate cyclase/cGMP
pathway. CO also suppressed the secretion of IL-2, and this suppressive effect of CO on IL-2 secretion mediated the antiprolif-

Downloaded from http://www.jimmunol.org/ by guest on December 27, 2016

erative action of CO. CO selectively inhibited the extracellular signal-regulated kinase pathway, which could explain the sup-
pressive effects of CO on T cell proliferation and IL-2 secretion. Based on these findings, we suggest that HO-1/CO suppresses T
cell proliferation and IL-2 secretion, possibly via its inhibition of extracellular signal-regulated kinase activation. The Journal of
Immunology, 2004, 172: 4744 4751.

H eme oxygenase-1 (HO-1)3 is the rate-limiting enzyme in
the conversion of heme to CO, free iron, and biliverdin,
the latter being reduced to bilirubin by biliverdin reduc-
tase. HO-1 is induced by a variety of stimuli in many types of cells,
including human CD4 T cells (1). Several studies have demon-
strated anti-inflammatory properties of HO-1 (2, 3), the existence
of which is further supported by the fact that HO-1-deficient mice
develop a progressive chronic inflammatory state (4, 5) and that a
human lacking HO-1 enzymatic activity died of an inflammatory
syndrome (6).
After the receipt of signals from APCs through TCR and CD28
costimulator, CD4 T cells are triggered to produce IL-2 and enter
the cell cycle. During or after several days of rapid cell division,
these cells differentiate into one of two classes of effector CD4 T
cells (Th1 and Th2 cells). It is generally accepted that activation
and subsequent proliferation of resting CD4 T cells are essential
*Department of Microbiology and Immunology, School of Medicine, and Genomic
Research Center for Immune Disorders, Wonkwang University, Iksan, Chonbuk, Re-
public of Korea; Vascular System Research Center and Department of Molecular and
Cellular Biochemistry, Kangwon National University School of Medicine, Chunchon,
Kangwon-Do, Republic of Korea; and Seoul National University Medical School,
Seoul, Republic of Korea
Received for publication October 13, 2003. Accepted for publication January
23, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by a grant from the Korea Health 21 R&D Project of the
Ministry of Health and Welfare (01-PJ3-PG6-01GN09-003).
2 Furthermore, the MAP kinase pathway has been shown to mediate
Address correspondence and reprint requests to Dr. Hun-Taeg Chung, Department
of Microbiology and Immunology, Wonkwang University Medical School, 344-2
Shinyong-Dong, Iksan, Chonbuk 570-749, Republic of Korea. E-mail address:
htchung@wonkwang.ac.kr
3
Abbreviations used in this paper: HO-1, heme oxygenase-1; MAP, mitogen-acti-
vated protein; ERK, extracellular signal-regulated kinase; JNK, Jun NH2-terminal
kinase; MEK, MAP/ERK kinase; CoPP, cobalt protoporphyrin; RuCO, tricarbonyl-
dichlororuthenium (II) dimer; Hb, hemoglobin; ODQ, 1H-[1,2,4]oxadiazolo-[4,3-
a]quinoxalin-1-one; DN-MEK1, dominant-negative MEK1; CA-MEK1, constitu-
tively active MEK1.
processes in the T cell-mediated immune response and that they
are associated with increased release of IL-2 (7).
The mitogen-activated protein (MAP) kinase cascade is one of
the most ancient and evolutionarily conserved signaling pathways,
which is also important for many processes occurring in immune
responses. Three major groups of MAP kinases have been de-
scribed in mammalian cells: they are the extracellular signal-reg-
ulated kinase (ERK), the Jun NH2-terminal kinase (JNK), and the
p38 kinase (8). ERK, JNK, and p38 pathways are rapidly up-reg-
ulated by engagement of the TCR in T cells and play a critical role
in the events leading to activation and increased IL-2 secretion (8).
There are two isoforms of ERK, ERK-1 and -2. They can be ac-
tivated by MAP/ERK kinase (MEK). ERK activation is dependent
on p56lck and coupling of the TCR/CD3 complex to p21ras, with
subsequent activation of the Raf-1/MEK/ERK kinase cascade (8,
9). JNK activation also requires p21ras and signals generated by
the CD28 costimulatory receptor (10). The activation of the Raf-
1/MEK/ERK pathway is essential for induction of IL-2 transcrip-
tion in T cells (11). After phosphorylation of c-jun by JNK, acti-
vated c-fos and c-jun combine to form the AP-1 protein required
for IL-2 synthesis (12). Interestingly, deficient ERK and JNK ac-
tivations have been reported to exist in clones that are anergized
(13). However, there is also evidence that ERK inhibition alone
suppressed T cell proliferation, but did not induce anergy (14).
CO, a reaction product of HO-1 activity, has been shown to be
highly protective in several rodent disease models (15, 16). It has
anti-inflammatory, antiapoptotic, and antiproliferative effects (17),
thereby conferring, at least in part, the protective effects of HO-1.
the biological effects of CO: the p38 MAP kinase activation me-
diates the cytoprotective effect of CO on ischemia-reperfusion lung
injury (18), CO prevents glucose deprivation-induced cytotoxicity
through ERK MAP kinase inactivation in rat hepatocytes (19), and
the antiproliferative effect of CO on smooth muscle cells and hu-
man airway smooth muscle cells requires p38 MAP kinase acti-
vation (20) and ERK MAP kinase inactivation (21), respectively.

Copyright 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00

The Journal of Immunology
We have recently demonstrated that human CD4 T cells ex-
press HO-1 and that HO-1-overexpressing Jurkat T cells tend to
display lower proliferative response (1). The aim of this study,
therefore, was to elucidate the mechanism(s) by which HO-1 could
mediate its antiproliferative effect on CD4 T cells, and we dem-
onstrated that CO, a reaction product of HO-1, could suppress IL-2
secretion, probably by inhibiting ERK activation, and thereby re-
sulted in suppression of T cell proliferation in response to anti-
CD3 plus anti-CD28 Abs.
Materials and Methods
Reagents, cytokines, and Abs
RPMI 1640 supplemented with 2 mM L-glutamine, 1% nonessential amino
acids, 1% pyruvate, 100 U/ml penicillin, 100 g/ml streptomycin (Life
Technologies, Grand Island, NY), and 10% FBS (HyClone Laboratories,
Logan, UT) was used as complete medium in all cultures. Anti-CD3 (clone
UCHT1) and anti-CD28 (clone CD28.2) Abs were purchased from Immu-
notech (Westbrook, ME) and BD PharMingen (San Diego, CA), respec-
tively. Phospho (p)-specific rabbit Abs to p-ERK-1/2, p-JNK-1/2, and p-
p38 were obtained from Cell Signaling Technology (Beverly, MA), and
ERK-1/2, JNK-1/2, and p38 Abs were from Santa Cruz Biotechnology
(Santa Cruz, CA). PE- and FITC-conjugated and HRP-conjugated second-
ary Abs were purchased from BD PharMingen and Santa Cruz Biotech-
nology, respectively, and U0126 and cobalt protoporphyrin (CoPP) were
from Promega (Madison, WI) and Porphyrin Products (Logan, UT), re-
spectively. SP600125 was purchased from Calbiochem (San Diego, CA).
CO gas (99%), tricarbonyldichlororuthenium (II) dimer (RuCO),
SB203580, hemoglobin (Hb), and 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-
1-one (ODQ) were purchased from Sigma-Aldrich (St. Louis, MO), and
anti-HO-1 and anti--actin Abs were from Santa Cruz Biotechnology.
cDNAs encoding constitutively active (S218E/S222E) and dominant neg-
ative (S218A/S222A) mutants of MEK1 were kindly provided by Dr. K. Y.
Choi (Yonsei University, Seoul, Korea). HO-1 cDNA was a kind gift from
Dr. A. M. K. Choi (University of Pittsburgh, Pittsburgh, PA). Recombinant
human IL-2, IL-10, IFN-, and anti-IL-10 neutralizing Ab were obtained
from R&D Systems (Minneapolis, MN). CFSE was purchased from Mo-
lecular Probes (Eugene, OR). The other reagents were from Sigma-Aldrich.
Isolations of resting CD4 T cells and CD8 T cells from
peripheral blood
PBMCs were isolated from healthy blood by Ficoll-Paque density gradient
centrifugation. After three washes in HBSS, CD4 T cells were isolated
from PBMCs using the MACS negative depletion system (Miltenyi Biotec,
Auburn, CA). No contamination with CD8 T cells, B cells, monocytes, or
NK cells was detected. Isolation of CD8 cells was performed using a
negative CD8 T cell isolation kit (Miltenyi Biotec).
Cell transfection
HO-1, dominant-negative MEK1 (DN-MEK1), and constitutively active
MEK1 (CA-MEK1) were cloned into pcDNA3 (Invitrogen, San Diego,
CA). Jurkat T cells (5 106) were transfected with 10 g of constructs by
electroporation at 270V, 950 F in serum-free RPMI 1640 using a Gene
Pulser (Bio-Rad, Richmond, CA) followed by culture in RPMI 1640 sup-
plemented with 10% FBS for 48 h and plating on 96-well plates at 5 105
cells/well in the presence of 1.25 mg/ml G418. Single stable clones of each
transfectant were isolated and expanded.
CO exposure
T cells were exposed to compressed air or varying concentrations of CO
(200 ppm), as previously described (22). Briefly, 1% CO in compressed air
was mixed with compressed air with or without 5% CO2 in a stainless steel
mixing cylinder before being delivered into the exposure chamber. The cell
culture chamber was humidified and maintained at 37C. A CO analyzer
(CM-525HB; Gastec, Kanagawa, Japan) was used to monitor CO levels
continuously in the chambers. T cells were grown in RPMI 1640 medium
containing 10% FBS in a humidified atmosphere of 5% CO2 in air or 200
ppm CO and 5% CO2 in air.
T cell activation, cytokine analysis, and proliferation
The human acute T cell leukemia Jurkat clone E6-1 was obtained from the
American Type Culture Collection (Rockville, MD). Purified CD4 T cells
or Jurkat T cells were cultured in RPMI 1640 supplemented with 10%
heat-inactivated FBS at 37C in 5% CO2. CD4 T cells (2 105/well)
4745
were activated in flat-bottom, 96-well plates precoated with the anti-CD3
Ab (1 g/ml) in the presence of soluble anti-CD28 Ab (1 g/ml). CO gas,
CO donor, CO scavenger, and ERK inhibitor at the appropriate concen-
trations were preincubated for 1 h before being added to plates and stim-
ulated with anti-CD3 plus anti-CD28 Abs. Culture supernatants were har-
vested after 48 h of incubation for measurements of cytokine
concentrations. Cytokine analysis was performed by analysis of super-
natants with commercially available ELISA kits for human IL-2, IFN-,
and IL-10 (R&D Systems). After 4 days of culture, proliferation was
assessed by [3H]TdR (0.5 Ci/well; Amersham Pharmacia Biotech,
Piscataway, NJ) uptake for the next 16 h.
Evaluation of cGMP
Different pools of CD4 T cell samples were incubated for 1 h at 37C in
the presence of 20 M CO or air. To inhibit phosphodiesterase activity,
31-isobutyl-1-methyl xanthine (10 M) was added to the cell suspension.
The concentration of cGMP was determined by a radioimmunoassay kit
using 125I-labeled cGMP (Amersham Pharmacia Biotech). Briefly, after
incubation, 500 l of 10% TCA was added to the cell suspensions. The
samples were then centrifuged and TCA was extracted with 0.5 M tri-n-
octylamine dissolved in TCA, and the samples were then acetylated with
acetic anhydride and the amount of cGMP in the aqueous phase was
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measured.
Western blotting analysis
Cells were solubilized in ice-cold 1% Triton X-100 lysis buffer supple-
mented with protease and phosphatase inhibitors as described previously
(21). After 30 min on ice, the lysates were clarified by centrifugation and
the protein concentration was determined with the Pierce bicinchoninic
acid protein assay reagent. Proteins (20 g) were resolved by SDS-PAGE
(10% acrylamide), transferred to nitrocellulose membranes, and probed
with specific Abs (diluted 1/1,000), followed by incubation with secondary
HRP-conjugated Ab (1/100,000). Bands were detected using the Luminol
chemiluminescent detection reagents (New England Biolabs, Beverly,
MA). Blots were exposed to autoradiographic film (DuPont Merk Chem-
istry Department, Wilmington, DE) for 12 min for detection.
Flow cytometry
Cells were suspended in HBSS containing 5% FBS, fixed by the drop-wise
addition of ice-cold 70% ethanol to a final 50% concentration, and held on
ice for 1 h. After extensive washing, the cells were suspended in HBSS
containing 50 g/ml propidium iodide (Sigma-Aldrich) and 50 g/ml
RNase A (Boehringer Mannheim, Indianapolis, IN) and were incubated for
1 h at room temperature. In some experiments, the states of ERK activation
and HO-1 expression were determined by intracellular staining with either
anti-p-ERK or anti-HO-1 Abs labeling either FITC- or PE-conjugated sec-
ondary Ab, as described previously (23). Stained cells were analyzed by
flow cytometry on a FACSVantage with CellQuest software (BD Bio-
sciences, Franklin Lakes, New Jersey). G0/G1, S, and G2/M populations
were quantified using the ModFiT program (BD Biosciences).
CFSE dilution assay
Naive CD4 T cells were labeled with 1.5 M CFSE for 10 min at 37C
in serum-free RPMI 1640. Cells were washed twice in RPMI 1640 con-
taining 10% FBS and were stimulated as indicated. Cell division was as-
sessed at 4 days by determining the pattern of CFSE dilution using flow
cytometry.
Statistics
Data were expressed as mean SEM of the individual titer. Levels of
significant differences between groups were determined by the Student t
test. Values of p 0.01 were considered statistically significant.
Results
HO-1 expression in T cells is antiproliferative
We found that pharmacological expression of HO-1 by the HO-1
inducer CoPP in PBMCs containing monocytes and lymphocytes
was antiproliferative in response to anti-CD3 plus anti-CD28 Abs
(Fig. 1A). This suggested that HO-1 expression in monocytes
and/or lymphocytes could suppress T cell proliferation. Therefore,
we examined whether CoPP could induce HO-1 expression in hu-
man CD4 T cells purified from PBMCs and also whether HO-1
expression by CoPP could be antiproliferative. Preincubation of
4746 CO SUPPRESSES T CELL PROLIFERATION VIA IL-2 INHIBITION

FIGURE 1. Effects of HO-1 expression on T

cell proliferation. PBMCs (A) and CD4 T cells
(B) purified from PBMCs were incubated for 12 h
with CoPP, and Jurkat T cells (C) were transfected
with human HO-1 gene (Jurkat/HO-1) or empty
vector (Jurkat/pcDNA). HO-1 expressions (top
panels) were confirmed by Western blot analysis
after 12-h incubation of either PBMCs or CD4 T
cells with CoPP (20 M) or after stable transfec-
tion of Jurkat T cells with either HO-1 gene or
empty vector. One of three experiments is shown.
T cell proliferation (bottom panels) was deter-
mined after 4-day stimulation of T cells with anti-
CD3 plus anti-CD28 Abs. Values are the mean
SEM of six triplicate experiments. , p 0.01.

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CD4 T cells with CoPP for 12 h resulted in an apparent increase
in HO-1 expression and a significant decrease in T cell prolifera-
tion (Fig. 1B). CoPP preincubation also suppressed the prolifera-
tive response of CD8 T cells (data not shown). To further inves-
tigate the contribution of HO-1 expression to T cell proliferation,
we transfected the HO-1 gene into human Jurkat T cells (Fig. 1C).
In agreement with the CoPP data, overexpression of HO-1 sup-
pressed T cell proliferation (Fig. 1C). These data demonstrate that
HO-1 expression in T cells is antiproliferative.
Exogenous and endogenous CO can suppress human T cell
proliferation, mimicking antiproliferative action of HO-1
We next determined which of the HO-1 metabolites could mediate
the antiproliferative effect of HO-1 on human CD4 T cells. Thus,
we preincubated CD4 T cells with CO gas, bilirubin, or free iron
(Fe2) for 1 h before stimulation of the T cells with anti-CD3 plus
anti-CD28 Abs. Only CO gas had an antiproliferative effect on
CD4 T cells (Fig. 2A). Similarly, the CO-releasing compound
RuCO suppressed the proliferative response of CD4 T cells in a
dose-dependent manner (IC50 20 M), but not in the presence
of the CO scavenger Hb (Fig. 2B). In addition, a 12-h preincuba-
tion of CD4 T cells with CoPP significantly suppressed the pro-
liferative response, whereas a 12-h preincubation of the cells with
CoPP in the presence of Hb showed no suppressive effect (Fig.
2C). Similarly, CoPP in the presence of Hb did not suppress the
proliferation of either CD8 T cells or Jurkat T cells (data not
shown). CO gas, RuCO, CoPP, or Hb at concentrations used in
these experiments showed no effect on T cell viability (data not
shown). These results suggest that exogenous and endogenous CO
can mimic the antiproliferative action of HO-1.
CO blocks the cell cycle entry of human T cells, independent of
the guanylate cyclase/cGMP pathway
We observed that addition of CO to CD4 T cells 24 h after stimu-
lation with anti-CD3 plus anti-CD28 Abs had no effect on prolifera-
tion (Fig. 3A). This suggested that CO was blocking early events
leading to T cell activation. Therefore, we examined the effect of
RuCO on the cell cycle and could show that CD4 T cells did not
progress past G0/G1 phase, if the cells were stimulated after preincu-
bation with RuCO for 1 h (Fig. 3B, lower left panel). We could also
show that CO was not inhibitory to cell cycle progression, if the ac-
tivation was already progressed beyond TCR signaling (Fig. 3B,
lower right panel). Furthermore, a kinetic CFSE analysis with in-
tracellular HO-1 staining revealed that CO was not inhibitory to T

FIGURE 2. Suppressive effects of CO on the proliferation of CD4 T cells. A, CD4 T cells were preincubated for 1 h with CO gas (200 ppm), bilirubin
(20 M), or Fe2 (20 M) before stimulation. B, CD4 T cells were preincubated for 1 h without (control) or with indicated concentrations of RuCO in
the absence or presence of Hb (80 M) before stimulation. C, CD4 T cells were preincubated for 12 h with CoPP (20 M) in the absence or presence
of Hb (80 M). The CD4 T cells were then stimulated for 4 days with anti-CD3 plus anti-CD28 Abs. Proliferation was measured, as described in Materials
and Methods. Data (B) are expressed as percent of control. Values are the mean SEM of six triplicate experiments. , p 0.01.
The Journal of Immunology 4747

FIGURE 3. Blocking effects of CO on the cell cy-

cle progression of human T cells. A, CD4 T cells
labeled with CFSE were cultured for 1 h with medium
(upper left panel) or were stimulated for 4 days with
anti-CD3 plus anti-CD28 Abs (upper right panel).
CD4 T cells labeled with CFSE were preincubated
for 1 h with RuCO (20 M) and stimulated for 4 days
with anti-CD3 plus anti-CD28 Abs (lower left panel).
RuCO (20 M) was added after 24-h stimulation of
CD4 T cells with anti-CD3 plus anti-CD28 Abs
(lower right panel), and the T cells were further cul-
tured for 3 days. CFSE fluorescence was measured by
flow cytometry. B, CD4 T cells were cultured for
48 h with medium (upper left panel) or were stimu-
lated with anti-CD3 plus anti-CD28 Abs (upper right
panel). CD4 T cells were preincubated for 1 h with
RuCO (20 M) and were stimulated for 48 h with

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anti-CD3 plus anti-CD28 Abs (lower left panel).
RuCO (20 M) was added after 24-h stimulation of
CD4 T cells with anti-CD3 plus anti-CD28 Abs
(lower right panel), and the T cells were further cul-
tured for 24 h. Cell cycle analysis was performed, as
described in Materials and Methods. Cells with
sub-2N amounts of DNA were not gated and com-
prised from 10 to 25% of the population. C, CD4 T
cells labeled with CFSE were stimulated for 4 days
with anti-CD3 plus anti-CD28 Abs. Cells were har-
vested and stained with anti-HO-1-PE. Dotted box in
one of three representative dot plots of HO-1 expres-
sion vs CFSE represents the population of HO-1-pos-
itive cells. D, CD4 T cells were preincubated for 1 h
with medium or RuCO (20 M) in the presence or
absence of ODQ (30 M) and were stimulated for 48 h
with anti-CD3 plus anti-CD28 Abs. Each bar repre-
sents the percentage of cells belonging to each cell
cycle in flow cytometric analysis. Values are the
mean SEM of six triplicate experiments.

cell cycle progression after a certain stage of activation because
HO-1/CO was induced during T cell activation (Fig. 3C).
Most of the biological effects of CO are attributed to its abilities
to modulate the activity of guanylate cyclase and to increase the
cellular cGMP levels (17). Therefore, we examined whether the
blockage of cell cycle progression by RuCO could be due to ele-
vation of cGMP and found that ODQ, a potent inhibitor of the
soluble form of guanylyl cyclase, had no effect on RuCO-induced
blockage of the cell cycle (Fig. 3D). However, ODQ significantly
blocked RuCO-induced cGMP synthesis in T cells (data not
shown).
CO inhibits IL-2 secretion by activated T cells, contributing
antiproliferative effect of CO
IL-2 is an important regulator of T cell proliferation and is released
by activated T cells (8 14). We observed that, among three HO-1
reaction products, only CO gas inhibited IL-2 secretion by acti-
vated CD4 T cells (Fig. 4A). Similarly to CO gas, RuCO also
inhibited the secretion of IL-2 by CD4 T cells in a dose-depen-
dent manner (IC50 20 M), but not in the presence of Hb (Fig.
4B). Additionally, RuCO at higher concentrations reduced the se-
cretion levels of IFN- and IL-10 (Table I). Moreover, preincu-
bation of CD4 T cells with CoPP inhibited IL-2 secretion,
whereas preincubation with CoPP in the presence of Hb showed no
inhibitory effect (Figs. 4C).
To test whether the observed CO-induced suppression of T cell
proliferation could be due to reduction of secretion levels of IL-2,
we added recombinant human IL-2 to the cultures of T cells stim-
ulated with anti-CD3 plus anti-CD28 Abs in the presence of
RuCO. The exogenous IL-2 effectively reversed the antiprolifera-
tive effects of CO in T cell culture (Fig. 4D). Unlike IL-2, neither
IFN- nor IL-10 reversed the antiproliferative effects of CO (Fig.
4D). These data indicate that the suppression of T cell proliferation
by CO could be due to its inhibition of IL-2 secretion.
CO inhibits ERK phosphorylation in activated T cells, probably
mediating the inhibitory effect of CO on IL-2 secretion
The ERK MAP kinases, which have been implicated as an intra-
cellular target to contribute to certain CO-induced biological ac-
tions (19, 21), also play critical roles in T cell proliferation and
IL-2 secretion (13, 14). Therefore, we examined the effects of CO
gas and RuCO on ERK-1/ERK-2 activation in CD4 T cells. CO
4748
FIGURE 4. Inhibitory effects of CO on IL-2 secretion. A, CD4 T cells
were preincubated for 1 h with CO gas (200 ppm), bilirubin (20 M), or
Fe2 (20 M) before stimulation. B, CD4 T cells were preincubated for
1 h without (control) or with indicated concentrations of RuCO in the
absence or presence of Hb (80 M) before stimulation. C, CD4 T cells
were preincubated for 12 h with CoPP (20 M) in the absence or presence
of Hb (80 M). D, CD4 T cells were preincubated for 1 h with RuCO (20
M) in the presence or absence of IL-2 (50 U/ml), IFN- (50 U/ml), IL-10
(50 U/ml), or anti-IL-10 neutralizing Ab (2 g/ml) before stimulation. The
CD4 T cells were then stimulated for 2 days (AC) or for 4 days (D) with
anti-CD3 plus anti-CD28 Abs. IL-2 secretion (AC) and proliferation (D)
were measured, as described in Materials and Methods. Data (B) are ex-
pressed as percent of control. Values are the mean SEM of six triplicate
experiments. , p 0.01.
gas and RuCO significantly inhibited ERK phosphorylation in the
activated CD4 T cells (Figs. 5, A and B). In contrast, RuCO had
no effect on either p38 phosphorylation or JNK phosphorylation
(Fig. 5B). In Jurkat T cells, RuCO also inhibited ERK phosphor-
ylation in a dose-dependent manner (data not shown). Next, we
were interested in determining the effects of blocking the ERK,
p38, and JNK pathways on T cell proliferation. U0126, a selective
inhibitor of ERK pathway, SB20358, a selective inhibitor of p38
pathway, and SP600125, a selective inhibitor of JNK pathway,
were used to block each pathway. U0126 significantly suppressed
T cell proliferation, but SB20358 and SP600125 did not (Fig. 5C).
Suppression of T cell proliferation by U0126 was most likely due
to reduction of IL-2 levels, because U0126-treated CD4 T cells
could proliferate when IL-2 was exogenously added to the cultures
(Fig. 5C). In addition, RuCO at 20 M reduced proliferative re-
sponse even when p38 and JNK pathways were blocked by
SB20358 and SP600125, respectively (Fig. 5C). However, RuCO
CO SUPPRESSES T CELL PROLIFERATION VIA IL-2 INHIBITION
Table I. Effects of RuCO on IFN- and IL-10 secretions in CD4 T
cellsa
Treatment IFN- (pg/ml) IL-10 (pg/ml)
Medium 300 20 350 10
RuCO (10 M) 320 10 330 9
RuCO (20 M) 290 12 300 13
RuCO (40 M) 210 5b 240 5b
RuCO (80 M) 150 7b 170 12b
a
Cells were preincubated for 1 h with medium (control for either IFN- or IL-10)
or RuCO at indicated concentrations and were stimulated for 48 h with anti-CD3 plus
anti-CD28 Abs. IFN- and IL-10 secretions were measured as described in Materials
and Methods. Results are the means SEM of six experiments.
b
Significance of difference from each control, p 0.01.
did not further reduce T cell proliferative response when ERK
pathway was effectively blocked by U0126 (Fig. 5C). These results
further suggest that the ERK pathway might be involved in the
suppressive effects of CO on T cell proliferation.
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CO may block upstream signaling cascades of MEK1 in
activated T cells, thereby resulting in ERK inactivation
Several studies have brought evidence that the intracellular signals
that mediate activation of transcription factors regulating IL-2
gene transcription in human T cells involve p21ras-mediated sig-
naling pathways (24 27). These studies obtained with T cell lines
collectively suggest that IL-2 gene transcription might require the
p21ras/Raf-1/MEK/ERK phosphorylation cascade.
To further evaluate the role of the ERK pathway in CO-induced
inhibition of IL-2 secretion, we transfected DN-MEK1 and CA-
MEK1 genes into Jurkat T cells to selectively inhibit or activate
the ERK1 pathway. DN-MEK1 expression suppressed ERK1 ac-
tivation and IL-2 secretion by the simultaneous ligation of CD3
and CD28 Abs (Fig. 6), which was similar to RuCO effects on IL-2
secretion. Conversely, CA-MEK1 expression enhanced ERK1 ac-
tivation and IL-2 secretion by the same stimuli (Fig. 6). No sig-
nificant inhibition of IL-2 secretion by RuCO was observed in
DN-MEK1- or CA-MEK1-transfected cells (Fig. 6). These results
suggest that CO might block or inactivate upstream signaling cas-
cades of MEK1, thereby inhibiting ERK pathway.
IL-10 may be involved in HO-1 expression in activated CD4 T
cells
It has been reported that IL-10 is able to induce HO-1 expression
and to exert its anti-inflammatory effects via the HO-1-dependent
pathway in monocytes (28). In human CD4 T cells, IL-10 secre-
tion (see Table I) as well as HO-1 expression (see Fig. 3C) was
induced by the simultaneous ligation of CD3 and CD28 Abs. Thus,
one may ask about whether IL-10 could be involved in HO-1 ex-
pression in CD4 T cells. Although IL-10 itself did not induce
HO-1 expression in naive CD4 T cells, this cytokine further en-
hanced HO-1 expression in CD4 T cells stimulated with CD3
plus CD28 Abs (Fig. 7). Moreover, anti-IL-10 neutralizing Ab
reduced the level of HO-1 expression in activated CD4 T cells
(Fig. 7). It is most likely that there are many complex signals,
including an IL-10 signal, to sufficiently induce HO-1 expression
in human CD4 T cells.
Discussion
The antiproliferative and anti-inflammatory HO-1 may play im-
portant roles in regulating T cell responses. It has been reported
that HO-1-deficient mice contract a progressive chronic inflamma-
tory disease, demonstrated by enlarged spleen and lymph nodes,
high peripheral white blood cell counts, and high splenic and
The Journal of Immunology
FIGURE 5. Inhibitory effects of CO on ERK phosphorylation in CD4
T cells. A, CD4 T cells were preincubated for 1 h with either medium or
CO gas (200 ppm) and were stimulated for 10 min with anti-CD3 plus
anti-CD28 Abs. The cells were harvested, stained with either isotype con-
trol (open histogram) or anti-p-ERK Abs (filled histogram) and were an-
alyzed by flow cytometry. B, CD4 T cells were preincubated for 1 h with
RuCO (20 M) and stimulated for 5 or 10 min with anti-CD3 plus anti-
CD28 Abs. Whole-cell lysates were prepared and the amounts of phos-
phorylated ERK1/2 (first panel), p38 (third panel), and JNK (fifth panel)
were assessed by Western blot. C, CD4 T cells were preincubated for 1 h
with medium, the ERK inhibitor U0126 (20 M), the p38 inhibitor
SB20358 (20 M), the JNK inhibitor SP600125 (20 M), RuCO (20 M), raising an interesting question of whether HO-1 expression could
or inhibitors plus RuCO and were stimulated for 4 days with anti-CD3 plus
anti-CD28 Abs in the presence or absence of IL-2 (50 U/ml). Proliferation
assay was performed, as described in Materials and Methods. Values are
the mean SEM of six triplicate experiments.
4749
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FIGURE 6. Effects of RuCO on IL-2 secretion in Jurkat T cells trans-
fected with DN-MEK1 or CA-MEK1. Jurkat T cells were transfected with
empty vector (Vector), DN-MEK1, or CA-MEK1. The effects of DN-
MEK1 and CA-MEK1 expression on ERK phosphorylation were deter-
mined by Western blot (top panel). Transfected cells were preincubated for
1 h with RuCO (20 M) and stimulated for 48 h with anti-CD3 plus
anti-CD28 Abs (bottom panel). IL-2 secretion was measured, as described
in Materials and Methods. Values are the mean SEM of six triplicate
experiments.
lymph node CD4:CD8 T cell ratios with numerous activated
CD4 T cells (4, 5). It has also been reported that splenocytes
isolated from HO-1-overexpressing mice tend to display lower
proliferation indices against allogeneic stimulation in both CD4
and CD8 subsets and markedly increased allotransplant survival
by inhibiting infiltrations of inflammatory cells and CD4 T cells
(29). The administration of a HO-1 inducer to normal mice results
in suppressions of T cell-mediated cytotoxicity and Th1-mediated
cytokine production and decreases in the lymphoproliferative al-
loresponse and differentiation of CTLs (30). HO-1 inducer also
prevents the induction of T cell-mediated experimental auto-
immune encephalomyelitis in rats (31). In accord with these ob-
servations, we recently demonstrated that human CD4 T cells
express HO-1 and that HO-1-overexpressing Jurkat T cells tend to
display lower proliferative response. In the present study, we ex-
plored the mechanism(s) by which HO-1 mediated its antiprolif-
erative effect on CD4 T cells and found that the HO-1/CO system
was an important regulator of T cell responses. CO suppressed
proliferation and IL-2 secretion of CD4 T cells, most likely by
inhibiting ERK activation.
The exact mechanisms of HO-1 expression in activated T cells
are currently unknown. However, the antiproliferative and anti-
inflammatory IL-10 produced by activated CD4 T cells was in-
volved at least in part in HO-1 expression in T cells (Fig. 7),
be involved in modulating T cell responses. Pharmacological in-
duction or gene transfer of HO-1 in human T cells was antiprolif-
erative (Fig. 1). Among the three HO-1 byproducts, only both ex-
ogenously added and endogenously generated CO-suppressed T
cell proliferation (Fig. 2), mimicking the antiproliferative action of
4750
FIGURE 7. Effects of IL-10 on HO-1 expression in CD4 T cells.
CD4 T cells were preincubated with medium, IL-10 (50 U/ml), or anti-
IL-10 neutralizing Ab (2 g/ml) and were stimulated for 48 h with anti-
CD3 plus anti-CD28 Abs. The cells were harvested, stained with either
isotype control (dotted histogram) or anti-HO-1 Abs (solid histogram), and
analyzed by flow cytometry. One of three experiments is shown.
HO-1. Neither bilirubin nor free iron showed any effect on prolif-
erative response under our experimental conditions. CO blocked
the cell cycle entry of T cells, which was independent of guanylate
cyclase/cGMP pathway (Fig. 3). However, it was of great interest
to observe that CO was not inhibitory to cell cycle progression if
the T cell activation already progressed beyond TCR signaling
(Fig. 3, AC). This suggests that CO blocks early events in T cell
activation. CO also inhibited IL-2 secretion in CD4 T cells stim-
ulated with anti-CD3 plus anti-CD28 Abs (Fig. 4). This inhibitory
effect of CO on the IL-2 secretion appears to be responsible for the
antiproliferative action of CO, because T cell proliferation oc-
curred when IL-2 was exogenously added to the culture (Fig. 4D).
At high doses, CO also inhibited both IFN- and IL-10 secretions
(Table I), but the additional inhibitions of these cytokines by CO
were not associated with the antiproliferative effect of CO (Fig.
4D). MAP kinase plays an important role in IL-2 secretion, and
there exists a strong correlation between decreased IL-2 secretion
and the inhibition of ERK activation in T cells (13, 14). In support
of these findings, our data clearly showed that CO selectively in-
hibited the ERK pathway in the activated T cells (Fig. 5). Further-
more, U0126, a selective inhibitor of ERK activation, suppressed
CO SUPPRESSES T CELL PROLIFERATION VIA IL-2 INHIBITION
T cell proliferative response, but not in the presence of exog-
enously added IL-2 (Fig. 5C). Similarly, an expression of DN-
MEK1 inhibited IL-2 secretion (Fig. 6). These findings led us to
suggest that CO might be able to inhibit the ERK activation, which
leads to inhibition of IL-2 secretion, eventually resulting in sup-
pression of T cell proliferation in response to the simultaneous
ligation of CD3 and CD28 Abs.
Naive CD4 T cells could be potentially exposed to endogenous
CO produced by CD4CD25 regulatory T cells, which consti-
tutively express HO-1 (1), as well as activated CD4CD25 re-
sponder T cells, which can express HO-1 after stimulation. Thus,
we speculate that CO may directly and/or indirectly affect T cell
responses in vivo. It is of interest that CO shows cGMP-indepen-
dent suppressive effects on T cell responses and it can inhibit ERK
activation in activated CD4 T cells. CO could indirectly inhibit
ERK phosphorylation, probably by blocking upstream signals of
MEK (Fig. 6). It could be possible that CO activates the small G
protein, Rap1, which has been shown to inhibit ERK activation by
blocking Ras-dependent activation of the MAP kinase kinase ki-
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nase, Raf-1 (32). Further studies are in progress to explore poten-
tial mechanisms of CO effects on ERK signaling pathways.
In summary, our findings suggest that HO-1/CO induces sup-
pressive effects on T cell proliferation and IL-2 secretion, possibly
via its inhibition of the ERK MAP kinase pathway, which is cur-
rently believed to be an important signaling pathway for mediating
T cell activation. Our findings may contribute not only to our
deeper understanding of the basic roles of HO-1 in the immune
system, but also to our search for novel targets for new therapeutic
approaches to modulate T cell-mediated immune responses.
Acknowledgments
We thank Dr. W. K. Paik for critical reading of the manuscript,
Dr. Augustine M. K. Choi for helpful discussions on a CO incubator, and
Drs. K. Y. Choi and J. S. Chun for the kind gifts of DN-MEK1 and CA-
MEK1 genes.
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