ORIGINAL RESEARCH PAPER

Inhibiting mast cell degranulation by HO-1 affects dendritic cellmaturation in vitro

Yuan-yuan Ma • Mu-qing Yang • Chun-feng Wang •

Jing Ding • Ji-yu Li

Received: 11 June 2013 / Revised: 5 February 2014 / Accepted: 12 February 2014 / Published online: 7 March 2014

� Springer Basel 2014

Abstract

Objective and design Mast cell (MC) degranulation can

break peripheral immune tolerance. However, its mecha-

nism remains unclear. Our goal was to study the

stabilization of MC membranes by heme oxygenase-1

(HO-1) in order to influence dendritic cell (DC) function.

Material Mast cells and dendritic cells were prepared

from 8-week-old to 10-week-old C57BL/6 mice; spleen

mononuclear cells (SMCs) were prepared from 8-week-old

to 10-week-old C57BL/6 and Balb/c mice.

Treatment Mast cells were pretreated with PBS, DMSO,

Hemin (50 ll/ml), and Znpp (50 ll/ml) for 8 h.

Method Real-time PCR and western-blot tested the HO-1

of MC mRNA and protein. The co-stimulatory molecules

of DCs (CD80, CD86, CD40) were measured by flow

cytometry, and levels of TNF-a, IL-6, and IFN-c were

measured by ELISA. We set up a one-way mixed lym-

phocyte reaction (MLR) model to test the proliferation of

SMCs after MC/DC interaction. *P \ 0.05 (t test) was

taken as the level of statistical significance.

Result MCs pretreated with hemin induced HO-1 mRNA

and protein expression, then interacted with DCs; expres-

sion of the co-stimulatory molecules was attenuated. The

TNF-a, IL-6, and IFN-c levels in the co-culture system

were decreased. These DCs couldn’t stimulate the prolif-

eration of SMCs.

Conclusion Inhibiting MC degranulation by HO-1

restrained DC maturation and attenuated the proliferation

of SMCs.

Keywords Mast cells � Heme oxygenase-1 �Degranulation � Dendritic cells � Immune tolerance

Introduction

Mast cells (MCs) have been well-established as mediators

of a variety of pathological conditions. For example, MCs

are the principal effector cells responsible for hives,

eczema, hay fever, and asthma associated with IgE-medi-

ated inflammatory reactions to allergen exposure [1]. As

long-lived cells, MCs can have enormous impacts on the

tissue microenvironment through release of a wide variety

of preexisting and cell-synthesized mediators such as pro-

teases, cytokines, chemokines, and arachidonic acid

metabolites. The important role of MCs in response to

certain parasite infections is well-accepted. In recent years,

the contribution of MCs in many other aspects of host

defense has been recognized [2–4]. Rapidly activated by a

variety of mechanisms in response to bacterial infection,

MCs can be crucial for the early recruitment of effector

cells such as neutrophils. During certain viral infections,

MCs might also be an important early source of specific

cytokines and chemokines. Recent studies suggest a com-

plex role for MCs in regulating both the nature and

intensity of immune responses to infection [5, 6], including

modulation of dendritic cells (DCs), B-cells, T-cells, and

regulatory T-cell responses. In 2006, Lu et al. discovered

that induction of immune tolerance in skin grafts was

Responsible Editor: Bernhard Gibbs.

Y. Ma � M. Yang � J. Li (&)

Department of General Surgery, Shanghai Tenth People’s

Hospital of Tong Ji University, Shanghai, China

e-mail: leejiyu@sina.com

M. Yang � C. Wang � J. Ding

Department of General Surgery, Xinhua Hospital Affiliated

Shanghai Jiao Tong University School of Medicine, Shanghai,

China

Inflamm. Res. (2014) 63:527–537

DOI 10.1007/s00011-014-0722-8 Inflammation Research

123

impaired in MC-deficient mice, whereas reconstitution of

bone marrow-derived MCs into the W/Wv mouse suc-

cessfully induced immune tolerance. These results

indicated that MCs might play an important role in immune

tolerance [7], but the underlying mechanism is not com-

pletely understood. As the polarization of T-helper cell

responses is driven by DCs, we asked whether MCs could

influence DC maturation and function. MCs have a bidi-

rectional role in immune responses. MCs induce regulatory

T cells (Tregs) in Th17-mediated autoimmune disease [8,

9]. MC degranulation also breaks peripheral immune tol-

erance, resulting in immune rejection [10]. Understanding

how to stabilize MC membranes and induce MC devel-

opment is, therefore, a major focus of attention in efforts to

promote immune tolerance.

Heme oxygenase-1 (HO-1) plays a pivotal protective

role in many disease models via its anti-inflammatory, anti-

apoptotic, and anti-proliferative actions [11–13]. HO-1 also

plays an important role in allograft immune response.

Takamiya et al. have demonstrated that HO-1 stabilizes

MCs in order to exercise an anti-inflammatory effect

through bilirubin [14]. We initially found that upregulation

of HO-1 in a donor liver could stabilize MC membranes to

ease ischemia reperfusion injury. We, therefore, hypothe-

sized that HO-1 could stabilize the MC membranes to

protect grafts from immune rejection.

Dendritic cells (DCs), the most potent of the antigen-

presenting cells (APCs), are known to play critical roles in

triggering immunity to many types of antigens. Mature

DCs present antigens to T cells to promote immune

rejection, whereas immature DCs (imDCs) have immune

tolerance [15]. ImDCs encountering antigens undergo a

maturation process that is associated with enhanced

expression of the co-stimulatory molecules CD40, CD80,

and CD86. We found that resting/stimulated MCs can

interact with DCs in an immune response and subsequent

T-cell activation. Thus we assumed that HO-1 upregulation

in MCs could ultimately affect DCs’ role in immune

responses.

Materials and methods

Mice

8-week-old to 10-week-old male C57BL/6 and BALB/c

mice were purchased from Schleck Experimental Animals

Co. (Shanghai, China). All mice were housed in a specific

pathogen-free facility and used in accordance with the

National Institutes of Health Guide for the Care and Use

of Laboratory Animals of the Chinese Academy of

Sciences.

Antibodies and reagents

Mouse antibodies directed against the following murine

antigens were used: CD117-FITC (clone ACK45), CD86-

FITC (clone PO3), CD40-APC (clone 3/23), CD80-PE

(clone 16-10A1), CD11c-FITC (clone N418), and FcaRI-

PE (clone MAR-1) from eBioscience (San Diego, CA,

USA). MHCII-FITC was obtained from eBioscience. The

metalloporphyrins hemin (an HO-1 inducer) and zinc

protoporphyrin (ZnPP, an HO-1 inhibitor) [16] were pur-

chased from Alexis Biochemicals, Inc. (San Diego, CA,

USA). Sodium cromoglicate and polyclonal antibodies

against b-actin were obtained from Sigma-Aldrich (St.

Louis, MI, USA). Antibodies against HO-1 were acquired

from Abcam (Cambridge, MA, USA).

Cell preparation

Murine bone marrow-derived mast cells (BMMCs)

Murine BMMCs were obtained as described previously.

Briefly, BM cells were isolated from femurs of C57BL/6

mice, cultured in complete RPMI (Gibco, USA), and sup-

plemented with 10 % FBS (Gibco, USA), 100U/mL

penicillin/streptomycin (Gibco, USA), 10 ng/mL recom-

binant murine IL-3 (Pretech, Rocky Hill, NJ), and 10 ng/

mL recombinant murine SCF (Pretech, Rocky Hill, NJ) at

37 �C and 5 % CO2. Four days later, non-adherent cells

were carefully removed and replaced with fresh culture

medium to enhance the purity of the MCs. This last step

was repeated every 6 days until adherent cells disappeared

after five to six passages. The purity of BMMCs was

assessed by expression of c-kit (CD117) and FCeRIa using

flow cytometry. BMMCs at a purity of 95 % were used.

Murine bone marrow-derived dendritic cells (BMDCs)

BM was extracted from tibias and femurs of C57bl/6 mice,

and cell suspensions were cultured for 6 days in RPMI

containing 10 % heat-inactivated FBS (Gibco, USA),

100U/mL penicillin/streptomycin (Gibco, USA), and

10 ng/mL recombinant murine GM-CSF (Peprotech,

Rocky Hill, NJ). Fresh medium was given every other day.

On day six, loosely adherent cells were harvested, washed,

and replated in fresh medium for an additional 24 h. Phe-

notypic analysis and functional assays were performed at

day seven. These cells were immature; the mature cells

could be acquired after stimulation with 100 ng/mL lipo-

polysaccharide (LPS) (Sigma-Aldrich, Munich, Germany)

for 24–48 h. Purity of DCs was analyzed by CD11c

expression using flow cytometry. For our experiments, DCs

at a purity higher than 95 % were used.

528 Y. Ma et al.

123

Murine spleen mononuclear cells (SMCs)

SMCs were obtained by homogenization of spleens from

BALB/c mice with Ficoll (Sigma-Aldrich) by the manu-

facturer’s protocol.

Reverse transcription (RT) real-time polymerase chain

reaction (PCR)

Total RNA was isolated from MCs using TRIzol (Sigma-

Aldrich) according to a standard protocol. Thereafter, 2 lg

of total RNA was reverse transcribed using the Superscript

III Transcription Kit (Invitrogen, Carlsbad, CA, USA) and

random primers (Roche, Basel, Switzerland). Real-time

PCR was performed on an ABI Prism 7700 (Applied

Biosystems, Foster City, CA, USA). For linear amplifica-

tion, Glyceraldehyde-3-phosphate dehydrogenase (Gapdh)

was used as an internal control for HO-1 (Hmox1). The

following PCR primers were synthesized by Shanghai

Sangon Biological Engineering Technology & Services

Co., Ltd:

Hmox1:

Forward primer, 50-ACGCATATACCCGCTACCTG-30

Reverse primer, 50-TGCTGATCTGGGATTTTCCT-30

Gapdh:

Forward primer, 50-TCCCTCAAGATTGTCAGCAA-30

Reverse primer, 50-AGATCCACAACGGATACATT-30.

Protein extraction and Western blotting

Proteins were extracted from BMMCs and assayed by

Western blot. Briefly, 2.5 to 6 9 106 cells were incubated

for 15 min on ice in lysis buffer (50 mM Tris HCl, pH

8.0, 120 mM NaCl, 0.25 % Nonidet P40, 0.1 % SDS;

Sigma-Aldrich) containing the protease inhibitors phe-

nylmethanesulfonylfluoride (PMSF), aprotinin, leupeptin,

and pepstatin (Roche; each at a final concentration of

10 ng/mL), and 1 mM dithiothreitol (DTT) (Sigma-

Aldrich). A quantity of 60 mg of each protein sample was

applied to a 15 % SDS-PAGE gel. Following electro-

phoresis, proteins were blotted onto a nitrocellulose

membrane (Amersham Pharmacia Biotech, USA). Mem-

branes were blocked with 5 % non-fat dry milk in TBS–

0.5 % Tween and then probed with either rabbit anti-

mouse HO-1 monoclonal antibody (2 mg/mL; Abcam) or

rabbit anti-mouse b-actin monoclonal antibody (Bioworld,

USA) followed by HRP-conjugated anti-rabbit IgG anti-

body (Amersham Pharmacia Biotech). Immunoreactive

protein bands were detected with the ECL detection kit

(Amersham Pharmacia Biotech).

One-way mixed lymphocyte reaction (MLR)

We set up MLR as previously described [17]. Briefly,

BALB/c and C57BL/6 mice SMCs were harvested, incu-

bated with mitomycin C (50 lg/ml, Sigma-Aldrich), and

used as responders separately. Varying ratios of BMMCs

were co-cultured with 2 9 105 allogeneic SMCs (10:1,

50:1, and 100:1) in rounded-bottom 96-well plates in a

total volume of 200 lL medium. Triplicate wells were set

up for each reaction and ratio. The reactions were incu-

bated for 6 days at 37 �C and 5 % CO2 in a humidified

incubator. [methyl-3H] thymidine (1 lCi/well; Bhaba

Atomic Research Centre, Mumbai, India) was added 18 h

before the end of the incubation period. The incorporation

of [methyl-3H] thymidine in proliferating SMCs was

measured using a beta-counter (Beckman-Coulter, Brea,

CA, USA) and expressed as counts per minute (cpm).

BMMC degranulation assay After treating MCs with

PBS, DMSO, hemin, or ZnPP for 8 h, BMMCs were pre-

incubated with anti-DNP-IgE (100 ng/mL) for 24 h then

challenged by DNP-HAS (200 ng/mL). After 1 h, condi-

tional media from these IgE/Ag-degranulated BMMCs

were mixed into the culture system. After solubilization

with 0.5 % Triton X-100 in Tyrode’s buffer, the enzymatic

activities of b-hexosaminidase in supernatants and cell

pellets were measured with p-nitrophenyl N-acetyl-b-D-

glucosaminide in 0.1 M sodium citrate (pH 4.5) for 60 min

at 37 �C. The reaction was stopped by addition of 0.2 M

glycine (pH 10.7). The release of the product 4-p-nitro-

phenol was detected by absorbance at 405 nm. The extent

of degranulation was represented as the percentage of 4-b-

nitrophenol absorbance in the supernatants over the sum of

absorbance in the supernatants and in cell pellets solubi-

lized in detergent [18]. To test how the upregulation of

HO-1 in MCs impacted MLR, MCs were treated with PBS,

DMSO, hemin, or ZnPP for 8 h, washed twice, then added

to MLR with an anti-DNP-IgE mAb associated with DNP-

HAS.

Analysis of pretreated MC–DC interaction and DC

maturation

BMMCs pretreated for 8 h stimulated MC degranulation

with anti-DNP IgE mAb associated with DNP-HAS.

BMDCs were cultured for 24 h at 2.5 9 105 cells/200 uL/

well in 24-well cell culture plates and afterwards incubated

with: i) imDC; ii) LPS (100 ng/mL) (imDC ? LPS); iii)

BMMCs pretreated with PBS ? imDC; iv) BMMCs pre-

treated with DMSO ? imDC; v) BMMCs pretreated with

hemin ? imDC; vi) BMMCs pretreated with ZnPP ?

imDC. In each sample, DCs and MCs were plated at a 1:1

ratio. Supernatants were harvested and bioactive IFN-c,

Inhibiting mast cell degranulation 529

123

TNF-a, and IL-6 were quantified by ELISA (eBiosciences)

according to the manufacturer’s instructions. DCs were

incubated with several immunofluorescent (PE-CD80,

APC-CD40, FITC-CD86, CD11c-PerCP-Cy5.5) antibodies

for 30 min on ice. Flow cytometry analysis was performed

using a FACS Calibur flow cytometer (BD BioSciences)

equipped with Cell Quest software.

SMCproliferation assays

C57BL/6 DCs were incubated with or without C57BL/6-

derived BMMCs for 24 h as described above. Subse-

quently, the DCs were removed and washed. The DCs were

co-cultured with SMCs which were inactivated by mito-

genic enzyme in 96-well cell culture plates. SMC

proliferation was measured following the addition of

1 lCi/well of [methyl-3H] thymidine as above.

Statistical analysis

All statistical analyses were done using SPSS9.13 for

Windows and expressed as the mean ± SEM. One-way

analysis of variance (ANOVA) was performed to deter-

mine the statistical significance of the data. Each sample

was tested for homogeneity of variance with the Student–

Newman–Keuls multiple comparison test for two-variable

analysis. If the variance was not homogeneous, we per-

formed nonparametric statistical tests. Differences were

considered as significant at *P \ 0.05.

Results

Hemin inhibits MC degranulation through upregulation

of HO-1 expression

We examined expression levels of HO-1 mRNA and pro-

tein in MCs treated with PBS, DMSO, hemin, or ZnPP by

real-time PCR, and Western blot, respectively. Figure 1a

shows HO-1 (Hmox1) mRNA expression in each MC group

after 8 h of treatment. The PCR analysis showed that HO-1

mRNA level was only increased in the hemin-treated group

(Fig. 1a). Consistent with the mRNA level, HO-1 cellular

protein level was also evaluated after hemin treatment

(Fig. 1b, c). HO-1 mRNA and protein were expressed at a

low level following incubation with PBS, DMSO, or ZnPP.

These results showed that the expression levels of HO-1

increased and HO-1 accumulated within MCs due to the

presence of hemin.

We next examined the effect of HO-1 overexpression on

the degranulation of MCs (Fig. 2). After stimulation with

Fig. 1 Expression of HO-1

mRNA and protein after MC

pretreatment

Fig. 2 Effects of hemin-dependent HO-1 induction on MC degran-

ulation. a HO-1 was evaluated by real-time polymerase chain reaction

(quantitative real-time PCR) after pretreatment of MCs with PBS,

DMSO, hemin(50 um/l), or ZnPP(50 um/l). The expression of HO-1

(Hmox1) mRNA in the hemin group was significantly increased.

b The protein level of HO-1 was evaluated by Western blotting after

pretreatment, with similar results to real-time PCR, b-actin was used

as an internal control. c The bands corresponding to HO-1 and b-actin

protein were densitometrically scanned. The results are expressed as a

full-length HO-1/b-actin protein ratio. Data are shown as the

mean ± SD of three independent experiments performed in triplicate

(a) or a representative result (b and c). *P \ 0.05. MC degranulation

induced by anti –DNP-IgE mAb and DNP-HAS was evaluated by

4-Nitrophenyl N-acetyl-b-D-glucosaminide after pretreatment. Com-

pared with the control group, pretreatment with PBS, DMSO, or

ZnPP(50 um/l) obviously elicited MC degranulation, whereas the

hemin(50 um/l) and sodium cromoglicate groups were not signifi-

cantly different than the control group. Data are shown as the

mean ± SD of three independent experiments performed in triplicate.

*P \ 0.05.

530 Y. Ma et al.

123

anti-DNP-IgE mAb and DNP-HAS, we measured the b-

hexosaminidase release. Pretreatment of MCs with hemin

for 8 h led to a decrease in b-hexosaminidase release.

Furthermore, there was no difference between the sodium

cromglicate group and the unstimulated control group.

These results demonstrate that HO-1 is a potent inhibitor of

MC degranulation and stabilizes MC membranes.

Resting MCs restrain the proliferation of SMCs in MLR

To determine how resting MCs influence the MLR, Fig. 3

shows that using spleen mononuclear cells (SMCs) derived

from BALB/c mice as stimulator cells weakly elicited the

proliferation of SMCs from C57BL/6 mice, whereas SMCs

derived from C57BL/6 mice as responder cells distinctly

elicited the proliferation of SMCs from BALB/c mice.

These ex vivo findings indicate that when using C57BL/6

mice as donors, recipient BALB/c mice set up the MLR to

induce immune rejection. In an in vitro MLR model with

cocultured resting MCs, the proliferation of the responder

cells was dampened as shown by [methyl-3H] thymidine

incorporation, suggesting that resting MCs induce immune

tolerance to a certain degree. At the same time, we found

that when the ratio of MCs and SMCs is 1:100, MCs sig-

nificantly restrained one-way MLR.

MC degranulation prompts proliferation of SMCs

to break up immune tolerance, whereas HO-1

upregulation reduces MC degranulation

Since the proliferation of SMCs is significantly restrained

in the ratio of 1:100 in one-way MLR, cells cultured in that

ratio were used as control. Compared with control, induc-

tion of MC degranulation with anti-DNP-IgE mAb

associated with DNP-HAS significantly stimulated prolif-

eration of SMCs in one-way MLR (Fig. 4a). Pretreatment

of MCs was done with PBS, DMSO, hemin, or ZnPP in

one-way MLR. After MC activity was elicited, SMC pro-

liferation was found inhibited in the hemin-treated group,

which indicated the effect of HO-1 in MC degranulation

(Fig. 4b). These results demonstrate that upregulation of

HO-1 in MCs could reduce degranulation and lessen the

proliferation of SMCs in the immune rejection model of

one-way MLR.

The interaction between stabilized MCs and imDCs

blocks DC maturation by decreasing the immune

mediators IFN-c, TNF-a, and IL-6

How does MC degranulation prompt SMC proliferation? It

is known that proliferation of SMCs requires the presence

of the APC activation signal for stimulation. Given that

DCs are well-characterized, important, common APCs, we

hypothesized that MCs might interact with DCs to influ-

ence SMCs. After pretreatment with PBS, DMSO, hemin,

or ZnPP, we cocultured MCs with imDCs to induce MC

Fig. 3 Effect of resting MCs on the proliferation of reactive

lymphocytes in a one-way MLR model. A one-way MLR model

was set up in vitro with (1) BALB/c-derived SMCs; (2) C57BL/6-

derived SMCs; (3) mitomycin C-inactivated SMCs from BALB/c

mice as stimulator cells and SMCs from C57BL/6 mice as responder

cells; (4) mitomycin C-inactivated SMCs from C57BL/6 mice as

stimulating cells and SMCs from BALB/c mice as responder cells; (5)

a mixture of BMMCs from C57BL/6 mice in (3) at a MC:SMC ratio

of 1:10; (6) same as (5) but with MC:SMC 1:50; (7) same as (5) but

with MC:SMC 1:100; (8) a mixture of BMMCs from C57BL/6 mice

in (4) at a MC:SMC ratio of 1:10; (9) a mixture of BMMCs from

C57BL/6 mice in (4) at a MC:SMC ratio of 1:50; (10) a mixture of

BMMCs from C57BL/6 mice in (4) at a MC:SMC ratio of 1:100.

[methyl-3H] thymidine (1 lCi/well) was used to test proliferation.

Proliferation of SMCs was obviously highest in group (4) in the MLR

model compared with (1)(2)(3). On the basis of these findings,

different ratios of resting MCs were added to the MLR model. The

role of immune suppression is strongest in group (10)(MC:SMC

1:100). Statistical significance was calculated using Student’s t test

with *P \ 0.05

Fig. 4 Effect of activated MCs on proliferation of reactive lympho-

cytes in the one-way MLR model. a Anti-DNP-IgE associated with

DNP-HAS stimulated MC activation in the following groups: (1)

SMCs from BALB/c mice cultured alone; (2) mitomycin C-inacti-

vated SMCs from C57BL/6 mice as stimulator cells and SMCs from

BALB/c mice as responders; (3) added resting MCs from C57BL/6 to

(2), MC:SMC = 1:100;(4) added anti-DNP-IgE mAb associated with

DNP-HAS in (3). The active MC group could stimulate the

proliferation of SMCs vs (1), (2), and (3). Statistical significance

was calculated using the Student–Newman–Keuls test. *P \ 0.05.

b MCs were pretreated with (4) PBS, (5) DMSO, (6) hemin (50 um/l),

and (7) Znpp (50 um/l) before stimulation of degranulation with anti-

DNP-IgE and DNP-HAS. Compared with (3), which added resting

MCs in the MLR model, pretreatment with PBS, DMSO, and ZnPP

obviously enhanced proliferation, whereas induction of HO-1

expression with hemin significantly reduced proliferation. Statistical

significance calculated using Student’s t test. *P \ 0.05

Inhibiting mast cell degranulation 531

123

degranulation. To determine whether cells interact through

direct contact or cytokine-dependent pathways, both cells

were cocultured directly or in separated transwell cham-

bers. After 24 h, co-stimulation of CD80, CD86, and CD40

on the DC surface was measured by flow cytometry and

compared between imDCs and mature DCs induced by

LPS. We found that after LPS stimulation, co-stimulation

was enhanced (Fig. 5). MCs pretreated with PBS, DMSO,

or ZnPP primed imDCs to increase the surface expression

of CD80, CD86, and CD40 to different degrees. The ZnPP

group, in which the expression of HO-1 was decreased,

most obviously increased co-stimulation. However, there

was no significant difference between the hemin group and

imDCs. The same results were observed whether the MCs

and DCs were in contact or separated by a transwell. These

findings indicated that MC degranulation induced DC

maturation. Upregulation of HO-1 in pretreated MCs could

stabilize the MC membrane and restrain MC degranulation;

thus the capacity for stimulation of DC maturation was

attenuated. We, therefore, hypothesized that MC-mediated

maturation of DCs was dependent on MC degranulation.

To assess whether DC maturation was affected by soluble

MC mediators released following degranulation, we col-

lected the supernatant from our coculture system to detect

which MC-derived cytokines could prompt the maturation

of DCs. The cytokines were measured by an ELISA kit

(Fig. 6). We found that, compared with resting MCs (either

when cocultured together or separately), degranulated MCs

could produce more IFN-c, TNF-a, and IL-6. However,

these cytokines were kept to a low level in MCs which

were pretreated with hemin to upregulate HO-1 and sta-

bilized MC membranes to interact with DCs. In order to

Fig. 5 DC surface expression of co-stimulatory molecules was

decreased following crosstalk with MCs pretreated with hemin. The

expression of the co-stimulatory signals CD80, CD86, and CD40 on

DC surfaces were analyzed after 24 h coculture of imDCs with MCs

in direct contact [a (c, d, e, f), b (c, d, e, f), and c (c, d, e, f)] compared

with imDCs [a (a), b (a), and c (a)] and LPS-stimulated (100 ng/mL)

DCs [a (b), b (b), c (b)]. The co-stimulatory molecules of the hemin

group (DC & HMC) expressed at low levels. a (g, h, i, j), b (g, h, i, j),

and c (g, h ,i, j) showed that separated coculture of imDCs and MCs

had the same results as coculture with direct contact. d, e, and f show

the expression of the co-stimulatory signals CD80, CD86, and CD40

on DC surfaces as mean fluorescence intensity (MFI) measured by

flow cytometry after 24 h of the respective incubation (separate or

direct contact). MFI of the respective isotype control was subtracted.

Data are presented as the percentage of MFI of DCs alone, set as

100 %. Data are the mean ± SEM of three independent experiments.

Statistical significance was calculated using Student’s t test.

*P \ 0.05

532 Y. Ma et al.

123

verify whether these specific cytokines released by MC

degranulation played a role in maturation of DCs, we

added these cytokines to the supernatant of DCs, then

compared with the groups of i) DCs; ii) LPS (100 ng/mL)

(DCs ? LPS); iii) MCs pretreated with Znpp ? DCs. We

found that LPS, cytokines, and MCs pretreated with ZnPP

groups primed DCs to increase the expression of surface

molecular CD80, CD86, and CD40 to different degrees

(Fig 7a, b, c). These results indicated that DC maturation

might be a result of stimulation by the cytokines which

were released by MC degranulation.

Upregulation of HO-1 in MCs sustained the imDC state

to restrain SMC proliferation in one-way MLR

On the basis of the previous experiment we cocultured

different pretreated MCs and DCs at a 1:1 ratio then

stimulated MC degranulation. After 24 h, this coculture

system elicited the proliferation of SMCs, as shown by the

addition of 3H-TdR 18 h before the end of the incubation

period. Regardless of whether the MCs and DCs were in

direct contact or separated, Figure 8 shows that mature

DCs stimulated by LPS cocultured with MCs pretreated

with PBS, DMSO, or ZnPP stimulated proliferation of

SMCs more than imDCs. The mature DC group, and the

ZnPP groups had the greatest capacity for stimulation and

were not significantly different. The hemin and imDC

groups weakly elicited proliferation of SMCs and were not

significantly different. These results indicate that MC

degranulation induces DC maturation and, thus, enhances

the ability of DCs to stimulate the proliferation of SMCs.

Discussion

MCs have long been thought to only possess effecter cell

functions, given that they store an armada of pro-inflam-

matory cytokines and chemokines in their granules. More

recently, it has been suggested that MCs can also function

as an immunosuppressive cell population [5]. Consistent

with this possibility, MC-deficient mice subjected to

nephrotoxic serum nephritis (NTS) were found to develop

significantly increased disease indices compared with wild-

type control animals [19, 20]. Gri and coworkers first

provided evidence that Tregs might interact with MCs in

allergic disorders by limiting the degranulation of MCs

in vitro [18]. de Vriesa and colleagues showed that regional

or systemic antibody- or chemically-induced MC degran-

ulation could lead to a T-cell-dependent loss of tolerant

skin allograft, and the ‘‘natural’’ loss of tolerant allograft

could be reversed by blocking MC degranulation in vivo

[10]. These findings have underscored the enormous plas-

ticity of the MC compartments and their impact on

acquired immunity and acquired immune privilege [21].

Previous reports have shown that HO-1-transfected MCs

had decreased degranulation and anti-allergic activity [14–

22]. HO-1 is a rate-limiting enzyme that breaks down heme

into iron biliverdin, which is further converted to carbon

monoxide (CO) and the antioxidant bilirubin by biliverdin

reductase [23, 24]. Our current understanding of HO-1 is

that it is important in a wide variety of physiological and

pathological processes in addition to heme catabolism and

even maintains cellular homeostasis. Data showing that

HO-1 expression in a transplanted organ could inhibit the

rejection of that organ led to the more general concept that

expression of a ‘‘protective gene’’, i.e., HO-1, in a trans-

planted graft could promote its survival [25]. Therefore, we

wondered whether HO-1 upregulation could stabilize MC

membranes to affect immune tolerance. We used hemin

and ZnPP, which respectively upregulate and downregulate

HO-1, to treat MCs. We detected HO-1 protein and mRNA

levels and confirmed their up-regulation after hemin

treatment. After stimulating the MCs with IgE associated

with anti-DNP, we found that upregulation of the HO-1 in

MCs could stabilize their membranes. Resting MCs could

not stimulate the proliferation of SMCs in the MLR model,

but MCs activated to degranulate potently stimulated the

proliferation of SMCs. Furthermore, hemin-pretreated MCs

could upregulate HO-1 and reduce the proliferation of

SMCs.

Fig. 6 Cytokine changes in coculture supernatants. a TNF-a, b IFN-

c, and c IL-6 levels in the supernatant of the coculture system of each

group are presented as the mean ± SEM of at least four independent

experiments. Statistical significance compared with control group

(CMC) was calculated using Student’s t test. *P \ 0.05

Inhibiting mast cell degranulation 533

123

Fig. 7 The effect of cytokines

on DC maturation. i) DCs; ii)

LPS (100 ng/mL)

(DCs ? LPS); iii) MCs

pretreated with Znpp ? DCs;

iv) cytokines [TNF-a (10 ng/

mL), IFN-c (10 ng/mL), or IL-6

(20 ng/mL)] ? DCs. Then DCs

were incubated with several

immunofluorescent (PE-CD80,

FITC-CD40, FITC-CD86)

antibodies for 30 min on ice.

Flow cytometry analysis was

performed using a FACSCalibur

flow cytometer (BD

Biosciences) equipped with Cell

Quest software. Co-stimulatory

molecules of CD80, CD86, and

CD40 on DC surfaces were

compared between each group.

We found that LPS, cytokines,

and MCs pretreated with ZnPP

groups primed DCs to increase

the surface expression of CD80,

CD86, and CD40 to different

degrees.There was no

significant difference between

these three groups (*P \ 0.05)

(a, b, c). These findings

indicated that MC degranulation

might release the cytokines

IFN-c, TNF-a, and IL-6 to

stimulate DC maturation

534 Y. Ma et al.

123

How do MCs interact with SMCs to exert immune tol-

erance? In recent years, MCs have been recognized to

influence or be influenced by DCs, T cells, and B cells, thus

functioning as regulatory and/or effector cells [26].

Although previous studies have shown that MCs promote

the migration of DCs to draining lymph nodes during

bacterial infection [27, 28], the impact of MCs on the

regulation of the T cell-activating capacity of DCs remains

puzzling. To identify the underlying mechanisms, Dudeck

et al. found that the effect of MCs on DC function is to

promote Th1 and Th17 responses [29]. MCs have also been

shown to induce the release of various cytokines and

chemokines, enabling them to recruit other inflammatory

cells and to affect DCs [30]. The cytokines TNF-a, IL-6,

and IFN-c secreted from activated MCs play a pivotal role

[31, 32]. TNF-a and exosomes induced functional matu-

ration of DCs by upregulating expression of a6b4 and

a6b1 integrins along with the MHC class II molecules

CD80, CD86, and CD40 [33], whereas DCs, as the most

important APCs, were potent immunostimulators. Matu-

ration of DCs was characterized by increasing expression

of co-stimulatory molecules and improved capacity to ac-

tivatie antigen-specific T cells [34]. We, therefore, studied

the MC role that influenced the DC function on immunity

in vitro. We upregulated HO-1 in MCs, stimulated their

Fig. 7 continued

Fig. 8 Effect of DCs cocultured with activated pretreated MCs on the

proliferation of allogeneic lymphocytes. SMC proliferation was

reduced by stimulation with hemin-pretreated MCs that could

upregulate HO-1 expression to prime DCs. SMCs were incubated

alone or were incubated with imDCs, mDCs, PBS-pretreated MCs

(PMC), DMSO-pretreated MCs (DMC), hemin-pretreated MCs

(HMC), or ZnPP-pretreated MCs (ZMC) for 24 h. Pretreated MCs

were activated by anti DNP-IgE associated with DNP-HAS.

[methyl-3H] thymidine (1 lCi/well) was added before analysis. The

proliferation of SMCs was measured using a beta counter and

expressed as counts per minute (cpm). Compared with imDCs, mDCs,

and PMCs, DMCs and ZMCs had an enhanced capability to induce

SMC proliferation. However, SMC proliferation was not induced in

the HMC group. Data are represented as the mean ± SEM of five

experiments. Statistical significance was calculated using nonpara-

metric statistics. *P \ 0.05. b MCs and DCs were cocultured while

separated by a transwell. The results were similar to a

Inhibiting mast cell degranulation 535

123

degranulation, cocultured them with imDCs, and used

MLR to induce SMC proliferation. We found that the

group in which HO-1 upregulation stabilized MC mem-

branes showed reduced expression of DC co-stimulatory

molecules, maintained the immature state of DCs, and did

not have a substantial effect on SMC proliferation. Con-

versely, downregulation of HO-1 expression promoted MC

degranulation, expression of DC co-stimulatory molecules,

DC maturation, and SMC proliferation. These MC/DC

cocultures showed a significant release of TNF-a, IL-6, and

IFN-c, which could stimulate DC maturation. In vitro, we

used the one way-MLR to test immune responses. To avoid

the impact of cell-to-cell contact, we separated MCs and

DCs but found that the results were the same. Taken

together, these findings show that upregulation of HO-1 in

MCs could stabilize MC membranes and prevent their

degranulation to maintain the immature DC state and

ultimately alleviate the immune response. We thus dem-

onstrate a possible mechanism by which regulation of MC

degranulation induces immune tolerance in vitro.

Acknowledgments This study has been supported by Grants from

Young Scientist Project, National Natural Science Foundation of

China, No. 30600598; ‘‘Qi Ming Star for Young Scientist’’ Project,

Science and Technology Commission of Shanghai Municipality,

No.10QH1401800; ‘‘Shu Guang Scholar’’ Project, Shanghai Muni-

cipal Educational Commission, No.10SG20; the Key Medical Project

of Science and Technology Commission of Shanghai Municipality,

No. 09411952500; National Natural Science Foundation of China,

No. 81270555; Program for New Century Excellent Talents in Uni-

versity,No.NECT-13-0422.

We thank Dr. Zhang-yanyun and members of laboratory of the

Chinese Academy of Sciences. We would also thank Dr. Liu-jianwen,

Shen-ke.

Yuan-yuan Ma and Mu-qing Yang contributed equally. Responsi-

ble editor: Bernhard Gibbs.

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