The experimental protocol was approved by the institutional animal ethics committee of Shanghai Tenth People Hospital of Tong Ji University (Shanghai, China).

A total of 18 male C57BL/6 mice, 8–10-weeks-old, were purchased from Schleck Experimental Animals Co. (Shanghai, China). All mice were housed in a pathogen-free facility, maintained at 26°C under 12 h light/dark cycle and had access to food and water ad libitum. They were used in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals of the Chinese Academy of Sciences (5).

KIT proto-oncogene receptor tyrosine kinase (CD117)-fluorescein isothiocyanate (FITC) (dilution 1:200; cat. no. 48-1171-80; eBioscience, Inc., San Diego, CA, USA), anti-mouse F4/80-allophycocyanin (APC) (dilution 1:20; cat. no. 47-4801-80; eBioscience, Inc.), anti-mouse CD11b-FITC (dilution 1:40; cat. no. 47-0118-41; eBioscience, Inc.), Fc fragment of IgE receptor Ia (FCεRIα) -phycoerythrin (PE) (dilution 1:10; cat. no. ab124529; Abcam, Cambridge, UK) and anti-mouse CCR7 (dilution 1:200; cat. no. 25-1971-63; eBioscience, Inc.) antibodies were used. The metalloporphyrins, hemin (an HO-1 inducer) and zinc protoporphyrin (Znpp; an HO-1 inhibitor), were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Sodium cromoglicate (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) β-actin (1:2,000; cat. no. A2228; Sigma-Aldrich; Merck Millipore) and antibody against HO-1 (1:1,000; cat. no. ab13248; Abcam).

C57/BL6 mice were sacrificed by anesthesia with intraperitoneal injection of ketamine (90 mg/kg) (Sigma-Aldrich; Merck Millipore) and xylazine (10 mg/kg) (Sigma-Aldrich; Merck Millipore) solution and test for loss of reflexes to ensure deep narcotization. Then, non-parenchymal cell suspensions were acquired from C57/BL6 mice using in situ collagenase perfusion of liver and KCs were isolated by sedimentation in a two-step Percoll gradient with selective adherence of cells to plastic flasks as previously described (3). Cell viability was determined by trypan blue exclusion, and the purity of the KC fraction was determined using anti-mouse F4/80-APC (dilution 1:20) and anti-mouse CD11b-FITC antibodies (dilution 1:40). Murine bone marrow-derived mast cells (BMMCs) and DCs (BMDCs) were obtained as described previously (17,18). The cells were collected 8×10⁵ and centrifuged at 135 × g 5 min at 4°C, then the cells were resuspended in PBS and 2% fetal bovine serum 200 µl. All cells were incubated with the antibody for 30 min on ice, then washed twice with PBS and 2% serum and centrifuged at 1,200 × g for 5 min, analysis was performed using FlowJo 7.6. The purity of BMMCs was assessed by measuring the expression of CD117 and FCεRIα using flow cytometry. BMMCs were used at a purity of 95%. The purity of DCs was analyzed by measuring CD11c expression using flow cytometry.

Total RNA was isolated from KCs using TRIzol (Sigma-Aldrich; Merck Millipore) according to standard procedures. Thereafter, 2 µg of total RNA was reverse transcribed to cDNA using the Superscript III Transcription kit (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with Genomic DNA Eraser (Takara Biotechnology Co., Ltd., Dalian, China) according to manufacturer's protocol. PCR was performed on a Px2 Thermal Cycler, using the following conditions: 1 cycle of denaturation at 95°C for 5 min, 35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 40 sec and an additional cycle of extension at 72°C for 10 min. Then 2% gel was used. GAPDH was used as an internal control. qPCR was performed on a Chromo4 Four-Color Real-Time PCR Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using the SYBR Premix Ex Taq II (Tli RNaseH Plus) kit (Takara Biotechnology Co., Ltd.). Using the following conditions: Initial the cycle of denaturation at 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec, annealing 60°C for 30 sec and extension 70°C for 15 sec. Data was normalized using the 2^−ΔΔCq method (19). PCR was performed on an ABI Prism 7700 (Applied Biosystems; Thermo Fisher Scientific, Inc.). For linear amplification, GAPDH was used as an internal control. The following PCR primers were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China): HO-1, 5′-ACGCATATACCCGCTACCTG-3′ (forward) and 5′-TGCTGATCTGGGATTTTCCT-3′ (reverse); GAPDH, 5′-TCCCTCAAGATTGTCAGCAA-3′ (forward) and 5′-AGATCCACAACGGATACATT-3′ (reverse).

Reagents were purchased from Sigma-Aldrich unless otherwise indicated. Proteins were extracted from KCs and western blotting was performed. Briefly, 2.5–6×10⁶ cells were incubated for 15 min on ice in lysis buffer [50 mM TrisHCl pH 8.0; 120 mM NaCl; 0.25% Nonidet P40; 0.1% SDS; and protease inhibitors phenylmethylsulfonyl fluoride, aprotinin, leupeptin, and pepstatin (Roche Diagnostics) at a final concentration of 10 ng/ml, 1 mM DTT]. A total of 60 mg, of each protein sample was subjected to 15% SDS-PAGE and blotted onto a nitrocellulose membrane (GE Healthcare Life Sciences, Chalfont, UK). The protein quantification performed using a BCA kit according to the manufacturer's protocol (Sigma-Aldrich; Merck Millipore). Membranes were blocked with 5% non-fat dry milk in TBS-Tween (0.5%) 4°C overnight and probed with either rabbit anti-mouse HO-1 monoclonal antibody (2 mg/ml; Abcam) or rabbit anti-mouse β-actin monoclonal antibody (1:2,000) followed by horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:3,000; cat. no. RPN4301; GE Healthcare Life Sciences, Logan, UT, USA). Immunoreactive protein bands incubated with the enhanced chemiluminescence (ECL) reagent 30 sec according to the manufacturer's protocol using an ECL detection kit (cat. no. RPN998; GE Healthcare Life Sciences) and were visualized with image lab 4.0 software.

After treating KCs with PBS, dimethyl sulfoxide (DMSO), 50 µM/l hemin or 50 µM/l Znpp for 8 h, cells were collected and cultured in 24-well cell culture plates at a density of 2.5×10⁵ cells per 200 µl, either with direct contact with MCs or without MCs (5×10⁵ cells), separated by a Transwell chamber 0.4 µm. The 50 µM/l sodium cromoglicate was used to pretreat the MC as the stabilization control. After 24 h, each group of MCs were pre-incubated with anti-dinitrophenol (DNP)-IgE (100 ng/ml) (1:1,000, cat. no. D8406; Sigma-Aldrich; Merck Millipore) for 24 h and subsequently challenged using 100 ng/ml dinitrophenol-human serum albumin DNP-HSA. After 1 h, the cell supernatant of the co-culture system was collected. Following solubilization with 0.5% Triton X-100 in Tyrode's buffer, the enzymatic activity of β-hexosaminidase in supernatants and cell pellets was measured using p-nitrophenyl-N-acetyl-β-D-glucosaminide in 0.1 M sodium citrate, pH 4.5, at 37°C for 60 min. The reaction was halted by addition of 0.2 M glycine (pH 10.7) and the amount of p-nitrophenol released was measured by absorbance at a wavelength of 405 nm using a spectrophotometer. The extent of degranulation was calculated as the p-nitrophenol absorbance of the supernatant/the total absorbance of the supernatant and detergent-solubilized cell pellet.

MCs were cultured with KCs in 10% serum that were pretreated with PBS, DMSO, hemin or Znpp 24 h, using 50 µM/l sodium cromoglicate to pretreat the MCs as the stabilization control, then MC degranulation was stimulated with anti-DNP-IgE associated with DNP-HSA. DC migration was assessed using Transwell assays (8 µm pore size). DCs were incubated in 10% serum in upper chambers at a density of 5×10⁵ cells per well, MCs by pretreated KCs in the lower chamber. After 48 h, cells in the upper chamber were discarded and DCs that had migrated to the lower chamber harvested. Cells in the lower chamber were fixed with 10% methyl alcohol, stained with 0.1% crystal violet for 20 min, washed with double distilled H₂O and counted under an inverted microscope, at a magnification of ×200.

Additionally, DCs were cultured with stimulated MCs for 24 h in Transwell plates Supernatants were harvested from lower chamber of the co-culture system, and prostaglandin E2 (PGE2) (cat. no. H6-KA0324; eBioscience USA), C-C motif chemokine ligand 19 (CCL-19) (cat. no. RK-KOA0264; eBioscience USA) and CCL21 (cat. no. 85-88-58214-22; eBioscience USA) were quantified by ELISA (eBioscience, Inc.) according to the manufacturer's instructions. DCs were incubated with immunofluorescent CD16/31 (dilution, 3 µl for 10⁶ cells) and anti-mouse CCR7 antibodies for 30 min on ice then washed twice. Flow cytometry was performed using Flow Jo 7.6 software.

All statistical analysis was carried out using SPSS 9.13 (SPSS, Inc., Chicago, IL, USA) and results are presented as the mean ± standard deviation. One-way analysis of variance along with Dunnett's post hoc test was performed to determine the statistical significance of the data. P<0.05 was considered to indicate a statistically significant difference.

HO-1 mRNA and protein levels in KCs cultured with PBS, DMSO, hemin or Znpp were measured by RT-PCR, RT-qPCR and western blotting. HO-1 mRNA was significantly increased 8 h after exposure to hemin compared with incubation with PBS, DMSO or Znpp (Fig. 1). Consistent with these results, western blot analysis demonstrated that HO-1 protein levels were lower in PBS, DMSO and Znpp-treated groups after 8 h, compared with the hemin-treated group (Fig. 2).

KCs were pretreated with PBS, DMSO, hemin or Znpp for 8 h and cultured either in contact with MCs or separately (Fig. 3). After 24 h, MC degranulation was stimulated with anti-DNP-IgE and DNP-HSA, and enzymatic activity of β-hexosaminidase was used to estimate the level of MC degranulation usually. Following treatment with hemin, a decrease in β-hexosaminidase release was observed in KCs that were in contact with MCs, and also those that were separate from MCs. There was no difference between the sodium cromoglicate group and hemin group demonstrating that upregulation of HO-1 in KCs may inhibit MC degranulation and stabilize MC membranes.

DC migration from peripheral tissues to secondary immune organs, particularly lymph nodes, is a prerequisite for initiating an effective immune response, and CCR7 expression at the surface of DCs facilitates homing (20,21). MC membranes were stabilized by hemin-pretreated KCs, whether cultured in contact or separately as mentioned above, therefore MCs cultured separately were selected for use in further experiments. Degranulation was stimulated and they were co-cultured with DCs. After 24 h, expression of CCR7 at the DC surface was decreased in hemin and sodium cromoglicate-treated groups compared with PBS, DMSO and Znpp-treated groups (Fig. 4), and there was no difference between the sodium cromoglicate group and hemin group.

Transwell plates were used to investigate DC migration (Fig. 5), and five areas were chosen randomly for cell counting under the inverted microscope at magnification of ×200. Pretreatment of KCs with hemin (Fig. 5D) upregulated HO-1, stabilized MC membranes and decreased migration of DCs to the lower Transwell chamber, compared with KCs pretreated with PBS (Fig. 5B), DMSO (Fig. 5C) or Znpp (Fig. 5E). Again, there was no difference between the sodium cromoglicate group (Fig. 5F) and hemin group, confirming that MC degranulation stimulated DC migration.

Stimulation of MC degranulation may result in the release of cytokines that influence DC migration. The levels cytokines PGE2, CCL19 and CCL21 were measured in supernatants from co-cultures using ELISA (Fig. 6). Compared with membrane-stabilized MCs, represented by hemin and sodium cromoglicate-treated groups, degranulated MCs produced significantly increased levels of all three cytokines. Due to the fact that increased MC degranulation led to increased DC migration, these cytokines therefore potentially contributed to the changes observed in DC migration.