Endogenous sulfur dioxide is a novel inhibitor of hypoxia-induced mast cell degranulation

Abstract

Introduction: Mast cell (MC) degranulation is an important step in the pathogenesis of inflammatory reactions and allergies; however, the mechanism of stabilizing MC membranes to reduce their degranulation is unclear.

Methods: SO₂ content in MC culture supernatant was measured by HPLC-FD. The protein and mRNA expressions of the key enzymes aspartate aminotransferase 1 (AAT1) and AAT2 and intracellular AAT activity were detected. The cAMP level in MCs was detected by immunofluorescence and ELISA. The release rate of MC degranulation marker β-hexosaminidase was measured. The expression of AAT1 and cAMP, the MC accumulation and degranulation in lung tissues were detected.

Objectives: To exam whether an endogenous sulfur dioxide (SO₂) pathway exists in MCs and if it serves as a novel endogenous MC stabilizer.

Results: We firstly show the existence of the endogenous SO₂/AAT pathway in MCs. Moreover, when AAT1 was knocked down in MCs, MC degranulation was significantly increased, and could be rescued by a SO₂ donor. Mechanistically, AAT1 knockdown decreased the cyclic adenosine monophosphate (cAMP) content in MCs, while SO₂ prevented this reduction in a dose-independent manner. Pretreatment with the cAMP-synthesizing agonist forskolin or the cAMP degradation inhibitor IBMX significantly blocked the increase in AAT1 knockdown-induced MC degranulation. Furthermore, in hypoxia-stimulated MCs, AAT1 protein expression and SO₂ production were markedly down regulated, and MC degranulation was activated, which were blunted by AAT1 overexpression. The cAMP synthesis inhibitor SQ22536 disrupted the suppressive effect of AAT1 overexpression on hypoxia-induced MC degranulation. In a hypoxic environment, mRNA and protein expression of AAT1 was significantly reduced in lung tissues of rats. Supplementation of SO₂ elevated the cAMP level and reduced perivascular MC accumulation and degranulation in lung tissues of rats exposed to a hypoxic environment in vivo.

Conclusion: SO₂ serves as an endogenous MC stabilizer via upregulating the cAMP pathway under hypoxic circumstance.

Keywords: Degranulation; Endogenous sulfur dioxide; Mast cells; Stabilization; cAMP.

Graphical abstract

Fig. 1

The presence of endogenous SO₂/AAT pathway in MCs. (A) The SO₂ content in the supernatant of MCs, VECs and VSMCs detected by HPLC-FD; (B) Relative AAT1 mRNA levels in MC, VEC and VSMC detected by real time RT-PCR; (C) Relative AAT2 mRNA levels in MC, VEC and VSMC detected by real time RT-PCR; (D) Relative AAT1 protein expression in MC, VEC and VSMC detected by western blot; (E) Relative AAT2 protein expression in MC, VEC and VSMC detected by western blot; (F) AAT activity in MC, VEC and VSMC detected by colorimetric analysis. *P < 0.05, **P < 0.01. The data are presented as the mean ± SD. The results in Fig. 1B-1C were normalized with those in MCs. All the research was carried out for three times independently.

Fig. 2

Knockdown of AAT1 promoted the degranulation of MCs. (A) The SO₂ content in the MC supernatant detected by HPLC-FD. NaHSO₃/Na₂SO₃ mixture (1:3 M/M) at a dose of 100 μM was used as a SO₂ donor; (B) cAMP level in MCs detected by immunofluorescence. NaHSO₃/Na₂SO₃ mixture (1:3 M/M) at a dose of 100 μM was used as a SO₂ donor; (C) The release rate of β-hexosaminidase in MCs detected by colorimetric analysis. NaHSO₃/Na₂SO₃ mixture (1:3 M/M) at a dose of 100 μM was used as a SO₂ donor; (D) The release rate of β-hexosaminidase in MCs treated with different concentration of SO₂ donor NaHSO₃/Na₂SO₃ mixture (50–200 μM) detected by colorimetric analysis; (E) The cAMP content in MCs treated with different concentration of SO₂ donor NaHSO₃/Na₂SO₃ mixture (50–200 μM) detected by ELISA. *P < 0.05 and the data are presented as the mean ± SD. All the research was carried out for three times independently.

Fig. 3

The elevation of cAMP levels blocked AAT1 knockdown-induced MC degranulation. (A) The SO₂ content in MC supernatant detected by HPLC-FD; (B) The cAMP level in MCs detected by immunofluorescence; (C) The release rate of β-hexosaminidase in MCs detected by colorimetric analysis. *P < 0.05 and the data are presented as the mean ± SD. All the research was carried out for three times independently.

Fig. 4

AAT1 overexpression significantly inhibited the hypoxia-induced degranulation of MCs in vitro. (A-B) Western blot analysis of protein expression of AAT1 (A) and AAT2 (B) in MCs in normoxic and hypoxic groups in vitro; (C) The SO₂ content in supernatant of MCs was detected by HPLC in vitro; (D) The cAMP content in MCs detected by ELISA in vitro; (E) Colorimetric analysis of the release rate of β-hexosaminidase in MCs in vitro ; *P < 0.05, **P < 0.01 and data are presented as the mean ± SD. All the research was carried out for three times independently.

Fig. 5

SO₂ donor inhibited the hypoxia-driven downregulation of cAMP and increase in MC degranulation in vivo. (A) Western blot analysis of AAT1 protein expression in rat lung tissue; (B) Real time RT-PCR analysis of AAT1 mRNA levels in rat lung tissue; (C) Immunohistochemistry analysis of AAT1 protein expression in rat lung tissue; (D) Immunohistochemistry analysis of cAMP protein expression in rat lung tissue; (E) Toluidine blue staining was used to detect MC accumulation and degranulation (red arrows) around rat pulmonary vascular tissue. The rats in the hypoxic + SO₂ group were injected with NaHSO₃/Na₂SO₃ mixture (0.18 mmol and 0.54 mmol per kilogram body weight) before hypoxia exposure each day. The rats of the normoxic and hypoxic groups were injected with the same volume of physiological saline. **P < 0.01 and data are presented as the mean ± SD, n = 6 each group.

Supplementary Fig. 1

Figure S1: Effects of AAT2 knockdown on the cAMP level in MC and MC degranulation. (A) Western blot analysis of AAT2 protein expression; (B) HPLC-FD analysis of SO₂ content in the MC supernatant; (C) ELISA analysis of cAMP content in MCs; (D) Colorimetric analysis of the release rate of β-hexosaminidase in MCs. **P < 0.01 and data are presented as the mean ± SD. All the research was carried out for three times independently.

Supplementary Fig. 2

Figure S2: The effects of hypoxia on AAT2 mRNA and protein expression in rat lung tissue. (A) Real time RT-PCR analysis of AAT2 mRNA levels in rat lung tissue; (B) Western blot analysis of AAT2 protein expression in rat lung tissue; (C) Immunohistochemistry analysis of AAT2 protein expression in rat lung tissue. The rats in the hypoxic + SO₂ group were injected with NaHSO₃/Na₂SO₃ mixture (0.18 mmol and 0.54 mmol per kilogram body weight) before hypoxia exposure each day. The rats of the normoxic and hypoxic groups were injected with the same volume of physiological saline. **P < 0.01 and data are presented as the mean ± SD. All the research was carried out for three times independently.