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. 2018 Jul 5;20(1):134.
doi: 10.1186/s13075-018-1632-x.

The roles of neutrophil serine proteinases in idiopathic inflammatory myopathies

Siming Gao  1 , Xiaoxia Zuo  1 , Di Liu  1 , Yizhi Xiao  1 , Honglin Zhu  2 , Huali Zhang  3 , Hui Luo  4
Affiliations

The roles of neutrophil serine proteinases in idiopathic inflammatory myopathies

Siming Gao et al. Arthritis Res Ther. .

Abstract

Background: Dermatomyositis and polymyositis are the best known idiopathic inflammatory myopathies (IIMs). Classic histopathologic findings include the infiltration of inflammatory cells into muscle tissues. Neutrophil serine proteinases (NSPs) are granule-associated enzymes and play roles in inflammatory cell migration by increasing the permeability of vascular endothelial cells. In this study, we aimed to find the roles of NSPs in pathogenesis of IIMs.

Methods: RNA and DNA were isolated to measure the relative expression of NSPs and their methylation levels. The expression of NSPs in serum and muscle tissues was tested by enzyme-linked immunosorbent assay, immunohistochemistry, and immunofluorescence, respectively. Serum from patients was used to culture the human dermal microvascular endothelial cells (HDMECs), and then we observed the influence of serum on expression of VE-cadherin, endothelial cell tube formation, and transendothelial migration of peripheral blood mononuclear cells (PBMCs).

Results: We found that the expression of NSPs was increased in PBMCs, serum, and muscle tissues of IIM patients; these NSPs were hypomethylated in the PBMCs of patients. Serum NSPs were positively correlated with clinical indicators of IIM patients, including lactic dehydrogenase, erythrocyte sedimentation rate, C-reactive protein, immunoglobulin G, immunoglobulin M, and immunoglobulin A. Patients with anti-Jo-1, with anti-Ro-52, or without interstitial lung disease had lower levels of proteinase 3. Serum NSPs degraded the VE-cadherin of HDMECs, and serum NSP application increased the permeability of HDMECs.

Conclusions: Our studies indicate, for the first time, that NSPs play an important role in muscle inflammatory cell infiltration by increasing the permeability of vascular endothelial cells in IIM patients.

Keywords: Dermatomyositis; Inflammatory cell migration; Neutrophil serine proteinases; Polymyositis; Vascular permeability.

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Conflict of interest statement

Ethics approval and consent to participate

This study was approved by the institutional review board at Xiangya Hospital, Central South University of Changsha (Changsha, Hunan, China). All of the participants in the study signed a written informed consent form prior to participation.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Expression of CTSG, NE, and PR3 in DM/PM and their methylation. Relative expression of CTSG, NE, and PR3 increased in DM and PM PBMCs compared to controls at RNA level (a–c). CTSG, NE, and PR3 hypomethylated in PBMCs of DM/PM patients (d–f). No significant difference in serum levels of CTSG in DM, PM, and controls (g). Serum levels of NE and PR3 higher in DM patients and PR3 higher in PM patients than in controls (h, i). Values are mean ± SEM. CTSG cathepsin G, DM dermatomyositis, GAPDH glycerol-3-phosphate dehydrogenase, NC normal control, NE neutrophil elastase, PM polymyositis, PR3 proteinase 3
Fig. 2
Fig. 2
Expression of CTSG, NE, and PR3 in DM/PM muscle tissues. DM sections displayed typical perifascicular atrophy and inflammatory cell infiltration in perimysial area, while inflammatory cells mainly infiltrated around or invaded non-necrotic muscle fibers in HE (a). Immunohistochemistry showed an increase of CTSG protein in DM/PM muscle tissues, especially around muscle bundles (b). Expression of NE (green) and PR3 (green) also significantly higher in muscle bundles and perivascular areas of DM/PM patients shown by immunofluorescence (c, d). Nuclei (blue) stained by DAPI. CTSG cathepsin G, DM dermatomyositis, HE hematoxylin and eosin, NC normal control, NE neutrophil elastase, PM polymyositis, PR3 proteinase 3
Fig. 3
Fig. 3
Correlations between serum CTSG, NE, and PR3 levels and clinical indicators. Serum levels of CTSG in DM/PM correlated positively with levels of LDH, ESR, IgG, and IgA (a). Serum levels of NE in DM/PM correlated positively with CRP and IgM (b). Serum levels of PR3 correlated positively with CRP, ESR, IgG, and IgM in DM/PM patients (c). Patients with anti-Jo-1, with anti-Ro-52, or without ILD had lower levels of PR3 (d). CRP C-reactive protein, CTSG cathepsin G, ESR erythrocyte sedimentation rate, Ig immunoglobulin, ILD interstitial lung disease, LDH lactic dehydrogenase, NE neutrophil elastase, PR3 proteinase 3. *represents compared with patients, whose anti-Ro-52 antibody or anti-Jo-1 antibody are negative, or patients with ILD P < 0.05
Fig. 4
Fig. 4
Serum NSPs degraded VE-cadherin, disrupted tube formation, and increased permeability of HDMECs. After stimulating HDMECs with 20% serum from patients for 24 h, expression of VE-cadherin (110 kDa) decreased. VE-Cadherin expression could be neutralized by nonspecific inhibitor PMSF (a, b). After treating HDMECs with 20% serum from patients, tube formation ability of HDMECs decreased. This function of serum could be alleviated by PMSF (c, d). After treating HDMECs with 20% serum from patients, permeability of HDMECs to human PBMCs was significantly increased. This increase could be lessened by PMSF (e). Values are mean ± SEM. DM dermatomyositis, NC normal control, PM polymyositis, PMSF phenylmethylsulphonyl fluoride
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