The roles of neutrophil serine proteinases in idiopathic inflammatory myopathies

Idiopathic inflammatory myopathies (IIMs) are a group of heterogeneous autoimmune diseases that are accompanied by progressive symmetric muscle weakness, elevated serum levels of muscle enzymes, electromyographic abnormalities, and inflammatory infiltrates observed via muscle biopsy [1, 2]. Dermatomyositis (DM; juvenile, adult), polymyositis (PM), sporadic inclusion body myositis (sIBM), and immune-mediated necrotizing myopathy (IMNM) are the most common subtypes of IIMs [3], of which DM and PM are probably the best known [4].

Classic histopathologic findings for DM are CD4⁺ T cells, B cells, and plasmacytoid dendritic cells (pDCs) infiltrating muscle fibers. Unlike DM, the infiltrating inflammatory cells of PM are CD8⁺ T cells and macrophages [5, 6]. The infiltrating CD4⁺ T cells can differentiate into Th1 and Th17 cells; Th1 can activate macrophages by producing IFN-γ, and Th17 can facilitate the migration of mononuclear cells to the muscle by producing IL-17 [7, 8]. The infiltrating CD8⁺ T cells mediate myocytotoxicity via release of perforin [9]. The infiltrating macrophages can present antigens to T cells and produce multiple cytokines and chemokines [10]. Additionally, infiltrating pDCs may be the main source of type I interferon [11]; however, the exact mechanism by which these inflammatory cells migrate into muscles is not fully understood and requires further study.

Neutrophil serine proteinases (NSPs) are granule-associated enzymes that play an essential role in blood coagulation, apoptosis, inflammation, and immune responses [12–14]. Recent studies have shown that three NSPs—cathepsin G (CTSG), neutrophil elastase (NE), and proteinase 3 (PR3)—play roles in inflammatory cell migration. These NSPs can cleave the endothelial VE-cadherin, are involved in the possible disruption of endothelial integrity [15, 16], and can disturb the endothelial cell cytoskeletal architecture to increase vascular permeability [17, 18]. Moreover, CTSG is a chemoattractant for mononuclear cells and neutrophils [19]. There are few studies about NSPs and their roles in DM/PM.

In our previous microarray analysis, we used total RNA from peripheral blood mononuclear cells (PBMCs) of DM and PM patients, and Illumina HumanHT-12 v4.0 Expression Beadchips (Illumina, Inc.) for mRNA transcription profiling. The platform contained 47,323 transcripts. We identified a total of 2006 differentially expressed genes in patients with DM/PM compared to normal controls (NC) (P < 0.05 and fold change ≥ 2), among which 908 genes were upregulated and 1098 genes were downregulated. CTSG, NE, and PR3 were increased in DM and PM patients (data not shown).

In our previous genome-wide DNA methylation analysis, we used the Illumina Human-Methylation 450 K BeadChip array for DNA methylation profiling as described previously [20]. This platform contains 485,000 CpG sites across the whole genome and covers 99% of RefSeq genes with an average of 17 CpG sites per gene region distributed across the promoter, 5′ UTR, first exon, gene body, and 3′ UTR. It covers 96% of CpG islands with additional coverage in island shores and the flanking regions. The results showed that CTSG, NE, and PR3 were hypomethylated in DM and PM patients; the exact methylation sites are presented in Additional file 1: Table S1.

Previous studies have proven that purified NSPs can increase vascular permeability by cleaving the endothelial VE-cadherin. We hypothesize that in DM and PM patients the upregulation of NSPs cleaves the endothelial VE-cadherin, thus disrupting endothelial integrity and increasing the permeability of vascular tissue and the migration of inflammatory cells to extravascular tissue. To better understand the function of these proteinases, we measured their expression in PBMCs, serum, and muscle tissues, and further explored serum NSPs in the pathogenesis of DM and PM. We found that the expression of NSPs was increased in PBMCs, serum, and muscle tissues of DM and PM patients; additionally, the NSPs were hypomethylated in PBMCs in patients. Moreover, serum NSPs from DM/PM patients cleaved the endothelial VE-cadherin, disrupted endothelial integrity, and increased the migration of PBMCs.

We reported that the relative expression of NSPs was significantly higher and hypomethylated in the PBMCs of DM/PM patients. NE and PR3 were elevated in DM/PM serum, and there was positive correlation between serum NSPs and clinical indicators, such as ILD, LDH, ESR, CRP, IgG, IgM, IgA, anti-Jo-1 autoantibody, and anti-Ro-52 autoantibody. Serum NSPs degraded the VE-cadherin of HDMECs while increasing the permeability of HDMECs. This study is the first to measure the expression of CTSG, NE, and PR3 in DM/PM patients and to show human dermal microvascular endothelial junctional protein degradation and increased permeability.

In our study, DM patients have the higher incidence of ILD. The serum level of LDH was significantly higher in PM patients than in DM patients, and the level of CK had an increasing trend. These findings were in accordance with previous studies because PM patients typically have more severe muscle disease [26, 27]. DM can be distinguished from PM by its typical cutaneous features, which include heliotrope rash, Gottron’s papules, V-sign rash, shawl sign rash, and so on [28, 29]. Thalidomide has biologic effects on cytokines and cell-mediated responses, and is beneficial in systemic lupus erythematosus, pyoderma gangrenosum, erythema nodosum leprosum, and skin manifestation of DM [30, 31]. Hydroxychloroquine also has a positive effect on the cutaneous manifestations of DM [32]. Our DM patients had higher utilization rates of thalidomide and hydroxychloroquine for the treatment of skin lesions related to DM.

CTSG, NE, and PR3 are three serine proteinases that are stored in azurophil granules of neutrophils, monocytes, mast cells, and so on [33]. When the aforementioned cells were stimulated by immune complexes, certain pharmacological agents, or phagocytosis, the three NSPs were either released to the extracellular space or bound to the surface of those cells [33, 34]. These three NSPs exert several effects, such as processing inflammatory mediators and extracellular matrix proteins, thrombus formation, and engaging protease-activated receptors [14]. Our study proves for the first time that NSPs were increased in PBMCs, serum, and muscle tissues of DM/PM patients; that the serum levels of NSPs had a positive correlation with the levels of LDH, ESR, CRP, IgG, IgA, and IgM; and that patients with ILD have higher levels of PR3. It has been reported that the serum LDH level has significant correlation with the degree of muscle weakness, muscle destruction, and the degree of muscle inflammation [35], and that the levels of LDH tend to indicate disease activity [27]. Serum levels of ESR and CRP are risk factors of interstitial lung disease (ILD) in DM/PM [36–39], elevated ESR is associated with increased mortality in patients with DM [37, 40], and CRP has positive correlation with the global activity scores in DM [41]. So, to an extent, these parameters can reflect the activity or prognosis of DM/PM. Further study is needed to identify the exact functions of NSPs in DM/PM patients.

Recently, studies have shown that NSPs can enter the endothelial cell, disturb the endothelial cytoskeletal architecture, increase endothelial cell apoptosis, and reduce viability [17, 42]. NE can disrupt junctional proteins such as E-cadherin and VE-cadherin [43] and β-catenin [44], and can break down components of the extracellular matrix [45]. NE also activates matrix metalloproteinase 9 [46], thus potentiating the destructive effects on the vasculature and facilitating neutrophil transmigration [47]. PR3 has been shown to induce the release of CXCL-8 from endothelial cells [48], to activate matrix metalloproteinases [49] and to potentiate neutrophil transmigration across the endothelium [50]. Serum NSPs degraded VE-cadherin in the HDMECs in vitro. VE-cadherin is a critical component of the adherens junction [51]. Homogeneous VE-cadherin interactions with adjacent cells maintain cell–cell adhesion and contribute to endothelial permeability. Loss of VE-cadherin function greatly reduces microvascular stability and leads to increased permeability and hemorrhage [52, 53]. Degradation of protein junctions would result in increased permeability of endothelial cells.

What is the clinical relevance of our findings? The infiltration of inflammatory cells into muscles plays a key role in the pathogenesis of DM/PM. We identified NSPs as key molecules that degrade primary junctional proteins of the HDMECs, thereby increasing permeability of the HDMECs and facilitating the migration of inflammatory cells. In light of these findings, selective NSP antagonists, with or without antioxidants, may offer future vascular protection and new treatment for DM/PM.

Despite the novel and clinically relevant findings in this study, there are some limitations. First, the patients included in our study differed in the course and severity of their disease, and they had different treatment options. Second, we used the serum of patients to stimulate HDMECs, which would not completely rule out the influence of other elements in the serum and the NSPs produced by other cells. Third, the function of NSPs was not confirmed in vivo. Thus, further studies should be done to confirm whether the expression and roles of NSPs in patients have relationships with the course and/or severity of the diseases, and the function of NSPs in the model of DM/PM.

Our study showed that NSPs were elevated in DM/PM patients both at RNA and protein levels. The serum NSPs had some relation with clinical indicators, although the exact relationship was not fully understood. Most importantly, serum NSPs degraded the VE-cadherin of HDMECs and increased the permeability of HDMECs to PBMCs. These interactions may play key roles in the development and progression of DM/PM.