Increased Levels of VCAM-1 in Sera and VLA-4 Expression on Neutrophils in Dermatomyositis with Interstitial Lung Disease

ABSTRACT Vascular cell adhesion molecule-1 (VCAM-1) and its ligand very late antigen (VLA-4) play important roles in many autoimmune diseases. Our study aimed to investigate the serum level of VCAM-1 and VLA-4 expression on peripheral blood neutrophil surface in patients with dermatomyositis (DM), especially focusing on patients with interstitial lung disease (ILD). Blood specimens of 42 patients with DM and 42 healthy controls matched for age and gender were recruited. Total serum VCAM-1 level was measured using commercial enzyme-linked immunosorbent assay (ELISA) and the percentages of VLA-4 expression on neutrophils were analyzed by flow cytometry. We divided patients into subgroups according to whether they had ILD and whether they exhibited diffuse alveolar damage (DAD) via high-resolution computed tomography (HRCT). sVCAM-1 was increased in classical DM (cDM) and clinical amyopathic dermatomyositis (CADM) compared with healthy controls (both p < .01). DM-ILD had higher sVCAM-1 levels than the none-ILD group (p < .01). sVCAM-1 was also significantly increased in the DAD group compared to the none-DAD group (p < .01). The percentages of VLA-4 expression on neutrophils in cDM and CADM patients were significantly elevated than that in healthy controls (both p < .01). The percentage of VLA-4 expression on neutrophils in DM patients with ILD was higher than none-ILD group (p < .01). In the patients with ILD, DAD group had a higher percentage of VLA-4 expression on neutrophils than none-DAD group (p < .01). Our findings indicated that serum VCAM-1 levels combined with VLA-4 expression on neutrophils might be useful for detecting the severity of lung disease in patients with DM.


Introduction
Dermatomyositis (DM) is characterized by myositis and rash with complications in other vital organs such as the lung and heart. Interstitial lung disease (ILD) is one of the most common and life-threatening complications of DM, with a prevalence of up to 86% (Ning et al. 2019). The one-year survival rate for patients with DM-ILD is 56.7%, and even lower in patients with acute ILD (Chen et al. 2009). However, the pathogenesis of DM-ILD remains unclear.
VCAM-1 (CD106) is a 90-kDa glycoprotein predominantly expressing on endothelial cells. Its main ligand is VLA-4 (α4β1 integrin) which plays a major role in mediating rolling and firm adhesion of leukocytes to the endothelium, as well as leukocyte transmigration (Mitroulis et al. 2015). The soluble ectodomain of VCAM-1 (sVCAM-1) is released from the cell surface into the circulation by proteolysis, a process that is upregulated in inflammatory diseases (Singh et al. 2005). The expression of VCAM-1 is closely related to tumor angiogenesis and metastasis in gastric carcinoma and breast cancer (Byrne et al. 2000;Ding et al. 2003). In addition, VCAM-1 and VLA-4 are major factors in promoting the survival of endothelial and mural cell during angiogenesis (Garmy-Susini et al. 2005). VLA-4 is expressed mainly on lymphocyte, monocytes, eosinophils, and neutrophils (Van Staveren et al. 2018). VCAM-1/VLA-4 pathway has been proved to be associated with inflammatory and autoimmune diseases by recruiting leukocytes to tissues (Mitroulis et al. 2015;Yusuf-Makagiansar et al. 2002). For example, VCAM-1/VLA-4 pathway seems to be critical for the infiltration in rheumatoid arthritis (RA) (Klimiuk et al. 2002;Wang et al. 2015). In addition, it has been demonstrated that VLA-4/VCAM-1 pathway is important in mediating leukocyte adherence to the inflamed endothelium in the central nervous system of multiple sclerosis (MS) patients and experimental autoimmune encephalomyelitis (EAE) model (Rice et al. 2005;Wan et al. 2018).
In DM, it had been found increased expression of VCAM-1 on blood vessels and muscle fibers (Jain et al. 2009;Lundberg et al. 2000). In addition, circulating levels of sVCAM-1 were significantly higher in juvenile dermatomyositis (JDM) (Kim et al. 2012). However, for adult DM with ILD, the serum level of VCAM-1 and its ligand expression are still not clear. Bronchoalveolar lavage had revealed elevated levels of lymphocytes and neutrophils in patients with DM accompanied by rapidly progressive interstitial lung disease (RP-ILD) (Chino et al. 2016). It had been reported that increased VLA-4 expression on lymphocytes may promote lymphocyte transmigration, leading to the onset of ILD (Taooka et al. 2016). On the other side, the significantly increased neutrophils in BALF were found in those DM-ILD patients with poor prognosis (Schnabel et al. 2003). Neutrophil extracellular traps (NETs) released by neutrophils are thought to be important for promoting neutrophil infiltration and leading to lung damage Peng et al. 2018). However, the mechanism of pathological recruitment of neutrophils is unclear. Thus, we investigated VCAM-1 levels in sera and VLA-4 expression on neutrophils in DM, especially focusing on the patients with ILD.

Patients and controls
Blood specimens of 42 untreated patients with DM, 29 classical DM and 13 clinical amyopathic dermatomyositis (CADM), were recruited from the Department of Rheumatology of the First Affiliated Hospital of China Medical University from May 2019 to May 2020, as well as 42 healthy blood donors. Other autoimmune diseases, current or chronic infections, and other severe concomitant diseases were excluded. The study was supported by the Ethics Committee of the First Affiliated Hospital of China Medical University (No. 2018-214-3) and was conducted according to the principles expressed in the Declaration of Helsinki. All participants signed an informed consent prior to the start of the study. All the patients fulfilled the European League Against Rheumatism/American College of Rheumatology (EULAR/ACR) classification criteria for IIM (idiopathic inflammatory myopathies) (Lundberg et al. 2017). DM patients with ILD were diagnosed by two trained rheumatologists based on high-resolution computed tomography (HRCT) and pulmonary function tests. All blood samples were centrifuged to obtain serum immediately and stored at −80°C. The fresh anticoagulated blood samples with ethylenediamine tetra acetic acid (EDTA) were disposed with polymorphonuclear leucocyte separation medium (Polymorphprep™, Axis-Shield) to get granulocytes.

Radiological patterns for DM-ILD
The most frequently described patterns in DM are non-specific interstitial pneumonia (NSIP, ground-glass opacity with little honeycombing) and organizing pneumonia (OP, irregular consolidation with a predominantly basal/peripheral or peri-bronchovascular distribution). Usual interstitial pneumonia (UIP) pattern can also be observed (basal, subpleural reticulation and honeycombing), but at a lower frequency. Another pattern is diffuse alveolar damage (DAD, diffuse ground-glass opacity and extensive consolidation) which is associated with a poor prognosis and rapidly developing dyspnoea (De Lauretis et al. 2011;Ferguson and Berkowitz 2012;Ishikawa et al. 2018).

Measurement of serum VCAM-1 level by ELISA
Total serum VCAM-1 level was measured using ELISA kits according to the manufacturer's instructions (Cat. BMS232, eBioscience, San Diego, CA, USA). All assays were performed in duplicate, and the data are presented as ng/ml.

Statistical analysis
Continuous variables were expressed as mean ± standard deviation or median (interquartile range). Differences between two groups were compared using Student's t-test or Mann-Whitney U-test. A one-way analysis of variance (ANOVA) or Kruskal-Wallis H test was used for multiple comparisons, followed by Bonferroni correction t-test or Mann-Whitney U-test to compare differences between each two groups. The correlation of serum VCAM-1 level and VLA-4 expression on neutrophils with laboratory parameters was performed by Pearson's or Spearman's rank correlation coefficient. Analysis was conducted by the SPSS 20.0 software and GraphPad Prism for Windows version 8.00 (Graph Pad Software, La Jolla, CA, USA). Two-tailed p values less than 0.05 were statistically significant.

Participant characteristics
A total of 42 patients with DM were enrolled, while 42 healthy controls were also recruited matched for age and gender. Demographic and clinical characteristics of DM patients and healthy controls are shown in Table 1.
The relationship between clinical entities of DM and DM-specific autoantibodies was described in Table 2. As shown in Table 2, anti-SSA/Ro52 antibody was the most frequent antibody in DM (13 cDM and 6 CADM). Eleven patients (9 cDM and 2 CADM) were antisynthetase antibodies positive. Six patients (1 cDM and 5 CADM) were anti-MDA5 antibody positive.
The clinical characteristics between cDM and CADM are displayed in Table 3. As shown in Table 3, no significant differences were found for the clinical indexes except CK and LDH.

VLA-4 expression on the surface of neutrophils
The percentage of VLA-4 expression on the surface of neutrophils was described as CD16+ VLA-4 +. Figure 2 shows an example of FACS dot plot of CD16+ VLA-4 +.

The clinical significance of serum VCAM-1 level and VLA-4 expression on neutrophils with laboratory parameters
Serum VCAM-1 levels and VLA-4 expression on neutrophils were significantly negatively correlated with DLCO, PaO2 and significantly positively correlated with immunoglobulin and D-D (Table 4).

Discussion
In our study, cDM and CADM patients exhibited increased sVCAM-1 level in sera and VLA-4 expression on neutrophils surface compared with healthy controls. To our knowledge, this is the first time to detect VLA-4 expression on the surface of neutrophils in DM patients. In addition, these two indicators were elevated in patients with ILD compared to non-ILD. Patients with DAD patterns had higher VCAM-1 levels in sera and VLA-4 expression on neutrophils than those without DAD. Wolters et al. showed that the pathogenesis of connective tissue disease (CTD)-ILD and idiopathic pulmonary fibrosis (IPF) was different. The pathogenesis of IPF was mainly caused by epithelial cell dysfunction, while CTD-ILD was driven by inflammatory invading into interstitial and alveolar spaces (Wolters et al. 2018). The migration of leukocytes to sites of injury or infection is tightly regulated by the leukocyte adhesion cascade. In the beginning, rolling of leukocytes is medicated by selectins (Zarbock et al. 2011). Leukocyte    The soluble ectodomain of VCAM-1 can be released from the cell surface into the circulation via proteolytic cleavage (Garton et al. 2003;Singh et al. 2005). The soluble forms of cellular adhesion molecules (sCAMs) were observed to correlate with the endothelial surface expression of CAMs and can be used as potential biomarkers for endothelial activation (Kjaergaard et al. 2013). In our study, we found a positive correlation between serum sVCAM-1 and D-D which confirmed that VCAM-1-induced vascular endothelium damage may lead to hypercoagulability and hyperfibrinolysis. Recent studies have shown that the lung endothelium was permeable to inflammatory cells owing to the VCAM-1 expression. Neutrophil and mononuclear phagocytes recruited following up-regulation of pulmonary endothelium VCAM-1 contributed to lung injury and integrin antagonists reduced alveolar epithelial cell injury (Parmley et al. 2007;Wang et al. 2019). Soluble VCAM-1 in serum can reveal neutrophil adhesion in the lung microvasculature and lung injury (Laudes et al. 2004). In our study, serum levels of sVCAM-1 were significantly increased in DM-ILD and sVCAM-1 levels were significantly negatively correlated with lung function, including FVC, DLCO and PaO2. This result revealed that the pathogenesis of DM-ILD may be associated with endothelial cell damage. Soluble VCAM-1 plays a key role in the onset of synovitis in RA, which is accompanied by the infiltration of T cells and monocytes (Kitani et al. 1998;Tokuhira et al. 2000). Multiple factors, including increased production of proinflammatory cytokines, the presence of autoantibodies, and increased oxidative stress activate endothelial cells, leading to increased expression of VCAM-1 (Da Rosa Franchi Santos et al. 2019). sVCAM-1 recruits pathological levels of neutrophils to injury sites and amplifies lung inflammation during acute lung injury (Mishra et al. 2016). Bronchoalveolar lavage fluid from a patient with DM-ILD revealed neutrophil infiltration (Chino et al. 2016). It has been reported that excessive formation of NETs by neutrophils in DM caused damage to pulmonary vascular endothelial cells and infiltration of inflammatory cells, leading to the occurrence of ILD (Zhang et al. 2014). Therefore, we hypothesized that the pathogenesis of DM-ILD may be related to VCAM-1 and neutrophil infiltration. Ibbotson et al. first discovered that neutrophil recruitment was dependent on the VLA-4/VCAM-1 pathway in human disease (Ibbotson et al. 2001). We thus explored VLA-4 expression on the surface of neutrophils in patients with DM. Our findings manifested that VLA-4 expression on neutrophils was elevated in DM-ILD and significantly negatively correlated with DLCO, PaO2. Patients with DAD patterns had higher VCAM-1 levels in sera and VLA-4 expression than those without DAD. The results may confirm the hypothesis that neutrophils infiltrated via VCAM-1/VLA-4 pathway in DM-ILD. In addition, our results indicated that increased sVCAM-1 and VLA-4 expression on neutrophils were more likely to be related to ILD in DM. Whether they are also involved in idiopathic ILD needs to be investigated in our further study.
Our study found that anti-SSA/Ro-52 antibodies were the most common myositis antibodies in both cDM and CADM, which was consistent with previous studies (Gan et al. 2020;Temmoku et al. 2019). It has been confirmed that anti-SSA/Ro-52 antibodies were associated with an increased frequency of RP-ILD and the cumulative 24-month survival rate tended to be lower in patients with anti-SSA/Ro52 antibodies (Sabbagh et al. 2019;Temmoku et al. 2019;Xu et al. 2020). In our study, anti-MDA5 antibodies were the second most common myositis antibodies in CADM. According to our clinical experience, the positive rate of anti-MDA5 antibodies in patients with CADM in Chinese was usually lower than 50% (Gan et al. 2020). We finally compared the potential association between the antibodies profile and VCAM-1/VLA-4, and the results showed that anti-SSA/Ro-52 antibodies-positive group and anti-MDA5 antibodies-positive group had higher sVCAM-1 levels and VLA-4 expression on neutrophils. These results further indicated that VCAM-1/VLA-4 may play an important role in DM patients with ILD.
The present study also has limitations. One is that our results are based on a small sample size and may be biased. Furthermore, we will enlarge the sample size to validate the conclusions. The other limitation is that the population in our study is Chinese. Other studies from the different ethnic or geographic populations are needed to clarify whether the present results are generally applicable.
In conclusion, serum sVCAM-1 levels and VLA-4 expression on neutrophils may play an important role in neutrophil chemotaxis in DM-ILD. It is possible that serum sVCAM-1 level may be a useful biological marker to reflect the severity of lung disease in patients with DM. Our results highlight the involvement of neutrophils in the pathogenesis of DM-ILD.