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Abstract
Background: Elafin is a serine protease inhibitor with anti-inflammatory properties. It is expressed in various epithelial tissues with increased production under inflammatory conditions. Increased tissue elafin expression in Behçet's disease (BD) lesions has previously been demonstrated. Aims and Objectives: We hypothesised that serum elafin might be increased in patients with BD and aimed to assess the relationship of serum elafin with disease activity and organ involvement in BD. Materials and Methods: Fifty-four BD patients (29 active, 25 inactive) and 30 healthy controls were included in this single-centre case-control study. Serum elafin was measured using ELISA. Complete blood count, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) tests were also performed. Results: Serum elafin levels were significantly higher in patients with BD (1.58 ± 0.47 ng/ml, range: 0.67–2.96) compared to controls (1.10 ± 0.28 ng/ml, range: 0.65–1.49) (P < 0.001). Patients with active BD had higher elafin levels than patients with inactive BD (P = 0.008). Active arthritis was associated with an increase in elafin (P = 0.012), while the presence of mucocutaneous symptoms was not. Serum elafin correlated significantly with ESR (P = 0.001). The ideal cut-off value for the diagnosis of BD was determined as 1.24 ng/ml with a sensitivity and specificity of 72.2% and 70.0%, respectively. Conclusion: Serum elafin is significantly increased in patients with BD. It may serve as a marker of disease activity, especially articular involvement.
Keywords: Behcet's disease, inflammatory diseases, vasculitis
Introduction 
Behçet's disease (BD) is a chronic, multisystemic, inflammatory disease characterised by recurrent oral aphthous ulcers, genital ulcers, and uveitis. An aberrant immune reaction triggered by environmental factors in the presence of a genetic predisposition is implicated in the pathogenesis. Both innate and adaptive immunity come into play, with T-cell hypersensitivity, increased production of proinflammatory cytokines and neutrophil activation having a pivotal role. Increased neutrophil activity causes tissue damage, which in turn enhances the immune response. Lesions demonstrate marked perivascular neutrophil and lymphocyte infiltration, resulting in endothelial dysfunction.[1]
Elafin is a serine protease inhibitor produced mainly by epithelial cells. Its expression is upregulated by proinflammatory cytokines such as IL-1β and TNF-α that are potent stimulants of neutrophil activation, and it acts as an 'alarm antiprotease' that counteracts the destructive effects of neutrophil elastase.[2] An increased amount of epidermal elafin has been detected in skin biopsies taken from patients with BD.[3] Elafin deposition in the vascular wall in neutrophil-mediated cutaneous vasculitis has also been demonstrated by immunohistochemistry.[4] Based on the central role of neutrophils in the pathogenesis of BD, we hypothesised that serum elafin levels may be altered in patients with BD and may be associated with disease activity.
Materials and Methods We included 54 adult BD patients (29 in active, 25 in inactive phase) diagnosed according to the International Study Group criteria and 30 healthy controls in this single-centre, prospective case-control study.[5] On retrospective evaluation, all patients also met the International Criteria for BD.[6] Exclusion criteria were defined as receiving systemic treatment for BD in the last 3 months (except for colchicine and penicillin), presence of another inflammatory disease, active infection, pregnancy, history of transplantation or malignancy, renal or hepatic dysfunction (glomerular filtration rate <60 ml/min, serum alanine/aspartate aminotransferase >x3 the upper reference limit).
BD was considered to be active if one or more of the following criteria were present at the examination: oral/genital/cutaneous ulcers or ocular symptoms (uveitis) that persisted for 2 weeks or more within the past 4 weeks, or subcutaneous venous thrombosis, arthralgia, intestinal ulceration, progressive central nervous system lesions, progressive vasculitis and epididymitis in the past 4 weeks, according to the criteria proposed by the BD Research Committee of Japan.[7] The study was approved by the Istanbul Medical Faculty Review Board and conducted according to the principles of the Helsinki Declaration. All patients were informed, and written consent was obtained before the study.
Demographic data and a detailed medical history were obtained during the examination and from patient files. Venous blood samples were obtained from each patient under aseptic conditions after 12 hours of fasting. A complete blood count and routine biochemical measurements were performed. Erythrocyte sedimentation rate (ESR) was measured by the Westergren method, and C-reactive protein (CRP) was measured by the turbidimetric method (Cobas C702 system, Roche Diagnostics, Indianapolis, USA), with reference ranges of 0–20 mm/h and 0–5 mg/dl, respectively. Serum samples were stored at -20°C until the elafin assay. Serum elafin level was measured using an enzyme-linked immunosorbent assay according to the manufacturer's instructions (catalogue number: EK1117, Boster Biological Technology, California, USA).
Data were analysed using SPSS version 23.0 (IBM Corp, New York, USA). Quantitative data were represented as mean ± SD, and median, where appropriate. Categorical variables were compared with the Chi-Square test. The Shapiro–Wilk test was used to assess the normality of distribution, and the Levene test was used to determine the homogeneity of variances. Depending on sample distribution, the Student t-test or Mann–Whitney U test was used to compare two groups, and the ANOVA or Kruskal–Wallis test was used to compare three groups. The correlation among parameters was assessed through Spearman analysis. A receiver operating characteristic (ROC) curve was used to determine the best cut-off value for elafin and its sensitivity and specificity in the diagnosis of BD. A P value of <0.05 was considered significant.
Results There were 54 BD patients included in the study; 29 with active, 25 with inactive disease, and 30 healthy controls with a mean age of 38.4 ± 10.2 and 38,4 ± 9.6 respectively. There was no significant difference between groups in regards to age and sex [Table 1]. The mean age at BD diagnosis was 30.5 ± 8.5 with a median disease duration of 6 years. Forty-five patients (83.3%) were using colchicine, and five patients were receiving periodic penicillin injections.
Table 1: The demographic data and laboratory measurements in Behçet's disease and control groupsSerum elafin levels were significantly higher in patients with BD (1,58 ± 0,47 ng/ml, range: 0.67–2.96) compared to the control group (1.10 ± 0.28 ng/ml, range: 0.65-1.49) (P < 0,001) [Table 1]. There was also a significant difference between active BD, inactive BD patients and controls, with the highest levels in active BD patients and the lowest levels in controls [Table 2]. When these three groups were compared with each other in pairs, a significant difference was observed between each pair [Figure 1]. Serum elafin level was not associated with age, sex, smoking or alcohol usage. Mucocutaneous involvement, such as oral aphthous ulcers, genital ulcers, papulopustular lesions and erythema nodosum, was not correlated with elafin levels. On the other hand, a significant increase was observed in patients with arthritis (P = 0,012) [Table 3]. Although patients with uveitis had higher elafin levels, it was not statistically significant. One patient presented with epididymitis and another with thrombophlebitis; statistical analysis could not be performed due to the low number of patients.
Table 2: Laboratory measurements in active Behçet's disease, inactive Behçet's disease and control groups
Figure 1: Comparison of serum elafin levels between active Behçet's disease, inactive Behçet's disease and control groups. BD = Behçet's disease
Table 3: Clinical findings in patients with Behçet's disease and their relation with elafin levelsPatients with BD had higher ESR (20.2 ± 17.7 mm/h and 0.1–9.7 mm/h, respectively, P < 0.001) and CRP levels (9.9 ± 11.7 mg/dl and 2.1 ± 2.7 mg/dl, respectively, P < 0.001) compared to the control group. Two inflammatory parameters derived from the complete blood count, the neutrophil/lymphocyte ratio (NLR) and the thrombocyte/lymphocyte ratio (TLR), were also higher in BD patients compared to controls [Table 1]. The difference between active BD, inactive BD patients and controls was also significant for these four parameters [Table 2]. Serum elafin correlated significantly with ESR (P < 0.001), but not with CRP, NLR and TLR (P = 0.052, P = 0.642, and P = 0.075, respectively).
Through the ROC curve analysis, the optimal cut-off value for elafin was calculated as 1.24 ng/ml. The sensitivity and specificity of elafin for the diagnosis of BD were 72.2% and 70.0%, respectively, with a positive predictive value (PPV) of 81.3% and a negative predictive value (NPV) of 58.3% [Figure 2].
Figure 2: Receiver operating characteristic (ROC) curve demonstrating the sensitivity and specificity of elafin in the diagnosis of Behçet's disease. The dot represents the cut-off point. PPV = Positive predictive value, NPV = Negative predictive value, AUC = Area under curve Discussion Activation of the innate immune system against infectious or self-antigens, mainly through neutrophils, NK cells and γδ+ T lymphocytes, is thought to be the initial step in the pathogenesis of BD, which is then sustained by the adaptive immune response. Increased production of proinflammatory cytokines leads to recruitment and activation of neutrophils, which act as the main driver of tissue damage and vasculitis through the release of proteinases and elastases and increased production of reactive oxygen species.[1]
Elafin, also known as peptidase inhibitor-3 (PI3) or skin-derived antileukoproteinase, is a serine protease inhibitor that was first isolated from bronchial mucus in 1981.[8] The initial product of the PI3 gene, pre-elafin, also named trappin-2, consists of an N-terminal cementoin domain with transglutaminase activity that binds extracellular matrix components and a C-terminal elafin domain with antiprotease activity. Trappin-2 is cleaved by proteases after secretion, yielding active elafin, which directly inhibits neutrophil elastase, pancreatic elastase and proteinase 3. Elafin also inhibits elastin-mediated neutrophil chemotaxis and elastase-dependent activation of matrix metalloproteinases, limiting tissue destruction caused by neutrophils and macrophages. Furthermore, it has antiproliferative and immunomodulatory properties and is considered an antimicrobial peptide due to its role in innate immunity.[2],[9]
Elafin is constitutively expressed in respiratory, gastrointestinal, and genitourinary epithelia, placenta and fetal membranes. While it is virtually absent in normal skin, it is produced under inflammatory conditions.[10] Elafin is mainly synthesised by keratinocytes and, to a lesser extent, by fibroblasts, endothelial cells and γδ+ T lymphocytes.[11],[12] Its expression has been shown to increase in vitro in the presence of proinflammatory cytokines such as IL-1β, IL-8 and TNF-α, all of which have increased serum levels in BD, especially during disease activation.[3],[4],[13]
It is likely that elafin is produced in response to neutrophilic activation to counteract its deleterious effects in diseases including, but not limited to, BD. A strong epidermal reaction to anti-elafin antibodies was demonstrated by Tanaka et al. in skin biopsies with dermal neutrophilic infiltration caused by BD, Sweet's syndrome, psoriasis and cellulitis. On the other hand, no immunoreactivity was seen in normal skin or in lesions with dermal lymphocytic infiltration caused by discoid lupus erythematosus and chronic prurigo.[3] In another study, elafin deposition was observed in the vascular wall of cutaneous neutrophil-mediated vasculitis, which is commonly observed, especially in early BD lesions.[4] Elafin expression has also been shown to increase during wound healing, which is an important aspect of mucocutaneous involvement in BD.[14]
Evidence suggests that elafin expression is not exclusive to disorders characterised by neutrophilic infiltration; it is also involved in various vascular inflammatory processes. Miyagawa et al. reported similar serum trappin-2 levels among patients with diffuse cutaneous or limited cutaneous systemic sclerosis (SSc) and controls. However, they observed increased serum trappin-2 levels in SSc patients with digital ulcers and elevated right ventricular systolic pressure, as well as enhanced trappin-2 expression in small vessels of SSc lesional skin, suggesting that trappin-2, the precursor of elafin, plays a role in the vasculopathy in SSc.[15] Furthermore, there is substantial evidence from pre-clinical studies that indicates elafin has beneficial effects on various vascular injury and inflammation models.[16] These findings may also have implications on the role of elafin in BD, considering the role of endothelial dysfunction and vasculitis in its pathogenesis.
In our study, elafin levels were markedly higher in BD patients, especially those with active disease. It can be inferred from our results that low elafin levels are not sufficient to rule out BD due to the relatively low NPV (58.3%), but high elafin levels may aid in BD diagnosis due to the higher PPV (81.3%).
While no single mucocutaneous lesion was associated with increased elafin levels, a significant increase was noted in patients with arthritis. The mean serum elafin was also higher in patients with uveitis, although statistically insignificant. We partially attributed this to the low number of patients with uveitis. Of note, most patients included in the study had predominantly mucocutaneous symptoms, while fewer had systemic involvement. During the course of the disease, 16 patients had experienced arthritis, 10 had uveitis and 8 had deep venous thrombosis/thrombophlebitis. At the time of the study, nine had active arthritis, five had uveitis and only one had thrombophlebitis. There were no patients with a history of neurologic, gastrointestinal, or arterial vascular involvement. We mainly attributed this to the exclusion of patients taking immunosuppressives, which is usually warranted in systemic disease.
Increased serum elafin levels have been reported in smokers compared to non-smokers previously, as well as increased elafin in the sputum and nasal lavage of smokers.[17],[18] Elafin is thought to play a regulatory role in the inflammatory process initiated in response to irritant cigarette smoke. However, we did not observe a significant difference in serum elafin between these two groups (P = 0.786). The difference between smoker and non-smoker BD patients was also insignificant (P = 0.746). The family history of BD was not associated with a difference in serum elafin either (P = 0.545).
Increased ESR and CRP levels in BD have been demonstrated repeatedly, although their use in predicting active disease manifestations is controversial.[19] Our results showed higher ESR and CRP levels in BD patients, in accordance with the literature. Elafin correlated significantly with ESR. NLR and TLR, two novel nonspecific hematologic markers of inflammation that have been associated with a variety of medical conditions, including BD, were also higher in BD patients compared to controls.[20]
The main limitation of our study is the relatively low number of patients with a history of organ involvement. Future studies that include BD patients with a more varied range of systemic manifestations may shed light on the association between elafin and specific organ involvement. Nevertheless, we think it is noteworthy that elafin levels were increased in BD patients even in the absence of organ involvement in most cases. Finally, there are insufficient data on whether colchicine, which was used by the majority of the patients, influences elafin levels.
Conclusion This study demonstrates that serum elafin is significantly increased in BD patients compared to healthy controls. Considering that patients with active disease had significantly higher levels than those with inactive disease, elafin may serve as a marker of disease activity, especially indicating articular involvement. Elafin may play an important role in the pathogenesis of BD by limiting tissue destruction and vascular inflammation.
Acknowledgement
We would like to thank Dr. M. Osman Köksal for his contribution in performing biochemical assays.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References 
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