Rheumatol Int DOI 10.1007/s00296-014-3020-4
Original Article
Increased serum APRIL differentially correlates with distinct cytokine profiles and disease activity in systemic lupus erythematosus patients Ghada Boghdadi · Enass A. Elewa
Received: 8 February 2014 / Accepted: 5 April 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Cytokines play an important role in the pathogenesis of systemic lupus erythematosus (SLE). Among the cytokines that regulate B cell homeostasis is a proliferation-inducing ligand (APRIL). This study aimed to determine whether serum levels of APRIL are raised in patients with SLE and correlate with disease activity or proinflammatory cytokines production, or both. Serum APRIL, interleukin-17 (IL-17), IL-4 and interferon gamma (IFN-γ) levels were measured in forty patients with SLE and 30 healthy controls. Disease activity was assessed by SLE disease activity index (SLEDAI), and results were correlated with serum APRIL levels. Serum APRIL levels were significantly higher in patients with SLE than in healthy controls. Positive correlation was found between serum APRIL levels and total SLEDAI score and anti-dsDNA antibody titers. Moreover, serum APRIL levels was significantly higher in patients with arthritis, mucocutaneous manifestations and proteinuria. APRIL is increased in patients with active SLE accompanying the increase of IL-17 and IFNγ. Significant positive correlations between serum levels of APRIL and IL-17 and IFN-γ and a negative correlation between serum levels of APRIL and IL-4 were found. The results suggest that APRIL may be an important marker of disease activity in patients with SLE. We provide the
G. Boghdadi (*) Immunology Research Lab, Department of Microbiology and Immunology, Faculty of Medicine, Zagazig University, Zagazig, Egypt e-mail: [email protected]; [email protected] E. A. Elewa Department of Rheumatology and Rehabilitation, Faculty of Medicine, Zagazig University, Zagazig, Egypt
analyses of APRIL levels in patients with SLE, suggesting new tools for the diagnosis, prognosis and possible therapeutic management of SLE. Keywords Systemic lupus erythematosus (SLE) · APRIL · IL-17 · IL-4 · IFN-γ
Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease associated with chronic immune activation and tissue damage, and this damage results from deposition of immune complexes and infiltration of activated T cells into susceptible organs [1]. Routine methods of assessing disease activity include acute phase markers and autoimmune serology are of limited sensitivity and specificity [2]. Although a relationship between double-stranded DNA antibodies has been shown in several studies, organ involvement in SLE cannot be accurately predicted [3]. It is interesting to speculate if newer tests can help predict disease course. Thus, cytokine measures have been studied for associations with organ involvement as well as their potential ability to monitor disease [1]. Multiple cytokines have been implicated in the disease activity or organ involvement in SLE. Among these, interleukin-6 (IL-6), interferon (IFN), B lymphocyte stimulator (BlyS), IL-10, IL-17 are thought to play an important role in the creation of the characteristic milieu in SLE, which promote B cell survival and autoantibody production [4]. So that, the knowledge of cytokines not only provides new insight into pathogenesis of SLE, but also it has allowed the development of clinical applications such as monitoring of disease activity and as potential therapeutic targets with the use of several biologic agents, targeting different cytokines or their receptors.
13
Consequently, many trials of anti-cytokine therapies for SLE are underway [5]. A proliferation-inducing ligand (APRIL, also called TNFSF13), a member of TNF ligand family, is a type II membrane-binding protein of 250 amino acids [6]. APRIL is a close sequence homolog of B cell activation factor (BAFF, also known as BlyS/zTNF4/TALL-1), also a member of TNF family [7]. At the receptor level, BAFF binds to receptor from the TNF-R family, BAFF-R [8], while APRIL uses heparan sulfate proteoglycans (HSPGs) as co-receptors [9, 10]. They are important regulators of B cell maturation, survival and function. In addition, they regulate the size and composition of B cell compartment and act as important driving factors for B cell hyperplasia and autoantibody production in autoimmune processes [11]. The treatment of lupus prone (NZB6W) F1 mice with a soluble TACI-immunoglobulin fusion protein (soluble decoy receptor for BAFF and APRIL) inhibits the development of proteinuria and prolongs survival of the animals [12]. Moreover, it has also been reported that gene polymorphism of APRIL is associated with systemic lupus erythematosus [13]. These findings indicate that APRIL as well as BAFF may be involved in the development of SLE, whereas many previous studies have evaluated the role of BAFF and APRIL in various immune based diseases; rheumatoid arthritis [14], Sjögren syndrome [15] and SLE [16] yet little information is available about serum APRIL levels in SLE and its role in SLE pathogenesis. For these reasons, this study was designed to determine whether serum levels of APRIL are raised in patients with SLE and correlated with other proinflammatory cytokines or disease activity.
Patients and methods Patients This study was carried out at Department of Rheumatology and Rehabilitation and the Immunology Research Lab, Department of Microbiology and Immunology, Faculty of Medicine, Zagazig, Egypt. Forty patients with SLE were included in the study, who all fulfilled the updated American College of Rheumatology (ACR) classification criteria for SLE [17]. Thirty healthy age- and gender-matched volunteers served as controls. The volunteers were seronegative for human immunodeficiency virus, hepatitis B or C virus, without evidence of cancer, congenital heart disease or connective tissue disorder. The study was approved by the ethical committee of the hospital and written informed consent was obtained from each patient. A detailed clinical history and a complete physical examination including
13
Rheumatol Int
general, locomotor system, skin, cardiovascular, chest, neurological and vascular examinations were carried out for each patient. Disease activity The disease activity was assessed in SLE patients by Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) [18]. Activity categories were defined on the basis of SLEDAI scores [19]; no activity (SLEDAI 0), mild activity (SLEDAI 1–5), moderate activity (SLEDAI 6–10), high activity (SLEDAI 11–19) and very high activity (SLEDAI 20). Investigations In the study group, the following laboratory parameters were analyzed: complete blood cell count (CBC), erythrocyte sedimentation rate (ESR), urine analysis, blood urea, creatinine and fibrinogen levels, C reactive protein (CRP), partial thromboplastin time (PTT), liver function tests (GOT, GPT, bilirubin), immunoglobulins (IgG, IgA, IgM), complement (C3, C4), anti-dsDNA antibody and antinuclear antibodies (ANA). Determination of IL‑17, IL‑4, IFN‑γ and APRIL Blood samples taken from the patients and healthy controls were kept at room temperature for 30 min till they coagulated. They were then centrifuged at 2,000 rpm for 10 min and serums were obtained. The serum samples were kept at −70 °C until the study. IL-17, IL-4, IFN-γ and APRIL were measured in serum samples by use of ELISA method according to the kit manufacturer’s instructions (WKEA MED supplies, WH-082, USA, R&D Quantikine HS400, UK, Invitrogen, KAC1231, Nivelles, Belgium and Affymetrix eBioscience BMS2008, USA, respectively). Standards as well as samples were assayed as duplicates and the interassay variations were shown to be within the range given by the manufacture. The analytic sensitivity was IL-4 = 0.22 pg/ml; IFN-γ = 0.03 IU/ml; and APRIL = 0.4 pg/ml. Statistical analysis It was performed using SPSS statistical software, version 11.0 (SPSS, Chicago, IL). Quantitative variables were given as mean ± SD, medians, range and categorical variables in frequencies and percents. t test or the Wilcoxon’s rank-sum tests were used for continuous variables according to the distribution of the variable and the v2 test for categorical variables. . Measure the correlation between two quantitative variables by Pearson’s correlation
Rheumatol Int
Kruskall–Wallis one-way analysis of variance (KW) test was used to compare median for >2 independent samples that are not related. p value 0.05). In the group of the 40 SLE patients, 38 people were diagnosed as having active and 2 with inactive disease. The median SLEDAI of SLE Table 1 Demographic data of patients and control groups Patients (n = 40)
Controls (n = 30)
Age (years) Gender (F/M)
31.1 ± 9.95 38/2
27 ± 10.4 29/1
Disease duration
5.1 ± 4.9
Data expressed as mean ± SD
Table 2 Clinical and laboratory characteristics of the patients with SLE
A proliferation-inducing ligand was detectable in all patients with SLE and in all healthy individuals. Table 3 shows that the APRIL level in patients with SLE (8.81 ± 3.1) was significantly higher than that in healthy individuals (1.58 ± 0.8), (p
Original Article
Increased serum APRIL differentially correlates with distinct cytokine profiles and disease activity in systemic lupus erythematosus patients Ghada Boghdadi · Enass A. Elewa
Received: 8 February 2014 / Accepted: 5 April 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Cytokines play an important role in the pathogenesis of systemic lupus erythematosus (SLE). Among the cytokines that regulate B cell homeostasis is a proliferation-inducing ligand (APRIL). This study aimed to determine whether serum levels of APRIL are raised in patients with SLE and correlate with disease activity or proinflammatory cytokines production, or both. Serum APRIL, interleukin-17 (IL-17), IL-4 and interferon gamma (IFN-γ) levels were measured in forty patients with SLE and 30 healthy controls. Disease activity was assessed by SLE disease activity index (SLEDAI), and results were correlated with serum APRIL levels. Serum APRIL levels were significantly higher in patients with SLE than in healthy controls. Positive correlation was found between serum APRIL levels and total SLEDAI score and anti-dsDNA antibody titers. Moreover, serum APRIL levels was significantly higher in patients with arthritis, mucocutaneous manifestations and proteinuria. APRIL is increased in patients with active SLE accompanying the increase of IL-17 and IFNγ. Significant positive correlations between serum levels of APRIL and IL-17 and IFN-γ and a negative correlation between serum levels of APRIL and IL-4 were found. The results suggest that APRIL may be an important marker of disease activity in patients with SLE. We provide the
G. Boghdadi (*) Immunology Research Lab, Department of Microbiology and Immunology, Faculty of Medicine, Zagazig University, Zagazig, Egypt e-mail: [email protected]; [email protected] E. A. Elewa Department of Rheumatology and Rehabilitation, Faculty of Medicine, Zagazig University, Zagazig, Egypt
analyses of APRIL levels in patients with SLE, suggesting new tools for the diagnosis, prognosis and possible therapeutic management of SLE. Keywords Systemic lupus erythematosus (SLE) · APRIL · IL-17 · IL-4 · IFN-γ
Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease associated with chronic immune activation and tissue damage, and this damage results from deposition of immune complexes and infiltration of activated T cells into susceptible organs [1]. Routine methods of assessing disease activity include acute phase markers and autoimmune serology are of limited sensitivity and specificity [2]. Although a relationship between double-stranded DNA antibodies has been shown in several studies, organ involvement in SLE cannot be accurately predicted [3]. It is interesting to speculate if newer tests can help predict disease course. Thus, cytokine measures have been studied for associations with organ involvement as well as their potential ability to monitor disease [1]. Multiple cytokines have been implicated in the disease activity or organ involvement in SLE. Among these, interleukin-6 (IL-6), interferon (IFN), B lymphocyte stimulator (BlyS), IL-10, IL-17 are thought to play an important role in the creation of the characteristic milieu in SLE, which promote B cell survival and autoantibody production [4]. So that, the knowledge of cytokines not only provides new insight into pathogenesis of SLE, but also it has allowed the development of clinical applications such as monitoring of disease activity and as potential therapeutic targets with the use of several biologic agents, targeting different cytokines or their receptors.
13
Consequently, many trials of anti-cytokine therapies for SLE are underway [5]. A proliferation-inducing ligand (APRIL, also called TNFSF13), a member of TNF ligand family, is a type II membrane-binding protein of 250 amino acids [6]. APRIL is a close sequence homolog of B cell activation factor (BAFF, also known as BlyS/zTNF4/TALL-1), also a member of TNF family [7]. At the receptor level, BAFF binds to receptor from the TNF-R family, BAFF-R [8], while APRIL uses heparan sulfate proteoglycans (HSPGs) as co-receptors [9, 10]. They are important regulators of B cell maturation, survival and function. In addition, they regulate the size and composition of B cell compartment and act as important driving factors for B cell hyperplasia and autoantibody production in autoimmune processes [11]. The treatment of lupus prone (NZB6W) F1 mice with a soluble TACI-immunoglobulin fusion protein (soluble decoy receptor for BAFF and APRIL) inhibits the development of proteinuria and prolongs survival of the animals [12]. Moreover, it has also been reported that gene polymorphism of APRIL is associated with systemic lupus erythematosus [13]. These findings indicate that APRIL as well as BAFF may be involved in the development of SLE, whereas many previous studies have evaluated the role of BAFF and APRIL in various immune based diseases; rheumatoid arthritis [14], Sjögren syndrome [15] and SLE [16] yet little information is available about serum APRIL levels in SLE and its role in SLE pathogenesis. For these reasons, this study was designed to determine whether serum levels of APRIL are raised in patients with SLE and correlated with other proinflammatory cytokines or disease activity.
Patients and methods Patients This study was carried out at Department of Rheumatology and Rehabilitation and the Immunology Research Lab, Department of Microbiology and Immunology, Faculty of Medicine, Zagazig, Egypt. Forty patients with SLE were included in the study, who all fulfilled the updated American College of Rheumatology (ACR) classification criteria for SLE [17]. Thirty healthy age- and gender-matched volunteers served as controls. The volunteers were seronegative for human immunodeficiency virus, hepatitis B or C virus, without evidence of cancer, congenital heart disease or connective tissue disorder. The study was approved by the ethical committee of the hospital and written informed consent was obtained from each patient. A detailed clinical history and a complete physical examination including
13
Rheumatol Int
general, locomotor system, skin, cardiovascular, chest, neurological and vascular examinations were carried out for each patient. Disease activity The disease activity was assessed in SLE patients by Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) [18]. Activity categories were defined on the basis of SLEDAI scores [19]; no activity (SLEDAI 0), mild activity (SLEDAI 1–5), moderate activity (SLEDAI 6–10), high activity (SLEDAI 11–19) and very high activity (SLEDAI 20). Investigations In the study group, the following laboratory parameters were analyzed: complete blood cell count (CBC), erythrocyte sedimentation rate (ESR), urine analysis, blood urea, creatinine and fibrinogen levels, C reactive protein (CRP), partial thromboplastin time (PTT), liver function tests (GOT, GPT, bilirubin), immunoglobulins (IgG, IgA, IgM), complement (C3, C4), anti-dsDNA antibody and antinuclear antibodies (ANA). Determination of IL‑17, IL‑4, IFN‑γ and APRIL Blood samples taken from the patients and healthy controls were kept at room temperature for 30 min till they coagulated. They were then centrifuged at 2,000 rpm for 10 min and serums were obtained. The serum samples were kept at −70 °C until the study. IL-17, IL-4, IFN-γ and APRIL were measured in serum samples by use of ELISA method according to the kit manufacturer’s instructions (WKEA MED supplies, WH-082, USA, R&D Quantikine HS400, UK, Invitrogen, KAC1231, Nivelles, Belgium and Affymetrix eBioscience BMS2008, USA, respectively). Standards as well as samples were assayed as duplicates and the interassay variations were shown to be within the range given by the manufacture. The analytic sensitivity was IL-4 = 0.22 pg/ml; IFN-γ = 0.03 IU/ml; and APRIL = 0.4 pg/ml. Statistical analysis It was performed using SPSS statistical software, version 11.0 (SPSS, Chicago, IL). Quantitative variables were given as mean ± SD, medians, range and categorical variables in frequencies and percents. t test or the Wilcoxon’s rank-sum tests were used for continuous variables according to the distribution of the variable and the v2 test for categorical variables. . Measure the correlation between two quantitative variables by Pearson’s correlation
Rheumatol Int
Kruskall–Wallis one-way analysis of variance (KW) test was used to compare median for >2 independent samples that are not related. p value 0.05). In the group of the 40 SLE patients, 38 people were diagnosed as having active and 2 with inactive disease. The median SLEDAI of SLE Table 1 Demographic data of patients and control groups Patients (n = 40)
Controls (n = 30)
Age (years) Gender (F/M)
31.1 ± 9.95 38/2
27 ± 10.4 29/1
Disease duration
5.1 ± 4.9
Data expressed as mean ± SD
Table 2 Clinical and laboratory characteristics of the patients with SLE
A proliferation-inducing ligand was detectable in all patients with SLE and in all healthy individuals. Table 3 shows that the APRIL level in patients with SLE (8.81 ± 3.1) was significantly higher than that in healthy individuals (1.58 ± 0.8), (p
