Peptides 57 (2014) 17–19
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Short communication
Serum antimicrobial peptides in patients with familial Mediterranean fever Abdurrahman Tufan a,∗ , Rıdvan Mercan a , Ozge Tugce Pasaoglu b , Hatice Pasaoglu b , Mehmet Akif Ozturk a , Berna Goker a , Seminur Haznedaroglu a a b
Gazi University, Department of Internal Medicine, Division of Rheumatology, Ankara, Turkey Gazi University, Department of Biochemistry, Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 14 December 2013 Received in revised form 9 March 2014 Accepted 10 March 2014 Available online 18 April 2014 Keywords: Antimicrobial peptide Cathelicidin Defensin Familial Mediterranean fever Inflammation Pathogenesis
a b s t r a c t Familial Mediterranean fever (FMF) is characterized by recurrent inflammation of serosal and synovial membranes. Despite the fact that it is a genetic disease, environmental factors, including infections, are shown to be triggering factors associated with the precipitation of attacks in FMF. Antimicrobial peptides (AMPs) are components of innate immunity which exert antimicrobial activity against many microorganisms. Human AMPs; cathelicidin (LL37) and defensins have immunomodulatory properties and are involved in the pathogenesis of many inflammatory disorders. Hence, we investigated serum AMPs in 23 newly diagnosed FMF patients. Blood samples were obtained at baseline, 6 months after initiation of colchicine and during an attack. Twenty-four healthy individuals constituted the control group. The concentrations of LL37, alpha-1, beta-1 and beta-2 defensins were determined by ELISA. Serum AMPs did not change during attacks and did not correlate with acute phase reactants. However, serum LL37 and defensins were found to be remarkably higher in FMF patients compared to healthy individuals both at baseline and 6 months after initiation of colchicine therapy which suggest that AMPs might have a role in the pathogenesis of FMF. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Antimicrobial peptides (AMPs) are a wide group of defense proteins that have involved in the innate immune system [8]. In humans, there are three main groups of these proteins: cathelicidin (LL37), defensins and histatins [4]. AMPs show nonspecific antimicrobial activity against many microorganisms including bacteria, viruses and fungi. Among these AMPs, cathelicidin (LL37) and alpha defensins (HNP) located to azurophilic granules of neutrophils where they employed in direct killing of phagocytosed microorganisms [14,19]. LL37 and beta defensins are located to skin and epithelia of respiratory, gastrointestinal and genitourinary systems, where the body first contacts with microbes and noxious agents [13]. On epithelia, most AMPs, such as LL37 and human beta defensin 1 (hBD1) are constitutively secreted, however some
Abbreviations: AMPs, antimicrobial peptides; CRP, C-reactive protein; FMF, familial Mediterranean fever; HNP, alpha defensins; hBD1, human beta defensin 1; hBD2, human beta defensin 2; LL37, cathelicidin. ∗ Corresponding author at: Gazi Universitesi Hastanesi, Romatoloji Bolumu, 06500 Besevler, Ankara, Turkey. Tel.: +90 312 2025825. E-mail address: [email protected] (A. Tufan). http://dx.doi.org/10.1016/j.peptides.2014.03.010 0196-9781/© 2014 Elsevier Inc. All rights reserved.
of them, including human beta defensin 2 (hBD2), are secreted upon induction [8]. AMPs are not only involved in the elimination of microbes, they also have important immunomodulatory properties, including cytokine and chemokine activation, chemotaxis, angiogenesis, wound repair and resolution of inflammation [5,19,20]. Hence, AMPs can initiate an inflammatory response or activate adaptive immune system [5]. To date, the roles of AMPs have been clearly demonstrated in the pathogenesis of various inflammatory disorders, like psoriasis, atopic dermatitis, Wegener’s granulomatosis and inflammatory bowel diseases [7,12,17,18]. Familial Mediterranean fever (FMF) is characterized by recurrent episodes of fever, serositis and arthritis [16]. The exact mechanism of FMF is yet to be defined; however, mutational defects in MEFV gene impair the function of pyrin protein which is crucial in the regulation of interleukin (IL)-1 and nuclear factor-B (NFB) pathways [1,2]. Although there is substantial progress in our understanding of its pathogenesis, there are still many unanswered questions like; why the disease occurs with attacks and how are the attacks triggered. FMF is a disorder of innate immune system and characterized by self-limiting attacks. Some environmental factors induce an attack on a permanent genetic defect. Interestingly, most of these factors
18
A. Tufan et al. / Peptides 57 (2014) 17–19 Table 1 Clinical features of FMF patients. n (%)
Fig. 1. Flowchart for blood sampling of study subjects.
also influence expression of AMPs, which are components of innate immunity. Moreover, AMPs, particularly cathelicidin (LL37) and alpha defensin, are able to stimulate IL-1 secretion [3,6,15]. Hence, we investigated the role of these peptides in patients with FMF. 2. Methods 2.1. Patients and controls
Fever Peritonitis Pleuritis Arthritis Erysipeloid erythema Standing myalgia Family history Unrevealing surgery Age at onset of symptoms, years Age at diagnosis, years CRP, pre-treatment, mg/L CRP, post-treatment, mg/L CRP, during attack, mg/L
17 (73.9) 21 (91.3) 15 (65.2) 13 (56.5) 8 (34.8) 8 (34.8) 16 (69.6) 7 (30.4) 17 (6–46) 31 (19–56) 16.2 (7.4–25.0) 3.9 (1.7–6.3) 65.5 (12.6–123)
CRP: C-reactive protein, all values were presented as n (%) or median (min–max).
Twenty three newly diagnosed, consecutive FMF patients fulfilling Livneh criteria were included [11]. A detailed clinical interview including attack sites, patterns and frequencies were performed for each patient. Patients with active infections, other inflammatory conditions and those with co-morbidities such as spondylitis, inflammatory bowel disease and diabetes were excluded. All patients were prescribed colchicine 1.5 mg/day initially and the dose was then adjusted, according to the clinical response and side effects. Only minority of patients could not tolerate 1.5 mg/day colchicine due to its gastrointestinal side effects. Twenty-four, ageand sex-matched healthy subjects constituted the control group. The study was approved by the institutional ethics committee and all subjects gave their written informed consent. 2.2. Blood samples and measurement of AMPs Blood samples were collected from each patient at the time of diagnosis prior to the initiation of colchicine therapy and 6 months later, while on colchicine in an attack free condition. If a patient had an attack during the follow up period, an additional blood sample was drawn, within the first 24 h of the attack (Fig. 1). All sera were stored at −80 ◦ C until studied. HNP1, hBD1, LL37 (Cusabio Life Sciences) and hBD2 (Phoenix Pharmaceuticals, CA) commercial ELISA test kits were used for the measurement of serum AMPs. All measurements were carried out in duplicate according to the manufacturers’ instructions. 2.3. Statistical analysis All serum AMP levels are presented as median (minimum– maximum). Serum levels of AMPs in healthy subjects and FMF patients were compared with Mann–Whitney U test. Among FMF patients, baseline, attack and at 6-month attack free levels were compared with Friedman test. For two group post hoc comparisons Wilcoxon test was used. Categorical data comparisons were performed with chi-square test. Spearman rank test was carried out for the correlation analyses. All p values are two-sided and values less than 0.05 are considered as significant.
3. Results Clinical characteristics of study patients are presented in Table 1. Median ages at the onset of symptoms, as well as at the diagnosis were remarkably higher in FMF patients in this study compared to the literature. This is due to the inclusion criteria of this study, i.e. inclusion of only the newly diagnosed adult patients. Median serum levels of alpha defensin, beta defensin-1 and LL37 were found to be significantly higher in pre-treatment FMF patients compared to healthy subjects (Table 2). Alpha defensin-1 and LL37, remained higher after treatment with colchicine. However, median serum level of beta defensin 2, an epithelia-derived inducible AMP, did not differ between FMF patients and healthy controls. Colchicine therapy resulted in a significant reduction only in the serum levels of alpha defensin, but did not have a significant effect on other AMPs. During the 6-month follow up period, fifteen patients had attacks, but blood samples could be obtained in thirteen of them. The most common triggering factor reported was psychological stress (69.2%). Infection was not reported as a triggering factor in any patient experiencing an attack while on colchicine. Interestingly, there was no significant change in the serum levels of any of the AMPs during an attack, compared to pre- or post-treatment attack free periods. Serum levels of AMPs did not correlate with C reactive protein (CRP) levels or erythrocyte sedimentation rates, during an attack period or attack free pre- and post-treatment periods. 4. Discussion FMF is characterized by self limiting episodes of peritonitis, pleuritis and arthritis accompanied by fever. FMF is caused by mutations in MEFV gene which encodes pyrin, a critical molecule in the regulation of IL-1 and NF-B pathways [2]. Impaired control of IL-1 activation by defective pyrin is believed to be responsible for most of the clinical manifestations of FMF. Seemingly, attacks do occur spontaneously, however a trigger can be identified in
Table 2 Serum antimicrobial peptides in healthy subjects and patients.
Alpha defensin, ng/mL Cathelicidin, ng/mL Beta 1-defensin, pg/mL Beta 2-defensin, pg/mL
Healthy subjects n = 24
FMF pre-treatment n = 23
FMF post-treatment n = 20
P1
P2
1.22 (0.01–3.98) 43.7 (39.7–58.5) 701 (607–1240) 1131 (216–2037)
6.6 (2.2–12.7) 49.9 (42.1–85.1) 751 (640–1740) 1407 (380–2202)
2.5 (0.12–15.7)* 51.5 (43.7–60.1) 729 (440–1051) 1322 (132–2142)
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Short communication
Serum antimicrobial peptides in patients with familial Mediterranean fever Abdurrahman Tufan a,∗ , Rıdvan Mercan a , Ozge Tugce Pasaoglu b , Hatice Pasaoglu b , Mehmet Akif Ozturk a , Berna Goker a , Seminur Haznedaroglu a a b
Gazi University, Department of Internal Medicine, Division of Rheumatology, Ankara, Turkey Gazi University, Department of Biochemistry, Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 14 December 2013 Received in revised form 9 March 2014 Accepted 10 March 2014 Available online 18 April 2014 Keywords: Antimicrobial peptide Cathelicidin Defensin Familial Mediterranean fever Inflammation Pathogenesis
a b s t r a c t Familial Mediterranean fever (FMF) is characterized by recurrent inflammation of serosal and synovial membranes. Despite the fact that it is a genetic disease, environmental factors, including infections, are shown to be triggering factors associated with the precipitation of attacks in FMF. Antimicrobial peptides (AMPs) are components of innate immunity which exert antimicrobial activity against many microorganisms. Human AMPs; cathelicidin (LL37) and defensins have immunomodulatory properties and are involved in the pathogenesis of many inflammatory disorders. Hence, we investigated serum AMPs in 23 newly diagnosed FMF patients. Blood samples were obtained at baseline, 6 months after initiation of colchicine and during an attack. Twenty-four healthy individuals constituted the control group. The concentrations of LL37, alpha-1, beta-1 and beta-2 defensins were determined by ELISA. Serum AMPs did not change during attacks and did not correlate with acute phase reactants. However, serum LL37 and defensins were found to be remarkably higher in FMF patients compared to healthy individuals both at baseline and 6 months after initiation of colchicine therapy which suggest that AMPs might have a role in the pathogenesis of FMF. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Antimicrobial peptides (AMPs) are a wide group of defense proteins that have involved in the innate immune system [8]. In humans, there are three main groups of these proteins: cathelicidin (LL37), defensins and histatins [4]. AMPs show nonspecific antimicrobial activity against many microorganisms including bacteria, viruses and fungi. Among these AMPs, cathelicidin (LL37) and alpha defensins (HNP) located to azurophilic granules of neutrophils where they employed in direct killing of phagocytosed microorganisms [14,19]. LL37 and beta defensins are located to skin and epithelia of respiratory, gastrointestinal and genitourinary systems, where the body first contacts with microbes and noxious agents [13]. On epithelia, most AMPs, such as LL37 and human beta defensin 1 (hBD1) are constitutively secreted, however some
Abbreviations: AMPs, antimicrobial peptides; CRP, C-reactive protein; FMF, familial Mediterranean fever; HNP, alpha defensins; hBD1, human beta defensin 1; hBD2, human beta defensin 2; LL37, cathelicidin. ∗ Corresponding author at: Gazi Universitesi Hastanesi, Romatoloji Bolumu, 06500 Besevler, Ankara, Turkey. Tel.: +90 312 2025825. E-mail address: [email protected] (A. Tufan). http://dx.doi.org/10.1016/j.peptides.2014.03.010 0196-9781/© 2014 Elsevier Inc. All rights reserved.
of them, including human beta defensin 2 (hBD2), are secreted upon induction [8]. AMPs are not only involved in the elimination of microbes, they also have important immunomodulatory properties, including cytokine and chemokine activation, chemotaxis, angiogenesis, wound repair and resolution of inflammation [5,19,20]. Hence, AMPs can initiate an inflammatory response or activate adaptive immune system [5]. To date, the roles of AMPs have been clearly demonstrated in the pathogenesis of various inflammatory disorders, like psoriasis, atopic dermatitis, Wegener’s granulomatosis and inflammatory bowel diseases [7,12,17,18]. Familial Mediterranean fever (FMF) is characterized by recurrent episodes of fever, serositis and arthritis [16]. The exact mechanism of FMF is yet to be defined; however, mutational defects in MEFV gene impair the function of pyrin protein which is crucial in the regulation of interleukin (IL)-1 and nuclear factor-B (NFB) pathways [1,2]. Although there is substantial progress in our understanding of its pathogenesis, there are still many unanswered questions like; why the disease occurs with attacks and how are the attacks triggered. FMF is a disorder of innate immune system and characterized by self-limiting attacks. Some environmental factors induce an attack on a permanent genetic defect. Interestingly, most of these factors
18
A. Tufan et al. / Peptides 57 (2014) 17–19 Table 1 Clinical features of FMF patients. n (%)
Fig. 1. Flowchart for blood sampling of study subjects.
also influence expression of AMPs, which are components of innate immunity. Moreover, AMPs, particularly cathelicidin (LL37) and alpha defensin, are able to stimulate IL-1 secretion [3,6,15]. Hence, we investigated the role of these peptides in patients with FMF. 2. Methods 2.1. Patients and controls
Fever Peritonitis Pleuritis Arthritis Erysipeloid erythema Standing myalgia Family history Unrevealing surgery Age at onset of symptoms, years Age at diagnosis, years CRP, pre-treatment, mg/L CRP, post-treatment, mg/L CRP, during attack, mg/L
17 (73.9) 21 (91.3) 15 (65.2) 13 (56.5) 8 (34.8) 8 (34.8) 16 (69.6) 7 (30.4) 17 (6–46) 31 (19–56) 16.2 (7.4–25.0) 3.9 (1.7–6.3) 65.5 (12.6–123)
CRP: C-reactive protein, all values were presented as n (%) or median (min–max).
Twenty three newly diagnosed, consecutive FMF patients fulfilling Livneh criteria were included [11]. A detailed clinical interview including attack sites, patterns and frequencies were performed for each patient. Patients with active infections, other inflammatory conditions and those with co-morbidities such as spondylitis, inflammatory bowel disease and diabetes were excluded. All patients were prescribed colchicine 1.5 mg/day initially and the dose was then adjusted, according to the clinical response and side effects. Only minority of patients could not tolerate 1.5 mg/day colchicine due to its gastrointestinal side effects. Twenty-four, ageand sex-matched healthy subjects constituted the control group. The study was approved by the institutional ethics committee and all subjects gave their written informed consent. 2.2. Blood samples and measurement of AMPs Blood samples were collected from each patient at the time of diagnosis prior to the initiation of colchicine therapy and 6 months later, while on colchicine in an attack free condition. If a patient had an attack during the follow up period, an additional blood sample was drawn, within the first 24 h of the attack (Fig. 1). All sera were stored at −80 ◦ C until studied. HNP1, hBD1, LL37 (Cusabio Life Sciences) and hBD2 (Phoenix Pharmaceuticals, CA) commercial ELISA test kits were used for the measurement of serum AMPs. All measurements were carried out in duplicate according to the manufacturers’ instructions. 2.3. Statistical analysis All serum AMP levels are presented as median (minimum– maximum). Serum levels of AMPs in healthy subjects and FMF patients were compared with Mann–Whitney U test. Among FMF patients, baseline, attack and at 6-month attack free levels were compared with Friedman test. For two group post hoc comparisons Wilcoxon test was used. Categorical data comparisons were performed with chi-square test. Spearman rank test was carried out for the correlation analyses. All p values are two-sided and values less than 0.05 are considered as significant.
3. Results Clinical characteristics of study patients are presented in Table 1. Median ages at the onset of symptoms, as well as at the diagnosis were remarkably higher in FMF patients in this study compared to the literature. This is due to the inclusion criteria of this study, i.e. inclusion of only the newly diagnosed adult patients. Median serum levels of alpha defensin, beta defensin-1 and LL37 were found to be significantly higher in pre-treatment FMF patients compared to healthy subjects (Table 2). Alpha defensin-1 and LL37, remained higher after treatment with colchicine. However, median serum level of beta defensin 2, an epithelia-derived inducible AMP, did not differ between FMF patients and healthy controls. Colchicine therapy resulted in a significant reduction only in the serum levels of alpha defensin, but did not have a significant effect on other AMPs. During the 6-month follow up period, fifteen patients had attacks, but blood samples could be obtained in thirteen of them. The most common triggering factor reported was psychological stress (69.2%). Infection was not reported as a triggering factor in any patient experiencing an attack while on colchicine. Interestingly, there was no significant change in the serum levels of any of the AMPs during an attack, compared to pre- or post-treatment attack free periods. Serum levels of AMPs did not correlate with C reactive protein (CRP) levels or erythrocyte sedimentation rates, during an attack period or attack free pre- and post-treatment periods. 4. Discussion FMF is characterized by self limiting episodes of peritonitis, pleuritis and arthritis accompanied by fever. FMF is caused by mutations in MEFV gene which encodes pyrin, a critical molecule in the regulation of IL-1 and NF-B pathways [2]. Impaired control of IL-1 activation by defective pyrin is believed to be responsible for most of the clinical manifestations of FMF. Seemingly, attacks do occur spontaneously, however a trigger can be identified in
Table 2 Serum antimicrobial peptides in healthy subjects and patients.
Alpha defensin, ng/mL Cathelicidin, ng/mL Beta 1-defensin, pg/mL Beta 2-defensin, pg/mL
Healthy subjects n = 24
FMF pre-treatment n = 23
FMF post-treatment n = 20
P1
P2
1.22 (0.01–3.98) 43.7 (39.7–58.5) 701 (607–1240) 1131 (216–2037)
6.6 (2.2–12.7) 49.9 (42.1–85.1) 751 (640–1740) 1407 (380–2202)
2.5 (0.12–15.7)* 51.5 (43.7–60.1) 729 (440–1051) 1322 (132–2142)
