- Email: [email protected]
Profiles of serum cytokine levels in Takayasu arteritis patients: Potential utility as biomarkers for monitoring disease activity
G Model
JJCC-1423; No. of Pages 8 Journal of Cardiology xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Journal of Cardiology journal homepage: www.elsevier.com/locate/jjcc
Original article
Profiles of serum cytokine levels in Takayasu arteritis patients: Potential utility as biomarkers for monitoring disease activity Natsuko Tamura (MD)a, Yasuhiro Maejima (MD, PhD)a, Daisuke Tezuka (MD, PhD)a,b, Chisato Takamura (MD, PhD)a, Shunji Yoshikawa (MD)a, Takashi Ashikaga (MD, PhD, FJCC)a, Kenzo Hirao (MD, PhD)a, Mitsuaki Isobe (MD, FJCC)a,* a b
Department of Cardiovascular Medicine, Tokyo Medical and Dental University, Tokyo, Japan Advanced Imaging Center Yaesu Clinic, Department of Cardiovascular Medicine, Tokyo, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 June 2016 Received in revised form 24 October 2016 Accepted 28 October 2016 Available online xxx
Background: Takayasu arteritis (TA) is an autoimmune arteritis of unknown etiology. Currently, the erythrocyte sedimentation rate (ESR) and serum C-reactive protein (CRP) levels are widely used to monitor disease activity of TA. However, sometimes it is difficult to reflect inflammatory symptoms in either CRP or ESR values, especially in TA patients with immunosuppressive therapies. Therefore, higheraccuracy biomarkers for evaluating disease activity need to be explored. Methods and results: We examined 21 Japanese patients diagnosed with TA; 17 TA patients were treated with prednisone with or without additional immunosuppressive therapies and the remaining 4 patients were treated with infliximab, a human monoclonal anti-tumor necrosis factor (TNF)-a antibody. In active phase, the serum levels of both TNF-a and interleukin (IL)-6 were significantly higher than in healthy subjects, as is the case with both the levels of CRP and ESR. In contrast, the levels of both IL-12 and IL-23 remained in the normal range. Both TNF-a and IL-6 levels were markedly decreased in response to therapies, on equality with both CRP and ESR levels. Regarding the TA patients treated with infliximab, both CRP and IL-6 levels tended to be decreased after infliximab therapy. Conversely, TNF-a level after infliximab therapy was higher than before therapy. Conclusion: Both TNF-a and IL-6 levels, but not IL-12 or IL-23 levels, in the serum could be potent biomarkers that can reflect the activity of TA. ß 2016 Published by Elsevier Ltd on behalf of Japanese College of Cardiology.
Keywords: Takayasu arteritis Cytokine Biomarker Autoimmune disease
Introduction Takayasu arteritis (TA) is an autoimmune systemic arteritis whose etiology remains unclear. A growing body of evidence suggests that there are particular characteristics of gender, race, and geographic distribution in TA patients. A large majority of TA patients are female and distribution of age of TA onset is from 10 to 40 years [1]. Prevalence of TA is much higher in Asia than in the Western world [2–4]. The annual incidence of TA in Japan is estimated to be around 150 patients per year [5]. The lesions of TA mainly include the aorta and its major arterial branches, including left subclavian artery [6]. As the inflammation of arteries in TA is
* Corresponding author at: Department of Cardiovascular Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, 13rd Floor of M&D Tower, Bunkyoku, Tokyo 113-8519, Japan. Fax: +81 3 5803 0133. E-mail address: [email protected] (M. Isobe).
progressive, all vessels of TA patients would be involved eventually. Increasing lines of evidence suggest that single nucleotide polymorphisms (SNPs) of some specific human leukocyte antigen (HLA), including HLA-Bw52, HLA-B39, and HLA-B67 are associated with genetic susceptibility to TA [7–9]. Recently, we have demonstrated that SNPs of IL12B and HLA-B reveal a synergistic role in the susceptibility to TA in Japanese patients [10]. These findings suggest that SNP-mediated alteration of cellular immunity mechanisms would be critical in the prevalence of TA. Currently, the major way of monitoring TA is based on the evaluation of conventional biomarkers, including serum C-reactive protein (CRP) level, erythrocyte sedimentation rate (ESR), and the imaging tests, such as angiography, computed tomography (CT) and magnetic resonance imaging (MRI). However, there are cases that have obvious inflammatory symptoms with no significant increase in either CRP or ESR values, such as relapsed TA patients. We previously reported that there was no significant difference in the values of inflammatory markers, including CRP and ESR,
http://dx.doi.org/10.1016/j.jjcc.2016.10.016 0914-5087/ß 2016 Published by Elsevier Ltd on behalf of Japanese College of Cardiology.
Please cite this article in press as: Tamura N, et al. Profiles of serum cytokine levels in Takayasu arteritis patients: Potential utility as biomarkers for monitoring disease activity. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.10.016
G Model
JJCC-1423; No. of Pages 8 N. Tamura et al. / Journal of Cardiology xxx (2016) xxx–xxx
2
between the monophasic TA patients and the relapsing-remitting ones [11]. Conventional biomarkers often fail to identify the recurrence of TA activity after intensive drug therapy using steroids and/or other immunosuppressive agents, including methotrexate, azathioprine, anti-tumor necrosis factor (TNF)-a agents (infliximab), and anti-interleukin (IL)-6 receptor (IL-6R) antibody (tocilizumab) [12–17]. Previously, we demonstrated that fluorodeoxyglucose positron emission tomography and computed tomography (FDG-PET/CT), a novel imaging modality, is useful not only for the detection of active inflammatory lesions at diagnosis of TA, but also for the determination of TA recurrence despite receiving immunosuppressive agents [17]. However, it is limited where the FDG-PET/CT examinations can undergo. Thus, more sensitive biomarkers are required to catch the subtle flare up of TA. We hypothesized that the serum levels of the following cytokines (TNF-a, a target of infliximab; IL-6, a target of tocilizumab; IL-12 and IL-23, the products of IL12B gene) could be sensitive biomarkers for the TA patients whose inflammatory status is active even in the absence of an increase in CRP nor ESR.
committee in our university and performed under the informed consent of all patients. Analyses of blood samples The measurements of serum CRP level and ESR were conducted at clinical laboratory of University Hospital in Tokyo Medical and Dental University. For the evaluation of serum levels of various cytokines, including TNF-a, IL-6, IL-12, and IL-23, the venous blood was centrifuged at 3000 rpm for 5 min at room temperature. Plasma was stored in microtubes at 20 8C until the assays were performed. The measurements of these cytokines were conducted by SRL (Tokyo, Japan) using enzyme-linked immunosorbent assay kits. We used the normal levels of typical values in CRP and ESR (CRP is less than 0.3 mg/dl, and ESR is from 2 mm/h to 15 mm/h in women) which are used in our hospital. We used the reference values of TNF-a, IL-6, IL-12, and IL-23 which are provided from SRL laboratory. The normal level of TNF-a (high sensitivity TNF-a) is from 0.6 pg/ml to 2.8 pg/ml, IL-6 is less than 4.0 pg/ml, IL-12 is less than 7.8 pg/ml, and IL-23 is less than 39.0 pg/ml.
Methods Statistical analysis Study subjects Twenty-one Japanese TA patients who visited University Hospital of Tokyo Medical and Dental University between April 2007 and Jan 2014 were investigated. All patients were diagnosed with TA according to the 1990 American College of Rheumatology criteria for classification of TA [1]. Serum levels of CRP, TNF-a, IL-6, IL-12, and IL-23 and/or ESR were evaluated in TA patients in both active and inactive phases. Among 17 TA patients who did not receive infliximab therapy, 12 patients were diagnosed as having new-onset TA, and the remaining 5 patients were diagnosed with flared-up TA. Flared-up TA is defined as follows: The TA patients who (1) showed sustained elevation of CRP concentration which could not be explained by other reasons and/or (2) had clinical signs or symptoms due to arteritis regardless of corticosteroid and/ or some immunosuppressive therapies. We defined the ‘‘active phase’’ as the state of disease that are new-onset and flared-up. Concurrently, we defined the ‘‘inactive phase’’ as the state of disease not flared-up. This study was approved by the ethics
All results were analyzed by using Wilcoxon signed-rank test. Because all our data were not distributed normally, we analyzed all data in a non-parametric way. The cut-off values were determined for the biomarkers at the points which are the closest to the upper left corner in the ROC curves. All figures were presented as median and interquartile range. The significant difference was set at the 5% level. Results Clinical characteristics of TA patients The clinical characteristics of 21 TA patients are shown in Table 1. Two male and 19 female TA patients were included. The mean age of these patients was 36.6 years in the range of 22–69 years old. All patients received corticosteroid therapy. During active phase, the mean prednisone dosage was 32.1 15.1 mg/day, ranging from 10 to 60 mg/day. During inactive phase, the mean
Table 1 The clinical characteristics of TA patients (N = 21). Pt No.
Age
Sex
Inflammatory bowel disease
Immunosuppressive therapies
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
24 54 47 53 55 22 35 40 25 22 33 43 35 36 44 28 69 24 24 28 28
F F F F F F F F F M F F F F F M F F F F F
– – – – – – – – – + + – – – – – – – – – –
– MTX – – MTX MTX – – – – Tacrolimus – – MTX – – – MTX Ciclosporin + MTX MTX + tacrolimus MTX + ciclosporin + azathioprine + tacrolimus
Infliximab
Clinical situations
+ + + +
New onset Flare up New onset New onset New onset Flare up New onset New onset New onset Flare up Flare up New onset New onset Flare up New onset New onset New onset Flare up Flare up Flare up Flare up
TA, Takayasu arteritis; F, female; M, male; MTX, methotrexate.
Please cite this article in press as: Tamura N, et al. Profiles of serum cytokine levels in Takayasu arteritis patients: Potential utility as biomarkers for monitoring disease activity. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.10.016
G Model
JJCC-1423; No. of Pages 8 N. Tamura et al. / Journal of Cardiology xxx (2016) xxx–xxx
3
Table 2 The clinical characteristics of TA patients who were treated with infliximab (N = 4). Pt No.
Age
Sex
Immunosuppressive therapy
BW, kg
Infliximab initial dose, mg/body/month
mg/kg/month
Prednisone at the infliximab start, mg/day
Immunosuppressive therapies at the infliximab start, mg/week
18 19 20 21
24 24 28 28
F F F F
MTX Ciclosporin + MTX MTX + tacrolimus MTX + ciclosporin + azathioprine + tacrolimus
60 50 56 45
180 150 170 135
3 3 3.04 3
20 22.5 25 9.5
MTX 15 mg MTX 10 mg Tacrolimus 6 mg Tacrolimus 2 mg
TA, Takayasu arteritis; F, female; BW, body weight; MTX, methotrexate.
Table 3 The serum levels of indicated biomarkers in TA patients (N = 17). Pt No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Mean SD
CRP, mg/dl
ESR, mm (h)
TNF-alpha, pg/ml
IL-6, pg/ml
IL-12, pg/ml
IL-23, pg/ml
Prednisone, mg
Active
Inactive
Active
Inactive
Active
Inactive
Active
Inactive
Active
Inactive
Active
Inactive
Active
Inactive
0.21 0.53 0.03 0.31 1.51 4.34 5.78 0.37 0.23 3.41 1.29 0.84 1.86 1.41 1.83 3.65 0.31 1.64 1.64
0.03 0.54 0.03 0.07 0.07 0.44 0.88 0.11 0.03 0.04 0.60 0.16 0.07 0.03 0.06 0.02 1.70 0.29 0.43
39 19 8 39 ND ND 115 75 16 53 36 45 73 83 ND ND 37 49.08 29.06
2 ND 7 15 22 20 14 11 2 2 24 14 16 10 6 ND 12 11.80 6.86
1.5 35.0 1.0 1.6 2.6 0.9 3.2 1.5 0.8 1.8 0.8 1.5 1.0 0.8 1.9 2.3 1.3 3.50 7.90
0.9 1.7 1.0 1.1 0.8 0.7 1.8 1.0 0.6 1.0 0.8 1.0 0.8 0.7 1.4 1.5 0.5 1.02 0.36
3.5 52.4 1.4 2.7 8.4 25.5 41.0 2.8 3.9 14.5 2.0 6.1 3.8 2.5 6.3 13.9 5.4 11.54 14.28
1.1 4.7 1.4 2.4 5.6 6.2 1.9 2.5 2.0 4.0 4.9 3.4 5.2 2.6 2.3 0.8 6.5 3.38 1.78
2.3 2.1 2.0 2.1 2.1 2.1 2.6 1.6 2.8 1.9 2.5 1.7 1.9 2.2 ND ND ND 2.14 0.32
2.1 ND 1.9 1.6 1.9 1.8 2.0 1.6 2.5 ND ND 2.1 ND ND ND ND ND 1.94 0.26
15.4 16.3 19.2 13.2 12.8 15.7 22.7 17.3 14.8 14.5 17.7 12.8 15.4 17.1 ND ND ND 16.06 2.59
20.2 ND 16.4 15.4 12.2 18.0 17.6 15.7 16.4 ND ND 18.1 ND ND ND ND ND 16.67 2.10
30 10 45 30 50 15 40 55 40 10 20 15 35 20 40 60 30 32.06 15.05
13 10 10 16 20 12 12 12 10 10 16 6 22.5 12 30 40 20 15.97 8.27
TA, Takayasu arteritis; SD, standard deviation; CRP, C-reactive protein; TNF, tumor necrosis factor; IL, interleukin; ESR, erythrocyte sedimentation rate.
prednisone dosage was 16.0 8.3 mg/day, ranging from 6 to 40 mg/ day. During inactive phase, on the other hand, the mean dosage of prednisone was greatly reduced than during active phase (p < 0.001). Among 21 TA patients, 9 patients received immunosuppressive therapies in addition to corticosteroid therapy. Immunosuppressive therapies included methotrexate (n = 8), tacrolimus (n = 3), cyclosporine (n = 2) and azathioprine (n = 1). Four patients were dosed with infliximab in addition to prednisone and immunosuppressive therapies. Two of 21 TA patients also had inflammatory bowel disease (IBD). The clinical characteristics of 4 TA patients who were treated with infliximab are shown in Table 2. These TA patients had to be treated with infliximab because the condition of these patients deteriorated despite receiving intensive therapies using both prednisone and other immunosuppressive agents. The levels of biomarkers in TA patients The serum levels of CRP, TNF-a, IL-6, IL-12, and IL-23, and ESR in 17 of the TA patients are shown in Table 3. As expected, both serum CRP level and ESR of these patients in active phase were significantly elevated above normal values. On the other hand, both serum CRP level and ESR of these patients in inactive phase were significantly decreased than those in active phase (p < 0.01) (Fig. 1a and b). Similarly, both serum TNF-a and IL-6 levels of these patients in active phase were significantly higher than the normal levels of these cytokines and both TNF-a and IL-6 of these patients in inactive phase were significantly decreased than those in active phase (TNF-a: p < 0.001, IL-6: p < 0.05) (Fig. 1c and d). These
results suggest that not only CRP and ESR, the established biomarkers reflecting the disease activity of TA, but also serum levels of TNF-a and IL-6 sensitively reflect TA condition as well. The sensitivity of CRP was higher than ESR, TNF-a, and IL-6. On the other hand, the specificity of CRP was the lowest among four serological parameters (Table 4). Both serum IL-12 and IL-23 levels of these patients both in active and inactive phase were ranged within the normal limits. Changes in biomarker levels of patients who received infliximab therapy The serum levels of CRP, TNF-a, and IL-6 of 4 patients who underwent infliximab therapy were evaluated just before and after infliximab administration (Fig. 2). Although these analyses did not demonstrate any significance, both of serum CRP and IL-6 levels in TA patients who were administered infliximab showed the tendency of decrease (Fig. 2a and c). On the other hand, serum level of TNF-a in these patients tended to increase after infliximab therapy (Fig. 2b). The levels of CRP, TNF-a, and IL-6 in the patients who received infliximab treatment are shown in Table 5 and Fig. 3. As these 4 patients could not achieve remission by the treatment with prednisone and immunosuppressive agents, they had no choice but to undergo infliximab therapy. Unlike in the cases of Pt No. 18–20, the serum CRP level of Pt No. 21 did not increase before infliximab administration. Despite such a state, both TNF-a and IL-6 levels of Pt No. 21 were much higher than those of threshold values determined in this study (TNF-a > 1.3 pg/ml, IL-6 > 5.4 pg/ml).
Please cite this article in press as: Tamura N, et al. Profiles of serum cytokine levels in Takayasu arteritis patients: Potential utility as biomarkers for monitoring disease activity. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.10.016
G Model
JJCC-1423; No. of Pages 8 N. Tamura et al. / Journal of Cardiology xxx (2016) xxx–xxx
4
p < 0.01
p < 0.01
( b)
7
140
6
120
5
100
ESR, mm/h
CRP, mg/dl
(a)
4
80
3
60
2
40
1
20
0
0 inacve
acve
acve
p < 0.001
(c)
p < 0.05
(d)
35
inacve
60
5 50
4
IL-6, pg /ml
TNF-α, pg/ml
4.5
3.5 3 2.5
40
30
2 20 1.5 1
10
0.5 0
0
acve
inacve
acve
inacve
Fig. 1. The serum levels of CRP, TNF-a, IL-6, and ESR in the TA patients are shown (N = 17). (a and b) Both serum CRP level and ESR in the majority of TA patients in active phase were elevated beyond their normal ranges. In inactive phase, these values were significantly decreased compared to those in the active phase (p < 0.01). (c and d) Both serum TNF-a and IL-6 levels in all of TA patients in active phase were elevated beyond their normal ranges. In inactive phase, these values were significantly decreased compared to those in the active phase (TNF-a: p < 0.001, IL-6: p < 0.05). CRP, C-reactive protein; TNF, tumor necrosis factor; IL, interleukin; ESR, erythrocyte sedimentation rate; TA, Takayasu arteritis.
Table 4 Threshold value, sensitivity, specificity, positive predictive value, negative predictive value and accuracy of the indicated biomarkers.
Threshold value Sensitivity Specificity Positive predictive value Negative predictive value Accuracy
CRP
ESR
TNF-a
IL-6
0.21 mg/dl 94.1 70.6 76.2 92.3 82.4
36 mm/h 76.9 100.0 100.0 83.3 89.3
1.3 pg/ml 64.7 76.5 73.3 68.4 70.6
5.4 pg/ml 52.9 82.4 75.0 63.6 67.6
CRP, C-reactive protein; TNF, tumor necrosis factor; IL, interleukin; ESR, erythrocyte sedimentation rate.
Please cite this article in press as: Tamura N, et al. Profiles of serum cytokine levels in Takayasu arteritis patients: Potential utility as biomarkers for monitoring disease activity. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.10.016
G Model
JJCC-1423; No. of Pages 8 N. Tamura et al. / Journal of Cardiology xxx (2016) xxx–xxx
N.S.
(a)
( b)
N.S.
8
16
7
14
6
12
5
TNF-α, pg/ml
CRP, mg/dl
5
4 3 2
10 8 6 4
1 2 0 aer
before
0 before
aer
(c) N.S. 18 16
IL-6, pg/ml
14 12 10 8 6 4 2 0 before
aer
Fig. 2. Serum levels of CRP, TNF-a, and IL-6 of 4 patients who received infliximab are shown. (a) Serum CRP level of these patients tended to decrease by the infliximab therapy. (b) Serum level of TNF-a tended to increase after infliximab therapy. (c) Serum IL-6 level of these patients tended to reduce after infliximab therapy. CRP, C-reactive protein; TNF, tumor necrosis factor; IL, interleukin.
Discussion Here we show that both TNF-a and IL-6 are potent biomarkers for TA activity comparable to those of both ESR and CRP. On the other hand, we could not find any significant correlation between TA activity and serum level of IL-12 and/or IL-23. Thus, both TNF-a and IL-6, but not IL-12 and IL-23, could be potent biomarkers for TA on inflammatory active status. Our current data revealed that the serum level of IL-6 perceptively indicated the TA disease activity. The specificity of IL-6 was higher than CRP. Therefore, it can be useful for assessing disease activity. Previously, Noris et al. and Saadoun et al. reported that the alteration of the serum IL-6 level might be correlated with TA disease activity [18–20]. We also confirmed that the serum IL-6 level was correlated with disease activity of Japanese TA patients. A growing body of evidence suggests that IL-6 is abundantly secreted not only from immune cells but also from the cells of aortic tissue, such as vascular smooth muscle cells and endothelium, in TA patients [21,22]. Nishimoto et al. and Salvarani et al. reported that
treatment with tocilizumab was effective for the patients with TA. In parallel, they also found that tocilizumab therapy caused transient elevation of the serum IL-6 level in those patients [23,24]. In another investigation by Nishimoto et al., the authors demonstrated that the transient elevation of soluble IL-6 receptor (sIL-6R) after administration of tocilizumab increased in patients with rheumatoid arthritis (RA) and Castleman disease, possibly through forming tocilizumab/sIL-6R complex, thereby prolonging elimination half-life of sIL-6R, which, in turn, increased serum sIL6R level. They also revealed that capture of IL-6R by the treatment with tocilizumab results in the increase of free serum IL-6 level through the suppression of IL-6R-mediated IL-6 consumption in patients with RA and Castleman disease [25]. Interestingly, our current data indicate that infliximab therapy for TA patients caused elevation of serum TNF-a level even though the symptoms and the serum levels of both CRP and IL-6 were relieved. But of course, it should not be interpreted that the mechanisms how to elevate serum TNF-a level during infliximab therapy by citing the case of serum IL-6 level on tocilizumab-treated RA in a
Please cite this article in press as: Tamura N, et al. Profiles of serum cytokine levels in Takayasu arteritis patients: Potential utility as biomarkers for monitoring disease activity. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.10.016
G Model
JJCC-1423; No. of Pages 8 N. Tamura et al. / Journal of Cardiology xxx (2016) xxx–xxx
6
Table 5 The serum levels of biomarkers in TA patients who were treated with infliximab (N = 4).
Pt No
infliximab Before
1.08
3.2
13.4
18
Before
0.75
3.5
31.3
19
CRP, mg/dl
TNF-α, pg/ml
IL-6, pg/ml
Aer
0.90
14.6
12.0
Before
1.49
1.2
ND
Before
0.37
0.8
12.6
Before
0.50
0.8
4.5
Before
0.38
0.7
5.4
Before
7.88
0.9
18.6
Aer
0.03
8.5
0.8
Before
7.60
1.2
15.8
Before
1.08
1.2
9.3
Before
3.46
1.1
9.6
20
21
Aer
0.03
9.9
4.2
Before
0.09
1.7
10.8
Before
0.05
1.9
8.7
Before
1.49
1.7
18.7
Before
0.44
1.2
11.8
Before
0.08
1.3
6.8
Before
0.12
1.2
5.3
Aer
0.03
9.7
4.7
Red numbers indicate that it is higher than each threshold value. TA, Takayasu arteritis; CRP, C-reactive protein; TNF, tumor necrosis factor; IL, interleukin.
straightforward way because infliximab binds to TNF-a, but not to the receptor [26]. Indeed, an investigation by Takeshita et al. suggested that the difficulty of the evaluation of TNF-a level in an accurate fashion after infliximab treatment is due to interference from drug-cytokine complexes [27]. Also, as we have not evaluated the serial profile of TNF-a in the TA patients during infliximab therapy, further investigations are needed to assess this issue. Basically, measurement of serum CRP level in combination with ESR evaluation is the most useful for assessing the disease activity in the majority of TA patients as indicated in Table 4. On the other hand, we found that there are some TA cases that need to be evaluated by serum TNF-a and IL-6 levels on behalf of CRP and ESR values. Among the patients who underwent infliximab therapy, there was a patient (Pt No. 21) whose values of both ESR and CRP remained within normal range. However, both serum TNF-a and IL-6 levels in this patient were remarkably higher than those of cut-off values (Table 5 and Fig. 3). Our collaborators and we recently demonstrated that combination of SNPs on IL12B and HLA-B52:01 is significantly associated with severity of aortic regurgitation, a representative severe complication of TA [10]. As the TA-associated SNP region of IL12B (rs6871626) locates on the intron of chromosome 5, it may not affect the amount of IL12-p40, a product of IL12B organizing both IL-12 and IL-23 with another subunit. We recently elucidated whether SNP of IL12B was associated with TA disease severity by examining the relationship between genotype and clinical profiles of TA patients (n = 90) and we found that there is a strong association between the number of risk alleles and the frequency of severe cases as defined by the age at onset, steroid resistance and a
relapse of TA disease [28]. In contrast to TNF-a and IL-6, however, our current data indicated that either IL-12 or IL-23 did not reflect the activity of TA. In patients with psoriasis, a chronic autoimmune inflammatory disease of the skin, there is a significant association with SNP of IL12B [29]. Contrary to the case of TA, the serum IL-12 levels of the psoriasis patients who had IL12B risk haplotype were higher than in those who had no risk haplotype. However, similar to our current findings, the serum level of IL-23 in the psoriasis patients who had IL12B risk haplotype was lower than in those who had no risk haplotype [30]. Therefore, we guess that anti IL12p40 monoclonal antibody, ustekinumab, may have a good effect on psoriasis, but may not have any effect on TA. These results suggest that alteration of IL12B demonstrate unexpected effects on the pathogenesis of both TA and psoriasis without significant elevation of the level of gene products in the serum. Thus, we need to conduct further investigations to address the issue regarding the correlation between genetic alteration of IL12B and clinical manifestations of both TA and psoriasis, including serum IL-12 and IL-23 levels. There are some limitations to this study. First, there were many patients lacking data of IL-12 and IL-23 both in active and inactive phases because of the nature of this study as a retrospective analysis. Second, we could analyze only 4 patients who were treated with infliximab. Finally, the relatively small number of patients and serological parameters used to evaluate the activity of TA included in our study were also limiting factors. Thus, we suppose that our findings require confirmation by large-scale prospective clinical study with more TA patients treated with infliximab.
Please cite this article in press as: Tamura N, et al. Profiles of serum cytokine levels in Takayasu arteritis patients: Potential utility as biomarkers for monitoring disease activity. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.10.016
G Model
JJCC-1423; No. of Pages 8 N. Tamura et al. / Journal of Cardiology xxx (2016) xxx–xxx
7
8
(a)
7
CRP, mg/dl
6 5
Pt No.18 Pt No.19
4
Pt No.20 Pt No.21
3 2 1 Threshold value 0.21 mg/dl
0
Infliximab administraon 14
(b)
TNF-α, pg/ml
12 10 Pt No.18 Pt No.19
8
Pt No.20 6
Pt No.21
4 Threshold value 1.3 pg/ml
2 0
Infliximab administraon
(c)
30
IL-6, pg/ml
25 20
Pt No.18 Pt No.19
15
Pt No.20 Pt No.21
10 Threshold value 5.4 pg/ml
5 0
Infliximab administraon Fig. 3. Serum levels of CRP, TNF-a, and IL-6 of 4 patients who received infliximab therapy were measured several times (from 2 to 5 times) before infliximab therapy. (a) Serum CRP levels of Pt No. 21 did not so much increase before the infliximab therapy, although the patient had symptoms. (b) Serum TNF-a levels of Pt No. 21 before infliximab therapy were higher than the cut off value (1.3 pg/ml). (c) Serum IL-6 levels of Pt No. 21 before infliximab therapy were higher than the cut off value (5.4 pg/ml). CRP, C-reactive protein; TNF, tumor necrosis factor; IL, interleukin.
In conclusion, our current study revealed that both serum TNF-a and IL-6 levels could be potent biomarkers that can be useful to monitor the disease activity of Japanese TA patients. Concurrently, these results suggest that the drugs targeting these cytokines, namely infliximab and tocilizumab, should be potential therapeutic agents in TA. Funding This work was supported in part by Japan Society for the Promotion of Science KAKENHI Grant-in-Aid for Exploratory Research [26670399 to M.I.]. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgment The authors would like to thank Ms Noriko Tamura for her excellent contributions.
References [1] Arend WP, Michel BA, Bloch DA, Hunder GG, Calabrese LH, Edworthy SM, Fauci AS, Leavitt RY, Lie JT, Lightfoot RW. The American College of Rheumatology 1990 criteria for the classification of Takayasu arteritis. Arthritis Rheum 1990;33:1129–34. [2] Dabague J, Reyes PA. Takayasu arteritis in Mexico: a 38-year clinical perspective through literature review. Int J Cardiol 1996;54(Suppl.):S103–9. [3] Hall S, Barr W, Lie JT, Stanson AW, Kazmier FJ, Hunder GG. Takayasu arteritis. A study of 32 North American patients. Medicine (Baltimore) 1985;64:89–99. [4] Ishikawa K. Natural history and classification of occlusive thromboaortopathy (Takayasu’s disease). Circulation 1978;57:27–35. [5] Koide K. Takayasu arteritis in Japan. Heart Vessels Suppl 1992;7:48–54. [6] Hata A, Noda M, Moriwaki R, Numano F. Angiographic findings of Takayasu arteritis: new classification. Int J Cardiol 1996;54(Suppl.):S155–63. [7] Kimura A, Kitamura H, Date Y, Numano F. Comprehensive analysis of HLA genes in Takayasu arteritis in Japan. Int J Cardiol 1996;54(Suppl.):S61–9. [8] Weyand CM, Goronzy JJ. Medium- and large-vessel vasculitis. N Engl J Med 2003;349:160–9. [9] Takamura C, Ohhigashi H, Ebana Y, Isobe M. New human leukocyte antigen risk allele in Japanese patients with Takayasu arteritis. Circ J 2012;76: 1697–702. [10] Terao C, Yoshifuji H, Kimura A, Matsumura T, Ohmura K, Takahashi M, Shimizu M, Kawaguchi T, Chen Z, Naruse TK, Sato-Otsubo A, Ebana Y, Maejima Y, Kinoshita H, Murakami K, et al. Two susceptibility loci to Takayasu arteritis reveal a synergistic role of the IL12B and HLA-B regions in a Japanese population. Am J Hum Genet 2013;93:289–97.
Please cite this article in press as: Tamura N, et al. Profiles of serum cytokine levels in Takayasu arteritis patients: Potential utility as biomarkers for monitoring disease activity. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.10.016
G Model
JJCC-1423; No. of Pages 8 8
N. Tamura et al. / Journal of Cardiology xxx (2016) xxx–xxx
[11] Ohigashi H, Haraguchi G, Konishi M, Tezuka D, Kamiishi T, Ishihara T, Isobe M. Improved prognosis of Takayasu arteritis over the past decade – comprehensive analysis of 106 patients. Circ J 2012;76:1004–11. [12] Hoffman GS, Leavitt RY, Kerr GS, Rottem M, Sneller MC, Fauci AS. Treatment of glucocorticoid-resistant or relapsing Takayasu arteritis with methotrexate. Arthritis Rheum 1994;37:578–82. [13] Valsakumar AK, Valappil UC, Jorapur V, Garg N, Nityanand S, Sinha N. Role of immunosuppressive therapy on clinical, immunological, and angiographic outcome in active Takayasu’s arteritis. J Rheumatol 2003;30:1793–8. [14] Hoffman GS, Merkel PA, Brasington RD, Lenschow DJ, Liang P. Anti-tumor necrosis factor therapy in patients with difficult to treat Takayasu arteritis. Arthritis Rheum 2004;50:2296–304. [15] Molloy ES, Langford CA, Clark TM, Gota CE, Hoffman GS. Anti-tumour necrosis factor therapy in patients with refractory Takayasu arteritis: long-term follow-up. Ann Rheum Dis 2008;67:1567–9. [16] Mekinian A, Ne´el A, Sibilia J, Cohen P, Connault J, Lambert M, Federici L, Berthier S, Fiessinger JN, Godeau B, Marie I, Guillevin L, Hamidou M, Fain O. Efficacy and tolerance of infliximab in refractory Takayasu arteritis: French multicentre study. Rheumatology (Oxford) 2012;51:882–6. [17] Tezuka D, Haraguchi G, Ishihara T, Ohigashi H, Inagaki H, Suzuki J, Hirao K, Isobe M. Role of FDG PET-CT in Takayasu arteritis: sensitive detection of recurrences. JACC Cardiovasc Imaging 2012;5:422–9. [18] Noris M, Daina E, Gamba S, Bonazzola S, Remuzzi G. Interleukin-6 and RANTES in Takayasu arteritis: a guide for therapeutic decisions? Circulation 1999;100(1):55–60. [19] Park MC, Lee SW, Park YB, Lee SK. Serum cytokine profiles and their correlations with disease activity in Takayasu’s arteritis. Rheumatology (Oxford) 2006;45:545–8. [20] Saadoun D, Garrido M, Comarmond C, Desbois AC, Domont F, Savey L, Terrier B, Geri G, Rosenzwajg M, Klatzmann D, Fourret P, Cluzel P, Chiche L, Gaudric J, Koskas F, et al. Th1 and Th17 cytokines drive inflammation in Takayasu arteritis. Arthritis Rheumatol 2015;67:1353–60. [21] Arnaud L, Haroche J, Mathian A, Gorochov G, Amoura Z. Pathogenesis of Takayasu’s arteritis: a 2011 update. Autoimmun Rev 2011;11:61–7.
[22] Seko Y, Sato O, Takagi A, Tada Y, Matsuo H, Yagita H, Okumura K, Yazaki Y. Restricted usage of T-cell receptor Valpha-Vbeta genes in infiltrating cells in aortic tissue of patients with Takayasu’s arteritis. Circulation 1996;93: 1788–90. [23] Salvarani C, Magnani L, Catanoso M, Pipitone N, Versari A, Dardani L, Pulsatelli L, Meliconi R, Boiardi L. Tocilizumab: a novel therapy for patients with largevessel vasculitis. Rheumatology (Oxford) 2012;51:151–6. [24] Nishimoto N, Nakahara H, Yoshio-Hoshino N, Mima T. Successful treatment of a patient with Takayasu arteritis using a humanized anti-interleukin-6 receptor antibody. Arthritis Rheum 2008;58(4):1197–200. [25] Nishimoto N, Terao K, Mima T, Nakahara H, Takagi N, Kakehi T. Mechanisms and pathologic significances in increase in serum interleukin-6 (IL-6) and soluble IL-6 receptor after administration of an anti-IL-6 receptor antibody, tocilizumab, in patients with rheumatoid arthritis and Castleman disease. Blood 2008;112:3959–64. [26] Ma X, Xu S. TNF inhibitor therapy for rheumatoid arthritis. Biomed Rep 2013;1:177–84. [27] Takeshita M, Suzuki K, Kikuchi J, Izumi K, Kurasawa T, Yoshimoto K, Amano K, Takeuchi T. Infliximab and etanercept have distinct actions but similar effects on cytokine profiles in rheumatoid arthritis. Cytokine 2015;75:222–7. [28] Matsumura T, Amiya E, Tamura N, Maejima Y, Komuro I, Isobe M. A novel susceptibility locus for Takayasu arteritis in the IL12B region can be a genetic marker of disease severity. Heart Vessels 2016;31:1016–9. [29] Cargill M, Schrodi SJ, Chang M, Garcia VE, Brandon R, Callis KP, Matsunami N, Ardlie KG, Civello D, Catanese JJ, Leong DU, Panko JM, McAllister LB, Hansen CB, Papenfuss J, et al. A large-scale genetic association study confirms IL12B and leads to the identification of IL23R as psoriasis-risk genes. Am J Hum Genet 2007;80:273–90. [30] Johnston A, Xing X, Swindell WR, Kochkodan J, Riblett M, Nair RP, Stuart PE, Ding J, Voorhees JJ, Elder JT, Gudjonsson JE. Susceptibility-associated genetic variation at IL12B enhances Th1 polarization in psoriasis. Hum Mol Genet 2013;22:1807–15.
Please cite this article in press as: Tamura N, et al. Profiles of serum cytokine levels in Takayasu arteritis patients: Potential utility as biomarkers for monitoring disease activity. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.10.016
JJCC-1423; No. of Pages 8 Journal of Cardiology xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Journal of Cardiology journal homepage: www.elsevier.com/locate/jjcc
Original article
Profiles of serum cytokine levels in Takayasu arteritis patients: Potential utility as biomarkers for monitoring disease activity Natsuko Tamura (MD)a, Yasuhiro Maejima (MD, PhD)a, Daisuke Tezuka (MD, PhD)a,b, Chisato Takamura (MD, PhD)a, Shunji Yoshikawa (MD)a, Takashi Ashikaga (MD, PhD, FJCC)a, Kenzo Hirao (MD, PhD)a, Mitsuaki Isobe (MD, FJCC)a,* a b
Department of Cardiovascular Medicine, Tokyo Medical and Dental University, Tokyo, Japan Advanced Imaging Center Yaesu Clinic, Department of Cardiovascular Medicine, Tokyo, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 June 2016 Received in revised form 24 October 2016 Accepted 28 October 2016 Available online xxx
Background: Takayasu arteritis (TA) is an autoimmune arteritis of unknown etiology. Currently, the erythrocyte sedimentation rate (ESR) and serum C-reactive protein (CRP) levels are widely used to monitor disease activity of TA. However, sometimes it is difficult to reflect inflammatory symptoms in either CRP or ESR values, especially in TA patients with immunosuppressive therapies. Therefore, higheraccuracy biomarkers for evaluating disease activity need to be explored. Methods and results: We examined 21 Japanese patients diagnosed with TA; 17 TA patients were treated with prednisone with or without additional immunosuppressive therapies and the remaining 4 patients were treated with infliximab, a human monoclonal anti-tumor necrosis factor (TNF)-a antibody. In active phase, the serum levels of both TNF-a and interleukin (IL)-6 were significantly higher than in healthy subjects, as is the case with both the levels of CRP and ESR. In contrast, the levels of both IL-12 and IL-23 remained in the normal range. Both TNF-a and IL-6 levels were markedly decreased in response to therapies, on equality with both CRP and ESR levels. Regarding the TA patients treated with infliximab, both CRP and IL-6 levels tended to be decreased after infliximab therapy. Conversely, TNF-a level after infliximab therapy was higher than before therapy. Conclusion: Both TNF-a and IL-6 levels, but not IL-12 or IL-23 levels, in the serum could be potent biomarkers that can reflect the activity of TA. ß 2016 Published by Elsevier Ltd on behalf of Japanese College of Cardiology.
Keywords: Takayasu arteritis Cytokine Biomarker Autoimmune disease
Introduction Takayasu arteritis (TA) is an autoimmune systemic arteritis whose etiology remains unclear. A growing body of evidence suggests that there are particular characteristics of gender, race, and geographic distribution in TA patients. A large majority of TA patients are female and distribution of age of TA onset is from 10 to 40 years [1]. Prevalence of TA is much higher in Asia than in the Western world [2–4]. The annual incidence of TA in Japan is estimated to be around 150 patients per year [5]. The lesions of TA mainly include the aorta and its major arterial branches, including left subclavian artery [6]. As the inflammation of arteries in TA is
* Corresponding author at: Department of Cardiovascular Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, 13rd Floor of M&D Tower, Bunkyoku, Tokyo 113-8519, Japan. Fax: +81 3 5803 0133. E-mail address: [email protected] (M. Isobe).
progressive, all vessels of TA patients would be involved eventually. Increasing lines of evidence suggest that single nucleotide polymorphisms (SNPs) of some specific human leukocyte antigen (HLA), including HLA-Bw52, HLA-B39, and HLA-B67 are associated with genetic susceptibility to TA [7–9]. Recently, we have demonstrated that SNPs of IL12B and HLA-B reveal a synergistic role in the susceptibility to TA in Japanese patients [10]. These findings suggest that SNP-mediated alteration of cellular immunity mechanisms would be critical in the prevalence of TA. Currently, the major way of monitoring TA is based on the evaluation of conventional biomarkers, including serum C-reactive protein (CRP) level, erythrocyte sedimentation rate (ESR), and the imaging tests, such as angiography, computed tomography (CT) and magnetic resonance imaging (MRI). However, there are cases that have obvious inflammatory symptoms with no significant increase in either CRP or ESR values, such as relapsed TA patients. We previously reported that there was no significant difference in the values of inflammatory markers, including CRP and ESR,
http://dx.doi.org/10.1016/j.jjcc.2016.10.016 0914-5087/ß 2016 Published by Elsevier Ltd on behalf of Japanese College of Cardiology.
Please cite this article in press as: Tamura N, et al. Profiles of serum cytokine levels in Takayasu arteritis patients: Potential utility as biomarkers for monitoring disease activity. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.10.016
G Model
JJCC-1423; No. of Pages 8 N. Tamura et al. / Journal of Cardiology xxx (2016) xxx–xxx
2
between the monophasic TA patients and the relapsing-remitting ones [11]. Conventional biomarkers often fail to identify the recurrence of TA activity after intensive drug therapy using steroids and/or other immunosuppressive agents, including methotrexate, azathioprine, anti-tumor necrosis factor (TNF)-a agents (infliximab), and anti-interleukin (IL)-6 receptor (IL-6R) antibody (tocilizumab) [12–17]. Previously, we demonstrated that fluorodeoxyglucose positron emission tomography and computed tomography (FDG-PET/CT), a novel imaging modality, is useful not only for the detection of active inflammatory lesions at diagnosis of TA, but also for the determination of TA recurrence despite receiving immunosuppressive agents [17]. However, it is limited where the FDG-PET/CT examinations can undergo. Thus, more sensitive biomarkers are required to catch the subtle flare up of TA. We hypothesized that the serum levels of the following cytokines (TNF-a, a target of infliximab; IL-6, a target of tocilizumab; IL-12 and IL-23, the products of IL12B gene) could be sensitive biomarkers for the TA patients whose inflammatory status is active even in the absence of an increase in CRP nor ESR.
committee in our university and performed under the informed consent of all patients. Analyses of blood samples The measurements of serum CRP level and ESR were conducted at clinical laboratory of University Hospital in Tokyo Medical and Dental University. For the evaluation of serum levels of various cytokines, including TNF-a, IL-6, IL-12, and IL-23, the venous blood was centrifuged at 3000 rpm for 5 min at room temperature. Plasma was stored in microtubes at 20 8C until the assays were performed. The measurements of these cytokines were conducted by SRL (Tokyo, Japan) using enzyme-linked immunosorbent assay kits. We used the normal levels of typical values in CRP and ESR (CRP is less than 0.3 mg/dl, and ESR is from 2 mm/h to 15 mm/h in women) which are used in our hospital. We used the reference values of TNF-a, IL-6, IL-12, and IL-23 which are provided from SRL laboratory. The normal level of TNF-a (high sensitivity TNF-a) is from 0.6 pg/ml to 2.8 pg/ml, IL-6 is less than 4.0 pg/ml, IL-12 is less than 7.8 pg/ml, and IL-23 is less than 39.0 pg/ml.
Methods Statistical analysis Study subjects Twenty-one Japanese TA patients who visited University Hospital of Tokyo Medical and Dental University between April 2007 and Jan 2014 were investigated. All patients were diagnosed with TA according to the 1990 American College of Rheumatology criteria for classification of TA [1]. Serum levels of CRP, TNF-a, IL-6, IL-12, and IL-23 and/or ESR were evaluated in TA patients in both active and inactive phases. Among 17 TA patients who did not receive infliximab therapy, 12 patients were diagnosed as having new-onset TA, and the remaining 5 patients were diagnosed with flared-up TA. Flared-up TA is defined as follows: The TA patients who (1) showed sustained elevation of CRP concentration which could not be explained by other reasons and/or (2) had clinical signs or symptoms due to arteritis regardless of corticosteroid and/ or some immunosuppressive therapies. We defined the ‘‘active phase’’ as the state of disease that are new-onset and flared-up. Concurrently, we defined the ‘‘inactive phase’’ as the state of disease not flared-up. This study was approved by the ethics
All results were analyzed by using Wilcoxon signed-rank test. Because all our data were not distributed normally, we analyzed all data in a non-parametric way. The cut-off values were determined for the biomarkers at the points which are the closest to the upper left corner in the ROC curves. All figures were presented as median and interquartile range. The significant difference was set at the 5% level. Results Clinical characteristics of TA patients The clinical characteristics of 21 TA patients are shown in Table 1. Two male and 19 female TA patients were included. The mean age of these patients was 36.6 years in the range of 22–69 years old. All patients received corticosteroid therapy. During active phase, the mean prednisone dosage was 32.1 15.1 mg/day, ranging from 10 to 60 mg/day. During inactive phase, the mean
Table 1 The clinical characteristics of TA patients (N = 21). Pt No.
Age
Sex
Inflammatory bowel disease
Immunosuppressive therapies
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
24 54 47 53 55 22 35 40 25 22 33 43 35 36 44 28 69 24 24 28 28
F F F F F F F F F M F F F F F M F F F F F
– – – – – – – – – + + – – – – – – – – – –
– MTX – – MTX MTX – – – – Tacrolimus – – MTX – – – MTX Ciclosporin + MTX MTX + tacrolimus MTX + ciclosporin + azathioprine + tacrolimus
Infliximab
Clinical situations
+ + + +
New onset Flare up New onset New onset New onset Flare up New onset New onset New onset Flare up Flare up New onset New onset Flare up New onset New onset New onset Flare up Flare up Flare up Flare up
TA, Takayasu arteritis; F, female; M, male; MTX, methotrexate.
Please cite this article in press as: Tamura N, et al. Profiles of serum cytokine levels in Takayasu arteritis patients: Potential utility as biomarkers for monitoring disease activity. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.10.016
G Model
JJCC-1423; No. of Pages 8 N. Tamura et al. / Journal of Cardiology xxx (2016) xxx–xxx
3
Table 2 The clinical characteristics of TA patients who were treated with infliximab (N = 4). Pt No.
Age
Sex
Immunosuppressive therapy
BW, kg
Infliximab initial dose, mg/body/month
mg/kg/month
Prednisone at the infliximab start, mg/day
Immunosuppressive therapies at the infliximab start, mg/week
18 19 20 21
24 24 28 28
F F F F
MTX Ciclosporin + MTX MTX + tacrolimus MTX + ciclosporin + azathioprine + tacrolimus
60 50 56 45
180 150 170 135
3 3 3.04 3
20 22.5 25 9.5
MTX 15 mg MTX 10 mg Tacrolimus 6 mg Tacrolimus 2 mg
TA, Takayasu arteritis; F, female; BW, body weight; MTX, methotrexate.
Table 3 The serum levels of indicated biomarkers in TA patients (N = 17). Pt No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Mean SD
CRP, mg/dl
ESR, mm (h)
TNF-alpha, pg/ml
IL-6, pg/ml
IL-12, pg/ml
IL-23, pg/ml
Prednisone, mg
Active
Inactive
Active
Inactive
Active
Inactive
Active
Inactive
Active
Inactive
Active
Inactive
Active
Inactive
0.21 0.53 0.03 0.31 1.51 4.34 5.78 0.37 0.23 3.41 1.29 0.84 1.86 1.41 1.83 3.65 0.31 1.64 1.64
0.03 0.54 0.03 0.07 0.07 0.44 0.88 0.11 0.03 0.04 0.60 0.16 0.07 0.03 0.06 0.02 1.70 0.29 0.43
39 19 8 39 ND ND 115 75 16 53 36 45 73 83 ND ND 37 49.08 29.06
2 ND 7 15 22 20 14 11 2 2 24 14 16 10 6 ND 12 11.80 6.86
1.5 35.0 1.0 1.6 2.6 0.9 3.2 1.5 0.8 1.8 0.8 1.5 1.0 0.8 1.9 2.3 1.3 3.50 7.90
0.9 1.7 1.0 1.1 0.8 0.7 1.8 1.0 0.6 1.0 0.8 1.0 0.8 0.7 1.4 1.5 0.5 1.02 0.36
3.5 52.4 1.4 2.7 8.4 25.5 41.0 2.8 3.9 14.5 2.0 6.1 3.8 2.5 6.3 13.9 5.4 11.54 14.28
1.1 4.7 1.4 2.4 5.6 6.2 1.9 2.5 2.0 4.0 4.9 3.4 5.2 2.6 2.3 0.8 6.5 3.38 1.78
2.3 2.1 2.0 2.1 2.1 2.1 2.6 1.6 2.8 1.9 2.5 1.7 1.9 2.2 ND ND ND 2.14 0.32
2.1 ND 1.9 1.6 1.9 1.8 2.0 1.6 2.5 ND ND 2.1 ND ND ND ND ND 1.94 0.26
15.4 16.3 19.2 13.2 12.8 15.7 22.7 17.3 14.8 14.5 17.7 12.8 15.4 17.1 ND ND ND 16.06 2.59
20.2 ND 16.4 15.4 12.2 18.0 17.6 15.7 16.4 ND ND 18.1 ND ND ND ND ND 16.67 2.10
30 10 45 30 50 15 40 55 40 10 20 15 35 20 40 60 30 32.06 15.05
13 10 10 16 20 12 12 12 10 10 16 6 22.5 12 30 40 20 15.97 8.27
TA, Takayasu arteritis; SD, standard deviation; CRP, C-reactive protein; TNF, tumor necrosis factor; IL, interleukin; ESR, erythrocyte sedimentation rate.
prednisone dosage was 16.0 8.3 mg/day, ranging from 6 to 40 mg/ day. During inactive phase, on the other hand, the mean dosage of prednisone was greatly reduced than during active phase (p < 0.001). Among 21 TA patients, 9 patients received immunosuppressive therapies in addition to corticosteroid therapy. Immunosuppressive therapies included methotrexate (n = 8), tacrolimus (n = 3), cyclosporine (n = 2) and azathioprine (n = 1). Four patients were dosed with infliximab in addition to prednisone and immunosuppressive therapies. Two of 21 TA patients also had inflammatory bowel disease (IBD). The clinical characteristics of 4 TA patients who were treated with infliximab are shown in Table 2. These TA patients had to be treated with infliximab because the condition of these patients deteriorated despite receiving intensive therapies using both prednisone and other immunosuppressive agents. The levels of biomarkers in TA patients The serum levels of CRP, TNF-a, IL-6, IL-12, and IL-23, and ESR in 17 of the TA patients are shown in Table 3. As expected, both serum CRP level and ESR of these patients in active phase were significantly elevated above normal values. On the other hand, both serum CRP level and ESR of these patients in inactive phase were significantly decreased than those in active phase (p < 0.01) (Fig. 1a and b). Similarly, both serum TNF-a and IL-6 levels of these patients in active phase were significantly higher than the normal levels of these cytokines and both TNF-a and IL-6 of these patients in inactive phase were significantly decreased than those in active phase (TNF-a: p < 0.001, IL-6: p < 0.05) (Fig. 1c and d). These
results suggest that not only CRP and ESR, the established biomarkers reflecting the disease activity of TA, but also serum levels of TNF-a and IL-6 sensitively reflect TA condition as well. The sensitivity of CRP was higher than ESR, TNF-a, and IL-6. On the other hand, the specificity of CRP was the lowest among four serological parameters (Table 4). Both serum IL-12 and IL-23 levels of these patients both in active and inactive phase were ranged within the normal limits. Changes in biomarker levels of patients who received infliximab therapy The serum levels of CRP, TNF-a, and IL-6 of 4 patients who underwent infliximab therapy were evaluated just before and after infliximab administration (Fig. 2). Although these analyses did not demonstrate any significance, both of serum CRP and IL-6 levels in TA patients who were administered infliximab showed the tendency of decrease (Fig. 2a and c). On the other hand, serum level of TNF-a in these patients tended to increase after infliximab therapy (Fig. 2b). The levels of CRP, TNF-a, and IL-6 in the patients who received infliximab treatment are shown in Table 5 and Fig. 3. As these 4 patients could not achieve remission by the treatment with prednisone and immunosuppressive agents, they had no choice but to undergo infliximab therapy. Unlike in the cases of Pt No. 18–20, the serum CRP level of Pt No. 21 did not increase before infliximab administration. Despite such a state, both TNF-a and IL-6 levels of Pt No. 21 were much higher than those of threshold values determined in this study (TNF-a > 1.3 pg/ml, IL-6 > 5.4 pg/ml).
Please cite this article in press as: Tamura N, et al. Profiles of serum cytokine levels in Takayasu arteritis patients: Potential utility as biomarkers for monitoring disease activity. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.10.016
G Model
JJCC-1423; No. of Pages 8 N. Tamura et al. / Journal of Cardiology xxx (2016) xxx–xxx
4
p < 0.01
p < 0.01
( b)
7
140
6
120
5
100
ESR, mm/h
CRP, mg/dl
(a)
4
80
3
60
2
40
1
20
0
0 inacve
acve
acve
p < 0.001
(c)
p < 0.05
(d)
35
inacve
60
5 50
4
IL-6, pg /ml
TNF-α, pg/ml
4.5
3.5 3 2.5
40
30
2 20 1.5 1
10
0.5 0
0
acve
inacve
acve
inacve
Fig. 1. The serum levels of CRP, TNF-a, IL-6, and ESR in the TA patients are shown (N = 17). (a and b) Both serum CRP level and ESR in the majority of TA patients in active phase were elevated beyond their normal ranges. In inactive phase, these values were significantly decreased compared to those in the active phase (p < 0.01). (c and d) Both serum TNF-a and IL-6 levels in all of TA patients in active phase were elevated beyond their normal ranges. In inactive phase, these values were significantly decreased compared to those in the active phase (TNF-a: p < 0.001, IL-6: p < 0.05). CRP, C-reactive protein; TNF, tumor necrosis factor; IL, interleukin; ESR, erythrocyte sedimentation rate; TA, Takayasu arteritis.
Table 4 Threshold value, sensitivity, specificity, positive predictive value, negative predictive value and accuracy of the indicated biomarkers.
Threshold value Sensitivity Specificity Positive predictive value Negative predictive value Accuracy
CRP
ESR
TNF-a
IL-6
0.21 mg/dl 94.1 70.6 76.2 92.3 82.4
36 mm/h 76.9 100.0 100.0 83.3 89.3
1.3 pg/ml 64.7 76.5 73.3 68.4 70.6
5.4 pg/ml 52.9 82.4 75.0 63.6 67.6
CRP, C-reactive protein; TNF, tumor necrosis factor; IL, interleukin; ESR, erythrocyte sedimentation rate.
Please cite this article in press as: Tamura N, et al. Profiles of serum cytokine levels in Takayasu arteritis patients: Potential utility as biomarkers for monitoring disease activity. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.10.016
G Model
JJCC-1423; No. of Pages 8 N. Tamura et al. / Journal of Cardiology xxx (2016) xxx–xxx
N.S.
(a)
( b)
N.S.
8
16
7
14
6
12
5
TNF-α, pg/ml
CRP, mg/dl
5
4 3 2
10 8 6 4
1 2 0 aer
before
0 before
aer
(c) N.S. 18 16
IL-6, pg/ml
14 12 10 8 6 4 2 0 before
aer
Fig. 2. Serum levels of CRP, TNF-a, and IL-6 of 4 patients who received infliximab are shown. (a) Serum CRP level of these patients tended to decrease by the infliximab therapy. (b) Serum level of TNF-a tended to increase after infliximab therapy. (c) Serum IL-6 level of these patients tended to reduce after infliximab therapy. CRP, C-reactive protein; TNF, tumor necrosis factor; IL, interleukin.
Discussion Here we show that both TNF-a and IL-6 are potent biomarkers for TA activity comparable to those of both ESR and CRP. On the other hand, we could not find any significant correlation between TA activity and serum level of IL-12 and/or IL-23. Thus, both TNF-a and IL-6, but not IL-12 and IL-23, could be potent biomarkers for TA on inflammatory active status. Our current data revealed that the serum level of IL-6 perceptively indicated the TA disease activity. The specificity of IL-6 was higher than CRP. Therefore, it can be useful for assessing disease activity. Previously, Noris et al. and Saadoun et al. reported that the alteration of the serum IL-6 level might be correlated with TA disease activity [18–20]. We also confirmed that the serum IL-6 level was correlated with disease activity of Japanese TA patients. A growing body of evidence suggests that IL-6 is abundantly secreted not only from immune cells but also from the cells of aortic tissue, such as vascular smooth muscle cells and endothelium, in TA patients [21,22]. Nishimoto et al. and Salvarani et al. reported that
treatment with tocilizumab was effective for the patients with TA. In parallel, they also found that tocilizumab therapy caused transient elevation of the serum IL-6 level in those patients [23,24]. In another investigation by Nishimoto et al., the authors demonstrated that the transient elevation of soluble IL-6 receptor (sIL-6R) after administration of tocilizumab increased in patients with rheumatoid arthritis (RA) and Castleman disease, possibly through forming tocilizumab/sIL-6R complex, thereby prolonging elimination half-life of sIL-6R, which, in turn, increased serum sIL6R level. They also revealed that capture of IL-6R by the treatment with tocilizumab results in the increase of free serum IL-6 level through the suppression of IL-6R-mediated IL-6 consumption in patients with RA and Castleman disease [25]. Interestingly, our current data indicate that infliximab therapy for TA patients caused elevation of serum TNF-a level even though the symptoms and the serum levels of both CRP and IL-6 were relieved. But of course, it should not be interpreted that the mechanisms how to elevate serum TNF-a level during infliximab therapy by citing the case of serum IL-6 level on tocilizumab-treated RA in a
Please cite this article in press as: Tamura N, et al. Profiles of serum cytokine levels in Takayasu arteritis patients: Potential utility as biomarkers for monitoring disease activity. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.10.016
G Model
JJCC-1423; No. of Pages 8 N. Tamura et al. / Journal of Cardiology xxx (2016) xxx–xxx
6
Table 5 The serum levels of biomarkers in TA patients who were treated with infliximab (N = 4).
Pt No
infliximab Before
1.08
3.2
13.4
18
Before
0.75
3.5
31.3
19
CRP, mg/dl
TNF-α, pg/ml
IL-6, pg/ml
Aer
0.90
14.6
12.0
Before
1.49
1.2
ND
Before
0.37
0.8
12.6
Before
0.50
0.8
4.5
Before
0.38
0.7
5.4
Before
7.88
0.9
18.6
Aer
0.03
8.5
0.8
Before
7.60
1.2
15.8
Before
1.08
1.2
9.3
Before
3.46
1.1
9.6
20
21
Aer
0.03
9.9
4.2
Before
0.09
1.7
10.8
Before
0.05
1.9
8.7
Before
1.49
1.7
18.7
Before
0.44
1.2
11.8
Before
0.08
1.3
6.8
Before
0.12
1.2
5.3
Aer
0.03
9.7
4.7
Red numbers indicate that it is higher than each threshold value. TA, Takayasu arteritis; CRP, C-reactive protein; TNF, tumor necrosis factor; IL, interleukin.
straightforward way because infliximab binds to TNF-a, but not to the receptor [26]. Indeed, an investigation by Takeshita et al. suggested that the difficulty of the evaluation of TNF-a level in an accurate fashion after infliximab treatment is due to interference from drug-cytokine complexes [27]. Also, as we have not evaluated the serial profile of TNF-a in the TA patients during infliximab therapy, further investigations are needed to assess this issue. Basically, measurement of serum CRP level in combination with ESR evaluation is the most useful for assessing the disease activity in the majority of TA patients as indicated in Table 4. On the other hand, we found that there are some TA cases that need to be evaluated by serum TNF-a and IL-6 levels on behalf of CRP and ESR values. Among the patients who underwent infliximab therapy, there was a patient (Pt No. 21) whose values of both ESR and CRP remained within normal range. However, both serum TNF-a and IL-6 levels in this patient were remarkably higher than those of cut-off values (Table 5 and Fig. 3). Our collaborators and we recently demonstrated that combination of SNPs on IL12B and HLA-B52:01 is significantly associated with severity of aortic regurgitation, a representative severe complication of TA [10]. As the TA-associated SNP region of IL12B (rs6871626) locates on the intron of chromosome 5, it may not affect the amount of IL12-p40, a product of IL12B organizing both IL-12 and IL-23 with another subunit. We recently elucidated whether SNP of IL12B was associated with TA disease severity by examining the relationship between genotype and clinical profiles of TA patients (n = 90) and we found that there is a strong association between the number of risk alleles and the frequency of severe cases as defined by the age at onset, steroid resistance and a
relapse of TA disease [28]. In contrast to TNF-a and IL-6, however, our current data indicated that either IL-12 or IL-23 did not reflect the activity of TA. In patients with psoriasis, a chronic autoimmune inflammatory disease of the skin, there is a significant association with SNP of IL12B [29]. Contrary to the case of TA, the serum IL-12 levels of the psoriasis patients who had IL12B risk haplotype were higher than in those who had no risk haplotype. However, similar to our current findings, the serum level of IL-23 in the psoriasis patients who had IL12B risk haplotype was lower than in those who had no risk haplotype [30]. Therefore, we guess that anti IL12p40 monoclonal antibody, ustekinumab, may have a good effect on psoriasis, but may not have any effect on TA. These results suggest that alteration of IL12B demonstrate unexpected effects on the pathogenesis of both TA and psoriasis without significant elevation of the level of gene products in the serum. Thus, we need to conduct further investigations to address the issue regarding the correlation between genetic alteration of IL12B and clinical manifestations of both TA and psoriasis, including serum IL-12 and IL-23 levels. There are some limitations to this study. First, there were many patients lacking data of IL-12 and IL-23 both in active and inactive phases because of the nature of this study as a retrospective analysis. Second, we could analyze only 4 patients who were treated with infliximab. Finally, the relatively small number of patients and serological parameters used to evaluate the activity of TA included in our study were also limiting factors. Thus, we suppose that our findings require confirmation by large-scale prospective clinical study with more TA patients treated with infliximab.
Please cite this article in press as: Tamura N, et al. Profiles of serum cytokine levels in Takayasu arteritis patients: Potential utility as biomarkers for monitoring disease activity. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.10.016
G Model
JJCC-1423; No. of Pages 8 N. Tamura et al. / Journal of Cardiology xxx (2016) xxx–xxx
7
8
(a)
7
CRP, mg/dl
6 5
Pt No.18 Pt No.19
4
Pt No.20 Pt No.21
3 2 1 Threshold value 0.21 mg/dl
0
Infliximab administraon 14
(b)
TNF-α, pg/ml
12 10 Pt No.18 Pt No.19
8
Pt No.20 6
Pt No.21
4 Threshold value 1.3 pg/ml
2 0
Infliximab administraon
(c)
30
IL-6, pg/ml
25 20
Pt No.18 Pt No.19
15
Pt No.20 Pt No.21
10 Threshold value 5.4 pg/ml
5 0
Infliximab administraon Fig. 3. Serum levels of CRP, TNF-a, and IL-6 of 4 patients who received infliximab therapy were measured several times (from 2 to 5 times) before infliximab therapy. (a) Serum CRP levels of Pt No. 21 did not so much increase before the infliximab therapy, although the patient had symptoms. (b) Serum TNF-a levels of Pt No. 21 before infliximab therapy were higher than the cut off value (1.3 pg/ml). (c) Serum IL-6 levels of Pt No. 21 before infliximab therapy were higher than the cut off value (5.4 pg/ml). CRP, C-reactive protein; TNF, tumor necrosis factor; IL, interleukin.
In conclusion, our current study revealed that both serum TNF-a and IL-6 levels could be potent biomarkers that can be useful to monitor the disease activity of Japanese TA patients. Concurrently, these results suggest that the drugs targeting these cytokines, namely infliximab and tocilizumab, should be potential therapeutic agents in TA. Funding This work was supported in part by Japan Society for the Promotion of Science KAKENHI Grant-in-Aid for Exploratory Research [26670399 to M.I.]. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgment The authors would like to thank Ms Noriko Tamura for her excellent contributions.
References [1] Arend WP, Michel BA, Bloch DA, Hunder GG, Calabrese LH, Edworthy SM, Fauci AS, Leavitt RY, Lie JT, Lightfoot RW. The American College of Rheumatology 1990 criteria for the classification of Takayasu arteritis. Arthritis Rheum 1990;33:1129–34. [2] Dabague J, Reyes PA. Takayasu arteritis in Mexico: a 38-year clinical perspective through literature review. Int J Cardiol 1996;54(Suppl.):S103–9. [3] Hall S, Barr W, Lie JT, Stanson AW, Kazmier FJ, Hunder GG. Takayasu arteritis. A study of 32 North American patients. Medicine (Baltimore) 1985;64:89–99. [4] Ishikawa K. Natural history and classification of occlusive thromboaortopathy (Takayasu’s disease). Circulation 1978;57:27–35. [5] Koide K. Takayasu arteritis in Japan. Heart Vessels Suppl 1992;7:48–54. [6] Hata A, Noda M, Moriwaki R, Numano F. Angiographic findings of Takayasu arteritis: new classification. Int J Cardiol 1996;54(Suppl.):S155–63. [7] Kimura A, Kitamura H, Date Y, Numano F. Comprehensive analysis of HLA genes in Takayasu arteritis in Japan. Int J Cardiol 1996;54(Suppl.):S61–9. [8] Weyand CM, Goronzy JJ. Medium- and large-vessel vasculitis. N Engl J Med 2003;349:160–9. [9] Takamura C, Ohhigashi H, Ebana Y, Isobe M. New human leukocyte antigen risk allele in Japanese patients with Takayasu arteritis. Circ J 2012;76: 1697–702. [10] Terao C, Yoshifuji H, Kimura A, Matsumura T, Ohmura K, Takahashi M, Shimizu M, Kawaguchi T, Chen Z, Naruse TK, Sato-Otsubo A, Ebana Y, Maejima Y, Kinoshita H, Murakami K, et al. Two susceptibility loci to Takayasu arteritis reveal a synergistic role of the IL12B and HLA-B regions in a Japanese population. Am J Hum Genet 2013;93:289–97.
Please cite this article in press as: Tamura N, et al. Profiles of serum cytokine levels in Takayasu arteritis patients: Potential utility as biomarkers for monitoring disease activity. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.10.016
G Model
JJCC-1423; No. of Pages 8 8
N. Tamura et al. / Journal of Cardiology xxx (2016) xxx–xxx
[11] Ohigashi H, Haraguchi G, Konishi M, Tezuka D, Kamiishi T, Ishihara T, Isobe M. Improved prognosis of Takayasu arteritis over the past decade – comprehensive analysis of 106 patients. Circ J 2012;76:1004–11. [12] Hoffman GS, Leavitt RY, Kerr GS, Rottem M, Sneller MC, Fauci AS. Treatment of glucocorticoid-resistant or relapsing Takayasu arteritis with methotrexate. Arthritis Rheum 1994;37:578–82. [13] Valsakumar AK, Valappil UC, Jorapur V, Garg N, Nityanand S, Sinha N. Role of immunosuppressive therapy on clinical, immunological, and angiographic outcome in active Takayasu’s arteritis. J Rheumatol 2003;30:1793–8. [14] Hoffman GS, Merkel PA, Brasington RD, Lenschow DJ, Liang P. Anti-tumor necrosis factor therapy in patients with difficult to treat Takayasu arteritis. Arthritis Rheum 2004;50:2296–304. [15] Molloy ES, Langford CA, Clark TM, Gota CE, Hoffman GS. Anti-tumour necrosis factor therapy in patients with refractory Takayasu arteritis: long-term follow-up. Ann Rheum Dis 2008;67:1567–9. [16] Mekinian A, Ne´el A, Sibilia J, Cohen P, Connault J, Lambert M, Federici L, Berthier S, Fiessinger JN, Godeau B, Marie I, Guillevin L, Hamidou M, Fain O. Efficacy and tolerance of infliximab in refractory Takayasu arteritis: French multicentre study. Rheumatology (Oxford) 2012;51:882–6. [17] Tezuka D, Haraguchi G, Ishihara T, Ohigashi H, Inagaki H, Suzuki J, Hirao K, Isobe M. Role of FDG PET-CT in Takayasu arteritis: sensitive detection of recurrences. JACC Cardiovasc Imaging 2012;5:422–9. [18] Noris M, Daina E, Gamba S, Bonazzola S, Remuzzi G. Interleukin-6 and RANTES in Takayasu arteritis: a guide for therapeutic decisions? Circulation 1999;100(1):55–60. [19] Park MC, Lee SW, Park YB, Lee SK. Serum cytokine profiles and their correlations with disease activity in Takayasu’s arteritis. Rheumatology (Oxford) 2006;45:545–8. [20] Saadoun D, Garrido M, Comarmond C, Desbois AC, Domont F, Savey L, Terrier B, Geri G, Rosenzwajg M, Klatzmann D, Fourret P, Cluzel P, Chiche L, Gaudric J, Koskas F, et al. Th1 and Th17 cytokines drive inflammation in Takayasu arteritis. Arthritis Rheumatol 2015;67:1353–60. [21] Arnaud L, Haroche J, Mathian A, Gorochov G, Amoura Z. Pathogenesis of Takayasu’s arteritis: a 2011 update. Autoimmun Rev 2011;11:61–7.
[22] Seko Y, Sato O, Takagi A, Tada Y, Matsuo H, Yagita H, Okumura K, Yazaki Y. Restricted usage of T-cell receptor Valpha-Vbeta genes in infiltrating cells in aortic tissue of patients with Takayasu’s arteritis. Circulation 1996;93: 1788–90. [23] Salvarani C, Magnani L, Catanoso M, Pipitone N, Versari A, Dardani L, Pulsatelli L, Meliconi R, Boiardi L. Tocilizumab: a novel therapy for patients with largevessel vasculitis. Rheumatology (Oxford) 2012;51:151–6. [24] Nishimoto N, Nakahara H, Yoshio-Hoshino N, Mima T. Successful treatment of a patient with Takayasu arteritis using a humanized anti-interleukin-6 receptor antibody. Arthritis Rheum 2008;58(4):1197–200. [25] Nishimoto N, Terao K, Mima T, Nakahara H, Takagi N, Kakehi T. Mechanisms and pathologic significances in increase in serum interleukin-6 (IL-6) and soluble IL-6 receptor after administration of an anti-IL-6 receptor antibody, tocilizumab, in patients with rheumatoid arthritis and Castleman disease. Blood 2008;112:3959–64. [26] Ma X, Xu S. TNF inhibitor therapy for rheumatoid arthritis. Biomed Rep 2013;1:177–84. [27] Takeshita M, Suzuki K, Kikuchi J, Izumi K, Kurasawa T, Yoshimoto K, Amano K, Takeuchi T. Infliximab and etanercept have distinct actions but similar effects on cytokine profiles in rheumatoid arthritis. Cytokine 2015;75:222–7. [28] Matsumura T, Amiya E, Tamura N, Maejima Y, Komuro I, Isobe M. A novel susceptibility locus for Takayasu arteritis in the IL12B region can be a genetic marker of disease severity. Heart Vessels 2016;31:1016–9. [29] Cargill M, Schrodi SJ, Chang M, Garcia VE, Brandon R, Callis KP, Matsunami N, Ardlie KG, Civello D, Catanese JJ, Leong DU, Panko JM, McAllister LB, Hansen CB, Papenfuss J, et al. A large-scale genetic association study confirms IL12B and leads to the identification of IL23R as psoriasis-risk genes. Am J Hum Genet 2007;80:273–90. [30] Johnston A, Xing X, Swindell WR, Kochkodan J, Riblett M, Nair RP, Stuart PE, Ding J, Voorhees JJ, Elder JT, Gudjonsson JE. Susceptibility-associated genetic variation at IL12B enhances Th1 polarization in psoriasis. Hum Mol Genet 2013;22:1807–15.
Please cite this article in press as: Tamura N, et al. Profiles of serum cytokine levels in Takayasu arteritis patients: Potential utility as biomarkers for monitoring disease activity. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.10.016