http://informahealthcare.com/mor ISSN 1439-7595 (print), 1439-7609 (online) Mod Rheumatol, 2014; 24(1): 78–85 © 2013 Japan College of Rheumatology DOI 10.3109/14397595.2013.852837
ORIGINAL ARTICLE
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Pentraxin 3 is associated with disease activity but not atherosclerosis in patients with systemic lupus erythematosus Yuki Shimada • Yu Funakubo Asanuma • Kazuhiro Yokota Yoshihiro Yoshida • Hiroshi Kajiyama • Kojiro Sato • Yuji Akiyama • Toshihide Mimura
•
Received: 10 October 2012 / Accepted: 28 December 2012 Ó Japan College of Rheumatology 2013
Abstract Objectives Pentraxin 3 (PTX3) plays an important role in inflammation, immunity, and atherosclerosis. Plasma PTX3 level has drawn attention as a marker that responds to local inflammation. Systemic lupus erythematosus (SLE), a chronic inflammatory disorder which can affect multiple organs, develops atherosclerosis prematurely. We examined the hypotheses that the concentration of plasma PTX3 increases in patients with SLE and that PTX3 is associated with the disease activity and premature atherosclerosis. Methods Plasma PTX3 concentrations were measured in 65 patients with SLE and 53 control subjects. The patients were also evaluated with respect to their clinical characteristics, disease activity indices, and corticosteroid therapy. We performed carotid ultrasonography to measure subclinical atherosclerosis in patients with SLE. Results Plasma PTX3 concentration of the SLE patients was significantly higher than that of the healthy controls (median 3.9 vs. 2.0 ng/mL, p \ 0.001). In patients with SLE, PTX3 concentrations were correlated with SLEDAI (p = 0.011), BILAG index (p \ 0.001), C-reactive protein (p \ 0.001), anemia (p = 0.020), hypoalbuminemia (p = 0.022), and daily dose of prednisolone (p = 0.008) after adjustment for age and sex. PTX3 was not associated with disease duration, anti-ds DNA antibody, CH50, or carotid atherosclerosis.
Y. Shimada Y. F. Asanuma (&) K. Yokota Y. Yoshida H. Kajiyama K. Sato Y. Akiyama T. Mimura Department of Rheumatology and Applied Immunology, Faculty of Medicine, Saitama Medical University, 38 Morohongo, Moroyama-machi, Iruma-gun, Saitama 350-0495, Japan e-mail: [email protected] Published online: 03 February 2013
Conclusions Patients with SLE have increased concentrations of PTX3 compared with control subjects. PTX3 was significantly associated with disease activity but not with carotid atherosclerosis. Keywords Pentraxin 3 Systemic lupus erythematosus Disease activity Inflammation Atherosclerosis
Introduction Pentraxins (PTXs) are a superfamily of multifunctional conserved molecules. Based on the primary structure of the protomers, PTXs are divided into two groups, which are known as long and short PTXs [1]. C-reactive protein (CRP) belongs to the short PTX family and is produced in the liver after stimulation by interleukin-6 (IL-6) [2]. PTX3 is produced by cells involved in innate immunity such as polymorphonuclear leukocytes, macrophages, and dendritic cells, and this molecule interacts with several ligands such as interleukin-1 or tumor necrosis factor-a to play a role in innate immunity [3]. PTX3 does not react to IL-6 and is rarely expressed in hepatic cells. Therefore, unlike CRP, PTX3 might be useful as an index directly reflecting local inflammation. PTX3 binds to several ligands, including complement component C1q, which is known to play a role in innate humoral immunity as well as in the clearance of apoptotic cells [1]. PTX3 also acts as a modulator of inflammatory processes and is involved in the development of atherosclerotic lesions. PTX3 levels are known to be increased in rheumatic diseases such as rheumatoid arthritis [4], Takayasu arteritis [5], small vessel vasculitis [6], infectious diseases such as fungal infection [7] and sepsis [8], and cardiovascular diseases such as acute myocardial infarction [9]. In patients with
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Patients and methods
measurements obtained 5 min apart after the subject had rested quietly in a supine position for at least 10 min. Subjects were considered to have hypertension if they took antihypertensive agents or if they had a systolic blood pressure of at least 140 mmHg or a diastolic pressure of at least 90 mmHg. The activity of SLE was measured with the SLE disease activity index (SLEDAI) [16] and the British Isles lupus assessment group index (BILAG index) [17]. The systemic lupus international collaborating clinics (SLICC)/ ACR damage index was used to estimate organ damage [18]. The ACR has formulated case definitions, reporting standards, and diagnostic testing recommendations for the 19 neuropsychiatric SLE syndromes [19]. Central nervous system (CNS) lupus was defined as being present if patients had some of the 12 neuropsychiatric manifestations of CNS involvement (aseptic meningitis, cerebrovascular disease, demyelinating syndrome, headache, movement disorder, seizure disorder, myelopathy, acute confusional state, anxiety disorder, cognitive dysfunction, mood disorder, or psychosis). Lupus nephritis was defined as present if the clinical and laboratory findings met the ACR criteria (persistent proteinuria[0.5 mg/day or greater than 3? by dipstick, and/ or cellular casts, including red blood cells, hemoglobin, granular, tubular, or mixed casts) [14]. Renal biopsy was performed in patients who had evidence of renal involvement in order to establish the diagnosis of lupus nephritis according to the ISN/RPS 2003 classification [20].
Subjects
Measurement of plasma PTX3 concentrations
We studied 65 patients with SLE and 53 controls. We enrolled consecutive eligible patients aged more than 18 years old who met the ACR criteria for SLE and attended Saitama Medical University Hospital between July 2006 and July 2011 [14, 15]. Patients who had infectious diseases were excluded. Control subjects who did not meet the criteria for SLE or any other inflammatory disease and did not have infectious diseases were recruited by advertising. This study was approved by the Institutional Review Board of Saitama Medical University Hospital and each subject gave written informed consent.
Blood samples were collected from all SLE patients and healthy controls after an overnight fast. Plasma was separated by centrifugation and was stored at -20 °C until assay. The plasma PTX3 concentration was determined by enzyme-linked immunosorbent assay using a commercially available kit, which was a solid-phase sandwich immunoassay based on the F(ab0 )2 fragment of a monoclonal antihuman PTX3 antibody (Human pentraxin3/TSG-14 ELISA system, Perseus Proteomics, Tokyo, Japan). The assay was performed as reported previously [21]. Inoue et al. [21] have confirmed that this ELISA does not cross-react with short pentraxins such as CRP and serum amyloid P.
autoimmune disorders such as rheumatoid arthritis and systemic vasculitis, the plasma PTX3 concentration increases in correlation with disease activity [4–6]. PTX3 is produced by macrophages and endothelial cells in human atherosclerotic plaques [10]. In patients with acute myocardial infarction, the PTX3 concentration increases more rapidly than that of CRP [9]. Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disorder that can affect multiple organs and is characterized by persistent systemic inflammation. Patients with SLE develop premature atherosclerosis and the traditional coronary risk factors do not fully explain their accelerated atherosclerosis [11, 12]. There is growing evidence to suggest that inflammation plays a key role in the pathogenesis of atherosclerosis [13], so inflammation has been suggested as a mechanism mediating the acceleration of atherosclerosis in SLE patients. Since SLE is characterized by chronic inflammation, immune dysfunction, and accelerated atherosclerosis, PTX3 may play a role in the pathogenesis of this disease as well as in the development of accelerated atherosclerosis. In the present study, we investigated whether SLE patients have increased PTX3 levels and whether PTX3 reflects disease activity and accelerated atherosclerosis in these patients.
Clinical assessment Other laboratory tests Demographic and clinical data were collected. Clinical information was obtained through a structured interview, physical examination, review of the medical records, and laboratory tests. Current use of prednisolone was determined by combining the information provided by patients and data from the medical records. Height and weight were measured, and the body mass index (BMI) was calculated as the weight (in kilograms) divided by the square of the height (in meters). Blood pressure was determined as the average of two
In the SLE patients, blood was collected after an overnight fast for measurement of the complete blood count, creatinine, albumin, total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, fasting blood sugar, HbA1c, and uric acid. CRP and the erythrocyte sedimentation rate (ESR) were also measured as markers of inflammation, while anti double-stranded (ds) DNA antibody, complement
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component 3 (C3), and total hemolytic complement (CH50) were determined as SLE-related markers.
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Carotid ultrasonography A series of scans of the carotid artery were obtained by experienced technicians using an identical protocol in 59 patients with SLE. This study was done using high-resolution B-mode ultrasound (Aplio MX, Toshiba Medical Systems, Tochigi, Japan; or ProSound II SSD-6500, Aloka, Tokyo, Japan). The technicians (who were blinded to PTX3 data) scanned the right and left common carotid arteries (CCAs), the carotid bulb, and the internal carotid arteries. Trained readers measured the mean intima-media thickness (IMT) at 1 cm segments of the near and far walls of both CCAs and the far walls of the bilateral carotid bulbs and internal carotid arteries. The upper limit of normal for the IMT was set as 1.0 mm. Atherosclerotic plaque was defined as a lesion protruding into the vessel lumen by at least 50 % beyond the diameter of the surrounding wall. Plaques were measured on the near and far walls of the right and left CCAs, at the bifurcation, and in the internal carotid arteries. All scans were performed by experienced laboratory technicians and physicians with no prior knowledge of each patient’s clinical profile. Statistical analysis Demographic characteristics are presented as the mean ± SD (median) for continuous variables and as frequencies and percentages for categorical variables. PTX3 concentrations were compared between SLE patients and healthy controls by using Wilcoxon’s rank sum test, while Fisher’s two-tailed exact test was employed for comparison of frequencies between groups. The associations between the PTX3 level and CRP, ESR, anti-ds DNA antibody, C3, CH50, SLEDAI, BILAG index, SLICC damage index, or clinical characteristics were examined in the SLE patients using Spearman’s rank correlation analysis. Spearman’s rank correlation coefficients were calculated to assess univariate associations between plasma PTX3 and continuous variables, and then multiple linear regression analysis was done to adjust for age and sex. All analyses in this study employed a level of significance of p \ 0.05 (two-sided) and were performed with IBM SPSS statistics software ver. 18.0 (IBM SPSS Japan, Tokyo, Japan).
Results Baseline characteristics of the SLE patients and control subjects Table 1 shows the baseline characteristics of the SLE patients and the control subjects. In this study, 65 patients
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with SLE and 53 healthy controls were matched for age (mean ± SD 44 ± 16 years vs. 43 ± 10 years, p = 0.871) and sex (percentage of women 90.8 vs. 98.1 %, p = 0.096). The median duration of disease for the SLE patients was 6 years. In the patients’ group, eight patients had CNS lupus and 24 patients had lupus nephritis. Two of these patients had both CNS lupus and lupus nephritis. There was no significant difference of either systolic blood pressure or diastolic blood pressure between the two groups. Patients with SLE were more likely to have hypertension (p \ 0.001) and they had a higher BMI (p = 0.020). There were no significant differences with respect to smoking, diabetes, history of coronary heart disease, and postmenopausal status (women only) between the two groups. The median SLEDAI score of the SLE patients was 9 and the BILAG index was also 9. Plasma PTX3 concentration Figure 1 shows the plasma PTX3 concentrations of the SLE patients (n = 65) and the controls (n = 53). In the SLE patients, PTX3 levels ranged from 1.1 to 71.6 ng/mL, with a mean ± SD of 6.9 ± 9.7 ng/mL and a median value of 3.9 ng/mL. In the control subjects, PTX3 ranged from 0.7 to 7.1 ng/mL, with a mean ± SD of 2.2 ± 1.1 ng/mL and a median value of 2.0 ng/mL. The plasma PTX3 concentration was significantly higher in the SLE patients than in the healthy controls (p \ 0.001). In addition, 12 of the 65 SLE patients had extremely high PTX3 levels over 10 ng/mL. Eight of these 12 outliers were patients with neuropsychiatric disorders and three of the remaining four patients had lupus nephritis. The remaining one patient had neuropsychiatric disorders and lupus nephritis. Correlation between PTX3 and SLE-related markers, coronary risk factors, or carotid atherosclerosis in SLE patients We examined correlations between the plasma PTX3 concentration and SLE-related markers, coronary risk factors, and other laboratory parameters in the 65 patients with SLE (Table 2). PTX3 was negatively correlated with hemoglobin (q = -0.504, p \ 0.001), serum albumin (q = -0.342, p = 0.005), and C3 (q = -0.389, p = 0.005). Conversely, there was a positive correlation between PTX3 and SLEDAI (q = 0.323, p = 0.009), the BILAG index (q = 0.471, p \ 0.001), and the SLICC/ACR damage index (q = 0.331, p = 0.007). After adjustment for age and sex, PTX3 was still significantly associated with anemia (p = 0.020), hypoalbuminemia (p = 0.022), elevation of CRP (p \ 0.001), increased disease activity according to the SLEDAI (p = 0.011) or the BILAG index (p \ 0.001), and the daily dose of prednisolone (p = 0.008). However, PTX3 was not
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Table 1 Characteristics of patients with SLE and healthy controls
Plus-minus values are noted as mean ± SD (median). Unless otherwise noted, Wilcoxon rank sum tests were used for comparing continuous variables between groups NA not applicable
Fisher’s two-tailed exact test was used
SLE patients (n = 65)
Characteristics
Healthy controls (n = 53)
p value
Age (years)
44 ± 16 (42)
43 ± 10 (41)
0.871
Sex (% female)
90.8
98.1
0.096
Disease duration (years)
9 ± 9 (6)
NA
NA
Systolic blood pressure (mmHg)
120 ± 18 (117)
114 ± 12 (115)
0.082
Diastolic blood pressure (mmHg)
72 ± 13 (72)
70 ± 12 (70)
0.382
Body mass index
23.3 ± 5.0 (21.9)
20.9 ± 2.5 (20.3)
0.020
Current smoker (%)
27.7
28.3
0.552
Hypertension (%)
33.8
5.7
\0.001
Diabetes (%)
6.2
3.8
0.441
Past history of coronary heart disease (%)
14.1
9.6
0.331
Postmenopausal (% in female)
30.5
20.8
0.169
Current Statin use (%)
7.7
7.5
0.977
SLEDAI
10 ± 8 (9)
NA
NA
BILAG index
11 ± 9 (9)
NA
NA
SLICC Erythrocyte sedimentation rate (mm/h)
2 ± 2 (1) 55 ± 42 (41)
NA NA
NA NA
C-reactive protein (mg/dL)
0.9 ± 1.7 (0.2)
NA
NA
Anti-ds-DNA antibody (IU/mL)
232.8 ± 1037.5 (29.0)
NA
NA
C3 (mg/dL)
67.9 ± 22.8 (62.0)
NA
NA
CH50 (U)
28.8 ± 10.2 (29.1)
NA
NA
cholesterol, HDL cholesterol, LDL cholesterol), fasting blood suger, HbA1c, and uric acid. PTX3 was positively correlated with triglycerides (q = 0.298, p = 0.024), but the association disappeared after adjustment for age and sex. Moreover, plasma PTX3 was not correlated with the mean IMT (q = -0.054, p = 0.684) measured by carotid ultrasound, and there was no significant difference of PTX3 between SLE patients with carotid plaques (n = 19) and those without carotid plaques [n = 40; plasma PTX3 concentration: 6.4 ± 6.8 (median 3.4) ng/mL vs. 7.5 ± 11.5 (median 4.0) ng/mL, p = 0.581]. Relationship between PTX3 and organ damage in SLE patients Fig. 1 Comparison of plasma PTX3 concentrations between patients with SLE and healthy controls. Each dot represents plasma PTX3 levels in individuals among patients and controls. The Wilcoxon rank sum test was used to determine the p value for differences in concentration between patients with SLE and healthy controls
correlated with the disease duration, ESR, anti-ds DNA antibody titer, CH50, white blood cell count, lymphocyte count, platelet count, or serum creatinine. The plasma PTX3 level did not differ significantly between SLE patients with corticosteroid therapy (n = 46) and those without it (n = 19), being 7.4 ± 11.2 (median 3.8) ng/mL versus 5.6 ± 4.5 (median 4.1) ng/mL (p = 0.988), respectively. There was no correlation between the PTX3 concentration and various coronary risk factors, including age, blood pressure, BMI, serum cholesterol levels (total
Since there was a positive correlation between the plasma concentration of PTX3 and the disease activity indices, we suspected that patients with major organ damage such as CNS lupus or lupus nephritis would have higher PTX3 levels. Therefore, we compared plasma PTX3 concentrations between eight patients with CNS lupus and 57 patients without it. We found that the plasma PTX3 concentration of the SLE patients with CNS lupus (mean 23.0 ± 20.4 ng/mL, median 15.2 ng/mL) was significantly higher than that of the patients without it (mean 4.6 ± 3.7 ng/mL, median 3.7 ng/mL; p \ 0.001). Table 3 shows the details of the eight SLE patients with CNS lupus. Neuropsychiatric manifestations detected in these patients were psychosis, acute confusional state, and myelopathy.
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Table 2 Factors associated with plasma PTX3 levels in patients with SLE
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Variables
Correlation coefficient
p values
Age and sex adjusted p value
General characteristics Age (years)
0.051
0.687
Systolic blood pressure (mmHg)
0.047
0.715
NA 0.869
Diastolic blood pressure (mmHg)
-0.190
0.132
0.139
Body mass index (kg/m2)
-0.158
0.213
0.980
-0.133
0.291
0.865
0.131
0.314
0.583
C3 (mg/dL)
-0.389
0.005
0.268
CH50 (U)
-0.211
0.095
0.682
SLEDAI BILAG index
0.323 0.471
0.009 \0.001
0.011 \0.001
SLICC damage index
0.331
0.007
0.158
0.226
0.070
0.008
Disease characteristics Disease duration (years)
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Anti ds-DNA antibody (IU/mL)
Treatment Daily dose of prednisolone (mg) Laboratory results White blood cell counts (/lL)
-0.026
0.839
0.188
Lymphocytes counts (/lL)
-0.168
0.185
0.433
Hemoglobin (g/dL)
-0.504
\0.001
0.020
Platelets counts (/lL)
-0.112
0.374
0.769
0.076
0.548
0.951
Albumin (mg/dL)
-0.342
0.005
0.022
Total cholesterol (mg/dL)
-0.002
0.987
0.710
HDL cholesterol (mg/dL)
-0.092
0.511
0.868
LDL cholesterol (mg/dL)
0.046
0.736
0.762
0.298 -0.047
0.024 0.721
0.244 0.809
Creatinine (mg/dL)
Triglycerides (mg/dL) Fasting blood sugar (mg/dL) HbA1c (%)
0.167
0.195
0.699
-0.031
0.810
0.977
Erythrocyte sedimentation rate (mm/h)
0.234
0.061
0.160
C-reactive protein (mg/dL)
0.168
0.182
\0.001
-0.054
0.684
0.112
Uric acid (mg/dL) Inflammatory markers Significance values were calculated using Spearman’s correlations. Multivariable linear regression was used for adjustment of age and sex
Carotid atherosclerosis Mean intima-media thickness (mm)
Five of the eight patients with CNS lupus were positive for anti-ds DNA antibody, and half of them had hypocomplementemia. Five of the eight patients were positive for anti-ribosomal P antibody, which is known as a marker of CNS lupus, and one patient was positive for antiphospholipid antibody. Plasma PTX3 concentrations did not show a significant difference between the SLE patients with lupus nephritis (n = 24; mean ± SD 8.9 ± 14.3 ng/mL, median 3.9 ng/ mL) and those without it (n = 41; mean ± SD 5.7 ± 5.4 ng/mL, median 3.9 ng/mL; p = 0.488). Plasma PTX3 concentration was significantly higher in patients with CNS lupus (n = 6; mean ± SD
16.3 ± 6.6 ng/mL, median 15.2 ng/mL) than in patients with lupus nephritis (n = 22; mean ± SD 5.8 ± 5.2 ng/ mL, median 3.7 ng/mL; p \ 0.001). PTX3 levels in two patients overlapping with both CNS lupus and lupus nephritis were 71.6 and 14.5 ng/mL, respectively.
Discussion This study showed that the plasma concentration of PTX3 was significantly higher in SLE patients than in healthy control subjects. Among the SLE patients, PTX3 was
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Table 3 Characteristics of patients with CNS lupus (n = 8)
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Patient no.
Age
Sex
Disease duration (years)
NP disorder
Plasma PTX3 level (ng/mL)
AntidsDNA antibody
Low compliment
Antiribosomal P antibody
Antiphospholipid antibody
SLEDAI
BILAG index
1
52
F
25
Psychosis
10.93
-
?
?
?
2
12
2
36
F
4.5
Acute confusional state
11.082
?
-
-
-
10
14
3
68
M
0.1
Myelopathy
14.386
?
?
?
-
27
33
4
73
F
2
Acute confusional state
15.205
?
-
?
-
36
31
5
26
F
0.1
Myelopathy
15.288
?
?
-
-
9
24
6
36
F
14
Acute confusional state
16.528
?
-
?
-
19
28
7
74
M
0.25
Acute confusional state, Psychosis
28.95
-
-
?
-
27
37
8
31
M
13
Psychosis
71.58
-
?
-
-
23
34
correlated with disease activity, but not with coronary risk factors or carotid atherosclerosis. According to Yamasaki et al. [22], the mean plasma PTX3 concentration was 2.00 ng/mL (95 % confidence interval 1.95–2.04) for 1749 healthy Japanese people who underwent health checks, excluding those with malignancy, renal dysfunction, or rheumatic disease (mean age 59.6 ± 11.4 years, 818 men and 931 women). The mean plasma PTX3 concentration was higher in women than in men (2.12 vs. 1.87 ng/mL). The ELISA kit that we used in this study to measure plasma PTX3 levels was the same as that employed by Yamasaki.Of the 53 healthy controls in our study 52 were women and their mean plasma PTX3 concentration was 2.2 ± 1.1 ng/mL (median 2.0 ng/mL), which was nearly identical to that of the healthy subjects in Yamasaki’s study. Fazzini et al. [6] and Hollan et al. [23] reported serum PTX3 concentrations of 28 patients with SLE (six men and 22 women) and three patients with SLE, respectively. The mean serum PTX3 concentration of the 28 SLE patients reported by Fazzini et al. was 0.38 ± 0.50 ng/mL, which was lower than that of 1.00 ± 0.47 ng/mL in their healthy controls (n = 59). However, 12 of their 28 SLE subjects (42.8 %) did not have active disease (SLEDAI = 0), while only three subjects (4.6 %) in our larger group of 65 SLE patients had a SLEDAI of 0. Kim et al. [24] reported that PTX3 levels were higher in febrile SLE patients with disease flare-up than in healthy controls. Since we found a positive correlation between the plasma PTX3 concentration and SLEDAI or the BILAG index in our SLE patients, we considered that they had a higher mean PTX3 level
compared with the control group because of their relatively high disease activity. PTX3 was correlated with CRP, which is a traditional marker of inflammation, in our patients with SLE after adjustment for age and sex. PTX3 was also related to anemia and hypoalbuminemia, which are two parameters known to reflect the persistence of systemic inflammation. In SLE patients, CRP does not always reflect disease activity, even if they have serious organ damage [25, 26]. CRP is usually normal or slightly elevated in SLE patients, although it can rise much higher in patients with serositis. CRP is synthesized by the liver in response to IL-6. In contrast, PTX3 is synthesized by a wide variety of cells and tissues in response to multiple inflammatory mediators, including IL-1b and TNF-a [1]. Thus, the increased levels of PTX3 may be an indicator of persistent systemic inflammation in patients with SLE. An increase of anti-ds DNA antibodies and/or a decrease of complement is associated with increased renal and hematologic involvement in SLE patients. However, antids DNA antibodies are not always present in SLE, with approximately 60 % of patients being positive during the course of their disease [27]. A decrease of complement is not consistently associated with flare-up of SLE [28]. Moreover, neuropsychiatric disorders in SLE patients do not always develop and worsen in correlation with anti-ds DNA antibody, serum complement CH50 and the inflammatory marker serum CRP. PTX3 was associated with the two disease activity indices, but was not correlated with anti-ds DNA antibodies or complement in our patients with SLE. All of the eight patients with CNS lupus had an
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abnormally high level of PTX3 (C10 ng/mL). A prospective study of 1047 recently diagnosed SLE patients, who were followed for up to 10 years for neuropsychiatric events, showed that autoantibodies such as anti-ribosomal P antibodies and antiphospholipid antibodies were not associated with the first occurrence of neuropsychiatric events [29]. Therefore, increased concentration of plasma PTX3 may provide additional information about disease activity or presence of active CNS involvement that is not obtained from the usual markers for SLE. Doni et al. [30] investigated the effect of glucocorticoid hormones on PTX3 production by different cell types in vitro and in humans. The in vitro study showed that glucocorticoid treatment inhibited PTX3 production by myeloid dendritic cells. In contrast, glucocorticoid treatment induced and (under inflammatory conditions) enhanced PTX3 production by fibroblasts and endothelial cells. In vivo, low-dose glucocorticoid treatment augmented circulating PTX3 levels in healthy donors. Our results showed that plasma PTX3 concentration was correlated with current dose of prednisolone after adjustment for age and sex. Although corticosteroid treatment may partly affect increase of PTX3 in patients with SLE, there was no significant difference of plasma PTX3 levels between SLE patients with and without corticosteroid therapy (median of plasma PTX3 level 3.9 vs. 3.9 ng/mL, p = 0.68). PTX3 is also strongly correlated with SLEDAI and BILAG index after adjustment for age, sex and use of corticosteroid in SLE patients (both p \ 0.005). Thus, PTX3 may reflect disease activity rather than influence of corticosteroid therapy in patients with SLE. Longitudinal studies would warrant the concentration of PTX3 correlates with disease activity through the treatment course in each patient with SLE. Accelerated atherosclerosis in SLE is probably caused by a complex interplay among traditional risk factors, SLErelated factors such as disease activity, and the influence of chronic inflammation. Mediators related to both inflammation and atherosclerosis are attractive candidates to explain the pathogenesis of accelerated atherosclerosis in SLE, and one such candidate mediator is PTX3. However, the plasma PTX3 level was not related to the presence of carotid plaque or to the carotid IMT measured by ultrasonography in our SLE patients. There was also no relation of PTX3 with coronary risk factors, including serum lipids, glucose intolerance, hypertension, obesity, and aging. Since this study was cross-sectional, a longer follow-up of patients would presumably be necessary to determine whether atherosclerosis progresses and/or the incidence of cardiovascular disease are higher in SLE patients with high plasma PTX3 levels. In conclusion, the plasma PTX3 concentration is significantly higher in SLE patients than in healthy controls.
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The PTX3 level is correlated with disease activity and surrogate markers of persistent inflammation, but is not associated with carotid atherosclerosis. In patients with SLE, PTX3 may provide additional information about disease activity that is not obtained from the usual inflammatory and SLE-related markers. Acknowledgments The authors thank Dr. Daisuke Ikuma, Ms. Yukari Yamada and Ms. Kozue Watanabe for their technical support. We also appreciate Ms. Yoshimi Aizaki for her secretarial assistance. Dr. Asanuma was partly supported by research grants from The Ministry of Education, Culture, Sports, Science and Technology of Japan. Conflict of interest
None.
References 1. Cies´lik P, Hrycek A. Long pentraxin 3 (PTX3) in the light of its structure, mechanism of action and clinical implications. Autoimmunity. 2012;45:119–28. 2. Mantovani A, Garlanda C, Doni A, Bottazzi B. Pentraxins in innate immunity: from C-reactive protein to the long pentraxin PTX3. J Clin Immunol. 2008;28:1–13. 3. Deban L, Jaillon S, Garlanda C, Bottazzi B, Mantovani A. Pentraxins in innate immunity: lessons from PTX3. Cell Tissue Res. 2011;343:237–49. 4. Luchetti MM, Piccinini G, Mantovani A, Peri G, Matteucci C, Pomponio G, et al. Expression and production of the long pentraxin PTX3 in rheumatoid arthritis (RA). Clin Exp Immunol. 2000;119:196–202. 5. Dagna L, Salvo F, Tiraboschi M, Bozzolo EP, Franchini S, Doglioni C, et al. Pentraxin-3 as a marker of disease activity in Takayasu arteritis. Ann Intern Med. 2011;155:425–33. 6. Fazzini F, Peri G, Doni A, Dell’Antonio G, Dal Cin E, Bozzolo E, et al. PTX3 in small-vessel vasculitides: an independent indicator of disease activity produced at sites of inflammation. Arthritis Rheum. 2001;44:2841–50. 7. Garlanda C, Hirsch E, Bozza S, Salustri A, De Acetis M, Nota R, et al. Non-redundant role of the long pentraxin PTX3 in antifungal innate immune response. Nature. 2002;420:182–6. 8. Muller B, Peri G, Doni A, Torri V, Landmann R, Bottazi B, et al. Circulating levels of the long pentraxin PTX3 correlate with severity of infection in critically ill patients. Crit Care Med. 2001;29:1404–7. 9. Peri G, Introna M, Corradi D, Iacuitti G, Signorini S, Avanzini F, et al. PTX3, a prototypic long pentraxin, is an early indicator of acute myocardial infarction in humans. Circulation. 2000;102: 636–41. 10. Rolph MS, Zimmer S, Bottazzi B, Garlanda C, Mantovani A, Hansson GK. Production of the long pentraxin PTX3 in advanced atherosclerotic plaques. Arterioscler Thromb Vasc Biol. 2002;22: 10–4. 11. Asanuma Y, Oeser A, Shintani AK, Turner E, Olsen N, Fazio S, et al. Premature coronary-artery atherosclerosis in systemic lupus erythematosus. N Engl J Med. 2003;349:2307–15. 12. Esdaile JM, Abrahamowicz M, Grodzicky T, Li Y, Panaritis C, du Berger R, Coˆte R, et al. Traditional Framingham risk scores fail to fully account for accelerated atherosclerosis in systemic lupus erythematosus. Arthritis Rheum. 2001;44:2331–7. 13. Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med. 1999;340:115–26.
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14. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1982;25:1271–7. 15. Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1997;40:1725. 16. Gladman DD, Iban˜ez D, Urowitz MB. Systemic lupus erythematosus disease activity index 2000. J Rheumatol. 2002;29: 288–91. 17. Simmons DP, Coppock JS, Bacon PA, Bresnihan B, Isenberg DA, Maddison P, et al. Development and assessment of a computerized index of clinical disease activity in systemic lupus erythematosus. Members of the British Isles Lupus Assesment Group (BILAG). Q J Med. 1988;69:927–37. 18. Gladman DD, Urowitz MB. The SLICC/ACR damage index: progress report and experience in the field. Lupus. 1999;8:632–7. 19. ACR AD HOC Committee on Neuropsychiatric Lupus Nomenclature. The American College of Rheumatology nomenclature and case definitions for neuropsychiatric lupus syndromes. Arthritis Rheum. 1999;42:599–608. 20. Weening JJ, D’Agati VD, Schwartz MM, Seshan SV, Alpers CE, Appel GB, et al. The classification of glomerulonephritis in systemic lupus erythematosus revised. J Am Soc Nephrol 2004;15: 241–50 (Erratum in: J Am Soc Nephrol. 2004;15:835–6). 21. Inoue K, Sugiyama A, Reid PC, Ito Y, Miyauchi K, Sagara M, et al. Establishment of a high sensitivity plasma assay for human pentraxin3 as a marker for unstable angina pectoris. Arterioscler Thromb Vasc Biol. 2007;27:161–7. 22. Yamasaki K, Kurimura M, Kasai T, Sagara M, Kodama T, Inoue K. Determination of physiological plasma pentraxin 3 (PTX3) levels in healthy populations. Clin Chem Lab Med. 2009;47: 471–7. 23. Hollan I, Bottazzi B, Cuccovillo I, Forre OT, Mikkelsen K, Saatvedt K, et al. Increased levels of serum pentraxin 3, a novel
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ORIGINAL ARTICLE
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Pentraxin 3 is associated with disease activity but not atherosclerosis in patients with systemic lupus erythematosus Yuki Shimada • Yu Funakubo Asanuma • Kazuhiro Yokota Yoshihiro Yoshida • Hiroshi Kajiyama • Kojiro Sato • Yuji Akiyama • Toshihide Mimura
•
Received: 10 October 2012 / Accepted: 28 December 2012 Ó Japan College of Rheumatology 2013
Abstract Objectives Pentraxin 3 (PTX3) plays an important role in inflammation, immunity, and atherosclerosis. Plasma PTX3 level has drawn attention as a marker that responds to local inflammation. Systemic lupus erythematosus (SLE), a chronic inflammatory disorder which can affect multiple organs, develops atherosclerosis prematurely. We examined the hypotheses that the concentration of plasma PTX3 increases in patients with SLE and that PTX3 is associated with the disease activity and premature atherosclerosis. Methods Plasma PTX3 concentrations were measured in 65 patients with SLE and 53 control subjects. The patients were also evaluated with respect to their clinical characteristics, disease activity indices, and corticosteroid therapy. We performed carotid ultrasonography to measure subclinical atherosclerosis in patients with SLE. Results Plasma PTX3 concentration of the SLE patients was significantly higher than that of the healthy controls (median 3.9 vs. 2.0 ng/mL, p \ 0.001). In patients with SLE, PTX3 concentrations were correlated with SLEDAI (p = 0.011), BILAG index (p \ 0.001), C-reactive protein (p \ 0.001), anemia (p = 0.020), hypoalbuminemia (p = 0.022), and daily dose of prednisolone (p = 0.008) after adjustment for age and sex. PTX3 was not associated with disease duration, anti-ds DNA antibody, CH50, or carotid atherosclerosis.
Y. Shimada Y. F. Asanuma (&) K. Yokota Y. Yoshida H. Kajiyama K. Sato Y. Akiyama T. Mimura Department of Rheumatology and Applied Immunology, Faculty of Medicine, Saitama Medical University, 38 Morohongo, Moroyama-machi, Iruma-gun, Saitama 350-0495, Japan e-mail: [email protected] Published online: 03 February 2013
Conclusions Patients with SLE have increased concentrations of PTX3 compared with control subjects. PTX3 was significantly associated with disease activity but not with carotid atherosclerosis. Keywords Pentraxin 3 Systemic lupus erythematosus Disease activity Inflammation Atherosclerosis
Introduction Pentraxins (PTXs) are a superfamily of multifunctional conserved molecules. Based on the primary structure of the protomers, PTXs are divided into two groups, which are known as long and short PTXs [1]. C-reactive protein (CRP) belongs to the short PTX family and is produced in the liver after stimulation by interleukin-6 (IL-6) [2]. PTX3 is produced by cells involved in innate immunity such as polymorphonuclear leukocytes, macrophages, and dendritic cells, and this molecule interacts with several ligands such as interleukin-1 or tumor necrosis factor-a to play a role in innate immunity [3]. PTX3 does not react to IL-6 and is rarely expressed in hepatic cells. Therefore, unlike CRP, PTX3 might be useful as an index directly reflecting local inflammation. PTX3 binds to several ligands, including complement component C1q, which is known to play a role in innate humoral immunity as well as in the clearance of apoptotic cells [1]. PTX3 also acts as a modulator of inflammatory processes and is involved in the development of atherosclerotic lesions. PTX3 levels are known to be increased in rheumatic diseases such as rheumatoid arthritis [4], Takayasu arteritis [5], small vessel vasculitis [6], infectious diseases such as fungal infection [7] and sepsis [8], and cardiovascular diseases such as acute myocardial infarction [9]. In patients with
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Patients and methods
measurements obtained 5 min apart after the subject had rested quietly in a supine position for at least 10 min. Subjects were considered to have hypertension if they took antihypertensive agents or if they had a systolic blood pressure of at least 140 mmHg or a diastolic pressure of at least 90 mmHg. The activity of SLE was measured with the SLE disease activity index (SLEDAI) [16] and the British Isles lupus assessment group index (BILAG index) [17]. The systemic lupus international collaborating clinics (SLICC)/ ACR damage index was used to estimate organ damage [18]. The ACR has formulated case definitions, reporting standards, and diagnostic testing recommendations for the 19 neuropsychiatric SLE syndromes [19]. Central nervous system (CNS) lupus was defined as being present if patients had some of the 12 neuropsychiatric manifestations of CNS involvement (aseptic meningitis, cerebrovascular disease, demyelinating syndrome, headache, movement disorder, seizure disorder, myelopathy, acute confusional state, anxiety disorder, cognitive dysfunction, mood disorder, or psychosis). Lupus nephritis was defined as present if the clinical and laboratory findings met the ACR criteria (persistent proteinuria[0.5 mg/day or greater than 3? by dipstick, and/ or cellular casts, including red blood cells, hemoglobin, granular, tubular, or mixed casts) [14]. Renal biopsy was performed in patients who had evidence of renal involvement in order to establish the diagnosis of lupus nephritis according to the ISN/RPS 2003 classification [20].
Subjects
Measurement of plasma PTX3 concentrations
We studied 65 patients with SLE and 53 controls. We enrolled consecutive eligible patients aged more than 18 years old who met the ACR criteria for SLE and attended Saitama Medical University Hospital between July 2006 and July 2011 [14, 15]. Patients who had infectious diseases were excluded. Control subjects who did not meet the criteria for SLE or any other inflammatory disease and did not have infectious diseases were recruited by advertising. This study was approved by the Institutional Review Board of Saitama Medical University Hospital and each subject gave written informed consent.
Blood samples were collected from all SLE patients and healthy controls after an overnight fast. Plasma was separated by centrifugation and was stored at -20 °C until assay. The plasma PTX3 concentration was determined by enzyme-linked immunosorbent assay using a commercially available kit, which was a solid-phase sandwich immunoassay based on the F(ab0 )2 fragment of a monoclonal antihuman PTX3 antibody (Human pentraxin3/TSG-14 ELISA system, Perseus Proteomics, Tokyo, Japan). The assay was performed as reported previously [21]. Inoue et al. [21] have confirmed that this ELISA does not cross-react with short pentraxins such as CRP and serum amyloid P.
autoimmune disorders such as rheumatoid arthritis and systemic vasculitis, the plasma PTX3 concentration increases in correlation with disease activity [4–6]. PTX3 is produced by macrophages and endothelial cells in human atherosclerotic plaques [10]. In patients with acute myocardial infarction, the PTX3 concentration increases more rapidly than that of CRP [9]. Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disorder that can affect multiple organs and is characterized by persistent systemic inflammation. Patients with SLE develop premature atherosclerosis and the traditional coronary risk factors do not fully explain their accelerated atherosclerosis [11, 12]. There is growing evidence to suggest that inflammation plays a key role in the pathogenesis of atherosclerosis [13], so inflammation has been suggested as a mechanism mediating the acceleration of atherosclerosis in SLE patients. Since SLE is characterized by chronic inflammation, immune dysfunction, and accelerated atherosclerosis, PTX3 may play a role in the pathogenesis of this disease as well as in the development of accelerated atherosclerosis. In the present study, we investigated whether SLE patients have increased PTX3 levels and whether PTX3 reflects disease activity and accelerated atherosclerosis in these patients.
Clinical assessment Other laboratory tests Demographic and clinical data were collected. Clinical information was obtained through a structured interview, physical examination, review of the medical records, and laboratory tests. Current use of prednisolone was determined by combining the information provided by patients and data from the medical records. Height and weight were measured, and the body mass index (BMI) was calculated as the weight (in kilograms) divided by the square of the height (in meters). Blood pressure was determined as the average of two
In the SLE patients, blood was collected after an overnight fast for measurement of the complete blood count, creatinine, albumin, total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, fasting blood sugar, HbA1c, and uric acid. CRP and the erythrocyte sedimentation rate (ESR) were also measured as markers of inflammation, while anti double-stranded (ds) DNA antibody, complement
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component 3 (C3), and total hemolytic complement (CH50) were determined as SLE-related markers.
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Carotid ultrasonography A series of scans of the carotid artery were obtained by experienced technicians using an identical protocol in 59 patients with SLE. This study was done using high-resolution B-mode ultrasound (Aplio MX, Toshiba Medical Systems, Tochigi, Japan; or ProSound II SSD-6500, Aloka, Tokyo, Japan). The technicians (who were blinded to PTX3 data) scanned the right and left common carotid arteries (CCAs), the carotid bulb, and the internal carotid arteries. Trained readers measured the mean intima-media thickness (IMT) at 1 cm segments of the near and far walls of both CCAs and the far walls of the bilateral carotid bulbs and internal carotid arteries. The upper limit of normal for the IMT was set as 1.0 mm. Atherosclerotic plaque was defined as a lesion protruding into the vessel lumen by at least 50 % beyond the diameter of the surrounding wall. Plaques were measured on the near and far walls of the right and left CCAs, at the bifurcation, and in the internal carotid arteries. All scans were performed by experienced laboratory technicians and physicians with no prior knowledge of each patient’s clinical profile. Statistical analysis Demographic characteristics are presented as the mean ± SD (median) for continuous variables and as frequencies and percentages for categorical variables. PTX3 concentrations were compared between SLE patients and healthy controls by using Wilcoxon’s rank sum test, while Fisher’s two-tailed exact test was employed for comparison of frequencies between groups. The associations between the PTX3 level and CRP, ESR, anti-ds DNA antibody, C3, CH50, SLEDAI, BILAG index, SLICC damage index, or clinical characteristics were examined in the SLE patients using Spearman’s rank correlation analysis. Spearman’s rank correlation coefficients were calculated to assess univariate associations between plasma PTX3 and continuous variables, and then multiple linear regression analysis was done to adjust for age and sex. All analyses in this study employed a level of significance of p \ 0.05 (two-sided) and were performed with IBM SPSS statistics software ver. 18.0 (IBM SPSS Japan, Tokyo, Japan).
Results Baseline characteristics of the SLE patients and control subjects Table 1 shows the baseline characteristics of the SLE patients and the control subjects. In this study, 65 patients
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with SLE and 53 healthy controls were matched for age (mean ± SD 44 ± 16 years vs. 43 ± 10 years, p = 0.871) and sex (percentage of women 90.8 vs. 98.1 %, p = 0.096). The median duration of disease for the SLE patients was 6 years. In the patients’ group, eight patients had CNS lupus and 24 patients had lupus nephritis. Two of these patients had both CNS lupus and lupus nephritis. There was no significant difference of either systolic blood pressure or diastolic blood pressure between the two groups. Patients with SLE were more likely to have hypertension (p \ 0.001) and they had a higher BMI (p = 0.020). There were no significant differences with respect to smoking, diabetes, history of coronary heart disease, and postmenopausal status (women only) between the two groups. The median SLEDAI score of the SLE patients was 9 and the BILAG index was also 9. Plasma PTX3 concentration Figure 1 shows the plasma PTX3 concentrations of the SLE patients (n = 65) and the controls (n = 53). In the SLE patients, PTX3 levels ranged from 1.1 to 71.6 ng/mL, with a mean ± SD of 6.9 ± 9.7 ng/mL and a median value of 3.9 ng/mL. In the control subjects, PTX3 ranged from 0.7 to 7.1 ng/mL, with a mean ± SD of 2.2 ± 1.1 ng/mL and a median value of 2.0 ng/mL. The plasma PTX3 concentration was significantly higher in the SLE patients than in the healthy controls (p \ 0.001). In addition, 12 of the 65 SLE patients had extremely high PTX3 levels over 10 ng/mL. Eight of these 12 outliers were patients with neuropsychiatric disorders and three of the remaining four patients had lupus nephritis. The remaining one patient had neuropsychiatric disorders and lupus nephritis. Correlation between PTX3 and SLE-related markers, coronary risk factors, or carotid atherosclerosis in SLE patients We examined correlations between the plasma PTX3 concentration and SLE-related markers, coronary risk factors, and other laboratory parameters in the 65 patients with SLE (Table 2). PTX3 was negatively correlated with hemoglobin (q = -0.504, p \ 0.001), serum albumin (q = -0.342, p = 0.005), and C3 (q = -0.389, p = 0.005). Conversely, there was a positive correlation between PTX3 and SLEDAI (q = 0.323, p = 0.009), the BILAG index (q = 0.471, p \ 0.001), and the SLICC/ACR damage index (q = 0.331, p = 0.007). After adjustment for age and sex, PTX3 was still significantly associated with anemia (p = 0.020), hypoalbuminemia (p = 0.022), elevation of CRP (p \ 0.001), increased disease activity according to the SLEDAI (p = 0.011) or the BILAG index (p \ 0.001), and the daily dose of prednisolone (p = 0.008). However, PTX3 was not
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Table 1 Characteristics of patients with SLE and healthy controls
Plus-minus values are noted as mean ± SD (median). Unless otherwise noted, Wilcoxon rank sum tests were used for comparing continuous variables between groups NA not applicable
Fisher’s two-tailed exact test was used
SLE patients (n = 65)
Characteristics
Healthy controls (n = 53)
p value
Age (years)
44 ± 16 (42)
43 ± 10 (41)
0.871
Sex (% female)
90.8
98.1
0.096
Disease duration (years)
9 ± 9 (6)
NA
NA
Systolic blood pressure (mmHg)
120 ± 18 (117)
114 ± 12 (115)
0.082
Diastolic blood pressure (mmHg)
72 ± 13 (72)
70 ± 12 (70)
0.382
Body mass index
23.3 ± 5.0 (21.9)
20.9 ± 2.5 (20.3)
0.020
Current smoker (%)
27.7
28.3
0.552
Hypertension (%)
33.8
5.7
\0.001
Diabetes (%)
6.2
3.8
0.441
Past history of coronary heart disease (%)
14.1
9.6
0.331
Postmenopausal (% in female)
30.5
20.8
0.169
Current Statin use (%)
7.7
7.5
0.977
SLEDAI
10 ± 8 (9)
NA
NA
BILAG index
11 ± 9 (9)
NA
NA
SLICC Erythrocyte sedimentation rate (mm/h)
2 ± 2 (1) 55 ± 42 (41)
NA NA
NA NA
C-reactive protein (mg/dL)
0.9 ± 1.7 (0.2)
NA
NA
Anti-ds-DNA antibody (IU/mL)
232.8 ± 1037.5 (29.0)
NA
NA
C3 (mg/dL)
67.9 ± 22.8 (62.0)
NA
NA
CH50 (U)
28.8 ± 10.2 (29.1)
NA
NA
cholesterol, HDL cholesterol, LDL cholesterol), fasting blood suger, HbA1c, and uric acid. PTX3 was positively correlated with triglycerides (q = 0.298, p = 0.024), but the association disappeared after adjustment for age and sex. Moreover, plasma PTX3 was not correlated with the mean IMT (q = -0.054, p = 0.684) measured by carotid ultrasound, and there was no significant difference of PTX3 between SLE patients with carotid plaques (n = 19) and those without carotid plaques [n = 40; plasma PTX3 concentration: 6.4 ± 6.8 (median 3.4) ng/mL vs. 7.5 ± 11.5 (median 4.0) ng/mL, p = 0.581]. Relationship between PTX3 and organ damage in SLE patients Fig. 1 Comparison of plasma PTX3 concentrations between patients with SLE and healthy controls. Each dot represents plasma PTX3 levels in individuals among patients and controls. The Wilcoxon rank sum test was used to determine the p value for differences in concentration between patients with SLE and healthy controls
correlated with the disease duration, ESR, anti-ds DNA antibody titer, CH50, white blood cell count, lymphocyte count, platelet count, or serum creatinine. The plasma PTX3 level did not differ significantly between SLE patients with corticosteroid therapy (n = 46) and those without it (n = 19), being 7.4 ± 11.2 (median 3.8) ng/mL versus 5.6 ± 4.5 (median 4.1) ng/mL (p = 0.988), respectively. There was no correlation between the PTX3 concentration and various coronary risk factors, including age, blood pressure, BMI, serum cholesterol levels (total
Since there was a positive correlation between the plasma concentration of PTX3 and the disease activity indices, we suspected that patients with major organ damage such as CNS lupus or lupus nephritis would have higher PTX3 levels. Therefore, we compared plasma PTX3 concentrations between eight patients with CNS lupus and 57 patients without it. We found that the plasma PTX3 concentration of the SLE patients with CNS lupus (mean 23.0 ± 20.4 ng/mL, median 15.2 ng/mL) was significantly higher than that of the patients without it (mean 4.6 ± 3.7 ng/mL, median 3.7 ng/mL; p \ 0.001). Table 3 shows the details of the eight SLE patients with CNS lupus. Neuropsychiatric manifestations detected in these patients were psychosis, acute confusional state, and myelopathy.
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Table 2 Factors associated with plasma PTX3 levels in patients with SLE
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Variables
Correlation coefficient
p values
Age and sex adjusted p value
General characteristics Age (years)
0.051
0.687
Systolic blood pressure (mmHg)
0.047
0.715
NA 0.869
Diastolic blood pressure (mmHg)
-0.190
0.132
0.139
Body mass index (kg/m2)
-0.158
0.213
0.980
-0.133
0.291
0.865
0.131
0.314
0.583
C3 (mg/dL)
-0.389
0.005
0.268
CH50 (U)
-0.211
0.095
0.682
SLEDAI BILAG index
0.323 0.471
0.009 \0.001
0.011 \0.001
SLICC damage index
0.331
0.007
0.158
0.226
0.070
0.008
Disease characteristics Disease duration (years)
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Anti ds-DNA antibody (IU/mL)
Treatment Daily dose of prednisolone (mg) Laboratory results White blood cell counts (/lL)
-0.026
0.839
0.188
Lymphocytes counts (/lL)
-0.168
0.185
0.433
Hemoglobin (g/dL)
-0.504
\0.001
0.020
Platelets counts (/lL)
-0.112
0.374
0.769
0.076
0.548
0.951
Albumin (mg/dL)
-0.342
0.005
0.022
Total cholesterol (mg/dL)
-0.002
0.987
0.710
HDL cholesterol (mg/dL)
-0.092
0.511
0.868
LDL cholesterol (mg/dL)
0.046
0.736
0.762
0.298 -0.047
0.024 0.721
0.244 0.809
Creatinine (mg/dL)
Triglycerides (mg/dL) Fasting blood sugar (mg/dL) HbA1c (%)
0.167
0.195
0.699
-0.031
0.810
0.977
Erythrocyte sedimentation rate (mm/h)
0.234
0.061
0.160
C-reactive protein (mg/dL)
0.168
0.182
\0.001
-0.054
0.684
0.112
Uric acid (mg/dL) Inflammatory markers Significance values were calculated using Spearman’s correlations. Multivariable linear regression was used for adjustment of age and sex
Carotid atherosclerosis Mean intima-media thickness (mm)
Five of the eight patients with CNS lupus were positive for anti-ds DNA antibody, and half of them had hypocomplementemia. Five of the eight patients were positive for anti-ribosomal P antibody, which is known as a marker of CNS lupus, and one patient was positive for antiphospholipid antibody. Plasma PTX3 concentrations did not show a significant difference between the SLE patients with lupus nephritis (n = 24; mean ± SD 8.9 ± 14.3 ng/mL, median 3.9 ng/ mL) and those without it (n = 41; mean ± SD 5.7 ± 5.4 ng/mL, median 3.9 ng/mL; p = 0.488). Plasma PTX3 concentration was significantly higher in patients with CNS lupus (n = 6; mean ± SD
16.3 ± 6.6 ng/mL, median 15.2 ng/mL) than in patients with lupus nephritis (n = 22; mean ± SD 5.8 ± 5.2 ng/ mL, median 3.7 ng/mL; p \ 0.001). PTX3 levels in two patients overlapping with both CNS lupus and lupus nephritis were 71.6 and 14.5 ng/mL, respectively.
Discussion This study showed that the plasma concentration of PTX3 was significantly higher in SLE patients than in healthy control subjects. Among the SLE patients, PTX3 was
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Table 3 Characteristics of patients with CNS lupus (n = 8)
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Patient no.
Age
Sex
Disease duration (years)
NP disorder
Plasma PTX3 level (ng/mL)
AntidsDNA antibody
Low compliment
Antiribosomal P antibody
Antiphospholipid antibody
SLEDAI
BILAG index
1
52
F
25
Psychosis
10.93
-
?
?
?
2
12
2
36
F
4.5
Acute confusional state
11.082
?
-
-
-
10
14
3
68
M
0.1
Myelopathy
14.386
?
?
?
-
27
33
4
73
F
2
Acute confusional state
15.205
?
-
?
-
36
31
5
26
F
0.1
Myelopathy
15.288
?
?
-
-
9
24
6
36
F
14
Acute confusional state
16.528
?
-
?
-
19
28
7
74
M
0.25
Acute confusional state, Psychosis
28.95
-
-
?
-
27
37
8
31
M
13
Psychosis
71.58
-
?
-
-
23
34
correlated with disease activity, but not with coronary risk factors or carotid atherosclerosis. According to Yamasaki et al. [22], the mean plasma PTX3 concentration was 2.00 ng/mL (95 % confidence interval 1.95–2.04) for 1749 healthy Japanese people who underwent health checks, excluding those with malignancy, renal dysfunction, or rheumatic disease (mean age 59.6 ± 11.4 years, 818 men and 931 women). The mean plasma PTX3 concentration was higher in women than in men (2.12 vs. 1.87 ng/mL). The ELISA kit that we used in this study to measure plasma PTX3 levels was the same as that employed by Yamasaki.Of the 53 healthy controls in our study 52 were women and their mean plasma PTX3 concentration was 2.2 ± 1.1 ng/mL (median 2.0 ng/mL), which was nearly identical to that of the healthy subjects in Yamasaki’s study. Fazzini et al. [6] and Hollan et al. [23] reported serum PTX3 concentrations of 28 patients with SLE (six men and 22 women) and three patients with SLE, respectively. The mean serum PTX3 concentration of the 28 SLE patients reported by Fazzini et al. was 0.38 ± 0.50 ng/mL, which was lower than that of 1.00 ± 0.47 ng/mL in their healthy controls (n = 59). However, 12 of their 28 SLE subjects (42.8 %) did not have active disease (SLEDAI = 0), while only three subjects (4.6 %) in our larger group of 65 SLE patients had a SLEDAI of 0. Kim et al. [24] reported that PTX3 levels were higher in febrile SLE patients with disease flare-up than in healthy controls. Since we found a positive correlation between the plasma PTX3 concentration and SLEDAI or the BILAG index in our SLE patients, we considered that they had a higher mean PTX3 level
compared with the control group because of their relatively high disease activity. PTX3 was correlated with CRP, which is a traditional marker of inflammation, in our patients with SLE after adjustment for age and sex. PTX3 was also related to anemia and hypoalbuminemia, which are two parameters known to reflect the persistence of systemic inflammation. In SLE patients, CRP does not always reflect disease activity, even if they have serious organ damage [25, 26]. CRP is usually normal or slightly elevated in SLE patients, although it can rise much higher in patients with serositis. CRP is synthesized by the liver in response to IL-6. In contrast, PTX3 is synthesized by a wide variety of cells and tissues in response to multiple inflammatory mediators, including IL-1b and TNF-a [1]. Thus, the increased levels of PTX3 may be an indicator of persistent systemic inflammation in patients with SLE. An increase of anti-ds DNA antibodies and/or a decrease of complement is associated with increased renal and hematologic involvement in SLE patients. However, antids DNA antibodies are not always present in SLE, with approximately 60 % of patients being positive during the course of their disease [27]. A decrease of complement is not consistently associated with flare-up of SLE [28]. Moreover, neuropsychiatric disorders in SLE patients do not always develop and worsen in correlation with anti-ds DNA antibody, serum complement CH50 and the inflammatory marker serum CRP. PTX3 was associated with the two disease activity indices, but was not correlated with anti-ds DNA antibodies or complement in our patients with SLE. All of the eight patients with CNS lupus had an
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abnormally high level of PTX3 (C10 ng/mL). A prospective study of 1047 recently diagnosed SLE patients, who were followed for up to 10 years for neuropsychiatric events, showed that autoantibodies such as anti-ribosomal P antibodies and antiphospholipid antibodies were not associated with the first occurrence of neuropsychiatric events [29]. Therefore, increased concentration of plasma PTX3 may provide additional information about disease activity or presence of active CNS involvement that is not obtained from the usual markers for SLE. Doni et al. [30] investigated the effect of glucocorticoid hormones on PTX3 production by different cell types in vitro and in humans. The in vitro study showed that glucocorticoid treatment inhibited PTX3 production by myeloid dendritic cells. In contrast, glucocorticoid treatment induced and (under inflammatory conditions) enhanced PTX3 production by fibroblasts and endothelial cells. In vivo, low-dose glucocorticoid treatment augmented circulating PTX3 levels in healthy donors. Our results showed that plasma PTX3 concentration was correlated with current dose of prednisolone after adjustment for age and sex. Although corticosteroid treatment may partly affect increase of PTX3 in patients with SLE, there was no significant difference of plasma PTX3 levels between SLE patients with and without corticosteroid therapy (median of plasma PTX3 level 3.9 vs. 3.9 ng/mL, p = 0.68). PTX3 is also strongly correlated with SLEDAI and BILAG index after adjustment for age, sex and use of corticosteroid in SLE patients (both p \ 0.005). Thus, PTX3 may reflect disease activity rather than influence of corticosteroid therapy in patients with SLE. Longitudinal studies would warrant the concentration of PTX3 correlates with disease activity through the treatment course in each patient with SLE. Accelerated atherosclerosis in SLE is probably caused by a complex interplay among traditional risk factors, SLErelated factors such as disease activity, and the influence of chronic inflammation. Mediators related to both inflammation and atherosclerosis are attractive candidates to explain the pathogenesis of accelerated atherosclerosis in SLE, and one such candidate mediator is PTX3. However, the plasma PTX3 level was not related to the presence of carotid plaque or to the carotid IMT measured by ultrasonography in our SLE patients. There was also no relation of PTX3 with coronary risk factors, including serum lipids, glucose intolerance, hypertension, obesity, and aging. Since this study was cross-sectional, a longer follow-up of patients would presumably be necessary to determine whether atherosclerosis progresses and/or the incidence of cardiovascular disease are higher in SLE patients with high plasma PTX3 levels. In conclusion, the plasma PTX3 concentration is significantly higher in SLE patients than in healthy controls.
Mod Rheumatol, 2014; 24(1): 78–85
The PTX3 level is correlated with disease activity and surrogate markers of persistent inflammation, but is not associated with carotid atherosclerosis. In patients with SLE, PTX3 may provide additional information about disease activity that is not obtained from the usual inflammatory and SLE-related markers. Acknowledgments The authors thank Dr. Daisuke Ikuma, Ms. Yukari Yamada and Ms. Kozue Watanabe for their technical support. We also appreciate Ms. Yoshimi Aizaki for her secretarial assistance. Dr. Asanuma was partly supported by research grants from The Ministry of Education, Culture, Sports, Science and Technology of Japan. Conflict of interest
None.
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