Hepatol Int (2013) 7:539–547 DOI 10.1007/s12072-012-9345-8
ORIGINAL ARTICLE
B cell-activating factor is associated with the histological severity of nonalcoholic fatty liver disease Teruki Miyake • Masanori Abe • Yoshio Tokumoto • Masashi Hirooka • Shinya Furukawa Teru Kumagi • Maho Hamada • Keitarou Kawasaki • Fujimasa Tada • Teruhisa Ueda • Yoichi Hiasa • Bunzo Matsuura • Morikazu Onji
•
Received: 21 September 2011 / Accepted: 25 January 2012 / Published online: 9 February 2012 Ó Asian Pacific Association for the Study of the Liver 2012
Abstract Purpose B cell-activating factor (BAFF) is expressed in adipocytes and affects lipogenesis and insulin sensitivity. In addition, the BAFF receptor is expressed in visceral adipose tissue and liver. The aim of this study was to analyze serum BAFF levels in patients with nonalcoholic steatohepatitis (NASH) and simple steatosis (SS) and to compare their respective clinical and histological findings. Methods A total of 96 patients with nonalcoholic fatty liver disease (20 with SS and 76 with NASH) were enrolled and their serum BAFF levels were analyzed. Comprehensive blood chemistry analysis and histological examination of liver samples were also conducted. Results Serum BAFF levels were higher in patients with NASH than in those with SS (p = 0.016). NASH patients with ballooning hepatocytes and advanced fibrosis had higher levels of BAFF in sera (p = 0.016 and p = 0.006, respectively). In addition, the prevalence of NASH increased significantly as the serum BAFF level increased (p = 0.004). Higher serum BAFF levels were found to be an independent risk factor for development of NASH (OR 1.003, 95% CI 1.0003–1.006; p = 0.047). Conclusions Nonalcoholic steatohepatitis patients had higher levels of serum BAFF than patients with SS, and higher levels were associated with the presence of hepatocyte ballooning and advanced fibrosis. The serum BAFF
T. Miyake M. Abe Y. Tokumoto M. Hirooka S. Furukawa T. Kumagi M. Hamada K. Kawasaki F. Tada T. Ueda Y. Hiasa B. Matsuura M. Onji (&) Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Toon, Ehime 791-0295, Japan e-mail: [email protected]
level may be a useful tool for distinguishing NASH from SS. Keywords Adipokine B cell-activating factor Fibrosis Hepatocyte ballooning Nonalcoholic fatty liver disease Nonalcoholic steatohepatitis
Introduction Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in the world [1–3], and is recognized as the hepatic manifestation of metabolic syndrome [3]. The majority of patients with NAFLD have simple steatosis (SS), which has a good prognosis. However, some patients develop nonalcoholic steatohepatitis (NASH) with a further risk of developing cirrhosis, liver failure, and hepatocellular carcinoma (HCC) [4–10]. Therefore, it is important to distinguish between NASH and SS. Discrimination between these conditions is considered to be difficult without histological evaluation [9, 10]. Patients with NAFLD are usually diagnosed based on the accumulation of fat in the liver. Although the pathogenesis of NASH remains unclear, the ‘‘second hit,’’ such as inflammatory cytokines, adipokines, oxidant stress, or lipid peroxidation may be required to develop NASH [11–15]. B cell-activating factor (BAFF; CD257) is a tumor necrosis factor (TNF) family member that is essential for the development of mature B cell populations [16]. BAFF is expressed on the surface of monocytes, macrophages, dendritic cells, neutrophils, activated T cells, malignant B cells, and epithelial cells [17–19]. Most expressed BAFF is cleaved from the cell surface and circulates as a soluble active homotrimer and specially binds to the BAFF
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receptor (BAFF-R; CD268) [20]. It has been reported that serum BAFF levels are elevated in patients with autoimmune diseases, such as systemic lupus erythematosus, Sjo¨gren’s syndrome, and rheumatoid arthritis [19]. Recently, a relationship between BAFF and adipocytes has been noted [21–24]. It has been previously reported that BAFF is expressed in adipocytes and affects insulin sensitivity in vivo and in vitro [24]. In addition, it has been demonstrated that BAFF-R is expressed in visceral adipose tissue (VAT) and liver [24]. However, it is unknown whether BAFF is associated with the pathogenesis of NAFLD. In the present cross-sectional study, serum BAFF levels in patients with NASH and SS are analyzed and comparisons of their respective clinical and histological findings are conducted.
Patients and methods Patients A total of 96 patients (48 men and 48 women), aged 19–80 years, diagnosed with NAFLD by histological examination from December 2005 to May 2011 at Ehime University Hospital were enrolled. The Ethics Committee of Ehime University Hospital approved the study protocol (Approval ID# 1012004), which conformed to the ethical guidelines of the 1975 Declaration of Helsinki. The inclusion criteria were (1) liver biopsy showing steatosis in at least 5% of the hepatocytes, (2) appropriate exclusion of liver diseases of other etiology, including viral hepatitis, autoimmune hepatitis, drug-induced liver disease, primary biliary cirrhosis, biliary obstruction, hemochromatosis, Wilson’s disease, and a-1-antitrypsin deficiency-associated liver disease, (3) consumption of alcohol \20 g/day; and (4) no evidence of decompensated liver cirrhosis or HCC. Written informed consent was obtained from all study participants. Measurements and evaluation Examinations included a history of prescribed medication, physical examination, anthropometric measurements, and biochemical measurements. Patients were diagnosed as having diabetes mellitus (DM) if they had documented use of hypoglycemic medication, a random glucose level [200 mg/dL, or fasting plasma glucose (FPG) [126 mg/ dL [25]. Dyslipidemia was diagnosed if they had been treated with medication to improve lipidemic levels, their total cholesterol (TC) level [220 mg/dL, triglyceride (TG) level [150 mg/dL, high-density lipoprotein cholesterol (HDL-c) \40 mg/dL, or low-density lipoprotein
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cholesterol (LDL-c) [140 mg/dL [26]. Hypertension was diagnosed if the patient was taking antihypertensive medication and/or had a resting recumbent blood pressure of C140/90 mmHg on at least two occasions [27]. Venous blood samples were taken on the morning of the second day of hospitalization after a 12 h overnight fast. A portion of the serum sample was frozen at -80°C within 4 h of collection and then thawed at the time of measurement. Data collection included blood count, prothrombin time, and blood chemistry: biomarkers for diseases and liver enzymes, including total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gammaglutamyl transpeptidase (GGT), alkaline phosphatase, TC, TG, HDL-c, LDL-c, uric acid, FPG, hemoglobin A1c, ferritin, hyaluronic acid (HA), and type IV collagen. Serum levels of interleukin (IL)-1b, IL-6, IL-8, and TNFa were estimated using a commercial kit (BD Biosciences Pharmingen, San Jose, CA, USA) using the cytometric bead array method as described by Miyake et al. [28]. Serum levels of BAFF, adiponectin, leptin, resistin, and IL-18 were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits, including Quantikine human BAFF, adiponectin, and leptin immunoassays (R&D Systems, Minneapolis, MN, USA), human resistin ELISA kit (BioVendor, Brno, Czech Republic), and human IL-18 kit (MBL, Nagoya, Japan). Visceral fat area (VFA) was evaluated by computed tomography (CT). The interval of the single scan was 5 mm in the abdominal area. Visceral fat volume was calculated at the fourth lumbar vertebra as previously described by Hirooka et al. [29]. Histological evaluation All patients enrolled in the study underwent a percutaneous liver biopsy under ultrasonic or laparoscopic guidance. The liver specimens were embedded in paraffin and stained with hematoxylin and eosin and reticulin silver stain. Two hepatopathologists (Masanori Abe, Yoshio Tokumoto) who were blinded to the clinical data reviewed the liver biopsy specimens. Adequate liver biopsy samples were defined as being 1.5-cm long and/or having more than six portal tracts. SS was defined as steatosis or steatosis with nonspecific inflammation only. NASH was defined as a combination of steatosis, lobular inflammation, and ballooning degeneration with or without perisinusoidal/pericellular fibrosis [7, 8]. The NAFLD Activity Score (NAS) proposed by Kleiner et al. [30] was recorded as the unweighted sum of the scores for steatosis (0–3), lobular inflammation (0–3), and ballooning degeneration (0–2). If the liver histology was too atypical to make a judgment, cases with NAS C5 were considered to be NASH. The severity of hepatic fibrosis (stage) was defined as: stage 1, zone 3
Hepatol Int (2013) 7:539–547 Table 1 Patient profile and laboratory data at baseline for the NAFLD patients
541 pa
SS (n = 20)
NASH (n = 76)
Gender (M/F)
9/11
39/37
Age (years)
47 (20–75)
56 (20–80)
0.250
BMI (kg/m2)
25.9 (14.8–36.5)
27.7 (19.0–48.2)
0.070
VFA (cm2)
117.8 (60.4–193.0)
146.7 (42.0–312.0)
0.048
Demographic data 0.615
Laboratory data White blood cells (/lL)
5,600 (3,300–10,500)
6,100 (2,600–11,600)
0.451
Hemoglobin (g/dL)
14.9 (10.3–17.4)
14.7 (10–19.3)
0.797
Platelets (104/lL)
22.2 (13–34)
20.3 (6.5–36.4)
0.710
PT (%)
112.0 (75.9–138.6)
105.6 (57.4–158.3)
0.130
TB (mg/dL)
0.7 (0.5–2.6)
0.8 (0.2–3.7)
AST (U/L)
25.5 (13–66)
44.5 (15–208)
ALT (U/L)
34 (19–129)
58.5 (8–371)
GGT (U/L)
39 (13–654)
51 (16–328)
0.263
ALP (U/L) TC (mg/dL)
225.5 (101–651) 204.5 (104–290)
246.5 (111–640) 199 (97–384)
0.412 0.996
0.605 \0.001 0.009
TG (mg/dL)
118 (39–185)
139 (47–590)
0.059
HDL-c (mg/dL)
52 (36–141)
47 (22–75)
0.024
LDL-c (mg/dL)
116 (34–193)
125 (48–285)
0.585
UA (mg/dL)
6 (3–8.5)
6.1 (2.9–10.1)
0.564
HbA1c (%)
5.5 (4.5–10.8)
6.1 (4.3–14.1)
0.256
FPG (mg/dL)
99.5 (76–212)
104 (66–353)
0.631
Ferritin (ng/mL)
130 (11–327)
192 (3–1290)
0.034
HA (ng/mL)
18.5 (8–148)
41 (8–667)
0.002
Data are presented as numbers or median (range)
Type IV collagen (ng/mL)
3.9 (2.8–5.1)
4.8 (3.1–10)
0.003
IL-1b (pg/mL)
71.1 (2.2–343.8)
78.3 (3.1–645.3)
0.463
ALP alkaline phosphatase, ALT alanine aminotransferase, AST aspartate aminotransferase, FPG fasting plasma glucose, GGT gamma-glutamyl transpeptidase, HA hyaluronic acid, HbA1c hemoglobin A1c, HDL-c high-density lipoprotein cholesterol, IL-1b interleukin1b, IL-6 interleukin-6, IL-8 interleukin-8, IL-18 interleukin18, LDL-c low-density lipoprotein cholesterol, NAFLD nonalcoholic fatty liver disease, NASH nonalcoholic steatohepatitis, PT prothrombin time, SS simple steatosis, TB total bilirubin, TC total cholesterol, TG triglycerides, TNF-a tumor necrosis factor-a, UA uric acid
IL-6 (pg/mL)
132.3 (2.3–1220.7)
149.1 (2.8–3685.2)
0.591
IL-8 (pg/mL)
713.1 (11.8–27111.3)
1080.9 (13.5–66913)
0.397
IL-18 (pg/mL)
327.3 (195.6–576.7)
382.6 (131.3–1552.5)
0.142
TNF-a (pg/mL)
80.0 (1.89–181.8)
91.5 (1.7–555.3)
0.159
Complications (%) Hypertension
5 (25)
35 (46.1)
0.126
Diabetes mellitus
7 (35)
47 (61.8)
0.042
Dyslipidemia
15 (75)
59 (77.6)
0.772
Insulin
2 (10)
9 (11.8)
1.0
Sulfonylurea
1 (5)
7 (9.2)
1.0
Phenylalanine derivative
0 (0)
3 (4.0)
a-Glucosidase inhibitor
1 (5)
3 (3.9)
1.0
Biguanide
2 (10)
6 (7.9)
0.670
a
v2 test or Mann–Whitney U test
Prescribed medication (%)
Thiazolidine derivative
1 (5)
7 (9.2)
1.0
Angiotensin receptor blocker
2 (10)
13 (17.1)
0.730 1.0
Statin
3 (15)
12 (15.8)
Fibrates
0 (0)
2 (2.6)
perisinusoidal fibrosis; stage 2, zone 3 perisinusoidal fibrosis with portal fibrosis; stage 3, zone 3 perisinusoidal fibrosis and portal fibrosis with bridging fibrosis; and stage 4, cirrhosis [31].
Statistical analysis The data are expressed as medians and ranges. The Mann– Whitney U test was used to analyze continuous variables.
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The v2 test was used for analysis of categorical data. Group comparisons involving more than two independent groups were performed using the Kruskal–Wallis test. The Cochran–Armitage test for trends was used to assess trends in the incidence rates of outcomes. Multivariate analysis was performed using a stepwise logistic regression model. The cutoff points for continuous variables were determined by receiver operating characteristic (ROC) curve analysis; p \ 0.05 was considered statistically significant. Statistical analyses were performed using JMP version 9 software (SAS Institute Japan, Tokyo, Japan).
Results Patient profiles Consecutive subjects who met the inclusion criteria were enrolled in the study. The baseline characteristics of the study population are shown in Table 1. Of the 96 participants, 76 (79.2%, 39 males and 37 females) met the diagnostic criteria for NASH. Patients with NASH had a higher incidence of DM and had higher levels of VFA, AST, ALT, ferritin, HA, and type IV collagen (p \ 0.05 for all variables) than patients with SS. HDL-c levels were lower in NASH patients. Among the cytokines and adipokines (Table 1; Fig. 1), the serum BAFF levels were higher in patients with NASH than in those with SS (p = 0.016) (Fig. 1a). BAFF levels and liver histopathology Figure 2 shows serum BAFF levels with respect to liver histological findings. The steatosis score, lobular inflammation, portal inflammation, Mallory–Denk bodies, lipogranulomas, and NAS were not associated with the serum BAFF level (Fig. 2a–f). In contrast, serum BAFF levels were significantly higher in NAFLD patients with ballooning hepatocytes and in those with advanced fibrosis stage (p = 0.016 and p = 0.006, respectively) (Fig. 2g, h). Association of BAFF levels with the prevalence of NASH To understand the relationship between BAFF levels and NAFLD, the prevalence rate (PR%) of NASH with serum BAFF level (Table 2) were investigated. All of the subjects were classified into quartiles according to their BAFF levels. The PR% of NASH in the subjects in each quartile of BAFF levels was analyzed. Quartile 1 was defined as BAFF \938 pg/mL, quartile 2 had BAFF 938 to \1,175 pg/mL, quartile 3 had BAFF 1,175 to\1429 pg/mL, and quartile 4 had BAFF [1,429 pg/mL. The PR% for the
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subjects in quartiles 1, 2, 3, and 4 were 58.2, 79.2, 87.5, and 91.7%, respectively (Table 2). The PR% of NASH increased significantly as the serum BAFF level increased (p = 0.004). These results suggest that patients with higher serum BAFF levels are more likely to develop NASH. Risk factor analysis for NASH Univariate analysis identified that body mass index (BMI), VFA, AST, ALT, ferritin, HA, type IV collagen, and BAFF were associated with significant risks of NAFLD (Table 3). Correlations among the risk factors for NASH are shown in Table 4. Multiple combinations of multivariate analyses were subsequently carried out and four factors (VFA, AST, HDL-c, and BAFF) were identified as independent risk factors for NASH. As shown in Table 5, stepwise multivariate logistic regression analysis confirmed that AST, HDL-c, and BAFF were associated with risk of NASH. A high serum BAFF level was found to be an independent risk factor for developing NASH (OR 1.003, 95% CI 1.0003–1.006; p = 0.047). BAFF as a predictor of NASH A cutoff value for diagnosis of NASH was then determined. The area under the ROC curve (95% CI), cutoff value, sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy for predicting NASH were 0.737 (0.637–0.821), 831.1 pg/mL, 93.4, 50, 87.6, 66.7, and 84.3%, respectively (Table 6). The use of serum BAFF levels for prediction of NASH demonstrated high sensitivity, positive predictive value, and accuracy.
Discussion The pathogenesis of NASH is currently thought to be a multi-step process closely associated with visceral fat [11– 15]. In the livers of NAFLD patients, 57% of the accumulated fat is derived from lipolysis of VAT, particularly in obesity with insulin resistance [32–34]. In addition, hypertrophied adipocytes of VAT release several cytokines and chemokines. These factors induce inflammation in VAT and are believed to affect the liver through the portal vein. Therefore, these factors may play a role in the pathogenesis of NASH [35–37]. Recently, BAFF has been reported to be expressed in mature adipocytes [21–24]. The expression of BAFF was changed under pro-inflammatory and anti-inflammatory conditions in vitro and in vivo [24]. In addition, Alexaki et al. [23] reported that BAFF enhanced lipogenesis in vitro. It has been also demonstrated that BAFF levels were
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Fig. 1 Serum adipokine levels in SS and NASH patients. a The serum B cell-activating factor (BAFF) level was significantly higher in NASH patients than in SS patients (p = 0.016). b Adiponectin, c leptin, and d resistin in sera were not significantly different between NASH and SS (p = 0.185, p = 0.926, and p = 0.303, respectively). Group comparisons were performed using the Mann–Whitney U test. Data are shown as medians and ranges
preferentially increased in VAT and sera in obese mice. In addition, BAFF plays a role in induction of impaired insulin sensitivity [24]. These data indicate that BAFF is an adipokine capable of inducing insulin resistance, and may predispose obese individuals to development of metabolic syndrome. In this study, the serum levels of BAFF in histologically diagnosed NAFLD patients were examined. It was found that NASH patients had higher serum BAFF levels than SS patients (Fig. 1), and that levels of BAFF were associated with the PR% of NASH (Table 2). In addition, patients with ballooned hepatocytes and advanced fibrosis in the liver had higher serum BAFF levels (Fig. 2). Furthermore, Hamada et al. [24] demonstrated that BAFF-R was preferentially expressed in liver as well as VAT [24]. These results indicate that BAFF is a significant factor associated with the development of NASH. BAFF has been reported to be involved in the development of autoimmune diseases [19]. In addition, nonspecific antibody production has been observed in patients with liver diseases, such as NAFLD [38]. However, serum BAFF levels were not significantly different in patients
with antinuclear antibodies [1,163.9 (654.1–2,384.7) pg/ mL] and those without [1,210.1 (572.2–3,249.1) pg/mL]. The results of this study provide new insights into potential roles of BAFF in clinical diagnosis and treatment of NASH. Clinically, one of the most important goals of therapy for NASH patients is prevention of progression to cirrhosis. Therefore, diagnosis of NASH at an early stage is important. However, discriminating between NASH and SS is considered to be difficult. A liver biopsy is the gold standard method for diagnosis and staging of fibrosis in patients with NASH [1, 10, 31]. However, this procedure is invasive and associated with a risk of complications [39]. In the present study, BAFF demonstrated reasonable accuracy in diagnosing NASH and was found to be a strong predictive factor for NASH. The second area in which BAFF may be valuable is in the treatment of NASH. Ballooned hepatocytes and fibrosis of the liver were most closely associated with BAFF in NASH (Fig. 2). Ballooned hepatocytes are caused by lipotoxicity and oxidant stress [40]; fibrosis is associated with the activation of hepatic stellate cells [41]. Therefore, BAFF may induce the ‘‘second hit’’ thought to be required
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544 Fig. 2 Serum B cell-activating factor (BAFF) levels according to histological findings in the liver specimens. a Steatosis score, b lobular inflammation, c portal inflammation, d Mallory–Denk bodies, e lipogranulomas, and f nonalcoholic fatty liver disease activity score (NAS) were not affected by the serum BAFF level. In contrast, the serum BAFF level was significantly higher in NAFLD patients with g the presence of ballooning hepatocytes and h advanced fibrosis stage (p = 0.016 and p = 0.006, respectively). Two group comparisons were performed using the Mann– Whitney U test. Three group comparisons were performed using the Kruskal–Wallis test. Data are shown as medians and ranges
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Table 2 Association of the BAFF level with prevalence of NASH BAFF level
Quartile 1
Quartile 2
Quartile 3
Quartile 4
NASH/total
14/24
19/24
21/24
22/24
PR (%)
58.2
79.2
87.5
91.7
pa
0.004
BAFF B cell-activating factor, NASH nonalcoholic steatohepatitis, PR prevalence rate a
Cochran–Armitage test
Table 3 Risk factors for NASH identified by univariate analysis (n = 96)
Factor
Odds ratio (95% CI)
Gender (M = 1, F = 2)
1.135 (0.692–1.881)
0.616
Age (years)
1.020 (0.789–1.053)
0.209
BMI (kg/m2)
1.126 (1.010–1.276)
0.045
VFA (cm2)
1.011 (1.0003–1.0223)
0.043
1.00007 (0.9998–1.0004)
0.622
0.989 (0.745–1.303)
0.937
White blood cells (/lL) Hemoglobin (g/dL) 4
ALP alkaline phosphatase, ALT alanine aminotransferase, AST aspartate aminotransferase, BAFF B cell-activating factor, CI confidence interval, FPG fasting plasma glucose, GGT gamma-glutamyl transpeptidase, HA hyaluronic acid, HbA1c hemoglobin A1c, HDL-c high-density lipoprotein cholesterol, IL-1b interleukin1b, IL-6 interleukin-6, IL-8 interleukin-8, IL-18 interleukin18, LDL-c low-density lipoprotein cholesterol, NASH nonalcoholic steatohepatitis, PT prothrombin time, TB total bilirubin, TC total cholesterol, TG triglycerides, TNF-a tumor necrosis factor-a, UA uric acid
p
Platelets (10 /lL)
0.990 (0.924–1.061)
0.779
PT (%)
0.981 (0.951–1.011)
0.215
TB (mg/dL)
1.175 (0.664–3.270)
0.671
AST (U/L)
1.078 (1.035–1.138)
0.002
ALT (U/L)
1.019 (1.005–1.040)
0.029
GGT (U/L) ALP (U/L)
0.997 (0.992–1.002) 0.99998 (0.995–1.005)
0.254 0.992
TC (mg/dL)
1.0004 (0.990–1.012)
0.943
TG (mg/dL)
1.010 (1.002–1.020)
0.039
HDL-c (mg/dL)
0.945 (0.902–0.982)
0.010
LDL-c (mg/dL)
1.004 (0.991–1.017)
0.579
UA (mg/dL)
1.120 (0.795–1.60)
0.520
HbA1c (%)
1.076 (0.837–1.443)
0.592
FPG (mg/dL)
1.003 (0.993–1.015)
0.606
Ferritin (ng/mL)
1.005 (1.001–1.011)
0.039
HA (ng/mL)
1.020 (1.004–1.045)
0.048
Type IV collagen (ng/mL)
3.468 (1.548–9.944)
0.008
IL-1b (pg/mL)
1.001 (0.996–1.009)
0.698
IL-6 (pg/mL)
1.0003 (0.999–1.003)
0.679
IL-8 (pg/mL)
1.00003 (0.99998–1.00012)
0.453
IL-18 (pg/mL)
1.003 (0.9995–1.0076)
0.155
TNF-a (pg/mL) BAFF (pg/mL)
1.007 (0.997–1.018) 1.003 (1.001–1.005)
0.223 0.004
Adiponectin (ng/mL) Leptin (pg/mL) Resistin (ng/mL)
for NASH development. Blockade of BAFF may be a potential therapeutic strategy for preventing disease progression. However, further research is needed to confirm this hypothesis. The levels of circulating adipokines and cytokines in NAFLD have been reported to be affected by other risk factors for NASH, such as DM, hypertension, and
0.9999 (0.9998–1.0001)
0.286
1.00001 (0.99996–1.00008)
0.747
1.056 (0.936–1.228)
0.427
dyslipidemia and may simply reflect an association between NALFD and other risk factors rather than a true causal relationship [42–44]. In our study, the levels of other adipokines and cytokines were not significantly different between patients with NASH and those with SS. The specific reasons for this are not clear. Although several studies have reported differences in serum cytokine levels
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Table 4 Correlations (R2) among risk factors for NASH BMI BMI
X
VFA
VFA
AST
ALT
TG
HDL-c
Ferritin
HA
Type IV collagen
BAFF
0.340**
0.010
0.020
0.045*
0.015
0.0002
0.004
0.009
0.035
X
1.7e-5
2.6e-5
0.017
0.014
0.037
0.007
0.007
0.010
X
0.672**
0.102*
0.005
0.258
6.1e-6
0.052*
0.05*
AST ALT
X
TG
0.136**
0.031
0.20**
0.03**
0.0009
0.003
X
0.134*
0.082**
0.0001
0.0001
0.002
X
0.070*
0.006
2.4 e-5
0.001
X
0.001
0.009
1.1e-5
0.305**
0.270**
X
0.188**
HDL-c Ferritin HA
X
Type IV collagen BAFF
X
ALT alanine aminotransferase, AST aspartate aminotransferase, BAFF B cell-activating factor, BMI body mass index, HA hyaluronic acid, HDL-c high-density lipoprotein cholesterol, NASH nonalcoholic steatohepatitis, TG triglycerides, VFA visceral fat area * p \ 0.05; **p \ 0.01 Table 5 Risk factors for NASH identified by multivariate analysis (n = 96) Factor
Odds ratio (95% CI)
p
AST (U/L)
1.104 (1.040–1.203)
0.007
HDL-c (mg/dL)
0.934 (0.876–0.980)
0.019
BAFF (pg/mL)
1.003 (1.0003–1.006)
0.047
Only variables that achieved statistical significance (p \ 0.05) in the stepwise multivariate logistic regression are shown; the complete set of variables included VFA, AST, HDL-c, and BAFF AST aspartate aminotransferase, BAFF B cell-activating factor, CI confidence interval, HDL-c high-density lipoprotein cholesterol, NASH nonalcoholic steatohepatitis Table 6 Sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of BAFF for prediction of NASH AUC (95% CI)
Cutoff value (U/L)
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
Diagnostic accuracy (%)
0.737 (0.637–0.821)
831.1
93.4
50
87.6
66.7
84.3
Each value was determined by ROC curve analysis AUC area under the curve, BAFF B cell-activating factor, CI confidence interval, NASH nonalcoholic steatohepatitis, NPV negative predictive value, PPV positive predictive value
between NAFLD patients and controls, others have shown contradictory results. In particular, several studies found no significant differences in serum cytokine levels between SS and NASH patients [45], consistent with the present study. In addition, most of the NAFLD patients in the present study had metabolic diseases and took several medications, which may have affected the results. There were several limitations of our cross-sectional study. For example, we were unable to confirm a relationship between serum BAFF levels and insulin resistance (HOMA-R), because many patients were being treated with several medications and/or had high levels of plasma glucose. Future studies are necessary to evaluate this relationship using a prospective validation design. In conclusion, we found that NASH patients had higher serum BAFF levels than SS patients, and higher BAFF
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levels were associated with hepatocyte ballooning and advanced fibrosis. The serum BAFF level may be a useful tool for distinguishing NASH from SS. Blockade of BAFF may be a promising therapeutic strategy for patients with NASH. Acknowledgements This work was supported in part by a grant-inaid for scientific research from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (KAKENHI No. 23700907), and a research grant from Ehime University.
References 1. Angulo P. Nonalcoholic fatty liver disease. N Engl J Med 2002;346:1221–1231 2. Farrell GC. Non-alcoholic steatohepatitis: what is it, and why is it important in the Asia-Pacific region? J Gastroenterol Hepatol 2003;18:124–138
Hepatol Int (2013) 7:539–547 3. Marchesini G, Bugianesi E, Forlani G, et al. Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome. Hepatology 2003;37:917–923 4. Day CP. Natural history of NAFLD: remarkably benign in the absence of cirrhosis. Gastroenterology 2005;129:375–378 5. Teli MR, James OF, Burt AD, et al. The natural history of nonalcoholic fatty liver: a follow-up study. Hepatology 1995;22: 1714–1719 6. Adams LA, Lymp JF, St Sauver J, et al. The natural history of nonalcoholic fatty liver disease: a population-based cohort study. Gastroenterology 2005;129:113–121 7. Matteoni CA, Younossi ZM, Gramlich T, et al. Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology 1999;116:1413–1419 8. Rafiq N, Bai C, Fang Y, et al. Long-term follow-up of patients with nonalcoholic fatty liver. Clin Gastroenterol Hepatol 2009;7: 234–238 9. Diehl AM, Goodman Z, Ishak KG. Alcohol like liver disease in nonalcoholics. A clinical and histologic comparison with alcoholinduced liver injury. Gastroenterology 1988;95:1056–1062 10. Ludwig J, Viggiano TR, McGill DB, et al. Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed disease. Mayo Clin Proc 1980;55:434–438 11. Day CP, James OF. Steatohepatitis: a tale of two ‘‘hits’’? Gastroenterology 1998;114:842–845 12. Neuschwander-Tetri BA, Caldwell SH. Nonalcoholic steatohepatitis: summary of an AASLD single topic conference. Hepatology 2003;37:1202–1219 13. Chitturi S, Abeygunasekera S, Farrell GC, et al. NASH and insulin resistance: insulin hypersecretion and specific association with the insulin resistance syndrome. Hepatology 2002;35:373– 379 14. Stefan N, Kantartzis K, Ha¨ring HU. Causes and metabolic consequences of fatty liver. Endocr Rev 2008;29:939–960 15. Wigg AJ, Roberts-Thomson IC, Dymock RB, et al. The role of small intestinal bacterial overgrowth, intestinal permeability, endotoxaemia, and tumour necrosis factor alpha in pathogenesis of non-alcoholic steatohepatitis. Gut 2001;48:206–211 16. Tilg H, Moschen AR. Inflammatory mechanisms in the regulation of insulin resistance. Mol Med 2008;14:222–231 17. Litinskiy MB, Nardelli B, Hilbert DM, et al. DCs induce CD40independent immunoglobulin class switching through BLyS and APRIL. Nat Immunol 2002;3:822–829 18. Nardelli B, Belvedere O, Roschke V, et al. Synthesis and release of B-lymphocyte stimulator from myeloid cells. Blood 2001;97: 198–204 19. Mackay F, Silveira PA, Brink R. B cells and the BAFF/APRIL axis: fast-forward on autoimmunity and signaling. Curr Opin Immunol 2007;19:327–336 20. Bossen C, Schneider P. BAFF, APRIL and their receptors: structure, function and signaling. Semin Immunol 2006;18:263–275 21. Kim YH, Choi BH, Cheon HG, et al. B cell activation factor (BAFF) is a novel adipokine that links obesity and inflammation. Exp Mol Med 2009;41:208–216 22. Pelekanou V, Kampa M, Kafousi M, et al. Expression of TNFsuperfamily members BAFF and APRIL in breast cancer: immunohistochemical study in 52 invasive ductal breast carcinomas. BMC Cancer 2008;8:76 23. Alexaki VI, Notas G, Pelekanou V, et al. Adipocytes as immune cells: differential expression of TWEAK, BAFF, and APRIL and their receptors (Fn14, BAFF-R, TACI, and BCMA) at different stages of normal and pathological adipose tissue development. J Immunol 2009;183:5948–5956 24. Hamada M, Abe M, Miyake T, et al. B cell-activating factor controls the production of adipokines and induces insulin resistance. Obesity 2011;19:1915–1922
547 25. Sasai H, Sairenchi T, Iso H, et al. Relationship between obesity and incident diabetes in middle-aged and older Japanese adults: the Ibaraki Prefectural Health Study. Mayo Clin Proc 2010;85:36–40 26. Teramoto T, Sasaki J, Ueshima H, et al. Diagnostic criteria for dyslipidemia. Executive summary of Japan Atherosclerosis Society (JAS) guideline for diagnosis and prevention of atherosclerotic cardiovascular diseases for Japanese. J Atheroscler Thromb 2007;14:155–158 27. Ogihara T, Kikuchi K, Matsuoka H, et al. The Japanese Society of Hypertension Guidelines for the management of hypertension (JSH 2009). Hypertens Res 2009;32:3–107 28. Miyake T, Akbar SM, Yoshida O, et al. Impaired dendritic cell functions disrupt antigen-specific adaptive immune responses in mice with nonalcoholic fatty liver disease. J Gastroenterol 2010;45:859–867 29. Hirooka M, Kumagi T, Kurose K, et al. A technique for the measurement of visceral fat by ultrasonography: comparison of measurements by ultrasonography and computed tomography. Intern Med 2005;44:794–799 30. Kleiner DE, Brunt EM, Van Natta M, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005;41:1313–21 31. Brunt EM, Janney CG, Di Bisceglie AM, et al. Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J Gastroenterol 1999;94:2467–2474 32. Donnelly KL, Smith CI, Schwarzenberg SJ, et al. Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Invest 2005;115:1343–1351 33. Nielsen S, Guo Z, Johnson CM, et al. Splanchnic lipolysis in human obesity. J Clin Invest 2004;113:1582–1588 34. Lewis GF, Carpentier A, Adeli K, et al. Disordered fat storage and mobilization in the pathogenesis of insulin resistance and type 2 diabetes. Endocr Rev 2002;23:201–229 35. Edmison J, McCullough AJ. Pathogenesis of non-alcoholic steatohepatitis: human data. Clin Liver Dis 2007;11:75–104 36. Baranova A, Gowder SJ, Schlauch K, et al. Gene expression of leptin, resistin, and adiponectin in the white adipose tissue of obese patients with non-alcoholic fatty liver disease and insulin resistance. Obes Surg 2006;16:1118–1125 37. Lutchman G, Promrat K, Kleiner DE, et al. Changes in serum adipokine levels during pioglitazone treatment for nonalcoholic steatohepatitis: relationship to histological improvement. Clin Gastroenterol Hepatol 2006;4:1048–1052 38. Vuppalanchi R, Gould RJ, Wilson LA et al. Clinical significance of serum autoantibodies in patients with NAFLD: results from the nonalcoholic steatohepatitis clinical research network. Hepatol Int (Epub ahead of print). 2011. doi:10.1007/s12072-011-9277-8 39. Poynard T, Ratziu V, Bedossa P. Appropriateness of liver biopsy. Can J Gastroenterol 2000;14:543–548 40. Neuschwander-Tetri BA. Hepatic lipotoxicity and the pathogenesis of nonalcoholic steatohepatitis: the central role of nontriglyceride fatty acid metabolites. Hepatology 2010;52:774–788 41. Marra F, Romanelli RG, Giannini C, et al. Monocyte chemotactic protein-1 as a chemoattractant for human hepatic stellate cells. Hepatology 1999;29:140–148 42. Hui JM, Hodge A, Farrell GC, et al. Beyond insulin resistance in NASH: TNF-alpha or adiponectin? Hepatology 2004;40:46–54 43. Chitturi S, Farrell G, Frost L, et al. Serum leptin in NASH correlates with hepatic steatosis but not fibrosis: a manifestation of lipotoxicity? Hepatology 2002;36:403–409 44. Angulo P, Alba LM, Petrovic LM, et al. Leptin, insulin resistance, and liver fibrosis in human nonalcoholic fatty liver disease. J Hepatol 2004;41:943–949 45. Tsochatzis EA, Papatheodoridis GV, Archimandritis AJ. Adipokines in nonalcoholic steatohepatitis: from pathogenesis to implications in diagnosis and therapy. Mediators Inflamm 2009;2009: 831670
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ORIGINAL ARTICLE
B cell-activating factor is associated with the histological severity of nonalcoholic fatty liver disease Teruki Miyake • Masanori Abe • Yoshio Tokumoto • Masashi Hirooka • Shinya Furukawa Teru Kumagi • Maho Hamada • Keitarou Kawasaki • Fujimasa Tada • Teruhisa Ueda • Yoichi Hiasa • Bunzo Matsuura • Morikazu Onji
•
Received: 21 September 2011 / Accepted: 25 January 2012 / Published online: 9 February 2012 Ó Asian Pacific Association for the Study of the Liver 2012
Abstract Purpose B cell-activating factor (BAFF) is expressed in adipocytes and affects lipogenesis and insulin sensitivity. In addition, the BAFF receptor is expressed in visceral adipose tissue and liver. The aim of this study was to analyze serum BAFF levels in patients with nonalcoholic steatohepatitis (NASH) and simple steatosis (SS) and to compare their respective clinical and histological findings. Methods A total of 96 patients with nonalcoholic fatty liver disease (20 with SS and 76 with NASH) were enrolled and their serum BAFF levels were analyzed. Comprehensive blood chemistry analysis and histological examination of liver samples were also conducted. Results Serum BAFF levels were higher in patients with NASH than in those with SS (p = 0.016). NASH patients with ballooning hepatocytes and advanced fibrosis had higher levels of BAFF in sera (p = 0.016 and p = 0.006, respectively). In addition, the prevalence of NASH increased significantly as the serum BAFF level increased (p = 0.004). Higher serum BAFF levels were found to be an independent risk factor for development of NASH (OR 1.003, 95% CI 1.0003–1.006; p = 0.047). Conclusions Nonalcoholic steatohepatitis patients had higher levels of serum BAFF than patients with SS, and higher levels were associated with the presence of hepatocyte ballooning and advanced fibrosis. The serum BAFF
T. Miyake M. Abe Y. Tokumoto M. Hirooka S. Furukawa T. Kumagi M. Hamada K. Kawasaki F. Tada T. Ueda Y. Hiasa B. Matsuura M. Onji (&) Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Toon, Ehime 791-0295, Japan e-mail: [email protected]
level may be a useful tool for distinguishing NASH from SS. Keywords Adipokine B cell-activating factor Fibrosis Hepatocyte ballooning Nonalcoholic fatty liver disease Nonalcoholic steatohepatitis
Introduction Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in the world [1–3], and is recognized as the hepatic manifestation of metabolic syndrome [3]. The majority of patients with NAFLD have simple steatosis (SS), which has a good prognosis. However, some patients develop nonalcoholic steatohepatitis (NASH) with a further risk of developing cirrhosis, liver failure, and hepatocellular carcinoma (HCC) [4–10]. Therefore, it is important to distinguish between NASH and SS. Discrimination between these conditions is considered to be difficult without histological evaluation [9, 10]. Patients with NAFLD are usually diagnosed based on the accumulation of fat in the liver. Although the pathogenesis of NASH remains unclear, the ‘‘second hit,’’ such as inflammatory cytokines, adipokines, oxidant stress, or lipid peroxidation may be required to develop NASH [11–15]. B cell-activating factor (BAFF; CD257) is a tumor necrosis factor (TNF) family member that is essential for the development of mature B cell populations [16]. BAFF is expressed on the surface of monocytes, macrophages, dendritic cells, neutrophils, activated T cells, malignant B cells, and epithelial cells [17–19]. Most expressed BAFF is cleaved from the cell surface and circulates as a soluble active homotrimer and specially binds to the BAFF
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receptor (BAFF-R; CD268) [20]. It has been reported that serum BAFF levels are elevated in patients with autoimmune diseases, such as systemic lupus erythematosus, Sjo¨gren’s syndrome, and rheumatoid arthritis [19]. Recently, a relationship between BAFF and adipocytes has been noted [21–24]. It has been previously reported that BAFF is expressed in adipocytes and affects insulin sensitivity in vivo and in vitro [24]. In addition, it has been demonstrated that BAFF-R is expressed in visceral adipose tissue (VAT) and liver [24]. However, it is unknown whether BAFF is associated with the pathogenesis of NAFLD. In the present cross-sectional study, serum BAFF levels in patients with NASH and SS are analyzed and comparisons of their respective clinical and histological findings are conducted.
Patients and methods Patients A total of 96 patients (48 men and 48 women), aged 19–80 years, diagnosed with NAFLD by histological examination from December 2005 to May 2011 at Ehime University Hospital were enrolled. The Ethics Committee of Ehime University Hospital approved the study protocol (Approval ID# 1012004), which conformed to the ethical guidelines of the 1975 Declaration of Helsinki. The inclusion criteria were (1) liver biopsy showing steatosis in at least 5% of the hepatocytes, (2) appropriate exclusion of liver diseases of other etiology, including viral hepatitis, autoimmune hepatitis, drug-induced liver disease, primary biliary cirrhosis, biliary obstruction, hemochromatosis, Wilson’s disease, and a-1-antitrypsin deficiency-associated liver disease, (3) consumption of alcohol \20 g/day; and (4) no evidence of decompensated liver cirrhosis or HCC. Written informed consent was obtained from all study participants. Measurements and evaluation Examinations included a history of prescribed medication, physical examination, anthropometric measurements, and biochemical measurements. Patients were diagnosed as having diabetes mellitus (DM) if they had documented use of hypoglycemic medication, a random glucose level [200 mg/dL, or fasting plasma glucose (FPG) [126 mg/ dL [25]. Dyslipidemia was diagnosed if they had been treated with medication to improve lipidemic levels, their total cholesterol (TC) level [220 mg/dL, triglyceride (TG) level [150 mg/dL, high-density lipoprotein cholesterol (HDL-c) \40 mg/dL, or low-density lipoprotein
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cholesterol (LDL-c) [140 mg/dL [26]. Hypertension was diagnosed if the patient was taking antihypertensive medication and/or had a resting recumbent blood pressure of C140/90 mmHg on at least two occasions [27]. Venous blood samples were taken on the morning of the second day of hospitalization after a 12 h overnight fast. A portion of the serum sample was frozen at -80°C within 4 h of collection and then thawed at the time of measurement. Data collection included blood count, prothrombin time, and blood chemistry: biomarkers for diseases and liver enzymes, including total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gammaglutamyl transpeptidase (GGT), alkaline phosphatase, TC, TG, HDL-c, LDL-c, uric acid, FPG, hemoglobin A1c, ferritin, hyaluronic acid (HA), and type IV collagen. Serum levels of interleukin (IL)-1b, IL-6, IL-8, and TNFa were estimated using a commercial kit (BD Biosciences Pharmingen, San Jose, CA, USA) using the cytometric bead array method as described by Miyake et al. [28]. Serum levels of BAFF, adiponectin, leptin, resistin, and IL-18 were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits, including Quantikine human BAFF, adiponectin, and leptin immunoassays (R&D Systems, Minneapolis, MN, USA), human resistin ELISA kit (BioVendor, Brno, Czech Republic), and human IL-18 kit (MBL, Nagoya, Japan). Visceral fat area (VFA) was evaluated by computed tomography (CT). The interval of the single scan was 5 mm in the abdominal area. Visceral fat volume was calculated at the fourth lumbar vertebra as previously described by Hirooka et al. [29]. Histological evaluation All patients enrolled in the study underwent a percutaneous liver biopsy under ultrasonic or laparoscopic guidance. The liver specimens were embedded in paraffin and stained with hematoxylin and eosin and reticulin silver stain. Two hepatopathologists (Masanori Abe, Yoshio Tokumoto) who were blinded to the clinical data reviewed the liver biopsy specimens. Adequate liver biopsy samples were defined as being 1.5-cm long and/or having more than six portal tracts. SS was defined as steatosis or steatosis with nonspecific inflammation only. NASH was defined as a combination of steatosis, lobular inflammation, and ballooning degeneration with or without perisinusoidal/pericellular fibrosis [7, 8]. The NAFLD Activity Score (NAS) proposed by Kleiner et al. [30] was recorded as the unweighted sum of the scores for steatosis (0–3), lobular inflammation (0–3), and ballooning degeneration (0–2). If the liver histology was too atypical to make a judgment, cases with NAS C5 were considered to be NASH. The severity of hepatic fibrosis (stage) was defined as: stage 1, zone 3
Hepatol Int (2013) 7:539–547 Table 1 Patient profile and laboratory data at baseline for the NAFLD patients
541 pa
SS (n = 20)
NASH (n = 76)
Gender (M/F)
9/11
39/37
Age (years)
47 (20–75)
56 (20–80)
0.250
BMI (kg/m2)
25.9 (14.8–36.5)
27.7 (19.0–48.2)
0.070
VFA (cm2)
117.8 (60.4–193.0)
146.7 (42.0–312.0)
0.048
Demographic data 0.615
Laboratory data White blood cells (/lL)
5,600 (3,300–10,500)
6,100 (2,600–11,600)
0.451
Hemoglobin (g/dL)
14.9 (10.3–17.4)
14.7 (10–19.3)
0.797
Platelets (104/lL)
22.2 (13–34)
20.3 (6.5–36.4)
0.710
PT (%)
112.0 (75.9–138.6)
105.6 (57.4–158.3)
0.130
TB (mg/dL)
0.7 (0.5–2.6)
0.8 (0.2–3.7)
AST (U/L)
25.5 (13–66)
44.5 (15–208)
ALT (U/L)
34 (19–129)
58.5 (8–371)
GGT (U/L)
39 (13–654)
51 (16–328)
0.263
ALP (U/L) TC (mg/dL)
225.5 (101–651) 204.5 (104–290)
246.5 (111–640) 199 (97–384)
0.412 0.996
0.605 \0.001 0.009
TG (mg/dL)
118 (39–185)
139 (47–590)
0.059
HDL-c (mg/dL)
52 (36–141)
47 (22–75)
0.024
LDL-c (mg/dL)
116 (34–193)
125 (48–285)
0.585
UA (mg/dL)
6 (3–8.5)
6.1 (2.9–10.1)
0.564
HbA1c (%)
5.5 (4.5–10.8)
6.1 (4.3–14.1)
0.256
FPG (mg/dL)
99.5 (76–212)
104 (66–353)
0.631
Ferritin (ng/mL)
130 (11–327)
192 (3–1290)
0.034
HA (ng/mL)
18.5 (8–148)
41 (8–667)
0.002
Data are presented as numbers or median (range)
Type IV collagen (ng/mL)
3.9 (2.8–5.1)
4.8 (3.1–10)
0.003
IL-1b (pg/mL)
71.1 (2.2–343.8)
78.3 (3.1–645.3)
0.463
ALP alkaline phosphatase, ALT alanine aminotransferase, AST aspartate aminotransferase, FPG fasting plasma glucose, GGT gamma-glutamyl transpeptidase, HA hyaluronic acid, HbA1c hemoglobin A1c, HDL-c high-density lipoprotein cholesterol, IL-1b interleukin1b, IL-6 interleukin-6, IL-8 interleukin-8, IL-18 interleukin18, LDL-c low-density lipoprotein cholesterol, NAFLD nonalcoholic fatty liver disease, NASH nonalcoholic steatohepatitis, PT prothrombin time, SS simple steatosis, TB total bilirubin, TC total cholesterol, TG triglycerides, TNF-a tumor necrosis factor-a, UA uric acid
IL-6 (pg/mL)
132.3 (2.3–1220.7)
149.1 (2.8–3685.2)
0.591
IL-8 (pg/mL)
713.1 (11.8–27111.3)
1080.9 (13.5–66913)
0.397
IL-18 (pg/mL)
327.3 (195.6–576.7)
382.6 (131.3–1552.5)
0.142
TNF-a (pg/mL)
80.0 (1.89–181.8)
91.5 (1.7–555.3)
0.159
Complications (%) Hypertension
5 (25)
35 (46.1)
0.126
Diabetes mellitus
7 (35)
47 (61.8)
0.042
Dyslipidemia
15 (75)
59 (77.6)
0.772
Insulin
2 (10)
9 (11.8)
1.0
Sulfonylurea
1 (5)
7 (9.2)
1.0
Phenylalanine derivative
0 (0)
3 (4.0)
a-Glucosidase inhibitor
1 (5)
3 (3.9)
1.0
Biguanide
2 (10)
6 (7.9)
0.670
a
v2 test or Mann–Whitney U test
Prescribed medication (%)
Thiazolidine derivative
1 (5)
7 (9.2)
1.0
Angiotensin receptor blocker
2 (10)
13 (17.1)
0.730 1.0
Statin
3 (15)
12 (15.8)
Fibrates
0 (0)
2 (2.6)
perisinusoidal fibrosis; stage 2, zone 3 perisinusoidal fibrosis with portal fibrosis; stage 3, zone 3 perisinusoidal fibrosis and portal fibrosis with bridging fibrosis; and stage 4, cirrhosis [31].
Statistical analysis The data are expressed as medians and ranges. The Mann– Whitney U test was used to analyze continuous variables.
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The v2 test was used for analysis of categorical data. Group comparisons involving more than two independent groups were performed using the Kruskal–Wallis test. The Cochran–Armitage test for trends was used to assess trends in the incidence rates of outcomes. Multivariate analysis was performed using a stepwise logistic regression model. The cutoff points for continuous variables were determined by receiver operating characteristic (ROC) curve analysis; p \ 0.05 was considered statistically significant. Statistical analyses were performed using JMP version 9 software (SAS Institute Japan, Tokyo, Japan).
Results Patient profiles Consecutive subjects who met the inclusion criteria were enrolled in the study. The baseline characteristics of the study population are shown in Table 1. Of the 96 participants, 76 (79.2%, 39 males and 37 females) met the diagnostic criteria for NASH. Patients with NASH had a higher incidence of DM and had higher levels of VFA, AST, ALT, ferritin, HA, and type IV collagen (p \ 0.05 for all variables) than patients with SS. HDL-c levels were lower in NASH patients. Among the cytokines and adipokines (Table 1; Fig. 1), the serum BAFF levels were higher in patients with NASH than in those with SS (p = 0.016) (Fig. 1a). BAFF levels and liver histopathology Figure 2 shows serum BAFF levels with respect to liver histological findings. The steatosis score, lobular inflammation, portal inflammation, Mallory–Denk bodies, lipogranulomas, and NAS were not associated with the serum BAFF level (Fig. 2a–f). In contrast, serum BAFF levels were significantly higher in NAFLD patients with ballooning hepatocytes and in those with advanced fibrosis stage (p = 0.016 and p = 0.006, respectively) (Fig. 2g, h). Association of BAFF levels with the prevalence of NASH To understand the relationship between BAFF levels and NAFLD, the prevalence rate (PR%) of NASH with serum BAFF level (Table 2) were investigated. All of the subjects were classified into quartiles according to their BAFF levels. The PR% of NASH in the subjects in each quartile of BAFF levels was analyzed. Quartile 1 was defined as BAFF \938 pg/mL, quartile 2 had BAFF 938 to \1,175 pg/mL, quartile 3 had BAFF 1,175 to\1429 pg/mL, and quartile 4 had BAFF [1,429 pg/mL. The PR% for the
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subjects in quartiles 1, 2, 3, and 4 were 58.2, 79.2, 87.5, and 91.7%, respectively (Table 2). The PR% of NASH increased significantly as the serum BAFF level increased (p = 0.004). These results suggest that patients with higher serum BAFF levels are more likely to develop NASH. Risk factor analysis for NASH Univariate analysis identified that body mass index (BMI), VFA, AST, ALT, ferritin, HA, type IV collagen, and BAFF were associated with significant risks of NAFLD (Table 3). Correlations among the risk factors for NASH are shown in Table 4. Multiple combinations of multivariate analyses were subsequently carried out and four factors (VFA, AST, HDL-c, and BAFF) were identified as independent risk factors for NASH. As shown in Table 5, stepwise multivariate logistic regression analysis confirmed that AST, HDL-c, and BAFF were associated with risk of NASH. A high serum BAFF level was found to be an independent risk factor for developing NASH (OR 1.003, 95% CI 1.0003–1.006; p = 0.047). BAFF as a predictor of NASH A cutoff value for diagnosis of NASH was then determined. The area under the ROC curve (95% CI), cutoff value, sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy for predicting NASH were 0.737 (0.637–0.821), 831.1 pg/mL, 93.4, 50, 87.6, 66.7, and 84.3%, respectively (Table 6). The use of serum BAFF levels for prediction of NASH demonstrated high sensitivity, positive predictive value, and accuracy.
Discussion The pathogenesis of NASH is currently thought to be a multi-step process closely associated with visceral fat [11– 15]. In the livers of NAFLD patients, 57% of the accumulated fat is derived from lipolysis of VAT, particularly in obesity with insulin resistance [32–34]. In addition, hypertrophied adipocytes of VAT release several cytokines and chemokines. These factors induce inflammation in VAT and are believed to affect the liver through the portal vein. Therefore, these factors may play a role in the pathogenesis of NASH [35–37]. Recently, BAFF has been reported to be expressed in mature adipocytes [21–24]. The expression of BAFF was changed under pro-inflammatory and anti-inflammatory conditions in vitro and in vivo [24]. In addition, Alexaki et al. [23] reported that BAFF enhanced lipogenesis in vitro. It has been also demonstrated that BAFF levels were
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Fig. 1 Serum adipokine levels in SS and NASH patients. a The serum B cell-activating factor (BAFF) level was significantly higher in NASH patients than in SS patients (p = 0.016). b Adiponectin, c leptin, and d resistin in sera were not significantly different between NASH and SS (p = 0.185, p = 0.926, and p = 0.303, respectively). Group comparisons were performed using the Mann–Whitney U test. Data are shown as medians and ranges
preferentially increased in VAT and sera in obese mice. In addition, BAFF plays a role in induction of impaired insulin sensitivity [24]. These data indicate that BAFF is an adipokine capable of inducing insulin resistance, and may predispose obese individuals to development of metabolic syndrome. In this study, the serum levels of BAFF in histologically diagnosed NAFLD patients were examined. It was found that NASH patients had higher serum BAFF levels than SS patients (Fig. 1), and that levels of BAFF were associated with the PR% of NASH (Table 2). In addition, patients with ballooned hepatocytes and advanced fibrosis in the liver had higher serum BAFF levels (Fig. 2). Furthermore, Hamada et al. [24] demonstrated that BAFF-R was preferentially expressed in liver as well as VAT [24]. These results indicate that BAFF is a significant factor associated with the development of NASH. BAFF has been reported to be involved in the development of autoimmune diseases [19]. In addition, nonspecific antibody production has been observed in patients with liver diseases, such as NAFLD [38]. However, serum BAFF levels were not significantly different in patients
with antinuclear antibodies [1,163.9 (654.1–2,384.7) pg/ mL] and those without [1,210.1 (572.2–3,249.1) pg/mL]. The results of this study provide new insights into potential roles of BAFF in clinical diagnosis and treatment of NASH. Clinically, one of the most important goals of therapy for NASH patients is prevention of progression to cirrhosis. Therefore, diagnosis of NASH at an early stage is important. However, discriminating between NASH and SS is considered to be difficult. A liver biopsy is the gold standard method for diagnosis and staging of fibrosis in patients with NASH [1, 10, 31]. However, this procedure is invasive and associated with a risk of complications [39]. In the present study, BAFF demonstrated reasonable accuracy in diagnosing NASH and was found to be a strong predictive factor for NASH. The second area in which BAFF may be valuable is in the treatment of NASH. Ballooned hepatocytes and fibrosis of the liver were most closely associated with BAFF in NASH (Fig. 2). Ballooned hepatocytes are caused by lipotoxicity and oxidant stress [40]; fibrosis is associated with the activation of hepatic stellate cells [41]. Therefore, BAFF may induce the ‘‘second hit’’ thought to be required
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544 Fig. 2 Serum B cell-activating factor (BAFF) levels according to histological findings in the liver specimens. a Steatosis score, b lobular inflammation, c portal inflammation, d Mallory–Denk bodies, e lipogranulomas, and f nonalcoholic fatty liver disease activity score (NAS) were not affected by the serum BAFF level. In contrast, the serum BAFF level was significantly higher in NAFLD patients with g the presence of ballooning hepatocytes and h advanced fibrosis stage (p = 0.016 and p = 0.006, respectively). Two group comparisons were performed using the Mann– Whitney U test. Three group comparisons were performed using the Kruskal–Wallis test. Data are shown as medians and ranges
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545
Table 2 Association of the BAFF level with prevalence of NASH BAFF level
Quartile 1
Quartile 2
Quartile 3
Quartile 4
NASH/total
14/24
19/24
21/24
22/24
PR (%)
58.2
79.2
87.5
91.7
pa
0.004
BAFF B cell-activating factor, NASH nonalcoholic steatohepatitis, PR prevalence rate a
Cochran–Armitage test
Table 3 Risk factors for NASH identified by univariate analysis (n = 96)
Factor
Odds ratio (95% CI)
Gender (M = 1, F = 2)
1.135 (0.692–1.881)
0.616
Age (years)
1.020 (0.789–1.053)
0.209
BMI (kg/m2)
1.126 (1.010–1.276)
0.045
VFA (cm2)
1.011 (1.0003–1.0223)
0.043
1.00007 (0.9998–1.0004)
0.622
0.989 (0.745–1.303)
0.937
White blood cells (/lL) Hemoglobin (g/dL) 4
ALP alkaline phosphatase, ALT alanine aminotransferase, AST aspartate aminotransferase, BAFF B cell-activating factor, CI confidence interval, FPG fasting plasma glucose, GGT gamma-glutamyl transpeptidase, HA hyaluronic acid, HbA1c hemoglobin A1c, HDL-c high-density lipoprotein cholesterol, IL-1b interleukin1b, IL-6 interleukin-6, IL-8 interleukin-8, IL-18 interleukin18, LDL-c low-density lipoprotein cholesterol, NASH nonalcoholic steatohepatitis, PT prothrombin time, TB total bilirubin, TC total cholesterol, TG triglycerides, TNF-a tumor necrosis factor-a, UA uric acid
p
Platelets (10 /lL)
0.990 (0.924–1.061)
0.779
PT (%)
0.981 (0.951–1.011)
0.215
TB (mg/dL)
1.175 (0.664–3.270)
0.671
AST (U/L)
1.078 (1.035–1.138)
0.002
ALT (U/L)
1.019 (1.005–1.040)
0.029
GGT (U/L) ALP (U/L)
0.997 (0.992–1.002) 0.99998 (0.995–1.005)
0.254 0.992
TC (mg/dL)
1.0004 (0.990–1.012)
0.943
TG (mg/dL)
1.010 (1.002–1.020)
0.039
HDL-c (mg/dL)
0.945 (0.902–0.982)
0.010
LDL-c (mg/dL)
1.004 (0.991–1.017)
0.579
UA (mg/dL)
1.120 (0.795–1.60)
0.520
HbA1c (%)
1.076 (0.837–1.443)
0.592
FPG (mg/dL)
1.003 (0.993–1.015)
0.606
Ferritin (ng/mL)
1.005 (1.001–1.011)
0.039
HA (ng/mL)
1.020 (1.004–1.045)
0.048
Type IV collagen (ng/mL)
3.468 (1.548–9.944)
0.008
IL-1b (pg/mL)
1.001 (0.996–1.009)
0.698
IL-6 (pg/mL)
1.0003 (0.999–1.003)
0.679
IL-8 (pg/mL)
1.00003 (0.99998–1.00012)
0.453
IL-18 (pg/mL)
1.003 (0.9995–1.0076)
0.155
TNF-a (pg/mL) BAFF (pg/mL)
1.007 (0.997–1.018) 1.003 (1.001–1.005)
0.223 0.004
Adiponectin (ng/mL) Leptin (pg/mL) Resistin (ng/mL)
for NASH development. Blockade of BAFF may be a potential therapeutic strategy for preventing disease progression. However, further research is needed to confirm this hypothesis. The levels of circulating adipokines and cytokines in NAFLD have been reported to be affected by other risk factors for NASH, such as DM, hypertension, and
0.9999 (0.9998–1.0001)
0.286
1.00001 (0.99996–1.00008)
0.747
1.056 (0.936–1.228)
0.427
dyslipidemia and may simply reflect an association between NALFD and other risk factors rather than a true causal relationship [42–44]. In our study, the levels of other adipokines and cytokines were not significantly different between patients with NASH and those with SS. The specific reasons for this are not clear. Although several studies have reported differences in serum cytokine levels
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Table 4 Correlations (R2) among risk factors for NASH BMI BMI
X
VFA
VFA
AST
ALT
TG
HDL-c
Ferritin
HA
Type IV collagen
BAFF
0.340**
0.010
0.020
0.045*
0.015
0.0002
0.004
0.009
0.035
X
1.7e-5
2.6e-5
0.017
0.014
0.037
0.007
0.007
0.010
X
0.672**
0.102*
0.005
0.258
6.1e-6
0.052*
0.05*
AST ALT
X
TG
0.136**
0.031
0.20**
0.03**
0.0009
0.003
X
0.134*
0.082**
0.0001
0.0001
0.002
X
0.070*
0.006
2.4 e-5
0.001
X
0.001
0.009
1.1e-5
0.305**
0.270**
X
0.188**
HDL-c Ferritin HA
X
Type IV collagen BAFF
X
ALT alanine aminotransferase, AST aspartate aminotransferase, BAFF B cell-activating factor, BMI body mass index, HA hyaluronic acid, HDL-c high-density lipoprotein cholesterol, NASH nonalcoholic steatohepatitis, TG triglycerides, VFA visceral fat area * p \ 0.05; **p \ 0.01 Table 5 Risk factors for NASH identified by multivariate analysis (n = 96) Factor
Odds ratio (95% CI)
p
AST (U/L)
1.104 (1.040–1.203)
0.007
HDL-c (mg/dL)
0.934 (0.876–0.980)
0.019
BAFF (pg/mL)
1.003 (1.0003–1.006)
0.047
Only variables that achieved statistical significance (p \ 0.05) in the stepwise multivariate logistic regression are shown; the complete set of variables included VFA, AST, HDL-c, and BAFF AST aspartate aminotransferase, BAFF B cell-activating factor, CI confidence interval, HDL-c high-density lipoprotein cholesterol, NASH nonalcoholic steatohepatitis Table 6 Sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of BAFF for prediction of NASH AUC (95% CI)
Cutoff value (U/L)
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
Diagnostic accuracy (%)
0.737 (0.637–0.821)
831.1
93.4
50
87.6
66.7
84.3
Each value was determined by ROC curve analysis AUC area under the curve, BAFF B cell-activating factor, CI confidence interval, NASH nonalcoholic steatohepatitis, NPV negative predictive value, PPV positive predictive value
between NAFLD patients and controls, others have shown contradictory results. In particular, several studies found no significant differences in serum cytokine levels between SS and NASH patients [45], consistent with the present study. In addition, most of the NAFLD patients in the present study had metabolic diseases and took several medications, which may have affected the results. There were several limitations of our cross-sectional study. For example, we were unable to confirm a relationship between serum BAFF levels and insulin resistance (HOMA-R), because many patients were being treated with several medications and/or had high levels of plasma glucose. Future studies are necessary to evaluate this relationship using a prospective validation design. In conclusion, we found that NASH patients had higher serum BAFF levels than SS patients, and higher BAFF
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levels were associated with hepatocyte ballooning and advanced fibrosis. The serum BAFF level may be a useful tool for distinguishing NASH from SS. Blockade of BAFF may be a promising therapeutic strategy for patients with NASH. Acknowledgements This work was supported in part by a grant-inaid for scientific research from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (KAKENHI No. 23700907), and a research grant from Ehime University.
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