Immunological Investigations, 2015; 44(1): 13–22 ! Informa Healthcare USA, Inc. ISSN: 0882-0139 print / 1532-4311 online DOI: 10.3109/08820139.2014.914530

S100A12 expression in patients with primary biliary cirrhosis Danxu Ma,1,2 Xi Li,1,3 Lei Zhang,1 Chuiwen Deng,1 Ting Zhang,1 Li Wang,1 Chaojun Hu,1 Yongzhe Li,1 and Fengchun Zhang1

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Department of Rheumatology and Clinical Immunology, Peking Union Medical College Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing, China, 2 Department of Anesthesiology, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China, 3 Department of Clinical Laboratory, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China Objectives: S100 calcium binding protein A12 (S100A12) has been supposed to be a proinflammatory factor associated with non-infectious inflammatory diseases. However, whether S100A12 is involved in the inflammatory process of primary biliary cirrhosis (PBC) has not been shown. Methods: The levels of S100A12 mRNA transcripts in peripheral mononuclear blood cells (PBMCs) of 66 Chinese patients with primary biliary cirrhosis (PBC), 62 healthy controls (HC) and 55 chronic hepatitis B (CHB) were measured by qRT-PCR. S100A12 serum concentrations in 34 PBC patients were measured by ELISA. Results: The levels of S100A12 mRNA transcripts in PBMCs of patients with PBC were significantly higher than healthy controls (p50.01) and that of patients with CHB (p50.01). Importantly, the levels of S100A12 mRNA in PBMCs and S100A12 protein levels in serum were positively correlated with biochemical indicators of bile duct and hepatocyte damage. Conclusion: S100A12 might participate in the damage of biliary epithelial cells and hepatocytes in PBC, and analysis of S100A12 expression could be useful as a surrogate marker for the evaluation of PBC activity. Keywords Enzyme-linked immunosorbent assay, peripheral blood mononuclear cells, primary biliary cirrhosis, quantitative real-time reverse transcription polymerase chain reaction, S100 calcium binding protein A12

INTRODUCTION Primary biliary cirrhosis (PBC) is an autoimmune disease histopathologically characterized by chronic inflammatory destruction of the small and medium intrahepatic bile ducts. It predominantly affects middle-aged females (Bao et al., 2010; Invernizzi et al., 1997, 2010). In the absence of treatment, Correspondence: Yongzhe Li, Department of Rheumatology and Clinical Immunology, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, #41 Damucangxidan, Beijing, 100032, China. Tel: 86-10-69158795. Fax: 86-10-69158795. E-mail: [email protected] or Fengchun Zhang, Department of Rheumatology and Clinical Immunology, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, #41 Damucangxidan, Beijing, 100032, China. Tel: 86-10-69158794. Fax: 86-10-69158794. E-mail: [email protected]

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PBC can result in liver fibrosis, cirrhosis, and eventually hepatic failure. Patients with PBC frequently present with pruritus, fatigue, jaundice, and osteoporosis (Invernizzi et al., 1997, 2010). The serum antimitochondrial antibody (AMA) is widely accepted as a diagnostic hallmark of PBC. However, it is not clinically helpful for follow-up, as several studies have demonstrated that its levels do not correlate with stage or disease activity (Invernizzi et al., 1997, 2010; Lindor et al., 2009). Thus, much work is needed to find a reliable biomarker for the determination of disease severity. Human S100 calcium binding protein A12 (S100A12) is expressed abundantly in neutrophils and monocytes/macrophages. S100A12 is a member of the S100 family of EF-hand calcium-binding proteins. It is a chemo-attractant for monocytes and is a ligand for the receptor for advanced glycation end products (RAGE)(Pietzsch & Hoppmann, 2009). Ligation of S100A12 with RAGE is followed by activation of intracellular signal cascades including MAP-kinase and NF-kB. This induces secretion of cytokines (e.g. tumor necrosis factor-a and interleukin-1b) and expression of adhesion molecules, such as intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, and thereby mediates proinflammatory effects on lymphocytes, endothelial cells, neutrophil and mononuclear phagocytes (Hofmann et al., 1999; Pietzsch & Hoppmann, 2009; Yang et al., 2001). S100A12 appears to be of clinical value in the evaluation and assessment of a number of inflammatory disorders, such as Kawasaki’s disease (Abe et al., 2005; Foell et al., 2003a; Fu et al., 2010; Qi et al., 2012), rheumatoid arthritis (Baillet et al., 2010; Bovin et al., 2004), Behcet’s disease (Han et al., 2011), and inflammatory bowel disease (Foell et al., 2003b; Brinar et al., 2010; van de Logt & Day, 2013). Given that PBC is also an autoimmune inflammatory disease, we hypothesized that S100A12 might participate in the inflammatory process of PBC, and levels might be useful for the evaluation of PBC activity. The aim of this study was to measure S100A12 gene expression in peripheral blood mononuclear cells, and serum soluble protein levels in PBC patients, and further to analyze their relationships with biochemical indicators of bile duct and hepatocyte damage.

MATERIALS AND METHODS Study subjects A total of 66 patients with PBC, 62 healthy controls, and 55 patients with chronic hepatitis B (CHB) were recruited. PBC patients were definitively diagnosed at Peking Union Medical College Hospital according to the AALD 2009 PBC diagnostic guidelines (Lindor et al., 2009). All enrollees met 2 out of 3 criteria for positive serum AMA, had two times or greater than the upper limit of normal concentration of serum alkaline phosphatase (ALP) for more than 6 months and/or compatible liver histology. Patients were excluded if they had viral hepatitis, focal intrahepatic or extrahepatic lesions or obstruction, alcohol or drug-related liver injury or liver

S100A12 expression in PBC

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Table 1. Clinical characteristics of primary biliary cirrhosis(PBC) patients and controls.

Characteristics

PBC patients (n ¼ 66)

Healthy controls (n ¼ 62)

CHB patients (n ¼ 55)

Female, n (%) Male, n (%) Age, years Edema, n (%) ANA, n (%) AMA, n (%) ALT (U/L) AST (U/L) GGT (U/L) ALP (U/L) Tbil (umol/L) PT(s) MRS

62(93.9%) 4(6.1%) 55(29–81) 14(21.2%) 46(69.7%) 61(92.4%) 42.12 ± 31.82 47.68 ± 26.84 175.86 ± 215.62 197.71 ± 159.07 30.32 ± 42.80 11.09 ± 1.72 4.57 ± 1.54

58(93.6%) 4(6.4%) 52(37–78) NT NT NT 18.53 ± 9.05 19.24 ± 6.34 20.48 ± 11.42 59.26 ± 16.96 11.30 ± 3.76 NT –

51(92.7%) 4(7.3%) 54(28–64) NT NT NT 151.51 ± 243.56 106.80 ± 176.04 70.44 ± 75.61 79.07 ± 34.78 19.89 ± 19.07 NT –

Values are expressed as mean ± SD or real number of cases and percentage. CHB, chronic hepatitis B; ANA, anti-nuclear antibody; AMA, anti-mitochondrial antibody; ALP, alkaline phosphatase; GGT, gamma-glutamyltransferase; ALT, alanine aminotransferase; AST, Aspartate aminotransferase; TBil, total bilirubin; PT, prothrombin time; MRS, Mayo risk score; NT, not tested.

cancer. Sixty-two healthy age- and sex-matched subjects were recruited from blood donors, and 55 CHB patients served as disease controls. The diagnosis of CHB was made based on clinical, laboratory and imaging findings. The demographic and clinical characteristics of participants are presented in Table 1. Written informed consent was obtained from all subjects, and the study was approved by the Ethics Committee of Peking Union Medical College Hospital, China. Laboratory assessments and mayo risk score Fasting peripheral blood samples were obtained from individual participants and their sera were prepared. The levels of serum gamma-glutamyltransferase (GGT), alanine transaminase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBil) were measured by routine laboratory assays. The concentrations of serum anti-nuclear antibodies (ANA) and anti-mitochondrial antibodies (AMA) in PBC patients were determined by indirect immunofluorescence assay (IFA) using the specific kits, according to the manufacturers’ instructions (Euroimmun Medical Laboratory Diagnostics, Lubeck, Germany). The prognosis of individual patients was predicted by the Mayo Risk Score (MRS) (Dickson et al., 1989). PBMCs preparation and RNA extraction Fresh heparinized peripheral blood samples were obtained. PBMCs were isolated from peripheral venous blood by Ficoll-Hypaque density gradient centrifugation. Total cellular RNA was extracted using Trizol reagent (Invitrogen, California, USA) in accordance with the manufacturer’s instructions. RNA yield and purity were determined spectrophotometrically at 260/280 nm.

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D. Ma et al. Table 2. Primer sequences used for amplification of S100A12 and GAPDH. Name S100A12

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GAPDH

Forward Primer Reverse Primer Forward Primer Reverse Primer

Sequence

Amplicon size (bp)

ATTGAGGGGTTAACATTAGGCTG GATATTCTTGATGGTGTTTGCAAGC TCGGAGTCAACGGATTTGGTC GCCATGGGTGGAATCATATTGG

172 146

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) The cDNA was synthesized by using total RNA and reverse transcription reagents (DRR037, TakaRa, Dalian, China). For qRT-PCR, primers were designed according to DNAMAN and Primer 5 software and synthesized by Invitrogen, USA. Primers are shown in Table 2. Human GAPDH gene was used as an endogenous control for sample normalization. qRT-PCR analysis was applied with 2 mL cDNA per reaction (SYBR PrimeScript RT-PCR kit DRR041, TakaRa, Dalian, China) with the following cycling parameters: 95  C for 30 s, followed by 40 cycles of 95  C for 5 s and 60  C for 30 s. At the end of the PCR cycle, a dissociation curve was generated to ensure the amplification of a single product, and the threshold cycle time (De Sanctis et al., 2009) for each gene was determined. The relative mRNA expression of target gene and housekeeping gene were determined by comparing the 2(DDCt). Enzyme-Linked Immunosorbent Assay (ELISA) Concentration of S100A12 in the serum of patients with PBC were determined by an ELISA method using a commercially available kit (S100A12/EN-RAGE ELISA Kit, Circulex, Japan). Statistical analysis All data were presented as mean ± SD or median and percentile. Data were analyzed with SPSS17.0 software using the non-parametric Mann–Whitney U-test to determine the statistical significance of differences. The correlation of two different parameters was calculated with Spearman’s rank correlation coefficient (r). p Values less than 0.05 were considered significant.

RESULTS S100A12 mRNA expression in PBC patients Our study demonstrated that the relative levels of S100A12 mRNA transcripts in PBMCs of PBC patients were significantly higher than those of healthy controls (p50.01, Figure 1) and CHB patients (p50.01, Figure 1). Importantly, the levels of S100A12 mRNA in PBMCs were positively correlated with serum levels of AST, GGT and ALP (Table 3 and Figure 2). S100A12 serum concentration in PBC patients Furthermore, we analyzed the S100A12 levels in serum of 34 PBC patients, and found that S100A12 serum levels were positively correlated with levels of GGT, ALP and TBil (Table 3 and Figure 3).

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S100A12 expression in PBC

Figure 1. The relative levels of S100A12 mRNA transcripts in PBMCs. PBMCs were isolated from individual subjects and the relative levels of S100A12 mRNA transcripts to control GAPDH in PBMCs from 66 PBC patients, 62 healthy controls and 55 CHB patients were determined by qRT-PCR. **p50.01 versus healthy controls.

Table 3. The correlation of S100A12 with ALT, AST, GGT, ALP, Tbil and MRS in primary biliary cirrhosis (PBC) patients. PBC patients mRNA

ALT AST GGT ALP Tbil MRS

Serum

r

p

r

p

0.231 0.280 0.305 0.243 0.144 0.021

0.061 0.023 0.013 0.050 0.250 0.869

0.132 0.095 0.419 0.349 0.357 0.307

0.458 0.595 0.014 0.043 0.038 0.077

Data were analyzed with Spearman’s rank correlation coefficient analysis. Bold values are statistically significant (p50.05).

DISCUSSION From the current results, PBC patients had increased relative levels of S100A12 mRNA, and the mRNA and protein levels of S100A12 were correlated positively with biochemical indicators of bile duct and hepatocyte damage.

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Figure 2. The correlation of levels of S100A12 mRNA transcripts with levels of serum ALP in primary biliary cirrhosis (PBC) patients. The values of relative levels of S100A12 mRNA transcripts were plotted against the concentrations of serum ALP in individual PBC patients, and analyzed by Spearman’s rank correlation coefficient.

S100A12 has been reported to be relevant for diagnostic use in various non-infectious inflammatory diseases (Meijer et al., 2012). Primary biliary cirrhosis is also an inflammatory autoimmune disease that mainly targets the cholangiocytes of the interlobular bile ducts in the liver. Therefore, S100A12 might be involved in the inflammation of biliary epithelial cells and hepatocytes in PBC and its level could be useful as a surrogate marker for disease activity. There have been no reports on the function of S100A12 in liver injury. We suspect that S100A12 might lead to the damage of biliary epithelial cells and hepatocytes through the following mechanisms. S100A12 enhances the migration and activation of monocytes/macrophages (Pietzsch & Hoppmann, 2009). In PBC patients, the areas around injured intrahepatic biliary epithelial cells are frequently infiltrated with various immune cells such as T lymphocytes, monocytes, neutrophils, NK cells (Liang et al., 2008). PBC monocytes have been shown to have enhanced pro-inflammatory activities in PBC. Monocytes activated by pathogen-associated molecular patterns (PAMPs) through TLRs, can release pro-inflammatory cytokines such as IL-1, IL-6, IL-12, IL-18, and TNF-a, can then amplify the adaptive T cell-mediated immune response. Monocytes from patients with PBC appear to be more sensitive to signaling by select TLRs, resulting in secretion of selective proinflammatory cytokines integral to the inflammatory response. This may be

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S100A12 expression in PBC

Figure 3. The correlation of serum S100A12 concentrations with the serum ALP levels in 34 primary biliary cirrhosis (PBC) patients. The values of S100A12 serum concentrations were plotted against the concentrations of serum ALP in individual PBC patients, and analyzed by Spearman’s rank correlation coefficient.

critical to the breakdown of self-tolerance (Invernizzi et al., 2010; Mao et al., 2005; Selmi et al., 2010). Therefore, high levels of S100A12 expression may enhance the activation of monocytes which participate in the inflammatory process of PBC. S100A12 activates nuclear factor-kB, a master regulator of inflammation and cell death. NF-kB activates several hundred pro-inflammatory genes. The present studies have shown that cytokines which are regulated by NF-kB, such as IL-1, IL-2a, IL-6, IL-8, IFN, MCP-1 play an important role in the inflammatory process of PBC (Sasaki et al., 2010; Shindo et al., 1996; Zhao et al., 2011). On the other hand, NF-kB is a critical immediate early response gene involved in modulating cellular responses and apoptosis following diverse environmental injuries. The initial histology of PBC is characterized by a distinctive pattern of bile duct damage including chronic non-suppurative destructive cholangitis, bile duct loss and chronic cholestasis most prominent in the early stages. Fas-mediated mechanisms of apoptosis are thought to be involved in the bile duct loss in PBC. The early activation of NF-kB may be critical for the induction of Fas-L expression (Afford et al., 2001; Harada et al., 1997). It has been reported that ursodeoxycholic acid (UDCA), which is the current mainstay of treatment for PBC, suppresses NF-kB dependent transcription pathway (Miura et al., 2001). Thus, it is possible that S100A12 participates in the damage of biliary epithelial cells and hepatocytes through the NF-kB pathway.

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S100A12 up-regulates adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1(VCAM-1)(Meijer et al., 2012). PBC is characterized by a highly selective destruction of small and medium-size intra-hepatic bile ducts. Lymphocytes and other inflammatory cells penetrate the peribiliary vascular plexus and portal venules, and then migrate into the perivenular tissue toward the bile ducts. In this process, increased expression of ICAM-1 and VCAM-1 on the endothelial cells of blood vessels are significant. UDCA therapy attenuates the overexpression of ICAM1 around lymphocytes and among damaged bile ducts (Invernizzi et al., 2010; Yokomori et al., 2003a; Yokomori et al., 2003b). In summary, it is possible that high levels of S100A12 expression may affect the expression of inflammatory cell adhesion molecules which participate in the damage of biliary epithelial cells in PBC. In the current study, we used CHB patients as the disease control group. Unlike PBC, CHB patients show mild bile duct damage. The occurrence of bile duct loss or cholestasis is very rare. Apoptotic cells are frequently found among biliary epithelial cells, particularly around the more cholangitic bile ducts in PBC. However, very few occur in control livers (Harada & Nakanuma, 2006; Liang et al., 2008). S100A12 has been reported to be a pro-inflammatory factor in non-infectious inflammatory diseases. That could explain the downregulation of S100A12 mRNA levels in CHB patients. The altered levels of S100A12 mRNA in PBC and CHB patients might be useful in the diagnosis of PBC. In summary, the current data indicate that levels of PBMC S100A12 mRNA were increased markedly in the PBC group, but decreased in the CHB group compared to the healthy control group. Moreover, the mRNA and protein levels of S100A12 were positively correlated with biochemical indicators of bile duct and hepatocyte damage. All these results suggest that S100A12 might participate in the damage of biliary epithelial cells and hepatocytes in PBC, and its level could be useful as a surrogate marker for the evaluation of PBC activity. Further studies are needed for a more precise elucidation of the proinflammatory activities of S100A12.

ACKNOWLEDGEMENTS This work was carried out at Department of Rheumatology and Clinical Immunology, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, #41 Damucangxidan, Beijing, 100032, China. Authors Danxu Ma, Xi Li, Lei Zhang, and Chuiwen Deng made equal contributions to this paper.

DECLARATION OF INTEREST This study was supported by grants from the National Natural Science Foundation of China (81072486 and 81172857), the Research Special Fund for Public Welfare Industry of Health (No. 201202004), the National High Technology Research and Development Program of China (863 Program) (No. 2011AA02A113) and Key Clinical Program of the Ministry of Health (2010–2012).

S100A12 expression in PBC

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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S100A12 expression in patients with primary biliary cirrhosis.

S100 calcium binding protein A12 (S100A12) has been supposed to be a pro-inflammatory factor associated with non-infectious inflammatory diseases. How...
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