Journal of Pediatric Surgery 50 (2015) 297–300
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
Immunolocalization of surfactant protein D in the liver from infants with cholestatic liver disease Ryuta Saka a,b,⁎, Itaru Yanagihara c, Takashi Sasaki a, Satoko Nose a, Makoto Takeuchi d, Masahiro Nakayama d, Hiroomi Okuyama a,b a
Department of Pediatric Surgery, Hyogo College of Medicine Department of Pediatric Surgery, Osaka University Graduate School of Medicine Department of Developmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health d Department of Pathology and Laboratory Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health b c
a r t i c l e
i n f o
Article history: Received 25 October 2014 Accepted 2 November 2014 Key words: Surfactant protein D Liver Cholestasis Biliary atresia
a b s t r a c t Purpose: Surfactant protein D (SP-D) is one of specific surfactant proteins constituting pulmonary surfactant. Recent studies have revealed that SP-D is detected in various non-pulmonary tissues and is involved in the host defense and immunomodulation. However, the relationship between SP-D and liver diseases has not yet been investigated. The aim of this study was to detect the immunolocalization of SP-D in the livers of infants with cholestatic liver disease. Methods: The expression of immunoreactive SP-D was assessed in infants with cholestasis, including biliary atresia (BA, n = 7), neonatal hepatitis (NH, n = 2), and paucity of the intrahepatic bile duct (PIBD, n = 4). Immunoreactive SP-D was also assessed in six infants who died of non-liver disease as controls. Tissue samples were obtained at liver biopsy, or by post-mortem sampling. The tissue sections were incubated with anti-SP-D polyclonal antibodies and were counterstained with hematoxylin. Results: In the normal livers, SP-D was detected in the intrahepatic bile ducts, but was not detected in hepatocytes. In contrast, intense SP-D staining was noted in the hepatocytes from infants with BA, NH, and PIBD. Although SP-D was detected in the intrahepatic bile ducts in the infants with NH, negative or weak staining was seen in the intrahepatic bile ducts in infants with BA. Conclusion: Our data showed that SP-D is present in the bile ducts of the normal infant liver, and it was found to accumulate in the hepatocytes of cholestatic livers. These results suggest that SP-D is produced in hepatocytes and is secreted into the bile ducts. © 2015 Elsevier Inc. All rights reserved.
Pulmonary surfactant is a complex of lipids and proteins. To date, four specific surfactant proteins (SPs) have been defined, named SP-A, SP-B, SP-C and SP-D. SP-D, as well as SP-A, has important roles in innate immunity. SP-D binds to various pathogenic microorganisms and works as an opsonin to enhance the uptake of these cells and particles by immune cells [1]. SP-D also regulates the cellular response by affecting the production of inflammatory mediators such as tumor necrosis factor-α (TNF α) via an interaction with toll-like receptor (TLR)-4. Although the expression of SP-A is limited to the lung in human [2], SP-D can be detected in many extrapulmonary organs, including the liver [3]. However, little is known about the role of extrapulmonary SP-D in humans [4]. Cholestatic liver disease in infants, which requires urgent diagnosis and treatment, results from various causes, and the incidence is approximately 1 in 2500 live births [5]. Biliary atresia (BA) is the most common ⁎ Corresponding author at: Department of Pediatric Surgery, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya-shi, Hyogo, Japan 6638501. Tel.: +81 798 45 6582; fax: +81 798 45 6581. E-mail address: [email protected] (R. Saka). http://dx.doi.org/10.1016/j.jpedsurg.2014.11.020 0022-3468/© 2015 Elsevier Inc. All rights reserved.
surgical cause of cholestatic liver disease in infants. Recurrent cholangitis is also one of the most important postoperative complications of BA, which may aggravate liver function. We hypothesized that SP-D may contribute to innate immunity in bile duct, and relate to the pathogenesis of postoperative cholangitis in BA. To the best of our knowledge, the relationship between SP-D and liver disease has not yet been investigated. The aim of this study was to clarify the distribution of SP-D in the hepatocytes and bile ducts of infants with cholestatic liver disease including BA, neonatal hepatitis (NH) and paucity of the intrahepatic bile duct (PIBD) and to compare it with that in normal control (NC) infants without liver disease. 1. Materials and methods This study was approved by the institutional committees of Hyogo College of Medicine and Osaka Medical Center for Maternal and Child Health. Specimens were collected during surgery and liver biopsy from infants with cholestatic liver diseases. Bile duct paucity was defined histologically, when the ratio of interlobular bile ducts to the number of portal tracts was b0.5 (normal range, 0.9–1.8) in at least
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R. Saka et al. / Journal of Pediatric Surgery 50 (2015) 297–300
Table 1 Demographics of infants with cholestatic liver diseases. BA (n = 7) Age (day) T-Bil (mg/dl) D-Bil (mg/dl) AST (IU/L) ALT (IU/L)
62 (16–90) 7.8 (7.4–11.1) 4.9 (3.9–7.9) 150 (83–263) 58 (41–193)
NH (n = 2)
PIBD (n = 4)
63, 70
49 (24–68) 6.2 (5.4–8.5) 4.9 (3.5–5.6) 104 (84–123) 88 (54–103)
3.4, 9.0 1.5, 6.1 109, 110 70, 141
The data are expressed as the median (range), except for the NH group. BA: biliary atresia, NH: neonatal hepatitis, PIBD: paucity of intrahepatic bile duct.
five portal areas [6]. All the diagnosis of Paucity of Intrahepatic Bile Ducts (PIBD) was made by a pediatric pathologist (MN). As a control, fetal/neonatal autopsy cases without any liver dysfunction were used.
The immunoreactive hepatocytes were classified as follows: 0 (negative); 1 (rare periportal hepatocytes staining); 2 (multifocal to diffuse periportal staining); 3 (more diffuse staining extending into the lobules); 4 (staining of the majority of hepatocytes). The immunoreactivity of the lumens of the interlobular bile ducts and proliferating bile ductules (surface of biliary epithelial cells) was scored based on the number of positive structures present per portal tract as follows: 0 (absent); 1 (b25%); 2 (25%–50%); 3 (N50%). Then, the average scores were calculated for each slide. The interlobular bile ducts were recognized as biliary ductal elements in the central region of the portal tract with round and open lumina on hematoxylin and eosin (HE) staining. The proliferating bile ductules were recognized as biliary elements at the edge of the portal tract, which often had irregular lumina on HE staining. 1.3. Statistical analysis Patient demographics and the results of the immunohistological evaluation are expressed as the median (range). Scores were statistically analyzed using the Wilcoxon rank sum test. All statistical analyses were performed using the JMP®8 software package (SAS Institute Inc., Cary, NC). A P value b 0.05 was considered to be significant.
1.1. Immunohistochemistry 2. Results Formalin-fixed samples were embedded in paraffin and cut into 3 μm sections. After deparaffinization in xylene and rehydration in a graded series of ethanol, the sections were incubated with 0.3% H2O2 in methanol for 30 min to block the endogenous peroxidase activity. After three washes with PBS, the sections were incubated with Protein Block, Serum-Free (Dako, Glostrup, Denmark) to inhibit the non-specific staining for 30 min, and were washed with PBS. Then, the sections were incubated overnight at 4 °C with an anti-SP-D polyclonal Ab (1:1000, Chemicon #AB3434). After three washes with PBS, the sections were incubated with EnVision ™ + Rabbit/HRP (Dako) for 1 h at room temperature. The tissue sections were then incubated with DAB substrate (Nichirei Biosciences, Tokyo, Japan) for the chromogen reaction and were counterstained with hematoxylin. The slides were mounted with malinol (Muto Pure Chemicals, Tokyo, Japan) and visualized by light microscopy. 1.2. Evaluation of immunoreactivity Immunoreactivity of the samples was assessed using a semiquantitative histopathological score [7]. Immunostained sections were evaluated by two authors who were unaware of the diagnosis or clinical outcome.
2.1. Patients The age and the results of the liver function tests of the patients are shown in Table 1. As normal controls, a total of six autopsy cases (0–2 months old), without any liver diseases clinically and histologically, were evaluated. For the cholestasis patients, two had neonatal hepatitis (NH), four had PIBD and seven had BA. All cholestasis cases were younger than three months of age, and there were no significant differences in the age, bilirubin level (total and direct), AST and ALT among the cholestatic etiologies. 2.2. Immunohistochemistry In the normal controls (Fig. 1), the luminal surface of the bile duct was immunostained for SP-D, however, no intracellular immunostaining was found in the biliary epithelial cells. Hepatocytes showed almost no immunoreactivity. By contrast, in cholestatic livers (Figs. 2–4), the hepatocytes showed strong immunoreactivity, regardless of the disease. Although the luminal surface of the bile duct was strongly immunostained in the NH cases, the immunoreactivity of the proliferating bile
Fig. 1. Immunohistological findings of the normal controls (NC). Although the hepatocytes showed no immunoreactivity (A: original magnification ×100), the luminal surface of the intrahepatic bile duct (black arrow) was immunostained for SP-D (B: ×200, inset: ×400).
R. Saka et al. / Journal of Pediatric Surgery 50 (2015) 297–300
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Fig. 2. The immunohistological findings of the patients with biliary atresia. Hepatocytes were positively and diffusely immunostained, and the proliferating bile ductules (black arrow) showed a variety of immunoreactivities, ranging from vague (A: original magnification ×200, inset: ×400) to clear (B: original magnification ×200, inset: ×400) in the luminal surface of the bile ductules.
Fig. 3. The immunohistological findings of the patients with neonatal hepatitis. (A) The hepatocytes were diffusely immunostained (original magnification ×100) (B) The luminal surface of the bile duct (black arrow) was immunostained (×200, inset: ×400).
duct in BA varied from weak to strong. In the PIBD cases, the bile duct could not be evaluated because the number of bile duct was too small. Statistical analyses of the immunohistological scores were performed only between NC and BA, because the other groups were too small (Fig. 5). In hepatocytes, the NC showed a significantly lower score than
the BA [0.25 (0–1.5) vs. 3.5 (3.0–4.0); P b 0.01]. In the bile duct, the BA cases showed a significantly lower score compared with the NC [1.5 (1.0–2.5) vs. 2.5 (2.0–3.0), P = 0.02). Although the statistical analysis could not be applied to NH and PIBD, NH and PIBD showed high hepatocytes score and NH showed high bile duct score.
Fig. 4. The immunohistological findings of the cases with a paucity of the intrahepatic bile duct. Although the bile duct could not be evaluated, hepatocytes showed strong immunoreactivity in diffuse. (A: original magnification ×100, B: ×200).
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Fig. 5. The immunohistological scores. The scores of hepatocytes (0–4) BA showed significantly higher scores than NC (P b 0.01). The scores of the bile ducts (0–3). BA showed lower scores in the bile ducts compared to NC (P = 0.02). NC: normal control, BA: biliary atresia.
3. Discussion Surfactant protein (SP)-D belongs to a subtype of mammalian lectins termed collectin, which is composed of an N-terminal cysteine-rich region, a collagen-like region, an alpha helical coiled neck region and a carbohydrate recognition domain. Collectin is known to have an important role in innate immunity, especially in the airway [8]. Although SP-A is almost exclusively localized in the airway [2], extrapulmonary expression of SP-D has been reported in humans [3]. Recently, the functions of SP-D, especially its contribution to innate immunity, have been investigated in the lung. SP-D enhances the uptake of pathogens by opsonizing them, functioning as a molecule that causes the activation of ligands and regulating cell surface receptor expression [1]. SP-D directly binds a variety of microorganisms, including bacteria (gram-positive/negative) and viruses, and functions as an opsonin to enhance their uptake [9]. SP-D also regulates the cellular response by immune cells. SP-D inhibits the binding of smooth and rough LPS, which is found in the membrane of gram-negative bacteria and elicits immune responses by immune cells, especially TLR4/MD-2 expressing cells, attenuates the MD-2 binding to LPS and inhibits LPS-induced inflammatory responses (TNF-α secretion and NF-κB activation) [10]. SP-D null mice showed increased lung inflammation and decreased macrophage phagocytosis during bacterial infection in the lungs [11]. Although the function of SP-D in the lungs has been relatively well investigated, its function in the liver has been unclear. Tang et al. reported a relationship between SP-D and autophagy in the liver [12], however, this study is one of only a few regarding the function of SP-D in the liver. We speculate that SP-D may contribute to innate immunity against pathogens, especially gram-negative bacteria, in the biliary system and bloodstream. To investigate the function and localization of SP-D in the liver, we compared cholestatic livers with normal control livers. In this study, immunoreactivity for SP-D was found in the luminal surface of bile duct, but not in the hepatocytes and biliary epithelial cells in the normal controls. In contrast, the hepatocytes showed strong immunoreactivity in the infants with cholestasis, regardless of the etiology. Interestingly, the BA cases showed relatively weak immunoreactivity in the luminal surface of the bile duct compared with the NC and other cholestatic patients. These results suggest that SP-D is produced in the hepatocytes and secreted into the bile juice in the normal liver. SP-D accumulation in the hepatocytes may reflect cholestasis; therefore, SP-D may be a possible clinical marker of cholestasis. By performing immunostaining for SP-D, it may be possible to diagnose the degree and location of cholestasis, and to clarify the pathogenesis of such cholestasis. The fact that SP-D, which is important in innate immunity, exists in the bile duct, implies that SP-D may play an important role in the postoperative cholangitis in BA, which is associated with the obstruction of
the secretion of bile juice, which includes SP-D. Postoperative cholangitis in BA patients after Kasai portoenterostomy is a troubling complication, which sometimes leads to the need for liver transplantation. The incidence of cholangitis is higher in BA cases than in those with other etiologies requiring hepaticojejunostomy, such as choledochal cysts. The cholangitis in BA patients after the Kasai procedure is due to intestinal flora, especially gram-negative bacteria. Considering the effect of SP-D against gram-negative bacteria, SP-D may have an important role against cholangitis in BA patients. The major limitation of this study is the small number of specimens of postoperative BA. We do not regularly perform liver biopsies after the Kasai operation but comparisons of liver specimens immunostained for SP-D between patients with recurrent cholangitis and those without cholangitis may further clarify the relationship between SP-D and cholangitis. Further investigations on the relationship between the clinical symptoms (jaundice, cholangitis and prognosis) and the localization of SP-D are also necessary. Acknowledgement This study was supported by research grants from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan. References [1] Wright JR. Immunoregulatory functions of surfactant proteins. Nat Rev Immunol 2005;5:58–68. [2] Madsen J, Tornoe I, Nielsen O, et al. Expression and localization of lung surfactant protein A in human tissues. Am J Respir Cell Mol Biol 2003;29:591–7. [3] Stahlman MT, Gray ME, Hull WM, et al. Immunolocalization of surfactant protein-D (SP-D) in human fetal, newborn, and adult tissues. J Histochem Cytochem 2002;50: 651–60. [4] Kurimura Y, Nishitani C, Ariki S, et al. Surfactant protein D inhibits adherence of uropathogenic Escherichia coli to the bladder epithelial cells and the bacteriuminduced cytotoxicity: a possible function in urinary tract. J Biol Chem 2012;287: 39578–88. [5] De Bruyne R, Van Biervliet S, Vande Velde S, et al. Clinical practice: neonatal cholestasis. Eur J Pediatr 2011;170:279–84. [6] Kocak N, Gurakan F, Yuce A, et al. Nonsyndromic paucity of interlobular bile ducts: clinical and laboratory findings of 10 cases. J Pediatr Gastroenterol Nutr 1997;24:44–8. [7] Ernst LM, Spinner NB, Piccoli DA, et al. Interlobular bile duct loss in pediatric cholestatic disease is associated with aberrant cytokeratin 7 expression by hepatocytes. Pediatr Dev Pathol 2007;10:383–90. [8] Sano H, Kuroki Y. The lung collectins, SP-A and SP-D, modulate pulmonary innate immunity. Mol Immunol 2005;42:279–87. [9] Kuroki Y, Takahashi M, Nishitani C. Pulmonary collectins in innate immunity of the lung. Cell Microbiol 2007;9:1871–9. [10] Yamazoe M, Nishitani C, Takahashi M, et al. Pulmonary surfactant protein D inhibits lipopolysaccharide (LPS)-induced inflammatory cell responses by altering LPS binding to its receptors. J Biol Chem 2008;283:35878–88. [11] LeVine AM, Whitsett JA, Gwozdz JA, et al. Distinct effects of surfactant protein A or D deficiency during bacterial infection on the lung. J Immunol 2000;165:3934–40. [12] Tang Z, Ni L, Javidiparsijani S, et al. Enhanced liver autophagic activity improves survival of septic mice lacking surfactant proteins A and D. Tohoku J Exp Med 2013;231:127–38.
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
Immunolocalization of surfactant protein D in the liver from infants with cholestatic liver disease Ryuta Saka a,b,⁎, Itaru Yanagihara c, Takashi Sasaki a, Satoko Nose a, Makoto Takeuchi d, Masahiro Nakayama d, Hiroomi Okuyama a,b a
Department of Pediatric Surgery, Hyogo College of Medicine Department of Pediatric Surgery, Osaka University Graduate School of Medicine Department of Developmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health d Department of Pathology and Laboratory Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health b c
a r t i c l e
i n f o
Article history: Received 25 October 2014 Accepted 2 November 2014 Key words: Surfactant protein D Liver Cholestasis Biliary atresia
a b s t r a c t Purpose: Surfactant protein D (SP-D) is one of specific surfactant proteins constituting pulmonary surfactant. Recent studies have revealed that SP-D is detected in various non-pulmonary tissues and is involved in the host defense and immunomodulation. However, the relationship between SP-D and liver diseases has not yet been investigated. The aim of this study was to detect the immunolocalization of SP-D in the livers of infants with cholestatic liver disease. Methods: The expression of immunoreactive SP-D was assessed in infants with cholestasis, including biliary atresia (BA, n = 7), neonatal hepatitis (NH, n = 2), and paucity of the intrahepatic bile duct (PIBD, n = 4). Immunoreactive SP-D was also assessed in six infants who died of non-liver disease as controls. Tissue samples were obtained at liver biopsy, or by post-mortem sampling. The tissue sections were incubated with anti-SP-D polyclonal antibodies and were counterstained with hematoxylin. Results: In the normal livers, SP-D was detected in the intrahepatic bile ducts, but was not detected in hepatocytes. In contrast, intense SP-D staining was noted in the hepatocytes from infants with BA, NH, and PIBD. Although SP-D was detected in the intrahepatic bile ducts in the infants with NH, negative or weak staining was seen in the intrahepatic bile ducts in infants with BA. Conclusion: Our data showed that SP-D is present in the bile ducts of the normal infant liver, and it was found to accumulate in the hepatocytes of cholestatic livers. These results suggest that SP-D is produced in hepatocytes and is secreted into the bile ducts. © 2015 Elsevier Inc. All rights reserved.
Pulmonary surfactant is a complex of lipids and proteins. To date, four specific surfactant proteins (SPs) have been defined, named SP-A, SP-B, SP-C and SP-D. SP-D, as well as SP-A, has important roles in innate immunity. SP-D binds to various pathogenic microorganisms and works as an opsonin to enhance the uptake of these cells and particles by immune cells [1]. SP-D also regulates the cellular response by affecting the production of inflammatory mediators such as tumor necrosis factor-α (TNF α) via an interaction with toll-like receptor (TLR)-4. Although the expression of SP-A is limited to the lung in human [2], SP-D can be detected in many extrapulmonary organs, including the liver [3]. However, little is known about the role of extrapulmonary SP-D in humans [4]. Cholestatic liver disease in infants, which requires urgent diagnosis and treatment, results from various causes, and the incidence is approximately 1 in 2500 live births [5]. Biliary atresia (BA) is the most common ⁎ Corresponding author at: Department of Pediatric Surgery, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya-shi, Hyogo, Japan 6638501. Tel.: +81 798 45 6582; fax: +81 798 45 6581. E-mail address: [email protected] (R. Saka). http://dx.doi.org/10.1016/j.jpedsurg.2014.11.020 0022-3468/© 2015 Elsevier Inc. All rights reserved.
surgical cause of cholestatic liver disease in infants. Recurrent cholangitis is also one of the most important postoperative complications of BA, which may aggravate liver function. We hypothesized that SP-D may contribute to innate immunity in bile duct, and relate to the pathogenesis of postoperative cholangitis in BA. To the best of our knowledge, the relationship between SP-D and liver disease has not yet been investigated. The aim of this study was to clarify the distribution of SP-D in the hepatocytes and bile ducts of infants with cholestatic liver disease including BA, neonatal hepatitis (NH) and paucity of the intrahepatic bile duct (PIBD) and to compare it with that in normal control (NC) infants without liver disease. 1. Materials and methods This study was approved by the institutional committees of Hyogo College of Medicine and Osaka Medical Center for Maternal and Child Health. Specimens were collected during surgery and liver biopsy from infants with cholestatic liver diseases. Bile duct paucity was defined histologically, when the ratio of interlobular bile ducts to the number of portal tracts was b0.5 (normal range, 0.9–1.8) in at least
298
R. Saka et al. / Journal of Pediatric Surgery 50 (2015) 297–300
Table 1 Demographics of infants with cholestatic liver diseases. BA (n = 7) Age (day) T-Bil (mg/dl) D-Bil (mg/dl) AST (IU/L) ALT (IU/L)
62 (16–90) 7.8 (7.4–11.1) 4.9 (3.9–7.9) 150 (83–263) 58 (41–193)
NH (n = 2)
PIBD (n = 4)
63, 70
49 (24–68) 6.2 (5.4–8.5) 4.9 (3.5–5.6) 104 (84–123) 88 (54–103)
3.4, 9.0 1.5, 6.1 109, 110 70, 141
The data are expressed as the median (range), except for the NH group. BA: biliary atresia, NH: neonatal hepatitis, PIBD: paucity of intrahepatic bile duct.
five portal areas [6]. All the diagnosis of Paucity of Intrahepatic Bile Ducts (PIBD) was made by a pediatric pathologist (MN). As a control, fetal/neonatal autopsy cases without any liver dysfunction were used.
The immunoreactive hepatocytes were classified as follows: 0 (negative); 1 (rare periportal hepatocytes staining); 2 (multifocal to diffuse periportal staining); 3 (more diffuse staining extending into the lobules); 4 (staining of the majority of hepatocytes). The immunoreactivity of the lumens of the interlobular bile ducts and proliferating bile ductules (surface of biliary epithelial cells) was scored based on the number of positive structures present per portal tract as follows: 0 (absent); 1 (b25%); 2 (25%–50%); 3 (N50%). Then, the average scores were calculated for each slide. The interlobular bile ducts were recognized as biliary ductal elements in the central region of the portal tract with round and open lumina on hematoxylin and eosin (HE) staining. The proliferating bile ductules were recognized as biliary elements at the edge of the portal tract, which often had irregular lumina on HE staining. 1.3. Statistical analysis Patient demographics and the results of the immunohistological evaluation are expressed as the median (range). Scores were statistically analyzed using the Wilcoxon rank sum test. All statistical analyses were performed using the JMP®8 software package (SAS Institute Inc., Cary, NC). A P value b 0.05 was considered to be significant.
1.1. Immunohistochemistry 2. Results Formalin-fixed samples were embedded in paraffin and cut into 3 μm sections. After deparaffinization in xylene and rehydration in a graded series of ethanol, the sections were incubated with 0.3% H2O2 in methanol for 30 min to block the endogenous peroxidase activity. After three washes with PBS, the sections were incubated with Protein Block, Serum-Free (Dako, Glostrup, Denmark) to inhibit the non-specific staining for 30 min, and were washed with PBS. Then, the sections were incubated overnight at 4 °C with an anti-SP-D polyclonal Ab (1:1000, Chemicon #AB3434). After three washes with PBS, the sections were incubated with EnVision ™ + Rabbit/HRP (Dako) for 1 h at room temperature. The tissue sections were then incubated with DAB substrate (Nichirei Biosciences, Tokyo, Japan) for the chromogen reaction and were counterstained with hematoxylin. The slides were mounted with malinol (Muto Pure Chemicals, Tokyo, Japan) and visualized by light microscopy. 1.2. Evaluation of immunoreactivity Immunoreactivity of the samples was assessed using a semiquantitative histopathological score [7]. Immunostained sections were evaluated by two authors who were unaware of the diagnosis or clinical outcome.
2.1. Patients The age and the results of the liver function tests of the patients are shown in Table 1. As normal controls, a total of six autopsy cases (0–2 months old), without any liver diseases clinically and histologically, were evaluated. For the cholestasis patients, two had neonatal hepatitis (NH), four had PIBD and seven had BA. All cholestasis cases were younger than three months of age, and there were no significant differences in the age, bilirubin level (total and direct), AST and ALT among the cholestatic etiologies. 2.2. Immunohistochemistry In the normal controls (Fig. 1), the luminal surface of the bile duct was immunostained for SP-D, however, no intracellular immunostaining was found in the biliary epithelial cells. Hepatocytes showed almost no immunoreactivity. By contrast, in cholestatic livers (Figs. 2–4), the hepatocytes showed strong immunoreactivity, regardless of the disease. Although the luminal surface of the bile duct was strongly immunostained in the NH cases, the immunoreactivity of the proliferating bile
Fig. 1. Immunohistological findings of the normal controls (NC). Although the hepatocytes showed no immunoreactivity (A: original magnification ×100), the luminal surface of the intrahepatic bile duct (black arrow) was immunostained for SP-D (B: ×200, inset: ×400).
R. Saka et al. / Journal of Pediatric Surgery 50 (2015) 297–300
299
Fig. 2. The immunohistological findings of the patients with biliary atresia. Hepatocytes were positively and diffusely immunostained, and the proliferating bile ductules (black arrow) showed a variety of immunoreactivities, ranging from vague (A: original magnification ×200, inset: ×400) to clear (B: original magnification ×200, inset: ×400) in the luminal surface of the bile ductules.
Fig. 3. The immunohistological findings of the patients with neonatal hepatitis. (A) The hepatocytes were diffusely immunostained (original magnification ×100) (B) The luminal surface of the bile duct (black arrow) was immunostained (×200, inset: ×400).
duct in BA varied from weak to strong. In the PIBD cases, the bile duct could not be evaluated because the number of bile duct was too small. Statistical analyses of the immunohistological scores were performed only between NC and BA, because the other groups were too small (Fig. 5). In hepatocytes, the NC showed a significantly lower score than
the BA [0.25 (0–1.5) vs. 3.5 (3.0–4.0); P b 0.01]. In the bile duct, the BA cases showed a significantly lower score compared with the NC [1.5 (1.0–2.5) vs. 2.5 (2.0–3.0), P = 0.02). Although the statistical analysis could not be applied to NH and PIBD, NH and PIBD showed high hepatocytes score and NH showed high bile duct score.
Fig. 4. The immunohistological findings of the cases with a paucity of the intrahepatic bile duct. Although the bile duct could not be evaluated, hepatocytes showed strong immunoreactivity in diffuse. (A: original magnification ×100, B: ×200).
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Fig. 5. The immunohistological scores. The scores of hepatocytes (0–4) BA showed significantly higher scores than NC (P b 0.01). The scores of the bile ducts (0–3). BA showed lower scores in the bile ducts compared to NC (P = 0.02). NC: normal control, BA: biliary atresia.
3. Discussion Surfactant protein (SP)-D belongs to a subtype of mammalian lectins termed collectin, which is composed of an N-terminal cysteine-rich region, a collagen-like region, an alpha helical coiled neck region and a carbohydrate recognition domain. Collectin is known to have an important role in innate immunity, especially in the airway [8]. Although SP-A is almost exclusively localized in the airway [2], extrapulmonary expression of SP-D has been reported in humans [3]. Recently, the functions of SP-D, especially its contribution to innate immunity, have been investigated in the lung. SP-D enhances the uptake of pathogens by opsonizing them, functioning as a molecule that causes the activation of ligands and regulating cell surface receptor expression [1]. SP-D directly binds a variety of microorganisms, including bacteria (gram-positive/negative) and viruses, and functions as an opsonin to enhance their uptake [9]. SP-D also regulates the cellular response by immune cells. SP-D inhibits the binding of smooth and rough LPS, which is found in the membrane of gram-negative bacteria and elicits immune responses by immune cells, especially TLR4/MD-2 expressing cells, attenuates the MD-2 binding to LPS and inhibits LPS-induced inflammatory responses (TNF-α secretion and NF-κB activation) [10]. SP-D null mice showed increased lung inflammation and decreased macrophage phagocytosis during bacterial infection in the lungs [11]. Although the function of SP-D in the lungs has been relatively well investigated, its function in the liver has been unclear. Tang et al. reported a relationship between SP-D and autophagy in the liver [12], however, this study is one of only a few regarding the function of SP-D in the liver. We speculate that SP-D may contribute to innate immunity against pathogens, especially gram-negative bacteria, in the biliary system and bloodstream. To investigate the function and localization of SP-D in the liver, we compared cholestatic livers with normal control livers. In this study, immunoreactivity for SP-D was found in the luminal surface of bile duct, but not in the hepatocytes and biliary epithelial cells in the normal controls. In contrast, the hepatocytes showed strong immunoreactivity in the infants with cholestasis, regardless of the etiology. Interestingly, the BA cases showed relatively weak immunoreactivity in the luminal surface of the bile duct compared with the NC and other cholestatic patients. These results suggest that SP-D is produced in the hepatocytes and secreted into the bile juice in the normal liver. SP-D accumulation in the hepatocytes may reflect cholestasis; therefore, SP-D may be a possible clinical marker of cholestasis. By performing immunostaining for SP-D, it may be possible to diagnose the degree and location of cholestasis, and to clarify the pathogenesis of such cholestasis. The fact that SP-D, which is important in innate immunity, exists in the bile duct, implies that SP-D may play an important role in the postoperative cholangitis in BA, which is associated with the obstruction of
the secretion of bile juice, which includes SP-D. Postoperative cholangitis in BA patients after Kasai portoenterostomy is a troubling complication, which sometimes leads to the need for liver transplantation. The incidence of cholangitis is higher in BA cases than in those with other etiologies requiring hepaticojejunostomy, such as choledochal cysts. The cholangitis in BA patients after the Kasai procedure is due to intestinal flora, especially gram-negative bacteria. Considering the effect of SP-D against gram-negative bacteria, SP-D may have an important role against cholangitis in BA patients. The major limitation of this study is the small number of specimens of postoperative BA. We do not regularly perform liver biopsies after the Kasai operation but comparisons of liver specimens immunostained for SP-D between patients with recurrent cholangitis and those without cholangitis may further clarify the relationship between SP-D and cholangitis. Further investigations on the relationship between the clinical symptoms (jaundice, cholangitis and prognosis) and the localization of SP-D are also necessary. Acknowledgement This study was supported by research grants from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan. References [1] Wright JR. Immunoregulatory functions of surfactant proteins. Nat Rev Immunol 2005;5:58–68. [2] Madsen J, Tornoe I, Nielsen O, et al. Expression and localization of lung surfactant protein A in human tissues. Am J Respir Cell Mol Biol 2003;29:591–7. [3] Stahlman MT, Gray ME, Hull WM, et al. Immunolocalization of surfactant protein-D (SP-D) in human fetal, newborn, and adult tissues. J Histochem Cytochem 2002;50: 651–60. [4] Kurimura Y, Nishitani C, Ariki S, et al. Surfactant protein D inhibits adherence of uropathogenic Escherichia coli to the bladder epithelial cells and the bacteriuminduced cytotoxicity: a possible function in urinary tract. J Biol Chem 2012;287: 39578–88. [5] De Bruyne R, Van Biervliet S, Vande Velde S, et al. Clinical practice: neonatal cholestasis. Eur J Pediatr 2011;170:279–84. [6] Kocak N, Gurakan F, Yuce A, et al. Nonsyndromic paucity of interlobular bile ducts: clinical and laboratory findings of 10 cases. J Pediatr Gastroenterol Nutr 1997;24:44–8. [7] Ernst LM, Spinner NB, Piccoli DA, et al. Interlobular bile duct loss in pediatric cholestatic disease is associated with aberrant cytokeratin 7 expression by hepatocytes. Pediatr Dev Pathol 2007;10:383–90. [8] Sano H, Kuroki Y. The lung collectins, SP-A and SP-D, modulate pulmonary innate immunity. Mol Immunol 2005;42:279–87. [9] Kuroki Y, Takahashi M, Nishitani C. Pulmonary collectins in innate immunity of the lung. Cell Microbiol 2007;9:1871–9. [10] Yamazoe M, Nishitani C, Takahashi M, et al. Pulmonary surfactant protein D inhibits lipopolysaccharide (LPS)-induced inflammatory cell responses by altering LPS binding to its receptors. J Biol Chem 2008;283:35878–88. [11] LeVine AM, Whitsett JA, Gwozdz JA, et al. Distinct effects of surfactant protein A or D deficiency during bacterial infection on the lung. J Immunol 2000;165:3934–40. [12] Tang Z, Ni L, Javidiparsijani S, et al. Enhanced liver autophagic activity improves survival of septic mice lacking surfactant proteins A and D. Tohoku J Exp Med 2013;231:127–38.
