Journal of Immunological Methods, 141 (1991) 73-79 © 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 0022175991002300
73
JIM 05994
Mannan-binding protein in human liver N . G . R y l e y 1, A . R . H e r y e t 1, j. L u 2, K.B.M. R e i d 2 and K.A. F l e m i n g 1 1Nuffield Department of Pathology and Bacteriology, John Radcliffe Hospital, Headington, Oxford, U.K., and 2 MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, U.K. (Received 22 January 1991, revised received 22 March 1991, accepted 2 April 1991)
Mannan-binding protein (MBP) is a Ca2+-dependent lectin which was first described in 1978 in rabbit liver, and subsequently in serum and liver tissue from humans and a range of animal species. MBP structurally resembles Clq, and may act both as a focus for complement activation on the surface of microorganisms and as an opsonin in its own right. Low serum levels of serum MBP have been described in a group of children known to suffer from severe recurrent infections. MBP has also been reported to behave as an acute phase reactant. This preliminary study has investigated the localisation of MBP in human tissues using material obtained both at post mortem and from diagnostic liver biopsies. Using the IgG fraction of rabbit anti-human MBP, immunoperoxidase staining showed no evidence of significant MBP in a wide range of normal tissues, including liver taken both at post mortem and needle biopsy. However, there was a significant degree of staining for MBP in liver biopsies showing a variety of different pathologies, in particular severely damaged alcoholic livers, and those harbouring metastatic tumour. Moderate degrees of staining were also seen in liver biopsies from patients suffering from chronic biliary disease. It is uncertain whether this localisation of MBP in abnormal liver is an acute phase response, or represents a more fundamental link with liver disease. This question could be the focus for future studies. Key words: Mannan-binding protein; Liver; Complement; Lectin
Introduction Mannan-binding protein (MBP) is a Ca2+-de pendent lectin which was first described by Kawasaki, T., et al. (1978), who isolated it from rabbit liver, and which has now been isolated from human, bovine, rabbit and rat sera (Kozutsumi et al., 1980; Kawasaki, N., et al.,
Correspondence to: N.G. Ryley, Nuffield Department of Pathology, Level 4, Academic Centre, John Radcliffe Hospital, Headington, Oxford OX3 9DU, U.K.
1983, 1985; Oka et al., 1988). It has been shown to have a specificity for terminal non-reducing N-acetylglucosamine, mannose, fucose and glucose residues (Childs et al., 1989), and therefore has the potential to bind to these moieties on the cell wails of pathogenic gram negative bacteria, mycobacteria and yeasts. In serum it is present as a polydisperse mixture of mainly trimers, tetramers, pentamers and hexamers of an approximately 90 kDa structural unit (Lu et al., 1990). The 90 kDa structural unit is composed of three chains of 32 kOa, each chain being composed of three distinct sections: a short N terminal section of 20 residues; a collagen-like
74 region of 60 residues; and a C terminal domain of 150 residues (Drickamer et al., 1986; Ezekowitz et al., 1988; Taylor et al., 1989), which contains the framework of conserved amino acids characteristics of the 'C-type' carbohydrate binding domains of mammalian lectins (Drickamer et al., 1987). MBP has an overall structural similarity to Clq. The p e n t a m e r / h e x a m e r forms of MBP are able to interact with the C l r e C l s 2 proenzyme complex, and after binding to suitable carbohydrate structures, promote the formation of C1 esterase activity (Lu et al., 1990; Ohta et al., 1990). Selective concentration of MBP on the surface of microorganisms could act as a focus for complement activation through the classical pathway (Super et al., 1989). MBP may itself be opsonic, playing a role in the first line of defence against certain pathogenic microorganisms such as Salmonella spp. (Kuhlmann et al., 1989). It has also been found to prevent infection of H9 lyrephoblasts by H I V in vitro, by binding to the high mannose glycans expressed on the envelope glycoprotein of H I V (Ezekowitz et al., 1989). Low serum levels of MBP have been described in children whose sera are unable to opsonise bakers' yeast (Saccharomyces cereL'isiae), who are known to suffer from severe recurrent infections (Super et al., 1989). Purified MBP corrected this defect in a dose-dependent way in vitro. MBP has also been reported to behave as an acute phase reactant (Ezekowitz et al., 1988), although the normal serum level of MBP varies widely from 1 to 870 /zg/1, with 90% of the values lying between 2 and 1 6 0 / z g / l (Lu et al., 1990). MBP has been isolated from rabbit (Kawasaki et al., 1978), rodent (Mizuno et al., 1981) and human (Wild et al., 1983) liver, which has therefore been the presumed site of synthesis. In the rat more than 99% of the MBP activity recovered from whole livers was shown to be derived from hepatocytes (Mori et al., 1983). We undertook to investigate the localisation of MBP in human tissues, concentrating on liver biopsies from patients with a variety of hepatic abnormalities, but including normal liver biopsies and a range of other tissues, so as to ascertain whether synthesis may also occur at other sites, both in health and disease.
M a t e r i a l s and methods
Patients The liver sections were taken from diagnostic biopsies passing through the department. The average age of the patients was 54 (range 11-85), the sex ratio F : M = 1.4: 1. In some groups there were different proportions of female and male due to clinical features of the disease groupings (e.g., 12 of 15 were female in the chronic biliary disease group). The racial origin of the vast majority of the patients was Caucasian.
Tissues Small portions of fresh liver biopsies from a wide variety of patients were snap frozen and stored in liquid nitrogen, the rest of the tissue being processed routinely (paraffin sections) for diagnostic purposes. The tissue was frozen and stored within 30 min of the biopsy procedure. Control tissues from three post mortems were similarly frozen and stored (within 6 h of death). Frozen sections (5 ~ m ) were cut onto gelatine
TABLE I ALCOHOLIC LIVER DISEASE Anti-MBPstaining of liver biopsies showing alcoholic liver disease.'MBP grade' is strength of staining with anti-MBP on a scale of 0-3 (3 representing strong cytoplasmic staining). Case
Liver biopsy appearance
MBP grade
Case 8 Case 9 Case 10
Fatty change Fatty change Fatty change Fatty change
0 0 0/1 1
Case 11 Case 12
Hepatitis
(I
Case 13
Hepatitis
0/1
Case 14
Fat+fibrosis
Case 15 Case 16 Case 17 Case 18 Case 19 Case 20 Case 21 Case 22 Case 23 Case24 Case 25 Case 26 Case 27
Hepatitis + fibrosis Hepatitis + fibrosis Hepatitis+fibrosis Hepatitis+fibrosis Hepatitis+fibrosis
Cirrhosis Cirrhosis Cirrhosis Cirrhosis+ hepatitis Cirrhosis+hepatitis Cirrhosis+ hepatitis Cirrhosis+hepatitis
Cirrhosis+ hepatitis
1
2 2 2 2
2/3
1 2 3 0 1/2
2 3 3
75 TABLE II
cols. Briefly, the slides were p r e t r e a t e d with 10%
MALIGNANT DISEASE
n o r m a l swine s e r u m in T r i s / H C l p H 7.6. P r i m a r y
Anti-MBP staining of liver biopsies showing either primary or secondary malignant disease. 'MBP grade' is strength of staining with anti-MBP on a scale of 0-3 (3 representing strong cytoplasmic staining),
a n t i b o d y ( a n t i - M B P ) was a p p l i e d d i l u t e d in 10% n o r m a l swine s e r u m (in T r i s / H C 1 p H 7.6) at final
Case
Liver biopsy appearance
MBP grade
Case 28 Case 29 Case 30
Hepatocellular carcinoma Hepatocellular carcinoma Cholangiocarcinoma + large duct obstruction Cholangiocarcinoma + sclerosing cholangitis Metastatic adenocarcinoma Metastatic adenocarcinoma Metastatic adenocarcinoma Metastatic adenocarcinoma Metastatic adenocarcinoma
0 2
Case 31 Case 32 Case 33 Case 34 Case 35 Case 36
dilutions of 1 in 50 and 1 in 25. The secondary a n t i b o d y used was peroxidase labelled swine a n t i - r a b b i t i m m u n o g l o b u l i n s (Dako), the substrate b e i n g H z O z / d i a m i n o b e n z i d i n e . Two negative controls ((a) n o p r i m a r y antibody; (b) rabbit a n t i - h u m a n chorionic g o n a d o t r o p h i n (Dako) diluted 1 in 200 as p r i m a r y antibody) were i n c l u d e d o n each slide. O n e case k n o w n to be positive from p r e l i m i n a r y studies was i n c l u d e d with all the staining batches as a positive control.
1 2 1 2 2/3 2/3 3
Grading protocol
- c h r o m e a l u m coated m u l t i s p o t slides, a n d air dried for 2 - 1 8 h. Fixation was achieved by treatm e n t with a c e t o n e for 10 m i n at r o o m t e m p e r a ture.
G r a d i n g of staining density was assessed o n a scale of 0-3, grade 3 b e i n g d e f i n e d as the most i n t e n s e degree of staining (strong cytoplasmic stain) f o u n d within this sample of 71 liver biopsies. Direct c o m p a r i s o n s were m a d e b e t w e e n a n t i - M B P at 1 / 5 0 a n d the H C G control p r e s e n t o n each m u l t i s p o t slide.
Antibody T h e a n t i b o d y used was the I g G fraction (1.1 m g / m l in 70 m M p h o s p h a t e buffer at p H 7.4) of r a b b i t a n t i - h u m a n m a n n a n - b i n d i n g p r o t e i n , pre-
TABLE IV
p a r e d as described by L u et al. (1990).
CHRONIC BILIARY DISEASE
Staining S t a i n i n g was carried out by the indirect imm u n o p e r o x i d a s e t e c h n i q u e using s t a n d a r d proto-
Anti-MBP staining of liver biopsies from patients with chronic biliary disease (PBC = primary biliary cirrhosis, PSC = primary sclerosing cholangitis, SSC =secondary sclerosing cholangitis). 'MBP grade' is strength of staining with anti-MBP on a scale of 0-3 (3 representing strong cytoplasmic staining).
TABLE III
Case
Liver biopsy appearance
MBP grade
LARGE DUCT OBSTRUCTION
Case 42 Case 43 Case 44
?PBC ?PBC ?PBC
0 1 1
Case 45 Case 46 Case 47 Case 48 Case 49 Case 50 Case 51 Case 52 Case 53 Case 54 Case 55 Case 56
PBC stage 1 PBC stage 1 PBC stage 1/2 PBC stage 2 PBC stage 2 PBC stage 2 PBC stage 3 PBC stage 3 PSC PSC PSC ?PSC ?SSC
0/1 2 2 1 1 2 1 1/2 0 0 1 2
Anti-MBP staining in liver biopsies showing evidence of large duct obstruction. 'MBP grade' is strength of staining with anti-MBP on a scale of 0-3 (3 representing strong cytoplasmic staining). CBD = common bile duct. Case
Liver biopsy appearance
Case 37 Case 38
Large duct obstruction Largeduct obstruction (choledochal cyst) Largeduct obstruction Large duct obstruction Largeduct obstruction
Case 39 Case 40 Case 41
MBP
(CBD calculus)
grade 0 1
(CBD calculus) (CBD calculus) (CBD calculus)
2 2 1/2
76
Results
TABLE V MISCELLANEOUS
(1) T h r e e p o s t m o r t e m cases w e r e u s e d as ' n o r m a l ' c o n t r o l s ( m a l e 78 y e a r s d i e d o f m y o c a r dial infarction; f e m a l e 90 y e a r s d i e d o f b r o n c h o p -
Anti-MBP staining in a miscellaneous group of abnormal liver biopsies. 'MBP grade' is the strength of staining with anti-MBP on a scale of 0-3 (3 representing strong cytoplasmic staining).
n e u m o n i a ; m a l e 82 y e a r s d i e d with d i s s e c t i n g aortic aneurysm). Tissues s a m p l e d i n c l u d e d liver,
Case
Liver biopsy appearance
MBP grade
kidney, spleen, l y m p h - n o d e , p a n c r e a s , a d r e n a l , thyroid, p r o s t a t e , lung, h e a r t , s k e l e t a l muscle, aorta, o e s o p h a g u s , s t o m a c h , c e r e b r u m a n d c e r e bellum. N o n e s h o w e d e v i d e n c e o f significant staining with a n t i - M B P , i n c l u d i n g the liver. A
Case 57 Case58 Case 59 Case60 Case 61 Case 62 Case 63 Case 64 Case 65 Case 66 Case 67 Case 68 Case 69 Case 70 Case 71
Cholestasis (?drug, ?hepatitis C) Cholestasis(?hepatitis C) Cholestasis (?dextropropoxyphene) Cholestasis (?drug, ?calculi) Chronic active hepatitis(cryptogenic) Chronic active hepatitis (auto-immune) Fatty change (non specific) Fat and necrosis (?aetiology) Drug related hepatitis(methotrexate) Granulomatous hepatitis Granulomatons hepatitis Haemosiderosis Haemochromatosis with cirrhosis Fibrosis(?aetiology) Diabetic steatonecrosis (hepatitis)
1 1/2 2 2/3 2 2 0 2 1 1 1 0 l 1 2/3
very m i n o r d e g r e e of positive staining was n o t e d in the i n t e r s t i t i a l tissues o f the kidney, p a n c r e a s a n d a d r e n a l , a n d in t h e vessel l u m i n a (which c o n t a i n e d a fibrin m e s h ) o f the s p l e e n in case P M 2. A similar intensity of staining was n o t e d in t h e aortic wall of case P M 1. (2) T h e seven n o r m a l liver b i o p s i e s s t u d i e d s h o w e d no e v i d e n c e o f significant staining with
Fig. 1. Liver biopsy from 57-year-old alcoholic cirrhotic, lmmunoperoxidase staining of MBP in the cytoplas'm of hepatocytes (large arrow). Note contrast with unstained fibrous portal area adjacent to nodule (small arrow). Magnification 200 ×.
77
anti-MBP, with only an equivocal positive result (graded 0/1) being seen in two of the seven cases (cases 1-7). (3) Ten of the 20 cases showing alcoholic liver disease exhibited moderate to strong anti-MBP staining (grades 2 or 3), all of these being at the more severely damaged end of the alcoholic liver disease spectrum (Table 1). One of these cases (alcoholic cirrhotic) is illustrated in Fig. 1. (4) There was moderate to strong anti-MBP staining in four of the five cases taken from livers with metastatic disease (adenocarcinoma), the most prominent cytoplasmic staining being seen in hepatocyte areas immediately adjacent to the metastatic deposits in some of these cases. One of these cases is illustrated in Fig. 2. Three of the four cases of primary hepatic
!
.
.
malignancy showed a mild to moderate degree of staining (Table II). (5) The liver biopsies from patients with large duct obstruction (showing variable degrees of cholangitis and portal sclerosis) showed a mild to moderate degree of anti-MBP staining (grades 1-2) in four of the five cases (Table III). (6) 12 of the 15 cases of chronic biliary disease (primary biliary cirrhosis or primary sclerosing cholangitis) showed some degree of hepatocyte staining with anti-MBP, but none showed the intensity of staining seen in some of the other groups. There was no obvious relationship with severity of disease (Table IV). (7) In the group classified as 'miscellaneous', there were occasional cases showing moderate/ intense positive staining (one of the cases of
.
.
.
Fig. 2. Liver biopsy from 67-year-old man with metastatic colonic adenocarcinoma (immunoperoxidase staining for MBP on frozen section). Note contrast between strong positive staining for MBP in hepatocytes (large arrow), and lack of positive staining in tumour cells (small arrow). Magnification 420 ×.
78 cholestasis of unknown aetiology, and a case of diabetic hepatitis), but there were insufficient numbers in any of the disease categories to highlight any trend (Table V).
Discussion In this study we have attempted to address the question of tissue localisation of MBP, which has not been studied in a systematic manner previously. Initial attempts at staining fresh tissue revealed a number of biopsies positive for MBP in hepatocytes, but repeated attempts on the corresponding formalin fixed tissue were less successful, hence the use of fresh tissue throughout this study, Our survey of a wide range of fresh tissues from three post mortems showed very little in the way of positive staining, with only a small amount of staining of interstitial/connective tissue in a few organs (this feature may well represent circulating MBP deposited in the soft tissues). In particular there was no significant staining of the liver samples (there was no evidence of primary liver disease in any of the three cases). The normal liver biopsies (seven cases) showed only an equivocal positive result in two cases. Moderate or strong staining for MBP was found in all the different categories of liver disease studied. In alcoholic liver disease, the cells were more strongly stained in cases with severe inflammation and fibrosis/cirrhosis than in those with less marked pathological changes. This is an interesting observation in that it is well known that alcoholic liver disease predisposes to bacterial infection. The presence of increased amounts of MBP in the liver could be taken to suggest increased synthesis and possibly release of MBP, although by analogy with endocrine tumours and staining for hormones, presence within the cells of a substance does not necessarily imply release into the circulation. Bearing in mind the putative role for MBP in host defence, this might represent an attempted response (effective or not) to increased numbers of infections in those with severe liver disease/cirrhosis, or may just be part of an acute phase response (Taylor et al., 1989).
The biliary disease groups (large duct obstruction and P B C / P S C ) showed mild to moderate positive staining in 14 of 20 cases, though none showed the strong staining of some of the alcoholic cirrhotics. An unexpected finding was mode r a t e / dense staining in four of the five biopsies from the livers of patients with metastatic disease (the tumours themselves being negative), although the fifth showed less intense positive staining (grade 1). Whether this represents a specific response to metastatic deposits needs further investigation (together with concomitant serum MBP levels, see later). It is known that some patients with severe extra-hepatic inflammatory processes (such as rheumatoid arthritis) have high levels of MBP serologically (Reid, unpublished observations). It is of interest that although a few of these were included in the normal and miscellaneous groups of liver pathology, they were not found to have particularly high levels of staining. Although the number of cases in our study is small, the results indicate that: (1) MBP is not detectable to any significant degree by immunocytochemistry in normal liver or in any other tissue examined. In contrast, diseased liver tissue often shows increased MBP. (2) When positive staining is seen in liver tissue, it is in the form of diffuse cytoplasmic staining within hepatocytes, with some groups of cells being more positive than others within the small amount of tissue included in liver biopsies (i.e., some focality, but no obvious relationship to acinar architecture). (3) Cases with alcoholic cirrhosis or severe fibrosis/hepatitis tend to show marked MBP staining. (4) Livers affected by metastatic disease also show strong staining for MBP within hepatocytes, particularly those immediately adjacent to the tumour. Comparison of results for liver MBP staining with serum MBP levels from the same patients would give further information as to whether the presence of detectable MBP in abnormal liver is simply an acute phase response (such as with a I anti-trypsin), or whether there is a more fundamental link with hepatic pathology.
79
References Childs, R.A., Drickamer, K., Kawasaki, T., Thiel, S., Mizouchi, T. and Feizi, T. (•989) Neoglycolipids as probes of oligosaccharide recognition by recombinant and natural mannose-binding proteins of rat and man. Biochem. J. 262, 131. Drickamer, K., Dordal, M.S. and Reynolds, L. (1986) Mannose-binding proteins isolated from rat liver contain carbohydrate-recognition domains/inked to collagenous tails. Complete primary structures and homology with pulmonary surfactant apoprotein. J. Biol. Chem. 26•, 6878. Drickamer, K. (1987) Membrane receptors that mediate glycoprotein endocytosis: structure and biosynthesis. Kidney Int. 23 (suppl.), s167. Ezekowitz, R.A.B., Day, L.E. and Herman, G.E. (1988) A human mannose-binding protein is an acute-phase reactant that shares sequence homology with other vertebrate lectins. J. Exp. Med. 167, 1034. Ezekowitz, R.A.B., Kuhlman, M., Groopman, J. and Byrn, R. (1989) A human serum mannose-binding protein inhibits in vitro infection by the human immunodeficiency virus. J. Exp. Med. 169, 185. Kawasaki, N., Kawasaki, T. and Yamashura, I. (1983) Isolation and characterisation of a mannose-binding protein from human serum. J. Biochem. 94, 937. Kawasaki, N., Kawasaki, T. and Yamashura, I. (1985) Mannan-binding protein and couglutinin in bovine serum. J. Biochem. 98, 1309. Kawasaki, T., Eton, R. and Yamashura, I. (1978) Isolation and characterisation of a mannose-binding protein from rabbit liver. Biochem. Biophys. Res. Commun. 81, 1018. Kozutsumi, Y., Kawasaki, T. and Yamashina, T. (1981) Kinetical properties of the serum mannan-binding protein from rabbit. A comparison with those of the liver mannan-binding protein. J. Biochem. 90, 1799.
Kuhlman, M., Joiner, K. and Ezekowitz, R.A.B. (1989) The human mannose-binding protein functions as an opsonin. J. Exp. Med. 169, 1733. Lu, J., Thiel, S., Wiedemann, H.,. Timpl, R. and Reid, K.B.M. (1990) Binding of the pentamer/hexamer forms of mannan-binding protein to zymosan activates the proenzyme Clr2Cls 2 complex of the classical pathway of complement, without involvement of Clq. J. Immunol. 144, 2287. Mizuno, Y., Kotzutsumi, Y., Kawasaki, T. and Yamashina, I. (1981) Isolation and characterisation of a mannose-binding protein from a rat liver. J. Biol. Chem. 256, 4247. Mori, K., Kawasaki, T. and Yamashina, I. (1983) Identification of the mannan-binding protein from rat livers as a hepatocyte protein distinct from the mannan receptor on sinusoidal ceils. Arch. Biochem. Biophys. 222, 542. Obta, M., Okada, M., Yamashina, I. and Kawasaki, T. (1990) The mechanism of carbohydrate mediated complement activation by the serum mannan binding protein. J. Biol. Chem. 265, 1980. Oka, S., Ikeda, K., Kawasaki, T. and Yamashina, I. (1988) Isolation and characterization of two distinct mannanbinding proteins from rat serum. Arch. Biochem. Biophys. 260, 257. Super, M., Thiel, S., Lu, J., Levinsky, R.J. and Turner, M.W. (1989)Association of low levels of mannan-binding protein with a common defect of opsonisation. Lancet, 1236. Taylor, M.E., Brickel, P.M., Craig, R.K. and Summerfield, J.A. (1989) Structure and evolutionary origin of the gene encoding a human serum mannose-binding protein. Biochem. J. 262, 763. Wild, J., Robinson, D. and Winchester, B. (1983) Isolation of mannose-binding proteins from human liver. Biochem. J. 210, 167.
73
JIM 05994
Mannan-binding protein in human liver N . G . R y l e y 1, A . R . H e r y e t 1, j. L u 2, K.B.M. R e i d 2 and K.A. F l e m i n g 1 1Nuffield Department of Pathology and Bacteriology, John Radcliffe Hospital, Headington, Oxford, U.K., and 2 MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, U.K. (Received 22 January 1991, revised received 22 March 1991, accepted 2 April 1991)
Mannan-binding protein (MBP) is a Ca2+-dependent lectin which was first described in 1978 in rabbit liver, and subsequently in serum and liver tissue from humans and a range of animal species. MBP structurally resembles Clq, and may act both as a focus for complement activation on the surface of microorganisms and as an opsonin in its own right. Low serum levels of serum MBP have been described in a group of children known to suffer from severe recurrent infections. MBP has also been reported to behave as an acute phase reactant. This preliminary study has investigated the localisation of MBP in human tissues using material obtained both at post mortem and from diagnostic liver biopsies. Using the IgG fraction of rabbit anti-human MBP, immunoperoxidase staining showed no evidence of significant MBP in a wide range of normal tissues, including liver taken both at post mortem and needle biopsy. However, there was a significant degree of staining for MBP in liver biopsies showing a variety of different pathologies, in particular severely damaged alcoholic livers, and those harbouring metastatic tumour. Moderate degrees of staining were also seen in liver biopsies from patients suffering from chronic biliary disease. It is uncertain whether this localisation of MBP in abnormal liver is an acute phase response, or represents a more fundamental link with liver disease. This question could be the focus for future studies. Key words: Mannan-binding protein; Liver; Complement; Lectin
Introduction Mannan-binding protein (MBP) is a Ca2+-de pendent lectin which was first described by Kawasaki, T., et al. (1978), who isolated it from rabbit liver, and which has now been isolated from human, bovine, rabbit and rat sera (Kozutsumi et al., 1980; Kawasaki, N., et al.,
Correspondence to: N.G. Ryley, Nuffield Department of Pathology, Level 4, Academic Centre, John Radcliffe Hospital, Headington, Oxford OX3 9DU, U.K.
1983, 1985; Oka et al., 1988). It has been shown to have a specificity for terminal non-reducing N-acetylglucosamine, mannose, fucose and glucose residues (Childs et al., 1989), and therefore has the potential to bind to these moieties on the cell wails of pathogenic gram negative bacteria, mycobacteria and yeasts. In serum it is present as a polydisperse mixture of mainly trimers, tetramers, pentamers and hexamers of an approximately 90 kDa structural unit (Lu et al., 1990). The 90 kDa structural unit is composed of three chains of 32 kOa, each chain being composed of three distinct sections: a short N terminal section of 20 residues; a collagen-like
74 region of 60 residues; and a C terminal domain of 150 residues (Drickamer et al., 1986; Ezekowitz et al., 1988; Taylor et al., 1989), which contains the framework of conserved amino acids characteristics of the 'C-type' carbohydrate binding domains of mammalian lectins (Drickamer et al., 1987). MBP has an overall structural similarity to Clq. The p e n t a m e r / h e x a m e r forms of MBP are able to interact with the C l r e C l s 2 proenzyme complex, and after binding to suitable carbohydrate structures, promote the formation of C1 esterase activity (Lu et al., 1990; Ohta et al., 1990). Selective concentration of MBP on the surface of microorganisms could act as a focus for complement activation through the classical pathway (Super et al., 1989). MBP may itself be opsonic, playing a role in the first line of defence against certain pathogenic microorganisms such as Salmonella spp. (Kuhlmann et al., 1989). It has also been found to prevent infection of H9 lyrephoblasts by H I V in vitro, by binding to the high mannose glycans expressed on the envelope glycoprotein of H I V (Ezekowitz et al., 1989). Low serum levels of MBP have been described in children whose sera are unable to opsonise bakers' yeast (Saccharomyces cereL'isiae), who are known to suffer from severe recurrent infections (Super et al., 1989). Purified MBP corrected this defect in a dose-dependent way in vitro. MBP has also been reported to behave as an acute phase reactant (Ezekowitz et al., 1988), although the normal serum level of MBP varies widely from 1 to 870 /zg/1, with 90% of the values lying between 2 and 1 6 0 / z g / l (Lu et al., 1990). MBP has been isolated from rabbit (Kawasaki et al., 1978), rodent (Mizuno et al., 1981) and human (Wild et al., 1983) liver, which has therefore been the presumed site of synthesis. In the rat more than 99% of the MBP activity recovered from whole livers was shown to be derived from hepatocytes (Mori et al., 1983). We undertook to investigate the localisation of MBP in human tissues, concentrating on liver biopsies from patients with a variety of hepatic abnormalities, but including normal liver biopsies and a range of other tissues, so as to ascertain whether synthesis may also occur at other sites, both in health and disease.
M a t e r i a l s and methods
Patients The liver sections were taken from diagnostic biopsies passing through the department. The average age of the patients was 54 (range 11-85), the sex ratio F : M = 1.4: 1. In some groups there were different proportions of female and male due to clinical features of the disease groupings (e.g., 12 of 15 were female in the chronic biliary disease group). The racial origin of the vast majority of the patients was Caucasian.
Tissues Small portions of fresh liver biopsies from a wide variety of patients were snap frozen and stored in liquid nitrogen, the rest of the tissue being processed routinely (paraffin sections) for diagnostic purposes. The tissue was frozen and stored within 30 min of the biopsy procedure. Control tissues from three post mortems were similarly frozen and stored (within 6 h of death). Frozen sections (5 ~ m ) were cut onto gelatine
TABLE I ALCOHOLIC LIVER DISEASE Anti-MBPstaining of liver biopsies showing alcoholic liver disease.'MBP grade' is strength of staining with anti-MBP on a scale of 0-3 (3 representing strong cytoplasmic staining). Case
Liver biopsy appearance
MBP grade
Case 8 Case 9 Case 10
Fatty change Fatty change Fatty change Fatty change
0 0 0/1 1
Case 11 Case 12
Hepatitis
(I
Case 13
Hepatitis
0/1
Case 14
Fat+fibrosis
Case 15 Case 16 Case 17 Case 18 Case 19 Case 20 Case 21 Case 22 Case 23 Case24 Case 25 Case 26 Case 27
Hepatitis + fibrosis Hepatitis + fibrosis Hepatitis+fibrosis Hepatitis+fibrosis Hepatitis+fibrosis
Cirrhosis Cirrhosis Cirrhosis Cirrhosis+ hepatitis Cirrhosis+hepatitis Cirrhosis+ hepatitis Cirrhosis+hepatitis
Cirrhosis+ hepatitis
1
2 2 2 2
2/3
1 2 3 0 1/2
2 3 3
75 TABLE II
cols. Briefly, the slides were p r e t r e a t e d with 10%
MALIGNANT DISEASE
n o r m a l swine s e r u m in T r i s / H C l p H 7.6. P r i m a r y
Anti-MBP staining of liver biopsies showing either primary or secondary malignant disease. 'MBP grade' is strength of staining with anti-MBP on a scale of 0-3 (3 representing strong cytoplasmic staining),
a n t i b o d y ( a n t i - M B P ) was a p p l i e d d i l u t e d in 10% n o r m a l swine s e r u m (in T r i s / H C 1 p H 7.6) at final
Case
Liver biopsy appearance
MBP grade
Case 28 Case 29 Case 30
Hepatocellular carcinoma Hepatocellular carcinoma Cholangiocarcinoma + large duct obstruction Cholangiocarcinoma + sclerosing cholangitis Metastatic adenocarcinoma Metastatic adenocarcinoma Metastatic adenocarcinoma Metastatic adenocarcinoma Metastatic adenocarcinoma
0 2
Case 31 Case 32 Case 33 Case 34 Case 35 Case 36
dilutions of 1 in 50 and 1 in 25. The secondary a n t i b o d y used was peroxidase labelled swine a n t i - r a b b i t i m m u n o g l o b u l i n s (Dako), the substrate b e i n g H z O z / d i a m i n o b e n z i d i n e . Two negative controls ((a) n o p r i m a r y antibody; (b) rabbit a n t i - h u m a n chorionic g o n a d o t r o p h i n (Dako) diluted 1 in 200 as p r i m a r y antibody) were i n c l u d e d o n each slide. O n e case k n o w n to be positive from p r e l i m i n a r y studies was i n c l u d e d with all the staining batches as a positive control.
1 2 1 2 2/3 2/3 3
Grading protocol
- c h r o m e a l u m coated m u l t i s p o t slides, a n d air dried for 2 - 1 8 h. Fixation was achieved by treatm e n t with a c e t o n e for 10 m i n at r o o m t e m p e r a ture.
G r a d i n g of staining density was assessed o n a scale of 0-3, grade 3 b e i n g d e f i n e d as the most i n t e n s e degree of staining (strong cytoplasmic stain) f o u n d within this sample of 71 liver biopsies. Direct c o m p a r i s o n s were m a d e b e t w e e n a n t i - M B P at 1 / 5 0 a n d the H C G control p r e s e n t o n each m u l t i s p o t slide.
Antibody T h e a n t i b o d y used was the I g G fraction (1.1 m g / m l in 70 m M p h o s p h a t e buffer at p H 7.4) of r a b b i t a n t i - h u m a n m a n n a n - b i n d i n g p r o t e i n , pre-
TABLE IV
p a r e d as described by L u et al. (1990).
CHRONIC BILIARY DISEASE
Staining S t a i n i n g was carried out by the indirect imm u n o p e r o x i d a s e t e c h n i q u e using s t a n d a r d proto-
Anti-MBP staining of liver biopsies from patients with chronic biliary disease (PBC = primary biliary cirrhosis, PSC = primary sclerosing cholangitis, SSC =secondary sclerosing cholangitis). 'MBP grade' is strength of staining with anti-MBP on a scale of 0-3 (3 representing strong cytoplasmic staining).
TABLE III
Case
Liver biopsy appearance
MBP grade
LARGE DUCT OBSTRUCTION
Case 42 Case 43 Case 44
?PBC ?PBC ?PBC
0 1 1
Case 45 Case 46 Case 47 Case 48 Case 49 Case 50 Case 51 Case 52 Case 53 Case 54 Case 55 Case 56
PBC stage 1 PBC stage 1 PBC stage 1/2 PBC stage 2 PBC stage 2 PBC stage 2 PBC stage 3 PBC stage 3 PSC PSC PSC ?PSC ?SSC
0/1 2 2 1 1 2 1 1/2 0 0 1 2
Anti-MBP staining in liver biopsies showing evidence of large duct obstruction. 'MBP grade' is strength of staining with anti-MBP on a scale of 0-3 (3 representing strong cytoplasmic staining). CBD = common bile duct. Case
Liver biopsy appearance
Case 37 Case 38
Large duct obstruction Largeduct obstruction (choledochal cyst) Largeduct obstruction Large duct obstruction Largeduct obstruction
Case 39 Case 40 Case 41
MBP
(CBD calculus)
grade 0 1
(CBD calculus) (CBD calculus) (CBD calculus)
2 2 1/2
76
Results
TABLE V MISCELLANEOUS
(1) T h r e e p o s t m o r t e m cases w e r e u s e d as ' n o r m a l ' c o n t r o l s ( m a l e 78 y e a r s d i e d o f m y o c a r dial infarction; f e m a l e 90 y e a r s d i e d o f b r o n c h o p -
Anti-MBP staining in a miscellaneous group of abnormal liver biopsies. 'MBP grade' is the strength of staining with anti-MBP on a scale of 0-3 (3 representing strong cytoplasmic staining).
n e u m o n i a ; m a l e 82 y e a r s d i e d with d i s s e c t i n g aortic aneurysm). Tissues s a m p l e d i n c l u d e d liver,
Case
Liver biopsy appearance
MBP grade
kidney, spleen, l y m p h - n o d e , p a n c r e a s , a d r e n a l , thyroid, p r o s t a t e , lung, h e a r t , s k e l e t a l muscle, aorta, o e s o p h a g u s , s t o m a c h , c e r e b r u m a n d c e r e bellum. N o n e s h o w e d e v i d e n c e o f significant staining with a n t i - M B P , i n c l u d i n g the liver. A
Case 57 Case58 Case 59 Case60 Case 61 Case 62 Case 63 Case 64 Case 65 Case 66 Case 67 Case 68 Case 69 Case 70 Case 71
Cholestasis (?drug, ?hepatitis C) Cholestasis(?hepatitis C) Cholestasis (?dextropropoxyphene) Cholestasis (?drug, ?calculi) Chronic active hepatitis(cryptogenic) Chronic active hepatitis (auto-immune) Fatty change (non specific) Fat and necrosis (?aetiology) Drug related hepatitis(methotrexate) Granulomatous hepatitis Granulomatons hepatitis Haemosiderosis Haemochromatosis with cirrhosis Fibrosis(?aetiology) Diabetic steatonecrosis (hepatitis)
1 1/2 2 2/3 2 2 0 2 1 1 1 0 l 1 2/3
very m i n o r d e g r e e of positive staining was n o t e d in the i n t e r s t i t i a l tissues o f the kidney, p a n c r e a s a n d a d r e n a l , a n d in t h e vessel l u m i n a (which c o n t a i n e d a fibrin m e s h ) o f the s p l e e n in case P M 2. A similar intensity of staining was n o t e d in t h e aortic wall of case P M 1. (2) T h e seven n o r m a l liver b i o p s i e s s t u d i e d s h o w e d no e v i d e n c e o f significant staining with
Fig. 1. Liver biopsy from 57-year-old alcoholic cirrhotic, lmmunoperoxidase staining of MBP in the cytoplas'm of hepatocytes (large arrow). Note contrast with unstained fibrous portal area adjacent to nodule (small arrow). Magnification 200 ×.
77
anti-MBP, with only an equivocal positive result (graded 0/1) being seen in two of the seven cases (cases 1-7). (3) Ten of the 20 cases showing alcoholic liver disease exhibited moderate to strong anti-MBP staining (grades 2 or 3), all of these being at the more severely damaged end of the alcoholic liver disease spectrum (Table 1). One of these cases (alcoholic cirrhotic) is illustrated in Fig. 1. (4) There was moderate to strong anti-MBP staining in four of the five cases taken from livers with metastatic disease (adenocarcinoma), the most prominent cytoplasmic staining being seen in hepatocyte areas immediately adjacent to the metastatic deposits in some of these cases. One of these cases is illustrated in Fig. 2. Three of the four cases of primary hepatic
!
.
.
malignancy showed a mild to moderate degree of staining (Table II). (5) The liver biopsies from patients with large duct obstruction (showing variable degrees of cholangitis and portal sclerosis) showed a mild to moderate degree of anti-MBP staining (grades 1-2) in four of the five cases (Table III). (6) 12 of the 15 cases of chronic biliary disease (primary biliary cirrhosis or primary sclerosing cholangitis) showed some degree of hepatocyte staining with anti-MBP, but none showed the intensity of staining seen in some of the other groups. There was no obvious relationship with severity of disease (Table IV). (7) In the group classified as 'miscellaneous', there were occasional cases showing moderate/ intense positive staining (one of the cases of
.
.
.
Fig. 2. Liver biopsy from 67-year-old man with metastatic colonic adenocarcinoma (immunoperoxidase staining for MBP on frozen section). Note contrast between strong positive staining for MBP in hepatocytes (large arrow), and lack of positive staining in tumour cells (small arrow). Magnification 420 ×.
78 cholestasis of unknown aetiology, and a case of diabetic hepatitis), but there were insufficient numbers in any of the disease categories to highlight any trend (Table V).
Discussion In this study we have attempted to address the question of tissue localisation of MBP, which has not been studied in a systematic manner previously. Initial attempts at staining fresh tissue revealed a number of biopsies positive for MBP in hepatocytes, but repeated attempts on the corresponding formalin fixed tissue were less successful, hence the use of fresh tissue throughout this study, Our survey of a wide range of fresh tissues from three post mortems showed very little in the way of positive staining, with only a small amount of staining of interstitial/connective tissue in a few organs (this feature may well represent circulating MBP deposited in the soft tissues). In particular there was no significant staining of the liver samples (there was no evidence of primary liver disease in any of the three cases). The normal liver biopsies (seven cases) showed only an equivocal positive result in two cases. Moderate or strong staining for MBP was found in all the different categories of liver disease studied. In alcoholic liver disease, the cells were more strongly stained in cases with severe inflammation and fibrosis/cirrhosis than in those with less marked pathological changes. This is an interesting observation in that it is well known that alcoholic liver disease predisposes to bacterial infection. The presence of increased amounts of MBP in the liver could be taken to suggest increased synthesis and possibly release of MBP, although by analogy with endocrine tumours and staining for hormones, presence within the cells of a substance does not necessarily imply release into the circulation. Bearing in mind the putative role for MBP in host defence, this might represent an attempted response (effective or not) to increased numbers of infections in those with severe liver disease/cirrhosis, or may just be part of an acute phase response (Taylor et al., 1989).
The biliary disease groups (large duct obstruction and P B C / P S C ) showed mild to moderate positive staining in 14 of 20 cases, though none showed the strong staining of some of the alcoholic cirrhotics. An unexpected finding was mode r a t e / dense staining in four of the five biopsies from the livers of patients with metastatic disease (the tumours themselves being negative), although the fifth showed less intense positive staining (grade 1). Whether this represents a specific response to metastatic deposits needs further investigation (together with concomitant serum MBP levels, see later). It is known that some patients with severe extra-hepatic inflammatory processes (such as rheumatoid arthritis) have high levels of MBP serologically (Reid, unpublished observations). It is of interest that although a few of these were included in the normal and miscellaneous groups of liver pathology, they were not found to have particularly high levels of staining. Although the number of cases in our study is small, the results indicate that: (1) MBP is not detectable to any significant degree by immunocytochemistry in normal liver or in any other tissue examined. In contrast, diseased liver tissue often shows increased MBP. (2) When positive staining is seen in liver tissue, it is in the form of diffuse cytoplasmic staining within hepatocytes, with some groups of cells being more positive than others within the small amount of tissue included in liver biopsies (i.e., some focality, but no obvious relationship to acinar architecture). (3) Cases with alcoholic cirrhosis or severe fibrosis/hepatitis tend to show marked MBP staining. (4) Livers affected by metastatic disease also show strong staining for MBP within hepatocytes, particularly those immediately adjacent to the tumour. Comparison of results for liver MBP staining with serum MBP levels from the same patients would give further information as to whether the presence of detectable MBP in abnormal liver is simply an acute phase response (such as with a I anti-trypsin), or whether there is a more fundamental link with hepatic pathology.
79
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