Hepatic sinusoidal endothelial cells can store and metabolize serum immunoglobulin

Hepatic Sinusoidal Endothelial Cells Can Store and Metabolize Serum Immunoglobulin SHINICHI

IWAMURA,~ HIDEAKI

ENZAN, 2 TOSHIJI SAIBARA, ~ SABURO ONISHI, ~ AND YASUTAKE YAMAMOTO 1

Sinusoidal i n c l u s i o n - c o n t a i n i n g e n d o t h e l i a l cells in t h e liver w e r e i n v e s t i g a t e d w i t h particular interest in their c a p a c i t y o f m e t a b o l i z i n g i m m u n o g l o b u l i n . Formalin-fixed deparaffinized liver s p e c i m e n s w e r e u s e d for i m m u n o h i s t o c h e m i s t r y , a n d p r o n a s e digestion w a s p r o v e d to be effective for a n t i g e n retrieval of i m m u n o globulin. The i n c l u s i o n s in s i n u s o i d a l e n d o t h e l i a l cells w e r e strongly i m m u n o s t a i n e d w i t h a n t i - i m m u n o g l o b u lin (Ig)G, IgA, a n d IgM a n t i b o d i e s in p r e d i g e s t e d sections. The c o m p l e m e n t s w e r e n o t identified i m m u n o h i s t o c h e m i c a l l y in the i n c l u s i o n s e v e n after p r o n a s e treatment. T w o w o m e n w i t h a u t o i m m u n e liver disease, w h o initially r e p r e s e n t e d high levels o f s e r u m 3/globulin a n d a b u n d a n t i n c l u s i o n - c o n t a i n i n g e n d o t h e l i a l cells, w e r e studied. The s u b s e q u e n t biopsy after effective corticosteroid t h e r a p y d e m o n s t r a t e d significant histological i m p r o v e m e n t as well as t h e d i s a p p e a r a n c e of inclus i o n - c o n t a i n i n g e n d o t h e l i a l cells (ICECs). D u r i n g a n d after treatment, their s e r u m 3/globulin levels w e r e drastically reduced. In c o n c l u s i o n , t h e h e p a t i c sinusoidal endothelial cells c a n t a k e up s e r u m i m m u n o g l o b u l i n , probably t h r o u g h a r e c e p t o r - m e d i a t e d p a t h w a y , a n d its e x c e s s i v e storage results in the f o r m a t i o n o f c y t o p l a s m i c i n c l u s i o n s that are easily r e c o g n i z e d by a light microscope. The stored i m m u n o g l o b u l i n m a y be d e g r a d e d in t h e cytoplasm, a n d t h e i n c l u s i o n s w o u l d disappear in a s s o c i a t i o n w i t h the r e d u c t i o n o f s i n u s o i d a l 3/globulin content. In o t h e r words, t h e intralobular d e n s i t y o f inc l u s i o n - c o n t a i n i n g e n d o t h e l i a l cells is a m o r p h o l o g i c a l predictor for t h e s e r u m ~/globulin level. (HEPATOLOGY 1995;22:1456-1461.)

The liver plays an important role in the uptake and clearance of circulating micromolecules and macromolecules. Although liver parenchymal cells are widely involved in maintaining homeostasis in the body, nonparenchymal cells are also known to have their unique functions.

Abbreviations: SEC, sinusoidal endothelial cells; Ig, immunoglobulin; FcR, receptors for Fc portion of IgG; ICEC, inclusion-containing endothelial cell; PBC, primary biliary cirrhosis; PBS, phosphate-buffered saline; ALT, alanine transaminase. From the 1First Department of Internal Medicine and the 2First Department of Pathology, Kochi Medical School, Okoh-cho, Nankoku, Kochi, Japan. Received March 6, 1995; accepted July 10, 1995. Address reprint requests to: Hideaki Enzan, MD, First Department of Pathology, Kochi Medical School, Okoh-cho, Nankoku, Kochi 783, Japan. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2205-001853.00/0

For instance, the Ito cells, also known as fat-storing cells, play a central part in storage and metabolism of vitamin A (retinoids) 1'2 as well as collagen synthesis in hepatic fibrosis. 3-5 Dietary retinoids are initially absorbed by intestinal mucosal cells and endocytosed as chylomicron remnants by liver parenchymal cells, and then transferred to cytoplasmic small fat droplets of Ito cells. 1'2 Kupffer cells act as liver resident macrophages and demonstrate many immunologic functions, including phagocytosis, antigen presentation, and cytokine release. Sinusoidal endothelial cells (SECs) are known to express receptors for the Fc portion of immunoglobulin G (IgG) (FcR) 6'7 and are thought to be responsible for removing soluble immune complex like Kupffer cells. 8'9 In cirrhotic liver, FcR is reported to decrease in number, and impaired clearance of immune complex is also discussed. 1°'1~ Hyaluronic acid/chondroitin sulfate proteoglycans are also mainly removed by the SECs from the circulation. ~2 The SEC may possibly be involved in some other unknown functions, considering its structural characteristics. We have recently demonstrated sinusoidal inclusioncontaining endothelial cell (ICEC), characterized by the cytoplasmic eosinophilic granules, in h u m a n livers biopsy specimens. 18Because the serum T globulin level tended to increase in relation to the density of the ICEC, we have considered that ICECs may be involved in the metabolism of serum immunoglobulin, despite negative immunoreactivity with anti-immunoglobulins using conventional immunohistochemistry. In the current study, we have reevaluated immunohistochemical findings on the ICEC with an antigen retrieval technique, to confirm our speculation on its pathological significance. In addition, two patients with autoimmune liver disease who initially represented abundant ICECs were presented, and the alteration of the density of ICEC was discussed in relationship with the serum T globulin level. PATIENTS AND METHODS P a t i e n t s . A total 0f 47 needle liver bi0psy specimens, representing 3+ ICEC positivity score, 13 were studied. Of these specimens, there were 16 cases with hepatitis B surface antigen-positive chronic hepatitis, 28 cases with hepatitis C virus antibody-positive chronic hepatitis, one case with autoimmune hepatitis, one case with primary biliary cirrhosis

1456

HEPATOLOGYVol. 22, No. 5, 1995 (PBC) and one case with chronic active hepatitis-PBC mixed type. 14,15The histological grading and staging of chronic hepatitis was based on the classification described by Desmet et al. TM Among the patients, five hepatitis B surface antigen-positive chronic hepatitis and five hepatitis C virus antibodypositive chronic hepatitis eases that demonstrated extremely abundant ICECs were further selected and examined for immunohistochemistry. Two eases of autoimmune liver disease were specially focused on the appearance of ICECs in relation to the serum T globulin level. Their liver histology were examined before and after corticosteroid therapy. Tissue Preparation. Liver biopsy tissues were routinely fixed in 10% neutralized formaldehyde and processed for paraffinized sections. Hematoxylin-eosin stain was applied for determination of ICEC density. The additional other staining methods including reticulin silver impregnation, Azan-Mallory, periodic acid-Schiff, Berlin blue, and Victoria blue stains were applied for histological diagnosis. Immunohistochemistry. Immunostaining was applied to deparaffinized 4-#m thick sections. Polyclonal antibodies (rabbit anti-human IgG, IgM, IgA, C3, C4, albumin, transferrin) and monoclonal antibodies (anti-K and k light chain) were purchased from DAKO, Glostrup, Denmark. Monoclonal antibodies against FcR-T II (CDw32) and FcR-T III (CD16) were purchased from Immunotech (Boston, MA) and Becton Dickinson (Mountain View, CA), respectively. Deparaffinized sections were primarily digested for antigen-unmasking by 0.1% pronase-E (Sigma, St Louis, MO) dissolved in 0.01 mol/L phosphate-buffered saline (PBS, pH 7.4) for 20 minutes at 37°C. After inactivation of endogenous peroxidase, the sections were incubated for 1 hour at room temperature on the slide glass with the primary antibody, at a 100-fold dilution for anti-C3, C4, albumin, transferrin, K and k light chain, or a 500-fold dilution for anti-IgG, IgA, IgM, in 0.01 mol/L PBS. Then the sections were rinsed three times for 5 minutes each in PBS and incubated with the biotin-labeled secondary antibody (Nichirei, Tokyo, Japan) for 1 hour. After three more washes in PBS, peroxidase-labeled streptoavidin (Nichirei, Tokyo, Japan) was poured on the slide glasses, and incubation was carried out for 30 minutes. Then the slides were rinsed in 0.05 mol/L Tris-HC1 buffer (pH 7.6), and color was developed with 0.05% diaminobenzidine tetrachloride and 0.01% hydrogen peroxide dissolved in the same buffer, followed by nuclear counterstain with Meyer's hematoxylin. After dehydration in a graded alcohol series, the specimens were mounted for examination under a light microscope. RESULTS

lmmunohistochemical Findings. The inclusions in ICEC were clearly positive for antibodies against hum a n IgG, IgA, IgM, both K and k light chain (Fig. la, b, c, d, and e), whereas they were not immunoreactive for anti-C3, C4, albumin, and transferrin. The periphery of inclusions displayed stronger immunoreactivity t h a n the central area. The cytoplasm of infiltrating plasma cells in the portal tracts and sinusoidal lumen was strongly immunostained with anti-immunoglobulins and served as a positive control. The SECs, including ICECs, were negative for anti-CDw32 and CD16 in formalin-fixed, deparaffinized sections even after pronase digestion.

IWAMURA ET AL 1457 Above results were consistently observed in all the examined specimens. Disappearance of lCEC After Corticosteroid Therapy in Two Cases of Autoimmune Liver Disease. The first case was a 33-year-old woman with autoimmune hepatitis. In December 1993, she complained of h a n d arthralgia and shoulder stiffness and consulted a near clinic. Although her symptoms relieved shortly, a blood analysis incidentally revealed abnormal liver function tests. She was referred to Kochi Medical School and admitted on J a n u a r y 5, 1994 (Fig. 2). There was no history of blood transfusion, excessive alcohol consumption, or exposure to any hepatotoxic drugs. On examination, her liver was palpable 3 cm below the right costal margin without tenderness. Laboratory findings showed: alanine transaminase (ALT), 355 IU/ L; aspartate transaminase, 342 IU/L; total bilirubin, 2.5 mg/dL; alkaline phosphatase, 438 IU/L; g a m m a glutamyltransaminase, 187 IU/L. Total serum protein was as high as 11.8 g/dL, and the fractionation revealed 52.7% of ~/ globulin (6.22 g/dL). Serological tests for viral hepatitis A, B, and C were all negative, whereas anti-nuclear and anti-DNA autoantibodies" were positive with a titer of 1:640 and 1:5,120, respectively. Antimicrosome and SS-A antibodies were also positive, and antimitochondrial and a n t i - s m o o t h muscle antibodies were negative. Her major histocompatibility complex haplotype was A2/A24(9), B46/B70, Cwl/Cw7, DR8/ DR4. The sialography and lip biopsy showed t h a t she was complicated with SjSgren's syndrome. Liver biopsy specimen demonstrated severe piecemeal necrosis with bridging necrosis, dense infiltration of lymphocytes mixed with prominent plasma cells in the portal tracts, and a b u n d a n t ICECs (Fig. 3a and 3b). Histology activity index 17was scored 17 points. She was diagnosed as autoimmune hepatitis with severe activity and severe fibrosis. The t r e a t m e n t was started with 40 mg of oral prednisolone daily on J a n u a r y 10 (Fig. 2). Immunosuppressive t r e a t m e n t was effective, as rapid normalization of serum aminotransferase and drastic reduction of serum T globulin level were achieved. She had been taking prednisolone tapered in stepwise fashion, and she was discharged on April 5. However, because her serum ALT level deteriorated soon after discharge, she was required to re-admit and the dose of prednisolone was resumed up to 30 mg/day. Her serum ALT again returned to the normal level sharply and sustained. A second biopsy was done on J u n e 9, when her serum T globulin level was 1.08 g/ dL. The liver histology represented significant improvement of both portal inflammation and hepatocyte necrosis, and no ICECs were observed (Fig. 3c). The second case was a 64-year-old woman with chronic active h e p a t i t i s - PBC mixed type. 14 Since 1992, she had been consulting an ophthalmologist about her recurrent iritis. She was referred to another clinic to be checked for systemic disorders in March 1993, and was incidentally pointed out as having abnormal liver function without symptoms. She was transferred to Kochi Medical School on April 16 (Fig. 4). There was

1458 IWAMURA ET AL

HEPATOLOGYNovember 1995

FIG. 1. Immunostaining of ICEC. Inclusions in ICEC are intensely immunostained with anti-human immunoglobulins (arrows). The margin of inclusions shows stronger reactivity than inner region. (a) anti-IgG, (b) anti-IgM, (c) anti-IgA, (d) anti-K light chain, and (e) anti-X light chain. S: sinusoidal lumen. Chronic hepatitis B with severe activity and severe fibrosis. Serum 7 globulin level: 2.05 g/ dL. (Formalin-fixed, deparaffinized section; original magnification xl,000.) Bar = 10 #m.

no h i s t o r y of blood t r a n s f u s i o n , excessive alcohol consumption, or exposure to hepatotoxic drugs. She h a d n e v e r complained of systemic itching. On e x a m i n a t i o n , she did not d e m o n s t r a t e a p p a r e n t jaundice, and h e r liver was not palpable. L a b o r a t o r y findings showed: ALT, 60 IU/L; a s p a r t a t e t r a n s a m i n a s e , 56 IU/L; total bilirubin, 0.5 mg/dL; alkaline p h o s p h a t a s e , 340 IU/L; g a m m a glutamic t r a n s a m i n a s e 170 IU/L. Total s e r u m protein level was 8.8 g/dL, a n d the fractionation indi-

cated 30.5% of 7 globulin (2.68 g/dL). Serological t e s t s for viral h e p a t i t i s A, B, a n d C w e r e all negative, a n d a n t i n u c l e a r a n d a n t i m i t o c h o n d r i a l antibodies were positive with t i t e r s of 1:1280 a n d 1:640, respectively. Antimicrosome, SS-A, SS-B a n d a n t i - s m o o t h muscle antibodies were negative. H e r m a j o r histocompatibility complex-haplotype was A26(10)/A33(19), B60(40)/ B75(15), Cw3, DR9. As far as studied, she did not h a v e o t h e r a u t o i m m u n e diseases.

HEPATOLOGYVol. 22, No. 5, 1995

Case 1: 33 y.o. female

IWAMURA ET AL

Autoimmune hepatitis

(mg/day)

"f /

700 . ~ l s t

2nd biopsy

biopsy

11500

7

S ~

400

4

300

3

2OO

2

100

1

o

z

m

~"

o

FIG. 2. Clinical course of case 1. ALT, serum ALT (alanine aminotransferase) level; ~, Glb, serum T globulin level.

1459

Liver biopsy specimen histology demonstrated the loss ofinterlobular bile ducts and atypical ductular proliferation with lymphocytic infiltration in portal tracts. The lobular architecture was partially distorted with bridging fibrosis. Piecemeal necrosis and intralobular hepatocyte necrosis were more prominent as compared with typical PBC, and many ICECs were observed. Although we had no chance to examine M2 or M4 antimitochondrial antibodies, 14 both clinical and histological features were compatible with chronic active hepatitisPBC mixed type. 14'15 The treatment was started with 20 mg of oral prednisolone daily on May 18 (Fig. 4). During immunosuppressive therapy, serum alkaline phosphatase and ~/globulin level were decreased and sustained within normal range, although the serum ALT level was somehow fractuated. Oral ursodesoxycholic acid administration was additionally started on July 13 and was effective until March 1994. A second biopsy was performed on March 3, when her serum ~, globulin level was 1.09 g/dL. Liver histology represented significant improvement of both portal inflammation and hepatocyte necrosis, and bile duct damage was still present. There were no ICECs observed in the specimen.

FIG. 3. Light micrographs of liver biopsy specimen before and after corticosteroid therapy in case 1. (a) Note severe piecemeal necrosis and bridging necrosis with marked plasma cell infiltration. The lobular architecture is partly distorted. P, portal tract. Case 1 (hematoxylin-eosin stain; original magnification ×800), Bar = 100 #m. (b) Many ICECs are observed before treatment. They are characterized by strongly eosinophilic cytoplasmic granules (arrows). Case 1 (hematoxylin-eosin stain; original magnification )<800), Bar = 10 #m. (c) Liver biopsy specimen after treatment shows significant improvement of the portal inflammation and intralobular hepatocyte necrosis. There are no ICEC observed. P, portal tract. Case 1 (hematoxylin-eosin stain; original magnification ×80), Bar = 100 #m.

1460

IWAMURA ET AL

Case 2:64 y.o. female

HEPATOLOGYNovember 1995

CAH-PBC mixed type

(mgtclay)

lO

A

4°° I

33.S!

3OO •

25O

2.5 •

2OO

2

150

1.5

100

1

50

o.s

o

FIG. 4. Clinical course of case 2. ALP, serum ALP (alkaline phosphatase) level; T Glb, serum T globulin level; UDCA, ursodesoxycholic acid.

DISCUSSION

We have recently demonstrated the morphological feature of the ICEC by examining a large number of human liver biopsy specimens in various liver diseases. 13 In our previous report the good correlation between the ICEC density and the serum T globulin level, together with the electron microscopic appearance of ICECs, strongly suggested that the cytoplasmic inclusions would be composed of immunoglobulin content. However, we had not been able to clarify the nature of the inclusions in ICECs. They had appeared to be negative for various antibodies against serum proteins, including immunoglobulin. Recently the antigen retrieval technique has been widely applied for immunohistochemistry. It is reported that fixation does not alter the secondary protein structure per se, but masks the antigenicity.lS Cattoretti et al described that microwave heating or proteolytic enzymes, such as trypsin and pronase, can restore the accessibility of antibodies to epitopes masked by formalin fixation. 19 Using this procedure, it became possible to study the immunolocalization of many antigens in deparaffinized sections in which the tissue architecture was much better preserved than cryostat sections. Moreover, it was evident that a large number of primary antibodies, previously thought to be applicable only on cryostat sections were successfully employed in formalin-fixed specimens. According to Cattoretti et al, 19 polyclonal antibodies against IgG, IgA, and IgM do not stain untreated formalin-fLxed and deparaffinized sections. This would be the main reason why we could not obtain positive results for any immunoglobulins in the previous examination, 13 because we used untreated sections. Because anti-CDw32 and -CD16 were only applicable

to cryostat sections, the changes of FcR expression on the ordinary SEC and ICEC are not completely discussible in this study. However, the SECs would take up immunoglobulin, at least partly, through FcR expressed on their surface. In ICECs, the unbound FcR could be reduced in number by enhanced receptor-mediated uptake to a high level of serum immunoglobulin. Thus, the immunoreactivity against FcRs would be negative. It is still unclear whether SECs take up immunoglobulin as an immune complex or not. At least, we have not identified complements in ICECs. Among the subclasses of immunoglobulins, IgA plays a major role for the mucosal immune system. 2° In rats, mice, and rabbits, circulating polymeric forms of IgA are largely transported into bile by binding the receptor, secretory component, which is expressed on the sinusoidal plasma membrane of hepatocytes. 2° Unlike the rodents, human liver parenchymal cells are not involved in transcytosis of polymeric IgA. 21 The majority of polymeric IgA in bile are derived from the plasma cells located around biliary epithelial cells, which express the secretory components. Nagura et a122'23 demonstrated the immunohistochemical deposition of IgA along the sinusoidal surface of hepatocytes, SECs, and Kupffer cells in alcohol-induced and non-alcohol-induced liver diseases, but they did not observe a cytoplasmic inclusion-like structure in the SECs. Although the presence of IgA in human hepatocytes is reported to be in a degradative form rather than a transport pathway into bile, the involvement of SECs in the transcytosis or degradation of IgA has not been discussed. Our study suggests that the SECs would be the sites of catabolism of the serum IgA. We believe the morphological and functional examination of hepatic sinusoidal endothelial cells would make a significant contribution to studying the endotheliology indispensable for better understanding of liver pathophysiology.

Acknowledgment: We wish to thank M. Shirota and E. Miyazaki for their excellent technical assistance. REFERENCES 1. Hendricks FJ, Brouwer A, Knook DL. The role of hepatic fatstoring (stellate) cells in retinoid metabolism. HEPATOLOGY 1987;7:1368-1371. 2. Yamada M, Blaner WS, Soprano DR, Dixon JL, Kjeldbye HM, Goodman DS. Biochemical characteristics of isolated rat liver stellate cells. HEPATOLOGY1987;7:1224-1229. 3. Kent G, Gay S, Inouye T, Bahu R, Minick OT, Popper H. Vitamin A-containing lipocytes and formation of type III collagen in liver injury. Proc Natl Acad Sci U S A 1976;73:3719-3722. 4. Minato Y, Hasumura Y, Takeuchi J. The role of fat-storing cells in Disse space fibrogenesis in alcoholic liver disease. HEPATOLOGY 1983;3:559-566. 5. Senoo H, Hata R, Nagai Y, Wake K. Stellate cells (vitamin Astoring cells) are the primary site of collagen synthesis in nonparenchymal cells in the liver. Biomed Res 1984;5:451-458. 6. Muro H, Shirasawa H, Maeda M, Nakamura S. Fc receptors of liver sinusoidal endothelium in normal rats and humans: a histologic study with soluble immune complexes. Gastroenterology 1987;93:1078-1095. 7. Scoazec JY, Feldmaun G. In situ immunophenotyping study of

HEPATOLOGYVol. 22, No. 5, 1995

8. 9. 10. 11. 12. 13. 14.

15.

endothelial cells of the human hepatic sinusoid: results and functional implications, HEPATOLOGY1991; 14:789-797. Kosugi I, Muro H, Shirasawa H, Ito I. Endocytosis of soluble IgG immune complex and its transport to lysosomes in hepatic sinusoidal endothelial cells. J Hepatol 1992; 16:106-114. Skogh T, Blomhoff R, Eskild W, Berg T. Hepatic uptake of circulating IgG immune complexes. Immunology 1985;55:585-594. Muro H, Shirasawa H, Kosugi I, Nakamura S. Defect of Fc receptors and phenotypical changes in sinusoidal endothelial cells in human liver cirrhosis. Am J Pathol 1993; 143:105-120. Muro H, Shirasawa H, Kosugi I, Ito I. Defect of sinusoidal Fc receptors and immune complex uptake in CC14-inducedliver cirrhosis in rats. Gastroenterology 1990;99:200-210. McGary CT, Raja RH, Weigel PH. Endocytosis ofhyaluronic acid by rat liver endothelial cells. Evidence for receptor recycling. Biochem J 1989;257:875-884. Iwamura S, Enzan H, Saibara T, Onishi S, Yamamoto Y. Appearance of sinusoidal inclusion-containingendothelial cell in liver disease. HEPATOLOGY1994;20:604-610. Berg PA, Wiedmann KH, Sayers T, K15ppel G. Serological classification of chronic cholestatic liver disease by the use of two different types of antimitochondrial antibodies. Lancet II 1986; 1329-1332. K15ppel G, Seifert G, Lindner H, Dammermann R, Sack H, Berg PA. Histopathological features in mixed types of chronic aggressive hepatitis and primary biliary cirrhosis, correlations of liver histology with mitochondrial antibodies of different specificity. Virchows Arch A Pathol Anat Histol 1977;373:143-160.

IWAMURA ET AL

1461

16. Desmet VJ, Gerber M, Hoofnagle JH, Manns M, Poulsen H, Scheuer PJ. Classification of chronic hepatitis: diagnosis, grading and staging. HEPATOLOGY1994; 19:1513-1520. 17. Knodell RG, Ishak KG, Black WC, Chen TS, Craig R, Kaplowitz N, Kiernan TW, et al. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. HEPATOLOGY1981;1: 431-435. 18. Mason JT, O'Leary TJ. Effects of formaldehyde fixation on protein secondary structure: a calorimetric and infrared spectroscopic investigation. J Histochem Cytochem 1987;35:225-229. 19. Cattoretti G, Pileri S, Parravicini C, Becker MHG, Poggi S, Bifulco C, Key G, et al. Antigen unmasking on formalin-fixed, paraffin-embedded tissue sections. J Pathol 1993; 171:83-98. 20. Brown WR, Kloppel TM. The liver and IgA: immunological, cell biological and clinical implications. HEPATOLOGY 1989;9:763784. 21. Tomana M, Kulhavy R, Mestecky J. Receptor-mediated binding and uptake of immunoglobulinA by human liver. Gastroenterology 1988;94:762-770. 22. Amano K, Tsukada K, Takeuchi T, Fukuda Y, Nagura H. IgA deposition in alcoholic liver disease: an immunoelectron microscopic study. Am J Clin Pathol 1988;89:728-734. 23. Nagura H, Sumi Y, Fukuda Y, Hasegawa H, Watanabe K, Brown WR. Studies on the relationships of IgA to human liver. IgA deposition in non-alcoholic liver diseases. Acta Pathol Jpn 1989;39:363-372.