J Clin Pathol 1983;36:751-755

Immune complex nephritis in alcoholic cirrhosis:detection of Mallory body antigen in complexes bymeans of monoclonal antibodies to Mallory bodiesJ BURNS, AJ D'ARDENNE, JA MORTON, J O'D McGEE

From the University of Oxford, Nuffield Department of Pathology John Radcliffe Hospital, Oxford OX29DU

SUMMARY A Mallory body (alcoholic hyaline) antigen (JMB2) which is also present in intermedi-ate filaments of epithelial origin was demonstrated immunohistochemically in renal glomeruli ofthree out of eleven patients with alcoholic liver damage. In two of these patients, both of whomhad alcoholic cirrhosis with Mallory bodies, it was associated with mesangial deposits of IgA andC3. JMB2 was not found in glomeruli of normal controls, nor in a series of cases ofglomerulonephritis in non-alcoholic patients. It is concluded that JMB2 is present in immunecomplexes in renal glomeruli of patients with renal disease consequent on alcoholic liver disease.

Glomerular abnormalities, in particular mesangialexpansion, are frequently found in patients withalcoholic liver disease and these are associated withmesangial deposits of IgA.' 2 They are sometimesbut not always associated with clinical manifesta-tions of glomerulonephritis and renal failure.24 Theaetiology of these changes is not established but theyare often accompanied by high concentrations ofIgA in serum.- The latter is present in both mono-meric and polymeric forms, and in immune com-plexes.67 It has been suggested that patients withcirrhosis develop circulating immune complexes as aresult of defective hepatic sequestration of gutassociated antigens.8 Alternatively, it has beenspeculated that these complexes may be derivedfrom IgA aggregates combining with liver cell mem-brane proteins or with Mallory body (MBs) proteinsfrom alcohol damaged liver.9

In this paper two monoclonal antibodies (anti-JMB1 and anti-JMB2) directed against MBs/intermediate filaments were used to determinewhether MB-related antigens are present in theglomeruli of patients with alcoholic liver disease.

It is demonstrated by immunohistochemistry thatJMB2 is present in glomerular mesangium and capil-laries in some cases together with IgA and C3 inpatients with alcoholic cirrhosis.

Accepted for publication 15 February 1983

Material and methods

Samples of liver and kidney were obtained from 11cases of alcoholic liver disease at postmortem, andfrom six controls. The controls consisted of onepatient with primary biliary cirrhosis, one with a his-tory of excessive alcohol intake but no liver pathol-ogy, and four patients without evidence of eitherhepatic or renal disease. Renal biopsies wereobtained from patients with Berger's IgA neph-ropathy (1), segmental glomerulonephritis (1),membranoproliferative glomerulonephritis (4),membranous glomerulonephritis (5), crescenticglomerulonephritis (2); four morphologically nor-mal renal biopsies were also included in the series.Part of the tissue from each case was processed forroutine histology and the remainder for immunohis-tochemistry. Immunoglobulins and complementcomponents were detected by directimmunofluorescence, and MB antigens by indirectimmunoperoxidase.'0 All immunohistochemistrywas performed on cryostat sections of frozen tissue.Two Mallory body derived monoclonal antibodieswere used in the form of culture supematant. Thesewere anti-JMB1, which reacts preferentially withMallory bodies," and anti-JMB2 which reacts withMallory bodies and epithelial intermediatefilaments. " The preparation and immunohistochem-ical staining characteristics of these antibodies havebeen documented previously.'" I

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Table 1 Hepatic and glomerular pathology in patients with alcoholic liver disease

Case No Liver pathology Glomerular pathology*

Diagnosis Mallory bodies IgA IgG IgM C3 Clq JMB2

1 AC ++++ ++ + + + 0 ++2 AC + ++ + + + 0 +3 AC + + + 0 + 0 + 04 AC 0 0 0 + 0 + 05 AC + + 0 0 0 0 0 06 AC + 0 0 + + + 07 AC 0 0 0 + + + 08 AC +++0 0 0 0 0 09 AH ++++0 0 0 0 0

10 AH ++++0 0 0 0 0 011 AH +++0 0 0 0 0 0

AC = alcoholic cirrhosis AH = alcoholic hepatitis* + to + + + + is an arbitrary score based on subjective assessment of the quantity of immunofluorescence or immunoperoxidase deposits.

Fig. 1 IgA deposition in mesangium ofpatient withalcoholic cirrhosis. Immunofluorescence x 350

Results

Of the 11 cases of alcoholic liver disease, eight hadcirrhosis and three had severe alcoholic hepatitis(Table 1). The majority had MBs within hepatocytesdetectable on both routine light microscopy and onstaining with anti-JMB1 and anti-JMB2. There wasa variable degree of fatty infiltration.By light microscopy the kidneys from patients

with alcoholic liver disease showed only minorchanges. These included mesangial expansion in afew cases and occasional basement membranethickening. Three cases of alcoholic cirrhosis hadextensive deposition of IgA in the mesangium ofglomeruli (Fig. 1) accompanied by varying amounts

- w_~ ~ :ff lc* A

Fig. 2 JMB2 deposition in mesangium and capillary loopsin patient with alcoholic cirrhosis. Immunoperoxidase x400

of IgG, IgM, C3 and Clq (Table 1). In two of thesepatients mesangial and capillary JMB2 (Fig. 2) wasdemonstrated. JMB2 was also found in glomeruli ofone of the three cases of alcoholic hepatitis butwithout accompanying immunoglobulin or comple-ment. Two cases of alcoholic cirrhosis had smallglomerular deposits of IgM, C3 and Clq withoutevidence of either IgA or JMB2. Glomerular stain-ing for JMB1 was negative in all patients. JMB1 andJMB2 were not detected in the glomeruli of onepatient with primary biliary cirrhosis, in onealcoholic without liver pathology and in four caseswith normal liver and renal structure and function.

In normal kidney JMB2 was present in renal tubu-lar epithelium and in parietal cells lining Bowman's

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Table 2 Clinical details ofpatients with alcoholic liver disease

Case No Diagnosis Cause of death Blood urea Serum IgA Urine(mmol/l) (gil)

1 AC Hepatic failure 9.43 - N2 AC Bronchopneumonia and meningitis N 17-2 RBC3 AC Bronchopneumonia N - N4 AC Ruptured oesophageal varices N - N5 AC Hepatic failure 8-0 - N6 AC Bronchopneumonia, carcinoma of pharynx N - N7 AC Lobarpneumonia N - N8 AC Hepatic failure 26.3 N N9 AH Cerebral haemorrhage N 7.0 N

10 AH Bro chopneumonia N - N11 AH Ruptured abdominal aortic aneurysm - - -

N= nonnal.- not recorded.RBC = red blood cells.

I.

'A

.N N*. I.14..

Fig. 3 JMB2 distribution innormal kidney. This antigen is inrenalproximal and distal tubulesand in Bowman's capsule.Immunoperoxidase x 250

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Fig. 4 JMB2 deposition in.J. - crescentic nephritis. JMB2 is only

detectable in proliferating epithelialE cells. Immunoperoxidase x 250

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capsule (Fig. 3) and JMB1 to a lesser extent. JMB1and JMB2 were not fouind in glomerular tufts. In theseries of cases of glomerulonephritis unrelated toliver disease, JMB2 was only detected in proliferat-ing epithelial cells of crescentic nephritis (Fig. 4).The principal clinical details and causes of death

in the alcoholic patients are given in Table 2. Onlyone patient (case 2) had clinical evidence ofglomerular disease in that he had microscopichaematuria. This patient also had mesangial IgAand JMB2. Three other cases (1, 5 and 8) hadimpaired renal function with raised plasma urea andcreatinine, but at no time was blood or proteinfound in the urine. Serum IgA was not measured inall patients. Where it was recorded it was raised intwo cases (2 and 9). Hepatitis B antigen was notdetected in serum of any of the patients.

Discussion

The finding of IgA in glomerular mesangium ofalmost half the patients with alcoholic cirrhosis is inaccord with previous observations, as are thefindings on light microscopy and clinical investiga-tion.' 24 The observation that the antigen JMB2 issometimes present in association with this mesangialIgA raises the possibility that they are present asimmune complexes. Proof of such a relation wouldrequire elution of antibody from kidney andidentification of its specificity; the circumstantialevidence is nevertheless strong. The finding ofJMB2 without immunoglobulin in one case ofalcoholic hepatitis does not preclude its presence inimmune complexes in cirrhosis, since glomerularbinding of antigen may precede stimulation of anantibody response.

If JMB2 is indeed involved in immune complexproduction in cirrhosis, the question arises as to itsorigin. The most obvious source would be alcoholdamaged hepatocytes. JMB2 is a constituent of Mal-lory bodies and these were present in the majority ofcases examined. Circulating alcoholic hyaline andantibody to alcoholic hyaline have been reportedpreviously,'2 although these finding have not beenconfirmed. An alternative explanation is thatglomerular bound JMB2 is derived from renal tubu-lar epithelial cells, gut or pancreas" since it is anormal constituent of the epithelial cells of thesetissues. Immune complex glomerulonephritisinduced by renal tubular antigen has been reportedin animals'3 and renal tubular antigen has beendemonstrated in membranous glomerulonephritis inman. 14 This antigen was derived from proximal tubu-lar brush border and did not have the cytoplasmicdistribution of JMB2. Furthermore no normal kid-ney nor the cases of glomerulonephritis examined,

including five cases of membranous glomerulone-phritis and one of Berger's IgA nephropathy, haddetectable JMB2 in glomerular tufts.Only one of the patients with alcoholic liver dis-

ease in this series had clinical evidence ofglomerulonephritis as manifest by microscopichaematuria. This corresponds with previous obser-vations5 and lends weight to the hypothesis thatrenal immune complex deposition per se does notnecessarily give rise to clinical renal disease. Otherimportant factors are probably "nephritogenicity"of bound antigen and host immune response.'5Three alcoholic patients had small deposits of IgM

and complement but no JMB2 or IgA. Circulatingantigens other than those directly related to liverdamage may have been present terminally and it isnoteworthy that several died with overwhelminginfections.The possibility that JMB2 is involved in the pro-

duction of immune complexes in alcoholic cirrhosismay be clinically significant not only because of thepotentially damaging effect of the complexes them-selves, but also because it provides evidence of anautoimmune response in these patients. Such a reac-tion may be important in the perpetuation of liverdamage initiated by alcohol and in the genesis ofrenal complications in patients with alcoholic liverdisease.

Mrs V Macintosh typed this manuscript.

References

Berger J, Yaneva H, Nabarra B. Glomerular changes in patientswith cirrhosis of the liver. Adv Nephrol 1977;7:3-14.

2Nakamoto Y, Lida H, Kobayashi K, Dohi K, Kida H, Hattori N,Takeuchi J. Hepatic glomerulonephritis. Virchows Archiv[A]1981;392:45-54.

3Nochy D, Caliard P, Bellon B, Bariety J, Druet P. Association ofovert glomerulonephritis and liver disease: a study of 34patients. Clin Nephrol 1976;6:422-7.

4Caliard P, Feldmann G, Prandi D, et al. Immune complex typeglomerulonephritis in cirrhosis of the liver. Am J Pathol1975;80:329-40.

5 Iturriaga H, Perede T, Estevez A, Ugarte G. Serum immuno-globulin A changes in alcoholic patients. Ann Clin Res1977;9:3943.

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7Sancho J, Egido J, Sanchez-Crespo M, Blasco R. Detection ofmonomeric and polymeric IgA containing immune complexesin serum and kidney from patients with alcoholic liver disease.Clin Exp Immunol 1981;47:327-35.

Gormley AA, Smith PS, Seymour AE, Clarkson AR, WoodroffeAJ. IgA glomerular deposits in experimental cirrhosis. Am JPathol 1981;104:50-4.

9Swerdlow MA, Chowdhury LN, Horn T. Patterns of IgA deposi-tion in liver tissues in alcoholic liver disease. Am J Clin Pathol1982;77:259-66.

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'° Morton JA, Bastin J, Fleming KA, McMichael A, Burns J,McGee J O'D. Mallory bodies in alcoholic liver disease:identification of cytoplasmic filament/cell membrane and uni-que antigenic determinants by monoclonal antibodies. Gut1981;22:1-7.

McGee J O'D, Morton JA, Barbatis C et al. Monoclonal anti-bodies to Mallory Bodies/Intermediate Filaments and HLA(Class 1) Antigen in human liver disease. McMichael A, FabreJW, eds. Monoclonal antibodies in clinical medicine London:Academic Press, 1982:431-55.

12 Kanagasundaram N, Kakumu S, Chen T, Leevy CM. Alcoholichyalin antigen (AHAg) and antibody (AHAb) in alcoholichepatitis. Gastroenterology 1977;73:1368-73.

3 Edgington TS, Glassock RJ, Dixon FJ. Autologous immunecomplex nephritis induced with renal tubular antigen. I.

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Identification and Isolation of the pathogenetic antigen. J ExpMed 1968;127:555-72.

4 Naruse T, Kitamura K, Miyakawa Y, Shibata S. Deposition ofrenal tubular epithelial antigen along the glomerular capillarywalls of patients with membranous glomerulonephritis. JImmunol 1973;110: 1163-6.

s5 Williams DG. New ideas in the pathogenesis of nephritis. J ClinPathol 1981;34:1223-7.

Requests for reprints to: Professor J O'D McGee, Univer-sity of Oxford, Nuffield Department of Pathology, JohnRadcliffe Hospital, Oxford OX2 9DU, England.

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