Clin. exp. Immunol. (1975) 19, 209-217.
THE INTERACTION OF RHEUMATOID FACTOR WITH HEPATITIS B SURFACE ANTIGEN-ANTIBODY COMPLEXES J. A. MARKENSON,* C. A. DANIELS,t A. L. NOTKINS,t J. H. HOOFNAGLE,§ J. GERETY§ AND L. F. BARKER§ tDepartment of Pathology, Duke University Medical Center, Durham, North Carolina; I Laboratory of Oral Medicine, National Institute of Health, Bethesda, Maryland; § Division of Blood and Blood Products, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland, U.S.A. (Received 3 July 1974) SUM MARY
A solid phase radioimmunoassay was developed in which the hepatitis B surface antigen was adsorbed to the surface of plastic beads. When the antigen-coated beads were incubated with human IgG antibody against hepatitis B surface antigen, immune complexes were formed on the solid phase surface. IgM rheumatoid factor was found to bind to the hepatitis B surface antigen-antibody complexes but not to the antigen or the IgG antibody alone. Since both hepatitis B surface antigenantibody complexes and rheumatoid factor are commonly present in type B viral hepatitis, it is suggested that rheumatoid factor may play a role in the pathogenesis of this viral disease in man.
INTRODUCTION An antigen which forms the outer coat or surface of the hepatitis B virus (HBV), termed hepatitis B surface antigen (HB.Ag), is found transiently in the acute phase sera of most patients with type B viral hepatitis (Blumberg, Alter & Visnich, 1965; Shulman, 1970). In the chronic HBV carrier state, HBSAg may be detected in the serum for an extensive period of time (Krugman & Giles, 1970). Antigen-antibody (HBAg-Ab) complexes (Shulman & Barker, 1969) have also been found in the sera of patients with acute type B viral hepatitis with renal (Nowoslawski et al., 1972) and vascular (Gocke et al., 1970) lesions attributed to the deposition of these immune complexes. Infectious virus-antibody (VA) complexes can be found in the circulation of animals chronically infected with a number of other viruses (Hirsch, Allison & Harvey, 1969; Notkins et al., 1966; Oldstone & Dixon, 1970). Similarly renal (Oldstone & Dixon, 1971), * Present address: Department of Medicine, Cornell University Medical College, New York, New York 10021, U.S.A. Correspondence: Dr Charles A. Daniels, Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, U.S.A.
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hepatic, and vascular (Henson et al., 1969) lesions have been attributed to the deposition of these immune complexes. Infectious VA complexes can be neutralized by anti-immunoglobulin antisera (Notkins et al., 1966; Ashe & Notkins, 1966; Porter & Larsen, 1967) including rheumatoid factor (RF) (Ashe et al., 1971; Gipson, Daniels & Notkins, 1974). Recently we have reported that RF is found in the sera of 58.6% of patients with type B viral hepatitis (Hoofnagle et al., 1974). Since both RF and HBAb-Ag complexes have been shown to occur in type B viral hepatitis, the possibility exists that RF might interact with HBSAg-Ab complexes in vivo and play a role in this human disease. The present study was initiated to determine if RF would attach to HBSAg-Ab complexes in vitro and what serum factors would potentiate or inhibit this interaction. MATERIALS AND METHODS RF assay RF was detected by agglutination of human gamma-globulin-coated latex particles (RA Test, Hyland Laboratories). All assays were done in triplicate and sera with titres of 1 :20 or greater were considered positive for RF.
HBSAg The presence of HB.Ag was detected by counter electrophoresis (Gocke & Howe, 1970) (CEP), and radioimmunoassay (Ling & Overby, 1972) (RIA). To prepare purified HB.Ag, antigen-containing sera were layered on the top of a 25-50% sucrose gradient in a BXIV zonal rotor and centrifuged for 24 hr at 30,000 rev/min at 250C. The antigen-positive fractions were pooled and banded two additional times on 13-60% sucrose gradients (30,000 rev/min for 6 hr at 250C). This purified HBAg preparation was stored in aliquots at -20'C until used. Unless otherwise stated, dilutions were made in 0 14 M sodium chloride solution containing 0 005 M phosphate buffer (PBS), pH 7 4. Hepatitis B antibody Antibody to hepatitis B surface antigen (anti-HB,) was measured by passive haemagglutination (Vyas & Shulman, 1970). Unless otherwise specified, sera from multipletransfused haemophiliac patients were used as the source of anti-HBs. The IgG and IgM, serum fractions containing anti-HBs activity were obtained from haemophiliac sera by DEAE-cellulose chromatography. The immunoglobulin fractions were lyophilized and used in experiments at 10 mg/ml in PBS. Two sources of paired sera (preinfection and convalescent) also were studied. One source was a physician (R.G.), who seroconverted prior to the study; the other source was two chimpanzees experimentally infected with HBSAg-positive serum (Markenson et al., 1973). Prior to use, sera were heated to 56°C for 30 min and were shown to be free of RF. Isolation of RF RF was isolated from the sera of patients with rheumatoid arthritis by Sephadex G-200 chromatography (Schrohenloher, Kunkel & Tomasi, 1964). The peak of IgM fraction (10 mg/ml) had a latex titre of 1: 1000 and did not contain anti-HBs activity. In this study, RF was isolated from three different patients and each preparation exhibited the properties described in the experiments. RF, HBsAg and anti-HBs were labelled with 1251 by the chloramine T method (Hunter & Greenwood, 1962).
NWy~I
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Solid phase radioimmunoassay Six millimetre, round, plastic beads (Geiger's Outlet, Pasadena, California) were used as the solid phase immunoadsorbent. The beads were coated by incubating overnight at 250C with HBSAg, or IgM anti-HBs, or normal human serum (containing neither the HBAg nor anti-HBs). The coated beads then were washed five times with PBS and used the same day. Beads, coated with either dilutions of HBSAg or normal human serum (control), were placed in separate 75 x 100 mm glass tubes and incubated overnight at 250C with 0-2 ml of 125I-labelled anti-HBs (200,000 ct/min). The beads were washed five times with PBS and the results expressed as ct/min of labelled antibody bound. Each value represents the average of two or more determinations on duplicate samples.
Attachment of 125'-labelled RF to immune complexes HBSAg-coated beads were incubated at 250C overnight with 0-2 ml dilutions of anti-HBS or normal (nonimmune) serum or PBS (control). The fluid was removed and the beads washed five times with PBS. 0-2 ml of 125I-labelled RF (200,000 ct/min) was incubated with each bead overnight at 250C, and the washed beads were counted for radioactivity. The results were expressed either as the counts of 125I-labelled RF attached or as a binding ratio (ct/min of 125I-labelled RF bound to beads treated with antibody divided by ct/min bound to beads treated with nonimmune serum). Blocking studies were performed by using unlabelled RF to inhibit the attachment of 125I-labelled RF. Beads containing complexes on their surface were incubated overnight first with unlabelled RF, followed by incubation with the same preparation labelled with 125i. RESULTS
Formation of immune complexes To demonstrate that RF could attach to HBSAg-Ab complexes a technique was first developed for forming immune complexes in vitro. Plastic beads were coated with various concentrations of purified HBsAg and then incubated with 125I-labelled anti-HBS. The data in Fig. 1 show that beads exposed to a 1 :10 dilution of the HBSAg preparation bound an appreciable quantity of 125I-labelled anti-HB.Ag (5000 ct/min). At lower concentrations 0~~~~~~~~~~~~~~~~~~ mDX0 _
2
0
Control
10-6
10-5
FIG. 1. Attachment of 125I-labelled
10-4
10-3
10lo-2
Dilution of HBSAg
anti-HB. to beads coated with dilutions of HBAg.
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of HBSAg the beads bound considerably less anti-HB,. Beads coated with normal human serum did not bind appreciable amounts of 125I-labelled anti-HBs. Attachment of RF to immune complexes When different concentrations of HBSAg coated beads were incubated with excess unlabelled anti-HBS and then exposed to '25I-labelled RF, greater than 1000 ct/min of
/
E
_0
DO 4 _-A .0
Contl
10-4
0-5
10-3
1o-2
10-
Dilution of HBsAg
FIG. 2. Attachment of `25I-labelled RF to beads coated with dilutions of HBAg after incubation with either anti-HBs-containing (@) or non-immune human serum (0). TABLE 1. Binding of 125I-labelled RF to HBsAg-coated beads exposed to either preinfection or convalescent serum
I25I-labelled RF bound (ct/min) after exposure of HBSAg-coated bead* to: Sera examined Human (R.G.)
Chimpanzee (196) Chimpanzee (959)
Pre-infection serat
Convalescent serat
250 369 232
995 1045 682
Binding ratio+ 4-0 2-8 2-9
* Coated with a 1 :10 dilution of HbsAg in PBS. t Incubated with a 1 :10 dilution of serum.
I Ratio determined by dividing ct/min bound in presence of convalescent serum by ct/min bound in presence of preinfection serum.
radioactivity attached to the immune complexes on the bead surface (Fig. 2). At lower concentrations of HBSAg, less anti-HB, bound to the beads (see Fig. 1), and in turn the binding of RF decreased (see Fig. 2). Neither uncoated beads exposed to anti-HB, (control), nor HB.Ag-coated beads incubated with dilutions of normal (nonimmune) human serum, bound significant quantities of 1251-labelled RF (Fig. 2).
Specificity of attachment of RF Since the anti-HBS used in the previous experiments was from haemophiliac serum (which
Rheumatoid factor and HB5Ag
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might contain a number of isoantibodies), paired sera from HBV infections were employed to establish the specificity of the RF attachment. HBAg-coated beads incubated with the human pre-infection serum and then 125"-labelled RF bound little radioactivity (Table 1). In contrast, HBSAg-coated beads exposed to convalescent (immune) serum from the same individual bound four times as much '251-labelled RF. Similarly, experiments employing paired sera from two chimpanzees showed that significant amounts of RF attached only after exposure of the beads to the convalescent sera (binding ratios of 2-8 and 2 9). TABLE 2. Inhibition* of 1251-labelled RF attachment to HBAg-anti-Hbs complexes by unlabelled RF
HBsAg bead-exposed to: First incubation
12 1-labelled RF bound
Second incubation Third incubation
Immune serum Immune serum Non-immune serum
Buffer Unlabelled RF Buffer
(ct/min)
1251-labelled RF 1251-labelled RF 1251-labelled RF
1184 253 274
* HBAg coated beads were sequentially exposed to: (1) immune or non-immune serum; (2) buffer (PBS) or unlabelled RF; and (3) 125I-labelled RF.
0
a]2.0 A v
1/1024
1/2560
A--A-- A-
1/640 1/160 1/40 Dilution of anti-HBs
4A 1/10 Undiluted
FIG. 3. Attachment of 125I-labelled RF to HBAg-coated beads after incubation with dilutions of whole serum (e), or IgG (a), or IgM anti-HB, (A). Binding ratio = (ct/min of 1251. labelled RF attached to anti-Hb,-treated beads)/(ct/min of 1251-labelled RF attached to NHStreated beads).
To further establish the specificity of RF attachment to HB.Ag-anti-HB, complexes, inhibition studies with unlabelled RF were done. HBSAg-coated beads sequentially incubated with immune sera, buffer, and then 125I-labelled RF bound greater than 1100 ct/min (Table 2). HBSAg-coated beads exposed to immune sera, unlabelled RF, and then 1251-labelled RF bound less than 300 ct/min. When compared to the non-immune serum control, the unlabelled RF completely inhibited the attachment of 125I-labelled RF. Interaction of RF with different classes of antibody In order to determine which class of antibody in serum potentiated RF attachment,
J. A. Markenson et al.
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anti-HBS-containing serum was chromatographed and the IgG and IgM fractions tested. HBSAg-coated beads were incubated with serial dilutions of: (1) anti-HBs whole serum; (2) the IgG fraction; or (3) the IgM fraction. 125I-labelled RF was added and the amount of attachment was expressed as a binding ratio (Fig. 3). With undiluted whole serum the maximal binding ratio occurred at a 1:20 dilution of whole serum. Significantly less binding occurred at higher and lower dilutions of serum. When the IgG antibody was used, the maximum RF binding ratio also occurred at about a 1:20 dilution, but in contrast to whole serum, the binding ratio did not decrease at the higher concentrations of IgG. The IgM fraction of anti-HBS did not potentiate the binding of RF at any of the nine concentrations tested. To determine if the IgM fraction contained any anti-HB, activity, HBAg-coated beads were incubated with serial dilutions of the IgM fraction or normal human serum and then 11 0 _
90
70 570
co/
,, 50_/
30_ 1.0~~~~~~~~~~~~~ I E
1/80
1/40
~
~ ~
1/20 1/10 Dilution of IgM onti-HBs
~ ~ ~~~~~~~~~~~~~~~I I
t
Undiluted
FIG. 4. Attachment of 'l25-labelled HBAg to beads coated with dilutions of IgM anti-HB,. Binding ratio = (ct/min of 'l25-labelled HbAg attached to IgM-treated beads)/(ct/min of 1251-labelled HbsAg attached to NHS-treated beads).
were exposed to 125I-labelled HBsAg. The data in Fig. 4 show that the IgM fraction contained anti-HBs activity. Eleven times as much 125I-labelled HBsAg bound to beads treated with undiluted immune IgM as to beads treated with non-immune serum. Whether the IgM anti-HBS is responsible for sterically blocking the binding of RF noted with whole serum, remains to be determined.
DISCUSSION RF is found in the sera of patients with rheumatoid arthritis (Singer & Plotz, 1956), as well as with a number of chronic diseases, e.g. tuberculosis (Singer et al., 1962), sarcoidosis (Kunkel, Simon & Fudenberg, 1958), leprosy (Cathcart et al., 1961), syphilis (Peltier & Christian, 1959), leishmaniasis (Kunkel et al., 1958), and bacterial endocarditis (Williams & Kunkel, 1962). A higher than normal incidence of RF also has been found in various types of liver disease (Bouchier, Rhodes & Sherlock, 1964) including hepatitis (State et al., 1973; Dudley, O'Shea & Sherlock, 1973; Howell, Malcolm and Pike, 1960), biliary cirrhosis, nutritional cirrhosis, hepatocellular carcinoma, and metastatic carcinoma of the
Rheumatoidfactor and HBAg
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liver. We have reported that RF is present in 58 6% of the patients with HBAg-positive hepatitis as compared to 3%/O of controls (Hoofnagle et al., 1974). Since, however, 58 6% of patients with acute liver disease in the absence of HBAg also had elevated RF titres, RF cannot be used in the diagnosis of acute hepatitis B virus infection as has been suggested by others (Ziegenfuss, Miller & Rossman, 1971). What is the reason for the elaboration of RF in these chronic human diseases? One of the most reliable methods of inducing RF-like antibodies is by repeated immunization of animals with killed bacteria (Abruzzo & Christian, 1961) or soluble proteins (Aho and Wagner, 1967). Antibodies first develop against the exogenous antigens and then it is thought that RF-like antibodies are elaborated against the host's own altered IgG which has attached to the antigen in the form of an immune complex. Indeed, injections of antigenantibody complexes into guinea-pigs and rabbits stimulate RF production (Adler, 1956). From these animal studies, the presence of RF in human diseases has been regarded as evidence for the presence of immune complexes (Gell & Kelus, 1967). In acute type B hepatitis, viral antigens may be present in the circulation for many weeks. Immune complexes then appear in the serum (Shulman & Barker, 1969) and finally the antigen disappears upon the emergence of free antibody. As in the animal RF models, patients may be first forming antibodies against the HB.Ag and then elaborating RF against the IgG present in the immune complexes (Shirodaria, Fraser & Stanford, 1973). Since, however, our prior study (Hoofnagle et al., 1974) showed that the same percentage of HBSAg-positive and HBAg-negative patients with acute liver disease had RF in their serum it is evident that antigens other than HBsAg must be responsible for RF formation in HBSAg-negative patients. In fact, a variety of autoimmune antibodies (antinuclear, antimitochondrial, antismooth muscle, and antihepatic) have been found in acute and chronic liver diseases. Presumably, normally hidden antigens are exposed or derepressed during hepatic injury and RF is made to the resulting immune complexes. RF has been found in the convalescent sera of patients with a number of viral infections, including influenza A (Svec & Dingle, 1965), cytomegalovirus (Langenhuysen, 1971), Rubella virus (Johnson & Hall, 1958), Epstein-Barr virus (Capra, Winchester & Kunkel, 1969), and transiently during the acute phase of several other 'viral' infections (Dresner & Trombly, 1959). Our previous studies showed that at least in vitro, RF could attach to infectious virus-antibody complexes, e.g. herpes simplex virus (Ashe et al., 1971) and vaccinia virus (Gipson et al., 1974) and assist in their neutralization. The present study shows that RF can attach to HBSAg-IgG complexes, but whether this enhances neutralization cannot be determined until a more convenient assay for infectivity of HBV is developed. In patients with viral hepatitis (Nowoslawski et al., 1972; Ekoyan et al., 1972; Kater, Van Gorp & Borst-Eilers, 1973) granular deposits of IgG, complement, and IgM have been demonstrated within vessel walls, on hepatocyte plasma membranes, and in Kupfer cells. Immunofluorescent studies (Bonomo, Tursi & Minerva, 1966) have revealed collections of RF-containing plasma cells and lymphocytes in the portal triad. In vitro studies with sensitized erythrocytes have shown that RF can either potentiate or inhibit the amount of complement-mediated cell lysis depending on the amount of antibody bound to the surface of the sensitized cells (Schmid, Roitt & Rocha, 1973). Whether the interaction of RF with immune complexes plays a protective or destructive role, or no role whatsoever in type B viral hepatitis is unknown and may depend on when in the stage of the disease (antibody excess or antigen excess) RF is elaborated. B
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J. A. Markenson et al. ACKNOWLEDG MENTS
We are grateful to Dr D. Gordon Sharp of the University of North Carolina School of Medicine for preparing purified HBAg by zonal ultracentrifugation. This study was supported by contract number NIH-NIDR-2401 awarded to C.A.D. by the National Institute of Dental Research. We thank Miss Christine Artley for the help in the preparation of the manuscript. REFERENCES
ABRUZZO, J.L. & CHRISTIAN, C.L. (1961) The induction of a rheumatoid factor-like substance in rabbits J. exp. Med. 114, 791. ADLER, F. (1956) Antibody formation after injection of heterologous immune globulin. J. Immunol. 76, 217. AHO, K. & WAGNER, 0. (1961) Production of anti-antibodies in rabbits. Ann. Med. exp. Fenn. 39, 79. ASHE, W.K., DANIELS, C.A., SCOrr, G.S. & NOTKINS, A.L. (1971) Interaction of rheumatoid factor with infectious herpes simplex virus-antibody complexes. Science, 172, 176. ASHE, W.K. & NOTKINS, A.L. (1966) Neutralization of an infectious herpes simplex virus-antibody complex by anti-y-globulin. Proc. nat. Acad. Sci. (Wash.), 56, 447. BLUMBERG, B.S., ALTER, H.J. & VIsNicH, S. (1965) A 'new' antigen in leukemia sera. J. Amer. med. Ass. 191, 541. BONOMO, L., TuRsi, A. & MINERVA, V. (1966) Immunofluorescence study of rheumatoid factor in liver tissue of patients with rheumatoid arthritis and hepatic diseases. J. Path. Bact. 92, 423. BOUCHIER, I.A.D., RHODES, K. & SHERLOCK, S. (1964) Serological abnormalities in patients with liver disease. Brit. med. J., i, 592. CAPRA, J.D., WINCHESTER, R.J. & KUNKEL, H.J. (1969) Cold-reactive rheumatoid factors in infectious mononucleosis and other diseases. Arthr. and Rheum. 12, 67. CATHCART, E.S., WILLIAMS, R.C., Ross, H. & CALKINS, E. (1961) The relationship of the latex fixation test to the clinical and serological manifestations of leprosy. Amer. J. Med. 31, 758. DRESNER, E. & TROMBLY, P. (1959) Latex-fixation reaction in non-rheumatic diseases. New Engl. J. Med. 261, 981. DUDLEY, F.J., O'SHEA, M.J. & SHERLOCK, S. (1973) Serum autoantibodies in hepatitis associated antigen (HAA) positive patients. A possible index of cell mediated immunity to the associated-infective agent. Clin. exp. Immunol. 13, 367. EKNOYON, G., GYORKEY, F., DICHOSO, C., MARTINEZ-MALDONADO, M., SUKI, W.N. & GYORKEY, P. (1972) Renal morphological and immunological changes associated with acute viral hepatitis. Kid. Internat. 1, 413. GELL, P.G.H. & KELUS, A.S. (1967) Anti-antibodies. Advanc. Immunol. 6,461. GIPSON, T.G., DANIELS, C.A. & NOTKINS, A.L. (1974) Interaction of rheumatoid factor with infectious vaccinia virus-antibody complexes. J. Immunol. 112, 2087. GOCKE, D.J. & HOWE, C.J. (1970) Rapid detection of Australia antigen by counterimmunoelectrophoresis. J. Immunol. 104,1031. GOCKE, D.J., Hsu, K., MORGAN, C., BOMBARDIERI, S., LOCKSHIN, M. & CHRISTIAN, C.L. (1970) Association between polyarteritis and Australia antigen. Lancet, ii, 1149. HENSON, J.B., LEADER, R.W., GORHAM, J.R. & PADGETT, G.A. (1966) The sequential development of lesions in spontaneous aleutian disease of mink. Pathol. Vet. 3, 289. HIRSCH, M.S., ALLISON, A.C. & HARVEY, J.J. (1969) Immune complexes in mice infected neonatally with Maloney leukemogenic and marine sarcoma viruses. Nature (Lond.), 223, 739. HOOFNAGLE, J.H., MARKENSON, J.A., DANIELS, C.A., GERETY, R.J., NOTKINS, A.L. & BARKER, L.F. (1974) The lack of association between rheumatoid factor and hepatitis B antigen. Amer. J. med. Sci. 268, 23. HOWELL, D.S., MALCOLM, J.M. & PIKE, R. (1960) FII agglutinating factors in serums of patients with nonrheumatic diseases. Amer. J. Med. 29, 662. HUNTER, W.M. & GREENWOOD, F.C. (1962) Preparation of iodine 1131 labelled human grown hormone of high specific activity. Nature (Lond.), 194, 495. JOHNSON, R.E. & HALL, A.P. (1958) Rubella arthritis-report of cases studied by latex tests. New Engl. J. Med. 258, 743.
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KATER, L., VAN GORP, L.H.M. & BORST-EILERS, E. (1973) Hepatitis: intrahepatic expression of au antigen and immune complexes in liver tissue. Vox Sang. (Basel), 24, supplement 27. KRUGMAN, S. & GILES, J.P. (1970) Viral hepatitis. New light on an old disease. J. Amer. med. Ass. 212, 1019. KUNKEL, H.G., SIMON, H.J. & FUDENBERG, H. (1958) Observations concerning positive serological reactions for rheumatoid factor in certain patients with sarcoidosis and other hyperglobulinemic sera. Arthr. and Rheum. 1, 289. LANGENHUYSEN, M.M.A.C. (1971) Antibodies against y-globulin after blood transfusion and cytomegalovirus infection. Clin. exp. Immunol. 9, 393. LING, C.M. & OVERBY, L.R. (1972) Prevalence of hepatitis B virus antigen as revealed by direct radioimmune assay with I125 antibody. J. Immunol. 109, 834. MARKENSON, J.A., GERETY, R.J., HOOFNAGLE, J.H., DALGARD, D.W., MCGRATH, P.P. & BARKER, L.F. (1973) Effects of cyclophosphamide on hepatitis B virus infection and challenge in chimpanzees. Bact. Proc. 73, 222. NOTKINS, A.L., MAHAR, S., SCHEELE, C. & GOFFMAN, J. (1966) Infectious virus-antibody complex in the blood of chronically infected mice. J. exp. Med. 124, 81. NOWOSLAWSKI, A., KRAWCZYNSKI, K., BRZOSKO, W.J. & MADALINSKI, K. (1972) Tissue localization of Australiaan tigen immune complexes in acute and chronic hepatitis and liver cirrhosis. Amer. J. Path. 68, 31. OLDSTONE, M.B. & DIXON, F.J. (1970) Persistent lymphocytic choriomeningitis viral infection. III. Virusanti-viral antibody complexes and associated chronic disease following transplacental infection. J. Immunol. 105, 829. OLDSTONE, M.B. & DIXON, F.J. (1971) Immune complex disease in chronic viral infections. J. exp. Med. 134, supplement 32. PELTIER, A. & CHRISTIAN, C.L. (1959) The presence of the 'rheumatoid factor' in sera from patients with syphilis. Arthr. and Rheum. 2, 1. PORTER, D.D. & LARSEN, A.E. (1967) Aleutian disease of mink: infectious virus-antibody complexes in the serum. Proc. Soc. exp. Biol. (N. Y.), 126, 680. SCHMID, F.R., ROITT, I.M. & ROCHA, M.J. (1970) Complement fixation by a two-component antibody system: immunoglobulin G and immunoglobulin M anti-globulin (rheumatoid factor) J. exp. Med. 132, 673. SCHROBENLOHER, R.E., KUNKEL, H.G. & TOMASI, R.B. (1964) Activity of dissociated and reassociated 19s anti-y-globulins. J. exp. Med. 120, 1215. SHIRODARIA, P.V., FRASER, K.B. & STANFORD, F. (1973) Secondary fluorescent staining of virus antigens by rheumatoid factor and fluorescein-conjugated anti-IgM. Ann. rheum. Dis. 32, 53. SHULMAN, N.R. (1970) Hepatitis-associated antigen. Amer. J. Med. 49, 669. SHULMAN, N.R. & BARKER, L.F. (1969) Virus-like antigen, antibody and antigen-antibody complexes in hepatitis measured by complement fixation. Science, 165, 304. SINGER, J.M. & PLOTZ, C.M. (1956) Latex fixation test. I. Application to serologic diagnosis of rheumatoid arthritis. Amer. J. Med. 21, 888. SINGER, J.M., PLOTZ, C.M., PERALTA, F.M. & LYONS, H.C. (1962) The presence of anti-gamma globulin factors in sera of patients with active pulmonary tuberculosis. Ann. intern. Med. 56, 545. STATE, I., RUNGAN, V., VOLEULESCU, M. & STATE, D. (1973) Rheumatoid factor and Australia antigen in acute and chronic hepatitis. Rev. Roum. Med. interne, 10, 29. SVEC, K.H. & DINGLE, J.H. (1965) The occurrence of rheumatoid factor in association with antibody response to influenza A2 (Asian) virus. Arthr. and Rheum. 8, 524. VYAS, G.N. & SHULMAN, N.R. (1970) Hemagglutination assay for antigen and antibody associated with viral hepatitis. Science, 170, 332. WILLIAMS, R.C. & KUNKEL, H.G. (1962) Rheumatoid factor, complement and conglutinin aberrations in patients with sub-acute bacterial endocarditis. J. clin. Invest. 41, 666. ZIEGENFUSS, J.F., MILLER, J. & ROSSMAN, D. (1971) Rheumatoid factor and Australia antigen. New Engl. J. Med. 284,1104.
THE INTERACTION OF RHEUMATOID FACTOR WITH HEPATITIS B SURFACE ANTIGEN-ANTIBODY COMPLEXES J. A. MARKENSON,* C. A. DANIELS,t A. L. NOTKINS,t J. H. HOOFNAGLE,§ J. GERETY§ AND L. F. BARKER§ tDepartment of Pathology, Duke University Medical Center, Durham, North Carolina; I Laboratory of Oral Medicine, National Institute of Health, Bethesda, Maryland; § Division of Blood and Blood Products, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland, U.S.A. (Received 3 July 1974) SUM MARY
A solid phase radioimmunoassay was developed in which the hepatitis B surface antigen was adsorbed to the surface of plastic beads. When the antigen-coated beads were incubated with human IgG antibody against hepatitis B surface antigen, immune complexes were formed on the solid phase surface. IgM rheumatoid factor was found to bind to the hepatitis B surface antigen-antibody complexes but not to the antigen or the IgG antibody alone. Since both hepatitis B surface antigenantibody complexes and rheumatoid factor are commonly present in type B viral hepatitis, it is suggested that rheumatoid factor may play a role in the pathogenesis of this viral disease in man.
INTRODUCTION An antigen which forms the outer coat or surface of the hepatitis B virus (HBV), termed hepatitis B surface antigen (HB.Ag), is found transiently in the acute phase sera of most patients with type B viral hepatitis (Blumberg, Alter & Visnich, 1965; Shulman, 1970). In the chronic HBV carrier state, HBSAg may be detected in the serum for an extensive period of time (Krugman & Giles, 1970). Antigen-antibody (HBAg-Ab) complexes (Shulman & Barker, 1969) have also been found in the sera of patients with acute type B viral hepatitis with renal (Nowoslawski et al., 1972) and vascular (Gocke et al., 1970) lesions attributed to the deposition of these immune complexes. Infectious virus-antibody (VA) complexes can be found in the circulation of animals chronically infected with a number of other viruses (Hirsch, Allison & Harvey, 1969; Notkins et al., 1966; Oldstone & Dixon, 1970). Similarly renal (Oldstone & Dixon, 1971), * Present address: Department of Medicine, Cornell University Medical College, New York, New York 10021, U.S.A. Correspondence: Dr Charles A. Daniels, Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, U.S.A.
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J. A. Markenson et al.
hepatic, and vascular (Henson et al., 1969) lesions have been attributed to the deposition of these immune complexes. Infectious VA complexes can be neutralized by anti-immunoglobulin antisera (Notkins et al., 1966; Ashe & Notkins, 1966; Porter & Larsen, 1967) including rheumatoid factor (RF) (Ashe et al., 1971; Gipson, Daniels & Notkins, 1974). Recently we have reported that RF is found in the sera of 58.6% of patients with type B viral hepatitis (Hoofnagle et al., 1974). Since both RF and HBAb-Ag complexes have been shown to occur in type B viral hepatitis, the possibility exists that RF might interact with HBSAg-Ab complexes in vivo and play a role in this human disease. The present study was initiated to determine if RF would attach to HBSAg-Ab complexes in vitro and what serum factors would potentiate or inhibit this interaction. MATERIALS AND METHODS RF assay RF was detected by agglutination of human gamma-globulin-coated latex particles (RA Test, Hyland Laboratories). All assays were done in triplicate and sera with titres of 1 :20 or greater were considered positive for RF.
HBSAg The presence of HB.Ag was detected by counter electrophoresis (Gocke & Howe, 1970) (CEP), and radioimmunoassay (Ling & Overby, 1972) (RIA). To prepare purified HB.Ag, antigen-containing sera were layered on the top of a 25-50% sucrose gradient in a BXIV zonal rotor and centrifuged for 24 hr at 30,000 rev/min at 250C. The antigen-positive fractions were pooled and banded two additional times on 13-60% sucrose gradients (30,000 rev/min for 6 hr at 250C). This purified HBAg preparation was stored in aliquots at -20'C until used. Unless otherwise stated, dilutions were made in 0 14 M sodium chloride solution containing 0 005 M phosphate buffer (PBS), pH 7 4. Hepatitis B antibody Antibody to hepatitis B surface antigen (anti-HB,) was measured by passive haemagglutination (Vyas & Shulman, 1970). Unless otherwise specified, sera from multipletransfused haemophiliac patients were used as the source of anti-HBs. The IgG and IgM, serum fractions containing anti-HBs activity were obtained from haemophiliac sera by DEAE-cellulose chromatography. The immunoglobulin fractions were lyophilized and used in experiments at 10 mg/ml in PBS. Two sources of paired sera (preinfection and convalescent) also were studied. One source was a physician (R.G.), who seroconverted prior to the study; the other source was two chimpanzees experimentally infected with HBSAg-positive serum (Markenson et al., 1973). Prior to use, sera were heated to 56°C for 30 min and were shown to be free of RF. Isolation of RF RF was isolated from the sera of patients with rheumatoid arthritis by Sephadex G-200 chromatography (Schrohenloher, Kunkel & Tomasi, 1964). The peak of IgM fraction (10 mg/ml) had a latex titre of 1: 1000 and did not contain anti-HBs activity. In this study, RF was isolated from three different patients and each preparation exhibited the properties described in the experiments. RF, HBsAg and anti-HBs were labelled with 1251 by the chloramine T method (Hunter & Greenwood, 1962).
NWy~I
Rheumatoid factor and HBsAg
211
Solid phase radioimmunoassay Six millimetre, round, plastic beads (Geiger's Outlet, Pasadena, California) were used as the solid phase immunoadsorbent. The beads were coated by incubating overnight at 250C with HBSAg, or IgM anti-HBs, or normal human serum (containing neither the HBAg nor anti-HBs). The coated beads then were washed five times with PBS and used the same day. Beads, coated with either dilutions of HBSAg or normal human serum (control), were placed in separate 75 x 100 mm glass tubes and incubated overnight at 250C with 0-2 ml of 125I-labelled anti-HBs (200,000 ct/min). The beads were washed five times with PBS and the results expressed as ct/min of labelled antibody bound. Each value represents the average of two or more determinations on duplicate samples.
Attachment of 125'-labelled RF to immune complexes HBSAg-coated beads were incubated at 250C overnight with 0-2 ml dilutions of anti-HBS or normal (nonimmune) serum or PBS (control). The fluid was removed and the beads washed five times with PBS. 0-2 ml of 125I-labelled RF (200,000 ct/min) was incubated with each bead overnight at 250C, and the washed beads were counted for radioactivity. The results were expressed either as the counts of 125I-labelled RF attached or as a binding ratio (ct/min of 125I-labelled RF bound to beads treated with antibody divided by ct/min bound to beads treated with nonimmune serum). Blocking studies were performed by using unlabelled RF to inhibit the attachment of 125I-labelled RF. Beads containing complexes on their surface were incubated overnight first with unlabelled RF, followed by incubation with the same preparation labelled with 125i. RESULTS
Formation of immune complexes To demonstrate that RF could attach to HBSAg-Ab complexes a technique was first developed for forming immune complexes in vitro. Plastic beads were coated with various concentrations of purified HBsAg and then incubated with 125I-labelled anti-HBS. The data in Fig. 1 show that beads exposed to a 1 :10 dilution of the HBSAg preparation bound an appreciable quantity of 125I-labelled anti-HB.Ag (5000 ct/min). At lower concentrations 0~~~~~~~~~~~~~~~~~~ mDX0 _
2
0
Control
10-6
10-5
FIG. 1. Attachment of 125I-labelled
10-4
10-3
10lo-2
Dilution of HBSAg
anti-HB. to beads coated with dilutions of HBAg.
J. A. Markenson et al.
212
of HBSAg the beads bound considerably less anti-HB,. Beads coated with normal human serum did not bind appreciable amounts of 125I-labelled anti-HBs. Attachment of RF to immune complexes When different concentrations of HBSAg coated beads were incubated with excess unlabelled anti-HBS and then exposed to '25I-labelled RF, greater than 1000 ct/min of
/
E
_0
DO 4 _-A .0
Contl
10-4
0-5
10-3
1o-2
10-
Dilution of HBsAg
FIG. 2. Attachment of `25I-labelled RF to beads coated with dilutions of HBAg after incubation with either anti-HBs-containing (@) or non-immune human serum (0). TABLE 1. Binding of 125I-labelled RF to HBsAg-coated beads exposed to either preinfection or convalescent serum
I25I-labelled RF bound (ct/min) after exposure of HBSAg-coated bead* to: Sera examined Human (R.G.)
Chimpanzee (196) Chimpanzee (959)
Pre-infection serat
Convalescent serat
250 369 232
995 1045 682
Binding ratio+ 4-0 2-8 2-9
* Coated with a 1 :10 dilution of HbsAg in PBS. t Incubated with a 1 :10 dilution of serum.
I Ratio determined by dividing ct/min bound in presence of convalescent serum by ct/min bound in presence of preinfection serum.
radioactivity attached to the immune complexes on the bead surface (Fig. 2). At lower concentrations of HBSAg, less anti-HB, bound to the beads (see Fig. 1), and in turn the binding of RF decreased (see Fig. 2). Neither uncoated beads exposed to anti-HB, (control), nor HB.Ag-coated beads incubated with dilutions of normal (nonimmune) human serum, bound significant quantities of 1251-labelled RF (Fig. 2).
Specificity of attachment of RF Since the anti-HBS used in the previous experiments was from haemophiliac serum (which
Rheumatoid factor and HB5Ag
213
might contain a number of isoantibodies), paired sera from HBV infections were employed to establish the specificity of the RF attachment. HBAg-coated beads incubated with the human pre-infection serum and then 125"-labelled RF bound little radioactivity (Table 1). In contrast, HBSAg-coated beads exposed to convalescent (immune) serum from the same individual bound four times as much '251-labelled RF. Similarly, experiments employing paired sera from two chimpanzees showed that significant amounts of RF attached only after exposure of the beads to the convalescent sera (binding ratios of 2-8 and 2 9). TABLE 2. Inhibition* of 1251-labelled RF attachment to HBAg-anti-Hbs complexes by unlabelled RF
HBsAg bead-exposed to: First incubation
12 1-labelled RF bound
Second incubation Third incubation
Immune serum Immune serum Non-immune serum
Buffer Unlabelled RF Buffer
(ct/min)
1251-labelled RF 1251-labelled RF 1251-labelled RF
1184 253 274
* HBAg coated beads were sequentially exposed to: (1) immune or non-immune serum; (2) buffer (PBS) or unlabelled RF; and (3) 125I-labelled RF.
0
a]2.0 A v
1/1024
1/2560
A--A-- A-
1/640 1/160 1/40 Dilution of anti-HBs
4A 1/10 Undiluted
FIG. 3. Attachment of 125I-labelled RF to HBAg-coated beads after incubation with dilutions of whole serum (e), or IgG (a), or IgM anti-HB, (A). Binding ratio = (ct/min of 1251. labelled RF attached to anti-Hb,-treated beads)/(ct/min of 1251-labelled RF attached to NHStreated beads).
To further establish the specificity of RF attachment to HB.Ag-anti-HB, complexes, inhibition studies with unlabelled RF were done. HBSAg-coated beads sequentially incubated with immune sera, buffer, and then 125I-labelled RF bound greater than 1100 ct/min (Table 2). HBSAg-coated beads exposed to immune sera, unlabelled RF, and then 1251-labelled RF bound less than 300 ct/min. When compared to the non-immune serum control, the unlabelled RF completely inhibited the attachment of 125I-labelled RF. Interaction of RF with different classes of antibody In order to determine which class of antibody in serum potentiated RF attachment,
J. A. Markenson et al.
214
anti-HBS-containing serum was chromatographed and the IgG and IgM fractions tested. HBSAg-coated beads were incubated with serial dilutions of: (1) anti-HBs whole serum; (2) the IgG fraction; or (3) the IgM fraction. 125I-labelled RF was added and the amount of attachment was expressed as a binding ratio (Fig. 3). With undiluted whole serum the maximal binding ratio occurred at a 1:20 dilution of whole serum. Significantly less binding occurred at higher and lower dilutions of serum. When the IgG antibody was used, the maximum RF binding ratio also occurred at about a 1:20 dilution, but in contrast to whole serum, the binding ratio did not decrease at the higher concentrations of IgG. The IgM fraction of anti-HBS did not potentiate the binding of RF at any of the nine concentrations tested. To determine if the IgM fraction contained any anti-HB, activity, HBAg-coated beads were incubated with serial dilutions of the IgM fraction or normal human serum and then 11 0 _
90
70 570
co/
,, 50_/
30_ 1.0~~~~~~~~~~~~~ I E
1/80
1/40
~
~ ~
1/20 1/10 Dilution of IgM onti-HBs
~ ~ ~~~~~~~~~~~~~~~I I
t
Undiluted
FIG. 4. Attachment of 'l25-labelled HBAg to beads coated with dilutions of IgM anti-HB,. Binding ratio = (ct/min of 'l25-labelled HbAg attached to IgM-treated beads)/(ct/min of 1251-labelled HbsAg attached to NHS-treated beads).
were exposed to 125I-labelled HBsAg. The data in Fig. 4 show that the IgM fraction contained anti-HBs activity. Eleven times as much 125I-labelled HBsAg bound to beads treated with undiluted immune IgM as to beads treated with non-immune serum. Whether the IgM anti-HBS is responsible for sterically blocking the binding of RF noted with whole serum, remains to be determined.
DISCUSSION RF is found in the sera of patients with rheumatoid arthritis (Singer & Plotz, 1956), as well as with a number of chronic diseases, e.g. tuberculosis (Singer et al., 1962), sarcoidosis (Kunkel, Simon & Fudenberg, 1958), leprosy (Cathcart et al., 1961), syphilis (Peltier & Christian, 1959), leishmaniasis (Kunkel et al., 1958), and bacterial endocarditis (Williams & Kunkel, 1962). A higher than normal incidence of RF also has been found in various types of liver disease (Bouchier, Rhodes & Sherlock, 1964) including hepatitis (State et al., 1973; Dudley, O'Shea & Sherlock, 1973; Howell, Malcolm and Pike, 1960), biliary cirrhosis, nutritional cirrhosis, hepatocellular carcinoma, and metastatic carcinoma of the
Rheumatoidfactor and HBAg
215
liver. We have reported that RF is present in 58 6% of the patients with HBAg-positive hepatitis as compared to 3%/O of controls (Hoofnagle et al., 1974). Since, however, 58 6% of patients with acute liver disease in the absence of HBAg also had elevated RF titres, RF cannot be used in the diagnosis of acute hepatitis B virus infection as has been suggested by others (Ziegenfuss, Miller & Rossman, 1971). What is the reason for the elaboration of RF in these chronic human diseases? One of the most reliable methods of inducing RF-like antibodies is by repeated immunization of animals with killed bacteria (Abruzzo & Christian, 1961) or soluble proteins (Aho and Wagner, 1967). Antibodies first develop against the exogenous antigens and then it is thought that RF-like antibodies are elaborated against the host's own altered IgG which has attached to the antigen in the form of an immune complex. Indeed, injections of antigenantibody complexes into guinea-pigs and rabbits stimulate RF production (Adler, 1956). From these animal studies, the presence of RF in human diseases has been regarded as evidence for the presence of immune complexes (Gell & Kelus, 1967). In acute type B hepatitis, viral antigens may be present in the circulation for many weeks. Immune complexes then appear in the serum (Shulman & Barker, 1969) and finally the antigen disappears upon the emergence of free antibody. As in the animal RF models, patients may be first forming antibodies against the HB.Ag and then elaborating RF against the IgG present in the immune complexes (Shirodaria, Fraser & Stanford, 1973). Since, however, our prior study (Hoofnagle et al., 1974) showed that the same percentage of HBSAg-positive and HBAg-negative patients with acute liver disease had RF in their serum it is evident that antigens other than HBsAg must be responsible for RF formation in HBSAg-negative patients. In fact, a variety of autoimmune antibodies (antinuclear, antimitochondrial, antismooth muscle, and antihepatic) have been found in acute and chronic liver diseases. Presumably, normally hidden antigens are exposed or derepressed during hepatic injury and RF is made to the resulting immune complexes. RF has been found in the convalescent sera of patients with a number of viral infections, including influenza A (Svec & Dingle, 1965), cytomegalovirus (Langenhuysen, 1971), Rubella virus (Johnson & Hall, 1958), Epstein-Barr virus (Capra, Winchester & Kunkel, 1969), and transiently during the acute phase of several other 'viral' infections (Dresner & Trombly, 1959). Our previous studies showed that at least in vitro, RF could attach to infectious virus-antibody complexes, e.g. herpes simplex virus (Ashe et al., 1971) and vaccinia virus (Gipson et al., 1974) and assist in their neutralization. The present study shows that RF can attach to HBSAg-IgG complexes, but whether this enhances neutralization cannot be determined until a more convenient assay for infectivity of HBV is developed. In patients with viral hepatitis (Nowoslawski et al., 1972; Ekoyan et al., 1972; Kater, Van Gorp & Borst-Eilers, 1973) granular deposits of IgG, complement, and IgM have been demonstrated within vessel walls, on hepatocyte plasma membranes, and in Kupfer cells. Immunofluorescent studies (Bonomo, Tursi & Minerva, 1966) have revealed collections of RF-containing plasma cells and lymphocytes in the portal triad. In vitro studies with sensitized erythrocytes have shown that RF can either potentiate or inhibit the amount of complement-mediated cell lysis depending on the amount of antibody bound to the surface of the sensitized cells (Schmid, Roitt & Rocha, 1973). Whether the interaction of RF with immune complexes plays a protective or destructive role, or no role whatsoever in type B viral hepatitis is unknown and may depend on when in the stage of the disease (antibody excess or antigen excess) RF is elaborated. B
216
J. A. Markenson et al. ACKNOWLEDG MENTS
We are grateful to Dr D. Gordon Sharp of the University of North Carolina School of Medicine for preparing purified HBAg by zonal ultracentrifugation. This study was supported by contract number NIH-NIDR-2401 awarded to C.A.D. by the National Institute of Dental Research. We thank Miss Christine Artley for the help in the preparation of the manuscript. REFERENCES
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