Clin. exp. Immunol. (1979) 37, 213-220.
Autoimmune implications of immune complexes in clinical variants of hepatitis B H. DAUGHARTY, K. KELLEY, C. MOORE * & T. HERSH * Diagnostic Products Evaluation Branch, Biological Products Division, Bureau of Laboratories, Center for Disease Control, Public Health Service, US Department of Health, Education and Welfare, Atlanta, and * Department ofGastroenterology, Emory University Clinic and Emory University School of Medicine, Atlanta, Georgia 30322, USA
(Accepted for publication 22 December 1978)
SUMMARY
Immune complexes (IC) were investigated in sera from 208 individuals with various clinical types ofviral hepatitis diagnosed by clinical and laboratory criteria, including liver biopsy. Immune complexes were assessed by platelet aggregation (P1 A) and by radioimmunoassay (RIA). The data were related to autoimmune phenomena (especially rheumatoid factors) and to the role that the IgM class of hepatitis B (HB) antibody might have in IC formation. Although the highest frequency of P1 A was in the few sera from patients with cirrhosis or hepatoma, the next highest was in sera from acute hepatitis patients (71%), and the lowest in sera from chronic active (570%) and chronic persistent (46%) hepatitis patients. A proportional number of patients with IC's were positive for hepatitis B surface antigen (HBs). A parallel prevalence was noted between P1 A and autoantibodies, with anti-Ig's being found more frequently in sera from acute hepatitis and chronic active hepatitis patients. The relationship between RIA results for complexes and RIA results for anti-IgG was inverse, as though anti-IgG interfered with IC reactivity by RIA. Anti-IgM pre-incubated with sera increased the amount of P1 A in sera from patients with acute hepatitis as well as in those from patients with chronic persistent hepatitis, suggesting a more frequent IgM involvement in IC's in these diseases than in chronic active hepatitis. Whereas liver cell damage in acute and active hepatitis may reflect elevated autoantibodies, the IgM class of HBs antibody may be involved in acute as well as chronic persistent hepatitis.
INTRODUCTION Immune complexes affect the host immune response and vary with antigen levels (Daugharty & Gogel, 1975; Houston et al., 1974). Many laboratories are currently concerned with relating immune complexes to various pathological parameters of disease. The incidence, phase of occurrence and possible roles in pathogenesis for complexes have been delineated in some diseases, including leprosy (Taverne et al., 1976), type A and B hepatitis, myocardial infarction (Farrell et al., 1977) and cancer (Strobel, Daugharty & Hicklin, 1976). Autoantibodies against immunoglobulin G (IgG) have been implicated in altering the clinical course of disease. Chronicity of hepatitis B has implicated autoantibody against homologous IgG (Paronetto & Popper, 1976). This function of autoantibodies has been hypothesized to result from immune complexes which stimulate autoantibody production (Daugharty & Gogel, 1975) and take part in forming double antibody complexes (Daugharty & Gogel, 1976; Markenson et al., 1975). Correspondence: Dr H. Daugharty, Diagnostic Products Evaluation Branch, Biological Products Division, Bureau of Laboratories, Center for Disease Control, Public Health Service, US Department of Health, Education and Welfare, Atlanta, Georgia 30333, USA. 0099-9104/79/0800-0213$02.00 © 1979 Blackwell Scientific Publications C
213
214
H. Daugharty et al.
We have demonstrated previously (Daugharty & Gogel, 1976) the HBs antigen-antibody composition of immune complexes in HBs antigen-positive sera, as measured by the microtechnique of the platelet aggregation (P1 A) method (Myllyla, 1973). The P1 A and the liquid phase radioimmunoassay (RIA) with rheumatoid factor (Cowdery, Treadwell & Fritz, 1975) are both highly sensitive for detecting immune complexes. Equally successful methods involve complement components (Svehag, 1975) and cellular mechanisms (Theofilopoulos, Wilson & Dixon, 1976). One disadvantage of these procedures is that they detect some Ig classes preferentially. IgM-containing complexes which appear early in the disease are often not detected. Elevated IgM levels are associated with various phases of diseases (Dietz et al., 1976; Peters & Johnson, 1972) and can bind with antigen as either the primary or secondary antibody in a complex (Markenson et al., 1975; Dietz et al., 1976). We have attempted to increase the PI A sensitivity for IgM-containing complexes by pre-incubating specimens with anti-IgM. Immune complexes were quantified in sera from 208 patients with clinical variants of hepatitis B. The data obtained by immune complexes quantified by PI A and RIA were compared with anti-IgG levels and the IgM class of HB antibody involved in immune complex formation. Sera from patients with acute and chronic active hepatitis, in that order, had the highest incidence of immune complexes by P1 A. Whereas the clinical types may reflect the elevated anti-IgG in acute and chronic active hepatitis, the IgM class of HBs antibody is more evident from the P1 A enhancement in chronic persistent as well as acute hepatitis.
MATERIALS AND METHODS Specimens and reagents. The HBs antigen- and antibody-positive sera were obtained from patients with a clinical or serological history of hepatitis B infection. Positivity for antigen or antibody was determined by RIA. Positive sera for cytomegalovirus (CMV) antibody were obtained from the Center for Disease Control (CDC), Virology Division, Perinatal Virology Branch. Rocky Mountain spotted fever sera (RMSF) were obtained from the serology laboratory of the Viral and Rickettsial Products Branch, Biological Products Division, CDC. All serological specimens were maintained at - 20'C or lower until tested. The class-specific antiserum against IgM heavy chain used in the PI A pre-incubation experiments was obtained lyophilized from Cappel Laboratories, Incorporated, Downington, Pennsylvania. Standard rheumatoid serum for reaction with heat-aggregated IgG (AIgG) was obtained from the Rheumatology. Division of Internal Medicine, Grady Memorial Hospital, Atlanta, Georgia. Lyophilized human IgG, Cohn Fraction II, was obtained from the American Red Cross National Fractionation Center, Bethesda, Maryland. The globulin was suspended in borate-saline buffer (0-15 M NaCl, 0-002 M H3BO3, pH 7 4) (BSB) at a concentration of 20 mg/ml. After heating in a 630C waterbath for 12 min, the opalescent solution was filtered through a bed of Sephadex G200 equilibrated in the same buffer. The protein in the initial peak of eluted protein, monitored spectrophotometrically at 280 nm, was made more homogeneous in size by differentially centrifuging the solution at 7000 g for 30 min. The sediment was discarded to eliminate aggregates of highly polymerized IgG. Solubilized IgG aggregate molecules ranged in molecular weight from 1 to approximately 40 million (Gaarder & Michaelsen, 1974). The aggregated globulin was then radioiodinated with sodium l25iodide by the method of Greenwood, Hunter & Glover (1963). The Sephadex-purified IgG aggregates (0 5 mg) were mixed with sodium l25iodide (0.5 mCi). Oxidation was effected with chloramine-T (0 05 mg) and stopped with sodium metabisulphite (0-1 mg). The reaction mixture was filtered through Sephadex G25 to separate bound and unbound 125I. Platelet aggregation test. (i) Platelet preparation. The method of Myllyla (1973) was used for the PI A test in this study, with modifications for the hepatitis antigen-antibody system as described by Daugharty & Gogel (1976). Platelet concentrates were obtained from the American Red Cross on the day they were used. A standard suspension of washed platelets was prepared in Dulbecco phosphate buffer (0.15 M NaCl, 2-7 mM KCI, 1-5 mM KH2PO4, 8-0 mM Na2HPO4, 1-0 mm glucose, pH 7 4) without Ca and Mg. The final concentration of platelets was 1-34 x 105 per pl as measured in a Coulter counter. (ii) Platelet assay for complexes. The standard platelet suspension was added in a 0 05 ml aliquot to each well of a microtitre plate already containing two-fold dilutions of serum specimens diluted in Dulbecco buffer. After the reagents were mixed, microtitre plates were incubated at 40C for 15 hr and read in indirect lighting against a dark background. End point titres of specimens were determined as the maximum specimen dilution at which a positive agglutination settling pattern was observed. A PI A titre was considered positive if the end point was greater than 1:8. (iii) Pre-incubation with anti-IgM. When serum specimens were pre-incubated with IgM heavy chain antiserum, 25 ul of serial two-fold dilutions of test serum were mixed with an equal volume of the antiserum at a 1: 32 dilution. The mixture was held in a 370C incubator 3j hr before 50 Ip1 of the standard suspension of washed human platelets were added. After the solution had been mixed again, the microtitre plates were incubated for 15 hr at 40C. Controls of the test specimen at corresponding dilutions, without anti-IgM added, were run simultaneously to determine the enhancement or inhibition of platelet aggregating activity of the serum.
215
Immune complexes, hepatitis B, autoimmune
Radsoimmunoassay for immune complexes. The RIA method used was that of Cowdery et al. (1975) with two minor modifications. Briefly, the procedure consisted of a standard reaction between human rheumatoid factor and human AIgG radiolabelled with sodium '25iodide. Pre-incubating the specimen with the rheumatoid factor inhibits binding between the rheumatoid factor and the radiolabelled indicator. A test specimen was considered positive when inhibition of the standard reaction was > 2 standard deviations (s.d.) from the mean for ten healthy donor sera run in duplicate. Sodium sulphate precipitation of the IgG was omitted and carrier IgM was not added to the diluent for rheumatoid factor. Radioimmunoassay for anti-IgG. A liquid phase RIA similar to that of Hallgren & Wide (1977) was used to measure antiIgG in the serum. The 125I-labelled AIgG indicator reactive by direct precipitation by anti-IgG was determined as follows: the reaction between 0-2 ml of the serum specimen at 1: 20 dilution and 0-2 ml of a dilution of the 125I-IgG with 2-3000 cpm was allowed to proceed for 40-48 hr at 40C. BSB was added to the lOx 75 mm glass tubes and centrifuged at 7000 g for 30 min. Aspirated supernatants and corresponding precipitates were counted for radioactivity. The isotope precipitated from the total amount initially present in the mixture was calculated as a percentage. Samples in which these percentages differed > 2 s.d. from the mean for negative control sera from healthy donors were considered positive for anti-IgG. Aggregated IgG with selected molecular weight heterogeneity was used as a standard positive control for the PI A test and as the radiolabelled indicator in the RIA for complexes, and in the RIA for anti-IgG was ten to fifteen times that of conventional latex agglutination methods. Viral antigen and antibody assays. Tests for HBs antigen and antibody were performed by radioimmunoassay. Antibody against Rocky Mountain spotted fever was detected by the Laboratory Branch Complement Fixation method for complement fixation (CF) (Casey, 1965). The micromethod for CF was used for block (or grid) titration of varying dilutions of antigen with varying dilution of antibody. The method of titrating cytomegalovirus (CMV) antibodies has been reported previously (Knez et al., 1976). Ig class-specific radiolabelled indicators were used in the RIA for CMV antibody.
RESULTS Platelet aggregation activity in clinical variants of hepatitis B Sera from clinical variants of hepatitis B cases confirmed by serological positivity were evaluated by PI A for the presence of immune complexes. The range of cases varied from the acute through to the active and persistent (but inactive) chronic types, including several cases of hepatoma and cirrhosis. (a) 100
(b)
(C)
i
80 70.? 60 0 OL
5 0[
E
401)30
20
I0 0I
Autoimmune implications of immune complexes in clinical variants of hepatitis B H. DAUGHARTY, K. KELLEY, C. MOORE * & T. HERSH * Diagnostic Products Evaluation Branch, Biological Products Division, Bureau of Laboratories, Center for Disease Control, Public Health Service, US Department of Health, Education and Welfare, Atlanta, and * Department ofGastroenterology, Emory University Clinic and Emory University School of Medicine, Atlanta, Georgia 30322, USA
(Accepted for publication 22 December 1978)
SUMMARY
Immune complexes (IC) were investigated in sera from 208 individuals with various clinical types ofviral hepatitis diagnosed by clinical and laboratory criteria, including liver biopsy. Immune complexes were assessed by platelet aggregation (P1 A) and by radioimmunoassay (RIA). The data were related to autoimmune phenomena (especially rheumatoid factors) and to the role that the IgM class of hepatitis B (HB) antibody might have in IC formation. Although the highest frequency of P1 A was in the few sera from patients with cirrhosis or hepatoma, the next highest was in sera from acute hepatitis patients (71%), and the lowest in sera from chronic active (570%) and chronic persistent (46%) hepatitis patients. A proportional number of patients with IC's were positive for hepatitis B surface antigen (HBs). A parallel prevalence was noted between P1 A and autoantibodies, with anti-Ig's being found more frequently in sera from acute hepatitis and chronic active hepatitis patients. The relationship between RIA results for complexes and RIA results for anti-IgG was inverse, as though anti-IgG interfered with IC reactivity by RIA. Anti-IgM pre-incubated with sera increased the amount of P1 A in sera from patients with acute hepatitis as well as in those from patients with chronic persistent hepatitis, suggesting a more frequent IgM involvement in IC's in these diseases than in chronic active hepatitis. Whereas liver cell damage in acute and active hepatitis may reflect elevated autoantibodies, the IgM class of HBs antibody may be involved in acute as well as chronic persistent hepatitis.
INTRODUCTION Immune complexes affect the host immune response and vary with antigen levels (Daugharty & Gogel, 1975; Houston et al., 1974). Many laboratories are currently concerned with relating immune complexes to various pathological parameters of disease. The incidence, phase of occurrence and possible roles in pathogenesis for complexes have been delineated in some diseases, including leprosy (Taverne et al., 1976), type A and B hepatitis, myocardial infarction (Farrell et al., 1977) and cancer (Strobel, Daugharty & Hicklin, 1976). Autoantibodies against immunoglobulin G (IgG) have been implicated in altering the clinical course of disease. Chronicity of hepatitis B has implicated autoantibody against homologous IgG (Paronetto & Popper, 1976). This function of autoantibodies has been hypothesized to result from immune complexes which stimulate autoantibody production (Daugharty & Gogel, 1975) and take part in forming double antibody complexes (Daugharty & Gogel, 1976; Markenson et al., 1975). Correspondence: Dr H. Daugharty, Diagnostic Products Evaluation Branch, Biological Products Division, Bureau of Laboratories, Center for Disease Control, Public Health Service, US Department of Health, Education and Welfare, Atlanta, Georgia 30333, USA. 0099-9104/79/0800-0213$02.00 © 1979 Blackwell Scientific Publications C
213
214
H. Daugharty et al.
We have demonstrated previously (Daugharty & Gogel, 1976) the HBs antigen-antibody composition of immune complexes in HBs antigen-positive sera, as measured by the microtechnique of the platelet aggregation (P1 A) method (Myllyla, 1973). The P1 A and the liquid phase radioimmunoassay (RIA) with rheumatoid factor (Cowdery, Treadwell & Fritz, 1975) are both highly sensitive for detecting immune complexes. Equally successful methods involve complement components (Svehag, 1975) and cellular mechanisms (Theofilopoulos, Wilson & Dixon, 1976). One disadvantage of these procedures is that they detect some Ig classes preferentially. IgM-containing complexes which appear early in the disease are often not detected. Elevated IgM levels are associated with various phases of diseases (Dietz et al., 1976; Peters & Johnson, 1972) and can bind with antigen as either the primary or secondary antibody in a complex (Markenson et al., 1975; Dietz et al., 1976). We have attempted to increase the PI A sensitivity for IgM-containing complexes by pre-incubating specimens with anti-IgM. Immune complexes were quantified in sera from 208 patients with clinical variants of hepatitis B. The data obtained by immune complexes quantified by PI A and RIA were compared with anti-IgG levels and the IgM class of HB antibody involved in immune complex formation. Sera from patients with acute and chronic active hepatitis, in that order, had the highest incidence of immune complexes by P1 A. Whereas the clinical types may reflect the elevated anti-IgG in acute and chronic active hepatitis, the IgM class of HBs antibody is more evident from the P1 A enhancement in chronic persistent as well as acute hepatitis.
MATERIALS AND METHODS Specimens and reagents. The HBs antigen- and antibody-positive sera were obtained from patients with a clinical or serological history of hepatitis B infection. Positivity for antigen or antibody was determined by RIA. Positive sera for cytomegalovirus (CMV) antibody were obtained from the Center for Disease Control (CDC), Virology Division, Perinatal Virology Branch. Rocky Mountain spotted fever sera (RMSF) were obtained from the serology laboratory of the Viral and Rickettsial Products Branch, Biological Products Division, CDC. All serological specimens were maintained at - 20'C or lower until tested. The class-specific antiserum against IgM heavy chain used in the PI A pre-incubation experiments was obtained lyophilized from Cappel Laboratories, Incorporated, Downington, Pennsylvania. Standard rheumatoid serum for reaction with heat-aggregated IgG (AIgG) was obtained from the Rheumatology. Division of Internal Medicine, Grady Memorial Hospital, Atlanta, Georgia. Lyophilized human IgG, Cohn Fraction II, was obtained from the American Red Cross National Fractionation Center, Bethesda, Maryland. The globulin was suspended in borate-saline buffer (0-15 M NaCl, 0-002 M H3BO3, pH 7 4) (BSB) at a concentration of 20 mg/ml. After heating in a 630C waterbath for 12 min, the opalescent solution was filtered through a bed of Sephadex G200 equilibrated in the same buffer. The protein in the initial peak of eluted protein, monitored spectrophotometrically at 280 nm, was made more homogeneous in size by differentially centrifuging the solution at 7000 g for 30 min. The sediment was discarded to eliminate aggregates of highly polymerized IgG. Solubilized IgG aggregate molecules ranged in molecular weight from 1 to approximately 40 million (Gaarder & Michaelsen, 1974). The aggregated globulin was then radioiodinated with sodium l25iodide by the method of Greenwood, Hunter & Glover (1963). The Sephadex-purified IgG aggregates (0 5 mg) were mixed with sodium l25iodide (0.5 mCi). Oxidation was effected with chloramine-T (0 05 mg) and stopped with sodium metabisulphite (0-1 mg). The reaction mixture was filtered through Sephadex G25 to separate bound and unbound 125I. Platelet aggregation test. (i) Platelet preparation. The method of Myllyla (1973) was used for the PI A test in this study, with modifications for the hepatitis antigen-antibody system as described by Daugharty & Gogel (1976). Platelet concentrates were obtained from the American Red Cross on the day they were used. A standard suspension of washed platelets was prepared in Dulbecco phosphate buffer (0.15 M NaCl, 2-7 mM KCI, 1-5 mM KH2PO4, 8-0 mM Na2HPO4, 1-0 mm glucose, pH 7 4) without Ca and Mg. The final concentration of platelets was 1-34 x 105 per pl as measured in a Coulter counter. (ii) Platelet assay for complexes. The standard platelet suspension was added in a 0 05 ml aliquot to each well of a microtitre plate already containing two-fold dilutions of serum specimens diluted in Dulbecco buffer. After the reagents were mixed, microtitre plates were incubated at 40C for 15 hr and read in indirect lighting against a dark background. End point titres of specimens were determined as the maximum specimen dilution at which a positive agglutination settling pattern was observed. A PI A titre was considered positive if the end point was greater than 1:8. (iii) Pre-incubation with anti-IgM. When serum specimens were pre-incubated with IgM heavy chain antiserum, 25 ul of serial two-fold dilutions of test serum were mixed with an equal volume of the antiserum at a 1: 32 dilution. The mixture was held in a 370C incubator 3j hr before 50 Ip1 of the standard suspension of washed human platelets were added. After the solution had been mixed again, the microtitre plates were incubated for 15 hr at 40C. Controls of the test specimen at corresponding dilutions, without anti-IgM added, were run simultaneously to determine the enhancement or inhibition of platelet aggregating activity of the serum.
215
Immune complexes, hepatitis B, autoimmune
Radsoimmunoassay for immune complexes. The RIA method used was that of Cowdery et al. (1975) with two minor modifications. Briefly, the procedure consisted of a standard reaction between human rheumatoid factor and human AIgG radiolabelled with sodium '25iodide. Pre-incubating the specimen with the rheumatoid factor inhibits binding between the rheumatoid factor and the radiolabelled indicator. A test specimen was considered positive when inhibition of the standard reaction was > 2 standard deviations (s.d.) from the mean for ten healthy donor sera run in duplicate. Sodium sulphate precipitation of the IgG was omitted and carrier IgM was not added to the diluent for rheumatoid factor. Radioimmunoassay for anti-IgG. A liquid phase RIA similar to that of Hallgren & Wide (1977) was used to measure antiIgG in the serum. The 125I-labelled AIgG indicator reactive by direct precipitation by anti-IgG was determined as follows: the reaction between 0-2 ml of the serum specimen at 1: 20 dilution and 0-2 ml of a dilution of the 125I-IgG with 2-3000 cpm was allowed to proceed for 40-48 hr at 40C. BSB was added to the lOx 75 mm glass tubes and centrifuged at 7000 g for 30 min. Aspirated supernatants and corresponding precipitates were counted for radioactivity. The isotope precipitated from the total amount initially present in the mixture was calculated as a percentage. Samples in which these percentages differed > 2 s.d. from the mean for negative control sera from healthy donors were considered positive for anti-IgG. Aggregated IgG with selected molecular weight heterogeneity was used as a standard positive control for the PI A test and as the radiolabelled indicator in the RIA for complexes, and in the RIA for anti-IgG was ten to fifteen times that of conventional latex agglutination methods. Viral antigen and antibody assays. Tests for HBs antigen and antibody were performed by radioimmunoassay. Antibody against Rocky Mountain spotted fever was detected by the Laboratory Branch Complement Fixation method for complement fixation (CF) (Casey, 1965). The micromethod for CF was used for block (or grid) titration of varying dilutions of antigen with varying dilution of antibody. The method of titrating cytomegalovirus (CMV) antibodies has been reported previously (Knez et al., 1976). Ig class-specific radiolabelled indicators were used in the RIA for CMV antibody.
RESULTS Platelet aggregation activity in clinical variants of hepatitis B Sera from clinical variants of hepatitis B cases confirmed by serological positivity were evaluated by PI A for the presence of immune complexes. The range of cases varied from the acute through to the active and persistent (but inactive) chronic types, including several cases of hepatoma and cirrhosis. (a) 100
(b)
(C)
i
80 70.? 60 0 OL
5 0[
E
401)30
20
I0 0I
