Clin. exp. Immunol. (1978) 34, 318-325.
Serial studies on circulating immune complexes in post-streptococcal sequelae I. VAN DE RIJN, H. FILLIT, W. E. BRANDEIS, H. REID, T. POON-KING, M. McCARTHY, N. K. DAY & J. B. ZABRISKIE The Rockefeller University and the Sloan-Kettering Institute, New York, and the Caribbean Epidemiology Center and Streptococcal Disease Unit, San Fernando General Hospital, Trinidad and Tobago
(Received 29 June 1978) SUMMARY
Levels of circulating immune complexes were determined in the Trinidadian population with a high incidence of acute post-streptococcal sequelae, acute rheumatic fever and acute poststreptococcal glomerulonephritis. The studies were done on acute phase and serial bleedings up to 6 months after the acute illness. Clq solid phase assay employing radiolabelled protein A and the Raji cell radioimmunoassay were used in the detection of immune complexes. Both groups of patients had significantly elevated levels of immune complexes during the acute phase and showed an equivalent decline to slightly elevated levels during the 6 months of observation. The incidence and molecular size of the immune complexes in the two post-streptococcal sequelae were equivalent. The Raji cell assay tended to show much higher levels of complexes on an absolute basis, but both assays demonstrated an equivalent decline from the acute phase. These studies suggest that the presence of equivalent levels of complexes in two entirely different post-streptococcal sequelae indicates that other factors, such as the nature of the antigen in the complex, are more important in the pathogenesis of the post-streptococcal sequelae than the actual presence or the absolute levels of complexes.
INTRODUCTION The development of a number of assays for the detection of immune complexes in man has led to the observation that circulating immune complexes may be detected in many disease states (Luthra et al., 1975; Nydegger et al., 1974; Gabay et al., 1977; Stagno et al., 1977; Ward & Kibukamusoke, 1969; Bayer et aL, 1976; Gabriel & Agnello, 1977; Stuhlinger, Verroust & Morel-Maroger, 1976; Ooi, Ooi & Pollak, 1977). In particular, it has been established that immune complexes are present in a majority of cases of patients with acute post-streptococcal glomerulonephritis (PSGN) (Ooi, Vallota & West, 1977; Digeon et al., 1977; Woodroffe et al., 1977; Rossen et al., 1976). These reports have generally covered a small number of patients and have employed sera taken during the acute stage of the disease. In the studies reported, sera obtained from patients with other post-streptococcal sequelae were not included. In acute experimental immune complex disease in animals, it has been demonstrated that the titres of immune complexes rise during the acute phase and return to normal levels during the succeeding days (Wilson & Dixon, 1970). In an attempt to elaborate on the study of circulating immune complexes in acute PSGN, we studied forty-three of these patients in Trinidad, employing acute phase and serial sera for a period of 6 months following the acute stage of the disease. In addition, patients with acute rheumatic fever (ARF), impetigo (with documentation of Group A streptococcal infection) and normal healthy controls were also part of this study. Correspondence: Dr. I. van de Rijn, The Rockefeller University, New York, NY 10021, USA. 0099-9104/78/0120-0318$02.00 (© 1978 Blackwell Scientific Publications
318
Post-streptococcal sequelae
319
Finally, we determined the sedimentation coefficient of the immune complexes in the two poststreptococcal sequelae by sucrose density centrifugation.
MATERIALS AND METHODS Clinical material. All patients and normal controls (n = 117) used in the study were from the island of Trinidad. Previous work there has demonstrated that approximately 200 cases of acute rheumatic fever (ARF) and 400 cases of acute poststreptococcal glomerulonephritis (PSGN) occur simultaneously each year in the island population. Acute rheumatic fever. Acute rheumatic fever was diagnosed on the basis of the American Heart Association modified Jones criteria (American Heart Association, Council on Rheumatic Fexer and Congenital Heart Disease, 1965). The thirty-six patients with ARF used in this study had clinical evidence of either carditis and/or arthritis and were aged 7-15 years. Laboratory tests demonstrated elevated anti-streptolysin 0 (ASO) antibody titres, positive C-reactixe protein (CRP) and elevated sedimentation rates. None of these patients had proteinuria. Acute post-streptococcal glomerulonephritis. Forty-three patients were studied. The clinical signs and symptoms of poststreptococcal 'skin' glomerulonephritis haxe been described by Poon-King et al. 1967. The common site of infection is the skin with streptococcal infection developing secondary to mosquito bites or scabies. In our studies, more than 80% of patients had Group A streptococci isolated from skin lesions. Clinical signs of acute PSGN were edema, hypertension, oliguria, proteinuria and haematuria. In addition, serum C3 levels were less than 70 mg 0 0 in this population of patients, and the average age of onset was slightly lower than that of acute rheumatic fever patients (aged 3-10 years). Impetigo. The third group was composed of seventeen children with impetigo (aged 5-9 years). In these children, Group A streptococci were isolated from skin sores and, in some cases, from the throat. No protein was found in the urine of these children. Controls. The final group was composed of twenty-one normal, healthy children who had no signs of streptococcal infections (aged 5-9 years). Visible skin sores were absent and ASO levels were normal. Collection and storage of sera. Blood samples were obtained by routine venipuncture and collected in glass Vacutainer tubes and allowed to clot at 24°C for 1-2 hr. The serum was then separated by centrifugation at 800 g for 10 min, aliquoted and stored at both - 20'C and -70'C until tested in the immune complex assay. For determination of C3, only serum kept at -70'C was used. Solid phase Clq assay. The procedure used was basically that of Hay, Nineham & Roitt (1976), as modified by Walker et al. (1978) with the following changes. 1 0 ml volumes of human Clq, isolated by the method of Yonemasu & Stroud (1971) at 10 mg per litre in 0 05 M phosphate buffered saline (PBS) (pH 7-6), were incubated in polystyrene tubes (10 x 75 mm Falcon) for 18 hr at 40C. Subsequently, the tubes were washed three times with PBS and incubated at room temperature for 2 hr with 0.2% gelatin in 0 01 M PBS, pH 7-6. After a further wash with 0-0500 Tween 20 in PBS (PBS/Tween), the tubes were used immediately in the immune complex assay. During the above 2 hr incubation, 50 p1 of test serum were mixed with 100 P1 of 0-2 M (ethylenedinitrilo)-tetraacetic acid pH 7.5 (EDTA) and incubated for 30 min at 37 C and then transferred to an ice-bath. Duplicate samples (50 pI) were placed in Clq coated plastic tubes together with 950 Al of PBS/Twxeen and incubated for 60 min at 370C, followed by 30 min at 4 C. Unbound proteins were then removed by washing three times with cold PBS/Tween. Finally, the immune complexes were detected by adding approximately 50 ng of 125I-labelled protein A in 1 0 ml PBS/ Tuween to the tubes. The tubes were then incubated at 37°C for 1 hr, followed by incubation at 4 C for 30 min. Unbound radioactixely-labelled protein A was removed by washing fixve times with cold PBS/Txxeen. The tubes were then counted in a gamma spectrophotometer. Each time the assay was run, a standard curve of aggregated human gamma globulin was included as well as fixe normal serum controls. Values x ere considered valid only if the duplicate counts were within 5 0%0of each other. All X alues which had a greater variability than 5% were repeated. The immune complex levels in the serum samples are expressed as ug of aggregated human gamma globulin equivalent per ml of serum sample. Serum samples were considered positive if their values were greater than 2 standard deviations above that of normal control values. The Raji cell radioimmunoassay. The Raji cell radioimmunoassay for the determination of circulating immune complexes was carried out according to the method of Theofilopoulos, Wilson & Dixon (1976). 2 x 106 Raji cells in 50ul Eagle's minimum essential medium (MEM) were reacted with 25 pl of a 1:4 dilution in saline (NSS) of the serum to be tested. After an incubation period of 45 min at 37°C with gentle shaking, the cells were washed three times with MEM, reacted (30 min, 4 C) with an optimum amount of a 1:2 dilution of '25I-rabbit anti-human IgG in MEM containing 1% human serum albumin (MEM-HSA), and gently shaken. The cells were then washed three times with MEM-HSA, and radioactivity in the cell pellet determined in a gamma counter. The amount of uptake was referred to a standard curve of radioactive antibodv uptake by cells incubated with 25 p1 of a 1:4 final dilution of normal human serum (NHS), freshly obtained or stored at - 70'C, or of a pool of twenty NHS (sources of complement) to which various amounts (from 4 pug to 10 ng) of aggregated human gamma globulin (AHG) had been added. The mixture of NHS or pooled NHS with various amounts of AHG were pre-incubated for 30 min at 37°C before being added to cells. The amount of complexes in each serum tested was expressed as pg AHG equivalent per ml of serum. A serum was considered positive if it contained a value of immune complexes greater than 16 ,ug/ml. Cell viability was determined by trypan blue exclusion. The cells in suspension used in our experiments were always passaged 48-72 hr in cell culture.
I. van de Rijn et al.
320
Preparations of aggregated human gamma globulin. Human IgG was obtained from Cohn Fraction II (Sigma Chemical Co., St. Louis, Missouri) followed by fractionation on diethyl-amino-ethyl (DEAE)-52 cellulose chromatography using 0-02 M sodium phosphate buffer pH 7-2. The IgG isolated by this method was centrifuged at 40,000 g for 90 min to remove aggregated IgG and the upper third was used for heat aggregation. The concentration of the human gamma globulin was then adjusted to 5-7 mg/ml and finally heated at 630C for 30 min and aliquoted for storage at - 70'C. Aliquots of the aggregated human gamma globulin were used for up to 1 month after preparation, whereupon new batches were made. Radioiodination ofprotein A. Protein A was obtained from Pharmacia Fine Chemicals (Piscataway, New Jersey) and radiolabelled with chloramine T according to the method of McConahey & Dixon (1966). Free iodine was removed from the radiolabelled protein A by chromatography on Sephadex G25 columns. The radiolabelled protein A was then used within 2 weeks of the time of labelling. Radioiodination ofrabbit anti-human IgG. Radioiodination of rabbit anti-human IgG 125I (New England Nuclear, Boston, Massachussetts) was performed using the chloramine T method according to McConahey & Dixon (1966). The labelled anti-human IgG was extensively dialyzed against PBS, pH 7-2 for 38 hr. The specific activity of the 125I-rabbit anti-human IgG (0 3 mg/ml) was 0 05-02 ,pCi/pug protein. Sucrose-density gradient centrifugation. 200 p1 of selected sera were applied to 5 0 ml of 10-35% sucrose gradient in 0-02 M sodium phosphate buffer pH 7-2. Radiolabelled standards 7S (IgG), 11S (Clq) and 19S (IgM) were run in addition to the test samples. All samples were run in duplicate. The gradients were centrifuged at 150,000 g in a Beckman 50-1 rotor for 17 hr at 7°C. Fractions (7 drops) were collected and assayed for the presence of immune complexes. Detection ofimmune complexes in the sucrose density ultracentrifugation gradient fractions. The fractions were tested by the Raji cell radioimmunoassay described above. The only modification was the use of 75 p1 NHS plus NSS, rather than NSS alone, to dilute 25 p1 of the fractions 1:4. Assay fir serum C3 levels. Serum C3 levels were determined using the Mancini radial immunodiffusion technique (Mancini, Carbonara & Heremans, 1965). All samples were done in duplicate using the aliquots stored at - 70°C for the immune complex assay.
RE SULTS Analysis ofcontrol and acute phase sera for immune complexes Sera from patients with acute rheumatic fever, acute post-streptococcal glomerulonephritis, impetigo and normal controls were assayed for the presence of immune complexes by both the solid phase Clq assay and Raji cell radioimmunoassay (Table 1). Immune complexes were found in one out of twenty-one sera from normal controls by the solid phase Clq assay and in none by the Raji cell radioimmunoassay, whereas in children with Group A streptococcal associated impetigo, three out of seventeen sera were positive by the Raji cell radioimmunoassay and none in the solid phase Clq assay. When the sera from patients with post-streptococcal sequelae were tested, both assays indicated that more than 740 of the sera in both groups contained immune complexes. In patients with acute PSGN, thirty-two out of forty-three (74%0) sera were positive for immune complexes by the Clq assay, and seventeen out of twenty-one (81%) in the Raji cell radioimmunoassay. The incidence of circulating immune complexes TABLE 1. Analysis of control and acute phase sera for immune complexes
Assay
Solid phase Clq*
Raji cellt
Normals
Impetigo
Nephritics
1/21 (5%) 0/20 (0%)
0/17 (0%) 3/17 (18%)
32/43 (74%) 29/36 (81%) 17/21 (81%) 17/19 (89%)
Rheumatics
* A serum was considered positive if it had a value of immune complexes greater than 2 s.d. above the mean. t A serum was considered positive if it contained a value of immune complexes greater than 16 pg/ml.
in sera of patients with ARF by the Clq assay and the Raji cell assay were 81% and 89% respectively. It was surprising that patients with ARF had a similar incidence of immune complexes in their serum to the nephritis group, since rheumatic fever is not considered an immune complex disease. In spite of the presence of complexes in ARF patients, none of these patients had proteinuria.
Post-streptococcal sequelae
321
Analysis of serum C3 levels Since it has been demonstrated that patients with elevated circulating immune complexes have decreased levels of complement in their serum, this parameter was investigated next in the various patient populations. By using the Mancini technique, levels of C3 were assayed in the sera ofnormal controls and in patients with acute disease and 6 months later (Table 2). Both normal controls and patients with TABLE 2. Mean complement levels (C3) in patients with post-streptococcal sequelae
Rheumatic
Nephritic Normal
Impetigo
AP*
6 months
AP
6 months
(mg %)
(mg %)
(mg %)
(mg %)
(mg %)
(mg %)
115-9+4-2t
116-3+2-3
35-5+ 2-4
140-4+7-4
207-0+ 11-7 136-0+ 8-3
* Acute phase. t Standard error of the mean.
impetigo had mean C3 levels of approximately 116 mg 00. Patients with acute PSGN had decreased levels (35.5 mg %,) whereas patients with ARF had markedly increased levels (207.0 mg 0%). In both groups of patients, C3 serum levels returned to the upper limit of normal during the 6 months after the acute phase of the disease process. Analysis of absolute levels of immune complexes In an attempt to account for the extreme differences in the levels of C3 in the two post-streptococcal sequelae, absolute elevations of immune complexes in patients with post-streptococcal sequelae were analysed. There was essentially no difference in the mean levels of immune complexes in the two assays between normal controls and patients with impetigo (Table 3). By the Clq assay both groups had less TABLE 3. Mean levels of immune complexes in patients with post-streptococcal sequelae
Assay
Nephritic
Rheumatic
Acute phase 6 months
Acute phase 6 months
Normal
Impetigo
(pg/ml)
(pg/ml)
(pg/ml)
(pg/ml)
(pg/ml)
(pg/ml)
44 0 184-0
17-6 21-8
42-3
166-0
17-5 34-4
Clq
3-4
3-4
Raji
< 16-0
< 16-0
than 4-0 ug/ml, whereas by the Raji cell technique both groups had less than 16 pg of immune complexes per ml of serum. When sera from patients with acute PSGN or ARF were studied, levels of 44-0 pg and 42-3 pg, respectively, of immune complexes per ml of serum during the acute phase were demonstrated using the Clq assay. 184 pg/ml and 166 pg/ml were demonstrated using the Raji cell technique. Six months after the acute phase, both patient populations contained slightly elevated levels of immune complexes, but these were still dramatically reduced compared to the levels during the acute phase. The Raji cell radioimmunoassay was able to detect greater amounts of immune complexes in the serum of the patient groups than the Clq assay, but in both assays equivalent absolute levels were found in the two populations. Thus, serum from 740 of patients with ARF or acute PSGN contained immune complexes and these immune complexes were essentially at the same absolute level.
I. van de Rijn et al.
322
Serial study of immune complexes The kinetics of the decline of immune complexes from the patients' sera were examined in the two patient groups by assaying serum samples from both, first during the acute phase and then serially at 1 month, 3 month and 6 month intervals. The mean levels of immune complexes from the two patient groups declined at roughly the same rate so that at the end of 6 months both were slightly elevated (Fig. 1). For this set of experiments, only the data for the Clq solid phase assay is shown as similar kinetics were obtained with the Raji cell technique. ( b)
(a)
110 _
100 _
too
E 90_
._
@80_ \E 70 < 60
50 40* 01
-30 20
"
*
4
10~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. 0
AP
I
3
t 6
AP
3
I 6
Months
FIG. 1. Quantification of circulating immune complexes in serial studies of (a) acute rheumatic fever and (b) acute post-streptococcal glomerulonephritis sera using the solid phase Clq assay. Two standard deviations above the mean for the normal controls in this assay is 8 ug equivalent AHG/ml serum. Serum samples were monitored at the acute phase (AP) and 1, 3 and 6 months thereafter.
Sucrose density gradient studies The sera of four patients, two from each post-streptococcal sequelae, were studied using sucrose density gradients in order to determine the molecular size of the immune complexes. Immune complexes were detected in the sucrose gradients by the Raji cell radioimmunoassay (Fig. 2a) and the Clq assay (Fig. 2b). Radiolabelled standards 7S (IgG), 1 iS (Clq), and 19S (IgM) were run in addition to the test samples. In all of the sera immune complexes of 19S in size or greater were demonstrated by both the assay systems. In addition, the Clq assay also showed immune complex-like material in the size range of 10-12S. Normal serum (not shown), separated by sucrose gradient and assayed by the Raji cell radioimmunoassay for immune complexes, demonstrated increased binding only in the 7S region while the Clq solid phase assay showed no significant binding throughout the gradient. The increased binding of material by the Raji cell radioimmunoassay in the 7S range is believed to be due to Fc receptors on the Raji cell (Theofilopoulos et al., 1976).
DISCUSSION Our studies suggest that sera from patients with either acute rheumatic fever or acute post-streptococcal glomerulonephritis contain comparable levels of immune complexes in the sera and that the
Post-streptococcal sequelae (a)
7S
19S
IS
323
ARF
1816- 4p
0
0~~~~~~~~
01
012 c 22 E_ i
PSGN
20 r
/\
18
/
16 14 1
l
16
(b)
l
7S
19S
IS
PSGN 2 0
no 4 a/\jr x22 E2:. I'I'I' I
I
I
I
ARF 2
16
Serial studies on circulating immune complexes in post-streptococcal sequelae I. VAN DE RIJN, H. FILLIT, W. E. BRANDEIS, H. REID, T. POON-KING, M. McCARTHY, N. K. DAY & J. B. ZABRISKIE The Rockefeller University and the Sloan-Kettering Institute, New York, and the Caribbean Epidemiology Center and Streptococcal Disease Unit, San Fernando General Hospital, Trinidad and Tobago
(Received 29 June 1978) SUMMARY
Levels of circulating immune complexes were determined in the Trinidadian population with a high incidence of acute post-streptococcal sequelae, acute rheumatic fever and acute poststreptococcal glomerulonephritis. The studies were done on acute phase and serial bleedings up to 6 months after the acute illness. Clq solid phase assay employing radiolabelled protein A and the Raji cell radioimmunoassay were used in the detection of immune complexes. Both groups of patients had significantly elevated levels of immune complexes during the acute phase and showed an equivalent decline to slightly elevated levels during the 6 months of observation. The incidence and molecular size of the immune complexes in the two post-streptococcal sequelae were equivalent. The Raji cell assay tended to show much higher levels of complexes on an absolute basis, but both assays demonstrated an equivalent decline from the acute phase. These studies suggest that the presence of equivalent levels of complexes in two entirely different post-streptococcal sequelae indicates that other factors, such as the nature of the antigen in the complex, are more important in the pathogenesis of the post-streptococcal sequelae than the actual presence or the absolute levels of complexes.
INTRODUCTION The development of a number of assays for the detection of immune complexes in man has led to the observation that circulating immune complexes may be detected in many disease states (Luthra et al., 1975; Nydegger et al., 1974; Gabay et al., 1977; Stagno et al., 1977; Ward & Kibukamusoke, 1969; Bayer et aL, 1976; Gabriel & Agnello, 1977; Stuhlinger, Verroust & Morel-Maroger, 1976; Ooi, Ooi & Pollak, 1977). In particular, it has been established that immune complexes are present in a majority of cases of patients with acute post-streptococcal glomerulonephritis (PSGN) (Ooi, Vallota & West, 1977; Digeon et al., 1977; Woodroffe et al., 1977; Rossen et al., 1976). These reports have generally covered a small number of patients and have employed sera taken during the acute stage of the disease. In the studies reported, sera obtained from patients with other post-streptococcal sequelae were not included. In acute experimental immune complex disease in animals, it has been demonstrated that the titres of immune complexes rise during the acute phase and return to normal levels during the succeeding days (Wilson & Dixon, 1970). In an attempt to elaborate on the study of circulating immune complexes in acute PSGN, we studied forty-three of these patients in Trinidad, employing acute phase and serial sera for a period of 6 months following the acute stage of the disease. In addition, patients with acute rheumatic fever (ARF), impetigo (with documentation of Group A streptococcal infection) and normal healthy controls were also part of this study. Correspondence: Dr. I. van de Rijn, The Rockefeller University, New York, NY 10021, USA. 0099-9104/78/0120-0318$02.00 (© 1978 Blackwell Scientific Publications
318
Post-streptococcal sequelae
319
Finally, we determined the sedimentation coefficient of the immune complexes in the two poststreptococcal sequelae by sucrose density centrifugation.
MATERIALS AND METHODS Clinical material. All patients and normal controls (n = 117) used in the study were from the island of Trinidad. Previous work there has demonstrated that approximately 200 cases of acute rheumatic fever (ARF) and 400 cases of acute poststreptococcal glomerulonephritis (PSGN) occur simultaneously each year in the island population. Acute rheumatic fever. Acute rheumatic fever was diagnosed on the basis of the American Heart Association modified Jones criteria (American Heart Association, Council on Rheumatic Fexer and Congenital Heart Disease, 1965). The thirty-six patients with ARF used in this study had clinical evidence of either carditis and/or arthritis and were aged 7-15 years. Laboratory tests demonstrated elevated anti-streptolysin 0 (ASO) antibody titres, positive C-reactixe protein (CRP) and elevated sedimentation rates. None of these patients had proteinuria. Acute post-streptococcal glomerulonephritis. Forty-three patients were studied. The clinical signs and symptoms of poststreptococcal 'skin' glomerulonephritis haxe been described by Poon-King et al. 1967. The common site of infection is the skin with streptococcal infection developing secondary to mosquito bites or scabies. In our studies, more than 80% of patients had Group A streptococci isolated from skin lesions. Clinical signs of acute PSGN were edema, hypertension, oliguria, proteinuria and haematuria. In addition, serum C3 levels were less than 70 mg 0 0 in this population of patients, and the average age of onset was slightly lower than that of acute rheumatic fever patients (aged 3-10 years). Impetigo. The third group was composed of seventeen children with impetigo (aged 5-9 years). In these children, Group A streptococci were isolated from skin sores and, in some cases, from the throat. No protein was found in the urine of these children. Controls. The final group was composed of twenty-one normal, healthy children who had no signs of streptococcal infections (aged 5-9 years). Visible skin sores were absent and ASO levels were normal. Collection and storage of sera. Blood samples were obtained by routine venipuncture and collected in glass Vacutainer tubes and allowed to clot at 24°C for 1-2 hr. The serum was then separated by centrifugation at 800 g for 10 min, aliquoted and stored at both - 20'C and -70'C until tested in the immune complex assay. For determination of C3, only serum kept at -70'C was used. Solid phase Clq assay. The procedure used was basically that of Hay, Nineham & Roitt (1976), as modified by Walker et al. (1978) with the following changes. 1 0 ml volumes of human Clq, isolated by the method of Yonemasu & Stroud (1971) at 10 mg per litre in 0 05 M phosphate buffered saline (PBS) (pH 7-6), were incubated in polystyrene tubes (10 x 75 mm Falcon) for 18 hr at 40C. Subsequently, the tubes were washed three times with PBS and incubated at room temperature for 2 hr with 0.2% gelatin in 0 01 M PBS, pH 7-6. After a further wash with 0-0500 Tween 20 in PBS (PBS/Tween), the tubes were used immediately in the immune complex assay. During the above 2 hr incubation, 50 p1 of test serum were mixed with 100 P1 of 0-2 M (ethylenedinitrilo)-tetraacetic acid pH 7.5 (EDTA) and incubated for 30 min at 37 C and then transferred to an ice-bath. Duplicate samples (50 pI) were placed in Clq coated plastic tubes together with 950 Al of PBS/Twxeen and incubated for 60 min at 370C, followed by 30 min at 4 C. Unbound proteins were then removed by washing three times with cold PBS/Tween. Finally, the immune complexes were detected by adding approximately 50 ng of 125I-labelled protein A in 1 0 ml PBS/ Tuween to the tubes. The tubes were then incubated at 37°C for 1 hr, followed by incubation at 4 C for 30 min. Unbound radioactixely-labelled protein A was removed by washing fixve times with cold PBS/Txxeen. The tubes were then counted in a gamma spectrophotometer. Each time the assay was run, a standard curve of aggregated human gamma globulin was included as well as fixe normal serum controls. Values x ere considered valid only if the duplicate counts were within 5 0%0of each other. All X alues which had a greater variability than 5% were repeated. The immune complex levels in the serum samples are expressed as ug of aggregated human gamma globulin equivalent per ml of serum sample. Serum samples were considered positive if their values were greater than 2 standard deviations above that of normal control values. The Raji cell radioimmunoassay. The Raji cell radioimmunoassay for the determination of circulating immune complexes was carried out according to the method of Theofilopoulos, Wilson & Dixon (1976). 2 x 106 Raji cells in 50ul Eagle's minimum essential medium (MEM) were reacted with 25 pl of a 1:4 dilution in saline (NSS) of the serum to be tested. After an incubation period of 45 min at 37°C with gentle shaking, the cells were washed three times with MEM, reacted (30 min, 4 C) with an optimum amount of a 1:2 dilution of '25I-rabbit anti-human IgG in MEM containing 1% human serum albumin (MEM-HSA), and gently shaken. The cells were then washed three times with MEM-HSA, and radioactivity in the cell pellet determined in a gamma counter. The amount of uptake was referred to a standard curve of radioactive antibodv uptake by cells incubated with 25 p1 of a 1:4 final dilution of normal human serum (NHS), freshly obtained or stored at - 70'C, or of a pool of twenty NHS (sources of complement) to which various amounts (from 4 pug to 10 ng) of aggregated human gamma globulin (AHG) had been added. The mixture of NHS or pooled NHS with various amounts of AHG were pre-incubated for 30 min at 37°C before being added to cells. The amount of complexes in each serum tested was expressed as pg AHG equivalent per ml of serum. A serum was considered positive if it contained a value of immune complexes greater than 16 ,ug/ml. Cell viability was determined by trypan blue exclusion. The cells in suspension used in our experiments were always passaged 48-72 hr in cell culture.
I. van de Rijn et al.
320
Preparations of aggregated human gamma globulin. Human IgG was obtained from Cohn Fraction II (Sigma Chemical Co., St. Louis, Missouri) followed by fractionation on diethyl-amino-ethyl (DEAE)-52 cellulose chromatography using 0-02 M sodium phosphate buffer pH 7-2. The IgG isolated by this method was centrifuged at 40,000 g for 90 min to remove aggregated IgG and the upper third was used for heat aggregation. The concentration of the human gamma globulin was then adjusted to 5-7 mg/ml and finally heated at 630C for 30 min and aliquoted for storage at - 70'C. Aliquots of the aggregated human gamma globulin were used for up to 1 month after preparation, whereupon new batches were made. Radioiodination ofprotein A. Protein A was obtained from Pharmacia Fine Chemicals (Piscataway, New Jersey) and radiolabelled with chloramine T according to the method of McConahey & Dixon (1966). Free iodine was removed from the radiolabelled protein A by chromatography on Sephadex G25 columns. The radiolabelled protein A was then used within 2 weeks of the time of labelling. Radioiodination ofrabbit anti-human IgG. Radioiodination of rabbit anti-human IgG 125I (New England Nuclear, Boston, Massachussetts) was performed using the chloramine T method according to McConahey & Dixon (1966). The labelled anti-human IgG was extensively dialyzed against PBS, pH 7-2 for 38 hr. The specific activity of the 125I-rabbit anti-human IgG (0 3 mg/ml) was 0 05-02 ,pCi/pug protein. Sucrose-density gradient centrifugation. 200 p1 of selected sera were applied to 5 0 ml of 10-35% sucrose gradient in 0-02 M sodium phosphate buffer pH 7-2. Radiolabelled standards 7S (IgG), 11S (Clq) and 19S (IgM) were run in addition to the test samples. All samples were run in duplicate. The gradients were centrifuged at 150,000 g in a Beckman 50-1 rotor for 17 hr at 7°C. Fractions (7 drops) were collected and assayed for the presence of immune complexes. Detection ofimmune complexes in the sucrose density ultracentrifugation gradient fractions. The fractions were tested by the Raji cell radioimmunoassay described above. The only modification was the use of 75 p1 NHS plus NSS, rather than NSS alone, to dilute 25 p1 of the fractions 1:4. Assay fir serum C3 levels. Serum C3 levels were determined using the Mancini radial immunodiffusion technique (Mancini, Carbonara & Heremans, 1965). All samples were done in duplicate using the aliquots stored at - 70°C for the immune complex assay.
RE SULTS Analysis ofcontrol and acute phase sera for immune complexes Sera from patients with acute rheumatic fever, acute post-streptococcal glomerulonephritis, impetigo and normal controls were assayed for the presence of immune complexes by both the solid phase Clq assay and Raji cell radioimmunoassay (Table 1). Immune complexes were found in one out of twenty-one sera from normal controls by the solid phase Clq assay and in none by the Raji cell radioimmunoassay, whereas in children with Group A streptococcal associated impetigo, three out of seventeen sera were positive by the Raji cell radioimmunoassay and none in the solid phase Clq assay. When the sera from patients with post-streptococcal sequelae were tested, both assays indicated that more than 740 of the sera in both groups contained immune complexes. In patients with acute PSGN, thirty-two out of forty-three (74%0) sera were positive for immune complexes by the Clq assay, and seventeen out of twenty-one (81%) in the Raji cell radioimmunoassay. The incidence of circulating immune complexes TABLE 1. Analysis of control and acute phase sera for immune complexes
Assay
Solid phase Clq*
Raji cellt
Normals
Impetigo
Nephritics
1/21 (5%) 0/20 (0%)
0/17 (0%) 3/17 (18%)
32/43 (74%) 29/36 (81%) 17/21 (81%) 17/19 (89%)
Rheumatics
* A serum was considered positive if it had a value of immune complexes greater than 2 s.d. above the mean. t A serum was considered positive if it contained a value of immune complexes greater than 16 pg/ml.
in sera of patients with ARF by the Clq assay and the Raji cell assay were 81% and 89% respectively. It was surprising that patients with ARF had a similar incidence of immune complexes in their serum to the nephritis group, since rheumatic fever is not considered an immune complex disease. In spite of the presence of complexes in ARF patients, none of these patients had proteinuria.
Post-streptococcal sequelae
321
Analysis of serum C3 levels Since it has been demonstrated that patients with elevated circulating immune complexes have decreased levels of complement in their serum, this parameter was investigated next in the various patient populations. By using the Mancini technique, levels of C3 were assayed in the sera ofnormal controls and in patients with acute disease and 6 months later (Table 2). Both normal controls and patients with TABLE 2. Mean complement levels (C3) in patients with post-streptococcal sequelae
Rheumatic
Nephritic Normal
Impetigo
AP*
6 months
AP
6 months
(mg %)
(mg %)
(mg %)
(mg %)
(mg %)
(mg %)
115-9+4-2t
116-3+2-3
35-5+ 2-4
140-4+7-4
207-0+ 11-7 136-0+ 8-3
* Acute phase. t Standard error of the mean.
impetigo had mean C3 levels of approximately 116 mg 00. Patients with acute PSGN had decreased levels (35.5 mg %,) whereas patients with ARF had markedly increased levels (207.0 mg 0%). In both groups of patients, C3 serum levels returned to the upper limit of normal during the 6 months after the acute phase of the disease process. Analysis of absolute levels of immune complexes In an attempt to account for the extreme differences in the levels of C3 in the two post-streptococcal sequelae, absolute elevations of immune complexes in patients with post-streptococcal sequelae were analysed. There was essentially no difference in the mean levels of immune complexes in the two assays between normal controls and patients with impetigo (Table 3). By the Clq assay both groups had less TABLE 3. Mean levels of immune complexes in patients with post-streptococcal sequelae
Assay
Nephritic
Rheumatic
Acute phase 6 months
Acute phase 6 months
Normal
Impetigo
(pg/ml)
(pg/ml)
(pg/ml)
(pg/ml)
(pg/ml)
(pg/ml)
44 0 184-0
17-6 21-8
42-3
166-0
17-5 34-4
Clq
3-4
3-4
Raji
< 16-0
< 16-0
than 4-0 ug/ml, whereas by the Raji cell technique both groups had less than 16 pg of immune complexes per ml of serum. When sera from patients with acute PSGN or ARF were studied, levels of 44-0 pg and 42-3 pg, respectively, of immune complexes per ml of serum during the acute phase were demonstrated using the Clq assay. 184 pg/ml and 166 pg/ml were demonstrated using the Raji cell technique. Six months after the acute phase, both patient populations contained slightly elevated levels of immune complexes, but these were still dramatically reduced compared to the levels during the acute phase. The Raji cell radioimmunoassay was able to detect greater amounts of immune complexes in the serum of the patient groups than the Clq assay, but in both assays equivalent absolute levels were found in the two populations. Thus, serum from 740 of patients with ARF or acute PSGN contained immune complexes and these immune complexes were essentially at the same absolute level.
I. van de Rijn et al.
322
Serial study of immune complexes The kinetics of the decline of immune complexes from the patients' sera were examined in the two patient groups by assaying serum samples from both, first during the acute phase and then serially at 1 month, 3 month and 6 month intervals. The mean levels of immune complexes from the two patient groups declined at roughly the same rate so that at the end of 6 months both were slightly elevated (Fig. 1). For this set of experiments, only the data for the Clq solid phase assay is shown as similar kinetics were obtained with the Raji cell technique. ( b)
(a)
110 _
100 _
too
E 90_
._
@80_ \E 70 < 60
50 40* 01
-30 20
"
*
4
10~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. 0
AP
I
3
t 6
AP
3
I 6
Months
FIG. 1. Quantification of circulating immune complexes in serial studies of (a) acute rheumatic fever and (b) acute post-streptococcal glomerulonephritis sera using the solid phase Clq assay. Two standard deviations above the mean for the normal controls in this assay is 8 ug equivalent AHG/ml serum. Serum samples were monitored at the acute phase (AP) and 1, 3 and 6 months thereafter.
Sucrose density gradient studies The sera of four patients, two from each post-streptococcal sequelae, were studied using sucrose density gradients in order to determine the molecular size of the immune complexes. Immune complexes were detected in the sucrose gradients by the Raji cell radioimmunoassay (Fig. 2a) and the Clq assay (Fig. 2b). Radiolabelled standards 7S (IgG), 1 iS (Clq), and 19S (IgM) were run in addition to the test samples. In all of the sera immune complexes of 19S in size or greater were demonstrated by both the assay systems. In addition, the Clq assay also showed immune complex-like material in the size range of 10-12S. Normal serum (not shown), separated by sucrose gradient and assayed by the Raji cell radioimmunoassay for immune complexes, demonstrated increased binding only in the 7S region while the Clq solid phase assay showed no significant binding throughout the gradient. The increased binding of material by the Raji cell radioimmunoassay in the 7S range is believed to be due to Fc receptors on the Raji cell (Theofilopoulos et al., 1976).
DISCUSSION Our studies suggest that sera from patients with either acute rheumatic fever or acute post-streptococcal glomerulonephritis contain comparable levels of immune complexes in the sera and that the
Post-streptococcal sequelae (a)
7S
19S
IS
323
ARF
1816- 4p
0
0~~~~~~~~
01
012 c 22 E_ i
PSGN
20 r
/\
18
/
16 14 1
l
16
(b)
l
7S
19S
IS
PSGN 2 0
no 4 a/\jr x22 E2:. I'I'I' I
I
I
I
ARF 2
16
