Serial Studies of Circulating Immune Complexes in Poststreptococcal Glomerulonephritis

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Serial

Studies

of Circulating

Immune

PEDIATRICS

Vol. 70 No. 5 November

1982

725

Complexes

in Poststreptococcal

Glomeruloneph

ntis

Ching-Yuang Lin,

MD

From the Department of Pediatrics, Mackay Memorial Hospital, Taipei, and China Medical College, Taiwan, Republic of China

ABSTRACT. Levels of circulating immune complexes were determined in 30 children who suffered from acute

poststreptococcal glomerulonephritis. The study was started in the acute phase of the disease and continued

for one year. Raji cell radioimmune assay was used for

the detection of immune complexes. The patients had significantly elevated levels of immune complexes during the acute phase, especially in the first three days after

the onset of hematuria. Six months later the levels of

immune complexes declined to slightly elevated levels, and nine months after the initial attack, no immune complexes were detectable. However, the patients who had persistent hematuria and proteinuna continued to

have detectable immune complexes during this time. Pediatrics 70:725-727, 1982.

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. Some

studies’8 have confirmed that immune complexes

(IC)

are present in a majority of patients with acute

poststreptococcal glomerulonephritis (PSGN). Serial studies of sera obtained from patients with PSGN have been reported infrequently, at1d in such

studies the follow-up time has often been less than six months.5 In this report serial sera collected for

a period of one year from 30 patients with acute

PSGN were studied. All patients were admitted to

Mackay Memorial Hospital in the acute phase of

the disease. Normal healthy control subjects were

also studied for comparison.

Received for publication Sept 16, 1981; accepted Dec 17, 1982. Reprint requests to (C-Y.L.) Department of Pediatrics, Mackay

Memorial Hospital, China Medical College, Ho-Ping East Road,

Taipei, Taiwan 106, Republic of China.

MATERIALS AND METHODS

Patient Population

From July 1979 to February 1980, 30 children

suffering from PSGN were admitted to the

Depart-ment of Pediatrics, Mackay Memorial Hospital.

Their ages ranged from 3 to 14 years (mean 7.6

years); 22 were boys and eight were girls. The

criteria for diagnosis of PSGN were (1) presence of clinical signs of edema, hypertension, oliguria, pro-teinuria, and hematuria; (2) serum C3 level <70

mg/100 ml; (3) history of recent upper respiratory

tract infection, the causative agent of which was

proved to be Streptococcus by serologic test includ-ing elevated Antistreptolysin 0 (ASLO) antibody

titer (333 Todd units) at onset of disease; (4)

positive C-reactive protein and elevated ESR; (5)

no evidence or history of previous renal disease; and (6) if biopsy was taken, light microscopic

changes showed typical acute diffuse proliferative

glomerulonephritis and electron microscopy re-vealed demonstrable humps. The 30 patients were followed up for more than one year. Sera were serially collected from onset up to 12 months, and

the C3 levels were checked immediately. Blood

samples for IC detection were stored at -70 C until

use.

Control Subjects

Twenty normal healthy children (aged 3 to 14

years) who had no signs of streptococcal infection

were included in the control group. No visible skin

sores were present and their ASLO levels were

normal.

Raji Cell Radioimmunoassay

The Raji cell radioimmunoassay for the deter-mination of circulating immune complexes (CIC) was modified according to the method of Theofilo-poulos et al.9 The Fc receptor of Raji cell was first

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15 3O 3 12

day. month,

Figure. Analysis and serial study of immune complex in poststreptococcal glomerulonephritis (PSGN). Abbre-viations used are: CIC, circulating immune complexes; AHG, aggregated human y-globulin.

TABLE 1. Mean (PSGN)*

Level of Immune Complex in Serial Sera of Patients with Poststreptococcal Glomerulonephritis

Acute Phase (1st 30 Days)

3 6 9 12 Normal

3rd 5th 10th 15th 30th

Day Day Day Day Day Month Month Month Month Control

CIC (jsg AHG/m1)t 234.0 164.2 74.0 52.4 32.5 22.6 18.4 12.0 <10 <10

No. of patients 24/30 20/30 19/30 18/30 16/30 13/30 5/30 1/30 0/30 0/20

Detectable cases 80% 66.6% 63.3% 60% 53.3% 43.3% 16.6% 3.3% 0% 0% * Abbreviation used is: AHG, aggregated human -y-globulin.

t Mean value was average circulating immune complexes (CIC) of detectable cases.

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Serum was considered detectable if it contained >10 jg/ml of immune complex.

726 CIRCULATING IMMUNE COMPLEXES IN GLOMERULONEPHRITIS

blocked for an incubation period of 30 minutes at 37 C with 75 immunoglobulin (Ig)G. Then the cells were washed three times with minimum essential medium (MEM), and allowed to react (40 C 37

minutes) with anti-IgG Fab’; 2 x 106 Raji cells in 50 /Ll of Eagle’s MEM were allowed to react with 25

jd of a 1:4 dilution in normal saline solution of the

serum to be tested. After an incubation period of 45

minutes at 37 C with gentle shaking, the cells being washed three times with MEM, were allowed to react (30 minutes, 4 C) with an optimum amount of a 1:2 dilution of ‘25I-rabbit antihuman IgG in MEM containing human serum albumin (MEM-HSA). The mixture was shaken gently. Then the cells were washed three times with MEM-HSA, and radioac-tivity in the cell pellet was determined in a gamma counter. The amount of uptake was compared with a standard curve of radioactive antibody uptake by cells incubated with 25 tl of a 1:4 final dilution of normal human serum (NHS), which was freshly obtained, or stored at -70 C, or came from a pool of 20 NHS (sources of complement) to which

var-ious amounts (from 4 .tg to 10 ng) of aggregated human -y-globulin (AHG) had been added. The mixture of NHS or pooled NHS with various

amounts of AHG were preincubated for 30 minutes at 37 C before being added to the cells. The amount of complexes in each serum tested was expressed in micrograms of

AHG

equivalent per milliliter of serum. The serum was considered positive if it contained a value of immune complexes greater than 16 zg/m1.5 Cell viability was determined by

trypan blue exclusion. The cells in suspension used in this experiment were passaged 72 hours in cell culture.

Assay for Serum C3 Levels

Serum C3 levels were determined using the Man-cmi radial immunodiffusion technique with com-mercialiy available immunoplates (Oxford).

RESULTS

Analysis and Serial Study of Immune Complex

With the Raji cell technique, the children in the control group had <10 tg of immune complexes per

milliliter of serum. Serial sera from patients with

PSGN were tested with Raji cell radioimmune

as-say, and a high incidence of CIC was detected in the acute stage (Table 1). CIC was detected in 80% of the sera taken by the third day after the onset of hematuria. From the serial sera taken during the one-year follow-up, the detectable rate of CIC grad-ually decreased, but remained detectable in more than half of the patients one month later. After six

months, the detectable rate was only 16.6% and became undetectable 12 months after the onset.

During the acute phase of the disease process, high concentration and easily detectable CIC was present in the initial three days after the onset of hematuria. The level ranged from 160 to 340 tg/mi (mean 234 tg/mi). However, it decreased rapidly to 52.4 jig/mi on the fifth day and 32.5 ig/ml on the thirtieth day. Serial studies were performed at one,

three, nine, and 12 months after onset of hematuria. The results are shown in Table 1 and in the Figure.

Relation between circulating immune complexes and patients having persistent hematuria and

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TABLE 2. Analysis of Relation Between Circulating Immune Complexes (CIC) and Persistant Hematuria and

Proteinuria Six Months After Onset of Disease

CIC Positive* CIC Negative Patients with proteinuria 5/7 (71.4%) 2/7 (28.5%)

and microscopic hema-tuna

Patients with normal un- 0/23 (0%) 23/23 (100%)

nalysis

S Serum was considered positive if it contained >10 tg/ ml of immune complex.

teinuria six months after the onset of the disease is

shown in Table 2. Of the patients having persistent hematuria and proteinuria, 71.4% had continuous presence of immune complexes.

By using the Mancii technique, levels of C3 were

assayed in the sera of normal control subjects and in patients with acute disease and six months later.

Normal control subjects had mean C3 levels of 120

± 5.6 [SD] mg/100 ml. Patients with acute PSGN had decreased levels (31.2

± 10.6 [SD]

mg/100 ml).

Six months after the acute phase of the disease

process, C3 serum levels returned to normal (132

± 8.6 [SD]

mg/100 ml).

DISCUSSION

In this study, sera from children with acute PSGN contained immune complexes that declined sharply after the initial attack. High CIC level and detectability were noted in the initial three days after the onset of hematuria. The incidence of im-mune complexes in patients with acute PSGN in this series is comparable to those of other

rts51

The level of immune complexes is elevated in the acute phase of PSGN, but decreases dramatically6 in the first week, and becomes undetectable nine

months after the onset. In our study, those patients who had suffered from persistent hematuria and proteinuria for 6 months always showed the pres-ence of immune complexes in the sera during this period. Follow-up renal biopsy was shown to have a crescent formation in 20% of the total 5 glomeruli and electron microscopy showed subepithelial elec-tron-dense deposits along several capillary loops with membranous transformation and

intramem-ARTICLES 727 branous deposits in one of those five patients with proteinuria and microscopic hematuria. This phe-nomenon has been generally explained by residual glomerular basement membrane damage after the acute immune complex insult.5 The titers of ASLO dropped during this period. It was also noticed that the level of ASLO titers was not related to the continued presence of immune complexes. Finally, the decreased C3 level in patients with acute PSGN returned to normal six months later. This was not related to the continued presence of immune com-plexes.

ACKNOWLEDGMENT

The author thanks Shunji Matsuda, MD, Professor Hyakuji Yabuuchi, Osaka University, Japan for their

kind advice on Raji cell radioimmune assay, and Dr Hey-chi Hsu, Department of Pathology, National Taiwan University Hospital, for pathologic examination of the renal biopsy.

REFERENCES

1. Franco 5, Moalem T, Becker M, et al: Circulating immune

complexes in children with acute poststreptococcal glomer-ulonephritis. Isr J Med Sci 16:357, 1980

2. Rod? Iguez-Iturbe B, Carr RI, Garc’Ia R, et al: Circulating

immune complexes and serum immunoglobulins in acute poststreptococcal glomerulonephritis. Clin Nephrol 13:1,

1980

3. Nissenson AR, Baraff U, Fine RN, et al: Poststreptococcal

acute glomerulonephritis: Fact and controversy. Ann Intern Med9l:76, 1979

4. Villarreal H Jr, Rico E, Zabriskie JB: Streptococcus-related acute glomerulonephritis. Arch Inst CardiolMex 49:89, 1979 5. Van De Rijn I, Fillit H, Brandcis WE, et al: Serial studies on circulating immune complexes in poststreptococcal sequelae. Clin Exp Immunol 34:318, 1978.

6. Ooi YM, Vailota EH, West CD: Serum immune complexes in membranoproliuerative and other glomerulonephritides. Kidney

mt

11:278, 1977

7. Teornroth T: The fate of subepithelial deposits in acute

poststreptococcal glomerulonephritis. Lab Invest 35:461,

1976

8. Humair L: Experimental glomerulonephritis: Immunological

and pathogenetic mechanism. Praxis 65:510, 1976

9. Theofilopoulos AN, Wilson CB, Dixon FJ: The Raji cell radioimmune assay for the detecting of immune complexes in human sera. J Clin Invest 57:169, 1976

10. Woodroffe AJ, Border WA, Theofilopoulos AN, et al:

Detec-tion of circulating immune complexes in patients with

gb-merubonephritis. Kidney Int 12:268, 1977

11. Ooi YM, Vallota EH, West CD: Serum immune complexes in membranoproliferative and other glomerubonephritides.

Kidney Int 11:275, 1977

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1982;70;725

Pediatrics

Ching-Yuang Lin

Glomerulonephritis