Aug 28, 1986 - anti-BSA antibodies to the severity of glomerulonephritis in rats with ... BSA antibodies and the severity of glomerulonephritis at each stage.
Clin. exp. Immunol. (1987) 67, 277-282
Relationship of the quality and quantity of circulating anti-BSA antibodies to the severity of glomerulonephritis in rats with chronic serum sickness BERNICE NOBLE, M. W. STEWARD, A. VLADUTIU & J. R. BRENTJENS Departments of Microbiology, Pathology and Medicine, School of Medicine, State University of New York at Buffalo, Buffalo, New York, 14214, and Immunology Unit, London School of Hygiene and Tropical Medicine, Keppel Street, London, WCIE 7HT
(Acceptedfor publication 28 August 1986)
SUMMARY Chronic serum sickness glomerulonephritis, induced in hyperimmunized rats by daily intravenous administration of bovine serum albumin, occurs in three stages, mild, moderate and severe, with abrupt onsets and distinctive features of kidney pathophysiology and immunopathology. We have studied the relationship between circulating antiBSA antibodies and the severity of glomerulonephritis at each stage. The total amount of antibodies declined gradually during the course of disease, to low concentrations in the most severe stage of kidney inflammation. High levels of immune complexes were present in the circulation while precipitating antibodies were maintained, and rats remained in the mill stage of disease, exhibiting no abnormalities of kidney function and only mesangial immunopathology. The start of the moderate stage of chronic serum sickness, identified by proteinuria and the accumulation of immune deposits along the glomerular basement membrane, was associated with the disappearance of precipitating antibodies from circulation. With the onset of the severe stage of disease, marked by depressed glomerular filtration and sodium excretion, circulating antibodies of high affinity were no longer detected and circulating immune complex levels were only marginally elevated above normal. The experiments reported here demonstrate that, in chronic serum sickness glomerulonephritis of rats, transitions from one stage of kidney disease to another can be inferred from changes in the population of circulating antibodies. Kidney histopathology, therefore, can be predicted reliably from serological data alone.
Keywords antibody affinity circulating immune complexes nephritis serum sickness kidney disease
immune complex
INTRODUCTION In Lewis (LEW) rats with chronic serum sickness there are three discrete stages of glomerulonephritis, mild, moderate and severe (Van Liew, Brentjens & Noble, 1983; Park et al., 1985; Van Liew, Noble & Brentjens, 1985) which occur in a synchronous and highly reproducible manner. In mild chronic serum sickness, immune deposits are limited to the mesangium; histopathology is slight or absent. In moderate chronic serum sickness, there is abnormal proteinuria and significant immunopathology of the glomeruli. A disturbance of whole kidney function, including decreased
Correspondence: Dr Bernice Noble, Department of Microbiology, Sherman Hall, School of Medicine, SUNY at Buffalo, Buffalo, New York 14214
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sodium excretion and decreased glomerular filtration rate, is detected only in the severe stage of serum sickness, in association with diffuse, necrotizing proliferative glomerulonephritis. We have studied the relationship of the quality and quantity of circulating anti-BSA antibodies to the severity of immune complex-mediated glomerular injury exemplified by each of the three stages of chronic active serum sickness. MATERIALS AND METHODS Animals. Female LEW rats (Charles River Breeding Laboratories), weighing approximately 125 g, were used. Induction of chronic serum sickness. Rats were given three subcutaneous injections of 3 0 mg BSA in incomplete Freund's adjuvant at 2-week intervals, followed by daily intravenous injections of 2 0 mg BSA in 0-4 ml isotonic saline, according to a scheme described elsewhere (Arisz et al., 1979; Noble et al., 1981). Controls received subcutaneous injections of BSA in adjuvant followed by intravenous injections of saline alone. Monitoring the course of chronic serum sickness. Urine and plasma samples were collected weekly from the time of the first subcutaneous immunization. Kidney biopsies were performed at 3week intervals, and autopsies were done. Stages of chronic serum sickness were identified by the following criteria (Van Liew, Brentjens & Noble, 1983; Park et al., 1985). Mild serum sickness: protein excretion normal (< 10 mg/24 h/100 g body weight); immune deposits and histopathology limited to the glomerular mesangium; usually lasts 3-4 weeks from the introduction of intravenous injections of BSA. Moderate serum sickness: protein excretion abnormally elevated; fluid balance normal; immune deposits located primarily in the peripheral capillary wall; diffuse proliferative glomerulonephritis. Severe serum sickness: oedema and ascites; proliferative glomerulonephritis with capillary loop necrosis, immune deposits in peripheral capillary wall; death from renal failure occurs usually within 2 weeks. Collection ofplasma samples. Every 7-10 days, 0-5-1 0 ml samples of blood were collected from tail veins into heparinized hematocrit capillaries. Unless otherwise indicated, blood was collected just before the daily BSA injection, 24 h after the previous dose. Determination of urinary protein excretion. Urine was collected from animals kept in metabolism cages for 16 h with free access to water but no food. Urinary protein concentration was determined by means of a biuret test and protein excretion was expressed as mg/24 h/100 g body weight. Assays for antibodies to BSA. The titre of precipitating anti-BSA antibodies was measured by means of double diffusion tests performed in Ouchterlony plates made with 10% agarose gel (Arisz et al., 1979). Rat anti-BSA serum of high titre was used as a positive control and expressed as log2 of the highest dilution forming a clearly discernible precipitation line. The total concentration (Ab, pmol binding sites/1O pi) and affinity (K, 1 M- I x 106) of anti-BSA antibodies were determined by the double isotope ammonium sulphate precipitation method of Gaze, West & Steward (1973). Assay for circulating immune complexes. The levels of immune complexes in plasma were measured by a Raji cell radioimmunoassay (Theophilopoulos, Wilson & Dixon, 1976). Briefly, affinity purified rabbit anti-rat IgG was labelled with 1251 by the chloramine-T method (McConahey & Dixon, 1966). Purified rat IgG was alkali-aggregated and samples of rat plasma and aggregated rat IgG with added complement were incubated with Raji cells. After washing, the cells were treated with '251-labelled anti-rat IgG. The percentage of binding was calculated for each plasma sample and the level of immune complexes was determined from a standard curve constructed by using various known amounts of aggregated rat IgG and expressed as microgram equivalents of rat IgG per millilitre. Immunopathology. Direct immunofluorescence tests were performed on frozen sections of normal rat kidney to determine the sites of deposition of immune reactants (Brentjens et al., 1974a,b; 1975). FITC-conjugated antisera to rat immunoglobulins and rat complement were purchased from Cappel Laboratories. Kidney tissue samples, fixed in 10% buffered formalin and embedded in paraffin, were stained with hematoxylin-eosin and periodic acid-Schiff reagent for examination by light microscopy.
Antibodies in immune complex nephritis
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RESULTS
Relationship ofcirculating antibodies to the severity of immune complex glomerulonephritis. With the exception of the drop in titre of precipitating antibodies, the anti-BSA antibody responses in rats with mild chronic serum sickness were not different from those in animals given saline alone after priming with BSA in adjuvant (Table 1). Moderate serum sickness was associated with lower total anti-BSA antibodies and with the reduction of precipitating antibodies to undetectable levels in most rats (7/10). Antibody affinity values did not differ from control values. The severe stage of disease was characterized by a significant fall in antibody affinity; total anti-BSA antibody concentration was approximately 1/4 of the amount detected in controls, and no precipitating antibodies were detected. Relationship of proteinuria to antibody affinity. Data in Table 2 were obtained from three separate groups of animals with chronic serum sickness and include all measurements of affinity for which a closely paired measurement of protein excretion was also available. Antibody affinity values were similar to those observed in controls over a wide range of abnormal protein excretion, corresponding to mild and moderate stages of kidney disease, but were significantly lower in rats with highly elevated protein excretion and severe chronic serum sickness. Relationship of circulating immune complexes to the severity of immune complex glomerulonephritis. In mild serum sickness, the levels of circulating immune complexes, detected by the Raji cell radioimmunoassay, were highest 24 h after each intravenous BSA injection. At 10 min (data not shown) and 8 h afterwards, immune complex levels were only 20 25o% of those maximum values. In the moderate and severe stages, circulating immune complex levels were not significantly altered by the daily antigen injection. The immune complex level in normal animals was only 2% of that detected in mild serum sickness and 15% of the amount in the moderate stage of disease. However, immune complex levels in the severe stage, though significantly raised, were less than 2-fold above normal.
Table 1. Relationship of anti-BSA antibodies to the severity of immune complex glomerulonephritis (mean + s.e.)
Stage of disease* Controlt Mild
Protein Antibody Total antibody (Ab,) Precipitating excretion pmol binding antibody titre affinity (K) n (I/mol x 1O6) sites/ 10 l serum (10g2) (mg/24 h/ 00 gBW)
10 10 Moderate 10 Severe 8
1 18±0-06 1 21+0-20 0 98+007 0 54+0l10t
1116+115 1038+191 684+ 149§ 256+ 141+
27±0-3 1 7+0-2
03 ±+02§
