DEMONSTRATION OF CIRCULATING ANTIBODIES TO THE GIDMERULAR BASEMENT MEMBRANE D. J . Evans and G. Manganella Department of Histopathology Royal Postgraduate Medical School London, England
It is well known that many species, including sheep, rabbit, and rat, are susceptible t o a Masugi-type nephritis. In this experimental model system, an animal is sensitized t o basement membrane1 or given an antibody directed against the basement membrane* and after this procedure, it develops glomerular disease. T h e development of disease depends on the fixation of adequate amounts of antibody on the glomerular basement membrane (GBM), and this antibody can be demonstrated by indirect immunofluorescence, when it shows a smooth linear pattern of distribution along the glomerular capillary loops. The existence of this type of pathogenetic mechanism in human renal disease has been clearly established. Linear patterns of glomerular staining with fluorescent anti-IgG, elution, refixation of the antibody t o glomerular basement membrane, and in vivo induction of glomerular disease in primates by antibody eluted from the diseased human kidney3 constitute the impressive array of evidence. The frequency with which this disease occurs is still not known or alternatively may differ widely from country t o country. Berger found only one case in 600 ~ t u d i e d whereas ,~ Wilson and Dixons currently quote a figure of 5%. In our 500 cases, the figure stands at 2%.6 There are several difficulties in diagnosing this disease. The patient with anti-GBM antibody disease usually exhibits a brightly positive smooth staining of the GBM with anti-IgG that is not difficult to recognize. Sometimes, positivity is also present t o other immunoglobulins. However, the normal glomerulus may not be negative with fluorescent antisera t o IgG and may display what has been euphemistically called a “linear accentuation of fluorescence.” This phenomenon has been noted by many investigators and seems t o be due t o nonantibody IgG present in the membrane. The presence of IgG in isolated membranes was conclusively shown by Westberg and Michael.’ The frequency with which this linear accentuation is observed varies greatly; it was, for instance, seen in most of t h e cases of minimal change glomerulonephritis investigated by Roy et aL,8 and although Wilson and Dixons find it in only 10% of renal biopsy specimens, in our material the incidence is substantially greater than this if cases with obvious granular deposits are excluded. In any event, a subjective element is introduced into the evaluation of linear fluorescence, and the observer must estimate t h e intensity of the fluorescence and decide whether it is significant. Even the intensity may not be a reliable guide as t o whether the IgG in the membrane is specific anti-GBM antibody. Linear patterns of fluorescence have ~ in diabetes,1° and in the been described in systemic lupus e r y t h e m a t o s ~ sand
600
60 1
Evans & Manganella: Circulating Antibodies
latter case, t h e eluted IgG did not refix to t h e GBM. It seems likely that these linear patterns are due t o an unusually high content of nonantibody IgG in the membrane. In confirming the suspected diagnosis of anti-GBM disease, it becomes necessary t o find a system that will sensitively detect antibodies t o GBM and t o elute them from biopsies or t o demonstrate their presence in blood. For their detection, three systems can be used: radioimmunoassay, hemagglutination techniques, and indirect immunofluorescence. The major problems with the first two systems are preparation and purification of the antigen, because it cannot be solubilized without treatment with enzymes, such as collagenase, and since such treatment yields a complex mixture of breakdown products, it is difficult t o formulate criteria of purity. We were able t o obtain reproducible titers with hemagglutination techniques, but sometimes the antigen preparations were unsatisfactory for no obvious reason. We have therefore evaluated the indirect immunofluorescence system. For these tests, we used a Leitz Ortholux microscope with incident illumination. The ultraviolet lamp was an HBO 200. For excitation, a fluorescein interference filter and BG 38 filter combination was used. The suppression filter was a K 510. The anti-IgC sera employed were from sheep (Wellcome) and were tested and titered against known standards. We first used human kidney sections as the substrate but found that in the autopsy material available, linear accentuation of the basement membrane present in the controls usually made evaluation impossible. Wilson and Dixon found this problem could be solved b y utilizing fresh normal kidney:5 we did not have access t o much of this material, and what we obtained showed linear accentuation of IgG on the membrane. None of the sera from patients with suspected anti-GBM disease gave positive reactions when applied t o rodent kidney. We therefore decided to use monkey kidney, and this tissue yielded satisfactory results that correlated well with those obtained by hemagglutination (see TABLE 1). It appears important for this test that the fluorescent antisera d o not cross react with monkey IgG; otherwise, linear accentuation of the basement membrane may again constitute a problem.
TABLE 1
Case A B C
D (i) (ii) (iii) 4/12 post nephr. ( i v )
E F G
M
Passive Hemagglutination Titer 1 : 32
Indirect Immunofluorescence Titer
1 : 16 1 : 64 1 : 32 1:8 1:8
1:2 1:2 1 : 16 1 : 16 1:8 1:4
1 : 64 1 : 32 1:8 1 : 16 1 : 16
0 1:4 1:2 0 0
602
Annals New York Academy of Sciences
The genesis of anti-GBM antibodies is still not clear. In Goodpasture’s syndrome, it has been suggested that sensitization t o the lung basement membrane occurs and that because the glomerular basement membrane is closely related antigenically, renal disease may follow.’ In one case that we studied, focal nephritis preceded linear GBM staining.6.’ Moreover, about half the patients with anti-GBM disease exhibit no specific lung pathologic features. As we have previously shown, leukocyte sensitization t o GBM is a regular feature of “proliferative glomerulonephritis,” and it seems likely that antibodies t o GBM may develop during the course of a nephritis, thus adversely altering its natural history: t h e development of antibodies t o CBM during t h e progression of other types of nephritis has also been noted by Klassen et ~ l . and b y Wilson and D i x ~ n . ~ In conclusion, it would appear that anti-GBM disease is a rather uncommon renal condition. Most patients have circulating antibodies against GBM. Indirect immunofluorescence with monkey kidney is a rapid and simple method for detection and monitoring of antibody levels, and though it lacks the sensitivity of alternate methods, it avoids the difficulties implicit in isolating wellcharacterized GBM antigen.
REFERENCES STEBLAY, R. W. 1962. J. Exp. Med. 116: 253. MASUGI, M. 1934. Beitr. Pathol. Anat. 92: 429. LERNER, R. A., R. J. GLASSOCK & F. J. DIXON. 1967. J. Exp. Med. 126: 989. BERGER, J. 1973. Personal communication. WILSON, C. B. & F. J. DIXON. 1974. Kidney Int. 5: 389. 6. SISSONS, J. G. P., D. J. EVANS, D. K. PETERS, A. J. EISINGER, J. M. BOULTON-JONES, 1. J. SIMPSON & M. MACANOVIC. 1974. Brit. Med. J.
1. 2. 3. 4. 5.
4: 11. 7. WESTBERG, N. G . & A. F. MICHAEL. 1970. Biochemistry 9: 3837. 8. ROY, L. P., N. G. WESTBERG & A. F. MICHAEL. 1973. Clin. Exp. Immunol. 13: 553. 9. KOFFLER, D., V. AGNELLO, R. I. CARR & H. G. KUNKEL. 1969. Amer. J. Pathol. 56: 305. 10. GALLO, G. R. 1970. Amer. J. Pathol. 61: 377. 11. MACANOVIC, M., D. J. EVANS & D. K. PETERS. 1972. Lancet 1: 207. 12. HALCRIMSON, C. G., C. B. WILSON, F. J. DIXON, I. PENN, J. T. ANDERSON, D. A. OGDEN & T. E. STARZL. 1971. Arch. Surg. 103: 283. 13. EISINGER, A. J. 1973. Amer. J. Med. 55: 565. 14. KLASSEN, J., C. ELWOOD, A. GROSSBERG, F. MILGROM, M. MONTES, M. SEPULVEDA & G. ANDRES. 1973. Amer. SOC. Nephrol. 59.
It is well known that many species, including sheep, rabbit, and rat, are susceptible t o a Masugi-type nephritis. In this experimental model system, an animal is sensitized t o basement membrane1 or given an antibody directed against the basement membrane* and after this procedure, it develops glomerular disease. T h e development of disease depends on the fixation of adequate amounts of antibody on the glomerular basement membrane (GBM), and this antibody can be demonstrated by indirect immunofluorescence, when it shows a smooth linear pattern of distribution along the glomerular capillary loops. The existence of this type of pathogenetic mechanism in human renal disease has been clearly established. Linear patterns of glomerular staining with fluorescent anti-IgG, elution, refixation of the antibody t o glomerular basement membrane, and in vivo induction of glomerular disease in primates by antibody eluted from the diseased human kidney3 constitute the impressive array of evidence. The frequency with which this disease occurs is still not known or alternatively may differ widely from country t o country. Berger found only one case in 600 ~ t u d i e d whereas ,~ Wilson and Dixons currently quote a figure of 5%. In our 500 cases, the figure stands at 2%.6 There are several difficulties in diagnosing this disease. The patient with anti-GBM antibody disease usually exhibits a brightly positive smooth staining of the GBM with anti-IgG that is not difficult to recognize. Sometimes, positivity is also present t o other immunoglobulins. However, the normal glomerulus may not be negative with fluorescent antisera t o IgG and may display what has been euphemistically called a “linear accentuation of fluorescence.” This phenomenon has been noted by many investigators and seems t o be due t o nonantibody IgG present in the membrane. The presence of IgG in isolated membranes was conclusively shown by Westberg and Michael.’ The frequency with which this linear accentuation is observed varies greatly; it was, for instance, seen in most of t h e cases of minimal change glomerulonephritis investigated by Roy et aL,8 and although Wilson and Dixons find it in only 10% of renal biopsy specimens, in our material the incidence is substantially greater than this if cases with obvious granular deposits are excluded. In any event, a subjective element is introduced into the evaluation of linear fluorescence, and the observer must estimate t h e intensity of the fluorescence and decide whether it is significant. Even the intensity may not be a reliable guide as t o whether the IgG in the membrane is specific anti-GBM antibody. Linear patterns of fluorescence have ~ in diabetes,1° and in the been described in systemic lupus e r y t h e m a t o s ~ sand
600
60 1
Evans & Manganella: Circulating Antibodies
latter case, t h e eluted IgG did not refix to t h e GBM. It seems likely that these linear patterns are due t o an unusually high content of nonantibody IgG in the membrane. In confirming the suspected diagnosis of anti-GBM disease, it becomes necessary t o find a system that will sensitively detect antibodies t o GBM and t o elute them from biopsies or t o demonstrate their presence in blood. For their detection, three systems can be used: radioimmunoassay, hemagglutination techniques, and indirect immunofluorescence. The major problems with the first two systems are preparation and purification of the antigen, because it cannot be solubilized without treatment with enzymes, such as collagenase, and since such treatment yields a complex mixture of breakdown products, it is difficult t o formulate criteria of purity. We were able t o obtain reproducible titers with hemagglutination techniques, but sometimes the antigen preparations were unsatisfactory for no obvious reason. We have therefore evaluated the indirect immunofluorescence system. For these tests, we used a Leitz Ortholux microscope with incident illumination. The ultraviolet lamp was an HBO 200. For excitation, a fluorescein interference filter and BG 38 filter combination was used. The suppression filter was a K 510. The anti-IgC sera employed were from sheep (Wellcome) and were tested and titered against known standards. We first used human kidney sections as the substrate but found that in the autopsy material available, linear accentuation of the basement membrane present in the controls usually made evaluation impossible. Wilson and Dixon found this problem could be solved b y utilizing fresh normal kidney:5 we did not have access t o much of this material, and what we obtained showed linear accentuation of IgG on the membrane. None of the sera from patients with suspected anti-GBM disease gave positive reactions when applied t o rodent kidney. We therefore decided to use monkey kidney, and this tissue yielded satisfactory results that correlated well with those obtained by hemagglutination (see TABLE 1). It appears important for this test that the fluorescent antisera d o not cross react with monkey IgG; otherwise, linear accentuation of the basement membrane may again constitute a problem.
TABLE 1
Case A B C
D (i) (ii) (iii) 4/12 post nephr. ( i v )
E F G
M
Passive Hemagglutination Titer 1 : 32
Indirect Immunofluorescence Titer
1 : 16 1 : 64 1 : 32 1:8 1:8
1:2 1:2 1 : 16 1 : 16 1:8 1:4
1 : 64 1 : 32 1:8 1 : 16 1 : 16
0 1:4 1:2 0 0
602
Annals New York Academy of Sciences
The genesis of anti-GBM antibodies is still not clear. In Goodpasture’s syndrome, it has been suggested that sensitization t o the lung basement membrane occurs and that because the glomerular basement membrane is closely related antigenically, renal disease may follow.’ In one case that we studied, focal nephritis preceded linear GBM staining.6.’ Moreover, about half the patients with anti-GBM disease exhibit no specific lung pathologic features. As we have previously shown, leukocyte sensitization t o GBM is a regular feature of “proliferative glomerulonephritis,” and it seems likely that antibodies t o GBM may develop during the course of a nephritis, thus adversely altering its natural history: t h e development of antibodies t o CBM during t h e progression of other types of nephritis has also been noted by Klassen et ~ l . and b y Wilson and D i x ~ n . ~ In conclusion, it would appear that anti-GBM disease is a rather uncommon renal condition. Most patients have circulating antibodies against GBM. Indirect immunofluorescence with monkey kidney is a rapid and simple method for detection and monitoring of antibody levels, and though it lacks the sensitivity of alternate methods, it avoids the difficulties implicit in isolating wellcharacterized GBM antigen.
REFERENCES STEBLAY, R. W. 1962. J. Exp. Med. 116: 253. MASUGI, M. 1934. Beitr. Pathol. Anat. 92: 429. LERNER, R. A., R. J. GLASSOCK & F. J. DIXON. 1967. J. Exp. Med. 126: 989. BERGER, J. 1973. Personal communication. WILSON, C. B. & F. J. DIXON. 1974. Kidney Int. 5: 389. 6. SISSONS, J. G. P., D. J. EVANS, D. K. PETERS, A. J. EISINGER, J. M. BOULTON-JONES, 1. J. SIMPSON & M. MACANOVIC. 1974. Brit. Med. J.
1. 2. 3. 4. 5.
4: 11. 7. WESTBERG, N. G . & A. F. MICHAEL. 1970. Biochemistry 9: 3837. 8. ROY, L. P., N. G. WESTBERG & A. F. MICHAEL. 1973. Clin. Exp. Immunol. 13: 553. 9. KOFFLER, D., V. AGNELLO, R. I. CARR & H. G. KUNKEL. 1969. Amer. J. Pathol. 56: 305. 10. GALLO, G. R. 1970. Amer. J. Pathol. 61: 377. 11. MACANOVIC, M., D. J. EVANS & D. K. PETERS. 1972. Lancet 1: 207. 12. HALCRIMSON, C. G., C. B. WILSON, F. J. DIXON, I. PENN, J. T. ANDERSON, D. A. OGDEN & T. E. STARZL. 1971. Arch. Surg. 103: 283. 13. EISINGER, A. J. 1973. Amer. J. Med. 55: 565. 14. KLASSEN, J., C. ELWOOD, A. GROSSBERG, F. MILGROM, M. MONTES, M. SEPULVEDA & G. ANDRES. 1973. Amer. SOC. Nephrol. 59.
