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Studies on the placenta
Studies on the placenta II. Nephrotoxicity of antibodies produced against a chorionic glycoprotein
NICHOLAS F. GANG, M.Sc., PH.D. ESTHER S. SCHWARTZ, B.Sc. ANNA MAJEROVICS MARIE-LAURE
DE
CHAMPLAIN, M.D.
ESTHER TRACHTENBERG Montreal, Quebec, Canada, and New York, New York The effect of rabbit antibodies produced against human and rat chorionic glycoprotein (GP) was investigated in relation to induction of nephritis and/or interruption of pregnancy in the rat. In virgin female and male rats used for the study of nephrotoxicity, proteinuria gradually increased with time. The glomeruli of rats examined 3 hours after injection of the antibodies revealed the presence of anti-GP immunoglobulin G (lgG) and of autologous C'3 in a linear, diffuse pattern in the capillary walls. Electron microscopy showed swelling and displacement of the endothelium from the basement membrane by neutrophils and fusion of the foot processes. In the glomeruli of rats killed on the fourteenth day of nephritis, the presence of autologous IgG could be observed by immunofluorescence. The ultrastructural pathology of the rats with chronic nephritis consisted of marked thickening of the basement membrane, mesangial proliferation, and distortion of the foot processes. The injection of antibodies into pregnant rats during the third trimester of gestation was without effect on the outcome of pregnancy. It is concluded that: (1) at least one of the nephrotoxic antigens common to the placenta and kidney is a GP of trophoblast basement membrane origin; (2) the pathogenesis of the glomerulopathy is similar to that produced with antibodies against glomerular basement membrane; and (3) antibodies other than anti-GP antibodies are responsible for immune abortion, which can be induced when the whole placenta is used as the source of antigen.
I T I s w E L L established that heterologous antibodies against crude placental homagenates, or definable fractions prepared by differential centrifugation, cross react with renal and extrarenal basement membranes. 1 The injection of heterologous antiplacental serum will lead to a glomerulopathy similar to that of Masugi nephritis12 • 13 and/or termination of pregnancy. 1 Recently, we isolated a noncollagenous glycoprotein (G P) from chorionic villi. 6 • 11 Immunofluorescent studies revealed that antibodies produced against the GP localized in vitro on the trophoblast basement membrane in the chorion and on the Bowman's capsule, glomerular, tubular, and small blood vessel basement membranes in the kidney. 11 Previously, whole
From the Laboratories of Experimental Pathology, Lady Davis Institute for Medical Research, the Department of Pathology, Jewish General Hospital, and the Department of Pathology, Mount Sinai School of Medicine of the City University of New York. Supported in part by Experimental Immunobiology of the Reproductive System Fund, Continental Pharma/ DAMON CORP., and by Operating Grant MA-4647 from the Medical Research Council of Canada. Received for publication October 30, 1973.
Revised February 4, 1974. Accepted February 20, 1974. Reprint requests: Dr. Nicholas F. Gang, Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 C8te St. Catherine Rd., Montreal 249, Quebec, Canada.
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74 Gang et al.
placental homogenates, or fractions thereof were used as the source of antigen(s) instead of purified glycoprotein. 1 • "· ' 0 • 14 The present study was undertaken to determine whether heterologous antibodies against the purified GP antigen are responsible for the induction of immune nephritis and/or interruption of pregnancy in the rat. Material and methods
Experimental animals. Sprague-Dawley virgin female and male rats, weighing 150 to 175 grams each, were used for the study of nephrotoxicity. Pregnant rats of the same strain in the eighteenth day of gestation, weighing 300 to 340 grams each, were used to determine the abortive effect of anti-GP antibodies. Rabbits of the New Zealand strain, weighing 2.5 to 3.0 kilograms each, were immunized with the purified GP for the production of antiserum. Immunologic procedures. The chorionic GP antigen was isolated from term human placentas obtained at delivery and rat placentas removed by surgical means on the twenty-first day of gestation, as previously described.6· 11 The antigen was obtained and purified as follows: The chorionic and decidual plates were removed, and the middle zone of the placenta was cut into small fragments; the terminal chorionic villi (CVI) were then scraped off from the minor villous stem with a razor blade. The CVI preparation was pressed through a 200 mesh stainless steel sieve with a spatula, washed through the same sieve, poured onto a 325 mesh wire cloth, and washed with an excess of isotonic saline. The CVI which were retained on top of the 325 mesh sieve were then collected with the aid of a Pasteur pipet, centrifuged, washed in distilled water, and lyophilized. These procedures enabled us to remove large connective tissue, blood vessel, cell, and cellular debri contaminants. The chorionic GP antigen was then prepared from CVI by sonication in 1M saline for 7 minutes to remove cells and cellular membranes, extraction with 0.3M acetic acid to remove residual connective tissue contaminants, and, subsequently, treatment with
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September I, 1974 Obstet. Gynecol.
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deoxyribo- and ribo-nuclease to remove nucleic acids. The resultant insoluble fraction was then washed with distilled water and lyophilized. 11 Chemical analyses of the final preparation revealed a collagen-free GPY Antisera against the human and rat purified chorionic GP were produced in rabbits. The rabbits were injected in the footpads with 2 mg. of lyophilized GP suspended in 1 mi. of physiologic saline and emulsified with 1 mi. of Freund's complete adjuvant. Two further injections were given intramuscularly during the fifth and sixth weeks of immunization. Blood was drawn, by cardiac puncture, 8 days after the last injection. The antisera were then decomplemented by heating at 56° C. for 1 hour and absorbed with freshly harvested, washed human and rat erythrocytes, and the immunoglobulin G ( IgG) fraction was prepared. 8 Normal rabbit IgG was used as the control. For immunofluorescent microscopy, small pieces of chorionic tissues from the placenta and cortical tissues from the kidney were removed in situ and processed according to conventional methods. 5 Fluorescein-conjugated goat antirabbit IgG* and a fluoresceinconjugated IgG fraction of rabbit antirat IgG and C'3t were absorbed first with activated charcoal and subsequently with liver powder* to remove nonspecific staining materials. To locate the heterologous (rabbit) IgG in the kidney and the chorion and the autologous (rat) IgG and 0'3 in the kidney, the direct immunofluorescent procedure was used 7 on uniform sections of 3 p, thickness. Control sections were treated with the appropriate unlabeled antibodies prior to the conjugated ones. All of the sections were photographed on Kodak type B high-speed Ektachrome film,t with a uniform exposure time of 2 minutes. Chemical procedures. Total urinary protein was determined by a micromodification of the biuret method. 7 *Difco Labs., 920 Henry St., Detroit, Michigan 48201. tCappel Laboratories, Inc., Downingtown, Penlli'Ylvania. tEaatman Kodak Co., 343 State St., Rochester, New York 14650.
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Fig. 1. Electron micrograph of part of a glomerulus of a male rat killed 3 hours after injection of normal rabbit lgG. The capillary lumen (C) is patent; the endothelium (END), basement membrane (arrows), and epithelial foot processes ( EP) are unaltered. (Original magnification x6,000.)
Morphologic procedures. For electron microscopy, cortical biopsies were taken in situ alternately from the left and right kidneys and placed in a drop of ice-cold 3 per cent glutaraldehyde in cacodylate buffer (pH 7.25). The specimens were then cut into small fragments, transferred into vials, and fixed in the cold solution for 2 hours. After washing in the buffer solution, the specimens were postosmicated with 1 per cent Os04 in cacodylate buffer, dehydrated in graded alcohol solutions, treated with propylene oxide, and embedded in Epon. 9 Thin sections were cut on an LKB Ultratome III with diamond knives,* placed on uncoated copper grids ( 200 mesh) , and examined with Hitachi 8 and 12 electron microscopes. For determining the number of polymorphonuclear leukocytes (PMN), tissues were fixed in 4 per cent formaldehyde, embedded *E. I. DuPont de Nemours &: Co., Instrument Products Div., Wilmington, Delaware 19898.
in paraffin, and stained with hematoxylin and eosin. PMN's were counted in 30 glomeruli from each rat. Experimental procedures and results Nephrotoxicity of rabbit antibodies to human and rat chorionic GP. Ninety-six male and 96 virgin female rats were placed in individual metabolic cages. Forty-eight rats of each sex, serving as the experimental animals, were further divided into 2 equal groups. The first group received an intravenous (tail vein) injection of rabbit antihuman GP IgG and the second group received rabbit antirat GP IgG, at a dose of 400 p.g per 100 Gm. of body weight in 1 mi. of physiologic saline. Twelve male and 12 female rats were killed 3 hours after injection of the antibodies, and the remaining animals were killed 14 days later. An equal number of rats, serving as the
76 Gang et al.
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Fig. 2. Electron micrograph of part of a glomerulus of a female rat killed 3 hours after administration of rabbit antihuman chorionic GP IgG. Note obliteration of capillary lumen, neutrophils lying directly on the basement membrane (arrows) , and swelling of visceral epithelium ( EP) with obliteration of urinary spaces. (Original magnification x5,400.)
controls, were injected with normal rabbit IgG at the same dose and by the same route as those of the experimental animals. They were killed in corresponding numbers and at the same time intervals as those of the experimental groups. Urine was collected at 24 hour intervals from each animal from the time of injection of the IgG preparation until they were killed. All urine samples were made up to 30 ml. with distilled water, and, after filtration through Whatman No. 2 filter paper, aliquots were taken for total urinary protein determinations. An additional 12 rats received antihuman GP IgG and 12 others received antirat GP IgG, adsorbed before injection with purified human and/or rat GP or glomerular basement membrane. Proteinuria. The protein excretion in all of the animals prior to injection of the appropriate IgG preparation was less than 20
mg. per day. The control animals, injected with normal rabbit IgG, did not develop proteinuria during the course of the study. In all of the experimental animals, male and female alike, injected with either rabbit antihuman or rabbit antirat GP antibodies, proteinuria gradually increased and was 400 ± 20 mg. per day by the fourteenth day. No proteinuria was observed in rats injected with the antibodies adsorbed with GP or glomerular basement membrane. PMN leukocyte infiltration of the glomeruli. In the control animals, the number of PMN's was 2.5 ± 0.5 per glomerulus. In rats killed 3 hours after administration of the anti-GP IgG, the number of neutrophils increased to 24 ± 2 per glomerulus. The glomeruli of rats killed 14 days after injection of the nephrotoxic antibodies showed no increase in the number of PMN's over those of the controls. General morphology. The renal glomeruli
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Fig. 3. Electron micrograph of part of a glomerular capillary wall of a male rat killed 14 days after injection of rabbit antirat chorionic GP IgG. Note detachment of the endothelium from the basement membrane (arrow), uneven thickening of the basement membrane ( BM), and fusion and distortion of the foot processes of the visceral epithelial cell ( EP). (Original magnification x6,000.)
of the virgin female and male control rats showed no abnormalities (Fig. 1). The general features of the glomerular lesions, as observed with the electron microscope, were identical in the male and female animals killed 3 hours after administration of either the antihuman or antirat GP antibodies. General features were swelling and detachment of the endothelium from the glomerular basement membrane, increase in the mesangial rna trix, focal fusion of the foot processes with swelling of the visceral epithelial cells, and obliteration of urinary spaces (Fig. 2). In addition, PMN's could be visualized in most of the capillary loops, lying directly on the basement membrane and displacing the endothelium (Fig. 2) . In rats killed 14 days after administration of normal rabbit IgG, the glomeruli appeared unaltered. The capillary lumen was patent. Neither swelling of the endothelium nor an increase in the thickness of the glomerular
basement membrane was apparent. No change in the visceral epithelial cells could be visualized. The glomeruli of male and female rats killed 14 days after injection of antihuman or antirat GP antibodies exhibited identical ultrastructural alterations. They consisted of swelling and separation of the endothelium from the basement membrane, an increase in mesangial cells and matrix with consequent partial obliteration of the capillary lumen (Figs. 3 and 4), and marked thickening and fraying of the glomerular basement membrane (Fig. 5 ). An amorphous, granular, electron-dense substance could be visualized in the subendothelial region, closely associated with the lamina densa (Fig. 5). There were marked fusion and distortion of the foot processes, together with moderate swelling of the visceral epithelial cells (Figs. 3, 4, and 5) . lmmunofiuorescent studies. In the control
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Fig. 4. Electron micrograph of part of a glomerulus of a female ra t killed 14 days after administration of rabbit antirat chorionic GP IgG. In addition to changes described in Fig. 3, note the significant increase in the mesangial cells (MC) and matrix (M), togeth er with swelling of the visceral epithelial cells (EP ). (Original magnification >'6,000. )
animals killed at 3 hours, or 14 days after injection of normal rabbit IgG, no localization of the heterologous antibodies could be detected in any of the structures of the kidney . In both male and female rats injected with either antihuman or antirat GP IgG and killed at the third hour of nephritis, the heterologous antibodies appeared in a diffuse, smooth pattern on the basement membranes of the glomerular capillaries (Fig. 6) . There was no localization of the antibodies in any other structures of the kidney. Fluoresceinconjugated rabbit antirat C'3 showed a staining pattern identical to that of the heterologous antibodies (Fig. 7) . In rats killed on the fourteenth day of nephritis, there was a significant decrease in the amount of heterologous antibodies in the glomerular basement membrane (Fig. 8 ) . On the other hand, as illustrated in Fig. 9, autologous IgG showed an intense, diffuse fluorescence. The effect of antihuman and antirat GP
antibodies on the outcome of pregnancy in the rat. The purpose of the second experiment was to determine whether a nti-G P antibodies could interrupt pregnancy in the rat. A total of +8 rats , in the eighteenth day of gestation, were used . The procedures employed with respect to injection of the antibodies and immunofluorescent procedures were as described in the preceding experiment. The administration of normal rabbit IgG or rabbit antihuman or antirat GP IgG had no effect on the course of pregnancy. All rats delivered on the twenty-first day of gestation and gave birth to healthy litters. Furthermore, no localization of rabbit antirat or antihuman GP antibodies could be detected on the trophoblast basement membrane. Comment
It has been demonstrated repeatedly tha t the administration of heterologous antipla-
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Fig. 5. Electron micrograph of part of a capillary wall of a female rat killed 14 days after injection of rabbit antihuman chorionic GP antibodies. Note uneven swelling of the basement membrane (BM) together with fraying of the membrane (large arrows) and the presence of a granular electron-dense substance in the subendothelial regions (small arrows). (Original magnification x6,000.)
cental sera has a striking nephrotoxic effect and can interrupt pregnancy in the species from which the antigen was obtained.H However, the exact nature of the antigen(s) common to the placenta and kidney, which operate in the two pathogenetic mechanisms described above, has not yet been estab· lished. 1 • 3 • 10 • 14 We have previously reported that antibodies produced against a purified chorionic noncollagenous glycoprotein cross reacted in vitro not only with the basal lamina of the trophoblasts but also with the basement membrane of Bowman's capsule, glomerulus, and tubules of the kidneyY The purpose of this investigation was to establish whether antibodies produced against human and rat GP antigens are responsible for the development of nephritis and/or abortion. Contrary to in vitro immunologic studies, upon intravenous injection, the anti-GP antibodies localized exclusively in the glomerular basement membrane in virgin female, male,
and pregnant female rats. This could have been caused by the inability of the antibodies to cross the cellular layer of the chorion and, hence, to reach their respective antigenic sites on the basal lamina. These findings lend support to the hypothesis, based on studies with crude placental homogenates, that the antigen ( s) common to the placenta and kidney are of trophoblast basement membrane origin. 3, 1o, 14 No difference in the localization of the antibodies in the glomerular capillary walls of rats was apparent, whether the antigen was of human or rat origin. This finding is of interest since the cross-reaction of human tissue to that of rat tissue is most unusual. The only explanation that one might offer is that at least some of the chorionic GP antigens are non-species specific and share common antigenic sites with the glomerular basement membrane. However, it must be emphasized that this conclusion is based solely on immunofluorescent studies which,
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Fig. 6. Fluorescent micrograph of a glomerulus of a female rat killed 3 hours after administration of rabbit antihuman chorionic GP lgG and stained with fluorescein-conjugated goat antirabbit lgG. Note intense diffuse, linear fluorescence of the capillary walls. (Original magnification x450.)
Fig. 7. Fluorescent micrograph same as Fig. 6 but stained with fluorescein-conjugated rabbit antirat C'3. Note linear staining of the capillary walls indicating the presence of complement. (Original magnification x450.)
at best, are semiquantitative. The presence of C'3 in the capillary walls and the infiltration of the glomeruli by the neutrophils following injection of the nephrotoxic antibodies were similar to the pathogenesis of Masugi nephritis. 4 The general ultrastructural alterations of the glomeruli of rats killed 3 hours after administration of anti-GP IgG, namely, swelling and displacement of the endothelial cells by neutrophils and fusion of the foot processes, were also similar to the changes described for the early stages of immune nephritis. 2 Taken together, these results indi-
cate that the onset of nephritis produced with anti-GP antibodies is also a complement- and neutrophil-mediated process. The development of massive proteinuria noted during the course of this study; the presence of autologous IgG in the glomerular capillary walls observed in rats 14 days following injection of the antibodies, together with the swelling of endothelium; the significant increase in the thickness of the basement membrane; and fusion of the foot processes were the same ultrastructural and functional alterations as those described for
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Fig. 8. Fluorescent micrograph of a glomerulus of a male rat killed 14 days after administration of rabbit antirat chorionic GP lgG and stained with fluorescein-conjugated goat antirabbit lgG. Note the decrease in intensity of fluorescence, indicating a decrease in the amount of heterologous antibodies remaining in the basement membrane. (Original magnification x450.)
Fig. 9. Fluorescent micrograph of glomeruli of a female rat killed 14 days after administration of rabbit antirat chorionic GP and stained with goat antirat lgG. Note intense linear fluorescence of the capillary walls, indicating the presence of autologous antibodies. (Original magnification x200.)
the autologous phase of immune nephritis. 2 • 4 Therefore, we might conclude that immune nephritis induced with the use of anti-GP antibodies is a disease comprised of two phases. The first or "heterologous" phase is characterized by the interaction of the antiGP antibodies with the glomerular basement membrane, followed by complement fixation, chemotaxis, and neutrophil infiltration. The second or "autologous" phase develops later and is the result of the host's response to the heterologous anti-GP IgG, causing further injury.
The observation that antihuman or antirat GP IgG antibodies had no effect on the outcome of pregnancy in rats injected during the third trimester suggests that either the antibodies were without effect because of their inability to cross the trophoblast layer and react at their respective antigenic sites on the trophoblast basement membrane or antibodies against the mitochondrial and microsomal fractions of trophoblast homogenates are involved in immune abortion. 1 On the basis of these studies, it is concluded that the collagen-free GP isolated from
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the chorion represents one of the common antigens shared between the trophoblast and the glomerular basement membranes. Furthermore, antibodies produced against the purified GP are nephrotoxic but not productive of abortion.
September 1, 1974 1\m. J, Ohstet. Gyncrol.
Gynecology of the Jewish General Hospital and especially to Dr. M. M. Gelfand for supplying us with the placentas. We would also like to express our appreciation to Mr. David Saxe for preparation of the illustrations and to Miss Hoda Karam for preparation of the manuscript.
The authors wish to express their gratitude to the staff of the Department of Obstetrics and
REFERENCES
I. Beer, A. E., and Billingham, R. E.: Immunobiology of mammalian reproduction, in Dixon, F. J., and Kunkel, P. L., editors: Advances in Immunology, New York, 1971, vol. 14, Academic Press, Inc. 2. Bencosme, S. A., and Morrin, P. A. F.: Ultrastructural pathology of the glomerulus, in Dalton, A. J., and Haguenau, F., editors: Ultrastructure in Biological Systems, Ultrastructure of the Kidney, New York, 1967, vol. 2, Academic Press, Inc., p. 143. 3. Boss, J. H.: AM. J. 0BSTET. GYNECOL. 93: 574, 1965. 4. Cochrane, C. G., Unanue, E. R., and Dixon, F. J.: J. Exp. Med. 122: 99, 1965. 5. Coons, A. H., Leduc, E. H., and Connolly, I.: J. Exp. Med. 102: 49, 1955.
6. Gang, N. F., and Gelfand, M. M.: Proc. Soc. Exp. Bioi. Med. 140: 188, 1972. 7. Gang, N. F., and Kalant, N.: Lab. Invest. 22: 531, 1970. 8. Katz, D. H., Unanue, E. R., and Dixon, F. J.: J. lmmunol. 98: 260, 1967. 9. Luft, J. H.: J. Biophys. Biochem. Cytol. 9: 409, 1961. 10. Pressman, D., and Korngold, L.: J. lmmunol. 78: 75, 1957. 11. Schwartz:, E. S., Gang, N. F., and Gelfand, M. M.; AM. J. 0BSTET. GYNECOL. 118: 857, 1974. 12. Seegal, B. C., and Loeb, E. N.: Proc. Soc. Exp. Bioi. Med. 45: 248, 1940. 13. Seegal, B. C., and Loeb, E. N.: Fed. Proc. 2: 101, 1943. 14. Steblay, R. W.: ]. Immunol. 88: 434, 1962.
NICHOLAS F. GANG, M.Sc., PH.D. ESTHER S. SCHWARTZ, B.Sc. ANNA MAJEROVICS MARIE-LAURE
DE
CHAMPLAIN, M.D.
ESTHER TRACHTENBERG Montreal, Quebec, Canada, and New York, New York The effect of rabbit antibodies produced against human and rat chorionic glycoprotein (GP) was investigated in relation to induction of nephritis and/or interruption of pregnancy in the rat. In virgin female and male rats used for the study of nephrotoxicity, proteinuria gradually increased with time. The glomeruli of rats examined 3 hours after injection of the antibodies revealed the presence of anti-GP immunoglobulin G (lgG) and of autologous C'3 in a linear, diffuse pattern in the capillary walls. Electron microscopy showed swelling and displacement of the endothelium from the basement membrane by neutrophils and fusion of the foot processes. In the glomeruli of rats killed on the fourteenth day of nephritis, the presence of autologous IgG could be observed by immunofluorescence. The ultrastructural pathology of the rats with chronic nephritis consisted of marked thickening of the basement membrane, mesangial proliferation, and distortion of the foot processes. The injection of antibodies into pregnant rats during the third trimester of gestation was without effect on the outcome of pregnancy. It is concluded that: (1) at least one of the nephrotoxic antigens common to the placenta and kidney is a GP of trophoblast basement membrane origin; (2) the pathogenesis of the glomerulopathy is similar to that produced with antibodies against glomerular basement membrane; and (3) antibodies other than anti-GP antibodies are responsible for immune abortion, which can be induced when the whole placenta is used as the source of antigen.
I T I s w E L L established that heterologous antibodies against crude placental homagenates, or definable fractions prepared by differential centrifugation, cross react with renal and extrarenal basement membranes. 1 The injection of heterologous antiplacental serum will lead to a glomerulopathy similar to that of Masugi nephritis12 • 13 and/or termination of pregnancy. 1 Recently, we isolated a noncollagenous glycoprotein (G P) from chorionic villi. 6 • 11 Immunofluorescent studies revealed that antibodies produced against the GP localized in vitro on the trophoblast basement membrane in the chorion and on the Bowman's capsule, glomerular, tubular, and small blood vessel basement membranes in the kidney. 11 Previously, whole
From the Laboratories of Experimental Pathology, Lady Davis Institute for Medical Research, the Department of Pathology, Jewish General Hospital, and the Department of Pathology, Mount Sinai School of Medicine of the City University of New York. Supported in part by Experimental Immunobiology of the Reproductive System Fund, Continental Pharma/ DAMON CORP., and by Operating Grant MA-4647 from the Medical Research Council of Canada. Received for publication October 30, 1973.
Revised February 4, 1974. Accepted February 20, 1974. Reprint requests: Dr. Nicholas F. Gang, Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 C8te St. Catherine Rd., Montreal 249, Quebec, Canada.
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74 Gang et al.
placental homogenates, or fractions thereof were used as the source of antigen(s) instead of purified glycoprotein. 1 • "· ' 0 • 14 The present study was undertaken to determine whether heterologous antibodies against the purified GP antigen are responsible for the induction of immune nephritis and/or interruption of pregnancy in the rat. Material and methods
Experimental animals. Sprague-Dawley virgin female and male rats, weighing 150 to 175 grams each, were used for the study of nephrotoxicity. Pregnant rats of the same strain in the eighteenth day of gestation, weighing 300 to 340 grams each, were used to determine the abortive effect of anti-GP antibodies. Rabbits of the New Zealand strain, weighing 2.5 to 3.0 kilograms each, were immunized with the purified GP for the production of antiserum. Immunologic procedures. The chorionic GP antigen was isolated from term human placentas obtained at delivery and rat placentas removed by surgical means on the twenty-first day of gestation, as previously described.6· 11 The antigen was obtained and purified as follows: The chorionic and decidual plates were removed, and the middle zone of the placenta was cut into small fragments; the terminal chorionic villi (CVI) were then scraped off from the minor villous stem with a razor blade. The CVI preparation was pressed through a 200 mesh stainless steel sieve with a spatula, washed through the same sieve, poured onto a 325 mesh wire cloth, and washed with an excess of isotonic saline. The CVI which were retained on top of the 325 mesh sieve were then collected with the aid of a Pasteur pipet, centrifuged, washed in distilled water, and lyophilized. These procedures enabled us to remove large connective tissue, blood vessel, cell, and cellular debri contaminants. The chorionic GP antigen was then prepared from CVI by sonication in 1M saline for 7 minutes to remove cells and cellular membranes, extraction with 0.3M acetic acid to remove residual connective tissue contaminants, and, subsequently, treatment with
Am.
September I, 1974 Obstet. Gynecol.
J.
deoxyribo- and ribo-nuclease to remove nucleic acids. The resultant insoluble fraction was then washed with distilled water and lyophilized. 11 Chemical analyses of the final preparation revealed a collagen-free GPY Antisera against the human and rat purified chorionic GP were produced in rabbits. The rabbits were injected in the footpads with 2 mg. of lyophilized GP suspended in 1 mi. of physiologic saline and emulsified with 1 mi. of Freund's complete adjuvant. Two further injections were given intramuscularly during the fifth and sixth weeks of immunization. Blood was drawn, by cardiac puncture, 8 days after the last injection. The antisera were then decomplemented by heating at 56° C. for 1 hour and absorbed with freshly harvested, washed human and rat erythrocytes, and the immunoglobulin G ( IgG) fraction was prepared. 8 Normal rabbit IgG was used as the control. For immunofluorescent microscopy, small pieces of chorionic tissues from the placenta and cortical tissues from the kidney were removed in situ and processed according to conventional methods. 5 Fluorescein-conjugated goat antirabbit IgG* and a fluoresceinconjugated IgG fraction of rabbit antirat IgG and C'3t were absorbed first with activated charcoal and subsequently with liver powder* to remove nonspecific staining materials. To locate the heterologous (rabbit) IgG in the kidney and the chorion and the autologous (rat) IgG and 0'3 in the kidney, the direct immunofluorescent procedure was used 7 on uniform sections of 3 p, thickness. Control sections were treated with the appropriate unlabeled antibodies prior to the conjugated ones. All of the sections were photographed on Kodak type B high-speed Ektachrome film,t with a uniform exposure time of 2 minutes. Chemical procedures. Total urinary protein was determined by a micromodification of the biuret method. 7 *Difco Labs., 920 Henry St., Detroit, Michigan 48201. tCappel Laboratories, Inc., Downingtown, Penlli'Ylvania. tEaatman Kodak Co., 343 State St., Rochester, New York 14650.
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Fig. 1. Electron micrograph of part of a glomerulus of a male rat killed 3 hours after injection of normal rabbit lgG. The capillary lumen (C) is patent; the endothelium (END), basement membrane (arrows), and epithelial foot processes ( EP) are unaltered. (Original magnification x6,000.)
Morphologic procedures. For electron microscopy, cortical biopsies were taken in situ alternately from the left and right kidneys and placed in a drop of ice-cold 3 per cent glutaraldehyde in cacodylate buffer (pH 7.25). The specimens were then cut into small fragments, transferred into vials, and fixed in the cold solution for 2 hours. After washing in the buffer solution, the specimens were postosmicated with 1 per cent Os04 in cacodylate buffer, dehydrated in graded alcohol solutions, treated with propylene oxide, and embedded in Epon. 9 Thin sections were cut on an LKB Ultratome III with diamond knives,* placed on uncoated copper grids ( 200 mesh) , and examined with Hitachi 8 and 12 electron microscopes. For determining the number of polymorphonuclear leukocytes (PMN), tissues were fixed in 4 per cent formaldehyde, embedded *E. I. DuPont de Nemours &: Co., Instrument Products Div., Wilmington, Delaware 19898.
in paraffin, and stained with hematoxylin and eosin. PMN's were counted in 30 glomeruli from each rat. Experimental procedures and results Nephrotoxicity of rabbit antibodies to human and rat chorionic GP. Ninety-six male and 96 virgin female rats were placed in individual metabolic cages. Forty-eight rats of each sex, serving as the experimental animals, were further divided into 2 equal groups. The first group received an intravenous (tail vein) injection of rabbit antihuman GP IgG and the second group received rabbit antirat GP IgG, at a dose of 400 p.g per 100 Gm. of body weight in 1 mi. of physiologic saline. Twelve male and 12 female rats were killed 3 hours after injection of the antibodies, and the remaining animals were killed 14 days later. An equal number of rats, serving as the
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Fig. 2. Electron micrograph of part of a glomerulus of a female rat killed 3 hours after administration of rabbit antihuman chorionic GP IgG. Note obliteration of capillary lumen, neutrophils lying directly on the basement membrane (arrows) , and swelling of visceral epithelium ( EP) with obliteration of urinary spaces. (Original magnification x5,400.)
controls, were injected with normal rabbit IgG at the same dose and by the same route as those of the experimental animals. They were killed in corresponding numbers and at the same time intervals as those of the experimental groups. Urine was collected at 24 hour intervals from each animal from the time of injection of the IgG preparation until they were killed. All urine samples were made up to 30 ml. with distilled water, and, after filtration through Whatman No. 2 filter paper, aliquots were taken for total urinary protein determinations. An additional 12 rats received antihuman GP IgG and 12 others received antirat GP IgG, adsorbed before injection with purified human and/or rat GP or glomerular basement membrane. Proteinuria. The protein excretion in all of the animals prior to injection of the appropriate IgG preparation was less than 20
mg. per day. The control animals, injected with normal rabbit IgG, did not develop proteinuria during the course of the study. In all of the experimental animals, male and female alike, injected with either rabbit antihuman or rabbit antirat GP antibodies, proteinuria gradually increased and was 400 ± 20 mg. per day by the fourteenth day. No proteinuria was observed in rats injected with the antibodies adsorbed with GP or glomerular basement membrane. PMN leukocyte infiltration of the glomeruli. In the control animals, the number of PMN's was 2.5 ± 0.5 per glomerulus. In rats killed 3 hours after administration of the anti-GP IgG, the number of neutrophils increased to 24 ± 2 per glomerulus. The glomeruli of rats killed 14 days after injection of the nephrotoxic antibodies showed no increase in the number of PMN's over those of the controls. General morphology. The renal glomeruli
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Fig. 3. Electron micrograph of part of a glomerular capillary wall of a male rat killed 14 days after injection of rabbit antirat chorionic GP IgG. Note detachment of the endothelium from the basement membrane (arrow), uneven thickening of the basement membrane ( BM), and fusion and distortion of the foot processes of the visceral epithelial cell ( EP). (Original magnification x6,000.)
of the virgin female and male control rats showed no abnormalities (Fig. 1). The general features of the glomerular lesions, as observed with the electron microscope, were identical in the male and female animals killed 3 hours after administration of either the antihuman or antirat GP antibodies. General features were swelling and detachment of the endothelium from the glomerular basement membrane, increase in the mesangial rna trix, focal fusion of the foot processes with swelling of the visceral epithelial cells, and obliteration of urinary spaces (Fig. 2). In addition, PMN's could be visualized in most of the capillary loops, lying directly on the basement membrane and displacing the endothelium (Fig. 2) . In rats killed 14 days after administration of normal rabbit IgG, the glomeruli appeared unaltered. The capillary lumen was patent. Neither swelling of the endothelium nor an increase in the thickness of the glomerular
basement membrane was apparent. No change in the visceral epithelial cells could be visualized. The glomeruli of male and female rats killed 14 days after injection of antihuman or antirat GP antibodies exhibited identical ultrastructural alterations. They consisted of swelling and separation of the endothelium from the basement membrane, an increase in mesangial cells and matrix with consequent partial obliteration of the capillary lumen (Figs. 3 and 4), and marked thickening and fraying of the glomerular basement membrane (Fig. 5 ). An amorphous, granular, electron-dense substance could be visualized in the subendothelial region, closely associated with the lamina densa (Fig. 5). There were marked fusion and distortion of the foot processes, together with moderate swelling of the visceral epithelial cells (Figs. 3, 4, and 5) . lmmunofiuorescent studies. In the control
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Fig. 4. Electron micrograph of part of a glomerulus of a female ra t killed 14 days after administration of rabbit antirat chorionic GP IgG. In addition to changes described in Fig. 3, note the significant increase in the mesangial cells (MC) and matrix (M), togeth er with swelling of the visceral epithelial cells (EP ). (Original magnification >'6,000. )
animals killed at 3 hours, or 14 days after injection of normal rabbit IgG, no localization of the heterologous antibodies could be detected in any of the structures of the kidney . In both male and female rats injected with either antihuman or antirat GP IgG and killed at the third hour of nephritis, the heterologous antibodies appeared in a diffuse, smooth pattern on the basement membranes of the glomerular capillaries (Fig. 6) . There was no localization of the antibodies in any other structures of the kidney. Fluoresceinconjugated rabbit antirat C'3 showed a staining pattern identical to that of the heterologous antibodies (Fig. 7) . In rats killed on the fourteenth day of nephritis, there was a significant decrease in the amount of heterologous antibodies in the glomerular basement membrane (Fig. 8 ) . On the other hand, as illustrated in Fig. 9, autologous IgG showed an intense, diffuse fluorescence. The effect of antihuman and antirat GP
antibodies on the outcome of pregnancy in the rat. The purpose of the second experiment was to determine whether a nti-G P antibodies could interrupt pregnancy in the rat. A total of +8 rats , in the eighteenth day of gestation, were used . The procedures employed with respect to injection of the antibodies and immunofluorescent procedures were as described in the preceding experiment. The administration of normal rabbit IgG or rabbit antihuman or antirat GP IgG had no effect on the course of pregnancy. All rats delivered on the twenty-first day of gestation and gave birth to healthy litters. Furthermore, no localization of rabbit antirat or antihuman GP antibodies could be detected on the trophoblast basement membrane. Comment
It has been demonstrated repeatedly tha t the administration of heterologous antipla-
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Fig. 5. Electron micrograph of part of a capillary wall of a female rat killed 14 days after injection of rabbit antihuman chorionic GP antibodies. Note uneven swelling of the basement membrane (BM) together with fraying of the membrane (large arrows) and the presence of a granular electron-dense substance in the subendothelial regions (small arrows). (Original magnification x6,000.)
cental sera has a striking nephrotoxic effect and can interrupt pregnancy in the species from which the antigen was obtained.H However, the exact nature of the antigen(s) common to the placenta and kidney, which operate in the two pathogenetic mechanisms described above, has not yet been estab· lished. 1 • 3 • 10 • 14 We have previously reported that antibodies produced against a purified chorionic noncollagenous glycoprotein cross reacted in vitro not only with the basal lamina of the trophoblasts but also with the basement membrane of Bowman's capsule, glomerulus, and tubules of the kidneyY The purpose of this investigation was to establish whether antibodies produced against human and rat GP antigens are responsible for the development of nephritis and/or abortion. Contrary to in vitro immunologic studies, upon intravenous injection, the anti-GP antibodies localized exclusively in the glomerular basement membrane in virgin female, male,
and pregnant female rats. This could have been caused by the inability of the antibodies to cross the cellular layer of the chorion and, hence, to reach their respective antigenic sites on the basal lamina. These findings lend support to the hypothesis, based on studies with crude placental homogenates, that the antigen ( s) common to the placenta and kidney are of trophoblast basement membrane origin. 3, 1o, 14 No difference in the localization of the antibodies in the glomerular capillary walls of rats was apparent, whether the antigen was of human or rat origin. This finding is of interest since the cross-reaction of human tissue to that of rat tissue is most unusual. The only explanation that one might offer is that at least some of the chorionic GP antigens are non-species specific and share common antigenic sites with the glomerular basement membrane. However, it must be emphasized that this conclusion is based solely on immunofluorescent studies which,
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Fig. 6. Fluorescent micrograph of a glomerulus of a female rat killed 3 hours after administration of rabbit antihuman chorionic GP lgG and stained with fluorescein-conjugated goat antirabbit lgG. Note intense diffuse, linear fluorescence of the capillary walls. (Original magnification x450.)
Fig. 7. Fluorescent micrograph same as Fig. 6 but stained with fluorescein-conjugated rabbit antirat C'3. Note linear staining of the capillary walls indicating the presence of complement. (Original magnification x450.)
at best, are semiquantitative. The presence of C'3 in the capillary walls and the infiltration of the glomeruli by the neutrophils following injection of the nephrotoxic antibodies were similar to the pathogenesis of Masugi nephritis. 4 The general ultrastructural alterations of the glomeruli of rats killed 3 hours after administration of anti-GP IgG, namely, swelling and displacement of the endothelial cells by neutrophils and fusion of the foot processes, were also similar to the changes described for the early stages of immune nephritis. 2 Taken together, these results indi-
cate that the onset of nephritis produced with anti-GP antibodies is also a complement- and neutrophil-mediated process. The development of massive proteinuria noted during the course of this study; the presence of autologous IgG in the glomerular capillary walls observed in rats 14 days following injection of the antibodies, together with the swelling of endothelium; the significant increase in the thickness of the basement membrane; and fusion of the foot processes were the same ultrastructural and functional alterations as those described for
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Fig. 8. Fluorescent micrograph of a glomerulus of a male rat killed 14 days after administration of rabbit antirat chorionic GP lgG and stained with fluorescein-conjugated goat antirabbit lgG. Note the decrease in intensity of fluorescence, indicating a decrease in the amount of heterologous antibodies remaining in the basement membrane. (Original magnification x450.)
Fig. 9. Fluorescent micrograph of glomeruli of a female rat killed 14 days after administration of rabbit antirat chorionic GP and stained with goat antirat lgG. Note intense linear fluorescence of the capillary walls, indicating the presence of autologous antibodies. (Original magnification x200.)
the autologous phase of immune nephritis. 2 • 4 Therefore, we might conclude that immune nephritis induced with the use of anti-GP antibodies is a disease comprised of two phases. The first or "heterologous" phase is characterized by the interaction of the antiGP antibodies with the glomerular basement membrane, followed by complement fixation, chemotaxis, and neutrophil infiltration. The second or "autologous" phase develops later and is the result of the host's response to the heterologous anti-GP IgG, causing further injury.
The observation that antihuman or antirat GP IgG antibodies had no effect on the outcome of pregnancy in rats injected during the third trimester suggests that either the antibodies were without effect because of their inability to cross the trophoblast layer and react at their respective antigenic sites on the trophoblast basement membrane or antibodies against the mitochondrial and microsomal fractions of trophoblast homogenates are involved in immune abortion. 1 On the basis of these studies, it is concluded that the collagen-free GP isolated from
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the chorion represents one of the common antigens shared between the trophoblast and the glomerular basement membranes. Furthermore, antibodies produced against the purified GP are nephrotoxic but not productive of abortion.
September 1, 1974 1\m. J, Ohstet. Gyncrol.
Gynecology of the Jewish General Hospital and especially to Dr. M. M. Gelfand for supplying us with the placentas. We would also like to express our appreciation to Mr. David Saxe for preparation of the illustrations and to Miss Hoda Karam for preparation of the manuscript.
The authors wish to express their gratitude to the staff of the Department of Obstetrics and
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I. Beer, A. E., and Billingham, R. E.: Immunobiology of mammalian reproduction, in Dixon, F. J., and Kunkel, P. L., editors: Advances in Immunology, New York, 1971, vol. 14, Academic Press, Inc. 2. Bencosme, S. A., and Morrin, P. A. F.: Ultrastructural pathology of the glomerulus, in Dalton, A. J., and Haguenau, F., editors: Ultrastructure in Biological Systems, Ultrastructure of the Kidney, New York, 1967, vol. 2, Academic Press, Inc., p. 143. 3. Boss, J. H.: AM. J. 0BSTET. GYNECOL. 93: 574, 1965. 4. Cochrane, C. G., Unanue, E. R., and Dixon, F. J.: J. Exp. Med. 122: 99, 1965. 5. Coons, A. H., Leduc, E. H., and Connolly, I.: J. Exp. Med. 102: 49, 1955.
6. Gang, N. F., and Gelfand, M. M.: Proc. Soc. Exp. Bioi. Med. 140: 188, 1972. 7. Gang, N. F., and Kalant, N.: Lab. Invest. 22: 531, 1970. 8. Katz, D. H., Unanue, E. R., and Dixon, F. J.: J. lmmunol. 98: 260, 1967. 9. Luft, J. H.: J. Biophys. Biochem. Cytol. 9: 409, 1961. 10. Pressman, D., and Korngold, L.: J. lmmunol. 78: 75, 1957. 11. Schwartz:, E. S., Gang, N. F., and Gelfand, M. M.; AM. J. 0BSTET. GYNECOL. 118: 857, 1974. 12. Seegal, B. C., and Loeb, E. N.: Proc. Soc. Exp. Bioi. Med. 45: 248, 1940. 13. Seegal, B. C., and Loeb, E. N.: Fed. Proc. 2: 101, 1943. 14. Steblay, R. W.: ]. Immunol. 88: 434, 1962.