I,~;nlutluch¢'mistrt 1970. Vol. 13. pp 295-298
Pergamon Press.
l~nted in Great Bntain
AGE RELATED CARBOHYDRATE CONTENT OF MOUSE KIDNEY GLOMERULAR BASEMENT MEMBRANE AND ITS REACTIVITY TO ANTISTREPTOCOCCAL MEMBRANE ANTISERA WILLIAM T. BLUE and CHARLES F. LANGE Loyola University Stritch School of Medicine. Department of Microbiology. Maywood, IL 60153. U.S.A. (First receioed 6 July 1975; in ret, ised form 20 October 1975)
Alaatraet--Rabbit antisera to group A, type 12 streptococcal cell membrane (SCM) and human glornerular or glomerular basement membrane (GBM) preparations were found to immunologically react with adult mouse GBM by an indirect fluorescent antibody test. Such cross-reactivity was enhanced by enzymatic cleavage of GBM carbohydrate. In comparative tests with neonatal and adult kidneys, the antisera were more reactive With the former than the latter. GBM carbohydrate cleavage had little or no effect on the reactivity of neonatal GBM With the antisera. Quantitative carbohydrate analyses revealed that adult mouse GBM contained a greater content of carbohydrate than did neonatal GBM. Thus the differential reactivities of adult and neonatal mouse GBM with anti-SCM antisera are directly related to their respective carbohydrate contents.
INTRODUCTION
nature, and few conclusions as to the nature of the disease stemmed from the works.
Nephrotoxic antigens located in the kidney glomerular basement membrane (GBM) of a variety of animals, including man, are glycoproteins (Huang & MATERIALS AND METHODS Kalant, 1968; Marquardt et al., 1973; Misra, 1973). Antisera Glomerulonephritis following infections with certain group A streptococci may be facilitated by a crossAll antisera were prepared in rabbits. Twenty-two SCM reaction between such glycoproteins and antibodies antisera, which were made against 7 chemically defined to cell membrane antigens of the streptococcal preparations of group A, type 12 SCM or soluble extracts organisms (Markowitz & Lange, 1964; Blue & Lange, of SCM (Lange, 1969), and 10 glon~rular antisera, pre. 1975), which are also glycoproteins (Lange. 1969). pared against whole human glomerulL GBM, or soluble Antisera to the cell membrane of nephritogenic type or insoluble extracts of GBM, were used in all fluorescent antibody tests. 12, group A streptococci have been reported to crossreact with a wide variety of animal tissues, including Kidneys kidney antigens of dogs (Rapaport et al., 1969). rats Fresh white albino mouse.(Swiss outbred strain, Mus (Markowitz et aL, 1960), guinea pigs (Lyampert et al., musculus) kidneys were used for all experiments. Adult 1968) and Rhesus monkeys 0Markowitz, 1969). guinea mouse kidneys were those taken from mice greater than pig skin (Lange, 1973; Rapaport et al., 1969), skeletal two months of age, while neonatal kidneys were those from muscle and heart (Lyampert et al., 1968) as well as mice less than seven days old. Kidney sections were cut human heart tissue (Zabriskie & Freimer, 1966). It two microns thick from frozen cortex segments on an IEC has recently been shown that antigens of adult human cryostat, and fixed on microscrope slides with acetone. GBM are 'masked' by carbohydrate units, and enzy- Glomeruli and G B M were isolated by standard methods (Greenspon & Krakower, 1950; Krakower & Greenspons, matic cleavage of such units increases the antigenicity 1951), using 200 mesh wire screening and sonicatio~ of GBM (Quish & Lange, 1973). Also, the cross-reaction between group A, type 12 streptococcal cell Carbohydrases membrane (SCM) antisera and adult human GBM The carbohydrase (CHOase) preparation,freefrom prois potentiated by cleavage of GBM carbohydrate tease activity, was previously characterized (Quish & (Blue & Lange, 1975). To further characterize this Lange, 1973). The lyophihzed mixture contained the phenomenom the extent of cross-reactivity between enzymes glucosidase,~galactosidase, neuraminidase, fucomouse kidney GBM and antisera to group A, type sidase and hexosaminidase, and was used in a 0"2°,osolu12 SCM, as well as the nature of antigenic masking tion in phosphate buffered saline,pH 7.0. in adult and neonatal mouse GBM, were examined. Fhtorescent antil~ly tests Also, the carbohydrate and amino acid contents of ,Fluorescent antibody tests, on kidney cortex sections. adult and neonatal mouse GBM were analyzed and were performed by an indirect method, with the final overcompared. Although mice have been used as exper- lay being fluorescc4n-conjugatedsheep anti-rabbit gamma imental models for the study of post-streptococcal globulin. Fluorescent intensities were graded on a scale glomerulonephritis (Kelly & Wirm, 1958; Sharp, of 0--4+, and initially, all antisera were evaluated and 1964). the systems used were non-immunological m graded on a blind basis. 295
296
WILLIAM T. BLUE and CHARLES F. LANGE Table 1. Indirect fluorescent antibody tests. Rabbit anti-membrane sera vs mouse kidney GBM Number positive/number tested Antisera
Untreated
CHOase treated
Number showing increased antibody binding after CHOase
3/22
6:22
5
Neonatal Adult
19/22 9/10
19/22 10/10
0 6
Neonatal
10/10
10/10
2
Tissue sections Adult
SCM GBM
Carbohydrate analyses Quantitative carbohydrate analyses were carried out using spectrophotometric prooedun~ Total h~xos~ were determined by the Orcinoi pro¢~unt (Rmewear & Smith. 1961), fu¢,ose by the cyst¢in¢--hydrochlorid© method (Dische & Shettles, 1948), si~lic ~ bythe thiobarbituri¢ acid prooedurc ~ a r r e a , 1959) and hcxowninm by a modified Elan-Morgan procedure fWinzler, 1955). Amino acid analyses Quantitative amino acid analyses were performed with a Beckman model 120C amino acid analyzer.
RESULTS
Fluorescent antibody tests The twenty-two anti-SCM and ten anti-human glomerular antisera were evaluated by indirect fluorescent antibody tests, and their staining intensities graded, on both neonatal and adult mouse kidney cortex sections. All tests were run in duplicate, on sections which were either untreated or pretreated o ~ m i g h t with a 0.2% solution of CHOase. A summary of the results is presented in Table 1. Only 3 of the 22 anti-SCM antisera gave positive GBM fluorescence on adult mouse kidney sections. The number of positive sera increased to 6 when they were tested on sections of adult mouse kidney cortex which had been pre-treated with carbohydrates. Of the 6 positive sera, 5 showed increased antibody binding, as m c a s u t ~ by incrcmed fluor,~c~nt intensities, due to the CHOase tr~tment. In comparison, on neonatal mouse sections, 19 of the 22 antisera were positive, and CHOase treatment had no effect on the fluorescent intensities observed. With the glorr~rular antisera, all 10 gave positive GBM fluorescence. However, 6 of the aatisera showed increased antibody binding after C H O a ~ treatment on adult sections, as compared to only 2 on neonatal sections.
Chemical analyses Since the differential reactivities of adult and neonatal mouse kidney GBM with the SCM and glomcrular antiscra seemed in part del~mdent upon GBM carbohydrate, as wimesscd by the effect of 'unmaskrag' adult GBM antigcm with C H O a ~ while enzyme treatment had tittle or no effc,'t on m GBM, qmmtitative carbohydrate amdys(m of isohted adult and neonatal GBM were carried out. The resdts are presented m Table 2. All individual carbohydrates were found to be higher in quantity in adult GBM as compared to neonatal. Total carbohydrate was approximately 1-4% higher in adult GBM. Also shown is the quantitative carbohydrate analyses of whole type 12 SCM (Markowitz & L a n t ~ 1964). Amino acid analyses of adult and neonatal GRM's were performed and compared. The most strikin s differences in composition were tho~ of lysinc and hydroxylsine" and proline and hydroxyproline. Lysin¢ and proline contents dropped s ~ t l y from neonatal to adult G B M (5.2-4,8*/0,and 5.2--4.65:,,respectively), while hydroxylysme and hydroxyproline were found to increase signilicantlyin content (I~70--O95"~ and 2-8--4.3*/o).Most other amino acids remained fairly constant in content.
DISCUSSION
Although many consider post-streptococcal glomerulonephritis an immune com#cx type d i ~ and often make this statement, the evidence is far from conclusive. To date no definitivedata exists to confirm this concept. While many investigators are willing to accept the idea that the subepithelial electron dense areas seen on electron microscopy represent "immune complexes' no one has actually isolated or identified both an antibody and its corresponding antigma as has been done for example m the case of systemic lupus ¢rythematosis ($1.E) with DNA and
Table 2. Quantitative carbohydrate determinations of adult and neonatal mouse GBM and streptococcal membranes (SCM)
GBM Adult Neonatal SCM
Total hexose 4.77 4.18 8.3
Carbohydrate (% dry weight) Sialic acid Fucose 1.19 0-78 None
0'68 0.38 0-08 (Rhamnose)
Hexosamines
Total carbohydrate
0-68 0-54 2.7
7.32 5.88 1I. 1
Antigenicity of Mouse GBM an anti-DNA antibody. It is felt that the data presented in the present report offer an alternative explanation to the underlying mechanisms in the disease process. The reactivity of group A, type 12 anti-SCM antisera with mouse kidney GBM is viewed as support for the cross-reactive theory of post-streptococcal glomerulonephritis, since such sera also cross-react with human kidney GBM (Lange, 1969; Lange, 1973; Blue & Lange, 1975). In addition, antisera to group A, type 12 SCM have been reported to be nephrotoxic for dogs (Rapaport et al., 1969). Rhesus monkeys (Markowitz, 1969) and rats (Markowitz et at., 1960), and have been shown to cause accelerated (while graft) skin graft rejection in guinea pigs (Rapapon, 1969) as well as hemorrhagic necrosis (Rapaport et al, 1969; Lange, 1973), all apparently due to a cross-reaction with mammalian tissue antigens. The pathogenic mechanisms which lead to poststreptococcal glomerulonephritis in humans are as yet poorly understood. One theory envisions an immunologic cross-reaction between human GBM and SCM antigens of the invading streptococcus. Since such a cross-reaction has been demonstrated (Markowitz & Lange, 1964; Blue & Lange, 1975) it is of interest that a similar antigenic similarity exists with mice. Thus a possible animal model to test the theory is available. Also, the observation that post-streptococcal giomerulonephritis occurs in young children to a greater extent than in adults may find an explanation in the reported findings. To what extent the lack of 'masking' of cross-reactive GBM epitopes by carbohydrate units in earlier ages plays in the pathogenesis of the disease remains to be determined but should be considered. Thus the changes which occur in amino acid and carbohydrate content of kidney GBM with aging may be significant. The differential reactivities, with SCM antisera~ of adult and neonatal mouse GBM is apparently due to their differential carbohydrate contents. As reported earlier (Quish & Lange, 1973; Blue & Lange, 1975) human GBM protein antigens are 'masked' by carbohydrate units, which was shown by the increased antigenicity of GBM following CHOase treatment. The report of Quish & Lange (1973) established by quantitative precipitin curves that the supernatants obtained with the native antigen could react producing a new quantitative precipitin curve with the same antigen treated to be 100°/0 free of its sialic acid and fucose and approximately 50% free of its other carbohydrates. The clear implication here was that the carbohydrate was definitely not involved in the immunologic reactions but primarily only the protein and especially those protein epitopes in close proximity to the point of attachment of the carbohydrate. Of course to get the complete answer to the role carbohydrate does play will depend on the isolation of these oligosaccharides free of their protein backbone and the preparation of specific antiserum to them. But the disparity seen (Table 2) in the carbohydrate contents of the SCM and the adult or neonatal GBM do not seem to suggest any immunochemical relatedness between these two diverse substances. Thus the results found in the present report with adult mouse GBM, wherein carbohydrate cleavage apparently 'unmasked' some cross-reactive anti-
297
genie sites further enhance these concepts. The effect of such cleavage was not found, in comparison, with neonatal GBM. However, untreated neonatal sections were much more reactive with SCM antisera than adult sections, acting as if their cross-reactive sites were already 'unmask/xl'. Quantitative carbohydrate analyses of neonatal and adult mouse GBM's support the immunological findings, in that neonatal GBM had less carbohydrate content than adult GBM. Apparently the 34~ less slalic acid along with reduced % content of fucose (44~), hexose (12%), hexosamines (20°/0) was sufficient to manifest the immunologic findings. Thus during the maturation of the mouse kidney, carbohydrate must be added to the GBIVL becoming part of the GBM matrix and masking underlying antigenic sites. Such a hypothesis is supported by the amino acid analyses, which showed that as the kidney matures, hydroxylation of lysine and proline residues occurs, and as shown by Spiro (1967), hydroxylysine is an important linkage point to GBM carbohydrate. Although the results presented here are from studies with one strain of mice, it is the opinion of the investigators that these same results would be obtained in all strains of mice. An extension of these findings is the possibility of recognizing the specific biochemical changes which occur in the kidney with aging, i.e. neonatal to adult, as detected by changes in immunologic reactivity. Since the original streptococcal immunogens have been chemically characterized fMarkowitz & Lange, 1964; Lange, 1969; Lange, 1973)` comparative chemistries between these two diverse biological organisms present interesting findings. Acknowledgements--This work was supported in part by the following USPHS grants: AM 14622 and GRSG RR 05368.
REFERENCES
Blue W. T. & Lange C. F. (1975) J. lnmnm. 114, 306. Disehe Z. & Sherries L. B. (1948) J. biol. Chem. 175, 595. Greenspon S. A. & Krakower C. A. (1950) Arch. Path. 49, 291.
Huang F. & Kalant N. (1968) Can. d. Biochem. 46, 1523. Kelly D. K. & Winn J. F. (1958) Science 127, 1337. Krakower C. A. & Greenspon S. A. (1951) Arehs Path. 51, 626. Lange C. F. (1969) Transplant. Proc. 1, 959. Lange C. F. (1973) Res. Commun. Chem. Path. Pharm 6, 263. Lyampert I. M, Bordiyuk N. H. & Ugryumona G. A. (1968) Immunology 15, 845. Markowitz A. S. (1969) Transplant. Proc. 1,985. Markowitz A. S., Armstrong S. H. & Kushner D. S. (1960) Nature, New Biol. lg7, 1095. Markowitz A. S. & Lange C. F. (1964) J. lmmun. 92, 565. Marquardt H., Wilson C. F. & Dixon F. J. (1973) Kidney Int. 3, 57. Misra R. P. (1973) Immunology 25, 967. Quish T. B. & Lange C. F, (1973) Res. Commun. Chem. Path. Pharm. 5, 574. Rapaport F. T., Markowitz A. S. & McCluskey R. T. (1969) J. exp. Med. 129, 623. Rapaport F. T., Markowitz A. S., McCluskey R. T., Hanaoka T. & Shemada T. (1969) Transplant. Proc. 1, 981.
298
WILLIAM
T. B L U E
and C H A R L E S
Rosewear J. W. & Smith E. L. (1961) J. bioL Chem. 236, 425. Sharp J. T. (1964) J. Lab. clin. Med. 63, 232. Spiro R. G. (1967) J. biol. Chem. 242, 1923.
F. L A N G E
Warren L. (1959) J. bJol. Chem. 134, 1971. Winzler R. J. (1955) Methods bi~hem. AnalysJs 2, 279. Zabriskie J. B. & Freimer E. H. (1966) J. exp. Med. 124, 661.
Pergamon Press.
l~nted in Great Bntain
AGE RELATED CARBOHYDRATE CONTENT OF MOUSE KIDNEY GLOMERULAR BASEMENT MEMBRANE AND ITS REACTIVITY TO ANTISTREPTOCOCCAL MEMBRANE ANTISERA WILLIAM T. BLUE and CHARLES F. LANGE Loyola University Stritch School of Medicine. Department of Microbiology. Maywood, IL 60153. U.S.A. (First receioed 6 July 1975; in ret, ised form 20 October 1975)
Alaatraet--Rabbit antisera to group A, type 12 streptococcal cell membrane (SCM) and human glornerular or glomerular basement membrane (GBM) preparations were found to immunologically react with adult mouse GBM by an indirect fluorescent antibody test. Such cross-reactivity was enhanced by enzymatic cleavage of GBM carbohydrate. In comparative tests with neonatal and adult kidneys, the antisera were more reactive With the former than the latter. GBM carbohydrate cleavage had little or no effect on the reactivity of neonatal GBM With the antisera. Quantitative carbohydrate analyses revealed that adult mouse GBM contained a greater content of carbohydrate than did neonatal GBM. Thus the differential reactivities of adult and neonatal mouse GBM with anti-SCM antisera are directly related to their respective carbohydrate contents.
INTRODUCTION
nature, and few conclusions as to the nature of the disease stemmed from the works.
Nephrotoxic antigens located in the kidney glomerular basement membrane (GBM) of a variety of animals, including man, are glycoproteins (Huang & MATERIALS AND METHODS Kalant, 1968; Marquardt et al., 1973; Misra, 1973). Antisera Glomerulonephritis following infections with certain group A streptococci may be facilitated by a crossAll antisera were prepared in rabbits. Twenty-two SCM reaction between such glycoproteins and antibodies antisera, which were made against 7 chemically defined to cell membrane antigens of the streptococcal preparations of group A, type 12 SCM or soluble extracts organisms (Markowitz & Lange, 1964; Blue & Lange, of SCM (Lange, 1969), and 10 glon~rular antisera, pre. 1975), which are also glycoproteins (Lange. 1969). pared against whole human glomerulL GBM, or soluble Antisera to the cell membrane of nephritogenic type or insoluble extracts of GBM, were used in all fluorescent antibody tests. 12, group A streptococci have been reported to crossreact with a wide variety of animal tissues, including Kidneys kidney antigens of dogs (Rapaport et al., 1969). rats Fresh white albino mouse.(Swiss outbred strain, Mus (Markowitz et aL, 1960), guinea pigs (Lyampert et al., musculus) kidneys were used for all experiments. Adult 1968) and Rhesus monkeys 0Markowitz, 1969). guinea mouse kidneys were those taken from mice greater than pig skin (Lange, 1973; Rapaport et al., 1969), skeletal two months of age, while neonatal kidneys were those from muscle and heart (Lyampert et al., 1968) as well as mice less than seven days old. Kidney sections were cut human heart tissue (Zabriskie & Freimer, 1966). It two microns thick from frozen cortex segments on an IEC has recently been shown that antigens of adult human cryostat, and fixed on microscrope slides with acetone. GBM are 'masked' by carbohydrate units, and enzy- Glomeruli and G B M were isolated by standard methods (Greenspon & Krakower, 1950; Krakower & Greenspons, matic cleavage of such units increases the antigenicity 1951), using 200 mesh wire screening and sonicatio~ of GBM (Quish & Lange, 1973). Also, the cross-reaction between group A, type 12 streptococcal cell Carbohydrases membrane (SCM) antisera and adult human GBM The carbohydrase (CHOase) preparation,freefrom prois potentiated by cleavage of GBM carbohydrate tease activity, was previously characterized (Quish & (Blue & Lange, 1975). To further characterize this Lange, 1973). The lyophihzed mixture contained the phenomenom the extent of cross-reactivity between enzymes glucosidase,~galactosidase, neuraminidase, fucomouse kidney GBM and antisera to group A, type sidase and hexosaminidase, and was used in a 0"2°,osolu12 SCM, as well as the nature of antigenic masking tion in phosphate buffered saline,pH 7.0. in adult and neonatal mouse GBM, were examined. Fhtorescent antil~ly tests Also, the carbohydrate and amino acid contents of ,Fluorescent antibody tests, on kidney cortex sections. adult and neonatal mouse GBM were analyzed and were performed by an indirect method, with the final overcompared. Although mice have been used as exper- lay being fluorescc4n-conjugatedsheep anti-rabbit gamma imental models for the study of post-streptococcal globulin. Fluorescent intensities were graded on a scale glomerulonephritis (Kelly & Wirm, 1958; Sharp, of 0--4+, and initially, all antisera were evaluated and 1964). the systems used were non-immunological m graded on a blind basis. 295
296
WILLIAM T. BLUE and CHARLES F. LANGE Table 1. Indirect fluorescent antibody tests. Rabbit anti-membrane sera vs mouse kidney GBM Number positive/number tested Antisera
Untreated
CHOase treated
Number showing increased antibody binding after CHOase
3/22
6:22
5
Neonatal Adult
19/22 9/10
19/22 10/10
0 6
Neonatal
10/10
10/10
2
Tissue sections Adult
SCM GBM
Carbohydrate analyses Quantitative carbohydrate analyses were carried out using spectrophotometric prooedun~ Total h~xos~ were determined by the Orcinoi pro¢~unt (Rmewear & Smith. 1961), fu¢,ose by the cyst¢in¢--hydrochlorid© method (Dische & Shettles, 1948), si~lic ~ bythe thiobarbituri¢ acid prooedurc ~ a r r e a , 1959) and hcxowninm by a modified Elan-Morgan procedure fWinzler, 1955). Amino acid analyses Quantitative amino acid analyses were performed with a Beckman model 120C amino acid analyzer.
RESULTS
Fluorescent antibody tests The twenty-two anti-SCM and ten anti-human glomerular antisera were evaluated by indirect fluorescent antibody tests, and their staining intensities graded, on both neonatal and adult mouse kidney cortex sections. All tests were run in duplicate, on sections which were either untreated or pretreated o ~ m i g h t with a 0.2% solution of CHOase. A summary of the results is presented in Table 1. Only 3 of the 22 anti-SCM antisera gave positive GBM fluorescence on adult mouse kidney sections. The number of positive sera increased to 6 when they were tested on sections of adult mouse kidney cortex which had been pre-treated with carbohydrates. Of the 6 positive sera, 5 showed increased antibody binding, as m c a s u t ~ by incrcmed fluor,~c~nt intensities, due to the CHOase tr~tment. In comparison, on neonatal mouse sections, 19 of the 22 antisera were positive, and CHOase treatment had no effect on the fluorescent intensities observed. With the glorr~rular antisera, all 10 gave positive GBM fluorescence. However, 6 of the aatisera showed increased antibody binding after C H O a ~ treatment on adult sections, as compared to only 2 on neonatal sections.
Chemical analyses Since the differential reactivities of adult and neonatal mouse kidney GBM with the SCM and glomcrular antiscra seemed in part del~mdent upon GBM carbohydrate, as wimesscd by the effect of 'unmaskrag' adult GBM antigcm with C H O a ~ while enzyme treatment had tittle or no effc,'t on m GBM, qmmtitative carbohydrate amdys(m of isohted adult and neonatal GBM were carried out. The resdts are presented m Table 2. All individual carbohydrates were found to be higher in quantity in adult GBM as compared to neonatal. Total carbohydrate was approximately 1-4% higher in adult GBM. Also shown is the quantitative carbohydrate analyses of whole type 12 SCM (Markowitz & L a n t ~ 1964). Amino acid analyses of adult and neonatal GRM's were performed and compared. The most strikin s differences in composition were tho~ of lysinc and hydroxylsine" and proline and hydroxyproline. Lysin¢ and proline contents dropped s ~ t l y from neonatal to adult G B M (5.2-4,8*/0,and 5.2--4.65:,,respectively), while hydroxylysme and hydroxyproline were found to increase signilicantlyin content (I~70--O95"~ and 2-8--4.3*/o).Most other amino acids remained fairly constant in content.
DISCUSSION
Although many consider post-streptococcal glomerulonephritis an immune com#cx type d i ~ and often make this statement, the evidence is far from conclusive. To date no definitivedata exists to confirm this concept. While many investigators are willing to accept the idea that the subepithelial electron dense areas seen on electron microscopy represent "immune complexes' no one has actually isolated or identified both an antibody and its corresponding antigma as has been done for example m the case of systemic lupus ¢rythematosis ($1.E) with DNA and
Table 2. Quantitative carbohydrate determinations of adult and neonatal mouse GBM and streptococcal membranes (SCM)
GBM Adult Neonatal SCM
Total hexose 4.77 4.18 8.3
Carbohydrate (% dry weight) Sialic acid Fucose 1.19 0-78 None
0'68 0.38 0-08 (Rhamnose)
Hexosamines
Total carbohydrate
0-68 0-54 2.7
7.32 5.88 1I. 1
Antigenicity of Mouse GBM an anti-DNA antibody. It is felt that the data presented in the present report offer an alternative explanation to the underlying mechanisms in the disease process. The reactivity of group A, type 12 anti-SCM antisera with mouse kidney GBM is viewed as support for the cross-reactive theory of post-streptococcal glomerulonephritis, since such sera also cross-react with human kidney GBM (Lange, 1969; Lange, 1973; Blue & Lange, 1975). In addition, antisera to group A, type 12 SCM have been reported to be nephrotoxic for dogs (Rapaport et al., 1969). Rhesus monkeys (Markowitz, 1969) and rats (Markowitz et at., 1960), and have been shown to cause accelerated (while graft) skin graft rejection in guinea pigs (Rapapon, 1969) as well as hemorrhagic necrosis (Rapaport et al, 1969; Lange, 1973), all apparently due to a cross-reaction with mammalian tissue antigens. The pathogenic mechanisms which lead to poststreptococcal glomerulonephritis in humans are as yet poorly understood. One theory envisions an immunologic cross-reaction between human GBM and SCM antigens of the invading streptococcus. Since such a cross-reaction has been demonstrated (Markowitz & Lange, 1964; Blue & Lange, 1975) it is of interest that a similar antigenic similarity exists with mice. Thus a possible animal model to test the theory is available. Also, the observation that post-streptococcal giomerulonephritis occurs in young children to a greater extent than in adults may find an explanation in the reported findings. To what extent the lack of 'masking' of cross-reactive GBM epitopes by carbohydrate units in earlier ages plays in the pathogenesis of the disease remains to be determined but should be considered. Thus the changes which occur in amino acid and carbohydrate content of kidney GBM with aging may be significant. The differential reactivities, with SCM antisera~ of adult and neonatal mouse GBM is apparently due to their differential carbohydrate contents. As reported earlier (Quish & Lange, 1973; Blue & Lange, 1975) human GBM protein antigens are 'masked' by carbohydrate units, which was shown by the increased antigenicity of GBM following CHOase treatment. The report of Quish & Lange (1973) established by quantitative precipitin curves that the supernatants obtained with the native antigen could react producing a new quantitative precipitin curve with the same antigen treated to be 100°/0 free of its sialic acid and fucose and approximately 50% free of its other carbohydrates. The clear implication here was that the carbohydrate was definitely not involved in the immunologic reactions but primarily only the protein and especially those protein epitopes in close proximity to the point of attachment of the carbohydrate. Of course to get the complete answer to the role carbohydrate does play will depend on the isolation of these oligosaccharides free of their protein backbone and the preparation of specific antiserum to them. But the disparity seen (Table 2) in the carbohydrate contents of the SCM and the adult or neonatal GBM do not seem to suggest any immunochemical relatedness between these two diverse substances. Thus the results found in the present report with adult mouse GBM, wherein carbohydrate cleavage apparently 'unmasked' some cross-reactive anti-
297
genie sites further enhance these concepts. The effect of such cleavage was not found, in comparison, with neonatal GBM. However, untreated neonatal sections were much more reactive with SCM antisera than adult sections, acting as if their cross-reactive sites were already 'unmask/xl'. Quantitative carbohydrate analyses of neonatal and adult mouse GBM's support the immunological findings, in that neonatal GBM had less carbohydrate content than adult GBM. Apparently the 34~ less slalic acid along with reduced % content of fucose (44~), hexose (12%), hexosamines (20°/0) was sufficient to manifest the immunologic findings. Thus during the maturation of the mouse kidney, carbohydrate must be added to the GBIVL becoming part of the GBM matrix and masking underlying antigenic sites. Such a hypothesis is supported by the amino acid analyses, which showed that as the kidney matures, hydroxylation of lysine and proline residues occurs, and as shown by Spiro (1967), hydroxylysine is an important linkage point to GBM carbohydrate. Although the results presented here are from studies with one strain of mice, it is the opinion of the investigators that these same results would be obtained in all strains of mice. An extension of these findings is the possibility of recognizing the specific biochemical changes which occur in the kidney with aging, i.e. neonatal to adult, as detected by changes in immunologic reactivity. Since the original streptococcal immunogens have been chemically characterized fMarkowitz & Lange, 1964; Lange, 1969; Lange, 1973)` comparative chemistries between these two diverse biological organisms present interesting findings. Acknowledgements--This work was supported in part by the following USPHS grants: AM 14622 and GRSG RR 05368.
REFERENCES
Blue W. T. & Lange C. F. (1975) J. lnmnm. 114, 306. Disehe Z. & Sherries L. B. (1948) J. biol. Chem. 175, 595. Greenspon S. A. & Krakower C. A. (1950) Arch. Path. 49, 291.
Huang F. & Kalant N. (1968) Can. d. Biochem. 46, 1523. Kelly D. K. & Winn J. F. (1958) Science 127, 1337. Krakower C. A. & Greenspon S. A. (1951) Arehs Path. 51, 626. Lange C. F. (1969) Transplant. Proc. 1, 959. Lange C. F. (1973) Res. Commun. Chem. Path. Pharm 6, 263. Lyampert I. M, Bordiyuk N. H. & Ugryumona G. A. (1968) Immunology 15, 845. Markowitz A. S. (1969) Transplant. Proc. 1,985. Markowitz A. S., Armstrong S. H. & Kushner D. S. (1960) Nature, New Biol. lg7, 1095. Markowitz A. S. & Lange C. F. (1964) J. lmmun. 92, 565. Marquardt H., Wilson C. F. & Dixon F. J. (1973) Kidney Int. 3, 57. Misra R. P. (1973) Immunology 25, 967. Quish T. B. & Lange C. F, (1973) Res. Commun. Chem. Path. Pharm. 5, 574. Rapaport F. T., Markowitz A. S. & McCluskey R. T. (1969) J. exp. Med. 129, 623. Rapaport F. T., Markowitz A. S., McCluskey R. T., Hanaoka T. & Shemada T. (1969) Transplant. Proc. 1, 981.
298
WILLIAM
T. B L U E
and C H A R L E S
Rosewear J. W. & Smith E. L. (1961) J. bioL Chem. 236, 425. Sharp J. T. (1964) J. Lab. clin. Med. 63, 232. Spiro R. G. (1967) J. biol. Chem. 242, 1923.
F. L A N G E
Warren L. (1959) J. bJol. Chem. 134, 1971. Winzler R. J. (1955) Methods bi~hem. AnalysJs 2, 279. Zabriskie J. B. & Freimer E. H. (1966) J. exp. Med. 124, 661.
