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Protection of kidneys by pepsin-degraded immunoglobulin
Journal
of Surgical Research
Clinical
and Laboratory
Investigation
Volume 11 Number 2, February 1971 PROTECTION GEORGE
V.
SMITH,
OF KIDNEYS M.D.,
F.A.C.S.,
BY PEPSIN-DEGRADED ALLEN
AND
GUY
J. KENPON, OWENS,
M.D.,
AND
MATERIALS
Healthy mongrel dogs were given intramuscular injections of fresh allogeneic kidney cells and stroma mixed with Freund’s adjuvant weekly for 3 weeks followed by intravenous injection of 5 x lOlo cells 1 and 2 weeks later. Bilateral nephrectomy was performed 24 hours prior to exsanguination 1 week following the last intravenous dose of kidney tissue. The serum collected was separated into two fractions and one-half frozen at -20°C. for Eurther use in perfusion studies. The other half was used as source of pepsin-degraded dog an;idog kidney immunoglobulin G (DADKG) %nd its pepsin-degraded fragment F (ab) 2. Methods used involved precipitation of globulins with ammonium sulfate, separation )f gamma G globulins on DEAE-cellulose collmns, and elution with 0.01 M. phosphate mffer [21]. Pepsin digestion was carried out at pH 4.0, It 27-3O”C. using twice crystallized pepsin
1971 by Academic
Press,
Inc.
J. LOVETT
III,
B.S.,
Bilateral nephrectomy was performed and kidneys cIeared of blood with 300 cc. of warm 0.01 M. phosphate-buffered normal saline at pH 7.6. Kidneys were then perfused with immune dog serum for 20 minutes or with the pepsin-degraded gamma globulin F (ab) 2 of normal or immune serum for 0.5 hour at room temperature, followed by perfusion and incubation with the immune serum for 30 minutes using the perfusion system designed and produced by Folkman [5]. The kidneys were reimplanted in the contralateral iliac fossae as autografts and observed for changes in color, turgor, urine output, and bleeding from cut surface of the cortex. Biopsies were taken 5, 30, 90, and 240 minutes following revascularization of the kidney autograft. 57
0
EDMUND
Perfusion Studies
From the Department of Surgery, University of 3onnecticut Medical School, Hartford, Connecticut,, ?ewington Veterans Administration Hospital, Newngton, Connecticut. Submitted for publication June 8, 1970. :opyright
Ph.D.,
F.A.C.S.
(Grade B, Calbiochem) at weight ratios of 1:50 pepsin to protein concentration of 8 mg./ml. [27]. The F (ab) 2 fraction was recovered by differential ammonium sulfate precipitation at 0.6 saturation, redissolved in phosphate-buffered saline, and used at a concentration of 12 mg./ml. in perfusion studies. Identification of the gamma G globulin was by identity of electrophoretic migration and reaction with specific antibodies. Cytotoxicity studies using trypan blue uptake as evidence of cell death demonstrated death of 50% of the cells at 1: 256 dilutions of the crude immune serum. Evidence of antibodies was also demonstrated by gross and microscopic appearance of the kidneys of the dogs injected with kidney tissue. The F (ab) 2 fragments proved cytotoxic to only 5% of the cells at 1: 16 dilution.
THE OCCURRENCE of the hyperacute rejection process [9, 22, 291 accounts for the loss of human kidney allografts and while various explanations are plausible, adequate explanation evades complete understanding. This report presents additional evidence that the acute rejection process as produced by Dubernard [4] and Robertshaw [18] is immunologic and that it may be prevented by pretreatment of a kidney with partially degraded antibodies. METHODS
D.V.M.,
IXIMUXOGLOBULIN
58
JOURNAL
OF
SURGICAL
RESEARCH,
RESULTS The difference in color of the two experimental groups of kidneys was striking 5-10 minutes following revascularization following an initial 5 minutes of apparent normalcy. The kidneys perfused with immune serum became mottled, dark and soft. Incision of the cortex of the kidneys perfused with immune serum alone produced a sluggish flow of dark blood. The kidneys perfused with the pepsin-degraded gamma G globulin prior to perfusion with immune serum exhibited excellent color, and firm consistency throughout the experiment. Incision of the cortex of the kidneys perfused with the pepsin-degraded gamma G globulin followed by perfusion with immune serum resulted in a quick issue of bright red blood. Microscopic appearance of kidneys was most striking at 4 hours following transplantation. Kidneys perfused wit,h the immune serum alone exhibited shrunken glomeruli, marked deposition of PTAH-positive material in glomeruli and tubules in contrast to nearly normal appearance of the kidneys perfused with the pepsin-degraded gamma G globulin prior to perfusion with the immune dog serum. Prior perfusion with serum or F (ab) 2 from normal serum provided no detectable protection in two kidneys subsequently perfused with immune serum. DISCUSSION The phenomenon of blocking by antibody activity is not new. However, the application of such a concept to the allograft system would require a noncytotoxic, noncomplement-binding, and perhaps nonplatelet-agglutinating system. Nissonoff’s [13] isolation of the products of pepsin digestion is accompanied by work of many others with the papain pepsin-digested globulins [l, 3, 6, 7, 11, 12, 14-17, 21, 23-281. The work of Hassek with gamma globulin and fragments thereof in skin grafts in ducks lends support to this system as a means of prevention or amelioration of the hyperacute rejection process. Whitehouse and Brode [28] have demon-
VOL.
11,
NO.
2,
FEBRUARY
1971
strated the blocking activity of F(ab)z antibody fragments in tumor systems. The work of Kano and Milgrom [S] and of Williams [29] suggests that the 7s antibodies react with recipient anti-7S antibodies and thereby protect vascular endothelium from further immunologically induced damage. The work of Waller [27] and Blaylock working with anti-Rh antibodies and rheumatoid factors found that the certain antiglobulins react with the partially digested globulins but not with the intact 7s globulin, suggesting a means by which kidneys might be coated with the pepsin or pepsin-degraded noncytotoxic immunoglobulin fragment followed by the antibody to the 7s globulin producing a kidney protected by the same globulin system apparently protecting the kidney as described by Kano and Milgrom’s work. Work is under way to delineate the role of antiplatelet antibodies and vasospastic factors in this system in light of the work of Belzer [2], Kissmeyer-Nielsen [lo], and work in this laboratory [20]. The ident’ity of the factor responsible for the interference with the immune serum is not certain, although studies with fluorescein-labeled antibodies indicate blocking of the intact 75 immunoglobulins by the pepsin-degraded fragments. How long such a protective action lasts is not known and would be important if applied to a system where a continued supply of antikidney globulins were available in the intact animal. The influence of the antikidney globulin on cellular activity of platelets or leucocytes may be decreased by the degraded antibodies [28] but the amount adherent to the kidney would be small and the rapidity of action required very great. The likelihood of antilymphoid activity having a role in the protection reported here has little rationale especially in light of the relative inactivity of the pepsin-degraded ATG [17]. SUMMARY Dog kidneys perfused with allogeneic antikidney serum undergo changes of acute rejection when reimplanted as autografts. Perfusion of the kidney with the pepsin-degraded fragment of immunoglobulin G prior to perfu-
SMITH
ET
AL.:
PROTECTION
sion with the immune serum prevents development of the acute rejection lesion. Implications are discussed. REFERENCES 1. Baxter, J. H., and Small, P. A., Jr. Antibody to rat kidney: In vivo effects of univalent and divalent fragments. Science 140 :1406-1407, 1963. 2. Belzer, F. O., Reed, T. F., Pryor, 0. P., Kountz, S., and Dunphy, T. E. Cause of renal injury obtained from cadaveric donors. Surg. Gynec. Obstet. 130:467477, 1970. 3. Dolby, D. E. The action of pepsin on protein fraction from horse antiserum to diphtheria toxin. B&hem. J. 92 : 112-119, 1964. 4. Dubernard, J. M., Carpenter, C. V., Busch, G. J., Diethelm, A. G., and Murray, J. E. Rejection of canine renal allografts by passive transfer of sensitized serum. Surgery 64 :752-760, 1968. S. 5. Folkman. M. J., Cole, P., and Zimmerman, Tumor behavior in isolated perfused organs. Ann. fhrg. 164 :491-494, 1966. 6. Hasek, M., et al. Protective action of homologous serum component in graft rejection. TranspZant. hoc. 1:527-529, 1969. 7. Ishizaka. K., Ishizaka, T., and Sugahara, T. Biological activity of soluble antigen-antibody complexes. J. Immun. %X:690-701, 1962. F. Relation of anti8. Kano, K., and Milgrom, gamma globulin antibodies to transplantation antibodies in human allograft recipient. Transplantation 7 : 181-219,1969. 9. Kissmeyer-Nielsen, N. F., Olsen, S., Peterson, V. P., and Fjeldborg, 0. Hyperacute rejection of kidney allografts associated with pre-existing humoral antibodies against donor cells. Lancet 2:662, 1966. Kissmeyer-Nielsen, F., and Svejgaard, A. Blocking effect of rheumatoid factor on complement fixation by thrombocyte antibodies. VOX Sung. 12 : 199-210, 1967. Kolker, P., Hampers, C. L., Hager, E. D., and Lear, P. D. Characterization of immunologically active substances from canine renal allotransplants. Transplantation 6:1-3, 1968. 2. Mandel, M. A., and Asofsky, R. The effects of heterologous anti-thymocyte sera in mice. J. Immun. 100:1259-1267, 1968. 3. Nissinoff, A., Wissler, F. C., Lipman, L. N., and Woe&y. D. C. Separation of univalent fragments from the bivalent rabbit antibody molecule by reduction of sulfide bonds. Arch. Biochem. Biophys. 89:230-244, 1960. 4. Nussenzweig, V., and Benacerraf, B. J. Presence
OF
KIDNEYS
59
identification antigens the determinants Fd fragments of gamma1 or gamma2 guinea pig immunoglobulins. Immunology 97:171, 1966. 15. Ovary, Z. The structure of various immunoglobulins and their biologic activities. Ann. N.Y. Acud. Sci. 129 :776-786, 1966. 16. Porter, R. R. A chemical study of rabbit antiovalbumin. Biochemistry 46:31-47, 1950. 17. Riethmuller, G., Riethmuller, D., Stein, H., and Hansen, P. In vivo and in vitro properties of intact and pepsin-digested heterologous antimouse thymus antibodies. J. Zmmun. 100:969-973, 1968. 18. Robertshaw, G. E., Madge, G. E., Williams, G. M., and Hume, D. M. Hyperacute rejection of dog renal allografts. Surg. Forum Amer. Coil. fhTg. 20:291-292, 1969. 19. Shur, I?. H., and Becker, E. L. Complement fixing properties of pepsin-treated rabbit and sheep antibodies. Science 143 :36&361, 1963. 20. Smith, G. V., Lovett, E. J., Rogers, J., and Owens, G. Prot.ection of transplanted kidneys by alpha adrenergic blockade. Submitted for publication. 21. Speigelberg, H. K., and Weigle, W. 0. The catabolism of homologous and heterologous 75 gamma globulin fragments. J. Exp. Med. 121:323-327, 1965. 22. Starzl, T. E., Lerner, R. A., Dixon, F. J., Groth, C. G., Brettschneider, L., and Terasaki, P. Schwartzman reaction after human renal homotransplantation. New Eng. J. Med. 278:642,1968. 23. Tao, T.-W., and Uhr, H. Capacity of pepsin digested antibody to inhibit antibody formation. Nature London 212 :208-209, 1966. 24. Taranta, A., and Franklin, E. C. Complement fixation by antibody fragments. Science 134:19811982, 1961. 25. Thunold, S. Globulin coating in vivo of Ehrlich’s ascites carcinoma cells. Transplantation 6 :7167272,1968. 26. Uhr, J. W., and Muller, G. Regulatory effect of antibody on the immune response. Advun. Immun. 8:81-126, 1968. 27. Wailer, M., and Blaylock, K. Further studies on the anti-globulin factors in human serum to the pepsin-digested fragments of the Ri- anti-Rh antibody. J. Immun. 97:438-443, 1969. 28. Whitehouse, F., and Broder, S. Blocking activity of non-cytotoxic F(abJz tumor cell antibody fragments. Proc. Sot. Ezp. Biol. Med. 127:1064-1066, 1968. 29. Williams, M., Hume, D. M., Hudson, R. P., Kane, K., and Milgrom, F. Hyperacute renal homograft rejection in man. New Eng. J. Med. 279:611-618, 1968.
of Surgical Research
Clinical
and Laboratory
Investigation
Volume 11 Number 2, February 1971 PROTECTION GEORGE
V.
SMITH,
OF KIDNEYS M.D.,
F.A.C.S.,
BY PEPSIN-DEGRADED ALLEN
AND
GUY
J. KENPON, OWENS,
M.D.,
AND
MATERIALS
Healthy mongrel dogs were given intramuscular injections of fresh allogeneic kidney cells and stroma mixed with Freund’s adjuvant weekly for 3 weeks followed by intravenous injection of 5 x lOlo cells 1 and 2 weeks later. Bilateral nephrectomy was performed 24 hours prior to exsanguination 1 week following the last intravenous dose of kidney tissue. The serum collected was separated into two fractions and one-half frozen at -20°C. for Eurther use in perfusion studies. The other half was used as source of pepsin-degraded dog an;idog kidney immunoglobulin G (DADKG) %nd its pepsin-degraded fragment F (ab) 2. Methods used involved precipitation of globulins with ammonium sulfate, separation )f gamma G globulins on DEAE-cellulose collmns, and elution with 0.01 M. phosphate mffer [21]. Pepsin digestion was carried out at pH 4.0, It 27-3O”C. using twice crystallized pepsin
1971 by Academic
Press,
Inc.
J. LOVETT
III,
B.S.,
Bilateral nephrectomy was performed and kidneys cIeared of blood with 300 cc. of warm 0.01 M. phosphate-buffered normal saline at pH 7.6. Kidneys were then perfused with immune dog serum for 20 minutes or with the pepsin-degraded gamma globulin F (ab) 2 of normal or immune serum for 0.5 hour at room temperature, followed by perfusion and incubation with the immune serum for 30 minutes using the perfusion system designed and produced by Folkman [5]. The kidneys were reimplanted in the contralateral iliac fossae as autografts and observed for changes in color, turgor, urine output, and bleeding from cut surface of the cortex. Biopsies were taken 5, 30, 90, and 240 minutes following revascularization of the kidney autograft. 57
0
EDMUND
Perfusion Studies
From the Department of Surgery, University of 3onnecticut Medical School, Hartford, Connecticut,, ?ewington Veterans Administration Hospital, Newngton, Connecticut. Submitted for publication June 8, 1970. :opyright
Ph.D.,
F.A.C.S.
(Grade B, Calbiochem) at weight ratios of 1:50 pepsin to protein concentration of 8 mg./ml. [27]. The F (ab) 2 fraction was recovered by differential ammonium sulfate precipitation at 0.6 saturation, redissolved in phosphate-buffered saline, and used at a concentration of 12 mg./ml. in perfusion studies. Identification of the gamma G globulin was by identity of electrophoretic migration and reaction with specific antibodies. Cytotoxicity studies using trypan blue uptake as evidence of cell death demonstrated death of 50% of the cells at 1: 256 dilutions of the crude immune serum. Evidence of antibodies was also demonstrated by gross and microscopic appearance of the kidneys of the dogs injected with kidney tissue. The F (ab) 2 fragments proved cytotoxic to only 5% of the cells at 1: 16 dilution.
THE OCCURRENCE of the hyperacute rejection process [9, 22, 291 accounts for the loss of human kidney allografts and while various explanations are plausible, adequate explanation evades complete understanding. This report presents additional evidence that the acute rejection process as produced by Dubernard [4] and Robertshaw [18] is immunologic and that it may be prevented by pretreatment of a kidney with partially degraded antibodies. METHODS
D.V.M.,
IXIMUXOGLOBULIN
58
JOURNAL
OF
SURGICAL
RESEARCH,
RESULTS The difference in color of the two experimental groups of kidneys was striking 5-10 minutes following revascularization following an initial 5 minutes of apparent normalcy. The kidneys perfused with immune serum became mottled, dark and soft. Incision of the cortex of the kidneys perfused with immune serum alone produced a sluggish flow of dark blood. The kidneys perfused with the pepsin-degraded gamma G globulin prior to perfusion with immune serum exhibited excellent color, and firm consistency throughout the experiment. Incision of the cortex of the kidneys perfused with the pepsin-degraded gamma G globulin followed by perfusion with immune serum resulted in a quick issue of bright red blood. Microscopic appearance of kidneys was most striking at 4 hours following transplantation. Kidneys perfused wit,h the immune serum alone exhibited shrunken glomeruli, marked deposition of PTAH-positive material in glomeruli and tubules in contrast to nearly normal appearance of the kidneys perfused with the pepsin-degraded gamma G globulin prior to perfusion with the immune dog serum. Prior perfusion with serum or F (ab) 2 from normal serum provided no detectable protection in two kidneys subsequently perfused with immune serum. DISCUSSION The phenomenon of blocking by antibody activity is not new. However, the application of such a concept to the allograft system would require a noncytotoxic, noncomplement-binding, and perhaps nonplatelet-agglutinating system. Nissonoff’s [13] isolation of the products of pepsin digestion is accompanied by work of many others with the papain pepsin-digested globulins [l, 3, 6, 7, 11, 12, 14-17, 21, 23-281. The work of Hassek with gamma globulin and fragments thereof in skin grafts in ducks lends support to this system as a means of prevention or amelioration of the hyperacute rejection process. Whitehouse and Brode [28] have demon-
VOL.
11,
NO.
2,
FEBRUARY
1971
strated the blocking activity of F(ab)z antibody fragments in tumor systems. The work of Kano and Milgrom [S] and of Williams [29] suggests that the 7s antibodies react with recipient anti-7S antibodies and thereby protect vascular endothelium from further immunologically induced damage. The work of Waller [27] and Blaylock working with anti-Rh antibodies and rheumatoid factors found that the certain antiglobulins react with the partially digested globulins but not with the intact 7s globulin, suggesting a means by which kidneys might be coated with the pepsin or pepsin-degraded noncytotoxic immunoglobulin fragment followed by the antibody to the 7s globulin producing a kidney protected by the same globulin system apparently protecting the kidney as described by Kano and Milgrom’s work. Work is under way to delineate the role of antiplatelet antibodies and vasospastic factors in this system in light of the work of Belzer [2], Kissmeyer-Nielsen [lo], and work in this laboratory [20]. The ident’ity of the factor responsible for the interference with the immune serum is not certain, although studies with fluorescein-labeled antibodies indicate blocking of the intact 75 immunoglobulins by the pepsin-degraded fragments. How long such a protective action lasts is not known and would be important if applied to a system where a continued supply of antikidney globulins were available in the intact animal. The influence of the antikidney globulin on cellular activity of platelets or leucocytes may be decreased by the degraded antibodies [28] but the amount adherent to the kidney would be small and the rapidity of action required very great. The likelihood of antilymphoid activity having a role in the protection reported here has little rationale especially in light of the relative inactivity of the pepsin-degraded ATG [17]. SUMMARY Dog kidneys perfused with allogeneic antikidney serum undergo changes of acute rejection when reimplanted as autografts. Perfusion of the kidney with the pepsin-degraded fragment of immunoglobulin G prior to perfu-
SMITH
ET
AL.:
PROTECTION
sion with the immune serum prevents development of the acute rejection lesion. Implications are discussed. REFERENCES 1. Baxter, J. H., and Small, P. A., Jr. Antibody to rat kidney: In vivo effects of univalent and divalent fragments. Science 140 :1406-1407, 1963. 2. Belzer, F. O., Reed, T. F., Pryor, 0. P., Kountz, S., and Dunphy, T. E. Cause of renal injury obtained from cadaveric donors. Surg. Gynec. Obstet. 130:467477, 1970. 3. Dolby, D. E. The action of pepsin on protein fraction from horse antiserum to diphtheria toxin. B&hem. J. 92 : 112-119, 1964. 4. Dubernard, J. M., Carpenter, C. V., Busch, G. J., Diethelm, A. G., and Murray, J. E. Rejection of canine renal allografts by passive transfer of sensitized serum. Surgery 64 :752-760, 1968. S. 5. Folkman. M. J., Cole, P., and Zimmerman, Tumor behavior in isolated perfused organs. Ann. fhrg. 164 :491-494, 1966. 6. Hasek, M., et al. Protective action of homologous serum component in graft rejection. TranspZant. hoc. 1:527-529, 1969. 7. Ishizaka. K., Ishizaka, T., and Sugahara, T. Biological activity of soluble antigen-antibody complexes. J. Immun. %X:690-701, 1962. F. Relation of anti8. Kano, K., and Milgrom, gamma globulin antibodies to transplantation antibodies in human allograft recipient. Transplantation 7 : 181-219,1969. 9. Kissmeyer-Nielsen, N. F., Olsen, S., Peterson, V. P., and Fjeldborg, 0. Hyperacute rejection of kidney allografts associated with pre-existing humoral antibodies against donor cells. Lancet 2:662, 1966. Kissmeyer-Nielsen, F., and Svejgaard, A. Blocking effect of rheumatoid factor on complement fixation by thrombocyte antibodies. VOX Sung. 12 : 199-210, 1967. Kolker, P., Hampers, C. L., Hager, E. D., and Lear, P. D. Characterization of immunologically active substances from canine renal allotransplants. Transplantation 6:1-3, 1968. 2. Mandel, M. A., and Asofsky, R. The effects of heterologous anti-thymocyte sera in mice. J. Immun. 100:1259-1267, 1968. 3. Nissinoff, A., Wissler, F. C., Lipman, L. N., and Woe&y. D. C. Separation of univalent fragments from the bivalent rabbit antibody molecule by reduction of sulfide bonds. Arch. Biochem. Biophys. 89:230-244, 1960. 4. Nussenzweig, V., and Benacerraf, B. J. Presence
OF
KIDNEYS
59
identification antigens the determinants Fd fragments of gamma1 or gamma2 guinea pig immunoglobulins. Immunology 97:171, 1966. 15. Ovary, Z. The structure of various immunoglobulins and their biologic activities. Ann. N.Y. Acud. Sci. 129 :776-786, 1966. 16. Porter, R. R. A chemical study of rabbit antiovalbumin. Biochemistry 46:31-47, 1950. 17. Riethmuller, G., Riethmuller, D., Stein, H., and Hansen, P. In vivo and in vitro properties of intact and pepsin-digested heterologous antimouse thymus antibodies. J. Zmmun. 100:969-973, 1968. 18. Robertshaw, G. E., Madge, G. E., Williams, G. M., and Hume, D. M. Hyperacute rejection of dog renal allografts. Surg. Forum Amer. Coil. fhTg. 20:291-292, 1969. 19. Shur, I?. H., and Becker, E. L. Complement fixing properties of pepsin-treated rabbit and sheep antibodies. Science 143 :36&361, 1963. 20. Smith, G. V., Lovett, E. J., Rogers, J., and Owens, G. Prot.ection of transplanted kidneys by alpha adrenergic blockade. Submitted for publication. 21. Speigelberg, H. K., and Weigle, W. 0. The catabolism of homologous and heterologous 75 gamma globulin fragments. J. Exp. Med. 121:323-327, 1965. 22. Starzl, T. E., Lerner, R. A., Dixon, F. J., Groth, C. G., Brettschneider, L., and Terasaki, P. Schwartzman reaction after human renal homotransplantation. New Eng. J. Med. 278:642,1968. 23. Tao, T.-W., and Uhr, H. Capacity of pepsin digested antibody to inhibit antibody formation. Nature London 212 :208-209, 1966. 24. Taranta, A., and Franklin, E. C. Complement fixation by antibody fragments. Science 134:19811982, 1961. 25. Thunold, S. Globulin coating in vivo of Ehrlich’s ascites carcinoma cells. Transplantation 6 :7167272,1968. 26. Uhr, J. W., and Muller, G. Regulatory effect of antibody on the immune response. Advun. Immun. 8:81-126, 1968. 27. Wailer, M., and Blaylock, K. Further studies on the anti-globulin factors in human serum to the pepsin-digested fragments of the Ri- anti-Rh antibody. J. Immun. 97:438-443, 1969. 28. Whitehouse, F., and Broder, S. Blocking activity of non-cytotoxic F(abJz tumor cell antibody fragments. Proc. Sot. Ezp. Biol. Med. 127:1064-1066, 1968. 29. Williams, M., Hume, D. M., Hudson, R. P., Kane, K., and Milgrom, F. Hyperacute renal homograft rejection in man. New Eng. J. Med. 279:611-618, 1968.