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Kidney Lymphatics: An Important Network in Transplantation
Vol. 111, February
THE JOURNAL OF UROLOGY
Copyright© 1974 by The Williams & Wilkins Co.
Printed in USA.
KIDNEY LYMPHATICS: AN IMPORTANT NETWORK IN TRANSPLANTATION A.T. K. COCKETT,A. SAKAI ANDI. C. V. NETTO
From the Division of Urology, University of Rochester School of Medicine and Dentistry, Rochester, New York
sufficient lymph was collected, usually it to b hours. Rejection was assessed gross appearance of the graft, cessation of urine and sections. Cell samples collected from capsu-· lar and cistema chyli lymph, which had been at 4C during collection, were washed twice Eagle's medium. Viability of these cells was proximately 95 per cent, measured by the trypan blue exclusion test. One part was stained with May-Giemsa rabbit anti-dog IgG serum using the direct m1.mu.nofluorescent method. The number of rosetteforming cells among cell samples was counted Biozzi's original methods with a slight modifica tion. 6 The effect of azathioprine (2 to 4 mg. per lymphoblast formation was determined in 10 harboring an allograft. All renal lymph cannulations were undertaken 4 days after The 8 animals with allografts that did not receivs-; any azathioprine served as controls and underwent cannulation on day 4. Four experimental animrJs received azathioprine 1 day before the renal im plant. The drug was continued in the animal for an additional 4 days before cannulation. Dosage was adjusted to maintain the white count at levels of 5,000 per cu. mm. Tvm animals received azathioprine before treatment for 3 days and a third subgroup of 4 animals received azathioprine for 7 days before the allograft was implanted. Again the drug was continued for 4 additional days until lymph cannulation i.n the 4-day allograft.
A kidney allograft is rejected immunologically delayed hypersensitivity when mononuclear cells infiltrating the graft a major role. Host lymphocytes in response to foreign tissue transform from so-called small lymphocytes to larger pyroninophilic cells labeled immunoblasts. The immunoblast is readily seen in the kidney allograft. 1 The immunological properties and fate of these cells are unknown since qualitative and quantitative studies of the cell have not been possible until recently when a technique for cannulating the renal lymphatic became available. 2 Pedersen and Morris first reported in the sheep that lymphocytes in the hilar lymphatic of the kidney are able to transform into immunoblasts when transplanted to allogeneic hosts. 3 We have reported the same phenomenon in the dog• and Hamburger and associates have confirmed these findings in man. 5 The nature of cells found in renal capsular and hilar lymphatics during canine renal allograft rejection is described herein. The effects of immunosuppression azathioprine are also detailed. MATERIALS AND METHODS
Thirty-eight mongrel dogs undergoing renal lymph cannulation-12 autografts, 18 allografts and 8 normal kidneys-were used in this study. A kidney was transplanted heterotopically to the neck of the host in the allografted animals. The host's own kidney, in the allograft or autograft experiment, was not removed to eliminate uremia which might influence the experiment. Immunosuppressive drugs were withheld in 3 of the 4 animal groups. On successive days after transplantation the grafted kidney was explored. A polyethylene tube (PE50) was cannulated into hilar and capsular lymphatics of the graft. Because of difficulty in maintaining the cannulation for more than 12 hours the animals were sacrificed after Accepted for publication July 6, 1973. Supported by the Buswell-Urology Research Fund. 1 Kountz, S. L., Williams, M. A., Williams, P. L., Kapros, C. and Dempster, W. J.: Mechanism of rejection of homotransplanted kidneys. Nature, 199: 257, 1963. 2 Cockett, A. T. K., Moore, R. S. and Kado, R. T.: The renal lymphatics and therapy of pyelonephritis. Brit. J. Urol., 37: 650, 1965. 3 Pedersen, N. C. and Morris, B.: The role of the lymphatic system in the rejection of homografts: a study of lymph from renal transplants. J. Exp. Med., 131: 936, 1970. 'Cockett, A. T. K., Sakai, A. and Netto, I. C. V.: Host lymphocyte transformation in lymph of the canine kidney allograft. Invest. Urol., in press. 5 Hamburger, J., Dimitriu, A., Bankir, L., DebraySachs, M. and Auvert, J.: Collection of lymph from kidneys homotransplanted in man: cell transformation in vivo. Nature, 232: 633, 1971.
RESULTS
Total number of leukocytes was markedly creased with time in the renal lymph of both autographs and allografts (table 1). The counts from normal dog kidney lymph were .05 plus or minus .04 times 10 6 per hour. The count was seen in allografts (12.2 plus or minus 8. times 10 6 per hour at day 4), compared to auto grafts (1.2 plus or minus 1.0 times 10 6 per hour) The cells were exclusively of the mononuclear type in the allograft whereas in the autograft the cells were predominantly polymorphonuclear in type Cytological changes of mononuclear cells renal lymph of the allograft were impressive 1, A). Within 2 days changes were evident in many lymphocytes. They were increased in size, the 6 Biozzi, G., Stiffel, C. and Mouton, D.: A antibody-containing cells in the course of ""'""""""V In: Immunity, Cancer, and Chemotherapy: Basic Relationships on the Cellular Level. Edited by E. Mihich. New York: Academic Press, p. 103, 1967.
141
142
COCKETT, SAKAI AND NETTO TABLE
Adult normal kidney (7 dogs) 4-week autografts (5 dogs) 4-day allografts (8 dogs)
1. Cellular changes in renal hilar lymph Lymph Flow (ml./hr.)
WBC (No./hr. x 10')
% oflmmunoblast
1.0 ± 0.8 2.5 ± 1.3 10.6 ± 4.7
0.05 ± 0.04 1.4 ± 1.0 12.2 ± 8.1
0.1 ± 0.1 1.2 ± 1.4 21.6 ± 7.4
% Polymorpho-
nuclear
18.4 ± 14.9 55.0 ± 27.7 19.7 ± 11.2
Fm. 1. A, cells from renal hilar lymphatic 4 days after implanting allograft. Note large transformed cells with vacuoles in cytoplasm and small pyroninophilic cells. B, kidney autograft. Majority of cells are polymorphonuclear in autograft. May-Giemsa, reduced from x450.
cytoplasm was somewhat more extensive and more basophilic and the nucleus often possessed a nucleolus. Four days after allografting nearly a fourth of lymphoid cells in both renal lymphatic networks (capsular and hilar) were transformed. The ratio of mononuclear cells to transformed cells was 63.9 plus or minus 13.3 to 21.6 plus or minus 7.4. Of these, about 10 per cent were in mitosis. The lymphoblasts (14 to 21 µ in diameter) were considerably larger than the lymphocytes (6 to 13 µ). The nucleus had a leptochromatic chromatin pattern and contained one or more well developed basophilic nucleoli (fig. 1, A). The cytoplasm was greatly increased in volume. Clear vacuoles were present in the cytoplasm of the blast cells. These cells were strikingly similar to the cells transformed by phytohemagglutinin stimulation. 7 By day 8 the number of transformed cells was significantly reduced (5 per cent oflymphoid cells). The transformed lymphocytes could only be found in both renal lymphatic networks of the allograft and such cells were never found in renal vein during the allograft period (up to 8 days). The immunoblast was not seen in renal lymph of autografts. The majority of cells were polymorphonuclear leukocytes (fig. 1, B). Table 2 lists the white cell counts and types of cells seen in our various allograft models. These numbers are compared with autografted kidneys. Pre-treatment schedules with azathioprine administration to selected animals are also listed. Lymph flow is reduced in the animals pre-treated for 7 days with azathioprine. Immunoblasts were not seen in the lymph from this group. The percentage of polymorphonuclears are also listed. 7 Elves, M. W.: The Lymphocytes. Philadelphia: J.B. Lippincott Co., p. 198, 1966.
The table also compares 2 other dosage schedules using azathioprine. The differences in renal lymph flow and white cell counts are also documented. Approximately 5 per cent of these large transformed blast cells (less than 1 per cent of mononuclear cells) were shown to contain IgG by direct immunofluorescent staining (fig. 2, A). A test for hemolysin producing cells among the immunoblasts failed to show any specific immune cells against donor red cells. However, the number of rosette-forming cells rose significantly 4 days after allograft implantation. Rosette-forming cell counts per 1,000 lymphoid cells were 2.1 plus or minus 0.5, compared to 0.02 plus or minus 0.01 in the renal autograft. The latter appears to be a background level (fig. 2, B). DISCUSSION
Our technique for cannulating the lymphatics and analyzing lymph from canine renal allografts provides the most useful information regarding the immunological properties of entrapped host lymphocytes stimulated by the allograft. Our work herein demonstrates that circulating host lymphocytes can be transformed in the renal lymphatics by alloantigens. Lymphocyte transformation takes place mainly in renal capillaries in the early stages of immunity. This is supported by the fact that as early as 2 days after grafting a moderate increase in number of immunoblasts (5 per cent of mononuclear cells) already appears in both renal lymphatic networks, whereas in none of the lymphoid organs or pathways tested-including spleen, regional lymph node, cisterna chyli and blood-was a significant elevation of immunoblasts demonstrated. Circulating lymphocytes, particularly antigen
KIDNEY LYMPHATICS TABLE
2. Effect of immunosuppression (imuran) on cells from renal hilar lymph in 4-day allografts Pre-Treatment
With Imuran Autograft Allograft Allograft ( 4 dogs) Allograft (2 dogs) Allograft ( 4 dogs)
0 ()
1 day -:J days 7 days
Lymph Flow (ml./hr.)
WBC (No./hr. x 10')
2.5 ± 1.3 10.6 1_ 4.7 4.5 ± 0.5 2.0 ± 1.5
1.4 ± 1.0 12.2 L 8.1 4.0 J_ 1.2 4.6 ± 0.6 0.15 TO 5
0.2 ± (J.5
%, oflmmunoblasts
1.2 ± 1.4 21.6 -" 7.4 27 .0 _c 3.2 5.S ± 0.5 ()
q) Polymorpho~
nuclear 55.0±27.7 19.7 ± 11.:2 19.0 J_ 5.4 J"i.O + 6.0 46.o ± :i.s
B FIG. 2. A. immunoblast stained by rabbit anti-dog IgG fluorescent antibody. About 5 per cent of immunoblasts (approximately 1 per cent of mononuclear cells) contain antibody. Allograft, 5 days after implant. B, immuno-adherent (rosette-forming) cell. Note 10 donor red cells firmly binding to cell surface of immunoblast, 4 days after allograft implanted. May-Giemsa, reduced from x4.SO.
sens1t1ve cells, penetrate the capillary wall and make contact with kidney tissue antigens. The data suggest that there is an immune selection mechanism operating only for specific antigen sensitive cell which recognizes the antigen. The fact that this selection mechanism did not become apparent until 4 days after allograft implantation further suggests that the cells being selected by the mechanism may be the progeny of transformed lymphocytes. The question as to whether the fate of the immunoblast is the plasma cell, as a result of further differentiation, still remains conjectural. However, contrary to our assumption that the progeny of the immunoblast may be small pyroninophilic cells responsible for celi mediated immunity, the data indicate that some immunoblasts contain IgG antibody. Horowitz previously demonstrated by fluorescent antibody methods the presence of gamma globulin containing cells in dog kidney grafts within 24 hours of transplantation. 8 The presence of antibody and its functional importance are still matters of speculation. The data show that host serum alone may have no effect on the cytolysis of cultured kidney cells nor does it contain detectable antibodies. The immunoblasts interacted with donor red
cells forming rosette-forming cells. This may be accord with the early observation of Simonsen who demonstrated a rise of hemagglutinin antibody in the dog kidney allograft. 9 A calculation made from the present data indicates that at day 4 or 5 about 25 per cent of mononuclear cells are transformed and approximately 1 and 0.2 per cent of mononuclear cells contain IgG antibody and stimulate rosette formation. These findings are seen in the capsular and hilar lymphatic networks 4 days after allotrarn,plantation. Azathioprine prevented the transformation of host lymphocytes when administered at least 7 days before implanting the allograft. The drug was continued during the 4 days following the transplant at a dosage approximating 2 mg. per kg. The dosage was adjusted after peripheral white blood counts were obtained. This immunosuppressive drug was not capable of preventing immunoblast formation in the renal lymph when it was 2 days before allograft implantation. Pre-treatment with the drug for 5 days was moderately effective (table 2).
8 Horowitz, R. E., Burrows, L., Paronetto, F. and Wildstein, W.: Immunocytochemical observations on canine kidney homografts. Fed. Proc., 22: 274, 1963.
'Simonsen, M.: Biological incompatibility in kidney transplantation in dogs; serological investigations, Acu:c Path. Microbiol. Scand., 32: 36, 1953.
SUMMAKY
Immunoblasts found in the renal networks of a canine allograft 1)
144
COCKETT, SAKAI AND NETTO
hemagglutinin stimulated cells in morphology and H' thymidine uptake, 2) are entrapped by the graft from the host circulation, 3) have cells of 2 populations, one (less than 10 per cent) being IgG producing cells and the majority being effectors killing donor cells by direct contact and 4) cause adherence of red cells on their cell surface. Azathi-
oprine when given at least 7 days prior to implantation of the allograft will prevent transformation of host lymphocytes. Dr. Eric Schenk helped with the immunofluorescent staining.
THE JOURNAL OF UROLOGY
Copyright© 1974 by The Williams & Wilkins Co.
Printed in USA.
KIDNEY LYMPHATICS: AN IMPORTANT NETWORK IN TRANSPLANTATION A.T. K. COCKETT,A. SAKAI ANDI. C. V. NETTO
From the Division of Urology, University of Rochester School of Medicine and Dentistry, Rochester, New York
sufficient lymph was collected, usually it to b hours. Rejection was assessed gross appearance of the graft, cessation of urine and sections. Cell samples collected from capsu-· lar and cistema chyli lymph, which had been at 4C during collection, were washed twice Eagle's medium. Viability of these cells was proximately 95 per cent, measured by the trypan blue exclusion test. One part was stained with May-Giemsa rabbit anti-dog IgG serum using the direct m1.mu.nofluorescent method. The number of rosetteforming cells among cell samples was counted Biozzi's original methods with a slight modifica tion. 6 The effect of azathioprine (2 to 4 mg. per lymphoblast formation was determined in 10 harboring an allograft. All renal lymph cannulations were undertaken 4 days after The 8 animals with allografts that did not receivs-; any azathioprine served as controls and underwent cannulation on day 4. Four experimental animrJs received azathioprine 1 day before the renal im plant. The drug was continued in the animal for an additional 4 days before cannulation. Dosage was adjusted to maintain the white count at levels of 5,000 per cu. mm. Tvm animals received azathioprine before treatment for 3 days and a third subgroup of 4 animals received azathioprine for 7 days before the allograft was implanted. Again the drug was continued for 4 additional days until lymph cannulation i.n the 4-day allograft.
A kidney allograft is rejected immunologically delayed hypersensitivity when mononuclear cells infiltrating the graft a major role. Host lymphocytes in response to foreign tissue transform from so-called small lymphocytes to larger pyroninophilic cells labeled immunoblasts. The immunoblast is readily seen in the kidney allograft. 1 The immunological properties and fate of these cells are unknown since qualitative and quantitative studies of the cell have not been possible until recently when a technique for cannulating the renal lymphatic became available. 2 Pedersen and Morris first reported in the sheep that lymphocytes in the hilar lymphatic of the kidney are able to transform into immunoblasts when transplanted to allogeneic hosts. 3 We have reported the same phenomenon in the dog• and Hamburger and associates have confirmed these findings in man. 5 The nature of cells found in renal capsular and hilar lymphatics during canine renal allograft rejection is described herein. The effects of immunosuppression azathioprine are also detailed. MATERIALS AND METHODS
Thirty-eight mongrel dogs undergoing renal lymph cannulation-12 autografts, 18 allografts and 8 normal kidneys-were used in this study. A kidney was transplanted heterotopically to the neck of the host in the allografted animals. The host's own kidney, in the allograft or autograft experiment, was not removed to eliminate uremia which might influence the experiment. Immunosuppressive drugs were withheld in 3 of the 4 animal groups. On successive days after transplantation the grafted kidney was explored. A polyethylene tube (PE50) was cannulated into hilar and capsular lymphatics of the graft. Because of difficulty in maintaining the cannulation for more than 12 hours the animals were sacrificed after Accepted for publication July 6, 1973. Supported by the Buswell-Urology Research Fund. 1 Kountz, S. L., Williams, M. A., Williams, P. L., Kapros, C. and Dempster, W. J.: Mechanism of rejection of homotransplanted kidneys. Nature, 199: 257, 1963. 2 Cockett, A. T. K., Moore, R. S. and Kado, R. T.: The renal lymphatics and therapy of pyelonephritis. Brit. J. Urol., 37: 650, 1965. 3 Pedersen, N. C. and Morris, B.: The role of the lymphatic system in the rejection of homografts: a study of lymph from renal transplants. J. Exp. Med., 131: 936, 1970. 'Cockett, A. T. K., Sakai, A. and Netto, I. C. V.: Host lymphocyte transformation in lymph of the canine kidney allograft. Invest. Urol., in press. 5 Hamburger, J., Dimitriu, A., Bankir, L., DebraySachs, M. and Auvert, J.: Collection of lymph from kidneys homotransplanted in man: cell transformation in vivo. Nature, 232: 633, 1971.
RESULTS
Total number of leukocytes was markedly creased with time in the renal lymph of both autographs and allografts (table 1). The counts from normal dog kidney lymph were .05 plus or minus .04 times 10 6 per hour. The count was seen in allografts (12.2 plus or minus 8. times 10 6 per hour at day 4), compared to auto grafts (1.2 plus or minus 1.0 times 10 6 per hour) The cells were exclusively of the mononuclear type in the allograft whereas in the autograft the cells were predominantly polymorphonuclear in type Cytological changes of mononuclear cells renal lymph of the allograft were impressive 1, A). Within 2 days changes were evident in many lymphocytes. They were increased in size, the 6 Biozzi, G., Stiffel, C. and Mouton, D.: A antibody-containing cells in the course of ""'""""""V In: Immunity, Cancer, and Chemotherapy: Basic Relationships on the Cellular Level. Edited by E. Mihich. New York: Academic Press, p. 103, 1967.
141
142
COCKETT, SAKAI AND NETTO TABLE
Adult normal kidney (7 dogs) 4-week autografts (5 dogs) 4-day allografts (8 dogs)
1. Cellular changes in renal hilar lymph Lymph Flow (ml./hr.)
WBC (No./hr. x 10')
% oflmmunoblast
1.0 ± 0.8 2.5 ± 1.3 10.6 ± 4.7
0.05 ± 0.04 1.4 ± 1.0 12.2 ± 8.1
0.1 ± 0.1 1.2 ± 1.4 21.6 ± 7.4
% Polymorpho-
nuclear
18.4 ± 14.9 55.0 ± 27.7 19.7 ± 11.2
Fm. 1. A, cells from renal hilar lymphatic 4 days after implanting allograft. Note large transformed cells with vacuoles in cytoplasm and small pyroninophilic cells. B, kidney autograft. Majority of cells are polymorphonuclear in autograft. May-Giemsa, reduced from x450.
cytoplasm was somewhat more extensive and more basophilic and the nucleus often possessed a nucleolus. Four days after allografting nearly a fourth of lymphoid cells in both renal lymphatic networks (capsular and hilar) were transformed. The ratio of mononuclear cells to transformed cells was 63.9 plus or minus 13.3 to 21.6 plus or minus 7.4. Of these, about 10 per cent were in mitosis. The lymphoblasts (14 to 21 µ in diameter) were considerably larger than the lymphocytes (6 to 13 µ). The nucleus had a leptochromatic chromatin pattern and contained one or more well developed basophilic nucleoli (fig. 1, A). The cytoplasm was greatly increased in volume. Clear vacuoles were present in the cytoplasm of the blast cells. These cells were strikingly similar to the cells transformed by phytohemagglutinin stimulation. 7 By day 8 the number of transformed cells was significantly reduced (5 per cent oflymphoid cells). The transformed lymphocytes could only be found in both renal lymphatic networks of the allograft and such cells were never found in renal vein during the allograft period (up to 8 days). The immunoblast was not seen in renal lymph of autografts. The majority of cells were polymorphonuclear leukocytes (fig. 1, B). Table 2 lists the white cell counts and types of cells seen in our various allograft models. These numbers are compared with autografted kidneys. Pre-treatment schedules with azathioprine administration to selected animals are also listed. Lymph flow is reduced in the animals pre-treated for 7 days with azathioprine. Immunoblasts were not seen in the lymph from this group. The percentage of polymorphonuclears are also listed. 7 Elves, M. W.: The Lymphocytes. Philadelphia: J.B. Lippincott Co., p. 198, 1966.
The table also compares 2 other dosage schedules using azathioprine. The differences in renal lymph flow and white cell counts are also documented. Approximately 5 per cent of these large transformed blast cells (less than 1 per cent of mononuclear cells) were shown to contain IgG by direct immunofluorescent staining (fig. 2, A). A test for hemolysin producing cells among the immunoblasts failed to show any specific immune cells against donor red cells. However, the number of rosette-forming cells rose significantly 4 days after allograft implantation. Rosette-forming cell counts per 1,000 lymphoid cells were 2.1 plus or minus 0.5, compared to 0.02 plus or minus 0.01 in the renal autograft. The latter appears to be a background level (fig. 2, B). DISCUSSION
Our technique for cannulating the lymphatics and analyzing lymph from canine renal allografts provides the most useful information regarding the immunological properties of entrapped host lymphocytes stimulated by the allograft. Our work herein demonstrates that circulating host lymphocytes can be transformed in the renal lymphatics by alloantigens. Lymphocyte transformation takes place mainly in renal capillaries in the early stages of immunity. This is supported by the fact that as early as 2 days after grafting a moderate increase in number of immunoblasts (5 per cent of mononuclear cells) already appears in both renal lymphatic networks, whereas in none of the lymphoid organs or pathways tested-including spleen, regional lymph node, cisterna chyli and blood-was a significant elevation of immunoblasts demonstrated. Circulating lymphocytes, particularly antigen
KIDNEY LYMPHATICS TABLE
2. Effect of immunosuppression (imuran) on cells from renal hilar lymph in 4-day allografts Pre-Treatment
With Imuran Autograft Allograft Allograft ( 4 dogs) Allograft (2 dogs) Allograft ( 4 dogs)
0 ()
1 day -:J days 7 days
Lymph Flow (ml./hr.)
WBC (No./hr. x 10')
2.5 ± 1.3 10.6 1_ 4.7 4.5 ± 0.5 2.0 ± 1.5
1.4 ± 1.0 12.2 L 8.1 4.0 J_ 1.2 4.6 ± 0.6 0.15 TO 5
0.2 ± (J.5
%, oflmmunoblasts
1.2 ± 1.4 21.6 -" 7.4 27 .0 _c 3.2 5.S ± 0.5 ()
q) Polymorpho~
nuclear 55.0±27.7 19.7 ± 11.:2 19.0 J_ 5.4 J"i.O + 6.0 46.o ± :i.s
B FIG. 2. A. immunoblast stained by rabbit anti-dog IgG fluorescent antibody. About 5 per cent of immunoblasts (approximately 1 per cent of mononuclear cells) contain antibody. Allograft, 5 days after implant. B, immuno-adherent (rosette-forming) cell. Note 10 donor red cells firmly binding to cell surface of immunoblast, 4 days after allograft implanted. May-Giemsa, reduced from x4.SO.
sens1t1ve cells, penetrate the capillary wall and make contact with kidney tissue antigens. The data suggest that there is an immune selection mechanism operating only for specific antigen sensitive cell which recognizes the antigen. The fact that this selection mechanism did not become apparent until 4 days after allograft implantation further suggests that the cells being selected by the mechanism may be the progeny of transformed lymphocytes. The question as to whether the fate of the immunoblast is the plasma cell, as a result of further differentiation, still remains conjectural. However, contrary to our assumption that the progeny of the immunoblast may be small pyroninophilic cells responsible for celi mediated immunity, the data indicate that some immunoblasts contain IgG antibody. Horowitz previously demonstrated by fluorescent antibody methods the presence of gamma globulin containing cells in dog kidney grafts within 24 hours of transplantation. 8 The presence of antibody and its functional importance are still matters of speculation. The data show that host serum alone may have no effect on the cytolysis of cultured kidney cells nor does it contain detectable antibodies. The immunoblasts interacted with donor red
cells forming rosette-forming cells. This may be accord with the early observation of Simonsen who demonstrated a rise of hemagglutinin antibody in the dog kidney allograft. 9 A calculation made from the present data indicates that at day 4 or 5 about 25 per cent of mononuclear cells are transformed and approximately 1 and 0.2 per cent of mononuclear cells contain IgG antibody and stimulate rosette formation. These findings are seen in the capsular and hilar lymphatic networks 4 days after allotrarn,plantation. Azathioprine prevented the transformation of host lymphocytes when administered at least 7 days before implanting the allograft. The drug was continued during the 4 days following the transplant at a dosage approximating 2 mg. per kg. The dosage was adjusted after peripheral white blood counts were obtained. This immunosuppressive drug was not capable of preventing immunoblast formation in the renal lymph when it was 2 days before allograft implantation. Pre-treatment with the drug for 5 days was moderately effective (table 2).
8 Horowitz, R. E., Burrows, L., Paronetto, F. and Wildstein, W.: Immunocytochemical observations on canine kidney homografts. Fed. Proc., 22: 274, 1963.
'Simonsen, M.: Biological incompatibility in kidney transplantation in dogs; serological investigations, Acu:c Path. Microbiol. Scand., 32: 36, 1953.
SUMMAKY
Immunoblasts found in the renal networks of a canine allograft 1)
144
COCKETT, SAKAI AND NETTO
hemagglutinin stimulated cells in morphology and H' thymidine uptake, 2) are entrapped by the graft from the host circulation, 3) have cells of 2 populations, one (less than 10 per cent) being IgG producing cells and the majority being effectors killing donor cells by direct contact and 4) cause adherence of red cells on their cell surface. Azathi-
oprine when given at least 7 days prior to implantation of the allograft will prevent transformation of host lymphocytes. Dr. Eric Schenk helped with the immunofluorescent staining.