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Lymphoreticular Tissue Lesions in Mice after Immunization with Typhoid Fever Vaccine and Immunosuppression with Azathioprine
Department of Pathology, Medical School, Gdansk, Poland (Head: Doz. Dr. Ewa Boj)
Lymphoreticular Tissue Lesions in Mice after Immunization with Typhoid Fever Vaccine and Immunosuppression with Azathioprine LymphoretikuHire Gewebeveranderungen bei Mausen nach Immunisierung mit Typhus-Impfstoff und Immunosuppression mit Azathioprin ALBINA Z6t TOWSKA With 4 Figures and 2 Tables· Received August 12, I974 . Accepted August 26, 1975
III
revised form
Key words: Murine lymphoreticular tissue - Typhoid fever vaccme Immunization - Immunosuppression - Malignant Lymphoma
Summary C57BL and Balblc mice were immunized, were immunized and then administered immunosuppressives, were immunized with concurrent immunosuppression, and were immunized after prior administration of immunosuppressive agents. Typhoid fever vaccine used clinically was used for immunization. Immunosuppression was accomplished with azathioprine (Imuran). Control groups received azathioprine or no treatment. The experiment lasted 5 months. In mice immunized with typhoid fever vaccine and subsequent azathioprine administration, malignant lymphomas of a lymphoblastic type appeared in 20010 of C57BL mice and in 70010 of Balblc mice. In mice receiving antigenic vaccine together with azathioprine, malignant lymphomas appeared in only 5 to 10010 of cases. However, mice that first received azatioprine and were later immunized, died after the second administration of typhoid fever vaccine and showed pictures that brought to mind blastic transformation of lymphocytes in vitro. This proliferation was primarily found in subcortical areas of lymph nodes. Analysis of presented results indicates that chronic immunization of animals with typhoid fever vaccine may serve as experimental model of malignant lymphoma morpho· logically similar to that appearing in human pathology.
25 8 . A. i6ltowska
The genesis of malignant lymphomas in animals under the influence of various stimulating agents has been described by many authors (Doell et al., I967; Metcalf, I96I; O'Connor, I970; Potter, I970). It has been reported that immunosuppre~sion favors this proliferation (Allison, I970; Vredevoe and Hays, I969). Krueger and coIl. (I97I and I974) state that only prolonged immunosuppression in combination with persistent antigenic stimulation causes excessive lymphoreticular stem cell proliferation and malignant lymphoma. Many authors support the virus theory (Armstrong et al., I972; Gleichmann et al., I972; Huebner, I969; Meyers et al., I970; Schmidt, I973). The virus supposedly transforms the T lymphocytes and in this way initiates the graft-versus-host reaction (Schmidt, I973) or becomes active during immunological disorders of the graft-versus-host type (Armstrong et al., I972; Gleichmann et al., I972). Our own experiments showed formation of malignant growths of the lymphoreticular system in 4 species of rodents after stimulation with typhoid fever vaccine (ZOltowska and Kalinowski, I 973; Z6ltowska and Ciesielski, I974; Z6howska, I975). In animals immunized shortly after birth these growths were more frequent (Z6ltowska, I975, in press), which is probably due to immunodeficiency. To examine this more closely morphological effects of typhoid fever immunization in adults with accompanying immunosuppression were studied.
Material and Methods One hundred and twenty Balb/c mice and 12 0 C57BL mice, 6 weeks of age, were used. Experiments were carried out on both males and fem ales. The animals were divided into 6 groups consisting of 20 mice each. Group I received only typhoid fever vaccine. Group II received typhoid fev er vaccine alone for the first four weeks, after which typhoid fever vaccine was given together with azathioprine. Group III received both typhoid fever vaccine and azathioprine from the beginning of the experiment. Group IV for the first 2 weeks received azathioprine only, typhoid fever vaccine plus azathioprine after this period. Group V was given only azathioprine. Group VI received no treatment. The animals were immunized with typhoid fever vaccine produced by the Warsaw Serum and Vaccine Production Laboratory, I ml of the vaccine containing 1,000 bacilli Salmonella typhi, 0.45010 phenol and saline solution. Mice were injected with the vaccine twice weekly subcutaneously in the inguinal region with 0.1 ml doses for a period of 5 months. Immunosuppression was achieved by continuous administration of 15 mg/kg/day of azathioprine "Imuran" (Well come Foundation Ltd. London) in drinking water.
Lymphoreticular Tissue Lesions after Azathioprine. 259 Animals were sectioned immediately after natural death or after being sacrificed when found extremely ill. After 5 months control and remaining experimental mice were killed using ether anesthesia. Routinely, sections from all organs were taken for histopathological examination and stained with hematoxylin and eosin.
Results Group I - Mice recelvlllg only typhoid fever vaccine showed excess lymphocyte proliferation with scattered histiophagocytes in cortical regions and plasmocytic proliferation in the medullary cord of lymph nodes. In subcapsular, trabecular and medullary sinuses of lymph nodes numerous aggregates of macrophages were found. Around splenic central arterioles hyperplastic lymphohistiocytic regions were present, while peripheral areas of Malpighian follicles were composed of plasma cells and macrophages. Changes in both Balblc and C57BL strain mice were similar. In indivudual cases in this experimental group of animals generalized malignant lymphomas of lymphoreticular leukemic type with infiltration liver and spleen were observed. Group II - Mice of this group showed lymphohistiocytic proliferation in lymph nodes, Peyer's patches, thymus and spleen. In the spleen this proliferation was present round the central arterioles; peripheral portions of the Malpighian follicles consisted of fibrous tissue with individual plasma cells, lymphocytes and macrophages. The Malpighian follicles of the spleen were thus formed by lymphocytes with scattered histiocytes and enveloped by fibrous tissue. Similar changes were observed in lymph nodes, mainly submandibular ones in the area of the salivary glands (Fig. I). It seems likely that in the spleen as well as in the lymph nodes sclerotic areas corresponded to regions of connective tissue trabeculae and sinuses. In these sclerotic areas, among which declining numbers of macrophages and numerous mast cells could be seen, cells containing basophilic cytoplasm and a hyperchromatic nucleus with am irregular nucleolus were identifical. These cells proliferated in areas where fusiform cells were arranged similarly to angioblasts, and depending on the plane of sectioning follicular or trabecular arrangements could be observed. These views were reminiscent early preneoplastic malignant lymphoma (Fig. 2). In some cases in the lymph nodes these processes were more advanced and less differentiated cells of the lymphoreticular system were predominant, which allowed regognition of lymphoblastic malignant lymphoma (Fig. 3). This type of proliferation in three out of five tested C57BL strain mice when transplanted in syngeneic hosts grew rapidly and caused death of animals in 14-18 days in 10-300/0.
260 .
A. Z6!towska
Fig.!. Submandibular lymph node of C57 BL mouse from group II of experiment. (Mice were immunized with typhoid fever vaccine and then administered azathioprine.) Large histiocytes are scattered throughout well-differentiated lymphocyte proliferation in the cortical region. HE; X 120.
Fig. 2. Submandibular lymph node of C57BL mouse from group II of experiment. Note areas of fibrous tissue among which are macrophages and large cells containing basophilic cytoplasm and hyperchromatic nuclei resembling lymphoblastic cells. These cells border fusiform cells arranged similarly to angioblasts. HE; X 360.
Lymphoreticular Tissue Lesions after Azathioprine. 261
Fig. 3. Lymph node of C57BL mouse from group II of experiment. The lymph node is replaced by pleomorphic proliferation with lymphoblasts. HE; X 360.
Fig. 4. Lymph node of Balblc mouse from group IV of experiment. (Mice received for the first 2 weeks azathioprine only, typhoid fever vaccine plus azathioprine after this period.) Note lymphocytes and lymphoblasts and also mitotic figures. HE; X 360. 18 Beitr. Path. Bd. 156
262 .
A. Z61towska
In mice of the Balb/c strain, malignant lymphomas occurred more frequently. All animals in this experimental group died or were extremely ill and sacrificed within 6-15 weeks. Group III - Microscopically only in a few indivudual cases a picture of malignant proliferation was seen in lymph nodes. In the remaining animals of this group excess disseminated lymphohistiocytic and plasmacytic growth was found. Group IV - Animals of this group began dying after the second injection of vaccine with symptoms of diarrhea. Submandibular lymph nodes were swollen and whitish, microscopically blastic transformation of lymphocytes similar in response to immunogen in vitro was observed (Fig. 4). Bald/c strain mice were more sensitive. In the rest of the lymphoreticular system of mice in this experimental group lymphohistiocytic proliferation could be seen. Group V - Mice receiving only azathioprine showed atrophy within the entire lymphoreticular system or proliferation of plasma cells and single stem cells in medullary areas of lymph nodes and peripheral parts of Malpighian follicles. C57BL strain mice died within 4-8 weeks, while Balblc strain mice lived 10 weeks or longer. Group VI - Animals of this control group showed no deviation from normal. In all groups of animals female mice showed similar changes as male mice. The results in the experimental and control groups are given in Table I and 2.
T able
1.
Morphologic changes of lymphoreticular tissue in C57BL mice
No. of mice Groups of animals investigated
- _ ..
_
- - - - - - _... -
16
I1TF
17
II1TF
19
+ IM/ III/TF + IM/ IV/1M + TF/
17
V/IM/
19
VIIN/
17
Malignant Preneoplastic lymphoma lesions
_ . _ - - - _ ._-
--_ .. _ - - - - - --
-
Atrophy
--
14
2
4
Blastic Hypertransfor- plasia mation
6
7
2
10
4
9 8
9
Lymphoreticular Tissue Lesions after Azathioprine . 26 3 Table
2.
Morphologic changes of lymphoreticular tissue in Balb!c mice
No. of mice Groups of animals investigated
Malignant Preneolymplastic phoma lesions
Blastic Hyertransfor- plasia mation
Atrophy
----~----.--"'------
18
I1TFi
18
II1TF
16
III!TF
+
17
IV!IM
+ TF!
16
V!IM!
19
VIIN!
4
+
1M! 1M!
13
14 2
2
9
4
7
9
14
1M - imuran, TF - typhoid fever vaccine, N - without treatment
Discussion Numerous theories and hypotheses concerning the genesis of malignant lymphomas, although differing in some aspects of the pathogenesis, emphasize the meaning of immunological surveillance in the formation of malignancies of the lymphoreticular system) Armstrong et al., 1972; Gleichmann et al., 1972; Kruger, 1974; Schwartz, 1972). Support for these opinions can be drawn from our experiments. In experiments concerning chronic immunization with typhoid fever vaccine, the genesis of malignant lymphomas can be closely followed. Morphological examinations indicate that under the influence of typhoid fever vaccine a proliferation of lymphocytes taken place in areas where normally localized thymus-dependent cells are present. Among such proliferated lymphocytes scattered histiophagocytes containing cellular debris were present. Besides this, diffuse hyperplasia of plasma cells with Russell bodies and local drainage of lymphocytes was found in the medullary cords of lymph nodes. Subcapsular, medullary and trabecular sinuses, however, were filled with macrophages. The above description is similar to pictures reported by other authors who obtained malignant lymphomas by grafting allogenic lymphocytes. These changes are classified as preneoplastic (Gleichmann et al., 1972). In 20-40% of these animals malignant lymphomas developed, often a few months after discontinuing injections of vaccin€ (Z6ltowska and Kalinowski, 1973; Ultowska and Cieselski, 1974; z61towska, 1975).
264 . A. Z6ltowska
Animals receiving only typhoid fever vaccine showed excess lymphohistiocytic and plasmatic proliferation, in individual cases pictures of lymphoreticular leukemia were seen in mice. If after a few weeks of typhoid fever vaccine administration an immunosuppressive drug was added, excess lymphohistiocytic proliferation in thymus-dependent areas and also nodular fibrosis was observed. In lymph nodes thus altered malignant lymphomas appeared in up to 700/0 of the cases. Simultaneous application of both typhoid fever vaccine and immunosuppressive drug in this experiment did not give an effect similar to that evoked by prior stimulation with vaccine. Animals died mainly as a result of post-azathioprine anemia. Mice that received azathioprine for two weeks prior to typhoid vaccination showed blastic proliferation of the lymphoreticular system after two doses of vaccine. This seems to indicate an increased sensitivity of animals with an immunodeficiency to antigenic stimulation by typhoid fever vaccine. A similar effect could be achieved with typhoid fever vaccine in newborn animals with physiological immunodeficiency (Z6ltowska, 1975, in press). In these conditions lymphoreticular proliferation concerned mainly subcortical areas of lymph nodes and reminded one of blastic transformation of lymphocytes in response to differing immunogens in vitro. This indicates that typhoid fever vaccine in vivo can be an immunogen stimulating lymphocytes and probably in some conditions may be responsible for overcoming the areactivity of these cells to their antigenic determinants.
Zusammenfassung Nach chronischer Immunisierung mit Typhusvaccine entwickeln C-57-Bl- und BalbC-Mause maligne Lymphome. Bei nachtraglicher Azathioprin-Verabreichung treten diese Tumoren haufiger auf. Tiere, welche das Azathioprin vor der Typhusvaccine-Immunisierung erhalten hatten, zeigten ahnliche Phanomene wie die Blastentransformation der Lymphozyten in vitro.
References Allison, A. c.: Effects of antilymphocytic serum on bacterial and viral infections and virus oncogenesis. Fed. Proc. 29, 167-168 (1970) 2 . Armstrong, M. Y., Black, F. L., and Richars, F.: Tumour induction by cell-free extracts derived from mice with graft versus host disease. Nature New Biol. 235 , 153-154 (197 2 ) 3. Doell, R. G., Devaux St. Cyr. c., and Graber, P.: Immune reactivity prior to development of thymic lymphoma in C57BL mice. Int. J. Cancer 2 , 103-108 (1967) I.
Lymphoreticular Tissue Lesions after Azathioprine. 26 5 4. Gleichmann, E., Gleichmann, H., and Schwartz, R.: Immunologic induction of malignant lymphoma: genetic factors in the graft-versus-host model. ]. Nat. Cancer Inst. 49, 793- 801 (I97 2) 5. Huebner, R. ].: Inherited "switchedoff" RNA tumor virus oncogenes as determinants of cancer. U.I.e.e. Bull. Int. Union Against Cancer 7, 1-3 (I969) 6. Krueger, G. R. F., Malmgren, R. A., and Berard, e. W.: Malignant lymphomas and plasmocytosis in mice under prolonged immunosuppression and persistant antigenic stimulation. Transplantation II, 138-144 (I97I) 7. Krueger, G. R. F.: Lymphoreticular neoplasia in immunosuppression: Facts and Fancies. Beitr. Path. 151, 221-233 (I974) 8. Metcalf, D.: Reticular tumours in mice subjected to prolonged antigenic stimulation. Brit.]. Cancer 15, 769-779 (I96I) 9. Meyers, D. D., Meier, H., and Huebner, R. J.: Prevalence of murine C-type RNA virus specific antigen. Life Sci. 9, I07I-I080 (I970) IO. O'Connor, G. T.: Persistent immunologic stimulation as a factor in oncogenesis, with special reference to Burkitt's tumor. Amer. ]. Med. 48, 279-285 (I970) I1. Potter, M.: Mouse IgA myeloma proteins that bind polysaccharide antigens of enterobacterial origin. Fed. Proc. 29, 85-91 (1970) 12. Schmidt, e. G.: (I973), ref. according Krueger (I974) I3. Schwartz, R. S.: Immunoregulation, oncogenic viruses, and malignant lymphomas. Lancet I, I266-I269 (I972) 14. Vredevoe, D. L., and Hays, E.: Effect of antylymphocytic and antithymocytic sera on the development of mouse lymphoma. Cancer Res. 29, 1685-1690 (I969) I 5. Z6!towska, A., and Kalinowski, M.: Morphology of peripheral blood, bone marrow, lymph nodes, spleen and liver in animals subjected to long-term stimulation with typho-tetanic vaccine. Ann. Immuno!. 1-2,69-83 (1973) 16. Z6!towska, A., and Ciesielski, D.: Pathomorphology and serum protein electrophoresis in animals intensivelly stimulated with typhoid fever vaccine. Arch. Immuno!. Ther. Exp. 22, 315-325 (I974) 17. Z6!towska, A.: Stud on chronic immunization of animals. Morphology of changes of lymphoreticular system in three generations of animals chronically immunized with typhoid fever and typhoid-tetanus vaccine. Arch. Immuno!. Ther. Exp. 23, 29-53 (I 975) 18. Z6!towska, A.: Lymphoreticular tissue lesioms in animals immunized with typhoid fever vaccine from suckling period. Arch. Immuno!. Ther. Exp. 23, (I975) in press. Dr. Albina Z6!towska, Dept. of Pathology, Medical Sool, Gdansk, Poland
Lymphoreticular Tissue Lesions in Mice after Immunization with Typhoid Fever Vaccine and Immunosuppression with Azathioprine LymphoretikuHire Gewebeveranderungen bei Mausen nach Immunisierung mit Typhus-Impfstoff und Immunosuppression mit Azathioprin ALBINA Z6t TOWSKA With 4 Figures and 2 Tables· Received August 12, I974 . Accepted August 26, 1975
III
revised form
Key words: Murine lymphoreticular tissue - Typhoid fever vaccme Immunization - Immunosuppression - Malignant Lymphoma
Summary C57BL and Balblc mice were immunized, were immunized and then administered immunosuppressives, were immunized with concurrent immunosuppression, and were immunized after prior administration of immunosuppressive agents. Typhoid fever vaccine used clinically was used for immunization. Immunosuppression was accomplished with azathioprine (Imuran). Control groups received azathioprine or no treatment. The experiment lasted 5 months. In mice immunized with typhoid fever vaccine and subsequent azathioprine administration, malignant lymphomas of a lymphoblastic type appeared in 20010 of C57BL mice and in 70010 of Balblc mice. In mice receiving antigenic vaccine together with azathioprine, malignant lymphomas appeared in only 5 to 10010 of cases. However, mice that first received azatioprine and were later immunized, died after the second administration of typhoid fever vaccine and showed pictures that brought to mind blastic transformation of lymphocytes in vitro. This proliferation was primarily found in subcortical areas of lymph nodes. Analysis of presented results indicates that chronic immunization of animals with typhoid fever vaccine may serve as experimental model of malignant lymphoma morpho· logically similar to that appearing in human pathology.
25 8 . A. i6ltowska
The genesis of malignant lymphomas in animals under the influence of various stimulating agents has been described by many authors (Doell et al., I967; Metcalf, I96I; O'Connor, I970; Potter, I970). It has been reported that immunosuppre~sion favors this proliferation (Allison, I970; Vredevoe and Hays, I969). Krueger and coIl. (I97I and I974) state that only prolonged immunosuppression in combination with persistent antigenic stimulation causes excessive lymphoreticular stem cell proliferation and malignant lymphoma. Many authors support the virus theory (Armstrong et al., I972; Gleichmann et al., I972; Huebner, I969; Meyers et al., I970; Schmidt, I973). The virus supposedly transforms the T lymphocytes and in this way initiates the graft-versus-host reaction (Schmidt, I973) or becomes active during immunological disorders of the graft-versus-host type (Armstrong et al., I972; Gleichmann et al., I972). Our own experiments showed formation of malignant growths of the lymphoreticular system in 4 species of rodents after stimulation with typhoid fever vaccine (ZOltowska and Kalinowski, I 973; Z6ltowska and Ciesielski, I974; Z6howska, I975). In animals immunized shortly after birth these growths were more frequent (Z6ltowska, I975, in press), which is probably due to immunodeficiency. To examine this more closely morphological effects of typhoid fever immunization in adults with accompanying immunosuppression were studied.
Material and Methods One hundred and twenty Balb/c mice and 12 0 C57BL mice, 6 weeks of age, were used. Experiments were carried out on both males and fem ales. The animals were divided into 6 groups consisting of 20 mice each. Group I received only typhoid fever vaccine. Group II received typhoid fev er vaccine alone for the first four weeks, after which typhoid fever vaccine was given together with azathioprine. Group III received both typhoid fever vaccine and azathioprine from the beginning of the experiment. Group IV for the first 2 weeks received azathioprine only, typhoid fever vaccine plus azathioprine after this period. Group V was given only azathioprine. Group VI received no treatment. The animals were immunized with typhoid fever vaccine produced by the Warsaw Serum and Vaccine Production Laboratory, I ml of the vaccine containing 1,000 bacilli Salmonella typhi, 0.45010 phenol and saline solution. Mice were injected with the vaccine twice weekly subcutaneously in the inguinal region with 0.1 ml doses for a period of 5 months. Immunosuppression was achieved by continuous administration of 15 mg/kg/day of azathioprine "Imuran" (Well come Foundation Ltd. London) in drinking water.
Lymphoreticular Tissue Lesions after Azathioprine. 259 Animals were sectioned immediately after natural death or after being sacrificed when found extremely ill. After 5 months control and remaining experimental mice were killed using ether anesthesia. Routinely, sections from all organs were taken for histopathological examination and stained with hematoxylin and eosin.
Results Group I - Mice recelvlllg only typhoid fever vaccine showed excess lymphocyte proliferation with scattered histiophagocytes in cortical regions and plasmocytic proliferation in the medullary cord of lymph nodes. In subcapsular, trabecular and medullary sinuses of lymph nodes numerous aggregates of macrophages were found. Around splenic central arterioles hyperplastic lymphohistiocytic regions were present, while peripheral areas of Malpighian follicles were composed of plasma cells and macrophages. Changes in both Balblc and C57BL strain mice were similar. In indivudual cases in this experimental group of animals generalized malignant lymphomas of lymphoreticular leukemic type with infiltration liver and spleen were observed. Group II - Mice of this group showed lymphohistiocytic proliferation in lymph nodes, Peyer's patches, thymus and spleen. In the spleen this proliferation was present round the central arterioles; peripheral portions of the Malpighian follicles consisted of fibrous tissue with individual plasma cells, lymphocytes and macrophages. The Malpighian follicles of the spleen were thus formed by lymphocytes with scattered histiocytes and enveloped by fibrous tissue. Similar changes were observed in lymph nodes, mainly submandibular ones in the area of the salivary glands (Fig. I). It seems likely that in the spleen as well as in the lymph nodes sclerotic areas corresponded to regions of connective tissue trabeculae and sinuses. In these sclerotic areas, among which declining numbers of macrophages and numerous mast cells could be seen, cells containing basophilic cytoplasm and a hyperchromatic nucleus with am irregular nucleolus were identifical. These cells proliferated in areas where fusiform cells were arranged similarly to angioblasts, and depending on the plane of sectioning follicular or trabecular arrangements could be observed. These views were reminiscent early preneoplastic malignant lymphoma (Fig. 2). In some cases in the lymph nodes these processes were more advanced and less differentiated cells of the lymphoreticular system were predominant, which allowed regognition of lymphoblastic malignant lymphoma (Fig. 3). This type of proliferation in three out of five tested C57BL strain mice when transplanted in syngeneic hosts grew rapidly and caused death of animals in 14-18 days in 10-300/0.
260 .
A. Z6!towska
Fig.!. Submandibular lymph node of C57 BL mouse from group II of experiment. (Mice were immunized with typhoid fever vaccine and then administered azathioprine.) Large histiocytes are scattered throughout well-differentiated lymphocyte proliferation in the cortical region. HE; X 120.
Fig. 2. Submandibular lymph node of C57BL mouse from group II of experiment. Note areas of fibrous tissue among which are macrophages and large cells containing basophilic cytoplasm and hyperchromatic nuclei resembling lymphoblastic cells. These cells border fusiform cells arranged similarly to angioblasts. HE; X 360.
Lymphoreticular Tissue Lesions after Azathioprine. 261
Fig. 3. Lymph node of C57BL mouse from group II of experiment. The lymph node is replaced by pleomorphic proliferation with lymphoblasts. HE; X 360.
Fig. 4. Lymph node of Balblc mouse from group IV of experiment. (Mice received for the first 2 weeks azathioprine only, typhoid fever vaccine plus azathioprine after this period.) Note lymphocytes and lymphoblasts and also mitotic figures. HE; X 360. 18 Beitr. Path. Bd. 156
262 .
A. Z61towska
In mice of the Balb/c strain, malignant lymphomas occurred more frequently. All animals in this experimental group died or were extremely ill and sacrificed within 6-15 weeks. Group III - Microscopically only in a few indivudual cases a picture of malignant proliferation was seen in lymph nodes. In the remaining animals of this group excess disseminated lymphohistiocytic and plasmacytic growth was found. Group IV - Animals of this group began dying after the second injection of vaccine with symptoms of diarrhea. Submandibular lymph nodes were swollen and whitish, microscopically blastic transformation of lymphocytes similar in response to immunogen in vitro was observed (Fig. 4). Bald/c strain mice were more sensitive. In the rest of the lymphoreticular system of mice in this experimental group lymphohistiocytic proliferation could be seen. Group V - Mice receiving only azathioprine showed atrophy within the entire lymphoreticular system or proliferation of plasma cells and single stem cells in medullary areas of lymph nodes and peripheral parts of Malpighian follicles. C57BL strain mice died within 4-8 weeks, while Balblc strain mice lived 10 weeks or longer. Group VI - Animals of this control group showed no deviation from normal. In all groups of animals female mice showed similar changes as male mice. The results in the experimental and control groups are given in Table I and 2.
T able
1.
Morphologic changes of lymphoreticular tissue in C57BL mice
No. of mice Groups of animals investigated
- _ ..
_
- - - - - - _... -
16
I1TF
17
II1TF
19
+ IM/ III/TF + IM/ IV/1M + TF/
17
V/IM/
19
VIIN/
17
Malignant Preneoplastic lymphoma lesions
_ . _ - - - _ ._-
--_ .. _ - - - - - --
-
Atrophy
--
14
2
4
Blastic Hypertransfor- plasia mation
6
7
2
10
4
9 8
9
Lymphoreticular Tissue Lesions after Azathioprine . 26 3 Table
2.
Morphologic changes of lymphoreticular tissue in Balb!c mice
No. of mice Groups of animals investigated
Malignant Preneolymplastic phoma lesions
Blastic Hyertransfor- plasia mation
Atrophy
----~----.--"'------
18
I1TFi
18
II1TF
16
III!TF
+
17
IV!IM
+ TF!
16
V!IM!
19
VIIN!
4
+
1M! 1M!
13
14 2
2
9
4
7
9
14
1M - imuran, TF - typhoid fever vaccine, N - without treatment
Discussion Numerous theories and hypotheses concerning the genesis of malignant lymphomas, although differing in some aspects of the pathogenesis, emphasize the meaning of immunological surveillance in the formation of malignancies of the lymphoreticular system) Armstrong et al., 1972; Gleichmann et al., 1972; Kruger, 1974; Schwartz, 1972). Support for these opinions can be drawn from our experiments. In experiments concerning chronic immunization with typhoid fever vaccine, the genesis of malignant lymphomas can be closely followed. Morphological examinations indicate that under the influence of typhoid fever vaccine a proliferation of lymphocytes taken place in areas where normally localized thymus-dependent cells are present. Among such proliferated lymphocytes scattered histiophagocytes containing cellular debris were present. Besides this, diffuse hyperplasia of plasma cells with Russell bodies and local drainage of lymphocytes was found in the medullary cords of lymph nodes. Subcapsular, medullary and trabecular sinuses, however, were filled with macrophages. The above description is similar to pictures reported by other authors who obtained malignant lymphomas by grafting allogenic lymphocytes. These changes are classified as preneoplastic (Gleichmann et al., 1972). In 20-40% of these animals malignant lymphomas developed, often a few months after discontinuing injections of vaccin€ (Z6ltowska and Kalinowski, 1973; Ultowska and Cieselski, 1974; z61towska, 1975).
264 . A. Z6ltowska
Animals receiving only typhoid fever vaccine showed excess lymphohistiocytic and plasmatic proliferation, in individual cases pictures of lymphoreticular leukemia were seen in mice. If after a few weeks of typhoid fever vaccine administration an immunosuppressive drug was added, excess lymphohistiocytic proliferation in thymus-dependent areas and also nodular fibrosis was observed. In lymph nodes thus altered malignant lymphomas appeared in up to 700/0 of the cases. Simultaneous application of both typhoid fever vaccine and immunosuppressive drug in this experiment did not give an effect similar to that evoked by prior stimulation with vaccine. Animals died mainly as a result of post-azathioprine anemia. Mice that received azathioprine for two weeks prior to typhoid vaccination showed blastic proliferation of the lymphoreticular system after two doses of vaccine. This seems to indicate an increased sensitivity of animals with an immunodeficiency to antigenic stimulation by typhoid fever vaccine. A similar effect could be achieved with typhoid fever vaccine in newborn animals with physiological immunodeficiency (Z6ltowska, 1975, in press). In these conditions lymphoreticular proliferation concerned mainly subcortical areas of lymph nodes and reminded one of blastic transformation of lymphocytes in response to differing immunogens in vitro. This indicates that typhoid fever vaccine in vivo can be an immunogen stimulating lymphocytes and probably in some conditions may be responsible for overcoming the areactivity of these cells to their antigenic determinants.
Zusammenfassung Nach chronischer Immunisierung mit Typhusvaccine entwickeln C-57-Bl- und BalbC-Mause maligne Lymphome. Bei nachtraglicher Azathioprin-Verabreichung treten diese Tumoren haufiger auf. Tiere, welche das Azathioprin vor der Typhusvaccine-Immunisierung erhalten hatten, zeigten ahnliche Phanomene wie die Blastentransformation der Lymphozyten in vitro.
References Allison, A. c.: Effects of antilymphocytic serum on bacterial and viral infections and virus oncogenesis. Fed. Proc. 29, 167-168 (1970) 2 . Armstrong, M. Y., Black, F. L., and Richars, F.: Tumour induction by cell-free extracts derived from mice with graft versus host disease. Nature New Biol. 235 , 153-154 (197 2 ) 3. Doell, R. G., Devaux St. Cyr. c., and Graber, P.: Immune reactivity prior to development of thymic lymphoma in C57BL mice. Int. J. Cancer 2 , 103-108 (1967) I.
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