Journal of Neuroscience Research 3:275-280 ( 1 9 7 7 )
Transplantation Immunity in the Brain Zdengk Lodin, Milan HaSek, Jitka Chutna, MiloS SIadeEek, and Vladimir Holah Institute of Physiology, Czechoslovak Academy of Sciences, and Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Prague
The resistance of animals to allogeneic tumor cells injected into the brain increases with age. The second-set reaction in the brain of rats appears t o be equally effective after preimmunization into the brain and preimmunization into the leg. Also, the presence of cytotoxic antibodies is demonstrable after preimmunization into the brain. These findings suggest that both arcs of the transplantation reaction, the afferent and efferent, are involved in tumor-cell allotransplantation in the brain. Key words: brain immunity, transplantation, resistance to tumors
I N T R 0DUCT 10N
The immunologic privilege within the brain has long been considered. The views of the brain as a privileged transplantation site where allografts or even xenographs survive have been based on earlier findings of the success of mainly tumor tissue transplanted t o the brain. More recent studies, however, have not provided unequivocally positive results, as has been stressed b y Woodruff (1960) and Lance ( I 967). In a previous paper wc have shown that some degree of transplantation immunologic resistance to tumor allographs was demonstrable in rats. This resistance could be abolished b y rendering the recipient animals immunologically tolerant (Has’ek et al, 1977). The present study was undertaken t o determine the effect of a local preimmunization in the brain as compared with a systemic immunization whose effectiveness was investigated by Medawar (1948). MATERIALS AND METHODS
Strain AVN rats were used as recipients. The donors were Lewis strain rats, differing from the AVN recipinets in H-1 plus non-H-1 antigens. Tumor cells which were inoculated into the brains and legs were obtained from the culture of a virogenic sarcoma RSL. The RSL sarcoma was induced in a newborn Lewis rat b y injection of the Schmidt-Ruppin strain of Rous sarcoma virus. Tumor cells in 5 X l o 3 , 5 X lo4, 5 X l o 5 ,l o 6 , and 5 X lo6 in a volume of 0.05 m l of Eagle’s medium Address reprint requests to Z. Lodin, Institute of Physiology, Czechoslovak Academy of Sciences, Budgjovicka 1083, 142 20 Prague 4, Czechoslovakia.
0 1977 Alan R. Liss, Inc, 150 Fifth Avenue, New York, NY 10011
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Lodin et a1 TABLE I. Deaths in the Recipients ar Various Ages After Inoculation of Different Numbers of Tumor Cells Number of injected Cells
5 x 103 5 x 10' 5 x 105 10' 5
x 10'
Recipients at the age of 4 weeks
8/22 9/10
6 weeks
4 months
015 317
0110
1/24
15/35 617
1/10
Oil0 0130 19/31
were injected with a microsyringe into the occipital lobe in the right cerebral hemisphere to a depth of 1.5-2 mm (after preimmunization also in the left hemisphere) of the AVN recipients. After tumor-cell inoculation, the animals were inspected daily, and the effect of tumor growth was evaluated by death of the animals. The brains of 4-5 animals from individual groups were taken for histologic examination. The presence of cytotoxic anitbodies in the serum of AVN rats injected with RSL cells into the brain was assayed by the Cr release from labeled Lewis lymphocytes by the standard technique (Sanderson, 1965; Wigzell, 1965). RESULTS
From the results obtained it appears that the resistance increases with age of animals inoculated with tumor-cell suspensions into the brains when tested at 1-4 months of age. For example, 9 out of 10 4-week-old rats died following injection of lo6 tumor cells, whereas none of the 30 4-month-old rats did so. Furthermore, the mortality of animals increased with higher numbers of tumor cells (Table I). Most of the animals died 6-21 days after transplantation of tumor cell, and deaths in the recipients of the smaller doses occurred at a slower rate. Furthermore, the second-set reaction was followed after both intraleg and intrabrain preimmunization. Four-month-old rats were 1st inoculated with lo6 tumor cells, which did not kill the recipients even when the inoculum was injected into the brain. Fourteen days later, preimmunized animals received 5 X lo6 tumor cells which killed 19 out of 31 control untreated 4-month-old recipients. After intrabrain preimmunization, only 3 out of 30 animals died, and no deaths occurred after intraleg preimmunization (Table 11). Figure 1 illustrates a brain section from the animal which showed no massive tumor within 36 days of inoculation. However, lymphocytic infiltration and, in some cases, cells resembling in shape the tumor cells were seen in the immediate vicinity of capillaries in the brain parenchyma, especially near the choroid plexus vessels and along the meninges. Serial sections permitted the location of the injection site (Fig. 2). Thirty-six days after injection, the necrotic canal resulting from the insertion of the needle was still visible. Aggregates of lymphocytes and remaining tumor cells were seen near this canal. When deaths occurred, massive foci of tumor cells in the hemisphere inoculated with the tumor were found in serial blain sections. The growth of tumor varied considerably. In some cases the tumor tissue occupied a larger part of the cerebral hemisphere (Fig. 3) 8 days after inoculation. Solid foci were largely present, but clusters of tumor cells were seen even outside the foci. The brain was considerably edematous, and the occipital lobe and the cerebellum were compressed. Edematous changes were obvious also in the medulla oblongata and the pons. Most probably the nuclei in the medulla oblongata
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TABLE 11. Deaths in 4-Month-Old Rats Inoculated With RSL Tumor Into the Brain After Preimmunization and in Control Animals Preimmunization with 10' RSL cells into Brain Leg No pretreatment (controls)
Deaths after inoculation of 5 X 10' RSL cells into the brain 3/30 0129 19/31
Fig. 1. Sagittal section through a rat brain 36 days after inoculation of tumor cells. No signs of a tumorous focus are visible. Serial brain sections were made in all animals taken for histological examination. Hematoxylin-eosin. Overall view.
and pons were damaged, and this caused the death of animals. Figure 4 shows clusters of cells from the middle part of the tumor at high magnification. In preimmunized animals, cytotoxic antibody formation was simultaneously investigated in the serum collected 14 days after the 2nd tumor inoculation (Table 111). Cytotoxic antibody titers were produced following both preimmunization in the brain and preimmunization in the leg, but they were higher after the intrabrain preimmunization. D ISCUSS ION
The present results show that allogeneic tumor transplanted to the brain may kill the recipient when injected in massive doses. Because allogeneic RSL tumors never kill the recipients when injected into the leg and regress even if large doses of as many as lo7 tumor cells are used, it may be considered that immunity in the brain is less effective than in other sites of the body. However, it is also possible that immunity develops at a slower rate in the brain compared to other sites. It seems probable that even the smaller tumorous foci in the brain are lethal and that a transiently growing allogeneic tumor may kill the recipient rather rapidly before specific immunity has sufficiently developed. Also, the results of Raju and Grogan (1977) showing that an efferent arc blockade is a major
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L o d i n et a1
Fig. 2. The area of the brain of a rat which did not die from the tumor ( 3 6 days after inoculation). Necrotic canal is recognized at the site of injection. Clusters of lymphocytes and tumor cells are seen at the periphery of the canal. Edematous changes are observed in the neighborhood of the canal (X 10).
operative mechanism responsible for the protection of brain implants speak only for the quantitative immunologic deficiency of the brain. The phenomenon of the second-set reaction is, however, well demonstrable in the brain, both after preimmunization into the leg, which is in aggreement with the early findings of Medawar (1948), and preimmunization into the brain. Therefore we believe that the vascularization of the brain is sufficient for systemic immunity, including immunity in the brain, to develop. This view is also supported by the finding that immunologic tolerance abolishes the resistance to allogeneic tumor transplants (Haskk et al, 1977). Consistent with the finding of the second-set reaction following intrabrain immunization is also the presence of cytotoxic antibodies which were formed in higher titers after intrabrain preimmunization compared to the intraleg preimmunizaSion. The immune reactions in the brain were demonstrated in previous experiments of Sterzl and Lodin (1976) in which the administration of antigen to the brain led to marked antibody formation by the migrating cells localized in the brain. Also, the results obtained in man
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Fig. 3. Brain section f r o m a rat which died at 8 weeks of age 8 days after inoculation (1 X 10' tumor cells). A large solid tumorous focus is visible in the middle of the hemisphere. Hematoxylin-eosin. Overall view.
Fig. 4. Detail from the same section as in Figure 3. Aggregate of cells of a tumorous focus destroying the brain parenchyma. Hematoxylin-eosin (X 25).
where tumor-specific cell-mediated immune response can be demonstrated following the primary intracranial tumor (Levy, Mahaley, and Day, 1972) are in agreement with the above notion.
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Lo d in e t a1 TABLE 111. Presence of Cytotoxic Antibodies in the Serum of 4-Month-Old Rats After Reimmunization With AUogeneic Tumor Cells Into the Brain Group
1 2 3
Injection of RSL cells 1st dose 2nd dose
Antibody titer median (range)
brain 1%
brain brain
128 (8-256) 4(0-32)
-
-
-
Mean % 51Cr release
?
SEa
38.1 t 6.3 14.4 * 1.5 9.9 r 1.4
aSerum diluted 1:2.
The results with the second-set reaction presented here show that both arcs, the afferent and efferent, are involved in the allotransplantation in the brain and that the immunologic privilege of the brain for allotransplants is only quantitative in nature.
REFERENCES Hakk M, Chutni J, SlideFek M, Lodin Z (1977): Immunological tolerance and tumour allografts in the brain. Nature 268:68. Lance EM (1967): A functional and morphological study of intracranial parathyroid allowgrafts in the dog transplantation, 5: 147 1-1483. Levy NL, Mahaley MS, Day ED (1972): In vitro demonstration of cell-mediated immunity to human brain tumors. Cancer Res 32:477-482. Medawar PB (1948): Immunity to homologous grafted skin. 111. The fate of skin homografts transplanted to the brain, to subcutaneous tissue, and to the anterior chamber of the eye. Br J Exp Pathol 29: 5 8-69. Raju S, Grogan JB (1977): Immunologic study of the brain as a privileged site. Transplant Proc 9: 1187-1 191. Sanderson AR (1965): Quantitative titration, kinetic behavior and inhibition of cytotoxic mouse isoantisera. Immunology 9: 287 - 300. Y Sterzl J, Lodin Z (1976): Immune reactions localized in the nervous system. Immunol News 7:89. Wigzell H (1965): Quantitative titrations of mouse H-2 antibodies using ”Cr-labeled target cells. Transplantation 3:423-431. Woodruff MFA (1960): “The Transplantation of Tissues and Organs.” Springfield, Illinois: CC Thomas.