Toxicology Letters, 6465 (1992185-92 0 1992 Elsevier Science Publishers B.V., All rights reserved 03784274/92/$5.00
85
Athymic experimental animals in pharmaco-immunological research P. Juulat, H.B. Christensen’, H.P. Hougenb, 0. Svendsenc, P. Thygesen’ and J. Rygaardb ‘Dept. ofBiological Sciences, The Royal Danish School ofpharmacy, Copenhqen, bBartiwlin Instituttet, Kommunehospitalet, Copenhagen and ‘Scantox, Lille Skensved (Denmark)
Key words: Nude mice; Nude rats; Streptozotocin; Diabetes mellitus; Guanethidine; Sympathectomy; Toxicology
SUMMARY Immunodeficient animals - the nude mouse and the nude rat - allow studies of drug action and possible side effects without interference from the immune system. Comparative investigations in athymic and euthymic animals allowed us to elucidate the role of T-lymphocytes in the pathogenesis of streptozotocin-induced diabetes mellitus in mice, and the importance of NK-cells as effecters in guanethidine-induced sympathectomy in the rat. It is suggested that immunodeficient animals should be included in toxicological studies of xenobiotics.
INTRODUCTION
The purpose of this presentation is to call attention to athymic experimental animals, the nude mouse and the nude rat, as potential tools to study secondary phenomena, due to the immune system or other protective systems. For more than two decades various strains of immunodefkient mice and rats have been used as biological tools in exploration of immune and non-immune mechanisms, in particular the athymic nude mouse and rat devoid of functional T-cells, and in recent years the SCID (severe combined immuno-deficiency) mouse with impaired differentiation of both T and B lymphocytes. The athymic mouse and rat have also been extensively used in the study of the effects of chemotherapeutics on transplanted human tunlours. Correspondence to: J. Rygaard, Copenhagen K, Denmark.
Bartholin
Instittutet,
Kommunehospitalet,
DK-1399
86
Such immunodeficient animals may have an equally important place in toxicology, allowing studies of drug action and possible side effects of xenobiotics without interference from the immune system or other defence mechanisms. In the following the athymic mouse and rat are briefly described, and studies are reported on their use in clarification of the mechanisms behind streptozotocin-induced diabetes mellitus in mice and guanethidine-induced sympathectomy in the rat. THE ATHYMIC MOUSE AND RAT
The athymic nude mouse The athymic condition of the nude mouse was first described by Pantelouris in 1968 Ill. The athymic condition is due to a recessive autosomal mutation, nude, the symbol for which is nu. Based on the genetics of the mutation, various breeding schemes can be set up. Nude female are fertile in proper microbiologic conditions, but they have difficulties in weaning their young. Nude male are generally fertile, although infections in the genital tract may cause infertility. Breeding of nude mice can be based on mating of heterozygous male and female, or - preferably - on the mating of homozygous male with heterozygous haired female, which will give 50% nude offspring in contrast to heterozygous/heterozygous matings, where only an average of 25% are athymic. Kept under strict specified pathogen-free (spfl conditions nude mice may live for more than 18 months. A breeding colony was established in Copenhagen in December 1968, and at the end of 1969 we published the first reports on immune functions in nude mice and the first successful heterotransplantation of a human malignant tumor to nude mice [21. The immune defect of the nude mice is, of course, primarily a defect in T-cell functions. In the athymic animal, special interest has naturally focused on the appearance of mononuclear cells showing T-cell markers. The number of T-cell marker positive cells increases with age. The complement of B lymphocytes seems to be normal. Immunoglobulin production is sparse, the only immunoglobulin that is produced in uiuo in high amounts being IgM. The macrophage system in the nude mouse is highly active. The natural killer (NK-) cell was first described in the nude mouse in 1975. This new cell type which appeared in various percentages in nude mice depending on their genetic background, was able to kill a range of cultured cell lines, first of all YAC-cells. In analogy with the so-called natural antibodies, the cell was called the natural killer cell, because it appeared to be present without any previous priming of the animal. For reviews, see [3,41.
The mu/mu rat The athymic nude rat appeared in 1953 but died out, reappearing in 1975 El. The mutant was signed mu/mu (Rowett nude) rat. If kept in strict pathogen-free conditions, life-spans exceeding two years have been noted. Female athymic nude rats are normally fertile and produce the usual number of offspring, but can only rear a few of them. Therefore the normal breeding procedure is to backcross the heterozygous offspring. Inbred nude animals are now available in several rat strains. B lymphocytes are present in normal quantities in the nude rat. A few mononuclear cells with T-cell markers can be demonstrated with increasing age as in the nude mouse in spite of its athymic condition, but they are not functional in vivo. Isogeneic thymus grafting is an effective way of inducing normal immune reactivity in nude rats. For review, see C61. IMMUNOTOXICOLOGIC
STUDIES IN ATHYMIC MICE AND RATS
Streptozotocin-induced diabetes mellitus in normal and athymic (nude) mice In preparation for heterotransplantation studies of rat pancreas to nude mice we made an interesting observation in relation to the effect of streptozotocin in athymic mice as compared to normal mice of the same genetic background (Balb/c/A/Bom). Given a single dose of 200 m&g bw or 5 daily injections of 40 mg/kg bw twenty percent of nude mice did not become diabetic, whereas all normal mice in two groups developed significant hyperglycaemia, blood glucose >200mg/lOO ml (Fig. 1). The islets of Langerhans showed slight mononuclear cell infiltration in NUDE
MICE
BALBlc
‘ i
c&8
.cc%.~
1 NORMAL
MICE
BALBk
NORMAL
MICE
“NSH”
a000808
ot?co 0030
‘ i 100
200
300
400
500
> 600 mg11OOml
Fig. 1. Blood glucose values of nude and two strains of normal mice in single dosage experiments. Each circle, open or filled, represents one animal.
88
some of the normal mice, but not in the nude animals. This prompted us to attempt transfer of spleen cells from diabetic haired mice to non-diabetic animals, both normal and nude. Both types of recipients developed significant hyperglycaemia after transfer of 103-IO7 T-cell enriched spleen cells, most pronounced for recipients of small inocula (103-105), and after low dose irradiation of inocula, probably due to high sensitivity of the suppressor cells [7,8,91. The mononuclear cell infiltration in the islets of Langerhans might have made us conclude that an immune response was involved in the pathogenetic process, but the differential responses in normal and nude mice gave a very clear indication. We have made similar observations when trying to establish virus-induced diabetes in normal and nude mice with the EMCvirus: virus propagates to the same degree in normal and nude mice, but whereas all normal mice develop diabetes, nude mice are unaffected. Guanethidine-induced sympathectomy in normal and athymic rats For nearly 25 years guanethidine has been known to produce destruction of sympathetic neurones in rats following chronic high dose administration. The first report by Jensen-Holm [IO] was followed by several others dealing with the cytotoxic effect of guanethidine on rat sympathetic neurones, and it soon became evident that this was both an organ- and species-specific toxic reaction [11,121. This lead to the use of guanethidine in experimental pharmacology and physiology as a tool in studying various physiological and pathological processes, involving the peripheral sympathetic nervous system. The mechanism behind the neurotoxic effect of guanethidine was still not fully understood, although many efforts were made to determine how guanethidine destroyed sympathetic neurones. Several hypotheses concerning the mechanism have been presented, but within the last 10 years more evidence has accumulated suggesting involvement of the immune system. Today guanethidine-induced sympathectomy offers an attractive model of an autoimmune disorder: in appropriate rat strains the effect of guanethidine is highly reproducible, and - in contrast to some other models of autoimmunity - the general condition of the animals is unaffected. Elucidation of the involved mechanisms will provide a better knowledge of autoimmune disorders, in particular of the peripheral nervous system. The guanethidine-induced neurotoxicity has only been reported in the rat and occurs apparently only in certain strains. The lack of neurotoxic effect of guanethidine in other species is not a result of exclusion of guanethidine from the neurones. In both cats and mice guanethidine accumulates in the ganglia to the same extent as that necessary to cause neuronal destruction in rats.
89
The ultrastructural changes, such as mitochondrial swelling, observed in rat ganglionic neurones following guanethidine treatment, are also observed in cats and mice, but do not in themselves lead to neurotoxicity, since neuronal destruction does not occur in cats or mice. Manning et al Cl31 claimed, that guanethidine sympathectomy represented a drug-induced autoimmune disorder similar in nature to the low dose streptozotocin-induced diabetes. A thymus-dependent immunological effector mechanism in the guanethidine-induced neurotoxicity was suggested following detection of T-cells in the mononuclear cell infiltrate in the ganglia after guanethidine treatment. These observations prompted a series of experiments in our laboratories, using normal rats and athymic rats on the same genetic background (Lewis +/+ and Lewis mu/mu/Mel) as our experimental models in order to establish the role of thymus-dependent and -independent immune responses. Guanethidine treatment in athymic nude rats induced chromatolysis and neuronal destruction in a similar way as previously reported in normal euthymic rats. This unexpectedly demonstrated the same cytotoxic effect of guanethidine in the athymic as in the euthymic rats, while the infiltration of mononuclear cells was significantly smaller in the athymic rats. The neuronal destruction of the sympathetic ganglia induced by guanethidine must therefore involve a thymus-independent immune response. Treatment with immunosuppressive drugs such as cyclophosphamide, cyclosporin A and methylprednisolone concurrently with guanethidine in adult athymic rats gave similar results as observed in normal euthymic animals. The effect of the immunosuppressive drug cyclosporin A was investigated due to its mode of action, primarily suppressing the CD4 T-cells by blocking the interleukin-2 production. The CD4 T-helper cells play an important role in regulation of the thymus-dependent response. Therefore the lack of effect in preventing guanethidine-induced neurotoxicity in both normal euthymic and athymic rats can easily be explained by involvement of T-cell-independent immunological reactions. The mononuclear cell infiltrate in sympathetic ganglia was studied in both euthymic and athymic rats. Four different subpopulations of mononuclear cells were identified by means of monoclonal antibodies: MHC class II cells, pan T-cells, CD8 T-cells/NK-cells and CD4 T-cells/macrophages. The results confirmed that athymic nude rats did not possess T-marker positive cells to any significant extent, neither before nor after guanethidine treatment, whereas the number of T-cells in sympathetic ganglia of euthymic littermates increased substantially following guanethidine treatment [14,X1. A more interesting observation in this study was that the number of CD8 T-cells/NK-cells increased significantly in both athymic nude and euthymic rats following guanethidine administration.
90
TABLE I PERCENTAGES
OF NK-CELLS IN RAT SUPERIOR CERVICAL GANGLION
Treatment
N
% asialo GMl+ cells
% 0X8+ cells
Saline Anti-asialo GM1 Guanethidine Anti-asialo GM1 + guanethidine
5 5 5 5
0 0 7.2 f 1.5’ Ob
0.4 f 0.2 0 6.7 + 1.1’ 0.4 * o.3b
Mean + SEM. $
85
Athymic experimental animals in pharmaco-immunological research P. Juulat, H.B. Christensen’, H.P. Hougenb, 0. Svendsenc, P. Thygesen’ and J. Rygaardb ‘Dept. ofBiological Sciences, The Royal Danish School ofpharmacy, Copenhqen, bBartiwlin Instituttet, Kommunehospitalet, Copenhagen and ‘Scantox, Lille Skensved (Denmark)
Key words: Nude mice; Nude rats; Streptozotocin; Diabetes mellitus; Guanethidine; Sympathectomy; Toxicology
SUMMARY Immunodeficient animals - the nude mouse and the nude rat - allow studies of drug action and possible side effects without interference from the immune system. Comparative investigations in athymic and euthymic animals allowed us to elucidate the role of T-lymphocytes in the pathogenesis of streptozotocin-induced diabetes mellitus in mice, and the importance of NK-cells as effecters in guanethidine-induced sympathectomy in the rat. It is suggested that immunodeficient animals should be included in toxicological studies of xenobiotics.
INTRODUCTION
The purpose of this presentation is to call attention to athymic experimental animals, the nude mouse and the nude rat, as potential tools to study secondary phenomena, due to the immune system or other protective systems. For more than two decades various strains of immunodefkient mice and rats have been used as biological tools in exploration of immune and non-immune mechanisms, in particular the athymic nude mouse and rat devoid of functional T-cells, and in recent years the SCID (severe combined immuno-deficiency) mouse with impaired differentiation of both T and B lymphocytes. The athymic mouse and rat have also been extensively used in the study of the effects of chemotherapeutics on transplanted human tunlours. Correspondence to: J. Rygaard, Copenhagen K, Denmark.
Bartholin
Instittutet,
Kommunehospitalet,
DK-1399
86
Such immunodeficient animals may have an equally important place in toxicology, allowing studies of drug action and possible side effects of xenobiotics without interference from the immune system or other defence mechanisms. In the following the athymic mouse and rat are briefly described, and studies are reported on their use in clarification of the mechanisms behind streptozotocin-induced diabetes mellitus in mice and guanethidine-induced sympathectomy in the rat. THE ATHYMIC MOUSE AND RAT
The athymic nude mouse The athymic condition of the nude mouse was first described by Pantelouris in 1968 Ill. The athymic condition is due to a recessive autosomal mutation, nude, the symbol for which is nu. Based on the genetics of the mutation, various breeding schemes can be set up. Nude female are fertile in proper microbiologic conditions, but they have difficulties in weaning their young. Nude male are generally fertile, although infections in the genital tract may cause infertility. Breeding of nude mice can be based on mating of heterozygous male and female, or - preferably - on the mating of homozygous male with heterozygous haired female, which will give 50% nude offspring in contrast to heterozygous/heterozygous matings, where only an average of 25% are athymic. Kept under strict specified pathogen-free (spfl conditions nude mice may live for more than 18 months. A breeding colony was established in Copenhagen in December 1968, and at the end of 1969 we published the first reports on immune functions in nude mice and the first successful heterotransplantation of a human malignant tumor to nude mice [21. The immune defect of the nude mice is, of course, primarily a defect in T-cell functions. In the athymic animal, special interest has naturally focused on the appearance of mononuclear cells showing T-cell markers. The number of T-cell marker positive cells increases with age. The complement of B lymphocytes seems to be normal. Immunoglobulin production is sparse, the only immunoglobulin that is produced in uiuo in high amounts being IgM. The macrophage system in the nude mouse is highly active. The natural killer (NK-) cell was first described in the nude mouse in 1975. This new cell type which appeared in various percentages in nude mice depending on their genetic background, was able to kill a range of cultured cell lines, first of all YAC-cells. In analogy with the so-called natural antibodies, the cell was called the natural killer cell, because it appeared to be present without any previous priming of the animal. For reviews, see [3,41.
The mu/mu rat The athymic nude rat appeared in 1953 but died out, reappearing in 1975 El. The mutant was signed mu/mu (Rowett nude) rat. If kept in strict pathogen-free conditions, life-spans exceeding two years have been noted. Female athymic nude rats are normally fertile and produce the usual number of offspring, but can only rear a few of them. Therefore the normal breeding procedure is to backcross the heterozygous offspring. Inbred nude animals are now available in several rat strains. B lymphocytes are present in normal quantities in the nude rat. A few mononuclear cells with T-cell markers can be demonstrated with increasing age as in the nude mouse in spite of its athymic condition, but they are not functional in vivo. Isogeneic thymus grafting is an effective way of inducing normal immune reactivity in nude rats. For review, see C61. IMMUNOTOXICOLOGIC
STUDIES IN ATHYMIC MICE AND RATS
Streptozotocin-induced diabetes mellitus in normal and athymic (nude) mice In preparation for heterotransplantation studies of rat pancreas to nude mice we made an interesting observation in relation to the effect of streptozotocin in athymic mice as compared to normal mice of the same genetic background (Balb/c/A/Bom). Given a single dose of 200 m&g bw or 5 daily injections of 40 mg/kg bw twenty percent of nude mice did not become diabetic, whereas all normal mice in two groups developed significant hyperglycaemia, blood glucose >200mg/lOO ml (Fig. 1). The islets of Langerhans showed slight mononuclear cell infiltration in NUDE
MICE
BALBlc
‘ i
c&8
.cc%.~
1 NORMAL
MICE
BALBk
NORMAL
MICE
“NSH”
a000808
ot?co 0030
‘ i 100
200
300
400
500
> 600 mg11OOml
Fig. 1. Blood glucose values of nude and two strains of normal mice in single dosage experiments. Each circle, open or filled, represents one animal.
88
some of the normal mice, but not in the nude animals. This prompted us to attempt transfer of spleen cells from diabetic haired mice to non-diabetic animals, both normal and nude. Both types of recipients developed significant hyperglycaemia after transfer of 103-IO7 T-cell enriched spleen cells, most pronounced for recipients of small inocula (103-105), and after low dose irradiation of inocula, probably due to high sensitivity of the suppressor cells [7,8,91. The mononuclear cell infiltration in the islets of Langerhans might have made us conclude that an immune response was involved in the pathogenetic process, but the differential responses in normal and nude mice gave a very clear indication. We have made similar observations when trying to establish virus-induced diabetes in normal and nude mice with the EMCvirus: virus propagates to the same degree in normal and nude mice, but whereas all normal mice develop diabetes, nude mice are unaffected. Guanethidine-induced sympathectomy in normal and athymic rats For nearly 25 years guanethidine has been known to produce destruction of sympathetic neurones in rats following chronic high dose administration. The first report by Jensen-Holm [IO] was followed by several others dealing with the cytotoxic effect of guanethidine on rat sympathetic neurones, and it soon became evident that this was both an organ- and species-specific toxic reaction [11,121. This lead to the use of guanethidine in experimental pharmacology and physiology as a tool in studying various physiological and pathological processes, involving the peripheral sympathetic nervous system. The mechanism behind the neurotoxic effect of guanethidine was still not fully understood, although many efforts were made to determine how guanethidine destroyed sympathetic neurones. Several hypotheses concerning the mechanism have been presented, but within the last 10 years more evidence has accumulated suggesting involvement of the immune system. Today guanethidine-induced sympathectomy offers an attractive model of an autoimmune disorder: in appropriate rat strains the effect of guanethidine is highly reproducible, and - in contrast to some other models of autoimmunity - the general condition of the animals is unaffected. Elucidation of the involved mechanisms will provide a better knowledge of autoimmune disorders, in particular of the peripheral nervous system. The guanethidine-induced neurotoxicity has only been reported in the rat and occurs apparently only in certain strains. The lack of neurotoxic effect of guanethidine in other species is not a result of exclusion of guanethidine from the neurones. In both cats and mice guanethidine accumulates in the ganglia to the same extent as that necessary to cause neuronal destruction in rats.
89
The ultrastructural changes, such as mitochondrial swelling, observed in rat ganglionic neurones following guanethidine treatment, are also observed in cats and mice, but do not in themselves lead to neurotoxicity, since neuronal destruction does not occur in cats or mice. Manning et al Cl31 claimed, that guanethidine sympathectomy represented a drug-induced autoimmune disorder similar in nature to the low dose streptozotocin-induced diabetes. A thymus-dependent immunological effector mechanism in the guanethidine-induced neurotoxicity was suggested following detection of T-cells in the mononuclear cell infiltrate in the ganglia after guanethidine treatment. These observations prompted a series of experiments in our laboratories, using normal rats and athymic rats on the same genetic background (Lewis +/+ and Lewis mu/mu/Mel) as our experimental models in order to establish the role of thymus-dependent and -independent immune responses. Guanethidine treatment in athymic nude rats induced chromatolysis and neuronal destruction in a similar way as previously reported in normal euthymic rats. This unexpectedly demonstrated the same cytotoxic effect of guanethidine in the athymic as in the euthymic rats, while the infiltration of mononuclear cells was significantly smaller in the athymic rats. The neuronal destruction of the sympathetic ganglia induced by guanethidine must therefore involve a thymus-independent immune response. Treatment with immunosuppressive drugs such as cyclophosphamide, cyclosporin A and methylprednisolone concurrently with guanethidine in adult athymic rats gave similar results as observed in normal euthymic animals. The effect of the immunosuppressive drug cyclosporin A was investigated due to its mode of action, primarily suppressing the CD4 T-cells by blocking the interleukin-2 production. The CD4 T-helper cells play an important role in regulation of the thymus-dependent response. Therefore the lack of effect in preventing guanethidine-induced neurotoxicity in both normal euthymic and athymic rats can easily be explained by involvement of T-cell-independent immunological reactions. The mononuclear cell infiltrate in sympathetic ganglia was studied in both euthymic and athymic rats. Four different subpopulations of mononuclear cells were identified by means of monoclonal antibodies: MHC class II cells, pan T-cells, CD8 T-cells/NK-cells and CD4 T-cells/macrophages. The results confirmed that athymic nude rats did not possess T-marker positive cells to any significant extent, neither before nor after guanethidine treatment, whereas the number of T-cells in sympathetic ganglia of euthymic littermates increased substantially following guanethidine treatment [14,X1. A more interesting observation in this study was that the number of CD8 T-cells/NK-cells increased significantly in both athymic nude and euthymic rats following guanethidine administration.
90
TABLE I PERCENTAGES
OF NK-CELLS IN RAT SUPERIOR CERVICAL GANGLION
Treatment
N
% asialo GMl+ cells
% 0X8+ cells
Saline Anti-asialo GM1 Guanethidine Anti-asialo GM1 + guanethidine
5 5 5 5
0 0 7.2 f 1.5’ Ob
0.4 f 0.2 0 6.7 + 1.1’ 0.4 * o.3b
Mean + SEM. $
