Br. J. exp. Path. (1979) 60, 507
RESISTANCE AND SUSCEPTIBILITY TO THE INDUCTION OF RAT ADJUVANT DISEASE. DIVERGING SUSCEPTIBILITY AND SEVERITY ACHIEVED BY SELECTIVE BREEDING A. R. MAcKENZIE, P. R. SIBLEY AND B. P. W-HITE From the Department of Biochemistry, Glaxo Group Research Ltd (Ware) Received for publication April 11, 1979
Summary.-Rats selected for their ability to develop or resist adjuvant disease were used to establish 2 inbred lines of rat over 20 generations. A resistant line was rapidly established with almost 100% non -responsiveness by the sixth generation. A line show ing 100% susceptibility was also established very rapidly but throughout the course of the breeding programme the severity continued to increase in intensity to a level considerably above that to be seen in strains normally considered to be high responders. At the thirteenth generation and beyond, the susceptible line showed a marked sex difference in the secondary lesions, females being more severely affected than the males. The 2 lines of rat were also tested for their ability to develop experimental allergic encephalomyelitis (EAE) in selected generations. There was no clear correlation between the 2 diseases although those animals developing the most severe adjuvant disease also had the most severe EAE.
RAT ADJUVANT DISEASE, first described
by Stoerk, Bielinski and Budzilovich (1954) and Pearson (1956), has been known for some time to be markedly straindependent. Not only does the strain influence the incidence of the disease (Glenn and Gray, 1965; Swingle, Jaques and Kvam, 1969; Currey, 1970; Rosenthale, 1970; Walz, di Martino and Mishel, 1971; Zidek and Perlik, 1971; Perlik and Zidek, 1973) but also the form of the disease, some strains developing a nodular form and others an oedematous arthritis (Glenn and Gray, 1965; Swingle et al., 1969). The most thorough studies so far have been those of Zidek and Perlik (1971) and Perlik and Zidek (1973) in which 6 inbred strains of rat were compared (Lewis, Wistar, BP, Brown Norway, Long Evans and AVN). Their main conclusions were that a polygenic system was involved which had an additive character giving a "quasi-continuous" incidence to the disease. Genetic experiments between high-
responder (Lewis) and low-responder (AVN) rats suggested that resistance to the disease was dominant. No sex difference was detected in this work and the incidence and severity of the disease appeared to be controlled by the same genetic system. In a further study Perlik and Zidek (1974) compared the incidence of adjuvant disease with experimental allergic encephalomyelitis (EAE) in 3 inbred strains of rat. They found no clear correlation, although of the strains studied only the Lewis rat, which is a very high adjuvant-disease responder, developed EAE. By contrast Long Evans rats, despite developing adjuvant disease in approximately 050% of individuals, showed almost total failure to develop EAE. First filial hybrids of Lewis x AVN rats failed to develop EAE, suggesting that resistance to disease is dominant in this situation also. The earlier findings of Gasser et al. (1973), however, do not agree with this conclusion. A systematic attempt to select for res-
Correspondence: Dr A. R. MacKenzie, Cell Biology, Glaxo Group Research Ltd, Greenford Road, Greenford, Middlesex UB6 OHE.
050 8
A. R. MAcKENZIE, P. R. SIBLEY AND B. P. WHITE
ponsiveness to adjuvant was made by Awouters, Lenaerts and Niemegeers ( 1976). Starting with a moderately responsive inbred Wistar strain they were able to show increased incidence and severity in the F1 generation obtained from highresponding parents. The present work reports selective breeding experiments over 20 generations in which an attempt was made to establish 2 strains of rat, one highly susceptible and the other resistant to adjuvant disease. Subsequently these animals were tested for their ability to develop EAE. MATERIALS AND METHODS
Animals. The original breeding pairs for both lines were selected from a single litter from an outbred colony of albino rat, originally derived from a Wistar/Sprague-Dawley cross in 1969 and subsequently maintained as a closed colony at Glaxo Group Research Ltd (Ware). Although the incidence of adjuvant disease was fairly high (60-70%), the secondary lesions varied from very mild to severe. Selection of breeding stock was made by injecting each rat at 6-10 weeks of age with a standard amount of adjuvant (initially 300 ,ug of Mycobacterium tuberculosis in 0 05 ml liquid paraffin, but reduiced to 100 ,tg M. tuberculosis from the seventh generation onward because of increased response). Responder rats were brother/sister-mated when the disease had largely subsided. Rats were sometimes given short couirses of treatment with 1 mg/kg indomethacin to control the inflammation. Rats were considered to be responders if marked se3ondary lesions were present in the uninjected paw 21 days after injection. The most severely affected were used to produce the next genera-
(Allen & Hanburys-Adjuvant-Responder) for the susceptible strain and AH-AN (Allen & Hanburys-Adjuvant-non-responder) for the resistant strain. The strains were maintained as single lines with expansion at the above generations to provide sufficient experimental animals. These animals were cousins of those taken on to produce the next generation. The detailed studies were made on animals 6-12 weeks of age. Induction of adjuvant disease. Each rat received an intradermal injection of 300 ,ug of heat-killed ]1lycobacterium tuberculosis human strains CD, P and N (Central Veterinary Laboratory, Weybridge) in 0-05 ml of liquid paraffin (B.P.) into the plantar surface of the left hind paw. The course of the disease was assessed by measuring the volume of the 2 hind paws using a mercury plethysmograph connected to an air-pressure transducer. Induction of experimental allergic encephalomyelitis (EAE). EAE was induiced by a modification of the method of Greig, Gibbons and Elliott (1970). A homogenate of 40% w/v guinea-pig brain and spinal cord in 0-5% aqueous phenol was prepared and emulsified with an equal volume of FCA (Difco). Each rat received an intradermal injection of 0 1 ml of this emulsion in the base of the tail. This was followed immediately by an s.c. injection of 0 1 ml of Bordetella pertussis vaccine, containing 4 x 109 organisms/ml (Wellcome Reagents Ltd for thirteenth-generation study, Lister Institute for the eighteenth generation). A scoring system was used, based on 3 aspects of the clinical symptoms of disease, as shown in the Table. The maximum score was 6, i.e. paralysis of both hind limbs, limp tail and fore limb involvement. RESULTS
Adjuvant disease Male and female AH-AR and AH-AN rats, taken from the seventh or eighth, tion. fifteenth and nineteenth Non-responder rats were similarly selected thirteenth, or twentieth generations, were given and brother/sister-mated. Non-responsiveness was defined as a total lack of secondary lesions and minimal primary lesions in the injected paw 21 days after adjuvant. This procedure was followed in the 2 lines until the fourteenth generation was reached, by which time the severity of the arthritis was so great in the responder line that it became very difficult to breed from the tested animals. At this stage brother/sister-mating was continued without previously testing the parents. More detailed studies of the time course and severity of the disease in the 2 strains was made at the seventh or eighth, thirteenth, fifteenth and nineteenth or twentieth generations. The strains are designated AH-AR
TABLE.-Scoring system for clinical assessIntent of rats developing EAE Symptom Score Limp tail 1 Mild ataxia 1 Hind limb weakness 2 Paralysis of 1 hind limb 3 Paralysis of 2 hind limbs 4 1 (f) Any forelimb involvement The score was additive except that (c) excludes (b), (d) excludes (c) and (b), and (e) excludes (d) (c) and (b). Any animal that died was scored as 7 for the remainder of the experiment. (a) (b) (c) (d) (e)
509
RESISTANCE AND SUSCEPTIBILITY TO RAT ADJUVANT DISEASE
adjuvant and the course of the disease followed for at least 28 days. Although detailed studies were not carried out on earlier generations it was clear from the selection process that a high degree of uniformity of response was produced after only 2 generations. In addition male and female rats of the original stock were tested. Marked differences between the responses of AH-AR and AH-AN rats to mycobacterial adjuvant were observed. All generations of A H-AR rats tested showed an incidence of 100% but the severity continued to increase throughout the study. In the case of the eighth generation AH-AN rats, only one animal out of 9 showed swelling in the opposite hind paw. In this generation moderate swelling occurred in the injected paws of all rats at about Day 14, reaching a maximum at Day 18. By the thirteenth generation, however, althought a weak primary swelling occurred in the injected paw there was no exacerbation at Day 14. The time course of swelling in the uninjected hind paw at these generations is shown in Figs 1 and 2. When the arthritic response was examined in the fifteenth generation AHAR rats (Fig. 3), a further increase fin severity was observed which was main300-
300 l
(a)
200T
,100
< + ~~~~1020
D
30
(b)
100-
20 10 Days after adjuvant
30
FIG. 2.-The time course of the development of adjuvant disease in selectively bred rats at the 13th generation. (a)=AH-AR, (b)= AH-AN. The mean percentage increase (+ s.e.m.) of the uninjected paw volume is shown (s.e.m. not shown where < 10%). * = female, 0 = male.
9.c C
,
Days after adjuvant
FIG. 3. The time course of the development of adjuvant disease in 15th generation AHAR rats. The mean percentage increase (± s.e.m.) of the uninjected paw volume is shown (s.e.m. not shown where < 10%). * = female, 0 = male.
(a)
zuu
2100
300 n
a
200a, U 2.
-11 100-
(b)
' 100CL
1-
20 Days after adjuvant
10
FIG. 1. The time course of the development of adjuvant disease in (a) 8th generation AH-AR rats and (b) 7th generation AHAN rats. The mean percentage increase (± s.e.m.) of the uninjected paw volume is shown (s.e.m. not shown where < 10%). * = female, 0 = male.
34
30
10
20
30
Days after adjuvant
-FIG. 4.--The-duvelopment of adjuvant disease in rats from the same stock as the initial breeding pairs used to produce the selected lines. The mean percentage increase (s.e.m.) of the uninjected paw volume (s.e.m. not shown where < IO%). 0 =female, 0 = male.
510 I
A. R. MACKENZIE, P. R. SIBLEY AND B. P. WHITE
tained in the twentieth generation. For comparison the response of rats from the original outbred colony is shown in Fig. 4. Fig. 5 shows the percentage increase at Day 28 in uninjected paw volume against the generation, and demonstrates how the 2 lines rapidly diverged and continued to do so throughout the study. The growth of normal rat paws in 28 days accounts for 500
4004) a 0C
4)
cn a) a1)
Generation
FiG. 5.
Divergence of the arthritic response betwx eeii (lifferent generations of AH-AR and A H-AN rats. The mean percentage increase (+ s.e.m.) of the uninjected paw xolume, 28 (lays after adjuvant is shown where AH10).* (s.e.m. inot AR female, 0 = AH-AR male, *=AHAN female, A = AH-AN male, * = original stock female, j-I=original stock male. shown
up to 200/ groups.
RESISTANCE AND SUSCEPTIBILITY TO THE INDUCTION OF RAT ADJUVANT DISEASE. DIVERGING SUSCEPTIBILITY AND SEVERITY ACHIEVED BY SELECTIVE BREEDING A. R. MAcKENZIE, P. R. SIBLEY AND B. P. W-HITE From the Department of Biochemistry, Glaxo Group Research Ltd (Ware) Received for publication April 11, 1979
Summary.-Rats selected for their ability to develop or resist adjuvant disease were used to establish 2 inbred lines of rat over 20 generations. A resistant line was rapidly established with almost 100% non -responsiveness by the sixth generation. A line show ing 100% susceptibility was also established very rapidly but throughout the course of the breeding programme the severity continued to increase in intensity to a level considerably above that to be seen in strains normally considered to be high responders. At the thirteenth generation and beyond, the susceptible line showed a marked sex difference in the secondary lesions, females being more severely affected than the males. The 2 lines of rat were also tested for their ability to develop experimental allergic encephalomyelitis (EAE) in selected generations. There was no clear correlation between the 2 diseases although those animals developing the most severe adjuvant disease also had the most severe EAE.
RAT ADJUVANT DISEASE, first described
by Stoerk, Bielinski and Budzilovich (1954) and Pearson (1956), has been known for some time to be markedly straindependent. Not only does the strain influence the incidence of the disease (Glenn and Gray, 1965; Swingle, Jaques and Kvam, 1969; Currey, 1970; Rosenthale, 1970; Walz, di Martino and Mishel, 1971; Zidek and Perlik, 1971; Perlik and Zidek, 1973) but also the form of the disease, some strains developing a nodular form and others an oedematous arthritis (Glenn and Gray, 1965; Swingle et al., 1969). The most thorough studies so far have been those of Zidek and Perlik (1971) and Perlik and Zidek (1973) in which 6 inbred strains of rat were compared (Lewis, Wistar, BP, Brown Norway, Long Evans and AVN). Their main conclusions were that a polygenic system was involved which had an additive character giving a "quasi-continuous" incidence to the disease. Genetic experiments between high-
responder (Lewis) and low-responder (AVN) rats suggested that resistance to the disease was dominant. No sex difference was detected in this work and the incidence and severity of the disease appeared to be controlled by the same genetic system. In a further study Perlik and Zidek (1974) compared the incidence of adjuvant disease with experimental allergic encephalomyelitis (EAE) in 3 inbred strains of rat. They found no clear correlation, although of the strains studied only the Lewis rat, which is a very high adjuvant-disease responder, developed EAE. By contrast Long Evans rats, despite developing adjuvant disease in approximately 050% of individuals, showed almost total failure to develop EAE. First filial hybrids of Lewis x AVN rats failed to develop EAE, suggesting that resistance to disease is dominant in this situation also. The earlier findings of Gasser et al. (1973), however, do not agree with this conclusion. A systematic attempt to select for res-
Correspondence: Dr A. R. MacKenzie, Cell Biology, Glaxo Group Research Ltd, Greenford Road, Greenford, Middlesex UB6 OHE.
050 8
A. R. MAcKENZIE, P. R. SIBLEY AND B. P. WHITE
ponsiveness to adjuvant was made by Awouters, Lenaerts and Niemegeers ( 1976). Starting with a moderately responsive inbred Wistar strain they were able to show increased incidence and severity in the F1 generation obtained from highresponding parents. The present work reports selective breeding experiments over 20 generations in which an attempt was made to establish 2 strains of rat, one highly susceptible and the other resistant to adjuvant disease. Subsequently these animals were tested for their ability to develop EAE. MATERIALS AND METHODS
Animals. The original breeding pairs for both lines were selected from a single litter from an outbred colony of albino rat, originally derived from a Wistar/Sprague-Dawley cross in 1969 and subsequently maintained as a closed colony at Glaxo Group Research Ltd (Ware). Although the incidence of adjuvant disease was fairly high (60-70%), the secondary lesions varied from very mild to severe. Selection of breeding stock was made by injecting each rat at 6-10 weeks of age with a standard amount of adjuvant (initially 300 ,ug of Mycobacterium tuberculosis in 0 05 ml liquid paraffin, but reduiced to 100 ,tg M. tuberculosis from the seventh generation onward because of increased response). Responder rats were brother/sister-mated when the disease had largely subsided. Rats were sometimes given short couirses of treatment with 1 mg/kg indomethacin to control the inflammation. Rats were considered to be responders if marked se3ondary lesions were present in the uninjected paw 21 days after injection. The most severely affected were used to produce the next genera-
(Allen & Hanburys-Adjuvant-Responder) for the susceptible strain and AH-AN (Allen & Hanburys-Adjuvant-non-responder) for the resistant strain. The strains were maintained as single lines with expansion at the above generations to provide sufficient experimental animals. These animals were cousins of those taken on to produce the next generation. The detailed studies were made on animals 6-12 weeks of age. Induction of adjuvant disease. Each rat received an intradermal injection of 300 ,ug of heat-killed ]1lycobacterium tuberculosis human strains CD, P and N (Central Veterinary Laboratory, Weybridge) in 0-05 ml of liquid paraffin (B.P.) into the plantar surface of the left hind paw. The course of the disease was assessed by measuring the volume of the 2 hind paws using a mercury plethysmograph connected to an air-pressure transducer. Induction of experimental allergic encephalomyelitis (EAE). EAE was induiced by a modification of the method of Greig, Gibbons and Elliott (1970). A homogenate of 40% w/v guinea-pig brain and spinal cord in 0-5% aqueous phenol was prepared and emulsified with an equal volume of FCA (Difco). Each rat received an intradermal injection of 0 1 ml of this emulsion in the base of the tail. This was followed immediately by an s.c. injection of 0 1 ml of Bordetella pertussis vaccine, containing 4 x 109 organisms/ml (Wellcome Reagents Ltd for thirteenth-generation study, Lister Institute for the eighteenth generation). A scoring system was used, based on 3 aspects of the clinical symptoms of disease, as shown in the Table. The maximum score was 6, i.e. paralysis of both hind limbs, limp tail and fore limb involvement. RESULTS
Adjuvant disease Male and female AH-AR and AH-AN rats, taken from the seventh or eighth, tion. fifteenth and nineteenth Non-responder rats were similarly selected thirteenth, or twentieth generations, were given and brother/sister-mated. Non-responsiveness was defined as a total lack of secondary lesions and minimal primary lesions in the injected paw 21 days after adjuvant. This procedure was followed in the 2 lines until the fourteenth generation was reached, by which time the severity of the arthritis was so great in the responder line that it became very difficult to breed from the tested animals. At this stage brother/sister-mating was continued without previously testing the parents. More detailed studies of the time course and severity of the disease in the 2 strains was made at the seventh or eighth, thirteenth, fifteenth and nineteenth or twentieth generations. The strains are designated AH-AR
TABLE.-Scoring system for clinical assessIntent of rats developing EAE Symptom Score Limp tail 1 Mild ataxia 1 Hind limb weakness 2 Paralysis of 1 hind limb 3 Paralysis of 2 hind limbs 4 1 (f) Any forelimb involvement The score was additive except that (c) excludes (b), (d) excludes (c) and (b), and (e) excludes (d) (c) and (b). Any animal that died was scored as 7 for the remainder of the experiment. (a) (b) (c) (d) (e)
509
RESISTANCE AND SUSCEPTIBILITY TO RAT ADJUVANT DISEASE
adjuvant and the course of the disease followed for at least 28 days. Although detailed studies were not carried out on earlier generations it was clear from the selection process that a high degree of uniformity of response was produced after only 2 generations. In addition male and female rats of the original stock were tested. Marked differences between the responses of AH-AR and AH-AN rats to mycobacterial adjuvant were observed. All generations of A H-AR rats tested showed an incidence of 100% but the severity continued to increase throughout the study. In the case of the eighth generation AH-AN rats, only one animal out of 9 showed swelling in the opposite hind paw. In this generation moderate swelling occurred in the injected paws of all rats at about Day 14, reaching a maximum at Day 18. By the thirteenth generation, however, althought a weak primary swelling occurred in the injected paw there was no exacerbation at Day 14. The time course of swelling in the uninjected hind paw at these generations is shown in Figs 1 and 2. When the arthritic response was examined in the fifteenth generation AHAR rats (Fig. 3), a further increase fin severity was observed which was main300-
300 l
(a)
200T
,100
< + ~~~~1020
D
30
(b)
100-
20 10 Days after adjuvant
30
FIG. 2.-The time course of the development of adjuvant disease in selectively bred rats at the 13th generation. (a)=AH-AR, (b)= AH-AN. The mean percentage increase (+ s.e.m.) of the uninjected paw volume is shown (s.e.m. not shown where < 10%). * = female, 0 = male.
9.c C
,
Days after adjuvant
FIG. 3. The time course of the development of adjuvant disease in 15th generation AHAR rats. The mean percentage increase (± s.e.m.) of the uninjected paw volume is shown (s.e.m. not shown where < 10%). * = female, 0 = male.
(a)
zuu
2100
300 n
a
200a, U 2.
-11 100-
(b)
' 100CL
1-
20 Days after adjuvant
10
FIG. 1. The time course of the development of adjuvant disease in (a) 8th generation AH-AR rats and (b) 7th generation AHAN rats. The mean percentage increase (± s.e.m.) of the uninjected paw volume is shown (s.e.m. not shown where < 10%). * = female, 0 = male.
34
30
10
20
30
Days after adjuvant
-FIG. 4.--The-duvelopment of adjuvant disease in rats from the same stock as the initial breeding pairs used to produce the selected lines. The mean percentage increase (s.e.m.) of the uninjected paw volume (s.e.m. not shown where < IO%). 0 =female, 0 = male.
510 I
A. R. MACKENZIE, P. R. SIBLEY AND B. P. WHITE
tained in the twentieth generation. For comparison the response of rats from the original outbred colony is shown in Fig. 4. Fig. 5 shows the percentage increase at Day 28 in uninjected paw volume against the generation, and demonstrates how the 2 lines rapidly diverged and continued to do so throughout the study. The growth of normal rat paws in 28 days accounts for 500
4004) a 0C
4)
cn a) a1)
Generation
FiG. 5.
Divergence of the arthritic response betwx eeii (lifferent generations of AH-AR and A H-AN rats. The mean percentage increase (+ s.e.m.) of the uninjected paw xolume, 28 (lays after adjuvant is shown where AH10).* (s.e.m. inot AR female, 0 = AH-AR male, *=AHAN female, A = AH-AN male, * = original stock female, j-I=original stock male. shown
up to 200/ groups.
