- Email: [email protected]
Genetic and epigenetic control of levamisole-induced immunostimulation
Int. J. Immunopharmac., Vol. I, pp. 43-48 Pergamon Press Ltd. 1979. Printed in Great Britain.
GENETIC A N D EPIGENETIC CONTROL OF LEVAMISOLE-INDUCED IMMUNOSTIMULATION GERARD RENOUX, MICHELINE RENOUX and JEAN-MAURICE GUILLAUMIN Laboratoire d'Immunologie, Facult~ de M~decine, 37032 Tours C~dex, France (Received 1Sep~mber 1978)
Abstract--Antibody responses to a T-cell dependent antigen, sheep red blood cells, were evaluated in mice of various inbred strains, treated or untreated, with levamisole. These responses appear to be under polygenic control, not associated with the H-2 complex, and modified by a Y-linked component and epigenetic factors revealed by aging. Strain, sex, age and the dose of levamisole all influenced in an interrelated manner the activity of levamisole. Effects varied from inhibited to unchanged or increased antibody-forming cell numbers, without a direct relationship between the genetic regulation of levamisole effectiveness and a genotypic capacity to respond to the antigenic signal. Therefore, a complex relationship between host, antigen and immunopotentiator appears to be responsible for modifying the production of suppressor or helper influences. The present findings may serve as a warning against the uncritical use of levamisole.
The activities of levamisole as an immunopotentiator are well documented (Renoux, 1978). However, conflicting findings exist. Evidence both for nonresponder animals not effected by levamisole and for responders demonstrating a significant increase in their immune response (Renoux, G. & Renoux, M., 1974), indicate a peculiar reactivity of some species, including man, to the administration of levamisole. In other words, immunomodulation could be more a characteristic of the animal strain, than a drugdependent feature. To test this hypothesis, we measured the magnitude of immune responses to sheep red blood cells (SRBC) in young and adult male or female mice of various inbred strains, treated with levamisole. The present report shows that immunopotentiation of an antigen response by levamisole is modulated by the amount of levamisole administered, non H-2 linked genetic factors and by non genetic factors associated with age.
EXPERIMENTAL PROCEDURES
Mice
The Centre de S~lection des Animaux de Laboratoire, CNRS, Orl6ans, France, provided mice of the following strains: A / O r l (H-2 a, Ige); C57BL/6 (H-2 b, Iga2); BALB/c (H-2 a, lga); D B A /2 (H-2 a, IgC); C 3 H / H e (H-2 k, Iga), C57BR/cd (H-2 k, Iga). They were maintained in plastic cages in an air-conditioned room at 24°C; antibiotic-free food pellets and water were available a d libitum. 43
Reagents
Pyrogen-free sterile water was employed to prepare all reagents that were injected with sterile syringes. Levamisole (LMS) was obtained through the courtesy of Laboratoires Lebrun, Paris, France. Doses were expressed in milligrams of the salt per kilogram body weight. Assessment o f the responses to sheep red blood cells (SRBC)
Mice were immunized with intravenous administration of l0 s SRBC (evaluated by photometric measurement at 541 nm) and simultaneously treated by injecting subcutaneously either 2.5 mg/kg or 25 mg/kg of levamisole. Direct (IgM) plaque-forming cells (PFC) were evaluated 48 h later by a modification of the limited hemolysis in agar technique (Renoux, G. & Renoux, M. 1974) and each hemolytic plaque carefully marked. IgG antibody producing cells were evaluated on the same plate. After three washes with cold normal complement buffer, each plate was flooded with 2 ml of a 1 : 10 dilution of goat antiserum to mouse gammaglobulin (Meloy, Springfield, VA). After a 45 min incubation at 37°C, the antiserum was discarded and plates flooded again with 1.5 ml of guinea pig complement diluted 1 : 8 in normal complement buffer, and the dishes were again incubated for 45 min. Newly developed plaques were counted and their frequency per l0 s nucleated spleen cells was calculated. Statistical analysis was performed by Student's t-test for small samples.
44
G. RENOUX et al. RESULTS
Influences o f strain, sex and age on antibody-producing cells in response to sheep red blood cells
For the six inbred strains u n d e r study, young animals were o f 6 - 8 weeks o f age a n d adult mice were 1 8 - 2 0 weeks old. C o m p a r i s o n s m a d e o f adult male mice (Tables 1 a n d 2) show that based o n the ability to r e s p o n d to SRBC, these animals can be classified as low r e s p o n d e r B A L B / c mice; intermediate r e s p o n d e r strain C 5 7 B L / 6 ; a n d high resp o n d e r s A / O r l , D B A / 2 a n d C 3 H / H e strains, for b o t h IgM a n d I g G - P F C . All adult female mice u n d e r study could be classified as intermediate IgG resp o n d e r animals a n d all but the C 5 7 B L / 6 were high Igm responders. A Y-linked c o m p o n e n t seemed o f i m p o r t a n c e in regulating the m a g n i t u d e o f the responses to SRBC. Table 3 summarizes the c o m p a r i s o n between 1 8 - 2 0 week old male a n d female mice. Males had significant increases o f b o t h IgM a n d I g G - P F C n u m b e r s a b o v e females in A / O r l a n d C 3 H / H e s t r a i n s , a n d impaired responses in the low r e s p o n d e r B A L B / c strain. In addition, epigenetic factors associated with age modified a n t i b o d y p r o d u c t i o n (Tables 1 a n d 2). Indeed, adult male A / O r l , D B A / 2 a n d C 3 H / H e mice showed significant ( P < 0 . 0 1 ) 2- to 4-fold increases in n u m b e r s of I g G - P F C a b o v e young male mice o f the same strain. Age increased the IgM Table 1.
A B6 C D2 C3 BR
i m m u n e response o f males in three strains ( A / O r l , D B A / 2 a n d C 3 H / H e ) , impaired it in B A L B / c mice, a n d was without influence on C 5 7 B L / 6 mice. IgGP F C n u m b e r increased with aging only in female o f B A L B / c strain, while age exerted a positive influence o n I g M - P F C responses o f female mice, with the exception o f A / O r l animals. T h e r e was n o clear-cut relationship between the ability to produce I g G - P F C a n d the m a g n i t u d e o f I g M - P F C responses. For example, adult male A / O r l a n d C 3 H / H e mice b o t h p r o d u c e d identical numbers o f I g G - P F C , while SRBC induced 2.6 time more l g M - P F C in C 3 H / H e t h a n in A / O r l mice ( P < 0.01). To sum up, the capacity of mice to respond in terms o f b o t h IgG- a n d I g M - P F C after i m m u n i z a t i o n with SRBC varied f r o m strain to strain without a clear association with H-2 or Ig loci, a n d b o t h sex a n d age influenced the degree o f responsiveness. Influences o f strain, sex and age in modulating levamisole activities on immune responses to sheep red blood cells
In previous experiments, a 2.5 m g / k g dose of LMS increased the n u m b e r of I g M - P F C to SRBC in female o u t b r e d mice, while a 25 m g / k g dose was inactive (Renoux, G. & Renoux, M., 1974). Therefore, these two doses were employed t h r o u g h o u t to study the influences of genetic and epigenetic factors on LMS-induced immunopotentiation.
Levels of specific IgG-PFC in spleens of mice immunized with 108 SRBC and effects of levamisole (LMS) treatments
Mice
Male responses to SRBC + SRBC + SRBC 2.5 LMS 25 LMS
Female responses to SRBC + SRBC + SRBC 2.5 LMS 25 LMS
young adult young adult young adult young adult young adult young adult
21~_4 71+_7 10~L4 10_+_4 2+-+_0 4+-0 27+-+_0 50+-4 17+_7 763-_10
20A:_1 31~3 17~_4 5_+__1 NT 19+-4* 20~k_2 46~_5 22+_6 111+_35"
31±5" 37-L5 19+_1 6+- 1 20±4* 28i3" 40_+_0* 39J~4 43+_18" 112~E_31"
NT
NT
NT
45~L15 39~z_1 1,~Az_1 162:_2 17A_3 35+-2 16±1 28dr_3 27+-1 19+_5 22+_5 26~1
47±13 26_+__10 20&_5 17+_0 27±1" 72_+__2* 28i3" 18i_2 27:L7 75_+_14" 35+-4 21 + 7
48+_27 36±6 14+_2 26+-4 46+_9* 78+_2* 24~_2" 342_3 49~_5" 33~- 3" 16~L2 34+_6
A, A/Orl mice; B6, C57BL/6 mice; C, BALB/c mice; D2, DBA/2 mice; C3, C3H/He mice; BR, C57BR/cd mice. PFC were counted on day 2 post immunization and treatment. Means of 5±S.E.; 2.5 LMS or 25 LMS: mouse treatment with, respectively 2.5 mg/kg or 25 mg/kg of LMS. Young = 6 - 8 week old mice; Adult = 18-20 week old mice. *Significant at P ~< 0.01 to controls. NT = not tested.
Genetic and Epigenetic Control of Levamisole-Induced Immunostimulation Table 2.
Mice
A B6 C D2 C3 BR
young adult young adult young adult young adult young adult young adult
Table 3.
Mouse strain
A/Orl C57BL/6 BALB/c DBA/2 C3H/He
45
Levels of specific IgM-PFC in spleens of mice immunized with l08 SRBC and effects of levamisole (LMS) treatments. For captions, see Table 1 Male responses to SRBC + SRBC + SRBC 2.5 LMS 25 LMS 124+ 17 290±30 140+30 198±28 120±9 84±6 160/_4 476+33 552&81 765±109
169±28 330+35 215±38 145+18 160+19" 133+19" 255+35* 435 ± 6 0 716+70" 1171±400
276±54* 246±28 165±15 170+8 169+15" 167+21" 412±11" 390±~ 568~69 1136+150"
NT
NT
NT
Influences of sex on mouse response to SRBC Male to female responses lgG IgM + O -+ +
+ + -O +
*significantly increased ( + ) or impaired (--) responses of males compared to females at P~<0.01; O, no difference between sexes. As s h o w n in Tables l a n d 2, L M S h a d negligible or inhibiting effects to m o d i f y responses to S R B C in A / O r l or C 5 7 B L / 6 mice. In the other m o u s e strains, the m a g n i t u d e o f L M S - i n d u c e d stimulation varied with dose, age, sex, a n d the type o f a n t i b o d y test. Y o u n g C 3 H / H e mice irrespective o f sex were responsive in terms o f I g G - P F C to the high dose while in adults the lower dose was equally effective. LMS stimulated I g M - P F C p r o d u c t i o n o f young but not adult male D B A / 2 mice, but was ineffective in females irrespective o f age. T h e influence o f strain, sex a n d age o n the ability o f L M S to stimulate did not a p p e a r to be related to the m a g n i t u d e o f the response in u n t r e a t e d animals. Indeed, n o n r e s p o n d e r male B A L B / c mice were i m m u n o e n h a n c e d by LMS, while intermediate-high r e s p o n d e r A / O r l mice were depressed by the drug. A relationship between L M S - i n d u c e d IgG stimu-
Female responses to SRBC + SRBC + SRBC 2.5 LMS 25 LMS 180+62 220±7 58±9 91+18 221 +21 340+18 163+5 376±56 182±21 170±27 42±4 72+5
172+35 153±25" 55±2 86+1 231±21 287+1" 139±36 434+30 304±29* 387+50* 85± 10" 145± 13"
181 ±45 221 +27 23±9* 93+13 406±25* 130±2" 142±20 460+90 365 ± 18" 415±57" 214±24" 174&58"
lation a n d I g M - P F C responses might depend on the dose or on some yet u n k n o w n factors elaborated by LMS to modify the switch from IgM to IgG. Indeed, b o t h IgG a n d IgM responses were unaffected by LMS in female A / O r l mice a n d male or female C 5 7 B L / 6 mice, whereas LMS stimulated female C 5 7 B R / c d mice I g M - P F C responses, in c o n t r a s t with negligible changes in IgG responses. T a b l e 4 summarizes the foregoing findings. For the sake o f clarity, LMS doses were grouped, since mice responded, or not, to one or b o t h doses o f the agent. This table emphasizes the lack o f relationship between L M S - i n d u c e d stimulation a n d H-2 or Ig genetic control, as evidenced by c o m p a r i n g I g G - P F C n u m b e r s in treated female D B A / 2 a n d B A L B / c , or C 3 H / H e a n d C 5 7 B R / c d mice. T h e influences o f age to m o d u l a t e the activities of LMS were also particularly n o t a b l e in young or adult male D B A / 2 or C 3 H / H e mice, as well as the role o f sex in A / O r l , C 3 H / H e a n d D B A / 2 strains. Depressing effects o f LMS were unexpectedly observed in some of the a b o v e experiments. O n e o f the i m p o r t a n t factors in the regulation of a n t i b o d y production is the action o f suppressor T cells (Basten, Miller, Sprent & Cheers, 1974). It has been d e m o n strated with h u m a n peripheral b l o o d lymphocytes that LMS modified the n u m b e r or f u n c t i o n o f suppressor T cells in a critically d o s e - d e p e n d e n t activity ( S a m p s o n & Lui, 1976). O u r data suggest that the effectiveness of L M S on suppressor or helper functions o f T cells might be associated with strain, sex a n d dose influences. Indeed, one or the o t h e r doses o f LMS evoked impaired IgG responses in male A / O r i or D B A / 2 mice, a n d diminished IgM responses in female C 5 7 B L / 6 or B A L B / c .
46
G. RENOUX et al. Table 4.
A summary of strain, sex and age influences on levamisole activities
CH H-2 type allotype
A/Orl
a
Levamisole response Male Female lgM IgG IgM IgG
e
young
_+_
0
O
O
adult
0
--
0
0
0
0
--
0
O
O
O
O
40
+ --
0 0
± 0
~ +
+ +
0 --
+ +
O +
O O
+ +
+ +
+ +
0 O
C57BL/6 young adult DBA/2
young adult BALB/c young adult C3H/He young adult C57BR/cd young adult
b
d
d
k
k
a2
a
a
a
a NT NT
+, stimulation; O , no effect; --, inhibition, in comparison with untreated immunized controls at P ~< 0.01. NT, not tested. Thus, host factors, strain, sex a n d age, control a n d m o d u l a t e the effects o f LMS on the i m m u n e system o f n o r m a l healthy animals. These d a t a emphasize that precise statements a b o u t strain, age a n d sex of animals are needed to u n d e r s t a n d the results o f i m m u n o l o g i c testing, including experimental a n d clinical i m m u n o t h e r a p y . DISCUSSION Regulation o f the i m m u n e response is a complex p h e n o m e n o n u n d e r the control o f m a n y genes (Dorf, D u n h a m , J o h n s o n & Benacerraf, 1974; Renoux, M. & Renoux, G., 1975). Present findings extend the r61e o f genetic controls to i m m u n o m o d u l a t i o n . We have assayed the response o f m u r i n e spleen cells from inbred strains i m m u n i z e d with SRBC a n d treated with levamisole. T h e experimental results presented here indicate a polygenic control o f levamisole i m m u n o e n h a n c e m e n t , without correlation with the H-2 or CH specificities. A d m i n i s t r a t i o n of levamisole evokes a significant increase o f I g G - P F C n u m b e r s in adult female B A L B / c mice a n d not in adult female D B A / 2 , b o t h H-2 d, Cfi strains. Female C 3 H / H e mice exhibit e n h a n c e d I g G - P F C responses a n d not female
C 5 7 B R / c d mice, o f the same H-2 k, Cfi genotype. T r e a t m e n t s with levamisole are inactive, even inhibitory, for A / O r l or C 5 7 B L / 6 mice. However, there is no direct relationship between the effect o f levamisole a n d a genotypic capacity o f mice to respond to the antigenic signal, as exemplified by A / O r l mice, a strain highly responsive to SRBC. T h e action o f levamisole is further m o d u l a t e d by sex factors a n d epigenetic factors associated with age. Sex-associated factors behave differently in that impaired I g G - P F C responses might represent a delayed B-cell m a t u r a t i o n t h r o u g h inhibited T-cell helper functions in levamisole-treated male A / O r l or D B A / 2 mice. In contrast, an increased n u m b e r , or function, o f suppressor T cells might account for I g M - P F C inhibition in female C 5 7 B L / 6 or B A L B / c mice. Age can modify levamisole action as exemplified by the decreased lgG response, in adult male D B A / 2 mice, or by the increase of I g M - P F C n u m b e r s in male C 3 H / H e mice. These findings should not be confused with the well-known effects o f aging on immunity, namely a decreased i m m u n e response in old animals ( M a k i n o d a n , G o o d & Kay, 1977). We assume the changes in the efficacy o f levamisole, induced in male mice by an age difference o f 3
Genetic and Epigenetic Control of Levamisole-Induced Immunostimulation months, are associated with age-dependent enzyme adaptations which have already been related with sex and strain (Adelman, 1975). The in vitro action of levamisole on lymphocyte proliferation is mimicked by its component imidazole (Hadden, J. W., Hadden, E. M., Coffey, CorralesLopez & Sunshine, 1975) but not by dithiocarbamate. However, dithiocarbamate like levamisole is a potent immunopotentiator (Renoux, G., Renoux, M. & Guillaumin, 1977). These findings suggest a complex mechanism for the in v i v o modulation of levamisole activities. Factors which influence the availability of levamisole metabolites might explain genetic modulation of levamisole action. Inducible aryl hydrocarbon hydroxylase (AHH) would be among the enzymatic candidates for further study, since C57BL/6 and DBA/2 vary in AHH induction (Nebert & Gielen, 1972) as they vary in responsiveness to levamisole stimulation. Another aspect involves the effect of levamisole, or its metabolites, on macrophage activities. It is well-documented that macrophages play a crucial r61e in the regulation of immune responses both by removing excess antigen, presenting antigen in adequate form to immunocompetent cells for a given response, and releasing soluble factors stimulating or suppressing lymphocyte proliferation and differentiation. Theses physiologic activities of macrophages are produced through genetically controlled enzymes (Renoux, G. & Renoux, M., 1973). It is well documented also that the effects of levamisole on macrophages are critically dose-dependent (for review, see Renoux, G., 1978) implying a modulation of the enzymatic action involved in the-presentation of different immunogens. Another aspect to be considered is the effect of levamisole on the number or function of suppressor or helper T cells (Sampson & Lui, 1976), and the recruitment of T cells from precommitted precursor cells through the production by a nonthymic cellular system of a thymus-like factor (Renoux, G. & Renoux, M., 1977a,b). This action may be a critical one since our current studies have indicated that levamisole is unable to increase significantly the
47
amount of thymus-like factor in the resistant C57BL/6 strain of mice. A common assumption derived from chemotherapy is that of a linear dose-response relationship. However, this dogma cannot be applied to immunoenhancement with levamisole. Levamisole immunopotentiation is under a complex genetic control involving both the catabolism of the drug and the effects of levamisole on enzymes such as alkaline phosphatases (Van Belle, 1972) and cyclic nucleotides-related enzymes (Hadden et al., 1975) to regulate, depending on the host and the dose, a variety of cellular activities, resulting in pluripotential influences to modify immune responses. Unlike antibodies or cytologic agents, the activity of levamisole is strongly host-dependent and therefore the effects of this agent may appear to be either beneficial or harmful depending on the experimental situation. For example, an increased number of suppressor T cells could favorably modify the lupus-like disease of NZB/W F~ mice (Zulman, Michalski, McCoombs, Greenspan & Talal, 1978) and be deleterious in some cancer patients. Conversely, a levamisole-induced helper effect, as in C3H/He mice, might be of some use in cancer and certain other diseases. Therefore, one should carefully select experimental models according to the desired immunomanipulation. The random and uncritical use of levamisole may produce inconsistent and uninterpretable, if not adverse, results, and unjustifiably dampen the enthusiasm for a rational, scientificallybased immunopharmacology and immunotherapy. A careful appraisal of the immunological defect which allowed the malignancy or the auto-immune disease to develop should be undertaken prior to choosing the appropriate immunopotentiating agent. These agents will be useful in clinical situations only after the development of available tests to measure which type of cell-mediated or humoral immunity a given immunotherapeutic agent is modifying in individual patients. A c k n o w l e d g e m e n t s - - W e are indebted to Dr. John W. Hadden for helpful advice in the preparation of this manuscript.
REFERENCES
ADELMAN,R. C. (1975). Impaired hormonal regulation of enzyme activity during aging. Fedn Proc. Fedn A m . Socs exp. Biol., 34, 179--182. BASTEN, A., MILLER, J. F. A. P., SPRENT,J. & CHEERS, C. (1974). Cell-to-cell interaction in the immune response--X. T-cell-dependent suppression in tolerant mice. J. exp. Med.. 140, 199-217. DORF, M. E., DUNHAM,E. K., JOHNSON,J. P. & BENACERRAF,B. (1974). Genetic control of the immune response : the effect of non-H-2 linked genes on antibody production. J. Immun. 112, 1329--1336. HADDEN,J. W., HADDEN,E. M., COEFEY,R. F., CORRALES-LOPEZ,E. & SUNSHINE,G. (1975). Levamisoleand imidazole : in vitro effects on lymphocyte proliferation and cyclic nucleotide levels. Cell. Immun. 20, 98-103.
48
G. RENOUXet al.
MAKINODAN,T., GOOD, R. A. & KAY, M. M. (1977). Cellular basis, of Immunosenescence. In Immunology andaging (eds. MAKINODAN, T. and YUNIS, E.) pp. 9--22. Plenum Publishing Corp., New York. NEaERT, D. W. & GIELEN, J. E. (1972). Genetic regulation or aryl hydrocarbon hydroxylase induction in the mouse. Fedn Proc. 31, 1315--1325. RENOUX, G. 0978). Modulation of immunity by levamisole. Pharmac. Ther., 2, 397-423. RENOUX, G. & RENOUX, M. (1973). Liens entre les 6v6nements immunologiques et le contr61e g~n6tique de l'activit6 enzymatique des macrophages et des lymphocytes. C. r. hebd. S~anc. Acad. Sci., Paris, 276 D, 861-864. RENOUX, G. & RENOUX, M. (1974). Modulation of immune reactivity by phenylimidothiazole salts in mice immunized by sheep red blood cells. J. Immun., 113, 779-790. RENOUX, G. & RENOUX, M. (1977a). Thymus-like activities of sulphur derivatives on T-cell differentiation. J. exp. Med., 145, 466--471. RENOU×, G. & RENOUX, M. (1977b). Roles of the imidazole or thiol moiety on the immunostimulant action of levamisole. In Control o f Neoplasia by Modulation o f the Immune System (ed. CHmlGOS, M. A.) pp. 67 80. Raven Press, New York. RENOUX, G., RENOUX, M. & GUILLAUMIN, J. M. (1977). Le diethyldithiocarbamate de soude est un stimulant de l'immunit~. C. r. S~anc. Soc. Biol., 171,313-318. RENOUX, M. & RENOUX, G. (1975). Genetic control of immune responsiveness to a soluble Brucella abortus antigen. In Gram-Negative Bacterial Infections and Mode o f Endotoxins Actions (eds. URBASCHEK,B., URBASCHEK,R. and NETER, E.) pp. 155--159. Springer-Verlag, New York. SAMPSON, D. & Lul, A. (1976). The effect of levamisole on cell-mediated immunity and suppressor cell function. Cancer Res., 36, 952--955. VAN BEt LE, H. (1972). Kinetics and inhibition of alkaline phosphatases from canine tissues. Biochem. Biophys. Acta, 289, 158--168. ZULMAN, J., MICHALSKI, J., McCOOMBS, C., GREENSPAN, J. & TALAL, N. (1978). Levamisole maintains cyclophosphamide-induced remission in murine lupus erythematosus. Clin. exp. lmmun., 31,321-327.
GENETIC A N D EPIGENETIC CONTROL OF LEVAMISOLE-INDUCED IMMUNOSTIMULATION GERARD RENOUX, MICHELINE RENOUX and JEAN-MAURICE GUILLAUMIN Laboratoire d'Immunologie, Facult~ de M~decine, 37032 Tours C~dex, France (Received 1Sep~mber 1978)
Abstract--Antibody responses to a T-cell dependent antigen, sheep red blood cells, were evaluated in mice of various inbred strains, treated or untreated, with levamisole. These responses appear to be under polygenic control, not associated with the H-2 complex, and modified by a Y-linked component and epigenetic factors revealed by aging. Strain, sex, age and the dose of levamisole all influenced in an interrelated manner the activity of levamisole. Effects varied from inhibited to unchanged or increased antibody-forming cell numbers, without a direct relationship between the genetic regulation of levamisole effectiveness and a genotypic capacity to respond to the antigenic signal. Therefore, a complex relationship between host, antigen and immunopotentiator appears to be responsible for modifying the production of suppressor or helper influences. The present findings may serve as a warning against the uncritical use of levamisole.
The activities of levamisole as an immunopotentiator are well documented (Renoux, 1978). However, conflicting findings exist. Evidence both for nonresponder animals not effected by levamisole and for responders demonstrating a significant increase in their immune response (Renoux, G. & Renoux, M., 1974), indicate a peculiar reactivity of some species, including man, to the administration of levamisole. In other words, immunomodulation could be more a characteristic of the animal strain, than a drugdependent feature. To test this hypothesis, we measured the magnitude of immune responses to sheep red blood cells (SRBC) in young and adult male or female mice of various inbred strains, treated with levamisole. The present report shows that immunopotentiation of an antigen response by levamisole is modulated by the amount of levamisole administered, non H-2 linked genetic factors and by non genetic factors associated with age.
EXPERIMENTAL PROCEDURES
Mice
The Centre de S~lection des Animaux de Laboratoire, CNRS, Orl6ans, France, provided mice of the following strains: A / O r l (H-2 a, Ige); C57BL/6 (H-2 b, Iga2); BALB/c (H-2 a, lga); D B A /2 (H-2 a, IgC); C 3 H / H e (H-2 k, Iga), C57BR/cd (H-2 k, Iga). They were maintained in plastic cages in an air-conditioned room at 24°C; antibiotic-free food pellets and water were available a d libitum. 43
Reagents
Pyrogen-free sterile water was employed to prepare all reagents that were injected with sterile syringes. Levamisole (LMS) was obtained through the courtesy of Laboratoires Lebrun, Paris, France. Doses were expressed in milligrams of the salt per kilogram body weight. Assessment o f the responses to sheep red blood cells (SRBC)
Mice were immunized with intravenous administration of l0 s SRBC (evaluated by photometric measurement at 541 nm) and simultaneously treated by injecting subcutaneously either 2.5 mg/kg or 25 mg/kg of levamisole. Direct (IgM) plaque-forming cells (PFC) were evaluated 48 h later by a modification of the limited hemolysis in agar technique (Renoux, G. & Renoux, M. 1974) and each hemolytic plaque carefully marked. IgG antibody producing cells were evaluated on the same plate. After three washes with cold normal complement buffer, each plate was flooded with 2 ml of a 1 : 10 dilution of goat antiserum to mouse gammaglobulin (Meloy, Springfield, VA). After a 45 min incubation at 37°C, the antiserum was discarded and plates flooded again with 1.5 ml of guinea pig complement diluted 1 : 8 in normal complement buffer, and the dishes were again incubated for 45 min. Newly developed plaques were counted and their frequency per l0 s nucleated spleen cells was calculated. Statistical analysis was performed by Student's t-test for small samples.
44
G. RENOUX et al. RESULTS
Influences o f strain, sex and age on antibody-producing cells in response to sheep red blood cells
For the six inbred strains u n d e r study, young animals were o f 6 - 8 weeks o f age a n d adult mice were 1 8 - 2 0 weeks old. C o m p a r i s o n s m a d e o f adult male mice (Tables 1 a n d 2) show that based o n the ability to r e s p o n d to SRBC, these animals can be classified as low r e s p o n d e r B A L B / c mice; intermediate r e s p o n d e r strain C 5 7 B L / 6 ; a n d high resp o n d e r s A / O r l , D B A / 2 a n d C 3 H / H e strains, for b o t h IgM a n d I g G - P F C . All adult female mice u n d e r study could be classified as intermediate IgG resp o n d e r animals a n d all but the C 5 7 B L / 6 were high Igm responders. A Y-linked c o m p o n e n t seemed o f i m p o r t a n c e in regulating the m a g n i t u d e o f the responses to SRBC. Table 3 summarizes the c o m p a r i s o n between 1 8 - 2 0 week old male a n d female mice. Males had significant increases o f b o t h IgM a n d I g G - P F C n u m b e r s a b o v e females in A / O r l a n d C 3 H / H e s t r a i n s , a n d impaired responses in the low r e s p o n d e r B A L B / c strain. In addition, epigenetic factors associated with age modified a n t i b o d y p r o d u c t i o n (Tables 1 a n d 2). Indeed, adult male A / O r l , D B A / 2 a n d C 3 H / H e mice showed significant ( P < 0 . 0 1 ) 2- to 4-fold increases in n u m b e r s of I g G - P F C a b o v e young male mice o f the same strain. Age increased the IgM Table 1.
A B6 C D2 C3 BR
i m m u n e response o f males in three strains ( A / O r l , D B A / 2 a n d C 3 H / H e ) , impaired it in B A L B / c mice, a n d was without influence on C 5 7 B L / 6 mice. IgGP F C n u m b e r increased with aging only in female o f B A L B / c strain, while age exerted a positive influence o n I g M - P F C responses o f female mice, with the exception o f A / O r l animals. T h e r e was n o clear-cut relationship between the ability to produce I g G - P F C a n d the m a g n i t u d e o f I g M - P F C responses. For example, adult male A / O r l a n d C 3 H / H e mice b o t h p r o d u c e d identical numbers o f I g G - P F C , while SRBC induced 2.6 time more l g M - P F C in C 3 H / H e t h a n in A / O r l mice ( P < 0.01). To sum up, the capacity of mice to respond in terms o f b o t h IgG- a n d I g M - P F C after i m m u n i z a t i o n with SRBC varied f r o m strain to strain without a clear association with H-2 or Ig loci, a n d b o t h sex a n d age influenced the degree o f responsiveness. Influences o f strain, sex and age in modulating levamisole activities on immune responses to sheep red blood cells
In previous experiments, a 2.5 m g / k g dose of LMS increased the n u m b e r of I g M - P F C to SRBC in female o u t b r e d mice, while a 25 m g / k g dose was inactive (Renoux, G. & Renoux, M., 1974). Therefore, these two doses were employed t h r o u g h o u t to study the influences of genetic and epigenetic factors on LMS-induced immunopotentiation.
Levels of specific IgG-PFC in spleens of mice immunized with 108 SRBC and effects of levamisole (LMS) treatments
Mice
Male responses to SRBC + SRBC + SRBC 2.5 LMS 25 LMS
Female responses to SRBC + SRBC + SRBC 2.5 LMS 25 LMS
young adult young adult young adult young adult young adult young adult
21~_4 71+_7 10~L4 10_+_4 2+-+_0 4+-0 27+-+_0 50+-4 17+_7 763-_10
20A:_1 31~3 17~_4 5_+__1 NT 19+-4* 20~k_2 46~_5 22+_6 111+_35"
31±5" 37-L5 19+_1 6+- 1 20±4* 28i3" 40_+_0* 39J~4 43+_18" 112~E_31"
NT
NT
NT
45~L15 39~z_1 1,~Az_1 162:_2 17A_3 35+-2 16±1 28dr_3 27+-1 19+_5 22+_5 26~1
47±13 26_+__10 20&_5 17+_0 27±1" 72_+__2* 28i3" 18i_2 27:L7 75_+_14" 35+-4 21 + 7
48+_27 36±6 14+_2 26+-4 46+_9* 78+_2* 24~_2" 342_3 49~_5" 33~- 3" 16~L2 34+_6
A, A/Orl mice; B6, C57BL/6 mice; C, BALB/c mice; D2, DBA/2 mice; C3, C3H/He mice; BR, C57BR/cd mice. PFC were counted on day 2 post immunization and treatment. Means of 5±S.E.; 2.5 LMS or 25 LMS: mouse treatment with, respectively 2.5 mg/kg or 25 mg/kg of LMS. Young = 6 - 8 week old mice; Adult = 18-20 week old mice. *Significant at P ~< 0.01 to controls. NT = not tested.
Genetic and Epigenetic Control of Levamisole-Induced Immunostimulation Table 2.
Mice
A B6 C D2 C3 BR
young adult young adult young adult young adult young adult young adult
Table 3.
Mouse strain
A/Orl C57BL/6 BALB/c DBA/2 C3H/He
45
Levels of specific IgM-PFC in spleens of mice immunized with l08 SRBC and effects of levamisole (LMS) treatments. For captions, see Table 1 Male responses to SRBC + SRBC + SRBC 2.5 LMS 25 LMS 124+ 17 290±30 140+30 198±28 120±9 84±6 160/_4 476+33 552&81 765±109
169±28 330+35 215±38 145+18 160+19" 133+19" 255+35* 435 ± 6 0 716+70" 1171±400
276±54* 246±28 165±15 170+8 169+15" 167+21" 412±11" 390±~ 568~69 1136+150"
NT
NT
NT
Influences of sex on mouse response to SRBC Male to female responses lgG IgM + O -+ +
+ + -O +
*significantly increased ( + ) or impaired (--) responses of males compared to females at P~<0.01; O, no difference between sexes. As s h o w n in Tables l a n d 2, L M S h a d negligible or inhibiting effects to m o d i f y responses to S R B C in A / O r l or C 5 7 B L / 6 mice. In the other m o u s e strains, the m a g n i t u d e o f L M S - i n d u c e d stimulation varied with dose, age, sex, a n d the type o f a n t i b o d y test. Y o u n g C 3 H / H e mice irrespective o f sex were responsive in terms o f I g G - P F C to the high dose while in adults the lower dose was equally effective. LMS stimulated I g M - P F C p r o d u c t i o n o f young but not adult male D B A / 2 mice, but was ineffective in females irrespective o f age. T h e influence o f strain, sex a n d age o n the ability o f L M S to stimulate did not a p p e a r to be related to the m a g n i t u d e o f the response in u n t r e a t e d animals. Indeed, n o n r e s p o n d e r male B A L B / c mice were i m m u n o e n h a n c e d by LMS, while intermediate-high r e s p o n d e r A / O r l mice were depressed by the drug. A relationship between L M S - i n d u c e d IgG stimu-
Female responses to SRBC + SRBC + SRBC 2.5 LMS 25 LMS 180+62 220±7 58±9 91+18 221 +21 340+18 163+5 376±56 182±21 170±27 42±4 72+5
172+35 153±25" 55±2 86+1 231±21 287+1" 139±36 434+30 304±29* 387+50* 85± 10" 145± 13"
181 ±45 221 +27 23±9* 93+13 406±25* 130±2" 142±20 460+90 365 ± 18" 415±57" 214±24" 174&58"
lation a n d I g M - P F C responses might depend on the dose or on some yet u n k n o w n factors elaborated by LMS to modify the switch from IgM to IgG. Indeed, b o t h IgG a n d IgM responses were unaffected by LMS in female A / O r l mice a n d male or female C 5 7 B L / 6 mice, whereas LMS stimulated female C 5 7 B R / c d mice I g M - P F C responses, in c o n t r a s t with negligible changes in IgG responses. T a b l e 4 summarizes the foregoing findings. For the sake o f clarity, LMS doses were grouped, since mice responded, or not, to one or b o t h doses o f the agent. This table emphasizes the lack o f relationship between L M S - i n d u c e d stimulation a n d H-2 or Ig genetic control, as evidenced by c o m p a r i n g I g G - P F C n u m b e r s in treated female D B A / 2 a n d B A L B / c , or C 3 H / H e a n d C 5 7 B R / c d mice. T h e influences o f age to m o d u l a t e the activities of LMS were also particularly n o t a b l e in young or adult male D B A / 2 or C 3 H / H e mice, as well as the role o f sex in A / O r l , C 3 H / H e a n d D B A / 2 strains. Depressing effects o f LMS were unexpectedly observed in some of the a b o v e experiments. O n e o f the i m p o r t a n t factors in the regulation of a n t i b o d y production is the action o f suppressor T cells (Basten, Miller, Sprent & Cheers, 1974). It has been d e m o n strated with h u m a n peripheral b l o o d lymphocytes that LMS modified the n u m b e r or f u n c t i o n o f suppressor T cells in a critically d o s e - d e p e n d e n t activity ( S a m p s o n & Lui, 1976). O u r data suggest that the effectiveness of L M S on suppressor or helper functions o f T cells might be associated with strain, sex a n d dose influences. Indeed, one or the o t h e r doses o f LMS evoked impaired IgG responses in male A / O r i or D B A / 2 mice, a n d diminished IgM responses in female C 5 7 B L / 6 or B A L B / c .
46
G. RENOUX et al. Table 4.
A summary of strain, sex and age influences on levamisole activities
CH H-2 type allotype
A/Orl
a
Levamisole response Male Female lgM IgG IgM IgG
e
young
_+_
0
O
O
adult
0
--
0
0
0
0
--
0
O
O
O
O
40
+ --
0 0
± 0
~ +
+ +
0 --
+ +
O +
O O
+ +
+ +
+ +
0 O
C57BL/6 young adult DBA/2
young adult BALB/c young adult C3H/He young adult C57BR/cd young adult
b
d
d
k
k
a2
a
a
a
a NT NT
+, stimulation; O , no effect; --, inhibition, in comparison with untreated immunized controls at P ~< 0.01. NT, not tested. Thus, host factors, strain, sex a n d age, control a n d m o d u l a t e the effects o f LMS on the i m m u n e system o f n o r m a l healthy animals. These d a t a emphasize that precise statements a b o u t strain, age a n d sex of animals are needed to u n d e r s t a n d the results o f i m m u n o l o g i c testing, including experimental a n d clinical i m m u n o t h e r a p y . DISCUSSION Regulation o f the i m m u n e response is a complex p h e n o m e n o n u n d e r the control o f m a n y genes (Dorf, D u n h a m , J o h n s o n & Benacerraf, 1974; Renoux, M. & Renoux, G., 1975). Present findings extend the r61e o f genetic controls to i m m u n o m o d u l a t i o n . We have assayed the response o f m u r i n e spleen cells from inbred strains i m m u n i z e d with SRBC a n d treated with levamisole. T h e experimental results presented here indicate a polygenic control o f levamisole i m m u n o e n h a n c e m e n t , without correlation with the H-2 or CH specificities. A d m i n i s t r a t i o n of levamisole evokes a significant increase o f I g G - P F C n u m b e r s in adult female B A L B / c mice a n d not in adult female D B A / 2 , b o t h H-2 d, Cfi strains. Female C 3 H / H e mice exhibit e n h a n c e d I g G - P F C responses a n d not female
C 5 7 B R / c d mice, o f the same H-2 k, Cfi genotype. T r e a t m e n t s with levamisole are inactive, even inhibitory, for A / O r l or C 5 7 B L / 6 mice. However, there is no direct relationship between the effect o f levamisole a n d a genotypic capacity o f mice to respond to the antigenic signal, as exemplified by A / O r l mice, a strain highly responsive to SRBC. T h e action o f levamisole is further m o d u l a t e d by sex factors a n d epigenetic factors associated with age. Sex-associated factors behave differently in that impaired I g G - P F C responses might represent a delayed B-cell m a t u r a t i o n t h r o u g h inhibited T-cell helper functions in levamisole-treated male A / O r l or D B A / 2 mice. In contrast, an increased n u m b e r , or function, o f suppressor T cells might account for I g M - P F C inhibition in female C 5 7 B L / 6 or B A L B / c mice. Age can modify levamisole action as exemplified by the decreased lgG response, in adult male D B A / 2 mice, or by the increase of I g M - P F C n u m b e r s in male C 3 H / H e mice. These findings should not be confused with the well-known effects o f aging on immunity, namely a decreased i m m u n e response in old animals ( M a k i n o d a n , G o o d & Kay, 1977). We assume the changes in the efficacy o f levamisole, induced in male mice by an age difference o f 3
Genetic and Epigenetic Control of Levamisole-Induced Immunostimulation months, are associated with age-dependent enzyme adaptations which have already been related with sex and strain (Adelman, 1975). The in vitro action of levamisole on lymphocyte proliferation is mimicked by its component imidazole (Hadden, J. W., Hadden, E. M., Coffey, CorralesLopez & Sunshine, 1975) but not by dithiocarbamate. However, dithiocarbamate like levamisole is a potent immunopotentiator (Renoux, G., Renoux, M. & Guillaumin, 1977). These findings suggest a complex mechanism for the in v i v o modulation of levamisole activities. Factors which influence the availability of levamisole metabolites might explain genetic modulation of levamisole action. Inducible aryl hydrocarbon hydroxylase (AHH) would be among the enzymatic candidates for further study, since C57BL/6 and DBA/2 vary in AHH induction (Nebert & Gielen, 1972) as they vary in responsiveness to levamisole stimulation. Another aspect involves the effect of levamisole, or its metabolites, on macrophage activities. It is well-documented that macrophages play a crucial r61e in the regulation of immune responses both by removing excess antigen, presenting antigen in adequate form to immunocompetent cells for a given response, and releasing soluble factors stimulating or suppressing lymphocyte proliferation and differentiation. Theses physiologic activities of macrophages are produced through genetically controlled enzymes (Renoux, G. & Renoux, M., 1973). It is well documented also that the effects of levamisole on macrophages are critically dose-dependent (for review, see Renoux, G., 1978) implying a modulation of the enzymatic action involved in the-presentation of different immunogens. Another aspect to be considered is the effect of levamisole on the number or function of suppressor or helper T cells (Sampson & Lui, 1976), and the recruitment of T cells from precommitted precursor cells through the production by a nonthymic cellular system of a thymus-like factor (Renoux, G. & Renoux, M., 1977a,b). This action may be a critical one since our current studies have indicated that levamisole is unable to increase significantly the
47
amount of thymus-like factor in the resistant C57BL/6 strain of mice. A common assumption derived from chemotherapy is that of a linear dose-response relationship. However, this dogma cannot be applied to immunoenhancement with levamisole. Levamisole immunopotentiation is under a complex genetic control involving both the catabolism of the drug and the effects of levamisole on enzymes such as alkaline phosphatases (Van Belle, 1972) and cyclic nucleotides-related enzymes (Hadden et al., 1975) to regulate, depending on the host and the dose, a variety of cellular activities, resulting in pluripotential influences to modify immune responses. Unlike antibodies or cytologic agents, the activity of levamisole is strongly host-dependent and therefore the effects of this agent may appear to be either beneficial or harmful depending on the experimental situation. For example, an increased number of suppressor T cells could favorably modify the lupus-like disease of NZB/W F~ mice (Zulman, Michalski, McCoombs, Greenspan & Talal, 1978) and be deleterious in some cancer patients. Conversely, a levamisole-induced helper effect, as in C3H/He mice, might be of some use in cancer and certain other diseases. Therefore, one should carefully select experimental models according to the desired immunomanipulation. The random and uncritical use of levamisole may produce inconsistent and uninterpretable, if not adverse, results, and unjustifiably dampen the enthusiasm for a rational, scientificallybased immunopharmacology and immunotherapy. A careful appraisal of the immunological defect which allowed the malignancy or the auto-immune disease to develop should be undertaken prior to choosing the appropriate immunopotentiating agent. These agents will be useful in clinical situations only after the development of available tests to measure which type of cell-mediated or humoral immunity a given immunotherapeutic agent is modifying in individual patients. A c k n o w l e d g e m e n t s - - W e are indebted to Dr. John W. Hadden for helpful advice in the preparation of this manuscript.
REFERENCES
ADELMAN,R. C. (1975). Impaired hormonal regulation of enzyme activity during aging. Fedn Proc. Fedn A m . Socs exp. Biol., 34, 179--182. BASTEN, A., MILLER, J. F. A. P., SPRENT,J. & CHEERS, C. (1974). Cell-to-cell interaction in the immune response--X. T-cell-dependent suppression in tolerant mice. J. exp. Med.. 140, 199-217. DORF, M. E., DUNHAM,E. K., JOHNSON,J. P. & BENACERRAF,B. (1974). Genetic control of the immune response : the effect of non-H-2 linked genes on antibody production. J. Immun. 112, 1329--1336. HADDEN,J. W., HADDEN,E. M., COEFEY,R. F., CORRALES-LOPEZ,E. & SUNSHINE,G. (1975). Levamisoleand imidazole : in vitro effects on lymphocyte proliferation and cyclic nucleotide levels. Cell. Immun. 20, 98-103.
48
G. RENOUXet al.
MAKINODAN,T., GOOD, R. A. & KAY, M. M. (1977). Cellular basis, of Immunosenescence. In Immunology andaging (eds. MAKINODAN, T. and YUNIS, E.) pp. 9--22. Plenum Publishing Corp., New York. NEaERT, D. W. & GIELEN, J. E. (1972). Genetic regulation or aryl hydrocarbon hydroxylase induction in the mouse. Fedn Proc. 31, 1315--1325. RENOUX, G. 0978). Modulation of immunity by levamisole. Pharmac. Ther., 2, 397-423. RENOUX, G. & RENOUX, M. (1973). Liens entre les 6v6nements immunologiques et le contr61e g~n6tique de l'activit6 enzymatique des macrophages et des lymphocytes. C. r. hebd. S~anc. Acad. Sci., Paris, 276 D, 861-864. RENOUX, G. & RENOUX, M. (1974). Modulation of immune reactivity by phenylimidothiazole salts in mice immunized by sheep red blood cells. J. Immun., 113, 779-790. RENOUX, G. & RENOUX, M. (1977a). Thymus-like activities of sulphur derivatives on T-cell differentiation. J. exp. Med., 145, 466--471. RENOU×, G. & RENOUX, M. (1977b). Roles of the imidazole or thiol moiety on the immunostimulant action of levamisole. In Control o f Neoplasia by Modulation o f the Immune System (ed. CHmlGOS, M. A.) pp. 67 80. Raven Press, New York. RENOUX, G., RENOUX, M. & GUILLAUMIN, J. M. (1977). Le diethyldithiocarbamate de soude est un stimulant de l'immunit~. C. r. S~anc. Soc. Biol., 171,313-318. RENOUX, M. & RENOUX, G. (1975). Genetic control of immune responsiveness to a soluble Brucella abortus antigen. In Gram-Negative Bacterial Infections and Mode o f Endotoxins Actions (eds. URBASCHEK,B., URBASCHEK,R. and NETER, E.) pp. 155--159. Springer-Verlag, New York. SAMPSON, D. & Lul, A. (1976). The effect of levamisole on cell-mediated immunity and suppressor cell function. Cancer Res., 36, 952--955. VAN BEt LE, H. (1972). Kinetics and inhibition of alkaline phosphatases from canine tissues. Biochem. Biophys. Acta, 289, 158--168. ZULMAN, J., MICHALSKI, J., McCOOMBS, C., GREENSPAN, J. & TALAL, N. (1978). Levamisole maintains cyclophosphamide-induced remission in murine lupus erythematosus. Clin. exp. lmmun., 31,321-327.