Physiological and antigenic changes in Trypanosoma lewisi mediated by epinephrine

Comp gen. Pharmac., I973, 4, 327-332. [Scientechnica (Publishers) Ltd.]

327

PHYSIOLOGICAL AND ANTIGENIC CHANGES IN

TRYPANOSOMA LEWISI MEDIATED BY EPINEPHRINE* GILBERT SANCHEZ Biology Department,New Mexico Institute of Mining and Technology, Socorro, New Mexico 878o x,U.S.A.

(Received 23 January, 1973) ABSTRACT I. The effect of epinephrine treatment of Trypanosoma lewisi-infected rats on the physiology and antigenicity of the parasites was determined. 2. Trypanosomes isolated from epinephrlne-treated rats had accelerated rates of respiration and growth. 3. The antigenic make-up of trypanosomes isolated from epinephrine-treated rats early during the infection was similar to that o f ' adult ' trypanosomes from control rats. 4. Metabolic changes in the host by epinephrine treatment was shown to induce physiological and antigenic changes in the parasites. THE physiological and biochemical transformations of Trypanosoma lewisi during the course of infection have been a subject of interest for m a n y years. Taliaferro (I924, x932 ) reported that the reproduction of 2-. lewisi was inhibited by a serum factor, ablastin, which he considered to be an antibody. Ablastin has also been implicated as being responsible for various physiological and biochemical alterations in the trypanosomes. Moulder (i 948) reported that suspensions of washed, non-dividing trypanosomes had a higher rate of oxidative metabolism than the reproducing parasites. Similar changes in the rates of oxygen uptake have been reported by other investigators (Zwisler and Lysenko, 1954; Lincicome and Hill, I965; Sanchez and Dusanie, i968 ). D'Alesandro and Sherman (x 964) reported that the lactate dehydrogenase activity of reproducing trypanosomes was about three and one-half times greater than in non-reproducing ' adults '. Entner (i968) *This work was supported by a research contract (DADA I7-2o-C-2o2o) from the U.S. Army Medical Research and Development Command, Office of the Surgeon General. The findings in this report are not to be construed as an official Department of the Army position unless so designated by other authorized documents. The author would like to acknowledge the excellent technical assistance of Stephen A. Knight.

reported increased activities of various Krebs cycle enzymes in ' adult ' trypanosomes compared with reproducing forms. There have also been reports of antigenic changes during various phases of T. lewisi infections. Entner and Gonzalez (i966) showed changes in antigens by gel-diffusion techniques during various phases of the infection. This has also been demonstrated by other investigators (Lincicome and Watkins, I965; D'Alesandro, i966 ). With the exception of a few investigators (e.g., Solomon, i969; and Hauschka, I947) , the role of hormones in host-parasite interactions has been largely overlooked. In m a m m a l i a n systems hormones are considered to be the ' p r i m a r y messengers' of m a n y physiological and biochemical responses (Bitensky and Gorman, i972 ). Intracellular responses of target organs are effected by ' s e c o n d a r y messengers' released by hormonal interaction with the cell membrane. The best studied example of this phenomenon is the role of epinephrine in liver glycogenolysis (Sutherland, x972 ). Epinephrine activates adenyl cyclase in the m e m b r a n e of liver cells, resulting in the production of adenosine-3',5'-monophosphate (cAMP), which converts phosphorylase b to phosphorylase a. The activated phosphorylase results in the conversion of glycogen to glucose. In addition to the activation of phosphorylase

328

SANCHEZ

activity, c A M P is k n o w n to r e g u l a t e i m p o r t a n t activities in several different cells a n d to be the essential a c t i o n o f m a n y hormones. T h e s e regulations r a n g e from a c t i v a t i o n o f various enzymes in different cell types to gene i n d u c t i o n in certain b a c t e r i a l species ( S u t h e r l a n d , i972 ). I t is 'possible t h a t the release o f host h o r m o n e s d u r i n g the stress o f t r y p a n o s o m a l infection is p a r t i a l l y responsible for the various physiological changes in the parasites w h i c h take p l a c e d u r i n g the course o f infection. T h e suggestion t h a t h o r m o n e levels do c h a n g e in the host d u r i n g infection is s t r e n g t h e n e d b y the a p p e a r a n c e o f h y p o g l y c a e m i a in the host d u r i n g infections (Sanchez a n d Dusanic, I968 ). H y p o g l y c a e m i a in m a m m a l s usually results in i n c r e a s e d a d r e n a l a c t i v i t y a n d the release o f e p i n e p h r i n e , w h i c h aids in restoring the blood-glucose to n o r m a l levels ( S u t h e r l a n d , i972 ). O t h e r hormones, such as the glueocorticoids a n d glucagon, p l a y similar roles. F u r t h e r , h y p e r t r o p h y of the a d r e n a l g l a n d has been observed as a result o f infections b y the A f r i c a n t r y p a n o s o m e s (Seed, I972 ). D u r i n g 7-. lewisi infections there is convincing i n f o r m a t i o n t h a t t r y p a n o c i d a l a n t i b o d i e s p l a y a n i m p o r t a n t role in the decline phase ( D ' A l e s a n d r o , I966). T h e d a t a t h a t a n a n t i b o d y - - a b l a s t i n - - h a s a role in the inhibition o f r e p r o d u c t i o n a n d changes in physiology, however, are n o t convincing. T h e possibility t h a t these parasites m a y be r e g u l a t e d b y host factors, such as h o r m o n e s a n d h o r m o n e action, has m u c h m o r e a p p e a l . O t h e r organisms have b e e n r e p o r t e d to be subject to such regulation. N o o t h e r o r g a n isms have b e e n r e p o r t e d to be physiologically and biochemically regulated by an antibody. T h e p u r p o s e of this investigation has been to d e m o n s t r a t e t h a t physiological a n d antigenic changes in 77. lewisi c a n be i n d u c e d to occur b y e p i n e p h r i n e stress in the r a t host. M A T E R I A L S AND M E T H O D S PREPARATION OF TRYPANOSOMES

7-. lewisi used in these experiments is the strain that has been maintained in our laboratory in female albino rats (Holtzman Co., Madison, Wise.) by weekly subinoculations of tail-vein blood dilution in o'85 per cent saline.

Comtx gen. Pharmac.

All rats used in the experiment were randomly selected and were of about I8O g. weight. The experimental animals were inoculated with a dose of i × io 6 washed trypanosomes. The parasites were harvested at various days post-inoculation as previously described (Sanchez and Read, I969). EPINEPHRINE TREATMENT Rats were inoculated with 7-. lewisi and separated into 2 groups of IO rats each. One group served as the control and was given daily intraperitoneal injections of i.o ml. physiological saline. The experimental group was given daily intraperitoneal injections of 0. 5 rag. epinephrine (Sigma Chemical Company) suspended in I .o ml. physiological saline. The control and epinephrinetreated infected rats were exsanguinated by cardiac puncture at 4 and 8 days post-inoculation; the trypanosome suspensions were harvested and prepared as previously described (Sanchez and Read, I969). RESPIRATION

The rate of oxygen uptake was determined by standard manometric techniques on a Gilson Differential Respirometer as described by Sanchez and Dusanic (1968). ANTIGENIC ANALYSIS

Antisera against 4- and 8-day post-inoculation control trypanosomes were prepared in New Zealand White rabbits. Three rabbits were used for the post-inoculation trypanosomes of both ages. Each animal was inoculated subcutaneously at 4 sites on the back with 5.o rag. trypanosomc protein giving a total of 2o.o mg. The antigen consisted of T. lewisi homogenized by freezing and thawing repeatedly until no intact organisms could bc found upon microscopic examination. The homogenate was combined in a i : i (v/v) mixture with sterile, complete Frcund's adjuvant. The rabbits wcrc bled by cardiac puncture before immunization and at 3o days after the initial inoculations. The pooled pre-immunization bleeding provided the normal rabbit sera (NRS) used in the control experiments. Antiscra from the 3o-day blecdings were pooled and used in the agglutination reactions. Aliquots of the rabbit antisera against 4- and 8-day trypanosomes were adsorbed with 4- and 8-day post-inoculation trypanosomcs by incubating x.o-ml, suspensions of i × Ios trypanosomes with I .o ml. of the appropriate antisera at 37 ° C. for 15 minutes. The trypanosomes were then removed from the antiscra by ccntrifugation at 3ooo g for 15 minutes at 4 ° C. The adsorption procedure was repeated to increase the removal of homologous antibody. Unadsorbed antitrypanosome sera were diluted accordingly with buffer to maintain a comparable dilution to the adsorbed sera.

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P H Y S I O L O G I C A L A N D A N T I G E N I C C H A N G E S IN T .

The degree of agglutination oftrypanosomes was determined by macro- and microscopic comparison and was rated as follows: 4+ = complete agglutination, 3 + = s t r o n g agglutination, 2 + = moderate agglutination, and x+ = weak agglutination. Controls for spontaneous non-specific agglutinations were included using normal rabbit sera treated in the same manner as the antisera.

329

lewisi

from control rats a n d at 4 days post-inoculation from epinephrine-treated rats is shown in Fig. 2. T h e difference in the rate of

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PARASITAEMIA The growth rate of 7-. lewisi in control and epinephrine-treated rats was determined by preparing daily blood smears from tail-vein ,=4 -I bJ U

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4 .5 6 7 8 9 DAYS POST" INOCULATIOK

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FIG. i.--Parasitaemia of T. lewisi in control rats ( O w - - - O ) and epinephrine-treated rats

( O - - - 0).

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TIME ( M I N .)

blood. The blood smears were stained with Wright's stain and the ratio of trypanosomes to erythrocytes was determined by microscopic examination.

FIG. 2.--Comparative oxygen uptake by 7".

lewisi isolated

from control rats at 4 ( l ~ - - - - O ) and 8 (A A) days post-inoculation and 4-day post-inoculation trypanosomes isolated from epinephrine-treated rats ( O - - - O ) .

RESULTS PARASITAEMIA

Fig.

I illustrates a comparison of the parasitaemia in control versus epinephrinetreated rats infected with 7-. lewisi. T h e parasitaemia is significantly higher throughout the infection in epinephrine-treated rats than in the controls. At a b o u t day 5 postinoculation the concentration of parasites in the epinephrine-treated rats is c o m p a r a b l e to that of an 8-day infection in the control rats. RESPIRATION

T h e rate o f oxygen uptake by trypanosomes isolated at 4 and 8 days post-inoculation

oxygen uptake between 4- a n d 8-day control trypanosomes is typical of the change that has been shown previously (Moulder, I948 ) to occur as the infection progresses. However, the rate of oxygen uptake by 4-day trypanosomes isolated from epinephrinetreated rats is m u c h higher than that observed for 4-day control trypanosomes. F o u r - d a y trypanosomes isolated from epinephrine-treated rats consume oxygen at a rate comparable to that observed in 8-day control trypanosomes; i.e., epinephrine treatm e n t of the host appears to accelerate this aspect o f the parasite's metabolism. Epinephrine tested directly against the

Comp. gen. Pharmac.

SANGHEZ

33 °

trypanosomes of various ages in vitro had no detectable effect. AGGLUTINATION STUDIES

Table I shows the results of the agglutination studies performcd to determine the antigenic relationships between trypanosomes isolated from epinephrine-treated rats at 4 days post-inoculation and control rats at 4 and 8 days post-inoculation. The control experiments for spontaneous non-specific agglutination were all negative. Rabbit anti-4-day trypanosome serum reacted completely ( 4 + ) with 4-day control trypanosomes (isolated from saline-treated rats). A moderate ( 2 + ) reaction was observed with 4-day epinephrine trypanosomes (isolated from epinephrinc-treated rats) and 8-day control trypanosomcs. Adsorption of the anti-4-day serum with 4-day trypanosomes removed all of the agglutinating activity against 4-day control trypanosomes, but had no effect on 4-day epinephrine and 8-day control trypanosomes. Adsorption of rabbit anti- 4 day serum with 8-day control trypanosomcs removed some activity against 4-day control trypanosomcs and a major portion of the agglutinating activity of 4-day epinephrine and 8-day control trypanosomes. Rabbit anti-8-day serum tested against 4and 8-day control trypanosomcs and 4-day epinephrine trypanosomes resulted in a

complete ( 4 + ) reaction in all cases. When the anti-8-day-serum was adsorbed with 4-day trypanosomes most of the activity against 4-day control trypanosomes was removed. However, the effect on 4-day epinephrine trypanosomes and 8-day controls was minimal. Adsorption of the antiserum with 8-day trypanosomes removed some activity against 4-day control trypanosomes and all of the agglutinating activity against 4-day epinephrine and 8-day control trypanosomes. Similar tests conducted on 8-day post-inoculation trypanosomes isolated from epinephrine-treated rats resulted in no measurable difference from 8-day control trypanosomes. DISCUSSION The metabolic changes that occur in 7-. lewisi during the course of infection in the rat have been the subject of much controversy. Moulder (1948) proposed that the alterations that occur in the respiration of the parasite were due to ablastin. Lincicome and Hill (x965) considered this proposal in their work, but added the possibility of host substrates playing a role in the changing activities of the parasite. Sanchez (i973) showed that certain amino-acids, in particular phenylalanine, lysine, and glutamine, interact with glucose and glucosamine uptake by T. lewisi. These observations appear to

Table L--AGGLUTINATION REACTIONS OF 4- AND 8-DAY POST-INOCULATION Trypanosoma lewisi FROM CONTROL AND EPINEPHRINE-TREATED RATS RABBIT

ANTI-T. lewisi SE~

7-. lewisi ADSORBING SERA

T. lewisi AOOLUTINOGEN$

4 c*

4et

8c

4-day P-I 7-. lewisi 8-day P-I T. lewisi

Neg. Neg. Neg.

Neg. Neg. Neg.

Neg. Neg. Neg.

4-day P-I 7-. lewisi 8-day P-I 2-. lewisi

Neg. 3+

4+

2+

2+

4-day P-I T. lewisi 8-day P-I 2-. lewisi

4+ i+ 3+

4+ 3+ i-~

4+ 3+ i~-

NRS

Anti-4-day P-I

Anti-8-day P-I

2+ i+

2+ i+

Abbreviation: NRS, normal rabbit serum; P-I, post-inoculation trypanosomes; Neg., negative. * Trypanosomes from control rats. $ Trypanosomes from epinephrine-treated rats.

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P H Y S I O L O G I C A L A N D A N T I G E N I C C H A N G E S IN

support Lincicome and Hill's proposal on the role of other substrates in T. lewisi's metabolism. Sanchez and Dusanic (i968) showed that when the trypanosomes had the highest respiratory activity the levels of serum ADP and Pi were also high. When the levels of ADP and Pl were low, as in 4- and 12-day post-inoculation sera, the respiratory rates of the post-inoculation trypanosomes of corresponding age were also low. They suggested that general environmental factors (pH, inorganic ions, nucleotides, amino-acids, etc.) may have a regulatory role in the life processes of these parasites. The present study indicates that physiological and antigenic transformations can be induced in T. lewisi by increasing the epinephrine levels of the host. From the data presented it is evident that the parasitaemia of T. lewisi in epinephrine-treated rats at 5 days is comparable to 8-day infections in the controls (Fig. I). The rate of multiplication of the parasites is thus greatly accelerated. Although the growth rate is greater in epinephrine-treated rats, there is a levelling of growth at about days 6 and 7. Therefore, except for differences in magnitude, the growth curve is similar to the growth curve of the controls, indicating that the role of trypanocidal antibodies is unaffected. The data on oxygen uptake by 4- and 8-day post-inoculation controls and trypanosomes isolated at 4 days from epinephrine-treated rats (Fig. 2) indicate increased metabolism in the parasite when the titre of epinephrine in the host is raised. This observation supports the concept of host factor-regulated metabolism. The shift from glycolytic to oxidative metabolism is accelerated by epinephrine treatment of the host; this is a process that has been observed (Moulder, x948; and others) in these parasites to take place gradually between days 4 and 8 during a typical T. lewisi infection. It is of interest that a hypoglycaemic condition is most prevalent between day 4 and day I o during infection (Sanchez and Dusanic, I968 ). Hypoglycaemic conditions, as previously cited, are known to result in increases of

T. lewisi

331

epinephrine. Therefore, products of its action may be involved in the trypanosomal respiratory transformation during a normal course of infection. The agglutination reactions (Table I) strongly support the concept that 4-day trypanosomes isolated from epinephrinetreated rats are similar antigenically as well as physiologically to 8-day post-inoculation control trypanosomes. It appears, therefore, that the overall metabolic regulation of these parasites may also be reflected in the surface antigens, since the agglutination reactions by 4-day epinephrine-treated animals and 8-day controls were identical. An attractive hypothesis for the phenomena of trypanosome metabolic transformation is that increased hormonal activity induced by the stress of trypanosomal infections trigger a series of events which ultimately result in metabolic regulation of the parasites. Since epinephrine was found not to have a direct action on the trypanosomes, it would follow that other factors may be involved. Changes in the blood-levels of nucleotides during trypanosomal stress could very well play an important role in the regulation of the parasite's physiology and biochemistry. Partial evidence in support of this was provided by Sanchez and Dusanic (I968), when they showed that the levels of ADP and Pl correlated with the respiration of T. lewisi. Additional studies by Sanchez, Strickler, and Knight (1973) further support this concept. We have shown that T. lewisi and other species of trypanosomes (i.e., T. brucei and T. rhodesiense) are capable of transporting nucleotides, such as adenosine triphosphate, adenosine diphosphate, adenosine-5'-monophosphate, and adenosine-3'5'-monophosphate. In addition, preincubation of the trypanosomes with some of these nucleotides causes significant changes in the activity of various enzymes of the glycolytic pathway (Sanchez and Knight, i973). In some cases there is an activation of enzymes and in others an inactivation or repression of activity. The metabolic regulation is subsequently reflected in altered rates of protein synthesis. The results of these studies are currently in preparation for publication.

332

SANCHEZ

T h e b i o c h e m i c a l transformations effected on these parasites c o u l d u l t i m a t e l y be reflected in a n t i g e n i c variations. W i l l i a m s o n a n d B r o w n (i964) suggested: ' Since the v a r i a b l e antigens o f t r y p a n o s o m e s a r e p r o teins, a n t i g e n i c v a r i a t i o n should be a b l e to be a n a l y s e d in terms o f i n t r a c e l l u l a r control of p r o t e i n synthesis '. A n a l t e r n a t i v e a n d p e r h a p s similar a p p r o a c h is to s t u d y p r o t e i n synthesis in terms o f control o f the cell's i n t e r m e d i a r y m e t a b o l i s m as a whole. C u r r e n t d o g m a on the physiological a n d b i o c h e m i c a l alterations in 7-. lewisi i n d i c a t e t h a t a n ' a n t i b o d y ', t e r m e d ablastin, is responsible. T h e c o n c e p t o f a n a n t i b o d y h a v i n g m e t a b o l i c r e g u l a t o r y f u n c t i o n is u n i q u e to T. lewisi a n d some closely r e l a t e d species, such as Trypanosoma duttoni. S u c h a regulatory p h e n o m e n o n has n o t been r e p o r t e d to occur in a n y o t h e r physiological system. A b e t t e r a p p r o a c h is suggested b y e x a m i n a t i o n of the roles o f h o r m o n e s (prim a r y messengers) a n d the p r o d u c t s o f t h e i r a c t i o n ( s e c o n d a r y messengers) in the regulation o f p a r a s i t e m e t a b o l i s m . I n conclusion, as a result o f the d a t a p r e s e n t e d in this r e p o r t as well as w o r k c u r r e n t l y u n d e r w a y on the r e g u l a t o r y role o f host h o r m o n e s a n d nucleotides, a c o m p l e t e r e - e x a m i n a t i o n o f the a b l a s t i n p h e n o m e n o n in 7-. lewisi is w a r r a n t e d . T h e role o f hormones a n d o t h e r host factors in h o s t - p a r a s i t e interactions have for the most p a r t been overlooked and require additional attention. REFERENCES BITENSKY, M. W., and GORM.AN, R. E. (I972), 'Chemical mediation of hormone action ', Ann. Rev. Med., 23, 263-284. D'ALESANDRO, P. A. (I966), ' Immunological and biochemical studies ofablastin, the reproduction inhibiting antibody to Trypanosoma lewisi', Ann..IV. Y. Acad. Sci., xa9, 834--85~. D'ALESANDRO, P. A., and SHERMAN,I. W. (I964), 'Changes in lactic dehydrogenase levels of Trypanosoma lewisi associated with appearance of ablastic immunity ', Expl Parasit., x5, 43 ° 438. ENTNER, N. (x968), ' Comparison ofmitochondrial enzymes between young and adult forms of 7". lewisi ', 07. Protozool., XL, 636-638.

ENTNER,N., and GONZALEZ,C. (I966), ' Changes in antigenicity of Trypanosoma lewisi during the course of infections in rats ', 07. ProtozooL, x3, 642-645. HAUSCHKA, T. S. (1947), ' Sex of host as a factor in Chaga's disease ', 07. Parasit., 33, 399-4o4 • LINCICOME, D. R., and HILL, G. C. (I965), ' Oxygen uptake by T. lewisi complex cells. I. Lisolate ', Comp. Biochem. Physiol., x4~ 425-435. LINCICorum, D. R., and WATKINS, R. C. (I965) , 'Antigenic relationships among Trypanosoma lewisi--complex cells ', Parasitology, 55, 365-373 • MOULDER, J. W. (x948), ' Changes in the glucose metabolism of Trypanosoma lewisi during the course of infection in the rat ', 07. infect. Dis., 83, 42-49 • SANCHEZ, G. (1973), ' T h e effect of some amino acids on carbohydrate uptake by Trypanosoma lewisi ', Comp. Biochem. Physiol., in the press. SANCHEZ,G., and DUSANIC,D. G. (x968), ' Respiratory activity of Trypanosoma lewisi during several phases of infection in the rat ', Expl Parasit., 23, 36 I-37 °. SANCHEZ, G., and KNIGHT,S. (I973), ' Metabolic regulation of Tr2panosoma brucei and Tr2panosoma rhodesiense during the course of infection ', in preparation. SANCHEZ, G., and READ, C. P. (I969), ' Carbohydrate transport in Trypanosoma lewisi ', Comp. Biochem. Physiol., 28, 931-937. SANCHEZ, G., STRICKLER, J., and KNIGHT, S. (I973), 'Nucleotide transport by parasitic trypanosomes ', in preparation. SEED, J. R. (I972), personal communication. SOLOMON, G. B. (I969), ' H o s t hormones and parasitic infections ', Int. Rev. trop. Med., 3,

IOI--I58. SUTHE~LAND, E. W. (I972), 'Studies on the mechanisms of hormone action ', Science, N.Y., x77, 4o I-4O7. TALIAFERRO,W. H. (I924), ' A reaction product in infections with Trypanosoma lewisi which inhibits the reproduction of the trypanosomes ', 07. exp. Med., 39, I 7 I - I 9 ° . TALIAFERRO, W. H. (1932), ' Trypanocidal and reproduction-inhibiting antibodies to Trypanosoma lewisi in rats and rabbits ', Am. 07. Hyg., IS, 32-84 . WILLIAMSON,J., and BROWN, K. N. (I964), ' The chemical composition of trypanosomes. III. Antigenic constituents of Brucei group trypanosomes ', Expl Parasit., XL, 44-68. ZWlSLER, J. B., and LYSENKO,K. G. (I954), ' The oxidative metabolism of Trypanosoma lewisi from salicylate treated infected white rats ', 07. Parasit., 40, 531-535 •

Key Word Index: Trypanosoma lewisi, physiology, antigenic studies, hormones, epinephrine.