WITH NOTES ON HEART- RATE TEMPERATURE RELATTONSHIPS Thesis Tor TM Degree 0‘? Db D _‘jj’ji’: I MICHIGAN STATE UNIVERSE“ Alex D Belt: ' 1966 ***“"'“‘ IHEQS 0-169 Date This is to certifg that the thesis entitled Thyroid function in the neonatal rat with notes on heart-rate temperature relationships presented by Alex D. Beltz has been accepted towards fulfillment of the requirements for $2; degree in Meg Y ,r j ‘ ".- >{, .- / [.7 0' I / /< .9 _, (I f” ( , Major professor 5/20/66 (11 d; (I) ABSTRACT THYROID FUNCTION IN THE NEONATAL RAT WITH NOTES ON HEART-RATE TEMPERATURE RELATIONSHIPS by Alex D. Beltz Experiments to determine thyroid function Were con— ducted with neonatal rats ranging in age from birth to twenty— days of age. The direct output method was used for the estimation of thyroid secretion rate (TSR). This method involved output slope determinations, per cent dose uptake, and direct measurement of thyroid iodine. The rats were labeled with 1131 in two ways: (1) Intraperitoneal injection of 15 microcuries into pregnant rats near term, and (2) Single direct, subcutaneous injections of one microcurie per infant. It was found that the rat thyroid glands are function— al at birth, but at a low level which persists until the infant weighs about 20 gramso At this time, the TSR and the thyroid iodine content abruptly increase as a straight— line function of the log body weight° This continues, in this manner, at least until a weight of 244 grams is reached at an age of about sixty dayso The existence of an iodine cycle between the mother and infant rat was confirmed. Several litters of rats Alex D. Beltz were labeled with 1131, by a single subcutaneous dose of l microcurie per rat on the day of birth. At that time, an untagged newborn infant was placed with the litters In vivo counts were taken every forty~eight hours, and after each count the foster litter mate was removed and another untagged infant of the same age substituted. The counts of the substitutes were compared with the counts of the tagged litter mates,and it was found that approximately 9°35% of the average individual count was transferred to the substituted rat every forty—eight hours from his tagged litter mates through the mother's mammary gland. The number in the litter, age of the rats, and body Weight, did not affect the amount of I131 transferred to the foster ratsu Heart-rate temperature relationships were investi— gated by placing one rat at a time in an immobilization apparatus which held the rat immersed to the neck in a water bath; The water was cooled by the addition of icea Intraperitoneal temperatures, ambient temperatures, and an ECG record were taken simultaneously with a Grass Polygraph. It was found that the relation between the neonatal rat heart rate and body temperature can be best described by the Arrhanius equation within the limits of l6O - 300C. The previously published data of six other investigators were replotted by the author, and it was shown that the heart rate of several species of animals (both poikilotherms and homeotherms) conform to the Arrhenius equations It was Alex D . Bel tz also found that the age of the rat was inversely proportional to survival if the heart was stopped by coldo Mass and age did not influence the response of the heart to variations in body temperature. It is hypothesized that many more species of animals may have similar heart—rate temperature relationships and that some of the current concepts of the influence of exercise on heart rate may have to be re—evaluated° THYROID FUNCTION IN THE NEONATAL RAT WITH NOTES ON HEART-RATE TEMPERATURE RELATIONSHIPS Alex D. Beltz A THESIS Submitted to Michigan State University in partial fulfillment of the requirements . for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1966 DEDICATION This thesis is dedicated to my wife Caryl in deepest appreciation for her patience, forbearance, assistance,and firm support throughout the entire course of study at Michigan State University. ii ACKNOWLEDGEMENTS The author wishes to express his appreciation for the privilege of having been able to work in association with Dr. E. P. Reineke. The completion of this thesis, as Well as any possible future success of the author, can be attributed to the friendship and support given him by Dr. Reineke. Gratitude is also expressed to Mrs. Judy Anderson who cheerfully assisted with infant injections and radio— active counting, Miss Barbara Brace, who contributed all the art work, Mrs. Beverly Wandel, who bred and cared for the rats used in these experiments, and Mrs. Linda Allison, who made infant thyroid iodine analyses. Appreciation is also expressed to the Michigan Agricultural Experiment Station for a Special Research Fellowship, as well as laboratory expenses through the course of the experiments. iii TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . REVIEW OF LITERATURE Thyroid Function in the Neonatal Rat Heart—Rate Temperature Relationships MATERIALS AND METHODS . Thyroid Function in the Neonatal Rat Heart—Rate Temperature Relationships DATA . Thyroid Function in the Neonatal Rat Heart-Rate Temperature Relationships DISCUSSION Thyroid Function in the Neonatal Rat Heart—Rate Temperature Relationships SUMMARY AND CONCLUSIONS LITERATURE CITED . iv Page ON 15 15 21 23 23 29 33 33 36 4O 50 Fig LIST OF FIGURES Figure Page 1. Micrograms thyroxine secreted daily plotted against weight . . . . . . . . . . 24 2. Total micrograms of thyroid iodine plotted against weight . . . . . . . . . . . . . . 25 3. Neonatal heart—rate temperature relationship conforms to the Arrhenius equation . . . . 30 v Table Table LIST OF TABLES Page Neonatal thyroid function. A listing of rat weight, times of maximum uptake, release slopes (Ka and K4), thyroid iodine content, micrograms of daily thyroxine secretion rates . . . . . . . . 42 Mother infant iodine cycle average individual per cent dose, number of labeled rats in the litter, foster per cent counts from labeled litter, age of litter and substitute,days counted . . . . . . . . . . . . . . . . . 44 Heart-rate temperature relationships. Animals whose heart—rate response to cold fits the Arrhenius equation, temperature ranges for the application of the Arrhenius plot, Q10, names of original investigators whose data was replotted by the present author . 45 vi Table Table Table Compal Linea: Immob Arrhe LIST OF APPENDICES Page Table l . . . . . . . . . . . . . . . . . . . . . . . 42 Table 2 . . . . . . . . . . . . . . . . . . . . . . . 44 Table 3 . . . . . . . . . . . . . . . . . . . . . . . 45 Comparison of K4 and Ka Values . . . . . . . . . . . . 46 Linear Regression Equations . . . . . . . . . . . . . 47 Immobilization Apparatus . . . . . . . . . . . . . . . 48 Arrhenius Plots of Heart—Rate Versus Temperature . . . 49 Dog Rabbit Rat Adult Intact Rat Adult Swimming Rat Infant Turtle Frog Fish vii its be more a tions to det and wh thyroi thyroj with 1 that ( that 1‘ These neona‘ INTRODUCTION The newborn rat is known to have little control of its body temperature. As it matures, it becomes more and more able to regulate its body temperature against fluctua- tions in the ambient temperature. This study was initiated to determine the level of function of the neonatal thyroid and whether any parallel exists between the development of thyroid function and age. Immobility of the test animals was required for thyroid hormone output determinations, and was achieved with the application of cold. Subsequently, it was observed that complete immobility resulted from the treatment and that the time required for quiescence increased with age. These observations, and others, prompted a study of the neonatal heart rate as it is related to temperature. The literature on cooling as it affects the heart rate, is quite extensive and no attempt will be made to list every reference. The author sincerely hopes that the selected lists of references will not, by omission, mis— represent knOWn Work as it pertains to this study. Under each of the major divisions of this paper there will be tWO sections: one having to do with neonatal thyroid function, and the other with temperature heart rate relation— ships. d2 te C( a re Ii SURVEY OF LITERATURE Thyroid Function in the Neonatal Rat The function of the neonatal rat thyroid has been, as yet, relatively unexplored. Jacobson _t al (1959) reported that the fetal thyroid first concentrates iodine at the fourteenth to fifteenth day of gestation in mice, whose gestational period is nine— teen days. This concentrating ability precedes the formation of follicles and colloid by more than one day. The fetal thyroid gland of the rat shows functional differentiation about the eighteenth day of gestation (Gorbman and Evans, 1943), and one—day-old rats have thyroid gland follicles, colloid, mitotic figures, and colloid droplets in the acinar cells (Phillips and Gordon, 1954). Later (1955) they reported that the thyroid glands of two to four—day—old rats, according to histological evidence after administration of TSH and blood chromatograms, are capable of function. In the fetal rabbit the first uptake of radioactive iodine by the thyroid gland was detected at the fifteenth day of gestation (Waterman and Gorbman, 1956) when the gland itself was made up of epithelial cords and plates inter— spersed with mesenchyme. At seventeen to eighteen days the first follicles had appeared and indications of the formation of da Ho of in ti wi ge 19 si la‘ na la ra' ten TSJ Tht im of thyroxine and its precursors were found. At nineteen days the precursors and thyroxine had reached adult levels. However, it was not until twenty—one to twenty-two days of gestation that the thyroid abruptly began to accumulate increasing amounts of iodide. This level of activity con- tinued until term. Sheep also seem to follow this pattern with greater variation which is probably due to the longer gestation time (Gorbman §t_§l, 1957, Wright and Sinclair, 1959)° Pituitary—thyroid relationships in the rat were studied by Phillips and Gordon (1954). They found that significant quantities of thyrotropin (TSH) did not circu— late in the blood until the eleventh to twelfth post- natal day. They also noted a greater metamorphosis—stimu- lating activity in the pituitaries of seven to nine—day—old rats than in those of newborn rats, and that rats of four— teen to fifteen days of age with open eyes had less pituitary TSH than those of the same age whose eyes had not yet opened. These findings Were associated with an obServed gradual increase in rat thyroid cell heights from one to fifteen days of age, with a sudden spurt when the eyes opened. In their next report (1955), they stated that the pituitaries of one to fifteen—day—old rats store relatively little TSH but synthesize and release it at a fairly constant rate. Hwang and Wells (1959) gave evidence that the hypophysis— thyroid system begins to function by birth. They did this by retarding the fetal thyroid with subcutaneous injections of tre ef: V61 5m 11: oc< in de; ove anc‘ SE1 fet th) thy int 91a of thyroxine (T4) and triiodothyronine (T ), the combined 3 treatment of T and TSH in which the TSH prevents the usual 3 effects of T3, and hypophyseoprivia, whose effects are pre— vented by injected TSH. Hypophyseoprivia was accomplished by subtotal decapitation of near term feti which had been surgically removed from the mother and decapitated with the line of severance extending from approximately the external occipital protruberance dorso—ventrally through the mouth in a transverse plane. Fifteen feti survived this hypophysis deprivation for 40 - 77 hours. After the experiment was over the vasculature of the thyroid glands was examined and found to be intact. Myant (1963) also believes that the servo mechanism of thyroid—pituitary control acts during fetal life. Feldman (1960) presumed that the neonatal rat thyroid was responsive to TSH after noting hyperactive thyroids in newborn pups from thyroidectomized dams. In intact dams, he found that exogenous TSH either did not traverse the placenta or did so to a very slight degree. His reasons for this view Were that the injected TSH was a foreign protein and, therefore, might have been rejected by the animal, and secondly, that TSH has a very brief half— 1ife and with tWO injections per day, might have been eliminated too rapidly to affect the fetal thyroid. In vitro studies with incubated fetal and newborn rat thyroid glands, showed a much greater response to thyrotropin (TSH) than did the thyroid glands of adult rats (Nataf and Chaikoff, 1964). Th the mother (1958): an states tha the placen Th developmen Gorbman (1 usual prin operated i then an in existed, a The passage of thyroxine and triiodothyronine from the mother to the fetus in rabbits was reported by Myant (1958), and in rats by Hamburgh et a1, (1962). Myant (1963) states that the maternal thyroid hormone can readily cross the placenta in the later stages of pregnancy. The function of the thyroid hormones in the fetal development of mammals has yet to be fully explored. Gorbman (1958) states that it must be assumed that if the usual principles of evolutionary adaptation and selection operated in the phylogeny of the vertebrate thyroid system, then an important use or value of the system must have existed, and in fact still exists. Myant (1963) suggests that the thyroid hormone is probably required during fetal life for the normal development of some tissues whOSe earlier development from anlage is independent of it. This, he states, is why thyroid deficiency has a more marked effect in those species in which the young are born in a relatively mature state. Hamburgh gt a1, (1964) worked on the thyroid hormone requirements of the rat for normal maturation. He divided the experimental animals into several groups: (1) Hypothyroid neonatal rats were prepared by giving the mothers goitrogen (phenyl-thio—uracil or PTU) on the fifteenth day of gestation and continuing the treatment for three to four weeks postparturition, (2) Hyperthyroid neonatal rats Were produced by daily injections of 1—thyroxine (T4). The daily dosages which Were given varied with the age of the rat. Newborn rats were given one microgram T4 per day. The dosage was increa: given to r: establishe< neonatal re given with that thyrox cerebral an regulatory It hyperactive gators have Gorbman (19] by the thyn this experin was increased gradually until three micrograms T4 were given to rats twenty—one days and older. This amount was established as the maximum non—lethal dose, (3) Control neonatal rats were injected with normal saline in the amount given with thyroxine to the experimental animals. He found that thyroxine was needed for the normal development of cerebral and cerebellar cortical tissue and the thermo— regulatory mechanism, as well as conditioning and behavior. It is commonly believed that the thyroid gland is hyperactive during fetal and neonatal life, and many investi— gators have reported this (Review by Waterman, 1958). Gorbman (1952) published data which showed high 1131 uptake by the thyroid gland of the bovine fetus near term. For this experiment, he injected Il3l into two near—term pregnant cows and sacrificed them twenty—four hours later. The distribution of I131 was determined in thyroids, blood serum, and various organs of the adult and fetal animals. The blood Sera of the feti contained only slightly more radioactivity than those of the cows, and in both cows and their feti, this was in the form of iodide, with a small Fetal thyroids, on the other 131 amount of thyroxine (T4). hand, had a concentration of I 6 to 7 times greater per unit weight than did those of the mothers. Histologically, the maternal thyroids appeared much less active than those of the feti. These findings were confirmed in mice by Jacobson and Brent (1959). Van Middlesworth (1954), with data from seven newborn human infants which had received microcurie Gorbman Wit} thyroid glaI for I131 de extrapolati obtained a thyroid gla tively higl’ newborn ini lated that uterine ho that the P 4 or 5, th levels no: that thesa tthoid b: rePOrted : Seven-day over rate It was fc of I131 j more thy] maternal hOWeVer-I turnoVeI. haVQ bEE 1 microcurie of 1131 came to the same conclusion as had Gorbman with bovine feti. He made lg vivo counts of the thyroid glands 24 hours after injection. With the corrections for 1131 decay, he obtained a release slope (K), and by extrapolating this release slope line to Zero time, he obtained a total theoretical per cent dose uptake by the thyroid glands. Fisher and Oddie (1964) observed a rela— tively high rate of thyroidal radioiodine clearance in the newborn infant during the "first hour of life,“ and Specu- lated that this may be related to a relatively high intra- uterine hormone secretion rate. Marks gg _1 (1960) found that the PBI values of human infants rose from birth to day 4 or 5, then slowly fell during the first year of life to levels normal for older children and adults. They found that these values are not merely the result of increased thyroid binding globulin (TBG). Slebodzinski (1965) also reported neonatal hyperactivity of the thyroid gland in seven—day—old pigs. He based his conclusions on the turn- over rates of labeled thyroxine in the serum of these animals. It was found that one hour after intraperitoneal injection of I131 into pregnant rats, the fetal thyroid contained more thyroxine and triiodothyronine per mg. than the maternal thyroid gland (Waterman, 1958). Myant (1963), however, states that in the fetal rat the thyroid hormone turnover is much slower than in the adult animal. Most of the studies with neonatal thyroid glands have been based on radioactive iodine uptake rates, protein— bound iodi: turnover r1 the functi‘ of the ass do not tak‘ activity 0 release by factors su immature r This would gland with values (sa; findings 0 by JacobSO Van Middle factors in question, of meaSurez bound iodine (PBI) values, and injected 1131 thyroxine turnover rates. Values which consider only one phase of the functioning thyroid gland are open to question because of the assumptions that have to be made. These assumptions do not take into account sudden increase or decrease in activity of the iodine trapping mechanisms, inhibition of release by various exogenous.and endogenous factors, or other factors such as the apparently reduced ability of some immature rat kidneys to excrete iodine at an adult rate. This would account for the high 1131 uptake by the thyroid gland with high serum iodide as well as high thyroid iodine values (Samel and Caputa, 1965)° This may explain the findings of Gorbman (1952) in the bovine feti, and in mice by Jacobson and Brent (1959). The release slopes (K) of Van MiddleSWOrth (1954), because of insufficient correction factors in determination of the output slope, are open to question, as are all his assumptions based on only one type of measurement. In the experiments reported herein, the function of the neonatal thyroid will be shown by application of the direct output method (worked out by Reineke, and reported by Bhatnagar, 1963). This method includes the combination of a number of measurements for the determination of TSR. I131 They are: (1) per cent dose uptake, (2) release Slope (K4 and K corrected for decay, and (3) total thyroid 4) iodine determination. With a combination of theSe factors an estimate of TSR can be made in terms of the actual amount Of thyroid hormone secreted daily. The times, to tl that metabol did admit t1 neonatal ra‘ other inves Hill, 1947) temperature c atures (25 and at ambi 0f the inf; These data by the inf; adds that l ature and h(iterother early in l Temperature Heart Relationships The term "poikilothermic' has been applied, many times, to the neonatal rat. Gulick (1937), however, reported that metabolic studies did not support this concept but he did admit that, all things taken into consideration, the neonatal rat is "essentially poikilothermic." Several other investigators concurred in this View (Brody, 1943; Hill, 1947). Poczopko (1961), however, reported that the temperatures of even one—day—old rats, at low ambient temper- atures (250— 300C), were higher than those of the environment, and at ambient temperatures of 350C, the body temperatures of the infant rats were I“’at most” equal to the environment. These data show the existence of body temperature control by the infant rat even though it is of a low order. Poczopko adds that human infants also exhibit a variable body temper— ature and he suggests that such neonatal individuals be termed heterothermic to indicate incomplete temperature control early in life. The first investigator to work with temperature and heart rates was Hall (1832). Hamilton _g,_; (1937) measured heart rates as related to rectal temperatures of restrained kittens and adult rats in an ice box with ambient temperature conditions of 350— 42oF. He reported a linear relationship between heart rate and rectal temperature. Fairfield (1948), on the other hand, showed that intraperitoneal temperatures Should be taken in order to more closely approximate "core temperature." She worked with neonatal rats of zero to seventeen da dual suscept rates Were l and that new or lower whi lower than 1 lowered colo His temperat by Fairfield (Hannon, 195 1964). Rich to rectal te were taken 0 old, and sho of SiX to se 10 seventeen days of age and found much variability in indivi— dual susceptibility to cold. She also found that heart rates Were lower in the younger as compared with older infants, and that newborn rats recover from body temperatures of 7°C or lower while adult rats seldom recover from temperatures loWer than 140— 160C. Adult rat heart rates in relation to loWered colonic temperatures were reported by Adolph (1950). His temperature heart—rate curve Was similar to that plotted by Fairfield, as are the curves of many other investigators (Hannon, 1958; Lipp and Folk, 1960; Baker and Horvath, 1964). Richards g3 g1 (1953) reported heart rates in relation to rectal temperatures of newborn mice. The ECG recordings were taken of three litter mates from birth to twenty—days- old, and showed a marked increase of heart rate at the age of six to seven days as the animals developed hair. Bidrek and Johansson (1955) Worked with dogs, hedgehogs, South African frogs, and fish using rectal temperatures for the homeotherms, cloacal temperatures for the frogs, and water temperatures for the fish. Three of the curves plotted (dogs, frogs, and fish) Were similar in shape to those of Adolph and the other Workers cited earlier. Spontaneous activities of isolated auricles of hedgehogs, hamsters, and rats were reported by Hirvonen (1955). He stated that the auricles of hibernating mammals stopped beating at 1.50— 6OC while the nonhibernator heart beat ceased at 160 — 1800. The rate of the hamster auricle was reported as a linear logarithmic function of the temperature. The rabbit and rat auricle did others (Lyma rat auricle even the ban ventional A] step-wise d‘ colonic term that if‘the temperature effect of c of the rabh using recta the result: (1958) fou: rate of th were taken Showed a h to a recte more abrur Bullard (; but Point heart rat) cOlonic i on isolat “‘9 by 1 gm 11 auricle did not fit the same pattern. This was taken by others (Lyman and Blinks, 1959) to mean that the rabbit and rat auricle did not follow the Arrhenius equation. However, even the hamster data were not plotted according to the con— ventional Arrhenius Method. Nardone (1955b) reported a step—wise decrease in heart rate with a decrease in the colonic temperatures of the opossum. He stated, in addition, that if the mean heart rates are plotted against mean colonic temperatures a straight-line relationship is obtained. The effect of cold on the isolated as well as the intact heart of the rabbit was examined by Covino and Beavers (1958) using rectal temperatures for the intact rabbit. Both of the resulting curves resembled those of Adolph. Hannon (1958) found that the effect of anesthesia on the heart rate of the rat was quite significant. Rectal temperatures Were taken and it was found that rats with less anesthesia showed a higher heart rate at all temperatures ranging dOWn to a rectal temperature of 1800 where the heart rate dropped more abruptly than in the deeply anesthesized subjects. Bullard (1959) agreed with the results of Hamilton et al, but pointed out that a true linear relationship betWeen heart rate and temperature could not be established since colonic instead of heart temperatures Were measured. Studies on iSOlated hearts of hibernators and nonhibernators were done by Lyman and Blinks (1959). They stated that of the seven species studied (Sciurus carolinensis—grey squirrel, Sigmodon hispidus—cotton rat, Aplodontia rufa—mountain beaver, thirteen-lir and W over the en‘ not present relationshi did not agr relationshi plotted age with hamsts which were that hiber killed non relationst Baker and With Adel} heart rati 12 Mescoricetus auratus—golden hamster, Citellus tridecimlineatus— thirteen—lined ground squirrel, Tamius striatus—chipmunk, and Marmota monax—woodchuck), none fit the Arrhenius equation over the entire temperature range. However, actual data were not presented. The graph showing the heart rate temperature relationships for hamsters in the above group of animals did not agree with that of Hirvonen (1955) in that no linear relationship was found when the logarithm of heart beat was plotted against temperature. Lipp and Folk (1960) Worked with hamsters, white rats, and thirteen—lined ground squirrels which were restrained and put into a cold box. They found that hibernators recovered from body temperatures which killed nonhibernators. Heart rate and rectal temperature relationships in swimming adult rats Were determined by Baker and Horvath (1964) who reported curves which agreed with Adolph. Dahlen (1964) found that with cold, the cat heart rate decreased in a linear fashion. In these experi- ments the body temperature was taken from a thermistor placed in the eSOphagus at the level of the heart. The linearity of this decrease was measured from 350 to 200C, and the heart beat was reduced from one—hundred and seventy—five per minute to thirty—eight per minute. The heart rates of iSQlated bat hearts was found to be a function of temper— ature in both summer and winter animals, and, in general, agreed with thOSe of isolated hearts of several species of hibernators as reported by Michael and Menaker (1963). The bat heart rate temperature curves fit the Arrhenius equation — over the ent 45°C): and _t _1 (1960} in the tort metically at plotted for (1960) conc as well as and reheati time in mir. jected to c' digested wj and culture elaborated other hear a 910 of 3 Th by Wekstei first thre 13 over the entire temperature range of the study (-l.OOC — —250C), and can be described by a Q10 of 3.5. Hutton at a1 (1960) reported a temperature heart rate relationship in the turtle (Pseudemys scripta) which, when plotted arith- metically against temperature, is similar to that previously plotted for rats and several animals. Ldfgren et a1 (1960) concerned themselves with times to cardiac arrest as well as heart rates under conditions of extreme cold and reheating. Heart rates were stated in relation to the time in minutes the animals (rat, frog, and toad) Were sub— jected to different ambient temperatures. Rat hearts were digested with trypsin and single beating cells were isolated and cultured by Harary and Farley (1960). These cells elaborated protoplasmic extensions which, when they contacted other heart cells, synchronized the beat. These cells had a Q of about three. 10 The control of the rat neonatal heart beat is said by Wekstein (1965) to be divided into two periods: (1) the first three days of life when the heart rate is independent of sympathetic nervous system activity, and (2) days six to sixteen “when the observed increase in heart rate appears to be a function of the level of activity of the sympathetic nervous system.“ This is in agreement with the conclusions reached by Alimukhamedov (1962) about the phases of thermo- regulation development in young rodents. In this Work he states that during the first days of post natal development thermoregulation is accomplished by muscle action with later development of chemical thermoregulation. Pr oc the heart it state that primarily o of ion tran stated that linked to c and time, a depolariza‘ fibers. V cold appar diffusion Says that exposure 1 time lag. not aboli 0f potent amounted increase 14 Production of the impulse and its conduction over the heart is not well understood. Hollander and Webb (1955) state that the duration of the action potential depends primarily on the rate of repolarization and thus on the rate of ion transport through the membrane. Hecht (1965) has stated that the self—excitation of pacemaker tissues is linked to changes in membrane currents depending on voltage and time, and that threshold, resting potential, and diastolic depolarization interact in controlling automaticity of cardiac fibers. Van Harreveld and Christensen (1957) report that cold apparently reduces nerve metabolism so that the ionic diffusion gradient cannot be maintained. Deleze (1960) says that on rapid heating of Purkinje fibers after cold exposure there is a rapid rise in their potential with no time lag. He also reports that temperature sensitivity is not abolished by sodium-free solutions, and that the drop of potential at a body temperature below 200C cannot be accounted for by extracellular potassium accumulation or an increase of inward sodium current. Th were bred Al ml Tubercx needle in: the muscu the outer a small b technique through t ., MATERIALS AND METHODS Thyroid Function of the Neonatal Rat The rats used Were all of CarWOrth CFN strain and were bred in this laboratory. A11 injections of infant rats were given with a one ml Tuberculin syringe through a 1/2 inch twenty—seven gauge needle inserted at the base of the tail and pushed through the musculature of the hind leg, emerging subcutaneously on the outer thigh. Injections were of 0.1 m1 volume and raised a small bleb on the outer surface of the skin. With this technique there was very little loss of injected material through the puncture wound. In these experiments I131 was administered in two ways: subcutaneous injection of the infants with one micro— curie carrier—free 1131, and (2) intraperitoneal injection of fifteen microcuries of carrier—free 1131 into pregnant rats one or two days before parturition. Since 1131 uptake by the thyroid gland took place in the infants at an extremely slow rate (in relation to adult uptake rates), the method No. 2 was used to determine the thyroid 1131 turnover rates in young rats of one to ten days of age. On the day of birth, the young of tagged mothers were trans— ferred to untagged lactating rats. 15 _g l forty-eifi cold. A inch NaI analyzer sealer-a Model 16 counting on the s neath a1 25 mm d: four in. 35 cm. inch NaI crystal, was attached to a transistorized scaler- analyzer (Nuclear-Chicago, Model 8725). Connected to the sealer—analyzer was a count rate meter (Nuclear—Chicago, Model 1620 CS) which was used to determine the optimal counting geometry before recording the count for each rat on the scaler. The scintillation probe was mounted be— neath an aperature 19 mm in diameter for infant rats, and 25 mm diameter for thirty- and sixty—day—old rats, located four inches from the end of a lead plate 2.5 cm x 38 cm x 35 cm. The in vivo counts Were td +++ + A 0‘ + 2 H+ 2 Ce = H2AsO3 + H20 (catalysis 2 Ce H2 5 4 (yellow) (colorless) Iodine catalyzes the reduction of cerium causing decolori- zation of the solution. If time and temperature are constant, the extent of declolorization measures the quantity of iodine present. The 100 mm tube and its cor overnight. were incint an electri up to 1100 ml of 2 N tilled wat tube were at 2000 R] For infan' 4ml of t the dupli water. 1 SOlution 18 The gland from each rat was placed in a pyrex 25 x 100 mm tube with one m1 of 4 N sodium carbonate. The tube and its contents Were dried in an oven at 900 to 95°C overnight. The next day the tube and its dried contents were incinerated for two to three hours at 6250 to 6500C in an electric muffle furnace equipped with a pyrometer ranging up to llOOOC. After the tubes Were removed and cooled, tw0 ml of 2 N HCl, two m1 of 7 N H SO 2 4’ tilled water were added to the ash. The contents of the and 6 m1 of glass dis— tube were mixed well and centrifuged for twenty minutes at 2000 RPM (International Centrifuge, Model UV, Head 240). For infant rat thyroids, tw0 duplicates were made with 2 and 4 m1 of the supernatent Solution from each tube. Each of the duplicates was made up to 5 ml with glass distilled water. To each tube was added 0.5 m1 of arsenious acid solution with a blow—out pipette. The tube was then placed in a warming bath. The temperature of the water bath and incubation time varied with the age of the rats from which the thyroids were taken. For example, for one—day—old rats 20 minutes at 500C and for ten-day—old rats 15 minutes at 270C Were used. After a fifteen minute warming period, 0.5 m1 of Ceric ammonium sulfate was added. At the end of the given time period, 0.5 ml of a 0.1% Solution of Brucine was added to each tube to stop the reaction. The tubes were removed from the water bath and read directly on a Coleman Universal Spectrophotometer (Model 11, with a PC—4 blue filter) at a wave length of 480 millimicrons. The curves pre been ploti T thyroxine plied by iS the mi each day. Worked 01 Multiply, Wlll giv Parts Potenci 19 instrument was set for 100% transmittance with distilled water. In each age group, two reagent blanks were prepared as described above excepting the thyroid tissue. The values for the reagent blanks Were subtracted from the readings of the standards and unknOWns. The concentrations of iodine in each of the tubes were read directly from the standard iodide curves prepared under identical conditions and which had been plotted on arithmetic graph paper. To calculate the daily thyroid secretion rate in thyroxine (T4) equivalents, the output rate (K4) was multi- plied by the total iodine content of the gland. The result is the micrograms of hormonal iodine secreted by the rat each day. Conversion of this value involved calculations worked out by Reineke, and cited by Bhatnagar (1963). Multiplying the micrograms of daily iodine secretion by 1.529 will give thyroxine equivalents. HOWever, the gland also Secretes triiodothyronine (T3) which has been determined in this laboratory as being 4.85 times as potent per unit of iodine as T in suppressing TSH release in the rat. The rat 4 thyroid contains 6 parts of T4 to 1 part T3 iodine (Pitt- Rivers and Rall, 1961). Then the relative potency of the released compounds are calculated in relation to the amounts secreted as follows: T4 T3 Totals Parts 6.00 1.00 7.00 Potency 1.00 4.85 6.00 4.85 10.85 To adjust potencies andTlvi 3 The values 1.529 x 1 This will activity. T iodinated with I131 thyroid i T3), and same prop 20 To adjust the T4 equivalents in proportion to the relative potencies of the tWO compounds assumed to be released (T4 10.85 7 The values then are combined in the equation below: = 1.55. and T3), the factor Which is used is 1.529 x 1.55 x Thyroid iodine content = ug daily of secreted T4 This will express the total hormonal effect in terms of T4 activity. The aSSumptions of this method are: (1) that the iodinated compounds in the thyroid are uniformly labeled with I131, and (2) that all the iodine released from the thyroid is in the form of thyroxine (T4) or triiodothyronine T3), and (3) that these two compounds are released in the same proportions in which they occur in the thyroid. Experiments were conducted to determine the iodine cycle between mothers and infants. It is knOWn that micturition of the very young infant is controlled by the mother who, at regular intervals, voids the young by stimu- lation of the perineal region with her tongue (Capek and Jelinek, 1956). The mother, while stimulating the perineum of her young also ingests at least some of the urine (Samel and Caputa, 1965). In this way, iodine is cycled from the infant to the mother and partitioned between the mother's thyroid and mammary glands. Thence, by way of the milk, a portion of the iodine is given back to the infant. Young rats nurse about every hour. Therefore, every hour, the iodine coming into the infant would slightly increase the Specific activity of the thyroid iodine, and concomittantly, reduce th would the However, so that a method wo experimen E subcutane injectior to each i young, we describe: and anotl infants v magnitude 21 reduce the output slope (K4). The direct output method would then underestimate the daily thyroid secretion rate. HOWever, the infants nurse throughout the experimental period so that any estimate of the thyroid secretion rate by this method Would vary by a fairly constant amount from one experiment to another. Forty—five rats in several litters Were injected subcutaneously with one microcurie of 1131. On the day of injection, a nonradioactive infant of the same age was added to each tagged litter. The litters, with the substituted young, were then counted every other day as heretofore described. After each count, the Substituted rat was killed and another of the same age put in its place. Fifteen infants Were examined in this way to determine the approximate magnitude of the iodide cycle between the infant and mother. Heart—Rate Tempeature Relationships Seventy—two neonatal rats were used in this study, ranging in age from one-day to twenty—days. The subjects were placed in an apparatus devised by the author (see diagram, Appendix:p.48) and gradually cooled in a water bath until the heart stopped. The data Were recorded simulateously on three channels of a Grass Model Five Polygraph (Grass Medical Instrument Company, Quincy, Mass.). Two of the recording needles traced the ambient and intraperitoneal temperatures and the third recorded the ECG. All three channels had Grass Model Five Driver Amplifiers. Temperatures were take probe (No Chicago, with Poin gauge). temperatu experimen intraperi a Tele-th Model 840 these, in Preamplif made with the head, to the di The above which lec‘ The EEG p 0f SUffic Were taken by two thermistors: a general purpose flexible probe (No. 8430 Cole—Parmer Instrument and Equipment Company, Chicago, Illinois), and a Semi—solid Insertion Thermistor with Pointed Tip (No. 8481 Cole—Parmer Instrument Co. 20 gauge). The general purpose thermistor took the ambient temperature being placed next to, but not touching, the experimental animal. The pointed—tip thermistor took the intraperitoneal readings. Each thermistor was attached to a Tele-thermometer (Yellow Springs Instrument Co., Inc. Model 8400 Cole-Parmer Instrument and Equipment Co.) and these, in turn, were attached to Model 5 P 1 Low Level Preamplifiers of the Grass Polygraph. ECG recordings were made with a subcutaneous needle electrode at the back of the head, and a second lead connected with an alligator clip to the distal end of the intraperitoneal thermistor probe. The abovementioned leads were connected to an ECG cable which led to a Model 5 P 5 EEG Preamplifier in the Polygraph. The EEG Preamplifier Was found necessary to produce tracings of sufficient amplitude to be counted. T and it wa iodine an equations secretior iodine ar Secreted shown in Secretio There is order, a reaches abrupt a conclqu COEffiCj is not efficie] lEVel. DATA Thyroid Function<1fthe Neonatal Rat The raw data were plotted Several different ways and it was found that weight is related more clearly to thyroid iodine and daily thyroid secretion rate than to age. The equations for the lines on both the graph for daily thyroid secretion rate with weight, and the graph for total thyroid iodine and weight are shown in Figures 1 and 2, respectively. Thyroxine secretion, in terms of micrograms T4 Secreted per day Was plotted against body Weight in gm, as Shown in Figure 1. Slope I represents daily thyroxine secretion rate (TSR) for infant rats weighing up to 22 gm. There is a TSR for these rats although it is of a very low order, and it continues at the same level until the infant reaches a weight of 22 grams. At this time there is an abrupt and continuous increase in the TSR extending to the conclusion of the experiment. It will be noted that the coefficient of correlation (r) for slope I is 0.479 which is not significant at the 5% level. Slope II has a co— efficient of correlation (r) of 0.9586 which is at the 1% level. Figure 2 shows total micrograms of thyroid iodine in compariSQn With grams body Weight. Slope I represents the total thyroid iodine of neonatal rats Weighing up to 23 0 75.3.. ,0 5 0 . 0 Z a l W >|: 2.5 r=-O.4794 / g SLOPE 11 / Y=2.2332 LOG x-2.9oe . 52.0 I/ z“ 9 F. LL] 0: t3 LO 0) F3m? 0.5 0.3 I OI /' I | I I I0 20 4o 60 so ICC 200 300 BODY WEIGHT, GMS Figure l. Micrograms thyroxine secreted daily plotted against Weight. 0. 0. O. O. 9 7 5 3 XXMZEO. n:OK>I._. JdFOL. 0. 0 allllLl Figure ILO SLOPE I Y= O.l64O + O.l348 LOG X r= 0.0983 9.0 SLOPE II Y= 6.5054 LOG x— 8.4I86 r=O.7624 7.0 3.0 TOTAL THYROID IODINE, 7 (n O L_____L___J___;_____J_____J___4___L_________L_______J | ID 20 4O 60 80 ICC 200 BODY WEIGHT, GMS Total micrograms of thyroid iodine plotted Figure 2. against weight. 22 gm. It functional the level gm, there contained ' Slope II, ' line funct The coeffi which is n is 0.7624 Th Figuresl a Appendix, was applie The TSR, p at the 10% Weight rel iodine con at the 0.1 related, w 26 22 gm. It must be pointed out that, although there is a functional thyroid gland in the very young rats of Group I, the level of function is quite low. When the rat reaches 22 gm, there is a marked increase in the amount of iodine contained by the thyroids of Groups II, III, and IV. Slope II, which shows this increase, is again a straight— line function throughout the remaining time of the experiment. The coefficient of correlation (r) for slope I is 0.0983 which is not significant at the 5% level,while r for slope II is 0.7624 which is significant at the 1% level. The equation for the lines of regression plotted in Figuresl and 2 were determined with formulae shown in the Appendix, p. 47. The non parametric Corner Test of Association was applied to the data to find if other trends existed. The TSR, plotted against age was found to be non—significant at the 10% level, as were age and weight, and both age and Weight relationships to slope (K4). However, the thyroid iodine content and age values were significantly correlated at the 0.1% level. Since age and TSR were not significantly related, weight relationships for both TSR and iodine content were used. Table l was arranged in an ascending order of rat Weight, and arbitrarily divided into four groups for com- parative purposes. Group I represents data from rats ' k Weighing up to 20 gm, with a mean Weight of 14.08 gm i 0.039 gm. Rats in Group II weighed 20-32 gms With a mean of 27.41 + 0.414 gm. Rats in Group III weighed 60—70 gm with *Standard error of the mean. an wei toi pu1 zei H be ai hi ca t< Vi 27 a mean value of 63.98 i 1.84 gm, and rats in Group IV Weighed 152.4 — 244 gm with a mean of 186.95 i 12.36 gm. The U values of Table 1 represent the theoretical total maximum Il3l uptake and are taken from the true out- put slope line (on the per cent dOSe graph) extrapolated to zero time. There are no U values for Group I since all rats in this group gained their radioactive iodine from a preparturition injection of their mothers. After birth, these animals were transferred to a nonradioactive lactating rat. Group II values are from .62 —3.0%, having a mean value of 1-544 i 0.72%, Group III U values range between 2.2 and 3.3%with a mean of 3.044_:0.l72%. Group IV values vary between6.42nui9~8%with a mean of7.43 i 0.39%. The ”time to maximum uptake" is the length of time, after injection, at which the thyroid count reaches its highest point. Group I again can have no values in this category for the following reasons: (1) fetal proximity . . 131 . . . to the needle used for the intraperitoneal I injection . . . 131 varied greatly, (2) the unknOWn partitioning of the I from the blood stream between the developing mammary gland, thyroid, and feti. Group II times for maximal uptake varied between four and six days, with a mean of 4.25 i 0.11 days. All of the animals in Group III and IV had maximal uptake in tWo days. requir lation). Therefore, Ki values Which are similar to, but not identic the dai iodine large 1 tion 1 of the the fC about mates. rat i1 perio mmm had n was c by t1 Whid is c Neon 28 identical with K4 estimations, had to be used to calculate the daily secretion of thyroxine (T4) equivalents. In Table 2 are the results of the mother—infant iodine cycle experiment. The individual variation is quite large but is approximately the same as the individual varia— tion in all of the neonatal thyroid studies. The mean value of the iodine transferred from the radioactive litter via the foster mother to the untagged, substituted infant is about 9.36 i 0.20%, of the mean thyroid count of the litter mates. This means that 9.36% of the thyroid count of a single rat in the litter was transferred, in a forty—eight hour period, to the substituted, untagged infant through the mammary gland of the foster mother. Inasmuch as the mother had not been given 1131, it is clear that the radioactivity was obtained by ingestion of urine of the tagged infants by the untagged mother, Who cycles it through her milk, which, in turn, is ingested by the untagged infant. This is consistent with data obtained by Samel and Caputa (1965). Neonatal age, weight, and the number in the litter apparently have no effect on the amount of iodine received by each infant. The average 1131 counts in the thyroids of the injected litters were compared with the counts obtained from the thyroids of the substituted, untagged young which had been with the litter for forty—eight hours. Even though the counts from the substituted young were of a lower order, they paralleled those of the average litter count. That is to say in suc of the ciateé ature shown is an rate absol who, atior in te 29 to say, the thyroid counts of the rats that were substituted in succession maintained the same slope as the average counts of the 1131 injected litter with which they had been asso— ciated for forty—eight hours. Heart—Rate Temperature Relationships The relationship between the intraperitoneal temper— ature and heart rate of infant rats used in this study is shOWn on Figure 3. It will be noted that this illustration is an Arrhenius plot in which the logarithm of the heart rate is plotted against the reciprocal of the intraperitoneal absolute temperature multiplied by one thousand. The Arrhenius equation is named after the originator who, in 1889, proposed it as an explanation for the acceler— ation in the rate of thermochemical reactions with a rise in temperature. For the equation to be valid a straight line must result when the logarithm of activity is plotted against the reciprocal of the absolute temperature. From this line one can compare the rate of activity at a given temperature to the rate at a temperature 100C lower. This ratio is called the temperature coefficient, and is designated Q Two limits to the Arrhenius plot in this experiment 10' must be noted: (1) a loWer limit of 289 K (160C) below which the heart approaches a complete cessation of the beat as a result of cold, and (2) an upper limit of 312 K (290C) above which the heart rapidly approaches its upper rate limit. lll‘cll‘ 3.0 2.6 2.2 |.8 L4 LOG BEAT LO 0.6 0.2 3.0 Figure 3. Y= 3.8493 - 0.2209 LOG X 3.! 3.2 3.3 3.4 3.5 l/TXIO3 COLD IMMOBILIZATION Neonatal heart—rate temperature relationship conforms to the Arrhenius equation. An pl< wa: wa an ra pe tt 01 (11 i1 31 The raW data were plotted in several different ways: (1) Arithmetic, (2) Log—linear, (3) Log—Log, and (4) the Arrhenius equation where the logarithm of the beat was plotted against the reciprocal of the absolute temperature x 1030 The only Way in which a straight—line relationship was obtained, was by application of the Arrhenius plot. In this experiment the Q10 of the neonatal heart was found to be 3.05. Age and weight of the experimental animals had no influence on the reduction of the heart rate in response to cooling. For this reason an attempt was made to recalculate pertinent published experiments to determine whether or not the results agree with those of the present work. The data of six investigators were replotted in accordance With re- quisites for the Arrhenius equation, Appendix p. 49. As cited from the review of literature, numerous investigators have reported relationships betWeen heart rate and temperature. In only two cases, Were the temperature and heart rate data described by the Arrhenius equation (Michael and Menaker, 1963,and Harary and Farley, 1960). In another case (Lyman and Blinks, 1959), there seemed to be some confusion as to the criteria for an Arrhenius plot. It was noted that all of the infant rat hearts stopped between 11.50 and 160C, and that the period of time the hearts could remain stopped, with survival after re— warming, was inverSely proportional to the age of the rat. Rats between seventeen and twenty days of age did not survive f0 C0 ra 32 for longer than one hour after rewarming if the heart stopped completely. This was attributed to CNS damage since the rats Were quite uncoordinated and exhibited primitive swim- ming motions in their attempts at locomotion. ra pc te DISCUSSION Thyroid Function of the Neonatal Rat The ages of the rats in this experiment were highly variable in relation to their weights. Group I represented rats from the day of birth to between the ninth to twelfth post natal day. Group II ranged in age from nine to nine- teen days, Group III were tWenty—nine day males, and Group IV rats were males which were sixty—days old. It was noted that 1131 injections of pregnant rats must be made Within three days of parturition or they Were not effective in labeling the fetal thyroids. This is con- sistent with published tables which state that the fetal thyroid gland becomes functional for both iodide uptake and hormonal release on the eighteenth to nineteenth day of gestation (Gorbman, 1958). As is shOWn in Figure 1, the TSR of neonatal rats is quite low until the rat weighs approximately 22 gm. At this time, it is abruptly increased. This increase con- tinues as a straight—line function of log weight, at least until the rat weighs about 250 gm and is about sixty days of age. Figure 2 shows that the neonatal rat thyroid con— tains relatively little iodine until the rat weighs about 22 gm. It then increases its iodine content as a straight— line function of log Weight for the remainder of the 33 ¥ LL 34 experimental period. It would be interesting to determine the physiological mechanisms that trigger these sudden changes. The I131 per cent dose uptake (U) for newborn rats could not be measured since they obtained their I131 from a preparturition injection of the mother. The times of uptake for these rats are quite variable. This is probably due to the undigested milk from their first feeding after birth and before they could be transferred to an untagged lactating rat. The degree of individual variability is probably due to the time of birth in relation to the time of transferral. The times to maximal 1131 uptake by infants which Were injected on the day of birth varied from four to six days with most of them reaching maximum uptake in five days. For Group II, however, the maximal uptake took four days and had values of seven to nineteen per cent. This is the point at which thyroid iodine uptake by the gland, as well as hormone release, is greatly accelerated. It is of interest to note that the release rates as represented by the K5 and K4 values are approximately the same for all groups when each group is considered by itself. Apparently, the rat, as it attains mass and surface area, increases the total amount of iodine in the gland, thereby increasing its secretion of thyroid hormone with little or no change in output rate. This is one reason for the utilization of the direct output method to determine the nl 35 neonatal TSR, since there is a direct determination of total thyroid iodine as a part of the procedure. As has been shOWn by the data, neonatal rats, upon reaching a body weight of 22 gm abruptly alter their iodide trapping mechanism and there is no way, other than a total thyroidal iodine determination, to discover when this phenomenon occurs. It has been mentioned in the literature survey (Gorbman, 1952) that there is an increased 1131 uptake by the bovine fetus near term. Marks at al (1960), states that the PBI of newborn infants rose from birth to day 4 or 5 and then slowly fell over the first year of life to levels normal for older children and adults. Neonatal pigs have alSO been reported to have high neonatal thyroid acti— vities on the basis of injected thyroxine Il3l degradation values (Slebodzinski, 1965). On the other hand, Myant (1963) states that the fetal rat thyroid turnover is much slower than that of the adult animal. The present experi— ments show that this is also the situation in rats weighing up to twenty—tWo grams. The rat, at birth, is less mature than other animals on which Work has been done (bovine, swine, and human infants). It can be speculated that the thyroid secretion of the fetal and very young neonatal rat, even though small, has a definite purpose. It is knOWn that the thyroid hormone is specifically responsible for the differentiation of cerebellar and cerebral cortical tissue, the development of the thermoregulatory mechanism, and con— ditioning behavior (Hamburgh, 1964). It has been stated 36 that, in rats of eight to ten days of age, chemical thermo— regulation augments the muscular movements which up to that time served as the sole mechanism of thermoregulation (Alimukhamedov, 1962). If the thyroid secretion is concerned in the development of this chemical thermoregulating mechanism, then the data of these experiments lend weight to this hypo— thesis. The iodine cycle between the infant, mother, and back to the infant has an effect on the determination of TSR of the infant. Iodine in the mother“s milk acts to reduce the infant thyroidal release rate. Since the infant nurses continually, this flattens the output slope and gives an underestimation of K“ and K4 values, with a corresponding 4 underestimation of TSR With the direct output method. How— ever, since the Group I and Group II rats nursed throughout the experimental period, this value would be constant for different age and weight groups and comparative secretion rates could be, and were, determined. An attempt was made to apply the substitution method Reineke and Singh, 1955) for neonatal TSR determination but consistent data were not obtained. Whether this was due to a low level of function of the hypothalamo—pituitary— thyroid axis or a lack of sufficiently sensitive experimental technique cannot be stated. Heart Rate Temperature Relationships The individual rats in this study shoWed much varia— tion in the reduction of heart rate as the body cooled. This ¥h 37 is in agreement with the Work of Fairfield (1948), Adolph (1950), Lipp and Folk (1960), and Baker and Horvath (1964). The heart rate of the neonatal rat to twenty-days of age conforms to the Arrhenius equation within temperature limits of 160 - 300C. The data of six original investigators was replotted by this author to show heart rate temperature conformity, by both poikilotherms and homeotherms, with the Arrhenius equation. Possibly many more examples may be found that have this temperature—heart relationship. The correlation betWeen the points on the graphs and the line drawn through them is admittedly not the best. It is very difficult to obtain accurate points from a published curve for several reasons: (1) Published curves have a minimal number of reference points of the ordinate and abcissa, and (2) The curves have usually been photographed and reduced in size. In this process the scale to which they Were originally drawn is reduced necessitating a rough measure— ment for each point. It can readily be seen that the heart rate 010 can roughly divide the homeotherms from the poikilotherms, although there is some overlap with certain animals. Generally, in accordance with Table 3, homeotherms have a 010 of 2.85 and above while poikilothermic animals are below 2.7. It appears from the results of this study that the heart rate is more dependent on temperature than previously supposed. It is believed that the pacemaker tissue of the _ 38 heart is that tiSSue which can intracellularly induce depolari— zation. The means by which these tissues accomplish depolari— zation is not known, although recently Hecht (1965), has stated that most cardiac investigators agree on an ionic theory of excitation in vertebrates. This is based on the interplay of sodium, potassium, and chloride ions as intra— cellular current carriers, and on the cellular membrane with calcium ions as intermediates. The passage of the nerve impulse is alSO known to be related to ionic flux. There— fore, the initiation of the impulse, as well as its passage over the heart, is essentially chemical in nature. This apparently, is especially true of the atrial conduction of the impulSe since no histological evidence has been intro— duced to show specific transmitting structures. Baker and Horvath (1964) in reporting on Work done with swimming un— anesthetized rats, stated that if the circulation of the limbs of the rats swimming in 200C water was sufficiently reduced as a consequence of cold vasoconstriction, then a build—up of chemical by—products of muscular contraction should have increased the heart rate. The heart rate apparently was not affected. This could be interpreted to mean that the increase in heart rate in response to exercise either is temperature dependent, or that the effects of a cooling body temperature override other reflex actions. Ebert et a1, (1964) state that in their experiments, hypothermia directly slowed the heart but no reflex effects were noted. It seems that further Work should be done to explore this problem. — 39 In planning both phases of these experiments it seemed likely that some correlation Would be found between the level of thyroid function in the neonatal rat and the responses of heart—rate to body temperature with increasing age and size. While interesting changes were observed in both of these functions, no clear—cut relationship between them has been found. SUMMARY AND CONCLUSIONS Thyroid Function in the Neonatal Rat With these experiments, it was found that the neo- natal rat has a low level of thyroid function at birth, and this condition persists until the animal weighs 22 gms. At this time there is an abrupt increase in the thyroxine secretion rate (TSR) and thyroid iodine content, both of which, from that time on, have a straight-line relationship with the logarithm of body weight. This rate of increase is maintained at least until a weight of 244 grams is reached and an age of about sixty days. The existence of a mother infant iodine cycle was confirmed and it was found that approximately 9.36% of the 1131 thyroid gland of the individual tagged in the average was transferred to an untagged rat of the same This rat received this 1131 from his litter mates, age within 48 hours. litter mates Whose urine was ingested (at least in part) by the mother and partitioned between her thyroid and mammary glands. The portion of 1131 which was taken up by the mammary tissue was subsequently released to the infants via the milk. The number of rats in the litter, their age, and Weight had no influence on the amount of the cycled iodine. 4O 41 The neonatal heart rate in relation to intraperitoneal temperatures was studied. The data show it to be best des— cribed by the Arrhenius equation within temperature limits of 160 and 300C. The published work of six other investi— gators was replotted by the author and Several different species of animals (both poikilothermic and homeothermic) were shown to have the same heart rate temperature relation— ship. The Q10 of the neonatal rat heart was also calculated, and it has a value of 3.05. It was also found that the age of the rat is inversely proportional to survival if the heart is stopped by cold. Mass and age do not affect the Arrhenius plot for infants 1 — 20 days of age. It is possible With the data shOWn in this paper and certain publications of others, to speculate that many more animals may show this relationship. It may alSO mean that some of the current concepts of the heart—rate relationship to exercise may have to be re—evaluated° 42 Table 1. Neonatal thyroid function. A listing of rat weight, times of maximum uptake, release slopes (Ki or K4), thyroid iodine content, micrograms of daily thyroxine secretion rates. Time to Micrograms Micrograms T Rat Maximum Kg or K4* Thyroid Secreted 4 Wt. U** Uptake Iodine Daily 10.0 Mother injected One day Ki 0.0788 0.2732 0.0510 10.2 " Three days 0.1427 0.2500 0.0846 10.3 " One day 0.1555 0.1656 0.0574 10.35 " One day 0.1488 0.3150 0.1111 10.35 " One day 0.1371 0.2101 0.0661 10.45 ” One day 0.1319 0.3800 0.1188 10.9 " One day 0.1523 0.5450 0.1967 11.85 " One day 0.1319 0.3102 0.0970 15.0 " Four days 0.1144 0.4525 0.1227 15.2 ” Four days 0.1590 0.1313 0.4949 15.5 " Two days 0.0861 0.1063 0.2169 15.5 " Two days 0.1488 0.1550 0.0489 15.6 " SiX days 0.1627 0.3163 0.1220 15.9 “ Two days 0.1026 0.4282 0.1041 16.0 ” Four days 0.0969 0.1519 0.0348 17.8 " Four days 0.1225 0.1538 0.0447 18.55 " Four days 0.1627 0.2013 0.0776 19.0 " Two days 0.1184 0.5825 0.1634 19.1 “ Two days 0.1344 0.1688 0.0526 Group II 22.9 .017 Six days K4 0.1319 0.4500 0.1715 24.0 .018 Four days 0.1427 0.3325 0.1372 24.0 .0185 Four days 0.2204 0.6838 0.3572 24.7 .0155 Four days 0.1762 0.6625 0.2599 25.4 .0126 Four days 0.1310 0.0125 0.0039 25.7 .0195 Four days 0.1932 0.6275 0.2873 26.7 .013 Four days 0.1606 1.1350 0.4319 26.7 .030 Four days 0.2566 1.2110 0.7366 26.8 .012 Six days 0.1498 0.6000 0.2131 27.1 .0258 Four days 0.2302 0.5360 0.2924 27.2 .0125 Four days 0.1353 0.6088 0.1952 27.4 .0218 Four days 0.2184 1.4250 0.7379 27.4 .0175 Four days 0.1540 1.0738 0.2924 27.4 .0170 Four days 0.1917 0.4913 0.2232 27.8 .0170 Four days 0.1451 0.9550 0.3286 28.0 .0095 Four days 0.1758 0.1485 0.0619 28.2 .0093 Four days 0.1025 0.6300 0.1530 28.7 .0062 Four days 0.0403 0.9350 0.0377 29.0 .013 Four days 0.1621 0.6500 0.2497 43 Table 1. Continued. Time to Micrograms Micrograms T4 Rat Maximum K4 or K4* Thyroid Secreted Wt. U** Uptake Iodine Daily 29.1 .019 Four days 0.2006 2.7938 0.3920 29.4 .0074 Four days 0.0804 0.7400 0.1410 29.4 .012 Four days 0.1418 0.8300 0.2790 29.8 .012 Four days 0.1301 0.6350 0.1958 30.0 .0115 Four days 0.1338 0.5525 0.1752 30.4 .0175 Four days 0.1365 1.8550 0.6002 31.3 .0165 Four days 0.1395 1.8875 0.3824 Group III 60.8 .0265 Two days 0.1894 2.5532 1.0977 61.0 .0370 Two days 0.1774 3.8250 1.5401 63.0 .0220 Two days 0.1555 2.7344 0.9654 63.1 .0305 Two days 0.1604 3.4000 1.2379 64.9 .0350 Two days 0.2018 3.0063 1.3772 65.6 .0285 Two days 0.2062 2.5782 1.2066 66.1 .0340 Two days 0.1541 2.7219 0.9523 67.3 .0300 Two days 0.1852 2.6063 1.0956 Group IV 152.4 0.680 Two days 0.1415 6.4625 2.1669 155.3 0.640 Two days 0.1287 5.9875 1.8256 162.3 0.725 Two days 0.1016 9.4813 2.2837 172.8 0.670 Two days 0.2211 4.2250 1.9925 175.6 0.800 Two days 0.1287 9.3125 2.8400 195.2 0.980 Two days 0.1763 4.2250 1.7655 238.5 0.780 TWO days 0.1404 6.1500 2.0461 243.5 0.670 Two days 0.2211 4.1000 2.1482 _______________________________________________'_______,______ *from semi-log slope. **Ca1cu1ated theoretical value from intercept of zero time from true release slope. .mmomgo HOMHG ©HMUCmum smonm H COHUMH>®U Uhmwcmpm mmm.m H x awn pcsoo zuhsom ham MCSOU WHHSB ham ucsoo wcoomm hmm #CSOU uma 5H mm.MH m owum ma maowa @ Nmam ma moaw h mfinw Ha waoa w mmam ma mmob h Hmom ma mm.» m bmflm m omuw w omen NH mfiom m mm.m OH abam m magw w mnema m maow h vmom w wasp OH mauv v moam m vooma m 000% w Hwaoa w Nhuw v «ohm m o.HH ®m< .®><.#wflq u®>< mmfl um>< eupfiq u®>¢ ®m< .w>¢ Hmufiflq 9®>< mm< o®>¢ owpflq om>¢ 9>H©CH CH o>fi©QH Q>HEQH CH 0>H©cH °>H©CH CH .>H©QH H>HUQH CH .>H©CH X muwm mmom X X mpmm mmow X X mumm mmOU R X mpmm mmom fi Mmumom .oz prufiq kumom .Oz meUflq Hmpmom .OZ prpflq Hmpmom .oz Hmpuflq ovmucsoo mwmv §m#5#H#mQSm Ucm Mmuwfla mo mmm §Mwugfla wwamgma Eogm mucsoo pcmo Mma Hmumow .kuufla mag Qfl mpmh vwamfima mo Hmnfisc §mmov ucwu me HMSUH>H©Cfl mmmnm>m maoho mcHGOH pcmwsfl Hmsuoz 0N magma 45 .HOflpsm Hmcflmfino hQ pOHQ wSHcmflHHm*% .Moswsm #Qmmem ha m#mw UmppOHQ®M* mmmfi §c0mmcmg0b ¢ Moamflm ooom I ooa anew Egmguoaflxflom h.q mflmmwfiw mm¥0®COH5®HmV gmHmI mmma Icommsmgon ¢ xouwflm ooom I OOH mmoa anmgpofiflxaom Amfl>mma msmocmxv mong coma Imm.mw coupsm ooom I oom wmom EymguoHHxflom Amumfluom mxaowswmmv ®H#HSE% Axmspm pqwmmumv .Npaom Doom I oofi mo.m = pqmwcHI mqma ~©Hmflmuflmm ooOm I 00H om.m anmmsuogmpom ucmmcHI coma Iamagmm w humumm Aamm pucmflwv o.m :usogw: m mfiamo “Hams mmpmaomH*¥ woma §£pm>Hom w memm Doom I Oom om.m : mGHEEHBm wadcmfi omma .gmflow< uoom I ooa mm.w pomucfl pagan : Awdoflmm>moq m5##mmv #mM¥ mama Iumxmcmz ¢ HmmgUfls comm I o.HI m.m UmpMHOmH wagon = Amsm5MHosH mflpoxzv pmm%% uoom I ooa Noam = mmpmaomfi pagan mmma NVmM®>mwm % OGH>OU Doom I 00H oa.m Euwspomfiom gompcfl UHSGM pflpnmm* mmma “comeMLOb d MUHme Uoom I OQN mwom EH®£¢O®EOE aamm mHQmUV mOQ% Hepmmflpmm>qH Hmcflmflmo mmcmm .QEQB 0H0 mme HmEHcfl UmppoammH mmB mwmw wmos3 mHOpmmflpwm>cH Hmcflmflno mo mmEmC § wag mo Coaumoflagmm map Mom mwmcmn ®H5pMHmQE®w UHOU Op mmcommwn mwmmluhmms mmOSS mHmEHcm .Homwsm pammmhm msp >9 0 §pOHm mSchghnm NCOHpMme msflcmauh< mfiw muflw \wQHSwCOmeamM ohsumnmmfiwu mpmulpummm .m magma 46 A comparison of K values with K; values 0.693 , -X 1/2 One—day comparison of values of release for different slopes (I, II, III, IV) K = 0.693 or 69.3% of the total value K: Kg = 0.49993 or 49.993% of the total value K = 0.5290 or 52.90% of the total value Kg = 0.4993 or 49.93% of the total value K = 0.3465 or 34.65% of the total value K: = 0.2928 or 29.28% of the total value I I III IV K = 0.26653 or 26.653% of the total value Kg = 0.23395 or 23.395% of the total value Plot out the supposedly identical values (K and KA) and compare. Read Materials and Methods for an explanation of how to convert K to K5. 47 Equations for the calculation of a linear regression line from a graphed logrithmic—linear plot. y = linear values logarithmic values x N = number of subjects or observations y = a + blog x ————— the values of a and b are determined by the following simultaneous equations: E (y) Na ' + b E ((109 x) a E (log x) + b E (log X2) E (log xy) The Coefficient of Correlation is calculated by the following equation: 2 _ a E ) + b E lo x — N r.— 2 2 E(y)—Ny Then — . 2 The Coefficient of Correlation (r) = J r 2 — — b E lo x The Standard of y2 E ‘ ) ) a N — 2 48 Immobilization apparatus. l. MASKING TAPE 2. RAT BODY 3. PLATE BASE 4.3LIDE 5. SLIDE WING NUT (FOR TIGHTENING SLIDE) 6. WASHER Z DIRECTION RAT FACES 8. ANCHORING POSTS 9. INTRAABDOMINAL THERMISTOR PROBE IO. CORK HOLDING PROBE II. HOLE FOR CORK I2. SUPPORTING RODS l3. WIRE HOLDING SUPPORTING RODS l4. WATER AND ICE TO BASE OF PLATE vm.m mm.m Om.m mw.m ©¢.m wvnm He.m I one Ammmac commcmaoe paw MUMme mo mama EOHM Umwpoammm wnmwm mom mo #OHm mDHCwQMH< 49 pmmm moq __ _. __J OH oooa x B\H ow.m n O ©¢.m vv.m mw.m ov.m mm.m mm.m vm.m mm.m Om.m mm.m o.H lf / .1 . .momHSH 43.053 .e .35 / I / . 20mm: 53.694 mo mpmp Eoum pwupoammu Show: umn wasp/w III! II III .Awomav gpm>uom paw M®¥mm mo mumw .l / .FI/ EOHM pmppoamwh 50.3 mSHQwQHH< 35mm: pmu mCHEEHBm uflsma . . . I I. .mmmlmmmumma ///.3 43.325 .6 922 84m: Emflfimm mo 3% 50E o.m / Umppoammn $0.3 mdflzoxamufiw ”5mm: “EH ucmmcH I emaImmvume .Hoamsam .Hmma .HdOb Ammmdv am .3 Lme>mmm UCM .0 .m §OCT/00 mo meow EOMM UmuOHmwm #Hmwfl #HQQQM GDMM pummm / /: I .. .- m3 . C I. , D ”Comm pmm “EmmcH / o.m IIIIII . DomeH [fill/III .uMHOmH pummm pHQQmm pummm umm adopm IIll/I pom mcHEEflBm waspm . Ill/ll I ‘4x / oooa x B\H mm.m u o w¢.m Nw.m o@.m mm.m om.m «m.m mm.m om.m o.H .mmIbmhmMIMEOHOHmhsm QEOU Ugo .Hawo .b Aoomav .Hm um coppsm no mume some owuuoemmm poam mSHC®£MH¢ pHmmn meHSB n.a wpmm bumwm moq LITERATURE CITED Adolph, E. F., 1950. Heat production in hypothermia. Amer. J. Physiol. 161:369. Alimukhamedov, A. A., 1962. Phases of thermo—regulation development in early ontogenesis in rodents. Voprosy ekologii (Problems of ecology) Vysshaya Shkola: Moscow 6:10—11. Referat.Zhur., Biol. 1962 No. 201275 (from Biol. Abs.). Baker, Mary Ann, and Steven M. Horvath. 1964. Heart rate and rectal temperature of swimming rats. Amer. J. Physiol. 270:1073-1076. Barker, S. B., Humphrey, M. J., and Soley, M. H., 1951. The chemical determination of protein—bound iodine. Jour. Clin. Invest. 30:55e62. Bhatnager, R. K., 1963. Interactions of Reserpine, Serotonin and Related drugs, as indicated by energy metabolism, thyroid and adrenal function of the rat. Master"s Thesis. Michigan State University. Bi6rck, Gunnar, and Bengt JohanSSOn, 1955. Comparative studies on temperature effects on the ECG in some vertebrates. Acta Physiologica Scandinavica, 34:257—272. Brody, E. B., 1943. Metabolism and growth rate of rats. J. Nutrition 24:461—468. BrOWnell, S. Z. 1951. Analysis of techniques for the determination of thyroid function with radioiodine. J. Clin. Endocrinology 11:1095—1105. Bryce—Smith, R., H. G. Epstein, and Po Glees. 1960. Physiological studies during hypothermia on monkeys. Jour. Appl. Physiol. 15:440—444. Bullard, Robt. W. 1959. Cardiac output of the hypothermic rat. Amer. J. Physiol. 196:415—419. Capek, K., and J. Jelinek. 1955. The development of the control of water metabolism. I. The excretion of urine in young rats. Physiol. Bohemoslov. 5:76. 52 53 Covino, Benjamin G., and W. R. Beavers. 1958. Effect of cold on the isolated, intact rabbit heart. Jour. Appl. Physiol. 13:422—424. Dahlen, R. W., 1964. Some effects of hypothermia on the cardiovascular system and ECG of cats. Proc. Soc. Expl. Biol. and Med. 115:1—4. Dawson, Wm. R., and George A. Bartholomew. 1958. Effects of temperature on Dipsosaurus (desert iguana). Physiol. Zool. 31:100-111. Deleze, J. 1960. Possible reasons for drop of resting potential of mammalian heart preparations during hypothermia. Circulation Research, 8:553—557. Draper, M. H., and M. Mya—Tu. 1959. A comparison of conduc— tion velocity in cardiac tissues of various mammals. Quar. Jour. EXpl. Physiology 44:91—109. Ebert, Paula A., W. Gerald Austin, L. J. Greenfield, H. W. Bender, and Andrew G. Morrow. 1964. Amer. J. Med. Electron. 3:162—168. Feldman, Joseph D. 1960. Iodine deficiency in newborn rats. Amer. Jour. Physiol. 199:1081-1083. Fisher, D. A., and T. H. Oddie. 1963. Thyroxine secretion rate during infancy: Effect of estrogen. J. Clin. Endocrinology and Metabolism 23:811—819. Giese, Arthur C. 1962. Cell Physiology. W. B. Saunders Co., Philadelphia. Gorbman, A., 1958. Thyroid development. In: Comparative Endocrinology, A. Gorbman (ed.), John Wiley & Sons, New York. ., Evans, H., 1943. Beginning of function in the thyroid of the fetal rat. Endocrinology 32:113—115. ., Lissitsky, S., 0. Michel, and J. Roche. 1952. Metabolism of radioiodine by the near term bovine fetus. Endocrinology 51:546—561. ., Allyn Waterman, C. M. Barnes, and L. K. Bustad. 1957. Thyroidal function in fetal and pre nant sheep given chronic low level dosages of I 31. Endocrinology 60:565—566. Gulick, Addison. 1937. Development of temperature control in the intact rat. Amer. J. Physiol. 119:322. 54 Hall, M. 1832. Phil. Trans. B (1832) 335 (as referred to by Ldfgren (1960). Hamburgh, Max, Edward Lynn, and Earl P. Weiss. 1964. Analysis of the influence of thyroid hormone on prenatal and postnatal maturation of the rat. Anat. Record 150:147—162. ., Edna Sobel, Robert Koblin, Alan Rinestone. 1962. Passage of thyroid hormone across the placenta in intact hypophysectomized rats. Anat. Record 144:219—228. Hamilton, James B., Melvin Dresback, and Ruth S. Hamilton. 1937. Cardiac changes during progressive hypothermia. Amer. J. of Physiol. 161:369. Hannon, John P. 1958. Effect of temperature on the heart rate, electro—cardiogram, and certain myocardial oxidations of the rat. Circ. Res. 6:771—778. Harary, Isaac, and Barbara Farley. 1960. .In vitro organi— zation of single beating rat heart cells into beating fibers. Science 132:1839—1840. Hecht, Hans H. 1965. Comparative physiology and morpho— logical aspects of pacemaker tissues. Ann. N. Y. Acad. Sci. 127:113—116. Heggestad, C B., and L. J. Wells. 1954. Lack of compen- satory changes in the developing thyroid in fetal rats from hypophysectiomized mothers. Anat. Record 118:390-398. Hill, R. M., 1947. The control of body temperature in white rats. Amer. Jour. Physiol. 149:650—656. Hirvonen, Leo. 1955. Temperature range of spontaneous activity of isolated hedgehog, hamster, and rat auricle. Acta Physiologica Scandinavica 36:38—46. Hollander, Phillip B., and J. Leyden Webb. 1955. Cellular membrane potentials and contractility of normal rat atrium and the effects of temperature, tension, and stimulus frequency. Cir. Research 3:604—612. Hutton, Kenneth E., Don R. Boyer, James C. Williams, and Peter M. Campbell. 1960. Metabolic and heart rates in turtles. Jour. Cell. and Comp. Physiol. 55:87-93. HWang, Ung Kee, and Lemen J. Wells. 1959. Hypophysis— thyroid system in the fetal rat: Thyroid after hypophyseoprivia, thyroxine, triiodothyronine, thyrotropin, and growth hormone. Anat. Record 134:125—141. 55 Jacobson, Antone G., and Robert L. Brent. 1959. Radioiodine concentration by the fetal mouse thyroid. Endocrinology 65:408—416. Lepeshkin, Eugene. 1951. Modern Electrophysiology. vol. I. Williams and Wilkins Co., Baltimore. Lindsay, H. A., Kullman, V. S. 1966. Pentobarbitol Sodium: variation in toxicity. Science 151:576—577. Lipp, James A., and G. Edgar Folk Jr. 1960. Cardiac response to cold of two species of mammalian hibernators. Ecology 41:377—378. Lister, John W., Brian F. Hoffman, and Frederick Kavaler. 1964. Reversible cold block of the specialized cardiac tissues of the unanesthetized dog. Science 145:723—725. Lofgren, L., T. Pettersson, and G. Hjelmman. 1960. The effect of extreme cooling followed by rewarming on the cardiac rate in some test animals. Cardiologica 37:85—98. Lyman, Charles P., and Doris Blinks. 1959. The effects of temperature on the i801ated hearts of cloSely re— lated hibernators and nonhibernators. Jour. Cell. Comp. Physiol. 54:53—64. Marks, J., J. Wolfson, and R. Klein. 1960. Neonatal thyroid function. Amer. Journ. Dis. Children 100:549. Michael, C° R. and M. Menaker. 1963. Effect of temperature on the isolated heart of the bat (Myotis lucifugus). Jour. Cell. Comp. Physiol. 62:355—358. Middlesworth, L. van. 1954. Radioactive iodide uptake of newborn infants. Amer. Jour. Dis. Children 88: 439-442. Moore, C. R. 1950. The role of the fetal endocrine glands in development. J. Clin Endocrinology 10:942—985. Moore, E. Niel. 1965. Atrioventricular transmission in newborn calves. Ann. N.Y. Acad. Sci., 727:113—116. Myant, N. B. 1958. The passage of thyroxine and triiodothy— ronine from mother to foetus in pregnant rabbits, with a note on the concentration of protein—bound iodine in foetal serum. Jour. Physiol. 142:329—342. ____1 56 . 1963. Tarczyca a roswoj ploda (the thyroid and fetal development [Lampetra planeri]. Postepy Biochemii 9:19—34. (English summary in Biol. Abs.) Nardone, Roland M. 1955a. Electrocardiogram of the Arctic Ground squirrel during hibernation and hypothermia. Amer. J. Physiol. 182:364—368. , Charles G. Wilker, and X. J. Musacchia. 1955b. Electrocardiograms of the opossum during exposure to cold. Amer. J. of Physiol. 181:352—356. Nataf, B. M. and I. L. Chaikoff. 1964. Effect of injected thyrotropic hormone on in vitro uptake and metabolism of 1131 by thyroid glands of fetal and newborn rats. Endocrinology 75:547—553. Phillips, J. B., and A. S. Gordon. 1954. A study of the pituitary—thyroid relations in the 1— to 15-day—old rat. Anat. Record 120:702—703. . 1955. A study of pituitary—thyroid relations in immature rats. Anat. Record 123:487—508. Pitt—Rivers, R., and Rall, J. E. 1961. Radioiodine equili— brium studies of thyroid and blood. Endocrinology 68:308—316. Poczopko, Piotr. 1961. A continuation to the studies on changes of energy metabolism during prenatal develop— ment. I. Development of mechanisms of body temperature regulation in rats. J. Cell. Comp. Physiol. 57:175—184. Reineke, E. P., and Singh, 0. N. 1955. Estimation of thyroid hormone secretion rate of intact rat. Proc. Soc. Expl. Biol. and Med. 88:203—207. Richards, A. G., E. Simonsen, and M. B. Visscher. 1953. Electrocardiogram and phonogram of adult and newborn mice in normal conditions and under the effect of cooling, hypoxia, and potassium. Amer. J. Physiol. 1742293—298. Samel, M., and Caputa, A. 1965. The role of the mother in I131 metabolism of suckling and weanling rats. Canad. J. Physiol. and Pharmacol. 43:431-436. Sandell, E. B., Kolthoff, I. M. 1944. Nfikrochem. acta., 1:9 Cited by Salter, W. T., and McKay, E. A. Iodine in blood and thyroid of man and small animals. Endocrinology 35:380—390. 57 Slebodzinski, A. 1965. Physiological significance of thyroxine—binding globulin capacity and the daily utilization of thyroxine in the newborn pig. Jour. Endocrinology 32:65—75. Torres, Joseph C., Rachelle Warschaw, and Evangelos T. Angelakos. 1962. Influence of hypothermia on atrial rhymicity and excitability. Circulation 26:797. Van Harreveld, A., and Eleanor Christensen. 1957. Effects of temperature on the membrane potential of mammalian nerve. Physiol. et Pharmacol. Neerland 6:597—607. Walker, H. M., and Joseph Lev. 1953. Statistical Inference. Henry Holt and Company, New York, p. 447—449. Waterman, Allyn J. 1958. Development of thyroid—pituitary Systems in warm—blooded amniotes. In: Comparative Endocrinology. Gorbman, A. (ed.). John Wiley and Sons, New York, pp. 351—367. ., and A. Gorbman. 1956. Development of the thyroid gland of the rabbit. Jour. Exp1.ZooL.l32:509—538. Wekstein, David R. 1965. Heart rates of the pre—weanling rat and its autonomic control. Amer. J. Physiol. 208:1259—1262. Wiedmann, S.» 1961. Membrane excitation in cardiac muscle. Circulation 24:499—505. Wolff, J. 1951. Some factors that influence the release of iodine from the thyroid gland. Endocrinology 48:284. Wright, Evan, and D. P. Sinclair. 1959. The concentration of radioiodine by the foetal thyroid gland and its relation to congenital goitre in sheep. New Zealand Jour. Agri. Res. 2:933—937. f5 a'u'u'u'u'n'u'u'n' ll - I I I I I I _. .. .3,- ._ I! - 1 In“. F. ‘35 .155“. IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII (WINHIWNWWWWIII/WWW!!!)WWW! 293 00056 7911 202,7 5'5qu . LIBRARY ' Michigan State University This is to certify that the has beenaccepted towards fulfillment f th requir rsement for M J degreein WA MSU LIBRARIES RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. SUPPRESSION OF HUMRN T'IBTEIKKNTE RESPONSES BY Erypgngggma_grgzi By Lisa Ann Beltz A DISSEREETION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHIIOSOEHY Department of Microbiology and Public Health 1988 ABSTRACT SUPPRESSION OF HUMAN T LYMPHOCYTE RESPONSES BY W BY Lisa Ann Beltz W is a parasitic protozoan of man which causes a debilitating and often fatal disease whose early stages are accompanied by decreased immune reactivity, the extent and underlying causes of which were unlmown. In order to address these issues, we utilized an in vitro system in which T. cruzi trypcmastigotes were co-cultured with normal human peripheral blood mononuclear cells (PH/1C) . In this system, T.- cruzi reduced PEMC proliferation following stirmrlation by several mitogenic lectins or antibodies to either the T cell rweptor complex or CD2. This reduction was not due to inadequate levels of nutrients or mitogens or to a loss of PEMC viability after co—culture with the parasite. T. cruzi also inhibited the growth of several but not all immortalized cell lines. While monocytes were not required for decreased PEMC responsiveness, parasite viability was necessary. Similar results were obtained when T. cruzi was separated from cells by a Millipore filter, demonstrating that suppression occurred via a factor secreted by the parasite. Maximal inhibition was noted only when T. cruzi was added to cultures within 24 hr of stimulation; therefore, early stages of activation were affected. Interleukin 1 and interleukin 2 (IL2) are products of stimulated monocytes and T cells, respectively, required early during T cell activation. Following Optimal stimulation of human PMC, production of these lymphokirm and interferon-r was unaffected by T. cruzi while the Lisa Ann Beltz parasite decreased I12 and interferon-T production by mouse splenocytes. IL2 restored proliferation of suppressed mouse but not human lympiocytes. The inability of human T cells to respond to endogenous or exogenous IL2 correlated with inhibited expression of IL2 receptors. Both the number of cells bearing IL2 receptors and receptor density were decreased by T. cruzi within 12 hr. low and high affinity receptors were both affected. The expression of T113, an early activation marker, and the transferrin receptor, a growth factor receptor appearing late in activation, were also inhibited by T. cruzi while EAl, the earliest reported activation marker of T cells, was unaffected. Suppression of human T cell functions by T. cruzi is thus selective, with the key events lying not in altered lymphokine production but rather in decreased expression of crucial growth factor receptors . Copyright by Lisa Ann Beltz 1988 To my father, whose life has been my inspiraton, and my mother, who helped me to transform my dreams into reality AW I would like to thank Dr. Felipe Kierszenbaum for his guidance and patience during the years I spent training in his laboratory. He believed in me and gave me encwragement and support when the inevitable problems arose. I wish to thank the members of my guidance committee, Drs. Jeffrey Williams, Pamela Fraker, Walter Esselman, and John Wang, for their time and efforts on my behalf and for helping me to develop a broader view of science. Additionally, I wish to express my gratitude to two researchers with whom I was fortunate to collaborate, Dr. Gerald Sonnenfeld of the University of louisville and Dr. Marcelo Sztein of George Washington University. A special word of thanks must be given to Dr. Sztein for not only reading the countless number of samples whichIsenttohim, butalso forhissenseofhumorandhis stimulating conversations. I want to thank Drs. Julia Wirth, Alfred Ayala, and Mark Connelly for many interesting discussions and occasionally, for the use of their materials, but most importantly, for their camaraderie. I must also express my appreciation for the excellent technical assistance of Mr. James Kidder, Mr. William Morgan, Mrs. Patricia Hoops, and Mrs. Lisa Santangelo. The extent of their contribution bwomes more apparent daily. AnothergroupofpeopletowhomIgivemywarmestthanksaremy blood donors who literally gave of themselves for this work. vi No of my associates deserve special mention. Drs. Fernando Villalta and Maria de Fatima Lima were instrumental in shaping me as a researdier. Their love and dedication to science were highly contagious. They, and their daughter Rachel, have been as a second familytomeandthedebtwhichlowethemcanneverberepaid. I am deeply irdebted as well to my family - my mother, my brother, and my grandparents - whose support never wavered. Without their love and enowragement, my goals would have been attainable but empty. Finally, I give my deepest thanks to my God, Who is the ultimate source of all knowledge and strength. 'Ihrough Him this work was done, and to Him, I now dedicate it. vii TABIEOFGDNI'EN'I’S Page List of Figures xi List of Tables xii Abbreviations xv Inmction one...tocoo-0.0.0.0000DOC.000.000....tooonuooooooco-o l 1.1mmcruz1anoverv1ew 2 II. ImmunosuppressioncausedbyT. cruzr 4 III.Anoverviewochell stimulation 8 IV. Human interleukinz 13 V. The interleukin 2 receptor (IL2R) 15 VI. Interferon-T ....... 20 pterl Stlppreesion of Human Lymphocyte Responses by W 47 Abstract 48 Introduction ...... 49 MaterialsandMethods 50 Results ..................... ...... .. 55 Discussion 67 Refereroesmmm... ...... 70 viii Chapterz Page W Inhibits Interferon-r Production by Mouse Spleen Cells but not Human Peripheral Blood meytes oucoovoeo. oooooo o ..... eoooooeoooostucco-cocooeoooo 72 Abstract ..... 73 Introduction 75 Materials andMethods ........................................ 77 Discussion ..... 86 diapter'B Novel Mechanism for Wi-irduced Suppression of mitten Lymphocytes: Inhibition of Interleukin 2 Receptor Expression........ ..... 94 Abstract.... ...... .......... ..... .... ....... 95 Introduction ...... 97 Results.............. ....... ............. .......... .......... 102 Discussion 113 References 118 pter4 W Decreases the Expression of Both the Interleukin 2 and Transferrin Receptors but not EAl, anEarlyMarkerofLymphocybeActivation.... ..... 121 Abstract ....... ...... 122 Introduction.. ...... 123 MaterialsandMethods ..... 125 Discussion 134 Refm I.0..UOOOI.OO.IOI...OIOO.IOQOIOOOOOIOOOIIOOIOOOC.0. 138 ix Chapter 5 Page Suppression of the CD2 Pathway of Hmnan T Cell Activation by g;ypgggggmg_grug; ............................................ 142 Abstract ..................................................... 143 Introduction ................................................. 144 Materials aniMethods ........................................ 146 Results ...................................................... 149 Dismissim...” ....... ........................................ 152 Referaxtesmu... ..... . ................. 156 AppendixI W Mediates its Suppressive Effect via a Soluble Factor..... ...... . .......... 159 AppendixII W Inhibits the Growth of Several but not all ImbrtalizwcelleI‘eSOO-aocooo ..... oouoooocooooo-ooncoe 168 Summaryand Conclusions 175 LIST OF FIGURES Page Effects of T. cruzi on IL2R expression by human lymphocytes 105 Table LIST OFTABLES Page Chapterl Suppression of Con A—induced PHVIC responses by blood forms of T. cruzi 56 Suppression of PHVIC responses induced with subopt- imal, optimal and supraoptimal concentrations of ConA,PHAorPWMbyblood formsofT.cruzi.......... 57 Mitogenic capacity of Con A solutions before and after absorption with a suppressive concentration of T. cruzi 59 Ability of RHII+5%FBS medium to support Con A—induced responses after incubationwithT.cruzi.............. 60 Effects of addition of T. cruzi at different times afterPEMCstimulationwithConA ..... ........ 62 Reduced production of IL2 by PEMC incubated with Failure of exogenous new to restore Con A respon- siveness of PEMC 65 T. cruzidoes notabsorborconsmne IL2 66 diapterz T. cruzi-induced inhibition of IFN-r production by Hm-Stjmlataltsc OOOOIOOOCDOOOCCO00.0.0.0...000000000 81 Lack of restoration by exogenous IFN-T of the cap- acity of PHA-stiimlated mSC to proliferate after T. cruzi-induced suppressmn 83 Lack of effect of T. cruzi on IFN-T production byhmIO.QIOOIIOOOOIOOOIIOOOOOIOOOOICOCCIIOOOOOU..CO 85 ‘able IV Chapter 3 T. cruzi-induced suppression of IL2R expression by 1mm COICOOOIOOI.....IOIIOIIIIOOCOIOCIOOOOOOOOII Effects of T. cruzi on the capacity of human lymphocytes to incorporate 3H—thymidine and secrete IL2 in response to stimulation with HiA or anti-CD3 . . . Effects of T. cruzi on 3H—thymidine incorporation and IL2R expression by human lymphocytes stimulated with FHA or anti-CD3 in the presence or absence of exogerxmsIIZ ............ ...... . ......... Restoration by exogenous IL2 of the capacity of mouse but not htmian lymphocyte responses (3H-thymidine incorporation) suppressedby T. cruzi IL1 production by human monocytes/macrophages in the presence or absence of T. cruzi Chapter4 The effect of T. cruzi on the expression of the IL2R bystimulatedPEMC ..... . ........ .. The effect of T. cruzi on the birding of 125I-IL2 to the 112R under high affinity conditions Iack of effect of T. cruzi on the expression of EM by Stjmllatapmc oooooo-ooou-oooteoooooonce-coon...o. The effect of T. cruzi on the expression of the TfR bystmflatwmOOo...I.0.DOD...COCO'OOIOOCOIOOIOOIO Chapters T. cruzi inhibits blastogenesis by both the T cell The effect of T. cruzi on IL2 production and IL2R expression after stimulation by either PHA or anti-m O...COOOOOOOOIOOOOCCOOOOOIOIIIOOIIOOCOOIOIOOOI Appendixl T. cruzi suppresses htman Pmc proliferation in the absence of direct contact with the cells The suppressive affect of SSF is reversible xiii 103 106 109 111 112 129 130 132 133 150 151 161 163 Lble EamosureomectotheSSFdidmtirmibitIIZ production ........... . ........ . ................ . ..... ILZRexpression is decreased bythe T. cruzi SSF AppendixII T. cruzi decreases the growth of crib-2 and U937 cell lines.... ............................ . ........... T. cruzi does not effect the ability of HUT 10232 cells to proliferate or express IL2R ............... . . . Page 165 166 171 172 Con A IL1 ILZ ABBREVIATIONS Concanavalin A counts per minute early activation antigen 1 fetal bovine serum interleukin 1 interleukin 2 crude human interleukin 2 purified human interleukin 2 interleukin 2 receptor crude rat interleukin 2 interferon-1 lymphocyte function-associated antigen 2 lymphocyte function-associated antigen 3 lipopolysaocharide mean channel number of the logarithm of the fluoresence intensities determined by flow cytometry mouse spleen cells human peripheral blood mononuclear cells phosphate-buffered saline containing 1% bovine serum albumin phytohemagglutinin pokeweed mitogen RIMI 1640 medium containing FBS REM 1640 medium containing 2.5% FBS REVII 1640 medium containing 5% FBS XV REMI+10%FBS RM 1640 medium containing 10% FBS SSF secreted suppressive factor Tm transferrin receptor xvi IN'I'ROIUCI‘ION I. W: an overview W is the hemoflagellated protozoan which is the causative agent of Chagas' disease. Fifteen to twenty million people are estimated to be infected with this parasite and an additional forty to forty-five million are at risk of acquiring the infection (1) . While the vast majority of the cases have been confined to the tropical and subtropical regions of South and Central America, several reports have demonstrated iretaroes of human infection acquired in the United States (2-4) , where a high percentage of intermediate invertebrate and vertebrate hosts harboring T. cruzi has been found in some geographical ‘ regions (5) . Cnagas' disease can be divided into three phases: acute, latent, and chronic. The early, acute phase may be asymptomatic and occurs most frequently in children (5) . Diagnosis may be made by the presence of an indurated skin lesion (chagoma) or an unilateral edema of the eyelid, conjunctivitis, and enlarged satellite lymph node (Romana's sign) (5,6) . Parasitemia may also be demonstrated at this time and within two to three months. Other possible manifestations lude fever, hepatosplenomegaly, lymphadenopatth lymphocytosis, ms terations, heart failure, and meningoencegmalitis (5,6). Mortality ing the acute stage of infection is five to ten percent (5,7). 3 After a latent period of variable length, lasting up to twenty or thirty years, a percentage of those infected pass into the more severe, chronic stage of the disease. This stage is characterized by damage to the cardiovascular system (myocarditis, cardiac failure) or the digestive system (megacolon or megaesophagus) , as well as nervous tissue (5,8—10). Ten percent of the deaths among adults may be due to chronic Chagas' disease in some regions of Central and South America (11) . Most of the early descriptions of parasite morphology and life cycle are the work of Carlos Chagas (reviewed in 5) . The life cycle of this parasite involves transmission betweem an invertebrate host of the family Reduviidae, subfamily Triatominae, and a vertebrate host. A wide range of mammals serve as suitable hosts: these include man, derestic animals, and rodents, as well as sylvatic reservoirs. Amphibians and birds are refractory to infection (12,13) . Infection of the mammalian host begins when the elongated, flagel- lated metacyclic trypomastigotes from the insect feces are rubbed into mucosa or the site of a reduviid bite. The trypomastigotes invade nearby cells (especially those of the muscular tissues or the reticulo- othelial system) and transform into the amastigote form. The latter tiply by binary fission in the host cell's cytoplasm and then form into nondividing bloodstream trypomastigotes which are eased from the bursting host cell. These trypomastigotes may either de other cells to continue the mammalian cycle or may be ingested a reduviid bug during a blood meal. In the vector's midgut, tigotes transform into epimastigotes, the dividing form in the Mt 4 insect. After passage to the hindgut, the epimastigotes transform into the infective metacyclic trypomastigotes. II. Ixmmmosuppression caused by T. cruzi Both the cellular and hLmoral arms of the immune system play important roles in host defense during the latent and chronic phases of Chagas' disease (reviewed in 5 and 14). The early, acute stage of infection in humans and mice, however, is characterized by a state of specific and nonspecific immnos11ppression. This condition is not unique to T. cruzi infection, occurring in several other parasitic diseases as well. Several reports have demonstrated the occurrence of suppressed cell—mediated responses in humans during the acute phase of the disease (15,16). This phenomenon is accompanied by an increase in the absolute number of CD8+ T suppressor/cytotoxic cells and a decrease ‘ in the number of CD4+ T helper lymphocytes (16) . Cellular immunity, as measured by lymphocyte blastogenesis and delayed-type hypersensitivity reactions, returns to normal levels during the chronic stage (17-19) . Studies of the underlying medianisms) of this acute phase immunosup— pression have not been undertaken in human infection, perhaps due in part to the difficulty in obtaining and/or diagnosing patients during this phase of the disease. » The vast majority of studies of T. cruzi-induced immmmosuppression ve utilized the mouse model system. Cell-mediated immune responses inhibited in mice during the acute phase of infection. Splenocytes rom these mice exhibit decreased blastogenic responses to the T cell 'togens ooncanavalin A (Con A) and phytohenagglutinin (FHA) and to the 1 ( I\ I ... t n I.\ II\ ”U a \m a u \m .m m Cm n 5 B cell mitogens lipopolysaocharide (LPS) and dextran sulfate (20-27) . Partial to complete recovery of these responses occurs during the chronic phase (24—26) . Proliferative responses to trypanosomal antigens are also inhibited during the acute but not the chronic stage of the disease in the moderately susceptible CBA/J and resistant C57 131/6 mice (25,26) , whereas in the more susceptible C3H/He1 mice, the suppression extends into the chronic phase as well (26) . In addition to decreased proliferative responses, T cells from T. cruzi—infected mice are also defective in providing helper activity to B lymphocytes (28). When either epimastigotes (27,29) or bloodstream trypcmastigotes (29,30) are added to cultures of Splenocytes from uninfected mice, there is a significant reduction in the blastogenic response to Con A and LPS. This decrease is produced only whe’) the parasite is present during the initial 24 hours of stimulation (29,30) , suggesting that the suppressive event occurs at an early stage of lymphocyte activation. T. cruzi also inhibits the delayed—type hypersensitivity reaction to skin sensitizing agents (21,31) and trypanosomal antigens (21,32) during the acute phase of the disease. The inhibition in the response to T. cruzi antigens persists into the chronic phase while reponsiveness to an unrelated antigen is restored (33) . Spleen cells from acutely but not from chronically infected mice are also unable to Droduce migration inhibitory factor in vitro (32,34). Thehumoralarmofthe immmesystenisalsoaffectedbyT. cruzi mfection. Splenocytes from awtely infected mice display deficient lumbers of plaque-forming cells (PFC) to both T cell-dependent 6 (heterologous erythrocytes, trinitrophenyl—bovine serum albumin) and -independent (di- and trinitrophenyl—Ficoll, Brucella abortus, IPS) antigens in vitro (26,35-41) . The decrease in IgG but not IgM PFC persists well into the chronic phase (40,41). Both primary and secondary IgG responses are affected, while only the primary IgM response is reduced (36,38) . A restriction in the IgG isotype profile in the sera of chronically infected mice has also been noted; the predominant isotype being IgG2, with deficient production of IgG1 and 1963 (42). A mnmber of mechanisms have been suggested to play a causative role in the above noted immunosuppression. Several researchers have reported the presence of suppressor T cells which decreased T and B cell proliferation (22) , delayed—type hypersensitivity reactions ( 33) , and IgG production (40). Other workers, however, have shown that the removal of Lyt 2.1 or 'Ihyl positive cells does not lead to a decrease in suppressive activity (20,25,43,44) . Another cell type which has been detonstrated to play a role in T. cruzi-irduced immunosuppression is the suppressor macrophage, which has been shown to decrease blasto- genic responses (20,26,39,45) and the number of PFC (39,46). Irxiometh— acin was shown to increase responsiveness (45) , suggesting the involvement of PGE2. other macrophage functions which are required for immune responses, such as antigen uptake and presentation, expression of major histocompatibility complex (MHC) antigens, and release of interleukin 1, are not altered by I._c_ru_z_i infection (35,36,47). Decreased numbers of splenic T cells (24,44) and polyclonal activation of B (38,48,49) and T (49-51) cell responses, leading to clonal fo [2‘3 56; 51111 7 depletion, have also been suggested to have a part in causing the immunosuppression. Interleukin 2 (IL2) production is also decreased in stimulated spleiocytes from infected mice (47,52). Since this lymphokine plays a vital role in both T and B cell responses (see Interleukin 2), the decrease in I12 levels may be partially responsible for the deficiency in lymphocyte responsiveness. Various soluble factors have also been suggested to play a role in T. cruzi-irduced immunodeficieicy. The first such factor to be reported was found in the serum of acutely infected animals (37,50,53- 56): this work was not reproducible and was later retracted by the authors (57) . Another factor was reported by this group of researchers to be present in the culture supernatant of Splenocytes from infected mice (54,58) and acts only on syngeneic Splenocytes. Recently, another suppressive factor from these culture supernatants has been reported (59). This factor has a molecular weight of 14 to 15 Kd, a pit of 6.6, is not haplotype—restricted and is believed to be of host cell origin. _Finally, cultures of infected Splenocytes are reported to produce a suppressive factor when incubated with epimastigotes, trypcmastigotes, or the 104,000 x g supernatant fraction of epimastigotes (60). Several attempts have been made to overcome T. cruzi-induced immunosuppression. Since IL2 production/secretion is decreased in spleen cells from infected mice and this lymphokine is required for T cell proliferation as well as for B cell differentation (see Inter- leukin 2), several groups of researchers have tried to overcame the suppressive effect of the parasite by the addition of exogenous IL2 to cultmres cf Splenocytes from infected mice. T cell blastogenic mmh-m.m 8 responses to Con A stimulation were not restored (47) , while a recovery of B cell responses was produced by either crude (52,61) or purified IL2 (62) . IL2 was also able to restore the ability of T cells to provide helper activity to B cells (28) . When administered to infected mice either alone (63) or in combination with parasite antigens (64) , IL2 restored the in vivo humoral responses of these mice with a subsequent decrease in parasitenia and a slight increase in longevity. The addition of IL2 and parasite antigens is most effective in restora- tion (64) . This group has also found that the administration of parasite antigens alone is able to overcome the suppressive effect of T. cruzi if administered more than once and given at the appropriate time intervals (65) . III. An Overview of T Cell Stimulation T lymphocyte stimulation, with the subsequent synthesis and release of factors involved in macrophage, B lymphocyte, and natural killer cell (NK) activation as well as in clonal expansion of antigen- specific T cells, plays a crucial role in the host immune response. ’Several patlmays of T cell activation have been reported. The most common means of in vivo stimulation occurs via engagetent of the T cell antigen receptor complex (CD3-Ti) . The first event to occur in this pathway is the phagocytosis and processing of the antigen by macro- phage/monocytes, B cells, and dendritic cells, followed by its presen- tation to T cells in the context of the correct MHC antigen (reviewed in 66). The T4+ subset which consists of helper and suppressor inducer cells recognizes processed antigen in the context of MHC class ti< DI! 9 II antigens (67) . The T8+ subset to which both suppressor—effector and cytotoxic T cells belong responds to antigen plus class I MHC antigens (67). The processed antigen and MHC molecule are recognized by the CD3-Ti complex on T cells (67). CD3 (T3) is a molecule which is fotmd on all mature human T lymphocytes and consists of a membrane- bound heterotrimer (68) which is non-ccnvalently linked to Ti (69). Ti is the clonotypically unique structure which allows specific antigen recognition (70,71) . It is a membrane—bard heterodimer belonging to the immunoglobulin superfamily (72) whose individual chains each undergo somatic rearrangement to provide the large diversity of antigen-recognizing structures required by the host (73—76) . Recogni— tion of antigen plus MHC or the addition of antibodies to either Ti or CD3 leads to a removal of the complex from the cell surface (70,77) and provides the first signal in T cell activation (70,78-80) . In addition to their role in antigen presentation, macrophages, B cells, and dendritic cells also synthesize and secrete interleukin 1 (IL1) upon stimulation (81,82) . This lymphokine elicits a large variety of responses in a number of different cell types (83) . One of its actions is to provide a second signal in T cell stimulation (81) . Phorbol myristyl acetate is able to mimick ILl activity (84,85) , possibly through the activation of protein kinase C. The combination of signals provided by CD3-Ti and IL1 lead to the production of interleukin 2 (13:2) and its surface receptor (ILZR) (83,86). IL2 is a lymphokine synthesized and released by activated T helper :ells (87). Upon binding to its receptor, IL2 transmits an intra- fa 1e 10 cellular signal for cell progression from the early to the late G1 stage of the cell cycle (88,89) . Resting cells bear only very low numbers of a low affinity form of the IL2R, but upon contact with processed antigen and IL1, T cells express greatly enhanced levels of membrane-bound receptors, including some with a high affinity for I122 (see Interleukin 2 Receptors). Receptor phosphorylation (90) , activation of a Na+/H+ pimp (91) , protein kirase C mobilization (92,93) , activation of an unique protein kinase (94), increased levels of cytosolic Ca+2 (95,96), inositol triphosphate generation (97), and the inhibition of cAMP accumulation (98) have been reported to be involved in the signal transmission. The synthesis of both 112 and the IL2R occurs early during T cell activation and is transcriptionally regulated. ILZ mRNA is first seen at 9 hours after stimulation and peaks at 24 hours (99,100) , while IL2R mRNAisfirstdetectableat3hoursandismaximalbetween6and24 hours (100). Release of IL2 by the cells occurs by 12 hours of activation and is maximal at 48 hours (101) . The expression of the IL2R on the surface of the cells begins approximately 6 hours after (stimulation and peaks at 48 hours (102) . The synthesis of both IL2 and its receptor are subsequently dwnregulated (100,102,103) . Other events occurring during T cell activation include the synthesis of IFN-T (See Interferon-7) , the expression of several growth factor rmeptors (104—108) , oncogene transcription (103,109) , DNA synthesis, and cell division. Most of these events are regulated at least partially by the interaction of IL2 with the IL2R. The induction of IFN—‘r transcription occurs approximately 3 hours after stimulation, I ac ll peaks at 9 to 15 hours, and begins to decrease at 24 hours (100). While several reports show that I12 may upregulate IFN-T production, these kinetic studies suggest that IFN—r synthesis is at least partially independent of I12 regulation (see Interferon-7) . Transferrin is required for lymphocyte proliferation, and anti— transferrin receptor (TfR) antibodies block thymidine incorporation in T cells (106,110,111), indicating the vital role of this receptor in lymphocyte blastogenesis. This growth factor receptor is expressed late during 1mm activation, with its me first being detectable at 6 to 14 hours and peaking between 14 and 48 hours (100,103). Expression of the T131 on the cell surface is detectable at 48 hours and is maximal 72 to 96 hours later (112) . The expression of this receptor appears to be dependent on the presence of I12, and antibodies to the 112R block the appearance of the TfR on file cell surface (106) , suggesting that the II2—I12R interaction regulates the expression of the TfR. Other growth factor receptors which are expressed at higher levels on activated T cells include the IL]. rweptor (both high and low affinity foms; 113) , the insulin receptor (104) and the type I and II I insulin—like growth factor receptors (107). The transcription of protooncongenes also occurs during T cell activation (103,109) . Some of oncogene mRNA, such as c-myc and c-fos, appear early, prior to the induction of the IL2 mRNA, while others, (such as c~myb, N—ras, and p53, are transcribed later. The expression of the latter group is enhanced by the addition of I12 (103) , suggesting a regulatory effect of this lymphokine. 12 Ultimately, the stimulated lymphocyte passes through the s and 62 phases of the cell cycle to the M phase where it undergoes division. Thus, activation of the T cells by the CD3-Ti pathway leads to lympho- kine production and expansion of antigen-reactive cells. Mitogenic lectins are able to mimick this process but produce polyclonal lympho— cyte activation. In addition to the above—mentioned antigen—dependent pathway, several antigen-independent pathways of T lymphocyte stimulation have been reported, involving (:02 (114), 171344 (115—117), and Tp90 (118). Of these, the 032 pathway has been best characterized (reviewed in 114) . The first demonstration that CD2 [the sheep erythrocyte receptor, T11, lymphocyte function-associated antigen 2 (LFA-Z) , Leu5] may be involved in an alternative pathway of lymphocyte activation came from the finding that a pair of antibodies directed against two distinct epitopes of 032, T112 and T113, were able to induce blastogenesis. This stimulation is monocyte—independent (112) . Upon stimulation with FHA, the number of 032 molecules on the cell surface increases, and T113 becomes detectable within 24 hours. This epitope is not expressed on resting cells and its expression is believed to result from a change in molecular conformation (112) . An antibody directed against the T112 epitope, which is found on all T cells, also rapidly induces T113 expression, in as little as 30 minutes (112) . The ligand of 032 has rwently been identified as LFA-3 (119,120) , a molecule expressed in endothelial, epithelial, and connective tissues, as well as on many blood cells (121) . While all T cells may bind to an LFA-like molecule on sheep erythrocytes via 032, only Si ac l3 activated T cells bind to human erythrocytes, which express a much lower level of LFA—3 than sheep erythrocytes (122,123). No forms of LEA-3 have been characterized, one form attached to the cell membrane by a hydrophobic C-terminus and the other via a phosphatidylinositol tail (124,125). Both forms show significant homology to 032 (124). The binding of LFA—3 to CD2 allows T‘ cells to become responsive to stimulation by anti—T113 (126) . It is possible, therefore, that the interaction of 032 with LEA—3 on accesory cells triggers T113 expres— sion and that the subsequent binding of this epitope to its ligand induces antigen-irflendent proliferation. This pathway may be of particular importance for immature thymocytes which lack the CD3-Ti ccmplex (114) . The putative ligand of T113 has yet to be identified. 032 and the CD3-Ti complex are separate entities and are not associated on the cell surface (127). Moreover, CD3-Ti is not required for C02 activation since the latter pathway is operative in CD3” thymocytes (128) . Nevertheless, the removal of CD3-Ti from the cell surface inhibits 032—induced proliferation (112) , suggesting that the antigen—dependent pathway may regulate 032 responsiveness. Like stimulation via CD3—Ti, triggering by C02 also involves the synthesis of IL2 and the expression of IL2R (128) . Furthermore, CD2 activation induces phosphorylation of 033 (129) . Taken together, these data indicate that at least two pathways of T cell activation exist, either antigen—dependent or ~indepernient. These pathways involve separate signaling molecules interacting with separate receptors but may merge subsequent to receptor binding, with each pathway regulating the activity of the other. VG IV. Human Interleukin 2 IL2 is a lymphokine secreted by activated T cells which allows progression from the early to the late 61 phase of the cell cycle (see An Overview of T Cell stimulation) . It has been well characterized, at the amino acid as well as the DNA level. This lymphokine has a molecular weight of 15 RD and consists of a 133 amino acid polypeptide containing one intramolecular disulfide bridge (87) . Although one 0- 1inked glycosylation site is present, carbohydrate is not neccessary for biologin activity (130-132) . X—ray crystallography studies indicate a significant amount of a helical secondary structure (133) . 1 There exists only a single copy of the ILZ gene, located on chromosome 4q (134). This gene contains 4 exons separated by inter— vening sequences (135,136) . The CDNA for IL2 has also been cloned and sequenced (137) , and encodes a polypeptide of 153 amino acids, with a putative signal sequence of 20 N-terminal residues. IL2 has been found to have a variety of activities in several lifferent cell types. In T lymphocytes, IL2 triggers the production of ther lymphokines, the expansion of reactive clones and the generation »f cytotoxic activity (101) . In B lymphocytes, IL2 has been reported 0 play a role in both differentiation and division (138,139), although he former function is still a matter of controversy. IL2 enhances the ytotoxicity of monocytes and NK cells (140,141) , as well as stimu- iting the respiratory burst and degranulation of neutrophils (142) . Deficient IL2 production is found in several pathological condi— .ons. These include infection with T. cruzi (47,62) , W Irv 164 ho] 15 m (143,144), W copgolense (145), Ieishmania donovani (146), and Mycobacterium bovis (147), ard in leprcmatous leprosy (148), pulmonary tuberculosis (149), certain types of cancer (150), systemic lupus erythematosus (151), Hodgkin‘s disease (152), ard AIDS (153- 155) . In the first four of these infections, exogenous IL2 has restorative effects either in vivo or on in vitro lymphocyte functions (28,47, 61-64, 143—146) . V. The Interleukin 2 Receptor (IL2R) The biological activities of 112 are mediated through the ILZR, which, after binding its ligand, is internalized ard transported to the lysoscmal compartment where it is degraded (156) . The IL2R is expressed on activated T and B lymphocytes (156-158) , with the former expressing approximately twice as many receptors as the latter (159) . Immature thymocytes (160) ard IFN-T- or LPS-irduced monocytes (161,162) also hear the receptor. The expression of the IL2R on T cells may be upregulated through several agents: these include IL1 (163) , IL2 (164- 166), IFN-‘r (167,168), phorbol myristic acetate (169) ard thymic hormones (170). The initial birding studies using radiolabeled—ILZ detected 200- 11,000 receptors on activated T cells with a Kd of 10‘11 to 10'12 (170) . Studies with 3H-anti-Tac, an antibody to the receptor which glooks the birding of I12 (172,173), however, detected 30,000 to 60,000 LLZR per cell (169) . This discrepancy in receptor number was resolved by studies which used a broader range of radiolabeled—IL2 concentra— :ions. These studies demonstrated the presence of two classes of fr 10 (1 thl alt 16 receptors: the first class bourd ligard with the previously noted high affinity (Kd = 10'11 to 10'12) , while the second class had a m of approximately 10‘8 and was represented by 40,000 to 50,000 molecules per cell (171) . Anti—T‘ac birds both classes of the receptor (173) , while physiological levels of IL2 are believed to interact with only the high affinity form (172) . In order to further characterize the IL2R, several groups of investigators made use of various cell lines from patients with cutarecus T lymphomas transformed by the human T cell lymphotropic virus I (arm—1) (174). These cell lines include HUT 102, YT, and MP-l and constitutively produce ard express membrane-bourd IL2R at 5 to 10 times higher levels than activated normal T cells (169,175) . Additionally, several of these lines spontaneously release IL2 (175) . Since these cells have greatly elevated numbers of IL2R, they were used to perform the initial purification ard characteriza- tions of the rweptor (176-178) . Subsequent studies have shown that the genes encoding this molecule as well as the amino acid sequences are the same in both normal and the HTLV-l-infected lines (179,180), though differences in molecular weight have been reported (177,178) are due to variations in post—translatioral processing (180). Information gleaned from the study of both HI‘LV-l-infected cell ines ard normal lymphoblasts have revealed that the low affinity form f the receptor is composed of a single polypeptide chain that reacts ith the anti—Tao antibody (176-178,181). It has a molecular weight of pproximately 55 kD on lymphoblasts (50 kD on HUT102) with a pI of 5.6- .0, contains N— ard O—linked glycosylation, ard is phosphorylated and fated (HG—178,180) . Proteolytic analysis of this molecule suggests 17 the existence of two disulfide-linked domains, with the IL2 birding site in the N-terminal region (182) . Complementary DNA for the above-mentioned p55 polypeptide has been cloned and sequenced, ard encodes a protein consisting of 251 amino acids with a N—terminal extracellular region, a 19 amino acid trans- membrane segment, and a predicted cytoplasmic region of 13 residues (183—185) . This last firding was unexpected since most growth factor receptors have more extensive cytoplasmic tails, which frequently contain tyrosine kinase activity. This information suggested that the p55 polypeptide may be urable to generate an intracellular signal itself ard may be associated with a separate molecule which is able to do so. Genomic 111A for the p55 polypeptide has also been examined. There existsonlyasinglecopyofthegeneencodingthismolecllearditis located on chromosome 10 (179) . The gene consists of 8 exons, of which two, exons 2 and 4, are believed to have arisen from a gene duplica— :ion. Interestingly, alternatively spliced mRNAs which lack the second 3f these sequences do not produce functional receptors (179) . The IL2R gene has two transcription initiation sites in normal T cells (three in lTLV-I-infected lines) ard three different polyadenylation sites (179) . .‘wo major size groups of mRNA have been found, of 1500 and 3500 base airs, with the 1500 base pair moieties making use of the 5'-most olyadenylation site (183) . The former group is believed to contain at easttwokirdsomeNAandthelatter, at least four, althoughthe ctualnlmlberofspeciesineachgroupmaybegreaterduetothe resence of several transcription start sites as well as to alternative Mmmam mole % m Mm .1 18 RNA splicing (186) . Both of these groups contain RNA which give rise to furotional receptors (183) . When the chNA encoding the human form of the p55 polypeptide was transfected into a mouse T—lymphocytic line, both low and high affinity forms of the IL2R were expressed ard the cells were able to respord to human I12 (187) . When mouse L cells were the recipients of the cDIA for either human (187,188) or mouse (181,189) p55, low but not high affinity IL2R were produced. These low affinity receptors could be converted to the high affinity form following the fusion of the transfected L cells with membranes of human T cells (189) . Together, these results suggested that p55 is responsible for low affinity birding ard acts in concert with a secord molecule, found in the membranes of T cells, to produce high affinity binding. Evidence for the existence of the putative secord chain of the IL2R was provided by studies in which 125I—IL2 was cross-linked to its receptor using the bifunctional agent disuccinimidyl suberate ard analyzed on SDS-polyacrylamide gels (190-194) . When the cross—linking was performed with either normal T lymphoblasts or HUI' 102 cells, two bands Of 55 ard 70-75 kD were detected (190-192,194,195) . The 55 kD molecule is precipitable by anti-Tao (190—192,195) and is also demon- strable on cells transfected with p55 cum (191) . This polypeptide thus appears to correspond to the previously characterized chain of the receptor. The 70—75 kD polypeptide (henceforth referred to as p75) , however, does not react with anti-Tao (190-192,194,195) ard represents a novel 112-birding molecule. I mole 19 Further clarification of the roles of p55 ard p75 in 11.2 birding were obtained by performing cross-linking studies with YT cells, a NK- like HI'LV—l-infected cell line (196) . Normally, these cells bird IL2 with a Kd of lo-9 (intermediate affinity birding) ard this binding is not inhibitable by anti-Tao. Cross-linking studies using YT cells revealed a single 112-birding bard of 75 kD (192,195). These cells can also be irduwd to express the high affinity form of the receptor (196,197) . Cross-linking of the induced YT cells to IL2 yields both the p75 ard p55 chains (192,195). Taken together, these firdings irdicate that p55 alone is capable of low affinity I12 birding, p75 alone produces birding of an intermediate affinity, and together, p55 and p75 form the high affinity IL2R. Since the number of low affinity receptors far exweds the number of those of high affinity, the levels of p75 are believed to be the limiting factor in the formation of high affinity rmeptors. The respective contributions of p55 ard p75 to high affinity ainding were examined rwently (198,199) . The p55 chain allows rapid association (5 sec) ard dissociation (6-10 sec), while both the association (42-47 min) ard dissociation (250-330 min) of IL2 with p75 is much slower. Together, they form a receptor with the rapid associa- :ion (37 sec) characteristic of p55 ard the slow dissociation (285 min) >f p75. As previously noted, p55 contains an extremely short cytoplasmic egion which may be unable to function in signal transmission. The p75 olecule, on the other hard, is able to internalize IL2 (193) ard ransmit a signal for cell division (200,201) or immunoglobulin let 21C (16 20 synthesis (202,203) in the absence of p55. Additionally, low levels of p75 but not p55 are present on resting T cells (194,200) ard thus may explain how high levels of IL2 are able to activate unstimulated cells (204). In addition to the membrane—bard form of the 112R, a soluble form also exists. These soluble receptors are released from activated normal T cells and HTLV-l-infected lines (205) and this release is enhanced by IL2 (206) . The soluble form is approximately 10 kD less than the membrane-associated rweptor ard may thus arise by either alterrative RNA splicing or by proteolytic cleavage from the cell surface (205,207) . The serum levels of the soluble IL2R are enhanced in certain disease states, including Hodgkin's disease, adult T—cell leukemia, chronic lymphocytic leukemia, Sezary syrdrome, ard AIIB (208— 210) . Since the soluble receptor is able to bird 112, it may act as a cmpetitive inhibitor of the membrane-bourd form, decreasing the IL2- responsiveness of T cells in these diseases (211) . Decreased expression of the membrane-associated IL2R has also been demmtrated in certain pathological corditions: pulmonary tuberculosis 1(149), Hodgkin's disease (152), AIDS (154,212), ard infection with L drug (144). In the latter case, this decrease is the result of ‘sllppressor cell activity ard not directly induced by the parasite. II. Interferon-1 IFN-T is another lymphokine produced by activated T cells. Its synthesis is regulated, at least in part, by IL2 since anti-Tao ’164,213) ard culture conditions which inhibit IL2 production (164,214) 21 also decrease IFN—T synthesis. The addition of IL2 to these cultures restores IFN-T production (214). IL2 is also able to irduce IFN-T synthesis in unstirmllated lymphocytes and this effect is enhanced by phorbol myristate acetate (213) . While these reports show the ability of I12 to upregulate IFN—T production, the presence of IL2 is not an absolute requirement since normal IFN-r production occurs in T. brucei infections in the face of deficient levels of 112 (144) . Temporally, IFN-‘r mRNA is produced prior to that of IL2. (see An Overview of T Cell Stilmlaticn) , again suggesting that IL2 is not the sole factor regulating IFN—T production. IFN-‘r has a wide range of functions in a number of different cell types (reviewed in 215) . In addition to its antiviral effects, this lymphokine irduces the expression of MHC ard LFA-l antigens (215,216) and the Fc receptor for IgG (215) , activates neutrophils (217) , increases tumoricidal activity in monocytes and NK cells (141,218) , (activates macrophages ard monocytes for antimicrobial activity (219) ard irduces differentiation of myelo-monocytic ard B cells (215) . In T cells, IFN-T may either increase or decrease growth, deperding on the dosage ard time of administration (219,220) . This enhancement may be due, in part, to the ability of IFN-T to increase IL1 (221) and IL2 (222,223) production as well as the expression of IL2R on T cells (167,168) and monocytes (161,224). IFN-7 is released from the stimulated T cells as a glycoprotein with a pI of 8.6 (225) which exists in three monomeric forms with molecular weights of 15, 20, ard 25 kD, in increasing order of occur- rence (226) . These forms have identical amino acid sequences, with the 22 5 kD molecule containing two N—linked glycosylation sites and the 20 form having only one (227) . ‘ IFN—‘r cmAhasbeenclonedardsequenced, ardencodesa lypeptide of 146 amino acids, 20 of which are believed to function as sigral sequence (228) . There exists only a single copy of the gene or IFN-r, located on dlromosome 12 (229). This gene is composed of our exons ard contains a repetitive element (230). Receptors for IFN-T have been demonstrated on monocytes and onocyte-like cell lines (231-234), fibroblasts (235), lymphoblastoid ells (236,237) , and WISH amniotic cells (238) . This rweptor birds FN-r with high affinity (Kd ranging from 10‘9 to 10'10) (231-235,237, 38) . Cross-linking of radiolabeled IFN-T to the cell membranes, ollowed by analysis by SIB-PAGE, shows a receptor with a molecular eight of loo-150 kD (234,236-238) , whereas isolation of the receptor sing anti-receptor antibodies produces two molecules of 50 ard 90 kD, 3th of which can bind IFN—r (239) . Thus, the receptor appears to be of two subunits. Deficient production of IFN—‘r is fourd in several disease states, luding acute tuberculosis (240), Ieishmania infections (241,242), lepromatous but not tuberculoid leprosy (243) . Since leprosy is a of disease states with the lepromatous form exhibiting greater thogenicity than the tuberculoid form, increased pathology correlates defective IIN—‘r production in this cordition. Additiorally, the in IFN-r synthesis in Ieishmania infections is noted in ptible but not resistant strains of mice (241,242). Thus, the 23 ability of a host to produce IFN-T may determine the subsequent severity of some diseases. VII. Research Goals During the initial phase of Chagas' disease, there exists a state of suppressed responsiveness in both the humoral ard the cellular branches of the immune system. All of the previously reported studies examining the underlying mechanisms of this phenomenon have utilized themousemcdel system. Thegoals ofthisresearchweretosuldyL _cru_zi—irduced immunosuppression of human T lymphocyte responses ard to examine at which stage of lymphocyte activation this suppression first is seen. Chapter one describes the ability of T. cruzi trypcmastigotes to inhibit the proliferative response of normal hlman T lymphocytes stimulated by a variety of mitogens. The ability of activated human ard mouse lymphocytes to produce ard respond to IFN-‘r is described in chapter two while the synthesis of IL1 ard IL2 ard the expression of the 112R are the topics of chapter three. Several markers of T cell activation have been described which appear in a definite temporal order. These markers include early activation antigen 1, the IL2R, ard the transferrin rweptor. Chapter four explores the ability of T. cruzi to affect each of these markers over time in order to study the specificity of the immunosuppression as well as to determine the earliest stages at which lymphocytes are inhibited. The expression of both the high ard the low affinity form oftheILZRareexamined. 24 The CDZ pathway of lymphocyte activation provides an alternative :e to the better chacterized CDB-T‘i pathway of stimulation. The Lity of T. cruzi to inhibit T cell stimulation through this pathway mired in Chapter five. Appendix 1 examines whether cell—to-parasite contact is required the induction of immunosuppression and whether this event is arsible. lymphocyte activation involves a series of stages as the cell as from the resting stage of Go into the cell cycle; the parasite 1d exert its inhibitory effect at any of these stages. Imtmrtalized 1 lines, however, are already in the cell cycle and thus bypass eral of the events which occur during activation, perhaps even the ges which are acted upon by T. cruzi. The question of whether L a; is able to suppress the growth of several established cell lines examinedinAppendixz. 25 REFERENCES Science at work. 1984. UNDP/World Bank/WHO, Geneva, Switzerland. Woody, N.C. and Woody, H.B. 1955. American trypanoscmiasis (duagas' disease): first indigenous case in the USA. J. Amer. PM. Assn. 159:676. Schiffler, R.J., Mansur, G.P., Navin, T.R., and Limpakarnianarat, K. 1983. Indigenous Chagas' disease (American trypanosardasis) in (blifornia. J. Amer. Med. Assn. 25:2983. Navin, T.R., Roberto, R.R., Jaranek, D.D., Limpalar'njanarat, K., Mortenson, E.W., Clover, J.R., Yescott, R.E., Taclindo, C., Stem, F., and Allain, D. 1985. Human and sylvatic W cruzi infection in California. Amer. J. Public Healfll. 75:366. Teixeira, A.R.L. 1987. The stercorarian trypanosomes. In Immme responses in parasitic infections: Immunology, immumo— pathology, and immmoproghylaxis. p. 25-117. E. J. L. Soulsby ed. CRC Press, Boca Raton, FL. Rassi, A. 1979. Clinica: Fase Aguda. In Win—cruzi e Doenca de Chagas. p. 249-264. Z. Brener and Z. Andrade eds. Guanabara Koogan, Rio de Raneiro, Brazil. Miles, M.A. 1983. The epidemiology of South American trypanosomiasis - biochemical and immunological approaches and their relevance to control. Trans. Roy. Soc. ‘I‘r'op. Med. Hyg. 77:5. Amorim, D.S., Manco, J.C., Gallo, L., Jr., and Neto, J.A.M. 1979. Clinica: Forma Cardiaca. In W e Doenca de Chagas. p. 265-311. Z. Brener and Z. Andrade eds. Gianabara Koogan, Rio de Raneiro, Brazil. De Rezende, J.M. 1979. Clinica: Manifestacoes Digestivas. In W e Doenca de Chagas. p. 312-361. 2. Brener and Z. Andrade eds. Guanabara Koogan, Rio de Raneiro, Brazil. 26 10. Koberle, F. 1968. Chagas' disease and Chagas' syndromes: the pathology of American trypanoscmiasis. Advances in Parasitology 6:63. 11. Gomez Pereira, M. 1984. caractericticas da mortalidade urbana pcr Doenca de Chagas, Distrito Federal, Brazil. Boletin da Oficina Sanitaria Pan Americana 96:213. 12. Brener, Z. 1979. O parasito: Relacoes hospedeiro-parasito. In Mama e Doenca de magas. p. 1—41. Z. Brener and Z. Andrade eds. Guanabara Koogan, Rio de Raneiro, Brazil. 13. Kierszenbaum, F., Ivany, J. and Budzko, D.B. 1976. Mechanisms of natural resistance to trypanoscmal infection. Role of complement in avian resistance to W infection. Immunology 30:1. 14. Scott, M.T., and Snary, D. 1982. lI_‘ry;§nosoma cruzi and Chagas disease. In Immunology of Parasitic Infections. p.262-298. Cohen and Warren eds. Blackwell Scientific, Boston, MA. 15. Teixeira, A.R.L., 'I‘eueira, G., Maoedo, V., and Prata, A. 1978. Acquired cell-mediated imtmnodepression in acute Chagas' disease. J. Clin. Invest. 62:1132. 16. Voltarelli, J.C., Donadi, E.A., and Falcao, R.P. 1987. Immnosuppression in human acute Chagas disease. Trans. Roy. Soc. Trop. Md. Hyg. 81:169. 17. Tshudi, E.I., Anziano, D.F., and Dalmasso, A.P. 1972. Lymphocyte transformation in (magas disease. Infect. Immun. 6:905. 18. Montufar, O.M.B., Musatti, C.C., Mendes, B., and Mendes, N.F. 1977. Cellular immunity in chronic Chagas' disease. J. Clin. Microbiol. 5:401. 19. Gusmao, R., Rassi, A., Rezende, J.M., and Neva, F.A. 1984. Specific and nonspecific lymphocyte blastogenic responses in individuals infected with Momma cruz'. Am. J. Trop. Md. Hyg. 33:827. 20. Rowland, E.C., and Kuhn, R.E. 1978. Supression of cellular responses in mice during Wow—21 infections. Infect. Imm. 20:393. 21. Rowlard, E.C., and Kuhn, R.E. 1978. Suppression of anamnestic cellular responses during experimental American Trypanosamiasis. J. Parasitol. 64:741. 22. Euros, C., Schadtler—Siwon, I., and Ortiz-Ortiz, L. 1979. Suppressor cells present in the spleens of W— infected mice. J. Immmnol. 122:1243. 27 1. Cumingham, D.S., and Kuhn, R.E. 1980. Lymphoblast transformation as a measure of immune conpetence during experimental Chagas' disease. J. Parasitol. 66:390. . Hayes, M.M., and Kierszenbaum, F. 1981. Experimental magas' disease: kinetics of lymphocyte responses and immunological control of the transition from acute to chronic W infection. Infect. Immun. 31:1117. Kierszenbaum, F. 1981. On evasion of W from the host immune response. Iynpl'moproliferative responses to trypanosomal antigens during acute and chronic experimental diagas' disease. Immunol. 44:641. O'Daly, J.A., Simonis, 3., Role, N.D., ani Caballero, H. 1984. Suppression of humoral immunity and lymphocyte responsiveness during experimental rI_’r‘_ypa_goscma cruzi infections. Rev. Inst. Med. trop. Sao Paulo. 26:67. Ialonde, R.G., Ali-Khan, Z., and Tancwitz, H.B. 1985. W: Regulation of mitogenic responses during infection in genetically resistant and susceptible inbred mouse strains. limp. Parasitol. 59:33. Tarleton, R. L., and Kuhn, R. E. 1985. Measurement of parasite- specific immune responses in vitro: evidence for suppression of the antibody responsetolrypajgsgm cruzi. Eur. J. Imlmmol. 15: 845. Maleckar, J.R., and Kierszenbaum, F. 1984. Suppression of mouse lymphocyte responses to mitogens in vitro by Meme—cruzi. Internat. J. Parasitol. 14:45. Maleckar, J .R., and Kierszenbaum, F. 1983. Inhibition of mitogen-induced proliferation of mouse T and B lymphocytes by bloodstream forms of W. J. Immunol. 130:908. Reed, S.G., Larson, C.L., and Speer, C.A. 1977. Suppression of cell-mediated immmity in experimental Chagas' disease. Z. Parasitenk. 52:11. ‘ Maleckar, J.R., and Kierszenbaum, F. 1983. Variation in cell— mediated immunity to whosoma cruzi during experimental Chagas' disease. Ann. Trop. Med. Parasitol. 77:247. Scott, M.T. 1981. Delayed hypersensitivity to W in mice: specific suppressor cells in chronic infection. Immunol. 44:409. Schmunis, G.A., Vattuone, H., A. Szarfman, and Pesce, U.J. 1973. Cell mediated immunity in mice inocculated with epimastigotes or trypcmastigotes of W. z. Tropemed. Parasit. 24:81. 28 Clinton, E.A., Ortiz-Ortiz, L., Garcia, W., Martinez, T., and Capin, R. 1975. W: Early immune responses in infected mice. Exper. Parasitol. 37:417. Ramos, C., Iamoyi, B., Feole, M., Rodriguez, M., Peirez, M., and Ortiz-Ortiz, L. 1978. W: Immunosuppressed responses to different antigens in the infected mouse. Exp. Parasit. 45:190. Qmmingham, D.S., Kuhn, R.E., and Rowland, E.C. 1978. Suppression of humoral responses during W infecticms in mice. Infect. Imrmm. 22:155. Oorsini, A.C., Oliveira, 0.L.P., and Costa, M.G. 1980. Humoral suppression in W infection in relation to the timing of antigen presentation. Z. Parasitenk. 64:85. Cunningham, D.S., and Kuhn, R.E. 1980. W- induced suppression of the primary immune response in murine cell cultures to T—cell—dependent and —independent antigens. J. Parasitol. 66:16. Reed, S.G., Roters, S.B., and Goidl, E.A. 1983. Spleen cell- mediated suppression of IgG production to a non-parasite antigen during chronic W cruzi infection in mice. J. Immunol. 131:1978. Jeng, G. K. C., and Kierszenbaum, F. 1984. Alterations in production of imrmmoglobulin classes and subclasses during experimental Mom cruzi infection. Infect. Imam. Scott, M. T., and Goes—Sampson, M. 1984. Restricted IgG profiles in T_.__ cruzi infected mice and C‘hagas' disease patients Clin. exp. Immunol. 58: 372. - Kierszenbaum, F., and Budzko, D.B. 1982. Wan—21: deficient lymphocyte reactivity during experimental acute Chagas' disease in the absence of suppressor T cells. Parasite Immunol. 4:441. Harel—Bellan, A., Joskowitz, M., Fradelizi, D., and Eisen, H. 1985. T lymphocyte function during experimental Chagas' disease: ' production of and response to interleukin 2. Eur. J. Immunol. - 15:438. Kierszenbaum, F. 1982. Immmologic deficiency during experimental Chagas' disease (Wm—2i infection): role of adherent, nonspecific esterase-positive cells. J. Immunol. 129:2202. Tarleton, R.L., and Scott, D.W. 1987. Initial induction of ‘ immmity, followed by suppression of responses to parasite 29 antigens during W infection of mice. Parasite Immunol. 9:579. Harel—Bellan, A., Joskcwitz, M., Fradelizi, D., and Eisen, H. 1983. Modification of lI‘-cell proliferation and interleukin 2 production in mice infected with W. Proc. Natl. Acad. Sci. USA 80:3466. Ortiz—Ortiz, L., Parks, D.B., Rodriguez, M., and Weigle, w.o. 1980. Polyclonal B lymphocyte activation during W cruzi infection. J. Immunol. 124:121. Minoprio, P.M., Eisen, H., Forni, L., D'Imperio Lima, M.R., Joskcwicz, M., and Coutinho, A. 1986. Polyclonal lymphocyte responses to murine W infection. I. giantitation of both T- and B—cell responses. Scand. J. Immunol. 24:661. Qmmingham, D.S., and Kuhn, R.E. 1980. W- induced suppressor substance. I. Cellular irwolvement and partial characterization. J. Immunol. 124:2122. Minoprio, P.M., Coutinho, A., Joskowicz, M., D'Imperio Lima, M.R., and Eisen, H. 1986. Polyclonal lymphocyte responses to marine W infection. II. Cytotoxic T lymphocytes. Scand. J. Immunol. 24:669. Tarleton, R. L., and Kuhn, R. E. 1984. Restoration of in __v_itro immune responses of spleen cells from mice infected with W by supernatants containing interleukin 2. J. Immunol. 133:1570. Qumingham, D.S., and Kuhn, R.E. 1980. Mamm- , induced suppressor substance. II. Regulatory activity. Immmmogeneties 10:557. Curmingham, D.S., and Kuhn, R.E. 1980. W- induced suppressor substance. III. Activation of suppressor cells. J. Parasitol. 66:881. Cunningham, D. S., Benavides, G. R., and Kuhn, R. E. 1980. Differences in the regulation of humoral responses between mice infected with W and mice administered T. cruz—_i- induwd suppressor substance. J. Immunol. 125: 2317. Cunningham , D.S., Benavides, G.R., and Kuhn, R.E. 1980. Suppression of mitogen-induced blastogenesis by the W cruzi-irduce}. suppressor substance. J. Parasitol. 66:722. Tarleton, R.L., and Kuhn, R.E. 1984. loss of suppressor activity in the serum of mice infected with W. J. Parasitol. 70:253. 30 Ctmningham, D.S., Kuhn, R.E., and Hatcher, F.M. 1981. W: responses by cells from infected mice to alloantigens. Exp. Parasitol. 51:141. Serrano, L.E., and O'Daly, J.A. 1987. Protein fraction from W infected spleen cell supernatants with jimmonsmppressive activity in vitro. Internat. J. Parasitol. 17:851. lIarleton, R.L., Schafer, R., and Kuhn, R.E. 1983. Effects of extracts of W_ cruzi on imrmme responses: induction of a nonsmcific suppressor factor. Infect. Immun. 41:978. Reed, S.G., Inverso, J.A., and Roters, 5.3. 1984. Heterologous antibody responses in mice with chronic T. cruzi infection: depressed T helper function restored with supernatants containing interleukin 2. J. Immunol. 13321558. Reed, S.G., Inverso, J.A., and Roters, 8.8. 1984. Suppressed antibody responses to sheep erythrocytes in mice with chronic W infections are restored with interleukin 2. J. Iimmol. 133:3333. Chorcmanski, L., and Kuhn, R.E. 1985. Interleukin 2 enhances specific and nonspecific immune responses in experimental Chagas' disease. Infect. Immun. 50:354. Chorcmanski, L., and Kuhn, R.E. 1987. Use of parasite antigens and interleukin—2 to enhance suppressed immune responses during W infection in mice. Infect. Immun. 55:403. C‘hor'cmanski, L., and Kuhn., R. E. 1986. Repeated antigenic stimilation overcomes imninosuppression in experimental Chagas' disease. Immunol. 59:289. Unanue, E.R. 1984. Antigen-presenting function of the necrnphage. Ann. Rev. Immunol. 2:395. Meuer, S.C., Acute, 0., Hercend, T., Schlossman, S.F., and Reinherz, E.L. 1984. The human T—cell receptor. Ann. Rev. Immunol. 2:23. Horst, J., Alexander, 5., Elder, J., and 'I'erhorst, C. 1983. The T3 complex on human T lymphocytes involves four structurally distinct glycoproteins. J. Biol. Chem. 25825135. Brenner, M.B., Trcwbridge, 1.8., and Strcxninger, (LL. 1985. Cross-linking of human T cell receptor proteins: association between the T cell idiotype [3 subunit and the T3 glycoprotein heavy subunit. Cell 40:183. 71 31 '0. Meuer, S.C., Fitzgerald, K.A., Hussey, R.E., Hodgdon, J.C., Schlossman, S.F., and Reinherz, E.L. 1983. Clonotypic structures involved in antigen—specific human T cell function. Relationship to the T3 molecular complex. J. Exp. Med. 157:705. 1. Reinherz, E.L., Acute, 0., Fabbi, M., Bensussan, A., Milanese, C., Rcyer, H.D., Meuer, S.C., and Schlosanan, S.F. 1984. Clonotypic surface structure on human T lymphocytes: Functional and biochemical analysis of the antigen receptor complex. Immunological Rev. 81:95. 2. Hood, L., Krcnenberg, M.,. and Hurflmpillar‘, T. 1985. T cell antigen rwptors and the inmunoglobulin supergene family. Cell 40:225. 3. Yanagi, Y., Yoshikai, Y., Leggett, K., Clark, S.F., Alelusamler, I. , and Mak, T.W. 1984. A human T cell-specific ch clone encodes a protein having extensive hamlogy to immmoglobuin chains. Nature 308:145. L. HEdriCk, S.M., Cohen, D.I., Nielsen, E.A., and Davis, M.M. 1984. Isolation of cDNA clones encoding T cell—specific membrane- associated proteins. Nature 308:149. i. Hedrick, S.M., Nielsen, E.A., Kavaler, J., Cohen, D.I., and Davis, M.M. 1984. Sequence relationships between putative 'F cell receptor polypeptides and immnoglobulins. Nature 308:153. . Chien, Y., Gascoigne, N.R.J., Kavaler, J., Iee, N.E., and Davis, M.M. 1984. Somatic recombination in a murine T—cell receptor gene. Nature 309:322. . Reirherz, E.L., Meuer, 3., Fitzgerald, K.A., Hussey, R.E., Levine, K., and Schlossman, S.F. 1982. Antigen rwognition by human T lymphocytes is linked to surface expression of the T3 molecular complex. Cell 30:735. . Meuer, S.C., Hodgdon, J.C., Hussey, R.E., Protentis, J.P., ' Schlossman, S.F., and Reinherz, E.L. 1983. Antigen-like effects of monoclonal antibodies directed at receptors on human T cell clones. J. Exp. Med. 158:988. Meuer, S.C., Hussey, R.E., Cantrell, D.A., Hodgdon, J.C., Schlossman, S.F., Smith, K.A., and Reinherz, E.L. 1984. Triggering of the T3-Ti antigen-receptor ccmplex results in clonal T—cell proliferation through an interleukin 2-dependent autocrine pathway. Proc. Natl. Acad. Sci. USA 81:1509. Iandegren, U., Andersson, J., and Wigzell, H. 1984. Mechanism of T lymphocyte activation by OKI'3 antibodies. A general model for T cell induction. Eur. J. Immunol. 14:325. 32 31. Mizel, S.B. 1982. Interleukin 1 and T cell activation. ' Immunological Rev. 63:51. 32. Matsushima, K., Procopio, A., Abe, H., Scalla, G., Ortaldo, J.R., . and Qapenheim, J .J . 1985. Production of interleukin 1 activity by normal human peripheral blood B lymphocytes. J. Inmmmol. 135:1132. .3. Drum, S.K., Schmidt, J.A., and Oppenheim, J.J. 1985. Interleukin 1: an immmmological perspective. Ann. Rev. Immnol. 3:263. 4. Rosenstreich, D.L., and Mizel, S.B. 1979. Signal requirements for T lymphocyte activation. I. Replacement of macrophage function with phorbol myristic acetate. J. Immunol. 123:1749. 5. Farrar, J.J., Mizel, S.B., Fuller-Farrar, J., Farrar, W.L., and Hilfiker, M.L. 1980. Macrophage-independent activation of helper T cells I. Production of interleukin 2. J. Immunol. 125:793. 5. Smith, K.A., Gilbride, K.J., and Favata, M.F. 1980. Lymphocyte activating factor promotes lIhcell growth factor production by cloned murine lymphoma cells. Nature 287:853. ’. Robb, R.J. 1984. Interleukin 2: the molecule and its function. Immunol. Today 5:203. ;_ Kristensen, F., Bettens, F., Walker, C., Jancourt, F., and de Weck, A.L. 1983. HLmen lymphocyte proliferation: relationship between cell cycle events and interleukin—2 (IL-2) production. In Interleukins, Iymphokines, and Cytokines. p. 43-49. J. J. Oppenheim and S. Cohen eds. Academic Press, New York. . Stern, J.B., and Smith, K.A. 1986. Interleukin-2 induction of T—cell G1 progression and c—myb expression. Science 233:203. «. Farrar, W.L., and ‘I‘aguchi, M. 1985. Interleukin 2 stimulation of protein kinase C membrane association: evidence for IL1-2 receptor phosphorylation. Lymphokine Res. 4:87. Mills, G.B., Cragoe, R.J., Jr., Gelfand, E.W., and Grinstein, s. 1985. Interleukin 2 induces a rapid increase in intracellular pH through activation of a Na+/H+ antiport. J. Biol. Chem. 260:12500. Farrar, W.L., and Arderscn, W.B. 1985. Interleukin-2 stimulated association of protein kinase C with plasma membrane. Nature 315:233. Farrar, W.L., and Rusoetti, F.W. 1986. Association of protein kinase C with interleukin 2 receptor expression. Immunol. 136:1266. 9! 99 100 101 102. 103. 33 EVans, S.W., and Farrar, W.L. 1987. Identity of cannon phospholylation substrates stimulated by interleukin 2 and diacylglycerol suggests a role of protein kinase C for IL 2 signal transduction. J. Cell. Biochem. 34:47. Gearing, A.J.H., Wadhwa, M., and Perris, A.D. 1985. Interleukin 2 stimulates T cell proliferation using a calcium flux. Immunol. Lett. 10:297. Rossio, J.L., and Ruscetti, F.W. 1986. Ligand—specific calcium mobilization in IL 2 and IL 3 dependent cell lines. Lympholdne Res. 5:163. Farrar, W.L., andRuscetti, F.W. 1986. Developments concerning the biochemical and molecular basis of IL—2 mediated T cell growth. In The Year in Immmology. p. 90-96. J.M. Cruse, R.E. Lewis, Jr. eds. Karger, Basel, Switzerland. Becknar, S.K., and Farrar, W.L. 1986. Interleukin 2 modulation of adenylate cyclase: potential role of protein kinase C. J. Biol. Chem. 261:3043. Efrat, S., Pilo, S., and Kaempfer, R. 1982. Kinetics of induction and molecular size of mRNAs encoding human inter- leukin-2 and r—interferon. Nature 297:236. Kronke, M., Ieonard, W.J., Depper, J.M., and Greene, W.C. 1985. Sequential expression of genes involved in human lymphocyte growth and differentiation. J. Exp. Med. 161:593. Farrar, J.J., Benjamin, W.R., Hilfiker, M.L., Howard, M., Farrar, W.L., and Fuller-Farrar, J. 1982. The biochemistry, biology, and role of interleukin 2 in the induction of cytotoxic T cell and antibody-forming B cell responses. Immunological Rev. 63:129. Depper, J.M., Ieonard, W.J., Kronke, M., Noguchi, P.D ., Qnmingham, R. E., Waldmann, T. A., and Greene, W.C. 1984. Regulation of interleukin 2 receptor expression: effects of phorbol diester, phospholipase C, and reexposure to lectin or antigen. J. Immunol. 133: 3054. , J. C., Alpers, J. D., Nowell, P.C., andHoover, R.G. 1986. ential expression of protooncogenes during lectin—stimulated togenesis of normal hman lymphocytes. Proc. Natl. Acad. Sci. SA 83:3982. , U., Krug, F., and (matrecasas, P. 1972. Emergence of in receptors on hmren lymphocytes during in vitro fonretion. Proc. Natl. Acad. Sci. USA 69:2604. 10! 106 V 107 108 109. 110. 111. 112. 113. 34 Cottner, T., Williams, J.M., Christenson, L., Shapiro, H.M., Strom, T.B., and Strominger, J. 1983. Simultaneous flow cytometric analysis of human T cell activation antigen expression and DNA content. J. Ebcp. Med. 157:461. Neckers, L.M., and Cossman, J. 1983. Transferrin receptor induction in mitogen-stimulated human T lymphocytes is required for DNA synthesis and cell division and is regulated by interleukin 2. Proc. Natl. Acad. Sci. USA 80:3494. Kozak, R.W., Haskell, J.P., Greenstein, L.A., Rechler, M.M., Waldmann, T.A., and Nissley, S.F. 1987. Type I and II insulin- like growth factor receptors on hmnan phytohemagglutinin— activated T lymphocytes. Cell. Immmol. 109:318. Konttinen, Y.T., Berfroth, V., Nordstrom, D., Segerberg— Konttinen, M., and Tblvanen, E. 1986. Expression of MHC class II antigen, interleukin-2 rweptor, transferrin receptor and GP 40/80 glyccprctein during different phases of a normal PHA- driven lymphocyte activation in vitro. Acta Path. Microbiol. Immmol. Scand. 94:181. Kelly, K., Cochran, B.H., Stiles, C.D., and Ieder, P. 1983. Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell 35:603. Dillner—Centerlind, M.-L., Mamerstrom, S., and Perlmann, P. 1979. Transferrin can replace serum for in vitro growth of mitogen-stimulated T lymphocytes. Eur. J. Immunol. 9:942. Brock, J.H., and Mainou-Fowler, T. 1983. The role of iron and transferrin in lymphocyte transformation. Immunol. Today 4:347. Meuer, S.C., Hussey, R.E., Fabbi, M., Fox, D., Acute, 0., Fitzgerald, K.A., Hodgdon, J.C., Protentis, J.P., Schlossman, S.F., and Reinherz, E.L. 1984. An alternative pathway of T-cell activation: a functional role for the 50 kD T11 sheep erythrocyte rmeprtor protein. Cell 36:897. Iowenthal, J. W., and MacDonald, H. R. 1986. Binding and internalization of interleukin 1 by T cells. J. Exp. Med. 614: 1060. Springer, T.A., Dustin, M.L., Kisnimto, T.K., and Marlin, 5.0. 1987. The lymphocyte {Emotion-associated LFA-l, CD2, and LFA-3 molecules: cell adhesion receptors of the immune system. Ann. Rev. Immurol. 5:223. Moretta, A., Pantaleo, G, Lopex—Botet, M., and Moretta, L. 1985. Involvement of T44 molecules in an antigen-ii‘xdepadent pathway of T cell activation. Analysis of the correlations to the T cell antigen—receptor complex. J. Exp. Med. 162:823. 116. 117. 118. 119. 35 Hara, T., Fu, S.M., and Hanson, J. 1985. Human T cell activation. II. A new activation pathway used by a major T cell population via a disulfide-bended dimer of a 44 kilodalton polypeptide (9.3 antigen). J. Exp. Med. 161:1513. Weiss, A., Manger, B., and Imboden, J. 1986. Synergy between the T3/antigen receptor complex and 'Ip44 in the activation of human T cells. J. Immunol. 137:819. Chrrel, S., Salvi, S., Giuffre, L., Isler, P., and Cerottini, J.- C. 1987. A novel 90—kDa polypeptide (Tp90) possibly involved in an antigen—independent pathway of T cell activation. Eur. J. Immunol. 17:835. Plunkett, M.L., Sanders, M.E., Selvaraj, P., mstin, M.L., and Springer, T.A. 1987. Resetting of activated human T lymphocytes with autologous erythrocytes. Definition of the receptor and ligand molecules as CD2 and lymphocyte function-associated antigen 3 (LEA-3). J. Exp. Med. 165:664. Distin, M.L., Sanders, M.E., Shaw, 8., and Springer, T.A. 1987. Purified lymphocyte function-associated antigen 3 binds to CDZ and mediates T lymphocyte adhesion. J. Exp. Med. 165:677. Krensky, A.M., Sanchez-Madrid, F., Robbins, B., Nagy, J.A., Springer, T.A., and Burakoff, SJ. 1983. The functional significance, distribution, and structure of LFA—l, LFA—Z, and LEA-3: cell surface antigens associated with CID-target interactions. J. Immunol. 131:611. Makogoba, M.W., Shaw, 8., Gugel, E.A., and Sanders, M.E. 1987. Human T cell rosetting is mediated by LFA-3 on autologous erythrocytes. J. Immunol. 138:3587. Selvara, P., Dustin, M.L., Mitnacht, R., Hunig, T., Springer, , T.A., and Plunkett, M.L. 1987. Resetting of human T lymphocytes with sheep and hLmnan erythrocytes: comparison of hlmlan and sheep , ligand binding using purified E receptor. J. Immunol. 138:2690. Seed, B. 1987. An LFA—3 cum encodes a phospholipid—linked ‘_ membrane protein homologous to its receptor C02. Nature 329:840. Dustin, M.L., Selvarj, P., Mattaliano, R.J., and Springer, T.A. * 1987. Anchoring mechanisms for LFA-3 cell adhesion glycoprotein at membrane surface. Nature 329:846. _Hunig, T., Tiefenthaler, G., zmm Buscherfelde, K.-H.M., and Meuer, S.C. 1987. Alternative pathway of activation of T cells by binding of CDZ to its cell-surface ligand. Nature 326:298. 127. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137, . 36 Meuer, S.C., Acute, 0., Hussey, R.E., Hodgdon, J.C., Fitzgerald, K.A., Schlossman, S.F., and Reinherz, E.L. 1983. Evidence for the T3-associated 90 kd heterodimer as the T cell antigen receptor. Nature 303:808. Fox, D.A., Hussey, R.E., Fitzgerald, K.A., Bensussan, A., Daley, J.P., Schlossman, S.F., and Reinherz, E.L. 1985. Activation of human thymocytes via the 50kD T11 sheep erythrocyte binding protein irduces the expression of interleukin 2 receptors on both T3+ and T3- populations. J. Immmol. 134:330. Brei‘tmeyer, J.B., Daley, J.P., Levine, H.B., and Schlossman, S.F. 1987. The T11 (C02) molecule is functionally linked to the T3/Ti T cell receptor in the majority of T cells. J. Immunol. 139:2899. Robb, R.J., and Smith, K.A. 1981. Heterogeneity of human T- cell growth factor(s) due to variable glycosylation. Mol. Immunol. 18:1087. Robb, R.J., Kutny, R.M., Panico, M., Morris, H.R., and Chowdhry, V. 1984. Amino acid sequence and post—translational modification of human interleukin 2. Proc. Natl. Acad. Sci. USA 81:6486. Roifman, C.M., Mills, S.B., cnu, M., and Gelfand, E.W. 1985. Functional comparison of recombinant interleukin 2 (IL-2) with IL-2—containing preparations derived from cultured cells. Cell. Immunol. 95:146. Brarflhubert, B.J., Boone, T., Kenney, W.C., and McKay, D.B. 1987. Crystals and a low resolution structure of interleukin-2. J. Biol. Chem. 262:12306. Seigel, L.J., Harper, M.E., Wang-Staal, F., Gallo, R.C., Nash, W.G., and O'Brien, SJ. 1984. Gene for T—cell growth factor: location on hmman chromosome 4g and feline chromosome Bl. Science 223:175. Holbrook, N.J., smith, K.A., Fornace, A.J., Jr., Ccmeau, C.M., ‘ Wiskocil, R.L., and Crabtree, G.R. 1984. IIbcell growth factor: complete nucleotide sequence and organization of the gene in normal and malignant cells. Proc. Natl. Acad. Sci. USA 81:1634. Fujita, T., Takoaka, C., Matsui, H., and Taniguchi, T. 1983. , Structure of the human interleukin 2 gene. Proc. Natl. Acad. Sci. USA 80:7437. Taniguchi, T., Matsui, H., Fujita, T., 'I‘akaoka, C., Kabukazu, N., ‘Yoshiimoto, R., and Hammiro, J. 1983. Structure and expression of m a cloned chNA for human interleukin-2. Nature 302:305. 138. 139. 1 141. 142. 143. 144. . 145. 146. 147. P'!‘ {DH‘O< Human: 01(an ran—mm h-'-lrn(n ESE: STU-£5 37 Muraguchi, A., Kenrl, J.H., Butler, J.L., and Fauchi, A.S. 1984. Regulation of human B-cell activation, proliferation, and differentiation by soluble factors. J. Clin. Invest. 4:337. Miedema, F., and Melief, C.J.M. 1985. T-cell regulation of human B-cell activation. A reappraisal of the role of interleukin 2. Immunol. Today. 6:258. Malkcvsky, M., Lovelam, E., North, M., Asherson, G.L., Gao, L., Ward, P., and Fiers, W. 1987. Recombinant interleukin-2 directly augments the cytotoxicity of htmen monocytes. Nature 325:262. Frey, J.R., Kamber, M., and Peck, R. 1987. Recombinant interferons or interleukin-2 increase cytotoxicity by human monocytes and NK cells. Lymphokine Res. 6:215. chanko, I.C., and Ferrante, A. 1987. Interleukin 2 inhibits migration and stimulates respiratory burst and degranulation of human neutrophils in vitro. Immunol. Iett. 15:285. Sileghem, M. Hamers, R., and De Baetseiler, P. 1986. Active suppression of interleukin 2 secretion in mice infected with Emano—soma brucei Arfl‘aT 1.1.E. Parasite Immunol. 8:641. Sileghem, M., Hamers, R., and De Baetseiler, P. 1987. Ebcperimental 'I_’rypan_gs_oma brucei infections selectively suppress both interleukin 2 production and interleukin 2 receptor expression. Eur. J. Iimmmol. 17:1417. Mitchell, L.A., Pearson, T.W., and Gauldie, J. 1986. Interleukin-1 and interleukin—2 production in resistant and susceptible mice infected with 1mm congolense. Immunol. 57:291. 'Reiner, N.E., and Finke, J.H. 1983. Interleukin 2 deficiency in murine Ieishmaniasis donovani and its relationship to depressed spleen cell responses to phytohemagglutinin. J. Immunol. 131:1487. Vismara, D., Icmbardi, G., Piccolella, E., and Colizzi, V. 1985. Dissociation between interleukin—1 and interleukin—2 production in proliferative response to microbial antigens: restorative effects of exogenous interleukin-2. Infect. Immun. 49:298. Haregewain, A., Godal, T., Mustafa, A.S., Belehu, A., and Yemnaneberhan, T. 1983. T—cell conditioned media reverse T-cell unresponsiveness in lepromatous leprosy. Nature 303:342. 'I‘ocssi, Z., Kleinhenz, M.E., and Ellner, J.J. 1986. Defective interleukin 2 production and responsiveness in human pilmonary tuberculosis. J. Earp. Med. 163:1162. 151 152 153 156 157 156 16( 161 38 Mertelsmann, R., and Welte, K. 1986. Human interleukin 2: molecular biology , physiology and clinical possibilities. Immmobiol. 172:400. Murakawa, Y., Takeda, S., Ueda, Y. Suzuki, N., Hasina, T., and Sakema, T. 1985. Characteristics of T lymphocyte subpcpulation responsible for deficient interleukin 2 actitiy in patients with systemic lupus erythematosus. J. Immunol. 134:187. Zamkoff, K.W., Reeves, W.G., Paelozzi, B.J., Comis, R.L., and Tamar, R.H. 1985. Impaired interleukin regulation of the phytohemagglutinin response in Hodgkin's disease. Clin. Immurol. Immmmopathol. 35:111. Welte, K., and Mertelsmann, R. 1985. HLmen interleukin 2: biochemistry, physiology, and possible pathogenetic role in immunodeficiency syndromes. Canc. Invest. 3. 35. G1pta, S. 1986. Study of activated T cells in man. II. Interleukin 2 receptor and transferrin receptor expression on T cells and production of interleukin 2 in patients with acquired immune deficiency syndrome (AIDS) and AIDS-related complex. Clin. Immunol. Immminopath. 38. 93. Borzy, M.S. 1987. Interleukin 2 production and responsiveness in individuals with acquired immunodeficiency syndrome. Cell. Immunol. 104:142. Robb, R.J., Munck, A., and smith, K.A. 1981. T cell growth factor receptors: quantitation, specificity, and biological relevance. J. Exp. Med. 154:1455. 'ISudi, M., Uchiyama, T., and Uchino, H. 1984. Expression of Tac antigen on activated normal hmean B cells. J. Exp. M. 160:612. Waldmann, T.A., Goldman, C.K., Robb, R.J., Depper, J.M., Ieonard, W.J., Sharrow, S.0., Bongicvanni, K.F., Korsmeyer, S.J., and Greene, W.C. 1984. Expression of interleukin 2 receptors on activated human B cells. J. Exp. Md. 160:1450. Icwenthal, J.W., Zubler, R.H., Nabholz, M., and MacDonald, H.R. 1985. Similarities between interleukin-2 receptor number and affinity on activated B and T lymphocytes. Nature 315:669. Raulet, D.B. 1985. Expression and function of interleukin-2 receptors on immature thymocyes. Nature 314:101. Herrmarm, F., Cannistra, S.A., Levine, H., and Griffin, J.D. 1985. Expression of interleukin 2 receptors and binding of interleukin 2 by gamma interferon—induced human leukemic and normal monocytic cells. J. Exp. Med. 162:1111. 162 . 163 . 164. 165. 166. 167. 168 . 169. 170. 171; 39 Halter, W., Goldman, C.K., Gasbo, L., Nelson, D.L., Greene, W.C., and Waldmann, T.A. 1987. Eb9995. ‘I‘He final suspensions of 52 these cells were prepared in RPMI+5%FBS. 'Ihe IL2—dependent HT-Z cell line (kindly provided by Dr. Phillippa Marrack from the University of Colorado Health Sciences Center, Denver, CD) was used to neasure IL2 activity in biological fluids. These cells were maintained in mavens at 37°C by mixing equal volumes of cell culture and the Karat preparation (see above). Depletion of nomific—esterase—msitive cells Suspensions of name (3.5 ml at 5 x 106 cells/ml) were incubated at 37°C (5% C132 incubator) for 1 hr in a 60—mm diameter sterile plastic petri dish. 'Ihe nonadherent cells were removed and subjected to the same procedure once more, and then centrifuged (280 X G, 10 min, 4°C) . 'Ihe adherent, nonspecific—esterase— positive cells were further depleted by chromatography over a Sephadex G~10 (Pharmacia, Piscetaway, NJ) column (Mishell, Mishell & Shigii, 1980). The nonspecific esterase test has (been described (Yam, Li & Crosby, 1971). Blmenesis assay Cell cultures were set up in triplicate in 96-well microculture plates. Each culture contained 1.25 X 105 PBMC and the appropriate mitogen concentration (see Results) in a total vollme of 0.1 m1. When parasites or other reagents were to be present, they were contained in 0.025 ml and substituted for the equivalent volume of RM+5%FBS. All cultures were incubated at 37°C and 5% C02 for 96 hr (unless otherwise stated) and pulsed with 1 (Lei 3H—thymidine (specific activity 2 QmCi/mmole, Amersham, Arlington Heights, IL) during the last 24 hr. Cultures were interrupted by harvesting (MASH II, FLA. Bicprcducts, lb 01 fl‘ b] of [5? mai (5% 53 Walkersville, MD) and radioactivity was neasured in a liquid scintillation spectrometer. Absorption of Con A solutions with T. cruzi Solutions of Con A (concentrations described under Results) were incubated with 5 x 105 blood forms of T. cruzi per milliliter at 37°C (C02 incubator) for 24 hr. The parasites were then removed by filtration through sterile 0.22-um Millipore filters (Bedford, MA). The filtrate was used as the culture nedium in blastogenesis assays to test PBVIC responses to the residual amount of mitogen. Incubation of RHVII+5%FBS with T. cruzi After incubating RPMI+5%FBS nedium with or without 5 X 106 blood forms of T. cruzi per milliliter at 37°C ((1)2 incubator) for 4 days and filtration (0.22 um pore size), the filtrates were used in blastogenesis assays to test PHVIC responses to various concentrations of Con A. Measurement of IL2 activng Cultures of HPZ cells were set up in triplicate in microculture wells, each containing 4 X 103 cells. The final volume of these cultures was 0.2 m1, including 0.1 ml of two-fold dilutions of the biological I, material to be tested. The alltures were incubated at 37°C for 48 hr (5% C112) and pulsed with 1 uCi 3H-thymidine during the last 24 hr. Cell harvesting and measurement of radioactivity incorporated into 1 synthesized IIIA was as described above. T. cruzi incubation with IL2ch . Solutions of Inch were incubated with purified blood trypcmastigotes ; at final concentrations varying from 1.25 X 106 to 2 X 107 organisms/ml at: 54 at 37°C for 48 hr. After removing the parasites by filtration through sterile 0.22-mn—pore—size filters, the filtrates were tested for IL2 activity as described above. For control purposes, aliquots of new were subjected to the same conditions except that the parasites were absent. Presentation of results and statistical analy_s_is Each set of results presented in the tables is typically repre— sentative of two to four experiments with a similar protocol. The results represent the nean of triplicate determinations i 1 SEM. Differences between neans were considered to be statistically significant if P50.05 by Student's "t" test. S. u] e mmuw 55 S_uppression of MC wnses to mitgens El T. cruzi When present in the cultures, purified blood fonts of T. cruzi suppressed PHdC responses to Con A (Table 1). The concentration of Con A producing optimal responses varied among repeat experiments (data not shown), probably due to the use of PBMC from different donors and different babies of the mitogen. Meyer, significant suppression by T. cruzi was observed in all experiments. Altl'milgh in some experiments a significant reduction of PHVIC responses to the tested mitogens was produced with 2.5 x 105 blood forms/ml, the minimal concentration of parasites causing such effect in most experiments was 5 x 106 organisms/ml and was used in all subsequent experiments. Of interest, tissue culture-derived trypcmastigotes and epimastigotes grown in an axenic medium also suppressed Con A-induced lymphoproliferative Pmc responses (data not shown). The suppressive effect of blood trypcmastigotes was also seen when either PHA or PWM were used to stimulate the PEMC and occurred over a wide range of mitogen concentrations, including suboptimal, optimal and supraoptimal doses (Table 2). Since T. cruzi can bind Con A and PHA (Pereira i114, 1980), we considered the possibility that the parasite might have reduced the concentration of these mitogens to suboptimal levels. To test this pOSSibility, Pmc were stimulated with solutions of Con A or PHA which had been either absorbed with 5 x 106 organisns/ml for 24 hr or mock- absorbed without parasites. Absorption of Con A solutions with (:5. 1.45. < ...... t: :cuuvarccam -H m3...» 6 5 ..«commta «openness occ ocec> _otecou or“ as humane; res: .mo.QWa a .eomouse or“ route xpwue_umeew ..w.. .msme oLwN ea coupe mum: mmuwmctaa use ”L; «N umc_ are m:_t=u uc_nwsxso-:m _u: H rum: umm_aa who: are L; mm to» accesses? are: morawpau use uIIIIIIIIIIIIIIIIllllllatillrzlllllllnluiIIIrIIIIIIIIIIIIIiIIIIIIII1IIIIIIIIIIIIIiIIIIIIIIIIIIIIIIIIIIIIII as“; .3.“ 73 .3“; New; .03 x m; menus .3“ I: .33 as manor. a3 x on so“ ”.2 Tauonm acne? New: e2 x 2 manna acumen sent: some: c uH m e o A~s\msm_cmmtov 0H xv .s.a.o :o_acssemucoo m«_matca 57 Table 2. Suppression of PEMC responses induced with suboptimal, optimal and supraoptimal concentrations of Con A, PHA or PWM by blood forms of T. cruzi Mitogen Mitogen concent. 3H—Thymidine incorporation (cpm X 10'3) ___usfl)_Parasitesabsent_—_____D_Pamsitesresent con A 0 1.4 i 0.2 1.6 i 0.4 0.4 14.4 i 1.0 1.4 i 0.11 4 45.2 i 2.9 2.0 i 0.11 8 40.5 i 1.0 2.9 i 0.01 16 1.9 i 0.6 1.1 i 0.1 PHA 0 0.7 i 0.1 2.2 i 0.2 6.3 27.6 i 1.0 1.3 i 0.11 12.5 18.7 i 0.2 0.3 i 0.11 25 42.9 t 1.6 5.7 i 0.21 50 26.8 i 0.3 2.3 i 0.31 PWM 0 0.2 i 0.0 0.1 i 0.0 2.5 3.3 i 0.4 0.4 i 0.02 5 2.9 i 0.1 0.9 i 0.12 10 2.8 i 0.2 1.3 i 0.12 The experiments with each mitogen were conducted separately. 1'2 150.001 and p50.05, respectively, for reductions in cpm with respect to the corresponding control value (parasites absent). T— '77 .msmmm 0 Thu Significant PBMC stimulation was also produced by solutions of BIA after absorption with T. cruzi. Also considered were the possibilities a) that T. cruzi consumed nutrients required for optimal lymphocyte proliferation and b) that reduced levels of 3H-thymidine incorporation resulted from a greater loss of PHVIC viability due to the presence of T. cruzi. A conditioned incorporation by PEMC as mock-absorbed medium (Table 4). When the proportions of trypan-blue-excluding PEMC were determined in Con A— stilmllated cultures at the end of the 96-hr incubation period, the values obtained in the absence of T. cruzi in repeat experiments were 77 to 83% whereas in the presence of the organisms the corresponding values were 72 to 74%. We also investigated whether monocytes/macrophages, whose aocesory cell function may have been altered upon their infection by T. cruzi, were a requirement for parasite-induced suppression to occur. When PEVIC populations wl'iose monocyte/macroyiage contents had been reduced from 6-9.7% to <0.7'%'. were stimulated with Con A or HIA in the presence of T. cruzi, their responses were still significantly suppressed. Thus, the lymphocyte responses in the presence of medium alone, 8 [lg Con A/ml and 25 pg FHA/ml were 4002 i 1618, 29,768 i 900 and 52,743 i mmmw 59 Table 3. Mitogenic capacity of solutions before and after absorption with a suppressive concentration of T. cruzi Mitogen 3H-Thymidine incorporation (cpm x 10'3) after Mock absorption T. cruzi absorption None 7.6 i 0.4 Con A 4 ug/ml 21.1 _-t 2.3 5.1 i- 1.4 Con A 6 lag/ml 6.3 i 0.3 19.5 i 1.0 Con A 8 pg/ml 6.5 i- 0.3 17.0 i 1.0 None 7.7 1- 0.5 BIA 5 ug/ml 55.5 i 0.2 50.6 i 3.3 PHA 7.5 )ug/ml 55.9 i 1.2 45.7 i 3.6 FHA 10 nag/ml 55.4 i- 1.5 42.4 i- 3.1 The solutions of Con A and PHA were mock-absorbed (same physical treatments, no parasites) or absorbed with 5 x lo6 parasites/ml for 24 hr, filtered through 0.22-pm-pore-size filters, and then used to stimulate PEVIC in 96-hr cultures in the absence of parasites. The cultures were pulsed with 1 uci 3H-thymidine during the last 24 hr. .--e». 4 E Com (#9 60 Table 4. Ability of RPMI+5%FBS medium to support Con A—induced responses after incubation with T. cruzi Con A 3H-thymidine incorporation (cpm X 10'3) in (pg/ml) Untreated medium Medium preincubated with parasites 0 3.5 i- 0.1 3.3 i 0.1. 4 24.9 i 0.6 21.9 i- 0.3 6 31.5 1- 0.9 23.1 i 0.4 8 31.1 i 3.9 19.0 i 0.6 The 96-hr PBMC cultures were performed in the absence of T. cruzi. The culture media consisted of filtered (0.22-pm filter) RIMI+5%FBS which had been incubated in the absence ("Untreated") or presence of 5 x 106 parasites/ml. Con A was added at zero time. The cultures were sed with 1 uCi 3H-thymidine during the last 24 hr. DI 61 1443 cpm, respectively, whereas in the presence of 5 x 106 parasites/ml the responses amounted to 3832 _-+_- 1133, 17,248 i 496 and 15,367 i 348 cpm, respectively. No suppression was seen when gluteraldehyde-killed blood trypcmastigotes were substituted for living organisms in the PEMC cultures (data not shown). Experiments were then designed to establish the period of time during which the suppressive effect of the parasite was exerted. In these PEMC cultures, trypcmastigotes were added at various times after mitogenic stimulation. Maximal suppression was produced when the organisms were present in the cultures from the beginning (zero time), although significant suppression occurred when the parasite was incorporated into the PHVIC cultures 24, 48 or 72 hr later (Table 5) . Effect of T. cruzi on IL2 production and utilization Since IL2 is produced by stimulated T cells and plays a key role in lymphocyte proliferation, we set out to establish whether T. cruzi suppressed PEVIC responses by affecting IL2 production. levels of IL2 were measured after 48 hr of PBMC incubation with 25 lag/ml PHA in the presence or absence of 5 x 106 blood trypcmastigotes per ml. As shown in Table 6, the levels of IL2 activity found in the filtrates of PHA- stimulated PEMC cultures were significantly smaller when the parasites were present. If T. cruzi suppressed mitogen-irduced responses by PBMC merely by impairing IL2 production, exogenous IL2 should correct the deficiency, as was seen by investigators who studied antibody production to T. cruzi-unrelated antigens by lymphocytes from infected mice (Reed et al, 1984,1984a; Tarleton & Kuhn, 1983). 62 Table 5. Effects of addition of T. cruzi at different times after PHTC stimulation with Con A Time of addition 3H—thymidine incorporation (cpm X 10'3) of T. cruzi (hr) PBMC alone name + T. cruzi (95ml 0 43.4 i 1.2 11.2 i- 0.9 (74.2) 24 45.0 i- 2.2 30.0 i 0.7 (33.3) 48 47.4 i 1.3 31.1 1- 0.7 (33.4) 72 41.5 i 0.4 27.7 1- 1.7 (33.3) Ninety-six-hr cultures; stimulated with 6 0g Con A/ml and pulsed with 1 uci 3H-thymidine during the last 24 hr. 1 96R, percent reduction in cpm due to the presence of parasites. All %R values represent statistically significant reductions in cpm (P50.05). 63 .<:a+uzma gum: oozmnuao ~5_a> or“ as «cannon ram: mucmtmeemo .apm>w«uoammt .mO.QWa ore Hoo.oww «... .t; .r; cw «we? use accuse mcmuwsac«-:m .r; mv toe mco_u:~om whore sum: nouuaauc_ who: mutaupao p—oo Nae: an» ._E\mmu_muraa me" x m co “_Naro .eeaxaouzmac oucmmmra to qua+uzmac mucmmaa are c_ —s\ are ob “common 632:: see .53 .ez new: mocmeucewu .Hoo.owa .eowrma uraopao rc-mm use access “camera we: :a x v N: . Lance amour.“ s5. 2H9: .~.e . _.H .H.c + s.o N.“ . ~.s~ a.“ + e.~N Nouns Nouns "swam manna sense socuem méume sane? scum; escue~ mnumam amuse Tensw tens; douse tone; vHH vHH wfifi od ofl m OH O H ca lllllllv :lllllll N i_TLNDLU .h+< cou mNaLu .h+< com sum l:+< cou c. < can so coconuts as“ :. uxma an Am-oH x .s.a.oc co_ecrcarooc_ mcmuwsxru-z “_E\m«_c:c “_Exmnc m N n. a sec .cz.axu uxmd co mmmcm>wmcoammr 1 :03 chain». a. gal 66 .o «_bah race: ooaerummc «a max sumac N n_ are .uowa creeps mama—_c uNwm-Utoa.Es-NN.o success puree—_e one or we roe _Natu .» r«_r accesses, new: combataamra soN hm use to “Hosa_p< acute.” son 0.: To” 2: a: 64” um Women 2322 NM: in see can a: sense. 2: A—s\msmwcamrov as: x m 62 x 2 as: x a; «a _tho .e sb_3 co_uaeomae tween N n_ cocuatcmaa whoeoa N n_ so co coconuts was cw storm N-FI so Am-o~ x .E.a.oc cowsaroatoucc assumessu-:m comes—Tu N 4" N he msamcou to aromas so: moon w~aro .e .m usage 67 DISCIJSSION Our work produced results showing that living but not killed L g-u_zi can suppress human lymphocyte responses to a variety of mitogens and established experimental conditions to explore the possible mechanism(s) . AlthoughT. cruzi islmowntoabsorbConAandPHA (Pereiragc a_l., 1980), two independent findings indicated that the parasite did not reduce PIMC responses by making less mitogen available to them. First, the suppressive effect of T. cruzi was seen over a wide range of mitogen concentrations, including supraoptimal levels (Table 2), minimizing the possibility that mitogen absorption solely accounted for the reduced responses. Second, optimally stimulatory levels of Con A and PHA remained in solutions of these mitogens after absorption with 5 x 106 parasites/ml (Table 3). We could also rule out the possibility that T. cruzi competed with the Pmc for essential nutrients because culture medium incubated with a suppressive parasite concentration supported blastogenisis as well as mock-treated medium (Table 4). Since parasite-induced suppression was observed with PEMC preparations before and after depletion of nonspecific—esterase- positive cells, which includes monocytes and macrophages, it would appear that the parasite could directly affect lymphoid cells. However, the possibility that diminished accessory cell activity resulting from monocyte/macrophage infection contributes to the noted suppression can not be ruled out. The extent of suppression of Con A-induced PBMC responses was greater when the parasites were incorporated into the cultures at zero 68 time -i.e. , together with the mitogen— than when added after 24, 48 or 72 hr (Table 5) . These observations suggested that T. cruzi interfered with the early stages of lymphocyte activation and that cells which had lmdergone activation in the absence of the parasites were less or no longer susceptible to the suppressive effect. However, the reduced PEMCrespcxxsesseenwhen these cellsweremixedwiththe trypcmastigotes 24, 48 or 72 hr after imitation of the cultures were statistically significant, and probably reflected a suppressive effect of the parasites on lymphocytes activated by the mitogen at times later than 0 time, including a sword generation of cells responding to the mitogen. We found lower levels of IL2 activity in the supernatants of PHA- stimulated PEMC when T. cruzi was present (Table 6). Reduced IL2 activity has also been reported by investigators studying suppressed _i_n vi_tro antibody production by lymphocytes from T. cruzi-infected mice (Reed gt_a_l, 1984, 1984a; Tarleton & Kuhn, 1983). However, we were unable to restore PHVIC responses to Con A when exogenous Iszh was added even at a relatively high concentration (114 units/ml) (Table 7). Inthisrespect, wrresultswithhuman PBMCwouldseemtodiffer from those obtained by others with spleen cells from T. cruzi-infected mice (Reed Lal., 1984, 1984a; Tarleton & Kuhn, 1983) , with which a certain , degree of restoration was afforded by adding IL2. This apparent disagreement might be rooted in differences between species or be due to the use of different assay systems: the induction of antibody- , forming cells by antigens administered to infected hosts was measured in the work with the mouse cells (Reed et al. , 1984, 1984a: Tarleton & 69 Kuhn, 1983) whereas our assay neasured proliferative responses of normal human lymphocytes to mitogens. It should be noted, however, that Harel-Bellan _et__al_. (1983) , who used T. cruzi—infected mouse spleen cells to neasure Con A—induced lymphoproliferative responses could not restore the responsiveness of these cells with exogenous I12. Whether IL2 can correct some but not all types of lymphocyte alterations caused by T. cruzi infection or by the addition of this parasite to cell cultures is an interesting question deserving further attention. The failure of exogenous IL2 to restore the lymphoproliferative capacity of the PBMC might have been due to a) absorption or consumption of IL2 by T. cruzi, b) an irreversible PEMC alteration, c) a reduced capacity of PBMC to bind or respond to IL2, and/or d) a need for additional cytokines whose production might have also been altered. Since absorption with up to 2 x 107 parasites/ml did not remove significant amounts of IL2 activity, removal of this lymphokine by T. cruzi seemed unlikely. On this-basis, reduced levels ‘ of IL2 activity in PIE-stimulated PHVIC cultures containing the parasite would probably be due to reduced production. The other three possibilities remain open subjects for further studies. AW This work was supported by grant AI 14848 from the United States Public Health Service. 70 REFERENCES ERENER Z. (1980) Immunity tow. Adv. Parasitol. 18, 247. EJDZKD D. B. & HERSZFNBAIM F. (1974) Isolation of W from blood. J. Parasitol. 60, 1037. CLJNION B. A., ORI‘IZ-ORI‘IZ L., GARCIA W., MARTINEZ T. & CEPIN R. (1975) W: early immune response in infected mice. Emit-.01. 3‘7, 417. CUNNINGHAM D. s. & KUHN R.E. (1980) gzypggosoma cruzi-induced suppression of the primary immme response in mine cell cultures to T cell—dependent and -i_ndependent antigens. L Parasitol. 66, 16. HARELr-BEZLIAN A., JOSKOWICZ M., FRADEIIZI D. & EISEN H. (1983) Modification of T cell proliferation and interleukin 2 production in mice infected with gurgosoma cruz' . Proc. Natl. Acad. Sci. (U.S.A.) 80, 3466. HAYES M. M. & HERSZENBAUM F. (1981) Experimental Chagas' disease: kinetics of lymphocyte responses and immmological control of the transition from acute to chronic W infection. Infect. Immun. 31, 1117. LIMA M. F. & KIERSZENBAIM F. (1982) Biochemical requirements for intracellular invasion by Mosoma cruzi: protein synthesis. J. Protozool. 29, 566. KIERSZENBALM F. (1982) Immunologic deficiency during experimental Chagas' disease (Mom cruzi infection): role of adherent, nonspecific esterase-positive splenic cells. J. Immunol. 129, 2202. KUHN R. E. (1981) Immunology of Mom cruzi infections. In: Parasitic Diseases Vol. 1 (ed. by J. M. Mansfield), p. 137. Marcel Dekker, New York. MISHELL B. B., MISHEIL R. I. & SHIGII J. M. (1980) Adherence. In: Selected Me’tlwds in Cellular Immunole (eds. B. B. Mishell and S. M. Shigii), p. 175. Freeman, San Francisco. MAIECKAR J. R. & HERSZENBAUM F. (1983) Inhibition of mitogen-irduced proliferation of mouse T and B lymphocytes by bloodstream forms of W cruzi. J. Immunol. 130, 908. PERIERAM. E. A., WIRES M. A., VIIIALTA F. & ANmADE A. F. B. (1980) Iectin receptors as markers for W. Developmental stages and a study of the interaction of wheat germ agglutinin 71 with sialic acid residues on epimastigote cells. J. . Med. 152, 1375. RAMOS C., SCHAEEIERrSIWON I. & ORTIZ-ORTIZ L. (1979) Suppressor cells present in the spleens of w-mm mice. L Immunol. 122, 1243. REED s. G., INVERSO J. A. & ROTERS s. B. (1984) Heterologous antibody responses in mice with chronic T. cruzi infection: depressed T helper function restored with supernatants containing interleukin 2. J. Immunol. 133, 1558. RED 8. G., INVERSO J. A. & ROI'ERS s. B. (1984b) Suppressed antibody responsestosheeperythrocytes inmicewithchroniclfirmmia cruzi infections are restored with interleukin 2. J. Immunol. 133, 3333. TARIEION R. L. & KUHN R. E. (1983) Restoration of in vitro immune responses of spleen cells from mice infected with W cruzi by supernatants containing interleukin 2. J. Immmol. 133, 1570. TEIXERIRA A. R. L., TEIXEIRA G., MACEDo v. & PRATA A. (1978) Acquired cell-mediated immunodepression in acute Chagas' disease. J. Clin. Inv. 62, 1132. TIIDEN A. B. & BAICH C. M. (1982) A comparison of PGE2 effects on human suppressor cell function and on interleukin 2 function. L m1. 129, 2469. VIIIAIII‘A F. & LEON W. (1979) Effect of purification by DEAE—cellulose column on infectivity of W blood forms. L Parasitol. 65, 88. WARREN L. G. (1960) Metabolism of Schizogaflmm cruzi Chagas. I. Effects of culture age and substrate concentration on respiratory rate. J. Parasitol. 46, 529. YAM L. T., II c. Y. & CROSBY w. H. (1971) Cytochemical identification of monocytes and granulocytes. Am. J. Clin. Pathol. 55, 283. 72 CHAPI'ERZ W inhibits interferon-T production by mouse spleen cells but not human peripheral blood lymphocytes 73 ABS".T.'RI-\CII Acute w; infection is accompanied by immunosuppression and co—culture of the parasite with mouse or human lymphocytes curtails the proliferative capacity of these cells. As a part of our studies to define how T. cruzi affects lymphocyte functions, we examined in this work the interferon—1 (IFN-r)—producing capacity of phytohemagglutinin—stimulated mouse spleen cells and human peripheral blood mononuclear cells in the presence and absence of the parasite. The levels of IIEN-T in the supernatants of parasite-mouse spleen cell co—cultures were significantly lower than those found in the supernatants of control cultures lacking the parasite. This decrease was observed at both 48 and 72 h after mitogenic stimulation and was not due to absorption or inactivation of the lymphokire by the parasite since incubation of a solution of recombinant IFN-T with L and did not reduce antiviral activity. The T. cruzi-induced suppression of mouse spleen cell proliferation was not overcome by the addition of IFN-T to these cultures nor was exogenous IFN-‘r able to enhance the restorative effect of interleukin 2. Thus, deficient IFN-‘r production did not appear to play a causative role in the reduced proliferative response of the spleen cells. In contrast with the mouse cell cultures, no significant decrease in IFN-r production was seen in human peripheral blood mononuclear cell cultures containing a parasite concentration which decreased murine IFN-T levels; yet the prolifera- tive capacity of the human cells in response to phytohemagglutinin was reduced. These results denote the ability of T. cruzi to inhibit IFN-T 74 production by mouse spleen cells and reveal a notable difference in the process of immmosxppression of mouse spleen cells and human peripheral blood mononuclear cells by T. cruzi. 75 INIRDIIJCI‘ION The acute phase of Mam—i infection in mice is accompanied by several manifestations of immmosuppression, including decreased interleukin 2 (IL2) production and a reduction in mitogen— induced lymphocyte proliferation (8-10,13,22,23,32) . The parasite also suppresses the capacity of normal mouse spleen cells to divide in vitro after mitogen stimulation (14). The birding of I12 to its receptor on T. lymphocytes triggers a number of intracellular events, including stimulation of IFN—r production by T cells (5,24,35) and the transduction of a signal for cell division (reviewed in 29) . Since IL2 production by mouse spleen cells is decreased by T. cruzi, it is thus possible that IFN-T synthesis may also be inhibited. IFN-T plays an important role in host defense against microbial invasion (reviewed in Ref. 7 and 34), and enhances the in vitro killing of intracellular parasites, such as T‘oxoplasma gondii (17,19) , leishmania donovani (16) and T. cruzi (21,37), by macrophages. I_n vii), IFN-T acts synergistically with anti-T. cruzi antibodies to decrease parasitemia and prolong the survival of infected mice (20). This lymphokine also affects the proliferative response of lymphocytes to mitogens. In addition to its well-known growth inhibitory functions (reviewed in Refs. 7 and 34) , IFN—‘r may also enhance T cell activity (2,6,12,25,27), depending on the dosage and time of administration (31) . Given the roles of IFN-T in host defense and lymphoproliferation, we have examined whether T. cruzi can alter production of this 76 important lymphokine by normal murine or human lymphocytes and, if so, whether this alteration plays a role in the suppression of lymphocyte prol iferation . 77 MATERIALSANDMEIHOIB Parasites. T‘rypcmastigotes of T. cruzi (Tulahuen isolate) were purified from the blood of Crl—CD1(ICR)BR Swiss mice (Charles River laboratory, Portage, MI) infected intraperitoneally two weeks previously with 2 x 105 organisms. The parasites were purified by density gradient centrifugation over a mixture of Ficoll—Hypaque of density 1.077 (3) followed by diethylaminoethyl—cellulose chromatography (36) . After two washings by centrifugation (800 x g, 20 min, 4°C) , the parasites were resuspended at the desired concentration in RPMI 1640 medium (Gibco, Grand Island, NY) containing 100 units penicillin and 100 pg streptomycin per ml, and either 2.5 or 5% heat- inactivated fetal bovine serum (56°C, 20 min) (RPMI+2.5%FBS or RM+5%FBS, respectively). Murine spleen cells (nSC) . suspensions of mSC from inbred CBA/J mice (Jackson laboratories, Bar Harbor, ME) were prepared as described previously (14) and resuspended at a final concentration of 2.5 x 106 mSC/ml in RM+2.5%FBS. mmanperirieralbloodmnmxclearcells (hPBJC). ThehPH/ICwere isolated from the blood of healthy donors by centrifugation over Ficoll-Hypaque (350 x g, 45 min, 20°C). After three washings (350 x g, 10 min, 4°C) in serum-free RPMI 1640 medium, the W were resuspended at 1.25 x lo6 cells/ml in RPMI+5%FBS. Cell viability, determined by trypan blue dye exclusion, was always >99%. (Jo-cllture conditiors. Suspensions of mSC were incubated in the presence or absence of 5 pg/ml phytdiemagglutinin (FHA-P; Sigma 78 Chemical Co., st. Iouis, MD) with or without 2.5 x 106 T. cruzi/ml, Lmless otherwise noted. The cultures were incubated at 37°C (5% (1)2) for the desired periods of time (see Results). ollturee of hPBMC were treated similarly except that the final parasite concentration was 5 x 106 organisms/ml. These concentrations of T. cruzi were selected because they represent the minimal levels which consistently produce immmoappressioml under optimal stimulatory corditions for me and hpmc (l; Beltz and Kierszenbaum, unpublished results). Heasurarerrt of IFN-‘r. Cultures of mSC or hPEMC were incubated in 24-well plates in a volume of 1 ml for 48 or 72 h under the conditions described in the preceding paragraph. Cells and parasites were removed by passage through 0.22-mm—pore—size filters and the filtrates were stored at ~70°C until assayed for TEN-r. Murine TEN-1 activity was determined by a plaque reduction assay using mouse L-929 cells and the Indiana strain of bovine vesicular stomatitis virus (30). The titer was expressed as units/ml corresponding to the reciprocal of the highest dilution to reduce plaques by 50%. In this assay, one unit was equivalent to 0.88 mm 9002—9041511 reference units. Identification of the antiviral activity as IFN-T was provided by its lability at pH 2 and inhibition by anti—mile IEN—ur antibodies (a gift of Dr. E. Havell, Trudeau Institute, Saranac, NY). Human IFN— was assayed using a radioimmmlnoassay kit (Centocor, Malvern, NY). This system uses two antibodies directed at different epitopes of IFN-T and is designed to detect only biologically active IFN-T. Absorpticn of murine TIN—1. Recombinant murine IFN-r (specific activity = 2.3 X 107 units/mg protein; a gift from Genentech Inc. , 79 South San Francisco, 0A) was incubated in 24-well plates at a concentration of 500 units/ml in RPMI+2.5%FBS in the presence or absence of 5 or 10 x 106 T. cruzi/ml at 37°C for 48 h. After passage of the supernatants through 0.22-um-pore-size filters, residual IFN-T activity was determined as described above. W proliferation assays. Cultures of mSC or hm were incubated in the preseree or absence of T. cruzi in 96-well plates in a volume of 0.1 ml in the manner described under Clo-culture conditions. mcgenous rwombinant murine IFN-T, partially purified h1m1an IFN-r (Meloy laboratories, Springfield, VA), and/or recombinant glycosylated hmman IL2 (Genzyme, Boston, MA) were added to some of the cultures at the desired concentrations (see Results). The lymphokines, when added to the cultures, replaced an equivalent volume of medium. The cultures were pulsed with 1 uCi 3H—thymidine (specific activity = 2.0 Ci/mmole; New England Nuclear Biotechnology Systems, Wilmington, DE) at 48 h (mSC) or 72 h (hPEMC) and terminated 24 h later by automated harvesting. Incorporated radioactivity was determined in a liquid scintillation counter. All determinations were performed in triplicate anitheresultswereexpressedasmeancmmtsperminngmistaniard deviation . _ sen—77%? 7~ , - 80 The levels of IFN-‘r activity in the supernatants of BIA—stimulated mouse spleel cells containing T. cruzi were found to be significantly lower than those in parasite-free cultures (Table 1). This reduction was demonstrable 48 h after the initiation of the cultures (decrease of 47%) but was more pronounced at 72 h (decrease of 259%) . Similar resultswere foundwhen2 pug/ml ofconcanavalinAwasusedas‘Ule mitogen (data not shown). T‘. cruzi neither secreted an IFN-r-like activity nor did it induce unstimulated mSC to do so (Table 1). The noted decrease in the levels of IFN-T in the culture supernatants might have resulted from a reduction in the production/ secretion of this lymphokine or from its removal by the parasite. Tb determine which of these possibilities explained our observations, solutions of recombinant mmlrine IFN-T were incubated in the presence or absence of T. cruzi for 48 h and the amount of residual antiviral activity was then determined. The amounts of IFN-r remaining in the supernatants of caltures after the absorption with T. cruzi did not differ significantly from that in the mock-treated controls (p50.1) . Thus, for example, in one of the experiments, the IFN-T activities of the solutions after incubation with 5 x 106 or 1 x 107 trypcmastigotes/ ml were 357 i 140 and 303 j: 51 units /ml, respectively, whereas 284 i 74 units/ml were detectable in control cultures to which parasites had not been added. It should be noted that the concentrations of parasites used for these absorptions represented two and four times, respectively, the level which was sufficient to reduce IFN—r activity 81 TABLE 1. T. cruzi—induced Inhibition of IFN-‘r Production by PHA—stimulated m'SCa Material tested IFN-‘r (unitsgml) 48 h 72 h mSC 530 530 T. cruzi 530 530 me + T.cruzi 530 530 T. cruzi + H-IA 530 530 mSC + HIA 58 67 misc + FHA + T. cruzi 31 530 a The tested materials consisted of the culture supernat- ants of me (2.5 x 106 cells/ml) and/or T. cruzi (2.5 x 106 organisms/ml) in the presence or absence of 5 pg/ml FHA. The supernatants were collected at the indicated times after initiation of the cultures. This set of results is typically representative of two separate repeat experiments. 82 in stimulated mSC cultures (see Table 1). The addition of T. cruzi to cultures of stimulated mSC decreases lympl'loproliferation (14) . Because IFN-T is able to modify the proliferation of T cells, either reducing or enhancing it depending on the dose and the time of administration (30), the possibility that the observed suppression in lymphocyte growth may have resulted from the inhibition of IFN—T production by T. cruzi was explored. Varying amounts of exogems recombinant murine IFN-T were added to the cultures and their effects on 3H-thymidine incorporation by T. cruzi— suppressed mSC were determined. As shown in Table 2, the addition of IFN-T at concentrations ranging from 8 to 250 units/ml did not overcome the suppressive effect. Higher IFN—r concentrations were not tested in these experiments because preliminary results (data not shown) had indicated that levels greater than 188 units/ml exert an inhibitory effect on lymphoproliferation. This can also be seen in Table 2 for the control result obtained with 250 units IFN-‘r/ml. mogenous IL2 has been shown to restore the proliferative response of FHA-stimulated mSC suppressed in vitro by T. cruzi (L. A. Beltz, M. B. Sztein and F. Kierszenbaum, J. Immunol., in press). Since IFN-‘r has been reported to affect the interaction of IL2 with lymphocytes by increasing the expression of IL2 receptors, we tested whether IFN-r would act synergistically with IL2 and enhance the restorative effect of the latter lymphokine. The results indicated that treatment with 16 or 125 units/ml TEN-4 did not overcome T. cruzi-induced suppression when added together with suboptimal IL2 levels (50 units/ml) and did not improve mSC responsiveness when a restorative concentration of IL2 83 mm 2. lack of Restoration by Exogenous IFN-‘r of the capacity of WA- stimulated mSC to Proliferate after T. cruzi—induced Slppressiona IFN-r 3H-thym_idine incorporation (m x 10'3) by % Decreaseb (units/ml) sc + FHA so + PHA + T. cruzi 0 15.2 i 1.0 0.8 1 0.0c 95 8 14.6 i 1.0 0.8 : 0.2C 95 16 15.9 i 0.8 0.7 i 0.20 95 32 14.6 i 0.4 1.0 i 0.40 93 63 14.7 i 1.7 0.9 i 0.5C 94 125 13.9 i 0.5 0.9 : 0.1C 94 250 4.6 i 0.7C 0.9 : 0.0C 94 a Recombinant murine IFN-T was added at the indicated concentrations to cultures of use (2.5 x 106 cells/ml) containing 5 mg lam/ml in the presence or absence of 2.5 x 106 T. cruzi/ml. The cultures were incubated for 72 h and 1 pCi 3H-thymidine was present during the last 24 h. This set of results is typically representative of three separate repeat experiments. b Percent decrease with respect to the corresponding control (mSC + PHA, no IFN-T) . c p50.05, for the reductions in cm with respect to either control, i.e., mSC + PHA with or without IFN-T, as calculated by Student's "t" test. 84 (100 units/ml) was used (data not shown). We next examined whether T. cruzi would inhibit IFN-r production or secretion by hPETC. The presence of 5 x 106 parasites/ml in cultures of FHA-stimulated hPHVIC did not lead to a significant reduction in the levels of I‘r'N-T in the supernatants (decrease of 5% at 72 h; Table 3). This parasite comentration was twice that found to consistently decrease murine IFN—‘r production (Table 1) and suppress reproducibly mitogen-induced lymphoproliferation of hPHvIC (1) . The addition of exogenous human IFN-‘r did not restore the suppressed proliferative response of WC exposed to T. cruzi whether or not IL2 was present (data not shown). 85 Table 3. Iack of Effect of T. cruzi on IFN—T Production by hPEMC Material testeda IFN-‘r (unitszml) 48 h 72 h hPEMC 55 S5 T. cruzi S5 $5 hPHTC + T. cruzi 55 S5 T. cruzi + FHA 55 55 hPEVIC + PHA 120 125 hPHVIC + HiA + T. cruzi 96 119 a The tested materials consisted of the culture supernatants of WC (1.25 x 106 cells/ml) and/or T. cruzi (5 x 106 organisms/ml) incubated in the presence or absence of 5 lag/ml PHA. The supernatants were collected at the indicated time of culture and IFN-T activity was assayed by mdioimmwmssay. This set of results is typically representative of two separate repeat experiments . 86 DISCUSSION These results showed that the presence of T. cruzi in cultures of FHA—stimulated mouse spleen cells decreased the levels of IFN-‘r activity in the supernatants (Table 1). This decrease was not due to absorption, consumption, or inactivation of the lymphokine by the parasite since incubation of recombinant IFN-T with T. cruzi did not lead to a loss in antiviral activity even when incubated with four times as many parasites as were necessary to consistently suppress lymphoproliferation and reduce the levels of IFN-‘r in our culture system. Therefore, the decrease in IFN-T levels was due to reduced production or secretion of the lymphokine. We have previously reported that the incubation of PEMC with T. cruzi does not lead to losses in white cell numbers or viability and that the parasite does not remove significant amounts of nutrients or mitogen from the cultures (1) . Decreased proliferation by mSC from infected mice or by normal mSC incubated with T. cruzi in vitro is paralleled by decreases in I122 (8,32; Beltz and Kierszenbaum, unpublished results) and IFN-T production (Table 1). In contrast, T. cruzi is unable to decrease IL2 production by hPBMC urrier optimal culture conditions (L. A. Beltz, M. B. Sztein and F. Kierszenbaum, J. Immunol., in press) and, as reported herein, also has no significant effect on IFN—‘r production by hPHTC (Table 3). Thus, there appear to exist notable differences in how the parasite affects msc and hPEMC responses to PHA. IFN-r and IL2 are elements of a complex regulatory retwork and are able to affect each other's synthesis and utilization (5,11,12,24,27, 35) , with IFN-T production being upregulated by IL2 (5,24 ,35) . 87 Accordingly, antibodies to the IL2 receptor and dexamethasone, a drug which blocks IL2 synthesis, decrease IFN-1 production by mitogen- stimulated hPEdC (24). Furthermore, the addition of exogenous IL2 to macrophage-depleted mixed lymphocyte cultures (5) and unstimulated mo (35) induces IFN—r synthesis. Since T. cruzi decreases the production of both I12 and IFN-T by mSC while having no effect on the production of either lymphokine by hPBMC, it is thus possible that the parasite's ability to inhibit synthesis of the former lymphokine is at least partially responsible for the decrease in the latter. It should be noted, however, that lymphocytes from mice infected with 11W mop; have an impairment in IL2 but not IFN-T secretion (28) , demonstrating that normal IL2 levels may not be an absolute requirement for optimal IFN-r synthesis and opening the alternative possibility that T. cruzi may exert several independent suppressive effects on the T cells. A decreased capacity to produce IFN-T is characteristic of lymphocytes from patients with lepromatous, but not tuberculoid, leprosy (18) . Since leprosy is a spectrum of disease states with the lepromatous and tuberculoid forms being the most and least pathogenic, respectively, increased pathology in this condition appears to correlate with a reduced capacity to produce IFN-T. A similar defect is seen in susceptible, but not resistant, strains of mice infected with Leishmania donovani (15) or L. major (26). Thus, the ability of the host to produce IFN-r may determine the subsequent severity of the disease. 88 Inthecaseongr—ugi, severalen_\Li_tr_oand_iLy_iygfiniings suggest a possible role of IFN-r in host defense. Thus, the addition of IFN-r to cultures of both murine fibroblasts and macrophages increases their resistance to m infection by L__cru_zi trypc- mastigotes (21,37) . Furthermore, exogenous IFN-‘r acts synergistically with anti-trypanoscmal antibodies to decrease parasitemia and prolong survival of infected mice (20). The ability of Low; to reduce IFN—r production by use thus might decrease the host's capacity to eliminate the parasite. T. cruzi has been reported to decrease mitogen-induced proliferation of lymphocytes from either infected mice (8-10,13) or humans (33) . This defect is also observed when the parasite is co— cultured with mSC or hPHVIC from uninfected donors (1,14) . While IFN-T has been found to amplify lymphocyte responses to mitogenic stimulation under certain circumstances (7,12,27,31), the data presented in Table 2 show that exogenous IFN-r could not overcome the suppressive effect of T. cruzi. Therefore, it seems unlikely that reduced IFN-T production lead to the T. cruzi-induced reduction in lymphoproliferation. The capacity of mSC to produce IL2 is decreased following either in vivo or in vitro exposure to T. cruzi (8,32; L. Beltz and F. Kierszenbaum, unpublished results). The addition of IL2 to these Suppressed cultures restores their ability to secrete immunoglobulin (22,23,32) and to proliferate in response to mitogen stimulation (L. A. Beltz, M. B. Sztein and F. Kierszenbaum, J. Immunol., in press). Because IFN—r has been reported to increase the expression of IL2 receptors on both T cells (12,27) and monocytes (11) and higher levels 89 of the 11.2 receptor allow cells to respond to lower concentrations of I12 (4) , we tested whether IFN—r would enhame the restorative capacity of I12. This, however, was not the case: IFN-‘r did not act synergist- ically with IL2 or lower the concentration of IL2 required to achieve nSC recovery (data not shown). In conclusion, we have demonstrated a deficient capacity of nSC to produce or secrete IFN—r after exposure to T. cruzi. While this deficiency does not appear to be involved in the suppression of lymphocyte proliferation, it nevertheless may decrease the resistance of other host cells, such as macrophages, to parasite invasion and growth. Furthezmore, these results demonstrate a salient difference in the suppressive activities of T. cruzi toward mSC and hPHVIC. Whether this difference stems from the use of different populations of lymphocytes or from an actual difference in mouse and human lymphocyte responses to T. cruzi remains to be resolved. AW Thisworkwas supportedbyNASAgrantNAG9-181arxiaBimedical Research Support Grant from the College of Osteopathic Medicine, Michigan State University. 90 REFERENCES 1. Beltz, L. A., and F. Kierszaibaum. 1987. Suppression of human lymphocyte responses by W. Inmnmology 60:309—315. 2. Boraschi, D., S. Censini, andA. Tagliabue. 1984. Interferon—r reduces nacrophage—suppressive activity by inhibiting prostaglandin E2 release and inducing interleukin 1 production. J. Immunol. 133:764-768. 3. Brizko, D. B., an! F. Kierszerbamn. 1974. Isolation of W cruzi from blood. J. Parasitol. 60:1037—1038. 4. (antrell, D. A., and K. A. Smith. 1984. The interleukin-2 T-cell system: A new cell growth model. Science 224:1312-1316. 5. Farrar, W. L., H. M. Jchrem, arfl J. J. Farrar. 1981. Regulation of the production of immme interferon and cytotoxic T lynphocytes by interleukin 2. J. Immmol. 126:1120-1125. 6. Frasca, D., L. Adorini, S. Landolfo, and G. Doria. 1985. Enhancing effect of IFN-T on helper T cell activity and IL2 production. J. Imrmmol. 134:3907-3911. 7. Friedman, R. M., and S. N. Vogel. 1983. IFN's with special emphasis on the immune system. Adv.ImImJnol. 34:97-140. 8. Harel—Bellan, A., M. Joskowicz, D. Fradelizi, and H. Eisen. 1983. Modification of T-cell proliferation and interleukin 2 production in mice infected with W cruzi. Proc. Natl. Acad. Sci. USA 80:3466-3469 . 9. Harel-Bellan, A., M. Joskowicz, D. Fradelizi, and H. Eisen. 1985. T lymphocyte function during experimental Chagas' disease: production of and response to interleukin 2. Eur. J. Imrmmol. 15:438-442. 10. Haya, M. M., and F. Kierszenbalm. 1981. Ecperimental (hagas' disease. kinetics of lymphocyte responses and immunological control of the transition from acute to chronic W infection. Infect. Immun. 31:1117-1124. 11. Hermann, F., S. A. Cannistra, H. Levine, andJ. D. Griffin. 1985. quressicn of interleukin 2 receptors and binding of interleukin 2 by gamma interferon-induced himIan leukemic and normal monocytic cells. J. Exp. Med. 162:1111-1116. 12. Johnsm, H. M., and W. L. Farrar. 1983. The role of a gamma interferon-like lymphokine in the activation of T cells for expression of interleukin 2 receptors. Cell. Immunol. 75:154-159. 13. 14. 16. 17. 18. 19. 20. 21. 22. 23. 91 Kierszaibaum, F. 1981. On evasion of gygen_osgma_cru_z_i from the host immune response. Lymphoproliferative responses to trypanoscmal antigens during acute and chronic experimental Chagas' disease. Immunology. 44:641-648. Inleckar, J. R., and F. Kierszenbaum. 1983. Inhibition of mitogen- induced proliferation of mouse T and B lymphocytes by bloodstream forms of W. J. Immunol. 130:908-911. Fin-ray, H. W., H. Masur, and J. S. Keiflily. 1982. Cell—mediated immune response in experimental viseral leishmaniasis. 1. Correlation between resistance to Ieishmania donovani and lymphokine-generating capacity. J. Irmminol. 129:344-350. Murray, H. W., B. Y. Rubin, ard C. D. Rothemel. 1983. Killing of intracellular Ieishmania donovani by lymphokine-stimulated human mononuclear phagocytes. Evidence that interferon-1' is the activating lymphokine. J. Clin. Invest. 72:1506-1510. Nathan, C. F., H. W. Murray, M. E. Wiebe, and B. Y. Rubin. 1983. Identification of interferon—T as the lymphokine that activates human macrophage oxidative metabolism and anti-microbial activity. J. Exp. Med. 158:670-689. Nogueira, N.,G.Kap1an,E.Ievy, E.N.Sarno, P.Bhshner, A. Granelli—Piperno, L. Vieira, V. C. Gould, W. Ievis, R. Steinmn, Y. K. Yip, and z. A. (John. 1983. Defective T-interferon production in leprosy. Reversal with antigen and interleukin 2. J. Exp. Med. 158:2165-2170. Pfefferkorn, E. R., and P. M. Guyre. 1983. Recombinant human gamma interferon blocks the growth of Tbxmlasma gondii in cultured human fibroblasts. Fed. Proc. 42:964-967. Plata, F., F. Garcia-Pore, andJ. Wietzerbin. 1987. Immme resistance to W: synergy of specific antibodies and recombinant interferon gamma in vivo. Ann. Inst. Pasteur/Immunol. 138:397-415. Plata, F., J. Wietzerbin, F. Garcia-Pas, E. Falcoff, am H. Eisen. 1984. Synergistic protection by specific antibodies and interferon against infection by W in vitro. Eur. J. Immunol. 14:930-935. Reed, S. G., J. A. Inverso, ard S. B. Raters. 1984. Heterologous antibody respcmes in mice with chronic T. cruzi infection: depressed '1‘ helper function restored with supernatants containing interleukin 2. J. Immunol. 133:1558-1563. Reed, S. G., J. A. Inverso, and S. B. Raters. 1984. Suppressed antibody responses to sheep erythrocytes in mice with chronic W1 infections are restored with interleukin 2. J. Imummol. 1.33:3333—3337. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 92 Ream, G. H., ard N.-H. Yeh. 1984. Interleukin 2 regulates expression of its receptor and synthesis of gamma interferon by human T lymphocytes. Science 225:429-430. Rosztoczy, I., o. Siroki, and I. Beladi. 1986. Effects of interferons—a, -[3, and -1 on human interleukin—2 production. J. Interferon Res. 6:581- 589. Sadrick, M. D., R. M. Iocksley, C. Table, and H. V. luff. 1986. Murine cutaneous leishmaniasis: resistance correlates with the capacity to generate interferon-r in response to Ieislmani_____a antigens in vitro. J. Immunol. 136: 655-661. Sdraxrich, P., U. Ucer, M. Killian, and K. Pfizermnaier.1985. Differential effects of gamma-interferon on human T cells during primary activation in vitro, p. 63-67. _I_n C. Sorg, A. Schimpl, and M. Iandy (eds) , Cellular and molecular biology of lymphokines. Proceedings of International Workshop in W. Germany, Academic Press, Orlando. Sileghan, M., R. Haulers, and P. De Baetselier. 1987. Experimental Momma brucei infections selectively suppress interleukin 2 production and interleukin 2 receptor expression. Eur. J. Immunol. 17:1417-1421. Smith, K. A. 1984. Interleukin 2. Ann. Rev. Immunol. 2:319—333. Samenfeld, G., A. D. Mandel, and T‘. C. Merigan. 1977. The immmosuppressive effect of type II mouse interferon preparations on antibody production. Cell. Immunol. 34:193—206. Sonnenfeld, G., A. D. Mandel, and T. C. Merigan. 1978. Time and dosage dependence of immancenl’mncement by murine type II interferon preparations. Cell. Immunol. 40:285—293. Tarleton, R. L., and R. E. Kuhn. 1984. Restoration of in __v__itrc immune responses of spleen cells from mice infected with 1M cruzi by supernatants containing interleukin 2. J. Immunol. 133:1570-1575. T'eixeira, A. R. L., G. Teixeira, V. Macedo, and A. Prata. 1978. Acquired cell-mediated immunodepression in acute Chagas' disease. J. Clin. Invest. 62:1132-1141. Trirrhieri, G., and B. Perussia. 1985. Immune interferon: a pleiotropic lymphokine with multiple effects. Immunol. Today. 6:131-136. Vilcek, J ., D. mitiksen—Destefam, D. Siegel, A. Klim, R. J. Rdab, and J. 12. 1985. Regulation of IFN—‘r induction in human peripheral blood cells by exogenous and endogenously produced interleukin 2. J. Immunol. 135:1851-1856. 93 36. Villalta, F., ard W. Iecn. 1979. Effect of purification by DEAE- cellulose column on infectivity of W blood fonts. J. Parasitol. 65:188-189. 37. Wirth, J. J., F. Kierszenbaum, G. Samenfeld, and A. Zlotnik. 1985. Enhancing effects of gamma interferon on phagocytic cell association with and killing of W. Infect. Immun. 49:61-66. 94 m3 NOVEL MECHANISM FOR W-NWCED SUPPRESSION OF HUMAN IXMHTOCYTES: INHIBITION OF M 2 RECEPIUR EXPRESSION 95 ABS'I'RAC’II Co-culture of blood forms of W - the causative agent of Chagas' disease - with human peripheral blood mononuclear cells impaired the capacity of T lymphocytes to express surface receptors for interleukin 2 (IL2R) . This effect was evidenced by marked reductions in both the proportion of Tac+ cells and the density of T‘ac antigen on the surface of the positive cells, determined by flow cytometry. The extent of the inhibition increased with increasing parasite concentrations. Under optimal or suboptimal conditions of stimulation with either phytohemagglutinin or monoclonal anti-CD3 - specific for an epitope of the CD3-’I'i human T cell antigen receptor complex- the presence of T. cruzi curtailed the capacity of T lymphocytes to proliferate and express IL2R but did not affect IL2 production. Furthermore, the addition of exogenous IL2 did not restore the responsiveness of suppressed human lymphocytes but did when mouse lymphocytes were used instead. Therefore, unlike mouse lymphocytes, human lymphocyte suppression by T. cruzi did not involve deficient IL2 production and was accompanied by impaired 112 utilization. Co-culture of human monocytes/macrophages with suppressive concentrations of '_I'_. M increased interleukin 1 (IL1) production and the parasite did not decrease IL1 sweretion stimulated by a bacterial lipopolysaocharide. Therefore, the suppression of 112R expression and lymphoproliferation is not likely to have been an indirect consequence of insufficient IL1 production due to infection of monocytes or macrophages. We have previously shown that suppression of human lymphocyte proliferation by 96 T. cruzi is not caused by nutrient consumption, absorption of IL2, lymphocyte killing or mitogen removal by the parasite. Therefore, these resultsuncoveranovel suppressivemedianisminducedbyL @111, involving inhibited expression of IL2R following lymphocyte activation and rendering T‘ cells unable to rweive the IL2 signal required for continuation of their cell cycle and mounting effective immune responses. 97 INI‘KDIIJCI‘ION Chagas' disease - caused by the protozoan W—is a major health problem in South and Central America. Its acute phase, bothinlaboratcryanimalsand inhumans, isawompaniedbyastate of suppressed immunity believed to facilitate the establishment and dissemination of the etiologic agent in the host (1-15) . Several immunological abnormalities have been identified in mice infected with T. cruzi, including increased levels of suppressor lymphocytes and macrophages (1—4) and diminished levels of T cells (5) in the spleen, impaired lymphocyte proliferation in response to mitogens (2,4-9) or parasite antigens (10), suppressed antibody-forming capacity (11) and impaired IL2 production (4,7,12) . How these abnormalities are induced is not known and, unfortunately, differences in how mouse and human lymphocytes are affected by T. cruzi make it difficult to extrapolate these findings to human infection. Thus, murine splenic lymphocytes - whether from infected animals (12, 16, 17) or co-cultured with the parasite in vitro (Beltz and Kierszenbaum, unpublished results)- display reduced interleukin 2 (IL2)—producing capacity, the consequences of which are overcome by the addition of exogenous IL2 (12, 16, 17); this is not the case for human lymphocytes suppressed by the parasite (15). To study the early alterations that T. cruzi irduces in hrmen lymphocytes and to explore the medianisms) involved, weusedaninvitrosysteminwhich lymphocytesarxi monocytes/macrophages were incubated with the parasite in the presence of lymptocyte—activating stimuli. We report that T. cruzi inhibits the 98 capacity of human T lymphocytes to express surface interleukin 2 receptors (II.2R) upon activation. This effect may render T lymphocytes unable to receive the IL2 signal required to proceed with their cell division cycle and mount significant levels of immunity. 99 MATERIAISANDMEIHOIB Parasites. Blood (trypcmastigote) forms of Tulahuen strain L _cin were isolated from the blood of Crl-CD-1(ICR) Swiss mice (Charles River Breeding laboratories) infected 2 weeks previously with 2 X 105 parasites intraperitoneally. The parasites were purified by centrifugation over a mixture of Ficcll-Hypaque of density 1.077 (18) , followed by chromatography through DEAE—cellulcse (19) . The eluted organisms (100% trypcmastigotes) were concentrated by centrifugation (800 X g, 20 min, 4°C) and resuspended at the desired concentration in RPME 1640 medium supplemented with 5% (vol/vol) heat-inactivated fetal bovine serum and containing streptomycin at 100 pg/ml and penicillin at 100 units/ml (henceforth referred to as RPMHS) . Perile blood mononuclear cells (PHVIC) . Normal PBMC from healthy volunteers were purified by density gradient centrifugation through a mixture of Ficcll-Hypaque of density 1.077 (350 x g, 20°C, 45 min). After three washings with serum-free RPMI 1640 medium, the cells were resuspended at the desired concentration in RHVII+S. Cell viability, determined by trypan-blue-dye exclusion, was >99%. Mouse spleen cells. Single cell suspensions of inbred CPA/J mouse (Jackson laboratory) spleen cells were prepared in RFMHS as described (13) . Recombinant ILZ. Recombinant, glycosylated human IL2 was purchased from Genzyme (Boston. MA) - Lw proliferation assay. PEMC were incubated in RIMES (5% (132; 96-well plates) at 37°C for 96 hours with or without 0.6 or 5 100 pg/ml PHA (Sigma Chemical Co.) or 6 or 25 ng/ml anti-C03 (Ortho Diagnostics) [a monoclonal antibody specific for an epitope of the T cell antigen receptor complex CD3-Ti (20)] in the presence or absence of T. cruzi. The culture volume was 0.1 ml. All conditions were tested in triplicate. Each culture received 1 ”Ci 3H—thymidine 24 hours before termination by automated harvesting. Radioactivity was determined in a liquid scintillation counter; the results were expressed as mean counts per minute (cpm) i” 1 standard deviation. The concentrations of PIMC and parasites at zero time, and after 48 and 96 hr of culture are given under Results. Viability was established by trypan blue exclusion. Measurement of IL2 activity. ILZ activity was determined in the supernatants of 48—hour PBMC cultures set up as described above except that the final volume was 1.5 ml and 24-well plates were used. The supernatants were passed through a 0.22-um-pcre—size filter to remove parasites if present and stored at -20°C until tested. The IL2- dependent HT—2 cell line was used to determine IL2 activity (15) and theresultswereexpressedasunits/ml inreferencetoastandardIL‘Z preparation (concanavalin A—stimulated rat spleen cell culture super- ernatants) which was arbitarily assigned a value of 100 units/ml (21) . Flow mule determinations. PEMC (1.25 x 105 cells/ml) were incubated in RIMI+S (5% (132; 24—well plates) at 37°C for 48 hours with FHA or anti—CD3 monoclonal antibody in the presence or absence of L orig. After 48 hours, the cells were washed three times with phosphate-buffered saline pH 7.2 containing 1% bovine serum albumin (Sigma) and were stained by treatment with anti-T‘ac monoclonal antibody 101 [which recognizes an epitope of the human ILZR (22) and was kindly provided by Dr. T. A. Waldmann, National Institutes of Health] followed, after washing, by flourescein-conjugated F(ab')2 derived from goat anti—mouse IgG antibody (Tago Immunodiagnostics) . The cells were fixed in 1% formaldehyde and were analyzed in a FMS IV flow cytometer. Ten thousand PEMC, gated to exclude T. cruzi and cell debris, were accumulated for each histogram. The percentage of IL2R+ cells [i.e., %T‘ac+ cells (22)] in each preparation was calculated after subtracting the background of nonspecific labeling with MOPC—21 (a nonspecific IgG derived from a mouse myeloma cell culture) and flucrescein—conjugated F(ab')2 anti-mouse IgG. The mean channel number of the logarithm of the fluorescence intensities (MFCh) was the parameter used to compare the relative density of 'I‘ac antigen on the different Tac+ cell populations. The logarithm of fluorescence intensities was distributed over 256 channels. Production and bicassay for interleukin 1 (IL1) . One ml of PEMC suspension at 2.5 x 106 PEMC/ml was incubated at 37°C for 2 hours in 24-well plates; the adherent cells (>98% monocytes/macrophages by both morphological criteria and positive staining for non—specific esterase) were incubated with medium alone or containing 5 X 106 or 1 X 107 trypcmastigotes/ml in the presence or absence of 20 pg/ml bacterial lipopolysaocharide (LPS, Difco) at 37°C for 24 hours (5% CD2). Culture supernatant dilutions were added to mouse thymocyte cultures stimulated with a suboptimal PHA concentration (1 pg/ml) as described in detail by Meltzer and Oppenheim (23) . The results were expressed as cpm representing 3I-I--thymidine incorporation by proliferating thymocytes. 102 RESUIE’S T. cruzi inhibits IL2R egression El human 1%. In the presence of bloodstream forms of T. cruzi, Pmc stimulated with FHA showed a markedly decreased capacity to express surface 112R. This effect was parasite concentration dependent (Table 1) and was evidenced byadecreaseinthepropartion ofIL2R+cellsaswellasinthe I density of Tac antigen on the surface of the positive lymphocytes whether optimal or suboptimal PHA concentrations were used (Fig. 1). To establish whether this effect was also produced under conditions known to mimick antigen-induced lymphocyte activation (23) , we carried out similar experiments using anti-CD3 as the stimulant. The results demonstrated that T. cruzi also impaired IL2R expression in this case whether the lymphocytes were stimulated with optimal or suboptimal amounts of anti-CD3 (Fig. 1). T. cruzi did not stain positively for Tao antigen [i.e. , did not bind anti-Tac or file fluorescein—labeled F(ab')2 anti-mouse IgG] whether or not co-cultured withPBMCinthepresenceorabsenceofPHAoranti—Cm, anddidnot respond to recombinant IL2 (20 to 250 units/ml) with altered levels of 3I-I-thymidine incorporation (data not shown). T. cruzi has been shovm to suppress 3H—thymidine incorporation by Pmc stimulated with suboptimal or optimal HiA concentrations (15) . TheresultspresentedinTablezindicatedthatthiswasalsothecase when the lymphocytes were triggered with suboptimal or optimal concentrations of anti-CD3. Under the suboptimal or optimal stimulatory conditions used in the experiments described above (0.6 and 103 'IABIEI T. cruzi-induced mressicn of 112R expression by human 1W3 T. cruzi/ml PHA % T‘ac+ cells (%V) MK!) 0 Absent <2 0 Presemt 46.3 114 1 x lo6 Present 34.9 (—25) 112 5 x 106 Present 25.5 (~43) 67 10 x 106 present 4.2 (-91) 86 a Olltures of PEVIC (1.25 X 106 cells/ml) with or without T. cruzi were incubated with or without 5 pg/ml PHA for 48 hr. The cells were stained with anti-Tac and fluorescein-labeled goat F(ab')2 anti—mouse IgG. The percentage of Tac+ cells was calculated after subtracting the background of nonspecific labeling (see Materials and Methods). Percent variation (%V) with respect to the value obtained with FHA alone = [(value with parasites - value without parasites) / value without parasites X 100]. This set of results is typically representative of two separate repeat experiments. 104 Figure 1. Effects of T. cruzi on IL2R expression by human lymphocytes. PEMC were incubated at 37°C for 48 hours with FHA or anti-CD3 monoclonal antibody in the presence or absence of 5 X 106 L _cru_zi/ml. The cells were processed for flow cytometric analysis as described under Table 1. (A) Responses to an optimal FHA concentration (5 pg/ml): PHA, 55.0% Tac+ cells, mm: 120; new. cruzi, 40.3% Tac+ cells, MFCh 103. (B) Responses to a suboptimal PHA concentration (0.6 cells pg/ml): PHA, 28.6% Tac+ cells, MFCh 126; new. cruzi, 19.6% 'Iac+, mm 109. (C) Responses to an optimal anti-C03 concentration (25 ng/ml): anti—CDB, 41.5% Tac+ cells, MFCh 137; anti— CD3+JI‘. cruzi, 27.0% Tac+ cells, MFCh 121. (D) Responses to a suboptimal anti—CD3 concentration (6 ng/ml): anti-CD3, 20.5% Tac+ cells, mm 133; anti-CD3+T. cruzi, 16.0% Tac+ cells, MFCh 117. In control PBMC cultures (no PHA or anti-CD3) , the proportion of Tac+ cells never exweded 4%. The sets of data for PHA— and anti-CD3— induced responses are representative of five and three separate repeat experiments, respectively. ME‘Ch is the mean channel number of the logarithm of the fluorescence intensity. 105 >tmzwhz. ucaoemscam‘l wczmcmwecie was nascemfimml .55 H . S. necctmfimmx 3A|1V138 33 BEBWDN 106 TABLE 2 Effects of T. cruzi on the cagcity of human 1% to proliferate and secrete IL2 in rfimnse to PHA or anti-CD3a Mitogen 3Iii-thymidine incorporation IL2 (units/m1) PEHC PHVIC+T. cruzi (%V) PEMC PEMC+’I‘. cruzi PHA, 5 ug/ml 88.51-6.0 31.8125 (-64) 353 435 HIA, 0.6 pg/ml 54.5111 23.51-0.6 (—57) 4 25 Anti-CD3, 25 ng/ml 36.7108 16.2i1.2 (-56) 31 18 Anti-CD3, 6 ng/ml 6.23:2.4 3.4106 (—45) 1 7 a PEMC (1.25 x 106 cells/ml) were incubated at 37°C for 96 hours with FHA or anti-CD3 monoclonal antibody in the presence or absence of 5 x 106 parasites/ml. Each culture received 1 ”Ci 3H-thymidine 24 hours before termination. All values of 3H—thymidine incorporation are expressed as thousand cpm. The background values obtained without mitogen (0.2 and 7.0 for PEMC cultures without and with parasites, respectively) were subtracted from those obtained in the presence of mitogen. All differences between values obtained with and without parasites were statistically significant (250.02, Student's "t" test). IL2 activity was concurrently determined in the supernatants of 48-hour PEMC cultures set up as described above. The differences between IL2 levels in the absence and presence of T. cruzi were not significant. %V, see legend to Table 1. This set of data is typically represent— ative of three repeat experiments. 107 5 lug/ml HIA, respectively, and 6 and 25 ng/ml anti-CD3, respectively, with 1.25 x 106 PBMC/ml) , the levels of IL2 activity found in the supenlatantswerenotdecreasedbythetrypanosaresarxiinoccasional instances ILZ levels were, in fact, greater in the presence of the parasite than in its absence. We previously reported that supraoptimal culture conditions [i.e., higher mitogen (25 or 50 pg/ml FHA) and PBMC (5 x 106 cells/ml) concentrations] result in reduced IL2 production in the presence of T. cruzi (15) . When we tested the IL2R—expressing capacity of human lymphocytes under these supraoptimal conditions in the presence of the parasite, the proportions of Tac+ cells in cultures stimulated with 25 and 50 [lg/ml H—IA were 22 and 45% lower, respectively, than those found in parasite-free cultures; IL2 activity was reduced by 24 and 32%, respectively (data not shown). A comparison of the concentrations and viability of the PEMC revealed that the presence of the parasite caused no significant difference in these parameters 48 hr after culture initiation. Thus, for example, in a representative experiment, the PEMC concentrations measuredat48hrintheabsenceandpresenceof'l‘. cruziwereleo6 (99% viable) and 0.9 x lo6 (99% viable) PHVIC/ml, respectively, when no mitogen was present. The corresponding values in the presence of 5 ug/ml PHA were 0.9 x lo6 (99% viable) and 1 x 106 (99% viable) panic/ml. 'Ihe values obtained after 96 hr, in the absence of mitogen, were 6.5 X 105 (99% viable) and 7.8 x 105 (92% viable) PBMC/ml, in the absence and presence of parasites, respectively. FHA-induced lymphoproliferation and the suppressive effect of the parasite were evident after 96 hr; 1.5 X 106 (96% viable) and 7.8 X 105 (90% viable) PEMC/ml were present 108 in cultures lacking and containing the organisms, respectively. Although there were some fluctuations in the 48- and 96-hr PHJC concentrations from experiment to experiment, the differences or lack of difference summarized in the preceding sentences were consistently deserved. hiring the 96-hr culture there were minimal variations in total parasite concentration. However, the proportion of trypcmastigotes (100% at the initiation of the cultures)- was reduced to approximately 50% and 40% after 48 and 96 hr, respectively, whether mitogen was present or not, the rest being amastigote-like organisms. Epimastigote forms were not detectable. In the various experiments, 3H—thymidine incorporation by the parasites alone represented 5 to 15% of the cpm obtained with parasites plus PEMC stimulated with 31A or anti-CD3. This contribution, part of the background, was subtracted from the experimental values presented in Tables 2 and 3. We have previously sham that T. cruzi does not absorb, consume or inactivate IL2 (15) , ruling out removal of this lymphokine by the parasite as the suppressive mechanism. However, because lymphocytes from infected mice recover their responsiveness to mitogens or antigens after addition of exogenous IL2 (12, 16, 17) , and IL2 lip—regulates the expression of its own receptor (24), we tested the possibility that the lymphocytes affected by the parasite might have required more IL2 than was produced by mitogen-stimulated PHVIC to normally express IL2R and proliferate. As shown in Table 3, neither IL2R expression nor the level of 3H-thymidine uptake returned to normality after addition of 250 units/ml IL2. Similar results were obtained in experiments in which doses of ILZ up to 600 units/ml were used and 3H—t‘nymidine 109 .— opens ucmmop mum .>x .mbmmams or“ so m>wnmsemmmtcmt aspmcmcmu use mou-_s:c use <:e toe once to news wee. .Aemme as: m.«cmuaem .No.owes usec_eecm_m sepmceammeesn new: mcsmmmtec access: new sew: cccmebac mmsF~> cmmzeme mccewtmeemc spa .s—m>_ecccmoe .NnH see: use “screw: Wtho .e me_emeaecu mutaepsc toe “.0 can o.~ use .apm>_eooammt .NnH new: use «sores: mmtsupau toe m.N use ~.o norm: ”capes use «muemctma to oucomam use cw mou-eeec sue: semewtmaxw use see .x_u>mucmammt .~b_ sue: use seesaw: w~=ec .e mcmcwmecco cuteness to» ¢.m can v.¢ can unanswered «access meumemmt .Nnm ss_: use «noses: motes—so toe o.H can m.o “chooses to mucmmmtc use cm noc_mbco whose sore unsceteeam «to: cmmcu_s eschew: cccwuucc nm=P~> use .eeo scanners no cmnmmeaxm wen cc_uatoatcuce ucmueezse-z so nus—m> PP< m .ummamcs assume so “cacao «cmpmswacm cm um>mmumt mossy—:0 Fetacoo “cmooeme we“ tween a—cecsumsem A_e\mewca omm ems motsupsu mucmtcotecm use ca emcee we: NnH case; cmeaexmcoarm .ecec_necome ._~ open» as ccmmw— use cw ucn_tcmmc he cassettes chm: motsesau uzma use a nIIIIIIIIIIIIIIIIIIIIllIItIIIIIIIIIIIIIIIlIIIIIIHIIIlIIIIIIJHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIlllllllllll ms- Hse\m.HN ems\o.mm Ase-c m.c . e.e m.m . e.w~ .+ _e\ee ms .mec-esaa em- ess\s.es NNsxm.om Aem-c e.~ n m.m e.H H e.em - _e\me mm .mec-.se< Hm- ses\e.me mms\m.me Ame-e a.o H m.ma s.c H e.am . Fe\ae m .exa em- aca\m.am eNH\e.am imm-c e.c u a.e ~.m H c.se - Fe\ee m .eza .......III..y.lrlI.III.IIlalIIIIIIIII.IIIIII..uIIlIxlI.I.IaIuInIII..I.IIIunIIIIlIiI...1.......|n..........1.n a_smu acls\.caea ecus\.oaen I'll!!! +oae _Naro .e.uzma sea—a csma sees _Nsro .e.czma cease czma Nh_ aaoeamo.c fill-l toe >x cowmmmecxm mmn_ coeectocecccs mc_csesre-xm cc combeucq IIIIIIIIIIIi1eIlIIIiIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIlllzllllllllll embH macaw cxe so moccnne to mocmmctc ces e_ mou-_ecc to are see: ccwcsse_em Iiiiii m abscesses_ cases so coemwctnxc mNnH use somectcctcoem ccscmesse-xm mm e~sto .H cc meshes“ -~ unm99%. Proliferaticm assay. PEMC were incubated in 96-well plates in a volume of 0.1 ml in the presence or absence of either 5 pug/ml phytohemagluttinin (FHA; Sigma Chemical Co., St. Iouis, MD), 25 ng/ml OKI'3 (Ortho Diagnostics, Raritan, 1U) , an antibody reactive with CD3, or a 1:100 dilution of anti—Tllz and anti-’I'113 monoclonal antibodies (reactive with two distinct epitopes of 0132; generous gifts of Dr. S. F. Sdflossman, Dana-Farber Cancer Institute, Boston, MA). Some wells also contained T. cruzi at concentrations ranging from 2.5 to 10 X 106 147 parasites/ml, which replaced an equal volume of RHdI+5%FBS. Cultures were incubated at 37°C, 5% 002 for 96 hrs, with 1 uci of 3H-thymidine (specific activity = 2.0 Ci/mmole; New England Nuclear Biotechnology Systems, Wilmington, DE) being present during the final 24 hrs. oiltures were terminated by automated harvesting and the levels of incorporated radioactivity were determined in a liquid scintillation counter. Resultswere expressedasmeancountsperminute (con) i 1 standard deviation of triplicate cultures. IL2 Assay. PHVIC were incubated in 24-well plates in a volume of 1.5 ml in the presence or absence of 5 pg FHA/ml or a 1:100 dilution of anti-T112 + anti—T113 antibodies with or without 5 X 106 T. cruzi/ml. At 48 of incubation, cultures were centrifuged (350 X g, 10 min, 4°C) and the supernatant was clarified by filtration through 0.22 inn-pore- size filters and stored at -20° C until assayed for IL2. IL2 activity was determined using the IL2-dependent CELL-2 cell-line as previously described (10). Results are expressed as units IL2/ml in reference to a standard IL2 preparation of 48-hr concanavalin A—stimulated rat spleen cell supernatants which was assigned a value of 1000 units/ml (20). Expression of the 112R. The cell pellet of the above described cultures was washed three times with phosphate-buffered saline containing 1% bovine serum albumin (PBS-BSA) and subsequently incubated for 30 min with a fluorescein—conjugated antibody directed against the IL2R, anti-2A3 (Becton—Dickinson, Mountain View, CA), following the manufacturer's instructions. After fixation in 19s formalin, PE’IC (10,000 cells per condition) were analyzed in a FACS IV flow cytometer 148 gated to exclude T. cruzi and cell debris. The percentage of IL2R+ cells was calculated after subtracting the background of nonspecific labeling with fluorescein-conjugated anti-keyhole limpet hemacyanagin ' (Becton—Dickinson) . The mean channel number of the logarithm of the fluorescence intensities (MF‘Oi), distributed over 256 channels, was used to compare the relative density of the IL2R on the different positive pqnlations. Determination of the expressim of T112 and 'I'll3. Cultures of PE’ICsetupin24-wellplatesinthepresenceorabsenceofPHAas described above were incubated for periods of time ranging from 6 to 24 hrs, washed with PBS-BSA, and incubated for 30 min with a 1:150 dilution of T112 or T113, or with control mouse IgG (C‘albicchem, La Jolla, CA) . After two washes with PBS—BSA, cells were incubated for 30 min with fluorescein—conjugated F(ab')2 goat anti-mouse IgG and analysed by flow cytometry as described above. The percentages of PEMC expressing T112 and T113 were calculated after subtracting the background of nonspecific labeling obtained with the control mouse IgG, and the MFCh was used to determine the densities of these epitopes. 149 T. cruzi decreased the proliferative capacity of PEMC stimulated via either the CD3-Ti or the CD2 pathways of T lymphocyte activation (Table 1). The decreased responsiveness was dependent upon parasite concentration, with low levels of suppression being observed when 2.5 X 106 T. cruzi/m1 were used, and the extent of suppression increased when the parasite concentrations were raised to 7.5 or 10 X 105 organisms/ m1. T. cruzi did not reduce IL2 production by PEIIC after stimulation ' with ETA or anti-C02. Indeed, IL2 levels in the anti-CDz-stimulated cultures were increased by the presence of T. cruzi (data not shown). A small increase in IL2 concentrations is occassionally also noted when FHA or anti-CD3 is the stimulant (Chapter 3). The expression of the IL2R was decreased by T. cruzi following activation by PHA or anti—CD2 (Table 2). This decrease was seen both in the number of IL2R+ cells and in the density of the receptor on the positive cells. When anti-CD2 was used in activation, the decrease was of a lesser extent than that observed with FHA. It should be noted that T. cruzi does not bind anti—IL2R antibodies (Chapters 3 and 4), therefore, the decrease in the levels of IL2R observed on the PBMC was not due to the parasite reducing the availability of antibodies to these cells. 150 Table 1. T. cruzi Inhibits Blastcgenesis by Both the T Cell Receptor and C132 Pathwaysa 3H—‘ItIR incorporation (cpm x 10'3) in the presence of the followg' concentrations of T. cruzi ( x 10'61m1): Stimulus 0 2.5 5.0 7.5 10.0 PHA 61.7 _+_- 3.0 20.9 i 1.1 6.3 i 0.1 3.1 i 0.5 1.8 i- 0.3 anti-CD3 37.2 i 2.6 22.1 i 1.4 12.0 i 0.5 5.6 i 0.3 2.3 i 0.4 anti—(:02 14.2 i- 0.5 6.5 i 0.2 2.4 i 0.1 0.9 i 0.3 0.4 _t 0.1 a PMCwereincubated for96hr~sinthepresenceorabsenceof PHA, anti-C03, or anti-Tllz + anti-41113 (anti-CD2 antibodies) with or without T. cruzi in 96-well plates. One uci of 3H-thymidine (3114mm was present per well during the last 24 hrs. All differences between values obtained for cultures with and without T. cruzi are statist- ically significant (P50.05, Student's "t" test). These results are typically representative of three separate repeat experiments. 151 Table 2. The Effect of T. cruzi on IL2R Expression After Stimlation by either KIA or anti-CD2":I Stimlus T. cruzi % IL2R+ cells W HIA ' 55 . 7 161 H'XA + 35 u 9 112 anti-C132 - 48 . 9 167 anti-CD2 + 41 . 0 124 a PMCwereincubatedfortIShrsinthepresenceorabsenceof FHA or anti-Tllz + anti-'I‘113 (anti-CD2 antibodies) and/or T. cruzi (5 X 106 organisms/m1). IL2R expression was determined as described under Materials and Methods. less than 6% of the Pmc incubated in the absence of PHA or anti-C02 expressed IL2R. These results are typically representative of three separate repeat experiments. 1" m, mean channel number of the logarithm of the fluorensence intensities of positive cells. 152 DISGJSSION T. cruzi decreased the proliferative responses of PEVIC stimulated by either FHA, anti-CD3, or anti-CDZ (Table 1). This suppression was observed with concentrations of T. cruzi ranging from 2.5 to 10 X 106 parasites/ml in a dose-dependent fashion. Since the (:02 pathway has recently been demonstrated to be only active in memory T cells and not in naive T cells (J. A. Byne, J., L. Butler, E. L. Reinherz, and M. D. Cooper, Abst. Ann. Nbet. Fed. Amer. Soc. Exper. Biol. 1988, FASEB J., vol.2, p. A1240.) , the data in Table 1 are the first demonstration of the ability of T. cruzi to suppress responses of memory T lymphocytes. Anti-CD3 is an antibody directed against the CD3-Ti antigen receptor complex and triggers T cell activation through this molecule (11). (DZ is a marker found on all thymocytes and mature T lymphocytes whose cell surface expression is upregulated upon lymphocyte activation (21) . Two antibodies directed against the T112 and T113 epitopes of CDZ are able to act in concert to stimulate IL2 production (16) , ILZR expression (16), and cell division (12). The antibody to T112, an epitope found on all resting T cells, is able to induce a confirma- tional change in CDZ which allows the expression of the T113 epitope (12) . The subsequent engagement of T113 by antibody then leads to the above noted events. Since CD2 is not associated with the CD3-Ti ccmplex on the cell surface (17) and since it is operative in the stimulation of CD3” thyrmcytes (16), it thus constitutes an alternative pathway of T cell activation. Since this pathway does not require the presence of monocytes (12) , the data in Table 1 demanstrate that L 153 M is able to exert its suppressive effect directly upon the T 1W- T. cruzi was previously found not to decrease the production or secretion of ILZ by PEVIC stimulated by FHA or anti—CD3 under optimal culture conditions (Chapter 3). The presence of T_._cruLi in Pmc cultures increased the levels of IL2 following stimulation by anti—C132. This is occassionally also seen when PEA or anti—CD3 is used as the mitogen (Chapter 3). The underlying cause of the argumentation in IL2 levels is not clear at this time, but may have resulted from a decrease in the ability of the PHVIC to internalize and degrade this lymphokine. Alternatively, IL2 production may be increased by the presence of L guz_i_ inthe cultures. Inthisregard, it shouldbenotedthatL _cru_z_i does not release IL2 nor does it induce resting lymphocytes to do so (data not shown). It has also been found that the parasite does cause a reduction in IL2 production under supraoptimal stimulatory conditions (5 x 106 PBMC/ml, 2 25 pg FHA/ml; 10). Since T cells exposed to T. cruzi demonstrate a reduced capacity to proliferate in the face of normal or above normal levels of ILZ, we next examined the effect of the parasite on the expression of the ILZR. As was previously reported (10) , _T_‘.__cru_zi inhibited the expression of the ILZR on Hn-stbmuated mac (Table 2). Both the number of mm‘ cells and the density of the receptor on the positive cells was decreased. Similar results were obtained when anti—C02 was used as the stimulant, although the reduction was less pronounced than that seen in FHA—stimulated cultures (Table 2) . Preliminary results indicate that the upregulation of the T112 154 epitope of CDZ ard the expression of the T113 epitope of this molecule which occur during the first 6 to 12 hrs of lymphocyte activation were inhibitedbythepresence of T. cruzi intheculmres (datanot shown). Work is currently in progress to determine the kinetics of the suppressed expression of these epitopes. Since T. cruzi decreases both the mn'nber of activated. cells bearizg T112 and T113 and the densities of these epitopes on positive cells (data not shown), it is thus possible that these events are at least partially responsible for the suppressed proliferative responses of lymphocytes triggered via the CD2 activation pathway. The ligand for the T112 epitope of CDZ has recently been identified as lymphocyte function-associated antigen—3 (LFA—3) , a glycoprotein present in endothelial, epithelial, ard connective tissues, as well as on most blood cells (15,22,23). While resting T cells bird to a lFA-3-like molecule on sheep erythrocytes, resulting in rosetting (23) , only activated T cells with enhanced expression of CD2 are able to bird autologous erythrocytes which bear lower levels of LEA-3 than their cvine counterparts (23,24) . The birding of T lymphocytes to the LFA—3-like molecule on sheep erythrocytes allows subsequent activation by anti—T113 (25) . The putative natural ligard for the T113 epitope awaits identification. The role of the C132 alternative pathway of T cell activation is not completely understood at this time. However, this pathway may be of particular importance for the activation of CD3' thymocytes by LFA- 3+ thymic epithelial cells during ontogeny (26-28) . The upregulation of CDZ ard the expression of the T113 epitope on activated mature T 155 lymphocytes may enhance activation via CD3-Ti and increase the avidity of T cell interactions with other mo+ hematopoietic cells (15,29). While the CD3-Ti carplex and CD2 are distinct entities ard not associated on the cell surface (17) , these two activation pathways do have mutmal regulatory interactions. For example, the removal of CD3 from the cell surface blocks activation via CDZ (12) . Furthermore, stimulation by the C02 pathway irduces phosphorylation of C03 (30). It appears, therefore, that the CD3-Ti and C132 pathways involve separate signals transmitted through separate ard distinct receptors, each system, nevertheless, being able to exert regulatory effects upon the other. air firdings irdicate that T. cruzi is able to inhibit T lymphocyte activation through both of these pathways. 1. 10. ll. 156 REFERENCES Teixeira, A. R. L., G. Teixeira, V. O. Macedo, ard A. Prata. 1978. Acquired cell-mediated imumlncdepression in acute Chagas' disease. J. Clin Invest. 62:1132. Voltarelli, J. C., E. A. Donadi, ard R. P. Falcao. 1987. Ilmmmcsuppression in human acute Chagas disease. Trans. 39y. Soc. 1139. Med. Egg. 81:169. Hayes, M. M., ard F. Kierszenbamn. 1981. Experimental Chagas' disease: kinetics of lymphocyte responses ard immunological control of the transition from acute to chronic W infection. Infect. Immun. 31:1117. Kierszenbaum, F. 1981. On evasion of W from the host immune response. Lymphoprcliferative responses to trypanosmal antigens during acute ard chronic experimental Chagas' disease. Immunclgy 44:64 . Rowland, E. C., and R. E. Kuhn. 1978. Suppression of cellular responses in mice during W infection. Infect. Immun. 20:393. Reed, S. G., C. L. larscn, and C. A. Speer. 1977. Suppression of cell-mediated immunity in experimental Chagas' disease. A Parasitenk. 52:11. Rowland, E. C., ard R. E. Kuhn. 1978. Suppression of anamnestic cellular responses during American trypanosomiasis. J. Parasitol. 64:741. Clinton, B. A., L. Ortiz-Ortiz, W. Garcia, T. Martinez, ard R. Capin. 1975. Momma cruzi: early immune responses in infected mice. _Egzp. Parasitol. 37:41 . Maleckar, J. R., ard F. Kierszenbaum. Inhibition of mitogen- irduced proliferation of mouse T and B lymphocytes by bloodstream Forms of W. J. Immunol. 130:908. Beltz, L. A., and F. Kierszenbaum. 1987. Suppression of human lymphocyte responses by Wow—2i. Immunolggy 60:309. Meuer, S. C., K. A. Fitzgerald, R. E. Hussey, J. C. Hodgdon, S. F. Schlossman, and E. L. Reinherz. 1983. Clonotypic structures involved in antigen specific human T cell function: relation to the CD3 molecular emplac- W Mailer, S. C., R. E. Hussey, M. Fabbi, D. Fox, 0. Acuto, K. A. Fitzgerald, J. C. Hodgdon, J. P. Protentis, S. F. Schlossman, ard E. L. Reinherz. 1984. An alternative pathway of T—cell 157 activation: a functional role for the 50 kd T11 sheep erythrocyte receptor protein. Cell 36:897. 13. Hara, T., S. M. Fu, ard J. A. Hansen. 1985. Hmman T cell activation. II. A new activation pathway used by a major T cell population via a disulfide—borded dimer of a 44 kilodalton polypeptide (9.3 antigen). J. F_:xp. Med. 161:1513. 14. Carrel, S., S. Salvi, L. Giuffre, P. Isler, ard J.-C. Oerottini. 1987. A novel 90-kDa polypeptide (Tp90) possibily involved in an antigen-independent pathway of T cell activation. Eur. J. Immunol. 17:835. 15. Springer, T. A.,. M. L. Distin, T. K. Kishimoto, and S. D. Marlin. 1987. The lymphocyte function—associated LFA—l, CDZ, and LFA—3 moleclles: cell adhesion receptors of the immune system. Ann. Rev. Immunol. 5:223. 16. Fox, D. A., R. E. Hussey, K. A. Fitzgerald, A. Bensussan, J. F. Daley, S. F. Schlossman, ard E. L. Reinherz. 1985. Activation of human thymocytes via the SOKD T11 sheep erythrocyte birding protein irduces the expression of interleukin 2 receptors on both 13+ and T3- populations. J. Immunol. 134: 330. 17. Meuer, S. C., O. Acuto, R. E. Hussey, J. C. Hodgdon, K. A. Fitzgerald, S. F. Schlossman, ard E. L. Reinherz. 1983. Evidence for the T3~asscciated 90 kd heterodimer as the T cell antigen receptor. Nature 303: 808. 18. Budzko, D. B. ard F. Kierszenbaum. 1974. Isolation of W from blood. J. Parasitol. 60:1037. l9. Villalta, F., and W. Leon. 1979. Effect of purification by DEAE- cellulose column on infectivity of 11m cruzi blood forms. J. Parastiol. 65:188. 20. Gillis, S., M. M. Ferm, W. Cu, and K. Smith. 1978. T cell growth factor: parameters of production ard quantitative microassy for activity. J. Immunol. 120:2027. 21. Bernard, A., C. Gelin, B. Raynal, D. Elam, C. Gosse, ard L. Boumsell. 1982. Phenomenon of human T cell rosetting with sheep erythrocytes analysed with monoclonal antibodies. J. Eng. Med. 155:1317. 22. li‘unig, T. R. 1986. The ligard of the erythrocyte receptor of T lymphocytes: expression on white blood cells and possible involvement in T cell activation. J. Immunol.136: 2103. 23. Selvaraj, P., M. L. Dustin, R. Mitnacht, T. Hunig, T. A. Springer, and M. L. Plunkett. 1987. Rosetting of human T lymphocytes with sheepardhtmanerythrocytes: oomparisoncfhumanandsheepligand birding using purified E receptor. J. Immunol. 139:2690. 158 24. Makgoba, M. W., S. Shaw, E. D. Gugel, and M. E. Sanders. 1987. Human T cell rosetting is mediated by IFA-3 on autolcgous erythrocytes. J. Immunol. 1238:3587. 25. Hunig, T., G. Tiefenthaler, K.-H. M. zum Buschenfelde, ard S. C. Meuer. 1987. Alternative pathway of activation of T cells by binding of CDZ to its cell-surface ligand. Nature 326:298. 26. Vollger, L. W., D. T. Tuck, T. A. Springer, B. F. Hayes, ard K. H. Singer. 1987. Thymccyte birding to human thymic epithelial cells is inhibited by monoclonal antibodies to C02 ard LFA—3 antigens. J. Immunol. 138:358. 27. Blue, M.-L., J. F. Daley, H. Levine, K. A. Craig, ard S. F. Schlossman. 1987. Activation of immature cortical thymocytes through the T11 sheep erythrocyte birding protein. J. Imrmmol. 13833108. 28. Denning, S. M., D. T. Tuck, L. W. Vollger, T. A. Springer, K. H. Singer, ard B. F. Haynes. Monoclonal antibodies to CDZ ard lymphocyte function-associated antigen 3 inhibit human thymic epithelial cell-depedent mature thymocyte activation. L Immunol. 139:2573. 29. Shaw, S., ard G. E. G. Luce. 1987. The lymphocyte function associated antigen (LFA)-1 and CDZ/LFA—3 pathways of antigen- indeperdemt human T cell adhesion. J. Immunol. 139:1037. 30. Breitmeyer, J. B. 1987. How T cells ccmmunicate. Nature 329:760. 159 AppedixI T. cruzi Mediates its Suppressive Effect Via a Secreted Factor The addition of T. cruzi trypcmastigotes to cultures of normal hmmPRflhasbeeishwnintheprwdingchapterstosuppressthe proliferative response as well as the expression of the IL2R by these cells while the production of IL2 was unaltered. In order to determine whether this immunosuppression requires direct cell-to-parasite contact or whether a secreted suppressive factor exists, we tested the immunosuppressive ability of T. cruzi in the presence or absence of direct cell-to—parasite contact. T. cruzi trypcmastigotes were purified from the blood of infected mice as previously described (1) ard resuspended at a final concentration of 5 x 106 parasites/ml in RM 1640 medium (Gibco, Grand Island, NY) containing 100 units penicillin ard 100 pg streptomycin per ml and 596 heat-inactivated (56°C, 20 min) fetal bovine serum (RPMI+5%FBS) . Human PBMC were isolated as previously described (1) and resuspended at a final concentration of 1.25 x 106 cells/ml in RPMI+5%FBS. In order to test whether direct cell—to—parasite contact is required for T. cruzi to inhibit the proliferative response of PEMC to mitogens, me were placed in the wells of 24—well plates in the presence or absence of 5 lug/ml phytohemagglutinin (FHA: Sigma Chemical Company, St. I_ouis, MO). To each well, a Millicell—HA insert was added (Millipore, Bedford, MA). This insert contains a 0.45 um—pore—size filter which allows only the passage of soluble material between the 160 two compartments. The volumes of medium on the inside and outside of the insert were 0.4 and 0.5 ml, respectively. T. cruzi was present in some of the cultures either on the outside of the insert (allowing direct contact with the PMC) or within the insert (no direct contact) ard replaced an equal volume of RIMI+5%FBS. Cultures were pulsed with 5 uCi of 3H—thymidine 72 hr after initiation and harvested at 78 to 96 hr. Insomeexperiments, the insertswereremvedatvariwstimes of culture ard replaced with new inserts containing 0.4 ml of RPMI+5%FBS ard 5 pg/ml PHA. The abilityof Pmctoproduce IIZwastestedbymeasuringthe IL2 activity in 48-hr supernatants of cultures set up as described above. 112 activity was determined using the IL2-dependent CI'LL-z cell line (1) . The expression of the ILZR was measured on PETC in the presence or absence of direct cell-to-parasite contact 48 hr after mitogenic stimulation (Chapter 3). Results were expressed as the percentage of T‘ac+ cells ard mean channel number of the logarithm of the fluorescence intensites of the positive cells. The data presented in Tables 1 through 3 are typically represent— ative of two to six separate repeat experiments of similar design. T. cruzi was able to suppress the proliferative responses of PM to PHA whether or not the cells ard parasites were separated by a Millicell filter (Table 1). Irdeed, the suppressive capacity of L _cnii was the same in both corditions. It thus appears that the immunosuppressive effects of T. cruzi are mediated by a secreted suppressive factor(s) (SSF) . Whether this factor(s) originated 161 Table l. T. cruzi Suppresses Human PBMC Proliferation in the Absence of Direct Contact with the Cellsa PHA (5 ug/ml) T. cruzi 3H-thymidine incorporation (qm x 10'3) - - 1.3 i- 0.9 + - 51.6 i 5.9 + contactb 21.3 i 4.1 + no contact 28.7 i 2.3 a Ninety-six hr cultures pulsed with 5 pCi 3H-thymidine at 72 hr. b "Contact" refers to the presence of direct contact between PBMC and T. cruzi. "No contact" denotes that T. cruzi was separated frcm the P340 by a Millicell filter. 162 intracellularly or was released from the parasite's plasma membrane is not known. Next, the suppressive effects of 24 to 96 hr supernatants of L _CI'L_IZi trypcmastigote cultures were tested. While 5 x 106 parasites/ml were able to decrease mitogen-induced lymphoproliferation, the supernatants of these cultures as well as the supernatant of 1 x 107 L _c_r_u__z_i/ml did not effect the incorporation of 3H—thymidire by PHA- stimllatedPEMC (datanotslmn). SincebothPBMCandPI-lAwere also present in the cultures and PHA binds to and agglutinates T. cruzi (2) , it is thus possible that this mitogen or a cell product induces the release of SSF. To examine this possibility, the suppressive activity of 24 to 96 hr culture supernatants of 5 x 106 T. cruzi/m1 alone, _T_. gig; + 5 ug/ml FHA, T. cruzi + 1.25 x 106 PEMC/ml, or T. cruzi + PH’IC + BIA were tested; none of them suppressed mitogen-induced prolifer- ation of Pmc (data not shown). Thus, the SSF appears to be labile or degraded, complicating attempts to purify and characterize this molecule(s) . Further support for the lability of SSF were the results of studies inwhichthe insertcontainingT. cruziwasremovedand replaced with a new insert lacking parasites at 24, 48, or 72 hr. These cultures were pulsed with 3H-thymidine at 72 hr and harvested 6, 12, or 24 hr later. men-1 the inserts containing T. cruzi remained in the cultures for the duration of the experiment, the proliferative responses were decreased approximately 85% compared to PM stimulated in the absence of the parasite (Table 2). When the inserts were removed at 72 hr (at the time of the pulse) and harvested 6 hr later, 163 Table 2. The Suppressive Effect of SSF is Reversiblea time of 3H—tl‘qm'dine incommtion (w x 10'3) T. cruzi insert removal 6 hrb 12 hr 24 hr none - 61.4 i 6.5 61.7 510.2 77.4 i 22.2 contact - 9.2 i 0.8 8.2 1 1.6 15.9 i 1.2 (85)C (87) (79) no contact - 8.4 _t 1.2 7.4 i 1.6 11.6 1 2.4 (86) (88) (85) no contact 72 hr 11.5 i 3.2 14.9 i 4.6 36.6 i 2.7 (81) (76) (53) no contact 48 hr 38.7 i 4.0 50.4 i 6.1 85.3 i 5.1 (37) (18) (‘10) no contact 24 hr 45.1 i 4.2 40.3 i 8.0 82.5 i 1.9 (26) (35) (-6) _____________’_—___———————— a PHdeereincubatedwiflTSug/mlPHAinthepresenceorabsence of T. cruzi. All cultures were pulsed with 5 mi 3H—thymidii'le 72 hr after initiation. "Contact“ refers to the presence of direct contact between PBMC and T. cruzi. ”No contact" denotes that T. cruzi was separated from the PBMC by a Millicell filter. b Cultures were harvested 6, 12, or 24 hr after the pulse. 164 the extent of the suppression (81%) was approximately the same as in those cultures still containing T. cruzi, but as the time after parasite removal increased (12 and 24 hr pulses), the suppressive effect decreased (76 and 53%, respectively). This decrease in suppressionovertimewasseentoagreaterextentinthoseculturesin which the inserts containing parasites were removed at 48 or 24 hr of culture (Table 2) . Since ally the parasites themselves and not the SSF was removed, these results suggest that the suppression is reversible and that SSF is labile and must be continuously produced in order to be effective. The lability of SSF may either be intrinsic or may be due to PHVIC internalizing and degrading the molecule. In order to determine whether the immnosuppression caused by L m and the SSF are similar, several other parameters of lymphocyte activation were examined. No decrease in the production of IL2 under optimal culture conditions was caused by either w or SSF (Table 3). In contrast, SSF was able to inhibit the expression of the ILZR (Table 4) as had been previously shown for T._cru_z_i (Chapter 3) . In summary, L_c;u_zi appears to secrete a soluble suppressive factor which is labile and whose effects are reversible. Both L_cruz_i and its SSF decrease mitogen-induced proliferation and the expression of IL2R while not affecting the production of IL2. 165 Table 3. Ecposure of PHTC to the SSF did not Inhibit IL2 Productiona Supernatant 3H--thymidine incorporation (qzm x 10'3) by CI'LL—Z tested in the presence of the wants diluted: 1:2 1:4 1:8 1:16 PM 0.4 1' 0.0 0.3 1 0.0 0.3 1' 0.0 0.3 1 0.0 PHVIC'tH‘IA 20.6 1' 1.7 12.0 1' 1.7 5.1 1 0.8 1.7 1 0.4 FETCH-HEW. cruz1 29.3 1 1.6 16.4 1 0.6 8.7 1' 1.8 3.2 1' 0.3 PEMC+PHA+SSF 27.0 1' 3.2 12.7 1 0.7 7.1 1' 2.5 2.6 1' 0.5 a Forty-eight-hr supernatants of the indicated cultures were tested for 112 activity using the 11.2—dependent CI‘LL—Z cell line. 3H- thymidine incorporation by CI‘LL—2 cells incubated in RPMI+1O96FBS = 0.4 i- 0.2. Similar incorporation was produced by CI‘LL—Z incubated in the culture supernatants of unstimulated PEMC cultured in the presence of T. cruzi.‘ 166 Table 4. ILZRExpression is DecreasedbytheT. cruzi SSFa T. cruzi % T‘ac+ cells W — 51.3 140 no contact 46.2 130 a PMCwere incubated for 48 hrwithmA (5 ug/ml) in the presence or absence of T. cruzi prior to staining for the Tac antigen. "No contact" denotes that T. cruzi was separated frcxm the PHVIC by a Millicell filter. less than 5% ofthePEMCincubatedaloneorexposedtoSSFintheabsence of PEA were positive for Tac. b The density of the T‘ac antigen expressed as the mean channel number of the logarithm of the fluorescence inten— sities distributed over 256 channels. 167 REFERENCE 1. Beltz, L.A., and F. Kierszenbaum. 1987. Suppression of human lymphocyte responses by W. Immunology 60:309. 2. Pereira, M.E.A., M.A. Loures, F. Villalta, and A.P.B. Andrade. 1980. lectin receptors as markers for W. Developmental stages and a study of the interaction of wheat germ agglutinin with sialic acid residues on epimastigote cells. J. Exp. Med. 152:1375. 168 APPENDH II T. cruziInhibitstheerthofSeveralbutnotall Immortalized Cell Lines Lymphocyte activation involves a series of temporally distinct events as the cells move from the Go resting stage into the 91 stage of the cell cycle and thence onward to nuclear and cytoplasmic divisions (1,2) . The ability of T. cruzi to suppress T cell proliferative responses to mitogenic stimulation may lie in the inhibition of any one or more of these events. Immortalized cell lines, however, have already entered the cell cycle and therefore bypass several of the activation requirements. It is thus possible that these cell lines are no longer dependent upon the activation event(s) which T. cruzi inhibits and may subsequently escape the antiproliferative effects of the parasite. In order to explore this possibility, we have tested the ablility of T. cruzi to decrease the growth of several cell lines. T. cruzi trypcmastigotes were isolated from the blood of mice at two weeks post-infection as previously described (3) and resuspended at the desired concentrations in RM 1640 medium (Gibco, Grand Island, NY) containing 100 units penicillin and 100 pg streptomycin per ml and 10% heat-inactivated (56°C, 20 min) fetal bovine serum (RHVlI+1O96FBS) , or supernatants of concanavalin A—stimulated rat spleen cells (rat IL2) for the studies using CELL-2 cells. The murine 112-dependent CI'LL-2 cell line (American Type Culture Collection), maintained by passage in rat IL2 in a similar fashion to that previously described for HIL-z 169 cells (3) , was centrifuged once prior to use in the proliferation assay and was resupended at a final concentration of 2 X 104 cells/ml in rat IL2. The human nonadherant myelocytic U937 cell line (American Type Culture collection) and the human Thlymphotropic virus type 1 (mm-1)- infected HUT 10232 cell line (4; provided by Dr. Warren Leonard, National Institutes of Health, Bethesda, MD) were maintained by passage in menus and brought to final concentrations of l x 105 and 5 x 105 cells/ml, respectively, in the same medium. To test the ability of T. cruzi to inhibit the proliferation of these cell lines, cells were incubated at flue previously indicated concentrations for 24 or 48 hr in the presence or absence of serial dilutions of the parasite. CI'Ilr-Z and HUT cells were incubated in 96- well plates in a volume of 0.2 and 0.1 ml, respectively, while U937 cultures were set up in 24-well plates containing Millicell filter inserts (Millipore, Bedford, MA) in a volume of 0.9 ml (as described in Appendix I) to avoid cell infection. Cultures containing GILL—2 were pulsed with 1 “Ci 3H—thymidine (specific activity = 2.0 Ci/mmole; New England Nuclear, Wilmington, DE) 6 hr before harvest, while those containing U937 and HUT cells received a 24 hr pulse. Cultures were terminated by automated harvesting and the amounts of incorporated 3H— thymidine were determined with the use of a liquid scintillation counter. Results were expressed as mean cpm i 1 standard deviation. The staining of HUT cells for the expression of the IL2R was performedasdescribedindlapterB forPBVICandtheresaltswere expressed as the peroentage of IL2R+ cells and the mean channel number 170 of the logarithm of the fluorescence intensities (MFC‘h) distributed over 256 channels. ThedatainTablelshowthatT. cruziwasabletodecreasethe growth of CTLL—Z and U937 cells. Proliferation of CI‘LD-Z, an IL2- dependent murine T cell line, was reduced by the presence of 1 x 107 L _cru_2:_i/ml after 24 hr, while 2.5 X 106 T. cruzi/ml were effective at 48 hr. Growth of the human myelocytic U937 cells was suppressed by 5 X 106 T. cruzi/ml at 48 hr (Table 1), while 1 x 107 parasites/ml had an effect after only 24 hr (data not shown). Since the U937 cells and the parasites were separated by a 0.45-um-pore-size filter, the noted decrease can not be a result of infection of this monocyte-like cell line.‘ It should be noted that the suppressive ratio of parasites to cells was 125:1 and 500:1 for CI‘LL-Z at 48 and 24 hr, respectively, and 50:1 and 100:1 for the U937 cells. This is a much higher ratio than is required to inhibit the mitogen-induced proliferation of mouse spleen cells (1:1; Chapter 2) or human PHVIC (4:1; Chapter 1). In contrast to the results obtained with the CI'LL—z and U937 cell lines (Table 1) or normal human peripheral blood mononuclear cells (Chapters 3 and 4), '_I‘_. £1 (5 X 106 parasites/ml) was not able to affect the growth or the expression of the IL2R by HUT 102B2 cells after 48 hrs of co—culture (Table 2) . Similar results were seen when the parasite concentration was increased to 1 x 107 T. cruzi/ml (data not shown). Higher concentrations of T. cruzi were not tested due to the rapid acidification of the culture medium under these conditions (unpublished observation) . The failure of T. cruzi to inhibit growth or IL2R expression of 171 Table 1. T. cruzi Decreases the Growth of GILL-2 and U937 Cell Linesa Incomgration of 3H-thm‘dine at: Cell line T. cruzi (x105) 24 hr 48 hr crLL—z none 13.0 i 0.1 44.4 i- 0.6 1.25 14.2 r 0.6 44.5 r 3.3 2.5 15.3 i 0.5 27.6 i 0.1b 5.0 11.9 1- 0.4 6.3 r 0.610 10.0 7.4 r 0.213 3.1 i 0.210 U937 none NDC 70.9 r 11.1 1.25 ND 78.3 r 4.1 2.5 ND 70.1 i 3.1 5.0 ND 44.5 : 6.3b 10.0 ND 24.8 i 3.710 a The results with curl-2 and U937 cells were obtained in separate experiments, each of which was repeated on three occassions. b p_<_0.05, for the reductions in cpm with respect to the corresponding control which lacked T. cruzi, as calculated by Student's "t" test. 0 Not determined. 172 Table 2. T. cruzi does not Affect the Ability of HUT 10282 Cells to Proliferate or Express IL2Ra Material 3H—thymidine incorporation %ILZR+ cells log MFCh (m x 10'3) HUT 14.6 i 0.5 66.0 120 HUT-PT. cruzi 16.3 i: 0.1 69.0 117 a The proliferative response and the expression of the IL2R were tested separately in forty-eight hr cultures of HUT 102B2 cells incubated with or without 5 X 106 T. cruzi/ml. These results are representative of two separate repeat experiments. 1'73 HUT 10232 cells may lie in the mechanism of their transformation. This cell line is infected with and was immortalized by HI'LV—l (4,5). The initial stages of this immortalization appear to utilize an autocrine mechanism of growth, involving the constitutive production of IL2 and the IL2R, which is inhibitable by antibodies to the IL2R (5,6). later events lead to file loss of IL2-dependence and lack of IL2 production by some of the HILV-l-infected lines (reviewed in 7). The mechanism of theenhancedtranscriptionofmardllszRNAappearstomltfmm the interaction of the transactivator gene product (tat-I) of HI'LV—l with the prawters of these cellular genes (8,9) which bear sequence homology with the regulatory regions of HI'LV-l (10). In the case of the IL2R gene, the promoter engaged by tat-1 differs from that used in the normal activation process (8) . The differences between the growth of normal activated peripheral blood mononuclear cells and HITN—l— infected cell lines (loss of 11.2-dependent growth, constitutive expression of the ILZR, and the use of a different IL2R promoter) may explain the ability of T. cruzi to suppress growth and IL2R expression in the former but not the latter case. In summary, at high parasite to cell ratios T. cruzi is able to suppress the growth of a long—term IL2-dependent line and a monocyte- like cell line, but not mV—l-infected HUl‘ 102B2 cells under the conditions tested. The latter finding also suggests that T. cruzi- induwd growth—inhibition of immortalized cell lines is not merely the result of nutrient consmmption. l. 10. 174 REFERENCES Denhardt, D. T., D. R. Edwards, and C. L. J. Parfett. 1986. Gene expression during the mamalian cell cycle. Biochimica Biophysica Acta 865:83. Wedner, H. J. 1984. Biochemical events associated with lymphocyte activation. Survey of Immunologic Research 3:295. Beltz, L. A., ard F. Kierszenbaum. 1987. Suppression of human lymphocyte responses by W. Immunology 60:309. Poiesz, B. J., F. W. miscetti, A. F. Gazdar, P. A. an, J. D. Minna, and R. C. Gallo. 1980. Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T—cell lymphana. Proc. Natl. Acad. Sci. USA 77:7415. Gazzolo, L., and M. D. Dodon. 1987. Direct activation of resting T lymphocytes by human T—lymphotropic virus type I. Nature 326:714. Goctenberg, J. E., F. W. Ruscetti, J. W. Mier, A. Gadzar, and R. C. Gallo. 1981. Human cutaneous T cell lymphoma and leukemia cell lines produce and respond to T cell growth factor. J. Exp. Med. 154:1403. Greene, W. C., W. J. Leonard, J. M. Depper, D. L. Nelson, and T. A. Waldmann. 1986. The human interleukin-2 receptor: normal and abnormal expression in T cells and in leukemias induced by the human T—lymphotropic retroviruses. Annals Internal Led. 105:560. Cross, S. L., M. B. Feinberg, J. B. Wolf, N. J. Holbrook, F. Wong Staal, and W. J. Ieonard. 1987. Regulation of the human interleukin—2 receptor a chain promoter: activation of a nonfunctional promoter by the transactivator gene of HI‘LV—l. Cell 49:47. Siekivitz, M., M. B. Feinberg, N. Holbrook, F. Wong—Steal, and W.C. Greene. 1987. Activation of interleukin 2 and interleukin 2 receptor (Tac) promoter expression by the transactivator (tat) gene product of human T—cell leukemia virus, type 1. Proc. Natl. Acad. Sci. USA 84:5389. Fujita, T., H. Shibuya, T. Chashi, K. Yamanishi, and T. T‘aniguchi. 1986. Regulation of human interleukin-2 gene: functional INA sequences in the 5' flarflcing region for the gene expression in activated T lymphocytes. Cell 46:401. 175 SUMMARY AND (DNCIUSIONS A state of suppressed immune reactivity occurs during the early stages of infection with W. Both cellular and humoral arms of the immune system, hmrever, are functional during the infec- tion's later stages and play a vital role in host defense. It may thus be hypothesized that the initial state of immunosuppression enables L M to establish itself intracellularly and that overcoming this phenomenon may allow clearance of the parasite by the host's immnme system before the onset of pathology. It was our goal, therefore, to characterize the immune alterations which T. cruzi induces in human T lymphocytes with the ultimate goal of developing means to abrogate these events and the subsequent occurance of disease. Prior to this work, knowlege of the extent of the parasite-induced immmmosuppressive events in human lymphocytes was extremely limited. It was shown herein that T. cruzi was able to suppress the proliferation of human T cells following activation by either the T cell receptor or the CD2 antigen-independent stimulatory pathways and that the ability of the parasite to inhibit the expression of the interleukin 2 rweptor played a key role in this process. The addition of T. cruzi trypcmastigotes or epimastigotes to cultures of normal human peripheral blood mononuclear cells (Pmc) reduced the ability of the latter to proliferate in response to a variety of mitogenic lectins in a parasite-dose—dependent manner and over a wide range of mitogen concentrations. This reduction was not 176 due to consumption of essential nutrients or to a lowering of mitogen concentrations to suboptimal levels by T. cruzi nor to a loss of Pmc viability after co-culture with the parasite. T. cruzi was also able \ to suppress Pmc proliferative responses after stimulation by anti-CD3, a monoclonal antibody directed against the T cell receptor complex, and anti-T112 and anti-Tll3 antibodies whidi trigger T cells via the CDZ activation pathway. The presence of monocytes was not required for the decrease in PBMC responsiveness while parasite viabilty was necessary. T. cruzi additionally inhibited the growth of several but not all immortalized cell lines tested. T. cruzi was able to exert its suppressive effects when separated from the cells by a Millipore filter insert, demonstrating that a soluble factor released by the parasite was involved in the suppressive process. T cell responsiveness showed a partial recovery within 24 hr after removal of the insert containing T. cruzi, demonstrating the reversibility of the immune alterations and the lability of the suppressive factor. I ' Maximalsuppressionwasnotedwhen T. cruziwasaddedtocultmres within the first 24 hr of stimulation and decreased as the time of parasite addition was prolonged. Thus T. cruzi appeared to affect an early stage of lymphocyte activation. Interleukins (IL) 1 and 2 are produced by stimulated monocytes and T cells, respectively, and are required early during the T cell growth cycle. Under conditions of optimal stimulation, T. cruzi was unable to reduce the ability of human PEMC to produce or secrete either of these molecules or interferon-1 while mouse spleen cells were deficient in the production of both IL2 177 and interferon—T after co-culture with the parasite. In keeping with these results, IL2 was able to restore the mitogen—induced prolifer- ative responses of sqpressed mouse but not human lymphocytes. Thus, there are notable differences in the process of suppression of mouse spleen cells and human Pmc. These results indicate that caution must be exerted when extrapolating findings obtained with the murine model system to the human disease. The inability of human FEE! to respond to endogenous or exogenous IL2 correlates with the ability of T. cruzi to inhibit the expression of the I12 receptor (ILZR) on T cells. Both the number of cells bearingIIQRandthereceptordensityweredecreasedbyT. cruziina manner which was dependent upon the parasite concentration. This decrease was observed within 12 hr of stimulation and persisted until at least 60 hr. Both the low and the high affinity forms of the ‘ receptor were affected. Impression of the transferrin receptor, a molecule required for lymphoproliferation as well as a late activation marker, was also inhibited by T. cruzi while the levels of early activation antigen 1, the earliest reported activation marker of T cells, were unaffected by the parasite during the initial 6 to 24 hr of stimulation. T. cruzi additionally suppressed the up—regulation of the surface expression of the T112 epitope of CDZ as well the exposure of the T113 epitope of this molecule which occurs during activation. Thus, T. cruzi is selective in its inhibition of human T cell activation events and this specificity may provide the key in overcoming the parasite-induced suppression. 178 'meeventwhichappearstobeofthegreatest importanceisthe decreased expression of the 112R since triggering by this molecule allows progression from the early to the late G]. stage of the cell cycle and regulates many of the subsequent events of T cell activation. Additionally, this is the earliest process reported to be altered by L M. Future work in. this area might address the following questions: 1. Does T. cruzi cause an increase in the levels of the soluble ImRasisthecaseinAIlBandcertain formsofcaroer? 2. Is the expression of the membrane form of the IL2R on activated B cells and monocytes also affected? 3. Are the levels of ILZRmRNA decreased by T. cruzi, and if so, is this an expression of decreased transcription or of decreased mRNA stability? 4. Is the expression of cellular oncogenes altered by T. cruzi? 5. How may the stability of the parasite-induced suppressive factor be increased so as to allow its purification and character— ization? The answers to these questions will allow a greater understanding of the process of immtmosuppression by T. cruzi and may be of value in the study of immune alterations caused by other pathogens, in particular, the Immnan immunodeficiency virus. 'IICHIGAN STATE UNIV LIBRnRIES :y . v 9330056791 1