Universy of Arkansas LI
Immunoloiz= u~edicli
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Brown Adipose Tissue and Immunity EFFECT OF NEONATAL ADIPECTOMY ON HUMORAL AND CELLULAR IMMUNE REACTIONS IN THE RAT B. D. JANKOVIC, ALENKA JANEZIC AND LJILJANA POPESKOVIC Immunology Research Centre, Belgrade, Yugoslavia
(Received 12th July 1974; acceptedfor publication 20th September 1974) Summary. This work concerns the involvement of brown adipose tissue in the immune system of the rat. Wistar rats were thymectomized, adipectomized (surgical extirpation of the interscapular brown adipose tissue), thymectomized and adipectomized, and sham-operated at birth, only 8-week-old females being employed in the experiment. The production of antibody to bovine serum albumin (BSA) and sheep red blood cells (SRBC), delayed skin reactions to BSA, rejection of thyroid allograft implanted under the kidney capsule, and development of experimental allergic encephalomyelitis were investigated. Neonatal adipectomy did not affect the production of anti-BSA and anti-SRBC antibodies. On the other hand, delayed skin reactions to BSA, rejection of thyroid allograft, and incidence and severity of allergic encephalomyelitis were much more pronounced in adipectomized animals. It has been postulated that the immune function of brown adipose tissue is an expression of the secretory activity of the tissue. Since the immunosuppressive effect of neonatal thymectomy on demyelinating disease was neutralized by neonatal adipectomy, and vice versa, and since thymectomy rendered ineffective the immunopotentiating influence of adipectomy on this disease, as demonstrated in thymo-adipectomized rats, it was concluded that the brown adipose tissue is a natural antagonist of the thymus in cell-mediated
immunity. This paper also describes the extra thymuses which were situated in the vicinity of the thyroid and parathyroid lobes of 23-2 per cent of rats. INTRODUCTION Brown adipose tissue is a unique tissue in mammals (Lindberg, 1970). Morphologically, it is composed of typical adipocytes with multilocular distribution of fat, and contains a rich network of blood vessels and a sympathetic nerve supply (Smalley, 1970). Functionally, it represents the principal site of nonshivering thermogenesis (Smith and Horwitz, 1969). The importance of brown adipose tissue for the maintenance of temperature in hibernators, on the one hand, and the suppression of immune reactions by deep hypothermia (Petrik, 1922; Schmidt, 1967) on the other, suggested a relationship between brown adipose tissue and immunity. A few studies in this area have indicated that extracts obtained from brown adipose tissue of hibernators are capable of suppressing the production of antibody under conditions of tissue culture (Sidky, Draggett and Auerbach, Correspondence: Dr B. D. Jankovic, Immunology Research Centre, P.O. Box 979, Vojvode Stepe 458, 11000
Belgrade, Yugoslavia.
597
598 B. D. Jankovi6, Alenka jane6ic and Ljiljana Popeskovi6 1969; Sidky and Hayward, 1972). However, we have demonstrated both in vivo and in non-hibernators the immunosuppressive activity of brown adipose tissue; it was found that repeated injections of extracts prepared from the rat brown adipose tissue produced a considerable reduction of immune responses (Jankovic, Popeskovic, Janelic and Lukic, 1974a, b). In addition, we reported briefly that the removal of brown adipose tissue from newborn rats enhanced to a great extent the reactions of cell-mediated immunity (Jankovic, Janelic and Popeskovic, 1974). This paper provides some basic information about the immune capacity of rats from which the interscapular brown adipose tissue was removed at birth, and describes an initial effort to correlate the immune function of the brown adipose tissue and that of the thymus.
MATERIALS AND METHODS
Animals Rats used in this study were all random bred Wistar strain housed in a closed colony. The animals were adipectomized, thymectomized, thymo-adipectomized or shamoperated at birth, and only 8-week-old females were used in the experiment.
Adipectomy There are several masses of brown adipose tissue in the rat thorax and abdomen, but the largest mass of tissue is situated in a depression of the muscles between the shoulder blades. This interscapular brown adipose tissue was extirpated (adipectomy) within 24 hours from newborn rats under deep hypothermic anaesthesia. After an incision along the mid-dorsal line between the scapulae, the overlying white fat was dissected and retracted, and then the brown adipose tissue was freed from surrounding muscles and removed. The skin incision was closed with interrupted sutures. Neonatal adipectomy proved to be a simple microsurgical procedure with an extremely low mortality rate. Sham-operated newborn rats were treated in an identical manner, except that the interscapular brown adipose tissue was left intact. The extirpated interscapular brown adipose tissue was equivalent to 0-7O-0-80 per cent of the body weight (Barnard & Skala, 1970), and to 60-70 per cent of the total brown adipose tissue of the newborn rat. Therefore, adipectomy as performed in this study should be considered incomplete.
Experimental groups The first group consisted of neonatally adipectomized rats; the second group was composed of neonatally thymectomized rats; the third group included rats which were both neonatally thymectomized and adipectomized; and the fourth group of sham-operated rats served as control. Non-operated female rats were also used in the experiment. The completeness of adipectomy and thymectomy was checked at the end of the experiment both grossly and histologically by examining the interscapular and mediastinal region. In order to ascertain the presence of extra thymuses in neonatally thymectomized rats, serial cross-sections of the thyroid region of the neck were prepared and histologically inspected. Sensitization with bovine serum albumin (BSA) Rats were immunized with crystalline BSA incorporated in Freund's complete adjuvant
599 Brown Adipose Tissue and Immunity (1 volume of BSA in saline, 0-15 volumes of Arlacel A, 0X85 volumes of Draceol, and autoclaved M. tuberculosis at a final concentration of 3 mg/ml). A single 01 -ml dose of BSAadjuvant mixture containing 0 5 mg of BSA was injected subcutaneously into the left hind foot-pad. The animals were skin-tested for delayed hypersensitivity 10 and 20 days later with 30 yug of BSA injected intradermally in the depilated flank. Delayed reactions were read at 24 hours, and the intensity ofreactions was graded from 0 to + + + according to the diameter and degree of induration.
Immunization with sheep red blood cells (SRBC) Separate groups of adipectomized and sham-operated rats were given a single intravenous injection of 7 x 108 SRBC in 0-2 ml of saline. Antibody determination Rats injected with BSA-adjuvant mixture were bled 10 and 20 days after immunization, and the sera separated from clotted blood and treated with 2-mercaptoethanol (ME). The presence of ME-sensitive and ME-resistant anti-BSA antibodies was determined using Takatsy's passive microhaemagglutination technique. Rats immunized with SRBC were bled 6 days later, and the sera tested for MEsensitive and ME-resistant haemagglutinins by means of a microhaemagglutination reaction using 7 x 45 mm tubes, 0-025 ml of a 1 per cent suspension of SRBC and an equal volume of serial serum dilutions. After incubation for 2 hours at room temperature, the end-point of antibody activity of sera was detected microscopically.
Induction of experimental allergic encephalomyelitis One part of fresh rat spinal cord was homogenized with two parts of Freund's complete adjuvant, and 0-1 ml of this mixture was inoculated into the left hind foot-pad of adipectomized, thymectomized, thymo-adipectomized and sham-operated rats. Animals were observed daily for the appearance of clinical signs of disease, and those showing paralysis were killed. The rats which did not exhibit neurological symptoms were killed 21 days after sensitization. Cerebrum, cerebellum and several levels of spinal cord of all rats were processed for staining with haematoxylin and eosin. A minimum of twelve sections of nervous tissue (from each rat) were examined and histological lesions graded. An arithmetic score reflecting an overall response was calculated for each group of rats, depending on the degree of encephalomyelitis defined as follows: negative = 0; + = 1; + + = 2; and +++ = 3. Implantation of thyroid graft under the kidney capsule Female neonatally adipectomized and sham-operated rats were grafted with thyroid tissue from inbred female 6-week-old Lewis rats. The right kidney was exposed through a dorsal paramidline skin incision. The thyroid graft was inserted under the capsule of the upper part of the kidney by means of a glass needle. The skin incision was then closed with surgical clips. Grafted kidneys were extirpated 2, 3, 4, 5 and 6 days after transplantation, fixed in Carnoy's solution, embedded in paraffin wax, and sections were stained with haematoxylin and eosin. The thyroid grafts were qualitatively analysed and the degree of rejection graded from 0 to + + +.
600
B. D. Jankovic, Alenka jane&il and Ljiljana Popeskovi6
Histology Rats of all groups were autopsied at the end of experimentation, and the thymus, regional and remote lymph nodes, spleen and Peyer's patches were taken for histological appraisal. Tissue sections were stained with haematoxylin and eosin, and methyl green and pyronin.
RESULTS BODY WEIGHT
Adipectomized, thymectomized, thymo-adipectomized and sham-operated rats gained weight steadily and equally. A small number ofthymectomized animals which exhibited signs of wasting disease (Jankovic, Waksman and Arnason, 1962) were excluded from the experiment. It was noted that there was no wasting disease in thymo-adipectomized rats. ANTIBODY PRODUCTION IN ADIPECTOMIZED RATS
The amount of ME-sensitive and ME-resistant antibodies against BSA increased between 10 and 20 days after immunization in both adipectomized and control rats. The capacity of adipectomized animals to produce circulating anti-BSA and anti-SRBC antibodies was very similar to that of sham-operated rats (Table 1). DELAYED SKIN REACTIONS IN ADIPECTOMIZED RATS
Delayed skin hypersensitive reactions were markedly enhanced in adipectomized rats in comparison with reactions in shamr-operated animals (Table 1). Histological appraisal of skin reactions of six adipectomized and six sham-operated rats 10 days after immunization with BSA revealed that oedema and cellular infiltration of the dermis, and underlying muscles and connective tissue were much more extensive in adipectomized than in control rats. Polymorphonuclear leucocytes surrounding small arteries were regularly seen in all sections of the skin, indicating the Arthus component of the reaction. Nevertheless, in all reactions perivenous aggregates of mononuclear cells predominated. No attempts were made to search for the basophilic component of delayed cutaneous reactions (Dvorak, Dvorak, Simpson, Richerson, Leskowitz and Karnovsky, 1970) in adipectomized rats. DEVELOPMENT OF ALLERGIC ENCEPHALOMYELITIS
Clinical and histological features of experimental allergic encephalomyelitis in adipectomized, thymectomized, thymo-adipectomized and sham-operated rats are shown in Table 2. Only one out of twenty-five sham-operated rats developed paralysis. On the other hand, twelve of nineteen adipectomized animals showed clear neurological symptoms. Clear-cut neurological symptoms were first apparent at 14 days. No thymectomized and thymo-adipectomized rats became paralysed. The incidence of paralysis correlated well with the intensity of histological lesions in the central nervous system, and the most numerous and advanced lesions were observed in adipectomized rats. In these animals, the lesions were not restricted to the nervous parenchyma but in several instances extended to the choroid plexus. The lesions consisted of lymphocytes, histiocytes, and rare plasma cells and polymorphonuclears.
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TABLE 2 INCIDENCE OF PARALYSIS AND SEVERITY OF HISTOLOGICAL LESIONS IN SHAM-OPERATED (S-O), NEONATALLY ADIPECTOMIZED (BATx), NEONATALLY THYMECTOMIZED (Tx), AND NEONATALLY THYMO-ADIPECTOMIZED (Tx-BATx) RATS SENSITIZED WITH ALLOGENEIC SPINAL CORD
Number of rats with histological lesions
Rats with paralysis
Group S-O BATx Tx Tx-BATx
Number of rats
Number
Per cent
25 19 12 10
1 12 0 0
4 63 0 0
0
+
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4 4 0 1
Mean + + + score
1l0 2-8 0-1 09
1 15 0 0
TABLE 3
REJECTION
OF ALLOGENEIC THYROID IMPLANTED UNDER THE KIDNEY CAPSULE OF SHAM-OPERATED TALLY ADIPECTOMIZED (BATx) RATS
(S-O)
AND NEONA-
Days after transplantation
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S-O BATx
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2*0 30
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0 0
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2-6 30
The results are expressed in terms of a histological evaluation of rejection. * Five rats in group.
REJECTION OF THYROID ALLOGRAFTS
The course of rejection of allogeneic thyroid implanted under the renal capsule of sham-operated and adipectomized rats is indicated in Table 3. This comparative study demonstrated an accelerated rejection in adipectomized rats (Fig. la, c and e): at 2 days, the vascularization of allografts was very pronounced and many blood vessels were dilated (Fig. la); at 3 days, advanced destruction of thyroid follicles and cellular infiltration of graft bed was a characteristic feature of the rejection (Fig. 1c); and at 4 days, the follicles were completely destroyed and the cellular make-up of thyroid grafts was replaced by host cells (Fig. le). At 6 days the rejection of grafts in adipectomized rats was almost indistinguishable from that in controls, so far as the cellular infiltration of the graft was concerned. A preliminary attempt at studying the rejection of skin allografts in adipectomized rats had been made. However, the implantation of the thyroid under the kidney capsule was found to be a more suitable model for the purpose of this experiment since it allowed recognition of incipient signs of allograft rejection.
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FIG. 1. The course of rejection of thyroid allograft (G) implanted under the kidney capsule (K) of adipectomized (a, c and e) and sham-operated (b), (d) and (f) rats: 2 (a) and (b), 3 (c) and (d), and 4 (e) and (f) days after transplantation. Arrows indicate dilated blood vessels in the graft. (Haematoxylin and eosin; magnification x 80.)
INVOLUTION OF BROWN ADIPOSE TISSUE
Histological examination of the interscapular brown adipose tissue of rats reared and maintained at room temperature revealed that adipocytes of young adult rats, in contrast to adipocytes of newborn rats, contained large areas occupied by confluent droplets of
B. D. jankovi6, Alenka jane&6ic and Ljiljana Popeskovic 604 lipid (Fig. 2a, b). This finding should be regarded as general morphological information about alterations which take place in the involuting brown adipose tissue and which are associated with profound ultrastructural and biochemical changes (Barnard and Skala, 1970). Those involution changes may explain the failure of adipectomy performed on 6-week-old rats (this group is not included in the present study) to affect the reactions of cell-mediated immunity.
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FIG. 2. Morphology of the interscapular brown adipose tissue of (a) newborn and (b) 8-week-old rats. Note the involution in older animal. For comparison, (c) the interscapular white fat of newborn rat. (Haematoxylin and eosin; magnification x 300.)
EXTRA THYMUSES IN THE RAT
As mentioned before, this experiment included only those neonatally thymectomized rats which were proved histologically not to contain thymic remnants in the mediastinum and extra thymic lobes in the thyroid region of the neck. Since the thymus, thyroid and parathyroid are of the same embryonic origin, and since it was found that a number of correctly thymectomized rats still contained extra thymic lobes, it was deemed advisable to search for extra thymuses in a larger number of rats. For this purpose, the thyroid region of eighty-six non-operated and thymectomized Wistar rats was histologically examined, and it was found that 23-2 per cent of rats possessed extra thymuses of various size. Most of those thymuses were situated in the immediate vicinity of thyroid and parathyroid glands. However, some thymic lobes occupied the central part of the thyroid and were discovered only by serial cross-sectioning of the thyroid tissue. In some instances, thymic cells were distributed between thyroid follicles in such a manner as to give an impression of a 'spontaneous thyroiditis' (Fig. 3a). In fact, these cells belonged to the apical part of an intrathyroid thymic lobe (Fig. 3b). It is clear that extra thymuses should seriously be taken into account in studies in which thymectomized animals are prepared by surgical ablation of the mediastinal thymic lobes.
Brown Adipose Tissue and Immunity
605
FIG. 3. (a) Aggregates of thymus cells in the thyroid of normal rats. (b) Further cross-sectioning of the same sample of tissue revealed the presence of an intrathyroid thymus lobe. (Haematoxylin and eosin; magnification x 144.)
HISTOLOGICAL CHANGES IN LYMPHOID TISSUES
In spite of repeated and careful histological analyses, no apparent changes in cellular make-up of the thymus, spleen, lymph nodes and Peyer's patches were observed in neonatally adipectomized rats (Fig. 4a and c). In a number of adipectomized animals the thymus was larger than in sham-operated controls, but this difference in thymus weight was on the border of statistical significance (P> 0.05). On the other hand, thymectomized rats showed typical depletion of small lymphocytes (Waksman, Arnason and Jankovic, 1962) in thymus-dependent areas of the spleen, lymph nodes and Peyer's patches. The lymphoid dysplasia in thymus-less rats were not restored, or were restored only slightly, by neonatal adipectomy (Fig. 4b, d).
606
606B. D. Jankovi, Alenka ane&6 and Ljiljana Popeskovid
FIG. 4. (a) Spleen of adipectomized rat showing normal density of lymphocytes in periarteriolar white pulp, and (b) spleen of thymo-adipectomized rat lacking periarteriolar lymphocytes. (Methyl green and pyronin; magnification x 144.) Two right axillary lymph nodes, (c) one from an adipectomized rat with dense lymphocytes in paracortical area, and (d) another from a thymo-adipectomized rat with scattered lymphocytes in paracortical zone. In both lymph nodes germinal centres appear normal. (Haematoxylin and eosin; magnification x 144.)
DISCUSSION Since its initial description in 1551 by Gesner, various functions have been assigned to brown adipose tissue, inter alia it was regarded as part of the thymus and endocrine system (reviewed by Johansson, 1959). It is now widely recognized that the principal function of the tissue is the production of heat. This thermogenic function is under the control of the
607 6 Brown Adipose Tissue and Immunity sympathetic nervous system which, by means of catecholamines stimulates adenyl cyclase and thus accelerates lipolysis (Himms-Hagen, 1972). Brown adipose tissue is metabolically very active (Smith and Horwitz, 1969), and functional changes that occurred after surgical removal of the interscapular brown adipose tissue from cold-acclimatized rats led to the hypothesis that brown adipose tissue elaborates a product which alters metabolic processes in other tissues (Himms-Hagen, 1972). Since the regression of brown adipose tissue (Dawkins and Hull, 1964) coincides with involution of the thymus and with immune maturation, it may be deduced that the activity of the tissue is under the control of hormones (Johansson, 1959). With these premises in mind, it seems that the heat-producing activity of brown adipose tissue is only partially responsible for the significant decrease of immune potential in animals in deep hypothermia (Petrik, 1922; Andjus and Matic, 1959; Jaroslaw and Smith, 1961), and in animals with artificially lowered body temperature (Kopeloff and Stanton, 1942). The suppression of antibody response in such animals is most probably caused by a general decrease of the metabolic rate of the body (Sidky, Hayward and Ruth, 1972), including the synthesis of immunoglobulins. The present experiment differs conceptually from those mentioned in the preceding paragraph, as well as from the in vitro studies of brown adipose tissue (Sidky et al., 1969; Sidky and Hayward, 1972) since it was performed on non-hibernators, under in vivo conditions, at normal body temperature, and on animals lacking the interscapular brown adipose tissue. Recently, we have shown in rats that repeated injections of extracts from brown adipose tissue of newborn or 21-day-old cold-adapted rats produced a significant suppression of delayed skin hypersensitivity, whereas the formation of antibody was not greatly affected (Jankovic et al., 1974a, b). These findings are in complete agreement with the results presented here which show that neonatal adipectomy enhances the reactions of cell-mediated immunity (delayed hypersensitivity, experimental allergic encephalomyelitis and rejection of thyroid allograft) while leaving intact the production of antibody against BSA and SRBC. However, there is a discrepancy between our results and the observation that brown adipose tissue extract from hibernating animals inhibits the elaboration of antibody in vitro (Sidky et al., 1969; Sidky and Hayward, 1972). Several factors may account for this difference in results: experimental conditions, use of hibernators and non-hibernators, manner of preparation of tissue extracts, and amounts of extract employed in the study. In addition, since the brown adipose tissue of hibernators exerts an extraordinary metabolic activity, the content of a postulated immuno-inhibitory agent (vide infra) may be expected to be much higher in the extract of the tissue from a hibernator than from a non-hibernator. The discrepancy between the humoral and cell-mediated immune responses in adipectomized rats, as described here, could be explained in the following way: the neonatal excision of the interscapular brown adipose tissue represents, in fact, an incomplete adipectomy, which leaves intact other deposits of brown adipose tissue, so that the amount of the tissue in adipectomized rats is still sufficient to allow a normal production of antibody. Reasoning of this kind would imply that in non-hibernators the mechanisms which underlie cell-mediated immunity are more sensitive to the lack of brown adipose tissue than the mechanisms primarily involved in humoral immunity. The simplest construction that can be put upon these results is that the immuno-inhibitory activity of brown adipose tissue is probably the expression of a biologically active substance produced by the tissue (Himms-Hagen, 1972). Neonatal adipectomy,
B. D. jankovic5, Alenka janeei6 and Ljiljana Popeskovic 608 though incomplete, leads to a marked deficiency in this substance and thus to the potentiation of cell-mediated immune responses. Since neonatal adipectomy failed to reconstitute thymus-dependent areas in lymphoid tissues of thymo-adipectomized rats, it appears that the postulated immuno-inhibitory factor interferes rather with the function oflymphocytes than with the proliferation of lymphocytes. Bearing in mind the influence that the sympathetic nervous system exerts on brown adipose tissue (Steiner, 1972), it may be assumed that catecholamines (e.g. noradrenaline) may be mediators of the immune function of brown adipose tissue. Another possibility is that the immune effect ofbrown adipose tissue is due to a release of fatty acids from the tissue (Drahota, 1970). The interconnections between brown adipose tissue and hormones should also be taken into consideration (Johansson, 1959). Finally, two important aspects emerged from the study of experimental allergic encephalomyelitis in neonatally adipectomized, thymectomized, and thymo-adipectomized rats. First, adipectomy and thymectomy produced quite different effects on that disease: adipectomy potentiated whereas thymectomy suppressed its development. Secondly, adipectomy neutralized the immunosuppressive influence of thymectomy, and vice versa, thymectomy rendered ineffective the immunopotentiating influence of adipectomy on allergic encephalomyelitis. In view of these observations, it is tempting to conclude that the brown adipose tissue is a natural antagonist of the thymus in cell-mediated immunity.
ACKNOWLEDGMENTS This work was supported by grants from the Republic Fund for Research of SR Serbia, Belgrade. We acknowledge with thanks the technical assistance of Miss Anica Rankovic. REFERENCES
ANDJUS, R. K. and MATIC, 0. (1959). 'Hibernation and immunological reactivity.' Pftagers Arch. ges. Physiol., 270, 55. BARNARD, T. and SKALA, J. (1970). 'The development of brown adipose tissue.' Brown Adipose Tissue (ed. by 0. Lindberg), p. 33. American Elsevier, New York. DAWKINS, M. J. R. and HULL, D. (1964). 'Brown adipose tissue and the response of new-born rabbits to cold.'J. Physiol., 172, 216. DRAHOTA, Z. (1970). 'Fatty acid oxidation by brown adipose tissue mitochondria.' Brown Adipose Tissue (ed. by 0. Lindberg), p. 225. American Elsevier, New York. DVORAK, H. F., DvORAK, A. M., SIMPSON, B. A., RiCHERSON, H. B., LESKOWITZ, S. and KARNOVSKY, M. J. (1970). 'Cutaneous basophil hypersensitivity. II. A light and electron microscopic description.' J. exp. Med., 132, 558. HIMMs-HAGAN, J. (1972). 'Effects of catecholamines on metabolism.' Catecholamines (ed. by H. Blaschko and E. Muscholl), p. 363. Springer-Verlag, Berlin. JANKOVIC, B. D.,JANEzId, A. & POPESKOVI,6, L. (1974). 'Brown adipose tissue: potentiation of delayed skin hypersensitivity and experimental allergic encephalomyelitis in adipectomized rats.' Fed. Proc., 33, 804. JANKOVId, B. D., POPESKOVI6, L., JANEiId, A. and LUKI6, M. L. (1974a). 'Immune reactions in rats
treated with allogeneic brown adipose tissue and rats adipectomized at birth.' Proc. rugoslav Immunol. Soc., 3, 122. JANKOVI6, B. D., POPESKOVI6, L., JANEZI6, A. and LUKI6, M. L. (1974b). 'Brown adipose tissue: effect on immune reactions in the rat.' Naturwissenschaften, 61, 36. JANKOVIC, B. D., WAKSMAN. B. H. and ARNASON, B. G. (1962). Role of the thymus in immune reactions in rats. I. The immunologic response to bovine serum albumin (antibody formation, Arthus reactivity, and delayed hypersensitivity) in rats thymectomized or splenectomized at various times after birth.' J. exp. Med., 116, 159. JAROSLAW, B. N. and SMITH, D. E. (1961). 'Antigen disappearance in hibernating ground squirrels.' Science, 134, 734. JOHANSSON, B. (I1959). 'Brown fat: a review.' Metabolism, 8, 221. KOPELOFF, L. M. and STANTON, A. H. (1942). 'The effect of body temperature upon haemolysin production in the rat.'J. Immunol., 44, 247. LINDBERG, 0. (1970). Brown Adipose Tissue. American Elsevier, New York. PETRIK, J. (1922). 'Contribution A la serologie des mammiferes hibernants.' Publ. Fac. Med. Brno R.C.S., 1, 247. SCHMIDT, J. P. (1967). 'Response of hibernating
Brown Adipose Tissue and Immunity animals to physical, parasitic and infectious agents.' Mammalian Hibernation, volume 3 (ed. by K. C. Fischer, A. R. Dawe, C. P. Lyman, E. Schonbaum and F. E. South, Jr), p. 421. Oliver & Boyd, Edinburgh. SIDKY, Y. A., DAGGETr, L. R. and AUERBACH, R. (1969). 'Brown fat: its possible role in immunosuppression during hibernation.' Proc. Soc. exp. Biol. (N. r7.), 132, 760. SIDKY, Y. A. and HAYWARD, J. S. (1972). 'Immunosuppression by extracts of brown fat.' Fed. Proc., 31, 800. SIDKY, Y. A., HAYWARD, J. S. & RUTH, R. F. (1972). 'Seasonal variations of the immune response of ground squirrels kept at 22-24'C.' Canad. J. Physiol. 50, 203.
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SMALLEY, R. L. (1970). 'Changes in composition and metabolism during adipose tissue development.' Brown Adipose Tissue (ed. by 0. Lindberg), p. 73. American Elsevier, New York. SMITH, R. E. and HORWrrz, B. A. (1969). 'Brown fat and thermogenesis.' Physiol. Rev., 49, 330. STEINER, G. (1972). 'Immunosympathectomy as a tool in metabolic studies.' Immunosympathectomy (ed. by G. Steiner & E. Sch6nbaum), p. 159. Elsevier, Amsterdam. WAKSMAN, B. H., ARNASON, B. G. and JANKOVI6, B. D.
(1962). 'Role of the thymus in immune reactions in rats. III. Changes in the lymphoid organs of thymectomized rats.'J. exp. Med., 116, 187.
Immunoloiz= u~edicli
ARY Center
Brown Adipose Tissue and Immunity EFFECT OF NEONATAL ADIPECTOMY ON HUMORAL AND CELLULAR IMMUNE REACTIONS IN THE RAT B. D. JANKOVIC, ALENKA JANEZIC AND LJILJANA POPESKOVIC Immunology Research Centre, Belgrade, Yugoslavia
(Received 12th July 1974; acceptedfor publication 20th September 1974) Summary. This work concerns the involvement of brown adipose tissue in the immune system of the rat. Wistar rats were thymectomized, adipectomized (surgical extirpation of the interscapular brown adipose tissue), thymectomized and adipectomized, and sham-operated at birth, only 8-week-old females being employed in the experiment. The production of antibody to bovine serum albumin (BSA) and sheep red blood cells (SRBC), delayed skin reactions to BSA, rejection of thyroid allograft implanted under the kidney capsule, and development of experimental allergic encephalomyelitis were investigated. Neonatal adipectomy did not affect the production of anti-BSA and anti-SRBC antibodies. On the other hand, delayed skin reactions to BSA, rejection of thyroid allograft, and incidence and severity of allergic encephalomyelitis were much more pronounced in adipectomized animals. It has been postulated that the immune function of brown adipose tissue is an expression of the secretory activity of the tissue. Since the immunosuppressive effect of neonatal thymectomy on demyelinating disease was neutralized by neonatal adipectomy, and vice versa, and since thymectomy rendered ineffective the immunopotentiating influence of adipectomy on this disease, as demonstrated in thymo-adipectomized rats, it was concluded that the brown adipose tissue is a natural antagonist of the thymus in cell-mediated
immunity. This paper also describes the extra thymuses which were situated in the vicinity of the thyroid and parathyroid lobes of 23-2 per cent of rats. INTRODUCTION Brown adipose tissue is a unique tissue in mammals (Lindberg, 1970). Morphologically, it is composed of typical adipocytes with multilocular distribution of fat, and contains a rich network of blood vessels and a sympathetic nerve supply (Smalley, 1970). Functionally, it represents the principal site of nonshivering thermogenesis (Smith and Horwitz, 1969). The importance of brown adipose tissue for the maintenance of temperature in hibernators, on the one hand, and the suppression of immune reactions by deep hypothermia (Petrik, 1922; Schmidt, 1967) on the other, suggested a relationship between brown adipose tissue and immunity. A few studies in this area have indicated that extracts obtained from brown adipose tissue of hibernators are capable of suppressing the production of antibody under conditions of tissue culture (Sidky, Draggett and Auerbach, Correspondence: Dr B. D. Jankovic, Immunology Research Centre, P.O. Box 979, Vojvode Stepe 458, 11000
Belgrade, Yugoslavia.
597
598 B. D. Jankovi6, Alenka jane6ic and Ljiljana Popeskovi6 1969; Sidky and Hayward, 1972). However, we have demonstrated both in vivo and in non-hibernators the immunosuppressive activity of brown adipose tissue; it was found that repeated injections of extracts prepared from the rat brown adipose tissue produced a considerable reduction of immune responses (Jankovic, Popeskovic, Janelic and Lukic, 1974a, b). In addition, we reported briefly that the removal of brown adipose tissue from newborn rats enhanced to a great extent the reactions of cell-mediated immunity (Jankovic, Janelic and Popeskovic, 1974). This paper provides some basic information about the immune capacity of rats from which the interscapular brown adipose tissue was removed at birth, and describes an initial effort to correlate the immune function of the brown adipose tissue and that of the thymus.
MATERIALS AND METHODS
Animals Rats used in this study were all random bred Wistar strain housed in a closed colony. The animals were adipectomized, thymectomized, thymo-adipectomized or shamoperated at birth, and only 8-week-old females were used in the experiment.
Adipectomy There are several masses of brown adipose tissue in the rat thorax and abdomen, but the largest mass of tissue is situated in a depression of the muscles between the shoulder blades. This interscapular brown adipose tissue was extirpated (adipectomy) within 24 hours from newborn rats under deep hypothermic anaesthesia. After an incision along the mid-dorsal line between the scapulae, the overlying white fat was dissected and retracted, and then the brown adipose tissue was freed from surrounding muscles and removed. The skin incision was closed with interrupted sutures. Neonatal adipectomy proved to be a simple microsurgical procedure with an extremely low mortality rate. Sham-operated newborn rats were treated in an identical manner, except that the interscapular brown adipose tissue was left intact. The extirpated interscapular brown adipose tissue was equivalent to 0-7O-0-80 per cent of the body weight (Barnard & Skala, 1970), and to 60-70 per cent of the total brown adipose tissue of the newborn rat. Therefore, adipectomy as performed in this study should be considered incomplete.
Experimental groups The first group consisted of neonatally adipectomized rats; the second group was composed of neonatally thymectomized rats; the third group included rats which were both neonatally thymectomized and adipectomized; and the fourth group of sham-operated rats served as control. Non-operated female rats were also used in the experiment. The completeness of adipectomy and thymectomy was checked at the end of the experiment both grossly and histologically by examining the interscapular and mediastinal region. In order to ascertain the presence of extra thymuses in neonatally thymectomized rats, serial cross-sections of the thyroid region of the neck were prepared and histologically inspected. Sensitization with bovine serum albumin (BSA) Rats were immunized with crystalline BSA incorporated in Freund's complete adjuvant
599 Brown Adipose Tissue and Immunity (1 volume of BSA in saline, 0-15 volumes of Arlacel A, 0X85 volumes of Draceol, and autoclaved M. tuberculosis at a final concentration of 3 mg/ml). A single 01 -ml dose of BSAadjuvant mixture containing 0 5 mg of BSA was injected subcutaneously into the left hind foot-pad. The animals were skin-tested for delayed hypersensitivity 10 and 20 days later with 30 yug of BSA injected intradermally in the depilated flank. Delayed reactions were read at 24 hours, and the intensity ofreactions was graded from 0 to + + + according to the diameter and degree of induration.
Immunization with sheep red blood cells (SRBC) Separate groups of adipectomized and sham-operated rats were given a single intravenous injection of 7 x 108 SRBC in 0-2 ml of saline. Antibody determination Rats injected with BSA-adjuvant mixture were bled 10 and 20 days after immunization, and the sera separated from clotted blood and treated with 2-mercaptoethanol (ME). The presence of ME-sensitive and ME-resistant anti-BSA antibodies was determined using Takatsy's passive microhaemagglutination technique. Rats immunized with SRBC were bled 6 days later, and the sera tested for MEsensitive and ME-resistant haemagglutinins by means of a microhaemagglutination reaction using 7 x 45 mm tubes, 0-025 ml of a 1 per cent suspension of SRBC and an equal volume of serial serum dilutions. After incubation for 2 hours at room temperature, the end-point of antibody activity of sera was detected microscopically.
Induction of experimental allergic encephalomyelitis One part of fresh rat spinal cord was homogenized with two parts of Freund's complete adjuvant, and 0-1 ml of this mixture was inoculated into the left hind foot-pad of adipectomized, thymectomized, thymo-adipectomized and sham-operated rats. Animals were observed daily for the appearance of clinical signs of disease, and those showing paralysis were killed. The rats which did not exhibit neurological symptoms were killed 21 days after sensitization. Cerebrum, cerebellum and several levels of spinal cord of all rats were processed for staining with haematoxylin and eosin. A minimum of twelve sections of nervous tissue (from each rat) were examined and histological lesions graded. An arithmetic score reflecting an overall response was calculated for each group of rats, depending on the degree of encephalomyelitis defined as follows: negative = 0; + = 1; + + = 2; and +++ = 3. Implantation of thyroid graft under the kidney capsule Female neonatally adipectomized and sham-operated rats were grafted with thyroid tissue from inbred female 6-week-old Lewis rats. The right kidney was exposed through a dorsal paramidline skin incision. The thyroid graft was inserted under the capsule of the upper part of the kidney by means of a glass needle. The skin incision was then closed with surgical clips. Grafted kidneys were extirpated 2, 3, 4, 5 and 6 days after transplantation, fixed in Carnoy's solution, embedded in paraffin wax, and sections were stained with haematoxylin and eosin. The thyroid grafts were qualitatively analysed and the degree of rejection graded from 0 to + + +.
600
B. D. Jankovic, Alenka jane&il and Ljiljana Popeskovi6
Histology Rats of all groups were autopsied at the end of experimentation, and the thymus, regional and remote lymph nodes, spleen and Peyer's patches were taken for histological appraisal. Tissue sections were stained with haematoxylin and eosin, and methyl green and pyronin.
RESULTS BODY WEIGHT
Adipectomized, thymectomized, thymo-adipectomized and sham-operated rats gained weight steadily and equally. A small number ofthymectomized animals which exhibited signs of wasting disease (Jankovic, Waksman and Arnason, 1962) were excluded from the experiment. It was noted that there was no wasting disease in thymo-adipectomized rats. ANTIBODY PRODUCTION IN ADIPECTOMIZED RATS
The amount of ME-sensitive and ME-resistant antibodies against BSA increased between 10 and 20 days after immunization in both adipectomized and control rats. The capacity of adipectomized animals to produce circulating anti-BSA and anti-SRBC antibodies was very similar to that of sham-operated rats (Table 1). DELAYED SKIN REACTIONS IN ADIPECTOMIZED RATS
Delayed skin hypersensitive reactions were markedly enhanced in adipectomized rats in comparison with reactions in shamr-operated animals (Table 1). Histological appraisal of skin reactions of six adipectomized and six sham-operated rats 10 days after immunization with BSA revealed that oedema and cellular infiltration of the dermis, and underlying muscles and connective tissue were much more extensive in adipectomized than in control rats. Polymorphonuclear leucocytes surrounding small arteries were regularly seen in all sections of the skin, indicating the Arthus component of the reaction. Nevertheless, in all reactions perivenous aggregates of mononuclear cells predominated. No attempts were made to search for the basophilic component of delayed cutaneous reactions (Dvorak, Dvorak, Simpson, Richerson, Leskowitz and Karnovsky, 1970) in adipectomized rats. DEVELOPMENT OF ALLERGIC ENCEPHALOMYELITIS
Clinical and histological features of experimental allergic encephalomyelitis in adipectomized, thymectomized, thymo-adipectomized and sham-operated rats are shown in Table 2. Only one out of twenty-five sham-operated rats developed paralysis. On the other hand, twelve of nineteen adipectomized animals showed clear neurological symptoms. Clear-cut neurological symptoms were first apparent at 14 days. No thymectomized and thymo-adipectomized rats became paralysed. The incidence of paralysis correlated well with the intensity of histological lesions in the central nervous system, and the most numerous and advanced lesions were observed in adipectomized rats. In these animals, the lesions were not restricted to the nervous parenchyma but in several instances extended to the choroid plexus. The lesions consisted of lymphocytes, histiocytes, and rare plasma cells and polymorphonuclears.
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TABLE 2 INCIDENCE OF PARALYSIS AND SEVERITY OF HISTOLOGICAL LESIONS IN SHAM-OPERATED (S-O), NEONATALLY ADIPECTOMIZED (BATx), NEONATALLY THYMECTOMIZED (Tx), AND NEONATALLY THYMO-ADIPECTOMIZED (Tx-BATx) RATS SENSITIZED WITH ALLOGENEIC SPINAL CORD
Number of rats with histological lesions
Rats with paralysis
Group S-O BATx Tx Tx-BATx
Number of rats
Number
Per cent
25 19 12 10
1 12 0 0
4 63 0 0
0
+
++
5 15 0 0 10 2 2 7
4 4 0 1
Mean + + + score
1l0 2-8 0-1 09
1 15 0 0
TABLE 3
REJECTION
OF ALLOGENEIC THYROID IMPLANTED UNDER THE KIDNEY CAPSULE OF SHAM-OPERATED TALLY ADIPECTOMIZED (BATx) RATS
(S-O)
AND NEONA-
Days after transplantation
Group*
S-O BATx
0
+
++
2 0
3 2
0 2
5
4
3 Mean + + + score
0 1
0*6 1-8
0
+
++
0 0
1 0
3 0
Mean +++ score 1 5
2*0 30
0
+
++
0 0
0 0
2 0
Mean +++ score 3 5
2-6 30
The results are expressed in terms of a histological evaluation of rejection. * Five rats in group.
REJECTION OF THYROID ALLOGRAFTS
The course of rejection of allogeneic thyroid implanted under the renal capsule of sham-operated and adipectomized rats is indicated in Table 3. This comparative study demonstrated an accelerated rejection in adipectomized rats (Fig. la, c and e): at 2 days, the vascularization of allografts was very pronounced and many blood vessels were dilated (Fig. la); at 3 days, advanced destruction of thyroid follicles and cellular infiltration of graft bed was a characteristic feature of the rejection (Fig. 1c); and at 4 days, the follicles were completely destroyed and the cellular make-up of thyroid grafts was replaced by host cells (Fig. le). At 6 days the rejection of grafts in adipectomized rats was almost indistinguishable from that in controls, so far as the cellular infiltration of the graft was concerned. A preliminary attempt at studying the rejection of skin allografts in adipectomized rats had been made. However, the implantation of the thyroid under the kidney capsule was found to be a more suitable model for the purpose of this experiment since it allowed recognition of incipient signs of allograft rejection.
Brown Adipose Tissue and Immunity
603
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FIG. 1. The course of rejection of thyroid allograft (G) implanted under the kidney capsule (K) of adipectomized (a, c and e) and sham-operated (b), (d) and (f) rats: 2 (a) and (b), 3 (c) and (d), and 4 (e) and (f) days after transplantation. Arrows indicate dilated blood vessels in the graft. (Haematoxylin and eosin; magnification x 80.)
INVOLUTION OF BROWN ADIPOSE TISSUE
Histological examination of the interscapular brown adipose tissue of rats reared and maintained at room temperature revealed that adipocytes of young adult rats, in contrast to adipocytes of newborn rats, contained large areas occupied by confluent droplets of
B. D. jankovi6, Alenka jane&6ic and Ljiljana Popeskovic 604 lipid (Fig. 2a, b). This finding should be regarded as general morphological information about alterations which take place in the involuting brown adipose tissue and which are associated with profound ultrastructural and biochemical changes (Barnard and Skala, 1970). Those involution changes may explain the failure of adipectomy performed on 6-week-old rats (this group is not included in the present study) to affect the reactions of cell-mediated immunity.
a
O
L
FIG. 2. Morphology of the interscapular brown adipose tissue of (a) newborn and (b) 8-week-old rats. Note the involution in older animal. For comparison, (c) the interscapular white fat of newborn rat. (Haematoxylin and eosin; magnification x 300.)
EXTRA THYMUSES IN THE RAT
As mentioned before, this experiment included only those neonatally thymectomized rats which were proved histologically not to contain thymic remnants in the mediastinum and extra thymic lobes in the thyroid region of the neck. Since the thymus, thyroid and parathyroid are of the same embryonic origin, and since it was found that a number of correctly thymectomized rats still contained extra thymic lobes, it was deemed advisable to search for extra thymuses in a larger number of rats. For this purpose, the thyroid region of eighty-six non-operated and thymectomized Wistar rats was histologically examined, and it was found that 23-2 per cent of rats possessed extra thymuses of various size. Most of those thymuses were situated in the immediate vicinity of thyroid and parathyroid glands. However, some thymic lobes occupied the central part of the thyroid and were discovered only by serial cross-sectioning of the thyroid tissue. In some instances, thymic cells were distributed between thyroid follicles in such a manner as to give an impression of a 'spontaneous thyroiditis' (Fig. 3a). In fact, these cells belonged to the apical part of an intrathyroid thymic lobe (Fig. 3b). It is clear that extra thymuses should seriously be taken into account in studies in which thymectomized animals are prepared by surgical ablation of the mediastinal thymic lobes.
Brown Adipose Tissue and Immunity
605
FIG. 3. (a) Aggregates of thymus cells in the thyroid of normal rats. (b) Further cross-sectioning of the same sample of tissue revealed the presence of an intrathyroid thymus lobe. (Haematoxylin and eosin; magnification x 144.)
HISTOLOGICAL CHANGES IN LYMPHOID TISSUES
In spite of repeated and careful histological analyses, no apparent changes in cellular make-up of the thymus, spleen, lymph nodes and Peyer's patches were observed in neonatally adipectomized rats (Fig. 4a and c). In a number of adipectomized animals the thymus was larger than in sham-operated controls, but this difference in thymus weight was on the border of statistical significance (P> 0.05). On the other hand, thymectomized rats showed typical depletion of small lymphocytes (Waksman, Arnason and Jankovic, 1962) in thymus-dependent areas of the spleen, lymph nodes and Peyer's patches. The lymphoid dysplasia in thymus-less rats were not restored, or were restored only slightly, by neonatal adipectomy (Fig. 4b, d).
606
606B. D. Jankovi, Alenka ane&6 and Ljiljana Popeskovid
FIG. 4. (a) Spleen of adipectomized rat showing normal density of lymphocytes in periarteriolar white pulp, and (b) spleen of thymo-adipectomized rat lacking periarteriolar lymphocytes. (Methyl green and pyronin; magnification x 144.) Two right axillary lymph nodes, (c) one from an adipectomized rat with dense lymphocytes in paracortical area, and (d) another from a thymo-adipectomized rat with scattered lymphocytes in paracortical zone. In both lymph nodes germinal centres appear normal. (Haematoxylin and eosin; magnification x 144.)
DISCUSSION Since its initial description in 1551 by Gesner, various functions have been assigned to brown adipose tissue, inter alia it was regarded as part of the thymus and endocrine system (reviewed by Johansson, 1959). It is now widely recognized that the principal function of the tissue is the production of heat. This thermogenic function is under the control of the
607 6 Brown Adipose Tissue and Immunity sympathetic nervous system which, by means of catecholamines stimulates adenyl cyclase and thus accelerates lipolysis (Himms-Hagen, 1972). Brown adipose tissue is metabolically very active (Smith and Horwitz, 1969), and functional changes that occurred after surgical removal of the interscapular brown adipose tissue from cold-acclimatized rats led to the hypothesis that brown adipose tissue elaborates a product which alters metabolic processes in other tissues (Himms-Hagen, 1972). Since the regression of brown adipose tissue (Dawkins and Hull, 1964) coincides with involution of the thymus and with immune maturation, it may be deduced that the activity of the tissue is under the control of hormones (Johansson, 1959). With these premises in mind, it seems that the heat-producing activity of brown adipose tissue is only partially responsible for the significant decrease of immune potential in animals in deep hypothermia (Petrik, 1922; Andjus and Matic, 1959; Jaroslaw and Smith, 1961), and in animals with artificially lowered body temperature (Kopeloff and Stanton, 1942). The suppression of antibody response in such animals is most probably caused by a general decrease of the metabolic rate of the body (Sidky, Hayward and Ruth, 1972), including the synthesis of immunoglobulins. The present experiment differs conceptually from those mentioned in the preceding paragraph, as well as from the in vitro studies of brown adipose tissue (Sidky et al., 1969; Sidky and Hayward, 1972) since it was performed on non-hibernators, under in vivo conditions, at normal body temperature, and on animals lacking the interscapular brown adipose tissue. Recently, we have shown in rats that repeated injections of extracts from brown adipose tissue of newborn or 21-day-old cold-adapted rats produced a significant suppression of delayed skin hypersensitivity, whereas the formation of antibody was not greatly affected (Jankovic et al., 1974a, b). These findings are in complete agreement with the results presented here which show that neonatal adipectomy enhances the reactions of cell-mediated immunity (delayed hypersensitivity, experimental allergic encephalomyelitis and rejection of thyroid allograft) while leaving intact the production of antibody against BSA and SRBC. However, there is a discrepancy between our results and the observation that brown adipose tissue extract from hibernating animals inhibits the elaboration of antibody in vitro (Sidky et al., 1969; Sidky and Hayward, 1972). Several factors may account for this difference in results: experimental conditions, use of hibernators and non-hibernators, manner of preparation of tissue extracts, and amounts of extract employed in the study. In addition, since the brown adipose tissue of hibernators exerts an extraordinary metabolic activity, the content of a postulated immuno-inhibitory agent (vide infra) may be expected to be much higher in the extract of the tissue from a hibernator than from a non-hibernator. The discrepancy between the humoral and cell-mediated immune responses in adipectomized rats, as described here, could be explained in the following way: the neonatal excision of the interscapular brown adipose tissue represents, in fact, an incomplete adipectomy, which leaves intact other deposits of brown adipose tissue, so that the amount of the tissue in adipectomized rats is still sufficient to allow a normal production of antibody. Reasoning of this kind would imply that in non-hibernators the mechanisms which underlie cell-mediated immunity are more sensitive to the lack of brown adipose tissue than the mechanisms primarily involved in humoral immunity. The simplest construction that can be put upon these results is that the immuno-inhibitory activity of brown adipose tissue is probably the expression of a biologically active substance produced by the tissue (Himms-Hagen, 1972). Neonatal adipectomy,
B. D. jankovic5, Alenka janeei6 and Ljiljana Popeskovic 608 though incomplete, leads to a marked deficiency in this substance and thus to the potentiation of cell-mediated immune responses. Since neonatal adipectomy failed to reconstitute thymus-dependent areas in lymphoid tissues of thymo-adipectomized rats, it appears that the postulated immuno-inhibitory factor interferes rather with the function oflymphocytes than with the proliferation of lymphocytes. Bearing in mind the influence that the sympathetic nervous system exerts on brown adipose tissue (Steiner, 1972), it may be assumed that catecholamines (e.g. noradrenaline) may be mediators of the immune function of brown adipose tissue. Another possibility is that the immune effect ofbrown adipose tissue is due to a release of fatty acids from the tissue (Drahota, 1970). The interconnections between brown adipose tissue and hormones should also be taken into consideration (Johansson, 1959). Finally, two important aspects emerged from the study of experimental allergic encephalomyelitis in neonatally adipectomized, thymectomized, and thymo-adipectomized rats. First, adipectomy and thymectomy produced quite different effects on that disease: adipectomy potentiated whereas thymectomy suppressed its development. Secondly, adipectomy neutralized the immunosuppressive influence of thymectomy, and vice versa, thymectomy rendered ineffective the immunopotentiating influence of adipectomy on allergic encephalomyelitis. In view of these observations, it is tempting to conclude that the brown adipose tissue is a natural antagonist of the thymus in cell-mediated immunity.
ACKNOWLEDGMENTS This work was supported by grants from the Republic Fund for Research of SR Serbia, Belgrade. We acknowledge with thanks the technical assistance of Miss Anica Rankovic. REFERENCES
ANDJUS, R. K. and MATIC, 0. (1959). 'Hibernation and immunological reactivity.' Pftagers Arch. ges. Physiol., 270, 55. BARNARD, T. and SKALA, J. (1970). 'The development of brown adipose tissue.' Brown Adipose Tissue (ed. by 0. Lindberg), p. 33. American Elsevier, New York. DAWKINS, M. J. R. and HULL, D. (1964). 'Brown adipose tissue and the response of new-born rabbits to cold.'J. Physiol., 172, 216. DRAHOTA, Z. (1970). 'Fatty acid oxidation by brown adipose tissue mitochondria.' Brown Adipose Tissue (ed. by 0. Lindberg), p. 225. American Elsevier, New York. DVORAK, H. F., DvORAK, A. M., SIMPSON, B. A., RiCHERSON, H. B., LESKOWITZ, S. and KARNOVSKY, M. J. (1970). 'Cutaneous basophil hypersensitivity. II. A light and electron microscopic description.' J. exp. Med., 132, 558. HIMMs-HAGAN, J. (1972). 'Effects of catecholamines on metabolism.' Catecholamines (ed. by H. Blaschko and E. Muscholl), p. 363. Springer-Verlag, Berlin. JANKOVIC, B. D.,JANEzId, A. & POPESKOVI,6, L. (1974). 'Brown adipose tissue: potentiation of delayed skin hypersensitivity and experimental allergic encephalomyelitis in adipectomized rats.' Fed. Proc., 33, 804. JANKOVId, B. D., POPESKOVI6, L., JANEiId, A. and LUKI6, M. L. (1974a). 'Immune reactions in rats
treated with allogeneic brown adipose tissue and rats adipectomized at birth.' Proc. rugoslav Immunol. Soc., 3, 122. JANKOVI6, B. D., POPESKOVI6, L., JANEZI6, A. and LUKI6, M. L. (1974b). 'Brown adipose tissue: effect on immune reactions in the rat.' Naturwissenschaften, 61, 36. JANKOVIC, B. D., WAKSMAN. B. H. and ARNASON, B. G. (1962). Role of the thymus in immune reactions in rats. I. The immunologic response to bovine serum albumin (antibody formation, Arthus reactivity, and delayed hypersensitivity) in rats thymectomized or splenectomized at various times after birth.' J. exp. Med., 116, 159. JAROSLAW, B. N. and SMITH, D. E. (1961). 'Antigen disappearance in hibernating ground squirrels.' Science, 134, 734. JOHANSSON, B. (I1959). 'Brown fat: a review.' Metabolism, 8, 221. KOPELOFF, L. M. and STANTON, A. H. (1942). 'The effect of body temperature upon haemolysin production in the rat.'J. Immunol., 44, 247. LINDBERG, 0. (1970). Brown Adipose Tissue. American Elsevier, New York. PETRIK, J. (1922). 'Contribution A la serologie des mammiferes hibernants.' Publ. Fac. Med. Brno R.C.S., 1, 247. SCHMIDT, J. P. (1967). 'Response of hibernating
Brown Adipose Tissue and Immunity animals to physical, parasitic and infectious agents.' Mammalian Hibernation, volume 3 (ed. by K. C. Fischer, A. R. Dawe, C. P. Lyman, E. Schonbaum and F. E. South, Jr), p. 421. Oliver & Boyd, Edinburgh. SIDKY, Y. A., DAGGETr, L. R. and AUERBACH, R. (1969). 'Brown fat: its possible role in immunosuppression during hibernation.' Proc. Soc. exp. Biol. (N. r7.), 132, 760. SIDKY, Y. A. and HAYWARD, J. S. (1972). 'Immunosuppression by extracts of brown fat.' Fed. Proc., 31, 800. SIDKY, Y. A., HAYWARD, J. S. & RUTH, R. F. (1972). 'Seasonal variations of the immune response of ground squirrels kept at 22-24'C.' Canad. J. Physiol. 50, 203.
609
SMALLEY, R. L. (1970). 'Changes in composition and metabolism during adipose tissue development.' Brown Adipose Tissue (ed. by 0. Lindberg), p. 73. American Elsevier, New York. SMITH, R. E. and HORWrrz, B. A. (1969). 'Brown fat and thermogenesis.' Physiol. Rev., 49, 330. STEINER, G. (1972). 'Immunosympathectomy as a tool in metabolic studies.' Immunosympathectomy (ed. by G. Steiner & E. Sch6nbaum), p. 159. Elsevier, Amsterdam. WAKSMAN, B. H., ARNASON, B. G. and JANKOVI6, B. D.
(1962). 'Role of the thymus in immune reactions in rats. III. Changes in the lymphoid organs of thymectomized rats.'J. exp. Med., 116, 187.
