r 185 I Lymphology 10 (1977) 185-191 I ©Georg Thieme Verlag Stuttgart ,I The Effect of Steroids on the Circulating Lymphocyte I Population f I. Changes in the thoracic duct lymphocyte population of the rat I after neonatal thymectomy and prednisolone treatment. I Lars A. Hedman, Per M. Lundin Department of Pathology, University of Gothenburg, Sahlgren's Hospital, S-413 45 Gothenburg, Sweden

Summary The most pronounced morphological effect I The influence of prednisolone on the thoracic duct of steroids in sensitive species is seen in the r cells of rats was measured by the cell count in thymic cortex which, after large doses, can lymph at different times after a single injection and be totally depleted of small lymphocytes. In I correlated with lymphocytolysis in lymphoid tissues. peripheral lymphoid organs such as spleen, In both control and neonatally thymectomized lymph nodes and Peyer's patches the most i animals there was a marked fall in the cell count obvious effect occurs in the follicular germinal and flow at 3 hours and the percentage reduction I was greater in thymectomized animals. Restitution centers with pronounced lymphocytolysis and was rapid in both groups and pretreatment levels phagocytosis of pyknotic lymphocyte nuclei. I were regained in 17 hours. The blood mononuclear Germinal centres disappear in one or two cells followed the same pattern. In animals with an days. In other lymph node regions and in the intact thymus adrenalectomy causes a significant spleen a lesser effect can be seen with lysis of increase in thoracic duct cell counts but in neonat· I ally thymectomized animals thoracic duct lymph is a few cells in the periphery of follicles and unchanged after adrenalectomy. It ean be concluded subcapsularly in the lymph node cortex. A .I that in the rat lymphocyte level in the circulating minimal effect is seen in the so-called thymus pool is thymus dependent but restoration of the dependent areas described by Pa"ott et al . (3), circulating cell count after steroid induced involut· ! ion is independent of intact thymic function. which contain few steroid sensitive cells according to Lundin and Jiirplid ( 4). Bone J Our data support the hypothesis of lymphocy te trapping and redistribution as a major mechanism marrow lymphocytes or at least the inununo­ l after a si ngle cortisone dose. competent population is steroid resistant (5). The relationship of these changes to inunune .•I Key-words: Lymphocyte - Thoracic duct - Lymphoid tissue - Blood - Corticosteroid - system functions is not known. At least some ! Thymectomy - Adrenalectomy - Rat of the functional changes in cell mediated r inununity are also observed in steroid resistant species but large doses or prolonged treatment Introduction I are required (1). The significance of the steroid sensitivity of The modem classification of lymphocytes into r lymphocytes and lymphoid tissue in species T and B cells has been modified and clarified such as mice and rats is still unknown. j during recent years. Some authors have tried Neither is the steroid sensitive population to defme subpopulations ofT and B cell lines. I clearly defined relative to knowledge of Thymus dependent lymphocytes are, for functionally different lymphocyte popul­ I instance·; divided into· T 1 and T2 cells by ations, and li ttle is known about a possible i Cantor and Asofsky (6), and Th and Tp cells steroid effect on lymphocytes of so-called I by Kanda et al. (7). Defmition of these T cell steroid resistant species such as man and the i subgroups differ, but some common points guinea pig. For reviews see Claman ( l) and emerge. The cell of one subgroup (Tl, Th ) Bach (2). This work was supported by a grant fro m the Swedish Cancer Society (No. 311 -B74-04XC). j Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY. 186 Lars A. Hedman, Per M. Lundin respectively) is steroid and radiation sensitive However, the antibody forming cells precursor and morphologically represented by the in the bone marrow is not altered according thymus cortical cell, probably short-lived and to Cohen and Claman (5). The splenic poorly recirculating. The distribution and role precursor cells may be responsible for the in the peripheral pool is not clarified, No steroid sensitivity of the humoral antibody certain immunological function has been _ response ( 5, 16). An influence on T · B cell ascribed to it in vivo, but Kappler found that cooperation cannot, however, be excluded. it may function as a suppressor cell (8) Thus, in spite of the dramatic response in The other subgroup (T2, Tp respectively) is sensitive species, the steroid sensitive steroid and radiation resistent. It is probably population is vaguely defined concerning located in the thymic medulla and peri­ morphology, kinetics and function. The pherally at least some cells are long-Jived. possibility of a corresponding cell population These long-lived cells are the most numerous in steroid resistant species, such as man and thymus dependent cells of the circulating guinea pig, is not clarified. I pool and in the thymus dependent areas of The aim of the present work is to try to I lymph nodes, spleen and Peyer's patches. As define the steroid sensitive lymphocyte • judged by the capacity to induce a graft population morphologically and functionally I versus host reaction they are apparently in relation to modern concepts of lymphocyte 1 immunocompetent as found by Blomgren function. Thls paper describes the effect of I and Andersson (9). They may function as a prednisolone on the lymphocytes of the I helper or suppressor cell in antibody product­ circulating pool, (i.e. the thoracic duct and ion (8). The T memory cell (Tpm according blood), and in the lymphoid tissues of the to Kanda) may be included in this group (7). rat (a steroid sensitive species), during acute The relationshlp between these two T cell steroid involution and restitution. lines is unclear. They may represent two separate populations already in the thymus or Material and methods t the T l cell line may differentiate peripherally Rats of the Sprague-Dawley strain were , to the T2 cell (10). The role ofT lymphocyte thymectomized within 24 hours of birth. [ subsets in different phases of the immune Thymectomy was performed under hypo- 1 response have been discussed by, among thermia and ether anaesthesia with partial others, Cantor and Boyse ( 11 , 12) and Peck sternotomy and vacuum extraction of the l et al. (13). More recently, T cell subsets in thymic lobes using a dissection microsco~e. I mice has been characterized with the antigens Totally 143 animals aged 7-10 weeks, w1th 1 of the Ly-series (1 1). body weights of about 200-300 g were use~. [ The situation is as unclear for the B cell. In Sex distribution was equal. Shamthymectom1zed I the steroid sensitive species, the mouse, (anaesthesia and sternotomy) and nonthym­ lymphocytolysis is observed also in B cell ectomized rats served as controls. areas such as lymph follicles and especially in The corticosteroid used was a water soluble l the germinal centres in all secondary lymphoid preparation of prednisolone sodium succin~te i tissues (4). There is no evidence of more than (Precortalon Aquosum, Organon). It was g1ven '[ one B cell development line. The heterogeneity intramuscularly in the thlgh in a dose of of the B cell population is related to the 10 mg/ 100 g body weight. Some animals were I differentiation of a stem cell to a virgin cell injected with equal volumes of saline instead ' and further to an antibody producing cell or of prednisolone. a memory cell (14). The mature antibody forming cells (e.g. plaque-forming cells) are Three 17 or 40 hours later thoracic duct certainly steroid resistant, but with administ­ drain;ge was started under anaesthesia ration prior to antigen exposure a primary (M ebumal 60 mg/ml intraperitoneally at antibody response is easily depressed with 6 mg/ 100 g body weight). The open-neck­ correct timing as described by B erglund (1 5). technique of Reinhardt ( 17), was used. The Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY. The Effect of Steroids on the Circulating Lymphocyte PopulationfPart I 187

minimum requirements were drainage for 20 Total cell count in thoracic duct lymph in rats. minutes and 0.1 cc of lymph, but usually drainage continued 60 minutes. The lymph -- Non thymectomized was collected in heparinized glass tubes. 50 000 cells/J.ll ·-----· Thymectomized Lymph volume was measured and a sample. _ taken for mononuclear cell count. The total flow was then calculated in cells/hour. A 40 000 blood sample was taken from the lingual vein at the end of the drainage and mononuclear 30 000 and polymorphonuclear cells counted. Lymph and blood smears were also made for micro­ scopical examination. The animals were 20 000 sacrified and the spleen, lymph nodes (thymic, para-aortic, axillary and mesenterial) and !\. ____ .. --f ------1 I adrenals were dissected out, weighed and 10 00 \ r __ .. ------fiXed in neutral formalin for histology. I Some of the neonatally thymectomized 0 3 17 40 J animals and corresponding controls were hours after prednisolone injection adrenalectomized as adults. A posterior 1 approach was used under ether anaesthesia Fig. 1 The thoracic duct lymph cell count in with aseptic conditions. Postoperatively the normal and neonatally thymectomized rats at I animals had saline to drink. Three or four different times after a corticosteroid injection. r days after adrenalectomy thoracic duct drainage was started as described above.

Cell count, cell flow and organ weights are Total cell flow in t horacic duct lymph in rats. I given as the mean and standard error of the mean and Student's t-test is used to compare - - Non thymectomized I the different groups. Thymectomized l 106 cells/hour ------Results 25 I The average relative cell count and total l lymph cell flow in the thoracic duct of un­ T treated controls (nonthymectomized and 20 I shamthymectomized animals) and of I I thymectomized untreated rats are shown in figs 1 and 2. Thymectomized rats showed 15 I an obvious significantly reduced lymphocyte count in the thoracic duct. ! In both controls and thymectomized animals 10 I there was a marked fal l in the lymphocyte counts 3 hours after the steroid injection. 5 l For the controls the lymphocyte reduction r was abou t SO per cent and for the thymectom­ ized animals about 70 per cent of the original I levels expressed as cells/hour. Seventeen hours 0 3 17 40 I after the steroid injection the original cell hours after prednisolone injection level was restored in both groups. Fig. 2 The thoracic duct lymph cell now in normal i Mononuclear cells in venous blood followed and neonatally thymectomized rats at different ) a similar pattern in both groups with a times after a corticosteroid injection.

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188 Lars A. Hedman, Per M. Lundin

Total mononuclear cell count in venous blood in rats. Total po lynuclear cell count in venous blood in rats.

-- Non thymectomized --. Non thymectomized cells/Jll cells/J.ll ·-----· Thymectomized 3000 ·-----· IThymectomized 6000 l I l 2000 T 5000 I

1000 \ 4000 l

3000 0 3 17 40 I hours aher prednisolone injection i 2000 Fig. 4 The venous polymorphonucleai cell count in I normal and neonatally thymectomized rats at different times after a corticosteroid injection. l 1000 I marked fall 3 hours after steroid treatment and ' restoration within 17 hours. The polymorpho­ I 0 3 17 40 nuclear cells in venous blood showed a slight hours after prednisolone injection I similar tendency (figs 3 and 4). I Fig. 3 The venous mononucleai cell count in The body, spleen and adrenal weights were normal and neonatally thymectomized rats at the same in both untreated thymectomized different times after a corticosteroid injection. and untreated control animals (Table 1). Mesenterial and other lymph nodes were smaller in the thymectomized animals and !he

Table I Body weights (G.) and organ weights (MG.) of normal and thymectomized rats at different times after a corticosteroid injection (mean ± standaid error of the mean).

Mesenterial Other Rats B.W. Thymus Spleen Lymph Node Lymph Nodes* Adrenals

Nonthymectomized: untreated 28 262 ± 14 513 ± 40 792± 45 292 ± 13 151 ± 9 58 Nonthymectomized: 3 hours after steroid treatment 15 245 ± 8 421 ± 29 801 ± 110 290 ± 15 137± 10 64 Nonthymcctomized: 17 hours after steroid treatment 15 263 ± 13 372 ± 31 971 ± 183 244 ± 20 139 ± 12 62 Nonthymcctomized: 40 hours after steroid treatment 12 308 ± 18 323 ± 28 682:!: 40 310±45 116 ± 7 49

Thymectomized: untreated 11 267 ± 17 734 ± 77 220 ± 12 125 ± 12 62 Thymectomized: 3 hours after steroid treatment 10 247 ± 14 767 ± 120 216 ± 18 124 ± 9 70 Thymectomized: 17 hours after steroid treatment 11 280 ± 16 883 ± 156 186 ± 15 121 ± 12 51 Thymectomized: 40 ho urs after steroid treatment 12 316 ± 16 663 ± 36 265 ± 66 93 ± 8 54 * includes axillaiy, para-aiotic and thymic lymph nodes. Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY. TI1e Effect of Steroids on the Circulating Lymphocyte Population/Part I 189

difference was not significant only for Forty hours after injection the spleen and mesen terial lymph nodes. After steroid lymph nodes showed the same picture as at treatment body, spleen and adrenal weights 17 hours, with few pyknotic cells. Follicles I were unchanged, but 40 hours after injection and germinal centres were smaller than in I lymph node weights were lowered. In the untreated controls. control group the thymic weight was In the nonthymectomized animals there was I significantly decreased 40 hours after the a significant rise in the lymph cell count and corticosteroid injection (Table 1). flow 3 days after adrenalectomy. Neonatally I Histologically the spleen and lymph nodes thymectomized animals showed no significant I of the thymectomized untreated animals changes in the cell count in thoracic duct showed a depletion of small lymphocytes in lymph 3 or 4 days after adrenalectomy I the so-called thymus dependent areas. Three (Figs 5 and 6). I hours after the prednisolone injection controls showed a marked lymphocytolysis in the Discussion I thymic cortex, spleen and lymph nodes. In I The rat is regarded a typical steroid sensitive I the lymph nodes cytolysis was seen chiefly in animal with well documented morphological germinal centres and between follicles in the changes after steroid treatment as described l outer cortex. In the spleen lymphocytolysis by Dougherty (18). Using a corticosteroid was seen in the germinal centres. The thymus preparation without depot effect the changes I dependent areas of spleen and lymph nodes in the spleen and lymph nodes are quickly I showed few pyknotic cells. Neonatally restored. The thymus shows marked changes, thymectomized animals showed a similar especially the cortex, where regeneration i picture in the spleen and lymph nodes. Seven­ starts about the fifth or sixth day (19). teen hours after the injection most of the pyknotic cells in the spleen and lymph nodes The reduction of circulating lymphocytes as had disappeared in both grou-ps. In the effect of thymectomy performed at different I controls many pyknotic cells remained in the ages are described earlier (20, 2 1) and our I thymic cortex which was reduced in thickness. ' I Total cell flow in thoracic duct lymph in rats. I Total cell count in thoracic duct lymph in rats. Non thymectomized 1. -- Non thymectomized 106 cells/hour Thymectomized I ·-----· Thymectomized 40 r 90 oooj ""''"' 3 I 70 000- l 20 I 50 000

( 30 000 10 !------!------; :t------f------1 I 10 000

I 0 3 4 0 3 4 I days after adrenalectomy days after adrenalectomy I Fig. 5 The thoracic duct lymph cell count in Fig. 6 The thoracic duct lymph cell flow in normal normal and neonatally thymectomized rats at and neonatally thymectomized rats at different I different times after adrenalectomy. times afte.r adrenalectomy. Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY. 190 Lars A. Hedman, PerM. Lundin results with neonatally thymectomized rats population, but it is known that peripheral I agree with these earlier observations. The lymphocytes of the rat are sensitive to j effect of thymectomy concerning the steroids in vitro (21 ). completeness of T lymphocyte depletion is It is, however, difficult to connect a unclear. Animals neonatally thymectomized I pronounced cytolysis with the rapid restitut- -~ certainly lack most T cells but cannot be ion of the circulating lymphocyte level. A regarded as pure " B cell animals". Remaining possible alternative explanation could be a T cell numbers depend on the time of · - trapping of cells with redistribution to one or j thymectomy and the animals maturation. several organs. The bone marrow is one such Neonatally thymectomized mice have up to possible organ and an increase of theta positive I 20 per cent theta positive cell s in thoracic cells in bone marrow after steroid treatment duct lymph and with further treatment by in mice has been described by Moorhead, irradiation and bone marrow reconstitution Claman (24) and Cohen (25). Infusion of that level can be depressed to about 10 per st Cr-labelled lymphocytes in mice during cent according to Sprent (22). However, steroid treatment also showed an accumulat­ i T-B cell cooperation must be defective and ion in bone marrow (26). ' alteration of the B cells cannot be excluded. The reappearance of cells in the circulation l In the present work the changes in organ (both lymph and blood) can theoretically be I weights after neonatal thymectomy were the due to a return of the original cell population same as Reinhardt (23) found in 1945. In the after trapping, or to a rapid proliferation of i spleen and lymph nodes we found the same new cells, or by a mobilization of cells from effects as Parrott et al. (3) described in 1966, some depot. It can of course also be the i with lymphocyte depletion in the so-called result of a combination of these alternatives I thymus dependent areas. In thoracic duct and to clarify that a careful analysis of the lymph the steroid causes a rapid initial cell populations before, during and after decrease of lymphocytes with rapid restitution steroid treatment is needed. to pretreatment levels within 17 hours. This rapid reappearance of circulating cells is seen In the control animals with an intact thymus I both in thymectomized animals and animals there was a significant rise in lymph cell I with an intact thymus. Thus, the restitution number 3 days after adrenalectomy. This can , of the circula ting cell level after steroid treat­ be explained by the loss of endogenous I ment cannot be dependent on a direct cellular cortisone secretion that normally depresses a influence of the thymic cortex. In contrast steroid sensitive lymphocyte population in the [ the original level of lymphocytes in the circulating pool. Neonatal thymectomy com- r circulating pool is thymus dependent. bined with adult adrenalectomy produced no changes in lymph cell counts up to 4 day~ ( Steroid injection seems to cause cytolysis after adrenalectomy, which shows that this chiefly in the thymus independent areas of cell population is thymus dependent. I spleen and lymph nodes both i_n neon~tally thymectomized and control arumals wtth a Our dat-a support the hypothesis of lympho- l pronounced cytolysis seen after 3 hours. cyte trapping and redistribution as a major Thymus dependent areas of thymectomized mechanism after" a single cortisone dose. I animals are readily identified with only very Analysis of the lymphocyte populations [ few pyknotic cells after steroid treatment. during different phases of steroid action on the The microscopically observed effect of lymph of rats with a Coulter Counter and label I steroids on tissues, especially the thymus, is indexing after isotope administration sho~s 1 cytolysis, and the disappearance of circulating the same size distribution and same label mdex lymphoid cells has also been explained by proftle in all phases (Hedman and Lundin, to I cell death. be published). These experiments would I further support the hypothesis of lymphocyte Thjs predicted cytolysis can hardly be trapping and redistribution as a major ( visualized in the circulating lymphocyte mechanism of corticosteroid action. .

Permission granted for single print for individual use. 1 Reproduction not permitted without permission of Journal LYMPHOLOGY. l The Effect of Steroids on the Circulating Lymphocyte Population/ Part I 191 I R eferences I Claman, H.N. : Corticostero ids and lymphoid 13 Peck, A.B. et a!.: Environmental and genetic cells. New Engl. J . Med. 287 (1972) 388-397 control of T cell activation in vitro: A study I 2 Bach, J.F.: Frontiers of Biology. The mode of using isolated, alloantigen-activated T cell clones. action of immunosuppressive agents. North lmmunol. Rev. 35 (1977) 146- 180 i Holland Pub!. Co. Amsterdam 1975 14 Stro.ber, S. : Immune function cell surface 3 Parrott, D.M. V. et al. : Thymus-dependent areas characteristics and maturation of B cell sub· I in the lymphoid organs of neonatally populations. Transplant. Rev. 24 (1975) 84- 112 thymectomized mice. J . Exp. Med. 123 (1966) 15 Berglund, K. : Studies o n factors which condition I 191 - 210 the effect of cortisone o n antibody production. 4 Lundin, P., B. Jiirp/id: Effects of corticosteroid I. The significance of time of hormone administrat· I and radiation on lympho id tissue in mice. ion in primary hemolysin response. Acta Path. Comparisons and mutual interactions. Microbiol. Scand. 38 (1956) 311 - 328 t Lymphology 6 (1973) 158- 166 16 North , R.I.: The action of cortisone acetate on 5 Cohen, J.J., H.N. Claman: Thymus-marrow cell-mediated immunity of infection: Histogenesis I immunocompetence. V. Hydrocortisone-resistant of the lymphoid cell response and selective cells and processes in the hemolytic antibody elim ination of committed lymphocytes. Cell i response of mice. J. Exp. Med. 133 (1 971) 1 lmmunol. 3 (1972) 501-515 1026-1034 17 Reinhardt, 111.0.: Rate of now and cell count of l 6 Cantor, H., R. Asofsky: Synergy among rat thoracic duct lymph. Proc. Soc. Exp. Bioi. lymphoid cells mediating the graft-versus-host Med. 58- 59 (1945) 123- 124 i response. Ill. Evidence for interaction between 18 Dougherty, T.F. , A. White: Functio nal alterations two types of thymus-derived cells. J . Exp. Med. in lymphoid tissue induced by adrenal cortical i 135 (1972) 764- 779 secretion. Am. J. Anat. 77 (1945) 81- 116 7 Kanda, S. et al.: Immunologic properties of 19 Lundin, M., U. Sclrelirr : The effect of steroids on I mouse thymus cells: Identification ofT cell the histology and ultrastructure of lymphoid { functions within a minor, low-density sub­ tissue. II. Regeneration of the rat thymus after population. J. Exp. Med. 136 (1972) 1461- 1477 pronounced involution. Path. Eur. 3 (1968) 8 Kappler, J. W., P.C. Marrack: Functional 531- 541 heterogeneity among the T-derived lymphocytes 20 Schooley, J.C. , M.J\11. Shrewsbury: The lympho· of the mouse. Ill. Helper and suppressor T-cell cyte and hemopoiesis. The thymus and the activated by concanavalin A. Cell. lmmunol. 18 recirculating lymphocyte pool. Symp. Bristol, (1975) 9- 20 April 1966, Williams and Wilk ins, Baltimore 1966 9 Blomgren, H., B. Andersson: Evidence fo r a 21 Lundin, P.M., J.C. Schooley: The influence of small pool of immunocompetent cells in the prednisolone and thymecto my on the thoracic ! mouse thy mus. Exp. Cell. Res. 57 (1969) duct lymphocyte populatio n of the rat. 185- 192 Lymphology 6 (197 3) 90-97 I 10 Goldschneider, 1.: Antigenic relationship between 22 Sprent, J.: Circulating T and B lymphocytes of bone marrow lymphocytes, cortical thymocytes the mouse. I. Migratory properties. Cell. Immunol. I and a subpo pulation of peripheral T· cells in the 7 (1973) 10-39 rat: Description of a bone marrow lymphocyte 23 Reinhardt, W.O. : Effect of thymectomy in rats I antigen. Cell. lmmunol. 24 (1976) 289-307 on lymph nodes and spleen. Anat. Rec. 91 (1945) 11 Ca/ltor, H., E.A. Boyse: Functional subclasses of 295 I T lymphocytes bearing different ly-antigens. 24 Moorhead, J. Jl!., H.N. Cia man: Thymus-derived I. The generation of functio nally distinct T cell lymphocytes and hydrocortiso ne: Identification I subclasses is a differentiative process independent of subsets of theta-bearing cells and redistribution of antigen. J. Exp. Med. 141 (1975) 1376- 1389 to bone marrow. Cell. lmmunol. 5 (1972) 74-86 ! 12 Cantor, H., E.A. Boyse: Functional subclasses of 25 Cohen, J.J.: Thymus-derived lymphocytes T lymphocytes bearing different ly-antigens. sequestered in the bone marrow of hydrocortisone­ ) 1f. Cooperation between subclasses of ly+ cells in treated mice. J. Immunol. 108 (1972) 841 - 844 the generation of killer activity. J . Exp. Med. 141 26 Zatz, M.M.: Effects of cortisone on lymphocyte ( (1975) 139 0- 1399 homing. Israel J. Med. Sci. 11 (1975) 1368- 1375 .,I Lars A. Hedman, PerM. Lundi.'l, Department of Pathology, University of Gothenburg, Sahlgren 's Hospital, S-413 45 Gothenburg, Sweden l

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