Mechanisms of Ageing and Development, 4 (1975) 231-239 © Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

231

T H E L A T E E F F E C T S OF S E L E C T E D I M M U N O S U P P R E S S A N T S O N I M M U N O C O M P E T E N C E , DISEASE I N C I D E N C E , A N D M E A N L I F E - S P A N I. H U M O R A L I M M U N E A C T I V I T Y

E. H. PERKINS, W. J. PETERSON, C. F. GOTTLIEB, M. K. HALSALL*, L. H. CACHEIRO and T. MAKINODAN** Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. (U.S.A.) (Received May 13, 1975)

SUMMARY The effect of different immunosuppressive treatments during young adulthood on humoral immune competence late in life was determined. It was found that the marked reduction in humoral immune competence in aged mice is further compromised when severe insults are administered early in life. Thus, thymectomy, splenectomy, and sublethal X-irradiation produced lasting immunodepression as measured (1) in situ and (2) by the hemolysin, direct and indirect plaque forming cell responses of adoptively transferred spleen cells. In contrast, treatment with cyclophosphamide and cortisone acetate were without effect, indicating that drugdamaged cells of the immune system were replaced by competent cells during the course of time. Decrease in immune competence of aged thymectomized animals could not be correlated with a decrease in numbers of theta-bearing T or immunoglobulin receptor-bearing B lymphocytes. The significance of the observed unequal effects of these immunosuppressants on immune competence, as they relate to disease incidence and life expectancy, are dealt with in the third paper in this series.

INTRODUCTION It is well known that the incidence of certain types of cancer, autoimmune disease, and infection increases with age 1-3, and in mice, at least, the increase in the susceptibility to these diseases is correlated with a decrease in the activity of the immune system 4. Proponents1, 5 of the immune-surveillance theory of Thomas 6, have proposed that the relationship is causal and the age-related deterioration of the im-

* Present address: M. K. Halsall, Chemistry Department, University of Cincinnati, Cincinnati, Ohio 45112 (U.S.A.) ** Present address: Dr. T. Makinodan, Gerontology Research Center, Baltimore City Hospitals, Baltimore, Md. 21224, (U.S.A.)

232 mune system is primarily responsible for these diseases. If so, suppression of the immune system, especially by long-lasting irreversible insults, should increase the incidence and accelerate the onset of these diseases. This, in turn, should result in a decrease in life expectancy. Accordingly, a long-term study was initiated with two objectives in mind: (a) to evaluate methods for inducing lasting immunodeficiency by assessing immunocompetence late in life and (b) to determine what effect temporary and permanent immunosuppressive insults have on humoral and cell-mediated immunocompetence, disease incidence, and life expectancy. In this initial series of studies young adult (2- to 3-month-old) and adult (12- to 15-month-old) long-lived (C57BL/Cum ~ × C3H/Anf Cum c7) F1 (BC3F1) mice (mean life-span, 30 months; maximum life-span, 45 months) were subjected to surgical (thymectomy or splenectomy), chemical (cyclophosphamide or cortisone acetate), or radiation (400R, total-body) insults, and their immune activities were assessed 18-22 months later. In addition, the effects of these insults on the life-span and on the incidence of various diseases were assessed. We wish to present our findings in three separate reports. In this, the first report, we will discuss the late effects of these insults on the humoral immune activity. In the second we will discuss the late effects of these insults on the cell-mediated immune competence and in the third, disease incidence and mean life-span. MATERIALS AND METHODS BC3F1 mice of both sexes and 2-3 or 12-15 months of age were treated with surgical, chemical, or radiation insults known to be immunosuppressive.

Surgical insults Surgical insults were performed on mice anesthetized with Diabutal (1.501.80 mg of sodium pentobarbital per mouse). For thymectomy two midline incisions were made, the first through the skin above the sternum and the second through the manubrium sternum down to about the third rib. The thymus was exposed by prying the cut edges of the sternum apart with a pair of Curved forceps held in one hand, and at the same time the lobes of the thymus were freed and removed from tissue facia with a pair of small blunt forceps held in the other hand. The skin incision was subsequently closed with wound clips. In the case of splenectomy, the mouse was placed face down and a dorsal-ventral incision was made through the skin in the general area of the spleen. The spleen was located, exteriorized and dissociated from the connecting vessels and facia by heat cauterization. The incision was then closed with wound clips.

Chemical insults The reagents used as chemical insults were cortisone acetate (cortisone: sterile aqueous suspension, 25 mg/ml) and cyclophosphamide (Cytoxan: 100 mg per vial) purchased from the Upjohn Company, Kalamazoo, Michigan and Mead Johnson Laboratories, Evansville, Indiana, respectively. The dose of drugs used

233 was the maximum nonlethal dose administered on each of five consecutive days to 2-month-old BC3F1 mice. Expressed in mg per day per g of body weight, the dose for cortisone was 0.124 and for Cytoxan 0.021. These drugs were prepared at the desired concentration in 3 × distilled H20, and 0.5 ml doses were injected subcutaneously. Control mice were injected with 0.5 ml of distilled water only.

Radiation insult BC3F1 mice received 400R of whole-body irradiation from a G. E. Maxitron X-ray machine under the following irradiation conditions: 300 kVp at 30 mA; 168 R/min at a target-object distance of 70 cm; inherent filtration, 4.75 mm Be, added filtration, 3 mm A1; and a half-value layer of 0.470 mm Cu. After the administration of the various insults, the mice were caged in groups of 10, permitted free access to food and water, and set aside to age for 18-22 months. Cell-transfer studies At the time of experimentation donor mice from each insult group were weighed and killed by decapitation; then their spleens and femurs were removed. The spleens were weighed and placed in petri plates containing ice-cold MEMSpinner medium (Gibco, Grand Island, New York) fortified with penicillin (50 units/ml) and streptomycin (50 /~g/ml). Only spleens free of overt pathology were used, and a small portion of each spleen was fixed in Bouin's solution for further histological analysis. A pooled dispersed cell suspension was prepared from the remaining portions by freeing the cells into a volume of cold medium equivalent to 1 ml per spleen with a 5-ml syringe (22-gauge needle) in one hand and a pair of forceps in the other hand. Connective tissue debris was filtered out by passing the cell suspension through a piece of 200-mesh stainless steel screen previously sterilized in an autoclave. Routinely, 1.0 ml aliquots containing 25 x 106 spleen cells from immunosuppressed or age-control groups and 2.5 x l0 s sheep red blood cells (RBC) were injected intravenously into irradiated syngeneic recipients, and the hemolysin titers of the serum and the number of antibody-forming cells in the spleen were determined by the method of Jerne 7 seven days later. Statistical analysis was performed on logtransformed data, which provides the approximation closest to normality s. Hemolysin titration Hemolysin titration was carried out in microtiter plates. Hemolysin titer was defined as the highest dilution of a two-fold serially diluted serum that caused almost complete hemolysis (a 32- reading on a 42- scale) in the presence of excess guinea pig complement. Theta (6))-bearing cells The relative number of 6)-bearing cells was determined with a rabbit antibrain-associated 6) (BA6)) antiserum. Rabbit anti-mouse brain serum absorbed with mouse bone marrow and RBC in the presence of complement is cytotoxic for T but not B cellsg, 10. Routinely, 10 x 106 lymphoid cells in 0.1 ml Hanks' medium

234 containing 1 0 ~ decomplemented fetal calf serum was added to 0.1 ml of a 1:10 dilution of absorbed antiserum. After incubation for 30 min at 37°C in a 5 7O COg atmosphere, the cells were washed 3 times and 0.1 ml of 1:10 guinea pig complement (C) that had been absorbed 3 times with 0.1 g of Bacto agar/ml was added. After a 30-min incubation, the percentage of dead cells (O-bearing) was determined by eosin dye uptake. The percent cytotoxicity was determined by the following formula: ( ~ killed with anti-O ÷ C) - - ( 7o killed with anti-O only) ~,~cytotoxicity =

× 100. 1 0 0 - (7o killed with anti-O only)

Immunoglobulin receptor-bearing cells The relative number of immunoglobulin receptor-bearing cells was determined with goat anti-mouse IgG reagent (Cappel Labs, Downington, Pennsylvania). The serum was absorbed 3 times with equal volumes of BC3F1 thymus and RBC to remove nonspecific cytotoxicity. One million (106) of the cells being tested in 0.1 ml Hanks' medium containing 1 0 ~ decomplemented fetal calf serum were mixed with 0.1 ml of a 1:20 dilution of antiserum and 0.1 ml of 1:3 guinea pig complement (which had been absorbed 3 times with 0.1 g of Bacto agar/ml). After a 30-min incubation, the percentage of dead cells was determined from eosin dye uptake. Percent cytotoxicity was determined with the same formula used to determine it for O-bearing cells. RESULTS To test the humoral immune activity of 30-month-old mice that had received immunosuppressive treatments at between 12 and 15 months of age, the cell-transfer method was utilized. This method, which allows efficient utilization of immunocompetent tissues, was selected because about 50~o of the animals in both the control and the various immunosuppressed groups had died before reaching this age, thereby limiting the number of animals available for immunological assessment. With this method a small aliquot of a given spleen cell-suspension can be tested for humoral immunity, and the remaining suspension can be tested for cell-mediated immunity by similar cell-transfer methods and selected in vitro assay systems. This is not possible in in situ studies where animals are injected with antigen and sacrificed at the peak time of response, precluding the use of their immunocompetent tissues for further investigational procedures to assess primary immune responses. The results of this study are presented in Fig. 1. When the mice were 30 months of age, the activity of 25 × 106 spleen ceils was only 20 (hemolysin) or 2 1 ~ (number of 19S direct plaque-forming cells, or DPFC) of that of young adult spleen cells. The log2 hemolysin titers produced by spleen cells from mice that had received cortisone (4.1) or Cytoxan (3.8) did not differ significantly from those of age-control animals (4.0). However, a significant reduction occurred with spleen cells from thymectomized or irradiated donors (log2 titer of 1.7 and 1.1, respectively). Similarly, the mean number of DPFC per recipient spleen did not differ significantly between age-control

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(1803) and cortisone-treated (1324) or Cytoxan-treated (1766) animals, whereas a sharp reduction was seen with ceils from thymectomized (309) and irradiated mice (333). Comparable results were obtained with 24-month-old donor mice that had received the same insults at a much younger age (2-3 months). In these animals, as with the previously tested 30-month-old animals, immune assessment was made 18-22 months after immunosuppressive treatment. Thus, the magnitude of the response of spleen cells from 24-month-old mice was also reduced substantially (i.e., hemolysin titers and DPFC counts gave 27 and 32 ~ of the young adult response, respectively) and only spleen cells from animals that had been thymectomized or irradiated showed a significantly reduced humoral immune response when compared with age-control animals (Fig. 2). Except in the case of irradiated mice, mortality reduced normal or immunosuppressed animal populations by about only 10 ~ at 24 months of age. Therefore, sufficient numbers of both age-control and immunosuppressed animals were available so that in situ studies to determine total immune capacity could be made. Accordingly, mice were injected intraperitoneally with 2.5 × 108 sheep RBC and killed on days 4, 5, 6, 7, and 10 when serum hemolysin titers and the number of DPFC in the spleen were determined. The numbers of indirect (7S) plaque-forming cells (IPFC) were also determined on day 10. Figure 3 presents the peak hemolysin and DPFC responses and the 10-day IPFC response of mice free of macroscopically visible lymphoid tissue diseases. When the mean maximum response did not differ significantly (5 level) on 2 or more consecutive days, the individual values were combined in calculat-

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Fig. 3. In situ anti-sheep-RBC response by 24-month-old normal or 24-month-old old mice that were exposed to immunosuppressive insults at 2-3 months of age. Mean ± 95 ~ confidence limits. (E~) Peak hemolysin. ([]) DPFC response, 9-23 mice per group. ([~) Ten-day 7S indirect plaqueforming cells or IPFC response, 5 mice per group. Young adult hemolysin DPFC and IPFC/spleen responses were 11.1, 315,780 and 76,796, respectively.

237 ing the mean peak response. In these in situ studies the mean peak log2 hemolysin titer was 7.7 for age-control animals, 7.25 for cortisone- and 7.1 for Cytoxan treated animals. Thymectomy or splenectomy at 2-3 months of age resulted in markedly reduced titers (4.5 and 3.7, respectively). It should be noted that while most groups had detectable hemolysin titers by day 4, which was the earliest day tested, splenectomized animals first demonstrated hemolysin titers on day 7. These titers were only slightly lower by day 10. The response of both IPFC and DPFC closely paralleled the response measured by hemolysin titers. The in situ DPFC and IPFC responses by 24-month-old age-control animals free of overt disease were 9 and 36~ of the response by young adult mice, respectively. As stated previously, the X-ray-insulted mice were not available for this experiment. However, since the results of all other groups in the in situ study agree remarkably well with those of the cell-transfer study, it is expected that, had X-irradiated old mice been available, a marked reduction in in situ humoral immune response would have been noted. To determine if the decrease in immune competence seen in aged animals from which the thymuses had been removed at 2-3 months of age could be related to a decrease in the number of T and/or immunoglobulin receptor-bearing B lymphocytes, the relative numbers of these cells in the spleen, bone marrow, lymph nodes, thymus, and peritoneal cavity were determined. It can be seen from Table I that no significant difference in the percentage of T and B lymphocytes between age-control animals and aged thymectomized animals was detected. Furthermore, the mean numbers of cells recovered from the respective organs or tissues of age-control and

TABLE I EFFECT OF THYMECTOMY AT 2-3 MONTHS OF AGE ON THE RELATIVE N U M B E R OF T A N D B CELLS IN THE SPLEEN, BONE MARROW, PERITONEAL FLUID, L Y M P H NODE, A N D THYMUS OF 30-MONTH-OLD BC3F1 MICE*

Organ or % O-positive lymphocytes % Immunoglobulin receptortissue bearing lymphocytes Age Thymectomized control Age Thymectomized control Spleent 40.0 Femoral bone marrow 8.5 Peritoneal fluid 5.6 Lymph node 56.3 Thymus 89.4

4- 2.2

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* Results of 3 separate experiments, each using pooled cell-suspensions of 2 or 3 organs or tissues from pathology-free animals. t Spleen weights among age-control and thymectomized animals were 122 dz 8 and 136 4- 22 mg, respectively.

238 thymectomized animals were comparable. Thus, it would appear that the reduced immune activity of aged thymectomized mice is not due to loss of T or B lymphocytes. Finally, it should be noted that the relative numbers of colony-forming stem cells in the bone marrow of the age-control and the four treatment groups did not differ significantly (data not presented). This observation suggests that these insults, administered early in life, had no appreciable detrimental effect on the bone marrow progenitor cell compartment late in life. Additional support for this conclusion may be found in the second part of this report 11 where it is shown that the graft-versushost activities of bone marrow cells among young, old, and immunosuppressed old animals do not vary significantly either. DISCUSSION These findings demonstrate that the marked reduction in humoral immune competence in aged mice is further compromised when permanent insults (e.g., surgical) are administered early in life. The late effects of thymectomy and splenectomy were not unexpected, for sheep RBC are a thymus-dependent antigen requiring T cells for the production of antibody by B cells, and the spleen is the major producer of hemolysin in micelL It has also been found 1~ that thymectomy is the one immunosuppressive insult that consistently results in impaired cell-mediated immune activity in old mice. However, it should be noted that even with this severe insult, humoral and cell-mediated immune activities were still present. The effects of sublethal (400R) irradiation on humoral immune competence were clearly evident and, although early recovery occurs 13-15, these effects are accentuated late in life. Observations on cell-mediated immune competence were in striking concordance ix. Since X-irradiation is not selectively immunosuppressive, it would appear that the long-lasting immunosuppressive effect is due, in part, to damage incurred in other organ systems as will be discussed below. In contrast to the effects of X-irradiation, the level of humoral immunity 18-22 months after Cytoxan or cortisone treatments was always comparable to that of age-control animals. The lack of long-lasting immunosuppressive effects with Cytoxan and cortisone indicates that drug-damaged cells of the immune system were readily replaced by new cells during the course of time. Our observation that there is no appreciable difference late in life between the bone marrow (B cell) progenitor cell compartment of the age-control animals and that of animals receiving the insults leads one to conclude that the recovery of the thymus-dependent, T cell compartment after treatment with Cytoxan and cortisone is also complete, in contrast to the incomplete recovery after radiation insult and surgical ablation. One apparent explanation for the difference observed between the effects of chemical immunosuppressants (cortisone and Cytoxan) and those of X-irradiation on immune competence, apart from factors such as dose and length of treatment, is that drug treatment results primarily in cell death followed by recovery whereas Xirradiation results not only in cell death but also in somatic mutations that can give rise to immunologically incompetent cells. Furthermore, such detrimental effects

239 would n o t be limited to the i m m u n e system. X - i r r a d i a t i o n can be expected to affect other tissues a n d organ systems adversely also, possibly altering physiological processes that m a y be essential for a n optimally f u n c t i o n i n g i m m u n e system. The significance of the observed u n e q u a l effects of these i m m u n o s u p p r e s s a n t s o n i m m u n e competence, as they relate to the i m p o r t a n t parameters of disease incidence a n d life expectancy, are dealt with in the third paper 16 of this series. ACKNOWLEDGEMENT This research was sponsored by the Energy Research a n d D e v e l o p m e n t Adm i n i s t r a t i o n u n d e r contract with U n i o n Carbide C o r p o r a t i o n .

REFERENCES 1 R. L. Walford, The Immunologic Theory of Aging, Munksgaard, Copenhagen, 1969. 2 F. M. Burnet, The concept of immunological surveillance, Prog. Exp. Tumor Res., 13 (1970) 1. 3 M. M. Sigel and R. A. Good, Tolerance, Autoimmunity and Aging, C. C. Thomas, Springfield, lll., 1972, p. 181. 4 T. Makinodan, E. H. Perkins and M. G. Chen, Immunologic activity of the aged, Adv. Gerontol. Res., 3 (1971) 171. 5 F. M. Burnet, Immunological Surveillance, Pergamon Press, Sydney, 1970. 6 L. Thomas, in H. S. Lawrence (ed.), Cellular and Humoral Aspects of the Hypersensitive State, P. B. Hoeber, New York, 1959, p. 529. 7 N. K. Jerne, A. A. Nordin and C. Henry, in B. Amos and H. Koprowski (eds.), Cell-Bound Antibodies, Wistar Institute Press, Philadelphia, 1963, p. 109. 8 C. F. Gottlieb, Application of transformation to normalize the distribution of plaque-forming cells, J. Immunol., 113 (1974) 51. 9 A. E. Rief and J. M. V. Allen, The AKR thymic antigen and its distribution in leukemia and nervous tissue, J. Exp. Med., 120 (1964) 413. 10 E. S. Golub, Brain-associated 0 antigen: reactivity of rabbit anti-mouse brain with mouse lymphoid cells, Cell. Immunol., 2 (1971) 353. 11 W. J. Peterson, E. H. Perkins, S. A. Goodman, Y. Hori, M. K. Halsall, and T. Makinodan, The late effects of selected immunosuppressants on immunocompetence, disease incidences and mean life-span. II. Cell-mediated immune activity. Mech. Ageing Dev., 4 (1975) 241. 12 M. J. Bosma, E. H. Perkins and T. Makinodan, Further characterization of the lymphoid cell transfer system for the study of antigen-sensitive progenitor cells, J. ImmunoL, 101 (1968) 963. 13 T. Makinodan, Changes in immunobiological processes caused by radiation. In A. Zuppinger (ed.), Handbuch der Medizinischen Radiologie, vol. 11, part 2, Springer-Verlag, Berlin, 1966, p. 303. 14 P. Nettesheim, M. L. Williams and A. S. Hammons, Regenerative potential of immunocompetent cells. IlI. Recovery of primary antibody-forming potential from X-irradiation. The role of the thymus, J. Immunol., 103 (1969) 505. 15 G. B. Price and T. Makinodan, Radiosensitivity of immunologically competent cells, Trans. N.Y. Acad. Sci., Series II, 32 (1970) 453. 16 C. P. Peter, E. H. Perkins, W. J. Peterson, H. E. Walburg and T. Makinodan, The late effects of selected immunosuppressants on immunocompetence, disease incidence and mean life-span. IlI. Disease incidence and life expectancy, Mech. Ageing Dev., 4 (1975) 251.