Proc. Nati. Acad. Sci. USA Vol. 89, pp. 7752-7756, August 1992 Immunology Interleukin 1 regulates secretion of zinc- by human thymic epithelial cells and its action on T-lymphocyte proliferation and nuclear kinase C (interleukin 2/interleukln 2 receptor/metaflotbionen mRNA) J. A. COTO*t, E. M. HADDENt, M. SAUROt, N. ZORNt, AND J. W. HADDEN*t University of South Florida Medical College, Departments of *Internal Medicine and tPharmacology, Program of Immunopharmacology, Tampa, FL 33612 Communicated by Robert A. Good, May 8, 1992

ABSTRACT Thymic epithelial cells (TEC) are known to 2Ra) forms a multiunit high-affinity receptor required for secrete thymic that influence maturation of T lym- signal transduction. Recently, it has been shown that IL-1 phocytes. One of these , thymulin, requires zinc in an injection in rats enhances zinc uptake and metallothionein equlmolar ratio for biological activity. A previous study [Cous- mRNA levels in the , bone marrow, and (6). The ins, R. J. & Leinart, A. S. (1988) FASEB J. 2, 2884-2890] importance of zinc in immune function is well documented showed that interleukin 1 (IL-i) in vivo stimulates zinc uptake (7). Zinc deficiencies have been shown to be related to severe by the thymus. Both the a and J forms ofIL-1, which stimulate depression in cell-mediated immunity and serum thymulin proliferation ofhuman TEC, also stimulate their uptake ofzinc bioactivity (8). An important characteristic ofthymulin is that in vitro, and this latter stimulation is both dependent and it is biologically active only when coupled in an equimolar independent of proliferation. Zinc induces zinc accumulation ratio with zinc (9). without proliferation. Two other stimulants of proliferation, This study examines the effects in vitro of IL-1 on human bovine pituitary extract and , stimu- TEC to stimulate zinc uptake, metallothionein mRNA ex- late zinc uptake by TEC, but only in a manner dependent on pression, and secretion of thymulin, and it examines the proliferation. Utilizing in situ hybridization, we show that the action of thymulin with IL-1 on T lymphocytes to expand IL-1 a and P forms and zinc induce metallothionein mRNA IL-2R+ cells and to stimulate nuclear protein kinase C (PKC) expression in TEC. Metallothionein is thought to be involved in in isolated nuclei. the transfer of zinc to thymulin. IL-1 was shown to stimulate the secretion of thymulin as measured both by its ability to smulate induction ofIL-2 receptor-positive lymphocytes from MATERIALS AND METHODS human peripheral blood lymphocytes and by the azathioprine- Culture ofhuman TEC was performed, and proliferation was sensitive rosette assay. In addition, the zinc-thymul complex assessed as described (3). Recombinant IL-la and IL-2p, in the presence, but not absence, of IL-1 stimulates nuclear factor 12.5 and bovine protein kinase C in isolated lymphocyte nuclei. IL-1 apparently epidermal growth (EGF; ng/ml), zinc- pituitary extract (BPE; 50 ,ug/ml) were used as growth regulates the synthesis or secretion and delivery of stimulants; mitomycin C was used at 25 pg/ml for 2.5 hr as thymulin complex to the T-lymphocyte system. an inhibitor of proliferation (all from Sigma). Purity of the epithelial cell cultures (100%6) was monitored by immuno- Intense investigation into the function of the thymus has staining of TEC with mouse monoclonal antibody to human demonstrated its importance in cellular immunity (1). Thymic cytokeratin (AMAC, Westbrook, ME) detected by a second epithelial cells (TEC) are thought to play an important role in fluoresceinated goat polyclonal anti-mouse antibody (Coul- thymocyte differentiation through direct cell contact and ter). Thymulin presence was confirmed by a similar sandwich secretion of thymic hormones. Precursor cells in bone mar- technique with mouse monoclonal antibody to thymulin row and spleen, termed prothymocytes, migrate to the thy- New mus gland where they are processed to maturity in a stepwise provided by G. Incefy (Rockefeller University, York). manner involving complicated changes in surface pheno- Cell-free supernatants (TES) were prepared from TEC types. A number of putative thymic -like peptides grown in Lab-Tek chamber slides (Nunc) at a cell concen- that influence this maturity have been isolated from thymus, tration of 1.8 x 104 cells per chamber in serum-free Dulbec- including thymulin (2). co's modified Eagle's medium (DMEM) (GIBCO) supple- Interleukin 1 (IL-1) is a multitissue mediator central to the mented with 10 mM Hepes (GIBCO), 1 mM sodium pyruvate, body's response to microbial invasion, inflammation, immu- 4 mM glutamine, penicillin at 40 units/ml, and streptomycin nological reactions, and tissue injury. IL-1 is produced by at 40 ,ug/ml (last four from Sigma). Zinc uptake was assessed thymic epithelial and stromal cells and can stimulate prolif- in culture plates (Becton Dickinson) by using 65Zn [100 nCi eration of TEC (3) and their secretion of interleukin 6 (IL-6) (3.7 kBq)/ml; (NEN)]. After incubation the cells were and granulocyte/macrophage colony-stimulatingfactor (GM- washed twice with 10 mM EDTA to remove extracellular CSF) (4). Two biochemically distinct but structurally and 65Zn as described and validated (10), and the cells were functionally related IL-1 molecules, IL-la and IL-1,B, have transferred by cotton swabs to a Beckman Gamma 5500 been cloned (5) and found to have 26% amino acid homology. scintillation counter for measurement of radioactivity. Immunologically, IL-1 promotes interleukin 2 (IL-2) produc- tion from T lymphocytes and increases IL-2 receptor (IL-2R) Abbreviations: BPE, bovine pituitary extract; EGF, epidermal number and binding affinity by inducing the 75-kDa receptor growth factor; GM-CSF, granulocyte/macrophage colony- which, with the low-affinity 55-kDa receptor (IL- stimulating factor; IL-1, interleukin 1; IL-2, interleukin 2; IL-2R, (IL-2R,(), IL-2 receptor; IL-3, interleukin 3; IL-6, interleukin-6; IL-la, IL-1, IL-2a, and IL-2p, a and S forms of IL-1 and IL-2; MC, mitomycin The publication costs of this article were defrayed in part by page charge C; PKC, protein kinase C; TEC, thymic epithelial cells; TES, thymic payment. This article must therefore be hereby marked "advertisement" epithelial cell supernatants. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 7752 Downloaded by guest on September 26, 2021 Immunology: Coto et al. Proc. Natl. Acad. Sci. USA 89 (1992) 7753

In situ hybridization for metallothionein IIA mRNA was 20r- *-i performed in Lab-Tek chambers by using a 22-base DNA probe provided by 0. Serlupi (Ares Serono, Rome) and 161- labeled with radioactive adenosine triphosphate ([32P]ATP; N NEN) in a 90-min reaction with T4 polynucleotide kinase -7- (Boehringer Mannheim) and 10x T4 kinase buffer [0.5 M Tris x 12 -E. chloride, pH 9.5/0.1 M MgCl2/50 mM dithiothreitol/50%o (vol/vol) glycerol] at 370C. Radioactive DNA probe was then isolated on a G-25M Sephadex column (Pharmacia). The hybridization reaction with metallothionein mRNA included 10 the DNA probe at 0.5 ng/,ul; 1Ox yeast tRNA (Bethesda 4 Research Laboratories) for nonspecific site labeling; 5X solution of 0.5% polyvinyl pyrrolidone, 0.5% Ficoll, and 1.0% bovine serum albumin; and 20x standard sodium citrate C IL-1,8 EGF BPE MC IL-1,8 EGF BPE (SSC; lx SSC = 0.15 M NaCl/0.015 M sodium citrate, pH + + + 7). The hybridization reaction was terminated after 24 hr. The MC MC MC culture slides were washed three times with 2 x SSC at 430C FIG. 2. Stimulation of zinc uptake by human TEC treated with and were allowed to dry for 2 hr after the final wash. Culture IL-13, EGF, and BPE. TEC were stimulated with IL-18 (20 ng/ml), slides were subjected to autoradiography in a Kodak X-Omat EGF (12.5 ng/ml), or BPE (50 pg/ml) for 48 hr with or without cassette (Kodak Health Sciences, Rochester, NY), and the pretreatment with mitomycin C (MC) (25 pg/ml). Data are presented resulting intensity of labeling was measured with a Beckman as the mean ± SEM and represent one ofthree separate experiments. densitometer. *, P < 0.01 vs. control (C). **, P < 0.05 vs. MC. Thymulin biological activity was measured in TES as described by Bach and Dardenne (11) for the azathioprine- filtration. Nuclear PKC was assayed in murine splenocyte nuclei as described (13). sensitive sheep cell rosette formation assay. Thymulin ac- tivity in TES was also assayed by a IL-2R induction assay, a modification of the method of Leichtling et al. (12). In this RESULTS assay, human peripheral blood lymphocytes were cultured in RPMI 1640 medium in the absence of serum and in the IL-1 Augments Zinc Uptake in Human TEC. Initial studies presence of a suboptimal dose of phytohemagglutinin (0.1 showed that IL-la and IL-1,8 augment zinc uptake in TEC ,ug/ml; Burroughs Wellcome). After 48 hr the cells were progressively over time. Optimal effects of zinc uptake by removed and stained with fluoresceinated anti-CD25 (IL-2R) TEC stimulated with IL-la and IL-1P may be achieved with antibody (Becton Dickinson) and analyzed for the percentage a dose of 5 and 10 ng/ml measured at 48 hr. Fig. 1 shows the of positive cells in a fluorescence-activated cell sorter dose-response effect of IL-la and IL-lP on zinc uptake (FACS). The same sample was counted for a total number of compared with the proliferation of the cells. cells using a Coulter Counter. The total number of IL-2R+ Fig. 2 summarizes the zinc uptake studies with IL-1lp, cells in each sample was estimated assuming that all cells that EGF, and BPE. Cells were cultured for 48 hr with and proliferate bear the IL-2R. The data are expressed as total without pretreatment with mitomycin C. In the absence of number of IL-2R+ cells produced by IL-i-induced TES over mitomycin C, all three growth promoters stimulated zinc control TES or medium. Zinc-thymulin complex (Sigma, and uptake by TEC; however, in the presence of mitomycin C to kindly provided by J.-F. Bach, H6pital Necker, Paris) was inhibit DNA synthesis, only IL-113 was able to stimulate zinc used as a positive control in these assays. To confirm the uptake in vitro, independent of the proliferative response. specificity of thymulin as the inducer of IL-2R+ cells, the IL-la behaved similarly in three experiments. TES were filtered by using a Mr 10,000 cut-off filter (Cen- Fig. 3 shows the effect ofzinc on zinc accumulation in TEC triprep concentrator 10, Amicon) that retained IL-1, IL-6, at 48 hr in a dose-response manner. Fig. 3 illustrates a linear and GM-CSF (interleukin products of TEC that might be relationship between zinc concentration and zinc accumula- active in this assay), and to confirm the identity of thymulin tion over a physiologic concentration range. action in this assay, the TES were preincubated with poly- IL-1 and Zinc Induce Metaflothionein IIA mRNA in TEC. clonal anti-thymulin antibody (from J.-F. Bach) prior to Table 1 shows that IL-la and IL-1,8 stimulate the expression of metallothionein mRNA progressively at 6 and 48 hr in the ._ presence and absence of mitomycin C. Zinc (10 ,uM) in the I 3000 absence of IL-la and IL-13 (20 mg/ml) increased metal- oZ 9 2500 ', - lothionein mRNA expression at 24 hr (Table 2) and aug-

2000 - r. ._ " 0 -- 1500 C 4) c 30 ;ax NO 0 1000 m- .0 4- e 25 *gE3 cm coI 0 ;: c soo 5 *e125-4 20 .1 x u u 8 C 15 5 10 15 20 1 IL, 4 ng/ml S2 FIG. 1. Dose-response effect of IL-la (e) and IL-1(3 (o) on zinc (oE I I I I uptake compared with proliferation by human TEC at 48 hr of 0 5 10 15 20 25 culture. For 65Zn uptake, cells treated with mitomycin C (25 pg/ml) Zn, M x 10-7 were pulsed for 2.5 hr before treatment. Data are presented as the mean ± SEM and represent one of three separate experiments in FIG. 3. Dose-response effect of zinc on zinc accumulation by duplicate. [3H]Thymidine incorporation was adapted from Galy et al. human TEC. Samples were stimulated with zinc for 48 hr. Data are (3). presented as the mean of three separate experiments in duplicate. Downloaded by guest on September 26, 2021 7754 Immunology: Coto et A Proc. Natl. Acad. Sci. USA 89 (1992) Table 1. Effect of IL-1 on metallothionein IIA mRNA expression Mean absorbance Ratio to Condition units control 6-hr incubation Control 2.42 ± 0.23 IL-la 3.09 ± 0.40 1.22 2 IL-13 2.96 ± 0.69 1.28 X1C I 48-hr incubation x r- Control 4.58 + 0.50 0 Control Thymullin Thymulin + anti-thymulin IL-la 8.82 ± 0.63 1.74* C, medium mediur m medium IL-1,8 7.37 ± 0.94 1.45* . S Mitomycin C 3.10 ± 0.33 -B + IL-la 5.52 ± 0.53 1.78* + IL-1, 5.78 ± 0.17 1.86* Human TEC were incubated for 48 hr with IL-la or IL-1,8 (20 4 ng/ml). Data presented are the means ± SEM of 10 readings from 3 each treatment. *P < 0.01 vs. control. 2 mented the effect of IL-la and IL-1,8. Zinc did not increase 1 metallothionein mRNA expression at 48 hr (not shown). IL-I Induces Thymulin Secretion from TEC. TES were Control IL-la IL-la TES + anti-thymulin collected in serum-free media after 48 hr ofculture and tested TES TES for thymulin biological activity in the Bach assay (11), in FIG. 4. (A) Effect of thymulin on the expansion of IL-2R+ cells which results are expressed as the highest dilution ofthe TES and retention of thymulin activity by using anti-thymulin antibody. showing sensitivity to azothioprine inhibition. We confirmed Synthetic thymulin was incubated with anti-thymulin antibody for 2 the requirement for zinc in the assay of thymulin. IL-1,3 hr prior to filtration. Activity was determined by measuring induction induced a 4-fold increase in TES thymulin activity in this of IL-2R+ cells. *, P < 0.01 vs. control. (B) Stimulation of thymulin assay [control = 1:4 dilution (five experiments); IL-1X3 = 1:32 secretion by human TEC treated with IL-la (20 ng/ml) for48 hr. TES dilution (three experiments) P < 0.01]. Normal serum thy- were incubated with and without anti-thymulin antibodies prior to mulin levels have been reported to be 1-4 pg/ml (14), and filtration as above. Thymulin activity was measured by induction of IL-2R+ cells. Data are presented as the mean ± SEM and represent normal sera yield values of 1:16 dilution; thus, thymulin one of three separate experiments. *, P < 0.05 vs. control. levels induced by IL-1 are estimated to be in the low picogram range. measured in this assay. Pretreatment of the IL-i-induced TES thymulin activity was also assayed by the expansion TES with anti-thymulin antibody prior to filtration removed of IL-2R+ lymphocytes under serum-free conditions. It is the thymulin activity. IL-1,B had similar effects to induce important to note that this assay is dependent upon IL-1 thymulin assessed in this manner in three experiments. produced by adherent accessory cells. If accessory cells are Zinc-Thymulin Potentiates IL-1 Activation of PKC in Iso- removed, reconstitution with recombinant IL-1 in small lated Splenocyte Nudei. A recent study (15) suggested that amounts is necessary (up to 1 ng/ml) thus, zinc-thymulin IL-1 has a nuclear site of action through its endocytosis and effects on this response ofmature T lymphocytes requires the transport to the nucleus. Our preliminary study (16) showed presence of IL-1. that IL-1 induces a 4-fold increase in nuclear PKC in isolated IL-i-induced TES were prepared and compared to control splenocyte nuclei, and the effect reaches a plateau at 1 ng/ml TES for the presence of thymulin. Synthetic thymulin is as does the biological response in the IL-2R+ cell assay. Fig. active in the IL-2R+ lymphocyte assay (Fig. 4) only in the 5 shows the ability of zinc-thymulin complex to stimulate presence of equimolar zinc; zinc alone, up to 10 ,uM, is nuclear PKC in the presence of 1 ng of IL-1p. The effect of inactive. The effects of thymulin saturate above 1 pg/ml, IL-1 (1 ng/ml) alone is depicted on the ordinate (16). Zinc- making quantitation difficult. The effect of thymulin is pre- thymulin had no effect in the absence of IL-1; zinc-thymulin vented by preincubation of the TES with anti-thymulin A Thymulin-Zn2+ antibody prior to filtration. Fig. 4B shows the effect of IL-la 1.000 . * * A '4-4 Thymulin-Zn2+/IL-l to induce thymulin-like activity in the Mr < 10,000 fraction O n

0 o Table 2. Effect of IL-la and IL-1P in combination with zinc on Ei o 0.750- metallothionein IIA mRNA expression 04~ 0500 Mean absorbance Ratio to B s Condition units control '- (.u c 0.250 Control 9.68 ± 0.29 I

- - A------Zinc 13.56 ± 0.38 1.40* AT- - A IL-la 13.48 ± 0.16 1.39* + zinc 15.32 ± 0.37 1.58* -12 -11 -10 -9 IL-lP 15.60 ± 0.29 1.61* Thymulin, logM + zinc 17.50 ± 0.39 1.81* FIG. 5. Concentration response for thymulin activation of nu- Human TEC were incubated for 24 hr, a time in which significant clear PKC (nPKC) in rat splenic nuclei. Nuclei were incubated in the proliferation does not occur, with IL-la (20 ng/ml), IL-1,B (20 presence of thymulin alone (10-9-10-12 M) with and without IL-1 (1 ng/ml), or zinc (10 ,uM). Data presented are the means ± SEM of 10 ng/ml) added simultaneously to each tube. The data are expressed as readings from each treatment. the mean + SEM offour separate experiments. **, PKC activity that *P < 0.01 vs. control. is significantly different from basal levels (P < 0.01). Downloaded by guest on September 26, 2021 Immunology: Coto et al. Proc. Natl. Acad. Sci. USA 89 (1992) 7755 is therefore synergistic with IL-1. It is notable that the effects pg/ml range and saturates at this level in both assays used in of zinc-thymulin are observed at 1:10,000-1:100,000 serum this study. It would appear that like calcium, small amounts levels of zinc (1 pg/ml), pointing to an exquisitely sensitive ofzinc are active through what must be a delicately regulated delivery mechanism to nuclear PKC. uptake process whose mechanism is unknown. The action of zinc-thymulin on nuclear PKC was suggested by recent DISCUSSION reports showing that both and IL-1 stimulate nu- clear PKC in splenocytes (13, 16) and by our demonstration These results extend a prior report (6) showing that IL-1 of the IL-l-zinc-thymulin synergy in expanding IL-2R+ induces thymic uptake of zinc and metallothionein mRNA lymphocytes. The demonstration that zinc-thymulln shows expression in vivo by showing that IL-la and IL-1,8 induce synergistic action with IL-1 on nuclear PKC strongly sug- TEC to take up zinc and induce metallothionein mRNA in gests that nuclear PKC may mediate the response. As targets vitro. Since we have been unable to show that IL-1 alone ofPKC-related events, it seems likely that the IL-2 and IL-2R induces zinc uptake in lymphocytes, our results suggest that may be activated under these circumstances. TEC, and not thymocytes, are the target ofIL-i-induced zinc It is notable that zinc enhances PKC translocation in uptake and metallothionein expression in vivo. TEC lymphocytes (28) and is required for PKC activation (29) and have been implicated previously as the main endocrine cells that PKC contains two zinc fingers, possibly necessary for its of the thymus and have been involved in secretion, intranuclear action; thus, the associations of zinc, and including interleukins (IL-1, IL-3, IL-6, and GM-CSF; refs. IL-1, 4, 14, 17, 18) and thymic hormones (thymopoietin, PKC may be of great importance. al, and thymulin; refs. 19 and 20). Of the thymic hormones, It is also notable that thymosin a, has also been reported although thymopoietin and thymosin al have been detected to increase IL-2R and IL-2 in the Leichtling assay (12). in TEC by immunofluorescence (19, 20) and in our TEC by Several observations point to an intracellular, even nuclear, RIA (P. Naylor, G. Goldstein, and J.W.H., unpublished action ofthymosin al involving growth regulation (21, 30). In data), we have failed to show that regulated secretion occurs. conjunction with our results, it seems possible that thymosin Thymectomy does not remove their presence from serum (P. a, may participate in thymic hormone-related signaling pro- Naylor, personal communication), and their genes are ex- cesses in T lymphocytes. pressed in other nonthymic tissues (21, 37). Therefore, ofthe Prolactin is another substance associated with zinc- putative thymic hormones, only thymulin appears to be thymulin release from TEC in culture (31); it also promotes thymus-restricted (22) and regulated in its secretion (23-25). lymphocyte proliferation and IL-2R expression (31) and Since zinc and thymulin on an equimolar basis are essential activates nuclear PKC (13). Both IL-1 and a thymic peptide for biological activity (9) and since metallothionein is the zinc induce prolactin secretion from cells (32, donor for thymulin (19), it was logical to presume that IL-1 33), and prolactin-secreting pituitary cell implants reverse effect on zinc uptake and metallothionein mRNA expression thymic involution in aging (34). Thus, the pituitary via would translate into an effect upon zinc-thymulin secretion. prolactin appears to be another primary thymotrophic regu- It was confirmed that IL-1 induces thymulin secretion as lator linked to the IL-1-zinc-thymulin axis developed in this measured on T-cell precursors in the Bach assay and on work. mature T cells in an assay measuring IL-2R+ cell expansion. The present studies suggest that IL-1 or IL-1 plus its In both cases, the specificity of thymulin action was con- companion cytokines will be important in the development of firmed by the necessity for the copresence of zinc which was the thymus-dependent immune system and its restoration by itself inactive. In the latter assay, the specificity was following involution. Preliminary evidence support both confirmed further by the neutralization and removal of the these predictions (27, 35). Finally, zinc repletion in aged mice zinc-thymulin by specific polyclonal antisera. Similar tech- increases thymic weight, thymulin-containing cells, and se- niques previously confirm the specificity of thymulin detec- rum levels (36) and may, with IL-1 and prolactin, be key to tion in the Bach assay (11). cellular immune restoration in aging. We have shown (4) that IL-1 (a and f8) induces IL-6 and GM-CSF but not IL-1 secretion from TEC. Others have 1. Good, R. A. (1991) Immunol. Today 12, 283-286. shown that activation of the LFA-3 (leukocyte function- 2. Bach, J.-F., Dardenne, M., Pleau, J. M. & Bach, A. M. (1975) associated antigen 3) receptor on TEC leads to IL-1 secretion Ann. N.Y. Acad. Sci. 249, 186-210. (26). Also, thymic accessory cells may secrete IL-1. There- 3. Galy, A., Hadden, E. M., Touraine, J.-L. & Hadden, J. W. fore, IL-1 appears to be a major regulator in the intrathymic (1989) Cell. Immunol. 124, 13-27. environment by endocrine, autocrine, and paracrine mech- 4. Galy, A. M., Dinarello, C. A., Kupper, T. S., Kameda, A. & anisms. The induction by IL-1 Hadden, J. W. (1990) Cell. Immunol. 129, 161-175. of zinc-thymulin secretion 5. Dinarello, C. A. (1988) FASEB J. 2, 108-115. from thymus in vivo has yet to be demonstrated; however, it 6. Cousins, R. J. & Leinart, A. S. (1988) FASEB J. 2, 2884-2890. seems likely that IL-1 delivery of zinc-thymulin to T lym- 7. Good, R. A., Fernandez, G. & West, A. (1979) Clin. Bull. 9, phocytes is a central issue in the regulation ofT lymphocytes. 3-12. We have presented evidence for synergistic interactions of 8. Fraker, P. J., Gershwin, M. E., Good, R. A. & Prasad, A. IL-1 and IL-2 on the proliferation of prothymocytes, imma- (1986) Fed. Proc. Fed. Am. Soc. Exp. Biol. 45, 1474-1479. ture thymocytes as well as this well-known association on 9. Bach, J.-F. & Dardenne, M. (1989) Med. Oncol. Tumor Phar- mature T lymphocytes (27) and have postulated that acqui- macother. 6, 25-29. sition of the programmed response involving IL-1 induction 10. Failla, M. L. & Cousins, R. J. (1978) Biochem. Biophys. Acta of high-affinity IL-2Rs and enhanced response to IL-2 is 538, 435-444. central to the commitment of lymphocytes to the T-cell 11. Bach, J.-F. & Dardenne, M. (1973) Immunology 25, 353-366. lineage and, thus, a logical result of thymic influence. It is 12. Leichtling, K. D., Serrate, S. A. & Sztein, M. B. (1989) Int. 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(1991) Proceedings ofthe Fourth 25. Moccheggiani, E. & Amadio, L. (1990) Endocrinol. Invest. 13, International Conference on Biomedical Gerontology (ABG, 139-147. Ancona, Italy), abstr. 197, p. 279. 26. Le, P. T., Vollger, L. W., Haynes, B. F. & Singer, K. H. 37. Zevin-Sonkin, D., Ilan, E., Riss, J., Theodor, L. & Shoham, J. (1990) J. Immunol. 144, 4541-4547. (1991) Int. J. Immunopharmacol. 13, 798 (abstr.). Downloaded by guest on September 26, 2021