Proc. Nati. Acad. Sci. USAVol. 89, pp. 7752-7756, August 1992Immunology

Interleukin 1 regulates secretion of zinc-thymulin by human thymicepithelial cells and its action on T-lymphocyte proliferation andnuclear protein 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 tosecrete thymic hormones that influence maturation of T lym-phocytes. One of these peptides, thymulin, requires zinc in anequlmolar ratio for biological activity. A previous study [Cous-ins, R. J. & Leinart, A. S. (1988) FASEB J. 2, 2884-2890]showed that interleukin 1 (IL-i) in vivo stimulates zinc uptakeby the thymus. Both the a and J forms of IL-1, which stimulateproliferation ofhuman TEC, also stimulate their uptake of zincin vitro, and this latter stimulation is both dependent andindependent of proliferation. Zinc induces zinc accumulationwithout proliferation. Two other stimulants of proliferation,bovine pituitary extract and epidermal growth factor, stimu-late zinc uptake by TEC, but only in a manner dependent onproliferation. Utilizing in situ hybridization, we show that theIL-1 a and P forms and zinc induce metallothionein mRNAexpression in TEC. Metallothionein is thought to be involved inthe transfer of zinc to thymulin. IL-1 was shown to stimulatethe secretion of thymulin as measured both by its ability tosmulate induction of IL-2 receptor-positive lymphocytes fromhuman peripheral blood lymphocytes and by the azathioprine-sensitive rosette assay. In addition, the zinc-thymul complexin the presence, but not absence, of IL-1 stimulates nuclearprotein kinase C in isolated lymphocyte nuclei. IL-1 apparentlyregulates the synthesis or secretion and delivery of zinc-thymulin complex to the T-lymphocyte system.

Intense investigation into the function of the thymus hasdemonstrated its importance in cellular immunity (1). Thymicepithelial cells (TEC) are thought to play an important role inthymocyte differentiation through direct cell contact andsecretion of thymic hormones. Precursor cells in bone mar-row and spleen, termed prothymocytes, migrate to the thy-mus gland where they are processed to maturity in a stepwisemanner involving complicated changes in surface pheno-types. A number of putative thymic hormone-like peptidesthat influence this maturity have been isolated from thymus,including thymulin (2).

Interleukin 1 (IL-1) is a multitissue mediator central to thebody's response to microbial invasion, inflammation, immu-nological reactions, and tissue injury. IL-1 is produced bythymic epithelial and stromal cells and can stimulate prolif-eration of TEC (3) and their secretion of interleukin 6 (IL-6)and granulocyte/macrophage colony-stimulating factor (GM-CSF) (4). Two biochemically distinct but structurally andfunctionally related IL-1 molecules, IL-la and IL-1,B, havebeen cloned (5) and found to have 26% amino acid homology.Immunologically, IL-1 promotes interleukin 2 (IL-2) produc-tion from T lymphocytes and increases IL-2 receptor (IL-2R)number and binding affinity by inducing the 75-kDa receptor(IL-2R,(), which, with the low-affinity 55-kDa receptor (IL-

2Ra) forms a multiunit high-affinity receptor required forsignal transduction. Recently, it has been shown that IL-1injection in rats enhances zinc uptake and metallothioneinmRNA levels in the liver, bone marrow, and thymus (6). Theimportance of zinc in immune function is well documented(7). Zinc deficiencies have been shown to be related to severedepression in cell-mediated immunity and serum thymulinbioactivity (8). An important characteristic ofthymulin is thatit is biologically active only when coupled in an equimolarratio with zinc (9).

This study examines the effects in vitro of IL-1 on humanTEC to stimulate zinc uptake, metallothionein mRNA ex-pression, and secretion of thymulin, and it examines theaction of thymulin with IL-1 on T lymphocytes to expandIL-2R+ cells and to stimulate nuclear protein kinase C (PKC)in isolated nuclei.

MATERIALS AND METHODSCulture ofhuman TEC was performed, and proliferation wasassessed as described (3). Recombinant IL-la and IL-2p,epidermal growth factor (EGF; 12.5 ng/ml), and bovinepituitary extract (BPE; 50 ,ug/ml) were used as growthstimulants; mitomycin C was used at 25 pg/ml for 2.5 hr asan inhibitor of proliferation (all from Sigma). Purity of theepithelial cell cultures (100%6) was monitored by immuno-staining of TEC with mouse monoclonal antibody to humancytokeratin (AMAC, Westbrook, ME) detected by a secondfluoresceinated goat polyclonal anti-mouse antibody (Coul-ter). Thymulin presence was confirmed by a similar sandwichtechnique with mouse monoclonal antibody to thymulinprovided by G. Incefy (Rockefeller University, New York).

Cell-free supernatants (TES) were prepared from TECgrown in Lab-Tek chamber slides (Nunc) at a cell concen-tration of 1.8 x 104 cells per chamber in serum-free Dulbec-co's modified Eagle's medium (DMEM) (GIBCO) supple-mented with 10mM Hepes (GIBCO), 1 mM sodium pyruvate,4 mM glutamine, penicillin at 40 units/ml, and streptomycinat 40 ,ug/ml (last four from Sigma). Zinc uptake was assessedin culture plates (Becton Dickinson) by using 65Zn [100 nCi(3.7 kBq)/ml; (NEN)]. After incubation the cells werewashed twice with 10 mM EDTA to remove extracellular65Zn as described and validated (10), and the cells weretransferred by cotton swabs to a Beckman Gamma 5500scintillation counter for measurement of radioactivity.

Abbreviations: BPE, bovine pituitary extract; EGF, epidermalgrowth factor; GM-CSF, granulocyte/macrophage colony-stimulating factor; IL-1, interleukin 1; IL-2, interleukin 2; 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, mitomycinC; PKC, protein kinase C; TEC, thymic epithelial cells; TES, thymicepithelial cell supernatants.tTo whom reprint requests should be addressed.

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In situ hybridization for metallothionein IIA mRNA wasperformed in Lab-Tek chambers by using a 22-base DNAprobe provided by 0. Serlupi (Ares Serono, Rome) andlabeled with radioactive adenosine triphosphate ([32P]ATP;NEN) in a 90-min reaction with T4 polynucleotide kinase(Boehringer Mannheim) and 10x T4 kinase buffer [0.5 M Trischloride, pH 9.5/0.1 M MgCl2/50 mM dithiothreitol/50%o(vol/vol) glycerol] at 370C. Radioactive DNA probe was thenisolated on a G-25M Sephadex column (Pharmacia). Thehybridization reaction with metallothionein mRNA includedthe DNA probe at 0.5 ng/,ul; 1Ox yeast tRNA (BethesdaResearch Laboratories) for nonspecific site labeling; 5X

solution of 0.5% polyvinyl pyrrolidone, 0.5% Ficoll, and1.0% bovine serum albumin; and 20x standard sodium citrate(SSC; lx SSC = 0.15 M NaCl/0.015 M sodium citrate, pH7). The hybridization reaction was terminated after 24 hr. Theculture slides were washed three times with 2x SSC at 430Cand were allowed to dry for 2 hr after the final wash. Cultureslides were subjected to autoradiography in a Kodak X-Omatcassette (Kodak Health Sciences, Rochester, NY), and theresulting intensity of labeling was measured with a Beckmandensitometer.Thymulin biological activity was measured in TES as

described by Bach and Dardenne (11) for the azathioprine-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 thisassay, human peripheral blood lymphocytes were cultured inRPMI 1640 medium in the absence of serum and in thepresence of a suboptimal dose of phytohemagglutinin (0.1,ug/ml; Burroughs Wellcome). After 48 hr the cells wereremoved and stained with fluoresceinated anti-CD25 (IL-2R)antibody (Becton Dickinson) and analyzed for the percentageof positive cells in a fluorescence-activated cell sorter(FACS). The same sample was counted for a total number ofcells using a Coulter Counter. The total number of IL-2R+cells in each sample was estimated assuming that all cells thatproliferate bear the IL-2R. The data are expressed as totalnumber of IL-2R+ cells produced by IL-i-induced TES overcontrol TES or medium. Zinc-thymulin complex (Sigma, andkindly provided by J.-F. Bach, H6pital Necker, Paris) wasused as a positive control in these assays. To confirm thespecificity of thymulin as the inducer of IL-2R+ cells, theTES were filtered by using a Mr 10,000 cut-off filter (Cen-triprep concentrator 10, Amicon) that retained IL-1, IL-6,and GM-CSF (interleukin products of TEC that might beactive in this assay), and to confirm the identity of thymulinaction in this assay, the TES were preincubated with poly-clonal anti-thymulin antibody (from J.-F. Bach) prior to

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FIG. 2. Stimulation of zinc uptake by human TEC treated withIL-13, EGF, and BPE. TEC were stimulated with IL-18 (20 ng/ml),EGF (12.5 ng/ml), or BPE (50 pg/ml) for 48 hr with or withoutpretreatment with mitomycin C (MC) (25 pg/ml). Data are presentedas the mean ± SEM and represent one ofthree separate experiments.*, P < 0.01 vs. control (C). **, P < 0.05 vs. MC.

filtration. Nuclear PKC was assayed in murine splenocytenuclei as described (13).

RESULTS

IL-1 Augments Zinc Uptake in Human TEC. Initial studiesshowed that IL-la and IL-1,8 augment zinc uptake in TECprogressively over time. Optimal effects of zinc uptake byTEC stimulated with IL-la and IL-1P may be achieved witha dose of 5 and 10 ng/ml measured at 48 hr. Fig. 1 shows thedose-response effect of IL-la and IL-lP on zinc uptakecompared with the proliferation of the cells.

Fig. 2 summarizes the zinc uptake studies with IL-1lp,EGF, and BPE. Cells were cultured for 48 hr with andwithout pretreatment with mitomycin C. In the absence ofmitomycin C, all three growth promoters stimulated zincuptake by TEC; however, in the presence of mitomycin C toinhibit DNA synthesis, only IL-113 was able to stimulate zincuptake in vitro, independent of the proliferative response.IL-la behaved similarly in three experiments.

Fig. 3 shows the effect ofzinc on zinc accumulation in TECat 48 hr in a dose-response manner. Fig. 3 illustrates a linearrelationship between zinc concentration and zinc accumula-tion over a physiologic concentration range.

IL-1 and Zinc Induce Metaflothionein IIA mRNA in TEC.Table 1 shows that IL-la and IL-1,8 stimulate the expressionof metallothionein mRNA progressively at 6 and 48 hr in thepresence and absence of mitomycin C. Zinc (10 ,uM) in theabsence of IL-la and IL-13 (20 mg/ml) increased metal-lothionein mRNA expression at 24 hr (Table 2) and aug-

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FIG. 3. Dose-response effect of zinc on zinc accumulation byhuman TEC. Samples were stimulated with zinc for 48 hr. Data arepresented as the mean of three separate experiments in duplicate.

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Table 1. Effect of IL-1 on metallothionein IIAmRNA expression

Mean absorbance Ratio toCondition units control

6-hr incubationControl 2.42 ± 0.23IL-la 3.09 ± 0.40 1.22IL-13 2.96 ± 0.69 1.28

48-hr incubationControl 4.58 + 0.50IL-la 8.82 ± 0.63 1.74*IL-1,8 7.37 ± 0.94 1.45*Mitomycin C 3.10 ± 0.33+ 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 (20ng/ml). Data presented are the means ± SEM of 10 readings fromeach treatment.*P < 0.01 vs. control.

mented the effect of IL-la and IL-1,8. Zinc did not increasemetallothionein mRNA expression at 48 hr (not shown).

IL-I Induces Thymulin Secretion from TEC. TES werecollected in serum-free media after 48 hr of culture and testedfor thymulin biological activity in the Bach assay (11), inwhich results are expressed as the highest dilution of the TESshowing sensitivity to azothioprine inhibition. We confirmedthe requirement for zinc in the assay of thymulin. IL-1,3induced a 4-fold increase in TES thymulin activity in thisassay [control = 1:4 dilution (five experiments); IL-1X3 = 1:32dilution (three experiments) P < 0.01]. Normal serum thy-mulin levels have been reported to be 1-4 pg/ml (14), andnormal sera yield values of 1:16 dilution; thus, thymulinlevels induced by IL-1 are estimated to be in the low picogramrange.TES thymulin activity was also assayed by the expansion

of IL-2R+ lymphocytes under serum-free conditions. It isimportant to note that this assay is dependent upon IL-1produced by adherent accessory cells. If accessory cells areremoved, reconstitution with recombinant IL-1 in smallamounts is necessary (up to 1 ng/ml) thus, zinc-thymulineffects on this response ofmature T lymphocytes requires thepresence of IL-1.

IL-i-induced TES were prepared and compared to controlTES for the presence of thymulin. Synthetic thymulin isactive in the IL-2R+ lymphocyte assay (Fig. 4) only in thepresence of equimolar zinc; zinc alone, up to 10 ,uM, isinactive. The effects of thymulin saturate above 1 pg/ml,making quantitation difficult. The effect of thymulin is pre-vented by preincubation of the TES with anti-thymulinantibody prior to filtration. Fig. 4B shows the effect of IL-lato induce thymulin-like activity in the Mr < 10,000 fraction

Table 2. Effect of IL-la and IL-1P in combination with zinc onmetallothionein IIA mRNA expression

Mean absorbance Ratio toCondition units control

Control 9.68 ± 0.29Zinc 13.56 ± 0.38 1.40*IL-la 13.48 ± 0.16 1.39*+ zinc 15.32 ± 0.37 1.58*

IL-lP 15.60 ± 0.29 1.61*+ zinc 17.50 ± 0.39 1.81*

Human TEC were incubated for 24 hr, a time in which significant

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FIG. 4. (A) Effect of thymulin on the expansion of IL-2R+ cellsand retention of thymulin activity by using anti-thymulin antibody.Synthetic thymulin was incubated with anti-thymulin antibody for 2hr prior to filtration. Activity was determined by measuring inductionof IL-2R+ cells. *, P < 0.01 vs. control. (B) Stimulation of thymulinsecretion by human TEC treated with IL-la (20 ng/ml) for48 hr. TESwere incubated with and without anti-thymulin antibodies prior tofiltration as above. Thymulin activity was measured by induction ofIL-2R+ cells. Data are presented as the mean ± SEM and representone of three separate experiments. *, P < 0.05 vs. control.

measured in this assay. Pretreatment of the IL-i-inducedTES with anti-thymulin antibody prior to filtration removedthe thymulin activity. IL-1,B had similar effects to inducethymulin assessed in this manner in three experiments.Zinc-Thymulin Potentiates IL-1 Activation of PKC in Iso-

lated Splenocyte Nudei. A recent study (15) suggested thatIL-1 has a nuclear site of action through its endocytosis andtransport to the nucleus. Our preliminary study (16) showedthat IL-1 induces a 4-fold increase in nuclear PKC in isolatedsplenocyte nuclei, and the effect reaches a plateau at 1 ng/mlas does the biological response in the IL-2R+ cell assay. Fig.5 shows the ability of zinc-thymulin complex to stimulatenuclear PKC in the presence of 1 ng of IL-1p. The effect ofIL-1 (1 ng/ml) alone is depicted on the ordinate (16). Zinc-thymulin had no effect in the absence of IL-1; zinc-thymulin

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FIG. 5. Concentration response for thymulin activation of nu-clear PKC (nPKC) in rat splenic nuclei. Nuclei were incubated in thepresence of thymulin alone (10-9-10-12 M) with and without IL-1 (1ng/ml) added simultaneously to each tube. The data are expressed asthe mean + SEM offour separate experiments. **, PKC activity thatis significantly different from basal levels (P < 0.01).

proliferation does not occur, with IL-la (20 ng/ml), IL-1,B (20ng/ml), or zinc (10 ,uM). Data presented are the means ± SEM of 10readings from each treatment.*P < 0.01 vs. control.

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is therefore synergistic with IL-1. It is notable that the effectsof zinc-thymulin are observed at 1:10,000-1:100,000 serumlevels of zinc (1 pg/ml), pointing to an exquisitely sensitivedelivery mechanism to nuclear PKC.

DISCUSSIONThese results extend a prior report (6) showing that IL-1induces thymic uptake of zinc and metallothionein mRNAexpression in vivo by showing that IL-la and IL-1,8 induceTEC to take up zinc and induce metallothionein mRNA invitro. Since we have been unable to show that IL-1 aloneinduces zinc uptake in lymphocytes, our results suggest thatTEC, and not thymocytes, are the target ofIL-i-induced zincuptake and metallothionein gene expression in vivo. TEChave been implicated previously as the main endocrine cellsof the thymus and have been involved in peptide secretion,including interleukins (IL-1, IL-3, IL-6, and GM-CSF; refs.4, 14, 17, 18) and thymic hormones (thymopoietin, thymosinal, and thymulin; refs. 19 and 20). Of the thymic hormones,although thymopoietin and thymosin al have been detectedin TEC by immunofluorescence (19, 20) and in our TEC byRIA (P. Naylor, G. Goldstein, and J.W.H., unpublisheddata), we have failed to show that regulated secretion occurs.Thymectomy does not remove their presence from serum (P.Naylor, personal communication), and their genes are ex-pressed in other nonthymic tissues (21, 37). Therefore, of theputative thymic hormones, only thymulin appears to bethymus-restricted (22) and regulated in its secretion (23-25).Since zinc and thymulin on an equimolar basis are essentialfor biological activity (9) and since metallothionein is the zincdonor for thymulin (19), it was logical to presume that IL-1effect on zinc uptake and metallothionein mRNA expressionwould translate into an effect upon zinc-thymulin secretion.It was confirmed that IL-1 induces thymulin secretion asmeasured on T-cell precursors in the Bach assay and onmature T cells in an assay measuring IL-2R+ cell expansion.In both cases, the specificity of thymulin action was con-firmed by the necessity for the copresence of zinc which wasby itself inactive. In the latter assay, the specificity wasconfirmed further by the neutralization and removal of thezinc-thymulin by specific polyclonal antisera. Similar tech-niques previously confirm the specificity of thymulin detec-tion in the Bach assay (11).We have shown (4) that IL-1 (a and f8) induces IL-6 and

GM-CSF but not IL-1 secretion from TEC. Others haveshown that activation of the LFA-3 (leukocyte function-associated antigen 3) receptor on TEC leads to IL-1 secretion(26). Also, thymic accessory cells may secrete IL-1. There-fore, IL-1 appears to be a major regulator in the intrathymicenvironment by endocrine, autocrine, and paracrine mech-anisms. The induction by IL-1 of zinc-thymulin secretionfrom thymus in vivo has yet to be demonstrated; however, itseems likely that IL-1 delivery of zinc-thymulin to T lym-phocytes is a central issue in the regulation ofT lymphocytes.We have presented evidence for synergistic interactions of

IL-1 and IL-2 on the proliferation of prothymocytes, imma-ture thymocytes as well as this well-known association onmature T lymphocytes (27) and have postulated that acqui-sition of the programmed response involving IL-1 inductionof high-affinity IL-2Rs and enhanced response to IL-2 iscentral to the commitment of lymphocytes to the T-celllineage and, thus, a logical result of thymic influence. It isnotable then, that zinc-thymulin, but neither alone, inducesthe expansion of IL-2R+ lymphocytes in the presence, butnot absence, of IL-1. These findings suggest that zinc-thymulin maintains and expands the program once it isinitiated in T lymphocytes.

It is important to note that despite serum levels of zinc inthe pg/ml range, delivery to the T lymphocyte occurs in the

pg/ml range and saturates at this level in both assays used inthis study. It would appear that like calcium, small amountsof zinc are active through what must be a delicately regulateduptake process whose mechanism is unknown. The action ofzinc-thymulin on nuclear PKC was suggested by recentreports showing that both prolactin and IL-1 stimulate nu-clear PKC in splenocytes (13, 16) and by our demonstrationof the IL-l-zinc-thymulin synergy in expanding IL-2R+lymphocytes. The demonstration that zinc-thymulln showssynergistic action with IL-1 on nuclear PKC strongly sug-gests that nuclear PKC may mediate the response. As targetsofPKC-related events, it seems likely that the IL-2 and IL-2Rgenes may be activated under these circumstances.

It is notable that zinc enhances PKC translocation inlymphocytes (28) and is required for PKC activation (29) andthatPKC contains two zinc fingers, possibly necessary for itsintranuclear action; thus, the associations of zinc, IL-1, andPKC may be of great importance.

It is also notable that thymosin a, has also been reportedto increase IL-2R and IL-2 in the Leichtling assay (12).Several observations point to an intracellular, even nuclear,action ofthymosin al involving growth regulation (21, 30). Inconjunction with our results, it seems possible that thymosina, may participate in thymic hormone-related signaling pro-cesses in T lymphocytes.

Prolactin is another substance associated with zinc-thymulin release from TEC in culture (31); it also promoteslymphocyte proliferation and IL-2R expression (31) andactivates nuclear PKC (13). Both IL-1 and a thymic peptideinduce prolactin secretion from anterior pituitary cells (32,33), and prolactin-secreting pituitary cell implants reversethymic involution in aging (34). Thus, the pituitary viaprolactin appears to be another primary thymotrophic regu-lator linked to the IL-1-zinc-thymulin axis developed in thiswork.The present studies suggest that IL-1 or IL-1 plus its

companion cytokines will be important in the development ofthe thymus-dependent immune system and its restorationfollowing involution. Preliminary evidence support boththese predictions (27, 35). Finally, zinc repletion in aged miceincreases thymic weight, thymulin-containing cells, and se-rum levels (36) and may, with IL-1 and prolactin, be key tocellular immune restoration in aging.

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7756 Immunology: Coto et al.

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