Immunology 1990 69 209-214

Interleukin-2-dependent control of disease development inspontaneously diabetic BB rats

J. ZIELASEK, V. BURKART, P. NAYLOR,* A. GOLDSTEIN,* U. KIESEL & H. KOLBDiabetes Research Institute, University of Duisseldorf, Duisseldorf, FRG and *Department of Biochemistry and Molecular Biology,

George Washington University, School of Medicine and Health Sciences, Washington, District of Columbia, U.S.A.

Acceptedfor publication 10 October 1989

SUMMARY

Long-term treatment with recombinant interleukin-2 (IL-2) of diabetes-prone BB rats hadcontrasting effects in two different BB rat sublines. Diabetes development was enhanced in the sublinewith a low intrinsic diabetes risk and suppressed in the subline with a high diabetes risk. IL-2treatment started between 35 and 42 days of age and lasted for 3 months. In subline 1, the diabetesincidence increased from 23% to 53% (P<0-01), in subline 2 it decreased from 73% to 32%(P< 0-0 1). The two sublines differed in serum levels of factors controlling IL-2 synthesis and activity.Mean IL-2 inhibitory activity was higher in subline 2 (between 140% and 290% of levels in subline 1,P< 0-01). Conversely, mean concentrations of thymosin alpha 1 and beta 4 were higher in subline 1

(between 140% and 200% of levels in subline 2, P<0.01). Thus the two sublines differ in theirresponse to exogenous IL-2 and also in serum levels of mediators affecting availability of IL-2. Weconclude that an internal network of hormonal factors, including IL-2, contributes to the control ofdiabetes development in the BB rat.

INTRODUCTION

Animal models of insulin-dependent (type I) diabetes arecharacterized by several disorders of both humoral and cellularimmunological parameters (Marliss et al., 1982; Kolb, 1987;Mordes, Desemone & Rossini, 1987). The presence of auto-antibodies directed against pancreatic islet-cell antigens inpatients with type I diabetes is also reported from spontaneouslydiabetic Bio Breeding (BB) rats (Dyrberg et al., 1982; Pipeleerset al., 1987). Cellular immune abnormalities mainly comprisethe numbers and functional properties of leucocyte subtypes. Inlong-standing type I diabetic patients, a mild lymphopeniacould be observed (Pontesilli et al., 1986), whereas a pronouncedreduction of lymphocytes seems to be a general finding indiabetic and diabetes-prone BB rats (Jackson et al., 1981; Elder& MacLaren, 1983). One of the major functional alterations oflymphocytes seems to be their disturbed ability to produceinterleukin-2 (IL-2). In a single study of type I diabeticindividuals, three subgroups of patients could be distinguishedwith increased, decreased or normal IL-2 production by phyto-haemagglutinin (PHA)-stimulated peripheral blood mono-nuclear cells (Lang et al., 1987). Other studies, however,confirmed a decreased synthesis of IL-2 by mitogen-stimulatedlymphocytes from patients with type I diabetes (Zier et al., 1984;Kaye et al., 1986). A reduced IL-2 secretion is also reported

Correspondence: Dr J. Zielasek, Diabetes Research Institute,University of Dusseldorf, Auf'm Hennekamp 65, D-4000 Dusseldorf 1,FRG.

from lymphocytes of BB rats (Prud'homme et al., 1984).Furthermore, lymphocytes from newly diagnosed type I dia-betic patients exhibit a defective secretion of soluble IL-2receptor molecules, which can effectively bind circulating IL-2(Giordano et al., 1989). These findings indicate a disregulationof IL-2 production and/or availability as a common feature oftype I diabetic patients and spontaneously diabetic BB rats.

The availability of biologically active IL-2 is essential forclonal expansion of lymphocytes during an immune responseand critically depends on the presence of regulatory factors likethymosin fractions (Zatz & Goldstein, 1985), products of thearachidonate metabolism (Kramer & Koszinowsky, 1982) or acombination of both. IL-2 levels are also directly regulated byfactors with IL-2 inhibitory activity (Kucharz & Goodwin,1988). Interestingly, the levels of IL-2 inhibitors were found tobe altered in autoimmune disorders (Krdmer et al., 1985; Djeu etal., 1986). The presence of IL-2 inhibitors may be a limitingfactor for the efficacy of a therapeutic substitution of IL-2 inimmune disorders with IL-2 deficiency.

In a previous experiment we found that human recombinantIL-2 induces an acceleration ofthe development ofautoimmunediabetes in a subline ofBB rats with a low spontaneous diabetesincidence of 23% (Kolb et al., 1986). In the present study wedescribe the converse effect, namely a suppression of the diseaseby IL-2 in a subline of BB rats with a high incidence of diabetesof 73%. We found different serum levels of thymic hormonesand IL-2 inhibitory activity in these two BB rat sublines, whichmay explain their contrasting responsiveness to IL-2 treatment.

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MATERIALS AND METHODS

AnimalsThe two BB rat sublines used in our experiments were derivedfrom breeding pairs kindly provided by Dr A. A. Like,University of Massachusetts, Worcester, MA. The animals ofthe first subline (BB subline 1) were derived from the breedingpair R1OOC/R107A, and those ofthe second subline (BB subline2) from breeding pair 18BEl10/18BE43. The mean diabetesincidence was 20-40% in subline 1 and 60-80% in subline 2. Theanimals received tap water and standard rat chow (ssniff R,SSniff, Soest) ad libitum.

Interleukin-2Highly purified human recombinant interleukin-2 (IL-2) (Jur-kat-cell derived) was kindly provided by Dr Wrann, Sandoz,Vienna, Austria. The specific activity of the IL-2 preparationwas 8000 U/pg, as determined by a standard cytotoxic T-lymphocyte line (CTLL-2) proliferation assay.

IL-2 treatmentLitters of BB rats were divided into two groups. The ratsreceived twice daily intraperitoneal injections of saline or 20 pgrecombinant IL-2/kg body weight. IL-2 was freshly dissolved insaline before injection. The treatment lasted from 35-42 days ofage until 120 days of age, and the observation period ended atDay 150. The rats were monitored daily for the development ofdiabetes. The criteria for diabetes were weight loss, polyuria,glycosuria (+ +or+++ +; Gluketur Stix, Boehringer-Mann-heim, Mannheim) and hyperglycemia with blood glucose levelsof > 14mm for at least 5 days (Hexokinase method; Boehringer-Mannheim).

Thymosin RIAThe levels ofthymosin alpha 1 and beta 4 were determined in thesera of saline- and IL-2-treated non-diabetic BB rats at Day 60.The radioimmunoassays (RIA) were performed using a modifi-cation ofthe published, well-characterized methods (McClure etal., 1982; Naylor et al., 1984, 1986; Wada et al., 1988). Aliquotsof the sera were diluted to a final volume of400 ul in RIA buffer(0-01 M Na-hydrogenphosphate, 0 15 M NaCl, 0 05% NaN3, 1%bovine serum albumin). The concentrations of the thymosinswere determined in a competitive RIA by the addition of'25Iodine-labelled thymosins and specific antibodies raised inrabbits against thymosin alpha 1 and beta 4 (Alpha-1-Biomedi-cals, San Carlos, CA). The samples were incubated for 18 hr at4°. Goat anti-rabbit IgG bound to polystyrene beads (Kynar,Roche Diagnostics, Nutley, NJ) was added and the boundradioactivity was determined in a gamma counter (Beckman,Carlsbad, CA). The concentrations of thymosin alpha 1 andbeta 4 were determined from standard curves using syntheticpeptides.

Assay for IL-2 inhibitory activityAt Days 60, 90 and 120, IL-2 inhibitory activity was determinedin the sera of saline- and IL-2-treated rats. The sera were dilutedto a final concentration of 8% in culture medium RPMI-1640(Gibco-Europe, Heidelberg) supplemented with 25 mg/l ampi-cillin, 120 mg/l pencillin, 120 mg/l streptomycin (Serva, Heidel-berg), 1 mm sodium pyruvate, 2 mM L-glutamine, 10 ml/l non-

essential amino acids (100 x; Gibco-Europe), 2 g/l NaHCO3,

a 80aEc 600c 40.0a2 20

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Subline Subline1 2

(b)

I | I I I I I I I50 70 90 110 130 150

Age (days)

Figure 1. Effect of IL-2 administration on diabetes development.(a) Diabetes incidence in subline I and in subline 2. Hatched bars,BB rats treated with twice daily i.p. injections of IL-2 (20 pg/kg); openbars, sham-treated control (C). (b) Time-course of diabetes develop-ment in subline 2, sham-treated (0) or IL-2-treated (O).* P<0001compared to control.

2-383 g/l HEPES (Serva) and 10% FCS (Boehringer-Mann-heim).

CTLL-2, kindly provided by Dr A. Reske-Kunz, Universityof Mainz, were cultivated in 96-well flat-bottomed microtitreplates (1 x 104 cells/100 pl/well) in culture medium. The mediumwas supplemented with 4 U/ml recombinant IL-2, which causeshalf-maximal proliferation of the CTLL in our system. Thediluted sera (100 p1) were added in triplicate and the sampleswere incubated at 370 for 24 hr, the last 8 hr in the presence of 1pCi [3H]thymidine/well (specific activity 37MBq/ml; AmershamBuchler, Braunschweig). The cells were harvested with amultichannel cell-harvester (Flow Laboratories, Meckenheim)on filter paper strips and the incorporated radioactivity wasdetermined in a beta-counter (Kontron, Eching bei Munchen).Units of IL-2 inhibitory activity were calculated from thecapacity ofthe sera to inhibit the IL-2-driven proliferation oftheCTLL-2. One unit of IL-2 inhibitory activity was defined as theconcentration which neutralizes the proliferative effect of I unitof recombinant IL-2.

HistologyFor histological analysis of the pancreas, animals were killed5 days after diabetes onset or at Day 150. Pancreata were fixedin Bouin and embedded in paraffin. Sections (8-10 pm) werestained with haematoxylin and eosin.

Statistical analysisStatistical analyses were performed using the chi-square test fordiabetes incidence and the Mann-Whitney rank test (two-tailed) for mean age of diabetes manifestation.

RESULTS

The effect of IL-2 administration on diabetes development wascompared in BB subline I (low spontaneous diabetes incidence)and subline 2 (high spontaneous diabetes incidence). A contrast-ing effect of IL-2 was noted. Administration of the lymphokineincreased the diabetes incidence in subline 1 (17 of 32 versus 8of 35, P<001), as reported earlier (Kolb et al., 1986) butdecreased diabetes development in subline 2 (Fig. 1) from 73%(19/26) in the saline-treated group to 32% (6/19). This decreaseof incidence was statistically significant (P < 0.01). As in the firstexperiment, we found that IL-2-treatment significantly acceler-ated diabetes development from a mean age of 96 days in the

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IL-2 immunotherapy in autoimmune diabetes

Figure 2. Exocrine pancreatitis in IL-2-treated BB rats. The pancreaticsection of an IL-2-treated rat of subline 2 shows massive infiltration ofexocrine tissue (arrow heads) and large areas of fibrosis (stars).Magnification x 240.

? 60 - Subline I Subline 2

o3000 L210.

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Total LD Non- Total LD Non-diob. diab.

Figure 4. Comparison of serum thymosin alpha 1 levels (Day 60). Meanlevels + SEM in six IL-2-treated (IL-2) and six sham-treated (C) animalsof subline 1; three in each group developing diabetes later (LD). Insubline 2, eight IL-2-treated (IL-2) and eight sham-treated animals (C)were tested; four in each group developed diabetes later (LD)

.ihk1no I

I

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Time (days)

Figure 3. Analysis of IL-2 inhibitory activity in the serum. Serum levelsof IL-2 inhibitory activity in sham-treated (0) and IL-2-treated (U) ratsof BB subline 1 and in sham-treated (0) and IL-2-treated (0) rats ofsubline 2 were determined by the method described in the Materials andMethods. Mean +SEM of three to 12 animals.

control group to 85 days in the IL-2-treated group (P < 0-01). Asin the first experiment, no differences in the sex distribution ofdiabetic rats were observed (data not shown).

The same batch of IL-2 was used in the two experiments andits biological activity was verified in proliferation assays usingCTLL-2 cells (Gillis et al., 1978). Histological analysis ofpancreata revealed that IL-2 treatment enhanced inflammationof the exocrine tissue, in both sublines, as evidenced by largeinflammatory infiltrates in the exocrine portion of the pancreas(Fig. 2). Massive exocrine interstitial pancreatitis was observedin 100% of IL-2-treated rats and in none of the sham-treatedanimals. In IL-2-treated animals, islet inflammation was foundto be enhanced in comparison to saline-treated controls, asdescribed previously (Kolb et al., 1986). No differences werefound between the animals ofsubline 1 and subline 2 in regard toseverity of insulitis or inflammation of the exocrine pancreas.

We tested the hypothesis that a different immunoregulatorystate in the two sublines is responsible for the contrastingresponse to exogenous IL-2. Sera of the two sublines were testedfor the presence of IL-2 inhibitory activity. At Days 60, 90 and

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Figure 5. Comparison of serum thymosin beta 4 levels (Day 60). Meanlevels+SEM in six IL-2-treated (IL-2) and six sham-treated (C) animalsof subline 1; three in each group developing diabetes later (LD). Insubline 2, eight IL-2-treated (IL-2) and eight sham-treated animals (C)were tested; four in each group developed diabetes later (LD)

120 the levels of IL-2 inhibitory activity were significantly lower(P < 0-01) in sham-treated animals ofBB subline 1 (41-73 U/ml)than in subline 2 (101-120 U/ml) (Fig. 3). Modulation of theselevels by IL-2 treatment could only be observed at Day 120,when we found a slight increase ofIL-2 inhibitory serum activityin animals ofBB subline I and a slight decrease in animals ofBBsubline 2 (not significant).

Serum levels ofthymosin alpha 1 were determined on Day 60in IL-2- and sham-treated rats of both sublines and were foundto be significantly different between the two sublines (P< 0 01)(Fig. 4). Mean levels of thymosin alpha 1 in subline 1 were44 ng/ml compared to 25 ng/ml in subline 2. Within each sublineIL-2- and sham-treated animals had similar thymosin alpha 1concentrations in the serum. Also, no difference was seenbetween animals developing diabetes later and those stayingnormoglycemic throughout the observation period.

A parallel analysis of serum levels of thymosin beta 4 wasperformed. Again mean serum levels significantly differedbetween the two sublines (subline 1: 3270 ng/ml, subline 2:1630 ng/ml, P< 0 01) (Fig. 5). Within each subline IL-2 treat-ment did not alter thymosin beta 4 levels nor did later diabeticanimals differ from rats which did not develop diabetes.

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J. Zielasek et al.

DISCUSSION

It is well known that sublines of BB rats differ in their risk ofdeveloping diabetes (Kryspin-Sorensen, Dyrberg & Kastern,1986), as well as in the incidence of lymphocytic thyroiditis(Rajatanavin et al., 1989). We describe here that two sublines ofthe Dusseldorf colony of BB rats which widely differ in theirdiabetes risk show a contrasting response to long-term treat-ment with human recombinant IL-2: enhancement of diabetesdevelopment in the low risk subline 1 and suppression in thehigh risk subline 2. It has previously been shown that IL-2activates BB rat lymphoid cells to an islet cytotoxic state (Pukel,Baquerizo & Rabinovitch, 1987) and that a combination of amonoclonal anti-IL-2 receptor antibody and a subtherapeuticdose of cyclosporin A can cure BB rats from freshly manifesteddiabetes (Hahn et al., 1987). These observations support ourfinding of an involvement of IL-2 in the pathogenesis ofdiabetes. Interestingly, IL-2 has also been shown to inducesuppressor cells (Duclos, Maillot & Galanaud, 1986) and evento normalize defective suppressor T-cell function of patientswith systemic lupus erythematosus in vitro (Volk & Diamant-stein, 1986). Whether exogenous IL-2 has a similar effect onsuppressor cell function in BB rats is unknown.

IL-2 when injected into diabetes-prone BB rats accumulatesin the inflamed pancreas because of the high local concentrationof IL-2 receptors (Signore et al., 1987). Accordingly we observedenhanced inflammation of islets and exocrine pancreatic tissue,which was particularly marked in the exocrine tissue in bothsublines. The inflammatory response to exogenous IL-2 corre-lated with enhanced diabetes development in subline I but not insubline 2, which indicates a distinction between gross overallorgan inflammation and the specific immune reactions leadingto beta cell destruction and diabetes.

The disease-modulating effect of exogenous IL-2 was foundpreviously to be dose-dependent. Single instead of twice dailyinjections and/or reduction of the dose by a factor of 4 up to 20were accompanied by loss of effect (Zielasek, 1989). Lowerdoses given two to three times per week for a shorter period oftime were also ineffective (Burstein et al., 1987).

The contrasting effect of exogenous IL-2 on diabetesdevelopment in the two sublines suggests that differences in thecytokine network controlling autoimmunity exist and that suchdifferences contribute to the low versus high intrinsic diabetesrisk in sublines I and 2. This assumption is supported by ourobservation that endogenous modulators of IL-2 synthesis andactivity have different serum levels in the two sublines.

Inhibitors of IL-2 in serum have been described repeatedly(Hardt et al., 1981; Malkovsky et al., 1982; Honda, Chan &Shevach, 1985; Lelchuk & Playfair, 1985; Lotze et al., 1985;Male et al., 1985; Maki et al., 1986; Fujiwara et al., 1987), someof which may be due to soluble IL-2 receptors (Giordano et al.,1988). Levels of serum IL-2 inhibitors and soluble IL-2receptors were found to be disturbed in patients and animalswith autoimmune disorders (Djeu et al., 1986; Fukushima et al.,1987; Kromer et al., 1985). Kucharz & Goodwin (1988) suggestthat low levels of IL-2 inhibitors accelerate immune reactionswhereas increased levels result in immunosuppression. The lowlevels of IL-2 inhibitory activity in subline 1 thus may not besufficient to limit the stimulating effects of injected IL-2.Interestingly, untreated 100-day-old Wistar rats, from whomBB rats have been derived, had IL-2 inhibitory serum activity in

the range of saline-treated BB rats of subline 2 (data not shown).Kromer et al. (1988) have recently found that in autoimmunechicken the decreased IL-2 inhibitory activity was due to a lowerthymidine content leading to lower competition with radio-labelled thymidine in the assay system. Such an artefact isexcluded here by the use of high serum dilutions in the testsystem (final content 4%) and by the admixture of excess (10%)fetal calf serum.

IL-2 synthesis, high affinity IL-2 receptor expression andT-lymphocyte activity is enhanced by thymosin fraction 5 (Zatzet al., 1984; Zatz & Goldstein, 1985; Serrate et al., 1987;Skotnicki et al., 1988), of which thymosin alpha 1 and beta 4 aretwo defined constituents (Low & Goldstein, 1984). Thymosinalpha 1 enhances mitogen-induced IL-2 receptor expression ofhuman peripheral blood lymphocytes (Sztein, Serrate & Gold-stein, 1986) and increases production of IL-2 by spleen cells ofaged mice (Frasca et al., 1986). T lymphocytes thus activated bythymosin alpha 1 may act back on the thymus because IL-2promotes proliferation of thymocytes (Reem et al., 1985;Gearing, Wadhwa & Perris, 1986; Brocke et al., 1987). Inaddition, thymosin alpha 1 was shown to induce NK activity ofhuman large granular lymphocytes (LGL), and enhanced IL-2production by LGL and macrophage-supplemented T cellsstimulated with PHA (Serrate et al., 1987).

In our experiments high intrinsic levels of thymosin alpha 1and beta 4 were observed in animals with a low spontaneousdiabetes incidence, whereas low levels of these hormones seemto be associated with a high incidence of diabetes. Levels ofserum thymosins or of serum IL-2 inhibitory activity were notaffected by treatment with exogenous IL-2. This indicates thatthese factors are subject to a strict internal control not readilyaffected by the activity state of the immune system.

Thymosin alpha I and thymosin beta 4 levels may reflectimmune-endocrine differences since both have been associatedwith immune and endocrine alterations (Naylor & Goldstein,1988). Interestingly, Winteretal. (1988) found much lower basalthymosin alpha I serum levels in 5-month-old BB, Lewis andWistar Furth rats than we did in 2-month-old BB rats. Wesuggest that these differences are due to an age-related decline ofserum thymosins, as has previously been described in man(McClure et al., 1982).

It is of interest that treatment with thymosin fraction 5showed contrasting effects in a high risk autoimmune thyroiditisstrain compared to the low risk strain. Suppression of thedisease was seen in the high risk strain and enhancement in thelow risk strain if thymosins were administered 2-4 weeks afterimmunization with thyroglobulin (Tomazic, Suter & Chretien,1984). These results match our observations with recombinantIL-2 and support the hypothesis that thymosins and IL-2are important single factors in the control of autoimmunereactivity.

In conclusion, we found different effects of human recom-binant IL-2 on the development of diabetes in the BB rat, i.e.enhancement of diabetes if the spontaneous incidence was low,and suppression of diabetes if the spontaneous incidence washigh. This is the first observation of a protective effect of IL-2in an animal model of an autoimmune disease. Our resultsfurthermore indicate that thymosins and other serum factorsregulating IL-2 activity may play a role in the cytokine networkcontrolling the pathogenesis of type 1 diabetes and probablyother autoimmune diseases. Further studies are needed to

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IL-2 immunotherapy in autoimmune diabetes 213

correlate serum thymosin and IL-2 inhibitory activities with theprogression to overt diabetes in both man and animal models oftype 1 diabetes.

ACKNOWLEDGMENTS

This study was supported by the Deutsche Forschungsgemeinschaft, bythe Ministerin fur Jugend, Familie und Gesundheit, and by theMinisterin fur Wissenschaft und Forschung des Landes Nordrhein-Westfalen. J. Zielasek was supported by a grant from the GermanNational Scholarship Foundation. Drs Naylor and Golstein acknow-ledge the support through grants and gifts of the NCI (CA 24972) andAlpha 1 Biomedicals Inc.

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