0013-7227/92/1305-2851$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine Society

Vol. 130, No. 5 Printed

Interleukin-ID Increases the Activity Dismutase in Rat Pancreatic Islets* L. A. HAKAN BORG, ENRICO AND D&IO L. EIZIRIK

CAGLIERO,

STELLAN

SANDLER,

in U.S.A.

of Superoxide NILS

WELSH,

Department of Medical Cell Biology, Uppsala University, Uppsalu, Sweden; and the Eye Research Institute and Department of Ophtalmology (E.C.), Harvard Medical School, Boston, Massachusetts 02114

suggests that the effects of rIL-l@ on the islet SOD activities are dependent on binding to membrane receptors and activation of gene transcription. Northern blot analysis showed a 4-fold increase in islet MnSOD mRNA content after a 90-min incubation and a lo-fold increase after a 180-min incubation with rIL-10. Thus, the enhanced MnSOD activity in the islets reflects increased gene expression. To evaluate a possible role for free oxygen radicals as mediators of the early action of rIL-10 on the pancreatic B-cells, isolated islets were exposed to rIL-1@ only or to rIL-16 plus various free radical scavengers. None of the scavengers, single or in combinations, could counteract the suppressive action of rIL-10 on islet insulin secretion. The present data suggest that rIL-l/3 induces increased activity of SOD, in particular MnSOD, in pancreatic islets. This may be due to a direct action of rIL-10 that is mediated by an increase in gene transcription. (Endocrinology 130: 2851-2857, 1992)

ABSTRACT. The suppressive effects of interleukin-10 (ILlb) on the function of pancreatic islets may be related to induction of gene transcription and protein synthesis. Presently, the effects of human recombinant IL-l@ (rIL-l@) on the activities of superoxide dismutase (SOD) and the expression of corresponding genes were studied in rat pancreatic islets. Islets that were exposed to rIL-1B for 48 h showed a 2.6-fold greater activity of mitochondrial manganese containing SOD (MnSOD) than control islets. The cytosolic copper- and zinc-containing SOD (CuZnSOD) was, however, less affected by rIL-10. Also, brief exposure of the islets to rIL-lb induced an increase in SOD activities. Hence, 12 h after a l-h exposure of the islets to rIL18, there was a 1.4-fold increase in the activity of both MnSOD and CuZnSOD. The early induction of SOD by rIL-1P was inhibited by an interleukin-1 receptor antagonist protein and actinomycin-D, which is a blocker of gene transcription. This

I

NTERLEUKIN-1 (IL-l) may be involved in the destruction of the pancreatic islet B-cells that results in insulin-dependent diabetes mellitus (1, 2). Studies in vitro have shown that IL-l causes structural damage to the B-cells (3, 4) and a decreased content of insulin and DNA (5-7) in rat pancreatic islets. The cytokine inhibits both total protein biosynthesis (6, 8) and insulin secretion (5-7) in such islets. Also, IL-l has recently been shown to be toxic to human B-cells in tissue culture (9). The deleterious effects of the cytokine are potentiated by tumor necrosis factor (10-12) and interferon-y (12). Some of the molecular mechanisms behind the effects of IL-l on pancreatic B-cells have recently been unveiled. The action of the cytokine requires binding to membrane

receptors (13, 14) and subsequent protease activation (15, 16), gene transcription, and protein translation (15, 17, 18). This is followed by synthesis of nitric oxide (19, 20), which leads to an impairment of mitochondrial function and defective ATP production (4, 11, 20, 21). It remains, however, to be determined which genes are transcribed and which proteins are induced in response to IL-l. Recent observations suggest that the c-fos protooncogene is activated early after B-cell exposure to the cytokine (22). After longer periods of B-cell exposure to IL-l, synthesis of heat shock proteins is also induced (23-25). In the current series of experiments the effects of human recombinant IL-l@ (rIL-l@) on the gene expression and enzymatic activity of superoxide dismutase (SOD) in rat pancreatic islets were investigated. The mitochondrial manganese-containing SOD (MnSOD) and cytosolic copper- and zinc-containing SOD (CuZnSOD) were analyzed separately. The enzymes are involved in cellular protection against toxic oxygen radicals (26), and it has been shown in some cell types that they may be induced by IL-l (27-30). Since generation of free oxygen radicals has been suggested to mediate the action of IL-l on pancreatic B-cells (1, 24, 31), it is of special

Received November 27, 1991. Address all correspondence and requests for reprints to: Dr. Dicio L. Eizirik, Department of Medical Cell Biology, Uppsala University, Box 571, S-751 23 Uppsala, Sweden. *This work was supported by grants from the Swedish Diabetes Association, the Juvenile Diabetes Foundation International, the Family Ernfors Fund, the Torsten and Ragnar Soderberg Foundation, the Magnus Bergvall Foundation, the Aage-Louis Hansen Fund, the Swedish Society of Medical Sciences, the Nordisk Insulin Foundation Committee, the Swedish Hoechst Diabetes Fund, and the Swedish Medical Research Council (12X-109, 12X-6538,12)3-8273, 12X-9237, and 12X9886). 2851

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IL-lfl

2852

INDUCES

PANCREATIC

interest to investigate whether IL-l induces a defence mechanism against such radicals in these cells. Materials

and Methods

Muteri&

Human rIL-l@ was kindly provided by Dr. K. Bendtzen, Laboratory of Medical Immunology, Rigshospitalet (Copenhagen, Denmark). The cytokine was produced by Immunex (Seattle, WA) and had a biological activity of 5 x 10’ U/mg comparedwith an interim international standard rIL-1P preparation from National Institute for Biological Standards and Control (London, United Kingdom) (32). Human rIL-1 receptor antagonist protein was a kind gift from Dr. D. E. Tracey, Hypersensitivity DiseasesResearch, Upjohn Co. (Kalamazoo, MI), and it was prepared as describedrecently (33). Collagenase(EC 3.4.24.3), xanthine oxidase (EC 1.1.3.22), and sodium dodecyl sulfate (SDS) were purchasedfrom Boehringer Mannheim (Mannheim, Germany); tissue culture medium RPMI-1640 and fetal calf serum from Flow Laboratories (Irvine, United Kingdom); actinomycin-D, 1,1-dimethylurea, l,lO-phenantroline, citiolone (N-acetyl-r&L-homocysteine thiolactone), superoxidedismutase(EC 1.15.1.1;from bovine erythrocytes), and xanthine from Sigma Chemical Co. (St. Louis, MO); luminol (5-amino-2,3-dihydro-1,4-phthalazinedione), and purpald (4-amino-3-hydrazino-5-mercapto-1,2,4triazole) from Aldrich-Chemie (Steinheim, Germany); and agarose from FMC BioProducts (Rockland, ME). All other chemicals of analytical grade were obtained from E. Merck (Darmstadt, Germany). The MicroFastTrack mRNA Isolation Kit wasobtained from Invitrogen Corp. (San Diego, CA). GeneScreen membranes were purchasedfrom New England Nuclear (Boston, MA). The Multiprime DNA Labelling System and [a-“‘P]deoxy-CTP were obtained from Amersham International (Amersham, Aylesbury, United Kingdom). Incubation

of pancreatic

islets

Pancreatic islets were isolated from adult male SpragueDawley rats of a local colony by a collagenasedigestion method (6, 34). Before exposure to rIL-10, they were kept in tissue culture for 6 days. The islets were cultured free-floating in RPMI-1640, containing 11.1 mM glucose and supplemented with 10% (vol/vol) fetal calf serum under a gas phase of humidified air and 5% (vol/vol) carbon dioxide at 37 C (35). The medium waschangedon days 2 and 4. In someexperiments the islets were continuously exposedto 5 rig/ml rIL-lb in culture medium for 12 or 48 h. For studies of gene expression, the islets were incubated with 10 rig/ml rIL-1P for 90 or 120 min. In other experiments the islets were first incubated with 10 rig/ml rIL-1P for 1 h and subsequently maintained in culture without rIL-lfl for 12 h. In someof these experiments 1 pg/ml IL-1 receptor antagonist protein (IRAP), 5 rg/ml actinomycin-D, 61 mM dimethylurea, or 100pM phenantroline were added to the medium together with rIL-10. Citiolone at a concentration of 3 mM was present in the culture mediumduring 3 days preceding exposureto rIL-l@. The islet secretory responseto glucose after exposure to rIL-l@ was

ISLET

SOD

Endo. 1992 Vol 130. No 6

evaluated, asdescribedpreviously (18). The 12-h culture period after incubation with rIL-l@ was selected becauseprevious observations had shown that the decline in islet function reachesa nadir lo-12 h after initial exposure to the cytokine (15). After the incubations, the isletswere retrieved and disrupted by sonication (20 kHz; 30 W) for 10 set in 200 ~1 redistilled water. Their DNA content was detetermined by fluorophotometry (36). Enzyme

determination

For measurementsof SOD activities, batches of 200 islets were homogenizedby sonication (20 kHz; 30 W) for 10 set in 250 ~1 100 mM Na2HP04-KHzP04 buffer, pH 7.8, which contained 0.1 mM EDTA. The activity of SOD was measuredby its inhibition of the chemiluminescenceof luminol, which was induced by superoxide anions produced by the action of xanthine oxidase on xanthine (37). The activity of SOD causing 50% inhibition of the chemiluminescencewas defined as 0.01 U. This correspondsto 4.2 ng SOD from bovine erythrocytes. To estimate CuZnSOD and MnSOD activities in the islet homogenatesseparately, both the total SOD activity and the activity of MnSOD alone were determined. The latter determination was achieved by a specific inhibition of CuZnSOD with 5 mM KCN (38). It should be noted that the high pH used for these determinations may affect the activities of various SOD isoenzymes differently (39, 40), which may lead to an underestimation of the MnSOD activities. The activity of catalase was measuredby a spectrophotometric method (41). The method usesthe peroxidative function of catalase for determination of the enzyme activity by the production of formaldehyde from methanol. Samplesof tissue homogenateswere incubated in duplicate for 20 min with 5.9 M methanol and 4.2 mM H205 in a %o-mM KH,P04-NaOH buffer, pH 7.0. After termination of the enzymatic reaction with a concentrated KOH solution, a secondincubation with purpald was performed. To obtain a colored compound, the product of the reaction betweenformaldehyde and purpald was oxidized by KI04, and the absorbancewasmeasuredat 550 nm. Northern

blot analysis

For Northern blot analysis, poly(A)+ mRNA was isolated from groups of 500 islets using a MicroFastTrack mRNA Isolation Kit. The isolation was followed by electrophoresison a 1% (wt/vol) agarose-2.2M formaldehyde gel. The mRNA was transferred to GeneScreenmembranesand first hybridized with a [cY-“ZP]deoxy-CTP-labeled~04660 cDNA probe in 50% (vol/ vol) formamide and 0.1% (wt/vol) SDS at 42 C (42, 43). The ~04660 cDNA contains sequences encoding the human MnSOD gene(44), and it waslabeledby oligonucleotide-primed labeling using the Multiprime DNA Labelling System. The membraneswere washedin a solution of 15 mM NaCl, 1 mM NaH2P0.,, 0.1 mM EDTA, and 0.1% (wt/vol) SDS at 55 C. For autoradiography, Kodak XAR-5 films (Eastman Kodak, Rochester, NY) were exposedto the membranesat -80 C. Densitometric analysis of the autoradiograms was performed after nonsaturating exposureswith a Quick Scan Jr densitometer (Helena Laboratories, Beaumont, TX). After autoradiography, the membraneswere washedin 0.1% (wt/vol) SDS at 100 C

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IL-18 INDUCES

PANCREATIC

and sequentially rehybridized with pS61-10 cDNA and pHF-yA1 cDNA. The pS61-10 cDNA contains sequences encoding the human CuZnSOD gene (45), and the pHFyA-1 cDNA contains sequences encoding the human y-actin gene (46).

ISLET

SOD

TABLE 1. Effects of rIL-l& ities in rat pancreatic islets

Results

Pancreatic islets that were exposed to rIL-1P for 48 h had a 150% higher MnSOD activity than control islets (Fig. 1). The cytosolic CuZnSOD was less affected by rIL-1P than the mitochondrial MnSOD, since the total SOD activity was only 50% higher in the cytokinetreated islets than in the controls. Also, brief exposure of the rat islets to rIL-10 induced an increase in SOD activities (Table 1). Hence, 12 h after a l-h exposure of the islets to 10 rig/ml rIL-l& there was a 40% increase in their MnSOD activities. A similar increase in the activity of CuZnSOD occurred, which resulted in a total SOD activity in the islets exposed to IL-l@ that was also 40% higher than that in the control islets. When islets were cultured for 12 h in the presence of 5 rig/ml rIL-10, they also showed a 40-50% increase in SOD activities compared to those in islets cultured without the cytokine (data not shown). However, rIL-l/3 did not change the islet activity of catalase, which is another enzyme related to scavenging of free oxygen radicals. The islet catalase activity was 5.1 f 0.7 pkat/pg DNA (five observations) in control islets and 4.6 f 0.5 pkat/pg DNA (five observations) in islets exposed to 5 rig/ml rIL-l/3 for 48 h. The early induction of SOD activities by rIL-10 in the pancreatic islets was inhibited by IL-l receptor antago-

MllSOD

l **

CuZnSOD

**

Total SOD

I

50

1

I

100 Superoxide

150 dismutase

200 activity

(mu/m

250 DNA)

FIG. 1. Effect of rIL-lb on SOD activities in rat pancreatic islets. Isolated islets were incubated for 48 h in the absence (0) or presence (H) of 5 rig/ml rIL-10. Subsequently, SOD activities were determined in tissue homogenates. Results are given as the mean + SEM of five observations. Differences between islets exposed to rIL-1B and controls were evaluated by Student’s t test. *, P < 0.05; **, P c 0.01; ff*, P < 0.001.

IRAP,

and actinomycin-D

Superoxide Incubation

conditions

Statistical analysis

Data are presented as the mean f SEM, and groups of data were compared using a paired or unpaired Student’s t test, as appropriate.

2853

dismutase (mu/fig DNA)

on SOD

activ-

activity

MnSOD

CuZnSOD

Control IRAP Actinomycin-D

26 + 3 24 + 6 24 + 5

173 + 13 143 f 9 155 + 18

199 f. 15 167 f 14 180 + 21

rIL-l/3 rIL-10 rIL-10

37 * 4” 26 f 5 27 + 5

249 + 26 206 + 35 157 + 11

286 f 28” 232 k 37 184 + 14

+ IRAP + actinomycin-D

Total

SOD

Isolated islets were incubated for 1 h in the absence or presence of 10 rig/ml rIL-l@ and with 1 rg/ml IRAP or 5 fig/ml actinomycin-D, as indicated. Subsequently, the islets were maintained in culture without rIL-10, IRAP, or actinomycin-D for 12 h, and SOD activities were determined in tissue homogenates. Results are given as the mean + SEM of 5-11 observations. Differences between islets exposed to rIL10, IRAP, and actinomycin-D and controls were evaluated by Student’s t test. y P < 0.05.

nist protein (IRAP) or actinomycin-D (Table 1). Neither IRAP nor actinomycin-D had any significant effect on islet SOD activity in the absence of rIL-10. Insulin secretion in response to an acute glucose challenge of islets first exposed to 10 rig/ml IL-l/3 for 1 h and then maintained under culture conditions for 12 h was decreased by 75% compared to that in control islets (see Fig. 3). It is known that IRAP and actinomycin-D completely counteract such a suppressive effect of rIL-l/3 on rat pancreatic islet function (14, 15, 17), and these findings were reproduced in the current series of experiments (data not shown). To investigate whether the induction of SOD activities in the islets caused by rIL-10 was dependent on gene transcription, Northern blot analysis of poly(A)+ mRNA from islets incubated in the absence or presence of ILlp for 90 or 180 min was performed. When the blots were hybridized with a MnSOD cDNA (pO4660), two major bands, estimated to be 1 and 4 kilobases, were detected. This hybridization pattern has been described previously for MnSOD mRNA (30). For control islets there was a barely detectable signal for MnSOD mRNA, whereas this signal was much stronger for islets exposed to rIL-lfi (Fig. 2). Indeed, as shown by densitometry, there was a 4-fold increase in islet MnSOD mRNA after a 90-min exposure and a lo-fold increase after a 18O-min exposure to rIL-1P. Rehybridization of the blots with a y-actin cDNA (pHFyA-1) did not show any consistent rIL-l/3-dependent changes in y-actin mRNA, which indicates a specific induction of MnSOD mRNA by rIL-10 in the pancreatic islets. Hybridization of the same Northern blots with a CuZnSOD cDNA probe (pS61-10) failed to show CuZnSOD mRNA for both control and rIL-l/3treated islets.

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IL-l/3

2854

INDUCES

PANCREATIC

4 kb

1 kb 2 kb FIG. 2. Effect of rIL-16 on MnSOD mRNA content in rat pancreatic islets. Isolated islets were incubated for 90 or 180 min in the absence or presence of 10 rig/ml rIL-10, as indicated. Subsequently, Northern blot analysis was performed, including hybridizations with cDNA encoding the human MnSOD gene (~04660) and the human y-actin gene (pHFyA-1). The figure is representative of three separate experiments. kb, Kilobases.

To evaluate a possible role for free oxygen radicals as mediators of the early action of rIL-l/3 on pancreatic Bcells, isolated islets were exposed to either rIL-10 only or rIL-l/3 plus various free radical scavengers (Fig. 3). After a l-h incubation, followed by a 12-h culture, the islets exposed to rIL-l/3 had a 70-80% lower insulin response to a high glucose concentration than the control islets. None of the free oxygen radical scavengers, single or in combinations, could counteract the suppressive action of rIL-l/? on islet insulin secretion. Moreover, the addition of SOD to the medium during both islet exposure to rIL-l/3 and the subsequent culture also failed to protect the B-cells against the inhibitory effect of the cytokine (data not shown). However, these latter data must be interpretated with caution, since added SOD is likely to remain extracellularly. Discussion Eukaryotic cells have two types of cellular SOD. CuZnSOD is located in the cytosol, and MnSOD in the matrix of the mitochondria (47). Both belong to an enzymatic system that protects the cells against toxic oxygen radicals (26). SOD converts superoxide to molecular oxygen and hydrogen peroxide, and hydrogen peroxide is subsequently converted to water and oxygen by catalase and various peroxidases. By removing superoxide and hydrogen peroxide, SOD and catalase prevent the formation of the highly reactive hydroxyl radical,

ISLET

SOD

Endo. Voll30

l

1992 No 5

which is formed by the iron-catalyzed Haber-Weiss reaction (48). The reaction of hydroxyl radicals with cellular targets probably explains most of the tissue damage that accompanies superoxide and hydrogen peroxide formation (49). The present data show that treatment of rat pancreatic islets with rIL-1P increases the activity of both MnSOD and CuZnSOD, whereas the activity of catalase is not affected. Although the enhancement of MnSOD and CuZnSOD activities in the islets was similar in magnitude after a 12-h exposure to rIL-l& there was a greater stimulation of MnSOD activity after 48 h of exposure to the cytokine. The effect of rIL-1P on islet SOD activities seems, nevertheless, to result from early actions of the cytokine. Thus, incubation of the islets for 1 h with rIL18 induced increased SOD activities after 12 h. Also, a marked increase in the islet content of MnSOD mRNA was observed after 90 min of exposure to rIL-l@. Similar observations have been made in other cell types, in which exposure to the cytokine for 1-12 h has induced an increase in MnSOD mRNA content, and longer (24- to 72-h) periods of exposure to rIL-lfi have resulted in increased MnSOD enzymatic activity (27-30). It is noteworthy that while induction of the enzymatic activity of islet MnSOD was associated with an increase in islet MnSOD mRNA content, no corresponding effect of rILl@ on islet CuZnSOD mRNA content could be detected. Indeed, it has been reported that IL-l fails to induce CuZnSOD mRNA or CuZnSOD enzymatic activity in various cell lines (27-30). However, in line with the present data, it has been shown that rIL-10 slowly increases CuZnSOD activity in lung fibroblasts (50). The increased activities of SOD in the pancreatic islets caused by rIL-1P probably require binding of the cytokine to membrane receptors and induction of gene transcription. This notion is supported by the observations that IRAP, which is a competitive antagonist to the IL-l receptor (33), and actinomycin-D, which is a blocker of gene transcription, inhibited the effects of rIL-la on islet SOD activities. However, the details of the molecular mechanisms for induction of MnSOD mRNA by rIL-lb are not yet known. One possibility is that rIL-l/3 induces an early generation of reactive oxygen species that may explain both the induction of SOD and the inhibition of B-cell function. It has been shown in endothelial cells and fibroblasts that IL-l or tumor necrosis factor induces the production of superoxide and hydrogen peroxide (51, 52). Scavengers of free oxygen radicals protect pancreatic islet cells in monolayer cultures against the cytotoxic effects of IL-l in combinations with tumor necrosis factor and interferon-y (31). In the present experiments isolated rat islets were exposed to rIL-1P in the presence of high concentrations of dimethylurea, phenantroline, and citiolone. Dimethylurea is a hydroxyl radical scav-

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IL-l/I

INDUCES

PANCREATIC

ISLET

SOD

2855

No scavenger

DMU FIG. 3. Effects of free oxygen radical scavengers on rIL$-induced suppression of glucose-stimulated insulin secretion in rat pancreatic islets. Isolated islets were incubated for 1 h in the absence (0) or presence (m) of 10 rig/ml rIL-P and with 61 mM dimethylurea (DMU), 100 ELM phenantroline (PHT), or 3 mM citiolone (CIT), as indicated. Subsequently, the islets were maintained in culture without rIL-1@, DMU, PHT, or CIT for 12 h, and insulin secretion in response to 16.7 mM glucose was measured during a further 2-h incubation. Results are given as the mean + SEM of 5-10 observations. Differences between islets exposed to rIL-10 and controls were evaluated at each of the incubation conditions by paired Student’s t test. ***, P c 0.001.

PHT

I

CIT

***

DMU+PHT

***

DMU+CIT

***

DMU+PHT+CIT

I

I

I

I

I

I

I

I

I

0

50

100

150

200

250

300

350

400

Insulin

enger, which counteracts both alloxan-induced (53, 54) and streptozotocin-induced (55) diabetes. Phenantroline is an iron chelator that also inhibits hydroxyl radical formation and prevents alloxan-induced diabetes (56). Citiolone has been shown to induce increased intracellular SOD activity (37,57), to scavenge hydroxyl radicals (58), and to protect against streptozotocin-induced diabetes (57). However, none of these free radical scavengers, singly or in combinations, could protect the pancreatic islets from the early functional impairment caused by rIL-1P. These results substantiate earlier observations showing no beneficial effect of dimethylurea, phenantroline (4), SOD, or catalase (59) on isolated rat islets or of dimethylurea on insulin-producing HIT cells (13) when exposed to rIL-1P alone. Taken together, the data suggest that an initial and massive generation of free oxygen radicals cannot explain the suppressive ac-

secretion

(ng/2h.

pg DNA)

tion of rIL-l@ on the pancreatic B-cells. Also, increased MnSOD gene expression, an early effect of rIL-l@ on the pancreatic islets, may hardly result from intracellular generation of free oxygen radicals. The induction of MnSOD is probably a more direct effect of rIL-lb. Studies on human melanoma cell lines indicate that IL-l induces an increase in SOD activity in both cells that are sensitive and resistant to the toxic action of the cytokine (27), and in a pulmonary epithelial-like cell line it has been shown that IL-l induces an immediate MnSOD gene expression, whereas hyperoxia, which would result in free oxygen radical production, does not (29). Since a short exposure of isolated islets to rIL-l@ also increases the expression of the c-fos gene (22), the expression of both SOD and c-fos may be part of the initial response of the islet B-cells to the cytokine. Recently, it has been described that rIL-l@ stimulates

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2856

IL-1B

INDUCES

PANCREATIC

the production of nitric oxide in pancreatic islets (19, 20). Moreover, it has been shown that inhibitors of nitric oxide synthase completely counteract the inhibitory effects of rIL-10 on pancreatic B-cells (19, 20), which suggests that nitric oxide may be an important mediator of the toxic actions of rIL-lfi. Superoxide may react with nitric oxide (60), and it has been proposed that this reaction occurs in uivo and may be of relevance for regulation of the intracellular concentrations of nitric oxide (61). Thus, an increased SOD activity in the pancreatic islets, induced by rIL-lP, could favor accumulation of nitric oxide in B-cells by increasing superoxide removal. On the other hand, recent data (62) suggest that part of the nitric oxide cell toxicity may derive from its combining with superoxide, leading to the formation of peroxynitrite anion, which decomposes when protonated into the toxic hydroxyl radical. In this context, ILl-induced activation of SOD and consequent removal of superoxide could reduce the accumulation of nitric oxidederived toxic products. Thus, it still remains to be established whether a putative interaction between IL-l-induced nitric oxide accumulation and SOD activation will lead to increased cell toxicity or will contribute to cell defence by neutralizing two potentially toxic radicals (i.e. superoxide and nitric oxide).

ISLET

9.

10.

11.

12.

13.

14.

15

16.

17.

18.

Acknowledgments

We thank Drs. U. J. Eriksson and M. Welsh for helpful discussions. The excellent technical assistance of I.-B. Hallgren, E. TGmelius, E. Forsbeck, A. Nordin, and S. Svanholm is gratefully acknowledged.

19.

20.

References

5.

6.

7.

8.

Nerup J, Mandrup-Poulsen T, M$lvig J, Helqvist S, Wogensen L, Egeberg J 1988 Mechanisms of pancreatic P-cell destruction in type 1 diabetes. Diabetes Care [Suppl 11 11:16-23 Bendtzen K 1989 Immune hormones (cytokines): pathogenic role in autoimmune rheumatic and endocrine diseases. Autoimmunity 2:177-189 Mandrup-Poulsen T, Egeberg J, Nerup J, Bendtzen K, Nielsen JH, Dinarello CA 1987 Ultrastructural studies of time-course and cellular specificity of interleukin-1 mediated islet cytotoxicity. Acta Path01 Microbial Immunol Stand [Sect C] 95:55-63 Sandler S, Bendtzen K, Borg LAH, Eizirik DL, Strandell E, Welsh N 1989 Studies on the mechanism causing inhibition of insulin secretion in rat pancreatic islets exposed to interleukin lfl indicate a perturbation in the mitochondrial function. Endocrinology 124:1492-1501 Bendtzen K, Mandrup-Poulsen T, Nerup J, Dinarello CA, Svenson M, Nielsen JH 1986 Cytotoxicity of human p1 7 interleukin-1 for pancreatic islets of Langerhans. Science 232:1545-1547 Sandler S, Andersson A, HellerstrBm C 1987 Inhibitory effects of interleukin 1 on insulin secretion, insulin biosynthesis and oxidative metabolism of isolated rat pancreatic islets. Endocrinology 121:1424-1431 Eizirik DL, Strandell E, Bendtzen K, Sandler S 1988 Functional characteristics of rat pancreatic islets maintained in culture following exposure to human interleukin 1. Diabetes 37:916-919 Spinas GA, Hansen BS, Linde S, Kasten W, Mprlvig J, MandrupPoulsen T, Dinarello CA, Nielsen JH, Nerup J 1987 Interleukin-1

21.

22.

23.

24.

25.

26. 27.

28.

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