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Endocrinology Vol. 143, No. 9 3490-3497
Copyright © 2002 by The Endocrine Society

ARTICLE

Polyethylene Glycolated Recombinant TNF Receptor I Improves Insulitis and Reduces Incidence of Spontaneous and Cyclophosphamide-Accelerated Diabetes in Nonobese Diabetic Mice

Jin-Lin Wang, Xueming Qian, Narumol Chinookoswong, John Lu, Gwyneth Chow, Lars E. Theill and Zhi-Qing Shi

Departments of Metabolic Disorders (J.-L.W., N.C., Z.-Q.S.), Inflammation (X.Q., L.E.T.), Pathology (G.C.), and Cancer Biology (J.L.), Amgen, Inc., Thousand Oaks, California 91320

Address all correspondence and requests for reprints to: Dr. Zhi-Qing Shi, Department of Metabolic Disorders, Amgen, Inc., Thousand Oaks, California 91320. E-mail: jshi{at}amgen.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have conducted three studies to examine the role of TNF{alpha} in islet destruction in female nonobese diabetic mouse (NOD) mice, a model of human autoimmune diabetes, using polyethylene glycolated (PEGylated) soluble TNF receptor type I (PEG sTNF-RI) as TNF{alpha} antagonist. PEG sTNF-RI (3 mg/kg, sc) was given every other day to NOD mice from age wk 8 for 12 wk (study 1), from age wk 12 for 8 wk (study 2), or from age wk 8 for 3 wk, with cyclophosphamide (6 mg/mouse) injected at wk 9 to accelerate the onset of diabetes (study 3). Diabetic incidence was reduced (control vs. PEG sTNF-RI) from 68.7% (11 of 16) to 18.3% (3 of 16) in study 1, from 84.6% (11 of 13) to 28.5% (4 of 14) in study 2, and from 66.6% (8 of 12) to 23.1% (3 of 13) in study 3, respectively. The incidence of insulitis was also reduced from 91.6% (11 of 12) to 12.5% (2 of 16) in study 1 and from 100% (7 of 7) to 16.6% (2 of 12) in study 2 by PEG sTNF-RI. PEG sTNF-RI also largely preserved islet insulin content, reduced mRNA of inducible nitric oxide synthase and IL-6 in pancreases, and lowered plasma corticosterone, glycerol, and free fatty acid levels. These results confirm a pathogenic role of TNF{alpha} in mediating insulitis in NOD mice and suggest the prophylactic and therapeutic potential of PEG sTNF-RI for human autoimmune diabetes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE NONOBESE diabetic (NOD) mouse is a model of autoimmune diabetes mellitus (1). The disease process in NOD mice is remarkably similar to human autoimmune type 1 diabetes (2). Diabetes development in NOD mice is characterized by insulitis, a leukocytic infiltration of the pancreatic islets, and a marked decrease in the insulin content of pancreatic islet ß-cells (3, 4). It is demonstrated by ample literature that the susceptibility to autoimmune diabetes in NOD mice is polygenic and affected by numerous environmental factors (5). TNF{alpha} is a proinflammatory cytokine that has been shown to have a strong genetic linkage to several autoimmune disorders, including type 1 diabetes (6). TNF{alpha} is a key mediator in lymphocyte activation, in regulating the expression of MHC class I and II molecules, and in inducing the release of secondary inflammatory mediators. It also acts as a direct effector in mediating pancreatic islet ß-cell damage (7, 8). Thus, TNF{alpha} has been considered to be a critical cytokine mediating the pathogenic destruction of islet ß-cells (9, 10). The cells expressing TNF{alpha} mRNA are found in the islets of Langerhans of NOD mice during early stages of the disease (11, 12). Treatment of NOD mice with anti-TNF{alpha} monoclonal antibodies inhibited the development of insulitis and diabetes (13, 14). Paradoxically, in vivo administration of TNF{alpha} reportedly has very divergent effects on the development of diabetes in NOD mice. In female newborn NOD mice, injection of TNF{alpha} accelerates the development of diabetes mellitus (15). However, in adult NOD mice, repeated injections of NOD mice with TNF{alpha} protected mice from the development of diabetes (16, 17). Localized expression of TNF{alpha} as a transgene in islet ß-cells induced insulitis without progression to diabetes (18). These reports suggest that TNF{alpha} has complex effects on the development of autoimmune diabetes.

Inhibitions of TNF{alpha} activity in autoimmune diabetes studies have provided some interesting insight into the biological actions of TNF{alpha}. Studies of TNF receptor I (TNF-RI)-deficient NOD mice have shown that autoimmune diabetes is caused by two independent and yet synergistic mechanisms, including CD8+ T cell-mediated, perforin-dependent ß-cell lysis and a TNF-RI-dependent cytotoxic mechanism involving CD4+, dendritic cells, and macrophages (19). The development of autoimmune diabetes in NOD mice was reportedly prevented by transgenic expression of soluble TNF-R p55 (20). The infiltration-promoting effects of TNF{alpha} are not restricted to the pancreatic islets, as neutralization of TNF{alpha} by transgenic expression of soluble TNF-R (sTNF-R) p55 also effectively inhibits inflammatory infiltration to the salivary glands of NOD mice (21).

Recombinant human sTNR-R type I (sTNF-RI) has been cloned and isolated by recombinant DNA techniques using Escherichia coli as a host (22). In the present study, we used a recombinant form of a natural inhibitor of TNF{alpha}, recombinant human sTNF-RI. A high molecular weight polyethylene glycol (PEG) molecule is conjugated to the N terminus to form the long-acting PEG sTNF-RI intended for clinical evaluation (23). PEG sTNF-RI offers the advantages of minimal immunogenicity, greater efficacy in animal models of rheumatoid arthritis and Crohn’s disease (24, 25, 26), and less frequent dosing due to prolonged circulating half-life with the addition of PEG to the sTNF-RI. We use PEG sTNF-RI as a tool in the current study to examine the role of TNF{alpha} in the preonset and perionset stages of autoimmune diabetes in NOD mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female NOD/LtJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and were maintained in 12-h light, 12-h dark cycled and temperature-controlled animal facility. The NOD mice were fed normal rodent chow with water ad libitum. All animal procedures were approved by the institutional animal care and use committee of Amgen, Inc. (Thousand Oaks, CA), in accord with accepted standards of humane animal care. There were three separate studies using the NOD mice, described as follows.

Study 1.
A 12-wk treatment of NOD mice was initiated at 8 wk of age using the PEGylated sTNF-RI (PEG sTNF-RI; Amgen, Inc.) freshly prepared in an aqueous solution. PEG sTNF-RI (3 mg/kg) was given sc every other day to the prediabetic NOD mice (n = 16). PBS solution, which was used in making up the PEG sTNF-RI dose solution, was given as vehicle control to the age-matched control group (n = 16). The treatments lasted for 12 wk and were terminated at the age of 20 wk, at which point the animals were killed with isoflurane, and blood and tissue samples were taken.

Study 2.
An 8-wk PEG sTNF-RI treatment in NOD mice was initiated at 12 wk of age. PEG sTNF-RI (3 mg/kg) was given sc every other day to the prediabetic NOD mice (n = 14). PBS was given sc to the control group (n = 13). The treatment lasted for 8 wk and was terminated at the age of 20 wk. Animals were killed, and tissue and blood samples were taken.

Study 3.
Overt diabetes was accelerated by cyclophosphamide (CY; Sigma, St. Louis, MO) in NOD mice in this study. The NOD mice at 8 wk of age were treated with either PEG sTNF-RI (3 mg/kg; n = 13) or PBS as the vehicle control (n = 12) every other day for 1 wk. CY was administered ip as a bolus of 300 mg/kg at wk 9. PEG sTNF-RI (3 mg/kg) or PBS vehicle administration was continued every other day for another 2 wk after the injection of CY. Blood glucose levels were monitored twice weekly, and the NOD mice were killed 2 wk after CY injection (at 11 wk of age).

Immunohistochemistry and assessment of insulitis
Pancreatic tissue samples were harvested from all surviving NOD mice at the end of each study and were processed for hematoxylin-eosin and insulin immunohistochemical staining. The severity of insulitis was assessed as periinsulitis (islets surrounded by few lymphocytes), intermediate insulitis (lymphocytic infiltration into interior of islets), and severe insulitis (lymphocytic infiltration account for >50% of the islets). For the purpose of simplifying the data presentation, significant insulitis is defined by the number of islets with intermediate and severe lymphocytic infiltration that are more than 50% of the total islets examined in each pancreas. For insulin immunohistochemistry of pancreatic endocrine cells, the formalin-fixed paraffin sections were deparaffinized and immunostained for insulin by using an immunoperoxidase technique. Deparaffinized tissue sections were blocked with Cas Block (Zymed Laboratories, Inc., South San Francisco, CA), incubated with guinea pig antiswine insulin antibody (DAKO Corp., Carpinteria, CA). The antibody was detected with biotinylated goat antiguinea pig Igs (Vector Laboratories, Inc., Burlingame, CA). Slides were quenched with peroxidase blocking solution (DAKO Corp.), followed by avidin-biotin complex (Vector Laboratories, Inc.). Reaction sites were visualized with diaminobenzidine and counterstained with hematoxylin.

Measurement of blood glucose
Blood glucose concentrations were measured twice weekly with a Glucometer Elite (Bayer Corp., Elkhart, IN) using tail vein blood obtained by a needle lancet in conscious, unrestrained mice. The onset of diabetes was deemed when two consecutive blood glucose readings exceeded 200 mg/dl, or any single measurement exceeded 250 mg/dl.

mRNA analysis
The total RNA was isolated from pancreatic tissues of NOD mice using a Total RNA Isolation Kit from Ambion, Inc. (Austin, TX). RT-PCR was performed using a mouse inducible nitric oxide synthase (iNOS) relative RT-PCR kit (Ambion, Inc.). The iNOS primers yielded a gene-specific product of 349 bp and multiplexed efficiently with the supplied 18S rRNA primers, as an internal control. Amplification of 18S rRNA in addition to the mRNA under study allowed us to normalize samples for differences in loading. The specific primers for IL-6 are 5'-TCTTTCTCGAATGTACCAGG and 3'-CATGGTGGCTCAGTACTACG. PCR amplification was performed with 50-µl PCR reactions containing 5 µl first strand cDNA in buffer (5 mM KCl and 1 mM Tris); 0.01% Triton X-100; 25 mM MgCl2; 10 mM each of deoxy-ATP, deoxy-CTP, deoxy-GTP, and deoxy-TTP; 20 µM of each of the 5' and 3' primers; and 25 U AmpliTaq (PE Applied Biosystems/Cetus Corp., Foster City, CA). Reactions were assembled at room temperature and then loaded directly into the heating block of a Perkin-Elmer Thermal Cycler 9600 (PE Applied Biosystems) at 80 C. PCRs were heat-denatured at 95 C for 3 min, followed by 30 cycles at 95, 55, and 72 C for 30 sec each. Negative control reactions containing water instead of DNA and positive controls containing the standard DNA were also included for each primer pair. PCR products were subjected to electrophoresis on a 2% agarose gel, followed by reading with a FluorChem image system (Alpha Innotech, Inc., San Leandro, CA). Image analysis was performed using AlphaEaseFc software. The integrated density value is the sum of all of the pixel values after background correction. The ratio value is the spot density value normalized by 18S signals in the same loading lane.

Biochemical measurements
The blood samples were obtained from animals by cardiac puncture at the end of the experiments. The biochemical parameters cholesterol, triglyceride, glycerol, ß-hydroxybutyrate, free fatty acids, lactate, and corticosterone were measured spectrophotometrically on a Hitachi 717 Clinical Chemistry Autoanalyzer (Roche Molecular Biochemicals, Indianapolis, IN). Plasma insulin concentrations were determined with a rat insulin RIA kit from Linco Research, Inc. (St. Charles, MO).

Statistical analyses
All data are expressed as the mean ± SE. Differences in incidence of spontaneous or CY-induced diabetes between groups were analyzed using {chi}2 test (Fisher’s exact test). The Tukey multiple factors ANOVA test was used to determine statistical differences in biochemical values between the experimental groups. A significant difference is assumed at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of PEG sTNF-RI on incidence of spontaneous diabetes
In study 1, two groups of female NOD mice, 8 wk of age, received PEG sTNF-RI or PBS sc injections over a period of 12 wk. The results are shown in Fig. 1Go. PEG sTNF-RI treatment significantly reduced the incidence of diabetes to 18.8% (3 of 16) from that in the PBS control group (68.7%, 11 of 16) by 20 wk of age. The mortality rate was reduced from 25% (4 of 16) in the PBS control group to 0% (0 of 16) by PEG sTNF-RI treatment (Fig. 1Go).



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  		Figure 1. The effect of PEG sTNF-RI treatment for 12 wk on diabetic incidence (upper panel) and mortality rate (lower panel) in NOD mice (study 1). The NOD mice were treated with PEG sTNF-RI (3 mg/kg·2 d; {bullet}; n = 16) or PBS ({circ}; n = 16) sc injection for a period of 12 wk starting at 8 wk of age (as indicated by arrow). *, P < 0.05 compared with PBS control group.

 
In study 2, the PEG sTNF-RI treatment was started at the age of 12 wk and lasted for 8 wk. The results are shown in Fig. 2Go. The incidence of diabetes at 20 wk of age in the PBS-treated NOD mice was 84.6% (11 of 13), but was reduced to 28.6% (4 of 14) in the PEG sTNF-RI-treated group (P < 0.05). PEG sTNF-RI treatment also reduced the mortality rate in NOD mice from 46.2% (6 of 13) in the control group to 14.3% (2 of 14) in the PEG sTNF-RI-treated group.



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  		Figure 2. The effect of PEG sTNF-RI treatment for 8 wk on diabetic incidence (upper panel) and mortality rate (lower panel) in NOD mice (study 2). The NOD mice were treated with 3 mg/kg PEG sTNF-RI ({bullet}; n = 13) or PBS ({circ}; n = 14) sc injection for a period of 8 wk starting at 12 wk of age (as indicated by arrow). *, P < 0.05 compared with PBS control group.

 
In study 3, a single dose of 300 mg/kg CY caused a rapid onset of overt diabetes in young (8 wk of age) NOD mice, as shown in Fig. 3Go. The incidence of diabetes in the PBS control group was 66.6% (8 of 12). In contrast, only 23.1% (3 of 13) of PEG sTNF-RI-treated mice became overtly diabetic during the same period. The difference was significant by {chi}2 test (P < 0.01).



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  		Figure 3. Incidence of diabetes in NOD mice after injection of CY to accelerate the onset of diabetes. The PEG sTNF-RI (3 mg/kg, sc; n = 13) and PBS (n = 12) injections were given for 1 wk in the 8-wk-old NOD mice before CY injection. The CY (300 mg/kg, ip) was then injected on d 0. The PEG sTNF-RI and PBS treatments were continued for another 2 wk after CY injection. *, P < 0.05 compared with PBS control group.

 
Histological examination
To determine the effect of PEG sTNF-RI on insulitis in NOD mice, we examined histological sections of pancreases from PBS control and PEG sTNF-RI-treated NOD mice. As shown in Table 1Go, the incidence of insulitis was significantly reduced in NOD mice treated with PEG sTNF-RI for 12 wk (starting at 8 wk of age) or 8 wk (starting at 12 wk of age). In study 1, the incidence of insulitis decreased from 91.6% (PBS group) to 12.5% (PEG sTNF-RI group; P < 0.05). In study 2, treatment of NOD mice with PEG sTNF-RI for 8 wk reduced the incidence of insulitis to 16.6% (2 of 12) compared with that in the PBS control group (100%, 7 of 7), although statistical significance was not achieved due to a substantial loss of animals (6 of 13) in the PBS group. Figure 4Go shows the representative histology photographs of the pancreatic islets after treatment with PEG sTNF-RI or PBS (data from study 2). The PBS control mice had developed severe insulitis, with a high degree of lymphocytic infiltration and cell necrosis in pancreatic islets (Fig. 4AGo). However, most of the PEG sTNF-RI-treated mice presented near-normal islets, with mild inflammatory infiltration and tissue damage (Fig. 4BGo). Immunohistochemical staining of serial sections revealed that the islets of PBS control mice had a substantially reduced number of insulin-positive ß-cells (Fig. 4CGo), but insulin staining was dense and defuse across most islets after treatment with PEG sTNF-RI (Fig. 4DGo). Therefore, the massive infiltration of lymphocytes and the high incidence of diabetes in the control group are in sharp contrast to those in PEG sTNF-RI-treated mice.


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  		Table 1. Systemic administration of PEG sTNF-RI attenuated insulitis in NOD mice

 


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  		Figure 4. These representative figures show the hematoxylin-eosin stain (top panel) and the insulin immunohistochemical staining (bottom panel) of the same islets from study 2. In the left column are the islets collected from the PBS control animals at 20 wk of age. The PBS control mice showed heavy lymphocytic infiltration of the islets and substantially reduced insulin content in islets (A and C). In the right column are islets collected from PEG sTNF-RI-treated NOD mice. With PEG sTNF-RI, the lymphocytic infiltration was mild and the islets showed well-preserved insulin-containing ß-cells (B and D).

 
Effect of PEG sTNF-RI on iNOS and IL-6 mRNA expression
Using amplification of mRNA by RT-PCR, the iNOS message could be detected in 20-wk-old PBS-treated NOD mice. A strong expression of iNOS mRNA could be detected 14 d after a single injection of CY (Fig. 5Go). Quantification of the signal intensities showed 13% and 39% decreases in iNOS mRNA expression after treatment with sTNF-RI in studies 1 and 2, respectively. There was also a significantly decrease (58%) in iNOS mRNA expression in sTNF-RI-treated NOD mice with CY-accelerated diabetes. IL-6 gene expression was decreased in NOD mice treated with PEG sTNF-RI in all three studies; this was especially more pronounced in study 3 in mice with CY-accelerated diabetes.



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  		Figure 5. RT-PCR analysis of iNOS and IL-6 mRNA expression in NOD mice. RT-PCR were performed to measure levels of iNOS and IL-6 mRNA in NOD mice treated with PEG sTNF-RI (Tr) or PBS (C). The representative results are shown in the upper panel with the specific bands for iNOS (349 bp) or IL-6 (308 bp). The lower panel shows the integrated densitometric quantification values (IDV) of each band normalized against the value of 18S rRNA (as the control) from each study. *, P < 0.05 compared with the PBS control group.

 
Effect of PEG sTNF-RI on plasma hormones and metabolites
Table 2Go shows the results obtained from study 2. After a 12-wk treatment, plasma corticosterone levels were significantly reduced from 43.6 ± 9.7 µg/dl in the control group to 23.5 ± 1.4 µg/dl in the PEG sTNF-RI-treated group. The plasma insulin and T4 levels did not show significant differences between PEG sTNF-RI and control groups. Plasma glycerol levels were 49.0 ± 10.9 mg/dl in the PBS control group, but were reduced to 25.7 ± 2.0 in the PEG sTNF-RI-treated group. The plasma free fatty acid level was 40% lower after PEG sTNF-RI treatment (0.5 ± 0.1 mEq/liter) than that in the PBS control group (0.9 ± 0.2 mEq/liter). The plasma cholesterol, triglyceride, lactate, and ß-hydroxybutyrate levels were not significantly different from those in the PEG sTNF-RI-treated group.


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  		Table 2. The effect of PEGsTNF-RI on plasma hormones and metabolites in NOD mice

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TNF{alpha} is a powerful mediator of inflammation with potent immune regulatory functions. The broad tissue distribution of its receptors suggests that TNF{alpha} has a number of biological activities in a wide variety of tissues (27, 28). TNF{alpha} has been considered to be a key cytokine responsible for mediating the pathogenic destruction of pancreatic islet ß-cells (29, 30). TNF{alpha} may be a direct cytotoxic factor targeting pancreatic ß-cells in vitro by generation of nitric oxide (31), either alone or in synergy with other cytokines (29, 32, 33). TNF{alpha} has also been shown to up-regulate the expression of MHC class I and II molecules (7) and cell adhesion molecules, such as intracellular adhesion molecule I, and vascular cell adhesion molecule I (34). Pharmacological inhibition of TNF{alpha} has yielded encouraging results in models of collagen-induced arthritis and experimental autoimmune encephalomyelitis (24, 35). Paradoxically, in diabetes research, blockade of TNF{alpha} with a specific antibody or soluble receptor elicited either islet-protecting or islet-damaging effects (7). The mechanistic distinction between the two opposing effects of TNF{alpha} has yet to be resolved. In the present study, we report that systemic administration of PEG sTNF-RI molecule has a protective effect in preventing autoimmune diabetes in NOD mice when treatments were initiated at early stages of the disease process. The protective effect of PEG sTNF-RI is evidenced by a significant reduction in the incidence of diabetes and mortality rate in NOD mice. Histological examination demonstrates a marked improvement in lymphocytic infiltration in pancreatic islets of NOD mice treated with PEG sTNF-RI. PEG sTNF-RI treatment also reduced the diabetic incidence in CY-accelerated diabetes in NOD mice. Our results were consistent with the studies in which TNF{alpha} was permanently neutralized in NOD mice by transgenic expression of a sTNF-R p55 human FcIgG3-fusion protein (20). Those NOD mice also had decreased insulitis and were completely protected from the development of spontaneous or CY-accelerated diabetes (20).

Paradoxically, the systemically administrated TNF{alpha} appears to exert dichotomous effects on pancreatic islet ß-cells depending on the age of the NOD mice at the start of treatment. When TNF{alpha} treatment was started at birth or at 2 wk of age, diabetes onset was accelerated by TNF{alpha}. However, if TNF{alpha} treatment was started at or after 4 wk of age, the onset of diabetes was delayed. These results suggest that the effects of TNF{alpha} on the development of autoimmune diabetes are complex and highly dependent on developmental age and localization of its action (16, 17). This study examines the effects of blockade of TNF{alpha} on the progression of autoimmune diabetes at two time points of early stage diabetes development, i.e. 8 or 12 wk of age, which are known to feature progressive islet inflammation in NOD mice (1). Although our study did not provide direct evidence of histological improvement of islets at these early stages, our observation of marked improvement of diabetic incidence from wk 12–20 suggests that an islet protective effect may be present even at an earlier stage.

To gain further insight into the mechanisms of the protective effects of PEG sTNF-RI, we examined pancreatic histological sections of both PBS- and PEG sTNF-RI-treated NOD mice at 20 wk of age. In both studies 1 and 2, the incidence of insulitis was markedly reduced from 92–100% in the PBS control groups to 13–17% in the PEG sTNF-RI-treated groups. Thus, the PEG sTNF-RI-induced protective effect on spontaneous autoimmune diabetes may result from its inhibitory effect on lymphocytes trafficking into the pancreatic islets. There have been controversial reports on the effects of sTNF-RI on the course of insulitis. Local expression of TNF{alpha} as a transgene in ß-cells induced massive insulitis without progression to diabetes. Despite infiltration of a considerable number of lymphoid cells in islets, the expression of TNF{alpha} protected NOD mice from the development of diabetes (36). However, our data suggested that blockade of TNF{alpha} in vivo not only inhibits lymphocytic migration into pancreatic islets, but also protects islet ß-cells from inflammatory destruction.

The effect of PEG sTNF-RI on CY-accelerated diabetes in NOD mice was also intriguing. A single injection of CY (300 mg/kg) to 9-wk-old NOD mice reportedly triggered an early onset of diabetes in most animals within 10–15 d (37). However, simultaneous initiation of treatment with PEG sTNF-RI (3 mg/kg, every other day) for 15 d in our study resulted in a substantial reduction in diabetic incidence. It has been shown that pancreatic islets from NOD mice contain more TNF{alpha} after CY injection (38, 39). When spontaneous progression to diabetes was accelerated by CY injection, there is a significant increased production of cytokines, including TNF{alpha} and IL-1ß, which could contribute to the acceleration of diabetes onset (37). Furthermore, the incidence of CY-induced diabetes in the TNF-RI knockout NOD mice was markedly reduced compared with the control NOD mice (32). In the present study systemic administration of sTNF-RI lead to a reduction in incidence of CY-induced diabetes similar to those TNF-RI-deficient NOD mice. These observations indicate an important role of TNF{alpha} in CY-accelerated diabetes. The present results are consistent with the hypothesis that ß-cell destruction is dependent upon the increased production of certain proinflammatory cytokines such as TNF{alpha}.

Plasma insulin levels were not different in the surviving mice of both control and PEG sTNF-RI-treated groups. The reasons are not clear. It could be partly due to a higher mortality rate in the control mice, and insulin levels were measured in the surviving animals, which were less diabetic than those that died earlier in the course of study. Thus, the insulin levels tend to be biased higher in the surviving control mice and diminish any possible difference vs. the PEG sTNF-RI-treated animals. The higher levels of corticosterone in the control mice indicate stress hormone response related to a severe diabetic state, which was alleviated with PEG sTNF-RI treatment. The elevated plasma glycerol and free fatty acid levels in the control mice indicate diabetes-induced increase in lipolysis, which was also improved by PEG sTNF-RI.

This study analyzed the association of iNOS and IL-6 gene expression in the pancreas of NOD mice treated with PBS or PEG sTNF-RI. The PEG sTNF-RI-treated mice showed a significant reduction in both iNOS and IL-6 gene expression compared with PBS control mice. The differences between the treatment and control groups become even greater when diabetes development was accelerated by CY injection. The iNOS mRNA is induced in macrophages, endothelial cells, and islet ß-cells by inflammatory cytokines such as IL-1ß and TNF{alpha} (39, 40). Many previous studies have suggested a possible role of TNF{alpha} in ß-cell destruction that activated macrophages lysed islet cells in vitro or in vivo via iNOS-dependent nitric oxide overproduction (41, 42). NOD mice given anti-IL-6 antibody had a significant reduction of incidence of diabetes and decreased severity of insulitis (43). The decreased expression of iNOS and IL-6 mRNA in the pancreas of NOD mice receiving PEG sTNF-RI indicates an inhibition of inflammatory processes and suggest a contribution of IL-6 and intraislet NO production to ß-cell destruction.

In summary, our results indicate that prophylactic treatment using PEGylated sTNF-RI in prediabetic NOD mice resulted in significant reductions in spontaneous diabetic incidence and mortality. Treatment using PEGylated sTNF-RI also reduced diabetic incidence in CY-accelerated diabetes in NOD mice. The protective effects of PEGylated sTNF-RI on autoimmune diabetes are associated with histological improvements and suppression of iNOS, IL-6 gene expression in pancreatic islets. We conclude that inhibition of the proinflammatory actions of TNF{alpha} by PEG sTNF-RI treatment provides strong protection of pancreatic ß-cells against cytokine destruction in NOD mice and that PEGylated sTNF-RI offers an alternative of early intervention in attenuating or even arresting the cytokine-induced islet destruction in autoimmune diabetes.


   Acknowledgments
 
We are grateful to Larry Ross and Sylvia Copon for their assistance in conducting the biochemical and hormonal assays.


   Footnotes
 
This work was supported by Amgen, Inc.

Abbreviations: CY, Cyclophosphamide; iNOS, inducible nitric oxide synthase; NOD, nonobese diabetic mouse; PEG, polyethylene glycol; sTNF-RI, soluble TNF receptor type I.

Received April 17, 2002.

Accepted for publication June 5, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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