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Transcriptional Regulation of Type I Diabetes by NF-B ¹

Salah-Eddine Lamhamedi-Cherradi^2,*, Shijun Zheng^*, Brendan A. Hilliard^*, Lingyun Xu^*, Jing Sun^*, Saaib Alsheadat^*, Hsiou-Chi Liou and Youhai H. Chen^3,*

^* Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; and Department of Medicine, Cornell University Medical College, New York, NY 10021

	Abstract

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Development of type I diabetes requires coordinated expression of myriad genes responsible for the initiation and progression of the disease. Expression of these genes are regulated by a small number of transcription factors including the Rel/NF-B family. To determine the roles of the Rel/NF-B family in type I diabetes, we studied multiple low-dose streptozotocin-induced diabetes in mice deficient in either c-Rel or NF-B1. We found that mice deficient in each of these NF-B subunits were resistant to streptozotocin-induced diabetes. However, the mechanisms of the disease resistance may differ in different cases. Deficiency in c-Rel selectively reduced Th1, but not Th2 responses, whereas NF-B1 deficiency had little effect on T cell responses to anti-CD3 stimulation. Death of dendritic cells was accelerated in the absence of NF-B1, whereas death of macrophages and granulocytes was affected primarily by c-Rel deficiency. Furthermore, Stat-1 expression was significantly reduced in macrophages deficient in NF-B1, but not c-Rel. These results indicate that both c-Rel and NF-B1are essential for the development of type I diabetes and that strategies targeting each of these subunits would be effective in preventing the disease.

	Introduction

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Type 1 diabetes is an autoimmune inflammatory disease of the pancreatic islets (1). In both human type I diabetes and rodent models of the disease, pancreatic cells that produce insulin are selectively destroyed by infiltrating inflammatory cells (2, 3). This is accompanied by coordinated expression of a myriad of inflammatory genes in the islets of Langerhans. Transcription factors that regulate the expression of inflammatory genes may, therefore, play critical roles in the development of diabetes.

The Rel/NF-B family of transcription factors comprises a group of related proteins that are expressed in various cell types including lymphocytes, dendritic cells (DCs), ⁴ monocytes/macrophages, granulocytes, and cells of the pancreatic islets. The mammalian Rel/NF-B members (NF-B1, NF-B2, RelA, RelB, and c-Rel) share a conserved amino terminus (Rel homology domain) that encompasses sequences essential for DNA binding, dimerization, and nuclear localization (4). In unstimulated cells, the majority of Rel/NF-B dimers reside in the cytoplasm as inactive complexes bound to inhibitor proteins, IBs. Upon stimulation, IB proteins can be phosphorylated by the Ikk complex (5) and degraded through the proteasome pathway (6, 7). Rel/NF-B dimers can then translocate into the nucleus and bind to specific decameric DNA sequences (the B elements) of target genes (8).

Recent studies using gene knockout (KO) mice suggest that different members of the Rel/NF-B family may be endowed with different functions. Thus, c-Rel KO mice develop normally and acquire a structurally normal immune system (9, 10). However, T cells derived from these mice produce reduced levels of IL-2, IL-3, IFN-, and GM-CSF, whereas B cells from these mice are more susceptible to apoptotic stimuli (10, 11, 12). Similarly, NF-B1 (p50/p105)-deficient mice do not suffer from developmental defects, but are more susceptible to intracellular and extracellular Gram-positive bacterial infections, and are partially compromised in their B cell response to LPS (13). Surprisingly, they are resistant to viral and Gram-negative bacterial infections (13). By contrast, mice deficient in RelA die in utero, presumably due to enhanced hepatocyte apoptosis (14, 15). RelB-deficient mice develop normally, but suffer from severe disorders ranging from splenomegaly to chronic microbial infections (11, 16). Similarly, NF-B2-deficient mice also suffer from severe immune disorders. Both their spleens and lymph nodes are bereft of B lymphocytes, undermining their capacity to form germinal centers (17, 18). These observations strongly suggest that members of the Rel/NF-B family perform non-overlapping functions, and that a loss-of-function mutation of a Rel/NF-B gene may not be fully compensated for by other members of this family.

Low-dose streptozotocin (STZ)-induced diabetes is an animal model for human type I diabetes (19). The disease can be induced in susceptible strains of mice by multiple injections of low-dose STZ (19, 20). Low-dose STZ-induced diabetes shares many clinical and histological features of human type I diabetes, and requires the participation of both T cells and macrophages (21). Although STZ may release NO upon degradation (22, 23), it is unclear whether the concentrations of reaction oxygen species produced by low-dose STZ are high enough to induce oxidative damage to islet cells. The molecular mechanisms of low-dose STZ-induced diabetes remain to be established. To explore the roles of Rel/NF-B in diabetes, we studied STZ-induced diabetes in c-Rel and NF-B1 deficient mice. We found that although both c-Rel and NF-B1 are essential for the development of diabetes, they are involved in regulating different arms of immune responses. These findings may have important ramifications for the treatment and prevention of type I diabetes.

	Materials and Methods

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Mice

C57BL/6 (B6) mice that carry a c-Rel gene mutation were generated as described previously (9). Normal B6 mice (H-2b), B6 x 129 F₂ (B6,129) mice (H-2b), and NF-B1-deficient B6,129 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were housed in the University of Pennsylvania animal care facilities under pathogen-free conditions.

Reagents and ELISA

STZ was purchased from Sigma-Aldrich (St. Louis, MO). The following reagents were purchased from BD PharMingen (San Diego, CA): purified rat anti-mouse IL-2, IL-4, IL-6, IL-10, IL-12, TNF-, and IFN- mAbs; recombinant mouse IL-2, IL4, IL-6, IL-10, IL-12, TNF-, and IFN-. ELISA for IL-2, IL-4, IL-6, IL-10, IL-12, TNF-, and IFN- was performed using paired mAbs specific for corresponding cytokines per manufacturer’s recommendations.

Induction and clinical evaluation of diabetes

Autoimmune diabetes was induced using multiple low doses of STZ as described (24). Briefly, 7- to 9-wk-old male mice were injected i.p. once a day for five consecutive days with 40 mg/kg of body weight of STZ dissolved in citrate buffer, pH 4.5. Mice were tested once every other day for urinary glucose levels using the keto-Diastix kit (Bayer, Elkhart, IN). Mice were considered diabetic if the urinary glucose levels equaled or exceeded 500 mg/dl on two consecutive tests. To determine the degree of insulitis, pancreata were fixed in 10% formalin, sectioned, stained with H&E, and examined by microscopy.

Bone marrow cell cultures

To prepare cells from bone marrow, femurs and tibiae were surgically removed and the marrow was flushed with complete RPMI medium. After removing erythrocytes, bone marrow cells were cultured at 10⁶/ml in complete RPMI medium containing 100 ng/ml GM-CSF and 500 U/ml IL-4 (R&D Systems, Minneapolis, MN). Five days later, cells in suspension were harvested, washed, and used for the studies described here.

Apoptosis studies

Bone marrow cells were first cultured in the presence of IL-4 and GM-CSF for 5 days as described above. Cells were then washed and incubated overnight without IL-4 or GM-CSF. Following staining with anti-CD11c, anti-Gr1, anti-Mac-1, and annexin V5, the degree of apoptosis of each cell type was determined by flow cytometry.

Measurement of nitrite

NO was determined by measuring the end product nitrite, using a method based on the Griess reaction. Briefly, aliquots of culture supernatant (100 µl) were mixed with 100 µl of Griess reagent at room temperature for 10 min. Absorbance was measured at 540 nm in an automated microplate reader. The concentration of nitrite was determined by reference to a standard curve of sodium nitrite. Culture medium was used as the blank.

Western blot

The cytosolic lysates of macrophages were fractionated by electrophoresis on a 12% PAGE and transferred to a nitrocellulose membrane. After blocking with 5% nonfat milk, the membrane was incubated with rabbit anti-Stat-1 (Upstate Biotechnology, Lake Placid, NY) or anti--actin, and HRP-labeled secondary Abs (Amersham Pharmacia Biotech, Piscataway, NJ). Color was developed using ECL Western blotting detection reagents (Amersham Pharmacia Biotech).

Statistical analysis

The significance of the differences in disease severity and immune parameters was determined by the Mann-Whitney U test and ANOVA, respectively.

	Results

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c-Rel^-/- and NF-B1^-/- mice are resistant to low-dose STZ-induced diabetes

To determine whether NF-B1 and c-Rel are required for the development of type I diabetes, wild-type control mice and mice homozygous for the c-Rel or NF-B1 gene mutation were treated daily with low-dose STZ (40 mg STZ per kg body weight) for 5 consecutive days (days 0–4) (21, 25, 26, 27, 28, 29). Diabetes was monitored by both urine glucose test and pancreatic islet histochemistry. Fig. 1 illustrates disease courses in c-Rel^-/-, NF-B1^-/-, and their respective control mice. Diabetes developed in the majority of control mice (>70%), starting 8 and 12 days after the first STZ injection in B6,129 and B6 mice, respectively. Remarkably, mutation in either c-Rel or NF-B1 gene strongly reduced the incidence of diabetes. Only 23% (3 of 13) of NF-B1-deficient mice and 29% (5 of 17) of c-Rel-deficient mice developed diabetes (Fig. 1).

View larger version (22K): [in this window] [in a new window]   FIGURE 1. c-Rel-/- and NF-B1-/- mice are resistant to STZ-induced diabetes. A, Normal (•, c-Rel+/+, n = 16) and c-Rel-deficient (, c-Rel-/-, n = 17) mice were injected with STZ to induce diabetes as described in Materials and Methods. Data presented are means ± SEM of diabetes incidence. The differences between the two groups are statistically significant (p < 0.0004) as determined by Mann-Whitney U test. B, Normal (, NF-B1+/+) and NF-B1-deficient (, NF-B1-/-) mice (n = 13) were injected with STZ to induce diabetes as described in Materials and Methods. Data presented are means ± SEM of diabetes incidence. The differences between the two groups are statistically significant (p < 0.0001) as determined by Mann-Whitney U test.

View larger version (152K): [in this window] [in a new window]   FIGURE 2. Histological profiles of pancreas. Mice were treated as in Fig. 1 and sacrificed at the end the experiments. Pancreata were collected, fixed in 10% formalin, and embedded in paraffin. Paraffin sections 5 µm thick were stained with H&E. A and B, Pancreata of c-Rel+/+ and NF-B1+/+ mice, respectively, with significant infiltration surrounding the islets. C and D, Pancreata of c-Rel-/- and NF-B1-/- mice, respectively, with little infiltration.

Development of low-dose STZ-induced diabetes requires participation of both T cells and APCs (21). To determine whether activation and differentiation of T cells were affected by c-Rel or NF-B1 deficiency, splenocytes from control, c-Rel-, or NF-B1-deficient mice were tested in vitro for their responses to anti-CD3 mAb or anti-CD3 plus anti-CD28 mAb. As shown in Fig. 3, splenocytes of control animals proliferated vigorously in response to anti-CD3 mAb or anti-CD3 plus anti-CD28 mAb, and produced both Th1 (IL-2 and IFN-) and Th2 (IL-4 and IL-10) cytokines. By contrast, splenocytes from c-Rel-deficient mice produced much reduced levels of IFN- and IL-2, and proliferated less vigorously than those of control mice (Fig. 3). However, both IL-4 and IL-10 production was significantly increased in c-Rel^-/- cultures (Fig. 3), suggesting that c-Rel may selectively inhibit Th1 but promote Th2 responses (30). In contrast to c-Rel^-/- splenocytes, NF-B1^-/- cells mounted relatively normal responses to anti-CD3 mAb or anti-CD3 plus anti-CD28 mAb (data not shown), suggesting that c-Rel, but not NF-B1, is essential for anti-CD3 responses. It should be pointed out that both c-Rel and NF-B1 are required for the development of T cell responses in immunized animals (30, 31). However, T cell priming in vivo requires not only T cells but also APCs (which express both c-Rel and NF-B1). Thus, c-Rel and NF-B1 may regulate T cell priming in vivo by either directly acting on T cells or indirectly acting on APC. By contrast, in vitro priming of T cells as shown in Fig. 3 may not require APC. This may explain why NF-B1 is required for T cell priming in vivo (31), but not in vitro.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. c-Rel-/- T cells are defective in their Th1 but not Th2 responses. Normal () and c-Rel-deficient () mice, were treated as in Fig. 1 and sacrificed at the end of the experiments. Splenocytes, 5 x 105/ml, were cultured in complete DMEM with or without 2 µg/ml anti-CD3 and 2 µg/ml anti-CD28. Culture supernatants were collected 40 h later and tested for cytokines by ELISA. For proliferation assays, cells were pulsed with [3H]thymidine at 48 h and radioactivity determined 16 h later. Results shown are means+SD of individual groups with five to seven mice per group. The differences between c-Rel+/+ and c-Rel-/- groups are statistically significant for all the cultures with anti-CD3 (p < 0.009). The experiments were repeated three times with similar results.

c-Rel^-/- and NF-B1^-/- DCs are defective in their cytokine responses

To determine whether c-Rel and NF-B1 gene mutations affect the functions of DCs, bone marrow-derived DCs were stimulated with different amounts of LPS and their cytokine responses determined by ELISA. As shown in Fig. 4, c-Rel^-/- and NF-B1^-/- DCs produced significantly less amounts of IL-12p40 and TNF- than control cells. Since IL-12 is required for Th1 cell differentiation, this finding may partially explain the selective defect of Th1 responses in c-Rel-deficient mice.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Both c-Rel-/- and NF-B1-/- DCs are defective in their cytokine responses. DC-enriched bone marrow cell cultures were prepared as described in Materials and Methods. All cells were cultured at 106/ml with or without 10–100 ng/ml of LPS. Culture supernatants were collected at 48 h, and IL-12p40 and TNF- concentrations were determined by ELISA. Results are representative of two experiments.

c-Rel^-/- and NF-B1^-/- macrophages have selective defects in their responses to IFN- and LPS

Since cell destruction in type I diabetes requires the participation of macrophages, we also determined the effect of c-Rel and NF-B1 deficiency on macrophage functions. As shown in Fig. 5, following IFN- and/or LPS stimulation, c-Rel^-/- peritoneal macrophages produced significantly less amounts of IL-6, IL-12p40, TNF-, and NO than control cells. Similar defects in IL-12 and NO production were observed in NF-B1^-/- macrophages, but the production of IL-6 and TNF- was not significantly affected by NF-B1 deficiency. Thus, c-Rel and NF-B1 deficiency altered not only the functions of T cells, but also those of DCs and macrophages.

View larger version (28K): [in this window] [in a new window]   FIGURE 5. Both c-Rel-/- and NF-B1-/- macrophages have selective defects in their cytokine responses. Macrophages from peritoneal cavity were prepared as described (39 ). All cells were cultured at 106/ml with or without 10 U/ml IFN- and 100 ng/ml LPS. Culture supernatants were collected at 48 h, and IL-6, IL-12p40, TNF-, and NO concentrations determined as described in Materials and Methods. Results are representative of two experiments.

Selective defect in apoptosis of c-Rel^-/- and NF-B1^-/- cells

To determine whether c-Rel and NF-B1 deficiencies affected the death of hematopoietic cells, we studied growth factor withdrawal-induced apoptosis of normal, c-Rel^-/-, and NF-B1^-/- cells. As shown in Fig. 6, >15% of bone marrow-derived DCs, macrophages, and granulocytes underwent apoptosis upon IL-4 and GM-CSF withdrawal. c-Rel deficiency significantly accelerated the death of granulocytes and macrophages but not DCs. By contrast, deficiency in NF-B1 had little effect on the death of granulocytes and macrophages but significantly enhanced apoptosis of DCs (Fig. 6). In contrast to these results, apoptosis of myeloid cells activated by 1–10 µg/ml LPS was not affected by either c-Rel or NF-B1 gene mutation (data not shown). Thus, the roles of c-Rel/NF-B1 in myeloid cell death appear to be cell and context dependent.

View larger version (15K): [in this window] [in a new window]   FIGURE 6. Regulation of leukocyte apoptosis by c-Rel and NF-B1. DCs, granulocytes, and macrophages were generated from bone marrow of c-Rel-/-, NF-B1-/-, and their respective control mice as described in Materials and Methods. Cells were then cultured overnight in the absence of GM-CSF and IL-4, and analyzed for apoptosis by annexin-V staining. Data shown are means + SD of individual groups with three mice per group.

NF-B1 regulates Stat-1 expression in macrophages

Both Rel/NF-B and Stat-1 are latent cytoplasmic transcription factors that can be activated by a variety of cytokines. IFN-, upon engaging its receptors, causes autophosphorylation of receptor-associated Janus kinases that phosphorylate and activate Stats. To explore the potential mechanisms of inhibition of cytokine secretion in c-Rel^-/- and NF-B1^-/- macrophages treated with IFN-/LPS (Fig. 5), we examined whether Stat-1 expression was normal in these cells. As shown in Fig. 7, resting macrophages expressed low levels of Stat-1 which were markedly increased by IFN-/LPS stimulation. NF-B1, but not c-Rel, deficiency significantly decreased the levels of Stat-1 expression. This result indicates that NF-B1 may act upstream of Stat-1 in macrophages to modulate cytokine production following IFN-/LPS stimulation. Since its deficiency does not significantly affect Stat-1 expression, c-Rel may regulate cytokine secretion through Stat-1-independent mechanisms. More investigation is needed to clarify this issue.

View larger version (39K): [in this window] [in a new window]   FIGURE 7. NF-B1 regulates Stat-1 expression in macrophages. c-Rel-/-, NF-B1-/-, and their respective control peritoneal macrophages were cultured with or without IFN- (10 U/ml) and LPS (100 ng/ml) for 48 h. The macrophages were then harvested following trypsinization, and the cytosolic protein extracts were analyzed by Western blot for their Stat-1 and -actin expression.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Development of low-dose STZ-induced diabetes requires the participation of macrophages and DCs as well as T cells. c-Rel and NF-B1 may regulate diabetes through acting on all these cell types. Recent reports indicate that both c-Rel and NF-B1 can modulate T cell responses to conventional Ags (30, 31). Results shown in Fig. 3 suggest that c-Rel also regulates polyclonal anti-CD3 responses with a strong bias toward Th1 cells, a finding consistent with our report in conventional Ag systems (30). With respect to DCs, a recent report demonstrated that p50 and p65 are involved in their development, whereas p50 and c-Rel are necessary for IL-12 production and survival in response to CD40 and TRANCE (34). In another report, c-Rel deletion in DC also disrupts the production of the p35 subunit of IL-12, but interestingly does not affect p40 levels (35). Additionally, although both c-Rel and p65 bind to the IL-12p40 promoter with comparable affinity, p40 expression is only affected in c-Rel KO but not in p65 KO macrophages in response to LPS and/or IFN- treatment (36, 37). Using freshly isolated cells, we showed that c-Rel deficiency had a general effect on IL-12, IL-6, TNF-, and NO production by DCs and macrophages (Figs. 4 and 5). Similarly, NF-B1-deficient myeloid cells also suffer from certain deficiencies in their cytokine responses. Thus, in addition to directly affecting T cells, c-Rel or NF-B1 deficiency may also indirectly diminish autoreactive T cell activation via APC. Our demonstration that NF-B1 and c-Rel may regulate apoptosis of different myeloid cells reinforces the view that members of the Rel/NF-B family play distinct roles that may not be compensated for by other members of this family. Thus, p50 protein appears to be more important in mediating anti-apoptotic effect in DCs, whereas c-Rel protein is more effective in protecting granulocytes and macrophages from cytokine deprivation-induced death.

Aside from promoting T cell activation, macrophages also play a direct role in the pathogenesis of STZ-induced diabetes. It has been suggested that STZ may induce NO production by macrophages through inhibiting NF-B activation (38). To explore the potential roles of c-Rel and NF-B1 in macrophage function, we studied peritoneal macrophage responses to IFN- and LPS. We found that loss of NF-B1 or c-Rel selectively reduced the production of IL-6 and NO. The increase in NO production during macrophage activation is most likely due to the induction of inducible NO synthase, one of the downstream targets of NF-B. However, although inducible NO synthase is known to be up-regulated during inflammatory or immune responses, it remains to be determined whether inflammatory cells and pancreatic islet cells both produce NO during type I diabetes.

Results presented in this paper support the following model of c-Rel/NF-B1 action in autoimmune diabetes. By directly binding to the corresponding DNA sequence (the B site) located in the promoter regions of target genes, c-Rel/NF-B1expressed by lymphocytes and cells of the innate immune system orchestrate autoimmune diabetes through two distinct pathways. First, c-Rel/NF-B1 expressed by the innate immune system regulate the activation, cytokine secretion, and apoptosis of myeloid cells. Second, c-Rel/NF-B1 expressed by the adaptive immune system regulates the activation and differentiation of islet-specific T cells. The combined effect of c-Rel/NF-B1 action in the immune system is, therefore, to promote formation, and to prevent resolution of inflammatory lesions in the islet. However, although c-Rel and NF-B1 may synergize with each other in this process, they also perform non-overlapping functions and deficiency in one cannot be compensated for by the other. Thus, effective treatment of autoimmune diabetes may require abrogation of all Rel/NF-B activities.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants AI50059, NS40188, and NS40447.

² Current address: Tanox, Inc., Houston, TX 77025.

³ Address correspondence and reprint requests to Dr. Youhai H. Chen, 653 BRB-II/III, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 421 Curie Boulevard, Philadelphia, PA 19104. E-mail address: yhc{at}mail.med.upenn.edu.

⁴ Abbreviations used in this paper: DC, dendritic cell; p50, NF-B1; KO, knockout; STZ, streptozotocin.

Received for publication June 3, 2003. Accepted for publication August 19, 2003.

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