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The Journal of Immunology, 2001, 167: 1461-1468.
Copyright © 2001 by The American Association of Immunologists

Dendritic Cells from Nonobese Diabetic Mice Exhibit a Defect in NF-{kappa}B Regulation Due to a Hyperactive I{kappa}B Kinase1

Donald J. Weaver, Jr.2,*, Brian Poligone2,{ddagger},§, Thi Bui*, Ussama M. Abdel-Motal*, Albert S. Baldwin, Jr.{dagger},{ddagger},§ and Roland Tisch3,{ddagger},*

* Department of Microbiology and Immunology, School of Medicine; {dagger} Department of Biology, {ddagger} Lineberger Comprehensive Cancer Center, and § Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC 27599


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin-dependent diabetes mellitus (IDDM) is characterized by the T cell-mediated destruction of insulin-producing {beta} cells. Accordingly, APCs, such as macrophage, have also been shown to be important in the disease process. However, the role(s) of dendritic cells (DCs) that exhibit potent APC function remains undefined in IDDM. Here we demonstrate that DCs derived from nonobese diabetic (NOD) mice, a model for IDDM, are more sensitive to various forms of stimulation compared with those from C57BL/6 and BALB/c mice, resulting in increased IL-12 secretion. This property is a consequence of hyperactivation of NF-{kappa}B, a transcription factor known to regulate IL-12 gene expression. Specifically, NOD DCs exhibit persistent hyperactivation of both I{kappa}B kinase and NF-{kappa}B in response to stimuli, in addition to selective degradation of I{kappa}B{epsilon}. Transfection of NOD DCs with a modified form of I{kappa}B{alpha} significantly reduced IL-12 secretion, suggesting that hyperactivation of NF-{kappa}B was in part responsible for increased IL-12 production. An enhanced capacity of NOD DCs to secrete IL-12 would be expected to contribute to the development of pathogenic Th1 (Tc1) cells during the diabetogenic response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Insulin-dependent diabetes mellitus (IDDM)4 is an autoimmune disease characterized by the T cell-mediated destruction of the pancreatic {beta} cells found within the islets of Langerhans. Studies in the nonobese diabetic (NOD) mouse, a model of IDDM, have revealed that both CD4+ and CD8+ T cells are required for infiltration and destruction of pancreatic islets (1). The events linked to defective {beta} cell tolerance within the T cell compartment remain largely ill defined, but clearly are influenced by multiple genetic and environmental factors. One contributing factor appears to be the breakdown of peripheral immunoregulation and subsequent development of {beta} cell-specific CD4+ Th1 and possibly CD8+ Tc1 cells. The events that drive this apparent skewing toward {beta} cell-specific CD4+ Th1 cell (and CD8+ Tc1 cell) differentiation are unclear (2). However, it is well established that APCs have a significant impact on Th cell subset differentiation by providing and determining the level of costimulatory signals and establishing the cytokine milieu at the time of T cell priming (3, 4, 5). Furthermore, a number of studies have demonstrated that macrophages from NOD mice exhibit several developmental and functional defects that are believed to influence disease progression (6, 7, 8, 9, 10, 11). These defects include an inability to effectively elicit regulatory T cell function in a syngeneic mixed lymphocyte reaction, and a propensity to secrete high levels of IL-12 relative to other strains of mice (6, 11).

Dendritic cells (DCs) are specialized APCs exhibiting a potent capacity to activate and influence the differentiation of naive T cells (12). DC development begins with progenitors found in the bone marrow that give rise to precursors that traffic to peripheral nonlymphoid tissues. As immature cells, DCs exhibit a high capacity to capture Ag. Upon activation by a host of stimuli, such as pathogens, inflammatory cytokines, or necrotic cells, immature DCs migrate into T cell-rich areas of the lymph nodes for final maturation (13, 14, 15). The maturation process results in a decreased ability to process Ag and a concomitant increase in the ability of DCs to activate naive T cells. The latter is enhanced by engagement of CD40 by CD40 ligand-expressing T cells, which leads to increased expression of MHC class II, costimulatory molecules CD80 and CD86, and secretion of cytokines and chemokines (12). IL-12 has also been reported to synergize in an autocrine manner with CD40 signaling to enhance the APC function of DCs (16). However, the molecular events that govern DC maturation and APC function are poorly understood. Because of the Th1 (and Tc1) cell dependence upon IL-12, DCs may contribute to the preferential development of pathogenic T effector cells in IDDM.

The NF-{kappa}B/Rel family of transcription factors is a potent mediator of inflammatory responses (17) and has been associated with various aspects of DC development and immunobiology (18, 19, 20, 21, 22). Interestingly, several studies have proposed a link between NF-{kappa}B and chronic inflammatory diseases such as rheumatoid arthritis (23). The NF-{kappa}B transcription factor is a dimer composed of the rel family of proteins. The prototypical NF-{kappa}B transcription factor is a heterodimeric complex consisting of a 50-kDa protein and a 65-kDa protein. In unstimulated cells, NF-{kappa}B is sequestered in the cytoplasm by the inhibitory proteins, I{kappa}B{alpha}, I{kappa}B{beta}, and I{kappa}B{epsilon} (17, 24). Following stimulation by inflammatory signals, the inhibitory proteins are phosphorylated by a multisubunit I{kappa}B kinase (IKK) and degraded by the 26S proteasome (17, 25). NF-{kappa}B then translocates to the nucleus and binds to consensus sequences in the promoters of several genes to initiate transcription (17, 21, 22).

Differences in the roles of the I{kappa}B proteins are likely to be important in the regulation of NF-{kappa}B. For example, degradation of I{kappa}B{alpha} is typically associated with transient activation of NF-{kappa}B, while degradation of I{kappa}B{beta} is associated with persistent activation of this transcription factor (26, 27). Although I{kappa}B{epsilon} is similar in structure to I{kappa}B{alpha} and I{kappa}B{beta}, its role in NF-{kappa}B regulation is poorly understood. I{kappa}B{epsilon} is known to bind p65/p50 heterodimers in the cytoplasm, and with certain stimuli is degraded, resulting in an increased nuclear translocation of NF-{kappa}B (28). However, unlike I{kappa}B{alpha}, which upon resynthesis removes NF-{kappa}B from the nucleus, I{kappa}B{epsilon} is unable to terminate NF-{kappa}B activity in this manner (29, 30).

A role for NF-{kappa}B in DC development has been shown by targeted deletion of the relB gene in mice, which significantly reduces the number of myeloid DCs (21, 31). In addition, NF-{kappa}B binding sites are found in the promoters of genes critical for DC function, such as IL-12 p40 and MHC class II molecules (32, 33). Furthermore, NF-{kappa}B is activated by a number of stimuli encountered by DCs, including engagement of CD40, IL-1, TNF-{alpha}, and bacterial products such as LPS. The current study demonstrates that DCs isolated from NOD mice are more sensitive to activational signals resulting in elevated IL-12 secretion compared with DCs prepared from C57BL/6 and BALB/c mice. The increased IL-12 secretion by NOD DCs was a direct result of enhanced nuclear translocation and transcriptional activity of NF-{kappa}B relative to DCs from C57BL/6 and BALB/c mice. In addition, hyperactivation of the IKK was detected in activated NOD DCs, resulting in enhanced degradation of I{kappa}B{epsilon}. Therefore, hyperactivation of NF-{kappa}B and increased production of IL-12 by NOD DCs may be a contributing factor in the apparent skewing toward Th1 (and Tc1) subset development observed in IDDM.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6J and BALB/cJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were maintained in specific pathogen-free conditions. NOD/LtJ mice were similarly housed and bred. Currently in our colony, IDDM develops in ~80% of NOD/LtJ female mice by 1 yr of age.

Preparation of primary DC

Male and female mice between 8 and 12 wk of age were used for the isolation of bone marrow- and spleen-derived DCs. Bone marrow DC precursors were obtained from the femurs of NOD, C57BL/6, and BALB/c mice. Following lysis of RBC, CD4 (mAb M1/42.3.9), CD8 (mAb HO2.2), MHC class II (mAb B21.2, anti-I-Ab,d and mAb 10.2.36, anti-I-Ag7), and B220 (mAb RA3-3A1/6.1)-positive cells were removed via complement-mediated lysis. The remaining cells were plated on six-well, low cluster plates in RPMI 1640 medium containing 10% FBS and penicillin/streptomycin (complete medium), 10 ng/ml murine IL-4 (PeproTech, Rocky Hill, NJ), and 10 ng/ml murine GM-CSF (PeproTech). On the second day of culture, nonadherent cells were replated and cultured as described above for 8 days. Splenic DCs were established by plating a spleen cell homogenate depleted of RBC on six-well low cluster plates in complete medium containing 10 ng/ml GM-CSF and 1 ng/ml murine TGF-{beta} (PeproTech). The culture medium was changed on day 7. Both bone marrow- and spleen-derived DCs were harvested on day 10 of the culture. Flow cytometric analysis demonstrated that both types of DCs expressed DEC-205, MHC class I, MHC class II, CD11c, CD80, CD86, and CD40, but not CD8{alpha}.

Fluorescence staining

The following mAbs used for fluorescence staining were purchased from PharMingen (San Diego, CA): anti-CD40 (clone HM40-3), FITC-anti-CD86 (clone GL1), FITC-anti-CD80 (clone 16-10A1), FITC anti-B220 PE-anti-H-2Db (clone KH95), PE-anti-H-2Kd (clone SF1-1.1); FITC-anti-CD11c (clone HL3), PE-anti-CD4 (clone L3T4), PE-anti-CD8 (clone 53-6.7), FITC-anti-CD44 (clone IM7), biotinylated anti-CD69 (clone H1.2F3), biotinylated anti-CD62L (clone MEL-14). mAb anti-I-Ad (clone MK-D6), and mAb anti-I-Ag7 (clone 10.2.36) were provided by E. P. Reich (Immunologic, Palo Alto, CA). mAb anti-DEC 205 clone NLDC-145 was provided by Dr. R. Johnston (University of North Carolina, Chapel Hill, NC). PE-anti-mouse and PE-streptavidin secondary reagents were purchased from PharMingen. Following staining, analysis was conducted on a FACScan (BD Biosciences, San Jose, CA) using Summit software (Cytomation, Ft. Collins, CO).

EMSA and Western blotting

DCs cultured in complete medium at 5 x 105 cells/well in a 24-well plate were stimulated with 100 ng/ml IL-12, 1 µg/ml anti-CD40 (mAb HM40-3; PharMingen), 1 µg/ml purified hamster IgM isotype control (G235-11; PharMingen), 50 µg/ml LPS, and 10 ng/ml murine TNF-{alpha} for specific periods of time. Nuclear and cytoplasmic extracts were prepared as described previously (31). For all experiments protein concentrations were determined using the BCA Protein Assay Reagent A following the manufacturer’s protocol (Pierce, Rockford, IL). EMSA were performed as described previously using the DNA probe: 5'-CAGGCTGGGGATTCCCATCTCCACAGTTTCACTTC-3', which contains the NF-{kappa}B binding site from the MHC class I H-2Kb gene (32). As controls, EMSAs were also performed using a double-stranded Oct-1 DNA probe (5'-TGTCGAATGCAAATCACTAGAA-3') from Santa Cruz Biotechnology (Santa Cruz, CA). Bands were visualized using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and densitometry was performed using ImageQuant 4.0 (Molecular Dynamics). For Western blotting, 100 µg whole cell lysate or nuclear extract was analyzed by SDS-PAGE using a 10% separating gel. Proteins were transferred to Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ) using a semidry transfer system and blocked overnight with 5% nonfat dried milk in PBS. Blots were probed with anti-I{kappa}B{alpha} (sc-371 and sc-847), anti-I{kappa}B{beta} (sc-969), or anti-I{kappa}B{epsilon} (sc-7155) rabbit polyclonal Abs obtained from Santa Cruz Biotechnology. Following incubation with HRP-labeled goat anti-rabbit secondary Ab (Amersham Pharmacia Biotech), blots were developed using ECL reagents (Amersham Pharmacia Biotech).

Transient transfections and luciferase assays

DCs were transfected using SuperFect transfection reagent (Qiagen, Valencia, CA) according to the manufacturer’s instructions for six-well plates. Briefly, a transfection mixture containing 1.0 µg DNA and 8 µl SuperFect reagent in a volume of 0.4 ml RPMI 1640 was added to 5 x 106 DCs for 3 h at 37°C. DCs were then washed and cultured overnight in complete medium. For luciferase assays, cells were transfected with the 3x{kappa}B-LUC wild-type and 3x{kappa}B-LUC mutated reporter plasmids containing three repeats of the wild-type or mutated {kappa}B sites from the MHC class I enhancer described previously (34). After 24 h cells were stimulated with 100 ng/ml IL-12, 1 µg/ml anti-CD40, or 1 µg/ml isotype control for specific time periods, and luciferase assays were performed as described previously (35).

IL-12 secretion assay

Bone marrow or splenic DCs were harvested on day 10 of culture and plated at 106 cells/well in a 24-well plate with 0.1 ml complete medium. Cells were stimulated with 100 ng/ml IL-12, 1 µg/ml anti-CD40, 1 µg/ml isotype control, 10 ng/ml TNF-{alpha}, or 1 µg/ml LPS for 24 h. DCs were washed five times with complete medium and incubated for 1 h. Supernatants were collected and assayed for the presence of IL-12 to insure that residual IL-12 from stimulation did not influence results. An additional 0.1 ml complete medium was added to DCs and incubated for 48 h before monitoring IL-12 production. In some experiments 5 x 106 DCs were transfected with either 1 µg pCMV4-I{kappa}B{alpha}-SR or control pCMV4 as described above. After 24 h DCs were washed, plated, and stimulated with 100 ng/ml IL-12 or 1 µg/ml anti-CD40 Ab as described above. An anti-IL-12 p70 ELISA kit (Endogen, Woburn, MA) was used to detect the presence of biologically active IL-12 p70 in culture supernatants.

IKK assay

DCs (5 x 106) were stimulated for various periods of time with 1 µg/ml anti-CD40 Ab and whole cell lysates were prepared. IKK signalsome was immunoprecipitated using rabbit polyclonal anti-IKK-1 (sc-1783; Santa Cruz Biotechnology) or mAb CT-2 anti-IKK-2 (Signal Pharmaceuticals, San Diego, CA). In vitro kinase experiments were performed by incubating 0.25 µg immunoprecipitated proteins with 4 µg I{kappa}B{alpha}-GST or I{kappa}B{beta}-GST substrates. Reactions were incubated for 60 min at 30°C with 0.4 µCi [{gamma}-32P]ATP in kinase buffer. Kinase reactions were terminated upon addition of SDS-PAGE sample buffer and were analyzed by SDS-PAGE. Bands were visualized by autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Enhanced secretion of IL-12 by NOD DCs

Currently, the events that preferentially promote {beta} cell-specific CD4+ Th1 (and possibly CD8+ Tc1) subset development in IDDM are poorly understood. The potent capacity of DCs to activate naive T cells while providing a source of IL-12 prompted us to investigate a possible role for these APCs in {beta} cell-specific T cell development. Bone marrow was isolated from NOD, C57BL/6, and BALB/c mice and was cultured short term under conditions that promote the development of immature myeloid DCs as determined by morphology and flow cytometry (see Materials and Methods). DCs from all strains expressed high levels of CD11c and CD11b. In more than five independent flow cytometry analyses cells were 95–99% CD11c+CD11b+CD8{alpha}-. There were no significant differences in expression levels of various cell surface proteins such as CD80, CD86, and CD40 on DCs prepared from the three strains of mice (Table IGo). DCs prepared from NOD, C57BL/6, and BALB/c mice were then stimulated with IL-12, anti-CD40 Ab, TNF-{alpha}, or LPS, and biologically active IL-12 p70 secretion was measured. NOD-derived DCs secreted significantly higher levels of IL-12 than either C57BL/6 or BALB/c DCs when stimulated with IL-12, anti-CD40 Ab, and LPS (Fig. 1Go). NOD DCs also exhibited an increase in IL-12 secretion vs C57BL/6 and BALB/c DCs when stimulated with TNF-{alpha} (Fig. 1GoC). In addition, the sensitivity of NOD DCs to anti-CD40 Ab and IL-12 stimulation was enhanced (Fig. 1Go, A and B). For example, 1.5 ng/ml IL-12 was detected in cultures of NOD DCs treated with 2.0 µg/ml anti-CD40 Ab (Fig. 1GoB). In contrast, 20 µg/ml anti-CD40 Ab was required to detect ~1.5 ng/ml IL-12 in the C57BL/6 and BALB/c DC cultures (Fig. 1GoB). To determine whether NOD DCs found in the periphery also exhibited enhanced IL-12 secretion upon stimulation, myeloid-derived DCs were cultured short term from the spleen. As demonstrated in Fig. 1GoD, NOD splenic DCs secreted 5.2-fold more IL-12 than BALB/c splenic DCs following LPS stimulation.


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  		Table I. Phenotypic analysis of bone marrow-derived DCs1

 


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  		FIGURE 1. NOD DCs exhibit enhanced IL-12 secretion, Bone marrow-derived (A–C) or splenic-derived (D) DCs prepared from 8- to 10-wk-old NOD, C57BL/6, and BALB/c female mice and cultured for 10 days were stimulated with various concentrations of murine IL-12 (A), anti-CD40 Ab or isotype control (B), 10 ng/ml TNF-{alpha} and 1.0 µg/ml LPS (C), or 50 µg/ml of LPS (D). Supernatants were harvested, and IL-12 p70 secretion was determined by ELISA. Data represent the mean values (±SEM) of triplicate wells and are representative of two independent experiments. *, A t test confirmed that differences between NOD DCs stimulated with LPS vs C57BL/6 and BALB/c DCs (C) or BALB/c DCs (D) were significant (p < 10-3).

 
Increased and persistent NF-{kappa}B nuclear translocation in NOD-derived DCs

The promoter for the IL-12 p40 gene contains two NF-{kappa}B binding sites, including an NF-{kappa}B half-site plus an NF-{kappa}B binding site within the Ets element (32, 36). Accordingly, we investigated whether the enhanced IL-12 secretion exhibited by NOD DCs was due to differences in regulation of NF-{kappa}B. The DNA binding activity of NF-{kappa}B in nuclear extracts isolated from DCs stimulated with IL-12, anti-CD40 Ab, and TNF-{alpha} was assessed using an EMSA. The different treatments induced NF-{kappa}B nuclear translocation at 15 min (Fig. 2GoA). At this time point, DNA binding of nuclear NF-{kappa}B was 3- to 6-fold higher in extracts prepared from NOD DCs than in extracts isolated from C57BL/6 or BALB/c DCs regardless of the type of stimulation (Fig. 2GoA). As an internal control, OCT-1 DNA binding activity was also monitored via EMSA. In contrast to NF-{kappa}B DNA binding activity, the amount of OCT-1 binding was not altered upon stimulation (Fig. 2GoA). More importantly, OCT-1 binding activity was similar when DC lysates from NOD, C57BL/6, and BALB/c mice were compared (Fig. 2GoA). Elevated NF-{kappa}B activation in NOD DCs was observed in male and female mice and was independent of age and disease progression. Treatment of NOD splenic DCs with LPS also led to significantly enhanced levels of NF-{kappa}B nuclear translocation at various time points compared with splenic DCs prepared from BALB/c mice (Fig. 2GoB).



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  		FIGURE 2. Increased nuclear translocation of NF-{kappa}B in NOD DCs. A, Bone marrow-derived DCs from NOD, C57BL/6, and BALB/c were stimulated with 10 ng/ml TNF-{alpha}, 100 ng/ml IL-12, or 1 µg/ml anti-CD40 Ab for 15 min. Nuclear extracts were prepared and analyzed by EMSA using an NF-{kappa}B DNA probe. As a control, extracts were also analyzed using an OCT-1 DNA probe. Data are representative of at least three independent experiments. B, Splenic-derived DCs from NOD and BALB/c mice were stimulated with 50 µg/ml LPS for various times, and nuclear extracts were examined via EMSA.

 
The kinetics of NF-{kappa}B activation were assessed following stimulation by IL-12 or anti-CD40 Ab by EMSA. Nuclear translocation of NF-{kappa}B was evident even 48 h after the addition of IL-12 or anti-CD40 Ab in lysates prepared from NOD DCs (Fig. 3Go). In contrast, a decrease in the levels of nuclear NF-{kappa}B in lysates prepared from C57BL/6 and BALB/c mice was detected at 4 and 12 h following IL-12 and anti-CD40 Ab stimulation, respectively (Fig. 3Go). Using Abs specific for each Rel family member, the components of NF-{kappa}B complexes induced by IL-12, anti-CD40 Ab, and TNF-{alpha} stimulation were determined by supershift analysis. NF-{kappa}B complexes were composed of multiple members of the Rel/NF-{kappa}B family of proteins, including RelB, p50, p52, and RelA. Furthermore, no qualitative differences in these nuclear components were observed among DCs from the three strains (data not shown).



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  		FIGURE 3. Persistent activation of NF-{kappa}B in NOD DCs. Bone marrow-derived DCs from NOD, C57BL/6, and BALB/c mice were stimulated with 100 ng/ml IL-12 or 1 µg/ml anti-CD40 Ab for various periods of time. Nuclear translocation of NF-{kappa}B was analyzed by EMSA as described in Materials and Methods.

 
Increased and persistent transcriptional activity of NF-{kappa}B in NOD-derived DCs

To correlate the differences in NF-{kappa}B DNA binding activity with transcriptional activity, DCs were transfected with luciferase reporter plasmids to monitor the transcriptional activity of NF-{kappa}B. Following transfection, DCs were stimulated with IL-12 or anti-CD40 Ab, and luciferase activity was monitored over a 48-h period. IL-12 (Fig. 4GoA) as well as anti-CD40 Ab (Fig. 4GoB) stimulation induced transcriptional activity of NF-{kappa}B, and in agreement with EMSA results, luciferase activity was consistently 3- to 4-fold higher throughout the experiment in NOD DCs compared with C57BL/6 and BALB/c DCs (Fig. 4Go). Twelve hours following IL-12 stimulation, the transcriptional activity of NF-{kappa}B declined 2-fold in C57BL/6 and BALB/c DCs, whereas activity remained elevated in NOD DCs (Fig. 4GoA).



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  		FIGURE 4. Increased NF-{kappa}B-dependent transcription in NOD DCs. A luciferase reporter plasmid containing four upstream NF-{kappa}B sites was transfected into NOD, C57BL/6, and BALB/c bone marrow-derived DCs. After 24 h, DCs were stimulated with either 100 ng/ml IL-12 (A) or 1 µg/ml anti-CD40 Ab (B) for varying times, and luciferase activity was measured on a luminometer. The luciferase activity of DCs transfected with reporter plasmids containing mutated NF-{kappa}B sites was between 600 and 800 relative luciferase units. Data represent the mean values (±SEM) of triplicate experiments.

 
NF-{kappa}B regulates enhanced IL-12 secretion of NOD DCs

The above observations suggest that dysregulation of NF-{kappa}B activation in NOD DCs may contribute to increased IL-12 secretion. To provide evidence that NF-{kappa}B indeed has a direct role in the secretion of IL-12 from DCs, NOD DCs were transfected with an expression vector encoding I{kappa}B{alpha} (SS32/36AA) super-repressor (I{kappa}B{alpha}-SR), in which serines 32 and 36 have been substituted with alanines. This form of I{kappa}B{alpha} cannot be phosphorylated and degraded by the 26S proteasome, preventing NF-{kappa}B nuclear translocation and transcriptional activity (17). Cells expressing the I{kappa}B{alpha}-SR that were treated with TNF-{alpha} exhibited a luciferase activity similar to or below that in untreated cells (data not shown). Typically, transfection efficiencies of up to 60% were obtained as determined by flow cytometric analysis of DCs transfected with plasmid DNA encoding green fluorescent protein. As demonstrated in Fig. 5Go, transfection of the I{kappa}B{alpha}-SR significantly reduced IL-12 secretion upon anti-CD40 Ab or IL-12 stimulation relative to DCs transfected with the control pCMV4 or not and treated with IL-12.



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  		FIGURE 5. I{kappa}B{alpha}-SR inhibits IL-12 production by DCs. Bone marrow-derived DCs were transfected with 1 µg I{kappa}B{alpha}-SR (pCMV4-SR) or control pCMV4. After 24 h DCs were stimulated with 100 ng/ml IL-12 (A) or 1 µg/ml anti-CD40 mAb (B). Supernatants were harvested, and IL-12 production measured by ELISA. Data represent the mean values (±SEM) of triplicate wells and are representative of duplicate experiments. *, A t test confirmed that differences between DCs transfected with pCMV4-SR vs nontransfected or pCMV4-transfected DCs were significant (p < 10-3).

 
I{kappa}B{epsilon} exhibits enhanced degradation after stimulation in NOD DCs

To determine the mechanism responsible for hyperactivation of NF-{kappa}B in NOD DCs, whole cell extracts were analyzed by Western blot using Abs specific for the I{kappa}B inhibitory proteins. I{kappa}B{alpha} and I{kappa}B{beta} levels were similar among DCs prepared from the three strains of mice. I{kappa}B{alpha} showed a characteristic degradation at 30 min and was resynthesized by 4 h, whereas I{kappa}B{beta} was unaltered throughout the time period (data not shown). In contrast, I{kappa}B{epsilon} was degraded after anti-CD40 Ab and IL-12 stimulation in NOD DCs, whereas C57BL/6 and BALB/c DCs showed no loss of expression (Fig. 6Go). Degradation of I{kappa}B{epsilon} was observed at 30 min following stimulation and was completely resynthesized after 12–24 h. These data indicate that stimulation of NOD DCs with anti-CD40 Ab and IL-12 leads to an initial degradation of I{kappa}B{alpha} and I{kappa}B{epsilon}, and that only I{kappa}B{alpha} is degraded in C57BL/6 and BALB/c DCs. Therefore, persistent degradation of I{kappa}B{epsilon} may contribute to the increased NF-{kappa}B activity in NOD DCs.



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  		FIGURE 6. I{kappa}{beta}{epsilon} is degraded after stimulation in NOD DCs. Bone marrow-derived DCs from NOD, C57BL/6, and BALB/c mice were stimulated with 1 µg/ml anti-CD40 mAb or 100 ng/ml IL-12 for various periods of time. NOD, C57BL/6, and BALB/c DC lysates were probed with Abs specific for I{kappa}B{epsilon} via Western blot. Data are representative of three independent experiments.

 
IKK isolated from stimulated NOD DCs exhibits enhanced and persistent activity in vitro

Because EMSAs suggested that the nuclear levels of NF-{kappa}B were higher in NOD DCs relative to BALB/c- and C57BL/6-derived DCs after stimulation, and because I{kappa}B{epsilon} was degraded in NOD, but not other strains, the activities of the IKK complex in DCs from each mouse strain were compared. In this experiment DCs were stimulated with 1 µg/ml anti-CD40 Ab for specific periods of time, and IKK-1 or IKK-2 was immunoprecipitated from whole cell lysates. The kinase activity of the respective IKKs was assessed by measuring phosphorylation of an I{kappa}B{alpha}-GST substrate. Consistent with hyperactivation of NF-{kappa}B and increased I{kappa}B{epsilon} degradation, both IKK-1 and IKK-2 immunoprecipitated from NOD DCs demonstrated higher activity than complexes isolated from C57BL/6 and BALB/c DCs (Fig. 7Go). Furthermore, the activity of IKK complexes derived from C57BL/6 and BALB/c DCs declined after 30 min, whereas the activity of NOD-derived kinases remained elevated after 60 min.



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  		FIGURE 7. Elevated levels of IKK kinase activity in NOD DCs following activation. Bone marrow-derived DCs from NOD, C57BL/6, and BALB/c mice were stimulated with 1 µg/ml anti-CD40 Ab for various periods of time. IKK signalsome was immunoprecipitated using mAb anti-IKK1 or mAb anti-IKK2. In vitro kinase reactions were performed by preincubating 250 µg immunoprecipitated protein with 4 µg I{kappa}B{alpha}-GST and [{gamma}-32P]ATP. Kinase reactions were analyzed by SDS-PAGE and visualized by autoradiography.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As critical mediators of T cell activation and differentiation, APCs are likely to contribute to the immune dysregulation observed in IDDM by shaping the local microenvironment through the secretion of cytokines and by providing crucial cell-to-cell contacts, including CD80, CD86, and CD40. The role of macrophages in IDDM has been studied extensively, and several reports suggest that macrophages isolated from NOD mice possess multiple developmental and functional defects (6, 7, 8, 10, 11). In part, these defects have been associated with an inability of NOD macrophages to provide efficient costimulation required by regulatory T cells, and an increased capacity to secrete IL-12 relative to other strains of mice. In contrast to macrophages, the role of DCs in IDDM has only recently been addressed. In a transgenic model system, adoptively transferred DCs pulsed with a neo-self-Ag expressed specifically in the {beta} cells of recipient mice activated the corresponding T cells and promoted the development of diabetes (9). Furthermore, DCs primed with TNF-{alpha} accelerated the progression of diabetes in neonatal NOD mice (37). Additional experiments have shown that DCs are an early source of TNF-{alpha} in the NOD islet infiltrate, which may be a key contributing factor for the initiation of {beta} cell destruction (38).

The present study provides the first evidence that NOD DCs prepared from the bone marrow and spleen have specific signaling defects. Namely, NOD DCs were found to be more sensitive to IL-12, anti-CD40 Ab, and LPS stimulation, resulting in enhanced levels of IL-12 secretion compared with DCs from BALB/c and C57BL/6 mice (Fig. 1Go). Interestingly, the greatest difference in IL-12 sensitivity and subsequent secretion of IL-12 was observed between NOD and BALB/c DCs. It is well established that BALB/c mice have a genetic propensity toward the development of Th2 cell reactivity (36, 39, 40). Consequently, the relative insensitivity of BALB/c DCs to IL-12 may in part limit Th1 cell subset development.

The observation that NOD DCs are more sensitive than BALB/c and C57BL/6 DCs to various forms of stimulation resulting in elevated levels of IL-12 secretion is consistent with the observed hyperactivation of NF-{kappa}B. Previous analysis has identified multiple NF-{kappa}B binding sites within the IL-12 p40 chain gene promoter (32). Consequently, enhanced NF-{kappa}B activation and subsequent nuclear translocation would be expected to increase transcription of the IL-12 p40 chain gene. Indeed, NF-{kappa}B-dependent transcription of a luciferase reporter construct was significantly higher in NOD vs BALB/c or C57BL/6 DC transfectants (Fig. 4Go). Furthermore, transfection of the I{kappa}B{alpha}-SR resulted in a marked reduction in IL-12 secretion, demonstrating that NF-{kappa}B has a direct role in the observed increase in IL-12 expression by activated NOD DCs (Fig. 5Go, A and B). Interestingly, peritoneal exudate macrophages prepared from NOD mice have been reported to secrete elevated levels of IL-12 upon activation relative to macrophages prepared from a variety of mouse strains (6). Consistent with this finding, we have preliminary data indicating that NOD macrophages prepared from the spleen also exhibit significantly enhanced levels of NF-{kappa}B activation following stimulation compared with splenic BALB/c and C57BL/6 macrophages (R. Tisch, unpublished observations). Currently studies are underway to determine whether the defects in the regulation of NF-{kappa}B activity detected in DCs and macrophage are NOD specific, or whether strains of mice, such as NOR or SWR, that are genetically more similar also exhibit similar defects. Interestingly, preliminary results indicate that bone marrow-derived DCs prepared from NOD mice have significantly enhanced levels of NF-{kappa}B compared with NOR DCs following stimluation with 50 µg/ml LPS or 5 µg/ml anti-CD40 Ab. Furthermore, nuclear levels of NF-{kappa}B detected in NOR DCs treated with LPS or anti-CD40 Ab are similar to those in BALB/c and C57BL/6 DCs. Moreover, NOD DCs secrete increased levels of IL-12 after 50 µg/ml LPS stimulation compared with NOR DCs. These results suggest that dysregulation of NF-{kappa}B and enhanced IL-12 secretion may be common properties of NOD APCs.

Associated with the increased nuclear translocation of NF-{kappa}B in NOD DCs was the preferential degradation of I{kappa}B{epsilon}. Currently, the roles of the individual I{kappa}B inhibitory proteins in regulating NF-{kappa}B activity are not fully understood. Previous reports indicate that I{kappa}B{epsilon} is degraded after stimulation, resulting in nuclear translocation of NF-{kappa}B. However, I{kappa}B{epsilon} is inefficient in terminating NF-{kappa}B transcription (27, 29). The susceptibility of I{kappa}B{epsilon} to enhanced kinase activity could contribute to the observed hypersensitivity of NOD DCs to external stimuli (Fig. 1Go). Consequently, not only may NOD DCs be predisposed to heightened levels of activation, but they are also less efficient at controlling NF-{kappa}B activity due to the inability of I{kappa}B{epsilon} to translocate to the nucleus and dislodge NF-{kappa}B from consensus sites (Figs. 1Go, 2Go, and 6Go). These results support the idea that although I{kappa}B inhibitory proteins are structurally similar, subtle biochemical differences, such as additional residues found in the amino terminus of I{kappa}B{epsilon}, may mediate distinct regulatory effects (26, 29).

We are uncertain why IKK hyperactivation results in enhanced degradation of I{kappa}B{epsilon}, but not I{kappa}B{alpha} or I{kappa}B{beta}. The defect resulting in IKK hyperactivity may, for example, lead to preferential phosphorylation of I{kappa}B{epsilon}. Alternatively, persistent hyperactivation of NF-{kappa}B in NOD DCs may have resulted in an adaptive response in the regulation of I{kappa}B{alpha} or I{kappa}B{beta}. It is also possible that the sensitivities of our assays are unable to detect a difference in I{kappa}B{alpha} degradation or recovery after stimulation. Interestingly, a recent study proposed that NF-{kappa}B activation is suppressed in NOD splenocytes due to a defect in large multifunctional proteasome (LMP) 2 and associated proteasome activity (41, 42). Although our current data do not agree with these findings, it is possible that the degradation I{kappa}B{alpha} or I{kappa}B{beta} is preferentially reduced due to a defect in the LMP2-deficient proteasome. However, others have questioned whether NOD splenocytes do, in fact, exhibit reduced levels of LMP2 (43, 44). We are currently exploring whether a defect in LMP2 in NOD DCs affects DC function.

The elevated levels of NF-{kappa}B activation in NOD DCs following stimulation coincided with increased and persistent activation of the IKK complex (Fig. 5Go). The upstream event(s) leading to increased NOD IKK complex activation is currently not clear. Mitogen-activated protein kinase/extracellular signal regulatory kinase kinase, NF-{kappa}B-inducing kinase, and TNFR-associated factor proteins have been proposed to activate the IKK complex (17, 45). However, a recent study has demonstrated that DCs prepared from alymphoplasia mice that lack functional NF-{kappa}B-inducing kinase continue to secrete IL-12 and stimulate T cells indistinguishable from mice heterozygous for the genetic mutation when stimulated via engagement of CD40 (46). Therefore, a defect(s) in mitogen-activated protein kinase/extracellular signal regulatory kinase kinase- or TNFR-associated factor-dependent signal transduction may be associated with IKK complex activation in NOD DCs following CD40 engagement and possibly other NF-{kappa}B-dependent signaling events.

The function of the IKK signalosome is another important consideration that must be explored. It has been recently shown that fibroblasts from relB-/- mice have enhanced IKK activity after stimulation with LPS (47). These findings suggest that RelB may inhibit IKK activity after stimulation. Although we have observed no deficiencies in RelB levels in gel shift experiments (data not shown), it is still possible that functional defects in RelB could contribute to the hyperactivation of IKK in NOD DCs. Additionally, A20 has been shown to associate with the IKK signalosome and is an inhibitor of NF-{kappa}B activity (48, 49). Therefore, examination of the various proteins important in IKK activity will be necessary to determine whether an alleleic variant in NOD is responsible for the hyperactivation of IKK.

Overall, our results are consistent with the idea that NOD DCs may contribute to the development of {beta} cell-specific Th1 and Tc1 cells in vivo. The enhanced sensitivity of NOD DCs to various conditions, but specifically to IL-12, could significantly amplify existing levels of IL-12 within the local immune environment, preferentially favor Th1 (and Tc1) differentiation, and contribute to the development of pathogenic CD4+ and CD8+ effector T cells (16). A higher capacity of NOD DCs to secrete IL-12 and other NF-{kappa}B-dependent cytokines may also compensate for relatively weak binding interactions that occur between I-Ag7 and {beta} cell-specific peptides, which, in turn, would be predicted to limit the efficiency of T cell activation. Indeed, preliminary experiments have demonstrated that NOD DCs have an enhanced capacity to stimulate naive CD8+ T cells in a peptide-specific manner (B. Poligone and R. Tisch, manuscript in preparation). A protective role for NOD DCs cannot be ruled out. For example, high levels of IL-12 may feed into an immunoregulatory loop, resulting in increased levels of IFN-{gamma} secretion by peripheral NK and NKT cells, which has been proposed to have a protective effect in IDDM (50). In addition, recent studies have shown that adoptive transfer of bone marrow (51) or splenic (52) DCs into young NOD recipients can prevent diabetes. It is also important to consider whether other DC subsets have defects in NF-{kappa}B signaling. Myeloid DCs secrete less IL-12 than lymphoid DCs, yet appear to be more immunostimulatory (53). It is possible that the defect in NF-{kappa}B we have uncovered is myeloid specific, enhancing their immunostimulatory function. Nevertheless, the roles of the various subsets remain uncertain, and possible defects in NF-{kappa}B must be explored.

Initiation and progression of IDDM involve a complex set of events regulated by a number of largely unidentified genes and environmental factors. We have found that DCs prepared from the bone marrow and spleen of NOD mice exhibit dysregulation of NF-{kappa}B activation, which results in enhanced IL-12 secretion. The precise events associated with the observed hyperactivation of NF-{kappa}B remain to be defined, but such studies should provide further insight into the general regulation of NF-{kappa}B and the IKK complex. Furthermore, our results provide evidence that NOD DCs may be key contributors to the apparent skewing of {beta} cell-specific Th1 and Tc1 cell development. However, whether specific subsets of DCs exhibit this defect and, in turn, the relative contribution of NOD DCs to disease progression have yet to be ascertained. Nevertheless, based on these results and our preliminary findings in NOD macrophages, hyperactivation of NF-{kappa}B may prove to be an important factor impacting on the function of APCs in autoimmune diabetes.


   Acknowledgments
 
We thank Drs. Denis Guttridge and Arndt Schottelius for their technical assistance.


   Footnotes
 
1 This work was supported in part by Grant 1-P60-DE 13079 from the National Institute of Dental and Craniofacial Research and Grant 5RO1-DK-52365 from the National Institute of Diabetes and Digestive and Kidney Diseases. D.J.W. was supported in part by National Institute of Allergy and Infectious Disease Training Grant 5-T32-AI07273. A.S.B. and B.P. were supported in part by Grant AI35098. Back

2 D.J.W. and B.P. contributed equally. Back

3 Address correspondence and reprint requests to Dr. Roland Tisch, Department of Microbiology and Immunology, 635 Mary Ellen Jones Building, CB#7290, University of North Carolina, Chapel Hill, NC 27599-7290. E-mail address: rmtisch{at}med.unc.edu Back

4 Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; DC, dendritic cell; IKK, I{kappa}B kinase; NOD, nonobese diabetic; LMP, large multifunctional proteasome. Back

Received for publication March 2, 2001. Accepted for publication May 17, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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