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NF-B Hyperactivation Has Differential Effects on the APC Function of Nonobese Diabetic Mouse Macrophages¹

Pradip Sen^*, Sandip Bhattacharyya^2,*, Mark Wallet^2,*, Carmen P. Wong^*, Brian Poligone, Maitreyee Sen^*, Albert S. Baldwin, Jr. and Roland Tisch^3,*,

^* Department of Microbiology and Immunology, School of Medicine, and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599

	Abstract

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Type 1 diabetes is characterized by a chronic inflammatory response resulting in the selective destruction of the insulin-producing cells. We have previously demonstrated that dendritic cells (DCs) prepared from nonobese diabetic (NOD) mice, a model for spontaneous type 1 diabetes, exhibit hyperactivation of NF-B resulting in an increased capacity to secrete proinflammatory cytokines and stimulate T cells compared with DCs of nondiabetic strains of mice. In the current study, the activational status of NF-B and its role in regulating the APC function of macrophages (M) prepared from NOD, nonobese resistant (NOR), and BALB/c mice was investigated. Independent of the stimulus, splenic and bone marrow-derived M prepared from NOD mice exhibited increased NF-B activation relative to NOR and BALB/c M. This hyperactivation was detected for different NF-B complexes and correlated with increased IB degradation. Furthermore, increased NF-B activation resulted in an enhanced capacity of NOD vs NOR or BALB/c M to secrete IL-12(p70), TNF-, and IL-1, which was inhibited upon infection with an adenoviral recombinant encoding a modified form of IB. In contrast, elevated NF-B activation had no significant effect on the capacity of NOD M to stimulate CD4⁺ or CD8⁺ T cells in an Ag-specific manner. These results demonstrate that in addition to NOD DCs, NOD M exhibit hyperactivation of NF-B, which correlates with an increased ability to mediate a proinflammatory response. Furthermore, NF-B influences M APC function by regulating cytokine secretion but not T cell stimulation.

	Introduction

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Type 1 diabetes (T1D)⁴ is characterized by the selective destruction of the insulin-producing cells found in the islets of Langerhans. Both CD4⁺ and CD8⁺ T cells have been shown to be the primary mediators of cell destruction in the nonobese diabetic (NOD) mouse, a spontaneous model of T1D (1, 2, 3). This chronic inflammatory response is in part driven by skewing of cell-specific CD4⁺ T cells toward Th1 differentiation, and presumably Tc1 development for CD8⁺ T cells. Currently, the key events promoting preferential differentiation of autoreactive Th1 (and Tc1) cells in the periphery are poorly understood. It is well established that professional APCs such as macrophages (M), dendritic cells (DCs), and B cells have a critical role influencing the nature and magnitude of T cell immunity. Processing and presentation of Ag and secretion of cytokines by APCs influence T cell activation and subset differentiation (4, 5, 6). Indeed, various reports have provided evidence that all three types of APC contribute to the initiation and progression of cell autoimmunity (7, 8, 9, 10). A key role for M in T1D, for example, has been demonstrated by studies in which disease progression is blocked in NOD mice by either depleting M or inactivation of M (11, 12, 13). In addition, M were shown to be essential for the in vivo diabetogenicity of CD8⁺ T cells expressing a transgenic TCR specific for a cell Ag (14).

Notably, M prepared from NOD mice exhibit developmental and functional defects that are thought to promote the development of pathogenic T cells (15, 16, 17, 18, 19, 20). For instance, fewer mature M develop in vitro from the bone marrow of NOD vs nonautoimmune strains of mice, which in turn has been correlated with an inability of NOD M to effectively stimulate a syngeneic mixed lymphocyte reaction (17, 18). The latter has been interpreted to indicate a reduced capacity of NOD M to stimulate regulatory T cell differentiation and/or effector function. Furthermore, peritoneal exudate cells (PECs) prepared from thioglycollate-treated NOD mice secrete elevated levels of IL-12 compared with nonautoimmune strains of mice (15, 20). High levels of IL-12 secreted by NOD M would be expected to enhance Th1/Tc1 cell differentiation.

M have also been implicated in directly mediating cell destruction. For instance, studies have shown that M and DCs are the first cells to infiltrate the pancreas of NOD mice (12, 21, 22, 23). Furthermore, proinflammatory cytokines such as IL-1, IFN-, and TNF- and other soluble mediators including oxygen free radicals produced by activated M are directly or indirectly cytotoxic to cells in vitro (24, 25, 26). Moreover, M have been shown to be the primary source of TNF- detected in the pancreas at early stages of disease progression in NOD mice (23). Together, these observations suggest that M contribute to the pathogenesis of T1D in multiple ways.

The family of NF-B transcription factors is known to regulate the development and function of immune effector cells (27, 28). NF-B is found as a homo- or heterodimer composed of the Rel family of proteins. In resting cells, NF-B is typically sequestered in the cytoplasm through a complex with the inhibitory proteins IB, IB, and IB. After stimulation, the IB proteins are phosphorylated by a multisubunit IB kinase and then degraded in a ubiquitin and proteasome-dependent manner (27, 28). Various stimuli such as LPS, TNF-, and CD40 engagement activate NF-B in M and DCs (29, 30, 31, 32, 33, 34). NF-B then translocates to the nucleus and activates transcription of a broad and diverse group of genes, including those encoding IL-12(p40) (20), TNF- (29), and IL-1/ (32, 35).

Recently, we found that myeloid DCs cultured from NOD bone marrow or spleen exhibit hyperactivation of NF-B in response to several stimuli (34). Importantly, elevated levels of NF-B activation resulted in increased secretion of IL-12(p70) and TNF- and an enhanced capacity to stimulate T cells relative to DCs prepared from C57BL/6, BALB/c, and nonobese resistant (NOR) mice (36). These results demonstrated that a defect in regulation of NF-B activation exists in NOD DCs and that NF-B has a key role modulating the APC function of DCs. With this in mind, the current study was initiated to examine 1) whether hyperactivation of NF-B also occurs in NOD M and 2) how NF-B influences the APC function of M. Here we demonstrate that NOD M exhibit enhanced NF-B activation relative to M prepared from BALB/c and NOR mice after different stimuli. This elevated NF-B activation in NOD M resulted in increased IL-12(p70), TNF-, and IL-1 secretion but had no effect on the T cell stimulatory capacity of the M.

	Materials and Methods

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Mice

BALB/cJ and NOR/Lt mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained under specific pathogen-free conditions at the University of North Carolina animal facilities. NOD/LtJ mice were similarly housed and bred. Currently in our colony, T1D develops in 80% of NOD/LtJ female mice by 1 year of age. The NOD.CL4 transgenic mouse line was established by back-crossing transgenes encoding the CL4 clonotypic TCR derived from BALB/c.CL4 mice (provided by R. Liblau, Institut National de la Santé et de la Recherche Médicale, Paris, France) onto the NOD/LtJ genotype for 20 generations. The CL4 clonotypic TCR is H2K^d-restricted and specific for an influenza hemagglutinin (HA) peptide spanning amino acid residues 512–520. TCR transgenic BDC2.5 NOD mice, originally obtained from C. Benoist and D. Mathis (Harvard University, Cambridge, MA) were bred in our colony and represent the 20th NOD back-cross generation. The IA^g7-restricted BDC2.5 clonotypic TCR is specific for an unidentified cell Ag. Approximately 25% of BDC2.5 transgenic mice develop overt diabetes by 25 wk of age in our colony.

Preparation of splenic M and bone marrow-derived M

Splenic- and bone marrow-derived M were prepared from male or female mice between 8 and 12 wk of age. After lysis of RBCs, splenocytes were depleted of CD4 (mAb GK1.5), CD8 (mAb HO2.2), CD11c (mAb HL3), and B220 (mAb RA3-3A1/6.1)-expressing cells via complement-mediated lysis. The remaining cells were plated in six-well tissue culture plates (BD Biosciences, San Jose, CA) in RPMI 1640 medium containing 10% FBS and penicillin/streptomycin (base medium) and 25 ng/ml murine M-CSF (R&D Systems, Minneapolis, MN). On the second day of culture, nonadherent cells were removed after thorough washing with PBS (four to five times), and the remaining adherent cells were cultured as above for 9 days. M-CSF containing medium was added on days 4, 6, and 8 of culture. On day 9, M-CSF containing medium was removed, adherent cells were washed with PBS, and base medium (lacking M-CSF) was added, after which cultures were maintained for an additional 2 days. Bone marrow M were prepared from the femurs of male or female mice. RBCs were lysed and B220, CD11c, CD4, and CD8-expressing cells were depleted by complement lysis. The remaining cells were plated on six-well low cluster plates (Corning Glass, Corning, NY) in base medium containing 25 ng/ml murine M-CSF and were cultured as above for splenic M. Flow cytometric analysis demonstrated that splenic- and bone marrow-derived M expressed F4/80, CD11b, MHC class I (H2K^d), MHC class II, CD80, CD86, and CD40.

Direct isolation of splenic M

Splenic M were prepared directly from male or female mice between 8 and 12 wk of age. After lysis of RBCs, splenocytes were depleted of CD4 (mAb GK1.5), CD8 (mAb HO2.2), CD11c (mAb HL3), and B220 (mAb RA3-3A1/6.1)-expressing cells via complement-mediated lysis. A second step of purification entailed removal of any residual CD4⁺, CD8⁺ T cells, B cells, and DCs via the MACS system using anti-CD4, anti-CD8, anti-B220, and anti-CD11c magnetic beads (Miltenyi Biotech, Auburn, CA). Approximately 80% and 30% of the remaining cells were found to be CD11b and F4/80-positive, respectively.

Fluorescence staining

The following mAbs used for fluorescence staining were purchased from BD PharMingen (San Diego, CA): FITC anti-CD40 (clone HM40-3), FITC anti-CD86 (clone GL1), FITC anti-CD80 (clone 16-10A1), PE anti-CD11c (clone HL3), PE anti-H2K^d (clone SF1-1.1), and PE CD11b (clone M1/70). Anti-IA^d (clone MK-D6) and anti-IA^g7 (clone 10.2.16) were provided by E.P. Reich (Immunologic, Palo Alto, CA). FITC anti-F4/80 (clone Cl:A3-1) was purchased from Caltag Laboratories (Burlingame, CA). PE anti-mouse IgG and FITC anti-mouse IgG secondary reagents were purchased from BD PharMingen. Stained cells were analyzed on a FACScan (BD Biosciences) using Summit Software (Cytomation, Ft. Collins, CO).

EMSA and Western blotting

M cultured in base medium at 5 x 10⁵ cells/well in 24-well low cluster plates (Corning Glass) were stimulated with 50 µg/ml LPS as described previously (34, 37), 20 µg/ml anti-CD40 mAb (clone HM40-3; BD PharMingen), 20 µg/ml purified hamster IgM isotype control (clone G235-11; BD PharMingen), or 20 ng/ml murine TNF- (R&D Systems) for the specified times. Anti-CD40 mAb and TNF--treated M were found to exhibit optimal activation at a concentration of 20 µg/ml and 20 ng/ml, respectively. Nuclear and cytoplasmic extracts were prepared as described previously (38). EMSA was performed as described using ³²P-labeled dsDNA probes containing NF-B binding sites derived from the MHC class I H2K promoter (5'-CAGGCTGGGGATTCCCATCTCCACAGTTTCACTTC-3') (34), the murine IL-12(p40) promoter (5'-CTTCTTAAAATTCCCCCAGA-3'), or the murine TNF- promoter (5'-CCTCTGGGGCTGCCCCATA-3') (20, 39). A double-stranded OCT-1 DNA probe (5'-TGTCGAATGCAAATCACTAGAA-3'; Santa Cruz Biotechnology, Santa Cruz, CA) was used as a control for the EMSAs. Bands were visualized using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). For supershift experiments, 2 µg of anti-RelA (sc-109), anti-c-Rel (sc-71X), anti-RelB (sc-226X), anti-p52 (sc-298X), and anti-p50 (sc-1190) Abs (Santa Cruz Biotechnology) were added to each sample and incubated for 20 min at room temperature before the addition of ³²P-labeled probe.

For Western blotting, 80 µg of cytoplasmic extract was resolved via 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 were blocked overnight with 5% dried milk in PBS plus 0.05% Tween 20. Blots were probed with anti-IB (sc-371; Santa Cruz Biotechnology) and anti--actin (A2066; Sigma-Aldrich, St. Louis, MO) rabbit polyclonal Abs. Subsequent binding of an HRP-labeled goat anti-rabbit Ab (Promega, Madison, WI) was determined using ECL development reagents (Amersham Pharmacia Biotech).

Measurement of M secretion of IL-12(p70), TNF-, and IL-1

M were harvested after 2 days of culture in the absence of M-CSF, plated at 10⁶ cells/ml in a 24-well plate with 1 ml of base medium, and stimulated with 50 µg/ml LPS, 20 µg/ml anti-CD40, or 20 µg/ml isotype-matched Ab for 48 h. In addition, M were stimulated with 20 ng/ml of TNF- for 6 h, washed with PBS, and then cultured for 48 h. Supernatants were pooled and 100-µl aliquots assayed using ELISA kits (BD PharMingen) specific for IL-12(p70), TNF-, and IL-1 per the manufacturer’s instructions.

Preparation of pancreatic islets

Pancreatic islets were isolated from young NOD mice as described previously (40). Briefly, pancreases from groups of 8–10 mice were perfused with 2 mg/ml collagenase P (Roche, Indianapolis, IN) and digested for 20 min at 37°C. Islets were purified from digested tissue using a Ficoll gradient and then were handpicked. Purified islets were dissociated into a single cell suspension of cells using enzyme-free cell dissociation solution (Sigma-Aldrich) and then were washed and counted.

M: T cell assays

CD8⁺ T cells were purified via the MACS system from the spleens of (NOD.CL4 x BALB/c)F₁ (F1.CL4) mice using anti-CD8 magnetic beads (Miltenyi Biotech). Flow cytometric analysis confirmed that T cells were CD62L^highCD44^lowCD69^low and 90% CD8⁺. CD4⁺ T cells were similarly purified from the spleens of NOD.BDC2.5 mice using anti-CD4 magnetic beads. Greater than 90% of cells were CD4⁺, exhibiting a naive phenotype (CD62L^highCD44^lowCD69^low) based on flow cytometry analysis.

M pulsed with dissociated islets. Varying numbers of M were incubated with a specified number of cells in 200 µl of base medium supplemented with 50 µM 2-ME, 1 mM sodium pyruvate, 1x nonessential amino acids, and 1 mM glutamine (complete medium) in a 96-well microtiter plate. After 4 h, M were washed four times with medium and 10⁵ CD4⁺ T cells were added to each well in a total of 250 µl of complete medium. Culture supernatants were harvested after 48 h, and 100-µl aliquots were analyzed for levels of IL-2 and IFN- via capture ELISA per the manufacturer’s instructions (BD PharMingen). A 1:1 ratio of M to dissociated islets was found to be optimal for T cell activation. A total of 10⁵ bone marrow M in 200 µl of complete medium were used for the assay.

M pulsed with influenza virus. Varying numbers of M were incubated with 100 hemagglutination activity units (hau) of heat-inactivated influenza strain A/Puerto Rico/8/34 (PR8; provided by J. Frelinger, University of North Carolina, Chapel Hill, NC) in 200 µl of complete medium in a 96-well microtiter plate. After 4 h, M were washed with PBS, and 10⁵ naive CD8⁺ T cells were added to each well in a final volume of 250 µl. At 48 h, culture supernatants were harvested, and levels of IL-2 and IFN- secretion were determined as above. The 100 hau of heat-inactivated influenza virus was found to elicit maximum T cell activation.

To confirm that processing of whole virus was proteasome mediated, M were pretreated with 20 µM of the proteasome-specific inhibitor lactacystin (Calbiochem, San Diego, CA) prepared in 0.1% DMSO for 12 h and then incubated with 100 hau of heat-inactivated PR8 influenza virus for 4 h. A total of 20 µM lactacystin was found to be the lowest concentration that mediated maximum inhibition of T cell stimulation without causing M death.

Infection of M with adenoviral recombinants

Adenoviral recombinants encoding either IB superrepressor (Ad-SR) or -galactosidase (Ad-LacZ) were prepared by the University of North Carolina Center of Gene Therapy. Construction of the IB superrepressor in which serines at positions 32 and 36 of IB have been substituted with alanines has been described elsewhere (41). M prepared from 9-day cultures were washed in PBS, resuspended in base medium, and then infected for 24 h with Ad-SR or Ad-LacZ at a multiplicity of infection (moi) of 200. Maximum inhibition of nuclear translocation of NF-B was detected in LPS-stimulated M infected with 200 moi of Ad-SR. Greater than 90% of M were found to be infected by 200 moi of Ad-LacZ as determined by a -galactosidase assay.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Enhanced NF-B activation in NOD M

To investigate aspects of NF-B activation in NOD M, conditions were established to culture mature M from splenic progenitors. Splenocytes depleted of T and B cells were cultured for 9 days in murine M-CSF and then for an additional 2 days in medium lacking M-CSF. The latter eliminates the priming effect of residual M-CSF found in culture. Under these conditions, cultures consisted of 95% M exhibiting a mature phenotype based on flow cytometric analysis of CD11b and F4/80 expression, respectively. Furthermore, M displayed a reduced proliferative capacity in the absence of M-CSF. For example, a 3.4-fold difference in [³H]thymidine incorporation was observed between cultures supplemented with (3961.2 ± 282 cpm) or without (1154 ± 122 cpm) M-CSF. Splenic M were also prepared from BALB/c and diabetes-resistant NOR mice. The NOR mouse is a NOD-related MHC-syngeneic recombinant strain that contains 12% C57BL/KsJ-derived genes, exhibits peri-insulitis, only minimal intrainsulitis, and fails to develop overt diabetes (42). LPS-stimulated M prepared from the three mouse strains exhibited similar levels of CD40 and H2K^d expression (Table I). However, NOD M expressed elevated H2K^d expression relative to NOR and BALB/c M in untreated cultures (Table I). Levels of CD80 and CD86 expression were also found to be somewhat reduced on BALB/c vs NOD or NOR M stimulated with LPS (Table I). Furthermore, comparable phenotypes were detected between bone marrow and splenic M prepared from the respective strains of mice.

View this table: [in this window] [in a new window]   Table I. Flow cytometric analysis of cell surface proteins expressed by splenic Ma

View larger version (40K): [in this window] [in a new window]   FIGURE 1. NOD splenic M exhibit increased nuclear translocation of NF-B compared with NOR and BALB/c M after stimulation. Splenic M prepared from NOD, NOR, and BALB/c mice were stimulated with 50 µg/ml LPS and 20 µg/ml anti-CD40 mAb for 30 min or 20 ng/ml TNF- for 1 h. A, Nuclear extracts were prepared and analyzed by EMSA using H2K NF-B consensus binding oligonucleotide. A double-stranded OCT-1 DNA probe was used as an internal control. The same nuclear extracts from untreated and LPS-stimulated splenic M were analyzed using mIL-12(p40) (B) and TNF--specific (C) NF-B binding oligonucleotides. Right panels represent corresponding densitometric analysis, which was measured as a ratio of intensity of NF-B to OCT-1 binding per unit area. The NF-B complexes labeled here are based on supershift analysis in Fig. 3. Data are representative of at least three independent experiments.

View larger version (54K): [in this window] [in a new window]   FIGURE 2. Kinetic analysis of NF-B activation upon LPS stimulation in splenic and bone marrow M. Splenic (A) and bone marrow (B) M prepared from NOD, NOR, and BALB/c mice were stimulated with 50 µg/ml LPS for varying times. Nuclear extracts were prepared and analyzed by EMSA using H2K- or mIL-12(p40)-specific oligonucleotides. A double-stranded OCT-1 DNA probe was used as an internal control. Data are representative of at least three independent experiments.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. NOD M isolated directly from spleen exhibit increased nuclear translocation of NF-B compared with NOR and BALB/c M after LPS and TNF- stimulation. Splenic M prepared directly from NOD, NOR, and BALB/c mice were stimulated with 50 µg/ml LPS (A) or 20 ng/ml TNF- (B) for 30 min or 1 h, respectively. Nuclear extracts were prepared and analyzed by EMSA using H2K-specific oligonucleotide. Data are representative of at least three independent experiments.

Similar complexes of NF-B are activated in NOD, NOR, and BALB/c M

To determine whether NF-B hyperactivation in splenic NOD M extracts reflected preferential activation of specific complexes, a supershift analysis using Abs specific for each Rel family member was conducted. As demonstrated in Fig. 4, NF-B binding to the H2K-specific probe consisted largely of the p50 and p65 subunits, whereas p65, c-Rel, and p50 were found to bind the mIL-12(p40)-specific probe in NOD, NOR, and BALB/c M treated with LPS. These results indicate that the increased NF-B activity detected in NOD M was not associated with selective induction of specific NF-B complexes.

View larger version (93K): [in this window] [in a new window]   FIGURE 4. NF-B subunit binding to H2K- and mIL-12(p40)-specific oligonucleotides in splenic M. NOD, NOR, and BALB/c splenic M were stimulated with 50 µg/ml LPS for 30 min. Nuclear extracts incubated with the H2K- or mIL-12(p40)-specific oligonucleotides and Abs specific for different NF-B subunits were examined via EMSA. In untreated M, no NF-B activation was detected. Data are representative of at least three independent experiments.

IB exhibits enhanced degradation after stimulation in NOD M

To gain insight into the mechanism of increased NF-B DNA binding activity in NOD M, degradation of the IB inhibitory protein was examined in cytoplasmic extracts via Western blot. NOD M displayed 3- to 16-fold increased IB degradation compared with NOR and BALB/c M after stimulation with LPS, anti-CD40 Ab, or TNF- (Fig. 5). In contrast, similar levels of -actin were detected in cytoplasmic extracts prepared from M of all three strains of mice, independent of the type of activation (Fig. 5). This observation indicates that hyperactivation of NF-B in NOD M was associated with increased IB degradation compared with NOR and BALB/c M.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Enhanced IB degradation in NOD vs NOR or BALB/c splenic M. Splenic M prepared from NOD, NOR, and BALB/c mice were stimulated with 50 µg/ml LPS, 20 µg/ml anti-CD40 mAb for 30 min, or 20 ng/ml TNF- for 1 h. A, Cytoplasmic extracts were prepared and probed on a Western blot with an anti-IB Ab. Expression of -actin was used as an internal control. B, Densitometric analysis that was measured as a ratio of intensity of IB to -actin expression. Data are representative of at least three independent experiments.

NF-B regulates enhanced cytokine secretion in NOD M

Next, splenic M prepared from NOD, NOR, and BALB/c mice were assessed for IL-12(p70), TNF-, and IL-1 secretion after stimulation. It is well established that NF-B in part regulates the transcription of the IL-12(p40) (20), TNF- (29), and IL-1 (32, 35) genes. Consistent with the observed hyperactivation of NF-B, NOD M secreted significantly increased levels of IL-12(p70), TNF-, and IL-1 after LPS stimulation compared with NOR or BALB/c M (Table II). Anti-CD40 Ab treatment also elicited significantly elevated IL-12(p70) and TNF- secretion by NOD M (Table II). The effect of TNF- treatment was more restricted, and IL-12(p70) was markedly increased in NOD M but only background levels of TNF- or IL-1 secretion was detected for M from all three strains of mice (Table II).

View this table: [in this window] [in a new window]   Table II. Cytokine secretion after stimulation by NOD, NOR, and BALB/c splenic M

View this table: [in this window] [in a new window]   Table III. Inhibition of cytokine secretion by NOD M infected with Ad-SR

NF-B activation is not required for M to stimulate CD4⁺ or CD8⁺ T cells in vitro

We previously reported that elevated NF-B activation significantly increased the capacity of NOD DCs to stimulate T cells (36). To determine whether increased NF-B activation influenced NOD M APC function in a similar manner, splenic M from the different strains of mice were examined for the ability to stimulate naive CD4⁺ and CD8⁺ T cells. The BDC2.5 TCR (43) transgenic mouse was used as a source of IA^g7-restricted cell-specific CD4⁺ T cells, permitting a direct comparison between IA^g7 expressing NOD and NOR M. BDC2.5 CD4⁺ T cells were cultured with varying numbers of NOD and NOR M pulsed with dissociated islets at a ratio of 1:1. As demonstrated in Fig. 6, there was no overall difference in the levels of IL-2 and IFN- secreted by BDC2.5 CD4⁺ T cells stimulated by NOD vs NOR spleen or bone marrow M.

View larger version (17K): [in this window] [in a new window]   FIGURE 6. Increased NF-B activation in NOD M does not correlate with an elevated capacity to stimulate CD4+ T cells. A and B, Varying concentrations of NOD () and NOR splenic M () were pulsed with dissociated islets at a ratio of 1:1 and cultured with 105 naive BDC2.5 CD4+ T cells for 48 h. As a control, NOD () and NOR M () were cultured with 105 naive BDC2.5 CD4+ T cells in the absence of dissociated islets. C and D, A total of 105 NOD (filled bar) or NOR (open bar) bone marrow M were pulsed with dissociated islets and cultured with 105 naive BDC2.5 CD4+ T cells as above. Culture supernatants were analyzed for the production of IL-2 and IFN- by ELISA. Data represent mean ± SD of triplicate wells and are representative of at least three independent experiments.

View larger version (11K): [in this window] [in a new window]   FIGURE 7. Increased NF-B activation in NOD M does not correlate with an elevated capacity to stimulate CD8+ T cells. Varying concentrations of NOD (), NOR (), and BALB/c () M were pulsed with 100 hau of heat-inactivated influenza virus for 4 h. A and B, M were washed and cultured with 1 x 105 naive F1.CL4 CD8+ T cells for 48 h. As a control, NOD (), NOR (), and BALB/c () M were cultured with 105 naive F1.CL4 CD8+ T cells in the absence of virus. Culture supernatants were analyzed for the production of IL-2 and IFN- by ELISA. Data represent mean ± SD of triplicate wells and are representative of at least three independent experiments.

View larger version (18K): [in this window] [in a new window]   FIGURE 8. T cell stimulation by NOD M is independent of NF-B activation. A total of 105 splenic M of NOD were infected with Ad-SR or Ad-LacZ at 200 moi. After 24 h, M were cultured with 1 x 105 naive BDC2.5 CD4+ T cells plus dissociated islets (A and B) or with F1.CL4 CD8+ T cells plus heat-inactivated influenza (C and D) as described for Fig. 6. Culture supernatants were analyzed for the production of IL-2 and IFN- by ELISA. Data represent mean ± SD of triplicate wells and are representative of at least three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, M precursors from spleen and bone marrow were cultured in the presence of M-CSF to standardize the phenotype and activational status of the cells. In this way, a homogeneous population (>95%) of M exhibiting a mature and relatively quiescent phenotype was obtained. The phenotype of these cultured APCs likely varies to some degree compared with M found in vivo on a tissue-to-tissue basis. M display a broad range of phenotypes and effector functions (47, 48, 49, 50). This heterogeneity is in part due to the origin of the precursor cells and the nature of the signals that induce M maturation in vivo. Nevertheless, the fact that M prepared from both spleen and bone marrow exhibited similar phenotypes argues that dysregulation of NF-B is an intrinsic defect of NOD M that influences the ability of these cells to respond to stimulation. This conclusion is further supported by the observation that hyperactivation of NF-B was also detected in NOD M isolated directly from the spleen (Fig. 3).

Similar to our findings made with bone marrow and splenic-derived NOD DCs, NOD M exhibited a 2.1- to 5.6-fold increase in the level of NF-B activation compared with NOR and BALB/c M (Figs. 1–3). Although there was no marked difference in the kinetics of activation between M prepared from the three strains of mice, the level of NF-B activation was significantly increased in NOD M at all time points examined (Fig. 2). Furthermore, elevated DNA binding by NF-B was detected in NOD M extracts for the three oligonucleotides used for EMSA, namely the H2K-, mIL-12(p40), and TNF--specific probes. It is noteworthy that the oligonucleotide probes are bound by distinct NF-B complexes. The H2K probe, which contains a consensus NF-B binding site, was bound by the p65 and p50 subunits (Fig. 4). In contrast, complexes binding to the mIL-12(p40) probe, which contains a unique NF-B binding site (51), consisted of the c-Rel, p65, and p50 subunits (Fig. 4). Previous studies have shown that NF-B complexes binding to the IL-12(p40) promoter consist primarily of the c-Rel/p50, p65/p50 trans-activating heterodimers and the p50/p50 homodimer (52). These results indicate that NOD M are hyperresponsive to stimulation, resulting in elevated activation of multiple NF-B complexes.

A recent study by Liu and Beller (20) reported that elevated IL-12 secretion detected in PECs prepared from thioglycolate-treated NOD vs other strains of mice corresponded to differences in the ratio between c-Rel/p50 and p50/p50 complexes bound to the mIL-12(p40). In this model, elevated transcription of the IL-12(p40) gene is mediated by an increase in the trans-activating c-Rel/p50 heterodimer relative to the p50/p50 homodimer, which functions as an NF-B-specific inhibitor. This contrasts with our findings in which an increase in both heterodimer (p65/p50, c-Rel/p50) and homodimer (p50/p50) complexes was observed in splenic and bone marrow NOD M independent of the probe used for EMSA or the type of stimulation. Key differences between the two studies include the subsets of M investigated and the activational status of the cells. For example, the PECs examined by Liu and Beller (20) were highly activated after thioglycolate treatment, whereas in this study the bone marrow and splenic-derived M (in the absence of M-CSF) or M isolated directly from the spleen typically exhibited a resting phenotype. These disparities may result in distinct profiles of NF-B activation. Nevertheless, together these results indicate that multiple mechanisms can be invoked to regulate the activational and transcriptional status of NF-B.

Our findings that NOD M cultured from the bone marrow or spleen or isolated directly from the spleen exhibit NF-B hyperactivation upon stimulation are inconsistent with recent work by Hayashi and Faustman (53). These investigators reported a proteasome defect in NOD Kupfer cells and splenocytes that resulted in impaired NF-B activation after stimulation with TNF- for 4 h (53). Currently, it is not clear why marked differences exist between the two studies. Hayashi and Faustman (53) assessed NF-B activation in a bulk splenocyte preparation and used an HIV-specific probe to measure NF-B DNA binding, which could in part explain the discrepancies between the respective studies. Nevertheless, our findings made with different NOD M preparations and NF-B DNA binding probes, coupled with results reported by Liu and Beller (20), clearly demonstrate that NF-B is activated in various subsets of NOD M upon stimulation.

The level of secretion of proinflammatory cytokines was the most notable effect NF-B hyperactivation had on the APC function of NOD M. For example, NOD M exhibited significantly elevated levels of IL-12(p70), TNF-, and IL-1 secretion in comparison with NOR and BALB/c M after LPS stimulation. NOD M also exhibited enhanced proinflammatory cytokine secretion upon anti-CD40 Ab or TNF- treatment, albeit to a lesser extent than with LPS (Table II). The observed increase in IL-12(p70) and TNF- secretion by NOD M was consistent with the elevated binding of NF-B to the IL-12(p40)- and TNF--specific probes, respectively (Fig. 1). Furthermore, these findings are consistent with studies demonstrating that PECs prepared from thioglycolate-treated NOD mice exhibit increased levels of IL-12 secretion (15, 20). In contrast with our results, however, NOD PECs were found to secrete reduced levels of IL-1 and TNF- relative to NOR PECs after LPS stimulation (15). As mentioned above, differences in the activation status and/or mode of M maturation may explain the disparate profile of cytokine secretion observed in the respective studies. Importantly, transfer of the IB superrepressor gene demonstrated a direct role for NF-B in the increased secretion of proinflammatory cytokines by NOD M. Inhibiting NF-B activation with Ad-SR (but not Ad-LacZ) significantly suppressed levels of IL-12(p70), TNF-, and IL-1 secretion after LPS, TNF-, or anti-CD40 Ab treatment of NOD M (Table III).

Interestingly, the level of NF-B activation had no significant effect on M to stimulate either CD4⁺ or CD8⁺ T cells. Comparable levels of IL-2 and IFN- secretion by BDC2.5 clonotypic CD4⁺ T cells were detected in cultures of splenic or bone marrow M prepared from NOD or NOR mice and pulsed with dissociated islets. Furthermore, NOD and NOR peritoneal M pulsed with a mimotope peptide exhibited similar capacities to stimulate BDC2.5 CD4⁺ T cells (P. Sen and R. Tisch, unpublished observations). Similarly, splenic M prepared from NOD and NOR pulsed with heat-inactivated influenza virus stimulated similar IL-2 and IFN- secretion by F1.CL4 CD8⁺ T cells. Although BALB/c M typically exhibited a reduced capacity to stimulate F1.CL4 CD8⁺ T cells, this could be due to lower levels of CD80 and CD86 expression relative to NOD and NOR M (Table I). The fact that infection of NOD M with Ad-SR under conditions that clearly suppressed NF-B activation did not alter the profile of cytokine secretion by either BDC2.5 CD4⁺ or F1.CL4 CD8⁺ T cells (Fig. 8) provided further evidence that the level of NF-B activation had no significant effect on the T cell stimulatory capacity of the M. This result contrasts with our previous work demonstrating that inhibition of NF-B activation effectively blocked T cell stimulation by DCs, despite up-regulation of costimulatory molecules on the cell surface (36). This latter observation indicates a broader role for NF-B in modulating the APC function of DCs compared with M. In addition, these findings indicate that NF-B predominantly influences the APC function of M by regulating cytokine gene expression.

The apparent hyperresponsiveness of NOD M derived from bone marrow and splenic precursors and the linked ability to secrete elevated levels of proinflammatory cytokines is consistent with a role in establishing an extracellular milieu promoting cell autoimmunity. The fact that NOD vs NOR M differ in this regard further suggests that the properties of NOD M described herein contribute to disease progression. M are among the first cells found infiltrating the pancreas and along with DCs have been shown to be the primary source of TNF- in young NOD mice (12, 21, 22, 23). Elevated levels of TNF- secretion would be expected to have pleiotropic effects, including up-regulation of adhesion molecules on endothelial cells and promotion of cell trafficking to the pancreas (54, 55, 56). M and CD4⁺ and CD8⁺ T cell trafficking to the pancreas would be further augmented by elevated levels of IL-12(p70) (57). Relatively high levels of both TNF- and IL-12(p70) secreted by M in the pancreas would also be expected to efficiently activate and mature resident DCs (58, 59). These DCs in turn would function to activate and mediate differentiation of cell-specific CD4⁺ and CD8⁺ T effector cells within either the pancreas or draining lymph nodes. Increased levels of TNF- and IL-1 could also induce cell injury as reported in vitro. Finally, elevated IL-12(p70) secretion by M would be expected to efficiently induce Th1 and Tc1 cell differentiation. Indeed, depletion of M in NOD mice inhibits the differentiation of cell-specific Th1 cells and prevents the development of insulitis and overt diabetes (12).

In summary, this study demonstrates that dysregulation of NF-B activation is a property of NOD M in addition to NOD DCs. Whether similar mechanisms are used promoting NF-B hyperactivation in M vs DCs is currently being examined. Furthermore, the general role of NF-B in regulating APC function differs between M and DCs. In M, NF-B is primarily involved in regulating the expression of proinflammatory cytokines, whereas NF-B has a broader role in DCs. This work also provides evidence that, under the appropriate conditions, splenic and bone marrow-derived M can effectively stimulate T cells and promote conditions necessary for the development of cell-specific autoimmunity. The task at hand is to determine how conditions influence the phenotype and effector function of M in vivo.

	Footnotes

 
¹ This work was supported by Grant 1-P60-DE 13079 from the National Institute of Dental and Craniofacial Research. C.P.W. was supported in part by National Institute of Allergy and Infectious Disease Training Grant 5-T32-AI07273.

² S.B. and M.W. contributed equally to this paper.

³ Address correspondence and reprint requests to Dr. Roland Tisch, Department of Microbiology and Immunology, 804 Mary Ellen Jones Building, CB#7290, University of North Carolina, Chapel Hill, NC 27599-7290. E-mail address: rmtisch{at}med.unc.edu

⁴ Abbreviations used in this paper: T1D, type 1 diabetes; NOD, nonobese diabetic; M, macrophage; DC, dendritic cell; PEC, peritoneal exudate cell; NOR, nonobese resistant; HA, hemagglutinin; hau, hemagglutination activity units; Ad-SR, adenoviral recombinant encoding IB superrepressor; Ad-LacZ, adenoviral recombinant encoding -galactosidase; moi, multiplicity of infection.

Received for publication September 12, 2002. Accepted for publication December 6, 2002.

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