INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

INSULIN-DEPENDENT DIABETES mellitus (IDDM) is a T cell-mediated autoimmune disease, with the prediabetic state characterized by the production of autoantibodies that react with specific proteins expressed by pancreatic -cells. The insulin-producing -cells are also selectively destroyed by the autoreactive immune response in rodent models that spontaneously develop IDDM, such as the nonobese diabetic (NOD) mouse (38). Similar to that for diverse human autoimmune diseases, NOD female mice preferentially express autoimmunity. Normal lymphoid cell development, proper T cell selection after antigen presentation, and maintenance of cytokine balance are thought to be important for the prevention of autoimmunity. Dysregulation of these processes thus culminates in a diverse group of diseases that are defined clinically by the target organ destroyed, and, for unknown reasons, female cohorts are preferentially affected.

Various interacting cellular and humoral immune events underlie the pathogenesis of autoimmunity. Improper T cell education is reflected in the T cell-promoted production of autoantibodies by B cells and typically accompanied by marked imbalances in cytokine expression. Lymphoid immaturity is often associated with autoimmunity, and apoptotic defects in proper lymphoid selection are genetically determined in certain spontaneous mouse models of autoimmune disease (37, 67, 68).

A strong genetic association exists between certain autoimmune disease and the expression of certain kinds (haplotypes) of major histocompatibity complexes (MHC) (3, 49, 60). MHC gene encodes cell-surface molecules that display peptides for immune recognition. Although autoimmune target organs are diverse, the MHC region of the genome contributes multiple, poorly defined risk factors to the development of autoimmunity (3, 43, 44, 49, 60). This region of the genome contains immune response genes that are important for T cell education and for antigen presentation by MHC class I and class II molecules. Two such genes encoding the proteasome subunits (Lmp2 and Lmp7) are located in this high-risk genomic region (3, 49, 60).

The proteasome is a multiprotein complex that catalyzes the ATP- and ubiquitin-dependent processing or degradation of intracellular proteins (13, 23). Proteasome-mediated protein cleavage plays an important role in the regulation of cell growth, metabolism, and function. The cleavage of endogenous proteins by the proteasome also generates self-peptides that contribute to T cell education after presentation by MHC class I molecules (18). Although, in general, the proteasome exhibits minimal variability in substrate selectivity and subunit composition, interferon- markedly increases the expression of the LMP2 and LMP7 subunits. Incorporation of these subunits into the proteasome alters its specificity for self-proteins such that the suitability of the generated peptides for presentation in the peptide-binding groove of MHC class I molecules is increased (8, 17, 22). Studies with LMP2^/ mice demonstrate a lymphoid system with improper T cell selection. T cells from these animals exhibit cytotoxicity both in vitro and in vivo toward syngeneic cells with intact presentation of self-peptides by MHC class I (55, 62, 70).

The proteasome also mediates the processing and activation of the transcription factor nuclear factor-B (NF-B). NF-B is activated in response to various extracellular stimuli, including interleukin-1, lipopolysaccharide, and tumor necrosis factor- (TNF-) (4, 5, 58, 63). Activated NF-B, in turn, regulates the expression of genes that contribute to cytokine generation, cell adhesion, lymphocyte maturation, protection from apoptosis, and processing and presentation of antigens by MHC class I (7, 9, 14, 57, 61, 71).

Active NF-B exists predominantly as a heterodimer composed of either a p50 or p52 subunit and p65 (RelA). The p50 and p52 subunits are produced by proteasome-mediated processing of p105 and p100 precursors, respectively (12, 16, 47, 52, 53); the production of p50 by the proteasome can occur cotranslationally (36). In the cytoplasm of resting cells, NF-B associates with the inhibitor protein IB. Cell stimulation results in the phosphorylation and subsequent ubiquitination and proteasome-mediated degradation of IB. This allows the p50-p65 or p52-p65 dimers to translocate to the nucleus and to induce the transcription of target genes (11, 39, 41, 59, 73). The phosphorylation of IB requires the IB kinases IKK and IKK (15, 42, 48, 50, 64). Sequestration of Rel complexes by p105 or p100 deters rapid mobilization to the nucleus in response to cell stimulation, rendering cells susceptible to TNF--induced apoptosis as a result of the consequent failure to activate the expression of protective genes (36, 56).

Both humans with Type 1 diabetes and NOD mice exhibit a defect in antigen presentation by MHC class I molecules that results from impaired generation or transport of self-peptides (18, 33). Moreover, humans with Type 1 diabetes and NOD mice are defective in the production of LMP2 mRNA (21, 72), and adult NOD mouse splenocytes exhibit a reduced abundance of LMP2 protein (26, 28), which may result in altered generation of self-peptides by the proteasome. Therefore, humans and animals with autoimmune diabetes exhibit symptomatology indicative of possible errors in proteolytic activation of diverse substrates, including interrupted NF-B by the proteasome. The proteasome-mediated NF-B activation in female NOD mice is significantly defective, and, in the NF-B subunit, proteolytic p50 generation is impaired. The proteasomes from male NOD mice produce a detectable cleavage product but of an incorrect size (26). It is likely that humans and rodents with autoimmune diabetes also exhibit defects in cytokine gene regulation and secretion due to impaired NF-B activation.

We recently investigated proteasome-mediated-NF-B activation, which plays an important role in the expression of genes involved in immune response. The activity of NF-B in NOD mouse lymphocytes was shown to be markedly reduced as a result of a defect in the proteolytic generation of p50 and p52 by the ubiquitin-proteasome pathway in female mice (26, 28). The virtual inability of TNF- to activate NF-B in adult NOD mouse spleen cells was associated with an increased susceptibility to TNF--induced apoptosis. The defect in proteasome function in NOD mouse splenocytes was indirectly attributed to the lack of the LMP2 protein.

We directly investigated the role of the LMP2 proteasome subunit in autoimmune disease by comparing the characteristics of LMP2^/ mice and autoimmune NOD mice. Disruption of the Lmp2 gene, even in the nonautoimmune B6 mouse strain, was shown to be sufficient to impair NF-B activation and to render cells more susceptible to TNF--induced apoptosis of lymphocytes. TNF--induced apoptosis was detected in spleen cells from 7-wk-old or elder female and male NOD mice, an age that exhibits insulitis (38). Furthermore, the effect of TNF- on NOD mouse spleen cells appeared more pronounced for female than for male animals in both dose and time course exposure studies. With the artificially disrupted Lmp2 gene, no gender specificity of defective proteasome phenotypes is shown. In marked contrast, spontaneously defective LMP2 production preferentially occurs in female NOD mice, conferring a marked developmental and cell-specific lineage restriction that may bias the spontaneous NOD model to actual disease expression. Our observations thus define the contributions of LMP2 disruption to diverse autoimmune phenotypes of NF-B disruption and apoptosis.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Mouse and preparation of nuclear and cytosolic extracts. BALB/c and NOD mice were purchased from Jackson Laboratories (Bar Harbor, ME); LMP2^/ mice were a generous donation from Dr. Luc Van Kaer (Nashville, TN). Nuclear and cytosolic extracts were prepared from spleen cells of 7-wk-old BALB/c mice, NOD mice, or LMP2^/ mice as well as from human T cell lymphoma MOLT-4 cells (American Type Culture Collection, Manassas, VA). Spleen cells were harvested, centrifuged for 15 min at 1,500 g, washed in 10 ml of ice-cold phosphate-buffered saline, and again collected by centrifugation. The cell pellets were resuspended in 4 ml of solution A [10 mM HEPES-NaOH (pH 7.8), 10 mM KCl, 2 mM MgCl₂, 1 mM dithiothreitol (DTT), 0.1 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF)] and incubated for 15 min at 4°C. After the addition of 250 µl of 10% NP-40 detergent, the cell suspension was vigorously mixed, incubated for 30 min at 4°C, and centrifuged for 15 min at 1,500 g. The resulting supernatant was adjusted to a protein concentration of 35 µg/µl and saved as the cytosolic extract. The nuclear pellet was resuspended in 1.5 ml of a solution containing 50 mM HEPES-NaOH (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, and 10% glycerol, mixed for 30 min at 4°C, and centrifuged for 15 min at 1,500 g. The resulting supernatant was adjusted to a protein concentration of 20 µg/µl and saved as the nuclear extract.

Oligonucleotides and electrophoretic mobility-shift assay analysis. A double-stranded oligodeoxynucleotide corresponding to the B binding motif of human immunodeficiency virus-type 1 (5'-GATCTAGGGACTTTCCGCTGGGGACTTTCCAG) was synthesized with a DNA synthesizer by the phosphoramidate method and purified on an OPC cartridge (Life Technologies, Grand Island, NY). The oligonucleotide was end-labeled with [-²P]dCTP and Klenow polymerase (Promega, Madison, WI). For electrophoretic mobility-shift assay (EMSA) of B-binding activity, nuclear extract (2 µl) was incubated at 37°C for 30 min in a total volume of 10 µl containing 10 mM HEPES-NaOH (pH 7.9), 50 mM KCl, 5 mM Tris · HCl (pH 7.0), 1 mM DTT, 15 mM EDTA, 10% glycerol, 1.0 µg of poly(dI:dC), and 4 ng of ³²P-labeled B oligonucleotide. The resulting DNA-protein complexes were resolved by PAGE on nondenaturing 8% gels with 0.5× Tris-borate-EDTA buffer at 4°C (29, 30). Control assays were also performed with oligonucleotides containing AP1 or SP1 binding sites (34, 45).

Immunoblot analysis. Whole cell, nuclear, and cytosolic extracts of spleen cells from various aged mice were subjected to SDS-PAGE on 12.5% gels under nonreducing conditions. The separated proteins were transferred electrophoretically to a polyvinylidene difluoride membrane, which was then incubated for 2 h at room temperature with TBS-T [20 mM Tris · HCl (pH 7.6), 137 mM NaCl, and 0.05% Tween 20] containing 8% bovine serum albumin. The membrane was then incubated for 12 h at 4°C with TBS-T containing the appropriate primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), washed four times with TBS-T for 15 min each time at room temperature, incubated for 2 h at room temperature with TBS-T containing alkaline phosphatase-conjugated secondary antibodies, washed five times with TBS-T, and subjected to the alkaline phosphatase color reaction by standard method.

Immunoprecipitation. Immunoprecipitation was performed with antibodies to the COOH terminus of IB (Santa Cruz Biotechnology) in RIPA 1640 buffer [50 mM Tris · HCl (pH 8.0), 150 mM NaCl, 0.2% NP-40, and 0.5% sodium deoxycholate] containing 0.1% SDS and 5 mM N-ethylmaleimide. Immune complexes were precipitated with protein A-Sepharose beads (Pharmacia, Piscataway, NJ) that had been equilibrated in the same buffer and were then washed with solution B [10 mM HEPES-NaOH (pH 7.4), 1 mM EDTA, 10 mM KCl, 50 mM NaF, 50 mM glycerol 2-phosphate, 1 mM sodium orthovanadate, PMSF (0.1 mg/ml), 0.2% NP-40, and 90 mM NaCl]. The washed complexes were eluted from the beads with solution B containing 0.1 M Capso (pH 11.2) and were then subjected to immunoblot analysis with antibodies to ubiquitin or to phosphoserine (Santa Cruz Biotechnology).

Cell survival assay. Spleen cells were prepared from male and female BALB/c, LMP2^/, and NOD mice, with and without diabetes, and were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and were exposed for various times to mouse TNF- (R&D Systems, Minneapolis, MN). Embryonic macrophages harvested from the liver of 13.5-day-old embryo and embryonic fibroblasts harvested from 14.5-day-old embryo were obtained from BALB/c, NOD, or LMP2^/ mice as described (6). Macrophage and fibroblasts were incubated with TNF- (10 ng/ml) for various times or with the indicated concentrations of TNF- for 24 h, after which the number of viable cells was determined by trypan blue exclusion, as described (6).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Impaired production of LMP2 protein and activation of NF-B in adult female LMP2^/ and NOD mouse spleen cells. We first examined the activation of NF-B in response to TNF- in spleen cells derived from 7-wk-old female NOD mice and LMP2^/ mice as well as in control human T cell lymphoma MOLT-4 cells and BALB/c mouse spleen cells. Incubation of MOLT-4 cells or BALB/c mouse spleen cells with TNF- (10 ng/ml) for 4 h resulted in a marked increase in NF-B activity, as determined by EMSA (Fig. 1A). In contrast, TNF- induced only a slight increase in NF-B activity in spleen cells from NOD mice or LMP2^/ mice. The specificity of the DNA-binding activity detected in the nuclear extracts of TNF--treated MOLT-4 cells or splenocytes from all three mouse strains was confirmed by the cold competition assay with unlabeled wild-type B and mutant B oligonucleotides. NF-B binding to the B probe was prevented by preincubation of the nuclear extracts with a 100-fold molar excess of unlabeled wild-type B oligonucleotide but not with an excess of a mutant B (data not shown). We conclude that the DNA-binding activities are due to the activity of NF-B; the specificity of the defect in NF-B DNA-binding activity in NOD and LMP2^/ mice was confirmed with control DNA-binding activities of the transcription factors SP1 and AP1. Exposure of splenocytes or MOLT-4 cells to TNF- had no effect on the binding of the transcription factors SP1 or AP1 to corresponding specific oligonucleotide probes, and the DNA-binding activities of these factors did not differ among the four cell types examined (Fig. 1A).

View larger version (68K): [in this window] [in a new window]   Fig. 1.   Comparison of nuclear factor-B (NF-B) activity, expression of NF-B subunits, and degradation of IB among LMP2/, nonobese diabetic (NOD), and BALB/c mouse spleen cells. A: MOLT-4 cells or spleen cells from 7-wk-old LMP2/, NOD, or BALB/c mice were incubated for 4 h in the absence () or presence (+) of tumor necrosis factor- (TNF-; 10 ng/ml), after which the DNA-binding activity of NF-B was examined by electrophoretic mobility shift assay (EMSA) of nuclear extracts (N.E.) with a 32P-labeled B oligonucleotide. Control assays were also performed with oligonucleotides containing SP1 or AP1 binding sites. The positions of specific DNA-protein complexes are indicated by arrowheads. Lane 1, negative control in which no nuclear extract was added to the reaction mixture. B: mouse spleen cells were incubated for 4 h in the absence () or presence (+) of TNF- (10 ng/ml), after which nuclear extracts were subjected to immunoblot analysis with antibodies to the indicated proteins. C: cell lysates prepared from BALB/c, NOD, and LMP2/ mice were derived from splenocytes from 18.5-day-old embryo (E18.5) and 7-day-old, 7-wk-old, and 5-mo-old animals and subjected to immunoblot analysis with antibodies to the indicated proteins. D: mouse spleen cells were incubated for the indicated times in the presence of TNF- (10 ng/ml), after which cytosolic extracts were subjected to immunoblot analysis with antibodies to IB or to the indicated CDKs. Top two IB bands correspond to the phosphorylated protein. E: whole cell lysates of mouse splenocytes were subjected to immunoblot analysis with antibodies to the indicated proteins. IKK, IB kinase; NIK, NF-B-inducing kinase. B, BALB/c; N, NOD; L, LMP2/.

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Impaired degradation of IB in response to TNF- in adult LMP2^/ and NOD female mouse spleen cells. The degradation of IB by the ubiquitin-proteasome pathway is required for TNF--induced NF-B activation (11, 39, 41, 59, 73). Therefore, we investigated the role of LMP2 in IB degradation with spleen cells from NOD and LMP2^/ mice compared with BALB/c mice. IB had virtually disappeared from the cytosol of BALB/c spleen cells after exposure to TNF- for 40 min (Fig. 1D); this decrease in the amount of cytosolic IB was not accompanied by an increase in the amount of this protein in the nucleus (data not shown). The abundance of IB in the cytosol of BALB/c spleen cells had begun to recover after treatment with TNF- for 240 min (Fig. 1D). In contrast, the amount of IB in the cytosol of NOD and LMP2^/ mouse spleen cells was not markedly affected by TNF- at any of the time points examined (Fig. 1D).

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Impaired TNF--induced IB degradation due to immature proteasome activity in adult female NOD or LMP2^/ mouse lymphocytes. We next investigated whether the impaired degradation of IB in spleen cells from NOD and LMP2^/ mice is attributable to defective biological activity of the proteasome or to upstream defects in the phosphorylation or ubiquitination of IB. Spleen cells were treated for various times with TNF-, and cell extracts were then prepared in the presence of 0.1% SDS and 5 mM N-ethylmaleimide to inhibit isopeptidase activities that might affect the detection of ubiquitinated proteins. Extracts were first subjected to SDS-PAGE and immunoblot analysis with antibodies to IB, which revealed a ladder of high-molecular-mass immunoreactive proteins that accumulated as early as 5 min after stimulation of cells with TNF- (Fig. 2A). These high-molecular-mass proteins as well as the bands corresponding to unmodified and phosphorylated IB were no longer apparent after exposure of BALB/c spleen cells to TNF- for 40 min; however, IB reappeared in these cells after incubation with TNF- for a total of 120 min (Fig. 2A, left). In contrast, the abundance of the high-molecular-mass immunoreactive proteins was decreased after incubation of NOD and LMP2^/ mice spleen cells with TNF- for 40 or 120 min, but they did not disappear until 240 min (Fig. 2A).

View larger version (48K): [in this window] [in a new window]   Fig. 2.   Defective TNF--induced degradation of IB in LMP2/ and NOD mouse spleen cells. A: spleen cells isolated from 7-wk-old BALB/c (left), NOD (middle), and LMP2/ (right) mice were incubated in the absence () or presence (+) of TNF- (10 ng/ml) for the indicated times. Cell extracts were then prepared in RIPA 1640 buffer containing 0.1% SDS and 5 mM N-ethylmaleimide and subjected to immunoblot analysis with antibodies to IB. Positions of molecular size standards (in kDa) are shown on the left and those of polyubiquitinated [(Ub)n-IB], phosphorylated (P-IB), and unmodified IB are indicated on the right. B and C: extracts of BALB/c (left), NOD (middle), and LMP2/ (right) mouse spleen cells (prepared as in A) were subjected to immunoprecipitation with antibodies to IB; the resulting precipitates were then subjected to immunoblot analysis with antibodies to ubiquitin (B) or to phosphoserine (C).

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Lineage and developmental susceptibility of LMP2^/ and NOD mouse cells to TNF--induced apoptosis. The activation of NF-B by the ubiquitin-proteasome pathway protects cells from TNF--induced apoptosis (6, 51, 61, 66, 71). Furthermore, inhibition of the nuclear translocation of NF-B enhances the apoptotic effect of TNF-. Lymphoid-specific apoptotic defects are characteristic phenotypes of autoimmunity, but the histological evidence of autoimmunity-induced target cell destruction is not apparent in NOD mice until after 5 wk of age. We investigated the developmental time course of the sensitivity of cell viability to TNF- with mouse embryonic fibroblasts (MEFs), embryonic macrophages, and spleen cells from 1-day-old, 7-day-old, 7-wk-old, and 5-mo-old female mice. Whereas TNF- had no effect on the viability of cultured MEFs from either NOD or BALB/c mice, it induced a dose- and time-dependent decrease in the survival of LMP2^/ MEFs (Fig. 3A). TNF- also had no effect on the viability of cultured macrophages derived from BALB/c or NOD mouse liver on embryonic day 13.5, but it reduced the survival of such cells prepared from LMP2^/ mouse embryos in a dose- and time-dependent manner (Fig. 3B). These observations suggest that Lmp2 expression is not defective in MEFs and macrophages derived from NOD mouse embryos, consistent with the lack of histological evidence of autoreactivity at this early stage of development.

View larger version (74K): [in this window] [in a new window]   Fig. 3.   Effects of TNF- on the survival of fibroblasts from embryonic day 14.5 and macrophages from embryonic day 13.5 BALB/c, NOD, and LMP2/ mouse embryos as well as of spleen cells isolated from BALB/c and NOD mice. A and B: fibroblasts and macrophages, respectively, derived from embryonic day 14.5 or 13.5 BALB/c, NOD, and LMP2/ mouse embryos were incubated either with various concentrations of TNF- for 24 h (left) or in the presence of TNF- (10 ng/ml) for the indicated times (right), after which cell viability was assessed by trypan blue exclusion. Data are means ± SD of 4 replicates from representative experiments and expressed as a percentage of the survival value for the corresponding cells not exposed to TNF-. C and D: spleen cells isolated from BALB/c or NOD mice at postnatal day 1 (C) or postnatal day 7 (D) were treated as in A and B. E: various cell types analyzed in A-D were incubated for 24 h in the absence or presence of TNF- (10 ng/ml), after which DNA fragmentation was evaluated by agarose gel electrophoresis and ethidium bromide staining. MEF, mouse embryonic fibroblast.

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Gender- and disease-specific susceptibility of adult NOD mouse cells to TNF--induced apoptosis. Whereas incubation of spleen cells from 7-wk-old BALB/c mice with TNF- had virtually no effect on cell survival, TNF- induced a marked dose- and time-dependent decrease in the viability of spleen cells from 7-wk-old female and male NOD mice, an age at which insulititis is exhibited (Fig. 4A). The effect of TNF- on NOD mouse spleen cells appeared more pronounced for female than for male animals for both dose and time course exposure studies. NOD mice exhibit a sex difference with regard to the onset of diabetes. In females, the onset of diabetes is first apparent at ~20 wk of age, and the number of animals that develop overt diabetes increases with age, with the cumulative incidence of diabetes being 70-80% by 30 wk of age (38). In contrast, <30% of male NOD mice typically have diabetes by 30 wk of age. Agarose gel electrophoresis again confirmed that TNF- induced internucleosomal fragmentation of DNA in spleen cells from 7-wk-old NOD mice but not in those from age-matched BALB/c mice (Fig. 4B). Furthermore, TNF- induced a dose- and time-dependent decrease in the viability of spleen cells derived from 5-mo-old NOD mice with IDDM but had no such effect on spleen cells from 5-mo-old BALB/c mice (Fig. 4C). Again, internucleosomal fragmentation of DNA characteristic of apoptosis was apparent in TNF--treated spleen cells from 5-mo-old NOD mice with IDDM but not in those from age-matched BALB/c mice (Fig. 4D).

View larger version (68K): [in this window] [in a new window]   Fig. 4.   Effects of TNF- on the survival of spleen cells derived from BALB/c and NOD mice at 7 wk and 5 mo of age. A and C: spleen cells isolated from male or female BALB/c or NOD mice at 7 wk (A) or 5 mo (C) of age were incubated either for 24 h with various concentrations of TNF- (top) or for the indicated times with TNF- (10 ng/ml) (bottom), after which cell viability was assessed by trypan blue exclusion. The 5-mo-old NOD mice studied exhibited symptoms of autoimmune diabetes. Data are means ± SD of 4 replicates from representative experiments and expressed as a percentage of the survival value for the corresponding cells not exposed to TNF-. IDDM, insulin-dependent diabetes mellitus. B and D: spleen cells from male (M) or female (F) BALB/c or NOD mice at 7 wk (B) or 5 mo (D) of age were incubated for 24 h in the absence or presence of TNF- (10 ng/ml), after which DNA fragmentation was analyzed by agarose gel electrophoresis and ethidium bromide staining.

Gender-specific susceptibility of adult LMP2^/ mouse cells to TNF--induced apoptosis. TNF- induced a dose- and time-dependent decrease in the viability of both male and female spleen cells derived from 7-wk-old LMP2^/ mice but had no such effect on spleen cells from 7-wk-old BALB/c mice (Fig. 5A). Again, internucleosomal fragmentation of DNA characteristic of apoptosis was apparent in TNF--treated spleen cells from 7-wk-old LMP2^/ mice but not in those from age-matched BALB/c mice (Fig. 5B). Thus the LMP2 defect in proteasome function in these cells was associated with no gender, developmental, or cellular specificity in TNF--induced apoptosis in contrast to the spontaneous LMP2 defect in NOD. Furthermore, anti-nuclear autoantibodies (ANA) were significantly detected in the serum from LMP2^/ mice (data not shown) as well as NOD mice (32). In recent years, it has become clear that ANA has proven to be a useful serological test in the diagnosis of various autoimmune disease.

View larger version (42K): [in this window] [in a new window]   Fig. 5.   Effects of TNF- on the survival of spleen cells derived from BALB/c and LMP2/ mice at 7 wk of age. A: spleen cells isolated from male or female LMP2/ or BALB/c mice at 7 wk of age were incubated either for 24 h with various concentrations of TNF- (top) or for the indicated times with TNF- (10 ng/ml) (bottom), after which cells viability was assessed by trypan blue exclusion. Data are means ± SD of 4 replicates from representative experiments and are expressed as percentage of the survival value for the corresponding cells not exposed to TNF-. B: spleen cells from male (M) or female (F) BALB/c or LMP2/ mice at 7 wk of age were incubated for 24 h in the absence or presence of TNF- (10 ng/ml), after which DNA fragmentation was analyzed by agarose gel electrophoresis and ethidium bromide staining.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We show here that LMP2 ablation in the context of adult NOD mouse or LMP2^/ mouse confers a unique collection of phenotypes, some of which are associated with autoimmune symptomalogy. The LMP2 protein plays an important role in the proteasomal processing of self-antigens for T cell education and prevention of T cell autoreactivity (1, 2, 8, 22, 24, 55, 70). In this study, we now show that interrupted proteasomes lacking the LMP2 subunit either through a spontaneous defect (NOD) or induced defect (LMP2^/) impairs activation of NF-B, promotes TNF--induced cell death, and impairs degradation-induced IB. In contrast, the basal expression of NF-B-inducing kinase, which phosphorylates and activates IB kinase, and the upstream phosphorylation and ubiquitination of IB are intact in NOD and LMP2^/ splenocytes, thus implementing specific defects in the proteasome as responsible for the overlapping phenotypes. These findings document the feasibility of detecting important intermediary disease phenotypes by expressing genetically interrupted candidate genes on normal background mice. Remarkably, diverse autoimmune phenotypes are still clearly expressed.

The induced genetic disruption of the Lmp2 gene in LMP2^/ mice differs in three important aspect from the defect in LMP2 protein expression in NOD mice. First, LMP2 expression is deficient in all tissues of the LMP2^/ mice throughout embryonic development and postnatal life. In contrast, the expression of this gene is impaired only in cells of the lymphoid and myeloid lineage and only after ~7 wk of age in NOD mice (Figs. 3-5). This difference is likely central to the fact that restricted target destruction is apparent in NOD mice but not in LMP2^/ mice. Although spontaneous autoimmunity in the NOD mouse and humans with Type 1 diabetes is associated with disruption of peptide presentation by lymphoid cells, pancreatic -cells show increased expression of MHC class I molecules and self-peptides at early stages of the disease before lymphoid invasion and increased LMP2 and NF-B activation (19, 25, 46, 65). These discordant expressions of MHC class I molecules, the proteasome, and NF-B likely underlie target selection and direct cytotoxic T cell attack. Second, the defects in proteasome-mediated NF-B activation and TNF--induced apoptosis in LMP2^/ mice are not gender restricted. Third, for yet fully defined reasons, the apoptotic defects in adult NOD mouse splenocytes are of a greater magnitude than those in LMP2^/ mice, suggesting that additional genetic mechanisms may contribute to the increased apoptosis sensitivity.

The defect in proteasome function in NOD and LMP2^/ adult mouse splenocytes was evident from impaired IB degradation by ubiquitin-proteasome signal pathways (Figs. 1, C and D, and 2). The increased sensitivity of NOD mouse spleen cells to TNF--induced apoptosis was one consequence of the failure of TNF- to induce activation of NF-B in these cells (Figs. 3 and 4). The role of LMP2 in NF-B activation was confirmed by two approaches. First, the use of the LMP2^/ spleen cells, similarly missing the MHC encoded proteasome subunit, demonstrates the new obligatory role of LMP2 in TNF--induced IB degradation (Figs. 1, C and D, and 2). Second, only NOD tissues lacking LMP2 protein as well as LMP2^/ MEFs and embryonic macrophages have impaired NF-B activation and are observed to have TNF--induced apoptosis (Figs. 3 and 4). Macrophages and fibroblasts obtained from 13.5-day-old and 14.5-day-old NOD embryos have normal levels of LMP2 protein expression and intact resistance to TNF- exposure (Fig. 3, A, B, and E). In contract, 7-wk-old adult NOD splenocytes, granulocytes, and macrophages have missing LMP2 protein and/or dysfunctional NF-B with TNF--induced cell death (26, 28) (Fig. 4). Islet cells, erythrocytes, and liver cells derived from adult NOD mice did not exhibit a defect in LMP2 expression, as revealed by immunoblot analysis and/or TNF- sensitivity (data not shown). Dysfunction of Lmp2 gene in the MHC region of the genome thus virtually abolishes the activity of a transcription factor NF-B that plays important roles in immune and other functions. The NOD mouse is thus a newly defined developmental stage- and tissue-specific mosaic model of discordant MHC gene expression and proteasome dysfunction. Furthermore, the LMP2^/ mouse is a model of autoimmune phenotypes controlled by a single gene.

The ubiquitin-proteasome pathway plays an essential role for various key biological processes, including cell cycle progression, transcription, and signal transduction (40). Although proteasome subunits vary minimally among eukaryotic cells, interferon- increases the expression of LMP2 and LMP7, which are both encoded by genes located in the MHC region of the genome. Most cells express basal amounts of each of these proteins in the absence of interferon- (20, 24, 31, 62). The incorporation of LMP2 and LMP7 into proteasomes has been viewed only as an immune function that promoted the generation of endogenous peptides compatible with the peptide-binding cleft of MHC class I molecules (1, 2, 8). Our data now suggest that LMP2 plays an obligatory role in TNF--induced NF-B activation and cell survival (27).

The transcription factor NF-B requires complex processing as well as IB degradation by ubiquitin-proteasome signal pathway for functional activity. Once activated, NF-B protects cells from TNF--induced apoptosis, promotes lymphocyte maturation and antigen processing, and regulates the expression of various cytokine genes. Indeed, the phenotypes of knockout mice lacking Rel family proteins or LMP2 itself show partial overlap in symptomatology to those of NOD mice (7, 10, 35, 54, 62, 69). However, LMP2^/ mice do not develop diabetes by 32 wk of age, consistent with the fact that the well-established genetic requirements of multiple chromosomal regions appear to determine disease penetrance in NOD mice and humans. In addition, the homogenous nature of the gene defects in all tissues in LMP2^/ mice does not mirror the mosaic nature of developmental stage and tissue-specific dysregulation of the NOD proteasome cleavage errors. Significantly, impaired LMP2 expression at select developmental stages and in a tissue-specific manner in the NOD proteasome could confer target selection and disease expression. Restoration or continuous normal expression of endogenous peptide presentation by cell surface MHC class I molecules on select NOD islet tissue could elicit an ensuing response of the immature and improperly educated immune system. Furthermore, ANA were significantly detected in the serum from LMP2^/ mice (data not shown) and NOD mice (32). In recent years, ANA has clearly proven to be useful in the diagnosis of various autoimmune disease. Importantly, the published studies have reported similar phenotypes of knockout mice lacking the NF-B subunits or LMP2 relative to NOD mice.

In conclusion, we have shown that Lmp2, which is located in the MHC region of the genome, is an important gene in the control and prevention of diverse symptoms of autoimmunity that are apparent in both NOD and LMP2^/ mice. The diabetes in NOD mice appears to be a gender-related autoimmune disease (38); therefore, the proteasome dysfunction in NOD mice results from the age-related and preferential gender-specific lack of Lmp2 expression. This abnormality results in the inability of lymphoid cells to activate NF-B. The abnormal processing of intracellular proteins thus may contribute to a portion of the autoimmune phenotypes of the NOD mice and possibly play a role in the pathogenesis of autoimmunity.