Autoimmune kidney disease and lymphadenopathy in NODlpr mice are not modified by deficiency in tumor necrosis factor receptor 1 or ß2-microglobulin
Tara Catterall1,
Dina Stockwell1,
Vikki Marshall2,
Andreas Strasser2 and
Janette Allison1
1 Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia 2 Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Parkville, Victoria 3050, Australia
Correspondence to: J. Allison; E-mail: janettea{at}unimelb.edu.au
Transmitting editor: D. Tarlinton
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Abstract
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Fas and TNFRI, two members of the tumor necrosis factor receptor family with an intracellular death domain, each play critical roles in apoptotic death of lymphocytes and certain other cell types. We determined the overlapping functions of Fas and TNFRI by breeding non-obese diabetic (NOD) mutant mice that lacked both receptors. NODlpr mice developed extensive lymphadenopathy, splenomegaly, CD4CD8 B220+
ßTCR+ T cells and autoimmune kidney disease. This pathology was not modified by concomitant deficiency in TNFRI as was reported for lpr mice on a B6 background. NODlpr mice lacking CD8+ T cells, because of a null mutation in ß2-microglobulin (ß2m), also developed a similar disease profile to NODlpr animals, but the CD4CD8 B220+
ßTCR+ T cells now derived from a CD4+ T cell lineage. These results demonstrate that, as in the autoimmune-prone MRL stain, the NOD genetic background promotes lupus nephritis-like pathology and extensive lymphadenopathy when lpr is present. Loss of TNFRI does not exacerbate the pathology caused by deficiency in Fas and loss of ß2m does not reduce it.
Keywords: autoimmunity, lupus nephritis, lymphadenopathy, non-obese diabetic
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Introduction
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Fas (CD95/APO-1) and TNFRI belong to the death receptor subgroup of the tumor necrosis factor receptor family (Fas, TNFRI, DR3, DR6, TRAILR1 and TRAILR2). These receptors contain an intracellular death domain and signal via adapter proteins (FADD and TRADD) resulting in recruitment and activation of caspase-8 and -10 (in humans) with subsequent initiation of the caspase cascade leading to apoptotic cell death (13). Death receptors can also promote cell proliferation and differentiation, a function well characterized for TNFRI, but more controversial for Fas (4).
In mouse, the lpr mutation in the Fas gene results in a 10-fold decrease in expression of the protein, leading to a striking phenotype that varies depending on the genetic background (1,5). Common to all strains is an age- and thymus-dependent increase in the number of lymphocytes (lymphadenopathy) as well as splenomegaly, hypergammaglobulinemia and autoantibody production. This phenotype is most pronounced on the MRL background, but of slower onset and severity on the C57BL/6 (B6) background. For example, in the autoimmune-prone MRL strain, the lpr mutation promotes severe kidney pathology, skin disease and arthritis, whereas it does not in non-autoimmune strains (e.g. C3H, BALB/c and B6). Lymphadenopathy can occur in the absence of CD8+ T cells (6) or CD4+ T cells (7), is reduced but present in the absence of B cells (8) and does not require CD28 or B7-1/B7-2 signals to develop (9,10). Autoimmunity in the MRLlpr mouse, on the other hand, depends on B cells (11), CD4+ T cell help for the B cells (6,7) and class I-restricted T cells (12). If
ß T cells are removed, 
T cells or some other T cell subset can provide the necessary help for B cells to allow Ig class switching and progression to autoimmunity (13). Since transgenic expression of Fas in T cells of MRLlpr mice can rescue them from T cell lymphadenopathy, but not autoimmune kidney disease (14), B cells are primarily responsible for the kidney pathology.
All lymphocyte types (
ß T, 
T, B and NK) contribute to the lymphadenopathy, but a major player is the unusual TCR
ß+, B220+ T cell population that is double-negative for CD4 and CD8 (hereafter called B220+ T cells). This population can represent up to 80% of all lymphocytes in aged (>5 months) lpr mice. It has the phenotype of chronically activated T cells with up-regulated levels of CD69, Ly6C, CD44, CD28, B7-1 and surface Fas ligand (FasL) (1517), and an abnormally phosphorylated CD3
chain (18). These cells are inert and cannot be activated in vitro with certain mitogens or antigens, but proliferate in response to phorbol myristate acetate and ionomycin. Studies with lpr mice that lack ß2-microglobulin (ß2m) showed that the unusual B220+ T cell subset was derived from peripheral naive CD8+ T cells that had somehow become activated (12,1922). In some studies, lpr/ß2m deficiency resulted in an overall decrease in lymphadenopathy (12,20). In other studies, where mice were of mixed genetic background, lymphadenopathy was unaffected and the 10- to 15-fold decrease in B220+ T cells was accompanied by an increase in other lymphocyte subsets such as B cells (21), CD4+ T cells (19), 
T cells (20) or CD4loB220+ T cells (19).
It is still unclear what drives lymphadenopathy in lpr mice. The previously popular idea, that the lpr mutation prevented the deletion of T cells activated by exogenous microbial antigens, is not supported by data from Maldonado showing that germ-free MRLlpr mice still developed lymphadenopathy, B220+ T cells and autoimmunity (23). Other studies also showed that Fas plays little if any role in the death of activated T cells responding to viral infection (LCMV) (24), exogenous peptide (LCMV or hen egg lysozyme) (25,26), exogenous superantigen (staphylococcal enterotoxin B) (27,28), in vivo administration of anti-CD3 (29) or a physiological T cell stimulus (H-Y antigen) (30). Other mechanisms such as death by neglect controlled the T cell response in these situations (27,28), although it has been proposed that Fas may be important in regulating the T cell response to weak stimuli, such as low-level anti-CD3 administration (29) or suboptimal peptide concentrations (24).
It seems possible, therefore, that lymphadenopathy in lpr mice is driven by self-antigens and/or growth factors produced by activated lymphocytes. Senju et al. found that even naive T and B cells (CD44lo) contributed to lymphadenopathy in Fas knockout chimeric mice (31), implying that cognate TCR interactions were not essential for expansion or survival of lymphocytes. Conventional CD4+ and CD8+ T cells in lpr mice acquire an activated phenotype with age, and produce large amounts of cytokines such as IFN-
, tumor necrosis factor (TNF)-
and IL-2 in response to TCR cross-linking (15). Evidence that such cytokines may contribute to lymphadenopathy comes from experiments with transgenic mice overexpressing the B cell-activating factor, BAFF (a member of the TNF family) (32). These animals accumulate large numbers of B cells and develop systemic lupus erythematosus-like autoimmune disease. Interestingly, BAFF was reported to be overexpressed in lpr mice undergoing autoimmune disease (33).
The other well-studied death receptor, TNFRI (p55-TNFR), can promote lymphocyte death or survival. Deficiency in this receptor does not lead to lympho-accumulation, however, but impairs responses to intracellular parasites and causes problems in lymphoid tissue architecture (34). It was reported that TNFRI deficiency could synergize with lpr to increase lymphadenopathy in B6 mice (35). We show here that in the autoimmune-prone non-obese diabetic (NOD) mouse, the lpr mutation induces massive lymphadenopathy and autoimmunity, but this is not increased by removal of TNFRI nor decreased by loss of ß2m.
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Methods
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Mice
Animals were bred under specific pathogen-free conditions at the Walter and Eliza Hall Institute (Melbourne) and the Department of Microbiology, University of Melbourne. Experiments using animals were performed with the permission of the University of Melbournes Animal Experimentation Ethics Committees (project no. 01110). NODlpr mice were produced by backcrossing the lpr mutation from the C3H/HeJ strain to NOD/Lt for seven generations and then intercrossing (36). At the seventh backcross, NOD DNA was identified at D19Mit60 (15 cM) and D19Mit88 (34 cM) around the faslpr locus (
24 cM). Homozygosity for the NOD MHC locus (H-2g7) was set at the fourth backcross by screening for the absence of H-2Kk using flow cytometry. NOD mice lacking TNFRI (NODt mice) were produced by backcrossing the targeted mutation in the TNFRI gene (37) from a mixed 129/Ola x C57BL/6 strain to NOD for seven generations. NOD DNA was then identified at D6Mit149 (46.3 cM) and D6Mit14 (63.4 cM) around the TNFRI locus (60.4560.75 cM). Homozygosity for the NOD MHC locus (H-2g7) was determined by absence of H-2Kb using flow cytometry. Microsatellite and gene position data were obtained from the UCSC Genome Bioinformatics Site (http://genome.ucsc.edu). Neither NODlpr nor NODt mice develop diabetes. NODlprt mice were produced by intercrossing these two strains at the seventh backcross and selecting for double-mutant animals that were then interbred. NODlpr, NODlprt and NODt mice were also bred to the 10th backcross generation. NODlprt mice lacking ß2m were produced by mating NODlprt animals to the backcross 9 NOD ß2m knockout mice described by Kay et al. (38) which carried a targeted mutation in the ß2m gene, originally performed in 129/Sv D3 embryonic stem cells (39). Mice lacking CD8+ T cells in peripheral blood (and so homozygous-null for ß2m) were selected and littermates carrying the following genotypes analyzed: (i) NODlpr0/0 TNFRIw/0 (or w/w) ß2m0/0, (ii) NODlprw/0 (or w/w) TNFR0/0 ß2m0/0 and (iii) NODlpr0/0 TNFRI0/0 ß2m0/0. There was no difference between mice that were w/0 or w/w. Spleen weights were determined for some of the other genotypes, not carrying the lpr mutation, at 14 weeks of age and ranged from 0.08 to 0.12 g.
Screening of mice
Screening of mice for the wild-type Fas gene or the lpr mutation was performed by PCR (36). Screening of the wild-type TNFRI and TNFRI knockout genes was performed using Southern hybridization of BamHI-digested DNA (from tail tips) probed with a cDNA probe to TNFRI. Screening for deficiency in ß2m was performed by testing for the absence of CD8+ T cells in peripheral blood by flow cytometry.
Coulter counting of peripheral blood cells
Blood (20 µl) from the tail vein of mice was collected on ice through heparinized capillaries and then lysed in Coulter lysis buffer (Becton Dickinson, San Jose, CA) and the number of leukocytes measured on a Coulter Counter. Duplicate counts were performed. Mice were tail bled at 6 weeks of age then at 2-weekly intervals until 22 weeks old. Some mice that became unwell (defined by weight loss, hunching and ruffled fur) before 22 weeks were culled.
Cell preparation and counting
Mice were weighed and killed by CO2 asphyxiation. Lymph nodes (inguinals and axillaries) and spleen were removed and weighed. Lymph nodes were finely minced with scissors in 10 or 50 ml of PBS/10% FCS on ice and then homogenized in a glass homogenizer as gently as possible. In spite of this, isolation of cells from large lymph nodes could result in as much as 40% lysed cells which appeared as large pale blue cells after Trypan blue staining. Cells were counted in duplicate by Trypan blue exclusion and the number of live cells recorded. Cells were washed twice and then stained for flow cytometry.
Flow cytometry
Lymph node cells (106) were stained in 50 µl of antibody mix after blocking of Fc receptors with anti-FcR
II/III (24G2) supernatant. Antibodies (from PharMingen, San Diego, CA) were anti-CD4phycoerythrin (GK1.5), anti-CD8FITC (53.6.7), anti-B220FITC (RA3-6B2), anti-TCRß chainphycoerythrin (H57-597), anti-CD25FITC (PC61), anti-CD44FITC (IM7), anti-CD45RBphycoerythrin (16A), anti-CD69FITC (H1.2F3) and anti-CD4biotin (GK1.5) detected with allophycocyaninstreptavidin. Flow cytometry was performed using a Becton Dickinson FACSort upgraded with a red diode laser. A live forward/side scatter gate encompassing lymphocytes, granulocytes and macrophages (but excluding smaller dead cells) was set. Counts (50,000100,000) were collected.
Histology and immunofluorescence staining
Three or more male mice from each genotype (NOD, NODlpr, NODlprt and NODt) were analyzed at 5, 8, 12 and 16 weeks for pathology in the pancreas, salivary gland, liver, kidney and lung. Female NODlpr and NODlprt mice were likewise analyzed at 2025 weeks, as were male NODtriple mice. Bouins solution fixed, paraffin-embedded, sections were used and stained with hematoxylin & eosin. For detection of C3 complement, cryostat frozen sections of kidney were cut and fixed in acetone at room temperature for 5 min. They were stained with goat anti-C3 (1:200 dilution) conjugated to FITC (Cappel ICN, Costa Mesa, CA) for 1 h at room temperature.
Statistical analysis
Comparison between experimental groups was performed using an unpaired, two-tailed, Students t-test with unequal variance.
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Results
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Lymphadenopathy in NODlpr mice
NODlpr mice were screened every 2 weeks for lymphadenopathy over a 22-week period by analyzing peripheral blood lymphocytes (Fig. 1). Starting at 6 weeks of age, lymphadenopathy in male and female mice progressed with a similar steady increase over time, with up to 4-fold variation in blood cell counts in older mice. NODlpr animals at 22 weeks of age had anywhere from 3- to 10-fold more blood cells than NOD controls.

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Fig. 1. Progress of lymphadenopathy in NODlpr mice. NODlpr female and male mice (A and B, N = 34 and 26) were assessed every 2 weeks for peripheral blood lymphocytes by Coulter counting, as were peripheral blood lymphocytes from control NOD female and male mice (C and D, N = 10 and 9) and B6 females (E, N = 5). Error bars represent the SD. NODlpr mice for this experiment were from a 10th backcross to NOD.
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Activation status of CD4+ and CD8+ T cells in NODlpr mice
Lymph node cells from young (7 week) and old (21 week) NODlpr mice were analyzed by flow cytometry for the activation markers CD25, CD44, CD69 and CD45RB. About 7% of CD4+ T cells, 1% of CD8+ T cells and <1% of B220+ T cells from all mice expressed CD25 (not shown). CD44 expression was high on CD4+ and CD8+ T cells from both young and old NODlpr mice when compared to control NOD and C57BL/6 animals (Fig. 2). CD4+ and CD8+ T cells from young NOD and NODlpr mice showed similar profiles for the activation markers CD69 and CD45RB (a marker which is usually down-regulated on activated CD4+ T cells). CD4+ T cells from old NODlpr mice had more cells expressing CD69 (
50%) and an unusually uniform pattern of high level CD45RB (Fig. 2). B220+ T cells from old NODlpr mice typically expressed CD69 and did not down-regulate CD45RB (not shown). This pattern of activation marker expression on CD4+ and CD8+ T cells is similar to that reported for other lpr mouse strains (15,29,31,40).

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Fig. 2. Expression of activation markers on CD4+ and CD8+ T cells from NODlpr mice. Lymph node cells from young male NODlpr (7 week) and old NODlpr (21 week) mice were triple stained for CD4, CD8 and one of CD44, CD69 or CD45RB. T cells were gated for CD4+ or CD8+ and activation markers expressed by them displayed as a histogram. Control male mice were aged 7 (NOD) and 12 (C57BL/6) weeks.
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Lymphadenopathy in NODlprt mice
Previous reports found that, in B6 mice, lymphadenopathy induced by the lpr mutation was greatly accelerated by deficiency in TNFRI (35). NODlpr mice were therefore mated to NOD mice lacking TNFRI (NODt mice) to generate NODlprt knockout animals. A time-course analysis of lymph nodes and spleen from lines of male NODlpr, NODt and NODlprt animals was performed. At 5 weeks of age lymphadenopathy was already detectable in NODlpr and NODlprt lymph nodes and spleen, with the weights of these organs being almost double that of NODt and NOD controls (Fig. 3). At 8 weeks there was a modest increase in the weights of both experimental groups and at 12 weeks there was large increase. NODlpr and NODlprt animals at 12 weeks of age showed greatly increased lymph node weight (up to 60-fold) and spleen weight (up to 15-fold) when compared to NOD or NODt controls. There was no difference between male NODlpr and NODlprt mice with respect to lymph node or spleen weight at any age, even out to 1720 weeks. Because Zhou et al. (35) analyzed lymph node weight and cellularity in female (not male) mice, we performed a limited analysis of spleen and lymph node weights from NODlpr and NODlprt females at 1525 weeks of age (Fig 4). As with males, there did not appear to be any significant difference in lymph node and spleen weight between NODlpr and NODlprt female animals. Zhou et al. reported a 5- to 10-fold increase in lymph node weight and total cell numbers in B6lprt mice compared to B6lpr mice at all ages (35). For instance, at 8 weeks of age they found a 5-fold weight difference between B6lprt and B6lpr lymph nodes. However, we saw no difference in our NODlpr and NODlprt mice at 8 weeks, even though the NOD genetic background increased the overall lymphadenopathy in both groups when compared to B6lpr mice at this age.

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Fig. 3. Lymphadenopathy in NODlpr and NODlprt mice at different ages. Male NODlpr, NODlprt, NODt and NOD mice (at the seventh backcross) were analyzed for lymph node cellularity, lymph node weight (inguinals and axillaries; expressed as mg/g body wt) and spleen weight at different time points (cellularity was not done for the 1520 weeks time points). Each bar represents an individual mouse. Comparison of NODlpr and NODlprt spleen or lymph node weights at 12, 15 and 1720 weeks did not show a statistically different result except for spleen weights at 12 weeks (P < 0. 01). However, given the large variation in organ weights and the small sample number, this difference is unlikely to be biologically significant. Similar results were found for male and female NODlpr, NODlprt and control mice at the 10th backcross, which were analyzed for spleen weights only (N > 6 per time point performed at 8, 13,17 and 21 weeks of age).
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Fig. 4. Lymphadenopathy in NODlpr and NODlprt female mice. Female NODlpr and NODlprt mice (at the seventh backcross) were analyzed for lymph node weight (inguinals and axillaries; expressed as mg/g body wt) and spleen weight at different time points. Each bar represents an individual mouse.
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The cell types making up the lymphadenopathy were analyzed by flow cytometry. The B220+ T cell subset was similar in both NODlpr and NODlprt mice, and comprised
50% of the cellularity of the lymph nodes from 12-week-old animals (Fig. 5, rows 1 and 2). The remaining lymphadenopathy was distributed between CD4+ plus CD8+ T cells and B cells (Fig. 5). At 5 weeks of age, when B220+ T cells were <6% of the total lymphocyte population, the T:B ratio was
1.6 in NODlpr animals compared to 4:1 in control NOD mice (Fig. 6), indicating that the B cell pool had expanded more than the T cell pool. The T:B cell ratio in NODt mice was
8:1, but this reduced to 4:1 when lpr was present (Fig. 6), again indicating a preferential expansion of the B cell pool over the T cells pool in NODlprt animals.

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Fig. 5. Flow cytometric analysis of cell types from NODlpr and NODlprt animals. Lymph node cells from seventh backcross male NODlpr, NODlprt, NODt and NOD mice were analyzed for B220+ T cells, ßTCR+ T cells and B cells at different time points. The number of mice analyzed is given above the data points in the cell count plot. Plots for NODlpr animals (row 1) are displayed separately from NODlprt (row 2) to clearly show the overlap of data. Antibodies used were anti-B220 (RA3-6B2) and anti-TCRß (H57-597). Error bars represent the SD. (Note the 2-fold difference in scale for cell counts and cell type numbers.)
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Fig. 6. T:B cell ratio of cells from NODlpr, NODlprt, NODt and NOD male animals at different ages. Data from Fig. 5 was analyzed for the T:B cell ratio. The T cell population includes CD4+ T, CD8+ T and B220+ T cells. Error bars represent the SD.
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To summarize, deficiency in TNFRI did not potentiate lymphadenopathy in NODlpr mice. Lymphadenopathy occurred in conventional T and B cells as well as the B220+ T cell subset.
Lymphadenopathy in NODlpr mice lacking CD8+ T cells
Studies investigating the effect of the lpr mutation in different genetic backgrounds (B6, MRL and C3H/129 mixed) have clearly shown that the B220+ T cell population derives from CD8+ T cells. In some studies, deficiency in CD8+ T cells diminished the overall lymphadenopathy normally seen in lpr mice (12,20), whilst in others little effect on lymphadenopathy was observed (19,21,22). We bred NODlprt mice to NOD mice deficient in ß2m (and therefore lacking CD8+ T cells) and analyzed the littermates. Lymph node and spleen weights were, in general, similar for NODlprt mice and NODlpr mice deficient in ß2m (NODlprbeta) or deficient in both ß2m and TNFRI (NODtriple) (Fig. 7). However, lymph node weights from NODlprbeta mice at 8 and 1819 weeks were statistically different from those of NODlprt mice (P < 0. 01). This might reflect a true increase due to deficiency in ß2m or result from the influence of non-NOD genes linked to the mutated loci.

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Fig. 7. Lymphadenopathy in NODlprbeta and NODtriple mice at different ages. NOD and NODlprt male mice (at the seventh backcross), as well as littermates with the following mutations: (a) lpr and ß2m (lprbeta), (b) lpr, TNFRI and ß2m (triple), and (c) TNFRI and ß2m (tbeta) were analyzed for lymph node weight, lymph node cellularity and spleen weight at different time points. Each bar represents an individual mouse. Data at 5 weeks of age were only obtained for lprbeta mice. To perform the statistical analysis, NODlprt data from Fig. 3 was included since the sample size for NODlprt mice in Fig. 7 was too small.
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The cell types making up the lymphadenopathy were analyzed by flow cytometry. The B220+ T cell subset was similar in NODlprbeta and NODlprt mice (Fig. 8, rows 2 and 3), although the variation in cell numbers at 19 weeks of age was larger amongst NODlprbeta animals. It appeared that the B220+ T cell subset was reduced in NODtriple mice (Fig. 8, row 1). This subset thus comprised
50% of the cellularity of the lymph nodes from19-week-old NODlprbeta and NODlprt animals, but only
2030% of that from NODtriples. The remaining lymphadenopathy was distributed between CD4+ or CD8+ T cells and B cells. Similar results were found for male NODtriple mice at 23 weeks of age (N = 5), where the B220+ T cell population comprised
2030% compared to >80% for NODlprt mice (N = 5) (P < 0. 01; not shown).

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Fig. 8. Flow cytometric analysis of cell types from NODlprbeta and NODtriple animals. Lymph node cells from male NOD and NODlprt as well as NODlprbeta, NODtriple and NODtbeta littermates were analyzed for B220+ T cells, ßTCR+ T cells and B cells at different time points. The number of mice analyzed is given above the data points in the cell count plot. Plots for NODtriple (row 1) and NODlprbeta (row 2) animals are displayed separately from controls (row 3) to clearly show the overlap of data. Antibodies used were anti-B220 (RA3-6B2) and anti-TCRß (H57-597). Error bars represent the SD. (Note the 3-fold difference in scale for cell counts and cell type numbers.)
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Analysis of the CD8+ T cells in ß2m-sufficient and -deficient groups was also performed. In all groups of mice lacking ß2m, the CD8+ T cells comprised <1% of the total leukocyte population regardless of whether the mice also carried the lpr or lpr and TNFRI double mutations (not shown). Thus, the lpr-induced lymphadenopathy resulted in the relative expansion of the few CD8+ T cells that normally remain in mice lacking ß2m, maintaining them at
1% of the total population.
It seemed, therefore, that in the absence of CD8+ T cells, lymphadenopathy was unaltered in NODlpr mice, and was still comprised of the B220+ T cell population as well as T and B cell subsets. Giese and Davidson (19) reported that although B220+ T cells persisted in C3H/129 lpr mice lacking ß2m, they were in fact derived from a CD4+ T cell lineage. We therefore examined the B220+ T cell subset for expression of the CD4 molecule, and found that the majority (8090%) of these cells in NODlprbeta and NODtriple mice did in fact express low levels of CD4, while a smaller number (<17%) of B220+ T cells from NODlpr mice did so (Fig. 9).

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Fig. 9. B220+ T cells in NODtriple mice express CD4. Lymph node cells from male NODlpr, NODtriple and NOD mice (aged 2225 weeks) were co-stained for B220, TCRß and CD4. Cells expressing B220 and TCRß were gated on and analyzed for expression of CD4 (histogram). Antibodies used were anti-B220FITC (RA3-6B2), anti-TCRßphycoerythrin (H57-597) and anti-CD4biotin (GK1.4) detected with allophycocyaninstreptavidin. Four mice were analyzed per group.
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In summary, NODlpr mice lacking CD8+ T cells developed lymphadenopathy at a similar rate to NODlpr and NODlprt mice. The B220+ T cell subset was still present, but in the absence of CD8+ T cells these B220+ T cells now derived from a CD4+ T cell lineage. The B220+ T cell subset was reduced in NODtriple mice.
Autoimmunity in NODlpr mice
Introduction of the lpr mutation into the autoimmune-prone MRL genetic background leads to severe kidney pathology. We therefore analyzed the effect of the lpr mutation on kidney disease in autoimmune-prone NOD mice. At 5 weeks of age NODlpr, NODlprt, NODt and NOD control male mice (minimum number analyzed = 3) had no infiltrates in the pancreas, salivary gland, liver, kidney or lung (not shown). A similar phenotype was apparent at 8 and 12 weeks (N > 3), although NODt and NOD mice had developed islet infiltrates, and a small amount of infiltration was seen in kidneys from all groups (not shown). By 16 weeks of age, infiltrates were seen in liver and kidney of NODlpr and NODlprt male mice; however, in spite of the extensive lymphadenopathy, very little infiltration was seen in the pancreas or salivary gland (Fig. 10). Kidneys from NODlpr and NODlprt animals at 16 weeks had peri-vascular infiltrates, although the glomeruli looked similar to those of NOD controls (Fig. 11C and D). There was no obvious difference in kidney pathology between NODlpr (N = 8) and NODlprt (N = 5) animals at this age. By 2225 weeks of age, extensive infiltrates were seen in the liver around the portal vein (Fig. 11B), and in kidney and lung, but hardly at all in salivary gland or pancreas (not shown). Female NODlpr and NODlprt mice aged 25 weeks of age also had little or no infiltration in the pancreas or salivary glands, but, like males, had peri-arteriolar infiltrates in the kidney and severe glomerulonephritis (Fig. 11F). We also analyzed kidney from NODtriple male mice at 2225 weeks (Fig 10) (triples lack Fas, TNFRI and ß2m), and found intense peri-vascular infiltrates and severe glomerulonephritis with crescent formation (Fig. 11E) indicating autoimmunity. Serum urea measurements were performed for NOD (N = 6), NODlpr (N = 7) and NODlprt (N = 7) female mice between 15 and 19 weeks of age. NOD values ranged from 6.7 to 14.3 mmol/l. One NODlprt mouse had a raised level (28.8 mM) at 19 weeks of age, but the remaining NODlpr and NODlprt mice had levels similar to NOD controls (range = 8.710.9 mmol/l).

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Fig. 10. Infiltration into organs of male NODlpr, NODlprt and NOD triple mice. Infiltration of cells into liver, kidney and salivary gland was assessed using an arbitrary scoring system as follows: 0 (no infiltrate), 0.5 (a few infiltrating cells), 1 (small amount of infiltrate), 2 (moderate amount of infiltrate) and 3 (large amount of infiltrate). For pancreas the scoring system was as follows: 0 (no infiltrate), 0.5 (a few ductal infiltrating cells), 1 (peri-islet infiltrate in <50% of islets) and 2 (peri-islet infiltrate in >50% of islets).
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Fig. 11. Pathology in NODlpr and NODtriple mice. Livers of 22-week-old male NOD (A) and NODlpr (B) mice showing infiltrates around the portal vein in NODlprs. Kidney glomeruli of 16-week-old male NOD (C) and NODlpr (D) mice showing similar morphology. Kidney glomeruli of 25-week-old male NODtriple (E) and female NODlpr (F) mice showing crescent formation (arrows). Magnification: A and B = x200; CF = x400.
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To confirm the presence of autoimmune kidney disease in NODlpr and NODlprt mice, kidney glomeruli were tested for the presence of complement (C3) by immunofluorescence staining. At 20 weeks of age prominent staining was seen in the glomeruli of NODlpr (N = 4) and NODlprt (N = 3) animals (Fig. 12C and D), but not in those of NOD controls (N = 3) (Fig. 12A and B). C3 staining in the glomeruli was also apparent in NODtriple mice (Fig. 12E), and was also seen in NODlpr and NODlprt mice at 15 weeks of age (not shown).

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Fig. 12. Detection of complement (C3) in glomeruli of NODlpr and NODlprt mice by immunofluorescence. Cryostat frozen kidney sections were stained with goat anti-C3 antibody directly conjugated to FITC. (A and B) NOD kidney, (C) NODlpr kidney, (D) NODlprt kidney, (E) NODtriple kidney and (F) NODlprt kidney (low magnification). Magnification: AE = x300; F = x80.
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Discussion
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Defects in expression or function of Fas or its ligand (FasL) can cause lymphadenopathy, autoimmune disease and lymphoid malignancy in humans and mice (1). The extent and penetrance of these abnormalities is influenced by the genetic background. We investigated the impact of loss of Fas, plus or minus loss of TNFRI and ß2m in the autoimmune-prone NOD background.
Lymphadenopathy in both male and female NODlpr mice occurred at a steady rate similar to that seen in lpr mice of other genetic backgrounds. Lymphadenopathy and splenomegaly were particularly extensive in NODlpr mice with spleen weights of >1 g at 1820 weeks of age in contrast to B6lpr mice (spleen
0.3 g at 6 months) and MRLlpr (spleen
0.75 g at 18 weeks) (20,41,42). When compared to B6 mice, lymphocytes from NOD mice show resistance to apoptosis induced by cyclophosphamide, dexamethasone,
-irradiation or growth factor withdrawal-induced cell death in culture, and candidate loci for these phenotypes have been mapped to Idd5 and Idd6 (43,44). These loci (and others) may contribute to the extensive lymphadenopathy seen in NODlpr mice. For example, Vidal et al. (42) mapped four loci (chromosome 4, 5, 7 and 10) for lymphoid hyperplasia in an MRLlpr x B6lpr F2 cross.
It has been reported that B6lpr mice, deficient in TNFRI, developed autoimmunity and enhanced lymphadenopathy when compared to B6lpr controls (35). NODlprt mice, on the other hand, were not different from NODlpr animals. It is possible that, in the NOD genetic background, lpr does not cooperate with TNFRI deficiency to increase lymphadenopathy. Alternatively, the B6 double-mutant mice used by Zhou et al. still contained genes from the 129 strain background which may have promoted the lymphadenopathy and autoimmunity rather than deficiency in TNFRI per se. In support of this idea, Korner et al. found greatly reduced (not increased) lymphadenopathy in pure bred B6 mice, doubly-deficient in ligands for these death receptors (FasL and TNF-
) (45), and in vitro studies did not find synergy between Fas and TNFRI signaling in a lymphoblastoid B cell line (46).
Lymphadenopathy in NODlpr and NODlprt mice also occurred in the absence of CD8+ T cells (NODlprbeta and NODtriple mice). B220+ T cells were still present in these animals, but now derived mostly from a CD4+ T cell lineage as previously described for C3H/129lpr mice deficient in ß2m (19). Thus, the derivation of B220+ T cells from the CD8+ T cell subset also holds true for the NOD genetic background; however, in the absence of CD8+ T cells, B220+ T cell lymphadenopathy can still proceed from a CD4+ T cell lineage.
Korner et al. found that B6 mice doubly deficient in the ligands for TNFRI and Fas (TNF-
and FasL) had greatly reduced lympho-accumulation in spleen, lymph nodes and blood, and, in addition, had a reduced proportion of B220+ T cells specifically in blood (45). Reduction in B220+ T cells was seen in lymph nodes of NODtriple mice (lacking Fas, TNFRI and CD8+ T cells), although not in NODlprt lymph nodes (blood was not analyzed). Since the majority of B220+ T cells in NODtriple mice derive from a CD4lo T cell lineage, this latter population appears to be more affected by loss of TNFRI, whereas the CD4CD8B220+ T cell population that predominates in NODlprt lymph nodes does not. Deficiency in TNFRI or TNF is known to influence localization of B cells to the B cell follicles of lymphoid organs in B6 mice (34). NOD mice deficient in TNFRI had a lymph node T:B ratio of >8:1 (compared to a NOD value of 4:1), indicating that TNFRI deficiency in the NOD background also influences B cell migration/localization. B220+ T cells have been reported to localize to the follicular (B cell) area of the lymph nodes and spleen rather than the T cell area (47), and may therefore be affected by deficiencies in TNFTNFRI signaling.
Analysis of activation markers on T cells from young NODlpr mice showed that CD4+ and CD8+ T cells expressed high levels of CD44, but had normal profiles for markers of effector T cells (CD69 and CD45RB). This pattern is not unlike that found for normal T cells undergoing homeostatic proliferation in a lymphopoenic environment (48). It seems unlikely that environmental pathogens are responsible for the lymphadenopathy in lpr mice, but endogenous self-antigens or food antigens (23) as well as cytokine overexpression (32) may be relevant. T cells with weak cross-reactivity on self-antigens may proliferate and not be down-regulated in the absence of Fas. However, although crmA expression in T and B cells inhibits Fas-induced apoptosis (49,50), it does not induce lymphadenopathy, so some other factor must be involved to drive the lymphoproliferation. For example, Fas may also be needed for down-regulation of antigen-presenting cells (APC) by T cells during the immune response, similar perhaps to the killing of APC by perforin-producing CD8+ T cells (51).
Autoimmune kidney disease was apparent in male and female NODlpr, NODlprt and NODtriple mice at
2225 weeks of age. In contrast, although extensive infiltrates were also found in lung and liver at this time, little or none were seen in pancreas, as previously shown by Itoh et al. (52), or salivary gland, two tissues normally subject to autoimmunity in the NOD mouse. Presumably, the antigen-specific response to these organs was affected by the extensive lymphadenopathy and alteration in the T cell repertoire. The presence of kidney disease in NODtriple mice at the same age (2225 weeks) as NODlpr mice was not expected. Deficiency in ß2m has been shown to reduce the stability of serum IgG (53) and, as a result, lupus-like autoimmune syndrome in MRLlpr mice (12). It is possible that the extensive lymphadenopathy induced by lpr in the NOD background masked any protective effect of ß2m deficiency and we are now performing a time-course analysis on NODtriple mice to see if there is any protection against autoimmune kidney disease at earlier time points.
These results demonstrate that, in the autoimmune-prone NOD background, lpr induces massive lymphadenopathy and autoimmune kidney disease, as it does in MRL mice. Loss of TNFRI does not exacerbate the pathology caused by deficiency in Fas and loss of ß2m does not reduce it. NOD mice may, in fact, be prone to kidney disease, since administration of BCG to NOD mice can prevent diabetes, but induce lupus nephritis (54). Given that NOD mice spontaneously develop autoimmune lesions in a number of endocrine and exocrine organs (55), it is perhaps not unusual that the NOD genetic background should exacerbate the lpr effect compared to a non-autoimmune genetic background, such as C57BL/6. Mapping studies of modifier loci using the NODlpr mouse strain may prove useful for identifying a common set of susceptibility genes for immune-related traits in the NOD genetic background.
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Acknowledgements
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We are very grateful to Dr H. Bluethmann (Roche, Basel) for providing the TNFRI targeted mice, Dr T. W. Kay (Hall Institute, Melbourne) for backcrossed NODß2m knockout mice, Dr Simon Foote (Hall Institute, Melbourne) for kind support, The Royal Melbourne Hospital for performing the serum urea assays and Dr A Harris (Hall Institute, Melbourne) for use of the Coulter Counter. Kind thanks to Professor P. Bhathal for help with histology and S. Mihajlovic for histology preparation. This work was supported with grants and fellowships from the NHMRC (Australia), The Juvenile Diabetes Fund, Diabetes Australia, The Rebecca Cooper Foundation, The Clive and Vera Ramaciotti Foundation, The Dr Josef Steiner Cancer Research Institute (Bern, Switzerland), The Cancer Research Institute (New York), and The Leukemia and Lymphoma Society of America.
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Abbreviations
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APCantigen-presenting cell
B220+ T cellsCD4CD8 double-negative TCR
ß+ B220+ T cells
ß2mß2-microglobulin
FasLFas ligand
TNFRItumor necrosis factor receptor 1
NODnon-obese diabetic
NODtNODtnfrI mutant mice
NODlprtNODlpr tnfrI double-mutant mice
NODtbetaNODtnfrI ß2m double-mutant mice
NODlprbetaNODlpr ß2m double-mutant mice
NODtripleNODlpr tnfrI ß2m triple-mutant mice
TNFtumor necrosis factor
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