Intrathymic kidney cells delay the onset of lupus nephritis in MRL-lpr/lpr mice
Roy D. Bloom1,
Timothy OConnor2,
Borut Cizman1,
Raghu Kalluri1,
Ali Naji2 and
Michael P. Madaio1
Departments of 1 Medicine and 2 Surgery, University of Pennsylvania, Philadelphia, PA 19104-6144, USA
Correspondence to: M. P. Madaio, Renal Electrolyte and Hypertension Division, 700 Clinical Research Building, University of Pennsylvania, Philadelphia, PA 19104, USA. E-mail: madaio{at}mail.med.upenn.edu
Transmitting editor: J. V. Ravetch
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Abstract
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Introduction of antigens to maturing T cells in the neonatal thymus is an effective means of inducing tolerance; however, it is uncertain whether developing, pathogenic, autoreactive T cells can be selectively modulated during systemic autoimmunity. To address this issue, syngeneic cells, derived from the kidneys of pre-diseased, lupus-prone, MRL-lpr/lpr mice were administered intrathymically to either neonatal or young MRL-lpr/lpr mice. For comparison, littermates were injected with splenocytes, islet cells or saline. Kidney cells administered to neonatal mice resulted in attenuation of nephritis, despite elevated serum autoantibody levels, IgG deposits and lymphadenopathy. This effect was not observed with administration of either the renal cell preparations to older mice or islet cells to younger mice, although splenocytes provided some benefit in younger mice. The results indicate that a subset of autoreactive T cells, distinct from those that augment autoantibody production and lymphadenopathy, are necessary for the expression of severe nephritis in MRL-lpr/lpr mice, and they provide further support for a renal antigen-specific component to the phenotype,
Keywords: autoimmunity, lupus, nephritis, thymus, tolerance
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Introduction
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Intrathymic (i.t.) administration of antigens to neonatal mice has been used to modulate immune responses to exogenously administered antigens, for induction of allograft tolerance and for attenuation of organ-specific autoimmune diseases (16). Nevertheless, the effects of i.t. administration of autoantigens in spontaneously occurring, systemic autoimmune disease has not been evaluated. The participation of both pathogenic antibodies and autoreactive T cells within lesions of multiple organs provide additional layers of complexity (7), and it is unclear whether tolerance can be induced to individual autoantigens during the course of systemic autoimmunity, with B and T cell reactivity against multiple autoantigens. It is also uncertain whether individual disease manifestations (e.g. nephritis) can be manipulated in the context of autoimmunity involving multiple organ systems.
To address this issue, our approach involved manipulation of MRL-lpr/lpr mice. This strain spontaneously develops a fulminate and rapidly progressive form of systemic lupus erythematosus, characterized by autoantibody production and massive lymphadenopathy (8). Autoantibody deposition and T cell infiltration lead to inflammatory lesions in many organs. Nephritis is a particularly useful disease marker, because severe disease is predictable. The lesions are multiple, and include glomerulonephritis, vasculitis and interstitial nephritis. Although both B and T cells are required, the ontogeny, quantity and composition of immune deposits and cellular infiltrates vary considerably among these lesions (9,10). In the present studies, developing T cells in young MRL-lpr/lpr mice were exposed to renal cells within the thymus. The aging mice were then evaluated for autoimmune disease, including assessment of serum autoantibody levels, severity of nephritis and the extent of lymphadenopathy.
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Methods
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Mice
Autoimmune MRL/MpJ-lpr/lpr (MRL-lpr) (H-2k) mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and bred in our virus-free facility, on standard laboratory chow, to provide animals under weaning age. Female mice were used in all experiments to control for variation between sexes.
Intrathymic administration of cells
Bloodless glomeruli and tubules (intact segments) were isolated from the kidneys derived from pre-diseased, 6-week-old MRL-lpr/lpr mice, under sterile conditions, using differential sieving (11). After extensive washing in Ca2+ and Mg2+-free HBSS to remove isolated cells and debris, intact preparations of glomeruli and tubules were obtained. (This method effectively removes all intravascular blood cells and circulating Ig.) The cellular fractions from the kidney were combined prior to i.t. injection. Single-cell suspensions of islet cells were prepared from the pancreases of 3-month-old female MRL-lpr/lpr mice using a discontinuous Ficoll gradient (as previously described), after removal of acinar, vascular and other elements (12). Single-cell suspensions of freshly isolated splenocytes from 6-week-old MRL-lpr/lpr mice were prepared, after mincing tissue, lysine red blood cells, and washing the cells with Ca2+ and Mg2+-free HBSS. Freshly isolated cell preparations were resuspended in FCS-free complete RPMI prior to use. Intrathymic transplantation was performed by direct injection into the thymus as described (13). For comparative purposes some animals received the preparations i.p. as a single injection.
Evaluation of disease activity
Previously described methods were utilized to measure (ELISA) and compare serum anti-DNA antibody activity (14). Proteinuria was evaluated semiquantitatively by a dipstick in urine samples collected over 18 h. Each urine sample was diluted to 15 ml prior to testing. In preliminary studies, after equalization of volume, there was excellent correlation of this method with quantitation of total protein excretion using the BioRad (Hercules, CA) method. Spleen size and lymph node size was graded using a semiquantitative scale (04+), independently by one observer, who was blinded to the treatment regimen administered. For light microscopic evaluation, after coding, all samples were evaluated without knowledge of the origin of the kidney by two investigators (R. B. and B. C.). Overall the findings were concordant; however, in a few cases when discrepancies arose, the sections were re-evaluated. Multiple levels of whole kidneys were visualized and graded. A five-point scale was used to assess the severity of glomerulonephritis as follows: 0 = normal; 1+ = mild hypercellularity; 2+ moderate hypercellularity ± early crescents in <50% glomeruli; 3+ = advanced cellular crescents in >50% glomeruli; 4+ advanced crescents with sclerosis/fibrosis in >50% glomeruli. The extent of interstitial disease and vasculitis was graded on a five-point scale as follows: 0= normal; 1+ = small perivascular infiltrates only; 2+ = perivascular infiltrates and small interstitial infiltrates independent from vessels; 3+ = large peri vascular infiltrates completely surrounding the vessels and large interstitial infiltrates independent from vessels, associated with interstitial fibrosis and/or tubular atrophy. Direct immunofluorescence microscopy was used to determine the extent of immune deposits using fluoresceinated rabbit antisera versus either murine IgG (total), murine IgG2a or murine IgG3 on individual 4-µm sections of kidney from the individual mice (10). The intensity of staining was graded by a single observer (M. P. M.) without knowledge of the origin of the sections.
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Results
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Intrathymic administration of kidney cells to 2-week-old MRL-lpr/lpr mice was associated with a reduction in nephritis (Table 1). At 4 months of age, these animals had relatively mild disease. The extent of glomerulonephritis and tubulointerstitial nephritis/vasculitis in the mice was reduced compared to the animals that received either islets i.t., kidney cells i.p. or saline i.t. at 2 weeks. The histology of the animals that received kidney cells i.t. at 2 weeks could not be distinguished from age-matched, non-autoimmune mice. Representative light microscopic sections of the kidney from animals that received either the kidney cells i.t. at 2 weeks or splenocytes i.p. at 2 weeks are illustrated in Fig. 1. MRL-lpr/lpr mice that received splenocytes i.t. at 2 weeks had an intermediate response, with reduced but moderate disease (Table 1).

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Fig. 1. Intrathymic inoculation of kidney cells into 2-week-old MRL-lpr-lpr mice delays the onset of proteinuria. Two-week-old MRL/lpr mice were injected i.t. with either spleen cells (SC), islets or kidney cells (KC), or they were inoculated i.p. with spleen cells. The control group (solid squares) consisted of both sham-treated and unmanipulated mice (proteinuria was indistinguishable in the sham and unmanipulated mice). The percentage of mice with proteinuria in each group was determined at 8, 12, 16, 20 and 24 weeks of age respectively. *P < 0.05 kidney cells versus control and versus splenocytes i.p.
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In contrast, all of the mice that received i.t. injections of either kidney cells or splenocytes at 4 weeks of age developed severe nephritis (i.e. 34+ glomerulonephritis, interstitial nephritis and vasculitis), and the severity of disease could not be distinguished from either the untreated animals or among the groups. Similarly, the mice that received i.t. saline or islets at 4 weeks also developed severe disease and the severity of nephritis was indistinguishable from the animals that received either the kidney cells or splenocytes at 4 weeks. The severity of nephritis in these groups was indistinguishable from neonatal mice (2 week old) that received either the islets i.t. or the kidney cells i.p. (data not shown).
To determine whether the lack of nephritis was associated with decreased deposition of Ig, the kidneys of the mice with and without nephritis were examined by direct immunofluorescence. At 12 weeks of age, diffuse IgG deposits were present within the capillary walls and glomeruli of all the animals, in all of the groups (Table 1). Particularly noteworthy, the quantity and location of IgG deposits in mice with relatively normal light microscopic findings (i.e. mice that received kidney cells i.t. at 2 weeks, see above) were indistinguishable from mice with severe disease. We reasoned that a potential explanation for the disparity between the light microscopic and the immunofluorescence findings was that there were differences in the isotypes of the deposited IgG (e.g. perhaps due to lack of T cell help). However, further analysis of the kidney sections using isotype-specific antisera for IgG2a and IgG3 (the most nephritogenic isotypes in MRL-lpr/lpr mice) yielded indistinguishable results among the groups. Particularly noteworthy, there were abundant deposits (23+) of both IgG2a and IgG3 in all mice, including mice with and without light microscopic evidence of nephritis; the mice with nephritis could not be distinguished from mice without nephritis by the quantity of glomerular IgG2a and IgG3 deposits.
The presence of proteinuria correlated with the light microscopy. Overall, the number of animals that developed proteinuria was significantly reduced in the mice that received the kidney cells i.t. at 2 weeks (Fig. 1). Animals injected i.t. with either splenocytes or islets had more proteinuria than the animals that received kidney cells and less proteinuria than the controls; however, the latter differences were not statistically significant. All of the mice that received i.t. injections at 4 weeks developed heavy proteinuria (i.e. >50% of mice in each group had heavy proteinuria), regardless of the inoculum, and this response was indistinguishable from unmanipulated mice.
Serum levels of anti-DNA IgG were elevated in all groups and increased with age (Fig. 2); the titers were indistinguishable among the groups. At the time of sacrifice, the extent of lymphoproliferation was not different among treatment groups, when differences in nephritis were observed (Fig. 3).

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Fig. 2. Anti-DNA antibody titers in MRL-lpr/lpr mice are not affected by i.t. manipulation. MRL-lpr/lpr mice were inoculated i.t. at 2 weeks of age with either with splenocytes (SC), islets or kidney cells (KC), or i.p. with spleen cells. Anti-DNA antibody titers (ELISA mean ± SEM) in sera from all mice at two timepoints illustrated. There were no statistically significant differences among the groups at any timepoint and the autoantibody levels were not significantly different from sham-treated, unmanipulated mice.
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Fig. 3. Intrathymic inoculation of 2-week-old MRL-lpr/lpr mice does not influence lymphadenopathy. Mice were injected i.t. with either spleen cells (SC), islets (solid circles) or kidney cells (KC), or were inoculated i.p. with spleen cells (open triangles). The control group (solid squares) consisted of both sham-treated and unmanipulated mice. Mice either were inoculated i.t. with splenocytes (open squares), islets (open triangles), or kidney cells (open diamonds), or they were inoculated i.p. with splenocytes (open circles). Lymphadenopathy was evaluated at weekly intervals by two observers, blinded to the treatment group and graded from 04+ according to the size of the lymph nodes. P > 0.05 for all timepoints.
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Discussion
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The results indicate that there is a specific subset of autoreactive T cells that participates in the pathogenesis of lupus nephritis in MRL-lpr/lpr mice. Particularly noteworthy, these T cells are necessary for the rapid and fulminant expression of all aspects of nephritis, including the glomerular, interstitial and vascular components. The findings are consistent with previous reports, where maneuvers or therapies directed at elimination and/or modulation of T cells and T cell subsets reduce disease activity in this strain (1517). Furthermore, they extend these observations by implicating an important direct role for a particular subpopulation of nephritogenic, autoreactive T cells.
A few aspects of this study warrant further discussion. The relative efficacy of kidney cells in modulating lupus nephritis suggests that autoreactive T cells directed at renal antigens are involved in disease initiation. In this regard, the beneficial effect was not due to a generalized effect on T cell development, since peripheral T cell expansion was not effected. Lymphadenopathy was similar among the groups, skin lesions did not appear to be different among the groups (not shown) and serum autoantibody levels were indistinguishable. Lack of significant benefit with islet cells makes non-specific inhibition (e.g. due to release of intracellular enzymes or necrotic cells) unlikely. Thus, the populations of T cells that mediate nephritis were particularly influenced by exposure to renal antigen during their ontogeny. The beneficial effects of i.t. splenocytes are analogous to studies in autoimmune diabetes (and transplantation), where congenic splenocytes modulate insulinitis (and graft survival) (1823), and they imply that nephritis-relevant autoantigens are present among them. Whether this is due to the increased frequency of antigen-presenting cells in the spleen and/or the presence of pathogenically relevant, renal antigens in both organs requires further investigation.
These observations are most easily understood in the context of the pathogenesis of interstitial nephritis in this model (7). This aspect of disease is considered to be primarily T cell mediated, because T cells are present within the interstitium early during disease onset and prior to IgG deposition along tubular basement membranes. Impaired tolerance, due to the fas defect, is a major factor in the activation, proliferation and renal infiltration of T cells (24). Nevertheless, despite fas deficiency, exposure of developing T cells to high amounts of antigen in the thymus may over-ride the lpr defect (25) and our results confirm these observations. Although the quantity of antigen was not precisely determined in the present studies, exposure of appropriate quantities of renal antigen to developing, nephritogenic T cells, likely circumvented the underlying defect, modulating the subpopulation of T cells destined to infiltrate the kidney.
The beneficial effects of renal cells indicate that autoantigens expressed by these cells, per se, are pathogenically relevant. These findings are consistent with the observations of Diaz-Gallo et al., who isolated kidney-specific, autoreactive T cells from MRL-lpr/lpr mice that proliferated and produced cytokines in response to renal antigens (26). These T cells engage both renal tubular epithelial cells, along with infiltrating macrophages, to augment inflammation. Modulation of developing T cells specific for kidney antigens, as in the present study, provides further support for a specific, renal, antigenic component to the cellular infiltration. However, professional antigen-presenting cells, derived from the kidneys of pre-diseased animals, expressing both renal and more ubiquitous autoantigens most likely contributed to the beneficial effects observed, since i.t. splenocytes inhibited disease activity. Modulation of autoreactive T cells, reactive with neo-antigens expressed during inflammation (e.g. heat shock proteins derived from kidney cells or splenocytes), may have also contributed to the reduction in disease activity observed (27).
Intrathymic injection of renal cells were beneficial only when the cells were administered to 2-week-old mice; there was no benefit when they were administered to older animals. Presumably, nephritogenic T cells had already undergone maturation at this stage. Whether the observed benefit was due to deletion/anergy of autoreactive T cells, induction of non-inflammatory T cell subsets and/or induction of regulatory T cells was not directly addressed in these experiments. However, the presence the more nephritogenic isotypes (IgG2a and IgG3) in the glomerular lesions of mice, without histological evidence of nephritis, is consistent with the conclusion that the i.t. cellular injections had a direct effect on T cells destined to infiltrate the kidney (i.e. the benefit was not primarily due to either suppression of pathogenic autoantibody production or modulation of isotype switch).
The lack of glomerulonephritis in mice with immune deposits is consistent with previous reports involving the progeny of MRL-lpr/lpr backcrosses, congenitally deficient in either T cell subsets or individual cytokines. For example, nephritis was ameliorated in MRL-lpr/lpr without
ß T cells, consistent with the notion that lpr T cells accelerate disease in this strain (9). Although delayed, the animals eventually produced high levels of autoantibody and substantial glomerular IgG deposits. However, there was minimal histologic evidence of nephritis, suggesting that
ß T cells play a direct role in glomerulonephritis. Similar conclusions were derived from analysis of MRL-lpr/lpr mice deficient in IFN-
(28). Our findings extend these observations, since light microscopic evidence of nephritis was minimal, despite abundant glomerular deposition of IgG2a and IgG3. Recent studies of anti-glomerular basement membrane antibody disease also support the notion that T cells are required for antibody-mediated nephritis in mice (29). Precisely how T cells exert these effects is not addressed by our studies. However, modulation of disease-relevant T cells could raise the threshold for the quantity of deposited antibody to induce disease. Alternatively, T cells in contiguous areas (e.g. within interstitium and vessels) may provide inflammatory mediators (i.e. cytokines and chemokines) that migrate into nearby glomeruli, priming and activating cells, thereby lowering the threshold for inflammation.
In summary, the results provide further support for a renal antigen-specific T cell component in the development of lupus nephritis. Furthermore, they raise the possibility that modulation of this particular population of T cells may ameliorate disease activity.
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Acknowledgements
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The authors thank Dr Jan Erikson for critical review of the manuscript and advice. This work was supported by a George M. OBrien Kidney and Urological Research Center Grant (DK45191), PHS Award DK 33694 and PHS Training Grant DK-07006.
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Abbreviations
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i.t.intrathymic
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