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The Journal of Immunology, 1999, 163: 6884-6891.
Copyright © 1999 by The American Association of Immunologists

IL-12 Drives IFN-{gamma}-Dependent Autoimmune Kidney Disease in MRL-Faslpr Mice1

A. Schwarting*, G. Tesch*, K. Kinoshita*, R. Maron{dagger}, H. L. Weiner{dagger} and V. Rubin Kelley2,*

* Laboratory of Molecular Autoimmune Disease, Renal Division, and {dagger} Center for Neurological Disease, Brigham and Women’s Hospital, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-12 is secreted by kidney tubular epithelial cells in autoimmune MRL-Faslpr mice before renal injury and increases with advancing disease. Because IL-12 is a potent inducer of IFN-{gamma}, the purpose of this study was to determine whether local provision of IL-12 elicits IFN-{gamma}-secreting T cells within the kidney, which, in turn, incites injury in MRL-Faslpr mice. We used an ex vivo retroviral gene transfer strategy to construct IL-12-secreting MRL-Faslpr tubular epithelial cells (IL-12 "carrier cells"), which were implanted under the kidney capsule of MRL-Faslpr mice before renal disease for a sustained period (28 days). IL-12 "carrier cells" generated intrarenal and systemic IL-12. IL-12 fostered a marked, well-demarcated accumulation of CD4, CD8, and double negative (CD4-CD8- B220+) T cells adjacent to the implant site. We detected more IFN-{gamma}-producing T cells (CD4 > CD8 > CD4-CD8- B220+) at 28 days (73 ± 14%) as compared with 7 days (20 ± 8%) after implanting the IL-12 "carrier cells;" the majority of these cells were proliferating (60–70%). By comparison, an increase in systemic IL-12 resulted in a diffuse acceleration of pathology in the contralateral (unimplanted) kidney. IFN-{gamma} was required for IL-12-incited renal injury, because IL-12 "carrier cells" failed to elicit injury in MRL-Faslpr kidneys genetically deficient in IFN-{gamma} receptors. Furthermore, IFN-{gamma} "carrier cells" elicited kidney injury in wild-type MRL-Faslpr mice. Taken together, IL-12 elicits autoimmune injury by fostering the accumulation of IFN-{gamma}-secreting CD4, CD8, and CD4-CD8- B220+ T cells within the kidney, which, in turn, promote a cascade of events culminating in autoimmune kidney disease in MRL-Faslpr mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The MRL-Faslpr mouse has a complex systemic autoimmune disease, including nephritis, arthritis, massive lymphadenopathy and splenomegaly, that mimics systemic lupus erythematosus (1, 2, 3). MRL-Faslpr kidneys undergoing autoimmune destruction are infiltrated by T cell populations that are largely TCR {alpha}ß T cells and include CD4, CD8, and a unique T cell subset termed double negative (DN)3 (4). Multiple T cell populations are responsible for autoimmune disease in MRL-Faslpr mice. While MRL-Faslpr strains genetically deficient in broad T cell populations (TCR {alpha}ß) are protected from nephritis, elimination or blockade of CD4, CD8, or DN T cells halts, or at least retards, progressive renal injury in MRL-Faslpr mice (4, 5, 6, 7, 8). For example, genetically MRL-Faslpr mice deficient in CD4 or MHC class II (devoid of CD4 T cells), or blockade of CD4 with mAbs, spares the kidney from injury (5, 6, 7). Furthermore, elimination of T cells selected by class I molecules (CD8 and DN, derived from the CD8) by constructing a class I (ß2-microglobulin)-deficient MRL-Faslpr strain thwarts progressive kidney disease (8). Thus, kidney disease in MRL-Faslpr mice requires CD4, CD8, and the DN T cell populations.

Cytokines, including IFN-{gamma}, promote autoimmune tissue destruction in MRL-Faslpr mice (9, 10, 11, 12, 13, 14). We have previously established that IFN-{gamma} provides a positive amplification loop responsible for escalating autoimmune kidney destruction. In this scheme, kidney-infiltrating CD4, CD8, and DN T cells secrete IFN-{gamma}, which, in turn, induces the expression of CSF-1 and TNF-{alpha} within the kidney (15). CSF-1, in turn, fosters the intrarenal influx and expansion of macrophages (M{phi}) and T cells (15). Because MRL-Faslpr mice lacking IFN-{gamma}R are protected from fatal lupus nephritis (15, 16, 17, 18), we and others suggested that T cells secreting IFN-{gamma} within the kidney are required for kidney injury (15, 16, 17, 18).

IL-12 released from stimulated kidney parenchymal cells may be required to convert naive T cells into T cells that foster autoimmune disease. In support of this concept, IL-12 1) generated by APCs regulates T cells (19, 20, 21), 2) promotes T cell proliferation (22, 23), 3) stimulates T cells to generate IFN-{gamma} (24), and 4) enhances CD8 T cell cytotoxicity (25). Furthermore, IL-12 T cell stimulation is a prerequisite for committing CD4 T cells into a T cell subset (Th1 phenotype) associated with autoimmune disease in MRL-Faslpr mice (26, 27, 28). Furthermore, IL-12 is up-regulated in tumor epithelial cells (TEC) and M{phi} in MRL-Faslpr mice commencing before and continuing throughout the progressive loss of kidney function (29). Thus, IL-12 released within the kidney of MRL-Faslpr mice may be responsible for providing the signal for T cells to destroy the kidney.

In this study, we determined that local and systemic provision of IL-12 promotes IFN-{gamma}-dependent renal injury in MRL-Faslpr mice. Using an ex vivo gene transfer strategy, we delivered IL-12 under the kidney capsule and determined that local and/or systemic IL-12 elicited nephritis. We now report that intrarenal and systemic IL-12 promotes autoimmune kidney disease by fostering the intrarenal accumulation of IFN-{gamma}-secreting CD4, CD8, and DN T cells.


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

MRL/MpJ-++ (MRL-++), MRL/MpJ-Faslpr/Faslpr (MRL-Faslpr) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). MRL-Faslpr mice lacking the IFN-{gamma}R were derived by a series of six genetic intercross/backcross matings as previously described (15). Mice were bred and housed in our pathogen-free facility. Female mice were used in all experiments.

Retrovirus-mediated cytokine gene transfer into cultured TEC

We isolated and cultured TEC derived from MRL-Faslpr mice 1–2 mo of age as previously reported (30). Before retroviral infection, TEC were removed by trypsinization and plated at 1 x 106 cells. For the retroviral gene transfer, we used a helper-free retrovirus packaging cell line (CRIP) as established by Danos and Mulligan (31). A recombination-incompetent retroviral vector containing the gene of interest was introduced into the CRIP cells containing proviral sequences that were necessary for the "packing" (e.g., encapsidation) of the virus (31). Thus, these producer cells were shedding infectious retrovirus particles encoding the gene of interest. In this study, we have used CRIP-packaging cell lines producing helper-free recombinant retroviruses expressing the murine IL-12 gene (CRIP-IL-12) (generously provided by Dr. H. Tahara, University of Pittsburgh School of Medicine, Pittsburgh, PA). The CRIP-IL-12 cells were based on the TFG-mIL-12-Neo retroviral vector, which can express both IL-12 subunits (p35 and p40), and the neomycin phosphotransferase marker gene as described by Tahara et al. (32). Using the supernatant of CRIP-IL-12 cells in culture, we infected TEC with the recombinant retroviruses in the presence of polybrene and grew the infected cells to confluence. To verify gene transfer into TEC, 1) neomycin-resistant TEC were selected using G418 (1 mg/ml) and 2) IL-12 p70 or IFN-{gamma} were measured in supernatants collected 6 days after passage using an ELISA (33). CRIP-packaging cell lines producing retroviruses encoding the murine IFN-{gamma} gene (CRIP-IFN-{gamma}) were kindly provided by Dr. R. Mulligan (Children’s Hospital, Boston, MA). The CRIP-IFN-{gamma} cell lines were constructed using the MFG Moloney murine leukemia virus (MoMuLV) vector. Because our previous results establish that IFN-{gamma} induces apoptosis in MRL-Faslpr TEC, we used TEC derived from IFN-{gamma}R-deficient MRL-Faslpr mice to construct IFN-{gamma}-producing TEC. For control experiments, TEC were infected with LacZ coding MoMuLV (LacZ as reporter gene replaces the cytokine cDNA) (provided by Dr. R. Mulligan). We stained TEC for the presence of ß-galactosidase to verify LacZ gene transfer into TEC (34). TEC genetically modified to express a gene are termed "carrier cells," e.g., TEC-producing IL-12 are IL-12 "carrier cells."

Detecting IL-12 and IFN-{gamma}

To quantify the levels of IL-12 and IFN-{gamma} in the cell-culture supernatants and in serum, we evaluated samples according to our published methods (33). Briefly, Maxisorp (Nunc, Naperville, IL) were coated with capture mAbs (1–5 µg/ml 4°C overnight). After washing the plates, standards and samples (1:5 diluted) were added and incubated (4°C overnight). Wells were washed, and IL-12 and IFN-{gamma} levels were determined by mAbs and a peroxide visualization system. The Abs and reagents used in these assays were purchased from PharMingen (San Diego, CA) with the exception of avidin-peroxidase, which was from Sigma (St. Louis, MO).

Delivery of TEC under the renal capsule

We placed IL-12 "carrier cells," IFN-{gamma} "carrier cells," LacZ "carrier cells," or uninfected TEC under the renal capsule of IFN-{gamma}R-deficient and intact MRL-Faslpr mice at 1.5–2 mo of age (34). The viability of the TEC immediately before implantation (Ix) using trypan blue exclusion staining was >90%. We anesthetized mice with ether, and the left kidney was exposed through a flank incision. We injected IL-12 "carrier cells," IFN-{gamma} "carrier cells," LacZ "carrier cells," or uninfected TEC, 1 x 106 in 50 µl of HBSS (Sigma) under the capsule of the dorsal surface of the left kidney.

Proteinuria

Urine protein levels were assessed semiquantitatively using albumin reagent strips (Albustix; Miles Scientific, Naperville, IL) (0 = none; 1 = 30–100 mg/dl; 2 = 100–300 mg/dl; 3 = 300-1000 mg/dl; 4 = >1000 mg/dl). We compared IL-12 "carrier cell" or LacZ "carrier cell" implanted MRL-Faslpr mice with age, strain, and sex-matched unmanipulated mice prior to and 28 days post-Ix.

Renal pathology

We removed the implanted kidney and the contralateral (right) kidney at 7 or 28 days post-Ix. The kidneys were divided and one portion snap-frozen in OCT-compound (Miles Scientific) for cryostat sectioning, while the other portion was fixed in 10% neutral-buffered formalin and paraffin embedded. Tissue sections (4 µm) were stained with hematoxylin and eosin (H&E) and evaluated by light microscopy. To find the maximal lesion, we serial sectioned the kidney (6 µm). We evaluated at least 40 sections per specimen. The cell accumulation in the Ix site was assessed by counting the number of cell layers in the subcapsular space and adjacent renal cortex. To determine the percentage of M{phi} and T cells at the Ix site, we stained cryostat-cut sections with Abs to F4/80, CD4, CD8, and B220 determinants using the immunoperoxidase method as previously described (34). Because B220 determinants are expressed by DN T cells and B cells, we distinguished B and T cells by staining for the presence of B220 determinants and B cell-specific CD21 and CD35 epitopes (7G6; PharMingen). After blocking endogenous peroxidase activity with 0.6% H2O2 and 0.2% sodium azide and endogenous avidin and biotin using an avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA), tissue sections were incubated with purified rat anti-murine F4/80, CD4, CD8, or B220 Abs (5 µg/ml), followed by biotinylated goat anti-rat IgG (mouse adsorbed). Sections were incubated with avidin-peroxidase complex (Vector Elite Kit; Vector Laboratories), and immunoperoxidase labeling was detected using 3–3'-diaminobenzidine (DAB) as substrate. We counter-stained sections with methyl green/alcian blue. Specificity controls included replacement of primary Ab with normal rat IgG. We enumerated M{phi} and T cells within the renal lesion as a cell index (maximum cell layers x % cell phenotype). The infiltrate was enumerated by counting glomerular cells/glomerulus for >10 glomeruli and interstitial cells/100 µm2 field for >20 fields by two blinded observers. The mean ± SEM cells/glomerulus, and cells/100 µm2 field were determined, respectively. Finally, we evaluated kidney-infiltrating leukocytes in the contralateral kidney using the same methods as detailed above for the implanted kidney.

Detection of IFN-{gamma} in the kidney

We detected IFN-{gamma} in cryostat-sectioned kidneys using a modified immunoperoxidase technique with monoclonal rat anti-mouse IFN-{gamma} (10 µg/ml) (PharMingen) and saponin (35). Specificity controls included substituting primary Ab with normal rat IgG and neutralization by incubating the anti-mouse IFN-{gamma} Ab with a 20-fold molar excess of rmIFN-{gamma} (R&D Systems, Minneapolis, MN). The amount of IFN-{gamma} was graded from 0 to 3 (0 = none; 1 = mild; 2 = moderate; 3 = maximum) in >20 random glomeruli or >20 random interstitial fields. To determine the intrarenal IFN-{gamma}-producing leukocytic phenotype, we dual-stained sections for the presence of IFN-{gamma} and T cells or M{phi} markers. Cryostat-cut sections were incubated with 10 µg/ml FITC-conjugated rat anti-IFN-{gamma} Ab, and the bound primary Ab was detected with FITC-conjugated sheep anti-IgG with alkaline phosphatase (1:500; Boehringer Mannheim, Mannheim, Germany). The amount of Ab-bound alkaline phosphatase was detected with fast blue BB salt (Sigma) in the presence of levamisole. We subsequently detected CD4, CD8, F4/80, and B220 determinants using immunoperoxidase staining with DAB (9, 15, 30). Cell nuclei were counter-stained with methyl green/alcian blue. We enumerated M{phi}, T cells, or renal parenchymal cells expressing IFN-{gamma} in >20 interstitial/perivascular fields/100 µm2 and >10 glomeruli using coded slides assessed by two observers.

In situ detection of proliferating cells

We detected cell proliferation by staining for the presence of proliferating cell nuclear Ag (PCNA), as previously described (30). We subsequently identified CD4, CD8, or B220 determinants using immunoperoxidase and labeling with DAB. The amount of cell proliferation within the elicited lesion was assessed by counting the CD4, CD8, or B220 PCNA cells/100 µm2 field. In addition, paraformaldehyde-fixed cryostat-cut sections were stained for F4/80 and PCNA. Two observers examined more than five fields in each specimen using coded slides. Data are expressed as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gene transfer of IL-12 into MRL-Faslpr kidney induces mononuclear cell infiltration

To determine whether IL-12 incites interstitial nephritis in MRL-Faslpr mice, we compared IL-12 "carrier cells" with LacZ "carrier cells" infused under the renal capsule. Before infusion, IL-12 "carrier cells" constitutively secreted high levels of IL-12 in culture (1150 ± 550 pg/ml, n = 6). By contrast, LacZ "carrier cells" did not produce IL-12 (0 ± 0 pg/ml, n = 4). IL-12 "carrier cells," and not LacZ "carrier cells," implanted under the renal capsule increased circulating IL-12 (Table IGo). IL-12 "carrier cells" incited a massive accumulation of leukocytes within the Ix site, which extended into the adjacent intrarenal area (28 days post-Ix; Fig. 1GoA). In contrast, LacZ "carrier cells" did not elicit a leukocytic infiltration in MRL-Faslpr kidneys (28 days post-Ix; Fig. 1GoB).


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  		Table I. IL-12 "carrier cells" increase IL-12 in the circulation of MRL-Faslpr micea

 


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  		FIGURE 1. IL-12 "carrier cells" induce a large infiltration of leukocytes in MRL-Faslpr mice. A, Note the enhanced accumulation of infiltrating leukocytes extending from the subcapsular site to the renal cortex 28 days post-Ix (arrows). B, In comparison, LacZ "carrier cells" did not elicit a renal lesion 28 days post-Ix; magnification, x330; H&E. C, The renal lesion was evaluated in the subcapsular space at the implant site and adjacent renal cortex with the most extensive pathology by counting the number of cell layers; n = 5, *, p < 0.001 (cell layers of IL-12 "carrier cells" implant at day 7 vs day 28).

 
To evaluate time-dependent progressive leukocyte infiltration elicited by IL-12, we examined the kidney at 7 and 28 days post-Ix. IL-12 "carrier cells" incited a lesion (21 ± 10 cell layers, n = 4; 7 days post-Ix) extending from the renal capsule into the outer cortex adjacent to the Ix site. The numbers of infiltrating cells increased and expanded into the outer and inner cortex (51 ± 10 cell layers, n = 8; 28 days post-Ix). In comparison, LacZ "carrier cells," or uninfected TEC, did not incite renal injury in MRL-Faslpr mice (0 ± 0 cell layers, n = 8) (Fig. 1GoC). Thus, we established that IL-12 delivered into the kidney of MRL-Faslpr mice elicits a marked influx of leukocytes.

To determine whether the IL-12 "carrier cells" fostered an accumulation of T cells within the MRL-Faslpr kidneys, we assessed the phenotype of the kidney-infiltrating leukocytes at 7 and 28 days post-Ix (Fig. 2Go). Initially (7 days), the most prominent leukocytes in the IL-12-incited lesions were CD4 T cells. These CD4 T cells were accompanied by DN < CD8 T cells < M{phi}. We detected five times more infiltrating CD4 T cells as compared with DN T cells (7 days post-Ix; Fig. 2Go). The number of CD4 T cells continued to increase and remained the most prominent leukocytic population from 7 to 28 days. Similarly, CD8 and DN T cells also increased during this time frame, while the number of M{phi} remained stable during this period (Fig. 2Go). Of note, we did not detect B cells (B220 and CD21/35) in the induced renal lesions; therefore, we categorized cells bearing B220 determinants as DN T cells. To determine whether cell proliferation contributed to the intrarenal expansion of T cells, we evaluated the number of proliferating cells (PCNA) within the implant area at 28 days post-Ix. PCNA leukocytes were readily identified (60–70%) within the induced kidney lesion (Fig. 3Go). We determined that most of the proliferating cells were CD4 T cells (60%) using dual and sequential staining for PCNA and CD4. By comparison, few M{phi}, CD8, or DN T cells were proliferating (M{phi}, 8%; CD8, 16%; B220, 1%). Thus, IL-12 incites renal injury by fostering the proliferation and accumulation of CD4 T cells.



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  		FIGURE 2. IL-12 "carrier cells" induce an accumulation of CD4 T cells. Cell phenotype assessed by immunostaining for cell surface marker (CD4, CD8, F4/80) at 7 and 28 days post-Ix. DN T cells defined as CD4-, CD8-, B220+, CD21/35-. Cell index = % cells x number of cell layers; n = 5/group, *, p < 0.001.

 


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  		FIGURE 3. Gene transfer of IL-12 in the kidney of MRL-Faslpr mice elicits proliferation of CD4 T cells within the implant area. Proliferating cells (black nuclei) were detected by immunostaining for PCNA and evaluated for the cell phenotype by dual and sequential staining. Arrows indicate cells infiltrating the cortex; magnification, x800.

 
IL-12 "carrier cells" elicit IFN-{gamma} production in kidney-infiltrating T cells, which is required to elicit renal pathology

We detected IFN-{gamma} in the majority of kidney-infiltrating leukocytes (58 ± 11%, n = 5) at 28 days post-Ix (Fig. 4Go, A–C). Most IFN-{gamma}-producing cells were CD4, while lesser numbers were either CD8 or DN T cells (Fig. 4GoB). To determine whether IL-12 generated IFN-{gamma}-secreting kidney-infiltrating T cells, we determined the IFN-{gamma}-secreting T cell increase (%) within the kidney at 7 and 28 days. The percentage of T cells producing IFN-{gamma} elicited by IL-12 within the kidney dramatically increased from 7 days as compared with 28 days (20 ± 8%, n = 4–73 ± 14%, n = 6, respectively, p < 0.0001) (Fig. 4GoC). It should be noted that IFN-{gamma} was not detectable in the kidney of mice implanted with LacZ "carrier cells." Thus, IL-12 promoted an increase in T cells secreting IFN-{gamma} within the kidney. In addition to an increase of T cells secreting IFN-{gamma} within the IL-12-incited lesions, we detected an increase in the amount of IFN-{gamma} in the serum of MRL-Faslpr mice. In contrast to LacZ "carrier cells," which did not cause an increase in IFN-{gamma} in the circulation (0 ± 0 pg/ml, n = 4; 28 days post-Ix), IFN-{gamma} released from IL-12 "carrier cell" implants was abundant in the sera (313 ± 138 pg/ml, n = 4; 28 days post-Ix). Of note, we did not detect IL-4 in the kidney-infiltrating cells within the IL-12-elicited lesion, nor within the MRL-Faslpr kidneys during progressive spontaneous disease (immunostaining, data not shown).



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  		FIGURE 4. IL-12 "carrier cells" elicit IFN-{gamma}-producing kidney-infiltrating T cells. A, IFN-{gamma}-secreting cells within the IL-12-incited renal lesion were evaluated by immunostaining for IFN-{gamma} at 28 days post-Ix. Arrows indicate kidney-infiltrating cells. B, Dual staining for cell phenotype and IFN-{gamma} revealed the majority of IFN-{gamma}-producing kidney-infiltrating cells as CD4 T cells; n = 5, mean ± SEM, IFN-{gamma} cell index = % IFN-{gamma} cells x cell layers. C, The number of IFN-{gamma}-secreting T cells (CD4, CD8, and DN) in the IL-12-induced lesion increased between 7 and 28 days post-Ix; n = 4/group, mean ± SEM, *, p < 0.001 T cells at day 7 vs day 28; **, p < 0.001 IFN-{gamma}-positive cells at day 7 vs day 28.

 
IFN-{gamma} is critical for the induction of nephritis by IL-12 "carrier cells"

To ensure that the induction of IFN-{gamma} by IL-12 is required for IL-12-incited nephritis, we infused IL-12 "carrier cells" into IFN-{gamma}R-deficient and IFN-{gamma}R-intact MRL-Faslpr mice. IL-12 "carrier cells" incited an influx of leukocytes in IFN-{gamma}R-intact MRL-Faslpr kidneys (48 ± 12 cell layers, n = 4), notably absent in IFN-{gamma}R-deficient MRL-Faslpr kidneys (3 ± 3 cell layers, n = 4)(Table IIGo). Thus, signaling through the IFN-{gamma}R is required for the induction of nephritis by IL-12.


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  		Table II. IL-12-elicited leukocyte infiltrates in MRL-Faslpr kidneys require IFN-{gamma}a

 
To further test the hypothesis that cells secreting IFN-{gamma} or enhanced by IFN-{gamma} elicits renal injury in MRL-Faslpr mice, IFN-{gamma} "carrier cells" were implanted under the kidney capsule. IFN-{gamma} "carrier cells," which constitutively secreted IFN-{gamma} (serum levels: 420 ± 135 pg/ml, n = 4), as compared with TEC infected with LacZ, which did not secrete IFN-{gamma} (0 ± 0 pg/ml, n = 4), elicited renal injury (Table IIGo). Thus, delivery of IFN-{gamma} into the kidney incites renal injury in MRL-Faslpr mice.

The Faslpr mutation facilitates IL-12-elicited kidney disease

To establish whether IL-12 elicits renal injury in Fas-intact MRL-++ mice, we implanted IL-12 "carrier cells" into Fas-intact MRL-++ normal kidneys (2 mo of age) and assessed renal pathology. Implanting IL-12 "carrier cells" provided abundant levels of IL-12 in the circulation (Table IIIGo). Nevertheless, IL-12 did not elicit nephritis in MRL-++ mice. Thus, the Faslpr mutation is a prerequisite for IL-12-elicited kidney disease. IFN-{gamma} was not detectable in MRL-++ implanted with IL-12 "carrier cells," despite the high IL-12 serum levels. This differs from the MRL-Faslpr strain (age and sex matched), in which IL-12 "carrier cells" increase serum IFN-{gamma} levels.


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  		Table III. Fas-Intact MRL-++ mice are protected from IL-12-elicited kidney disease

 
Systemic IL-12 exacerbates pathology in the contralateral kidney in MRL-Faslpr mice

IL-12 is not detected in the circulation of MRL-Faslpr mice before renal injury (2 mo; 0 pg/ml, n = 3), but is abundant (670 ± 550, n = 5) in mice with advanced kidney disease (5 mo of age). IL-12 "carrier cells" implanted into the kidneys of MRL-Faslpr mice increased IL-12 serum levels into the range consistent with advanced kidney disease (Table IGo). Therefore, we explored whether the increase in circulating IL-12 promoted nephritis in MRL-Faslpr mice by examining the kidneys contralateral to those implanted with IL-12 "carrier cells." Glomerular, interstitial, and perivascular cell infiltrates were markedly increased in kidneys contralateral to those receiving IL-12 "carrier cells" as compared with LacZ "carrier cells" (28 days post-Ix; Fig. 5Go, A, C, and D). In fact, MRL-Faslpr kidneys contralateral to those implanted with IL-12 "carrier cells" for 28 days, which were 2.5 mo of age, histologically resembled spontaneous nephritis in MRL-Faslpr mice, which were 4 mo of age. This is impressive, because renal pathology in MRL-Faslpr kidneys is minimal at 2.5 mo of age and severe by 4 mo of age. In addition, IL-12 "carrier cells," which delivered IL-12 into the kidney and circulation, increased the loss of renal function in MRL-Faslpr mice. Proteinuria was increased 2-fold in MRL-Faslpr mice receiving IL-12 "carrier cells" as compared with LacZ "carrier cells" or unmanipulated MRL-Faslpr mice (Fig. 5GoB). The increased loss of renal function in these mice was consistent with the extent of glomerular pathology (Fig. 5Go, C and D). Thus, an increase in circulating IL-12 exacerbates the progression of kidney disease in MRL-Faslpr mice.



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  		FIGURE 5. Gene transfer of IL-12 exacerbates renal pathology in the contralateral MRL-Faslpr kidney. A, Renal pathology was assessed by counting intra- and periglomerular, interstitial, and perivascular cells. The numbers of glomerular, interstitial, and perivascular cells were markedly increased in the contralateral kidney at 28 days post-Ix as compared with renal pathology in the contralateral kidneys of LacZ "carrier cell"-implanted mice; mean ± SEM, n = 4, *, p < 0.001. B, Proteinuria was determined in MRL-Faslpr mice implanted with IL-12 "carrier cells" and LacZ "carrier cells" as described in Materials and Methods. Mice with increased levels of IL-12 generated by IL-12 "carrier cells" after 28 days had more severe proteinuria (n = 6/group, *, p < 0.01). C, Kidney contralateral to the IL-12 "carrier cell"-implanted kidney at 28 days post-Ix. Note the infiltrating leukocytes surrounding glomeruli (arrowheads) and in the interstitium. D, Kidney contralateral to the LacZ "carrier cell"-implanted kidney at 28 days post-Ix; magnification, x330, H&E.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We now report that IL-12 promoted T cell-mediated kidney disease in MRL-Faslpr mice. Introduction of IL-12 into the kidney of MRL-Faslpr mice via a gene transfer approach using IL-12 "carrier cells" elicited a local influx of leukocytes, elevated systemic levels of IL-12, accelerated kidney pathology in the contralateral kidney, and proteinuria. We identified several IL-12-mediated pathogenic mechanisms in MRL-Faslpr mice linked to eliciting kidney disease: 1) recruitment of CD4, CD8, and DN T cells into the kidney, 2) kidney-infiltrating T cell activation resulting in IFN-{gamma} production, and 3) expansion of CD4 T cells proliferating within the kidney. In addition, we determined that kidney damage is dependent on IFN-{gamma}: 1) IL-12 "carrier cells" failed to elicit kidney disease in IFN-{gamma}R-deficient MRL-Faslpr mice, and 2) delivery of IFN-{gamma} via IFN-{gamma} "carrier cells" into the kidney incites renal injury in MRL-Faslpr mice.

Up-regulation of IL-12 expression within TEC has been linked to the progression of autoimmune disease in MRL-Faslpr mice (29). A previous study established that up-regulation of IL-12, largely generated by TEC, was associated with progressive nephritis in MRL-Faslpr mice (29). While Huang et al. reported that daily injections of rIL-12 accelerated glomerular pathology, but reduced pyelonephritis, and the number of leukocytic infiltrates in the medulla (40), our results are consistent with an IL-12-dependent accelerated glomerulonephritis; however, we note an IL-12-dependent increase in leukocytes in the cortex and medulla. Because pyelonephritis is extremely rare in MRL-Faslpr mice, the discrepancy between our findings may be related to MRL-Faslpr-independent factors, for example an infection compounded by a limited sample size. In addition, daily injections of rIL-12 barely increased serum IFN-{gamma} levels; the amount of serum IL-12 was not reported. By comparison, our gene transfer approach resulted in a substantial elevation of serum IL-12 and IFN-{gamma}. Our present study using a gene transfer system that persistently delivers IL-12 locally and/or systemically into the kidney uniquely establishes: 1) the IL-12-dependent intrarenal pathogenic events responsible for autoimmune kidney disease, and 2) the local impact of IL-12 on T cell phenotypes within the kidney.

The release of IL-12 by TEC in MRL-Faslpr mice promotes intrarenal T cell commitment, proliferation, and "self-destruction." This concept is based on our findings that: 1) activated MRL-Faslpr TEC generate IL-12 (29); 2) intrarenal IL-12 induces kidney-infiltrating T cells to secrete IFN-{gamma}; and 3) T cells secreting IFN-{gamma} promote the destruction of renal parenchymal cells in MRL-Faslpr mice (26). The concept that IL-12 mediates "self-destruction" is further supported by other findings. It has previously been established that a high proportion of MRL-Faslpr CD4 T cells are activated at 4–6 wk, before any overt tissue pathology (36). Because IL-12 induces proliferation of activated, but not resting, T cells, it is conceivable that preactivated/autoreactive T cells respond more readily to IL-12 than CD4 T cells from normal strains (3, 4, 37, 38). In contrast, peripheral deletion of activated/autoreactive T cells is impaired by the Faslpr mutation (39). Thus, the IL-12 may foster an uncontrolled expansion of activated T cells leading to autoimmune tissue destruction. Moreover, we suggest that the availability of autoreactive T cells is a prerequisite for IL-12-elicited renal injury, because at 2 mo of age, MRL mice defective in Fas (MRL-Faslpr) have autoreactive T cells, and these cells are lacking in the MRL-++ strain until they are substantially older (1 year of age) (A.S. et al., manuscript in preparation). Therefore, IL-12 "carrier cells" do not elicit renal pathology in Fas-intact MRL-++ mice, and IFN-{gamma} is not detectable in the circulation of MRL-++ mice implanted with IL-12 "carrier cells" despite high IL-12 serum levels. Clearly, the interaction of "autoimmune background genes" and IL-12 is complex. We are currently exploring this issue using MRL-Faslpr mice genetically deficient in IL-12 receptors.

Systemic increases in IL-12 are characteristic of progressive nephritis in MRL-Faslpr mice (29). We now report that increasing systemic IL-12 into the range of MRL-Faslpr with advanced renal injury, via a gene transfer approach, accelerates kidney disease in MRL-Faslpr mice. Although the systemic levels of IL-12 were not measured, this finding is consistent with a prior study noting that IL-12 injections into MRL-Faslpr for 9 wk increased glomerular injury (40). We suggest that circulating IL-12 facilitates the recruitment of leukocytes into the kidney via multiple mechanisms: 1) IL-12 is a chemoattractant that recruits M{phi} and activated T cells (41); 2) IL-12 induces other chemokines including monocyte chemoattractant protein-1 and RANTES (these chemokines are, in turn, responsible for amplifying the influx of M{phi} and T cells (42, 43, 44)); and 3) IL-12 induces adhesion molecules. For example, IL-12 induces VCAM-1, ICAM-1, and E-selectin on the vascular endothelium, which anchors circulating lymphocytes to the vessel wall (41, 45). It is also noteworthy that we have used this gene transfer approach to deliver numerous cytokines and chemokines (CSF-1, GM-CSF, IL-6, TNF-{alpha}, RANTES) into the kidney and circulation. None of these circulating cytokines/chemokines resulted in an increase in kidney disease (34, 46, 47, 48). Of note, the systemic effects of IL-12 on pathology were not restricted to the kidney. We detected an increase (2-fold) of infiltrating leukocytes within the lungs (surrounding bronchioli and vessels) in MRL-Faslpr mice implanted with IL-12 "carrier cells" (data not shown). Thus, systemic IL-12 is responsible for mediating autoimmune disease in multiple tissues in the MRL-Faslpr mouse. In addition, the impact of systemic IL-12 on the kidney is magnified by local delivery. Implanting IL-12 "carrier cells" incites a well-demarcated massive leukocytic infiltrate (500–800 cells/field, equal to 20–50 cell layers/field; Fig. 1GoA) adjacent to the implant site. By comparison, systemic IL-12 results in a diffuse increase in infiltrating leukocytes in the interstitium, glomerular, and perivascular areas (40–80 cells/field; Fig. 5Go). Irregardless of the exact mechanism responsible for IL-12-mediated kidney disease, designing therapeutic strategies to combat kidney disease must include blocking intrarenal and circulating IL-12.

IL-12-elicited renal injury is characterized by proliferating IFN-{gamma}-secreting T cells within the kidney. IFN-{gamma}R signaling is crucial for the initiation of nephritis, because IL-12 did not induce renal injury in IFN-{gamma}R-deficient MRL-Faslpr mice. This is in agreement with our previous data indicating that IFN-{gamma}R-deficient MRL-Faslpr mice were protected from spontaneous autoimmune renal injury and the failure of T cells to proliferate in IFN-{gamma}R-deficient as compared with IFN-{gamma}R-intact MRL-Faslpr mice (15). In contrast, IFN-{gamma} also limits alloimmune responses by down-regulating the proliferation of activated T cells in murine cardiac and skin transplantation (49). One possible explanation for this dichotomy is that the action of IFN-{gamma} is dependent on the subset of T cells involved. For example, we have previously reported that DN T cells secreting IFN-{gamma} are self-regulatory. These DN T cells release IFN-{gamma}, which blocks proliferation of this subset following stimulation by TEC (50). In the present study, the DN T cells are not proliferating (1%) in the kidney. By comparison, CD4 IFN-{gamma}-secreting T cells are proliferating (60%). Thus, the DN and CD4 T cells have different sensitivities to the anti-proliferative signal delivered by IFN-{gamma}.

Although, we detected T cell proliferation in the kidney in MRL-Faslpr mice receiving IL-12 "carrier cells," because IL-12 is elevated in the circulation and in the kidney in these mice, several mechanisms are possible. T cells could be stimulated by systemic IL-12 within the lymphatic tissues and then recruited to the kidney as activated T cells. Alternatively, naive T cells could be attracted to the kidney by an increase in intrarenal IL-12 and then induced to proliferate and secrete IFN-{gamma}. Furthermore, these mechanisms are not necessarily mutually exclusive. Additional experiments are required to decipher the exact sequence and location of IL-12-elicited T cell proliferation.

Local IL-12 increases the expansion of activated infiltrating T cells within the kidney. However, we have identified T cells within the IL-12-elicited kidney lesion, which did not generate IFN-{gamma}. There are several possible explanations for the "unresponsiveness" of these T cells to generate IFN-{gamma} following stimulation with IL-12. First, they may belong to the Th2 T cell subset. Th2 cells produce IL-4, but not IFN-{gamma}, and do not respond to IL-12, because their IL-12 receptor is down-regulated (51). However, we have not detected IL-4-secreting cells within the inflamed kidney. Thus, it is unlikely that the IFN-{gamma}-negative T cells within the IL-12-elicited lesion contain Th2 cells. Another possibility is that these T cells require further signals, perhaps B7/CD28, which optimize IL-12-driven IFN-{gamma} generation (52). In support of this concept, IL-12 requires the costimulatory molecules B7-1 and B7-2 to reverse tolerance in a model of high-dose hapten-induced tolerance in contact sensitivity (53). Thus, IFN-{gamma} production of autoreactive T cells in the kidney of MRL-Faslpr mice may require IL-12 and costimulatory molecules. We are currently planning experiments to clarify which T cells remain resistant to IL-12 stimulation.

IL-12-driven kidney disease may involve multiple T cell populations. We have identified multiple T cells, CD4, CD8, and DN, within the IL-12 "carrier cell"-elicited kidney lesion in MRL-Faslpr mice. In addition, we have established that CD4, CD8, and DN T cells are required for kidney disease in MRL-Faslpr mice using strains deficient in CD4 and ß2-microglobulin, a class I molecule required to select CD8 and DN (originates from CD8 T cells) (Ref. 54 and manuscript in preparation). We now report that in the absence of IFN-{gamma}R signaling, IL-12 fails to elicit renal injury. Therefore, autoimmune kidney destruction in MRL-Faslpr mice is dependent on T cells generating IFN-{gamma} (5, 15). It is important to appreciate that multiple T cell populations including CD4, CD8, and DN T cells secrete IFN-{gamma} in the MRL-Faslpr mouse. In the present study, IL-12 fostered an accumulation of IFN-{gamma}-secreting kidney-infiltrating T cells that were predominantly CD4 T cells. Furthermore, these CD4 T cells were proliferating in the kidney, as opposed to the CD8 and DN subsets. Therefore, we speculate that IL-12 elicits renal injury by fostering the intrarenal expansion of IFN-{gamma}-secreting CD4 T cells. In addition, we speculate that IL-12 also stimulates other T cells (CD8 and DN) that participate in promoting kidney destruction. For example, IL-12 induces DN T cells in normal mice, and we have reported that DN T cell clones secreting IFN-{gamma} derived from the MRL-Faslpr kidneys placed under the kidney capsule induce MHC and ICAM on adjacent TEC responsible for kidney disease (55, 56). Future studies will define the impact of the DN and the CD8 T cells in the IL-12-elicited renal injury in MRL-Faslpr mice.

In conclusion, we suggest that therapeutic strategies that target local and systemic IL-12 offer a powerful approach to combat progressive autoimmune kidney disease.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants DK36149 and DK 52369 (to V.R.K.), the German Ernst Jung Foundation (to A.S.), and the National Kidney Foundation (to G.T.). Back

2 Address correspondence and reprint requests to Dr. Vicki Rubin Kelley, Renal Division, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: Back

3 Abbreviations used in this paper: DN, double negative; M{phi}, macrophage; TEC, tumor epithelial cell; MoMuLV, Moloney murine leukemia virus; Ix, implantation; DAB, 3–3'-diaminobenzidine; H&E, hematoxylin and eosin; PCNA, proliferating cell nuclear Ag. Back

Received for publication July 2, 1999. Accepted for publication September 22, 1999.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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