IFN-γ Receptor Signaling Is Essential for the Initiation, Acceleration, and Destruction of Autoimmune Kidney Disease in MRL-Faslpr Mice

Andreas Schwarting, Takashi Wada, Koji Kinoshita, Gregory Tesch and Vicki Rubin Kelley
J Immunol July 1, 1998, 161 (1) 494-503;

Abstract

CSF-1 and TNF-α in the kidney of MRL-Faslpr mice are proximal events that precede and promote autoimmune lupus nephritis, while apoptosis of renal parenchymal cells is a feature of advanced human lupus nephritis. In the MRL-Faslpr kidney, infiltrating T cells that secrete IFN-γ are a hallmark of disease. To examine the impact of IFN-γ on renal injury in MRL-Faslpr mice, we constructed a IFN-γR-deficient strain. In MRL-Faslpr mice lacking IFN-γR, circulating and intrarenal CSF-1 were absent, TNF-α was markedly reduced, survival was extended, lymphadenopathy and splenomegaly were prevented, and the kidneys remained protected from destruction. Mesangial cells (MC) that were signaled through the IFN-γR induced CSF-1 and TNF-α in MRL-Faslpr mice. We detected a large number of apoptotic renal parenchymal cells in advanced nephritis and determined that signaling via the IFN-γR induces apoptosis of tubular epithelial cells (TEC), but not MC. By comparison, TNF-α induces apoptosis in MC, but not TEC, of the MRL-Faslpr strain. Thus, IFN-γ is directly and indirectly responsible for apoptosis of TEC and MC in MRL-Faslpr mice, respectively. In conclusion, IFN-γR signaling is essential for the initiation (CSF-1), acceleration (CSF-1 and TNF-α), and apoptotic destruction of renal parenchymal cells in MRL-Faslpr autoimmune kidney disease.

Autoimmune disease in MRL-Faslpr mice is multifaceted, targeting the kidney, joints, eyes, and skin. In MRL-Faslpr mice, kidney destruction is determined by the MRL+/+ background genes interacting with a single gene mutation in Fas (1). The MRL genes are responsible for autoimmune kidney disease; the Faslpr (Fas deficiency) mutation converts a latent, mild nephritis into a rapid and fulminant kidney (2). Conceptually, the absence of Fas is responsible for defective peripheral deletion of autoreactive T cells, which are targeted to the kidney and initiate damage. Renal injury in MRL-Faslpr mice is complex and involves interstitial, glomerular, tubular, and perivascular pathology and is mediated by macrophages (Mφ),3 T cells, and cytokines (3, 4, 5, 6).

Many cytokines are increased in the kidneys undergoing autoimmune destruction in MRL-Faslpr (7, 8, 9, 10, 11). The specific cytokines known to initiate or promote kidney injury in MRL-Faslpr mice are CSF-1 and TNF-α (7, 8, 10). CSF-1 is responsible for eliciting and promoting autoimmune kidney destruction. We established that CSF-1 is expressed in the MRL-Faslpr kidney before disease, increases in proportion with advancing kidney damage, and is tightly linked to the extent of kidney pathology (7, 8). Furthermore, gene transfer of CSF-1 into MRL-Faslpr kidney elicits renal injury (12). Although TNF-α increases in parallel to CSF-1, gene transfer of TNF-α fails to elicit autoimmune kidney injury (13). Rather, dual gene transfer of TNF-α and CSF-1 amplifies the extent of renal damage produced by CSF-1 alone (13). Similarly, injecting TNF-α fails to incite renal injury in autoimmune mice before the spontaneous onset of renal injury, but accelerates renal pathology in mice with nephritis (14). Taken together, we envision that CSF-1 elicits autoimmune kidney destruction, while the actions of TNF-α are more distal and require other molecules, notably CSF-1, to accelerate established disease.

T cells are required for autoimmune kidney disease in MRL-Faslpr mice (5, 15, 16, 17). During renal injury, CD4+ and double-negative (DN) CD4−CD8−B220+CD21/35−, and a small amount of CD8+ cells infiltrate and accumulate within glomeruli, the interstitium, and perivascular compartments. The DN, CD4+ and CD8+ T cells secrete IFN-γ, a cytokine that has a broad array of functions capable of promoting tissue injury (18, 19). Since IFN-γ is increased in MRL-Faslpr kidneys, and blockade of the IFN-γR signaling prevents glomerulonephritis and prolongs survival (20), we constructed an IFN-γR-deficient MRL-Faslpr strain and determined whether IFN-γ is responsible for CSF-1-, TNF-α-, Mφ-, and T cell-dependent kidney damage. In addition, since apoptotic renal parenchymal cells are evident in human lupus nephritis (21, 22), we explored the possibility that IFN-γ directly or indirectly is responsible for apoptotic parenchymal cell death. We now report that IFN-γ is required for the production of CSF-1 and TNF-α and induces apoptosis of renal parenchymal cells. MRL-Faslpr mice lacking IFN-γR are protected from fatal lupus nephritis. We propose that IFN-γ released by kidney-infiltrating T cells induces CSF-1 and TNF-α, which, in turn, recruit Mφ and T cells. This influx of additional T cells into the kidney provides a positive amplification loop that results in apoptotic renal parenchymal cells and culminates in kidney destruction. In conclusion, IFN-γR signaling is essential for the initiation, acceleration, and destruction of the autoimmune kidney in MRL-Faslpr mice.

Materials and Methods

Mice

MRL/MpJ+/+ (MRL+/+) and MRL/MpJ-Faslpr/Faslpr (MRL-Faslpr) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). IFN-γR-deficient mice (129/Sv/Ev × C57BL/6) were provided by Dr. M. Aguet (ISREC, University of Lausanne, Lausanne, Switzerland). The IFN-γR gene was inactivated in these mice by transfecting embryonic stem cells with a replacement vector containing a disrupted murine IFN-γR gene (23). All mice were housed and bred in our pathogen-free animal facility.

Generation of IFN-γR-deficient MRL-Faslprmice

MRL-Faslpr mice lacking the IFN-γR were derived by a series of genetic backcrosses using the cross-backcross-intercross scheme. MRL-Faslpr mice were mated with IFN-γR-deficient (129/Sv/Ev × C57BL/6) mice to yield heterozygous F1 offspring. We intercrossed F1 mice and screened the progeny by PCR amplification of tail genomic DNA for the Faslpr mutation and IFN-γR using specific primers (23, 24). Double homozygous (Faslpr/Faslpr, IFN-γR−/−) N1F1 progeny were backcrossed to MRL-Faslpr/Faslpr mice. B1 progeny, homozygous for the Faslpr mutation and heterozygous for the IFN-γR (IFN-γR+/−), were intercrossed, and mice homozygous for the IFN-γR-deficient mutation were selected by PCR typing for continued backcrossing. After three generations of backcross-intercross matings, this breeding scheme generated a colony of MRL-Faslpr mice (95–97.8% MRL background) homozygous and heterozygous for the mutated IFN-γR (25). We analyzed this generation. In this manuscript, we describe IFN-γR−/− as IFN-γR-deficient and IFN-γR+/− as IFN-γR-intact MRL-Faslpr mice.

Clinical and histologic evaluation

We assessed urinary protein levels weekly using albumin reagent strips (Albustix, Miles, Naperville, IL) and graded them semiquantitatively (0 = none; 1 = 30–100 mg/dl; 2 = 100–300 mg/dl; 3 = 300–1000 mg/dl; 4 = >1000 mg/dl). Serum samples were taken by cardiac puncture at the time of sacrifice. Spleen weight and lymphadenopathy were compared among female IFN-γR-deficient, IFN-γR-intact, and wild-type MRL-Faslpr mice at 6 mo of age. Lymphadenopathy was scored on a scale of 0 to 4 by evaluating the number of nodes (0 = none; 1 = one; 2 = a few; 3 = generalized; 4 = massive). Kidneys were either snap-frozen in OCT compound (Miles) for cryostat sectioning or fixed in 10% neutral-buffered formalin. Formalin-fixed tissue was embedded in paraffin, and 4-μm sections were stained with hematoxylin and eosin and evaluated by light microscopy. We evaluated renal pathology by counting nuclei in the perivascular, glomerular, and interstitial areas. Cryostat-sectioned kidneys were stained for Mφ with F4/80 (American Type Culture Collection, Rockville, MD; HB198), for T cells with anti-CD4, anti-CD8, and anti-B220 rat anti-mouse mAb according to a previously described immunoperoxidase method (9) (PharMingen, San Diego, CA). To distinguish B220-positive DN T cells from B cells, we performed additional sequential staining using B220 and an Ab against a B cell epitope shared by CD21 and CD35 (7G6, PharMingen). Thus, the unique DN T cells characteristic of MRL-Faslpr mice were defined as CD4−, CD8−, CD21/35−, B220+. Specificity controls included replacement of primary Ab with normal rat IgG or rabbit serum. The number of Mφ and T cells within the renal lesion was reported as cells per glomerulus and cells per interstitial field, respectively.

To assess intrarenal Ig deposits, 4-μm frozen sections were stained with FITC-conjugated goat anti-murine IgG (Cappel Laboratories, Malvern, PA) for 30 min at 37 C. The amount and extent of IgG were evaluated in at least 50 glomeruli graded from 0 to 3 using coded slides by two investigators.

In situ detection of apoptotic cells

We detected apoptotic cells by enzymatic in situ labeling of apoptosis-induced DNA strand breaks (terminal deoxynucleotidyltransferase-mediated UTP end labeling; TUNEL method). Frozen sections were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice, and then labeled with TUNEL reaction mixture containing terminal deoxynucleotidyl transferase and fluorescein-labeled nucleotides (Boehringer Mannheim, Indianapolis, IN) for 60 min at 37°C. Incorporated nucleotides were subsequently labeled with sheep anti-fluorescein Fab conjugated with horseradish peroxidase (1/5; Boehringer Mannheim) for 30 min at 37°C. Bound Ab was detected by development with diaminobenzidene (Vector, Burlingame, CA) to obtain a brown color. To determine the cell phenotype of the apoptotic cells, we performed dual staining. After labeling apoptotic cells with the TUNEL reaction, tissue sections were stained for the presence of F4/80 (Mφ), CD4, CD8, B220 (T cells), and CD21/35 B220 (B cells) determinants using an avidin-biotin-phosphatase complex-alkaline phosphatase kit (Vector). Phosphatase was developed with Fast Blue reagent (Sigma, St. Louis, MO) and then counterstained with methyl green/alcian blue. The number of apoptotic Mφ, T cells, B cells, or renal parenchymal cells was evaluated by counting the double-stained cells (TUNEL positive and F4/80+, CD4+, CD8+, CD21/35+, B220+) in individual glomeruli or in the interstitial/perivascular field (100 μm2). In addition, TUNEL-positive cells were assessed morphologically by light microscopy for features of apoptosis, including condensed and fragmented nuclei. Two blinded observers scored at least 20 random glomeruli and 5 fields (100 μm2)/specimen.

Cell survival assay

Tubular epithelial cells (TEC) and mesangial cells (MC) from IFN-γR-deficient and intact MRL-Faslpr kidneys at 2 mo of age, before renal injury, were cultured on 96-well flat-bottom tissue culture plates (Costar, Cambridge, MA) at 10 × 104 cells/well and incubated with medium, IFN-γ (50 U/ml), or TNF-α (30 ng/ml) for 12 h, then washed with PBS and exposed to medium, IFN-γ (50 U/ml), TNF-α (30 ng/ml), or TNF-α plus IFN-γ for another 24 h. Cells were stained with 0.75% crystal violet for 15 min. Nonadherent cells were removed by two washes with ice-cold PBS, and extinction of stained adherent cells was measured at 490 nm in a Bio-Rad ELISA reader (Bio-Rad, Hercules, CA). Data were reported as a percentage of cell death (OD adherent cells in medium alone minus OD adherent cells plus cytokine)/(OD adherent cells in medium alone). To evaluate whether cell death was mediated by necrosis or apoptosis, nonadherent cells in the supernatant were harvested on slides by cytocentrifugation and examined for apoptosis by the TUNEL method as described above. In addition, nonadherent cells were stained with annexin V and propidium iodide (PI) to distinguish between apoptosis and necrosis. Annexin V gives a membraneous staining pattern in apoptotic and necrotic cells (26), whereas PI only stains nuclei from necrotic cells (27). Unfixed cells were washed with PBS and incubated with a 1/50 dilution of annexin V-fluos (Boehringer Mannheim) and a 1/50 dilution of PI in HEPES buffer for 15 min. Staining was evaluated by fluorescence microscopy (488-nm filter for excitation, 515-nm filter for annexin V detection, and >515-nm filter for PI detection).

In vitro detection of apoptotic cells

To confirm that apoptosis occurs in TEC and MC after cytokine stimulation (see cell survival assay), we assessed adherent TEC and MC after exposure to IFN-γ or TNF-α for apoptotic cells using the TUNEL method, morphologic criteria, and annexin V and PI staining. TEC and MC from IFN-γR-deficient and intact MRL-Faslpr kidneys were cultured on 24-well tissue culture plates (Costar, Cambridge, MA) at 20 × 105 cells/well and incubated with medium, IFN-γ (50 U/ml), TNF-α (30 ng/ml), or IFN-γ plus TNF-α for 24 h. Medium was analyzed for endotoxin by amebocyte lysate test (Sigma). We detected apoptosis in adherent-fixed and unfixed cells by TUNEL method, annexin V, and PI staining as described above.

Detection of CSF-1 and TNF-α in serum samples

We quantitated biologically active serum CSF-1 using a previously described Mφ colony-stimulating assay (28). Briefly, 1 × 105 bone marrow cells from C3H+/+ mice were added to test serum (30 μl) and plated in Noble agar in supplemented McCoy’s 5A medium. Cells were incubated at 37°C in humidified 5% CO2 atmosphere. Colonies were counted on day 10. The results were reported as CFU per 105 bone marrow cells. Test sera or culture supernatants were preincubated with 30 μl of polyclonal rabbit anti-murine CSF-1 Ab for 30 min at room temperature before the assay to establish the CSF-1 specificity.

We measured TNF-α in serum samples from IFN-γR-deficient and IFN-γR-intact MRL-Faslpr mice using the ELISA technique (Genzyme, Cambridge, MA) and reported duplicate samples as picograms per milliliter (mean ± SD). The detection limit of this assay is 15 pg/ml.

CSF-1 and TNF-α in the kidney

We detected CSF-1 and TNF-α in the kidney by the immunoperoxidase technique using polyclonal rabbit anti-human CSF-1 (10 μg/ml) on frozen sections and rabbit anti-murine TNF-α Ab (1/150 dilution) on paraffin sections. Specificity controls included the replacement of primary Ab with normal rabbit IgG and neutralization experiments incubating the anti-human CSF-1 Ab or the anti-murine TNF-α Ab with a 20-fold molar excess of CSF-1 (Genetics Institute, Cambridge, MA) or TNF-α (Genentech, South San Francisco, CA), respectively. CSF-1 and TNF-α were scored from 0 to 3 (0 = none, 1 = mild, 2 = moderate, 3 = maximum) in >20 random glomeruli and >20 random interstitial fields.

MC in vitro expression of CSF-1

MC were isolated, cultured, and characterized as previously described (29). MC monolayers from IFN-γR-deficient or intact MRL-Faslpr mice 2 mo of age, before renal disease, were treated with 0.25% trypsin and 1 mM EDTA and subcultured into petri dishes containing growth medium (20% heat-inactivated FCS in DMEM, 10 mM HEPES, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin). MC were cultured between the 5th and the 10th passage onto coverslides in a six-well plate (Falcon 3046) for CSF-1 immunostaining. MC were rendered quiescent by incubation in medium without serum for 12 h. We then stimulated MC with serum (20–50%) from MRL-Faslpr, MRL+/+, or C3H+/+ mice 6 mo of age with or without IFN-γ (50 U/ml) or IFN-γ (50 U/ml) alone. CSF-1 was evaluated 24 h later using the immunoperoxidase method as described above.

MC in vitro expression of TNF-α

MC from IFN-γR-deficient and IFN-γR-intact MRL-Faslpr kidneys were stimulated with medium, IFN-γ (50 U/ml), or LPS (1 μg/ml) for 24 h. Supernatants were harvested and measured using the ELISA technique. Duplicate data are reported as picograms per milliliter (mean ± SD).

Northern blot analysis

We isolated total RNA from dissected renal cortexes using RNAzol B (Tel-Test, Friendswood, TX), a modification of the guanidium thiocyanate-phenol-chloroform method (30). Total RNA (20 μg) was electrophoresed through a 1% agarose-formaldehyde gel, blotted to nylon membrane, and hybridized in 50% formamide with 32P-labeled nick-translated probes at 42 C. Hybridized membranes were washed in 2× SSC/0.1% SDS at room temperature and in 0.2× SSC/0.1% SDS at 60 C. The CSF-1 probe was provided by Dr. R. Stanley (Albert Einstein College, Bronx, NY) and consisted of a 594-bp fragment of the plasmid containing the cDNA. The TNF-α probe was a gift from Dr. K. Matsushima (Kanazawa University, Kanazawa, Japan) and consisted of a 500-bp fragment of the plasmid containing the insert. Blots were reprobed with β-actin (PstI fragment of pBA-1) as an internal control for the quantity and integrity of RNA.

Results

Renal pathology in IFN-γR-deficient MRL-Faslprmice is prevented

Renal pathology was diminished in IFN-γR-deficient compared with IFN-γR-intact MRL-Faslpr mice. The numbers of cells in the glomerular, interstitial, and perivascular areas were reduced in IFN-γR-deficient MRL-Faslpr mice compared with those in the IFN-γR intact MRL-Faslpr strain (Fig. 1⇓A). By comparison, the numbers of glomerular cells in IFN-γR-deficient MRL-Faslpr mice remained similar to those in normal MRL+/+ mice, while the number of cells in the interstitial and perivascular area, although dramatically reduced compared with that in IFN-γR intact MRL-Faslpr mice, was greater than the number in MRL+/+ mice. Thus, MRL-Faslpr mice lacking the IFN-γR are protected from renal damage. In addition, we noted that renal pathology in IFN-γR-intact MRL-Faslpr mice (B3 generation) was only modestly less than that in the wild-type MRL-Faslpr strain (Fig. 1⇓A). Thus, the degree of renal injury in the B3 generation IFN-γR-intact MRL-Faslpr strain is similar to that in the MRL-Faslpr strain.

FIGURE 1.
FIGURE 1.

A, Reduction in kidney-infiltrating cells in IFN-γR-deficient vs IFN-γR-intact MRL-Faslpr mice. Mononuclear cells in glomerular, tubulointerstitial, and perivascular areas are reduced in B3F1 generation IFN-γR-deficient (R−/−) compared with B3F1 generation IFN-γR-intact (R+/−) or wild-type (R+/+) MRL-Faslpr mice. Values are the mean ± SD of infiltrating and parenchymal cells (glomerular, cell counts in 20 random glomeruli; interstitial, 10 random fields (magnification, ×400); perivascular, 10 random inter- and intralobular arteries (magnification, ×400); n = 5 in each group; 6 mo of age). * indicates p < 0.01, IFN-γR intact vs IFN-γR deficient; ** indicates p < 0.01, MRL+/+ vs IFN-γR deficient (by Mann-Whitney test). B, The kidney infiltration of T cells and Mφ in MRL-Faslpr mice is dependent on IFN-γ. Mφ were detected by staining for the presence of F4/80, and T cells were detected by staining for CD4, CD8, and B220 (DN). Values are the mean ± SD. F4/80, CD4, CD8, and B220 cells were counted in 20 glomeruli, 10 random fields (magnification, ×400), and 10 random inter- and intralobular arteries (magnification, ×400; score: 0 = none, 1 = <5 layers surrounding less than a half-circle of the vessels, 2 = 5–10 cell layers surrounding more than a half-circle of the vessels, 3 = >10 layers surrounding vessels; n = 4/group; 6 mo of age). p < 0.01, IFN-γR-deficient (R−/−) vs IFN-γR-intact (R+/−) for all phenotypes.

Kidney-infiltrating T cells and Mφ were diminished in IFN-γR-deficient mice compared with those in IFN-γR intact MRL-Faslpr mice (Fig. 1⇑B). In particular, glomerular CD4+ and B220+ cells were markedly decreased (p < 0.001; Fig. 1⇑B). In addition, we established that kidney-infiltrating cells bearing B220 determinants are DN (CD4−CD8−) T cells, since these cells did not express B cell determinants (CD21/35; data not shown). Furthermore, we noted a distinctive cluster pattern of uniform infiltrating mononuclear cells. These distinct mononuclear clusters of CD4+ T cells, noted in the interstitial and perivascular areas in IFN-γR-deficient MRL-Faslpr mice, were not a feature of wild-type or IFN-γR-intact MRL-Faslpr mice (not shown).

There was a reduction in the amount of IgG in the IFN-γR-deficient compared with the IFN-γR-intact or wild-type MRL-Faslpr kidney. Granular deposits of IgG were detected in the mesangium and capillary walls in IFN-γR-intact (2.17 ± 0.57; n = 5) and wild-type (2.53 ± 0.45; n = 3) MRL-Faslpr mice. In contrast, IgG deposits in the kidney were dramatically reduced in IFN-γR-deficient MRL-Faslpr mice (0.75 ± 0.37; n = 6; p < 0.001).

Loss of renal function is prevented and mortality is reduced in IFN-γR-deficient MRL-Faslpr mice

IFN-γR-deficient MRL-Faslpr are protected from proteinuria. IFN-γR-intact and wild-type MRL-Faslpr mice became proteinuric at 4 mo of age. By comparison, IFN-γR-deficient MRL-Faslpr maintained normal renal function (Fig. 2⇓). The increase in proteinuria was modestly slower in IFN-γR-intact MRL-Faslpr mice compared with that in wild-type MRL-Faslpr mice (Fig. 2⇓). Since the mortality in the wild-type strain is caused by the rapid progressive renal disease, survival was dramatically prolonged in IFN-γR-deficient compared with IFN-γR-intact MRL-Faslpr mice (80 vs 10%, respectively, at 11 mo of age; Fig. 2⇓). We noted that in the B3F1 generation of IFN-γR-intact MRL-Faslpr mice, the 50% mortality was only minimally prolonged compared with that in the wild-type strain (8 vs 6 mo of age, respectively; Fig. 2⇓). Thus, the B3F1 IFN-γR-intact MRL-Faslpr generation has sufficient MRL genes to cause renal disease and is similar to the wild-type MRL-Faslpr strain.

FIGURE 2.
FIGURE 2.

Renal injury is prevented and mortality is reduced in IFN-γR-deficient MRL-Faslpr mice. Urinary protein levels were assessed weekly using albumin reagent strips (Albustix, Miles) and were graded semiquantitatively (0 = none; 1 = 30–100 mg/dl; 2 = 100–300 mg/dl; 3 = 300–1000 mg/dl; 4 = >1000 mg/dl). Data given are the mean ± SD (n = 6/group; p < 0.001, by Mann-Whitney test). Spleen weight was reduced fivefold and lymphadenopathy was reduced twofold in the IFN-γR-deficient MRL-Faslpr strain (n = 6/group; p < 0.001). The IFN-γR-deficient strain survived longer than the IFN-γR-intact strain. Survival is modestly shorter (50% mortality at 5.5 mo of age) in the wild-type MRL-Faslpr mice (X) than in the IFN-γR-intact MRL-Faslpr strain (50% mortality at 8 mo of age). * indicates p < 0.05 (by Mann-Whitney test).

Splenomegaly and lymphadenopathy are reduced in IFN-γR-deficient MRL-Faslpr mice

Splenomegaly and lymphadenopathy, hallmarks of disease in MRL-Faslpr mice, were diminished in IFN-γR-deficient MRL-Faslpr mice. Spleen weight was reduced fivefold in IFN-γR-deficient compared with IFN-γR-intact MRL-Faslpr mice (0.25 ± 0.1 g vs 1.1 ± 0.4 g, respectively; n = 6; p < 0.001). In addition, lymphadenopathy was reduced twofold in IFN-γR-deficient compared with IFN-γR-intact MRL-Faslpr mice (1.2 ± 0.8 vs 2.7 ± 0.4, respectively; n = 6; p < 0.001). It should be noted that the splenomegaly and lymphadenopathy were only modestly increased in the wild-type MRL-Faslpr mice compared to those in B3F1 generation IFN-γR-intact MRL-Faslpr mice (40 and 22%, respectively).

CSF-1 is absent in the circulation and kidney of IFN-γR-deficient MRL-Faslpr mice

We previously established that CSF-1 in the circulation and kidney is detected before renal injury and increases proportionally with progressive renal damage in MRL-Faslpr mice (8). CSF-1 was not detected in the sera of IFN-γR-deficient MRL-Faslpr mice at 6 mo of age, whereas CSF-1 (28 ± 4 and 36 ± 3 CFU, respectively; n = 5) was increased in sera from age-matched IFN-γR-intact and wild-type MRL-Faslpr mice. Similarly, we did not detect CSF-1 transcripts nor did we identify CSF-1 in the kidney sections of IFN-γR-deficient MRL-Faslpr mice. In contrast, CSF-1 was abundant in IFN-γR-intact or wild-type MRL-Faslpr kidneys using either method (Table I⇓ and Fig. 3⇓).

FIGURE 3.
FIGURE 3.

CSF-1 is absent in IFN-γR-deficient MRL-Faslpr mice. A, Biologically active CSF-1 was abundant in the sera of IFN-γR intact or wild-type MRL-Faslpr mice, but was notably absent in the IFN-γR-deficient MRL-Faslpr strain, as determined by colony-stimulating assay. Values are the mean ± SD (n = 4/group; p < 0.0001). B, Northern blot analysis. CSF-1 transcripts were absent in IFN-γR-deficient (−/−) MRL-Faslpr kidneys and up-regulated in IFN-γR-intact (+/−) MRL-Faslpr kidneys. C, Representative examples illustrating the CSF-1 in glomeruli of IFN-γR-intact MRL-Faslpr kidneys (C) and the absence of CSF-1 in IFN-γR-deficient MRL-Faslpr kidneys (D), as determined by the immunoperoxidase method. Magnification, ×1000.

View this table:
Table I.

CSF-1 is not expressed and TNF-α is down-regulated in IFN-γR-deficient MRL-Faslprkidneysa

TNF-α is down-regulated in the kidney of IFN-γR-deficient MRL-Faslpr mice

Our previous studies determined that the increase in TNF-α in the circulation parallels the increase in CSF-1 (10). In the present study, serum TNF-α levels in IFN-γR-deficient MRL-Faslpr mice were reduced compared with those in the IFN-γR-intact MRL-Faslpr strain. In addition, we noted a modest increase in TNF-α in the circulation of wild-type MRL-Faslpr mice compared with that in the IFN-γR-intact MRL-Faslpr strain (Fig. 4⇓), which reflected the slightly slower pace of renal disease as a result of the hybrid genetics. Similarly, we detected a decrease in TNF-α mRNA transcripts in the kidney of IFN-γR-deficient compared with IFN-γR-intact MRL-Faslpr mice at 6 mo of age by Northern blot analysis (Fig. 4⇓). Furthermore, we identified a decrease in TNF-α expression in TEC and infiltrating mononuclear cells in the IFN-γR-deficient compared with the IFN-γR-intact MRL-Faslpr kidney sections (Table I⇑ and Fig. 4⇓).

FIGURE 4.
FIGURE 4.

TNF-α is down-regulated in IFN-γR-deficient MRL-Faslprmice. A, Serum TNF-α was diminished in IFN-γR-deficient MRL-Faslpr compared with that in IFN-γR-intact or wild-type MRL-Faslpr mice, as detected using the ELISA technique. Values are the mean ± SD (n = 4/group). * indicates p < 0.01, IFN-γR-deficient MRL-Faslpr (R−/−) vs IFN-γR-intact MRL-Faslpr (R+/−); ** indicates p < 0.01, IFN-γR-intact MRL-Faslpr vs wild-type MRL-Faslpr. B, Northern blot. TNF-α transcripts were reduced in IFN-γR-deficient MRL-Faslpr (−/−) compared with those in IFN-γR-intact MRL-Faslpr (+/−) kidneys. Blots were rehybridized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). C, Immunoperoxidase staining of TNF-α. TNF-α was abundant in cortical TEC from IFN-γR-intact MRL-Faslpr kidneys (C), whereas TNF-α was reduced in the renal cortex of IFN-γR-deficient MRL-Faslpr (D). Paraffin sections; magnification, ×220.

IFN-γ induces CSF-1 in MC from MRL-Faslprmice

Since CSF-1 was absent in the circulation and kidney of IFN-γR-deficient MRL-Faslpr mice, we investigated whether signaling through the IFN-γR is directly responsible for the up-regulation of CSF-1. We stimulated cultured IFN-γR-deficient and -intact MRL-Faslpr MC with rIFN-γ (50 U/ml) for 24 h. IFN-γ induced CSF-1 in IFN-γR-intact MRL-Faslpr MC, whereas unstimulated MC did not express CSF-1 (Table II⇓). In addition, stimulation of IFN-γR-deficient and intact MRL-Faslpr MC with increasing concentrations of serum (20–50%) from nephritic MRL-Faslpr or normal (C3H+/+) mice did not induce CSF-1 (Table II⇓). To exclude the possibility that IFN-γ is neutralized by serum factors, we spiked MRL-Faslpr serum with ample IFN-γ to induce CSF-1. Since IFN-γ-supplemented MRL-Faslpr serum induced CSF-1, we suggest that circulating IFN-γ is not responsible for inducing CSF-1 in MRL-Faslpr.

View this table:
Table II.

IFN-γ, but not MRL-Faslpr sera, induces CSF-1 in MRL-Faslprmesangial cellsa

IFN-γ induces TNF-α in MC from MRL-Faslprmice

Primary cultured MC from IFN-γR-deficient or -intact MRL-Faslpr mice produced minimal levels of TNF-α constitutively (Table III⇓). Stimulation with IFN-γ (50 U/ml) increased TNF-α in IFN-γR-intact MRL-Faslpr MC (0 to 265 pg/ml), but did not induce TNF-α in MC isolated from the IFN-γR-deficient MRL-Faslpr strain.

View this table:
Table III.

IFN-γ induces TNF-α in MRL-Faslpr mesangial cellsa

Renal parenchymal cells from IFN-γR-deficient MRL-Faslpr mice are protected from apoptosis

Since priming cells with IFN-γ induces apoptosis of hemopoietic cells and may contribute to the extent of Mφ or T cell accumulation, we investigated the amount of apoptotic cells in situ (31, 32). We detected up to 2% apoptotic cells in IFN-γR-intact MRL-Faslpr mice (Table IV⇓ and Fig. 5⇓). In contrast, the number of apoptotic cells within glomerular, interstitial, and perivascular areas was markedly decreased (0.3%) in IFN-γR-deficient MRL-Faslpr mice (Table IV⇓ and Fig. 5⇓). By comparison, apoptotic cells were not detected in the kidney of age-matched normal (MRL+/+) mice (Table IV⇓). We determined the phenotype of the apoptotic cells by dual and sequential staining. Calculating the percentage of different apoptotic cell types per total number of apoptotic cells revealed that the majority of apoptotic cells (∼75%) were renal parenchymal cells, while few T cells (CD4, CD8, DN), Mφ (F4/80), or B cells (CD21/35) were apoptotic (data not shown).

FIGURE 5.
FIGURE 5.

IFN-γR deficiency in MRL-Faslpr mice reduces the number of apoptotic cells in the kidneys. Apoptotic cells were determined by TUNEL staining. Representative examples of apoptotic cells (condensed nuclei, marked with arrows) in a glomerulus (A) and in the interstitium (C) of IFN-γR-intact MRL-Faslpr kidneys are shown. By comparison, the number of apoptotic cells was reduced in glomeruli (B) and interstitium (D) of IFN-γR-deficient MRL-Faslpr kidneys. Magnification, ×330.

View this table:
Table IV.

IFN-γR signaling is required for increased apoptosis in MRL-Faslprkidneya

IFN-γ and TNF-α induce cell death in TEC and MC, respectively, in vitro

TEC.

Exposure of MRL-Faslpr TEC to IFN-γresulted in as much as 20% cell death after 24 h (Fig. 6⇓A). Coincubation of IFN-γ and TNF-α induced the same number of dead TEC as IFN-γ alone, while stimulation with TNF-α alone did not induce TEC death (Fig. 6⇓A). Priming of TEC with IFN-γ for 12 h with subsequent exposure to TNF-α resulted in a higher increase in TEC death than stimulation with IFN-γ alone (Fig. 6⇓A). By comparison, IFN-γ did not induce cell death in IFN-γR-deficient MRL-Faslpr TEC. Thus, signaling through the IFN-γR is responsible for TEC death.

FIGURE 6.
FIGURE 6.

IFN-γ and TNF-α induce renal parenchymal cell death in MRL-Faslpr mice. TEC and MC from IFN-γR-deficient and IFN-γR-intact MRL-Faslpr mice were primed with IFN-γ (50 U/ml) or TNF-α (30 ng/ml) for 12 h and exposed to IFN-γ, TNF-α, or IFN-γ plus TNF-α for 24 h. The proportion of dead cells determined by the crystal violet assay was expressed as the percentage of adherent cells cultured in medium minus the percentage of adherent cells after stimulation. A, Exposure of IFN-γR-intact MRL-Faslpr TEC to IFN-γ resulted in as much as 30% cell death after 24 h (A). In contrast, IFN-γ did not induce death in MRL-Faslpr MC (A). Serum deprivation caused the death of 40 to 50% TEC or MC (A). TNF-α induced MC death (18%) in IFN-γR-deficient and IFN-γR-intact MRL-Faslpr strains (B). Priming of TEC with IFN-γ enhanced the induction of death by TNF-α (B). Values are the mean ± SD (eight wells per experiment) for three separate experiments. * indicates p < 0.001 compared with cells in medium alone; ** indicates p < 0.05 compared with IFN-γ alone (by analysis of variance).

MC.

While IFN-γ did not induce death in MRL-Faslpr MC, TNF-α induced MC death (18%) in IFN-γR-deficient and IFN-γR-intact MRL-Faslpr strains (Fig. 6⇑B). Taken together, IFN-γ induces TEC death, and TNF-α induces MC death in MRL-Faslpr mice.

Apoptosis is involved in IFN-γ- and TNF-α-induced death of renal parenchymal cells

Serum deprivation is considered to efficiently initiate cell death by apoptosis (33). Therefore, serum deprivation was used as a positive control for apoptotic cell death in MRL-Faslpr TEC and MC. Serum deprivation of MRL-Faslpr TEC resulted in twofold more dead TEC in IFN-γR-deficient and IFN-γR-intact MRL-Faslpr strains compared with TEC death induced by IFN-γ (Fig. 6⇑A). Similarly, serum starvation of MRL-Faslpr MC induced more MC death than that induced by TNF-α (Fig. 6⇑B). This finding prompted us to examine whether apoptosis is involved in the mechanisms of TEC death induced by IFN-γ and TNF-α in MRL-Faslpr mice. We studied thenumber of apoptotic cells in dead (nonadherent) TEC by a combination of the TUNEL method, morphologic criteria, and annexin V and PI staining (Table V⇓). We detected 29% apoptotic (TUNEL- and annexin V-positive, but PI-negative) TEC among nonadherent TEC after incubation with IFN-γ (Table V⇓). In contrast, cell death was rarely detected in IFN-γR-deficient and IFN-γR-intact TEC after incubation with medium alone (Table V⇓). To ensure that apoptosis is involved in IFN-γ-induced TEC death, we also assessed adherent TEC and MC after exposure to IFN-γ or TNF-α by the TUNEL method, morphologic criteria, and annexin V and PI staining. We detected 2 to 4% apoptotic TEC in cultured monolayers after 12 h. Adding IFN-γ to TEC increased apoptosis in adherent IFN-γR-intact MRL-Faslpr TEC five- to sixfold, whereas IFN-γ did not induce apoptosis in IFN-γR-deficient MRL-Faslpr TEC (Table V⇓).

View this table:
Table V.

IFN-γ induces apoptosis in MRL-Faslpr TECa

Discussion

The IFN-γR-deficient MRL-Faslpr strains constructed by this and another laboratory (20) both show prolonged survival and the kidneys are protected from destruction compared with MRL-Faslpr strains with intact IFN-γR (20). This study is an in-depth analysis that focuses on the intrarenal pathologic events within the entire kidney (glomerular, interstitial, tubular, and perivascular areas) and analyzes the renal parenchymal and kidney-infiltrating cells and cytokines known to promote kidney destruction. By comparison, Le Hir’s laboratory restricted their study to the Ab-mediated immune events and reported a decrease in circulating autoantibodies implicated in glomerular damage (IgG2a and IgG3) (34, 35). We now report that the kidney infiltration of T cells and Mφ is prevented in IFN-γR-deficient MRL-Faslpr mice compared with that in the IFN-γR-intact MRL-Faslpr strain. In addition, signaling through the IFN-γR is responsible for CSF-1, TNF-α, and apoptosis in renal parenchymal cells during autoimmune kidney disease in MRL-Faslpr mice. Thus, several mechanisms contribute to the impact of IFN-γ during distinct points in the pathogenesis of autoimmune renal injury: the induction phase, since CSF-1 is responsible for inciting and promoting injury (7, 8, 10); the accelerating phase, since TNF-α is known to promote established kidney injury (10, 13); and the advanced phase, since apoptosis of renal parenchymal cells is a feature of severe disease.

We suggest that signaling through the IFN-γR is responsible for CSF-1 production by MC in MRL-Faslpr mice. This is based on the following: 1) CSF-1 is absent in the kidney and circulation of IFN-γR-deficient MRL-Faslpr mice; 2) CSF-1 is primarily a MC product (7); and 3) IFN-γ stimulates MRL-Faslpr MC to generate CSF-1. On the other hand, IFN-γ also indirectly induces CSF-1. TNF-α and/or circulating Ig complexes could increase intrarenal CSF-1, since TNF-α and Ig complexes are abundant in the serum of MRL-Faslpr mice, and each is capable of inducing CSF-1 in MC (36). However, it is intriguing that incubation of MC with sera from nephritic MRL-Faslpr mice did not induce CSF-1. This could indicate that systemic exposure of TNF-α or Ig complexes is insufficient to cause MC to secrete CSF-1. On the other hand, transplanting a normal MRL+/+ kidney into a MRL-Faslpr recipient after removal of nephritic kidneys induces CSF-1 in the kidney, suggesting that a circulating component is required for triggering CSF-1 production (37). It is also possible that this experiment was technically flawed. It is possible that the level of any of these components (IFN-γ, TNF-α, and Ig complexes) in the serum sample was insufficient or inactivated and thus failed to induce CSF-1. Alternatively, it is possible that the circulating component in the transplant experiment (37) was an autoreactive T cell that released IFN-γ locally within the kidney. We favor this explanation, since TEC genetically modified to secrete CSF-1 and/or TNF-α implanted in the kidney, delivering these cytokines locally and not systemically, elicited renal injury in MRL-Faslpr kidneys (12, 13). This suggests that high titers of cytokines localized within the kidney are required for promoting renal injury.

TNF-α was markedly reduced, but unlike CSF-1 was still detectable in the absence of IFN-γR signaling in MRL-Faslpr mice. This is not surprising, since TNF-α is regulated by two distinct mechanisms: neonatal up-regulation related to the Faslpr mutation and an increase in mature mice that is proportional to the severity of lupus nephritis (9, 10). In the IFN-γR-deficient MRL-Faslpr strain, the Faslpr mutation alone may be sufficient to sustain some TNF-α production.

We established that IFN-γ halts the expansion of Mφ in CSF-1-incited interstitial nephritis in MRL-Faslpr mice by blocking proliferation and enhancing apoptosis (32). Therefore, we initially anticipated that IFN-γR-deficient MRL-Faslpr mice would have more Mφ in the kidney during spontaneous renal disease. On the contrary, we identified fewer Mφ in the IFN-γR-deficient than in the IFN-γR-intact MRL-Faslpr strain. How can we explain this paradox? We suggest that IFN-γ overrides CSF-1-induced proliferation of Mφ in the kidneys of MRL-Faslpr mice. Since CSF-1 is absent in the IFN-γR-deficient MRL-Faslpr strain, Mφ are not recruited and do not proliferate in the kidney, and therefore the impact of IFN-γ is not evident.

It is also important to appreciate that T cells did not proliferate in IFN-γR-deficient MRL-Faslpr kidneys. This would seem counterintuitive. We previously reported that DN T cells secreting IFN-γ reduced the proliferation of T cells stimulated by TEC and are thus self regulatory (38). Furthermore, IFN-γ prevents proliferation of the murine Th2 cell (39) and limits alloimmune responses in murine cardiac and skin transplantation by down-regulating the proliferation of activated T cells (47). Yet we did not find an increase in T cell proliferation in IFN-γR-deficient MRL-Faslpr kidneys. This is understandable and analogous to our finding with Mφ. T cells, like Mφ, are not drawn into the kidney in IFN-γR-deficient MRL-Faslpr mice; thus, the antiproliferative impact of IFN-γ on T cells cannot be appreciated.

It has been reported by Le Hir’s laboratory that targeted deletion of IFN-γR- and IFN-γR-intact MRL-Faslpr mice developed lymphadenopathy (20). However, we note a difference in the lymphadenopathy comparing IFN-γR-deficient and IFN-γR-intact MRL-Faslpr mice. We suggest that this apparent discrepancy is related to the criteria used to evaluate lymphadenopathy. In our study using a semiquantitative analysis, lymphadenopathy in IFN-γR-deficient MRL-Faslpr mice was reduced compared with that in IFN-γR-intact or wild-type MRL-Faslpr mice, but did not return to the baseline. In contrast, Le Hir’s study used an all or none criteria. In agreement with our data, a recent study claimed that lymphadenopathy was reduced in IFN-γ-deficient MRL-Faslpr mice compared with that in IFN-γ-intact MRL-Faslpr mice (40). Furthermore, since DN T cells are responsible for lymphadenopathy in MRL-Faslpr mice, we suggest that IFN-γ regulates this T cell population. While these data support the concept that IFN-γ fosters the accumulation of DN T cells in lymph nodes, the mechanism remains to be elucidated.

Most importantly, we report for the first time an increase in apoptosis in renal parenchymal cells in MRL-Faslpr mice. Renal parenchymal cells can express Fas and Fas ligand in MRL+/+ mice (41). However, Fas/Fas ligand interactions cannot be responsible for mediating apoptosis in the Fas-deficient MRL-Faslpr strain. We detected apoptosis in the kidneys of 2% of the MRL-Faslpr mice. This is remarkable, since apoptotic cells are rapidly phagocytosed by Mφ, epithelial cells, and fibroblasts and are therefore extremely difficult to detect in tissue (42). The extensive amount of apoptosis in MRL-Faslpr kidneys may be the result of increased apoptosis of renal parenchymal cells overwhelming the phagocytic capacity of the kidney and/or impaired phagocytosis of apoptotic cells. We have now also established the novel finding that IFN-γ is responsible for the in vitro apoptosis of renal parenchymal cells. This was determined using several criteria that distinguish apoptosis (morphology, TUNEL, and annexin V) from necrosis (morphology, annexin V, and PI) (26, 27, 33). In addition, we have reported that Mφ from MRL-Faslpr mice proliferate more readily and are induced to undergo apoptosis more easily than Mφ from other strains (manuscript in preparation). Thus, the increase in apoptotic cells in the MRL-Faslpr kidney represents renal parenchymal cells and includes Mφ. Taken together, we propose that the intrarenal increase in IFN-γ in MRL-Faslpr mice released by T cells in the kidney triggers apoptosis of renal parenchymal cells. We speculate that the combined increase in apoptosis and defective clearance of these dead cells explains the substantial number of apoptotic cells in the MRL-Faslpr nephritic kidney.

We suggest that IFN-γ-induced apoptosis of renal parenchymal cells is detrimental to the kidney. In the IFN-γR-deficient MRL-Faslpr kidney protected from renal destruction we identified only few apoptotic cells. By comparison, apoptotic renal parenchymal cells were readily present in the IFN-γR-intact MRL-Faslpr kidneys in advanced renal injury. This is in agreement with studies in human lupus nephritis reporting a correlation between the increase in apoptotic renal parenchymal cells and the extent of glomerulosclerosis and deterioration of renal function (21). On the other hand, it has also been proposed that apoptosis is a protective mechanism that removes excess proliferating renal parenchymal cells and resolves proliferative glomerulonephritis (22). However, this putative homeostatic mechanism fails to prevent fatal lupus nephritis in MRL-Faslpr mice. By extension, it would be interesting to determine whether IFN-γ is responsible for inducing apoptosis in patients with lupus nephritis.

The focus of our study was the impact of IFN-γ at different stages of kidney disease in MRL-Faslpr mice. Nevertheless, renal injury in MRL-Faslpr mice is complex and involves cytokine/growth factors and Ab-dependent mechanisms (43). It is important to note that IFN-γ promotes the switch to the IgG subclasses implicated in glomerular injury (IgG2a and IgG3) (34, 35). Therefore, the decrease in circulating IgG2a and IgG3 anti-DNA Abs in IFN-γ-deficient and IFN-γR-deficient MRL-Faslpr mice may be responsible for protecting renal injury in MRL-Faslpr mice (20, 40, 44). In addition, circulating IgG may induce CSF-1 expression by mesangial cells, which, in turn, elicits renal injury (36). However, T and B cells are abnormal in MRL strains (45, 46); therefore, the regulation of autoantibodies by cytokines is unclear. Thus, the interrelationship between cytokines and autoantibodies in MRL-Faslpr mice requires additional studies.

In conclusion, we have identified several pathogenic mechanisms regulated by IFN-γ that are central to autoimmune kidney disease in MRL-Faslpr mice: the inductions of CSF-1, TNF-α, and apoptosis in renal parenchymal cells. We propose that kidney-infiltrating T cells release IFN-γ, which induces CSF-1 and TNF-α and recruits Mφ and autoreactive T cells. Autoreactive kidney-infiltrating T cells in MRL-Faslpr mice continue to supply IFN-γ and thus fosters a positive amplification loop responsible for renal parenchymal cell apoptosis and culminates in kidney destruction.

Acknowledgments

We are grateful to Dr. Charles B. Carpenter for critically reviewing this manuscript. We thank Josh Rosebrook for technical support.

Footnotes

  • ↵1 This work was supported by National Institutes of Health Grant DK36149, the Baxter Extramural Research Program, the German Ernst Jung Foundation for Science and Research (to A.S.), and a National Kidney Foundation Research Fellowship (to G.T.).

  • ↵2 Address correspondence and reprint requests to Dr. Vicki Rubin Kelley, Brigham and Women’s Hospital, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115. E-mail address: vicki{at}bustoff.bwh.harvard.edu

  • ↵3 Abbreviations used in this paper: Mφ, macrophage; DN, double negative; TUNEL, terminal deoxynucleotidyltransferase-mediated UTP end labeling; TEC, tubular epithelial cells; MC, mesangial cells; PI, propidium iodide.

  • Received November 6, 1997.
  • Accepted March 3, 1998.

References

  1. ↵
    Watanabe-Fukunaga, R., C. I. Brannan, N. G. Copeland, N. A. Jenkins, S. Nagata.. 1992. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356: 314
    OpenUrlCrossRefPubMed
  2. ↵
    Kelley, V. E., J. B. Roths. 1985. Interaction of mutant lpr gene with background strain influences renal disease. Clin. Immunol. Immunopathol. 37: 220
    OpenUrlCrossRefPubMed
  3. ↵
    Theofilopoulos, A. N., F. J. Dixon. 1981. Etiopathogenesis of murine SLE. Immunol. Rev. 55: 179
    OpenUrlCrossRefPubMed
  4. ↵
    Moyer, C. F., J. D. Strandberg, C. L. Reinisch. 1987. Systemic mononuclear-cell vasculitis in MRL/Mp-lpr/lpr mice: a histologic and immunocytochemical analysis. Am. J. Pathol. 127: 229
    OpenUrlPubMed
  5. ↵
    Wofsky, D., J. A. Ledbetter, P. L. Hendler, W. E. Seaman. 1985. Treatment of murine lupus with monoclonal anti-T cell antibody. J. Immunol. 134: 852
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Marshak-Rothstein, A., K. B. Elkon. 1996. B cells in systemic autoimmune disease: recent insights from Fas-deficient mice and men. Curr. Opin. Immunol. 8: 852
    OpenUrlCrossRefPubMed
  7. ↵
    Bloom, R. D., S. Florquin, V. R. Kelley. 1993. Colony stimulating factor-1 in the induction of lupus nephritis. Kidney Int. 43: 1000
    OpenUrlCrossRefPubMed
  8. ↵
    Yui, M. A., W. H. Brissette, D. C. Brennan, R. P. Wuthrich, V. E. Kelley. 1991. Increased macrophage colony stimulating factor in neonatal and adult autoimmune MRL-lpr mice. Am. J. Pathol. 139: 255
    OpenUrlPubMed
  9. ↵
    Boswell, J. M., M. A. Yui, D. W. Burt, V. E. Kelley. 1988. Increased tumor necrosis factor and IL-1β expression in the kidneys of mice with lupus nephritis. J. Immunol. 141: 3050
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Yokoyama, H., B. Kreft, V. Rubin Kelley. 1995. Biphasic increase in circulating and renal TNF-α in MRL-lpr mice with differing regulatory mechanisms. Kidney Int. 47: 122
    OpenUrlCrossRefPubMed
  11. ↵
    Fan, X., R. P. Wuthrich. 1997. Upregulation of lymphoid and renal interferon-γ mRNA in autoimmune MRL-Fas(lpr) mice with lupus nephritis. Inflammation 21: 105
    OpenUrlCrossRefPubMed
  12. ↵
    Naito, T., H. Yokoyama, K. J. Moore, G. Dranoff, R. C. Mulligan, V. R. Kelley. 1996. Macrophage growth factors introduced into the kidney initiate renal injury. Mol. Med. 2: 297
    OpenUrlPubMed
  13. ↵
    Moore, K. J., K. Yeh, T. Naito, V. Rubin Kelley. 1996. TNF-α enhances colony-stimulating factor-1-induced macrophage accumulation in autoimmune renal disease. J. Immunol. 137: 427
    OpenUrl
  14. ↵
    Brennan, D. C., M. A. Yui, R. P. Wuthrich, V. E. Kelley. 1989. Tumor necrosis factor and IL-1 in New Zealand Black/White mice. J. Immunol. 143: 3470
    OpenUrlAbstract
  15. ↵
    Jevnikar, A. M., M. J. Grusby, L. H. Glimcher. 1994. Prevention of nephritis in major histocompatibility complex class II-deficient MRL-lpr mice. J. Exp. Med. 179: 1137
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Koh, D. R., A. Ho, A. Rahemutulla, W. P. Fung-Leung, H. Griesser, T. W. Tak. 1995. Murine lupus in MRL/lpr mice lacking CD4 or CD8 T cells. Eur. J. Immunol. 25: 2558
    OpenUrlCrossRefPubMed
  17. ↵
    Seaman, W. E., D. Wofsy, J. S. Greenspan, J. A. Ledbetter. 1983. Treatment of autoimmune MRL/lpr mice with monoclonal antibody to Thy-1.2: a single injection has sustained effects on lymphoproliferation and renal disease. J. Immunol. 130: 1713
    OpenUrlAbstract
  18. ↵
    Diaz-Gallo, C., A. M. Jevnikar, D. C. Brennan, S. Florquin, A. Pacheco-Silva, V. Rubin Kelley. 1992. Autoreactive kidney-infiltrating T-cell clones in murine lupus nephritis. Kidney Int. 42: 851
    OpenUrlCrossRefPubMed
  19. ↵
    Manolios, N., L. Schrieber, M. Nelson, C. L. Geczy.. 1989. Enhanced interferon-γ (IFN) production by lymph node cells from autoimmune (MRL/l, MRL/n) mice. Clin. Exp. Immunol. 76: 301
    OpenUrlPubMed
  20. ↵
    Haas, C., B. Ryffel, M. Le Hir. 1997. IFN-γ is essential for the development of autoimmune glomerulonephritis in MRL/lpr mice. J. Immunol. 158: 5484
    OpenUrlAbstract
  21. ↵
    Sugiyama, H., N. Kashihara, H. Makino, Y. Yamasaki, Z. Ota. 1996. Apoptosis in glomerular sclerosis. Kidney Int. 49: 103
    OpenUrlCrossRefPubMed
  22. ↵
    Soto, H., J. Mosquera, B. Rodriguez-Iturbe, C. Henriquez La Roche, A. Pinto. 1997. Apoptosis in proliferative glomerulonephritis: decreased apoptosis expression in lupus nephritis. Nephrol. Dial. Transplant. 12: 273
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Huang, S., W. Hendriks, A. Althage, S. Hemmi, H. Bluethmann, R. Kamijo, J. Vilcek, R. M. Zinkernagel, M. Aguet. 1993. Immune response in mice that lack the interferon-γ receptor. Science 259: 1742
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Wu, J., T. Zhou, J. He, J. D. Mountz. 1993. Autoimmune disease in mice due to integration of an endogenous retrovirus in an apoptosis gene. J. Exp. Med. 178: 461
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Green, E. L. 1985. Biology of the Laboratory Mouse. Dover Publishers, New York, p. 18.
  26. ↵
    Vermes, I., C. Haanen, H. Steffens-Nakken, C. Reutelingsperger. 1995. A novel assay for apoptosis: flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. Immunol. Methods 184: 39
    OpenUrlCrossRefPubMed
  27. ↵
    Koopman, G., C. P. Reutelingsperger, G. A. Kuijten, R. M. Keehnen, S. T. Pals, M. H. van Oers. 1994. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84: 1415
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Bradley, T. R., D. Metcalf. 1966. The growth of mouse bone marrow cells in vitro. Aust. J. Exp. Biol. Med. 44: 287
    OpenUrlCrossRefPubMed
  29. ↵
    Brennan, D. C., A. M. Jevnikar, F. Takei, V. E. Rubin-Kelley. 1990. Mesangial cell accessory functions: mediation by intercellular adhesion molecule-1. Kidney Int. 38: 1039
    OpenUrlCrossRefPubMed
  30. ↵
    Chomczynski, P., N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156
    OpenUrlCrossRefPubMed
  31. ↵
    Munn, D. H., A. C. Beall, D. Song, R. W. Wrenn, D. C. Throckmorton. 1995. Activation-induced apoptosis in human macrophages: developmental regulation of a novel cell death pathway by macrophage colony-stimulating factor and interferon-γ. J. Exp. Med. 181: 127
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Schwarting, A., K. Moore, T. Wada, G. Tesch, H.-J. Yoon, V. R. Kelley. 1998. IFN-γ limits macrophage expansion in MRL-Faslpr autoimmune interstitial nephritis: a negative regulatory pathway. J. Immunol. 160: 4074
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Lieberthal, W., J. Levine. 1996. Mechanisms of apoptosis and its potential role in renal tubular epithelial cell injury. Am. J. Physiol. 271: F477
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Takahashi, S., M. Nose, J. Sasaki, T. Yamamoto, M. Kyogoku. 1991. IgG3 production in MRL/lpr mice is responsible for development of lupus nephritis. J. Immunol. 147: 515
    OpenUrlAbstract
  35. ↵
    Pankewycz, O. G., P. Migliorini, M. Madaio. 1987. Polyreactive autoantibodies are nephritogenic in murine lupus nephritis. J. Immunol. 139: 3287
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Satriano, J. A., K. Hora, Z. Shan, E. R. Stanley, T. Mori, D. Schloendorff. 1993. Regulation of monocyte chemoattractant protein-1 and macrophage colony-stimulating factor-1 by IFN-γ, tumor necrosis factor-α, IgG aggregates, and cAMP in mouse mesangial cells. J. Immunol. 150: 1971
    OpenUrlAbstract
  37. ↵
    Wada, T., T. Naito, R. C. Griffiths, T. M. Coffman, V. R. Kelley. 1997. Systemic autoimmune nephritogenic components induce CSF-1 and TNF-α in MRL kidneys. Kidney Int. 52: 934
    OpenUrlCrossRefPubMed
  38. ↵
    Diaz-Gallo, C., V. Rubin Kelley. 1993. Self-regulation of autoreactive kidney-infiltrating T-cells in MRL-lpr nephritis. Kidney Int. 44: 692
    OpenUrlCrossRefPubMed
  39. ↵
    Gajewski, T. F., F. W. Fitch. 1988. Anti-proliferative effect of IFN-γ in immune regulation: IFN-γ inhibits the proliferation of Th2 but not Th1 murine helper T lymphocyte clones. J. Immunol. 140: 4252
    OpenUrl
  40. ↵
    Peng, S. L., J. Moslehi, J. Craft. 1997. Roles of interferon-γ and interleukin-4 in murine lupus. J. Clin. Invest. 99: 1936
    OpenUrlCrossRefPubMed
  41. ↵
    Ortiz-Arduan, A., T. M. Danoff, R. Kalluri, S. Gonzalez-Cuadro, S. L. Karp, K. Elkon, J. Egido, E. G. Neilson. 1996. Regulation of Fas and Fas ligand expression in cultured murine renal cells and in the kidney during endotoxemia. Am. J. Physiol. 271: F1193
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Wyllie, A. H., J. F. R. Kerr, A. R. Currie.. 1980. Cell death: the significance of apoptosis. Int. Rev. Cytol. 68: 251
    OpenUrlCrossRefPubMed
  43. ↵
    Reininger, L., T. Berney, T. Shibata, F. Spertini, R. Merino, S. Izui. 1990. Cryoglobulinemia induced by a murine IgG3 rheumatoid factor: skin vasculitis and glomerulonephritis arise from distinct pathogenic mechanisms. Proc. Natl. Acad. Sci. USA 87: 10038
    OpenUrlAbstract/FREE Full Text
  44. ↵
    Balomenos, D., R. Rumold, A. N. Theofilopoulos. 1998. Interferon-γ is required for lupus-like disease and lymphoaccumulation in MRL-lpr mice. J. Clin. Invest. 101: 364
    OpenUrlCrossRefPubMed
  45. ↵
    Sobel, E. S., T. Katagiri, K. Katagiri, S. C. Morris, P. L. Cohen, R. A. Eisenberg. 1991. An intrinsic B cell defect is required for the production of autoantibodies in the lpr model of murine systemic autoimmunity. J. Exp. Med. 173: 1441
    OpenUrlAbstract/FREE Full Text
  46. ↵
    Katagiri, T., P. L. Cohen, R. A. Eisenberg. 1988. The lpr gene causes an intrinsic T cell abnormality that is required for hyperproliferation. J. Exp. Med. 167: 741
    OpenUrlAbstract/FREE Full Text
  47. ↵
    Konieczny, B. T., Z. Dai, E. T. Elwood, S. Saleem, P. S. Linsley, F. K. Baddoura, C. P. Larsen, T. C. Pearson, F. G. Lakkis. 1998. IFN-γ is critical for long-term allograft survival induced by blocking the CD28 and CD40 ligand T cell costimulation pathways. J. Immunol. 160: 2059
    OpenUrlAbstract/FREE Full Text
View Abstract
Back to top

In this issue

The Journal of Immunology
Vol. 161, Issue 1
1 Jul 1998
Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section