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IFN- Receptor Signaling Is Essential for the Initiation, Acceleration, and Destruction of Autoimmune Kidney Disease in MRL-Fas^lpr Mice¹

Laboratory of Molecular Autoimmune Disease, Renal Division, Brigham and Women’s Hospital, Boston, MA 02115

	Abstract

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CSF-1 and TNF- in the kidney of MRL-Fas^lpr 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-Fas^lpr kidney, infiltrating T cells that secrete IFN- are a hallmark of disease. To examine the impact of IFN- on renal injury in MRL-Fas^lpr mice, we constructed a IFN-R-deficient strain. In MRL-Fas^lpr mice lacking IFN-R, circulating and intrarenal CSF-1 were absent, TNF- 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-R induced CSF-1 and TNF- in MRL-Fas^lpr mice. We detected a large number of apoptotic renal parenchymal cells in advanced nephritis and determined that signaling via the IFN-R induces apoptosis of tubular epithelial cells (TEC), but not MC. By comparison, TNF- induces apoptosis in MC, but not TEC, of the MRL-Fas^lpr strain. Thus, IFN- is directly and indirectly responsible for apoptosis of TEC and MC in MRL-Fas^lpr mice, respectively. In conclusion, IFN-R signaling is essential for the initiation (CSF-1), acceleration (CSF-1 and TNF-), and apoptotic destruction of renal parenchymal cells in MRL-Fas^lpr autoimmune kidney disease.

	Introduction

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Autoimmune disease in MRL-Fas^lpr mice is multifaceted, targeting the kidney, joints, eyes, and skin. In MRL-Fas^lpr 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 Fas^lpr (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-Fas^lpr mice is complex and involves interstitial, glomerular, tubular, and perivascular pathology and is mediated by macrophages (M),³ T cells, and cytokines (3, 4, 5, 6).

Many cytokines are increased in the kidneys undergoing autoimmune destruction in MRL-Fas^lpr (7, 8, 9, 10, 11). The specific cytokines known to initiate or promote kidney injury in MRL-Fas^lpr mice are CSF-1 and TNF- (7, 8, 10). CSF-1 is responsible for eliciting and promoting autoimmune kidney destruction. We established that CSF-1 is expressed in the MRL-Fas^lpr 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-Fas^lpr kidney elicits renal injury (12). Although TNF- increases in parallel to CSF-1, gene transfer of TNF- fails to elicit autoimmune kidney injury (13). Rather, dual gene transfer of TNF- and CSF-1 amplifies the extent of renal damage produced by CSF-1 alone (13). Similarly, injecting TNF- 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- are more distal and require other molecules, notably CSF-1, to accelerate established disease.

T cells are required for autoimmune kidney disease in MRL-Fas^lpr 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-, a cytokine that has a broad array of functions capable of promoting tissue injury (18, 19). Since IFN- is increased in MRL-Fas^lpr kidneys, and blockade of the IFN-R signaling prevents glomerulonephritis and prolongs survival (20), we constructed an IFN-R-deficient MRL-Fas^lpr strain and determined whether IFN- is responsible for CSF-1-, TNF--, M-, 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- directly or indirectly is responsible for apoptotic parenchymal cell death. We now report that IFN- is required for the production of CSF-1 and TNF- and induces apoptosis of renal parenchymal cells. MRL-Fas^lpr mice lacking IFN-R are protected from fatal lupus nephritis. We propose that IFN- released by kidney-infiltrating T cells induces CSF-1 and TNF-, which, in turn, recruit M 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-R signaling is essential for the initiation, acceleration, and destruction of the autoimmune kidney in MRL-Fas^lpr mice.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

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

Generation of IFN-R-deficient MRL-Fas^lprmice

MRL-Fas^lpr mice lacking the IFN-R were derived by a series of genetic backcrosses using the cross-backcross-intercross scheme. MRL-Fas^lpr mice were mated with IFN-R-deficient (129/Sv/Ev x 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 Fas^lpr mutation and IFN-R using specific primers (23, 24). Double homozygous (Fas^lpr/Fas^lpr, IFN-R-/-) N1F1 progeny were backcrossed to MRL-Fas^lpr/Fas^lpr mice. B1 progeny, homozygous for the Fas^lpr mutation and heterozygous for the IFN-R (IFN-R^+/-), were intercrossed, and mice homozygous for the IFN-R-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-Fas^lpr mice (95–97.8% MRL background) homozygous and heterozygous for the mutated IFN-R (25). We analyzed this generation. In this manuscript, we describe IFN-R-/- as IFN-R-deficient and IFN-R^+/- as IFN-R-intact MRL-Fas^lpr 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-R-deficient, IFN-R-intact, and wild-type MRL-Fas^lpr 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 M 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-Fas^lpr 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 M 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 (M), 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 M, 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-R-deficient and intact MRL-Fas^lpr kidneys at 2 mo of age, before renal injury, were cultured on 96-well flat-bottom tissue culture plates (Costar, Cambridge, MA) at 10 x 10⁴ cells/well and incubated with medium, IFN- (50 U/ml), or TNF- (30 ng/ml) for 12 h, then washed with PBS and exposed to medium, IFN- (50 U/ml), TNF- (30 ng/ml), or TNF- plus IFN- 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- or TNF- for apoptotic cells using the TUNEL method, morphologic criteria, and annexin V and PI staining. TEC and MC from IFN-R-deficient and intact MRL-Fas^lpr kidneys were cultured on 24-well tissue culture plates (Costar, Cambridge, MA) at 20 x 10⁵ cells/well and incubated with medium, IFN- (50 U/ml), TNF- (30 ng/ml), or IFN- plus TNF- 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- in serum samples

We quantitated biologically active serum CSF-1 using a previously described M colony-stimulating assay (28). Briefly, 1 x 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- in serum samples from IFN-R-deficient and IFN-R-intact MRL-Fas^lpr 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- in the kidney

We detected CSF-1 and TNF- 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- 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- Ab with a 20-fold molar excess of CSF-1 (Genetics Institute, Cambridge, MA) or TNF- (Genentech, South San Francisco, CA), respectively. CSF-1 and TNF- 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-R-deficient or intact MRL-Fas^lpr 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-Fas^lpr, MRL^+/+, or C3H^+/+ mice 6 mo of age with or without IFN- (50 U/ml) or IFN- (50 U/ml) alone. CSF-1 was evaluated 24 h later using the immunoperoxidase method as described above.

MC in vitro expression of TNF-

MC from IFN-R-deficient and IFN-R-intact MRL-Fas^lpr kidneys were stimulated with medium, IFN- (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 2x SSC/0.1% SDS at room temperature and in 0.2x 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- 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 ß-actin (PstI fragment of pBA-1) as an internal control for the quantity and integrity of RNA.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Renal pathology in IFN-R-deficient MRL-Fas^lprmice is prevented

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

View larger version (49K): [in this window] [in a new window]   FIGURE 1. A, Reduction in kidney-infiltrating cells in IFN-R-deficient vs IFN-R-intact MRL-Faslpr mice. Mononuclear cells in glomerular, tubulointerstitial, and perivascular areas are reduced in B3F1 generation IFN-R-deficient (R-/-) compared with B3F1 generation IFN-R-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, x400); perivascular, 10 random inter- and intralobular arteries (magnification, x400); n = 5 in each group; 6 mo of age). * indicates p < 0.01, IFN-R intact vs IFN-R deficient; ** indicates p < 0.01, MRL+/+ vs IFN-R deficient (by Mann-Whitney test). B, The kidney infiltration of T cells and M in MRL-Faslpr mice is dependent on IFN-. M 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, x400), and 10 random inter- and intralobular arteries (magnification, x400; 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-R-deficient (R-/-) vs IFN-R-intact (R+/-) for all phenotypes.

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

Loss of renal function is prevented and mortality is reduced in IFN-R-deficient MRL-Fas^lpr mice

IFN-R-deficient MRL-Fas^lpr are protected from proteinuria. IFN-R-intact and wild-type MRL-Fas^lpr mice became proteinuric at 4 mo of age. By comparison, IFN-R-deficient MRL-Fas^lpr maintained normal renal function (Fig. 2). The increase in proteinuria was modestly slower in IFN-R-intact MRL-Fas^lpr mice compared with that in wild-type MRL-Fas^lpr 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-R-deficient compared with IFN-R-intact MRL-Fas^lpr mice (80 vs 10%, respectively, at 11 mo of age; Fig. 2). We noted that in the B3F1 generation of IFN-R-intact MRL-Fas^lpr 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-R-intact MRL-Fas^lpr generation has sufficient MRL genes to cause renal disease and is similar to the wild-type MRL-Fas^lpr strain.

View larger version (16K): [in this window] [in a new window]   FIGURE 2. Renal injury is prevented and mortality is reduced in IFN-R-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-R-deficient MRL-Faslpr strain (n = 6/group; p < 0.001). The IFN-R-deficient strain survived longer than the IFN-R-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-R-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-R-deficient MRL-Fas^lpr mice

Splenomegaly and lymphadenopathy, hallmarks of disease in MRL-Fas^lpr mice, were diminished in IFN-R-deficient MRL-Fas^lpr mice. Spleen weight was reduced fivefold in IFN-R-deficient compared with IFN-R-intact MRL-Fas^lpr 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-R-deficient compared with IFN-R-intact MRL-Fas^lpr 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-Fas^lpr mice compared to those in B3F1 generation IFN-R-intact MRL-Fas^lpr mice (40 and 22%, respectively).

CSF-1 is absent in the circulation and kidney of IFN-R-deficient MRL-Fas^lpr 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-Fas^lpr mice (8). CSF-1 was not detected in the sera of IFN-R-deficient MRL-Fas^lpr 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-R-intact and wild-type MRL-Fas^lpr mice. Similarly, we did not detect CSF-1 transcripts nor did we identify CSF-1 in the kidney sections of IFN-R-deficient MRL-Fas^lpr mice. In contrast, CSF-1 was abundant in IFN-R-intact or wild-type MRL-Fas^lpr kidneys using either method (Table I and Fig. 3).

View this table: [in this window] [in a new window]   Table I. CSF-1 is not expressed and TNF- is down-regulated in IFN-R-deficient MRL-Faslprkidneys1

View larger version (99K): [in this window] [in a new window]   FIGURE 3. CSF-1 is absent in IFN-R-deficient MRL-Faslpr mice. A, Biologically active CSF-1 was abundant in the sera of IFN-R intact or wild-type MRL-Faslpr mice, but was notably absent in the IFN-R-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-R-deficient (-/-) MRL-Faslpr kidneys and up-regulated in IFN-R-intact (+/-) MRL-Faslpr kidneys. C, Representative examples illustrating the CSF-1 in glomeruli of IFN-R-intact MRL-Faslpr kidneys (C) and the absence of CSF-1 in IFN-R-deficient MRL-Faslpr kidneys (D), as determined by the immunoperoxidase method. Magnification, x1000.

TNF- is down-regulated in the kidney of IFN-R-deficient MRL-Fas^lpr mice

Our previous studies determined that the increase in TNF- in the circulation parallels the increase in CSF-1 (10). In the present study, serum TNF- levels in IFN-R-deficient MRL-Fas^lpr mice were reduced compared with those in the IFN-R-intact MRL-Fas^lpr strain. In addition, we noted a modest increase in TNF- in the circulation of wild-type MRL-Fas^lpr mice compared with that in the IFN-R-intact MRL-Fas^lpr 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- mRNA transcripts in the kidney of IFN-R-deficient compared with IFN-R-intact MRL-Fas^lpr mice at 6 mo of age by Northern blot analysis (Fig. 4). Furthermore, we identified a decrease in TNF- expression in TEC and infiltrating mononuclear cells in the IFN-R-deficient compared with the IFN-R-intact MRL-Fas^lpr kidney sections (Table I and Fig. 4).

View larger version (112K): [in this window] [in a new window]   FIGURE 4. TNF- is down-regulated in IFN-R-deficient MRL-Faslprmice. A, Serum TNF- was diminished in IFN-R-deficient MRL-Faslpr compared with that in IFN-R-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-R-deficient MRL-Faslpr (R-/-) vs IFN-R-intact MRL-Faslpr (R+/-); ** indicates p < 0.01, IFN-R-intact MRL-Faslpr vs wild-type MRL-Faslpr. B, Northern blot. TNF- transcripts were reduced in IFN-R-deficient MRL-Faslpr (-/-) compared with those in IFN-R-intact MRL-Faslpr (+/-) kidneys. Blots were rehybridized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). C, Immunoperoxidase staining of TNF-. TNF- was abundant in cortical TEC from IFN-R-intact MRL-Faslpr kidneys (C), whereas TNF- was reduced in the renal cortex of IFN-R-deficient MRL-Faslpr (D). Paraffin sections; magnification, x220.

IFN- induces CSF-1 in MC from MRL-Fas^lprmice

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

View this table: [in this window] [in a new window]   Table II. IFN-, but not MRL-Faslpr sera, induces CSF-1 in MRL-Faslprmesangial cells1

IFN- induces TNF- in MC from MRL-Fas^lprmice

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

View this table: [in this window] [in a new window]   Table III. IFN- induces TNF- in MRL-Faslpr mesangial cells1

Renal parenchymal cells from IFN-R-deficient MRL-Fas^lpr mice are protected from apoptosis

Since priming cells with IFN- induces apoptosis of hemopoietic cells and may contribute to the extent of M or T cell accumulation, we investigated the amount of apoptotic cells in situ (31, 32). We detected up to 2% apoptotic cells in IFN-R-intact MRL-Fas^lpr 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-R-deficient MRL-Fas^lpr 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), M (F4/80), or B cells (CD21/35) were apoptotic (data not shown).

View this table: [in this window] [in a new window]   Table IV. IFN-R signaling is required for increased apoptosis in MRL-Faslprkidney1

View larger version (170K): [in this window] [in a new window]   FIGURE 5. IFN-R 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-R-intact MRL-Faslpr kidneys are shown. By comparison, the number of apoptotic cells was reduced in glomeruli (B) and interstitium (D) of IFN-R-deficient MRL-Faslpr kidneys. Magnification, x330.

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

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

View larger version (14K): [in this window] [in a new window]   FIGURE 6. IFN- and TNF- induce renal parenchymal cell death in MRL-Faslpr mice. TEC and MC from IFN-R-deficient and IFN-R-intact MRL-Faslpr mice were primed with IFN- (50 U/ml) or TNF- (30 ng/ml) for 12 h and exposed to IFN-, TNF-, or IFN- plus TNF- 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-R-intact MRL-Faslpr TEC to IFN- resulted in as much as 30% cell death after 24 h (A). In contrast, IFN- did not induce death in MRL-Faslpr MC (A). Serum deprivation caused the death of 40 to 50% TEC or MC (A). TNF- induced MC death (18%) in IFN-R-deficient and IFN-R-intact MRL-Faslpr strains (B). Priming of TEC with IFN- enhanced the induction of death by TNF- (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- alone (by analysis of variance).

Apoptosis is involved in IFN-- and TNF--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-Fas^lpr TEC and MC. Serum deprivation of MRL-Fas^lpr TEC resulted in twofold more dead TEC in IFN-R-deficient and IFN-R-intact MRL-Fas^lpr strains compared with TEC death induced by IFN- (Fig. 6A). Similarly, serum starvation of MRL-Fas^lpr MC induced more MC death than that induced by TNF- (Fig. 6B). This finding prompted us to examine whether apoptosis is involved in the mechanisms of TEC death induced by IFN- and TNF- in MRL-Fas^lpr 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- (Table V). In contrast, cell death was rarely detected in IFN-R-deficient and IFN-R-intact TEC after incubation with medium alone (Table V). To ensure that apoptosis is involved in IFN--induced TEC death, we also assessed adherent TEC and MC after exposure to IFN- or TNF- 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- to TEC increased apoptosis in adherent IFN-R-intact MRL-Fas^lpr TEC five- to sixfold, whereas IFN- did not induce apoptosis in IFN-R-deficient MRL-Fas^lpr TEC (Table V).

View this table: [in this window] [in a new window]   Table V. IFN- induces apoptosis in MRL-Faslpr TEC1

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We suggest that signaling through the IFN-R is responsible for CSF-1 production by MC in MRL-Fas^lpr mice. This is based on the following: 1) CSF-1 is absent in the kidney and circulation of IFN-R-deficient MRL-Fas^lpr mice; 2) CSF-1 is primarily a MC product (7); and 3) IFN- stimulates MRL-Fas^lpr MC to generate CSF-1. On the other hand, IFN- also indirectly induces CSF-1. TNF- and/or circulating Ig complexes could increase intrarenal CSF-1, since TNF- and Ig complexes are abundant in the serum of MRL-Fas^lpr 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-Fas^lpr mice did not induce CSF-1. This could indicate that systemic exposure of TNF- or Ig complexes is insufficient to cause MC to secrete CSF-1. On the other hand, transplanting a normal MRL^+/+ kidney into a MRL-Fas^lpr 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-, TNF-, 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- locally within the kidney. We favor this explanation, since TEC genetically modified to secrete CSF-1 and/or TNF- implanted in the kidney, delivering these cytokines locally and not systemically, elicited renal injury in MRL-Fas^lpr kidneys (12, 13). This suggests that high titers of cytokines localized within the kidney are required for promoting renal injury.

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

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

It is also important to appreciate that T cells did not proliferate in IFN-R-deficient MRL-Fas^lpr kidneys. This would seem counterintuitive. We previously reported that DN T cells secreting IFN- reduced the proliferation of T cells stimulated by TEC and are thus self regulatory (38). Furthermore, IFN- 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-R-deficient MRL-Fas^lpr kidneys. This is understandable and analogous to our finding with M. T cells, like M, are not drawn into the kidney in IFN-R-deficient MRL-Fas^lpr mice; thus, the antiproliferative impact of IFN- on T cells cannot be appreciated.

It has been reported by Le Hir’s laboratory that targeted deletion of IFN-R- and IFN-R-intact MRL-Fas^lpr mice developed lymphadenopathy (20). However, we note a difference in the lymphadenopathy comparing IFN-R-deficient and IFN-R-intact MRL-Fas^lpr 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-R-deficient MRL-Fas^lpr mice was reduced compared with that in IFN-R-intact or wild-type MRL-Fas^lpr 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--deficient MRL-Fas^lpr mice compared with that in IFN--intact MRL-Fas^lpr mice (40). Furthermore, since DN T cells are responsible for lymphadenopathy in MRL-Fas^lpr mice, we suggest that IFN- regulates this T cell population. While these data support the concept that IFN- 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-Fas^lpr 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-Fas^lpr strain. We detected apoptosis in the kidneys of 2% of the MRL-Fas^lpr mice. This is remarkable, since apoptotic cells are rapidly phagocytosed by M, epithelial cells, and fibroblasts and are therefore extremely difficult to detect in tissue (42). The extensive amount of apoptosis in MRL-Fas^lpr 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- 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 M from MRL-Fas^lpr mice proliferate more readily and are induced to undergo apoptosis more easily than M from other strains (manuscript in preparation). Thus, the increase in apoptotic cells in the MRL-Fas^lpr kidney represents renal parenchymal cells and includes M. Taken together, we propose that the intrarenal increase in IFN- in MRL-Fas^lpr 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-Fas^lpr nephritic kidney.

We suggest that IFN--induced apoptosis of renal parenchymal cells is detrimental to the kidney. In the IFN-R-deficient MRL-Fas^lpr kidney protected from renal destruction we identified only few apoptotic cells. By comparison, apoptotic renal parenchymal cells were readily present in the IFN-R-intact MRL-Fas^lpr 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-Fas^lpr mice. By extension, it would be interesting to determine whether IFN- is responsible for inducing apoptosis in patients with lupus nephritis.

The focus of our study was the impact of IFN- at different stages of kidney disease in MRL-Fas^lpr mice. Nevertheless, renal injury in MRL-Fas^lpr mice is complex and involves cytokine/growth factors and Ab-dependent mechanisms (43). It is important to note that IFN- 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--deficient and IFN-R-deficient MRL-Fas^lpr mice may be responsible for protecting renal injury in MRL-Fas^lpr 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-Fas^lpr mice requires additional studies.

In conclusion, we have identified several pathogenic mechanisms regulated by IFN- that are central to autoimmune kidney disease in MRL-Fas^lpr mice: the inductions of CSF-1, TNF-, and apoptosis in renal parenchymal cells. We propose that kidney-infiltrating T cells release IFN-, which induces CSF-1 and TNF- and recruits M and autoreactive T cells. Autoreactive kidney-infiltrating T cells in MRL-Fas^lpr mice continue to supply IFN- 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:

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

Received for publication November 6, 1997. Accepted for publication March 3, 1998.

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				D. Neumann, E. Del Giudice, A. Ciaramella, D. Boraschi, and P. Bossu Lymphocytes from Autoimmune MRL lpr/lpr Mice Are Hyperresponsive to IL-18 and Overexpress the IL-18 Receptor Accessory Chain J. Immunol., March 15, 2001; 166(6): 3757 - 3762. [Abstract] [Full Text] [PDF]

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