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IFN- Limits Macrophage Expansion in MRL-Fas^lpr Autoimmune Interstitial Nephritis: A Negative Regulatory Pathway¹

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

	Abstract

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IFN- is capable of enhancing and limiting inflammation. Therefore, an increase in IFN- in autoimmune MRL-Fas^lpr mice could exacerbate or thwart renal injury. We have established a retroviral gene transfer approach to incite interstitial nephritis in MRL-Fas^lpr mice that is rapid, enduring, and circumscribed. Renal tubular epithelial cells (TEC) were genetically modified to secrete macrophage (M) growth factors (CSF-1-TEC, GM-CSF-1-TEC) and infused under the renal capsule. To determine the impact of IFN- in M growth factor-incited renal injury, we constructed a MRL-Fas^lpr IFN--receptor (IFN-R)-deficient strain. Gene transfer of CSF-1 or GM-CSF incited more severe interstitial nephritis in IFN-R-deficient than in IFN-R-intact MRL-Fas^lpr mice, consisting of an increase of M. To determine the mechanism responsible for the increase in M in IFN-R-deficient MRL-Fas^lpr mice, we evaluated M proliferation, apoptosis, and recruitment. Proliferation of bone marrow M from IFN-R-intact MRL-Fas^lpr costimulated with CSF-1 or GM-CSF and IFN- was reduced twofold, while the IFN-R-deficient MRL-Fas^lpr bone marrow M remained stable. Furthermore, we detected more proliferating and fewer apoptotic M within the interstitium in IFN-R-deficient MRL-Fas^lpr mice. Using unilateral ureteral ligation we established that IFN-R signaling does not alter M recruitment into the kidney. Thus, the increase in M elicited by M growth factors in IFN-R-deficient MRL-Fas^lpr mice is a result of enhanced proliferation and decreased apoptosis, and is independent of recruitment. Taken together, we suggest that IFN- provides a negative regulatory pathway capable of limiting M-mediated renal inflammation.

	Introduction

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Interferon- is a pleiotropic cytokine with a broad spectrum of actions capable of enhancing and suppressing immune reactions (1). Studies identifying the role of IFN- in autoimmune disease support the concept that IFN- promotes tissue destruction (2, 3). In fact, IFN- is the hallmark of CD4 Th1 cells, which dominate immune responses in many autoimmune diseases (4, 5). IFN- is a regulator of the activation of self-reactive T cells mediated by accessory cell interactions and promotes immune tissue destruction through the induction of MHC classes I and II on APCs and by increasing M³ surface Fc receptors (6, 7, 8, 9). On the other hand, IFN- is a potent inhibitor of M and T cell proliferation (10, 11) and, therefore, could be responsible for dampening immune-mediated tissue injury. Binding of the IFN-R blocks the early events of growth factor-stimulated signal transduction, resulting in inhibition of DNA synthesis and cell proliferation (12). Since IFN- can provide a negative regulatory pathway to quench local immune/inflammatory tissue damage or, conversely, incite destructive events, the impact of removing IFN- from tissues programmed for autoimmune destruction would be difficult to predict.

Renal injury in MRL-Fas^lpr (formerly designated MRL-lpr) mice is determined by several factors. The MRL background genes are responsible for kidney disease (13). The MRL-+/+ strain develops a latent, mild autoimmune renal injury. The interaction of MRL with the single gene mutation in Fas (MRL-Fas^lpr) converts this mild renal injury into a rapid and fulminant tissue-destructive process (14). Renal pathology in MRL-Fas^lpr mice is complex; involves interstitial, glomerular, tubular, and perivascular lesions; and is mediated by Ab-dependent and cellular mechanisms (15, 16). Therefore, renal disease in MRL-Fas^lpr is a result of immune complexes and cytokine/growth factor-related events. Conceptually, the absence of Fas in this strain results in the failure to delete autoreactive T cells via apoptosis by the engagement of Fas with its cognate ligand. Undeleted autoreactive T cells are targeted to the kidney and initiate kidney disease. CSF-1 and other M growth factors can recruit autoreactive T cells (17). Based on previous experiments, we have established that CSF-1 is instrumental in promoting renal disease in MRL-Fas^lpr mice. Expression of CSF-1 and infiltration of M in the kidney of MRL-Fas^lpr mice precede renal injury and increase proportionally with the severity of destruction (18, 19). CSF-1 is required for the survival and proliferation of M propagated from MRL-Fas^lpr glomeruli (18). In addition, we determined in congenic transplant experiments that CSF-1, M, and renal injury are linked in MRL-Fas^lpr mice. Transplanting a nephritic MRL-Fas^lpr kidney bearing M and CSF-1 into a congenic MRL-+/+ mouse after the removal of normal kidneys resulted in the disappearance of CSF-1 and M and the resolution of renal injury (20). Conversely, transplanting a MRL-+/+ normal kidney into a MRL-Fas^lpr mouse following the removal of nephritic kidneys expressing CSF-1 and M induced CSF-1, the accumulation of M, and renal injury (20). Moreover, using a gene transfer approach, we established that CSF-1 incited local renal injury in mice with the Fas^lpr mutation (19). Taken together, these studies establish that CSF-1 in the kidney is responsible for autoimmune tissue destruction in MRL-Fas^lpr mice.

IFN- is increased in the spleen, lymph nodes, and kidney of MRL-Fas^lpr mice (21, 22). One probable source of IFN- in MRL-Fas^lpr kidney is the double negative (DN) T cell, which does not bear CD4 or CD8 determinants (23). These DN T cells spontaneously secrete IFN- and represent a major portion of kidney-infiltrating cells (24). We established that kidney-infiltrating DN T cells release IFN- in the kidney and increase MHC class II and intercellular adhesion molecules on tubular epithelial cells (TEC) in MRL-Fas^lpr mice (25). Conversely, the release of IFN- by kidney-infiltrating DN T cells provides a self-regulatory mechanism to limit autoreactive T cell expansion in the kidney (6). The purpose of this study was to determine whether IFN- in MRL-Fas^lpr mice promotes or thwarts renal damage. For this purpose, we selected a rapid, reliable approach to incite local renal interstitial nephritis in MRL-Fas^lpr mice. Gene transfer of M growth factors into the kidney of MRL-Fas^lpr mice induced interstitial nephritis mediated by M and T cells. In IFN-R-deficient compared with IFN-R-intact MRL-Fas^lpr mice, M growth factors incited a four- to fivefold expansion of M, resulting in extensive renal injury. In vitro and in situ studies establish that signaling through the IFN-R reduced M proliferation, enhanced apoptosis, and did not alter recruitment. In conclusion, IFN- provides a negative regulatory pathway to limit M growth factor-incited renal damage in MRL-Fas^lpr mice by inhibiting M proliferation and increasing M apoptosis.

	Materials and Methods

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Mice

MRL/MpJ-+/+ (MRL-+/+), 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)F₂ were provided by Dr. M. Aguet (Institut Suisse de Recherches Experimentales sur le Cancer, University of 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 (26). All mice were housed and bred in our pathogen-free animal facility.

Generation of IFN-R deficient MRL-Fas^lpr mice

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)F₂ mice to yield heterozygous F₁ offspring. F₁ mice were intercrossed, and progeny were screened by PCR amplification of genomic DNA obtained from the tail for the Fas^lpr mutation and the IFN-R gene using specific primers (26, 27). N1F1 progeny that were double homozygotes (Fas^lpr/Fas^lpr, IFN-R^-/-) were backcrossed to MRL-Fas^lpr/Fas^lpr mice. All B₁ progeny were homozygous for the Fas^lpr mutation and heterozygous for the IFN-R (IFN-R^+/-). B₁ offspring 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 mating, this breeding scheme generated a colony of MRL-Fas^lpr mice (90–95% MRL background) homozygous and heterozygous for the IFN-R mutation (28). In this report we describe IFN-R^-/- as IFN-R-deficient and IFN-R^+/- as IFN-R-intact MRL-Fas^lpr mice.

Retrovirus-mediated cytokine gene transfer into cultured TEC

We isolated and cultured TEC derived from MRL-Fas^lpr mice, 1 to 2 mo of age, as previously described (29). Briefly, kidneys were removed, and renal cortexes were minced, dispersed in collagenase solution, and passed through a series of steel sieves. Cells that passed through the smallest sieve (38 µm) were collected and resuspended in DMEM/F12 medium containing 10% FCS and hormone supplements (KI medium) (29). Nonadherent cells were transferred to collagen (type IV)-coated plates and grown to confluence. Before retroviral infection, TEC were removed by trypsinization and plated at 1 x 10⁶ cells.

CRIP packaging cell lines producing helper-free recombinant retroviruses carrying cytokine genes were generated as previously described (30). Briefly, DNA sequences encoding GM-CSF (bp 174–619) or CSF-1 (bp 160–1874) were subcloned into the Moloney murine leukemia virus (MoMuLV)-based MFG vector. The MFG vector carrying a cytokine gene was introduced into a mammalian packaging cell line (CRIP) containing proviral sequences. These recombinant retrovirus-producing cells were grown in DMEM complete medium (10% FCS, 100 U/ml each of penicillin and streptomycin, and 10 mM HEPES). The virus-containing cell culture supernatant was harvested, and viral stocks were applied to TEC (1 x 10⁶ cells) in the presence of polybrene. TEC were then replenished with KI medium and grown to confluence, and culture supernatants were collected for verification of cytokine secretion. Successful cytokine gene transfer into TEC was verified by immunohistochemistry and also by measuring GM-CSF and CSF-1 in supernatants collected 6 days after passage by colony-stimulating assay (31). For control experiments, TEC were infected with lacZ coding MoMuLV (lacZ as reporter gene replaces the cytokine cDNA; provided by Dr. Richard Mulligan, Boston, MA). Successful gene transfer into TEC was verified by 1) selecting neomycin-resistant infected TEC using G418 (1 mg/ml) and 2) staining the selected TEC with X-Gal for the presence of ß-galactosidase, as described previously (19). In this report, we denote TEC genetically modified to express GM-CSF and CSF-1 as GM-CSF-TEC and CSF-TEC, respectively, and TEC infected with lacZ-MoMuLV as lacZ-TEC.

Delivery of TEC under the renal capsule

We placed genetically modified (CSF-1-TEC, GM-CSF-TEC, lacZ-TEC) or uninfected TEC under the renal capsule of IFN-R-deficient and IFN-R-intact MRL-Fas^lpr mice at 12 wk of age as previously described (19). Mice were anesthetized with ether, and the left kidney was exposed through a flank incision. A cell suspension of 1 x 10⁶ GM-CSF-TEC, CSF-TEC, lacZ-TEC, or uninfected TEC in 50 µl of HBSS was injected under the capsule of the dorsal surface of the kidney, and the peritoneum and skin were closed. The viability of the TEC was >90% by trypan blue staining immediately before implantation.

M proliferation assay

Bone marrow M (BMM) extracted from IFN-R-deficient or IFN-R-intact MRL-Fas^lpr mice (6–8 wk of age) were cultured, and proliferation was assessed by [³H]thymidine uptake as previously described (32). Briefly, quiescent 6-day-old BMM were seeded at 2 x 10⁴ cells/well in flat-bottom 96-well plates and treated with increasing concentrations of recombinant human CSF-1 (1, 5, 10, and 20 ng/ml), recombinant human GM-CSF (5, 10, 20, and 50 U/ml), and recombinant murine IFN- (1, 10, 50, and 100 U/ml). After 48-h incubation, BMM were pulsed with 1 µCi/well of [³H]thymidine for an additional 18 h. The cells were then harvested (Wallac, Gaithersburg, MD), and [³H]thymidine uptake (counts per minute) was measured. Data from triplicate samples are reported as the difference between the uptake of [³H]thymidine (counts per minute) of stimulated and unstimulated BMM ± SEM.

Renal pathology

The implanted (left) kidney and the contralateral kidney were removed either 14 or 28 days postimplantation. Kidneys were either snap-frozen in OCT compound (Miles Scientific, Naperville, IL) for cryostat sectioning or fixed in 10% neutral-buffered formalin. Formalin-fixed tissue was embedded in paraffin. Sections (4 µm) were stained with hematoxylin and eosin and evaluated by light microscopy. To find the maximal lesion, the kidney lesion was serial sectioned (6 µm) and evaluated every 20 µm. The cell accumulation at the implant site was assessed by counting the number of cell layers in the subcapsular space and adjacent renal cortex. To determine the percentage of M and T cells at the implant site, we stained frozen sections by the immunoperoxidase method using Abs to F4/80, CD4, CD8, and B220 determinants as previously described (33). 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, San Diego, CA). After blocking endogenous peroxidase activity with 0.6% H₂O₂ and 0.2% sodium azide for 10 min and blocking endogenous avidin and biotin using an avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA), tissue sections were incubated with purified rat anti-murine F4/80, CD4, CD8, or B220 Abs (5 µg/ml) for 2 h at room temperature, followed by biotinylated goat anti-rat IgG (mouse adsorbed) for 1 h at room temperature. Tissue sections were then incubated for 1 h with avidin-peroxidase complex (Vector Elite Kit, Vector Laboratories) at room temperature. Peroxidase was developed using 3,3'-diaminobenzidine (DAB) to produce a brown color. Tissue sections were counterstained with methyl green/Alcian blue. Specificity controls included replacement of primary Ab with normal rat IgG or rabbit sera. The number of M and T cells within the renal lesion was reported as a cell index (maximum subcapsular and intrarenal cell layers x percentage of cell phenotype).

In situ detection of proliferating cells

Cell proliferation was detected by immunostaining for proliferating cell nuclear Ag (PCNA). Paraffin sections (4 µm) were heated in a microwave oven for 12 min in 10 mM sodium citrate buffer, pH 6.0, and labeled with 1 µg/ml anti-PCNA Ab, FITC conjugated (clone 19F4, Boehringer Mannheim, Indianapolis, IN), overnight at 4°C. Bound primary Ab was detected with sheep anti-FITC IgG conjugated with alkaline phosphatase (1/500; Boehringer Mannheim) for 1 h at room temperature. Alkaline phosphatase was developed with Fast Blue BB salt (Sigma, St. Louis, MO) to produce a blue color. Subsequently, sections were stained for CD4, CD8, and B220 determinants using the immunoperoxidase method with DAB to produce a brown color as described above. Cell proliferation within the elicited lesion was assessed by counting the number of PCNA-positive CD4, CD8 or B220-positive cells per 100-µm² field. In addition, sequential staining for F4/80 and PCNA on frozen sections fixed in paraformaldehyde was performed using the immunoperoxidase method as described above. At least five fields were scored for each lesion. Scoring was performed by two blinded observers.

Detection of apoptotic cells

Apoptotic cells were detected by enzymatic in situ labeling of apoptosis-induced DNA strand breaks (TUNEL method). Frozen sections were fixed in 4% paraformaldehyde in PBS, permeabilized in 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) 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. Peroxidase was developed with DAB (Vector Laboratories), and tissue sections were counterstained with methyl green/Alcian blue. The number of apoptotic cells within the implant area was assessed by counting the number of TUNEL-positive cells per 100-µm² field. At least five fields were scored by two blinded observers. In addition, TUNEL-positive cells were assessed by light microscopy for morphologic characteristics of apoptosis, such as condensed and fragmented nuclei.

Detection of serum CSF-1 and GM-CSF

Serum samples were collected 14 and 28 days after implanting CSF-1-TEC or GM-CSF-TEC and evaluated for CSF-1 and GM-CSF by the colony-stimulating assay as previously described (31).

Unilateral ureteral ligation

Hydronephrosis was induced by unilateral ureteral obstruction. C57BL/6 and IFN-R-deficient (129/Sv/Ev x C57BL/6)F₂ mice were anesthetized, and the left ureter was identified through midline incision and ligated with 4/0 silk. After 1 wk, the obstructed and contralateral kidneys were harvested and prepared for M and PCNA immunostaining as described above. Interstitial M accumulation was quantitated by counting the number of F4/80-positive cells per 100-µm² field. Cell proliferation within the interstitium was assessed by counting interstitial PCNA-positive cells per 100-µm² field. At least 10 fields were scored for each kidney. Scoring was performed by two blinded observers.

Statistical analysis

Data are reported as the mean ± SEM. Statistical significance was determined by the Mann-Whitney U test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Gene transfer of CSF-1 or GM-CSF in the kidney of IFN-R-deficient MRL-Fas^lpr amplifies renal injury

To determine whether IFN- signaling is required for the induction of renal injury in MRL-Fas^lpr mice, we infused retrovirally transfected TEC producing CSF-1 or GM-CSF under the renal capsule of IFN-R-deficient and IFN-R-intact MRL-Fas^lpr mice. Both IFN-R-deficient and IFN-R-intact MRL-Fas^lpr TEC constitutively secreted CSF-1 or GM-CSF into the circulation (Table I). By comparison, uninfected TEC did not express endogenous CSF-1 or GM-CSF (data not shown). Thus, we established that gene transfer of CSF-1 or GM-CSF into the kidney resulted in constitutive, sustained cytokine expression, and similar amounts of each molecule were secreted in IFN-R-intact and -deficient MRL-Fas^lpr mice. CSF-1-TEC and GM-CSF-TEC incited a massive accumulation of mononuclear cells at the implant site and in the adjacent interstitium in IFN-R-deficient MRL-Fas^lpr mice 28 days after implantation (73 ± 17 cell layers; n = 8; Fig. 1). In contrast, in IFN-R-intact MRL-Fas^lpr mice, CSF-1-TEC and GM-CSF-TEC incited a smaller lesion (34 ± 13 cell layers; n = 7; Fig. 1). Renal injury was not detected in MRL-Fas^lpr kidneys infused with either lacZ-TEC or uninfected TEC (0 ± 0 intrarenal cell layers; n = 4/group; Fig. 1).

View larger version (82K): [in this window] [in a new window]   FIGURE 1. Gene transfer of CSF-1 or GM-CSF in the kidney of IFN-R-deficient MRL-Faslpr amplifies renal injury. The renal lesion was evaluated by counting the number of cell layers in the subcapsular space and in the adjacent renal cortex at the implant site with the most extensive pathology. Note the enhanced accumulation of mononuclear infiltrating cells extending from the subcapsular site to the renal cortex (arrows) in IFN-R-deficient (A) compared with IFN-R-intact (B) MRL-Faslpr mice. By comparison, infusion of lacZ-TEC or uninfected TEC into IFN-R-intact or -deficient MRL-Faslpr kidneys did not induce renal lesions. Uninfected TEC infused into IFN-R-deficient MRL-Faslpr kidney are shown in C. Representative examples are shown. Magnification, x330; hematoxylin-eosin stain.

Increased M accumulation is responsible for the larger renal injury in IFN-R-deficient MRL-Fas^lpr mice

We compared the phenotype of infiltrating cells in the induced renal injury of IFN-R-intact and -deficient MRL-Fas^lprmice. M, not T cells, comprise the vast majority of cells accumulating in the renal lesion 28 days after implanting CSF-1-TEC or GM-CSF-TEC in IFN-R-deficient MRL-Fas^lpr mice (Fig. 2). Immunostaining revealed a four- to fivefold greater accumulation of M in CSF-1- and GM-CSF-induced renal injury in IFN-R-deficient compared with IFN-R-intact MRL-Fas^lpr mice (Table II). In contrast, the number of T cells did not differ in the induced lesion in the presence or the absence of IFN-R. In addition, we established that kidney-infiltrating cells bearing B220 determinants belong to the unique DN (CD4^-, CD8^-) T cell subset in MRL-Fas^lpr mice, since these cells did not express B cell determinants (CD21/CD35; data not shown).

View larger version (103K): [in this window] [in a new window]   FIGURE 2. Gene transfer of CSF-1 or GM-CSF in the kidney of IFN-R-deficient MRL-Faslpr mice increases the accumulation of M. The number of M within the renal lesion was evaluated by immunoperoxidase labeling with F4/80 Ab and is indicated as a cell index (maximum subcapsular and intrarenal cell layers x percentage of F4/80-positive cells). A, Placing CSF-1-TEC under the renal capsule of IFN-R-deficient MRL-Faslpr mice causes a dramatic accumulation of M in the implant area. B, In contrast, CSF-1-TEC cause fewer M to accumulate in the renal lesion of IFN-R-intact (IFN-R+/-) MRL-Faslpr mice. Magnification, x660.

View this table: [in this window] [in a new window]   Table II. IFN-R signaling does not alter the amount of T cells in Mø growth factor incited renal injury in MRL-Faslpr kidney1

Circulating CSF-1 or GM-CSF does not exacerbate renal pathology in IFN-R-deficient MRL-Fas^lpr mice

CSF-1-TEC and GM-CSF-TEC placed under the renal capsule delivered CSF-1 and GM-CSF into the circulation for 28 days (Table I). To investigate whether circulating M growth factors influenced renal pathology in IFN-R-deficient MRL-Fas^lpr kidneys, we evaluated the contralateral kidneys in CSF-1-TEC- and GM-CSF-TEC-implanted mice at 28 days. Despite sustained expression of CSF-1 and GM-CSF in the circulation, we did not detect an increase in the numbers of M or T cells (CD4, CD8, B220) in the glomeruli, interstitium, or perivascular lesions of the contralateral kidney of IFN-R-deficient MRL-Fas^lpr mice compared with that in untreated age-matched MRL-Fas^lpr kidneys (data not shown).

IFN- blocks proliferation of M to CSF-1 or GM-CSF and enhances apoptosis

To investigate whether the increase in M in IFN-R-deficient MRL-Fas^lpr mice was related to the loss of IFN- blockade of proliferation, we evaluated the number of proliferating cells within the implant area of IFN-R-deficient and IFN-R-intact MRL-Fas^lpr mice. Immunostaining for PCNA-positive cells revealed that blockage of IFN-R signaling resulted in an increase in cell proliferation within the induced renal lesion (Table III and Fig. 3). We identified the majority of proliferating cells as M (80%) by dual and sequential staining for PCNA and CD4, CD8, B220, and F4/80 determinants. By comparison, few T cells were proliferating (CD4, 7%; CD8, 6%; B220, 1%). Furthermore, since IFN- primes cells to undergo apoptosis (11), we investigated whether a decrease in apoptosis contributed to the enhanced M accumulation in IFN-R-deficient MRL-Fas^lpr kidneys. The loss of IFN-R signaling resulted in a decrease in apoptotic cells within the induced renal lesion (Table III and Fig. 3), which were identified as M in sequential sections (5 µm). Thus, we established that in M growth factor-induced renal injury, signaling through the IFN-R limits the expansion of M by inhibiting proliferation and increasing apoptosis. We therefore investigated whether IFN- is directly responsible for blocking CSF-1- or GM-CSF-mediated cell proliferation in vitro. M are rare in normal kidneys (34). An increase in glomerular M is indicative of renal disease. Since we established that glomerular M and BMM from MRL-Fas^lpr mice respond similarly to CSF-1 and GM-CSF (32) and are indistinguishable, we evaluated BMM from IFN-R-deficient and -intact MRL-Fas^lpr mice. Proliferation of BMM in response to CSF-1 or GM-CSF was dose dependent as assessed by [³H]thymidine uptake, with a maximum at 20 ng/ml CSF-1 and 50 U/ml GM-CSF, and was independent of the presence of a functional IFN-R (Fig. 4). GM-CSF caused a greater proliferative response than CSF-1, confirming our previous data (32). Dose-response curves for the coincubation of CSF-1 or GM-CSF with different concentrations of IFN- established that the antiproliferative action of IFN- was maximal at 50 U/ml. Costimulation with IFN- reduced M proliferation from 1.0 x 10⁴ cpm (CSF-1) and 1.3 x 10⁴ cpm (GM-CSF) to 0.5 x 10⁴ cpm (CSF-1) and 0.63 x 10⁴ cpm (GM-CSF; Fig. 4). IFN- alone did not alter the proliferation of IFN-R-intact or -deficient BMM. The BMM viability was >90%, indicating that IFN- was not cytotoxic (data not shown). Thus, these in vitro data support our in vivo findings that IFN- inhibits M proliferation to CSF-1 or GM-CSF.

View larger version (173K): [in this window] [in a new window]   FIGURE 3. Gene transfer of CSF-1 or GM-CSF in the kidney of IFN-R-deficient MRL-Faslpr mice increases the number of proliferating cells and decreases the number of apoptotic cells within the implant area. Proliferating cells (black nuclei) were detected by immunostaining for PCNA. The number of PCNA-positive cells within the implant area of IFN-R-deficient MRL-Faslpr mice (A) was reduced in IFN-R-intact MRL-Faslpr kidneys (B). Apoptotic cells within the implant area (black condensed nuclei, marked with arrows) were evaluated by TUNEL staining. Placing CSF-1-TEC under the renal capsule of IFN-R-intact MRL-Faslpr mice resulted in the formation of more apoptotic cells (C) compared with the effect of CSF-1-TEC treatment in IFN-R-deficient MRL-Faslpr kidneys (D). Magnification, x800.

View larger version (27K): [in this window] [in a new window]   FIGURE 4. A, BMM from IFN-R-deficient and IFN-R-intact MRL-Faslpr mice proliferate similarly to recombinant human CSF-1 (rhCSF-1) in a dose-dependent manner. BMM from IFN-R-deficient and IFN-R-intact MRL-Faslpr were incubated with different concentrations of rhCSF-1 (1, 5, 10, and 20 ng/ml) for 48 h. Proliferation was measured by uptake of [3H]thymidine. Data are reported as the difference between the uptake of [3H]thymidine (counts per minute) of stimulated and unstimulated BMM ± SEM. The results shown are representative of five experiments. B, IFN- can block CSF-1- or GM-CSF-induced proliferation of BMM derived from IFN-R-intact MRL-Faslpr mice. BMM from IFN-R-deficient and IFN-R-intact MRL-Faslpr were treated with either 20 ng/ml rhCSF-1 or 50 U/ml rhGM-CSF and coincubated with 50 U/ml rmIFN- for 48 h. Proliferation was measured as described above. The results shown are representative of at least five experiments.

Signaling through the IFN-R does not alter the recruitment of M into the kidney in experimental hydronephrosis

To ensure that the increase in M in IFN-R-deficient MRL-Fas^lpr mice was not related to a decrease in M recruitment to the implant site, we evaluated M recruitment in an experimental hydronephrosis model. One week after unilateral ureteral ligation, there was a florid infiltration of cells in the ligated kidney. We did not detect a difference between interstitial M infiltration in IFN-R-deficient compared with IFN-R-intact mice (M = 65 ± 13/field in the IFN-R-deficient kidney vs 62 ± 19/field in the IFN-R-intact kidney). In addition, we established that only a few infiltrating M were proliferating (M = 1–3 PCNA⁺ M/field) compared with those in M growth factor-incited lesion (M = 50–100 PCNA⁺ M/field; Table III). The few interstitial cells expressing PCNA 1 wk after unilateral ureteral ligation was not dependent on IFN-R signaling (M = 2.0 ± 0.4 PCNA⁺ M/field in IFN-R-deficient mice vs 2.0 ± 0.6 PCNA⁺ M/field in IFN-R-intact mice; n = 6). Thus, we have established that M accumulation in the kidney following unilateral ureteral ligation is a result of M recruitment, not proliferation.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
M are abundant in the kidney undergoing autoimmune attack in MRL-Fas^lpr mice. IFN-, a pleiotropic cytokine abundant in MRL-Fas^lpr mice, is capable of either enhancing or limiting inflammation. We now report that in the absence of IFN- signaling in MRL-Fas^lpr mice, 1) CSF-1- or GM-CSF-incited autoimmune renal injury is amplified; 2) M accumulation is accelerated and is responsible for the increased kidney destruction; and 3) M proliferation increases, apoptosis decreases, and recruitment is not altered. We suggest that IFN- limits the expansion of M in response to CSF-1 or GM-CSF in the kidney of MRL-Fas^lpr by decreasing proliferation and increasing apoptosis.

We have previously established that gene transfer of CSF-1-TEC or GM-CSF-TEC into the kidney of MRL-Fas^lpr mice fosters the accumulation of M and autoreactive T cells and initiates autoimmune renal injury (19). We are confident that the biologic impact of CSF-1-TEC and GM-CSF-TEC is a result of local delivery of CSF-1 or GM-CSF and is not related to the retroviral vector. Each genetically modified TEC secreted the inserted gene product (CSF-1 and GM-CSF) and did not produce other cytokines implicated in renal injury (TNF-, IL-6, and IL-2; data not shown). In addition, infusion of lacZ-TEC (containing the retroviral vector but no inserted cytokine cDNA) did not induce interstitial nephritis. Furthermore, we used the same retroviral vector to genetically modify TEC to secrete IL-6, TNF-, and IL-2. We previously reported that infusion of these genetically modified TEC did not elicit kidney-infiltrating cells or injury (35). Thus, the biologic impact of CSF-1-TEC or GM-CSF-TEC is a result of CSF-1 or GM-CSF, respectively.

M are a prime cellular target for IFN-. IFN- activates M to produce proinflammatory molecules (36, 37). Thus, we might have reasoned that CSF-1- or GM-CSF-driven autoimmune renal injury would be prevented in the absence of IFN-. However, we determined that IFN- did not block CSF-1- or GM-CSF-incited interstitial nephritis. In contrast, CSF-1- and GM-CSF-incited renal injury is increased in the absence of IFN-R signaling. Thus, IFN- down-regulates the accumulation of CSF-1- or GM-CSF-stimulated M in the kidney of MRL-Fas^lpr mice. How can we explain these discrepancies? In vitro experiments indicate that IFN- down-regulates the expression of nuclear proto-oncogenes (38). Thus, it is possible that down-regulation of the proto-oncogene c-fms, which encodes CSF-1R, might cause a decrease in proliferation. However, IFN- alone does not down-modulate c-fms in normal mice BMM (39). Further studies will determine whether the IFN-R signaling and the c-fms pathway in M derived from MRL-Fas^lpr are interrelated.

Since we did not detect any difference in M proliferation of IFN-R-deficient or IFN-R-intact MRL-Fas^lpr mice in response to CSF-1 or GM-CSF in vitro, we established that the mutation in the IFN-R did not alter CSF-1 binding and function. On the other hand, IFN- inhibited the M proliferation induced by CSF-1 or GM-CSF in vitro. Thus, we propose that the enhanced accumulation of M in IFN-R-deficient MRL-Fas^lpr mice is due to increased M proliferation. In support of this concept, we detected more proliferating cells within the implant area in IFN-R-deficient compared with IFN-R-intact MRL-Fas^lpr mice.

An increase in M in the kidney could result from more rapid cell division and/or a decrease in apoptosis. Recent studies indicate that the sensitivity to activation-induced apoptosis in human M is down-regulated by CSF-1 and is easily up-regulated by IFN- (11). The detection of apoptotic cells in tissue sections, however, is difficult due to their rapid clearance (40). In the present report we identified 3% apoptotic cells in IFN-R-intact MRL-Fas^lpr mice, which was reduced to 1% in the IFN-R-deficient strain. However, even if as few as 1% of the cells are identified as apoptotic in tissue sections, the overall cellular loss due to apoptosis over a few days may be substantial (41). Thus, we suggest that IFN--primed apoptosis can contribute to the extent of M accumulation.

Another possible mechanism for the increase in M in IFN-R-deficient MRL-Fas^lpr kidney could be a decreased recruitment of M to the implant site. However, in an experimental hydronephrosis model, characterized by a florid M infiltration, signaling through the IFN-R did not influence the number of M. Since infiltrating M did not show enhanced cell proliferation, we conclude that the recruitment of M is not altered by IFN-.

Our previous studies determined that kidney-infiltrating DN T cells are self regulatory (6). These DN T cells secrete IFN-, which reduces T cell proliferation to stimulating TEC (6). Furthermore, IFN- limits alloimmune responses in murine cardiac and skin transplantation by down-regulating the proliferation of activated T cells.⁴⁵ However, in this report, signaling through the IFN-R did not reduce T cell proliferation. How can we explain this paradox? It is important to realize that in M growth factor-incited renal injury, M are recruited well in advance of DN T cells (14 days). Thus, in the absence of IFN-R signaling, the increase in M proliferation may restrict an accumulation of T cells. Additional studies, such as gene transfer of primarily T cell-stimulating factors in IFN-R-deficient and -intact MRL-Fas^lpr kidneys, are required to determine whether IFN- will limit T cell expansion in induced interstitial nephritis.

Finally, IFN- is essential for spontaneous autoimmune glomerulonephritis in MRL-Fas^lpr mice (42). In addition, we have established that IFN- is required for the expression of CSF-1 in the kidney and, hence, the initiation of autoimmune nephritis (manuscript in preparation). Based on the present study we would have anticipated an increase in M in the kidney during spontaneous renal disease in IFN-R-deficient MRL-Fas^lpr mice. On the contrary, we identified fewer M and reduced renal injury in the absence of signaling through the IFN-R (manuscript in preparation). To explain this apparent paradox, we suggest that IFN- inhibition of M proliferation is restricted to CSF-1 or GM-CSF stimulation. Unless the M receives a signal to turn on proliferation, the IFN-R cannot provide a signal to turn off proliferation. Since CSF-1 is absent in the kidney in the spontaneous autoimmune nephritis of IFN-R-deficient MRL-Fas^lpr mice, IFN- cannot limit M proliferation. In addition, IFN- has diametric actions on autoimmune tissue destruction depending on the time of administration during the pathogenesis of disease (43, 44). Clearly, the role of IFN- in autoimmune disease is complex. It will be important to rigorously establish the point in the pathogenesis and the amount of IFN- that provide protection or, alternatively, facilitate kidney disease.

Our findings identify a negative regulatory mechanism in which IFN- limits the expansion of M in autoimmune inflammation. We suggest that IFN- produced by kidney-infiltrating T cells is responsible for halting M proliferation. Thus, even though M enter the lesion in advance of T cells, T cells prevail, since M expansion is limited by IFN-. In conclusion, IFN- provides a negative regulatory pathway to restrict M expansion in M growth factor-elicited acute inflammatory/immune reactions. We suggest that reduction of M proliferation and enhancement of apoptosis by IFN- limit acute inflammation.

	Acknowledgments

 
We are grateful to Dr. Charles B. Carpenter for critically reviewing this manuscript.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant DK36491, the German Ernst Jung Foundation for Science and Research (to A.S.), and the Natural Sciences and Engineering Research Council of Canada (to K.M.).

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

³ Abbreviations used in this paper: M, macrophage; DN, double negative; IFN-R, interferon- receptor; TEC, tubular epithelial cells; GM-CSF, granulocyte-macrophage colony-stimulating factor; MoMuLV, Moloney murine leukemia virus; BMM, bone marrow macrophage; DAB, 3,3'-diaminobenzidine; PCNA, proliferating cell nuclear antigen; TUNEL, terminal deoxynucleotidyltransferase-mediated UTP end labeling.

Received for publication July 28, 1997. Accepted for publication December 19, 1997.

	References

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