IFN-γ Production by Intrinsic Renal Cells and Bone Marrow-Derived Cells Is Required for Full Expression of Crescentic Glomerulonephritis in Mice

Jennifer R. Timoshanko, Stephen R. Holdsworth, A. Richard Kitching and Peter G. Tipping
J Immunol April 15, 2002, 168 (8) 4135-4141; DOI: https://doi.org/10.4049/jimmunol.168.8.4135

Abstract

The contribution of IFN-γ from bone marrow (BM) and non-BM-derived cells to glomerular and cutaneous delayed-type hypersensitivity (DTH) was studied in mice. Chimeric IFN-γ mice (IFN-γ+/+ BM chimera), in which IFN-γ production was restricted to BM-derived cells, were created by transplanting normal C57BL/6 (wild-type (WT)) BM into irradiated IFN-γ-deficient mice. BM IFN-γ-deficient chimeric mice (IFN-γ−/− BM chimera) were created by transplanting WT mice with IFN-γ-deficient BM. WT and sham chimeric mice (WT mice transplanted with WT BM) developed crescentic glomerulonephritis (GN) with features of DTH (including glomerular T cell and macrophage infiltration) in response to an Ag planted in their glomeruli and skin DTH following subdermal Ag challenge. IFN-γ-deficient mice showed significant protection from crescentic GN and reduced cutaneous DTH. IFN-γ+/+ BM chimeric and IFN-γ−/− BM chimeric mice showed similar attenuation of crescentic GN as IFN-γ-deficient mice, whereas cutaneous DTH was reduced only in IFN-γ−/− BM chimeras. In crescentic GN, IFN-γ was expressed by tubular cells and occasional glomerular cells and was colocalized with infiltrating CD8+ T cells, but not with CD4+ T cells or macrophages. Renal MHC class II expression was reduced in IFN-γ+/+ BM chimeric mice and was more severely reduced in IFN-γ-deficient mice and IFN-γ−/− BM chimeric mice. These studies show that IFN-γ expression by both BM-derived cells and intrinsic renal cells is required for the development of crescentic GN, but IFN-γ production by resident cells is not essential for the development of cutaneous DTH.

Glomerular crescent formation is a feature of rapidly progressive human glomerulonephritis (GN)3 and indicates severe disease with a poor prognosis for renal function. Evidence from experimental models of GN and from human disease strongly suggests that crescentic GN results from a Th1-predominant, delayed-type hypersensitivity (DTH)-like response to nephritogenic Ags (1). Inhibition of Th1 cytokines (IL-12 and IFN-γ) or administration of Th2 cytokines (IL-4 and IL-10) attenuates crescentic anti-glomerular basement membrane (GBM)-induced GN in mice by attenuating the systemic Th1 immune responses to the nephritogenic Ag. The contribution of cytokines produced by intrinsic renal cells to the effector phase of immune renal injury is poorly defined. We have recently demonstrated that local IL-12 production by intrinsic renal cells as well as production by bone marrow (BM)-derived cells contribute to inflammatory renal injury in this model (2).

IFN-γ is a potent proinflammatory molecule that activates macrophages and up-regulates MHC class I and MHC class II (MHC II) expression. It augments Th1 responses by inducing IL-12R expression on T cells (3) and enhancing IL-12 production by macrophages (4). IFN-γ is produced predominately by CD8+ T cells, CD4+ Th1 cells, and NK cells (5), but a variety of other cells, including endothelial cells (6, 7, 8) and smooth muscle cells (9), have also been demonstrated to be capable of producing this cytokine. IFN-γ-deficient mice produced by targeted mutation of the IFN-γ gene show normal development of the immune system and are healthy in the absence of infection (10). However, they are highly susceptible to intracellular pathogens and show markedly reduced MHC II expression, NO and superoxide production by macrophages (10), and reduced Th1 Ab isotypes and cutaneous DTH following Ag challenge (11, 12, 13).

IFN-γ is expressed in glomeruli in human (14, 15) and experimental GN (16, 17). The functional contribution of IFN-γ to the development of glomerular injury has been demonstrated by attenuation of crescentic GN by in vivo Ab inhibition of IFN-γ (16) and by using IFN-γ-deficient mice (11). IFN-γ receptor-deficient mice also show attenuation of renal injury induced by anti-GBM Ab (17). An important mechanism for the protection afforded by blocking or the absence of IFN-γ is via attenuation of the systemic Th1-biased immune response to the nephritogenic Ag, manifest by changes in the Ab isotype profile and diminution of cutaneous DTH response (11). This effect was not observed in a noncrescentic model of anti-GBM GN associated with mild glomerular injury (18). In this model IFN-γ-deficient mice were more prone to injury (31%) than wild-type (WT) mice (7%), but IFN-γ deficiency did not alter the Th1 bias of the immune response to the nephritogenic Ag. In addition to its ability to direct the systemic Th subset toward Th1, IFN-γ has the potential to augment GN by local effects on intrinsic renal cells. IFN-γ stimulates MHC class I (19) and II (20), IL-1 (21), and monocyte chemoattractant protein-1 (22) expression by mesangial cells in vitro. It induces MHC II expression on tubular epithelial cells (23, 24, 25, 26, 27) and induces MHC class I and II and ICAM-1 on glomerular epithelial cells (28, 29).

Although IFN-γ is expressed in the human kidney, and experimental GN and intrinsic renal cells respond to IFN-γ in vitro, their capacity to produce IFN-γ has not been demonstrated. The demonstration of IFN-γ production by endothelial cells (6, 7, 8) and smooth muscle cells (9) that are from a similar lineage as some renal cells (renal endothelial cells and mesangial cells) suggests the potential for IFN-γ production by intrinsic renal cells during inflammation. The current study demonstrates this capacity and examines the contribution of IFN-γ from BM-derived and non-BM-derived cells to the development of DTH responses in the kidney and skin. It demonstrates a role for both sources of IFN-γ in the development of Th1-dependent glomerular DTH, but only for BM cell-derived IFN-γ in cutaneous DTH.

Materials and Methods

Mice

Breeding pairs of mice with a targeted disruption of the IFN-γ gene (10) were obtained from The Jackson Laboratory (Bar Harbor, ME). These mice have been back-crossed for nine generations onto a C57BL/6 background. Mice were housed and bred under specific pathogen-free conditions at Monash University (Clayton, Australia).

BM transplantation

Five- to 6-wk-old male homozygous IFN-γ-deficient (IFN-γ−/−) or C57BL/6 (WT) recipient mice received 1100-rad total body irradiation. BM cells were harvested aseptically from the femurs and tibiae of WT or IFN-γ−/− donor mice and depleted of RBCs. Recipient mice were injected i.v. with 5 × 106 cells within 6 h of irradiation. Mice were housed under specific pathogen-free conditions for 8 wk to allow BM reconstitution. Circulating leukocyte numbers and lymphocyte subsets were assessed by flow cytometry as previously described (2) and were normal 8 wk after BM transplantation. The splenic distribution of T cells and macrophages, assessed by immunohistology, also appeared normal at this time, as previously reported (2). The efficiency of BM replacement following transplantation was assessed using congenic CD45 mouse strains (Ly5.1 and Ly5.2). Eight weeks after irradiation and transplantation 92 ± 1% of circulating leukocytes, 98 ± 1% of BM leukocytes, and 90 ± 2% of splenic leukocytes expressed the phenotypic marker of the transplanted BM (n = 6), similar to previous reports (30, 31). Resident CD45-positive cells of either strain were rarely detectable by immunofluorescence in kidneys of normal mice or mice that had undergone BM transplantation before induction of GN.

Induction of crescentic GN

Crescentic anti-GBM was induced in male mice by i.v. injection of a total of 14.4 mg sheep anti-mouse GBM globulin (in 900 μl PBS) divided equally into two doses given 3 h apart. The development of crescentic GN and cutaneous DTH was assessed in WT and IFN-γ−/− mice and the three groups of irradiated BM transplanted chimeric mice. These included sham chimeras in which WT BM was transplanted into WT mice, IFN-γ+/+ BM chimeras in which WT BM was transplanted into IFN-γ-deficient mice, and IFN-γ−/− BM chimeras in which IFN-γ−/− BM was transplanted into WT mice. All mice were 13–14 wk of age at the time of administration of anti-GBM globulin. Renal injury and cutaneous DTH were assessed 21 days later.

Histological assessment of glomerular injury

Glomerular crescent formation.

Kidney tissue was fixed in Bouin’s fixative and embedded in paraffin, and 3-μm sections were stained with periodic acid-Schiff reagent. Glomeruli were considered to exhibit crescent formation when two or more layers of cells were observed in Bowman’s space. A minimum of 50 glomeruli were assessed to determine the crescent score for each animal.

Glomerular CD4+ cell, CD8+ cell, B cell, NK cell, and macrophage accumulation.

Kidney tissue and spleen were fixed in periodate/lysine/paraformaldehyde. Six-micrometer cryostat-cut sections were stained to demonstrate CD4+ cells, CD8+ cells, B cells, and macrophages with mAbs, GK1.5, 53-6.7, 145-2C11, and M/170, respectively, using a three-layer immunoperoxidase technique (32). NK cells were detected with a polyclonal anti-asialo GM Ab (Bio-Scientific, Gymea, Australia) followed by a three-layer immunoperoxidase technique. A minimum of 20 equatorially sectioned glomeruli were assessed per animal, and results were expressed as cells per glomerular cross-section (c/gcs).

Tubulointerstitial infiltration.

The number of interstitial cells in periodic acid-Schiff-stained renal tissue sections was counted using a 10-mm2 graticule fitted in the eyepiece of the microscope. Five randomly selected cortical areas, which excluded glomeruli, were counted for each animal. Each high-powered field represented an area of 1 mm2. Data are expressed as cells per square millimeter and are the mean ± SEM for four animals in each group.

Functional assessment of glomerular injury

Proteinuria.

Mice were housed individually in cages to collect urine before administration of anti-GBM globulin and over the final 24 h of the experiment. Urinary protein concentrations were determined by a modified Bradford method. Before induction of GN, all groups of mice had 24-h urinary protein excretion in the normal range (0.5–2 mg/24 h).

Serum creatinine.

Serum creatinine concentrations were measured by the alkaline picric acid method using an autoanalyzer (Roche Diagnostic Systems, Melbourne, Australia).

Demonstration of renal and splenic MHC II and IFN-γ expression by confocal microscopy

Protein G-purified rat anti-mouse MHC II mAb (clone Y3P, a gift from Prof. K. Shortman, Walter and Eliza Hall Institute, Melbourne, Australia) was conjugated with Alexa Fluor dye 488 (Molecular Probes, Eugene, OR) as previously described (2). Protein G-purified anti-mouse CD4 mAb (GK1.5) and rat anti-mouse macrophage Ab (M170) were conjugated to Alexa Fluor dye 594 (Molecular Probes). Allophycocyanin-conjugated rat anti-mouse CD8a (Ly-2) was obtained from BD PharMingen (San Diego, CA). FITC-conjugated rat anti-mouse IFN-γ (clone XMG1.2) was obtained from Caltag Laboratories (Burlingame, CA). Cryostat-cut, snap-frozen kidney tissue sections (6 μm) were blocked with 5% normal rat serum in 1% BSA/PBS and then incubated with both Abs at a final dilution of 1/50 for 60 min at room temperature. Confocal images were collected using a Bio-Rad confocal inverted Nikon microscope (Bio-Rad, Hercules, CA) equipped with an air-cooled 25-mW argon/krypton laser, as previously described (2).

Induction of cutaneous DTH to sheep globulin

Mice developing GN were challenged 24 h before the end of the experiment by intradermal injection of 500 μg sheep globulin in 50 μl PBS into one hind footpad. A similar dose of an irrelevant Ag (horse globulin) was injected into the other footpad as a control. Footpad swelling was quantified 24 h later using a micrometer (Mitutoyo, Kawasaki-shi, Japan). Ag-specific DTH was taken as the difference in skin swelling between sheep globulin- and horse globulin-injected footpads and expressed as change in footpad thickness (Δ millimeters).

Humoral immune responses to sheep globulin

Mouse anti-sheep globulin Ab titers were measured by ELISA on serum collected at the end of each experiment. Assays were performed using microtiter plates coated with sheep globulin at a concentration of 10 μg/ml, and bound mouse Ig was detected using HRP-conjugated sheep anti-mouse Ig (Amersham, Little Chalfont, U.K.) as the detecting Ab, as previously described (33).

Experimental design and statistical analysis

BM engraftment and lymphocyte subset reconstitution were assessed on two separate occasions in a total of 16 sham chimeric mice, 13 IFN-γ+/+ BM chimeric mice, and 5 IFN-γ−/− BM chimeric mice. Age-matched WT mice served as controls. The development of GN was studied in a total of 21 WT mice, 19 IFN-γ−/− mice, 17 sham chimeric mice, 13 IFN-γ+/+ BM chimeric mice, and 11 IFN-γ−/− BM chimeric mice. Results are expressed as the mean ± SEM. Statistical significance was determined by one-way ANOVA, followed by Newman-Keuls post-hoc test.

Results

Cresentic GN develops normally in sham chimeras but is attenuated in IFN-γ-deficient mice

WT mice developed proliferative GN with crescent formation (Fig. 1⇓). Sham chimeras developed disease of similar severity, indicating that BM transplantation per se did not affect the development of this disease. The incidences of crescentic glomeruli (WT, 22.3 ± 1.4%; sham chimeras, 23.5 ± 2.6%), glomerular infiltration of CD4+ cells (WT, 1.1 ± 0.1 c/gcs; sham chimeras, 1 ± 0.1 c/cgs), and macrophages (WT, 2.4 ± 0.1 c/gcs; sham chimeras, 2.3 ± 0.3 c/gcs; Fig. 2⇓) were equivalent in both groups. CD8+ cells were also observed in glomeruli (0.9 ± 0.1c/gcs) and in interstitial areas; however, NK cells (0.05 ± 0.01 c/gcs) and B cells (0.01 ± 0.01 c/gcs) were rarely observed. The interstitial cellular infiltrate (WT, 146 ± 6.2 cells/mm2; sham chimeras, 149 ± 7.5 cells/mm2), consisting mainly of macrophages with occasional CD4+ and CD8+ cells, was also unaffected by BM transplantation. NK cells and B cells were not observed in the interstitium. Similarly, there was no difference in functional renal injury between the two groups, indicated by proteinuria (WT, 9.6 ± 1.4 mg/24 h; sham chimeras, 7.8 ± 0.6 mg/24 h) and serum creatinine (WT, 23.9 ± 2.1 μmol/l; sham chimeras, 17.4 ± 1.5 μmol/l; Fig. 3⇓).

FIGURE 1.
FIGURE 1.

Histological appearances of GN 21 days after the administration of sheep anti-mouse GBM globulin. A, WT mice developed proliferative GN with glomerular crescents (∗). Proliferative changes and crescents were attenuated in IFN-γ-deficient mice (B) and chimeric mice with IFN-γ+/+ BM (C) and IFN-γ−/− BM (D) (periodic acid-Schiff-stained sections; original magnification, ×400).

FIGURE 2.
FIGURE 2.

The incidence of crescent formation (percentage of glomeruli affected), interstitial cell counts (cells per square millimeter), and glomerular infiltration of CD4+ cells and macrophages (cells per glomerular cross-section) in normal (WT), sham chimeric, IFN-γ-deficient (IFN-γ−/−), IFN-γ+/+ BM chimeric, and IFN-γ−/− BM chimeric mice developing crescentic GN. ★★, p < 0.001; ★, p < 0.01; •, p < 0.05 (compared with WT).

FIGURE 3.
FIGURE 3.

Proteinuria (milligrams per 24 h) and serum creatinine (micromoles per liter) in normal (WT), sham chimeric, IFN-γ-deficient (IFN-γ−/−), IFN-γ+/+ BM chimeric, and IFN-γ−/− BM chimeric mice developing crescentic GN. ★★, p < 0.001; ★, p < 0.01; •, p < 0.05 (compared with WT).

Significant protection against the development of crescentic GN was observed in IFN-γ-deficient mice. Glomerular crescent formation (7.4 ± 0.8% of glomeruli) was attenuated compared with that in WT mice (p < 0.001), as was glomerular infiltration of CD4+ cells (0.5 ± 0.1 c/gcs; p < 0.001), macrophages (0.8 ± 0.2 c/gcs; p < 0.001), and the interstitial cell infiltrate (103 ± 6 cells/mm2; p < 0.001; Fig. 2⇑). Glomerular infiltration of CD8+ cells (0.7 ± 0.3 c/gcs), NK cells (0.04 ± 0.01 c/gcs), and B cells (0.04 ± 0.02 c/gcs) was unaffected. A reduction in the histological severity of injury (Fig. 1⇑) was associated with significant protection of renal function, indicated by reduction in proteinuria (3.3 ± 0.7 mg/24 h; p < 0.01) and serum creatinine (14 ± 0.9 μmol/l; p < 0.01) in IFN-γ mice compared with WT mice (Fig. 3⇑).

IFN-γ production by intrinsic renal cells and BM-derived cells is required for full expression of crescentic GN

Compared with WT mice and sham chimeras, the development of crescentic GN was significantly attenuated in IFN-γ+/+ BM chimeric and IFN-γ−/− BM chimeric mice, indicating that IFN-γ from both intrinsic renal cells and BM-derived cells is required for full expression of immune renal injury in this model. The incidence of crescentic glomeruli was significantly reduced (IFN-γ+/+ BM chimeras, 10.8 ± 1.2% (p < 0.001); IFN-γ−/− BM chimeras, 12 ± 1% (p < 0.001)). Glomerular accumulation of CD4+ cells (IFN-γ+/+ BM chimeras, 0.6 ± 0.1 c/gcs (p < 0.001); IFN-γ−/− BM chimeras, 0.1 ± 0.03 c/gcs (p < 0.001)), CD8+ cells (IFN-γ+/+ BM chimeras, 0.4 ± 0.06 c/gcs (p < 0.05); IFN-γ−/− BM chimeras, 0.2 ± 0.03 c/gcs (p < 0.001)), and macrophages (IFN-γ+/+ BM chimeras, 1.1 ± 0.2 c/gcs (p < 0.01); IFN-γ−/− BM chimeras, 0.4 ± 0.1 (p < 0.001)) and the interstitial inflammatory infiltrate (IFN-γ+/+ BM chimeras, 118 ± 5 cells/mm2 (p < 0.01); IFN-γ−/− BM chimeras, 67 ± 4 cells/mm2 (p < 0.001)) were all significantly reduced (Fig. 2⇑). The sparse appearance of NK cells and B cells was unaffected. Renal function was preserved in chimeric mice with significantly less proteinuria (IFN-γ+/+ BM chimeras, 5.3 ± 0.7 mg/24 h (p < 0.01); IFN-γ−/− BM chimeras, 2.7 ± 0.4 mg/24 h (p < 0.01)) and lower serum creatinine levels (IFN-γ+/+ BM chimeras, 14.6 ± 1.5 μmol/l (p < 0.01); IFN-γ−/− BM chimeras, 15.7 ± 1.4 μmol/l (p < 0.05)) compared with WT mice with GN (Fig. 3⇑).

Splenic and renal expression of IFN-γ (Fig. 4⇓)

FIGURE 4.
FIGURE 4.

Confocal images demonstrating IFN-γ expression (green), CD4+ or CD8+ expression (both red), and colocalization of IFN-γ with CD4+ or CD8+ (yellow) in spleens and kidneys of mice with GN. IFN-γ expression was prominent in the spleens of WT mice, and coexpression with CD4+ cells was observed on many cells (A). In the kidneys of WT mice (B and C) IFN-γ expression was observed on glomerular cells, tubular cells, and interstitial cells. Coexpression of IFN-γ on CD4+T cells was not observed (B), but coexpression was detectable on CD8+ cells. In IFN-γ-deficient mice, CD4+ cells were present in the spleen (D) and in CD4+ cells (E) and CD8+ cells (F) in the kidney; however, there was no detectable staining in either tissue with the anti-IFN-γ mAb. In IFN-γ+/+ BM chimeric mice, IFN-γ colocalized on numerous cells in the spleen (G). In the kidney, CD4+ T cells were observed in glomeruli, but colocalization with IFN-γ was not detected (H). In this panel an IFN-γ-positive, BM-derived cell (probably a CD8+ cell) can been seen. However, CD8+ cells in the kidney expressed IFN-γ (I). In the IFN-γ−/− BM chimeric mice, CD4+ T cells in the spleen did not express IFN-γ (J). In the kidney, IFN-γ expression by tubular cells and occasional glomerular cells was detected, but no expression on CD4+ cells (K) or CD8+ cells (L) was observed. Confocal immunofluorescence under oil immersion; original magnification, ×200 (spleen) and ×400 (kidney). Scale bars in the lower right corner of J, K, and L are representative for all figures in that column.

IFN-γ (Fig. 4⇑, green) was strongly expressed in the spleen and kidneys of WT mice with GN. In the spleen, coexpression (Fig. 4⇑A, yellow) of IFN-γ with CD4+ cells was observed on many cells, although some CD4+ cells (Fig. 4⇑A, red) did not express IFN-γ. In the kidney (Fig. 4⇑B), IFN-γ expression was observed on glomerular cells, tubular cells, and interstitial cells. CD4+ cells were prominent in glomeruli, but coexpression of IFN-γ with CD4+ was not observed. However, IFN-γ expression was colocalized (Fig. 4⇑C, yellow) on CD8+ cells in interstitial areas of WT mice. In IFN-γ-deficient mice CD4+ cells were observed in the spleen (Fig. 4⇑D), but no staining for IFN-γ was observed, confirming the specificity of the anti-IFN-γ Ab. In the kidneys of IFN-γ-deficient mice, CD4+ cells were present in glomeruli (Fig. 4⇑E), and CD8+ cells were present in interstitial areas (Fig. 4⇑F), but IFN-γ was absent. In IFN-γ+/+ BM chimeric mice, IFN-γ colocalized with numerous CD4+ cells in the spleen (Fig. 4⇑G). In the kidney (Fig. 4⇑H), CD4+ cells were observed in glomeruli, and occasional IFN-γ-positive cells (Fig. 4⇑H, green, possibly CD8+ cells) were observed in glomeruli, but no colocalization of IFN-γ with CD4+ was observed. IFN-γ colocalized with CD8+ cells in the interstitial areas; however, IFN-γ expression was not observed on any other cells within the kidney (Fig. 4⇑I). In the spleen of IFN-γ−/− BM chimeras, no CD4+ cells expressed IFN-γ, although some IFN-γ expression on non-BM cells (similar to WT mice) was observed (Fig. 4⇑J). In the kidney (Fig. 4⇑, K and L), IFN-γ expression by tubular cells and occasional glomerular cells was detected, but expression on CD4+ cells (Fig. 4⇑K) or CD8+ cells (Fig. 4⇑L) was not observed. Colocalization of IFN-γ on macrophages in the kidney was not detectable in any of the groups of mice (data not shown).

Renal expression of MHC II in GN

MHC II expression (Fig. 5⇓A, green) was prominent in interstitial areas and tubules, and to a lesser extent in glomeruli in WT mice. Occasional coexpression (Fig. 5⇓A, yellow) with macrophages was observed. In IFN-γ-deficient mice (Fig. 5⇓B) and in IFN-γ−/− BM chimeric mice (Fig. 5⇓D), MHC II was very sparsely expressed in kidneys, and no colocalization with macrophages (Fig. 5⇓, B and D, red) was observed. In IFN-γ+/+ BM chimeric mice (Fig. 5⇓C), renal MHC II expression was markedly reduced compared with that in WT mice, but expression was observed on some renal tubular cells and interstitial cells. Colocalization on macrophages was not apparent.

FIGURE 5.
FIGURE 5.

Confocal images demonstrating MHC II expression in the kidney. MHC II expression (green) was prominent in interstitial areas and tubules in WT mice with GN (A). Occasional coexpression (yellow) was observed with macrophages (red). In IFN-γ-deficient mice (B) and in IFN-γ−/− BM chimeric mice (D) MHC II was sparsely expressed in kidneys, with no expression observed on macrophages. In IFN-γ+/+ BM chimeric mice (C) MHC II expression was reduced compared with that in WT mice but was sparsely expressed by some tubular and interstitial cells. No expression on macrophages was observed. Confocal immunofluorescence, under oil immersion; original magnification, ×400.

Cutaneous DTH and serum Ab titers to sheep globulin (Fig. 6⇓)

FIGURE 6.
FIGURE 6.

Serum titers of mouse anti-sheep globulin in WT (□), IFN-γ-deficient (○), IFN-γ+/+ BM chimeric (▪), and IFN-γ−/−BM chimeric (•) mice developing crescentic GN. WT, sham, and IFN-γ chimeric mice developed DTH following cutaneous challenge with sheep globulin. DTH was significantly reduced in IFN-γ-deficient and IFN-γ−/− BM chimeric mice (★★, p < 0.001 compared with WT mice).

WT and sham chimeric mice developed DTH following cutaneous challenge with sheep globulin. Cutaneous DTH was unaffected in IFN-γ+/+ BM chimeric mice but was markedly reduced in IFN-γ−/− BM chimeric mice and IFN-γ-deficient mice (Fig. 6⇑A). Serum mouse anti-sheep globulin Ab titers were equivalent in all groups and were unaffected by complete or partial IFN-γ deficiency (Fig. 6⇑B).

Discussion

The contribution of IFN-γ production by BM-derived cells and resident (non-BM-derived) cells to the development of DTH in kidney and skin was studied by creating chimeric mice with the capacity for IFN-γ production restricted to one of these compartments. The technique of irradiation and BM transplantation has previously been successfully used to create mice with genotypically and functionally distinct BM derived cells (2, 34, 35, 36). In the current study IFN-γ+/+ BM chimeric mice were created by transplanting WT BM into IFN-γ-deficient mice. After 8 wk, these mice had normal circulating lymphocyte subsets and baseline proteinuria, and their BM cells were 98% of the transplanted phenotype. Splenic distribution of lymphocytes and macrophages was normal, with numerous BM-derived cells expressing IFN-γ, confirming successful engraftment. In IFN-γ−/− BM chimeras, created by transplanting WT mice with IFN-γ-deficient BM, there was no detectable coexpression of IFN-γ with CD4+ T cells in their spleens, indicating depletion of the recipient’s splenic lymphocytes. Flow cytometric analysis of CD45-positive spleen cells in transplanted congenic strains confirmed that 90% of splenic leukocytes expressed the phenotype of the transplanted BM. Studies in rats show that irradiation totally depletes the small numbers of resident renal BM-derived MHC II (Ia)-positive cells in 3 days (37), suggesting that this small resident BM-derived population has the same radiosensitivity as splenic leukocytes.

Previous studies have demonstrated that IFN-γ is required for the development of crescentic GN and cutaneous DTH (11, 13), both of which are manifestations of Th1-dependent cell-mediated injury. Studies in CD4+- and CD8+-deficient mice show a key role for CD4+ cells in the development of crescentic GN but demonstrate that injury is independent of CD8+ cells (38). The results of the current study demonstrate that while BM cell-derived IFN-γ alone is sufficient for cutaneous DTH, contributions from both BM and intrinsic renal cells are required for full expression of crescentic GN. Crescent formation, glomerular recruitment of macrophages and CD4+ T cells, and indices of functional renal injury (proteinuria and serum creatinine) were significantly reduced in the absence of IFN-γ production by either BM cells or intrinsic renal cells. The development of glomerular and cutaneous DTH was unaffected by BM transplantation per se, as WT mice transplanted with WT BM (sham chimeras) developed crescentic GN and skin swelling following Ag challenge equivalent to those in nontransplanted WT mice.

Previous studies have demonstrated that IL-12 production by intrinsic renal cells contributes to the development of crescentic GN (2). The current study provides further evidence of the contribution Th1 cytokines from intrinsic renal cells to this DTH-like renal inflammation. Confocal microscopy demonstrated IFN-γ expression on tubular cells, interstitial cells, and intrinsic glomerular cells. However, IFN-γ production by CD4+ cells and macrophages in the kidney could not be demonstrated despite prominent IFN-γ expression by CD4+ cells in the spleen. CD8+ cells were the only BM-derived cells that could be demonstrated to express IFN-γ at the site of the effector immune/inflammatory response in the kidney. This suggests that IFN-γ from BM-derived cells in secondary lymphoid tissue is important for development of the Th1 response to the nephritogenic Ag, but that intrinsic renal cells are an important source of IFN-γ for the effector response in the kidney. In contrast, cutaneous DTH is dependent on BM cell-derived IFN-γ and is unaffected by the absence of IFN-γ from resident cells. These observations indicate a proinflammatory role for renal parenchymal cells in the development of renal DTH that does not appear to be shared by keratinocytes in cutaneous DTH.

In vitro, IFN-γ has been shown to up-regulate MHC II expression on mesangial (24, 39, 40), glomerular epithelial (27, 28), renal proximal tubular (24), and endothelial cells (41). Up-regulation of MHC II allows murine mesangial cells to stimulate the proliferation of T cells (42) and to present Ag to T cells (43) in vitro. Transformed renal tubular epithelial cells can present Ag to MHC II in vitro (24). In the current in vivo experiment MHC II was prominently expressed on tubular cells and to a lesser extent on intraglomerular macrophages in WT mice with crescentic GN. The expression on other cells within glomeruli was rarely observed, suggesting that, despite the capacity of mesangial cells to express MHC II in vitro, they do not necessarily do so in response to inflammatory stimuli in vivo. These observations in crescentic GN suggest that proximal tubular cells are more likely than mesangial cells to be involved in the recognition of Ags in the kidney via MHC II-dependent mechanisms.

In vivo renal expression of MHC II was markedly reduced (although not entirely absent) in IFN-γ-deficient mice with GN, indicating an important, but not exclusive, role for IFN-γ in MHC II induction. Residual MHC II expression appeared to be mainly on interstitial cells and periglomerular cells. Similar findings were reported in MRL/lpr mice, in which IFN-γ deficiency was associated with a dramatic reduction of MHC II expression by tubular cells but some persistent expression in periglomerular and interstitial cells (44). In IFN-γ−/− BM chimeric mice, renal MHC II expression was reduced to a similar extent as in mice that were totally deficient in IFN-γ, suggesting that leukocyte-derived IFN-γ plays a pivotal role in up-regulating renal MHC II. Chimeric mice with absent intrinsic renal cell IFN-γ production also showed substantially reduced MHC II expression compared with WT mice with GN, indicating that intrinsic renal cell-derived IFN-γ is also required for full expression of MHC II in this disease. The induction of tubular cell MHC II in the presence of intrinsic renal cell-derived IFN-γ may indicate an autocrine role for proximal tubular cell-derived IFN-γ. These studies demonstrate that inflammatory cell and intrinsic renal cell IFN-γ together play a major role in the induction of renal MHC II.

The numbers of infiltrating CD4+ T cells and macrophages were reduced to a greater extent in the IFN-γ−/− BM chimeras than in IFN-γ+/+ BM chimeric mice, suggesting that systemic or BM cell-derived IFN-γ plays a dominant role in leukocyte recruitment. IFN-γ up-regulates the production of macrophage inflammatory protein-1α, monocyte chemoattractant protein-1, CSF-1, and RANTES by mesangial cells (45). In MRL/Faslpr mice IFN-γ receptor signaling is required for the induction of renal CSF-1 and TNF-α. The observation that CSF-1- and TNF-α-inducing bioactivity could not be detected in the sera of these mice suggested that the IFN-γ responsible for these effects was produced locally within the kidney in this immune complex-induced model of renal inflammation (46). In the current studies of a planted Ag-induced, DTH-like model of glomerular inflammation, it would appear that IFN-γ from BM-derived cells plays a predominant role in glomerular and interstitial leukocyte recruitment. This may be attributed to its important role in driving Th1 cell development in secondary lymphoid organs.

In conclusion, these studies show that intrinsic renal cells, in particular tubular cells, produce IFN-γ in murine crescentic GN. IFN-γ from both leukocytes and intrinsic renal cells contributes to glomerular DTH associated with crescentic GN. IFN-γ from BM-derived cells plays a pivotal role in directing the initial Th1-biased immune response to the nephritogenic Ag, whereas IFN-γ from resident cells plays an important role in the effector immune/inflammatory response in the kidney. Both leukocytes and renal resident cells are important sources of IFN-γ, which directs the immune response and mediates renal injury in crescentic DTH.

Acknowledgments

We thank J. Sharkey, A. Wright, D. Calleci, and T. Niklou for their technical assistance, and Prof. K. Shortman for the anti-MHC II mAb.

Footnotes

  • ↵1 This work was supported by the National Health and Medical Research Council of Australia and the Australian Kidney Foundation.

  • ↵2 Address correspondence and reprint requests to Dr. Peter G. Tipping, Monash University, Department of Medicine, Monash Medical Center, 246 Clayton Road, Clayton, 3168 Victoria, Australia. E-mail address: peter.tipping{at}med.monash.edu.au

  • ↵3 Abbreviations used in this paper: GN, glomerulonephritis; BM, bone marrow; c/gcs, cells per glomerular cross-section; DTH, delayed-type hypersensitivity; GBM, glomerular basement membrane; MHC II, MHC class II; WT, wild type.

  • Received June 21, 2001.
  • Accepted February 20, 2002.

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