Basic Immunology and Pathology

Inducible Co-Stimulator Null MRL-Faslpr Mice: Uncoupling of Autoantibodies and T Cell Responses in Lupus

Geraldine C. Zeller, Junichi Hirahashi, Andreas Schwarting, Arlene H. Sharpe and Vicki R. Kelley
JASN January 2006, 17 (1) 122-130; DOI: https://doi.org/10.1681/ASN.2005080802

Abstract

MRL/MpJ-Tnfrsf6lpr (MRL-Faslpr) mice develop a spontaneous T cell–dependent autoimmune disease that shares features with human lupus, including fatal nephritis, systemic pathology, and autoantibodies (autoAb). The inducible co-stimulator (ICOS) is upregulated on activated T cells and modulates T cell–mediated responses. To investigate whether ICOS has an essential role in regulating autoimmune lupus nephritis and the systemic illness in MRL-Faslpr mice, ICOS null (−/−) MRL Faslpr and ICOS intact (+/+) MRL-Faslpr strains (wild-type [WT]) were generated and compared. It was determined that in ICOS−/− MRL-Faslpr as compared with the WT strain, (1) there is a significant reduction in circulating IgG and double-stranded DNA autoantibody isotype titers, and (2) there is an amplification of the frequency of intrarenal T cells generating IFN-γ and TNF-α in ICOS−/− versus WT mice. Of note, eliminating ICOS in the MRL-Faslpr strain does not alter renal pathology or function. Despite the reduction in circulating IgG and autoantibody isotypes (G1, G2a, and G2b), the amount of these IgG isotypes depositing in kidneys is similar. Furthermore, the systemic illness (skin, salivary and lacrimal glands, lungs, lymphadenopathy, and splenomegaly) is equivalent in ICOS−/− MRL-Faslpr and WT mice. These findings highlight the danger of relying on individual parameters, such as quantitative serum Ig levels and T cell functions, as prognostic indicators of lupus.

Animal models of human illness provide tools to dissect pathogenesis at a molecular level. The MRL-Faslpr model is notably valuable because autoimmune disease in this strain is spontaneous, predictable, and steadily progressive and shares features with human lupus (1–3). The hallmarks of disease in this strain consist of a profound lymphoproliferation; a massive increase in immunoglobulins (Ig), including a multitude of autoantibodies (autoAb); and rapid, fatal autoimmune kidney destruction (50% mortality, 6 mo of age) (4). As in human lupus, multiple tissues, kidney, salivary and lacrimal glands, skin, and lungs are infiltrated by leukocytes (1,5). Kidney damage in the MRL-Faslpr mice is complex and dependent on T cells. Several T cell populations promote injury in this strain, including CD4, CD8, and the unique double-negative T cells expressing B cell determinants (DN) (6). These autoreactive T cell populations that promote lupus nephritis are highly regulated (7). Thus, identifying the signals that control autoreactive T cell activation and clonal expansion is central to understanding mechanisms that are responsible for lupus nephritis.

T cells require at least two distinct signals for full activation (8,9). The first signal is provided by the engagement of the T cell receptor (TCR) with MHC plus peptide complex on antigen-presenting cells (APC), and the second “co-stimulatory” signal is collectively provided by engagement of one or more T cell co-stimulatory receptors with their specific ligands on APC (10,11). Signaling through the TCR alone, without a co-stimulatory signal, can lead to prolonged T cell unresponsiveness, i.e., anergy (9). The co-stimulatory signal provided by interaction of CD28 on T cells with either B7–1 or B7–2 on APC is well characterized (12,13). The CD28/CTLA4–B7–1/B7–2 T cell co-stimulatory pathway is a complex pathway that positively and negatively regulates T cell activation (14). Blocking this pathway is beneficial in mouse lupus: (1) B7–1/B-2 null MRL-Faslpr mice are protected from lupus nephritis (15), (2) CD28 null MRL-Faslpr mice have diminished nephritis as compared with WT strains (16), and (3) provision of CTLA4Ig (a fusion protein that binds to B7 and blocks this pathway) in the MRL-Faslpr and NZB × NZW F1 hybrid female strains reduces kidney disease (17,18). Thus, the B7–1/B7–2–CD28 pathway governs the tempo of renal disease in strains with lupus.

The CD28 receptor family of molecules is expanding. ICOS is a recently identified CD28 homolog that is upregulated on activated T cells and resting memory T cells (19–21). The ligand for ICOS, ICOS-L (also named B7h, LICOS, B7RP-1, B7H-2, and GL-50) does not bind to CD28 or CTLA4 (22–24). Similarly, B7–1 and B7–2 are not ligands for ICOS. ICOS-L is more broadly expressed than B7–1 and B7–2. ICOS-L is expressed on professional APC (including dendritic and B cells) and parenchymal cells such as endothelial and epithelial cells, most notably, renal tubular epithelial cells (TEC) (25–28).

The consequences of ICOS–ICOS-L signaling in autoimmune diseases are complex. ICOS imparts signals that can either promote or inhibit T cell activation. For example, blocking ICOS during the initiation phase of induced experimental autoimmune encephalomyelitis exacerbates disease, whereas blocking this pathway during the effector phase prevents experimental autoimmune encephalomyelitis (29). Thus, it is possible that the impact of ICOS blockade may vary depending on the stages of autoimmune disease.

ICOS-L expression displayed on parenchymal cells may regulate T cells within tissues such as the kidney. For example, human endothelial cells that express ICOS-L and class II MHC stimulate resting memory CD4 T cells in the presence of superantigen to generate Th1 and Th2 cytokines (30). Conversely, (1) blocking ICOS and ICOS-L in renal TEC (expressing Ia+) co-cultured with antigen-specific T cell clones (Th1 and Th2) pulsed with antigen increases IFN-γ and IL-4 in these T cell clones (31), and (2) blocking ICOS and ICOS-L enhances IL-2 production of T cell hybridomas after antigen presentation via TEC (28). However, it remains unclear whether the ICOS pathway in the kidney inhibits or enhances immune responses in vivo, because activation in T cell clones may be regulated differently than in freshly isolated T cells, and in vitro conditions do not entirely recapitulate in vivo immune responses.

Lupus nephritis is dependent on T cell and Ab mechanisms. Because genetic deletion of ICOS results in defective T cell–dependent B cell responses, germinal center formation, and Ig isotype class switching (32), elimination of ICOS may reduce immune complex–mediated lupus nephritis. Taken together, it is plausible that eliminating ICOS may inhibit or, contrastingly, exacerbate lupus nephritis.

In this study, we tested the hypothesis that the ICOS–ICOS-L pathway regulates lupus nephritis and the systemic illness in the MRL-Faslpr mouse. To test this hypothesis, we constructed and compared lupus nephritis and the systemic illness in the ICOS−/− MRL-Faslpr and WT strains. It is interesting that genetic deletion of ICOS did not alter the expression of autoimmune disease (nephritis, systemic inflammation, and lymphoproliferative disorder) in the MRL-Faslpr strain, despite having a profound impact on Ab production and T cell functions.

Materials and Methods

Mice

MRL/MpJ-++ (MRL++), MRL/MpJ-Faslpr/Faslpr, (MRL-Faslpr), C57BL/6J, and C3H/Fej were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed and bred in our pathogen-free animal facility. ICOS null mice were generated as described previously (32). The use of mice in our study was reviewed and approved by the Standing Committee on Animals in the Harvard Medical School in adherence to the National Institutes of Health Guide for the care and use of laboratory animals.

Generating ICOS Null MRL-Faslpr Mice

We constructed the ICOS null MRL-Faslpr strain (ICOS−/− MRL-Faslpr) using a backcross-intercross scheme. MRL-Faslpr mice were mated with ICOS−/− mice to yield heterozygous F1 offspring. We intercrossed F1 mice and screened the progeny by PCR amplification of tail genomic DNA for the Faslpr mutation and ICOS, using specific primers. After five generations of backcross matings (N5), we analyzed the ICOS−/− MRL-Faslpr with littermate ICOS+/+ MRL-Faslpr mice (referred to as WT mice). We compared these strains at the N5 generation, because we established previously that there are sufficient MRL-Faslpr background genes to result in phenotypic changes that are characteristic of MRL-Faslpr mice (33). Identification of the disrupted and intact ICOS in experimental mice was determined by PCR using tail genomic DNA. The DNA was assessed by PCR using oligonucleotide primers that recognized the normal ICOS gene sense (5′-GGT TTT GTT TGC TTG GCT ATA-3′) and antisense (5′-GGT CTT GGT GAG TTC GCA GAG-3′) and Neo gene sense (5′-ATT GAA CAA GAT GGA TTG CAC-3′) and antisense (5′-TCT TCG TCC AGA TCA TCC T-3′). Gel analysis of the PCR products identified the ICOS at 298 bp and Neo gene fragments at 479 bp. Screening for the expression of Faslpr was preformed as described previously (34). Because the tempo of disease is modestly more severe (several weeks) in MRL-Faslpr female than male mice, we analyzed similar numbers of female and male mice.

Urinary Protein and Blood Urea Nitrogen

Urine proteins were assessed semiquantitatively by dipstick analysis as described previously (35) and blood urea nitrogen (BUN) levels were measured using a colorimetric analysis kit (Infinity; Thermo Electron, Melbourne, Australia).

Histopathology

Renal.

We fixed the kidneys in 10% formalin and prepared and stained sections with hematoxylin-eosin and periodic acid-Schiff reagent. We evaluated renal pathology on a scale of 0 to 3 using coded slides as described previously (35). We scored glomerular pathology in 20 glomerular cross-sections/kidney, tubular pathology in 200 randomly selected renal cortical tubules/kidney (×400), and perivascular cell accumulation in 10 random inter- and intralobular arteries/kidney (×400) by counting the number of cell layers surrounding the vessel.

Other Tissues.

We fixed, prepared, and analyzed the lungs and salivary and lacrimal glands as described previously (35).

Gross Pathology: Lymphadenopathy, Skin Lesions, and Splenomegaly

We evaluated lymphadenopathy and skin lesions monthly beginning at 2 mo of age and spleen enlargement at the time of killing or death as detailed in previous reports (36).

Immunohistochemistry

We stained cryostat-cut kidney sections for the presence of macrophages with anti-CD68 Ab (Serotec, Oxford, UK) and for T cells with anti-CD4, anti-CD8, and anti-B220 rat anti-mouse mAb (PharMingen, San Diego, CA) according to a previously described immunoperoxidase method (37). The immunostaining was analyzed by counting for the presence of CD68-, CD4-, CD8-, and B220-positive cells in 10 randomly selected high-power fields. Of note, we have determined that the B220-positive cells in the kidney are the unique DN T cells and are not B cells (38).

IgG Isotypes and C3 Deposits within Renal Glomeruli

Kidney cryostat sections (4 μm) were stained with FITC-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR); FITC-conjugated goat IgG fraction of mouse C3 (Cappel Laboratory, Malvern, PA); and FITC-conjugated rat anti-mouse IgG1, IgG2a, IgG2b, and IgG3 (PharMingen) for 30 min at 37°C. The amount of IgG and C3 lodged within glomeruli was titrated by diluting (1:1000, 1:5000, and 1:10000) the FITC-conjugated IgG. The FITC-conjugated IgG isotypes were titrated (two-fold from 1:50 through 1:1600). Fluorescence intensity within the peripheral glomerular capillary loops and within the mesangium was scored on a scale of 0 to 3 (0, none; 1, weak; 2, moderate; 3, strong). At least 10 randomly selected glomeruli/section were analyzed.

Serum Ig Profiles

We analyzed circulating polyclonal total IgG and IgM and IgG1, IgG2a, IgG2b, and IgG3 isotypes by ELISA. Plates were coated overnight at 4°C with goat anti-mouse Ig capture Ab (Southern Biotechnology Associates, Birmingham, AL) in PBS. The wells were blocked for 2 h with 3% BSA/PBS. We added Ig standards to the plate, using a series of three-fold dilutions, and assessed serum samples using serial dilutions starting at 1:100. Standards and serum samples were incubated overnight at 4°C, and bound Ig was detected with goat anti-mouse detection Ab conjugated with horseradish peroxidase (Southern Biotechnology Associates) and detected with TMB peroxidase substrate (Kirkegaard Perry Laboratories, Gaithersburg, MD). The absorbance was measured at 450 nm.

We analyzed the anti-dsDNA autoAb isotypes (total IgG, IgG1, IgG2a, IgG2b, IgG3, and IgM) in the sera of ICOS−/− and ICOS+/+ MRL-Faslpr mice at five dilutions (1:50, 1:150, 1:450, 1:1350, 1:4050, and 1:12.150) as previously reported (39).

Flow Cytometry

Measuring the intracellular cytokine production of leukocytes has been described previously (40). Briefly, we gently dissociated kidneys into single-cell suspensions and lysed the red blood cells. To detect ICOS and ICOS-L on cells in the kidney, we stained the cell suspensions with anti-ICOS PE Ab (0.5 μg/ml; eBioscience, San Diego, CA) and anti–ICOS-L PE Ab (0.5 μg/ml; eBioscience), respectively. To detect intracellular cytokines, we cultured cells for an additional 4 h in the presence of PMA (10 ng/ml; Sigma-Aldrich, St. Louis, MO) and ionomycin (500 ng/ml; Sigma-Aldrich). Monensin (3 μM; Sigma-Aldrich) was added during the last 3 h. We then stained for cell surface markers, including anti-CD4, anti-CD8, anti-B220 Ab-FITC, and anti–TCR-β chain Ab-APC (eBioscience). We fixed and permeabilized these cells and stained them with PE-labeled anti–IFN-γ or anti–TNF-α Ab (eBioscience) and fixed them with 1% paraformaldehyde. We analyzed 10,000 cells by flow cytometry.

Statistical Analyses

The data represent the mean ± SEM and were performed by GraphPad Prism Version 3.0 for Macintosh (GraphPad, San Diego, CA). The data shown in Figures 1 and 5 were evaluated using the one way ANOVA (Fisher), and for all other figures, we used the Mann-Whitney U Test. Values of P ≤ 0.05 were considered significant unless otherwise stated.

Figure 1.
Figure 1.

Inducible co-stimulator (ICOS) and ICOS ligand (ICOS-L) expression during lupus nephritis in MRL-Faslpr mice. (a and c) ICOS is upregulated in the kidney cortex of MRL-Faslpr mice between 2 and 5 mo of age as compared with age-matched C3H/Fej and C57BL/6J mice: (a) Real-time PCR; (c) flow cytometry (% of total cells in kidney). *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.001). (b and d) ICOS-L is constitutively expressed in MRL-Faslpr mice and is not substantially upregulated in the MRL-Faslpr strain with advancing renal injury, as compared with MRL-Faslpr, C3H/Fej, and C57BL/6J mice with normal kidneys: (b) Real-time PCR; (d) flow cytometry.

Results

ICOS Is Upregulated and ICOS-L Is Constitutively Expressed during Lupus Nephritis in MRL-Faslpr Mice

To determine whether ICOS expression in kidney cortex is upregulated in MRL-Faslpr mice with lupus nephritis, we compared ICOS expression in normal kidneys, from MRL-Faslpr and C3H/Fej, C57BL/6J mice at 2 mo of age, and ICOS expression in nephritic kidneys that were taken from MRL-Faslpr at 5 to 6 mo of age (Figure 1). We detected an increase in ICOS expression at the transcriptional (real-time PCR; Figure 1a) and protein levels (percentage of total renal cells, flow cytometry; Figure 1c) in MRL-Faslpr mice with nephritis as compared with MRL-Faslpr mice or other strains with normal kidneys.

We determined that ICOS-L transcripts (Figure 1b) and protein (Figure 1d) are constitutively expressed in the renal cortex of 2-mo-old MRL-Faslpr and C57BL/6J mice that have normal kidneys. However, expression is not significantly increased in the MRL-Faslpr mice at 5 to 6 mo of age. Thus, intrarenal ICOS-L does not vary with advancing lupus nephritis.

Intrarenal Macrophages and T Cells Are not Diminished in ICOS−/− MRL-Faslpr Kidneys

We previously determined that macrophages and T cells accumulate in the MRL-Faslpr kidney with nephritis (35,38). We found that the accumulation of macrophages (CD68) and T cells (CD4, CD8, and DN) in the glomerular and interstitial regions are similar in the ICOS−/− MRL-Faslpr as compared with the ICOS+/+ MRL-Faslpr mice at 6 mo of age (Figure 2). Therefore, genetic disruption of ICOS in MRL-Faslpr mice does not prevent the infiltration of multiple T cell populations and macrophages within the renal glomeruli and interstitium.

Figure 2.
Figure 2.

The number of macrophages and T cell populations in the renal glomeruli and interstitium are equivalent in ICOS−/− MRL-Faslpr and the wild-type (WT) strain. Macrophages (CD68) and CD4, CD8, and double-negative T cells expressing B cell determinants (DN) infiltrate the glomerular and interstitial area in ICOS−/− MRL-Faslpr and WT kidneys similarly (a and b). Macrophages and T cell populations (arrows) in ICOS−/− MRL-Faslpr and WT kidneys are similar at 6 mo of age. Magnification, ×200.

Frequency of Intrarenal Th1 and Other T Cell Subsets that Express IFN-γ and TNF-α Is Enhanced in ICOS−/− MRL-Faslpr Mice

To determine whether ICOS regulates T cells within the kidney, we compared the amounts of Th1 cytokines, IFN-γ, and TNF-α expressed by T cell populations (CD4) and other T cell subsets (CD8 and DN) that were freshly isolated from ICOS−/− MRL-Faslpr and WT kidneys. We found that the frequency of intrarenal CD4 T cells that express IFN-γ and TNF-α in ICOS−/− MRL-Faslpr mice was significantly higher than in the WT strain (Figure 3, 1). Similarly, the frequency of CD8 T cells and DN (B220+ TCR+) cells that generate IFN-γ increased significantly as compared with the WT (Figure 3, 2 and 3). Although the frequency of TNF-α in CD8 T cells seemed to be greater than the WT, it was NS (P = 0.19; Figure 3, 2). The frequency of B220+TCR+ cells that expressed TNF-α in ICOS−/− MRL-Faslpr and WT remained similar (P = 0.77; Figure 3, 3). By comparison, the frequency of T cells (CD4, CD8, and B220+TCR+) that generated IFN-γ and TNF-α in normal kidneys (C57BL/6J) was almost negligible (≤0.14% of total kidney cells, n = 4/group). Thus, eliminating ICOS in MRL-Faslpr mice increases the frequency of intrarenal T cell populations that produce IFN-γ and TNF-α.

Figure 3.
Figure 3.

There is an increased frequency of intrarenal Th1 cells in ICOS−/− MRL-Faslpr mice as compared with the WT strain (6 mo of age). The frequency of intrarenal CD4 cells that generate IFN-γ and TNF-α is increased as compared with the WT strain (1; *P ≤ 0.05). Similarly, the frequency of CD8 and B220 T cells that generate IFN-γ is enhanced in ICOS−/− MRL-Faslpr as compared with WT mice (2, 3; *P ≤ 0.05). While the frequency of CD8 that generate TNF-α tends to be higher, it is NS (2; P = 0.19). Of note, because the frequency of intrarenal T cell populations that generate IFN-γ and TNF-α was similar in age-matched ICOS+/+ MRL-Faslpr (N5) and MRL-Faslpr mice (n = 3 and n = 5, respectively), we combined the data.

The expression of Th2 cytokines such as IL-4 and IL-10 are increased in T cells after ICOS co-stimulation (41). Therefore, we compared the frequency of CD4, CD8, and B220+TCR+ cells that generate IL-4 and IL-10 in the kidney and spleen of ICOS−/− MRL-Faslpr and WT mice (n = 3/group). We were unable to detect T cell subsets that expressed IL-4 or IL-10 in the kidney of either strain; thus, renal disease in the MRL-Faslpr strain is Th1 and not Th2 driven. However, we determined that the frequency of IL-4–and IL-10–producing CD4, CD8, and B220+TCR+ cells in the spleen was similar in the ICOS−/− MRL-Faslpr and WT mice at 6 mo of age (data not shown). We conclude that eliminating ICOS increases the frequency of Th1 and other T cell subsets that produce IFN-γ and TNF-α but does not alter Th2 cells in the MRL-Faslpr strain.

Eliminating ICOS Suppresses Serum Ig and AutoAb Titers in MRL-Faslpr Mice

Elevated Ig and Ig class switching are hallmarks of autoimmune disease (4,42). B cells are responsible for the production of these Ab after help from autoreactive T cells (43,44). To determine whether ICOS alters these parameters in MRL-Faslpr mice, we compared circulating Ig and anti-dsDNA isotype titers in ICOS−/− MRL-Faslpr and WT strains (n = 5/group, 6 mo of age). Serum titers of total IgG and IgG isotypes (IgG1, IgG2a, and IgG2b) but not IgG3 and IgM are markedly reduced (69, 74, and 77%, respectively) in ICOS−/− MRL-Faslpr mice as compared with the WT strain at 6 mo of age (Figure 4a). Similarly, ICOS−/− MRL-Faslpr mice had reduced titers of serum anti-dsDNA isotypes (total IgG, IgG1, IgG2a, IgG2b, and IgM) but not IgG3 as compared with ICOS+/+ MRL-Faslpr mice evaluated at five dilutions (1:50 through 1:12,150; 1:150 dilution; Figure 4b). Of note, circulating anti-dsDNA Ab were undetectable in C57BL/6J mice, and the levels of these autoAb in the ICOS+/+ MRL-Faslpr (N5) and age-matched MRL-Faslpr were similar (n = 3/group; data not shown). Taken together, these findings indicate that ICOS stimulates the production of circulating IgG and autoAb (dsDNA) isotypes in MRL-Faslpr mice.

Figure 4.
Figure 4.

Serum Ig isotypes and anti-dsDNA antibody (Ab) isotypes are decreased in ICOS−/− MRL-Faslpr mice as compared with the WT strain, whereas IgG isotypes that are lodged in the kidney are similar. (a) Total IgG, IgG1, IgG2a, and IgG2b isotypes but not IgG3 or IgM are suppressed in ICOS−/− MRL-Faslpr mice as compared with ICOS+/+ MRL-Faslpr mice (6 mo of age; *P ≤ 0.01). (b) In addition, the dsDNA-specific Ig isotypes (total IgG, IgG1, IgG2a, IgG2b, and IgM) were diminished in the ICOS−/− Faslpr strain as compared with the WT strain (*P ≤ 0.01, **P ≤ 0.05 and +P ≤ 0.09). Ig were determined by ELISA (five dilutions); the 1:150 dilution is displayed. (c) IgG, C3, and IgG isotypes (IgG1, IgG2a, IgG2b, and IgG3) that were lodged in the mesangium and peripheral capillary loops (PCL) of renal glomeruli are similar in ICOS−/− MRL-Faslpr kidneys and WT mice.

Despite Reduction in Serum IgG Isotype Titers, Similar Amounts of These Isotypes and Complement Lodge in ICOS−/− MRL-Faslpr and WT Kidneys

The deposition of IgG and complement (C3) within glomeruli is a characteristic feature of renal disease in MRL-Faslpr mice (45,46). To determine whether the decrease in circulated Ig and dsDNA autoAb titers (total IgG, IgG1, IgG2a, and IgG2b) translated into a decrease in the deposition of these polyclonal and autoAb Ig in glomeruli, we compared ICOS−/−MRL-Faslpr and WT kidneys. The fluorescence intensity of IgG, C3, and IgG isotypes (IgG1, IgG2a, and IgG2b) within the glomerular peripheral capillary loops and the mesangium was similar in ICOS−/− MRL-Faslpr and WT strains (Figure 4c). In addition, the titers of IgG3 in glomeruli, not reduced in the serum of ICOS−/− MRL-Faslpr mice, was equivalent in ICOS−/− MRL-Faslpr mice and WT mice (Figure 4c). Thus, despite a decrease in serum IgG and dsDNA autoAb isotype titers in the ICOS−/− MRL-Faslpr strain, the amount of these IgG and C3 lodged within glomeruli was equivalent.

Eliminating ICOS in MRL-Faslpr Mice Does not Alter Severity or Tempo of Renal Pathology

To evaluate whether genetic disruption of ICOS prevents or exacerbates lupus nephritis, we compared renal pathology in ICOS−/− MRL-Faslpr and WT strains at 6 mo of age. ICOS−/− MRL-Faslpr and WT mice had severe glomerular, tubular, and perivascular pathology as compared with age and gender-matched MRL+/+ and C57BL/6J mice with normal kidneys (Figure 5, arrowheads). Eliminating ICOS did not have an impact on renal pathology. The severity of glomerular (proliferation, sclerosis, crescents; Figure 5, a and d), tubular (atrophy, dilation, casts; Figure 5, b and d), and perivascular pathology (Figure 5, c and e) were similar in ICOS−/− MRL-Faslpr and WT mice. Of note, the extent of renal pathology in the ICOS+/+ MRL-Faslpr strain at the N5 generation was similar to the MRL-Faslpr strain, confirming that there are sufficient MRL background genes at this generation to evaluate the impact of ICOS (Figure 5, a through c) on renal pathology. Strains with normal renal histology, MRL+/+, and C57Bl/6J served as controls. To determine whether genetic deletion of ICOS alters the tempo of renal pathology, we evaluated kidneys of ICOS−/− MRL-Faslpr and WT mice at an earlier point (3 to 4 mo of age; n = 6/group). The severity of glomerular, tubular, and perivascular pathology was equivalent in these strains (data not shown). Therefore, eliminating ICOS in MRL-Faslpr mice does not affect the severity or the progression of lupus nephritis.

Figure 5.
Figure 5.

Eliminating ICOS in MRL-Faslpr mice does not alter the severity and the tempo of renal pathology. The severity and the tempo of glomerular, tubular, and perivascular pathology were similar in the ICOS−/− MRL-Faslpr strain as compared with the WT strain. The extent of pathology was dramatically increased in both of these strains as compared with the MRL+/+ and C57BL/6J mice (*P ≤ 0.001). (d) Note the severe tubular damage including dilation and casts (*), glomerular crescents (arrows), and profound perivascular infiltrates (v, vessel) in both ICOS−/− MRL-Faslpr and WT kidneys. Magnification, ×400 in d; ×200 in e, periodic acid-Schiff staining.

Proteinuria and BUN Levels Are Similar in ICOS−/− MRL-Faslpr as Compared with WT Mice

To determine whether genetic disruption of ICOS in the MRL-Faslpr strain alters renal function, we evaluated the urinary protein and BUN levels in ICOS−/− MRL-Faslpr and WT strains monthly from 2 to 6 mo of age. The urinary protein levels in ICOS−/− and WT mice, undetectable at 2 mo of age, increased from 2 to 6 mo of age (Figure 6a) in both strains. Similarly, the BUN rose from 3 to 6 mo in both the ICOS−/− MRL-Faslpr and WT strains (Figure 6b). However, we did not detect a difference in the proteinuria or BUN levels between ICOS−/− MRL-Faslpr and WT strains. Thus, eliminating ICOS−/− in MRL-Faslpr mice does not alter proteinuria and BUN levels.

Figure 6.
Figure 6.

Proteinuria and blood urea nitrogen (BUN) are similar in ICOS−/− MRL-Faslpr mice as compared with the WT strain. Proteinuria (2 to 6 mo of age; a) and BUN (3 and 6 mo of age; b) were similarly elevated in ICOS−/− and the WT strain.

Systemic Pathology Is Similar in ICOS−/− MRL-Faslpr Mice and WT Mice

Several tissues in addition to the kidney, including lungs, salivary and lacrimal glands, and skin, are targeted for autoimmune destruction in the MRL-Faslpr strain (5). We detected similar numbers of infiltrating peribronchiolar and perivascular leukocytes in the lungs of ICOS−/− MRL-Faslpr and WT strains (6 mo of age; Figure 7a). In addition, the extent of leukocytic infiltration in the salivary and lacrimal glands in ICOS−/− and WT mice was equivalent (6 mo of age; Figure 7, b and c). The incidence (50% at 4 mo of age, 75% at 5 mo of age, and 90% at 6 mo of age) and severity of skin lesions also did not differ in the ICOS−/− MRL-Faslpr and WT strains. Finally, the lymphadenopathy and splenomegaly were similar in the ICOS−/− MRL-Faslpr and WT strains (n = 12 to 15/group; data not shown). Taken together, these data indicate that genetic disruption of ICOS is not sufficient to alter systemic pathology in MRL-Faslpr mice.

Figure 7.
Figure 7.

Lung and salivary and lacrimal gland pathology in ICOS−/− MRL-Faslpr and WT mice are similar (6 mo of age). The extent of lung and gland (salivary and lacrimal) pathology is illustrated in a, b, and c, respectively.

Discussion

Consistent with the known requirement of ICOS for T cell–dependent primary B cell responses (32), we have now determined that disruption of the ICOS gene in MRL-Faslpr mice serves to suppress serum polyclonal Ig and dsDNA autoAb isotype (G1, G2a, and G2b) titers that rise with advancing disease. Importantly, this suppression in circulating IgG and autoAb isotypes did not translate into improved renal or systemic pathologic consequences. Renal disease (pathology and function) and the systemic illness (inflammation and the lymphoproliferative disorder) were not deterred in the ICOS−/− MRL-Faslpr strain. Thus, serum IgG and dsDNA autoAb titers and lupus nephritis are uncoupled in ICOS−/− MRL-Faslpr mice. These findings complement our previous data in which nephritis is suppressed in MCP-1 null and IL-12 null MRL-Faslpr mice, although the serum IgG and autoAb remain elevated (33,36). Circulating autoAb are frequently monitored as prognostic indicators of autoimmune diseases. Our findings draw attention to the potential danger in relying on serum Ig and autoAb titers to monitor the course of autoimmune disease.

What are the consequences of a reduction in serum polyclonal IgG and autoAb isotype titers on Ig deposition in glomeruli in MRL-Faslpr mice that lack ICOS? Despite a reduction of these serum Ab titers, the amounts of these specific IgG isotypes that are lodged in glomeruli are not diminished in ICOS null MRL-Faslpr. There are several plausible explanations. Given that there is a finite amount of Ab that can lodge in glomeruli, it is possible that the serum Ab titers in ICOS−/− MRL-Faslpr mice exceed this finite amount; hence, the glomeruli are saturated with Ab despite the reduction in serum Ab titers as compared with WT mice. Alternatively, we favor the possibility that the pathogenic Ab that deposit in glomeruli are a relatively small subpopulation of those in the circulation. In either case, it is clear that the titers of Ig isotypes are not necessarily linked to the quantity of Ab that deposit in renal glomeruli.

In keeping with the concept that ICOS stimulation enhances Th2 differentiation, we observed an increased frequency of intrarenal Th1 cells in ICOS null MRL-Faslpr. The frequency of IFN-γ–and TNF-α–expressing CD4 T cells in the kidney was enhanced in ICOS−/− MRL-Faslpr as compared with WT mice. In addition, we detected a substantial rise in the frequency of IFN-γ and TNF-α expression in other intrarenal T cell populations (CD8 and DN). On the basis of this finding and coupled with evidence that (1) IFN-γ and TNF-α are nephritogenic in lupus mice (38,47), (2) T cells (CD4, CD8, and DN) are required for lupus nephritis (6), and (3) ICOS may inhibit tubular epithelial cell–mediated immune activation in the kidney in vitro (31), we might have anticipated that the elevated frequency of Th1 and other IFN-γ–and TNF-α–expressing T cells would hasten the onset and worsen lupus nephritis in the absence of ICOS. This is not the case. Clearly, the lupus syndrome is accompanied by a variety of autoreactive T cell and autoAb parameters. The precise role and contribution to lupus and renal disease of each parameter are uncertain. Taken together, our findings underscore the danger of relying on measurements of individual parameters to predict disease outcome.

Before this report, there was conflicting evidence regarding the importance of the ICOS pathway as a therapeutic target for systemic autoimmune diseases such as lupus. For example, in a mercury chloride–induced model of systemic autoimmune disease, as defined by a rise in serum anti-nucleolar Ab (48), ICOS blockade with anti-ICOS Ab suppressed the rise in these serum autoAb. This suggested that targeting the ICOS pathway had therapeutic promise for systemic autoimmune illnesses. However, a quantitative reduction of serum autoAb alone, as we have shown, does not necessarily lead to amelioration of tissue injury, and chronic mercury chloride–induced immune mediated nephritis was not explored. However, treatment of a spontaneous model of systemic autoimmune lupus, the NZB/W, with anti-ICOS-L Ab but not with anti-ICOS Ab before and during disease resulted in a reduction in anti-dsDNA Ab, suppression of nephritis, and extended survival (49). The different outcomes of administration of anti-ICOS and anti–ICOS-L Ab might reflect differences in affinity or half-life of these Ab. However, our studies using ICOS−/−MRL-Faslpr mice are consistent with the anti-ICOS Ab studies, suggesting that there are different biologic roles of ICOS and ICOS-L. Furthermore, studies are needed to understand these biologic differences. Further studies are also required to validate whether ICOS-L is a therapeutic target including ICOS-L null and Ab blockade approaches in the MRL-Faslpr strain. Finally, we compared nephrotoxic serum nephritis, another T cell–dependent immune-mediated renal disease, in ICOS−/− mice and WT mice. Renal pathology and function were similar in the ICOS−/− as compared with WT mice (data not shown). Thus, on the basis of our studies using ICOS null MRL-Faslpr mice and nephrotoxic serum nephritis, we suggest that ICOS monotherapy is not a promising therapeutic target for lupus or immune-mediated renal disease. More important, we conclude that the therapeutic validity of individual treatments should not be based on the assessment of a single immune compartment in a multicompartmental disease process such as lupus.

Acknowledgments

This work was supported by National Institutes of Health Grants DK 52369 (V.R.K.), DK 56848 (V.R.K.), DK 36149 (V.R.K.), and AI 38310 (A.H.S.) and the Deutsche Forschungsgemeinschaft ZE-711/1 (G.Z.).

We thank Dr. Kevin C. O’Connor for assistance with the dsDNA Ab isotype analysis and Dr. Gary Curhan and Dr. Surya M. Nauli for assistance with the statistical analysis.

Footnotes

  • Published online ahead of print. Publication date available at www.jasn.org.

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Inducible Co-Stimulator Null MRL-Faslpr Mice: Uncoupling of Autoantibodies and T Cell Responses in Lupus
Geraldine C. Zeller, Junichi Hirahashi, Andreas Schwarting, Arlene H. Sharpe, Vicki R. Kelley
JASN Jan 2006, 17 (1) 122-130; DOI: 10.1681/ASN.2005080802
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