Contributions of IL-1β and IL-1α to Crescentic Glomerulonephritis in Mice

Mice

Mice homozygous for targeted deletions of IL-1β (27) and IL-1RI (28) genes backcrossed onto a C57BL/6 background were bred and housed under specific pathogen-free conditions at Monash University (Clayton, Victoria, Australia). Twenty-four C57BL/6 (WT) controls, 11 IL-1β−/−, and 9 IL-1RI−/− male mice weighing 20 to 26 g and aged between 13- and 14-wk-old were used.

Induction of Crescentic GN

Crescentic GN was initiated by an intravenous injection of a total of 14.4 mg of sheep anti-mouse GBM globulin, given as two equally divided doses 3 h apart, as described previously (29,30⇓). Twenty days later, mice were placed individually in metabolic cages to collect urine over the final 24 h of the experiment. Twenty-four hours later (day 21), mice were anesthetized by inhalation of methoxyflurane, bled by cardiac puncture, and killed to collect kidney and splenic tissue.

Histologic Assessment of Glomerular Injury

Glomerular crescent formation was assessed in periodic acid-Schiff reagent (PAS)–stained 3-μm sections cut from kidney tissue that was fixed in Bouin fixative and embedded in paraffin. 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 was assessed to determine the crescent score for each animal.

Glomerular CD4⁺ T cell and macrophage accumulation was assessed in kidney and spleen tissue that was fixed in periodate/lysine/paraformaldehyde (PLP) for 4 h, washed in 7% sucrose, and then frozen in liquid nitrogen. Six-micrometer cryostat cut sections were stained to demonstrate CD4⁺ T cells and macrophages with monoclonal antibodies (GK1.5 and M1/70, respectively) using a three-layer immunoperoxidase technique (31). A minimum of 20 equatorially sectioned glomeruli were assessed per animal, and were results expressed as cells per glomerular cross section (c/gcs).

Glomerular deposition of sheep globulin, mouse Ig, and complement was detected by direct immunofluorescence on 6-μm cryostat cut sections from five animals from each experimental group. Glomerular deposition of sheep IgG was assessed by determining the end point titer on sections stained with FITC-conjugated rabbit anti-sheep IgG antibody (Silenus, Melbourne, Australia) in serial doubling dilutions from 1:200 to 1:3200. The highest dilution of detecting antibody at which staining above background levels could be detected was taken as the end point titer for each animal. Glomerular mouse Ig was quantified using a FITC-conjugated sheep anti-mouse Ig (Silenus). Sections were stained at a range of dilutions from 1:100 to 1:1,600 to find a titer in which deposition of mouse Ig was detectable in all groups (1 in 800). Representative sections from five animals per group were then stained at 1:800, and glomerular mouse Ig was scored 0 to 3+ in a blinded protocol. Glomerular complement deposition was detected using FITC-conjugated goat anti-mouse C3 (Cappel, Durham, NC) antibody at a dilution of 1:100. In a blinded protocol, a minimum of 50 glomeruli per mouse were scored semiquantitatively (0 to 3+), with 0 being equivalent to background and 3+ equal to the most intense fluorescence in any glomerulus.

Functional Assessment of Glomerular Injury

Proteinuria was determined from 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. The 24-h urinary protein excretion rate was calculated from the 24-h urine volume and the urinary protein concentration. Before induction of GN, all groups of mice had 24-h urine protein excretion in the normal range (0.5 to 2.0 mg/24 h).

Serum creatinine concentrations were measured by an enzymatic creatininase assay.

Measurement of Urinary Nitric Oxide Levels

Concentrations of urinary nitrite plus nitrate were determined in a Griess assay. Urine samples were centrifuged at 2000 g for 10 min at 4°C, and 50-μl aliquots were added to 50 μl of Griess reagent (1.5% sulfanilamide/0.15% naphthyl ethylene diamine) in a 96-well microtiter plate. The plate was incubated for 10 min at room temperature, and the absorbance was read on a microplate reader at 530 nm. The samples were run in duplicate, and the concentration of nitrite was calculated by comparison with absorbance values NaNO₂ standards of known concentration, and results are expressed as mg/ml.

Assessment of Systemic Immune Responses to Sheep Globulin

Cutaneous delayed-type hypersensitivity (DTH) to subdermal sheep globulin challenge was determined in mice developing GN over the final 24 h of the experimental protocol. Mice were challenged with a subdermal injection of 500 μg of sheep globulin in 50 μl of PBS into one hind footpad. A similar dose of an irrelevant antigen (horse globulin) was injected into the other footpad as a control. Measurements of footpad swelling were quantified 24 h later using a micrometer (Mitutoyo Corporation, Kawasaki, Japan). Antigen-specific DTH was taken as the difference in skin swelling between sheep globulin– and horse globulin–injected footpads and expressed as Δ footpad thickness (mm).

Total anti-sheep globulin Ig and anti-sheep globulin IgG1 and IgG2a isotypes were measured by ELISA on serum collected at the end of the experiment. Assays were performed using microtiter plates coated with sheep globulin at a concentration of 10 μg/ml. Plates were washed and blocked with 1%BSA in PBS for total Ig and IgG1 assays, or with 2% casein in PBS for the IgG2a assay. Plates were then washed and incubated with a 1:800 dilution of serum for 1 h at room temperature (or overnight at 4°C in the case of the IgG2a assay). Bound mouse Ig was detected using horseradish peroxidase (HRP)–conjugated sheep anti-mouse Ig (Amersham, Little Chalfont, UK) for total Ig and goat anti-mouse IgG1 or IgG2a, (Southern Biotechnology Assoc, Birmingham, AL) for IgG subclasses. 3,3′5,5′ tetramethyl-benzidine (TMB; Sigma Aldrich Pty Ltd, NSW, Australia) was added as a chromogenic substrate, and the absorbance was read at 450 nm, as described previously (32).

Measurement of IL-1α

Serum, kidney, and brain tissue samples were collected from normal WT (n = 3) and IL-1β−/− (n = 3) mice and WT (n = 5) and IL-1β−/− (n = 5) mice with GN. Kidney and brain samples were homogenized in 0.1 M NaCl, 0.05 M TrisCl, 2% vol/vol Triton X-100 together with protease inhibitors (Roche Diagnostics, Melbourne, Australia). The homogenate was spun at 14000 rpm for 15 min, and the supernatant was collected. Tissue extracts and serum were stored at −70° until assayed. IL-1α was measured by ELISA, with a detection limit of 0.03125 ng/ml. Microtiter plates were coated with anti-mouse IL-1α antibody as the capture Ab (R&D Systems, NSW, Australia) at a concentration of 3 μg/ml. Plates were washed in 0.05% Tween/PBS and blocked with 1%BSA in PBS for 2 h at 37°C. Plates were then washed and incubated with recombinant murine IL-1α (R&D Systems) at serial dilutions between 2.0 and 0.0625 ng/ml dilutions or with serum, brain, and kidney extracts at 4°C overnight. Plates were washed, and bound mouse IL-1α was detected using biotinylated anti-mouse IL-1α Ab (R&D Systems) at a concentration of 200 ng/ml for 1 h at room temperature. Plates were washed; streptavidin-HRP (Chemicon, Melbourne, Australia; 1:2000 in 1% BSA/PBS) for 20 min at room temperature followed by TMB (Sigma Aldrich Pty Ltd) was used as a chromogenic substrate. All samples were assayed in duplicate, and results were calculated as IL-1α ng/g wet weight or ng/ml for serum.

Statistical Analyses

Results are expressed as the mean ± SEM. The statistical significance was determined by one-way ANOVA, followed by Newman-Keuls post hoc test.

IL-1β Deficiency Reduces Cellular Mediators of Glomerular Injury and Attenuates Crescentic GN Independent of IL-1α

Crescentic GN in this murine model has been well characterized as a CD4⁺ Th1-dependent model (33–36⇓⇓⇓). It demonstrates features similar to human crescentic GN with crescent formation, glomerular accumulation of T cells, and macrophages with severe impairment of renal function. In the current study, WT mice developed severe proliferative GN (Figure 1A) with prominent crescent formation (25.1 ± 1.7% of glomeruli) and glomerular accumulation of CD4⁺ T cells (1.12 ± 0.09 c/gcs) and macrophages (2.12 ± 0.15 c/gcs) (Figure 2). Functional renal injury in WT mice was indicated by proteinuria (9.1 ± 1.0 mg/24 h; normal <2.0 mg/24h) elevated serum creatinine (25.2 ± 2.0 μmol/L; normal 9.8 ± 1.2 μmol/L) and increased urinary nitric oxide production (9.5 ± 1.6 mg/ml; normal 0.26 ± 0.03 mg/ml), which is a product of activated macrophages (Figure 3).

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Figure 1. Twenty-one days after the administration of anti-glomerular basement membrane (GBM) globulin, the histologic appearance of crescentic glomeruli in WT with severe proliferative glomerulonephritis (GN) (A), with increased interstitial infiltrate and glomerular deposition of periodic acid-Schiff (PAS)–positive material. In contrast, IL-1β−/− (B) and IL-1RI−/− (C) mice have mild histologic changes 21 d after anti-GBM globulin, crescentic glomeruli are occasional, and interstitial infiltrate is reduced. Immunofluorescence demonstrating glomerular mouse Ig deposition in WT (D), IL-1β−/− (E), and IL-1RI−/− (F) animals with GN. Intense glomerular staining was observed for WT and IL-1β−/− mice; however, IL-1RI−/− mice had significantly decreased glomerular deposition of mouse Ig. This observation correlates with glomerular complement deposition; similar to the glomerular mouse Ig pattern, observed WT (G) and IL-1β−/− (H) mice had intense immunofluorescence in glomeruli, with tubular C3 deposition also apparent, whereas IL-1RI−/− mice had a reduced level of C3 (I). A–C, PAS-stained sections, original magnification, ×200; D–I, immunofluorescence, original magnification, ×400).

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Figure 2. Histologic changes observed in crescentic GN, specifically the incidence of crescent formation (% glomeruli affected), glomerular infiltration of CD4⁺ T cells, and macrophages (cells/glomerular cross section [c/gcs]) in WT, IL-1β−/−, and IL-1RI−/− mice with crescentic GN. ★ indicates P < 0.05 compared with WT mice.

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Figure 3. Functional renal indices of injury. Proteinuria (mg/24 h), serum creatinine (μmol/L), and urinary nitric oxide levels (mg/ml) in WT, IL-1β−/−, and IL-1RI−/− mice with crescentic GN. ★ indicates P < 0.05 compared with WT mice. ★★ indicates P < 0.05 compared with IL-1β−/− mice. The broken line indicates the urinary protein excretion, serum creatinine, and urinary nitric oxide in normal WT mice.

Significant protection from development of crescentic GN was observed in IL-1β−/− mice. Glomerular crescent formation (15.3 ± 2.2% of glomeruli in IL-β−/−; P < 0.01 versus WT) was reduced, as was glomerular infiltration of CD4⁺ T cells (0.29 ± 0.04 c/gcs; P < 0.001 versus WT) and macrophages (0.42 ± 0.27 c/gcs; P < 0.001 versus WT) (Figure 2). Reduction in the histologic injury (Figure 1B) corresponded with significant protection of renal function, indicated by reduction in proteinuria (6.3 ± 0.9 mg/24 h; P < 0.05 versus WT) and urinary nitric oxide (4.5 ± 0.3 mg/ml; P < 0.05 versus WT). The reduction in serum creatinine (20.6 ± 0.8 μmol/L) was not statistically significant (P = 0.12) compared with WT mice (Figure 3).

Previous reports suggest that IL-1α levels in the brain following turpentine treatment and macrophage IL-1α production following LPS stimulation are significantly attenuated in IL-1β–deficient mice (27,37⇓). To address the possibility that decreased renal IL-1α levels may contribute to the protection against anti-GBM GN in IL-1β−/− mice, we measured renal, serum, and brain IL-1α levels in IL-1β–deficient mice before and after induction of GN. IL-1α in the kidney and brain from normal WT and IL-1β−/− mice were equivalent. Serum IL-1α levels were below the level of detection of the assay. Following induction of GN, IL-1α in the kidney or brain remained equivalent in WT and IL-1β−/− mice and serum levels were still undetectable. Although there was a trend to an increase in IL-1α in the kidneys of IL-1β−/− mice following induction of GN, this was not statistically significant (Figure 4).

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Figure 4. IL-1α levels were measured in brain and kidney by ELISA in WT and IL-1β−/− mice with and without GN. Tissue samples were homogenized and protein extracted, results expressed as concentration (ng) over tissue wet weight (g). No significant differences were observed in IL-1β−/− compared with WT mice in any of the tissues examined.

IL-1RI Deficiency Provides Additional Protection from Antibody-Mediated Injury

IL-1RI–deficient mice were used to determine the combined contribution IL-1β and IL-1α to crescentic GN. Histologically, IL-1RI−/− mice show similar protection to IL-1β−/− mice (Figure 1C) with regard to their development of crescents (14.0 ± 1.7% of glomeruli in IL-1RI−/−; P < 0.05 versus WT) and glomerular accumulation of CD4⁺ T cells (0.28 ± 0.03 c/gcs; P < 0.001 versus WT) and macrophages (0.33 ± 0.12 c/gcs; P < 0.001 versus WT) (Figure 2). However, IL-1RI−/− mice demonstrated additional preservation of renal function in comparison to IL-1β−/− mice, indicated by significantly less proteinuria (2.8 ± 1.4 mg/h; P < 0.01 versus IL-1β−/−) and serum creatinine (16.0 ± 1.4 μmol/L; P < 0.05 versus IL-1β−/−). Urinary nitric oxide levels in IL-1RI−/− mice (4.5 ± 0.4 mg/ml; P < 0.05 versus WT) were also reduced compared with WT mice (Figure 3) but were not different from IL-1β−/− mice, paralleling the patterns of glomerular macrophage accumulation.

Humoral Immunity to Sheep Globulin in IL-1β–Deficient and IL-1RI–Deficient Mice

Circulating titers of mouse anti-sheep globulin antibodies were measured as an index of systemic humoral immune responses to sheep globulin. Total anti-sheep globulin Ig was unaffected in IL-1β−/− but was significantly reduced in IL-1RI−/− mice compared with WT controls (Figure 5). Analysis of mouse anti-sheep globulin IgG subclasses showed increased IgG1 (Th2-dependent) and reduced IgG2a (Th1-dependent) isotypes in IL-1β−/− mice (IgG1, 2.45 ± 0.14; IgG2a, 0.76 ± 0.13) with an IgG1/IgG2a ratio of 3.22 compared with a ratio of 0.40 in WT mice (IgG1, 0.73 ± 0.09; IgG2a, 1.81 ± 0.32) with GN, suggesting deviation of immune response to sheep globulin from Th1 to Th2 in the absence of IL-1β. IL-1RI–deficient mice showed higher levels of both IgG1 (2.82 ± 0.09) and IgG2a (3.27 ± 0.03) IgG subclasses; however, their ratio was (0.68) similar to WT mice.

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Figure 5. (Top panel) Cutaneous DTH responses to sheep globulin in WT, IL-1β−/−, and IL-1RI−/− mice with GN (21 d after intravenous sheep globulin). ★ indicates P < 0.05 compared with WT mice. (Bottom panel) Serum titers of mouse anti-sheep globulin Ig in WT (▪), IL-1β−/− (▴), and IL-1RI−/− (•) mice developing crescentic GN (n = 5). ★ indicates Ig titers significantly reduced (P < 0.05) compared with WT mice and IL-1β−/− mice.

Linear glomerular deposition of sheep Ig was the same in all groups, with a mean end point titer of 1 in 1600, indicating equivalent glomerular deposition of the nephritogenic antigen. Glomerular deposition of mouse Ig was similar in WT (Figure 1D) and IL-1β−/− mice (Figure 1E) (mean score at 1:800 dilution: 2.00 ± 0.0 and 1.5 ± 0.28, respectively). In IL-1RI−/− (Figure 1F), glomerular mouse Ig was significantly reduced (mean score at 1:800 dilution 0.63 ± 0.13; P < 0.01 versus WT and IL-1β−/− mice; Figure 6). Glomerular deposition of C3 was unaffected in IL-1β−/− mice (Figure 1H; 1.71 ± 0.09) compared with WT mice (Figure 1G; 1.92 ± 0.08), but it was significantly reduced in IL-1RI−/− mice (Figure 1I; 1.54 ± 0.09; P < 0.01 versus WT; Figure 6), corresponding to the reduced circulating mouse anti-sheep globulin antibody titers.

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Figure 6. Deposition of mouse Ig and C3 in glomeruli of WT, IL-1β−/−, and IL-1RI−/− mice developing crescentic GN (n = 5). ★ indicates P < 0.05 compared with WT mice.

Reduced DTH Responses in IL-1β−/− and IL-1RI−/− Mice

Mice given sheep globulin demonstrated antigen-specific skin swelling after cutaneous challenge with sheep globulin. Cutaneous DTH responses were equivalent in WT (0.55 ± 0.07 mm) mice and IL-1β−/− (0.40 ± 0.05 mm) mice. Cutaneous DTH responses to sheep globulin were reduced by approximately 55% in IL-1RI−/− mice (0.25 ± 0.04 mm; P < 0.01 versus WT).