Although proliferative and crescentic glomerulonephritides occur in different primary renal diseases and are an important component of several systemic diseases, features of human renal biopsies suggest some common effector pathways. In most cases of rapidly progressive GN there is evidence for an important role for cellular immune effectors: T cells, macrophages, and neutrophils,1–3 a role confirmed in animal models.4–7 CD4+ T cells are key components of renal injury.4,8 When activated, CD4+ cells tend to differentiate into subsets (T helper cells—Th1, Th2 and Th17) that engage immune effectors in different ways. In proliferative forms of GN, T cells direct adaptive immune responses that drive glomerular disease, but also, in rapidly progressive GN, CD4+ cells themselves accumulate in glomeruli as effectors. These effector T helper cells activate innate effector cells, predominantly neutrophils and macrophages, and activate and damage intrinsic renal cells.
In GN, the variable Th1-Th2 predominance of responses influences the histologic patterns and severity of GN.9 Th1 cells, which secrete IFNγ and activate macrophages, are important in some forms of experimental proliferative GN. Th2 cells, characterized by IL-4 production, promote humoral immunity and are important in several forms of GN, but there is little evidence that Th2 cells play primary roles as effector cells within glomeruli in rapidly progressive GN. A binary Th1/Th2 model explains many of the differences in the patterns of immune responses in GN. However, there are discrepancies10 that might be explained by defining a role for a third major subset, Th17 cells, characterized by the production of IL-17A. Its biology has recently been comprehensively reviewed.11 Th17 subset effects potentially relevant to rapidly progressive GN include direct effects on neutrophils and stimulating the production of neutrophil chemoattractants by tissue cells. Thus, in most rapidly progressive types of GN, cell-mediated injury, a key component of injury, may be directed by the Th17 subset, the Th1 subset, or both subsets.
Although antigen-specific T cells are critical to adaptive immune responses, the cells themselves are relatively infrequent. T cell receptor (TcR) transgenic mice help define the contributions of different antigen-specific T helper cell subsets in organ-specific disease.12–14 In the studies presented here we have established a new antigen-specific model of GN. Ovalbumin (OVA)-specific OT-II TcR transgenic CD4+ cells15 are polarized ex vivo under Th1- or Th17-inducing conditions. Effector cells are transferred into recombination activating gene-1 deficient (Rag1−/−) mice with OVA planted in their glomeruli by injecting an OVA conjugate. OVA is conjugated to a mouse IgG1 mAb binding to α3(IV) collagen in murine glomerular basement membrane (GBM). The mAb-OVA conjugate dose is capable of planting significant OVA in glomeruli as an antigen to induce effector responses, but is insufficient to induce significant histologic or functional injury as an antibody. This model allows us to understand effector CD4+ T cell and Th subset-induced injury, with no effects from CD8+ cells or B cells.
CD4+ cells, isolated from OVA-specific TcR transgenic (OT-II) mice and cultured under Th1 or Th17 priming conditions (see Concise Methods), were confirmed to be Th1 or Th17 by cytokine production before transfer. Th1 polarized cells expressed IFNγ, whereas no IL-17A or IL-4 production was detected (Figure 1A). Th17 polarized cells were strong IL-17A producers, showing only weak IFNγ production, with >20% of cells producing IL-17A, but few IFNγ or double-positive cells (Figure 1B). To plant OVA in glomeruli, the mAb 8D1, a non-nephritogenic, murine IgG1 binding to mouse α3(IV)NC1,16 was conjugated to OVA and purified by size-exclusion chromatography so that no free OVA or unconjugated 8D1 mAb remained, confirmed by Western blotting (Figure 1C). Antigen-specific CD4+ cells recognized OVA bound to the 8D1 anti-GBM mAb. Culture of CFSE-labeled naive OT-II cells incubated with the 8D1-OVA conjugate enhanced their survival (30% to 40% survival after 72 h versus 5% to 6% with unconjugated antibody) and induced OT-II cell proliferation (72 h: 27% of cells, up to 4 cycles, Figure 1D). OT-II cells incubated with unconjugated 8D1 did not proliferate (Figure 1E). After intravenous injection, 8D1-OVA conjugates bound to the GBM in a linear manner; no fluorescence signal was observed after transfer of Th1 cells without 8D1-OVA (Figure 1G). Western blotting showed OVA in the kidney after injection of 8D1-OVA conjugate (Figure 1H). Lungs from 8D1-OVA-injected mice were weakly positive for mouse IgG, whereas no IgG was detected in the spleen or liver (detecting antibody titer 1:100) 3 or 21 d after injection. Mouse IgG was not detected in sera (ELISA, dilution 1:100) at day 3 or day 21. As expected (given the transfer of only CD4+ cells to Rag1−/− mice), no anti-OVA antibodies in sera were detected in recipient mice (data not shown).
To determine whether transfer of either Th1 or Th17 antigen-specific effector cells induces glomerular injury, 8D1-OVA conjugate was administered intravenously to Rag1−/− mice (lacking adaptive immunity). Three hours later, 5 × 106 Th1 or Th17 cells were injected intravenously. Groups of mice injected with 8D1-OVA alone (without cells) or Th1 cells alone (without 8D1-OVA) served as controls. At 21 d, the injected T cells largely maintained their initial phenotype, because host splenocytes from mice given Th1 cells showed enhanced OVA-stimulated IFNγ production whereas IL-17A production was enhanced in mice given Th17 cells (Figure 1I). Dermal-delayed-type hypersensitivity (DTH) was induced by footpad injection of OVA and measured after 24 h. Only mice that received the 8D1-OVA conjugate and Th1 polarized cells developed dermal DTH (Figure 1J), a classical Th1 response.17
After planting OVA in glomeruli, administration of Th1 or Th17 cells induced glomerular disease. Urinary albumin excretion was not increased in mice given 8D1-OVA conjugate alone or Th1 cells alone, but Th1 or Th17 cells with 8D1-OVA induced significant albuminuria (Figure 2A). Albuminuria was consistent throughout the time course of the study in the Th17 group, whereas in the Th1 group there was a progressive increase in albuminuria until day 21 (Figure 2B). Control mice given Th1 cells alone or the 8D1-OVA conjugate alone exhibited only mild histologic changes (no crescent formation, fibrinoid necrosis, or hyalinosis). Analysis of histologic injury demonstrated substantially more abnormal glomeruli in the mice given 8D1-OVA conjugate with Th1 or Th17 cells compared with control groups (Figure 2C). Th1 and Th17 (+8D1-OVA) recipients developed proliferative GN, [glomerular hypercellularity: 8D1-OVA and Th1 cells: 32.1 ± 1.0 cells/glomerular cross section (c/gcs), 8D1-OVA and Th17 cells: 29.8 ± 1.1 c/gcs, 8D1-OVA alone: 21.3 ± 0.2 c/gcs, Th1 cells alone: 18.9 ± 2.0 c/gcs; P < 0.001]. Representative kidney sections from each group are shown (Figure 2, D through G). Crescent formation and fibrinoid necrosis, although seen in only a few glomeruli, was observed exclusively in mice given 8D1-OVA conjugate and Th1 cells (5.5 ± 0.9% at day 21; Figure 2, H and I). No crescent formation was observed in mice receiving 8D1-OVA conjugate and Th17 cells. Mice did not develop significant renal impairment (measured by BUN; data not shown).
Recruitment and activation of leukocyte subpopulations differed in mice administered Th1 or Th17 cells (Figure 3A). Although glomerular CD4+ cell and macrophage numbers were similarly increased in mice given 8D1-OVA conjugate and either Th1 or Th17 cells at day 21, more neutrophils were found in mice given 8D1-OVA and Th17 cells compared with mice given 8D1-OVA and Th1 cells. Interstitial leukocyte infiltrates followed a similar pattern (Figure 3B). Consistent with the finding of increased neutrophils in kidneys of mice receiving Th17 cells, renal mRNA expression of the primary neutrophil attracting chemokine CXCL1 was elevated (Figure 3C). Th17 cells attract neutrophils18 and in vitro studies have shown that neutrophil recruitment is achieved via production of CXCL8, the human homologue of CXCL1, by Th17 cells.19 It is therefore likely that at least some of the Th17-induced renal injury is mediated by neutrophils. In mice receiving 8D1-OVA and Th1 cells, macrophages were likely to be more activated; only these mice developed dermal DTH and increased expression of mRNA for the macrophage chemoattractants CCL2 and CCL5 (Figure 3, D and E), which have been associated with experimental crescentic GN.20 Furthermore, type 2 nitric oxide synthase (NOS2/iNOS) mRNA, a marker of macrophage activation21 and urinary nitrate, a marker of intrarenal macrophage NOS2 production, were increased in this group (Figure 3, F and G).
Further studies were performed 3 d after cell transfer. At this time point, albuminuria was present in mice receiving 8D1-OVA conjugate and Th17 cells, but not 8D1-OVA conjugate and Th1 cells (Figure 4A), and a higher proportion of glomeruli were abnormal in mice that had received Th17 cells (Figure 4B). Therefore, Th17-induced glomerular injury occurred earlier than Th1-induced injury. Leukocytes were present in glomeruli (Figure 4C) with increased numbers of neutrophils in glomeruli of mice receiving Th17 cells (compared with Th1 cell recipients), whereas Th1 cell recipients exhibited more macrophages. At day 3, these findings were glomerulo-specific; differences between Th1 and Th17 cell recipients were not seen in the interstitium (Figure 4D).
These studies used Rag1−/− mice as recipients of effector antigen-specific Th1 or Th17 cells. Because these mice do not possess T or B cells, OVA planted in glomeruli cannot induce CD8+ or B cell responses, and regulatory T cells are unable to influence the pattern of injury. A major advantage of this strategy is that Th1- and Th17-mediated injury can be assessed in a pure experimental system. However, T cells transferred into Rag1−/− mice can undergo homeostatic expansion, and it is possible that the transferred Th1 cells might have expanded more rapidly than Th17 cells. Recently, studies in experimental type 1 diabetes induced by transfer of cells from a TcR transgenic mouse specific for an islet autoantigen showed conversion of Th17 cells to a Th1 phenotype after transfer.22,23 Although our Th17 polarized OT-II cells, specific for a foreign antigen, showed some IFNγ production after 21 d, they were still capable of producing IL-17A. Furthermore, dermal DTH and renal disease were different in Th1 recipients compared with the Th17 recipients at 21 d, supporting the maintenance of separate phenotypes after transfer. Although the studies presented here are the first to demonstrate a role for Th17 and Th1 cells in the same experimental system, other studies24–26 have used genetically deficient mice to implicate Th17 cells in experimental renal disease.
These studies describe a novel model of cell-mediated proliferative GN for which adaptive components are only effector antigen-specific CD4+ T cells. They demonstrate that both Th1 and Th17 cells can induce proliferative GN. Th17 cells induce albuminuria early, with persistent accumulation of leukocytes. Administration of Th1 cells lead to a slower rise in albuminuria, but more macrophage activation and DTH-like injury, including, in some glomeruli, crescent formation and fibrinoid necrosis. It is likely that Th1 and Th17 responses play a role in proliferative forms of GN and both represent potential therapeutic targets.
Antibody Conjugation and T Cell Polarization
The 8D1 mAb (5 mg/ml in PBS) was reacted with 0.1 mg/ml N-succinimidyl-6-maleimido-caproate (Sigma-Aldrich, St. Louis, MO) for 2 h at room temperature. OVA (10 mg/ml in PBS; Sigma-Aldrich) was reduced with 5 nM trin-butyl phosphine (Sigma-Aldrich) for 2 h at 50°C. The reduced OVA and activated antibody solutions were buffer-exchanged into degassed PBS, then mixed with OVA in a 10-fold molar excess over mAb. The conjugation reaction was allowed to proceed for 3 h at room temperature before stopping with 2 mM cysteine. The OVA-conjugated 8D1 was purified from free OVA and nonconjugated antibody by size exclusion chromatography (Superdex 200 column, GE Healthcare). Eluted fractions, which corresponded with peaks, were collected and stored at 4°C.
To generate Th1 and Th17 cells, OT-II cells from lymph nodes of OT-II transgenic mice were harvested. Initial cell preparations (containing 30% to 50% OT-II cells) were cultured with antigen presenting cells (4 × 105 OT-II+ cells/ml with 4 × 106 antigen presenting cells/ml) under Th1 or Th17 inducing conditions. For Th1, 1μM OVA323-339 peptide, 2 ng/ml rmIL-12 with 10 μg/ml of anti IL-4 (11B11, ATCC), for Th17 20 ng/ml rmIL-6, 50 ng/ml rmIL-23, 5 ng/ml rmTGF-β, 10 μg/ml anti-IL-4 mAb, and 10 μg/ml anti-IFNγ. After 48 h, cells were adjusted to a density of 4 × 105 cells/ml in fresh media, For Th1 cells, 5 μg/ml rmIL-2 was added, for Th17 cells 50 μg/ml of IL-2 and 20 ng/ml of rmIL-23; cells were incubated for 5 additional days. On day 7, cells were harvested and the proportions of IFNγ+ and IL-17A+ cells were determined by flow cytometry. (BD FACS Canto flow cytometer, BD Biosciences, North Ryde, Australia). IFNγ and IL-17A concentrations in OVA-stimulated splenocyte cultures from recipients were measured by ELISA.20 Mouse IgG and anti-OVA IgG were assessed by ELISA as described previously.25
To determine the activity and binding of the 8D1-OVA conjugate, OT-II cells from splenocytes were labeled with CFSE. Cells (5 × 105) were stimulated with 8D1-OVA or unconjugated 8D1 mAb (0.84 mg/ml) After 72 h later flow cytometric analysis was performed on the cells to determine their survival and proliferation. 8D1-OVA and unconjugated 8D1 mAb were tested for the presence of OVA and IgG using mAb to OVA (Abcam, Cambridge, MA) or sheep anti-mouse IgG (Amersham Biosciences, Rydalmere, Australia). The secondary antibody used was streptavidin horseradish peroxidise (Amersham). To detect the presence of OVA in the kidney, kidneys from Rag1−/− mice (specific pathogen-free facility, Monash Medical Centre, Melbourne Australia) were studied 24 h after receiving 8D1-OVA. Kidneys were homogenized and supernatants and cell lysates were analyzed by SDS-PAGE.
In Vivo Experiments and Assessment of Renal Injury
C57Bl/6.Rag1−/− mice were injected intravenously with 150 μg of 8D1-OVA or unconjugated 8D1 mAb. Groups that received cells were intravenously injected 3 h later with 5 × 106 Th1 or Th17 polarized cells. 24 h before the end of experiments (at 21 d after induction of disease), mice were injected with 100 μg of OVA (in 20 μl of sterile-filtered PBS) in the left hind-foot and 20 μl of sterile-filtered PBS alone in right hind-foot. The difference between footpad thicknesses was assessed using a micrometer. Studies were performed in accordance with National Health and Medical Research Council of Australia guidelines and approved by the Monash University Animal Ethics Committee. Results are expressed as mean ± SEM. Recipient group numbers were as follows: day 21 8D1-OVA alone, n = 7; Th1 cells alone, n = 3; 8D1-OVA + Th1 cells, n = 9; 8D1-OVA + Th17 cells, n = 11; day 3 8D1-OVA alone, n = 6; 8D1-OVA + Th1 cells, n = 6; 8D1-OVA + Th17 cells, n = 7. Statistical analysis was by ANOVA with post hoc analysis by Tukey's test (GraphPad Prism; Graphpad Software, San Diego, CA).
Glomerular abnormalities were assessed on Periodic acid-Schiff-stained, 3-μm-thick, paraffin-embedded sections on coded slides. The percentage of abnormal glomeruli was determined by examining a minimum of 50 glomeruli/mouse for abnormalities according to previously published protocols.27,28 Abnormalities included glomerular hypercellularity, crescent formation, fibrinoid necrosis, segmental proliferation, hyalinosis, and capillary wall thickening. Cellularity was quantified by counting total glomerular cell nuclei (minimum 20 glomeruli/mouse) expressed as c/gcs. Glomerular, lung, liver, and splenic IgG deposition was assessed on 6-μm-thick frozen sections using FITC-sheep anti-mouse Ig (1:100, Silenus, Hawthorn, Victoria, Australia). For albuminuria, 24-h urine collections were obtained on days 2 to 3, 7, 14, and 21 after injection of 8D1-OVA and/or cells. Albuminuria was measured using a Mouse Albumin ELISA Quantification Kit (Bethyl Laboratories Inc, Montgomery, TX). Glomerular leukocytes were enumerated by immunohistochemistry (minimum of 20 glomeruli/mouse),20 and interstitial leukocytes counted in a minimum of ten high-powered cortical fields. Urinary nitrate was measured using the Greiss assay, and measurement of CXCL1, CCL2, and CCL5 mRNA was by real-time PCR as described previously.20 Results for the 8D1-OVA alone group are presented as 1.
These studies were supported by a Program Grant from the National Health and Medical Research Council of Australia (NHMRC; A.R.K.), an NHMRC Postgraduate Research Scholarships (S.S.), and by grant DK080799 from the National Institutes of Health (D.B.B.).
Published online ahead of print. Publication date available at www.jasn.org.
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