Abstract
Increasing evidence suggested that Toll-like receptors (TLRs) were critically involved in immune responses of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The current study aimed to investigate the expression of TLR-2, TLR-4 and TLR-9 in kidneys of patients with ANCA-associated vasculitis. Renal biopsy specimens were collected from 24 patients with AAV. The expression of TLR-2, TLR-4 and TLR-9 in kidneys was detected by immunohistochemistry. Double immunofluorescence staining was performed to detect the expression of TLRs on various kinds of cells. In renal specimens, immunohistochemical examination revealed that expression of TLR-2 and TLR-4 could be detected in the glomeruli of AAV patients, while TLR-2 and TLR-4 were scarcely detected in the glomeruli of normal controls. Double immunofluorescence staining of TLR-2, TLR-4 and CD31 indicated that TLR-4 and TLR-2 were expressed on endothelial cells in the glomeruli. In the tubulointerstitial compartment, expression of TLR-2, TLR-4 and TLR-9 could be detected in both AAV patients and normal controls. The mean optical density of TLR-2 and TLR-4 in the tubulointerstitial compartment in AAV patients were significantly higher than that in normal controls. Among AAV patients, correlation analysis showed that the mean optical density of TLR-4 in the glomeruli correlated inversely with the initial serum creatinine, the proportion of total crescents and the proportion of cellular crescents in renal specimens (r = −0·419, P = 0·041; r = −0·506, P = 0·012; r = −0·505, P = 0·012, respectively). The expression of TLR-2 and TLR-4 was dysregulated in kidneys of AAV patients. The expression of TLR-4 in glomeruli was associated with the severity of renal injury.
Keywords: ANCA, Toll-like receptor, vasculitis
Introduction
Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) comprises a group of autoimmune disorders, including granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA), which are characterized by necrotizing inflammation of the small blood vessels [1]. ANCAs, the serological markers of AAV, are predominantly directed against neutrophil cytoplasmic constituents, in particular proteinase 3 (PR3) and myeloperoxidase (MPO) [2,3].
Although the aetiology of AAV is not yet fully clear, the capacity for prophylactic antibiotic therapy to prevent relapses [4,5] has suggested a close link between infection and AAV. Some studies showed the onset of AAV to vary by season, with the incidence peaking in winter [6,7], supporting an underlying infectious factor, although other investigations failed to observe any significant seasonal variation [8,9]. Toll-like receptors (TLRs), which are crucial in the innate immune response to invading pathogens through the recognition of conserved pathogen-associated molecular patterns [10], play an important role as a first defence mechanism linking the innate with the adaptive immune system. There is increasing evidence that TLR activation could exacerbate autoimmunity in renal diseases and vasculitis [11,12]. TLRs comprise a family of at least 11 known members in humans [12]. Of the several identified TLRs, TLR-2, TLR-4 and TLR-9 were proved to be critically involved in immune responses of ANCA-associated vasculitis. In animal studies, it has been demonstrated that lipopolysaccharide (LPS) aggravates anti-MPO antibody-induced necrotizing glomerulonephritis in a TLR-4-dependent manner [13,14]. TLR-2 and TLR-9 ligands can induce the development of anti-MPO autoimmunity by directing T helper type 1 (Th1) autoimmunity and Th17 autoimmunity, respectively [15]. In-vitro studies revealed that the TLR-9 ligand could induce ANCA production by peripheral blood mononuclear cells from AAV patients [16,17]. A recent study showed that the levels of TLR expression on peripheral leucocytes of patients with AAV were dysregulated [18]. However, until now, the role of TLRs in the progression of lesions associated with AAV remains largely unknown. In addition, TLRs are not confined to cells of the immune system, but are also expressed by resident cells of various tissues, including the kidneys [19]. Local expression of TLRs at sites of inflammation, such as the kidneys, remains to be investigated in AAV. The aim of this study was to investigate the expression of TLR-2, TLR-4 and TLR-9 in kidneys of patients with ANCA-associated vasculitis.
Materials and methods
Patients and samples
Renal biopsy specimens from 24 patients with AAV, diagnosed at Peking University First Hospital from January 2007 to April 2011, were collected randomly in this study. All the patients had a positive test for perinuclear ANCA (P-ANCA) by indirect immunofluorescence and MPO-ANCA by antigen-specific enzyme-linked immunosorbent assay (ELISA). All the patients met the Chapel Hill Consensus Conference (CHCC) definition of AAV [1] and had complete clinical and pathological data. Patients with secondary vasculitis or co-existence of other renal disease were excluded. Urinary tract infection was excluded according to urinalysis as well as patients' signs and symptoms. Other types of infection were also excluded. Six patients with biopsy-proven lupus nephritis (LN), diagnosed in the same period in our centre, were enrolled as the disease control. All the patients with lupus nephritis fulfilled the 1997 American College of Rheumatology revised criteria for systemic lupus erythematosus (SLE) [20]. Six renal tissues were obtained from the normal part of nephrectomized (because of renal carcinoma) kidneys and were used as normal controls; they were considered normal using light microscopy, immunofluorescence and electron microscopy.
The research was in compliance with the Declaration of Helsinki and approved by the ethics committee of our hospital. Written informed consent was obtained from each participant.
Renal histology
Renal histology of patients with AAV was evaluated according to the previous standardized protocol [21–23]. The presence of glomerular lesions, including fibrinoid necrosis, crescents and glomerulosclerosis, were calculated as the percentage of the total number of glomeruli in a biopsy. Interstitial and tubular lesions were scored semiquantitatively on the basis of the percentage of the tubulointerstitial compartment that was affected: interstitial infiltrate (– for 0%, + for 0–20%, ++ for 20–50% and +++ for > 50%), interstitial fibrosis (– for 0%, + for 0–50% and ++ for > 50%) and tubular atrophy (– for 0%, + for 0–50% and ++ for > 50%).
Detection of TLR expression in kidneys by immunohistochemistry
Immunohistochemical staining was performed for TLR-2, TLR-4 and TLR-9 on 4 μm deparaffinized sections of formaldehyde-fixed renal tissue using rabbit anti-human TLR-2 polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc10739), mouse anti-human TLR-4 monoclonal antibodies (Abcam, Cambridge, UK; ab22048) and mouse anti-human TLR-9 monoclonal antibodies (Abcam; ab12121) as primary antibodies. Antibodies against TLR-2, -4 and -9 were diluted to 20 μg/ml, 5 μg/ml and 5 μg/ml, respectively, in 0·01 mol/l phosphate-buffered saline (PBS), pH 7·4. After deparaffinization in xylene-ethanol at room temperature and rehydration in PBS, sections were immersed in freshly prepared 3% hydrogen peroxide for 20 min at room temperature to quench endogenous peroxidase activity. An antigen retrieval technique was then performed by heating the slides in citrate buffer (0·01 M, pH 6·0) in an 800 W microwave oven for 2 min and then at 200 W for 9 min. Following antigen retrieval the slides were cooled to room temperature, washed, and the sections were incubated with 3% bovine serum albumin (BSA) in PBS at room temperature for 30 min to block non-specific staining. Primary antibodies were added to each section after the removal of blocking BSA without washing. Primary antibodies were incubated for 16 h at 4°C. Secondary antibodies from the detection system, Dako EnVision horseradish peroxidase (HRP) (Dako A/S, Copenhagen, Denmark), were incubated for 30 min at 37°C. Next, sections were developed in fresh hydrogen peroxide plus 3-3′-diaminobenzidine tetrahydrochloride solution for 1 min, respectively. As negative controls, primary antibodies were replaced by normal rabbit immunoglobulin (Ig)G or normal mouse IgG. Finally, the sections were incubated with haematoxylin and dehydrated through alcohol and xylene. The sections were examined by light microscopy. Lymph node slides were used as positive controls.
Renal staining of TLR-2, TLR-4 and TLR-9 was evaluated by the Image Pro Plus analysis software version 6·0. Positive signals were quantified as the mean optical density (integrated option density/area). All the glomeruli in a section at ×400 and at least 15 fields of tubulointerstitial vision per kidney section at ×200 were observed blindly as a semiquantitive assessment of immunohistochemical staining.
Detection of TLR expression on various cell types
In order to determine which cell types displayed increased TLR expression, we performed double immunofluorescence of TLRs and specific markers of various cell types, including endothelial and infiltrating cells. Endothelial cells were identified by immunofluorenscence staining with primary antibodies against CD31. Infiltrating cells were identified by immunofluorenscence staining with antibodies against CD3 (T lymphocytes), CD20 (B lymphocytes), CD68 (monocytes/macrophages) and neutrophil elastase (neutrophils). The primary antibodies used are listed in Supporting information, Table S1.
Renal specimens of AAV patients were embedded on optimum cutting temperature (OCT) compound (Miles Laboratories, Elkhart, IN, USA) and frozen in an acetone–dry ice mixture. The frozen sections were cut into 2–3 μm sections on a cryostat and stored at −80°C until use. Non-specific binding sites were blocked in PBS containing 5% donkey serum for 1 h at room temperature. For double staining, we incubated the specimens with the first primary antibody overnight at 4°C. After washing with PBS, AF488-labelled donkey anti-rabbit IgG (1:500, Jackson ImmunoResearch, West Grove, PA, USA) was applied for 1 h. Sections were then incubated with the second primary antibody overnight at 4°C. After washing with PBS, cyanin 3 (Cy3)-labelled donkey anti-mouse IgG (1:500, Jackson ImmunoResearch) was applied for 1 h. The samples were washed with PBS, stained with 4′,6-diamidino-2-phenylindole (DAPI) (Zhongshan Golden Bridge Biotechnology, Beijing, China) and mounted with Mowiol. In negative controls, primary antibodies were replaced by PBS. Confocal images were captured with a Zeiss LSM 710 confocal microscope (Zeiss, Jena, Germany). Images were exported from the ZEN 2012 (blue edition) microscopy software.
Statistical analysis
For normally distributed data, differences of quantitative parameters between groups were assessed using the t-test, and descriptive statistics for these data are presented as mean ± standard deviation. For non-normally distributed data, differences of quantitative parameters between groups were assessed using the non-parametric test, and descriptive statistics for these data are presented as median and interquartile range (IQR). The association between two continuous variables was analysed using Pearson's correlation (for two parametric variables) or Spearman's rank correlation (for two non-parametric variables or one non-parametric variable with one parametric variable). A P-value of 0·05 or less was considered statistically significant. Analysis was performed with spss version 13·0 (SPSS Inc., Chicago, IL, USA).
Results
Demographic and general data
Among the 24 patients with AAV in active stage, 10 were male and 14 were female. According to the Chapel Hill Consensus Conference definition [1], nine of 24 patients were classified as GPA, while the other 15 patients were classified as MPA. The level of Birmingham Vasculitis Activity Scores (BVAS) [24] was 21·1 ± 6·4 (range 11–36). The clinical and histopathological data are listed in Table 1.
Table 1.
Clinical and histopathological data of 24 patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis
| Parameters | Number |
|---|---|
| Number | 24 |
| Male/female | 10/14 |
| Average age at onset of the disease (years) | 58·8 ± 11·8 |
| Scr (μmol/l) | |
| Mean ± s.d. | 380·3 ± 275·2 |
| Range | 78–1007 |
| ESR (mm/1 h) | 67·0 ± 38·7 |
| MPO-ANCA level (RU/ml) | 152·1 ± 47·7 |
| Renal insufficiency at diagnosis | 17 (70·8%) |
| Urinary protein (g/24 h) | |
| Mean ± s.d. | 2·3 ± 1·7 |
| Range | 0–7·4 |
| Skin rash | 1 (4·2%) |
| Arthralgia | 8 (33·3%) |
| Muscle pain | 5 (20·8%) |
| Pulmonary | 18 (66·7%) |
| ENT | 9 (37·5%) |
| Ophthalmic involvement | 6 (25·0%) |
| Gastrointestinal involvement | 2 (8·3%) |
| Nervous system | 2 (8·3%) |
| BVAS | 21·1 ± 6·4 |
| Average glomeruli per biopsy | 27·0 ± 9·5 |
| Glomerular lesions (%) | |
| Total crescents | 55·0 ± 32·8 |
| Cellular crescents | 42·3 ± 31·4 |
| Fibrous crescents (median, IQR) | 6·6 (0–19·3) |
| Tubulointerstitial lesions | |
| Interstitial infiltration (–/+/++/+++) | 0/4/11/9 |
| Interstitial fibrosis (–/+/++) | 2/7/15 |
| Tubular atrophy (–/+/++) | 0/9/15 |
ANCA = anti-neutrophil cytoplasmic antibody; BVAS = Birmingham Vasculitis Activity Scores; ENT = ear, nose and throat; ESR = erythrocyte sedimentation rate; IQR = interquartile range; s.d. = standard deviation.
Immunohistochemistry for TLR-2
In renal specimens of patients with AAV, immunohistochemical examination revealed that expression of TLR-2 was detected in the glomeruli, while TLR-2 was scanty in the glomeruli of normal controls (Fig. 1a,d). TLR-2 positive staining was also seen in proximal tubules, distal tubules and collecting ducts of the patients with AAV and normal controls (Fig. 2a,d). The mean optical density of TLR-2 in the tubulointerstitial compartment in patients with AAV was significantly higher than that in normal controls [0·027 (0·014–0·08) versus 0·002 (0–0·006), P < 0·001]. Patients with lupus nephritis exhibited weak expression of TLR-2 in glomeruli and diffuse expression in renal tubules and interstitium (Supporting information, Fig. S1).
Figure 1.
Immunohistochemistry staining for Toll-like receptor (TLR)-2, TLR-4 and TLR-9 in glomeruli of renal specimens. (a) Immunohistochemical staining of TLR-2 in glomerulus of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis. (b) Immunohistochemical staining of TLR-4 in glomerulus of ANCA-associated vasculitis. (c) Immunohistochemical staining of TLR-9 in glomerulus of ANCA-associated vasculitis. (d) Immunohistochemical staining of TLR-2 in glomerulus of a normal control. (e) Immunohistochemical staining of TLR-4 in glomerulus of a normal control. (F) Immunohistochemical staining of TLR-9 in glomerulus of a normal control.
Figure 2.
Immunohistochemistry staining for Toll-like receptor (TLR)-2, TLR-4 and TLR-9 in the tubulointerstitial compartment of renal specimens. (a) Immunohistochemical staining of TLR-2 in the tubulointerstitial compartment of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis. (b) Immunohistochemical staining of TLR-4 in the tubulointerstitial compartment of ANCA-associated vasculitis. (c) Immunohistochemical staining of TLR-9 in the tubulointerstitial compartment of ANCA-associated vasculitis. (d) Immunohistochemical staining of TLR-2 in the tubulointerstitial compartment of a normal control. (e) Immunohistochemical staining of TLR-4 in the tubulointerstitial compartment of a normal control. (f) Immunohistochemical staining of TLR-9 in the tubulointerstitial compartment of a normal control.
Among the patients with AAV, correlation analysis showed that the mean optical density of TLR-2 in the glomeruli was correlated inversely with the initial serum creatinine (r = −0·429, P = 0·037).
Immunohistochemistry for TLR-4
In renal specimens, immunohistochemical examination revealed high deposition of TLR-4 in the glomeruli and peritubular capillaries of patients with AAV, while TLR-4 was scarcely detected in the glomeruli and peritubular capillaries of normal controls (Fig. 1b,e). TLR-4 expression was also detected in the tubules of both normal controls and AAV patients (Fig. 2b,e). The mean optical density of TLR-4 in the tubulointerstitial compartment in patients with AAV was significantly higher than that in normal controls [0·051 (0·038–0·076) versus 0·005 (0·002–0·010), P < 0·001]. Patients with lupus nephritis exhibited weak expression of TLR-4 in glomeruli and diffuse expression in renal tubules and interstitium (Supporting information, Fig. S2).
Among the patients with AAV, correlation analysis showed that the mean optical density of TLR-4 in the glomeruli correlated inversely with the initial serum creatinine, the proportion of total crescents and the proportion of cellular crescents in renal specimens (r = −0·419, P = 0·041; r = −0·506, P = 0·012; r = −0·505, P = 0·012, respectively (Fig. 3a–c).
Figure 3.
Association between the mean optical density of Toll-like receptor (TLR)-4 in the glomeruli and clinicopathological parameters of patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis. (a) Association between the mean optical density of TLR-4 in the glomeruli and the serum creatinine. (b) Association between the mean optical density of TLR-4 in the glomeruli and the proportion of total crescents in renal specimens. (c) Association between the mean optical density of TLR-4 in the glomeruli and the proportion of cellular crescents in renal specimens.
Immunohistochemistry for TLR-9
In renal specimens, immunohistochemical examination revealed that expression of TLR-9 was scanty in glomeruli of patients with AAV and normal controls (Fig. 1c,f). Expression of TLR-9 was detected in proximal tubules, distal tubules and collecting ducts of patients with AAV and normal controls (Fig. 2c,f). There was no significant difference in the mean optical density of TLR-9 in the tubulointerstitial compartment in patients with AAV and that in normal controls. Patients with lupus nephritis exhibited weak expression of TLR-9 in glomeruli and diffuse expression in renal tubules and interstitium (Supporting information, Fig. S3).
Co-localization of TLRs and specific markers of various cell types
In the renal specimens of AAV patients, immunohistochemical assay revealed that expression of TLR-2 and TLR-4 was mainly in the glomeruli, while expression of TLR-9 was scanty in the glomeruli. We performed double staining to further determine which types of cells displayed increased TLR expression. We found that TLR-2 and TLR-4 could co-localize with CD31 in the glomeruli (Fig. 4), TLR-2 could co-localize with CD68 (Supporting information, Fig. S4) and TLR-4 and TLR-9 could co-localize with neutrophil elastase (Supporting information, Fig. S5). There were 2·56 ± 0·14 neutrophils per glomeruli in the renal specimens of AAV patients. Collectively, we determined that TLR-4 and TLR-2 were expressed mainly on endothelial cells in the glomeruli, TLR-4 and TLR-9 were expressed mainly on neutrophils, while TLR-2 were expressed on monocytes.
Figure 4.
Double immunofluorescence staining of Toll-like receptor (TLR)-2, TLR-4 and CD31 in glomeruli in renal specimens of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) patients. (a) Co-localization of TLR-2 (green) and CD31 (red) in glomerulus of AAV patients. (B) Co-localization of TLR-4 (green) and CD31 (red) in glomerulus of AAV patients. The specific co-staining for TLR-4, TLR-2 and CD31 was apparent in the merged picture as yellow/orange in glomeruli.
Discussion
TLRs play a crucial role in the activation and regulation of innate and adaptive immune responses through recognition of pathogen-associated molecular patterns and endogenous peptides [25,26]. They have been involved in the initiation of autoreactivity and amplification of tissue injury in autoimmune diseases, such as ANCA-associated vasculitis [15,27,28]. Among the several known TLRs, TLR-2, TLR-4 and TLR-9 were found to be associated closely with the development of AAV [13–15]. However, the role of TLRs in tissue injury of AAV is not yet clear. Data on TLR expression in ANCA-associated glomerulonephritis are lacking. Therefore, the current study aimed to characterize the expression of TLR-2, TLR-4 and TLR-9 in kidneys of patients with AAV.
In the present study, we observed remarkable expression of TLR-4 in glomeruli and peritubular capillaries in renal specimens from AAV patients, while patients with lupus nephritis exhibited weak expression of TLR-4 in glomeruli. Double immunofluorescence staining of TLR-4 and CD31 indicated that TLR-4 was expressed mainly on endothelial cells in the glomeruli of AAV. These results were consistent with the anti-MPO antibody-induced glomerulonephritis animal model by Summers et al. [14], which indicated an increase in TLR-4 expression in glomerular endothelial cells in mice. Co-localization of TLRs with neutrophils and monocytes was consistent with the studies by Tadema et al. [18] and Holle et al. [29]. Because all the patients recruited in our study were MPO-ANCA-positive, even in GPA patients [30,31], selection bias could not be excluded. Therefore, we further stained TLRs in renal specimens of two patients with PR3-ANCA-positive GPA. The expression of TLR-2, TLR-4 and TLR-9 in PR3-ANCA-positive GPA patients was very similar to that in MPO-ANCA-positive patients (Supporting information, Fig. S6).
Accumulating evidence suggests that damage-associated molecular patterns induce signalling via TLRs, resulting in the release of proinflammatory cytokines and chemokines with subsequent recruitment of inflammatory cells [32], and TLR-4 in glomerular endothelial cells was found to contribute to neutrophil recruitment in anti-MPO antibody-induced glomerulonephritis in mice [14]. Interestingly, in the present study, correlation analysis showed that the mean optical density of TLR-4 in the glomeruli correlated inversely with the initial serum creatinine, the proportion of total crescents and the proportion of cellular crescents in renal specimens of patients with AAV, which indicated an association between the expression of TLR-4 and the severity of renal injury. In a renal ischaemia–reperfusion injury model, TLR-4 was found to express on different cells at different times. At the early stage of reperfusion TLR-4 was expressed on endothelial cells, which contributed to early injury by regulating adhesion molecule expression and thus inflammation; at the later stage, it was expressed on tubular cells [33]. We speculated that the glomerular expression of TLR-4 in AAV was involved in endothelial activation in the early stage of the disease course. However, whether or not TLR-4 expression is consistent with the stage of disease needs further study.
To the best of our knowledge, this is the first study to characterize TLR expression in kidneys of patients with AAV. Increased expression of TLR-2 and TLR-4 in the glomeruli and tubulointerstitial compartment was observed, which indicated that local expression of TLRs in kidneys might be involved in the development of AAV. Summers et al. suggested that the TLR-2 ligand could initiate the development of anti-MPO autoimmunity by directing Th1 autoimmunity [15]. The role of TLR-2 on immune cells is well defined [34,35], but the precise role of TLR-2 in renal cells in AAV patients remains less defined. The dysregulation of the expression level of TLR-2 warrants further investigation of its function in AAV.
In conclusion, the expression of TLR-2 and TLR-4 was dysregulated in kidneys of patients with AAV. The expression of TLR-4 in glomeruli was associated with severity of renal injury of patients with AAV.
Acknowledgments
This study is supported by a grant from Chinese 973 project (no. 2012CB517700), two grants from the National Natural Science Fund (no. 81370829 and no. 81321064), and the ‘National Key Technology Research and Development (R&D) Program’ of the Ministry of Science and Technology of China (no. 2011BAI10B04).
Disclosure
All the authors declare no competing interests.
Author contributions
H. W. and S.-J. G. conducted the patient recruitment, collected samples and performed the experiment, analysed the data and wrote the manuscript. M. C. and M.-H. Z. designed and directed the study. M. C. is responsible for the interpretation of the data.
Supporting information
Additional Supporting information may be found in the online version of this article at the publisher's web-site:
Fig. S1. Immunohistochemistry staining for Toll-like receptor (TLR)-2 in renal specimens of lupus nephritis (LN) patients. (a) Immunohistochemical staining of TLR-2 in the tubulointerstitial compartment of renal specimens of LN patients. (b) Immunohistochemistry staining for TLR-2 in glomeruli of renal specimens of LN patients.
Fig. S2. Immunohistochemistry staining for Toll-like receptor (TLR)-4 in renal specimens of lupus nephritis (LN) patients. (a) Immunohistochemical staining of TLR-4 in the tubulointerstitial compartment of renal specimens of LN patients. (b) Immunohistochemistry staining for TLR-4 in glomeruli of renal specimens of LN patients.
Fig. S3. Immunohistochemistry staining for Toll-like receptor (TLR)-9 in renal specimens of patients with lupus nephritis (LN). (a) Immunohistochemical staining of TLR-9 in the tubulointerstitial compartment of renal specimens of LN patients. (b) Immunohistochemistry staining for TLR-9 in glomeruli of renal specimens of LN patients.
Fig. S4. Co-localization of Toll-like receptor (TLR)-2 and CD68 in renal specimens of AAV patients.
Fig. S5. Co-localization of Toll-like receptor (TLR)-4 as well as TLR-9 and elastase in renal specimens of AAV patients. (a) Co-localization of TLR-4 (red) and elastase (green). (b) Co-localization of TLR-9 (red) and elastase (green).
Fig. S6. Immunohistochemistry staining for Toll-like receptor (TLR)-2, TLR-4 and TLR-9 in renal specimens of proteinase 3 (PR3)-anti-neutrophil cytoplasmic antibody (ANCA)-positive granulomatosis with polyangiitis (GPA). (a) Immunohistochemical staining of TLR-2 in glomerulus of PR3-ANCA-positive GPA (b). Immunohistochemical staining of TLR-4 in glomerulus of PR3-ANCA-positive GPA. (c) Immunohistochemical staining of TLR-9 in glomerulus of PR3-ANCA-positive GPA. (d) Immunohistochemical staining of TLR-2 in the tubulointerstitial compartment of PR3-ANCA-positive GPA. (e) Immunohistochemical staining of TLR-4 in the tubulointerstitial compartment of PR3-ANCA-positive GPA. (f) Immunohistochemical staining of TLR-9 in the tubulointerstitial compartment of PR3-ANCA-positive GPA.
Table S1. Primary antibodies used for double immunofluorescence.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Fig. S1. Immunohistochemistry staining for Toll-like receptor (TLR)-2 in renal specimens of lupus nephritis (LN) patients. (a) Immunohistochemical staining of TLR-2 in the tubulointerstitial compartment of renal specimens of LN patients. (b) Immunohistochemistry staining for TLR-2 in glomeruli of renal specimens of LN patients.
Fig. S2. Immunohistochemistry staining for Toll-like receptor (TLR)-4 in renal specimens of lupus nephritis (LN) patients. (a) Immunohistochemical staining of TLR-4 in the tubulointerstitial compartment of renal specimens of LN patients. (b) Immunohistochemistry staining for TLR-4 in glomeruli of renal specimens of LN patients.
Fig. S3. Immunohistochemistry staining for Toll-like receptor (TLR)-9 in renal specimens of patients with lupus nephritis (LN). (a) Immunohistochemical staining of TLR-9 in the tubulointerstitial compartment of renal specimens of LN patients. (b) Immunohistochemistry staining for TLR-9 in glomeruli of renal specimens of LN patients.
Fig. S4. Co-localization of Toll-like receptor (TLR)-2 and CD68 in renal specimens of AAV patients.
Fig. S5. Co-localization of Toll-like receptor (TLR)-4 as well as TLR-9 and elastase in renal specimens of AAV patients. (a) Co-localization of TLR-4 (red) and elastase (green). (b) Co-localization of TLR-9 (red) and elastase (green).
Fig. S6. Immunohistochemistry staining for Toll-like receptor (TLR)-2, TLR-4 and TLR-9 in renal specimens of proteinase 3 (PR3)-anti-neutrophil cytoplasmic antibody (ANCA)-positive granulomatosis with polyangiitis (GPA). (a) Immunohistochemical staining of TLR-2 in glomerulus of PR3-ANCA-positive GPA (b). Immunohistochemical staining of TLR-4 in glomerulus of PR3-ANCA-positive GPA. (c) Immunohistochemical staining of TLR-9 in glomerulus of PR3-ANCA-positive GPA. (d) Immunohistochemical staining of TLR-2 in the tubulointerstitial compartment of PR3-ANCA-positive GPA. (e) Immunohistochemical staining of TLR-4 in the tubulointerstitial compartment of PR3-ANCA-positive GPA. (f) Immunohistochemical staining of TLR-9 in the tubulointerstitial compartment of PR3-ANCA-positive GPA.
Table S1. Primary antibodies used for double immunofluorescence.