
Antimyeloperoxidase antibodies can cause crescentic glomerulonephritis and pulmonary hemorrhage. Toll-like receptors (TLRs) respond to infectious agents activating host defenses, whereas infections potentially initiate disease and provoke relapses. Neutrophils were found to be key effector cells of injury in experimental models, as disease does not occur in their absence and injury is enhanced by lipopolysaccharide (LPS). In this study, highly purified LPS (a pure TLR4 ligand) acted with antimyeloperoxidase antibodies to synergistically increase kidney and lung neutrophil recruitment and functional injury; effects abrogated in TLR4-deficient mice. Increased kidney TLR4 expression after stimulation predominantly occurred in glomerular endothelial cells. Enhanced glomerular neutrophil recruitment correlated with increased kidney mRNA expression of CXCL1 and CXCL2 (homologs of human CXCL8), whereas their preemptive neutralization decreased neutrophil recruitment. Disease induction in bone marrow chimeric mice showed that TLR4 in both bone marrow and renal parenchymal cells is required for maximal neutrophil recruitment and glomerular injury. Further studies in human glomerular cell lines stimulated with LPS found that glomerular endothelial cells were the prominent sources of CXCL8. Thus, our results define a role for TLR4 expression in bone marrow-derived and glomerular endothelial cells in neutrophil recruitment and subsequent functional and histological renal injury in experimental antimyeloperoxidase glomerulonephritis. Small vessel vasculitis and pauci-immune necrotizing glomerulonephritis (GN) induced by antineutrophil cytoplasmic antibodies (ANCAs) target specific neutrophil cytoplasmic antigens, myeloperoxidase (MPO), and proteinase 3.

1,2 Combined renal and pulmonary disease is common in ANCA vasculitis and has considerable morbidity and mortality. Experimental animal studies have shown that ANCA are pathogenic. Passive transfer of ANCA can induce necrotizing GN and/or pulmonary capillaritis. [3] [4] [5] [6] [7] Links between infection and ANCA vasculitis are well established. Seasonal variation in patients presenting with the disease suggests a correlation with microbial infection, 8 infection may predate disease initiation and/or relapse [9] [10] [11] [12] and prophylactic antibiotic therapy has also been shown to successfully decrease disease relapses in ANCA vasculitis. 13 Infection is likely to be important in experimental ANCA models and lipopolysaccharide (LPS) dose dependently increases renal injury after the passive transfer of MPO-ANCA. 6 Neutrophils are amongst the first immune cells to traffic to inflamed sites. In experimental ANCA-induced GN neutrophils are the primary effector cells and neutrophil depletion protects mice from renal injury. 4 In humans, the chemokine CXCL8 (interleukin-8) is a potent neutrophil chemoattractant.

14 Renal biopsies from patients with ANCA disease demonstrated positive CXCL8 immunostaining in crescentic glomerular lesions, suggesting that CXCL8 contributes to glomerular injury seen in ANCA-associated GN. 15 The murine chemokines CXCL1 (KC) and CXCL2 (MIP-2), that bind to CXCR2, are homologs of human CXCL8 16 and serve as major chemoattractants for neutrophils in mice. 17 Toll-like receptors (TLRs) recognize pathogen associated molecular patterns from infectious agents and after ligation activate immune cells. TLR4 is expressed on neutrophils Starting from the known capacity of LPS to enhance the activity of anti-MPO antibodies in experimental systems, 6, 22 we aimed to define and explore a pathogenic and mechanistic role for TLR4 in experimental ANCA-induced glomerular neutrophil recruitment. We studied the effect of LPS and anti-MPO antibodies on glomerular and pulmonary neutrophil recruitment, which develops early in the diseases process and is TLR4 dependent. Functional and histological renal injury, which develops later, is also TLR4 dependent. TLR4 expression in the glomerulus increases after LPS and anti-MPO antibody stimulation. TLR4 is produced by GEnCs, which are also positive for CXCL1 and CXCL2. In vitro LPS stimulation of human glomerular cell lines implicates GEnC as a major source of TLR4. We demonstrate a functional relationship between CXCL1 and CXCL2, glomerular neutrophil recruitment and subsequent renal injury, and demonstrate that full expression of these chemokines is TLR4 dependant. Furthermore, we demonstrate in human cell lines that CXCL8 mRNA and protein production increases considerably after LPS stimulation, predominantly mediated through GEnC. Finally using bone marrow (BM) chimeric mice we identify the individual contributions of BM and tissue cell (TC) TLR4 to neutrophil recruitment and glomerular injury.
RESULTS

ANCA/LPS-induced glomerular neutrophil recruitment and lung MPO activity
Glomerular and pulmonary neutrophil recruitment was induced by administering either highly purified LPS (hpLPS, which specifically engages TLR4) with anti-MPO antibodies, anti-MPO antibodies alone, or hpLPS and control (antiovalbumin (OVA)) antibodies to genetically intact wild-type (WT) C57BL/6 mice. WT mice were injected with hpLPS, then anti-MPO or anti-OVA antibodies at 2 h and killed after a further 3 h. LPS, anti-MPO antibodies, or both induced glomerular leukocyte recruitment (Figure 1a ). Control anti-OVA antibodies alone had no effect on glomerular neutrophil recruitment compared with untreated mice (untreated mice 0.25 ± 0.01 neutrophils/glomerular cross-section (n/gcs), mice given anti-OVA antibodies 0.40±0.02 n/gcs). Mice injected with anti-MPO antibodies alone exhibited a significant glomerular neutrophil influx, similar to that observed in mice treated with hpLPS and anti-OVA antibodies. Together, hpLPS and anti-MPO antibodies synergistically increased glomerular neutrophil recruitment. Representative photomicrographs of glomerular neutrophil recruitment are shown (Figure 1c-e) . In the same studies, neutrophil accumulation in lung tissue was assessed by measuring pulmonary MPO activity (Figure 1b ). Untreated WT mice had 0.62±0.03 U of MPO activity per gram of lung tissue. Administration of either hpLPS and anti-OVA antibodies, or anti-MPO antibodies alone increased MPO activity to a similar degree. Administration of hpLPS and anti-MPO antibodies led to a further increase in lung MPO activity. Anti-OVA antibodies alone had minimal effect (1.04 ± 0.29 U/g).
ANCA/LPS-induced glomerular neutrophil recruitment and lung MPO activity
The requirement for TLR4 in maximal neutrophil recruitment was confirmed by comparing TLR4 À/À mice with WT mice (Figure 1f and g ). Glomerular neutrophil recruitment in untreated TLR4
ANCA/LPS-induced glomerular neutrophil recruitment and lung MPO activity
À/À mice (0.23 ± 0.06n/gcs) was similar to untreated WT controls. Neutrophil recruitment in TLR4 À/À mice given hpLPS (with anti-OVA antibodies) was similar to untreated TLR4 À/À mice. TLR4 À/À mice given anti-MPO antibodies had similar glomerular neutrophil numbers to WT mice given anti-MPO antibodies, but TLR4 À/À mice given hpLPS and anti-MPO antibodies recruited fewer neutrophils to glomeruli compared with WT given hpLPS and anti-MPO antibodies. Similar TLR4-dependent patterns were present in the pulmonary leukocyte recruitment, although in the absence of TLR4 (baseline activity in untreated TLR4 À/À mice 0.61 ± 0.07 U/g), pulmonary MPO activity was reduced in all three groups of mice: those given hpLPS and anti-OVA antibodies, hpLPS and anti-MPO antibodies, as well as those injected with anti-MPO antibodies alone.
ANCA/LPS-induced glomerular neutrophil recruitment and lung MPO activity
TLR4 is expressed in murine glomeruli and produced by murine GEnCs and human intrinsic glomerular cells
ANCA/LPS-induced glomerular neutrophil recruitment and lung MPO activity
We then examined TLR4 production in murine kidneys and human glomerular cells. TLR4 protein was readily detected in glomeruli of WT mice treated with LPS and anti-MPO antibodies. TLR4
ANCA/LPS-induced glomerular neutrophil recruitment and lung MPO activity
À/À mice treated with anti-MPO antibodies were a negative control. Using confocal microscopy TLR4 was colocalized with GEnCs. Other glomerular cell types, probably podocytes and possibly mesangial cells also expressed TLR4. Illustrative photomicrographs are shown in Figure 2a -d. TLR4 mRNA expression from glomerular and tubular interstitial compartments was assessed using laser capture microdissection ( Figure 2e ; the mean value for the tubulointerstitium was assigned a value of 1). Baseline TLR4 mRNA expression in glomeruli was 20-fold higher than the tubulointerstitium and increased further 24 h after LPS and anti-MPO antibodies. There was no change in tubulointerstitial TLR4 expression. We then analyzed TLR4 mRNA expression in human conditionally immortalized GEnC (ciGEnC), podocytes, and mesangial cell lines after stimulation with LPS ( Figure 2f ; the mean value for podocyte basal TLR4 mRNA expression was assigned a value of 1). Basal TLR4 mRNA expression was highest in ciGEnC and not detected in mesangial cells. Flow cytometric analysis of ciGEnC for TLR4 protein confirmed the expression of TLR4 protein (Figure 2g ). After LPS, TLR4 mRNA expression was increased at 24 h in ciGEnC, but podocyte and mesangial cell TLR4 mRNA expression was unchanged.
ANCA/LPS-induced glomerular neutrophil recruitment and lung MPO activity
CXCL1 and CXCL2 is induced in kidney tissue after hpLPS and anti-MPO antibodies
ANCA/LPS-induced glomerular neutrophil recruitment and lung MPO activity
To investigate mechanisms of glomerular neutrophil recruitment we studied the neutrophil chemoattractants CXCL1 and CXCL2 in renal tissue. CXCL1 and CXCL2 expression was assessed 5 h after both hpLPS and anti-MPO antibodies. Compared with untreated WT mice (means for untreated WT CXCL1 or CXCL2 mRNA expression were assigned a value of 1), CXCL1 mRNA expression increased 5 h after hpLPS and anti-MPO antibodies, in a partly TLR4-dependent manner (Figure 3a) . A similar pattern was seen with CXCL2 mRNA expression, but the reduction in gene expression in the absence of TLR4 was more profound (Figure 3b ). Immunohistochemical examination of glomeruli for CXCL1 and CXCL2 demonstrated minimal signal in untreated WT mice, with increased expression in all experimental groups (data not shown). WT mice given hpLPS and anti-MPO antibodies exhibited increased glomerular (with surrounding tubular) staining of CXCL1 and CXCL2 when compared with mice given either hpLPS with anti-OVA antibodies, or anti-MPO antibodies alone. As hypothesized, compared with WT mice, CXCL1 and CXCL2 staining was CXCL1 and CXCL2 colocalize with GEnCs, which also express TLR4
ANCA/LPS-induced glomerular neutrophil recruitment and lung MPO activity
To determine whether murine GEnCs are a source of CXCL1 and CXCL2 production, we immunostained kidneys from WT mice treated with LPS and anti-MPO antibodies for an endothelial marker (CD31), TLR4, CXCL1, and CXCL2 using confocal microscopy CXCL1 colocalized to GEnC and to 
Neutrophil recruitment is CXCL1 and CXCL2 dependent
Given the enhanced expression of CXCL1 and CXCL2 in experimental anti-MPO antibody-induced glomerular neutrophil recruitment, we neutralized either protein by administering a monoclonal anti-CXCL1 antibody, an anti-CXCL2 antibody, or isotype control 1 h before hpLPS and anti-MPO antibodies and assessed neutrophil recruitment. Compared with isotype control antibody, mice given either anti-CXCL1 or anti-CXCL2 antibody before hpLPS and anti-MPO antibodies showed significant decreases in glomerular neutrophil recruitment and lung MPO activity (Figure 5a-b) , confirming a functional role for both CXCL1 and CXCL2 in glomerular and pulmonary neutrophil recruitment.
TC TLR4 contributes to neutrophil recruitment
Given the expression of TLR4 in glomeruli, we defined the contributions of BM and TC TLR4 (glomerular and lung) expression in neutrophil recruitment by injecting LPS and anti-MPO antibodies into TLR4 BM chimeric mice. Chimeric mice were generated by injecting intact or deficient . Glomeruli from WT mice were stained with anti-CD31 antibodies to identify endothelial cells (red staining) (c). Merged image of TLR4 and endothelial cell staining that identifies endothelial cell TLR4 production (yellow) (d). After stimulation with LPS and anti-MPO (aMPO) antibodies there was an increase in TLR4 mRNA expression in microdissected murine glomeruli, and little change in TLR4 expression was seen in the tubulointerstitium (n ¼ 4) (e). In human conditionally immortalized ciGEnC lines, baseline TLR4 expression was increased compared with both podocytes and mesangial cells (which did not express TLR4), n ¼ 6 for all experimental groups (f). After LPS stimulation TLR4 expression increased only in endothelial cells. (g) TLR4 protein expression by ciGEnC. Flow cytometric analysis of cultured ciGEnC incubated with no antibody (i), isotype control antibody (ii), and anti-human TLR4 antibody (iii) demonstrated TLR4 protein expression. *Po0.05, **Po0.01, ***Po0.001. Original magnification x800. Ab, antibody; ciGEnC, conditionally immortalized glomerular endothelial cell, LPS, lipopolysaccharide; MFI, mean florescence intensity; MPO, myeloperoxidase; TLR, Toll-like receptor; WT, wild type.
TC TLR4 contributes to neutrophil recruitment
BM into irradiated mice. WT BM transplanted into WT mice (BM þ TC þ ; 'sham' chimeras) were a positive control, and TLR4 TC intact, BM TLR4 deficient (BM-C þ ) and TC TLR4 deficient, BM intact (BM þ TC-) chimeras were studied. Both BM and TC TLR4 are required for maximal neutrophil recruitment. Compared with BM þ TC þ chimeras, glomerular neutrophil recruitment was reduced in either TLR4 BM-TC þ chimeras or TLR4 BM þ TC-mice (Figure 6a ), but BM-derived TLR4 has a more prominent role. Both BM and TC TLR4 are required for maximum pulmonary neutrophil recruitment (Figure 6b) . Glomerular CXCL1 and CXCL2 production was assessed in kidneys of chimeric mice. Compared with 'sham' chimeras (BM þ TC þ ), semiquantitative assessment of CXCL1 and CXCL2 showed a reduction in both chemokines in TLR4 BM-TC þ mice and BM þ TC-mice (Figure 6c and d) , corresponding with the reduction in neutrophil recruitment. The trend towards decreased CXCL1 and CXCL2 mRNA expression in kidneys of BM-TC þ and BM þ TC-chimeric mice did not reach statistical significance (data not shown).
CXCL8 is produced by human GEnCs after LPS stimulation
Having established a role for TLR4 on intrinsic renal cells in inducing CXCL1 and CXCL2 dependant experimental glomerular neutrophil recruitment, we analyzed human glomerular cells for CXCL8 production (CXCL1 and CXCL2 are the murine homologs of CXCL8). Baseline mean ciGEnC mRNA expression was assigned a value of 1. Although CXCL8 mRNA expression was increased in ciGEnC, podocyte, and mesangial cells (Table 1) , the relative increase was most pronounced in ciGEnC (Figure 7a ). Basal CXCL8 production was highest in podocytes (Table 1) , however, after stimulation the increase is most pronounced in ciGEnC, with significant increases at 2, 4, and 24 h (Figure 7b ). These increases in both mRNA expression and protein production demonstrate that GEnC are the cell type most responsible for enhanced CXCL8 production. 
CXCL8 is produced by human GEnCs after LPS stimulation
As CXCL1 and CXCL2 were important in glomerular neutrophil recruitment, we defined their role in the development of histological and functional renal injury by extending studies to day 6. Compared with mice given ANCA/LPS and isotope control antibodies, albuminuria and hematuria were decreased after the administration of anti-CXCL2 antibody and ANCA/LPS, whereas a trend to decreased injury was seen after the administration of anti-CXCL1 antibody (Figure 9a and b) . Albuminuria, measured in the initial 24 h in mice administered control antibody and ANCA/LPS (170±39 mg/24 h) was decreased in mice given anti-CXCL1 antibody and ANCA/LPS (55 ± 14 mg/24 h, Po0.001) and anti-CXCL2 and ANCA/LPS (87 ± 10 mg/ 24 h, Po0.01). Glomerular histological injury (Figure 9c and d) was attenuated after the administration of CXCL1 or CXCL2 neutralizing antibodies, though reduced hypercellularity after anti-CXCL1 antibody did not reach statistical significance.
CXCL8 is produced by human GEnCs after LPS stimulation
Having demonstrated a role for both BM and glomerular TLR4 in neutrophil recruitment, we confirmed that both are required for maximal renal injury. Compared with BM þ TC þ 'sham' chimeras, albuminuria was decreased CXCL1 and TLR4 also colocalized (yellow) (e), and a merged three-color image showed that some endothelial cells were positive for both CXCL1 and TLR4 (white) (f). To assess CXCL2 production kidneys were stained for (g) CD31 (blue), (h) TLR4 (green), and (i) CXCL2 (red). CXCL2 colocalized with glomerular endothelial cells (magenta) (j) and TLR4 (yellow) (k). Merged three-color image showing that some endothelial cells were positive for both CXCL2 and TLR4 (white) (l). Original magnification x800. LPS, lipopolysaccharide; MPO, myeloperoxidase; TLR, Toll-like receptor; WT, wild type.
CXCL8 is produced by human GEnCs after LPS stimulation
in both BM-TC þ and BM þ TC-mice, (Figure 10a and b; trends to reduction in hematuria did not reach significance). Histological injury was reduced in BM-TC þ and BM þ TCmice with less glomerular fibrin deposition and hypercellularity (Figure 10c and d) .
DISCUSSION
These studies define roles for both BM cell and TC-derived TLR4 in the pathogenesis of neutrophil recruitment in LPS/ anti-MPO antibody renal and lung injury, and the roles of 
and WT-TLR4
À/À mice (BM þ TC-, n ¼ 8) injected with hpLPS and anti-MPO (aMPO) antibodies. Glomerular neutrophil recruitment (a) was decreased in both BM-TC þ and BM þ TC-compared with BM þ TC þ mice. There was decreased glomerular neutrophil recruitment in BM-TC þ mice compared with BM þ TC-mice. Lung MPO activity was decreased in both BM-TC þ and BM þ TC-mice compared with BM þ TC þ mice (b). Kidney CXCL1 (c) and CXCL2 (d) immunostaining was decreased in BM-TC þ and BM þ TC-mice compared with BM þ TC þ mice. *Po0.05, **Po0.01, ***Po0.001. gcs, glomerular cross-section; MPO, myeloperoxidase. GEnC are largely responsible for the increase in CXCL8 expression and protein production. In vivo studies at a later time point confirmed the functional relevance of both immune cell-and TC-derived TLR4, and of neutrophil chemoattractants, especially CXCL2.
and WT-TLR4
Experimental anti-MPO antibody-induced GN is neutrophil dependent. 4 Neutrophils express TLR4, and LPS that engages TLR4, is a potent stimulus for neutrophil activation. TLR4 ligation has pleiotropic effects on neutrophils including neutrophil adhesion, 23, 24 delayed apoptosis, enhanced chemokine production and increased superoxide generation. 25 In the current studies, the first series of experiments showed that administering both hpLPS and anti-MPO antibodies led to increased glomerular neutrophil recruitment and lung MPO activity. The effects of hpLPS on neutrophil recruitment were not seen in TLR4
and WT-TLR4
À/À mice. These studies are in accordance with previous work showing that LPS had the capacity to markedly increase anti-MPO antibodyinduced injury in mice. 6, 26 In the current studies, we used three separate techniques to determine the glomerular cell types responsible for TLR4 production. First, using confocal microscopy, we demonstrated that TLR4 is present in murine glomeruli and colocalizes with GEnC. Other glomerular cells also express TLR4 in this model. Second, using microdissected glomeruli, TLR4 mRNA expression was quantitated in glomeruli and the tubulointerstitium. After the administration of LPS and anti-MPO antibodies glomerular, but not tubulointerstitial TLR4 expression was increased. Third, using isolated human glomerular cells, we demonstrated that GEnC express significant amounts of TLR4, and that enhanced mRNA expression after stimulation is attributable to GEnC.
and WT-TLR4
Therefore, GEnC are significant sources of glomerular TLR4 expression after the administration of LPS and anti-MPO antibodies. The sites of intrarenal TLR4 production, both in the glomerulus and the tubulointerstitium have been addressed in several studies in other experimental models. TLR4 has been localized to the glomerulus in other models of renal disease. 20, 27, 28 In situ hybridization showed that mesangial and epithelial cells can express TLR4, 27 whereas in experimental cryoglobulinemic GN podocytes express TLR4. 20 Studies assessing TLR4 in murine models of tubulointerstitial injury have demonstrated TLR4 mRNA production from primary renal tubular epithelial cells, 29 whereas confocal microscopy has suggested TLR4 is present in proximal collecting tubules. 30 From the existing literature and the current studies, it is clear that TLR4 expression from intrinsic renal cells can vary according to the nature of the injurious stimulus. In the current studies, the results of a combination of in vivo and in vitro studies imply a role for the glomerular endothelium in TLR4 responses in the context of LPS and anti-MPO antibodies as initiators of injury.
and WT-TLR4
In vivo murine models have shown that the chemokines CXCL1 and CXCL2 directs neutrophil recruitment to the cornea, 31 peritoneum, 32, 33 and the joint. 34 The addition of LPS to antiglomerular basement membrane (GBM) globulin enhances heterologous renal injury. 27, 35 Furthermore, Brown et al., 27 demonstrated that neutralizing CXCL1 and CXCL2, which was TLR4 mediated and produced by renal cells, resulted in decreased glomerular injury. We have demonstrated that renal CXCL1 and CXCL2 expression (mRNA and protein) increases in a TLR4-dependent manner, both of which are produced by GEnC. The receptor for CXCL1 and CXCL2, CXCR2, is present on neutrophils, but LPS does not induce its expression or alter migration induced by neutrophil chemoattractants. 23, 36 The current studies show that CXCL1 and CXCL2 direct anti-MPO antibody glomerular and pulmonary neutrophil recruitment, as neutralizing CXCL1 and CXCL2 decreased glomerular neutrophil recruitment and lung MPO activity early, and functional and histological renal injury later in the disease.
and WT-TLR4
Both BM-derived cells 37 and an activated glomerular endothelium 38, 39 are thought to be important in glomerular neutrophil recruitment in GN induced by anti-MPO antibodies. The current studies demonstrate that both BM and TC TLR4 are required for maximal glomerular and lung neutrophil requirement, underlying separate roles in the disease process. These effects within the kidney extended out to at least 6 days, wherein mice deficient in either BM cell or TC TLR4 exhibited less injury, even in the face of more profound initial decrease in glomerular neutrophil recruitment in BM-TC þ mice. As CXCL1 and CXCL2 staining was decreased in BM-TC þ mice and BM þ TC-mice, both BM and TC-derived TLR4 are important in the renal production of CXCL1 and CXCL2, required for glomerular neutrophil recruitment. Neutralizing CXCL2 at the induction of injury resulted in attenuated functional and histological glomerular injury after 6 days; the effects of CXCL1 blockade were less prolonged. Although the current studies did not assess the cell type in the lung that produces neutrophil chemoattractants, previous studies have demonstrated that CXCL1 and CXCL2 are produced by Clara cells (non-ciliated bronchoalveolar epithelial cells in the distal airways). 40 Previous studies analyzing lung MPO activity to quantitate neutrophil recruitment in TLR4 chimeric mice have yielded conflicting results, with one study implicating TCs, 41 whereas another showed BM cell TLR4 to be important. 42 In experimental anti-MPO antibody-induced neutrophil recruitment MPO activity is decreased in both BM-TC þ and BM þ TCchimeric mice.
and WT-TLR4
Observations in human renal biopsies suggest a pathogenic role for CXCL8, the key neutrophil-attracting chemokine in ANCA GN. 15 Previous studies have suggested that CXCL8 can be produced by 'generic' macrovascular endothelial cells (HUVECs), 43 and cultured human mesangial cells. 44 We compared CXCL8 production by different human glomerular cells, including ciGEnC, concurrently, in a single study. Although baseline expression of CXCL8 mRNA and protein production was higher in podocytes; ciGEnC showed the most significant increase in expression and production after stimulation, suggesting that during inflammation GEnC produce CXCL8, which is responsible for neutrophil recruitment.
and WT-TLR4
We have demonstrated a pivotal role for both BM and intrinsic renal cell TLR4 in glomerular and lung neutrophil recruitment and injury in experimental ANCA disease. Maximal neutrophil recruitment is dependant on CXCL1 and CXCL2, TC expression, which is TLR4 dependant. Therefore, in addition to immune cell TLR4-mediated activation and recruitment, the current studies demonstrate a role for the glomerular endothelium, which involves GEnC TLR4 expression, and CXC chemokine production, that enhances neutrophil recruitment. Results from these studies add to evidence linking infection to autoimmune GN and provide evidence for possible benefits of TLR inhibition in immune glomerular disease.
MATERIALS AND METHODS
Generation of mouse anti-MPO immunoglobulin G (IgG), control mouse anti-OVA IgG, and hpLPS Murine MPO (mMPO) was generated as described previously. 45 Globulin was precipitated (50% ammonium sulfate) and IgG affinity purified by fast protein liquid chromatography and dialyzed against PBS. For anti-OVA antibodies, Mpo À/À mice were immunized with OVA using the same protocol. Endotoxin concentration measured o0.01 ng/ml, Limulus Amebocyte Lysate E-TOXATE (Sigma-Aldrich, St Louis, MO). For in vivo experiments LPS-L2654 (Sigma-Aldrich) was repurified to ensure TLR4 specificity. 46 Experimental design and statistical analyses Eight week old male WT (C57BL/6 (CD45.2) and for some chimeric studies congenic CD45.1 mice) and TLR4 À/À mice were used. Mice were from Monash University Animal Services (Melbourne, Australia), TLR4
MATERIALS AND METHODS
À/À mice originally from Professor S Akira. 47 Studies adhered to the National Health and Medical Research Council of Australia guidelines for animal experimentation. For neutrophil accumulation, mice were injected intraperitonally with hpLPS (10 mg), followed 2 h later by intravenous injection of IgG (anti-MPO antibodies 50 mg/anti-OVA (control) antibodies 50 mg), 3 h later animals were killed. For studies analyzing functional and histological renal injury, mice were injected with hpLPS 10 mg followed by anti-MPO antibodies (100 mg/g) and killed on day 6. Neutralizing monoclonal antibodies directed against CXCL1 or CXCL2 (MAB 453 and MAB 452 respectively; R&D systems, Minneapolis, MN, USA) were used with appropriate control antibodies, (rat IgG2a and IgG2b). At 1 h before the disease induction, mice were administered; anti-CXCL1-Ab (100 mg), anti-CXCL2-Ab (100 mg), or control Ab (100 mg) intravenous. BM (BM) chimeric mice were generated as previously described. 48 Flow cytometry demonstrated 492% reconstitution. Data are expressed as mean ± s.e.m. Groups of data were analyzed using student's ttest for analysis of two groups and one-way ANOVA (Tukey's post test) for more than two groups of data. GraphPad Prism software (GraphPad Software, San Diego, CA, USA) was used to analyze the data. A P-value of o0.05 was considered significant.
MATERIALS AND METHODS
Assessment of glomerular neutrophil accumulation, CXCL1 and CXCL2, histological and functional renal injury and pulmonary MPO Neutrophils were demonstrated by immunoperoxidase staining of periodate lysine paraformaldehyde fixed tissue as previously described, 49 a minimum of 50 glomeruli were assessed. Results are expressed as neutrophils per glomerular cross-section (n/gcs). CXCL1 and CXCL2 immunohistochemical staining was performed as previously described. 50 To assess the intensity of glomerular and juxtaposed tubular CXCL1 and CXCL2 staining a semiquantative intensity staining scale was used, previously described and published. 50 Values are expressed as numbers ranging from 0-4, where 0: no staining seen; 1: staining o25%; 2: 25-50%; 3: 51-75%; and 4: 475% of the glomerulus and adjacent tubules.
MATERIALS AND METHODS
For histological assessment, 40 consecutive glomeruli/mouse were examined. Kidneys were fixed with Bouin's fixative, embedded in paraffin and stained with Periodic Acid-Schiff reagent. Fibrin staining was performed as previously described. 51 and results expressed as percentage of glomeruli with fibrin deposition. Albuminuria was measured on 24 h urine collections using a Mouse Albumin ELISA Quantification Kit (Bethyl Laboratories, Montgomery, TX, USA). Hematuria was measured using urinary dipstix (Combur Tests, Roche, Basel, Switzerland). Lung MPO was used to assess pulmonary neutrophil accumulation as previously described. 52 One unit of MPO activity was defined as a change in D 460 of 1.0 after 2 min; results are expressed as U of MPO activity/g of lung tissue (U/g).
MATERIALS AND METHODS
For colocalization experiments using confocal microscopy, kidneys were perfused and fixed with zinc fixative, paraffin embedded and sectioned (3 mm). For endothelial cell/TLR4 colocalization, sections were blocked (10% chicken and donkey serum in 5% bovine serum albumin), then incubated with rabbit anti-mouse TLR4 (10 mg/ml, Invitrogen, Carlsbad, CA, USA) and rat anti-mouse CD31 (1:50, BD Biosciences, San Jose, CA, USA), followed by chicken anti-rabbit Alexa Fluor 488 and donkey anti-rat Alexa Fluor 594 (both 1:200, Molecular Probes, Invitrogen, Carlsbad, CA, USA). For colocalization of neutrophil chemokines, TLR4 and endothelial cells, anti-CD31 antibodies were followed by donkey anti-rat Alexa Fluor 647 (1:200, Molecular Probes). For CXCL1 colocalization, antibodies were rabbit anti-mouse TLR4 (20 mg/ml), and goat anti-mouse CXCL1 (20 mg/ml, R&D Systems), then chicken anti-rabbit Alexa Fluor 488 and chicken anti-goat Alexa Fluor 594 (1:200, Molecular Probes). For CXCL2 co-localization, antibodies were goat anti-mouse TLR4 (20 mg/ml, Santa Cruz Biotechnology, Santa Cruz, CA, USA), with chicken anti-goat Alexa Fluor 488, and rabbit anti-mouse CXCL2 (10 mg/ml, R&D Systems). CXCL2 signal was amplified with swine anti-rabbit HRP conjugated antibody (1:100, DAKO, Glostrup, Denmark) with a Cyanine-3-Tyramide Signal Amplification kit (PerkinElmer, Waltham, MA, USA). For all samples concurrent negative controls included substituting the primary antibodies for non-immune goat, rat or rabbit IgG, and additionally for TLR4, using TLR4 À/À mice receiving hpLPS and anti-MPO antibodies. Images were acquired using a Nikon C1 confocal laser scan head attached to a Nikon Ti-E inverted microscope (Nikon, Tokyo, Japan) using 488, 561, and 637 nm lasers. Single plane 512 Â 512 Â 12-bit images were captured in a line sequential manner (three line averaging). Confocal images were converted using Macbiophotonics Image J software (NIH, Bethesda, MD, USA).
MATERIALS AND METHODS
Culture of human glomerular cells and assessment of TLR4, interleukin-8, CXCL1 and CXCL2 mRNA and protein Human ciGEnC, 53 podocytes 54, 55 and human immortalized glomerular mesangial cells, kindly provided by Dr Banas (LudwigMaximilians University, Munich, Germany), 56 were cultured. Cells were treated with 1 mg/ml LPS (Escherichia Coli, serotype O26:B6; Sigma-Aldrich) for 2, 4, or 24 h, medium, collected and cells lysed for RNA isolation. For laser microdissection, glomeruli (2.72±0.29 Â 10 6 mm 2 ) and surrounding tubulointerstitial tissue (2.88±0.25 Â 10 6 mm 2 ) were dissected from renal cryosections of anti-MPO IgG/LPS treated mice from a previous study 26 using the Laser Robot Microbeam System (P.A.L.M. Micro laser Technology, Bernried, Germany) described previously. 57 Real-time PCR analysis on complementary DNA was carried out using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands) using assay on demand primer-probe sets for TLR4 (Hs00152939_m1), interleukin-8 (Hs00174103_m1), and GAPDH (Hs99999905_m1; glyceraldehyde 3-phosphate dehydrogenase) as the house keeping gene. For measurement of CXCL1 and CXCL2 real-time PCR was as previously described, standardized to 18S, expressed as a fold increase relative to untreated WT mice. 58 For fluorescence-activated cell sorting analysis of TLR4 protein expression on ciGEnC 2.3 Â 10 5 cells were cultured for 5 days, subsequently, TLR4 protein was detected using PE-Cy7 anti-human TLR4 antibody (eBioscience, San Diego, CA, USA). Mean fluorescence intensity of PE-Cy7 was measured using a LSR-II flow cytometer. CXCL8 was measured in culture medium from ciGEnC, podocytes and mesangial cells using ELISA (R&D systems).
DISCLOSURE
All the authors declared no competing interests. Figure S1 . Further negative controls for immunofluorescence, showing substitution of primary antibodies with appropriate non-immune IgG. Supplementary material is linked to the online version of the paper at http://www.nature.com/ki
SUPPLEMENTARY MATERIAL


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