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• W2043123870 abstract "A role for toll-like receptor 4 (TLR4) has been suggested in previous studies of glomerulonephritis, but the complex integration of these effects has not been explored. To separate effects on the innate and adaptive immune responses, we use the autologous nephrotoxic nephritis model with two disease induction protocols. First, we give a TLR4 ligand at the time of immunization and show the effects are mediated via TLR4 by comparing wild-type and TLR4-deficient mice. In wild-type mice histological measures of disease and serum creatinine are all at least twice as high as TLR4-deficient mice, due to an enhanced immune response to the nephritogenic sheep IgG. Second, we stimulate TLR4 later in the course of disease development and construct four groups of bone marrow chimeric or sham chimeric mice to study the role of TLR4 on bone marrow or renal cells. The most striking finding is that renal cell TLR4 stimulation increases glomerular crescent formation, with a mean of 21% and 25% in the two groups of mice with renal cell TLR4 compared with 0.1% and 0.6% in the two groups without, with differences mirrored by changes in serum creatinine. These findings, in a single disease model, illustrate that TLR4 stimulation triggers crescentic glomerulonephritis by effects on both the adaptive and innate immune response, with a crucial direct effect on renal cells. A role for toll-like receptor 4 (TLR4) has been suggested in previous studies of glomerulonephritis, but the complex integration of these effects has not been explored. To separate effects on the innate and adaptive immune responses, we use the autologous nephrotoxic nephritis model with two disease induction protocols. First, we give a TLR4 ligand at the time of immunization and show the effects are mediated via TLR4 by comparing wild-type and TLR4-deficient mice. In wild-type mice histological measures of disease and serum creatinine are all at least twice as high as TLR4-deficient mice, due to an enhanced immune response to the nephritogenic sheep IgG. Second, we stimulate TLR4 later in the course of disease development and construct four groups of bone marrow chimeric or sham chimeric mice to study the role of TLR4 on bone marrow or renal cells. The most striking finding is that renal cell TLR4 stimulation increases glomerular crescent formation, with a mean of 21% and 25% in the two groups of mice with renal cell TLR4 compared with 0.1% and 0.6% in the two groups without, with differences mirrored by changes in serum creatinine. These findings, in a single disease model, illustrate that TLR4 stimulation triggers crescentic glomerulonephritis by effects on both the adaptive and innate immune response, with a crucial direct effect on renal cells. Toll-like receptors (TLRs) are a family of pattern recognition receptors that play a central role in the cellular response to microbes. They are likely to be central to any link between glomerulonephritis and infection. It was first discovered that TLR4 recognized lipopolysaccharide (LPS), otherwise known as Gram-negative endotoxin. This was found when two mouse strains were observed to have mutations in the tlr4 gene.1Poltorak A He X Smirnova I Liu MY Van Huffel C Du X Birdwell D Alejos E Silva M Galanos C Freudenberg M Ricciardi-Castagnoli P Layton B Beutler B Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene.Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6421) Google Scholar Gene targeting has been used to construct TLR4-deficient mice, which has confirmed the role of TLR4 in the recognition of LPS.2Takeuchi O Hoshino K Kawai T Sanjo H Takada H Ogawa T Takeda K Akira S Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components.Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2779) Google Scholar Subsequently, other members of the TLR family have been shown to recognize a range of microbial components and 11 mammalian TLRs have been described.3Takeda K Akira S Toll-like receptors in innate immunity.Int Immunol. 2005; 17: 1-14Crossref PubMed Scopus (2700) Google Scholar As well as leukocytes, TLRs are also widely expressed on nonbone marrow-derived cells.4Zarember KA Godowski PJ Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines.J Immunol. 2002; 168: 554-561PubMed Google Scholar These tissue-cell TLRs have been shown to contribute to disease in several nonrenal experimental models5Andonegui G Bonder CS Green F Mullaly SC Zbytnuik L Raharjo E Kubes P Endothelium-derived Toll-like receptor-4 is the key molecule in LPS-induced neutrophil sequestration into lungs.J Clin Invest. 2003; 111: 1011-1020Crossref PubMed Scopus (342) Google Scholar, 6Schilling JD Mulvey MA Vincent CD Lorenz RG Hultgren SJ Bacterial invasion augments epithelial cytokine responses to Escherichia coli through a lipopolysaccharide-dependent mechanism.J Immunol. 2001; 166: 1148-1155PubMed Google Scholar in addition to models of renal tubulointerstitial disease.7Patole PS Schubert S Hildinger K Khandoga S Khandoga A Segerer S Henger A Kretzler M Werner M Krombach F Schlondorff D Anders HJ Toll-like receptor-4: renal cells and bone marrow cells signal for neutrophil recruitment during pyelonephritis.Kidney Int. 2005; 68: 2582-2587Crossref PubMed Scopus (97) Google Scholar, 8Wu H Chen G Wyburn KR Yin J Bertolino P Eris JM Alexander SI Sharland AF Chadban SJ TLR4 activation mediates kidney ischemia/reperfusion injury.J Clin Invest. 2007; 117: 2847-2859Crossref PubMed Scopus (680) Google Scholar, 9Shigeoka AA Holscher TD King AJ Hall FW Kiosses WB Tobias PS Mackman N McKay DB TLR2 is constitutively expressed within the kidney and participates in ischemic renal injury through both MyD88-dependent and -independent pathways.J Immunol. 2007; 178: 6252-6258PubMed Google Scholar, 10Leemans JC Stokman G Claessen N Rouschop KM Teske GJ Kirschning CJ Akira S van der Poll T Weening JJ Florquin S Renal-associated TLR2 mediates ischemia/reperfusion injury in the kidney.J Clin Invest. 2005; 115: 2894-2903Crossref PubMed Scopus (470) Google Scholar TLR2 and TL4 use the adaptor molecule TIRAP to activate the Myd88 dependent signaling pathway that includes IRAK4 and TRAF6. In addition, TLR4 activates Myd88 independent pathways via TRIF. Hence one would expect similarities in the effects of these two receptors on disease severity. We have recently established that stimulation of TLR2 and TLR4 can trigger disease in the heterologous phase of nephrotoxic nephritis.11Brown HJ Lock HR Sacks SH Robson MG TLR2 stimulation of intrinsic renal cells in the induction of immune-mediated glomerulonephritis.J Immunol. 2006; 177: 1925-1931Crossref PubMed Scopus (56) Google Scholar, 12Brown HJ Lock HR Wolfs TG Buurman WA Sacks SH Robson MG Toll-like receptor 4 ligation on intrinsic renal cells contributes to the induction of antibody-mediated glomerulonephritis via CXCL1 and CXCL2.J Am Soc Nephrol. 2007; 18: 1732-1739Crossref PubMed Scopus (94) Google Scholar This is a model of neutrophil-mediated acute glomerular inflammation induced by passively administered glomerular binding antibody.13Cochrane CG Unanue ER Dixon FJ A Role of polymorphonuclear leukocytes and complement in nephrotoxic nephritis.J Exp Med. 1965; 122: 99-119Crossref PubMed Scopus (243) Google Scholar For both TLR2 and TLR4, we demonstrated that stimulation of renal cell TLRs played an important role. The autologous phase of nephrotoxic nephritis is due to an immune response to the foreign glomerular bound antibody, which acts as a planted antigen.14Unanue ER Dixon FJ Experimental glomerulonephritis: the autologous phase of nephrotoxic seum nephritis.J Exp Med. 1965; 121: 715-725Crossref PubMed Scopus (59) Google Scholar It is a more clinically relevant model of glomerulonephritis as the pathology shows proliferation, macrophage and T cell infiltration, and thrombosis and crescent formation,15Tipping PG Kitching AR Glomerulonephritis. Th1 and Th2: what's new?.Clin Exp Immunol. 2005; 142: 207-215Crossref PubMed Scopus (66) Google Scholar which are all features found in human disease. We have recently demonstrated that TLR2 ligands given at the time of priming to the planted antigen can exacerbate disease in this model by modulating the nephritogenic immune response.16Brown HJ Sacks SH Robson MG Toll-like receptor 2 agonists exacerbate accelerated nephrotoxic nephritis.J Am Soc Nephrol. 2006; 17: 1931-1939Crossref PubMed Scopus (52) Google Scholar In addition to our own studies, Fu et al17Fu Y Xie C Chen J Zhu J Zhou H Thomas J Zhou XJ Mohan C Innate stimuli accentuate end-organ damage by nephrotoxic antibodies via Fc receptor and TLR stimulation and IL-1/TNF-alpha production.J Immunol. 2006; 176: 632-639PubMed Google Scholar also gave endotoxin and other TLR ligands to mice at the same time as nephrotoxic antibody. However, in this work there was no measurement of the immune response to the nephritogenic antigen sheep IgG, and so the relative roles of effects on the adaptive immune response or on innate mechanisms were not clear. The current study had two main aims. First, we wanted to assess whether stimulation of TLR4 at the time of priming could cause crescentic glomerulonephritis by augmenting the adaptive immune response to sheep IgG, as we had shown for TLR2. Second, we planned to study the effect of TLR4 stimulation on crescentic glomerulonephritis in the context of an established nephritogenic immune response, including an assessment of the role of TLR4 on renal cells. This would provide an in-depth understanding of the effects of TLR4 stimulation on both innate and adaptive immune mechanisms in a single clinically relevant model of glomerulonephritis. Wild-type C57BL6 mice were from Harlan UK. TLR4-deficient mice were kindly provided by S. Akira. These were backcrossed seven generations to C57BL/6. All experiments were performed according to UK home office and local regulations. Nephrotoxic serum (NTS) was raised in sheep as described,11Brown HJ Lock HR Sacks SH Robson MG TLR2 stimulation of intrinsic renal cells in the induction of immune-mediated glomerulonephritis.J Immunol. 2006; 177: 1925-1931Crossref PubMed Scopus (56) Google Scholar and two disease inducing protocols were used (LPS at priming, and LPS given later, denoted protocols A and B, respectively). Sheep IgG used for pre-immunization was prepared in house from normal sheep serum by using DEAE sepharose with purity more than 90% on SDS-polyacrylamide gel electrophoresis. In protocol A, mice were immunized intraperitoneally with 200 μg of normal sheep IgG that was given with 80 μl of aluminum hydroxide gel (Sigma, Poole, UK) in 310 μl of PBS (endotoxin free from Sigma), with 10 μg of highly purified LPS from Escherichia coli R515 (581-007-L002; Alexis Biochemicals, Nottingham, UK). All of these components were given as a single intraperitoneal injection. Five days later, mice received an injection of 160 μl of NTS via the tail vein to induce disease. In protocol B, disease was induced with 10 μl/g of serum intravenously. At days 5 and 7, 10 μg LPS was given intraperitoneally in 400 μl PBS, with PBS alone as a control. These protocols are illustrated in Figure 1. Endotoxin levels were measured by using a chromogenic kinetic Limulus Amoebocyte Lysate assay (Cambrex Bioscience, Verviers, Belgium). NTS used in all experiments contained <0.5 eU/ml. Eighty microliters aluminum hydroxide contained <0.004 eU. The 200-μg sheep IgG used for pre-immunization contained <0.0025 eU. Only mice surviving to the experiment end were included in the analysis. For the experiment depicted in Figure 2, Figure 3, Figure 4, one wild-type mouse was sacrificed on day 6 due to ill-health and one on day 7, with one TLR4-deficient mouse sacrificed on day 12, and the remainder on day 14. In addition, one wild-type mouse was not placed in the metabolic cage at the end of the experiment due to ill health. For the experiment in Figure 5, Figure 6, Figure 7, two mice given LPS were sacrificed on day 10 due to ill health, and the remainder on day 12. For the experiment in Figure 8, Figure 9, Figure 10, one wild-type → wild-type mouse was sacrificed on day 12 due to ill health, and the remainder on day 14 (with blood and urine not obtained from one wild-type → TLR4−/− mouse, which was ill on day 14).Figure 2TLR4 stimulation at immunization exacerbates crescentic glomerulonephritis. Wild-type and TLR4-deficient mice were pre-immunized with sheep IgG and LPS and disease was induced as described in protocol A. Disease parameters were assessed at day 14 (albuminuria at day seven as well). A–D: In wild-type, compared with TLR4-deficient mice, there were significantly more crescents, thrombosis, and glomerular CD68+ macrophages, but no difference in glomerular CD4+ cell numbers. E: Albuminuria was not different at day seven and higher in TLR4-deficient mice at day 14. F: Serum creatinine (day 14) was higher in wild-type mice. Each point is an individual mouse n = 7 (wild-type) and n = 8 (TLR4-deficient). Mice not surviving to day 14 were not included in the analysis and are listed in Materials and Methods.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3TLR4 stimulation at immunization exacerbates crescentic glomerulonephritis. Representative histology from wild-type and TLR4-deficient mice in the experiment shown in Figure 2. PAS stained sections showing a crescent and thrombosis in a wild-type but not a TLR4-deficient mouse. More macrophages were seen in the wild-type mice, as shown by CD68 immunoperoxidase staining. A crescent is shown by an arrow.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4A–D: TLR4 stimulation at immunization augments the humoral immune response to sheep IgG. Subclass-specific ELISAs were performed on serum from wild-type and TLR4-deficient animals pre-immunized with sheep IgG and LPS in the experiment in Figure 2. Normal serum from three untreated mice was included as a control (NMS). Wild-type mice had an increase in all subclasses at day five when compared with TLR4-deficient mice. This difference was maintained at day nine and 14 for IgG1 and IgG2c. Each point is an individual mouse. E and F: Spleens were taken at the end of the experiment and cytokines were measured in supernatants from splenocytes restimulated in the presence of sheep IgG, with controls cultured without sheep IgG. The data demonstrate an antigen-specific response, but there were no significant differences between the groups. Each point is the mean of triplicate cultures from an individual mouse. At the end of the experiment n = 7 (wild-type) and n = 8 (TLR4-deficient) for all data sets, with additional surviving mice included at earlier time points.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5LPS given after disease induction does not affect the immune response to sheep IgG. Wild-type mice were given LPS or control at days five and seven after NTS and sacrificed at day 12 according to protocol B. Subclass-specific ELISAs were performed on serum, with normal mouse serum from three nonimmunized mice as a control (NMS). There were no significant differences. Each point is an individual mouse, with n = 7 (LPS) and n = 8 (control). Mice not surviving to day 12 were not included in the analysis and are listed in Materials and Methods.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Nephrotoxic nephritis is exacerbated by LPS given after disease induction. Wild-type mice were given LPS or control at days 5 and 7 after NTS as described in Materials and Methods (protocol B). Data are from the same experiment as the ELISA data in Figure 4. A–D: There were significantly more crescents, thrombosis, and glomerular CD68+ macrophages but no difference in glomerular CD4+ cell numbers in mice given LPS compared with controls. E and F: There was no difference in albuminuria, but a higher serum creatinine in LPS treated mice. Each point is an individual mouse with n = 8 (control) and n = 7 (LPS).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Nephrotoxic nephritis is exacerbated by LPS given after disease induction. Representative histology at day 14 from the experiment shown in Figure 5. This shows crescent formation and thrombosis in mice given LPS (left column) compared with controls (right column). There were also more glomerular CD68+ macrophages, but no difference in CD4+ cell numbers. A crescent is shown by an arrow.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8The immune response to sheep IgG in chimeric and sham chimeric mice with nephrotoxic nephritis given LPS after disease induction. Mice were given LPS at days five and seven after NTS and sacrificed at day 14 according to protocol B. Serum was analyzed by using subclass-specific ELISAs, with normal mouse serum from three nonimmunized mice as a control (NMS). Several differences were shown between mice with wild-type (WT) or TLR4 bone marrow with P values shown for those that were significant. Importantly, there we no significant differences between mice that had the same bone marrow (between wild-type → wild-type and wild-type → TLR4−/−, or between TLR4−/− → TLR4−/− and TLR4−/− → wild-type). Each point is an individual mouse with n = 14, 15, 13, and 10 for wild-type → wild-type, TLR4−/− → TLR4−/−, wild-type → TLR4−/−, and TLR4−/− → wild-type mice, respectively. Mice not surviving to day 14 were not included in the analysis and are listed in Materials and Methods.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 9Stimulation of renal cell TLR4 exacerbates crescentic glomerulonephritis. Histological disease parameters assessed in chimeric and sham chimeric mice 14 days after disease induction with LPS or control given at days five and seven as described in Materials and Methods (protocol B). Statistical significance was only assessed between groups with the same bone marrow as discussed in the text. A and B: wild-type → wild-type (WT) mice had significantly more crescents and thrombosis than wild-type → TLR4−/− animals. TLR4−/− → wild-type mice had significantly more crescents and thrombosis than TLR4−/− → TLR4−/− animals. C and D: There were no differences in glomerular CD68+ cell numbers when comparing mice with the same marrow, and no differences between any groups in glomerular CD4+ cell numbers. E: There were no differences in albuminuria. F: wild-type → wild-type mice had significantly higher serum creatinines than wild-type → TLR4−/− animals. TLR4−/− → wild-type mice had significantly higher serum creatinines than TLR4−/− → TLR4−/− animals. Each point is an individual mouse with n = 14, 15, 13, and 10 for wild-type → wild-type, TLR4−/− → TLR4−/−, wild-type → TLR4−/−, and TLR4−/− → wild-type mice, respectively. Mice not surviving to day 14 were not included in the analysis and are listed in Materials and Methods.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 10Stimulation of renal cell TLR4 exacerbates disease. Representative histology from the experiment in Figure 8, showing crescents in wild-type → wild-type (WT) and TLR4−/− → wild-type mice, with a degree of thrombosis in all groups except TLR4−/− → TLR4−/− animals. Crescents are shown by arrows.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Disease was assessed as previously described.16Brown HJ Sacks SH Robson MG Toll-like receptor 2 agonists exacerbate accelerated nephrotoxic nephritis.J Am Soc Nephrol. 2006; 17: 1931-1939Crossref PubMed Scopus (52) Google Scholar In brief, crescents and thrombosis were scored on samples fixed in Bouin’s solution and stained with PAS. For thrombosis, 50 glomeruli were each scored out of 4. Neutrophils were identified by their characteristic nuclear morphology on PAS stained sections. CD68+ and CD4+ cells were detected on phosphate-lysine periodate fixed sections, with anti-CD68 (clone FA11; Serotec, Oxford, UK) and anti-CD4 (clone L3T4; BD Pharmingen, Oxford, UK), respectively. For the experiments in wild-type mice, the avidin-biotin complex method was used with a biotinylated mouse anti-rat (Jackson’s Immunoresearch, Westgrove, PA) and avidin-biotin complex kit (Vectorlabs, Peterborough, UK). For the chimera experiments, we changed to an immunofluorescence-based method. Detection was with fluorescein isothiocyanate conjugated mouse anti-rat IgG (Jacksons Immunoresearch), followed by Alexa Fluor 488 conjugated goat anti-fluorescein isothiocyanate (Molecular Probes, Paisley, UK). All histological scoring was done without knowledge of the sample identity. Albuminuria was measured by radial immunodiffusion by using rabbit anti-mouse albumin (Biogenesis, Poole, UK) as described previously.11Brown HJ Lock HR Sacks SH Robson MG TLR2 stimulation of intrinsic renal cells in the induction of immune-mediated glomerulonephritis.J Immunol. 2006; 177: 1925-1931Crossref PubMed Scopus (56) Google Scholar Normal C57BL/6 mice have less than 50 μg/ml of albuminuria using this assay, as previously reported (n = 12).16Brown HJ Sacks SH Robson MG Toll-like receptor 2 agonists exacerbate accelerated nephrotoxic nephritis.J Am Soc Nephrol. 2006; 17: 1931-1939Crossref PubMed Scopus (52) Google Scholar Serum creatinine was measured by mass spectrometry as described.16Brown HJ Sacks SH Robson MG Toll-like receptor 2 agonists exacerbate accelerated nephrotoxic nephritis.J Am Soc Nephrol. 2006; 17: 1931-1939Crossref PubMed Scopus (52) Google Scholar Sandwich enzyme-linked immunosorbent assays (ELISAs) to measure the immune response to sheep IgG were performed as before16Brown HJ Sacks SH Robson MG Toll-like receptor 2 agonists exacerbate accelerated nephrotoxic nephritis.J Am Soc Nephrol. 2006; 17: 1931-1939Crossref PubMed Scopus (52) Google Scholar with modification for IgG2c. Detecting antibodies for IgG1, IgG2b, and IgG3 were from Southern Biotechnology (Birmingham, Alabama). The IgG2c antibody was from Bethyl Laboratories (Montgomery, TX). The relative concentrations were measured by using a standard curve that was constructed from pooled serum samples from mice that were immunized with sheep IgG. Normal serum from unimmunized mice served as a control. With disease induced according to protocol A, single cell suspensions were prepared from spleens at the end of the experiment. After red cell lysis, these were cultured at 106 cells/ml for 5 days in X-Vivo 15 serum-free medium, containing 1% glutamax (Invitrogen, Paisley, UK) and 0.1% mercaptoethanol, in the presence or absence of sheep IgG at 100 μg/ml. The sheep IgG preparation was the same as that used for pre-immunization. Interleukin-4 and interferon-γ were measured in supernatants by using ELISA kits (R&D Systems, Oxford, UK) with limits of detection of 15.6 pg/ml and 31.2 pg/ml, respectively. Chimeric mice were made by irradiation with a dose of 9 Gy and reconstitution with 5 × 106 donor bone marrow cells. Experiments were performed 10 weeks after this. Confirmation of chimerism was obtained by using real-time PCR for the neomycin resistance gene (present in TLR4-deficient mice) and glyceraldehyde-3-phosphate dehydrogenase, using genomic DNA extracted from peripheral blood, as described previously.11Brown HJ Lock HR Sacks SH Robson MG TLR2 stimulation of intrinsic renal cells in the induction of immune-mediated glomerulonephritis.J Immunol. 2006; 177: 1925-1931Crossref PubMed Scopus (56) Google Scholar DNA from a TLR4-deficient mouse was used as a standard, and results were expressed as a percentage of this value. This confirmed that bone marrow was at least 90% donor derived in all mice tested (n = 5 per group). These were performed by using Graphpad Prism Software (GraphPad Software, Inc., La Jolla, CA). An unpaired Student’s t-test was used to compare two groups. If the variances were significantly different, a logarithmic transformation was used. More than two groups were compared by using a Kruskall-Wallis test with Dunn’s post test. Significance was taken as P < 0.05. We began by exploring the effect of TLR4 stimulation at priming on the adaptive immune response and disease severity in nephrotoxic nephritis to discover if our previously described results for TLR216Brown HJ Sacks SH Robson MG Toll-like receptor 2 agonists exacerbate accelerated nephrotoxic nephritis.J Am Soc Nephrol. 2006; 17: 1931-1939Crossref PubMed Scopus (52) Google Scholar were generally applicable to other TLRs. Disease was induced by using protocol A (as shown in Figure 1 and described in Materials and Methods) in wild-type and TLR4-deficient mice. Mice were bled from the tail vein at days 5 and 9 after injection of NTS for assessment of the humoral immune response. Mice were placed in metabolic cages for assessment of albuminuria at days 7 and 14. This experiment showed that TLR4 stimulation at the time of priming with sheep IgG exacerbated disease. Figure 2, A–F, shows that there were more glomerular macrophages, crescents, and thrombosis in wild-type mice compared with TLR4-deficient mice. Neutrophil numbers were low, and there was no difference between the groups. These were 0.086 ± 0.012 and 0.0725 ± 0.026 (mean ± SEM cells per glomerular cross section) in wild-type and TLR4-deficient mice, respectively. There was also evidence of a functional effect as serum creatinine was higher in wild-type mice. There was no difference in albuminuria at day 7, and there was higher albuminuria in TLR4-deficient mice at day 14, which just reached statistical significance. This may have been a reflection of the renal failure developing in wild-type but not TLR4-deficient mice. Figure 3 shows representative histology from this experiment. These data established that crescentic glomerulonephritis is exacerbated by TLR4 stimulation when this occurs at the time of priming with an antigen that is then planted in the glomerulus. To explore the mechanism of more severe disease in wild-type mice compared with TLR4-deficient mice, we assessed the immune response to sheep IgG in the above experiment. Using subclass-specific ELISAs, we analyzed serum at days 5, 9, and 14 after disease induction (corresponding to days 10, 14, and 19 after priming). As shown in Figure 4 (A–D), the peak response was seen at day 5, and there was an increase in all four IgG subclasses in wild-type compared with TLR4-deficient mice. The difference in IgG1 and IgG2c was maintained at later time points. To further assess any change in Th1 or Th2 skewing, we restimulated splenocytes with sheep IgG at the end of this experiment and measured interleukin-4 and interferon-γ in supernatants. As shown in Figure 4 (E and F), there were no significant differences. We also performed immunofluorescence staining to assess glomerular deposition of mouse IgG subclasses and found increased IgG2c and IgG3 (see Supplemental Figure 1 at http://ajp.amjpathol.org). Because this is a less sensitive technique, the absence of a difference for IgG1 and IgG2b does not invalidate the ELISA data. Additional factors regarding interpretation of immunofluorescence data are discussed below. These data established that the increase in disease severity in wild-type mice compared with TLR4-deficient mice was accompanied by a global augmentation of the adaptive immune response to sheep IgG with no change in Th1 or Th2 skewing. We had established that TLR4 stimulation at the time of priming to the relevant antigen could augment disease by modulating the adaptive immune response. Our next aim was to establish an experimental protocol to test the effect of TLR4 stimulation later in disease development, with minimal effect on the nephritogenic immune response. We therefore induced disease by using protocol B (shown in Figure 1 and described in Materials and Methods) in wild-type C57BL/6 mice. We assessed the immune response to sheep IgG at the end of the experiment (day 14) by using subclass-specific ELISAs. As shown in Figure 5, no significant differences were seen between mice given LPS or control. Antigen-specific levels of all IgG subclasses were similar in both groups. We also performed immunofluorescence staining for IgG subclasses and performed a semiquantitative analysis of the inten" @default.
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• W2043123870 title "Toll-Like Receptor 4 Stimulation Triggers Crescentic Glomerulonephritis by Multiple Mechanisms Including a Direct Effect on Renal Cells" @default.
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