Toll-Like Receptor 4 Ligation on Intrinsic Renal Cells Contributes to the Induction of Antibody-Mediated Glomerulonephritis via CXCL1 and CXCL2
Heather J. Brown*,
Helen R. Lock*,
Tim G.A.M. Wolfs,
Wim A. Buurman,
Steven H. Sacks* and
Michael G. Robson*
* Department of Nephrology and Transplantation, King's College London School of Medicine, Guy's Hospital, London, United Kingdom; and Department of Surgery, Nutrition and Toxicology Research Institute Maastricht, Academic Hospital Maastricht and Maastricht University, Maastricht, Netherlands
Address correspondence to: Dr. Heather Brown, Department of Nephrology and Transplantation, 5th Floor Thomas Guy House, Guy's Hospital, St. Thomas Street, London SE1 9RT, UK. Phone: +207-188-5659; Fax: +207-188-5660; E-mail: heather.brown{at}kcl.ac.uk
Received for publication June 19, 2006.
Accepted for publication March 18, 2007.
Autoimmune diseases such as glomerulonephritis are exacerbatedby infection. This study examined the effect of the Toll-likereceptor 4 (TLR4) ligand lipid A on the development of heterologousnephrotoxic nephritis. Administration of nephrotoxic antibodyresulted in significant glomerular neutrophil infiltration andalbuminuria only when a TLR4 ligand was administered simultaneously.The contribution of TLR4 on renal cells and circulating leukocyteswas assessed. Bone marrow chimeras were constructed with TLR4only on renal cells or bone marrow–derived cells. Theadministration of nephrotoxic serum and lipid A caused a neutrophilinflux in both chimeric groups greater than in sham chimerasthat were totally TLR4 deficient but significantly less thanin sham chimeras that were totally TLR4 sufficient. Both chimericgroups had greater albuminuria than totally TLR4-deficient shamchimeras; however, the chimeras with TLR4 only on intrinsicrenal cells had significantly less than the sham positive group.In situ hybridization showed expression of TLR4 mRNA in mesangialcells and glomerular epithelial cells. For investigation ofthe potential mechanism by which renal cells could contributeto disease exacerbation, mesangial cells were cultured and foundto express mRNA for TLR4, and stimulation of wild-type and TLR4-deficientmesangial cells with LPS caused production of CXC chemokinesby wild-type cells only. Treatment of chimeras with TLR4 presentonly on intrinsic renal cells with anti-CXCL1 and anti-CXCL2antibody before disease induction significantly reduced renalneutrophil infiltration. These results show that TLR4 on bothcirculating leukocytes and intrinsic renal cells contributesto the inflammatory effects of antibody deposition within theglomerulus, which depends at least in part on the productionof CXC chemokines by intrinsic renal cells.
Autoimmune diseases including glomerulonephritis can be exacerbatedby systemic infection. In IgA nephropathy, an upper respiratorytract infection commonly precedes disease presentation (1);relapses of ANCA-associated vasculitis are associated with nasalcarriage of Staphylococcus aureus (2,3); and in anti–glomerularbasement membrane disease, exacerbations during treatment areoften related to infection (4). Toll-like receptors (TLR) area family of receptors that recognize a variety of ligands thatare derived from pathogens. They influence both the inflammatoryand the adaptive immune response to invading pathogens and arelikely to be important in the exacerbation of autoimmune diseaseby infection.
TLRs are widely distributed on professional immune cells butare also found on non–bone marrow (BM)-derived cells (5).In the mouse, TLR4 expression in the kidney has been shown tobe predominantly tubular with sparse glomerular expression (6).TLR4 has also been shown to be expressed in a mesangial cellline (7). Infection may exacerbate glomerulonephritis in a numberof ways by activating TLR. The adaptive immune response maybe influenced to promote renal tissue injury. TLR agonists couldact on neutrophils, macrophages, or other cells of the innateimmune system to increase glomerular inflammation and exacerbatedisease. Alternatively, inflammation could be mediated by TLRsthat are present on intrinsic renal cells such as mesangialcells.
In this study, we used heterologous nephrotoxic nephritis, apassive model of antibody-mediated glomerular inflammation (8,9).Disease is characterized by a glomerular neutrophil influx,which peaks at approximately 2 h and is largely resolved by24 h with accompanying proteinuria. Previous studies have showna role for LPS in this model in both the rat and the mouse (10,11).Endotoxin preparations that were used previously were crude,and repurification has been shown to reduce signaling by TLR2(12). The precise role for pure TLR4 ligands in disease exacerbationtherefore cannot be established from these studies. We useda synthetic analogue of the active part of endotoxin calledlipid A and highly purified LPS derived from Escherichia coli,both of which are known to ligate TLR4 and contain undetectablelevels of other TLR ligands. Heterologous nephrotoxic nephritisis ideally suited to investigate mechanisms independent of anyeffects of TLR stimulation on the adaptive immune response.We show that for glomerular inflammation to occur in responseto antibody deposition, TLR4 ligation is also required. By theproduction of BM chimeras, we show that TLR4 on both intrinsicrenal cells and circulating leukocytes contribute toward theproduction of disease. We show by in situ hybridization thatTLR4 mRNA is expressed in mesangial and epithelial cells withinthe glomerulus. We also show that cultured mesangial cells expressTLR4 and can produce the CXC chemokines that are responsiblefor neutrophil chemotaxis in response to TLR4 ligation, andwe show that blocking these chemokines in vivo reduces diseaseseverity in chimeric mice expressing TLR4 only on intrinsicrenal cells.
Preparation of Nephrotoxic Serum and TLR Agonists
A mouse glomerular extract was made and nephrotoxic serum (NTS)was prepared as described previously (13). The endotoxin contentof the serum was measured using a chromogenic kinetic LimulusAmoebocyte Lysate assay (Cambrex Bioscience, Walkersville, MO).The amount of endotoxin received by each mouse was <0.1 EU.Synthetic lipid A ONO-4007 (Ono Pharmaceutical Co., Ltd., Osaka,Japan) was used as a pure TLR4 ligand. It was dissolved in 50%ethanol. The final concentration of ethanol in injected NTSwas 0.5%. The synthetic lipopeptide Pam3CysSK4 (EMC Microcollections,Tübingen, Germany) was used as a pure TLR2 ligand. Thiswas dissolved in DMSO. The final concentration of DMSO in injectedNTS was 0.1%. For mesangial cell stimulation experiments, thehighly purified TLR4 agonist LPS from E. coli R515 was used(581-007-L002; Alexis, San Diego, CA). Synthetic lipopeptidewas used as a positive control to show that TLR4-deficient animalsand TLR4-deficient mesangial cells do not have an inherent inabilityto recruit neutrophils or secrete chemokines, respectively.
Induction of Glomerulonephritis
TLR4-deficient mice (backcrossed seven generations to C57BL/6)were obtained from S. Akira (14), and wild-type C57BL/6 micewere from B&K Universal Ltd (Hull, UK). Animal experimentswere performed according to Home Office regulations. Glomerulonephritiswas induced by giving a single injection of 200 µl ofNTS with or without TLR agonist (10 µg/mouse) or vehicle,or the TLR agonist was given alone.
Histologic Assessment of Glomerular Inflammation
The glomerular histology was assessed at 2 h after disease inductionwith NTS. Kidneys were fixed in Bouin's solution and stainedwith periodic acid-Schiff reagent. At 2 h, the number of neutrophils(identified by their characteristic nuclear morphology) per50 glomerular cross-sections was counted. All sections scoredwere done so blindly. We compared this method with quantitativeimmunofluorescence. Frozen sections were incubated with ratanti-mouse neutrophil antibody (MCA771G; Serotec Ltd., Oxford,UK) and then with FITC-conjugated mouse anti-rat antibody (JacksonImmunoresearch Laboratories, West Grove, PA) Mean glomerularimmunofluorescence scores were calculated and correlated withscores that were obtained when neutrophils were counted on periodicacid-Schiff–stained sections. The correlation coefficientwas 0.78.
Urinary Albumin Measurement
Mice were housed singly in glass metabolic cages for 24 h immediatelyafter disease induction with NTS to measure the 24-h albuminuria.The urinary albumin concentration was measured by radial immunodiffusionas described previously (13). The sensitivity of the radialimmunodiffusion assay for albumin was 0.05 mg/ml.
Peripheral Neutrophil Counts and Measurement of Glomerular Sheep IgG Deposition
The total white cell number was counted on tail-vein blood afterlysing red cells in 2% acetic acid, and a blood film was madeto assess the percentage of neutrophils. Frozen sections werestained with FITC-conjugated donkey anti-sheep IgG (JacksonImmunoresearch Laboratories), and images of 20 glomeruli foreach sample were saved. The mean fluorescence intensity wasthen measured using Adobe Photoshop software version 8.0 (AdobeSystems, San Jose, CA).
Construction of BM Chimeras
Mice at 6 to 10 wk of age were used to construct chimeric miceby irradiation with a dosage of 9 Gy and reconstitution with5 x 106 donor BM cells. We previously established that thismethod gives >95% reconstitution with donor BM cells (15).
In Situ Hybridization In situ hybridization was carried out on 3-µm paraffin-embeddedkidney sections as described previously (6). Control samplesof normal kidney from this previous study on interstitial diseasewere used and reexamined for glomerular expression.
Mesangial Cell Culture
Wild-type and TLR4-deficient mesangial cells were isolated andcultured as described previously(15) and used after four passages.Cells were grown in 24-well plates to generate supernatantsfor ELISA. All cultures were performed in triplicate. Cellswere stimulated with TLR agonists (prepared as in Preparationof Nephrotoxic Serum and TLR Agonists) for 24 h, after whichsupernatants were taken for ELISA for CXCL1 and CXCL2 (R&DSystems, Minneapolis, MN). The sensitivity of the assays were156 and 31 pg/ml for CXCL1 and for CXCL2, respectively. Forreverse transcription–PCR, the primer sequences and conditionswere as described previously (16).
CXCL1 and CXCL2 Blocking Studies
Monoclonal neutralizing antibodies to CXCL1 and CXCL2 (MAB453and MAB452, respectively) were purchased from R&D Systems,along with rat IgG2a and IgG2b isotype controls. The antibodiesand isotypes were diluted in sterile endotoxin-free PBS. Onehour before disease induction with NTS and lipid A, each mousewas given a single intravenous injection that contained a combinationof 100 µg of anti-CXCL1 and 100 µg of anti-CXCL2or a single intravenous injection that contained a combinationof 100 µg of rat IgG2a and 100 µg of rat IgG2b isotypecontrols.
Statistical Analyses
The groups of data were compared using the unpaired t test andGraphPad Prism software (GraphPad Software, San Diego, CA);when there were more than two groups of data, a one-way ANOVAwas used. When the F test suggested that variances were significantlydifferent, data were analyzed after a logarithmic transformation.
Lipid A Exacerbates Disease in Wild-Type But Not TLR4-Deficient Mice
The role of lipid A in glomerulonephritis was assessed by givingNTS with or without 10 µg of lipid A, or lipid A was givenalone, to wild-type and TLR4-deficient mice. In each case, thecomponents were given together as a single intravenous injection.At 2 h, a significant glomerular neutrophil infiltrate was seenonly in wild-type mice that were given lipid A and NTS together(P < 0.001 versus any other group). Data are shown graphicallyin Figure 1A, with representative histology in Figure 1C. Toestablish that the TLR4-deficient mice were able to mount aneutrophil influx, their response to the TLR2 agonist lipopeptidewhen given with NTS was assessed as a control. Nephritis wasinduced in TLR4-deficient mice by giving the lipopeptide (10µg per mouse) and NTS together. The TLR4-deficient micedeveloped a significant glomerular neutrophil influx at 2 hin response to NTS and lipopeptide (Figure 1A). At 24 h afterinjection of lipid A and NTS, the renal neutrophil influx hadvirtually completely resolved, and there was no significantdifference between TLR4-deficient and wild-type groups at thispoint (Figure 1B). The 24-h albuminuria was measured after diseaseinduction in wild-type and TLR4-deficient mice that were givenboth lipid A and NTS together. Only wild-type mice producedsignificant albuminuria, with TLR4-deficient mice being protectedagainst disease (P < 0.0001; Figure 1D). These data showthat both antibody deposition and lipid A are necessary forthe neutrophil influx in this model. When both NTS and lipidA are given together, there is a strong synergistic effect througha mechanism that is entirely dependent on TLR4.
Figure 1. (A) Glomerular neutrophil influx in wild-type and Toll-like receptor 4 (TLR4)-deficient mice at 2 h after intravenous lipid A (10 µg) and nephrotoxic serum (NTS) given alone or together. There is a significantly greater neutrophil influx in the wild-type group that was given lipid A and NTS compared with any other group that was given either NTS alone, lipid A alone, or NTS and lipid A together (P < 0.001 for all comparisons). In addition, to show that TLR4-deficient mice can mount a neutrophil influx in response to stimulation with a different TLR ligand, the glomerular neutrophil influx at 2 h is shown after administration of synthetic lipopeptide (TLR2 ligand) and NTS in a TLR4-deficient group. (B) Glomerular neutrophil influx at 24 h in wild-type and TLR4-deficient mice after intravenous lipid A (10 µg/mouse) and NTS. (C) Representative glomerular histology at 2 h after the intravenous injection of NTS and lipid A in a wild-type and TLR4-deficient mice. At 2 h, there is a significant glomerular neutrophil influx in the wild-type group (neutrophil influx is highlighted by arrows) that is not seen in the TLR4-deficient group. (D) Twenty-four-hour albuminuria in wild-type and TLR4-deficient mice after lipid A and NTS given together. There was a significant difference between the two groups (P < 0.0001).
Differences in Disease Severity between Wild-Type and TLR4-Deficient Mice Cannot Be Explained by Differences in Circulating Numbers of Neutrophils
For investigation of whether the observed differences in glomerularneutrophil influx were related to changes in circulating numbers,peripheral blood neutrophil counts were taken at baseline and2 h after disease induction. There was no significant differencein neutrophil numbers at baseline between wild-type and TLR4-deficientmice. The TLR4-deficient group had a greater circulating neutrophilcount than wild-type mice at 2 h after disease induction (P< 0.05). The circulating neutrophil count remained unchangedin the wild-type group that was given NTS and lipid A when baselinenumbers were compared with those seen at 2 h. These data areshown in Figure 2A. Therefore, TLR4-deficient mice had lesssevere disease despite having a larger number of circulatingperipheral blood neutrophils.
Figure 2. (A) Peripheral neutrophil counts at baseline and 2 h after intravenous lipid A and NTS in wild-type and TLR4-deficient mice. P < 0.05 for the TLR4-deficient mice compared with the wild-type mice at 2 h. (B) Immunofluorescence for glomerular deposition of sheep IgG at 2 h after NTS and lipid A in a wild-type mouse (left) and a TLR4-deficient mouse (right). The number given is the mean fluorescence ± SEM for each group of mice.
Differences in Disease Severity between Wild-Type and TLR4-Deficient Mice Cannot Be Explained by Differences in Glomerular Sheep IgG Binding
For examination of whether the decreased neutrophil influx inTLR4-deficient mice was related to less glomerular antibodybinding, the amount of sheep IgG deposited within the glomeruluswas quantified. Kidney sections were taken at 2 h after diseaseinduction with NTS and lipid A in wild-type and TLR4-deficientmice. There was no difference between the two groups, as shownin Figure 2B. TLR4-deficient mice therefore had less severedisease, even though they had a similar amount of sheep IgGin their glomeruli at 2 h.
TLR4 on Both BM-Derived and Non–BM-Derived Cells Is Required for Maximal Neutrophil Influx
For investigation of the relative contribution of TLR4 on leukocytesand non–BM-derived cells to the neutrophil influx, BMchimeras were constructed. Mice with TLR4 present on leukocytesbut absent on non–BM-derived cells or with TLR4 presenton non-BM cells but absent on leukocytes (wild-type BM intoTLR4-deficient host [WTTLR4–/–] and TLR4-deficientBM into wild-type host [TLR4–/–WT]) were constructed.Two sham chimeric groups were also produced with TLR4 presenton all cells (WTWT) or with a complete absence of TLR4 (TLR4–/–TLR4–/–).These sham chimeras were made using identical methods to thoseused for the chimeric mice. At 8 wk after reconstitution, thechimeric and sham chimeric groups were given NTS and lipid A(10 µg per mouse). A significant glomerular neutrophilinflux at 2 h was seen in the WTWT group compared with the TLR4–/–TLR4–/–group (P < 0.001), consistent with the results that we hadpreviously seen in nonchimeric mice as shown in Figure 1. Inaddition, mice from both chimeric groups (WTTLR4–/–and TLR4–/–WT) developed significant glomerularneutrophil influx when compared with the sham chimeric TLR4–/–TLR4–/–group (P < 0.001 for both comparisons) but significantlyless disease than that seen in the WTWT sham group (P < 0.05and P < 0.001, respectively). Data are shown in Figure 3A,with representative histology shown in Figure 3C.
Figure 3. (A) Glomerular neutrophil influx at 2 h after intravenous lipid A and NTS in chimeric mice with TLR4 present on intrinsic renal cells but absent on BM-derived cells (TLR4–/–WT) and vice versa (WTTLR4–/–) and sham chimeric mice. (B) Albuminuria at 24 h after intravenous lipid A and NTS in sham WTTLR4–/– and TLR4–/–WT chimeric mice. The data illustrated are from two independent experiments indicated by and •. Statistics given for the WTTLR4–/– group compare only with data from the same experiment. (C) Representative histology taken at 2 h after NTS and lipid A given together. Neutrophil influx is highlighted by arrows.
TLR4 on Both BM-Derived and Non–BM-Derived Cells Contributes to Albuminuria
Similar to the experiments outlined in the previous section,chimeric and sham chimeric mice were constructed to assess therelative importance of TLR4 on BM-derived and non–BM-derivedcells toward the development of tissue injury as measured byalbuminuria. At 10 wk after reconstitution, the chimeric andsham chimeric mice were given NTS and lipid A (10 µg permouse). Significant albuminuria developed in the WTWT groupcompared with the TLR4–/–TLR4–/– group(P < 0.001). Both chimeric groups developed a greater albuminuriathan the sham TLR4–/–TLR4–/– group (P< 0.05 for TLR4–/–WT and P < 0.001 for WTTLR4–/–).There was significantly less albuminuria in the TLR4–/–WTgroup than in the WTWT group (P < 0.001) but no significantdifference in albuminuria between the WTTLR4–/–and WTWT groups. These data are shown in Figure 3B.
TLR4 mRNA Is Expressed within the Glomerulus
To investigate the glomerular expression of TLR4, in situ hybridizationwas carried out on normal murine kidney sections. Sections wereincubated with the antisense riboprobe to assess the expressionof TLR4 mRNA, and as a control, they were incubated with a senseriboprobe. Results show that TLR4 mRNA is expressed within theglomerulus in mesangial cells and epithelial cells (both podocytesand Bowman's capsule). In total, kidneys from four mice wereexamined, and all showed an identical distribution and intensityof staining. Representative pictures from one of these fourmice are shown in Figure 4.
Figure 4. Localization of TLR4 mRNA in the glomerulus of normal mice by in situ hybridization. In total, kidneys from four mice were examined, and all showed an identical distribution and intensity of staining. Representative pictures from one of these four mice are shown. TLR4 localization is shown by incubation with the antisense riboprobe at low power (A) and high power (B). As a negative control, sections were incubated with the sense riboprobe (C). Positive staining is shown within the mesangium (m) and also in podocytes (p) and Bowman's capsule (b).
Mesangial Cells Express TLR4 and Produce CXC Chemokines in Response to Lipid A
Mesangial cells could potentially be one cell type within thekidney that contributes to disease exacerbation. Therefore,the expression of TLR4 by mesangial cells and their abilityto respond to TLR4 ligands was assessed. Primary cultures ofmouse mesangial cells were used. First we examined the expressionof TLR4 on mesangial cells by reverse transcription–PCR.Figure 5A shows that mRNA for TLR4 is expressed by mesangialcells. We then stimulated mesangial cells with LPS or vehicleand measured levels of CXCL1 and CXCL2 in the supernatant byELISA. These chemokines are known to be important for neutrophilchemotaxis. We established a dosage response for CXCL1 as shownin Figure 5B. A dosage of 1 µg/ml LPS was then used insubsequent studies. To show that the response was mediated byTLR4, we compared the response of wild-type and TLR4-deficientmesangial cells. Figure 5C shows that wild-type but not TLR4-deficientmesangial cells produced CXCL1 in response to LPS. Figure 5Dshows that wild-type but not TLR4-deficient mesangial cellsproduced CXCL2 protein in response to LPS. To establish thatthere was no intrinsic inability of TLR4-deficient mesangialcells to produce CXCL1 and CXCL2, these cells were also stimulatedwith the synthetic lipopeptide and mounted a good response.These data showed that mesangial cells produced CXCL1 and CXCL2protein in response to LPS, an effect entirely mediated by TLR4.
Figure 5. (A) Reverse transcription–PCR for TLR4 on mRNA that was extracted from mesangial cells using actin as a reference gene. Mesangial cell RNA and macrophage cDNA are included as negative and positive controls. (B) Dosage-response curve showing the concentration of CXCL1 in supernatant taken from mesangial cells that were stimulated with increasing concentrations of LPS. (C) CXCL1 concentration measured by ELISA in supernatant of wild-type and TLR4-deficient mesangial cells that were stimulated with LPS or lipopeptide. (D) CXCL2 concentration measured by ELISA in supernatant of wild-type and TLR4-deficient mesangial cells that were stimulated with LPS or lipopeptide.
CXCL1 and CXCL2 Blocking Studies
To investigate the importance of CXCL1 and CXCL2 productionby intrinsic renal cells in neutrophil recruitment in this model,we carried out blocking studies. Chimeric mice that had TLR4only on intrinsic renal cells but not on BM-derived cells wereconstructed. Mice were given 100 µg of anti-CXCL1 andanti-CXCL2 antibodies or the equivalent-dosage of isotype control.After 1 h, disease was induced by the administration of 0.2ml of NTS with 10 µg of lipid A. Assessment of diseaseseverity at 2 h is shown in Figure 6. There was significantlyless glomerular neutrophil influx in the group that was givenanti-CXCL1 and anti-CXCL2 antibodies than in the group thatwas given isotype control (P < 0.0001), showing that theglomerular neutrophil influx in this model depends on the productionof CXC chemokines in response to TLR4 ligation on intrinsicrenal cells.
Figure 6. Glomerular neutrophil influx at 2 h after disease induction in chimeric mice with TLR present only on intrinsic renal cells (TLR4–/–WT), which were given anti-CXCL1 and anti-CXCL2 antibody or isotype control at 1 h before disease induction with NTS and lipid A. Administration of anti-CXCL1 and anti-CXCL2 antibody before disease induction significantly reduced the renal neutrophil influx at 2 h (P < 0.001) when compared with the isotype control group.
We have shown that the pure TLR4 agonist lipid A is requiredin addition to glomerular antibody deposition to cause disease.Using TLR4-deficient mice, we showed that this is through amechanism that depends on the presence of TLR4. We showed thatthe TLR4-deficient mice are able to produce a glomerular neutrophilinfiltration in response to antibody deposition and stimulationwith a TLR2 ligand, demonstrating that the TLR4-deficient micedo not have an intrinsic inability to mount a neutrophil response.
The absence of significant glomerular inflammation in TLR4-deficientmice when compared with the wild-type group may have been dueto differences in the circulating leukocyte counts. At 2 h afterthe administration of lipid A and serum, the TLR4-deficientgroup had a significantly greater peripheral neutrophil countthan the wild-type group. This is probably due to the releaseof neutrophils from the BM in response to the presence of foreignserum, but they are unable to migrate to the kidney becauseof the lack of either tissue or neutrophil activation. We alsoshowed by quantitative immunofluorescence that the differencesin disease severity that were seen in the two groups cannotbe attributed to differences in sheep IgG binding within theglomerulus, because at 2 h, both groups had indistinguishableamounts.
We showed a role for TLR4 on both BM-derived and non–BM-derivedcells in this model by constructing BM chimeras. In the chimericmice, neutrophil influx was shown to be more variable than inpreliminary experiments using normal wild-type mice. Despitethis, sham chimeras that were totally TLR4 sufficient demonstratedsignificant glomerular neutrophil influx and albuminuria whencompared with those that were totally TLR4 deficient when exposedto NTS and lipid A, as we expected. Both chimeric groups demonstratedsignificant glomerular neutrophil infiltrate at 2 h after theadministration of NTS and lipid A when compared the sham-negativegroup but significantly less than the sham-positive group, showingthat TLR4 on both circulating leukocytes and intrinsic renalcells is important. With respect to albuminuria, both chimericgroups developed significantly greater albuminuria than thesham-negative group. However, only the chimeric group with TLR4present on intrinsic renal cells had significantly less albuminuriathan the sham group that was totally TLR4 sufficient. The chimericgroup with TLR4 present on neutrophils had similar albuminuriato the sham group that was totally TLR4 sufficient, despitelower neutrophil numbers at 2 h. Therefore, although TLR4 onboth intrinsic renal cells and circulating leukocytes contributedequally to glomerular neutrophil influx, it is TLR4 on circulatingleukocytes that contributed greatest to tissue injury as assessedby albuminuria.
The response of neutrophils to TLR4 ligation has been previouslyinvestigated. TLR4 ligation on human neutrophils causes changesin adhesion molecule expression, superoxide generation, andrelease of proinflammatory cytokines and augments neutrophilmigration and survival (17–19). Studies in the mouse haveshown that endotoxin causes L selectin shedding and CD11b upregulation,which is known to be essential for leukocyte adhesion and transmigration(20). In this study, we focused on the role of local renal cellsin disease exacerbation.
Three studies have highlighted the potential importance of TLR4on local tissue cells in disease. TLR4 on lung endothelial cellsand not circulating leukocytes was important in the sequestrationof neutrophils in a murine model of septic lung injury (21).Resistance to uropathogenic E. coli in the bladder and E. colipyelonephritis requires the presence of TLR4 on both stromalcells and circulating leukocytes (22,23). These studies showan important role for TLR4 in the control of infection, andour study differs from these because we show a role for TLR4stimulation of intrinsic renal cells in the expression of antibody-mediatedinflammation and tissue damage.
In situ hybridization showed expression of TLR4 in mesangialcells and epithelial cells within the glomerulus. Although bothcell types could contribute to the observed effects of ligationof TLR4 on intrinsic renal cells, mesangial cells were a particularlylikely candidate. They are immunologically active cells thatare known to be capable of producing a number of chemokinesand cytokines and have also previously been noted to respondto stimulation by endotoxin (24–26). No previous studyhas shown the expression of TLR4 in primary cultures of mesangialcells, and we have shown this. Furthermore, in response to stimulationwith pure LPS, wild-type but not TLR4-deficient mesangial cellsproduce CXC chemokines that are responsible for neutrophil chemotaxis(27,28). This provides a biologically plausible mechanism wherebyTLR4 agonists, acting on renal cells, could exacerbate disease.To support these findings, we carried out in vivo studies. Weshowed using chimeras that had TLR4 only on intrinsic renalcells that blocking CXCL1 and CXCL2 reduces the glomerular neutrophilinflux that is seen at 2 h. This demonstrates that the productionof CXC chemokines in response to ligation of TLR4 on intrinsicrenal cells contributes to glomerular neutrophil influx in thismodel.
It has been shown that TLR3 ligands exacerbate lupus nephritis.Although in this study TLR3 was expressed on mesangial cells,there was no direct demonstration of a contribution to diseaseby mesangial cell TLR3 (29). The same group investigated therole of TLR7 and TLR9 ligands in lupus nephritis, and both exacerbateddisease. Neither TLR7 nor TLR9 was expressed on intrinsic renalcells, and the mechanisms in these studies involved effectson the adaptive immune response (30,31). Taken together, thesestudies highlight the individual roles for each specific TLR;although they share the same common structure and signalingpathways, each has a unique distribution and function in relationto ligand stimulation. In the autologous phase of nephrotoxicnephritis, nephrotoxic antibody and LPS were required to producedisease at time points beyond 2 wk (32). It is possible thatdifferences in disease were related to an effect on the adaptiveimmune response in this study.
We have shown that TLR4 ligands promote renal injury by increasinginflammation, independent of any effects from the adaptive immuneresponse. Renal autoimmune disease is frequently worsened byinfection, in the absence of any measurable effect on the serumlevels of pathogenic autoantibodies, such as anti-GBM or ANCA(4), and we provide a molecular explanation for this clinicalobservation. Our data suggest that specific targeting of mesangialcell TLR4 may be a useful therapeutic strategy for preventingexacerbations of glomerulonephritis, because disease is significantlyless in chimeric mice with TLR4 absent on intrinsic renal cells.These findings contribute to our understanding of the diversemechanisms by which TLR may contribute to the exacerbation ofautoimmune disease and in particular glomerulonephritis.
We are grateful to S. Akira for supplying TLR4-deficient mice;to J. Mitchell, who helped us to obtain them; and to Ono PharmaceuticalCo. for supplying ONO-4007.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
TLR4–/–] and TLR4-deficient BM into wild-type host [TLR4–/–
and •. Statistics given for the WT
