| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,
,¶
* Renal and Vascular Research Laboratory, Fundación Jiménez Díaz, Autónoma University, Madrid, Spain;
Division of Nephrology, Department of Internal Medicine,
Department of Immunology, and
Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan; and
¶ Department of Molecular Cell Immunology and Allergology, Advanced Medical Research Center, Nihon University School of Medicine, Tokyo, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-/-) were performed. Severe glomerular damage was induced in WT and W chimeras (BM from WT to irradiated -/-), while it was absent in -/- and W chimeras (-/- BM to WT). Moreover, WT PMN transfer, but not -/- PMN, reconstituted the disease in -/-, indicating that FcR on resident cells is not a prerequisite for PMN recruitment in this disease. Surprisingly, transferred WT PMN were recruited coincidentally with NF-
B activation and TNF-
overexpression even in glomeruli with preformed IC (nephrotoxic Ab administered 3 days previously), suggesting that PMN can initially be recruited via its own FcR without previous chemoattractant release. Furthermore, H2O2 inhibition by catalase attenuated the acute WT PMN recruitment and the induction of NF-B and TNF- much more than integrin (CD18) blockade, indicating a role for the respiratory burst before integrin-dependent accumulation. In coculture experiments with IC-stimulated PMN and glomeruli, PMN caused acute glomerular TNF- expression predominantly via FcR-mediated H2O2 production. In conclusion, glomerular IC, even preformed, can cause PMN recruitment and injury through PMN FcR-mediated respiratory burst during initial PMN tethering to IC. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Leukocytes are well-established effector cells in IC diseases. Their emigration from microcirculation and activation in the focus of IC are key events in pathological conditions. In general, their adhesion and transmigration in venules are determined largely by multistep process of sequential engagement of distinct receptor molecules on the leukocytes and endothelial surfaces (6). The selectin family and its ligands mediate the initial contact between the circulating leukocytes and the vascular endothelium ("rolling"). Subsequently, rolling leukocytes encounter activating stimuli that trigger activation-dependent adhesion required for integrin-mediated firm arrest. In IC diseases, it is viewed that de novo synthesis of many structurally diverse inflammatory mediators (e.g., C5a, leukotriene B4, platelet-activating factor) from tissue-resident cells associated with IC formation may trigger hemodynamic alterations and an initial chemotaxis to endothelium of postcapillary venules adjacent to the site of IC (6, 7, 8). Those mediators may also enhance selectin expression, leading to initial leukocyte recruitment (6, 8). Although convincing evidence showed the requirement for integrins, studies addressing the role of selectins have yielded conflicting data in some models of IC GN (8, 9, 10, 11, 12, 13). Glomerular leukocyte recruitment was not attenuated in these diseases by anti-P- or E-selectin Abs (9, 10, 11) or oligosaccharide (FF7)-blocking L- and P-selectin (12) or in P-selectin-deficient mice (13). Interestingly, blockade of selectin by FF7 may affect postglomerular capillary venules, but not glomerular capillaries, in IC GN (12). This evidence reveals redundant roles for selectin in some IC diseases and indicates that different activation mechanisms on endothelial cells may underlie the regulation of the initial leukocyte recruitment.
A recent paper showed a new paradigm in leukocyte recruitment to the IC site (14). It demonstrated that the selectin-independent, but FcRIII-dependent, contact formation (tethering) of leukocytes is involved in its initial recruitment at sites of IC deposition under physiological flow. Previous studies also demonstrated that cooperation between FcR and 
2 integrins, particularly Mac-1 (CR3, CD11b/CD18), is required for polymorphonuclear cells (PMN) accumulation on IC (15, 16). In fact, these ideas are compatible with our previous findings (17) in acute passive anti-glomerular basement membrane GN (anti-GBM GN) in which immobilized GBM-anti-GBM IC trigger rapid glomerular PMN accumulation and PMN-dependent damage. In FcR-chain-deficient mice (-/- mice) lacking functional FcR (FcRI, FcRIII, Fc
RI) (4), lethal endothelial damage is completely abrogated because of the absence of acute PMN influx in this disease (17). These findings allow the possibility that intracellular signaling of FcR in PMN during its FcR-dependent tethering may be involved in and initiate the inflammatory cascade leading to its own accumulation (firm adhesion). This idea could be important for the interpretation of preformed IC injury, since this mechanism may not require soluble mediators and endothelial activation in advance of the initial leukocyte recruitment. In addition, it may suggest that the phenotype of FcR in effector cells (e.g., expression level) may influence the susceptibility to IC diseases (4). To address this hypothesis, in the current work we employed anti-GBM GN. We looked for the contribution of renal resident cells in the initial recruitment, since those cells, including resident macrophages, also possess functional FcR (18, 19, 20), and some are probably recruited from bone marrow (BM) lineage (21, 22). We therefore performed in vivo reconstitution by BM chimeras and specific myeloid lineage (PMN) transfer between -/- mice and their wild-type (WT) littermates, and additionally examined the leukocyte recruitment onto preformed IC. PMN are thought to damage glomeruli, including in this disease, due to a combination of reactive oxygen species and proteolytic enzymes (7). Previous studies with this model in cathepsin G/elastase deficient-mice (23, 24) and an injection of a cysteine proteinase inhibitor (25) demonstrated that only early proteinuria (within day 1) was dependent on proteinase, but PMN influx and development of glomerular injury were not. Thus, we postulated that the FcR-mediated PMN respiratory burst may be a rapid and powerful effector mechanism in the early cell recruitment. Additionally, we here focused on early tissue NF-B activation, a regulator of many proinflammatory mediators, and TNF- expression. Our present findings further extend the implication of FcR in the pathogenesis of IC diseases and support the idea that FcR may be a potential therapeutic target in the management of these conditions.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-/- mice were generated by a homologous recombination method with BL6/III ES cells. Construction of targeting vectors and generation of this strain have been previously described in detail (26). In all in vivo experiments, we used female animals weighing 18–23 g. -/- mice have a C57BL/6 background. Although WT littermates (+/+) and C57BL/6 mice were analyzed in this disease, no significant differences were found in the kinetics of proteinuria and glomerular/interstitial damage, as noted in our previous studies (17). Thus, the results for C57BL/6 mice matched for age were shown as representative controls of WT.
Generation of BM chimeras
BM transplantation was performed on 6- to 8-wk-old female -/- and WT mice. BM cells were collected from tibias and femurs of each mouse strain, treated with Gay’s solution to exclude RBC contamination for protection against vascular (thrombotic) injury, and then i.v. transplanted (3–5 x 107 BM cells) to mice that had been irradiated with 1000 or 600 rad of x-ray with renal protection by lead plates. Since they had the same genetic background, there were no symptoms of graft-vs-host disease in any of them. In the next 4 wk, transplanted animals were kept in air-conditioned clean cages. We generated two different BM chimeras as follows: W (BM from -/- to irradiated WT) and W (WT BM to -/-). As controls, WW (WT BM to WT) and (-/- BM to -/-) mice were also generated.
Genotype exchange in peripheral blood of each BM chimera (W and W) was determined by PCR with purified genomic DNA from peripheral blood and tissue (tail) before and 5 wk after BM transplantation (QIAamp Blood Kit: Qiagen, Hilden, Germany). Primers used for the murine FcR-chain were as follows: specific primer for exon 3 (5'-GGAATTCGCTGCCTTTCGGACCTGGAT-3') and exon 2 (5'-GGAATTCGATGC-TGTCCTGTTTTTGTA-3') and for the created neo -chain (26) in which exon 2 was replaced (5'-GCCAACGCTATGTCCTGATAG-3'). PCR was simultaneously performed with these primers under the following conditions: 94°C for 1 min, 57°C for 1 min, and 72°C for 1.5 min for 33 cycles. After confirmation of the genotype exchange, these chimeras were subjected to the general experiments.
Experimental protocol for anti-GBM GN
The method for preparation of nephrotoxic serum (NTS) has been previously described (17). Anti-GBM GN was induced by i.v. injection of NTS through the tail vein in mice that had been preimmunized with rabbit IgG and CFA 4 days before NTS administration and followed until day 100. Since a preliminary study showed that NTS at a dose of 20 µl/20 g BW was sufficient to cause proteinuria and severe renal damage in WT mice, we employed this dose in WT and -/- mice and their chimeras. No mice developed anaphylactic symptoms after the injection of NTS. Urinary protein was determined by Knight’s method, as previously described (17). Kidneys were perfused with cold saline and removed under general anesthesia.
For evaluation of the effect of reactive oxygen species, -/- mice were i.p. injected with bovine liver catalase (534,000 U/20 g body weight; Sigma-Aldrich, Madrid, Spain) (27) or anti-CD18 neutralizing Ab (250 µg/20 g body weight; BD PharMingen, Heidelberg, Germany) (11, 28) three times at 16, 8, and 2 h before the injection of PMN.
Preparation of PMN from BM
For in vitro differentiation of PMN, collected BM cells were incubated overnight in DMEM supplemented with 20% FCS, 15% cell culture supernatant derived from Wehi-3b cells (ATCC TIB-68; American Type Culture Collection, Manassas, VA), 1% glutamine, and antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin) in 5% CO2 at 37°C as previously described (29). Before each experiment, PMN were analyzed for the expression of CD18 and Gr-1 (BD PharMingen), a marker of mature PMN, using a flow cytometer.
For in vivo transfer, we prepared 5 x 106 (high dose) or 5 x 105 (low dose) PMN and injected them through the tail vein 3 h before or 3 days after the injection of NTS. Preliminarily, we confirmed that no mice developed anaphylactic symptoms until 1 wk after the injection of PMN alone.
Renal histopathologic studies
Kidney sections fixed in 4% paraformaldehyde were stained with periodic acid-Schiff (PAS) reagent or Masson’s Trichrome in 4-µm-thick sections to assess histological changes by light microscopy. We first examined the diameter of representative glomeruli in the renal cortex of mice with nephritis at 2 h (0.11 ± 0.014 mm; n = 6). The sizes of the glomeruli were not significantly different from those in normal mice of the same age (0.097 ± 0.03 mm; n = 3). Thus, glomeruli with a diameter larger than 0.1 mm (>15 glomeruli) were employed for the evaluation of PMN influx, and more than three animals per group were examined. The results were expressed as cells per glomerular cross-section.
Frozen renal sections were used for immunofluorescence for rabbit IgG and murine C3 and then stained with FITC-labeled Abs (Cappel/ICN, Aurola, OH; DAKO, Barcelona, Spain). The extent of glomerular immunostaining of IgG and C3 was semiquantitatively determined by means of a scale from 0 (none) to 4+ (very intensive), as previously described (30).
RNA extraction and RT-PCR
Total RNA was obtained by the TRIzol method (Life Technologies, Gaithersburg, MD). One microgram of RNA was reverse transcribed and then amplified with a commercial kit (Promega, Southampton, U.K.), with the use of 0.5 µCi of [-32P]dCTP (3000 Ci/mmol; Amersham International, Little Chalfont, U.K.) and 20 pmol of specific primers for mouse TNF- (sense, 5'-GCGACGTGGAACTGGCAGAAG-3'; antisense, 5'-GGTACAACCCATCGGCTGGCA-3'; fragment, 384 bp) (31) and mouse GAPDH (sense, 5'-CCGGTGCTGAGTATGTAGTG-3'; antisense, 5'-CAGTCTTCTGAGTGGCAGTG-3'; fragment, 289 bp; reference no. AK 013857). The amplifications were conducted with annealing temperatures of 63°C (TNF-) or 57°C (GAPDH). The optimum number of amplification cycles used for semiquantitative RT-PCR (in vivo, 30 and 25; in vitro, 32 and 25, respectively) was chosen on the basis of pilot experiments (data not shown). The expression of GAPDH was used as an internal control. Aliquots of each reaction were run on 4% acrylamide-bisacrylamide gels. The gels were dried and exposed to X-OMAT AS films (Eastman Kodak, Madrid, Spain). Autoradiograms were quantified by scanning densitometry (Molecular Dynamics, Sunnyvale, CA). The density of each gene was compared after individual correction by density of GAPDH.
Extraction of nuclear proteins and EMSA
Nuclear extracts were obtained as previously described (31), and the activity of transcription factors was evaluated by EMSA. Briefly, frozen samples of renal cortex were pulverized in a metallic chamber and resuspended in a cold extraction buffer (20 mM HEPES-NaOH (pH 7.6), 20% (v/v) glycerol, 0.35 M NaCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, and 1 µg/ml pepstatin A). The homogenate was vigorously shaken, and the insoluble materials were precipitated by centrifugation at 12,000 rpm for 30 min at 4°C. Supernatants were dialyzed overnight against a binding buffer containing 20 mM HEPES-NaOH (pH 7.6), 20% (v/v) glycerol, 0.1 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, and 0.5 mM PMSF. These dialysates were cleared by centrifugation at 10,000 x g for 15 min at 4°C and stored in aliquots at -80°C until use. The protein concentration was quantified by the bicinchoninic acid method (Pierce, Rockford, IL).
NF-B consensus oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3'; Promega) was 32P end-labeled by incubation for 10 min at 37°C with 10 U of T4 polynucleotide kinase (Promega) in a reaction containing 10 µCi of [-32P]ATP (3000 Ci/mmol; Amersham Pharmacia Biotech, Arlington Heights, IL), 70 mM Tris-HCl, 10 mM MgCl2, and 5 mM DTT. The reaction was stopped by the addition of EDTA to a final concentration of 0.05 M. Nuclear proteins (20 µg) were equilibrated for 10 min in a binding buffer containing 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), and 50 µg/ml poly(dI-dC). When competition assays were performed, the cold probe was added to this buffer 15 min before addition of the labeled probe. Labeled probe (0.035 pmol) was added to the reaction and incubated for 20 min at room temperature. The reaction was stopped by the addition of gel loading buffer (250 mM Tris-HCl, 0.2% bromophenol blue, 0.2% xylene cyanol, and 40% glycerol) and was run on a nondenaturing, 4% acrylamide gel at 100 V at room temperature in 89 mM Tris-borate and 2 mM EDTA, pH 8.0. The gel was dried and exposed to X-OMAT AS films. Autoradiograms were quantified by scanning densitometry.
Southwestern immunohistochemistry
This technique was developed to detect the in situ distribution and DNA-binding activity of transcriptional factors (32). NF-B consensus oligonucleotide was digoxigenin labeled with a 3'-terminal transferase (Roche, Mannheim, Germany). Paraffin-embedded tissue sections were fixed in 0.5% paraformaldehyde and incubated with 0.1 mg/ml DNase I. The DNA binding reaction was performed by incubation with 50 pmol of the labeled DNA probe in buffer containing 0.25% BSA and 1 µg/ml poly(dI-dC). The sections were then incubated with alkaline phosphatase-conjugated anti-digoxigenin Ab, and colorimetric detection was performed as described. Preparations without probe were used as negative controls, and an excess of unlabeled probe was used to test the specificity of the technique.
Preparation of substrate-coated coverslips
BSA-anti-BSA IgG IC were immobilized on tissue culture plastic coverslips (Thermanox, Miles Scientific, Evanston, IL) with minor modifications of a previous report (14). Round coverslips (13 mm in diameter) were coated with 1 mg/ml BSA for 30 min, washed twice in PBS, incubated with 0.1 M glycine for 2 h to quench aldehyde groups, and then incubated with 20 µg of rabbit anti-BSA IgG (Sigma-Aldrich) in 0.5 ml of PBS for 1 h. This concentration of anti-BSA IgG has been previously shown to yield maximum adhesion of murine PMN to IC and leukotriene B4 release from these cells (15). In addition to the BSA plate without the Ab, we prepared BSA-anti-BSA (Fab) (Fab IC) coverslips as a control to confirm the involvement of FcR. Fab were prepared by the digestion of anti-BSA IgG with papain (40/1 mg ratio, 30 min, 37°C). Undigested IgG and papain were subsequently removed by filtration through protein A-Sepharose and BSA-agarose, and Fab were dialyzed against PBS.
Respiratory burst assay
PMN from each mouse strain were incubated with BSA-IC coverslips for 30, 60, and 120 min. Concentrations of hydrogen peroxide (H2O2) in aqueous solutions were measured by Peroxi Detect (Sigma-Aldrich), which is based on the fact that peroxides at acidic pH convert Fe2+ to Fe3+. The Fe3+ ion forms a colored adduct with xylenol orange detectable at 560 nm. The standard of 100 µM H2O2 was prepared by observing the absorbance at 240 nm (10 mM = 0.436 OD240). A standard curve of nanomoles of H2O2 was plotted by 0, 10, 20, 40, 60, and 80 µl of the 100 µl of standard H2O2. The nanomoles of peroxide in the sample were calculated from the standard curve: nmol peroxide/ml = [A 560 sample - A 560 blank] x dilution factor/[A 560 1 nmol peroxide] x (vol of sample).
To examine the effect of IC-induced PMN respiratory burst on glomerular gene expression, a Transwell chamber (Costar, High Wycombe, U.K.) with 0.4-µm pore-sized membrane (24 mm in diameter), where PMN cannot pass through, was used. The lower wells were loaded in triplicate with 2 x 104 glomeruli isolated by a sieving method as previously described (33). IC-, Fab IC-, or BSA alone-coated coverslips (13 mm in diameter) were put on the membrane of the upper chamber. Then a 1 x 106 PMN suspension was loaded into the upper chamber. Glomeruli from WT and -/- mice were coincubated for 2 h and then collected for RNA. In some cases PMN or glomeruli were pretreated with 1 x 104 U/ml catalase 30 min before loading into the chamber.
Statistical analyses
Results are expressed as the mean ± SD and were analyzed by ANOVA (Figs. 1 and 2) and Mann-Whitney test (Figs. 3–6) for comparison of quantitative variables. Statistical significance was established as p < 0.05 (two-tailed curve). For statistical analysis of survival rate (Fig. 1d), Yates’s correction was employed.
|
|
|
|
|
|
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To clarify the relevant cell types expressing FcR, we generated BM chimeras between mice lacking functional FcR (-/-) and their WT littermates. We also generated control mice that were transplanted with BM from the same mouse strain ( and WW) under the same irradiation conditions. In all types of mice, anti-GBM GN was induced as mentioned in Materials and Methods. We failed to find any significant differences in urinary protein and renal lesions between -/- and or between WT and WW (data not shown), indicating that BM in recipient animals was functionally reconstituted by transplantation, and the irradiation condition did not elicit any significant alteration of this disease.
We next analyzed the evolution of anti-GBM GN in these animals. All WT mice fell into lethal states with massive ascites until 6 wk after the administration of NTS, with a peak of massive proteinuria and hematuria around day 10 (Fig. 1a). Renal lesions, mainly characterized by endothelial swelling and fibrin deposition, were observed in WT mice (Fig. 1b). On the other hand, although there were no significant differences in the extent of glomerular anti-GBM IgG/C3 deposition as in WT mice (WT vs -/-: rabbit IgG, 3 ± 0.71 vs 3.2 ± 0.45 (p = 0.61); mouse C3, 2 ± 0.71 vs 1.8 ± 0.84 (p = 0.69)), all -/- mice survived without pathological proteinuria and obvious renal lesions even on day 100 (Fig. 1a and Fig. 2, a and b). CD4+ T cell-dependent injury, characterized by mesangial proliferation as described in our recent study (34), was not induced at this dose of NTS in -/- mice.
At 5 wk after BM transplantation (day 0), the genotypes of peripheral blood in W and W chimeras were replaced with that of WT or -/- mice, respectively (Figs. 1c and 2c). These W chimeras showed massive proteinuria (Fig. 1a) and glomerular lesions characterized by severe endothelial injury and fibrin deposition (Fig. 1b) to the same extent as in WT mice. Although we detected no significant difference in the peak proteinuria between WT and W chimeras (Fig. 1a), W chimeras survived significantly longer than WT mice (day 21, p = 0.003056; day 49, p = 0.0000389; Fig. 1d). In contrast, W chimeras as well as -/- mice (W/1000 rad) were protected from glomerular injury (Fig. 1a and Fig. 2, a and b). These findings confirm that BM-derived cells expressing FcR are responsible for the induction of this disease.
Glomerular damage can be induced coincidentally with the recovery of recipient WT BM in W/600 rad BM chimeras
To determine whether FcR+ BM-derived cells can induce glomerular injury in response to preformed IC, we manipulated the recovery of the recipient (WT) BM in W chimeras with this disease by lower levels of irradiation (600 rad). As shown in Fig. 2c, the genotypes of peripheral blood in both groups of W chimeras irradiated with 1000 or 600 rad were replaced with that of -/- mice at the beginning of this experiment (day 0). Although the genotype in W/1000 rad chimeras did not change during the disease course, W/600 rad chimeras showed a heterogeneous genotype (-/- and WT) in peripheral blood after day 40, indicating that recipient WT BM gradually recovered and delivered certain amounts of FcR+ circulating cells. Interestingly, coincident with the recovery, moderate proteinuria and glomerular lesions with endothelial damage appeared even after day 40 following the injection of NTS and were sustained until day 100 (Fig. 2, a and b). This suggests that recovered FcR+ BM-derived cells can induce considerable severe glomerular damage even 40 days after IC formation.
PMN recruitment and renal injury in -/- mice are reconstituted by FcR-positive PMN transfer with both acute (freshly formed) and preformed IC
Recent papers have suggested that a certain population of glomerular cells is recruited from BM-derived precursor cells (21) and may contribute to the pathogenesis of GN (22). Therefore, we next prepared in vitro differentiated PMN from BM cells and transferred them in vivo to determine the potential contribution of intrinsic or BM-derived FcR+ renal cells to this disease. After the incubation of BM cells in the specific conditioned medium as previously described (29), we evaluated their differentiation to PMN by staining of surface Ags on surviving cells with anti-Gr-1 and anti-CD18 Abs. Gr-1 Ag is directly correlated with granulocyte differentiation and maturation (35), while CD18 can be expressed not only in granulocytes, but also in NK, T, and B cells (36). With this protocol, we obtained a double-positive (Gr-1+/CD18+) cell population (88.8 ± 3.0; n = 5) by flow cytometric analysis and employed those positive cells for in vivo transfer and in vitro assays.
Then we explored the reconstitution of the disease, as seen in W and W/600 rad chimeras, by WT PMN transfer to -/- mice. However, it is known that i.v. cell administration induces nonspecifically transient hematuria and proteinuria, partially due to the systemic activation of complement. We also found transient urinary alterations even in the case of PMN transfer to normal mice (data not shown) until 12 h, but not after day 1. Therefore, we analyzed proteinuria after day 1. Transferred WT PMN followed by serum administration (PMN
Ab) caused proteinuria after day 1 in a dose-dependent manner (Fig. 3a). PMN influx was also detected 2 h after serum injection (Fig. 3c). -/- PMN transfer to -/- mice failed to induce proteinuria after day 1 or sufficient PMN influx (
71% less influx than when transferred with WT PMN; Fig. 3, a and c). Those data indicate that pathological proteinuria is caused by renal injury associated with the FcR-dependent PMN influx and confirm that FcR on renal resident cells are not prerequisites for glomerular PMN recruitment in this disease. Consistently, WT PMN transfer to -/- mice 3 days after NTS administration (AbPMN) induced glomerular damage associated with PMN influx at 2 h, but not -/- PMN transfer (69% less influx than WT PMN transfer; Fig. 3, b and c). This result further suggests that PMN can be recruited into glomeruli via their own FcR, presumably without de novo synthesis of soluble mediators by IC deposition.
Early induction of inflammatory mediators (NF-B and TNF-) is markedly attenuated in -/- mice
To address potential mechanisms in the initial PMN recruitment, we analyzed renal expression levels of inflammatory mediators, such as the cytokine TNF- and the transcription factor that regulates its expression (NF-B). As shown in Fig. 4a, a marked renal NF-B activation was noted after 2 h in WT mice, but not in -/- mice. WT mice also showed overexpression of TNF-, peaking at 2 h, that was dramatically attenuated in -/- mice (Fig. 4b). These data confirm that early induction of these mediators is critical for the evolution of this disease.
We next examined these mediators in transfer models. In both models (PMNAb or AbPMN in -/- mice), the transferred WT PMN induced significantly greater renal NF-B activation (Fig. 5a) and TNF- expression (Fig. 5b) than the transferred -/- PMN. Southwestern immunohistochemistry showed that WT PMN transfer, but not -/- PMN transfer, induced a marked increase in NF-B activation, not only in glomerular PMN but also in glomerular capillary endothelial cells (Fig. 5c), suggesting that the PMN recruitment via FcR is able per se to trigger inflammation through the activation of NF-B and regulated cytokine production.
Pretreatment with catalase attenuated glomerular PMN influx, NF-B activation, and TNF- overexpression
The next series of in vivo experiments was undertaken to explore the potential role of the respiratory burst in the early induction of renal NF-B and TNF-, especially in the transfer model with preformed IC, since soluble mediators associated with acute IC formation may also influence it. In addition, we evaluated the relative contribution of the integrin-mediated cascade. As shown in Fig. 3c, H2O2 blockade by catalase reduced PMN recruitment at 2 h (51.5 ± 3.2% reduction) much more than integrin blockade by an anti-CD18 neutralizing mAb (30.8 ± 7.3% reduction). Similar protective effects were found in renal NF-B activation and TNF- expression (reductions: catalase, 47.1 ± 5.9 and 59.7 ± 12.6%; anti-CD18, 17.7 ± 7.7 and 40.1 ± 11.5%, respectively). These data indicate that local H2O2 production by PMN transfer may play an important role in its own further recruitment, leading to integrin-dependent adhesion and early activation of inflammatory mediators.
Glomerular TNF- expression can be enhanced by PMN FcR-mediated respiratory burst
To mimic the in vivo GBM-anti GBM IC interaction with PMN, some slides were coated with BSA-anti BSA IgG IC and incubated with resting BM-derived PMN from WT and -/- mice in the absence of serum to eliminate the potential effects of complement. H2O2 production was measured at 0, 30, 60, and 120 min. WT PMN incubated with IC-coated coverslips showed, at 60 and 120 min, a significantly higher H2O2 production than -/- PMN (Fig. 6a), although -/- PMN with IC- or BSA alone-coated coverslips weakly produced H2O2. Pretreatment of WT (Fig. 6a) and -/- PMN (data not shown) with catalase reduced IC-induced H2O2 production to <0.1 nmol/ml, confirming the extracellular release of H2O2 by BM-derived PMN. These findings suggest that under these conditions FcR were a major effector molecule for PMN H2O2 release.
Next, we examined whether H2O2 can directly stimulate glomerular TNF- expression. As shown in Fig. 6b, both WT and -/- PMN incubated with BSA alone-coated coverslips increased glomerular TNF- expression at 2 h compared with that in coverslips without BSA coating. However, WT PMN incubated with IC-coated plates elicited significantly higher TNF- expression in both WT and -/- glomeruli than that by -/- PMN. Pretreatment with catalase reduced WT PMN-induced TNF- expression (40.5 ± 7.2% reduction). The same tendency in glomerular TNF- expression was found between IC- and Fab IC-coated plates (fold increase to Fab IC-coated plates (n = 4): WT PMN vs -/- PMN on the IC-coated plate, 1.55 ± 0.18 vs 0.98 ± 0.1 (p < 0.01) with -/- glomeruli; 1.59 ± 0.19 vs 0.87 ± 0.07 (p < 0.01) with WT glomeruli). Together these data demonstrate that FcR-mediated H2O2 from PMN can directly enhance the early TNF- expression in glomeruli.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
At first the current work demonstrates that the PMN-dependent severe glomerular endothelial damage observed in WT mice could be reconstituted in W BM chimeras (FcR-deficient mice (-/-) transplanted with WT BM), but not in W chimeras. This finding clarifies the idea that FcR on BM-derived cells are essential for the induction of this disease. Emerging data have shown that a certain population of tissue cells (e.g., dendritic cells) is recruited from BM-derived precursor cells that are likely to mediate immunological responses, including endocytosis via FcR (37), in various diseases. Indeed, glomerular cells are also recruited from those precursors in GN (22). This evidence suggests that FcR+ cells were delivered into the glomeruli of W chimeras by BM transplantation. Taking into account the presence of FcR on intrinsic mesangial cells (18, 19, 38), it was argued that FcR on renal resident cells may contribute to PMN recruitment. However, the present results clearly demonstrate that WT PMN transfer alone can reconstitute this disease in -/- mice. Moreover, a striking absence of acute glomerular damage in W chimeras was observed, even though FcR+ glomerular resident cells may already exist in the glomeruli of this chimera. Accordingly, we conclude that FcR on BM-derived PMN are prerequisites for the induction of anti-GBM GN, whereas FcR on renal resident cells may not play an important role in the early recruitment. Of note, W chimeras survived significantly longer than WT mice, although there was no significant difference in the initial injury between W chimeras and WT mice. Since the genotype of circulating cells in all W chimeras did not change, we postulate that renal intrinsic cells expressing FcR may contribute to the progression of this disease through other mechanisms, such as the interaction with heterologous Ab deposited in the mesangial area.
It has been considered that hemodynamic alterations and soluble mediators, such as C5a and leukotriene B4, associated with glomerular IC formation may provoke the initial leukocyte contact (7). We do, however, note that transferred WT PMN, but not -/- PMN, were recruited into glomeruli with preformed IC in -/- mice and induced glomerular damage as well as glomeruli with acute (freshly formed) IC. Consistently, W/600 rad chimeras also showed glomerular injury in accordance with the restitution of recipient WT BM after day 40. Although glomerular injury in both cases showed relatively less severity than that in WT mice, the present data suggest that PMN initially can encounter IC exposed at the open endothelial fenestrae, predominantly via their FcR, and trigger their margination in glomerulus with neither chemoattractive mediators nor strong endothelial activation in advance. A recent study demonstrates that PMN can be tethered onto IC in an FcR-dependent manner under physiological flow (14) and thus further supports our idea, although hemodynamic forces in the glomerular capillary may be different from those in venules, and these forces can be altered in IC-deposited glomerulus. In addition, this idea is compatible with previous findings that C3, C4, and C5a deficiency have no significant influence on glomerular PMN accumulation in this disease (15, 39, 40). In humans, neutrophils express FcRIIA (41) and glycophosphatidyl inositol-linked FcRIIIB (14), which are adherent to IC under flow conditions, but only the latter receptor has been proven to mediate the adherence under physiological conditions (2.0 dynes/cm2). The topographic localization of receptors on microvilli is critical for contact formation (tethering) (14, 42). In fact, FcRIIIB of human leukocytes is present on the microvilli (14). On the other hand, murine neutrophils do not express these receptors (4), but our current findings underscore that -chain-associated FcR, presumably FcIIIA, may be responsible for the tethering to both acute and preformed IC in the murine system. Consistently, initial PMN recruitment onto acute IC is absent in FcRIII-deficient mice as well as in -/- mice (14, 17). Therefore, this evidence indicates the microvillous localization of murine FcRIIIA and suggests that the spatial proximity at microvillous protrusions may favor ligation of FcRIIIA, inducing effector functions during initial tethering.
Firm adhesion of leukocytes is known to be mainly dependent on integrins (6). The 2 leukocyte integrins share a common -chain (CD18). Three subunits are noncovalently associated with CD18: CD11a (LFA-1), CD11b (Mac-1), and CD11c (p150, 95). In anti-GBM GN, studies with knockout mice (15) and using mAb treatments with anti-CD18 and anti-CD11b (11, 28), but not anti-CD11a (11), showed a striking decrease in leukocyte infiltration and proteinuria, highlighting a critical role for integrins in their adhesion. Certain previous studies indicate that selectin-mediated rolling may be of limited importance in PMN influx into the glomerulus during this disease, beyond the species differences (8, 9, 10, 11, 12, 13). Sufficient PMN accumulation and PMN-dependent proteinuria were lacking in Mac-1-deficient mice with this disease (15), because Mac-1, through interactions with or signals from FcR, is required for the filamentous actin reorganization necessary for stabilizing PMN interactions with IC. However, in this mutant, an initial FcR-dependent PMN influx accompanied by denudation of fenestrated endothelium as their activation outcome were observed in WT mice as well (15), indicating some other selectin-independent mechanisms activating endothelial cells before or overlapping the integrin activation cascade. The present results revealed that early PMN recruitment into glomerulus with preformed IC was attenuated by catalase treatment (which degrades H2O2 to H2O) much more than by anti-CD18 neutralizing Ab. This protective effect by catalase was also found in NF-B activation and TNF- expression. It is known that renal TNF- expression is regulated through NF-B activation (43), and that NF-B activation in endothelial cells is critically involved in TNF--induced adhesion molecule expression (44). In this sense it is noteworthy that Southwestern immunohistochemistry showed strong activation of NF-B not only in recruited PMN, but also in endothelial cells of -/- mice in the case of WT PMN transfer. In addition, preconditioning of adherent PMN by TNF-, such as activation of several specific kinase pathways, is required for their Mac-1-mediated effector function (45, 46). Therefore, those findings suggest that the selectin- and CD18-independent, IC-mediated respiratory burst in this acute phase may play a certain role in endothelial activation leading to integrin-dependent accumulation. TNF- is the predominant cytokine regulating PMN recruitment via the expression of endothelial adhesion molecules from upstream of the cascade in IC diseases, including anti-GBM GN (6, 8, 47). Under the present experimental conditions without physiological flow, -/- PMN also bound to the IC-coated surface and produced H2O2. This is probably due to the interaction with BSA or aldehyde moieties incompletely quenched. However, WT PMN showed a markedly higher production of H2O2 and a stronger induction of glomerular TNF- than -/- PMN, indicating a predominant contribution of FcR-mediated signaling. Accordingly, our present findings indicate that recruited (tethered) PMN, even transferred, may rapidly cause NF-B activation in the same cells and in the kidney through FcR-induced respiratory burst. Then, up-regulation of NF-B-dependent inflammatory mediators in glomeruli, such as TNF-, may facilitate additional PMN recruitment, presumably via increased adhesion molecule expression. Moreover, Mac-1 on PMN enhances FcR-IgG effector function (15, 48) and elicits further respiratory burst with interaction of GBM matrix component accompanied by TNF- (49), suggesting that an acceleration loop by FcR and Mac-1 may be involved in the chain of events after this initial activation in transferred PMN. Indeed, we initially observed PMN accumulation after 2 h, similar to the findings of our previous study (17), in both WT PMN transfer models (data not shown). The remaining PMN recruitment, NF-B activation, and TNF- expression were also found in -/- PMN transfer models. Therefore, we have to carefully elucidate some other adhesion receptors (e.g., 41) (50) or other mediators (e.g., angiotensin II) (34) in addition to some alteration in the in vitro differentiated PMN.
In summary, the present study demonstrates that glomerular IC, even preformed, can cause PMN recruitment and glomerular damage in anti-GBM GN, partly by an FcR-mediated respiratory burst in a PMN-dependent, but resident cell-independent, manner. Importantly, once it occurs, even though this mechanism cannot induce full development of glomerular damage, inflamed endothelial surface may alter the disease course, as seen in W/600 rad BM chimeras, presumably through the facilitation of sensitized-lymphocyte infiltration, and therefore contribute to the perpetuation of IC diseases. This potential mechanism also postulates that the expression level of FcR on leukocytes may influence on the susceptibility and perpetuation of IC disease or its flare-up. This would partly explain clinical evidence that bouts of the disease are frequently associated with unspecific upper respiratory tract infection, because cytokines related to common infection, such as IFN-, are known to enhance the expression levels of FcR on leukocytes (51). Although systemic blockade of FcR confers beneficial effects in acute IC glomerulonephritis (52), FcR exert physiological effects during the infection. Thus, tissue-specific blockade of the FcR-mediated effector mechanism would probably be a better approach for the management of chronic IC diseases.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Jesús Egido, Renal and Vascular Research Laboratory, Fundación Jiménez Díaz, Autónoma University, Avenida de Reyes Católicos 2, 28040 Madrid, Spain. E-mail address: jegido{at}fjd.es
3 Abbreviations used in this paper: IC, immune complex; anti-GBM GN, anti-glomerular basement membrane glomerulonephritis; BM, bone marrow; Fab IC, prepared BSA-anti-BSA (Fab); -/-, FcR deficient; GN, glomerulonephritis; NTS, nephrotoxic serum; PAS, periodic acid-Schiff; PMN, polymorphonuclear cells; WT, wild type.
Received for publication September 9, 2002. Accepted for publication January 3, 2003.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RIIIa (CD16) alters receptor function and predisposes to autoimmune disease. J. Clin. Invest. 100:1059.[Medline]
receptor gene in patients with IgA nephropathy. Hum. Genet. 108:128.[Medline]
RIII mediates neutrophil recruitment to immune complexes. a mechanism for neutrophil accumulation in immune-mediated inflammation. Immunity 14:693.[Medline]
receptor-dependent neutrophil adhesion and complement-dependent proteinuria in acute glomerulonephritis. J. Exp. Med. 186:1853.M2 avidity in polymorphonuclear neutrophils. J. Biol. Chem. 273:10556.RIII-A) and the associated FcRI-chain. J. Immunol. 153:1281.[Abstract]
2 integrins (CD11/CD18) in the regulation of cytokine gene expression of polymorphonuclear neutrophils during the inflammatory response. FASEB J. 13:1855.B in cultured mesangial cells mainly via AT(2) receptors: studies with AT(1) receptor-knockout mice. J. Am. Soc. Nephrol. 13:1162. receptors of human mesangial cells activates transcription factor nuclear factor-B and induces expression and synthesis of monocyte chemoattractant protein-1, IL-8, and IFN-inducible protein 10. J. Immunol. 159:3474.[Abstract]
receptors in vivo. Curr. Opin. Nephrol. Hypertens. 8:293.[Medline]
B (NF-B) and renal disease. Kidney Int. 59:415.[Medline]
B and cytokine-inducible enhancers. FASEB J. 9:899.[Abstract]
elicits association of PI 3-kinase with the p60TNFR and activation of PI 3-kinase in adherent neutrophils. Biochim. Biophys. Acta 281:651.
activation of the c-Jun N-terminal kinase pathway in human neutrophils: integrin involvement in a pathway leading from cytoplasmic tyrosine kinases apoptosis. J. Biol. Chem. 276:2189.M2, CD11b/CD18) and FcRIII cooperate in generation of a neutrophil respiratory burst: requirement for FcRIII and tyrosine phosphorylation. J. Cell Biol. 125:1407. augments the pro-inflammatory interaction between PMN and GBM via a CD18 dependent mechanism. Kidney Int. 48:698.[Medline]
4-integrin: an alternative pathway for neutrophil recruitment?. Immunol. Today 20:545.[Medline]
This article has been cited by other articles:
|
|
 |
|
  T. N. Mayadas, G. C. Tsokos, and N. Tsuboi Mechanisms of Immune Complex-Mediated Neutrophil Recruitment and Tissue Injury Circulation, November 17, 2009; 120(20): 2012 - 2024. [Full Text] [PDF]
|
|
|
|
 |
|
 C. N. Paiva, R. H. Arras, E. S. Magalhaes, L. S. Alves, L. P. Lessa, M. H. Silva, R. Ejzemberg, C. Canetti, and M. T. Bozza Migration inhibitory factor (MIF) released by macrophages upon recognition of immune complexes is critical to inflammation in Arthus reaction J. Leukoc. Biol., May 1, 2009; 85(5): 855 - 861. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Jakus, E. Simon, D. Frommhold, M. Sperandio, and A. Mocsai Critical role of phospholipase C{gamma}2 in integrin and Fc receptor-mediated neutrophil functions and the effector phase of autoimmune arthritis J. Exp. Med., March 16, 2009; 206(3): 577 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Utomo, J. Hirahashi, D. Mekala, K. Asano, M. Glogauer, X. Cullere, and T. N. Mayadas Requirement for Vav Proteins in Post-Recruitment Neutrophil Cytotoxicity in IgG but Not Complement C3-Dependent Injury J. Immunol., May 1, 2008; 180(9): 6279 - 6287. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. G. Wong, Y. Suzuki, C. Tanifuji, H. Akiba, K. Okumura, T. Sugaya, T. Yamamoto, S. Horikoshi, S. Y. Tan, C. Pollock, et al. Peritubular Ischemia Contributes More to Tubular Damage than Proteinuria in Immune-Mediated Glomerulonephritis J. Am. Soc. Nephrol., February 1, 2008; 19(2): 290 - 297. [Full Text] [PDF]
|
|
|
|
|
|
Z. Jakus, T. Nemeth, J. S. Verbeek, and A. Mocsai Critical but Overlapping Role of Fc{gamma}RIII and Fc{gamma}RIV in Activation of Murine Neutrophils by Immobilized Immune Complexes J. Immunol., January 1, 2008; 180(1): 618 - 629. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Gallego-Delgado, A. Lazaro, J. I. Osende, V. Esteban, M. G. Barderas, C. Gomez-Guerrero, R. Vega, F. Vivanco, and J. Egido Proteomic Analysis of Early Left Ventricular Hypertrophy Secondary to Hypertension: Modulation by Antihypertensive Therapies J. Am. Soc. Nephrol., December 1, 2006; 17(12_suppl_3): S159 - S164. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Kishimoto A Novel Approach to the Suppression of Atherosclerosis by Fc{gamma} Receptor Blockade Circ. Res., November 24, 2006; 99(11): 1154 - 1155. [Full Text] [PDF]
|
|
|
|
|
|
P. Hernandez-Vargas, G. Ortiz-Munoz, O. Lopez-Franco, Y. Suzuki, J. Gallego-Delgado, G. Sanjuan, A. Lazaro, V. Lopez-Parra, L. Ortega, J. Egido, et al. Fc{gamma} Receptor Deficiency Confers Protection Against Atherosclerosis in Apolipoprotein E Knockout Mice Circ. Res., November 24, 2006; 99(11): 1188 - 1196. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Bergtold, A. Gavhane, V. D'Agati, M. Madaio, and R. Clynes FcR-Bearing Myeloid Cells Are Responsible for Triggering Murine Lupus Nephritis J. Immunol., November 15, 2006; 177(10): 7287 - 7295. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Suzuki, M. Ruiz-Ortega, C. Gomez-Guerrero, Y. Tomino, and J. Egido Angiotensin II, the immune system and renal diseases: another road for RAS? Nephrol. Dial. Transplant., August 1, 2003; 18(8): 1423 - 1426. [Full Text] [PDF]
|
|
|
|
|
|
Y. Suzuki, M. Ruiz-Ortega, C. Gomez-Guerrero, Y. Tomino, and J. Egido Angiotensin II, the immune system and renal diseases: another road for RAS? Nephrol. Dial. Transplant., August 1, 2003; 18(88): 1423 - 1426. [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
