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* Laboratory of Molecular Autoimmune Disease, Renal Division, Brigham and Women’s Hospital, Boston, MA 02115; and
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 10461
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) during inflammation. Our prior studies indicate that activated M release soluble mediators that incite TEC death, and reducing intrarenal M during kidney disease diminishes TEC apoptosis. CSF-1 is required for M proliferation and survival. We hypothesized that in the absence of CSF-1, M-mediated TEC apoptosis would be prevented during renal inflammation. To test this hypothesis, we evaluated renal inflammation during unilateral ureter obstruction in CSF-1-deficient (Csf1op/Csf1op) mice. We detected fewer M and T cells and less apoptotic TEC in the obstructed kidneys of Csf1op/Csf1op mice compared with wild-type (WT) mice. The decrease in intrarenal M resulted from diminished recruitment and proliferation, not enhanced apoptosis. CSF-1 enhanced M activation. There were far fewer activated (CD69, CD23, Ia, surface expression) M in obstructed CSF-1-deficient compared with WT obstructed kidneys. Similarly, bone marrow M preincubated with anti-CSF-1 receptor Ab or anti-CSF-1 neutralizing Ab were resistant to LPS- and IFN-
-induced activation. We detected fewer apoptotic-inducing molecules (reactive oxygen species, TNF-
, inducible NO synthase) in 1) M propagated from obstructed Csf1op/Csf1op compared with WT kidneys, and 2) WT bone marrow M blocked with anti-CSF-1 receptor or anti-CSF-1 Ab compared with the isotype control. Furthermore, blocking CSF-1 or the CSF-1 receptor induced less TEC apoptosis than the isotype control. We suggest that during renal inflammation, CSF-1 mediates M recruitment, proliferation, activation, and, in turn, TEC apoptosis. |
Introduction |
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) release toxic mediators that induce apoptosis in resident parenchymal cells. We have demonstrated that activated, but not inactivated, M release soluble molecules that induce apoptosis in TEC (1). Furthermore, reducing the influx of M by blocking chemokines (monocyte chemoattractant protein-1 (MCP-1)) proportionally diminishes the apoptotic TEC (1). Thus, limiting intrarenal M accumulation and blocking M activation offer two potentially attractive approaches to protecting the kidney.
CSF-1 is an important chemoattractant, survival, and proliferating factor for M produced by a variety of cell types, including endothelial, mesangial, and tubular epithelial cells and fibroblasts (2, 3, 4). The actions of this homodimeric factor are mediated entirely through a high affinity tyrosine kinase receptor encoded by the c-fms proto-oncogene (5, 6). M in inflamed tissues generate proinflammatory cytokines that are dependent on CSF-1 (7). CSF-1 primes M to produce cytokines in response to a second signal, such as LPS or IFN- (8, 9). This suggests that eliminating CSF-1 may lessen tissue damage during inflammation.
Obstructive nephropathy is caused by blocking urine flow and results in extensive kidney tubulointerstitial damage, inflammation, and ultimately renal failure (10, 11). Unilateral ureter obstruction in rodents provides a reliable, reproducible model to incite inflammation by recruiting M and T cells into the kidney. In this model, M infiltrate the kidney just hours after ligation and continue to increase with time (12). The tubular apoptosis during obstruction parallels intrarenal M accumulation (13). During obstruction, the tubules are a major source of CSF-1 and are responsible for increasing M proliferation (14). Therefore, using this rapid and reproducible model of renal inflammation, we investigated CSF-1-dependent, M-initiated TEC apoptosis.
To eliminate the effects of CSF-1 we 1) used osteopetrotic (Csf1op/Csf1op) mice, which have a spontaneous mutation in the CSF-1 gene rendering them deficient in biologically active CSF-1 (15, 16); and 2) blocked the CSF-1 receptor and neutralized CSF-1 with an Ab. Obstructed CSF-1-deficient kidneys had far fewer apoptotic TEC than wild-type (WT) kidneys. We now report that the decrease in TEC apoptosis was a result of 1) fewer M in the kidney due to reduced proliferation and MCP-1-dependent recruitment, and 2) a decrease in M activation. Blocking the CSF-1 receptor or neutralizing CSF-1 before stimulating cultured M resulted in reduced M activation (measured by the expression of activation markers and the generation of reactive oxygen species) and a reduction in the ability of the M supernatant to incite TEC apoptosis. We suggest that CSF-1 is a therapeutic target that prevents TEC apoptosis and, by extension, M-directed apoptosis in a multitude of resident cells during inflammation.
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Materials and Methods |
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Osteopetrotic (Csf1op/Csf1op) mice and littermate control (+/Csf1op or +/+) mice on the C57BL/6J x C3Heb/FeJ-a/a background were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred on this segregating background in our pathogen-free facilities.
Unilateral ureteral obstruction
Unilateral ureteral obstruction was performed on adult mice at 2–3 mo of age as previously described (17). Mice were sacrificed 3, 7, or 14 days after obstruction, at which time the obstructed and contralateral kidneys were collected and bisected. One-half of the kidney was fixed in 10% phosphate-buffered formalin for paraffin-embedded sections, and the other was snap-frozen in OCT compound (Miles Laboratories, Elkhart, IN) for cryostat sectioning. Portions of the kidney cortex were snap-frozen for RNA isolation.
Identification of kidney-infiltrating cells by immunostaining
Obstructed and contralateral kidney M and CD4 and CD8 T cells were assessed by the immunoperoxidase technique with rat anti-mouse CD68 Ab (Serotec, Raleigh, NC), purified anti-mouse CD4, or CD8 Ab (BD PharMingen, San Diego, CA), respectively as previously described (18). Interstitial cell infiltrates were evaluated by counting the number of immunostained cells in 10 randomly selected, high power fields. Glomerular cell accumulation was assessed in 20 randomly selected glomeruli by counting the number of labeled cell layers (18).
Proliferating M were determined by dual immunostaining; the proliferating cells were identified by staining for the presence of the proliferating cell nuclear Ag (PCNA), and M were identified by staining for the presence of F4/80 (a M marker). We detected F4/80, instead of CD68, in the dual-staining experiments, since F4/80 and PCNA, but not CD68, are detectable in paraffin sections. Briefly, paraffin-embedded sections were deparaffined, rehydrated, and trypsinized for proteolytic Ag retrieval. Endogenous peroxidase activity, avidin, and biotin were blocked with 0.3% H2O2 in methanol and with an avidin/biotin blocking kit, respectively (Vector Laboratories, Burlingame, CA). Tissue sections were incubated with rat anti-mouse F4/80 Ab (1/100; Serotec) overnight and developed with 3,3'-diaminobenzidine substrate kit for peroxidase (Vector Laboratories). Sections were placed in 10 mM sodium citrate buffer, pH 6, microwaved on high power for 12 min, washed, and incubated with biotinylated mouse monoclonal anti-PCNA (Animal Research Kit, peroxidase was used to biotinylate anti-PCNA according to the manufacturer’s instructions; DAKO, Carpinteria, CA). Sections were developed with Fast Red after incubation with Vector ABC-AP system (Vector Laboratories) and counterstained with hematoxylin. Proliferating cells and M were counted in 10 randomly selected fields, while dual-stained proliferating M were counted in 50 randomly selected fields.
Quantification of intrarenal MCP-1: real-time PCR
MCP-1 transcripts in the renal cortex were analyzed using real-time, two-step, quantitative RT-PCR. Total RNA was isolated from snap-frozen kidney cortexes using TRIzol (Life Technologies, Gaithersburg, MD). Residual DNA was removed by treatment with 1 U of DNase I (Invitrogen, Carlsbad, CA) at room temperature for 15 min, followed by inactivation at 65°C for 10 min. The RT reaction was performed on 1 mg of RNA using an oligo(dT) primer and Superscript II reverse transcriptase (Life Technologies). Relative quantitation with real-time, two-step RT-PCR was performed with SYBR Green PCR reagents (Qiagen, Valencia, CA) and an ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA), according to the manufacturer’s instructions. Reactions were performed using 1.0 µl of cDNA at a concentration of 100 ng/µl, in a reaction volume of 50 µl. The mRNA levels were normalized to those of GAPDH. The PCR primers used were as follows: GAPDH: sense, 5'-CATGGCCTCCAAGGAGTAAG-3'; antisense, 5'-CCTAGGCCCCTCCTGTTATT-3'; and MCP-1: sense, 5'-ACC AGC AAG ATG ATC CCA AT-3'; antisense, 5'-TGT CTG GAC CCA TTC CTT CT-3.
Quantification of apoptotic cells
The intrarenal apoptotic cells were detected and quantified 1) in situ using immunostaining, and 2) in freshly isolated kidney cells using flow cytometry.
Immunostaining. Apoptotic cells were detected in paraffin-embedded, obstructed and contralateral kidneys by enzymatic in situ labeling of apoptosis-induced DNA strand breaks (TUNEL method) using the TdT FragEL DNA Fragmentation Detection Kit (Oncogene, Boston, MA) according to the manufacturer’s instructions. The number of TUNEL-positive cells was counted in 10 random high power fields and categorized as either tubular or nontubular using morphological criteria.
Flow cytometry. Single-cell suspensions were prepared from obstructed kidneys. Isolated cells were fixed, permeabilized with saponin (Sigma-Aldrich, St. Louis, MO), and stained with anti-CD68-FITC Ab (Serotec) and annexin V-PE (BD PharMingen). To assess TEC apoptosis, we trypsinized TEC and resuspended these cells in annexin V binding buffer. We then incubated these TEC with annexin V-FITC, and propidium iodine for 15 min at room temperature. Cells (10,000) were analyzed by flow cytometry using a FACSCalibur.
Cell culture: bone marrow M
To isolate bone marrow M we removed the femur and tibia, cut both ends of the bone, and flushed out the bone marrow with M SFM medium (Life Technologies). Cells were centrifuged at 1500 rpm for 5 min and resuspended in M SFM medium containing 30% supernatant from L-929 cells (as a source of CSF-1), 10% FBS, 10 mM HEPES, 100 U/ml penicillin G, and 100 µg/ml streptomycin. The next day, nonadherent cells were collected and plated on either a 96-well plate (for the activation marker study and reactive oxygen species studies) or a 24-well plate (for the TEC death study) for 2–3 days. Once confluent, M were incubated for 24 h with 1) purified anti-mouse CD115 Ab (anti-CSF-1 receptor Ab; eBiosciences, San Diego, CA) or the isotype control IgG2a (eBiosciences), 2) rabbit anti-mouse CSF-1 Ab or the control rabbit serum (19), or 3) L cell medium alone. M were then stimulated with 5 µg/ml LPS (Sigma-Aldrich) and 500 U of IFN- for 24 h.
Detection of M activation
Activation markers: flow cytometry.
Stimulated (LPS plus IFN-) bone marrow M incubated with anti-CSF-1 receptor Ab, anti-CSF-1 Ab, or isotype controls were stained with the M activation markers anti-mouse CD69-PE, CD23-PE Ab (0.5 µg; BD PharMingen), purified anti-inducible NO synthase (anti-iNOS) Ab (0.5 µg; Transduction Laboratories, San Diego, CA), or a 1/1 mixture of biotinylated anti-mouse Iak and Iab Ab (0.5 µg; BD PharMingen), followed by streptavidin-PE (PharMingen). Cells stained for iNOS were incubated with biotinylated goat anti-rabbit (Vector Laboratories) followed by streptavidin-PE. We analyzed 10,000 cells using FACSCalibur. Controls consisted of unstimulated M, unblocked cells, and appropriate isotype controls for each Ab.
Reactive oxygen species (ROS).
Stimulated (LPS plus IFN-) bone marrow M incubated with anti-CSF-1 receptor Ab, anti-CSF-1 Ab, or their isotype controls were incubated with 20 µM 2',7'-dichlorofluorescein diacetate (Sigma-Aldrich) for 4 h. This dye freely permeates the cell membrane and, upon activation by ROS, is cleaved and becomes fluorescent. Plates were read at 485-nm excitation and 530-nm emission on a PerkinElmer fluorescent plate reader (PE Applied Biosystems). Controls consisted of stimulated and unstimulated M, with and without 2',7'-dichlorofluorescein diacetate.
Inducing TEC apoptosis.
The supernatant generated from stimulated (LPS plus IFN-) M preincubated in anti-CSF-1 receptor Ab or anti-CSF-1 Ab was collected, centrifuged, and added (1 ml/well) to plated TEC grown in 24-well plates for 48 h. Cell death was assessed by flow cytometry as described above. Controls consisted of supernatant from M that were not stimulated and from M incubated with anti-CSF-1 receptor Ab isotype control IgG2a and control serum for anti-CSF-1 Ab. To eliminate the possibility that residual LPS and IFN- in the supernatant contributed to TEC apoptosis, we incubated TEC with LPS and IFN- for 48 h and determined apoptosis by flow cytometry as described above.
Detection of M activation: flow cytometry
Obstructed kidneys were removed and gently dissociated into a single-cell suspension. RBC were lysed using ACK lysing buffer (BioSource International, Camarillo, CA), and the remaining cells were washed in PBS. Washes and Ab dilutions were performed in FACS buffer (PBS, 5% FBS, and 0.09% NaN3). Cells were stained for the presence of cell surface activation markers using PE-labeled anti-CD69 and anti-CD23 Ab and biotinylated Iak and Iab Ab (all at 0.5 µg), washed, permeabilized with saponin (Sigma-Aldrich), and stained with FITC-labeled anti-CD68 Ab (0.5 µg). To detect iNOS, cells were isolated, permeabilized with saponin, and stained with anti-iNOS Ab (0.5 µg) and FITC-labeled anti-CD68 Ab. To detect TNF-, cells were recultured for an additional 4 h in the presence of PMA (1 ng/ml; Sigma-Aldrich) and ionomycin (500 ng/ml; Sigma-Aldrich). Monensin (3 µM; Sigma-Aldrich) was added during the last 3 h. The cells were washed twice in saponin buffer and incubated with PE-labeled anti-TNF- Ab and FITC-labeled anti-CD68 Ab. After incubating for 30 min on ice in the dark, the cells were washed twice in saponin buffer and suspended in FACS buffer to allow the resealing of permeabilized membrane. The data from 20,000 cells were analyzed on a FACSCalibur.
Statistical analysis
Data are reported as the mean ± SEM. The Mann-Whitney U test was used to evaluate significance.
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and CD4 T cell accumulation is reduced in CSF-1-deficient, obstructed kidneys
During kidney obstruction, as M infiltration increases, there is parallel tissue loss in the renal pelvis (hydronephrosis). To determine the optimal point when intrarenal M accumulation is maximal, and hydronephrosis is minimal, we analyzed kidneys at 3, 7, and 14 days postobstruction. We concluded that the optimal points to study the impact of CSF-1 on kidney inflammation were at 3 and 7 days postobstruction. The contralateral kidneys remained essentially normal.
CSF-1-deficient mice have fewer M in the obstructed kidneys compared with WT mice. M decreased substantially in the interstitium (66%, 3 days; 66%, 7 days) and in glomeruli (33%, 3 days; 52%, 7 days) in CSF-1-deficient compared with WT controls. By comparison, there were few M in the contralateral kidneys in CSF-1-deficient and intact mice (Fig. 1, a and b). The numbers of M in the contralateral kidney and in normal C57BL/6 kidneys were similar (data not shown).
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are the prominent leukocytic infiltrate, T cells are present in the obstructed kidney, albeit far fewer in number. We detected fewer CD4 T cells in the CSF-1-deficient, obstructed kidneys than in the kidneys of the WT mice (Fig. 2a). In contrast, CD8 T cells did not differ in CSF-1-deficient and intact obstructed kidneys (Fig. 2b), and CD4 and CD8 T cells did not infiltrate the contralateral kidneys of CSF-1-deficient and intact mice. Thus, CSF-1 enhances intrarenal infiltration of M and CD4 T cells during obstruction.
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MCP-1 is a powerful chemoattractant for M that is up-regulated during obstruction (20). CSF-1-deficient, obstructed kidneys had reduced intrarenal MCP-1 transcripts compared with the WT kidneys (Fig. 3). This decrease may have contributed to the reduced recruitment of M into the kidney. Intrarenal MCP-1 transcripts in MRL-Faslpr mice (6 mo of age) were increased 6- to 8-fold above those in normal kidneys as determined by RT-PCR (21). Thus, MRL-Faslpr (6 mo of age) and C3H/Fej served as positive and negative controls, respectively (data not shown).
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and intrarenal cells are reduced in CSF-1-deficient, obstructed kidneys
Since the decrease in M in the CSF-1-deficient, obstructed kidneys could result from a decrease in M proliferation and/or an increase in M apoptosis, we explored these possibilities. We determined that the total number of proliferating cells in the CSF-1-deficient, obstructed kidney was reduced compared with that in WT mice (Fig. 4a). Proliferating renal M were dramatically diminished (88%) in CSF-1-deficient, obstructed kidneys compared with their numbers in WT mice (Fig. 4b). Of note, the number of apoptotic M in the obstructed kidney did not differ in the CSF-1-deficient and intact strains (5.0 ± 0.2 (n = 2) vs 4.6 ± 0.6 (n = 3); Csf1op/Csf1op and +/Csf1op, respectively). This suggests that CSF-1 increases M proliferation, but does not enhance or decrease M apoptosis.
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CSF-1 deficient, obstructed kidneys had fewer apoptotic intrarenal cells (50%) compared with their numbers in WT kidneys 7 days postobstruction, as evaluated by TUNEL assay (Fig. 5a). Using morphological criteria to identify TEC, we determined that apoptotic TEC accounted for 42% of the total intrarenal apoptotic cells in WT mice and 25% in CSF-1-deficient mice (Fig. 5b). Thus, TEC apoptosis is increased by CSF-1.
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are less activated than WT M
We have established that activated, but not resting, M release soluble factors that result in TEC apoptosis (1). Therefore, we investigated whether the protection from TEC apoptosis in CSF-1-deficient, obstructed kidneys resulted at least in part from the inability of M to become activated. To evaluate M activation we 1) freshly isolated renal cells and probed for activation markers, and 2) investigated the capacity of M to become activated in culture.
M freshly isolated from CSF-1-deficient, obstructed kidneys had a reduced expression of the cell surface activation markers CD69 (an early activation marker whose expression is rapidly induced upon activation) (22) and CD23 (a low affinity receptor for IgE expressed on M and shown to mediate activation and proinflammatory response, including release of NO (23, 24, 25) and MHC class II markers (Iak,b)). NO and TNF- induce apoptosis (26, 27). In CSF-1-deficient kidneys, iNOS, the enzyme that produces NO, and TNF- expression is reduced compared with that in WT kidneys (Fig. 6). These data suggest that during renal inflammation CSF-1 is required for robust M activation.
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activation was confirmed in vitro using cultured M isolated from WT bone marrow. The actions of CSF-1 are solely mediated through the CSF-1 receptor (6). Therefore, M from Csf1op/Csf1op mice and M from WT mice treated with anti-CSF-1 receptor Ab should be similar. Stimulating bone marrow M with LPS and IFN- that had previously been incubated with anti-CSF-1 receptor Ab decreased expression of the activation markers CD69 (70%) and CD23 (59%) (Fig. 7a). Similarly, iNOS expression, another index of M activation, was reduced (47%) in M preincubated with anti-CSF-1 receptor Ab compared with M preincubated with the isotype control. Furthermore, activated M released ROS that are highly reactive and damage neighboring cells. Cultured M preincubated with anti-CSF-1 receptor Ab and stimulated with LPS and IFN- produced less ROS than M incubated with isotype control (Fig. 7b). To confirm the role of CSF-1 in M activation, we repeated the experiments using a CSF-1 neutralizing Ab. Again, M preincubated with anti-CSF-1 Ab became less activated than those preincubated with the control serum, CD69 (47%), CD23 (46%), iNOS (45%), or ROS (42%). These data suggest that CSF-1 enhances M activation and generation of molecules known to initiate apoptosis.
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to release mediators that induce TEC apoptosis. Given that 1) M activation is dependent on CSF-1, and 2) activated M release soluble mediators that induce TEC apoptosis (1), we evaluated whether the induction of TEC apoptosis by activated M is dependent on CSF-1. The supernatant generated by stimulated (LPS plus IFN-) bone marrow M preincubated with anti-CSF-1 Ab or anti-CSF-1 receptor Ab were less capable of inducing TEC apoptosis than the supernatant from control M (Fig. 8, a and b). Fig. 8b illustrates a representative experiment using anti-CSF-1 receptor Ab to block the effects of CSF-1 on M-mediated apoptosis. Incubation with anti-CSF-1 receptor Ab reduced TEC apoptosis 54 ± 3%, from isotype control (p < 0.01; n = 3). Incubating TEC with LPS and IFN- alone did not increase TEC apoptosis compared with unstimulated supernatant (data not shown). Therefore, residual LPS plus IFN- that may have remained in the supernatant was not responsible for increasing TEC apoptosis. Taken together, we conclude that CSF-1 confers on M an increased capacity to generate mediators that induce TEC apoptosis.
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accumulating in the kidney as a result of decreased recruitment, proliferation, and not as a consequence of enhanced apoptosis; 2) M activation during inflammation is in large part dependent on CSF-1; 3) CSF-1-deficient M isolated from the inflamed kidney express fewer molecules (iNOS, ROS, and TNF-) known to induce apoptosis; and 4) blocking CSF-1 or its receptor reduces M activation and M-mediated TEC apoptosis.
M killing of target cells is an emerging concept in tumor biology, development, and inflammation (28, 29). It is now evident that M programmed cell death requiring cell-cell contact is instrumental in tissue remodeling, as illustrated by the elimination of unwanted capillaries in neonatal eye development (30). M-directed regulation of resident cells may be instrumental in inflammation-mediated kidney diseases. In support of this concept, we previously determined that in nephrotoxic serum nephritis, decreased TEC apoptosis correlated with a decrease in M adjacent to TEC (1), and we determined that activated, but not resting, M induce TEC apoptosis through cell contact and the release of soluble mediators (1). We have now extended this concept by establishing that CSF-1 augments M-induced apoptosis in nonimmunologically induced inflammation caused by urethral obstruction. It should be noted M-mediated apoptosis is not restricted to TEC in these inflamed kidneys. M propagated from nephrotoxic serum nephritic kidneys induce apoptosis in myofibroblast-like mesangial cells, although cell contact is required (31). In fact, it is likely that during inflammation CSF-1 is responsible for M-dependent apoptosis in a broad range of sites.
What are the consequences of M directed-apoptosis of resident cells? Although we assume that this is a harmful process, it may also lead to tissue repair. Since M are phagocytes responsible for eliminating unwanted cells, M-regulated resident cell survival may provide a well-orchestrated means to induce programmed cell death and remove these cells. This process could lead to tissue destruction or tissue remodeling. In addition, since M engulfment of apoptotic cells causes the release of the potent immunosuppressive IL-10, M initiated apoptosis may ultimately be responsible for turning off immune-mediated events (32). Regardless of the biologic ramifications, it is clear that M-directed apoptosis of resident TEC is largely dependent on CSF-1.
Soluble mediators released from activated M induce TEC apoptosis. In contrast to previous reports in other cell types, in our studies direct cell-cell contact is not required (31, 33, 34). Identification of the soluble mediators responsible for M-induced apoptosis is critical in designing a more selective therapeutic approach to halt tissue injury. In this study we identified a decreased expression of iNOS (the enzyme required to form NO), reduced ROS, and TNF- in CSF-1-deficient M with impaired TEC killing (apoptosis). These molecules may be responsible for M-mediated TEC apoptosis. NO and ROS (including superoxide and hydroxyl radical) are highly diffusable, relatively unstable apoptotic mediators that are capable of inducing apoptosis and damage neighboring cells (35, 36, 37). The instability of NO and ROS make them unlikely mediators of apoptosis in our in vitro system, since the soluble molecules responsible must be stable for at least a number of hours, if not days. However, NO and superoxide (ROS) rapidly combine to form the more stable and potent oxidant, peroxynitrite (38), which is capable of inciting tubular cell apoptosis (39) and generating longer-lived apoptosis-inducing mediators (40, 41). Therefore, it is more probable that a relatively stable by-product of ROS mediates TEC apoptosis. Furthermore, TNF- increases the generation of NO and ROS, and thus represents a mechanism for perpetuating tissue damage (42, 43). Alternatively, TNF- alone may induce TEC apoptosis. TNF- induces apoptosis in numerous cell types, including tumor, endothelial, and primary cells (44, 45, 46). Although TNF- is expressed on M as a membrane-bound cytokine, another form of TNF- is released (47, 48). In conclusion, the scope of M functions is broad, and there are undoubtedly a vast number of molecules released by M (i.e., proteases) potentially capable of inciting apoptosis. We are currently trying to identify and pin-point the relative contributions of the soluble mediators released by activated M responsible for apoptosis of resident cells in the kidney.
It is worth noting that TEC express the CSF-1 receptor. Intrarenal CSF-1 receptor expression increases in the obstructed kidney compared with that in the contralateral control (data not shown). Studies in our laboratory indicate that CSF-1-rich L cell supernatant does not increase TEC apoptosis in vitro (unpublished observation); therefore, it is unlikely that the engagement of CSF-1 with the CSF-1 receptor displayed on TEC contributes to TEC apoptosis during ureter obstruction.
There are multiple steps that could lead to a decrease in M in the obstructed kidney of CSF-1-deficient mice, including reduced M recruitment, proliferation, survival, or enhanced apoptosis. We noted reduced intrarenal M proliferation in CSF-1-deficient, obstructed kidneys compared with WT obstructed kidneys. This is consistent with previous findings in glomerulonephritis, acute allograft rejection, and obstruction, highlighting that an increase in local M proliferation correlates with enhanced CSF-1 expression (14, 49, 50). CSF-1 is recognized as a survival factor for monocytes/M (51, 52, 53, 54); however, we have eliminated this possibility, since we did not detect increased intrarenal apoptosis in obstructed kidneys lacking CSF-1. Since GM-CSF, IL-1
, and IFN- are M survival factors (55), these molecules may be available to offset the absence of CSF-1. Finally, diminished recruitment may contribute to the decrease in M accumulation. Although we did not specifically examine recruitment, previous studies document that CSF-1 is a potent chemoattractant that stimulates monocyte migration (56, 57) and acts as a chemoattractant for CSF-1 receptor-expressing cells (58). There is evidence that CSF-1 recruits M into the kidney. We have shown that implanting TEC, genetically modified to release CSF-1, under the kidney capsule, recruits M to the CSF-1-rich implant site in MRL-Faslpr mice (18, 59). This recruitment may be related to CSF-1 increasing MCP-1 during inflammation, since our results show that CSF-1-deficient mice have reduced intrarenal MCP-1. MCP-1 is a potent chemoattractant of monocytes (60) and increases proportionately to intrarenal M infiltration during protein overload and obstruction (20, 61). In addition, MCP-1-deficient mice have reduced intrarenal M accumulation and consequently less apoptotic TEC after inducing nephrotoxic serum nephritis (1). However, it is unclear whether the decrease in intrarenal MCP-1 is directly or indirectly related to the decreased inflammation in CSF-1-deficient, obstructed kidneys. Obviously, many other chemokines and other molecules contribute to this decreased M accumulation. Thus, it is premature to conclude that MCP-1 alone is responsible for decreased M recruitment during renal obstruction. Finally, it is important to recognize that CSF-1-deficient mice have a reduced number of circulating monocytes (6) and resident kidney M (62) that undoubtedly contribute to the decreased M accumulation. Taken together, we suggest that decreased M accumulation in CSF-1-deficient, obstructed kidneys is a result of decreased M availability, recruitment, and proliferation, but not enhanced apoptosis.
CSF-1 is a potential therapeutic target for inflammation in the kidney and other tissues. M are recruited into the kidney during nearly every kidney disease, including autoimmune diseases, polycystic kidney disease, renal transplant rejection, and even during altered blood flow (21, 50, 63, 64). Thus, eliminating molecules, such as CSF-1, that foster M accumulation should reduce M-dependent tissue injury. For example, advancing renal disease correlates with increasing CSF-1 in MRL-Faslpr kidneys, and gene transfer of CSF-1 into the kidney incites renal injury in the C3H/Fej-Faslpr strain that does not spontaneously develop nephritis (54, 59). We have determined that MRL-Faslpr mice genetically deficient in CSF-1 are protected from renal disease and have expanded survival (preliminary observations). By extension, M are prominent inflammatory cells in a vast number of tissues and in a broad range of life-threatening and debilitating diseases. Blocking CSF-1 may protect from cardiovascular disease. This is well illustrated in apolipoprotein E-deficient mice that are prone to spontaneous atherosclerosis. Genetic deletion of CSF-1 in the apolipoprotein E-deficient mice decreases aortic atherosclerosis (65, 66), and it has been suggested that the number of activated M adjacent to the vascular wall is instrumental in atherogenesis. Similarly, CSF-1 may be a therapeutic target for autoimmune diseases. Blocking CSF-1 is beneficial in experimental arthritis (67, 68). Provision of neutralizing CSF-1 Abs in collagen-induced arthritis decreases the arthritic severity, while CSF-1-deficient mice remain resistant to the induction of arthritis (67). As mentioned above, eliminating CSF-1 prevents experimental autoimmune lupus (our preliminary observations). Finally, CSF-1 may be a therapeutic target for Alzheimer’s disease. Since CSF-1 and CSF-1 receptor expression are increased along with enhanced microglia proliferation and inflammation, blocking CSF-1 may retard disease (69, 70, 71). Taken together, CSF-1 offers a promising therapeutic target for diverse M-mediated illnesses.
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Acknowledgments |
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Footnotes |
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1 This work was supported in part by grants from the National Institutes of Health (to D.M.L. (1F32DK-6029101), V.R.K. (DK36149, DK56848, and DK52369), and E.R.S. (CA32551 and 5P30-CA13330)) and by a grant from the Alliance for Lupus Research (to V.R.K.). 
2 Address correspondence and reprint requests to Dr. Vicki Rubin Kelley, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: vkelley{at}rics.bwh.harvard.edu
3 Abbreviations used in this paper: TEC, tubular epithelial cell; iNOS, inducible NO synthase; M, macrophage; MCP-1, monocyte chemoattractant protein-1; PCNA, proliferating cell nuclear Ag; ROS, reactive oxygen species; WT, wild type.
Received for publication September 19, 2002. Accepted for publication January 8, 2003.
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References |
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stimulates M-CSF gene expression and monocyte proliferation: evidence for involvement of protein kinase C and protein tyrosine kinase. Biochim. Biophys. Acta 1313:161.[Medline]
1 in experimental hydronephrosis. Am. J. Physiol. 266:F926. and lipopolysaccharide induce apoptotic cell death in bovine glomerular endothelial cells. Kidney Int. 55:2322.[Medline]
effect on the expression of iNOS in macrophage and cardiac myocytes. Perfusion 16:67.-induced apoptosis of rat mesangial cells. J. Biol. Chem. 275:12684. production and TNF--dependent apoptosis during simulated ischemia. Am. J. Physiol. 281:C563. from cells. Nature 385:729.[Medline]
. Nature 385:733.[Medline]
-amyloid-induced interleukin-1, interleukin-6, and nitric oxide production by microglial cells. J. Biol. Chem. 273:20967.PP(V717F) transgenic mouse model of Alzheimer’s disease. Am. J. Pathol. 157:895.This article has been cited by other articles:
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  F. Y. Ma, J. Liu, A. R. Kitching, C. L. Manthey, and D. J. Nikolic-Paterson Targeting renal macrophage accumulation via c-fms kinase reduces tubular apoptosis but fails to modify progressive fibrosis in the obstructed rat kidney Am J Physiol Renal Physiol, January 1, 2009; 296(1): F177 - F185. [Abstract] [Full Text] [PDF]
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 J. Menke, M.-Y. Hsu, K. T. Byrne, J. A. Lucas, W. A. Rabacal, B. P. Croker, X.-H. Zong, E. R. Stanley, and V. R. Kelley Sunlight Triggers Cutaneous Lupus through a CSF-1-Dependent Mechanism in MRL-Faslpr Mice J. Immunol., November 15, 2008; 181(10): 7367 - 7379. [Abstract] [Full Text] [PDF]
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V. W. S. Lee, Y. M. Wang, Y. P. Wang, D. Zheng, T. Polhill, Q. Cao, H. Wu, I. E. Alexander, S. I. Alexander, and D. C. H. Harris Regulatory immune cells in kidney disease Am J Physiol Renal Physiol, August 1, 2008; 295(2): F335 - F342. [Abstract] [Full Text] [PDF]
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 J. Menke, G. C. Zeller, E. Kikawada, T. K. Means, X. R. Huang, H. Y. Lan, B. Lu, J. Farber, A. D. Luster, and V. R. Kelley CXCL9, but not CXCL10, Promotes CXCR3-Dependent Immune-Mediated Kidney Disease J. Am. Soc. Nephrol., June 1, 2008; 19(6): 1177 - 1189. [Full Text] [PDF]
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 Y. Wang, Y. Wang, Q. Cai, G. Zheng, V. W.S. Lee, D. Zheng, X. Li, T. K. Tan, and D. C.H. Harris By Homing to the Kidney, Activated Macrophages Potently Exacerbate Renal Injury Am. J. Pathol., June 1, 2008; 172(6): 1491 - 1499. [Abstract] [Full Text] [PDF]
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 P T Yang, W G Xiao, L J Zhao, J Lu, L M He, H KASAI, and M Ito Increase in the level of macrophage colony-stimulating factor in patients with systemic lupus erythematosus Ann Rheum Dis, March 1, 2008; 67(3): 429 - 430. [Full Text] [PDF]
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 U. Sharma, N.-E. Rhaleb, S. Pokharel, P. Harding, S. Rasoul, H. Peng, and O. A. Carretero Novel anti-inflammatory mechanisms of N-Acetyl-Ser-Asp-Lys-Pro in hypertension-induced target organ damage Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1226 - H1232. [Abstract] [Full Text] [PDF]
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 P. Boor, K. Sebekova, T. Ostendorf, and J. Floege Treatment targets in renal fibrosis Nephrol. Dial. Transplant., December 1, 2007; 22(12): 3391 - 3407. [Full Text] [PDF]
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B. Lange-Sperandio, A. Trautmann, O. Eickelberg, A. Jayachandran, S. Oberle, F. Schmidutz, B. Rodenbeck, M. Homme, R. Horuk, and F. Schaefer Leukocytes Induce Epithelial to Mesenchymal Transition after Unilateral Ureteral Obstruction in Neonatal Mice Am. J. Pathol., September 1, 2007; 171(3): 861 - 871. [Abstract] [Full Text] [PDF]
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A. Y. Hoi, M. J. Hickey, P. Hall, J. Yamana, K. M. O'Sullivan, L. L. Santos, W. G. James, A. R. Kitching, and E. F. Morand Macrophage Migration Inhibitory Factor Deficiency Attenuates Macrophage Recruitment, Glomerulonephritis, and Lethality in MRL/lpr Mice J. Immunol., October 15, 2006; 177(8): 5687 - 5696. [Abstract] [Full Text] [PDF]
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M.-H. Jang, D. M. Herber, X. Jiang, S. Nandi, X.-M. Dai, G. Zeller, E. R. Stanley, and V. R. Kelley Distinct In Vivo Roles of Colony-Stimulating Factor-1 Isoforms in Renal Inflammation J. Immunol., September 15, 2006; 177(6): 4055 - 4063. [Abstract] [Full Text] [PDF]
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 K. M. Irvine, C. J. Burns, A. F. Wilks, S. Su, D. A. Hume, and M. J. Sweet A CSF-1 receptor kinase inhibitor targets effector functions and inhibits pro-inflammatory cytokine production from murine macrophage populations FASEB J, September 1, 2006; 20(11): 1921 - 1923. [Abstract] [Full Text] [PDF]
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T. Kipari, J.-F. Cailhier, D. Ferenbach, S. Watson, K. Houlberg, D. Walbaum, S. Clay, J. Savill, and J. Hughes Nitric Oxide Is an Important Mediator of Renal Tubular Epithelial Cell Death in Vitro and in Murine Experimental Hydronephrosis Am. J. Pathol., August 1, 2006; 169(2): 388 - 399. [Abstract] [Full Text] [PDF]
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 S. Nandi, M. P. Akhter, M. F. Seifert, X.-M. Dai, and E. R. Stanley Developmental and functional significance of the CSF-1 proteoglycan chondroitin sulfate chain Blood, January 15, 2006; 107(2): 786 - 795. [Abstract] [Full Text] [PDF]
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G. C. Zeller, J. Hirahashi, A. Schwarting, A. H. Sharpe, and V. R. Kelley Inducible Co-Stimulator Null MRL-Faslpr Mice: Uncoupling of Autoantibodies and T Cell Responses in Lupus J. Am. Soc. Nephrol., January 1, 2006; 17(1): 122 - 130. [Abstract] [Full Text] [PDF]
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J. S. Duffield, P. G. Tipping, T. Kipari, J.-F. Cailhier, S. Clay, R. Lang, J. V. Bonventre, and J. Hughes Conditional Ablation of Macrophages Halts Progression of Crescentic Glomerulonephritis Am. J. Pathol., November 1, 2005; 167(5): 1207 - 1219. [Abstract] [Full Text] [PDF]
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L. Bao, I. Osawe, M. Haas, and R. J. Quigg Signaling through Up-Regulated C3a Receptor Is Key to the Development of Experimental Lupus Nephritis J. Immunol., August 1, 2005; 175(3): 1947 - 1955. [Abstract] [Full Text] [PDF]
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D. M. Lenda, E. R. Stanley, and V. R. Kelley Negative Role of Colony-Stimulating Factor-1 in Macrophage, T Cell, and B Cell Mediated Autoimmune Disease in MRL-Faslpr Mice J. Immunol., October 1, 2004; 173(7): 4744 - 4754. [Abstract] [Full Text] [PDF]
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