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The Journal of Immunology, 2003, 170: 6280-6291.
Copyright © 2003 by The American Association of Immunologists

Differential Activation of NF-{kappa}B, AP-1, and C/EBP in Endotoxin-Tolerant Rats: Mechanisms for In Vivo Regulation of Glomerular RANTES/CCL5 Expression1

Johanna Pocock*, Carmen Gómez-Guerrero{dagger}, Sigrid Harendza*, Murwan Ayoub*, Purificación Hernández-Vargas{dagger}, Gunther Zahner*, Rolf A. K. Stahl* and Friedrich Thaiss2,*

* Department of Internal Medicine, Division of Nephrology and Osteology, University of Hamburg, Hamburg, Germany; and {dagger} Renal and Vascular Research Laboratory, Fundación Jiménez Díaz, Autonoma University, Madrid, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chemokines play a pivotal role in the regulation of inflammatory cell infiltration in glomerular immune injury. To characterize mechanisms relevant for the regulation of chemokine expression in vivo, the LPS-mediated model of renal inflammation in rats was used in which we have previously demonstrated that the chemokine RANTES/CCL5 is expressed and secreted in glomeruli. Glomerular RANTES/CCL5 expression in this model correlated with an increased glomerular binding activity of the transcription factors AP-1, C/EBP, and NF-{kappa}B. To gain further insight into the functional roles of these transcription factors in the regulation of glomerular RANTES/CCL5 expression, we cloned the rat RANTES/CCL5 promoter and established the model of in vivo LPS tolerance. In tolerant rats, LPS-induced glomerular RANTES/CCL5 expression and activation of the transcription factors AP-1 and C/EBP were significantly reduced using both consensus and rat RANTES/CCL5-specific oligonucleotides. Reduced glomerular NF-{kappa}B binding activity after LPS injection could be demonstrated in tolerant rats only when using rat RANTES/CCL5-specific oligonucleotides. Reduced binding activity to this RANTES/CCL5-specific NF-{kappa}B binding site in the context of broad NF-{kappa}B activation might be due to changes in transcription factor interactions or chromatin remodeling processes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chemokines play a pivotal role in the regulation of inflammatory cell infiltration in a variety of glomerular inflammatory lesions. Inhibition of chemokines by chemokine-specific Abs or receptor blockers significantly reduced inflammatory cell infiltration in the kidney and improved morphological and functional lesions in these inflammatory models of renal diseases. RANTES/CCL5 is a member of the {beta}-chemokine family that is predominantly chemotactic for monocytes/macrophages and lymphocytes. RANTES/CCL5 plays a pivotal role in the regulation of inflammatory cell infiltration in a variety of renal diseases and also after kidney transplantation (1, 2, 3). In an LPS-mediated model of renal inflammation in rats we have previously shown that RANTES/CCL5 is expressed and secreted in isolated glomeruli and that RANTES/CCL5 plays a role in the recruitment of monocytes/macrophages to the glomerulus in this model of glomerular inflammation (4, 5).

Molecular mechanisms that might be relevant in the regulation of glomerular RANTES/CCL5 expression have not been examined in vivo in detail to date. The promoter regions of mouse and human RANTES/CCL5 have been described, and several binding sites for known transcription factors have been characterized to regulate RANTES/CCL5 expression in vitro (6, 7, 8, 9, 10). It is, however, unclear which of these transcription factors participates in the regulation of RANTES/CCL5 expression in glomerular injury in rats. We therefore cloned the rat RANTES/CCL5 promoter and examined the in vivo activation of known transcription factors with putative binding sites in the promoter region. We focused on transcription factors that have been demonstrated previously to play a role in the regulation of RANTES/CCL5 gene expression: NF-{kappa}B, AP-1, and C/EBP (9, 10). To further characterize the functional relevance of these transcription factors in the regulation of glomerular RANTES/CCL5 expression in vivo, we established a model of LPS tolerance and evaluated glomerular RANTES/CCL5 expression and activation of the transcription factors NF-{kappa}B, AP-1, and C/EBP. The results of the present experiments might help to design in vivo strategies to suppress RANTES/CCL5 expression in different models of glomerular inflammatory lesions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
LPS application and induction of tolerance in vivo

Male Wistar rats (150–180 g body weight; Charles River, Wiga, Germany) were injected i.p. with LPS (Escherichia coli type O111:B4; Sigma-Aldrich, Munich, Germany). The dose used was 2 mg LPS/kg body weight. For control experiments PBS was injected i.p.

Animals were primed with a low dose of LPS to induce LPS tolerance. For preliminary experiments two LPS doses (0.04 and 0.2 mg/kg body weight) were tested. The time interval used for the second high dose LPS application ranged from 24–72 h for the preliminary experiments. The results of these preliminary studies indicated that a 0.04 mg/kg body weight LPS priming dose and a 48-h interval before application of the high LPS dose were the most suitable for tolerance induction with respect to glomerular RANTES/CCL5 mRNA expression.

RNA isolation and Northern blot analysis

Glomeruli from freshly harvested kidneys were isolated by a sieving technique as described previously (5). Total RNA from isolated glomeruli of pooled kidneys was extracted by phenol-chloroform after direct lysis of glomeruli in 4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7), 0.5% sarcosyl, and 0.1 M 2-ME. Twenty micrograms of total RNA was electrophoresed through a 1.2% agarose gel in running buffer (2.2 M formaldehyde, 0.02 M MOPS, and 1 mM EDTA). The RNA was transferred onto nylon membranes (Hybond-N; Amersham Pharmacia Biotech, Freiburg, Germany) and, after UV cross-linking, prehybridized at 65°C for 1 h in 0.1x SSC (20x SSC = 3 M NaCl/0.3 M sodium citrate) and 0.5% SDS. The cDNA probe used for RANTES/CCL5 was an EcoRI-XhoI fragment of the cDNA encoding for murine RANTES (4, 5) and a 2.0-kb insert of pMCI encoding the murine 18 S RNA band. The cDNA fragments were labeled with 50 µCi of [{alpha}-32P]dCTP (3000 Ci/mmol; Amersham Pharmacia Biotech) using a random priming kit (Oncor Appligene, Heidelberg, Germany). The membranes were hybridized with 106 cpm probe/ml in Rapid-Hyb buffer (Amersham Pharmacia Biotech) for 24 h at 70°C using a rotating drum. After hybridization, blots were washed for 20 min in 2x SSC (20x SSC = 3 M NaCl/0.3 M sodium citrate, pH 7.0) and 0.1% SDS at room temperature and for at least 15 min in 0.4x SSC and 0.1% SDS at 65°C, then autoradiographed. Membranes were stripped with 5 mM Tris-HCl, 0.2 mM EDTA, 0.5% sodium pyrophosphate, and 5x Denhardt’s solution for 30 min at 65°C and were subsequently rehybridized with the cDNA probe for the 18S band to account for small loading and transfer variations. Exposed films were scanned with the Fluor-STM MultiImager and analyzed with the computer program Multianalyst 1.1, or the hybridized membranes were subjected to the GS-363 Molecular Imager System and Molecular Analyst 1.5 (Bio-Rad, Munich, Germany). Relative changes in RNA were calculated after assigning hybridization in control lanes the relative value of 1. Samples were normalized for the signal intensity of the 18S ribosomal RNA hybridization.

Glomerular nuclear mini-extracts and gel-shift experiments: consensus binding sites

Kidneys were perfused with 150 ml of sterile PBS/animal before harvesting. The nuclear mini-extracts were prepared, and the EMSA was run according to the procedure described by Sakurai et al. with minor modifications (11, 12). Briefly, isolated glomeruli were resuspended in 400 µl of buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin), homogenized with 50 strokes in a 7-ml glass-homogenizer (Wheaton homogenizer; Roth, Karlsruhe, Germany), and chilled on ice for 15 min. After adding 25 µl of 10% Nonidet P-40, the homogenate was vigorously vortexed for 10 s and centrifuged at 15,000 x g for 5 min. To extract the nuclear proteins, the nuclear pellet was resuspended in 100 µl of buffer B (20 mM HEPES (pH 7.9), 0.4 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) and rocked at 400 rpm on ice on a shaking platform for 15 min. After centrifugation at 15,000 x g for 5 min, the nuclear extracts were stored in aliquots at -80°C. Protein concentration was measured using the DC protein assay reagent (Bio-Rad).

NF-{kappa}B (5'-AGTTGAGGGGACTTTCCCAGGC-3'; Promega, Heidelberg, Germany), AP-1 (5'-CGCTTGATGACTCAGCCGGAA-3') and C/EBP (5'-TGCAGATTGCGCAA TCTGCA-3'; Santa Cruz Biotechnology, Heidelberg, Germany) consensus oligonucleotide probes were end-labeled with [{gamma}-32P]ATP (3,000 Ci/mmol; Amersham Pharmacia Biotech). Five micrograms of nuclear protein was incubated for 30 min at room temperature with 100,000 cpm of the probe in 20 mM HEPES (pH 7.9), 0.3 mM EDTA, 0.2 mM EGTA, 80 mM NaCl, and 2 µg poly(dI-dC)poly(Di-dC) (Amersham Pharmacia Biotech) in a total volume of 20 µl. Where indicated, competition experiments were performed by adding unlabeled consensus oligonucleotides in a 100-fold molar excess to the binding reaction. The supershift assays were performed by adding 2 µg of Abs against NF-{kappa}B p65 nuclear localization site (NLS) subunit (Roche, Mannheim, Germany), NF-{kappa}B p50 (C-19), NF-{kappa}B p50 NLS, NF-{kappa}B p65 (C-20), NF-{kappa}B p52 (K-27), c-Rel (N)-G, Rel B (C-19), c-Jun (H-79), c-Fos (K-25), Jun D (D-11), Jun B (C-11), C/EBP{alpha} (14AA), {beta} (C-19), {gamma} (C-20), {delta} (C-22), and {epsilon} (C-22) (all from Santa Cruz Biotechnology) immediately after the binding reaction and were incubated overnight at 4°C. The DNA-protein complexes were subjected to electrophoresis on a 4% polyacrylamide gel that contains 2.5% glycerol, 6.7 mM Tris-HCl (pH 7.5), 3.3 mM sodium acetate, and 0.1 mM EDTA. After electrophoresis for 2.5 h at 15°C the gel was vacuum-dried and autoradiographed.

Exposed films were scanned with the Fluor-STM MultiImager and analyzed with the computer program Multianalyst 1.1. Relative changes in shifted bands were calculated after assigning hybridization in control lanes the relative value of 1.

Western blot analysis

Isolated glomeruli of one kidney were lysed in 100 µl of Laemmli buffer (13). Protein concentration was determined with the DC protein assay reagent (Bio-Rad Laboratories). One hundred micrograms of protein was loaded onto a 12.5% SDS-PAGE and blotted semidry onto a polyvinylidene difluoride membrane (Hybond-P; Amersham Pharmacia Biotech). The blots were blocked in 5% nonfat dry milk in PBS and 0.1% Tween 20 before incubating the blots for 1 h in a 1/1000 dilution of Abs directed against I{kappa}B{alpha} and phosphorylated I{kappa}B{alpha} (Santa Cruz Biotechnology and Biolabs (Heidelberg, Germany)). After washes in PBS and 0.1% Tween 20, the blots were incubated for another hour in a 1/2000 dilution of goat anti-rabbit HRP-linked IgG (Southern Biotechnology Associates, Birmingham, AL) as the secondary Ab. The Ab-labeled proteins were detected with ECL according to the manufacturer’s description (Amersham Pharmacia Biotech). Exposed films were scanned with the Fluor-STM MultiImager and analyzed with the computer program Multianalyst 1.1 when normalized for {beta} actin expression (Bio-Rad).

Southwestern histochemistry

Southwestern histochemistry was performed as described in detail previously (14). NF-{kappa}B and AP-1 consensus oligonucleotides or rat RANTES/CCL5-specific oligonucleotides (see below) were digoxigenin-labeled with a 3'-terminal transferase (Roche). 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 10 pmol of the labeled DNA probe in buffer containing 0.25% BSA and 0.5 µg/ml poly(dI-dC). The sections were then incubated with alkaline phosphatase-conjugated anti-digoxigenin Ab, and colorimetric detection was performed. Preparations without probe were used as negative controls, and mutant-labeled probe and an excess of unlabeled probe were used to test the specificity of the technique.

Cloning of rat RANTES promoter

The 5'-flanking region of the rat RANTES/CCL5 gene was cloned using the Rat Genome Walker kit (Clontech, Heidelberg, Germany) with adapter (upstream) and specific (downstream) primers from the rat RANTES/CCL5 cDNA (accession no. U06436) as follows: RANTES/CCL5-specific primers: GSP1, 5'-AGAGGGCGGCTGCAACGAGGATG AC-3'; and GSP2, 5'-TGAGGGATGCAGCTGCAGAGATCTTCAT-3'; external primers: AP1, 5'-GTAATACGACTCACTATAGGGC-3'; and AP2, 5'-ACTATAGGGCACGCGTGGT-3'.

The PCR fragments were cloned into the pGEM-T easy vector (Promega) and sequenced using the automated sequence analyzer ABI 377 (PE Applied Biosystems, Weiterstadt, Germany). The accession number of the RANTES/CCL5 gene promoter in the GenBank database is AF 282896.

Glomerular nuclear mini-extracts and gel-shift experiments: specific binding sites

Gel shift experiments were performed as described above for consensus binding sites. Putative specific binding sites for known transcription factors in the rat RANTES/CCL5 promoter were searched for using MatInspector from Genomatix (15). Single-stranded sense and reverse oligonucleotides for these gel-shift experiments were synthesized using a DNA synthesizer (Oligosynthesiszer, Oligo 1000; Beckman, Munich, Germany) as described in detail previously (13). For annealing, oligonucleotides were incubated at 80°C for 5 min in a water bath and cooled slowly to room temperature. Annealing was confirmed by agarose gel electrophoresis.

Gel shift experiments using specific double-stranded oligonucleotides were performed essentially as described above. For competition experiments nuclear protein were incubated with a 50-fold molar excess of unlabeled consensus binding oligonucleotides added to the radiolabeled oligonucleotides with specific binding sites.

Experimental design

The following groups of animals were examined: control rats injected once with PBS, controls injected once with a low dose of LPS (0.04 mg/kg body weight) before PBS application, LPS-treated (2 mg/kg body weight) animals, and rats made tolerant by low dose LPS application before the high dose of LPS was injected. Glomeruli were isolated from controls and 1, 3, and 6 h after LPS injection.

Glomerular RANTES/CCL5 expression

Glomeruli were isolated, and RNA was prepared for Northern blotting. At each time point three animals were examined. The experiments were repeated twice.

Activation of transcription factors in isolated glomeruli

Activation of the transcription factors NF-{kappa}B, AP-1, and C/EBP in isolated glomeruli was analyzed by gel-shift experiments. To establish the model, consensus binding sites for the transcription factors were used. Having cloned and sequences the rat RANTES/CCL5 promoter, specific oligonucleotides with putative binding sites for these transcription factors were used for additional gel-shift experiments. For the gel-shift experiments performed, the number of animals used are given in the figure legends. In additional experiments glomerular cytoplasmic proteins from two animals were prepared for Western blotting, and experiments were repeated twice.

Activation of transcription factors in tissue sections

Activation of the transcription factors NF-{kappa}B and AP-1 was also determined by Southwestern histochemistry in paraffin-embedded tissue sections. As for gel-shift experiments, both consensus and rat RANTES/CCL5-specific oligonucleotides were used. Two animals were examined in each group at each time point, and experiments were repeated twice.

Statistical analysis

Results are expressed as the mean ± SEM. Statistical significance between individual groups was tested using the Wilcoxon-Mann-Whitney test to compare two distinct groups of animals examined. A value of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Glomerular RANTES/CCL5 gene expression induced by LPS application

To examine the in vivo regulation of RANTES/CCL5 mRNA expression in glomeruli, the model of LPS-induced glomerular inflammation was used. After i.p. LPS injection (2 mg/kg body weight), RANTES/CCL5 mRNA expression was significantly increased in isolated glomeruli at 1, 3, and 6 h compared with that in control animals (Fig. 1A).



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  		FIGURE 1. A, Glomerular RANTES mRNA expression is significantly increased at 1, 3, and 6 h after LPS injection (2 mg/kg body weight) in rats compared with controls (1 h, p < 0.05; 3 and 6 h, p < 0.01). At each time point three animals were examined, and the experiments were repeated twice. B, LPS tolerance was induced by two consecutive LPS injections. When the time interval between low dose LPS (0.04 mg/kg body weight) and high dose LPS (2 mg/kg body weight) was 48 h, glomerular RANTES expression was almost completely abolished compared with nontolerant animals, as shown for the 6 h point after high dose LPS injection (compare lanes 2 and 5; p < 0.001). At each time point three animals were examined, and the experiments were repeated twice.

 
To gain insight into the functional regulation of glomerular RANTES/CCL5 expression, an in vivo model of LPS tolerance was established. Tolerance was induced by priming the animals with a low dose of LPS (0.04 mg/kg body weight) i.p., followed by a high dose of LPS (2 mg/kg body weight) at different time intervals ranging from 24–72 h. In tolerant animals glomerular RANTES/CCL5 mRNA expression was significantly reduced compared with that in nontolerant rats (Fig. 1B, compare lanes 3–6 with lane 2) and was almost completely abolished after LPS injection when the high dose of LPS was given 48 h after the low or priming LPS dose (Fig. 1B, compare lanes 2 and 5).

Activation of transcription factor binding in isolated glomeruli after in vivo LPS application: consensus binding sites

The regulation of RANTES/CCL5 mRNA expression depends on the binding of transcription factors to its promoter. Therefore, the binding activity of nuclear proteins isolated from glomeruli after LPS injection was examined using consensus binding sites of transcription factors, which were shown previously to play a pivotal role in the regulation of human and mouse RANTES/CCL5 mRNA expression.

In nuclear proteins isolated from glomeruli an increased AP-1 binding activity was detected in gel-shift experiments at 1, 3, and 6 h after LPS injection as shown in Fig. 2 for the 1 and 3 h points. Supershift experiments performed at 3 h demonstrated two major protein bands of c-Jun and JunD and two smaller bands of c-Fos and JunB (Fig. 2), indicating a predominance of c-Jun/Jun-D heterodimers.



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  		FIGURE 2. Gel-shift and supershift experiments were performed with nuclear proteins isolated from glomeruli at 1, 3, and 6 h after LPS injection. AP-1 binding activity significantly increases at 1, 3, and 6 h (see also Fig. 3A) after LPS injection (p < 0.01). In supershift experiments c-Jun, c-Fos, JunD, and JunB can be detected in these protein complexes. C/EBP binding activity significantly increases at 1 and 3 h (see also Fig. 3B) with a maximum at 6 h after LPS injection (p < 0.01) as shown. In supershift experiments {beta}, {gamma}, {delta}, and {epsilon} proteins are detected in the complexes, with a minor band for {alpha} protein. NF-{kappa}B binding activity significantly increases (p < 0.01) at all time points examined (see also Fig. 3C, left). In supershift experiments the NF-{kappa}B proteins p50 and p65 can be detected in the nuclear protein complexes (see also Fig. 3C, right). At each time point three animals were examined, and the experiments were repeated twice.

 
In addition, C/EBP binding activity was detected in nuclear proteins isolated from glomeruli at 1, 3, and 6 h after LPS injection, with a maximum binding activity at 6 h after LPS injection as shown in Fig. 2. At this time point a number of different proteins could be demonstrated in the complexes formed, predominantly {beta}, {gamma}, {delta}, and {epsilon} proteins, with {alpha} showing a minor band in the supershift experiments (Fig. 2).

Also, NF-{kappa}B binding activity was detected in gel-shift experiments at 1, 3, and 6 h after LPS injection, as shown for 3 h in Fig. 2. Supershift experiments at this time point showed that p50 and p65 proteins were detected in the nuclear extracts using anti-p50 (NLS) and anti-p65 (NLS) Abs, whereas p52, RelB, and c-Rel Abs did not shift the NF-{kappa}B band further (Fig. 2).

Effect of tolerance induction on transcription factor binding in isolated glomeruli: consensus binding sites

To understand the functional role transcription factor activation might play in the in vivo regulation of glomerular RANTES/CCL5 mRNA expression in rats, the model of LPS tolerance was used. In nontolerant rats AP-1 binding activity significantly increased in glomeruli (p < 0.01) at 1, 3, and 6 h after LPS injection compared with controls (Fig. 3A, compare lane 2 with lanes 4, 6, and 8). In tolerant animals, however, glomerular AP-1 binding activity was significantly reduced (p < 0.05) after LPS injection at all time points examined (Fig. 3A, compare lanes 4 and 5, 6 and 7, and 8 and 9). No AP-1 binding activity was detected in controls treated or not treated with low dose LPS.



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  		FIGURE 3. Gel-shift experiments with nuclear proteins isolated from glomeruli of controls and 1, 3, and 6 h after LPS application in tolerant and nontolerant rats using consensus binding sites. A, AP-1 binding activity is not detected in the control groups (lanes 2 and 3). AP-1 binding activity significantly increases (*, p < 0.01) in nontolerant animals ({square}) after LPS injection compared with controls at all time points examined (lane 2 compared with lanes 4, 6, and 8). AP-1 binding activity is significantly reduced (#, p < 0.05) after LPS injection in tolerant rats () compared with nontolerant animals after LPS application (compare lanes 4 and 5, 6 and 7, and 8 and 9). B, C/EBP binding activity significantly increases at all time points after LPS injection in nontolerant animals ({square}) compared with controls with a maximum at 6 h (compare lane 2 with lanes 4 and 6 (*, p < 0.05) and with lane 8 (**, p < 0.01])). In tolerant animals () C/EBP binding activity is almost completely abolished (#, p < 0.001) in both control rats (lane 3) and at all time points after LPS injection compared with nontolerant animals (compare lanes 4 and 5, 6 and 7, and 8 and 9). C, NF-{kappa}B binding activity (left) is significantly increased (*, p < 0.01) at all time points examined after LPS injection in both nontolerant ({square}) and tolerant () animals compared with controls (lanes 2 and 3). However, there are no significant differences when tolerant and nontolerant animals after LPS injection are compared (compare lanes 4 and 5, 6 and 7, and 8 and 9). Supershift experiments (right) do not reveal any differences in the p50/p65 heterodimer complexes formed at 3 h after LPS injection when nontolerant and tolerant animals are compared. Using Rel B, c-Rel, and p52 Abs, no supershifted band can be detected. At each time point three animals were examined, and experiments were repeated three times. tol-control, animals injected with the low dose LPS 48 h before the isolation of glomerular nuclear proteins.

 
Glomerular C/EBP binding activity significantly increased in nontolerant animals after LPS injection, with a maximum at 6 h (Fig. 3B). This increased C/EBP binding activity was almost completely abolished in tolerant animals after LPS injection (Fig. 3B, compare lanes 4 and 5, 6 and 7, and 8 and 9). Glomerular C/EBP binding activity detected in nontolerant controls was also significantly reduced in tolerant controls, e.g., 48 h after low dose LPS injection (Fig. 3B, compare lanes 2 and 3).

Glomerular NF-{kappa}B binding activity, however, was significantly increased (p < 0.01) even in tolerant animals after LPS injection compared with controls (Fig. 3C, left, compare lanes 4–9 with lanes 2 and 3) and was not different from the increased binding activity in nontolerant rats after LPS injection (Fig. 3C, left, compare lanes 4 and 5, 6 and 7, and 8 and 9). Supershift experiments performed at 3 h after LPS injection also did not reveal any major differences in p50 or p65 protein complexes between tolerant and nontolerant animals (Fig. 3C, right).

The activation of NF-{kappa}B in glomeruli was further supported by experiments in which an increased amount of phosphorylated, e.g., degradable, I{kappa}B{alpha} proteins could be demonstrated in protein extracts isolated from glomeruli (Fig. 4). The expression of phosphorylated I{kappa}B{alpha} protein was significantly increased after LPS injection in both tolerant and nontolerant rats compared with that in controls. No difference in expression was detected when tolerant and nontolerant animals were compared at 3 and 6 h after LPS injection. At 1 h after LPS injection, tolerant animals showed increased expression of phosphorylated I{kappa}B{alpha} compared with nontolerant rats. The significance of this finding, if any, is not clear from the present experiments (Fig. 4).



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  		FIGURE 4. Glomerular proteins were isolated from controls and at 1, 3, and 6 h after LPS injection from tolerant and nontolerant rats. The expression of the phosphorylated inhibitor protein I{kappa}B{alpha} is significantly increased (p < 0.01) as early as 1 h after LPS application in tolerant animals and at 3 and 6 h after LPS injection in both nontolerant and tolerant rats compared with controls. Two animals were examined at each time point in each group, and experiments were repeated twice.

 
Activation of transcription factors by Southwestern histochemistry: consensus binding sites

In addition to gel-shift experiments, activation of the transcription factors NF-{kappa}B and AP-1 was analyzed in situ by Southwestern histochemistry in paraffin-embedded tissue sections of tolerant and nontolerant rats using double-stranded oligonucleotides with consensus binding sites (Fig. 5). An increased binding activity of the transcription factors was demonstrated in glomeruli at 1, 3, and 6 h after in vivo LPS application compared with that in control animals, as shown for the 3 h experiments (Fig. 5). In tolerant rats the activation of both NF-{kappa}B and AP-1 was significantly reduced compared with that in nontolerant rats at each time point examined after LPS application.



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  		FIGURE 5. In addition to gel-shift experiments DNA binding activity for the transcription factors NF-{kappa}B and AP-1 was examined in situ by Southwestern histochemistry. Intense nuclear positive staining is detected for NF-{kappa}B and AP-1 at 3 h after LPS injection in nontolerant animals compared with the appropriate controls, which is almost completely suppressed for both transcription factors in tolerant rats. Two animals were examined in each group at each time point, and experiments were repeated twice.

 
Cloning of a 1.5-kb fragment of the rat RANTES/CCL5 promoter

Next, as a general assessment of transcription factor binding activity using consensus binding sites does not reflect the binding activities of these transcription factors at the promoter region of interest, the rat RANTES/CCL5 promoter was cloned using a rat Genome Walker kit (GenBank accession no. AF282896). By homology restriction analysis (BLAST analysis) the rat RANTES promoter was aligned with the known sequence of the mouse RANTES promoter (GenBank accession no. U02298) and the major transcription start site predicted by homology (Fig. 6A).



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  		FIGURE 6. A, BLAST alignment of rat (AF282896) and mouse (U02298) RANTES promoters. The major transcription start site (+1) of the rat RANTES promoter was predicted by homology to the mouse gene. B, Binding sites for transcription factors AP-1, C/EBP, and NF-{kappa}B, oligonucleotides of the rat RANTES promoter used as specific probes for gel-shift experiments (see Fig. 7) with putative binding sites for the transcription factors, are shown in comparison with the transcription factor consensus binding sites.

 
Putative binding sites for known transcription factors were localized using the MatInspector from Genomatix (http://genomatix.gfs.de) (15), and several binding sites for NF-{kappa}B, AP-1, and C/EBP were found within the first 1.5-kbp region (NF-{kappa}B: -55 to -46, -82 to -73, -1588 to -1579, AP-1: -114 to -100, -202 to -191, -242 to -233, -415 to -403, -463 to -449, -563 to -549, -637 to -626, -998 to -987, -1135 to -1125, -1237 to -1226, -1418 to -1407, -1447 to -1436, C/EBP: -89 to -75, -210 to -200, -375 to -365, -390 to -379, -479 to -468, -1200 to -1191, -1485 to -1471). The putative specific binding sites in the rat RANTES/CCL5 promoter for the three transcription factors AP-1, C/EBP, and NF-{kappa}B, which were used for the gel-shift experiments described below, are given in comparison to the known consensus binding sequences in Fig. 6B.

Transcription factor binding activity in isolated glomeruli of tolerant and nontolerant rats: putative specific binding sites

To examine the regulation of rat RANTES/CCL5 mRNA expression by transcription factor binding in a more specific manner, gel-shift experiments were repeated using oligonucleotides with sequences derived from the rat RANTES/CCL5 promoter, including putative binding sites for AP-1, C/EBP, and NF-{kappa}B.

To demonstrate glomerular AP-1 binding activity two sequences were used, one proximal (-120 to -90) and the other distal (-1149 to -1104) to the transcription start site. With the proximal sequence no shifted bands in the gel-shift experiments could be demonstrated (data not shown). Using the distal binding site, binding activity was significantly increased in nontolerant rats 3 and 6 h after LPS injection compared with controls (Fig. 7A, left, compare lanes 6 and 8 with lane 2). In tolerant animals binding activity was significantly reduced after LPS injection at these time points compared with nontolerant animals (Fig. 7A, left, compare lanes 5 and 6, and lanes 7 and 8).



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  		FIGURE 7. A, Gel-shift experiments using AP-1 binding sequences (-1149 to -1104) of the rat RANTES promoter (left); binding activity significantly increases (*, p < 0.01) at 3 and 6 h after LPS injection in nontolerant rats compared with controls (compare lanes 6 and 8 with lane 2). In tolerant animals binding activity is significantly reduced (#, p < 0.05) at 3 and 6 h compared with nontolerant rats after LPS injection (compare lanes 5 and 6, and 7 and 8). Gel-shift experiments using C/EBP binding sequences (-486 to -462) of the rat RANTES promoter (right); binding activity significantly increases (*, p < 0.01) at 3 and 6 h after LPS injection in nontolerant rats compared with controls (compare lanes 7 and 9 with lane 3). In tolerant animals binding activity is significantly reduced (#, p < 0.05) at 3 and 6 h compared with nontolerant rats after LPS injection (compare lanes 6 and 7, and 8 and 9). Three animals were examined at each time point studied in each group, and experiments were repeated twice. B, Gel-shift experiments using NF-{kappa}B binding sequences (-1593 to -1572 (left) and -100 to -62 (right)) of the rat RANTES promoter; with the distal promoter sequences (left) binding activity significantly increases (*, p < 0.01) at 3 and 6 h after LPS injection in nontolerant rats compared with controls (compare lanes 5 and 7 with lane 1). In tolerant animals binding activity is significantly reduced (#, p < 0.05) at 3 and 6 h compared with nontolerant rats after LPS injection (compare lanes 5 and 6, and 7 and 8). Using the proximal promoter sequences (right), binding activity is significantly increased (*, p < 0.01) in nontolerant rats after LPS injection at all time points studied compared with controls (compare lane 2 with lanes 4, 6, and 8). NF-{kappa}B binding activity in tolerant animals is also significantly (p < 0.05) reduced at 3 and 6 h after LPS injection compared with nontolerant rats at these time points (compare lanes 6 and 7, and 8 and 9). Three animals were examined at each time point studied in each group, and experiments were repeated twice. C, Competition experiments. Using rat RANTES promoter-specific oligonucleotides (-100 to -62) competition experiments were performed, with nuclear proteins isolated at 3 h after LPS injection in tolerant and nontolerant rats. Consensus NF-{kappa}B binding competitors (left) significantly reduce (*, p < 0.01) binding activity in nontolerant (compare lanes 4 and 5) as well as in tolerant (compare lanes 7 and 8) animals. Consensus C/EBP binding competitors (right) are without any effect on specific binding activity in both nontolerant (compare lanes 4 and 5) and tolerant (compare lanes 7 and 8) animals after LPS injection. Two animals were examined in each group, and experiments were repeated twice. {square}, nontolerant animals; , tolerant rats; tol-control, animals injected with the low dose LPS 48 h before the isolation of glomerular nuclear proteins.

 
Using specific oligonucleotides with putative binding sites for C/EBP (-486 to -462 of the rat RANTES/CCL5 promoter) an increased binding activity was demonstrated in glomeruli at 3 and 6 h after LPS injection in nontolerant rats compared with controls (Fig. 7A, right, compare lanes 7 and 9 with lane 3). This increased binding activity at these time points was significantly reduced after LPS injection in tolerant animals (Fig. 7A, right, compare lanes 6 and 7, and lanes 8 and 9). However, contrary to what would have been predicted, binding activity was significantly increased in tolerant compared with nontolerant animals in both controls and to a lesser degree at 1 h after LPS injection (Fig. 7A, right, compare lanes 2 and 3, and lanes 4 and 5), the significance of which is not clear from the present experiments.

A complex regulatory network also obviously exists at the rat RANTES promoter levels with respect to NF-{kappa}B binding. Using specific oligonucleotides proximal to the transcription start site (-60 to -30) no increased binding activity was demonstrated (data not shown). Using oligonucleotides with a distal location (-1593 to -1572), increased binding activity was seen at 3 and 6 h after LPS injection in nontolerant rats compared with controls (Fig. 7B, left, compare lanes 5 and 7 with lane 1). This increased binding activity after LPS injection was significantly reduced in tolerant rats compared with nontolerant animals (Fig. 7B, left, compare lanes 5 and 6, and lanes 7 and 8). With oligonucleotides between these two sites (-100 to -60) binding activity was significantly increased at 1, 3, and 6 h after LPS injection in nontolerant animals compared with controls (Fig. 7B, left, compare lanes 4, 6, and 8 with lane 2). This increased binding activity was significantly reduced in tolerant rats at 3 and 6 h after LPS injection, but not, however, at 1 h after LPS injection, compared with nontolerant rats (Fig. 7B, left, compare lanes 6 and 7, and lanes 8 and 9 contrary to lanes 4 and 5).

To gain further insight into the nature of proteins bound to the oligonucleotide probes used, competition experiments were performed. For these additional experiments the oligonucleotides -100 to -60 relative to the transcription start site were used, as in these sequences putative binding sites for C/EBP and NF-{kappa}B proteins are detected by MatInspector analysis (Fig. 7C). Using NF-{kappa}B consensus binding sites as competitor, specific binding activity was almost completely blocked in nontolerant as well as in tolerant animals 3 h after LPS injection (Fig. 7C, left, compare lanes 4 and 5, and lanes 7 and 8). Mutant NF-{kappa}B consensus binding sites, however, induced only a weak, nonsignificant reduction of specific binding activity (Fig. 7C, left, compare lanes 4 and 6, and lanes 7 and 9). Specific binding activity was, in contrast, not reduced by competitor oligonucleotides with consensus C/EBP binding sites in both nontolerant and tolerant rats 3 h after LPS injection (Fig. 7C, right, compare lanes 4 and 5, and lanes 7 and 8).

Activation of transcription factors by Southwestern histochemistry: putative specific binding sites

The activation of transcription factors for NF-{kappa}B and AP-1 was also analyzed in situ by Southwestern histochemistry in paraffin-embedded tissue sections of tolerant and nontolerant rats using double-stranded oligonucleotide probes of the rat RANTES/CCL5 promoter with putative specific binding sites (Fig. 8). As demonstrated in this figure increased binding activity of NF-{kappa}B and AP-1 was seen in glomeruli at all time points examined after LPS injection in nontolerant rats and was almost completely abolished in tolerant animals (Fig. 8, A and B).



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  		FIGURE 8. Southwestern histochemistry using rat RANTES promoter-specific oligonucleotides. Renal tissue was examined in controls and at 1, 3, and 6 h after LPS injection in tolerant and nontolerant animals. A, NF-{kappa}B binding activity (oligonucleotides -100 to -60) significantly increased after LPS application in nontolerant animals (pictures in the upper lane). This increased NF-{kappa}B binding activity is almost completely abolished in tolerant animals at all time points examined (pictures in the lower lane). B, AP-1 binding activity (oligonucleotides -1149 to -1104) significantly increases after LPS application in nontolerant animals (pictures in the upper lane); it is significantly reduced in tolerant animals at all time points examined (pictures in the lower lane). Two animals were examined in each group at each time point, and experiments were repeated twice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
RANTES/CCL5, a member of the {beta}-chemokine family, plays a pivotal role in the regulation of bone marrow-derived cell infiltration at inflammatory sites. Increased RANTES/CCL5 expression has been described in a variety of experimentally induced and human renal diseases, such as in glomerulonephritis, in protein overload nephropathy, or after angiotensin II infusion and kidney transplantation. The functional role renal RANTES/CCL5 expression might play to recruit inflammatory cells to the kidney and induce inflammatory lesions was examined by the use of RANTES/CCL5 receptor antagonists aminooxypentane- or Met-RANTES (3, 16, 17, 18, 19).

Inhibition of gene expression at inflammatory sites by interference with transcription factor activation is one of the promising new therapeutic strategies for specific gene targeting. In recent years studies performed to understand mechanisms relevant for the regulation of RANTES/CCL5 expression have concentrated on the role transcription factors might play. Human and mouse RANTES/CCL5 promoter regions have been cloned, and, depending on the cell system examined, interaction of the transcription factors NF-{kappa}B, AP-1, C/EBP, IFN regulatory factor-3, and Sp1 have been shown to play a pivotal role in the regulation of RANTES/CCL5 gene expression (10, 20, 21, 22, 23, 24).

To evaluate transcription factors that might be relevant in the regulation of glomerular RANTES/CCL5 expression in rats, we cloned the rat RANTES/CCL5 promoter, which demonstrates high homology to the mouse RANTES/CCL5 promoter (Fig. 6). Computer-assisted matrix analysis of the promoter sequence using MatInspector analysis revealed putative binding sites for the transcription factors AP-1, C/EBP, and NF-{kappa}B. To gain insight into the activation of these transcription factors in the LPS model of glomerular inflammation, gel-shift experiments were performed using oligonucleotides with consensus binding sites. These experiments demonstrate an increased binding activity of the transcription factors in nuclear extracts of isolated glomeruli during the 6-h observation period after LPS injection (Fig. 2). The maximum binding activity was seen early after LPS injection, within 1 and 3 h for AP-1 and NF-{kappa}B and, with some delay, at 6 h for C/EBP, which is consistent with the results of transcription factor activation published recently (25, 26, 27). Similar to our results, an increased binding activity of these transcription factors has also been demonstrated previously in different models of renal disease, such as in immune complex glomerulonephritis, ureteral obstruction, protein overload nephropathy, or hypertension (28, 29, 30, 31, 32).

To gain further insight into the functional relevance of the activation of these transcription factors with respect to glomerular RANTES/CCL5 mRNA expression, next an in vivo model of LPS tolerance was established. In this model we could demonstrate reduced glomerular expression of RANTES/CCL5 after LPS injection in tolerant animals compared with nontolerant rats (Fig. 1B).

Next, the binding of transcription factors was examined in this model of LPS tolerance. Using oligonucleotides with consensus binding sites, AP-1 and C/EBP binding activity was significantly reduced in tolerant rats after LPS injection compared with that in nontolerant animals (Fig. 3, A and B). NF-{kappa}B binding activity was, however, not reduced in tolerant animals. Also, in supershift experiments we could not demonstrate an increase in p50 protein complexes in tolerant animals, as expected from data published in macrophages rendered tolerant in vitro. Contrary to our gel-shift experiments we found reduced NF-{kappa}B binding activity in situ in renal tissue sections, as demonstrated by Southwestern histochemistry (Fig. 5).

Tolerance has been demonstrated previously to significantly influence transcription factor binding. As relevant mechanisms, suppression of mitogen-activated protein kinase activation, diminished NF-{kappa}B translocation and trans-activation, changes in the ratio between p50 and p65 subunit proteins, and also increased suppressor of cytokine signaling-1 expression have been described previously (33, 34, 35, 36, 37, 38, 39). Under the conditions used in our experiments a reduction in NF-{kappa}B binding, an increase in p50 protein complexes, or a reduction in phosphorylated I{kappa}B{alpha} did not occur in the glomeruli of tolerant rats and therefore could not explain the reduced glomerular RANTES/CCL5 expression in LPS tolerance.

Therefore, mechanisms other than or in addition to NF-{kappa}B binding must be responsible for the reduced glomerular RANTES expression. There is increasing evidence from in vitro studies that NF-{kappa}B DNA binding activity is regulated by c-Fos and c-Jun or by C/EBP and, more specifically, that regulation of human RANTES gene expression requires cooperation between NF-{kappa}B and other regulatory transcription factors (40, 41, 42, 43, 44, 45, 46). The interaction between the transcription factors AP-1 and NF-{kappa}B for tolerance induction has also been described recently in in vivo experiments (47, 48). Additionally, NF-{kappa}B DNA binding activity might be reduced by additional proteins complexed to the p50 or p65 NF-{kappa}B proteins. This possibility is highlighted by our Southwestern histochemistry experiments. In these experiments we could demonstrate reduced NF-{kappa}B DNA binding in situ in glomeruli of paraffin-embedded kidney sections (Fig. 5). During the isolation procedure of glomeruli for nuclear protein preparations for gel-shift experiments these proteins might dissociate, and NF-{kappa}B proteins might become accessible for DNA binding sites in gel-shift experiments. Candidate proteins that mask NF-{kappa}B DNA binding sites in situ have not been characterized by our in vivo experiments. However, there is increasing evidence that NF-{kappa}B-Rel proteins are complexed with additional nuclear proteins that change the interaction with other nuclear coregulators (23, 49). Thus, the regulation of glomerular RANTES expression under the experimental conditions employed is likely to depend on the interaction between AP-1 and NF-{kappa}B and/or C/EBP and NF-{kappa}B. The reason for the inhibited RANTES expression despite sufficient NF-{kappa}B DNA binding might be the reduced AP-1 and/or C/EBP binding, which seem to be essential for the ability of NF-{kappa}B to trans-activate to stimulate RANTES expression. Also, NF-{kappa}B might compete with other transcription factors for DNA binding sites and inhibit gene expression. Depending on the cross-talk between the transcription factors and the timely coordinated binding to the promoter region, activation of NF-{kappa}B might even be a crucial negative regulator step, as shown recently for IL-1{beta}/NF-{kappa}B-induced suppression of IL-6-stimulated {alpha}2-macroglobulin expression (50).

As activation of transcription factors examined by oligonucleotides with consensus binding sites does not strictly reflect transcription factor binding to the gene of interest, we repeated gel-shift experiments using specific oligonucleotides from the rat RANTES/CCL5 promoter with putative binding sites for the three transcription factors of interest (24, 51). Using specific oligonucleotides with AP-1 and C/EBP binding activity, we could, in principle, reproduce the findings described with consensus oligonucleotides. That is, AP-1 and C/EBP binding were significantly reduced in tolerant animals after LPS injection compared with that in nontolerant rats (Fig. 7, A and B). The data from the gel-shift experiments in nontolerant rats using specific oligonucleotides, however, showed additional nuclear protein binding compared with gel-shift experiments using consensus oligonucleotides. This might indicate binding of different components of the transcriptions factor proteins or binding of additional to date uncharacterized proteins in nontolerant rats after LPS injection to RANTES/CCL5 promoter-specific oligonucleotides. The data using gel-shift experiments could also be reproduced by Southwestern histochemistry for the AP-1 binding-specific oligonucleotide (Fig. 8B). However, contrary to the results using consensus oligonucleotides, rat RANTES/CCL5 promoter-specific oligonucleotides with putative NF-{kappa}B binding sites demonstrated reduced binding activity in tolerant animals after LPS injection compared with nontolerant rats (Fig. 7C). This reduced NF-{kappa}B binding demonstrated by gel-shift experiments could be reproduced with the same oligonucleotides in situ in renal tissue by Southwestern histochemistry (Fig. 8A).

The differences in NF-{kappa}B binding activity using consensus binding oligonucleotides and rat RANTES/CCL5-specific oligonucleotides might reflect gene-specific regulation. First, NF-{kappa}B activation takes place in tolerant animals, as demonstrated by phosphorylation of I{kappa}B{alpha} and binding to consensus sites. NF-{kappa}B complexes, however, might be masked by additional protein complexes, as discussed above. Second, although activated, NF-{kappa}B proteins do not bind to specific binding sites of the rat RANTES/CCL5 promoter, which was demonstrated by the experiments using specific oligonucleotides of the rat RANTES/CCL5 promoter. The NF-{kappa}B binding site in the rat RANTES/CCL5 promoter used is significantly different from the HIV-NF-{kappa}B, IFN{beta}-NF-{kappa}B, or consensus-NF-{kappa}B binding sites (Fig. 9) (52, 53). The substitution of two G:C to A:T base pairs in the p50 binding site (Fig. 9; positions -4 and -3) and a T:A to C:G base pair in the p65 binding site (Fig. 9; position +1) of the rat RANTES/CCL5 promoter might therefore significantly lower the binding affinity of NF-{kappa}B proteins to this promoter region. The additional A:T base pairs in the NF-{kappa}B p50 protein binding site of the rat RANTES/CCL5 promoter might recruit the transcription factor high mobility group protein I(Y) (HMG I(Y)) to this site and facilitate NF-{kappa}B binding, as shown by x-ray crystal structure analysis of the IFN-{beta}- and urokinase plasminogen activator-NF-{kappa}B binding sites (54). Induction of tolerance in our model might therefore influence HMG I(Y) expression and hence binding of NF-{kappa}B proteins to the rat RANTES/CCL5 promoter. Additionally, as described in recent years, the chromatin structure of a gene has been shown to be at the center of regulation of expressional changes (55, 56, 57). Chromatin remodeling at the IL-12 p40 promoter after LPS stimulation has been shown to be a Toll-like receptor 4-dependent, but a c-Rel protein-independent, pathway (58). Thus, in the present experiments induction of tolerance might also inhibit modifications in chromatin structure and therefore make rat RANTES/CCL5 promoter inaccessible to NF-{kappa}B, although activated, and thus explain the reduced glomerular RANTES/CCL5 mRNA expression in tolerant rats.



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  		FIGURE 9. NF-{kappa}B binding site of the rat RANTES promoter (-100/-60) in comparison with the consensus binding site and Ig/HIV1 and IFN-{beta} NF-{kappa}B binding sites. The p50 NF-{kappa}B subunit proteins bind to the 5'-DNA half-site (positions -5 through -1), and the p65 subunit proteins bind to the 3'-DNA half-site (adopted from Refs. 53 and 54 ). The rat RANTES/CCL5 NF-{kappa}B site differs from the consensus binding site at positions -4, -5, and +1.

 
Our experiments demonstrate that the expression of glomerular RANTES/CCL5 was reduced in endotoxin tolerance. Tolerance induction abolished LPS-stimulated AP-1 and C/EBP binding activity in total glomerular nuclear protein preparations. Although NF-{kappa}B binding activity is increased after LPS injection even after the induction of tolerance, specific binding activity for the rat RANTES/CCL5 promoter was reduced. In tolerance, the rat RANTES/CCL5 gene might be rendered inaccessible to NF-{kappa}B by changes in the chromatin remodeling process or the interactions of transcription factors at NF-{kappa}B binding sites. Further analysis of factors that reduce glomerular RANTES/CCL5 expression in tolerance should therefore enhance our understanding of the RANTES/CCL5 regulation pathway and help design approaches for specific gene therapy.


   Acknowledgments
 
We gratefully acknowledge the support and helpful discussion of Dr. Carol Stocking (Heinrich Pette Institute, Hamburg, Germany) when establishing the EMSA technique. We also acknowledge the excellent technical support of A. Dombrowski and M. Scharper. This article is dedicated to Prof. Arnold Vogt (Department of Immunology, University of Freiburg, Freiburg, Germany) on behalf of his 75th birthday.


   Footnotes
 
1 This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG TH 343/8-5) and the Werner-Otto-Stiftung (to F.T.) and from the Spanish Health Ministry (PI020539) and Comunidad de Madrid (08.4/0014/2001) (to C.G.-G.). Back

2 Address correspondence and reprint requests to Dr. Friedrich Thaiss, Department of Internal Medicine, Division of Nephrology and Osteology, University Hospital, Martinistrasse 52, 20246 Hamburg, Germany. E-mail address: thaiss{at}uke.uni-hamburg.de Back

Received for publication November 5, 2002. Accepted for publication April 8, 2003.


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 Materials and Methods
 Results
 Discussion
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