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Originally published In Press as doi:10.1074/jbc.M409138200 on October 14, 2004

J. Biol. Chem., Vol. 279, Issue 52, 54039-54045, December 24, 2004
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Toll-like Receptor 9 Regulates Tumor Necrosis Factor-{alpha} Expression by Different Mechanisms

IMPLICATIONS FOR OSTEOCLASTOGENESIS*

Alla Amcheslavsky, Wei Zou, and Zvi Bar-Shavit{ddagger}

From the H. Hubert Humphrey Center for Experimental Medicine and Cancer Research, The Hebrew University Faculty of Medicine, P. O. Box 12272, Jerusalem 91120, Israel

Received for publication, August 10, 2004 , and in revised form, September 22, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
CpG oligodeoxynucleotides (CpG-ODNs), mimicking bacterial DNA, stimulate osteoclastogenesis via Toll-like receptor 9 (TLR9) in receptor activator of NF-{kappa}B ligand (RANKL)-primed osteoclast precursors. This activity is mediated via tumor necrosis factor (TNF)-{alpha} induction by CpG-ODN. To further reveal the role of the cytokine in TLR9-mediated osteoclastogenesis, we compared the ability of CpG-ODN to induce osteoclastogenesis in two murine strains, BALB/c and C57BL/6, expressing different TNF-{alpha} alleles. The induction of osteoclastogenesis and TNF-{alpha} release by CpG-ODN was by far more noticeable in BALB/c-derived than in C57BL/6-derived osteoclast precursors. Unexpectedly, as revealed by Northern analysis, CpG-ODN induction of TNF-{alpha} mRNA increase was more efficient in C57BL/6-derived cells. The cytokine transcript abundance was increased due to both increased message stability and rate of transcription. The difference between the two cell types was the result of a higher transcription rate in CpG-ODN-induced C57BL/6-derived cells caused by a single nucleotide polymorphism in {kappa}B2a site within the TNF-{alpha} promoter sequence. CpG-ODN enhanced the rate of the cytokine translation in BALB/c-derived cells. Thus, CpG-ODN modulated both transcription and translation of TNF-{alpha}. The induction of transcription was more evident in C57BL/6-derived cells, while the induction of translation took place only in BALB/c-derived osteoclast precursors. Altogether the cytokine was induced to a larger extent in BALB/c-derived osteoclast precursors, consistent with the increased CpG-ODN osteoclastogenic effect in these cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial products are the cause of pathological bone loss in a variety of diseases including periodontitis, osteomyelitis, bacterial arthritis, and infected metal orthopedic implant failure (1–5). Bacterial factors have been studied extensively for their ability to activate the innate immune system via Toll-like receptors (TLRs)1 present on the host immune cells (6, 7). Bone cells also express TLRs enabling the bacteria-derived factors to exert their effects on the skeleton (8, 9). The most studied bacterial factor in this regard is lipopolysaccharide, recognized by both osteoclast and osteoblast lineage cells via TLR4 (8, 9). Bacterial DNA has been shown to be a pathogen-derived structure activating the innate immune system via TLR9 (10–14). This activity depends on unmethylated CpG dinucleotides in particular base contexts ("CpG motif") (12, 14). Synthetic oligodeoxynucleotides containing CpG motifs (CpG oligodeoxynucleotides (CpG-ODNs)) mimic the bacterial DNA immunostimulatory activity (15).

We have shown that CpG-ODNs inhibit the activity of the physiological osteoclast differentiation factor, receptor activator of NF-{kappa}B ligand (RANKL). Interestingly CpG-ODNs strongly increase osteoclastogenesis in RANKL-pretreated osteoclast precursors (16). Thus, CpG-ODNs exert a dual effect on osteoclast differentiation: inhibition of osteoclastogenesis in early precursors but enhancement of osteoclastogenesis in precursors exposed to an osteoclastogenic signal. TNF-{alpha} mediates the enhanced osteoclastogenic effect of CpG-ODN by an autocrine mechanism (16).

BALB/c and C57BL/6 express different TNF-{alpha} alleles and differ in the response of their innate immune system (17–24). In light of the pivotal role of TNF-{alpha} in mediating the osteoclastogenic effect of CpG-ODN, we studied the induction of osteoclastogenesis and TNF-{alpha} expression in cells derived from mouse strains expressing different alleles of the cytokine. We found that both osteoclastogenesis and TNF-{alpha} release induced by TLR9 activation were by far more evident in BALB/c-than in C57BL/6-derived cells, and we examined the mechanistic basis for the differential cytokine regulation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Mice—Seven- to 9-week-old male BALB/c and C57BL/6 mice were obtained from Harlan Laboratories Ltd. (Jerusalem, Israel).

Reagents—Glutathione S-transferase-RANKL (residues 158–316) was prepared as described previously (16). M-CSF and neutralizing anti-TNF-{alpha} antibodies (rat IgG) were purchased from R&D Systems, Inc. (Minneapolis, MN). Nuclease-resistant phosphorothioate oligodeoxynucleotide (5'-TCCATGACGTTCCTGACGTT-3' (ODN 1826)) (15) was purchased from BTG (Rehovot, Israel) and had undetectable endotoxin according to a limulus amoebocyte lysate assay (BioWhittaker, Walkersville, MD). 5,6-Dichloro-1-{beta}-D-ribofuranosylbenzimidazole was purchased from Sigma. Mouse monoclonal anti-{beta}-actin antibody was purchased from Sigma. Mouse monoclonal anti-phospho-extracellular signal-regulated kinase (ERK), anti-phospho-p38, and rabbit polyclonal anti-ERK antibodies were purchased from Cell Signaling Technology Inc. (Beverly MA). Mouse monoclonal anti-phospho-c-Jun N-terminal kinase and rabbit polyclonal anti-TNF-{alpha} antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-mouse TLR9 antibody was purchased from IMGENEX (San Diego, CA). Protein G-agarose was purchased from Oncogene Research Products (San Diego, CA). Brefeldin A (BFA) was purchased from Calbiochem. Media and sera were purchased from Biological Industries (Beth Haemek, Israel).

In Vitro Osteoclast Formation Assay—Primary bone marrow macrophages (BMMs) were isolated as described previously (25) and were plated in 96-well plates (1.3 x 105/well) in {alpha}-minimum Eagle's medium containing 10% FCS, M-CSF (50 ng/ml), and RANKL (10 ng/ml). After 3 days medium was changed to medium devoid of RANKL with or without CpG-ODN (100 nM), and osteoclast formation (tartrate-resistant acid phosphatase-positive cells with three or more nuclei) was evaluated 30 h later.

Northern Blot Analysis—BMMs were plated in 35-mm tissue culture plates (2 x 106/plate) as described above. Total cellular RNA was extracted, fractionated, and transferred to nylon membranes as described previously (26). 32P-Labeled mouse TNF-{alpha} or mouse ribosomal protein L32 cDNA probes were used for hybridization. The hybridized membranes were then subjected to autoradiography, and the density of each of the mRNA bands was quantified by densitometry.

Western Blot Analysis—Western analysis was performed as described previously (26). Bands were quantified by densitometry.

Electrophoretic Mobility Shift Assay—Oligonucleotides corresponding to the {kappa}B2a site of the TNF-{alpha} promoter of BALB/c or C57BL/6 and to the {kappa}B consensus sequence (5'-CATGCCCTCTGGGGCCCCATA-3', 5'-CATGCCCTCTCGGGCCCCATA-3', and 5'-AGTTGAGGGGACTTTCCCAGGC-3', respectively) were labeled, and nuclear extracts (5 µg) were incubated with labeled probes as described previously (9). Samples were then fractionated on a 7% polyacrylamide gel and visualized by exposing the dried gel to film.

Nuclear Run-on Assay—BMMs were incubated in 150-mm tissue culture plates (2 x 107/plate). Cells were washed with PBS and then incubated for 1 h with or without CpG-ODN. Nuclei were harvested, resuspended in storage buffer (50 mM Tris-HCl, pH 8.3, 40% (v/v) glycerol, 5 mM MgCl2, 0.1 mM EDTA), and frozen at –80 °C until labeling. Transcripts that were initiated in the cells were allowed to continue in the presence of [{alpha}-32P]UTP (PerkinElmer Life Sciences) at 30 °C for 30 min. Labeled RNA was extracted with TRI Reagent and hybridized to nylon membrane cross-linked plasmid DNA for 48 h at 42 °C. The membranes were washed and exposed to x-ray film.

ELISA—BMMs were incubated in 48-well plates (3.0 x 105 cells/well) for 3 days as above. Cell monolayers were washed with PBS and incubated in 0.4 ml of {alpha}-minimum Eagle's medium containing 10% FCS and M-CSF in the absence or presence of CpG-ODN (100 nM) for 4 or 6 h. TNF-{alpha} release was measured by ELISA using a DuoSet ELISA kit (R&D Systems, Inc.) according to the manufacturer's instructions. The relative cell number was estimated by the methylene blue uptake assay (27) using a Dynatech plate reader (Dynatech Laboratories, Inc., Chantilly, VA).

Metabolic Labeling and Pulse-Chase Experiments—BMMs were incubated in 35-mm tissue culture plates (2 x 106/plate) as above. On day 3 cells were washed with PBS and incubated in methionine- and cysteine-free medium. After 30 min the medium was changed, and cells were incubated in methionine- and cysteine-free medium in the presence of 10% dialyzed FCS and 5 µg/ml BFA for 10 min. Then M-CSF was added to the cultures together with 100 µCi/ml 35S-labeled methionine and cysteine mixture (Redivue Pro-Mix L-[35S]methionine and L-[35S]cysteine in vitro cell labeling mixture, Amersham Biosciences). In pulse-chase experiments (28, 29), after a 10-min incubation with BFA, 50 ng/ml lipopolysaccharide was added to the cultures together with 100 µCi/ml 35S-labeled methionine and cysteine mixture. After 2 h cells were washed twice with ice-cold PBS. Cells were incubated in {alpha}-minimum Eagle's medium containing 10% dialyzed FCS with or without of 100 nM CpG-ODN for the indicated time and then washed extensively with PBS and lysed in RIPA lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1.0% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS). We carried out immunoprecipitation with either polyclonal anti-TNF-{alpha} or anti-{beta}-actin antibodies followed by SDS-PAGE and autoradiography.

Protein Synthesis—BMMs were incubated in 48-well plates (3.0 x 105 cells/well). On day 3 cells were washed with PBS and incubated in 0.4 ml of leucine-free medium containing 10% dialyzed FCS. [3H]Leucine (5 µCi/well) was added to the cultures, and cells were incubated for 4 h in the absence or presence of CpG-ODN. Total protein was precipitated and dissolved (28). The amount of 3H incorporated into cell-associated macromolecules was determined by scintillation counting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Modulation of Osteoclastogenesis and TNF-{alpha} Release by CpG-ODN—Since TNF-{alpha} mediates the osteoclastogenic effect of CpG-ODNs we compared this activity and CpG-ODN-induced TNF-{alpha} production in cells derived from BALB/c and C57BL/6 expressing two different alleles of the cytokine. It can be seen in Fig. 1A, as shown by us previously (16), that CpG-ODN markedly increased osteoclast differentiation in RANKL-primed cells. In this experiment, cells were incubated for 72 h with M-CSF (50 ng/ml) and RANKL (10 ng/ml). Medium was then changed, and cells were incubated for an additional 30 h in the absence or presence of CpG-ODN (100 nM). As seen in the figure, the increase in osteoclast differentiation by CpG-ODN was more evident in BALB/c-than in C57BL/6-derived cells (~6.6-versus ~2.2-fold induction, respectively). To examine whether this difference is correlated with a differential TNF-{alpha} release from cells derived from the two strains in response to CpG-ODN we used ELISA. We found (Fig. 1B) that the release of TNF-{alpha} from BALB/c-derived cells was greater by ~3-fold than the release from C57BL/6-derived cells. We did not detect any release of the cytokine in the absence of CpG-ODN up to 6 h of incubation.



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  		FIG. 1.
Modulation of osteoclastogenesis and TNF-{alpha} release by CpG-ODN. BALB/c- and C57BL/6-derived BMMs were plated as described under "Experimental Procedures." A, osteoclasts were scored as detailed under "Experimental Procedures." B, monolayers were washed and incubated for 4–6 h in the presence or absence of CpG-ODN, and TNF-{alpha} release was measured by ELISA. TRAP + MNc, TRAP-positive multinucleated cells.

 
We next examined whether the correlation between CpG-ODN-induced osteoclastogenesis and TNF-{alpha} release represents cause and effect. To this end, we performed an osteoclastogenesis assay in the presence of anti-TNF-{alpha} neutralizing antibodies. As we showed previously for BALB/c-derived cells (16), these antibodies markedly inhibited CpG-ODN-induced osteoclastogenesis also in C57BL/6-derived cells. At a dilution of 1:100 of the anti-TNF-{alpha} neutralizing antibodies, 60 and 80% inhibition were obtained with BALB/c and C57BL/6-derived cells, respectively (not shown). In parallel, we examined whether the anti-TNF-{alpha} neutralizing antibodies reduce TNF-{alpha} levels in medium conditioned by the CpG-ODN-treated cells. In this experiment, BALB/c- and C57BL/6-derived cells were plated in tissue culture plates (60-mm diameter, 5 x 106 cells/ plate in 5 ml), and after RANKL priming (3 days, 10 ng/ml) cells were incubated with CpG-ODN (100 nM) in 3 ml. Media were collected after 6 h, and aliquots were incubated without or with neutralizing anti-TNF-{alpha} antibodies overnight at 4 °C. Immunocomplexes were removed by immunoprecipitation. Aliquots were then subjected to ELISA. As seen in Table I, the cytokine levels were reduced. Already at a 1:1000 dilution of anti-TNF-{alpha} neutralizing antibody a dramatic decrease of TNF-{alpha} levels in serum was observed. Replacing the neutralizing antibodies with rat IgG did not reduce the cytokine levels (not shown). The possibility that a differential TNF receptor responsiveness in cells derived from the two strains contributes to the difference in CpG-ODN-induced osteoclastogenesis was ruled out by experiments showing no difference in TNF-{alpha} induction of c-Jun N-terminal kinase, ERK, and p38 phosphorylation in these cells (not shown).


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  		TABLE I
Anti-TNF-{alpha} neutralizing antibodies reduce TNF-{alpha} levels

BALB/c- or C57BL/6-derived cells were plated and incubated as described under "Results." Aliquots (0.5 ml) were incubated with the indicated dilution of anti-TNF-{alpha} neutralizing antibody and were subjected to ELISA after removal of the immunocomplexes.

 
Modulation of TNF-{alpha} mRNA Abundance by CpG-ODN in BALB/c- and C57BL/6-derived Cells—We then attempted to identify the mechanism(s) responsible for the higher TNF-{alpha} release from BALB/c-derived cells in response to CpG-ODN. To this end, we used Northern analysis to test the modulation of TNF-{alpha} mRNA steady-state levels by CpG-ODN. Addition of CpG-ODN to RANKL-primed bone marrow osteoclast precursors increased the cytokine mRNA levels in cells derived from the two strains (Fig. 2). Unexpectedly TNF-{alpha} transcript induction was more evident in C57BL/6-derived cells despite the lower cytokine release in response to CpG-ODN in these cells. Densitometric analysis showed that basal TNF-{alpha} transcript abundance was low and similar in the cells derived from the two strains. CpG-ODN addition increased TNF-{alpha} mRNA levels up to ~5- and ~15-fold in cells harvested from BALB/c and C57BL/6, respectively.



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  		FIG. 2.
Modulation of TNF-{alpha} mRNA abundance in BALB/c- and C57BL/6-derived cells by CpG-ODN. BMMs were plated as described under "Experimental Procedures." Cells were then washed and treated with CpG-ODN as indicated. RNA was prepared and examined for transcript abundance of TNF-{alpha}. L32 was the loading control.

 
To understand the differential modulation of TNF-{alpha} mRNA we first test whether CpG-ODN inhibits the cytokine transcript degradation and whether this activity differs in cells derived from the different strains. BALB/c- or C57BL/6-derived cells were grown for 72 h in medium containing M-CSF (50 ng/ml) and RANKL (10 ng/ml). Monolayers were then washed and incubated in the presence or absence of CpG-ODN (100 nM). After 50 min the transcription inhibitor 5,6-dichloro-1-{beta}-D-ribofuranosylbenzimidazole was added, and at the indicated time points RNA was collected for Northern analysis. Fig. 3A shows a representative autoradiogram showing TNF-{alpha} and L32 mRNA abundance. The TNF-{alpha}/L32 ratio was calculated from densitometric analyses (average from five independent experiments). As seen in Fig. 3B the rate of TNF-{alpha} mRNA degradation did not differ between cells derived from the two strains (half-life of 8.0 ± 1.0 and 8.0 ± 1.2 min in BALB/c- and C57BL/6-derived cells, respectively). CpG-ODN addition stabilized the cytokine transcript to a half-life of 17.1 ± 2.07 and 15.5 ± 0.84 min in BALB/c- and C57BL/6-derived cells, respectively. Thus, while message stabilization by CpG-ODN contributes to the increased TNF-{alpha} transcript abundance, it cannot be the reason for the difference in mRNA induction in the two strains.



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  		FIG. 3.
Modulation of TNF-{alpha} mRNA stability by CpG-ODN in BALB/c- and C57BL/6-derived cells. BMMs were plated as described under "Experimental Procedures." Cells were then washed and incubated with or without CpG-ODN for 50 min. Then 5,6-dichloro-1-{beta}-D-ribofuranosylbenzimidazole (2.5 µg/ml) was added at the indicated time. TNF-{alpha} and L32 mRNA abundance was examined using Northern blot analysis. A representative autoradiogram (A) and a densitometric analysis (B) of an average of five experiments are shown. The TNF/L32 ratio at time 0 was normalized to 100 on both control and CpG-ODN-treated cells in each experiment. Dashed and solid lines represent cells in the absence or presence of CpG-ODN, respectively; closed and open squares represent C57BL/6 and BALB/c-derived cells, respectively.

 
To examine directly the rate of transcription we performed a nuclear run-on assay. Cells were incubated for 60 min in the presence or absence of CpG-ODN. Nuclei were prepared and incubated with [32P]UTP for 30 min. RNA was collected and hybridized to filters containing plasmids carrying TNF-{alpha} and L32 cDNAs as well as to the corresponding empty plasmids. Filters were then subjected to autoradiography. The autoradiogram shown in Fig. 4 reveals that CpG-ODN increased the transcription rate in cells derived from the two strains, and consistent with a more noticeable increase in TNF-{alpha} mRNA abundance in C57BL/6 derived cells, the cytokine transcription rate in these cells was ~3.3-fold greater than that observed in BALB/c cells.



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  		FIG. 4.
Nuclear run-on analysis of BALB/c- and C57BL/6-derived cells in the presence or absence of CpG-ODN. BMMs were plated as described under "Experimental Procedures." Monolayers were then washed, and cells were incubated for 60 min in the presence or absence of CpG-ODN. Nuclei were subjected to nuclear run-on assay. Labeled RNA transcripts were prepared and hybridized to the target DNA as indicated.

 
We examined whether the difference in CpG-ODN-induced TNF-{alpha} transcription rate in cells derived from the two strains is due to differences in TLR9 expression levels and/or intracellular signaling pathways known to mediate CpG-ODN cellular activities. Cells derived from BALB/c and from C57BL/6 express similar levels of TLR9, and addition of CpG-ODN did not change these levels as determined by Western analysis (Fig. 5A). CpG-ODN induced the phosphorylation of p38, ERK, and c-Jun N-terminal kinase in cells derived from the two strains (Fig. 5B). Although the kinetics was not identical in the different cells, this slight variability does not seem to be responsible for the marked difference in TNF-{alpha} transcription.



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  		FIG. 5.
TLR9 expression and CpG-ODN-induced signaling molecule phosphorylation in BALB/c- and C57BL/6-derived cells. BMMs were plated as described under "Experimental Procedures." Cells were incubated with CpG-ODN as indicated, and extracts were prepared for Western analyses. A, TLR9 levels were estimated using antibodies to this protein. Anti-actin antibody serves as the loading control. B, phosphorylation of ERK, p38, and c-Jun N-terminal kinase (JNK) was estimated using the corresponding antibodies. Anti-ERK antibody serves as the loading control. p-, phospho.

 
NF-{kappa}B activation plays a role in CpG-ODN-induced transcriptional modulation of TNF-{alpha} (30). Three of the four {kappa}B sites in the TNF-{alpha} promoter are identical in the two strains. However, there is a single nucleotide difference between one of the {kappa}B sites ({kappa}B2a) in C57BL/6 and in BALB/c (ctcggctgcccc versus ctgggctgcccc, respectively) (21, 31). We used electrophoretic mobility shift assay to examine the possibility that nuclear extracts derived from CpG-ODN-stimulated cells bind to the BALB/c-derived {kappa}B2{alpha} less efficiently than to the C57BL/6-derived {kappa}B2{alpha}. To this end, BALB/c- and C57BL/6-derived {kappa}B2{alpha} oligodeoxynucleotide sequences were labeled, and their interactions with nuclear extracts derived from the two strains were analyzed. A shift was observed when the C57BL/6-derived {kappa}B2{alpha} was used but not with BALB/c-derived {kappa}B2{alpha} (Fig. 6A). Moreover {kappa}B2a derived from C57BL/6, but not from BALB/c, inhibited the shift when labeled {kappa}B consensus site was used (Fig. 6B). In both experiments similar results were obtained with BALB/c- and C57BL/6-derived nuclear extracts. Thus, we conclude that the single nucleotide polymorphism in {kappa}B2a is responsible for the difference in CpG-ODN-induced TNF-{alpha} transcription between cells derived from the two strains.



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  		FIG. 6.
BALB/c- and C57BL/6-derived {kappa}B2a interactions with nuclear proteins. BMMs were plated as described under "Experimental Procedures." A, cells were incubated with CpG-ODN for the indicated times, and nuclear extracts (NE) from either BALB/c-derived cells or C57BL/6-derived cells (left and right 10 lanes, respectively) were prepared and subjected to electrophoretic mobility shift assay using {kappa}B2a derived from either BALB/c or C57BL/6 (B or C, respectively). B, cells were treated with CpG-ODN for 60 min (lanes 2–11; untreated control, lane 1), nuclear extracts were prepared from BALB/c- and C57BL/6-derived cells (upper and lower panels, respectively), and the ability of {kappa}B2a derived from either BALB/c (lanes 3–5) or C57BL/c (lanes 6–8) as well as unlabeled {kappa}B consensus sequence (lanes 9–11) to compete with the {kappa}B consensus sequence was measured using electrophoretic mobility shift assay.

 
Modulation of TNF-{alpha} Protein by CpG-ODN in BALB/c- and C57BL/6-derived Cells—We next examined the rate of TNF-{alpha} translation in BALB/c- and in C57BL/6-derived cells in the absence or presence of CpG-ODN. BMMs were grown for 3 days in medium containing M-CSF and RANKL. Cells were then washed briefly with PBS and incubated for 30 min in medium devoid of methionine and cysteine. Then 35S-labeled methionine and cysteine mixture (100 µCi/ml) and BFA (10 µg/ml) were added for 4 h in the absence or presence of CpG-ODN for the whole 4 h or for the last 2 h only. Cells were lysed and immunoprecipitated with anti-TNF-{alpha} and with anti-actin antibodies. The immunoprecipitates were subjected to PAGE and then to autoradiography (Fig. 7A). Using actin as a reference we found that TNF-{alpha} translation was enhanced in BALB/c- and C57BL/6-derived cells in response to CpG-ODN up to 17.0- and 2.6-fold, respectively. Taking into account TNF-{alpha} mRNA levels in naive and CpG-ODN-stimulated cells (see Fig. 2), we concluded that the rate of the cytokine translation rate was increased in BALB/c- but not in C57BL-derived cells. This difference does not reflect a differential effect of CpG-ODN on global protein synthesis since [3H]leucine incorporation into cellular macromolecules (Fig. 7B) was not markedly affected by CpG-ODN and was similar in cells derived from the two strains.



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  		FIG. 7.
Modulation of TNF-{alpha} protein translation by CpG-ODN in BALB/c- and C57BL/6-derived cells. A, cells were incubated in the presence or absence of CpG-ODN and 35S-labeled cysteine and methionine, harvested, and subjected to SDS-PAGE as detailed under "Experimental Procedures." B, cells were incubated in the presence or absence of [3H]leucine, and the incorporation to the trichloroacetic acid-insoluble fraction was measured as described under "Experimental Procedures."

 
To examine whether the mechanism of CpG-ODN-increased TNF-{alpha} protein includes modulation of degradation, cells were grown in medium in the presence of M-CSF and RANKL for 3 days. Cells were then washed with PBS and incubated for 30 min in the methionine- and cysteine-free medium. Lipopolysaccharide (50 ng/ml) was added for 2 h in the presence of 35S-labeled methionine and cysteine mixture (100 µCi/ml) and BFA (10 µg/ml). Cells were then washed extensively with PBS, incubated for the indicated time with or without CpG-ODN, and then analyzed as in the previous experiment (Fig. 7A). Fig. 8A shows an autoradiogram of an experiment examining the effect of CpG-ODN on TNF-{alpha} degradation in cells derived from BALB/c and C57BL/6. An average from two independent experiments shows that the rate of the cytokine degradation did not differ between cells derived from the strains (a half-life of 10.5 ± 4.8 and 10.4 ± 2.3 min in BALB/c- and C57BL/6-derived cells, respectively). Upon CpG-ODN addition, the half-life was slightly (not significantly) increased to 15.6 ± 3.0 and 15.1 ± 3.9 min in BALB/c- and C57BL/6-derived cells, respectively (Fig. 8B).



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  		FIG. 8.
Modulation of TNF-{alpha} protein stability by CpG-ODN in BALB/c- and C57BL/6-derived cells. BMMs were plated and treated as described under "Experimental Procedures" in the presence or absence of CpG-ODN for the indicated time. A representative autoradiogram (A) and a densitometric analysis (B) of an average of two experiments are shown. The TNF/actin ratio at time 0 was normalized to 100 on both control and CpG-ODN-treated cells in each experiment. Dashed and solid lines represent cells in the absence or presence of CpG-ODN, respectively; closed and open squares represent C57BL/6- and BALB/c-derived cells, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Although TNF-{alpha} is long known for its role in osteoclastogenesis and bone resorption (33–35), details of this involvement are controversial. For example, several investigators have found that TNF-{alpha} exhibits osteoclastogenic activity independently of RANKL (36, 37), while others have demonstrated that TNF-{alpha}-induced osteoclastogenesis depends on RANKL (26, 38). It is generally accepted, however, that the cytokine mediates the osteoclastogenic effect of lipopolysaccharide (5, 39), and we have found that TNF-{alpha} also mediates the osteoclastogenic activity of CpG-ODN (16). Bacterial products are involved in a variety of pathological bone loss situations in human at least in part because of increased TNF-{alpha} production (40, 41). The pleiotropic cytokine TNF-{alpha} is involved in a variety of diseases, and it is therefore not surprising that the influence of the cytokine polymorphisms on its production and disease have drawn attention.

In the present study we examined the interrelationships between induction of osteoclastogenesis and TNF-{alpha} production by TLR9 activation in cells expressing different alleles of the cytokine. Activation of TLR9 by CpG-ODN resulted in a more efficient osteoclastogenesis in BALB/c-than in C57BL/6-derived osteoclast precursors. Consistently higher levels of TNF-{alpha} were released from BALB/c-derived cells in response to CpG-ODN challenge. We have shown that in cultures containing both osteoclast and osteoblast-lineage cells CpG-ODN induces TNF-{alpha} in both cell types (9). It was therefore important for the present study that the cultures did not contain osteoblastic or mesenchymal cells. We confirmed this by the absence of alkaline phosphatase-positive cells (<0.2%) and the absence of RANKL (not shown).

TNF-{alpha} gene expression has been studied extensively and shown to be regulated on multiple levels: transcription, mRNA stability, translation, and protein stability (42–46). Despite a higher level of TNF-{alpha} release in response to CpG-ODN by cells derived from BALB/c, the induced cytokine mRNA levels were lower in these cells. Induction of TNF-{alpha} transcription is used by several factors, including CpG-ODN, as a mechanism to regulate the cytokine (30, 43, 47). We found that in C57BL/6 induction of TNF-{alpha} transcription by CpG-ODN was more evident, and we showed that this difference was caused by a single nucleotide polymorphism between the two strains in one of the NF-{kappa}B sites ({kappa}B2a) within the TNF-{alpha} promoter. We excluded the possibility that the differential regulation of the cytokine transcription in the two strains by CpG-ODN was also affected indirectly by differences in TLR9 levels and/or signal transduction events mediated by this receptor. It is of note that different results were obtained when dendritic cells from these strains were studied (48). In this work, a higher expression of TLR9 was demonstrated in C57BL/6, and no significant difference in TNF-{alpha} release in response to CpG-ODN was found. It is possible that the discrepancy in TLR9 level and TNF-{alpha} release is due to the different cell types studied. In addition, we estimated directly the receptor protein level (Western analysis), while Liu et al. (48) used reverse transcription-PCR. The cytokine release in the study by Liu et al. (48) was measured in medium collected after 24 h of incubation with CpG-ODN, while we measured TNF-{alpha} after 4 and 6 h. CpG-ODN also impacts the cytokine via message stabilization but to a similar degree in the two mice, consistent with 100% homology at the AU-rich elements known to play a role in the message stability (21, 31). It has been shown that the mitogen-activated protein kinase p38 cascade is involved in regulating mRNA stability via 3'-untranslated regions of several mRNAs including TNF-{alpha} mRNA (49). CpG-ODN induces p38 phosphorylation in a similar manner in cells derived from the two strains, consistent with a similar degree of TNF-{alpha} transcript stabilization observed. While CpG-ODN did not markedly affect TNF-{alpha} protein degradation, the TLR9 ligand enhanced the rate of the cytokine translation in BALB/c-derived cells. The mechanism(s) responsible for the higher TNF-{alpha} translation rate in BALB/c derived cells in response to CpG-ODN are not known currently. It was shown that GAU trinucleotide insertional mutation present in the AU-rich element of TNF-{alpha} mRNA of New Zealand White mice results in the hindered binding of RNA-binding proteins, thereby leading to a significantly reduced production of TNF-{alpha} protein (50). However, neither BALB/c nor C57BL/6 carry this mutation, and no other differences between the mice known to be involved in translational regulation are observed in the 3'-untranslated region. There are, however, five single nucleotide polymorphisms in sites not known for their involvement in translational regulation. We intend to examine the possibility that some or all of these sites play a role in regulating the cytokine translation. It is possible that the differential stimulation of TNF-{alpha} translation by CpG-ODN in the two strains is not caused by the cytokine polymorphism but by a differential effect on factor(s) modulating the cytokine translation. For example, the TNF-{alpha} translational silencer, TIA-1, is a reasonable candidate since this protein is regulated differently in BALB/c and in C57BL/6 (51, 52). We will explore the possible scenario that CpG-ODN modulates TIA-1 differently in the two strains, leading to a more effective silencing of TNF-{alpha} translation in C57BL/6-derived cells.

It was recently proposed that the CpG motif is an important active ingredient in bacterial extracts (53). Thus, our findings predict that pathological consequences of bacterial infections depend on the TNF-{alpha} allele expressed. It has indeed been shown that the two mouse strains we used respond in a different manner to bacterial infections (24, 54). As a cytokine with a broad range of proinflammatory and immunostimulatory actions, TNF-{alpha} is thought to play a pivotal role in a number of human diseases including rheumatoid arthritis and periodontitis and a number of infectious and autoimmune diseases (55, 56). Thus, the association between TNF-{alpha} polymorphisms, its production, and disease has been studied by a number of investigators. While in a number of studies it was concluded that such an association exists (57–61), other studies did not find evidence for this (62–64). This is not surprising since factors modulating the cytokine synthesis or molecules mediating its activity such as TNF receptors are also expected to impact TNF-{alpha}-dependent functions.

In conclusion, using two mouse strains expressing different TNF-{alpha} alleles, we clarified the mechanism of TLR9-mediated induction of TNF-{alpha}. Our study revealed the basis for a differential regulation of different TNF-{alpha} alleles. Further studies are required to examine the role of TNF-{alpha} polymorphism in bacteria-induced pathological bone loss. In this regard it is of interest to mention a recent study (65) showing association between bone mineral density of the lumbar spine with TNF gene polymorphism in early postmenopausal Japanese women. Finally, although this aspect is not directly linked to our experiments, our data might be of relevance to the clinical use of CpG-ODNs as vaccines for infectious disease and cancer (32, 66–68). It is tempting to speculate that the ability of the oligodeoxynucleotide to stimulate the patient's cells to produce TNF-{alpha} could predict the success of the therapy, and therefore the selection of this approach should take into consideration the specific TNF-{alpha} allele expressed.


   FOOTNOTES
 
* This work was supported by Israel Science Foundation Grant 662-02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 972-2-67588363; Fax: 972-2-6414583; E-mail: barsha{at}cc.huji.ac.il.

1 The abbreviations used are: TLR, Toll-like receptor; ODN, oligodeoxynucleotide; RANKL, receptor activator of NF-{kappa}B ligand; ERK, extracellular signal-regulated kinase; BMM, bone marrow macrophage; BFA, brefeldin A; TNF, tumor necrosis factor; M-CSF, macrophage colony-stimulating factor; FCS, fetal calf serum; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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