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J Dent Res 82(2): 96-100, 2003
© 2003 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Production of Colony-stimulating Factor in Human Dental Pulp Fibroblasts

Y. Sawa*, Y. Horie, Y. Yamaoka, N. Ebata, T. Kim, and S. Yoshida

Department of Oral Functional Science, Graduate School of Dental Medicine, Hokkaido University, N13 W7, Kita-ku, Sapporo 060-8586, Japan;

*corresponding author, sawa{at}den.hokudai.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Class II major histocompatilibity complex (MHC)-expressing cells are usually distributed in dental pulp, and it was postulated that the colony-stimulating factor (CSF) derived from dental pulp fibroblasts contributes to the migration of class II MHC-expressing cells into pulp tissue. This study aimed to investigate the CSF production of human dental pulp fibroblasts. In pulp tissue sections, granulocyte (G)-CSF was detected from normal teeth, while G-CSF, macrophage (M)-CSF, and granulocyte-macrophage (GM)-CSF were detected from teeth with dentinal caries. In cultured dental pulp fibroblasts, G-CSF was detected by immunostaining, immunoprecipitation, and ELISA, and mRNAs of G-CSF, M-CSF, and GM-CSF were detected by RT-PCR. The dental pulp fibroblasts cultured with TNF-{alpha} were found to increase the G-CSF expression and to produce M-CSF and GM-CSF. These findings suggest that dental pulp fibroblasts usually produce G-CSF. In the presence of TNF-{alpha}, dental pulp fibroblast express M-CSF and GM-CSF.

KEY WORDS: dental pulp • fibroblasts • M-CSF • GM-CSF • G-CSF


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The colony-stimulating factor (CSF) regulates the recruitment of mature leukocytes. Granulocyte (G)-CSF aids leukocyte migration across vascular endothelium, independent of an effect on adhesion, as a powerful stimulator of the migration (Rajavashisth et al., 1990; Yong, 1996; Pulendran et al., 2000). It has been reported that macrophage (M)-CSF initiates an influx of monocytes into the dental follicle and plays a role in odontoclast differentiation and recruitment during root resorption of deciduous teeth (Wise et al., 1996; Niida et al., 1997). Granulocyte-macrophage (GM)-CSF allows granulocytes to induce the expression of class II major histocompatibility complex (MHC) in macrophages (Fischer et al., 1988) and to inhibit the recruitment of granulocytes (Stanley et al., 1994). Recently, the presence of class II MHC-expressing cells in the odontoblastic layer was reported (Yoshiba et al., 1996; Ohshima et al., 1999) and observed in uninflamed dental pulp without blood vessels expressing multiple leukocyte adhesion molecules which play a key role in leukocyte migration from the blood stream into tissue (Springer, 1994; Sawa et al., 1998). Therefore, we postulated CSF which may be produced by pulpal fibroblasts as a candidate for cytokines which allow monocytes to migrate into pulp tissue from blood vessels and to differentiate into class II MHC-expressing cells. The study was designed to investigate the CSF production of dental pulp fibroblasts.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Human dental pulp tissue was obtained from third molars with dentinal caries (n = 16) and normal premolars extracted for orthodontic reasons (n = 20) from 18- to 40-year-old individuals. All procedures were performed with appropriate informed consent, and the protocol was approved by the Institutional Review Board on the Use of Human Subjects of Hokkaido University Graduate School of Dental Medicine. The pulp tissue was removed from the pulp cavity with tweezers after the teeth were divided parallel to the tooth axis by means of a chisel, and the removed pulp tissue was divided into two halves; one half was frozen and cut into 8-µm sections for immunostaining, and the other was cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco Life Technologies, Inc., Grand Island, NY, USA) as described elsewhere (Yamaoka et al., 2000). The dental pulp fibroblast clones were confirmed to be fibroblasts by hematoxylin-eosin (H-E) staining and immunostaining with mouse anti-human vimentin (Dako Japan, Inc., Kyoto, Japan), and were used in all examinations when 2 x 105 of cells (P2 and PD 2.0) had formed a 50% confluent monolayer in a 100-mm dish. The monolayer was further cultured in DMEM with 0.05 ng/mL of TNF-{alpha} or without for 24 hrs for protein analysis, or in RPMI 1640 (Gibco) without serum and phenol red for 96 hrs for reverse-transcription/polymerase chain-reaction (RT-PCR). Human cutaneous microvascular endothelial cells (VEC; CC-2505, Clonetics Corporation, Walkersville, MD, USA) and human lung fibroblasts (IMR-90; ATCC CCL-186) were used as controls (Sawa et al., 2000).

Immunostaining
Procedures were performed as described elsewhere (Yamaoka et al., 2000). Mouse anti-human M-CSF, GM-CSF, and G-CSF IgGs (Ancell Co., Bayport, MN, USA) were used (anti-CSFs). Alexa Fluor (AF) 488-conjugated goat anti-mouse IgG (Molecular Probes, Inc., Eugene, OR, USA) as second antibody was used with the tissue sections. A cocktail of anti-CSFs (1 µg/mL; Ancell) and rabbit anti-human Ki-67 (Dako), and a cocktail of AF 488-conjugated goat anti-mouse IgG and AF 568-conjugated goat anti-rabbit IgG (0.1 µg/mL; Molecular Probes) were used with dental pulp fibroblast clones as second antibodies.

Immunoprecipitation and Immunoblot
The dental pulp fibroblast monolayer was solubilized in 1 mL of cell lysis buffer [50 mM HEPES (pH 7.3), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 100 mM NaF, 10 mM Na pyrophosphate, and 1% Triton X, and 5% glycerol]. The lysate was centrifuged at 12,500 x g for 20 min at 4°C, and the supernatant (2 mg/mL) was mixed with 1 µg of anti-CSFs (Ancell) and 30 µL of Protein G-agarose beads (Roche Diagnostics GmbH, Mannheim, Germany). After the mixture was gently shaken at 4°C for 12 hrs, the beads were mixed in 30 mL of sample buffer and centrifuged. The supernatant was loaded on 15% polyacrylamide gel by electrophoresis, and immunoblot with anti-CSFs was performed (Ancell) as described elsewhere (Yamaoka et al., 2000).

RT-PCR
The extraction of total RNA from monolayers of dental pulp fibroblast clones (n = 20) and VEC was achieved with a QIAshredder column and an RNeasy kit (Qiagen, Inc., Tokyo, Japan). The RT-PCR was performed on 30 ng of total RNA with 50 pM of primer sets for G-CSF, M-CSF, and GM-CSF genes according to the manufacturer’s instructions (Stratagene, La Jolla, CA, USA). The PCR was also performed on human ß-actin (Stratagene), following each term of PCR for CSFs. The products were separated on 2% agarose gel (NuSieve; FMC, Rockland, ME, USA) and visualized by Syber Green (Takara Shuzo Co., LTD, Tokyo, Japan).

Enzyme-linked Immunosorbent Assay (ELISA)
The dental pulp fibroblast monolayer was solubilized in cell lysis buffer as described above. A 40-fold dilution with 0.1 M carbonate buffer (pH 9.6) was placed into a 96-well microtitration plate for 12 hrs at 4°C, and the reaction of cell lysate (1 µg protein per well) with anti-CSFs (Ancell) was performed as described elsewhere (Sawa et al., 2000). Wells without treatment by anti-CSFs were used as a control, indicating the non-specific IgG binding activity of the second antibody. The CSF production was evaluated by Student’s t test on the anti-CSF binding activity, expressed as the mean A415 of 5 wells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunohistochemical Analysis on Pulp Tissue Sections
The dental pulp fibroblasts were immunostained by anti-G-CSF in all sections from normal teeth and teeth with dentinal caries. The dental pulp fibroblasts were also immunostained by anti-M-CSF (6 positives of 16) and by anti-GM-CSF (11 positives of 16) in sections from teeth with dentinal caries, while not immunostained in sections from normal teeth. There was tissue with the odontoblastic layer which strongly immunostained by anti-G-CSF and anti-M-CSF (not by GM-CSF) from teeth with dentinal caries (3 positives of 16), while there was no tissue with the immunoreactivity in the odontoblastic layer from normal teeth (Fig. 1Go).



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  		Figure 1. Immunostaining of dental pulp sections for CSFs. The reaction products are visualized by green fluorescence. (a,c,e,g) Serial sections from a normal tooth. (b,d,f,h) Serial sections from a tooth with dentinal caries. (a,b) H-E staining. (c,d) G-CSF. (e,f) M-CSF. (g,h) GM-CSF. In sections from the normal tooth, dental pulp fibroblasts (arrowheads) are stained by anti-G-CSF, but not by anti-M-CSF and anti-GM-CSF. In sections from the tooth with dentinal caries, dental pulp fibroblasts (arrowheads) are stained by anti-G-CSF and anti-M-CSF, especially strongly in the odontoblast layer (arrow), and by anti-GM-CSF. Bar = 100 µm.

 
Immunohistochemical Analysis on Cultured Dental Pulp Fibroblasts
The dental pulp fibroblasts were immunostained by anti-G-CSF but not by anti-M-CSF and anti-GM-CSF, while dental pulp fibroblasts cultured with TNF-{alpha} were immunostained by anti-G-CSF, and by anti-M-CSF and anti-GM-CSF to a weaker extent than by anti-G-CSF. Both dental pulp fibroblasts expressing and those not expressing Ki-67 in the nuclei were immunostained by anti-CSFs. The IMR-90 was not immunostained by anti-CSFs (Fig. 2Go).



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  		Figure 2. Immunostaining of cultured dental pulp fibroblasts. The reaction products of anti-CSFs are visualized by green fluorescence, and of anti-Ki-67 by red fluorescence in the nuclei. (a1) G-CSF. (a2) G-CSF in IMR-90. (b) M-CSF. (c) M-CSF, with TNF-{alpha}. (d) GM-CSF, with TNF-{alpha}. Cells are stained by anti-G-CSF, while IMR-90 as a control was not stained. Cells are not stained by anti-M-CSF. Cells cultured with TNF-{alpha} are stained by anti-M-CSF and anti-GM-CSF to a weaker extent than anti-G-CSF. Both cells in the cell-division cycle expressing Ki-67 in the nuclei and dormant cells not expressing Ki-67 are stained by anti-CSFs. There are accumulated proliferative cells (d, arrowhead). Bar = 100 µm.

 
Immunoblot Analysis
The G-CSF was detected in dental pulp fibroblasts cultured in the absence or presence of TNF-{alpha}. The M-CSF was not detected in cells cultured without TNF-{alpha}, while it was detected in cells with TNF-{alpha}. The GM-CSF was not detected in cells cultured in the absence or presence of TNF-{alpha} (Fig. 3AGo).



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  		Figure 3. (A) Immunoblot analysis of immunoprecipitated products with anti-CSFs. In whole-cell proteins of dental pulp fibroblasts cultured without TNF-{alpha}, G-CSF (19 kDa) was detected, while M-CSF (85 kDa) and GM-CSF (22 kDa) were not detected. In whole-cell proteins of dental pulp fibroblasts cultured with TNF-{alpha}, G-CSF and M-CSF were detected, while GM-CSF was not detected. M1, M2 = molecular-weight markers of 19.3, 86 kDa. (B) Expressions of G-CSF, M-CSF, and GM-CSF genes. (a) Dental pulp fibroblasts. (b) VEC. PCR products for G-CSF and M-CSF mRNAs were detected in VEC at an intensity similar to that in a control. GM-CSF mRNA was detected at an intensity lower than that in VEC, and also lower than in G-CSF and M-CSF mRNAs. PCR products for ß-actin showed similar intensities in all samples.

 
Expression of CSFs mRNA
The mRNAs of G-CSF and M-CSF were detected in all dental pulp fibroblast clones at an intensity of PCR products in VEC similar to that in controls. The GM-CSF mRNA was detected in all dental pulp fibroblast clones, it was weaker than in VEC, and weaker than G-CSF and M-CSF mRNAs. The ß-actin was detected at similar intensities in all samples (Fig. 3BGo).

The Effect of TNF-{alpha} on CSF Production
In cells cultured without TNF-{alpha}, the reaction to anti-G-CSF was statistically significantly different from that of the control (p < 0.05), but the reactions to anti-M-CSF and anti-GM-CSF were not. In cells cultured with TNF-{alpha}, reactions to anti-CSFs were significantly different from those of the control and cells cultured without TNF-{alpha} (p < 0.005). There were no significant differences among reactions to anti-CSFs on cells cultured with TNF-{alpha}. Non-specific IgG binding activity of cells increased by culture with TNF-{alpha} (Fig. 4Go).



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  		Figure 4. Effect of TNF-{alpha} on CSF production, examined by anti-CSFs binding activity to lysate of cells cultured in the absence (open bar) or presence (filled bar) of TNF-{alpha}. Each bar, expressing absorbance change at 415 nm per min, represents the mean value (labeled) and standard deviation of 5 determinations. Controls are shown in the left margin (cont), indicating the non-specific IgG binding activity of second antibody. *Significantly different from control (p < 0.05). **Significantly different from control, and from cells without TNF-{alpha} (p < 0.005).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The G-CSF was detected from dental pulp fibroblasts of all sections and clones (Figs. 1-4GoGoGoGo), and G-CSF in cultured dental pulp fibroblasts increased by TNF-{alpha} (Fig. 4Go). These results suggest that dental pulp fibroblasts usually produce G-CSF and that the production is enhanced by inflammatory cytokines. The M-CSF and GM-CSF were not detected from dental pulp fibroblasts in sections from normal teeth or in culture (Figs. 1-4GoGoGoGo), while the mRNAs were detected here (Fig. 3Go). These results may suggest that dental pulp fibroblasts usually express M-CSF and GM-CSF mRNAs which had degraded without translation, or that the proteins are immediately degraded by the ubiquitin/proteasome pathway (Putland et al., 2002). Especially GM-CSF may easily degrade, because the gene expression was weaker than that of G-CSF and M-CSF. The M-CSF and GM-CSF were immunohistochemically detected in sections from teeth with dentinal caries (Fig. 1Go). Since the immunoreactivities varied among teeth with dentinal caries, the CSF expression may be dependent on the progress of dentinal caries. The M-CSF and GM-CSF were detected from dental pulp fibroblasts cultured with TNF-{alpha} (Figs. 2-4GoGoGo), suggesting that dental pulp fibroblasts produce M-CSF and GM-CSF when induced by TNF-{alpha}. The absence of GM-CSF in immunoprecipitation may be due to inactivation in the procedure. It is known that Ki-67 expresses in the nuclei of proliferating cells at all cell-division phases except G0 (Barnard et al., 1987). The CSF production was observed in both dental pulp fibroblasts expressing and those not expressing Ki-67 (Fig. 2Go), showing that CSF production would be independent of the signal transduction by growth factors.

The reactivity of dental pulp fibroblasts to anti-G-CSF was stronger than that to anti-M-CSF and anti-GM-CSF in immunostaining (Fig. 2Go), while there were no significant differences among reactivities to anti-CSFs in cells cultured with TNF-{alpha} in ELISA (Fig. 4Go). It is thought that anti-G-CSF reacted both to G-CSF and to other family molecules like IL-6 and oncostatin M (Rose and Bruce, 1991), and that molecules of this family were inactivated at pH 9.6 in coating buffer (Sawa et al., 1994). The non-specific IgG binding activity of dental pulp fibroblasts increased in culture with TNF-{alpha} (Fig. 4Go). The enhancement can be ascribed to the induction of immunoglobulin superfamily members in cells stimulated by TNF-{alpha}, which cause homophilic binding to the IgG Fc domain (Springer, 1994).

Since there was no specific expression of CSFs in the odontoblastic layer of uninflamed pulp (Fig. 1Go), there may be other factors that contribute to the migration of dendritic-like class II MHC-expressing cells in the odontoblastic layer (Ohshima et al., 1999). Interestingly, there was some inflamed pulp tissue where the odontoblastic layer strongly reacted with anti-G-CSF and anti-M-CSF (Fig. 1Go). In inflamed pulp, class II MHC-expressing cells are found in the pulp core, and such cells are dense in the odontoblastic layer (Sawa et al., 1998). Odontoblasts stimulated by inflammatory agents may produce G-CSF and M-CSF at a stronger level than fibroblasts in the pulp core, which contributes to the monocyte transmigration from blood vessels, to the differentiation into macrophages and dendritic cells, and also to the accumulation in the odontoblastic layer.

In conclusion, our results suggest that dental pulp fibroblasts usually produce G-CSF, and that dental pulp fibroblasts have the ability to produce M-CSF and GM-CSF induced by TNF-{alpha}. The CSF provided from dental pulp fibroblasts may contribute to the efficient recruitment of leukocytes for immunological surveillance to create pulp defense systems.


   ACKNOWLEDGMENTS
 
This work was supported by a grant-in-aid for Scientific Research (B)(2) from the Ministry of Education, Science, Sports, and Culture of Japan (No. 12470379).

Received April 23, 2002; Last revision September 3, 2002; Accepted October 24, 2002


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 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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