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Dentin-induced in vivo Inflammatory Response and in vitro Activation of Murine Macrophages

¹ Department of Stomatology, Pathology, Bauru Dental School, University of São Paulo-Bauru, Rua Sérvio Túlio Carrijo Coube, 3-33, Apto 91-C, Jardim Infante Dom Henrique, 17012-632-Bauru, São Paulo, Brazil;
² Catholic University of Brasília and Brasília Medicine School, University of Brasília-Brasília, Distrito Federal, Brazil; and
³ Department of Pharmacology, Ribeirão Preto Medicine School, University of São Paulo-Ribeirão Preto, São Paulo, Brazil;

*corresponding author, vanessa{at}fob.usp.br

	ABSTRACT

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The activation of inflammatory cells and consequent release of mediators play an important role in the resorption of mineralized tissues. In the present study, we examined the ability of dentin extracts to induce inflammatory cell recruitment and activation. We showed here that dentin extracts triggered an intense cell migration and progressive cell maturation, in a time- and dose-dependent manner. Expression of interleukin-1ß (IL-1ß), tumor necrosis factor- (TNF-), nitric oxide (NO), and hydrogen peroxide (H₂O₂) was also up-regulated by dentin extracts. These results show that inflammatory events can be elicited in response to dentin, which may suggest a possible involvement of dentin molecules in the inflammatory events, coupled with their release at the root resorption sites.

KEY WORDS: root resorption • nitric oxide • cytokines • dentin

	INTRODUCTION

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Inflammatory mediators locally released into the tooth/periodontal microenvironment, such as cytokines and reactive intermediates, are recognized as stimulating the resorption process of mineralized tissue. The principal cell types involved in this process are osteoclasts, highly specialized cells for resorption of hard tissues, osteoblasts, and inflammatory cells, which produce stimulators of osteoclastic activity such as cytokines, enzymes, and hormones (Flynn et al., 1999). It is well-established that interleukin-1ß (IL-1ß) and tumor necrosis factor- (TNF-) stimulate osteoclast development, while the precise effects of nitric oxide (NO) and hydrogen peroxide (H₂O₂) on osteoclastic function are contradictory. There is evidence that NO and H₂O₂ at low concentrations stimulate bone-resorption-inducing osteoclastic differentiation and cell motility (Garret et al., 1990; Brandi et al., 1995). On the other hand, in vitro studies have indicated that NO suppresses osteoblastic and osteoclastic formation and activity at either high (Brandi et al., 1995; Evans and Ralston, 1996) or low concentrations (Holliday et al., 1997).

The role is well-established of known stimuli that lead to the release of inflammatory mediators during root resorption, such as bacteria and their products. However, there are no studies investigating whether the release of dentin molecules could contribute to maintaining the resorption process during pathological exposure. For this reason, we examined the effects of dentin extracts on inflammatory cell recruitment and activation through stimulation of NO, H₂O₂, IL-1ß, and TNF- release. The participation of dentin constituents in the process of inflammatory root resorption is hypothesized.

	MATERIALS & METHODS

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All experimental procedures involving the use of animals were approved by the institutional Animal Welfare Committee.

Preparation of Dentin Extracts
Dentin was obtained from the crowns of impacted teeth recently extracted. The subjects’ rights have been protected, and a fully informed consent was obtained. After removal of soft tissue, dentin was ground by high-speed diamond rotation. The dentin powder was diluted in PBS and referred to as particulate extract (D-part). The particles’ sizes were determined by scanning electron microscopy as 2-10 µm. This preparation was centrifuged at 2000 x g for 10 min and the supernatant referred to as non-particulate extract (D-n-part). All fractions were sterilized in an autoclave at 121°C for 15 min. Alternatively, dentin powder, without being previously autoclaved, was demineralized with the use of 10% (w/v) EDTA. After 14 days, the EDTA-soluble fractions were concentrated by ultrafiltration (Amicon, YM10, W.R. Grace & Co., Danvers, MA, USA), dialyzed against water, diluted in PBS, filtered (0.22 µm), and referred to as d-Ext. The protein content of fractions was determined by the method of Bradford, with bovine serum albumin as the standard. The protein concentration of the D-part and D-n-part at 5 mg/mL (w/v) was below the detection level of method (1 to 2000 µg/mL). However, each mg (w/v) of dentin powder resulted in 0.18 µg/mL of protein after demineralization.

In vivo Inflammatory Response
Isogenic BALB/c mice were injected subcutaneously into the ventral region, or intraperitoneally (i.p.) with D-part or D-n-part at 0.1, 1, and 5 mg/mL. Control animals received PBS. Subcutaneous tissue from 2 to 24 days was subjected to histological evaluation. At intervals from 2 to 192 hrs after i.p. injections, peritoneal cells were prepared for differential counting and immunocytochemistry by centrifugation (Cytospin 3, Shandon, Lipshaw Inc., Pittsburgh, PA, USA). Slides for differential counting were stained by the May-Grünwald-Giemsa method (Woods and Ellis, 1996).

Mouse Macrophages
Peritoneal macrophages were harvested from naïve and thioglycolate-injected mice and suspended in RPMI 1640 (Flow Laboratories, Inc., McLean, VA, USA) containing 10% fetal bovine serum (Hy-Clone, Logan, UT, USA), 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma Chemical Co., St. Louis, MO, USA). We cultured 1 x 10⁶ cells/well at 37°C in an atmosphere of 5% CO₂.

Cell Culture
A 120-µL quantity of D-part (5 mg/mL) containing 1 x 10⁷ particles/mL was spread over glass coverslips before cell addition, resulting in 10 particles/cell. D-n-part and d-Ext were tested, respectively, at 5 mg/mL and 9 µg/mL. In some experiments, the cell culture was incubated with silica particles (SiO₂) (Sigma), which were 1 to 5 µm in diameter, at 30 particles/cell or 3 x 10⁷ particles/mL. Non-treated macrophages were used as a control group. After 24 hrs, adherent cells were cultured for 24 and 48 hrs alone or with IFN- (60 U/mL) (Genentech, Inc., San Francisco, CA, USA) and/or LPS (500 ng/mL) (Sigma), L-NMMA (200 µM), or aminoguanidine (AG) (200 µM) (Sigma), competitive inhibitors of NO synthase. The supernatants were assayed for NO and TNF- production and adherent cells for H₂O₂ production and immunocytochemistry. Dentin extract cytotoxicity was determined by either MTT assay (Mosmann, 1983) or trypan blue dye exclusion. Thioglycolate-stimulated macrophages were incubated with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] (5 mg/mL) (Sigma) during the last 6 hrs of culture. Cells were also incubated with trypan blue (1 mg/mL) for 5 min. Microscopic evaluation of dye exclusion determined the viable cells.

To test LPS contamination, we pre-incubated dentin extracts with polymyxin B at 500 ng/mL, 1 µg/mL, and 2 µg/mL, for 15 min at 37°C (Morrison and Jacobs, 1976) and then assayed them for NO production in macrophage cultures. LPS served as control.

Secretion of NO₂^-
We evaluated the secretion of NO by measuring NO₂^- accumulation in culture supernatants by the Griess reaction (Green et al., 1982).

Microassay for H₂O₂
H₂O₂ production was determined by means of a microassay for phenol red oxidation (Pick and Mizel, 1981). Cells were also stimulated with PMA [phorbol 12-myristate 13-acetate] (400 ng) (Sigma) to induce the maximum oxidative response. We used AG (200 µM) alone or in combination with PMA to assess the possible interference of NO levels in H₂O₂ measurement.

Measurement of TNF- Levels
TNF- was assayed by a sandwich enzyme-linked immunosorbent assay (ELISA) with anti-mouse TNF- (Santa Cruz Biotechnology) and alkaline phosphatase-labeled IgG antiserum (Vector Laboratories). Purified recombinant TNF- was used as standard.

Immunocytochemistry
Cells were processed for immunocytochemistry by means of an avidin-biotin-conjugated peroxidase technique. Five different antibodies were used: goat polyclonal antibodies to murine IL-1ß, M-20 (1:3000); to murine TNF-, M-18 (1:1000); rabbit polyclonal antibodies to murine inducible NO synthase (iNOS) or NOS 2, M-19 (1:200) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rat monoclonal antibodies to murine Mac-1 and Mac-2 (1:500) (Boehringer Mannheim Biochemicals, Indianapolis, IN, USA). The immunoproduct was visualized by 3,3' diaminobenzidine or 3-amino-9-ethyl-carbazole (Sigma). Negative controls consisted of sections in which primary antibodies were omitted and by the replacement of primary antibody by non-immune serum (DAKO, Glostrup, Denmark).

Statistical Analysis
Numerical values are means + standard error of mean (SEM). Data were analyzed by ANOVA followed by Tukey-Kramer’s test. The level of significance was set at P < 0.05.

	RESULTS

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Effects of Dentin Extracts on Cell Viability
Cell viability after dentin treatment alone or with IFN- and/or LPS, and L-NMMA, assessed by either Trypan blue dye exclusion or MTT reduction assay, was greater than 85%. However, 95% of cell viability was observed in the absence of dentin (Fig. 1A). Cells treated with 0.01% of sodium azide showed 35% of viability.

View larger version (25K): [in this window] [in a new window]   Figure 1. (A) Effect of dentin extracts on cell viability. Thioglycolate-elicited macrophages were treated with particulate (D-part) and non-particulate (D-n-part) dentin extracts (5 mg/mL) in the presence or absence of IFN- (60 U/mL), LPS (500 ng/mL), IFN-/LPS, and L-NMMA (200 µM), and cell viability was determined by MTT reduction assay after 48 hrs. *Significantly different from respective control values at P < 0.05. (B) Effect of polymyxin B on nitric oxide production induced by dentin extracts. Peritoneal macrophages were stimulated with particulate dentin extract (D-part) (5 mg/mL) and LPS (500 ng/mL) pre-incubated or not with polymyxin B as described in MATERIALS & METHODS. *Significantly different from respective control values at P < 0.05. #Indicates a level of significance (P < 0.01) relative to polymyxin B-untreated extracts. Data represent the mean + SEM of triplicates.

Effects of Dentin Extracts on Leukocyte Migration
All dentin extracts induced a dose- and time-dependent leukocyte migration in peritoneal cavities (Figs. 2A, 2B, 2C, 2D) that peaked at 12 hrs for D-part at 5 mg/mL. From this, the neutrophil migration declined and reached control levels after 72 hrs. Mononuclear cell numbers increased until 96 hrs and returned to control levels only after 192 hrs (Fig. 2D). Dentin-induced migrating cells from 12 hrs exhibited strong immunostaining for iNOS (data not shown). In subcutaneous tissue, we observed an initial, predominantly neutrophilic, infiltrate, followed by progressive cell maturation and formation of an epithelioid granuloma after 12 days (Figs. 2E, 2F). These findings are consistent with dentin-induced leukocyte migration in the peritonitis model.

View larger version (286K): [in this window] [in a new window]   Figure 2. (A,B,C) Effects of doses of dentin extracts on leukocyte recruitment. Peritoneal cavities were injected with 1 mL of indicated concentrations of dentin extracts and were aspirated with PBS 6 hrs post-injection. The number of leukocytes was assessed as described in MATERIALS & METHODS. Data points represent the mean + SEM from at least 5 mice. *Significantly different from control values at P < 0.05. (D) Time-dependency of dentin-extract-induced leukocyte extravasation. Mice were treated i.p. with particulate dentin extract (D-part) (5 mg/mL) at time zero or were left untreated (control group). Peritoneal cavities were washed at the indicated time points for quantification of polymorphonuclear leukocytes (PMNs) and mononuclear cells (MNs). Data are the mean + SEM of 5 mice. (E,F) Inflammatory responses to dentin extracts in subcutaneous tissue. Mice were injected s.c. with particulate dentin extract (D-part) (5 mg/mL), and the inflammatory cell recruitment was assessed after 5 (E) and 12 days (F) (original magnifications: E, 250x; and F, 400x).

View larger version (46K): [in this window] [in a new window]   Figure 3. (A,B,C,D) Time-course and effect of IFN- and LPS on nitric oxide production induced by dentin extracts. Thioglycolate-elicited (A,C) or naïve macrophages (B,D) were treated with particulate (D-part), non-particulate (D-n-part) (5 mg/mL), and demineralized (d-Ext) (9 µg/mL) dentin extracts, and silica (30 particles/cell) in the presence or absence of AG (200 µM), IFN- (60 U/mL), LPS (500 ng/mL), or L-NMMA (200 µM) for 48 hrs or at indicated times. *Significantly different from respective control values at P < 0.05. In (A) and (B), # indicates the significance at P < 0.05 vs. condition without AG. In (C) and (D), # indicates the significance at P < 0.05 vs. condition without additional stimulation. (E,F) Effect of dentin treatment on hydrogen peroxide and TNF- production by thioglycolate-elicited macrophages. Cells were stimulated with D-n-part and D-part (5 mg/mL), d-Ext (9 µg/mL), or silica (30 particles/cell) in the presence or absence of PMA (400 ng/mL) and AG (200 µM), and H2O2 production was assessed after 48 hrs. *Significantly different from respective control values at P < 0.05. In E, # indicates the significance at P < 0.05 vs. condition without PMA. In F, # indicates the significance at P < 0.05 vs. condition without additional stimulation. Data are the mean + SEM of triplicates.

TNF- Production
All dentin extracts, but not silica treatment, induced significant production of TNF-. The additional stimulation with IFN- and LPS induced a significant increase of TNF- production for all groups (Fig. 3F). Both thioglycolate-elicited and naïve macrophages displayed similar levels of TNF- release after dentin challenge (data not shown).

Immunocytochemical Expression of iNOS, IL-1ß, and TNF-
More than 95% of adherent cells, treated or not, were strongly immunoreactive for Mac-1 and Mac-2, consistent with the macrophage phenotype (Fig. 4A). For all markers, treatment with D-part or D-n-part for 48 hrs resulted in similar patterns of immunostaining, which was increased by additional stimulation (IFN-/LPS). The majority of dentin-treated macrophages exhibited moderate immunostaining for iNOS and IL-1ß (Figs. 4B, 4C), and strong immunostaining for TNF- (Fig. 4D), but not for all labeled cells. Control groups showed no evidence of immunostaining for iNOS (Fig. 4E) or IL-1ß, while TNF--positive cells were weakly immunostained (Fig. 4F).

View larger version (666K): [in this window] [in a new window]   Figure 4. Immunocytochemical expression of Mac-1 (A), iNOS (B), IL-1ß (C), and TNF- (D) by dentin-extract-treated macrophages. Thioglycolate-elicited macrophages were treated with particulate dentin extracts (D-part) (5 mg/mL) for 48 hrs. Sections in (E) and (F) are from control groups (original magnifications: A and F, 300x; B and C, 500x; D, 600x; E, 400x). (G) Postulated scheme of participation of dentin in the maintenance of external inflammatory root and bone resorption. In addition to bacteria and their products and inflammatory cell-derived cytokines, dentin may directly cause cell migration and activation (adapted from Susuki et al., 1999).

	DISCUSSION

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Moreover, H₂O₂ production was detected only in macrophages challenged with particulate dentin extracts, suggesting the principal involvement of particles in this pathway of activation.

When compared with controls, the treatment with particulate extracts induced an increase in cell death, which was significantly reduced with L-NMMA. Since it is an inducer of NO and H₂O₂, this result points to a role of these reactive intermediates in cell death, probably mediated by peroxynitrite. On the other hand, for non-particulate extract-treated macrophages, which are induced to produce NO but not H₂O₂, cell death worsens in the presence of L-NMMA, indicating a protective role of NO. In fact, there is evidence in the literature that NO could have dual effects depending on its concentration (Kröncke et al., 1997).

The chemical nature of the dentin factor which provokes the inflammatory response requires further study. The particulate extract used in this work can be phagocytosed by macrophages and may activate it. However, similar effects on macrophage activation were induced by demineralized and non-particulate extracts, corroborating the hypothesis that observed effects are mediated by dentin molecules, not due to particles. Besides, we were unable to detect NO, H₂O₂, IL-1ß, and TNF- in silica-treated macrophages, reinforcing the idea that phagocytosis per se is not sufficient to provide the additional signal for the induction of NO synthase activity in macrophages (Cunha et al., 1993).

Fig. 4G summarizes a hypothetical model of how dentin can participate in the maintenance of external inflammatory root resorption. We have suggested that dentin molecules set free as a result of matrix dissolution and trafficking pathways in osteoclasts during the resorption process (Nesbitt and Horton, 1997) may be able to act in a paracrine manner at resorption sites and function as chemotactic and activator signals for inflammatory cells. Inflammatory mediators IL-1ß, TNF-, and IFN- have already been implicated as up-regulators of bone and root resorption (Kabashima et al., 1998; Kawashima and Stashenko, 1999), but the effects of reactive intermediates are conflicting (Lowik et al., 1994; Holliday et al., 1997; Brandi et al., 1995).

This work demonstrates for the first time the ability of dentin to elicit inflammatory events. We believe that this property points to a possible role of dentin in the processes of inflammatory root resorption when dentin molecules are continually released into the tooth/periodontal microenvironment, or even in bone resorption associated with developing periapical lesions. A better understanding of factors that lead to the production of inflammatory mediators at this type of site and their in vivo effects on osteoclastic cells might facilitate the development of new therapies for inflammatory hard tissue resorption.

	ACKNOWLEDGMENTS

 
This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundação de Amparo à Pesquisa do Estado de São Paulo, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. We thank Maria Cristina Carrara Felippe, Prof. Dr. Lúcia Helena Faccioli, Emília E. Hayashi, Ana Paula Campanelli, Prof. Dr. Simone G. da Fonseca, and Marilena Heredia for their technical support; and Paul T. Shafee for his English language services in copy-editing the manuscript.

Received March 9, 2001; Last revision January 31, 2003; Accepted February 28, 2003

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