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Macrophages and Mast Cells Control the Neutrophil Migration Induced by Dentin Proteins

¹ Department of Stomatology, Faculty of Dentistry of Bauru;
² Department of Pharmacology and
³ Biochemistry and Immunology, School of Medicine of Ribeirão Preto, University of São Paulo, Brazil;
⁴ Department of Basic Sciences, School of Dentistry, Araçatuba State University of São Paulo; and
⁵ Department of Basic Sciences, University of Texas-Houston Health Science Center Dental Branch, Houston, TX, USA;

^* corresponding author, tarcilia{at}zipmail.com.br.

	ABSTRACT

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Dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), the major dentin proteins, have been shown to induce neutrophil migration through release of IL-1ß, TNF-, MIP-2, and KC. However, the sources of these mediators were not determined. Here, the roles of macrophages and mast cells (MC) in dentin-induced neutrophil accumulation were investigated. Peritoneal MC depletion or the enhancement of macrophage population increased DSP- and DPP-induced neutrophil extravasation. Moreover, supernatants from DSP- and DPP-stimulated macrophages caused neutrophil migration. The release of neutrophil chemotactic factor by macrophages was inhibited by dexamethasone or the supernatant of DSP-treated MC. Consistently, dexamethasone and the MC supernatant inhibited the production of IL-1ß, TNF-, and MIP-2 by macrophages. This inhibitory activity of the DSP-stimulated MC was neutralized by anti-IL-4 and anti-IL-10 antibodies. These results indicate that dentin induces the release of the neutrophil chemotactic substance(s) by macrophages, which are down-modulated by MC-derived IL-4 and IL-10.

KEY WORDS: neutrophil chemotaxis • DSP • DPP

	INTRODUCTION

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Molecules released from dentin, including numerous growth factors and proteins, are considered to play roles in signaling the chemotaxis and activity of tooth-surrounding cells (Ogata et al., 1997). Dentin phosphoprotein (DPP) and dentin sialoprotein (DSP) are the major non-collagenous dentin proteins (Butler, 1987; Butler et al., 1992). In addition to their role in dentinogenesis, they were shown to have the ability to stimulate neutrophil migration (Silva et al., 2004). From this, it is plausible that the release of proteins and other bioactive molecules, during processes associated with dentin matrix dissolution, could affect the course of dental diseases, such as root resorption. The leukocyte recruitment to these sites probably relies on the establishment of chemotactic and/or haptotactic gradients within this micro-environment. In investigating the mechanism by which DSP and DPP promote neutrophil recruitment, we demonstrated that they induce the production of IL-1ß, TNF-, MIP-2, and KC, which cause neutrophil recruitment in vivo (Silva et al., 2004). However, the cellular sources of these mediators were not identified.

Several studies have shown that resident cells may participate in leukocyte recruitment through the synthesis of inflammatory mediators. The importance of mast cells (MC) was previously demonstrated in the neutrophil migration induced by LTB₄ (Ribeiro et al., 1997), LPS, and zymosan (Ajuebor et al., 1999). Concerning macrophages, the chemotactic activity of zymosan, LPS, IL-1, and TNF has been shown to be dependent on these cells (Cunha and Ferreira, 1986; Ajuebor et al., 1999). Macrophages and MC have been found in inflamed pulp and periapical sites, being implied as sources of mediators and effectors of leukocyte extravasation (Kabashima et al., 2002). In this study, we investigated whether dentin-induced neutrophil chemotaxis is dependent on the resident macrophages and MC.

	MATERIALS & METHODS

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Animals
All experimental procedures involving the use of animals were reviewed and approved by the Institutional Animal Welfare Committee of the School of Medicine of Ribeirão Preto, University of São Paulo. All experiments were performed twice with 5 male BALB/c mice, from 6 to 8 wks old and weighing from 20 to 25 grams, per group.

Dentin Proteins
The purification of DSP and DPP from rat incisor dentin was performed by standard procedures as described (Butler, 1987; Butler et al., 1992).

Depletion of Resident MC
For MC depletion, mice received increasing concentrations of compound 48/80 (Sigma, St. Louis, MO, USA) every 12 hrs during 4 days (0.6 mg/kg, 1.0 mg/kg, 1.2 mg/kg, and 2.4 mg/kg, respectively). We confirmed MC depletion by examining peritoneal cells stained with toluidine blue dye. Twenty-four hrs later, test animals received DSP, DPP (1 µg/cavity), and N-formylmethionyl-leucyl-phenylalanine (fMLP, 100 nmol/cavity; Sigma) i.p., and control animals received PBS. Six hrs later, peritoneal cavities were washed with 3 mL PBS. The exudates were counted (Coulter^® A^CT Corporation, Miami, FL, USA), centrifuged onto slides (Cytospin, Shandon Lipshaw Inc., Pittsburgh, PA, USA), and stained by the May-Grünwald-Giemsa method for differential counting.

Increase in Peritoneal Macrophage Population
The increase in peritoneal macrophage numbers (Ribeiro et al., 1997) was obtained by i.p. injection of 3 mL of 3% (w/v) thioglycolate broth (Difco, Detroit, MI, USA) 3 days before the stimuli injection. Neutrophil migration was assessed 6 hrs after i.p. injection of DSP and DPP (1 µg/cavity), fMLP (100 nmol/cavity), or PBS.

Bone-marrow-derived MC
Primary MC cultures were derived from femoral bone marrow of BALB/c mice. Cells were incubated with Dulbecco's Modified Eagle's medium (Sigma) supplemented with 1 mM L-glutamine (Sigma), 10 mM HEPES (Sigma), 100 U/mL penicillin (Sigma), 100 µg/mL streptomycin (Sigma), 10 mL non-essential amino acid solution (Sigma), 110 mM pyruvate, 0.3 µg/mL fungizone (Gibco, Grand Island, NY, USA), 15% fetal bovine serum (Gibco), IL-3 (200 ng; Peprotech Inc., Rocky Hill, NJ, USA), IL-4 (50 ng; Peprotech), and increasing concentrations of stem cell factor (Peprotech; 200 ng, 300 ng, 400 ng, and 500 ng, respectively, from the first to fourth wks of culture). Afterward, cells were maintained for 24 hrs in the absence of cytokines. MC (5 x 10⁵ cells/well) were treated with DSP (10 µg/mL) for 1 hr, washed with PBS, and cultured for 1 hr in RPMI, and then the supernatants were filtered (0.22 µm). In some experiments, the MC supernatants were incubated with anti-IL-4 and anti-IL-10 antibodies (5 µg/mL) (PharMingen, San Diego, CA, USA).

Release of Neutrophil Chemotactic Factor by DSPand DPP-stimulated Macrophages
Neutrophil chemotactic factor was obtained as described (Cunha and Ferreira, 1986). Macrophages harvested from naïve peritoneal cavities were re-suspended in RPMI (Gibco), plated at 10⁶ cells/well, and allowed to adhere for 24 hrs. The adherent cells were stimulated with DSP or DPP at 0.3, 1, and 10 µg/mL for 1 hr at 37°C in an atmosphere of 5% CO₂. Next, cells were washed with PBS and cultured for 6 hrs in RPMI without the stimuli. Macrophage supernatants were then filtered (0.22 µm) and injected i.p. (1 mL/cavity), and neutrophil migration was assessed 6 hrs later. In some experiments, before stimulation, cells were incubated for 1 hr with dexamethasone (10 µM/well) (Sigma) or MC supernatants.

DSP-stimulated macrophage supernatant, alone and treated with MC, was also tested in vitro. Briefly, neutrophil chemotaxis with purified neutrophils from rat venous blood was assayed with the use of a 5-µm-pore-size polycarbonate membrane (Millipore, Bedford, MA, USA) in micro-Boyden chambers (Neuro Probe, Cabin John, MD, USA). The chamber was incubated for 1 hr, and number of cells in 6 high-power fields was counted.

ELISA
The concentrations of IL-10 and IL-4 in peritoneal exudates and MC supernatants, and IL-1ß, TNF-, and MIP-2 in macrophage supernatants were determined by ELISA according to the manufacturer's instructions. Mouse anti-IL-4 and anti-IL-10 antibodies were from PharMingen, and anti-IL-1ß, anti-TNF-, and anti-MIP-2 antibodies were from Peprotech. The concentration of each cytokine was calculated from a standard curve (from 4 to 4000 pg/mL).

Statistical Analysis
The data were analyzed by one-way ANOVA with a Bonferroni post-test. Statistical significance was considered to be achieved at P < 0.05.

	RESULTS

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Effect of Depletion of MC and Augmentation of Peritoneal Resident Macrophages
We observed a marked reduction in MC after pre-treatment with the MC activator compound 48/80 (Fig. 1A), whereas macrophages were increased five-fold by the previous injection of thioglycolate (Fig. 1B). MC depletion resulted in a significant increase in DSP- and DPP-induced neutrophil migration (Fig. 1A). Similarly, the augmentation of macrophage numbers led to a considerable increase in DSP- and DPP-induced neutrophil migration. The administration of thioglycolate or 48/80 compound did not affect the responsiveness of the peritoneal cavities to fMLP.

View larger version (18K): [in this window] [in a new window]   Figure 1. (A) Role of mast cells (MC) and (B) macrophages in DSP- and DPP-induced neutrophil migration. Resident MC were depleted, and the macrophage population was augmented by treatment of mice with 48/80 compound () (A) and thioglycolate () (B), respectively, as described in MATERIALS & METHODS. Control mice were pre-treated with PBS (). After pre-treatment, mice were injected i.p. with PBS (C), fMLP (100 nmol/cavity), DSP, and DPP (1 µg/mL), and neutrophil migration was assessed 6 hrs later. Results are mean ± SEM of 10 mice per group. The experiment was repeated twice. *P < 0.05 compared with respective control; #P < 0.05 compared with groups pre-treated with PBS.

View larger version (26K): [in this window] [in a new window]   Figure 2. (A) Effects of doses of DSP and DPP on the release of neutrophil chemotactic factor derived from macrophages stimulated by DSP (DSP-M) and DPP (DPP-M). Macrophages were incubated for 1 hr with DSP and DPP (0.3, 1, and 10 µg/mL). The cells were then washed and incubated for 6 hrs in RPMI medium, and the supernatant was injected i.p. for evaluation of neutrophil migration. (B) Inhibitory activity of dexamethasone (DEX) on the release of neutrophil chemotactic factor derived from macrophages stimulated by DSP (DSP-M) and DPP (DPP-M). Macrophages were pre-treated with dexamethasone (10 µM/well) before stimulation with DSP and DPP (10 µg/mL). (C) Inhibitory effect of mast cell (MC) supernatant on neutrophil migration induced by DSP-stimulated macrophages (DSP-M). MC cultures were treated with DSP (10 µg/mL) and PBS for 1 hr, and the supernatant was used to treat macrophages before DSP stimulation. (D) Effects of anti-IL-4 and anti-IL-10 antibodies on inhibitory activity of DSP-stimulated MC supernatant (DSP-MC supernatant). DSP-MC supernatants were incubated with PBS, anti-IL-4, and anti-IL-10 antibodies (5 µg/mL) and then were used to treat macrophages before DSP stimulation. For all experiments, neutrophil migration was evaluated after 6 hrs. Results are mean ± SEM of 10 mice per group. The experiment was repeated twice. *P < 0.05 compared with RPMI; #P < 0.05 compared with the group without DEX (B); #P < 0.05 compared with the groups treated with PBS (C,D).

View this table: [in this window] [in a new window]   Table. Effects of MC Supernatant and Dexamethasone on the Production of IL-1ß, TNF-, and MIP-2 by DSP-stimulated Macrophagesa

View larger version (18K): [in this window] [in a new window]   Figure 3. Presence of IL-4 and IL-10 in peritoneal exudates from mice injected with DSP (A) and DSP-stimulated mast cell supernatant (B). Peritoneal exudates of mice injected with DSP (1 µg/mL) () or PBS () were recovered after 6 hrs. MC (5 x 105 cells/well) were treated with DSP (10 µg/mL) and washed, and supernatant was recovered after 1 hr. Cytokine production was assessed by ELISA. Results are mean ± SEM of 10 mice per group. The experiment was repeated twice. *P < 0.05 compared with PBS.

	DISCUSSION

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We previously showed that DSP and DPP cause neutrophil recruitment in a model of acute inflammation via induction of IL-1ß, TNF-, KC, and MIP-2 release (Silva et al., 2004). Although the involvement of neutrophils in the root resorption process is not well-characterized, there are some studies suggesting that neutrophils could participate in this event, since they are present during the active phase of pathophysiologic conditions associated with tooth resorption (Sasaki et al., 1990). Moreover, these cells are an important source of inflammatory mediators in sites of periapical bone resorption (Takeichi et al., 1996) and periodontal disease (Pouliot et al., 2000). In this work, we have characterized the cells involved in dentin-induced neutrophil recruitment. We used the model of acute peritonitis, because it allows for the accurate quantification of cell recruitment (Cunha and Ferreira, 1986). A marked increase in resident macrophages, achieved with the use of a well-validated protocol (Ribeiro et al., 1997), intensified the dentin-induced neutrophil migration. On the other hand, fMLP-stimulated neutrophil migration, which occurs by a mechanism independent of resident cells (Ribeiro et al., 1997), was not affected. There is some evidence that macrophages are important sources of inflammatory mediators in periapical resorption sites and during physiologic root resorption (Sasaki et al., 1990), and may even have a key role in these processes. Consistently, dentin proteins caused the release of a neutrophil chemotactic factor from macrophages, which was inhibited by dexamethasone, a glucocorticoid. This result suggests the involvement of cytokines and eicosanoids in this process, since drugs in this class are well-known inhibitors of these mediators. Indeed, dexamethasone treatment attenuates the macrophage production of IL-1ß, TNF-, and MIP-2, described as chemotactic mediators of DSP- and DPP-induced neutrophil migration (Silva et al., 2004).

The in vivo assay used to demonstrate the neutrophil chemotactic activity of the DSP- and DPP-stimulated macrophage supernatants does not allow us to conclude that the substances present in the supernatants are acting directly or stimulating the resident peritoneal cells to release the secondary neutrophil chemotactic mediators. However, the ability of the DSP-stimulated macrophage to induce neutrophil migration in vitro reinforces that mediators present in the supernatant are able, at least in part, to stimulate the neutrophil chemotaxis directly.

In contrast to the stimulatory role of macrophages in dentin-induced neutrophil migration, MC were shown to function as negative regulators of this process. It is noteworthy that MC numbers were shown to be increased in inflamed pulp and periapical sites, being implicated in periapical resorption, possibly by interference in osteoclastic activity (Kabashima et al., 2002).

The MC-depleted mice showed a significant increase in neutrophil extravasation, and DSP-MC supernatant displayed an inhibitory effect on the release of macrophage-derived chemotactic substance, which was reversed by treatment with anti-IL-4 and anti-IL-10 antibodies. Furthermore, significant production of IL-4 and IL-10 was detected in exudates and in MC supernatants after DSP challenge. The ability of DPP to induce MC activation in vitro was not addressed in the present study and needs to be determined. The fact that anti-IL-4 and anti-10 antibodies reversed the inhibitory effect of the DSP-stimulated MC supernatants upon macrophage-derived neutrophil chemotactic mediators is consistent with in vivo effects of these cytokines. Indeed, intravenous administration of IL-10 inhibits IL-1ß-induced neutrophil migration (Perretti et al., 1995). Also, IL-4 overproduction has a protective role in a model of arthritis (Saidenberg-Kermanac'h et al., 2004). These effects may result in a repression of pro-inflammatory gene expression by these cytokines (Levings and Schrader, 1999). It is important to mention that, following DSP treatment, IL-4 and IL-10 may inhibit both the release of macrophage-derived neutrophil chemotactic mediators (see above) and the expression of leukocyte adhesion molecules (Thornhill and Haskard, 1990).

In conclusion, our results support the idea that there is a balance between cytokines released from macrophages and MC to regulate the neutrophil influx induced by dentin. The investigation of these activities may help in the further delineation of the possible involvement of dentin proteins in inflammatory events, coupled with their release at root resorption sites.

	ACKNOWLEDGMENTS

 
This work was supported by grants from FAPESP and CAPES. The authors are indebted to Giuliana Bertozi and Alessandra Cheraim for their helpful assistance.

Received February 2, 2004; Last revision October 5, 2004; Accepted October 20, 2004

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