Basic Research—Biology
CCR2 Deficiency Results in Increased Osteolysis in Experimental Periapical Lesions in Mice Thiago Pompermaier Garlet, DDS, MS,* Sandra Yasuyo Fukada, DDS, PhD,* Isabella Francisco Saconato, PharmD,* Mario Julio Avila-Campos, PhD,† Tarcı´lia Aparecida da Silva, DDS, PhD,‡ Gustavo Pompermaier Garlet, DDS, PhD,§ and Fernando de Queiroz Cunha, PhD* Abstract Introduction: Periapical lesions are chronic inflammatory disorders of periradicular tissues caused by etiologic agents of endodontic origin. The inflammatory chemokines are thought to be involved in the latter observed osteolysis. With a murine model of experimental periapical lesion, the objective of this study was to evaluate the role of the chemokine receptor CCR2 in the lesion progression, osteoclast differentiation and activation, and expression of inflammatory osteolysis-related mediators. Methods: For lesion induction, right mandibular first molars were opened surgically with a 1⁄4 carbine bur, and 4 bacterial strains were inoculated in the exposed dental pulp; left mandibular first molars were used as controls. Animals were killed at 3, 7, 14, and 21 days after surgeries to evaluate the kinetics of lesion development. Results: CCR2 KO mice showed wider lesions than WT mice. CCR2 KO mice also expressed higher levels of the osteoclastogenic and osteolytic factors, receptor activator of nuclear factor kappa B ligand (RANKL) and cathepsin K, of the proinflammatory cytokine tumor necrosis factor–alpha, and of the neutrophil migration related chemokine, KC. Conclusions: These results suggest that CCR2 is important in host protection to periapical osteolysis. (J Endod 2010;36:244–250)
Key Words Bone resorption, CCR2, chemokine receptors, OPG, periapical lesions, RANKL
From the *Department of Pharmacology, School of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo (FMRP/USP), Ribeira˜o Preto, SP, Brazil; †Department of Microbiology, Institute of Biomedical Sciences (ICB/USP), Sa˜o Paulo, SP, Brazil; ‡ Department of Oral Pathology and Surgery, Faculty of Dentistry, Federal University of Minas Gerais (UFMG), Belo Horizonte, MG, Brazil; and §Department of Histology, School of Dentistry of Bauru, University of Sa˜o Paulo (FOB/USP), Bauru, SP, Brazil. Address requests for reprints to Fernando de Queiroz Cunha, School of Medicine of Ribeira˜o Preto (FMRP/USP), Department of Pharmacology, Av. Bandeirantes, 3900, Ribeira˜o Preto, SP, 14049-900 Brazil. E-mail address:
[email protected]. 0099-2399/$0 - see front matter Copyright ª 2010 American Association of Endodontists. doi:10.1016/j.joen.2009.09.004
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eriapical lesions are prevalent skeletal diseases in the jaws, characterized by loss of mineralized tissue surrounding dental root apex. Tissue destruction and bone resorption are consequences of a local inflammatory response, triggered by microorganism infection of the dental pulp. Different leukocyte subsets, as well as many cytokines and chemokines, have been identified in periapical lesions (1, 2). Among the leukocytes, neutrophils and macrophages are involved in either the control of microorganism infection or tissue lesion development. Neutrophils are recruited to inflammatory site at the acute phase of inflammatory process, including microorganism infections, arthritis, and periodontal and periapical diseases (3–5). The end of the acute phase is characterized by the recruitment of macrophages, which also contributes to elimination of the inflammatory stimuli or to their restraining through granuloma formation (6). This event is crucial to the reestablishment of the host homeostasis. In fact, failure of macrophage recruitment during microorganisminduced inflammatory diseases leads to reduction of infection control, and, as consequence, an increment of neutrophil recruitment is observed that mediates an enhancement in the host tissue lesions (7–9). Therefore, defects of macrophage chemoattraction can result in aggravation of inflammation (10, 11). Along with different leukocyte subtypes, multiple cytokines and chemokines, which promote selective cell migration through the binding to their specific cell surface receptors, have been identified in periapical lesions. The CXC (family of chemokines that contains one amino acid between the cysteine residues) chemokines, including CXCL8 (interleukin 8) or its murine counterpart CXCL1 (keratocyte-derived chemokine) have been found in periapical lesions (12). They are chemokines related to selective neutrophil migration, mediating their effect through CXCR1 and CXCR2 receptors (1, 13). Although the macrophages might be stimulated by the CXC chemokines (14), they express mainly CC chemokine receptors such as CCR1, CCR2, and CCR5, which make them more responsive to CC chemokines such as CCL1 (macrophage inflammatory protein [MIP]–1a), CCL2 (macrophage chemotactic protein [MCP]1), and CCL5 (RANTES [regulated on activation, normal T-cell expressed and secreted]) (15). Genetic deficient mice to MCP-1 or MCP-3, mainly ligands to CCR2 in homeostatic conditions, exhibited reduced macrophage migration after Listeria monocytogenes administration, and as consequence, the animals are more susceptible to the infection (16). Similarly, the inhibition of CCR2 markedly enhances the susceptibility of mice to bacterial infection (17). Recently, in vitro studies have suggested that CCR2 is also involved in osteoclast biology (18). MCP-1 is able to induce osteoclast differentiation and promote their survival (18, 19). Moreover, it has also been described to be induced by receptor activator of nuclear factor kappa B ligand (RANKL), promotes osteoclast fusion, and favors the balance of osteoclast differentiation against other stimuli (20). CCR2 was also found to be up-regulated in human periapical lesions, and MCP-1 was more prevalent in cysts than in granulomas, suggesting that CCR2 activation could play a different role in the progression of these diseases (6). However, the activation of osteoclasts depends on RANK-RANKL engagement, an event inhibited by the decoy receptor osteoprotegerin (OPG) (5, 18, 19). In this context, recent studies suggested that RANKL/OPG expression ratio might determine lesion progression in human periapical disease (21).
JOE — Volume 36, Number 2, February 2010
Basic Research—Biology In the view of the potential role of the chemokine receptor CCR2 in the recruitment of macrophages, which plays an important role in the control of infection and as consequence of the inflammation magnitude, we hypothesized that CCR2 signaling might play an important regulatory role in periapical tissue destruction. Therefore, we analyzed whether the genetic disrupt of CCR2 receptor could lead with the aggravation of the experimental periapical lesions.
Material and Methods Animals All animal procedures were performed according to international and institutional guidelines, and this methodology was approved by FMRP local ethics committee. In this study, C57BL/6 wild-type (WT) and CCR2 knockout (CCR2-KO) mice, obtained from Jackson Laboratory (Bar Harbor, ME), were used. CCR2-KO animals are generally healthy and do not express any significant phenotype. All experiments were performed with 8-week-old mice, weighing around 22 g, with at least 5 animals in each experimental group. Mice were bred and maintained in FMRP animal house facilities. Periapical Lesion Induction Periapical lesion induction was performed as previously described (22, 23). Animals were anesthetized by intraperitoneal injection of tribromoethanol (250 mg/kg of body weight) and mounted on a jaw retraction board. The right mandibular first molar was surgically opened with a 1⁄4 carbide bur in a slow rotation handpiece, allowing exposure of dental pulp. Four endodontic pathogenic bacterial strains were inoculated: Porphyromonas gingivalis (ATCC 33277), Prevotella nigrescens (ATCC 33563), Actinomyces viscosus (ATCC 91014), and Fusobacterium nucleatum subsp. polymorphum (ATCC 10953). Bacteria were quantified with the McFarland scale, and same quantities of each strain were combined. A total of 1.106 bacteria were inoculated in 10 mL of medium. The cavity was not sealed after bacterial inoculation. During lesion development, animals were fed ad libitum. Animals were killed by cervical displacement after 3, 7, 14, and 21 days of infection, the jaws were dissected, and the samples were prepared to histologic or molecular analysis. Histopathologic Analysis The mandible samples were fixed in 10% phosphate-buffered formalin and demineralized in 4% ethylenediaminetetraacetic acid (EDTA), pH 7.2, at room temperature for 5 weeks. Once decalcified, the specimens were washed in running water for 24 hours, dehydrated in ascending concentrations of ethanol, cleared in xylol, and embedded in paraffin. Longitudinal 5-mm-thick sections were cut in mesiodistal orientation at the tooth apex level. Slides were stained with hematoxylin-eosin or submitted to a tartrate-resistant acid phosphatase (TRAP) detection assay for osteoclast identification, and examined under a light microscope (Leica DMR; Leica Microsystem Wetzlar Gmbh, Wetzlar, Germany). The sections of the jaw sample containing the distal root of the mandibular first molar and simultaneously showing the coronal and apical pulp through the apical foramen and the connecting periapical tissue were selected for quantitative measurements. For each data point, specimens were obtained from 5 mice. Morphometric analysis of periapical lesion size was performed in tissue sections stained with hematoxylin-eosin, with light microscopes. Only the sections in which the whole root canal including apical foramen could be seen were accepted for measurement to ensure that the section would represent the largest periapical lesion area. Measurements were performed with ImageJ 1.34 software (National Institutes of Health). JOE — Volume 36, Number 2, February 2010
TRAP Detection Assay Osteoclastogenesis was measured as the number of osteoclasts (TRAP-positive cells) per millimeter length of resorbed bone. Following manufacturer’s instructions, deparaffinized sections were incubated in a solution prepared by dissolving 8 mg of naphtol AS-BI (Sigma Chemical Co, St Louis, MO) in 500 mL of N-N-dimethylformamide followed by the addition of 50 mL of 0.2 mol/L sodium acetate buffer (pH 5.0) and 70 mg of Fast Red Salt TR (Sigma Chemical Co). Sodium tartrate dihydrate (50 mmol/L) was added to the solution. After incubation at 37 C, the sections were washed in distilled water and stained with hematoxylin. TRAP-positive cells appeared red. The quantitative analysis of the number of TRAP-positive osteoclasts was determined by counting multinucleated TRAP-positive cells in direct contact with bone on the light microscope and expressed as the number per millimeter of bone length. The surface of the alveolar bone was measured with the image analysis system. Real-time Polymerase Chain Reaction After death, jaws were removed and further manipulated over a cold Petri dish with diethyl pyrocarbonate–treated instruments to minimize RNA degradation. Soft tissues were removed, and a section of bone around the first molar distal root was collected. This sample was frozen in nitrogen and triturated. RNA was extracted from the triturated samples following the Trizol method. Real-time polymerase chain reaction (PCR) reactions were performed as previously described (24). Initially, complementary DNA (cDNA) was synthesized by using 3 mg of RNA through a reverse transcription reaction (Invitrogen Life Technologies, Carlsbad, CA). Realtime PCR quantitative mRNA analyses were performed in a MiniOpticon System (BioRad, Hercules, CA) by using the SYBR-green fluorescence quantification system for quantitation of amplicons. The standard PCR conditions were 95 C (10 minutes) and then 40 cycles of 94 C (1 minute), 56 C (1 minute), and 72 C (2 minutes), followed by the standard denaturation curve. The sequences of human primers were designed by using the PrimerExpress software (Applied Biosystems, Warrington, UK) with nucleotide sequences present in the GenBank database. The primer sequences, the predicted amplicon sizes, the annealing and melting temperatures are depicted in Table 1. PCR conditions for each target were conscientiously optimized with regard to primer concentration, absence of primer dimer formation, and efficiency of amplification of target genes and housekeeping gene control. SYBR Green PCR Master Mix (Invitrogen Life Technologies), 400 nmol/ L specific primers, and 2.5 ng of cDNA were used in each reaction. The samples were considered positive to the target gene expression when their fluorescence signals were higher than the threshold determined by negative controls’ fluorescence. Calculations for determining the relative level of gene expression were performed with the cycle threshold (Ct) method, in which the mean Ct values from triplicate measurements were used to calculate expression of the target gene, with normalization to the housekeeping gene b-actin in the samples, by using the 2–DCt formula. Negative controls without RNA and without reverse transcriptase were also performed. Statistical Analysis The lesion area and osteoclast number values regarding the kinetics of lesion development (eg, WT animals at days 0, 3, 7, 14, and 21) were submitted to the one-way analysis of variance statistical test, followed by Tukey post test. Differences in the relative intensity of mRNA expression between CCR2-KO and WT mice groups were analyzed with one-way analysis of variance, followed by Tukey post test. Values of P
