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RESEARCH REPORT |
1 Division of Immunology, Institute of Biomedical Sciences, Federal University of Uberlândia, Uberlândia (MG), Brazil;
2 Department of Clinic and Operative Dentistry and Endodontics, School of Dentistry, Federal University of Uberlândia, Uberlândia (MG), Brazil;
3 Division of Rheumatology, Health Sciences Center, University of Virginia, Charlottesville, VA, USA; and
4 Department of Oral and Maxillofacial Pathology, School of Dentistry, Federal University of Uberlândia, Av. Pará, 1720, CEP 38405-902, Uberlândia (MG), Brazil;
*corresponding author, passaro{at}ufu.br
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: odontogenic cyst • radicular cyst • periapical lesion • cytokines • bone resorption
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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), and tumor necrosis factor alpha (TNF-
) (Gemmell and Seymour, 1998; Honma et al., 1998; Barkhordar et al., 1999; Kawashima and Stashenko, 1999). IL-6 has been found in the epithelium and/or fluid from radicular cysts by immunohistochemistry and/or immunoenzymatic assays (Bando et al., 1993; Formigli et al., 1995; Takeichi et al., 1996). The colony-stimulating factors (CSF) are included in a wide class of hematopoietic cell differentiation and growth factors. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is synthesized by activated T-lymphocytes, activated mononuclear phagocytes, vascular endothelium cells, and fibroblasts. Studies using murine osteoblasts showed release of GM-CSF when those cells were stimulated with bacterial lipopolysaccharides (Horowitz et al., 1989). Growth-factor-dependent proliferation of human leukemia cell lines was found when IL-6 and GM-CSF were associated. The latter cytokine would act together with IL-6 in the bone resorption process of inflammatory sites (Caracciolo et al., 1989). IL-3 derived from CD4+ T-lymphocytes was studied by Barton and Mayer (1989) in mice bone marrow cell culture. These authors showed that IL-3 induced differentiation of osteoclast-like precursor cells. This cytokine has been involved in bone resorption that occurs in inflammatory diseases such as rheumatoid arthritis.
IL-3 and GM-CSF are factors that still have not been detected in the tissue and fluid of human cystic lesions. However, recently, Fujikawa et al. (2001) have shown the effects of GM-CSF and IL-3 in human monocyte culture with osteoblasts. Thus, M-CSF, an important factor in osteoclastogenesis, was detected in both the IL-3- and GM-CSF-stimulated monocyte cultures and was not detected in the supernatants from monocyte cultures to which IL-3 and GM-CSF were not added. This fact suggests the importance of GM-CSF and IL-3 for stimulating the release of M-CSF, with consequent cellular differentiation to osteoclasts and the occurrence of bone resorption.
The aim of this study was to identify and measure the levels of GM-CSF, IL-3, and IL-6 inflammatory cytokines in radicular cysts, which have been associated with the bone resorption process, as a contribution to the knowledge of the growth process of such lesions.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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A group of 26 impacted third molars recommended for extraction was included so that we could obtain a pool of pulpal tissues in which inflammation would be an unlikely event. We used these pulpal tissue specimens as negative controls to determine the specificity of the assays in cyst tissues as described by others (Wang and Stashenko, 1993; Barkhordar et al., 1999).
Specimen Collection and Tissue Preparation
Cyst fluid was collected from the lesions by aspirative puncture, immediately centrifuged at 2000 g for 10 min at 4°C, and then stored at -70°C. Cystic tissues were stored at -70°C until analyzed by immunoenzymatic assay (ELISA).
For extract preparation, pulpal and cystic tissues were weighed and finely minced with a scalpel, and the fragments were incubated 100 mg of tissue in 1 mL of digestion mixture consisting of 10 mM HEPES buffer in RPMI 1640 medium, 40 µg/mL gentamycin, 100 µg/mL bovine serum albumin (BSA, Sigma Chemical Co., St. Louis, MO, USA), protease inhibitors (50 µg/mL leupeptin + 1.6 mM phenylmethylsulfonyl fluoride [PMSF] + 10 µg/mL aprotinin; Sigma), and 100 µg/mL Zwittergent-12 (Calbiochem, San Diego, CA, USA). After 90 min at 37°C under continuous agitation, the digestate was immediately cooled in an ice bath and further subjected to a sonication cycle for 30 sec, and subsequently centrifuged at 2000 g for 10 min at 4°C. Supernatants were collected and stored in an ice bath until used in ELISA.
Cytokine Assays in Cyst Fluid and Tissue
ELISAs for GM-CSF, IL-3, and IL-6 were carried out according to the manufacturer's instructions (R&D Systems Inc., Minneapolis, MN, USA), with use of the respective capture antibodies: polyclonal antibody anti-GM-CSF (AF-215-NA) at 2 µg/mL, monoclonal antibody (mAb) anti-IL-3 (MAB203) at 8 µg/mL, and mAb anti-IL-6 (MAB206) at 8 µg/mL. Absorbance results were expressed in pg/mL as determined from a standard curve constructed with each assay by use of the respective human recombinant cytokines. The cytokine content in cystic tissue samples represents the amount of cytokine present in 100 mg of tissue. The lower level of cytokine detection ranged from 62.5 to 125 pg/mL for the GM-CSF assay, from 250 to 500 pg/mL for IL-3, and from 9.4 to 18.8 pg/mL for IL-6.
Statistical Analysis
We calculated coefficients of intra- and inter-assay variations to determine the reproducibility of each assay, considering values less than 20% as satisfactory results according to the manufacturer. Statistical analysis consisted of determinations of geometric means (gm) with 95% confidence intervals (CI). Comparisons of positivity rates for the different cytokines in the fluid and tissue samples were performed with use of the differences between two proportions by Z statistic. Levels of cytokines present in cystic fluid and tissue were analyzed by the Spearman correlation test. Values of p < 0.05 were regarded as statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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, the geometric means of the cytokine levels were 200.5 pg/mL (95% CI, 155.9 to 257.6 pg/mL) for GM-CSF, 369.2 pg/mL (95% CI, 255.8 to 533.3 pg/mL) for IL-3, and 92.4 pg/mL (95% CI, 48.1 to 177.4 pg/mL) for IL-6, with no statistically significant differences among them (p > 0.05).
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). The coefficients of intra- and inter-assay variation were within the limit value considered acceptable by the manufacturer (< 20%), thus demonstrating good reproducibility of the assays.
Fig. 2 shows the positivity rates of radicular cysts (fluid and tissue) for the IL-3, GM-CSF, and IL-6 cytokines. Among the 14 fluid samples analyzed, 13 (92.8%) were positive for IL-6, rates significantly higher than those obtained for GM-CSF (42.8%) and IL-3 (0.0%) (p < 0.01). In contrast, tissue samples showed no significant difference between the positive rates of IL-6 (86.4%) and GM-CSF (95.8%) (p > 0.05). On the other hand, positivity to IL-3 (28.6%) in these samples was significantly lower than that found for the two previously mentioned cytokines (p < 0.001).
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The Spearman test revealed no correlation between the levels of GM-CSF vs. IL-6 (r = 0.1345; p = 0.5507) and IL-3 vs. IL-6 (r = -0.0979; p = 0.6813) in tissue samples. However, a low but significant positive correlation was found between the levels of GM-CSF and IL-3 (r = 0.4937; p = 0.0229) in these samples (data not shown).
When analyzing the frequency of the results obtained in ELISAs for the detection of the respective cytokines in fluid and tissue samples (Table 1), we verified that IL-6 was found in both fluid and tissue in 84.6% of the samples, values significantly higher than the 15.4% obtained in either fluid or tissue (p < 0.005). On the other hand, IL-3 was found only in cystic tissue (21.4%), although in the vast majority of the samples (78.6%), detectable levels of IL-3 were not found in both fluid and tissue (p < 0.01). In contrast, GM-CSF was found in both fluid and tissue in 42.9% of the samples and in only the cystic tissue in half of them.
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. A high-positive association was found for GM-CSF and IL-6, with detectable levels of both cytokines in 86.4% of the tissue samples (p < 0.001). On the other hand, there was no association between IL-3 and IL-6, or GM-CSF and IL-3, with 65.0% and 66.7% of the samples showing significant non-association results for the respective paired cytokines (p < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Several bone-resorbing factors have been characterized, including prostaglandins (PGE2, PGI2), leukotrienes, and collagenases that were already isolated from radicular cysts (Harvey et al., 1984; Meghji et al., 1989; Hoenig et al., 1991). However, the bone resorption process has been only partially inhibited by the action of cyclooxygenase and lipoxygenase inhibitors (Hoenig et al., 1991), indicating the participation of other mediators in this regulation. IL-1 is the most active cytokine that would act on cyst expansion through its effects on a wide spectrum of cell types, such as fibroblast proliferation, prostaglandin production by the cystic capsule, and osteolysis (Meghji et al., 1989).
The mediators involved in the inflammatory process and bone resorption appear to be more complex. Thus, human and animal studies have demonstrated the active participation of other cytokines, such as IL-6, IL-3, GM-CSF, IL-11, IL-17, and IL-18, which have shown their potential role in the pathogenesis of osteolytic diseases (Barton and Mayer, 1989; Paul, 1998). These cytokines might be acting synergistically with IL-1, promoting activation/differentiation of osteoclasts and production/secretion of prostaglandins by many cell types, including fibroblasts and osteoblasts (Caracciolo et al., 1989; Torabinejad, 1994).
Our results demonstrating the presence of IL-6 in the vast majority of the fluid and tissue samples are in agreement with those of previous studies (Bando et al., 1993; Formigli et al., 1995; Meghji et al., 1996; Euler et al., 1998; Honma et al., 1998). It has been reported that cystic growth may be due to the autocrine stimulation of cyst epithelial cell proliferation by IL-1 and IL-6, and the osteolytic activity of these cytokines, causing local bone loss (Bando et al., 1993). The concentrations of IL-6 in the fluid samples, in the present study, were greater than the range of 48.08 to 92.46 pg/mL obtained by Formigli et al. (1995).
IL-3 was detected in the cystic tissue in about one-third of the samples. Its presence may be related to osteoclast precursor proliferation, similar to that which occurs in inflammatory diseases such as rheumatoid arthritis, since IL-3 produced by activated T-lymphocytes has increased the osteoclast number in bone marrow cell culture (Barton and Mayer, 1989). In addition, IL-3 associated with GM-CSF were capable of stimulating human osteoclast formation via M-CSF production (Fujikawa et al., 2001), and, interestingly, our results have shown a significant positive correlation between the levels of IL-3 and GM-CSF in tissue samples. Hence, IL-3 and GM-CSF might play an important role in the bone resorption seen in this kind of disease.
In the present study, high levels of GM-CSF were detected in cystic fluid and tissue samples. It has been shown that the role of GM-CSF and IL-3 in the bone resorption process is caused by synergistic interactions between these mediators (Einat et al., 1996). In particular, the effects of these two cytokines in mice with osteopetrosis have been analyzed by the injection of daily recombinant murine GM-CSF (5 ng) and/or IL-3 (100 ng) (Myint et al., 1999). An increase in the number of TRAP (tartrate-resistant acid phosphatase)-positive cells was verified, suggesting that GM-CSF and IL-3 might induce osteoclast development. Similarly, it is believed that there is a synergism between GM-CSF and IL-6 in this process (Caracciolo et al., 1989). However, such interactions appear to be incompletely understood. Recent evidence associates IL-18 with osteoclast-forming inhibition as mediated by GM-CSF (Horwood et al., 1998).
As demonstrated in this study, a high-positive association between GM-CSF and IL-6 in tissue samples was observed, and some samples positive for IL-3 were also positive for GM-CSF and IL-6. In fact, IL-6 and PGE2 were detected at high levels in radicular cyst fluids, and these cytokines are known to be potent stimulators of bone resorption (Formigli et al., 1995; Meghji et al., 1996). Therefore, their presence in the cystic fluids may also be explained by the local inflammatory and immunological processes taking place in the fibrous capsule of the cysts. In the growth of these lesions, variations may be occurring in the modulation of bone resorption, likely associated with changes in the interaction between injury and the host defense system. Our results demonstrated the presence of GM-CSF and IL-6 in the fluid and tissue from human radicular cysts, while IL-3 was found only in the tissue. These cytokines may contribute to the inflammation and bone resorption that characterize cystic growth.
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ACKNOWLEDGMENTS |
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Received February 13, 2001; Last revision November 5, 2001; Accepted November 14, 2001
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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