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Levels of GM-CSF, IL-3, and IL-6 in Fluid and Tissue from Human Radicular Cysts
1 Division of Immunology, Institute of Biomedical Sciences, Federal University of Uberlândia, Uberlândia (MG), Brazil; Correspondence: *corresponding author, passaro{at}ufu.br
Cytokines released by immune system cells play an important role in cyst enlargement. This study aimed to determine, by ELISA, the levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), and IL-6 in fluid and tissue from human radicular cysts. GM-CSF was found in 42.8% of the fluid samples (164.3 pg/mL) and IL-6 in 92.8% (641.4 pg/mL). No IL-3 was detected in any fluid samples. In the tissue samples, 28.6% were positive for IL-3 (369.2 pg/mL), 86.4% for IL-6 (92.4 pg/mL), and 95.8% for GM-CSF (200.5 pg/mL). It can be concluded that GM-CSF and IL-6 were widely found in the fluid and tissue samples. In contrast, IL-3 was found only in the cystic tissue, even though in few lesions. These cytokines may contribute to the inflammation, cystic growth, and bone resorption that characterize cystic lesions.
Key Words: odontogenic cyst • radicular cyst • periapical lesion • cytokines • bone resorption
Bone resorption is one of the damaging consequences of radicular cyst growth. Different cytokines have been identified in periapical lesions where bone resorption characterizes disease progression, such as interleukin 1 (IL-1), IL-4, IL-6, IL-8, IL-10, IL-12, interferon-gamma (IFN-  ), 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.
Patients and Tissue Samples Twenty-four radicular cysts were used in this study. Fluid and fresh tissue samples were obtained from 14 lesions, and only fresh tissue was collected from the other 10 lesions. From each specimen, part of the sample was subjected to routine histological processing. The diagnosis of radicular cyst was based on clinical, radiographic, and the WHO histological criteria (Kramer et al., 1991). All patients were asymptomatic, had a good performance status, and had not taken antibiotics during the previous 6 months. Surgical procedures were performed at the Dental Hospital of the Federal University of Uberlândia, after fully informed consent had been obtained from each patient, according to the Institutional Ethics Committee protocol. 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 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
Statistical Analysis
From a total of 24 radicular cysts, 21 tissue samples were assayed for IL-3, 22 for IL-6, and all of them for GM-CSF. Such variation occurred due to variations in the lesions' size, with some lesions presenting an insufficient amount to be tested in all cytokine assays. As demonstrated in Fig. 1  , 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).
In the 14 cystic fluid samples, the cytokine levels in gm were 641.4 pg/mL (95% CI, 320.6 to 1282.3 pg/mL) for IL-6 and 164.3 pg/mL (95% CI, 50.5 to 534.6 pg/mL) for GM-CSF, also with no statistically significant differences (p > 0.05). IL-3 was not detected in any tested sample, according to the detection limit for each assay (Fig. 1  ). 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
When analyzing the positivity for the same cytokine in the fluid and tissue samples, we found GM-CSF and IL-3 in significantly more tissue than fluid samples (p < 0.001, p < 0.05). For IL-6, the positivity was comparable in both samples (p > 0.05). 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
An association between positive and negative results for the three cytokines assayed in the tissue samples is demonstrated in Table 2 . 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).
The enlargement of radicular cystic lesions has been primarily attributed to an increase of intra-luminal hydrostatic pressure that might exert a tension on the adjacent bone walls. Nevertheless, periradicular inflammation has also been implicated in this process through the development of immunological events (Wang and Stashenko, 1993; Lerner, 1994; Torabinejad, 1994; Formigli et al., 1995; Zecchi-Orlandini et al., 1996). 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.
This study was supported by the National Research Council (CNPq), grant #523914/96-2. We are grateful to Dr. José Roberto Mineo and Dr. Mônica Sopelete for technical advices. Received for publication February 13, 2001. Revision received November 5, 2001. Accepted for publication November 14, 2001.
Journal of Dental Research, Vol. 81, No. 1,
64-68 (2002) Related articles in Journal of Dental Research:
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