Immunohistochemical localization of cyclooxygenase-2 in radicular cysts

AIM: The purpose of this study was to investigate the expression of cyclooxygenase-2 (COX-2) in radicular cysts. METHODOLOGY: Thirty biopsy specimens of radicular cysts were examined using immunohistochemistry. A peroxidase-labelled streptavidin-biotin technique was used for identification of the COX-2. Fisher's exact test (two-tail) was used for statistical analysis of the results. RESULTS: The result demonstrated that COX-2 expression was significantly higher in radicular cysts with higher levels of inflammatory infiltrates. COX-2 stain was detected in the lining epithelium, subepithelial fibroblasts, macrophages and endothelial cells in all specimens. CONCLUSIONS: COX-2 expression is significantly higher in radicular cysts. COX-2 may play an important role in the pathogenesis of radicular cysts.     

Immunolocalization of interstitial collagenase (MMP-1) and tissue inhibitor of metalloproteinases-1 (TIMP-1)in radicular cysts

To investigate the mechanisms involved in expansion of radicular cysts, monoclonal antibodies against interstitial collagenase (MMP-1) and tissue inhibitor of metalloproteinases-1 (TIMP-1) were used to localize the sites of MMP-1 and TIMP-1 expression in 30 radicular cysts. Positive MMP-1 staining was detected in the lining epithelium and subepithelial fibroblasts, macrophages, endothelial cells and osteoblasts/osteocytes in all specimens. Positive TIMP-1 staining was identified in osteoblasts/osteocytes and endothelial cells of all specimens, and in the lining epithelium and subepithelial fibrous connective tissue wall of five radicular cysts with an intense inflammatory cell infiltrate. The number and distribution of positive cells for MMP-1 or TIMP-1 varied widely among individual specimens, but strong immunostaining was constantly detected at sites with prominent subepithelial inflammation. Results here support the hypothesis that MMP-1 may play an important role in the expansion of radicular cysts. The absence of TIMP-1 expression in lining epithelium and subepithelial fibroblasts and macrophages in most cases studied indicated that an imbalance between MMP-1 and TIMP-1 production may lead to radicular cyst expansion.     

High IL-6 synthesis in cultured fibroblasts isolated from radicular cysts

OBJECTIVE: Inflammatory cytokines have been reported to be related with inflammation and expansion of jaw cysts. In this study, to examine the relationship between radicular cysts and inflammatory cytokines, it was found that there was notable unique evidence on cytokine synthesis from fibroblasts isolated from radicular cysts. METHODS: The expression of such cytokines, namely, interleukin-1beta, IL-1beta, IL-6, IL-8, IL-10, tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), transforming growth factor-beta1 (TGF-beta1), and granulocyte-macrophage colony-stimulating (GM-CSF) mRNA, in nine radicular cysts was examined and compared with that detected in six specimens of healthy gingival mucosa. Furthermore, separating all fibroblasts from their respective radicular cysts, healthy gingival mucosa, and healthy periodontal ligaments, these fibroblast groups were cultured without stimulators and a supernatant for each was obtained to analyse IL-1beta, IL-6, IL-8, TNF-alpha, and IFN-gamma by ELISA. RESULTS: Differences between radicular cysts and healthy gingival mucosa were not clearly shown by the expression of cytokine mRNA. Analysing inflammatory cytokine synthesis in fibroblast groups from these three kinds of tissues, surprisingly, the levels of IL-6 mRNA and protein were recognised to be higher in fibroblasts of radicular cysts than in those of control tissues by ELISA and a real-time RT-PCR. Significant differences in the cultured supernatants of these fibroblast groups were not recognised in the release of IL-1beta, IL-8, TNF-alpha, and IFN-gamma by ELISA. CONCLUSIONS: From these results, it was suggested that fibroblasts inducing IL-6 production might play important roles in the expansion of radicular cysts. It is considered that fibroblasts around radicular cysts may lead to high IL-6 synthesis over time in chronic inflammation.     

Prostaglandin synthesis in dental cysts

The synthesis of prostaglandins by dental squamous cell cysts was studied. Radiothinlayer-chromatography (RTLC) showed C14-labelled arachidonic acid to be converted into the prostaglandins PGE2, 6-oxo-PGF1 alpha, PGF2 alpha and PDG2. In addition, hydroxyfatty acid synthesis in excess of total prostaglandin production was observed. The substances mentioned are thought to be causally involved in the osteolytic activity of dental cysts.     

Immunocytochemical localization of inflammatory cytokines and vascular adhesion receptors in radicular cysts

Odontogenic cysts are one of the commonest bone destroying lesions of the maxillofacial skeleton, with the inflammatory radicular cyst being the commonest jaw cyst. Explains of radicular cysts produce an interleukin-1-like activity which could explain the osteolysis seen with these tumours though the cellular source of this osteolytic activity is unknown. In the present study, cytokines with known inflammatory and osteolytic activity: interleukin-1 (IL-1), tumour necrosis factor (TNF), inlerleukin-6 (IL-6). and the chemotactic cytokine interleukin-8 (IL-8) have been localized immunocytochemically in radicular cysts. The cellular adhesion receptors ICAM-1 and ELAM-1 have also been immunolocalized. All specimens showed positive staining for IL-1 (alpha and beta) and IL-6, with these cytokines being located in epithelial and vascular endothelial cells. Only two specimens demonstrated TNF and IL-8 staining, which was located in macrophages. All specimens demonstrated ELAM-1 staining in endothelium and ICAM-1 staining in epithelium, endothelium and mononuclear cells. These findings show that radicular cysts contain two bone-modulating cytokines. IL-1 and IL-6, and that these appear to be synthesized mainly by the epithelial cells. Cysts also contain a proportion of activated blood vessels whose endothelial cells express the cellular adhesion receptors ICAM-1 and ELAM-1.     

Evidence that PGI2-generation in human dental cysts is stimulated by leucotrienes C4 and D4

Various prostaglandins, particularly PGE2 and PGI2, appear to play a major role in osteolytic processes. The bone-destructive action of dental squamous cell cysts is thought to be caused or at least mediated by prostaglandins. Both leucotrienes and prostaglandins are synthesized from a common precursor substance, i.e. arachidonic acid, in a series of reactions. There is a special relation between leucotrienes and PGI2 in that PGI2 synthesis in endothelial cells can be enhanced by LTC4 and LTD4. R-TLC showed conversion of the arachidonic acid added into PGI2 in 8 out of 12 cases, as reflected by the stable degradation product 6-oxo-PGF1 alpha. In 8 cases bioassay showed PGI2 synthesis without addition of LTC4 and LTD4. Incremental LTC4 and LTD4 additions were found to cause a highly significant increase of PGI2 synthesis rates. In summary, LTC4 and LTD4 stimulate PGI2 synthesis in chronic inflammatory processes in vivo and may thus elicit or accelerate osteolysis.     

Interleukin-1alpha-dependent regulation of matrix metalloproteinase-9(MMP-9) secretion and activation in the epithelial cells of odontogenic jaw cysts

Interleukin-1alpha (IL-1alpha) and matrix metalloproteinase-9 (MMP-9) are thought to be involved in odontogenic cyst expansion. In this study, we investigated the effects of IL-1alpha on the secretion and activation of MMP-9 in odontogenic jaw cysts. An active form of MMP-9 was present in odontogenic keratocyst (6 of 8 cases) fluids more frequently than dentigerous cyst (3 of 10 cases) and radicular cyst (3 of 10 cases) fluids, although proMMP-9 was present in all cyst fluids. Odontogenic keratocyst fragments in explant culture secreted a larger amount of IL-1alpha than dentigerous cyst and radicular cyst fragments in explant culture, and spontaneously secreted both proMMP-9 and an active form of MMP-9. The fragments of dentigerous cysts and radicular cysts secreted a small amount of proMMP-9, but no active form of MMP-9. Exogenously added recombinant human IL-1alpha (rhlL-1alpha) increased the secretion and activation of proMMP-9 in the fragments of dentigerous cysts and radicular cysts. The epithelial cells isolated from odontogenic keratocysts secreted IL-1alpha and proMMP-9 without stimulation. Under the cultivation on a fibronectin-coated dish, rhIL-1alpha increased the secretion of proMMP-9 from the epithelial cells in a dose-dependent manner. Moreover, rhIL-1alpha induced the secretion of proMMP-3 and plasminogen activator urokinase (u-PA) from the epithelial cells, and converted the secreted proMMP-3 to the active form in the presence of plasminogen. The secreted proMMP-9 was also activated in the presence of rhIL-1alpha and plasminogen. Hence, our results suggest that IL-1alpha may up-regulate not only proMMP-9 secretion but also proMMP-9 activation by inducing proMMP-3 and u-PA production in the cyst epithelial cells by autocrine/paracrine regulatory mechanisms.     

Immunohistochemical localization of tissue-type plasminogen activator and type I plasminogen activator inhibitor in radicular cysts

BACKGROUND: The plasminogen/plasmin proteolytic system participates in a wide variety of extracellular matrix degradation. Detailed knowledge of plasminogen activators (PAs) and their inhibitors may be important for understanding the pathogenesis of radicular cysts. The purpose of this study was to investigate the in situ localization of tissue-type PA (t-PA) and type I PA inhibitor (PAI-1) in radicular cysts. METHODS: Thirty formalin-fixed, paraffin-embedded specimens of radicular cysts were examined using immunohistochemistry. In addition, another section from each radicular cyst specimen was stained with hematoxylin and eosin to assess the presence of inflammatory infiltrates. Differences in t-PA and PAI-1 expression between tissues with low and high levels of inflammation were subsequently analyzed using Fisher's exact test. RESULTS: Both t-PA- and PAI-1-positive cells were detected in the lining epithelium, connective tissue, inflammatory infiltrates, and endothelium. In addition, the t-PA signal was mainly expressed in epithelial cells. However, the PAI-1 signal was mainly expressed in fibroblasts. Moreover, significantly greater t-PA as well as PAI-1 expression was noted in radicular cysts with high levels of inflammation as compared to tissues with low levels of inflammatory cell infiltrates (P < 0.05). CONCLUSIONS: The present study confirms earlier indications of local production of PA and its inhibitor in radicular cysts. In addition, this study further shows the tissue localization of the antigens for t-PA as well as PAI-1, and demonstrates that the expression of both t-PA and PAI-1 increases with the grade of inflammation in radicular cysts.     

Microvessel density and vascular endothelial growth factor (VEGF) expression in human radicular cysts

PURPOSE: To assess vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) by immunohistochemistry and to relate them to the inflammatory status in a sample of radicular cysts. METHODS: Specimens of 24 human radicular cysts were examined by immunohistochemistry using antibodies anti-VEGF and anti-CD34 and to evaluate vascular density. Integrity of the epithelium and inflammatory state of the connective tissues were evaluated and related with the immunohistochemical findings. A Spearman correlation test was utilized to compare the means of each parameter. RESULTS: VEGF immunoreactivity was detected in both epithelial and connective tissues of radicular cysts. Stromal cells showed higher levels of VEGF expression when compared to epithelial cells. MVD proved to be related to VEGF expression levels (P < or = 0.01). In addition, increased MVD was associated with high levels of inflammation (P < or = 0.01). Most of the specimens showed a massive inflammatory infiltrate in the connective tissue. The integrity of the cystic lining tend to decrease with increased inflammation.     

Enhancement of human dental cyst PGI2 formation by platelet derived growth factor and its role in cyst growth and bone resorption

Various prostaglandins, particularly PGE2 and PGI2, appear to play a major role in osteolytic processes. Radiochromatographic studies have demonstrated that 6-oxo-PGF1-alpha is a major product of exogenously added arachidonic acid in human dental cysts. As platelets may also act as inflammatory cells, platelet-derived growth factor might also have a PGI2-stimulating influence in such cysts. Eleven human dental cysts were examined by a radioimmunoassay and bioassay which can show PGI2 synthesis in human dental cysts without addition of PDGF. Incremental PDGF addition caused a highly significant increase in the rate of PGI2 synthesis. PGDF thus stimulates PGI2 synthesis in chronic inflammatory processes in vitro and may thereby elicit or accelerate osteolysis.     

Evidence for fibroblasts as the major source of prostacyclin and prostaglandin synthesis in dental cyst in man

Periodontal cysts synthesize large amounts of prostaglandins and collagenase which probably cause the localized bone destruction essential for intraosseous cyst growth. Fragments of cyst wall, and fibroblasts cultured from them, synthesized prostacyclin (PGI2) in addition to prostaglandin E2 (PGE2), PGF2 alpha and collagenase in vitro. Soluble products from cultures of unstimulated and phytohaemagglutinin-stimulated blood mononuclear cells enhanced the synthesis of these prostaglandins in monolayer cultures of cyst-wall fibroblasts. It is therefore proposed that cyst capsule fibroblasts are the major source of these bone-resorbing factors, acting under the stimulus of lymphocytes and monocytes in chronically inflamed cysts. Cysts which were not infiltrated by chronic inflammatory cells (follicular cysts, a keratocyst, an ameloblastoma, and an aneurysmal bone cyst) also produced prostaglandins and collagenase, indicating that the stimulatory mechanism for the production of bone-resorbing factors in these cysts may differ from that in periodontal cysts.     

The concentration of TNF-alpha correlate with number of inflammatory cells and degree of vascularization in radicular cysts

Objective: To correlate values of tumor necrosis factor‐alpha (TNF‐α) depending on the count of inflammatory cells with degree of vascularization in cystic fluid of radicular cysts. Material and methods: We investigated TNF‐α concentration in 43 radicular cysts obtained from patients undergoing surgery, under local anaesthesia, and after aspiration of cystic fluid from non‐ruptured cysts by enzyme‐linked immunosorbent assay assay in respect of different clinical parameters as well as by histomorphometric analyses. Results: Significantly higher concentration of TNF‐α is associated with smaller radicular cysts, higher protein concentration in cystic fluid as well as with higher presence of inflammatory cells, and increased degree of vascularization in pericystic tissues and cyst wall thickness. Conclusions: We believe that determination of TNF‐α in cystic fluid simultaneously with other parameters can be an additional parameter for clinical diagnosis of inflammed cysts.     

6-oxo-PGF1 alpha -a new tumour marker for tumours in the maxillo-facial region

The diagnosis of secondary or recurrent tumour growth after operations on primary maxillo-facial tumours is still only possible at a late stage. On the other hand, the prostaglandins of the E series assume an important role in connection with tumour growth. The present investigation succeeded for the first time in detecting an increase in the level of 6-oxo-PGF1 alpha, the stable metabolite of PGI2, before planned radical operation in patients with maxillo-facial tumours. In the further course of the illness it was possible to establish a correlation between the plasma values and the occurrence of metastasis or recurrence of disease. These results indicate that a certain significance can be attributed to 6-oxo-PGF1 alpha as a tumour marker.     

Basement membrane-type heparan sulfate proteoglycan (perlecan) and low-density lipoprotein (LDL) are co-localized in granulation tissues: a possible pathogenesis of cholesterol granulomas in jaw cysts

BACKGROUND: As perlecan contains a low-density lipoprotein (LDL) receptor-like repeats in the second domain of its core protein, LDL may be bound to perlecan, which is rich in granulation tissues. We wanted to study if this is the case in the cyst wall of radicular cysts, which are often associated with cholesterol granuloma. METHODS: Thirty-three specimens of radicular cyst with cholesterol granulomas were immunohistochemically examined for comparative localizations of perlecan, apoprotein B (apo B), and oxidized LDL (Ox-LDL), and for mRNA expression levels for perlecan. RESULTS: Myxoid or edematous stroma of immature granulation tissues was strongly positive for perlecan and simultaneously for apo B and Ox-LDL. Macrophages including foamy cells scattered in the granulation tissues were also immunopositive for Ox-LDL and occasionally for apo B. In situ hybridization showed that fibroblasts, endothelial cells, and pericytes had strong signals for perlecan, which was also confirmed by RT-PCR. CONCLUSION: These results suggest that perlecan, which is abundantly produced and accumulated in the cyst wall of immature granulation tissue, traps Ox-LDL locally, and that Ox-LDL is phagocytosed by macrophages. Thus, LDL-laden foamy macrophages are aggregated in the granulation tissue, and free cholesterol from ruptured macrophages may be concentrated locally to be crystallized, which may induce foreign body granulomas in the cyst wall.     

Localization of mRNA for inflammatory cytokines in radicular cyst tissue by in situ hybridization, and induction of inflammatory cytokines by human gingival fibroblasts in response to radicular cyst contents

The expression of mRNA encoding the inflammatory cytokines interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor α (TNF-α) have been examined in radicular cysts by in situ hybridization. Furthermore, the biological activity of the contents of radicular cysts (RCC) has been assayed by adding extracts of RCC to cultured human gingival fibroblasts (HGFs) and analyzing the culture medium for the release of inflammatory cytokines. In the epithelial layer, keratinocytes expressed all cytokine mRNAs examined at various levels. Basal layer cells expressed mRNA for each cytokine. In the subepithelial granulation tissue of the cysts, fibroblasts and macrophages expressed mRNA for IL-6, IL-8, IL-1β and TNF-α mRNA at varying levels; especially clear expression of TNF-α and IL-1β mRNA was detected on macrophages. The infiltrating lymphoid cells, largely composed of T cells and plasma cells, expressed these cytokine mRNAs, especially those encoding IL-6 and IL-8, at various levels. In vitro analysis indicated dose-dependent release of both IL-6 and IL-8 by HGFs in response to RCC. After heating to 100°C for 10 min, RCC almost completely failed to stimulate IL-6 release from HGFs. Furthermore, anti-IL-1β antibody (neutralization test) did not prevent the stimulation of IL-6 release by RCC. Significant amounts of IL-6 and IL-8 were detected in RCC in two cases, and a trace amount of IL-1β was detected in one case. This study demonstrated the wide expression of mRNA encoding inflammatory cytokines in radicular cyst tissues, and RCC itself was capable of stimulating 1L-6 and 1L-8 production from HGFs.     

Immunohistochemical correlation between microvessel density and lymphoid infiltrate in radicular cysts

Oral Diseases (2012) 19 , 92–99 Objective: Radicular cysts occur as a result of the immunological response to continuous antigenic stimulation from root canals. We correlated the immunophenotypical composition of the lymphoid infiltrate to the microvessel density expressed by the count of CD34 reactive endothelial cells in radicular cysts. Subjects and methods: Thirty‐four cases of radicular cysts were evaluated by immunohistochemistry, using antibodies against B‐ and T‐cell antigens (CD20, CD3, CD4, CD8) and against the endothelial cell marker CD34. Statistical analysis was performed. Results: In the epithelium, we observed a low amount of lymphoid infiltrate in all 34 radicular cysts, and a strong significant negative correlation between T and B lymphocytes and between T‐helper and T‐cytotoxic/suppressor lymphocytes. In the cyst capsule, we observed a significant positive correlation between B and T lymphocytes, B and T‐cytotoxic/suppressor lymphocytes, T and T‐helper lymphocytes and between the number of CD34+ blood vessels and T and T‐helper lymphocytes, respectively. We observed a statistically significant correlation between percentage of CD34+ vessels and inflammatory infiltrate grade. Conclusions: Both humoral and cellular immune reactions and neovascularization are likely to occur in the complex events of tissue destruction. The inflammatory infiltrate has an important role in neoangiogenesis and consequently in radicular cysts development and growth.     

Detection of vascular endothelial growth factor/ vascular permeability factor in periapical lesions

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a multifunctional cytokine. It is overexpressed in several conditions, which are characterized by vascular hyperpermeability and angiogenesis. In this investigation, we have evaluated the possibility that VEGF/VPF could be expressed in periapical lesions. We studied 17 periapical granulomas and 6 periapical cysts by immunohistochemistry. An immunopositive reaction for VEGF/VPF was observed in all 23 periapical lesions; however, the intensity of immunostaining by anti-VEGF antibody varied according to histopathological findings. In periapical granulomas without epithelium, almost all of the inflammatory cells were immunoreactive to anti-VEGF/VIP antibody. In periapical granulomas, which had rests of Malassez in them, some inflammatory cells were stained. On the other hand, epithelial cells always were stained by VEGF/VPF antibody, both in periapical lesions with epithelium and in radicular cysts. This study demonstrated that periapical lesions express VEGF/VPF, although with some differences in cell immunolabeling, which correlated to the lesions' stages of development. Initially, VEGF/VPF would assure angiogenesis and vascular hyperpermeability, resulting in accumulation of inflammatory cells, later it could be involved in cyst fluid accumulation. We hypothesize, therefore, that VEGF/VPF expression plays an important role in the pathogenesis of periapical granulomas and enlargement of radicular cysts by several mechanisms.     

CD1a-positive cells in odontogenic cysts

Langerhans cells (LC) are bone marrow-derived cells that have a CD1a-positive immunophenotype and are an important portion of the cell-mediated immune response. The aim of this study was an immunohistochemical evaluation of CD1a positive cells in different types of oral cysts. Fifty-five cysts were studied: 18 odontogenic keratocysts (OKC), of which five were orthokeratotic and 13 parakeratotic; 19 radicular cysts; and 18 dentigerous cysts. Positive LC was 80% for orthokeratotic OKC, 33% for parakeratotic OKC, approximately 35% for radicular cysts, and approximately 20% for dentigerous cysts. The results show that OKC with well-differentiated epithelial linings presented a greater number of LC than the other cysts. However, when the cyst wall was inflamed there were no differences in LC expression in the different types of cysts. The data confirm that LC distribution seems to be associated with the degree of differentiation of the epithelia.     

Expression of transforming growth factor-beta 1 (TGF-beta 1) in odontogenic cysts

AIM: To evaluate the positivity to transforming growth factor-beta 1 (TGF-beta 1) in different types of odontogenic cysts. METHODOLOGY: A total of 30 radicular cysts (RCs), 27 follicular cysts (FCs) and 28 odontogenic keratocysts (OKCs) were evaluated for immunohistochemical analysis of TGF-beta 1. TGF-beta 1 was evaluated in blood vessels, stromal cells (fibroblasts) and pluristratified squamous epithelium. TGF-beta 1 expression was determined by evaluating the number of positive elements. TGF-beta 1 expression was determined by evaluating 1000 cells in the pluristratified squamous epithelium (500 in the basal and parabasal layers, and 500 in the superficial layer) and 500 cells (the fibroblasts in the stroma) for each specimen, and counting the number of positive cells. The number of positive vessels was evaluated in 10 high power fields (HPF). The Chi-square test was used to evaluate differences between the two groups (RC + FC and OKC). A P-value <0.05 was considered to indicate statistical significance. RESULTS: A higher and statistically significant positivity was found in the basal-suprabasal epithelial layers (P=0.0011), superficial epithelium (P=0.053) and stromal cells (P=0.0002) of orthokeratotic and parakeratotic OKC as compared with RC and FC. CONCLUSIONS: These differences suggest that control of the cell cycle may be abnormal in orthokeratotic OKCs. These OKCs may have an intrinsic growth potential not present in other cyst types.