Modulation of phagocytosis, chemotaxis, and adhesion of neutrophils by areca nut extracts

BACKGROUND: A higher prevalence of periodontal diseases among areca chewers than non-areca chewers has been demonstrated. Neutrophils, representing the first line of the host defense mechanism against microbial infection, play important roles in maintaining periodontal health. This study determined the possible effects of areca nut on phagocytosis, chemotaxis, and adhesion of human neutrophils. METHODS: Aqueous extracts of ripe areca nut without husk (rANE) and fresh and tender areca nut with husk (tANE) were examined for their effects on neutrophil phagocytosis using flow cytometry and confocal laser scanning microscopy. The effects of rANE and tANE on chemotaxis and adhesion of neutrophils to human aortic endothelial cells were examined using fluorescence-labeled neutrophils. RESULTS: Both rANE and tANE inhibited the phagocytic activity of neutrophils in a dose-dependent manner. The levels of internalized fluorescent bacteria in neutrophils decreased after ANE treatment. However, exposure of neutrophils to rANE and tANE stimulated the chemotaxis activity of neutrophils to N-formyl-Met-Leu-Phe (fMLP) and enhanced adhesion of neutrophils to human aortic endothelial cells in a dose-dependent manner. Moreover, treatment of neutrophils with rANE was more effective than incubation with tANE. CONCLUSIONS: Components of areca nut inhibited phagocytosis activity of neutrophils but enhanced chemotaxis and adhesion of neutrophils. Alterations in functions of neutrophils may lead to signs of clinical diseases associated with areca chewing. The components in ANEs that are responsible for these observations remain to be elucidated.     

Inhibitory effects of areca nut extracts on phagocytosis of Actinobacillus actinomycetemcomitans ATCC 33384 by neutrophils

BACKGROUND: Areca quid chewers have a higher prevalence of periodontal disease than non-chewers. Little is known about the influence of areca quid on the immune system. This study was to determine the possible effects of the areca nut on phagocytic activity of human neutrophils. METHODS: Aqueous extracts of ripe areca nut without husk (rANE), fresh and tender areca nut with husk (tANE), a major alkaloid (arecoline), and a phenolic component ([+]-catechin) of areca nut were examined for their effects on cellular viability using trypan blue exclusion assay. The possible effects on the phagocytic activity of neutrophils against a periodontal pathogen, Actinobacillus actinomycetemcomitans ATCC 33384, were determined using flow cytometry and confocal laser scanning microscopy. RESULTS: At the concentrations tested, rANE, tANE, arecoline, and (+)-catechin did not significantly affect viability of neutrophils. However, rANE, tANE, arecoline, and (+)-catechin inhibited the phagocytic activity of neutrophils in a dose-dependent manner. Approximately 50% of the relative phagocytic activity of neutrophils was affected when 50 microg/ml of rANE, 400 microg/ml of tANE, 20,000 microg/ml of arecoline, or 2,500 microg/ml of (+)- catechin was used. Decreased levels of internalized fluorescent bacteria were also demonstrated. However, arecoline or (+)-catechin alone could not be used to explain the inhibitory effects observed for rANE and tANE. CONCLUSIONS: Components of areca nut reduced the uptake of A. actinomycetemcomitans ATCC 33384 by human neutrophils. The inhibition of areca nut on phagocytosis of neutrophils may be one possible mechanism by which the areca nut compromises the periodontal health of areca quid chewers.     

Effects of areca nut extract on the apoptosis pathways in human neutrophils

Ho W‐H, Lee Y‐Y, Chang L‐Y, Chen Y‐T, Liu T‐Y, Hung S‐L. Effects of areca nut extract on the apoptosis pathways in human neutrophils. J Periodont Res 2010; 45: 412–420. © 2010 The Authors. Journal compilation © 2010 Blackwell Munksgaard Background and Objective: Areca nut, a major component in area quid, possesses genotoxic and carcinogenic activities. Areca nut extract (ANE) may affect the defensive functions of neutrophils. Recent studies suggest that areca nut chewing is associated with a higher prevalence of periodontal disease as a result of the detrimental effects of ANE on the host defense system. This study examined the effects of ANE on the apoptosis pathways in human neutrophils. Material and Methods: Apoptosis/necrosis of neutrophils was determined using flow cytometry. Proteins involved in the apoptosis pathway were determined using western blotting analysis. Results: The results indicated that ANE reduced early apoptosis, but increased the primary necrosis of neutrophils. ANE may arrest neutrophils in the G0/G1 phase and reduce the apoptotic hypodiploid DNA contents. The levels of cleaved forms of poly(ADP‐ribose) polymerase, and of caspase‐3 and caspase‐8 were decreased by treatment with ANE. Moreover, glycogen synthase kinase‐3α/β may be involved in the ANE‐modulated effects of neutrophils. Conclusion: Areca nut may regulate death pathways in neutrophils. This may be one mechanism by which areca nut compromises the periodontal health of areca nut chewers.     

Effects of areca nut extracts on the functions of human neutrophils in vitro

Aqueous extracts of ripe areca nut without husk (ripe ANE) and fresh and tender areca nut with husk (tender ANE) were examined for their effects on the defensive functions of human neutrophils. Exposure of peripheral blood neutrophils to ripe ANE and tender ANE inhibited their bactericidal activity against oral pathogens, including Actinobacillus actinomycetemcomitans and Streptococcus mutans, in a dose-dependent manner. At the concentrations tested, ripe and tender ANEs did not significantly affect the viability of neutrophils as verified by their ability to exclude trypan blue dye. However, both ANEs inhibited the production of bactericidal superoxide anion by neutrophils as measured by cytochrome c reduction. Moreover, the ripe ANE inhibited neutrophils more effectively than did tender ANE. Arecoline, a major alkaloid of areca nut, only exhibited an inhibitory effect on the functions of neutrophils when high concentrations were used. Therefore, arecoline could not be used to explain the inhibitory effects observed for ANEs. In conclusion, our results demonstrated that ripe and tender ANEs reduced the antibacterial activity and the superoxide anion production of neutrophils. This effect may contribute to a less efficient elimination of bacteria from the periodontal environment. Inhibition of the antimicrobial functions of neutrophils may alter the microbial ecology of the oral cavity, and this may be one possible mechanism by which areca nut compromises the oral health of users of areca nut products.     

Deficiency in collagen and fibronectin phagocytosis by human buccal mucosa fibroblasts in vitro as a possible mechanism for oral submucous fibrosis

Oral submucous fibrosis (OSF), a chronic oral mucosal condition commonly found in south Asians, is a disorder characterized by a quantitative as well as a qualitative alteration of collagen deposition within the subepithelial layer of the oral mucosa. Since degradation of collagen by fibroblast phagocytosis is an important pathway for physiological remodelling of soft connective tissues, we have investigated phagocytosis of collagen- and fibronectin-coated latex beads by fibroblast cultures with an in vitro model system. Coated fluorescent latex beads were incubated with human oral mucosa fibroblasts and the fluorescence associated with internalized beads was measured by flow cytometry. Cells from normal tissues that had been incubated with beads for 16 h contained a mean of 75% collagen phagocytic cells and 70% fibronectin phagocytic cells; however, about 15% and 10% of phagocytic cells individually contained more than twice the mean number of beads per cell. In contrast, cells from OSF tissues exhibited a 40% reduction of the proportions of collagen phagocytic cells (mean=35%) and a 48% decrease of the proportions of fibronectin phagocytic cells (mean=22%), none of the cells having a high number of beads as compared to normal fibroblasts. OSF lesions appear to contain fibroblasts with marked deficiencies in collagen and fibronectin phagocytosis. To investigate if inhibition of phagocytosis could be demonstrated in vitro, normal fibroblast cultures were incubated with areca nut alkaloids (arecoline, arecaidine). The cultures had a dose-dependent reduction in the proportions of phagocytic cells. On the other hand, corticosteroid used in the treatment of OSF exhibited a dose-dependent enhancement in the proportion of phagocytic cells. Therefore, our hypothesis for OSF, although over-simplified, is that betel nut alkaloids (arecoline, arecaidine) inhibit fibroblast phagocytosis and this provides a mechanism for the development of OSF. The benefit of a local intralesional injection of corticosteroid is also possibly, at least in part, through an enhancement of fibroblast collagen phagocytosis.     

Fc-binding components: a virulence factor in Actinobacillus actinomycetemcomitans?

Actinobacillus actinomycetemcomitans (ATCC 33384) can produce and release components that bind to the Fc part of IgG. Fc-binding components were observed in whole bacteria, capsular material and medium from broth cultures. The components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with biotinylated Fc-fragments and myeloma proteins. In a phagocytosis assay with human granulocytes and sheep erythrocytes, preincubation of opsonized erythrocytes with protein A reduced phagocytosis by 90%. In contrast, preincubation of the opsonizing antibody with medium components from a culture of A. actinomycetemcomitans enhanced the opsonizing effect of the antibody. The enhanced binding of erythrocytes may be caused by formation of aggregates between opsonizing antibody and bacterial Fc-binding components. Aggregated IgG can bind to low-affinity Fc gamma II and gamma III receptors that cannot bind monomeric IgG. Release of Fc-binding components from bacteria may contribute to the periodontal lesion through interference with the phagocytic activity of granulocytes and with the complement system. Fc-binding components may also interfere with downregulation of the B-cell response.     

槟榔提取物抑制人类口腔黏膜成纤维细胞生长的实验研究

目的通过比较正常口腔黏膜和口腔黏膜下纤维性变(OSF)组织中成纤维细胞(FB)增殖差异、检测槟榔提取物(ANE)对成纤维细胞增殖的影响,来探讨OSF的发病机理.方法对人类口腔黏膜成纤维细胞进行分离培养,然后用四唑盐(MTT)比色试验法检测OSF患者和正常人口腔黏膜FB增殖状况,并且观察ANE对FB增殖的影响.结果表示增殖水平的OD值在OSF-FB为0.254±0.045,高于NM-FB的OD值0.236±0.012(P<0.05),ANE以浓度-效应依赖关系抑制FB增殖.结论 OSF-FB细胞增殖率较NM-FB高;ANE对口腔黏膜FB有细胞毒作用,提示槟榔及其有效成分不完全是通过直接刺激FB增殖而诱发OSF.     

Fc(alpha) receptor I (CD89) on neutrophils in periodontal lesions

Aims: In this study, we have examined the occurence of FcαRI‐bearing cells in gingival tissue, gingival fluid and blood, in search for possible roles of IgA and FcαRI in periodontal lesions. Methods: Gingival biopsies from inflamed and healthy sites were obtained from patients with chronic marginal periodontitis. Sections of inflamed gingiva were examined by immunofluorescence techniques and compared to sections from healthy sites. Smears were made from blood and gingival crevicular fluid and similarly studied. Results: Dense infiltrates of neutrophils with strong expression of FcαRI (and FcγRIII) were found in connective tissue and epithelium of the apical part of periodontal pockets from diseased sites. In contrast, only few such cells were found in healthy gingiva from the same patients. Neutrophils in gingival fluid, tissue and blood expressed FcαRI with similar intensity, whereas the expression of FcγRIII was significantly decreased in gingival crevicular fluid. Considerable numbers of bacteria from gingival plaque were found to be covered by IgA. Conclusion: It is suggested that FcαRI on neutrophils may play an important rôle in elimination of IgA‐opsonized bacteria, both in periodontal tissue and the adjacent pockets.     

Effects of Areca Nut Extract on Lipopolysaccharides‐Enhanced Adhesion and Migration of Human Mononuclear Leukocytes

Background: Areca chewers have a higher prevalence of periodontitis than non‐chewers. Cell adhesion and movement (migration) are important for leukocyte recruitment to inflammation sites. This study investigates the effects of areca nut extract (ANE) on the adhesion and migration abilities of the human immune cells, peripheral blood mononuclear cells (PBMCs). The combined effects of nicotine and lipopolysaccharides (LPS) were also analyzed. Methods: Purified PBMCs obtained from healthy adults were treated with ANE, nicotine, and/or LPS. Cell adhesion ability was examined using fibronectin‐coated microslides, Liu stain, and light microscopy. Cell migration ability was evaluated using the transwell system followed by staining and fluorescence microscopy. Statistical difference was analyzed using the Mann‐Whitney U test. Results: When compared with the media‐treated control samples, PBMCs treated with ANE for 4 hours showed a significant reduction of the adherent cells on the microslides. Interestingly, LPS treatment increased cell adhesion, which could be reduced by simultaneous ANE plus nicotine treatment. The chemotactic migration of PBMCs was reduced by ANE treatment for 1, 4, or 24 hours in a dose‐dependent manner. LPS treatment increased PBMC migration, which could be reduced by simultaneous treatment with ANE or with ANE plus nicotine. Conclusions: ANE reduced the adhesion and migration abilities of PBMC. ANEs, with or without nicotine, also attenuated the migration of LPS‐stimulated PBMCs. The results implicated that the immune cell functions were impaired in areca chewers, which might increase the host susceptibility to oral and periodontal infection.     

Areca nut extracts suppress the differentiation and functionality of human monocyte-derived dendritic cells

Wang C‐C, Chen T‐Y, Wu H‐Y, Liu T‐Y, Jan T‐R. Areca nut extracts suppress the differentiation and functionality of human monocyte‐derived dendritic cells. J Periodont Res 2012; 47: 198–203. © 2011 John Wiley & Sons A/S Background and Objective: Areca quid chewing, a major risk factor contributing to the occurrence of oral cancer and precancer, has been reported to be associated with the severity and high prevalence of periodontal diseases in areca quid chewers. As dendritic cells are critically involved in the regulation of innate and adaptive immunity in oral mucosa, the objective of the present study was to investigate the effect of areca nut extracts (ANE) on the differentiation and reactivity of dendritic cells derived from monocytes. Material and Methods: Human peripheral blood monocytes were cultured in the presence of granulocyte–monocyte colony‐stimulating factor and interleukin‐4 for 7 d to generate dendritic cells. To examine the effect of ANE on the generation of dendritic cells, the monocytes were exposed to ANE throughout the 7 d culture period. In addition, the effect of ANE on the maturation of monocyte‐derived dendritic cells induced by lipopolysaccharide (LPS) was examined. Results: Monocytes cultured in granulocyte–monocyte colony‐stimulating factor and interleukin‐4 exhibited a typical phenotype of dendritic cells, as evidenced by the heightened expression of human leukocyte antigen (HLA)‐DR, CD11c and the co‐stimulatory molecules CD40, CD80 and CD86. Exposure of the monocytes to ANE did not influence the expression of HLA‐DR and CD11c, but markedly attenuated the proportion of CD40‐positive cells and the mean fluorescence intensity of CD86. The expression of co‐stimulatory molecules in LPS‐activated dendritic cells was not affected, whereas the mRNA expression of interleukin‐12 induced by LPS was markedly suppressed by ANE treatment in a concentration‐dependent manner. Conclusion: These results suggest that ANE exposure interfered with the differentiation of dendritic cells from monocytes. Moreover, the functionality of mature monocyte‐derived dendritic cells was attenuated in the presence of ANE.     

Areca nut extracts enhance the development of CD11b+Gr‐1+ cells with the characteristics of myeloid‐derived suppressor cells in antigen‐stimulated mice

J Oral Pathol Med (2011) 40 : 769–777 Background: Areca quid chewing is an etiological factor contributing to the development of oral cancer and pre‐cancers, whose pathophysiology has been linked to inflammation and immune deterioration. Myeloid‐derived suppressor cells (MDSC) play a key role in the regulation of immunity under certain pathological conditions, such as inflammation and cancer. As areca nut extracts (ANE) have been reported to induce a proinflammatory effect in antigen‐stimulated mice, we hypothesized that ANE might enhance the development of MDSC. Methods: Ovalbumin (OVA)‐sensitized BALB/c mice were daily administered with ANE (5–50 mg/kg), polyphenol‐enriched ANE (PANE; 25 mg/kg) or arecoline (5 mg/kg) by intraperitoneal injection for 10 doses. The mouse footpads were then subcutaneously challenged with OVA to induce local inflammatory responses. Results: ANE and PANE treatment significantly increased the spleen index and the population of CD11b⁺Gr‐1⁺ cells in the spleen and peripheral blood, whereas arecoline was inactive. In addition, ANE and PANE treatment enhanced the expression of cytokines and enzymes associated with the immunosuppressive function of MDSC, including IL‐10, arginase‐I and iNOS in splenic CD11b⁺ cells. Concordantly, ANE and PANE treatment augmented the infiltration of Gr‐1⁺IL‐10⁺ cells in the footpads challenged with OVA. Conclusions: Our results suggested that areca nut constituents, in particular, polyphenols enhanced the development of myeloid‐derived suppressor cells in vivo, which may be a critical mechanism linking inflammation and the compromised immunity reported to be associated with the pathophysiology of areca‐related oral diseases.     

Effects of periodontal therapy on phagocytic activity of peripheral blood neutrophils - evidence for an extrinsic cellular defect

Purpose: To compare phagocytic activity of peripheral blood neutrophils from subjects with and without periodontal disease and evaluate the effects of periodontal therapy in individuals with similar levels of resolution of inflammation at the end of treatment. Materials and Methods: To compare the phagocytic activity of neutrophils, peripheral blood was collected from 27 control subjects with a healthy periodontium and 28 periodontitis subjects before and after treatment. The phagocytosis of killed Saccharomyces cerevisiae, pre-sensitised or non-sensitised with fresh serum from the donor, was quantified and a phagocytic index was calculated as the mean number of yeast cells phagocytised by the percentage of neutrophils involved in phagocytosis. Results: Prior to periodontal treatment, subjects with periodontitis exhibited significantly lower neutrophil phagocytic activity than control subjects with a healthy periodontium. Periodontal treatment significantly improved in clinical periodontal status and resulted in significantly increased phagocytosis of both pre-sensitised (from 113.0 pre- to 157.0 post-treatment, P = 0.02) and non-sensitised S. cerevisiae (from 1.5 pre- to 3.5 post-treatment, P = 0.001), to levels observed in control subjects. Conclusions: The phagocytic activity of peripheral blood neutrophils from subjects with periodontal disease was lower than that of healthy controls. Subjects who underwent non-surgical periodontal treatment and strict supportive therapy for 6 months showed improved phagocytic activity in peripheral blood neutrophils. The phagocytic index values from subjects with periodontal disease after treatment achieved those found in the control group.     

Areca nut extract treatment elicits the fibroblastoid morphological changes, actin re-organization and signaling activation in oral keratinocytes

BACKGROUND: Areca (named as betel) is an important etiological factor linked with the high prevalence of oral squamous cell carcinoma (OSCC) in South-Asian countries. This in vitro study investigated the cellular changes and signaling activation in oral keratinocytes in response to areca nut extract (ANE) treatment. METHODS: Normal human oral keratinocyte (NHOK) and oral epidermoid carcinoma cell, Meng-1 (OECM-1) OSCC cell line were treated with variable dosages of ripen ANE. The morphological and cytoskeletal changes, as well as the activation of GTPase proteins and signaling kinases, were analyzed. RESULTS: Most NHOK cells in culture were polygonal, with only <5% cells exhibiting fibroblastoid morphology. However, 10 microg/ml ANE elicited fibroblastoid morphological change, genesis of lamellipodia, loss of subcortical actin, and stress-fiber formation in approximately 25% cultivated NHOK cells. Similar morphological changes were observed in nearly all OECM-1 cells following the ANE treatment. The activation of Rac and Rho GTPase, together with the prominent phosphorylation of a stress-activated kinases, particularly JNK1, was identified in treated OECM-1 cells. CONCLUSION: The novel evidences from the study that ANE impairs the actin organization and activates the signals in oral keratinocytes might bestow further insight into the impacts of ANE in oral pathogenesis.     

Differential capacity for complement receptor‐mediated immune evasion by Porphyromonas gingivalis depending on the type of innate leukocyte

The complement system plays a central role in immunity and inflammation, although certain pathogens can exploit complement to undermine protective immunity. In this context, the periodontal keystone pathogen Porphyromonas gingivalis was previously shown by our group to evade killing by neutrophils or macrophages through exploitation of complement C5a receptor 1 (C5aR1) and complement receptor 3 (CR3). Here, we examined whether P. gingivalis uses complement receptors to also subvert killing by dendritic cells. In line with earlier independent studies, intracellular viable P. gingivalis bacteria could be recovered from mouse bone‐marrow‐derived dendritic cells (BMDC) or human monocyte‐derived dendritic cells (MDDC) exposed to the pathogen. However, in the presence of C5a, the intracellular survival of P. gingivalis was significantly decreased in a C5aR1‐dependent way. Further work using wild‐type and receptor‐knockout BMDC showed that, in the presence of C3a, the C3a receptor (C3aR) similarly enhanced the intracellular killing of P. gingivalis. In contrast, C5aR2, an alternative receptor for C5a (G protein‐coupled receptor 77), was associated with increased intracellular P. gingivalis viable counts, consistent with the notion that C5aR2 functions as a negative regulator of C5aR1 activity. Moreover, P. gingivalis failed to use CR3 as a phagocytic receptor in BMDC, in contrast to our earlier findings in macrophages where CR3‐mediated uptake promotes P. gingivalis survival. Collectively, these data show that complement receptors mediate cell‐type‐specific effects on how innate leukocytes handle P. gingivalis, which appears to exploit complement to preferentially evade those cells (neutrophils and macrophages) that are most often encountered in its predominant niche, the periodontal pocket.     

Increased levels of soluble Fcγ receptor III in gingival fluid from periodontal lesions

Enzyme-linked immunosorbent assay was used for determination of the concentration of soluble Fcγ receptor III (FcγRIII) in 40 samples of gingival fluid obtained from periodontal pockets in 30 patients with periodontitis. The assay was based on a monoclonal immobilized antibody binding FcγRIII and a polyclonal FcγRIII rabbit antibody for its quantification. The results indicate a substantially increased concentration of soluble FcγRIII in gingival fluid as compared to the serum level. This increased concentration of soluble FcγRIII may interfere with phagocytosis and immune homeostasis in the periodontal lesions.     

Areca nut extract upregulates vimentin by activating PI3K/AKT signaling in oral carcinoma

J Oral Pathol Med (2011) 40 : 160–166 Background: Areca nut is a group I carcinogen. Areca nut extract (ANE) is known to activate signaling pathways in oral epithelial cells. Activation of the serine/threonine protein kinase AKT/pKB (AKT) signaling pathway is known to be important during the neoplastic process. Vimentin is a mesenchymal intermediate filament and a regulator of tumor progression. This study investigated the impact of ANE on PI3K/AKT activation during vimentin expression. Materials and methods: Oral carcinoma cells were treated with ANE to explore the signaling changes underlying vimentin expression. Oral carcinoma tissues were subjected to immunohistochemical analysis to study the implications that vimentin expression has on patient survival. Results: After ANE treatment, the OECM‐1 and Fadu cells developed a fibroblastoid morphology and there was an increase in vimentin expression. The treatment also induced the phosphorylation of AKT and glycogen synthase kinase 3β in OECM‐1 cells. Blockage of phosphatidylinositol 3‐kinase (PI3K)/AKT signaling attenuated vimentin expression when it was induced by ANE. However, it did not affect ANE‐mediated extracellular signal‐regulated kinase (ERK) activation or cyclooxygenase 2 (COX‐2) upregulation. Oral carcinoma tissue samples were found to have significantly higher levels of vimentin and pAKT expression than their controls. Tumors exhibiting no vimentin expression and weak AKT phosphorylation were found to be associated with better survival than groups with high levels of expression. Conclusion: Our results imply that PI3K/AKT activation and vimentin expression are important pathogenic cascades in areca‐associated oral carcinogenesis.     

Increased respiratory burst activity is associated with normal expression of IgG-Fc-receptors and complement receptors in peripheral neutrophils from patients with juvenile periodontitis

The respiratory burst activity in peripheral neutrophils from nine patients with localized juvenile periodontitis (LJP) and age- and sex-matched healthy controls was studied by measuring the intensity of luminol-enhanced chemiluminescence (CL) induced by unopsonized and three differently opsonized zymosan particles, formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA). The neutrophils from LJP patients showed in general more intense CL with all activators than did their controls. Particulary, the CL response induced by unopsonized zymosan particles and FMLP were significantly higher (p < 0.05 and 0.001). When comparisons were made between female LJP patients (n = 6) and matched controls, also serum-opsonized and IgG-opsonized zymosan particles produced CL was significantly increased (p < 0.05). In order to determine whether the elevated CL responses to zymosan particles were due to altered levels of the interacting receptors on neutrophil surface, an immunofluorescence analysis of the expression of IgG-Fc-receptors (FcR) and complement receptors (CR) was performed with flow cytometry. No significant difference in the expression of FcRII, FcRIII, CRl and CR3 was detected in LJP group compared to controls. Since the elevated CL responses can not be explained by changes in receptor numbers it is hypothesized that the increased respiratory burst activity in LJP may be caused by altered post receptor signalling pathway.     

Effect of priming in subpopulations of peripheral neutrophils from patients with chronic periodontitis

Background: Patients with periodontal disease are reported to generate more reactive oxygen species (ROS) than matched controls, suggesting increased inflammatory defense activity. The purpose of this study is to determine whether there are subpopulations of peripheral neutrophils in patients with chronic periodontitis (CP) that generate different levels of intracellular ROS when primed with tumor necrosis factor‐α (TNF‐α) or the chemokine interleukin‐8 (IL‐8, CXCL8) compared to controls. Methods: Venous blood was collected from 13 patients with CP despite careful maintenance over 2 to 8 years and from 13 healthy age‐ and sex‐matched controls. Neutrophils were separated from whole blood over a Percoll gradient and then activated via the Fcγ receptor with opsonized Staphylococcus aureus after priming with TNF‐α or IL‐8. The samples were analyzed by flow cytometry using the fluorescent probe dihydrorhodamine 123. Generation of ROS was measured as the intensity of fluorescence (IFL). Results: Two subpopulations were found in both patients and controls: one with low and one with high generation of IFL. The subpopulation with high generation of IFL in patients with CP was more responsive to IL‐8 (P <0.05) than the same subpopulation in healthy controls. No other differences in generation of ROS or priming effects were found between patients with CP and controls. Generation of ROS was dependent on nicotinamide adenine dinucleotide phosphate oxidase, and the intracellular ROS was primarily the oxygen anion. Conclusion: Patients with CP had a subpopulation of peripheral neutrophils that were more responsive to IL‐8 priming than controls.