Aspects of interleukin-8 gene expression by gingival and dermal fibroblasts stimulated with interleukin-1beta or tumour necrosis factor alpha

Fibroblast-derived interleukin (IL)-8 is thought to have an important role in the orchestration of immuno-participant cells infiltrating the skin and gingiva in response to continuously recurring bacterial infection. Therefore, the IL-8 gene expression should be under tight regulatory control and it might be temporally and spatially limited in inflammatory tissue. The purpose of this study was to examine the aspect of the IL-8 gene expression by fibroblasts stimulated with pro-inflammatory cytokines, IL-1beta and TNF-alpha. In situ hybridisation revealed that fibroblasts did not express IL-8 mRNA whereas keratinocytes and endothelial cells did in IL-1beta- or TNF-alpha-injected mice skin. However, cultured mouse dermal fibroblasts expressed not only IL-8 but also IL-1beta mRNA without stimulation by exogenous IL-1beta and TNF-alpha, and the expression was not enhanced by the exogenous cytokines. A similar result was obtained in late-passage human gingival fibroblasts. These results suggest that fibroblasts remain insensitive to IL-1beta and TNF-alpha so as to induce the IL-8 gene expression in non-inflammatory mice skin. Mouse dermal and late-passage human gingival fibroblasts in vitro are likely to be altered in phenotype into IL-8-producing cells along with the production of IL-1beta. In skin inflammation and periodontal diseases, fibroblasts may express the IL-8 gene even without an exogenous cytokine, IL-1beta or TNF-alpha, during their proliferation similar to the situation in our culture system.     

Lipid peroxidation caused by oxygen radicals from Fusobacterium-stimulated neutrophils as a possible model for the emergence of periodontitis

OBJECTIVE: The possible contribution of bacteria and polymorphonuclear neutrophils (PMN) to the disease process of periodontitis was evaluated. DESIGN: Fusobacterium nucleatum has been associated with chronic adult periodontitis. Intracellular production and extracellular release of reactive oxygen species (ROS) by PMN stimulated by fusobacteria were evaluated. To estimate the potential extracellular damage that might be caused by the ROS, the lipid peroxidation (LPO) of an exogenous phospholipid, Intralipid, was assayed. METHODS: The ROS production of PMN was studied by the nitroblue tetrazolium and chemiluminescence tests. The levels of malonaldehyde (MDA) and 4-hydroxyalkenals were used to indicate LPO. RESULTS: Fusobacterium nucleatum strains stimulated neutrophils to produce a large amount of ROS, independently of plasma complement factors. The two strains tested induced considerable intracellular, but no extracellular chemiluminescence responses during the first hour, indicating that ROS were released into phagosomes. However an incubation period of 4 h, in the presence of the extracellular lipid resulted in a high degree of LPO, presumably caused by ROS release from the Fusobacterium-stimulated PMN. ROS production and lipid peroxidation could be counteracted by vitamin E. CONCLUSION: In periodontitis local bacteria might stimulate PMN to release ROS, which cause inflammation and destruction.     

Ability of oral bacteria to induce tissue-destructive molecules from human neutrophils

Aim:  The induction of tissue-destructive molecules from neutrophils by periodontopathic bacteria has been suggested as one of the mechanisms of periodontal destruction. The aim of this study was to determine whether the ability to stimulate neutrophils is an authentic characteristic of periodontopathic bacteria.
Methods:  We evaluated, along with phagocytosis, the production of reactive oxygen species (ROS), matrix metalloproteinase-8 (MMP-8), and interleukin-1 β by neutrophils in response to non-periodontopathic Streptococcus sanguinis and periodontopathic bacteria Fusobacterium nucleatum and Treponema denticola , in the absence or presence of antibodies. Phagocytosis, the death of neutrophils, and intracellular ROS production were measured by flow cytometry and the concentrations of MMP-8 and interleukin-1 β secreted into medium were determined by enzyme-linked immunosorbent assay.
Results:  S. sanguinis and F. nucleatum induced greater production of ROS, MMP-8, and interleukin-1 β than did T. denticola. The levels of tissue-destructive molecules produced by neutrophils had a positive correlation with phagocytosis. Opsonization of bacteria with antibodies significantly increased phagocytosis and ROS production and release, thus increasing both bacterial clearance and potential tissue damage.
Conclusion:  The ability of oral bacteria to induce tissue-destructive molecules from neutrophils is not an inherent characteristic of periodontopathic bacteria, which would provide a new insight into the role of neutrophils in periodontal destruction.     

Increased release of elastase from in vitro activated peripheral neutrophils in patients with adult periodontitis

The main object of this study was to determine if there was a difference between patients with adult periodontitis and healthy controls in the release of elastase. We also wanted to test the release of alpha-1-antitrypsin and lactoferrin from in vitro-activated peripheral neutrophils. A leukocyte-rich preparation from venous blood was made by lysing the red blood cells. The leukocytes were stimulated for 1 h at 37 degrees C with opsonized Staphylococcus aureus and the released elastase was measured with a chromogenic substrate. The release of elastase after stimulation with bacteria was significantly higher in patients than in controls. The amounts of elastase from unstimulated cells, i.e., both released extracellularly and extracted from the pellet, were similar in the 2 groups. However, after stimulation, the amount of elastase in the patient group, but not in the control group, was significantly increased. Similar releases of alpha-1-antitrypsin (AIAT) and lactoferrin were found in both groups of subjects. In conclusion, this study shows that peripheral neutrophils from patients with adult periodontitis release more active elastase after in vitro activation compared to healthy controls. The release of A1AT and lactoferrin showed no differences, indicating that the increased elastase activity was not due to a impaired inhibition by A1AT and that the differences in degranulation were limited to the primary granula.     

Thymocyte activating factors in human gingival fibroblast cultures stimulated by oral Bacteroides lipopolysaccharides: induction, identification and modification by various cytokines

Normal human gingival fibroblasts stimulated in vitro by lipopolysaccharide (LPS) from black pigmented oral Bacteroides species produced cell-free (CF) and cell-associated (CA) thymocyte activating factors (TAF). The LPS from other bacteria, including Escherichia coli and Salmonella species, induced minimum levels of TAF in the cultures. The CF-TAF was partially inhibited by anti-human interleukin (HuIL)-1 beta or HuIL-6 antibody, but not by anti-HuIL-1 alpha antibody. However, complete inhibition of the CF-TAF was not observed upon addition of both anti-HuIL-1 beta and HuIL-6 antibodies. Fibroblasts stimulated with Bacteroides LPS released high levels of CF-IL-6 activity. Recombinant (r) HuIL-6 negligibly exhibited TAF activity even in high doses up to 500 U/ml, although it augmented the TAF activity of rHuIL-1 beta. These findings indicated that the CF-TAF consisted mainly of IL-1 beta, and that IL-6 enhanced TAF activity of IL-1 beta. However, other TAF factor (s) may be present in CF specimens. In contrast to CF-TAF, the CA-TAF was inhibited with anti-HuIL-1 alpha. Recombinant human tumor necrosis factor (rHuTNF) directly stimulated fibroblasts to produce CA-TAF, and it also primed them to enhance CA-TAF induction in response to Bacteroides LPS. On the other hand, natural human interferons (nHuIFN) alpha, beta, and gamma did not induce CF- or CA-TAF in fibroblasts. When fibroblasts were primed with nHuIFN beta or gamma, the CA-TAF production by the cells in response to LPS, but not rHuTNF, was markedly enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hyper-reactive mononuclear cells and neutrophils in chronic periodontitis

OBJECTIVES: Stimulated mono- and polymorphonuclear cells from patients with periodontitis have shown increased release of interleukin-1beta (IL-1beta) and oxygen radicals, respectively. The aim was to study whether this hyper-reactivity could be found both in mono- and polymorphonuclear cells from the same patient, and whether there was a relation to the gene coding for IL-1beta (IL-1beta(+3953)). MATERIAL AND METHODS: Peripheral mononuclear cells from 14 non-smoking and well-treated patients and pair-matched controls were incubated with opsonized Staphylococcus aureus and lipopolysaccharide (LPS). Released IL-1beta and tumour necrosis factor (TNF)-alpha were determined with ELISA. Generation of oxygen radicals from the Fcgamma-receptor-stimulated neutrophils was measured with chemiluminescence and the polymorphism at IL-1beta(+3953) was measured with polymerase chainreaction. RESULTS: The mononuclear cells from the patients released more IL-1beta after incubation with LPS (p<0.001) and with bacteria (p<0.05). The release of TNF-alpha tended to be higher in the patient group. The peripheral neutrophils from the patients generated more oxygen radicals (p<0.06). We found no differences between the study groups regarding the IL-1beta(+3953) polymorphism. CONCLUSION: The similarity in systemic inflammation between patients and controls suggests that the increased release/generation of IL-1beta and oxygen radicals from peripheral leukocytes in periodontitis patients is of a constitutional nature and of pathogenic relevance.     

Effect of neutrophil apoptosis on monocytic cytokine response to Porphyromonas gingivalis lipopolysaccharide

BACKGROUND: Neutrophil apoptosis may play a critical role in the resolution of inflammation by stimulating anti-inflammatory cytokine generation from monocytes. In this study, we investigated the effect of apoptotic neutrophils on interleukin (IL)-10 and IL-1beta production from monocytes in response to Porphyromonas gingivalis lipopolysaccharide. METHODS: Peripheral blood neutrophils from healthy individuals were isolated by sodium diatrizoate density gradient centrifugation. In order to induce apoptosis, neutrophils were cultured for 24 hours in modified Dulbecco's medium supplemented with 10% autologous serum. Cell apoptosis was quantified by Annexin V positivity and loss of CD16 expression on the cell surface. Peripheral blood mononuclear cells were isolated from the same subjects; monocytes were purified by magnetic cell sorting and cultured with or without apoptotic or fresh neutrophils. Lipopolysaccharide from Porphyromonas gingivalis was used for cell stimulation. IL-1beta and IL-10 levels in supernatants were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: IL-10 generation was significantly increased in monocytes cultured with apoptotic neutrophils compared to monocytes alone or cocultured with fresh neutrophils (P <0.05). IL-1beta was suppressed both in resting and lipopolysaccharide-stimulated monocytes in the presence of apoptotic neutrophils compared to monocytes alone or monocytes cultured with fresh neutrophils at all time points (P <0.05). CONCLUSION: Neutrophil apoptosis provides a signal to monocytes, changing the phenotype of the monocyte resulting in the production of anti-inflammatory cytokines and suppression of proinflammatory cytokines in response to lipopolysaccharide.     

Interleukin-1beta,clinical parameters and matched cellular-histopathologic changes of biopsied gingival tissue from periodontitis patients

OBJECTIVE: The aim of the present study was to investigate whether interleukin (IL)-1beta in diseased tissues adjacent to periodontal pockets can reflect the degree of inflammation and destruction of these tissues pathologically. BACKGROUND: IL-1beta-dependent mechanisms have been strongly implicated in contributing to inflammation and destruction of bone and attachment loss, which are characteristic features of periodontal disease. This biochemical mediator released during pro-inflammatory processes has not been objectively integrated with clinical and histopathologic features of periodontal disease. METHODS: Periodontitis-affected inflamed tissue and clinically nonaffected healthy gingivae were harvested from 14 periodontal patients, respectively. The severity of tissue inflammation was illustrated by clinical parameters and cellular histologic changes and quantified by histometric assessments. IL-1beta in these extracted specimens was measured with an enzyme-linked immunosorbent assay (ELISA) technique. Pathogenic roles that IL-1beta plays in gingival inflammation and pathologic tissue changes in tissue sections were analyzed statistically. RESULTS: The overall total tissue IL-1beta, tissue concentration of IL-1beta, and percentage of inflammatory cell infiltration (PICI) determined from diseased gingivae were obviously higher than those of controls from both healthy sites of periodontitis and non-periodontitis subjects. With increasing gingival index (GI), plaque index (PlI), and probing depth (PD), there was a marked elevation in total tissue IL-1beta. Total tissue IL-1beta was significantly correlated with GI, PlI, the PICI, and tissue alterations. Polymorphonuclear leukocytes (PMNs) and monocyte-macrophage cells seemed to predominate in heavily infiltrated areas of diseased gingiva. These cell types were confirmed by immunocytochemical localization with either monoclonal mouse antihuman neutrophil elastase antibody or monoclonal mouse antihuman macrophage (CD68) antibody, respectively. Total tissue IL-1beta and the PICI were also elevated in diseased gingivae near deeper PD, while neither total IL-1beta nor tissue concentration was statistically correlated with PD. Thus, correlation analysis indicates that IL-1beta level in inflamed periodontal tissues correlates highly with clinical parameters (GI and PlI) and PICI (the degree of inflammation). CONCLUSIONS: These observations suggest that IL-1beta plays a significant role in the pathogenic mechanisms of periodontal tissue destruction, and that measurement of tissue IL-1beta would be a valuable aid and useful for diagnostic markers of periodontal diseases.     

Actinobacillus actinomycetemcomitans-induced expression of IL-1α and IL-1β in human gingival epithelial cells: role in IL-8 expression

Gingival epithelial cells (GEC) are the first cells of the host that encounter the periodontal pathogens. and therefore their role in the initiation of the inflammatory response is critical. We aimed to: 1) characterize the expression of interleukin (IL)- Ialpha and IL-Ibeta in human gingiva and cultured GEC: 2) demonstrate the ability of A. actinomycetemcomitans extracts to upregulate IL-1alpha, IL-1beta and IL-8 expression in GEC in vitro: and 3) characterize the role of IL-1alpha and IL-1beta in the induction of IL-8 expression in GEC in vitro. Ten gingival biopsies (5 inflamed and 5 controls) and cultured GEC were examined for IL-1alpha and IL-Ibeta using immunohistochemical techniques. GEC were also challenged with A. actinomycetemcomitans extracts or IL-1alpha, and secretion of IL-1 and IL-8 was determined by ELISA. In vivo, IL-lalpha and IL-1beta were localized in the gingival epithelium and the infiltrating leukocytes. In vitro, A. actinomycetemcomitans extracts induced a time-dependent expression of IL-1alpha, IL-1beta and IL-8 in GEC. IL-1 inhibitors did not affect A. actinomycetemcomitans-induced IL-8. although they inhibited IL-8 induced by IL-1alpha or IL-1beta. In conclusion, GEC are a major source of IL-1alpha and IL-1beta in the periodontium, which in turn induce additional inflammatory mediators such as IL-8. Therefore GEC can be a potential target for therapeutic intervention in the future.     

Epithelial cell pro-inflammatory cytokine response differs across dental plaque bacterial species

Aim: The dental plaque is comprised of numerous bacterial species, which may or may not be pathogenic. Human gingival epithelial cells (HGECs) respond to perturbation by various bacteria of the dental plaque by production of different levels of inflammatory cytokines, which is a putative reflection of their virulence. The aim of the current study was to determine responses in terms of interleukin (IL)-1 β , IL-6, IL-8 and IL-10 secretion induced by Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum and Streptococcus gordonii in order to gauge their virulence potential.
Materials and Methods: HGECs were challenged with the four bacterial species, live or heat killed, at various multiplicity of infections and the elicited IL-1 β , IL-6, IL-8 and IL-10 responses were assayed by enzyme-linked immunosorbent assay.
Results: Primary HGECs challenged with live P. gingivalis produced high levels of IL-1 β , while challenge with live A. actinomycetemcomitans gave high levels of IL-8. The opportunistic pathogen F. nucleatum induces the highest levels of pro-inflammatory cytokines, while the commensal S. gordonii is the least stimulatory.
Conclusion: We conclude that various dental plaque biofilm bacteria induce different cytokine response profiles in primary HGECs that may reflect their individual virulence or commensal status.     

Secretion of IL-1β, TNF-α, IL-8 and IL-1ra by human polymorphonuclear leukocytes in response to lipopolysaccharides from periodontopathic bacteria

Polymorphonuclear leukocytes (PMN) are the first cells that migrate into periodontal tissues and gingival crevices in response to invading pathogens. It was recently demonstrated that PMN have the ability to synthesize and release cytokines following appropriate stimulation, while it is not clear whether these capacities are directly related to periodontal destructive processes. We therefore investigated the amounts of the cytokines interleukin-1β (IL-lβ), tumor necrosis factor α (TNF-α), IL-8 and IL-1 receptor antagonist (IL-lra) secreted by PMN from healthy donors following stimulation with lipopolysaccharide (LPS) from 4 periodontopathic bacteria, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Capnocytophaga ochracea and Fusobacterium nucleatum, and the non-oral bacterium Escherichia coli. A. actinomycetemcomitans, F. nucleatum and E. coli LPS stimulated the release of significantly greater amounts of IL-lβ, TNF-α and IL-8 than the control unstimulated PMN (p<0.01). The levels of IL-lβ, TNF-α and IL-8 released from cells stimulated with P. gingivalis or C. ochracea LPS were significantly lower than those of cells stimulated with A. actinomycetemcomitans or E. coli LPS (p<0.05). On the other hand, substantially greater amounts of IL-lra were released from PMN stimulated with each LPS and from control unstimulated PMN during the first 6 h, and then significantly greater amounts of IL-lra were secreted by PMN stimulated with A. actinomycetemcomitans and E.coli LPS during the following 12 h (p<0.01). The inhibitory effects of IL-lra on the biological activity of IL-1 in the supernatants of PMN were examined by the thymocyte comitogen proliferation assay. The supernatants of PMN stimulated with each LPS showed less biological IL-1 activity as compared with the same doses of recombinant human IL-lβ detected by enzyme-linked immunosorbent assay. Furthermore, no activity was detected in the supernatants of PMN stimulated with P. gingivalis or C. ochracea LPS. These findings demonstrated that LPS from periodontopathic bacteria were capable of stimulating PMN to release not only pro-inflammatory cytokines but also their inhibitors such as IL-lra. Different secretion levels of these cytokines and their biological activities induced by the various LPS might be important in the onset and progression of periodontal diseases.     

Expression of intracellular elastase activity in peripheral neutrophils from patients with adult periodontitis

This study aimed to investigate whether circulating neutrophils from patients with periodontitis contain more catalytically active elastase than neutrophils from healthy controls. The amount of IL-1beta in these cells was also analyzed and the correlation with elastase activity was tested. The periodontitis group consisted of 15 subjects with marked periodontal destruction. The healthy control group consisted of 15 subjects with no clinical signs of periodontal destruction. The elastase activity and the IL-1beta content in the cells were measured with flow cytometry using a specific substrate and antibodies, respectively. The plasma concentration of IL-1beta and the total content of antigenic elastase in the crushed cells were measured with an ELISA. The elastase activity per neutrophil was significantly higher in the patients than in the controls, while the total antigenic elastase content did not differ. The % of cells positively stained for IL- 1beta was somewhat higher in the patient group. Significantly higher amounts of IL-1beta per sample, estimated by multiplying the % of stained cell with the amount of staining per cell, were found in the patient group. A significant correlation between IL-Ibeta and elastase activity was noted in the patient group (R=0.6, p=0.001), but not in the control group. In conclusion, peripheral neutrophils from patients with adult periodontitis express more active elastase and total amounts of IL-1. The similar amounts of antigenic elastase suggests that this higher activity is possibly due to some kind of priming/activation already in the circulation.     

Effect of lipopolysaccharide from Porphyromonas gingivalis on prostaglandin E2 and interleukin-1β release from rat periosteal and human gingival fibroblasts in vitro

Lipopolysaccharide (LPS) was extracted from Porphyromonas gingivalis (W83) by the Westphal procedure, nuclease-digested and ultracentrifuged. Fibroblasts were obtained from human gingival tissue and rat periosteum, grown to confluence then stimulated in serum-free medium with 0.1, 1.0 and 10.0 micrograms/ml LPS. The levels of prostaglandin E2 (PGE2) and interleukin-1 beta (IL-1 beta) released were measured after 2, 4 and 6 d by specific radioimmunoassays. Unstimulated gingival fibroblasts produced low levels of PGE2 (24.5 +/- 1.5 (SD) ng/ml) and IL-1 beta (0.34 +/- 0.29 ng/ml). LPS stimulated statistically significant dose-related increases in PGE2 and IL-1 beta at the concentrations of LPS tested. At 10.0 micrograms/ml, LPS-stimulated fibroblasts produced 363.5 +/- 40.3 ng/ml PGE2 and 1.81 +/- 0.1 ng/ml IL-1 beta in 6 d. These results demonstrate that LPS from P. gingivalis is capable of stimulating PGE2 and IL-1 beta release from fibroblasts. This would appear to be an additional mechanism by which LPS can induce tissue breakdown in periodontal disease.     

Production of inflammatory cytokines by human gingival fibroblasts stimulated by cell-surface preparations of Porphyromonas gingivalis

Porphyromonas gingivalis is a gram-negative rod associated with the progression of human periodontal disease. Inflammatory cytokines are believed to be the major pathological mediators in periodontal diseases. We therefore investigated the productions of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha) in human gingival fibroblasts treated with lipopolysaccharide, polysaccharide and outer-membrane proteins from P. gingivalis ATCC 53977. Outer-membrane protein from P. gingivalis enhanced the production of IL-6 and IL-8 from the cells of periodontium in vitro as well as lipopolysaccharide did. The IL-8 production activity of polysaccharide from P. gingivalis was higher than that of other cell-surface components. The levels of IL-6 and IL-8 released from the P. gingivalis lipopolysaccharide-treated human gingival fibroblasts were lower than those of the same cells treated with lipopolysaccharides from Actinobacillus actinomycetemcomitans or Escherichia coli. Rabbit antisera against either outer-membrane protein or lipopolysaccharide inhibited the IL-6 and IL-8 production derived from human gingival fibroblasts stimulated sonicated supernatants from P. gingivalis. The present study suggests that, in addition to lipopolysaccharide, outer-membrane protein and polysaccharide of P. gingivalis are also pathological mediators in periodontal diseases.     

Oral submucous fibrosis patients have altered levels of cytokine production

Oral submucous fibrosis (OSF) is a pre-malignant fibrotic lesion of the mouth in betel quid chewers and is characterised by dense bands of collagen in the juxta-epithelial region preceded by inflammation. We have investigated the spontaneous and stimulated production of cytokines by peripheral blood mononuclear cells (PBMC) from OSF patients and compared them with genetically-related relatives, Indian and Caucasian control subjects. The cytokines studied included: interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). The results show: a) significant differences in the stimulated versus non-stimulated levels of IL-1beta, IL-6, IL-8 and TNF-alpha but not of IFN-gamma production by patients, and in the relatives' stimulated versus non-stimulated levels of IL-1beta, IL-6 and IFN-gamma; b) no difference in the spontaneous cytokine production between any two groups; and c) significant increases in the patients' stimulated cytokines compared to the Caucasian and Indian controls (P< or =0.050). These results demonstrate increased levels of proinflammatory cytokines and reduced anti-fibrotic IFN-gamma in patients with OSF, which may be central to the pathogenesis of OSF.     

Decreased interleukin-1beta and elastase in the gingival crevicular fluid of individuals undergoing anti-inflammatory treatment for rheumatoid arthritis

BACKGROUND: The primary aim of this study was to compare the inflammatory activity in the gingival crevicular fluid (GCF) in a group of patients with rheumatoid arthritis (RA) and a group of matched controls. Secondarily, we aimed to evaluate the effect of rheumatologic treatment on periodontal inflammation. METHODS: Seventeen individuals with RA with a mean duration of disease of 12.1 (+/- 9.9) years and the same number of systemically healthy individuals matched for age, gender, periodontal status, and tobacco use were selected. Medication data were registered, and GCF was collected by means of an intracrevicular washing method. Besides clinical registrations, periodontal inflammation was assessed by analysis of the cytokines interleukin (IL)-1beta and -18 and of elastase activity. RESULTS: Amounts of IL-1beta and total elastase were significantly lower in the patient group. IL-1beta and total elastase had a significant and strong correlation in the RA group (r(s) = 0.883). This correlation was not observed in the control group. CONCLUSION: The anti-inflammatory treatment taken by RA patients might influence the periodontal inflammation status represented by IL-1beta and elastase in the GCF.     

IL-8 and TNF-alpha from peripheral neutrophils and acute-phase proteins in periodontitis

OBJECTIVE: In other studies, we have found deviant functions in peripheral neutrophils in periodontitis. The aim here was to study (1) the release of cytokines, IL-8 and TNFalpha, from neutrophils in 15 treated periodontitis patients and pair-matched controls as well as (2) the effects of cigarette smoking. MATERIAL AND METHODS: Cytokines released in the incubation medium from un-stimulated and Fcgamma-R-stimulated neutrophils and some acute-phase reactants were measured with ELISA. RESULTS: Non-smoking patients had trends for lower TNFalpha release compared to non-smoking controls, while corresponding trends were rather similar for Il-8. Smoking had a moderate but inconsistent effect on the release of both cytokines. However, in patients, the ratio between stimulated/un-stimulated release of Il-8 was significantly lowered by smoking (p<0.03). The parameters of inflammation in plasma differed only slightly between patients and controls, indicating that periodontal disease in a quiet phase has a negligible systemic effect with the possible exception for a higher IL-8 level. In contrast, smoking had significant systemic effect on the neutrophil count and IgG levels. CONCLUSIONS: Release of IL-8 and TNF-alpha from peripheral neutrophils and various parameters of inflammation in plasma seem to be affected more by cigarette smoking than periodontal disease.