Objectives: Stimulated mono- and polymorphonuclear cells from patients with periodontitis have shown increased release of interleukin-1 beta (IL-1 beta) 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-1 beta (IL-1 beta(+3953)).Materials 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-1 beta and tumour necrosis factor (TNF)-alpha were determined with ELISA. Generation of oxygen radicals from the Fc gamma-receptor-stimulated neutrophils was measured with chemiluminescence and the polymorphism at IL-1 beta(+3953) was measured with polymerase chainreaction.Results: The mononuclear cells from the patients released more IL-1 beta 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-1 beta(+3953) polymorphism.Conclusion: The similarity in systemic inflammation between patients and controls suggests that the increased release/generation of IL-1 beta and oxygen radicals from peripheral leukocytes in periodontitis patients is of a constitutional nature and of pathogenic relevance.

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.

Host-defence defects in hidradenitis suppurativa patients have been suspected, but not proven. Activated neutrophils can destroy the surrounding tissues by a release of reactive oxygen species and active proteases. Peripheral neutrophils from 15 female patients (mean age 46, range 27-57 years) in an inactive state of their hidradenitis suppurativa, were studied and compared with 15 age-matched healthy female controls. There were no significant differences between patients and controls in the assessments of intracellular elastase activity, total content of antigenic elastase or release of elastase. Furthermore, no differences were found in total content and membrane expression of the receptors measured. The generation of free oxygen radicals, after stimulation with the protein kinase C activator phorbol myristate acetate, was significantly higher in the patients than in the controls, while there was no difference after Fc-receptor-mediated stimulation. Dysfunctional neutrophils might be involved in the pathogenesis of hidradenitis suppurativa, but the findings should be interpreted with caution because of the small number of observed cases.

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.

Stimulated neutrophils from subjects with various inflammatory tissue-destructive conditions, such as periodontal, pulmonary, gastrointestinal, and cardiovascular diseases, generate more oxygen radicals and proteases, implicated in tissue destruction, than neutrophils from healthy persons. Cigarette smoking aggravates these diseases. The aim of our study was to investigate the effect of cigarette smoking on the priming capacity of tumor necrosis factor-α, measured as generation of radicals from stimulated neutrophils, in smoking and non-smoking subjects with or without periodontitis. The priming effect was higher in neutrophils from smokers. In the group with periodontitis, smoking caused an even greater increase in the generation of radicals, indicating an additive effect of this local disease. The membrane expression of CD11b, CD15, and CD63 was significantly higher on neutrophils from smokers, indicating upregulated neutrophil functions. This increased priming effect of tumor necrosis factor-α in smokers subjects could be of importance in the aggravation of tissue-destructive inflammatory diseases.

OBJECTIVE:The aim was to investigate whether increased Fcgamma-receptor (FcgammaR)-induced chemiluminescence (CL) from peripheral neutrophils in periodontitis could be due to increased sensitivity to non-specific priming by the cell preparation.DESIGN:Two methods of preparation were used: (1) cells purified by density centrifugation and lysis of erythrocytes; and (2) cells in a leukocyte suspension obtained by lysis of erythrocytes alone.METHODS:Neutrophils from 17 subjects with periodontitis and pair-matched healthy controls were studied by CL and flow cytometry (CD16, CD15 and CD11b).RESULTS:Higher CL was found in the purified neutrophils, indicating higher non-specific priming by the Percoll density centrifugation. Priming with TNFalpha and lipopolysaccharide abolished this difference. The net effect of specific priming was significantly lower in the purified neutrophils, indicating a depletion of the priming sensitivity by the density centrifugation. Both preparations reduced CD15 in relation to whole blood, while there was no difference between the two methods in any of the membrane markers. Thus, in both preparations, lysis alone changed the membranes to the same extent. Both methods gave nearly the same relative CL increase in the periodontitis patients as in the controls.CONCLUSION:There was no difference between the neutrophils from patients and controls in sensitivity to non-specific priming by the preparation method. The higher FcgammaR-induced CL in patients with periodontitis is not related to such a non-specific priming by density centrifugation.

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.

The influence of smoking on the activity of the gingival neutrophils in young periodontally healthy adults was studied. The neutrophil activity was measured in terms of the gingival crevicular fluid (GCF) levels of elastase, lactoferrin (LF), alpha-1-antitrypsin (alpha-1-AT), alpha-2-macroglobulin (alpha-2 MG) and protein. 30 healthy dental students with no clinical signs of periodontitis, 15 smokers (8 women and 7 men) aged 20-32 years and 15 non-smokers (7 women and 8 men) aged 22-31 years, volunteered to take part in the investigation. The gingival inflammation was registered at 6 sites and the GCF volume was collected from the same sites. The GCF volume was measured with a Periotron 6000. The elastase activity was measured with a chromogenic low molecular substrate and the LF alpha-1-AT, alpha-2-MG levels were determined with ELISA. The protein concentration was measured by the Bradford method. The results showed a statistically significantly lower GCF volume among smokers as compared to non-smokers. No significant difference was found in the elastase activity/microl of the GCF supernatant between smokers and non-smokers but there was a large inter-individual variation. Nor did the concentrations of LF, alpha-1-AT, alpha-2-MG and protein per microl GCF differ significantly between the 2 groups. The results suggest that the influence of smoking on the examined factors associated with neutrophil activity is limited under healthy or slightly inflamed gingival conditions giving only small amounts of GCF.

We have earlier reported a higher Fc gamma-receptor (Fc gamma R)-mediated generation of reactive oxygen species, measured as luminol-enhanced chemiluminescence (CL) from peripheral neutrophils in adult periodontitis patients. The aims of this study were to confirm our previous results and to elucidate the mechanism of this phenomenon by measuring CL in parallel with the intracellular production of hydrogen peroxide, after stimulation with opsonized bacteria. To determine whether the higher CL was associated with altered responsiveness to priming, the cells were preincubated with tumor necrosis factor alpha (TNF alpha) and lipopolysaccharide (LPS). While CL was significantly higher in subjects with periodontitis, there was no difference in hydrogen peroxide production between the patients and the controls, indicating that the hyperreactivity is related to the generation of other oxygen species than H2O2 and/or to processes in the outer cell membrane. The responsiveness to priming with LPS on CL was slightly but not significantly higher in the periodontitis group, suggesting that priming could be of value for distinguishing subjects with periodontitis. When assaying intracellular production of H2O2, TNF alpha and LPS had both a priming and an activating effect. There were no significant differences between the two groups. In conclusion, this study shows a higher Fc gamma R-mediated CL of peripheral neutrophils from adult patients with periodontitis, thus confirming our earlier results. The hyperreactivity seems to be related to the outer cell membrane or to oxygen species other than H2O2.

The response of peripheral neutrophils was studied in 16 patients with allergic asthma after challenge with birch/grass pollen allergen, in order to identify inflammatory markers associated with only the early asthmatic reaction and those associated with both early and late asthmatic reactions. The allergen challenge proceeded until the patients had an early asthmatic reaction with 100% increase in specific airway resistance. Bronchoconstriction after allergen challenge was monitored hourly over 9 h and finally after 18 h, by measurement of the forced expiratory volume in 1 s. Seven patients had a late reaction, defined as a decrease in forced expiratory volume in 1 s of more than 15%. Blood samples were taken before and 18 h after challenge. After allergen challenge (18 h) the blood concentration of neutrophils in patients with a late asthmatic reaction was 1.4 times higher than before challenge and there was a tendency for increased Fcγ receptor-mediated chemiluminescence. Lewis X-antigen (CD 15), which is associated with endothelial adhesion and extravasation, significantly decreased at the same time. Neutrophils were incubated with the tetrapeptide arginine-glycine-aspartate-serine before and 18 h after allergen challenge. Both patient groups showed an increased Fcγ receptor-mediated chemiluminescence and a decreased Fcγ receptor membrane expression following allergen challenge, suggesting a preactivation. In conclusion, patients with a dual asthmatic reaction show a sustained primed inflammatory response and primed neutrophils compared with patients with only an early reaction when measured after the decline of clinical symptoms provoked by allergen challenge.

The 19 patients included in this study had all been successfully treated for total or partial edentulism with single crystal sapphire implants as retention for overdentures or fixed bridges. As there is a need for more reliable non-invasive parameters for detecting changes surrounding endosseous implants, the aim of this study was to assess the content and the activity of neutrophils in crevicular fluid samples from 3 categories of sites: (1) crevices around implants from edentulous patients (2) crevices around implants in partially edentulous patients, and (3) crevices surrounding teeth. Fluid samples were taken with paper strips from 9 partially edentulous and 10 edentulous patients and the volume measured with a Periotron 6000. Elastase activity was measured as a marker of neutrophil activity and lactoferrin concentration as a marker of the number of neutrophils. Elastase activity per microliter and lactoferrin concentration was, despite similar clinical and radiographic signs, significantly higher in samples from crevices surrounding teeth and implants in the partially edentulous patients compared to samples from crevices around implants in the totally edentulous patients. There were no differences between teeth and implants in partially edentulous patients. The increased elastase activity and lactoferrin concentration indicates a higher neutrophil activity in patients with remaining teeth.

We have earlier reported hyperreactive peripheral neutrophils in adult periodontitis, measured as respiratory burst after Fc gamma receptor-mediated activation in vitro, but we have not been able to relate this increased activity to aberrations in the expression of relevant membrane molecules. Various types of inflammatory conditions involving the gingiva should affect membranes differently. We therefore collected crevicular neutrophils from three types of inflammatory sites: (i) with and (ii) without tissue destruction in the same periodontitis patients and (iii) inflamed sites in controls with gingivitis alone and compared the expression of membrane molecules by flow cytometry. The % of positively stained cells and their mean intensities of fluorescence (IFL) were similar in the three types of sites for CD15, CD11a, CD11b and CD16. Peripheral neutrophils studied with the same markers were not activated. This was verified by similar plasma concentrations of lactoferrin and L-selectins in the periodontal and control groups. Compared to peripheral cells, the crevicular neutrophils showed a significantly lower percentage of stained cells, while the stained cells increased their IFL. In conclusion, hyperreactive peripheral neutrophils in periodontitis show the same expression of membrane molecules after migration through different types of inflammatory lesions as do normal neutrophils in gingivitis.

OBJECTIVES:To study the response to in vitro priming of peripheral neutrophils from patients with periodontitis compared to healthy controls. Peripheral neutrophils from these patients had shown increased production of oxygen radicals after activation with opsonized bacteria and a difference in priming response could suggest an explanation for this hyperreactivity.MATERIALS AND METHODS:Peripheral neutrophils from a group of patients with periodontitis and from age- and sex-matched controls were preincubated with tumor necrosis factor alpha, lipopolysaccharide (LPS), formyl-methionyl-leucyl-phenylalanine and the tetra peptide arginyl-glycyl-aspartate-serine. After preincubation, the cells were activated with gammaglobulin opsonized-bacteria, i.e., a Fc gamma R-stimulation. The priming effect was assessed as the production of oxygen radicals and as the degranulation or primary granules.RESULTS:Showed that the patients had a slightly lower responsiveness to priming than had the controls. This difference in priming response was most pronounced when measured as degranulation of primary granules after preincubation with LPS and 20 min of activation.CONCLUSIONS:This study shows no difference in response to priming, with optimal concentrations of inflammatory mediators, between peripheral neutrophils from patients with adult periodontitis and healthy controls.

The release of free oxygen radicals and degranulation was studied in neutrophils from 14 patients with adult periodontitis and 14 age- and sex-matched healthy controls. The neutrophils were activated by Fc gamma-receptor stimulation, using Staphylococcus aureus opsonized with gamma globulin. Release of oxygen radicals was measured as luminol-enhanced chemiluminescence. Degranulation was assessed as release of elastase, measured with a specific substrate and as release of lactoferrin, measured with ELISA. The neutrophils from the patients showed a significantly higher chemiluminescence and a slightly higher release of elastase, whereas the release of lactoferrin was the same in both groups.; In contrast, the ratio between the 2 degranulation products, elastase and lactoferrin, was significantly higher in the group with periodontitis. A flow cytometric analysis of the membrane expression of the adhesion molecules CD 11a, CD 11b, CD 15, CD 16, CD 35 and Mel 14 showed no differences in the median immunofluorescence between the 2 groups. This study showed a more than 2-fold higher release of free oxygen radicals from Fc gamma-receptor stimulated neutrophils compared with healthy controls, which indicates a specific neutrophil-associated host response in adult periodontitis.

Hyperactive or primed neutrophils which damage tissue via cytokines and membrane receptors may be implicated in the pathogenesis of inflammatory conditions. The purpose of this study was to elucidate the priming mechanism in neutrophils by assessing changes in membrane receptors and the Fc gamma R-mediated respiratory burst, measured as chemiluminescence. Purified neutrophilic granulocytes from healthy volunteers were preincubated with recombinant human tumor necrosis factor-alpha. This had a priming effect, increasing both the Fc gamma receptor-mediated luminol-enhanced chemiluminescence and the membrane expression of the C3bi receptor (CRB) (r = 0.843). The membrane densities of Fc gamma RII, Fc gamma RIII, and CR1 were unaffected by tumor necrosis factor-alpha. The mechanism of increased chemiluminescence may involve redistribution of the Fc gamma receptors and cooperation with upregulated CR3, facilitating crosslinking of the receptors. The experiments were performed in a buffer without divalent cations, since these increased the background activity and abolished the priming effect of tumor necrosis factor-alpha. In conclusion, a simultaneous increase in the Fc gamma R-mediated respiratory burst and CR3 density after priming with tumor necrosis factor-alpha indicates a cooperation between Fc gamma R and CR3.

Periodontitis is characterized by asymptomatic periodic collagen degradation, which is accompanied by the formation of granulation tissue induced by bacteria. The lesions sometimes contain micro-organisms and/or micro-abscesses that are of unknown significance. The aim of this study was to determine whether bacteria in a sterile granulation tissue could enhance its collagenolytic capacity. The formation of granulation tissue was induced by implanting a cellulose sponge in the subcutaneous tissue in the back of the rat. Bacteria were injected every other day into the sponge from day 8 to day 18. The cell-dependent degradation of a homologous 3H-collagen powder enveloped in the sponge was measured by the radioactivity of the urine excreted 8-18 days after the implantation. The injections increased the excretion of radioactivity by about 40% compared with the controls (n = 8, p < or = 0.005), but caused no clinical signs of acute infection or inflammation. On day 18, 2 days after the last injection of bacteria, no bacteria or increased cell infiltration were observed in the granulation tissue. The appearance of the latter could not be distinguished from that of the control tissues injected with buffer alone. It seems reasonable to assume that the increased collagen degradation results from enhanced activity of phagocytes, which may also be related to an increased release of tissue-destructive proteases and free oxygen radicals into the extracellular space. In conclusion, brief recurrent episodes of bacteria in granulation tissue can increase its collagen degrading-capacity. The latter may be due to augmented cell activity in the tissue. This response seems to have some features comparable to the pathogenesis of episodic periodontitis, e.g., by mimicking the collagen degradation.

To study the local inflammatory reaction associated with gingivitis and periodontitis, the protein concentrations were measured in gingival crevicular fluid (GCF) from 3 types of site: (i) inflamed sites in patients with gingivitis alone; (ii) inflamed sites with or (iii) without tissue destruction in patients with periodontitis. The GCF was sampled with paper strips and the protein concentration was measured with the Bradford protein assay. Higher protein-concentrations in GCF were found at inflamed sites in patients with gingivitis alone than at clinically similar sites without tissue destruction in patients with periodontitis. In contrast, the sites with various degrees of disease in the same periodontitis patient showed the same protein concentrations. These discrepancies indicate that the local inflammatory reaction in the gingiva is host- and not site-specific, i.e., the tissue destruction in periodontitis reflects a specific host response.

Granulocyte elastase activity and alpha-2-macroglobulin (alpha-2-MG) were studied in the gingival crevicular fluid (GCF) from 3 categories of sites in 6 patients with gingivitis and 6 patients with periodontitis. 6 inflamed sites in each gingivitis patient were sampled on paper strips and 12 sites, 6 with and 6 without attachment loss and periodontal pockets, were selected in each periodontitis patient. To avoid the influence of increase GCF volume from deep pockets, the elastase activity and the alpha-2-MG were calculated per microliters of GCF. The proteolytic activity of elastase was measured with a low molecular weight substrate and the antiprotease, alpha-2-MG, with ELISA. The measured activity could be ascribed to elastase that had been released into the gingival tissues and into the GCF prior to sampling. In the periodontitis patients, the sites with tissue destruction had a significantly higher elastase activity per site and per microliters GCF and a significantly lower alpha-2-MG per microliters than the 2 other categories of sites without tissue destruction. The destructive inflammation seems to be associated with increased release of elastase, either from more numerous or from more active granulocytes and with an increased proteolytic consumption of the inhibitor accompanied by the fast elimination of the protease-inhibitor-complex. In conclusion, the study shows a strong relationship between elastase activity and tissue destruction, a finding that supports the pathogenic theory of an involvement of granulocytes and their proteolytic enzymes in the mechanism of periodontal destruction.