Activation of the neutrophil respiratory burst by plasma from periodontitis patients is mediated by pro-inflammatory cytokines

Aim: To determine the effect of periodontitis patients' plasma on the neutrophil oxidative burst and the role of albumin, immunoglobulins (Igs) and cytokines. Materials and Methods: Plasma was collected from chronic periodontitis patients (n=11) and periodontally healthy controls (n=11) and used with/without depletion of albumin and Ig or antibody neutralization of IL-8, GM-CSF or IFN-a to prime/stimulate peripheral blood neutrophils, isolated from healthy volunteers. The respiratory burst was measured by lucigenin-dependent chemiluminescence. Plasma cytokine levels were determined by ELISA. Results: Plasmas from patients were significantly more effective in both directly stimulating neutrophil superoxide production and priming for subsequent formyl-met-leu-phe (fMLP)-stimulated superoxide production than plasmas from healthy controls (p<0.05). This difference was maintained after depletion of albumin and Ig. Plasma from patients contained higher mean levels of IL-8, GM-CSF and IFN-a. Individual neutralizing antibodies against IL-8, GM-CSF or IFN-a inhibited the direct stimulatory effect of patients' plasma, whereas the ability to prime for fMLP-stimulated superoxide production was only inhibited by neutralization of IFN-a. The stimulating and priming effects of control plasma were unaffected by antibody neutralization. Conclusions: This study demonstrates that plasma cytokines may have a role in inducing the hyperactive (IL-8, GM-CSF, IFN-a) and hyper-reactive (IFN-a) neutrophil phenotype seen in periodontitis patients.

The central role of the neutrophil in ischaemic/reperfusion injury

Inadequate blood flow to an organ, ischaemia, may lead to both local and remote tissue injury characterized by oedema, increased microvascular permeability to protein and degradation of connective tissue components. This damage is probably caused by the accumulation and inappropriate activation of neutrophils which occurs when the tissue is reperfused. To test this hypothesis a number of in vitro models of the sequential stages of ischaemia/reperfusion injury were examined. Methods were initially developed to examine the adhesion of neutrophils to monolayers of a cultured endothelial cell line (ECV304) after periods of hypoxia and reoxygenation. Neutrophil migration in response to factors secreted by the treated endothelial cells was then assessed. The genesis of an inappropriate oxidative burst by the neutrophil upon exposure to endothelial chemoattractants and adhesion molecules was also measured. Finally to appraise how tissue function might be affected by endothelial cell hypoxia the contractility of vascular smooth muscle was examined. Neutrophil adhesion to ECV304 cells, which had been hypoxic for 4 hours and then reoxygenated for 30 minutes, was significantly increased. This response was probably initiated by reactive oxygen species (ROS) generated by the endothelial cells. Blockage of their production by allopurinol reduced the heightened adhesion. Similarly removal of ROS by superoxide dismutase or catalase also attenuated adhesion. ROS generation in turn caused the release of a soluble factor (s) which induced a conformational change on the neutrophil surface allowing it to bind to the intercellular adhesion molecule 1 (ICAM-1) on the endothelial cell. Soluble factor (s) from hypoxia/reoxygenated endothelial cells also had a powerful neutrophil chemoattractant ability. When neutrophils were exposed to both hypoxic/reoxygenated endothelial cells and the soluble factor (s) released by them a large oxidative burst was elicited. This response was greatest immediately after reoxygenation and one hour later was diminishing suggesting at least one of the components involved was labile. Analysis of the supernatant from hypoxic/reoxygenated endothelial cell cultures and studies using inhibitors of secretion suggested platelet activating factor (PAF) may be a major component in this overall sequence of events. Lesser roles for IL-8, TNF and LTB4 were also suggested. The secretory products from hypoxia/reoxygenated endothelial cells also affected smooth muscle contractility having an anti-vasoconstrictor or relaxation property, similar to that exerted by PAF.

Macrophages and lymphocyte responiveness to mitogen

Purified B-cells fail to proliferate in response to the strong thymus-independent (TI) antigen Lipopolysaccharide (LPS) in the absence of macrophages (Corbel and Melchers, 1983). The fact that macrophages, or factors derived from them are required is supported by the inability of marginal zone B-cells in infants to respond to highly virulent strains of bacteria such as Neisseria meningitidis and Streptococcus pneumoniae (Timens, 1989). This may be due to the lack of CD21 expression on B-cells in infants which could associate with its co-receptor (C3d) on adjacent macrophages. It is not clear whether cell surface contacts and/or soluble products are involved in lymphocyte-macrophage interactions in response to certain antigens. This thesis describes the importance of the macrophage in lymphocyte responses to T-dependent (TD) and TI antigens. The major findings of this thesis were as follows: (1). Macrophages were essential for a full proliferative response to a range of T - and B-cell mitogens and TI-1 and TI-2 antigens, including Concanavalin A, LPS, Pokeweed mitogen (PWM), Dextran sulphate, Phytohaemagglutinin-P (PHA-P) and Poly[I][C]. (2). A ratio of 1 macrophage to 1000 lymphocytes was sufficient for the mitogens to exert their effects. (3). The optimal conditions were established for the activation of an oxidative burst in cells of the monocyte/macrophage lineage as measured by luminometry. The order of ability was OpZ >PMA/lonomycin >f-MLP >Con A >DS >PHA >Poly[I][C] >LPS >PWM. Responses were only substantial and protracted with OpZ and PMA. Peritoneal macrophages were the most responsive cells, whereas splenic and alveolar macrophages were significantly less active and no response could be elicited with Kupffer cells, thus demonstrating heterogeneity between macrophages. (4). Activated macrophages that were then fixed with paraformaldehyde were unable to restore mitogenic responsiveness, even with a ratio of 1 macrophage to 5 lymphocytes. (5). Although highly purified T- and B-cells could respond to mitogen provided live macrophages were present, maximum activation was only observed when all 3 cell types were present. (6). Supernatants from purified macrophage cultures treated with a range of activators were able to partially restore lymphocyte responses to mitogen in macrophage-depleted splenocyte cultures, and purified T - and B-cell cultures. In fact supernatants from macrophages treated with LPS for only 30 minutes could restore responsiveness. Supernatants from OpZ treated macrophages were without effect. (7). Macrophage supernatants could not induce proliferation in the absence of mitogen. They therefore provide a co-mitogenic signal required by lymphocytes in order to respond to mitogen. (8). Macrophage product profiles revealed that LPS and Con A-treated macrophage supernatants showed elevated levels of IL-1β, TNF -α L TB4 and TXB2. These products were therefore good candidates as the co-mitogenic factor. The possible inhibitory factors secreted by OpZ-treated macrophages were PGE2, IL-10 and NO. (9). The removal of cytokines, eicosanoids and TNF-α from LPS-treated macrophage supernatants using Cycloheximide, Dexamethasone and an MMPI respectively, resulted in the inability of these supernatants to restore macrophage-depleted lymphocyte responses to mitogen. (10). rIL-1β and rTNF-α are co-mitogenic factors, as macrophage-depleted lymphocytes incubated with rIL-1β and rTNF-α can respond to mitogen.

Oxidative modification of a specific apolipoprotein B lysine residue confers altered receptor specificity on LDL

During inflammation, many cell types release reactive oxygen species (ROS) via the respiratory burst. These ROS are potent oxidants of LDL and its major protein, apolipoprotein B. Whilst native LDL is taken up by endothelial cells via a feedback controlled receptor-regulated process, oxidative modification of LDL renders it a ligand for many scavenger receptors. Scavenger receptors include CD-36, LOX-1 and the prototypic macrophage SR A I/II, all of which are variably expressed. Uncontrolled uptake of oxidised LDL is implicated in the pathogenesis of atherosclerosis. In addition, oxidised LDL increases CCR2 protein and mRNA expression on monocytes, and thus may contribute to monocyte retention and perpetuation in inflammatory, unstable atherosclerotic lesions. However, little data are available on the effects of specific minor modifications to apolipoprotein B. In order to identify the sequence specificity and nature of oxidative modifications which confer altered properties on LDL, we have investigated the effects of modified peptides (which correspond to the putative LDLR binding domain) on LDL uptake by HUVECs and U937 monocytes.

Mechanisms contributing to the enhanced respiratory burst of neutrophils observed in periodontitis

The aim of this thesis is to investigate possible mechanisms that may contribute to neutrophil hyperactivity and hyper-reactivity. One possibility is the presence of a neutrophil priming factors within the peripheral circulation of periodontitis patients. To examine this possibility differentiated HL-60 cells and primary neutrophils were studied in the presence and absence of plasma from periodontitis patients. In independent experiments, plasma was depleted of IL-8, GM-CSF, interferon-a, immunoglobulins and albumin. This work demonstrated that plasma factors such as IL-8, GM-CSF, and interferon-a present during periodontitis may contribute towards the reported hyperactive neutrophil phenotype. Furthermore, this work demonstrated that products from Pg may regulate neutrophil accumulation at infected periodontal sites by promoting gingipain-dependent modification of IL-8-77 into a more biologically active chemokine. To elucidate whether the oxidatively stressed environment that neutrophils are exposed to in periodontitis could influence hyperactivity and hyper-reactivity, neutrophils were depleted of glutathione. This work showed that during oxidative stress, where cellular redox-levels have been altered, neutrophils exhibit an increased respiratory burst. In conclusion, this work highlights the multiple mechanisms that may contribute to neutrophil hyperactivity and hyperreactivity including gingipain-modulated activity of IL-8 variants, the effect of host factors such as IL-8, GM-CSF, interferon-a on neutrophils priming and activation, and the shift of neutrophil GSH:GSSG ratio in favour of a more oxidised environment as observed in periodontitis.