Macrophage phagocytosis of apoptotic neutrophils is critically regulated by the opposing actions of pro-inflammatory and anti-inflammatory agents : key role for TNF-α

Development of chronic inflammation or autoimmunity may be related to deregulated mechanisms orchestrating successful resolution of inflammation, especially apoptosis of inflammatory cells and their subsequent clearance by macrophages (Mφ). Chronically inflamed sites are characterised by an excess of the key pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) and importantly, TNF-α inhibitors, widely used in the clinical setting for the treatment of rheumatoid arthritis (RA), inflammatory bowel disease and psoriasis, significantly delay disease progression. TNF-α therefore may affect processes implicated in resolution of inflammation. Although TNF-α and pro-inflammatory bacterial products such as lipopolysaccharide (LPS) influence rates of inflammatory cell apoptosis, little is known about their effects on Mφ phagocytosis of apoptotic cells (efferocytosis). In this PhD thesis, the effects of several pro-inflammatory agents (i.e., LPS, lipoteichoic acid (LTA), peptidoglycan (PGN) and TNF-α) on efferocytosis by human blood monocytederived Mφ (MDMφ) have been investigated. LPS, LTA and PGN all inhibited MDMφ efferocytosis in a concentration- and time-dependent manner; however, LPS did not inhibit the uptake of immunoglobulin-G (IgG)-opsonized erythrocytes. Moreover, although TNF-α did inhibit efferocytosis, phagocytosis of IgG-opsonized erythrocytes was not inhibited. Furthermore, the LPS effect was attenuated by dimeric soluble human recombinant TNF receptor-1 (sTNFR1/ Fc), indicating a critical role of TNF-α. Concomitant treatments with monomeric soluble human recombinant TNF receptor-1 (sTNF-R1) or the TNF-α Converting Enzyme (TACE) inhibitor, TOPI-0, only partially reversed the inhibitory effect of LPS. Even though TNF-α release takes place within the first few hours following LPS stimulation, the LPS-induced inhibitory effect occurred only if treatment was performed for 96 hours or longer. Analysis of supernatants obtained from LPS-treated MDMφ revealed that there appears to be interplay between concentrations of TNF-α and interleukin-10 (IL-10) and that these cytokines exert opposing actions on efferocytosis. IL-10 per se increased MDMφ efferocytosis and addition of exogenous IL-10 to LPS-treated samples rescued phagocytosis. The latter effect was associated with the IL-10-induced, concentration-dependent inhibition of TNF-α release. Interestingly, when IL-10 was added to TNF-α-treated MDMφ, only slight augmentation of phagocytosis was observed. Furthermore, when IL-10-mediated effects were blocked by concomitant treatment with anti-human IL-10 receptor 1 antibody (anti-IL-10- R1Ab), the LPS inhibitory effect on phagocytosis was much greater and occurred at 24 hours after treatment. The role of IL-10 on efferocytosis was also investigated using IL-10 deficient murine bone marrow-derived Mφ (BMDMφ). IL-10 deficient BMDMφ, when compared to wild-type, were characterised by a much lower ability to phagocytose apoptotic neutrophils and this effect was independent of culture conditions (control samples and LPS or TNF-α treatments). Finally, effects of the synthetic steroid (dexamethasone) and nonsteroidal anti-inflammatory drugs (NSAID) on MDMφ phagocytosis were examined. Dexamethasone, like IL-10, augmented MDMφ efferocytosis, reversed the inhibitory effects of both LPS and TNF-α, and suppressed LPS-induced production of TNF-α. In contrast NSAID did not increase MDMφ efferocytosis per se. However, preliminary data suggest that aspirin blocks the inhibitory effect of TNF-α on phagocytosis. In summary, it has been determined that prolonged treatment with proinflammatory agents such as LPS, LTA and PGN inhibits MDMφ efferocytosis which may potentially postpone the resolution of inflammation in vivo. I have shown that TNF-α is a key mediator in this process and that IL-10 exerts an important regulatory effect on TNF-α production and consequently on efferocytosis. Furthermore, several approaches have been unveiled to successfully reverse LPS-mediated inhibition of efferocytosis by decreasing either TNF-α production or its inhibitory effect with sTNF-RI/Fc, exogenous IL- 10 or dexamethasone. These findings indicate that TNF-α and other agents which influence efferocytosis may have significance in the resolution phase of inflammation. In addition, presented findings provide important mechanistic information into the potential mode of action of anti-TNF-α agents and steroids and may help to explain their clinical success in the treatment of chronic inflammatory diseases.EThOS - Electronic Theses Online ServiceGBUnited Kingdo