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