Background Juvenile-onset systemic lupus erythematosus (JSLE) is a multisystem autoimmune disease characterised by autoantibodies directed against nuclear autoantigens. In the presence of JSLE sera, an imbalance of pro- and anti-apoptotic factors results in an increase in neutrophil apoptosis, a potential source of autoantigens. Type 1 and 2 interferons are increased in JSLE serum, however the specific effect of IFN alpha on neutrophil apoptosis has not been elucidated.
Objectives Investigate the effect of IFN alpha on neutrophil apoptosis.
Methods Neutrophils isolated from healthy control adult donors were incubated unstimulated, or IFN alpha (10ng) for ∼20hrs. GMCSF is known to delay neutrophil apoptosis, and was used as a positive control. The percentage of apoptotic neutrophils were measured using flow cytometry via annexin V FITC staining. Western blot analysis was used to measure caspase 3 activation. Cleaved caspase is the active protein and therefore caspase dependant apoptosis would result in increased caspase 3 expression from its inactive form (procaspase 3).
Results IFN alpha was observed to reduce neutrophil apoptosis. Flow cytometry (% annexin V positive cells, mean ± SEM) demonstrated 50%±4.9% apoptosis following IFNα exposure (n=5) compared to 74%±8% unstimulated (n=5, p=0.016). GMCSF was observed to delay neutrophil apoptosis (55%±4.4). Western blot analysis showed reduced caspase cleavage upon IFNα incubation. This was demonstrated by the observation the procaspase 3/caspase 3 ratio to be increased in the presence of IFNα (5.4±3.5, n=4) as compared to unstimulated neutrophils (2.4±1.8, n=4, p=0.149) and GMCSF stimulated (5.81±2.9, n=4).
Conclusions Modulation of neutrophil apoptosis by inflammatory cytokines may be associated with the outcome of inflammation. Understating the relationship between type 1 interferons and neutrophil apoptosis is important in systemic autoimmune diseases such as JSLE. We have shown that IFN alpha reduces neutrophil apoptosis which is either directly or indirectly due to reduced caspase 3 activation. The implications for this interaction on development and progression of inflammatory autoimmune diseases is currently being investigated paying particular focus on the both type 1 and type 2 interferons.
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Disclosure of Interest None declared