AB0073 Effects of Interferon Alpha on Neutrophil Apoptosis and Caspase 3 Cleavage
Authors: S. Irwin, A. Midgley, M.W. Beresford
Journal: Annals of the Rheumatic Diseases (2015)

<h3>Background</h3> 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. <h3>Objectives</h3> Investigate the effect of IFN alpha on neutrophil apoptosis. <h3>Methods</h3> 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). <h3>Results</h3> 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, <i>p</i>=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, <i>p</i>=0.149) and GMCSF stimulated (5.81±2.9, n=4). <h3>Conclusions</h3> 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. <h3>References</h3> Bengtsson, A.A., Sturfelt, G., Truedsson, L., Blomberg, J., Alm, G., Vallin, H., Rönnblom, L. (2000) Activation of type I interferon system in systemic lupus erythematosus correlates with disease activity but not with antiretroviral antibodies. Lupus. 9, 664-71. Bouts, Y.M., Wolthuis, D.F, Dirkx, M.F., Pieterse, E., Simons, E.M., van Boekel, A.M., Dieker, J.W., van der Vlag, J. (2012) Apoptosis and NET formation in the pathogenesis of SLE. Autoimmunity. 45, 597-601. Kim, T., Kanayama, Y., Negoro, N., Okamura, M., Takeda, T., Inoue, T. (1987) Serum levels of interferons in patients with systemic lupus erythematosus. Clin. Exp. Immunol. 70, 562-69. Midgley, A., McLaren, Z., Moots, R.J., Edwards, S.W., Beresford, M.W. (2009). The role of neutrophil apoptosis in juvenile-onset systemic lupus erythematosus. Arthritis Rheum. 60, 2390-2401 Sakamoto, E., Hato, F., Kato, T., Sakamoto, C., Akahori, M., Hino, M., Kitagawa, S. (2005). Type I and type II interferons delay human neutrophil apoptosis via activation of STAT3 and up-regulation of cellular inhibitor of apoptosis 2. J. Leukoc. Biol. 78, 301-9. <h3>Disclosure of Interest</h3> None declared