P59
ANCA STIMULATED NEUTROPHILS RELEASE BLYS AND PROMOTE B
CELL SURVIVAL IN VITRO
Holden, N¹, Harper, L¹, Williams, J², ¹Savage, C
¹Renal Immunobiology, School of Immunity and Infection, College of Medical
and Dental Sciences, University of Birmingham, ²Wellcome Trust Clinical
Research Facility, University Hospital Birmingham
The presence of B cell infiltrates within the kidney has been reported in a number of
inflammatory renal diseases, including the ANCA-associated vasculitides. Infiltrating
B cells are likely to promote continued inflammation within the kidney and may
contribute to niches for auto-antibody production. As B cells must receive survival
and activation signals from their microenvironment, we sought to determine a role for
ANCA-activated neutrophils in promoting B cell survival, through the release of B
cell survival factor, BLyS (B Lymphocyte Simulator).
Neutrophils isolated from healthy donors were assessed for BLyS expression
by flow cytometry and release by capture ELISA. Non-primed unstimulated
neutrophils expressed BLyS on their surface which was maintained in culture for over
2 hours. Neutrophils cultured in the presence of TNF-alpha (10ng/ml) or fMLP (1µM)
exhibited increased surface expression, within 30 minutes which reduced down to
basal levels following 2 hours culture. Cells treated with priming concentrations of
TNF-alpha (2ng/ml) also showed a similar tendency to change their expression of
surface BLyS, yet this was significantly enhanced when primed neutrophils were
treated with ANCA but not control IgG (200mg/ml). BLys was detected in cell
supernatants supporting the notion that BLyS can be shed from the surface of the
neutrophils following an inflammatory stimulus.
To determine whether ANCA-activated neutrophils support B cell survival in
vitro, supernatants collected from ANCA-treated neutrophils (3 hours) were added to
the ‘germinal centre’ like B cell lymphoma cell line L3055 (10%): L3055 cells were
cultured for 48 hours with low serum (2.5%) to induce apoptosis and cell death was
determined by measuring both active caspase-3 expression and annexin IV/propidium
iodide staining using flow cytometry. Neutrophil supernatants from ANCA IgG, but
not normal IgG, increased B cell survival (>20%), equivalent to a BLyS positive
control (100ng/ml) used in the same culture conditions. Neutrophil supernatants
added to L3055 cell culture were also able to increase cell proliferation over 72 hours.
Using CFSE to label actively dividing B cells, neutrophil supernatants from ANCA
treated cells induced an increase in cell proliferation (reduction of half the geometric
mean) above that of medium alone.
We have shown that ANCA specifically causes the release of BLyS from
activated neutrophils and that supernatants from activated neutrophils can support B
cell survival in vitro. ANCA induced release of neutrophil-derived BLyS may
promote B cell survival and prolonged production of auto-antibodies within the
inflamed kidney, thereby highlighting BLyS as a potential therapeutic target to inhibit
ANCA induced inflammation. The work may be particularly relevant in light of
proposed uses of novel anti-BLyS monoclonal antibody therapies to treat a number of
autoimmune diseases.