Role of Fas/FasL Pathway in Pediatric Idiopathic Neutropenia.

Idiopathic neutropenia in children is marked by low neutrophil counts (<1500/ul) circulating in the peripheral blood in patients whose disease spontaneously develops unrelated to drugs, cancers, specific antibodies, or known genetic deficiencies. Neutrophils, PBMCs and plasma were purified/obtained from 24 subjects (n=17 acute and n=7 chronic) with idiopathic neutropenia, aged 3 months to 11 yrs. After screening a number of cytokines (IL-2, IL-4 IL-6, IL-8, IL-10, IFN-γ, TNF-α), growth factors (GCSF), cell death factors (Fas, FasL, Granzymes/Perforin), and chemokines (CCL5, XCL1, XCR1), QT-PCR studies of neutropenic subjects’ neutrophils indicated an up-to-14-fold increase in Fas transcripts compared to age-matched healthy control neutrophils. FACS analysis on patient neutrophils demonstrated increased expression of Fas (93%+/− 15%) compared to healthy control neutrophils (41%+/−11%). No increase in Fas surface expression was seen on PBMCs or on CD4+T cells from neutropenia patients compared to healthy controls. Studies of patient plasma showed increased FasL in acute and chronic patients (up to 40-fold higher). Increased IL-6 and IL-10 levels in plasma were another distinctive characteristic of neutropenic patients. Healthy control neutrophils incubated in neutropenic patient plasma for 4 hrs showed greater rates of apoptosis (evaluated by PI/Annexin; up to 38-fold higher) compared to incubation with healthy control plasma. Heat denaturation, IgG exclusion, and size separation studies suggest that the killing factor(s) is a heat-sensitive protein which is not IgG and has a MW between 35 and 100 kD. Blocking with anti-FasL antibodies incubated with patient plasma caused a statistically significant 5-fold to 11-fold decrease in neutrophil apoptosis, approaching the rates of healthy controls, implicating FasL as a major mediator of neutrophil regulation. Thus, the Fas/FasL pathway may play an important role in idiopathic neutropenia.