Role of Fas/FasL Pathway in Pediatric Idiopathic Neutropenia.
Authors: Joh Jae Won , Callejas Angel , Wong Wendy , Cohen Harvey Joel , Nadeau Kari , Jeng Michael 
Published: October 2019

Abstract
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.
Patient number Chronicity Age FasL (pg/ml) Anti-neutrophil Ab Fold increase in neutrophil death over control plasma Fold increase in apoptosis over control plasma 1 chronic 2 yrs 7±1 positive 6.5±0.7 8.5±0.8 2 chronic 18 mo. 6±1 positive 7.3±0.5 8.1±0.2 3 chronic 23 mo. 5±0.5 wk positive/neg 7.8±0.6 8.5±0.4 4 chronic 1 yr 5±2 positive 6.5±0.5 7.3±0.6 5 chronic 5 yrs 7±1 wk positive 6.1±0.9 2.7±0.2 6 chronic 20 mo. 10±1 negative 11.3±1.4 2.1±0.5 7 chronic 11 mo. 6±1 negative 6.1±0.8 5.7±0.6 8 acute 2 yrs 16±2 N/A 18.7±1.3 22.4±2.9 9 acute 2 yrs 18±3 negative 12.0±2.1 19.1±2.5 10 acute 5 yrs 4±1 negative 2.5±0.5 2.6±0.4 11 acute 11 mo. 3±0.6 N/A 1.5±0.3 2.1±0.2 12 acute 3 yrs 17±1 negative 15.3±1.2 19.2±0.8 13 acute 2 yrs 40±3 negative 22.9±1.4 38.4±2.4 14 acute 2 yrs 21±2 N/A 14.3±1.0 20.5±1.2 15 acute 8 mo. 10±0.9 N/A 9.6±0.8 12.0±1.6 16 acute 17 mo. 19±3 negative 21.3±2.2 26.7±3.5 17 acute 6 yrs 8±1 N/A 6.7±1.3 11.5±2.1 18 acute 3 yrs 9±2 N/A 7.2±2.6 8.8±1.5 19 acute 11 yrs 12±2 negative 14.8±0.9 17.4±1.9 20 acute 5 yrs 6±1 negative 3.4±0.6 5.2±0.7 21 acute 4 mo. 8±2 N/A 6.2±1.0 8.1±2.2 22 acute 3 mo. 27±3 N/A 21.4±3.5 39.4±2.4 23 acute 11 yrs 22±1 negative 17.2±3.0 24.3±2.8 24 acute 2 yrs 18±2 positive 15.8±1.2 25.0±1.7 Representative control N/A 4 yrs 1.3±0.4 N/A 1.2±0.3 0.8±0.4