Identification of Specific Chemokines and Apoptosis Molecules in Pediatric Idiopathic Neutropenia.

In order to explore possible mechanisms for idiopathic neutropenia, molecular and functional analyses on neutrophils and plasma of pediatric neutropenic patients were performed. 22 subjects, 8 acute and 14 chronic neutropenia, aged 5 months to 12 years, were recruited through Lucile Salter Packard Children’s Hospital hematology clinics with an IRB-approved consent. Patients with known neutrophil elastase mutations were excluded. We first examined whether the neutrophils of neutropenia patients had increased expression of molecules associated with cell death. QT-PCR was performed and transcript levels in neutrophils of neutropenia subjects were compared against those of normal subjects. In addition, to assess if a factor(s) in the plasma of neutropenia patients induced neutrophil cell death, neutrophil survival assays were done by incubating neutrophils isolated from normal healthy controls with 200 ul of undiluted plasma from chronic neutropenic subjects. Neutrophil death was then analyzed via Annexin V-PI staining. Finally, to identify specific plasma proteins, which may have a potential role in neutrophil-mediated cell death, 2D gels were run on neutropenic and control plasma.

Results: Due to limited sample size, assays were not run on all subjects. Acute and chronic neutropenic patients (n=10) experienced an increase in FAS, an apoptotic marker and protein known to be associated with neutrophil cell death. There were no significant differences in fold expression between acute and chronic patients. Neutrophil survival assays (n=9) revealed a median of 6.5 and mean 6.8 fold (range 2–24 fold) higher level of plasma-mediated cell death with neutropenic plasma. Preliminary studies of 2D gels on plasma revealed differential expression of inflammatory proteins in neutropenia patients compared to normal healthy controls. Further investigation on these proteins is ongoing. In summary, rapid cell death can be induced in normal neutrophils through neutropenia plasma incubation. Further identification of these apoptosis-associated plasma proteins is in process. In addition, the increase in certain apoptosis-associated chemokines and cytokines could lead to further target identification for diagnostic and/or treatment purposes of neutropenia.