Pediatric idiopathic thrombocytopenic purpura (ITP) is a bleeding disorder characterized by immune-mediated platelet destruction. Current evidence suggests that childhood ITP is associated with an imbalance within the T cell regulatory network following an external immune trigger resulting in an excessive or sustained proinflammatory T helper cytokine response. In order to understand why certain children are at risk for sustained thrombocytopenia or chronic ITP, we investigated cytokine responses to peptides from a common platelet membrane autoantigen, glycoprotein (GP) Ib, and the relationship to the genotype frequencies of single nucleotide polymorphisms (SNPs) from genes associated with immune regulation. Methods: PBMC and DNA were collected from 114 children with ITP, (80 acute, 34 chronic) and 25 controls. PBMC were stimulated with Phytohemaglutinin-P (PHA) or GP Ib peptides and the resulting Interferon gamma (IFNγ), Interleukin 1 beta (IL-1β), and Interleukin 4 (IL-4) cytokine production was measured by ELISA. DNA was isolated and genotypes determined for inflammatory cytokine IL-1α (−889), Fcγ receptor FcγRIIIA (F158V), and Complement C1qc (exon 1, codon 42). Results: Previously, we have shown a large shift in the allele frequencies of the FcγRIIIA receptor SNP (F158V) in ITP patients (F=.36, V=.64 vs F=.70, V=.30; p<0.001) and IL-1α −889 SNP (C=.67, T=.33 vs C=.84, T=.16; p=0.005) compared to controls. Data from the current analysis is shown in table below. ITP patients produce significantly less IL-4 (54%; p=0.0001) and IL-1β (80%; p=0.0041) than controls when stimulated with the mitogen PHA. Conversely, when stimulated with GP Ib peptides, ITP patients produce more IFNγ (252%; p=0.0272) than controls. In particular, ITP patients with genotype FcγRIIIA V/V, or IL-1α C/C, or C1qc C/T produce more IFNγ than controls when stimulated with autoantigenic peptides. In addition to increased IFNγ production, only ITP patients with FcγRIIIA V/V genotype produce significantly more IL-1β and IL-4 than controls. These same patients were shown to have 100% response rate to IVIG in a previous report.

Differential Cytokine Production from ITP Patients’ PBMC Compared to Controls

Cytokine ComparisonPHAGP Ib peptidesFc γRIIIA V/VIL-1α C/C1qc C/T
P values in ( ), from nonparametric ANOVA, P<0.05 considered significant 
IFNγ 90% (.4977) 252% (.0272) 775% (.0324) 584% (.0002) 319% (.0079) 
IL-4 54% (.0001) 92% (.3674) 908% (.0048) 118% (.2451) 112% (.6266) 
IL-1β 80% (.0041) 74% (.7114) 272% (.0168) 97% (.0134) 52% (.0163) 
Cytokine ComparisonPHAGP Ib peptidesFc γRIIIA V/VIL-1α C/C1qc C/T
P values in ( ), from nonparametric ANOVA, P<0.05 considered significant 
IFNγ 90% (.4977) 252% (.0272) 775% (.0324) 584% (.0002) 319% (.0079) 
IL-4 54% (.0001) 92% (.3674) 908% (.0048) 118% (.2451) 112% (.6266) 
IL-1β 80% (.0041) 74% (.7114) 272% (.0168) 97% (.0134) 52% (.0163) 

Conclusions: Our results show that:

  1. ITP patients’ T cells have an abnormal response to non- specific immune stimulation (PHA) resulting in a lack of production of IL-4, which normally aids in extinguishing an autoimmune response, and

  2. these same T cells are hyper responsive to the platelet autoantigen GP Ib peptides, based on increased levels of the proinflammatory cytokine, IFNγ.

This polarization of ITP patients towards a proinflammatory immune response may result in sustained stimulation of antigen primed B cells leading to platelet destruction. In addition, to fully understand the etiology of childhood ITP, robust statistical analysis of cytokine abnormalities should be performed in the context of immune receptor polymorphisms, as these genetic differences appear to modulate the impact of the proinflammatory trigger in these patients.

Author notes

Disclosure: No relevant conflicts of interest to declare.

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