IRp60 is expressed and functional on human cord blood derived mast cells

Abstract

Rationale Allergic inflammatory responses are likely to be regulated by the cross-talk between activatory and inhibitory signals. Mast cells, via activation carried out by IgE-dependent mechanisms, and later on by interacting with eosinophils, are the initiators and perpetuators of these processes. IRp60 is a 60-kDa glycoprotein expressed on T cell subsets and monocytes. Upon sodium pervanadate treatment IRp60 becomes tyrosine-phosphorylated, associating with SHP-1 and SHP-2. We examined the expression and function of IRp60 on human cord blood-derived mast cells (CBMC). Methods Mature CBMC were obtained from cord blood mononuclear cells cultured with Stem Cell Factor, IL-6 and PGE2 for 9-12 weeks. Expression of IRp60 was assessed by FACS and Western blot. CBMC were incubated with human eosinophil major basic protein (MBP) (10-100nM) or poly-L-arginine (25-100nM) for 12-24 hrs and IRp60 expression was assessed. For activation, CBMC were cultured for 5 days (96-well plate) with chimeric IgE (2μg/ml). Cells were washed and incubated with goat anti-mouse anti-IgE (30min, 37°, 5% CO2) in 96-well plate pre-coated with the cross-linker sheep anti-mouse (25μg/ml) and mouse anti-human IRp60. β-hexosaminidase release was determined by enzymatic-colorimetric assay; IL-4 by ELISA. Results CBMC expressed IRp60 (90 ± 6.5%). MBP down-regulated IRp60 expression on CBMC (DMFI=12). Viability of CBMC with MBP was >98% at 24 hours. Cross-linking of IRp60 together with FcϵRI inhibited b-hexosaminidase release (85.50 ± 4.40% IgE-activated release vs. 52.46 ± 5.51% IgE+Irp60-activated, p<0.001) and IL-4 release (8.41 ± 1.19 pg/ml IgE-activated vs. 0.32 ± 0.09 pg/ml IgE+IRp60-activated, p<0.01). Conclusions The demonstration that CBMC express a functional IRp60 receptor suggests a novel pathway for the regulation of IgE-mediated responses.