Increased Expression of the Signaling Lymphocyte-Activation Molecule (mSLAM) in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH).

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell disorder characterized by intravascular hemolysis, thrombosis, and bone marrow failure; in PNH, mutations in the PIG-A gene prevent synthesis of glycosylphosphoinositol (GPI) anchors and therefore cell membrane expression of GPI-anchored proteins. PNH cell clonal expansion occurs in the setting of immune-mediated hematopoietic cell destruction. The mechanism responsible for PNH clonal expansion is uncertain, but there is experimental evidence for relative resistance of GPI-anchored protein negative cells to NK-cell mediated cytotoxicity. Signaling lymphocyte activation molecule (SLAM; CD150; SLAMF1) is a glycosylated type-I transmembrane protein of relative molecular mass 70 Kd; the protein has a cytoplasmic domain bearing a paired immunoreceptor tyrosine-based switch motif. SLAM is constitutively expressed on the surface of activated T-, B- lymphocytes, dendritic cells and macrophages with a high-affinity self ligand. SLAM functions include co-stimulation of T and B cells, augmentation of T-cell cytotoxicity, and regulation of proliferation and differentiation of B cells. The recruitment of SLAM or SLAM-associated proteins is important in modulating innate and adaptive immune responses. Engagement of CD244 (2B4), another SLAM family member, by CD48--a GPI-anchored protein--results in CD244 activation, leading to NK cell stimulation and NK cell mediated cytotoxicity. We examined SLAM and CD244 expression in peripheral blood mononuclear cells from patients with PNH and healthy controls to establish their involvement in PNH. We first analyzed SLAM expression by flow cytometry on CD56+ and CD3+ cells from 11 PNH patients (5 females, 6 males) and 10 healthy controls. In CD56+ cells membrane SLAM (mSLAM) was up-regulated in 10 of 11 patients compared to controls (p=0.04). mSLAM was more modestly elevated in CD3+ cells from patients (p=0.04). Expression of CD244 also showed increased expression in PNH patients’ CD56+ cells (p=0.0008) and in CD3+ (p= 0.05). mRNA levels, measured by RT-PCR, of mSLAM in patients (n=4) and controls (n=4) were consistent with flow cytometric data, PNH patients having increased mSLAM mRNA levels (p=0.18). In a single PNH patient who showed up-regulation by flow and RT-PCR we also detected increased mSLAM protein levels by immunoblot. RT-PCR was also employed to quantitate mRNA levels of soluble or cytoplasmic SLAM (sSLAM): sSLAM expression was decreased in patients (n=4) compared to controls (n=3) (p< 0.0001). All differences between groups were evaluated using the F test to compare variances. In summary, NK cells from PNH patients have increased levels of mSLAM and 2B4; up-regulation suggests involvement of SLAM family members in immunologically mediated selection of PNH clones in immune-mediated marrow failure syndromes and is consistent with a role for NK cells in PNH clonal expansion.