Evidence for a mechanosensitive calcium influx into red cells.

Red cells exposed to glycophorin A reactive antibodies and lectins develop a non-specific cation permeability. To determine if this might be due to the activation of a non-selective mechanosensitive channel we have subjected red cells, loaded with a calcium responsive fluorescent probe, to filtration through 5 and 3 microm pores. Calcium entered 28% of normal red cells at the moment of deformation when 3 microm filtered, a finding consistent with the transient activation of a mechanosensitive channel. Red cells containing hemoglobin AC and AS had enhanced calcium responses to filtration. An increased influx of calcium in hemoglobin disorders might play a role in providing protection against Falciparum malaria.

Clinical manifestations associated with the aberrant expression of the soluble granulocyte-macrophage colony-stimulating factor receptor in patients presenting with haematological malignancies.

The receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) can exist as both transmembrane (tmGMRalpha) and soluble (solGMRalpha) isoforms, and the latter, is a normal constituent of human plasma. We investigated if aberrant solGMRalpha expression occurs in haematopoietic malignancies and whether or not solGMRalpha expression levels correlated with clinical presentation. Compared with the normal population, patients with acute lymphoblastic leukaemia (ALL) had low levels of solGMRalpha whereas clonal disorders of the myeloid lineage demonstrated higher levels of solGMRalpha. Patients with acute myelogenous leukaemia (AML) and high levels of solGMRalpha presented with a distinct clinical picture. These patients were older, predominantly belonged to the M4 and M5 French-American-British (FAB) subtypes, and they had higher white blood cell counts at presentation including myeloid precursors and myeloblasts. They often presented with either unexplained lung infiltrates or hypoxia and lower rates of microbiologically defined infections. Elevated solGMRalpha levels were not associated with decreased relapse-free and overall survival in the AML population. On multivariate analysis, the correlation between elevated solGMRalpha levels and age, M4 and M5 FAB subtypes and decreased numbers of infections persisted. Our study is the first to describe that distinct clinical presentations are associated with aberrant solGMRalpha levels in haematological malignancies.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and inflammatory stimuli up-regulate secretion of the soluble GM-CSF receptor in human monocytes: evidence for ectodomain shedding of the cell surface GM-CSF receptor alpha subunit.

Soluble GM-CSF receptor alpha subunit (sGMRalpha) is a soluble isoform of the GMRalpha that is believed to arise exclusively through alternative splicing of the GMRalpha gene product. The sGMRalpha mRNA is expressed in a variety of tissues, but it is not clear which cells are capable of secreting the protein. We show here that normal human monocytes, but not lymphocytes, constitutively secrete sGMRalpha. Stimulation of monocytes with GM-CSF, LPS, PMA, or A23187 rapidly up-regulates the secretion of sGMRalpha in a dose-dependent manner, demonstrating that secretion is also regulated. To determine whether sGMRalpha arose exclusively through alternative splicing of the GMRalpha gene product, or whether it could also be generated through ectodomain shedding of GMRalpha, we engineered a murine pro-B cell line (Ba/F3) to express exclusively the cDNA for cell surface GMRalpha (Ba/F3.GMRalpha). The Ba/F3.GMRalpha cell line, but not the parental Ba/F3 cell line, constitutively shed a sGMRalpha-like protein that bound specifically to GM-CSF, was equivalent in size to recombinant alternatively spliced sGMRalpha (60 kDa), and was recognized specifically by a mAb raised against the ectodomain of GMRalpha. Furthermore, a broad-spectrum metalloprotease inhibitor (BB94) reduced constitutive and PMA-, A23187-, and LPS-induced secretion of sGMRalpha by monocytes, suggesting that shedding of GMRalpha by monocytes may be mediated in part through the activity of metalloproteases. Taken together, these observations demonstrate that sGMRalpha is constitutively secreted by monocytes, that GM-CSF and inflammatory mediators up-regulate sGMRalpha secretion, and that sGMRalpha arises not only through alternative splicing but also through ectodomain shedding of cell surface GMRalpha.

Glycophorin A-mediated haemolysis of normal human erythrocytes: evidence for antigen aggregation in the pathogenesis of immune haemolysis.

The inexplicable severity of anti-Pr autoimmune haemolytic anaemia led us to test the hypothesis that the haemolysis was primarily due to a change in the function of glycophorin A, on which the Pr antigen is located. The lectins Maclura pomifera and wheat germ agglutinin that bind to glycophorin A induced the haemolysis of normal erythrocytes in vitro. Lectin binding led to an increase in erythrocyte membrane permeability to sodium and potassium, the former resulting in an influx of water and subsequent haemolysis. The response was glycophorin A specific as Concanavalin A, which binds to band 3, did not cause haemolysis and peanut agglutinin only did so after removal of erythrocyte sialic acid. The lectin-induced cation leak was not mediated by activation of cation channels as the inhibitors, tetrodotoxin, amiloride and 4,4' disothiocyanate stilbene 2,2'disulphonate, had no effect, suggesting that the haemolysis was due to exacerbation of the inherent cation permeability of the erythrocyte membrane. A human IgAK anti-Pr autoantibody and a mouse anti-human glycophorin A antibody increased erythrocyte permeability to sodium. The role of glycophorin A in stabilizing and, upon aggregation, destabilizing the phospholipid bilayer is discussed. Our findings may help explain the severity of anti-Pr autoimmune haemolytic anaemia and other pathophysiological changes in human erythrocytes.