No abstract

Summary. 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′ diisothiocyanate 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.

The hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates its activity through binding to cell surface receptors. The receptor for GM-CSF belongs to a superfamily of cytokine receptors characterized by a conserved extracellular motif. The high affinity GM-CSF receptor (GMR) consists of two transmembrane anchored subunits; a ligand binding alpha subunit (transmembrane GMRalpha) and a signal transducing beta subunit (GMRbeta), both of which belong to the cytokine receptor superfamily. The human GM-CSF receptor alpha subunit also exists in a soluble form (solGMRalpha), which antagonizes GM-CSF activity in vitro. We directly tested the potential for solGMRalpha to interact with GMRbeta in vitro. Our experiments demonstrated that exogenous solGMRalpha, even in the presence of GM-CSF, does not interact with GMRbeta on the cell surface. However, when solGMRalpha and GMRbeta are co-expressed in baby hamster kidney cells, solGMRalpha is retained on the cell surface and forms a functional intermediate affinity GM-CSF binding complex (Kd = 331 pM). In addition, the cell surface expression of solGMRalpha is independent of the presence of GM-CSF as demonstrated using flow cytometry. Cells expressing only solGMRalpha do not show cell surface retention or form functional GM-CSF cell surface binding complexes. Sequencing of our GMRbeta clone revealed a nucleotide substitution (A --> C) resulting in the substitution of Ala for Glu at position 9 from the amino terminus of the mature GMRbeta peptide. Because the GMRbeta (A --> C) clone is capable of forming functional high affinity receptors with transmembrane GMRalpha (Kd = 64 pM), we feel that the cell surface retention of solGMRalpha is independent of the GMRbeta mutation. We suggest that the co-expression and interaction of solGMRalpha and GMRbeta represents a previously unrecognized GM-CSF receptor complex and a novel, ligand-independent mechanism of cytokine receptor assembly.

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

The high-affinity granulocyte-macrophage-colony-stimulating-factor (GM-CSF) receptor (R) is composed of at least two subunits, termed a and p. The a subunit is crucial for initiating ligand receptor interaction and for ligand specificity. The experiments reported in this study sought to identify the domains of human (h)GM-CSF which are responsible for interaction with hGM-CSFRa. Anti-(human-GM-CSF) mAb were used as competitors in a '"I-GM-CSF receptor-binding assay on cells expressing the recombinant hGM-CSFRa chain. Inhibition of "'I-GM-CSF binding to the GMCSFRa chain was demonstrated by mAb which mapped to the middle third of the third a helix (amino acids 78-83), the distal two-thirds of the third a helix and the initial 7 residues of the loop between the third and fourth helix (amino acids 78-94), and the extreme carboxy terminus. No inhibition of binding occured with an antibody whose domain begins in the first /?-pleated sheet (amino acids, 39-43), continues through the second helix (amino acids 55-64) and into the proximal third of the third helix (to amino acid 77). mAb which mapped to the distal half of the fourth helix and the carboxy-terminal tail (amino acids 11 0-127) increased the binding of '"I-GM-CSF to GM-CSFRa. Due to the known discontinous epitopes the possibility that the distal portion of the fourth helix contributes to binding cannot be eliminated. However, the domains of hGM-CSF most clearly involved in binding to the hGM-CSFRa are the distal two-thirds of the third a helix, the immediate downstream residues and the extreme carboxy terminus of hGM-CSF.The hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) exerts its biological activity by first binding to a specific high-affinity receptor on the surface of responsive cells. The native high-affinity receptor is composed of at least two subunits. The initially cloned a subunit imparts ligand specificity but binds GM-CSF with only low (nanomolar) affinity [l], The more recently described b subunit is incapable of binding to GM-CSF by itself but in association with the a subunit forms the highaffinity (picomolar) complex [2]. Current evidence suggests that it is the p subunit that transduces signal [3], a subunit that is shared with the receptors for interleukin-3 (IL-3) and IL-5 [4, 51. Thus, in this model, the initial interaction between GM-CSF and the a subunit of the receptor is crucial for the eventual formation of the high-affinity, GM-CSF-specific, signal-transducing complex. In this study we have sought to identify the domains of GM-CSF which interact with the GM-CSF-receptor a subunit (GM-CSFRa). Abbreviations. h, human; GM-CSF, granulocyte-macrophage colony-stimulating factor; GM-CSFR, granulocyte-macrophage colony-stimulating-factor receptor; GM-CSFRa, GM-CSFRa subunit; GM-CSFRP, GM-CSFR/3 subunit; IL, interleukin; BHK, baby hamster kidney.The crystallographic structure of GM-CSF has been determined [6]. The cytokine exhibits a barrel-shaped structure with four short, slightly twisted, a helices formin...