Expression of CD59, a regulator of the membrane attack complex of complement,on human skeletal muscle fibers

Control of complement deposition on autologous cells is mediated by a group of complement regulatory membrane proteins acting at different levels of the complement cascade. Decay accelerating factor (CD55) prevents the assembly of C3 convertases and CD59 membrane inhibitor of reactive lysis (MIRL) restricts homologous complement lysis by the membrane attack complex of complement (MAC) by inhibition of C5b-8 catalyzed insertion of C9. The aim of this work was to study the eventual expression of CD55 and CD59 on human skeletal muscle fibers. Highly sensitive immunoblotting using murine monoclonal antibodies showed that CD59, but not CD55, was present in skeletal muscle fibers. Immunocytochemistry with a monoclonal antibody against CD59 demonstrated a dense granular immunostaining mainly localized at the level of the sarcolemma. Thus, CD59, but not CD55, is expressed on normal skeletal muscle fibers. CD59 may play a prominent role in preventing MAC deposition and subsequent complement-mediated damage in myopathies where the complement system activation is involved. © 1997 John Wiley & Sons, Inc.     

X-linked vacuolated myopathy: Membrane attack complex deposition on the surface membrane of injured muscle fibers is not accompanied by S-protein

We have studied the expression of S-protein on the muscle from patients with X-linked vacuolated myopathy [characterized by the deposition of the complement C5b-9 membrane attack complex (MAC) over abnormal muscle fibers] and controls by immunocytochemistry and immunoblotting. No expression was detected on muscle from controls and patients with X-linked vacuolated myopathy. These findings suggest that S-protein does not render the MAC inactive in X-linked vacuolated myopathy. This situation may be due to the fact that the pathways of MAC activation and the expression of S-protein in X-linked vacuolated myopathy are different from the ones observed in ischemic and/or necrotic, or immune diseases. These results emphasize the role of the membrane complement regulatory proteins (i.e., CD59) in X-linked vacuolated myopathy. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:932–935, 1998.     

Association of nucleophosmin negatively regulates CXCR4-mediated G protein activation and chemotaxis

CXCR4, the primary receptor for CXCL12, plays a critical role in the development of hematopoietic, vascular, central nervous, and immune systems by mediating directional migration of precursor cells. This mechanism promotes homing of tumor cells to metastatic sites that secrete CXCL12, and CXCR4 expression is a negative prognostic factor in acute myelogenous leukemia (AML). To elucidate mechanisms that regulate CXCR4 signaling, we used a proteomic approach to identify proteins physically associated with CXCR4. Analysis of CXCR4 immune complexes identified nucleophosmin (NPM), which was confirmed by reciprocal coimmunoprecipitation for NPM. Constitutively active CXCR4 variants bound higher levels of NPM than the wild-type receptor, which was reversed by T140, an inverse agonist. NPM binding to CXCR4 localized interactions to the C terminus and cytoplasmic loop (CL)-3, but not CL-1 or CL-2. Alanine scanning mutagenesis demonstrated that positively charged amino acids in CL-3 were critical for NPM binding. Recombinant NPM decreased GTP binding in membrane fractions after activation of CXCR4 by CXCL12. Suppression of NPM expression enhanced chemotactic responses to CXCL12, and, conversely, overexpression of a cytosolic NPM mutant reduced chemotaxis induced by CXCL12. This study provides evidence for a novel role for NPM as a negative regulator of CXCR4 signaling induced by CXCL12 that may be relevant to the biology of AML.     

Expression of blood group i antigen and fetal hemoglobin in paroxysmal nocturnal hemoglobinuria

BACKGROUND: The mechanism by which the paroxysmal nocturnal hemoglobinuria (PNH) clone progressively takes over normal hematopoietic cells remains unknown. The respective in vivo differentiation of normal and PNH erythroid progenitors was investigated through the expression of two fetal erythroid markers (i antigen and fetal hemoglobin [HbF]) whose expression in adult red cells is associated with altered erythropoiesis. STUDY DESIGN AND METHODS: Murine monoclonal antibodies directed against HbF and i and CD59 antigens were used to phenotype red cells of 10 PNH patients. A multiparametric flow cytometry assay of red cells and reticulocytes was designed to assess a possible association of i and HbF with PNH or normal red cells. RESULTS: Most patients exhibited greater expression of i and HbF than did normal controls. In each case, the percentages of i-positive or HbF-positive cells within CD59-deficient and CD59- positive red cells were very close, clearly showing a lack of preferential association of these markers with normal or PNH cells. CONCLUSION: In PNH patients, normal and PNH erythroid progenitors have the same ability to promote HbF and i antigen expression, which suggests that normal and PNH erythroid progenitors (burst-forming units- erythroid, colony-forming units-erythroid, erythroblasts) behave similarly in response to bone marrow stress.     

Production of a New Murine Monoclonal Antibody with Fy6 Specificity and Characterization of the Immunopurified N-Glycosylated Duffy-Active Molecule

A murine monoclonal antibody (mAb i3A; IgG1, kappa light chain) was obtained using human red blood cells as immunogen. The antibody showed Fy6 specificity since it agglutinated all but Fy(a-b-)-untreated red cells and failed to agglutinate chymotrypsin-treated cells. An erythrocyte membrane protein of 42–46 kD was revealed as the major component recognized by the antibody on immunoblots. The antibody also bound to 92- to 95- and 200-kD proteins, tentatively identified as oligomers of the 42- to 46-kD monomeric form. The affinity-purified Fy6-active protein was converted to a sharp band of 35 kD after N-glycanase treatment. The molecule appeared as a slightly broadly band after neuraminidase treatment but was not further altered by O-glycanase. The i3A mAb bound to 6,000±1,000 receptor sites on either Fy(a-b+), Fy (a+b+) and Fy(a+b-) red cells with an affinity constant in the range of 3–6 times 10⁸ M^-1. No binding was observed to other blood cells nor to several cells (B, T, myelomonocytic and erythro-leukemia cell lines). Also, the bulk of i3A-Fy6 immune complexes could be dissociated from the red cell membrane with as low as 0.2% Triton X-100, showing that the Fy6-active glycoprotein is not tightly associated with the membrane skeleton. Our data obtained with a new monoclonal antibody directed to the Fy6 antigen demonstrate that the blood group Duffy-active component is a red cell-specific glycoprotein carrying one or more N-linked oligosaccharides.     

The metastasis suppressor KISS1 lacks antimetastatic activity in the C8161.9 xenograft model of melanoma

The objective of this study was to use the established xenograft model of human melanoma (C8161.9) to test a pharmacological approach to the effect of the metastasis suppressor KISS1. A KISS1 analog was used to inhibit the metastatic development of C8161.9 cells in nude mice. Further experiments were performed to test the validity of the C8161.9 model and test the connection between KISS1 expression and loss of metastatic potential. New clones of C8161.9 cells were obtained, with or without KISS1 expression, and were tested for metastasis formation. The absence of benefit in survival with the KISS1 analog compared with PBS prompted us to revisit the C8161.9 model. We found that the cells expressing KISS1, used in the previous study and obtained by transfection and single-cell cloning, were defective for both formation of orthotopic tumors and metastases. In mixing experiments, these cells could not suppress orthotopic tumor growth of KISS1-negative C8161.9 cells, suggesting that the suppression of metastasis by C8161.9-KISS1 cells may be intrinsic to the selected clone rather than related to KISS1 expression. Isolation of clones from parental C8161.9 cells in soft agar yielded cell populations that phenotypically and genotypically mimicked the KISS1-positive clone. In addition, new clones expressing KISS1 did not show any decrease in metastatic growth. These data demonstrate the heterogeneity of cell types in the C8161.9 cell line and the high risk of artifact linked to single-cell selection. A different xenograft model will be necessary to evaluate the use of KISS1 analogs as antimetastatic therapy.     

Investigation of the survival of paroxysmal nocturnal hemoglobinuria red cells through the immunophenotyping of reticulocytes

BACKGROUND: Complement-mediated lysis of red cells (RBCs) is a classic feature of paroxysmal nocturnal hemoglobinuria (PNH) that is traditionally studied with a combination of radiolabeling of RBCs and in vitro hemolysis tests. Phenotyping of reticulocytes was used as an alternative method for the evaluation of the relative life span of normal RBCs (PNH I) and RBCs that were partially (PNH II) or completely (PNH III) deficient in CD59. STUDY DESIGN AND METHODS: Murine monoclonal antibodies CD55, CD58, and CD59 and thiazole orange were used to phenotype reticulocytes by two-color flow cytometry in nine PNH patients. RBC survival could be calculated from the ratio of CD59- or CD59low mature RBCs to CD59- or CD59low reticulocytes obtained from these patients who had not received a transfusion. RESULTS: The life span of PNH III RBCs varied from about 17 to 60 days. PNH II reticulocytes were found in the four patients with PNH II RBCs. The life span of PNH II RBCs varied with their residual expression of CD59, and cells with 15 to 20 percent of the normal amount of CD59 were protected against in vivo hemolysis. CONCLUSION: Phenotyping of reticulocytes is a convenient and reliable tool for evaluating the relative survival of normal and PNH RBCs. PNH II and PNH III reticulocytes are phenotypically distinct, and some PNH II RBCs may be sensitive to complement-mediated lysis in vitro, but normally they are complement-resistant in vivo.     

Cytolytic effect of human anti-Gal IgM and complement on porcine endothelial cells: A kinetic analysis

Abstract: The binding of human natural antibodies to porcine endothelial cells is the first step leading to activation of complement and lysis of the cells. The kinetic of incorporation of propidium iodide into porcine endothelial cell line SVPAEC/6A or peripheral blood lymphocytes was investigated to monitor the permeabilization of target cells by membrane attack complex. In less than 5 min, more than 90% of porcine cells were highly fluorescent upon incubation with human sera, showing they incorporate propidium iodide. The lysis occurred neither with adult sera pretreated with dithiothreitol nor with umbilical cord sera, suggesting that lytic antibodies belong to the IgM class. By using antibodies purified from human sera, the bulk of lytic activity was found to be associated with IgM specific for the Galal-3Gal epitope, whereas IgG were not able to lyse the porcine cells. These antibodies could be readily absorbed on rabbit red blood cells known to express the main epitope target of xeno human antibodies. Finally, our results show that permeabilization of porcine endothelial cells is a very early phenomenon, probably directly associated with the biological process responsible for the hyperacute rejection.     

Flow cytometric monitoring of red blood cell chimerism after bone marrow transplantation

Chimerism after bone marrow transplantation (BMT) was investigated by flow cytometry analysis of red blood cells (RBCs) and of reticulocytes using a series of selected monoclonal or polyclonal antibodies directed against ABO, Rhesus, Kell, Duffy or MNSs antigens. The method allows the routine detection of less than 0.1% of positive cells in artificial mixed field populations. Blood samples from 135 patients undergoing BMT were investigated around days 15, 30, 45, 60, 90, 180, and then every 6 months after transplantation. Characteristic patterns showing expression of donor red blood cell antigens (expansion markers) and concomitant decrease of recipient specific antigens (depletion markers) within days 16-20 were observed for 125 successfully engrafted patients. Distinct patterns were obtained in 10 patients. A delay in engraftment was evidenced in four patients by the absence of chimerism during the first 6 months without any sign of relapse. Re-appearance of recipient RBCs and reticulocytes was observed in five patients; it was consistent with relapse that was later confirmed by clinical, haematological and cytogenetic studies. Finally, a stable and partial chimerism with 20% of RBCs expressing a marker from the recipient was observed in one patient without any sign of relapse. The reported investigation demonstrated that flow cytometry of RBCs and reticulocytes represents a powerful method to efficiently monitor bone marrow transplanted patients on a long-term basis.