The role of CS1 moiety of fibronectin in VLA mediated haemopoietic progenitor trafficking.

In vitro the integrin VLA4 mediates the adhesion of haemopoietic progenitors to bone marrow stroma through an interaction with its ligands VCAM-1 and the CS1 moiety of fibronectin. The VLA4/VCAM-1 pathway has been implicated in haemopoietic trafficking in vivo since antibodies to both VLA4 and VCAM-1 decrease the homing (lodgement) of transplanted progenitors and mobilize progenitors. However, the role of the CS1 domain of fibronectin in progenitor trafficking in vivo has not been explored. We studied the effect of competitive inhibition of the VLA4/CS1 pathway on progenitor homing and mobilization in mice. Pre-incubation of bone marrow cells with a CS1 inhibitor did not alter the number of CFU-C or CFU-S12 lodged to the bone marrow of lethally irradiated mice 3 h after transplantation. In addition, continuous administration of a CS1 inhibitor did not increase the number of CFU-C in the peripheral blood. In order to study the role of the VLA4/CS1 pathway in trafficking of more primitive progenitors we studied whether administration of a CS1 inhibitor mobilized radioprotective cells. In contrast to the effect of anti-VCAM-1 which mobilized cells capable of rescuing 100% of lethally irradiated mice, administration of a CS1 inhibitor did not increase the number of radioprotective cells in the peripheral blood. Haemopoietic progenitors also bind to the RGD motif of fibronectin through an interaction with VLA5 and we therefore also studied the effect of antibodies to VLA5 on progenitor homing and mobilization. Antibody to VLA5 did not alter bone marrow lodgement at 3 h or increase the number of circulating haemopoietic progenitors. These studies therefore imply that, in contrast to VCAM-1, the CS1 moiety of fibronectin is not a significant ligand in VLA4 mediated progenitor trafficking in vivo.

Adult and fetal human globin genes are expressed following chromosomal transfer into MEL cells.

Somatic cell hybridization of mouse erythroleukemia (MEL) cells and HEL cells, a human erythroleukemia line that produces fetal (gamma) but fails to express adult (beta) globin, was used to test whether the expression of the two human globin genes is regulated cis or trans. An experimental approach using anti-human globin monoclonal antibodies for detection, efficient cloning, and monitoring of hybrids of interest was employed. Further characterization of hybrids used isoelectric focusing for detection of human globins and S1 nuclease mapping. In contrast to the parental HEL line, all chromosome 11-retaining HEL-MEL hybrids expressed human beta-globin, suggesting that the HEL beta-globin genes (i) are transcriptionally competent, (ii) become activated in response to a positive trans-acting element within the MEL environment, and (iii) fail to express into the HEL environment because of either the absence of a positive trans-acting element or the presence of a trans-acting inhibitor of beta-globin gene expression. In addition to beta-globin, the primary HEL-MEL hybrids co-expressed gamma-globin; however, gamma-globin expression segregated by subcloning so that secondary and tertiary clones either expressed only beta-globin or co-expressed gamma- and beta-globin. The results of subcloning can be explained by assuming that gamma-globin gene expression is controlled by a HEL cell-derived transacting element encoded by a gene not syntenic to chromosome 11 or by postulating that the HEL gamma-globin genes become randomly modified during the continuous proliferation of hybrids.

Hemoglobin ontogenesis: test of the gene excision hypothesis.

The gene excision hypothesis of hemoglobin ontogenesis was tested in persons with HbSC disease, with the use of monospecific fluorescent antibodies for the identification of hemoglobins S, C, and F in individual erythrocytes. The results are incompatible with the prediction that only one gamma- or beta-globin gene may be active in any single chromosome and provide further evidence for incomplete repression of gamma-globin genes lying cis to active beta-globin genes.

Identification of Haemoglobin S in red cells and normoblasts, using fluorescent anti-Hb S antibodies.

Anti-haemoglobin S antibodies were raised in horses and purified by affinity chromatography. These antibodies recognize beta6 valine, while they fail to bind to haemoglobins with a glutamy1 (Hb A) or a lysy1 (Hb C) residue in this position. The purified anti-Hb S antibodies were composed of equine IgG(a, b) and IgG(T) subclasses and failed to cause precipitation with Hb S, evidently because of the bivalencey of both antibodies and antigen. Identification of Hb S in single erythrocytes was achieved by reacting the fluorescein isothiocyanate-conjungated anti-Hb S antibodies with the haemoglobin antigen in fixed blood smears. All the red cells from persons with sickle cell trait and sickle cell anaemia are labelled with the anti-Hb S-FITC, while red cells from persons with normal haemoglobin or other mutant haemoglobin genotypes are not. Erythrocytes from newborns with sickle cell trait bind the anti-Hb S antibody, although the intensity of labelling varies from cell to cell. This method of cellular identification of Hb S preserves the morphological characteristics of the reactive cells and permits observations of the pattern of haemoglobin synthesis among cells of various degrees of maturity. Fluorescent identification of Hb S in fixed blood smears may be applicable to prenatal diagnosis of haemglobinopathies.

Stimulation of fetal hemoglobin synthesis in bone marrow cultures from adult individuals.

The regulation of fetal hemoglobin in adult erythroid cells was investigated with bone marrow cultures. Fetal hemoglobin (Hb F) was identified in individual erythroid colonies with fluorescent antibodies against Hb F and synthesis of gamma chains was determined with analyses of radioactive globins. The appearance of fetal hemoglobin in erythroid colonies was clonal. All the cells of the Hb F synthesizing colonies contained fetal hemoglobin. The frequency of erythroid colonies showing Hb F was higher than expected compared to the frequency of Hb F containing cells in the blood. Production of Hb F in culture, as shown by analysis of the radioactive globins, was 5 to 14 times higher than baseline Hb F synthesis. These results suggest that the ability for gamma chain synthesis in erythroid cells is determined at or above the level of the precursor cell from which the erythroid colonies, in vitro, derive (probably an erythropoietin responsive stem cell), and that stimulation of fetal hemoglobin synthesis in adult erythroid cells is possible.