Alessandra Bassini

Stroma-derived factor 1α induces a selective inhibition of human erythroid development via the functional upregulation of Fas/CD95 ligand

CXC chemokine receptor 4 (CXCR4), the high‐affinity receptor for stroma‐derived factor 1α (SDF‐1α), shows distinct patterns of expression in human CD34+ haematopoietic progenitor cells induced to differentiate in vitro along the granulocytic and erythroid lineages. In serum‐free liquid cultures supplemented with stem cell factor (SCF), interleukin 3 (IL‐3) and granulocyte colony‐stimulating factor, the expression of surface CXCR4 progressively increased in cells differentiating along the granulocytic lineage. The addition in culture of 200 ng/ml of SDF‐1α, a concentration which maximally activated intracellular Ca2+ flux, only modestly affected the expression levels of CD15 and CD11b granulocytic antigens, as well as the total number of viable cells. On the other hand, in liquid cultures supplemented with SCF, IL‐3 and erythropoietin, SDF‐1α induced the downregulation of glycophorin A erythroid antigen, accompanied by a progressive decline in the number of viable erythroblasts. Moreover, in semisolid assays, SDF‐1α significantly reduced the number of plurifocal erythroid colonies (erythroid blast‐forming units; BFU‐E), whereas it did not affect granulocyte–macrophage colony‐forming units (CFU‐GM). We also demonstrated that the inhibitory effect of SDF‐1α on glycophorin A+ erythroid cell development was mediated by the functional upregulation of CD95L in erythroid cultures. These data indicate that SDF‐1α plays a role as a negative regulator of erythropoiesis.

Emerin presence in platelets.

Abstract Emerin is an almost ubiquitous protein which is abnormal in X-linked Emery-Dreifuss muscular dystrophy (EMD), a syndrome characterized by muscle weakness, joint contractures and cardiac arrhythmia. Emerin is localized in the cells at the nuclear rim and its function is still unknown. In some models, emerin has also been described in the cytoplasm; however, its presence outside the nucleus is still matter of debate. We report the presence of emerin in circulating normal human platelets and its absence in platelets from X-linked EMD patients. Since platelets are cytoplasmic fragments derived from megakaryocytes, the presence of emerin in platelets confirms cytoplasmic localization of this protein, probably related to specific functions. We found also that emerin is present in the cytoplasm of megakaryocytes, while it is absent in circulating granulocytes.

HIV‐1 gp120 induces the activation of both c‐fos and c‐jun immediate‐early genes in HEL megakaryocytic cells

We have previously demonstrated that the addition in culture of recombinant HIV‐1 IIIB envelope gp120 affects the survival/growth of pluripotent haemopoietic progenitors, and, in particular, of those committed towards the megakaryocytic lineage. To characterize some of the molecular mechanisms involved in this phenomenon, we investigated the expression of members of the activating protein‐1 (AP‐1) complex in the HEL megakaryoblastic cell line. Following the treatment of HEL cells with recombinant IIIB envelope gp120, we noticed: (i) increased levels of endogenous c‐fos and c‐jun mRNA and proteins, (ii) activation of both c‐fos and c‐jun promoters, and (iii) a very rapid stimulation of a MAPK/ERK pathway.

The CD4 receptor plays essential but distinct roles in HIV-1 infection and induction of apoptosis in primary bone marrow GPIIb/IIIa+ megakaryocytes and the HEL cell line.

Summary. We investigated whether cells belonging to the megakaryocyte lineage could be infected in vitro with human immunodeficiency virus type‐1 (HIV‐1). Primary GPIIb/IIIa+ bone marrow (BM) cells and HEL continuous cell line were first phenotypically characterized for the presence of megakaryocytic markers and CD4 antigen, then challenged in vitro with the laboratory strain IIIB of HIV‐1. Both GPIIb/IIIa+ BM and HEL cells expressed significant levels of CD4 receptor (<50%) and were efficiently infected with HIV‐1, as judged by the presence of proviral DNA after polymerase chain reaction analysis and by quantitative evaluation of gag p24 antigen in the culture supernatants. Of note, infection with HIV‐1 in both primary BM megakaryocytes and HEL cells was specifically blocked by soluble recombinant CD4. To ascertain whether the CD4 receptor was essential for infection of megakaryocytic cells, HEL were subcloned into CD4+ and CD4 cells. Although unfractionated and CD4+ HEL cells were productively infected with HIV‐1, CD4 HEL cells could not be infected. Infection of HEL cells did not induce gross cytotoxic effects or a significant increase of apoptosis. On the other hand, treatment of unfractionated or CD4+ HEL cells with cross‐linked recombinant env gp120 or Leu3a anti‐CD4 monoclonal antibody markedly (P< 0.01) increased the degree of apoptosis with respect to HEL cells infected with HIV‐1 or treated with cross‐linked gag p24 or anti‐GPIIb/IIIa antibody. Taken together, these data indicate that the CD4 receptor represents the main route of infection in cells belonging to the megakaryocytic lineage. Moreover, an inappropriate engagement of CD4 by either free env gpl20 or anti‐CD4 monoclonal antibody could be more relevant than a direct infection with HIV‐1 in the induction of the frequent BM megakaryocyte abnormalities found in HIV‐1 seropositive thrombocytopenic patients.

Selective enrichment in human megakaryocyte progenitors by the B203.13 surface differentiation antigen

The B203.13 monoclonal antibody specifically recognizes a subset (8–18.5%) of human CD34+ haemopoietic progenitors, which are significantly (P < 0.05) enriched in megakaryocyte progenitors (CFU‐meg). Moreover, B203.13 expression was progressively lost as maturation proceeded along the megakaryocytic lineage. In fact: (i) CFU‐meg derived from CD34+/B203.13+ showed a greater number of cells/colony with respect to CD34+/B203.13−; (ii) after 10 d of liquid cultures with 100 ng/ml of thrombopoietin, only a minority of CD34‐derived cells positive for CD41 coexpressed B203.13; (iii) in situ immunocytochemical staining revealed that B203.13 was expressed exclusively on immature megakaryoblasts; (iv) circulating platelets did not express B203.13.