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Leukopenia and anaemia are observed in about a fifth of all patients with systemic lupus erythematosus (SLE) and may be due either to the destruction of blood cells or their decreased production. The former may be humoral or cell-mediated or result from apoptosis of peripheral blood cells. Several observations suggest the occurrence of the latter reduced in vitro proliferation of pluripotent bone marrow progenitors from the bone marrow aspirates of SLE patients,reduced counts of CD34+ cells in bone marrow aspirates in SLE patients, apoptosis of lymphopoietic progenitors and apoptosis of bone marrow cells. The aim of our study was to investigate whether humoral factors may induce suppression of haematopoiesis by increased apoptosis of CD34+ cells. For this purpose, we incubated allogeneic CD34+-enriched cells with sera of 18 leukopenic SLE patients. Apoptosis was induced by four of 18 sera. This effect was independent of complement-inhibition and FAS-blockade. Although reduced proliferation of autologous pluripotent bone marrow progenitors has been attributed to an IgG serum inhibitor, removal of IgG from these four proapoptotic sera had no effect on apoptosis of allogeneic CD34+ cells. The proapoptotic effect was associated with high titres of anti-dsDNA antibodies and low haemoglobin concentrations, but not with high titres of antinuclear antibodies, TNF-alpha and IFN-alpha of the sera tested.

The cytotoxic effect of chlorodeoxyadenosine (CdA) on lymphocytes and monocytes requires phosphorylation by the enzyme deoxycytidine kinase and can be antagonized by coadministration of deoxycytidine (dCyt), a competitive substrate of deoxycytidine kinase. It has also been shown for lymphocytes that coadministration of 3-aminobenzamide (3-ABA), an inhibitor of the enzyme poly-(ADP ribose) synthetase, is activated by CdA-mediated DNA strand breaks, consumes intracellular nicotinamide-dinucleotide (NAD) and can antagonize the lethal effect of CdA. Recent in vitro studies have shown that not only growth of lymphocytes and monocytes, but also colony formation by erythroid and myeloid progenitors derived from normal human bone marrow, is inhibited by CdA in a dose-dependent manner. In this study we examined the effect of various doses of dCyt (10(-6) to 10(-3) M) on CdA-mediated growth inhibition of erythroid and myeloid progenitor cells in vitro. Our results show that colony formation by human bone marrow-derived progenitor cells--CFU-E (colony-forming unit erythroid), BFU-E (burst-forming unit erythroid) and CFU-GM (colony-forming unit granulocyte/macrophage)-in semisolid medium is protected by a high, but clinically achievable and non-toxic, concentration of dCyt (> 10(-4) M) against the inhibitory effects of coadministered high concentrations of CdA. The protective effect of dCyt was markedly different on the various subclasses of progenitor cells, however. Thus, with coadministration of 10(-4) M dCyt, the CFU-E colony formation could be restored to almost 100% despite the presence of high concentrations of CdA (160 nM) compared to control cultures, whereas the colony formation of BFU-E and CFU-GM was restored to only 50%. At a concentration of 10(-3) M dCyt, colony formation of BFU-E and CFU-GM was raised to 80% of control cultures even in the presence of high concentrations of CdA (160 nM). Further experiments in which 3-ABA was coadministered to CdA-treated cultures showed that in all concentrations tested (0.3 to 5 mM) 3-ABA was not able to prevent CdA-mediated cytotoxicity on bone marrow progenitors. Based on these studies, we suggest that the CdA toxicity on CFU-E is mainly mediated by phosphorylation by deoxycytidine kinase, whereas additional mechanisms may be operative in BFU-E and CFU-GM. Considerable biochemical differences seem to exist between hematopoietic stem cells on the one hand and lymphocytes and monocytes from peripheral blood on the other.

Like erythro- and granulopoiesis megakaryocytopoiesis (MK poiesis) is controlled by specific feed-back mechanisms. Platelet production has been found to be regulated predominantly by the number or mass of circulating platelets. Our present understanding of MK poiesis is based on data from 3 major experimental areas, which are individually discussed in this paper. In vivo experiments on thrombocytotic or thrombocytopenic mice. Radioisotopic assays for thrombopoietin determination. Investigation by in vitro culture methods of megakaryocyte colony-forming precursor cells (CFU-M).

In 13 patients with myelodysplastic syndrome (MDS) mature and immature erythropoietic (CFU-E, BFU-E), granulopoietic (CFU-GM) and megakaryopoietic (CFU-Meg) colony formation from human bone marrow mononuclear cells was evaluated in a microagar culture system. All but three patients exhibited abnormal CFU-Meg. The defect of CFU-Meg paralleled the reduction of BFU-E, whereas CFU-GM number declined to a lesser extent. Not only the CFU-Meg number, but also the number of megakaryocytes (Mk) per colony was reduced suggesting an additional functional CFU-Meg defect. Megakaryocytic growth factor (Meg-CSF) abnormalities in MDS patients were detected using normal nonadherent T-lymphocyte depleted bone marrow cells as target cells for serum testing. Even for sera from patients with a reduction of platelets and bone marrow megakaryocytes Meg-CSF levels were not increased. No cellular or humoral inhibition could be detected in an MDS patient with a 5q- karyotype, who had an isolated defect of the megakaryocytic cell lineage at presentation. Some patients revealed a spontaneous formation of mixed erythrocytic, granulocytic and megakaryocytic clusters in the presence of fetal calf serum or autologous patient serum, probably representing autonomous proliferation of the malignant clone. In conclusion, both cellular and humoral factors can cause abnormalities of the megakaryocytic cell lineage in MDS patients.

In the present study we evaluated the reactivity of monoclonal cytotoxic antibodies directed against myeloid differentiation antigens with hemopoietic precursor cells. VIM-D5 and VIM-2 inhibit the proliferation of clusters and colony formation after seven days of incubation. Day-14 CFU-GM are not affected by these antibodies. After complement-mediated cytolysis with VIM-2, the number of BFU-e was significantly reduced; however, this effect was largely abrogated by addition of leukocyte-conditioned medium to the cultures as an exogenous source of burst-promoting activity. Furthermore, the maturation of myeloid progenitor cells has been examined by delayed treatment with VIM-D5 and complement during the in vitro culture period. In these experiments a different maturation behavior of day-7 and day-14 CFU-GM was demonstrated. To study whether a cryptic carbohydrate structure is present on more immature CFU-GM, the effect of neuraminidase treatment of myeloid progenitor cells on reactivity with VIM-D5 was tested.

Cellular interactions responsible for regulating in vitro megakaryocytopoiesis were studied using a microagar culture system which permits the simultaneous proliferation of human megakaryocytic progenitor cells (CFU-M) and T-lymphocytic colonies (CFU-TL). The proliferation of these colony types depends mainly on two factors: phytohaemagglutinin (PHA) and erythropoietin (EP). The direct addition of increasing PHA concentrations to the liquid overlayer resulted in a parallel increase of CFU-M and CFU-TL. If the T-lymphocytes were removed by an E-rosetting technique a marked diminution of CFU-M and CFU-TL numbers was observed. However, monocyte depletion resulted in a marked augmentation of CFU-M proliferation compared to unfractionated mononuclear cells. In order to confirm that the reduction of CFU-M proliferation observed after T-depletion was primarily mediated by the absence of T-lymphocytes, we have co-cultured different concentrations of previously removed autologous T-lymphocytes with a constant number of T-depleted bone marrow cells. A parallel increase of CFU-TL and CFU-M was found if 0.75 - 7.5 X 10(4) T-lymphocytes were added to the culture. In conclusion, our results indicate that activated T-lymphocytes augment proliferation of human bone marrow CFU-M and that monocytes are less important for the growth of megakaryocytic colonies.