D. M. McCarthy

1,25-dihydroxyvitamin D3 inhibits proliferation of human promyelocytic leukaemia (HL60) cells and induces monocyte-macrophage differentiation in HL60 and normal human bone marrow cells

1,25-dihydroxyvitamin D3 induces monocyte-macrophage differentiation and inhibits proliferation of cells from the human promyelocytic leukaemia cell line HL60. Similarly human bone marrow progenitor cells differentiate preferentially along the monocyte-macrophage pathway when incubated in the presence of 1,25-dihydroxyvitamin D3. We suggest that the inhibition of growth which occurs after addition of the vitamin to HL60 might be paralleled in vivo by inhibition of proliferation of leukaemic cells; also we speculate that the vitamin may be involved in the control of both monocyte-macrophage and osteoclast production in vivo.

FIBROSIS OF THE BONE MARROW: CONTENT AND CAUSES

Fibrosis of the bone marrow accompanies many diseases (Table I). When excessive it presumably impedes haemopoiesis. The relative contributions of the fibrosis per se and of the underlying pathological state to the bone marrow failure are not clear: it is also unclear to what extent any future treatment would be beneficial if it were aimed specifically at limiting the development of fibrosis. The precise composition of the fibrous tissue present in the marrow in most haematological conditions has not been studied; similarly the mechanisms by which it develops are largely unknown. Progress has recently been made in two areas: firstly, the nature of the connective tissue matrix of normal marrow and of that from patients with idiopathic myelofibrosis and agnogenic myeloid metaplasia (IMF) has been largely elucidated; secondly, the sequence of events that leads to fibrosis in the myeloproliferative syndromes with a prominent megakaryoblastic/megakaryocytic component is now better understood.

Haematological reconstitution and severity of graft-versus-host disease after bone marrow transplantation for chronic granulocytic leukaemia: the influence of previous splenectomy

Summary. Eighteen patients with chronic granulocytic leukaemia (CGL) were treated by chemoradiotherapy and transplantation of bone marrow (BMT) collected from their HLA‐identical sibs; engraftment with donor marrow occurred in all cases. Ten of the patients had been subjected to splenectomy before BMT; recovery after BMT of granulocyte, lymphocyte and platelet numbers in the peripheral blood was more rapid in these patients than in the eight patients who retained their spleens. Acute graft‐versus‐host disease occurred in 12 of the 15 evaluable patients and appeared to be more severe in those who lacked their spleens at the time of transplant. Of the 12 patients surviving at follow‐up times ranging from 59 to 207 weeks, six had been subjected to splenectomy before BMT and six retained their spleens. We conclude that engraftment was more rapid in the splenectomized patients and splenectomy might have increased the chance of eradicating the leukaemia, but these considerations must be balanced against the short‐term and long‐term risks associated with splenectomy.

An extrinsic factor controls neutrophil alkaline phosphatase synthesis in chronic granulocytic leukaemia.

Summary The mechanism underlying the reduced neutrophil alkaline phosphatase (NAP) activity in chronic granulocytic leukaemia (CGL) has been investigated by in vivo manipulation of CGL neutrophils. These cells, which had almost no alkaline phosphatase activity following collection by leucapheresis, had greatly elevated activities 14–17 h after transfusion into three neutropenic recipients. It is postulated that NAP synthesis in mature neutrophils is controlled by an extrinsic factor and that the levels of this factor is reduced in patients with CGL with high neutrophil counts.

The concentration and subcellular localisation of zinc, magnesium and calcium in human polymorphonuclear leukocytes

The calcium, magnesium and zinc content of human polymorphonuclear leukocyte homogenates and their subcellular fractions has been determined by atomic absorption spectrophotometry. Neutrophils were homogenised in sucrose medium and subjected to analytical subcellular fractionation. The sucrose density gradient fractions were assayed for zinc, magnesium and the principal organelle marker enzymes. Zinc was found to be largely soluble, with small amounts in the alkaline phosphatase-containing granules (phosphasomes). Magnesium had a multimodal distribution with a soluble component, and peaks corresponding to plasma membrane, specific granules and azurophils. The level of calcium, expressed per mg DNA, was significantly decreased in both chronic granulocytic leukaemia (CGL) and pregnancy. Leucocyte zinc was only significantly reduced in CGL. Leukocyte magnesium showed no significant variation within the three groups of subjects. No significant correlation was noted between the zinc, calcium or magnesium content and leukocyte alkaline phosphatase activity in any of the three groups.

Biochemical Studies on Cryopreserved Granulocytes: Possible Explanations for the Defects Induced

Summary. The functional capacity of normal human granulocytes has been studied after cryopreservation by two different methods with dimethylsulphoxide as cryoprotectant. Both methods resulted in substantial disruption of organelle membranes and impaired cell function. Similar organelle disruption could be induced without cryopreservation following the addition of dimethylsulphoxide to cells, but the extent of this disruption was dependent on the temperature at which the dimethylsulphoxide was diluted out. The loss of cellular function also depended on the temperature and rate of dilution.

Granulocytic cryopreservation: further studies on the pathogenesis of impaired cellular function

Summary The effects of different methods of purifying peripheral blood granulocytes on their functional capacity after cryopreservation have been studied. The standard method of isolating granulocytes by centrifugation through Lymphoprep sensitizes cells to damage induced by subsequent cryopreservation. Some of this damage occurs during addition of cryoprotectant and we describe how to add and remove dimethyl sulphoxide without impairing cell function. The granulocytic function that suffers most during cryopreservation is granulocyte migration. This impairment may be partially due to uncontrolled entry into the cell of calcium. In contrast the bactericidal capacity in vitro is not so greatly reduced and ability to reduce nitroblue tetrazolium redox dye even less so.

Changes in surface antigens of HL-60 cells during differentiation in vitro

SummaryWe have examined the pattern of binding of monoclonal antibodies OKOM 1, FMC 10, FMC 12, FMC 13, FMC 17 and FMC 33 to human promyelocytic leukaemia (HL-60) cells. We found that the expression of antigens detectable with FMC 17 and FMC 33 (specific for monocytes and macrophages) was increased by exposure of HL-60 cells to 1,25-dihydroxyvitamin D3 but not by exposure of HL-60 cells to 12-tetradecanoyl phorbol-13-acetate (TPA). The antigen detected with the OKM 1 antibody was highly induced by TPA. The expression of granulocyte-specific antigens detected by FMC 10 and FMC 13 was increased during induction of granulocytic maturation; these antigens were retained during monocytemacrophage differentiation of HL-60 cells. We conclude that in some cases the expression of particular antigens during maturation of malignant cells proceeds normally while in other cases antigenic differences between leukaemic and normal cells at equivalent levels of maturation can be detected.