Shoshana Merchav

The role of interleukin-1 and tumour necrosis factor-α in human multiple myeloma

This study investigates the capacity of interleukin‐1α (IL‐1α), interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α) to induce interleukin‐6 (IL‐6) production in freshly isolated myeloma cells (MC) and bone marrow‐derived stromal cells (MSC). Recombinant human (rh) IL‐1α, IL‐1β and TNF‐α augmented production of IL‐6 in human MC.IL‐6 was determined on a factor‐dependent Cess cell line. This activity was completely abrogated by anti‐IL‐6 antibodies. Prior incubation of IL‐1α, IL‐1β and TNF‐α with their respective antibodies inactivated the ability of recombinant cytokines to stimulate the release of IL‐6 from myeloma cells. IL‐1α, IL‐1β and TNF‐α enhanced 3H‐TdR uptake in myeloma cells through IL‐6, as antibodies to IL‐6 completely abolished the DNA synthesis induced by culture supernatants of MC exposed to these cytokines. rhIL‐6 reversed the inhibitory action of anti‐IL‐6 antibodies and reinduced DNA synthesis in MC. Next we found that IL‐lα, IL‐1β and TNF‐α induced MSC to produce IL‐6. In contrast, supernatants of unstimulated MSC did not contain detectable IL‐6 biologic activity. Further data demonstrated that human MC were able to induce IL‐6 production in MSC. The stimulatory activities of MC appeared to be mediated through endogenously released IL‐1, as the addition of antibodies towards IL‐1 at the initiation of cocultures completely abrogated the IL‐6 production. We conclude from our data that IL‐1 and TNF‐α may play an important role in the pathogenesis of human multiple myeloma.

Enhancement of erythropoiesis in vitro by human growth hormone is mediated by insulin-like growth factor I

Insulin‐like growth factor I (IGF‐I) is the presumed paracrine or autocrine growth‐promoting mediator of growth hormone in peripheral tissues. In order to evaluate the role of IGF‐I as mediator of human growth hormone (hGH) in erythropoiesis, we compared the effects of both peptides upon in vitro colony formation by primitive (BFU‐E) and relatively mature (CFU‐E) human erythroid precursors. Biosynthetic IGF‐I (2 ng/ml) and hGH (25 ng/ml) induced a significant increase in the growth of both BFU‐E and CFU‐E. BFU‐E growth was maximally enhanced by 6 ng/ml IGF‐I and by 50 ng/ml hGH, resulting in an increase in burst numbers of 62 ± 12% and 52 ± 12%, respectively. Maximal enhancement of CFU‐E growth was detected at higher concentrations of IGF‐I (20 ng/ml) and hGH (150 ng/ml), with respective increases of 121 ± 35% and 137 ± 18% in colony numbers. Enhancement of bone marrow and peripheral blood erythroid progenitor cell growth by hGH required the presence of monocytes and was abrogated by specific monoclonal antibodies directed against IGF‐I membrane receptors. The in vitro growth‐promoting effect of hGH upon human erythroid precursors thus appears to be mediated by paracrine IGF‐I.

Regulatory abnormalities in the marrow of patients with myelodysplastic syndromes

Regulation of haemopoiesis in the marrow of patients with myelodysplastic syndromes (MDS) was evaluated by assaying (1) the production of haemopoietic regulators acting upon multipotent and committed progenitors by MDS marrow cells, and (2) the responsiveness of MDS marrow progenitors to stimulation with granulocyte colony‐stimulating factor (G‐CSF). The levels of multipotent progenitor cell colony‐stimulating activity (CFU‐GEMMCSA) in 7 d bone marrow‐conditioned medium (BMCM) from MDS patients were markedly reduced as compared to controls. MDS BMCM also exhibited reduced levels of burst‐promoting activity (BPA) for primitive erythroid (BFU‐E) progenitors. Both CFU‐GEMMCSA and BPA detected in BMCM were completely neutralized by antibodies directed against interleukin‐3. MDS BMCM exhibited markedly reduced levels of murine‐active CSA. This activity was partially neutralized by anti‐CSF‐1 antibodies. Levels of regulators in BMCM of refractory anaemia (RA), sideroblastic anaemia, RA with excess blasts, and chronic myelomonocytic leukaemia were virtually the same. CFU‐GEMM and BFU‐E growth in MDS marrow (n= 9) was markedly reduced. A 5‐fold saturating dose of G‐CSF induced an approximately 2‐fold increase in CFU‐GEMM in four of eight MDS and a 1.5‐fold increase of BFU‐E in five of nine MDS, but not in control (n= 5) marrow cell cultures. Impaired haemopoiesis in MDS marrow may be related to abnormalities both in regulator production by marrow accessory cells and in regulator responsiveness of multipotent and committed progenitors.

In‐vitro response of erythroid progenitors from children with thalassaemia major to human growth hormone and insulin‐like growth factor I

OBJECTIVE Most short‐statured children with β‐thalassaemia major have markedly reduced levels of circulating insulin‐like growth factor I (IGF‐I). Both human growth hormone (hGH) and IGF‐I enhance the in‐vitro growth of erythroid progenitors, with hGH exerting its effects via paracrine production of IGF‐I. The aim of this study was to characterize further the hGH‐IGF‐l axis abnormalities in thalassaemia major by evaluating the erythroid potentiating effects of both peptides in cultures of thalassaemic and control erythroid progenitors.

Production of human pluripotent progenitor cell colony stimulating activity (CFU-GEMMCSA) in patients with myelodysplastic syndromes

The present study was aimed at assessing the possible relationship between the T-lymphocyte abnormalities and the stem cell dysfunction in myelodysplastic syndromes (MDS), by investigating the production of specific stimulators of stem cell differentiation in such patients. Conditioned media from peripheral blood mononuclear cells (PBMNC) of MDS patients and healthy controls, prepared with or without phytohaemagglutinin (PHA), were assayed for their capacity to stimulate the in-vitro formation of multilineage colonies (CFU-GEMM) in target marrow cell cultures of healthy donors. Both PHA-induced DNA synthesis and T-cell subpopulation ratios (T4/T8) in patient cells were significantly lower than in controls. However, no impaired production of pluripotent progenitor cell colony stimulating activity (CFU-GEMMCSA) by PHA-stimulated and unstimulated PBMNC, could be found. Normal levels of activity were also produced by isolated T lymphocytes of MDS patients. Autologous serum neither enhanced nor suppressed the production of CFU-GEMMCSA. Our observations demonstrate that PHA-induced production of CFU-GEMMCSA is not directly correlated with DNA synthesis. Furthermore, we have found that both major T-cell subsets, defined by OKT4 and OKT8 monoclonal antibodies, are equally capable of producing CFU-GEMMCSA. The normal production of CFU-GEMMCSA by T cells of MDS patients suggests that this T-cell function is not an etiological factor in the stem cell disorder of myelodysplastic syndromes.

Aplastic anemia associated with in vitro inhibition of erythropoiesis by bone marrow-adherent cells*

A patient with aplastic anemia that evolved following pure red cell aplasia is described. Cultures of the patients marrow cells revealed greatly reduced numbers of primitive (BFU‐E) and relatively mature (CFU‐E) erythroid progenitors, but normal numbers of multipotential (CFU‐GEMM) precursors. The BFU‐E/CFU‐GEMM and CFU‐E/BFU‐E ratios in the patients marrow cell cultures were also reduced. T cell‐ or antibody‐mediated inhibition of in vitro erythropoiesis could not be demonstrated in this patient. However, the patients marrow‐adherent cells suppressed the growth of autologous and allogeneic BFU‐E and CFU‐E, without influencing the growth of CFU‐GEMM. Medium conditioned by the patients adherent cells failed to inhibit the growth of normal erythroid precursors. Our findings suggest a role for marrow‐adherent cells in the pathogenesis of aplastic anemia in this patient.