Fas antigen, a receptor molecule that mediates signals for programmed cell death, is involved in T-cell-mediated killing of malignant, virus-infected or allogeneic target cells. Interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), potent inhibitors of haemopoiesis, enhance Fas receptor expression on bone marrow (BM) CD34+ cells, and both cytokines render haemopoietic progenitor cells susceptible to Fas-mediated inhibition of colony formation due to the induction of apoptosis. Haemopoietic suppression in aplastic anaemia (AA) has been associated with aberrant IFN-gamma, increased TNF-beta expression, and elevated numbers of activated cytotoxic T-cells in marrow. We have now examined Fas antigen expression in fresh AA BM samples. In normal individuals few CD34+ cells expressed Fas antigen and normal marrow cells had low sensitivity to Fas-mediated inhibition of colony formation. In contrast, in early AA, BM CD34+ cells showed markedly increased percentages of Fas receptor-expressing CD34+ cells, which correlated with increased sensitivity of AA marrow cells to anti-Fas antibody-mediated inhibition of colony formation. The proportion of Fas antigen-bearing cells was lower in recovered patients' BM. Fas antigen was also detected in the marrow of some patients with myelodysplasia, especially the hypocellular variant. These results are consistent with the hypothesis that AA CD34+ cells, probably including haemopoietic progenitor cells, express high levels of Fas receptor due to in vivo exposure to IFN-gamma and/or TNF-alpha and are suitable targets for T-cell-mediated killing. Our results suggest that the Fas receptor/Fas ligand system are involved in the pathophysiology of BM failure.

Increased expression of interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in bone marrow failure disorders suggests a possible pathophysiologic role of these cytokines in disease. In this study, we tested the action of TNF-alpha and IFN-gamma on phenotypically and functionally defined stages of hematopoietic development using highly purified progenitor cell populations assayed in standardized culture systems. We hypothesized that the inhibitory effects of IFN-gamma and TNF-alpha might be related to the induction of programmed cell death. In methylcellulose colony assays, IFN-gamma and TNF-alpha inhibited the growth of early hematopoietic cells, including committed CD34+CD38+ progenitor cells and phenotypically less mature CD34+CD38- cells, with 50% decreased colony formation occurring in the range of 750-1,000 U/ml of IFN-gamma and 10-15 ng/ml of TNF-alpha. More potent suppressive effects were observed in cultures supplemented with the combination of both cytokines than in cultures treated with IFN-gamma or TNF-alpha alone. When used at these concentrations, IFN-gamma and TNF-alpha inhibited growth of CD34(+)-enriched long-term culture-initiating cells by 88% and 68%, respectively. IFN-gamma and TNF-alpha triggered apoptosis of total bone marrow and CD34+ cells, recognized by the presence of a characteristic pattern of DNA degradation after low molecular weight DNA extraction, and by detection of apoptotic cells by the in situ terminal deoxynucleotidyl transferase assay. We speculate that chronic exposure of hematopoietic tissue to TNF-alpha and IFN-gamma in vivo may result in broad depletion of the stem and progenitor cell pools. Death of these cells due to apoptosis rather than transient inhibition of proliferation may be responsible for long-lasting hematologic consequences.

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Clinical and laboratory studies have suggested involvement of interferon-gamma (IFN-gamma) in the pathophysiology of aplastic anemia. T cells from aplastic anemia (AA) patients secrete IFN-gamma in vitro, activated cytotoxic lymphocytes infiltrate aplastic bone marrow (BM), and IFN-gamma mRNA, not detected in normal BM, is present in BM from most AA patients. Many patients respond to immunosuppressive therapy with antithymocyte globulin and cyclosporine. Using long-term BM cultures (LTBMC) as a tissue culture model of hematopoiesis, we show that IFN-gamma is a potent inhibitor in the long-term culture- initiating cell (LTC-IC) assay, the best in vitro surrogate test for human hematopoietic stem cells, as well as of the output of committed progenitor cells (colony-forming unit-granulocyte-macrophage [CFU-GM] and burst-forming unit-erythroid [BFU-E]). In LTBMC, continuous addition of relatively high IFN-gamma concentrations (1,000 U/mL weekly or 200 U/mL every 2 days) was required for inhibition of secondary colony formation, a measure of LTC-IC number and clonogenicity. To mimick local production of IFN-gamma, human stromal cells were engineered by retroviral-mediated gene transfer to express a transduced IFN-gamma gene. IFN-gamma secreted by stromal cells was far more potent than exogenous IFN-gamma in its effects in the LTC-IC assay. For purified CD34+ cells culture in the presence of IFN-gamma stroma dramatically reduced secondary colony numbers as well as production of CFU-GM and BFU-E. Supernatants from these cultures contained only about 20 U/mL of IFN-gamma; this quantity of cytokine, when added to LTBMC, had little effect on hematopoiesis. The mechanism of hematopoietic suppression was related to the inhibition of cell cycle progression and induction of apoptosis of CD34+ cells. There was no apparent effect of local low-level IFN-gamma production on stromal cell function, as reflected in cell morphology, cell surface phenotype, or expression of hematopoietic growth factor genes. LTBMC with genetically altered stromal cells offers an in vitro model of immune suppression of hematopoiesis in AA and may be helpful in testing certain therapeutic modalities. We infer from our data that local production of low levels of inhibitory cytokine is sufficient to markedly inhibit hematopoiesis and to destroy stem cells and more mature progenitor cells.