Tadatsugu Sato

Increased expression of Fas antigen on bone marrow CD34+ cells of patients with aplastic anaemia

Summary 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‐γ (IFN‐γ) and tumour necrosis factored (TNF‐α), 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‐γ, increased TNF‐β 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‐γ and/or TNF‐α 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.

Nitric oxide suppression of human hematopoiesis in vitro. Contribution to inhibitory action of interferon-gamma and tumor necrosis factor-alpha.

IFN-gamma and TNF-alpha, potent inhibitors of hematopoiesis, induce nitric oxide synthase (NOS) in various cell types. When normal human bone marrow (BM) or CD34+ cells were exposed to NO, inhibition of colony formation was dose dependent and direct. NO induced apoptosis in BM progenitors, as shown by electrophoretic detection of DNA degradation and deoxynucleotidyl transferase assay. Using PCR and immunoprecipitation, we found inducible NOS (iNOS) mRNA and iNOS protein in BM after stimulation with IFN-gamma or TNF-alpha. iNOS mRNA was also detected by PCR in highly purified CD34+ cells; TNF-alpha or IFN-gamma increased iNOS expression. The presence of iNOS in CD34+ cells was confirmed in single cells by immunochemical staining. NG-Monomethyl-L-arginine (MM-Arg), an NOS inhibitor, partially reversed the effects of TNF-alpha and, to a lesser extent, IFN-gamma in methylcellulose culture of total BM and CD34+ cells, and inhibited apoptosis of BM cells induced by these cytokines. When the effects of competitive iNOS inhibition were tested on more immature progenitors, MM-Arg increased the number of long-term BM culture-initiating cells in control cultures but failed to protect these cells from the inhibitory action of IFN-gamma and TNF-alpha. Our results suggest that NO may be one mediator of cytokine-induced hematopoietic suppression.

Expression and modulation of cellular receptors for interferon-γ, tumour necrosis factor, and Fas on human bone marrow CD34+ cells

Interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α) and Fas‐ligand can mediate potent inhibitory signals in haemopoietic cells. Clinical and laboratory studies have suggested the involvement of these cytokines in the regulation of normal haemopoiesis and in the pathophysiology of bone marrow (BM) failure syndromes. As the effects of cytokines may also be regulated at the cellular receptor level, we studied the expression and modulation of TNF receptor (TNFR), IFN‐γR and Fas‐R on haemopoietic progenitor cells. In freshly isolated BM, using flow cytometry, TNFR1 (p55), TNFR2 (p75), IFN‐γR, and Fas‐R were detected on 5–12% of mononuclear cells. Two‐colour staining showed comparable receptor expression on a CD34+ population, which includes haemopoietic progenitor and stem cells. Using reverse transcriptase‐PCR (RT‐PCR) transcription of mRNA coding for these receptors was demonstrated in fresh, highly purified CD34+ cells. These findings indicate that the effects of these factors on progenitor cells may be directly mediated. In cultured BM cells, expression of TNFR1 was not influenced by IFN‐γ, TNF‐α or apoptosis‐inducing anti‐Fas monoclonal antibody (mAb). IFN‐γ decreased CD34+ cell TNFR2 expression. CD34+ cell Fas‐R expression was increased by IFN‐γ and TNF‐α. IFN‐γR expression was enhanced by anti‐Fas mAb and to lesser degree with TNF‐α. Similar results were obtained with RT‐PCR analysis in cultured CD34+ cells. Potentiation of anti‐Fas mAb‐mediated inhibition of haemopoietic colony formation by IFN‐γ and TNF‐α was observed. Similarly, anti‐Fas mAb enhanced the inhibitory effects of IFN‐γ. These results suggest that, in addition to interacting at the level of intracellular signalling pathways, IFN‐γ, TNF‐α or Fas‐ligand may potentiate or antagonize their effects through modulation of cytokine receptor expression.

Secondary colony formation after long-term bone marrow culture using peripheral blood and bone marrow of HIV-infected patients.

Objective:To determine if defective bone marrow function in HIV infection is associated with decreased numbers of progenitor cells or defective stromal cell function. Design:Defective bone marrow function may in part be responsible for the cytopenias so frequently seen in patients with AIDS. Although a number of investigators have reported impaired growth of committed hematopoietic progenitor cells in HIV-1-infected patients, few studies have examined the most primitive hematopoietic stem cells. Our study was designed to determine the function and quality of the most immature hematopoietic progenitor and stem cells in the peripheral blood and bone marrow of HIV-1-infected patients and to assess stromal cell function. Methods:For quantification of these cells we used a modified long-term culture-initiating cell (LTCIC) assay in which the number of secondary colony-forming cells after 5 weeks of stromal culture served as a measure of LTCIC. Stromal cells from normal controls and HIV-1-infected patients were used for cross-matching experiments. Normal CD34+ cells or those derived from HIV-infected patients were plated and colony growth assessed. Results:We found that HIV-1-infected patients had a mean of 0.8 secondary colony-forming cells/105 peripheral blood mononuclear cells (PBMC), whereas normal controls showed 1.2 secondary colony-forming cells/105 PBMC (P = not significant) after long-term culture. Asymptomatic patients showed well preserved numbers of secondary colony-forming cells after long-term culture of PBMC, but a significant reduction was seen in patients with a history of opportunistic infections (P < 0.01), low CD4+ cell count (< 200 × 106/l; P< 0.05), or leukopenia (P < 0.05). Decreased numbers of secondary colony-forming cells have also been found in bone marrow of HIV-1-infected patients with advanced disease. When normal CD34+ cells were cultured on stromal layers from bone marrow of HIV-1-infected patients or normal controls, no differences in the numbers of surviving progenitor cells were found. Conclusions:Our data indicate that the hematopoietic defect in HIV-1 infection involves the most immature hematopoietic cells and becomes evident in advanced disease. Stromal function of HIV-infected patients appears normal.