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Fas-mediated apoptosis is one of the immune effector pathways leading to the elimination of virus infected cells. In vivo, apoptotic signals are delivered to virus infected cells by Fas-L and other cytokines secreted by specific T lymphocytes. Cellular immune response appears to be essential in prevention of human cytomegalovirus (HCMV) disease. We have hypothesized that HCMV infection might directly or indirectly result in upregulation of Fas receptor and in the presence of Fas ligand, lead to apoptosis of infected cells. We show that infection of human fibroblasts with HCMV is associated with upmodulation of Fas-R process that could be further potentiated by interferon (IFN-gamma). Using DNA agarose gel electrophoresis, terminal dideoxy transferase reaction, and annexin assay, we demonstrated that in a productive HCMV infection of human fibroblasts, loss of cell viability was not only due to virus-mediated cell lysis but also to due to apoptosis. IFN-gamma induced relative HCMV resistance and prevented loss in cell viability. In contrast, anti-Fas monoclonal antibody CH11, serving as Fas agonist, resulted in an accelerated loss in viability of infected cells. IFN-gamma in combination with CH11 further increased the rate of apoptosis and compared to cultures with CH11 only, this effect was not restricted to only infected cells. While IFN-gamma did not affect the number of cells expressing immediate early antigen, it markedly reduced structural protein expression. IFN-gamma in combination with CH11, decreased the expression of HCMV matrix protein pp65, reduced the amount of HCMV DNA and infectious virus produced. Our results are consistent with the theory that cells infected with HCMV can be eliminated by immune effector cells via Fas-mediated apoptosis. IFN-gamma, in addition to its intrinsic antiviral activity, primes HCMV infected cells to the action of Fas ligand and Fas-mediated apoptosis.

Inhibitory and stimulatory cytokines regulate the function and survival of hematopoietic progenitor cells. Interactions between cytokines and progenitor cells may result in programmed cell death. Apoptosis of hematopoietic cells ultimately serves to diminish the size of the stem cell compartment in bone marrow (BM) failure, and this has frustrated efforts at ex vivo expansion of hematopoietic stem cells for BM transplantation. We previously demonstrated that triggering of the Fas-receptor, which is expressed on BM CD34+ cells, mediates apoptosis of progenitor cells. Although interleukin-1beta-converting enzyme (ICE) appears to be an important factor in the signaling cascade regulating Fas-mediated apoptosis of lymphoid cells, its role in apoptosis of CD34+ cells has not been explored. In this study, we determined whether ICE protein was present in CD34+ cells and assessed its role in limiting expansion of hematopoietic stem cells by apoptosis. We demonstrated that ICE mRNA was constitutively produced in CD34+ cells, although the active form of ICE protein was not detected in fresh, unstimulated CD34+ cells from healthy donors. ICE protein could be induced in these CD34+ cells when they were cultured for 24 hours in the presence of hematopoietic growth factors. Interferon (IFN)-gamma and Fas agonist (CH11 monoclonal antibody) enhanced ICE expression and triggered CD34+ cell apoptosis and cell death. In both short- and long-term BM cultures, hematopoietic colony-forming cell numbers were increased after ICE blockade with a synthetic ICE inhibitor (Ac-Tyr-Val-Ala-Asp-aldehyde), even in the absence of IFN-gamma, suggesting that ICE regulates the proliferation and cell death of committed and primitive progenitor cells. The suppressive effect of IFN-gamma and Fas agonist on colony formation was also antagonized by ICE inhibitor. The effects of ICE blockade on proliferation of hematopoietic progenitors cells were related to inhibition of apoptosis, as demonstrated by annexin staining and in situ terminal dideoxytransferase apoptosis assays. Our results suggest that ICE protein is present in CD34+ cells after exposure to cytokines, that regulation of the levels of ICE protein in CD34+ cells is posttranscriptional, and that ICE plays a role in the regulation of apoptosis and expansion of primitive hematopoietic cells. ICE inhibition could potentially be used to reverse intrinsic and cytokine-mediated apoptotic signals for the purpose of stem and progenitor cell expansion.

Patients with aplastic anemia (AA) respond to immunosuppressive therapy, and several lines of laboratory evidence support a role for cell-mediated immunity in the pathogenesis of marrow failure including expansion of cytotoxic T lymphocytes (CTL) in the blood of AA patients, overexpression of inhibitors such as IFN-gamma in the marrow of AA patients, and suppression of hematopoietic cells by CTL in vitro. However, the phenotype of immune effectors in the marrow of AA patients remains unknown. We examined severe (sAA) and moderate AA (mAA) patients and compared them to healthy volunteers and patients with myelodysplastic syndrome (MDS). Our study shows that percentages of HLA-DR+ CD8+ lymphocytes and natural killer (NK) cells, CD56+, were elevated in the marrow of AA patients. Peripheral blood (PB), in all instances, did not reflect changes seen in the bone marrow (BM). Increased percentages of activated CD8+ cells were found in marrow and blood in 43% of AA patients, but in 28% of AA patients, activation of CD8+ cells was only detectable in the marrow. During hematopoietic recovery, activated CD8+ cells and NK cells in marrow declined, but not to normal levels. T cells bearing the gamma delta-phenotype were elevated in the blood of sAA patients (p < 0.05) but were not significantly increased in BM from sAA and MDS patients. Percentages of activated immune effectors are increased in the marrow of AA patients as is consistent with a localized immune response in this disease. Marrow phenotyping may be more sensitive than peripheral blood analysis for detecting an abnormal cellular immune response.

Stem cell inhibitor (SCI) has been shown to inhibit the proliferation of primitive progenitors. The inhibitor, a product of bone marrow macrophages, activated lymphocytes, and monocytes, is identical to macrophage inflammatory protein (MIP-1 alpha). We report homologous (SCI/hMIP-1 alpha) sequences in freshly isolated lymphocytes, monocytes, and granulocytes and have found that SCI mRNA can be induced in monocytes by lipopolysaccharide (LPS) and interleukins 1, 2, and 6. In contrast, interferon gamma (IFN-gamma) decreases the expression of SCI/hMIP-1 alpha. Although only a low level expression of SCI/hMIP-1 alpha mRNA can be detected in normal human bone marrow nucleated cells (NCBM), very significant increases in the levels of SCI/hMIP-1 alpha RNA transcripts are observed in NCBM from patients with aplastic anemia (AA) and myelodysplastic syndrome (MDS). These data suggest that the expression of SCI/hMIP-1 alpha in bone marrow may reflect dysregulated cytokine production and activation of the immune system that may possibly contribute to disease progression.