In vitro effects of interferon-gamma and tumor necrosis factor-alpha on CD34+ bone marrow progenitor cells from aplastic anemia patients and normal donors

Acquired aplastic anemia is characterized by loss or dysfunction of hematopoietic stem and progenitor cells. The proinflammatory cytokines Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) may be responsible for the immune-mediated pathology observed in some patients. The CD34+ population of bone marrow mononuclear cells contains primitive cells responsible for hemopoiesis. We investigated the response of CD34+ cells from aplastic anemia patients to a combination of IFN-gamma and TNF-alpha, and compared them to cells from normal volunteer donors. This was to determine whether aplastic CD34+ cells are more sensitive than normal cells to IFN-gamma/TNF-alpha-mediated effects, and whether cytokine-induced CD95 expression can explain the high levels of apoptosis observed in CD34+ cells from aplastic patients. CD34+38- cells were most affected by overnight incubation with these cytokines, their proportion and numbers being reduced in both normal donors and patients. There was no evidence for increased apoptosis, suggesting that this effect may be due to differentiation. IFN-gamma/TNF-alpha induced upregulation of CD95 on both normal and aplastic CD34+ cells, although the basal level of CD95 expression was increased in aplastic cells. However, CD95 induction did not make cells from normal donors or aplastic anemia patients susceptible to induction of apoptosis by agonistic anti-CD95 antibodies, soluble CD95 ligand, or membrane-bound CD95L. In vivo CD95L is required for CD95 induced apoptosis. No forms of this protein were detectable in lymphocytes from aplastic patients. We conclude that increased apoptosis in aplastic CD34+ cells is not due to increased sensitivity to IFN-gamma/TNF-alpha. We further show that normal and aplastic CD34+ cells are resistant to CD95 apoptosis, even in the presence of mCD95L.     

Interferon-gamma constitutively expressed in the stromal microenvironment of human marrow cultures mediates potent hematopoietic inhibition

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.     

Further evidence for lymphokine overproduction in severe aplastic anemia [see comments]

Interferon-gamma (IFN-gamma) and tumor necrosis factor (TNF) are lymphokines with a potent hematopoietic progenitor cell suppressive capacity. In untreated and immunosuppressed patients with severe aplastic anemia (SAA) and in control individuals we measured (a) serum levels of IFN-gamma and TNF and its production by peripheral blood mononuclear cells (PBMNC); (b) serum levels of neopterin, a product that reflects endogenous IFN production; (c) resting and activated lymphocyte subpopulations; and (d) serum levels of soluble interleukin- 2 receptor (IL-2R). Serum levels of IFN and TNF did not differ significantly in untreated and treated SAA patients and control individuals. Spontaneous and phytohemagglutinin-induced production of IFN and TNF by PBMNC, however, were highly increased in both untreated and treated SAA patients. Increased and decreased neopterin serum levels in untreated and treated SAA patients, respectively, suggest modulation of endogenous lymphokine release subsequent to immunosuppression. HLA-DR+ antigen was mainly expressed by CD8 T cells. Circulating numbers of activated (CD4 and CD8) T cells and serum levels of IL-2R were not increased in both untreated and treated SAA patients. The proportion of HLA-DR+ T cells in the PBMNC of untreated SAA patients correlated with the extent of lectin-induced IFN production. Although we were unable to confirm previous reports in SAA on (a) detectable IFN in blood and bone marrow serum, (b) improvement of stem cell growth upon neutralization of endogenous IFN, (c) absolutely increased numbers of circulating activated T cells, and (d) normalization of these abnormalities subsequent to successful immunosuppression, our data clearly support previous reports on abnormal lymphokine production in severe aplastic anemia. Our failure to relate this phenomenon to the severity of disease states, however, further raises doubts on the pathogenetic significance of lymphokine overproduction in SAA.     

In vitro production of granulocyte-macrophage colony-stimulating factor in aplastic anemia: possible mechanisms of action of antithymocyte globulin

Various abnormalities of lymphokine production have been described in patients with aplastic anemia. To determine if abnormal production of hematopoietic growth factors could contribute to the process of aplastic anemia we studied the in vitro production of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) by phytohemagglutinin (PHA)- and antithymocyte globulin (ATG)-stimulated peripheral blood lymphocytes from 29 patients with aplastic anemia and 15 normal controls. GM-CSF production in response to 1% PHA was seen in nearly all samples (43 of 44) and similar amounts of GM-CSF were produced by patients with aplastic anemia and normal controls. Production of GM-CSF by ATG-stimulated lymphocytes was seen in 7 of 23 patients with aplastic anemia (30%); two of these patients also demonstrated low-level spontaneous production of GM-CSF. Production of GM-CSF in response to ATG was also seen in 2 of 11 normal controls (18%) and barely detectable spontaneous production of GM-CSF was seen in both. Biologically active IL-3 could also be detected in PHA- or ATG-stimulated peripheral blood mononuclear cells in several patients and normal controls. Our results indicate that lymphocytes from patients with aplastic anemia can be stimulated in vitro to produce normal quantities of GM-CSF, suggesting that impaired potential for production of T-cell derived hematopoietic growth factors is unlikely to account for the marrow hypoplasia seen. In several patients overproduction of GM-CSF was observed, consistent with the notion that some patients with aplastic anemia may have circulating activated T cells. We also demonstrate that ATG can stimulate the production of growth factors such as IL-3 and GM-CSF, supporting the role for ATG in stimulating hematopoiesis.     

Interferon is a mediator of hematopoietic suppression in aplastic anemia in vitro and possibly in vivo.

We have investigated interferon as a mediator of hematopoietic suppression in bone marrow failure. Interferon production by stimulated peripheral blood mononuclear cells from patients with aplastic anemia was significantly higher than that observed in controls; spontaneous interferon production by these cells was also high for more than half of aplastic anemia patients. Circulating interferon, not detectable in normal individuals, was detected in 10 of 24 patients. Interferon is a potent inhibitor of hematopoietic cell proliferation and, therefore, may be the mediator of suppression in many in vitro models employing patients' cells and sera. The possible pathogenic importance of interferon in aplastic anemia was suggested by an increase in hematopoietic colony formation in vitro after exposure of bone marrow cells to antiinterferon antisera (277 +/- 71% increase for patients compared to 1.6 +/- 1.6% for normal individuals). Interferon levels in the bone marrow sera of aplastic anemia patients were high (mean = 203 international units (IU)/ml, n = 8), even in comparison to circulating levels in the same patients. Normal bone marrow sera also contained measurable interferon but at lower levels (41 IU/ml, n = 16), indicating that interferon may be a normal bone marrow product. High concentration of bone marrow interferon, possibly due to abnormal immunologic activity or a reaction to virus infection of the bone marrow, may mediate hematopoietic suppression in aplastic anemia patients.     

Lymphokine abnormalities in aplastic anemia: implications for the mechanism of action of antithymocyte globulin

Anti-thymocyte globulin (ATG) provides effective therapy for many patients with aplastic anemia, and its mechanism of action has been presumed to be secondary to lymphocytotoxicity. However, our studies of lymphocyte function in aplastic anemia show marked abnormalities of lymphokine production, which ATG may modulate. In 12 of 17 patients with aplastic anemia, interleukin 2 (IL2) production was markedly elevated in vitro (P less than .01 by paired statistical analysis). Expression of the IL2 receptor, or Tac antigen, on peripheral lymphocytes assessed by flow microfluorometry was also increased above the normal range in 11 of 15 cases. Studies of ATG suggested that it might act to stimulate lymphocyte function. In vitro, ATG is a mitogen, as measured by incorporation of 3H-thymidine into blood mononuclear cells; the response of cells to ATG from patients with aplastic anemia was exaggerated in comparison with normals. Cell proliferation was accompanied by production of IL2 to levels that were, in some cases, similar to those obtained with lectin stimulation. Finally, supernatants from lymphocytes cultured in the presence of ATG were able to replace adherent cells in providing growth factors for the support of nonadherent cells in methylcellulose hematopoietic colony assays. These results provide a mechanism for an "immunostimulatory" action of ATG in effecting hematopoietic response in some patients with aplastic anemia.     

Hematopoietic growth factors in the pathogenesis and for the treatment of aplastic anemia

The production and release of hematopoietic growth factors from bone marrow stromas established in vitro from patients with aplastic anemia is normal or increased. Addition of hematopoietic growth factors to aplastic anemia bone marrow cells results in only modest increases in colony growth, with the exception of granulocyte colony-stimulating factor (G-CSF), which corrects their impaired cloning efficiency to normal. Most clinical data on the use of hematopoietic growth factors in aplastic anemia have derived from uncontrolled and small single-arm studies or case reports. Sustained trilineage hematologic responses have not been observed when hematopoietic growth factors have been used alone or in combination. Serious side effects have been reported for most of the hematopoietic growth factors in patients with aplastic anemia, with the exception of G-CSF. There is a major concern that they may further increase the risk of clonal disorders such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Hematopoietic growth factors should not be used alone in newly diagnosed patients as specific treatment for aplastic anemia, and their use in combination with immunosuppressive therapy should be confined to multicenter, prospective randomized studies.     

Interferon-gamma gene expression in unstimulated bone marrow mononuclear cells predicts a good response to cyclosporine therapy in aplastic anemia.

Cyclosporine (CyA) therapy has been shown to be effective in some patients with aplastic anemia. In an attempt to characterize aplastic patients likely to benefit from CyA therapy, we examined bone marrow mononuclear cells (BMMC) obtained before therapy from 23 patients with aplastic anemia, who were treated with CyA alone. Expression of four myelosuppressive cytokines, including tumor necrosis factor (TNF), lymphotoxin, macrophage inflammatory protein-1 alpha (MIP-1 alpha), and interferon-gamma (IFN-gamma) was examined using polymerase chain reaction (PCR)-assisted messenger RNA (mRNA) amplification. mRNA for TNF, lymphotoxin, and MIP-1 alpha was readily detectable at variable levels in BMMC from normal and transfused controls as well as in BMMC from aplastic patients. In contrast, IFN-gamma mRNA was only demonstrable in BMMC from some patients with aplastic anemia, irrespective of a history of transfusions. Of 13 patients who responded to CyA therapy and achieved transfusion-independence, IFN-gamma mRNA was detected in 12 patients, whereas the mRNA was only detectable in 3 of 10 patients refractory to CyA therapy (P = .003, Fisher's exact test). Follow-up examination of BMMC obtained from seven CyA-responding patients after hematologic remission showed disappearance of IFN-gamma mRNA in four patients. These results suggest that detection of IFN-gamma gene expression in pretreatment BMMC from aplastic patients using PCR may be helpful in predicting a good response to CyA therapy.     

Intracellular interferon-gamma in circulating and marrow T cells detected by flow cytometry and the response to immunosuppressive therapy in patients with aplastic anemia

Immunosuppressive therapy leads to meaningful hematologic improvement in most patients with aplastic anemia (AA). Failure to respond and a later relapse could be due to deficient numbers of hematopoietic stem cells, inadequate treatment of the immune process, or a nonimmunologic etiology. Interferon-gamma (IFN-gamma) has been implicated in the pathophysiology of hematopoietic failure in AA. On the basis of previous findings showing overexpression of IFN-gamma in bone marrow (BM) and peripheral blood (PB) in this disease, we hypothesized that quantitation of IFN-gamma might be applied to predict and monitor responses to immunosuppressive therapy. We measured expression of IFN-gamma in lymphocytes obtained from 123 AA patients, using intracellular 2-color fluorescent staining and flow cytometry. Of 70 patients with severe AA, 36 (51%) demonstrated increased IFN-gamma in circulating T cells. IFN-gamma was detected in only 4 of 53 patients who had recovered from AA. IFN-gamma was not found in PB lymphocytes of patients with other hematologic diseases and heavy transfusion burdens or in healthy volunteers. Among 62 AA patients who were assessed before first treatment with immunosuppressive drugs, 27 of 28 (96%) with circulating IFN-gamma-containing T cells subsequently responded to therapy; in contrast, only 11 of 34 (32%) patients whose PB lacked IFN-gamma lymphocytes improved to transfusion independence. IFN-gamma-containing lymphocytes declined following treatment in all cases. Of 17 patients assessed during relapse, IFN-gamma was present in T cells prior to the blood count decline in 13, and 12 responded to reinstitution of immunosuppressive drugs. Of 30 BMs tested prior to first treatment, 20, all in responding patients, were positive for IFN-gamma, whereas the negative tests were obtained in 10 nonresponding patients. IFN-gamma is increased in the PB lymphocytes of many patients with AA, and these cells decline with therapy. The presence of intracellular IFN-gamma may predict response to immunosuppressive treatment and also the onset of relapse.     

Regulation of the tyrosine kinase Itk by the peptidyl-prolyl isomerase cyclophilin A

Interleukin-2 tyrosine kinase (Itk) is a nonreceptor protein tyrosine kinase of the Tec family that participates in the intracellular signaling events leading to T cell activation. Tec family members contain the conserved SH3, SH2, and catalytic domains common to many kinase families, but they are distinguished by unique sequences outside of this region. The mechanism by which Itk and related Tec kinases are regulated is not well understood. Our studies indicate that Itk catalytic activity is inhibited by the peptidyl prolyl isomerase activity of cyclophilin A (CypA). NMR structural studies combined with mutational analysis show that a proline-dependent conformational switch within the Itk SH2 domain regulates substrate recognition and mediates regulatory interactions with the active site of CypA. CypA and Itk form a stable complex in Jurkat T cells that is disrupted by treatment with cyclosporin A. Moreover, the phosphorylation levels of Itk and a downstream substrate of Itk, PLCgamma1, are increased in Jurkat T cells that have been treated with cyclosporin A. These findings support a novel mode of tyrosine kinase regulation for a Tec family member and provide a molecular basis for understanding a cellular function of the ubiquitous peptidyl prolyl isomerase, CypA.     

Perforin gene mutations in patients with acquired aplastic anemia

Perforin is a cytolytic protein expressed mainly in activated cytotoxic lymphocytes and natural killer cells. Inherited perforin mutations account for 20% to 40% of familial hemophagocytic lymphohistiocytosis, a fatal disease of early childhood characterized by the absence of functional perforin. Aplastic anemia, the paradigm of immune-mediated bone marrow failure syndromes, is characterized by hematopoietic stem cell destruction by activated T cells and Th1 cytokines. We examined whether mutations in the perforin gene occurred in acquired aplastic anemia. Three nonsynonymous PRF1 mutations among 5 unrelated patients were observed. Four of 5 patients with the mutations showed some hemophagocytosis in the bone marrow at diagnosis. Perforin protein levels in these patients were very low or absent, and perforin granules were completely absent. Natural killer (NK) cell cytotoxicity from these patients was significantly decreased. Our data suggest that PRF1 genetic alterations help explain the aberrant proliferation and activation of cytotoxic T cells and may represent genetic risk factors for bone marrow failure.     

Cytokine gene polymorphisms in acquired bone marrow failure

Some acquired aplastic anemia (AA) results from immune-mediated destruction of hematopoietic stem cells. Cytokine gene polymorphisms are implicated in controlling cytokine production and increasing the susceptibility to some autoimmune diseases. We characterized the IL-6/-174, TNF-alpha/-308, IL-10/-1082, IFN-gamma/+874, TGFbeta1/-509 single nucleotide polymorphisms (SNP's) and the IL1-RA second intron variable number tandem repeat (VNTR) alleles in 73 patients with AA and compared the frequency of genotypes to established control populations. We found that some patients with acquired AA have polymorphisms which are linked to high production of proinflammatory cytokines, particularly TNF-alpha and IFN-gamma.