Evidence for a role of TNF-related apoptosis-inducing ligand (TRAIL) in the anemia of myelodysplastic syndromes

Abstract

Myelodysplastic syndromes (MDS) are characterized by impaired erythropoiesis, possibly caused by proapoptotic cytokines. We focused our study on the cytokine TRAIL (TNF-related apoptosis-inducing ligand), which has been shown to exhibit an anti-differentiation activity on erythroid maturation. Immunocytochemical analysis of bone marrow mononuclear cells (BMMC) showed an increased expression of TRAIL in MDS patients with respect to acute myeloid leukemia (AML) patients and normal BM donors. TRAIL expression was increased predominantly in myeloid precursors of granulocytic lineage and in a subset of monocytes and pro-erythroblasts. Significant levels of soluble TRAIL were released in 21 of 68 BMMC culture supernatants from MDS patients. On the other hand, TRAIL was detected less frequently in the culture supernatants of AML (4 of 33) and normal BMMC (0 of 22). Analysis of peripheral blood parameters revealed significantly lower levels of peripheral red blood cells and hemoglobin in the subset of patients whose BMMC released TRAIL in culture supernatants compared to the subgroup of patients who did not release TRAIL. Moreover, TRAIL-positive BMMC culture supernatants inhibited the differentiation of normal glycophorin A+ erythroblasts generated in serum-free liquid phase. Thus, increased expression and release of TRAIL at the bone marrow level is likely to impair erythropoiesis and to contribute to the degree of anemia, the major clinical feature of MDS.

Figure 1

TRAIL expression in normal and pathological BMMC. TRAIL expression was examined by immunohistochemistry by APAAP, using anti-TRAIL mAb. In A, BMMC slides, obtained from 6 to 10 different individuals per study group were examined. Data are expressed as mean ± SD of percentage of TRAIL-positive cells calculated by scoring at least six random fields for each slide. Representative fields of BMMC from normal donors (B), from MDS (C), and AML (D) patients, examined immunohistochemically for TRAIL are shown. Stainings with May-Gruenwald-Giemsa of the same samples are also shown. In B, some TRAIL-positive metamyelocytes and granulocytes (white asterisks, left panels) and megakaryocytes (white arrow, right panel) are indicated, while erythroblasts (black arrowhead, left panel) are negative for TRAIL expression. In C, several myeloid cells at different level of differentiation exhibit a bright positivity for TRAIL. While erythroblasts are negative (asterisks in left and right panels), a subset of proerythroblasts (arrowhead, right panel) are positive. In D, monocytoid blast cells show either negativity (bottom panel) or a dim positivity (top panel) for TRAIL. Original magnification, ×20.

Figure 2

Release of TRAIL in the supernatants of normal and pathological BMMC. Culture supernatants derived from BMMC of control individuals (ie, BM not involved by any pathology) and of patients affected by AML or MDS were analyzed for soluble TRAIL by ELISA. The absolute number of TRAIL-positive samples in each study group are reported. Each dot was determined in duplicate. *, Statistically significant differences in the frequency of TRAIL detection (P < 0.05).

Figure 3

Peripheral blood (PB) and BM hematological parameters. PB hematological parameters (A) and BM hematopoietic progenitors (B) were measured in the control group as well in the MDS patients. Comparisons between the patients in which soluble TRAIL was (TRAIL-positive) or was not (TRAIL-negative) detected in the supernatants of MDS BMMC are indicated. Horizonal bars are median, upper and lower edges of box are 75th and 25th percentiles, lines extending from box are 10th and 90th percentiles. Parameters are expressed as follow: RBC × 10³/μl; Hb g/dl; Platelets (Plts) × 10³/μl; WBC × 10³/μl; BFU-E/10⁵ BMMC; CFU-GM/10⁵ BMMC. *, Statistically significant differences (<0.05).

Figure 4

Effect of conditioned media of MDS BMMC on erythroid maturation. CB-derived CD34⁺ cells were cultured in the presence of EPO + IL-3 + SCF for 6 days. At this time point, cultures were supplemented with conditioned media of TRAIL-positive MDS BMMC, in the presence or absence of TRAIL-R2-Fc chimeric protein. At 12 days of cultures, cells were analyzed for GPA expression by flow cytometry. Shadowed histograms represent cells stained with mAb anti-GPA, while unshadowed histograms represent the background fluorescence obtained from the staining of the same cultures with isotype-matched control mAb. Mean fluorescence intensity (MFI) values are indicated. Similar results were observed in three independent experiments.