Mesenchymal stem cells contribute to the abnormal bone marrow microenvironment in patients with chronic idiopathic neutropenia by overproduction of transforming growth factor-ß1.

Chronic idiopathic neutropenia (CIN) is a granulopoiesis disorder associated with an inhibitory bone marrow (BM) microenvironment consisting of activated T-lymphocytes and pro-inflammatory mediators. In this study, we investigated the possible involvement of BM mesenchymal stem cells (MSCs) in the pathophysiology of CIN by assessing the frequency and function of BM MSCs in terms of the proliferative/clonogenic characteristics, the differentiation capacity, the potential to produce pro-inflammatory cytokines, and the ability to suppress T-cell proliferation. The frequency, differentiation capacity toward adipocytes, chondrocytes, or osteoblasts, and immunosuppressive potential to inhibit mitogen-induced T-cell proliferation did not differ significantly between patient (n = 14) and normal (n = 21) MSCs. Tumor necrosis factor-α, interleukin-1ß, and interleukin-6 levels in MSC supernatants did not differ significantly between patients and controls; however, transforming growth factor (TGF)-ß1 levels were significantly elevated in patients, particularly in those displaying the -509C/T TGF-ß1 polymorphism. Patient MSCs displayed defective proliferative/clonogenic potential, which could not be attributed to altered cellular survival characteristics or to increased TGF-ß1 production as TGF-ß1 neutralization did not restore the impaired colony formation by patient MSCs. We conclude that although BM MSCs do not exert a significant role in the immune deregulation associated with CIN, they contribute to the inhibitory microenvironment by overproducing TGF-ß1, at least in patients displaying the -509C/T polymorphism.

Biologic characteristics of bone marrow mesenchymal stem cells in myelodysplastic syndromes.

Myelodysplastic syndromes comprise a heterogeneous group of clonal hematopoietic stem cell malignancies characterized by ineffective bone marrow (BM) hematopoiesis, peripheral blood cytopenias and substantial risk for progression to acute myeloid leukemia. It is generally accepted that myelodysplastic syndromes originate as a result of multistep leukemogenesis, implicating genetic, epigenetic and immune-mediated alterations of an early hematopoietic stem cell. However, alterations in the BM microenvironment in terms of abnormal hematopoietic-to-stromal cell interactions, relative deficiency of hematopoietic growth factors and aberrant release of inhibitors may also have a role in myelodysplastic syndrome (MDS) pathogenesis. The possible involvement of the BM mesenchymal stem cells (MSC) in the pathogenetic/pathophysiologic process of MDS has been recently studied but existing data on MSCs' cytogenetic and functional integrity are controversial. Notably, in our study we did not find any significant quantitative or qualitative deficits in MDS-derived MSCs. As no conclusive data on the characteristics of BM MSCs have been reported so far, future studies should aim at elucidating whether BM MSCs belong primarily to the abnormal clone or whether they are indirectly damaged and whether they might be safely used for therapeutic purposes in MDS patients. This article aims to give an overview of the current state of the art on the quantitative, functional, immunoregulatory and cytogenetic properties of BM MSCs in MDS.

Reserves, functional, immunoregulatory, and cytogenetic properties of bone marrow mesenchymal stem cells in patients with myelodysplastic syndromes.

Defective hematopoiesis supporting capacity of bone marrow (BM) stroma has been implicated in the pathophysiology of myelodysplastic syndromes (MDS). The aim of this study is to explore whether the BM stroma progenitors, namely the mesenchymal stem cells (MSCs), are primarily affected in MDS by evaluating the reserves, the functional properties, as well as the cytogenetic characteristics, in comparison to BM hematopoietic cells, in patients with de novo MDS (n = 13). The number, differentiation potential toward adipocytes/chondrocytes/osteoblasts and immunosuppressive function in terms of inhibition of mitogen-induced T-cell proliferation did not differ significantly between patient and normal (n = 20) MSCs. Patient MSCs did not show any aberrations in the production of proinflammatory or growth-promoting cytokines and did not harbor the cytogenetic abnormalities present in hematopoietic cells. Occasional patient and normal MSC cultures, however, developed irrelevant chromosomal alterations (trisomies 5 and 7) with uncertain pathophysiologic significance. Compared to controls, patient MSCs displayed impaired proliferative and clonogenic potential through passages that might represent a nonspecific abnormality associated with the chronic inflammatory process present in patients' BM. These data suggest that BM MSCs from MDS patients do not belong to the abnormal clone and do not represent the main cellular source contributing to the inflammatory marrow microenvironment.

The -509C/T polymorphism of transforming growth factor-beta1 is associated with increased risk for development of chronic idiopathic neutropenia.

OBJECTIVE: Impaired granulopoiesis in chronic idiopathic neutropenia (CIN) has been associated with an inflammatory bone marrow (BM) microenvironment consisting of pro-inflammatory and pro-apoptotic mediators, such as tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1, and Fas-Ligand (Fas-L). In this study, we evaluated the frequency of TNF-alpha, TGF-beta1 and Fas-L gene polymorphisms in CIN patients and explored their role in excessive cytokine production and their association with CIN development. METHODS: The TNF-alpha-308G/A, TGF-beta1 -509C/T, +869T/C, +915G/C, and Fas-L -844T/C polymorphisms were studied in 57 CIN patients, and 100 healthy controls from Crete, a well-defined area with genetically homogeneous population, using a polymerase chain reaction-based restriction fragment length polymorphism assay. RESULTS: The mutant genotype C/T or T/T of TGF-beta1 -509C/T polymorphism was more common in CIN patients than in controls (P = 0.033). Compared to wild-type genotype, the TT genotype was associated with increased risk for CIN development (OR: 5.7; 95% CI: 1.18-27.26; P = 0.033). Compared to controls, patients with CT and TT genotypes displayed increased TGF-beta1 levels in serum (P < 0.0001 and P = 0.0002, respectively) and BM (P < 0.0001 and P = 0.0002, respectively). No significant difference was found between patients and controls in the frequency of TNF-alpha-308G/A, TGF-beta1 +869T/C and +915G/C and Fas-L -844T/C polymorphisms. CONCLUSIONS: The TGF-beta1 -509C/T polymorphism is associated with increased risk for CIN and contributes to the pathophysiology of the disorder by inducing TGF-beta1 overproduction. This is the first study providing evidence that genetic factors may predispose to CIN and may have a role in the pathophysiology of the disorder.

Increased levels of soluble flt-3 ligand in serum and long-term bone marrow culture supernatants in patients with chronic idiopathic neutropenia.

The levels of serum and long-term bone marrow culture supernatant soluble flt-3 ligand (sFL) were determined in 54 patients with chronic idiopathic neutropenia (CIN) and 16 normal controls. Both serum and supernatant sFL levels were significantly increased in the patients compared with controls. Individual sFL values inversely correlated with the number of circulating neutrophils and the proportion of bone marrow CD34+ cells. Supernatant sFL values positively correlated with the levels of supernatant G-CSF. These findings suggest that the impaired myelopoiesis in CIN patients is accompanied by a compensatory mechanism attempting to increase the neutrophil production at the myeloid progenitor cell level.

Soluble c-kit ligand production by bone marrow stromal cells is independent of the degree of neutropenia in patients with chronic idiopathic neutropenia.

The levels of soluble c-kit ligand (sKL), also known as Steel factor or stem cell factor, were measured in blood serum and long-term bone marrow culture supernatants of 81 patients with chronic idiopathic neutropenia (CIN) and 22 normal controls using a commercially available enzyme-linked immunosorbent assay (ELISA). We found that the levels of serum and culture supernatant sKL did not differ significantly between patients and control subjects and that both serum and supernatant values of the cytokine did not correlate with the number of circulating neutrophils. Furthermore, we found that the levels of the culture supernatant granulocyte colony-stimulating factor (G-CSF), also measured by ELISA, were significantly increased in the patients compared to controls but individual G-CSF values did not correlate with the values of supernatant sKL. These findings suggest that sKL-producing cells continuously secrete sKL and that cytokine secretion is independent of the degree of neutropenia or the levels of supernatant G-CSF in patients with CIN.

High prevalence of Helicobacter pylori infection and monoclonal gammopathy of undetermined significance in patients with chronic idiopathic neutropenia.

The prevalence of Helicobacter pylori infection was evaluated in 120 patients with chronic idiopathic neutropenia (CIN), 8 patients with monoclonal gammopathy of undetermined significance (MGUS) associated with CIN, and 74 age- and sex-matched normal volunteers, all derived from the same geographical area. The purpose of the study was to investigate the possible causal relationships of H. pylori infection with the development of MGUS in CIN patients. We found that the prevalence of H. pylori infection was elevated to 69.2% in the group of CIN patients, 100% in the group of patients with CIN-associated MGUS, and 32.4% in the group of control subjects. No statistically significant difference, however, was found in the prevalence of H. pylori infection between CIN patients with concomitant MGUS and CIN patients without MGUS, no resolution of the gammopathy after eradication of the bacterium, no significant rise in the titers of serum anti-H. pylori antibodies, and no formation of an abnormal precipitation line in immunoelectrophoresis using a saline extract of NCTC11367 H. pylori reference strain as antigen. We concluded that there is no evidence that H. pylori infection is the cause of MGUS in CIN patients.