Transforming growth factor-beta1 affects interleukin-10 production in the bone marrow of patients with chronic idiopathic neutropenia

Background: Chronic idiopathic neutropenia (CIN) is a bone marrow (BM) failure syndrome characterized by accelerated apoptosis of myeloid progenitor cells because of a local imbalance between pro‐inflammatory and anti‐inflammatory cytokines. In this study, we investigated the interplay among transforming growth factor‐beta1 (TGF‐β1), interleukin‐10 (IL‐10), and soluble flt‐3 ligand (sFL) within the BM of CIN patients and probed the role of these cytokines in the pathophysiology of CIN. Design: We used long‐term BM cultures (LTBMC) to evaluate TGF‐β1, IL‐10, and sFL levels in CIN patients (n = 70) and healthy subjects (n = 35). Cytokine levels in LTBMC supernatants were correlated with the number of circulating neutrophils and the proportion of BM CD34⁺/CD33⁺ myeloid progenitor cells. Results: CIN patients had increased TGF‐β1 and sFL levels in LTBMCs compared with controls and individual cytokine values were found to be correlated inversely with the number of neutrophils and the proportion of CD34⁺/CD33⁺ cells. Patients displayed low supernatant IL‐10 levels compared with controls and cytokine values were found to be correlated positively with the number of neutrophils and the proportion of CD34⁺/CD33⁺ cells. The levels of TGF‐β1 were found to be inversely correlated with IL‐10 and positively with sFL values in LTBMC, supernatants suggesting a possible interplay among these cytokines in CIN BM. Neutralization of TGF‐β1 in LTBMCs increased IL‐10 levels significantly in patients but not in controls, while neutralization had no effect on sFL levels. Conclusion: Excessive production of TGF‐β1 within the BM microenvironment of CIN patients results in downregulation of IL‐10 and reduction of myeloid progenitor cells. Overexpression of sFL probably represents a compensatory mechanism to the low myeloid progenitor cells.     

Chronic idiopathic neutropenia: Granulopoietic assessment by both marrow culture and granulocyte kinetics

Granulopoietic assessment was made in 14 patients with chronic idiopathic neutropenia (CIN) whose neutrophils were consistently less than 1.5 × 10⁹/ liter and in whom there was absence of splenomegaly and recent drug ingestion. Granulopoiesis was studied using a combination of bone marrow culture in nutrient agar and granulocyte kinetics. Agar colony growth assessed bone marrow concentration of granulocyte progenitor cells (GPC) and the proportion of GPC in DNA synthesis by in vitro ³HTdR suicide. Granulocyte kinetics with in vitro DF³²P labelling of patient granulocytes measured granulocyte half-life (T^1/2), turnover rate (GTR), and the circulating, marginated, and total blood granulocyte pools. The results indicated that either GPC concentration or the proportion of GPC in DNA synthesis was outside the normal range in all but one patient and decreased in ten out of 14 patients. CIN was also characterized by reduced total, circulating, and marginated blood granulocyte pools, reduced GTR, and normal granulocyte half-life. The neutropenia appeared to be due to a variety of intra-marrow causes, including either a reduction in the GPC compartment, a reduction in GPC proliferation, a maturation arrest, or a reduced amplication during granulopoiesis. Increased granulocyte utilization, intra-vascular destruction or excessive margination could be excluded as possible causes of CIN in this series. Although GPC parameters tended to be reduced, suggesting a production defect, there were signs in nine patients that the bone marrow was attempting to compensate for the peripheral neutropenia. It is suggested that for a complete assessment of granulopoiesis in man, granulocyte kinetic studies need to be combined with quantitative studies of the bone marrow granulocyte progenitor compartment using the agar colony system.     

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.     

Impaired megakaryopoiesis in patients with chronic idiopathic neutropenia is associated with increased transforming growth factor beta1 production in the bone marrow

Patients with chronic idiopathic neutropenia (CIN) display relatively low peripheral blood platelet counts and hypo-lobulated megakaryocytes in the bone marrow (BM). The underlying pathogenetic mechanismswere probed by studying the reserves and clonogenic potential of BM megakaryocytic progenitor cells using flow-cytometry and a collagen-based clonogenic assay for the identification of megakaryocyte colony-forming units (CFU-Meg). Thrombopoietin (TPO) and transforming growth factor-beta1 (TGFbeta1) levels were also evaluated in long-term BM culture supernatants using an enzyme-linked immunosorbent assay. CIN patients (n = 39) showed a low proportion of BM CD34(+)/CD61(+) megakaryocytic progenitor cells and low frequency of early and mixed CFU-Meg in the BM mononuclear, but not CD34(+), cell fraction, compared with healthy controls (n = 20). TPO and TGFbeta1 levels were significantly higher in patients compared with controls. TPO levels inversely correlated with platelet counts whereas TGFbeta1 values correlated inversely with CD34(+)/CD61(+) and CFU-Meg megakaryocytic progenitor cell numbers and positively with TPO levels. The addition of an anti-TGFbeta1 neutralising antibody significantly increased the numbers of CFU-Meg in CIN patients but not in controls, compared with baseline. These data suggest that increased local production of TGFbeta1 probably affects the BM megakaryocytic progenitor cell growth in CIN whereas the compensatory production of TPO finally balances the TGFbeta1-induced inhibitory effect.     

Serum granulocyte colony-stimulating factor levels in patients with chronic neutropenia of childhood: modulation of G-CSF levels by myeloid precursor cell mass

The serum G-CSF levels of eight patients with severe congenital neutropenia (SCN) were found to be significantly higher than those of 22 patients with chronic benign neutropenia (CBN). The relative number of cells expressing the G-CSF receptor in light density bone marrow cells (LDBMC) was lower in patients with SCN than in patients with CBN or in normal subjects. When recombinant human G-CSF was incubated with LDBMC, G-CSF levels were decreased by LDBMC from normal subjects and CBN patients, but not by those from SCN patients. Serum G-CSF concentrations, which are affected by mature neutrophils, may also be modulated by myeloid precursor cells in the bone marrow.     

Pro-inflammatory bone marrow milieu in patients with chronic idiopathic neutropenia is associated with impaired local production of interleukin-10

The levels of interleukin-10 (IL-10) were evaluated in long-term bone marrow (BM) culture supernatants from 54 patients with chronic idiopathic neutropenia (CIN) and 30 healthy volunteers using enzyme-linked immunoabsorbent assay. Cytokine levels were significantly reduced in patients, compared with controls, and strongly correlated with peripheral blood neutrophil counts. Low levels of supernatant IL-10 were associated with increased values of supernatant IL-1beta, tumour necrosis factor-alpha, IL-6 and transforming growth factor-beta(1). We suggest that the pro-inflammatory milieu in the BM of CIN patients may be causatively related to the impaired production of IL-10, a cytokine normally displaying strong anti-inflammatory properties.     

Phase II study of glycosylated recombinant human granulocyte colony-stimulating factor after HLA-identical sibling bone marrow transplantation

Background: The lengthy period of neutropenia which follows allogeneic bone marrow transplantation (BMT) results in significant morbidity and some mortality. Recombinant human granulocyte colony-stimulating factor (rhuG-CSF) effectively reduces neutropenia and morbidity when given after autologous BMT, but has not been adequately investigated in allografts. Aims: To assess the tolerability, safety and efficacy of rhuG-CSF after allogeneic BMT. Methods: rhuG-CSF was administered to 13 adult patients with haematological malignancies after HLA-identical sibling BMT. Five μg/kg of rhuG-CSF was given daily by subcutaneous bolus injection, commencing four hours after marrow infusion and continuing until the neutrophil count was ≥ 1.0 × 10⁹/L on three consecutive days. Graft-versus-host disease (GVHD) prophylaxis was cyclosporin and short-course methotrexate (days 1, 3, 6 and 11). Prophylactic intravenous (IV) antibiotics were administered from the onset of neutropenia. The control group consisted of patients with comparable diagnoses, transplanted before and after the current study using identical supportive care and GVHD prophylaxis policies. Results: Although time to recovery of the neutrophil count to >0.1 × 10⁹/L was similar, the rhuG-CSF-treated patients experienced accelerated recovery to > 0.5 × 10⁹/L, which occurred at a median of 15 days (range 11–21) after marrow infusion in study patients compared to 18.5 days (range 14–41) in the controls (p = 0.04). No significant differences were detected in any of the indices of transplant-related morbidity examined, including the number of days of fever, the incidence of culture-positive infections, the usage of antibiotics, the requirement for parenteral nutrition and IV morphine, the maximum severity of mucositis and GVHD, and the day of discharge. Conclusion: Within the context of this study, rhuG-CSF had limited impact on the clinical outcome of HLA-identical sibling BMT. (Aust NZ J Med 1994; 24: 541–546.)     

Increased cell apoptosis in bone marrow trephine biopsies and immunomagnetically isolated myeloid progenitor cells in patients with chronic idiopathic neutropenia

The frequency of apoptotic cells in bone marrow trephine biopsies and cytospins of immunomagnetically isolated myeloid progenitor cells was determined in 39 patients with chronic idiopathic neutropenia (CIN) and 12 hematologically normal individuals using the in situ end-labeling (ISEL) apoptosis detection method. We found that 66.7% of the patients but none of the normal controls displayed apoptotic cells equal to or higher than 5% of the total mononuclear cells in bone marrow biopsies (p<0.01). In the double stain, we also found that the proportion of apoptotic CD15⁺ myeloid precursor cells did not differ significantly between patients and control subjects, while the proportion of apoptotic CD34⁺ hemopoietic cells could not be estimated with accuracy because of the presence of CD34⁺ endothelial cells. Significantly increased apoptosis was noted in cytospins of immunomagnetically isolated patient CD34⁺ and CD34⁺/CD33⁺ cells but not CD34^-/CD33⁺ cells, compared to the controls (p<0.001, p<0.02 and p>0.05, respectively). These findings confirm and extend our previous observations in flow-cytometric studies of apoptosis in CIN, indicating that increased apoptosis in CIN bone marrow concerns mainly the CD34⁺ and CD34⁺/CD33⁺ progenitor cell compartments. We conclude that the accelerated apoptosis in these compartments may account for the impaired neutrophil production in CIN patients.     

Modification of human long-term bone marrow cultures: establishment of a functional stromal microenvironment devoid of myeloid progenitors.

Differences in the plastic adhesive properties of bone marrow (BM) cells were used to initiate modified stromal layers (MSL) from long-term cultures by removing non-adherent cells shortly (4 to 18 hours) after initial seeding. Following this early modification, adherent cells generated a confluent layer after 21 days of culture. Cellular characteristics of volume and spontaneous fluorescence determined by flow cytometry showed that the MSL included 82% fibroblastic stromal cells, 8% macrophages and 10% myelomonocytic cells. Furthermore, clonogenic assays revealed that the MSL were devoid of hematopoietic progenitor cells. MSL were found to sustain long-term myelopoiesis for at least 7 weeks from exogenously added hematopoietic progenitors isolated from bone marrow (CD34+ cells), thereby demonstrating their functionality. The present experimental model appears of interest for the study of interactions between defined populations of hematopoietic cells and cells of the adherent layer. Of importance, our present modifications of human long-term bone marrow culture are technically simple and do not involve manipulation of the stromal cells.     

Morphologically defined myeloid cell compartments, lymphocyte subpopulations, and histological findings of bone marrow in patients with nonimmune chronic idiopathic neutropenia of adults

P <0.001 and P<0.001, respectively). Individual proportions of these cells strongly correlated with the number of circulating neutrophils (r=−0.462, P<0.01 and r=0.495, P<0.01, respectively). However, in the great majority of patients (78.9%), no significant changes in marrow cellularity or the myeloid to erythroid cell ratio could be demonstrated. Patients also had increased proportions of CD19⁺B cells, CD20⁺B cells, and plasma cells with polytypic expression relative to controls (P<0.02, P<0.01, and P<0.001, respectively). Individual values of plasma cells were inversely correlated with the number of blood neutrophils (r=−0.414, P<0.01). Dispersed bcl-2⁺lymphocytic aggregates without germinal centers were seen in about one-third of the patients. T cells and natural killer (NK) cells did not show any significant change. Patients had increased proportions of CD57⁺, CD16⁺, and HLA-DR⁺ cells and, in a few cases, increased proportions of histiocytes and eosinophils. CD45RO⁺ cells were reduced only in patients with pronounced neutropenia. Expression of p53 protein has not been detected in any cell population. With the exception of some megaloblastoid features of erythroid lineage seen in two patients and the presence of some micromegacaryocytes seen in two others, no significant morphological abnormalities were noted. All of these findings are consistent with our previously reported suggestion for the possible existence of an underlying low-grade chronic inflammatory process in NI-CINA patients, which may be involved in the pathogenesis of neutropenia in the affected subjects. Received: 8 February 2000 / Accepted: 7 April 2000     

Effects of a preformed extracellular matrix on long-term serum-free bone marrow culture

Summary The extracellular matrix (ECM) produced by the stromal layer plays a key role in the regulation of commitment and differentiation of hematopoietic cells. Long-term bone marrow culture (LTBMC) allows analysis of the stromal microenvironment. Recently, serum-free LTBMC has been described, but the formation of a classical adherent layer was never observed under these conditions. We have evaluated the effect(s) of a chemically well defined ECM on serum-free and serum-dependent LTBMC. In serum-dependent cultures ECM did not induce a significant increase of hematopoiesis. In serum-free conditions, a marked improvement of hematopoiesis was observed, both in terms of CFU-GM and BFU-E yield and in duration of cultures. A confluent stromal layer was observed only in the presence of ECM. The present results indicate that the addition of ECM to serum-free cultures provides a standardized culture condition, while improving progenitor cell recovery and allowing formation of a confluent stromal layer. Moreover, ECM+ LTBMC may provide a model to study the effect(s) of adhesive proteins and hematopoietic growth factors normally present in serum.This work was supported by an AIRC grant and the Program Terapia dei Tumori,Istituto Superiore di Sanità, Rome     

Serum levels of lactoferrin and myeloperoxidase in chronic idiopathic and secondary neutropenia

In 20 patients with chronic neutropenia, serum lactoferrin (S-LF) and serum myeloperoxidase (S-MPO) levels were assessed. By immunofluorescence, granulocyte-bound immunoglobulins were detected in 12 patients, whereas circulating immune complexes were found in the blood of 8 patients by the ¹²⁵-I-Clq-binding test (Clq-BT). In both groups of patients, there was a relative increase of S-LF and a relative or sometimes absolute increase of S-MPO. In the latter group, results of the Clq-BT correlated positively with S-MPO but negatively with neutrophil counts. No correlations between S-LF or S-MPO and the results of the granulocyte immunofluorescence test were found. Our results suggest that S-LF and S-MPO levels may be helpful in the further study of patients with chronic neutropenia, to gain more insight into the pathogenetic mechanisms operative in this disease.     

Assessment of bone marrow stem cell reserve and function and stromal cell function in patients with severe congenital neutropenia

OBJECTIVE: To investigate further the cellular defect responsible for impaired granulopoiesis in severe congenital neutropenia (SCN), we have evaluated bone marrow (BM) stem cell reserve and function and BM stromal cell myelopoiesis supporting capacity in two patients with SCN. METHODS: BM primitive stem cells and myeloid progenitor cells were assessed using flow cytometry, limiting dilution assay, clonogenic assays, and long-term BM cultures (LTBMC). BM stroma function was assessed by evaluating the ability of irradiated stromal layers from the patients to induce granulocyte-macrophage colony formation (CFU-GM) by normal CD34+ cells. RESULTS: Compared to the normal controls (n = 37), SCN patients displayed a low percentage of CD34+/CD38+ cells (P < 0.05), low CFU-GM colony formation by highly purified CD34+ cells (P < 0.05), low CFU-GM recovery in LTBMC (P < 0.05), and normal primitive stem cells as indicated by the frequency of CD34+/CD38- cells and the number of long-term culture initiating cells. Patient BM stromal layers exhibited normal myelopoiesis supporting capacity as shown by the CFU-GM content of irradiated LTBMC recharged with normal CD34+ cells. In addition, patient LTBMC supernatants displayed 20-fold normal granulocyte colony stimulating factor and 2-fold normal granulocyte-macrophage colony stimulating factor levels. CONCLUSION: These data show that primitive BM stem cells and stromal cells are not affected in SCN patients, while they support further the concept of a primary defect at the myeloid progenitor cell level. To know the differentiation stage at which the underlying defect causes the malfunction will be relevant for further elucidation of its nature at the molecular level.     

Haemopoietic growth factor production by normal and aplastic anaemia stroma in long-term bone marrow culture

Summary. Defective marrow stroma, or microenvironment, have been proposed as one of several mechanisms to account for bone marrow failure in aplastic anaemia (AA). This could involve defects in positive- or negative-acting haemopoietic regulator expression by AA stroma, or alteration of normal stroma-stem cell interactions.
We have used a sensitive bioassay to investigate production of granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-3, IL-6 and stem cell growth factor (SCF), by normal and AA stroma In long-term bone marrow culture (LTBMC). LTBMC were grown to confluence, irradiated and harvested to yield a single cell suspension. These cells were cocultured with normal target bone marrow mononuclear cells (BMMC), or CD34⁺ cells, in clonogenic assays, in the absence of exogenous cytokines. Cytokines responsible for the colony-stimulating activity (CSA) and burst-promoting activity (BPA) produced by stromal cells were identified by neutralizing antibodies to specific cytokines. All normal stroma populations produced G-CSF and GM-CSF, 93% produced IL-3, 80% produced IL-6, and 70% produced SCF. Similarly, all AA stroma produced G-CSF and GM-CSF, and 71% produced SCF. In contrast, only 71% of AA stroma produced IL-3 and 36% produced IL-6. Target cell stimulation was not dependent on direct stroma-target cell contact, suggesting production of soluble cytokines. However, although both IL-6 and G-CSF were detected in LTBMC supernatants by enzyme-linked immunoassay (ELISA), IL-3 and GM-CSF were undetectable, perhaps indicating low-level local production of these factors.     

Low frequency of myeloid progenitor cells in chronic idiopathic neutropenia of adults may be related to increased production of TGF-beta1 by bone marrow stromal cells.

Previous studies in our laboratory have shown that patients with chronic idiopathic neutropenia of adults (CINA) have increased serum levels of inflammatory cytokines including IL-1beta. Since IL-1beta may affect bone marrow stromal cell function, a study was designed to investigate the capacity of patients' stromal cells to produce adequate amounts of haemopoietic growth factors or excessive amounts of inhibitors of myelopoiesis in long-term bone marrow cultures (LTBMCs). The study was carried out on 52 CINA patients and 19 normal controls. We found that CINA patients had significantly low numbers of marrow lineage-specific CD34+ cells, including CFU-GM and CD34+/CD33+ cells. Stromal cells from patients' LTBMCs failed to stimulate CFU-GM colony formation by normal marrow cells in a manner comparable to that of stromal cells of controls. Patients' LTBMC supernatants had normal or increased amounts of G-CSF. Detectable amounts of supernatant GM-CSF were found in 35% of patients and 19% of controls. IL-3 and MIP-1alpha were not detected in any supernatant fluid. Moreover, supernatants from patients' LTBMCs had increased concentrations of IL-6 and TGF-beta1, which strongly correlated with serum IL-1beta. About 82% of our patients had TGF-beta1 values higher than the upper limit of values found in the controls. Individual TGF-beta1 values inversely correlated with the number of circulating neutrophils and the frequency of marrow CD34+/CD33+ cells. We suggest that increased levels of serum IL-1beta, resulting from an underlying low-grade chronic inflammatory process, may stimulate marrow stromal cells to produce both haemopoietic growth factors and inhibitors of myelopoiesis. Since steady-state myelopoiesis results from a balance between negative- and positive-acting cytokines, it seems very probable that the increased production of TGF-beta1 by bone marrow microenvironment in CINA patients may suppress myelopoiesis and contribute, to some extent, to the pathogenesis of neutropenia in affected subjects.     

Use of granulocyte colony-stimulating factor (G-CSF) for mobilizing peripheral blood stem cells: risk of mobilizing clonal myeloma cells in patients with bone marrow infiltration

Peripheral blood stem cells have been used for autologous reconstitution of haemopoiesis after high dose cytotoxic therapy and produce similar disease response rates as autologous bone marrow transplants. Peripheral blood stem cell transplants are an especially attractive option for patients in whom marrow harvest is not feasible due to bone marrow damage or infiltration. Recombinant growth factors mobilize adequate numbers of stem cells from the marrow but their effect on tumour cell circulation kinetics is not known. We report a patient with multiple myeloma and bone marrow infiltration in whom the use of G-CSF for stem cell mobilization led to release of plasma cells into the peripheral circulation and contamination of the stem cell harvest.     

Long-term follow-up of granulocyte colony-stimulating factor receptor mutations in patients with severe congenital neutropenia: implications for leukaemogenesis and therapy

Severe congenital neutropenia (SCN) is characterized by profound neutropenia, recurrent severe bacterial infections and maturation arrest in the myeloid lineage. Granulocyte colony-stimulating factor (G-CSF) treatment results in clinical improvement in over 90% of cases. Point mutations of the G-CSF receptor (G-CSFR) have been implicated in the progression of SCN to acute myeloid leukaemia (AML). Data are presented here on the 9-year follow-up of seven patients and the further screening of 18 other cases. One of the two original cases with a G-CSFR mutation has improved clinically; nevertheless, mutant DNA could still be detected at a very low level > 8 years after identification. The second child with a mutation progressed to myelodysplasia/AML 5 years after her mutation was detected. No mutations were found in the 18 new cases. One of three transformed cases had a G-CSFR mutation. This work is in agreement with the suggestion that G-CSFR mutations may provide a survival advantage to haemopoietic stem cells, but argues against the inevitability of leukaemic progression in their presence. Furthermore, the low frequency of G-CSFR mutations in SCN and the importance of regular screening and close clinical and laboratory follow-up if a mutation is found were demonstrated.