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-beta1), 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-beta1, 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-beta1 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-beta1 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-beta1 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-beta1 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.

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.

Evidence for downregulation of erythropoietin receptor in bone marrow erythroid cells of patients with chronic idiopathic neutropenia.

OBJECTIVE: The aim of this study is to probe the mechanisms underlying anemia in patients with chronic idiopathic neutropenia (CIN) by evaluating parameters of bone marrow (BM) erythropoiesis. PATIENTS AND METHODS: Ten CIN patients fulfilling the criteria of anemia of chronic disease, 27 nonanemic CIN patients, and 30 healthy volunteers were enrolled in the study. Reserves and survival characteristics of BM erythroid cells were evaluated using flow cytometry and clonogenic assays. Serum erythropoietin (EPO) was measured with ELISA. Expression of EPO receptors (EPORs) on BM erythroid cells was evaluated by flow cytometry and reverse-transcription polymerase chain reaction. RESULTS: CIN patients display defective erythropoiesis in addition to previously reported impaired granulopoiesis. Patients have low number of CD34(+)/CD71(+) progenitor and CD36(-)/ Glycophorin A(+) (GlycoA(+)) precursor BM cells, and increased proportion of apoptotic cells within the CD34(+)/CD71(+) and CD36(+)/GlycoA(+) compartments. Burst-forming units erythroid (BFU-Es) in BM mononuclear or purified CD34(+) cells were significantly reduced in the patients. Patient BFU-Es increased significantly following in vitro treatment with tumor necrosis factor-alpha (TNF-alpha) and/or interferon gamma (IFN-gamma)-neutralizing antibodies. Local TNF-alpha and IFN-gamma production was higher in anemic than in nonanemic patients. EPO production was appropriate in the patients, but EPOR expression was significantly reduced in patient GlycoA(+) cells, especially in anemic patients. CONCLUSION: Impaired BM erythropoiesis in CIN patients is probably the result of increased local production of TNF-alpha and IFN-gamma that induce apoptosis, cell growth inhibition, and downregulation of EPOR expression on erythroid cells. We suggest that anemia in CIN patients displays overlapping pathophysiologic features with anemia of chronic disease.

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.

Effect of cA2 anti-tumor necrosis factor-alpha antibody therapy on hematopoiesis of patients with myelodysplastic syndromes.

PURPOSE: Tumor necrosis factor alpha (TNF-alpha) plays a prominent role in the pathophysiology of myelodysplastic syndromes (MDS). The aim of this study was to explore the biological and immunoregulatory effect of the treatment with the anti-tumor necrosis factor-alpha monoclonal antibody cA2 on bone marrow (BM) progenitor/precursor and stromal cells and lymphocyte subsets, as well as the clinical response in MDS patients. EXPERIMENTAL DESIGN: Ten low-intermediate risk MDS patients received i.v. cA2 (3 mg/kg) at weeks 0, 2, 6, and 12. The number, survival, and clonogenic potential of BM progenitor/precursor cells, the hematopoiesis-supporting capacity of BM stromal cells, and the lymphocyte activation status were investigated in the patients at baseline and following treatment using flow cytometry, clonogenic assays, and long-term BM cultures (LTBMC). Clinical response was evaluated according to standardized criteria. RESULTS: cA2 administration reduced the proportion of apoptotic and Fas+ cells in the CD34+ cell compartment (P = 0.0215 and P = 0.0344, respectively) and increased the clonogenic potential of BM mononuclear and CD34+ cells (P = 0.0399 and P = 0.0304, respectively) compared with baseline. The antibody reduced tumor necrosis factor-alpha levels in LTBMC supernatants (P = 0.0043) and significantly improved the hematopoiesis-supporting capacity of LTBMC adherent cells. The proportion of activated peripheral blood and BM T-lymphocytes decreased significantly after treatment, suggesting an immunomodulatory effect of cA2. Two patients displayed minor hematologic responses whereas the remaining patients displayed stable disease with no disease progression. CONCLUSIONS: The encouraging biological insights from cA2 administration may be useful in conducting further clinical trials using cA2 for selected MDS patients, particularly those with evidence of immune-mediated inhibition of hematopoiesis.

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.

Helicobacter pylori infection is probably the cause of chronic idiopathic neutropenia (CIN)-associated splenomegaly.

Splenic volume and Helicobacter pylori (H. pylori) infection were evaluated in 67 patients with chronic idiopathic neutropenia (CIN) and 39 healthy individuals. Using ultrasound, splenomegaly was found in 61.7% of H. pylori-infected subjects compared to only 8.7% noted in the group of H. pylori-non-infected individuals (P < 0.0001). Splenomegaly was also found in 47.8% of CIN patients compared to 12.8% in the group of non-CIN subjects (P = 0.0003). However, applying the two-way ANOVA test, a statistically significant effect on splenic volume was documented for "factor H. pylori " (F1(102) = 16.800, P < 0.0001) but not for "factor CIN" (F1(102) = 3.213, P = 0.0760), suggesting that CIN-associated splenomegaly is probably due to H. pylori infection.

Multiple myeloma involving the central nervous system: a report of two cases with unusual manifestations.

We describe two patients with multiple myeloma (MM) involving the central nervous system (CNS). In both patients, CNS involvement was manifested with extremely rare signs and symptoms. The first patient, a 64-year-old woman, developed obstructive hydrocephalus 10 years after the initial diagnosis of MM. The disease has been successfully treated with intrathecal chemotherapy, radiotherapy and systemic chemotherapy. The other patient, a 65-year-old man, developed hypopituitarism and diabetes insipidus 1 year after MM diagnosis. Disease in this patient proved resistant to both radiotherapy and chemotherapy; the patient died 2 years later. These rare complications add new information on the wide spectrum of clinical and laboratory manifestations of MM. It is obvious that descriptions of additional cases are required to better understand the mechanisms of CNS involvement in MM patients.

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.

Expression of the Tpl2/Cot oncogene in human T-cell neoplasias.

BACKGROUND: Tpl2/Cot oncogene has been identified in murine T-cell lymphomas as a target of MoMuLV insertion. Animal and tissue culture studies have shown that Tpl2/Cot is involved in interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha) production by T-cells contributing to T-cell proliferation. In the present report we examined a series of 12 adult patients with various T-cell malignancies, all with predominant leukemic expression in the periphery, for the expression of Tpl2/Cot oncogene in order to determine a possible involvement of Tpl2/Cot in the pathogenesis of these neoplasms. RESULTS: Our results showed that Tpl2/Cot was overexpressed in all four patients with Large Granular Lymphocyte proliferative disorders (LGL-PDs) but in none of the remaining eight patients with other T-cell neoplasias. Interestingly, three of the LGL-PD patients displayed neutropenia, one in association with sarcoidosis. Serum TNF-alpha levels were increased in all Tpl2/Cot overexpressing patients while serum IL-2 was undetectable in all subjects studied. Genomic DNA analysis revealed no DNA amplification at the Tpl2/Cot locus in any of the samples analyzed. CONCLUSIONS: We conclude that Tpl2/Cot, a gene extensively studied in animal and tissue culture T-cell models may be also involved in the development of human LGL-PD and may have a role in the pathogenesis of immune manifestations associated with these diseases. This is the first report implicating Tpl2/Cot in human T-cell neoplasias and provides a novel molecular event in the development of LGL-PDs.

Alendronate reduces serum TNFalpha and IL-1beta, increases neutrophil counts, and improves bone mineral density and bone metabolism indices in patients with chronic idiopathic neutropenia (CIN)-associated osteopenia/osteoporosis.

The current study was undertaken to investigate the effect of alendronate on bone mineral density (BMD), bone metabolism markers, and serum bone-resorbing cytokines in patients with chronic idiopathic neutropenia (CIN)-associated osteopenia/osteoporosis. Sixteen randomly selected women, 7 with CIN-associated osteoporosis and 9 with CIN-associated osteopenia, and 14 age- and menopausal status-matched healthy volunteers, were enrolled in the study. Patients received 10 mg alendronate daily per os for 360 days and studies were done before treatment (day 0) and at varying time points during the study. We found that patients' BMD measurements increased by 5.32% after treatment, and that the elevated serum osteocalcin (OC), a bone formation marker, decreased by day 30, normalized by day 90, and increased again by day 270 of treatment. Elevated values of patients' urine deoxypyridinoline (Dpd) and N-telopeptide of type I of collagen (NTx), two bone resorption markers, returned to the control range by day 30 and decreased thereafter. Increased levels of patients' serum tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta), two bone resorbing cytokines, returned to the control range by day 30 and decreased thereafter. Peripheral blood neutrophil counts increased by day 30 and continued to rise thereafter, reaching a mean value higher than 2650 neutrophils per microl of blood on day 360. Interestingly, alendronate-induced changes in the levels of both cytokines correlated inversely with the respective changes in neutrophil counts and BMD measurements, and positively with the changes in the respective means of urine NTx and Dpd values. All these findings indicate that alendronate is effective in treating CIN-associated osteopenia/osteoporosis, and that the beneficial effect of the compound may lie, at least in part, in its property to inhibit the production of TNFalpha and IL-1beta by cells of the monocyte/macrophage system, in which osteoclasts are included.

The role of apoptosis in the pathophysiology of chronic neutropenias associated with bone marrow failure.

Chronic neutropenia syndromes associated with bone marrow (BM) failure comprise distinct congenital and acquired hematologic disorders with varying degree of neutropenia due to decreased or ineffective BM neutrophil production. Recent evidence suggests that defective granulocytopoiesis in these neutropenia states is a consequence of accelerated apoptotic cell death of BM myeloid progenitor cells and/or their differentiated progeny. Inherited or spontaneously appearing mutations in the ELA2 gene encoding for neutrophil elastase have been implicated in the accelerated apoptotic process of the BM myeloid cells in patients with cyclic and severe congenital neutropenia. A disturbed balance between pro-apoptotic and anti-apoptotic intracellular or membrane molecules such as downregulation of the bcl-2 family members or upregulation of the death receptor Fas, have been implicated in neutropenia associated with myelokathexis, Shwachman-Diamond syndrome and acquired chronic idiopathic neutropenia of adult. In this review we summarize the available evidence suggesting that abnormally increased apoptosis and impaired proliferative and differentiating properties of neutrophil progenitor and precursor cells represent a common pathogenetic mechanism for impaired granulocytopoiesis in both acquired idiopathic and congenital neutropenia states. The underlying distinct cellular and molecular abnormalities and the role of the BM microenvironment are extensively analysed.

Impaired granulocytopoiesis in patients with chronic idiopathic neutropenia is associated with increased apoptosis of bone marrow myeloid progenitor cells.

To probe the pathophysiologic mechanisms underlying neutropenia in patients with chronic idiopathic neutropenia (CIN) with hypoplastic and left-shifted granulocytic series in the bone marrow (BM), we have studied granulocytopoiesis in 32 adults with CIN by evaluating the number and survival characteristics of cells in several stages of granulocyte differentiation using flow cytometry and BM culture assays. We found that patients with CIN displayed a low percentage of CD34(+)/CD33(+) cells, defective granulocyte colony-forming unit (CFU-G) growth potential of BM mononuclear or purified CD34(+) cells, and low CFU-G recovery in long-term BM cultures (LTBMCs), compared with controls (n = 46). A low percentage of CD34(+)/CD33(+) cells in patients was associated with accelerated apoptosis and Fas overexpression within this cell compartment compared with controls. No significant difference was documented in the percentage of apoptotic cells or the Fas(+) cells within the fractionated CD34(+)/CD33(-), CD34(-)/CD33(+), and CD34(-)/CD33(-)/CD15(+) BM subpopulations or the peripheral blood neutrophils, suggesting that the underlying cellular defect in CIN probably concerns the committed granulocyte progenitors. LTBMC stromal layers from patients produced abnormally high amounts of tumor necrosis factor alpha and cytokine levels in culture supernatants inversely correlated with the number of myeloid progenitor cells and positively with the proportion of apoptotic CD34(+) cells. Patient LTBMC stromal layers displayed pathologic interferon gamma and Fas-ligand mRNA expression and failed to support normal myelopoiesis. These data suggest that impaired granulocytopoiesis in CIN is probably due to overproduction of inflammatory cytokines by immune cells within the BM microenvironment that may exert an inhibitory effect on myelopoiesis by inducing Fas-mediated apoptosis in the granulocyte progenitors.

Bone marrow stem cells and stromal cells in autoimmune cytopenias.

High-dose immunosuppression followed by autologous haemopoietic stem cell transplantation (ASCT) is a promising practice for the treatment of severe, resistant autoimmune disorders. Patients with refractory autoimmune cytopenias (AIC), primary or secondary to systemic autoimmune diseases (AID) including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), have been proposed as potential candidates for such a therapeutic procedure. An abnormal immune milieu, however, may affect the number and functional characteristics of stem cells and/or stromal cells in the bone marrow (BM) and might impact on harvesting and engraftment potential of stem cells or on BM reconstitution following engraftment in patients with AIC undergoing ASCT. Using flow cytometry and in vitro culture assays we have shown that patients with primary AIC display increased number of BM CD34+ cells in response to abnormally high production of granulocyte-colony stimulating factor (G-CSF) by BM stroma. In contrast, patients with AIC secondary to systemic AID display increased apoptosis of BM progenitor cells resulting in low CD34+ cell numbers and abnormal haemopoiesis supporting capacity of BM stroma due to the aberrant, local or systemic, inhibitory cytokine production or to intricate interactions between haemopoietic and immune cells present within the BM microenvironment. In this review we summarize the available knowledge on BM stem cell reserve and function and stromal cell function in patients with primary and secondary AIC with special reference to SLE and RA. The underlying mechanisms possibly involved in the pathogenesis of the observed abnormalities are also discussed.

Anemia of chronic disease in rheumatoid arthritis is associated with increased apoptosis of bone marrow erythroid cells: improvement following anti-tumor necrosis factor-alpha antibody therapy.

Circumstantial evidence has implicated tumor necrosis factor alpha (TNF-alpha) in the pathogenesis of anemia of chronic disease (ACD) in rheumatoid arthritis (RA). We investigated the role of TNF-alpha in erythropoiesis of patients with active RA (n = 40) and the effect of anti-TNF-alpha antibody administration (cA2). Patients with RA had lower numbers of CD34+/CD71+ and CD36-/glycophorin A+ (glycoA+) bone marrow (BM) cells and increased proportions of apoptotic cells within the CD34+/CD71+ and CD36+/glycoA+ cell compartments, compared to healthy controls (n = 24). Erythroid burst-forming units (BFU-Es) obtained by BM mononuclear or purified CD34+ cells were significantly lower in RA patients compared to controls. These abnormalities were more pronounced among patients with ACD. Increased TNF-alpha levels in patient long-term BM culture supernatants inversely correlated with BFU-Es and hemoglobin levels and positively with the percentage of apoptotic CD34+/CD71+ and CD36+/glycoA+ cells. Following cA2 therapy, a normalization was documented in the number of CD34+/CD71+ and CD36-/glycoA+ cells, the number of BFU-Es, and the proportion of apoptotic CD34+/CD71+ and CD36+/glycoA+ cells, which was associated with a significant increase in hemoglobin levels compared to baseline. Recovery from anemia was more prominent in patients with ACD. The exogenous addition of an anti-TNF-alpha antibody in the cultures increased BFU-E number in patients prior to cA2 treatment but not after treatment, further substantiating the inhibitory role of TNF-alpha on patients' erythropoiesis. We conclude that TNF-alpha-mediated apoptotic depletion of BM erythroid cells may account for ACD in RA and that cA2 administration may ameliorate ACD in these patients by down-regulating the apoptotic mechanisms involved in erythropoiesis.

Bone marrow progenitor cell reserve and function and stromal cell function are defective in rheumatoid arthritis: evidence for a tumor necrosis factor alpha-mediated effect.

Based on previous reports for impaired hematopoiesis in rheumatoid arthritis (RA), and in view of the current interest in exploring the role of autologous stem cell transplantation (ASCT) as an alternative treatment in patients with resistant disease, we have evaluated bone marrow (BM) progenitor cell reserve and function and stromal cell function in 26 patients with active RA. BM progenitor cells were assessed using flow cytometry and clonogenic assays in short-term and long-term BM cultures (LTBMCs). BM stroma function was assessed by evaluating the capacity of preformed irradiated LTBMC stromal layers to support the growth of normal CD34(+) cells. We found that RA patients exhibited low number and increased apoptosis of CD34(+) cells, defective clonogenic potential of BM mononuclear and purified CD34(+) cells, and low progenitor cell recovery in LTBMCs, compared with healthy controls (n = 37). Patient LTBMC stromal layers failed to support normal hematopoiesis and produced abnormally high amounts of tumor necrosis factor alpha (TNF alpha). TNF alpha levels in LTBMC supernatants inversely correlated with the proportion of CD34(+) cells and the number of colony-forming cells, and positively with the percentage of apoptotic CD34(+) cells. Significant restoration of the disturbed hematopoiesis was obtained following anti-TNF alpha treatment in 12 patients studied. We concluded that BM progenitor cell reserve and function and BM stromal cell function are defective in RA probably due, at least in part, to a TNF alpha-mediated effect. The role of these abnormalities on stem cell harvesting and engraftment in RA patients undergoing ASCT remains to be clarified.