Lymphopenia in patients with chronic idiopathic neutropenia is associated with decreased number of T-lymphocytes containing T-cell receptor excision circles.

OBJECTIVES: Chronic idiopathic neutropenia (CIN) is a disorder of granulopoiesis characterized by the presence of activated T-lymphocytes that induce/sustain apoptosis of bone marrow (BM) granulocytic progenitors. T-cell lymphopenia is commonly found in CIN. The aim of the study is to probe the mechanisms underlying T-cell lymphopenia in CIN. METHODS: We investigated parameters of T-cell homeostasis namely the proliferation/apoptotic rate of naïve and memory T cells, the T-cell senescence by telomere measurement, the recent thymic T-cell production through quantification of T-cell receptor rearrangement excision circles (TRECs), and the production of interleukin (IL)-7. RESULTS: Patients with CIN (n = 44) displayed lower proportion of naïve CD45RA(+) cells within the CD4(+) and CD8(+) cells compared with controls (n = 15). The proportion of apoptotic cells within the CD8(+) fraction was higher in patients compared with controls and was correlated with the percentage of Ki-67(+) cells, indicating an activation-induced accelerated CD8(+) cell death. The TREC content of CD4(+) and CD8(+) cells was lower in patients compared with controls and was correlated with the proportion of CD45RA(+) CD4(+) and CD8(+) cells and with the levels of serum and BM IL-7, which were significantly decreased in the patients. The mean relative telomere length of CD4(+) and CD8(+) cells was significantly lower in patients with CIN compared with age-matched controls. CONCLUSIONS: The aberrant T-cell expansions associated with the pathogenesis of CIN result in increased proliferation/apoptosis and possibly exhaustion of peripheral blood T cells which, in association with the inadequate compensatory thymic export of new TREC expressing T cells partially because of IL-7 deficiency, may contribute to lymphopenia in 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.

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.

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.