Placental growth factor attenuates suppression of erythroid colony formation by interferon.

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor family and is associated with inflammation and with pathologic angiogenesis. PlGF is released from marrow erythroid cells, and serum PlGF concentrations have been reported to distinguish sickle cell patients from healthy controls. We observed that erythroid colony forming units (CFU-Es) from homozygous sickle cell (SS) patients are less sensitive to inhibition by rhgammaIFN than those from healthy controls, and the contribution of PlGF to this process was evaluated. At 10-1000-pg/mL concentrations, PlGF neither inhibits nor enhances CFU-E colony formation, and no differences were observed between the responses of SS patients or healthy controls. rhPlGF 100 pg/mL reversed the inhibitory effects of rhgammaIFN on CFU-E colony formation. rhPlGF significantly attenuated rhgammaIFN induction of the Fas ligand in an erythroid cell line (HCD57). Both HCD57 cells and CD36+ human marrow cells express Flt-1, which is a receptor for PlGF. A neutralizing antibody against Flt-1 partially attenuated the IFN-protective effect of rhPlGF, although this effect was not statistically significant. In conclusion, increased PlGF concentrations in the marrow of SS patients may protect erythroid progenitors from cytokine-induced inhibition of colony formation, and it may be a mechanism by which erythropoiesis in sickle cell disease is preserved despite concurrent inflammation.

Cytokine concentrations in bone marrow of stable sickle cell anemia patients.

Inflammation plays a significant role in the clinical manifestations of sickle cell anemia. In studies of anemic patients with other clinical syndromes, measurement of the concentrations of cytokine mediators of inflammation in bone marrow aspirates has provided unique correlations with clinical and laboratory parameters. We determined concentrations of interleukin (IL)-1, IL-6, tumor necrosis factor (TNF), and placental growth factor (PlGF) in bone marrow aspirates from six homozygous sickle cell (SS) patients who were not acutely ill and who were not receiving hydroxyurea, erythropoietin, or chronic transfusion and compared them with specimens from seven healthy controls. We also measured concentrations of soluble transferrin receptor (sTfR) and of marrow erythroid colony-forming units (CFU-E) as markers of erythropoietic activity. sTfR concentration was significantly higher in SS patients (p = .024). CFU-E concentration was not significantly different between the two groups. Bone marrow concentrations of IL-6 and IL-1 did not differ between the study groups. TNF was undetectable in all specimens, plasma or marrow. Bone marrow PlGF concentrations were significantly higher in SS patients (p = .004). Since PlGF is a product of erythroid cells, the ratio of marrow PlGF to marrow sTfR was determined and found to be significantly greater in SS patients. This suggests that the observed difference in marrow PlGF concentrations does not reflect increased erythropoiesis but rather represents increased PlGF production per erythroid unit.

Hepcidin inhibits in vitro erythroid colony formation at reduced erythropoietin concentrations.

The anemia of chronic disease (ACD) results from 3 major processes: slightly shortened red cell survival, impaired reticuloendothelial system iron mobilization, and impaired erythropoiesis. Hepcidin is an acute-phase protein with specific iron regulatory properties, which, along with the anemia seen with increased hepcidin expression, have led many to consider it the major mediator of ACD. However, if hepcidin is the major factor responsible for ACD, then it should also contribute to the impaired erythropoiesis observed in this syndrome. Erythroid colony formation in vitro was inhibited by hepcidin at erythropoietin (Epo) concentrations less than or equal to 0.5 U/mL but not at Epo 1.0 U/mL. At Epo concentrations of 0.3 U/mL, HCD57 erythroleukemia cells exposed to hepcidin exhibit decreased expression of the antiapoptotic protein pBad compared with controls. These studies suggest that hepcidin may contribute to anemia in ACD not only through effects on iron metabolism, but also through inhibition of erythroid progenitor proliferation and survival.

Serum hepcidin in clinical specimens.

The hepatic antimicrobial protein, hepcidin, is implicated in duodenal iron absorption and mobilization. Overexpression of the hepcidin gene is associated with a hypoferraemic, microcytic, iron-refractory anaemia. On the basis of these observations, it has been proposed that hepcidin is a mediator of the common clinical syndrome, anaemia of chronic disease (ACD), and recent findings evaluating urinary hepcidin production in patients support this hypothesis. In the present report, serum hepcidin concentrations were measured in 55 specimens submitted for ferritin determination, and in 37 specimens collected from anaemic patients undergoing diagnostic bone marrow examination. The serum hepcidin concentration exhibited a statistically significant correlation with serum ferritin concentrations in both patient subsets. No statistically significant correlations were observed between serum hepcidin and other laboratory markers of iron status or anaemia diagnosis. Serum hepcidin does not appear to correlate as well with clinical diagnosis as urinary hepcidin, suggesting that a better understanding of the clearance and metabolism of this protein is required to understand fully its potential contribution to the pathogenesis of ACD.

Effects of oxidative stress on human erythroid colony formation: Modulation by gamma-interferon.

Evidence of increased oxidative stress is a hallmark of many chronic diseases associated with anemia. The current study was undertaken to evaluate the effects of oxidative stress on erythroid colony formation in vitro by bone marrow light density mononuclear cells (LDMN) and by peripheral blood derived cells enriched for erythroid colony forming units (CFU-E), and how these effects can be modified by a cytokine implicated in the anemia of chronic disease. When blood-derived and marrow cells were cultured with 50 microM H(2)O(2), CFU-E colony formation by blood-derived cells but not by marrow cells was significantly inhibited, suggesting a protective effect of marrow accessory cells. This inhibitory effect on peripheral blood-derived CFU-E was shown to be caspase-dependent. rhgammaIFN at concentrations which did not inhibit CFU-E colony formation sensitized LDMN marrow cells to inhibition by H(2)O(2). Exposure of LDMN marrow cells to rhgammaIFN at concentrations of 10 U/mL or higher significantly decreased the concentration of thioredoxin (Trx) in cell supernatant. Addition of recombinant Trx to LDMN marrow cells cultured with H(2)O(2) and rhgammaIFN partially (although not completely) reversed inhibition of CFU-E colony formation. These findings suggest that inflammatory cytokines implicated in the pathogenesis of the anemia of chronic disease may exert their effects at least in part through modulation of oxidative stress.

Thioredoxin and protein nitrotyrosine in bone marrow supernatant from patients with human immunodeficiency virus infection.

BACKGROUND: Balance and imbalance between oxidant stress and antioxidants contributes to the manifestations of human immunodeficiency virus (HIV) infection. Previously, we demonstrated a characteristic cytokine pattern in marrow supernatant from HIV patients who underwent diagnostic examinations. METHODS: In this report, we have determined the protein nitrotyrosine (pNT) concentration (an indicator of nitric oxide-superoxide interaction) as well as the concentration of the redox enzyme thioredoxin (Trx) in marrow supernatant from HIV patients, healthy controls, and other patients, and in serum from comparable subjects. RESULTS: pNT concentrations were similar in serum and marrow supernatant and did not differ between subject subsets. Trx concentrations in both marrow supernatant and serum were higher for HIV patients than for other subjects; serum Trx concentrations were significantly higher than marrow Trx concentrations for the non-HIV patients and controls. The ratios of pNT/Trx concentrations in serum were similar in all subsets tested. In marrow aspirate, however, these ratios differed widely and significantly, with the highest values observed in non-HIV patients and the lowest in HIV patients. Only for HIV patients were serum and marrow supernatant pNT/Trx ratios similar. In HIV patients, marrow Trx concentrations correlated with CD4 count, CD4/CD8 ratio, and marrow colony-forming unit E (CFU-E) concentration; marrow pNT concentration correlated with the concentration of tumor necrosis factor in marrow supernatant, and with platelet count. No correlations were observed in other subject subsets. CONCLUSIONS: These findings suggest that there is a degree of local regulation of the redox state of the marrow microenvironment that varies with the patients' clinical statuses, and which is associated with effects on hematopoiesis.