Erythropoietin Elaboration Research Articles

Pro-inflammatory proteins S100A9 and tumor necrosis factor-α suppress erythropoietin elaboration in myelodysplastic syndromes

Accumulating evidence implicates innate immune activation in the pathobiology of myelodysplastic syndromes. A key myeloid-related inflammatory protein, S100A9, serves as a Toll-like receptor ligand regulating tumor necrosis factor-α and interleukin-1β production. The role of myelodysplastic syndrome-related inflammatory proteins in endogenous erythropoietin regulation and response to erythroid-stimulating agents or lenalidomide has not been investigated. The HepG2 hepatoma cell line was used to investigate in vitro erythropoietin elaboration. Serum samples collected from 311 patients with myelodysplastic syndrome were investigated (125 prior to treatment with erythroid-stimulating agents and 186 prior to lenalidomide therapy). Serum concentrations of S100A9, S100A8, tumor necrosis factor-α, interleukin-1β and erythropoietin were analyzed by enzyme-linked immunosorbent assay. Using erythropoietin-producing HepG2 cells, we show that S100A9, tumor necrosis factor-α and interleukin-1β suppress transcription and cellular elaboration of erythropoietin. Pre-incubation with lenalidomide significantly diminished suppression of erythropoietin production by S100A9 or tumor necrosis factor-α. Moreover, in peripheral blood mononuclear cells from patients with myelodysplastic syndromes, lenalidomide significantly reduced steady-state S100A9 generation (P=0.01) and lipopolysaccharide-induced tumor necrosis factor-α elaboration (P=0.002). Enzyme-linked immunosorbent assays of serum from 316 patients with non-del(5q) myelodysplastic syndromes demonstrated a significant inverse correlation between tumor necrosis factor-α and erythropoietin concentrations (P=0.006), and between S100A9 and erythropoietin (P=0.01). Moreover, baseline serum tumor necrosis factor-α concentration was significantly higher in responders to erythroid-stimulating agents (P=0.03), whereas lenalidomide responders had significantly lower tumor necrosis factor-α and higher S100A9 serum concentrations (P=0.03). These findings suggest that S100A9 and its nuclear factor-κB transcriptional target, tumor necrosis factor-α, directly suppress erythropoietin elaboration in myelodysplastic syndromes. These cytokines may serve as rational biomarkers of response to lenalidomide and erythroid-stimulating agent treatments. Therapeutic strategies that either neutralize or suppress S100A9 may improve erythropoiesis in patients with myelodysplastic syndromes.

The Proinflammatory Protein S100A9 Suppresses Erythropoietin Elaboration in Patients with Myelodysplastic Syndromes

Introduction: Accumulating evidence implicates innate immune activation in the pathobiology of myelodysplastic syndromes (MDS) and its inflammatory bone marrow microenvironment. Excess elaboration of S100A9 accompanied by secondary induction of TNFα and IL-1β, characterize the inflammatory milieu found in lower risk MDS. Erythroid stimulating agents (ESA) and lenalidomide (LEN) are erythropoietic promoters with known anti-inflammatory properties. To date, the role of such inflammation parameters in endogenous erythropoietin (Epo) regulation and response to such treatments has not been investigated. Herein, we investigated the role of these inflammatory cytokines on Epo elaboration and response to ESA and LEN in MDS.Materials and Methods: The HepG2 hepatoma cell line was used to investigate Epo elaboration in vitro. Serum collected from 311 MDS patients from 3 centers (AOU Careggi, University of Firenze; Saint Louis hospital in Paris; H. Lee Moffitt Cancer Center in Tampa) were investigated. 125 were obtained prior to ESA treatment and 186 prior to LEN or LEN+EPO treatment. ELISAs for S100A9, S100A8, TNFα, IL1β and EPO were performed. Clinical data were analyzed from all patients. Spearman's correlation, Mann-Withney and Jonckheere-Terpstra tests were used for analysis of continuous variables. Chi-square test was used for analysis of non-continuous variables.Results: Median age of the patients was 76 years [41-94]. IPSS was low, intermediate-1, intermediate-2 and high risk in 47%, 49%, 3% and 1% of patients, respectively; whereas 28%, 42%, 22%, 7% and 1% of patients were very low, low, intermediate, high and very high risk according to IPSS-R. Serum S100A9 concentration was significantly higher in patients with lower risk versus higher risk MDS (17,798 pg/ml vs 15,291 pg/ml, p=0.01). No significant difference was observed for TNFα and IL1 β according to IPSS or IPSS-R. ELISA quantitation following 24h in vitro stimulation of HepG2 cells with S100A9 (10 to 20 µg/ml), TNFα (10 to 100 ng/ml) or IL1β (10 to 100ng/ml) showed that each cytokine significantly suppressed Epo elaboration in a concentration-dependent manner. To validate these findings, we assessed the relationship between serum concentration of these inflammatory cytokines and Epo level in MDS patients. We observed a significant negative correlation between TNFα and Epo concentration (r=-0.158, p=0.01), and a corresponding significant negative correlation between S100A9 and Epo (r=-0.143, p=0.01). We also found a significant positive correlation between S100A9 and S100A8 (r=0.757, p<0.001), S100A9 and TNFα (r=0.222, p<0001), S100A9 and IL1β (r=175, p=0.004), and IL1β with TNFα concentration (r=257, p<0.001). Moreover, we found that ESA responders had a significant higher serum TNFα concentration prior to treatment than non-responders (12 pg/ml vs 6 pg/ml, p=0.02) with a corresponding significantly lower Epo concentration in ESA responders. There was no significant correlation between S100A9 serum concentration and EPO response. Finally, in patients treated with LEN + ESA, we observed a significant positive correlation between S100A9 and duration of LEN or LEN + ESA response (r=-0.418, p=0.03). In vitro, 1µM LEN significantly reduced suppression of Epo elaboration by S100A9 or TNFα in HepG2 cells. Magnitude of LEN's improvement in EPO elaboration was greater for S100A9 vs TNF (43% vs 17%, p=0.08).Moreover, in peripheral blood mononuclear cells (PBMC) from MDS patients, 1µM LEN significantly decreased steady state S100A9 generation, and LPS-induced TNFα elaboration.Conclusion: These findings demonstrate that S100A9 and its NFκ-B transcriptional target, TNFα, directly suppress Epo elaboration and endocrine response to anemia in MDS. These inflammatory proteins may serve as rational biomarkers for response to lenalidomide and/or ESA treatment and merit prospective validation. Therapeutic strategies that either neutralize S100A9 or suppress elaboration of this alarmin may improve erythropoiesis in MDS by restoring Epo response and suppressing innate immune effectors. DisclosuresFenaux:Janssen: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Santini:celgene, Janssen, Novartis, Onconova: Honoraria, Research Funding. List:Celgene Corporation: Honoraria, Research Funding.

Ureter obliteration transiently depresses erythropoietin production.

The erythropoietin (EPO) gene is activated in peritubular cells of the renal cortex in response to hypoxia and EPO is secreted into the circulation. Oxygen tension in the venous blood normally determine EPO elaboration, and, therefore, oxygen consumption in the kidney appears to be an essential part of the oxygen-sensing physiological mechanism. As renal oxygen consumption is closely linked to urine production, we have compared responsiveness of the EPO gene to diminished oxygen supply in the normal kidney with that of the hydronephrotic kidney, resulting from ureter ligation. Erythropoietin mRNA was determined in RNA extracted from murine kidneys at various times up to 61 days after ureter ligation. Mice were exposed to hypoxia for 4 h before being killed. Unilateral ureter ligation significantly decreased EPO mRNA levels in the affected kidney. However, the ability to increase EPO mRNA recovered between days 4 and 22, despite persisting ureter ligation. Our results demonstrate a dissociation between the excretory functions of the kidney and EPO production. Moreover, they show an early but transient suppression of EPO gene responsiveness to hypoxia after ureter ligation.

Hydronephrosis and polycythemia

Polycythemia is consistently produced in animals undergoing experimental hydronephrosis. This hematologic response is thought to be mediated by a microcirculatory insult and a more accelerated elaboration of erythropoietin by the hydronephrotic kidney. While an association between hy dronephrosis and polycythemia has been reported in humans, it is a relatively rare event. Further studies arc necessary to explain why this should be so, as well as to confirm that the polycytheimia in humans is secondary to an increased production of erythropoietin.

Case report: studies on the mechanism of erythrocytosis associated with a uterine fibromyoma.

Summary. Erythrocytosis has occasionally been associated with uterine fibromyoma and attributed to an inappropriate elaboration of erythropoietin (Ep) by the tumour. Whether Ep per se, its precursor substrate, or activator analagous to the renal erythropoietic factor (REF) is produced, has not been defined. In a 54‐yr‐old woman with erythrocytosis and uterine fibromyoma, plasma, urine and tumour extracts were examined to clarify these possible mechanisms. Preoperatively, elevated serum and urinary Ep were present with constant levels over a range of haematocrits induced by serial phlebotomy. Extracts of the tumour did not generate Ep when incubated with normal serum, nor did they contain substrate activity for known REP. Tumour extracts did, however, possess Ep activity which was abolished with anti‐Ep. The patient has remained haematologically normal more than 1 yr following surgery. The data show that the erythrocytosis associated with uterine fibromyoma is mediated through the direct production of Ep by the tumour and not by a substrate‐REF‐like mechanism as found in the normal kidney.

Relationship Between Renal Blood Flow and Erythropoietin Production In Dogs.

SummaryReduction in renal artery blood flow in dogs with the use of a modified Goldblatt clamp to a range of 22.4-35.4% of preconstriction values for 96 hours resulted in a significant increase in plasma levels of erythropoietin at 12 hours. When renal blood flow was reduced to a level 43.5-50% of initial flow erythropoietin titers were also elevated at 12 hours, whereas, reduction to a range of 57.5-71% of initial values only resulted in a slight rise in erythropoietin levels at 24 hours. The mechanism of enhanced erythropoietin elaboration following reduction of renal blood flow is postulated to be the result of renal hypoxia.

The Effects of Allografts of Thymic Epithelial Reticular Cells on the Lymphoid Tissues of Neonatally Thymectomized Mice

AbstractIsolated dog kidneys were perfused with blood at normal and reduced oxygen tensions with and without cobalt. Erythropoietin titers were found to be significantly increased in the blood perfusates from kidneys perfused with blood at lowered oxygen tensions, while no change in erythropoietin was seen when kidneys were perfused with blood at a normal oxygen tension. Cobalt produced significant increases in erythropoietin levels in the perfusates with normal as well as reduced oxygen tension blood, but its effect was more marked when the kidneys were perfused with hypoxic blood. Renal blood flows were higher when the dogs' own lungs were used to oxygenate the blood than when a bubble oxygenator was used in the system. However, hypoxic blood resulted in significant increases in erythropoietin production in both systems. In studies of the kidney histology from both perfusion systems with hypoxic and normal oxygen tension blood, most of the kidneys demonstrated varying degrees of glomerular congestion. No correlation was found between erythropoietin elaboration and renal congestion or other degenerative cellular changes in the kidney. Therefore, it does not seem likely that erythropoietin produced in the isolated perfused kidney is due to histologic damage to renal cells. These findings support the concept that increased erythropoietin production in the isolated perfused kidney in response to cobalt or hypoxic blood is a direct effect of these stimuli on the kidney.

The Influence of Hypoxemia and Cobalt on Erythropoietin Production in the Isolated Perfused Dog Kidney

AbstractIsolated dog kidneys were perfused with blood at normal and reduced oxygen tensions with and without cobalt. Erythropoietin titers were found to be significantly increased in the blood perfusates from kidneys perfused with blood at lowered oxygen tensions, while no change in erythropoietin was seen when kidneys were perfused with blood at a normal oxygen tension. Cobalt produced significant increases in erythropoietin levels in the perfusates with normal as well as reduced oxygen tension blood, but its effect was more marked when the kidneys were perfused with hypoxic blood. Renal blood flows were higher when the dogs’ own lungs were used to oxygenate the blood than when a bubble oxygenator was used in the system. However, hypoxic blood resulted in significant increases in erythropoietin production in both systems. In studies of the kidney histology from both perfusion systems with hypoxic and normal oxygen tension blood, most of the kidneys demonstrated varying degrees of glomerular congestion. No correlation was found between erythropoietin elaboration and renal congestion or other degenerative cellular changes in the kidney. Therefore, it does not seem likely that erythropoietin produced in the isolated perfused kidney is due to histologic damage to renal cells. These findings support the concept that increased erythropoietin production in the isolated perfused kidney in response to cobalt or hypoxic blood is a direct effect of these stimuli on the kidney.

ERYTHROCYTOSIS IN EMPHYSEMA.

Marked erythrocytosis is frequently seen in hypoxia due to high altitudes and cyanotic congenital heart disease (Hurtado, Merino and Delgado, 1945) but is relatively uncommon in association with chronic respiratory failure (Baldwin, Cournand and Richards, 1949). This discrepancy has exercised a number of writers. Wilson, Borden and Ebert (1951) have proposed that bronchial infection may depress the erythropoietic response of the bone marrow in patients with chronic bronchitis and emphysema and Ratto, Briscoe, Morton and Comroe (1955) have postulated depression of erythropoiesis by carbon‐dioxide retention. Simpson (1957) found evidence of bleeding in a number of his emphysematous subjects and suggested that concealed haemorrhage may be of importance in modifying haematological changes in respiratory hypoxia.More recently, Shaw and Simpson (1961) and Lertzman, Israels and Cherniack (1962) have proposed that expansion of plasma volume accompanying respiratory failure may account for the apparent infrequency of erythrocytosis in emphysema, but the view that the erythropoietic response to hypoxia is normal in emphysema is based on evidence which is not entirely satisfactory. Thus Shaw and Simpson compared the red‐cell mass in English subjects with emphysema with that of native Peruvian Indians living at high altitudes, a comparison which assumes similarity of nutrition and incidence of conditions such as intestinal infestation. Lertzman and associates compared the red‐cell mass in respiratory failure with that in patients with hypoxia due to other causes. In the majority of these latter patients, the hypoxia was due to obesity but the red‐cell mass was expressed per kilogram of body weight rather than as a function of height or lean‐body mass. In each of these studies the red‐cell mass in emphysema varied from within the normal range to more than double this figure in patients with hypoxia of comparable severity. The fact that many chronically cyanosed emphysematous patients show no significant increase in red‐cell mass remains unexplained and warrants further attention.In the present investigation a small group of severely disabled patients was chosen for study. All suffered from chronic bronchitis and emphysema with persistent cyanosis during remission from acute pulmonary infection. Four exhibited the conventional haematological changes of secondary polycythaemia, whilst another four were similarly disabled but showed no such changes in the peripheral blood. The two groups were compared in an attempt to account for the apparent failure of erythropoietic response to hypoxia in the latter. The investigation was designed to ascertain whether the defect lay in the elaboration of erythropoietin, the humoral mediator of the hypoxic stimulus to the bone marrow, or in the response of the bone marrow to this humoral stimulus.