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Published ahead of print on March 30, 2006, doi:10.1164/rccm.200504-561OC
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American Journal of Respiratory and Critical Care Medicine Vol 174. pp. 51-57, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200504-561OC

Original Article

Erythropoiesis Abnormalities Contribute to Early-Onset Anemia in Patients with Septic Shock

Yann-Erick Claessens, Michaëla Fontenay, Frédéric Pene, Jean-Daniel Chiche, Martine Guesnu, Cyrla Hababou, Nicole Casadevall, Jean-Francçois Dhainaut, Jean-Paul Mira and Alain Cariou

Medical Intensive Care Unit, INSERM U567, Department of Emergency Medicine, and Laboratory of Hematology, Hôpital Cochin; Faculté de Médecine, Université Paris-Descartes; Laboratory of Hematology, Hôtel-Dieu, Paris; and Ortho Biotech France, Inc., Issy-les-Moulineaux, France

Correspondence and requests for reprints should be addressed to Alain Cariou, M.D., Medical Intensive Care Unit, Cochin Hospital, APHP Université Paris-Descartes, 27 rue du Faubourg Saint-Jacques, F-75679 Paris, Cedex 14, France. E-mail: alain.cariou{at}cch.ap-hop-paris.fr


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: The intimate mechanisms of early onset anemia observed in critically ill patients with septic shock remain unclear.

Objectives: We investigated erythropoiesis abnormalities in this setting by studying morphologic, functional, and biochemical patterns of erythroid lineage.

Methods: Erythroid lineage in the bone marrow from patients with septic shock who developed early-onset anemia was compared with that of healthy control subjects. Survival and proliferation capacities were quantified in both groups. Biochemical and flow cytometry patterns of apoptosis were dissected by exploring antiapoptotic (erythropoietin [Epo] receptor–dependent) and proapoptotic (death receptor–dependent) pathways.

Measurements and Main Results: Erythroid lineage was morphologically similar in both groups. Apoptosis of glycophorin-A–positive erythroid precursors was increased in patients versus control subjects as assessed by labeling with annexin V (26.1 ± 8.8 vs. 3.1 ± 2.9%, p < 0.05) or 3–3'-dihexyloxacarbocyanine iodide (55.9 ± 10.5 vs. 19.1 ± 5.4%, p < 0.05), respectively. This was associated with significant overexpression of Fas on erythroid precursors and higher tumor necrosis factor-{alpha} plasma levels in patients with septic shock vs. control subjects. Moreover, growth capacities of late erythroid progenitors of burst-forming unit erythroids (BFU-Es) at Day 10 were impaired in the presence of serum from patients with septic shock as compared with the effect of serum from control subjects (27 ± 12 vs. 109 ± 27 per 105 seeded cells, respectively; p < 0.001). Saturating concentrations of recombinant human Epo (rHuEpo) restored growth capacity of patients' BFU-Es (72 ± 14 per 105 seeded cells) in autologous conditions of serum.

Conclusions: Early-onset anemia that may be observed in patients with septic shock is associated with defective erythropoiesis related to an excess of apoptosis that can be counterbalanced in vitro by rHuEpo.

Key Words: anemia • apoptosis • erythropoiesis • erythropoietin • septic shock

Anemia is common in critically ill patients who often require packed red blood cell (PRBC) transfusion (1). Patients with severe sepsis or septic shock usually have a hemoglobin level lower than other patients admitted to the intensive care unit (ICU) and are more likely to require transfusion of PRBCs (2). A number of mechanisms have been proposed to explain anemia in this population, including fluid-loading–related hemodilution, daily blood withdrawal for routine laboratory analysis (3), and inflammation with impaired iron metabolism (4). However, these mechanisms of anemia during septic shock remain overall insufficiently explored as erythropoiesis has never been investigated in this context.

Erythropoiesis results in the production of red blood cells from the bone marrow pluripotent stem cells. In adults, it is tightly regulated by erythropoietin (Epo), a glycoprotein hormone mainly produced by interstitial cells of the kidney. Epo binds itself to a specific Epo receptor (EpoR) to activate a signaling cascade that supports in vivo survival and proliferation of mature erythroid progenitors of colony-forming unit erythroids and their terminal differentiation. These positive effects of Epo are counterbalanced by ligands for the members of the tumor necrosis factor (TNF) receptor family, especially Fas ligand (FasL), TNF apoptosis-inducing ligand (TRAIL), and TNF-{alpha} (5–7). An imbalance between survival and death signals resulting in impaired red cell production has been previously observed in myelodysplasia and rheumatoid arthritis (8, 9). Apoptosis is also of dramatic importance during septic shock (10). More precisely, the involvement of apoptosis by the Fas pathway was previously suggested in various lineages during sepsis (10, 11) but had never been studied in the erythroid compartment. To investigate the erythropoiesis abnormalities that could contribute to the early-onset anemia during septic shock, morphologic and functional studies were conducted in erythroid cells derived from the bone marrow of critically ill patients with septic shock. Some of the results of this study were reported in the form of an abstract at the 100th meeting of the American Thoracic Society in 2004 (12).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Ten patients were enrolled in a prospective observational study and were compared with healthy control subjects. Eligible patients were adults (age > 18 yr) with septic shock related to a community-acquired infection (13) who developed early- onset anemia defined by a hemoglobin level below 100 g/L within 48 h after ICU admission. Patients could not be enrolled if any of the following criteria were present: history of bone marrow disease, chemotherapy or immunosuppressive treatments, chronic renal failure (defined as need for chronic renal replacement therapy or known serum creatinine level above 180 µmol/L before the septic event), infection by HIV or human T-cell lymphotropic virus type 1 (HTLV), hemolysis, underlying bleeding disorders, or recent surgical procedure (during the last 30 d). Data collected included demographics, clinical characteristics including Simplified Acute Physiologic Score (SAPS) 2 and logistic organ dysfunction (LOD) severity scores (14, 15), biological features, treatments, and outcome. Healthy control subjects were patients referred for exploration of thrombophilia, which includes bone marrow aspiration as a routine procedure. These control subjects were found to have normal hemogram and normal clonogenic culture study in methyl-cellulose medium and bone marrow pathologic analysis. The use of these control samples was granted with the agreement of the French Ministry of Health. The study was approved by our institution's ethics committee and all subjects or their surrogates gave written, informed consent.

Plasma and Serum Dosages of Epo, Interleukin-6, TRAIL, FasL, TNF-{alpha}, and IFN-{gamma}
Plasma and serum were obtained from peripheral blood at time of medullar aspiration. Cytokine levels were measured at inclusion using ELISA with specific reagents according to the manufacturer's instructions (Quantikine; R&D System, Minneapolis, MN).

Bone Marrow Samples and Cytologic Analysis
The mean delay between ICU admission and medullar aspiration was 30 ± 12 h. Bone marrow was collected by sternal aspiration in a dried sodium heparin–containing bottle and diluted in phosphate-buffered saline (PBS) containing 0.8% bovine serum albumin. Bone marrow mononuclear cells (BMMNCs) were isolated using Ficoll-Hypaque gradient. Cellular lineages were quantified using light microscopy (x100) after May-Grünwald-Giemsa staining.

Glycophorin A–positive Cell Separation
Cells that expressed membrane glycophorin A (GPA), a specific protein of mature erythroid elements, were isolated on MIDI-MACS immunomagnetic columns among the BMMNCs according to the manufacturer's instructions (Miltenyi Biotech, Bergisch Badgach, Germany [8]). GPA positivity quantified by flow cytometry was above 90% after isolation. Morphology was assessed by light microscopy (x100). This separation provided erythroid precursors that were tested for fluorescence-activated cell sorter analysis and biochemical assays.

Colony Formation Assay
BMMNCs were cultured to quantify clonogenic erythroid progenitors that form late burst-forming unit erythroids (BFU-Es) after 10 d of culture in semisolid medium. Cells were plated as previously described in 0.8% methyl-cellulose medium containing fetal calf serum 20% and a mixture of cytokines including 2 U/ml recombinant human (rHu) Epo provided by OrthoBiotech France, Inc. (Issy-les-Moulineaux, France) (8). The effect of control subjects' and patients' sera on BFU-E formation was also tested. BMMNCs from patients were plated in usual semisolid culture medium with 0.8% methyl-cellulose containing 0.5 U/ml rHuEpo, 50 ng/ml stem cell factor (SCF), and 40 ng/ml rHu IGF-1 added with 20% of control subjects' or patients' sera. In some experiments, addition of a higher concentration of rHuEpo (2 U/ml) tested the "rescue" properties of Epo. Late BFU-Es were numbered under an inverted microscope at Day 10 by two independent examinators blinded for the sample provenance.

Epo-induced Stimulation
For the analysis of EpoR signaling, BMMNCs were resuspended and serum starved for 4 h before stimulation with 10 U/ml rHuEpo for 10 min before lysis. Protein extracts were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblots were performed using phospho-ERK 1/2 and phospho-STAT5 from Promega Life Science (Madison, WI), anti-S473 phospho-AKT from New England Biolabs (Beverly, MA), and anti-threonine 32 phospho-FKHRL1 from Upstate Biotechnologies (Lake Placid, NY).

Immunophenotyping
GPA-positive BMMNCs were incubated (20 min, 4°C) in the presence of anti-Fas fluorescein isothiocyanate, anti–TNF-{alpha} receptor type 1 (TNFR1) phycoerythrin (PE)-conjugated antibodies (Becton-Dickinson, Palo Alto, CA). Cells were also incubated with unlabeled primary antibodies to TRAIL or TRAIL receptors (TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4) and with secondary PE-conjugated antibodies before analysis. A Coulter Epics XL device (Beckman Coulter, Inc., Miami, FL) was used to quantify fluorescence. Fas, TNFR1, TRAIL, TRAIL-R1, TRAIL-R2, TRAIL-R3, and TRAIL-R4 expression was reported as the ratio of mean fluorescence intensity to the irrelevant control antibody.

Analysis of Apoptosis
Apoptosis was quantified on GPA-positive cells by staining both with annexin V fluorescein isothiocyanate (Boehringer Mannhein, Mannhein, Germany) and propidium iodide (PI; Sigma-Aldrich, St. Louis, MO). Variation in the mitochondrial transmembrane potential ({Delta}{psi}m) was measured by labeling cells with both 3-3'-dihexyloxacarbocyanine iodide (DiOC6[3]; Sigma-Aldrich) and PI (16). Results were expressed as percentages of annexin V–positive/PI-negative cells and percentages of DiOC6(3) low/PI-negative cells.

Statistical Analysis
Values are expressed in mean ± SD. In the absence of a normal distribution, continuous variables of independent samples are compared by nonparametric Mann-Whitney's test. Paired data were analyzed by Wilcoxon rank sum test. p Values of less than 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patients
Between November 2002 and March 2003, 46 patients were admitted to the medical ICU for septic shock, of whom 10 (mean age, 51 ± 16 yr) met the inclusion criteria. The reasons for noninclusion were as follows: hemolysis (n = 1); HIV infection (n = 2); chemotherapy, immunosuppressive treatment, or hemopathy (n = 14); chronic renal failure (n = 3); and inability to obtain informed consent (n = 16).

Patients' characteristics are reported in Table 1. Mean SAPS 2 and LOD scores were, respectively, 68.7 and 7.8, highlighting a severely ill population with a predictable mortality rate higher than 80%. Mean duration of stay in the ICU was 15 d (ranging from 2 to 56 d). Primary infection was pneumonia (n = 7), peritonitis (n = 1), endocarditis (n = 1), and septicemia (n = 1). Bloodstream cultures were positive in four patients and pathogens were identified in seven. All patients enrolled in the study developed multiorgan failure related to sepsis and required supportive care with vasoactive drugs and mechanical ventilation. An elevated serum creatinine concentration was present at inclusion in 4 of 10 patients and 6 patients required renal replacement therapy after bone marrow and blood sampling. The in-ICU mortality rate was 60%. Biological data at inclusion plus hemoglobin level and transfusion requirement during ICU stay are reported in Table 2. All patients exhibited nonregenerative anemia and all 5-d survivors (n = 6) required PRBC transfusion because of a hemoglobin level below 70 g/L according to standard recommendations (17).


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  		TABLE 1. DEMOGRAPHICS, DIAGNOSIS, MICROBIOLOGICAL IDENTIFICATION, PROGNOSTIC SCORING SYSTEM, LENGTH OF STAY IN INTENSIVE CARE UNIT, AND OUTCOME FOR THE 10 PATIENTS IN THE STUDY

 

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  		TABLE 2. LABORATORY DATA ON ADMISSION AND RED CELL PACKED TRANSFUSIONS DURING INTENSIVE CARE UNIT HOSPITALIZATION

 
Erythroid Lineage Is Not Decreased in Bone Marrow of Patients with Septic Shock and Anemia
Morphologic study of the bone marrow aspiration revealed a normal distribution of the hematologic lineages in patients with septic shock (Table 3). No differences could be detected in the erythroid, granulomonocytic, and lymphoid lineages between patients with septic shock and control subjects, and bone marrow smears were equally rich in the two groups. Cytologic count of erythroid compartment in patients with septic shock revealed 7, 22, and 71% of basophilic, polychromatophilic, and acidophilic erythroblasts, respectively. Megakaryocytes were present in all bone marrow aspirations. In addition, no features of hemophagocytosis were detected in bone marrow from patients with septic shock. These results suggest that early-onset anemia observed during septic shock cannot be explained by a decreased number of erythroid precursors, abnormal differentiation, or erythrophagocytosis secondary to macrophagic activation.


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  		TABLE 3. DISTRIBUTION OF THE HEMATOLOGIC LINEAGES AFTER CYTOLOGIC EXAMINATION OF THE BONE MARROW ASPIRATION FROM CONTROL SUBJECTS AND PATIENTS WITH SEPTIC SHOCK

 
Apoptosis Is Increased in Bone Marrow Erythroid Precursors during Septic Shock
Erythroid precursors, which are distinguished from other BMMNCs by their GPA staining and are called GPA+ cells, were isolated and studied for apoptosis by flow cytometry. The percentage of annexin V–positive/PI-negative cells was significantly higher in erythroid precursors derived from patients with septic shock as compared with control subjects (26.1 ± 8.8 and 3.1 ± 2.9%, respectively; p < 0.05; Figure 1A). The variation of mitochondrial transmembrane potential ({Delta}{psi}m) was also measured as an early step of apoptosis. We found that the percentage of DiOC6(3) low/PI-negative cells was significantly higher in patients with septic shock than in control subjects (55.9 ± 10.5 vs. 19.1 ± 5.4%, respectively; p < 0.05; Figure 1B). This confirms that there was an increase of cells with features of apoptosis, either collapse of {Delta}{psi}m or plasma membrane modifications.


Figure 1
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  		Figure 1. Flow cytometry analysis for the quantification of apoptotic cells. Apoptotic cells were quantified by flow cytometry using membrane staining with annexin V–positive/propidium iodide (PI)–negative (A) and 3–3'-dihexyloxacarbocyanine iodide (DiOC6[3]) low/PI–negative (B). Results are presented as mean ± SD percentage of apoptotic cells for each staining. A p value < 0.01 is considered statistically significant and marked with an asterisk. C illustrates a representative experiment of the fluorescence-activated cell sorter analysis: annexin V (top) and {Delta}{psi}m (bottom) in a control (left) and a patient (right).

 
Epo Signaling Is Preserved in BMMNCs from Patients with Septic Shock
Because in vivo survival of the mature erythroid progenitors mainly depends on the presence of Epo, we quantified the Epo plasma level and the functionality of its receptor in patients with septic shock and control subjects. Plasma Epo concentration in patients with septic shock did not differ from that of control subjects (34 ± 15 vs. 38 ± 8 mU/ml, p = 0.965) and remained within normal range (Table 2). EpoR appeared to be functional as demonstrated by analysis of EpoR signaling pathway. Hence signals of phospho-ERK 1/2, phospho-STAT5, phospho-Akt and activation of its downstream substrate FKHRL1 were increased in BMMNCs from control subjects and patients after Epo stimulation (Figure 2A). This suggests that antiapoptotic signaling is normal in Epo-responsive cells. Then we quantified late erythroid progenitors giving BFU-Es at Day 10 of semisolid culture from the BMMNCs both in patients and control subjects. The number of BFU-Es was similar in control subjects and patients with septic shock (151 ± 65 vs. 174 ± 57 per 105 seeded cells, respectively; p = 0.58; Figure 2B). This result shows that the pool of late erythroid progenitors is not quantitatively impaired in patients' bone marrows.


Figure 2
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  		Figure 2. (A) EpoR signaling in bone marrow mononuclear cells (BMMNCs) from patients with septic shock and control subjects. Cells were extracted from total bone marrow and were incubated with Epo. Immunoblotting was used to reveal the activation of pathways downstream from EpoR. (B) Quantification of burst forming unit erythroids (BFU-Es) in semisolid clonogenic culture. A total of 2.5 x 105 BMMNCs were seeded in a methylcellulose medium containing 10% fetal calf serum with Epo, stem cell factor (SCF), interleukin (IL)-6, IL-3, and granulocyte-macrophage colony–stimulating factor. BFU-E–derived colonies were counted at Day 10. Results are presented as mean ± SD of the BFU-E colonies numbered for 105 seeded cells.

 
Fas Is Overexpressed at the Membrane of Erythroid Precursors during Septic Shock
We tested GPA+ cells for the expression of pro- and antiapoptotic proteins to investigate whether a specific death receptor pathway could be involved in the erythroid compartment apoptosis. Using flow cytometry, we quantified the expression of Fas, the main death domain receptor involved in the negative regulation of normal erythropoiesis. The expression of Fas was significantly higher in patients with septic shock than in control subjects (mean fluorescence intensity: 3.06 ± 1.20 vs. 1.06 ± 0.18, respectively; p < 0.01; Table 4). In contrast, the membrane expression of the type 1 TNF-{alpha} receptor and of TRAIL and its specific receptors (the death domain–containing TRAIL-R1 and TRAIL-R2 and the decoy receptors TRAIL-R3 and TRAIL-R4) were similar in both groups (Table 4).


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  		TABLE 4. GLYCOPHORIN A–POSITIVE ERYTHROID PRECURSORS MEMBRANE EXPRESSION OF Fas, TUMOR NECROSIS FACTOR (TNF) RECEPTOR 1, TNF APOPTOSIS-INDUCING LIGAND (TRAIL), AND TRAIL RECEPTORS

 
Serum from Patients with Septic Shock Impairs the Development of Erythroid Progenitors
To assess the role of serum from patients with septic shock on the development of late erythroid progenitors, BMMNCs from several patients with septic shock were plated in semisolid cultures containing 0.5 U/ml rHuEpo and 20% serum from either patients or control subjects. At Day 10, the number of BFU-Es was significantly lower in the presence of serum from patients with septic shock (27 ± 12 per 105 seeded cells) as compared with control serum (109 ± 27 per 105 seeded cells, p < 0.001; Figure 3) suggesting a strong growth inhibitory effect of septic serum. To test the ability of a saturating concentration of Epo to restore the development of erythroid progenitors in the presence of septic serum, 2 U/ml rHuEpo was added to clonogenic cultures. At Day 10, both in the presence of septic and control serum, the number of BFU-Es was significantly increased in the presence of an elevated concentration of Epo but remained higher when erythroid progenitors were cultured with control serum as compared with septic serum (182 ± 15 vs. 72 ± 14, respectively; p < 0.001), suggesting the presence of an inhibitory factor in the latter group (Figure 3).


Figure 3
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  		Figure 3. Results of semisolid clonogenic cultures: BFU-E formation using serum from control subjects and from patients with septic shock, in the presence of 0.5 or 2 U/ml rHuEpo. Comparisons between control and septic serums were performed using a Mann-Whitney test. Comparisons between the two Epo conditions (0.5 and 2 U/ml) in the control and septic serums were performed using a Wilcoxon rank sum test.

 
To investigate whether septic serum components contributed to impaired erythroid progenitor development, we measured plasma concentrations of IL-6 as a marker of inflammation and cytokines known to regulate apoptosis including TNF-{alpha}, FasL, TRAIL, and IFN-{gamma}. Plasma levels of IL-6 significantly differed between patients with septic shock and control subjects (1,332 ± 821 vs. 13 ± 11 ng/ml, respectively; p < 0.001). Also, TNF-{alpha} levels were higher in plasma of patients with septic shock (31 ± 17 pg/ml) as compared with control subjects (12 ± 9 pg/ml, p = 0.018). Concentrations of the remaining cytokines involved in the apoptotic response did not significantly differ between the two groups (Table 5).


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  		TABLE 5. PLASMA CONCENTRATION OF THE DIFFERENT CYTOKINES TESTED IN SERUM AND PLASMA FROM PATIENTS WITH SEPTIC SHOCK AND CONTROL SUBJECTS

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Erythropoiesis abnormalities have never been studied in the setting of early-onset anemia in patients with septic shock. Here we show that (1) erythroid lineage is not quantitatively decreased in the bone marrow of patients with septic shock with early anemia, (2) anemia is associated with apoptosis of bone marrow erythroid precursors despite the presence of a functional EpoR on BMMNCs, (3) membrane expression of the death domain receptor Fas is up-regulated, and (4) the number of late erythroid progenitors is decreased in the presence of serum from patients with septic shock.

This research presents some limits. The number of studied patients is low but may be sufficient for a preliminary study with well-defined characteristics in a specific subset of patients with septic shock as revealed by the homogeneity of bone marrow studies. In contrast, the nature of the control population (healthy ambulatory patients) may be criticized but ethical considerations precluded medullar aspirations in the best control population, which would have been patients with septic shock who had not developed anemia. Finally, we did not assess the quantification of colony-forming unit erythroids that correspond to the most mature erythroid progenitors that differentiate into erythroid precursors in vivo.

Anemia is a common feature in critically ill patients with septic shock. Among the numerous factors that may contribute to its development, some of them cannot be implicated in our population due to the design of the study. Only patients with early-onset anemia were studied, thus excluding frequent blood sampling and renal failure as potential causes. Even if some of our patients (6 of 10) required renal replacement therapy, these patients had no previous renal disorder and the renal failure was an acute phenomenon related to the septic shock. Furthermore, as patients with blood loss (surgical procedure, active bleeding) or hemolysis were excluded, erythropoiesis abnormalities can be suspected as a factor that contributes to early-onset anemia in patients with septic shock. Erythropoiesis is under control of antiapoptotic effects of Epo but also depends on proapoptotic signals from Fas and TRAIL pathways (6, 7, 18). Our results suggest that early-onset anemia in patients with septic shock is associated with a defective erythropoiesis related to an imbalance between anti- and proapoptotic signals. The patients exhibited inappropriately low plasma Epo concentrations for the degree of anemia as previously reported (19). This finding is in accordance with previously published data on low Epo levels in serum of critically ill patients with sepsis (20). This inadequacy could partly explain anemia because Epo signaling is pivotal for survival of late erythroid progenitors in vivo and in vitro (21, 22), through the phosphatidylinositol 3-kinase pathway (23). Another mechanism involved in early-onset anemia could be a blunted EpoR signaling. However, Western blot analysis of BMMNCs from patients with septic shock stated that this pathway was functional because Akt and FKHRL1, downstream substrates of phosphatidylinositol 3-kinase, were phosphorylated in response to Epo stimulation, suggesting a functional EpoR.

Apoptosis in sepsis can account for organ failure (11) and involves hematopoietic lineages including polymorphonuclear cells (24), lymphocytes, dendritic cells (25, 26), and endothelial cells (27). Our results suggest that apoptosis is increased in the erythroid lineage of patients with septic shock compared with control subjects. Indeed, mean percentages of DiOC6(3) low cells/PI-negative cells and of annexin V–positive/PI-negative cells were higher in patients with septic shock than in the control group. Because apoptosis was rare in the control group, we could exclude the possibility that GPA+ cells were stressed during the isolation step. The collapse of {Delta}{psi}m revealed by low DiOC6(3) labeling has been demonstrated to characterize cells ongoing early steps of apoptosis preceding DNA fragmentation and to be reversible when Bcl-2 is overexpressed (16). By contrast, annexin V–labeled cells correspond to apoptotic cells with DNA fragmentation and phosphatidylserine exposure, which is a phagocytic signal. Because the mean percentage of cells with a drop of {Delta}{psi}m was higher than the mean percentage of annexin V–positive cells both in the septic shock and control groups, it is very likely that only some of the GPA+ erythroid precursors enter the full apoptotic state. Apoptosis was associated with increased membrane expression of Fas and high TNF-{alpha} plasma levels. Physiologically, Fas allows the down-regulation of early erythroid progenitors by late erythroid progenitors that express FasL (7). In various conditions, including hematologic diseases, up-regulation of Fas-related apoptosis may explain anemia (8, 28). Anemia related to inflammatory disorders like rheumatoid arthritis has also been explained by an increased apoptosis of the erythroid lineage linked to TNF-{alpha} (9). Indeed, TNF-{alpha} is overexpressed in the acute phase of severe sepsis (reviewed in Reference 29). This cytokine is supposed to down-regulate erythropoiesis (30, 31) as it positively regulates the expression of Fas by a transcriptional mechanism. Thus, overexpression of Fas on erythroid precursors might be related to the high level of TNF-{alpha}. Soluble FasL plasma concentration, which is usually elevated in sepsis (32), was not increased in our septic population. Moreover FasL was not overexpressed in GPA+ erythroid precursors lysates of patients with septic shock (data not shown). The accurate mechanisms of erythroid progenitor apoptosis remain unclear in our study. The study of erythroid precursors isolated from TNF-{alpha} null mice and lpr mice (which encodes a nonfunctional Fas) subjected to septic shock may help to clarify the role of these signaling pathways in the genesis of septic anemia.

Even if proapoptotic factors still need to be precisely identified, we have clearly shown the negative effect of septic serum on late erythroid progenitor proliferation. Interestingly, addition of high-concentration rHuEpo partially allows the in vitro development of these erythroid progenitors even in the presence of septic serum. This result is in accordance with a previous study reporting that Fas cross-linking–induced apoptosis in erythroblasts was antagonized by Epo in a dose-dependent manner (7). We hypothesize that the relatively low level of plasma Epo in patients with septic shock may represent an insufficient antiapoptotic signal delivered to the erythroid lineage.

Recent clinical data also support our observations. Critically ill patients treated with rHuEpo required less transfusion than a control group (33) with dose regimen of rHuEpo allowing plasma concentration comparable to those used in culture medium (34). Unfortunately, the subgroup analysis did not specifically evaluate the subset of patients with septic shock. Our results support the need for further clinical research on the beneficial effects of high doses of rHuEpo in patients with septic shock.


   FOOTNOTES
 
Supported by OrthoBiotech France, Inc.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournal.org

Originally Published in Press as DOI: 10.1164/rccm.200504-561OC on March 30, 2006

Conflict of Interest Statement: Y.-E.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. F.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.-D.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.H. is an employee of Janssen Cilag division of Ortho Biotech. N.C. received {euro}2,000 in 2003 and {euro}2,000 in 2004 for speaking at conferences and participating in advisory boards for Ortho Biotech. J.-F.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.-P.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.C. has participated as a speaker in scientific meetings organized and financed by Orthobiotech France, Inc., and received honoraria from their firm. Orthobiotech France, Inc., had no involvement in the analysis and interpretation of the data, in the writing of the report, or in the decision to submit the paper for publication.

Contributors: The study was designed by Y.E. Claessens and A. Cariou. The experiments were realized by Y. E. Claessens, M. Guesnu, and N. Casadevall. C. Hababou contributed to the collection of the data. Y. E. Claessens, A. Cariou, F. Pene, M. Fontenay, and J. P. Mira produced the manuscript. J. D. Chiche and J. F. Dhainaut participated in its critical revision. Y. E. Claessens had full access to all the data in the study and had final responsibility for the decision to submit for publication.

Received in original form April 11, 2005; accepted in final form March 30, 2006


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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