Aged or abnormal red blood cells with exposed phosphatidylserine (PS-RBCs) are cleared from the circulation by splenic macrophages. In asplenic patients, other mononuclear phagocytic cells in tissues and in circulation may function in this capacity. To better understand these changes and the relationship among splenic status, PS-RBCs, blood monocytes, and serum tumor necrosis factor (TNF-α), a product of mononuclear phagocyte activation, patients with hemoglobin E/β-thalassemia (E/β-Thal) were studied. Whole blood of 20 nonsplenectomized, 20 splenectomized E/β-Thal patients, and 20 healthy subjects was assayed for PS-RBCs; for monocytes, activated monocytes, and monocyte response to lipopolysaccharide stimulation; and serum was assayed for TNF-α. Asplenic E/β-Thal patients had significantly increased (P < 0.05) amounts of PS-RBCs, monocytes, activated monocytes, and levels of serum TNF-α. The amount of PS-RBCs correlated with levels of serum TNF-α, but the amount of activated monocytes did not correlate with either the amount of PS-RBCs or levels of serum TNF-α. Monocyte response to lipopolysaccharide stimulation in asplenic patients was not as efficient as in the other patients or in normals (77 vs. 404, and 304 folds increment, respectively). The results suggest that splenectomy in E/β-Thal patients led to an increased amount of PS-RBCs and activation in the mononuclear phagocytic system.
Thalassemia (Thal), an autosomal recessive hereditary hemolytic anemia, is the most common known human genetic disorder. It is caused by mutations of the globin gene clusters resulting in varying degrees of decreased globin chain synthesis. It is classified into α and β according to the globin chain involved . It can be co-inherited with an abnormal hemoglobin (Hb), such as Hb E, resulting in Hb E/β-Thal (E/β-Thal), which is the most common form of the symptomatic thalassemic syndromes globally [2, 3]. The produced defective red blood cells (RBC) are cleared prematurely by various mechanisms during their passages through the spleen . One important mechanism is through splenic macrophages' recognition of phosphatidylserine (PS)-exposing cells . From overwork, the spleen may become very large, and splenectomy is usually performed for hypersplenism and/or symptomatic splenomegaly. These defective and pathologic RBCs must then be cleared by other mononuclear phagocytic or reticuloendothelial (RE) cells, such as Kupffer cells , whose function is not as effective as shown by an increased amount of such cells in circulation in splenectomized E/β-Thal patients [5, 7].
Monocyte activation has previously been reported in both Thal [8–10] and sickle cell disease (SCD) [11, 12]. Increased antibody-dependent cellular cytotoxicity  and up-regulation of Fcγ receptor I (FcγRI or CD64) expression  of monocytes in response to their clearance of thalassemic RBCs were reported in both α- and β-Thal. Macrophage colony-stimulating factor, interferon-γ, and tumor necrosis factor (TNF-α) also play roles as inducers and effectors of this activation [8, 9]. Increased serum TNF-α, interleukin-6, and interferon-γ levels were reported in E/β-Thal patients, particularly after splenectomy [13, 14]. Increased amounts of TNF-α and interleukin-1β per cell, indicative of monocyte activation, and increased serum C-reactive protein levels were also reported in SCD , who invariably have functional asplenia.
To better understand the inter-relationship among splenic status, PS-exposing RBCs (PS-RBCs), monocyte activation, and serum TNF-α, a product of mononuclear phagocyte activation, we studied these parameters in E/β-Thal patients with and without intact spleen, and compared them with those of normal controls (NC).
In total, 40 ambulatory and well E/β-Thal patients who attended the adult hematology clinic at Ramathibodi Hospital were studied. Their age ranged from 19 to 57 years, and 19 were men. They were free from medication and blood transfusion for at least four preceding weeks. Half of them had undergone splenectomy, with a median interval since splenectomy of 13.5 years (range, 5–35). In nonsplenectomized (NS) patients, median vertical measurement from splenic tip on palpation to left costal margin was 3.5 cm (range, 0.5–12.5). Blood transfusion and iron chelation therapy were usually modest, and at the discretion of the attending physicians. Controls were 20 consenting healthy subjects, who had not taken any drugs for at least four preceding weeks. Study protocol was approved by the institutional ethics committee for studies in humans (11-46-31). Written informed consents were obtained from all patients.
Patients' characteristics, mean (standard deviation) or median (range) of hematological data, absolute amounts of PS or annexin V (AV)+ RBCs, activated monocytes (CD14+ expressing intracellular TNF-α and CD14+/CD11b+), and levels of serum TNF-α of the patients and normal controls (NC) are shown in Table I. Total amount of packed RBCs transfusion in the two patients' groups almost reached statistically significant difference (P = 0.055, independence t-test). Values of platelet count in the NS group had a wide variation because of the varied spleen sizes. Splenectomized (S) patients had significantly lower amounts of RBCs, and significantly higher amounts of reticulocytes and NRBCs than the others (P < 0.05), indicating more severe hemolysis. They also had significantly higher amounts of white blood cells (WBCs), monocytes, platelets, PS-RBCs, activated monocytes, and levels of serum TNF-α than the others (P < 0.05). Similar to the amount of monocytes, values of the latter three in the NS and NC groups were not significantly different, suggesting an influential role of the spleen on these parameters.
|Characteristic||Hemoglobin E/β-thalassemia||Normal controls (n = 20)|
|Splenectomized (n = 20)||Nonsplenectomized (n = 20)|
|Age (year)||26.0 (20.0–48.0)a||33.0 (19.0–57.0)b||24.0 (18.0–44.0)|
|Total packed red blood cells transfusion (unit)||83.5 ± 111||22.4 ± 41.4||0|
|RBC (1012/L)||2.8 ± 0.4c,d||3.8 ± 1.0c||4.7 ± 0.6|
|Hb (g/L)||60 (45–80)a,b||71 (55–117)b||131 (106–176)|
|Hct (proportion of 1.0)||0.209 (0.164–0.288)a,b||0.229 (0.193–0.368)b||0.396 (0.324–0.547)|
|MCV (fL)||75.4 ± 7.0c,d||65.3 ± 7.8c||86.5 ± 5.9|
|MCH (fmol/cell)||1.4 ± 0.1c||1.3 ± 0.2c||1.8 ± 0.2|
|MCHC (mmol/L)||18.0 ± 1.6c||19.2 ± 1.2c||20.3 ± 1.9|
|Reticulocyte (109/L)||239.1 (154.0–509.6)a,b||75.9 (48.4–193.2)b||41.2 (8.5–76.7)|
|NRBC/100 WBC||598.0 (61.0–1,418.0)a,b||4.0 (0.0–66.0)b||0|
|WBC (109/L)||10.2 ± 2.2c,d||7.6 ± 2.2c||5.7 ± 1.2|
|Monocyte (106/L)||976.0 (223.0–2,650.0)a,b||295.0 (100.0–909.0)||372.0 (180.0–990.0)|
|Platelet (109/L)||735.0 (450.0–1,056.0)a,b||227.5 (31.0–423.0)||280.0 (163.0–381.0)|
|Absolute amount of annexin V + RBCs (109/L)||96.3 ± 44.9c,d||46.9 ± 21.6||40.2 ± 19.3|
|Absolute amount of CD14+-expressing intracellular TNF-α (106/L)||4.3 (1.0–67.9)a,b||0.5 (0.1–10.5)||1.2 (0.1–2.9)|
|MFI of CD14+/CD11b+||65.7 ± 28.2c,d||44.1 ± 12.0||51.4 ± 10.8|
|Serum TNF-α (ng/L)||11.3 ± 3.6c,d||8.4 ± 3.3||7.1 ± 1.6|
Correlation among absolute amounts of activated monocytes, PS-RBCs, and levels of serum TNF-α were assessed by multiple linear regression analysis after controlling for types of subjects. In consideration of skewness, data were first transformed to a log-scale. The only statistically significant correlation (coefficient = 0.914, standard error = 0.188, P < 0.001) was between levels of serum TNF-α and absolute amounts of PS-RBCs in the S group.
Values of median (range) of percentage of CD14+-expressing intracellular TNF-α before and after lipopolysaccharide (LPS) stimulation in each group of subjects are shown in Table II. Before stimulation, the S group had a significantly larger number of such cells than the others (P < 0.05). A significant increase in amounts of such cells after LPS stimulation was shown in all groups (P < 0.001). However, the increment in the S was less than those in the NS and NC groups (77 vs. 404, and 304 folds increment, respectively).
|Type of subjects||N||Percentage of CD14+-expressing intracellular TNF-α, median (range)||P-valuea|
|Before stimulation||After LPS stimulation|
|Splenectomized hemoglobin E/β-thalassemia||20||0.45 (0.15–3.42)b||34.65 (0.31–67.16)c||<0.001|
|Nonsplenectomized hemoglobin E/β-thalassemia||20||0.21 (0.02–3.50)||84.93 (42.94–94.83)||<0.001|
|Normal controls||20||0.26 (0.02–1.23)||78.93 (55.25–93.45)||<0.001|
The increased amount of circulating PS-RBCs in the S group (Table I) is in agreement with previous reports [5, 7] and likely due to the less-efficient function of other RE cells than splenic macrophages in clearing these pathologic cells. However, until we can do pre- and postsplenectomy studies on the same patient, there will always be an argument that the finding could also be due to a more severe disease in the splenectomized one. Findings of a nonstatistically significant difference between the amounts of PS-RBCs in the NS and NC groups (Table I) reinforce a recognition of the better performance by the spleen . Taken together, these findings in the S and NS groups in the context of overlapping disease severity between them suggest that the final amount of circulating PS-RBCs is determined more by status of the spleen than the inherent RBC defects alone.
Monocytosis in the S group could be due to reactive production and/or inability to home to the spleen. However, it was not found in another study despite elevated serum macrophage colony-stimulating factor levels, which were unrelated to splenic status . This discrepancy requires further studies.
Monocyte activation in the S group (Table I) was similar to previous reports in SCD patients [11, 12], who invariably have autosplenectomy, suggesting its close relationship with splenic absence in these two hereditary hemolytic anemia. The smaller amounts, but not statistically different, of activated monocytes in the NS compared with the NC groups (Table I) is quite interesting. It could be misleading because of a small number of patients causing skewness of data, or because of ancillary monocytes having limited roles in the presence of more efficiently functioning (in clearing defective RBCs) and abundant splenic macrophages in an enlarged hyperplastic spleen. Compared with the S group, the higher increment of CD14+-expressing intracellular TNF-α after an ex vivo LPS stimulation in the NS one (Table II) with reserved capacity would lend some support to the latter possibility. It also suggests that the impaired response of monocytes in splenectomized patients could be due to “overwork,” at least to help clear the now abundant circulating pathologic RBCs in the absence of splenic macrophages. Because chronic iron overload can also contribute to this impairment [15, 16], we searched for data on serum ferritin levels near the time of studies and found no statistically significant difference between the S and NS groups [median (range) = 2,599 (424–7,180) ng/mL and 1,150 (266–4,450) ng/mL, respectively. P = 0.136, Mann-Whitney U test].
Levels of serum TNF-α were significantly increased in the S compared with the other two groups (Table I). Their correlation with the amounts of activated monocytes and PS-RBCs were assessed by multiple linear regression analysis after controlling for types of subjects. The only correlation among the three variables in the three groups was between serum TNF-α levels and amounts of PS-RBCs in the S group (coefficient = 0.914, standard error = 0.188, P < 0.001). The results suggest that, after splenectomy, an abnormally large amount of PS-RBCs activated many types of RE cells, all of which can produce TNF-α . Finding hepatosplenomegaly in Thal disease and chronic hemolytic anemia suggests the Kupffer cell being the prominent one.
Our findings of monocyte activation in splenectomized E/β-Thal patients help explain certain clinical features pertaining to this group. Monocyte activation together with elevated serum TNF-α levels could activate vascular endothelial cells, leading to an expression of adhesion molecules and tissue factor on their surfaces . Together with increased amounts of thrombogenic PS-RBCs [5, 7] and activated platelets , this could further facilitate the formation of thrombotic pulmonary arteriopathy, the basis of pulmonary arterial hypertension, which is more prevalent after splenectomy . The blunted inflammatory cytokine response to LPS, in keeping with previous reports of reduced monocyte phagocytic activity because of a “compensatory” increased erythrophagocytic activity in splenectomized β-Thal patients [19, 20], may affect host defense against infection, which is known to be more prevalent and severe among these patients [21–23].
In conclusion, our studies suggest role of spleen in controlling the amount of PS-RBCs and mononuclear phagocytic activity in E/β-Thal patients. Validation in a larger cohort and preferably on the same patients before and after splenectomy would strengthen our conclusion. In the meantime, splenectomy must be judiciously applied in E/β-Thal patients to avoid the undesirable consequences.