The Egyptian Journal of Haematology

ORIGINAL ARTICLE
Year
: 2018  |  Volume : 43  |  Issue : 4  |  Page : 198--205

Influence of splenectomy on CXCL8 and T lymphocyte subsets in children with β-thalassemia major: single center Egyptian study


Nahed Hablas1, A Fakhereldin1, Sheren Awny2, Sarah A Hamam3,  
1 Pediatrics Departments, Tanta University, Tanta, Egypt
2 Internal Medicine, Tanta University, Tanta, Egypt
3 Clinical Pathology, Tanta University, Tanta, Egypt

Correspondence Address:
Sarah A Hamam
10 Dar Elsalam Street from Omar Ben Abdel Aziz Street, Tanta
Egypt

Abstract

Background After splenectomy, the immune system is modified; there are numerous quantitative and functional defects involving T and B lymphocytes and blood anomalies of serum level of cytokines. The present study aimed to evaluate immuneinflammatory status interactions with thalassemia pathogenesis, especially after splenectomy, which can provide new modalities for the management of the disease and its complications. Patients and methods The present study was conducted on 40 children with β-thalassemia major under follow-up at the Hematology Unit, Pediatric Department, Tanta University. There were 24 (60%) male and 16 (40%) female patients; they were blood transfusion dependent and underwent splenectomy for more than 6 months. Their ages ranged from 6 to 17 years. Besides, 40 healthy children served as the control group. All children included in the study were subjected to complete blood count, iron profile, T-cell subsets including CD3, CD4, and CD8 and serum interleukin (IL)-8 levels. Results There were significantly higher IL8, CD3, CD4, and CD8 and significantly lower CD4/CD8 ratio in patients than controls. There were significantly higher IL8, CD3, CD4, CD8, serum ferritin and iron levels and significantly lower total iron binding capacity (TIBC) level after splenectomy than before. Moreover, there was a significant positive correlation between serum ferritin and the immunological markers. Conclusion and recommendations Children with β-thalassemia major had significant abnormalities of the serum levels of CD3, CD4, CD8, and IL8 before and after splenectomy. These changes were associated with increased tendency to infections. Hence, evaluation of serum levels of IL8, CD4, and CD8 may be a useful tool and can help to provide new modalities for management.



How to cite this article:
Hablas N, Fakhereldin A, Awny S, Hamam SA. Influence of splenectomy on CXCL8 and T lymphocyte subsets in children with β-thalassemia major: single center Egyptian study.Egypt J Haematol 2018;43:198-205


How to cite this URL:
Hablas N, Fakhereldin A, Awny S, Hamam SA. Influence of splenectomy on CXCL8 and T lymphocyte subsets in children with β-thalassemia major: single center Egyptian study. Egypt J Haematol [serial online] 2018 [cited 2019 Dec 15 ];43:198-205
Available from: http://www.ehj.eg.net/text.asp?2018/43/4/198/255874


Full Text



 Introduction



β-Thalassemia major (BTM) represents a major health problem worldwide with extensive range of complications disturbing different organs [1]. TM is the most common inherited anemia in Egypt, resulting from impaired hemoglobin synthesis and ineffective erythropoiesis [2].

The mainstay of conventional therapy is chronic lifelong blood transfusions. The long-term complication of chronic transfusions is iron overload, which is considered as the main culprit responsible for various organ disabilities and increased incidence of infections, as it reduces phagocytosis, reduces opsonization and increases bacterial activity. Moreover, it causes alterations in T-lymphocyte subsets [3].

Splenectomy in BTM is one form of the conventional management, carried out at blood consumption greater than 50% above the mean requirement of the splenectomized population [4]. The spleen is a primary organ of immunological surveillance. After splenectomy, the immune system is modified; there are numerous quantitative and functional defects involving T and B lymphocytes, including impaired activity of monocytes and neutrophils, numerical or functional alternation of different peripheral lymphocytes and blood anomalies of serum level of cytokines [5],[6].

Interleukin-8 (IL8) is a cytokine produced by peripheral blood mononuclear cells, endothelial cells and fibroblasts. It has a complex function in the regulation of the inflammatory process, as it is a chemoattractant protein for neutrophils that stimulates chemotaxis and degranulation [7].

 Patients and methods



Patients

This is a prospective case–control study conducted at the Pediatric Department, Tanta University Hospital. It was carried out on 40 children with homozygous B-thalassmia major (they were diagnosed by clinical examination and hemoglobin electrophoresis) who were attending the Hematology Clinic for receiving blood transfusion and treatment. There were 24 (60%) male and 16 (40%) female patients. Their ages ranged from 6 to 17 years; they were blood transfusion dependent. Besides, 40 healthy children served as the control group. Samples were collected after research ethical committee approval and obtaining written informed consents from the parents of studied children, samples were collected for patients at the start then before surgery and 6 month after splenectomy. The study was carried out in the period between August 2015 and June 2017.

Inclusion criteria

Children with BTM with significant iron overload and who were maintained on regular chelation therapy, as per our protocol (all patients were treated with Deferasirox at a dose of 20–30 mg/kg/day once daily before meals). For patients with persistently high serum ferritin levels above 3000 ng/ml, Deferasirox is combined with Desferrioxamine 20–40 mg/kg for 8–12-h subcutaneous infusion using infusion pump or continuous intravenous infusion for 8–10 h per day for 10 days monthly [8]) and underwent splenectomy were included in the study.

Exclusion criteria

β-Thalassemia patients who were positive for viral hepatitis and HIV were excluded from the study.

Methods

All the laboratory tests were performed in the Clinical Pathology Department, Tanta University, Egypt

Routine laboratory tests

The complete blood count was analyzed with ERMA PCE-210 cell counter (Tokyo, Japan). Stained blood films with Gimsa were examined microscopically.

T-cell subsets (CD4+ and CD8+) were analyzed using Facs Canto flow cytometer (BD, Heidelberg, Germany). Briefly, 100 µl of EDTA blood was stained with 5 µl of the antibodies, anti-CD4-FTIC, anti-CD8-PE, and anti-CD45-V500 (all antibodies were provided by BD) in the staining tubes; the tubes were incubated in dark conditions for 20 min; thereafter, the samples were mixed with BD FACS lysing solution (1×) then incubated for 15 min in dark conditions. The samples were then centrifuged at 1250 rpm for 5 min; the supernatant was discarded to remove the lysed red blood cells. PBS was added; thereafter, the samples were centrifuged at 1250 rpm for 5 min; the supernatant was discarded to remove any remaining debris or red blood cells; thereafter, the pellets were resuspended in 350 µl of PBS. The absolute numbers of cells were calculated using the following formula: percentage of cells×total number of white blood cells/100 [9].

Estimation of serum interleukin-8 by enzyme-linked immunosorbent assay

Blood samples for the measurement of IL8 were collected in a serum separator tube, and samples were allowed to clot for 30 min at room temperature before centrifugation for 15 min at 1000g. Then serum samples were stored at −80°C until they were assayed. The concentrations of IL8 in the serum samples were measured using a commercial human IL8 ELISA kit Catalog Number D8000C (R and D System Inc., Minneapolis, Minnesota, USA), according to the manufacturer’s instructions. The intensity of the developed color was measured by reading optical absorbance at 450 nm using a microplate reader (Sunrise; Tecan Group Ltd, Mannedorf, Switzerland). The results were expressed as a pictogram of per milliliter plasma (pg/ml) [10].

Statistical methods

All statistical analyses were performed using SPSS software version 21 (SPSS Inc., Chicago, Illinois, USA); the differences were taken as significant when P value was less than 0.05. Data were expressed as mean±SD and were compared using Mann–Whitney U-test. Comparison of means before and after splenectomy are carried out using the paired t-test. Bivariate correlation test was used to test the association between variables.

 Results



Demographic data and clinical presentations of the studied groups are shown in [Table] 1 and [Table 2]. There were no significant differences between patients and controls with regard to age and sex, but there were significantly lower weight and height differences and significantly higher incidence of infective episodes in the patient group ([Table 1]).{Table 1}{Table 2}

Before splenectomy, there were significantly lower median hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and neutrophil percentages and platelet count, and significantly higher mean white blood cells count, reticulocyte, eosinophil, monocyte, and lymphocyte percentage in patients than in controls ([Table 3]). There were significantly higher serum ferritin, iron level and significantly lower total iron binding capacity (TIBC) in patients compared to control group with P<0.05 ([Table 4]). There were significantly higher IL8, CD3, CD4, and CD8 levels and significantly lower CD4/CD8 ratio in patients than in controls ([Table 5]).{Table 3}{Table 4}{Table 5}

After splenectomy, there were no significant differences with regard to MCV, MCH, reticulocyte % and platelets count and significantly higher mean HB, white blood cells count, monocyte, eosinophil, and lymphocyte percentage, while there was a significant decline in neutrophil percentage than before splenectomy ([Table 6]). There were significantly higher IL8, CD3, CD4, CD8, serum ferritin and iron levels and significantly lower TIBC after splenectomy than before ([Table 7]).{Table 6}{Table 7}

By correlating serum ferritin to the studied immunological markers, there were significant positive correlations between ferritin and CD8 and IL8. There was no correlation of IL8 level and neutrophils (r=−0.04, P=0.7) [Figure 1]a,b.{Figure 1}

 Discussion



Several immunological changes have been reported with BTM. Many factors such as path physiology of the disease, repeated antigen sensitization at the time of blood transfusions, iron overload, splenectomy, zinc deficiency, and chelating agents are known to have profound effects on the immune system [11].

Serum ferritin and iron levels were higher after splenectomy than before; this is in agreement with Mahmoud et al. [12]; these findings can be explained, as the spleen is a reservoir for iron, and spleen iron seems to be cleared faster than liver iron using effective chelation therapy [13].

Our results revealed that there was a significant increase in the serum IL8 level in the BTM children, with a profound increase after splenectomy. This result came in accordance with Shfik et al. [14]. It has been reported that macrophages and fibroblasts can be responsible for IL8 production, either directly or indirectly via tumor necrosis factor-α synthesis. Hence, serum IL8 level might be increased in thalassaemic children as a consequent macrophage activation due to the transfusion-related continuous antigenic stimulation and iron overload. Moreover, it was suggested that during erythrophagocytosis the activated monocytes may generate different cytokines to augment their phagocytic function and that IL8 may rise in response to endogenous stimuli such as tumor necrosis factor and IL1 [15].

Lymphocyte subpopulations include T cells, B cells, and NK cells. T lymphocytes contain CD4+ T cells, which become activated upon foreign antigen exposure, such as intracellular pathogens, fungi and protozoa, in addition to CD8+ T cells, which destroy virally infected cells. CD4+ cells along with CD8+ cells represent the majority of T lymphocytes, Blood transfusion causes chronic infections, and thus constant stimulation of the immune system, which might induce the production of T cells [16]. Anemia, reticuloendothelial system dysfunction, and ferritin levels affect immune response, which results in higher susceptibility to infections. Iron and its binding proteins have immune-modulating properties, and excess of iron may produce deleterious effects on the immune system [17]. Likewise, splenectomy has been correlated with quantitative lymphocyte changes and aggravation of the immunological effects of multiple transfusions due to the reduced clearance of immune cells [18].

The result of this study showed that CD3, CD4, and CD8 levels were significantly increased in patients than in the control group. Moreover, there was a significant increase in the CD3, CD4, and CD8 levels after splenctomy than before (P<0.05); these results are in agreement with Gharagozloo et al. [19], Mahmoud et al. [12], and Kadam et al. [20]. The increased T-lymphocyte count after splenectomy could be attributed to a higher frequency of acute infectious episodes in these patients and might be consecutively associated and could not be effectively filtered by the spleen [21] and lead to the suggestion that the spleen plays a role in lymphocyte regulation [20]. Contrary to our results, Samir et al. [21] found no significant difference in CD4 helper T lymphocytes, CD8 cytotoxic T lymphocytes, or CD4+/CD8+ ratio among both groups.

The result of our study showed a significant lower median of CD4/CD8 ratio after splenectomy than before The study by Ahluwalia [22] and Javad et al. [23] also had a lower CD4/CD8 ratio in splenectomized as compared with nonsplenectomized patients and controls.

By correlating serum ferritin to CD8 and IL8 levels, we found a significant positive correlation, which coincided with Kadam et al. [20]; their study also showed a significant positive correlation of serum ferritin with CD4 and CD8.

Conclusion and recommendations

Children with BTM have significant abnormalities of the serum levels of CD3, CD4, CD8, and IL8 before and after splenectomy. These changes might be associated with an increased tendency to infections. Hence, evaluation of serum levels of IL8, CD4, and CD8 may be a useful tool in improving our knowledge about these immunological defects in thalassemia, especially after splenectomy.

Acknowledgements

The authors would thank the children, patients, their parents, physician assistances, nurse practitioners, and fellows who acquired the specimens.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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