ORIGINAL ARTICLE

Leukocytes apoptosis and adipocytokines in children with betathalassemia major

Khalid I. Elsayh1 • Wafaa S. Mohammed2 • Asmaa M. Zahran3 • Khaled Saad1

Received: 20 April 2015 / Accepted: 14 May 2015

� Springer-Verlag Italia 2015

Abstract b-Thalassemia is a significant public health

problem in Egypt. Infectious complications represent the

second most common cause of mortality and the major

cause of morbidity in b-thalassemia major (BTM). The

increased susceptibility of these patients to infectious dis-

eases has been attributed to the abnormalities of the immune

system, which is evident by systemic inflammation and

immune deficiency. In a case control study, 35 patients with

BTM were compared with 30 sex- and age-matched chil-

dren who served as controls. Serum ferritin, high-sensitive

CRP (hsCRP), leptin and adiponectin levels were deter-

mined in all subjects. Apoptosis of neutrophils and

lymphocytes was measured by the Annexin V-fluoroisoth-

iocyanate binding assay. Serum leptin was significantly

lower in patients when compared to controls. In contrast,

adiponectin and hsCRP levels were significantly higher in

the patients than the controls. Positive correlation was

found between adiponectin and hsCRP. BTM patients had

significantly higher total leukocytes, neutrophils and lym-

phocytes compared with controls. BTM children exhibited a

significantly increased apoptosis in T-lymphocytes; how-

ever, there was no significant difference in the percentage of

apoptosis of B-lymphocytes and neutrophils between the

patients and the controls. There was a significant negative

correlation between serum leptin and the percentage of

apoptotic T-lymphocytes. Our BTM patients had a high

percentage of apoptotic T-lymphocyte in comparison with

controls. In addition, they had disturbed serum levels of

adipocytokines and inflammatory markers. These derange-

ments could have a role in the immunological disturbance

observed in thalassemic patients.

Keywords Adiponectin � Apoptosis � Leptin �Inflammatory markers � Thalassemia

Introduction

b-Thalassemia is a significant public health problem in

Egypt, where over one million newborns are expected to be

affected with this disorder, and it is considered the most

common genetically determined chronic hemolytic anemia

(85.1 %) in our locality. A high frequency of carriers has

been reported in Egypt, ranging from 4 to 10 %. This is due

to high rate of consanguineous marriage, which helps to

accumulate deleterious genes in Egyptian families [1, 2].

Infectious complications represent the second most

common cause of mortality and the major cause of mor-

bidity in b-thalassemia major (BTM), with a prevalence of

12–13 %. In addition to the high risk of blood-borne

infections associated with multiple transfusions, the

increased susceptibility of these patients to infectious dis-

eases has been attributed to the abnormalities of the

immune system, which is evident by systemic inflamma-

tion and immune deficiency [3–5].

Inflammation is known to play an important role in the

development of complications in BTM patients. A chronic

inflammatory state is present in BTM patients, with

increased levels of pro-inflammatory cytokines and

inflammatory markers such as C-reactive protein (CRP).

& Khaled Saad

ksaad8@yahoo.com; Khaled.ali@med.au.edu.eg

1 Pediatric Department, Faculty of Medicine, Assiut

University, Assiut 71516, Egypt

2 Clinical Pathology Department, Faculty of Medicine, Assiut

University, Assiut, Egypt

3 Clinical Pathology Department, South Egypt Cancer Institute,

Assiut University, Assiut, Egypt

123

Clin Exp Med

DOI 10.1007/s10238-015-0361-6

Adipocytokines are considered important players in the

pathogenesis of numerous metabolic, immune, vascular

and inflammatory disorders [6–8]. Among these cytokines,

much awareness has been paid to leptin and adiponectin,

both of which have substantial effects on the inflammatory

process [6, 7].

Leptin is characterized by multiple and evident effects

on metabolic and immune functions. Its immune effects

include the stimulation of hematopoiesis and lym-

phopoiesis, the activation of monocytes, dendritic cells and

macrophages, the activation of neutrophils and natural

killer (NK) cells and the modulation of the adaptive

immunity, by enhancing T cell survival and stimulating the

production of pro-inflammatory cytokines and suppresses

the production of anti-inflammatory Th2 cytokines such as

IL-4 in CD4 T cell [7–10].

Adiponectin is a protein hormone that modulates a

number of metabolic processes, including glucose regula-

tion and fatty acid catabolism, and also has anti-inflam-

matory, antiatherogenic and antidiabetic properties. In

some chronic inflammatory/autoimmune diseases, such as

rheumatoid arthritis and inflammatory bowel disease, adi-

ponectin may have pro-inflammatory effects and it reduces

inflammation, oxidative stress and cytokine production [6,

7, 11].

Studies investigating leukocyte apoptosis in BTM chil-

dren were few and had contradictory results; in addition, the

data on the relationships between leukocyte apoptosis with

adipocytokines and inflammatory markers are limited. The

present study aimed to analyze the neutrophil, T- and

B-lymphocyte apoptosis and to measure serum adiponectin

and leptin levels in a cohort of Egyptian children with BTM.

Patients and methods

Patients

Thirty-five Egyptian BTM children (21 males) with an

average age ± SD of 10.97 ± 3.81 year (5–13 years) were

voluntarily recruited to this case-controlled study in the

Pediatric Hematology Unit of the Assiut Children

University Hospital, Assiut, Egypt. Thirty socio-economic,

age- and sex-matched healthy children were included in

this study as controls. The study was approved by the local

ethics committee and was conducted in accordance with

the Declaration of Helsinki, and informed consent was

obtained in every case from their legal guardians.

Inclusion criteria

Thalassemia patients on regular blood transfusion and

chelation therapy, either oral chelation with defrasorix or

deferiprone or subcutaneous administration of deferoxam-

ine were included.

Exclusion criteria

Patients with known diabetes, cardiac, renal, infectious,

inflammatory or pulmonary diseases and newly diagnosed

BTM cases yet to receive a blood transfusion were exclu-

ded from the study. Splenectomized patients (to exclude

hematological and immunological effects of splenectomy)

were also excluded from the study.

Methods

All patients and controls were subjected to the following:

thorough medical history and examination, including age,

sex and duration of symptoms, blood transfusion and

chelation therapy. Samples were obtained prior to a

scheduled transfusion. All patients were abstained from

medications (e.g., corticosteroids, antimicrobials), chela-

tors and nutritional supplements for the previous 24 h.

Fasting blood samples were collected from resting subjects

through venipuncture in EDTA and plain tubes. The

complete blood count was done on Celltac E automated

hematology Analyzer (Nihon Kohden Corporation, Tokyo,

Japan). Samples were centrifuged for 15 min at 3000 rpm

at 4 �C, separated into aliquots, and immediately stored at

-70 �C until tested. Assays were performed in duplicate

according to the manufacturer’s instructions. Serum fer-

ritin, high-sensitive CRP (hsCRP), leptin and adiponectin

levels were determined in all participants using enzyme

immunosorbent assay (ELISA). Serum ferritin was deter-

mined using ELISA kit manufactured by Diametra SRL,

Italy, and hsCRP was determined using ELISA kit from

Monobind Industry (Lake Forest, CA, USA). For adipo-

nectin determination, we used the ELISA kit purchased

from Assay PRO, USA. Serum leptin was determined using

the ELISA kit manufactured by DBC Diagnostic Biochem

Canada Inc.

Flow cytometric detection of apoptosis

of neutrophils and lymphocytes

Apoptosis was measured by the Annexin V-fluoroisoth-

iocyanate (FITC) binding assay according to the manu-

facturer’s instructions (BD Biosciences, San Jose, CA).

Fifty lL of whole blood was stained with 5 lL of peri-

dinium-chlorophyll-protein (Per-CP)-conjugated anti-CD3

(T-Lymphocyte marker), phycoerythrin (PE)-conjugated

anti-CD13 (neutrophil marker) and allophycocyanin

(APC)-conjugated anti-CD19 (B-Lymphocyte marker),

washed twice with 2 mL of phosphate-buffered saline

Clin Exp Med

123

(PBS), and red blood cells were lysed. The cells were

washed and resuspended in 100 lL of the Annexin

V-conjugate binding buffer to which 5 lL of FITC-con-

jugated Annexin V was added. The mixture was incu-

bated in dark at room temperature for 15 min, after which

400 lL of the binding buffer was added, and 10,000 cells

were acquired and analyzed by FACSCalibur flow

cytometry. Antihuman IgG was used as an isotype-mat-

ched negative control for each sample. Forward and side

scatter histogram was used to define neutrophil and

lymphocyte populations. Then, the percentages of CD19?

(B-lymphocytes) and CD3? (T-lymphocytes) were asses-

sed in the lymphocyte populations. Then the expression of

Annexin V in T-lymphocytes, B-lymphocytes and neu-

trophils was detected (Fig. 1).

Statistical analysis

Data analysis was performed with the Statistical Package

for Social Sciences (SPSS version 16). Data are expressed

as mean ± standard deviation (SD) for all parameters. Due

to the small sample size and a propensity for outliers in

some of the variables, statistical differences between the

groups were examined using the Mann–Whitney test.

Spearman’s correlation was used to determine the corre-

lation between studied parameters. A value of p B 0.05

denoted a statistically significant difference.

Results

The clinical and demographic characteristics of the patients

and the controls were shown in Table 1. Hemoglobin,

height, weight and body mass index (BMI) of the patients

were significantly lower than their respective values in the

control group. Furthermore, as expected, serum ferritin

levels of all patients were significantly higher than those of

the healthy controls. Serum leptin was significantly lower

in patients when compared to controls. In contrast, adipo-

nectin and hsCRP levels were significantly higher in the

patients than the controls (Table 2). Leptin was negatively

correlated with ferritin levels (p\ 0.001, r = -0.56,

Fig. 2). Positive correlation of BMI leptin levels was sta-

tistically significant only in healthy controls (p\ 0.01,

r = 0.285). Positive correlation was found between adi-

ponectin and hsCRP (p\ 0.05, r = 0.25, Fig. 3).

Fig. 1 Flow cytometric

analysis of leukocytes apoptosis

in thalassemia patients.

a Forward and side scatter

histogram was used to define the

lymphocyte (R1) and

granulocyte population (R2). b,c The B cells (CD19?) and T

cells (CD3) were assessed in

lymphocyte population.

Neutrophils (CD13?) were

assessed in granulocyte

population compared with the

negative isotype control (not

shown). The B cells (CD19?),

T cells (CD3) and neutrophils

(CD13?) were gated for further

analysis of annexin V

expression. d Example of

annexin V expression on B cells

(CD19?), T cells (CD3) or

neutrophils. The positivity was

defined as fluorescence (green

histogram) higher than that of

the isotype control (blue

histogram) (color figure online)

Clin Exp Med

123

When leukocyte subsets were investigated, BTM

patients had significantly higher total leukocytes, neu-

trophils and lymphocytes compared with controls (Table 3).

The mean number of B-lymphocytes was increased in the

patients than controls, while T-lymphocytes and monocytes

were comparable in the patients and the controls. Compared

with the controls, BTM children exhibited an increased

apoptosis in T-lymphocytes; however, there was no sig-

nificant difference in the percentage of apoptosis of

B-lymphocytes and neutrophils between the patients and

the controls (Table 3). There was a significant negative

correlation between serum leptin and the percentage of

apoptotic T-lymphocytes (R = -0.613, p = 0.0001).

Discussion

The association of high adiponectin with elevated hsCRP

levels found in the present study could potentially explain

the pro-inflammatory effect of adiponectin that might be

involved in the inflammatory vascular process and eluci-

date the association of inflammation/adipose tissue linkage

in vivo [12]. Similar to our findings, previous studies

reported elevated adiponectin levels in thalassemic chil-

dren [6, 7]. Chaliasos et al. [7] reported that higher adi-

ponectin levels were positively correlated with the

increased levels of endothelin-1 (ET-1) in thalassemic

patients. Adiponectin may take part in the equilibrium

between the release of cytokines and adhesion molecules

from the endothelium. Elevated adiponectin may be the

expression of a counter-regulatory response aimed at

modifying the endothelial damage and cardiovascular risk

in BTM patients [7].

Lower leptin levels reported in our study and in previous

studies [7, 13–16] and its negative correlation with ferritin

possibly reflect the toxic effects of iron overload on cell

0 1 2 3 4 5Leptin "ng/ml"

0

500

1000

1500

2000

2500

3000

3500

4000

)lm/gn(

nitirreF

r = -0.56p<0.001

Fig. 2 Correlation between serum leptin and ferritin

Table 1 Demographic, clinical

and some laboratory

characteristics of thalassemia

patients and controls

Parameters Thalassemia patients (35) Control (30) p value

Age (year) 10.97 ± 3.81 11.60 ± 1.91 N.S

Weight (kg) 24.77 ± 7.99 37.20 ± 5.45 \0.001

Height (cm) 123.91 ± 15.88 142.0 ± 9.59 \0.001

BMI (kg/m2) 15.70 ± 2.13 18.38 ± 1.24 \0.001

Hemoglobin (g/dL) 9.31 ± 1.45 12.21 ± 2.34 \0.04

Ferritin (ng/mL) 2007.82 ± 646.63 81.0 ± 35.61 \0.001

Duration of blood transfusion (year) 7.86 ± 3.721 – –

Chelation therapy: number (%)

*Deferoxamine 18 (51.4) – –

*Deferiprone 9 (25.7) – –

*Deferasirox 8 (22.8) – –

Mann–Whitney test

Data are presented as mean ± SD. p\ 0.05 is significant

BMI body mass index

Table 2 High-sensitive CRP,

leptin and adiponectin levels in

both thalassemia patients and

controls

Parameters Thalassemia patients (35) Controls (30) p value

hsCRP (mg/L) 4.87 ± 1.89 1.49 ± 0.37 \0.05

Leptin (ng/mL) 2.92 ± 0.71 9.59 ± 5.31 \0.001

Adiponectin (ug/mL) 13.39 ± 7.59 7.89 ± 3.25 \0.001

Data are presented as mean ± SD. p\ 0.05 is significance difference

hsCRP high-sensitive C-reactive protein

Clin Exp Med

123

membranes and proteins in BTM patients, since free iron

causes peroxidative damage in lipid membrane and pro-

teins with the generation of free radicals. Thus, iron

overload (such as in BTM) results in the destructions of the

adipocyte. Along with the destruction of the fat cell

membrane and the dysfunction in adipose tissue, it leads to

a decrease in leptin serum level. Furthermore, the

replacement of red bone marrow with yellow bone marrow

which contains adipocytes can be the cause of this decrease

[7, 15, 16]. A positive correlation between leptin and BMI

was detected only in healthy controls, a finding in agree-

ment with previous reports [7, 12]. Since the BMI of the

patients is lower than that of the matched control group, it

could be concluded from our study and these reports [7, 12]

that the adipocytes of thalassemic patients are unable to

maintain adequate leptin production when a higher leptin

secretion is required, possibly due to toxic effect of iron

overload, suggesting that the adipose tissue dysfunction

can be considered one of the endocrinopathies affecting

thalassemic patients, and the consequent low leptin levels

might play a role in the neuroendocrine and hematopoietic

dysfunctions.

Few studies investigated the distribution of leukocyte

subsets and leukocyte apoptosis in thalassemic patients

[17–21]. A major cause of morbidity and mortality in

thalassemic patients is infection, which assumed to be the

result of immunological changes that could explain the

higher total leukocytes, lymphocytes and neutrophil counts

in our patients than the controls. The increased B-lym-

phocytes in our patients may be due to chronic stimulation

of immune system in response to infection and related to

increased production of immunoglobulin levels noted in

thalassemia patients [22].

A previous study [17] reported that thalassemic patients

had significantly higher total leukocytes and lymphocyte

counts compared with the controls, and also B-lymphocyte

percentage was significantly higher in patients with no

significant difference in T-lymphocytes. Another study

found that BTM patients showed a marked and persistent

lymphocytosis with an increase in the number of both T

and B cells with the chief increase in B cells [18]. Another

study [19] found that the absolute number of total leuko-

cytes, lymphocytes and neutrophils was increased in the

thalassemic patients.

In our study, despite the fact that the percentage of

apoptotic T-lymphocyte was more in the BTM patients

than the controls, the T-lymphocyte count was not

decreased. Thalassemia patients are chronically immuno-

stimulated by transfusions, non-transferrin-bound iron,

chelation and organ injury. This chronic stimulation of the

immune system not only may lead to increased number of

T-lymphocytes, but also lead to exhaustion of T-lympho-

cytes and increase their apoptosis. The negative correlation

between the level of serum leptin and the percentage of

apoptotic T-lymphocytes could indicate that the deficiency

of leptin in our thalassemia patients may be responsible for

the increased apoptosis of T-lymphocytes as leptin has

direct antiapoptotic effect on T cells [23, 24]. Walter et al.

[25] reported that thalassemic peripheral blood leukocytes

Table 3 leukocyte subsets and

leukocyte apoptosis in

thalassemia patients and

controls

Parameters Thalassemia patients (35) Control (30) P value

WBCs (109/L) 14.84 ± 3.70 9.32 ± 2.17 \0.001

Neutrophils (109/L) 11.21 ± 2.70 6.31 ± 2.30 0.03

Monocytes (109/L) 0.68 ± 0.34 0.73 ± 0.27 0.562

lymphocytes (109/L) 2.79 ± 0.44 2.36 ± 0.23 0.003

B-lymphocytes [CD19? (109/L)] 0.51 ± 0.10 0.33 ± 0.14 0.001

T-lymphocytes [CD3? (109/L)] 2.15 ± 0.09 2.12 ± 0.06 0.281

T-lymphocytes apoptosis % 37.24 ± 14.493 12.01 ± 2.82 \0.001

B-lymphocytes apoptosis % 10.98 ± 1.37 10.01 ± 2.45 0.669

Neutrophil apoptosis % 13.02 ± 5.71 11.60 ± 2.82 0.334

Mann–Whitney test

Data are presented as mean ± SD, p\ 0.05 is significance difference

% Percentage

0 5 10 15 20 25 30 35

hs.CRP (mg/l )

0

2

4

6

8

10

12

14

16

)lm/gu(

Anitc

enop

id

r = 0.250<0.05*

Fig. 3 Correlation between adiponectin and hsCRP

Clin Exp Med

123

had increased levels of pro-apoptotic marker Bax (an

inducer of mitochondrial dysfunction) and a high ratio of

Bax/Bcl-2, indicating decreased stability of the mitochon-

drial outer membrane and increased potential for mito-

chondrial dysfunction and apoptosis. They demonstrated

that markers of leukocyte apoptosis and mitochondrial

dysfunction were high in thalassemia patients compared

with controls [25]. Persistence or lack of immune cells at

inflammatory sites or the development of chronic inflam-

mation observed in b-thalassemia patients may result from

dysregulation of the apoptotic cell death pathway [25, 26].

Conclusion

Our BTM patients had a high percentage of apoptotic

T-lymphocyte in comparison with controls. In addition,

they had disturbed serum levels of adipocytokines and

inflammatory markers. These derangements could have a

role in the immunological disturbance observed in tha-

lassemic patients.

Conflict of interest None.

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