IOSR Journal of Dental and Medical Sciences (IOSR-JDMS)

e-ISSN: 2279-0853, p-ISSN: 2279-0861.Volume 16, Issue 11 Ver. II (Nov. 2017), PP 20-25
www.iosrjournals.org

Relationship Between Age, CD14+ Leukocytes And Inflammatory

Cytokines In Indonesian Children With Recurrent Tonsillitis

*Fatah Satya Wibawa1,2,*, Eryati Darwin3, Irza Wahid4,5, Netti Suharti6

1
Department of Otolaryngology-Head and Neck Surgery, Faculty of Medicine, University of Lampung, Bandar
Lampung, Indonesia
2
Department of Otolaryngology-Head and Neck Surgery, Dr. H. Abdoel Moeloek Hospital, Bandar Lampung,
Indonesia
3
Department of Histology, Faculty of Medicine, Andalas University, Padang, Indonesia
4
Department of Internal Medicine, Faculty of Medicine, Andalas University, Padang, Indonesia
5
Department of Internal Medicine, Dr. M. Djamil Hospital, Padang, Indonesia
6
Department of Microbiology, Faculty of Medicine, Andalas University, Padang, Indonesia
Corresponding Author: Fatah Satya Wibawa*

Abstract: Tonsillitis is common among children but the incidence and inflammatory responses varies
according to gender, race, and age of the patients. The present study was designed to determine association
strength between sex and age with immune response parameters, between expression of CD14 and
inflammatory cytokines, and between cytokines molecules IFN-γ and TNF-α in Indonesian children with
recurrent tonsillitis. Following informed concent, venous blood of the children (17 boys and 14 girls) were
taken and divided into two parts. The first part (1 ml) was subjected to CD14+ leukocytes (lymphocytes,
monocytes and neutrophils) analysis by whole-blood flowcytometry-based method. The second part (3 ml) was
prepared for serum cytokine measurement using sandwich ELISA method. The results showed that in children
with recurrent tonsillitis, the CD14 and cytokines content in their serum is not associated with sex.
Furthermore, neither CD14 leukocytes nor serum cytokines correlated with age. However, there was a strong
association between CD14 leukocytes with serum cytokines (P<0,031), and between IFN-γ with TNF-α
(P<0,001). It can be inferred that in Indonesian children with recurrent tonsillitis, immune response parameters
might not determined by sex and age. However, serum cytokines play a role in CD14+ expression, and
cytokines themselves, especially IFN-γ and TNF-α can positively predict each other.
Keywords: recurrent tonsillitis, inflammatory response, CD14 cells, leukocyte, cytokine, IFN-γ, TNF-α
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Date of Submission: 27 -10-2017 Date of acceptance: 04-11-2017
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I. Introduction
One of common infectious diseases in childhood is tonsillitis that involves parenchyma of the palatine
tonsils. When the symptoms appear more than three times a year the disease is called recurrent tonsillitis, and if
more than five times the tonsillitis is then called true recurrent tonsillitis [1]. Recurrent (chronic) tonsillitis is the
most common inflammatory lesions of the pharynx determining numerous local as well as distant evolutive
complications [2]. Tonsillar inflammation is immune responses that are evoked as a host defense against various
environmental stimuli including external stresses such as pathogens, foreign material, ionizing radiation and
internal stresses such as excessive accumulation of metabolites, autoimmune responses and cancer. The
inflammatory immune responses might involve both innate and acquired mechanisms in which all types of
leucocytes and their secretory products such as cytokines, growth factors and chemokines act as inflammatory
mediators [3].
Although tonsillitis is common among children but the incidence varies according to gender, race, and
age of the children [4, 5]. These phenomena may be a logical consequence of the fact that the factors involved in
the immune response are also determined by sex, race, and age. Children of all races who were under 6 years old

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 Relationship Between Age, CD14+ Leukocytes And Inflammatory Cytokines In ….

had a higher white blood cells (leukocytes) count than older persons had, and in both sexes of all ages, white
population was found to have higher leukocyte counts than the black population [6]. In black population, girls
three and four years of age had significantly higher mean lekocytes than boys in the same age group [7]. Age is
known not only determine leukocyte counts but also the immune cells responses. It has reported that
intracellular expression of interferon (IFN)-γ, tumour necrosis factor (TNF)-α and interleukin (IL)-2 protein
expression increased progressively with age [8].
The present study was designed to determine whether in Indonesian children with recurrent tonsillitis,
their immunological responses are determined by sex and age? Furthermore, does expression of CD14,
especially CD14+ lymphocytes, CD14+ monocytes, and CD14+ neutrophils, associated with inflammatory
cytokines, especially (IFN)-γ and (TNF)-α? Finally, it is no less important, whether the cytokines can predict
each other?

II. Materials And Methods

2.1 Patients
Patients included in the study were children, boys and girls aged 4– 15 years, with chronic tonsillitis
(tonsillitis symptoms occured more than three times a year) showing indications for tonsillectomy in Dustira
Hospital, Cimahi, West Java, Indonesia.

2.2 Blood Samples Preparation

Following informed consent signed by the patient's parent/guardian, 4 ml venous blood sample of the
children was collected by means of a venipuncture in a sterilized tube for blood collection containing heparin.
Blood samples then divided into two parts. The first part (1 ml) was subjected to CD14+ leukocytes
(lymphocytes, monocytes and neutrophils) analysis by whole-blood flowcytometry-based method. The second
part (3 ml) was prepared for serum cytokine measurement using sandwich ELISA method.

2.3 Whole-blood Flow Cytometry

Whole blood samples (100 μl) were firmly mixed with 10 μl of antibodies CD3 (SK7) PerCP (BD Cat.
No. CD0035-B17) and CD14 PE (BD Cat. No. CD0036-B17) for 30 second. After lysing solution was added
and erythrocytes are completely lysed, the samples were analysed within 6 hours. Both flow cytometer and
antibodies used in the study were BD Facs Calibur system from BD Bioscience (Becton Dickinson, San Jose,
CA, USA). The flowcytometry results are expressed in % gated and % total CD14+leukocytes (lymphocytes,
monocytes and neutrophils).

2.4 Cytokines mesurement

The second part of the blood sample (3 ml) to be analyzed for its cytokine content, centrifuged at 5000
rpm for 30 min. Human IFN-gamma Quantikine ELISA Kit and Human TNF-alpha Quantikine ELISA Kit,
both from R & D Systems Inc. (Minneapolis, MN 55413, USA) are consecutively used to measure tumor
necrosis factor α (TNF-α) and interferon γ (IFN-γ) using sandwich ELISA method.

2.5 Data Analysis

Student t-test and Simple Linear Regression and Correlation statistics from XLSTAT version 2017.1 was
used in data analysis. Student t-test was used to analyze whether patient’s sex determines their immune
response profile. Simple linear regression and correlation ststistics was used to measure the degree of linear
association between research variables including: patient’s age and observed immune response parameters; and
between CD14+leukocytes and cytokines.

III. Results
Descriptive data of all observed parameters of children with recurrent tonsillitis covering sex, age, and
percentage of CD + leukocytes and cytokines concentration are presented in Table 1. When the patient's
immunological data is grouped by sex, then the t-test results performed on the mean value of each parameter
yields statistical data as shown in Table 2. None of p-value of each immune response parameter in Table 2

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 Relationship Between Age, CD14+ Leukocytes And Inflammatory Cytokines In ….

showing a significant difference level. It is inferred that in children with recurrent tonsillitis, the CD14 and
cytokines content in their serum is not determined by sex.
Table 3 shows the degree of linear association, based on simple regression and correlation statistics
between immunological parameters and patient's age. Referring P-values of ANOVA for each correlation
strength between patient's age and observed immune responses, neither CD14 leukocytes nor serum cytokines
related to age in recurrent tonsillitis observed in the study.
Simple regression and correlation test between percentage of CD14 cells (CD14+ lymphocytes, CD14+
Monocytes, CD14+ Netutrophils) and serum cytokines (TNF-α and IFN-γ) of the research subjects results in the
correlation coefficients (r) presented in Table 4. Among the three types of CD14 leukocyte analyzed, only
lymphocytes appear to be positively associated with concentration of TNF-α (P < 0,020) and IFN-γ ( P < 0,031).
Furthermore, there is highly significant correlation between TNF-α and IFN-γ (r = 0,567; P<0,001). Thus it can
be asserted that IFN-γ and TNF-α are predictors of each other.

Table 1 Description of CD14+ leukocytes and cytokines in blood samples of each patient according to their sex
and age
Patients Sex Age CD14+ CD14+ CD14+ Cytokines
Lymphocytes Monocytes Neutrophils
% % % % % % TNF- IFN-γ
Gated Total Gated Total Gated Total α (pg) (pg)
1 Male 6 2,95 1,15 54,3 1,16 0,66 0,21 2,82 9,09
2 Male 10 5,13 1,19 63,59 1,43 0,64 0,31 1,46 11,15
3 Male 11 4,57 0,86 74,61 1,65 0,65 0,34 4,46 8,37
4 Male 8 6,09 1,52 71,32 1,89 0,39 0,19 1,05 8,55
5 Male 9 4,06 1,13 83,62 2,01 0,86 0,4 9,02 9,99
6 Male 8 6,19 1,34 71,65 2,23 0,35 0,19 1,63 7,46
7 Male 10 1,47 0,54 48,29 0,93 1,02 0,36 3,73 7,87
8 Male 15 0,61 0,22 60,37 0,45 1,47 0,35 2,41 8,73
9 Male 14 3,84 1,3 51,2 1,67 0,4 0,14 5,94 8,46
10 Male 4,5 3,66 1,38 48,84 1,06 0,53 0,09 7,96 8,17
11 Male 8 6,26 1,78 65,34 2,13 0,31 0,11 8,43 8,86
12 Male 10 9,17 2,39 62,57 3,06 0,24 0,09 6,35 9,00
13 Male 14 11,98 1,87 68,46 2,46 0,33 0,15 8,01 10,51
14 Male 10 10,84 2,43 64,17 2,37 0,33 0,11 5,62 9,55
15 Male 5 10,43 1,24 34,81 1,16 0,18 0,03 10,67 12,45
16 Male 5 7,71 1,33 35,17 1,35 0,22 0,07 23,92 11,76
17 Male 4 6,77 2,16 79,21 2,49 1,1 0,34 6,88 10,51
18 Female 13 0,68 0,14 69,39 1,62 0,47 0,18 4,95 11,20
19 Female 5 3,23 1,18 71,79 1,48 0,45 0,09 4,60 7,91
20 Female 8 3,78 1,04 66,24 1,74 0,68 0,17 2,13 8,72
21 Female 9 6,94 1,94 70,55 2,57 0,28 0,11 1,68 7,90
22 Female 11 0,14 0,05 81,01 2,84 0,77 0,17 0,26 7,39
23 Female 7 1,39 0,45 56,23 1,14 0,71 0,27 2,63 9,62
24 Female 6,5 4,27 0,9 54,82 1,06 0,7 0,19 3,85 9,48
25 Female 8 2,57 0,21 24,02 0,34 0,29 0,05 6,29 9,65
26 Female 13 1,97 0,7 42,56 1,1 0,78 0,11 1,11 8,15
27 Female 9 5,09 1,38 60,47 1,84 0,3 0,14 6,02 10,81
28 Female 10 4,48 1,27 24,3 1,32 0,83 0,13 3,07 10,00
29 Female 7 5,39 0,88 32,99 1,27 0,34 0,09 1,62 9,26
30 Female 5 5,21 1,5 21,8 1,39 0,71 0,13 6,51 10,13
31 Female 4 8,93 2,43 39,94 1,76 0,14 0,02 6,57 9,60

Table 2 Statistical summary of t-test for mean differences of all observed immune response parameters between
male and female children
t-test IFN-γ TNF-α CD14+ CD14+ CD14+
Parameters Lymphocytes Monocytes Neutrophils
% % % % % %
Gated Total Gated Total Gated Total
Difference 0,167 2,829 2,122 0,397 9,880 0,202 0,037 0,073

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t (Observed value) 0,358 1,853 2,022 1,726 1,611 0,850 0,334 2,035
|t| (Critical value) 2,045 2,045 2,045 2,045 2,045 2,045 2,045 2,045
DF 29 29 29 29 29 29 29 29
p-value (Two-tailed) 0,723 0,074 0,052 0,095 0,118 0,402 0,741 0,051
alpha 0,05 0,05 0,05 0,05 0,05 0,05 0,05 0,05

Table 3 The degree of linear association between immunological parameters and patient's age
Statistical TNF-α IFN-γ CD14+ CD14+ Monocytes CD14+
Parameters Lymphocytes Neutrophils
% % % % % %
Gated Total Gated Total Gated Total
r -0,321 -0,187 -0,229 -0,320 0,255 0,086 0,258 0,287
R² 0,103 0,035 0,052 0,102 0,065 0,007 0,067 0,082
Adjusted R² 0,072 0,002 0,020 0,071 0,033 -0,027 0,035 0,051
F 3,325 1,049 1,604 3,303 2,023 0,215 2,075 2,601
P 0,079 0,314 0,215 0,080 0,166 0,646 0,160 0,118

Table 4 Correlation Coefficient ( r ) resulted from correlation/regression test between

Cytokines (TNF-α and IFN-γ) and CD14+leukoytes
CD14+Lymphocytes CD14+ Monocytes CD14+ Neutrophyl
% Gated % Total % Gated % Total % Gated % Total
Cytokines TNF-α 0,4169* 0,263 -0,251 -0,027 -0,332 -0,262
IFN-γ 0,3883* 0,127 -0,296 -0,132 -0,198 -0,102
* Significant (P<0.05);

IV. Discussion
In general, this study did not manage to confirm previous works regarding relationship between human
haematological as well as immunological parameters and age. It was reported that in infancy and early
childhood, cytokine concentration correlate differentially with age [8]. In current study, besides the level of
correlation coefficient is not significant, the relationship trend between age and cytokines is also contrary to the
reports mentioned. It is clear from Table 3 that r-values of both IFN-γ and TNF-α are negatif. Furthermore,
disregarding the patient's age diversity, Todorovic at al. compared production of these cytokines in tonsillar
hypertrophy (TH) and recurrent tonsillitis (RT) and found that the concentration of TNF-α and IFN-γ is
significantly higher in RT than in TH [9]. These differences seem to be due to differences in the status of the
research subjects. In reference [8] the subjects were normal children while in this study the subjects were
patients with chronic tonsillitis. Additionally, neither reference [8] nor reference [9] describe what ethnic group
is their research subjects, while in this study all patients were Mongoloid.
As has been widely reported, the influence of ethnicity on various aspects of human health and
physiology is real and significant. Africans and Afrocaribbeans were known to have lower total white cell,
neutrophil and platelet counts than Caucasians [10]. In addition, blacks show significantly lower thyrotropin,
hematocrit, mean cell hemoglobin centration (MCHC), mean cell hemoglobin, and hemoglobin [11]. In
children vertically infected with HIV-1, E.R.Sharp and colleagues found that African American children
demonstrated significantly higher Gag responses than Hispanic children [12]. Blacks were also reported to have
higher EBV IgG compared to white [13]. Unfortunately, research data describing the comparison of
hematological and immunological parameters between blacks (Africans) or white (Caucasian) with Asians
(Monggoloid) is still hard to find. The differences in influence of age on immunological parameters yielded in
current study compared with other previous findings very likely related to the ethnicity of the patients.
Among the three types of CD14 leukocyte analyzed, only lymphocytes positively associated with
concentration of TNF-α and IFN-γ in a siginificant manner (Table 4). In contrast, the expression of CD14+
monocytes as well as CD14+ neutrophils are even negatively correlated with concentration levels of TNF-α and
IFN-γ, although statistically the correlation was not significant. However, as can be seen in Table 1, the current
research findings confirm the accepted trend that CD14 is most widely expressed by monocytes [14, 15]. Since
CD14 is predominantly derived from monocytes and monocytes are the primary source of TNF-α, then elevated
TNF alpha levels should be followed by increased CD14+ monocytes [16]. It is very difficult to find literature

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 Relationship Between Age, CD14+ Leukocytes And Inflammatory Cytokines In ….

reviewing the association of cytokines with the expression of CD14+ lymphocytes. However, in patients
infected with HIV-1 has been found a phenomenon of increased population of CD14-like positive-testing
lymphocyte [17]. Thus, the unique findings of this study may not be a deviation of the accepted trends, but
something reasonable given the differences in the ethnicity of the patient.
Lastly, current research findings found there is highly significant correlation between TNF-α and IFN-
γ (r = 0,567; P<0,001). Although the satisfactory explanation of the mechanism of mutual relations between the
two cytokines is not so clear, in certain cases the correlation between them is apparent. When cytokines were
used in combination with GM-CSF: IFN-γ down-regulated production of IL-lRa while up-regulating the
production of IL-lca/fl, IL-6 and TNF-α [18]. Also known in patients with recurrent tonsillitis, increased
intracellular deposition of microbial antigens accounted for the elevated incidence of IL-1β, TNF-α, IL-6, IL-8,
IL-2, IFN-γ, IL-10 and IL-4 expressing cells [19]. Thus the findings of this study reveal that in Indonesian
children with chronic tonsillitis, the level of a cytokine content can be used to estimate levels of other cytokines.

V. Conclusion
It can be inferred that in Indonesian children with recurrent tonsillitis, immune response parameters
might not determined by sex and age. However, serum cytokines play a role in CD14+ expression, and
cytokines themselves, especially IFN-γ and TNF-α can positively predict each other.

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*Fatah Satya Wibawa. "Relationship Between Age, CD14 Leukocytes And Inflammatory
Cytokines In Indonesian Children With Recurrent Tonsillitis." IOSR Journal of Dental and
Medical Sciences (IOSR-JDMS) 16.11 (2017): 20-25

DOI: 10.9790/0853-1611022025 www.iosrjournals.org 25 | Page