ORIGINAL RESEARCH - JOURNAL OF FUNCTIONAL VENTILATION AND PULMONOLOGY. VOLUME 7 - ISSUE 22. 2016

ABSTRACT

Background. Asthma is a respiratory disease characterized by chronic airway inflammation involving many cells and cellular components. Cytokines are proinflammatory protein regulated mainly from Th2 cells which  play a critical role in activation of airway inflammation. Measurements of   cytokine concentration in unselected peripheral blood can predict the inflammatory  prognosis in  children with asthma exacerbations.
Methods. This  was a prospective descriptive  study in 55 children  with acute asthma, 20 children with  stable asthma  and 15 healthy children.  All subjects were measured IL-4, IL-5 and IL-13 in unselected peripheral blood by flowcytometry-assisted immunoassay methods.
Results. The concentrations  of IL-4 and IL-5 were significantly higher  in the exacerbation periods in comparison with the stable periods (0.33pg/ml  versus 0.036 pg/ml, p=0.08; 2.3 pg/ml  versus 1.0 pg/ml, p=0.02; respectively) and healthy controls (0.33pg/ml  versus 0.02 pg/ml, p=0.002; 2.3 pg/ml  versus 0.75 pg/ml, p=0.029; respectively).  There were no difference in the concentrations of IL-4, IL-5 between patients with stable asthma and healthy controls. There were also no difference in  concentration of IL-13 in the three groups. 
Conclusions. The cytokines such as IL-4, IL-5 play an important role in the activation and  maintaining airway inflammation in children with asthma.

KEY-WORDS: Asthma; acute asthma exacerbation; cytokines; IL-4; IL-5; IL-13.

RÉSUMÉ

Contexte. L'asthme est une maladie respiratoire caractérisée par une inflammation chronique des voies aériennes impliquant de nombreuses cellules et des composants cellulaires. Les cytokines sont des protéines pro-inflammatoires régulées principalement à partir de cellules Th2 qui jouent un rôle critique dans l'activation de l'inflammation des voies aériennes . Les mesures de la concentration de cytokines dans le sang périphérique non sélectionné peuvent prédire le pronostic inflammatoire chez les enfants présentant des exacerbations de l'asthme.
Méthodes. Il s'agit d'une étude descriptive prospective chez 55 enfants atteints d' exacerbation aigue de l’asthme, 20 enfants atteints d'asthme stable et 15 enfants sains. Tous les sujets ont été mesurés IL-4, IL-5 et IL-13 dans le sang périphérique non sélectionné par cytofluorométrie assistée immunoessais.
Résultats. Les concentrations d'IL-4 et d'IL-5 étaient significativement plus élevées dans les périodes d'exacerbation que dans les périodes stables (0,33 pg / ml contre 0,036 pg / ml, p = 0,08, 2,3 pg / ml contre 1,0 pg / ml, p = 0,02, respectivement) et des témoins sains (0,33 pg / ml contre 0,02 pg / ml, p = 0,002, 2,3 pg / ml contre 0,75 pg / ml, p = 0,029, respectivement). Il n'y avait aucune différence dans les concentrations d'IL-4, IL-5 entre les patients avec l'asthme stable et les témoins sains. Il n'y avait pas non plus de différence dans la concentration d'IL-13 dans les trois groupes.
Conclusion. Les cytokines telles que IL - 4, IL - 5 jouent un rôle important dans l 'activation et le maintien de l' inflammation des voies aériennes chez les enfants asthmatiques.

MOTS CLÉS: Asthme; cytokines, IL-4; IL-5; IL-13.

INTRODUCTION

Cytokines play a key role in orchestrating the chronic inflammation and structural changes of the respiratory tract in asthma and have become important targets for the development of new therapeutic strategies in these disease. Cytokines are classified into lymphokines, proinflammatory cytokines, growth factors, chemokines and antiinflammatory cytokines, although many of these functions may overlap [1].
In patients with asthma, there is an increase in the number of CD4⁺ Th cells in the airways, which are predominantly of the Th2 subtype. Th2 cells are characterized by secretion of IL-4, IL-5,  and IL-13 [2]. These cytokines from Th2 cells play an important role in maintaining the inflammation in the airways, even if patients do not have clinical symptoms [3].  Measurements of  cytokine concentrations in peripheral blood in  children with acute and stable  asthma help to understand the airway inflammation in asthmatic children.

We conducted this study to compare these cytokine concentrations  from Th2 cells in peripheral blood between children in the period of the acute asthma and the asymptomatic period.

PATIENTS AND METHODS

This was a prospective descriptive study in 55 children in acute asthma, 20 children  with free symptoms  and 15 healthy children. Children with asthma were diagnosed according to international recommendation (GINA-2015) [4]. The severity of asthma was assessed according to Pediatrics Asthma Score (PAS) [5]. 
Blood samples (3ml) were collected from children with asthma for quantitative of IL-4, IL-5 and IL-13 cytokines at the time of admission and discharge. cyanose, et  un wheezing.
Healthy children admitted  to hospital for health checks were invited to be volunteer to participate in this study. Blood samples of healthy children were collected only one time. All blood samples collected were drawn into tubes containing 10% ethylenediaminetetra-acetic acid and kept refrigerated at -80⁰C. 

Cytokines were quantified by flowcytometry-assisted immunoassay technique with Bio-Plex system from Bio-Rad Laboratories production (USA). 
This study was approved by the Medical Ethics Committee of National Vietnam Children’s Hospital.  The study results were analyzed using SPSS 16.0 software.

RESULTS

The study enrolled 55 children with acute asthma, 20 children with stable asthma and 15 healthy children which  were collected blood samples for quantitative of  IL-4, IL-5 and IL-13 concentrations.
Children hospitalized due to  asthma attacks were mainly under 5 years old, with the boys were  64.58%. In acute period, 69% of children with asthma had viral infection, in which 55% having Rhinovirus  and 14% having  RSV infection.  43.75%  of children suffered from severe and very severe asthma exacerbations (Table 1).

The concentration of IL-4 in children with  acute asthma was  significantly higher than asthmatic children without symptoms (0.33pg/ml compared to 0.036 pg/ml, p =0.08). Similarly, concentration of IL-5 in acute period was higher than stable period (2.3 pg/ml versus 1.0 pg/ml, p =0.02). There was no difference in IL-13 concentrations  between children with  acute asthma and asymptomatic period.
The concentration of IL-4 in acute asthma were  significantly higher than the control group (0.33pg/mL  versus 0.02 pg/ml, p =0.002). Similarly, concentration of  IL-5 in acute asthma was higher than the control group (2.3 pg/mL versus 0.75 pg/ml, p = 0.029). The concentration of IL-13 in children with asthma  was  no difference in comparison with  healthy children (2.9pg/ml versus 2.0 pg/ml).  There  were no differences in the concentrations of IL-4, IL-5 and IL-13 between children with stable asthma and healthy  controls  (0.036  pg/mL versus  0.02 pg/ml; p = 0.39; 1.0pg / ml versus 0.75pg / ml; p = 0.79; 4.2pg / ml versus  2.0 pg / ml; p = 0.4; respectively). The concentrations of IL-4, IL-5 in children with acute asthma were significantly increased in comparison  with children with stable asthma and healthy controls. The concentration of IL-13 was no different in children with acute asthma, stable asthma and healthy controls.

DISCUSSION

Chronic airway inflammation is the basis pathology  of asthma. Studies in animal models  and humans indicate the important role of Th2 cells produce IL-4, IL-5, IL-13 [6],  the sustained inflammatory cytokines in allergic diseases  and allergic asthma. The primary inflammatory lesion of asthma consists of accumulation of CD4 (+) T helper type 2 (Th2) lymphocytes and eosinophils in the airway mucosa.
Th2 cells orchestrate the asthmatic inflammation through the secretion of a series of cytokines, particularly interleukin 4 (IL-4), IL-13, IL-5 [7]. IL-4 is an important cytokine in the progression  of allergic inflammation.   IL-4 is the major factor regulating IgE production by B cells, and is required for optimal Th2 differentiation [7] [8]. An increase in IL-4  is observed in blood and bronchoalveolar lavage in patients with allergic asthma [9].  IL-4   induces  airway hyperresponsiveness  in patients with asthma [10].

Interleukin-5 has long been associated with the cause of several allergic diseases including  allergic rhinitis and asthma. An  increase in  IL-5 have been observed in  circulating, airway tissue, and induced sputum in patients with asthma. IL-5 is crucial in regulating the eosinophilic response both in vitro and in vivo. IL-5 is a key factor for eosinophilia and could therefore be responsible for some of the tissue damage seen in chronic asthma [7].
Interleukin-13 is a pleiotropic Th2 cytokine that has been shown to be central to the pathogenesis of asthma. Some of the most prominent of the effects of IL-13 include increases in goblet cell differentiation, activation of fibroblasts, elevation of bronchial hyperresponsiveness, and switching of B cell antibody production from IgM to IgE. These data indicate that IL-13 antagonists may fulfill an important unmet need in patients with poorly controlled asthma and biologic evidence of persistent IL-13 activity [11].

Study of  Berry showed that the concentration of IL-13 is increased in children with asthma in comparison with healthy children [12]. Our study indicated that the concentrations of IL-14, IL-5 were significantly higher in children with acute asthma in compared to children with stable asthma and healthy controls. However, there were no difference in the concentration of IL-13 in three groups.
The concentration of IL-4   is an increase in blood, serum and bronchoalveolar lavage in patients  with asthma [10] [13]. In addition, concentration of   IL-4  is often increased in the presence of respiratory syncytial virus [14]. According to Hoekstra, the  difference in  the concentration  of IL-4 seems to depend on the severity of acute asthma [15].  Bogie reported that  children  having  moderate  and  severe   acute asthma had higher  IL-4 concentration compared to children with  mild acute asthma [13]. However, another study showed no difference in the concentration of IL-4 between the control group and children  with mild or moderate acute  asthma [16].

According to Charoenying, the concentrations of IL-4 are no difference between  patients with  acute asthma and patients with stable asthma  [17], as well as the control groups [18]. The different  results may be  explained by the  differences in the sample sizes,  and the difference of asthma severity  in different  studies.
The concentration of IL-5 in the serum of patients with asthma is  significantly higher than the control groups [13]. IL-5 concentration of  children with moderate or severe asthma attacks is significantly higher than the group with mild asthma [9]. However, study  by Al Daghri indicated that IL- 5  is reduced in  children with severe asthma compared with the control groups, but IL-4 concentration is  significantly increased [19].

Although IL-13 has been shown to be central to the pathogenesis of asthma,  Machura indicated that IL-13 concentration does not differ between children with asthma and control groups [18]. Al Daghri  showed a similar results [19]. Our study did not find the difference in IL-13 concentrations in children with acute asthma, children with stable asthma and healthy children. However, Machura also indicated that IL-13 concentration of CD4 Th2 cells  is an  increase in  children with severe acute asthma, but no variation in children with mild or moderate acute asthma and  positively correlation with the duration of asthma  [18].

CONCLUSION

These cytokines produced from Th2 cells  play an important role in maintaining inflammation in children with asthma. The cytokines such as IL-4, IL-5  are increased  in children with  acute asthma compared to children with stable  asthma and healthy children  demonstrated   an important role of these cytokines  in activating and maintaining  the acute period of asthma.

Acknowledgments

We thank the doctors, nurses and the staff of the Department of Allegy- Immunology and Rhematology of the National Vietnam Children’s Hospital had helped us to conduct this study. This study was supported by a grant from the National Foundation for Science & Technology Development (NAFOSTED no. 106.99-2012.12 ).
 

CONFLIT OF INTEREST
Non.

REFERENCES

1. Barnes PJ (2008). The cytokine network in asthma and chronic obstructive pulmonary disease. J Clin Invest 118: 3546-3556.

2. Barnes  PJ (2008). Immunology of asthma and chronic obstructive pulmonary disease. Nat. Rev. Immunol. 8:183-192.

3. Chiba Y, Nakazawa S, Todoroki M et al. (2009), Interleukin-13 augments bronchial smooth muscle contractility with an up-regulation of RhoA protein.  Am J Respir Cell Mol Biol 40: 159–67.

4. GINA 2015. Global strategy for asthma management and prevention.

5. Ducharme FM, Chalut D, Plotnick L, et al (2008).  The Pediatric Respiratory Assessment Measure: a valid clinical score for assessing acute asthma severity from toddlers to teenagers. J Pediatr 152: 476–480.

6. Larché M, Robinson DS  and  Kay AB (2003).  The role of T lymphocytes in the pathogenesis of asthma.  J Allergy  Clin Immunol 111(3) 450–463.

7. Renauld JC (2001). New insights into the role of cytokines in asthma, J Clin Pathol 54, 577–89.

8. Seder RA, Paul WE, Davis MM et al(1992). The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4 T from T cell receptor transgenic mice, J Exp Med;  176:1091–1098.

9. Walker C, Bauer W, Braun RK et al (1994), Activated T cells and cytokines in bronchoalveolar lavages from patients with various lung diseases associated with eosinophilia, Am J Respir Crit Care Med, 150 (4):1038–1048.

10. Shi HZ, Deng JM, Xu H et al (1998) Effect of inhaled interleukin-4 on airway hyperreactivity in asthmatics, Am JRespir Crit Care Med  157:1818–1821.

11. Corren J (2013). Role of interleukine-13 in asthma. Curr Allergy Asthma Rep. 13(5) 415-20.

12. Berry MA, Parker D, Neale N et al. (2004), Sputum and bronchial submucosal IL-13 expression in asthma and eosinophilic bronchitis. J Allergy Clin Immunol  114: 1106–9.

13. Bogie M, Savic N, Tomi V et al (2004)  Clinical significance of measurement of interleukin 4 and interleukine 5 serum concentrations in bronchial asthma.  Jugoslov  Med Biohem. 23: 51–54. 

14. Johnson TR,  Graham BS (1999).   Secreted respiratory syncytial virus Glycoprotein Induces Interleukin-5 (IL-5), IL-13, and Eosinophilia by an IL-4-Independent Mechanism.  J Virol, 73(10): 8485-8495.

15. Hoekstra MO, Hoekstra Y, De Reus D et al (1997). Interleukin-4, interferon-gamma and interleukin-5 in peripheral blood of children with moderate atopic asthma .  Clin Exp Allergy. 27:1254–1260.

16. Abdulamir AS, Hafidh RR, Abubakar F, Abbas KA (2008). Changing survival, memory cell compartment, and T-helper balance of lymphocytes between severe and mild asthma.  BMC Immunology, 9 (73) doi: 10.1186/1471-2172-9-73.

17. Charoenying Y, Kamchaisatian W, Atamasirikul K et al (2010). _.Cytokine responses during exacerbation compared with stable phase in asthmatic children.  Asian Pacific  J Allergy  Immunol  28: 35-40

18. Machura E, Mazur B,  Rusek-Zychma M,  Czarneck MB (2010)^. Cytokine production by peripheral blood CD4⁺ and CD8⁺T Cells in atopic  childhood asthma. Clin Develol Immunol. 2010. PMC 3004408.

19. Al- Daghri NM, Alokail MS, Abd-Alrahman SH (2014). Th1/Th2 cytokine pattern in Arab children with severe asthma.Int J Clin Exp Med. 7(8) 2286-91.

 TABLES AND FIGURES

 RFERENCES

1. Barnes PJ (2008). The cytokine network in asthma and chronic obstructive pulmonary disease. J Clin Invest 118: 3546-3556.

2. Barnes  PJ (2008). Immunology of asthma and chronic obstructive pulmonary disease. Nat. Rev. Immunol. 8:183-192.

3. Chiba Y, Nakazawa S, Todoroki M et al. (2009), Interleukin-13 augments bronchial smooth muscle contractility with an up-regulation of RhoA protein.  Am J Respir Cell Mol Biol 40: 159–67.

4. GINA 2015. Global strategy for asthma management and prevention.

5. Ducharme FM, Chalut D, Plotnick L, et al (2008).  The Pediatric Respiratory Assessment Measure: a valid clinical score for assessing acute asthma severity from toddlers to teenagers. J Pediatr 152: 476–480.

6. Larché M, Robinson DS  and  Kay AB (2003).  The role of T lymphocytes in the pathogenesis of asthma.  J Allergy  Clin Immunol 111(3) 450–463.

7. Renauld JC (2001). New insights into the role of cytokines in asthma, J Clin Pathol 54, 577–89.

8. Seder RA, Paul WE, Davis MM et al(1992). The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4 T from T cell receptor transgenic mice, J Exp Med;  176:1091–1098.

9. Walker C, Bauer W, Braun RK et al (1994), Activated T cells and cytokines in bronchoalveolar lavages from patients with various lung diseases associated with eosinophilia, Am J Respir Crit Care Med, 150 (4):1038–1048.

10. Shi HZ, Deng JM, Xu H et al (1998) Effect of inhaled interleukin-4 on airway hyperreactivity in asthmatics, Am J Respir Crit Care Med  157:1818–1821.

11. Corren J (2013). Role of interleukine-13 in asthma. Curr Allergy Asthma Rep. 13(5) 415-20.

12. Berry MA, Parker D, Neale N et al. (2004), Sputum and bronchial submucosal IL-13 expression in asthma and eosinophilic bronchitis. J Allergy Clin Immunol  114: 1106–9.

13. Bogie M, Savic N, Tomi V et al (2004)  Clinical significance of measurement of interleukin 4 and interleukine 5 serum concentrations in bronchial asthma.  Jugoslov  Med Biohem. 23: 51–54. 

14. Johnson TR,  Graham BS (1999).   Secreted respiratory syncytial virus Glycoprotein Induces Interleukin-5 (IL-5), IL-13, and Eosinophilia by an IL-4-Independent Mechanism.  J Virol,  73(10): 8485-8495.

15. Hoekstra MO, Hoekstra Y, De Reus D et al (1997). Interleukin-4, interferon-gamma and interleukin-5 in peripheral blood of children with moderate atopic asthma .  Clin Exp Allergy. 27:1254–1260.

16. Abdulamir AS, Hafidh RR, Abubakar F, Abbas KA (2008). Changing survival, memory cell compartment, and T-helper balance of lymphocytes between severe and mild asthma.  BMC Immunology, 9 (73) doi: 10.1186/1471-2172-9-73.

17. Charoenying Y, Kamchaisatian W, Atamasirikul K et al (2010). _.Cytokine responses during exacerbation compared with stable phase in asthmatic children.  Asian Pacific  J Allergy  Immunol  28: 35-40

18. Machura E, Mazur B,  Rusek-Zychma M,  Czarneck MB (2010)^. Cytokine production by peripheral blood CD4⁺ and CD8⁺T Cells in atopic  childhood asthma. Clin Develol Immunol. 2010. PMC 3004408.

19. Al- Daghri NM, Alokail MS, Abd-Alrahman SH (2014). Th1/Th2 cytokine pattern in Arab children with severe asthma. Int J Clin Exp Med. 7(8) 2286-91.

 ARTICLE INFO

DOI: 10.12699/jfvp.7.22.2016.55

Conflict of Interest
Non

Date of manuscript receiving
12/10/2016

Date of publication after correction
25/11/2016

Article citation
Le-Thi-Thu. H,  Nguyen-Thi-Dieu. T. Study of the cytokine concentrations in unselected peripheral blood   in children with asthma. J Func Vent Pulm 2016;22(7):49-53.