Журнал «Здоровье ребенка» 3 (46) 2013

Analysis of sputum induced by inhalation of hypertonic saline has recently been established as a useful non-invasive technique for measuring airway inflammation in patients [1-3]. We therefore used this technique to evaluate the presence of airway and lung inflammation in children with the acute bronchitis, pneumonia, chronic lung disease. Because the underlying mechanisms of lung inflammatory response in children with the chronic lung disease has not yet been completely elucidated [4-6].

Material and methods

The 105 patients were recruited from Regional Children Clinical Hospital (RCCH), Kharkiv, Ukrainian. The Head of RCCH is Muratov G.R., the head of  pediatric department of KNMU is prof. Senatorova G.S.   Children with the acute bronchitis (n=38) aged on average (6,9±2,4) years who had been admitted to the pulmonology department served as group 1.  The age distribution in the 1 group was as follows: 15(39,5±7,9%) children, 2-5 years, served as group 1a, and 23(60,5±7,9%) children, 6-14 years, served as group 2b. The patient with the acute pneumonia (n=35) aged on average (8,0±2,3) years  served as group 2.  The age distribution in the 2 group was as follows: 7(20,0±6,8%) children, 2-5 years, served as group 2a, and  28(80,0±6,8%) children, 6-14 years, served as group 2b. Fifteen children with the chronic lung disease (n=15), aged on average (8,0±2,3) years, which had lung fibrosis,  served as group 3.  The age distribution in the 3 group was as follows: 6(40,0±13,1%) children, 2-5 years,

served as group 3a, and  9(60,0±13,1%) children, 6-14 years, served as group 3b. Healthy controls (n =18) were negative for allergies and respiratory diseases. Control group consisted of 6 (33,3±11,4%) patients, 2-5 years and 12 (66,7±11,4%) patients, 6-14 years. Respiratory diseases was defined according to the Ukrainian protocol of diagnosis and treatment lung diseases in children. After clinical evaluation and immunology blood testing, induced sputum was collected.To determine the biochemical analysis of sputum induced after inhalation of hypertonic saline, we analyzed sputum induced in children subjects. The sputum was induced with inhalation of ultrasonically nebulized hypertonic (2,7-5%) saline solution. The study was approved by the ethics committee of the Kharkiv national medical university and all parents of children gave informed consent to participate in the study. Statistical analysis was performed using „Stadia-6” ,version „Рrof”, „Statistica-6”.

Results

Many researchers have observed that cytocine production in human beings is a process that is in some way influenced by the age of the individual [7]. To define the range for levels of cytokine (IL-4, IL-6, IL-8, IL-10) in induced sputum in a pediatric population were analyzed as described above. Results of the analysis are displayed graphically. The median and the interquartile range are presented for each age group. The cytokine profiles of the sputum samples are summarised in figure 1-4.

Local IL-4 levels in sputum were higher in the samples of the all cases than in their controls. When compared with sputum from normal subjects sputum of patients, 2-5 years, with bronchitis (p=0.0063), with pneumonia (p=0.0227), with chronic lung diseases (p=0.0019) contained a significantly higher levels of IL-4. In patients, 2-5 years, the median level of IL-4 in sputum of children with bronchitis, with pneumonia and with chronic lung disease and control group were 55,1 pg/ml, 62,4 pg/ml and 56,9 pg/ml and 33,4 pg/ml.

We found that induced sputum from subjects of patients, 6-14 years, with bronchitis (p=0.00014), with pneumonia (p=0.0091), with chronic lung diseases (p=0.0001) had a higher concentration of IL-4, compared to control. In patients, 6-14 years, the median level of IL-4 in sputum of children with bronchitis, with pneumonia and with chronic lung disease and control group were 47,8 pg/ml, 35,9 pg/ml and 55,9 pg/ml and 21,6 pg/ml. There was statistically significant difference between sputum IL-4 levels of patients with pneumonia and with chronic lung disease (p=0.039). This observation is inline with reports indicating a significant role of IL-4 in promoting inflammation in the lung. IL-4 increases the expression of other inflammatory cytokines from fibroblasts that might contribute to inflammation and lung remodelling in chronic respiratory diseases. In healthy control group to find age-depended increase of IL-4 levels was analysed. Subjects of healthy children, 2-5 years, had a significantly higher concentration of IL-4 (p˂0,05) in induced sputum than subjects of healthy children, 6-14 years.

Concentrations of IL-6 in the sputum from subjects of patients, 2-5 years, with bronchitis (p=0.0002), with pneumonia (p=0.0013), with chronic lung diseases (p=0.0019) were increased compared to control. There were significant differences in the sputum cytokine levels between the subjects of children, 2-5 years, with bronchitis and pneumonia (p=0.0109). In patients, 2-5 years, the median level of IL-6 in sputum of children with bronchitis, with pneumonia, with chronic lung disease and control group were 52,5 pg/ml, 32,2 pg/ml, 58 pg/ml and 12,2 pg/ml. The present data show that production of IL-6 indicating a significant role in the pathogenesis of acute  inflammation.

The increase in the IL-6 concentration of children, 6-14 years, with bronchitis (p=0.0001), with pneumonia (p=0.0018), with chronic lung diseases  (p=0.0001) was statistically significant compared to healty. In patients, 6-14 years, the median level of IL-6 in sputum of children with bronchitis, with pneumonia and with chronic lung disease and control group were 82,9 pg/ml, 67,0 pg/ml, 53,6 pg/ml and 20,2 pg/ml.

Subjects of healthy children, 6-14 years, had a significantly higher levels of IL-6 (p˂0,05) in induced sputum than subjects of healthy children, 2-5 years. The age-depended increase of IL-6 in healthy children is indicating a role of infection in the immunity responses in this age period.

In our study we compared IL-8 production in the sputum from control subjects and from all patients. We found that induced sputum from subjects of patients, 2-5 years and 6-14 years, with bronchitis (p=0.0019) and (p=0.0001), with pneumonia (p=0.0033) and (p=0.0001), with chronic lung diseases (p=0.0004) and (p=0.0001), had a higher concentration of IL-8 compared to healthy children, respectively.

Children, 2-5 years and 6-14 years, with lung fibrosis had the highest median levels of  IL-8 in the induced sputum (90,5pg/ml) and (89,4pg/ml) than children with bronchitis (76,6 pg/ml, p=0.0004) and (79,3 pg/ml, p=0.0043), with pneumonia (81,1 pg/ml, p=0.011) and (79,1 pg/ml, p=0.0017), respectively. The concentration of IL-8 in the induced sputum samples differentiated patients with bronchitis and pneumonia from patient with lung fibrosis, and indicated at risk for transformation acute diseases to chronic lung diseases.

There were no significant differences in the sputum cytokine levels between the subjects of healthy children, 2-5 years and 6-14 years. Healthy children had the median levels of IL-8 (30,2 pg/ml) and (34 pg/ml), respectively.

IL-10 were significantly increased in induced sputum sample from patients of all groups compared with normal subjects.

We studied that induced sputum from subjects of patients, 2-5 years and 6-14 years, with bronchitis (p=0.0012) and (p=0.0001), with pneumonia (p=0.0013) and (p=0.0001), with chronic lung diseases (p=0.0019) and (p=0.0001), had a higher concentration of IL-10 compared to control group, respectively.

There was statistically significant difference between sputum median IL-10 levels of patients, 2-5 years, with lung fibrosis (82,1 pg/ml), and patients, with bronchitis (49,3 pg/ml, p=0.0002) and patients, with pneumonia (66,2 pg/ml, p=0.016). There were statistically significant median levels of IL-10 in the sputum of patients, 6-14 years, with lung fibrosis (81,5 pg/ml), and patients, with bronchitis (49,9 pg/ml, p=0.0001) and patients, with pneumonia (72,6 pg/ml, p=0.0001).

In children with bronchitis we observed significant strong correlations between sputum IL -4 and the percentage of blood CD8-cells (r =0,605, p<0.05), and the percentage of blood CD16-cells (r =0,609, p<0.05), levels of blood IgG (r =0,658, p<0.05).The levels of IL-4 in the induced sputum of patients with the pneumonia correlated strongly with the percentage of blood CD22- lymphocytes (r =0,857, p<0.05).Sputum IL-4 levels in  children with the lung fibrosis correlated with percentage of blood CD2- lymphocytes (r =0,771, p<0.05), and with levels of blood CD4/CD8-cells(r =0,716 p<0.05).

In induced sputum of children with bronchitis, the concentrations of IL-6 and percentage of blood CD2- lymphocytes (r =-0,635), and the percentage of blood CD22- lymphocytes (r =-0,511), were significant correlated (p< 0.05). In the subjects with pneumonia the percentage of blood CD4- cells (r =-0,785, p< 0.05), was correlated with the IL-6. There were inversely correlations, which was associated with the dissociation of blood inflammatory cells in airway from  the development of inflammation and specific local and humoral response.

Conclusions

1. Induced sputum examination is non-invasive, economical, simple, easily accepted by children, widely can be used in clinical.

2. We found the change of sputum levels IL-4 and IL-6 in healthy children with age from 2 to 14 years. These results revealed the association between cytokine-producing and age.

3. Our results indicate that there is a predominant inflammation in the airways of patients with chronic lung diseases associated cytokines. Increasing sputum levels of IL-4 and IL-6 of all groups are indicating a role of cytokines in the remodeling process of the airways and lung.

4. The present data show that production of IL-8 and IL-10 in sputum, reflecting upper airway and lung inflammatory responses, was statistically significantly elevated  in children with lung fibrosis, as compared to children with bronchitis and pneumonia. The increase in the IL-8 and IL-10 concentration in induced sputum of patient with bronchitis and pneumonia we can use in clinical as the risk subgroup of patients with chronic lung diseases.