Clinical immunology
Early and late activation markers on thymus-dependent lymphocytes and natural killer cells in the blood of children with adenoid hypertrophy and concomitant otitis media with effusion

Introduction

The immune system provides protection against infection and regulates processes of destruction and regeneration of body tissues; it plays the role of “the inner doctor”. Our health depends on the efficiency of this “doctor”, which is the sum of efficient mechanisms of defense and regeneration; it enables proper function of individual organs and body systems.

Properly functioning system does not allow an assault against its own normal tissues (auto aggression) or against foreign neutral agents (allergy), it directs its assault against pathogenic infectious agents. It also has a positive influence on its own tissues, helping in the regeneration process. Destructive mechanisms are defensive instruments and constructive mechanisms are regenerative instruments of the immune system. The right address is necessary for these mechanisms to function appropriately.

The efficiency of the immune system is essentially conditioned by the degree of immune competence. This we define as the ability to recognize and properly respond to potentiation or inhibition of immunologic reaction depending on the identified needs.

Respiratory tract infections account for about 50% of illnesses in children up to 5 years of age and about 30% in children between 5 to 12 years of age. The highest average number of respiratory tract infections (7 to 9) is observed between 2 and 4 years of age, whereas in the age group 8-10 years average is about 4 to 6 recurrences of infection during the year [1]. One of the causes of recurrent infections, especially recurrent otitis media with effusion (OME) is adenoid hypertrophy. Pharyngeal adenoid hypertrophy in recurrent OME may be the cause and the result of chronic inflammation process within the middle ear. About recurrence of OME, to a large extent, decide the specifics of the mechanisms of infection, interaction between cells of the mucosa and the virulence of microorganisms. On the other hand, the intensity, the extent and duration of the infection is decided by the host immune response. In case of frequent infections, where antibiotics do not prevent recurrences one should consider the possibility of impaired immune mechanisms, such as immunoglobulin deficiency, impaired function of phagocytes or quantitive and functional disturbance of thymus dependent lymphocytes [2-4].

In view of this data, in this study we attempted to answer the question of how pharyngeal adenoid hypertrophy, and associated otitis media, are reflected in percentage rate and the expression of early and late activation antigens on subsets of peripheral blood lymphocytes. This evaluation may be the basis for expanding the indications for a possible adenoidectomy.

Material and methods

Patients





The survey involved 119 children of both sexes, aged from 2 to 6 years with pharyngeal adenoid hypertrophy. First group included 44 children, which had an incident of OME, in no more than 3 months preceding the study. Second group included 45 children who had at least 4 episodes of OME during 6 to 12 months preceding the study. Third group included 30 children, who had pharyngeal adenoid hypertrophy and OME was not observed. Patients were examined during remission of illness. University Bioethics Commission gave its consent to the study.

Morphological evaluation of peripheral blood

For the evaluation of the differential white blood cell count 2 ml of venous blood was taken to a test tube with EDTA. Differential white blood cell count analysis was performed in the hematological analyzer (MAXMEM, Coulter, Germany). Leukocytosis was given in the G/L, the element quantity of differential white blood cell count is given in percentage ratio (%) and absolute (BL, in the G/L) values.

Evaluation of peripheral blood lymphocyte subpopulations. With the rest of the samples of morphological venous blood a cytometric analysis was made. To 100 l of whole blood 10 l of following monoclonal antibodies were added (Dako): CD3-RPE-Cy5/CD4-FITC/CD8-RPE, CD4-RPE-Cy5/CD69-FITC/HLA-DR-RPE, CD8-RPE-Cy5/CD69-FITC/HLA-DR-RPE, CD16+56-RPE-Cy5/CD69-FITC/ HLA-DR-RPE. For each test a set of isotype negative controls was used. After 15 min of incubation at room temperature, each sample had undergone a rapid, automatic lyses (ImmunoPrep Work Station, Coulter). After thorough mixing, the samples were analyzed by flow cytometry method (Coulter Epics XL), counting 104 cells each time. The results were given as percentage ratio and based on the data from differential white blood cell count – absolute values.

Statistical analysis

Statistical analysis was calculated according to ANOVA/MANOVA procedures. The differences between the arithmetic means of each age group, for each of the evaluated parameters were analyzed by Mann-Whitney nonparametric ranking test, accepting p < 0.05 as significant difference.

Results

In the first stage, an evaluation of the number of leukocytes, percentage ratio and absolute value of peripheral blood lymphocytes was done. The results of the evaluation are presented in Table 1.

In the absence of significant differences between the statistical calculations performed for average percentage and absolute number of lymphocytes, in subsequent stages of the test, statistical analysis was done on the percentage ratio of leukocyte subpopulation only. In the next stage of the test, percentage ratio of peripheral blood lymphocyte subpopulations was assessed. The results of the evaluation are presented in Table 2. In the case of CD19, CD3, CD4 and CD8 positive cells no significant differences were observed between groups 1 (i.e. children with pharyngeal adenoid hypertrophy, who had 1 incident of acute otitis media) and children in group 3, who had pharyngeal adenoid hypertrophy without incidents of otitis media. A statistically significant difference from group 1 was observed in group 2, i.e. children with pharyngeal adenoid hypertrophy, who during the period from 6 months to 2 years had at least 4 incidents of acute otitis media with effusion. Within this group there was a significantly higher percentage ratio of CD19 cells, NK cells, a lower ratio of TH/TS cells and the lower percentage of CD3 and CD4 cells. There were no significant differences in regards to the percentage of CD8 cells.

The percentage ratio of CD4, CD8 and NK cells with co-expression of receptor CD69 (early) and HLA-DR (late) activation was subsequently assessed. In evaluating the sub-population of lymphocytes with HLA-DR co expression receptor, statistically significant differences were found in CD8 and NK lymphocyte subsets, where a group of children with pharyngeal adenoid hypertrophy and recurrent OME had significantly higher average values in relation to two other groups. The percentage leukocyte subpopulation with co-expression of receptor CD69 has the highest average values in both groups; particularly, significantly higher in group 2 for NK, CD4 and CD8 lymphocytes.

Discussion

The aim of these study was to evaluate the level of various lymphocyte subpopulations and the presence of early and late activation markers on T lymphocytes and NK cells in children with pharyngeal adenoid hypertrophy accompanied by OME.

The purpose of this evaluation were lymphocytes from peripheral blood of children with pharyngeal adenoid hypertrophy with incidental OME (group 1) and frequent OME (group 2). Obtained results were compared to a group of children with pharyngeal adenoid hypertrophy without OME (group 3). To optimalize conditions adopted in this study patients with OME were evaluated over a period of remission.

In the first stage of this test we characterized population of peripheral blood lymphocytes. The studies assessed the percentage ratio of – CD3+, CD4+, CD8+ – T cells; percentage ratio of – CD19+ – B cells and CD3-CD16+56+-NK cells. Activation of lymphocytes was evaluated by assessing surface expression of HLA-DR and CD69 antigens.

In the group of children with OME, at the same time there was a decrease in percentage of CD3+ cells and an increase in the percentage of CD19+ cells and NK cells. Based on the results, one can not determine whether the observed quantitative shortage of CD3+ cells is the cause or effect of the observed clinical symptomatology? In the case-by-case study, the factor that suppresses the immune system, particularly its component cells, appears to be chronic antigenic stimulation. On the other hand, the cause of this suppression could be antibiotics taken during the infection, which are highly ineffective, given the recurring nature of the process and deepens an existing immune deficiency [5-9].

Based on previous studies [10, 11] quantitative reduction and functional impairment of T lymphocyte populations by the use of most antibiotics, it can be assumed that adverse effects of antibiotics correspond to the term “stage thymectomy” [12].

The dominance of the process of inflammation in the group of children with OME states a significantly higher proportion of CD8+ cells and NK cell with co-expression receptor HLA-DR – late activation antigen. The increase of HLA-DR expression in the assessed cases is probably caused by chronic antigenic stimulation. It is difficult, on the basis of these tests to judge, the increase in the percentage of NK cells and NK cell with HLA-DR co-expression receptor, is by how much compensatory to T-cell deficiency, and to how this compensation meets the needs of the immune system in conditions of forced stimulation during recurrent OME infection in children. The question remains open of the quality of compensatory responses of NK cells, in their participation in an immune response to which they are biologically characterized and predisposed. Attempting to answer this question, one should assess the expression of early activation antigen – CD69, for the evaluated lymphocyte subsets.

In the studies of percentage ratios of lymphocytes with antigen CD69 co-expression, in a group of children with pharyngeal adenoid hypertrophy and recurrent OME in all lymphocyte subsets i.e. CD4+ cell, CD8+ and NK cells, significantly higher values were obtained in relation to both other groups.

It has been proven that the CD69 antigens manifest in vitro under the influence of a wide range of stimuli in most hemopoetic cell lines, in vivo CD69 antigen expression is mainly observed in inflammatory sites mainly in T lymphocytes [13]. In the phenotypes of dormant CD3+ lymphocytes manifestation of CD69 antigen in healthy subjects ranges from 5% to 12% [14]. In the cytoplasm of dormant T lymphocyte, CD69 antigen occurs in preformed stages, and thus its surface expression, in the initial phase of activation does not require mRNA synthesis de novo [13, 14].

In in vitro studies, a correlation was observed between the dose of antigen Alloiococcus otitidis and the degree of expression of CD69 antigen in T lymphocytes from peripheral blood. The highest antigenic stimulation of CD69 molecule was observed in CD8+ lymphocytes [15, 16].

Also, in the studies evaluating the lymphocytes from pharyngeal tonsil in patients with OME, an increase in expression of early and late activation antigens, which would correspond to chronic antigenic stimulation was reported [17-19]. Similar studies evaluating the subpopulations of leukocytes isolated from effusion fluid from the middle ear have shown a correlation between the increase in the inflammatory process in pharyngeal tonsil and local inflammatory process within the middle ear [20-23].

In the present study we observed that recurring OME with pharyngeal adenoid hypertrophy is accompanied by varying degree of immune deficiency, mainly in a lymphocytes T population. This deficit is compensated by the rise in NK cells and CD19 cells. Based on these studies, it is difficult to conclude the primary or secondary character of observed changes. These changes, however, seem to be a one more indication for surgery in cases where pharmacotherapy does not provide the expected results.

References

 1. Bousquet J, Fiocchi A (2006): Prevention of recurrent respiratory tract infections in children using a ribosomal immunotherapeutic agent: a clinical review. Paediatr Drugs 8: 235-243.  

2. Carrock Sewell WA, Webster AD (1998): Infection in patients with congenital immune deficiencies. Curr Opin Infect Dis 11: 419-423.  

3. Kamer V, Zeman K, Kamer-Kejna A, et al. (1998): Evaluation of certain immunological parameters in infants and small children with recurrent respiratory tract infections. Int Rev Allergol Clin Immunol 4: 150-154.  

4. Aicher A, Hayden-Ledbetter M, Brady WA, et al. (2000): Characterization of human inducible costimulator ligand expression and function. J Immunol 164: 4689-4696.  

5. Held W, Coudert JD, Zimmer J (2003): The NK cell receptor repertoire: formation, adaptation and exploitation. Curr Opin Immunol 15: 233-237.  

6. Lombardi G, Sidhu S, Batchelor R, Lechler R (1994): Anergic T cells as supressor cells in vitro. Science 264: 1587-1589.  

7. Noroski LM, Shearer WT (1998): Screening for primary immunodeficiencies in the clinical immunology laboratory. Clin Immunol Immunopathol 86: 237-245.  

8. Puck JM (1997): Primary immunodeficiency diseases. JAMA 278: 1835-1841.  

9. Shevach EM (2000): Regulatory T cells in autoimmunity. Annu Rev Immunol 18: 423-449.

10. Chaperon EA: Supression of lymphocytes by cephalosporins. In: The influence of antibiotics on the host-parasite relationship. Eickenberg HU, Hahn H (eds.). Opferbuch, Springer Verlag, Berlin 1982; 22-30.

11. Dąbrowski MP, Dąbrowska-Bernstein BK: Immunoregulatory role of thymus. CRC Press. Inc. Boca Raton, Florida, USA 1990.

12. Werfel T, Boeker M, Kapp A (1997): Rapid expression of the CD69 antigen on T cells and natural killer cells upon antigenic stimulation of peripheral blood mononuclear cell suspensions. Allergy 52: 465-469.

13. Mardiney M 3rd, Brown MR, Fleisher TA (1996): Measurement of T-cell CD69 expression: a rapid and efficient means to assess mitogen- or antigen-induced proliferative capacity in normals. Cytometry 26: 305-310.

14. Harimaya A, Himi T, Fujii N, et al. (2005): Induction of CD69 expression and Th1 cytokines release from human peripheral blood lymphocytes after in vitro stimulation with Alloiococcus otitidis and three middle ear pathogens. FEMS Immunol Med Microbiol 43: 385-392.

15. Sade K, Fishman G, Kivity S, et al. (2011): Expression of Th17 and Treg lymphocyte subsets in hypertrophied adenoids of children and its clinical significance. Immunol Invest 40: 657-666.

16. Mattila PS, Nykänen A, Eloranta M, Tarkkanen J (2000): Adenoids provide a microenvironment for the generation of CD4(+), CD45RO(+), L-selectin(–), CXCR4(+), CCR5(+) T lymphocytes, a lymphocyte phenotype found in the middle ear effusion. Int Immunol 12: 1235-1243.

17. Kotowski M, Niedzielski A, Niedzielska G, Lachowska-Kotowska P (2011): Dendritic cells and lymphocyte subpopulations of adenoid in the pathogenesis of otitis media with effusion. Int J Pediatr Otorhinolaryngol 75: 265-269.

18. Musiatowicz M, Wysocka J, Kasprzycka E, Hassmann E (2001): Lymphocyte subpopulations in hypertrophied adenoid in children. Int J Pediatr Otorhinolaryngol 59: 7-13.

19. Skotnicka B, Stasiak-Barmuta A, Hassmann-Poznańska E (2002): Subpopulacje limfocytów w płynach wysiękowych ucha środkowego. Otolaryngol Pol 56: 695-700.

20. Zhang Q, Liu C, Wang J, et al. (2009): Expression pattern of aquaporin 4 and 5 in the middle ear of guinea pigs with secretory otitis media. Acta Otolaryngol 28: 1-7.

21. Matković S, Vojvodić D, Baljosevic I (2007): Cytokine levels in groups of patients with different duration of chronic secretory otitis. Eur Arch Otorhinolaryngol 264: 1283-1287.

22. Cayé-Thomasen P, Stangerup SE, Jo/rgensen G, et al. (2008): Myringotomy versus ventilation tubes in secretory otitis media: eardrum pathology, hearing, and eustachian tube function 25 years after treatment. Otol Neurotol 29: 649-657.

23. Firat Y, Koç C, Olcay I, et al. (2006): The incidence of atopy in adults with recurrent secretory otitis media: screening with Phadiatop. Kulak Burun Bogaz Ihtis Derg 16: 11-17.