© Borgis - New Medicine 3/1999, s. 42-43
Marcin Musiatowicz1, Jolanta Wysocka2, Edwina Kasprzycka2, Elżbieta Hassmann-Poznańska1
Lymphocyte subpopulations in adenoids according to age
1 Department of Pediatric Otorhinolaryngology, Medical University of Białystok, Poland
Head of Dept.: Prof. Elżbieta Hassmann-Poznańska M.D.
2 Department of Pediatric Laboratory Diagnostic, Medical University of Białystok, Poland
Head of Dept.: Dr. hab. Jolanta Wysocka M.D.
Summary
The aim of this study was to investigate changes in lymphocyte subpopulations in adenoids according to age. Investigations were carried out on adenoids removed during adenoidectomy procedures performed on a laryngological basis (hypertrophy) in children in two age groups: 0-5 years old and 5-10 years old. Monoclonal antibodies against superficial antigens of lymphocytes: CD3, CD4, CD8, HLA-DR, CD25 and CD16+56 were used. Analyses were performed using a Coulter EPICS XL flow cytometer with 104 cells per count. No statistically significant differences between groups were found.
The adenoid together with lymphatic tissue associated with the bronchi (BALT) and guts (GALT) is a part of the lymphoepithelial system. The adenoid increases its size during the first years of life and then undergo spontaneous involution. The adenoid growth is mainly caused by antigen stimulation, while the mechanisms responsible for its natural involution still remain undiscovered. Not much is known about changes within the adenoid tissue caused by ageing, in comparison with tonsils.
The most common surgical procedure performed in childhood is adenotomy. This is why knolwledge of the adenoid function, which changes during the growth period, is essential for evaluation of indications for adenotomy and assessment of its results.
Flow cytometry was used for the first time by Hemlin et al. (3), for the quantitative analysis of lymphocyte subpopulations in adenoids. It was previously used in relation to the tonsils (6, 7). This method allows for a great number of cells to be analysed, giving reliable data.
The aim of this study was to assess changes in the lymphocyte subpopulations in adenoids according to age.
Materials and methods
The study was carried out on two age groups: 0-5 and 5-10 years old. The material used for analyses were adenoids removed during adenotomy performed due to hypertrophy. Once removed the adenoid was immediately put on an RPMI 1640 growth medium with 10% bovine foetal serum at a temp. of 4°C and transported to the laboratory. Tissue was cut and mechanically crumbled in order to obtain a monocellular suspension which was subsequently centrifuged at 100-500 g for 1 min. at a temp. of 4°C and rinsed twice (300 g for 8 min. at 4°C) using PBS with the addition of 0.1mM EDTA and 0.02% NaN3. Cell density was measured simultaneously with a Bürker´s camera and Coulter haematologic analyzer MAXM. Vitality of cells was assessed after dyeing with 0.5% trypan blue. Cellular suspensions were incubated with bicolour monoclonal antibodies by Becton Dickinson switched to the following superficial lymphocyte antigens: CD19, CD3, CD4, CD8, HLA-DR, CD25 and CD16 + 56. Negative controls were isotope consistent with the class of antibodies used in the study. After 20 minutes of incubation at room temperature each sample underwent fast lysis by means of an EPIC IMMUNOLOGY WORK STATION. The final analyses were performed using Coulter EPICS XL flow cytometer with 104 cells analyzed per count.
Results
In the group of children aged between 0 and 5 years the ratio of lymphocytes was about 77%. The proportion of B (CD19) and T (CD3) lymphocytes was 58.75 ? 4.84% and 35.54 ? 5.51 respectively. Among the T lymphocyte population the proportion while that of T helper cells Th (CD4) was 27,69 ? 4.34%), while the ratio of T suppresor lymphocytes Ts (CD8) was 6.51 ? 2.25%. The ratio of Th (CD4) to Ts (CD8) lympho- cytes in the above group was 5.1 ? 2.99. We found a relatively small proportion of natural killer cells NK (CD3-)/CD16++56+ in the analyzed adenoid tissue (0.85 ? 0.38%). The percentage of CD3+CD25+ was also low (0.88 ? 0.63). The proportion of CD3+ cells with the activation marker HLA-DR+ was 7.23 ? 5.2.
In the group of older children (from 5 to 10 years) the results were very similar and no statistically significant differences were found between the two groups.
Discussion
The study shows that the cell suspension prepared in the above way maintained its vitality. The preparation technique undoubtedly leads to the loss of a certain amount of cells but cytometrical and immunohistopatological analyses made by Hemlin et al. (3, 4) indicate that cell losses are similar among different types and are statistically comparable. The T and B cells distribution in the adenoid tissue measured by means of flow cytometry differs significantly from that in blood. There is a majority of B cells and an essentially higher proportion of CD4 to CD8 cells in adenoids. Similar results obtained by the same method were reported by Hemlin et al. (3). The results of the present study were also consistent with those of Van Nieuwkerk et al. (8), obtained by means of different techniques. The percentage of lymphocyte subpopulations in adenoid is almost identical to that observed in tonsils (6, 7).
Our data show that the ratio of T (CD3+) cells was 33-35% and the proportion of lymphocytes Th (CD4) to Ts (CD8) was 5.1, which corresponds to the results of Hemlin et al. (4).
The CD25 is a receptor marker for interleukine 2 (IL-2) in activated T lymphocytes. In our study the ratio of CD25+ cells was less than 1%, while Hemlin et al. (4) report 1.6%. These proportions indicate that adenoid tissue is not fully activated. In the present study this proportion was even smaller in the group of older children. Hemlin et al. (5), reported a statistically significant increase of CD25+ cells according to age. A low number of CD25+ was also reported in the activated T cells, which suggests confirmation of the hypothesis that activated T cells go through the CD25+ (IL-2 receptor) phase to the next stage very fast (6, 7). Bernstein et al. (1), suggest that tonsillar T cells have inhibited activating B cells to immunoglobulin production activity.
This study does not show a statistically significant difference between adenoid lymphocyte subpopulations between the two age groups. This may result from the fact that in both groups adenoid tissue showed features of hypertrophy, the reason for adenotomy. Hemlin et al. (4), also reported an absence of significant differences in the lymphocyte subpopulations apart from the CD25+ cell increase. Nadal et al. (5) reported a significant decrease of IgG producing cells in adenoids according to age.
Our data show that there are no significant changes in adenoid lymphocyte subpopulations according to age. Because of the small difference in age between the two examined groups, a group of older children (above 10 years of age) should be included in further studies.
Piśmiennictwo
1. Bernstein J.M. et al.: Are thymus-derived lymphocytes (T cells) defective in the nasopharyngeal and palatine tonsils in children? Otolaryngology Head and Neck Surgery 1993, 109:693-700. 2. Brandtzaeg P.: Immune functions and immunology of palatine and nasopharyngeal tonsils. [In:] Immunology of the ear (Ed. Bernstein J.M., Ogra L.P.), Raven Press, New York, 63-106, 1987. 3. Hemlin C. et al.: Adenoid tissue lymphocyte subpopulations - evaluation of a quantitative analysis with flow cytometry. APMIS 1993, 101:551-556. 4. Hemlin C. et al.: Flow cytometric quantification of lymphocyte subpopulations and immunoglobulin-containing cells in adenoid tissue in relation to secretory otitis media and age. Acta Otolaryngol (Stockh) 1995, 115:443-448. 5. Nadal D. et al.: Distribution characteristics of immunoglobulin-secreting cells in adenoids. Relationship to age and disease. Int. J. Pediatr. Otorhinolaryngol. 1992, 24(2):121-130. 6.Plum J. et al.: Phenotyping of mononuclear cells from tonsils and corresponding biopsies using a cytofluorimeter - a comparative study. Acta Otolaryngol (Stokh) 1986, 101:129-134. 7.Sugiyama M. et al.: Subsets of tonsillar lymphocytes and activated cells in each subset analysed by three - colour flow cytometry. Acta Otolaryngol. (Stockh) 1998, Suppl., 454:138-147. 8.Van Nieuwkerk E.B.Y. et al.: Lymphoid and non-lymphoid cells in the adenoid of children with otitis media with effusion: a comparative study. Clin. Exp. Immunol. 1990, 79:233-239.
