Quantification of cells expressing markers of proliferation and apoptosis in chronic tonsilitis

Abstract

During chronic tonsillitis, the relationship between proliferation and apoptosis of lymphocytes in tonsillar follicles can be disturbed, which gives rise to attenuation of tonsil immunocompetence and diminishing its contribution in systemic immunity. In this study, we have quantified the cells expressing the markers of proliferation and apoptosis in the follicles of the palatine tonsil. Six tonsils from patients aged 10-29 years with hypertrophic tonsillitis and five tonsils from patients aged 18-22 years with recurrent tonsillitis were studied. The sections of paraffin blocks of tonsillar tissue were stained by the immunohistochemical LSAB/HRP method with the utilisation of antibodies for: Ki-67 antigen-cell marker of proliferation; Bcl-2 and survivin anti-apoptotic factors and Fas/CD95, caspase-3 and Bax pro-apoptotic factors. The size of lymphoid follicles, i.e. mean follicle area and number of lymphoid follicle immunopositive cells per mm2 of a slice area, i.e. numerical areal density were determined by the quantitative image analysis. The localisation of Ki-67, Bcl-2, survivin, Fas/CD95, caspase-3 and Bax- immunopositive cells inside the palatine tonsil was similar in both types of tonsillitis. The number of Ki-67 immunopositive cells was significantly (p < 0.01) larger in the tonsils with hypertrophic tonsillitis (14681.4 ± 1460.5) in comparison to those with recurrent tonsillitis (12491.4 ± 2321.6), although the number of survivin and caspase-3 immunopositive cells was significantly (p < 0.05) larger in recurrent tonsillitis (survivin, 406.9 ± 98.4; caspase-3, 350.4 ± 119.4) when compared to those with hypertrophic tonsillitis (survivin, 117.4 ± 14.5; caspase-3, 210 ± 24). Our results show that the rate of the proliferation and apoptosis of follicular lymphocytes is different in various types of tonsillitis. This suggests that the immunological potential of the palatine tonsil varies in patients with hypertrophic and recurrent tonsillitis, which in practice poses a dilemma over the choice of conservative or surgical treatment.

Keywords: Apoptosis; Cell proliferation; Human palatine tonsil; Lymphoid follicle; Quantification.

Fig. 1.

Light micrograph of the palatine tonsil with: a) hypertrophic tonsillitis and b) recurrent tonsillitis. The pictures show the lymphoid follicles in which the pale-stained germinal centres and dark-stained mantle zones are visible. H&E × 100.

Fig. 2.

Immunohistochemistry of proliferative and anti-apoptotic activity in tonsillar lymphoid follicles. LSAB/HRP method: a) Expression of Ki-67 in germinal centre (GC) and mantle zone (MZ) of lymphoid follicle (× 200); b) Detail from the previous picture: Ki-67-imunopositive cells are the most numerous in the dark zone of germinal centre (GC) (× 400); c) Bcl-2 protein is expressed by the cells of the follicular mantle zone (MZ), (× 200); d) Absence of Bcl-2 expression in the germinal centre (GC) and strong expression in the mantle zone (MZ) is noticed (× 400); e) Survivin-expressing cells are present in the germinal centre (GC) of the lymphoid follicle (× 100); f) Survivin expression in a non-dividing cell (upper left corner) as well as the survivin-expressing cells with strongly stained mitotic figures (low central) are noticed (× 400).

Fig. 3.

Immunohistochemistry of apoptotic activity in tonsillar lymphoid follicles. LSAB/HRP method: a) A part of the lymphoid follicle germinal centre (GC) with numerous Fas/CD95- expressing cells (× 400); b) Detail from the previous picture (× 1000); c) Individual caspase- 3- expressing cells in the germinal centre (GC), (× 100); d) Detail from the previous picture: cytoplasm of the cell which morphologically corresponds to the follicular macrophage (upper left part), contains caspase-3 immunopositivity (× 1000); e) Strong Bax immunopositivity can be seen between the two lymphoid follicles (LyF), and weak immunopositivity in germinal centre cells (× 400); f) Germinal centre cells (upper lower part) show granular Bax immunopositivity, possibly phagocytosed apoptotic bodies (× 630); g) In the lower part of the picture a cell with strong Bax immunopositivity can be seen (× 1000).