Cell synthesis, proliferation and apoptosis in human dental periapical lesions analysed by in situ hybridisation and immunohistochemistry.

Abstract

The role of structural and host defensive cells in periapical lesions has been assessed previously by morphometric and immunohistochemical studies. The aim of this study was to investigate the function of peri- apical cells by employing molecular techniques to estimate the cell synthetic activity, proliferation and apoptosis in these lesions. We specifically sought answers to the following questions. Which cells of the periapical lesions are quiescent or actively synthesising proteins? Do immune cells proliferate in this region in the same way as epithelial cells proliferate? Furthermore do cells in peri- apical lesions undergo apoptosis, and if so which cells exhibit this programmed cell death? Twenty-five periapical tissue samples (15 granulomas and 10 radicular cysts) were assessed. Poly-adenosine (poly (A)) RNA and ribosomal RNA (rRNA) bearing cells in formalin-fixed/paraffin-embedded peri- apical tissues were analysed by in situ hybridization (ISH) using digoxigenin-labelled oligo d (T) and 28S rRNA probes respectively in order to estimate cell synthetic activity. Furthermore, S-phase proliferating and cycling cells were examined by ISH using a histone probe and Ki-67 immunostaining so as to assess cellular proliferation. Mononuclear cells were further differentiated by immunohistochemistry (IHC) as T cells, B cells and macrophages. Apoptotic cells were determined by in situ end-labelling methodology for detecting fragmented DNA. Poly (A) RNA (mostly messenger RNA) and 28S rRNA-expressing cells were detected in all samples. Plasma cells exhibited strongest staining for the two probes, with slight to moderate staining found in the epithelium, fibroblasts, macrophages, endothelial cells and lymphocytes, whereas almost all polymorphonuclear leucocytes (PMN) were negative for these probes. A few histone mRNA-expressing cells were detected in basal and suprabasal epithelial cells and mononuclear cells in 15/25 cases but their reactivity was weak. Ki-67 positive cells were found in all samples and their numbers were generally higher than histone mRNA positive cells. Apo- ptotic cells were detected in 23/25 cases and the majority of apoptotic cells were PMN which were engulfed by large cytophagocytic macrophages. This study indicates that in dental periapical lesions, apoptosis occurs predominantly in PMN. It is evident that most cells apart from PMN are exhibiting synthetic activity but only epithelial cells undergo proliferation which implies that immune cells must proliferate at distant lymph nodes and travel to the periapical lesion rather than proliferating within the lesion. These results suggest considerable advantages in estimating gene expression within cells in addition to the immunohistochemical detection of cells to determine cell activity at inflamed sites. Clearly, functional cell synthetic activity, resolution and clearance systems operate in peri- apical cystic and granuloma lesions.