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

OBJECTIVE: The role of structural and host defensive cells in periapical le sions has been assessed previously by morphometric and immunohistochemical studies. The aim of this study was to investigate the function of periapica l cells by employing molecular techniques to estimate the cell synthetic ac tivity, proliferation and apoptosis in these lesions. We specifically sough t answers to the following questions. Which cells of the periapical lesions are quiescent or actively synthesising proteins! Do immune cells prolifera te in this region in the same way as epithelial cells proliferate! Furtherm ore do cells in periapical lesions undergo apoptosis, and if so which cells exhibit this programmed cell death! MATERIALS: Twenty-five periapical tissue samples (15 granulomas and 10 radi cular cysts) were assessed. Polyadenosine (poly (A)) RNA and ribosomal RNA (rRNA) bearing cells in formalin-fixed/paraffin-embedded periapical tissues were analysed by in situ hybridization (ISH) using digoxigenin-labelled ol igo d (T) and 28S rRNA probes respectively in order to estimate cell synthe tic activity. Furthermore, S-phase proliferating and cycling cells were exa mined by ISH using a histone probe and Ki-67 immunostaining so as to assess cellular proliferation. Mononuclear cells were further differentiated by i mmunohistochemistry (IHC) as T cells, B cells and macrophages, Apoptotic ce lls were determined by in situ end-labelling methodology for detecting frag mented DNA. RESULTS: 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 f ew histone mRNA-expressing cells were detected in basal and suprabasal epit helial 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. Apoptotic cells were de tected in 23/25 cases and the majority of apoptotic cells were PMN which we re engulfed by large cytophagocytic macrophages. CONCLUSION: This study indicates that in dental periapical lesions, apoptos is occurs predominantly in PMN. It is evident that most cells apart from PM N are exhibiting synthetic activity but only epithelial cells undergo proli feration 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 gen e 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.