JE Boers, JL den Brok, J Koudstaal, JW Arends and FB Thunnissen
Department of Pathology, University of Limburg, Maastricht, the Netherlands.

Pulmonary neuroendocrine cells (PNECs) are ubiquitous in human adult airway epithelium, and are implicated in pulmonary disease as PNEC hyperplasia has been described in a great number of different pulmonary lesions. The purpose of this study was to determine the baseline quantity and proliferative fraction of adult PNECs in the conducting airway epithelium of normal lungs. Autopsy was performed within 6 h of death on 250 subjects. In 9 of 250 cases without pulmonary disease, seven sequential samples were taken from trachea down to respiratory bronchioles, fixed in formalin, and embedded in paraffin. After microwave pretreatment in citrate buffer, sequential immunohistochemical staining was carried out, using MIB-1 (antibody directed against recombinant parts of the Ki-67 antigen) as marker for the proliferation fraction and chromogranin-A (CgA) for the identification of PNECs. Quantification of all epithelial cells of the conducting airways, MIB-1, and CgA immunoreactive cells, and length of basement membrane was interactively performed using image analysis. The results show that microwave treatment significantly enhances the sensitivity of CgA without loss of specificity. In total, 326,500 epithelial cells and 105 cm of basement membrane (BM) length were counted (3,101 cells/cm BM). The proliferation fraction of all epithelial cells was 0.76 +/- 0.53% (mean +/- SD). No mitosis was seen. The mean quantity of PNECs was 0.41 +/- 0.17% of all epithelial cells (12.5 cells/cm BM). Many dendritic PNEC processes parallel to the basement membrane could be appreciated between adjacent epithelial cells. No difference in the number of PNECs was found between large and small conducting airways. PNECs were not observed in the alveoli. Only 13 PNECs were immunoreactive for MIB-1; the proliferative fraction of PNECs was 1 to 2%. In histologically normal adult human conducting airway epithelium, the average overall proliferation fraction is 0.76%. The proliferation fraction of PNECs is equal to the overall proliferative fraction. The fraction of PNECs is 0.41%, using anti-CgA antiserum after microwave pretreatment of slides, 12 times more than previously reported. Many dendritic neuroendocrine cell processes were seen between non-neuroendocrine epithelial cells adjacent to the basement membrane. The widely held belief that neuroendocrine cells are of little importance in the adult lung is contradicted by the relative high density of PNECs and its cytoplasmic processers.

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