Abstract
Pulmonary neuroepithelial bodies (NEBs), presumed polymodal airway sensors, consist of innervated clusters of amine (serotonin) and peptide-producing cells. While NEB responses to acute hypoxia are mediated by a membrane-bound O2 sensor complex, responses to sustained and/or chronic hypoxia involve a prolyl hydroxylase (PHD)–hypoxia-inducible factor-dependent mechanism. We have previously reported hyperplasia of NEBs in the lungs of Phd1−/− mice associated with enhanced serotonin secretion. Here we use a novel multilabel immunofluorescence method to assess NEB distribution, frequency, and size, together with the number and size of NEB cell nuclei, and to colocalize multiple cytoplasmic and nuclear epitopes in the lungs of Phd1−/−, Phd2+/−, and Phd3−/− mice and compare them with wild-type controls. To define the mechanisms of NEB cell hyperplasia, we used antibodies against Mash1 and Prox1 (neurogenic genes involved in NEB cell differentiation/maturation), hypoxia-inducible factor-1alpha, and the cell proliferation marker Ki67. Morphometric analysis of (% total lung area) immunostaining for synaptophysin (% synaptophysin), a cytoplasmic marker of NEB cells, was significantly increased in Phd1−/− and Phd3−/− mice compared to wild-type mice. In addition, NEB size and the number and size of NEB nuclei were also significantly increased, indicating that deficiency of Phds is associated with striking hyperplasia and hypertrophy of NEBs. In Phd2+/− mice, while mean % synaptophysin was comparable to wild-type controls, the NEB size was moderately increased, suggesting an effect even in heterozygotes. NEBs in all Phd-deficient mice showed increased expression of Mash1, Prox1, Ki67, and hypoxia-inducible factor-1alpha, in keeping with enhanced differentiation from precursor cells and a minor component of cell proliferation. Since the loss of PHD activity mimics chronic hypoxia, our data provide critical information on the potential role of PHDs in the pathobiology and mechanisms of NEB cell hyperplasia that is relevant to a number of pediatric lung disorders.
Highlights
The pulmonary neuroendocrine cell (PNEC) system is comprised of solitary cells and distinctive innervated clusters called neuroepithelial bodies (NEBs), which are localized within the airway mucosa of mammalian lungs
We examined the expression of an NEB cell cytoplasmic neuroendocrine cell marker, synaptophysin (SYP), together with nuclear immunolocalization of neurogenic genes: mammalian achete–scute homolog (Mash1), prospero homeobox 1 (Prox1), essential for NEB cell differentiation during development and postnatally;[15,16,17] a proliferation marker Ki67; and hypoxia-inducible factor (HIF)-1alpha, the transcriptional mediator of hypoxia-induced expression of numerous genes involved in O homeostasis.[18]
We show a similar degree and extent of NEB hyperplasia/ hypertrophy in Phd3−/− mice, while only moderate changes were observed in Phd2+/− mice, suggesting that partial deficiency is insufficient to induce a full NEB cell hyperplasia/ hypertrophy response
Summary
The pulmonary neuroendocrine cell (PNEC) system is comprised of solitary cells and distinctive innervated clusters called neuroepithelial bodies (NEBs), which are localized within the airway mucosa of mammalian lungs. Dovepress inflammation.[4] While solitary PNECs are thought to mediate local airway responses, NEBs are presumed to be polymodal airway sensors analogous to carotid body (CB) glomus cells and a part of the body’s O2 sensing homeostatic system.[5,6] The evidence supporting their role as airway O2 sensors includes: 1) structural features, including localization near airway branch points, an apical surface in contact with airway lumen, and extensive innervation by vagal afferent fibers; 2) expression of a membrane-bound O sensing molecular complex composed of the multicomponent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase coupled to an O sensitive K+ channel7; 3) the observation that exposure to acute hypoxia leads to activation of the O sensor via Ca2+2 mediated, dose-dependent 5-HT release[8]; and 4) the observation that 5-HT and other NEB cell-derived neurotransmitters (ie, ATP) activate their respective postsynaptic receptors on vagal afferents, transmitting the signals to the brain stem and affecting the control of respiration.[3] Earlier studies have shown that exposure to chronic, sustained hypoxia in both human and animal models leads to hyperplasia of NEBs similar to CB glomus cells.[9] We and others have previously reported striking hyperplasia of PNEC/NEBs in several pediatric lung disorders, including chronic lung disease of prematurity (bronchopulmonary dysplasia), sudden infant death syndrome, and neuroendocrine cell hyperplasia of infancy.[1,2] Chronic hypoxia likely plays an important role in the pathobiology of these disorders, the precise mechanisms are not known. Since PHDs are the principal cellular O2 sensors involved in the signal transduction of chronic/sustained hypoxia,[12,13] in the present study, we have performed detailed immunohistochemical and morphometric analysis of NEBs in the lungs of Phd1−/−, Phd2+/−, and Phd3−/− mice, and compared them with WT controls
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