Number Of Pulmonary Neuroendocrine Cells Research Articles

Airway basal stem cells generate distinct subpopulations of PNECs.

SUMMARYPulmonary neuroendocrine cells (PNECs) have crucial roles in airway physiology and immunity by producing bioactive amines and neuropeptides (NPs). A variety of human diseases exhibit PNEC hyperplasia. Given accumulated evidence that PNECs represent a heterogenous population of cells, we investigate how PNECs differ, whether the heterogeneity is similarly present in mouse and human cells, and whether specific disease involves discrete PNECs. Herein, we identify three distinct types of PNECs in human and mouse airways based on single and double positivity for TUBB3 and the established NP markers. We show that the three PNEC types exhibit significant differences in NP expression, homeostatic turnover, and response to injury and disease. We provide evidence that these differences parallel their distinct cell of origin from basal stem cells (BSCs) or other airway epithelial progenitors.

Pulmonary neuroendocrine cells: physiology, tissue homeostasis and disease.

ABSTRACTMammalian lungs have the ability to recognize external environments by sensing different compounds in inhaled air. Pulmonary neuroendocrine cells (PNECs) are rare, multi-functional epithelial cells currently garnering attention as intrapulmonary sensors; PNECs can detect hypoxic conditions through chemoreception. Because PNEC overactivation has been reported in patients suffering from respiratory diseases – such as asthma, chronic obstructive pulmonary disease, bronchopulmonary dysplasia and other congenital diseases – an improved understanding of the fundamental characteristics of PNECs is becoming crucial in pulmonary biology and pathology. During the past decade, murine genetics and disease models revealed the involvement of PNECs in lung ventilation dynamics, mechanosensing and the type 2 immune responses. Single-cell RNA sequencing further unveiled heterogeneous gene expression profiles in the PNEC population and revealed that a small number of PNECs undergo reprogramming during regeneration. Aberrant large clusters of PNECs have been observed in neuroendocrine tumors, including small-cell lung cancer (SCLC). Modern innovation of imaging analyses has enabled the discovery of dynamic migratory behaviors of PNECs during airway development, perhaps relating to SCLC malignancy. This Review summarizes the findings from research on PNECs, along with novel knowledge about their function. In addition, it thoroughly addresses the relevant questions concerning the molecular pathology of pulmonary diseases and related therapeutic approaches.

Regulation of Airway Progenitor Homeostasis and Cell Composition by Tight Junction Protein Claudin‐18

RationaleClaudin‐18 (Cldn18) is comprised of two isoforms, Cldn 18.1 and Cldn18.2. The lung‐specific isoform, Cldn18.1, regulates distal lung epithelial progenitor cell homeostasis. Its role in airway epithelium has not been elucidated, although Cldn18 expression is decreased in lungs of patients with chronic airway diseases (e.g., asthma and chronic obstructive pulmonary disease (COPD)). The airway epithelium is comprised of three progenitor populations, basal cells (BC), pulmonary neuroendocrine cells (PNEC) and club cells, and two non‐progenitor cell populations, namely goblet and ciliated cells. Club cells are secretory cells that are susceptible to naphthalene (NAP) injury. Club cell depletion occurs by day 3 post‐NAP, with spreading of ciliated cells to cover the denuded epithelial surface, and full reconstitution of the airway epithelium by day 14–21. Here we investigated the role of Cldn18.1 in airway epithelium using a previously developed global Cldn18 knockout (KO) mouse model.MethodsWild‐type (WT) and Cldn18 KO mice were injected intraperitoneally with NAP (225–275 mg/kg, corn oil as control). Lungs were harvested at baseline and 3‐days (maximal injury), 7‐days (beginning of regeneration) and 21‐days (full regeneration) post‐NAP, inflated with 4% paraformaldehyde and fixed overnight prior to embedding and sectioning. Hematoxylin and eosin, Periodic Acid‐Schiff (PAS) and immunofluorescence staining were performed.ResultsIn WT mice, Cldn18.1 was expressed in a small subset of airway ciliated cells. Cldn18 KO mice showed increased numbers of pulmonary neuroendocrine cells and neuroendocrine bodies, as well as ectopic localization of club cells and localization of keratin 5‐positive cells within the distal parenchyma. At day 3 post‐NAP in WT mice, Cldn18.1 was transiently expressed in all the ciliated cells covering the denuded epithelium, returning to being expressed in only a subset of cells by day 7. Cldn18 KO mice subjected to injury unexpectedly developed marked goblet cell hyperplasia at day 14–21 post‐NAP, with cells expressing markers for both club (CC‐10) and goblet (MUC5AC) cells.ConclusionsOur findings demonstrate an unexpected role for Cldn18.1 in regulating airway progenitor homeostasis and cell composition, implicating Cldn18.1 in regulation of downstream signaling pathways that inhibit goblet cell differentiation. A transient increase in the number of Cldn18.1‐positive ciliated cells in WT mice in the initial stages post‐injury suggests paracrine regulation of these inhibitory effects. Modulation of Cldn18 expression may be beneficial in the treatment of airway diseases characterized by goblet cell metaplasia.Support or Funding InformationFunding: American Lung Association, Hastings Foundation, NIH

PO-272 Leukemia-associated NPM mutations promote quiescence of hematopoietic stem cells and prevent their functional exhaustion upon oncogene-induced hyper-proliferation

IntroductionIntroduction: Lung neuroendocrine tumours (LNETs) arise from a rare lung cell population, called pulmonary neuroendocrine cells (PNECs), and account for 20%–25% of all lung cancers. Despite the involvement of PNECs in LNETs and a number of other respiratory diseases, relatively little is known about their normal function or contribution to cancer development and progression. To date, the study of PNECs has been limited by difficulties in isolating and culturing this cell population.Material and methodsBuilding on methods developed in our lab for the isolation and long-term expansion of healthy and diseased human airway cells, we have generated lung organoids derived from embryonic lung tissue. We have identified cultures conditions that allow for the enrichment of PNECs in embryonic lung organoids. In parallel, we have begun to generate a biobank of both low- and high-grade LNET organoids.ResultsPNEC-enriched embryonic lung organoids can be expanded indefinitely over multiple passages. Preliminary analysis by qPCR and whole mount immunofluorescence shows that PNEC-enriched organoids express a number of PNEC lineage markers including ASCL1, NEUROD1, and UCHL1. PNEC-enriched lung organoids also express PNEC-associated bioactive compounds such as SST and CGRP.ConclusionsWe have generated human embryonic lung organoids that contain significant numbers of PNECs. Applying the same principles, we have established organoid cultures of human LNETs. PNEC-enriched organoids and LNET organoids are currently being used for further molecular and genetic analyses.

PO-271 Using human lung organoids to study pulmonary neuroendocrine cells

Introduction Introduction: Lung neuroendocrine tumours (LNETs) arise from a rare lung cell population, called pulmonary neuroendocrine cells (PNECs), and account for 20%–25% of all lung cancers. Despite the involvement of PNECs in LNETs and a number of other respiratory diseases, relatively little is known about their normal function or contribution to cancer development and progression. To date, the study of PNECs has been limited by difficulties in isolating and culturing this cell population. Material and methods Building on methods developed in our lab for the isolation and long-term expansion of healthy and diseased human airway cells, we have generated lung organoids derived from embryonic lung tissue. We have identified cultures conditions that allow for the enrichment of PNECs in embryonic lung organoids. In parallel, we have begun to generate a biobank of both low- and high-grade LNET organoids. Results PNEC-enriched embryonic lung organoids can be expanded indefinitely over multiple passages. Preliminary analysis by qPCR and whole mount immunofluorescence shows that PNEC-enriched organoids express a number of PNEC lineage markers including ASCL1, NEUROD1, and UCHL1. PNEC-enriched lung organoids also express PNEC-associated bioactive compounds such as SST and CGRP. Conclusions We have generated human embryonic lung organoids that contain significant numbers of PNECs. Applying the same principles, we have established organoid cultures of human LNETs. PNEC-enriched organoids and LNET organoids are currently being used for further molecular and genetic analyses.

Quantitative evaluation of pulmonary neuroendocrine cells and the role of nitric oxide in experimentally induced renovascular hypertension in male albino rats

Background Renovascular hypertension is the most common cause of secondary hypertension that leads to disorders in many systems and organs including the lung. Aim of the work The present work was carried out to determine how experimentally induced renovascular hypertension affects rat lung tissue with a special focus on the disorders in nitric oxide (NO) production as well as the number of pulmonary neuroendocrine cells. Materials and methods Twenty-one adult male albino rats were used; they were divided into three groups. Group I included five control rats, group II included eight rats that were sham operated, and group III included eight rats that were subjected to a renovascular hypertension procedure. Lower segments of the right lung lobes were obtained and then subjected to H&E and Masson’s trichrome stains. Endothelial nitric oxide synthase (eNOS) and calcitonin antibody-2 were also used as immunohistochemical stains. Morphometric measurements included the area% of collagen fibers, the optical density of eNOS immunoreaction, and the number of calcitonin-positive neuroendocrine cells. Data were recorded and analyzed statistically. Results In group III, H&E-stained sections showed thickening of the tunica media of pulmonary arteries and alveolar walls with predominantly type II pneumocytes. Consequently, the thickened arteries showed a significant increase in the mean area% of collagen fibers, together with a significant decrease in the mean optical density of eNOS immunoreactivity when compared with the control group. The mean number of calcitonin immunoreactive neuroendocrine cells of group III showed a significant increase versus the control group. Conclusion The current study showed an increased number of pulmonary neuroendocrine cells with renovascular hypertension. This increase may represent a compensatory mechanism to overcome renovascular hypertension. Thus, clinical trials could be conducted to prove the possible role of calcitonin-producing pulmonary neuroendocrine cells in controlling renovascular hypertension.

Jagged1 is the major regulator of notch‐dependent cell fate in proximal airways

The Notch signaling pathway plays complex roles in developing lungs, including regulation of proximodistal fates, airway cell specification and differentiation. However, the specific Notch-mediated signals involved in lung development remain unclear. Here we report that Jagged1 is expressed in a subset of bronchial and bronchiolar epithelial cells, where it controls proximal airway cell fate and differentiation. In agreement with previous studies involving disruption of all Notch signaling, we found that deletion of Jagged1 in airway epithelium increased the number of ciliated cells at the expense of Clara cells, a phenotype associated with downregulation of Hes1. Deletion of Jagged1 also led to an increased number of pulmonary neuroendocrine cells (PNEC), suggesting that Jagged1/Notch signaling inhibits PNEC cell fate. As expected, Jagged1 deletion did not affect alveolar cell differentiation, although alveolar septation was impaired, likely an indirect effect of proximal airway defects. Finally, in the postnatal lung, Jagged1 deletion induced mucous metaplasia, accompanied by downregulation of Hes1 and Hes5. Our results demonstrate that Jagged1-mediated Notch signaling regulates multiple cell fate decisions as well as differentiation in the respiratory system to coordinate lung development and to maintain a balance of airway cell types in adult life.

Chronic inhalation of carbon monoxide: Effects on the respiratory and cardiovascular system at doses corresponding to tobacco smoking

Carbon monoxide (CO) is a dangerous poison in high concentrations, but the long-term effects of low doses of CO, as in the gaseous component of tobacco smoke, are not well known. The aims of our study were to evaluate the long-term effects of inhaled CO on the respiratory and cardiovascular system at doses corresponding to tobacco smoking and its effect on tumourigenesis and pulmonary neuroendocrine (NE) cells. Female Wistar rats were exposed to either CO (200 ppm) for 20 h/day ( n = 51) or air ( n = 26) for 72 weeks. Carboxyhaemoglobin was 14.7 ± 0.3% in CO exposed animals and 0.3 ± 0.1% in controls. In the lungs, no signs of pathology similar to that associated with cigarette smoking were observed, and no differences in number of pulmonary NE cells were observed between the groups. Chronic CO inhalation induced a 20% weight increase of the right ventricle ( p = 0.001) and a 14% weight increase of the left ventricle and interventricular septum ( p < 0.001). Histological examination of the myocardium did not reveal any signs of scarring. In the aorta and femoral artery, no signs of atherosclerosis were observed in CO exposed rats. No exposure related carcinogenic effects were observed. Spontaneous tumours were identified in 29% of CO exposed animals and in 28% of the controls. Our results suggest that low dose CO exposure is probably not responsible for the respiratory pathology associated with tobacco smoking. The effects on the cardiovascular system seem to involve myocardial hypertrophy, but not atherogenesis.

Modulation of pulmonary neuroendocrine cells in idiopathic interstitial pneumonia.

In order to reveal modulation of the number of pulmonary neuroendocrine cells (PNEC) in interstitial lung diseases and to clarify significance of cell proliferation activity in occurrence of PNEC, we counted airway PNEC of the patients of idiopathic interstitial pneumonia, secondary interstitial pneumonia and control lungs, and compared the number of PNEC with airway Ki-67 labeling. The lung tissue samples were obtained by video-assisted thoracoscopic surgery from 22 patients with usual interstitial pneumonia (UIP), 7 with non-specific interstitial pneumonia (NSIP), 8 with chronic hypersensitivity pneumonia (CHP), 13 with collagen vascular disease (CVD), and were compared with age-matched control lungs. The tissues were immunostained for chromogranin A and for Ki-67. Average incidence of bronchiolar PNEC in normal, UIP, NSIP, CHP, CVD lungs was 0.169%, 0.348%, 0.326%, 0.175% and 0.201%, respectively, and average Ki-67 labeling index in them was 0.241%, 1.186%, 1.605%, 1.058%, and 2.353%, respectively. And, in UIP lungs, PNEC incidence or Ki-67 labeling index was different according to pathological lesions. Thus, PNEC increase in the bronchiole of UIP, and the incidence of PNEC varies according to degree of activity of epithelial cell proliferation probably related to epithelial cell injury. Moreover, enhanced expression of human homolog of achaete-scute complex (hASH1) mRNA in UIP lungs suggests that hASH1 could play roles in the regulation of PNEC.

Impact of Mechanical Ventilation on the Developing Airway

After completing this article, readers should be able to: 1. Explain why immature infants are at a greater risk for mechanical ventilation-induced airway damage than are more mature infants. 2. Describe the effect of ventilatory pressures on components of the airway. 3. Delineate how mechanical ventilation disrupts airway mechanics. 4. Characterize how mechanical ventilation affects immature airway smooth muscle. 5. Describe techniques of optimizing ventilation to minimize respiratory damage. Although the air passages take on a mature appearance well before a fetus is viable, they undergo significant maturational changes in late gestation. During development, the airways are more susceptible to damage. In fact, although controversy still surrounds the pathogenesis of bronchopulmonary dysplasia (BPD) and other neonatal airway complications, it appears that the duration and higher pressures of mechanical ventilation, along with the use of oxygen, are related to the development of airway-related problems in preterm infants. As a result, because of their greater need for ventilatory assistance, very preterm infants are at substantially increased risk of airway damage. With recent and continuing advancements in neonatal care, a growing number of immature infants survive premature births. One of the primary hurdles that must be overcome in these infants is respiratory system immaturity. In this context, an understanding of the embryologic development and physiologic maturation of the respiratory system is imperative. The respiratory system can be subdivided grossly into the airways and the lung parenchyma. Developmental changes in the mechanical properties of these two components predispose the immature airway to injury. The immature lung is a relatively noncompliant structure. During neonatal development, lung compliance increases with lung maturity. However, the opposite is true for airways. Immature airways are relatively more compliant than their mature counterparts, with airway maturation resulting in a decrease in airway compliance. This produces an interesting predicament when contemplating the administration of mechanical ventilation …

Basic helix-loop-helix transcription factors regulate the neuroendocrine differentiation of fetal mouse pulmonary epithelium.

To clarify the mechanisms that regulate neuroendocrine differentiation of fetal lung epithelia, we have studied the expression of the mammalian homologs of achaete-scute complex (Mash1) (Ascl1 - Mouse Genome Informatics); hairy and enhancer of split1 (Hes1); and the expression of Notch/Notch-ligand system in the fetal and adult mouse lungs, and in the lungs of Mash1- or Hes1-deficient mice. Immunohistochemical studies revealed that Mash1-positive cells seemed to belong to pulmonary neuroendocrine cells (PNEC) and their precursors. In mice deficient for Mash1, no PNEC were detected. Hes1-positive cells belong to non-neuroendocrine cells. In the mice deficient in Hes1, in which Mash1 mRNA was upregulated, PNEC appeared precociously, and the number of PNEC was markedly increased. NeuroD (Neurod1 - Mouse Genome Informatics) expression in the lung was detected in the adult, and was enhanced in the fetal lungs of Hes1-null mice. Expression of Notch1, Notch2, Notch3 and Notch4 mRNAs in the mouse lung increased with age, and Notch1 mRNA was expressed in a Hes1-dependent manner. Notch1, Notch2 and Notch3 were immunohistochemically detected in non-neuroendocrine cells. Moreover, analyses of the lungs from the gene-targeted mice suggested that expression of Delta-like 1 (Dll1 - Mouse Genome Informatics) mRNA depends on Mash1. Thus, the neuroendocrine differentiation depends on basic helix-loop-helix factors, and Notch/Notch-ligand pathways may be involved in determining the cell differentiation fate in fetal airway epithelium.

Interaction of tobacco-specific toxicants with the neuronal α 7 nicotinic acetylcholine receptor and its associated mitogenic signal transduction pathway: potential role in lung carcinogenesis and pediatric lung disorders

Pulmonary neuroendocrine cells function as hypoxia-sensitive chemoreceptors, and they release peptides and biogenic amines that are important mediators of pulmonary neonatal adaptation. Some of these products additionally act as autocrine growth factors. Increased numbers of pulmonary neuroendocrine cells have been observed in several smoking-associated pediatric lung disorders such as bronchopulmonary dysplasia, cystic fibrosis, sudden infant death syndrome, and asthma. Disturbed pulmonary neuroendocrine function has been implicated in the etiology of this disease complex. One of the most common smoking-associated lung cancer types, small cell lung carcinoma, expresses phenotypic and functional features of pulmonary neuroendocrine cells. We, as well as others, have shown that the release of the autocrine growth factors 5-hydroxytryptamine (5-HT, serotonin) and mammalian bombesisn/gastrin releasing peptide (MB/GRP) by cell lines derived from human small cell lung carcinoma or fetal hamster pulmonary neuroendocrine cells are regulated by a neuronal nicotinic acetylcholine receptor comprised of α 7 subunits. In radio-receptor assays, nicotine and the nicotine-derived carcinogenic nitrosamines N′-nictrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) bound with high affinity to this receptor with affinitites NNK>nicotine>NNN. Binding of nicotine or NNK to the α 7 receptor resulted in calcium influx and overexpression and activation of the serine–threonine protein kinase Raf-1. In turn, this event lead to overexpression and activation of the mitogen activated (MAP) kinases extracellular signal regulated kinase 1 (ERK1) and extracellular signal regulated kinase 2 (ERK2) and stimulation of DNA synthesis accompanied by an increase in cell numbers in fetal pulmonary neuroendocrine cells and small cell carcinoma cells. Exposure of fetal pulmonary neuroendocrine cells for 6 days to NNK caused a prominant up-regulation of Raf-1. Our findings suggest that chronic exposure to nicotine and NNK in pregnant women who smoke may up-regulate the α 7 nicotinic receptor as well as components of its associated mitogenic signal transduction pathway, thus increasing the susceptibilities of the infants for the development of pediatric lung disorders. Similarly, up-regulation of one or several components of this nicotinic receptor pathway in smokers may be an important factor for the development of small cell lung carcinoma.

Alteration of Pulmonary Neuroendocrine Cells during Epithelial Repair of Naphthalene-Induced Airway Injury

Whole-mount airway preparations isolated from the lungs of mice treated by intraperitoneal injection of naphthalene and allowed to recover for 5 days were examined for the distribution and abundance of solitary pulmonary neuroendocrine cells (PNECs) and neuroepithelial bodies (NEBs) along the main axial pathway of the right middle lobe. Sham mice treated with corn oil vehicle were examined in a similar manner. An antibody to calcitonin gene-related peptide, a neuroendocrine cell marker, was used to identify the location, size, and number of PNECs and NEBs in the airways. After naphthalene treatment and epithelial repair, NEBs were significantly increased along the walls of the airways as well as on branch point ridges. The surface area covered by NEBs composed of 20 or fewer PNECs was significantly enlarged after naphthalene treatment compared with control NEBs of an equivalent cell number. The PNEC number per square millimeter was also increased more than threefold above control values after naphthalene treatment. These findings provide further support for a key role of neuroendocrine cells in the reparative process of airway epithelial cell renewal after injury.

INTERACTION OF TOBACCO-SPECIFIC TOXICANTS WITH NICOTINIC CHOLINERGIC REGULATION OF FETAL PULMONARY NEUROENDOCRINE CELLS: Implications for Pediatric Lung Disease

Pulmonary neuroendocrine cells (PNEC) produce neuropeptides and biogenic amines which regulate pulmonary vasoconstriction and bronchoconstriction. Increased numbers of PNEC along with elevated levels of their products have been consistently observed in a variety of pediatric pulmonary diseases, some of which are etiologically linked with prenatal exposure to cigarette smoke, and all of which are exacerbated by passive smoking. The objective of our studies was to characterize the autonomic regulation of these cells and to explore a potential direct interaction of tobacco-specific toxicants with these regulatory pathways. Using reverse transcription polymerase chain reaction (RT-PCR), radioreceptor assays, flow cytometry, enzyme-linked immunosorbent assay (ELISA), and cell proliferation assays, we found that the release of serotonin from fetal PNEC is regulated by a neuronalalpha7-nicotinic acetylcholine receptor (alpha7-nAChR) via a Ca2+-dependent mechanism. The tobacco-specific toxicants nicotine and 4-(methylnitrosamino)-1-(-3-pyridine)-1-butanone (NNK) bound with high affinity to this receptor, resulting in influx of Ca2+, release of serotonin, and stimulation of DNA synthesis. Chronic stimulation of the alpha7-nAChR in the fetal lungs by prenatal exposure to cigarette smoke may contribute to the development of smoking-related pediatric pulmonary disease, whereas postnatal chronic exposure to environmental smoke may exacerbate these pediatric diseases via direct interaction with this receptor.

Bombesin-like peptide mediates lung injury in a baboon model of bronchopulmonary dysplasia.

The etiology of bronchopulmonary dysplasia (BPD), a chronic lung disease of infants surviving respiratory distress syndrome, remains fundamentally enigmatic. BPD is decreasing in severity but continues to be a major problem in pediatric medicine, being especially prevalent among very premature infants. Increased numbers of pulmonary neuroendocrine cells containing bombesin-like peptide (BLP) have been reported to occur in human infants with BPD. We tested the hypothesis that BLP mediates BPD using the hyperoxic baboon model. Urine BLP levels increased soon after birth only in 100% O2-treated 140-d animals which developed BPD, correlating closely with severity of subsequent chronic lung disease. Similar elevations in urine BLP were observed in the 125-d baboon "interrupted gestation" model of BPD. Postnatal administration of anti-BLP antibody attenuated clinical and pathological evidence of chronic lung disease in the hyperoxic baboon model. Urine BLP could be a biological predictor of infants at risk for BPD, and blocking BLP postnatally could be useful for BPD prevention.

Number and proliferation of neuroendocrine cells in normal human airway epithelium.

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.

Increased pulmonary neuroendocrine cells with bombesin-like immunoreactivity in adult patients with eosinophilic granuloma.

Cigarette smoking is associated with hyperplasia of pulmonary neuroendocrine cells and variably increased levels of bombesin-like peptides in the lower respiratory tract. Because the neuropeptide bombesin is a chemoattractant for monocytes and a mitogen for 3T3 fibroblasts, we hypothesized that an excess of neuroendocrine cells and bombesin-like peptides could contribute to lung inflammation and fibrosis in certain cigarette smokers. Eosinophilic granuloma is a fibrotic lung disease of unknown etiology that in adults occurs almost invariably in cigarette smokers. We quantitated neuroendocrine cells with bombesin-like immunoreactivity in open lung biopsies from patients with eosinophilic granuloma (n = 6) and compared these with cigarette smokers (n = 6) who underwent lung resection for reasons other than primary lung disease. In addition, we compared them with patients with idiopathic pulmonary fibrosis (n = 8), a disease not associated with cigarette smoking. Finally, we also examined the mitogenic effect of bombesin on cultured human adult lung fibroblasts. The patients with eosinophilic granuloma exhibited a 10-fold increase in neuroendocrine cells with bombesin-like immunoreactivity compared to both smokers (P = 0.005) and patients with idiopathic pulmonary fibrosis (P = 0.005). In addition, bombesin produced a significant mitogenic effect on cultured human adult lung fibroblasts at concentrations of 1 nM and above. We conclude that increased numbers of pulmonary neuroendocrine cells with bombesin-like immunoreactivity are commonly found in patients with eosinophilic granuloma and, since bombesin-like peptides are chemotactic for monocytes and mitogenic for human lung fibroblasts, we speculate that neuroendocrine cell hyperplasia may be important in the pathogenesis of eosinophilic granuloma in adult cigarette smokers.

Abnormal Patterns of Pulmonary Neuroendocrine Cells in Victims of Sudden Infant Death Syndrome

Ventilatory dysfunction has become the main focus of current research in sudden infant death syndrome (SIDS). This has been correlated with structural abnormalities in the carotid body and respiratory nuclei of the brainstem. In recent studies, the denervating effect of asphyxial brainstem dysfunction on the pulmonary neuroendocrine cells, which probably function as chemoreceptors, was demonstrated and prompted the following study. The pulmonary neuroendocrine system was evaluated in 25 victims of SIDS and 20 control infants, ranging in age from 3 weeks to 7 months and 1 to 12 months, respectively. The pulmonary neuroendocrine cells were stained by the Churukian-Schenk method and the neuroendocrine cell-positive airway values expressed as a percentage of the total number of airways. The range of positive airway values for victims of SIDS was 2% to 97% with a median of 73%. In contrast, the range for the control infants was 1% to 44% with a median of 25.5%. The SIDS victims' percentage was significantly greater than the control infants' percentage (P less than .0001). The number of pulmonary neuroendocrine cells in positive airway was also increased among SIDS victims compared with control infants. The altered pulmonary neuroendocrine cell pattern could be attributable to either brainstem dysfunction or chronic hypoxia. These explanations are not, however, mutually exclusive of one another; in fact, it is possible that both mechanisms may be operative.

Stimulation of hamster pulmonary neuroendocrine cells and associated peptides by repeated exposure to cigarette smoke.

Although the normal function of pulmonary neuroendocrine (PNE) cells containing bioactive peptides is poorly understood, various pulmonary diseases are associated with hyperplasia of these cells, and they also may be progenitors for small cell lung cancer in humans. In this study we have investigated the effects of subchronic cigarette smoke exposure in the hamster on the PNE cells and their peptide content. Daily exposure to standard research cigarette smoke for as long as 90 days led to progressively higher levels of serum calcitonin (iCT) as well as higher lung tissue iCT and the gastrin releasing-like peptides or mammalian bombesin (MB). Subsequent to a 30-day period during which there was no further exposure to smoke, serum levels returned to control levels, but the lung levels of both iCT and MB remained higher than control levels. Also, after the 90 days of exposure, immunocytochemistry revealed an increase in the number of iCT-containing PNE cells. This increase in the number of PNE cells correlated well with the increased iCT content of the lung tissue. We conclude that subchronic cigarette smoke exposure causes an increase in pulmonary levels of iCT and MB, which may be linked to the observed proliferative response of the PNE cells.