Fluorescent human lung macrophages analyzed by spectral confocal laser scanning microscopy and multispectral cytometry

Abstract

Numerous highly fluorescent macrophages (MU), designated ''smoker cells,'' exist in the lungs of smokers and subjects who have quit smoking within 5 years. The brightly fluorescent MU, however, are not present in the lungs of never smokers. Some investigators have speculated that the intense fluorescence of the MU is due to smoke-induced changes in the autofluorescence of naturally occurring (i.e., endogenous) compounds (e.g., NADP). In contrast, other researchers have theorized that the fluorescence is due to the uptake of tobacco smoke particulates (i.e., ''tar''). Studies reported herein were undertaken to test the hypothesis that the origin of the MU fluorescence could be profiled with the novel technologies afforded by spectral confocal laser scanning microscopy (sCLSM) and multispectral cytometry (MSC). To this end, spectral emissions were obtained by sCLSM of optical sections of live MU isolated from fresh surgically excised human lung tissue and in air-dried lung tissue imprints. Confirmation of spectral profiles of these single cell observations was obtained in population studies with the use of high-throughput MSC in which multispectral analyses were performed with three different lasers. Proof of concept experiments demonstrated that relatively nonfluorescent MU from the lungs of nonsmokers became fluorescent upon short-term ex vivo exposure to tobacco smoke tar. Summarily, the studies reported herein document that the fluorescence of human lung MU is due to tobacco tar. Microsc. Res. Tech. 67:79-89, 2005. V V C 2005 Wiley-Liss, Inc.

Humanin vivofluorescence microimaging of the alveolar ducts and sacs during bronchoscopy

“…The alveolar entrances and alveolar ducts of smokers were usually filled with highly fluorescent cells, the movements of which could be clearly observed (see online video). Bronchoalveolar lavage analysis from smokers using fluorescence and conventional microscopy confirmed that these fluorescent cells correspond exclusively to alveolar macrophages [15].…”

Increased Human Buccal Cell Autofluorescence Is a Candidate Biomarker of Tobacco Smoking

“…Studies of laser-generated optical sections have shown that the greatest contributors are polycyclic aromatic hydrocarbons (see Discussion in ref. 17).…”

Inflammatory response of lung macrophages and epithelial cells to tobacco smoke: a literature review of ex vivo investigations

“…Notable is that the lung of a long-term smoker contains a large and unique population of ''pigmented'' MU that is readily seen in conventional white-light examinations of stained wax block sections [27,28]. Also, pigmented MU display a high level of fluorescence that is associated with the ingestion of tobacco smoke particulates (''tar'') [27][28][29]. The florescent MU, designated as ''smoker cells'' are not present in the lungs of non-smokers, including subjects who have been exposed to high levels of smoke and diverse airborne particulates in different environmental and occupational settings [27,28].…”

“…One of the primary clinically defined events of smoke-associated inflammation is the influx of macrophages (MU) [2][3][4][5][6][7][8]10]. Notable is that the lung of a long-term smoker contains a large and unique population of ''pigmented'' MU that is readily seen in conventional white-light examinations of stained wax block sections [27,28]. Also, pigmented MU display a high level of fluorescence that is associated with the ingestion of tobacco smoke particulates (''tar'') [27][28][29].…”