Abstract
BackgroundNeutrophils leave the bone marrow as terminally differentiated cells, yet little is known of the influence of nicotine or other tobacco smoke components on neutrophil differentiation. Therefore, promyelocytic HL-60 cells were differentiated into neutrophils using dimethylsulfoxide in the presence and absence of nicotine (3-(1-methyl-2-pyrrolidinyl) pyridine). Differentiation was evaluated over 5 days by monitoring terminal differentiation markers (CD11b expression and formazan deposition); cell viability, growth phase, kinetics, and apoptosis; assessing cellular morphology and ultrastructure; and conformational changes to major cellular components. Key neutrophil effector functions (oxidative burst, bacterial killing, matrix metalloproteinase release) were also examined.ResultsNicotine increased the percentage of cells in late differentiation phases (metamyelocytes, banded neutrophils and segmented neutrophils) compared to DMSO alone (p < 0.05), but did not affect any other marker of neutrophil differentiation examined. However, nicotine exposure during differentiation suppressed the oxidative burst in HL-60 cells (p < 0.001); inhibited bacterial killing (p < 0.01); and increased the LPS-induced release of MMP-9, but not MMP-2 (p < 0.05). These phenomena may be α-7-acetylcholine nicotinic receptor-dependent. Furthermore, smokers exhibited an increased MMP-9 burden compared to non-smokers in vivo (p < 0.05).ConclusionThese findings may partially explain the known increase in susceptibility to bacterial infection and neutrophil-associated destructive inflammatory diseases in individuals chronically exposed to nicotine.
Highlights
Neutrophils leave the bone marrow as terminally differentiated cells, yet little is known of the influence of nicotine or other tobacco smoke components on neutrophil differentiation
As our initial data showed that HL-60 cells express nicotinic receptors, because nicotine is a major component of tobacco smoke that is distributed throughout the body, and because the study of individual tobacco components allows a more efficient targeting of specific pathogenic mechanisms, we determined to examine the influence of physiologically relevant nicotine concentrations (10-7 to 10-4 M) on HL60 promyelocytes that were induced to differentiate into the neutrophil lineage with dimethylsulfoxide (DMSO). We report in this manuscript that while nicotine does not influence HL-60 proliferation, viability, cell cycle, ultrastructural characteristics and the expression of terminal differentiation markers, key effector functions, including superoxide generation, bacterial killing and Matrix metalloproteinase (MMP)-9 release, are dysregulated
We show that the oxidative burst in response to phorbol 12-myristate 13-acetate (PMA) is quantitatively diminished in HL-60 cells differentiated under the influence of nicotine (Figure 5), which is reflected in an impaired ability to kill the Gram-negative periodontal pathogen, P. gingivalis (Figure 6)
Summary
Neutrophils leave the bone marrow as terminally differentiated cells, yet little is known of the influence of nicotine or other tobacco smoke components on neutrophil differentiation. The World Health Organization recently estimated that there are 1.1 billion current smokers [1]. As this is equivalent to one third of the world's entire population greater than 15 years of age, it is difficult to over-estimate the enormity of this health problem. Neutrophils are key cells in combating microbial invasion, and smokers exhibit an increased risk of infectious diseases [7] including invasive pneumococcal disease [8], tuberculosis [7], meningitis, as well as exhibiting increased risk of infection with Helicobacter pylori, Porphyromonas gingivalis, Legionella pneumophila and other bacterial species [6,7]
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