Tobacco smoke is known to exert profound effects on neutrophils. For example, cigarette smoke and nicotine have been reported to suppress the respiratory burst17, 18
and the ability to kill pathogenic bacteria,17
while reports are conflicted on the influence of smoking on neutrophil chemotaxis.19
What is clear, however, is that neutrophil accumulation occurs in the lungs of smokers and that neutrophil numbers correlate with destructive pulmonary disease progression.4
The manner of cell death is a critical element in determining inflammatory intensity. However, the available evidence for an effect of tobacco smoke on neutrophil longevity is controversial. Some investigators have reported that nicotine can suppress neutrophil apoptosis; others have concluded that high nicotine doses did not influence apoptosis in freshly isolated peripheral neutrophils; while others have suggested showing nicotine to induce DNA cleavage in myeloid cells and to be a potent inducer of apoptosis in systemic neutrophils.19
An accurate composite picture of the influence of cigarette smoke and neutrophil longevity is difficult to generate because of a wide variation in experimental models and in methods of generating tobacco smoke preparations. In this study, therefore, we have examined multiple aspects of cell death in, and efferocytosis of, CSE-exposed neutrophils, all in a single experimental model employing primary human neutrophils, primary hMDMs and a readily reproducible CSE.
One recent report shows that total leukocytes isolated from smokers exhibit a significant suppression of multiple pro-apoptotic genes (C1D
), while activity of the anti-apoptotic gene, CTMP
, is induced compared with leukocytes from non-smokers.20
Herein, we show that CSE induces an atypical type of cell death in neutrophils that includes features of apoptosis (PI staining and PS relocalization; non-phlogistic response of hMDMs to neutrophil phagocytosis), autophagy (ultrastructural characteristics) and necrosis (leakage of granule and cytoplasmic components; persistence of CD31 and CD47 expression). Furthermore, such CSE-exposed neutrophils were effectively cleared by professional phagocytes, indicating the induction of cell surface molecular patterns identifying them as damaged and/or dying cells.
In light of these results, it is evident that the treatment of neutrophils with CSE triggered an atypical cell death pathway where some early features of apoptosis appeared but were not followed by executive phase. Even the spontaneous apoptotic program was dramatically inhibited. These data are in keeping with our previous study in which myeloid (HL-60) cells were differentiated into neutrophil-like cells in the presence of nicotine. Molecular signals consistent with early apoptosis were apparent, yet committed apoptosis did not occur.17
Our data are also in keeping with several clinical studies that have shown that apoptotic neutrophils represent only a small percentage of the total pulmonary population in several tobacco-exacerbated diseases, including acute respiratory distress syndrome (7.4 × 105
neutrophils/ml; 1.6% apoptotic cells)21
and pneumonia (1.1 × 106
neutrophils/ml; 0.3% apoptotic cells),22
while others have reported a dramatically reduced percentage of neutrophils undergoing spontaneous apoptosis in healthy smokers and subjects with COPD compared with non-smokers.10
Furthermore, recent studies have reported increased autophagy in mouse lungs subjected to chronic cigarette smoke exposure, and in pulmonary epithelial cells exposed to CSE,23
and may be induced by oxidative stress and mediated by sirtuin-1 (SIRT1).24
The gene products of bax-α and bak1, members of the bcl2 family are known to interact with mitochondria and promote apoptosis. Although present in significant amounts in control cells, bax-α and bak1 mRNAs were not detectable following 15-min incubation, indicating a suppression of intrinsic apoptosis in CSE-exposed neutrophils. Conversely, cells responded to CSE treatment with the sharp increase in expression of bcl-xl, the protein product of which prevents cytochrome C release within mitochondria and is thus anti-apoptotic. Thus, transcriptional profiling of key pro- and anti-apoptotic genes in CSE-exposed neutrophils reflects the suppression of the executive phase of apoptosis noted in biochemical assays and electron microscopy.
To estimate the response of general cytoprotective mechanisms to CSE, we analyzed the expression of major representatives of three families of heat shock proteins genes (hsp27
). Heat shock proteins are highly conserved often multi-functional proteins best characterized as molecular chaperones, which protect the native conformation of other proteins, guarding against structural inactivation and irreversible multimeric aggregation. Thus, induction of heat shock proteins in response to stress serves to help protect the cell against the initial insult and to augment recovery. Elevated heat shock protein expression can also provide tolerance to a wide array of stressors and resistance to apoptosis.25
In the case of hsp70
, in which mRNA was absent in control cells, we observed immediate and extensive expression throughout the CSE treatment. HSP 70 is anti-apoptotic and inhibits both caspase-dependent and -independent apoptotic pathways.26
Expression of hsp90α
increased their expression moderately with apparent maximum at 60
min. HSP27 is a small heat shock protein that suppresses cell death signaling in neutrophils.27
HSP90 has been shown to localize to mitochondria in tumor cells, with HSP90 inhibition leading to the induction selective apoptosis.28
Thus, the rapid CSE induction of hsp90α
transcripts is in keeping with suppression of overt apoptosis in smoke exposed neutrophils.
Although relocation of phosphatidylserine (PS) from the inner to the outer cell surface is a universal early marker of apoptotic cells and a prerequisite for their successful engulfment by phagocytes, several other cellular ‘eat me' signals are recognized, including altered carbohydrates, oxidized lipids, binding sites for thrombospondin and collectins (e.g., mannose-binding lectin, surfactant protein A and C1q), as well as calreticulin, an endoplasmic reticulum chaperone.29
These markers are recognized by an equally large variety of receptors on phagocytes, including CD14, CD68, CD91 (LRP), SR-A, ATP-binding cassette transporter 1 (ABC-1), complement receptors, integrin αvβ5
These receptors mediate the clearance of endogenous cells directly or indirectly via PS-binding proteins.5
Herein, through the use of oxLDL and blocking antibodies to selected scavenger receptors, we show that LOX1 and SR-A scavenger receptors were largely responsible for the recognition and engulfment of CSE-exposed neutrophils by hMDMs.
Interestingly, the addition of β-carotene or quercetin to CSE resulted in increased PI-staining as well as engulfment of the treated cells. In the case of β-carotene, this may be explained by the pro-oxidant properties of this lipid. However, no pro-oxidant activity has been reported for quercetin so far. The oxLDL blocking and pro-oxidant effect of β-carotene, taken together, strongly suggest that CSE-induced neutrophil surface ox-LDL-like sites thus providing ‘eat me signal' for macrophages.
Furthermore, it has been previously established that the type of cell death of ingested host cells can dictate the inflammatory response of professional phagocytes on engulfment. Macrophage ingestion of apoptotic cells has been shown to be non-phlogistic, while necrotic and autophagic cells generally seem to induce a pro-inflammatory reaction.30
As we show that CSE-exposed neutrophils exhibit atypical cell death patterns that include features of necrosis, autophagy and apoptosis, we decided to establish whether or not neutrophil phagocytosis elicited a pro-inflammatory response in hMDMs. CSE-treated neutrophils did not stimulate macrophages to produce significant amounts of TNF when compared with necrotic neutrophils and less, even, than measured for fresh neutrophils. This was perhaps surprising because, following longer treatment with CSE, a majority of neutrophils were PI-positive. The distinctive autophagosomes found in most of CSE-treated neutrophils also suggested a potential for a phlogistic response in hMDMs to phagocytosis.30
However, while phagocytosis of CSE-exposed primary human neutrophils was efficient and did not elicit inflammation, CSE-exposed neutrophils did exhibit degranulation. At lower doses of CSE, cytochalasin D-inhibited degranulation was specific to secondary granules, using lactoferrin release as a surrogate marker. Other secondary granule components presumably released alongside lactoferrin include, neutrophil collagenase (MMP-8), myeloperoxidase, NADPH oxidase, alkaline phosphatase and the anti-microbial molecules, lactoferrin, LL-37 and lysozyme. Increased MMP-8 has been associated with COPD.31
Thus, the tobacco-induced release of neutrophil secondary granule components could add to the pro-inflammatory and/or degradative burden and contribute to COPD progression and the exacerbation of other chronic, tobacco-induced inflammatory diseases.
We have previously reported that nicotine exposure inhibits the respiratory burst in myeloid cells, concomitant with a reduced capacity to clear periodontopathogenic bacteria, and that these phenomena are likely to be dependent on the α
7 acetylcholine receptor and linked to the cholinergic anti-inflammatory pathway.17
Here, we show that CSE-inhibits neutrophil uptake of another pathogen, S. aureus
. Although S. aureus
is a rare pathogen in COPD exacerbations, some strains are the exclusive cause of necrotizing pneumonia, an infectious disease with high mortality rate.32
Significantly, pneumonia is much more frequent in smokers than non-smokers.1
The observed dramatic inhibition of phagocytosis fits well into the overall picture of chronic and recurrent bacterial infections in COPD patients.
It should be noted that, compared with other similar studies, for example, Stringer et al.12
, high CSE concentrations (20–100% CSE) were employed herein. However, the high CSE concentrations were counterbalanced by short periods of neutrophil treatment, generally 30-min incubations. We hypothesize that such an approach reflects the peak ‘window of contact' between cigarette smoke and pulmonary neutrophils, that is, before the instable, reactive intermediates in smoke are diffused and/or metabolized to less active or inert molecules.
In summary, the highly effective LOX1 and SR-A-driven, non-phlogistic phagocytosis by hMDMs of CSE-exposed neutrophils that die by unconventional mechanisms, would be expected to have a major role in limiting pulmonary inflammation in smokers. In contrast, degranulation of secondary granules and – at long exposure times, leakage of primary, secondary and cytoplasmic contents – would be expected to enhance inflammation and tissue destruction. Furthermore, CSE-induced suppression of bacterial uptake by neutrophils is consistent with bacterial persistence in the lungs of smokers and is likely to promote further pulmonary recruitment of neutrophils responding to bacterial-derived chemotactic stimuli. Our finding of a highly complex, atypical cell death pattern in CSE-exposed neutrophils, which includes features of cellular stress, apoptosis, necrosis and autophagy, may help explain the conflicting literature in this area. Certainly, the consequences of neutrophil exposure to CSE are multifarious and a deeper understanding of their role in smoking-induced inflammatory pulmonary diseases, such as COPD, is required in order to develop improved preventative measures and therapeutic strategies.