Prolonged Neutrophil Survival Research Articles

A role for apoptosis in the control of neutrophil homeostasis in the circulation: insights from CD18-deficient mice

The control of neutrophil turnover in the circulation is a key event in homeostasis and inflammation. Using CD18- deficient (CD18(-/-)) mice that show a 19-fold increase of blood neutrophil counts when compared with wild-type animals (CD18(+/+)), we found that apoptosis of peripheral neutrophils was significantly reduced from 27.4% in the wild-type to 4.8% in CD18(-/-) mice within 4 hours after isolation as measured by analysis of DNA content. This was confirmed by detecting CD16 expression, nuclear morphology, and internucleosomal DNA degradation. In contrast, no difference in apoptosis was observed in neutrophils derived from the bone marrow. Neutrophilia and delayed neutrophil apoptosis were also present in CD18(-/-)/interleukin 6 (IL-6(-/-)) double knockout mice. Moreover, plasma of CD18(-/-) mice was not able to delay apoptosis of CD18(+/+) neutrophils and plasma of CD18(+/+) mice did not augment apoptosis of CD18(-/-) neutrophils. However, CD18(-/-) neutrophils revealed an up-regulation of the antiapoptotic gene bcl-X(l) and a down-regulation of the proapoptotic gene bax-alpha compared with CD18(+/+) neutrophils suggesting that this delayed apoptosis. Accordingly, down-regulation of Bax-alpha using antisense technique delayed apoptosis and prolonged neutrophil survival. The replacement of the hematopoietic system of CD18(+/+) mice by a 1:1 mixture of CD18(+/+) and CD18(-/-) hematopoietic cells abolished the delay of apoptosis in peripheral CD18(-/-) neutrophils and prevented neutrophilia. Altogether, this suggests that a delay of neutrophil apoptosis in CD18(-/-) mice causes an alteration of neutrophil homeostasis, which may induce the massive increase of peripheral neutrophil counts. Thus, apoptosis seems to be critically involved in the control of neutrophil turnover in the circulation.

Decreased Neutrophil Apoptosis in Patients with Sepsis is Related to the Activation of NF-κB

Background : Neutrophil-mediated inflammation is usually self-limiting, because neutrophils have a remarkably short life span. Prolonged neutrophil survival, which is caused by decreased spontaneous apoptosis, leads to persistent inflammation in sepsis. Because many inflammatory cytokines, which generate signals that delay apoptosis, are regulated by nuclear factor- κB transcription factor, we hypothesized that nuclear factor-κB might be related to the reduced neutrophil apoptosis observed in sepsis. Methods : Neutrophils of healthy volunteers and sepsis patients were freshly isolated from venous blood. Neutrophil apoptosis was assayed with two approaches : by counting apoptotic cells under a microscope and by flow cytometry using Annexin V. The activity of nuclear factor-κB was assessed by immunofluorescent staining or electrophoretic mobility shift assay. Expression of X-linked inhibitor of apoptosis was measured by western blot assay. Results : We confirmed reduced spontaneous neutrophil apoptosis in patients with sepsis. The number of apoptotic neutrophils in patients with sepsis increased to the level of that in healthy controls after

Neutrophil survival is prolonged in the airways of healthy infants and infants with RSV bronchiolitis.

Large numbers of neutrophils in the airway of infants infected by respiratory syncytial virus (RSV) are recruited by chemokines, such as interleukin-8, and specific inflammatory molecules can delay apoptosis increasing their longevity. The aim of this study was to investigate whether airway secretions in RSV bronchiolitis contain factors that influence neutrophil apoptosis. Nasal lavage fluid (NLF) was obtained from 24 infants with RSV bronchiolitis (31 infant controls and 12 adults). Neutrophils isolated from healthy adult volunteers were incubated with the NLF in Dulbecco modified Eagle medium (DMEM) for 24 h, and apoptosis and necrosis were quantified using Hoechst 33342 and propidium iodide viability dyes. The presence of putative factors that delay neutrophil apoptosis was investigated using inhibitors to leukotriene-B4, lipopolysaccharide and the IL-8 receptor CXCR2, and blocking antibodies to granulocyte-monocyte colony-stimulating factor. Characterisation of NLF involved tests of thermal instability, proteolysis, deoxyribonuclease digestion and molecular filtration. NLF from infants with RSV bronchiolitis and controls significantly delayed neutrophil apoptosis, whereas NLF from healthy adults did not. None of these inhibitor molecules blocked this delay in apoptosis but activity was heat liable and >3 kDa. The study showed that nasal lavage fluid from infants significantly delays neutrophil apoptosis. The speculation is that the prolonged survival of neutrophils in the infant airway contributes to the characteristic accumulation of neutrophils in the airways of infants with respiratory infections.

Modulation of Leukotriene B4 Receptor-1 Expression by Dexamethasone: Potential Mechanism for Enhanced Neutrophil Survival

Glucocorticoids can down-regulate many inflammatory and immune responses and constitute a powerful therapeutic tool in a number of diseases. However, they have a somewhat paradoxical effect on neutrophils, in that they prolong their survival. Because leukotriene B(4) (LTB(4)) can also extend neutrophil survival, we proposed that glucocorticoids could prevent neutrophil apoptosis by up-regulating their expression of the high-affinity LTB(4) receptor (BLT1). Here we show that, indeed, dexamethasone (DEX) up-regulates the steady-state levels of BLT1 mRNA in human neutrophils. The effect was time and concentration dependent, being maximal at 4 h and at 10-100 nM DEX. The effect was also dependent on transcriptional activity, whereas BLT1 mRNA stability was not affected. DEX-induced up-regulation of BLT1 expression was prevented by pretreatment with the LTB(4) antagonist LY255283. Moreover, LTB(4) itself up-regulated the expression of BLT1 mRNA. BLT1 protein expression on neutrophils exposed to DEX for 24 h was also up-regulated 2- to 3-fold, and DEX-treated as well as LTB(4)-treated cells showed enhanced responsiveness to LTB(4) in terms of intracellular Ca(2+) mobilization and chemotaxis. Whereas DEX and LTB(4) alone decreased neutrophil apoptosis by approximately 50%, neutrophils treated with both LTB(4) and DEX showed >90% survival at 24 h. Moreover, BLT1 antagonists prevented the increased neutrophil survival induced by DEX as well as by LTB(4). Taken together, our results suggest that DEX-induced up-regulation of BLT1 expression in neutrophils may be one mechanism through which glucocorticoids can prolong neutrophil survival, namely by enhancing cell responses to the antiapoptotic effect of LTB(4).

Acute endotoxemia prolongs the survival of rat lung neutrophils in response to 12-O-tetradecanoyl-phorbol 13-acetate.

Acute endotoxemia is associated with prolonged survival of adherent neutrophils in the lung vasculature. In the present studies, the effects of inflammatory mediators on signaling pathways regulating neutrophil survival were examined. We found that the protein kinase C activator, 12-O-tetradecanoyl-phorbol 13-acetate (TPA), but not interferon-gamma (IFN-gamma), prolonged the survival of adherent vasculature lung neutrophils from endotoxemic rats, a response that was correlated with reduced apoptosis. Although endotoxin administration to rats induced the expression of the anti-apoptotic protein Mcl-1 in lung neutrophils, TPA had no effect on this response. Endotoxin administration also induced expression of total p38 and p44/42 mitogen activated protein kinases (MAPK) in neutrophils, as well as phosphatidyl inositol 3 kinase (PI3K) and its downstream target protein kinase B (PKB). Treatment of the cells with TPA increased p38 MAPK expression in cells from both control and endotoxin treated animals. Cells from endotoxin treated, but not control animals, were found to exhibit constitutive binding activity of nuclear factor kappa B (NF-kappaB) which was blocked by TPA. In contrast, constitutive CCAAT/enhancer binding protein (C/EBP) nuclear binding activity evident in neutrophils from control animals was reduced following endotoxin administration. Moreover, this response was independent of TPA. These data suggest that NF-kappaB plays a role in TPA-induced signaling leading to prolonged survival of adherent vascular neutrophils in the lung during acute endotoxemia.

Inhibition of p38 mitogen activated protein kinase increases lipopolysaccharide induced inhibition of apoptosis in neutrophils by activating extracellular signal-regulated kinase

Background. Prolonged polymorphonuclear neutrophil (PMN) survival has been implicated in tissue injury after sepsis. Previously we reported that lipopolysaccharide (LPS) inhibits PMN apoptosis via the activation of the extracellular signal-regulated kinase (ERK). Conversely, the p38 mitogen activated protein kinase (MAPK) pathway is involved in the spontaneous apoptosis of PMNs. The interaction between these 2 pathways and their ability to regulate apoptosis during sepsis remain largely undefined. We hypothesize that there is interaction between the ERK and p38 pathways during sepsis. Methods. PMNs were isolated from healthy volunteers by Ficoll gradient centrifugation and red blood cell sedimentation. Cells were then pretreated for 1 hour with the ERK inhibitor (PD98059, 10 μmol/L), p38 inhibitor (SB203580, 1 μmol/L), or vehicle. After treatment with LPS, apoptosis and MAPK activity were correlated. Results. LPS stimulation significantly inhibits PMN apoptosis compared with unstimulated cells. Furthermore, inhibition of ERK significantly abrogates this effect, whereas inhibition of p38 augments LPS induced inhibition of apoptosis. Elk-1 phosphorylation (ERK target) is significantly increased by LPS alone and by inhibition of the p38 pathway during LPS stimulation. This correlates with ERK phosphorylation by Western blot. Conclusions. These data show that p38 inhibition enhances ERK activity during endotoxemia. Furthermore, these data suggest that cooperation between ERK and p38 MAPK pathways dictates the apoptotic potential of PMNs during inflammatory states. (Surgery 2001;130:242-8.)

Heat Shock Protein 27 Inhibits Apoptosis in Human Neutrophils

Background. Prolonged neutrophil(PMN) survival has been implicated in tissue injury following sepsis. A variety of bacterial products have been identified which inhibit PMN apoptosis including lipopolysaccharide(LPS). Extracellular heat shock proteins(Hsp) have recently been identified as potent regulatory signals for the innate immune system during the inflammatory response. We hypothesized that Hsp 27 can affect PMN phenotype with respect to apoptosis and cytokine profile.Materials and methods. PMN were isolated from the peripheral blood of healthy human volunteers by red blood cell sedimentation and gradient centrifugation. Cells were placed in media and cultured for 18 h with and without recombinant human Hsp 27 at various concentrations. In parallel experiments, PMN were stimulated with LPS, a known inhibitor of PMN apoptosis, for comparison. Apoptosis was quantified using annexin V and propidium iodide staining with flow cytometric analysis. Culture supernatants were assayed for secretion of TNF-α, IL-10, and IL-12.Results. Hsp 27 significantly inhibits PMN apoptosis [control; 81.8 ± 3.6%, vs Hsp 27, 60.4 ± 4.1% p < 0.05]. The reduction is similar to that signaled by LPS, alone. Together their effect is not synergistic. The Hsp 27 response is dose-dependent. Hsp 27 does not induce secretion of TNF-α, IL-10, or IL-12, whereas LPS does signal IL-12 and TNF-α secretion.Conclusion. These data demonstrate that exogenous Hsp 27 may play a role in neutrophil-mediated tissue injury during trauma and sepsis via its ability to inhibit neutrophil apoptosis. However, Hsp 27 does not significantly alter neutrophil phenotype with respect to cytokine production profile.

Eosinophil apoptosis--pathophysiologic and therapeutic implications.

Eosinophil apoptosis--pathophysiologic and therapeutic implications.

Effect of auranofin, an antirheumatic drug, on neutrophil apoptosis.

The effects of auranofin (AF) on apoptosis and on the biological functions of neutrophils were investigated. Neutrophils were incubated with various concentrations of AF for different periods. Cell viability was determined by the MTS assay and apoptosis was evaluated by flow cytometric analysis of propidium iodide (PI)- staining of the nuclei and annexin-V staining of phosphatidylserine in the cell membrane. The effect of AF on the expression of adhesion molecules (CD62L and CD11b/CD18) and on the generation of O2- by neutrophils was also determined. At a low concentration (1 microM), AF significantly prolonged neutrophil survival by delaying spontaneous apoptosis. Neutrophils incubated with AF for 12 and 24 hours maintained the capacity to express adhesion molecules and generate O2-. In contrast, a higher AF concentration (5 microM) shortened neutrophil survival by the induction of cell necrosis. Although the biological significance of inhibitory effect of AF on neutrophil apoptosis remains unclear, it seems to be unlikely that AF exerts the anti-inflammatory effect in vivo by directly suppressing neutrophil functions. Since AF has a wide range of effects on leukocytes, its therapeutic benefit in rheumatoid arthritis may be mediated in a complex manner.

Age-related effects of oxidative metabolism and cyclic AMP signaling on neutrophil apoptosis

Spontaneous as well as Fas-induced polymorphonuclear cell apoptosis is unchanged in the elderly. However, a weak responsiveness to antiapoptotic signals elicited by proinflammatory molecules has been reported in neutrophils isolated from aged humans. To gain insight into this field, here we have evaluated the role of oxidative metabolism and cyclic AMP (cAMP) signaling on age-related neutrophil apoptotic cell death. Results show that although superoxide dismutase (SOD), added exogenously to cell cultures, is able to prolong neutrophil survival in both young and aged individuals, high amounts of the enzyme are further effective in cell cultures of young donors only. Notably, the addition of catalase gives rise to a more striking, yet comparable, inhibition of neutrophil-programmed cell death in both groups of subjects. Furthermore, even low amounts of catalase are enough to restore a normal apoptotic outcome in SOD-treated cell cultures of old donors. Unlike the oxidative metabolism, cAMP signaling activation does not reveal any difference in the apoptotic response of neutrophils isolated from young and aged donors. Thus, supplementation of cell cultures with prostaglandin E2, dibutyryl cAMP or, to a lesser degree, forskolin results in a dose-dependent inhibition of DNA cleavage product appearance in both groups of subjects. The data outline that an impairment of neutrophil antioxidant shield, leading to an augmented cell oxidative load, is likely to occur as a feature of age. This may increase the apoptotic rate of stimulated cells, which may in turn account for the increased susceptibility of elderly individuals to life-threatening infections.

Multiple epithelial cell-derived factors enhance neutrophil survival. Regulation by glucocorticoids and tumor necrosis factor-alpha.

We examined the potential of several epithelial-derived factors to enhance neutrophil activation and survival. Neutrophils incubated in the presence of supernatants from nasal-derived primary epithelial cultures had significantly increased survival compared with neutrophils cultured in media alone. Of the cytokines reported to enhance neutrophil survival, transcripts for interleukin (IL)-1alpha, IL-1beta, IL-6, and granulocyte macrophage colony-stimulating factor (GM-CSF) (but not interferon-gamma or granulocyte colony-stimulating factor [G-CSF]) were detected by ribonuclease protection assay in basal and tumor necrosis factor (TNF)-alpha- stimulated epithelial cells. Of the eicosanoid products that enhance neutrophil survival, platelet-activating factor and leukotriene B(4) were not detected in the supernatants, whereas prostaglandin E(2) (PGE(2)) was produced in modest amounts. The levels of IL-6, GM-CSF, and PGE(2) in epithelial supernatants were significantly increased after transient TNF-alpha stimulation. This induction was suppressed if dexamethasone (Dex) was added during TNF-alpha stimulation. Only IL-6, GM-CSF, and PGE(2) promoted neutrophil survival over the range of concentrations detected in the supernatants, and a combination of neutralizing antibodies to GM-CSF and IL-6 completely inhibited the enhanced neutrophil survival in epithelial supernatants. Both the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique and morphologic scoring of apoptotic neutrophils confirmed that epithelial supernatants, as well as purified IL-6, GM-CSF, and PGE(2) all delayed neutrophil apoptosis. Finally, the effects of Dex on neutrophil survival and on epithelial cytokine production were investigated. Dex independently prolonged neutrophil survival but suppressed epithelial production of survival-enhancing factors in a dose-dependent manner. The net effect of Dex appeared to favor neutrophil survival.

Effects of glucocorticoids on apoptosis of infiltrated eosinophils and neutrophils in rats

The effects of glucocorticoids on the survival of rat eosinophils and neutrophils infiltrated into the peritoneal cavity were examined. Glucocorticoids including dexamethasone, prednisolone and hydrocortisone inhibited the survival of rat peritoneal eosinophils at 10 −6 M, whereas they prolonged survival of rat peritoneal neutrophils at 10 −8 M. Sex steroids including estradiol and progesterone did not affect cell survival. Dexamethasone decreased the viability of eosinophils after 3 days of incubation and maintained the viability of neutrophils until 4 days after incubation concentration dependently. The EC 50 of dexamethasone for inhibition of the survival of eosinophils was 1.5×10 −8 M, and that for the spontaneous death of neutrophils was 6.4×10 −10 M, suggesting that glucocorticoids at concentrations that inhibit eosinophil survival prolong neutrophil survival. Analysis of DNA fragmentation of cultured eosinophils and neutrophils revealed that glucocorticoids enhance eosinophil apoptosis but inhibit neutrophil apoptosis. The effects of dexamethasone on viability and DNA fragmentation were counteracted by the glucocorticoid receptor antagonist, mifepristone, concentration dependently. These findings indicate that glucocorticoids induce contradictory effects via the glucocorticoid receptor on rat eosinophils and neutrophils extravasated to an inflammatory locus such as the peritoneal cavity by modulating apoptosis.

Not with a Bang But a Whimper: Apoptosis in the Pathogenesis of Systemic Inflammation

Life, by its very de~nition, is the controlled growth and differentiation of embryonic stem cells. The fusion of sperm and ovum creates a unicellular entity that has the potential, through cell division and differentiation, to become an almost incomprehensibly complex collection of in excess of one trillion cells, interacting harmoniously as a unique individual. The precision and predictability of the process, occurring within a limited time span of days to years, is a source of in~nite curiosity and wonder for the biological scientist. The richness and passion that accompanies this process of biologic multiplication, provides the poet with an eternal source of inspiration. But it has only recently been appreciated that the controlled elimination of cells is as important to normal development as their initial growth. Development in utero is accompanied by biologic sculpting that, for example, transforms a _ipper into a hand with delicate ~ngers. Embryologic structures, necessary intermediates in the process of organ development, must be eliminated in the mature individual. And throughout life, controlled cell death plays a critical role in the turnover and renewal of epithelial surfaces, in the elimination of damaged or transformed cells, and in the involution of the massive cellular proliferation that accompanies in_ammation and tissue repair. This biologic process resulting in the tightly regulated death of living cells, has been termed apoptosis. First recognized more than a century ago, apoptosis has only recently captured scienti~c interest as a fundamental process with critical implications for disciplines as diverse as embryology, neurology, oncology, and infectious diseases. The basic template for the expression of apoptosis in multicellular organisms was established in studies of a nematode worm with a preposterous name—Caenorhabditis elegans. During its development to a mature individual comprising 1090 cells, C. elegans eliminates precisely 131 cells through apoptosis. Two genes, designated ced-3 and ced-4, induce apoptosis, while a third, ced-9 inhibits its expression [1]. Human counterparts of each of these genes have been identi~ed. The interleukin 1 b converting enzyme family, comprising 11 discrete enzymes that collectively have been called caspases, is the human homolog of ced-3 [2], while the bcl-2 family of proteins is the anti-apoptotic counterpart of ced-9 [3]. A human homolog of ced-4, designated Apaf-1, has recently been described [4]; its function remains to be elucidated. Apoptosis is not a pathologic state, but a normal physiologic process. Indeed, for many cell types, apoptosis appears to be the default program, with the result that the cell will die unless it receives a survival signal from the environment. Such survival signals can be transmitted by soluble mediators such as cytokines, or by adhesive interactions between the cell and its adjacent matrix [5,6]. Apoptosis in human cells represents a balance between the expression of proand anti-apoptotic genes, and both excessive and inadequate expression of apoptosis can cause human disease. The human immunode~ciency virus induces T cell apoptosis, producing the profound lymphopenia and enhanced susceptibility to opportunistic infection that is characteristic of AIDS [7], while the apoptotic death of neurons has been shown to be important in a variety of neurodegenerative disorders [8]. On the other hand, failure of expression of lymphocyte apoptosis contributes to the pathogenesis of autoimmune disease [9], and exaggerated in_ammation in patients with burn injury [10] or the systemic in_ammatory response syndrome [11] is associated with prolonged neutrophil survival, consequent to the inhibition of a constitutively-expressed cell death program. In this issue of Sepsis, seven investigators who have contributed to our growing understanding of apoptosis review its role in the pathogenesis of the sequelae of systemic in_ammation. Carmody and Cotter review the molecular events and intracellular mechanisms of apoptosis [12], while Slee and colleagues discuss the role played by the caspase cascade of pro-apoptotic enzymes in the execution of the apoptotic program [13]. Excessive expression of apoptosis can result in acute organ injury, and Bombeck and Billiar [14] and Faraco and co-workers [15] highlight the importance of apoptosis in the pathogenesis of hepatic and renal failure respectively. Cox discusses the critical role played by the anti-in_ammatory cytokine, interleukin 10, in reversing the delayed apoptosis of in_ammatory neutrophils [16], while Ayala and colleagues review lymphocyte apoptotic death occurring during experimental in_ammation [17]. Finally Fanning underlines the role of the heat shock response in the regulation of apop-

Effects of granulocyte-macrophage colony-stimulating factor and cyclic AMP interaction on human neutrophil apoptosis.

The current study was undertaken to evaluate the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and cyclic AMP (cAMP) signaling interaction on human neutrophil apoptosis, either occurring spontaneously or induced by Fas antigen activation. Results show that GM-CSF, dibutyryl cAMP (a cAMP analog) and forskolin (an adenylate cyclase activator) are all able to suppress spontaneous neutrophil cell death. Of note however, when GM-CSF is used in combination with cAMP-elevating agents, an additive effect on neutrophil survival is observed with dibutyryl cAMP only, whereas supplementation of cell cultures with GM-CSF and forskolin results in a progressive reduction of antiapoptotic effects exerted by the single compounds. Moreover, although dibutyryl cAMP and forskolin do not affect Fas-triggered apoptotic events, they are still able to modulate the GM-CSF capacity to prolong neutrophil survival following anti-Fas IgM cell challenge, with effects similar to those respectively exerted on spontaneous neutrophil apoptosis. The data indicate that GM-CSF may negatively modulate the cAMP-mediated antiapoptotic pathway in human neutrophils, likely via the inhibition of adenylate cyclase activity. This would prevent an abnormal neutrophil survival as a result of cAMP signaling stimulation, which provides a novel insight into the role of GM-CSF as a physiological regulator of myeloid cell turnover.

Dysregulated expression of neutrophil apoptosis in the systemic inflammatory response syndrome.

To study the effect of the systemic inflammatory response syndrome (SIRS) or major elective surgery on the apoptosis of circulating polymorphonuclear neutrophils because an activated inflammatory response is terminated, in part, through the programmed cell death, or apoptosis, of its effector cells. A prospective inception cohort study. A mixed surgical and medical intensive care unit of an adult tertiary care hospital. Sixteen patients with SIRS, 7 uninfected patients who had undergone elective aortic aneurysmectomy, and 8 healthy laboratory control subjects. Serial blood samples were drawn for evaluation of neutrophil apoptosis, activational state, and surface receptor expression by flow cytometry. Spontaneous apoptosis was significantly delayed in neutrophils from patients with SIRS (8.6%+/-6.8%) and patients who had undergone elective aortic aneurysmectomy (11.0%+/-5.0%) when compared with controls (34.9%+/-6.8%). These neutrophils were activated as evidenced by enhanced respiratory burst activity and augmented surface expression of CD11b. Apoptosis in response to engagement of cell surface Fas (also known as CD95 or APO-1) with an agonistic antibody was blunted. Plasma from patients with SIRS or patients who had undergone elective aortic aneurysmectomy suppressed the apoptotic responses of control neutrophils (plasma from patients with SIRS, 18.8%+/-10.3%; plasma from patients who had undergone elective aortic aneurysmectomy, 20.0%+/-6.1%; P<.01). Western blot analysis showed normal expression of the key proapoptotic proteases, interleukin 1beta converting enzyme and CPP32 (also known as YAMA, apopain, and caspase 3), indicating that delayed apoptosis was not a consequence of decreased levels of proapoptotic enzymes. Circulating neutrophils from patients with SIRS or from patients who have undergone major elective surgery show delayed expression of constitutive programmed cell death, and antiapoptotic factors are present in the general circulation. While prolonged neutrophil survival may represent an appropriate adaptive response to injury, the presence of activated and apoptosis-resistant cells in an antiapoptotic environment may contribute to the systemic inflammatory injury characteristic of SIRS and predispose to the development of the multiple organ dysfunction syndrome.

Nicotine prolongs neutrophil survival by suppressing apoptosis

Neutrophil accumulation in the lung is implicated in the pathogenesis of pulmonary emphysema and chronic bronchitis associated with cigarette smoking. To determine whether nicotine contributes to this accumulation through the prolongation of neutrophil survival, we examined the survival rates of isolated neutrophils cultured with or without nicotine. We found that nicotine prolonged neutrophil survival in a dose-dependent fashion, with a maximum effect at 10 −6 mol/L. The survival rate at 72 hours was 35.6% ± 1.2% in medium with 10 −6 mol/L nicotine, compared with 15.5% ± 0.5% in control medium (mean ± SEM; p < 0.01), as determined by trypan blue dye exclusion. This prolongation was brought about by suppression of apoptosis, as evidenced by both transmission electron and fluorescence microscopy, and was associated with the preservation of neutrophil functions such as chemotaxis and O 2 − generation. The prolongation of survival caused by nicotine was abrogated by the addition of Pro-Lys-Arg-NH 2, a competitive inhibitor of the specific binding of nicotine to noncholinergic receptors on neutrophils. However, the prolongation of survival caused by nicotine was not suppressed in the presence of K-252b, an inhibitor of protein kinase C. These findings suggest that nicotine prolongs neutrophil survival through noncholinergic nicotine receptors and new protein synthesis, without activation of protein kinase C.