The uncovering of ESE-1 in human neutrophils: implication of its role in neutrophil function and survival.

Epithelium-specific Ets transcription factor 1 (ESE-1) is a member of the E26 transformation-specific family of transcription factors that has an epithelial-restricted constitutive expression but is induced by inflammatory stimuli in non-epithelial cells. Here we report that ESE-1 is constitutively expressed in human, but not in murine, neutrophils and that ESE-1 is modestly upregulated in septic patient neutrophils. In normal human neutrophils, ESE-1 was detected at both RNA and protein levels but was found to be an unstable nuclear protein ex vivo. ESE-1 transcription was also induced during all-trans retinoic acid-mediated HL-60 differentiation, a human promyelocytic cell line often used as an in vitro model of human neutrophils. Elf3-/- mice had normal neutrophils but a reduced number of circulating B-lymphocytes. These findings indicate a potential role of ESE-1 in regulating human neutrophil differentiation and function, and that it has different roles in the immune system of different species.

Phagocytosis of Candida albicans induces apoptosis of human neutrophils.

Neutrophil-mediated inflammation is terminated through the programmed cell death or apoptosis of the neutrophil, a process that can be inhibited by soluble mediators released during an inflammatory response. It has been reported, however, that the phagocytosis of intact bacteria can accelerate apoptosis. We evaluated the effects of the phagocytosis of a common nosocomial pathogen, Candida albicans, on the expression of apoptosis. Phagocytosis of killed Candida induced a dose-dependent increase in the apoptosis of normal neutrophils after 18 h of in vitro culture, from 40.7+/-9.1% to 81.7+/-4.5%, while supernatants from neutrophil:Candida co-cultures actually inhibited apoptosis. Induction of apoptosis was not dependent on phagocytosis, since opsonization of yeast with serum failed to increase apoptosis, while inhibition of phagocytosis with latrunculin B resulted in a slightly increased apoptotic rate. Increased apoptosis induced by Candida was associated with increased activity of the membrane-associated apoptotic enzyme, caspase 8, and with increased expression of the active form of the key executioner caspase, caspase 3. Increased apoptosis was associated with depletion of intracellular glutathione (GSH), and could be inhibited by the addition of exogenous GSH. These data demonstrate an important physiologic role for host-pathogen interactions in the resolution of inflammation and suggest that the response to an invading pathogen is an important stimulus to the restoration of normal immunologic homeostasis.

Hypertonic inhibition of exocytosis in neutrophils: central role for osmotic actin skeleton remodeling.

Hypertonicity suppresses neutrophil functions by unknown mechanisms. We investigated whether osmotically induced cytoskeletal changes might be related to the hypertonic inhibition of exocytosis. Hyperosmolarity abrogated the mobilization of all four granule types induced by diverse stimuli, suggesting that it blocks the process of exocytosis itself rather than individual signaling pathways. Concomitantly, osmotic stress provoked a twofold increase in F-actin, induced the formation of a submembranous F-actin ring, and abolished depolymerization that normally follows agonist-induced actin assembly. Several observations suggest a causal relationship between actin polymerization and inhibition of exocytosis: 1) prestimulus actin levels were inversely proportional to the stimulus-induced degranulation, 2) latrunculin B (LB) prevented the osmotic actin response and restored exocytosis, and 3) actin polymerization induced by jasplakinolide inhibited exocytosis under isotonic conditions. The shrinkage-induced tyrosine phosphorylation and the activation of the Na(+)/H(+) exchanger were not affected by LB. Inhibition of osmosensitive kinases failed to prevent the F-actin change, suggesting that the osmotic tyrosine phosphorylation and actin polymerization are independent phenomena. Thus cytoskeletal remodeling appears to be a key component in the neutrophil-suppressive, anti-inflammatory effects of hypertonicity.

Redox manipulation using the thiol-oxidizing agent diethyl maleate prevents hepatocellular necrosis and apoptosis in a rodent endotoxemia model.

Manipulation of the intracellular redox state has been shown to alter cell activation pathways with resultant changes in cellular function. Previous studies have suggested that thiol oxidation, using the glutathione-depleting agent diethyl maleate (DEM), was able to inhibit endothelial cell activation. We hypothesized that this agent might exert beneficial effects following endotoxemia in the rat, a model in which transendothelial migration of neutrophils is central to the development of hepatocellular injury. Sprague-Dawley rats treated intraperitoneally with lipopolysaccharide (LPS) (200 microg/kg) plus D-galactosamine (GalN) (600 mg/kg) developed hepatocellular necrosis, as evidenced by liver enzyme release and morphological changes. Pretreatment with DEM abrogated this injury in a dose-dependent fashion. Histology revealed reduced neutrophil accumulation in both the parenchyma and sinusoids, consistent with reduced neutrophil sequestration and transendothelial migration. This effect appeared to be related to the ability of DEM to prevent LPS-induced up-regulation of both vascular cell adhesion molecule-1 (VCAM-1) mRNA and intercellular adhesion molecule-1 (ICAM-1) mRNA in the liver, as well as reducing tumor necrosis factor (TNF) mRNA expression. In addition, DEM prevented hepatocyte apoptosis following LPS treatment. The effect was reproduced when TNF was used as an inflammatory stimulus, suggesting a direct protective effect on the hepatocyte. Taken together, these studies show that redox manipulation through thiol oxidation may represent a novel approach to preventing liver necrosis and apoptosis in inflammatory conditions.

Maternal neutrophil apoptosis in normal pregnancy, preeclampsia, and normotensive intrauterine growth restriction.

OBJECTIVE: In normal pregnancy there is both a neutrophilia and a mild neutrophil activation. In preeclampsia both direct and indirect evidence supports further marked neutrophil activation. In the pathogenesis of preeclampsia peripheral blood neutrophils may play a vital role in communicating between the preeclamptic placenta and the maternal vascular endothelium and contribute to the endothelial cell dysfunction that characterizes the maternal syndrome of preeclampsia. Preeclampsia shares many elements with the systemic inflammatory response syndrome. Neutrophils, key effectors of the systemic inflammatory response syndrome, are associated with hepatic necrosis and adult respiratory distress syndrome, both of which most commonly kill women with preeclampsia. We hypothesized that delayed neutrophil apoptosis could explain (1) the neutrophilia of normal pregnancy and (2) the differential maternal responses to the shared placental abnormality of preeclampsia and normotensive intrauterine growth restriction. STUDY DESIGN: Neutrophils were isolated (dextran 500, Ficoll [Amersham Pharmacia Biotech AB, Uppsala, Sweden], and erythrocyte lysis) from (1) case patients with preeclampsia at

Hypertonic immunomodulation is reversible and accompanied by changes in CD11b expression.

BACKGROUND: In a two-hit model of hemorrhagic shock and lipopolysaccharide (LPS), we previously showed that hypertonic saline (HTS) resuscitation reduced lung sequestration of neutrophils and the accompanying injury. This effect was partially attributed to suppressed expression of the surface adhesion molecule CD11b. This study investigates the duration of this protective effect after a single HTS dose and the usefulness of repeated infusions. MATERIAL AND METHODS: The previous two-hit rodent model was used. Neutrophil lung sequestration was measured by bronchoalveolar fluid cell count. CD11b expression was followed by flow cytometry. In vitro studies used isolated human neutrophils. RESULTS: Eighteen hours following resuscitation, the protective effect of HTS was lost. At this time, LPS caused an increase in both neutrophil lung sequestration and CD11b expression, regardless of the resuscitation regimen used. A second infusion of HTS prevented these changes and restored the lung protection observed earlier. In vitro studies showed that the duration of hypertonic pretreatment is an important determinant of cell responsiveness under the isotonic conditions: Four but not 2 h hypertonic exposure was able to prevent upregulation of CD11b induced by LPS added immediately after reestablishing isotonicity. CONCLUSIONS: This study demonstrates that HTS resuscitation lessens lung neutrophil sequestration and CD11b surface expression induced by LPS. This protective effect is transient but can be restored by a second HTS infusion suggesting that maintenance of beneficial effect necessitates repeated HTS addition. The reversibility ensures rapid modulation of neutrophil functions, thereby preventing acute tissue damage without causing long-lasting immunosuppression.

Hypertonicity prevents lipopolysaccharide-stimulated CD11b/CD18 expression in human neutrophils in vitro: role for p38 inhibition.

BACKGROUND: Neutrophil sequestration in the lungs plays an important role in the development of acute respiratory distress syndrome. We previously reported that hypertonic saline resuscitation attenuated lung injury after hemorrhagic shock and lipopolysaccharide (LPS) by abolishing neutrophil CD11b up-regulation. We investigated the mechanism underlying this effect. METHODS: Human neutrophils were exposed to LPS in the presence or absence of hypertonicity or SB203580 (p38 inhibitor). CD11b and CD14 were studied by immunofluorescence and p38 phosphorylation by immunoblotting. RESULTS: Hypertonicity had no effect on CD11b or CD14, caused a weak p38 phosphorylation, and completely prevented the LPS-induced p38 phosphorylation and CD11b up-regulation. p38 inhibition also abrogated CD11b up-regulation by LPS. CONCLUSION: MAPKp38 is important in CD11b regulation by LPS. The inhibitory effect of hypertonicity on the LPS-mediated effect may contribute to its protective anti-inflammatory effect observed in vivo. Transient hypertonicity might minimize organ injury in diseases characterized by neutrophil-mediated damage such as ARDS.

The IL-1 beta-converting enzyme (caspase-1) inhibits apoptosis of inflammatory neutrophils through activation of IL-1 beta.

IL-1 beta-converting enzyme (ICE), also known as caspase-1, subserves two dichotomous biologic roles. It processes newly synthesized pro-IL-1 beta to yield the active cytokine and, as the human homologue of the Caenorhabditis elegans gene product, ced-3, it also induces cellular apoptosis through the cleavage of key intracellular structural and regulatory proteins and through the catalytic activation of other caspase family members. We show here that two different proinflammatory stimuli, LPS and granulocyte-macrophage-CSF, up-regulate the expression of both ICE and IL-1 beta in human polymorphonuclear neutrophils, and that the ICE-dependent cleavage of pro-IL-1 beta results in delayed expression of the constitutive cell death program. The apoptotic delay can be blocked by inhibiting tyrosine kinases or NF-kappa B activation and by inhibiting protein synthesis. Since an antisense oligonucleotide for IL-1 beta, a blocking Ab to IL-1 beta, and preincubation with the IL-1R antagonist all prevent the delay in apoptosis, we conclude that IL-1 beta acts in an autocrine manner to inhibit granulocyte programmed cell death. We conclude that caspase-1 (ICE) subserves both pro- and antiapoptotic roles; the latter role is evident during inflammation as an inhibition of spontaneous neutrophil apoptosis through the processing of IL-1 beta. The ICE-dependent activation of IL-1 beta may represent a common autocrine pathway for the divergent stimuli that inhibit the constitutive expression of neutrophil programmed cell death during inflammation.

Ribavirin inhibits viral-induced macrophage production of TNF, IL-1, the procoagulant fgl2 prothrombinase and preserves Th1 cytokine production but inhibits Th2 cytokine response.

Ribavirin, a synthetic guanosine analogue, possesses a broad spectrum of activity against DNA and RNA viruses. It has been previously shown to attenuate the course of fulminant hepatitis in mice produced by murine hepatitis virus strain 3. We therefore studied the effects of ribavirin on murine hepatitis virus strain 3 replication, macrophage production of proinflammatory mediators including TNF, IL-1, and the procoagulant activity (PCA), fgl2 prothrombinase; and Th1/Th2 cytokine production. Although ribavirin had inhibitory effects on viral replication (<1 log), even at high concentrations complete eradication of the virus was not seen. In contrast, at physiologic concentrations (up to 500 microg/ml), ribavirin markedly reduced viral-induced parameters of macrophage activation. With ribavirin treatment, the concentrations of PCA, TNF-alpha and IL-1beta all decreased to basal concentrations: PCA from 941 +/- 80 to 34 +/- 11 mU/10(6) cells; TNF-alpha from 10.73 +/- 2.15 to 2.74 +/- 0.93 ng/ml; and IL-1beta from 155.91 +/- 22.62 to 5.74 +/- 0.70 pg/ml. The inhibitory effects of ribavirin were at the level of gene transcription as evidenced by Northern analysis. Both in vitro and in vivo, ribavirin inhibited the production of IL-4 by Th2 cells, whereas it did not diminish the production of IFN-gamma in Th1 cells. In contrast, ribavirin had no inhibitory effect on TNF-alpha and IL-1beta production in LPS-stimulated macrophages. These results suggest that the beneficial effects of ribavirin are mediated by inhibition of induction of macrophage proinflammatory cytokines and Th2 cytokines while preserving Th1 cytokines.

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

OBJECTIVE: 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. DESIGN: A prospective inception cohort study. SETTING: A mixed surgical and medical intensive care unit of an adult tertiary care hospital. PATIENTS: Sixteen patients with SIRS, 7 uninfected patients who had undergone elective aortic aneurysmectomy, and 8 healthy laboratory control subjects. INTERVENTIONS: Serial blood samples were drawn for evaluation of neutrophil apoptosis, activational state, and surface receptor expression by flow cytometry. MAIN OUTCOME MEASURES: 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. CONCLUSIONS: 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.

Impaired apoptotic death signaling in inflammatory lung neutrophils is associated with decreased expression of interleukin-1 beta converting enzyme family proteases (caspases).

BACKGROUND: Fas and tumor necrosis factor receptor 1 (TNFR1) are membrane proteins that signal for apoptotic cell death by downstream activation of proteins of the interleukin-1 beta converting enzyme (ICE) family. Spontaneous apoptosis is delayed in neutrophils activated by transmigration into an inflammatory focus. In this study we evaluated the effects of transmigration on Fas and TNFR1-induced apoptosis and apoptotic gene expression. METHODS: Sprague-Dawley rats were killed 4 hours after intratracheal challenge with 500 micrograms lipopolysaccharide (LPS). Neutrophils isolated from the systemic circulation (circulation) or bronchoalveolar lavage fluid (lung) were incubated with or without an agonistic antibody to Fas (clone CH-11, 100 ng/ml) or TNF (10 ng/ml) for 24 hours. Apoptosis and Fas expression were assessed by flow cytometry. Expression of the antiapoptotic protein Bcl-2 and proapoptotic proteins ICE and CPP32 were measured by Western blots. RESULTS: Neutrophils transmigrating into the lung in response to LPS showed delayed apoptosis compared with circulating neutrophils and failed to undergo apoptosis in response to anti-Fas antibody or TNF-alpha. Fas expression was unaltered; however, TNFR1 expression was reduced. Bcl-2 was not detected in either group; both the pro- and active forms of ICE and active CPP32 were significantly decreased in lung neutrophils. The specific ICE inhibitor, YVAD-CMK, partially blocked the increased rates of apoptosis resulting from engagement of Fas or TNFR1. CONCLUSIONS: Neutrophil transmigration retards apoptosis through engagement of the death receptors Fas and TNFR1. This refractory state is associated with reduced levels of proapoptotic proteins. Blunted responsiveness to physiologic apoptotic stimuli prolongs neutrophil functional survival during acute inflammation and may contribute to the tissue injury associated with acute respiratory distress syndrome.

Granulocytic differentiation of HL-60 cells results in spontaneous apoptosis mediated by increased caspase expression.

HL-60 cells differentiating into neutrophil-like cells die an apoptotic death in vitro. Susceptibility to apoptosis is associated with decreased Bcl-2 protein and mRNA expression; however, the effect of differentiation on the expression of pro-apoptotic caspases is unknown. Spontaneous apoptosis occurred 6 days after retinoic acid treatment. Western blotting showed loss of Bcl-2 by day 7, and new expression of ICE (caspase 1) and CPP32 (caspase 3) protein by day 2. Northern analysis demonstrated loss of Bcl-2 mRNA and increases in ICE mRNA by day 2; CPP32 mRNA was unchanged. Differential Bcl-2 and ICE mRNA expression was also found when granulocytic differentiation was stimulated by DMSO. Differentiated HL-60 cell lysates exhibited functional ICE proteolytic activity. De novo caspase expression was responsible for the development of spontaneous apoptosis, since specific inhibitors of ICE (YVAD-CMK) and CPP32 (DEVD-CHO), inhibited retinoic acid induced spontaneous apoptosis. Functional maturation and susceptibility to apoptosis are both inducible and linked in this granulocyte precursor cell line.

Augmented intracellular glutathione inhibits Fas-triggered apoptosis of activated human neutrophils.

Agonist signals delivered through cell surface Fas induce apoptosis. However, the apoptotic program can be modulated by signals from the environment, and in particular, by signals delivered through adhesion molecules. Because neutrophil functional activity in inflammation is contingent on cell survival, and because circulating neutrophils normally die rapidly through a constitutively expressed apoptotic program, we evaluated Fas-mediated apoptosis in resting and inflammatory human neutrophils. We show that normal neutrophils respond to Fas engagement with accelerated rates of apoptosis, but cross-linking of beta2 integrins or priming with bacterial lipopolysaccharide (LPS) prevents this increase. Adhesion molecule cross-linking results in increased intracellular glutathione (GSH). Augmentation of intracellular GSH with exogenous GSH or N-acetylcysteine is sufficient to reduce the Fas-triggered increase in apoptotic rates. Prevention of the activation induced GSH increase by buthionine sulfoximine, a cell permeable inhibitor of GSH biosynthesis, restored Fas responsiveness in activated neutrophils, an effect that could be blocked with exogenous GSH. Taken together, these data show that Fas-induced signaling for neutrophil apoptosis is blocked in a redox sensitive manner by costimulatory signals delivered through beta2 integrins or activation by LPS, and provide a biologic explanation for sustained neutrophil survival in the inflammatory environment.

Neutrophil apoptosis is modulated by endothelial transmigration and adhesion molecule engagement.

Termination of a neutrophil-mediated inflammatory response occurs through the activation of the endogenous cell death program, apoptosis. Neutrophil apoptosis is a constitutive process that can be accelerated or delayed by signals from the microenvironment. Since cellular localization at the site of an inflammatory challenge is the critical first step in a neutrophil response, we investigated the effects of neutrophil transendothelial transmigration on the kinetic expression of apoptosis. Neutrophils isolated from rat lung following challenge with LPS demonstrated a significant delay in spontaneous apoptosis. This delay was a consequence of transmigration, since a comparable delay was seen when TNF-alpha, a potent inducer of apoptosis in vitro, was used as the inflammatory stimulus. Human neutrophils demonstrated comparable delays in apoptosis in vitro following migration across an endothelial monolayer in response to FMLP. Delayed apoptosis only occurred in cells that had first been primed by LPS, a stimulus shown to up-regulate beta2 integrins and down-regulate L-selectin. Finally, crosslinking of CD11a or CD11b, but not of CD18, with mAbs and F(ab')2 fragments produced a delay in spontaneous apoptosis, whereas crosslinking of L-selectin with mAb or its natural ligand, sulfatides, accelerated the apoptotic process. Cells in which apoptosis was inhibited demonstrated persistent functional respiratory burst activity. These observations establish a role for endothelial transmigration in the regulation of neutrophil apoptosis, and suggest that adhesion molecules serve a modulatory role in the expression of neutrophil programmed cell death.