Upregulated PD-L1 delays human neutrophil apoptosis and promotes lung injury in an experimental mouse model of sepsis.

PD-L1 is a ligand for PD-1, and its expression has been shown to be upregulated in neutrophils harvested from septic patients. However, the effect of PD-L1 on neutrophil survival and sepsis-induced lung injury remains largely unknown. In this study, PD-L1 expression correlated negatively with rates of apoptosis in human neutrophils harvested from patients with sepsis. Coimmunoprecipitation assays on control neutrophils challenged with interferon-γ and LPS showed that PD-L1 complexes with the p85 subunit of phosphatidyl 3-kinase (PI3K) to activate AKT-dependent survival signaling. Conditional CRE/LoxP deletion of neutrophil PD-L1 in vivo further protected against lung injury and reduced neutrophil lung infiltration in a cecal ligation and puncture (CLP) experimental sepsis animal model. Compared with wild-type animals, PD-L1-deficient animals presented lower levels of plasma tumor necrosis factor-α and interleukin-6 (IL-6) and higher levels of IL-10 after CLP, and reduced 7-day mortality in CLP PD-L1-knockout animals. Taken together, our data suggest that increased PD-L1 expression on human neutrophils delays cellular apoptosis by triggering PI3K-dependent AKT phosphorylation to drive lung injury and increase mortality during clinical and experimental sepsis.

Heat-shock protein-90 prolongs septic neutrophil survival by protecting c-Src kinase and caspase-8 from proteasomal degradation.

The brief lifespan of the polymorphonuclear neutrophil (PMN) is regulated through its capacity to undergo apoptosis, a constitutive process that is actively inhibited during sepsis. We sought to define the cellular mechanisms through which Heat Shock Protein 90 (Hsp90) prolongs the survival of inflammatory PMN. We evaluated Hsp90 expression and interaction with client proteins in PMNs from patients with sepsis and in healthy control PMNs treated with LPS (1 µg/mL). Hsp90 activity was inhibited pharmacologically using radicicol (Rad; 1 µM), and Hsp90 transcription was silenced in septic PMN using siRNA. PMN apoptosis was evaluated by flow cytometry and expression of cleaved caspase-8 and -3. Septic PMNs showed reduced rates of apoptosis compared with control PMNs 21 h after isolation, and Hsp90-α mRNA was significantly more abundant in septic PMN. Caspase-8 coimmunoprecipitated with Hsp90, c-Src, and the p85 inhibitory subunit of PI3K in both septic and LPS-treated PMN. Inhibition of Hsp90 activity with Rad or its translation using siRNA restored basal rates of apoptosis in both septic and LPS-treated PMN. Radicicol further reduced c-Src protein abundance, increased the ubiquitination of caspase-8 and c-Src, and enhanced the cleavage of caspase-8 and -3. We conclude that Hsp90 prolongs the survival of activated neutrophils by stabilizing a molecular complex of c-Src kinase and caspase-8, preventing their ubiquitination, and resulting in inhibition of the catalytic activity of caspase-8 and -3.

Tyrosine Phosphorylation of Caspase-8 Abrogates Its Apoptotic Activity and Promotes Activation of c-Src.

Src family tyrosine kinases (SFKs) phosphorylate caspase-8A at tyrosine (Y) 397 resulting in suppression of apoptosis. In addition, the phosphorylation of caspase-8A at other sites including Y465 has been implicated in the regulation of caspase-8 activity. However, the functional consequences of these modifications on caspase-8 processing/activity have not been elucidated. Moreover, various Src substrates are known to act as potent Src regulators, but no such role has been explored for caspase-8. We asked whether the newly identified caspase-8 phosphorylation sites might regulate caspase-8 activation and conversely, whether caspase-8 phosphorylation might affect Src activity. Here we show that Src phosphorylates caspase-8A at multiple tyrosine sites; of these, we have focused on Y397 within the linker region and Y465 within the p12 subunit of caspase-8A. We show that phosphomimetic mutation of caspase-8A at Y465 prevents its cleavage and the subsequent activation of caspase-3 and suppresses apoptosis. Furthermore, simultaneous phosphomimetic mutation of caspase-8A at Y397 and Y465 promotes the phosphorylation of c-Src at Y416 and increases c-Src activity. Finally, we demonstrate that caspase-8 activity prevents its own tyrosine phosphorylation by Src. Together these data reveal that dual phosphorylation converts caspase-8 from a pro-apoptotic to a pro-survival mediator. Specifically, tyrosine phosphorylation by Src renders caspase-8 uncleavable and thereby inactive, and at the same time converts it to a Src activator. This novel dynamic interplay between Src and caspase-8 likely acts as a potent signal-integrating switch directing the cell towards apoptosis or survival.

Pre-B cell colony enhancing factor induces Nampt-dependent translocation of the insulin receptor out of lipid microdomains in A549 lung epithelial cells.

Pre-B cell colony-enhancing factor (PBEF) is a highly conserved pleiotropic protein reported to be an alternate ligand for the insulin receptor (IR). We sought to clarify the relationship between PBEF and insulin signaling by evaluating the effects of PBEF on the localization of the IRß chain to lipid rafts in A549 epithelial cells. We isolated lipid rafts from A549 cells and detected the IR by immunoprecipitation from raft fractions or whole cell lysates. Cells were treated with rPBEF, its enzymatic product nicotinamide adenine dinucleotide (NAD), or the Nampt inhibitor daporinad to study the effect of PBEF on IRß movement. We used coimmunoprecipitation studies in cells transfected with PBEF and IRß constructs to detect interactions between PBEF, the IRß, and caveolin-1 (Cav-1). PBEF was present in both lipid raft and nonraft fractions, whereas the IR was found only in lipid raft fractions of resting A549 cells. The IR-, PBEF-, and Cav-1-coimmunoprecipitated rPBEF treatment resulted in the movement of IRß- and tyrosine-phosphorylated Cav-1 from lipid rafts to nonrafts, an effect that could be blocked by daporinad, suggesting that this effect was facilitated by the Nampt activity of PBEF. The addition of PBEF to insulin-treated cells resulted in reduced Akt phosphorylation of both Ser47³ and Thr³°8. We conclude that PBEF can inhibit insulin signaling through the IR by Nampt-dependent promotion of IR translocation into the nonraft domains of A549 epithelial cells. PBEF-induced alterations in the spatial geometry of the IR provide a mechanistic explanation for insulin resistance in inflammatory states associated with upregulation of PBEF.

Activated neutrophils induce epithelial cell apoptosis through oxidant-dependent tyrosine dephosphorylation of caspase-8.

Activated neutrophils can injure host cells through direct effects of oxidants on membrane phospholipids, but an ability to induce apoptotic cell death has not previously been reported. We show that neutrophils activated in vivo in patients who have sustained multiple trauma or in vitro by exposure to bacterial lipopolysaccharide promote epithelial cell apoptosis through SHP-1-mediated dephosphorylation of epithelial cell caspase-8. Epithelial cell apoptosis induced by circulating neutrophils from patients who had sustained serious injury depended on the generation of neutrophil-derived reactive oxygen intermediates and was blocked by inhibition of NADPH oxidase or restoration of intracellular glutathione. Caspase-8 was constitutively tyrosine phosphorylated in a panel of resting epithelial cells, but underwent SHP-1-dependent dephosphorylation in response to hydrogen peroxide, activated neutrophils, or inhibition of Src kinases. Cells transfected with a mutant caspase-8 in which tyrosine residues at Tyr397 or Tyr465 are replaced by nonphosphorylatable phenylalanine underwent accelerated apoptosis, whereas either mutation of these residues to phosphomimetic glutamic acid or transfection with the Src kinases Lyn or c-Src inhibited hydrogen peroxide-induced apoptosis. Exposure to either hydrogen peroxide or lipopolysaccharide-stimulated neutrophils increased phosphorylation and activity of the phosphatase SHP-1, increased activity of caspases 8 and 3, and accelerated epithelial cell apoptosis. These observations reveal a novel mechanism for neutrophil-mediated tissue injury through oxidant-dependent, SHP-1-mediated dephosphorylation of caspase-8 resulting in enhanced epithelial cell apoptosis.

Pre-B cell colony-enhancing factor (PBEF/Nampt/visfatin) primes neutrophils for augmented respiratory burst activity through partial assembly of the NADPH oxidase.

Pre-B cell colony-enhancing factor ([PBEF] also known as Nampt/visfatin) is a pleiotropic 52-kDa cytokine-like molecule whose activity has been implicated in multiple inflammatory disease states. PBEF promotes polymorphonuclear neutrophil (PMN) proinflammatory function by inhibiting constitutive PMN apoptosis. We investigated whether PBEF activates or primes for PMN respiratory burst. We found that although PBEF did not activate respiratory burst on its own, it primed for increased reactive oxygen species generation through the NADPH oxidase. PBEF promoted membrane translocation of cytosolic NADPH oxidase subunits p40 and p47, but not p67, induced p40 phosphorylation on Thr(154), and activated the small GTPase Rac. Priming, translocation, and phosphorylation were dependent on activation of p38 and ERK MAPKs, but not of PI3K. Priming by PBEF occurred independent of its NAD-generating capacity because neither nicotinamide mononucleotide or NAD could recapitulate the effects, and a specific inhibitor of PBEF, APO-866, could not inhibit priming. Taken together, these results demonstrate that PBEF can prime for PMN respiratory burst activity by promoting p40 and p47 translocation to the membrane, and this occurs in a MAPK-dependent fashion.

Regulation of apoptosis and priming of neutrophil oxidative burst by diisopropyl fluorophosphate.

BACKGROUND: Diisopropyl fluorophosphate (DFP) is a serine protease inhibitor that is widely used as an inhibitor of endogenous proteases in in vitro neutrophil studies. Its effects on neutrophil function are unclear. We sought to determine the biological effects of DFP on human neutrophil apoptosis and oxidative burst. METHODS: We isolated neutrophils from healthy volunteers, incubated them with DFP (2.5 mM), and evaluated neutrophil elastase (NE) activity, neutrophil degranulation, apoptosis as reflected in hypodiploid DNA formation and exteriorization of phosphatidylserine (PS), processing and activity of caspases-3 and -8, oxidative burst activity and hydrogen peroxide release. RESULTS: Consistent with its activity as a serine protease inhibitor, DFP significantly inhibited NE activity but not the degranulation of azurophilic granules. DFP inhibited constitutive neutrophil apoptosis as reflected in DNA fragmentation, and the processing and activity of caspases-3 and -8. DFP also inhibited priming of neutrophils for oxidative burst activity and hydrogen peroxide release. However, DFP enhanced the exteriorization of PS in a dose-dependent manner. CONCLUSION: We conclude that DFP exerts significant effects on neutrophil inflammatory function that may confound the interpretation of studies that use it for its antiprotease activity. We further conclude that endogenous proteases play a role in the biology of constitutive neutrophil apoptosis.

Interleukin-1beta mediates LPS-induced inhibition of apoptosis in retinoic acid-differentiated HL-60 cells.

Promyelocytic HL-60 cells differentiated to a neutrophilic phenotype by incubation with all-trans retinoic acid become constitutively apoptotic. Exposure to either LPS or IL-1beta inhibited the apoptosis of differentiated HL-60 cells. LPS induced the expression of pro-IL-1beta message, upregulated the activity of the interleukin-1beta converting enzyme (caspase-1), and increased the release of IL-1beta into the culture medium. Prevention of IL-1beta translation with an anti-sense oligonucleotide, or prevention of IL-1beta cellular binding with a blocking antibody, accelerated rates of spontaneous apoptosis, and abrogated the inhibitory effects of LPS. However inhibition of caspase-1 activity further inhibited constitutive apoptosis of mature HL-60 cells. These studies provide further evidence of a complex regulatory pathway that modulates the expression of granulocyte apoptosis during inflammation, and point to a specific role for IL-1beta as an autocrine survival factor.

Dynamic regulation of neutrophil survival through tyrosine phosphorylation or dephosphorylation of caspase-8.

Efficient expression of innate immunity is critically dependent upon the capacity of the neutrophil to be activated rapidly in the face of an acute threat and to involute once that threat has been eliminated. Here we report a novel mechanism regulating neutrophil survival dynamically through the tyrosine phosphorylation or dephosphorylation of caspase-8. Caspase-8 is tyrosine-phosphorylated in freshly isolated neutrophils but spontaneously dephosphorylates in culture, in association with the progression of constitutive apoptosis. Phosphorylation of caspase-8 on Tyr-310 facilitates its interaction with the Src-homology domain 2 containing tyrosine phosphatase-1 (SHP-1) and enables SHP-1 to dephosphorylate caspase-8, permitting apoptosis to proceed. The non-receptor tyrosine kinase, Lyn, can phosphorylate caspase-8 on Tyr-397 and Tyr-465, rendering it resistant to activational cleavage and inhibiting apoptosis. Exposure to lipopolysaccharide reduces SHP-1 activity and binding to caspase-8, caspase-8 activity, and rates of spontaneous apoptosis. SHP-1 activity is reduced and Lyn increased in neutrophils from patients with sepsis, in association with profoundly delayed apoptosis; inhibition of Lyn can partially reverse this delay. Thus the phosphorylation and dephosphorylation of caspase-8, mediated by Lyn and SHP-1, respectively, represents a novel, dynamic post-translational mechanism for the regulation of neutrophil apoptosis whose dysregulation contributes to persistent neutrophil survival in sepsis.

The role of the Src family of tyrosine kinases after oxidant-induced lung injury in vivo.

BACKGROUND: Patients sustaining major trauma are predisposed to the development of organ dysfunction. We have shown that oxidant stress generated by hemorrhagic shock/resuscitation (S/R) in rodents increases lipopolysaccharide (LPS)-induced lung injury and translocation of nuclear factor kappa B (NF-kappaB) in alveolar macrophages (AMs). In addition, using a cellular model, we have shown that priming with oxidants reprograms LPS signaling through an Src-dependent pathway. In the present studies, we hypothesize that oxidant priming by S/R in vivo involves Src family kinases. METHODS: Rats were bled to a mean arterial pressure of 40 mmHg and maintained for 1 hour, then resuscitated with shed blood and equal volume of Ringer's lactate. In some studies, animals received the antioxidant NAC (0.5 g/kg) or a Src family inhibitor, PP2 (0.1 or 0.2 mg/kg), before resuscitation. LPS was given intratracheally (30 mg/kg) for 4 hours. AMs were lavaged, and total cell counts were determined. AMs were also obtained at end resuscitation and exposed to LPS (0.1 microg/mL) from 0 to 60 minutes. Activation of Hck, an Src family kinase, was analyzed by Western blot using a phosphospecific antibody. Nuclear extracts were obtained to examine NF-kappaB translocation. RESULTS: S/R caused a rise in Src family activity compared with sham animals as shown by the phosphorylation of Hck. This was prevented by treating the animals during resuscitation with NAC. The LPS-induced NF-kappaB translocation in AMs after shock/resuscitation was 3-fold higher than in sham AMs treated with LPS. This augmented translocation was prevented by pretreating the animals with PP2 before resuscitation. In a parallel fashion, PP2 pretreatment reduced the absolute lung neutrophil sequestration. CONCLUSION: Oxidant stress generated during S/R in vivo causes Src family kinase activation in AMs. Inhibition of Src activation by PP2 attenuates AM priming for increased LPS responsiveness after hemorrhagic shock and causes a modest reduction in lung injury. Inhibition of the Src family kinases may be a novel approach for the treatment of lung injury after trauma.

Delayed neutrophil apoptosis in sepsis is associated with maintenance of mitochondrial transmembrane potential and reduced caspase-9 activity.

OBJECTIVE: The resolution of neutrophil (PMN)-mediated inflammation occurs through the apoptosis, or programmed cell death, of the neutrophil. PMN apoptosis is inhibited by a variety of inflammatory stimuli; moreover, PMN from critically ill septic patients show profoundly delayed rates of apoptosis in vitro. Since apoptosis is effected through the activity of intracellular cysteine proteases (caspases), we evaluated caspase expression and activity in neutrophils from septic patients and compared them with caspase expression and activity of resting or lipopolysaccharide-activated neutrophils from healthy volunteers. DESIGN: Prospective observational cohort study. SETTING: Tertiary level intensive care unit and associated research laboratory. SUBJECTS: Thirty-six intensive care unit patients with sepsis; ten healthy laboratory controls. INTERVENTIONS: Collection of up to 10 mL of whole blood for in vitro study of rates of apoptosis, expression and activity of caspases-1, -3, and -9, activation of nuclear factor-kappaB, and change in mitochondrial transmembrane potential. MEASUREMENTS AND MAIN RESULTS: Following 24 hrs of in vitro culture, 52 +/- 7.8% of control neutrophils, but only 29 +/- 5.4% of lipopolysaccharide-stimulated (1 microg/mL) PMN, showed nuclear changes of apoptosis. Only 6.2 +/- 1.1% of neutrophils from septic patients were apoptotic after 24 hrs. Significant nuclear translocation of nuclear factor-kappaB was evident in septic PMN, and inhibition of apoptosis was partially abrogated by prevention of nuclear factor-kappaB dissociation with pyrrolidine dithiocarbamate. Caspase-3 transcription and catalytic activity were significantly reduced in both patients' and lipopolysaccharide-treated PMN; caspase-1 transcription and activity were increased by lipopolysaccharide but reduced in septic patients. In contrast, caspase-9 transcription and activity were reduced in septic patients but not in lipopolysaccharide-treated PMN. Decreased caspase-9 activity was associated with sustained maintenance of mitochondrial transmembrane potential and reduced translocation of cytochrome c from the mitochondria to the cytosol. CONCLUSIONS: Apoptosis of circulating neutrophils from patients with clinical sepsis is profoundly suppressed, through a mechanism that involves activation of nuclear factor-kappaB that is associated with reduced activity of caspases-9 and -3 and maintenance of mitochondrial transmembrane potential and that differs in important respects from the inhibitory effects seen following the exposure of healthy neutrophils to inflammatory stimuli.

Pre-B cell colony-enhancing factor inhibits neutrophil apoptosis in experimental inflammation and clinical sepsis.

Pre-B cell colony-enhancing factor (PBEF) is a highly conserved 52-kDa protein, originally identified as a growth factor for early stage B cells. We show here that PBEF is also upregulated in neutrophils by IL-1beta and functions as a novel inhibitor of apoptosis in response to a variety of inflammatory stimuli. Induction of PBEF occurs 5-10 hours after LPS exposure. Prevention of PBEF translation with an antisense oligonucleotide completely abrogates the inhibitory effects of LPS, IL-1, GM-CSF, IL-8, and TNF-alpha on neutrophil apoptosis. Immunoreactive PBEF is detectable in culture supernatants from LPS-stimulated neutrophils, and a recombinant PBEF fusion protein inhibits neutrophil apoptosis. PBEF is also expressed in neutrophils from critically ill patients with sepsis in whom rates of apoptosis are profoundly delayed. Expression occurs at higher levels than those seen in experimental inflammation, and a PBEF antisense oligonucleotide significantly restores the normal kinetics of apoptosis in septic polymorphonuclear neutrophils. Inhibition of apoptosis by PBEF is associated with reduced activity of caspases-8 and -3, but not caspase-9. These data identify PBEF as a novel inflammatory cytokine that plays a requisite role in the delayed neutrophil apoptosis of clinical and experimental sepsis.

Oxidant-induced priming of the macrophage involves activation of p38 mitogen-activated protein kinase through an Src-dependent pathway.

BACKGROUND: Resuscitated hemorrhagic shock predisposes patients to the development of organ dysfunction, particularly to lung injury. Ischemia/reperfusion during shock is believed to prime the immune system for an exaggerated inflammatory response to a second delayed stimulus. We previously reported an in vitro model of oxidant-induced priming of the macrophage to lipopolysaccharide (LPS) involves the Src family of tyrosine kinases. Because the Src family has been shown to activate the p38 mitogen-activated protein kinase (MAPK) pathway, we hypothesize that LPS signaling after oxidant stress involves the p38 pathway and is activated by Src kinases. METHODS: The murine macrophage cell line, Raw 264.7, was first incubated with H(2)O(2) 100 micromol/L for 1 hour and then with low dose LPS 0.01 microg/mL for 5 to 45 minutes. In a separate experiment, the cells were pretreated with PP2 or SB203580, a specific inhibitor of the Src family and p38 respectively. The phosphorylation of p38, representative of its activation, was assessed in whole cell lysates by use of Western blotting. NF-kappaB translocation was detected by immunofluorescence with anti-p65 antibody. RESULTS: There is a time dependent earlier activation of p38 by oxidant stress. H(2)O(2) augmented the LPS-induced p38 phosphorylation. The Src inhibitor, PP2, prevented only the LPS-induced earlier phosphorylation after oxidant stress and had no effect on LPS activation of p38 alone. The p38 inhibitor had no effect in preventing NF-kappaB translocation in either the LPS- or H(2)O(2)/LPS-exposed cells. CONCLUSIONS: Oxidant stress generated during global ischemia/reperfusion activates p38 MAPK in an Src-dependent manner. Oxidants seem to alter the LPS-induced activation of p38. P38 does not seem to have a direct role in leading to oxidant-induced NF-kappaB translocation but may affect other oxidant-induced transcription factors. This altered pathway provides an alternative avenue to target therapy during the oxidant-induced priming of the macrophage induced by trauma resuscitation.

Injurious mechanical ventilation and end-organ epithelial cell apoptosis and organ dysfunction in an experimental model of acute respiratory distress syndrome.

CONTEXT: Recent clinical trials have demonstrated a decrease in multiple organ dysfunction syndrome (MODS) and mortality in patients with acute respiratory distress syndrome (ARDS) treated with a protective ventilatory strategy. OBJECTIVE: To examine the hypothesis that an injurious ventilatory strategy may lead to end-organ epithelial cell apoptosis and organ dysfunction. DESIGN AND SETTING: In vivo animals: 24 rabbits with acid-aspiration lung injury were ventilated with injurious or noninjurious ventilatory strategies. In vitro: rabbit epithelial cells were exposed to plasma from in vivo rabbit studies. In vivo human: plasma samples from patients included in a previous randomized controlled trial examining a lung protective strategy were analyzed (lung protection group, n = 9 and controls, n = 11). MAIN OUTCOME MEASURES: In vivo animals: biochemical markers of liver and renal dysfunction; apoptosis in end organs. In vitro: induction of apoptosis in LLC-RK1 renal tubular epithelial cells. In vivo human: correlation of plasma creatinine and soluble Fas ligand. RESULTS: The injurious ventilatory strategy led to increased rates of epithelial cell apoptosis in the kidney (mean [SE]: injurious, 10.9% [0.88%]; noninjurious, 1.86% [0.17%]; P<.001) and small intestine villi (injurious, 6.7% [0.66%]; noninjurious, 0.97% [0.14%]; P<.001), and led to the elevation of biochemical markers indicating renal dysfunction in vivo. Induction of apoptosis was increased in LLC-RK1 cells incubated with plasma from rabbits ventilated with injurious ventilatory strategy at 4 hours (P =.03) and 8 hours (P =.002). The Fas:Ig, a fusion protein that blocks soluble Fas ligand, attenuated induction of apoptosis in vitro. There was a significant correlation between changes in soluble Fas ligand and changes in creatinine in patients with ARDS (R = 0.64, P =.002). CONCLUSIONS: Mechanical ventilation can lead to epithelial cell apoptosis in the kidney and small intestine, accompanied by biochemical evidence of organ dysfunction. This may partially explain the high rate of MODS observed in patients with ARDS and the decrease in morbidity and mortality in patients treated with a lung protective strategy.