Downregulation of Mcl-1 has anti-inflammatory pro-resolution effects and enhances bacterial clearance from the lung.

Phagocytes not only coordinate acute inflammation and host defense at mucosal sites, but also contribute to tissue damage. Respiratory infection causes a globally significant disease burden and frequently progresses to acute respiratory distress syndrome, a devastating inflammatory condition characterized by neutrophil recruitment and accumulation of protein-rich edema fluid causing impaired lung function. We hypothesized that targeting the intracellular protein myeloid cell leukemia 1 (Mcl-1) by a cyclin-dependent kinase inhibitor (AT7519) or a flavone (wogonin) would accelerate neutrophil apoptosis and resolution of established inflammation, but without detriment to bacterial clearance. Mcl-1 loss induced human neutrophil apoptosis, but did not induce macrophage apoptosis nor impair phagocytosis of apoptotic neutrophils. Neutrophil-dominant inflammation was modelled in mice by either endotoxin or bacteria (Escherichia coli). Downregulating inflammatory cell Mcl-1 had anti-inflammatory, pro-resolution effects, shortening the resolution interval (Ri) from 19 to 7 h and improved organ dysfunction with enhanced alveolar-capillary barrier integrity. Conversely, attenuating drug-induced Mcl-1 downregulation inhibited neutrophil apoptosis and delayed resolution of endotoxin-mediated lung inflammation. Importantly, manipulating lung inflammatory cell Mcl-1 also accelerated resolution of bacterial infection (Ri; 50 to 16 h) concurrent with enhanced bacterial clearance. Therefore, manipulating inflammatory cell Mcl-1 accelerates inflammation resolution without detriment to host defense against bacteria, and represents a target for treating infection-associated inflammation.

Combined dysfunctions of immune cells predict nosocomial infection in critically ill patients.

BACKGROUND: Nosocomial infection occurs commonly in intensive care units (ICUs). Although critical illness is associated with immune activation, the prevalence of nosocomial infections suggests concomitant immune suppression. This study examined the temporal occurrence of immune dysfunction across three immune cell types, and their relationship with the development of nosocomial infection. METHODS: A prospective observational cohort study was undertaken in a teaching hospital general ICU. Critically ill patients were recruited and underwent serial examination of immune status, namely percentage regulatory T-cells (Tregs), monocyte deactivation (by expression) and neutrophil dysfunction (by CD88 expression). The occurrence of nosocomial infection was determined using pre-defined, objective criteria. RESULTS: Ninety-six patients were recruited, of whom 95 had data available for analysis. Relative to healthy controls, percentage Tregs were elevated 6-10 days after admission, while monocyte HLA-DR and neutrophil CD88 showed broader depression across time points measured. Thirty-three patients (35%) developed nosocomial infection, and patients developing nosocomial infection showed significantly greater immune dysfunction by the measures used. Tregs and neutrophil dysfunction remained significantly predictive of infection in a Cox hazards model correcting for time effects and clinical confounders {hazard ratio (HR) 2.4 [95% confidence interval (CI) 1.1-5.4] and 6.9 (95% CI 1.6-30), respectively, P=0.001}. Cumulative immune dysfunction resulted in a progressive risk of infection, rising from no cases in patients with no dysfunction to 75% of patients with dysfunction of all three cell types (P=0.0004). CONCLUSIONS: Dysfunctions of T-cells, monocytes, and neutrophils predict acquisition of nosocomial infection, and combine additively to stratify risk of nosocomial infection in the critically ill.

Cyclin-dependent kinases 7 and 9 specifically regulate neutrophil transcription and their inhibition drives apoptosis to promote resolution of inflammation.

Terminally differentiated neutrophils are short-lived but the key effector cells of the innate immune response, and have a prominent role in the pathogenesis and propagation of many inflammatory diseases. Delayed apoptosis, which is responsible for their extended longevity, is critically dependent on a balance of intracellular survival versus pro-apoptotic proteins. Here, we elucidate the mechanism by which the cyclin-dependent kinase (CDK) inhibitor drugs such as R-roscovitine and DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) mediate neutrophil apoptosis. We demonstrate (by a combination of microarray, confocal microscopy, apoptosis assays and western blotting) that the phosphorylation of RNA polymerase II by CDKs 7 and 9 is inhibited by R-roscovitine and that specific effects on neutrophil transcriptional capacity are responsible for neutrophil apoptosis. Finally, we show that specific CDK7 and 9 inhibition with DRB drives resolution of neutrophil-dominant inflammation. Thus, we highlight a novel mechanism that controls both primary human neutrophil transcription and apoptosis that could be targeted by selective CDK inhibitor drugs to resolve established inflammation.

Imaging inflammation: molecular strategies to visualize key components of the inflammatory cascade, from initiation to resolution.

Dysregulation of inflammation is central to the pathogenesis of innumerable human diseases. Understanding and tracking the critical events in inflammation are crucial for disease monitoring and pharmacological drug discovery and development. Recent progress in molecular imaging has provided novel insights into spatial associations, molecular events and temporal sequelae in the inflammatory process. While remaining a burgeoning field in pre-clinical research, increasing application in man affords researchers the opportunity to study disease pathogenesis in humans in situ thereby revolutionizing conventional understanding of pathophysiology and potential therapeutic targets. This review provides a description of commonly used molecular imaging modalities, including optical, radionuclide and magnetic resonance imaging, and details key advances and translational opportunities in imaging inflammation from initiation to resolution.

Cyclin-dependent kinase inhibitor drugs as potential novel anti-inflammatory and pro-resolution agents.

The cyclin-dependent kinase inhibitor (CDKi) drugs such as R-roscovitine have emerged as potential anti-inflammatory, pharmacological agents that can influence the resolution of inflammation. Usually, once an inciting inflammatory stimulus has been eliminated, resolution proceeds by prompt, safe removal of dominant inflammatory cells. This is accomplished by programmed cell death (apoptosis) of prominent effector, inflammatory cells typified by the neutrophil. Apoptosis of neutrophils ensures that toxic neutrophil granule contents are securely packaged in apoptotic bodies and expedites phagocytosis by professional phagocytes such as macrophages. A panel of CDKi drugs have been shown to promote neutrophil apoptosis in a concentration- and time-dependent manner and the archetypal CDKi drug, R-roscovitine, overrides the anti-apoptotic effects of powerful survival factors [including lipopolysaccharide (LPS) and granulocyte macrophage-colony stimulating factor (GM-CSF)]. Inflammatory cell longevity and survival signalling is integral to the inflammatory process and any putative anti-inflammatory agent must unravel a complex web of redundancy in order to be effective. CDKi drugs have also been demonstrated to have significant effects on other cell types including lymphocytes and fibroblasts indicating that they may have pleiotropic anti-inflammatory, pro-resolution activity. In keeping with this, CDKi drugs like R-roscovitine have been reported to be efficacious in resolving established animal models of neutrophil-dominant and lymphocyte-driven inflammation. However, the mechanism of action behind these powerful effects has not yet been fully elucidated. CDKs play an integral role in the regulation of the cell cycle but are also recognized as participants in processes such as apoptosis and transcriptional regulation. Neutrophils have functional CDKs, are transcriptionally active and demonstrate augmented apoptosis in response to CDKi drugs, while lymphocyte proliferation and secretory function are inhibited. This review will discuss current understanding of the processes of inflammation and resolution but will focus on CDKis and their potential mechanisms of action.

Relevance of granulocyte apoptosis to resolution of inflammation at the respiratory mucosa.

The respiratory mucosa is responsible for gas exchange and is therefore, of necessity, exposed to airborne pathogens, allergens, and foreign particles. It has evolved a multi-faceted, physical and immune defense system to ensure that in the majority of instances, potentially injurious invaders are repelled. Inflammation, predominantly mediated by effector cells of the granulocyte lineage including neutrophils and eosinophils, is a form of immune defense. Where inflammation proves unable to remove an inciting stimulus, chronic inflammatory disease may supervene because of the potential for tissue damage conferred by the presence of large numbers of frustrated, activated granulocytes. Successful recovery from inflammatory disease and resolution of inflammation rely on the clearance of these cells. Ideally, they should undergo apoptosis prior to phagocytosis by macrophage, dendritic, or epithelial cells. The outcome of inflammation can have serious sequelae for the integrity of the respiratory mucosa leading to disease. Therapeutic strategies to drive resolution of inflammation may be directed at the induction of granulocyte apoptosis and the enhancement of granulocyte clearance.

Modulation of granulocyte apoptosis can influence the resolution of inflammation.

Apoptosis of granulocytes and the subsequent clearance of apoptotic cells are important processes for the successful resolution of inflammation. Signalling pathways, including those involving NF-kappaB (nuclear factor kappaB), MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase) have been shown to be key regulators of inflammatory cell survival and apoptosis in vitro. In addition, manipulation of such pathways in vivo has indicated that they also play a role in the resolution of inflammation. Furthermore, manipulation of proteins directly involved in the control of apoptosis, such as Bcl-2 family members and caspases, can be targeted in vivo to influence inflammatory resolution. Recently, it has been shown that CDK (cyclin-dependent kinase) inhibitor drugs induce caspase-dependent human neutrophil apoptosis possibly by altering levels of the anti-apoptotic Bcl-2 family member, Mcl-1. Importantly, CDK inhibitor drugs augment the resolution of established 'neutrophil-dominant' inflammation by promoting apoptosis of neutrophils. Thus manipulation of apoptotic pathways, together with ensuring macrophage clearance of apoptotic cells, appears to be a viable pharmacological target for reducing established inflammation.

ECM overrides DNA damage-induced cell cycle arrest and apoptosis in small-cell lung cancer cells through beta1 integrin-dependent activation of PI3-kinase.

The emergence of resistance to chemotherapy remains a principle problem in the treatment of small-cell lung cancer (SCLC). We demonstrate that extracellular matrix (ECM) activates phosphatidyl inositol 3-kinase (PI3-kinase) signaling in SCLC cells and prevents etoposide-induced caspase-3 activation and subsequent apoptosis in a beta1 integrin/PI3-kinase-dependent manner. Crucially we show that etoposide and radiation induce G2/M cell cycle arrest in SCLC cells prior to apoptosis and that ECM prevents this by overriding the upregulation of p21(Cip1/WAF1) and p27(Kip1) and the downregulation of cyclins E, A and B. These effects are abrogated by pharmacological and genetic inhibition of PI3-kinase signaling. Importantly we show that chemoprotection is not mediated by altered SCLC cell proliferation or DNA repair. Thus, ECM via beta1 integrin-mediated PI3-kinase activation overrides treatment-induced cell cycle arrest and apoptosis, allowing SCLC cells to survive with persistent DNA damage, providing a model to account for the emergence of acquired drug resistance.

Kinetics of pulmonary neutrophil recruitment and clearance in a natural and spontaneously resolving model of airway inflammation.

BACKGROUND: Neutrophil apoptosis and phagocytic clearance have been proposed as key determinants affecting the resolution of airway inflammation. Objective To determine the kinetics of neutrophil priming, recruitment, activation and subsequent clearance in a naturally occurring equine disease model of neutrophilic pulmonary inflammation. METHODS AND RESULTS: A 5 h mouldy hay/straw challenge in hypersensitive horses induced transient pulmonary dysfunction lasting 4 days. At 24 h circulating neutrophils were primed and displayed delayed rates of spontaneous apoptosis in vitro. Neutrophil numbers in the airspaces peaked at 5 h and then fell abruptly, returning to pre-challenge levels by 4 days. Airspace neutrophils demonstrated increased respiratory burst activity compared with circulating cells and equine neutrophil elastase 2A concentrations increased in parallel with neutrophil numbers indicating in vivo priming and degranulation. The number of apoptotic neutrophils and proportion of alveolar macrophages containing phagocytosed apoptotic neutrophils increased significantly at 24 h and 4 days post-challenge corresponding to the period of most rapid neutrophil clearance. CONCLUSION: This is the first demonstration of spontaneous neutrophil apoptosis and phagocytic removal in a natural disease model of airway inflammation and provides critical kinetic data to support the hypothesis that this clearance pathway plays a central role in the resolution of neutrophilic inflammation.

Expression of V1A and GRP receptors leads to cellular transformation and increased sensitivity to substance-P analogue-induced growth inhibition.

Small-cell lung cancer (SCLC) is a particularly aggressive cancer, which metastasises early. Despite initial sensitivity to radio- and chemo-therapy, it invariably relapses, so that the 2-year survival remains less than 5%. Neuropeptides particularly arginine vasopressin (AVP) and gastrin-releasing peptide (GRP) act as autocrine and paracrine growth factors and the expression of these and their receptors are a hallmark of the disease. Substance-P analogues including [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance-P (SP-D) and [Arg6,D-Trp7,9,NmePhe8]-substance-P (6-11) (SP-G) inhibit the growth of SCLC cells by modulating neuropeptide signalling. We show that GRP and V1A receptors expression leads to the development of a transformed phenotype. Addition of neuropeptide provides some protection from etoposide-induced cytotoxicity. Receptor expression also leads to an increased sensitivity to substance-P analogue-induced growth inhibition. We show that SP-D and SP-G act as biased agonists at GRP and V1A receptors causing blockade of Gq-mediated Ca2+ release while directing signalling to activate ERK via a pertussis toxin-sensitive pathway. This is the first description of biased agonism at V1A receptors. This unique pharmacology governs the antiproliferative properties of these agents and highlights their potential therapeutic potential for the treatment of SCLC and particularly in tumours, which have developed resistance to chemotherapy.

Regulation of granulocyte apoptosis by NF-kappaB.

Granulocyte apoptosis is a crucial part of the successful resolution of inflammation. In vitro results show that activation of NF-kappaB (nuclear factor kappaB) in granulocytes is a survival mechanism. NF-kappaB inhibitors increase the rate of constitutive apoptosis in neutrophils and eosinophils and cause these cells to respond to the pro-apoptotic effects of TNF-alpha (tumour necrosis factor-alpha). Results from both in vivo and in vitro experiments suggest that there are at least two important waves of NF-kappaB activation in inflammatory loci, which increase the expression of COX-2 (cyclooxygenase-2), itself an NF-kappaB controlled gene. The first wave causes the production of inflammatory mediators such as PGE2 (prostaglandin E2), allowing the establishment of inflammation. The second wave causes the synthesis of PGD2 and its metabolites that induce granulocyte apoptosis by inhibiting NF-kappaB activation. These metabolites may therefore be important physiological mediators controlling the resolution of inflammation. Although NF-kappaB is an important target for anti-inflammatory therapy, the timing of inhibition in vivo may be crucial, to ensure that production of PGD2 and its sequential metabolites can occur.

Regulation of granulocyte apoptosis by hemopoietic growth factors, cytokines and drugs: potential relevance to allergic inflammation.

It has become apparent that the resolution of inflammation depends on the removal of unwanted inflammatory cells, a process governed by physiological apoptosis and non-inflammatory clearance of apoptotic cells. Granulocytes are central to many of the pathophysiological consequences of uncontrolled inflammatory reactions. Hemopoietic factors and cytokines play a critical role in regulating the longevity of these cells in vitro and in vivo. Here we review the progress that has been made in the understanding of granulocyte apoptosis and the implications for immunotherapy and pharmacological strategies in the treatment of allergic inflammatory diseases for therapeutic gain.

Glucocorticoid-mediated regulation of granulocyte apoptosis and macrophage phagocytosis of apoptotic cells: implications for the resolution of inflammation.

Glucocorticoids represent one of the most effective clinical treatments for a range of inflammatory conditions, including severe acute inflammation. Although glucocorticoids are known to affect processes involved in the initiation of inflammation, the influence of glucocorticoids on the mechanisms by which acute inflammation normally resolves have received less attention. Apoptosis of granulocytes present at inflamed sites leads to their rapid recognition and internalisation by macrophages, a process which may be important for resolution of inflammation. However, if clearance of either eosinophils or neutrophils is impaired, these cells rapidly undergo secondary necrosis leading to release of pro-inflammatory mediators from the phagocyte, potentially prolonging inflammatory responses. Physiologically relevant concentrations of glucocorticoids accelerate eosinophil apoptosis whilst delaying neutrophil apoptosis during in vitro culture. Here we discuss key pathways regulating the granulocyte apoptotic programme and summarise the effects of glucocorticoids on monocyte differentiation and the consequent changes to apoptotic cell clearance capacity. Definition of the mechanisms underlying resolution of inflammatory responses following glucocorticoid treatment may unveil new targets for modulation of inflammatory disease, allowing co-ordinated augmentation of granulocyte apoptosis together with increased macrophage capacity for clearance of apoptotic cells.

Regulation of pulmonary and systemic bacterial lipopolysaccharide responses in transgenic mice expressing human elafin.

The control of lung inflammation is of paramount importance in a variety of acute pathologies, such as pneumonia, the acute respiratory distress syndrome, and sepsis. It is becoming increasingly apparent that local innate immune responses in the lung are negatively influenced by systemic inflammation. This is thought to be due to a local deficit in cytokine responses by alveolar macrophages and neutrophils following systemic bacterial infection and the development of a septic response. Recently, using an adenovirus-based strategy which overexpresses the human elastase inhibitor elafin locally in the lung, we showed that elafin is able to prime lung innate immune responses. In this study, we generated a novel transgenic mouse strain expressing human elafin and studied its response to bacterial lipopolysaccharide (LPS) when the LPS was administered locally in the lungs and systemically. When LPS was delivered to the lungs, we found that mice expressing elafin had lower serum-to-bronchoalveolar lavage ratios of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), macrophage inflammatory protein 2, and monocyte chemoattractant protein 1, than wild-type mice. There was a concomitant increase in inflammatory cell influx, showing that there was potential priming of innate responses in the lungs. When LPS was given systemically, the mice expressing elafin had reduced levels of serum TNF-alpha compared to the levels in wild-type mice. These results indicate that elafin may have a dual function, promoting up-regulation of local lung innate immunity while simultaneously down-regulating potentially unwanted systemic inflammatory responses in the circulation.

Increased gastrin-releasing peptide (GRP) receptor expression in tumour cells confers sensitivity to [Arg6,D-Trp7,9,NmePhe8]-substance P (6-11)-induced growth inhibition.

[Arg(6),D-Trp(7,9),N(me)Phe(8)]-substance P (6-11) (SP-G) is a novel anticancer agent that has recently completed phase I clinical trials. SP-G inhibits mitogenic neuropeptide signal transduction and small cell lung cancer (SCLC) cell growth in vitro and in vivo. Using the SCLC cell line series GLC14, 16 and 19, derived from a single patient during the clinical course of their disease and the development of chemoresistance, it is shown that there was an increase in responsiveness to neuropeptides. This was paralleled by an increased sensitivity to SP-G. In a selected panel of tumour cell lines (SCLC, non-SCLC, ovarian, colorectal and pancreatic), the expression of the mitogenic neuropeptide receptors for vasopressin, gastrin-releasing peptide (GRP), bradykinin and gastrin was examined, and their sensitivity to SP-G tested in vitro and in vivo. The tumour cell lines displayed a range of sensitivity to SP-G (IC(50) values from 10.5 to 119 microM). The expression of the GRP receptor measured by reverse transcriptase-polymerase chain reaction, correlated significantly with growth inhibition by SP-G. Moreover, introduction of the GRP receptor into rat-1A fibroblasts markedly increased their sensitivity to SP-G. The measurement of receptor expression from biopsy samples by polymerase chain reaction could provide a suitable diagnostic test to predict efficacy to SP-G clinically. This strategy would be of potential benefit in neuropeptide receptor-expressing tumours in addition to SCLC, and in tumours that are relatively resistant to conventional chemotherapy.

Nitric oxide: a key regulator of myeloid inflammatory cell apoptosis.

Apoptosis of inflammatory cells is a critical event in the resolution of inflammation, as failure to undergo this form of cell death leads to increased tissue damage and exacerbation of the inflammatory response. Many factors are able to influence the rate of apoptosis in neutrophils, eosinophils, monocytes and macrophages. Among these is the signalling molecule nitric oxide (NO), which possesses both anti- and proapoptotic properties, depending on the concentration and flux of NO, and also the source from which NO is derived. This review summarises the differential effects of NO on inflammatory cell apoptosis and outlines potential mechanisms that have been proposed to explain such actions.

Dissociation of DNA fragmentation from other hallmarks of apoptosis in nitric oxide-treated neutrophils: differences between individual nitric oxide donor drugs.

The events of apoptotic cell death can be experimentally dissociated from each other in certain cell types. Here we demonstrate the ability of structurally diverse nitric oxide (NO) donating compounds to delay or enhance neutrophil apoptosis and to differentially influence distinct parameters of programmed cell death. We provide evidence that high concentrations of the NO donors GEA 3162, SPER/NO, and DEA/NO induce morphological and biochemical markers of neutrophil apoptosis, but that only DEA/NO causes a concomitant increase in DNA fragmentation as evidenced by nuclear propidium iodide intercalation and the classical laddering pattern of electrophoresed DNA. In contrast, both GEA 3162 and SPER/NO inhibit DNA cleavage in a time- and concentration-dependent manner. We are the first to show that DNA fragmentation can be dissociated from other changes of apoptosis in NO-treated neutrophils and that it may therefore be inappropriate to assess NO-induced apoptosis solely by measuring DNA fragmentation in this cell type.

Alveolar macrophage activity and the pulmonary complications of haematopoietic stem cell transplantation.

BACKGROUND: The success of haematopoietic (bone marrow or peripheral blood) stem cell transplantation (SCT) is compromised by pulmonary complications. We hypothesised that a proinflammatory alveolar microenvironment, reflected in alveolar macrophage (AM) cytokine production, would predispose to such complications. METHODS: AM were isolated from adult SCT recipients by bronchoalveolar lavage before SCT (n=32) and during post-transplant pancytopenia (n=23). Concentrations of tumour necrosis factor (TNF)alpha, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin (IL)-1 beta, IL-6, and IL-8 in 24 hour AM culture medium were measured by enzyme linked immunosorbent assay and compared with both the occurrence of post-SCT lung disease and with subjects' previous respiratory histories. RESULTS: Eleven subjects developed lung disease within 6 months of SCT. These subjects had higher median pre-transplant AM TNFalpha (8 (IQR 1-8) v 2 (1-5) ng/10(6)AM, p=0.01, median difference (D) = 3, 95% CI 0.1 to 7), GM-CSF (5 (0.7-8) v 0.2 (0.1-0.8), p=0.006, D = 4, 95% CI 0.5 to 7), and IL-6 (0.5 (0.1-1) v 0.1 (0.02-0.3), p=0.049, D = 0.3, 95% CI 0.0002 to 1) production than remaining subjects; IL-1 beta and IL-8 did not differ. During pancytopenia high AM GM-CSF production again predicted later lung disease (1 (0.7-9) v 0.1 (0.06-0.3), p=0.01, D = 1, 95% CI 0.1 to 6). A history of recent chest disease was associated with high AM TNFalpha and GM-CSF production and with post-SCT lung disease. Pre-SCT lung function was unrelated to post-SCT lung disease. CONCLUSIONS: Recent respiratory disease and persistent proinflammatory AM behaviour detectable before transplantation are associated with lung disease following SCT. These associations may prove useful in pre-transplant risk assessment.

The regulation of interleukin-8 by hypoxia in human macrophages--a potential role in the pathogenesis of the acute respiratory distress syndrome (ARDS).

BACKGROUND: The acute respiratory distress syndrome (ARDS) represents a form of severe acute inflammatory lung disease. We have previously demonstrated significantly raised interleukin-8 (IL-8) levels in the lungs of at-risk patients that progress to ARDS, and identified the alveolar macrophage as an important source of this chemokine. We wished to extend this study in a well-defined group of patients with major trauma, and to investigate potential mechanisms for rapid intrapulmonary IL-8 generation. MATERIALS AND METHODS: Patients with major trauma underwent bronchoalveolar lavage (BAL) and IL-8 levels were measured in BAL fluid by ELISA. Human macrophages were derived from peripheral blood monocytes from healthy volunteers. Rabbit alveolar macrophages were obtained from ex-vivo lavage of healthy rabbit lungs. Macrophages were culture under normoxic or hypoxic (PO2 26 mmHg) conditions. IL-8 and other proinflammatory mediator expression was measured by ELISA, northern blotting or multi-probe RNase protection assay. RESULTS: In patients with major trauma, IL-8 levels were significantly higher in patients that progressed to ARDS compared to those that did not (n = 56, P = 0.0001). High IL-8 levels negatively correlated with PaO2/FiO2 (r = -0.56, P < 0.001). In human monocyte derived macrophages hypoxia rapidly upregulated IL-8 protein (within 2 hours) and mRNA expression (within 30 mins). Acute hypoxia also increased rabbit alveolar macrophage IL-8 expression. Hypoxia increased DNA binding activity of AP-1 and C/EBP but not NF-kappaB. Hypoxia induced HIF-1 expression, but cobaltous ions and desferrioxamine did not mimic hypoxic IL-8 induction. Hypoxia downregulated a range of other proinflammatory mediators, including MCP-1 and TNF-alpha. Both the pattern of cytokine expression and transcription factor activation by hypoxia was different to that seen with endotoxin. CONCLUSIONS: Rapidly raised intrapulmonary IL-8 levels are associated with ARDS progression in patients with major trauma. Acute hypoxia, a clinically relevant stimulus, rapidly and selectively upregulates IL-8 in macrophages associated with a novel pattern of transcription factor activation. Acute hypoxia may represent one of potentially several proinflammatory stimuli responsible for rapid intrapulmonary IL-8 generation in patients at-risk of ARDS.

Cyclic AMP regulation of neutrophil apoptosis occurs via a novel protein kinase A-independent signaling pathway.

The second messenger molecule cyclic AMP dramatically modulates the apoptotic program in a wide variety of cells, accelerating apoptosis in some and delaying the rate of apoptosis in others. Human neutrophil apoptosis, a process that regulates the fate and numbers of these potentially histotoxic cells in inflammatory sites, is profoundly delayed by the cell-permeable analog of cyclic AMP, dibutyryl-cAMP. We have investigated the mechanisms underlying cyclic AMP-mediated delay of neutrophil apoptosis, and we show that cyclic AMP inhibits loss of mitochondrial potential occurring during constitutive neutrophil apoptosis. Furthermore, we demonstrate that cyclic AMP also suppresses caspase activation in these inflammatory cells. Despite increasing protein kinase A activity, this kinase is unlikely to mediate the effect of cyclic AMP on apoptosis because blockade of protein kinase A activation did not influence the survival effects of cyclic AMP. Further investigation of the signaling mechanism demonstrated that the delay of apoptosis is independent of phosphoinositide 3-kinase and MAPK activation. Our results suggest cyclic AMP delays neutrophil apoptosis via a novel, reversible, and transcriptionally independent mechanism. We show that proteasome activity in the neutrophil is vitally involved in this process, and we suggest that a balance of pro-apoptotic and anti-apoptotic proteins plays a key role in the powerful ability of cyclic AMP to delay neutrophil death.