Prolonged survival of neutrophils is essential for determining the progression and severity of inflammatory and immune-mediated disorders, including gouty arthritis. Survivin, an anti-apoptotic molecule, has been described as a regulator of cell survival. This study aims to examine the effects of YM155 treatment, a survivin selective suppressant, in maintaining neutrophil survival in vitro and in vivo experimental settings of neutrophilic inflammation.

BALB/c mice were injected with monosodium urate (MSU) crystals and treated with YM155 (intra-articularly) at the peak of inflammatory response. Leukocyte recruitment, apoptosis neutrophil and efferocytosis were determined by knee joint wash cell morphology counting and flow cytometry. Resolution interval (Ri) was quantified by neutrophil infiltration, monitoring the amplitude and duration of the inflammation. Cytokine production was measured by ELISA. Mechanical hypernociception was assessed using an electronic von Frey aesthesiometer. Efferocytosis was evaluated in zymosan-induced neutrophilic peritonitis. Survivin and cleaved caspase-3 expression was determined in human neutrophils by flow cytometry.

Survivin was expressed in neutrophils during MSU-induced gout, and the treatment with YM155 reduced survivin expression and shortened Ri from ∼8 h observed in vehicle-treated mice to ∼5.5 h, effect accompanied by increased neutrophil apoptosis and efferocytosis, both crucial for the inflammation resolution. Reduced IL-1β and CXCL1 levels were also observed in periarticular tissue. YM155 reduced histopathological score and hypernociceptive response. In human neutrophils, lipopolysaccharide (LPS) increased survivin expression, whereas survivin inhibition with YM155 induced neutrophil apoptosis, with activation of caspase-3.

Survivin may be a promising therapeutic target to control neutrophilic inflammation.

Delayed neutrophil apoptosis during sepsis may impact neutrophil organ accumulation and tissue immune homeostasis. Elucidating the mechanisms underlying neutrophil apoptosis may help identify potential therapeutic targets. Glycolysis is critical to neutrophil activities during sepsis. However, the precise mechanisms through which glycolysis regulates neutrophil physiology remain under-explored, especially those involving the non-metabolic functions of glycolytic enzymes. In the present study, the impact of programmed death ligand-1 (PD-L1) on neutrophil apoptosis was explored. The regulatory effect of the glycolytic enzyme, pyruvate kinase M2 (PKM2), whose role in septic neutrophils remains unaddressed, on neutrophil PD-L1 expression was also explored.

Peripheral blood neutrophils were isolated from patients with sepsis and healthy controls. PD-L1 and PKM2 levels were determined by flow cytometry and Western blotting, respectively. Dimethyl sulfoxide (DMSO)-differentiated HL-60 cells were stimulated with lipopolysaccharide (LPS) as an in vitro simulation of septic neutrophils. Cell apoptosis was assessed by annexin V/propidium iodide (annexin V/PI) staining, as well as determination of protein levels of cleaved caspase-3 and myeloid cell leukemia-1 (Mcl-1) by Western blotting. An in vivo model of sepsis was constructed by intraperitoneal injection of LPS (5 mg/kg) for 16 h. Pulmonary and hepatic neutrophil infiltration was assessed by flow cytometry or immunohistochemistry.

PD-L1 level was elevated on neutrophils under septic conditions. Administration of neutralizing antibodies against PD-L1 partially reversed the inhibitory effect of LPS on neutrophil apoptosis. Neutrophil infiltration into the lung and liver was also reduced in PD-L1-/- mice 16 h after sepsis induction. PKM2 was upregulated in septic neutrophils and promoted neutrophil PD-L1 expression both in vitro and in vivo. In addition, PKM2 nuclear translocation was increased after LPS stimulation, which promoted PD-L1 expression by directly interacting with and activating signal transducer and activator of transcription 1 (STAT1). Inhibition of PKM2 activity or STAT1 activation also led to increased neutrophil apoptosis.

In this study, a PKM2/STAT1-mediated upregulation of PD-L1 on neutrophils and the anti-apoptotic effect of upregulated PD-L1 on neutrophils during sepsis were identified, which may result in increased pulmonary and hepatic neutrophil accumulation. These findings suggest that PKM2 and PD-L1 could serve as potential therapeutic targets.

Neutrophils or polymorphonuclear leukocytes (PMN) are key participants in the innate immune response for their ability to execute different effector functions. These cells express a vast array of membrane receptors that allow them to recognize and eliminate infectious agents effectively and respond appropriately to microenvironmental stimuli that regulate neutrophil functions, such as activation, migration, generation of reactive oxygen species, formation of neutrophil extracellular traps, and mediator secretion, among others. Currently, it has been realized that activated neutrophils can accomplish their effector functions and simultaneously activate mechanisms of cell death in response to different intracellular or extracellular factors. Although several studies have revealed similarities between the mechanisms of cell death of neutrophils and other cell types, neutrophils have distinctive properties, such as a high production of reactive oxygen species (ROS) and nitrogen species (RNS), that are important for their effector function in infections and pathologies such as cancer, autoimmune diseases, and immunodeficiencies, influencing their cell death mechanisms. The present work offers a synthesis of the conditions and molecules implicated in the regulation and activation of the processes of neutrophil death: apoptosis, autophagy, pyroptosis, necroptosis, NETosis, and necrosis. This information allows to understand the duality encountered by PMNs upon activation. The effector functions are carried out to eliminate invading pathogens, but in several instances, these functions involve activation of signaling cascades that culminate in the death of the neutrophil. This process guarantees the correct elimination of pathogenic agents, damaged or senescent cells, and the timely resolution of the inflammation that is essential for the maintenance of homeostasis in the organism. In addition, they alert the organism when the immunological system is being deregulated, promoting the activation of other cells of the immune system, such as B and T lymphocytes, which produce cytokines that potentiate the microbicide functions.

Ischemic postconditioning (I/Post) is an endogenous protection mechanism that reduces injury induced by ischemia-reperfusion (I/R). It remains controversial whether I/Post protects against I/R injury to the aging heart. The long non-coding RNA, H19 protects H9c2 cells against hypoxia-induced injury. This study aimed to elucidate the role of H19 in the hypoxic postconditioning (H/Post) of aged cardiomyocytes.

Senescence induced by D-galactose in primary cardiomyocytes from neonatal Sprague-Dawley rats was measured by senescence-associated β-galactosidase staining. Hypoxic injury was evaluated by cell viability and apoptosis assays. H19 expression before and after hypoxia-reoxygenation (H/R) and H/Post was evaluated by real-time polymerase chain reactions. miR-29b-3p-binding sites in H19 and the cellular inhibitor of apoptosis protein 1 (cIAP1) were predicted by bioinformatics analysis, and interaction was verified by luciferase assay. The effects of altered H19, miR-29b-3p, and cIAP1 expression on the viability and apoptosis of senescent cardiomyocytes following H/Post were determined.

H/Post prevented H/R injury in normal but not senescent cardiomyocytes. H19 expression was remarkably down-regulated after H/Post in senescent compared with normal cardiomyocytes. Small interfering RNA-mediated knockdown of H19 in senescent cardiomyocytes increased H/Post-induced injury. miR-29b-3p was regulated by H19 and led to a greater injury. miR-29b-3p directly targeted the 3'-untranslated region of cIAP1 and suppressed its expression. Furthermore, knockdown of cIAP1 damaged senescent cardiomyocytes following H/Post.

These findings suggest that H19 mediated the antiapoptotic effect of H/Post against H/R-induced injury to aged cardiomyocytes by inhibiting miR-29b-3p expression.

The innate immune system is the first line of host defense against invading microorganisms. Polymorphonuclear leukocytes (PMNs or neutrophils) are the most abundant leukocyte in humans and essential to the innate immune response against invading pathogens. Compared to the acquired immune response, which requires time to develop and is dependent on previous interaction with specific microbes, the ability of neutrophils to kill microorganisms is immediate, nonspecific, and not dependent on previous exposure to microorganisms. Historically, studies of PMN-pathogen interaction focused on the events leading to killing of microorganisms, such as recruitment/chemotaxis, transmigration, phagocytosis, and activation, whereas postphagocytosis sequelae were infrequently considered. In addition, it was widely accepted that human neutrophils possessed limited capacity for new gene transcription and thus, relatively little biosynthetic capacity. This notion has changed dramatically within the past 20 years. Further, there is now more effort directed to understand the events occurring in PMNs after killing of microbes. Herein, we give an updated review of the systems biology-level approaches that have been used to gain an enhanced view of the role of neutrophils during host-pathogen interaction and neutrophil-mediated diseases. We anticipate that these and future systems-level studies will continue to provide information important for understanding, treatment, and control of diseases caused by pathogenic microorganisms. This article is categorized under: Physiology > Organismal Responses to Environment Physiology > Mammalian Physiology in Health and Disease Biological Mechanisms > Cell Fates.

Aberrant apoptosis is a disease susceptibility mechanism relevant for asthma, whereby fragility of the airway epithelium and enhanced survival of inflammatory cells, contributes to its pathogenesis and prolongation. Cellular Inhibitor of Apoptosis Proteins (cIAP) suppress apoptosis, and participate in the immune response. In this study, single nucleotide polymorphisms (SNP) in the BIRC2 (codes cIAP1) and BIRC3 (cIAP2) genes were evaluated for an association with asthma.

Caucasian asthmatic (n = 203) and control (n = 198) subjects were selected from participants in the North West Adelaide Health Study. SNPs (n = 9) spanning the consecutively positioned BIRC2 and BIRC3 genes, were selected using a haplotype tagging approach. Alleles and haplotype associations were analysed by logistic regression, assuming an additive genetic model, and adjusted for gender and atopy.

The frequency of the minor allele for the BIRC3 SNP rs3460 was significantly lower in asthmatics compared to the control cases (P = 0.046). BIRC3 SNPs rs7928663 and rs7127583 associated with a reduction in eosinophil and neutrophil abundance when assessed across the study population (multivariate P values = 0.002, and 0.005, respectively). Further, the frequency of a haplotype tagged by rs3460, rs7928663 and rs7127583 was reduced in the asthma sub group (P = 0.05), while the presence of the major allele for rs7928663 associated with an increased load of circulating eosinophils and neutrophils (multivariate P value = 0.001).

Polymorphisms in the BIRC3 gene, but not BIRC2, are associated with a protective effect with regards to asthma susceptibility, and a reduced load of inflammatory cells.

Neutrophils not only play a critical role as a first line of defense against bacteria and fungi infections but also contribute to tissue injury associated with autoimmune and inflammatory diseases. Neutrophils are rapidly and massively recruited from the circulation into injured tissues displaying an impressive arsenal of toxic weapons. Although effective in their ability to kill pathogens, these weapons were equally effective to induce tissue damage. Therefore, the inflammatory activity of neutrophils must be regulated with exquisite precision and timing, a task mainly achieved through a complex network of mechanisms, which regulate neutrophil survival. Neutrophils have the shortest lifespan among leukocytes and usually die via apoptosis although new forms of cell death have been characterized over the last few years. The lifespan of neutrophils can be dramatically modulated by a large variety of agents such as cytokines, pathogens, danger-associated molecular patterns as well as by pharmacological manipulation. Recent findings shed light about the complex mechanisms responsible for the regulation of neutrophil survival in different physiological, pathological, and pharmacological scenarios. Here, we provide an updated review on the current knowledge and new findings in this field and discuss novel strategies that could be used to drive the resolution of neutrophil-mediated inflammatory diseases.

The development of therapeutic resistance, after hormone or chemotherapy for example, is the underlying basis for most cancer deaths. Exposure to anticancer therapies induces expression of many stress related proteins, including small heat shock proteins (HSPs). HSPs interact with various client proteins to assist in their folding and enhance the cellular recovery from stress, thus restoring protein homeostasis and promoting cell survival. The vents of cell stress and cell death are linked, as the induction of molecular chaperones appears to function at key regulatory points in the control of apoptosis. On the basis of these observations and on the role of molecular chaperones in the regulation of steroid receptors, kinases, caspases, and other protein remodelling events involved in chromosome replication and changes in cell structure, it is not surprising that molecular chaperones have been implicated in the control of cell growth and in resistance to various anticancer treatments that induce apoptosis. Recently, several molecular chaperones such as Clusterin and HSP27 have been reported to be involved in development and progression of hormone-refractory prostate cancer. In this review, we address some of the molecular and cellular events initiated by treatment induced stress, and discuss the potential role of chaperone proteins as targets for prostate cancer treatment.

Neutrophil apoptosis is essential for the resolution of inflammation but is delayed by several inflammatory mediators. In such terminally differentiated cells it has been uncertain whether these agents can inhibit apoptosis through transcriptional regulation of anti-death (Bcl-X(L), Mcl-1, Bcl2A1) or BH3-only (Bim, Bid, Puma) Bcl2-family proteins. We report that granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-α prevent the normal time-dependent loss of Mcl-1 and Bcl2A1 in neutrophils, and we demonstrate that they cause an NF-κB-dependent increase in Bcl-X(L) transcription/translation. We show that GM-CSF and TNF-α increase and/or maintain mRNA levels for the pro-apoptotic BH3-only protein Bid and that GM-CSF has a similar NF-κB-dependent effect on Bim transcription and BimEL expression. The in-vivo relevance of these findings was indicated by demonstrating that GM-CSF is the dominant neutrophil survival factor in lung lavage from patients with ventilator-associated pneumonia, confirming an increase in lung neutrophil Bim mRNA. Finally GM-CSF caused mitochondrial location of Bim and a switch in phenotype to a cell that displays accelerated caspase-9-dependent apoptosis. This study demonstrates the capacity of neutrophil survival agents to induce a paradoxical increase in the pro-apoptotic proteins Bid and Bim and suggests that this may function to facilitate rapid apoptosis at the termination of the inflammatory cycle.

Neutrophil survival is regulated by a complex convergence of different pathways. The present review analyzes these pathways and discusses how neutrophil survival is modulated during the course of inflammatory reactions.

Although apoptosis appears to be the predominant cell death pathway in the neutrophil, recent data reveal that neutrophil survival is also regulated by a number of nonconventional pathways including NETosis, autophagic cell death, and other less characterized mechanisms. This supports an even more complex picture of the mechanisms involved in the regulation of neutrophil survival than previously thought.

The control of neutrophil survival is central to homoeostasis and resolution of inflammation. Cell death is usually discussed dichotomously in terms of apoptosis or necrosis. There are two main pathways responsible for the stimulation of apoptosis; a death receptor pathway triggered by Fas, tumor necrosis factor alpha, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and a mitochondrial pathway stimulated by a number of stressors such as DNA damage, growth factor deprivation, and chemotherapy drugs. Nonconventional pathways of neutrophil death include NETosis and autophagic cell death as well as a number of poorly characterized mechanisms. Understanding the integrated pathways responsible for the control of neutrophil survival holds therapeutic promise in infectious and inflammatory diseases.

The innate immune system is the first line of host defense against invading microorganisms. Polymorphonuclear leukocytes (PMNs or neutrophils) are the most abundant leukocyte in humans and essential to the innate immune response against invading pathogens. Compared with the acquired immune response, which requires time to develop and is dependent on previous interaction with specific microbes, the ability of neutrophils to kill microorganisms is immediate, non-specific, and not dependent on previous exposure to microorganisms. Historically, studies on PMN-pathogen interaction focused on the events leading to killing of microorganisms, such as recruitment/chemotaxis, transmigration, phagocytosis, and activation, whereas post-phagocytosis sequelae were infrequently considered. In addition, it was widely accepted that human neutrophils possessed limited capacity for new gene transcription and thus, relatively little biosynthetic capacity. This notion has changed dramatically within the past decade. Further, there is now more effort directed to understand the events occurring in PMNs after killing of microbes. Herein we review the systems biology-level approaches that have been used to gain an enhanced view of the role of neutrophils during host-pathogen interaction. We anticipate that these and future systems-level studies will ultimately provide information critical to our understanding, treatment, and control of diseases caused by pathogenic microorganisms.

The mammalian immune system comprises a complex network of physical and molecular elements that protect the individual from danger in the environment. An evolutionarily ancient innate immune system recognizes danger through pattern-recognition receptors that are encoded in the genome and mobilizes a rapid and potent but nonspecific response. This response is responsible for the clinical syndromes of sepsis and the multiple organ dysfunction syndrome. The adaptive immune system is highly selective in its targets and is endowed with memory but is slow in initial activation. Critical illness results in derangements of all components of the immune response, but the very complexity of the process has frustrated attempts to correct these derangements and to affect significantly the clinical course of sepsis.

The etiology of Crohn's disease (CD) remains unknown, and the defective function of neutrophils appears to be associated with this pathology. Neutrophils undergo spontaneous apoptosis which, if not tightly regulated, can induce the development of chronic inflammatory disease. The Bcl-2 protein family is also involved in the regulation of neutrophil apoptosis.

This study investigated the apoptosis and expression of some regulatory factors in CD patient and control polymorphonuclear neutrophils (PMN) in suspension and in adhesion on fibronectin, an extracellular matrix protein. These 2 conditions mimic circulating neutrophils before they are recruited at the intestinal levels, and their adhesion to tissue.

Apoptosis in CD patient PMN was delayed in suspension and accelerated in adhesion, which is the opposite of what happens in controls. Higher levels of Bax, Bcl-2, and Mcl-1 proteins were registered in freshly isolated CD patient PMN, in contrast to controls, in which Bcl-2 protein was undetectable. Among the studied pro- and antiapoptotic factors, Bax levels seem to be mainly related to the difference in apoptosis between PMN of CD patients and controls.

For the first time it has been demonstrated by direct experimental evidence that apoptosis in CD patient PMN is regulated differently from that of control PMN. Abnormal expression of regulating apoptosis proteins is shown in CD patient PMN. These data suggest that the defective functionality of neutrophils can be the early event responsible for the altered mucosal immune response in CD, and that neutrophil apoptosis may offer a new target for specific drugs and therapy tools.

During cellular stress, translation persists or increases for a number of stress-responsive proteins, including cellular inhibitor of apoptosis 2 (cIAP2). The cIAP2 transcript includes a very long (2.78-kb) 5' untranslated region (UTR) with an unusually high number of upstream AUGs (uAUGs), i.e., 64, and a stable predicted secondary structure (DeltaG congruent with -620 kcal/mol) that should completely block conventional scanning-dependent translation initiation. This region did not facilitate internal ribosome entry in vitro or when RNA reporter transcripts were transfected into cells. However, several structural features within the cIAP2 5' UTR were observed to be nearly identical to those required for ribosome shunting in cauliflower mosaic virus RNA and are well conserved in cIAP2 orthologs. Selective mutation revealed that the cIAP2 mRNA mediates translation exclusively via ribosome shunting that bypasses 62 uAUGs. In addition, shunting efficiency was altered by stress and was greatly facilitated by a conserved RNA folding domain (1,470 to 1,877 nucleotides upstream) in a region not scanned by shunting ribosomes. This arrangement suggests that regulation of cIAP2 shunting may involve recruitment of RNA binding proteins to modulate the efficiency of translation initiation.

Kupffer cells are important for bacterial clearance and cytokine production during infection. We have previously shown that severe infection with Pseudomonas aeruginosa ultimately results in loss of Kupffer cells and hepatic bacterial clearance. This was associated with prolonged hepatic inflammation. However, there is a period of time during which there is both preserved hepatic bacterial clearance and increased circulating TNF-alpha. We hypothesized that early during infection, Kupffer cells are protected against TNF-alpha-induced cell death via activation of survival pathways. KC13-2 cells (a clonal Kupffer cell line) were treated with P. aeruginosa (strain PA103), TNF-alpha, or both. At early time points, TNF-alpha induced caspase-mediated cell death, but PA103 did not. When we combined the two exposures, PA103 protected KC13-2 cells from TNF-alpha-induced cell death. PA103, in the setting of TNF exposure, stabilized the X-chromosome-linked inhibitor of apoptosis protein (XIAP). Stabilization of XIAP can occur via PI3K and Akt. We found that PA103 activated Akt and that pretreatment with the PI3K inhibitor, LY294002, prevented PA103-induced protection against TNF-alpha-induced cell death. The effects of LY294002 included decreased levels of XIAP and increased amounts of cleaved caspase-3. Overexpression of Akt mimicked the effects of PA103 by protecting cells from TNF-alpha-induced cell death and XIAP cleavage. Transfection with a stable, nondegradable XIAP mutant also protected cells against TNF-alpha-induced cell death. These studies demonstrate that P. aeruginosa delays TNF-alpha-induced Kupffer cell death via stabilization of XIAP.

Neutrophil apoptosis is impaired in neonates, and this contributes to prolonged inflammation and tissue injury in infants after infection or trauma. In the present studies, we investigated whether labor generates mediators that further suppress apoptosis. We found that neutrophil apoptosis was reduced in neonates exposed to labor, when compared with infants delivered by cesarean section before labor. This was not due to alterations in caspase-3 or inhibitor of apoptosis protein-2 (IAP-2). In contrast, labor primed neutrophils to express tumor necrosis factor alpha (TNF-alpha), suggesting that proinflammatory mediators contribute to reduced apoptosis after labor. Eicosanoids generated via cyclooxygenase-2 (Cox-2) and lipoxygenase (Lox) also regulate neutrophil apoptosis. 15-Lox, which generates proapoptotic lipoxins, but not Cox-2, was greater in neutrophils before labor, relative to cells exposed to labor. Anti-inflammatory eicosanoids exert their effects in part via peroxisome proliferator-activated receptor gamma (PPAR-gamma). Expression of gelatinase-associated lipocalin and catalase, two markers of PPAR-gamma activity, were increased in neonatal neutrophils before labor, relative to cells exposed to labor. These findings suggest that the anti-inflammatory environment is maintained before labor, in part, by eicosanoids. Although increased neutrophil longevity after labor is important for host defense in the immediate newborn period, it may contribute to inflammatory or oxidative injury in susceptible infants.

Delayed neutrophil apoptosis is characteristic of sepsis and may accentuate organ injury. It has been shown that PI-3K and MAPK pathways provide survival signaling in neutrophils. In this study, we demonstrate that neutrophils isolated from septic rats are resistant to apoptosis in comparison with the cells from normal animals. In contrast to normal serum, septic sera induced strong phosphorylation of AKT and p44/42 in neutrophils obtained from normal rats, resulting in marked resistance of these cells to apoptosis. Protection from apoptosis by septic sera was abrogated completely by inhibition of PI-3K and partially diminished by MEK inhibition. Increased neutrophil survival in septic rats was associated with increased levels of Bcl-xL in neutrophils and decreased levels of Bim expression. In vivo blockade of C5a in cecal ligation and puncture rats by anti-C5a antibody markedly restored the susceptibility of neutrophils to undergo apoptosis. C5a activated AKT and p44/42 and also enhanced X-linked inhibitor of apoptosis expression in neutrophils. LPS and C5a were able to induce Bcl-xL expression. Thus, neutrophil survival signals derived from effects of septic sera could be linked to activation of ERK1/2 and PI-3K, increased antiapoptotic protein expression, and ultimately, delayed neutrophil apoptosis.

Data are presented on pharmacodynamic (PD) method validation and preliminary clinical qualification of three PD biomarker assays. M65 Elisa, which quantitates different forms of circulating cytokeratin 18 (CK18) as putative surrogate markers of both apoptotic and nonapoptotic tumour cell death, was shown to be highly reproducible: calibration curve linearity r2 = 0.996, mean accuracy > 91% and mean precision < 3%, n = 27. Employing recombinant (r) CK18 and caspase cleaved CK18 (CK18 Asp396 neo-epitope) as external standards, kit to kit reproducibly was < 6% (n = 19). rCK18 was stable in plasma for 4 months at -20 degrees C and -80 degrees C, for 4 weeks at 4 degrees C and had a half-life of 2.3 days at 37 degrees C. Cytokeratin 18 Asp396 NE, the M30 Apoptosense Elisa assay antigen, was stable in plasma for 6 months at -20 degrees C and -80 degrees C, for 3 months at 4 degrees C, while its half-life at 37 degrees C was 3.8 days. Within-day variations in endogenous plasma concentrations of the M30 and M65 antigens were assessed in two predose blood samples collected from a cohort of 15 ovarian cancer patients receiving carboplatin chemotherapy and were shown to be no greater than the variability associated with methods themselves. Between-day fluctuations in circulating levels of the M30 and M65 antigens and in XIAP mRNA levels measured in peripheral blood mononuclear cells by quantitative (q) RT-PCR were evaluated in two predose blood samples collected with a 5- to 7-day gap from 23 patients with advanced cancer enrolled in a phase I trial. The mean variation between the two pretreatment values ranged from 13 to 14 to 25%, respectively, for M65, M30 and qRT-PCR. These data suggest that the M30 and M65 Elisa's and qRT-PCR as PD biomarker assays have favourable performance characteristics for further investigation in clinical trials of anticancer agents which induce tumour apoptosis/necrosis or knockdown of the anti-apoptotic protein XIAP.

There is now a growing awareness that infiltrating neutrophils play an important role in the molecular pathology of rheumatoid arthritis. In part, this arises from the fact that neutrophils have potent cytotoxic activity, but additionally from the fact that inflammatory neutrophils can generate a number of cytokines and chemokines that can have a direct influence on the progress of an inflammatory episode. Furthermore, the molecular properties of inflammatory neutrophils are quite different from those normally found in the circulation. For example, inflammatory neutrophils, but not blood neutrophils, can express cell surface receptors (such as MHC Class II molecules and FcgammaRI) that dramatically alter the way in which these cells can interact with ligands to modulate immune function. Cytokine/chemokine expression and surface expression of these novel cell surface receptors is dependent upon the neutrophil responding to local environmental factors to selectively up-regulate the expression of key cellular components via signalling pathways coupled to transcriptional activation. However, major changes in the expression levels of some proteins are also regulated by post-translational modifications that alter rates of proteolysis, and hence changes in the steady-state levels of these molecules.

Neutropenia is a common sequela of neonatal sepsis. Recent clinical trials have shown the beneficial effects of colony-stimulating factors (CSFs) on outcome in this group, but the exact mechanism remains unknown. Neonates and mothers who were at high-risk for infection were recruited for cord blood sampling in a university tertiary referral maternity hospital. Neonatal and adult neutrophils were evaluated for their ability to combat bacterial infection by examining their functional activity (CD11b and reactive oxygen intermediates) and their persistence at inflammatory sites (apoptosis). The mechanism for altered apoptotic responses was assessed by caspase activation assays, X chromosome-linked inhibitor of apoptosis protein expression, and cytosolic cytochrome c release. Although granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly delayed neutrophil apoptosis in normal adults, only G-CSF had a similar effect in normal neonates. Neutrophils from neonates who are at high risk for infection are unresponsive to the antiapoptotic effects of G-CSF or GM-CSF, unlike maternal neutrophils, which have delayed apoptosis in response to GM-CSF. However, CD11b expression and reactive oxygen intermediate production were significantly increased in normal neonatal neutrophils that were incubated with GM-CSF versus controls but not G-CSF or lipopolysaccharide. Decreased cytosolic cytochrome c release and caspases 3 and 9 activity are associated with the CSF-mediated delay in apoptosis in adults but not in newborns. The antiapoptotic X chromosome-linked inhibitor of apoptosis protein is up-regulated in neonates compared with adults and may mediate their differential spontaneous apoptosis. These results have important implications for the use of CSFs in neonatal sepsis, as responses differ from those seen in adults. Further delineation of neonatal neutrophil responses to CSFs may improve their therapeutic potential.