Type I interferon regulates cytokine-delayed neutrophil apoptosis, reactive oxygen species production and chemokine expression.

Interferons (IFNs) are key regulators of a number of inflammatory conditions in which neutrophils play an important role in pathology, such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), where type I IFNs are implicated in disease pathology. However, IFNs are usually generated in vivo together with other cytokines that also have immunoregulatory functions, but such interactions are poorly defined experimentally. We measured the effects of type I (IFN-α) IFN, elevated in both RA and SLE, on the functions of healthy neutrophils incubated in vitro in the absence and presence of proinflammatory cytokines typically elevated in inflammatory diseases [tumour necrosis factor (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF)]. IFN-α alone had no effect on neutrophil apoptosis; however, it abrogated the anti-apoptotic effect of GM-CSF (18 h, P < 0·01). The enhanced stability of the anti-apoptotic protein myeloid cell leukaemia 1 (Mcl-1) and delayed activation of caspase activation normally regulated by GM-CSF were blocked by IFN-α: this effect was mediated, in part, by activation of p38 mitogen-activated protein kinase (MAPK). IFN-α alone also primed reactive oxygen species (ROS) production and maintained the transient priming effect of TNF-α for up to 4 h: it also down-regulated GM-CSF- and TNF-α-activated expression of chemokine (C-X-C motif) ligand (CXCL)1, CXCL2, CXCL3, CXCL8, CCL3 and CCL4 but, in contrast, increased the expression of CXCL10. These novel data identify complex regulatory signalling networks in which type I IFNs profoundly alter the response of neutrophils to inflammatory cytokines. This is likely to have important consequences in vivo and may explain the complexity and heterogeneity of inflammatory diseases such as RA, in which multiple cytokine cascades have been activated.

APPA (apocynin and paeonol) modulates pathological aspects of human neutrophil function, without supressing antimicrobial ability, and inhibits TNFα expression and signalling.

Neutrophils are key players in the pathophysiological process underlying inflammatory conditions not only by release of tissue-damaging cytotoxic enzymes, reactive oxygen species (ROS) but also by secretion of important immunomodulatory chemokines and cytokines. Here, we report the effects of the novel agent APPA, undergoing formal clinical development for treatment of osteoarthritis, and its constituent components, apocynin (AP) and paeonol (PA) on a number of neutrophil functions, including effects on TNFα- expression and signalling. Neutrophils were treated with APPA (10-1000 µg/mL) prior to the measurement of cell functions, including ROS production, chemotaxis, apoptosis and surface receptor expression. Expression levels of several key genes and proteins were measured after incubation with APPA and the chromatin re-modelling agent, R848. APPA did not significantly affect phagocytosis, bacterial killing or expression of surface receptors, while chemotactic migration was affected only at the highest concentrations. However, APPA down-regulated neutrophil degranulation and ROS levels, and decreased the formation of neutrophil extracellular traps. APPA also decreased cytokine-stimulated gene expression, inhibiting both TNFα- and GM-CSF-induced cell signalling. APPA was as effective as infliximab in down-regulating chemokine and IL-6 expression following incubation with R848. Whilst APPA does not interfere with neutrophil host defence against infections, it does inhibit neutrophil degranulation, and cytokine-driven signalling pathways (e.g. autocrine signalling and NF-κB activation), processes that are associated with inflammation. These observations may explain the mechanisms by which APPA exerts anti-inflammatory effects and suggests a potential therapeutic role in inflammatory diseases in which neutrophils and TNFα signalling are important in pathology, such as rheumatoid arthritis.

The CDK inhibitor purvalanol A induces neutrophil apoptosis and increases the turnover rate of Mcl-1: potential role of p38-MAPK in regulation of Mcl-1 turnover.

Human neutrophils are terminally differentiated cells that do not replicate and yet express a number of enzymes, notably cell cycle-dependent kinases (CDKs), that are associated normally with control of DNA synthesis and cell cycle progression. In neutrophils, CDKs appear to function mainly to regulate apoptosis, although the mechanisms by which they regulate this process are largely unknown. Here we show that the CDK2 inhibitor, purvalanol A, induces a rapid decrease in myeloid cell leukaemia factor-1 (Mcl-1) levels in human neutrophils and peripheral blood mononuclear cells (PBMCs), but only induces apoptosis in neutrophils which are dependent upon expression on this protein for survival. This rapid decrease in cellular Mcl-1 protein levels was due to a purvalanol A-induced decrease in stability, with the half-life of the protein decreasing from approximately 2 h in control cells to just over 1 h after addition of the CDK2 inhibitor: it also blocked the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent stabilization of Mcl-1. Purvanalol A blocked GM-CSF-stimulated activation of extracellular-regulated kinase (Erk) and signal transducer and activator of transcription (STAT)-3, and stimulated an additive activation of protein kinase B (Akt) with GM-CSF. Purvalanol A alone stimulated a rapid and sustained activation of p38-mitogen-activated protein kinase (MAPK) and the pan p38-MAPK inhibitor, BIRB796, partly blocked the purvalanol A-induced apoptosis and Mcl-1 loss. These novel effects of purvalanol A may result, at least in part, from blocking GM-CSF-mediated Erk activation. In addition, we propose that purvalanol A-induced activation of p38-MAPK is, at least in part, responsible for its rapid effects on Mcl-1 turnover and acceleration of neutrophil apoptosis.

Cutaneous immunopathology of long-standing complex regional pain syndrome.

BACKGROUND: Both increased mast cells numbers and raised immune mediator concentrations indicate immune activation in the affected skin of patients with early complex regional pain syndrome (CRPS), but little is known about regional immune cell involvement in late-stage CRPS. The aim of the current study was to determine skin immune cell populations in long-standing CRPS. METHODS: Using 6-mm skin punch biopsies from CRPS-affected and non-affected tissues, and a combination of chemical and immunofluorescence staining, we examined the density and function of key cell populations including mast cells, epidermal Langerhans cells (LCs) and tissue resident T-cells. RESULTS: We found no significant differences in either overall immune cell infiltrates, or mast cell density between CRPS-affected and non-affected sub-epidermal tissue sections, contrasting recent findings in early CRPS by other groups. However, CD1a(+) LC densities in the epidermal layer were significantly decreased in affected compared to non-affected CRPS limbs (p < 0.01). T-cell clones isolated from CRPS-affected sub-epidermal tissues displayed a trend towards increased IL-13 production in ELISPOT assays when compared to T-cells isolated from non-affected areas, suggesting a Th2 bias. CONCLUSIONS: Immune cell abnormalities are maintained in late-stage CRPS disease as manifest by changes in epidermal LC density and tissue resident T-cell phenotype.

Human filarial Wolbachia lipopeptide directly activates human neutrophils in vitro.

The host inflammatory response to the Onchocerca volvulus endosymbiont, Wolbachia, is a major contributing factor in the development of chronic pathology in humans (onchocerciasis/river blindness). Recently, the toll-like pattern recognition receptor motif of the major inflammatory ligands of filarial Wolbachia, membrane-associated diacylated lipoproteins, was functionally defined in murine models of pathology, including mediation of neutrophil recruitment to the cornea. However, the extent to which human neutrophils can be activated in response to this Wolbachia pattern recognition motif is not known. Therefore, the responses of purified peripheral blood human neutrophils to a synthetic N-terminal diacylated lipopeptide (WoLP) of filarial Wolbachia peptidoglycan-associated lipoprotein (PAL) were characterized. WoLP exposure led to a dose-dependent activation of healthy, human neutrophils that included gross morphological alterations and modulation of surface expressed integrins involved in tethering, rolling and extravasation. WoLP exposure induced chemotaxis but not chemokinesis of neutrophils, and secretion of the major neutrophil chemokine, interleukin 8. WoLP also induced and primed the respiratory burst, and enhanced neutrophil survival by delay of apoptosis. These results indicate that the major inflammatory motif of filarial Wolbachia lipoproteins directly activates human neutrophils in vitro and promotes a molecular pathway by which human neutrophils are recruited to sites of Onchocerca parasitism.

Differential expression of factors involved in the intrinsic and extrinsic apoptotic pathways in juvenile systemic lupus erythematosus.

Dysregulated neutrophil apoptosis may result in the development of autoimmune disease by contributing to nuclear autoantigen exposure, leading to autoantibody generation and a breakdown in immune tolerance. It has previously been shown that neutrophil apoptosis is increased in juvenile-onset systemic lupus erythematosus (JSLE). This study aims to investigate the pathways involved in JSLE serum-induced apoptosis. Caspases 3, 7-9, IAP1/2, XIAP and FADD mRNA levels and TRAIL R2, BID/tBID, caspase 8 and 9 protein expression were measured in neutrophils from JSLE patients (n = 14) and controls (n = 10). The mRNA levels of caspases 7-9 were significantly higher in JSLE neutrophils than in controls, whereas the mRNA levels of IAP1, IAP2 and XIAP were decreased (p < 0.05). A decrease in neutrophil apoptosis induced by JSLE serum was observed in the presence of caspase 8 and 9 inhibitors (p < 0.05), and the activity of caspases 8 and 9 increased over time. tBID protein expression increased following incubation with JSLE serum. These data focus specifically on the expression and activity of the main caspases in the intrinsic and extrinsic apoptotic pathways. Increased expression of factors involved in the downstream signalling of the extrinsic apoptotic pathway indicates a prominent involvement of this pathway in JSLE serum-induced apoptosis.

Gastrin increases mcl-1 expression in type I gastric carcinoid tumors and a gastric epithelial cell line that expresses the CCK-2 receptor.

Elevated serum concentrations of the hormone gastrin are associated with the development of gastric carcinoid tumors, but the mechanisms of tumor development are not fully understood. We hypothesized that the antiapoptotic effects of gastrin may be implicated and have therefore investigated the role of antiapoptotic members of the bcl-2 family of proteins. AGS-G(R) human gastric carcinoma cells stably transfected with the CCK-2 receptor were used to assess changes in the expression of bcl-2 family members following gastrin treatment and the function of mcl-1 during apoptosis was investigated by use of small-interfering RNA (siRNA). Treatment of AGS-G(R) cells with 10 nM gastrin for 6 h caused maximally increased mcl-1 protein abundance. Gastrin-induced mcl-1 expression was inhibited by the transcription inhibitor actinomycin D and by the protein synthesis inhibitor cycloheximide. Downstream signaling of mcl-1 expression occurred via the CCK-2 receptor, protein kinase C, and MAP kinase pathways, but not via PI 3-kinase. Transfection with mcl-1 siRNA significantly suppressed mcl-1 protein expression and abolished the antiapoptotic effects of gastrin on serum starvation-induced apoptosis. Mcl-1 protein expression was also specifically increased in the type I enterochromaffin-like cell carcinoid tumors of 10 patients with autoimmune atrophic gastritis and hypergastrinemia. Gastrin therefore signals via the CCK-2 receptor, protein kinase C, and MAP kinase to induce expression of antiapoptotic mcl-1 in AGS-G(R) cells, and mcl-1 expression is also increased in human hypergastrinemia-associated type I gastric carcinoid tumors. Gastrin-induced mcl-1 expression may therefore be an important mechanism contributing toward type I gastric carcinoid development.

Oscillations in NF-kappaB signaling control the dynamics of gene expression.

Signaling by the transcription factor nuclear factor kappa B (NF-kappaB) involves its release from inhibitor kappa B (IkappaB) in the cytosol, followed by translocation into the nucleus. NF-kappaB regulation of IkappaBalpha transcription represents a delayed negative feedback loop that drives oscillations in NF-kappaB translocation. Single-cell time-lapse imaging and computational modeling of NF-kappaB (RelA) localization showed asynchronous oscillations following cell stimulation that decreased in frequency with increased IkappaBalpha transcription. Transcription of target genes depended on oscillation persistence, involving cycles of RelA phosphorylation and dephosphorylation. The functional consequences of NF-kappaB signaling may thus depend on number, period, and amplitude of oscillations.

Alternative splicing of Bcl-2-related genes: functional consequences and potential therapeutic applications.

Apoptosis is a morphologically distinct form of cell death. It is executed and regulated by several groups of proteins. Bcl-2 family proteins are the main regulators of the apoptotic process acting either to inhibit or promote it. More than 20 members of the family have been identified so far and most have two or more isoforms. Alternative splicing is one of the major mechanisms providing proteomic complexity and functional diversification of the Bcl-2 family proteins. Pro- and anti-apoptotic Bcl-2 family members should function in harmony for the regulation of the apoptosis machinery, and their relative levels are critical for cell fate. Any mechanism breaking down this harmony by changing the relative levels of these antagonistic proteins could contribute to many diseases, including cancer and neurodegenerative disorders. Recent studies have shown that manipulation of the alternative splicing mechanisms could provide an opportunity to restore the proper balance of these regulator proteins. This review summarises current knowledge on the alternative splicing products of Bcl-2-related genes and modulation of splicing mechanisms as a potential therapeutic approach.

An integrative genomics approach to the reconstruction of gene networks in segregating populations.

The reconstruction of genetic networks in mammalian systems is one of the primary goals in biological research, especially as such reconstructions relate to elucidating not only common, polygenic human diseases, but living systems more generally. Here we propose a novel gene network reconstruction algorithm, derived from classic Bayesian network methods, that utilizes naturally occurring genetic variations as a source of perturbations to elucidate the network. This algorithm incorporates relative transcript abundance and genotypic data from segregating populations by employing a generalized scoring function of maximum likelihood commonly used in Bayesian network reconstruction problems. The utility of this novel algorithm is demonstrated via application to liver gene expression data from a segregating mouse population. We demonstrate that the network derived from these data using our novel network reconstruction algorithm is able to capture causal associations between genes that result in increased predictive power, compared to more classically reconstructed networks derived from the same data.

Regulation of neutrophil apoptosis by Mcl-1.

Neutrophils rapidly undergo spontaneous apoptosis, but this process can be considerably delayed by exposure to a variety of agents such as pro-inflammatory cytokines. The anti-apoptotic protein of the Bcl-2 family, Mcl-1, plays a key role in the regulation of neutrophil apoptosis. The protein has some unusual properties compared with other family members, including an extremely high turnover rate. Many factors, such as cytokines and local oxygen concentrations, can regulate cellular levels of Mcl-1 via transcription and post-transcriptional modification, control the survival time of neutrophils within tissues and thereby influence the inflammatory response.

Regulation of neutrophil apoptosis via death receptors.

Human neutrophils constitutively undergo apoptosis, process which is critical for the successful resolution of inflammation by the safe removal of effete cells. A wide variety of agents can modulate neutrophil apoptosis and these act through multiple and complex receptor-signalling pathways. Whilst these pathways can be initiated via distinct cell surface receptors, many downstream intracellular pathways can converge, use common molecules or trigger similar cellular activities, such as activation of caspases and transcription factors. The cell surface receptors, TNFR and Fas both trigger apoptosis in certain cell types, including neutrophils. However, TNF receptors also activate survival mechanisms in human neutrophils. This review summarises current knowledge about the regulation of neutrophil apoptosis via death receptors, the molecular components involved in signalling and potential therapeutic targets that are based on death receptors or their signalling pathways.

Purification of ovine neutrophils and eosinophils: Anaplasma phagocytophilum affects neutrophil density.

Studies on the functions of ovine granulocytes require pure and functionally active populations of neutrophils and eosinophils. This report describes an improved technique for the separation of neutrophils and eosinophils from the peripheral blood of sheep infected with Anaplasma phagocytophilum and from normal sheep. After centrifugation and discarding the buffy coat layer, which contains the bulk of mononuclear cells, neutrophils with a high degree of purity (94.87 [+/-1.7]%, n=9) and good yield (69 [+/-9]%, n=9) were obtained by density gradient centrifugation on Percoll with a density of 1.09 g/ml (65%). However, this density was not suitable for neutrophils obtained from sheep during the peak period of A. phagocytophilum bacteraemia. Improved purity of infected neutrophils was obtained when the leucocytes were separated on Percoll with a density of 1.08 g/ml (55%). Relatively good purity of eosinophils was obtained when leucocytes from normal sheep were separated on Percoll with a density of 1.10 g/ml (70%). Ovine eosinophils formed a distinct band just below the band of mononuclear cells when a continuous Percoll gradient with a density of 1.10 g/ml was used. The purity of the eosinophils obtained was 87.7 (+/-12.5)% (n=6; range 64.1-97.6%), with a mean recovery rate of 61.9 (+/-20.3)%.

Cultivation of an ovine strain of Ehrlichia phagocytophila in tick cell cultures.

Ehrlichia phagocytophila (previously known as Cytoecetes phagocytophila) which causes tick-borne fever (TBF) in sheep and pasture fever in cattle in the UK and mainland Europe is transmitted by the temperate hard tick Ixodes ricinus. The disease in sheep is characterized by fever, leucopenia and immunosuppression. Studies on the pathogenesis and other aspects of the disease have been hampered because the organism has not been cultivated in continuous or primary cell culture systems. This paper describes the first successful cultivation of a European isolate of E. phagocytophila in two continuous cell lines, IDE8 and ISE6, derived from the temperate hard tick Ixodes scapularis. Once adapted to tick cell cultures the organism was serially sub-cultured in new cells by transferring small portions of infected cell suspension every 2 to 3 weeks. The identity of the organism was confirmed by polymerase chain reaction (PCR), with primers specific to the granulocytic ehrlichiae. Sequence analysis of the PCR products amplified from infected tick cells were shown to be identical with those amplified from the blood of sheep infected with the same strain of E. phagocytophila. A susceptible sheep inoculated with a third passage of the tick cell-adapted E. phagocytophila reacted with fever and rickettsiaemia 5 days later, thus satisfying Koch's postulates.

Insoluble and soluble immune complexes activate neutrophils by distinct activation mechanisms: changes in functional responses induced by priming with cytokines.

BACKGROUND: Rheumatoid synovial fluid contains both soluble and insoluble immune complexes that can activate infiltrating immune cells such as neutrophils. OBJECTIVES: To determine if these different complexes activate neutrophils through similar or different receptor signalling pathways. In particular, to determine the circumstances which result in the secretion of tissue damaging reactive oxygen metabolites and granule enzymes. METHODS: Blood neutrophils were incubated with synthetic soluble and insoluble immune complexes and the ability to generate reactive oxidants tested by luminescence or spectrophotometric assays that distinguished between intracellular and extracellular production. Degranulation of myeloperoxidase and lactoferrin was determined by western blotting. The roles of FcgammaRII (CD32) and FcgammaRIIIb (CD16) were determined by incubation with Fab/F(ab')(2) fragments before activation. The effect of cytokine priming was determined by incubation with GM-CSF. RESULTS: Insoluble immune complexes activated unprimed neutrophils, but most of the oxidants produced were intracellular. This activation required FcgammaRIIIb, but not FcgammaRII function. Soluble complexes failed to activate unprimed neutrophils but generated a rapid and extensive secretion of reactive oxygen metabolites when the cells were primed with granulocyte-macrophage colony stimulating factor (GM-CSF). This activity required both FcgammaRII and FcgammaRIIIb function. Insoluble immune complexes activated the release of granule enzymes from primed or unprimed neutrophils, but the kinetics of release did not parallel those of secretion of reactive oxygen metabolites. Only primed neutrophils released enzymes in response to soluble complexes. CONCLUSIONS: Soluble and insoluble immune complexes activate neutrophils by separate receptor signalling pathways. Profound changes in neutrophil responsiveness to these complexes occur after cytokine priming.

BCL-2 family expression in human neutrophils during delayed and accelerated apoptosis.

The human neutrophil spontaneously undergoes apoptosis, but this type of cell death can be delayed or accelerated by a wide variety of agents. There are wide discrepancies in the literature regarding the expression of the Bcl-2 family of proteins in human neutrophils. Here, we show that A1, Mcl-1, Bcl-X(L), and Bad are major transcripts in human neutrophils and that levels of these transcripts are cytokine regulated. However, no Bcl-X(L) protein was detected in Western blots. Protein levels for the proapoptotic proteins Bad, Bax, Bak, and Bik remained constant during culture, despite changes in the levels of mRNA for these gene products. These proapoptotic proteins were extremely stable, having very long half-lives. In contrast, A1 and Mcl-1 transcripts were extremely unstable (with approximately 3-h half-lives), and Mcl-1 protein was also subject to rapid turnover. These results indicate that neutrophil survival is regulated by the inducible expression of the short-lived Mcl-1 and possibly the A1 gene products. In the absence of their continued expression, these prosurvival gene products are rapidly turned over, and then the activity of the stable death proteins predominates and promotes apoptosis.

Fcgamma receptors in autoimmune diseases.

Fcgamma-receptors (Fcgamma-R) recognise the Fc portion of IgG and thus form a link between humoral and cellular immunity. These receptors are expressed by a variety of immune cells, and they function in the binding of immune complexes or IgG-opsonised particles, such as microbial pathogens. The are three major types of Fcgamma-R, namely Fcgamma-RI (CD64), Fcgamma-RII (CD32) and Fcgamma-RIII (CD16), and these differ in their ability to bind IgG and complexes. There are many isoforms of these receptors and a number of recently identified polymorphisms in their structure. This review describes the structure and function of these Fcgamma-Rs, and highlights how gene deficiencies and polymorphisms may contribute to the pathology of human diseases.

Molecular control of neutrophil apoptosis.

Human neutrophils constitutively undergo apoptosis and this process is critical for the resolution of inflammation. Whilst neutrophil apoptosis can be modulated by a wide variety of agents including GM-CSF, LPS and TNF-alpha, the molecular mechanisms underlying neutrophil death and survival remain largely undefined. Recent studies have shown the involvement of members of the Bcl-2 protein family (especially Mcl-1 and A1) and caspases in the regulation and execution of neutrophil apoptosis. Cell surface receptors and protein kinases, particularly mitogen-activated protein kinases, also play critical roles in transducing the signals that result in neutrophil apoptosis or extended survival. This review summarises current knowledge on the molecular mechanisms and components of neutrophil apoptosis.