Superoxide dismutase (SOD) as a potential inhibitory mediator of inflammation via neutrophil apoptosis

Superoxide dismutase (SOD) is supposed to be an effective agent for neutrophil-mediated inflammation in the area of critical medicine. We investigated the involvement of SOD in the regulation of neutrophil apoptosis. Exogenously added SOD effectively induced neutrophil apoptosis, and the fluorescence patterns determined using annexin-V and the 7-AAD were similar to those seen in Fas-mediated neutrophil apoptosis. Neutrophils are short-lived leukocytes that need to be removed safely by apoptosis. The clearance of apoptotic neutrophils from sites of inflammation is a crucial determinant of the resolution of inflammation. Catalase inhibited the neutrophil apoptosis and caspase-3 activation. Spontaneous apoptosis, hydrogen peroxide and anti-Fas antibody-induced apoptosis of neutrophils were accelerated in Down's syndrome patients, in whom the SOD gene is overexpressed. Hydrogen peroxide was thought to be a possible major mediator of ROS-induced neutrophil apoptosis in caspase-dependent manner. Neutrophil apoptosis represents a crucial step in the mechanism governing the resolution of inflammation and has been suggested as a possible target for the control of neutrophil-mediated tissue injury. SOD may be a potential inhibitory mediator of neutrophil-mediated inflammation.     

Nicotinamide enhances apoptosis of G(M)-CSF-treated neutrophils and attenuates endotoxin-induced airway inflammation in mice

Neutrophils constitute the first line of host defense against invading microorganisms. Yet their removal from the inflammatory environment is fundamental for injury restraint and resolution of inflammation. Nicotinamide, a component of vitamin B(3), is known to modulate cell survival. In this study, we assessed the influence of nicotinamide on neutrophil apoptosis, both in vitro and in vivo in a mouse model of endotoxin-induced lung inflammation. In vitro, nicotinamide promoted apoptosis of human blood neutrophils in a dose-dependent manner in the presence of the apoptosis inhibitors granulocyte colony-stimulating factor and granulocyte/macrophage colony-stimulating factor. The highest concentration of nicotinamide completely neutralized the pro-survival effect of granulocyte (macrophage) colony-stimulating factor. Nicotinamide proapoptotic effect was associated with enhanced caspase-3 activity. In addition, nicotinamide slightly reduced neutrophil chemotaxis in vitro. In vivo, pulmonary nicotinamide delivery decreased the levels of cellular and biochemical inflammation markers and increased the percentage of apoptotic neutrophils in bronchoalveolar lavages. Our findings suggest that nicotinamide is an apoptotic stimulus for neutrophils, thereby contributing to the resolution of neutrophilic inflammation in the lungs.     

Granulocyte-macrophage colony-stimulating factor delays neutrophil constitutive apoptosis through phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways

Activated neutrophils play an important role in the pathogenesis of sepsis, glomerulonephritis, acute renal failure, and other inflammatory processes. The resolution of neutrophil-induced inflammation relies, in large part, on removal of apoptotic neutrophils. Neutrophils are constitutively committed to apoptosis, but inflammatory mediators, such as GM-CSF, slow neutrophil apoptosis by incompletely understood mechanisms. We addressed the hypothesis that GM-CSF delays neutrophil apoptosis by activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI 3-kinase) pathways. GM-CSF (20 ng/ml) significantly inhibited neutrophil apoptosis (GM-CSF, 32 vs 65% of cells p < 0. 0001). GM-CSF activated the PI 3-kinase/Akt pathway as determined by phosphorylation of Akt and BAD. GM-CSF-dependent Akt and BAD phosphorylation was blocked by the PI 3-kinase inhibitor LY294002. A role for the PI 3-kinase/Akt pathway in GM-CSF-stimulated delay of apoptosis was indicated by the ability of LY294002 to attenuate apoptosis delay. GM-CSF-dependent inhibition of apoptosis was significantly attenuated by PD98059, an ERK pathway inhibitor. LY294002 and PD98059 did not produce additive inhibition of apoptosis delay. To determine whether PI 3-kinase and ERK are used by other ligands that delay neutrophil apoptosis, we examined the role of these pathways in IL-8-induced apoptosis delay. LY294002 blocked IL-8-dependent Akt phosphorylation. PD98059 and LY294002 significantly attenuated IL-8 delay of apoptosis. These results indicate IL-8 and GM-CSF act, in part, to delay neutrophil apoptosis by stimulating PI 3-kinase and ERK-dependent pathways.     

Oxidative and nitrative modifications of enkephalins by human neutrophils: effect of nitroenkephalin on leukocyte functional responses

Neutrophils play a major role in acute inflammation by generating reactive oxygen/nitrogen species. Opioid peptides, including enkephalins, are present at inflammation sites. Neutrophils contribute to protect against inflammatory pain by releasing opioid peptides. In this investigation, the ability of human polymorphonuclear cells to induce oxidative and nitrative modifications of Leu-enkephalin has been investigated in vitro. Activated human neutrophils mediate the oxidation of Leu-enkephalin resulting in the production of dienkephalin. In the presence of nitrite at concentrations observed during inflammatory and infectious process (10-50 μM), nitroenkephalin, a nitrated derivative of Leu-enkephalin, is additionally formed. The yield of nitroenkephalin increases with nitrite concentration and is significantly inhibited by the addition of catalase or 4-aminobenzoic acid hydrazide (ABAH), a specific inhibitor of peroxidases. These results suggest that neutrophils induce nitration of Leu-enkephalin by a mechanism that is dependent on myeloperoxidase activity and hydrogen peroxide. Oxidative/nitrative modifications of Leu-enkephalin have been also evidenced when cells were treated with the NO-donor molecule, DEANO. The nitrated enkephalin has been examined for its effect on leukocyte functional responses. The data reveal that nitroenkephalin at micromolar concentrations inhibits superoxide anion generation and degranulation of azurophilic granules of human polymorphonuclear cells. Moreover, nitroenkephalin inhibits spontaneous apoptosis of neutrophils, as evaluated by measuring caspase-3 activity. Collectively, our data indicate that the nitrated enkephalin attenuates neutrophil activation and promotes the short-term survival of these cells, suggesting a possible role of the nitrocompound in the efficiency and resolution of inflammatory processes.     

Regulation of neutrophil apoptosis: A role for protein kinase C and phosphatidylinositol-3-kinase

Neutrophils play a central role in host defense and are recruited in vast numbers to sites of infection where they phagocytose and kill invading bacterial pathogens. Neutrophils have a short half-life that is extended at the inflamed site by pro-inflammatory cytokines and contact with bacterial cell walls. Normal resolution of inflammation involves the removal of neutrophils and other inflammatory cells by the induction of apoptosis. Spontaneous neutrophil apoptosis does not require Fas ligation, but is mediated by caspases 3, 8 and possibly caspase 9 and also involves activation of protein kinase C-. With chronic inflammatory disease, neutrophil apoptosis is delayed by pro-inflammatory cytokines, leading to persistence of neutrophils at the inflamed site and non-specific tissue damage. Here we discuss the evidence for inhibition of neutrophil apoptosis via signaling though PI-3-kinase and downstream pathways, including PDK-1 and PKB. Therapeutic strategies to resolve chronic inflammation could therefore usefully target neutrophil apoptosis and the PI-3-kinase or PKC- signaling pathways.     

Ultrastructural observation of human neutrophils during apoptotic cell death triggered by Entamoeba histolytica

Neutrophils are important effector cells against protozoan extracellular parasite Entamoeba histolytica, which causes amoebic colitis and liver abscess in human beings. Apoptotic cell death of neutrophils is an important event in the resolution of inflammation and parasite's survival in vivo. This study was undertaken to investigate the ultrastructural aspects of apoptotic cells during neutrophil death triggered by Entamoeba histolytica. Isolated human neutrophils from the peripheral blood were incubated with or without live trophozoites of E. histolytica and examined by transmission electron microscopy (TEM). Neutrophils incubated with E. histolytica were observed to show apoptotic characteristics, such as compaction of the nuclear chromatin and swelling of the nuclear envelop. In contrast, neutrophils incubated in the absence of the amoeba had many protrusions of irregular cell surfaces and heterogenous nuclear chromatin. Therefore, it is suggested that Entamoeba-induced neutrophil apoptosis contribute to prevent unwanted tissue inflammation and damage in the amoeba-invaded lesions in vivo.     

Nonphlogistic clearance of late apoptotic neutrophils by macrophages: efficient phagocytosis independent of beta 2 integrins

Neutrophils undergo constitutive death by apoptosis, leading to safe nonphlogistic phagocytosis and clearance by macrophages. Recent work has shown that before secondary necrosis, neutrophils exhibiting classical features of apoptosis can progress to a morphologically defined late apoptotic state. However, whether such neutrophils could be safely cleared was unknown. We now report that human late apoptotic neutrophils could be purified from cultured neutrophil populations undergoing constitutive death and were subsequently ingested by human monocyte-derived macrophages by serum-independent mechanisms that did not trigger the release of IL-8 or TNF-alpha. Such ingestion was specifically inhibited by Abs to thrombospondin-1 and the alpha(v)beta(3) vitronectin receptor. Murine bone marrow-derived macrophage phagocytosis of late and early apoptotic neutrophils occurred by similar mechanisms, proceeding with the same efficiency as that observed for wild-type controls when macrophages from [alpha(m)](-/-) or [beta(2)](-/-) mice were used. We conclude that specific nonphlogistic, beta(2) integrin-independent mechanisms involving thrombospondin-1 and alpha(v)beta(3) allow macrophages to ingest late apoptotic neutrophils without eliciting inflammatory cytokine secretion.     

Lactoferrin inhibits neutrophil apoptosis via blockade of proximal apoptotic signaling events

Neutrophils are the most abundant leukocyte and have a short lifespan, dying by apoptosis approximately five days after leaving the bone marrow. Their apoptosis can be delayed at sites of inflammation to extend their functional lifespan, but inappropriate inhibition of apoptosis contributes to chronic inflammatory disease. Levels of the physiological iron chelator lactoferrin are raised at sites of inflammation and we have shown previously that iron-unsaturated lactoferrin inhibited human neutrophil apoptosis, but the mechanisms involved were not determined. Here we report that the anti-apoptotic effect of lactoferrin is dependent upon its iron saturation status as iron-saturated lactoferrin did not affect neutrophil apoptosis. We also show that the effect of lactoferrin is mediated at an early stage in apoptosis as it inhibited activation of sphingomyelinase, generation of ceramide, activation of caspase 8 and Bax and cleavage of Bid. Lactoferrin did not inhibit apoptosis induced by exogenous ceramide, supporting the proposal that it acts upstream of ceramide generation. We therefore conclude that raised lactoferrin levels are likely to contribute to chronic inflammation by delaying neutrophil apoptosis and that this is achieved by inhibiting proximal apoptotic signaling events.     

Regulation of neutrophil senescence by microRNAs

Neutrophils are rapidly recruited to sites of tissue injury or infection, where they protect against invading pathogens. Neutrophil functions are limited by a process of neutrophil senescence, which renders the cells unable to respond to chemoattractants, carry out respiratory burst, or degranulate. In parallel, aged neutrophils also undergo spontaneous apoptosis, which can be delayed by factors such as GMCSF. This is then followed by their subsequent removal by phagocytic cells such as macrophages, thereby preventing unwanted inflammation and tissue damage. Neutrophils translate mRNA to make new proteins that are important in maintaining functional longevity. We therefore hypothesised that neutrophil functions and lifespan might be regulated by microRNAs expressed within human neutrophils. Total RNA from highly purified neutrophils was prepared and subjected to microarray analysis using the Agilent human miRNA microarray V3. We found human neutrophils expressed a selected repertoire of 148 microRNAs and that 6 of these were significantly upregulated after a period of 4 hours in culture, at a time when the contribution of apoptosis is negligible. A list of predicted targets for these 6 microRNAs was generated from http://mirecords.biolead.org and compared to mRNA species downregulated over time, revealing 83 genes targeted by at least 2 out of the 6 regulated microRNAs. Pathway analysis of genes containing binding sites for these microRNAs identified the following pathways: chemokine and cytokine signalling, Ras pathway, and regulation of the actin cytoskeleton. Our data suggest that microRNAs may play a role in the regulation of neutrophil senescence and further suggest that manipulation of microRNAs might represent an area of future therapeutic interest for the treatment of inflammatory disease.     

Neutrophil apoptosis: A target for enhancing the resolution of inflammation

Neutrophils are essential for host defense and their programmed cell death and removal are critical for the optimal expression as well as for efficient resolution of inflammation. Delayed neutrophil apoptosis or impaired clearance of apoptotic neutrophils by macrophages contributes to the progression of chronic inflammation. Under most conditions, neutrophils are exposed to multiple factors and their fate would ultimately depend on the balance between pro‐survival and pro‐apoptotic signals. Life or death decisions are tightly controlled by a complex network of intracellular signaling pathways. Accumulating data indicate that receptors, such as the formyl peptide receptor 2/lipoxin receptor or β₂‐integrins can generate contrasting cues in neutrophils in a ligand‐specific manner and suggest a hierarchy among these signals. In this article, we review recent advances on how pro‐apoptosis and pro‐survival signals interact to determine the fate of neutrophils and the inflammatory response, and highlight novel pharmacological strategies that could be used to enhance the resolution of inflammation by redirecting neutrophils to apoptosis. J. Cell. Biochem. 108: 1039–1046, 2009. © 2009 Wiley‐Liss, Inc.     

Inhibition of neutrophil apoptosis by ATP is mediated by the P2Y11 receptor

Neutrophils undergo rapid constitutive apoptosis that is delayed by a range of pathogen- and host-derived inflammatory mediators. We have investigated the ability of the nucleotide ATP, to which neutrophils are exposed both in the circulation and at sites of inflammation, to modulate the lifespan of human neutrophils. We found that physiologically relevant concentrations of ATP cause a concentration-dependent delay of neutrophil apoptosis (assessed by morphology, annexin V/To-Pro3 staining, and mitochondrial membrane permeabilization). We found that even brief exposure to ATP (10 min) was sufficient to cause a long-lasting delay of apoptosis and showed that the effects were not mediated by ATP breakdown to adenosine. The P2 receptor mediating the antiapoptotic actions of ATP was identified using a combination of more selective ATP analogs, receptor expression studies, and study of downstream signaling pathways. Neutrophils were shown to express the P2Y11 receptor and inhibition of P2Y11 signaling using the antagonist NF157 abrogated the ATP-mediated delay of neutrophil apoptosis, as did inhibition of type I cAMP-dependent protein kinases activated downstream of P2Y11, without effects on constitutive apoptosis. Specific targeting of P2Y11 could retain key immune functions of neutrophils but reduce the injurious effects of increased neutrophil longevity during inflammation.     

Quercetin alleviates rheumatoid arthritis by inhibiting neutrophil inflammatory activities

Rheumatoid arthritis (RA) is a prototypical autoimmune disorder mainly characterized by joint inflammation and cartilage destruction. Neutrophils actively take part in the initiation and progression of RA. Neutrophils express inflammatory mediators, including cytokines and chemokines. Aberrant formation of neutrophil extracellular traps (NETs) has been demonstrated in the pathogenesis of RA. Thus, neutrophils are regarded as important therapeutic targets in RA treatment. Quercetin is one of the major flavonoids found in fruits and vegetables. Previous studies have demonstrated that quercetin is a potential agent for the treatment of RA. However, the underlying antiarthritic mechanism of quercetin has not been investigated clearly. In this study, we analyzed the therapeutic mechanism of quercetin for RA. Our results showed that quercetin ameliorates inflammation in RA mice by inhibiting neutrophil activities. Quercetin inhibited neutrophil infiltration and reduced the plasma levels of inflammatory cytokines. Quercetin promoted the apoptosis of activated neutrophils. In addition, quercetin inhibited NET formation by suppressing autophagy. These findings suggest that quercetin may be an alternative agent for the treatment of RA by inhibiting neutrophil activities.     

Cigarette smoke extract-induced suppression of caspase-3-like activity impairs human neutrophil phagocytosis

Neutrophils are the primary inflammatory cell in smokers' lungs, but little is known about the ability of cigarette smoke to modulate neutrophil function. Neutrophils undergo caspase-3-dependent spontaneous, as well as phagocytosis-induced, apoptosis. This study investigated the ability of cigarette smoke extract (CSE) to alter neutrophil caspase-3 activity, apoptosis, and phagocytosis. CSE treatment resulted in a dramatic suppression of neutrophil caspase-3-like activity, which correlated with reduced cleavage of glutamate-L-cysteine ligase catalytic subunit, a known target of active caspase-3. CSE did not affect procaspase-3 processing to its active fragment, suggesting a direct effect of CSE on active caspase-3. Consistent with this, CSE inhibited active recombinant caspase-3 activity, which was abolished by dithiothreitol, suggesting a redox-sensitive mechanism. CSE-induced suppression of caspase-3 activity did not alter spontaneous apoptosis but did impair phagocytic activity. Since CSE treatment resulted in profound suppression of caspase-3 activity but did not alter apoptosis, the possibility of a threshold level of caspase-3 activity was investigated. CSE reduced caspase-3 activity in a concentration-dependent manner. Despite near complete suppression of caspase-3 activity, spontaneous apoptosis was not altered. Conversely, treatment with the pan-caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone, reduced spontaneous apoptosis. These data demonstrate that CSE does not suppress caspase-3 activity below a threshold level to prevent spontaneous apoptosis, but the level of inhibition is sufficient to impair neutrophil phagocytic activity. These divergent functions of caspase-3 may contribute to the persistence of neutrophils in the lungs of smokers, as well as be a factor in their higher incidence of community-acquired pneumonia.     

Altered neutrophil homeostasis in kinin B1 receptor-deficient mice

The kallikrein-kinin system is activated during inflammation and plays a major role in the inflammatory process. One of the main mechanisms of kinin action includes the modulation of neutrophil function employing both receptors for kinins, B1 and B2. In this report we show by the use of B1 receptor-deficient mice that neutrophil migration in inflamed tissues is dependent on kinin B1 receptors. However, there is no change in circulating leukocyte number and composition after genetic ablation of this receptor. Furthermore, apoptosis of neutrophils necessary for the resolution of persistent inflammatory processes is impaired in mice lacking the B1 receptor. We also show that this receptor is expressed on neutrophils, thus it may be directly involved in the induction of apoptosis in these cells after prolonged activation at inflamed sites. In conclusion, our data show that the kinin B1 receptor modulates migration and the life span of neutrophils at sites of inflammation and may be therefore an important drug target in the therapy of inflammatory diseases.     

Apoptosis Is Essential for Neutrophil Functional Shutdown and Determines Tissue Damage in Experimental Pneumococcal Meningitis

During acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. Neutrophils show pro-inflammatory activity and may contribute to tissue damage. In pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. This damage may be due to excessive neutrophil activity. We here show that transgenic expression of Bcl-2 in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. The persistence of neutrophil brain infiltrates was accompanied by high levels of IL-1β and G-CSF as well as reduced levels of anti-inflammatory TGF-β. Significantly, Bcl-2-transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. In vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. The inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. In wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. These results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils.     

Nitrite generation and antioxidant effects during neutrophil apoptosis

Neutrophil apoptosis is important for the resolution of airway inflammation in a number of lung diseases. Inflammatory mediators, endogenous reactive oxygen and nitrogen species, and intracellular and extracellular antioxidants may all influence neutrophil apoptosis. This study investigated the involvement of these factors during apoptosis of neutrophils cultured in vitro. Neutrophils undergoing spontaneous apoptosis in culture as assessed by annexin V binding generated significant amounts of nitrite. Incubation with agonistic anti-Fas monoclonal antibody or tumor necrosis factor-alpha (TNF-alpha) enhanced neutrophil apoptosis at 6 h, although it decreased nitrite accumulation. Although granulocyte-macrophage colony-stimulating factor significantly reduced neutrophil apoptosis, this was also associated with decreased nitrite accumulation. In contrast, inhibition of apoptosis at 16 h by dibutyryl cyclic adenosine monophosphate was associated with increased nitrite accumulation. Exogenous glutathione (GSH) or N-acetylcysteine significantly enhanced neutrophil apoptosis at 6 h and stimulated the production of H(2)O(2), which may mediate apoptosis through intracellular hydroxyl radical production. Intracellular GSH concentrations decreased in neutrophils undergoing apoptosis, and this was more marked in neutrophils treated with anti-Fas or TNF-alpha. These results suggest a causal association between reduced endogenous nitric oxide production, reduced intracellular GSH, and Fas- and TNF-alpha-mediated neutrophil apoptosis, whereas enhanced neutrophil survival mediated by dibutyryl cyclic adenosine monophosphate is associated with increased nitrite generation and maintenance of intracellular GSH. The interaction of endogenous reactive oxygen species with extracellular antioxidants such as GSH could also contribute to the complex processes regulating neutrophil apoptosis and hence the resolution of inflammation in the lung.     

Reduced neutrophil apoptosis in diabetic mice during staphylococcal infection leads to prolonged Tnfα production and reduced neutrophil clearance

Diabetes is a frequent underlying medical condition among individuals with Staphylococcus aureus infections, and diabetic patients often suffer from chronic inflammation and prolonged infections. Neutrophils are the most abundant inflammatory cells during the early stages of bacterial diseases, and previous studies have reported deficiencies in neutrophil function in diabetic hosts. We challenged age-matched hyperglycemic and normoglycemic NOD mice intraperitoneally with S. aureus and evaluated the fate of neutrophils recruited to the peritoneal cavity. Neutrophils were more abundant in the peritoneal fluids of infected diabetic mice by 48 h after bacterial inoculation, and they showed prolonged viability ex vivo compared to neutrophils from infected nondiabetic mice. These differences correlated with reduced apoptosis of neutrophils from diabetic mice and were dependent upon the presence of S. aureus and a functional neutrophil respiratory burst. Decreased apoptosis correlated with impaired clearance of neutrophils by macrophages both in vitro and in vivo and prolonged production of proinflammatory tumor necrosis factor alpha by neutrophils from diabetic mice. Our results suggest that defects in neutrophil apoptosis may contribute to the chronic inflammation and the inability to clear staphylococcal infections observed in diabetic patients.     

Spontaneous neutrophil apoptosis involves caspase 3-mediated activation of protein kinase C-delta

Neutrophils are short-lived leukocytes that die by apoptosis. Whereas stress-induced apoptosis is mediated by the p38 mitogen-activated protein (MAP) kinase pathway (Frasch, S. C., Nick, J. A., Fadok, V. A., Bratton, D. L., Worthen, G. S., and Henson, P. M. (1998) J. Biol. Chem. 273, 8389-8397), signals regulating spontaneous neutrophil apoptosis have not been fully determined. In this study we found increased activation of protein kinase C (PKC)-beta and -delta in neutrophils undergoing spontaneous apoptosis, but we show that only activation of PKC-delta was directly involved in the induction of apoptosis. PKC-delta can be proteolytically activated by caspase 3. We detected the 40-kDa caspase-generated fragment of PKC-delta in apoptotic neutrophils and showed that the caspase 3 inhibitor Asp-Glu-Val-Asp-fluoromethylketone prevented generation of the 40-kDa PKC-delta fragment and delayed neutrophil apoptosis. In a cell-free system, removal of PKC-delta by immunoprecipitation reduced DNA fragmentation, whereas loss of PKC-alpha, -beta, or -zeta had no significant effect. Rottlerin and LY379196 inhibit PKC-delta and PKC-beta, respectively. Only Rottlerin was able to delay neutrophil apoptosis. Inhibitors of MAP-ERK kinase 1 (PD98059) or p38 MAP kinase (SB202190) had no effect on neutrophil apoptosis, and activation of p42/44 and p38 MAP kinase did not increase in apoptotic neutrophils. We conclude that spontaneous neutrophil apoptosis involves activation of PKC-delta but is MAP kinase-independent.     

Neutrophils recruited to the site of Mycobacterium bovis BCG infection undergo apoptosis and modulate lipid body biogenesis and prostaglandin E production by macrophages

Neutrophil influx to sites of mycobacterial infections is one of the first events of tuberculosis pathogenesis. However, the role of early neutrophil recruitment in mycobacterial infection is not completely understood. We investigated the rate of neutrophil apoptosis and the role of macrophage uptake of apoptotic neutrophils in a pleural tuberculosis model induced by BCG. Recruited neutrophils were shown to phagocyte BCG and a large number of neutrophils undergo apoptosis within 24 h. Notably, the great majority of apoptotic neutrophils were infected by BCG. Increased lipid body (lipid droplets) formation, accompanied by prostaglandin E(2) (PGE(2)) and TGF-beta1 synthesis, occurred in parallel to macrophage uptake of apoptotic cells. Lipid body and PGE(2) formation was observed after macrophage exposure to apoptotic, but not necrotic or live neutrophils. Blockage of BCG-induced lipid body formation significantly inhibited PGE(2) synthesis. Pre-treatment with the pan-caspase inhibitor zVAD inhibited BCG-induced neutrophil apoptosis and lipid body formation, indicating a role for apoptotic neutrophils in macrophage lipid body biogenesis in infected mice. In conclusion, BCG infection induced activation and apoptosis of infected neutrophils at the inflammatory site. The uptake of apoptotic neutrophils by macrophages leads to TGF-beta1 generation and PGE(2)-derived lipid body formation, and may have modulator roles in mycobacterial pathogenesis.     

Accumulation of 111In-neutrophils in rabbit skin in allergic and non-allergic inflammatory reactions in vivo. Inhibition by neutrophil pretreatment in vitro with a monoclonal antibody recognizing the CD18 antigen

The mAb 60.3 recognizes the neutrophil CD18 Ag. We have investigated the effect of in vitro pretreatment of radiolabeled neutrophils with mAb 60.3 on their accumulation in vivo. Further, we have compared the in vivo effects of mAb 60.3 with its effects on neutrophil adherence in vitro. Neutrophil accumulation in vivo was measured in response to: 1) exogenous mediators FMLP, C5a des Arg, LTB4 and IL-1; 2) endogenous mediators generated in a non-allergic inflammatory reaction induced by zymosan; and 3) endogenous mediators generated in two allergic inflammatory reactions, a passive cutaneous anaphylactic reaction and a reversed passive Arthus reaction in rabbit skin. Pretreatment of neutrophils with mAb 60.3 inhibited their accumulation in all the responses. The results demonstrate that there is a common mechanism mediating neutrophil accumulation in these inflammatory reactions. Neutrophils pretreated with mAb 60.3 were also unresponsive to chemoattractants in in vitro adherence assays. However, the antibody-treated neutrophils responded normally to FMLP and C5a with respect to granular enzyme release. These results suggest that the basal expression of CD18 Ag is important for the adherence of neutrophils to microvascular endothelial cells stimulated by the local generation, or administration, of chemical mediators in vivo. Despite the fact that mediators such as FMLP can increase CD18 expression in vitro, it appears more likely that such mediators act in vivo by inducing a conformational change in the basally expressed neutrophil adhesive molecules.