Respiratory Burst

shown yet for the product of the tobacco N gene (Whitham et al., 1994) and its putative ligand derived from the viral replicase (Padgett and Beachy, 1993) and for several of the Arabidopsis R gene products and their matching bacterial avirulence proteins . In Botanisches Institut contrast, a number of general peptide elicitors have Universitä t Basel been well characterized, but their receptor counterparts Helbelstrasse 1 have not been yet identified.

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According to the hitherto accepted view, neutrophils kill ingested microorganisms by subjecting them to high concentrations of highly toxic reactive oxygen species (ROS) and bringing about myeloperoxidase-catalysed halogenation. We show here that this simple scheme, which for many years has served as a satisfactory working hypothesis, is inadequate. We ®nd that mice made de®cient in neutrophil-granule proteases but normal in respect of superoxide production and iodinating capacity, are unable to resist staphylococcal and candidal infections. We also show that activation provokes the in¯ux of an enormous concentration of ROS into the endocytic vacuole. The resulting accumulation of anionic charge is compensated for by a surge of K + ions that cross the membrane in a pH-dependent manner. The consequent rise in ionic strength engenders the release of cationic granule proteins, including elastase and cathepsin G, from the anionic sulphated proteoglycan matrix. We show that it is the proteases, thus activated, that are primarily responsible for the destruction of the bacteria.

gp91phox is the catalytic subunit of the respiratory burst oxidase, an NADPH-dependent, superoxide generating enzyme present in phagocytes. In phagocytes, the enzyme functions in host defense, but reactive oxygen generation has also been described in a variety of non-phagocytic cells, including cancer cells. We previously reported the cloning of Nox1 (NADPH oxidase1), a homolog of gp91phox, its expression in colon and vascular smooth muscle, and its oncogenic properties when overexpressed . Nature 401, 79±82]. Herein, we report the cloning and tissue expression of three additional homologs of gp91phox, termed Nox3, Nox4 and Nox5, members of a growing family of gp91phox homologs. All are predicted to encode proteins of around 65 kDa, and like gp91phox, all show 5±6 conserved predicted transmembrane a-helices containing putative heme binding regions as well as a¯avoprotein homology domain containing predicted binding sites for both FAD and NADPH. Nox3 is expressed primarily in fetal tissues, and Nox4 is expressed in not only fetal tissues, but also kidney, placenta and glioblastoma cells. Nox5 is expressed in a variety of fetal tissues as well as in adult spleen and uterus. Nox isoforms are aberrantly expressed in several cells derived from human cancers, with Nox4 being the isoform most frequently expressed in the tumor cells investigated. Thus, expression of Nox family members is likely to account for some of the reactive oxygen generation seen in non-phagocytic cells. q

Exposure to particulate silica (most crystalline polymorphs) causes a persistent inflammation sustained by the release of oxidants in the alveolar space. Reactive oxygen species (ROS), which include hydroxyl radical, superoxide anion, hydrogen peroxide, and singlet oxygen, are generated not only at the particle surface, but also by phagocytic cells attempting to digest the silica particle. Two distinct kinds of surface centers-silica-based surface radicals and poorly coordinated iron ions-generate O 2

The macrophage cell line RAW 264.7 when activated with Escherichia coli lipopolysaccharide and interferon-y synthesized nitrite (NO-) and nitrate (NO-). Medium change after the activation showed that L-arginine was the only amino acid essential for this synthesis. D-Arginine would not substitute for L-arginine. Other analogues that could replace L-arginine were L-homoarginine, L-arginine methyl ester, Larginamide, and the peptide L-arginyl-L-aspartate. L-Argininic acid, L-agmatine, L-ornithine, urea, L-citrulline, and ammonia were among the nonprecursors, while L-canavanine inhibited this L-arginine-derived NO /NOsynthesis. When morpholine was added to the culture medium of the activated RAW 264.7 macrophages, N-nitrosation took place, generating Nnitrosomorpholine. GC/MS experiments using L-[guanido-

DNA fragmentation and cell membrane blebbing (Porter 1 Biologie des Interactions Neurone-glie and Jä nicke, 1999; Yuan and Yankner, 2000). The com-INSERM U.495 mitment to death of developing neurons is regulated by IFR 70 extracellular signals such as hormones, neurotransmit-UPMC ters, or secreted proteins that either promote survival 2 INSERM U.106 or favor the initiation of programmed cell death after IFR 70 binding to specific receptors expressed by neurons (Da-Hô pital de la Salpê triè re vies, 2003; de la Rosa and de Pablo, 2000; Oppenheim, 47 Bd de l'hô pital 1999). For instance, developing motoneurons or sensory 75013 Paris neurons deprived of neurotrophic factors undergo auto-France crine-or paracrine-triggered cell death through the neu-3 Department of Molecular Cell Biology ronal production of ligands that bind to receptors of the VUmc tumor necrosis factor (TNF) death receptor family (Raoul Van der Boechorststraat 7 et al., 1999; Barker et al., 2001). However, this receptor 1081 BT Amsterdam family also mediates the induction of neural cell death The Netherlands by mesoderm derivatives. In the embryonic chick retina before the period of synaptogenesis, the death of cells expressing the p75 NGF receptor results from exposure Summary to NGF produced by infiltrating macrophages (Frade and Barde, 1998). These early retinal macrophages originate The loss of neuronal cells, a prominent event in the from the yolk sac (Cuadros et al., 1991). They apparently development of the nervous system, involves reguvanish prior to a secondary invasion of the retina by lated triggering of programmed cell death, followed macrophage precursors that give rise to the resident by efficient removal of cell corpses. Professional phagoretinal mononuclear phagocytes, microglial cells. The cytes, such as microglia, contribute to the elimination latter are characterized by motile ameboid phenotypes of dead cells. Here we provide evidence that, in addithat progressively transform into resting cells with ramition to their phagocytic activity, microglia promote fied processes (Navascué s et al., 2002). Microglia occur the death of developing neurons engaged in synaptoin different regions of the developing CNS (Cuadros and genesis. In the developing mouse cerebellum, Purkinje Navascué s, 1998), where they contribute to the eliminacells die, and 60% of these neurons that already tion of cell corpses owing to their marked phagocytic expressed activated caspase-3 were engulfed or conbehavior, a function that has been well documented in tacted by spreading processes emitted by microglial several species (Ashwell, 1990; Marín-Teva et al., 1999; cells. Apoptosis of Purkinje cells in cerebellar slices Rakic and Zecevic, 2000; Upender and Naegele, 1999). was strongly reduced by selective elimination of micro-In vitro, purified microglial cells have been shown to glia. Superoxide ions produced by microglial respiraproduce neurotrophic factors (Mallat and Chamak, 1994) tory bursts played a major role in this Purkinje cell as well as potentially cytotoxic compounds including death. Our study illustrates a mammalian form of encytokines (Combs et al., 2001), proteases (Flavin et al., gulfment-promoted cell death that links the execution 2000), free radicals (Chao et al., 1992; Thery et al., 1991), of neuron death to the scavenging of dead cells. or glutamate agonists (Piani et al., 1991). However, aside from the retinal cell death induced by macrophage-Introduction produced NGF (Frade and Barde, 1998), the physiological role of microglia in the triggering of developmental neuron death has remained undetermined. During development, programmed cell death, with the Analysis of programmed cell death in the nematode features of apoptosis, affects numerous cells of the neu-C. elegans raises the possibility that the phagocytic beronal lineage, including proliferating neuroblasts and havior of microglia might favor apoptotic degeneration postmitotic neurons before or after their axons have of CNS cells. In the developing nematode, cells in the reached their target cells (de la Rosa and de Pablo, 2000; advanced caspase (CED-3)-dependent stage of degen-Oppenheim, 1999). This process implicates a proteolytic eration can recover, unless they are engulfed by neighcascade mediated by members of the caspase family of boring cells that promote the execution of death (Hoeppcysteine proteases that are present as relatively inactive ner et al., 2001; Reddien et al., 2001). Since vertebrate precursors in healthy cells (Nicholson, 1999). Cleavage caspase-3 and nematode CED-3 belong to the same caspase-death effector subgroup and share stringent specificity (Nicholson, 1999), we asked whether en-*Correspondence: [email protected] gulfing microglia might favor the developmental death 4 Present address: Dpto. de Biología Celular, Facultad de Ciencias, of differentiated neurons expressing caspase-3 and en-Universidad de Granada, Avda. Fuentenueva s/n, 18071 Granada, Spain. gaged in synaptogenesis. Neuron 536 We focused on Purkinje cells (PC) that form the effer-surrounded by microglial processes. A total of 1082 cell bodies in 155 sections of P3 cerebellar vermis were double ent pathway of the cerebellar cortex. Morphological alterations of PC, suggesting cell degeneration, have pre-stained with anti-CaBP and anti-active caspase-3. The proportion of these Purkinje cell bodies containing acti-viously been observed in the late chick embryo (Bertossi et al., 1986). The developmental death of mouse PC has vated caspase-3 and in close contact with spreading microglial processes was 63.9% Ϯ 4.21 (mean Ϯ SD been inferred from increases in the number of adult PC in transgenic mice either overexpressing the antiapoptotic from three animals). Bcl-2 protein or deficient in the proapoptotic bax gene (Fan et al., 2001; Zanjani et al., 1996). During the early Microglial Cells Promote the Death of PC postnatal period, mouse and rat PC that are initially in Cerebellar Slices distributed over several layers progressively form a To investigate the possible role of microglia in PC death, monolayer. They also undergo morphological and funcwe used cerebellar slices of P3 mice maintained in vitro. tional changes including dendrite remodelling and in-It has previously been shown that PC die in these slices tense synaptogenesis (Armengol and Sotelo, 1991; mostly during the first day in vitro (DIV), with the ex-Gardette et al., 1985; Hendelman and Aggerwal, 1980). pected features of programmed cell death, including At the same time, the number of cerebellar microglia caspase-3 activation, DNA fragmentation, ultrastrucrises sharply (Ashwell, 1990).

Salmonella pathogenicity island 2 (SPI-2), which is located at centisome 30.7 on the chromosome of Salmonella enterica serovar Typhimurium, is required for growth within macrophages and systemic infection in mice. We recently reported that the infection of macrophages with Salmonella induces the expression of cyclooxygenase-2 in a manner dependent on SPI-2 (K. Uchiya and T. Nikai, Infect. Immun. 72:6860-6869, 2004). In the present study, gene expression analysis using a cDNA array further showed the involvement of SPI-2 in the expression of suppressor of cytokine signaling 3 (SOCS-3), which is involved in the inhibition of cytokine signaling via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway. A high level of SOCS-3 expression was induced in J774 macrophages infected with wild-type Salmonella compared to that in macrophages infected with a strain carrying a mutation in the spiC gene within SPI-2. Other members of the SOCS family were no...

Fish have many non-specific and specific, humoral and cellular mechanisms to resist bacterial diseases. Non-specific humoral factors include growth inhibiting substances, e.g. transferrin and antiproteases; lysins, e.g. lysozyme, C-reactive protein (CRP), bactericidal peptides and most importantly, complement which has lytic, proinflammatory, chemotactic and opsonic activities thus making a link with non-specific phagocyte responses. These are primarily executed by neutrophils and macrophages. The phagocytes contain many hydrolytic enzymes and when stimulated by bacteria produce reactive oxygen species (ROS), especially the hydroxyl radical, via generation of superoxide anions and nitric oxide. ROS are highly bactericidal. In addition, the neutrophils possess large quantities of myeloperoxidase (MPO) which generates bactericidal hypohalite ions from halide ions and H 2 O 2 . Macrophages can receive MPO from neutrophils and this may enhance their bactericidal activity. Antibody constitutes a specific humoral defence by inhibiting bacterial adherence or invasion of non-phagocytic host cells and neutralising bacterial toxins. Antibody can act as an opsonin and by activating complement by the classical pathway may lyse the bacterial cell, activate inflammation, phagocyte influx and further enhance phagocytosis. Macrophage bactericidal activity can be markedly increased by cytokines released by T lymphocytes on contact with their specific antigen. Pathogenic bacteria have evolved many ways of subverting these defence mechanisms of fish. These include the in vivo production of high a$nity iron uptake systems (e.g. siderophores and their surface receptors, in Aeromonas salmonicida, Vibrio anguillarum). Some bacteria e.g. V. anguillarum strains of serotype 01, while being resistant to the alternative pathway of complement activation in normal serum, are killed in immune serum. In other bacteria, surface layers, capsules and elongated lipopolysaccharide (LPS) 0-antigens protect against classical complement-mediated killing (e.g. A. salmonicida, Photobacterium piscicida, V. anguillarum serotype 02 respectively). While some important pathogens (e.g. P. piscicida) are readily killed by macrophages, others (A. salmonicida) possess specialised superoxide dismutase and catalase to resist ROS and are toxic themselves to macrophages. Renibacterium salmoninarum uses antibody, complement and other serum components to activate the respiratory burst of macrophages on immediate contact thus exhausting this macrophage bactericidal mechanism before the bacterium is phagocytosed. Subsequently, R, salmoninarum is taken up by the macrophage where it multiplies as an intracellular pathogen. However, these two highly adapted pathogens, A. salmonicida and R. salmoninarum are both killed by activated macrophages indicating that the whole concerted e#orts of specific and non-specific, humoral and cellmediated immune responses are required for immunity against these two important pathogens. 1999 Academic Press 291

The roles of the NADPH phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS) in host resistance to virulent Salmonella typhimurium were investigated in gp91 phox Ϫ / Ϫ , iNOS Ϫ / Ϫ , and congenic wild-type mice. Although both gp91 phox Ϫ / Ϫ and iNOS Ϫ / Ϫ mice demonstrated increased susceptibility to infection with S . typhimurium compared with wildtype mice, the kinetics of bacterial replication were dramatically different in the gp91 phox Ϫ / Ϫ and iNOS Ϫ / Ϫ mouse strains. Greater bacterial numbers were present in the spleens and livers of gp91 phox Ϫ / Ϫ mice compared with C57BL/6 controls as early as day 1 of infection, and all of the gp91 phox Ϫ / Ϫ mice succumbed to infection within 5 d. In contrast, an increased bacterial burden was detected within reticuloendothelial organs of iNOS Ϫ / Ϫ mice only beyond the first week of infection. Influx of inflammatory CD11b ϩ cells, granuloma formation, and serum interferon ␥ levels were unimpaired in iNOS Ϫ / Ϫ mice, but the iNOS-deficient granulomas were unable to limit bacterial replication. The NADPH phagocye oxidase and iNOS are both required for host resistance to wild-type Salmonella, but appear to operate principally at different stages of infection.

Active oxygen species (AOS) are responsible for triggering defense responses in plants. Respiratory burst oxidase ho- mologs ( rboh genes) have been implicated in AOS generation. We have isolated two rboh cDNAs, NbrbohA and NbrbohB , from Nicotiana benthamiana leaves. NbrbohA was expressed constitutively at a low level and the transcripts were in- creased after mechanical stress of control leaf

preincubated with iberiotoxin, paxilline, 4-AP or NS1619 for 3 min in a rapidly stirred oxygenated chamber at 37 8C, then mixed with 10 8 IgG-opsonized microbes. Phagocytosis was measured as described 7 using fluorescent rather than radiolabelled bacteria.

The opportunistic human pathogen Pseudomonas aeruginosa is the predominant micro-organism of chronic lung infections in cystic fibrosis (CF) patients. P. aeruginosa colonizes the CF lungs by forming biofilm structures in the alveoli. In the biofilm mode of growth the bacteria are highly tolerant to otherwise lethal doses of antibiotics and are protected from bactericidal activity of polymorphonuclear leukocytes (PMNs). P. aeruginosa controls the expression of many of its virulence factors by means of a cell-cell communication system termed quorum sensing (QS). In the present report it is demonstrated that biofilm bacteria in which QS is blocked either by mutation or by administration of QS inhibitory drugs are sensitive to treatment with tobramycin and H 2 O 2 , and are readily phagocytosed by PMNs, in contrast to bacteria with functional QS systems. In contrast to the wild-type, QS-deficient biofilms led to an immediate respiratory-burst activation of the PMNs in vitro. In vivo QS-deficient mutants provoked a higher degree of inflammation. It is suggested that quorum signals and QS-inhibitory drugs play direct and opposite roles in this process. Consequently, the faster and highly efficient clearance of QS-deficient bacteria in vivo is probably a two-sided phenomenon: down regulation of virulence and activation of the innate immune system. These data also suggest that a combination of the action of PMNs and QS inhibitors along with conventional antibiotics would eliminate the biofilm-forming bacteria before a chronic infection is established.

Neutrophils play an essential role in the body's innate defense against pathogens and are one of the primary mediators of the inflammatory response. To defend the host, neutrophils use a wide range of microbicidal products, such as oxidants, microbicidal peptides, and lytic enzymes. The generation of microbicidal oxidants by neutrophils results from the activation of a multiprotein enzyme complex known as the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which is responsible for transferring electrons from NADPH to O 2 , resulting in the formation of superoxide anion. During oxidase activation, cytosolic oxidase proteins translocate to the phagosome or plasma membrane, where they assemble around a central membrane-bound component known as flavocytochrome b. This process is highly regulated, involving phosphorylation, translocation, and multiple conformational changes. Originally, it was thought that the NADPH oxidase was restricted to phagocytes and used solely in host defense. However, recent studies indicate that similar NADPH oxidase systems are present in a wide variety of nonphagocytic cells. Although the nature of these nonphagocyte NADPH oxidases is still being defined, it is clear that they are functionally distinct from the phagocyte oxidases. It should be noted, however, that structural features of many nonphagocyte oxidase proteins do seem to be similar to those of their phagocyte counterparts. In this review, key structural and functional features of the neutrophil NADPH oxidase and its protein components are described, including a consideration of transcriptional and post-translational regulatory features. Furthermore, relevant details about structural and functional features of various nonphagocyte oxidase proteins will be included for comparison.

This study describes a simple, reliable, highly sensitive and quantitative fluorescence microplate-assay of H O from 2 2 Ž . Ž . activated leukocytes using a novel horse radish peroxidase HRP substrate N-acetyl-3,7-dihydroxyphenoxazine A6550 . Unlike the widely used fluorescent HRP substrate scopoletin, A6550 is non-fluorescent and becomes highly fluorescent upon HRP-catalyzed H O oxidation. Using 50 mM A6550, the change in fluorescence due to H O generated from phorbol 2 2 2 2 12-myristate 13-acetate-activated human eosinophils and neutrophils is found to have a linear cell dose response up to 1.5 = 10 4 and 5 = 10 4 cells, respectively. The increase in fluorescence from A6550 is specifically due to H O generation 2 2 since it is inhibitable by catalase. Oxidized A6550 is found to be highly stable and the H O dose response is linear as long 2 2 as the ratio of A6550:H O in the reaction mixture is higher than five. Unlike scopoletin, A6550 has a very low 2 2 background, which changes little with time. In addition, the high fluorescent yield of oxidized A6550 results in an increased sensitivity for the detection of H O . When the concentrations of A6550 and HRP were 10 mM and 0.2 Urml, respectively, 2 2 as low as 2 pmol of H O could be reliably measured. The sensitivity of A6550rH O assay is found to be at least 10-fold 2 2 2 2 higher than with scopoletin as the HRP substrate. The protocol described in this study using A6550 to measure H O release 2 2 from activated granulocytes can be easily adapted to other cell types which generate H O .

Previous studies suggest that salicylic acid (SA) plays an important role in influencing plant resistance to ozone (O 3). To further define the role of SA in O 3-induced responses, we compared the responses of two Arabidopsis genotypes that accumulate different amounts of SA in response to O 3 and a SA-deficient transgenic Col-0 line expressing salicylate hydroxylase (NahG). The differences observed in O 3-induced changes in SA levels, the accumulation of active oxygen species, defense gene expression, and the kinetics and severity of lesion formation indicate that SA influences O 3 tolerance via two distinct mechanisms. Detailed analyses indicated that features associated with a hypersensitive response (HR) were significantly greater in O 3-exposed Cvi-0 than in Col-0, and that NahG plants failed to exhibit these HR-like responses. Furthermore, O 3-induced antioxidant defenses, including the redox state of glutathione, were greatly reduced in NahG plants compared to Col-0 and Cvi-0. This suggests that O 3-induced cell death in NahG plants is due to the loss of SA-mediated potentiation of antioxidant defenses, while O 3-induced cell death in Cvi-0 is due to activation of a HR. This hypothesis is supported by the observation that inhibition of NADPH-oxidases reduced O 3-induced H 2 O 2 levels and the O 3-induced cell death in Cvi-0, while no major changes were observed in NahG plants. We conclude that although SA is required to maintain the cellular redox state and potentiate defense responses in O 3 exposed plants, high levels of SA also potentiate activation of an oxidative burst and a cell death pathway that results in apparent O 3 sensitivity.

Heme is an ancient and ubiquitous molecule present in organisms of all kingdoms, composed of an atom of iron linked to four ligand groups of porphyrin. A high amount of free heme, a potential amplifier of the inflammatory response, is a characteristic feature of diseases with increased hemolysis or extensive cell damage. Here we demonstrate that heme, but not its analogs/precursors, induced tumor necrosis factor-␣ (TNF-␣) secretion by macrophages dependently on MyD88, TLR4, and CD14. The activation of TLR4 by heme is exquisitely strict, requiring its coordinated iron and the vinyl groups of the porphyrin ring. Signaling of heme through TLR4 depended on an interaction distinct from the one established between TLR4 and lipopolysaccharide (LPS) since anti-TLR4/MD2 antibody or a lipid A antagonist inhibited LPS-induced TNF-␣ secretion but not heme activity. Conversely, protoporphyrin IX antagonized heme without affecting LPS-induced activation. Moreover, heme induced TNF-␣ and keratinocyte chemokine but was ineffective to induce interleukin-6, interleukin-12, and interferoninducible protein-10 secretion or co-stimulatory molecule expression. These findings support the concept that the broad ligand specificity of TLR4 and the different activation profiles might in part reside in its ability to recognize different ligands in different binding sites. Finally, heme induced oxidative burst, neutrophil recruitment, and heme oxygenase-1 expression independently of TLR4. Thus, our results presented here reveal a previous unrecognized role of heme as an extracellular signaling molecule that affects the innate immune response through a receptor-mediated mechanism.

An assay was developed to study plant receptor kinase activation and signaling mechanisms. The extracellular leucine-rich repeat (LRR) and transmembrane domains of the Arabidopsis receptor kinase BRI1, which is implicated in brassinosteroid signaling, were ...

Acidification of phagosomes has been proposed to have a key role in the microbicidal function of phagocytes. Here, we show that in alveolar macrophages the cystic fibrosis transmembrane conductance regulator Clchannel (CFTR) participates in phagosomal pH control and has bacterial killing capacity. Alveolar macrophages from Cftr -/mice retained the ability to phagocytose and generate an oxidative burst, but exhibited defective killing of internalized bacteria. Lysosomes from CFTR-null macrophages failed to acidify, although they retained normal fusogenic capacity with nascent phagosomes. We hypothesize that CFTR contributes to lysosomal acidification and that in its absence phagolysosomes acidify poorly, thus providing an environment conducive to bacterial replication.

Summary A null mutation was prepared in the mouse for CD18, the b 2 subunit of leukocyte integrins. Homozygous CD18 null mice develop chronic dermatitis with extensive facial and subman- dibular erosions. The phenotype includes elevated neutrophil counts, increased immunoglobu- lin levels, lymphadenopathy, splenomegaly, and abundant plasma cells in skin, lymph nodes, gut, and kidney. Very few neutrophils were found

An NADPH oxidase analogous to that in mammalian phagocytes has been hypothesized to produce reactive oxygen species (ROS) in the plant defence response. A. thaliana contains at least six gp91 phox homologues, designated AtrbohA-F (A. thaliana Respiratory Burst Oxidase Homologues), which map to different positions. Transcripts of three of these genes can be detected in healthy plants by RNA gel blot analyses. The Atrboh gene products are closely related to gp91 phox and the intron locations suggest a common evolutionary origin. A putative EF-hand Ca 2⍣binding motif in the extended N-terminal region of the Atrboh proteins suggests a direct regulatory effect of Ca 2⍣ on the activity of the NADPH oxidase in plants.

Migration is a basic feature of many cell types in a wide range of species 1 . Since the 1800s, cell migration has been proposed to occur in the nervous and immune systems 2,3 , and distinct molecular cues for mammalian neurons and leukocytes have been identified. Here we report that Slit, a secreted protein previously known for its role of repulsion in axon guidance and neuronal migration, can also inhibit leukocyte chemotaxis induced by chemotactic factors. Slit inhibition of the chemokineinduced chemotaxis can be reconstituted by the co-expression of a chemokine receptor containing seven transmembrane domains and Roundabout (Robo), a Slit receptor containing a single transmembrane domain. Thus, there is a functional interaction between single and seven transmembrane receptors. Our results reveal the activity of a neuronal guidance cue in regulating leukocyte migration and indicate that there may be a general conservation of guidance mechanisms underlying metazoan cell migration. In addition, we have uncovered an inhibitor of leukocyte chemotaxis, and propose a new therapeutic approach to treat diseases involving leukocyte migration and chemotactic factors.

The identification of genes underlying quantitative-trait loci (QTL) for complex diseases, such as rheumatoid arthritis, is a challenging and difficult task for the human genome project. Through positional cloning of the Pia4 QTL in rats, we found that a naturally occurring polymorphism of Ncf1 (encoding neutrophil cytosolic factor 1, a component of the NADPH oxidase complex) regulates arthritis severity. The disease-related allele of Ncf1 has reduced oxidative burst response and promotes activation of arthritogenic T cells. Pharmacological treatment with substances that activate the NADPH oxidase complex is shown to ameliorate arthritis. Hence, Ncf1 is associated with a new autoimmune mechanism leading to severe destructive arthritis, notably similar to rheumatoid arthritis in humans.

Reactive oxygen species (ROS) produced by NADPH oxidase play critical roles in various cellular activities, including plant innate immunity response. In contrast with the large multiprotein NADPH oxidase complex of phagocytes, in plants, only the homologs of the catalytic subunit gp91 phox and the cytosolic regulator small GTPase Rac are found. Plant homologs of the gp91 phox subunit are known as Rboh (for respiratory burst oxidase homolog). Although numerous Rboh have been isolated in plants, the regulation of enzymatic activity remains unknown. All rboh genes identified to date possess a conserved N-terminal extension that contains two Ca 2þ binding EF-hand motifs. Previously, we ascertained that a small GTPase Rac (Os Rac1) enhanced pathogen-associated molecular pattern-induced ROS production and resistance to pathogens in rice (Oryza sativa). In this study, using yeast two-hybrid assay, we found that interaction between Rac GTPases and the N-terminal extension is ubiquitous and that a substantial part of the N-terminal region of Rboh, including the two EF-hand motifs, is required for the interaction. The direct Rac-Rboh interaction was supported by further studies using in vitro pulldown assay, a nuclear magnetic resonance titration experiment, and in vivo fluorescence resonance energy transfer (FRET) microscopy. The FRET analysis also suggests that cytosolic Ca 2þ concentration may regulate Rac-Rboh interaction in a dynamic manner. Furthermore, transient coexpression of Os Rac1 and rbohB enhanced ROS production in Nicotiana benthamiana, suggesting that direct Rac-Rboh interaction may activate NADPH oxidase activity in plants. Taken together, the results suggest that cytosolic Ca 2þ concentration may modulate NADPH oxidase activity by regulating the interaction between Rac GTPase and Rboh.

Superoxide dismutase (SOD) catalyzes the conversion of superoxide radical to hydrogen peroxide. Periplasmic localization of bacterial Cu,Zn-SOD has suggested a role of this enzyme in defense against extracellular phagocyte-derived reactive oxygen species. Sequence analysis of regions flanking the Salmonella typhimurium sodC gene encoding Cu,Zn-SOD demonstrates significant homology to λ phage proteins, reflecting possible bacteriophage-mediated horizontal gene transfer of this determinant among pathogenic bacteria. Salmonella deficient in Cu,Zn-SOD has reduced survival in macrophages and attenuated virulence in mice, which can be restored by abrogation of either the phagocyte respiratory burst or inducible nitric oxide synthase. Moreover, a sodC mutant is extremely susceptible to the combination of superoxide and nitric oxide. These observations suggest that SOD protects periplasmic or inner membrane targets by diverting superoxide and limiting peroxynitrite formation, and they demonstrate the ability of the respiratory burst and nitric oxide synthase to synergistically kill microbial pathogens in vivo.

Mice with X-linked chronic granulomatous disease (CGD) generated by targeted disruption of the gp91 phox subunit of the NADPH–oxidase complex (X-CGD mice) were examined for their response to respiratory challenge with Aspergillus fumigatus. This opportunistic fungal ...

Haemopoietic tissues exposed to ionizing radiation are shown to exhibit increased macrophage activation, defined by ultrastructural characteristics and increased lysosomal and nitric oxide synthase enzyme activities. Macrophage activation post-irradiation was also associated with enhanced respiratory burst activities and an unexpected neutrophil infiltration. Examination of p53-null mice demonstrated that macrophage activation and neutrophil infiltration were not direct effects of irradiation, but were a consequence of the recognition and clearance of radiation-induced apoptotic cells. Increased phagocytic cell activity was maintained after apoptotic bodies had been removed. These findings demonstrate that, contrary to expectation, recognition and clearance of apoptotic cells after exposure to radiation produces both a persistent macrophage activation and an inflammatory-type response. We also demonstrate a complexity of macrophage activation following radiation that is genotype dependent, indicating that the in vivo macrophage responses to radiation damage are genetically modified processes. These short-term responses of macrophages to radiation-induced apoptosis and their genetic modification are likely to be important determinants of the longer-term consequences of radiation exposure. Furthermore, in addition to any effects attributable to immediate radiation-induced damage, our findings provide a mechanism for the production of damage via a 'bystander' effect which may contribute to radiation-induced genomic instability and leukaemogenesis.

Increasing intakes of long-chain n-3 polyunsaturated fatty acids (PUFAs) can decrease markers of immunity. However, dose- and age-related responses have not been identified. The objective was to determine the effects of different amounts of eicosapentaenoic acid (EPA) on innate immune outcomes in young and older males. In a controlled, double-blind study, healthy young and older men consumed 1 of 4 supplements provided as capsules: placebo (corn oil) or different amounts of an oil providing 1.35, 2.7, or 4.05 g EPA/d for 12 wk. Blood samples were collected at baseline and after 12 wk. EPA was incorporated in a linear dose-response fashion into plasma and mononuclear cell (MNC) phospholipids; incorporation was greater in the older men. EPA treatment did not alter neutrophil or monocyte phagocytosis, monocyte respiratory burst, or the production of inflammatory cytokines by MNCs in the young or older men. EPA treatment caused a dose-dependent decrease in neutrophil respiratory burst o...

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Neutrophil responses to commercial LPS, a dual Toll-like receptor (TLR)2 and TLR4 activator, are regulated by TLR expression, but are amplified by contaminating monocytes in routine cell preparations. Therefore, we investigated the individual roles of TLR2 and TLR4 in highly purified, monocyte-depleted neutrophil preparations, using selective ligands (TLR2, Pam 3 CysSerLys 4 and Staphylococcus aureus peptidoglycan; TLR4, purified LPS). Activation of either TLR2 or TLR4 caused changes in adhesion molecule expression, respiratory burst (alone, and synergistically with fMLP), and IL-8 generation, which was, in part, dependent upon p38 mitogen-activated protein kinase signaling. Neutrophils also responded to Pam 3 CysSerLys 4 and purified LPS with down-regulation of the chemokine receptor CXCR2 and, to a lesser extent, down-regulation of CXCR1. TLR4 was the principal regulator of neutrophil survival, and TLR2 signals showed relatively less efficacy in preventing constitutive apoptosis over short time courses. TLR4-mediated neutrophil survival depended upon signaling via NF-B and mitogen-activated protein kinase cascades. Prolonged neutrophil survival required both TLR4 activation and the presence of monocytes. TLR4 activation of monocytes was associated with the release of neutrophil survival factors, which was not evident with TLR2 activation, and TLR2 activation in monocyte/neutrophil cocultures did not prevent late neutrophil apoptosis. Thus, TLRs are important regulators of neutrophil activation and survival, with distinct and separate roles for TLR2 and TLR4 in neutrophil responses. TLR4 signaling presents itself as a pharmacological target that may allow therapeutic modulation of neutrophil survival by direct and indirect mechanisms at sites of inflammation.

The reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is part of the microbicidal arsenal used by human polymorphonuclear neutrophils (PMNs) to eradicate invading pathogens. The production of a superoxide anion (O 2 -) into the phagolysosome is the precursor for the generation of more potent products, such as hydrogen peroxide and hypochlorite. However, this production of O 2 is dependent on translocation of the oxidase subunits, including gp91 phox , p22 phox , p47 phox , p67 phox , p40 phox , and Rac2 from the cytosol or specific granules to the plasma membrane. In response to an external stimuli, PMNs change from a resting, nonadhesive state to a primed, adherent phenotype, which allows for margination from the vasculature into the tissue and chemotaxis to the site of infection upon activation. Depending on the stimuli, primed PMNs display altered structural organization of the NADPH oxidase, in that there is phosphorylation of the oxidase subunits and/or translocation from the cytosol to the plasma or granular membrane, but there is not the complete assembly required for O 2 generation. Activation of PMNs is the complete assembly of the membrane-linked and cytosolic NADPH oxidase components on a PMN membrane, the plasma or granular membrane. This review will discuss the individual components associated with the NADPH oxidase complex and the function of each of these units in each physiologic stage of the PMN: rested, primed, and activated. J. Leukoc. Biol. 78: 1025-1042; 2005.

Nitric oxide ('NO) release, oxygen uptake and hydrogen peroxide (H202) production elicited by increasing phorbol12-myristate 13-acetate (PMA) concentrations were measured in human neutrophils. Half-maximal activities were sequentially elicited at about 0.0001-0.001 pg PMA/ml CNO) and O.OOl~.Ol pg PMA/ml (H,OJ. At saturated PMA concentrations, 'NO production, oxygen uptake and H,O, release were 0.56 ? 0.04, 3.32 f 0.52 and 1.19 f 0.17 nmol min-' 1 O6 cells-'. 'NO production accounts for about 30% of the total oxygen uptake. Luminol-dependent chemiluminescence, reported to detect NO reactions in other inflammatory cells, was also half-maximally activated at about 0.00-0.01 pg PMA/ml. Preincubation with A'"-monomethyl-L-arginine (L-NMMA) decreased 0, uptake and 'NO release but increased H,Oz production, while superoxide dismutase (SOD) increased 'NO detection by 30%. Chemiluminescence was also reduced by preincubation with L-NMMA and/or SOD. The results indicate that 'NO release is part of the integrated response of stimulated human neutrophils and that, in these cells, kinetics of-NO and O;-release favour the formation of other oxidants like peroxynitrite.

Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilms on tissues and other surfaces. We characterized the interaction of purified human neutrophils with P. aeruginosa, growing in biofilms, with regard to morphology, oxygen consumption, phagocytosis, and degranulation. Scanning electron and confocal laser microscopy indicated that the neutrophils retained a round, unpolarized, unstimulated morphology when exposed to P. aeruginosa PAO1 biofilms. However, transmission electron microscopy demonstrated that neutrophils, although rounded on their dorsal side, were phagocytically active with moderate membrane rearrangement on their bacteria-adjacent surfaces. The settled neutrophils lacked pseudopodia, were impaired in motility, and were enveloped by a cloud of planktonic bacteria released from the biofilms. The oxygen consumption of the biofilm/neutrophil system increased 6-and 8-fold over that of the biofilm alone or unstimulated neutrophils in suspension, respectively. H 2 O 2 accumulation was transient, reaching a maximal measured value of 1 M. Following contact, stimulated degranulation was 20 -40% (myeloperoxidase, ␤-glucuronidase) and 40 -80% (lactoferrin) of maximal when compared with formylmethionylleucylphenylalanine plus cytochalasin B stimulation. In summary, after neutrophils settle on P. aeruginosa biofilms, they become phagocytically engorged, partially degranulated, immobilized, and rounded. The settling also causes an increase in oxygen consumption of the system, apparently resulting from a combination of a bacterial respiration and escape response and the neutrophil respiratory burst but with little increase in the soluble concentration of H 2 O 2 . Thus, host defense becomes compromised as biofilm bacteria escape while neutrophils remain immobilized with a diminished oxidative potential.

The induction of hepatic CYP1A by selected polycyclic aromatic hydrocarbons (PAHs) was followed in Atlantic salmon (Salmo salar) by measurement of CYP1A messenger RNA (mRNA), CYP1A protein levels, and catalytically by the measurement of 7-ethoxyresorufin-O-deethylase activity. There was clear correspondence between all three methods of measurement both in terms of the specificity of response to five- and some four-ring PAHs and in terms of the dose-response relationship to methyl-cholanthrene. The level of induction was compared with that measured in salmon confined in sea pens around Shetland and exposed to crude oil spilled from the Braer in January 1993. This oil was rapidly dispersed by the extreme weather (turbulence) at the time of the spill. The time course of the hepatic CYP1A induction was followed and related to the levels of oil measured in water and the concentration of PAHs determined in the flesh of the fish. Again there was a good correspondence between the different methods of measuring CYP1A expression, and the results show a rapid induction response in fish at the most contaminated sites and small, insignificant changes occurring at the reference stations. There was a clear concentration response between CYP1A and catalytic activity and between the exposure observed.

Partial or total replacement of fish meal by a mixture of plant protein (PP) sources (corn gluten, wheat gluten, extruded peas, rapeseed meal and sweet white lupin) balanced with indispensable amino acids was examined in juvenile gilthead sea bream over the course of a 6-month growth trial. A diet with fish meal (FM) as the sole protein source was compared to diets with 50%, 75% and 100% of replacement (PP50, PP75, and PP100). The possible influence of diets on growth performance, plasma metabolites, gut integrity, liver structure, anti-oxidant and immune status was evaluated. Final body weight was progressively decreased with PP inclusion, but in PP50 and PP75-fed fish, feed efficiency (FE) was significantly improved and specific growth rates remained unchanged or slightly reduced in comparison to fish fed the FM diet. In fish fed PP100 diet, FE remained unchanged and feed intake and growth decreased dramatically. In this group of fish, liver fat deposition was also largely increased, enterocytes showed an increased number of lipidic vacuoles and/or deposition of protein droplets, and the submucosa of intestine was dilated/hypertrophied and infiltrated with eosinophilic granular cells. Plasma glucose levels did not differ among the four groups, but a significant and progressive decrease of plasma cholesterol and plasma protein levels was found with FM replacement. The glutathione redox status in blood and liver increased with the increase of PP content and this increment was statistically significant in the liver of the three PP-fed groups. Glutathione reductase and γ-glutamyl transferase were also enhanced by PP inclusion. Plasma lysozyme levels were not altered by the dietary treatment, but respiratory burst of head kidney leucocytes and plasma myeloperoxidase values were significantly increased in PP75 and PP100 fish, respectively. Complement (ACH50) was significantly increased in PP50 fed fish and decreased in PP75 and PP100 groups. As a general conclusion, substitution of FM by a mixture of PP sources exerted an anti-oxidative effect, compromised growth performance only at the 100% level, and decreased one of the immune defence mechanisms at above 75% level.