Phagocytosis and mechanisms of killing of Aspergillus fumigatus conidia by murine alveolar macrophages (AM), which are the main phagocytic cells of the innate immunity of the lung, were investigated. Engulfment of conidia by murine AM lasts 2 h. Killing of A. fumigatus conidia by AM begins after 6 h of phagocytosis. Swelling of the conidia inside the AM is a prerequisite for killing of conidia. The contributions of NADPH oxidase and inducible nitric oxide synthase to the conidicidal activity of AM were studied using AM from OF1, wild-type and congenic p47phox ؊/؊ 129Sv, and wild-type and congenic iNOS ؊/؊ C57BL/6 mice. AM from p47phox ؊/؊ mice were unable to kill A. fumigatus conidia. Inhibitors of NADPH oxidase that decreased the production of reactive oxidant intermediates inhibited the killing of A. fumigatus without altering the phagocytosis rate. In contrast to NADPH oxidase, nitric oxide synthase does not play a role in killing of conidia. Corticosteroids did not alter the internalization of conidia by AM but did inhibit the production of reactive oxidant intermediates and the killing of A. fumigatus conidia by AM. Impairment of production of reactive oxidant intermediates by corticosteroids is responsible for the development of invasive aspergillosis in immunosuppressed mice.

Phagocytes such as neutrophils play a vital role in host defense against microbial pathogens. The anti-microbial function of neutrophils is based on the production of superoxide anion (O 2 -), which generates other microbicidal reactive oxygen species (ROS) and release of antimicrobial peptides and proteins. The enzyme responsible for O 2 -production is called the NADPH oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of two transmembrane proteins (p22phox and gp91phox, also called NOX2, which together form the cytochrome b558) and four cytosolic proteins (p47phox, p67phox, p40phox and a GTPase Rac1 or Rac2), which assemble at membrane sites upon cell activation. NADPH oxidase activation in phagocytes can be induced by a large number of soluble and particulate agents. This process is dependent on the phosphorylation of the cytosolic protein p47phox. p47phox is a 390 amino acids protein with several functional domains: one phox homology (PX) domain, two src homology 3 (SH3) domains, an auto-inhibitory region (AIR), a proline rich domain (PRR) and has several phosphorylated sites located between Ser303 and Ser379. In this review, we will describe the structure of p47phox, its phosphorylation and discuss how these events regulate NADPH oxidase activation.

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Neutrophils play a key role in host defense by releasing reactive oxygen species (ROS). However, excessive ROS production by neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can damage bystander tissues, thereby contributing to inflammatory diseases. Tumor necrosis factor-␣ (TNF-␣), a major mediator of inflammation, does not activate NADPH oxidase but induces a state of hyperresponsiveness to subsequent stimuli, an action known as priming. The molecular mechanisms by which TNF-␣ primes the NADPH oxidase are unknown. Here we show that Pin1, a unique cis-trans prolyl isomerase, is a previously unrecognized regulator of TNF-␣-induced NADPH oxidase hyperactivation. We first showed that Pin1 is expressed in neutrophil cytosol and that its activity is markedly enhanced by TNF-␣. Inhibition of Pin1 activity with juglone or with a specific peptide inhibitor abrogated TNF-␣-induced priming of neutrophil ROS production induced by N-formylmethionyl-leucyl-phenylalanine peptide (fMLF). TNF-␣ enhanced fMLF-induced IntroductionNeutrophils play an important role in host defense against invading pathogens and in inflammation. In response to stimulating agents, such as the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF), neutrophils release large amounts of superoxide anions and other reactive oxygen species (ROS) in a phenomenon called the respiratory burst. ROS produced by the neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase play a key role in host defenses, 1-3 but excessive ROS production can damage healthy bystander tissues, thereby contributing to inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel diseases, and acute respiratory distress syndrome. 4,5 Neutrophil ROS production is mediated by the phagocyte NADPH oxidase, also called NOX2. NADPH oxidase is a multicomponent enzyme system that catalyzes NADPH-dependent reduction of oxygen to superoxide anion. 6,7 In resting cells, the NADPH oxidase is inactive and its components are distributed between the cytosol and membranes. When cells are activated, the cytosolic components (p47phox, p67phox, p40phox, and Rac2) migrate to the membranes, where they associate with the membranebound components (p22phox and gp91phox/NOX2, which form the flavocytochrome b558) to assemble the catalytically active oxidase. 7,8 During NADPH oxidase activation, p47phox, p67phox, p40phox, p22phox, and gp91phox/NOX2 become phosphorylated. 9-13 p47phox phosphorylation on several serines plays a pivotal role in oxidase activation in intact cells. 14,15 Neutrophil ROS production is enhanced or primed by a variety of mediators, including proinflammatory cytokines, such as tumor necrosis factor-␣ (TNF-␣). In vitro, TNF-␣ induces a very weak oxidative response by neutrophils but strongly enhances ROS release on exposure to a secondary stimulus, such as the bacterial peptide fMLF. [16][17][18] This "priming" of neutrophil ROS production plays a detrimental role in a variety of human inflammatory diseases, in which ROS hyperp...

Although the pathogenesis of the acute respiratory distress syndrome (ARDS) is complex and poorly understood, several observations point to an important role of interactions between polymorphonuclear neutrophils (PMN) and cytokines in this process. We therefore studied certain parameters involved in PMN transendothelial migration (adhesion molecule expression and cytoskeletal organization) in patients with ARDS (n = 14) in comparison with other ventilated patients (n = 15). We found that in the basal state, both whole-blood PMN and alveolar PMN obtained by bronchoalveolar lavage (BAL) were activated, as shown by decreased L-selectin CD62L and increased beta 2 integrin CD11b expression, as well as decreased F-actin content. The degree of PMN activation increased with the degree of lung injury and with the levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-8 (IL-8). Moreover, the capacity of ex vivo stimulation of alveolar PMN by a bacterial peptide was low in ARDS and could partly account for the high susceptibility of these patients to lung infection. Therefore, ARDS-associated lung injury could be caused, at least in part, by inappropriate adhesion and transendothelial migration of proinflammatory cytokine-primed PMN.

There is ample experimental evidence that polymorphonuclear neutrophils (PMN) play a critical role in the pathogenesis of the adult respiratory distress syndrome (ARDS). Since interleukin-8 (IL-8) is a strong chemotactic factor for PMN, we measured IL-8 levels in plasma and bronchoalveolar lavage (BAL) fluid of 18 patients, 12 with ARDS and 6 with severe pneumonia uncomplicated by ARDS, all of whom had an increased number of PMN in BAL fluid. Seven healthy subjects served as controls. We found elevated levels of IL-8 in the alveolar spaces of all patients tested. Elevated BAL IL-8 levels were related to a fatal outcome and the presence of shock and correlated with a general clinical severity index (simplified acute physiological score). BAL fluid levels of IL-8 were significantly higher in patients with ARDS than in patients with pneumonia. In plasma, IL-8 levels were increased similarly in all patients and did not correlate with survival or the presence of shock. The BAL fluid-to-plasma ratio of IL-8 was significantly greater than that of tumor necrosis factor alpha, indicating higher local production of IL-8. Moreover, the presence of a primed subpopulation of blood PMN with respect to H2O2 production indicates that IL-8 may contribute to the neutrophil-mediated process in the pathogenesis of ARDS and pneumonia.

To gain further insight into the pathogenesis of the adult respiratory distress syndrome (ARDS), we studied possible relationships among the activation status of circulating polymorphonuclear neutrophils (PMN), cytokine levels, and the severity of lung injury in 31 patients: 15 with ARDS, nine with severe pneumonia uncomplicated by ARDS, and seven mechanically ventilated with neither ARDS nor pneumonia. Nine healthy subjects served as controls. Using flow cytometry, we identified a subpopulation of PMN with an increased capacity to generate hydrogen peroxide after stimulation ex vivo in all three patient groups; significantly higher values were found in those with ARDS. The PMN stimulation index, a reflection of the degree of hyperresponsiveness, correlated with elevated levels of tumor necrosis factor-alpha (TNF alpha) in plasma, and both spontaneous and lipopolysaccharide-induced TNF alpha production by cultured monocytes. These biologic expressions of PMN activation and cytokine generation both correlated with indices of the severity of lung injury, but not with the overall clinical severity. In contrast, IL-6 and IL-1 beta showed little or no relationship with either the degree of lung injury or PMN hyperresponsiveness. We conclude that TNF-alpha-primed PMN may play a major role in the pathogenesis of ARDS-associated lung injury.

SUMMARY:Polymorphonuclear neutrophils (PMN) are involved in the pathogenesis of acute lung injury (ALI), secreting numerous mediators such as proteases, reactive oxygen species, and cytokines. Because we had recently observed the ability of normal human PMN to degranulate and synthesize oncostatin M (OSM), an IL-6 -family cytokine, we quantified OSM production ex vivo by highly purified blood and alveolar PMN from 24 ventilated patients with ALI, including some patients with severe pneumonia. Most of the patients had no detectable OSM in plasma, and OSM production by cultured blood PMN was similar to that of healthy controls. However, OSM was present in bronchoalveolar lavage (BAL) fluid supernatant, with significantly higher levels during pneumonia. In addition, alveolar OSM levels correlated with the number of PMN obtained by BAL, suggesting that PMN are an important source of OSM within the alveoli. Indeed, purified alveolar PMN from all of the patients, especially those with pneumonia, strongly produced OSM. Interestingly, in the latter patients, alveolar PMN always produced more OSM than autologous blood PMN. These results document the functional duality of PMN in ALI by showing the participation of PMN in the modulation of lung inflammation. (Lab Invest 2001, 81:133-141).