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It was recently demonstrated that nitric oxide (NO) inhalation improves arterial oxygenation in patients with the adult respiratory distress syndrome (ARDS). However, the potential adverse reaction of NO on inflammatory cells and mediators in the lung has not yet been investigated. In this study, we evaluated the impact of NO inhalation on lung polymorphonuclear neutrophil (PMN) activation and proinflammatory cytokine release, both of which are involved in the pathophysiology of ARDS. Two groups of patients with ARDS of similar etiologies were compared; one received NO (n=9) and the other did not (n=5). After 4 d of NO inhalation (18 ppm), PMN form bronchoalveolar lavage (BAL) showed a reduction in both spontaneous H2O2 production (p

Impaired polymorphonuclear neutrophil (PMN) function may contribute to the onset of certain bacterial and fungal infections and to tissue damage in human immunodeficiency virus (HIV)-infected patients. Published data on PMN function in HIV infection are controversial, possibly because most studies have involved PMNs isolated from the normal blood environment by various procedures that may modify PMN responses. We therefore used flow cytometry to study the expression of adhesion molecules at the PMN surface, actin polymerization, and the oxidative burst of whole-blood PMNs in 42 HIV-infected patients at different stages of the disease. These PMNs were activated in vivo, as shown by increased expression of the adhesion molecule CD11b/CD18, reduced L-selectin antigen expression, increased actin polymerization, and increased H2O2 production. The alterations were present in asymptomatic patients with CD4+ cell counts above 500/microliters and did not increase with progression of the disease. This PMN activation could contribute to the oxidative stress described in HIV infection. Stimulation by bacterial N-formyl peptides showed dysregulation of L-selectin shedding and decreased H2O2 production after cx vivo priming with tumor necrosis factor-alpha or interleukin-8. These latter impairments, which correlated with the decrease in CD4+ lymphocyte numbers, could contribute to the increased susceptibility of HIV-infected patients to bacterial infections.

Human polymorphonuclear neutrophils play a key role in host defenses against invading microorganisms. In response to a variety of stimuli, neutrophils release large quantities of superoxide anion (O2.-) in a phenomenon known as the respiratory burst. O2.- is the precursor of potent oxidants, which are essential for bacterial killing and also potentiate inflammatory reactions. Regulation of this production is therefore critical to kill pathogens without inducing tissue injury. Neutrophil production of O2.- is dependent on the respiratory burst oxidase, or NADPH oxidase, a multicomponent enzyme system that catalyzes NADPH-dependent reduction of oxygen to O2.-. NADPH oxidase is activated and regulated by various neutrophil stimuli at infectious or inflammatory sites. Proinflammatory cytokines such as GM-CSF, TNF and IL-8 modulate NADPH oxidase activity through a priming phenomenon. These cytokines induce a very weak oxidative response by PMN but strongly enhance neutrophil release of reactive oxygen species on exposure to a secondary applied stimulus such as bacterial N-formyl peptides. Priming phenomena are involved in normal innate immune defense and in some inflammatory diseases. The mechanisms underlying the priming process are poorly understood, although some studies have suggested that priming with various agonists is regulated at the receptor and post-receptor levels. Resolution of inflammation involves desensitization phenomena and cytokines are involved in this process by various mechanisms. A better understanding of phenomena involved in the regulation of NADPH oxidase could help to develop novel therapeutic agents for inflammatory diseases involving abnormal neutrophil superoxide production.

The most frequent form of chronic granulomatous disease (CGD) is caused by inactivation of the CYBB gene, which encodes the gp91-phox subunit of phagocyte NADPH oxidase. This defect prevents phagocytes from producing reactive oxygen species and thus from eradicating bacterial and fungal infections. We investigated 16 unrelated male patients with suspected X-linked CGD and gp91-phox deficiency. A mutation was found in the CYBB gene of all 16 patients, and 11 of these mutations were novel. Eleven patients (69%) had a point mutation (84GA in two unrelated patients, and 177CG, 217CT, 388CT, 676CT, 691CT, 868CT, 919AC, 1384GT and T1514G in one case each, yielding W28X, C59W, R73X, R130X, R226X, Q231X, R290X, T307P, E462X, L505R gp-91phox). One patient had an in-frame deletion removing two amino acids (R54 and A55). Finally, insertions or duplications were found in four patients (from +1 to +31 bases). Overall, 12 (75%) of the mutations led to the production of a truncated protein. No clear correlation was found between clinical manifestations and genomic/biochemical alterations. Thirteen mothers could be tested, and all were carriers. Hum Mutat 18:163, 2001.

Rare hereditary deficiencies have been described which affect each functional stage of polymorphonuclear neutrophils. They almost invariably lead to recurrent acute infection. Among the abnormalities involving adhesion and motility, the following can be noted: the Buckley syndrome; and leucocyte type 1 and 2 adhesion deficiencies, respectively caused by a deficiency in membrane expression of beta 2 integrin CD11/CD18, and sialyl lewis X. Granulation system abnormalities include relatively non-symptomatic myeloperoxidase deficiency, specific granulation deficiency or the Chediak-Higashi syndrome with the presence of giant lysosomal granulations. Chronic or familial septic granulomatosis constitutes the main disease described due to the oxidative PMN burst connected with the functional impairment of one of the constituents of NADPH oxidase (with an incidence of one in 5.10(6) to one in 10(6) births) The transmission is X-linked, or autosomal recessive depending on the mutation. The antenatal detection of the X-linked component, gp91 phox, can be made in suspected carrier mothers. In addition to the standard treatment (Bactrim and Itraconazole), bone marrow transplantation may also be carried out, and in future gene therapy may be introduced.

Cytokines such as IL-10, IL-4, IL-13 and TGF-beta play a major role in the regulation of immune responses and are considered as anti-inflammatory agents mainly due to their actions on monocytes. These cytokines are also known to participate in the regulation of PMN activities. However, few and contradictory results have been reported on their direct and priming effects on the PMN oxidative burst, which is essential for killing bacteria. We used a flow cytometry method to study the effects of these cytokines on the PMN oxidative burst; we also used whole blood to avoid PMN activation related to isolation procedures and in order to simulate the in vivo situation more closely. None of the cytokines tested had direct or priming effects on PMN H2O2 production. We also show for the first time that these cytokines do not modulate TNF priming of the PMN oxidative burst in response to N-formyl peptides (fMLP). These results show that the anti-bacterial activity of PMN, in terms of the PMN respiratory burst, is not down regulated by these anti-inflammatory cytokines in whole blood.

Neutrophils and monocytes are the major classes of phagocytes that migrate from the blood stream and accumulate in inflamed tissues in response to various chemoattractants. Because IL-10 is a potent anti-inflammatory cytokine, we analyzed its in vitro effect on chemotaxis using an under agarose method. We found that, as compared to prednisolone, IL-10 alone was a modest inhibitor of C5a, fMLP and IL-8-induced neutrophil chemotaxis, and C5a-induced monocyte chemotaxis. However, GM-CSF pretreatment of the cells potentiated this inhibitory effect. Similarly, the IL-10 induced modulation of the beta2 integrin CD11b/CD18 adhesion molecule expression was only observed on GM-CSF-preactivated neutrophils and monocytes. Taken together, these results suggest that the migration and accumulation of phagocytes at infection sites would not be significantly affected by IL-10 given as an immunomodulatory therapy.

It was recently proposed that nitric oxide (NO) inhalation interferes with polymorphonuclear neutrophil (PMN) activation status during acute pulmonary inflammation, although variable results have been observed considering timing of NO administration, species, and model differences. After intratracheal administration of lipopolysaccharide (LPS) in rats, we characterized pulmonary inflammatory reaction (lung wet, dry, and wet to dry weights) and, using flow cytometry, the activation status (H2O2 production and beta2 integrin CD11b/CD18 expression) of PMN obtained from blood and from bronchoalveolar lavage (BAL). Eight hours after LPS injection, rats received for an additional 10 h, at a same Fio2 (85%), either 15 parts per million NO or the same gas flow of nitrogen. We found that 18 h after LPS, lung wet, dry, and wet-to-dry weights, H2O2 production, and CD11b/CD18 expression were increased. PMN obtained from BAL were highly activated as evidenced by an already maximal expression of the beta2 integrin CD11b/CD18, whereas the high H2O2 production at basal state could be further enhanced after ex vivo stimulation. Blood PMN were not different from control cells at basal state; however, their increased capacity to be stimulated ex vivo suggested an in vivo priming effect of intratracheal LPS. In conclusion, inhaled NO, given with a high FiO2, in the presence of this established endotoxinic lung injury did not reverse the markers of PMN activation studied nor lung edema formation in this rat model.

Polymorphonuclear neutrophils (PMN) are the most abundant immune cells in inflammatory gingival sites of patients with early onset periodontitis, localized juvenile periodontitis, and rapidly progressive periodontitis (RPP). In the latter, the large number of PMN in connective tissue may explain the marked gingival destruction. Because interleukin-8 (IL-8) is a potent PMN chemoattractant, we evaluated circulating levels and gingival mRNA expression of IL-8. We found high IL-8 plasma levels as well as strong IL-8 mRNA expression in both epithelial and connective gingival cells from patients with RPP. Moreover, the gingival PMN themselves contained IL-8 mRNA, suggesting an autoamplification of PMN recruitment and activation in the gingiva. We also measured the expression of adhesion molecules at the PMN surface as well as the oxidative burst in whole blood from 14 patients with RPP, using flow cytometry to avoid irrelevant stimulations and to analyze single cells. In RPP patients, resting PMN showed reduced L-selectin, Lewis x, and sialyl Lewis x antigen expression as well as increased H2O2 production. These modifications of PMN adhesion molecule expression, together with their increased basal oxidative burst and excessive IL-8 production, may contribute to the noxious inflammatory reaction, which may in turn be autopotentiated by PMN production of IL-8. In addition, PMN showed a lack of increased response (H2O2 production) to formyl peptides after ex vivo priming with IL-8, possibly owing to IL-8 desensitization that may be involved in the increased susceptibility of RPP patients to infection. After appropriate treatment of RPP, the reduction in inflammation was associated with a return to control levels of both plasma IL-8 and PMN functions, suggesting that these features are linked.

The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen to superoxide anion (O-2) at the expense of NADPH. This multicomponent enzyme is dormant in resting cells but is activated on exposure to an appropriate stimulus. The phosphorylation-dependent mechanisms regulating the activation of the respiratory burst oxidase are unclear, particularly the phosphorylation status of the cytosolic component p67(phox). In this study, we found that activation of human neutrophils with formyl-methionyl-leucyl-phenylalanine (fMLP), a chemotactic peptide, or phorbol myristate acetate (PMA), a stimulator of protein kinase C (PKC), resulted in the phosphorylation of p67(phox). Using an anti-p67(phox) antibody or an anti-p47(phox) antibody, we showed that phosphorylated p67(phox) and p47(phox) form a complex. Phosphoamino acid analysis of the phosphorylated p67(phox) revealed only 32P-labeled serine residues. Two-dimensional tryptic peptide mapping analysis showed that p67(phox) is phosphorylated at the same peptide whether fMLP or PMA is used as a stimulus. In addition, PKC induced the phosphorylation of recombinant GST-p67(phox) in vitro, at the same peptide as that phosphorylated in intact cells. PMA-induced phosphorylation of p67(phox) was strongly inhibited by the PKC inhibitor GF109203X. In contrast, fMLP-induced phosphorylation was minimally affected by this PKC inhibitor. Taken together, these results show that p67(phox) is phosphorylated in human neutrophils by different pathways, one of which involves protein kinase C.

We used flow cytometry to study the effects of pentoxifylline (PTX) on the expression of adhesion molecules and fMLP receptors on whole-blood polymorphonuclear neutrophils (PMN) in response to TNF, together with the oxidative burst and actin polymerisation. This technique analyses cells individually and avoids PMN activation related to isolation procedures. PTX reduced CD11b upregulation induced by TNF. Moreover, PTX reduced both TNF-induced binding of bacterial formyl peptides (fMLP) by human PMN and TNF priming of the PMN oxidative burst in response to these peptides. PTX also reduced TNF-induced actin polymerisation, which has been reported to participate in receptor cycling. This phenomenon could account in part for the ability of PTX to reduce fMLP binding to the PMN surface and subsequently to inhibit the PMN oxidative burst in response to fMLP. In addition to the PTX-induced decrease of TNF production, these effects on PMN could be beneficial in pathological conditions where high TNF production may induce excessive PMN activation, leading to vascular damage and tissue injury.

Impaired polymorphonuclear neutrophil (PMN) function may contribute to the onset of certain life-threatening bacterial and fungal infections in human immunodeficiency virus (HIV)-infected patients. Published data on PMN functional activity in HIV infection are controversial, possibly because most studies have involved PMNs isolated from their blood environment by means of various procedures that may differently affect surface receptor expression and thereby alter cellular responses. We therefore used flow cytometry to study the expression of adhesion molecules at the PMN surface, actin polymerization, and the oxidative burst of whole-blood polymorphonuclear neutrophils in 42 HIV-infected patients at different stages of the disease. These PMNs were activated in vivo, as demonstrated by increased expression of the adhesion molecule CD11b/CD18, reduced L-selectin antigen expression, increased actin polymerization, and increased H2O2 production. The alterations were present in asymptomatic patients with CD4+ cell counts greater than 500/microL and did not increase with the progression of the disease. Stimulation by bacterial N-formyl peptides showed dysregulation of L-selectin shedding and decreased H2O2 production after ex vivo priming with tumor necrosis factor alpha or interleukin-8 (IL-8). These latter impairments, which correlated with the decrease in CD4+ lymphocyte numbers and with IL-8 and IL-6 plasma levels, could contribute to the increased susceptibility of HIV-infected patients to bacterial infections.

To gain further insight into the pathogenesis of the adult respiratory distress syndrome (ARDS), the authors 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, 9 with severe pneumonia uncomplicated by ARDS, and 7 mechanically ventilated patients with neither ARDS nor pneumonia. Nine healthy subjects served as controls. Using flow cytometry, the authors 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 (LPS)-induced TNF-alpha production by cultured monocytes. These biological 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.

In order to analyse the role of LFA1 and MO1 on neutrophil functions, the blocking effects of two monoclonal antibodies (MAb), one (anti-MO1) recognizing an epitope of the MO1-alpha chain and the other (25.31) an epitope of the LFA1-alpha chain, were measured. Adherence of 51Cr-labelled control neutrophils was 66 + 8% (mean +/- 1 SD) on plastic nuclon plates; this figure decreased to 33 +/- 5% and 23 +/- 6% of control adherence when the neutrophils had been pretreated with anti-LFA1-alpha (anti-alpha L) and anti-MO1-alpha (anti-alpha M), respectively. On another support (plastic culture chambers), 84 +/- 6% of control neutrophils adhered and the adherence of neutrophils pretreated with anti-alpha L or anti-alpha M was 10% and 43% of the control figure, respectively. These results show that adherence of neutrophils is dependent upon the plastic used. Moreover, inhibition of adhesion by the two MAbs was also dependent upon the support used for the assay, suggesting that MO1 and LFA1 may be surface proteins with different specificities. Both antigens capped upon adhesion, while they were randomly distributed in resting neutrophils. Anti-alpha L inhibited (congruent to 50%) locomotion more than did anti-alpha M (congruent to 25%), without altering chemoattractant-induced shape changes. These results suggest that the two MAbs inhibit chemokinesis but not chemotaxis. Many other adherence-associated functions, such as ingestion of opsonized Klebsiella pneumoniae, and cytotoxicity towards K/562 cells were decreased more by anti-alpha L than by anti-alpha M. In contrast, chemiluminescence and iodination induced by opsonized zymosan were inhibited more by anti-alpha M than by anti-alpha L. Degranulation induced by zymosan or opsonized zymosan was altered by anti-alpha M only, and this alteration involved azurophilic and not specific granules. Chemiluminescence induced by phorbol myristate acetate was inhibited to a greater extent by anti-alpha M than by anti-alpha L, while degranulation induced by phorbol myristate acetate was not altered by either of the two Mabs.

Mice exposed to pure oxygen develop a lethal edematous pulmonary disease. This Note demonstrates, in these Mice, a lymphoid involution with blood lymphopenia and severe decrease in thymic weight and cellularity. The thymus was completely destroyed following 96 hrs. exposure. The possible role of these leucocyte alterations in normobare oxygen toxicity should be considered.