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In response to a variety of stimuli, e.g. pathogens, phagocytes release reactive oxygen species which are essential for bacterial killing and also potentiate inflammatory reactions. We have used flow cytometry measurements to study the priming process of phagocyte oxidative burst in whole blood, in order to avoid introducing artefacts due to the purification process and to stimulate the in vivo situation more closely. In these conditions, we examined the in vitro effects of proinflammatory cytokines (TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, IL-8 and GM-CSF) on the PMN oxidative burst. We found that none of the cytokine tested directly activated the PMN oxidative burst. In contrast, TNF, GM-CSF and IL-8 strongly primed a subpopulation of PMN which produced large amounts of H2O2 in response to fMLP, suggesting that these cytokines may play a critical role in bactericidal killing in vivo. Furthermore, we reported a decreased H2O2 production by TNF or IL-8 primed PMN in HIV-infected patients. This impairment, which correlated with the clinical stage of the disease, could contribute to the increased susceptibility to bacterial infections in HIV-infected patients. In addition, we reported the case of a child with severe recurrent infections due to intracellular microorganisms which could be related to an impairment of the phagocyte priming process of the oxidative burst [corrected].

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.

We have tested the hypothesis that cellular activation events occurring in T lymphocytes and monocytes and mediated through translocation of the transcription factor NF-kappa B are dependent upon the constitutive redox status of these cells. We used phenolic, lipid-soluble, chain-breaking antioxidants (butylated hydroxyanisole (BHA), nordihydroquairetic acid, or alpha-tocopherol (vitamin E) to show that peroxyl radical scavenging in unstimulated and PMA- or TNF-stimulated cells blocks the functions depending on NF-kappa B activation. BHA was found to suppress not only PMA- or TNF-induced, but also constitutive, HIV-enhancer activity concomitant to an inhibition of NF-kappa B binding activity in both lymphoblastoid T (J.Jhan) and monocytic (U937) cell lines. This was also true for KBF (p50 homodimer) binding activity in U937 cells. Secretion of TNF, the product of another NF-kappa B-dependent gene, was abolished by BHA in PMA-stimulated U937 cells. The anti-oxidative effect of BHA was accompanied by an increase in thiol, but not glutathione, content in stimulated and unstimulated T cell, whereas TNF stimulation itself barely modified the cellular thiol level. Oxidative stress obtained by the addition of H2O2 to the culture medium of J.Jhan or U937 cells could not by itself induce NF-kappa B activation. These observations suggest that TNF and PMA do not lead to NF-kappa B activation through induction of changes in the cell redox status. Rather, TNF and PMA can exert their effect only if cells are in an appropriate redox status, because prior modification toward reduction with BHA treatment prevents this activation. It appears that a basal redox equilibrium tending toward oxidation is a prerequisite for full activation of transduction pathways regulating the activity of NF-kappa B-dependent genes.

Cystic fibrosis (CF), is the most common life-shortening autosomal recessive disorder in Caucasians. It is caused by mutations in a single gene on the long arm of chromosome 7 that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CF is characterized by abnormal Na+ and Cl- ion transport in several tissues, including the lungs, pancreas, gastrointestinal tract, liver, sweat glands, and male reproductive system. Progressive pulmonary disease is the dominant clinical feature of CF and accounts for morbidity and mortality. The inflammation characterized by an overabundance of activated neutrophils and macrophages on the respiratory epithelial surface is associated to a high production of reactive oxygen species (ROS) which contribute to the pathogenesis of cystic fibrosis. ROS could have different origins but the role of the NADPH oxidase system is essential. The "NADPH oxidases" (NOX/DUOX) family is an enzymatic complex formed by cytosolic and membrane subunits. Until now several homologues of the phagocytic NADPH oxidase have been identified in different tissues and it has been shown that the lungs preferentially expressed DUOX1-2. Thus, DUOX1-2 could be implicated in the anti-infectious defense system. The role of DUOX enzymes as a source of ROS in cystic fibrosis is examined as they could contribute to a better understanding of molecular mechanisms in CF. Moreover they could be a potential target for a new therapeutic approach.

Certain tumor cells generate factors that inhibit neutrophil chemotaxis. Our study was designed to explore whether such factors are produced by K 562 malignant cells and whether these have a broader effect in altering neutrophil functions. After 48 h of in vitro culture of K 562 cells, the culture medium and the cells were separated, lyophilized, and extracted with ethanol. These K 562 products, i.e., either the cell or supernatant extract, inhibited both nonstimulated locomotion and locomotion induced either by FMLP or activated serum. Furthermore, K 562 products inhibited neutrophil adherence and oxidative burst induced by opsonized zymosan, whereas oxidative burst induced by PMA or FMLP was not altered. K 562 products had an inhibitory effect on the PMN binding to iC3b-coated particles. They did not modify Mo1 expression of resting cells, did not alter the up-regulation of the receptor induced by FMLP but inhibited the FMLP-induced capping of Mo1 Ag. Con A capping was also inhibited. Actin polymerization in FMLP-stimulated PMN, as measured by flow cytometry and phalloidin binding to F-actin, was inhibited by K 562 products. The inhibitory factor present in K 562 products (cell and culture supernatant) was purified in three steps including gel filtration, ion-exchange chromatography, and IEF. The eluted active fraction corresponded to single band of about 8 kDa on SDS-PAGE. From these experiments, it is concluded that K 562 malignant cells in culture contain and release a low molecular mass factor (congruent to 8 kDa) that inhibits all adherence-related functions of neutrophils, whereas it does not alter FMLP- or PMA-induced oxidative burst. Further studies are needed to assess whether products of other tumor cells also act on the neutrophil by inhibiting adherence-related functions, Mo1 function and capping, and actin polymerization.

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.

Vitamin E (alpha-tocopherol), one of the most important natural antioxidants, is assumed to be beneficial in the prevention of cardiovascular diseases. alpha-Tocopherol exhibits acyl-peroxyl-radical scavenger properties and exerts cell-mediated actions in the hemovascular compartment, such as inhibition of superoxide anion (O-2) production by leukocytes. The aim of this study was to examine the mechanism underlying the inhibitory effect of alpha-tocopherol on O-2 production by human monocytes. In activated monocytes O-2 is produced by the NADPH-oxidase enzyme complex. The oxidase activation elicited by phorbol myristate acetate (PMA) requires membrane translocation of several cytosolic factors. We found that in human PMA-stimulated adherent monocytes, alpha-tocopherol (but not beta-tocopherol) inhibited O-2 production in intact cells but had no effect on a membrane preparation containing activated NADPH-oxidase, suggesting that alpha-tocopherol impairs the assembly process of the enzyme complex. We showed that translocation and phosphorylation of the cytosolic factor p47(phox) were reduced in monocytes preincubated with alpha-tocopherol. We verified that the tryptic phosphopeptide map of monocyte p47(phox) was similar to that of neutrophil p47(phox), indicating that several serine residues were phosphorylated. Peptides whose phosphorylation is dependent on protein kinase C (PKC) were phosphorylated to a lesser degree when p47(phox) was immunoprecipitated from alpha-tocopherol-treated monocytes. In vitro, the activity of PKC from monocytes was inhibited by alpha-tocopherol in a specific manner compared with that of beta-tocopherol or Trolox(R). Membrane translocation of PKC was not affected. These results show that alpha-tocopherol inhibits O-2 production by human adherent monocytes by impairing the assembly of the NADPH-oxidase and suggest that the inhibition of phosphorylation and translocation of the cytosolic factor p47(phox) results from a decrease in PKC activity.

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.

Despite numerous reports on IL-1 and the obvious importance of IL-1 in the cytokine network, very little is known regarding the molecular details of IL-1 regulation. While information is now emerging for IL-1 beta, mechanisms in IL-1 alpha gene regulation remain largely unknown. Examination of the 5'-flanking region of the human interleukin-1 alpha promoter (IL-1 alpha) and its first intron revealed several potential binding sites for known transcription factors. We thus studied this region by using an electrophoretic mobility shift assay (EMSA). EMSA studies showed that the region -12 to -6 of the human IL-1 alpha promoter contains an LPS-inducible AP-1-binding site composed of Jun and Fos proteins. On the other hand, no NF-kappa B binding within the IL-1 alpha promoter and its first intron was shown.

Several studies have implicated interleukin 1 alpha (IL-1 alpha) in the pathogenesis of rheumatoid arthritis (RA). We analysed IL-1 alpha intron 6 polymorphism in relation to RA (50 patients with RA and 50 healthy controls). The study of a healthy control population confirmed the existence of the different alleles with a frequency similar to that in the Caucasian populations of northern England. Allele and genotype distributions did not differ significantly between the normal and RA populations, although the allele corresponding to 8 repeats was over-represented in the RA population (8 and 14% in the healthy and RA populations respectively). This suggests that IL-1 alpha intron 6 polymorphism could be part of a complex process involving other unidentified genetic factors in the pathogenesis of RA.

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.

The "Centers for Disease Control" (CDC) recently published the guidelines for the performance of CD4+ T-cell determinations in persons with Human Immunodeficiency Virus (HIV) infection. Especially, a monoclonal antibody panel for lymphocyte immunophenotyping has been recommended i.e CD45/CD14, isotypic controls, CD3/CD4, CD3/CD8, CD3/CD19, CD3/CD56+ and/or CD16+. The authors compared, in 50 HIV+ patients, this method with the conventional method used in their laboratory i.e CD4/CD8 associated with isotypic controls. The mean values of CD4+ and CD8+ cells obtained using dual color immunophenotyping CD3/CD4 and CD3/CD8 did not differed significantly as compared with the values obtained using dual color immunophenotyping CD4/CD8. Especially, concerning CD4+ cells, differences did not exceed 4% considering each patient and 1% considering the mean values. However, in some patients, the differences between the levels of CD8+ cells obtained using the two methods were greater than 10%. These differences were due to an important percentage of CD3-CD8+ cells corresponding to NK cells. In another hand, there was no significant difference between the levels of CD4 and CD8+ cells obtained with or without correction using the gating reagent CD45/CD14. In conclusion, monoclonal antibody pannel recommended by CDC for lymphocyte immunophenotyping in HIV patients do not seem necessary in all cases. Analysis for CD4 and CD8 positive cells could be accomplish by three color simultaneous method (CD3/CD4/CD8), by first gating on CD3 positive T lymphocytes in order to eliminate both monocyte and NK cell contamination.

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.

Antibiotics do not act alone but in conjunction with the host defence system. In particular, it has been shown that antibiotics can modify cytokine production. The authors reported here the effects of antibiotics which penetrate inside the cells, such as quinolones and macrolides, on the capacity of blood monocytes to produce IL-I alpha, IL-1 beta, TNF alpha and IL-6 in response to endotoxin. Antibiotics can exert a differential effect on cytokine production: in fact, quinolones, in vitro, at concentrations higher than 25 micrograms/ml decreased IL-1 beta, TNF alpha and IL-6, while they do not modify IL-1 alpha. Moreover, ciprofloxacin orally administered (25 mg/kg for 7 days) transitory increased cytokine production. These results are discussed in terms of tissue concentration. Among the same family of antibiotics such as macrolides, differences on cytokine modulation were observed: spiramycin and erythromycin increased IL-6 production while roxithromycin did not exert any significant effect. All these results tend to prove that some antibiotics are immunomodulators; however interactions between antibiotics and immune responses are complex and studies with patients with infections will be necessary to a better understanding of these relationships.

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.

We have tested the hypothesis that cellular activation events occurring in T lymphocytes and monocytes and mediated through translocation of the transcription factor NF-kappa B are dependent upon the constitutive redox status of these cells. We used phenolic, lipid-soluble, chain-breaking antioxidants (butylated hydroxyanisole (BHA), nordihydroquairetic acid, or alpha-tocopherol (vitamin E) to show that peroxyl radical scavenging in unstimulated and PMA- or TNF-stimulated cells blocks the functions depending on NF-kappa B activation. BHA was found to suppress not only PMA- or TNF-induced, but also constitutive, HIV-enhancer activity concomitant to an inhibition of NF-kappa B binding activity in both lymphoblastoid T (J.Jhan) and monocytic (U937) cell lines. This was also true for KBF (p50 homodimer) binding activity in U937 cells. Secretion of TNF, the product of another NF-kappa B-dependent gene, was abolished by BHA in PMA-stimulated U937 cells. The anti-oxidative effect of BHA was accompanied by an increase in thiol, but not glutathione, content in stimulated and unstimulated T cell, whereas TNF stimulation itself barely modified the cellular thiol level. Oxidative stress obtained by the addition of H2O2 to the culture medium of J.Jhan or U937 cells could not by itself induce NF-kappa B activation. These observations suggest that TNF and PMA do not lead to NF-kappa B activation through induction of changes in the cell redox status. Rather, TNF and PMA can exert their effect only if cells are in an appropriate redox status, because prior modification toward reduction with BHA treatment prevents this activation. It appears that a basal redox equilibrium tending toward oxidation is a prerequisite for full activation of transduction pathways regulating the activity of NF-kappa B-dependent genes.

Ciprofloxacin, a new fluoroquinolone antibiotic, inhibits the in vitro production of interleukin 1 beta and tumor necrosis factor alpha by monocytes. We investigated its activity against type II collagen induced arthritis in rats. It exerted a dose dependent preventive effect at 50 and 75 mg/kg/day against clinical and histologic features of collagen induced arthritis without any influence on the production of anticollagen antibodies and alpha 1-acid glycoprotein. This effect was reversible after early removal of the treatment. Ciprofloxacin did not inhibit collagen induced arthritis in adrenalectomized rats but rather caused an exacerbation of the disease. Its effect was not modified by the simultaneous administration of an antiglucocorticoid, RU 40555.

Previous studies have shown that in lipopolysaccharide (LPS)--stimulated human monocytes, interleukin-1 (IL-1) production is altered by quinoline derivative antibiotics (quinolones), in a way which depends both on the dose and on the agents used. Given that IL-1 and tumor necrosis factor alpha (TNF) are produced in response to LPS and have some overlapping and synergistic activities, we sought to determine if TNF production was altered under the above-mentioned conditions. We investigated the effects of three quinolones: ciprofloxacin (Cip), pefloxacin (Pef) and ofloxacin (Ofl). These quinolones were found to decrease extracellular TNF production in a dose-dependent manner at concentrations higher than 25 micrograms/ml as previously described by our laboratory with regard to IL-1 production. Moreover, the order of the extracellular decrease in TNF and IL-1 induced by each drug was similar. However, in contrast to IL-1 activity, the quinolones studied also reduced cell-associated TNF. The kinetics of TNF production suggested that the quinolones affected TNF production at a very early step, probably during TNF synthesis rather than during its secretion into the extracellular medium. Furthermore, the quinolone-induced accumulation of intracellular cAMP could explain the extracellular decrease in both IL-1 and TNF production.

The existence of IL-1 activity on the cell surface of stimulated mononuclear phagocytes is a matter of controversy. In particular, fixation of IL-1-expressing cells for 15 min in 1% paraformaldehyde (PFA) is commonly used to evidence such "membrane-associated" IL-1 activity but other authors have attributed this to passive leakage of IL-1 alpha from the cells and report no activity with longer fixation times. Using specific IL-1 alpha and IL-1 beta assays, we found that after the mild standard PFA fixation procedure, not only IL-1 alpha but also IL-1 beta were released into the supernatants for up to 96 h following fixation; membrane IL-1 activity cannot thus be measured in these conditions. However, using conditions in which neither immunoreactive IL-1 molecules nor IL-1 activity are found in the supernatants (i.e. assay at 144 h, increased fixation time), we were still able to detect IL-1 activity on LPS-stimulated, PFA-fixed monocytes. This activity was independent of the duration of PFA fixation and was inhibited by anti-IL-1 alpha but not anti-IL-1 beta antibodies. Our data thus underline the importance of technical conditions in the study of membrane-associated IL-1 activity.

The effects of cytochrome P-450 inducers on O2 toxicity were studied in mice. We first examined three cytochrome P-450 inducers, which differ by their specific tissue affinity: phenobarbital sodium (PB), essentially active in the liver, and 3-methylcholanthrene (3-MC) and beta-naphthoflavone (BNF), which are also active in the lung. Both BNF and 3-MC increased the survival rate and significantly decreased pulmonary edema (pulmonary water and wet-to-dry weight ratio) in C57BL/6J mice exposed to hyperoxia (O2 greater than or equal to 95%), whereas PB had no protective effect. In the second part of this study, we compared the action of BNF in two strains of mice. In one (C57BL/6J), cytochrome P-450 can be induced by aromatic hydrocarbons, whereas in the other (DBA/2J) cytochrome P-450 is not inducible by these compounds. Protection against O2 toxicity was assessed in terms of lethality and pulmonary edema and of lung lipid peroxidation (assessed by measuring malondialdehyde). BNF only protected against O2 toxicity in the inducible strain. This protective effect of BNF on O2 toxicity in C57BL/6J mice was associated mainly with a large increase in the components of the cytochrome P-450 system (cytochrome P-450 and cytochrome b5) in the lung. The activity of pulmonary superoxide dismutase was also slightly increased, but the enhancement was not statistically significant. In contrast, in DBA/2J mice neither the components of the cytochrome P-450 system nor the activity of superoxide dismutase showed any increase.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo exposure of mice to normobaric O2 depresses the cellular immune response by a mechanism that remains unknown. In vitro oxidative injury leads to decreased sulfhydryl groups (SH) in lymphocytes. To determine whether in vivo exposure to O2 would have similar effects, we measured the SH content in spleen cells both from mice that had been exposed to normobaric O2 (O2 SC) and from controls exposed to ambient air (Air SC). The SH content of the fresh O2 SC was slightly decreased, whereas after 48 hr of culture, the SH content and the proliferative response of these cells were found to vary with the type and concentration of thiol or disulfide compounds added to the culture medium. Under standard culture conditions, i.e., RPMI 1640 medium containing 0.41 mM half-cystine, the SH content in O2 SC decreased sharply to about 10 and 20% that of Air SC in the absence or presence of Con A (2 micrograms/ml), respectively. Under these culture conditions, the proliferative response of O2 SC was 20.5% +/- 3.2 of Air SC. In cystine-free RPMI 1640 medium supplemented with various concentrations of L-cystine, L-cystine and 2-mercaptoethanol (2-ME), L-cysteine, or reduced glutathione (GSH), the proliferative response to Con A and the SH content of the O2 SC varied in parallel and were correlated (p less than 0.01). Half-cystine (0.41 mM) plus 2-ME (5 X 10(-5) M) or L-cysteine alone (4 mM) completely protected the SH content of O2 SC and induced a proliferative response 82% +/- 6 that of the controls. In cystine-free RPMI 1640 medium supplemented with GSH (4 mM), the SH content and proliferative response of O2 SC were 79 and 67.5% of Air SC, respectively. Other concentrations of these compounds were less effective. Oxygen scavengers such as SOD, catalase, mannitol, and vitamin E did not protect against the decrease of the O2 SC. The induced oxidative cellular damage might be related in part to a membrane lipid peroxidative process. These data show that in vivo exposure of mice to normobaric O2 induced lesions in splenic cells manifested under standard culture conditions by a decrease in both SH content and Con A proliferative response. The extent of these alterations could be modulated by variations of the thiol environment. Protection of the SH content correlated with protection of the proliferative response of the O2 SC.

Resume Les effets de la cyclosporine sur le nombre des lymphocytes circulants et des sous-populations lymphocytaires T ont ete evalues chez 10 malades atteints de SIDA, ayant au moins une infection opportuniste. Tous avaient avant le traitement, des lymphocytes T4 inferieurs a 400/μl. Onze cures de cyclosporine ont ete administrees en perfusion veineuse continue, pour une duree moyenne de 7, 9 jours, la dose (0,9–8 mg/kg/j) etant adaptee pour maintenir la concentration plasmatique entre 100 et 200 ng/ml. Les comptes des lymphocytes totaux, T4, T6, T8, effectues avant, a J4, J6, J8 et apres cyclosporine, n'ont pas montre globalement de modifications : une augmentation transitoire des lymphocytes T4 a J4 et une diminution des lymphocytes totaux a J8 et apres (p

Normobaric O2 exposure decreased spleen cell (SC) response to T cell mitogen Con A. 3H-TdR incorporation of SC from O2 exposed mice (O2SC) compared to those of control mice (Air SC) decreased significantly after 72 and 87 h O2 exposure. The dose response kinetics to Con A were identical in O2SC or Air SC. Increasing SC number did not restore the response to Con A and the depressed hyperoxic effect was not related to suppressor cells in the spleen of O2 exposed mice. Response of O2SC to Con A was restored by the thiol compound 2-mercaptoethanol (2-ME), and the degree of restoration by 2-ME, was inversely proportional to the depressed response. Addition of intact peritoneal cells (PC) induced restoration within the same range as 2-ME. Restoration of the mitogenic response by 2-ME involved antioxidant properties and suggested that macrophages were functionally injured by O2 exposure. In cases where mitogen response was highly depressed, restoration was only partial; in these conditions in vivo O2 injury probably involved both macrophages and splenic T cells. The mechanisms of O2 toxicity have been discussed in terms of free radical generation under hyperoxic conditions.

The use of normobaric exposure to O2 as a model for in vitro oxidative injury prevented phytohaemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC) from undergoing the G0 to G1 transition, but 5 x 10(-6) M 2-mercaptoethanol (2-ME) almost protected the cells from this blockade. The percentage of cells with IL-2 and transferrin-receptors was reduced by the O2 exposure and, like the cell cycle transition, was protected by 2-ME against oxidative injury. By contrast, IL-2 recovery in the supernatants of O2-exposed PHA-stimulated PBMC was enhanced. This enhancement may be due partly to the reduced IL-2 consumption caused by the decreases in IL-2 receptor expression and in proliferation. On the other hand, IL-2 recovery in the supernatants of O2-treated PBMC was always enhanced compared to the IL-2 control recovery after DNA synthesis was blocked in G1/S by mitomycin c, and the G0/G1 transition was protected by 2-ME. Furthermore, PHA-stimulated monocytes exposed to O2 produced more IL-1 than control cells. This enhanced IL-1 production was not modified by 2-ME. These results suggest that oxidative injury reduces the proliferation of PBMC by interfering with the cellular events that lead to the transition from the G0 to the G1 phase of the cell cycle. The protective effects of 2-ME suggest that thiol compounds have a critical role in the early events of the cell cycle. By contrast, exposure to O2 induced increases in the production of both IL-1 and IL-2 that may not be related to alterations in the thiol status of the cell.

Immunofluorescence and immunogold techniques yielded similar results when used to determine the T lymphocyte subsets (T4 and T8 positive cells) in patients with AIDS or AIDS-related complex and in healthy homosexual men seropositive for HTLV III/LAV antibody. In these pathological situations, the advantages of immunogold staining could render this technique useful in a clinical laboratory.

Rat spleen cells were cultivated in 1 ATA pure oxygen. The mitogenic responses with Con A were evaluated at different time of exposure (12 to 72 hrs). There was a stimulation of the cells after 12 hrs of exposure, which diminished at 18 hrs, in spite of the fact that the viability of the cells remained unchanged till 24 hrs of exposure. The kinetics of the mitogenic response of the splenic lymphocytes exposed to oxygen is biphasic as has been observed after irradiation.

Immunological cell functions were evaluated during 24, 48 and 96 h O2 exposure in C57Bl/6 mice. A normobaric O2 exposure resulted in depression of delayed type hypersensitivity (DTH) to oxazolone and Staphylococcus aureus antigens. This effect was proportional to the duration of O2 exposure. The antibody response of splenic cells was more rapidly (24 h O2 exposure) and markedly depressed using a T-dependent antigen (sheep red blood cell, SRBC) than with a T-independent antigen (trinitrophenylated lipopolysaccharide, TNP-LPS). While mitogen-induced proliferative responses of spleen cells to Con A and PHA were inhibited after 72 h of O2 exposure, proliferative responses to LPS were inhibited after 96 h. A dissociated antigen and mitogen responses was observed after a short time of O2 exposure (48 h): the antigen specific responses were impaired with a more pronounced effect on T lymphocytes, whereas the DNA synthesis in response to mitogen remained normal.

The effects of penicillin, macrolides (spiramycin and erythromycin), cephalosporins (cefaclor and cefadroxil), cycline (doxycycline) and quinolones (pefloxacin, ciprofloxacin and ofloxacin) on extracellular and cell-associated interleukin-1 activity from human monocytes were investigated in vitro. When cells were treated with 10 micrograms/ml of quinolones, cephalosporins or penicillin, no effect on IL-1 production could be detected. Using 100 micrograms/ml, extracellular IL-1 activity was found to be decreased by quinolones (about 35% of the control without antibiotic) without modification of the cell-associated IL-1 activity. Extra and intracellular IL-1 was only slightly decreased by cephalosporins, while penicillin did not alter the IL-1 activities. Spiramycin and doxycycline using 100 micrograms/ml increased extracellular IL-1 while cell-associated was decreased. A toxic effect may have been exerted by these antimicrobial agents.

Prolonged exposure of C57B16 mice to pure O2 at 1 ATA induced pulmonary edema associated with involution of lymphoid system and depressed immunity. The consequences of these toxic events were evaluated by 1) mortality rate, 2) determination of pulmonary water, 3) thymic and splenic cellularity, and 4) humoral (primary antibodies) and cellular (mitogenic) immune responses. Pretreatment of mice with 125 mg kg-1 of diethyldithiocarbamate (DDC) several days before exposure to O2 resulted in 1) an increase in animal survival (92-100% vs. 59% O2 controls), 2) a reduction in pulmonary edema, 3) partial stabilization of thymus and spleen lymphocyte populations, and 4) restoration of the humoral response (specific antibodies appeared earlier than in O2 control animals) and improvement of the mitogenic proliferative response of the spleen cells after hyperoxia. None of these effects were observed when DDC treatment coincided with the beginning of exposure. Our results indicated that DDC protects mice from both pulmonary and lymphoid hyperoxic injury, but only in a partial manner. It is suggested that the mechanism of this antioxidative property is indirect.

Rates of postoperative bacterial infections are high in patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) (1,2). These data suggest that host defence mechanisms may be impaired in the postoperative period. The purpose of this study is a reappraisal of the postoperative immune status in these patients since several investigations have reported its deficiency (3–5) while others found it normal (6,7).

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.

This study analyzes a method of indirect marking of T lymphocytes subpopulations using a 2nd antibody coupled to colloidal gold particles (Immunogold). There is an excellent correlation between this technique and the classical technique of indirect immunofluorescence. Immunogold enables an easy and rapid count of lymphocytes CD4+ and CD8+ during infections secondary to HIV virus (LAV/HTLV III). The permanent nature of the preparations and the cytological control make this method an interesting tool for the diagnosis and follow-up of patients suffering from AIDS, ARC or only asymptomatic seropositive individuals.

Inducers of cytochrome P450-linked mono-oxygenases increase the normobaric oxygen tolerance of the adult rat. Pulmonary inducers, as 3-methylcholanthrene and beta-naphthoflavone permit the rat survival and simultaneously a decrease of pulmonary edema. Phenobarbital, an hepatic inducer had lesser effects both on survival rate and on pulmonary and lymphoïd oxygen toxicity.

In vivo exposure of mice to normobaric O2 depresses the cellular immune response by a mechanism that remains unknown. In vitro oxidative injury leads to decreased sulfhydryl groups (SH) in lymphocytes. To determine whether in vivo exposure to O2 would have similar effects, we measured the SH content in spleen cells both from mice that had been exposed to normobaric O2 (O2 SC) and from controls exposed to ambient air (Air SC). The SH content of the fresh O2 SC was slightly decreased, whereas after 48 hr of culture, the SH content and the proliferative response of these cells were found to vary with the type and concentration of thiol or disulfide compounds added to the culture medium. Under standard culture conditions, i.e., RPMI 1640 medium containing 0.41 mM half-cystine, the SH content in O2 SC decreased sharply to about 10 and 20% that of Air SC in the absence or presence of Con A (2 micrograms/ml), respectively. Under these culture conditions, the proliferative response of O2 SC was 20.5% +/- 3.2 of Air SC. In cystine-free RPMI 1640 medium supplemented with various concentrations of L-cystine, L-cystine and 2-mercaptoethanol (2-ME), L-cysteine, or reduced glutathione (GSH), the proliferative response to Con A and the SH content of the O2 SC varied in parallel and were correlated (p less than 0.01). Half-cystine (0.41 mM) plus 2-ME (5 X 10(-5) M) or L-cysteine alone (4 mM) completely protected the SH content of O2 SC and induced a proliferative response 82% +/- 6 that of the controls. In cystine-free RPMI 1640 medium supplemented with GSH (4 mM), the SH content and proliferative response of O2 SC were 79 and 67.5% of Air SC, respectively. Other concentrations of these compounds were less effective. Oxygen scavengers such as SOD, catalase, mannitol, and vitamin E did not protect against the decrease of the O2 SC. The induced oxidative cellular damage might be related in part to a membrane lipid peroxidative process. These data show that in vivo exposure of mice to normobaric O2 induced lesions in splenic cells manifested under standard culture conditions by a decrease in both SH content and Con A proliferative response. The extent of these alterations could be modulated by variations of the thiol environment. Protection of the SH content correlated with protection of the proliferative response of the O2 SC.