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Twenty-two SLE sera were assessed for their stimulating properties for lymphocytes. After fixation of specific serum factors, lymphocyte cultures were performed and supernatant's of 24, 48, 72 and 96 h tested on two polymorphonuclear functions: chemotaxis and cyanide insensitive O2 consumption; 20 out of 22 SLE sera supernatant's led to enhanced chemotaxis. The factors involved were not destroyed by heating for 30 min at 56°C. Fifteen of the SLE sera supernatants increased the O2 consumption by PN. In most cases this second mediator was destroyed by heating. The buffer, the normal sera, the monoclonal antibodies stimulated lymphocytes supernatant's and the total lymphocyte lysate did not display such enhancing activities. So it clearly appears that SLE sera contain factors acting on lymphocyte membrane and induce them to secrete new lymphokines called PNAF (polymorphonuclear activating factors) which may play a role in pathogenesis of tissue lesions in SLE. [ABSTRACT FROM AUTHOR]

In order to analyse the role of LFA1 and MO1 on neutrophil functions, the blocking effects of two monoclonal antibodies (MAb), one (anti-MOI) recognizing an epitope of the MOI-α chain and the other (25.31) an epitope of the LFAI-α chain, were measured. Adherence of 51Cr-labelled control neutrophils was 66 + 8% (mean ± 1SD) on plastic nuclon plates; this figure decreased to 33 ± 5% and 23 ± 6% of control adherence when the neutrophils had been pretreated with anti-LFAI-α (anti-αL) and anti-MOl-α (anti-αM), respectively. On another support (plastic culture chambers), 84±6% of control neutrophils adhered and the adherence of neutrophils pretreated with anti-αL or anti-αM 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 MOI and LFAI may be surface proteins with different specificities. Both antigens capped upon adhesion, while they were randomly distributed in resting neutrophils. Anti-αL inhibited ([This symbol cannot be presented in ASCII format]50%) locomotion more than did anti-αM ([This symbol cannot be presented in ASCII format]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-αL than by anti-αM. In contrast, chemiluminescence and iodination induced by opsonized zymosan were inhibited more by anti-αM than by anti-αL. Dgranulation induced by zymosan or opsonized zymosan was altered by anti-αM only, and this alteration involved azurophilic and not specific granules. Chemiluminescence induced by phorbol myristate acetate was inhibited to a greater extent by anti-αM than by anti-αL, while degranulation induced by phorbol myristate acetate was not altered by either of the two Mabs. [ABSTRACT FROM AUTHOR]

Josamycin, a 16-membered ring macrolide is concentrated up to 20-fold in phagocytic cells compared with serum. We have studied the in-vitro interaction of this drug with human neutrophils (PMN) bactericidal function by using two strains resistant to this antibiotic, Pseudomonas aeruginosa and Klebsiella pneumoniae, and a sensitive one, Staphylococcus aureus 209P. It was shown that josamycin-pretreated adherent PMN displayed an increased phagocytic activity (about 30 to 40%) for S. aureus or K. pneumoniae, mainly due to the recruitment of an additional phagocytizing subset of PMN. Furthermore, the bacterial killing was enhanced in josamycin-treated PMN in a dose-dependent manner for K. pneumoniae (60-130% increase in the range of concentration 0.1-25 mg/l) and independently of the dose for S. aureus (about 425-460% increase for josamycin 0.1-10 mg/l). P. aeruginosa killing by whole blood was also significantly increased in the presence of 10 and 1 mg/l of josamycin. Other PMN functions were not much altered by josamycin except an enhancement of the formyl-methionyl-leucyl-phenylalanine-induced oxidative response. Chemotaxis was only increased by the presence of a high concentration (100 mg/l) of josamycin. These data suggest that the bactericidal synergy between PMN and josamycin could be related, partly at least, to a direct enhancing effect of josamycin on some PMN functions such as phagocytosis, chemotaxis and FMLP-induced chemiluminescence. On the other hand, alterations of bacteria, either inside the phagolysosome or in the extracellular medium, could lead to an enhanced susceptibility to the phagocytes' microbial mechanisms. [ABSTRACT FROM AUTHOR]

We have compared the in-vitro interaction of five macrolides (roxithromycin, erythromycin, spiramycin, oleandomycin and josamycin) with human neutrophils (PMN). Only roxithromycin strongly impaired the oxidative burst of PMN assessed by luminol amplified chemiluminescence, superoxide anion generation, and myeloperoxidase-mediated iodination of proteins. This effect was observed only for high concentrations of this drug (100 and 50 mg/l). Furthermore, the sensitivity of PMN to the depressive effect of roxithromycin permitted the definition of two kinds of PMN: in Highly Sensitive (HS)-PMN, the oxidative response was completely abolished while in Moderately Sensitive (MS)-PMN, a decreased, but yet measurable (20-50% of the control), response was obtained. The roxithromycin-induced depression of PMN was time-dependent and partly reversed by washing. Chemotaxis was also impaired by roxithromycin (100 mg/l) but phagocytosis of Klebsiella pneumoniae was unaltered even at high concentrations of the drug. Since roxithromycin displays the highest intracellular uptake, compared with the other macrolides assessed in this study, this could explain the results observed here. The relevance to the clinical situation needs further study. This effect of roxithromycin could be useful to control the inflammatory process associated in certain infectious diseases, in particular if high concentrations of the drug are obtained in tissues. [ABSTRACT FROM AUTHOR]

Ceftriaxone, a 2-aminothiazolyl cephalosporin does not alter human neutrophil (PMN) bactericidal function. However, low concentrations of ceftriaxone induce some bacterial strains to be more sensitive to PMN killing. We have studied the effect of a subinhibitory concentration of ceftriaxone (10 mg/l) on Pseudomonas aeruginosa (MIC greater than 128 mg/l). After an overnight exposure to this concentration of ceftriaxone, P. aeruginosa elongated into filaments. PMN killing of ceftriaxone-treated bacteria was better than killing of control bacteria. This enhanced killing was correlated with an increased sensitivity to oxygen-dependent bacterial killing. Furthermore, the altered bacteria induced a greater oxidative response of PMN which was independent of their chemiluminescence response after stimulation by control P. aeruginosa. This increased oxidative burst was attributable to both non-opsonodependent stimulation and to increased deposit of opsonins. [ABSTRACT FROM AUTHOR]

Two bacterial strains (Staphylococcus aureus and Klebsiella pneumoniae) were exposed to subinhibitory concentrations of ceftriaxone. After an overnight culture in presence of 1 MIC of ceftriaxone either in broth or on solid medium S. aureus showed enlarged forms which were better phagocytosed (increase about 40%) and killed (increase about 50%) than control staphylococci. Exposure of K. pneumoniae to 0.1 MIC ceftriaxone resulted in filamentation of bacteria. When grown in the presence of 0.01 MIC, K. pneumoniae did not elongate into filaments but were significantly more phagocytosed (increase about 40%) or killed (increase about 170%) than control bacilli. The mechanism of the greater sensitivity to PMN killing of the altered S. aureus and K. pneumoniae was assessed either with phenylbutazone-treated PMN or by in-vitro exposure to crude granule extracts of PMN. The altered bacteria displayed a significant susceptibility to the non-oxidative killing mechanism while untreated bacteria were unaffected by the non-oxidative system. These data could explain the synergy observed between ceftriaxone and leucocytes in the killing of some micro-organisms. [ABSTRACT FROM AUTHOR]

Ceftriaxone, an amino-2-thiazolyl cephalosporin, has been shown to cooperate in vitro with human neutrophils for the killing of some bacteria. In this work the direct interaction with human leucocyte bactericidal function has been studied. Ceftriaxone (1000 to 1 mg/l) did not alter neutrophil chemotaxis or superoxide anion production. It also did not interfere with the chemiluminescence response of isolated PMN although a paradoxical depressive effect was observed with whole human blood in the case of zymosan stimulation. The killing of Staphylococcus aureus and Klebsiella pneumoniae was not enhanced by ceftriaxone and phagocytosis was significantly depressed only with adherent neutrophils but not when using neutrophils in liquid medium. It is concluded that the synergy observed between leucocyte and ceftriaxone for bacterial killing cannot be related to a direct stimulation of neutrophil functions and should depend on bacterial alteration. [ABSTRACT FROM AUTHOR]

The enhanced bactericidal activity of human neutrophils induced by cefotaxime and cefodizime, two methoxy-imino-amino- 2-thiazolyl cephalosporins, is linked to the cell stimulation of oxygen-dependent and oxygen-independent killing systems, respectively. Cefotaxime enhances both the killing and the oxidative response of neutrophils to opsonized particulate stimuli (bacteria for both activities and opsonized zymosan for the oxidative burst). These effects were not observed with non-opsonized particles (bacteria or zymosan) or soluble stimuli. On the contrary, cefodizime enhances killing of opsonized and non-opsonized bacteria by neutrophils regardless of treatment with phenylbutazone which blocks neutrophil oxidative metabolism. Cefodizime does not universally alter the oxidative burst induced by various stimuli, but has been shown to enhance the bactericidal activity of crude extracts of neutrophil granules. The data suggest that cefodizime and non O2-dependent killing systems of neutrophils cooperate in killing bacteria. [ABSTRACT FROM AUTHOR]

, cefotaxime and cefodizime enhanced significantly the bactericidal activity of human neutrophils against P 209 A, but not phagocytosis. The increase was about 150% for cefotaxime and 400% for cefodizime at concentrations as low as 1 mg/1. Furthermore, by two different techniques (NBT and cytochrome C reduction tests) cefotaxime but not cefodizime significantly enhanced superoxide anion production by zymosan-stimulated neutrophils. Other neutrophil functions (chemotaxis and myeloperoxidase-mediated iodination of proteins) were not significantly altered by either antibiotic, even at concentrations as high as 1000 mg/1. [ABSTRACT FROM PUBLISHER]

We have studied the interference of roxithromycin with NADPH oxidase, the key enzymatic system for oxidant production by human neutrophils. Roxithromycin alters the reconstitution of an active enzyme and impairs the translocation to the outer membrane of the cytosolic components p47-phox and p67-phox. Interestingly, in resting cells roxithromycin directly triggers the translocation of these factors without stimulating the oxidative burst.

A classical velocity centrifugation technique was used to study the in vitro uptake of the new ketolide ABT-773 by human polymorphonuclear neutrophils (PMNs) and a myelomonoblastic cell line, PLB-985, which can be differentiated into PMNs under certain culture conditions, compared to that of HMR 3004. ABT-773 was rapidly taken up by PMNs (cellular concentration to extracellular concentration ratio [C/E], about 34 at 30 s and up to 207 at 5 min), and uptake plateaued from 30 to 180 min (C/E, about 300). ABT-773 was accumulated significantly better than HMR 3004 from 5 to 180 min. Nondifferentiated PLB-985 cells (ND-PLB) accumulated significantly less ABT-773 and HMR 3004 than PMNs and PLB-985 cells differentiated into PMNs (D-PLB). Whatever the cell type and in contrast to the results obtained with HMR 3004, ABT-773 was mainly located in the cytosol (about 75%) and was rapidly released from loaded cells (about 40% at 5 min), followed by a plateau, likely owing to avid reuptake. Verapamil and H89, an inhibitor of protein kinase A, increased drug efflux. Uptake was sensitive to external pH, and the activation energy was moderate (about 50 kJ/mol). The existence of an active transport system on the PMN membrane was suggested by the following findings: concentration-dependent and saturable uptake (V(max), about 10,000 ng/2.5 x 10(6) PMNs/5 min; K(m), about 60 microg/ml) the inhibitory effects of PMN activators or inhibitors (phorbol myristate acetate, verapamil, Ni(2+)) and the significantly decreased levels of accumulation by killed cells and cells treated at low temperatures. In addition, various macrolides impaired ABT-773 uptake, contrary to the findings for the quinolone levofloxacin. ND- and D-PLB also presented saturation kinetics that defined an active transport system (V(max) and K(m) values were similar to those obtained with PMNs), but the activation pathway of the carrier system did not seem to be fully functional in ND-PLB. As has been observed with other erythromycin A derivatives, ABT-773 impaired oxidant production by phagocytes in a time- and concentration-dependent manner. These data extend our previous results on the existence of an active transport system common to all macrolides and ketolides, at least in PMNs.

Macrolide antibiotics have recently triggered much interest owing to the immunomodulatory potential of some derivatives, particularly in the field of inflammatory diseases. Among the possible mechanisms underlying these anti-inflammatory effects, macrolide-induced inhibition of oxidant production by phagocytes has attracted much attention. We and others have previously reported that erythromycin A-derived macrolides impair the phagocyte oxidative burst, a property linked to the presence of L-cladinose. However, we have also demonstrated that other substituents can be involved in the modulation of phagocyte function. Here we have extended the analysis of structure-activity relationships by studying the effects of five 9-N-alkyl derivatives of erythromycylamine on oxidant production by human neutrophils in vitro. LY211397 (2-methoxyethyl derivative) neither altered cell viability nor superoxide anion production. LY281389 (n-propyl derivative) did not alter cell viability and was slightly more inhibitory than erythromycylamine for the production of superoxide anion; its IC50 (concentration that inhibits 50% of the neutrophil response) was about 18 and 24 microM (versus 72 and 74 pM for erythromycylamine) after 60 min of incubation following fMLP and PMA stimulation, respectively. LY80576 (N-phenyl-3-indolylmethyl derivative), LY281981 (3-phenyl-n-propyl derivative) and LY57843 (benzyl derivative) all displayed cellular toxicity at high pharmacological concentrations after 30 to 60 min of incubation. Interestingly, these latter three drugs exhibited a rapid (5 min incubation) and strong inhibitory effect on the neutrophil oxidative burst from either stimulus, with IC50 values of 3 to 10 pM. Further in-vitro and in-vivo investigations are required to analyze the anti-inflammatory potential of these three derivatives.

Objective: Reactive oxygen species are thought to play a role in rheumatoid arthritis (RA) in humans. We postulated that antioxidant treatment could have a beneficial effect in this disease. We therefore investigated the effects of vitamin E in the transgenic KRN/NOD mouse model of RA., Methods: Mice were treated by gavage with oral vitamin E (alpha-tocopherol). Clinical, histologic, and biochemical parameters were assessed for 6 weeks., Results: Vitamin E treatment did not modify the clinical features of the disease (date of onset or disease intensity, as measured by the articular index), but it did prevent joint destruction, as measured by qualitative and semiquantitative analyses. Redox status did not differ between treated and control mice. White blood cell chemiluminescence was higher in transgenic KRN/NOD mice than in controls, but vitamin E treatment attenuated this difference. Vitamin E treatment of the transgenic animals led to a significant decrease in the levels of interleukin-(IL-1beta) but not tumor necrosis factor alpha., Conclusion: Vitamin E seems to uncouple joint inflammation and joint destruction in this model of RA, with a beneficial effect on joint destruction. Since many investigations are currently in progress to evaluate the benefit of interventions targeted toward anti-IL-1beta, our findings suggest opportunities of therapeutic interest in human RA.

Objective: To assess the ability of anti-proteinase 3 (anti-PR3) classic antineutrophil cytoplasmic antibodies (cANCA) to stimulate endothelial expression of tissue factor (TF), which is the main initiator of the coagulation cascade that can lead to endothelial injury and thrombosis in patients with Wegener's granulomatosis., Methods: Human umbilical vein endothelial cells (HUVEC) were grown to confluence and stimulated with affinity-purified anti-PR3 antibodies, Igs from healthy subjects, and endotoxin (lipopolysaccharide) as positive control., Results: TF activity was generated in anti-PR3-stimulated cells, as shown by a chromogenic test. This activity was inhibited by specific anti-TF antibodies. TF messenger RNA (mRNA) was found in anti-PR3-stimulated cells, as detected by reverse transcriptase-polymerase chain reaction, but not in cells stimulated with irrelevant human Igs or Igs from normal control sera. TF expression reached maximum levels 12 hours after exposure to the anti-PR3 cANCA, and did not require complement. TF mRNA expression was inhibited by cycloheximide, suggesting a requirement for protein synthesis. When added to the incubation medium, interleukin-1 (IL-1) receptor antagonist inhibited the induced TF mRNA expression, suggesting that cANCA-stimulated cells initiate IL-1 synthesis. Moreover, cANCA induced IL-1alpha mRNA before TF mRNA., Conclusion: This study showed that anti-PR3 treatment of HUVEC induces sequential expression of IL-1alpha mRNA and TF mRNA, as well as their corresponding proteins. Both proteins could have pathogenic roles in the vasculitic process, since TF is the main initiator of the coagulation cascade.

Cultured human umbilical vein endothelial cells (HUVECs) treated with reactive oxygen species (ROS) show increased adherence of polymorphonuclear leukocytes (PMNs). Because pentoxifylline (PTX) is known to inhibit cell interactions, we studied PMN adherence to ROS-stimulated HUVECs pretreated with PTX. ROS were generated by the oxidation of hypoxanthine by xanthine oxidase, giving rise to superoxide anion and hydrogen peroxide. Human PMNs were then added to HUVEC monolayers. After various times, the cultures were washed and the number of adherent PMNs was estimated by measuring myeloperoxidase in the total cell homogenate. PTX inhibited adherence in a concentration-dependent manner. Moreover, the increase in intracellular cAMP content varied with the PTX concentration. Isobutylmethylxanthine (IBMX) and isoproterenol (ISO) which increase intracellular cAMP content, also inhibited the adherence of PMNs to ROS-stimulated HUVECs. We conclude that cAMP is probably involved in the intracellular regulation of ROS-mediated PMN adherence to endothelial cells.

Due to their high intracellular uptake, macrolides may interfere with phagocytes antibacterial system. We have studied the interaction of josamycin with bactericidal activity of human neutrophils (PMNs) or whole blood, in vitro. PMNs preincubated with josamycin (100-0.1 mg/l) display an increased phagocytic ability for K. pneumoniae, independently of the concentrations of josamycin; this effect appears to correlate with the recruitment of an additional population of phagocytizing PMNs without alteration of the mean number of PMN-associated bacteria, whatever the experimental conditions (adherent or non adherent PMNs). PMNs bactericidal function is also enhanced in the presence of josamycin in a dose-dependent manner (mean increase 60 to 370% for josamycin 0.1 to 10 mg/l). Bactericidal activity of whole human blood for P. aeruginosa (a strain resistant to the lytic effect of serum) is increased with a mean survival (CFUt min/CFU 0 min) of 24 and 71% at 60 and 120 min respectively, in the presence of josamycin 10 mg/l, and 42 and 128% in the presence of josamycin 1 mg/l, compared to the survival of the control, 68 and 166%. PMNs functions are not altered by josamycin with the exception of an enhancement of the oxidative burst induced by formylmethionyl-leucyl-phenylalanine, a synthetic compound similar to bacterial derivatives. In conclusion, we have shown a synergic interaction between phagocytes and josamycin for the killing of bacteria resistant to this antibiotic (MiC greater than 128 mg/l). This could be due either to an alteration of some PMN membrane receptors or to alterations of bacteria which render them more sensitive to natural bactericidal mechanisms and/or more able to activate these mechanisms.

Roxithromycin (RU 28965) a new semisynthetic macrolide has been reported to display an antibacterial spectrum and activity in vitro similar to those of others macrolides. However, roxithromycin seems more efficient than erythromycin in in vivo experimental infections (mice). We have previously reported that roxithromycin increases the ability of human neutrophils (PMN) for bactericidal activity (S. aureus) or phagocytosis (K. pneumoniae) in vitro without altering other PMN functional parameters. In this study, roxithromycin (single dose-300 mg) was given to 6 human volunteers. The neutrophils collected 90 min after ingestion display a significant increased ability to phagocytose and kill S. aureus and K. pneumoniae. Furthermore chemotaxis, oxidative burst and myeloperoxidase activity of the PMN after roxithromycin ingestion were enhanced compared to those of PMN before ingestion. This discrepancy between immunomodulating effect of roxithromycin in vitro and in vivo outlines the complexity of in vivo experimental models and requires further studies in vivo in particular in patients suffering from sepsis.

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.