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The fields of neutrophil and endothelial cell biology are being deeply revised. While lung marginated neutrophils have been identified decades ago, their roles in the healthy adult lung are still contentious. Furthermore, while it is now clear that the lung constitutes an important immunological niche, the role of lung endothelial cells has been neglected so far. A better understanding of the role of short-lived neutrophils in contributing to lung endothelial cell physiology will improve our understanding of lung endothelial cell fate and heterogeneity under homeostasis and inflammation. Furthermore, it will provide new mechanistic insights on lung marginated neutrophil function and crosstalk with endothelial cells and provide robust foundations for devising therapeutic approaches in which endothelial cell (dys)functions are involved.

Neutrophils play a key role in host defense against pathogens. They can contribute to pathological inflammation, and are thought to exacerbate tissue injury upon exposure to bacterial products, such as endotoxin (LPS). Recent findings suggest that neutrophils can also participate in adaptive immune responses and contribute to inflammation resolution. Many discoveries regarding the in vivo role of neutrophils were made possible by the use of genetically modified neutrophil-deficient mice, or by the use of neutrophil-depleting antibodies. Here we describe a new mouse model, PMN DTR mice, in which neutrophils can be selectively depleted upon injection of diphtheria toxin. Using this model, we have recently demonstrated that neutrophils play a protective role during lethal endotoxin-induced systemic shock. This new mouse model presents several major advantages over more classical models of neutropenia, which are discussed herein., (© 2018 médecine/sciences – Inserm.)

Neutrophils are well-recognized phagocytes in the first line of host defense, and are also a major source of pro-inflammatory cytokines, chemokines and lipid mediators, thereby contributing to the onset and early orchestration of the inflammatory response. In contrast, recent studies indicate that neutrophils have tools to limit the magnitude and length of an inflammatory response, and may take part in engaging the resolution process. This article describes endogenous signals that may transform the phenotype of a neutrophil: from a pro-inflammatory cell to one that promotes resolution. Adenosine, an autacoid which can be found at high concentrations in inflammatory sites, inhibits several inflammatory functions of the neutrophil via engagement of the A2A receptor and reshapes the profile of lipid mediators and cytokines released, causing cells to terminate the release of pro-inflammatory signals while progressing toward resolution. These endogenous resolution pathways may represent a key target for better treatments of inflammatory diseases.

Cancer chemotherapy is responsible for infections by decreasing the phagocytosis and chemotaxis of polymorphonuclear. We conducted a retrospective analysis during the period from 18/10/2006 to 21/05/2008, on all bacteria isolated from blood cultures performed in the department of clinical hematology at the hospital military instruction Mohamed V. One hundred and sixty two blood isolates were selected; Gram positive cocci (CGP) accounted for 60.34% and Gram negative bacilli (GNB) for 24.14%. Coagulase negative staphylococci (SNA) and S. aureus presented a resistance to methicilline respectively 54.55% and 22.22%. Prevalence of Gram positive cocci is consistent with the results of the EORTC (International Antimicrobial Therapy Cooperative Group). Analysis of resistance patterns of all species, except for staphylococci, showed phenotypes essentially community, sometimes wild. In conclusion probabilistic antibiotic treatement of bacteraemia in the haematology department should focus among other staphylococci resistant to methicilline.

After more than 20 years of hegemony, the Th1-Th2 paradigm was recently shaken by the discovery of a novel population of CD4 effector T cells, the Th17 cells. Th17 effector cells produce IL-17 and IL-22 and thus have pro-inflammatory properties notably favoring neutrophils recruitment and thus control of extracellular bacteria mainly at the epithelium surface. Th17 cells appear also as the major inducer of organ specific autoimmune pathologies such as EAE or rheumatoid arthritis, a function previously attributed to Th1 effector cells. The discovery of Th17 cells further supports the notion that effector CD4 T cells responses are diverse in vivo and that fine tuning of these different effector cells is critical to maintain tissue integrity.

Congenital neutropenia are extremely rare diseases, defined by a permanent or cyclic decrease of blood neutrophils. Molecular basis of several congenital neutropenia has been recently determined, involving gene coding for the neutrophil elastase gene (ELA2), GFI1, WAS protein and mitochondrial HAX1 protein. These mutations, dominant (ELA2, GFI1), X-linked (WAS) and autosomal recessive (HAX1), result in instability of the contents of the granules- particularly the neutrophil elastase- or in abnormalities of the cytoskeleton, and possibly, in an increased apoptosis. ELA2 mutations resulting both in profound and permanent neutropenia, and in cyclic--pseudo sinusoidal--neutropenia lead to consider that time pattern is very close in the two apparently distinct phenotypes. This observation suggests that temporal variations of neutrophils could be represented by non linear functions. Congenital neutropenia, specifically ELA2 mutated, are also characterized by a high rate of leukemia (about 15% at 20 years of age). Leukemia risk does not appear to be related to an oncogenic effect of ELA2 mutations, but much likely to the deepness of the neutropenia, and the intensity of G-CSF therapy.

Study Aims: The aim was to evaluate the in vitro effects of amoxicillin and its combination with clavulanic acid, two beta-lactams intravenously injected, on the oxidative metabolism of polymorphonuclear neutrophils. These cells play the major role in the "respiratory burst" as they produce superoxide anion to kill the infectious agent. An activation of this process by the injected antibiotics could enhance the bactericidal action or explain some of adverse effects., Materials and Methods: Two models were used to estimate the O(2)(-) amounts produced in the presence of the antimicrobial agents. In the cellular model, O(2)(-) was generated by neutrophils artificially stimulated or not (separated by a gradient centrifugation through Histopaque 1077). In the acellular model, O(2)(-) was produced by the xanthine-xanthine oxidase system. O(2)(-) was measured by spectrophotometry using the ferricytochrome C reduction., Results: The O(2)(-) production by polymorphonuclear neutrophils was increased when both antibiotics were added to the reaction mixture. A significant activation of the cell oxidative metabolism was observed with amoxicillin using various stimulating agents, that was higher without stimulation and lower when amoxicillin and clavulanic acid were associated., Conclusion: Amoxicillin could either activate polymorphonuclear neutrophils NADPH-oxidase or cause its activation by a membrane effect, or interfere with the zymosan activation way. It could then be supposed that this antimicrobial agent intensified the bactericidal effects.

Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack NADPH oxidase activity. Patients with CGD suffer from recurrent bacterial and fungal infections because of the absence of superoxide anions (O2- degrees ) generatingsystem. The NADPH oxidase complex is composed of a membranous cytochrome b558, cytosolic proteins p67phox, p47phox, p40phox and two small GTPases Rac2 and Rap1A. Cytochrome b558 consists of two sub-units gp91phox and p22phox. The most common form of CGD is due to mutations in CYBB gene encoding gp91phox. In some rare cases, the mutated gp91phox is normally expressed but is devoided of oxidase activity. These variants called X+ CGD, have provided interesting informations about oxidase activation mechanisms. However modelization of such variants is necessary to obtain enough biological material for studies at the molecular level. A cellular model (knock-out PLB-985 cells) has been developed for expressing recombinant mutated gp91phox for functional analysis of the oxidase complex. Recent works demonstrated that this cell line genetically deficient in gp91phox is a powerful tool for functional analysis of the NADPH oxidase complex activation.

Neutrophils are polynuclear cells essential to innate immunity. They are the first cells to migrate from the blood to the inflammatory site where they kill pathogens and secrete various mediators that regulate innate and adaptive immunity. Functional steps required for their microbicidal activity include: transendothelial migration, migration towards the invading pathogens, and then recognition, adhesion, engulfment, and killing of the target. Primary deficiencies of these stages are expressed by repeated and/or severe bacterial and fungal infections. These deficiencies include granule abnormalities and leukocyte adhesion deficiencies Type I and II, defective pathogen recognition and the defective oxidative burst that characterizes chronic granulomatous disease.

We have compared the interplay of several antimicrobial agents and aminoacids on the neutrophil respiratory burst in response to formylmethionyl-leucyl-phenylalanine (fMLP), a chemoattractant. Mainly, an inhibitory effect has been observed in the penicillin family of agents and an enhancing effect in the cephalosporin family of agents. The molecules in which the sulfur numbered 1 in the 6-APA or 7-ACA nucleus was replaced by a carbon or an oxygen, had a different effect as compared with the other members of the family. The modulatory effects of ampicillin and cephalothine were not significant at a concentration lower than 10 mg/l and the effect of cephalothine looked maximum at 20-40 mg/l. If studies in cell-free systems demonstrated that the inhibitory effect of some antimicrobials could be due to a direct oxidant-scavenger activity mainly of HOCl, only hypotheses are proposed to explain the enhancing activity of the others. It could be in relation with (i) a synergistic effect upon fMLP receptor leading to an increase in H(2)O(2)/HOCl production or (ii) the generation of new oxydant products originating in cephalosporin lysis under HOCl attack, which would be able to react with luminol. The interplay of antimicrobial agents with the respiratory burst measured outside the cells probably has no therapeutic consequences because the bactericidal activity of neutrophils is achieved inside phagosomes where few agents are known to come into and where chemical conditions are different. On the opposite, in clinical use, this interplay could be interesting to study for a prevention of side effects.

The immune response against a bacterial aggression involves the monocytes-macrophages and polymorphonuclear neutrophils (PMN) in the first line of defense. This natural or innate immunity controls the proliferation of micro-organisms while waiting for the development of aspecific immunity related to lymphocytes. Establishing a link between innate and specific immunity, interleukin-12 (IL-12) is an essential cytokine of the inflammatory response. In a first in vitro study, we showed that IL-12 potentiates the effect of LPS on the production of IL-8 by stimulated PMN, the main chemotactic and activating cytokine of neutrophils. IL-12 would thus support the local recruitment of PMN via an autocrine loop of amplification. In a second in vivo study in septic patients, we noted a defect in the pulmonary and systemic production of IL-12, suggesting a dysregulation of innate immunity during the course of sepsis.

Various biological events, such as cell differentiation, cell migration or gene expression, are controlled by cell-cell interactions or by cytokines, as well as by interactions between cells and extracellular matrix. The regulation of these events involves a directed and limited proteolysis of matrix macromolecules, that induces the release of proteic domains and peptides exhibiting biological activities. In this review, we summarise several data from our laboratory showing that peptides from type I and type IV collagens play an important role in the control of inflammation and tumor progression. Type I collagen peptides stimulate respiratory burst, granule exocytosis and cytokine secretion by human leukocytes (polymorphonuclear neutrophils or monocytes) for the detersion of inflammatory sites and then for the chemoattraction of various cell types needed for wound healing. A peptide of the NC1 domain of the alpha 3(IV) collagen chain prevents leukocyte activation. In addition, this peptide is also capable of limiting tumor progression by downregulating in vitro and in vivo invasive properties of melanoma cells.

Circulation of blood extracorporeally through plastic tubing causes severe shear stresses to blood cells and activates several regulatory cascades. These various pathways include the cytokine cascades, complement and coagulation. Interleukine-1, -6, -8, tumor necrosis factor-alpha have been implicated. Among various mediators of tissue injury released by activated neutrophils, elastase and metalloproteinases have been considered to be relevant in postoperative organ dysfunction in cardiac operations. Endothelial cells are extremely sensitive to insults that occur during cardiopulmonary bypass (CPB). These insults lead to disruption of barrier function and leukocyte adhesion. Immunoglobulins such as ICAM-1 and VCAM-1 are expressed on endothelium cells and act as ligands for integrins. It is also important to remember that during cardiac operations, the interest on the metabolism of in free radicals has focused on the heart and the lungs because they are exposed to ischemia and subsequent reperfusion. Increased production of free radicals during CPB is associated with myocardial and pulmonary dysfunctions. Now it is well recognized that the whole body inflammatory response induced by CPB is mainly responsible for postoperative organ dysfunctions.

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.

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.

Leukoagglutination is a rare EDTA-dependent phenomenon resulting in a spurious minoration of the leukocyte count performed using automated analyzers. We described seven cases. The leukocyte agglutination was detected by unstable WBC count, abnormal WBC histograms and presence of clusters of polymorphonuclears on the smear. PMN aggregates of 3 to 10 cells or bigger were observed. Discrepancies between the erroneous automated WBC count and the real count were moderate in most cases. Leukoagglutination was related to lymphoproliferative disorders, infections, alcoholic liver diseases, auto-immune diseases. Inflammatory context seemed to be requested. For few patients, the artefact occurred regardless of the type of anticoagulants (lithium heparin, buffered sodium citrate) and warming at 37 C did not always increase the WBC. Dilution in Unopette chambers was required. We confirmed that leukoagglutination of PMN was an in vitro artefact EDTA and/or temperature mediated.

Unlabelled: Polymorphonuclear neutrophil (PMN) adherence receptors expression varies with leukocyte activation state. Their quantification need accurate and inter-laboratories reproducible methods, without artefactual activation., Objective: The aim of this study was to study the influence of cell preparation on PMN adherence receptors expression., Materials and Methods: It was proposed to quantify, using immunolabeling standard (QIFIKIT(R), Dako), surface expression of the main adherence receptors (L-selectin and B(2) integrins), from different preparations of PMN: total blood collected with EDTA, isolated PMN by density gradient Polymorphprep(TM) 1,113 (Nycomed Pharma) and formaldehyde fixed PMN., Results: A decrease of all receptors was noted after isolation and fixation of PMN, in comparison with whole blood PMN analysis. These results differed from data previously reported since, in these studies, activated phenotypes (increased of B(2) integrins) were observed after isolation and fixation methods., Conclusion: The present study provides strong evidence that pre-analytical conditions are sources of biological variations and thus extreme care must be taken in the interpretation of results. It underlines the interest of consensual practices for these pre-analytic and analytic parameters in order to compare results in multicenter and longitudinal studies.

Adherence receptors are essential for heterotypic (endothelial cell, platelet) polymorphonuclear neutrophil (PMN) interaction. Determination of their expression level give information about activation state and functionality of PMN. Use of flow cytometry associated with an immunolabeling standard, represented by beads coated by a determined amount of immunoglobulins (Qifikit, Dako), allows analysis of specific antibody binding capacity and gives information about antigen density. Using this methodology, the exploration of surface adherence receptors, L-selectin (CD62L) and b2-integrins (CD11a-c/CD18) from PMN unstimulated and incubated with pro-inflammatory stimuli, formyl-methionyl-leucyl-phenylalanine (fMLP) and tumor necrosis factor a (TNFa), allows, on the one hand, the establishment of basal expression values on resting PNN and on the other hand, the study of PNN reactivity. This method of quantification can be applied to clinical studies as adherence receptor deficiency syndromes or inflammatory, infectious and vascular diseases.

Engraftment in relation to infused CD34+ cell number was retrospectively analysed in 66 patients with hematological diseases: non-Hodgkin's lymphoma (n = 33), multiple myeloma (n = 21), acute myelogenous leukemia (n = 7), Hodgkin's disease (n = 4) and myelodysplastic syndrome (n = 1). Progenitor cells were mobilized with rhG-CSF, alone or in association with chemotherapy. The cells were harvested by leukapheresis until at least 2 x 10(6) CD34+/kg body weight were obtained. A total of 194 leukaphereses were performed (median = 3 per patient, range 1-9). A median of 3.40 x 10(8) nucleated cells/kg (range 0.31-27.59) and a median of 7.15 x 10(6) CD34+ cell/kg (range 1.31-115.70) were transplanted. Regardless of transfusional support or patient diagnosis, engraftment was rapid in patients who had received > or = 5 x 10(6) CD34+ cell/kg. In this case, absolute neutrophil blood count > or = 0.5 x 10(9)/l was obtained on day 12 post graft (range 7-19) and platelet count > or = 20 x 10(9)/l was also reached after the same median time interval (range 8-121). From the present results, a minimal threshold of 5 x 10(6) CD34+ cell/kg appears to be suitable for providing rapid and complete hematopoieitc reconstitution in patients exposed to high doses of chemotherapy with or without total body irradiation. Furthermore, administration of rhG-CSF during post-graft period significantly decreased the neutrophil time recovery (P = 0.002) but not that of platelets (P > 0.05).

Polymorphonuclear neutrophils (PMN) and monocytes play a role in vascular diseases. Animal experimental models, using deleukocytation or injection of anti-CD11b-anti-CD18 monoclonal antibodies (inhibition of leukocytic adhesion and of interaction with the endothelial cell) have confirmed this role in the ischemia-reperfusion syndrome and in myocardial infarction. In man, increased production of oxygen radicals, PMN release of elastase, increased monocyte formation of tissue factor (TF) and integrins have been noted in coronary artery disease, in chronic arteriopathy of the lower limbs and in association with vascular risk factors such as diabetes. Pharmacological alteration of leukocyte hyperactivity therefore seems to be justified. Pentoxifylline, used with good effect in arteriopathy of the lower limbs, affects numerous leukocytic functions: diminution in adherence and in PMN production of free radicals, diminution in the formation of TF and cytokines (TNF). Gingkolides reduce leukocytic interactions and platelet activation through an anti-PAF (Platelet Activation Factor) action. Aspirin may interfere with free radicals and inhibit TF formation. alpha-tocopherol blocks the activation, by free radicals, of the transcription factor NF k B. By altering the TNF and IL-1 cytokines, leukocytic activation can be controlled. Other cytokines (IL-4, IL-10) have an immunosuppressive action and reduce the formation of TF. The pharmacological targets are therefore multiple. Their use in vascular diseases is only at a very preliminary stage.

Among the neutrophil polynuclear specific antigens (NA, NB, ND, NE, ...), NA antigen is the most common. It is a glycoprotein situated on the neutrophils FcRIIIb-receptor and presents 2 forms: NA1 and NA2. The epitope responsible of that polymorphism has got an amino acids composition that is unknown. The first techniques used for their analysis were the microagglutination and the granulocytotoxicity-later, the immunofluorescence, the chemiluminescence and the MAIGA (Monoclonal Antibody Immobilized granulocyte Antigen) were introduced. These last years, more efficient techniques appeared like Flow Cytometry and Polymerase Chain Reaction (PCR) that allowed phenotyping and genotyping of neutrophil polymorphonuclear specific antigens. The studies indicated that NA antigen frequency varies according to the populations and the ethnics. NA2 allelic form is more frequent than NA1 in the caucasian population (88% VS 46%). In human pathology, NA antigen is implicated in the physiopathological mechanisms of the alloimmune and probably auto-immune neutropenies.

Unlabelled: Autoimmune neutropenia (AIN) is a frequent cause of chronic neutropenia especially in youngest children. Its diagnosis is established by immunological proof of the autoimmune mechanism. The aim of this study is to better describe this autoimmune process and to show the contribution of bone marrow smears to this diagnosis., Patients and Methods: Ten children, six girls and four boys, were examined between 1990 and 1995. Eight of them had typical AIN, confirmed by the presence of antibodies against neutrophils. Two other patients were included on the basis of bone marrow pictures. Five non-neutropenic children with normal bone marrow smears were chosen as controls. Bone marrow analysis was always performed by the same cytologist according to a reproducible technique., Results: Six out of ten patients had important features of elective phagocytosis of neutrophils by marrow macrophages (unlike controls) without signs of dysgranulopoiesis or hemophagocytosis. Antibodies against neutrophils were detected in six patients with phagocytosis and in four patients without these cytological features. In two other children presenting the same bone marrow picture and clinical profile, an autoimmune process was probable, even in the absence of antibodies against neutrophils. Some patients had several infections and were given immunoglobulins and/or granulocyte colony-stimulating factor (G-CSF) therapy. The efficacy of Immunoglobulin was not constant, whereas G-CSF was effective at low doses and shortened the duration of infections., Conclusion: Prolonged neutropenia in childhood must lead to look for phagocytosis by marrow macrophages in bone marrow smears, as a possible sign of autoimmunity. Growth factors may temporarily be used associated with antibiotics therapy in severe and prolonged infections.

The inflammation is characterized by a multifrequency time structure described both in the edematous reaction and in the migration of neutrophilic polymorphonuclear (PMN) in the inflammatory site. The circadian rhythm of PMN migration appears to be similar when the migration was induced by BCG, LPS or carrageenan. The corticosteroids play an important role in the mechanisms in the circadian rhythm of PMN, but recent data in intact and castrated mice indicated that testosterone is also involved in these mechanisms. In arthritic patients, the signs and symptoms of the diseases varied as a function of time of day and of the type of arthritic diseases. Human data with indomethacin, ketoprofen and piroxicam indicated that it is possible to find an optimal time of day for the administration of these non-steroidal anti-inflammatory agents (NSAID). Clinicians can use these chronopharmacological data to maximize the analgesic effect and to minimize the side effects of the NSAID. The research on biological rhythms in inflammation and in the effects of NSAID lead to a better understanding of the mechanisms of inflammation and to the rational use of the drugs in arthritic patients.

The increasing interest in "warm" aerobic cardioplegia requires a critical reevaluation of the systemic effects of the associated normothermic cardiopulmonary bypass (CPB). As activated neutrophils seem to be essential mediators of the inflammatory response to CPB via the cytotoxicity of the products that are released during their adhesion to endothelial cells, the authors undertook a study of the influence of temperature on the interaction between the neutrophils and the endothelium in 95 patients undergoing warm (31-33.5 degrees C, n = 49) and cold (26-27 degrees C, n = 46) CPB surgery. Blood sampling was performed before, during and after CPB. The following markers of neutrophil-endocardium interaction were analysed: complement activation (C3a), cytokine production (tumor necrosis factor alpha, interleukines 1, 6 and 8, and interleukin-1 receptor antagonist); endothelial expression of cytokine-dependent [intercellular adhesion molecule (ICAM)] and cytokine-independent (P-selectin) adhesion molecules (P-selectin); expression of cytokine molecules on the surface of polynuclear neutrophils (CD11a, CD11b, CD11c); and finally, endothelial adhesion and transendothelial migration of neutrophils (interleukin 8 and elastase). The results showed that, irrespective of temperature, CPB was associated with changes strongly suggestive of phenomena of transendothelial adhesion and migration. Moreover, normothermia increased the intensity of the inflammatory response as shown by increased cytokine production, earlier expression of neutrophil adhesion molecules and increased elastase production.

New selenium-containing compounds behave as GPx mimics and protect endothelial cells (HUVEC) from damage upon exposure to 55 microM linoleic acid hydroperoxide or to 200 microM hydrogen peroxide. The simultaneous presence of the GPx mimic and the hydroperoxyde is not necessary, since a pre-treatment of endothelial monolayers with 1 to 10 microM of such compounds, preserves their morphology, their cell density and their longer-term viability. The compounds which are most efficient in this model of oxidative stress also protect endothelial monolayers which have been incubated with an excess (10:1) of polymorphonuclear neutrophils (PMN) and with 1 ng/ml of TNF-alpha, if such monolayers are pre- and co-treated (10 microM). They inhibit the adhesion of activated neutrophils which show-up as polymorphous and very dense particles, in the vicinity of which endothelial alterations can be seen. The inhibition of leucocyte adhesion and that of endothelial activation/alteration have been quantified by means of immunoassays of myeloperoxidase and von Willebrand factor (vWf). The lead-compound BXT-51072 is not a direct inhibitor of the NADPH oxidase of PMN. TNF-alpha alone induces the endothelial release of Interleukin-8 (Il-8) as well as the expression of P- and E-selectin. The extent and the kinetics of inhibition of such processes by compound BXT-51072 would explain several of the effects observed in the presence of PMN. The GPx mimics also inhibit the endothelial production of Il-8 which is induced by Interleukin-1 alpha. Finally, compound BXT-51072 inhibits the endothelial expression of the adhesion factor VCAM-1 which is more slowly induced by TNF-alpha. Such antioxidant catalysts therefore protect endothelial cells from the toxic effects of TNF-alpha through mechanisms which include a down-regulation of cytokines and cell-adhesion factors.

Unlabelled: Vascular complications in diabetic patients is a complex, probably multifactorial phenomena involving cellular phagocytosis. The aim of this study was to evaluate polymorphonuclear performance in 61 infection free diabetic patients based on tests of the different cell functions: 1) adhesion:adhesion molecule expression CD11a, CD11b, CD11c; adhesion test on nylon fibers. 2) chemotaxis:chemotaxis under aragose to FMLP (bacteria oligopeptide) and complement fractions. 3) Phagocytosis:latex beads. 4) Bacteriocidal power:chemoluminescence photometric oxidative potential before and after stimulation with opsonized zymosan and PMA; reduction of tetrazolium nitroblue. RESULTS were analyzed according to type of diabetes, glucose control, duration of the disease, history of infection and presence of vascular complications., Results: compared with a group of 30 controls, the diabetic patients had a significant impaired polynuclear chemotaxis function (p < 0.001) with both spontaneous adhesion and increased expression of adhesion molecules (CD 11b, CD 11c). The chemoluminescence test was increased at the baseline level due to spontaneous polynuclear adhesion and increased production of free radicals. This response decreased after stimulation compared with controls. The type of diabetes, Hb A1c level and history of infection did not appear to have an effect. Inversely, changes in chemotaxis and chemoluminescence were greater in patients with vascular complications. In summary, all the functions of polynuclear neutrophils tested were altered in diabetic patients and could favor vascular complications and infections episodes.

ANCA mainly recognize proteinase 3 (cytoplasmic fluorescence) in Wegener's granulomatosis and myeloperoxidase (perinuclear fluorescence) in other types of vasculitis. A causative role of ANCA seems not obvious because: a) active generalized Wegener's granulomatosis may exist with no detectable ANCA, b) ANCA titres don't reliably parallel clinical activity and c) of the increasing diversity of clinical situations where ANCA are described. Nevertheless, some lines of evidence point to a pathogenetic role of ANCA, especially in vascular lesions genesis: a) ANCA may prevent inactivation of antigenic proteases by antiproteases, b) translocation at the outer membrane of intracellular antigens make them accessible to ANCA, c) activation of polymorphonuclear leucocytes and increase of their aggregation and cytotoxicity to endothelial cells are specifically induced by ANCA, d) proteinase 3 and myeloperoxidase may adhere to the endothelial cell membrane and then be recognized by ANCA, e) a specific T-lymphocyte response to ANCA antigens could be elicited in Wegener's granulomatosis and f) an in vivo model of ANCA-related glomerulonephritis has recently been established.

The endothelial cells, because of their privileged situation between the tissues and the vessels, play an important role in the inflammatory reaction. Expression of adhesion molecules (ELAM 1 especially) allows adhesion of circulating neutrophils to the epithelial cell and thus initiates the reaction. During the reactions of immediate hypersensitivity, the flow of leucocytes observed in the late phase is dependent on mastocytes, the recruitment being under the positive or negative influence of several cytokines.

Pulmonary fibrosis corresponds to an accumulation of collagens and other proteins of the extracellular matrix in the interstitium and alveoli. Biochemical and cellular mechanisms of pulmonary fibrogenesis remain poorly understood. The cells of the alveolitis (macrophages, lymphocytes and neutrophils) play a key role in producing the factors which regulate the proliferation, chemotactism and secretory activity of the fibroblasts. Amongst these factors the cytokines (interleukins, interferons and growth factors) play a definite but very complex role. Certain cytokines stimulate in vitro the attraction and activation of cells of the alveolitis, as well as the multiplication, migration and secretory activity of fibroblasts. The following cytokines are involved: tumour necrosis factor alpha: (TNF alpha), interleukin 1 (IL-1), interleukin 6 (IL-6) interleukin 8 (IL-8) transforming growth factor beta (TGF beta), platelet derived growth factor (PDGF), insulin like growth factor 1 (IGF-1), fibronectin, monocyte chemotactic protein 1: (MCP-1). Other cytokines, principally the interferons (of alpha, beta or gamma type: IFN alpha, IFN beta, IFN gamma) inhibit in vitro and in vivo the proliferation and the production of collagen by fibroblasts. During the course of human pulmonary fibrosis or in experimental situations, the majority of the cytokines mentioned above are produced in excess in the lung. Without doubt they play an important role in the pathogenesis of fibrosis, even if it is not yet very well known how they interact and contribute in vitro to the process of fibrogenesis. Certain cytokines potentially regulating in the fibrosis are yet to be identified. In the future the use of cytokines and of their inhibitors will perhaps provide new therapies in pulmonary fibrosis.