Letteratura scientifica selezionata sul tema "Zymosan opsonisé"

Abstract After priming with cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, or IL-5, eosinophils are stimulated potently by opsonized particles like serum-treated zymosan (STZ), resulting in activation of the respiratory burst and production of lipid mediators, such as platelet-activating factor (PAF) and leukotriene C4 (LTC4). In the present study, the role of the opsonin receptors Fc gamma RII and CR3 during both STZ-induced activation of the respiratory burst and PAF release by human eosinophils was investigated. Inhibition studies with blocking mAbs (alpha hFc gamma RII: AT10, IV.3; alpha CR3: B2.12, 44a) showed that both Fc gamma RII and CR3 are important for STZ-induced PAF release by cytokine-primed eosinophils. In contrast, CR3 is involved in activation of the respiratory burst, whereas Fc gamma RII seems not to be important, because blocking anti-Fc gamma RII mAbs had no effect. Subsequently, experiments were performed with zymosan particles coated with IgG, iC3b, or a combination of both. IgG-coated particles poorly activated both responses in GM-CSF primed and unprimed cells. iC3b-Zymosan activated the respiratory burst as well as zymosan expressing both opsonins (IgG/iC3b-zymosan). In contrast, iC3b-zymosan induced significantly less PAF release by GM-CSF-primed eosinophils than did IgG/iC3b-zymosan, suggesting synergism between Fc gamma RII and CR3. This synergistic effect was not observed when IgG-zymosan and iC3b-zymosan were added simultaneously. Therefore, these data indicate that on human eosinophils, Fc gamma RII and CR3 act synergistically to activate PAF release, provided that their ligands are in close proximity.

A method was developed for testing the cytotoxicity of various bandage-like wound dressings and gel wound dressings. In this method, the ability of human polymor-phonuclear neutrophils (PMNs) to initiate a respiratory burst after exposure to the various wound dressings is used as a marker of cytotoxicity. Luminol-amplified chemiluminescence stimulated with opsonised zymosan or phorbol 12-myristate 13-acetate (PMA) is used to measure the degree of activation of the respiratory burst, i.e. the NADPH oxidase activity, after exposure to wound dressings. Opsonised zymosan (material from yeast cell walls) is a phagocytic stimulus that activates the NADPH oxidase by binding to Fc-receptors and complement receptors, and functions as an artificial bacterium, whereas PMA activates the NADPH oxidase by direct activation of protein kinase C. NADPH oxidase activity was inhibited by several wound dressings. The down-regulation of the respiratory burst is detrimental to the bactericial effect of PMNs, and can be used as a marker for the cytotoxicity of wound-dressing materials.

ABSTRACT Complement receptor type 3 (CR3) was initially described as an opsonic receptor. Subsequently, CR3-mediated lectin-sugar recognition mechanisms have been shown to play a major role in the nonopsonic phagocytosis of several pathogens, among them Mycobacterium tuberculosis. Little is known about the binding and signal transduction mechanisms operating during nonopsonic ingestion through CR3 of different microorganisms. In the present study, we used CHO cells stably transfected with CR3 to show that CR3 was able to mediate internalization of zymosan and pathogenic mycobacteria (Mycobacterium kansasii and Mycobacterium avium) but not that of nonpathogenic species (Mycobacterium smegmatis and Mycobacterium phlei). A combination of mannan and β-glucan inhibited the phagocytosis of zymosan but had no effect on M. kansasii ingestion. Among six monoclonal antibodies (MAbs) directed against the CD11b subunit of CR3 that decreased zymosan ingestion, only three inhibited M. kansasii phagocytosis. In particular, MAbs known to block the CR3 lectin site affected only internalization of zymosan. Using U937 macrophages, we observed that zymosan ingestion through CR3 induced superoxide production measured by cytochrome c reduction and by translocation of the NADPH oxidase cytosolic component p47phox to the phagosomal membrane, whereas phagocytosis of viable or heat-killed M. kansasii did not. Furthermore, lack of superoxide anion production during phagocytosis of M. kansasii was not due to inhibition of NADPH oxidase per se or superoxide anion scavenging. Together, our results indicate that (i) nonopsonic phagocytosis of zymosan and M. kansasii by CR3 implicates different molecular mechanisms involving multiple and distinct epitopes of CD11b and (ii) CR3 may transduce different cellular responses depending on the sites mediating nonopsonic phagocytosis.

Abstract Human recombinant tumor necrosis factor (TNF) was shown to be a weak direct stimulus of the neutrophil respiratory burst and degranulation. The stimulation, as measured by iodination, H2O2 production, and lysozyme release, was considerably increased by the presence of unopsonized zymosan in the reaction mixture, an effect which was associated with the increased ingestion of the zymosan. TNF does not act as an opsonin but, rather, reacts with the neutrophil to increase its phagocytic activity. TNF-dependent phagocytosis, as measured indirectly by iodination, is inhibited by monoclonal antibodies (Mab) 60.1 and 60.3, which recognize different epitopes on the C3bi receptor/adherence-promoting surface glycoprotein of neutrophils. Other neutrophil stimulants, namely N-formyl-methionyl-leucyl-phenylalanine, the Ca2+ ionophore A23187, and phorbol myristic acetate, also increase iodination in the presence of zymosan; as with TNF, the effect of these stimulants is inhibited by Mab 60.1 and 60.3, whereas, in contrast to that of TNF, their stimulation of iodination is unaffected by an Mab directed against TNF. TNF may be a natural stimulant of neutrophils which promotes adherence to endothelial cells and to particles, leading to increased phagocytosis, respiratory burst activity, and degranulation.

Abstract We have characterized and reported the immunostimulating properties of a novel polysaccharide - (1,4)-α-D-glucan (RR1)- isolated from the medicinal plant Tinospora cordifolia [23]. This novel glucan is water soluble and having (1, 4)-α-D-glycosidic linkages in the main chain and (1, 6)-α-D-glycosidic linked side chains at an interval of 6, 7 glucose units. The signaling mechanism of the novel (1,4)-a-D-glucan (RR1) was investigated in macrophages to evaluate its immunostimulating properties. When RAW264.7 macrophages were incubated with RR1 at 4°C, the novel glucan inhibited the phagocytosis of unopsonized zymosan A bioparticles in a dose-dependent manner. RR1 also inhibited the binding and internalization of opsonized zymosan A bioparticles, although at a lower level than laminarin. Incubation of macrophages with anti-CD11b mAb followed by RR1 failed to show any inhibitory effect on RR1-induced TNF-α synthesis confirming that complement receptor 3 (CR3) is not involved in the opsonic binding and internalization of RR1 in macrophages unlike zymosan A. The anti-CD11b mAb has significant inhibitory effect on the zymosan A-induced tumor necrosis factor (TNF)-α synthesis. RR1 induced TNF-α synthesis in macrophages in a dose-dependent manner which can be completely inhibited by the NF-κB inhibitor caffeic acid phenethyl ester (CAPE) or curcumin. RR1 activated NF-κB in a time- and dose-dependent manner and this modulation of nuclear NF-κB activity is associated with the degradation of I-κB a thus facilitating the translocation of NF-κB into the nucleus. RR1-induced NF-κB activity peaks at 8 h of RR1 stimulation while I-κB a degradation occurred within 1 h of stimulation. RR1-induced NF-κB activation occurred through TLR6 signaling as evidenced by the synthesis of IL-8 in TLR6-transfected HEK293 cells. These results show that the novel (1,4)-α-D glucan from Tinospora cordifolia activates the immune system through the activation of macrophages that occurs through TLR6 signaling, NF-κB translocation and cytokine production. (Supported by Miami Children’s Hospital Foundation research funds).

Abstract The adherence of serum-opsonized yeast to neutrophils results in phagocytosis of these particulate stimuli and activation of the respiratory burst. Both events are mediated or modulated in part by the surface receptors for IgG and complement. The link between the binding of complex particulate stimuli to the cell surface, and the triggering of these neutrophil functions, is not completely understood. We have previously described an anti-human neutrophil, murine monoclonal antibody PMN7C3, which specifically inhibits the respiratory burst of neutrophils stimulated with serum-opsonized yeast. In the present study, we show that the antigen recognized by PMN7C3 (PMN7 antigen) is present on a number of neutrophil proteins, including the recently described group of related leukocyte membrane glycoproteins CR3, LFA-1, and p150,95. The PMN-7 antigen differs from other antigens associated with the C3bi receptor complex (MAC 1, MO 1, OKM1, OKM10) in that it is present only on neutrophils among peripheral blood cells. Furthermore, the binding of PMN7C3 to the neutrophil surface inhibits the activation of the respiratory burst by serum opsonized zymosan without affecting phagocytosis of these particulate stimuli. The cross-linking of cell surface PMN7 antigen by multivalent antibody is associated with the capping and internalization of antigen-antibody complexes, and appears to be necessary for the expression of maximum inhibition of opsonized zymosan-triggered respiratory burst activity. PMN7C3 also binds to a group of granule-associated proteins biochemically distinct from CR3, LFA-1, and p150,95. These granule-associated proteins containing PMN7 antigen can be mobilized to the cell surface with secretion. PMN7 antigen-bearing proteins may play a role in modulating the activation of the respiratory burst associated with phagocytosis of serum-opsonize zymosan.

SUMMARY Receptors for opsonins, such as complement component C3b (CR1) and immunoglobulins, Fc receptors, interact with adhesion glycoproteins in mediating immune functions. Defects in expression of the adhesion glycoproteins CD11/CD18 results in severely hampered in vitro and in vivo adherence- related functions of leukocytes. Little is known regarding the effect of leukocyte adhesion deficiency (lad) on ligand binding and receptor expression. We investigated the binding and expression of CR1 and Fc receptors by bovine neutrophils isolated from dairy calves suffering from lad, compared with clinically normal (hereafter referred to as normal) age-matched calves. Neutrophils were also assayed for endogenously bound IgG and IgM and for exogenous binding of C3b, IgG1, IgG2, IgM, and aggregated IgG (aIgG), using flow cytometry. Luminol-enhanced chemiluminescence (cl) production in response to IgG2 opsonized zymosan was studied, and specific inhibition of cl was used to determine the specificity of IgG2 binding. Activation of protein kinase C with phorbol myristate acetate was used to determine the effect of cellular activation on expression of CR1. A greater percentage of neutrophils from normal calves bound C3b than did neutrophils from lad- affected calves. Receptor expression was similar. Activation with phorbol myristate acetate resulted in increased expression of CR1 on neutrophils from normal and lad-affected calves, but expression was almost twofold greater on neutrophils from normal calves. There was no difference between lad-affected and normal calves in percentage of neutrophils that bound endogenous IgG and IgM. A greater percentage of neutrophils from normal calves bound exogenous IgM than did neutrophils from lad-affected calves. Receptor expression for aIgG was greater on neutrophils from lad-affected calves than on those from normal calves. Luminol-enhanced cl of neutrophils in response to IgG2 opsonized zymosan was not different between lad-affected and normal calves. Results indicate increased binding and expression of Fc receptors for aIgG and decreased binding and expression for C3b and IgM on neutrophils from calves with lad. Leukocyte adhesion deficiency may be compounded by added defects in the expression and binding of receptors for opsonins, such as C3b and IgM.

To date, only limited information about the influence of ovarian hormones on canine immune system cells has been available. The study investigated the in vitro influence of progesterone and estradiol-17β on the activity of canine neutrophils. Treatment of cells by both hormones led to a significant decrease in phagocytosis-induced oxidative burst as detected using luminometry after stimulation with opsonised zymosan. The increase in oxidative burst, not connected with phagocytosis, was recorded after stimulation with a soluble stimulator. Using flow cytometry, the tendency of both hormones to decrease the production of reactive oxygen species associated with phagocytosis of Escherichia coli was also evident, although not significant. Suppression of canine neutrophil activity is not connected with pathogen recognition capabilities, since the expression of Toll-like receptor 4 was unaffected. This study reveals that both hormones have a suppressive effect on the activity of canine neutrophils and thus might contribute to the aetiology of pyometra.  

Les polymorphonucléaires neutrophiles, sont des acteurs essentiels du système immunitaire inné, responsables de la phagocytose des agents pathogènes. Lors de la phagocytose, les neutrophiles produisent des quantités substantielles d'anion superoxyde, qui génèrent ensuite des espèces réactives de l'oxygène (ERO) telles que le peroxyde d'hydrogène, les radicaux hydroxyles et l'acide hypochloreux, indispensables à la destruction des microbes. L'enzyme responsable de la production de superoxyde est le complexe NADPH oxydase, composé de protéines membranaires (gp91phox/NOX2 et p22phox) et de protéines cytosoliques (p47phox, p67phox, p40phox et Rac1/2). Lors de l'activation, ces composants cytosoliques transloquent vers la membrane, entraînant l'assemblage et l'activation de l'enzyme. Une régulation adéquate de l'activité de la NADPH oxydase est essentielle pour équilibrer une élimination efficace des pathogènes et éviter les dommages tissulaires excessifs dus aux ERO. L' objectifs de ma thèse vise à examiner la phosphorylation de la p47phox dans les neutrophiles humains stimulés par le zymosan opsonisé par le sérum (OZ), un agent connu pour induire la phagocytose. Nous nous concentrons sur l'identification des sites de phosphorylation spécifiques et l'élucidation des voies de signalisation impliquées dans ce processus. Les neutrophiles humains ont été isolés du sang veineux de volontaires sains par sédimentation au Dextran et centrifugation sur Ficoll. Le zymosan a été opsonisé en utilisant du sérum autologue riche en immunoglobulines (IgG) et en protéines du complément (C3b et C3bi). Les neutrophiles ont ensuite été stimulés avec du zymosan non opsonisé ou opsonisé, et la phosphorylation de p47phox a été évaluée par SDS-PAGE et Western blot avec des anticorps spécifiques. La production de ROS a été mesurée en utilisant la chimioluminescence amplifiée par le luminol. La microscopie confocale a été utilisée pour visualiser les interactions des neutrophiles avec le zymosan opsonisé fluorescent. Divers inhibiteurs de kinases ont été utilisés pour disséquer les voies de signalisation conduisant à la phosphorylation de p47phox. Les résultats montrent que le zymosan opsonisé par le sérum (OZ) a induit une phosphorylation rapide et transitoire de p47phox aux sites Ser304, Ser315, Ser320 et Ser328, détectable en 20 secondes et atteignant un pic à 40-60 secondes. Cette phosphorylation diminue sur 10 minutes, tandis que la production de ROS est restée soutenue pendant plus de 30 minutes. Le zymosan non opsonisé n'a pas induit de phosphorylation significative ni de production de ROS. La phosphorylation a eu lieu lors du contact avec le OZ, avant la phagocytose, et a été principalement induite par les opsonines IgG et C3bi via leurs récepteurs respectifs, Fc-gamma R et CR3. Les études utilisant des inhibiteurs ont révélé que les kinases tyrosine Src et Syk, PI3K, PLC, PLD, le calcium et PKC-beta2 sont essentiels pour la phosphorylation de p47phox et l'activation subséquente de la NADPH oxydase. Cette étude élucide les événements de phosphorylation spécifiques et les voies de signalisation qui régulent l'activation de la NADPH oxydase dans les neutrophiles humains pendant la phagocytose. Le zymosan opsonisé par le sérum induit une phosphorylation rapide de p47phox aux sites Ser304, Ser315, Ser320 et Ser328, nécessaire pour initier mais non maintenir l'activité de la NADPH oxydase. Les IgG et C3bi sont les principales opsonines conduisant ce processus via les récepteurs Fc-gamma R et CR3. Les voies de signalisation clés impliquent les kinases tyrosine Src et Syk, PI3K, PLC, PLD, le calcium et PKC-beta2. Ces résultats améliorent notre compréhension de l'activation des neutrophiles et offrent des cibles thérapeutiques potentielles pour moduler les réponses immunitaires
Neutrophils, also known as polymorphonuclear leukocytes (PMNs), are essential players in the innate immune system, responsible for the phagocytosis of pathogens. During phagocytosis, neutrophils produce substantial amounts of superoxide anion, which subsequently generates reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radicals, and hypochlorous acid, crucial for microbial killing. The enzyme responsible for superoxide production is the NADPH oxidase complex, composed of membrane-bound proteins (gp91phox/NOX2 and p22phox) and cytosolic proteins (p47phox, p67phox, p40phox, and Rac1/2). Upon activation, these cytosolic components translocate to the membrane, leading to the assembly and activation of the enzyme. Proper regulation of NADPH oxidase activity is essential to balance effective pathogen clearance and avoid excessive tissue damage due to ROS.The objectives of my thesis aims to investigate the phosphorylation of p47phox in human neutrophils stimulated by serum-opsonized zymosan (OZ), an agent known to induce phagocytosis. We focus on identifying the specific phosphorylation sites and elucidating the signaling pathways involved in this process. Human neutrophils were isolated from the venous blood of healthy volunteers using Dextran sedimentation and Ficoll centrifugation. Zymosan was opsonized using autologous serum rich in immunoglobulins (IgG) and complement proteins (C3b and C3bi). Neutrophils were then stimulated with either non-opsonized or opsonized zymosan, and p47phox phosphorylation was assessed using SDS-PAGE and Western blotting with specific antibodies. ROS production was measured using luminol-enhanced chemiluminescence. Confocal microscopy was employed to visualize neutrophil interactions with fluorescently labeled opsonized zymosan. Various kinase inhibitors were used to dissect the signaling pathways leading to p47phox phosphorylation. The results showed that serum-opsonized zymosan (OZ) induced rapid and transient phosphorylation of p47phox at Ser304, Ser315, Ser320, and Ser328, detectable within 20 seconds and peaking at 40-60 seconds. This phosphorylation declined over 10 minutes, while ROS production remained sustained for over 30 minutes. Non-opsonized zymosan did not induce significant phosphorylation or ROS production. Phosphorylation occurred upon contact with OZ, prior to phagocytosis, and was primarily induced by IgG and C3bi opsonins through their respective receptors, Fc-gamma R and CR3. Inhibitor studies revealed that Src and Syk tyrosine kinases, PI3K, PLC, PLD, calcium, and PKC-beta2 are crucial for p47phox phosphorylation and subsequent NADPH oxidase activation. This study elucidates the specific phosphorylation events and signaling pathways that regulate NADPH oxidase activation in human neutrophils during phagocytosis. Serum-opsonized zymosan induces rapid phosphorylation of p47phox at Ser304, Ser315, Ser320, and Ser328, which is necessary to initiate but not sustain NADPH oxidase activity. IgG and C3bi are the primary opsonins driving this process through Fc-gamma R and CR3 receptors. Key signaling pathways involve Src and Syk tyrosine kinases, PI3K, PLC, PLD, calcium, and PKC-beta2. These findings enhance our understanding of neutrophil activation and provide potential therapeutic targets for modulating immune responses