Literatura científica selecionada sobre o tema "Opsonized zymosan"

Summary Stimulatory effects of 6 zymosan preparations on luminol-dependent chemiluminescence (cl) responses of isolated bovine neutrophils were compared. Unopsonized zymosan particles and zymosan particles opsonized with bovine IgG1, IgG2, fresh serum, or serum from which zymosan-specific antibodies, but not complement, had been removed (C3-serum) induced strong cl responses, with nearly equal maximal peaks in the presence of extracellular Ca2+ and Mg2+, whereas the response to fetal bovine serum-opsonized zymosan particles was markedly low. Removal of extracellular divalent cations almost completely blocked the cl reaction triggered by unopsonized, IgG1-opsonized, C3-opsonized, and fetal bovine serum-opsonized zymosan particles. By contrast, no change in the respiratory burst activity induced by serum-opsonized zymosan and only partial reduction in the response to IgG2-opsonized zymosan were seen under these conditions. Further experiments were performed with 4 zymosan preparations on neutrophils isolated from 2 calves with a genetic deficiency of CD11/CD18 membrane antigens. The unopsonized zymosan-induced cl reaction was absent in these cells. A reduced, but clear, response was observed with C3-opsonized zymosan. Unexpectedly, in the absence of extracellular Ca2+ and Mg2+, serum-opsonized zymosan failed to generate the respiratory burst, whereas response to IgG2-opsonized zymosan was normal in the CD11/CD18-deficient neutrophils. These findings indicate that unopsonized zymosan may act in a divalent cation-dependent manner at the receptor for C3bi in bovine neutrophils, as it has been shown to do in the human system. In addition, it seems that IgG2-Fc receptors capable of signaling the respiratory burst in the absence of extracellular Ca2+ and Mg2+ exist on bovine neutrophils.

Abstract CD43, a prevalent white blood cell molecule distinguished by its mucin- like surface region, has been proposed as a “functional barrier” that prevents or negatively regulates a variety of cell surface interactions. Implicit in this hypothesis is the expectation that CD43 will be altered or removed when white blood cells are activated. To investigate alterations of CD43 in a dramatic example of functional cell activation, suspension neutrophils were challenged with opsonized zymosan, a characterized stimulator of phagocytosis and respiratory burst oxidase. Flow cytometry showed decreased surface density of CD43 in opsonized zymosan-treated neutrophils, and immune precipitation showed decreased cellular CD43 content, indicating that opsonized zymosan downregulates CD43 by a proteolytic mechanism. Based on densitometry of immune precipitates, CD43 levels were decreased 42% +/- 6% in neutrophils treated for 10 minutes with opsonized zymosan and decreased 70% +/- 3% in neutrophils treated with phorbol 12-myristate 13-acetate (PMA). CD43 downregulation in response to opsonized zymosan, like PMA-induced CD43 downregulation, was insensitive to the serine protease inhibitor diisopropylfluorophosphate (DFP). In contrast, CD43 downregulation in response to opsonized zymosan or PMA was prevented by 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF) and 3′4′- dichloroisocoumarin (3,4-DCI), both of which are characterized serine protease inhibitors. Activation of the neutrophil respiratory burst oxidase by opsonized zymosan or PMA was also insensitive to DFP and prevented by AEBSF and 3,4-DCI. These findings indicate a requirement for a proteolytic step in activation of the respiratory burst of intact suspension neutrophils by opsonized zymosan and PMA and suggest that CD43 cleavage may be a required proteolytic event.

CD43, a prevalent white blood cell molecule distinguished by its mucin- like surface region, has been proposed as a “functional barrier” that prevents or negatively regulates a variety of cell surface interactions. Implicit in this hypothesis is the expectation that CD43 will be altered or removed when white blood cells are activated. To investigate alterations of CD43 in a dramatic example of functional cell activation, suspension neutrophils were challenged with opsonized zymosan, a characterized stimulator of phagocytosis and respiratory burst oxidase. Flow cytometry showed decreased surface density of CD43 in opsonized zymosan-treated neutrophils, and immune precipitation showed decreased cellular CD43 content, indicating that opsonized zymosan downregulates CD43 by a proteolytic mechanism. Based on densitometry of immune precipitates, CD43 levels were decreased 42% +/- 6% in neutrophils treated for 10 minutes with opsonized zymosan and decreased 70% +/- 3% in neutrophils treated with phorbol 12-myristate 13-acetate (PMA). CD43 downregulation in response to opsonized zymosan, like PMA-induced CD43 downregulation, was insensitive to the serine protease inhibitor diisopropylfluorophosphate (DFP). In contrast, CD43 downregulation in response to opsonized zymosan or PMA was prevented by 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF) and 3′4′- dichloroisocoumarin (3,4-DCI), both of which are characterized serine protease inhibitors. Activation of the neutrophil respiratory burst oxidase by opsonized zymosan or PMA was also insensitive to DFP and prevented by AEBSF and 3,4-DCI. These findings indicate a requirement for a proteolytic step in activation of the respiratory burst of intact suspension neutrophils by opsonized zymosan and PMA and suggest that CD43 cleavage may be a required proteolytic event.

Abstract We determined the mechanism by which opsonized zymosan particles, which are derived from yeast and composed of carbohydrate polymers, stimulate platelet-activating factor (PAF) synthesis by monocytes. A role for CD11b/CD18 was demonstrated because antibodies to this integrin decreased PAF synthesis, zymosan bearing only a ligand for CD11b/CD18 (iC3b) induced the synthesis of PAF, and monocytes that did not express CD11b/CD18 produced much less PAF than control monocytes. Ligation of CD11b/CD18 was not sufficient for PAF synthesis suggesting that an additional receptor was involved. Monocytes are known to bind beta-glucan which is a major component of zymosan. Opsonized beta-glucan particles stimulated the synthesis of PAF, and a soluble form of beta-glucan partially inhibited PAF synthesis in response to opsonized zymosan. Two lines of evidence suggested that the beta-glucan receptor mediating this response was distinct from CD11b/CD18. First, CD11b/CD18-deficient monocytes produced PAF when stimulated by zymosan opsonized with isolated C3b, a molecule that binds to complement receptor type 1 (CD35). Second, inducing contact of monocytes with zymosan by centrifugation resulted in PAF synthesis that was not inhibited by antibodies to CD11b/CD18. The combination of soluble beta-glucan and antibodies to CD11b/CD18 completely blocked PAF synthesis in response to opsonized zymosan. Together, these results demonstrate that induction of maximal PAF synthesis by serum-opsonized zymosan requires the concerted interactions of monocyte receptors for iC3b and beta-glucan. Additionally, they suggest that CD11b/CD18 facilitates binding of the particle and that a beta-glucan receptor transduces the activation signal.

The purpose of the present work was to study effects of dehydroepiandrosterone (DHEA) on the oxygen-dependent microbicidal activity of blood leukocytes. The studies were performed on male white nonlinear rats aged 3-6 months (young rats) and adult animals over 1.5 years old (old rats) using an experimental model of zymosan peritonitis. DHEA was administered subcutaneously to old animals on the daily basis (a total of 4 injections of 2 mg/kg body weight, the last injection 24 hours before the end of the experiment). Rats of similar age served as controls and received DHEA solvent (officinal olive oil) according to the same time regimen. 12 hours before the end of the experiment, the animals were administered a sterile suspension of zymosan A intraperitoneally at a dose of 50 mg/kg. The oxygen-dependent microbicidal activity of blood leukocytes was assessed by luminol-dependent chemiluminescence using opsonized and non-opsonized zymosan at concentrations of 15 μg/mL, 150 μg/mL and 1500 μg/mL. In old male rats, compared to young ones, the chemiluminescence indexes showed a statistically significant increase when blood leukocytes were stimulated with both opsonized and non-opsonized zymosan. Administration of DHEA to old male rats leads to a pronounced decrease in chemiluminescence in samples at all concentrations of opsonized zymosan. When leukocytes of old animals are stimulated with non-opsonized zymosan in vitro, a statistically significant increase in the production of reactive oxygen species was observed, being more pronounced at lower concentrations of inducing agent (15 μg/mL and 150 μg/mL). Meanwhile, this effect was canceled at high concentrations of zymosan. The level of spontaneous chemiluminescence, reflecting in vivo cell activation, was higher in old rats treated with DHEA compared to young animals. The direction of changes in the production of reactive oxygen species in samples with low concentrations of the in vitro activation was similar to the effects of DHEA in old rats tested for spontaneous chemiluminescence. In general, our studies confirm the pronounced immunomodulatory activity of DHEA. In general, we have found that aging of male rats leads to pronounced pro-inflammatory activation of reactive oxygen species produced by blood leukocytes at the peak of inflammation induced by zymosan injections. Administration of DHEA to old male rats significantly reduced the production of reactive oxygen species by blood leukocytes, when the cells were in vitro activated by opsonized zymosan, but stimulates production of oxygen radicals in the samples without zymosan, as well as upon exposition to non-opsonized zymosan at concentrations of 15 μg/mL and 150 μg/ mL.

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.

AbstractThe protein tyrosine kinase Syk plays a central role in Fcγ receptor–mediated phagocytosis in the adaptive immune system. We show here that Syk also plays an essential role in complement-mediated phagocytosis in innate immunity. Macrophage-like differentiated HL60 cells and C3bi-opsonized zymosan comprised the pathogen-phagocyte system. C3bi-opsonized zymosan particles promptly attached to the cells and were subsequently engulfed via complement receptor 3. During this process, Syk became tyrosine phosphorylated and accumulated around the nascent phagosomes. The transfer of Syk-siRNA or dominant-negative Syk (DN-Syk) into HL60 cells resulted in impaired phagocytosis. Quenching assays using fluorescent zymosan revealed that most of the attached zymosan particles were located inside parental HL60 cells, whereas few were ingested by the mutant cells. These data indicated that Syk is required for the engulfment of C3bi-opsonized zymosan. During C3bi-zymosan–induced phagocytosis, actin accumulation occurred around phagosomes and was followed by depolymerization, and further RhoA was activated together with tyrosine phosphorylation of Vav. These responses including the actin remodeling were suppressed in Syk-siRNA– or DN-Syk–expressing cells. Our results demonstrated that Syk plays an indispensable role in complement-mediated phagocytosis by regulating both actin dynamics and the RhoA activation pathway and that these functions of Syk lead to phagosome formation and pathogen engulfment.

Abstract We have previously reported that K562, a chronic myelogenous leukemia cell line, releases a low molecular weight factor (6 to 8 Kd) that inhibits human polymorphonuclear neutrophil (PMN) adherence and adherence-related functions tested in vitro. We now report that this factor, which we have named K562 inhibitory factor (K562-IF), has potent anti-inflammatory activity in mice, associated with an inhibition of PMN functions. Its in vitro actions were less marked with mouse PMN than with human PMN. They included (1) an inhibition of both nonstimulated locomotion and locomotion induced by FMLP or serum; (2) an inhibition of the chemiluminescence induced by opsonized zymosan, but not that induced by phorbol myristate acetate or FMLP; (3) an inhibition of the degranulation stimulated by opsonized zymosan, as reflected by lactoferrin and lysozyme release; and (4) a decrease in arachidonic acid release and leukotriene B4 production by A23187- stimulated PMN. The in vivo actions of K562-IF after intraperitoneal injection included (1) an inhibition of subcutaneous PMN accumulation at the site of injection of opsonized zymosan (PMN accumulated neither outside the vessels nor intravascularly, as shown by means of histochemistry); (2) an inhibition of neutrophil accumulation in the peritoneum of mice having received sodium caseinate or opsonized zymosan intraperitoneally; and (3) lysozyme concentration in neutrophils having reached the peritoneum after opsonized zymosan treatment equal to that in blood, suggesting diminished release. PMN influx and degranulation in the peritoneum were reduced by 50% after 3 hours of treatment with 1 microgram of K562-IF (equivalent to the effect of 120 micrograms of prednisolone). Taken together, these results show that K562-IF is a potent anti-inflammatory agent that acts by inhibiting PMN functions.

We have previously reported that K562, a chronic myelogenous leukemia cell line, releases a low molecular weight factor (6 to 8 Kd) that inhibits human polymorphonuclear neutrophil (PMN) adherence and adherence-related functions tested in vitro. We now report that this factor, which we have named K562 inhibitory factor (K562-IF), has potent anti-inflammatory activity in mice, associated with an inhibition of PMN functions. Its in vitro actions were less marked with mouse PMN than with human PMN. They included (1) an inhibition of both nonstimulated locomotion and locomotion induced by FMLP or serum; (2) an inhibition of the chemiluminescence induced by opsonized zymosan, but not that induced by phorbol myristate acetate or FMLP; (3) an inhibition of the degranulation stimulated by opsonized zymosan, as reflected by lactoferrin and lysozyme release; and (4) a decrease in arachidonic acid release and leukotriene B4 production by A23187- stimulated PMN. The in vivo actions of K562-IF after intraperitoneal injection included (1) an inhibition of subcutaneous PMN accumulation at the site of injection of opsonized zymosan (PMN accumulated neither outside the vessels nor intravascularly, as shown by means of histochemistry); (2) an inhibition of neutrophil accumulation in the peritoneum of mice having received sodium caseinate or opsonized zymosan intraperitoneally; and (3) lysozyme concentration in neutrophils having reached the peritoneum after opsonized zymosan treatment equal to that in blood, suggesting diminished release. PMN influx and degranulation in the peritoneum were reduced by 50% after 3 hours of treatment with 1 microgram of K562-IF (equivalent to the effect of 120 micrograms of prednisolone). Taken together, these results show that K562-IF is a potent anti-inflammatory agent that acts by inhibiting PMN functions.

Abstract The effect of iC3b receptor (CR3)-mediated phagocytosis on the expression of CR (C3b receptor, CR3) and IgG FcR (FcRI, FcRII) has been investigated by using serum-opsonized zymosan as a multivalent ligand for CR3. Sixteen hours after a short (1-h) pretreatment of human monocyte monolayers with zymosan opsonized with human AB serum (250 micrograms/ml), CR3 expression (as assessed by flow cytometric analysis with mAb Mo1) was significantly reduced by 59 +/- 3% (mean +/- SEM, n = 15, p less than 0.001). Concomitant with CR3 down modulation, FcR binding activity (as assessed by binding of IgG-coated E) was also found to be decreased to 41 +/- 4% of control (n = 7, p less than 0.001). Reduced FcR function was paralleled by a decrease in the expression of FcRI (as assessed with mAb 32.2). This FcRI modulation was not caused by zymosan-bound IgG because zymosan opsonized with agammaglobulinemic serum equally down regulated CR3 and FcRI expression. Pretreatment with zymosan opsonized with human AB serum, however, did not change the expression of other IgG and C-binding sites such as FcRII (examined with mAb IV.3 and 2E1) and CR1 (assessed with mAb 57F) as well as of unrelated cell membrane structures (beta 2m, MHC class II). In contrast, co-modulation for FcR function and CR3 expression induced by polymeric IgG is accompanied by a decreased expression of FcRII. These data indicate that interaction of a specific receptor with its ligand not only changes the expression of the receptor triggered, but has also a modulating effect on other receptor systems on the same cell.

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

Macrophages are known to release active oxygen species during phagocytosis or in response to triggering with opsonized zymosan or phorbol myristate acetate. Chemiluminescence intensity from such active species is so low that it has been detected previously by utilizing luminol or lucigenin as a photoamplifier. Using a highly sensitive photon counting system developed recently by our group, we detected very weak chemiluminescence from mouse peritoneal macrophages without use of luminol or lucigenin.

Human PMNs in a suspension (2.5 - 3.5 × 106 cells/ml of PBS) were activated by opsonized zymosan (2.5 mg/ml) or by FMLP (22 jjg/ml) in presence or absence of prostaglandins (PG) E1 E2, D2, 6-keto-F2α, 6-keto-E1 prostacyclin and Iloprost (3nM -30 pM). The generation of superoxide anions was measured as a SOD-sensitive reduction of ferrocytochrome c. In FMLP-stimulated PMNs an average production of 0- 2 of 18 ± 3.2 nmoles/10- 2 PMNs/10 min was suppressed by 25% at following concentrations of PGD2, PGE2, PGE1s 6-keto-PGE! and PGF2 : 0.1, 0.2, 0.5, 0.8 and>30.0 pM, respectively. No significant inhibition occurred in the presence of prostacyclin, 6-keto-PGF2 and Iloprost at concentrations as high as 30 pM. In zymosan-stimulated PMNs prostaglandins of E series were less potent inhibitors than in FMLP-stimulated PMNs by following factors: PGE2 - 20, 6-keto-PGE2 - 13, PGE1 - 4, whereas PGD2 was equally potent inhibitor in FMLP- and zymosan-stimulated PMNs. It is concluded that PGE2 is an antagonist of FMLP-receptor-mediated events which are responsible for the generation of superoxide anions in PMNs, whereas its isomer PGD2 has another mechanism of the 0- 2-suppressive action. Perhaps, PGD2 is a direct inhibitor of membrane-bound NADPH-oxidase or an antagonist of oxygen activation by leukotrienes and lipoxins or a promotor of scavenging of 0- 2 by mitochondrial membranes. The latter mechanism has been proposed recently by Gryglewski as explanation for cytoprotective action of stable biologically active metabolites of prostacyclin. Mechanisms of anti-0- 2 action of PGE1 and 6-keto-PGE1 in PMNs are likely to be intermediate between these proposed for PGE2 and PGD2.

Background: Pseudomonas aeruginosa is a well-known opportunistic pathogen. The gram-negative bacillus, commonly associated with hospital-acquired infections, utilizes the host’s impaired immune responses to establish infection. Of its many virulence factors, pyocyanin is essential for P. aeruginosa to establish its full infectivity. Macrophages act as sentinels of the innate immune system, as well as play other roles in homeostasis, tissue remodeling, and bridging between the innate and adaptive immune systems. Aim: This study aimed to investigate the effects of pyocyanin on macrophage innate immune defenses by assessing the function of macrophages treated with pyocyanin and TLR ligands. Phagocytosis of opsonized zymosan, LPS-induced nitric oxide release and cytokine release were used as measures of functional responses. Results: This study found that pyocyanin inhibited phagocytosis-induced ROS release in a dose-dependent manner and reduced nitric oxide release from macrophages induced with P. aeruginosa LPS. In addition, pyocyanin modulated cytokines and chemokines release from macrophages exposed to P. aeruginosa LPS in a dose-dependent manner. Pyocyanin significantly enhanced IL-1β release as well as several chemokines. Therefore, pyocyanin facilitates Pseudomonas aeruginosa to persevere in the immunocompromised host through modulating macrophage’s innate immune defenses. Conclusion: Pyocyanin inhibits macrophage functional defense responses to facilitate Pseudomonas aeruginosa infection.

Interactions between platelets and neutrophils have been reported for the production of several mediators of inflammation such as hydrogen peroxides or leukotrienes. Another potential mediator of thrombosis and inflammation is paf-acether which is synthesized by activated platelets and neutrophils.Since platelets form and release large amounts of the paf-acether precursor lyso paf-acether, platelets and neutrophils cooperation for paf-acether biosynthesis was investigated. Purified human neutrophils (4 × 106 /ml) stimulated by opsonized zymosan (ZC, 1 mg/ml) formed 4.5 ± 2.5 ng/ml paf-acether. Human washed platelets (3 × 108 /ml) stimulated with thrombin (l IU/ml) formed 0.60 ± 0.43 ng/ml paf-acether. Platelets and neutrophils, incubated together and both stimulated by their specific agonist, formed more than twice as much paf-acether as platelets and neutrophils separately (10.90 ± 4.25 ng/ml, n = 6, p < 0.001). The formation of lyso paf-acether and the release of lysozyme and LDH were unchanged under the cooperation conditions. The formation of paf-acether almost doubled(10.24 ± 8.79 ng/ml paf-acether versus 5.30 ± 5.23, p < 0.05, n = 4) when ZC-stimulated neutrophils were incubated with supernatants from thrombin-stimulated platelets as well as with synthetic lyso paf-acether. Extracted and purified lyso paf-acether from thrombin-stimulated platelets led to increase of biosynthesis of paf-acether by neutrophils (13.86 ± 3.92 ng/ml paf-acether versus 5.76 ± 0.66, p < 0.05, n = 3). These results indicate that cooperation between platelets and neutrophils is likely to occur via acetylation by neutrophils of excess lyso paf-acether originating from platelets. Thus neutrophils can increase their biosynthesis of paf-acether by acetylating lyso paf-acether either synthetic or released from other cells.