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PubMed1998

Antimalarial drugs inhibit phospholipase A2 activation and induction of interleukin 1beta and tumor necrosis factor alpha in macrophages: implications for their mode of action in rheumatoid arthritis.

1. The effects of antimalarial drugs on the intracellular signaling leading to activation of the phospholipase C and phospholipase A2 pathways and the induction of proinflammatory cytokines have been studied in mouse macrophages. 2. Both chloroquine and quinacrine, and to a lesser extent hydroxychloroquine, inhibited arachidonate release and eicosanoid formation induced by phorbol diester. This inhibition was due to that of the activation of the arachidonate-mobilizing phospholipase A2. 3. All three antimalarials potently inhibited arachidonate release induced by zymosan. They also inhibited the zymosan-induced formation of inositol phosphates, which hints that an inhibitory effect at the phospholipase C level might explain the inhibition of the response to zymosan. 4. Quinacrine, and to a lesser extent chloroquine, has an inhibitory effect on the lipopolysaccharide- or zymosan-induced expression of interleukin 1beta and tumor necrosis factor alpha, both at the mRNA and protein levels. This, in particular, has important implications for the mode of action of these compounds in rheumatoid arthritis.

Animals↗
PubMed1997

Demonstration of functionally distinct human polymorphonuclear leukocyte fractions by simultaneous measurement of phagocytosis and oxygen radical generation.

Phagocytosis and oxygen radical generation by human polymorphonuclear leukocytes (PMNLs) were studied by a two-color flow cytometric analysis, where the red fluorescent product(s) of hydroethidine was used as an indicator of intracellular generation of oxygen radicals and opsonized zymosan (OZ) as an indicator of phagocytosis. Unstimulated cells formed a single population of cells without any significant fluorescence. PMNLs stimulated by OZ exhibited a high red fluorescence. Most PMNLs phagocytosed OZ and generated oxygen radicals when stimulated by zymosan particles opsonized with human AB serum at concentrations of more than 10%, whereas three distinct subpopulations (designated as R1, R2 and R3) appeared when stimulated by zymosan particles opsonized with 3% serum; R1 cells enhanced neither green nor red fluorescence, R2 cells enhanced green fluorescence but not red fluorescence, and R3 cells enhanced both green and red fluorescence. The R2 cells completely disappeared by the addition of Trypan blue. Most of the R3 cells disappeared by the addition of cytochalasin B. These findings on the three fractions were confirmed by the observation under fluorescence microscopy of cells in each fraction obtained by sorting. In conclusion, PMNLs could be separated into three functionally distinct subpopulations when stimulated by zymosan particles opsonized with a suboptimal concentration of serum.

Cell Differentiation↗
PubMed2000

Phosphorylation of p22phox is mediated by phospholipase D-dependent and -independent mechanisms. Correlation of NADPH oxidase activity and p22phox phosphorylation.

Human neutrophils participate in the host innate immune response, partly mediated by the multicomponent superoxide-generating enzyme NADPH oxidase. A correlation between phosphorylation of cytosolic NADPH oxidase components and enzyme activation has been identified but is not well understood. We previously showed that p22(phox), the small subunit of the membrane-bound oxidase component flavocytochrome b(558), is an in vitro substrate for both a phosphatidic acid-activated kinase and conventional protein kinase C isoforms (Regier, D. S., Waite, K. A., Wallin, R., and McPhail, L. C. (1999) J. Biol. Chem. 274, 36601-36608). Here we show that several neutrophil agonists (phorbol myristate acetate, opsonized zymosan, and N-formyl-methionyl-leucyl-phenylalanine) induce p22(phox) phosphorylation in intact neutrophils. To determine if phospholipase D (PLD) is needed for p22(phox) phosphorylation, cells were pretreated with ethanol, which reduces phosphatidic acid production by PLD in stimulated cells. Phorbol myristate acetate-induced phosphorylation of p22(phox) and NADPH oxidase activity were not reduced by ethanol. In contrast, ethanol reduced both activities when cells were stimulated by N-formyl-methionyl-leucyl-phenylalanine or opsonized zymosan. Varying the time of stimulation with opsonized zymosan showed that the phosphorylation of p22(phox) coincides with NADPH oxidase activation. GF109203X, an inhibitor of protein kinase C and the phosphatidic acid-activated protein kinase, decreased both p22(phox) phosphorylation and NADPH oxidase activity in parallel in opsonized zymosan-stimulated cells. Stimulus-induced phosphorylation of p22(phox) was on Thr residue(s), in agreement with in vitro results. Overall, these data show that NADPH oxidase activity and p22(phox) phosphorylation are correlated and suggest two mechanisms (PLD-dependent and -independent) by which p22(phox) phosphorylation occurs.

Adult↗
PubMed2003

In vitro effects of preserved or preservative-free antiglaucoma medications on human complement system.

PURPOSE: Antiglaucoma drugs have been associated with conjunctival and trabecular inflammatory cell infiltrates. However, the underlying mechanisms are still poorly understood. The aim of this study was to assess the effects of antiglaucoma medications on the complement system, an early mediator of the inflammatory response. METHODS: Human serum was first treated with a classical or alternative pathway activator (aggregated human IgG or zymosan, respectively) in the presence or the absence of preservative-free or benzalkonium (BAK)-preserved antiglaucoma drugs. CH50 assay was then performed to assess the functional activity of residual complement in treated serum. RESULTS: In the absence of complement activator, the antiglaucoma drugs tested in this study were all devoid of intrinsic complement-activating potency. Preserved and preservative-free carteolol as well as preserved latanoprost did not worsen or prevent complement activation induced by zymosan or aggregated IgG. Unexpectedly, both preserved and unpreserved timolol and betaxolol significantly counteracted the effects of complement activators. Timolol prevented activation triggered by both IgG and zymosan to the same extent (24% to 29%), despite the presence of BAK in the preserved formulation. Betaxolol was twice as effective at preventing the effect of IgG (34% to 37%) than that of zymosan (14%), regardless of the presence of BAK. However, BAK itself strongly aggravated complement activation by both activators. CONCLUSIONS: Carteolol, timolol, betaxolol and latanoprost did not activate complement system. On the contrary, the beta-blockers timolol and betaxolol exerted an anti-inflammatory effect by preventing complement activation. The deleterious effect of benzalkonium seems to have been neutralized within the preserved eyedrops through a mechanism that remains to be elucidated. Our study suggests that inflammatory signs in glaucoma patients should not be attributed to complement activation by antiglaucoma drugs.

Adrenergic beta-Antagonists↗
PubMed1998

Role of the epithelial layer in the generation of superoxide anion by the guinea-pig isolated trachea.

The lucigenin-dependent chemiluminescence generation by guinea-pig isolated tracheal two rings preparations was studied. Tracheal preparations stimulated with phorbol myristate acetate (PMA) or opsonized zymosan generated chemiluminescence. The total amount of chemiluminescence generated in 120 min was 754+/-63 mV x min for PMA and 4832+/-396 mV x min for zymosan. Generation of chemiluminescence was decreased by more than 50% when the tissues were co-incubated with superoxide dismutase (100 U/ml). Also, addition of direct donors of nitric oxide diminished chemiluminescence generation by zymosan-activated tracheal rings significantly by about 50%. However, the presence of the precursor or of inhibitors of nitric oxide synthase did not influence zymosan-induced chemiluminescence. Removal of the epithelial layer from tracheal rings caused an approximately 90% decrease in chemiluminescence response. However, isolated epithelial cell suspensions did not generate chemiluminescence. Histologic examination showed that the number of eosinophils in the tracheal tissue was reduced from 56+/-7 to 18+/-8 per mm basal membrane when the epithelial layer was removed. These results indicated that (1) superoxide anion formation can take place in the guinea-pig trachea, (2) eosinophils in the epithelial and submucosal layers of guinea-pig trachea are likely candidates for superoxide generation although other cell types can also be involved, and (3) besides relaxing airway smooth muscle, nitric oxide donors may also affect superoxide in the airways.

Animals↗
PubMed1987

Burn wound sepsis may be promoted by a failure of local antibacterial host defenses.

Little attention has been focused on the local burn wound environment, even though burn wound sepsis is a common cause of death in the burn victim. To characterize the effect of the local burn wound environment on neutrophil function and metabolism, the opsonic activity of blister fluid specimens against Pseudomonas aeruginosa was measured as was the effect of blister fluid on control neutrophil oxygen consumption using preopsonized zymosan and f-met-leu-phe (FMLP) as stimuli. Blister fluid did not support the killing of P. aeruginosa by normal neutrophils as well as normal serum. Additionally, blister fluid inhibited zymosan-stimulated, but not FMLP-stimulated, neutrophil oxygen consumption. The inhibitory effect of blister fluid on zymosan-stimulated oxygen consumption correlated with the extent of complement activation, measured as C3d or C3AI (p less than 0.01). That blister fluid did not inhibit the FMLP-mediated respiratory burst supports the concept that the blister fluid inhibitory effect on the zymosan-mediated respiratory burst was mediated through the complement receptor. These findings that blister fluid can affect the bactericidal and metabolic activity of normal neutrophils support the concept that cellular function can be altered by the microenvironment in which the cells are bathed. This potential impairment of host defenses within the burn wound could predispose the burn victim to burn wound sepsis.

Adolescent↗
PubMed2002

Does endotoxin-activated complement alter myocellular sodium homeostasis during sepsis?

BACKGROUND: Inappropriate complement activation is closely related to tissue injury and organ dysfunction during systemic infection. It is not clear, however, if endotoxin-induced complement activation is responsible for changes in myocellular sodium homeostasis during sepsis. METHODS: Rats underwent cecal ligation and puncture (CLP) or sham operation. Twenty-four hours after operation, fast-twitch extensor digitorum longus (EDL) muscles were isolated, incubated at 30 degrees C for 1 hour in Krebs-Henseleit buffer (KHB) (pH 7.4), and used to measure intracellular Na+ and K+ contents. Blood samples were collected to measure serum hemolytic complement activity and endotoxin levels. In addition, EDL muscles isolated from normal animals were incubated at 30 degrees C for 1 hour with zymosan-activated (10 mg/mL at 37 degrees C for 1 hour) rat sera, with lipopolysaccharide (LPS)-activated (LPS from Escherichia coli 055:B5, 10 or 200 microg/mL at 37 degrees C for 30 minutes) rat sera, with heat-inactivated (56 degrees C for 30 minutes) rat sera, with LPS (1 or 20 microg/mL), or in KHB. EDL muscles isolated from normal animals were also incubated with septic sera collected 6 or 24 hours after CLP with or without administration of soluble complement receptor type 1 (20 mg/kg, intraperitoneally). Myocellular Na+ and K+ contents ([Na+]i and [K+]i) were assayed using "washout" technique. Soluble C5b-9 complex levels in zymosan-activated or LPS-activated human sera were determined by enzyme-linked immunosorbent assay to evaluate the degree of complement activation induced by zymosan or LPS. RESULTS: Myocellular [Na+]i and [Na+]i/[K+]i ratios increased significantly 24 hours after CLP as compared with sham operation and were associated with decreased serum hemolytic complement activity and increased serum endotoxin levels. Zymosan-activated rat sera at sublytic concentrations markedly increased [Na+]i and [Na+]i/[K+]i ratios in isolated EDL muscles relative to heat-inactivated rat sera. LPS-activated rat sera, however, did not alter these two indices. In addition, myocellular [Na+]i and [Na+]i/[K+]i ratios were equivalent among normal EDL muscles incubated with septic sera, soluble complement receptor type 1-treated septic sera, or KHB. CONCLUSION: These results collectively suggest that polymicrobial sepsis, as produced by CLP, alters sodium homeostasis in fast-twitch skeletal muscles in association with changes in systemic complement activation and circulating endotoxin levels. Although endotoxin can activate the complement cascade, endotoxin-induced complement activation does not appear to be responsible for changes in myocellular sodium homeostasis observed during sepsis in rats.

Animals↗
PubMed1987

Influence of type and opsonization of ingested particle on intracellular free calcium distribution and superoxide production by human neutrophils.

Intracellular free calcium concentration [( Ca2+]i) was measured with the fluorescent Ca2+ indicator Fura 2 within individual human neutrophils during the phagocytosis of several types of particles, including serum-treated zymosan (STZ), immunoglobulin G (IgG)-coated zymosan (IGZ), C3b-coated zymosan (C3Z), nontreated zymosan (Z), and serum-treated similarly sized latex particles (STL). STZ was coated with both IgG and C3b. IGZ was coated with only IgG, and C3Z was coated with only C3b. STL was coated with only C3b but to a lesser extent than C3Z. The ingestion of particles was greatest for STZ and somewhat lower for C3Z. Ingestion of IGZ and STL was much less than ingestion of C3Z. The relative efficiencies of the particles for inducing superoxide production were as follows: STZ greater than IGZ = C3Z greater than Z = STL. [Ca2+]i significantly increased from the resting level of approximately 70 to greater than 240 nM (P less than 0.01) during phagocytosis of the particles. The increment in [Ca2+]i was greater in the paraphagosomal region than in the cell body after the ingestion of STZ or IGZ. The mean peak [Ca2+]i values in the paraphagosomal cytoplasm of neutrophils ingesting one particle of STZ, IGZ, C3Z, Z and STL were 536.1 +/- 57.6, 424.7 +/- 55.8, 373.8 +/- 62.7, 272.3 +/- 31.5, and 270.8 +/- 38.0 nM, respectively, which showed good correlation (r = 0.97) with the efficiency of the particles for inducing superoxide production. Depletion of extracellular Ca2+ by EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid] attenuated both [Ca2+]i increase and superoxide production induced by particles. Thus [Ca2+]i increased after ingestion of several types of particles, and the subcellular pattern of [Ca2+]i was different depending on the type of particle ingested. Greater increases in paraphagosomal [Ca2+]i were closely associated with greater increases in superoxide production by neutrophils.

Benzofurans↗
PubMed1989

Pentoxifylline modulation of plasma membrane functions in human polymorphonuclear leukocytes.

Pentoxifylline is known to have major effects on cell membrane function in mammalian cells, including human leukocytes. The protective effects of this agent in animal models of infection and inflammation may be due to alterations in phagocyte (neutrophil and macrophage) function. However, the exact mechanism of action of pentoxifylline is unknown. In this study, we evaluated the effect of the drug on several membrane-associated activities in human polymorphonuclear neutrophils and investigated possible mechanisms for the observed changes in neutrophil function. Pentoxifylline inhibited ingestion of microbial particles (Staphylococcus aureus and zymosan); decreased superoxide generation activated by zymosan, formyl-methionyl-leucyl-phenylalanine, and concanavalin A (but not phorbol myristate acetate); and decreased uptake (transport) of adenosine stimulated by formyl-methionyl-leucyl-phenylalanine and zymosan. In contrast, pentoxifylline actually increased clindamycin uptake in zymosan-stimulated polymorphonuclear neutrophils. However, pentoxifylline had no effect on uptake of adenosine or clindamycin in unstimulated neutrophils. In comparison with known inhibitors of nucleoside transport (nitrobenzylthioinosine and dipyridamole), the results suggested that pentoxifylline does not bind to membrane nucleoside transport receptors. At concentrations which inhibit neutrophil function, pentoxifylline activity is not mediated through external membrane nucleoside regulatory sites. Thus, pentoxifylline affects the activation signal chain at a point beyond the membrane receptors. Whatever its precise mechanism of action, pentoxifylline has a striking modulatory effect on cell membrane-associated responses in stimulated leukocytes and may prove useful for control of injurious inflammatory states.

Adenosine↗
PubMed1991

Activation of bovine neutrophils by partially purified Pasteurella haemolytica leukotoxin.

In this study we developed a new method for the partial purification of Pasteurella haemolytica leukotoxin by size-exclusion high-performance liquid chromatography. The partially purified leukotoxin had a molecular weight of 104,000, as estimated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reacted on an immunoblot with an antileukotoxin monoclonal antibody. As expected, high concentrations of the leukotoxin were inhibitory or lethal to bovine neutrophils. Incubation of bovine neutrophils with diluted leukotoxin, however, resulted in significant neutrophil activation that was comparable in magnitude to that obtained with standard activating agents such as opsonized zymosan or zymosan-activated serum. Dilute leukotoxin (1:128 to 1:8,192 dilutions) stimulated an oxidative burst (luminol-dependent chemiluminescence) by bovine neutrophils that was comparable in magnitude to that obtained with opsonized zymosan. Preincubation with leukotoxin did not significantly prime the neutrophils for an enhanced oxidative burst when they were then exposed to opsonized zymosan as a second stimulus. Dilute leukotoxin (1:100 to 1:1,000 dilutions) also stimulated cytoskeletal alterations in bovine neutrophils, as measured by a significant shape change response. Preferential release of secondary granule constituents (lactoferrin) occurred when neutrophils were incubated with 1:100 to 1:500 dilutions of leukotoxin. Significant release of primary granules, as measured by beta-glucosaminidase activity, was not observed except at low dilutions (1:20) of leukotoxin that resulted in significant release of cytosolic constituents (i.e., lactate dehydrogenase activity). The neutrophil-activating activity of the leukotoxin was heat labile, unaffected by polymyxin B, and abrogated by a leukotoxin-neutralizing monoclonal antibody. These data indicate that P. haemolytica leukotoxin, like the closely related Escherichia coli hemolysin, is a potent neutrophil-activating agent. Leukotoxin-stimulated release of neutrophil oxygen intermediates and granule constituents may contribute to the intense inflammation that characterizes bovine pulmonary pasteurellosis.

Animals↗
PubMed1992

Kinetic analysis of the amplification phase for activation and binding of C3 to encapsulated and nonencapsulated Cryptococcus neoformans.

Encapsulated and nonencapsulated cryptococci exhibit quantitative and qualitative differences in their activation of the complement system. We examined the kinetics for the rapid amplification phase in which C3 was activated and bound to encapsulated cryptococci, nonencapsulated cryptococci, and zymosan particles. Yeast cells were incubated in normal human serum containing 125I-labeled C3, and bound C3 fragments were measured after 1 to 64 min of incubation. A kinetic analysis showed that the apparent first-order rate constant (k') for binding of C3 to nonencapsulated cryptococci did not differ significantly from k' for binding of C3 to zymosan particles (P greater than 0.05). However, the rate constant for binding of C3 to encapsulated cryptococci was significantly (P less than 0.001) greater than k' for binding of C3 to nonencapsulated cryptococci and zymosan particles. A plot of C3 molecules bound to encapsulated cryptococci versus time cubed was nearly linear, suggesting that accumulation of C3 in the cryptococcal capsule follows the kinetics predicted by an expanding sphere. In contrast, the plot of C3 molecules bound to nonencapsulated cryptococci or zymosan particles against time was nearly linear, but those plots against time squared or time cubed were not. This result indicates that the rate-limiting step for the addition of C3 fragments to these latter yeast cells follows the kinetics of neither the perimeter of an expanding circle nor the surface of an expanding sphere. Taken together, the results indicate that the high rate of accumulation of C3 in the cryptococcal capsule is consistent with the expected geometry of an expanding sphere of bound C3 within the three-dimensional matrix of the capsule.

Complement Activation↗
PubMed1992

Reduced release of leukotrienes B4 and C4 from alveolar macrophages of rats with silicosis.

Silicosis leads to altered release of fibrogenic and immunomodulating mediators from alveolar macrophages (AM). Since 5-lipoxygenase metabolites have been shown to possess proinflammatory effects and to promote the release of cytokines such as tumor necrosis factor-alpha (TNF-alpha) from mononuclear phagocytes, we determined leukotriene secretion from silica-exposed AM. Rats were exposed to an aerosol of silica particles for 8 days and AM were harvested by bronchoalveolar lavage 5 to 7 mo after exposure. AM from both air-sham control and silica-exposed rats displayed minimal spontaneous leukotriene release upon in vitro culture. Stimulation with opsonized zymosan particles induced leukotriene B4 (LTB4) and leukotriene C4 (LTC4) secretion, which was much greater in control AM than in AM from silica-dusted rats. The reverse was found for zymosan-induced TNF-alpha production, which was higher in AM from silica-exposed than from control rats. To study the interrelation between leukotriene and TNF-alpha release, we incubated zymosan-stimulated AM with the 5-lipoxygenase inhibitor VZ 65. VZ 65 suppressed zymosan-induced TNF-alpha release from AM in a dose-dependent manner, and TNF-alpha production could be restored almost completely by addition of LTB4. These experiments demonstrate that silica exposure resulted in a decreased LTB4 and LTC4 production from AM, which may represent a regulatory mechanism to counterbalance enhanced TNF-alpha production during silicosis.

Animals↗
PubMed1978

Termination of the respiratory burst in human neutrophils.

Recent evidence has suggested that a particulate O(2) (-)-forming system is responsible for the respiratory burst in activated neutrophils. The respiratory burst is normally a transient event, lasting only 30-60 min. To investigate the mechanism by which the burst is terminated, we examined the O(2) (-)-forming activity of neutrophil particles as a function of time in the presence and absence of agents known to affect the function of intact cells. Measurements of the O(2) (-)-forming capacity of the particles against time of exposure of neutrophils to opsonized zymosan, a potent stimulating agent, revealed a rapid fall in activity when exposure was continued beyond 3 min. Exposure to zymosan under conditions in which the myeloperoxidase system was inactive (i.e., in the presence of myeloperoxidase inhibitors, or in the absence of oxygen) resulted in a substantial increase in the initial O(2) (-)-forming activity of particles from the zymosan-treated cells, but did not prevent the sharp fall in activity seen when zymosan exposure exceeded 10 min. The fall in activity was, however, prevented when activation took place in the presence of cytochalasin B (1.5 mug/ml), an agent thought to act largely by paralyzing the neutrophil through an interaction with its microfilament network.We conclude from these findings that the termination of the respiratory burst results at least in part from the inactivation of the particulate O(2) (-)-forming system. This inactivation involves at least two processes which probably act simultaneously. One is the destruction of the system through the action of myeloperoxidase. The other appears to require active cell motility and is independent of oxygen. The current view holds that the O(2) (-)-forming system of the neutrophil is located in the plasma membrane. It may be that the second process involves the internalization and degradation of this membrane-bound system.

Azides↗
PubMed1988

Functional characterization of macrophage receptors for in vitro phagocytosis of unopsonized Pseudomonas aeruginosa.

The phagocytic receptor for unopsonized Pseudomonas aeruginosa was characterized functionally using human monocyte-derived macrophages. Freshly isolated human peripheral blood monocytes were unable to ingest unopsonized P. aeruginosa; ingestion did not occur until the cells had been in culture for 2 d and it became maximal after 4 d. Macrophages plated on coverslips derivatized with anti-BSA IgG or with human gamma-globulin lost the capacity to phagocytose unopsonized P. aeruginosa, unopsonized zymosan, and EIgG but bound C3bi-coated erythrocytes normally. Each of the four human IgG subclasses and Fc fragments of anti-BSA IgG inhibited phagocytosis of both unopsonized P. aeruginosa and EIgG. Phagocytosis of P. aeruginosa and zymosan was markedly impaired and EIgG minimally inhibited if macrophages were plated on coverslips derivatized with mannan or when mannan was added to the phagocytosis buffer. Phagocytosis of P. aeruginosa and zymosan, and binding of EC3bi was dependent on the presence of divalent cations, but phagocytosis of EIgG was not. The macrophage phagocytic receptor for unopsonized P. aeruginosa was inactivated by proteolytic enzymes. Phagocytosis of P. aeruginosa was inhibited by D-mannose, L-fucose, and alpha methyl mannoside, but not by L-mannose, D-fucose, or D-glucose. The same sugars inhibited phagocytosis of unopsonized zymosan. We conclude that phagocytosis of unopsonized P. aeruginosa by human monocyte-derived macrophages is facilitated by mannose receptors.

Antibodies, Monoclonal↗
PubMedDate not supplied

[Phospholipid-deacylating activities of the mouse peritoneal macrophages during phagocytosis].

In the macrophages (M phi) obtained from mouse peritoneal exudates, five kinds of phospholipid-deacylating activities were detected using phosphatidylethanolamine (PE) and phosphatidylcholine (PC) labeled with [1-14C]oleic acid either in 1- or 2- position and 1- [1-14C]oleoyl-lysoPE, as substrates. Two types of phospholipipase A1 with pH optima of 4 to 6 and 8, respectively, and two types of phospholipase A2 activities with pH optima of 4 to 5 and 8, respectively, were identified. A detected lysophospholipase activity exhibited a broad pH optimum between 4 and 8. Both types of the phospholipase A1 and A2 of M phi hydrolyzed PE more than PC. Exogenously added Ca2+ did not increase the enzymatic activities. A comparison was made of three kinds of the M phi-phospholipid deacylating activities at pH8, after challenging the M phi with Mycobacterium lepraemurium, Escherichia coli, zymosan, or latex beads for 17 hours at 37 degrees C. The bacteria used to the phagocytosis were autoclaved. When the M phi were challenged with M. lepraemurium, the phospholipase A1, A2 and lysophospholipase activities were stimulated by about 160%, 150% and 140%, respectively. However, when challenged with E. coli, the phospholipase A1 activity remarkably decreased by about a third, although the phospholipase A2 activity was stimulated by about 150% that is similar to the challenge with M. lepraemurium. An inflammatory substance, zymosan seemed an effective inducer of the phospholipase A2, the enzymatic activity was remarkably stimulated by 260%, when challenged with 200 micrograms of zymosan. The increase in phospholipase A2 activity of the M phi pretreated with the bacteria or zymosan seems to result in an increase in the hydrolysis of arachidonic acid from the M phi-phospholipids to synthesize its inflammatory oxygenated metabolites. The lysophospholipase activity was not stimulated by the substances used to challenge the M phi, except for M. lepraemurium. No significant increase in three kinds of phospholipid-deacylating activities was observed after challenging the M phi with latex beads. It was considered from the above results that the M phi-phospholipid-deacylating activities at pH8 might be affected by sort of the ingested substances.

Animals↗
PubMed2001

Augmented TNF-alpha and IL-10 production by primed human monocytes following interaction with oxidatively modified autologous erythrocytes.

The presence of dysfunctional/damaged red blood cells (RBCs) has been associated with adverse clinical effects during the inflammatory response. The aim of this study was to elucidate whether oxidatively modified, autologous RBCs modulate monocyte cytokine responses in humans. Monocyte tumor necrosis factor alpha (TNF-alpha) and IL-10 production was measured in whole blood from healthy volunteers using ELISA and flow cytometry. Oxidatively modified RBCs (15 mM phenylhydrazine, 1 h, OX-RBC) or vehicle-treated RBCs (VT-RBC) opsonized by autologous serum were administered alone or in combination with one of three priming agents: E. coli lipopolysaccharide (LPS, 0.2 ng/ml), zymosan A (1 mg/ml), or phorbol 12-myristate 13-acetate (PMA, 50 ng/ml). OX-RBC or VT-RBC alone did not result in the release of TNF-alpha or IL-10. LPS, zymosan, and PMA caused marked and dose-dependent increases in TNF-alpha and IL-10 production. Addition of OX-RBC augmented the LPS-, zymosan-, and PMA-induced TNF-alpha release by approximately 100%. OX-RBC augmented LPS- and zymosan-induced IL-10 release by 400-600%. Flow cytometry analyses showed that monocytes were responsible for TNF-alpha and IL-10 production in whole blood. The presence of OX-RBC alone increased the complexity of CD14+ monocytes but caused no cytokine production. LPS alone induced cytokine production without altering cell complexity. After the combined (OX-RBC+LPS) treatment, monocytes of high complexity were responsible for TNF-alpha production. The presence of mannose or galactose (at 10-50 mM) did not alter the observed augmentation of cytokine production by OX-RBC, suggesting that lectin receptors are not involved in the response. These studies indicate that the interaction between damaged autologous erythrocytes and monocytes has a major impact on the cytokine responses in humans. An augmented cytokine production by the mononuclear phagocyte system may adversely affect the clinical course of injury and infections especially in genetic or acquired RBC diseases or after transfusions.

Adult↗
PubMed1992

Entamoeba histolytica alters arachidonic acid metabolism in macrophages in vitro and in vivo.

Entamoeba histolytica infections are associated with a state of transient suppression of cell-mediated immunity. Macrophages, the most important cells in host defence and control of invasive amoebiasis, in infected animals have been found to be deficient in effector functions and accessory cell potential. However, little is known of the cellular mechanisms responsible for the down-regulation. This study investigated whether macrophage dysfunction in amoebiasis is associated with altered macrophage arachidonic acid (AA) metabolism. Resident peritoneal macrophages (PMO) from naive gerbils produced enhanced levels of prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) in response to live E. histolytica trophozoites, to diffusible excretory/secretory products released from live amoebae in millicells and to freeze-thawed soluble amoebic proteins that were inhibitable by indomethacin (INDO) and nordihydroguaiaretic acid (NDGA), respectively. In contrast to PMO from naive gerbils, PMO from animals with amoebic liver abscesses at 10, 20 and 30 days post-infection (p.i.) released high basal levels of PGE2 and LTC4. In response to zymosan stimulation, PMO from infected animals produced two- and fourfold less PGE2 and LTC4, respectively, as compared to uninfected controls. AMO showed high constitutive basal release of PGE2 and LTC4. In response to amoebic and zymosan stimulation, AMO at 10 days p.i. produced significantly higher levels of PGE2 than AMO at 20 days p.i., while AMO at 30 days p.i. were refractory to stimulation to produce higher than basal levels of PGE2. Early (10 days) and late (20-30 days) AMO were refractory to amoebic and zymosan stimulation for enhanced LTC4 release. Pretreatment of AMO with AA substrate restored optimal PGE2 and LTC4 biosynthesis, but the cells were generally unresponsive to zymosan stimulation to produce augmented levels of LTC4. These results strongly suggest that intrinsic or secreted products or both from E. histolytica can induce profound alteration of eicosanoid formation in cyclooxygenase and lipoxygenase pathways in macrophages from naive and infected animals and that AA metabolism by AMO is sequentially modified during the course of the disease.

Animals↗
PubMed1991

[Experimental studies of proliferative vitreoretinopathy].

A new experimental rabbit model was developed to investigate vitreoretinal proliferation (PVR). PVR was initiated by injection of zymosan A from Saccharomyces cerevisiae into the vitreous. The experiments were performed in two groups. In group A zymosan was injected into the normal vitreous; in group B zymosan was injected after the vitreous body had been degraded by the previous injection of hyaluronidase. In group A only moderate phagozytotic activity was found up to the 5 h day. However, in group B excessive invasion of macrophages was observed within 20 h and phagozytotic activity increased markedly. This was confirmed by an increase of enzymatic activity of beta-n-acetyl-glucoseaminidase in the anterior chamber and in the vitreous space. Transmission electron microscopy revealed characteristic morphology in zymosan A, which can apparently only be digested very slowly in the phagocytes. Therefore, macrophages could be traced during their transformation into fibroblastlike cells forming the vitreoretinal membranes.

Acetylglucosaminidase↗