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PubMed1988

Characterization of murine bronchoalveolar macrophage respiratory burst: comparison of soluble and particulate stimuli.

Stimulation of the respiratory burst of murine bronchoalveolar macrophages obtained by lung lavage was studied using four different stimuli and different assay conditions. One soluble stimulus, phorbol myristate acetate (PMA), two intracellular particles, zymosan and Blastomyces dermatitidis conida, and one extracellular particle, B. dermatitidis yeast, were incubated with either freshly obtained macrophages in suspension or 2- and 48-hour macrophage monolayers. Suspension cultures were incubated with stimuli for 90 minutes and monolayers for 10 minutes before O2- was assayed. PMA did not elicit O2- production in macrophage suspensions or 2-hour macrophage monolayers, but 48-hour macrophage monolayers exhibited a 13-fold increase above control values (P = .0001). On the other hand, zymosan elicited an increase in O2- production in both suspensions and monolayers, although monolayers incubated for 48 hours produced almost fourfold more O2- than the other systems (P = .025). Opsonization had no effect on the ability of zymosan to elicit respiratory burst. B. dermatitidis conidia resulted in a two- to threefold increase in O2- production in macrophage suspensions and a five- to eightfold increase in 48-hour monolayers, representing significantly less respiratory burst stimulation than with either zymosan or PMA. Similarly, B. dermatitidis yeasts demonstrated similar submaximal stimulation of O2-, 3-4 times that over control values, and again this was less than zymosan and PMA. We conclude that 1) freshly obtained murine bronchoalveolar macrophages do not respond to PMA with an increase in O2- production, but that responsiveness is evident after 48 hours of incubation in monolayers; 2) B. dermatitidis conidia and yeasts do not stimulate respiratory burst activity to the same degree as zymosan or PMA; and 3) opsonization of zymosan is not necessary for stimulation of the murine bronchoalveolar macrophage oxidative burst, confirming previous data that functional complement receptors are not present on these cells.

Animals↗
PubMed1991

Polyphosphoinositide metabolism in polymorphonuclear cells from healthy and thermally injured rats: effect of the immunomodulator RU 41740.

Burn trauma is associated with alterations of various components of host defenses, including impaired neutrophil functions. In an animal model of experimental thermal injury, we studied if the modifications of cellular reactivity result from alterations in signalling systems by comparing polyphosphoinositide breakdown, particularly the production of inositol phosphates (IP, IP2 IP3), in healthy and burned rat polymorphonuclear neutrophil leukocytes (PMNs). Neutrophil activators such as N-formyl-methionyl-leucyl-phenylalanine (fMLP) and serum-opsonized zymosan increased in vitro production of inositol phosphates in PMNs from healthy rats. The immunomodulator RU 41740 had no effect by itself, but decreased the stimulating effect of fMLP and zymosan. In PMNs from burned rats, the stimulating effects of fMLP and zymosan were decreased, while RU 41740 stimulated inositol phosphate generation. In vivo treatment with RU 41740 inhibited the activation of phosphoinositide metabolism by fMLP or zymosan in healthy rat PMNs. Similar treatment of burned rats after injury restored the stimulating effect of fMLP and zymosan on inositol phosphate accumulation in PMNs. Thus, RU 41740 can modulate fMLP and zymosan receptor-mediated signal transduction, inducing an attenuation of the phosphatidylinositol hydrolysis response. After burn injury, when the activating effects of fMLP and zymosan are inhibited, RU 41740 can, on the contrary, stimulate phospholipase C-mediated polyphosphoinositide turnover and the formation of intracellular messengers such as IP3. These data show that RU 41740 has different effects on polyphosphoinositide metabolism in rat PMNs, according to the physiological and pathological state of the animals. Interestingly, it has a beneficial action on the post-burn decrease in PMN reactivity.

Animals↗
PubMed1998

A monoclonal antibody against tumour necrosis factor-alpha improves survival in experimental multiple organ dysfunction syndrome.

A single intraperitoneal administration of zymosan induces multiple organ dysfunction syndrome (MODS) in C57BL/6 mice. The authors investigated the effect of a monoclonal antibody V1q against murine tumour necrosis factor alpha (TNF-alpha) on the development of zymosan-induced MODS and on plasma concentrations and the production capacity of interleukin 6 (IL-6) by peritoneal cells. C57BL/6 mice received doses of V1q starting either simultaneously with administration of zymosan every four days, or from 4 or 8 days after administration of zymosan onwards. The animals were monitored for survival, condition, and body weight and temperature. Twelve days after zymosan all surviving animals were killed to obtain plasma, organs and peritoneal cells. Plasma concentrations of IL-6 and lipopolysaccharide-stimulated production of IL-6 by peritoneal cells were measured; organs were weighed as an indicator for organ damage and lung damage was assessed macroscopically. Survival improved when the animals were treated with V1q starting at either time point, and a subpopulation developed from the group receiving V1q from day 0 onwards that displayed improved body weight and temperature when compared to the animals receiving zymosan only. Also, the wet organ weights improved in this subgroup, indicating a beneficial effect of the monoclonal antibody. However, V1q administered could neither decrease the circulating IL-6 concentrations toward control values, nor did V1q treatment normalize IL-6 production capacity (stimulated or unstimulated). The development of zymosan-induced MODS can be attenuated by the monoclonal antibody V1q.

Animals↗
PubMed1984

Measurement of the respiratory burst in human monocytes and polymorphonuclear leukocytes by nitro blue tetrazolium reduction and chemiluminescence.

We compared measurements of chemiluminescence (CL) assessing the rate of production of oxygen intermediates at a given instance, and of nitro blue tetrazolium (NBT) reduction detecting total superoxide produced during the assay period, for the assessment of the respiratory burst of both human monocytes (M phi) and polymorphonuclear leukocytes (PMN). In the CL experiments, opsonized and non-opsonized zymosan was used to stimulate peripheral blood M phi and PMN. Opsonized zymosan yielded an earlier and 2-fold higher peak response than non-opsonized zymosan. The stimulatory action of zymosan in CL varied with the time of incubation and depended on the concentration of cells and zymosan. No light generation was observed in the absence of viable cells. In contrast to NBT reduction, incubation of PMN with dextran sulfate did not result in increased light generation but rather in a quenching of the response in CL. Opsonized zymosan also yielded a higher level of NBT reduction than non-opsonized zymosan. Comparing NBT reduction with CL in 21 healthy individuals, with 2 X 10(5) M phi and 2.5 X 10(5) PMN for NBT and 5 X 10(5) PMN for CL tests, we found that NBT brings about spontaneous oxidative metabolism, possibly reflecting the intracellular compartment, the chemiluminescent response of resting cells being only marginal. The data suggest higher sensitivity of the CL method. These results provide useful comparative data for 2 established methods used to document the respiratory burst in phagocytic cells.

Adolescent↗
PubMed1991

Activation of phospholipase C is not correlated to the formation of prostaglandins and superoxide in cultured rat liver macrophages.

This study evaluates the role of inositol phosphates as possible mediators of the activation of phospholipase A2 and NADPH oxidase in cultured rat liver macrophages. Inositol phosphate formation was achieved by zymosan, immune complexes, latex particles and calcium ionophore while the release of arachidonic acid and the formation of prostaglandin E2 was also elicited by phorbol ester and NaF, but not by latex particles; generation of superoxide was obtained by zymosan and phorbol ester only. The kinetics of the formation of inositol phosphates revealed that within the first few minutes after zymosan addition inositol trisphosphate was formed, followed by inositol bisphosphate and inositol monophosphate. Pre-treatment of the cells with dexamethasone or removal of extracellular calcium led to an inhibition of the zymosan-induced formation of inositol phosphates and prostaglandin E2 but had no effect on the generation of superoxide; inhibition of the Na+/H+ exchanger by removal of extracellular sodium ions led to a decrease of the zymosan-induced synthesis of prostaglandin E2, but did not affect the formation of inositol phosphates and superoxide. Pre-treatment of the cells with phorbol ester decreased the zymosan-induced synthesis of prostaglandin E2 and superoxide, but even enhanced the zymosan-induced formation of inositol phosphates. These data indicate that in cultured rat liver macrophages the formation of prostaglandins and superoxide cannot be correlated to an activation of phospholipase C.

Animals↗
PubMed1997

Loading with oxidised low density lipoprotein alters endocytic and secretory activities of murine macrophages.

Lipid-loaded macrophages were produced in vitro by incubation with acetylated or copper-oxidized LDL. In order to establish whether cellular membrane traffic is generally perturbed by such loading, we assessed endocytosis of fluid; cell surface binding, internalisation and degradation of a soluble ligand and of a particulate preparation; and exocytosis of lysosmal enzymes. Fluid-phase pinocytosis of sucrose was unaffected by either form of loading. Binding, uptake and degradation of soluble (mannosylated-BSA) and particulate (zymosan) ligands by these lipid-loaded and by non-loaded cells were compared. Loading with oxidized LDL decreased the processing of both ligands, while loading with acetylated LDL had little effect. Loading with oxidized LDL (Ox-LDL) also decreased zymosan binding at 4 degrees C; and the internalisation and degradation of ligands in Ox-LDL loaded and non-loaded cells reflected the extent of surface binding. Changes in binding and uptake of mannosylated-BSA and zymosan were not due to changes in viability or cell number. Zymosan stimulated release of lysosomal beta-N-acetyl-D-glucosaminidase from the cells. Loading with Ox- but not Ac-LDL decreased beta-N-acetyl-D-glucosaminidase secretion. After incubation with zymosan, intracellular levels of the enzyme were increased in the Ox-LDL loaded cells. Zymosan uptake and beta-N-acetyl-D-glucosaminidase secretion were correlated, but enzyme activity per culture rose more in the absence than in the presence of zymosan. We conclude that membrane traffic is perturbed in model foam cells, particularly those loaded with Ox-LDL.

Acetylglucosaminidase↗
PubMed1991

Complement activation retards resolution of acute ischemic renal failure in the rat.

We investigated the role of complement activation on the resolution of acute ischemic renal failure in the rat. Acute renal failure was induced by clamping of the renal arteries of Sprague-Dawley rats for 45 minutes (Day 0). On subsequent days, groups of rats with acute renal failure were exposed to daily zymosan infusion (an activator of the complement system), or to blood incubated with cuprophane (CUP) or polyacrylonitrile (PAN) dialysis membranes. We serially measured the change in BUN daily, glomerular filtration rate and 24-hour proteinuria on Day 3 and Day 5 following ischemia. On Day 6, the animals were sacrificed and their kidneys examined histologically. Zymosan and cuprophane exposed rats had a significant delay in the recovery of renal failure, reduced glomerular filtration rate, and histologically had more neutrophil infiltration than control or PAN exposed animals. To investigate the potential pathophysiology of these observations, we assessed the response of zymosan-exposed rats to infusion of deferoxamine (DFO), a potent inhibitor of hydroxyl radical formation (OH.). Infusion of DFO prior to zymosan significantly improved recovery of renal function. We also measured urinary thromboxane B2 levels in these groups of rats. While the groups of rats exposed to zymosan had the highest levels of thromboxane B2, these levels were not different between the groups exposed to zymosan alone, or to zymosan and DFO. These observations suggest a role for hydroxyl radicals in the prolongation of renal failure in this model. Taken together, these findings may have implications for the dialytic intervention in patients with acute renal failure.

Acute Kidney Injury↗
PubMed1978

Regulation of prostaglandin synthesis and of the selective release of lysosomal hydrolases by mouse peritoneal macrophages.

Macrophages isolated from the peritoneal cavity of untreated mice and maintained in tissue culture synthesize and release prostaglandins when challenged with zymosan. These cells also selectively release lysosomal acid hydrolases under the same conditions. The major prostaglandins released into the media are found to be prostaglandins E1, E2 and 6-oxoprostaglandin F1a, whereas prostaglandin F2a is not detected. Macrophages isolated from mice that have received an intraperitoneal injection of thioglycollate broth are far less responsive to zymosan challenge. These cells require 300 microgram of zymosan to synthesize and release one-third the amount of prostaglandins released from non-stimulated macrophages exposed to 50 microgram of zymosan. In addition, thioglycollate-stimulated macrophages release less than 10% of their lysosomal acid hydrolases when exposed to 300 microgram of zymosan whereas non-stimulated cells release approximately 50% of these enzymes after treatment with 50 microgram of zymosan. The zymosan-stimulated synthesis and release of prostaglandins are completely inhibited by indomethacin, whereas the increased selective release of lysosomal acid hydrolases is not affected. Macrophages, unlike fibroblasts, do not synthesize and release prostaglandins when exposed to serum or to bradykinin.

Animals↗
PubMed1987

Prostaglandin release but not superoxide production by rat Kupffer cells stimulated in vitro depends on Na+/H+ exchange.

The release of the prostaglandins E2 and D2, induced by zymosan and phorbol ester in cultured rat Kupffer cells, was found to depend on the extracellular concentration of Na+. Eicosanoid formation following the administration of the Ca2+ ionophore A23187 or of arachidonic acid, however, did not require the presence of sodium ions in the medium. A half-maximal rate of prostaglandin release by zymosan-treated Kupffer cells was obtained between 4 mM and 5 mM Na+; and a Na+ concentration of greater than or equal to 30 mM was required to maximally stimulate prostaglandin E2 and D2 formation in the cultured liver macrophages. In contrast, the superoxide production following the administration of zymosan or of phorbol ester was quite independent of extracellular Na+. The zymosan and phorbol-ester-stimulated release of prostaglandins E2 and D2 was inhibited by amiloride. Artificial intracellular alkalization enhanced the prostanoid production of unstimulated and of zymosan-stimulated cells whereas artificial intracellular acidification inhibited the zymosan-elicited prostaglandin synthesis. In contrast, the superoxide formation was independent of the pH changes. The data presented here suggest that the prostaglandin production elicited by zymosan or phorbol ester in cultured rat Kupffer cells requires an activated Na+/H+ exchange.

Amiloride↗
PubMed1994

Differential effects of the PAF receptor antagonist UK-74,505 on neutrophil and eosinophil accumulation in guinea-pig skin.

1. The effect of the dihydropyridine, platelet activating factor (PAF) receptor antagonist, UK-74,505, on leucocyte accumulation and oedema formation in guinea-pig skin was investigated. The inflammatory reactions studied were elicited by exogenous mediators, a passive cutaneous anaphylactic (PCA) reaction and zymosan particles. 2. Leucocyte accumulation and oedema formation were measured as the local accumulation of i.v. administered 111In-labelled neutrophils or eosinophils together with 125I-labelled albumin. UK-74,505 was either administered i.v. or used to pretreat the radiolabelled leucocytes in vitro prior to their last wash and injection into recipient animals. 3. In vitro, UK-74,505 inhibited PAF-induced elevations in cytoplasmic levels of Ca2+ ([Ca2+]i) in fura-2-loaded guinea-pig neutrophils and eosinophils with IC50 values of 10(-9) M and 7 x 10(-9) M respectively. Neutrophils and eosinophils pretreated with 10(-7) M and 10(-6) M UK-74,505 respectively, and maintained at 37 degrees C, were unresponsive to PAF for the 4 h period investigated. 4. In vivo, using 2 h test periods, i.v. UK-74,505 (0.5 and 2.5 mg kg-1) inhibited the accumulation of 111In-neutrophils, 111In-eosinophils and oedema formation induced by intradermal PAF, but had no effect on responses elicited by leukotriene B4 (LTB4) and zymosan-activated plasma (ZAP, used as a source of C5a des Arg). UK-74,505 (2.5 mg kg-1) was also without an effect on response induced by a PCA reaction but significantly suppressed the 111In-eosinophil accumulation following the intradermal administration of zymosan particles. The 111In-neutrophil accumulation induced by zymosan particles was not, however, affected by UK-74,505. 5. In a second series of in vivo experiments, "'In-leucocytes were pretreated in vitro with UK-74,505 prior to their last wash and injection into recipient animals. Radiolabelled neutrophils, and eosinophils were pretreated with 10-7 M and 10-6 M UK-74,505 respectively, concentrations previously shown to block the leucocyte responses to PAF in vitro for up to 4 h. The in vitro pretreatment of the cells with the PAF antagonist, whilst not affecting the responses to intradermally-injected PAF, suppressed the"'In-eosinophil accumulation response induced by zymosan particles.6. The results of this study indicate that PAF is not involved in neutrophil accumulation, eosinophil accumulation and oedema formation induced by LTB4, ZAP and a PCA reaction. Endogenous PAF does, however, appear to have a role in zymosan-induced eosinophil accumulation but not neutrophil accumulation, suggesting the existence of different inflammatory pathways in the induction of neutrophil and eosinophil accumulation in vivo. Furthermore, while leucocyte accumulation induced by exogenous PAF does not appear to involve leucocyte PAF receptors, the mechanism by which endogenous PAF mediates the zymosan-induced eosinophil accumulation appears dependent on the expression of PAF receptors on eosinophils.

Animals↗
PubMed1996

Comparative study of neutrophil activation by chemiluminescence and flow cytometry.

Neutrophil activation by phorbol 12-myristate 13-acetate (PMA) and zymosan was assessed by luminol-dependent chemiluminescence (CL) in a novel microtiter plate format and by flow cytometry (FC) based on the oxidation of dihydrorhodamine 123. The results of this comparison demonstrated striking differences in kinetic parameters between these two techniques for neutrophil activation by PMA and zymosan. PMA activation, as determined by FC, was found to be an all-or-none phenomenon in that below a critical concentration of PMA, few cells were positive. Above this concentration, almost all cells were positive; however, the fluorescence intensity of positive cells increased with an increasing PMA concentration until a plateau (maximal) level was reached. In contrast, increasing zymosan concentrations resulted in proportionate increases in the percentage of positive cells until close to 100% of cells were positive. However, the fluorescence intensity of positive cells remained about the same. CL activity increased proportionately with either PMA or zymosan concentration until a maximal level was achieved. The concentration of PMA required for half-maximal activity was about 10-fold higher for FC than for CL, whereas the analogous concentration of zymosan was about 30-fold higher for CL than for FC. In addition, opsonization had only a small negative effect on the ability of zymosan to activate neutrophils, as determined by FC, whereas it had a very large enhancing effect when determined by CL. The differences in kinetic parameters of activation suggest differential sensitivity to particulate (zymosan) versus soluble (PMA) stimulants for FC and CL.

Evaluation Studies as Topic↗
PubMed1984

Influence on plasma beta-N-acetyl-D-glucosaminidase of reticuloendothelial stimulation and depression: an experimental study in rats.

Plasma levels of the lysosomal enzyme, beta-N-acetyl-D-glucosaminidase (EC 3.2.1.30; 2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase ) were estimated 120 minutes after intravenous injection of zymosan in rats. Different groups of animals were investigated according to pretreatment with agents influencing reticuloendothelial activity. Pretreatment for two days with the reticuloendothelial stimulating agent, zymosan, or the reticuloendothelial suppressing agent, methyl palmitate, did not influence the basal plasma levels of beta-N-acetyl-D-glucosaminidase. In all groups after zymosan injection, plasma beta-N-acetyl-D-glucosaminidase activity was increased in comparison with basal levels. The increase of enzyme activity was most pronounced after pretreatment with zymosan and differed significantly from enzyme activity after pretreatment with methyl palmitate. Alcohol administration in combination with zymosan did not cause a further rise of beta-N-acetyl-D-glucosaminidase activity in normal rats nor in rats pretreated with zymosan. The observations suggest that plasma levels of beta-N-acetyl-D-glucosaminidase in rats after a standardized zymosan injection are related to the functional status of the reticuloendothelial system (RES).

Acetylglucosaminidase↗
PubMed2001

Serum amyloid P component and C-reactive protein mediate phagocytosis through murine Fc gamma Rs.

The pentraxins, serum amyloid P component (SAP) and C-reactive protein (CRP) are acute-phase serum proteins in mice and humans, respectively. Although SAP binds to DNA and chromatin and affects clearance of these autoantigens, no specific receptor for SAP has been identified. CRP is an opsonin, and we have shown that it binds to FcgammaR. Mice deficient in FcgammaR were used to assess the role of these receptors in phagocytosis by pentraxins using zymosan as a ligand. Phagocytosis of zymosan by bone marrow macrophages (BMM) was enhanced by opsonization with SAP or CRP. BMM from mice deficient in all three FcgammaR or in gamma-chain ingested unopsonized zymosan, but phagocytosis of SAP- or CRP-opsonized zymosan was not enhanced. SAP binding to BMM from gamma-chain-deficient mice was also greatly reduced, indicating little or no binding of SAP to FcgammaRII. SAP and CRP opsonized zymosan for phagocytosis by BMM from mice deficient in FcgammaRII or FcgammaRIII. SAP, but not CRP, opsonized zymosan for uptake by neutrophils that express only low levels of FcgammaRI. Together these results indicate that FcgammaRI and FcgammaRIII are receptors for SAP in the mouse. Opsonization of zymosan by CRP is mediated through FcgammaRI. Pentraxins are major proteins of the innate immune system and arose earlier in evolution than Igs. The use of FcgammaR by the pentraxins links innate and adaptive immunity and may have important consequences for processing, presentation, and clearance of the self-Ags to which these proteins bind.

Adjuvants, Immunologic↗
PubMed1984

Release of leukotrienes by human monocytes on stimulation of their phagocytic receptor for particulate activators.

The respective capacities of adherent human monocytes to metabolize endogenous arachidonic acid into leukotrienes C4 (LTC4) and B4 (LTB4) in response to activation with an ionophore, A23187, or to phagocytosis of unopsonized zymosan particles and IgG-sensitized sheep erythrocytes ( EsIgG ) were compared under optimal conditions for each stimulus. Resolution of the cellfree supernatant, after ionophore activation, by reverse-phase high performance liquid chromatography (RP-HPLC) identified only two products which eluted at the retention times of LTC4 and LTB4. There was correspondence between their quantitation by integrated optical density and radioimmunoassay, and the recoveries from the initial supernatant were 80% by radioimmunoassay. Activation of adherent monocytes from 12 donors by ionophore and by zymosan particles released 68.1 ng and 10.0 ng LTB4 and 29.5 ng and 2.1 ng LTC4, respectively. With trypsin pretreatment, the monocytes responded fully to ionophore activation but were inhibited in their response to zymosan particles as assessed by phagocytosis and leukotriene release, indicating that the zymosan stimulus acted through a trypsin-sensitive membrane receptor. When the response of adherent monocytes from nine donors to zymosan particles and to EsIgG were compared at identical particle concentrations and with similar numbers of ingesting monocytes, zymosan elicited LTB4 release (mean 6.7 ng) from all and LTC4 (mean 1.5 ng) from eight donors, while EsIgG caused low level release of LTB4 (mean 0.7 ng) from six and LTC4 from only one of the donors. Neither zymosan nor ionophore stimulation led to the metabolism of exogenously added [3H]LTB4 or [3H]LTC4 as assessed by RP-HPLC of the cellfree supernatants and by quantitation of the eluted labeled products. Thus, transmembrane activation of adherent monocytes by their receptor for particulate activators, in contrast to stimulation of their IgG-Fc receptor, reproducibly releases substantial quantities of LTB4 and LTC4, and may represent an important mechanism for regulating the microenvironment in the nonimmune host.

Arachidonic Acid↗
PubMed1994

Cooperation between Fc gamma receptor II and complement receptor type 3 during activation of platelet-activating factor release by cytokine-primed human eosinophils.

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.

Cytokines↗
PubMed1992

Production of leukotrienes and thromboxane by resident and activated rat alveolar macrophages: a possible role of protein kinase C.

Arachidonic acid metabolism in resident rat alveolar macrophages and in those activated with complete Freund's adjuvant (CFA) was studied. Adult Sprague-Dawley rats were injected with 0.05 ml CFA, and macrophages were harvested 10 days later. Macrophages were labeled overnight with carbon 14-labeled arachidonic acid, washed, and then stimulated with calcium ionophore A23187 (IoA), phorbol myristate acetate (PMA), or zymosan for 30 minutes. Prostaglandins, thromboxane, and leukotrienes were extracted from the medium and analyzed by radioimmunoassay or radio high-pressure liquid chromatography. Cell lipids were analyzed by radio thin-layer chromatography. Medium and cell beta-glucuronidase activity and protein kinase C activity of the membrane fraction were also assayed. We found (1) lower leukotriene B4 (LTB4) production in stimulated resident macrophages when compared with resident macrophages after IoA stimulation--the suppressed LTB4 production was reversed by PMA; (2) unchanged or higher LTB4 production in activated macrophages when compared with resident macrophages after zymosan stimulation; (3) inhibition of zymosan-stimulated LTB4 production by staurosporine, a protein kinase C inhibitor, in both groups; and (4) lower diacylglycerol (DAG) production in activated macrophages when compared with resident macrophages after IoA stimulation, but not after zymosan stimulation. These results suggest that the reduced response of activated macrophages to IoA is due to decreased production of an endogenous protein kinase C activator. This hypothesis was further supported by the observation that protein kinase C activation in response to IoA was lower in activated macrophages than in resident macrophages. In contrast, zymosan stimulation resulted in higher protein kinase C activation in activated macrophages when compared with resident cells. We hypothesize that protein kinase activation is necessary for leukotriene production and that the preserved ability of zymosan to activate PKC via DAG accounts for the high leukotriene production in zymosan-activated macrophages. We also found that stimulated thromboxane production was higher in activated than resident cells, regardless of the stimulus, and that thromboxane production was not affected by staurosporine. Thus alterations of eicosanoid metabolism in immunologically activated macrophages depend on the stimulus used and the type of eicosanoid examined. Furthermore, leukotriene biosynthesis in rat alveolar macrophages may be regulated by protein kinase C.

Animals↗
PubMed1986

Isolation of soluble yeast beta-glucans that inhibit human monocyte phagocytosis mediated by beta-glucan receptors.

The trypsin-sensitive receptor that mediates phagocytosis of unopsonized zymosan particles by human monocytes has been designated as a beta-glucan receptor because of its functional inhibition by specific algal and plant beta-glucans. Soluble ligands that are chemically and structurally identical to beta-glucan constituents of zymosan were isolated from a carbohydrate-enriched fraction of yeast extract by sequential chromatography on DE-cellulose, SP-Sephadex, and Con A-Sepharose. Preincubation of adherent human monocytes with 278, 210, and 2.5 micrograms/ml hexose equivalents in pooled chromatographic fractions from DE-cellulose, SP-Sephadex, and Con A-Sepharose, respectively, effected 50% reductions in subsequent phagocytosis of zymosan particles without affecting Fc-mediated ingestion of IgG-coated sheep erythrocytes (ESIgG). The purified yeast extract-derived beta-glucans, which contained 92% glucose and 8% mannose by gas chromatographic analysis and eluted from a Sephacryl S-200 column as a broad peak with a Kav of 0.39 and estimated molecular sizes of from 20,000 to 70,000 m.w., required only 3.5 +/- 0.9 micrograms/ml (mean +/- SD, n = 6), as compared with 31.5 micrograms/ml of the algal beta-glucan laminarin to achieve 50% decreases in zymosan ingestion. Alternatively, soluble yeast beta-glucans with estimated molecular sizes of from 2 X 10(5) to 2 X 10(6) were prepared from yeast glucan particles, which contained 98% glucose and 0% mannose, by sonication and sequential centrifugation at 15,000 and 100,000 X G for 30 and 60 min, respectively. Monocyte ingestion of zymosan was reduced by 50% by pretreatment with 60 ng/ml of the soluble beta-glucans in 15,000 X G supernatants, whereas ingestion of ESIgG was unaffected by as much as 50 micrograms/ml of this material. Partial acid hydrolysis of soluble glucan-derived beta-glucans in 15,000 X G supernatants followed by gel filtration on Bio-Gel P-4 revealed two well-defined peaks within the inclusion volume of the column with phagocytosis-inhibiting activity. Oligoglucosides that eluted at a Kav of 0.46 had an estimated molecular size of 2,000 m.w. and effected a 48% reduction in zymosan ingestion at inputs of 2 to 5 micrograms/ml, and smaller oligoglucosides with a Kav of 0.82 and an estimated molecular size of 1,000 m.w. effected a 50% reduction at inputs of 25 micrograms/ml. Preincubation of monocytes for 2 min with 25 micrograms/ml of the oligoglucosides with estimated molecular size of 1,000 m.w. and with 50 ng/ml of soluble glucan-derived beta-glucans in 100,000 X G supernatants reduced zymosan ingestion by 41% +/- 4 and 44% +/- 3 (mean +/- SD, n = 3), respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Complement Pathway, Alternative↗
PubMedDate not supplied

Quantification of C5a/C5a(desArg) in bovine plasma, serum and milk.

Complement activation generates two potent inflammatory mediators from C5, C5a and its derivative C5a(desArg), which results from the removal of the C-terminal arginine by ubiquitous carboxypeptidases. In this paper we describe the purification of milligram amounts of bovine C5a(desArg) by a simplified procedure, and the preparation of mouse monoclonal antibodies (MAbs) to C5a/C5a(desArg) which do not recognize native C5. A MAb was used to develop a sandwich ELISA which made it possible to quantify levels of C5a/C5adesArg in bovine biological fluids. Small amounts (means +/- SEM) of C5a/C5a(desArg) were found in EDTA-plasma (0.58 +/- 0.06 ng.mL-1). The anticoagulant EDTA was more efficient than citrate or heparin in inhibiting in vitro activation of the complement system. Complement activation occurred during coagulation since the baseline concentration of C5a/C5a(desArg) (15.4 +/- 4.1 ng.mL-1) was higher than in plasma. Zymosan, a potent activator of the complement cascade, was used to generate C5a/C5a(desArg). The time-course of the reaction and the dose-effect of zymosan were investigated. Optimal conditions were incubation at 39 degrees C for 1 or 2 h with 2 mg of zymosan per mL of serum. The maximal concentration of C5a/C5a desArg attained in zymosan-activated serum was 4.28 +/- 0.14 micrograms.mL-1. Normal milk (from healthy, uninflamed mammary glands) contained on average 0.12 ng of C5a/C5a(desArg).mL-1 (range 0.02-0.19 ng.mL-1). The maximal amount of C5a/C5a(desArg) which was generated in milk with zymosan was 1.1 ng.mL-1 (range 0.68-2.17 ng.mL-1). In milk from quarters with subclinical infections by coagulase-negative staphylococci, values were 0.18 ng.mL-1 and 2.37 ng.mL-1 for spontaneous and zymosan-generated C5a/C5a(desArg) concentrations, respectively. In milk from Escherichia coli endotoxin-induced mastitis, C5a/C5a(desArg) concentrations (means of four cows) before and after zymosan activation reached 6.5 ng.mL-1 and 55 ng.mL-1, respectively. These results indicate that a C5-convertase can operate in normal milk, that only minute amounts of C5a/C5a(desArg) can be generated (less than 1/1,000 of plasma potential), but that much higher concentrations are reached in milk during endotoxin-induced inflammation. The ELISA made it possible to determine normal ranges of C5a/C5a(desArg) in bovine blood plasma and in milk, and is a valuable tool to define the variations of its concentrations in exudates during inflammatory reactions.

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