A beta-glucan inhibitable receptor on human monocytes: its identity with the phagocytic receptor for particulate activators of the alternative complement pathway

The ligand specificity of the human monocyte receptor that mediates phagocytosis of particulate activators of the human alternative complement pathway was defined by inhibiting the phagocytic response with glycans known to be present in zymosan. When monocytes in monolayers were preincubated with 100 micrograms/ml of beta-glucan and then incubated with 1.25 to 2.5 X 10(6) zymosan particles, the percentage of cells that exhibited phagocytosis was inhibited in a time-dependent manner; maximal inhibition occurred within 20 min of preincubation. beta-Glucan inhibited monocyte phagocytosis of zymosan and rabbit erythrocytes (Er) in a similar dose-dependent fashion and at 100 micrograms/ml reduced monocyte ingestion of 5 X 10(6)/ml zymosan and 2 X 10(8)/ml Er by 63 +/- 8% and 68 +/- 16% (mean +/- SD, n = 3), respectively. The other glycan constituent of zymosan, mannan, was less than 1% as active, and 10 mg/ml of mannan reduced the number of monocytes ingesting zymosan and Er by 56 +/- 12% and 26 +/- 11%, respectively. At concentrations as high as 500 micrograms/ml, beta-glucan had no effect on monocyte Fc, C3b, or fibronectin receptor-mediated functions. Enzymatic hydrolysis of beta-glucan and alpha-mannan with beta-glucosidase or beta-glucanase before their incubation with monocytes abrogated their inhibitory capacity, whereas hydrolysis with alpha-mannosidase or alpha-glucosidase did not. Neither of the two alpha-glucans tested (dextran T-70 and nigeran) affected monocyte ingestion of zymosan particles or sheep erythrocytes (Es) sensitized with rabbit 7S anti-Es (EsIgG) at concentrations as high as 2 mg/ml. In contrast, a number of beta-glucans were active against zymosan but not EsIgG ingestion with a 75% reduction in the number of monocytes ingesting zymosan occurring with 100 micrograms/ml laminarin, 500 micrograms/ml soluble pachyman, and 900 micrograms/ml of soluble pustulan. The galactan, agarose, either in suspensions at 2 mg/ml or in a soluble portion at 600 micrograms/ml failed to affect monocyte ingestion of zymosan particles or Er. Thus, the monocyte receptor for particulate activators that is specifically inhibited by beta-glucan at a rate compatible with a phagocytic process and that recognizes beta-glucans but not alpha-glucans, mannan, or galactan is a beta-glucan receptor.     

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)     

Properties of glycans that activate the human alternative complement pathway and interact with the human monocyte beta-glucan receptor

The glycans used in an earlier study to define the ligand specificity of the human monocyte phagocytic receptor for unopsonized particulate activators were assessed for their capacities to activate the proteins of the human alternative complement pathway. Normal human serum was preincubated with glycans under conditions of chelation to prevent activation of the classical complement pathway, and the activation-depletion of the alternative complement pathway was determined by the subsequent capacity of the serum to lyse rabbit erythrocytes (Er). When serum was preincubated at a 1/2 dilution in 8 mM EGTA/2 mM Mg with increasing numbers of yeast glucan or zymosan particles, and was evaluated at final serum dilutions of 1/8, its capacity to lyse Er was found to be reduced by 50% with 1.9 X 10(6)/ml yeast glucan particles and 1.4 X 10(6)/ml zymosan particles. At 2 mg/ml of serum diluted 1/2 in 8 mM EGTA/2 mM Mg, nonturbid preparations of mannan, laminarin, or pyrogen-free inulin and turbid suspensions of cellulose, Sephadex, agarose, or purified inulin failed to activate the alternative complement pathway. In contrast, activation-depletion of the alternative pathway was induced by turbid preparations of crude inulin, nigeran, pachyman, barley beta-glucan, and pustulan, which at 700 micrograms/ml, 500 micrograms/ml, 350 micrograms/ml, 60 micrograms/ml, and 27 micrograms/ml, respectively, effected 50% reductions in the subsequent lysis of Er. After centrifugation of 2 mg/ml suspensions of barley beta-glucan at 1100 X G for 5 min and at 15,000 X G for 15 min, the supernatants contained 90 to 92% and 65% of the barley beta-glucan, respectively, as determined by the anthrone method. On a weight basis, the 1100 X G supernatant exhibited the same capacity to activate the alternative pathway as the corresponding original suspension, whereas the 15,000 X G supernatants had less than 3% of the original anti-complementary activity. Preincubation of adherent human monocytes with increasing concentrations of barley beta-glucan suspensions, 100,000 X G supernatants containing 64% of the original beta-glucan, and laminarin all decreased subsequent ingestion of 1.25 X 10(6) zymosan particles in a dose-related fashion. The numbers of monocytes from three different donors phagocytosing zymosan were reduced by 50% after pretreatment with 30 to 65 micrograms/ml, 25 to 48 micrograms/ml, and 12 to 15 micrograms/ml of barley beta-glucan suspensions, 100,000 X G supernatants of barley beta-glucan, and laminarin, respectively, even though the latter two preparations were fully soluble and had no capacity to activate the alternative pathway.(ABSTRACT TRUNCATED AT 400 WORDS)     

Perturbation of beta-glucan receptors on human neutrophils initiates phagocytosis and leukotriene B4 production

Normal human neutrophils were stimulated with the yeast cell wall product, zymosan, and examined for two biologic responses, ingestion of particles and production of leukotriene B4 (LTB4), under conditions that were comparable and optimal for the quantitation of each response. Monolayers of adherent neutrophils ingested unopsonized zymosan particles, at particle-to-cell ratios of 12.5:1 to 125:1, in a dose- and time-related manner. At a ratio of 125:1, the percentages of neutrophils ingesting greater than or equal to 1 and greater than or equal to 3 zymosan particles reached plateau levels of 55 +/- 6 and 32 +/- 9% (mean +/- SD, n = 8), respectively, within 30 min. At this same ratio, neutrophils during gravity sedimentation with zymosan particles synthesized LTB4 in a time-dependent manner for at least 45 min. The maximum amount of immunoreactive LTB4 released into supernatants was 3.8 +/- 1.2 ng per 10(6) neutrophils (mean +/- SD, n = 5) and the corresponding total immunoreactive LTB4 was 6.2 +/- 1.9 ng per 10(6) neutrophils. Treatment of 2 x 10(7) suspended neutrophils with 250 micrograms of trypsin for 20 min before concurrent assessment of neutrophil phagocytosis and LTB4 production reduced both of these responses by about 50%. Pretreatment of neutrophils with 800 micrograms/ml of soluble yeast beta-glucan inhibited their ingestion of zymosan by 84% (mean +/- SD, n = 3), with 50% inhibition occurring with 100 micrograms/ml of soluble beta-glucan; 800 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. Pretreatment of neutrophils with 400 micrograms/ml of soluble yeast beta-glucan inhibited neutrophil synthesis of LTB4 by 90%, with 50% occurring with 200 micrograms/ml; 400 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. The presence of 1.25 micrograms/ml of cytochalasin B during incubation with zymosan particles reduced neutrophil phagocytosis from 65 to 6%, and neutrophil synthesis of LTB4 from total levels of 6.0 +/- 0.3 ng/10(6) cells to zero (mean +/- SD, n = 3). Pretreatment with either cytochalasin B or vinblastine did not alter neutrophil generation of LTB4 induced by calcium ionophore. Neutrophils pretreated with vinblastine, at 4 x 10(-6) to 4 x 10(-4) M, and then maintained at one-half these concentrations during incubation with unopsonized zymosan particles exhibited no diminution in particle ingestion, but were markedly reduced in zymosan-induced synthesis of LTB4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Production and isolation of rabbit anti-idiotypic antibodies directed against the human monocyte receptor for yeast beta-glucans.

beta-Glucan receptors are present on mammalian phagocytic cells and provide an important physiologic mechanism for opsonin-independent clearance of yeasts and fungi. To prepare an immunologic probe to human monocyte beta-glucan receptors, an approach was taken that focused on the ligand specificity of the receptors as expressed by an anti-Id. The algal beta-glucan laminarin was chemically coupled to protein carriers to prepare an immunogenic beta-glucan. Three mouse IgG2a mAb were raised against laminarin, and one, mAb OEA10, exhibited specificity for the soluble unit ligand yeast heptaglucoside and the ligands present on zymosan and glucan particles that are recognized by monocyte beta-glucan receptors. These findings prompted usage of mAb OEA10 as immunogen for the preparation of an anti-Id. The resulting rabbit antiserum was subjected to sequential immunoaffinity chromatography to purify anti-idiotypic antibodies. The final product contained only IgG by SDS-PAGE and was shown to be specific by its selectively blocking binding of 125I-mAb OEA10 to laminarin. Pretreatment of adherent monocytes with 0.4 micrograms/ml of the anti-Id reduced the numbers of monocytes ingesting zymosan and glucan particles by 64 and 43%, respectively, whereas ingestion of IgG-coated SRBC was unaffected by as much as 16 micrograms/ml of the anti-Id. Incubation of adherent monocytes with increasing amounts of 125I-anti-Id in the absence and presence of 40-fold molar excess unlabeled anti-Id revealed dose-dependent specific binding, which approached plateau levels with 1 microgram/ml of labeled anti-Id. Thus, the anti-Id binds to monocytes and displays functional characteristics of soluble beta-glucan ligands, indicating that some of the anti-idiotypic antibodies recognize epitopes within monocyte beta-glucan receptors.     

Isolation of a yeast heptaglucoside that inhibits monocyte phagocytosis of zymosan particles

To isolate a unit ligand recognized by human monocyte beta-glucan receptors, acid-solubilized oligoglucosides were prepared by partial acid hydrolysis of purified yeast cell walls, gel filtered sequentially on Bio-Gel P-4 and P-2, derivatized with 2-aminopyridine, and separated by normal-phase HPLC. Ligand recognition was assessed by quantitating the effect of pretreatment with isolated materials on the capacities of adherent monocytes to phagocytose zymosan particles. Partial acid hydrolysis solubilized 23 +/- 4% (mean +/- SD; n = 7) of the cell wall glucans; at an input of 50 micrograms/ml, the solubilized products reduced the numbers of monocytes ingesting zymosan by an average of 44%. Gel filtration of acid-solubilized glucans on Bio-Gel P-4 revealed several peaks with phagocytosis-inhibiting activity, and fractions from the peak containing the smallest oligoglucosides, which accounted for 10 +/- 2% (mean +/- SD; n = 7) of the carbohydrate applied, were pooled. Further purification on Bio-Gel P-2 resolved this phagocytosis-inhibiting activity to a single peak that contained apparent heptaoses and accounted for 8 +/- 2% (mean +/- SD; n = 6) of the carbohydrate applied. At a concentration of 0.5 microgram/ml, the oligoglucosides pooled from the Bio-Gel P-4 and P-2 columns reduced the numbers of ingesting monocytes by 45 +/- 1% and 42 +/- 7% (mean +/- SD; n = 3), respectively. When derivatized with 2-aminopyridine, the oligoglucosides were resolved by HPLC to a number of peaks; a peak that eluted as an apparent heptaglucoside contained virtually all the inhibitory activity and accounted for only 6.6 +/- 0.7% (mean +/- SD, n = 7) of the carbohydrate applied. Gas chromatography analysis revealed only glucose and FAB-mass spectrometric analysis showed only heptaglucoside and no noncarbohydrate molecules. At a concentration of 1.6 ng/ml, the derivatized yeast heptaglucoside reduced the numbers of monocytes ingesting zymosan and glucan particles by 44 +/- 9% (mean +/- SD; n = 5) and 45 +/- 6% (n = 3), respectively. Thus, a heptaglucoside present in yeast cell walls is a unit ligand for human monocyte beta-glucan receptors.     

Characteristics of the beta-glucan receptor of murine macrophages

Phagocytosis of heat-killed yeast (HK-yeast), zymosan, and glucan particles by thioglycollate-elicited mouse peritoneal macrophages (Tg-macrophages) was inhibited by soluble glucan polymers/oligomers. The inhibitory capacity of soluble glucans decreased steeply with the decrease in the degree of polymerization (DPn); i.e., the concentration at which 50% inhibition of phagocytosis was attained was 0.23 microgram/ml for glucan 1 (DPn 24.8), 0.8 microgram/ml for glucan 2 (DPn 21.9), and greater than 40 micrograms/ml for glucan 3 (DPn 13.8). The glucan polymers were obtained by partial hydrolysis of glucan particles with formic acid (90%, 95 degrees C, 20 min) and fractionation according to solubility in ethanol water mixtures. A short preincubation (5 min, 4 or 37 degrees C) of Tg-macrophages with glucan 1 led to a subsequent inhibition of HK-yeast phagocytosis. Recovery of the phagocytic function was slow (27% in 3 h; 68% in 5 h) and required protein synthesis. beta-Glucan receptor expression was also suppressed by dexamethasone treatment. Mannan exerted at high concentrations (5 mg/ml) a partial inhibitory activity which was totally abrogated by beta-glucanase treatment. Treatment of macrophages with glucan together with mannan did not enhance the inhibitory capacity of glucan beyond the component abrogated by enzyme treatment. Contribution of local opsonization of HK-yeast to the phagocytic response (involvement of complement receptors) was indirectly negated; (a) glucan 1 which inhibits HK-yeast phagocytosis by up to 95% is not an activator of complement and therefore could not compete for the opsonizing proteins; (b) cycloheximide treatment in itself inhibited only partially HK-yeast phagocytosis whereas it inhibited the reexpression of the glucan receptors; (c) glucan 1 did not affect the phagocytosis of serum opsonized HK-yeast. Thus under the experimental conditions described, phagocytosis of HK-yeast by murine macrophages is mediated by and large by the beta-glucan receptors, while the mannose receptors and complement receptors do not contribute to the process.     

Regulation of cytosolic phospholipase A2 activation and cyclooxygenase 2 expression in macrophages by the beta-glucan receptor

Phagocytosis of non-opsonized microorganisms by macrophages initiates innate immune responses for host defense against infection. Cytosolic phospholipase A(2) is activated during phagocytosis, releasing arachidonic acid for production of eicosanoids, which initiate acute inflammation. Our objective was to identify pattern recognition receptors that stimulate arachidonic acid release and cyclooxygenase 2 (COX2) expression in macrophages by pathogenic yeast and yeast cell walls. Zymosan- and Candida albicans-stimulated arachidonic acid release from resident mouse peritoneal macrophages was blocked by soluble glucan phosphate. In RAW264.7 cells arachidonic acid release, COX2 expression, and prostaglandin production were enhanced by overexpressing the beta-glucan receptor, dectin-1, but not dectin-1 lacking the cytoplasmic tail. Pure particulate (1, 3)-beta-D-glucan stimulated arachidonic acid release and COX2 expression, which were augmented in a Toll-like receptor 2 (TLR2)-dependent manner by macrophage-activating lipopeptide-2. However, arachidonic acid release and leukotriene C(4) production stimulated by zymosan and C. albicans were TLR2-independent, whereas COX2 expression and prostaglandin production were partially blunted in TLR2(-/-) macrophages. Inhibition of Syk tyrosine kinase blocked arachidonic acid release and COX2 expression in response to zymosan, C. albicans, and particulate (1, 3)-beta-D-glucan. The results suggest that cytosolic phospholipase A(2) activation triggered by the beta-glucan component of yeast is dependent on the immunoreceptor tyrosine-based activation motif-like domain of dectin-1 and activation of Syk kinase, whereas both TLR2 and Syk kinase regulate COX2 expression.     

Phagocytosis by human monocytes of unopsonized particulate activators of the human alternative complement pathway: induction by cytokine

Monocytes isolated from human blood by centrifugal elutriation exhibited little ability to ingest rabbit erythrocytes (ER), zymosan particles, or desialated sheep erythrocytes. In contrast, 85 to 95% of these cells rosetted with C3b- or C3bi-bearing sheep erythrocytes (ES) or ingested IgG-coated ES. Preincubation of the monocytes with human lymphocytes increased their ability to ingest ER. the ER phagocytosis-inducing activity was contained in the 105,000 X G supernatant of lymphocyte lysates. These supernatants increased the percentage of ingesting monocytes from 5 to 15% to 80% within 60 min. The soluble factor was found to be relatively heat stable, inactivated by trypsin, and distinct from IFN-gamma. Its m.w. is less than 13,000. It was present in B and T lymphocytes and also in U937 cells. These results suggest that the ability of human monocytes to ingest nonopsonized particulate activators of the alternative complement pathway is a cytokine-inducible property and that the effect of the cytokine on complement receptor- or Fc receptor-dependent adherence or ingestion of opsonized particles is minor.     

Biochemical differences in the mechanism of macrophage lysosomal exocytosis initiated by zymosan particles and weak bases.

By utilizing compounds with different inhibitory properties, discrete biochemical differences were found in the mechanism of selective lysosomal enzyme secretion by macrophages in response to stimulation with zymosan particles and methylamine. Pretreatment of macrophages with trypsin markedly impaired the capacity of the cells to respond to stimulation with zymosan particles, but had no effect on methylamine-stimulated lysosomal enzyme secretion. Similarly, the addition of phenylmethanesulphonyl fluoride or EDTA to the incubation medium substantially inhibited zymosan-induced lysosomal enzyme secretion, whereas the methylamine-stimulated response was unaffected by these agents. The addition of 2-deoxyglucose to incubation media, however, strongly inhibited both zymosan- and methylamine-stimulated beta-galactosidase secretion. These findings are consistent with a mechanism for lysosomal enzyme secretion by macrophages, based on a receptor-dependent uptake of zymosan particles and a receptor-independent uptake of methylamine.     

Induction of macrophage lysosomal hydrolase synthesis and secretion by beta-1,3-glucan

Studies were undertaken to elucidate the active component in zymosan necessary to induce the delayed-onset synthesis and secretion of representative lysosomal hydrolases, hexosaminidase, and beta-glucuronidase in macrophages. Resident mouse peritoneal macrophages were challenged with zymosan particles and particulate beta-1,3-glucan, the major subcomponent of zymosan. Zymosan was found to induce a rapid secretion of preformed hexosaminidase with maximal release (75%) occurring 6 hr after the addition of zymosan. By contrast, beta-1,3-glucan was totally inactive in this respect. However, both zymosan and beta-1,3-glucan were found to induce the delayed-onset synthesis and secretion of hexosaminidase and beta-glucuronidase while maintaining constant cellular enzyme levels over a 5-day period following the addition of stimulus. These late responses were almost totally blocked by a noncytolytic concentration of cycloheximide, indicating their dependence on de novo protein synthesis. Mannan, the second major subcomponent of zymosan, had no effect on either immediate secretion or delayed-onset synthesis and secretion of hexosaminidase. These results suggest that the induction of the delayed-onset synthesis and secretion of the lysosomal hydrolases by zymosan may be dependent on the glucan subcomponent of zymosan. Moreover, it would also appear that the release of preformed lysosomal enzymes is not the trigger for the delayed-onset synthesis and secretion of hexosaminidase.     

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.     

Zymosan induces selective release of arachidonic acid from rabbit alveolar macrophages via stimulation of a beta-glucan receptor.

Zymosan, which is composed primarily of alpha-mannan and beta-glucan polymers, is a well recognized activator of macrophages. The type receptor by which unopsonized zymosan induces arachidonic acid release was investigated. It was found that particulate beta-glucan and zymosan stimulated an identical dose-dependent release of arachidonic acid. This release of arachidonic acid by zymosan was blocked by soluble beta-glucans whereas soluble mannan had no effect. This inhibition was not due to a general toxic effect of the soluble beta-glucans as they had no effect on calcium ionophore-induced release of arachidonic acid. Beta-glucan-induced fatty acid release from these cells was shown to be fairly specific for arachidonic acid. These data reveal that zymosan stimulates the specific release of arachidonic acid from rabbit alveolar macrophages, at least in part, via a beta-glucan receptor.     

Lysosomal enzyme release from human monocytes in response to particulate stimuli

Lysosomal enzyme release from human monocytes was evaluated in response to opsonized zymosan, opsonized sheep erythrocytes, and latex beads. Monocytes were found to release lysosomal enzymes immediately upon challenge with all three phagocytosable particles. Cytochalasin B enhanced beta-glucosaminidase release from mononuclear cells challenged with opsonized zymosan or opsonized red blood cells, but inhibited the response to latex particles. Lysosomal enzyme release was found to be independent of protein synthesis, and in the absence of cytochalasin B required the stimulus to be presented either as a phagocytosable particle or immobilized on a surface. The kinetics of enzyme release and phagocytosis were also examined and found to be different, lending support to the hypothesis that lysosomal enzyme release may be a physiologic response to a biologic stimulus in vivo and not simply an "accidental" consequence of an ongoing phagocytic event.     

Relationship of prostaglandin secretion by rabbit alveolar macrophages to phagocytosis and lysosomal enzyme release

The phospholipids of rabbit alveolar macrophages were pulse-labelled with [(14)C]-arachidonic acid, and the subsequent release of labelled prostaglandins was measured. Resting macrophages released measurable amounts of arachidonic acid, the prostaglandins E(2), D(2) and F(2alpha) and 6-oxoprostaglandin F(1alpha). Phagocytosis of zymosan increased the release of arachidonic acid and prostaglandins to 2.5 times the control value. In contrast, phagocytosis of inert latex particles had no effect on prostaglandin release. Indomethacin inhibited the release of prostaglandin, and, at high doses (20mug/ml), increased arachidonic acid release. Analysis of the cellular lipids showed that after zymosan stimulation the proportion of label was decreased in phosphatidylcholine, but not in other phospholipids or neutral lipids. Cytochalasin B, at a dose of 2mug/ml, inhibited the phagocytosis induced by zymosan but increased prostaglandin synthesis to 3.4 times the control. These data suggest that the stimulation of prostaglandin synthesis by zymosan is not dependent on phagocytosis. Exposure to zymosan also resulted in the release of the lysosomal enzyme, acid phosphatase. Furthermore, cytochalasin B augmented the zymosan-stimulated release of acid phosphatase at the same dose that stimulated prostaglandin synthesis. However, indomethacin, at a dose that completely inhibited prostaglandin synthesis, failed to block the lysosomal enzyme release. Thus despite some parallels between the release of prostaglandins and lysosomal enzymes, endogenous prostaglandins do not appear to mediate the release of lysosomal enzymes. The prostaglandins released from the macrophages may function as humoral substances affecting other cells.     

Adenylyl cyclase in yeast. Hydrodynamic properties and activation by trypsin

The adenylyl cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cAMP from ATP, and two RAS polypeptides, responsible for stimulation of cAMP synthesis by guanine nucleotides. We have determined hydrodynamic properties of yeast adenylyl cyclase in taurocholate extracts of wild type and RAS-deficient membranes. In taurocholate extracts of both kinds of membranes, the enzyme is insensitive to guanine nucleotide stimulation; in the presence of 0.5 M NaCl, the taurocholate-solubilized enzyme has a sedimentation coefficient of 12.5 S and a Stokes radius of 11 nm, consistent with a molecular weight of 594,000 for the protein-detergent complex. Treatment of particulate fractions with trypsin (less than 10 micrograms/ml) markedly activates membrane-bound adenylyl cyclase activity, abolishes stimulation by guanine nucleotides, and reduces the sedimentation coefficient of the detergent-solubilized enzyme; higher concentrations of trypsin release a still smaller water-soluble enzyme complex (7.5 S, 6.1 nm Stokes radius, calculated Mr = 190,000) from the membrane. In combination with genetic evidence (Kataoka, T., Broek, D., and Wigler M., (1985) Cell 43, 493-505), our data are consistent with a structural and functional model of yeast adenylyl cyclase in which GTP-activated RAS proteins stimulate cAMP synthesis by relieving an inhibitory constraint on the activity of the CYR1 gene product. This constraint may be mediated by the amino-terminal portion of the CYR1 polypeptide.     

Mechanisms of lysosomal enzyme secretion by human monocytes

Secretion of lysosomal enzymes by human monocytes in response to various stimuli and the effect of conditioned media from lymphocytes and neutrophils was studied. Monocytes were found to release β-glucosaminidase in response to NH₄Cl and to particles (zymosan, opsonised zymosan, asbestos and latex), but do not respond to some soluble stimuli like formyl-methionyl-leucyl-phenylalanine, phorbol myristate acetate, cytochalasin B, concanavalin A and $\text{N-}\text{acetylmuramyl-}\text{l}\text{-alanyl-}\text{d}\text{-isoglutamine}$. Neutrophil conditioned medium or neutrophil components did not have any effect on secretion. When treated with lymphokines the cells are more responsive, especially to zymosan. Even through there are similarities in the secretory activities of mouse macrophages and human monocytes, there are several differences both in the quantity of the response and in the mechanisms involved.     

Leukocyte complement: interleukin-like properties of factor Bb

It has been previously shown that the activated form of Factor B (Factor Bb) of the alternative pathway of complement activation stimulates monocyte spreading and killing of xenogenic erythrocytes and staphylococci. Factor Bb also stimulates lymphocyte blastogenesis in vitro, and native (uncleaved) Factor B is a major constitutive product of murine macrophages. To evaluate the possible "monokine" or "lymphokine"-like properties of Factor Bb, a radioimmunoassay was developed to measure the quantities of Factor B in phytohemagglutinin (PHA)-mitogen-stimulated cultures of human peripheral blood mononuclear cells. Nonstimulated mononuclear cell cultures from human peripheral blood (containing 10-14% monocytes and greater than 85% lymphocytes) at a density of 3 X 10(6) cells/ml (in serum-free medium) released less than 7 X 10(-10) M/liter (60 ng/ml) of Factor B antigen in 24 hr at 37 degrees C, and when mononuclear cells were stimulated with PHA mitogen in serum-free medium, the levels of Factor B antigen in media at 24 hr were significantly higher 1-3 X 10(-8) M/liter (0.9-2.8 micrograms/ml). The molecular size of Factor B in these media was 50-65 kDa by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a size appropriate for Factor Bb (60 kDa). Since pathological effects of macrophages in autoimmune disease may result from the release of lysosomal hydrolases, the effects of purified Factor Bb on mononuclear phagocytes were investigated in an in vitro system of murine peritoneal exudate macrophages. Factor Bb induced secretion of marker lysosomal hydrolases N-acetyl-beta-D-glucosaminidase (hexosaminidase) and beta-glucuronidase from thioglycollate-elicited murine peritoneal exudate macrophages in a dose-response and kinetic manner. Hydrolase release was induced in serum-free medium without a known particulate activator at a concentration of 80-200 nM (5-13 micrograms/ml) Factor Bb. Maximal release occurred in 3-5 hr at 37 degrees C and extracellular enzyme activity of hexosaminidase and glucuronidase increased as intracellular enzyme levels decreased, suggesting that Factor Bb triggers release of these enzymes from intracellular lysosomal pools. These results provide an example of a complement protein which is synthesized, released, and activated during mononuclear cell culture and which induces release of lysosomal enzymes from macrophages. In conventional terminology, Factor B or Factor Bb might be termed a "lymphokine," "monokine," or "interleukin".     

Down-modulation of lymphoproliferation and interferon-gamma production by beta-glucan derived from Saccharomyces cerevisiae

beta-glucan, one of the major cell wall components of Saccharomyces cerevisiae, has been found to enhance immune functions. This study investigated in vivo and in vitro effects of beta-glucan on lymphoproliferation and interferon-gamma (IFN-gamma) production by splenic cells from C57BL/6 female mice. All experiments were performed with particulate beta-glucan derived from S. cerevisiae. Data demonstrated that both, i.p. administration of particulate beta-glucan (20 or 100 micrograms/animal) and in vitro stimulation of splenic cells (20 or 100 micrograms/ml of culture) decreased lymphoproliferation and IFN-gamma production induced by concanavalin A. These results suggest that beta-glucan can trigger a down-modulatory effect regulating a deleterious immune system hyperactivity in the presence of a strong stimulus.     

Differential augmentation by recombinant human tumor necrosis factor-alpha of neutrophil responses to particulate zymosan and glucan

Zymosan (Z) and its major insoluble carbohydrate component beta-linked glucan activate human neutrophils (PMN) through a trypsin-sensitive recognition mechanism. This mechanism is believed to involve the PMN CR3R. Both Z and glucan generated dose and time-dependent release of the secondary lysosomal granule marker vitamin B12 binding protein, leukotriene B4 (LTB4) and superoxide from PMN and were phagocytosed with similar dose-dependent kinetics. The PMN superoxide and LTB4 responses to glucan; however, were consistently greater than those to the same doses of Z. The phagocytosis of both particles was significantly reduced after partial digestion with beta-laminarinase but not beta-glucosidase or alpha-mannosidase suggesting a recognition mechanism dependent on intact beta-1,3-glucosidic bonds in both particles. TNF-alpha (rhTNF-alpha) promoted a time- and dose-dependent increase in the expression of PMN CR3 up to 60 min. The increased expression of CR3 was paralleled by the release of the secondary lysosomal granule marker vitamin B12-binding protein. This granule contains a population of CR3R in its boundary membrane and it is the fusion of this membrane with the plasma membrane that may represent the mechanism by which CR3 expression is increased. Preincubation of PMN with 10(-9)M rhTNF-alpha augmented phagocytosis, LTB4, and superoxide generation by PMN in response to activation by Z. In contrast, none of the responses to glucan was significantly increased after incubation with rhTNF-alpha. These differences suggest a lack of absolute homology between the recognition mechanisms for zymosan and glucan and that there is a component of the recognition mechanism for zymosan that is independent of that for glucan and is up-regulated after rhTNF-alpha pretreatment.