RESUMO
This paper describes the cloning and characterization of five cDNA members of a novel family of mRNAs, termed hm-1, isolated from human U937 macrophage cells. Two family members (clones 46 and 11) show complete mRNA features [including ribosome binding sites (RBS), polyadenylation signals, and poly(A) tails], and encode the same protein (designated HM-1), but differ substantially in their 5' untranslated regions. The three other cDNAs (clones 20, 60, and 38) appear to represent partial cDNAs. The protein sequences deduced from the five hm-1 cDNAs are identical (some truncated), except for one Trp --> Cys substitution. Full-length HM-1 is 246 amino acids long, has a predicted MW of 29431, is rich in arginine residues, has a pI of 10.25, and a mean hydrophobicity index of -1.23. HM-1 contains no obvious hydrophobic N-terminal cleavable signal sequence, and no potential N-glycosylation sites, but does contain three highly conserved motifs present in U1-70K splicing factors, and contains numerous C-terminal Arg/Asp and Arg/Glu dipeptides characteristic of "RD" family members that function as regulators of mRNA splicing. Northern hybridizations indicate that hm-1 is a family of mRNAs differentially expressed in a variety of human tissues.
Assuntos
DNA Complementar/genética , Histiócitos/metabolismo , Proteínas/genética , Ribonucleoproteína Nuclear Pequena U1/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Expressão Gênica , Humanos , Dados de Sequência Molecular , Fatores de Crescimento Neural , Splicing de RNA , RNA Mensageiro/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Distribuição Tecidual , Células U937RESUMO
beta-Glucan receptors are present on mammalian leukocytes and initiate phagocytosis of particulate yeast beta-glucans, such as zymosan particles. Human monocytes and U937 cells express two membrane proteins of 180 and 160 kDa, each of which binds particulate yeast glucan through a 20-kDa polypeptide constituent. In this report, the structural composition of the two beta-glucan receptors and the biochemical properties of their polypeptide constituents were examined. The 180-kDa receptor was composed of three disulfide-linked polypeptides of 95, 60, and 20 kDa, whereas the 160-kDa receptor was a multimer of two polypeptides of 27 and 20 kDa. Unlike other receptor constituents, the 20-kDa polypeptide was nonglycosylated and focused at two distinct isoelectric points. Immunoblots of the focused polypeptides showed the two 20-kDa variants and the 95-kDa subunit to be constitutively tyrosine-phosphorylated, a feature not previously reported for receptors on human mononuclear phagocytes. Dephosphorylation of the receptor proteins resulted in the loss of antigenic phosphotyrosine without affecting the antigenicity of either 20-kDa variant for the anti-idiotypic antibody to beta-glucan receptors. Separate analysis of the 160-kDa receptor showed it contained both variants of the 20-kDa polypeptide. Thus, the 20-kDa subunit constituent of the two beta-glucan receptors is a functionally and chemically unique polypeptide with apparent microheterogeneity in its primary structure.
Assuntos
Glucanos/metabolismo , Monócitos/metabolismo , Receptores Imunológicos/química , Aminoácidos/análise , Sítios de Ligação , Linhagem Celular , Humanos , Ponto Isoelétrico , Glicoproteínas de Membrana/química , Peso Molecular , Fosfotirosina , Receptores Imunológicos/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Glucocorticoids are potent and diverse in their effects on mononuclear phagocytes, ranging from suppression to stimulation. To determine whether glucocorticoids affected functions mediated by monocyte beta-glucan receptors, human mononuclear cells (MNC) were incubated for 20 hr at 37 degrees with 20-2000 nM dexamethasone or hydrocortisone, and the monocytes were subsequently assayed for their ingestion of purified yeast glucan particles. Prior treatment with dexamethasone or hydrocortisone enhanced monocyte phagocytosis of glucan particles in a dose-dependent manner and both steroids effected a twofold increase at 200 nM. Monocytes from three different donors were assessed for secretion of beta-N-acetylglucosaminidase, and all were increased by exposure to 200 nM dexamethasone for 20 hr and subsequent stimulation with glucan particles for 2 hr; the average percentage net release was 16.2%. The enhancement in monocyte phagocytosis of glucan particles did not result by culture of MNC with beta-oestradiol, progesterone, testosterone or spironolactone, indicating a specificity for corticosteroids with glucocorticoid activity. The increases in phagocytic activity by monocytes that had been exposed during culture to either 200 nM dexamethasone or 200 nM hydrocortisone were both reduced by 40-50% by pretreatment of monocytes with the anti-idiotype (anti-Id) that recognizes beta-glucan receptors. Exposure of cells to 200 nM dexamethasone for 2 hr and washing before continued incubation for 18 hr in steroid-free media resulted in stimulation of monocyte beta-glucan receptors, whereas similar exposure without subsequent culture or with the addition of cycloheximide for the final 18 hr did not. Thus, glucocorticoids enhance monocyte functions mediated by beta-glucan receptors, and this stimulation is dependent on proteins that are newly synthesized during culture.
Assuntos
Dexametasona/farmacologia , Hidrocortisona/farmacologia , Monócitos/efeitos dos fármacos , Receptores Imunológicos/efeitos dos fármacos , Adulto , Proteínas Sanguíneas/biossíntese , Células Cultivadas , Relação Dose-Resposta a Droga , Glucanos/metabolismo , Humanos , Cinética , Monócitos/imunologia , Fagocitose/efeitos dos fármacosRESUMO
beta-glucans are pharmacologic agents that rapidly enhance host resistance to a variety of biologic insults through mechanisms involving macrophage activation. To determine whether stimulation of the beta-glucan receptors on human monocytes resulted in cytokine production, monolayers of monocytes were incubated with purified yeast glucan particles and measured for tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) mRNA and protein. By Northern blot analysis, TNF-alpha mRNA was detected within 30 min of incubation with glucan particles, peaked at 2 h, and remained elevated for at least 8 h. Glucan induction of IL-1 beta mRNA followed a similar time-course of initiation and accumulation. By enzyme-linked immunosorbent assays (ELISAs), significant levels of TNF-alpha and IL-1 beta were present in supernatants of glucan-treated cells within 1 h and plateau levels of both cytokines were approached within 4 h. At particle-to-cell ratios of from 0.4 to 18, glucan particles induced dose-dependent increases in TNF-alpha and IL-1 beta mRNA and corresponding increases in TNF-alpha and IL-1 beta proteins. Exposure of monocytes to glucan particles for 0-30 min and washing before continued incubation for 4 h in particle-free buffer induced production and secretion of TNF-alpha and IL-1 beta in a time-dependent fashion compatible with phagocytosis. The pretreatment of monocyte monolayers with trypsin reduced glucan-induced production of TNF-alpha and IL-1 beta in a dose-dependent manner with 5 micrograms/ml of trypsin effecting reductions of greater than 50%. Thus, glucan particles induce human monocyte production of TNF-alpha and IL-1 beta by a mechanism that is dependent on trypsin-sensitive beta-glucan receptors.
Assuntos
Glucanos/farmacologia , Interleucina-1/biossíntese , Monócitos/efeitos dos fármacos , Receptores de Superfície Celular/efeitos dos fármacos , Receptores Imunológicos , Fator de Necrose Tumoral alfa/biossíntese , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Interleucina-1/genética , Monócitos/metabolismo , RNA Mensageiro/análise , Transcrição Gênica/efeitos dos fármacos , Tripsina/farmacologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
A cofactor that selectively opsonizes particulate activators of the human alternative complement pathway and enhances their phagocytosis by human monocytes was identified in synovial fluids of patients with rheumatoid arthritis. The active material was present in fluids treated with protease inhibitors, was heat stable, and was unaffected by incubation with hyaluronidase. Chromatographic isolation of synovial fluid fibronectin by gelatin affinity and by immunoaffinity on antifibronectin monoclonal antibody BD4 yielded similar quantities of protein for each of 3 fluids. Synovial fluid proteins with the BD4 fibronectin epitope accounted for essentially all of the phagocytosis-enhancing activity and expressed this activity by opsonizing target activators. Additional chromatographic analyses of synovial fluid fibronectin with the BD4 epitope were carried out using Sepharose-bearing gelatin and 4 additional antifibronectin monoclonal antibodies. The opsonic materials were characterized as having 2 distinct fibronectin epitopes, which always mapped from the cell adhesive domain to the carboxyl-terminus of plasma fibronectin, but only rarely contained the gelatin binding domain.
Assuntos
Artrite Reumatoide , Fibronectinas/química , Proteínas Opsonizantes/química , Líquido Sinovial/química , Epitopos/análise , Fibronectinas/isolamento & purificação , Humanos , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Proteínas Opsonizantes/isolamento & purificação , FagocitoseRESUMO
beta-glucan receptors, with ligand specificity for yeast and fungal carbohydrate polymers, have been studied as phagocytic receptors of human monocytes. To characterize their structure, binding studies were carried out with human U937 cells and a rabbit IgG anti-Id that recognizes epitopes on monocyte beta-glucan receptors. Unstimulated U937 cells specifically bound large amounts of the anti-Id, but almost none of the control anti-isotype. At saturation, the number of anti-Id molecules bound per U937 cell was 2.6 x 10(6) with an apparent Ka of 1.9 x 10(7) M-1. Immunoprecipitates from detergent lysates of surface-radioiodinated U937 cells contained only two membrane proteins with antigenic specificity for the anti-Id, one having a mol wt of 180 kD and the other 160 kD. Both proteins were disulfide-linked and presented, after reduction, as five polypeptides of 95, 88, 60, 27, and 20 kD. Detergent lysates of unlabeled U937 cells, purified by affinity chromatography on anti-Id-Sepharose, yielded the same two nonreduced proteins and five reduction products in slab gels stained with Coomassie blue. In Western blots probed with the anti-Id, the most immunoreactive nonreduced and reduced affinity-purified products were the 160 and 20 kD molecules, respectively. Immunoblots of two-dimensional gels showed the 180 and 160 kD proteins to express a common epitope through disulfide linkage to the 20 kD polypeptide. By immunoblot analysis, U937 cell glucan-binding proteins from detergent lysates contained two cell proteins antigenic for the anti-Id that were indistinguishable from affinity-purified molecules in size and subunit composition. Studies of affinity-purified proteins from detergent lysed human monocytes were characterized by immunoblot analysis and found to be identical to U937 cell beta-glucan receptors. They consisted of two disulfide-linked proteins, with mol wt of 180 and 160 kD, and had in common a 20 kD polypeptide with the anti-Id epitope.
Assuntos
Monócitos/química , Receptores de Superfície Celular/isolamento & purificação , Receptores Imunológicos , Anticorpos Monoclonais/imunologia , Western Blotting , Linhagem Celular , Cromatografia de Afinidade , Eletroforese em Gel Bidimensional , Glucanos/metabolismo , Humanos , Peso Molecular , Testes de Precipitina , Receptores de Superfície Celular/químicaRESUMO
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.
Assuntos
Anticorpos Anti-Idiotípicos/biossíntese , Anticorpos Monoclonais/biossíntese , Glucanos/metabolismo , Monócitos/imunologia , Receptores de Superfície Celular/imunologia , Receptores Imunológicos , Leveduras/imunologia , Animais , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Epitopos/análise , Humanos , Polissacarídeos/imunologia , Coelhos , Soroalbumina Bovina/imunologia , Zimosan/imunologiaRESUMO
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.
Assuntos
Glucosídeos/isolamento & purificação , Glicosídeos/isolamento & purificação , Monócitos/imunologia , Fagocitose/efeitos dos fármacos , Saccharomyces cerevisiae/análise , Zimosan/imunologia , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Glucanos/metabolismo , Glucanos/farmacologia , Glucosídeos/fisiologia , Humanos , Hidrólise , Monócitos/metabolismo , Receptores Imunológicos/efeitos dos fármacos , Saccharomyces cerevisiae/imunologiaRESUMO
Human monocytes phagocytose particulate activators of the alternative complement pathway through beta-glucan receptors in the absence of opsonins. Recognition of soluble beta-glucans by monocytes selectively inhibits ingestion of particulate activators and has no effect on responses mediated by monocyte receptors for Fc-IgG, complement, or fibronectin. The smallest ligand unit recognized by monocyte beta-glucan receptors is an acid-resistant heptaglucoside present in yeast cell walls. Mouse monoclonal anti-beta-glucan antibodies have been prepared, one of which completely neutralizes the inhibitory capacity of the HPLC-purified heptaglucoside. This antibody has been used as immunogen for the preparation of an anti-Id. The pretreatment of monocytes with low concentrations of anti-Id inhibits monocyte ingestion of zymosan particles but not EsIgG, suggesting that this antibody has specificity for monocyte beta-glucan receptors and is a powerful probe for further receptor studies. Other receptors with specificities for carbohydrates are also present on mononuclear phagocytes. Receptors for mannose/fucose and those for galactose have been isolated and cloned. The development of probes, such as structural analogs of the active heptaglucoside and the anti-Id, will bring the beta-glucan receptors to a similar stage of definition. A major factor that adds a considerable degree of difficulty to studies of the beta-glucan receptors and is not shared by the other receptors for carbohydrates is the requirement for structural conformations provided by the alignment of several glucose units rather than the recognition of a hexose residue in, for example, a glycoconjugate. The designation of these receptors as beta-glucan receptors has inadvertently taken us into a new area of nonimmune defense. Animal studies indicate that beta-glucans with 1,3- and/or 1,6-linkages are active pharmacologic agents that rapidly confer protection to a normal host against a variety of biologic insults. The beta-glucan receptors provide a mechanism by which a heightened state of host responsiveness is initiated.
Assuntos
Monócitos/ultraestrutura , Fagocitose/fisiologia , Receptores de Superfície Celular/fisiologia , Receptores Imunológicos , Animais , Anticorpos Monoclonais/imunologia , Via Alternativa do Complemento , Glucanos/imunologia , Glucanos/metabolismo , Humanos , Idiótipos de Imunoglobulinas/biossíntese , Zimosan/fisiologiaRESUMO
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)
Assuntos
Glucanos/metabolismo , Leucotrieno B4/biossíntese , Neutrófilos/metabolismo , Fagocitose , Receptores de Superfície Celular/efeitos dos fármacos , Receptores Imunológicos , Citocalasina B/farmacologia , Glucanos/farmacologia , Humanos , Mananas/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Proteínas Opsonizantes , Fagocitose/efeitos dos fármacos , Receptores de Superfície Celular/fisiologia , Tripsina , Vimblastina/farmacologia , ZimosanRESUMO
Candida albicans is an opportunistic human pathogen with a cell wall that is qualitatively similar in carbohydrate composition to zymosan. Monolayers of human peripheral blood monocytes ingested 2.5 x 10(6)-2.5 x 10(7) heat-killed C. albicans blastospores in a dose-related manner. The percentage of monocytes ingesting at least one C. albicans reached a near plateau level of 68 +/- 6% (mean +/- SD, n = 3) when 2.5 x 10(7) particles per ml were incubated with monocytes for 30 min. Pretreatment of monocytes with 10 ng/ml to 100 micrograms/ml of soluble yeast beta-glucan inhibited C. albicans ingestion in a dose-dependent manner without affecting Fc-mediated ingestion of IgG-coated sheep erythrocytes (EsIgG). Pretreatment of monocytes with 100 micrograms/ml of soluble yeast beta-glucan inhibited the ingestion of 1 or more C. albicans by 80 +/- 11% (mean +/- SD, n = 4), with 50% inhibition occurring with approximately 7 micrograms/ml of soluble beta-glucan. Incubation of monocytes with 100 micrograms/ml to 1000 micrograms/ml of soluble yeast mannan resulted in no significant inhibition of C. albicans ingestion. The pretreatment of monolayers of monocytes for 30 min with 1 microgram/ml to 50 micrograms/ml of affinity-purified trypsin resulted in a dose-dependent inhibition of C. albicans ingestion with 10 micrograms/ml of trypsin inhibiting the percentage of monocytes ingesting 1 or more organisms by 71 +/- 6%. The inhibitory effects of soluble yeast beta-glucan and trypsin pretreatment on the ingestion of heat-killed C. albicans are comparable to the inhibitory effect of these agents on monocyte phagocytosis of zymosan and glucan particles. This relationship indicates that C. albicans contain a ligand that is recognized by the monocyte beta-glucan receptor of human monocytes.
Assuntos
Candida albicans/imunologia , Glucanos/imunologia , Monócitos/imunologia , Fagocitose , Receptores de Superfície Celular/imunologia , Receptores Imunológicos , Relação Dose-Resposta Imunológica , Glucanos/farmacologia , Temperatura Alta , Humanos , Mananas/farmacologia , Monócitos/efeitos dos fármacos , Esporos Fúngicos/imunologia , Tripsina/farmacologiaRESUMO
Human peripheral blood monocytes ingest particulate activators and generate leukotrienes via a trypsin-sensitive, beta-glucan-inhibitable receptor. The incubation of monolayers of monocytes with from 4 X 10(5) to 2 X 10(8) zymosan or glucan particles resulted in a dose-dependent release of up to 9% +/- 1.9 and 17.8% +/- 5.3 (mean +/- SD, n = 3) of the lysosomal enzyme, N-acetylglucosaminidase, into the culture medium. Lysosomal enzyme release occurred throughout the 2-hr period studied, with the greatest rate of N-acetyl-glucosaminidase release occurring during the first hour; the presence of 5 micrograms/ml of cytochalasin B accelerated this process when zymosan was the agonist. The preincubation of monocytes with from 0.5 to 500 micrograms/ml of soluble yeast beta-glucan inhibited N-acetylglucosaminidase release by 4 X 10(7) zymosan and glucan particles in a dose-dependent manner, with 50% inhibition occurring with 50 micrograms/ml of soluble yeast beta-glucan (mean +/- SD, n = 3). Preincubation with as much as 5 mg/ml of yeast mannan had no inhibitory effect on N-acetylglucosaminidase release. The pretreatment for 30 min of monolayers of monocytes with 50 micrograms/ml of affinity-purified trypsin, which selectively inactivates the monocyte-phagocytic response to particulate activators, also fully inhibited lysosomal enzyme release induced by zymosan and glucan particles. The inhibitory effects of a soluble ligand, yeast beta-glucan, and of trypsin pretreatment on lysosomal enzyme release correspond to the inhibitory effect of these agents on monocyte phagocytosis of zymosan and glucan particles and thus indicates ligand specificity for the beta-glucan receptor in the release of stored intracellular mediators.
Assuntos
Monócitos/enzimologia , Receptores de Superfície Celular/fisiologia , Receptores Imunológicos , Acetilglucosaminidase/metabolismo , Via Alternativa do Complemento , Relação Dose-Resposta a Droga , Glucanos/farmacologia , Humanos , Lisossomos/enzimologia , Fagocitose , Taxa Secretória/efeitos dos fármacos , Solubilidade , Tripsina , Zimosan/farmacologiaRESUMO
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)
Assuntos
Glucanos/isolamento & purificação , Monócitos/fisiologia , Fagocitose , Receptores de Superfície Celular/fisiologia , Receptores Imunológicos , Via Alternativa do Complemento , Glucanos/imunologia , Humanos , Peso Molecular , Oligossacarídeos/farmacologia , Receptores de Superfície Celular/efeitos dos fármacos , Saccharomyces cerevisiae/análise , SolubilidadeRESUMO
Murine bone marrow cells, plated at 4 X 10(4) cells/well and cultured in 50% fibroblast CM, yielded pure populations of large, individual, adherent cells that were phagocytic and morphologically indistinguishable from macrophages. Adherent macrophages appeared in small numbers with 24 h of culture, increased to maximal cell numbers within 10 days of culture, and remained at these cell densities for at least 11 weeks in culture. The capacities of adherent macrophages to ingest unopsonized zymosan particles and EsIgG, at inputs of 1.25 X 10(7) targets, were expressed by 7 and 40% of the cells derived from 24-hour cultures, respectively, were increased at nearly identical rates to comparable maximal levels within 10-14 days of culture and were exhibited by essentially all adherent cells derived from 2-11-week cultures. The percentage of adherent macrophages from twelve 3-6-week cultures ingesting greater than or equal to 1, greater than or equal to 6 and greater than or equal to 10 zymosan particles was 89 +/- 5, 47 +/- 11 and 14 +/- 9% (mean +/- SD, n = 12), respectively, and the percentage ingesting greater than or equal to 1, greater than or equal 6 and greater than or equal to 10 EsIgG was 86 +/- 5, 49 +/- 10 and 14 +/- 8%, respectively. Incubation of adherent macrophages with mannan-free ss-glucan particles at inputs of 5 X 10(5)-5 X 10(7)/ml initiated a phagocytic response comparable to that obtained with the same doses of zymosan particles which contained mannan and beta-glucan. Preincubation of adherent macrophages with 100 micrograms/ml of a fully soluble beta-glucan, laminarin, and solubilized barley beta-glucan reduced subsequent macrophage phagocytosis of greater than or equal to 6 zymosan particles by 53 and 40%, respectively. In contrast, yeast alpha-mannan was less than 1% as active, and 10 mg/ml reduced the number of adherent macrophages ingesting greater than or equal to 1 zymosan particles by 64%. At concentrations as high as 2 mg/ml, laminarin and barley beta-glucan had no effect on Fc receptor-mediated ingestion of EsIgG, and mannan at 20 mg/ml also failed to inhibit EsIgG ingestion. Pretreatment of adherent macrophages with 20 micrograms/ml of trypsin reduced the number of cells ingesting greater than or equal to 1 zymosan particles from 89 to 10% and those ingesting greater than or equal to 6 zymosan from 43 to 0%, whereas pretreatment with as much as 100 micrograms/ml of trypsin failed to decrease macrophage ingestion of EsIgG.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Glucanos/metabolismo , Macrófagos/imunologia , Fagocitose , Receptores de Superfície Celular/efeitos dos fármacos , Zimosan , Animais , Adesão Celular , Eritrócitos/imunologia , Glucanos/farmacologia , Humanos , Imunoglobulina G , Macrófagos/efeitos dos fármacos , Mananas/farmacologia , Camundongos , Receptores de Superfície Celular/imunologia , Fatores de Tempo , Tripsina/farmacologiaAssuntos
Ativação do Complemento , Via Alternativa do Complemento , Fibronectinas/fisiologia , Imunidade Celular , Monócitos/imunologia , Fagocitose , Animais , Anticorpos Monoclonais , Humanos , Macrófagos/imunologia , Camundongos , Proteínas Opsonizantes , Polissacarídeos/imunologia , Receptores de Complemento/imunologia , Receptores Imunológicos/imunologia , Zimosan/imunologiaRESUMO
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)
Assuntos
Ativação do Complemento/efeitos dos fármacos , Via Alternativa do Complemento/efeitos dos fármacos , Glucanos/farmacologia , Monócitos/metabolismo , Receptores de Complemento/efeitos dos fármacos , beta-Glucanas , Glucanos/metabolismo , Hordeum , Humanos , Mananas/farmacologia , Monócitos/imunologia , Fagocitose/efeitos dos fármacos , Polissacarídeos/farmacologia , Zimosan/metabolismo , Zimosan/farmacologiaRESUMO
Human monocytes possess a receptor for ingestion of particulate activators of the human alternative complement pathway that functions in the absence of plasma proteins and is distinct from the receptors for Fc-IgG and the major cleavage fragment of the third component of complement (C3b). Incubation of monolayers of monocytes with 1.1 X 10(6) to 2.2 X 10(7) glucan particles per ml initiated a phagocytic response comparable to that obtained with zymosan particles, of which beta-glucan is a constituent along with mannan. Maximal quantities of 4.93 +/- 3.43 ng of leukotriene B4 (LTB4) and 0.43 +/- 0.23 ng of leukotriene C4 (LTC4) (mean +/- SD, n = 3) were released by 10(6) monocytes stimulated with 1.1 X 10(7) glucan particles per ml. Preincubation of monocytes with 50 micrograms of soluble beta-glucan per ml reduced subsequent monocyte ingestion of 5 X 10(6) zymosan particles per ml and 2.2 X 10(6) glucan particles per ml by 52% and 55%, respectively, and diminished release of LTB4 by monocytes stimulated with 2 X 10(8) zymosan particles per ml and 8.6 X 10(6) glucan particles per ml by 73% and 61%, respectively. Preincubation with 1 mg of soluble mannan per ml had little effect on monocyte phagocytosis or LTB4 generation in response to either zymosan or glucan particles, and neither soluble beta-glucan nor mannan stimulated generation of LTB4 or LTC4. The effect of pretreatment of monocytes with soluble beta-glucan was time dependent, with the maximal effect being evident within 20 min of pretreatment, and was specific for zymosan or glucan particles in that the LTB4 and LTC4 release induced by 2.5 microM calcium ionophore A23187 was unaffected. That both phagocytosis and leukotriene generation are inhibited by soluble beta-glucan but not by mannan at a rate compatible with the phagocytic process of monocyte monolayers indicates ligand specificity for a beta-glucan receptor. As the beta-glucan receptor recognizes particulate activators of the alternative complement pathway, the nonimmune response to a single stimulus induces complement activation, phagocytosis, and leukotriene generation.
Assuntos
Leucotrieno B4/biossíntese , Monócitos/fisiologia , Fagocitose , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos , SRS-A/biossíntese , Via Alternativa do Complemento , Glucanos/farmacologia , Humanos , Técnicas In Vitro , Mananas/farmacologia , Monócitos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Zimosan/farmacologiaRESUMO
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.