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)     

Lysosomal enzyme release from human monocytes by particulate activators is mediated by beta-glucan inhibitable receptors

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.     

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)     

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.     

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)     

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.     

Shared idiotope on monoclonal anti-Ia.7 antibodies reactive with determinants in a structural domain of the I-E molecules

In previous studies, heterologous anti-idiotypic (anti-Id) antisera against the C3H.SW 14-4-4S or the A.TH 41.A anti-Ia.7 monoclonal antibodies (mAb) were shown to identify an interstrain cross-reactive idiotypic specificity (IdX.Ia.7) expressed on monoclonal or conventional anti-Ia.7 alloantibodies. The objective of the present investigation was to characterize further this IdX at the idiotopic level. To this end, 11 hybridomas producing IgG1, IgG2a, or IgM anti-Id mAb were derived from a rat immunized with a mixture of 10 A.TH or A.BY anti-Ia.7 mAb. The specificity of the latter anti-Id mAb was determined by direct Id binding radioimmunoassay (RIA) with the use of a panel of 52 anti-Ia mAb derived from hybridomas produced in various inbred mouse strains. These rat anti-Id mAb recognized idiotopes expressed on i) all anti-Ia.7 mAb against determinants in the topographic domain I of the I-Ek molecule but not on 18 other anti-I-Ek mAb directed at epitopes in domains II or III; ii) three of 19 anti-I-Ak mAb; and iii) one A.TL-derived anti-I-As mAb. Competitive Id binding assays revealed that among the 14 IdX+ anti-Ia.7 mAb, one (81.B) was bound to a lesser extent by various rat anti-Id mAb, suggesting that heterogeneity probably exists in this antibody family. By contrast, two isologous (B10.S(7R)) anti-Id mAb to the IdX.Ia.7+ mAb 41.A displayed a specificity restricted to 41.A individual idiotopes (IdI). Rat anti-IdX.Ia.7 and mouse anti-41.A IdI mAb inhibited the binding of 125I-labeled mAb 41.A to CBA spleen cells. These two sets of mAb bound in a noncompetitive fashion to mAb 41.A-coated plates, indicating that their corresponding public or private idiotopes were spatially distinct. These data may have implications for in vivo manipulations of anti-Ia immune responses.     

Idiotypic intramolecular help. Induction of tumor-specific antibodies by monoclonal anti-idiotypic antibody with the help of Fc-specific T helper clones

In this study, the mechanism of anti-Id vaccination was investigated by using cloned Th cells and an anti-idiotypic mAb. 2F10, an anti-idiotypic mAb derived from an Igh1-e allotype mouse strain, which induces protection against the L1210/GZL DBA/2 tumor, was used to prime DBA/2 mice. An Fc (Igh1-e)-specific syngeneic Th clone was cocultured in the presence of 2F10 anti-Id with 2F10-Fab-primed B cells. The Th clone responded with proliferation and also provided help for 2F10-Fab-primed B cells to produce antibodies that bind to L1210/GZL and not to P815 tumor cells. Intact 2F10 anti-Id was presented to Fc-specific Th cells by Fab (or Id) primed B cells more efficiently than the fragment mixture (Fab plus Fc) of 2F10 anti-Id, indicating that 2F10-Fab (or Id)-primed B cells capture 2F10 anti-Id through surface Ig receptors. Presenting B cells are sensitive to treatment with chloroquine and must come from H-2 matched mice, indicating that the Ag presentation by Fab-primed B cells to Fc-specific Th cells requires processing and is MHC restricted. Collectively these results outline a mechanism that may operate in anti-Id therapy of tumor-bearing animals by using tumor Ag mimicking anti-idiotypic antibodies. A similar mechanism could be effective in tumor patients immunized with xenogeneic anti-idiotypic antibodies operating under the "intra(Ag) molecular help."     

Dichotomous effect of monocyte Fc receptor interaction on anti-CD3-induced immunoglobulin synthesis

Monoclonal antibodies against the TCR/CD3 complex are capable of activating T cells which in turn may induce immunoglobulin synthesis in B cells under appropriate conditions. Here we present evidence that distinct immune responses, induced by four commonly used TCR/CD3 mAb (Leu4, OKT3, BMA030, BMA031) were related to the mAb interaction with monocyte Fc receptors for IgG. Depending on their isotype and on the technique by which they were crosslinked, TCR/CD3 mAb induced variable IgM and IgG synthesis in PBMC: If the mAb were crosslinked by monocyte IgG-Fc receptors they induced a high Ig production, while crosslinking the same mAb by plastic-bound goat anti-mouse antibodies (panning) failed to do so. Nevertheless, both crosslinking techniques triggered a strong proliferation and IL-2, IL-4, and IFN gamma lymphokine gene expression. The lack of Ig production under panning conditions was due to an additional IgG-Fc receptor interaction with monocytes: (a) If namely mAb F(ab')2 fragments, or mAb isotypes unable to bind to monocyte Fc receptors (IgG2b, IgG1 in nonresponders) were crosslinked by panning, both a good proliferation as well as Ig production ensued; (b) if TCR/CD3 mAb isotypes which could additionally bind to monocyte Fc receptor (IgG2a) were crosslinked, no Ig production occurred; (c) if mAb F(ab')2 fragments were crosslinked with a second anti-T cell antibody of IgG2a isotype, which could bind to monocyte Fc receptors, Ig synthesis was reduced. Interestingly enough, this diminishing effect, due to monocyte Fc receptor interaction, was only observed if CD4-positive cells were proliferating, but not if CD8-positive cells were activated.     

Leukocyte activation by (1-->3)-beta-D glucans

We studied the activities of several kinds of beta-glucans, including sonifilan, grifolan, Sclerotinia sclerotiorum glucan, laminarin and zymosan, on macrophages. Preculture of macrophages with inactive beta-glucans rendered the cells unresponsive to subsequent stimulation with grifolan, suggesting a specific pathway in the beta-glucan structure. The importance of protein C and phosphorylation of mitogen-activated protein kinase was demonstrated in the activation with grifolan or zymosan. Immunoprecipitation of complement receptor (CR3), coprecipitated other proteins carrying phosphotyrosine residues in stimulation with grifolan. These data suggest that protein kinase C and tyrosine kinases are essential for signal transduction, and that CR3 might participate in the activation through interaction with other intracellular proteins.     

Phagocytosis of serum-opsonized zymosan down-regulates the expression of CR3 and FcRI in the membrane of human monocytes

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

Human immune response to group A streptococcal carbohydrate (A-CHO). II. Antigen-independent stimulation of IgM anti-A-CHO production in purified B cells by a monoclonal anti-idiotopic antibody

The paper describes the induction by a monoclonal anti-idiotopic antibody (anti-Id mAb) of specific antibody production to group A streptococcal carbohydrate (A-CHO) in purified human B cells of several unrelated individuals. The anti-Id mAb, designated 16F498 (anti-Id498), recognizes a recurrent idiotope (Id 498) associated with the combining site of human antibodies to N-acetyl-D-glucosamine (GlcNAc), the immunodominant group of A-CHO. Id498 is expressed on IgM anti-GlcNAc antibodies but does not occur on IgG antibodies with the same specificity. It occurs also on a minor population of IgM antibodies without specificity for A-CHO. Id498 was found in 19 of 27 sera from unselected healthy donors and thus seems to be frequently expressed within the adult B cell repertoire. The in vitro induction of anti-A-CHO antibodies was analyzed in human B cells extensively depleted for T cells. Specific antibody secretion required cross-linked anti-Id which was achieved by coupling the mAb to agarose beads. No antibody secretion could be induced by soluble anti-Id (1 and 10 micrograms/ml). An optimal response required soluble T cell-derived factors which were added as a mixture of recombinant interleukin 2 with a T cell hybridoma supernatant that augments B cell growth and differentiation. Under these conditions an antigen-independent specific increase of IgM anti-A-CHO production (2.6- to 10-fold, or up to 2000 ng/ml respectively) could be induced in blood B cell populations of four of six normal individuals expressing the Id498 at serum level.     

Lipopolysaccharide exposure during purification of human monocytes by adherence increases their recovery and cytolytic activity

Exposure of monocytes to lipopolysaccharide (LPS) during, but not after, adherence purification increased their cytolytic activity in short-term 51Cr-release assays against K562 target cells. In the absence of LPS only a minority of monocytes could be recovered by adherence. With 1 ng/ml to 10 micrograms/ml LPS present during the 1-hr adherence procedure, however, monocytes spread more extensively on serum-coated plastic and glass surfaces and virtually all of the monocytes in a mononuclear leukocyte preparation were recovered in the adherent fraction. While increasing the recovery of monocytes threefold, LPS exposure during adherence also increased monocyte purity as assessed by peroxidase staining, morphology, and indirect immunofluorescence with monoclonal Mo2. The proportion of Leu-11-positive NK cells in the adherent fraction did not change. Depletion of NK cells by treatment with anti-Leu-11b and complement eliminated cytolytic activity from the nonadherent, but not from the adherent, fraction isolated with LPS. Thus, addition of LPS during adherence produced a monocyte preparation with enhanced cytolytic activity not attributable to NK contaminants. To test whether LPS caused production of lymphokines that activate monocytes, we tested supernatants of unseparated mononuclear leukocytes for the capacity to stimulate purified monocytes for cytolysis. Such supernatants stimulated monocytes more effectively than LPS alone. We conclude that LPS stimulates monocytes for cytolysis most effectively during adherence purification because LPS allows the recovery of weakly adherent monocytes with high cytolytic capacity; also, LPS may stimulate production of lymphokines that further augment monocyte cytolytic activity.     

Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3

We have examined the receptor-ligand interactions and the method of phagocytosis of virulent Mycobacterium tuberculosis by human monocytes. mAb against complement receptors (CR) inhibit adherence and phagocytosis of M. tuberculosis in fresh nonimmune serum. A mAb against the type 1 CR (CR1) inhibits adherence of M. tuberculosis by 40 +/- 5%, and three different mAb against the type 3 CR (CR3) each inhibit adherence by 39 +/- 5% to 47 +/- 4%. A mAb against CR1 used in combination with one of the three mAb against CR3 inhibits adherence by up to 64 +/- 7%. Most strikingly, two mAb used in combination against CR3 inhibit adherence by up to 81 +/- 2%. mAb against other monocyte surface Ag do not significantly influence adherence. In like fashion, mAb against CR but not other monocyte surface Ag inhibit adherence of preopsonized M. tuberculosis in the presence of heat-inactivated serum. By electron microscopy, monocytes ingest all M. tuberculosis that adhere in the presence of nonimmune serum; mAb against CR3 markedly inhibit ingestion. In contrast to CR, the FcR and the beta-glucan-inhibitable receptor for zymosan play little or no role in mediating M. tuberculosis adherence or ingestion. Adherence of M. tuberculosis is serum-dependent, requiring greater than or equal to 2.5% serum for optimal adherence. Heat inactivation of serum markedly reduces adherence of M. tuberculosis (75.5 +/- 7%) and preopsonization of bacteria enhances adherence by 2.9 +/- 0.4-fold. Adherence is also markedly reduced in C3- or factor B-depleted serum; repletion with C3 or factor B increases adherence by 2.1 +/- 0.4-fold and 1.86 +/- 0.05-fold, respectively. Fab anti-C3 IgG markedly inhibits monocyte adherence of preopsonized M. tuberculosis (71 +/- 1%). C component C3 is fixed to M. tuberculosis by the alternative C pathway as determined by a whole bacterial cell ELISA. Human monocytes ingest M. tuberculosis by conventional phagocytosis as viewed by electron microscopy. This study demonstrates that human monocyte CR1 and CR3 mediate phagocytosis of M. tuberculosis and C component C3 in serum is acting as the major bacterium-bound ligand.     

Prevention of murine cryoglobulinemia and associated pathology by monoclonal anti-idiotypic antibody

A murine IgG3 mAb, clone 6-19, derived from non-manipulated autoimmune MRL/MpJ-lpr/lpr mice is a rheumatoid factor specific for IgG2a and is able to generate cryoglobulins via nonspecific IgG3 Fc-Fc interaction. Intraperitoneal passive transfer of ascites containing the 6-19 mAb into BALB/c mice induces, within 18 h, remarkable pathology characterized by skin vasculitis and acute glomerulonephritis associated with cryoglobulinemia. In order to evaluate the possibility of modulating the development of tissue lesions by an anti-Id antibody, we have raised an IgG2b anti-Id mAb specific to the 6-19 mAb. The cryoprecipitation of 6-19 mAb was completely inhibited in the presence of excess amounts of anti-Id mAb in vitro. In vivo, pretreatment of BALB/c mice with anti-6-19 anti-Id mAb inhibited development of skin vasculitis and glomerulonephritis induced by the 6-19 mAb. The cryoglobulin formation was markedly diminished due to enhanced elimination of the 6-19 mAb from the circulation. In contrast, pretreatment with an IgM anti-IgG3 rheumatoid factor mAb neither protected nor aggravated the development of tissue lesions. These results suggest possible implications in the anti-Id treatment of similar vascular diseases in man.     

Characterization of a novel low affinity receptor for IFN-gamma on adherent human monocytes by radioligand binding studies and chemical cross-linking

Freshly isolated monocytes in suspension express 2000 to 4000 high affinity receptors for IFN-gamma. Because monocytes change phenotypically as they migrate out of the circulation and adhere to extracellular matrix, modulation of the expression of IFN-gamma receptors may occur. In order to determine if adherence alone modulates the receptor for IFN-gamma, we have studied receptor expression in adherent human peripheral blood monocytes. Elutriation-purified monocytes were allowed to adhere to polystyrene overnight at 37 degrees C. These cells now expressed 1 to 2 x 10(5) low affinity (Ka = 10(8) liters/M) receptors for [125I]rIFN-gamma. Binding to this receptor was specific and saturable. The expression of these receptors occurred rapidly (within 3 h) after adherence and was not inhibited by cycloheximide treatment. Binding to the receptor was abrogated by treating cells with trypsin, but was enhanced after treatment with alkaline protease or proteinase K. mAb against the high affinity receptor did not block binding to the low affinity receptor on adherent cells. The low affinity receptor transduced a signal to the cell as measured by the IFN-gamma-induced enhancement in FcR for human IgG1. The structure of the receptor on adherent cells was investigated by chemical cross-linking techniques. A receptor-[125I]rIFN-gamma complex was observed by SDS-PAGE to have a Mr of 180,000 to 200,000. Reduction of this complex with 2-ME resulted in the loss of the high Mr complex and the appearance of a doublet of lower Mr of 68,000 and 82,000. In contrast, cross-linking of monocytes in suspension yielded a complex of 110,000 to 120,000 Mr, which was unchanged upon reduction. Upon adherence, human monocytes express large numbers of a novel receptor for rIFN-gamma which is capable of stimulating the cell. This receptor appears to be composed of at least two components which are disulfide linked and structurally differs from the high affinity receptor on nonadherent monocytes.     

Characterization of monocyte adherence to human macrovascular and microvascular endothelial cells

The interaction of monocytes with cultured large vessel venous and arterial endothelial cells (EC) and with cultured microvascular EC was studied. Analysis of time-lapse microcinematographic video recordings showed that monocytes adhere rapidly to the surface of EC and subsequently remain spherical and fixed to the initial site of adherence. Some monocytes adherent to EC stretch out within 30 to 90 min and migrate over the EC surface or become stretched for about 10 to 30 min and then detach from the EC surface and move rapidly over the EC monolayer. It was shown that the interaction of monocytes with EC is dynamic, that the morphology of monocytes adherent to EC changes constantly, and that stretching of the monocytes over the surface of the EC is not an inevitable and irreversible consequence of binding. A quantitative adherence assay was developed in which both the morphology and the number of monocytes bound to EC were determined. For each type of EC the number of monocytes bound to a single EC was found to be linearly related to the number of monocytes added and was lower for smaller EC. The adherence of monocytes to venous and arterial EC followed a different time course than the adherence to capillary EC and adherence to both types of macrovascular EC was higher than adherence to microvascular EC was higher than adherence to microvascular EC. The percentage of adherent monocytes with a stretched morphology was lower when these cells were adherent to capillary EC than to both types of macrovascular EC and increased upon addition of serum. Adherence of monocytes to venous, arterial, and capillary EC was partially inhibited by mAb directed against the alpha-chain of lymphocyte function-associated Ag-1 or C3bi receptor (with mAb LM2/1, but not with mAb OKM1) and by mAb against the common beta-chain of the three leukocyte adhesion molecules. The degree of inhibition of monocyte adherence to EC by mAb against lymphocyte function-associated Ag-1 alpha and the common beta-chain was dependent on the type of EC and was higher for venous EC (57 to 70% inhibition) than for arterial (40 to 44% inhibition) and capillary (44 to 49% inhibition) EC. Inhibition of monocyte adherence obtained with anti-C3bi receptor-alpha mAb was similar for each EC type. mAb against p150, 95 did not affect adherence. None of the mAb could block binding completely; combinations of the mAb also did not result in increased inhibition of monocyte adherence to EC.     

Control of lipopolysaccharide (LPS) binding and LPS-induced tumor necrosis factor secretion in human peripheral blood monocytes.

We used flow cytometry to determine how LPS-binding protein (LBP) effects the binding of fluorescein-labeled LPS to human monocytes via receptor-dependent mechanisms. The addition of human, rabbit, mouse, or FCS strikingly increased the binding of LPS to monocytes compared with controls incubated in serum-free medium. This binding was totally prevented by preincubation of monocytes with MY4, an anti-CD14 mAb, or by enzymatic removal of CD14 from monocytes. Depletion of LBP from rabbit serum with anti-LBP antibodies also produced a similar suppression. Solutions of albumin did not support the enhanced binding observed in serum but the addition of purified rabbit LBP to albumin solutions resulted in binding similar to that observed in serum-containing medium. When type-specific anti-LPS mAb was added to human serum, LPS binding to monocytes occurred but was only partly inhibited by anti-CD14 mAb, suggesting that receptors other than CD14 (presumably Fc or complement receptors) were involved. Serum increased by 100- to 1000-fold the sensitivity of monocytes to the triggering by LPS resulting in TNF secretion. TNF secretion was inhibited by anti-CD14 mAb up to 100 ng/ml of LPS and by anti-LPS mAb up to 1 to 10 ng/ml. The inhibition of TNF secretion by anti-LPS mAb appeared to be the result of directing LPS to monocyte receptors other than CD14. In contrast, in medium containing normal as well as acute serum and in the absence of anti-LPS antibodies, the binding of LPS to monocytes and the triggering of TNF secretion appeared to be mediated mainly by interactions between CD14 and LBP-LPS complexes.     

Collagen-Induced Arthritis Suppressed with Monoclonal Anti-Idiotypic Antibody

In foregoing work, we identified at least 5 distinct epitopes on human type II collagen (CII), using 8 murine monoclonal antibodies (mAb) against human CII, and suggested that a species-nonspecific epitope on CII recognized by anti-CII mAb termed 1-5 is an arthritogenic epitope. We also found that antibody response against a selected epitope of human CII could be induced by immunization with rabbit anti-idiotypic (Id) antibody against anti-CII mAb. The author developed and characterized monoclonal anti-Id antibodies against 1-5 mAb recognizing a putative arthritogenic epitope. The author also investigated whether the anti-Id mAb could regulate antibody response directed against a selected epitope recognized by 1-5 mAb, and the induction of collagen-induced arthritis in DBA/1J mice. DBA/1J mice intravenously preinjected with anti-Id mAb to 1-5, did not produce anti-CII antibody expressing 1-5 Id upon immunization with human CII. Furthermore, as the development of collagen-induced arthritis (CIA) in DBA/1J mice pretreated with anti-Id mAb to 1-5 was significantly suppressed, anti-Id mAb will be a useful tool for studying the regulation of antibody response to a selected epitope. This study lends support to our hypothesis that the 1-5 epitope is an arthritogenic epitope.