Ultraweak sugar-sugar interactions for transient cell adhesion

Carbohydrate-carbohydrate interactions are rarely considered in biologically relevant situations such as cell recognition and adhesion. One Ca(2+)-mediated homotypic interaction between two Lewis(x) determinants (Le(x)) has been proposed to drive cell adhesion in murine embryogenesis. Here, we confirm the existence of this specific interaction by reporting the first direct quantitative measurements in an environment akin to that provided by membranes. The adhesion between giant vesicles functionalized with Le(x) was obtained by micropipette aspiration and contact angle measurements. This interaction is below the thermal energy, and cell-cell adhesion will require a large number of molecules, as illustrated by the Le(x) concentration peak observed at the cell membranes during the morula stage of the embryo. This adhesion is ultralow and therefore difficult to measure. Such small interactions explain why the concept of specific interactions between carbohydrates is often neglected.     

P-Selectin Glycoprotein Ligand-1 Forms Dimeric Interactions with E-Selectin but Monomeric Interactions with L-Selectin on Cell Surfaces

Interactions of selectins with cell surface glycoconjugates mediate the first step of the adhesion and signaling cascade that recruits circulating leukocytes to sites of infection or injury. P-selectin dimerizes on the surface of endothelial cells and forms dimeric bonds with P-selectin glycoprotein ligand-1 (PSGL-1), a homodimeric sialomucin on leukocytes. It is not known whether leukocyte L-selectin or endothelial cell E-selectin are monomeric or oligomeric. Here we used the micropipette technique to analyze two-dimensional binding of monomeric or dimeric L- and E-selectin with monomeric or dimeric PSGL-1. Adhesion frequency analysis demonstrated that E-selectin on human aortic endothelial cells supported dimeric interactions with dimeric PSGL-1 and monomeric interactions with monomeric PSGL-1. In contrast, L-selectin on human neutrophils supported monomeric interactions with dimeric or monomeric PSGL-1. Our work provides a new method to analyze oligomeric cross-junctional molecular binding at the interface of two interacting cells.     

Filamentation of Escherichia coli K12 elicited by some monomeric, dimeric and trimeric complexes of ruthenium in various oxidation states

A number of ruthenium complexes were tested for their ability to induce filamentation in Escherichia coli. These included monomeric and dimeric complexes with ruthenium in the II or III oxidation states, as well as mixed-valence complexes with ruthenium in the (II,III) oxidation states. In general, dimeric mixed-valence Ru(II,III) complexes were the most active class of compound, although some complexes of this type were relatively inactive. These were pyrazine- or bipyridyl-bridged complexes which are known to involve strong metal-ligand interaction, which stabilizes the Ru(II) oxidation state. Some Ru(III) complexes were also significantly active in induction of filamentous growth in E. coli. One of these was [Ru(NH3)5Cl]Cl2, which did not inhibit electron transport, Mg2+-ATPase activity or DNA synthesis in E. coli, but like [Ru2(NH3)6Br3]Br2 X H2O was a potent inhibitor of respiration-driven calcium transport in the organism. Filament-inducing activity of the complex was reduced in the presence of NaCl, but not in the presence of added Ca2+, ethanol, calcium pantothenate, or E. coli 'division promoting extract'. This behaviour is also similar to that of [Ru2(NH3)6Br3]Br2 X H2O. It is suggested that both complexes may induce filamentation in E. coli by a common mechanism, which may involve interference with calcium metabolism, or a wall or membrane target, rather than interaction with DNA.     

Expression of cell surface Lewis X and Y antigens and FUT4 mRNA is increased in Jurkat cells undergoing apoptosis

Cell surface molecules undergo specific changes during apoptosis, including the expression of phosphatidylserine (PS) and some proteins and alterations in sugar chains. Among the various sugar chains on the cell surface, Lewis X (Le(X)) and Lewis Y (Le(Y)) antigens are key determinants for a variety of biological processes. We studied the changes in Le(X) and Le(Y) expression in Jurkat cells, a human T cell line, during apoptosis. Flow cytometry showed that Le(X) and Le(Y) antigen expression was enhanced on the cell surface during apoptosis induced by anti-Fas antibody. To clarify the mechanism of enhanced Le(X) and Le(Y) expression, we assessed the expression levels of fucosyltransferase (FUT1, 2, 3-5-6, 4, and 9) mRNAs that are predominantly expressed in Jurkat cells and which are considered to form Le(X) and Le(Y). The expression of FUT4 mRNA was up-regulated after exposing cells to anti-Fas antibody. Moreover, the increase in Le(X) and Le(Y) antigen levels was significantly suppressed by caspase 3 or 8 inhibitors. These results indicated that the induction of FUT (mainly FUT4), the gene expression of which is mediated by signals downstream of caspase 3, increases Le(X) and Le(Y) expression in apoptotic cells.     

Structural basis of focal adhesion targeting domain-mediated signaling in cardiac hypertrophy

The focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK) exists in monomeric closed (c) or arm exchanged (ae) dimeric state. FAT interaction with Grb2 necessitates an intermediate open (o) state that interacts with Grb2 and activates signaling pathways leading to pathological cardiac hypertrophy. Targeted molecular dynamics (TMD) simulation was carried out in order to capture the structure of the intermediate formed by opening of Helix1 (H1) from monomeric cFAT leading to the formation of monomeric aeFAT. During TMD, H1 separated from the four helices bundle of cFAT, completely unfolded and performed a full turn before folding back to a helix inclined at an acute angle to the helical bundle in aeFAT. The entire transition can be described in six distinct intermediate structural stages. The most significant correlation of H1 motion was observed with Loop3 (L3) and is the likely reason for the complete disruption of the FAT interaction with paxillin during the transition. High-affinity analogs of the paxillin LD4 region can be a promising strategy to drive the equilibrium towards cFAT, thus antagonizing FAT-Grb2 association. During transition, the overall shift in orientation of all the four helices rejects paxillin binding and approves Grb2 association. Exposure and β-turn conformation of the YENV motif (residues 925-928) in oFAT-facilitated phosphorylation and Grb2 binding. Docking, MD simulation and conservation analysis of oFAT-Grb2 complex provided insight into the structural determinants of binding and specificity. Our work provides a structural basis for pharmacological modulation of dynamic conformational changes and interactions of FAT.     

Metal chelates of heterocyclic nitrogen-containing ketones. XX. Temperature dependence of magnetic susceptibility and electron spin resonance spectra of halogeno-copper(II) complexes of phenyl-2-picolyl ketone hydrazone and phenyl hydrazone

The interaction of hydrated copper(II) chloride or bromide with phenyl-2-picolyl ketone hydrazone and phenyl-2-picolyl ketone phenyl hydrazone, PPKhy and PPKPhy, respectively, have been studied employing elemental analyses, spectral studies (IR and electronic spectra) and the variable temperature magnetic susceptibility together with the electron spin resonance spectra. The complexes of the formulae [CuLX₂]₂, L = PPKhy or PPKPhy and X = Cl or Br, are found to be dimeric through a halide bridge while those of the stoichiometry [CuL₂]X₂ are monomeric in nature. The magnetic data showed that the dimeric complexes possess antiferromagnetic interaction. The mechanism of the magnetic interaction is discussed based on the variable temperature magnetic data and ESR spectra.     

Structural and energetic requirements for a second binding site at the dimeric β-lactoglobulin interface

Through experimental and theoretical approaches, it has been shown that bovine β-lactoglobulin (βlg) uses its hydrophobic cavity or calyx as the primary binding site for hydrophobic molecules, whereas the existence of a second ligand binding site at the dimeric interface has only been structurally identified for vitamin D3 (VD3). This binding exists even in the thermally denatured state, suggesting the prevalence of this secondary site. Although crystallographic experiments have suggested that VD3 can bind to both monomeric and dimeric states without significant structural differences, theoretical and experimental reports have proposed some structural requirements. Thus, in this study, based on known experimental data, the dynamic interaction of VD3 with the monomeric or dimeric forms of βlg was investigated through a protocol combining blind docking and 2 microsecond molecular dynamics simulations coupled with binding free energy and per-residue binding free energy decomposition analyses using the Molecular Mechanics Generalized Born Surface Area approach. Binding free energy calculations allowed us to estimate the energetic differences of coupling VD3 at the calyx and the dimeric interface for the monomeric or dimeric state, revealing that the dimeric structure is required to form a stable complex with VD3 at the dimeric interface. This also has an important impact on the dimerization process, whereas although the monomeric state also forms a stable complex with VD3 at the dimeric interface, the incorporation of the entropy component contributed to producing a marginally favorable binding free energy. Finally, the per-residue decomposition analysis provided energetic information about the most relevant residues in stabilizing the different systems.     

Inhibition of PDGF-stimulated and matrix-mediated proliferation of human vascular smooth muscle cells by SPARC is independent of changes in cell shape or cyclin-dependent kinase inhibitors

Interactions among growth factors, cells, and extracellular matrix regulate proliferation during normal development and in pathologies such as atherosclerosis. SPARC (secreted protein, acidic, and rich in cysteine) is a matrix-associated glycoprotein that modulates the adhesion and proliferation of vascular cells. In this study, we demonstrate that SPARC inhibits human arterial smooth muscle cell proliferation stimulated by platelet-derived growth factor or by adhesion to monomeric type I collagen. Binding studies with SPARC and SPARC peptides indicate specific and saturable interaction with smooth muscle cells that involves the C-terminal Ca2+-binding region of the protein. We also report that SPARC arrests monomeric collagen-supported smooth muscle cell proliferation in the late G1-phase of the cell cycle in the absence of an effect on cell shape or on levels of cyclin-dependent kinase inhibitors. Cyclin-dependent kinase-2 activity, p107 and cyclin A levels, and retinoblastoma protein phosphorylation are markedly reduced in response to the addition of exogenous SPARC and/or peptides derived from specific domains of SPARC. Thus, SPARC, previously characterized as an inhibitor of platelet-derived growth factor binding to its receptor, also antagonizes smooth muscle cell proliferation mediated by monomeric collagen at the level of cyclin-dependent kinase-2 activity.     

Expression of mutant and wild-type TIMP3 in primary gingival fibroblasts from Sorsby's fundus dystrophy patients

Gingival fibroblast cell lines were derived from Sorsby's fundus dystrophy (SFD) patients carrying the S181C TIMP3 and the E139X TIMP3 mutations. These cell lines were grown in culture to study expression of the wild-type and mutant tissue inhibitor of metalloproteinase 3 (TIMP3) alleles from a normal diploid cell type. Firstly, patient cells were found to co-express the wild-type and mutant TIMP3 alleles, S181C TIMP3 or E139X TIMP3, at the mRNA level using restriction fragment length polymorphism (RFLP) analysis. A SpeI RFLP for E139X TIMP3 is described. Low levels of endogenous TIMP3 protein expression were elevated using the natural polysaccharide calcium pentosan polysulfate (CaPPs) in combination with the cytokine IL-1alpha. Immunoblotting detected protein expression from both wild-type and mutant alleles, S181C TIMP3 or E139X TIMP3. S181C TIMP3 from these cells was found to dimerise and retain MMP2 inhibitory activity. To facilitate studies of the E139X TIMP3 protein, the allele was expressed using HighFive insect cells. In this cell type, the E139X TIMP3 was synthesised as a mixture of monomer and dimer. Both monomeric and dimeric E139X TIMP3 protein retained MMP2 inhibitory activity in gelatin zymography. Expression of mutant E139X or S181C TIMP3 protein from a normal diploid patient-derived fibroblast cell had no effect on either MMP2 or MMP9 expression or activation whilst transcribed from their normal promoter context.     

Carbohydrate-to-carbohydrate interaction, through glycosynapse, as a basis of cell recognition and membrane organization

Cell adhesion mediated by carbohydrate-to-carbohydrate interaction (CCI), or cell adhesion with concurrent signal transduction, are discussed in three contexts.1. Types of cell adhesion based on interaction of several combinations of glycosphingolipids (GSLs) at the surface of interfacing cells (" trans interactio") are reviewed critically, to exclude the possible involvement of GSL-binding proteins. Special emphasis is on: (i) autoaggregation of mouse teratocarcinoma F9 cells mediated by Le( x )-to-Le( x ) interaction, in which presence of Le( x )-binding protein is ruled out; (ii) adhesion of GM3-expressing cells to Gg3-expressing cells, in which involvement of GM3- or Gg3-binding protein is ruled out.2. Characteristic features and requirements of CCI, as compared with carbohydrate-to-protein interaction (CPI) and protein-to-protein interaction (PPI), are summarized, including: (i) specificity and requirement of bivalent cation; (ii) reaction velocity of CCI as compared to PPI; (iii) negative (repulsive) interaction; (iv) synergistic or cooperative effect of CCI and PPI, particularly GM3-to-Gg3 or GM3-to-LacCer interaction in synergy with integrin-dependent adhesion, or Le( x )-to-Le( x ) interaction in cooperation with E-cadherin-dependent adhesion.3. Microdomains at the cell surface are formed based on clustering of GSLs or glycoproteins organized with signal transducers. Among such microdomains, those involved in adhesion coupled with signal transduction to alter cellular phenotype are termed "glycosynaps". In some glycosynapses, growth factor receptors or integrin receptors are also involved, and their function is modulated by GSLs only when the receptor is N -glycosylated. This modulation may occur in part via interaction of GSLs with N -linked glycans of the receptor, termed " cis interactio".     

Divalent cations and redox conditions regulate the molecular structure and function of visinin-like protein-1

The NCS protein Visinin-like Protein 1 (VILIP-1) transduces calcium signals in the brain and serves as an effector of the non-retinal receptor guanylyl cyclases (GCs) GC-A and GC-B, and nicotinic acetyl choline receptors (nAchR). Analysis of the quaternary structure of VILIP-1 in solution reveals the existence of monomeric and dimeric species, the relative contents of which are affected but not exclusively regulated by divalent metal ions and Redox conditions. Using small-angle X-ray scattering, we have investigated the low resolution structure of the calcium-bound VILIP-1 dimer under reducing conditions. Scattering profiles for samples with high monomeric and dimeric contents have been obtained. The dimerization interface involves residues from EF-hand regions EF3 and EF4.Using monolayer adsorption experiments, we show that myristoylated and unmyristoylated VILIP-1 can bind lipid membranes. The presence of calcium only marginally improves binding of the protein to the monolayer, suggesting that charged residues at the protein surface may play a role in the binding process.In the presence of calcium, VILIP-1 undergoes a conformational re-arrangement, exposing previously hidden surfaces for interaction with protein partners. We hypothesise a working model where dimeric VILIP-1 interacts with the membrane where it binds membrane-bound receptors in a calcium-dependent manner.     

Biophysical studies of the membrane-embedded and cytoplasmic forms of the glucose-specific Enzyme II of the E. coli phosphotransferase system (PTS)

The glucose Enzyme II transporter complex of the Escherichia coli phosphotransferase system (PTS) exists in at least two physically distinct forms: a membrane-integrated dimeric form, and a cytoplasmic monomeric form, but little is known about the physical states of these enzyme forms. Six approaches were used to evaluate protein-protein and protein-lipid interactions in this system. Fluorescence energy transfer (FRET) using MBP-II(Glc)-YFP and MBP-II(Glc)-CFP revealed that the homodimeric Enzyme II complex in cell membranes is stable (FRET(-)) but can be dissociated and reassociated to the heterodimer only in the presence of Triton X100 (FRET(+)). The monomeric species could form a heterodimeric species (FRET(+)) by incubation and purification without detergent exposure. Formaldehyde cross linking studies, conducted both in vivo and in vitro, revealed that the dimeric MBP-II(Glc) activity decreased dramatically with increasing formaldehyde concentrations due to both aggregation and activity loss, but that the monomeric MBP-II(Glc) retained activity more effectively in response to the same formaldehyde treatments, and little or no aggregation was observed. Electron microscopy of MBP-II(Glc) indicated that the dimeric form is larger than the monomeric form. Dynamic light scattering confirmed this conclusion and provided quantitation. NMR analyses provided strong evidence that the dimeric form is present primarily in a lipid bilayer while the monomeric form is present as micelles. Finally, lipid analyses of the different fractions revealed that the three lipid species (PE, PG and CL) are present in all fractions, but the monomeric micellar structure contains a higher percentage of anionic lipids (PG & CL) while the dimeric bilayer form has a higher percentage of zwitterion lipids (PE). Additionally, evidence for a minor dimeric micellar species, possibly an intermediate between the monomeric micellar and the dimeric bilayer forms, is presented. These results provide convincing evidence for interconvertible physical forms of Enzyme-II(Glc).     

Functional importance of calcium binding sites in outer membrane phospholipase A

Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme that hydrolyses phospholipids requiring Ca(2+) as cofactor. In vitro studies have shown that OMPLA is only active as a dimer. The structures of monomeric and dimeric OMPLA provided possible clues to the activation process. In the inhibited dimeric species calcium ions are located at the dimer interface ideally suited to stabilise the oxyanion intermediates formed during catalysis. The side chain hydroxyl function of Ser152 is one of the ligands of this interfacial calcium. In the crystal structure of monomeric OMPLA the interfacial calcium site is lacking, but calcium was found to bind at a site involving the carboxylates of Asp149 and Asp184. In the current study the relevance of the identified calcium sites has been studied by site-directed mutagenesis. The Ser152Asn variant confirmed the importance of the interfacial calcium site for catalysis, and also demonstrated that this site is essentially involved in the dimerisation process. Replacements of the ligands in monomeric OMPLA, i.e. Asp149Asn, Asp149Ala and Asp184Asn, only showed minor effects on catalytic activity and dimerisation. A stronger effect observed for the variant Asp184Ala was explained by the proximity of Asp184 to the catalytically important Ser152 residue. We propose that Asp149 and Asp184 provide an electronegative funnel that may facilitate Ca(2+) transfer to the interfacial calcium site.     

Spectroscopic studies of the interaction between quercetin and G-quadruplex DNA

Quercetin is a kind of flavonoid which has been proved to exhibit anti-tumor activity. The interaction modes of quercetins with monomeric and dimeric G-quadruplexes were studied by absorption, fluorescence, CD, and (1)H NMR spectroscopies. The ligands were found to be stacked with terminal tetrads of monomeric G-quadruplexes by intercalation and bound to dimeric G-quadruplexes by groove binding.     

Recombinant dimeric IgA antibodies against the epidermal growth factor receptor mediate effective tumor cell killing

Dimeric IgA Abs contribute significantly to the humoral part of the mucosal immune system. However, their potential as immunotherapeutic agent has hardly been explored. In this article, we describe the production, purification, and functional evaluation of recombinant dimeric IgA against the epidermal growth factor receptor. Human joining chain-containing IgA was produced by nonadherent Chinese hamster ovarian (CHO)-K1 cells under serum-free conditions. Purification by anti-human κ and anti-His-tag affinity, as well as size exclusion chromatography, resulted in a homogenous preparation of highly pure IgA dimers. Functional studies demonstrated dimeric IgA to be at least as effective as monomeric IgA in triggering Ab-dependent cellular cytotoxicity by isolated monocytes or polymorphonuclear cell and in human whole-blood assays. Importantly, dimeric IgA was more effective in F(ab)-mediated killing mechanisms, such as inhibition of ligand binding, receptor downmodulation, and growth inhibition. Furthermore, only dimeric but not monomeric IgA or IgG was directionally transported by the polymeric Ig receptor through an epithelial cell monolayer. Together, these studies demonstrate that recombinant dimeric IgA Abs recruit a distinct repertoire of effector functions compared with monomeric IgA or IgG1 Abs.     

Leukocyte function-associated antigen 1-mediated adhesion stability is dynamically regulated through affinity and valency during bond formation with intercellular adhesion molecule-1

Neutrophil rolling and transition to arrest on inflamed endothelium are dynamically regulated by the affinity of the beta(2) integrin CD11a/CD18 (leukocyte function associated antigen 1 (LFA-1)) for binding intercellular adhesion molecule (ICAM)-1. Conformational shifts are thought to regulate molecular affinity and adhesion stability. Also critical to adhesion efficiency is membrane redistribution of active LFA-1 into dense submicron clusters where multimeric interactions occur. We examined the influences of affinity and dimerization of LFA-1 on LFA-1/ICAM-1 binding by engineering a cell-free model in which two recombinant LFA-1 heterodimers are bound to respective Fab domains of an antibody attached to latex microspheres. Binding of monomeric and dimeric ICAM-1 to dimeric LFA-1 was measured in real time by fluorescence flow cytometry. ICAM-1 dissociation kinetics were measured while LFA-1 affinity was dynamically shifted by the addition of allosteric small molecules. High affinity LFA-1 dissociated 10-fold faster when bound to monomeric compared with dimeric ICAM-1, corresponding to bond lifetimes of 25 and 330 s, respectively. Downshifting LFA-1 into an intermediate affinity state with the small molecule I domain allosteric inhibitor IC487475 decreased the difference in dissociation rates between monomeric and dimeric ICAM-1 to 4-fold. When LFA-1 was shifted into the low affinity state by lovastatin, both monomeric and dimeric ICAM-1 dissociated in less than 1 s, and the dissociation rates were within 50% of each other. These data reveal the respective importance of LFA-1 affinity and proximity in tuning bond lifetime with ICAM-1 and demonstrate a nonlinear increase in the bond lifetime of the dimer versus the monomer at higher affinity.     

Mouse M290 is the functional homologue of the human mucosal lymphocyte integrin HML-1: antagonism between the integrin ligands E-cadherin and RGD tripeptide.

Human mucosal lymphocyte antigen-1 (HML-1, alphaEbeta7) and E-cadherin, two members of unrelated cell adhesion superfamilies, have evolved to play cooperative roles in gut mucosal immunity. Human E-cadherin is self-ligand mediating intercellular adhesion of epithelial cells, as well as adhesion of intra-epithelial lymphocytes to intestinal enterocytes via an interaction with HML-1. Herein we report that both dimeric and monomeric forms of recombinant mouse E-cadherin-human immunoglobulin Fc chimera self-associate and support attachment of E-cadherin+ mouse colon epithelial cells. Both forms also support the adhesion of mouse MTC-1 T cells via M290, thereby establishing M290 as the functional mouse homologue of HML-1 and revealing that E-cadherin homophilic and heterophilic binding sites are distinct. Adhesion of MTC-1 cells to E-cadherin-Fc was inhibited by arginine-glycine-aspartate (RGD) peptides and vice versa cells bound to immobilized RGD polymer in an M290-dependent fashion, where adhesion was inhibitable with soluble E-cadherin-Fc. Hence, E-cadherin and RGD integrin ligands antagonize cell binding by one another, either by inducing integrin cross-talk or by binding to shared or overlapping sites within M290. Binding of E-cadherin-Fc by HML-1 costimulated the CD3-induced proliferation of purified CD4+ T cells, suggesting that E-cadherin expressed on dendritic cells may play a T cell costimulatory role in addition to facilitating dendritic cell-keratinocyte adhesion.     

Studies of Lewis antigens and H. pylori adhesion in CHO cell lines engineered to express Lewis b determinants

Many microbes bind and adhere via adhesins to host cell carbohydrates as an initial step for infection. Therefore, cell lines expressing Lewis b (Le(b)) determinants were generated as a potential model system for Helicobacter pylori colonization and infection, and their expression of blood group Lewis determinants was characterized. CHO-K1 cells were stably transfected with selected glycosyltransferase cDNAs, and two Le(b) positive clones, 1C5 and 2C2, were identified. Expression of Lewis (Le(a), Le(b), Le(x), and Le(y)) determinants was analyzed by flow cytometry of intact cells, SDS-PAGE/Western blot of solubilized glycoproteins, and thin layer chromatography immunostaining of isolated glycolipids (GL). Binding of H. pylori to cells was examined by microscopy and quantified. Flow cytometry showed that 1C5 and 2C2 were Le(a) and Le(b) positive. 1C5 expressed Le(b) on O-linked, but not N-linked, glycans and only weakly on GLs. In contrast, 2C2 expressed Le(b) on N-, O-glycans, and GLs. Furthermore, both clones expressed Le(a) on N- and O-glycans but not on GLs. 2C2, but not 1C5, stained positively for Le(y) on N-linked glycans and GLs. Both clones, as well as the parental CHO-K1 cells, expressed Le(x) on GLs. A Le(b)-binding H. pylori strain bound to the 1C5 and 2C2 cells. In summary, two glycosyltransferase transfected CHO-K1 cell clones differed regarding Lewis antigen expression on N- and O-linked glycans as well as on GLs. Both clones examined supported adhesion of a Le(b)-binding H. pylori strain and may thus be a useful in vitro model system for H. pylori colonization/infection studies.     

Cytophilic binding of IgE to the macrophage. III. Involvement of cyclic GMP and calcium in macrophage activation by dimeric or aggregated rat myeloma IgE

Discharge of lysosomal enzymes, measured by release of β-glucuronidase, was studied in uninduced rat macrophages stimulated in vitro with rat monoclonal IgE (IR 162) in different states of aggregation. Monomeric IgE showed negligible activity, while dimeric and aggregated IgE were shown to induce a rapid and selective release of β-glucuronidase as well as new synthesis of the enzyme, without change in the cytoplasmic marker, leucine aminopeptidase. Lysosomal enzyme release is related to the dose of dimeric IgE, becoming maximal above 2.5 μg/ml. β-Glucuronidase release from macrophages by dimers is competitively inhibited by monomeric IgE but only at high ratios, approximately 100-fold greater than those needed to block mast cell release of the same enzyme. The difference in inhibitability is consistent with the difference in binding affinity of macrophages and mast cells for monomeric IgE. This observation rules out the participation of the few remaining mast cells contained in the macrophage monolayer in β-glucuronidase release. Dimeric or aggregated IgE produced a rise in cyclic GMP coincident with the peak fixation of IgE by macrophages. Elevation of cyclic GMP by pharmacological means also stimulated β-glucuronidase release and new synthesis, as well as enhancing the effect on these of aggregated IgE. Enzyme release by IgE did not occur in the absence of extracellular calcium. We conclude that IgE, which has been cross-linked to form dimers before binding to specific macrophage receptors, triggers the cell and that cyclic GMP (and perhaps calcium) modulates the early step of macrophage activation.