Aggregation of complement receptors on human neutrophils in the absence of ligand

C3bi receptors (CR3) on human polymorphonuclear leukocytes (PMN) bind ligand-coated particles and promote their ingestion. The binding activity of CR3 is not constitutive but is transiently enabled by phorbol esters (Wright, S. D., and B. D. Meyer, 1986, J. Immunol. 136:1759-1764). Our observations indicate that the capacity of CR3 to bind ligand is tightly correlated with the degree of ligand-independent aggregation of the receptor in the plane of the membrane. Fixed PMN were labeled with anti-CR3 monoclonal antibodies and streptavidin colloidal gold before viewing in the electron microscope either en face or in thin section. On unstimulated PMN, gold particles marking CR3 were dispersed randomly. Stimulation of PMN for 25 min with phorbol myristate acetate (PMA) dramatically enhances binding of C3bi-coated particles, and the CR3 on such stimulated cells was observed in clusters containing more than six gold particles. CR3 was not aggregated over coated pits. After 50 min in PMA, the binding activity of CR3 falls, and the distribution of CR3 was again observed to be disperse. If a hydrophilic phorbol ester was washed away after a 20-min stimulation, binding activity remains elevated for at least 50 min, and CR3 remained aggregated. Thus, clustering of CR3 was temporally correlated with its ability to bind ligand and initiate phagocytosis. Unlike CR3, Fc receptors and HLA did not exhibit changes in their aggregation state in response to PMA. Treating PMN with formyl-methionyl-leucyl-phenylalanine, which enhances expression of CR3 but not its function, did not lead to aggregation of CR3. These observations suggest that a clustered configuration is a precondition necessary for binding ligand and signaling phagocytosis.     

Phorbol esters cause sequential activation and deactivation of complement receptors on polymorphonuclear leukocytes

Phorbol myristate acetate (PMA) exerts a biphasic effect on receptors for C3b and C3bi of human polymorphonuclear leukocytes (PMN). The addition of PMA for 10 min enhances the capacity of these receptors to promote binding and phagocytosis of C3b- and C3bi-coated erythrocytes. Upon additional incubation for 60 min, the capacity of these receptors to bind and ingest ligand-coated erythrocytes decreases to levels below those of resting cells. Although PMA does cause increased expression of cell surface C3b and C3bi receptors, the sequential rise and fall of receptor activity cannot be accounted for by alterations in the number of surface receptors. It appears, rather, that PMA causes qualitative changes in these receptors, first an increase in receptor activity (activation) and then a decrease in receptor activity (deactivation). In contrast with its effects on C3 receptors, PMA causes only a reduction in the capacity of Fc receptors to bind and ingest IgG-coated erythrocytes. Deactivation of Fc receptors also appears to involve a qualitative alteration in receptors, because the binding affinity of soluble immune complexes is sharply reduced upon stimulation of PMN with PMA. To explore the role of phosphorylation in the sequential activation and deactivation of phagocytosis-promoting receptors, we loaded PMN with thiophosphate (thioP). This compound is incorporated into cellular nucleotides and proteins, and the resultant (thio)phosphorylated proteins are resistant to phosphatases. thioP-loaded cells show enhanced receptor activity, suggesting that activation of receptors is mediated by a phosphorylation event. Cells loaded with thioP and treated with PMA for 70 min do not deactivate C3 or Fc receptors, suggesting that the deactivation is the result of a dephosphorylation event.     

Amino acid sequences within the alpha subunit of integrin alpha M beta 2 (Mac-1) critical for specific recognition of C3bi.

Phagocytosis of opsonized particles by neutrophils and monocytes plays a central role in host defense mechanisms against foreign pathogens. This process depends on the interaction between C3bi, a degradation product derived from activation of the complement system, and the alpha M beta 2 (CD11b/CD18, Mac-1) receptor, the major integrin on neutrophils. Previous studies had established a central role for the I domain, a stretch of approximately 200 amino acids within the alpha M subunit in the binding of C3bi, as well as many other alpha M beta 2 ligands. The present study was undertaken to establish the molecular basis of C3bi recognition by alpha M beta 2. The strategy employed the use of a series of mutant receptors in which short segments of the I domain of alpha M were switched to the corresponding segments of alpha L, which is structurally very similar but does not bind C3bi. We report three major findings: (1) The C3bi binding pocket is composed of three regions, P147-R152, P201-K217, and K245-R261 of alpha M, which surround the cation binding site within the MIDAS motif of the I domain. (2) Within the latter segment, K245 plays a critical role in mediating C3bi binding to alpha M beta 2. Mutation of K245 to Ala significantly reduced C3bi binding but had no effect on binding of another alpha M beta 2 I domain ligand, NIF. (3) Blocking of C3bi binding to alpha M beta 2 by monoclonal antibodies is achieved through two different mechanisms: direct competition for the ligand binding site or induction of conformational changes. Overall, these studies support the hypothesis that many of the ligands of alpha M beta 2 bind to overlapping but not identical sites within the I domain. Although the same short structural segments within the I domain may be involved in binding, different amino acids within these segments may contact different ligands.     

The effect of fibronectin on the processing of C1q- and C3b/bi-coated immune complexes by peripheral blood monocytes

In the past several years, it has been demonstrated that plasma fibronectin (Fn) binds to the C1q subunit of the complement system. The effect of Fn on the processing of immune complexes containing C1q and C3b by human peripheral blood monocytes was investigated. Preincubation of monocytes with Fn causes a significant increase in attachment of sheep erythrocytes coated with IgM and C1q (EIg-MC1q), but does not mediate their ingestion. EIg-MC1q attach to the Fn-treated monocytes via the C1q receptor because Fab anti-Fn antibodies do not inhibit their attachment to the monocytes. In addition, Fn-treated monocytes exhibit no change in C1q receptor number or affinity compared with monocytes treated with buffer. Fn mediates the phagocytosis of C3b/bi-coated particles, and C1q can enhance this process in two ways. First, phagocytosis of particles bearing C3b/bi and Fn is enhanced by the presence of C1q on the immune complex. Second, monocytes on Fn-coated surfaces ingest more particles if they are coated with both C3b/bi and C1q, compared with particles coated with either C3b/bi or C1q alone.     

Specific receptor sites for 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) on rabbit platelet and guinea pig smooth muscle membranes

By using tritiated 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (3H-PAF), we have directly identified its specific binding sites on rabbit platelet plasma membranes. The equilibrium dissociation constant for 3H-PAF is 1.36 (+/- 0.05) X 10(-9) M at 0 degrees C. The number of binding sites is 1.61 (+/- 0.34) X 10(12)/mg of membrane, which corresponds to approximately 150-300 receptors/platelet (depending on membrane vesicle orientation). Binding of 3H-PAF to rabbit platelet plasma membrane is rapid (t1/2 less than 5 min at 0 degrees C) and reversible. For a series of PAF analogues, their affinity for the receptor sites parallels with their relative potency to induce platelet aggregation. PAF can cause contraction of smooth muscle of heart, parenchymal strip, trachea, and ileum. Specific PAF receptor binding was demonstrated with purified plasma membrane from several smooth muscles and from polymorphonuclear leukocytes but not from presumably PAF nonresponsive cells such as erythrocytes and alveolar macrophages. It is likely that the interaction of PAF with these binding sites initiates the specific responses of platelets, polymorphonuclear leukocytes, and smooth muscles.     

Characterization of the C3 receptor induced by herpes simplex virus type 1 infection of human epidermal, endothelial, and A431 cells

Herpes simplex virus type 1 (HSV-1) infection induces the appearance of viral analogues of human Fc IgG and C3 receptors on the surface of human cells. The virally induced C3 receptor(s) has been broadly defined as a C3b receptor, but its ligand binding characteristics have not been rigorously defined. In this study, human epidermal cells, A431 cells, and human umbilical vein endothelial cells infected with HSV-1 demonstrated rosetting with sheep erythrocytes (E) coated with IgG (E-IgG) or the complement components C3b (EAC3b) or iC3b (EAC3bi), but not with E-IgM, C4 (EAC14), C3d (EAC3d), or E alone. Rosetting was markedly enhanced by pretreatment of HSV-1-infected cells with neuraminidase. Unlike human C3 receptors, the HSV-1-induced C3 receptor was found to be trypsin resistant. To determine whether HSV-1 induced CR1-like receptors or CR3-like receptors, infected cells were pretreated with EDTA, which is known to inhibit native CR3 function. EDTA failed to prevent rosetting with EAC3bi. Furthermore, blocking studies using monoclonal antibodies against CR1 and CR3 revealed that the anti-CR1 antibody 5C11 consistently blocked EAC3b and EAC3bi rosetting with HSV-1-infected cells in a dose dependent manner, but monoclonal antibodies against CR3 did not. This study indicates that the HSV-1-induced C3 receptor is an analogue of CR1.     

High affinity binding of immunoglobulin G fragments to human polymorphonuclear leukocytes

Human neutrophil elastase splits IgG into Fc, Fabc, and Fab fragments. The Fc and Fabc fragments bind with high affinity (KD 2.1 and 2.5 nM respectively) to a small number of binding sites (1175 and 1370 sites/cell respectively) on untreated human polymorphonuclear leukocytes. Molecular mass determination of the binding site by crosslinking of Fc fragments to the neutrophils followed by SDS electrophoresis yields one band corresponding to a molecular mass of 67 kDa for the binding site. Incubation of neutrophils with rIFN-gamma (50 ng/ml, 18 h, 37 degrees C) enhances the expression of binding sites by about 6 fold to about 14,500 sites/cell, while the binding affinity and the molecular mass of the ligand receptor complex remain constant. By comparison with known affinities of leukocyte Fc receptors it is concluded that IgG fragments bind to the high affinity FcRI receptor of human neutrophils.     

GalNAc/Gal-specific rat liver lectins: their role in cellular recognition

By investigating the presence and distribution of GalNAc/Gal-specific receptors on liver cells in vitro and in vivo, we provided evidence that the hepatocyte is not the only liver cell expressing receptor activity but that receptors of similar specificity are found on liver macrophages and also on endothelial cells. The receptor distribution in the plasma membrane is strinkingly different between the three cell types, as judged from the binding pattern of colloidal gold particles coated with asialofetuin or lactosylated serum albumin. Binding to hepatocytes occurs as single particles statistically distributed, binding to liver macrophages in a clustered arrangement all over the cell membrane and binding to endothelial cells also in a clustered arrangement but restricted to coated pits only. The different receptor distribution results in different binding and uptake abilities. Whereas hepatocytes bind and take up molecules and small particles (5 nm) only, the clustered receptor arrangement of endothelial cells and macrophages enables them to effectively bind and ingest larger particles. Ligands larger than 35 nm can be taken up by the macrophages only. The different receptor arrangement results also in different capacities of cell contact formation. Although in vitro liver macrophages and hepatocytes can both bind desialylated cells the macrophage needs much less galactosyl groups exposed on erythrocytes to establish stable contacts than the hepatocyte. The contacts formed by hepatocytes stay reversible for 30 min at 37 degrees C, whereas the contacts formed by the liver macrophages become irreversible after 10 min at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Native C3 does not bind to the C3b receptor (CR1) of human blood B lymphocytes or alter immunoglobulin synthesis

Complement receptors on lymphocytes were first described more than 12 yr ago (1-3) and have come to be used as a common marker for the identification of B cells (4). The function of these receptors on the lymphocyte and their possible role in induction and/or regulation of the immune response remain unclear. In particular, there continues to be controversy as to whether native C3 can bind to the C3b receptor of these cells without cleavage to C3b (5-10). The resolution of this question is critical in order to clarify the expected state of availability of the receptor in vivo, because in plasma, the C3 concentration is relatively high (1.1 to 1.5 mg/ml), whereas there is little or no circulating C3b due to efficient degradation by factor H and the C3-inactivator (11). With the recent development of an improved method for the isolation of C3 from human plasma, it has been possible to obtain biochemically and functionally pure C3 that has not undergone structural or conformational alteration during processing and fully retains the specific hemolytic activity of C3 in fresh serum (12). Berger et al. (13) were able to demonstrate that C3 prepared in this way failed to bind to the C3b receptor of human polymorphonuclear leukocytes or erythrocytes. Similar observations were made by Schreiber et al. (14), also with phagocytic cells and erythrocytes, and by Dixit et al. (15) with an isolated membrane receptor preparation from rabbit macrophages. In the present communication, we extend these observations to human peripheral blood B lymphocytes. Purified C3 in its native state fails to block B lymphocyte-EA (IgM) C4b3b rosettes, whereas C3b causes 50% inhibition at 5 to 6 micrograms/ml. Furthermore, C3 failed to alter polyclonal immunoglobulin (Ig) production by human B cells, whereas C3b inhibited this B cell function. These data suggest that native C3 does not bind to the C3b receptors of B lymphocytes, and thus they are not occupied under normal conditions in vivo.     

Receptor-mediated particle uptake by liver macrophages. The galactose-particle receptor mediates uptake via coated and also non-coated structures

The endocytosis pathways of particles with terminal beta-D-galactosyl groups were studied in isolated rat Kupffer cells by electron microscopy. Colloidal gold particles of sizes 5, 17 and 50 nm were coated with asialofetuin (ASF) and isolated liver macrophages were allowed to bind (at 4 degrees C) or take up (at 37 degrees C) these ligands. Particles of all three sizes were bound via the galactose-particle receptor as shown by carbohydrate inhibition experiments and were ingested effectively. But, whereas ASF-gold particles of sizes 5 and 17 nm are taken up via the coated pit/coated vesicle pathway, the 50 nm particles are not. These enter the cell via non-coated endocytic vacuoles. All three particle sizes are transported to the same lysosomal compartment. These observations demonstrate that at least in macrophages one receptor is capable to mediate endocytosis via two different pathways depending on ligand size and/or valency.     

Regulation of C5a and formyl peptide receptor expression on human polymorphonuclear leukocytes

Fluorescein conjugates of C5a (FL-C5a) and formyl methionine-leucine-phenylalanine-lysine (FL-FMLPL) have been used to determine how the expression of receptors for these peptides is regulated on human polymorphonuclear leukocytes (PMN). Video intensification microscopy showed that receptors for FL-C5a were homogeneously distributed on the surface of the PMN, but within minutes were mobilized into patches and internalized by the PMN. Internalization of C5a receptors was confirmed in studies in which external FL-C5a fluorescence was quenched by reducing the pH. A similar rapid internalization was observed with FL-FMLPL. This process was inhibited for both fluorescent ligands by monensin. Reexpression of C5a and formyl peptide receptors after internalization occurred with both receptors. By comparison, the rate of reexpression of formyl peptide receptors was much faster than that observed with C5a receptors with the half maximal reexpression time for each being 5 to 10 min and 18 to 60 min, respectively. C5a receptor reexpression was completely blocked by monensin suggesting receptor recycling, whereas monensin had little effect on FMLPL receptor reexpression. The reexpression of both receptors occurred in the presence of cycloheximide indicating that this process occurred independent of protein synthesis. Additional studies on formyl peptide receptor showed that when PMN were treated with ionomycin to fully mobilize the intracellular pool of FMLPL receptors, receptor reexpression failed to occur. These studies show that both C5a and formyl peptide receptors are internalized after binding ligand, but that their reexpression occurs through different mechanisms. C5a receptors appear to be recycled to the cell surface whereas formyl peptide receptors are reexpressed predominantly by translocation from an intracellular pool.     

Osmotic stress and the freeze-thaw cycle cause shedding of Fc and C3b receptors by human polymorphonuclear leukocytes

A major problem in the cryopreservation of human polymorphonuclear leukocytes (PMN) is the loss of phagocytic function in cryopreserved cells. This is not a problem with cryopreserved monocytes. To study the reasons for this difference in detail, PMN and monocytes were either osmotically stressed in hypertonic media or were frozen to various temperatures. Cells were then returned to conditions of physiologic osmolarity and temperature. All cells remained viable. However, the ability of PMN to phagocytize bacteria and to bind sheep erythrocytes (E) opsonized with IgG, C3b, or C3bi decreased sharply after exposure to media of 600 mOsM or greater and after freezing to -1.5 degrees C. In contrast, monocytes were unaffected until a concentration of 1500 mOsM or a freezing temperature of -5 degrees C was exceeded. To determine whether the functional losses of surface receptor activity in PMN resulted from a loss of receptors from the membranes or from inactivation or internalization of receptors, opsonized E were incubated in the supernatants from stressed PMN. On subsequent incubation with healthy PMN, these E made fewer rosettes than control opsonized E. The inhibitory effect of the supernatants on rosetting of IgG-sensitized E could be removed by preincubation with IgG bound to Sepharose 4B. Immunoprecipitation of C3b and C3bi receptors from surface-iodinated, osmotically stressed, and control PMN suggested that about 50% of cell surface complement receptors were lost from the cell surface during osmotic stress. These experiments suggest that receptors for IgG and C3 are extruded from PMN cell membranes as a result of hyperosmotic stress, which is associated with the freeze-thaw cycle. This may be an early event in the functional damage done to PMN during attempts at cryopreservation.     

Carbohydrates on human Fc gamma receptors. Interdependence of the classical IgG and nonclassical lectin-binding sites on human Fc gamma RIII expressed on neutrophils

To explore the molecular basis for the ability of aggregated IgG to block the phagocytosis by human polymorphonuclear leukocytes of Con A-opsonized E and of nonopsonized Escherichia coli with mannose-binding adhesins, we examined specific aspects of the glycoprotein structure of both the 40- to 43-kDa receptor for the Fc portion of IgG (Fc gamma RII) and the 50- to 78-kDa receptor for the Fc portion of IgG (Fc gamma RIIIPMN) from human polymorphonuclear leukocytes. Fc gamma RIIIPMN isolated by both mAb and ligand affinity chromatography, but not Fc gamma RII, binds Con A in Western blots. This binding is specifically inhibitable by alpha-methylmannoside. Digestion of Fc gamma RIIIPMN by recombinant endoglycosidase H, which is specific for high mannose-type (Con A-binding) oligosaccharides, alters the epitope recognized by mAb 3G8 in or near the IgG ligand-binding site of the receptor. Similarly, the ability of Fc gamma RIIIPMN to bind human IgG ligand is sensitive to endoglycosidase H digestion. Our data indicate that ligands other than the classical IgG opsonins can bind to human Fc gamma RIIIPMN per se through lectin-carbohydrate interactions. Furthermore, Fc gamma RIIIPMN contains a high mannose type oligosaccharide chain which contributes importantly to the integrity of the classical IgG ligand-binding site. Thus, specific glycosylations of the receptor are important for both classical and nonclassical engagement of Fc gamma RIII and may play a role in determining the properties of the ligand-binding site.     

Effect of membrane fluidizers on the number and affinity of chemotactic factor receptors on human polymorphonuclear leukocytes

Chemotaxis by leukocytes appears to be initiated by the binding of chemo-attractants to specific cell surface receptors. In other biological systems, the affinity and functional activity of membrane receptors are regulated by the local microviscosity. The present studies were undertaken to determine if the number and/or affinity of chemotactic factor receptors expressed on human polymorphonuclear leukocytes were similarly affected. Aliphatic alcohols and cis-vaccenic acid, agents known to decrease membrane microviscosity, were studied for their effects on the binding of the radiolabeled chemoattractant f-Met-Leu-[3H]Phe to human polymorphonuclear leukocytes. Butanol and propanol increased the number of f-Met-Leu-[3H]Phe binding sites approximately 1.5 fold. More dramatically, these same agents enhanced the affinity of the receptor by ten-fold, without affecting the specificity of the receptor. Similarly, cis-vaccenic acid enhanced both the number and affinity of this chemotactic factor receptor on human polymorphonuclear leukocytes contain cryptic receptors for the N-formylated peptide chemotactic factors, but more importantly that the affinity of these receptors can exist in more than one state and can be modulated by membrane microviscosity. Alterations of membrane fluidity in leukocytes during chemotaxis may be an important mechanism for regulating their sensitivity to chemoattractants.     

Binding of fluid-phase complement components C3 and C3b to human lymphocytes.

It is known that a population of B-lymphocytes has receptors for the third component of complement, C3, and that these lymphocytes may be identified by their ability to form rosettes with sheep erythrocytes coated with covalently bound fragments of complement component C3. Human tonsil lymphocytes, enriched for B-cells, form rosettes with sheep erythrocytes coated with antibody and complement components C1, C4b and C3b (EAC143b cells). Fluid-phase C3 will inhibit rosette formation between EAC143b and human tonsil lymphocytes over the same concentration range as fluid-phase C3b. C3 is not cleaved to C3b during incubation with lymphocytes or with lymphocytes and EAC143b cells. Fluid-phase 125I-labelled C3 and 125I-labelled C3b bind to lymphocytes in a specific manner. The characteristics of binding of both radioiodinated C3 and radioiodinated C3b are very similar, but the binding oc C3 is again not a result of cleavage to C3b. Salicylhydroxamic acid does not inhibit binding of 125I-labelled C3 to tonsil lymphocytes at concentrations that completely inhibit binding of 125I-labelled C3 to EAC142 cells via the nascent binding site of C3b. It is concluded that C3 and C3b share a common feature involved in binding to lymphocytes bearing receptors for the third component of complement.     

The interaction of small oligomers of complement 3B (C3B) with phagocytes. High affinity binding and phorbol ester-induced internalization by polymorphonuclear leukocytes

The binding of soluble, multimeric ligands of the major cleavage fragment of complement component 3 (C3b) to polymorphonuclear leukocytes (PMN) has been examined. The oligomers bound entirely via complement C3 receptor type 1 (CR1). There was a single affinity of binding (0.65 nM) at 37 degrees C, while this high affinity binding accounted for only a minority of ligand bound at 0 degree C, with the rest showing a 50-100-fold lower affinity. Azide, fluoride, cytochalasin B, and colchicine had no effect on oligomer binding to PMN. Binding of oligomers had no effect on CR1 expression by PMN. C3b oligomers were not spontaneously internalized by PMN, but were internalized in response to phorbol dibutyrate (PDBu). Both CR1 initially present on the PMN plasma membrane and CR1 initially present in the internal pool of receptors were able to participate in PDBu-induced ligand internalization. C3b oligomers attached to the detergent-insoluble cell cytoskeleton during incubation at 37 degrees C, but cytochalasin B did not inhibit PDBu-induced ligand internalization. Internalized ligand was no longer associated with the detergent-insoluble cytoskeleton. These data demonstrate that 1) some CR1 diffusion is required for optimal oligomer binding; 2) high affinity ligand is not a signal for plasma membrane expression of the internal pool of CR1; 3) CR1 cross-linking is not a sufficient signal for endocytosis; and 4) functional CR1 association with the cytoskeleton which occurs at the plasma membrane is not required for ligand internalization.     

Identification of CC chemokine receptor 7 residues important for receptor activation

The binding pocket of family A GPCRs that bind small biogenic amines is well characterized. In this study we identify residues on CC chemokine receptor 7 (CCR-7) that are involved in agonist-mediated receptor activation but not in high affinity ligand binding. The mutations also affect the ability of the ligands to induce chemotaxis. Two of the residues, Lys3.33(137) and Gln5.42(227), are consistent with the binding pocket described for biogenic amines, while Lys3.26(130) and Asn7.32(305), are found at, or close to, the cell surface. Our observations are in agreement with findings from other peptide and chemokine receptors, which indicate that receptors that bind larger ligands contain contact sites closer to the cell surface in addition to the conventional transmembrane binding pocket. These findings also support the theory that chemokine receptors require different sets of interactions for high affinity ligand binding and receptor activation.     

An immobile subset of plasma membrane CD11b/CD18 (Mac-1) is involved in phagocytosis of targets recognized by multiple receptors

CD11b/CD18 is a heterodimeric leukocyte surface receptor which functions in both C3bi-ligand binding and homotypic and heterotypic cell adherence. We have examined the effect of several anti-CD11b/18 mAb on phagocytosis of IgG (EIgG) or complement (EC4b) opsonized erythrocytes by polymorphonuclear leukocytes (PMN) and monocytes. F(ab')2 of two mAb (IB4, an anti-beta-chain mAb and Mo-1 an anti-alpha-chain mAb), inhibited both phagocytosis of EIgG and phorbol ester-stimulated phagocytosis of EC4b by PMN and monocytes. These F(ab')2 inhibited the binding of EIgG to monocytes, but they had no effect on binding of EIgG to PMN, or EC4b to either phagocyte. In addition, IB4 inhibited phorbol-ester stimulated phagocytosis of sheep E opsonized with C component 3bi (EC3bi) without inhibiting rosetting of these same targets. These data separate the anti-phagocytic effect of these mAb from effects on phagocyte-target adherence. When PMN were adherent to an anti-CD11b/CD18 F(ab')2-coated surface, EC3bi binding was abolished, but phagocytosis of EIgG or EC4b was unaffected. Subsequent addition of fluid- phase IB4 or Mo-1 F(ab')2 inhibited phagocytosis of EIgG or EC4b by the adherent cells. This suggested that the CD11b/CD18 involved in C3bi rosetting were mobile in the membrane, whereas those involved in phagocytosis of EIgG or EC4b were not. Cytochalasin treatment of PMN during adherence to F(ab')2-coated plates decreased both apical expression of CD11b/18 and subsequent ingestion of EIgG by 70%, suggesting that microfilaments are important in maintaining immobile CD11b/18 on the apical PMN surface. We conclude that there are functionally distinct populations of CD11b/CD18 on monocytes and PMN: one involved in C3bi rosetting and another involved in the process of phagocytosis mediated via several different receptors. CD11b/18 is not required for optimal target binding in all cases, but is always required for ingestion. As with several other integrins, the CD11b/18 molecules involved in phagocytosis have a functional association with the cell cytoskeleton.     

Identification of a second putative receptor of platelet-activating factor from human polymorphonuclear leukocytes

Due to multiple molecular species of platelet-activating factor (PAF) and the existence of high affinity binding sites in a variety of cells and tissues, possible existence of PAF receptor subtypes has been suggested. This report shows differences between specific PAF receptors in human leukocytes and platelets. Human polymorphonuclear leukocyte membranes showed high affinity binding sites for PAF with an equilibrium dissociation constant (KD) of 4.4 (+/- 0.3) x 10(-10) M. We compared the relative potencies of several PAF agonists and receptor antagonists between human platelet and human leukocyte membranes. One receptor antagonist (Ono-6240) was found to be 6-10 times less potent in inhibiting the specific [3H]PAF receptor binding, PAF-induced GTPase activity, as well as the PAF-induced aggregation in human leukocytes than in human platelets. Mg2+, Ca2+, and K+ ions potentiated the specific [3H]PAF binding in both systems. Na+ and Li+ ions inhibited the specific [3H]PAF binding to human platelets but showed no effects in human leukocytes. K+ ions decreased the Mg2+-potentiated [3H]PAF binding in human leukocytes but showed no effects in human platelets. PAF stimulates the hydrolysis of [gamma-32P] GTP with an ED50 of about 1 nM, whereas the biological inactive enantiomer shows no activity even at 10 microM in both human platelets and human leukocytes. The PAF-stimulated GTPase in human leukocytes can be abolished by the pretreatment of membranes with pertussis toxin and cholera toxin. However, the PAF-stimulated activity of GTPase in human platelets is insensitive to pertussis toxin and cholera toxin. These results suggest that there exists a second type of PAF receptor in human polymorphonuclear leukocytes, which is structurally different from the one characterized in human platelets, and that the guanine nucleotide-binding protein coupled to PAF receptors in human leukocytes is also different from the one in human platelets.     

Reversible Inactivation of Purified Leukocyte Integrin CR3 (CD11b/CD18, βmβ2) by Removal of Divalent Cations from a Cryptic Site

Integrins exhibit reversible changes in their ability to bind ligands and these changes enable transient cell adhesion. We recently showed that leukocyte integrin CR3 (complement receptor type three, CD11b/CD18, α_mβ₂) may be purified in a form that is either capable or incapable of binding soluble, monomeric ligand and that “inactive” CR3 may be rendered capable of binding ligand by addition of an anti-CR3 mAb known as KIM-127 (Cai and Wright, JBC. 270: 14358, 1995). Here, we demonstrate that active CR3 may be rendered inactive by treatment of immobilized receptor with EDTA. EDTA-treated CR3 failed to bind ligand even in the presence of mM Ca²⁺ and Mg²⁺, suggesting that EDTA-treatment caused a change in the receptor that is not readily reversed. EDTA-treated receptor did, however, bind ligand upon addition of KIM-127 plus Mg²⁺ with an affinity (17.8 ± 4.5 nM) similar to untreated, active receptor (12.5 ± 4.7 nM). EDTA-treated CR3 thus exhibits the properties of inactive CR3, in which the ligand binding site is cryptic but subject to exposure by KIM-127. A candidate for the cryptic ligand binding site is the I-domain, a Mg²⁺-binding region in the α chain of CR3. We found that monomeric C3bi binds directly to recombinant I-domain in a Mg²⁺-dependent fashion with an affinity of 300 ± 113 nM. These results thus suggest that CR3 may be inactivated by removing tightly bound divalent cation from a cryptic site in CR3.