Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14

CD14 is a 55-kD protein found as a glycosylphosphatidylinositol (GPI)- anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, and as a soluble protein in the blood. Both forms of CD14 participate in the serum-dependent responses of cells to bacterial lipopolysaccharide (LPS). While CD14 has been described as a receptor for complexes of LPS with LPS-binding protein (LBP), there has been no direct evidence showing whether a ternary complex of LPS, LBP, and CD14 is formed, or whether CD14 binds LPS directly. Using nondenaturing polyacrylamide gel electrophoresis (native PAGE), we show that recombinant soluble CD14 (rsCD14) binds LPS in the absence of LBP or other proteins. Binding of LPS to CD14 is stable and of low stoichiometry (one or two molecules of LPS per rsCD14). Recombinant LBP (rLBP) does not form detectable ternary complexes with rsCD14 and LPS, but it does accelerate the binding of LPS to rsCD14. rLBP facilitates the interaction of LPS with rsCD14 at substoichiometric concentrations, suggesting that LBP functions catalytically, as a lipid transfer protein. Complexes of LPS and rsCD14 formed in the absence of LBP or other serum proteins strongly stimulate integrin function on PMN and expression of E-selectin on endothelial cells, demonstrating that LBP is not necessary for CD14-dependent stimulation of cells. These results suggest that CD14 acts as a soluble and cell surface receptor for LPS, and that LBP may function primarily to accelerate the binding of LPS to CD14.     

Lipopolysaccharide (LPS) recognition in macrophages. Participation of LPS-binding protein and CD14 in LPS-induced adaptation in rabbit peritoneal exudate macrophages.

Exposure of rabbit peritoneal exudate macrophages (PEM) or whole blood to picomolar concentrations of LPS induces adaptation or hyporesponsiveness to LPS. Because of the importance of plasma LPS-binding protein (LBP) and the macrophage cell membrane protein CD14 in recognition of LPS, we examined the effect of LBP on LPS-induced adaptation in PEM. PEM exposed to LPS in the presence of LBP for 8 h were markedly less responsive to subsequent stimulation by LPS than monocytes/macrophages (M phi) adapted in the absence of LBP. LPS-induced expression of TNF was sharply reduced in LBP-LPS-adapted PEM, but in contrast these cells remained fully responsive to Staphylococcus aureus peptidoglycan. We considered that specific hyporesponsiveness in LPS-adapted M phi or in blood monocytes could be due to decreased expression of CD14 or diminished binding of LBP-LPS complexes to CD14. However, flow cytometry analysis revealed only minimal reduction of CD14 expression or CD14-dependent binding of a fluorescent LPS derivative when normo- and hyporesponsive cells were compared. These results show that complexes of LPS and LBP are more effective than LPS alone in inducing adaptation to LPS, and LPS-induced hyporesponsiveness probably results from changes in cellular elements distinct from CD14 that are involved in either LPS recognition or LPS-specific signal transduction.     

Activation of the adhesive capacity of CR3 on neutrophils by endotoxin: dependence on lipopolysaccharide binding protein and CD14

Tumor necrosis factor alpha, granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating factor, and formyl peptide were each found to cause a twofold increase in expression of CD14 on the surface of polymorphonuclear leukocytes (PMN). Upregulation of CD14 was complete by 20 min and thus appeared to result from expression of preformed stores of protein. The CD14 on the surface of PMN was shown to serve two biological functions. It bound particles coated with complexes of lipopolysaccharide (LPS) and LPS binding protein (LBP). This binding activity was enhanced by agonists that upregulated CD14 expression and may serve in the clearance of Gram-negative bacteria opsonized with LBP. Interaction of CD14 with LPS in the presence of LBP or serum also caused a dramatic, transient increase in the adhesive activity of CR3 (CD11b/CD18) on PMN. Enhanced activity of CR3 and other members of the CD11/CD18 family underlies many of the known physiological responses of PMN to LPS and may be a central feature of the in vivo responses of PMN to endotoxin.     

In the absence of other Fc receptors, Fc gamma RIIIA transmits a phagocytic signal that requires the cytoplasmic domain of its gamma subunit.

The transmembrane isoform of Fc gamma RIII, Fc gamma RIIIA, is found on NK cells, cultured monocytes, and tissue macrophages in association with a dimer of an accessory subunit, either gamma or zeta. Functions of individual Fc receptors have been difficult to analyze due to coexpression of the receptors on hematopoietic cells and permanent cell lines expressing Fc receptors. cDNAs for the alpha and gamma subunits of Fc gamma RIIIA were cotransfected into COS-1 cells, which lack endogenous Fc receptors, to evaluate receptor-mediated phagocytosis and changes in [Ca2+]i. Transfectants both bound and phagocytosed IgG-sensitized erythrocytes and, following activation of Fc gamma RIIIA, increased [Ca2+]i. The gamma subunit was essential both for the surface expression of the receptor and for transduction of the phagocytic signal. Truncation of the gamma subunit cytoplasmic domain (amino acids 65-80) eliminated phagocytic function. Phorbol ester inhibited phagocytosis in a concentration-dependent manner, but did not affect IgG-sensitized erythrocytes binding, suggesting that a protein kinase C-dependent pathway inhibits phagocytosis. The data indicate that a tyrosine containing cytoplasmic domain within the gamma subunit is required for phagocytosis by Fc gamma RIIIA.     

A regulatory role for Src homology 2 domain-containing inositol 5'-phosphatase (SHIP) in phagocytosis mediated by Fc gamma receptors and complement receptor 3 (alpha(M)beta(2); CD11b/CD18)

The Src homology 2 domain-containing inositol 5'-phosphatase (SHIP) is recruited to immunoreceptor tyrosine-based inhibition motif (ITIM)-containing proteins, thereby suppressing phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathways. The role of SHIP in phagocytosis, a PI 3-kinase-dependent pathway, is unknown. Overexpression of SHIP in macrophages led to an inhibition of phagocytosis mediated by receptors for the Fc portion of IgG (Fc gamma Rs). In contrast, macrophages expressing catalytically inactive SHIP or lacking SHIP expression demonstrated enhanced phagocytosis. To determine whether SHIP regulates phagocytosis mediated by receptors that are not known to recruit ITIMs, we determined the effect of SHIP expression on complement receptor 3 (CR3; CD11b/CD18; alpha(M)beta(2))-dependent phagocytosis. Macrophages overexpressing SHIP demonstrated impaired CR3-mediated phagocytosis, whereas macrophages expressing catalytically inactive SHIP demonstrated enhanced phagocytosis. CR3-mediated phagocytosis in macrophages derived from SHIP(-/-) mice was up to 2.5 times as efficient as that observed in macrophages derived from littermate controls. SHIP was localized to Fc gamma R- and CR3-containing phagocytic cups and was recruited to the cytoskeleton upon clustering of CR3. In a transfected COS cell model of activation-independent CR3-mediated phagocytosis, catalytically active but not inactive SHIP also inhibited phagocytosis. We conclude that PI 3-kinase(s) and SHIP regulate multiple forms of phagocytosis and that endogenous SHIP plays a role in modulating beta(2) integrin outside-in signaling.     

Transfection of CD14 into 70Z/3 cells dramatically enhances the sensitivity to complexes of lipopolysaccharide (LPS) and LPS binding protein

Bacterial endotoxin (lipopolysaccharide [LPS]) causes fatal shock in humans and experimental animals. The shock is mediated by cytokines released by direct LPS stimulation of cells of monocytic origin (monocyte/macrophage [MO]). Recent studies have supported the concept that the plasma protein, LPS binding protein (LBP), plays an important role in controlling MO responses to LPS. Specifically, evidence has been presented to suggest that CD14, a membrane protein present in MO, serves as a receptor for complexes of LPS and the plasma protein LPS binding protein (LBP). In this function CD14 mediates attachment of LPS-bearing particles opsonized with LBP and appears to play an important role in regulating cytokine production induced by complexes of LPS and LBP. The CD14-, murine pre-B cell line 70Z/3 responds to LPS by synthesis of kappa light chains and consequent expression of surface IgM. To better understand the role of CD14 in controlling cellular responses to LPS, we investigated the effect of transfection of CD14 into 70Z/3 cells on LPS responsiveness. We report here that transfection of human or rabbit CD14 cDNA into 70Z/3 cells results in membrane expression of a glycosyl-phosphatidylinositol-anchored CD14. When LPS is complexed with LBP, CD14-bearing 70Z/3 cells bind more LPS than do the parental or 70Z/3 cells transfected with vector only. Remarkably, the expression of CD14 lowers the amount of LPS required to stimulate surface IgM expression by up to 10,000-fold when LPS dose-response curves in the CD14-, parental and CD14-bearing, transfected 70Z/3 cells are compared. In contrast, the response of CD14-bearing 70Z/3 cells and the parental 70Z/3 cell line (CD14-) to interferon gamma is indistinguishable. LPS stimulation of the parental and CD14-bearing 70Z/3 cells results in activation of NF-kB. These data provide evidence to support the concept that the LPS receptor in cells that constitutively express CD14 may be a multiprotein complex containing CD14 and membrane protein(s) common to a diverse group of LPS-responsive cells.     

Autoimmune mice make anti-Fc gamma receptor antibodies

We demonstrate, using a recombinant truncated Fc gamma RII molecule as a probe, the presence of anti-Fc gamma R antibodies in several strains of autoimmune mice. Affinity chromatography on a truncated Fc gamma R column of pooled sera from aged NZB females resulted in isolation of 16 micrograms of IgM per ml of serum, approximately 2% of the total IgM; no anti-Fc gamma R IgM was found in sera from C58/J mice. Mice with high titers of anti-Fc gamma R IgM also had anti-Fc gamma R IgG. Affinity-purified anti-Fc gamma R IgG bound to Fc gamma R-bearing cells. A good correlation was found between the presence of anti-Fc gamma R Ig and impaired phagocytosis of immune complexes in autoimmune strains such as NZB or NZB/NZW F1. Sera with high titers of anti-Fc gamma R Ig from NZB and motheaten mice inhibited the binding of soluble immune complexes. Furthermore, BXSB, a lupus-prone mouse strain that does not produce anti-Fc gamma R Ig, shows normal macrophage binding and phagocytosis of immune complexes. A set of four IgM mAbs that bind to Fc gamma R was identified. These antibodies were polyspecific; some were directed against DNA, and others recognized a wide variety of antigens including histones, thyroglobulin, and transferrin, but all anti-Fc gamma R IgM antibodies effectively inhibited the binding of IgG1 anti-DNP/DNP20BSA complexes to J774 macrophages. The role of anti-Fc gamma R Ig in autoimmunity remains to be established. It may act to crosslink and activate Fc gamma Rs on neutrophils, macrophages, NK, and mesangial cells, or it may desensitize Fc gamma R function of Fc gamma R-bearing cells.     

CR3 (CD11b/CD18) expresses one binding site for Arg-Gly-Asp-containing peptides and a second site for bacterial lipopolysaccharide

Polymorphonuclear leukocytes (PMN) from three patients deficient in the CD18 family of receptors (LFA-1, CR3, and p150,95) exhibited an inability to bind erythrocytes coated with C3bi or bacterial LPS. These observations confirm that the CD18 family, and CR3 in particular, can bind the structurally dissimilar molecules C3bi and LPS. Further studies showed that LPS and C3bi bind to CR3 at distinct sites. mAb OKM10 against CR3 blocked binding of C3bi to PMN but did not block the binding of LPS. In contrast, mAb 904, directed against a different epitope on CR3, blocked binding of LPS to PMN but not binding of C3bi, thus suggesting that different regions of CR3 were involved in binding these two ligands. In addition, synthetic peptides based on the sequence in C3bi recognized by CR3 competitively blocked the binding of C3bi to CR3 but did not block the binding of LPS. Rather, occupation of the peptide binding site on CR3 by the synthetic peptides enhanced binding of LPS. These results indicate that CR3 has two distinct binding sites, one that recognizes ligands composed of protein and a second that recognizes LPS.     

Allelic polymorphisms of human Fc gamma receptor IIA and Fc gamma receptor IIIB. Independent mechanisms for differences in human phagocyte function.

Two different allelic polymorphisms among the isoforms of human Fc gamma receptors have been defined: the low-responder (LR)-high-responder (HR) polymorphism of huFc gamma RIIA expressed on both PMN and monocytes and the NA1-NA2 polymorphism of the neutrophil Fc gamma RIII (huFc gamma RIIIB). To address the issues of whether the LR-HR polymorphism has a significant impact on Fc gamma R-mediated functions in human blood cells and whether any differences in LR-HR might be related to higher Fc gamma R-mediated phagocytosis in NA1 donors, we examined Fc gamma R-specific binding and internalization by donors homozygous for the two huFc gamma RIIA alleles. PMN from LR homozygotes showed consistently higher levels of internalization of erythrocytes opsonized with pooled human IgG (E-hIgG). The absence of an LR-HR phagocytic difference with erythrocytes opsonized with either anti-Fc gamma RIIA MAb IV.3 or rabbit IgG, as opposed to E-hIgG, suggested that the Fc piece of the opsonin might be important for this LR-HR difference. Accordingly, we studied HR and LR homozygotes with human IgG subclass-specific probes. Both PMN (independent of huFc gamma RIIIB phenotype) and monocytes from LR donors bound and internalized erythrocytes coated with human IgG2 (E-hIgG2) efficiently, whereas phagocytes from HR donors did so poorly. E-hIgG2 internalization was completely abrogated by blockade of the ligand binding site of huFc gamma RIIA with IV.3 Fab, indicating that huFc gamma RIIA is essential for the handling of hIgG2 and that the mechanism of the LR-HR phagocytic difference is at the level of ligand binding to huFc gamma RIIA. In contrast, the difference in internalization of E-hIgG between NA1 and NA2 homozygous donors was independent of the huFc gamma RIIA phenotype and did not manifest differences in ligand binding. Thus, the two known allelic polymorphisms of human Fc gamma R have distinct and independent mechanisms for altering receptor function, which may influence host defense and immune complex handling.     

Human Fc gamma RII, in the absence of other Fc gamma receptors, mediates a phagocytic signal.

Fc gamma receptors are important components in the binding and phagocytosis of IgG-sensitized cells. Studies on the role of these receptors have been limited by the fact that most hematopoietic cells express more than one Fc gamma receptor. We studied the role of Fc gamma RIIA in isolation on a human erythroleukemia cell line (HEL) which expresses Fc gamma RIIA as its only Fc gamma receptor. HEL cells were observed to bind and phagocytose IgG-sensitized red blood cells (RBCs) in a dose-dependent manner. We then examined the role of Fc gamma RI and Fc gamma RII in isolation and in combination, in transfected COS-1 cells. Fc gamma RIIA-transfected COS cells also mediated both the binding and phagocytosis of IgG-sensitized RBCs. In contrast, phagocytosis was not observed in Fc gamma RI-transfected cells, although these cells avidly bound IgG-sensitized RBCs. Furthermore, coexpression of both receptors by doubly transfected cells did not affect the phagocytic efficiency of Fc gamma RIIA. These studies establish that Fc gamma RIIA can mediate phagocytosis and suggest that transfected COS-1 cells provide a model for examining this process. Since HEL cells exhibit characteristics of cells of the megakaryocyte-platelet lineage, including expression of Fc gamma RII as the only Fc gamma receptor, Fc gamma RIIA on megakaryocytes and platelets may be involved in the ingestion of IgG-containing immune complexes. Furthermore, these studies indicate that Fc gamma RI and Fc gamma RIIA differ in their requirements for transduction of a phagocytic signal.     

Inhibition of phagocytosis of complement C3- or immunoglobulin G-coated particles and of C3bi binding by monoclonal antibodies to a monocyte-granulocyte membrane glycoprotein (Mol).

Events that lead to phagocytosis of complement (C3)- or IgG-coated particles after their interaction with specific cell surface receptors are poorly understood. Two mouse monoclonal antibodies (an IgM and an IgG2a) to a human granulocyte-monocyte surface membrane differentiation antigen (Mol) inhibited ingestion by granulocytes both of oil Red O particles opsonized with normal human serum or with IgG and of sheep erythrocytes sensitized with IgG. In addition, they specifically inhibited rosetting between phagocytes and sheep erythrocytes coated with C3bi, a fragment of the complement component C3, generated by cleaving C3b with C3b inactivator and beta IH protein. These monoclonal anti-Mol antibodies did not inhibit IgG Fc, C3b or C3d receptor-mediated binding of erythrocytes coated with the respective proteins. The Fab fragment of the IgG2a monoclonal antibody inhibited noncytotoxic enzyme release from granulocytes when these cells were stimulated with zymosan coated with C3bi. Electrophoretic transfer of polymorphonuclear leukocyte detergent lysates to nitrocellulose, followed by immunofixation with monoclonal antibody, showed that these antibodies were directed to a 155,000-mol wt glycoprotein. This surface membrane structure appears to be involved in Fc and C3 receptor-dependent phagocytosis and closely associated with the C3bi receptor.     

A new Fc receptor on mouse macrophages binding IgG3

Monoclonal antibodies to sheep erythrocytes (SRBC) have proved useful in identifying two Fc receptors on mouse macrophages, one for IgG2a, and one for IgG1 and IgG2b. We have used monoclonal IgG3 anti-SRBC to identify a third Fc receptor on mouse macrophages which binds IgG3 uniquely. This receptor is present on primary resident and thioglycolate-induced peritoneal macrophages and on some macrophage cell lines. The binding of IgG3-coated SRBC is inhibited by aggregated byt not monomeric IgG3, and not by IgG1, IgG2a, and IgG2b aggregates. It is unaffected by treating the macrophages with trypsin or cytochalasin B and occurs at both 4 degrees and 37 degrees C. IgG3, like all other IgG subclasses, mediates phagocytosis. We have also generated a variant macrophage line which bears the receptors for IgG1 and IgG2b and for IgG2a, but not for IgG3.     

In vivo glucocorticoid modulation of guinea pig splenic macrophage Fc gamma receptors.

Although glucocorticoids are widely used in the treatment of immunohematologic disease, their relative efficacy is uncertain. We used an animal model, which has helped to elucidate the role of splenic macrophage Fc gamma receptors in the clearance of IgG-coated cells, to investigate whether each Fc gamma receptor is modulated by glucocorticoids to the same extent and to examine the relative potency of three commonly used glucocorticoids. Cortisol, prednisone, and dexamethasone all impaired the clearance of IgG-coated erythrocytes. However, dexamethasone was more effective than either prednisone or cortisol (P less than 0.001). Furthermore, splenic macrophages isolated from glucocorticoid-treated animals expressed impaired Fc gamma receptor function. This effect was greater in macrophages isolated from dexamethasone-treated animals, as compared to either cortisol- or prednisone-treated animals (P less than 0.001). To assess the effect of glucocorticoids on the two types of guinea pig splenic macrophage Fc gamma receptors, Fc gamma R1,2 and Fc gamma R2, specific immunoglobulin isotypes were used to measure macrophage binding of IgG-sensitized erythrocytes. Cortisol and prednisone primarily affected Fc gamma R2, whereas dexamethasone inhibited the function of both guinea pig Fc gamma receptors. Furthermore, dexamethasone was more effective (P less than 0.01) than either prednisone or cortisol in inhibiting the ability of both receptors to bind IgG-sensitized cells. Fluorescence-activated cell sorter analysis and fluorescence microscopy with monoclonal antibodies specific for each of these two receptors demonstrated that essentially all splenic macrophages expressed both receptors, and that these glucocorticoids decreased the level of each Fc gamma receptor protein expressed, rather than altering receptor mobility and clustering in the macrophage membrane. The effect on both Fc gamma receptors was greatest with dexamethasone and least with cortisol. These studies demonstrate the significant role of guinea pig splenic macrophage Fc gamma R2 in immune clearance and in the binding of IgG-coated cells. They demonstrate a differential effect of glucocorticoid hormones on Fc gamma receptor function and on surface receptor protein. Furthermore, they suggest that dexamethasone may be a more effective glucocorticoid than either prednisone or cortisol in inhibiting the clearance of IgG-coated cells by its effect on splenic macrophage Fc gamma receptors.     

Ontogeny of Fc receptors and complement receptor (CR3) during human myeloid differentiation.

Two different Fc receptors for IgG (Fc gamma R) have been identified on human leukocytes: a high avidity receptor (Fc gamma Rhi) present on monocytes but not on neutrophils, and a low avidity receptor (Fc gamma Rlo) present on neutrophils but not on monocytes. Fc gamma Rlo can be inhibited and the receptor precipitated by monoclonal antibody 3G8. We have used this monoclonal antibody to study the course of Fc gamma Rlo appearance on bone marrow cells, leukocytes of patients with chronic myelogenous leukemia (CML), and HL-60 and U937 cells induced to differentiate with agents such as dimethyl sulfoxide (DMSO), retinoic acid, phorbol myristate acetate, and lymphokine. We report that Fc gamma Rlo is a late differentiation antigen, first expressed at the metamyelocyte stage. Since precursors to metamyelocytes bear Fc gamma R, and the promyelocyte line HL-60 bears Fc gamma Rhi, there must be a progressive loss of Fc gamma Rhi during myeloid differentiation and the reciprocal expression of Fc gamma Rlo. Results of immunoprecipitation and polyacrylamide gel analysis of the proteins are consistent with these results. We have also studied the receptor for the C3bi complement component (CR3), which is blocked and immunoprecipitated by monoclonal antibody OKM10. During DMSO-driven differentiation of HL-60 cells, we find that CR3 is induced on all cells, whereas Fc gamma Rlo is induced on only 24% of cells, suggesting that CR3 appears earlier during differentiation than Fc gamma Rlo does.     

Polymorphisms in the lipopolysaccharide-binding protein and bactericidal/permeability-increasing protein in patients with myocardial infarction.

Gram-negative bacterial infection, namely Chlamydia pneumoniae has been recently discussed as a risk factor for myocardial infarction. The lipopolysaccharide-binding protein (LBP) and the bactericidal/permeability-increasing protein (BPI) play a role in the processes leading to recognition and neutralisation of the Chlamydia pneumoniae and their endotoxins lipopolysaccharides (LPS). LPS interact with plasma LBP, and LBP-LPS complex activates monocytes/macrophages, which can influence the atherosclerotic process. BPI is cytotoxic for Gram-negative bacteria and BPI-LPS complexes do not activate monocytes. We have analysed the polymorphisms in the LBP gene (Gly98-->Cys; Pro436-->Leu) and BPI gene (Lys216-->Glu; PstI polymorphism in intron-5; G545-->C) in 313 patients after myocardial infarction (MI) and in 302 control individuals. Genotype frequencies in the LBP gene and BPI gene did not differ between MI patients and control individuals. Our findings suggest that LBP and BPI polymorphisms do not influence the risk of MI.     

Phagocytosis of Legionella pneumophila is mediated by human monocyte complement receptors

We have examined receptors mediating phagocytosis of the intracellular bacterial pathogen, Legionella pneumophila. Three mAbs against the type 3 complement receptor (CR3), which recognizes C3bi, inhibit adherence of L. pneumophila to monocytes by 64 +/- 8% to 74 +/- 11%. An mAb against the type 1 complement receptor (CR1), which recognizes C3b, inhibits adherence by 68 +/- 1%. mAbs against other monocyte surface antigens do not significantly influence adherence. Monocytes plated on substrates of L. pneumophila membranes modulate their CR1 and CR3 receptors but not Fc receptors; such monocytes bind 70% fewer C3b- coated erythrocytes and 53% fewer C3bi-coated erythrocytes than control monocytes. Adherence of L. pneumophila to monocytes in nonimmune sera is dependent on heat-labile serum opsonins; adherence is markedly reduced in heat-inactivated serum (84% reduction) or buffer alone (97% reduction) compared with fresh serum. mAbs against CR1 and CR3 receptors also inhibit L. pneumophila intracellular multiplication and protect monocyte monolayers from destruction by this bacterium. This study demonstrates that human monocyte complement receptors, CR1 and CR3, mediate phagocytosis of L. pneumophila. These receptors may play a general role in mediating phagocytosis of intracellular pathogens.     

Effects of immobilized immune complexes on Fc- and complement-receptor function in resident and thioglycollate-elicited mouse peritoneal macrophages

We have examined the Fc- and complement-receptor function of resident and thioglycollate-elicited mouse peritoneal macrophages plated on surfaces coated with rabbit antibody-antigen complexes and with complement. We derive four major conclusions from these studies. (a) The trypsin-resistant Fc receptors of resident and thioglycollate-elicited macrophages are completely modulated when these cells are plated on rabbit antibody-antigen complexes. Residual Fc receptor activity is a result of the incomplete modulation of trypsin-sensitive IgG2a receptors. (b) The complement receptors of thioglycollate-elicited macrophages, but not of resident macrophages, are modulated when these cells are plated on complement-coated surfaces. The capacity of the two cell types to modulate their complement receptors is correlated with their ability to ingest complement-coated erythrocytes. (c) The complement and Fc receptors of both types of macrophages move independently of one another. (d) Complement masks the Fc segments of IgG in immune complexes thereby rendering them ineffective as ligands for macrophage Fc receptors.     

Phagocytic chimeric receptors require both transmembrane and cytoplasmic domains from the mannose receptor

Phagocytosis has traditionally been viewed as a specialized function of myeloid and monocytic cells. The mannose receptor (MR) is an opsonin-independent phagocytic receptor expressed on tissue macrophages. When human MR cDNA is transfected into Cos cells, these usually non-phagocytic cells express cell surface MR and bind and ingest MR ligands such as zymosan, yeast, and Pneumocystis carinii. Expression of cDNA for Fc gamma RI (CD64), the high-affinity Fc receptor, in Cos cells confers binding but barely detectable phagocytosis of antibody-opsonized erythrocytes (EA). We report here that chimeric receptors containing the ligand-binding ectodomain of the Fc receptor and the transmembrane and cytoplasmic domains of the MR ingest bound EA very efficiently, whereas chimeras with the Fc receptor ecto- and transmembrane domains and the MR tail, or the Fc receptor ecto- and cytoplasmic domains and the MR transmembrane region, are significantly less phagocytic. All of the chimeric receptors bind ligand with equal avidity, but gain of functional phagocytosis is only conferred by the MR transmembrane and cytoplasmic domains. Endocytosis of monomeric immunoglobulin G by chimeric receptors demonstrates a similar pattern, with optimal uptake by the chimera containing both tail and transmembrane regions from the MR. The chimeric receptors with only the transmembrane or the cytoplasmic domain contributed by the MR were less efficient. Site-directed mutagenesis of the single tyrosine residue in the cytoplasmic tail (which is present in a motif homologous to an endocytosis consensus motif in the LDL receptor cytoplasmic tail [Chen, W.-J., J. L. Goldstein, and M. S. Brown. 1990. J. Biol. Chem. 265:3116]) reduces the efficiency of phagocytosis and endocytosis to a similar extent.     

Selective phagocytic paralysis induced by immobilized immune complexes

The phagocytic recognition by peritoneal macrophages plated on glass- or plastic-bound immune complexes of bovine plasma albumin (BSA) and anti-BSA was examined. Ingestion but not the attachment of erythrocytes opsonized with an IgG rich antiserum (EA) was markedly inhibited. In contrast, macrophage interactions with complement-coated (EAC) red cells, or ingestion of latex particles, yeast cell walls or glutaraldehyde-treated erythrocytes was not inhibited. Complexes prepared with pepsin-treated anti-BSA IgG were ineffective indicating a requirement for the Fc region. Inhibition of ingestion of EA was not a consequence of macrophage spreading and did not appear to be mediated by solubilized complexes or by cell-derived inhibitors of phagocytosis. Significant restoration of the ability to ingest EA was obtained when macrophages on complex-coated substrates were incubated for 4-8 h in medium enriched with mouse or fetal bovine serum. Restoration was also attained by removing macrophages from complex-coated dishes and replating onto uncoated dishes. The selective inhibition of ingestion of EA may be due to blocking of Fc receptors by the complexes but depletion of receptors by endocytosis of complexes cannot be ruled out. Alternatively, the complexes may have induced selective failure of the interiorization mechanism.