The continuing saga of complement receptor type 3 (CR3)
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Cytotoxicity, Immunologic↗
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Soluble beta-glucan polysaccharide binding to the lectin site of neutrophil or natural killer cell complement receptor type 3 (CD11b/CD18) generates a primed state of the receptor capable of mediating cytotoxicity of iC3b-opsonized target cells.
When phagocyte CR3 binds to iC3b on bacteria or yeast, phagocytosis and degranulation are triggered because of simultaneous recognition of iC3b via a CD11b I-domain binding site and specific microbial polysaccharides via a lectin site located COOH-terminal to the I-domain. By contrast, when phagocyte or natural killer (NK) cell CR3 adheres to iC3b on erythrocytes or tumor cells that lack CR3-binding membrane polysaccharides, neither lysis nor cytotoxicity are stimulated. This investigation showed that soluble CR3-specific polysaccharides such as beta-glucan induced a primed state of CR3 that could trigger killing of iC3b-target cells that were otherwise resistant to cytotoxicity. Anti-CR3 added before sugars prevented priming, whereas anti-CR3 added after sugars blocked primed CR3 attachment to iC3b-targets. Polysaccharide priming required tyrosine kinase(s) and a magnesium-dependent conformational change of the I-domain that exposed the CBRM1/5 activation epitope. Unlike LPS or cytokines, polysaccharides did not up-regulate neutrophil CR3 expression nor expose the mAb 24 reporter epitope representing the high affinity ICAM-1-binding state. The current data apparently explain the mechanism of tumoricidal beta-glucans used for immunotherapy. These polysaccharides function through binding to phagocyte or NK cell CR3, priming the receptor for cytotoxicity of neoplastic tissues that are frequently targeted with iC3b and sparing normal tissues that lack iC3b.
Animals↗
Macrophage distribution in decidual tissue from early implantation to the periparturient period in mice as defined by the macrophage differentiation antigens F4/80, macrosialin and the type 3 complement receptor.
Antibodies to differentiation markers have made it possible to identify macrophages in murine tissues. Macrophages are potent mediators of immunological reactions and it has been proposed that they are pivotal cells at the maternal-fetal interface. Studies of macrophage distribution in murine decidual tissue have provided conflicting evidence for and against the presence of significant numbers of macrophages at the maternal-fetal interface. The study described here used three independent macrophage differentiation antigens to examine the macrophage distribution in decidual tissue from day 6 to day 19 of pregnancy. Macrophage distribution was defined initially using a polyclonal antiserum to the plasma membrane differentiation antigen F4/80. Macrophages were virtually absent from antimesometrial decidual tissue until degenerating tissue was invaded by macrophages from about day 15. The resident population of macrophages in the mesometrial stroma was retained when this area decidualized but these cells did not survive beyond day 13. Mesometrial decidual tissue was virtually devoid of macrophages after this time. The metrial gland contained many macrophages until degeneration set in, but few were seen by day 15 of pregnancy. These distributions were confirmed using monoclonal antibodies (mAb) to F4/80, macrosialin, a monocyte- and macrophage-specific membrane sialoprotein (mAb FA/11), and the leucocyte beta 2-integrin CR3 (CD11b/CD18; Mac-1), which is expressed on neutrophils as well as monocytes and some tissue macrophages. CR3+, F4/80- and FA/11- neutrophils were found to be more widely distributed in decidual tissue than were F4/80+ or FA/11+ macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
T-cell malignant lymphoma; T lymphoid cells with complement receptors and helper cell activity.
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Antigens, Surface↗
Cocapping of the leukoadhesin molecules complement receptor type 3 and lymphocyte function-associated antigen-1 with Fc gamma receptor III on human neutrophils. Possible role of lectin-like interactions.
We have tested the possible physical interactions between the iC3b receptor (CR3), lymphocyte function-associated Ag-1, and class III Fc gamma receptor (Fc gamma RIII) at neutrophil surfaces. Cells were labeled using fluorochrome-conjugated Fab or F(ab')2 fragments of antireceptor mAb. Labeled receptors were capped using second-step F(ab')2 fragments of goat anti-mouse Fab antiserum. After 20 min at 37 degrees C, 68% of the cells capped the anti-CR3 plus second-step complex. Capping was time, temperature, and cytochalasin B sensitive. When capped cells were probed with Fab' or F(ab')2 fragments of anti-Fc gamma RIII labeled with a distinct fluorochrome, 41% of the cells cocapped Fc gamma RIII. Indistinguishable results were obtained when potential antibody combining sites within caps were blocked with a large excess of Fab or F(ab')2 fragments. When Fc gamma RIII was capped, 49% of the cells cocapped CR3. Similarly, LFA-1 cocapped with both CR3 and Fc gamma RIII. Importantly, other membrane components including HLA class I, Mo5, CD13, CR type 1, and IL-8 receptors and N-4-nitrobenzo-2-oxa-1, 3-diszole L-alpha-dimyristoyl phosphatidylethanolamine did not cocap with CR3. However, the positive control Con A did cocap with CR3 and Fc gamma RIII. We next evaluated the effect of saccharides on CR3-Fc gamma RIII cocapping and found that 0.15 M N-acetyl-D-glucosamine (NADG), alpha-methyl-D-mannoside, and D-mannose significantly inhibited cocapping by 70, 58, and 48%, respectively. No inhibition was obtained using glucose, galactose, N-acetyl-neuraminic acid, fucose, sorbitol, fructose, or sucrose. Similarly, Fc gamma RIII-lymphocyte function-associated-1 cocapping was inhibited by NADG. However, the cocapping of CR3 with lymphocyte function-associated-1 or Con A were not affected by 0.15 M NADG, which suggests that NADG inhibition of leukoadhesin-Fc gamma RIII cocapping is not due to a general effect of NADG on capping. Inasmuch as Fc gamma RIII is a glycophospholipid-linked membrane protein, we speculate that it interacts with CR3 and/or lymphocyte function-associated-1 via lectin-like interactions.
Adult↗
Identification and characterization of a novel protein associated with macrophage complement receptor 3.
CR3 is a member of the beta 2 integrin family which functions as a bidirectional signaling receptor. The CR3 is composed of the alpha (CD11b) and beta (CD18) subunits, which contain a short intracytoplasmic domain devoid of catalytic activity. It was therefore postulated that CR3 is associated with intracellular molecules that link it to the cytoplasmic signal transduction apparatus. However, no direct association between such molecules and CR3 have been identified so far. We searched for CR3 co-associated molecules that might regulate the function of this receptor. For this purpose CR3 was immunoprecipitated from radiolabeled bone marrow macrophages using a combination of anti-CD11b and anti-CD18 mAbs. Two-dimensional isoelectric focusing analysis of the immunoprecipitates revealed the two CR3 subunits and an additional 16-kDa protein with an apparent isoelectric point of 5.1. This protein, designated p16/5.1, was intracellular, monomeric, nonglycosylated and noncovalently associated with CR3 but not with CR4. CR3-associated p16/5.1 was also detected in four of six macrophage lines as well as in thymic large macrophages, all of which express cell-surface CR3. We suggest that p16/5.1 may be involved in CR3-mediated function.
Animals↗
Human urokinase-type plasminogen activator primes neutrophils for superoxide anion release. Possible roles of complement receptor type 3 and calcium.
Urokinase-type plasminogen activator (uPA), which binds to cells via a specific receptor (uPAR), participates in pericellular proteolysis during leukocyte migration. Previous studies have indicated that uPAR is physically associated with CR3 (CD11b/CD18). To test the functional interactions of CR3 and uPAR, we have examined the ability of uPA to elicit changes in cytosolic calcium levels of normal neutrophils, neutrophils from a leukocyte adhesion deficiency (LAD) patient, and 3T3 transfectants expressing CR3, uPAR, or both. We found that calcium levels of neutrophils increased from 106 +/- 6 nM in untreated cells to 199 +/- 25 nM in the presence of uPA. In contrast, no significant change in calcium was observed when neutrophils from an leukocyte adhesion deficiency patient were examined. The uPA-dependent calcium rise was inhibited by mAb directed against either CR3 or uPAR and required intact uPA. To substantiate further these findings, we prepared transfectants expressing genes encoding uPAR, CR3, and both receptors; only cells expressing both receptors experienced a rise in intracellular calcium. Although uPA's calcium signal is insufficient to trigger superoxide production, FMLP dose-dependent superoxide production was greatly enhanced by incubating neutrophils with intact, but not fragmented, uPA. Flow cytometry experiments utilizing an FMLP analogue exclude the possibilities that urokinase binds to the FMLP receptor or up-regulates its expression. We suggest that calcium is a second messenger of uPA, that this message is mediated in a CR3-dependent fashion, and that this signal primes neutrophils for superoxide production.
3T3 Cells↗
Cytoplasmic tails of human complement receptor type 3 (CR3, CD11b/ CD18) regulate ligand avidity and the internalization of occupied receptors.
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Amino Acid Sequence↗
Physical association of complement receptor type 3 and urokinase-type plasminogen activator receptor in neutrophil membranes.
A previous study has shown that Fc gamma RIIIB (CD16), an extensively glycosylated glycosyl-phosphatidylinositol-linked neutrophil membrane protein, specifically co-caps with the iC3b R (CR3; CD11b/CD18). This study tests the possible physical interactions of another extensively glycosylated glycosyl-phosphatidylinositol-linked protein, the urokinase-type plasminogen activator receptor (uPAR), with CR3. Receptors were labeled using fluorochrome-conjugated F(ab')2 fragments of an anti-CR3 mAb. In some cases cells were capped using second step F(ab')2 fragments of an anti-mouse F(ab')2 antiserum. After 30 min at 37 degrees C, 65 +/- 4% of the cells exhibited CR3 caps whereas 61 +/- 2% demonstrated uPAR caps. When CR3-capped cells were probed with F(ab')2 fragments of anti-uPAR conjugated to a distinct fluorochrome, 45 +/- 3% of the cells co-capped uPAR. When uPAR was capped, 48 +/- 2% of the cells co-capped CR3. Similar levels of co-capping were observed using a DNP-conjugated anti-CR3 F(ab')2 and an anti-DNP second step F(ab')2 reagent for capping or using FITC-uPA as a probing reagent. Furthermore, CR3-uPAR co-capping and/or co-clustering was also observed using anti-CR3 IgM and Mn2+ as integrin aggregation stimuli. Significant co-capping of anti-CD14, anti-CD59, anti-Mo5, anti-HLA, or NBD-PE (a lipid probe) was not observed. Moreover, CR3 and uPAR co-capping was blocked by N-acetyl-D-glucosamine, but not by six other saccharides, suggesting that a lectin-like site may participate in co-capping. This suggests that CR3 may regulate adhesive events by several mechanisms, including the regulation of the spatial distribution of uPAR.
Adult↗
Complement receptor type 3 (CD11b/CD18) involvement is essential for killing of Listeria monocytogenes by mouse macrophages.
Recent work indicated that C receptor type 3 (CR3) mediates most phagocytosis of the facultative intracellular bacterium Listeria monocytogenes by mouse macrophages, which can kill it. In contrast, phagocytosis of Listeria by a population of nonlistericidal macrophages was largely CR3-independent. These findings suggested that CR3 binding during phagocytosis may be important in determining whether a macrophage kills Listeria, or is parasitized by the bacterium. The experiments reported here tested this hypothesis. When phagocytosis and killing were assayed separately, normally listericidal peritoneal macrophages still could phagocytose to some extent, but lost listericidal activity when CR3 was blocked by mAb. Anti-CR3 mAb inhibited killing in a dose-dependent fashion, and at high doses the cells became permissive hosts. Microbicidal function also was inhibited when active C components were absent during phagocytosis and during killing. Because Listeria are confined to phagosomes in listericidal macrophages but escape into the cytoplasm in nonlistericidal macrophages, we tested whether anti-CR3 mAb enhanced phagosomal escape. In fact, escape of Listeria into the cytoplasm was rare in both control and anti-CR3 mAb-treated macrophages. Moreover, electron microscopy of these cells demonstrated dividing intraphagosomal bacteria. Taken together, these results suggest that binding to CR3 during phagocytosis leads to bacterial killing, and that phagocytic pathways engaged when binding to CR3 is blocked do not trigger microbicidal activity. Furthermore, restriction of Listeria to the phagosome in the absence of CR3 engagement is not by itself sufficient for macrophage listericidal activity.
Animals↗
Complement receptor 3 of macrophages is associated with galectin-1-like protein.
We have previously identified a 16-kDa protein with a pI of 5.1 (P16/5.1) that is associated with macrophage CR3. Microsequencing of P16/5.1 indicated exclusive homology to the beta-galactoside-binding lectin, galectin-1. Abs specific to a galectin-1 unique peptide reacted with P16/5.1. The association of P16/5.1 with CR3 was specifically inhibited by lactose, which binds with high affinity to galectin-1. These data together with similarities in molecular mass and pI suggest that P16/5.1 is galectin-1. Two-color immunofluorescence staining revealed the expression of galectin-1 on the macrophage surface and its colocalization with CR3. However, a surplus of CR3 was free of galectin-1, and some galectin-1 molecules were associated with cell surface receptors other than CR3. Based on these results we propose two models depicting the functional significance of CR3-galectin-1 association: 1) homodimeric galectin-1 possessing a divalent sugar binding site may act as an extracellular adapter molecule that cross-links CR3 with other receptors; and 2) association of galectin-1 with beta-galactosides on the extracellular domain of CR3 may modify the binding affinity of the receptor to its ligand. These possibilities are not mutually exclusive and can clarify the mode by which CR3 transmits signals in macrophages.
Animals↗
Structure of the C3b binding site of CR1 (CD35), the immune adherence receptor.
Complement receptor type 1 (CR1 or CD35) is a multiple modular protein that mediates the immune adherence phenomenon, a fundamental event for destroying microbes and initiating an immunological response. It fulfills this role through binding C3b/C4b-opsonized foreign antigens. The structure of the principal C3b/C4b binding site (residues 901-1095) of CR1 is reported, revealing three complement control protein modules (modules 15-17) in an extended head-to-tail arrangement with flexibility at the 16-17 junction. Structure-guided mutagenesis identified a positively charged surface region on module 15 that is critical for C4b binding. This patch, together with basic side chains of module 16 exposed on the same face of CR1, is required for C3b binding. These studies reveal the initial structural details of one of the first receptor-ligand interactions to be identified in immunobiology.
Binding Sites↗
Functional differentiation of normal human neutrophils.
In the past differentiation of human neutrophils has been defined by morphology, cytochemistry, or surface markers. In our experiments we have sequenced the various events that occur during the functional differentiation of the normal human neutrophil and have also examined some of the functional properties in relationship to surface markers and biochemical events. Granulocytes were obtained from the bone marrow and blood of hematologically normal individuals. Cells were separated into different stages of maturation by their physical properties using counterflow centrifugal elutriation and density gradient separation. Three cell fractions were obtained that were enriched for either immature myeloid cells, band neutrophils, or segmented neutrophils. Since the enriched fractions were not entirely pure, methodologies for functional assays were chosen that allowed cytologic evaluation of the functional capacity of each cell type. The criteria used to classify the stages of differentiation included both morphology by light microscopy and DNA labeling with tritiated thymidine. Various neutrophilic properties were studied: Fc receptors, complement receptors (CR1, CR3), phagocytosis of both live and dead opsonized Staphylococcus aureus, microbial killing of S aureus, NBT dye reduction after cellular stimulation with endotoxin, and chemotaxis. Our results indicate that the functional properties of the neutrophil appear in a distinct order. The sequence for the functional differentiation of the human neutrophil appears to be the following: Fc receptors----immune phagocytosis----complement receptors----oxygen-independent microbial killing----oxygen-dependent microbial killing----chemotaxis.
Bacteriolysis↗
The role of receptors for complement in the induction of polyclonal B-cell proliferation and differentiation.
A panel of monoclonal antibodies and ligands that bind to the CR1 or CR2 complement receptors of B cells has been used to investigate the role of these membrane molecules in regulating B-cell proliferation and differentiation. When CR2 was modulated from the surface of B cells by treatment with the HB-5 antibody and a secondary goat anti-mouse immunoglobulin antibody, Epstein-Barr virus-induced polyclonal B-cell proliferation and immunoglobulin production were inhibited by 83 and 90%, respectively. In contrast, modulation of other cell surface molecules, HB-2, B1, and the C3b receptor (CR1), or pretreatment of B cells with C3d,g (a CR2 ligand) or HB-5 antibody, alone minimally inhibited these responses. Neither the HB-5 antibody C3d,g, nor a monoclonal antibody (YZ-1) reactive with CR1 induced resting B cells to proliferate, nor did they alter anti-mu antibody-induced proliferation. Similarly, treatment with C3d,g or with the HB-5 or YZ-1 antibodies did not induce B cells to secrete immunoglobulin or affect pokeweed mitogen-induced plasma-cell formation. Whereas CR2 appears to be the functionally relevant receptor for Epstein-Barr virus on B cells, the effects of ligand interactions with CR1 and CR2 on normal B-cell proliferation or differentiation remain unidentified.
Adult↗
Epstein-Barr virus (EBV)-containing nasopharyngeal carcinoma cells express the B-cell activation antigen blast2/CD23 and low levels of the EBV receptor CR2.
Anaplastic nasopharyngeal carcinoma (NPC) cells invariably harbor the Epstein-Barr virus (EBV) genome, an association that is unique among human virus-associated cancers. Although EBV is able to replicate in epithelial cells, results with expression of the EBV receptor (complement receptor type 2 [CR2]; also called CD21) in normal and malignant epithelial cells are conflicting. We grew five different EBV-associated NPC tumors in nude mice, and by using a sensitive transcriptional assay, we detected a very weak transcription signal of the EBV receptor CR2 gene in these cells. This suggests that low levels of EBV receptor may be expressed by malignant epithelial nasopharyngeal cells. The gene coding for Blast2/CD23, a B-cell activation molecule induced by EBV, was transcribed in three of the transplanted NPC tumors. The soluble form of the Blast2/CD23 protein was also detected in medium taken from short-term cultures of the same NPC cell lines. In contrast to the lymphoid system, in which Blast2/CD23 expression is associated with EBV nuclear antigen (EBNA2) expression, no EBNA2 protein could be detected in these NPC epithelial cells. Our study represents the first demonstration of Blast2/CD23 expression in epithelial cells. As the soluble form of the Blast2/CD23 protein possesses growth factor activity associated with EBV-induced B-cell immortalization, these results suggest a possible role for this molecule in the pathogenesis of NPC.
Animals↗
Epstein-Barr virus (EBV) induces expression of B-cell activation markers on in vitro infection of EBV-negative B-lymphoma cells.
A set of B-cell activation markers, including the EBV/C3d receptor [complement receptor type 2 (CR2) (CD21)], the 45-kDa lymphoblastoid cell-associated (Blast-2) antigen (CD23), and the B-cell restricted activation (Bac-1) antigen (which was recently identified as a potential B-cell growth factor receptor) can be turned on by infecting lymphoma cells that are genome negative for Epstein-Barr virus (EBV) with the B95-8 immortalizing strain of the virus. The nonimmortalizing EBV variant, strain P3HR-1, which possesses a deletion within the BamHI WYH region of the genome containing the coding sequence for the EBV-determined nuclear antigen 2, does not induce expression of these markers. Other lymphoblastoid cell-associated antigen markers can be activated by infection with either immortalizing or nonimmortalizing viruses. These results suggest that the immortalizing potential of EBV is correlated with its ability to induce expression of B-cell activation markers, which are suspected to play a major role in the physiological pathway leading to lymphoid cell proliferation. The viral genomic region deleted in the nonimmortalizing strain of EBV seems to be required for activation of some of these markers. Human lymphoma cell lines, such as those used in this study, can thus help identify the specific EBV genes involved in lymphoid B-cell proliferation and the mechanism of action of these genes.
Antigens, Neoplasm↗
Autoimmune New Zealand mouse. Alterations in C3 receptors, complement-induced clumps and follicular adherence of immune complexes.
Lymphocytes with free complement receptors are markedly diminished in autoimmune mice, probably as a reflection of a receptor blockage by immune complexes. There is also a slight decrease in Fc receptor-bearing cells and an increase in complement-induced clumps. These alterations may be related to a lack of immune complex fixation onto germinal centres and also may play a part in possible defects in T-B-cell co-operation, follicular localization of immune complexes and immune response control in autoimmune mice.
Animals↗
[Receptors for complement on rat mast cells and release of histamine].
Using rat complement-treated zymosan particles a rosetting of purified rat peritoneal mast cells could be demonstrated. The question was investigated whether the binding of activated complement could be a trigger of histamine release. Varying the degree of complement label on the zymosan particles, the time and temperature of incubation and the dependence on Ca2+ ions, we could not induce a release of histamine in any case. The addition of labeled zymosan increased slightly the mediator release induced by ATP. The immunologic significance of the complement receptors on mast cells is still unclear.
Animals↗