The role of lipooligosaccharide in Neisseria gonorrhoeae pathogenesis of cervical epithelia: lipid A serves as a C3 acceptor molecule

The use of primary, human, ecto- and endocervical epithelial cell cultures has increased our understanding of the pathogenesis of gonococcal infection in women. Primary cervical epithelial cells express complement (C') receptor type 3 (CR3) and C' proteins required for alternative pathway (AP) activity. Gonococcus -induced membrane ruffling and cellular invasion of primary cervical epithelia is mediated by CR3 and requires co-operative CR3 binding by gonococcus-bound iC3b, porin and pilus. We have extended these studies to identify the site of C3 deposition upon gonococci within the cervical microenvironment. By immunoprecipitation and ELISA we demonstrate that covalent and non-covalent associations occurred between gonococcal LOS and C' protein C3. Sialylation or LOS truncation did not alter the gonococcus-CR3 interaction. By Western blot analysis we observed comparable C3 opsonization patterns among a panel of LOS truncation mutants, sialylated wild-type gonococci, or wild-type bacteria that were not sialylated. Quantitative association/invasion assays performed in the presence or absence of LOS competimers support C3b deposition on the lipid A core structure. Our findings demonstrate a role for lipid A as a C3 acceptor site and suggest that multiple factors govern C3b deposition and its subsequent conversion to iC3b on the surface of the gonococcus within the cervical microenvironment.     

Neisseria gonorrhoeae PLD directly interacts with Akt kinase upon infection of primary, human, cervical epithelial cells

Neisseria gonorrhoeae secrets a phospholipase D (NgPLD), which augments complement receptor 3 (CR3)-mediated invasion of cervical epithelial cells. To elucidate the signalling pathways triggered with gonococcus CR3-engagement and the putative function of NgPLD in these events, we analysed the contribution of the phosphoinositide-Akt pathway to cervical infection. Our data indicated that Akt plays a critical role in cervical infection. Inhibition of myosin light chain kinase, PtdIns(4,5)P2, and Akt functions resulted in decreased gonococcus invasion of primary, human, cervical epithelial cells as well as Akt kinase activity. Akt activity was similarly impaired when cervical cells were challenged with NgPLD-mutant gonococci. Conversely, the PI3-kinase inhibitor, LY294002, enhanced gonococcal invasion of, and Akt activity within, primary cervical cells. We demonstrated that NgPLD directly binds to the Akt PH domain and can compete with a natural Akt ligand, PtdIns(3,4,5)P3, for Akt binding. Collectively, our data suggested that NgPLD augments gonococcus invasion of cervical epithelia by interacting with Akt kinase in a PI3-kinase-independent manner, which results in subversion of normal cervical cell signalling.     

Resistance to phagocytosis by group A streptococci: failure of deposited complement opsonins to interact with cellular receptors

The previous finding that phagocytosis-resistant M+ group A streptococci bear quantities of C3 which are sufficient for phagocytosis of their M- derivatives was investigated at two levels. It was first established that the C3 associated with M+ streptococci was not able to promote adherence to cells bearing the complement receptors CR1 and CR3 under conditions in which M- streptococci readily attached. The molecular form of C3 bound to M+ and M- streptococci was then defined by adding 125I-C3 to serum used for opsonization. C3 eluted from the bacteria by chaotropic and hydrolytic agents was analyzed by SDS-PAGE, and revealed that both cell types bound the opsonic forms of C3, C3b, and iC3b. Furthermore, approximately 80% of the C3b and iC3b associated with both cell types was covalently bound to a surface component, although most of the C3 bound to M+ streptococci was detergent-extractable, whereas greater than 50% of that bound to M- streptococci was not. These findings demonstrate that the M+ surface is interfering with the receptor binding of deposited C3b and iC3b, and that this contributes to resistance to phagocytosis by these organisms.     

The role of complement receptor 3 (CR3) in Neisseria gonorrhoeae infection of human cervical epithelia

Neisseria gonorrhoeae is an important sexually transmitted pathogen and a major cofactor in HIV-1 infection. This organism uses different mechanisms to infect male and female genital tract epithelia. Receptor-mediated endocytosis of N. gonorrhoeae is the principle mechanism of entry into male urethral epithelial cells. Infection in men leads to a pronounced inflammatory response. In contrast, N. gonorrhoeae infection in women induces ruffling of the cervical epithelia, allowing a macropinocytic mechanism of entry. Infection in women is frequently asymptomatic, suggesting suppression of the inflammatory response. N. gonorrhoeae -induced membrane ruffling and inflammation suppression are consistent with the ability of this bacterium to enter cervical epithelial cells, in vitro and in vivo , by interaction with complement receptor 3 (CR3), a receptor that does not trigger an inflammatory response. This receptor is present on cervical epithelial cells but not on male urogenital tract epithelia. N. gonorrhoeae engagement of CR3 initiates a unique mechanism of bacterial-induced membrane ruffling and internalization. These studies explain why the pathology of N. gonorrhoeae infection differs between males and females. Additionally, the observation that this receptor is present on cervical epithelia may provide insight into the pathogenesis of other sexually transmitted pathogens     

CEACAM is not necessary for Neisseria gonorrhoeae to adhere to and invade female genital epithelial cells

Neisseria gonorrhoeae has a repertoire of up to 11 opacity-associated (Opa) proteins that are adhesins. Most Opa proteins adhere to CEACAM antigens and when CEACAM molecules are present on the surface of transfected epithelial cells their binding by Opa is thought to induce invasion of these cells by gonococci. In this study, we investigated whether several malignant epithelial cell lines, normal cervical and fallopian tube epithelial cell cultures, as well as normal fallopian tube tissue express several of the CEACAM molecules, and whether gonococci use these molecules for adherence and invasion of these female genital epithelial cells. A primary cervical cell culture and metastatic cervical cell line ME180 both expressed CEACAM as shown by whole cell ELISA and flow cytometry, and increased the surface expression of total CEACAM during incubation with Opa⁺ gonococci. Opa⁺ gonococci both adhered to and invaded these cells; CEACAM-specific monoclonal antibody (MAb) partially abolished this interaction. Two primary fallopian epithelial tube cell cultures, a primary cervical cell culture and two malignant cell lines, HEC-1-B and HeLa, did not express CEACAM nor was CEACAM mRNA present. No evidence of either intracellular or secreted extracellular CEACAM was found with HEC-1-B and HeLa cells. Opa⁺ gonococci both adhered to and invaded CEACAM non-expressing cells; however, Opa⁺ gonococcal association with these non-expressing cell lines could not be inhibited with CEACAM-specific MAb. These data show that CEACAM is not always expressed on female genital epithelial cells and is not essential for gonococcal adherence and invasion. However, when CEACAM is expressed, Opa⁺ gonococci exploit it for the adherence to and invasion of these cells.     

Elevated NK-mediated lysis of Raji and Daudi cells carrying fixed iC3b fragments

Raji and Daudi cells were opsonized with C3b, iC3b, and C3d fragments by using purified complement components. The sensitivity of C3-opsonized cells to lysis mediated by low density blood lymphocytes was studied. Raji and Daudi cells carrying C3b or C3d fragments were lysed with similar efficiencies as the nonopsonized cells. The presence of iC3b on the target surface imposed elevated NK sensitivity. The iC3b-mediated enhancement of NK lysis was inhibited when iC3b fragments or rabbit anti-human C3 antibodies were included into the lytic assays. These results indicate that the iC3b fragments fixed on the targets bind to the CR3 on the lymphocytes. Results obtained in immobilized conjugate-lytic assays showed that iC3b-opsonized targets interact more readily with the lymphocytes. This was reflected by the elevated proportion of lymphocytes that were bound to the iC3b-carrying targets. The proportions of conjugates in which target damage occurred were similar with the control and with the iC3b-carrying cells. It seems therefore that opsonization of targets with iC3b leads to recruitment of effector lymphocytes due to contact with their CR3. However, once the effector-target contact is established, the triggering of lytic function does not seem to be influenced by the iC3b/CR3 bridge.     

Release of iC3b from apoptotic tumor cells induces tolerance by binding to immature dendritic cells in vitro and in vivo

Chemo- as well as immunotherapeutical approaches induce apoptosis in tumor cells. Apoptotic cells are known to activate homologous complement and to be opsonized with iC3b. Since maturation of dendritic cells (DC) can be inhibited by binding of iC3b to the complement receptor 3 (CR3, CD11b/CD18) and because immature DC induce tolerance, we investigated the induction of tolerance after pulsing DC with apoptotic cells in the presence or absence of native serum. Apoptosis in pancreatic carcinoma cells was induced either by heat-stress, chemotherapy or anti-Her2 antibody. Monocyte-derived DC were pulsed with apoptotic cells with or without native serum. DC were analyzed for the maturation state by flow cytometry and the cytotoxic activity was determined. Tolerance was prevented by addition of substances such as anti CD11b or N–acetyl-D-Glucosamine (NADG) which block iC3b binding to CR3. Furthermore, binding of iC3b from apoptotic cells to DC was blocked in a syngeneic pancreatic carcinoma mouse model. All of the former strategies for apoptosis induction resulted in iC3b release. Pulsing DC with apoptotic cells in the presence of serum prevents maturation of DC and induces finally tolerance. This tolerance could be prevented almost completely by blocking the interaction of iC3b with the CR3 receptor. This could be shown as well in an immunocompetent mouse model. Chemo- as well as immunotherapeutical approaches induce apoptosis in tumor cells. Release of iC3b from apoptotic tumor cells prevents fully maturation of DC and immature DC induce antigen-specific silencing or tolerance. Blocking of iC3b-binding could mostly prevent this effect.     

Kinetic analysis of the interactions of complement receptor 2 (CR2, CD21) with its ligands C3d, iC3b, and the EBV glycoprotein gp350/220

The molecular mechanisms involved in the interaction of complement receptor 2 (CR2) with its natural ligands iC3b and C3d are still not well understood. In addition, studies regarding the binding site(s) of the receptor on C3 as well as the affinities of the C3 fragments for CR2 have produced contradictory results. In the present study, we have used surface plasmon resonance technology to study the interaction of CR2 with its ligands C3d, iC3b, and the EBV surface glycoprotein gp350/220. We measured the kinetics of binding of the receptor to its ligands, examined the influence of ionic contacts on these interactions, and assessed whether immobilized and soluble iC3b bound with similar kinetics to CR2. Our results indicate that 1) gp350 binding to CR2 follows a simple 1:1 interaction, whereas that of the C3 fragments is more complex and involves more than one intramolecular component; 2) kinetic differences exist between the binding of C3d and iC3b to CR2, which may be due to an additional binding site found on the C3c region of iC3b; and 3) iC3b binds to CR2 with different kinetics, depending on whether the iC3b is in solution or immobilized on the surface. These findings suggest that binding of CR2 to iC3b and C3d is more complex than previously thought.     

Adherence of clinically isolated lactobacilli to human cervical cells in competition with Neisseria gonorrhoeae

Lactobacilli are normal inhabitants of our microbiota and are known to protect against pathogens. Neisseria gonorrhoeae is a human specific pathogenic bacterium that colonises the urogenital tract where it causes gonorrhoea. In this study we analysed early interactions between lactobacilli and gonococci and investigated how they compete for adherence to human epithelial cervical cells. We show that lactobacilli adhere at various levels and that the number of adherent bacteria does not correlate to the level of protection against gonococcal infection. Protection against gonococcal adhesion varied between Lactobacillus species. Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus reuteri were capable of reducing gonococcal adherence while Lactobacillus rhamnosus was not. Lactobacillus strains of vaginal origin had the best capacity to remain attached to the host cell during gonococcal adherence. Further, we show that gonococci and lactobacilli interact with each other with resultant lactobacilli incorporation into the gonococcal microcolony. Hence, gonococci bind to colonised lactobacilli and this complex frequently detaches from the epithelial cell surface, resulting in reduced bacterial colonisation. Also, purified gonococcal pili are capable of removing adherent lactobacilli from the cell surface. Taken together, we reveal novel data regarding gonococcal and lactobacilli competition for adherence that will benefit future gonococcal prevention and treatments.     

Selective induction of enhanced complement receptor 3 activity on murine macrophages

A high proportion of murine resident peritoneal macrophages bear complement receptors 1 and 3 (CR1, CR3) which bind C3b and iC3b components of complement, respectively. By contrast, macrophages derived from bone marrow, blood, and the elicited peritoneal exudate are predominantly CR1+3. To determine if the microenvironment of the normal peritoneal cavity influences CR3 phenotype, we studied the effects of lavage from the cavity on cultures of primary peritoneal exudate macrophages, and on macrophages derived from progenitors in the bone marrow, blood, and peritoneal exudate. The cell-free peritoneal lavage (CFPL), after 24 hr of culture, induced CR3 on primary and culture-derived populations of peritoneal exudate macrophages but had no effect on the CR3 phenotype of macrophages derived from bone marrow or blood. The CR3-inducing activity in CFPL was abolished by heating at 70 degrees C for 30 min and by trypsin, and was not affected by adsorption with EA(IgM)iC3b indicator cells, demonstrating that it is not soluble CR3. Finally, exudate macrophages exposed to CFPL required at least 24 hr before they expressed CR3; such macrophages regenerated CR3 after the receptors were removed by trypsin. The selective effect of the activity in CFPL for peritoneal exudate macrophages indicates that the local microenvironment of the peritoneal cavity can influence the expression of CR3.     

Contribution of complement component C3 and complement receptor type 3 to carbohydrate-dependent uptake of oligomannose-coated liposomes by peritoneal macrophages

Peritoneal macrophages (PEMs) preferentially and rapidly take up oligomannose-coated liposomes (OMLs) and subsequently mature to induce a Th-1 immune response following administration of OMLs into the peritoneal cavity. Here, we examine the contributions of complement component C3 and complement receptor type 3 (CR3) to carbohydrate-dependent uptake of OMLs by PEMs. Effective uptake of OMLs into PEMs in vitro was observed only in the presence of peritoneal fluid (PF), and OMLs incubated with PF were incorporated by PEMs in vitro in the absence of PF. These phenomena were inhibited by methyl-alpha-mannoside, N-acetylglucosamine or EDTA, but not by galactose. Pull-down analysis followed by peptide mass fingerprinting of PF-treated OMLs indicated that the OMLs were opsonized with complement fragment iC3b. In vivo uptake of OMLs by PEMs was inhibited by intraperitoneal injection of an antibody against CR3, a receptor for iC3b, and OML uptake by PEMs in the peritoneal cavity was not observed in C3-deficient mice. Thus, our results indicate that OMLs are opsonized with iC3b in a mannose-dependent manner in the peritoneal cavity and then incorporated into PEMs via CR3.     

Beta-glucan, a "specific" biologic response modifier that uses antibodies to target tumors for cytotoxic recognition by leukocyte complement receptor type 3 (CD11b/CD18).

beta-Glucans were identified 36 years ago as a biologic response modifier that stimulated tumor rejection. In vitro studies have shown that beta-glucans bind to a lectin domain within complement receptor type 3 (CR3; known also as Mac-1, CD11b/CD18, or alphaMbeta2-integrin, that functions as an adhesion molecule and a receptor for factor I-cleaved C3b, i.e., iC3b) resulting in the priming of this iC3b receptor for cytotoxicity of iC3b-opsonized target cells. This investigation explored mechanisms of tumor therapy with soluble beta-glucan in mice. Normal mouse sera were shown to contain low levels of Abs reactive with syngeneic or allogeneic tumor lines that activated complement, depositing C3 onto tumors. Implanted tumors became coated with IgM, IgG, and C3, and the absent C3 deposition on tumors in SCID mice was reconstituted with IgM or IgG isolated from normal sera. Therapy of mice with glucan- or mannan-rich soluble polysaccharides exhibiting high affinity for CR3 caused a 57-90% reduction in tumor weight. In young mice with lower levels of tumor-reactive Abs, the effectiveness of beta-glucan was enhanced by administration of a tumor-specific mAb, and in SCID mice, an absent response to beta-glucan was reconstituted with normal IgM or IgG. The requirement for C3 on tumors and CR3 on leukocytes was highlighted by therapy failures in C3- or CR3-deficient mice. Thus, the tumoricidal function of CR3-binding polysaccharides such as beta-glucan in vivo is defined by natural and elicited Abs that direct iC3b deposition onto neoplastic cells, making them targets for circulating leukocytes bearing polysaccharide-primed CR3. Therapy fails when tumors lack iC3b, but can be restored by tumor-specific Abs that deposit iC3b onto the tumors.     

Mechanism by which orally administered beta-1,3-glucans enhance the tumoricidal activity of antitumor monoclonal antibodies in murine tumor models

Antitumor mAb bind to tumors and activate complement, coating tumors with iC3b. Intravenously administered yeast beta-1,3;1,6-glucan functions as an adjuvant for antitumor mAb by priming the inactivated C3b (iC3b) receptors (CR3; CD11b/CD18) of circulating granulocytes, enabling CR3 to trigger cytotoxicity of iC3b-coated tumors. Recent data indicated that barley beta-1,3;1,4-glucan given orally similarly potentiated the activity of antitumor mAb, leading to enhanced tumor regression and survival. This investigation showed that orally administered yeast beta-1,3;1,6-glucan functioned similarly to barley beta-1,3;1,4-glucan with antitumor mAb. With both oral beta-1,3-glucans, a requirement for iC3b on tumors and CR3 on granulocytes was confirmed by demonstrating therapeutic failures in mice deficient in C3 or CR3. Barley and yeast beta-1,3-glucan were labeled with fluorescein to track their oral uptake and processing in vivo. Orally administered beta-1,3-glucans were taken up by macrophages that transported them to spleen, lymph nodes, and bone marrow. Within the bone marrow, the macrophages degraded the large beta-1,3-glucans into smaller soluble beta-1,3-glucan fragments that were taken up by the CR3 of marginated granulocytes. These granulocytes with CR3-bound beta-1,3-glucan-fluorescein were shown to kill iC3b-opsonized tumor cells following their recruitment to a site of complement activation resembling a tumor coated with mAb.     

Urokinase receptor (uPAR) regulates complement receptor 3 (CR3)-mediated neutrophil phagocytosis

Urokinase receptor (uPAR) associates in cis with complement receptor 3 (CR3). In the present study, we addressed whether this coupling regulates CR3-mediated phagocytosis. CR3-mediated attachment of iC3b-opsonized sheep red blood cells to human neutrophils and internalization of these cells were reduced by removal of cell-bound uPAR by phosphatidylinositol-specific phospholipase C and reconstituted in the presence of soluble uPAR. The attachment and internalization were suppressed in the presence of anti-uPAR polyclonal antibody, proteolytically inactive urokinase and saccharides that disrupt interaction of uPAR with CR3. Thus, uPAR acts as a cofactor for iC3b binding to CR3 and regulates CR3-mediated phagocytosis.     

Interaction of iC3b with recombinant isotypic and chimeric forms of CR2.

CR2 is a component of a signal transduction complex on B lymphocytes that augments B cell responses to Ag. We have quantitatively assessed binding by the two isotypic forms of CR2 for two of its ligands, the polymerized iC3b (p(iC3b)) fragment of C3, and gp350/220, the EBV membrane protein. The recombinant 15-SCR or 16-SCR forms of CR2 bound p(iC3b) with identical affinities. Full binding activity of CR2 for p(iC3b) was observed with a chimera comprised of SCR-1 and -2 of CR2 fused to SCR-17 through -30 of CR1. Therefore, the alternatively spliced SCR-10a has no role in binding p(iC3b), and the binding activity of wild type receptor for iC3b can be reconstituted with SCR-1 and -2 of CR2. The binding affinities of the two isoforms of CR2 for soluble gp350/220 were also similar. Additional sites in the C3c region of C3 have been postulated also to interact with CR2. However, monomeric iC3b and C3d were equally effective in inhibiting the binding of p(iC3b) to CR2, indicating that the C3c region of iC3b does not contribute to the interaction of iC3b with CR2. Finally, the relative abilities of C3b and iC3b to bind to CR1 and CR2 were compared. The conversion of C3b to iC3b generated a ligand with an approximate 100-fold decrease in affinity for CR1 and a 10-fold increased affinity for CR2, resulting in a 1000-fold greater likelihood for binding to the latter receptor that may then promote B cell activation.     

Factor I-mediated processing of complement fragments on HIV immune complexes targets HIV to CR2-expressing B cells and facilitates B cell-mediated transmission of opsonized HIV to T cells

Our study demonstrates that binding of complement-opsonized HIV to complement receptor type 1 on human erythrocytes (E) via C3b fragments is followed by a rapid normal human serum-mediated detachment of HIV from E. The release was dependent on the presence of factor I indicating a conversion of C3b fragments to iC3b and C3d on the viral surface. This in turn resulted in an efficient binding of opsonized HIV to CR2-expressing B cells, thus facilitating B cell-mediated transmission of HIV to T cells. These data provide a new dynamic view of complement opsonization of HIV, suggesting that association of virus with E might be a transient phenomenon and the factor I-mediated processing of C3b to iC3b and C3d on HIV targets the virus to complement receptor type 2-expressing cells. Thus, factor I in concert with CR1 on E and factor H in serum due to their cofactor activity are likely to be important contributors for the generation of C3d-opsonized infectious HIV reservoirs on follicular dendritic cells and/or B cells in HIV-infected individuals.     

Isolation of complement-fragment-iC3b-binding proteins by affinity chromatography. The identification of p150,95 as an iC3b-binding protein.

The proteins from labelled human spleen membranes and polymorphonuclear leucocytes which bind to the iC3b fragment of complement component C3 were prepared by iC3b-Sepharose chromatography in the presence of bivalent cations. Complement receptor type 3(CR3) was eluted from iC3b-Sepharose by removal of bivalent cations. Complement receptors type 1 and 2 (present in spleen but not in polymorphonuclear leucocytes) were sequentially eluted by an NaCl gradient. An additional protein of Mr 135 000 was eluted from iC3b-Sepharose under the same conditions as those used to elute CR3. Preabsorption of the starting material on an anti-(CR3 beta-subunit) antibody column before iC3b-Sepharose chromatography removed the alpha- and beta-chains of CR3 and the 135 000-Mr protein. Preabsorption with iC3b-Sepharose before the anti-(CR3 beta-subunit) antibody column showed that iC3b binds CR3 and p150,95, the smallest member of the group of three homologous proteins that share the same beta-subunit.     

Mutagenesis of the Arg-Gly-Asp triplet in human complement component C3 does not abolish binding of iC3b to the leukocyte integrin complement receptor type III (CR3, CD11b/CD18).

The leukocyte integrin complement receptor type III (CR3, CD11b/CD18) binds the C3 cleavage product iC3b. Many other integrins bind their ligands via an Arg-Gly-Asp (RGD) triplet. Both the RGD-containing C3 peptide 1390TRYRGDQDATMS1401 (pro-C3 numbering) and the RGD-like fibrinogen peptide GGAKQAGDV, which binds to the platelet integrin glycoprotein IIb-IIIa, were shown to inhibit the iC3b-CR3 interaction, suggesting that this binding is also RGD-mediated (Wright, S.D., Weitz, J.I., Huang, A. J., Levin, S.M., Silverstein, S.C., and Loike, J.D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7734-7738). However, unlike other integrin-ligand interactions, that of CR3 and iC3b is unaffected by the hexapeptide GRGDSP, and substitutions in the RGD triplet of C3 from other species appear to be tolerated. It was, therefore, proposed (Grossberger, D., Marcuz, A., du Pasquier, L., and Lambris, J.D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1323-1327) that the highly conserved DATMS portion of the inhibitory C3 peptide may have been responsible for its binding. To address these inconsistencies and directly assess the role of the 1390-1401 segment within the complete iC3b molecule in mediating binding to CR3, a human C3 cDNA was altered by site-directed mutagenesis and the expressed recombinant proteins were examined in a CR3-specific assay. Replacement of RGD by AAA did not abolish rosetting of the corresponding iC3b-coated erythrocytes to human CR3-bearing leukocytes. In addition, mutant iC3b molecules in which the positively charged R1391 (corresponding to K in the fibrinogen peptide) and the highly conserved 1397DATMS sequence were replaced by Q and NAAMA respectively, were still bound by CR3. We conclude that the iC3b-CR3 interaction is not mediated by the RGD triplet or its neighboring residues.     

The complement-mediated binding of soluble antibody/dsDNA immune complexes to human neutrophils

The complement-mediated binding of soluble antibody/3H-dsDNA immune complexes (prepared in vitro) to human polymorphonuclear leukocytes (PMN) has been investigated quantitatively. Studies with isolated complement components in conjunction with experiments on the binding of these complexes to human red blood cells suggest that the binding to both cell types is mediated predominantly by CR1 (C4b-C3b) receptors but that CR3 (iC3b or C3d-g) receptors may play a role in binding to PMN but probably not to RBC. Our results also indicate that under the standard conditions of these assays (37 degrees C, 20 to 40 min incubations) there is no significant internalization of the soluble antibody/dsDNA immune complexes after they are bound by the PMN.     

Membrane complement receptor type three (CR3) has lectin-like properties analogous to bovine conglutinin as functions as a receptor for zymosan and rabbit erythrocytes as well as a receptor for iC3b

Human leukocyte complement receptor type three (CR3) was shown to be lectin-like and to resemble bovine serum conglutinin (K) in that it bound to both iC3b and unopsonized yeast (Saccharomyces cerevisiae), and was inhibited by EDTA or N-acetyl-D-glucosamine (NADG). CR3 and K also bound to zymosan (Z), a yeast cell wall extract that contains primarily polysaccharide and no detectable protein. However, structural differences and the absence of K on bovine phagocytes indicated that CR3 was not the human homologue of bovine K. Phagocytic and respiratory responses to unopsonized Z were CR3 dependent because they were inhibited by monoclonal antibodies specific for the alpha-chain of CR3 and did not occur with phagocytes from patients with a genetic deficiency of CR3. The binding of CR3 to Z did not require opsonization of the Z with neutrophil-secreted C3, as Z binding and responses were not inhibited by Fab anti-C3. In addition, CR3-dependent binding of yeast occurred with neutrophils from which protein secretion was blocked by fixation with paraformaldehyde. Rabbit erythrocytes (RaE) also bound weakly to neutrophil CR3 and triggered ingestion. Anti-CR3 not only blocked the binding and ingestion of RaE but also blocked selectively the ingestion of RaEC3b without affecting the strong binding mediated by CR1. Even though sheep E and sheep EC3b were not ingested by neutrophils, a weak binding of CR3 to sheep E was suggested by the finding of 20 to 40% inhibition of sheep EAIgG ingestion by anti-CR3. Such inhibition was only observed in buffers that allowed activity of the CR3 binding site and not in buffers containing either EDTA or NADG. An apparently contradictory finding was that the weak CR3-dependent binding of Z triggered neutrophil ingestion and a superoxide burst, whereas the avid CR3-dependent binding of sheep EC3bi did not induce significant ingestion or a respiratory burst. Blocking studies with monoclonal antibodies specific for different epitopes of the alpha-chain of CR3 suggested that this might result from the presence of two distinct binding sites in CR3: one site for fixed iC3b that did not trigger functions, and a second function-triggering site for Z that did not bind to fixed iC3b.