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PubMed2001

Generation of recombinant human C3dg tetramers for the analysis of CD21 binding and function.

CD21 (complement receptor 2, CR2) binds the terminal proteolytic fragments of the third component of complement (C3) that have been covalently attached to immune complexes or other targets during the activation of complement. We used the technique of in vivo biotinylation to create a recombinant multivalent ligand for CD21. A sequence coding for a biotinylation signal peptide was added to the 3' end of the human C3dg cDNA. The modified C3dg was expressed in Escherichia coli and biotinylated intracellularly by the bacterial biotin holoenzyme synthetase (BirA) enzyme. Monomeric C3dg was unable to bind to CD21 as determined by flow cytometry, while biotinylated recombinant C3dg (rC3dg) complexed with fluorochrome-conjugated streptavidin bound tightly. Binding was observed using CD21 positive B cells but not seen on pre-B cells that do not express this complement receptor. Two assays were used to assess the functional capacity of the recombinant C3dg. First, multimeric C3dg caused the phosphorylation of the mitogen-activated kinase, p38, in mature B lymphoma cells. Second, C3dg greatly enhanced the activation of primary B cells in combination with a sub-stimulatory concentration of anti-IgM monoclonal antibody. These results illustrate the utility of the technique of in vivo biotinylation to generate ligands for cell surface receptors that require multimerization for high avidity binding and function.

Amino Acid Sequence↗
PubMed2002

Internalization of circulating apoptotic cells by splenic marginal zone dendritic cells: dependence on complement receptors and effect on cytokine production.

Under steady-state conditions, internalization of self-antigens embodied in apoptotic cells by dendritic cells (DCs) resident in peripheral tissue followed by DC migration and presentation of self-peptides to T cells in secondary lymphoid organs are key steps for induction and maintenance of peripheral T-cell tolerance. We show here that, besides this traffic of apoptotic cells mediated by peripheral tissue-resident DCs, splenic marginal zone DCs rapidly ingest circulating apoptotic leukocytes, process apoptotic cell-derived peptides into major histocompatibility complex class II (MHC-II) molecules, and acquire CD8alpha during their mobilization to T-cell areas of splenic follicles. Because apoptotic cells activate complement and some complement factors are opsonins for phagocytosis and play roles in the maintenance of peripheral tolerance, we investigated the role of complement receptors (CRs) in relation to phagocytosis of apoptotic cells by DCs. Apoptotic cell uptake by marginal zone DCs was mediated in part via CR3 (CD11b/CD18) and, to a lesser extent, CR4 (CD11c/CD18) and was reduced significantly in vivo in hypocomplementemic animals. Following phagocytosis of apoptotic cells, DCs exhibited decreased levels of mRNA and secretion of the proinflammatory cytokines interleukin 1alpha (IL-1alpha), IL-1beta, IL-6, IL-12p70, and tumor necrosis factor alpha (TNF-alpha), without effect on the anti-inflammatory mediator transforming growth factor beta1 (TGF-beta1). This selective inhibitory effect was at least partially mediated through C3bi-CD11b/CD18 interaction. Characterization of apoptotic cell/DC interaction and its outcome provides insight into the mechanisms by which apoptotic cells affect DC function without disrupting peripheral tolerance.

Animals↗
PubMed1989

Requirement of leukocytic cell adhesion molecules (CD11a-c/CD18) in the enhanced NK lysis of iC3b-opsonized targets.

Treatment of Raji or Daudi cells with human serum under conditions which allow the alternative pathway of C activation results in their C3-opsonization and enhanced sensitivity to NK-mediated lysis. The effector lymphocytes have low buoyant density, carry CD16 and HNK1 markers as well as the CD11a-c/CD18 leukocytic cell adhesion molecules. One of these molecules, made up of CD11b-CD18 (alpha- and beta-chains), is also the receptor for iC3b. We studied the role of the cell adhesion molecules in the NK effect on targets with and without C3-fragments. We focused on the E/T interaction of opsonized cells in the presence of anti CD18 mAb. mAb directed to the CD11a molecule caused 0 to 30% inhibition of the lysis of both non-opsonized and opsonized cells whereas the mAb antibody directed to the CD11c molecule had no effect. Reagents reactive with the iC3b binding site of CD11b (alpha-chain of the CR3) molecule did not alter the lysis of non-opsonized targets whereas they abrogated the C3-mediated increment of the Nk effect on opsonized cells. Two mAb preparations, 60.3 and IB4, directed to the CD18 chain shared by the three cell adhesion molecules abrogated in a dose-dependent way the lysis of both non-opsonized and opsonized targets. The 60.3 mAb inhibited the iC3b binding site of CR3 (despite its localization on the alpha-chain) and in accordance it inhibited the binding of lymphocytes to the opsonized target also. The IB4 did not affect this site and in accordance it inhibited only partially the binding of effectors to the C3 fragment carrying Raji, nevertheless it inhibited their lysis. This result indicates that the iC3b-CR3 bridge is insufficient for triggering the lysis in absence of the contact through the adhesion molecules.

Antibodies, Monoclonal↗
PubMed1995

Interaction of complement and specific antibodies with the external glycoprotein 120 of HIV-1.

Previously we have investigated the interaction of human complement as well as one polyclonal and three human monoclonal antibody preparations with the human immunodeficiency virus type-1 (HIV-1) transmembrane recombinant glycoprotein (rgp41). A strong competition was found between the antibodies and deposited complement proteins for the same binding sites located within the immunodominant region of rgp41. The aim of the present experiments was to see if the same type of antibody-complement-HIV-1 interactions could be observed with the outer envelope glycoprotein (rgp120) of HIV-1. Three different glycosylated rgp120 preparations, as well as a synthetic peptide corresponding to the V3 loop of the MN strain, were adsorbed to enzyme-linked immunosorbent assay (ELISA) plates and incubated with mixtures of anti-rgp120 antibodies and normal human serum (NHS) as a complement source. Fixed complement proteins and antibodies were detected with specific, peroxidase-labelled antibodies against different complement proteins (C1q, C4b, C3b) and the gamma-chain of antibodies. In the absence of anti-rgp120, high amounts of C3 were deposited to each rgp120 preparation tested (including the V3 peptide) but significant differences in the amounts of bound C1q and C4b were observed. Using sera deficient in different complement proteins, we found that both the classical and the alternative pathways contributed to the C3 binding to rgp120. Addition of specific antibodies did not increase complement activation by rgp120 and only in the case of a monoclonal antibody to the V3-loop could we see complement-dependent inhibition of antibody binding.

Antibodies, Monoclonal↗
PubMed1980

Modulation of human neutrophil function by monohydroxy-eicosatetraenoic acids.

The generation from arachidonic acid and purification of large quantities of a series of monohydroxy-eicosatetraenoic acids (HETEs) which differed only in the position of the hydroxyl group permitted an in vitro analysis of the relative effects of the HETEs on a variety of human neutrophil functions. All of the HETEs elicited maximal neutrophil chemotactic responses of comparable magnitude, but the chemotactic potencies exhibited a distinct rank order with 5-HETE greater than 8-HETE:9-HETE (85:15, w:w) greater than 11-HETE=12-L-HETE. Peak chemotactic responses were achieved at concentrations of 1 microgram/ml for 5-HETE, 5 microgram/ml for 8-HETE:9-HETE and 10 microgram/ml for 11-HETE and 12-L-HETE. In the absence of a concentration gradient, the HETEs were similar in potency with respect to the stimulation of neutrophil chemokinesis and the enhancement of the expression of neutrophil C3b receptors. At optimally chemotactic and chemokinetic concentrations, none of the HETEs stimulated the generation of superoxide by neutrophils, altered the expression of neutrophil IgG-Fc receptors, or evoked the release of lysosomal enzymes. Methyl esterification of 5-HETE and 12-L-HETE reduced the chemotactic activity to less than 12% of that of the parent compound. The HETE methyl esters competitively inhibited the chemotactic activity of the homologous free acids by approximately 50% at equimolar concentrations, without inhibiting the chemotactic activity of formyl-methionyl peptides or of chemotactic fragments of the fifth component of complement (C5fr). The stimulus specificity of the competitive inhibition of chemotaxis by HETE methyl esters and the functional selectivity of the HETEs as compared to the formyl-methionyl peptides and C5fr, which stimulate neutrophil oxidative metabolism and lysosomal enzyme release, suggest that HETEs activate human neutrophils by a unique mechanism.

Arachidonic Acids↗
PubMed1984

Phagocytosis by human monocyte-derived macrophages. Independent function of receptors for C3b (CR1) and iC3b (CR3).

Human monocytes and monocyte-derived macrophages were examined for their ability to bind and ingest C3-coated sheep erythrocytes (E). Greater than 90% of monocytes and macrophages formed rosettes with EC3b and EC3bi prepared with 15,000-20,000 molecules of C3 per E. Binding of EC3b to the monocyte or macrophage surface was inhibited by rabbit anti-C3b receptor (CR1), but was not inhibited by two different monoclonal anti-C3bi receptor (CR3) antibodies. EC3bi rosette formation was inhibited by monoclonal anti-CR3, but not by anti-CR1. Monocytes and macrophages did not form rosettes with similarly prepared EC3d,g or EC3d. Macrophages cultured for 7 days, but not freshly isolated monocytes, phagocytosed both EC3b and EC3bi. This ability was a consequence of macrophage maturation, as no external stimuli were present during in vitro culture. Experiments directed to determine if EC3b was converted to EC3bi before ingestion suggested that macrophage CR1 and CR3 mediated phagocytosis independently. No evidence was obtained that during the phagocytosis assay, macrophage factor I converted EC3b to EC3bi. The number of E bound or ingested by monocytes and macrophages was dependent on the number of molecules of C3b or iC3b bound per E. Monocytes and macrophages did not require the presence of either Ca++ or Mg++ for rosette formation with EC3b, whereas both divalent cations were required for optimum rosette formation with EC3bi. The presence of divalent cations was required for macrophage phagocytosis of EC3b and EC3bi. For ingestion of EC3b, Mg++ alone was sufficient, and the addition of Ca++ did not increase the number of EC3b ingested. For ingestion of EC3bi, both Ca++ and Mg++ were required for optimal phagocytosis, and their effect was concentration dependent and additive.

Calcium↗
PubMed1996

Internalin A can mediate phagocytosis of Listeria monocytogenes by mouse macrophage cell lines.

Listeria monocytogenes internalin A (InlA) is a surface protein that mediates the attachment of Listeria to, and invasion of, hepatocytes, epithelial, and endothelial cells. In this study, we tested whether InlA could also mediate phagocytosis of L. monocytogenes by the non-listericidal mouse macrophage cell lines J774A.1 and H36.12j. Recombinant InlA (rInlA) was used to derive mouse monoclonal anti-InlA antibodies (mAb) and rabbit anti-InlA antibodies. Fluorescence microscopy demonstrated that these anti-InlA antibodies reacted with wild-type L. monocytogenes, L. ivanovii, and L. innocua+, a mutant transformed with the inlAB operon that expresses surface InlA but failed to react with Bug 8, an InlA/InlB-negative transposon mutant of L. monocytogenes or with noninvasive Listeria sp. Fluorescence microscopy, radiolabeling, and flow cytometry showed that rInlA bound specifically to both macrophage cell lines. Incubation of macrophages and wild-type L. monocytogenes in the presence of rInlA or pretreatment of Listeria with anti-InlA antibodies specifically inhibited, by at least 50%, the phagocytosis of Listeria by both of these cells. By comparison, treatment with these reagents failed to affect the phagocytosis of Streptococcus pyogenes by either macrophage cell line nor did these reagents alter the ability of macrophages to internalize wild-type L. monocytogenes. We found that Bug 8, but not wild-type L. monocytogenes, failed to grow within both of these non-listericidal macrophage cell lines. In contrast to infection by wild-type L. monocytogenes, Bug 8 was rapidly eliminated from the spleens of both C57Bl/6 and DBA/2 mice. Data presented here show that only invasive Listeria sp. have surface InlA and that L. monocytogenes can enter non-listericidal macrophage cell lines by binding of bacterial InlA to the macrophage cell surface.

Animals↗
PubMed1985

Effect of complement on the viscoelastic properties of human erythrocyte membrane.

Using the micropipette technique, we examined the viscoelastic properties of the red blood cell (RBC) membrane which had been strongly coated with various components of complement (primary C3b, C3d, C4b, C4d) in vitro. The membrane elastic modulus (E), the viscosity index of the initial rapid phase of deformation (eta D1), the viscosity of the later slow phase of deformation (eta D2) and the viscosity of the recovery phase (eta R) were determined. Compared to control non-complement coated RBCs, RBCs coated with C3d, either alone or with other complement components, showed significant increase in the values for elasticity and viscosities. Thus C3d fixation resulted in decreased membrane deformability. Changes in membrane viscoelasticity due to bound C3d were not enhanced by bound C4b, C4d, C5, factor Bb or p; presence on RBC membrane of the latter two complement components may partially reverse the effect of C3d fixation. Lipid fluidity of RBC membrane, examined by fluorescence depolarization, increased with fixation of all complement components except C5. These complement-induced changes in membrane viscoelastic properties have potential pathophysiological and clinical implications. The data suggest that extravascular sequestration of human RBCs may be explained in part by increased membrane rigidity resulting from C3d fixation.

Complement C3↗
PubMed1982

Physicochemical and functional changes in human leukemic cell line HL-60.

The recently established human promyelocytic cell line HL-60 was induced to differentiate in the present of DMSO. During this process, physicochemical, and functional changes were detected simultaneously. After exposure to DMSO for more than 1 day, the cell volume decreased and the tendency for hydrophobic interaction increased. Using a hydrophobic two-phase system in counter current distribution fashion, it was then possible to separate more mature metamyelocytes and segmented granulocytes from immature myeloblasts and promyelocytes. Increased functional maturity was reflected by increased chemiluminescence (CL) response and phagocytic activity. Using yeast particles opsonized with IgG as stimulating agent, the CL response increased already after 1 day in DMSO, in parallel with increased phagocytosis of these particles. In contrast, C3b-opsonized yeast and phorbol 12-myristate 13-acetate (PMA) did not enhance the CL response conspicuosly until days 3-4. These data suggest that Fc receptor function linked to phagocytosis and the activation of oxidative metabolism develop earlier than that of C3b and PMA. The dissociation between Fc- and PMA-dependent stimulation of the oxidative metabolism may reflect different mechanisms of activation.

Cell Differentiation↗
PubMed2004

Engineering of human complement component C3 for catalytic inhibition of complement.

As a novel therapeutic approach in complement-mediated pathologies, we recently developed a human C3 derivative capable of obliterating functional complement by a catalytic, non-inhibitory mechanism. In this derivative, the C-terminal region of hC3 was substituted by a 275 amino acid sequence derived from the corresponding sequence of cobra venom factor (CVF), a complement-activating C3b homologue from snake venom. In this study, we replaced shorter C-terminal sequences of hC3 by corresponding CVF sequences to further reduce potential immunogenicity and to identify domains essential for the formation of functionally stable C3 convertases. In one of these derivatives that is still capable of obliterating functional complement in vitro, the non-human portion could be reduced to a small domain located in the C-terminus of different complement proteins. This conserved NTR/C345C motif is known to be involved in assembly of different convertases of the complement system. These results suggest a major role of the C345C domain in the regulation of the half-life of the C3 convertase. Moreover, its overall identity of 96% to human C3 renders this derivative a promising candidate for therapeutic intervention in complement-mediated pathologies.

Amino Acid Sequence↗
PubMed1983

Vitamin D metabolites change the phenotype of monoblastic U937 cells.

U937 is a human-derived lymphoma cell line that has monoblastic properties and high-affinity receptors for 1 alpha,-dihydroxyvitamin D3. Incubation of these cells with the vitamin D metabolite at 10 nM for 5 days produced marked stimulation in adherence and ingestion of Staphylococcus aureus (645% of control) and of C3b receptor (CR1) expression (292% of control) and a slight increase in hexose monophosphate shunt activity without changing cell growth rates or Fc fragment receptor expression. The changes in cellular association of S. aureus and the CR1 were detected as early as 48 hr of incubation and peaked between 3 and 5 days. Similar changes in the CR1 were induced by 25-hydroxy- and 24,25-dihydroxyvitamin D3 at micromolar concentrations. Dexamethasone, hydrocortisone, and progesterone had no effect on CR1 expression. U937 cells incubated in the presence of vitamin D metabolites exhibited a change in their phenotype. These results suggest that vitamin D metabolites may contribute to monocyte/macrophage differentiation.

Calcifediol↗
PubMed1990

The expression of C3b receptors in the differentiation of discoid lupus erythematosus and systemic lupus erythematosus.

We studied the expression of the C3b receptor, CR1, on erythrocytes (E-CR1) of patients who, in spite of having mild systemic symptoms, were diagnosed as having discoid lupus erythematosus and followed accordingly. We found that E-CR1 was markedly reduced in these patients, similar to that seen in patients with systemic disease. In contrast, those patients with completely asymptomatic discoid lupus erythematosus had the same expression of E-CR1 as the normal population.

Complement C3b↗
PubMed1984

Factors influencing immune complex localisation.

In systemic immune complex (IC) diseases such as SLE and rheumatoid vasculitis, IC accumulate in a number of tissues, either after deposition from the circulation or from in situ formation. The tissue localisation of IC depends on a delicate balance between the production of IC and the ability of the mononuclear phagocytic system (MPS) to remove them from blood. At times IC are cleared inefficiently, persist in the circulation and subsequently localise in tissues. This review evaluates the role of local tissue factors - anatomical, physiological, physical and immunological - in this process. We report on our studies examining the significance of C3b and IgG Fc receptors in tissues subject to IC deposition. No evidence for such receptors was found with the exception of a C3b receptor in human glomeruli. Our negative findings may be due to methodological difficulties in the identification of in situ receptors. Alternatively, immune receptors may not be present at these extra-glomerular sites and would therefore be unlikely to participate in IC localisation.

Animals↗
PubMed1985

A possible self-regulating mechanism mediated by C3b-acceptor-bound C3b generated by stimulated macrophages.

Macrophages have been shown to produce C3 and to bear Fc receptors (FcR), and besides the various C3 receptors, they possess C3b acceptors (C3bA) as well as surface proteases capable of cleaving C3. Using the immune adherence method, we demonstrated that the amount of covalently fixed (i.e., C3bA-bound) C3b is markedly increased upon cell stimulation by phorbol myristate acetate or aggregated IgG, even in the absence of C3. The enhancement of nascent C3b (C3bx) binding to C3bA on these cells could be reversed by inhibiting the process at different stages, using either cycloheximide, phenyl-methyl-sulphonyl-fluoride, salycil hydroxamic acid, or methylamine. On the basis of our present results and earlier results, we propose a self-regulatory mechanism by which activated, C3-producing macrophages cleave C3 by their surface proteases. C3bx generated in this way fixes covalently to C3bA of the producer cells, resulting in the inhibition of FcR on these cells.

Animals↗
PubMed2000

Amino acid residues in the PSI domain and cysteine-rich repeats of the integrin beta2 subunit that restrain activation of the integrin alpha(X)beta(2).

The leukocyte integrin alpha(X)beta(2) (p150,95) recognizes the iC3b complement fragment and functions as the complement receptor type 4. alpha(X)beta(2) is more resistant to activation than other beta(2) integrins and is inactive in transfected cells. However, when human alpha(X) is paired with chicken or mouse beta(2), alpha(X)beta(2) is activated for binding to iC3b. Activating substitutions were mapped to individual residues or groups of residues in the N-terminal plexin/semaphorin/integrin (PSI) domain and C-terminal cysteine-rich repeats 2 and 3. These regions are linked by a long range disulfide bond. Substitutions in the PSI domain synergized with substitutions in the cysteine-rich repeats. Substitutions T4P, T22A, Q525S, and V526L gave full activation. Activation of binding to iC3b correlated with exposure of the CBR LFA-1/2 epitope in cysteine-rich repeat 3. The data suggest that the activating substitutions are present in an interface that restrains the human alpha(X)/human beta(2) integrin in the inactive state. The opening of this interface is linked to structural rearrangements in other domains that activate ligand binding.

Amino Acid Sequence↗
PubMed1992

Polymorphism and proteolytic fragments of granulocyte membrane cofactor protein (MCP, CD46) of complement.

Human granulocytes (polymorphonuclear leucocytes, PMN) possess a membrane cofactor protein (MCP, CD46), which is structurally and functionally distinct from the MCPs of other cell types: it shows a single broad band of 56-80 kDa (without the doublet pattern characteristic of MCP) on SDS/PAGE and has less affinity for complement component C3b. We purified PMN MCP using monoclonal antibodies in order to study the molecular differences between it and other MCPs. Several forms of PMN MCP with size heterogeneity were noted on SDS/PAGE and by immunoblotting. O-Glycanase treatment decreased this heterogeneity, yielding a fast-migrating component identical in position on SDS/PAGE to the O-glycanase-treated MCP of other cells. The cell-specific variation of MCP, therefore, arises from post-translational glycosylation and not from a difference in primary structure. The Factor I cofactor activity of PMN MCP was more efficient in cleaving the methylamine-treated complement components C4/C3 than was MCP from other cells, which shared a similar potency of cofactor activity on a weight basis. Two types of small-form PMN MCP were identified during purification. These were 42 kDa and 30 kDa in size; the former was recognized by M177 (a monoclonal antibody against the active site marker), possessed N-linked sugars [located on the short consensus repeats (SCRs)] but not O-linked ones (on the Ser/Thr-rich region), and retained cofactor activity for C3b/C4b cleavage, similar in potency to that of other MCPs. The functionally active soluble form of MCP was observed specifically in PMN. Protease inhibitors did not inhibit liberation of the fragments, although the generated fragments became susceptible to serine proteases. The findings show that the SCRs are the functional domain of MCP and that the MCP proteolysis found only in PMN may modulate the properties of PMN MCP. In conclusion, the structural features of PMN MCP largely reflect a variability in the O-linked sugars, and the decreased affinity for C3b may be in part attributable to proteolysis.

Antigens, CD↗
PubMed1980

Alveolar macrophage lysosomal enzyme and C3b receptors in cryptogenic fibrosing alveolitis.

Levels of the lysosomal enzyme N-acetyl-beta-glucosaminidase were measured in alveolar macrophage extracts and lung lavage fluid obtained from patients with cryptogenic fibrosing alveolitis and a control group. Evidence is presented which suggests that macrophages from patients with cryptogenic fibrosing alveolitis secrete this enzyme in vivo. To investigate one of the possible mechanisms of stimulating this secretion, an assessment was made of the C3b receptor sites on alveolar macrophages. While there were fewer receptor sites on the macrophages from patients with cryptogenic fibrosing alveolitis, which might indicate that they were occupied in vivo, this difference did not reach statistical significance. It is suggested that the alveolar macrophage is not merely a phagocyte but that it also has a more active role in the pathogenesis of cryptogenic fibrosing alveolitis.

Acetylglucosaminidase↗