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PubMed2005

Critical role for complement receptor 3 (CD11b/CD18), but not for Fc receptors, in killing of Streptococcus pyogenes by neutrophils in human immune serum.

During phagocytosis, surface receptors on neutrophils interact with pathogens opsonized with complement factor C3b/iC3b and in some cases with antibodies. In human immune sera antibodies directed against surface-bound M proteins mediated killing of Streptococcus pyogenes by neutrophils. Surprisingly, blocking of the Fc receptors had little effect on the killing. In contrast, inhibition of C3b/iC3b generation, or blocking of the major neutrophil iC3b receptor CD11b/CD18, enabled S. pyogenes to grow efficiently in immune sera. Inhibition of CD11b/CD18, but not of CD32, the major neutrophil signaling Fc receptor, prevented Streptococcus-induced NADPH oxidase-dependent respiratory burst, and blocking of C3b/iC3b formation inhibited Streptococcus-induced activation of Cdc42, a small GTPase critically involved in transmitting pro-inflammatory signals to the cytoskeleton. Consequently, ligation of CD11b/CD18 by bacteria-bound iC3b is necessary for inducing a neutrophil response leading to elimination of S. pyogenes in immune human serum.

Animals↗
PubMed1991

The phagocytic process and the role of complement in host defense.

Three lines of defense play an integral role in host defense. The mechanical barrier (skin and mucous membrane: first line), non-specific defense (phagocytic cells non-specific humoral factors) and the specific line of defense. Phagocytic cells act in phases: chemotaxis, attachment of the bacteria to the membrane of the phagocytic cells, ingestion, killing and digestion. Attachment of the particle to receptors is only optimal when the bacteria are loaded with antibodies (IgG) and/or activated complement factors (C3b, C3Bbi), because the phagocyte has receptors for these opsonins (Fc, and C receptors). The late complement factors are also able to kill and lyse certain bacteria without phagocytes. The killing mechanism of the phagocyte is complex. It is oxygen dependent and oxygen independent. Presumably toxic oxygen species are most important to kill bacteria by phagocytes.

Bacterial Infections↗
PubMed1980

H1-receptor dependence of histamine-induced enhancement of human eosinophil C3b rosettes.

The pharmacological specificity of histamine-induced enhancement of human eosinophil C3b rosettes was studied using H1- and H2-receptor agonists and antagonists. The H1 agonist, 2-(2-aminoethyl) thiazole (2-2-AET), enhanced eosinophil C3b rosettes in a comparable fashion to that of histamine, whereas the H2 agonists, 4-methylhistamine and Dimaprit, were without effect. Similarly, rosette enhancement by histamine was inhibited by the H1 antagonists, chlorpheniramine and mepyramine, but not by the H2 antagonists, burimamide and metiamide. These experiments indicate that enhancement of eosinophil C3b rosettes by histamine, a mechanism which might be of importance in the amplification of complement-dependent killing of helminthic larvae, is predominantly H1-receptor-dependent.

Complement C3b↗
PubMed1984

Use of monoclonal antibodies against factor H to investigate the role of a membrane-associated protein antigenically related to H in C3b-receptor function.

Three murine monoclonal IgG1 kappa-antibodies, MAH-1, MAH-2, and MAH-3, were raised against factor H purified from human plasma. In cross-inhibition studies MAH-3 did not compete with MAH-1 and MAH-2, and vice versa, for the binding to H, whereas MAH-1 and MAH-2 inhibited each other. MAH-1 and MAH-2 inhibited the binding of H to C3b attached to an ELISA plate as well as to C3b bound to sheep erythrocytes by means of the classical pathway convertase and of C3b to H attached to an ELISA plate. The determinant defined by MAH-1 and MAH-2 was no longer accessible on H bound to C3b. In contrast, MAH-3 interfere with the binding of H to C3b or vice versa only to a smaller extent but recognized a determinant still accessible on H bound to C3b and was able to agglutinate EAC14o23b-H in an indirect Coombs test. All three antibodies were shown to bind to tonsil cells and Raji cells in an indirect cell ELISA. The membrane-associated molecule detected by these antibodies had an apparent m.w. of 140,000 D in SDS-PAGE. All three antibodies partially inhibited the binding of EAC14o23b to tonsil lymphocytes and, in the presence of 0.1 mM DFP, to Raji cells; binding of EAC14o23bi and EAC14o23d to tonsil cells was not affected. We conclude that MAH-3 recognizes a determinant distinct from the ones recognized by MAH-1 and MAH-2, the latter possibly defining identical epitopes that are located close to the binding site for C3b. The fact that these two distinct epitopes could be detected on a 140,000-D membrane-associated protein from lymphoid cells strongly suggests that this molecule is at least antigenically related to serum H and shares with H a region carrying the binding site for C3b. The rosette inhibition studies imply that this structure is important for the binding of C3b-coated particles to lymphoid cells.

Animals↗
PubMed1995

Study of the vesicles released during conservation of red cells.

The purpose of this study was to determine the protein composition of the vesicles released from senescent erythrocytes and its variation during their experimental conservation at +4 degrees C in citrate phosphate dextrose (CPD), over a period of 8 weeks. Techniques employed included electron microscopy, SDS-PAGE and immunoblotting. Electron microscopic observation of senescent erythrocytes showed the simultaneous release of one or several vesicles of varying size and shape, while close examination of individual vesicles revealed a slack membrane structure and the presence of band 3 protein. SDS-PAGE and immunoblotting showed the vesicles to be composed mainly of band 3 and its breakdown products and to be lacking in spectrin. Use of specific antibodies demonstrated the presence of free haemoglobin, immunoglobulin G (IgG) and fragment C3b of complement. During conservation for 8 weeks, the concentration of band 3 protein decreased, while the concentrations of IgG and C3b increased and there was no apparent variation in haemoglobin levels.

Complement C3↗
PubMed1994

The interaction of glycoprotein C of herpes simplex virus types 1 and 2 with the alternative complement pathway.

Glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) binds the human complement fragment C3b, but the two proteins differ in their ability to bind C3b on infected cell surfaces. In addition, gC-1, but not gC-2, accelerates the decay of the alternative pathway C3 convertase, thereby affecting later steps of the complement cascade. Previously, we constructed linker insertion and deletion mutants of gC-1 and gC-2 and used transient transfection to express mutant proteins in uninfected cells. In spite of the differences between gC-1 and gC-2, C3b binding was localized to residues within the central portion of both proteins, encompassing the first four cysteines. For gC-1, deletion mutants lacking amino acids 33 to 123 or 367 to 469 or lacking both regions still bound C3b. We recombined these deleted forms of gC-1 into gC-39, an HSV-1 strain lacking the gC gene. The altered forms of gC-1 were incorporated into virions, expressed on the surface of infected cells, and bound C3b. We used these proteins to investigate the structural basis for the inhibitory action of gC-1 on the complement cascade. We found that gC-1 does not inhibit formation of the alternative pathway C3 convertase. This convertase is stabilized by the serum protein properdin. Purified gC-1, but not gC-2, inhibits the binding of properdin to C3b, suggesting that this destabilizes the convertase. The mutant lacking amino acids 367 to 449 was able to inhibit properdin binding to a limited extent when present at high concentrations, although it bound to C3b more weakly than wild-type gC. In contrast, the protein lacking amino acids 33 to 123 was unable to inhibit properdin binding to C3b. Thus, gC-1 contains two structural domains, one for C3b binding, residues 124 to 366, and another, residues 33 to 133, which interferes with properdin binding to C3b.

Animals↗
PubMed1998

A monomeric human C4b-binding protein (C4bp) more efficiently inactivates C3b than natural C4bp: participation of C-terminal domains in factor I-cofactor activity.

We designed a cDNA construct encoding an artificial membrane molecule consisting of all 8 short consensus repeats (SCRs) of human monomeric C4b-binding protein (C4bp) followed by DAF's GPI anchor, named mC4bp, and expressed the protein on swine endothelial cells (SEC). At the same level of expression, mC4bp protected host cells as effectively as DAF, the most potent complement (C) regulator on the membrane. This result was unexpected from the reported functional properties of natural multimeric C4bp. Here, we investigated the mechanism whereby mC4bp has potent cell-protective activity. Our results were as follows: (1) mC4bp serves more efficiently as a methylamine-treated C3 (C3ma)-inactivating factor I-cofactor than natural C4bp and as efficiently as MCP as a methylamine-treated (C4ma)-inactivating cofactor by fluid-phase cofactor assay: (2) the potency of C3ma inactivation by mC4bp and factor I is quite high compared to those of other cofactors: (3)blocking studies using mAbs against C4bp suggested that both the 48 kDa N-terminal fragment and the C-terminal domain near the portion responsible for bundle formation participate in the high C3ma-inactivating capacity of mC4bp. Thus, acquiring high C3ma-inactivating capacity secondary to monomeric alteration leads to high C regulatory activity of mC4bp. These results infer that mC4bp differs from C4bp in its potent factor I-cofactor activity and is a good candidate as a safeguard against hyperacute rejection of xenografts.

Complement C3b Inactivator Proteins↗
PubMed1979

Metabolism of human beta 1H: studies in man and experimental animals.

Metabolic studies were performed with a purified, functionally-active preparation of human beta 1H. In seven normal human subjects, the half-life ranged from 66--87 hr with fractional catabolic rates (FCR) of 1.04--1.63%/hr. Synthesis rates were 0.22--0.57 mg/kg/hr and extravascular distribution ratios were 0.34--0.67. There was evidence of extra-vascular catabolism in each subject. In sixteen patients with immunological disease four showed hypercatabolism of beta 1H. However, three patients with C3 mephritis factor (NeF) had normal beta 1H turnover despite profound reduction in C3 concentration; it is suggested that the reaction of beta 1H with the C3b. Bb convertase exposes it to a catabolic site and that in the NeF patients the NeF stabilized convertase prevents such exposure. Studies of the acute phase response were carried out in nine patients following elective surgery, with C-reactive protein (CRP) used as the control protein: six patients showed no rise in beta 1H levels and three showed a small (20%) rise whereas all exhibited a gross rise in CRP. Pre-incubation of 125I- beta 1H with NHS, with NHS in the presence of NeF and with C3b+C3b 1NA caused no change in beta 1H turnover in animals despite demonstrable total C3 conversion with the NeF.

Animals↗
PubMed1981

Interaction of target cell-bound C3bi and C3d with human lymphocyte receptors. Enhancement of antibody-mediated cellular cytotoxicity.

The occurrence and distribution of distinct receptors for three C3 fragments on purified human blood lymphocytes were studied by rosette formation. Indicator cells were bovine, chicken, or sheep erythrocytes (E) bearing up to 100,000 molecules of human C3b (EC3b) without antibody. EC3b was converted to C3bi-bearing-E (EC3bi) with purified C3b inactivator (factor I) and beta1H (factor H), and to C3d-bearing E (EC3d) by treatment of EC3bi with trypsin. Using bovine E (Eb) as indicators, approximately 11% of the lymphocytes bound EbC3b, 6% bound EbC3bi and 2% bound EbC3d. Fractionation of the lymphocytes by adsorption to monolayers of C3-fragment-bearing Eb or by rosetting indicated that most of the cells with receptors for C3b were distinct from those having receptors for C3bi and/or C3d. Cells from two lymphoblastoid cell lines (Raji and Daudi) formed strong rosettes with EC3b, which were weak. 51Cr-labeled E was used as a target in antibody, C3-fragment-bearing E was not lysed by the lymphocytes. However, at suboptimal concentrations of IgG enhancing capacity of the fragments occurred in the order of C3bi greater than C3d greater than C3b. In addition, C3-fragment-bearing cells inhibited the lysis of antibody-coated cells not concluded that target cell bound C3 fragments enhance ADCC by improving contact between target cells and those effector cells which have C3 receptors. Cell-bound C3 effector cells. It is proposed that certain lymphocytes are capable of interacting with C3bi in addition to C3b and C3d and that C3bi and C3d have a greater regulatory effect on their cytolytic function than C3b.

Animals↗
PubMedDate not supplied

Interactions of the third component of human complement (C3) with tumour cells: evidence for presence of C3b acceptor sites and direct activation of C3 by tumour cells.

The interaction of C3 with different tumour cell lines was investigated. Our results indicated that nearly all the cell lines had C3b acceptors on their membranes. The binding of C3b was visualized by the detection of this protein on the cell surface with fluoresceinated anti-C3c serum. The involvement of proteases in the activation of C3 was demonstrated: complement-membrane fluorescence occurred in the presence of EDTA but was completely abolished with epsilon-aminocaproic acid. Experiments performed with purified C3 led to the conclusion that the protease activity might be the initial event for the activation of the alternative pathway by tumour cells.

Burkitt Lymphoma↗
PubMed1989

[Distribution of C3b receptors on polymorphonuclear leukocytes from normal healthy adults by scanning immunoelectron microscopy].

The distribution of C3b receptors on polymorphonuclear leukocytes cultured from normal healthy volunteers was examined by scanning immunoelectron microscopy with mouse anti-CR1 monoclonal antibody and 40 nm-goat anti-mouse IgG antibody. The distribution was categorized into three types; first, Au-particles clustered in numbers of several to ten or more on polymorphonuclear leukocytes (mean +/- S.D. = 59.5 +/- 36.8, n = 146), second, tens of the particles mostly on two or three ruffles of the cells (121.7 +/- 63.8, n = 45), and third, particles assembled at one part of the cells (74.1 +/- 47.4, n = 59). The distribution of Au-particles on all polymorphonuclear leukocytes showed a normal distribution curve (74.1 +/- 50.6, n = 250, p less than 0.02). These results show that polymorphonuclear leukocytes had some tendencies but no uniformity of distribution of C3b receptors on their plasma membrane when they were cultured.

Cells, Cultured↗
PubMed1983

Requirement for human red blood cells in inactivation of C3b in immune complexes and enhancement of binding to spleen cells.

We showed previously that soluble immune complexes (IC) added to normal human serum and unseparated autologous blood cells bind predominantly to red blood cells (RBC) bearing C3b receptors. RBC-associated IC are processed both by C3b/C4b inactivator (I) cleavage of C3b and by alternative pathway-dependent C3b generation, and are returned to the serum in a modified form. In the present study, soluble IC were prepared by using 125I-BSA and guinea pig anti-BSA, and were treated with diluted human serum to optimize sustained C3b-dependent binding to human RBC. This binding to RBC could not be abrogated by preincubation of IC with heat-inactivated human serum or purified serum regulatory proteins, I and beta 1H (H), but after binding, pretreated IC dissociated. When IC were prepared with serum containing 125I-C3, treatment with I + H did not release labeled C3 from the complexes. Analysis by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) of 125I-C3b in IC showed similar labeled peaks whether IC were pretreated with buffer or with I + H or were bound to RBC without pretreatment. In contrast, when IC containing 125I-C3b were exposed to I + RBC, nearly 70% of the label was released from the IC. The released material analyzed by SDS-PAGE showed major 125I-C3 peaks corresponding in m.w. to free beta-chain and alpha 1 cleavage products of 30,000 to 33,000 daltons. IC in undiluted serum after incubation with RBC gave less binding to human polymorphonuclear leukocytes than after incubation without RBC, but notably more binding to guinea pig spleen cells and human lymphoblastoid cells. These findings argue that exposure to serum regulatory proteins does not prevent subsequent binding of experimental IC to RBC, that RBC binding promotes degradation of C3b in IC which proceeds beyond iC3b, and that this I-mediated breakdown renders IC reactive with spleen cells.

Animals↗
PubMed2005

Comparison of surface recognition and C3b binding properties of mouse and human complement factor H.

Factor H (FH) is a central complement regulator both in plasma and on certain cellular and acellular surfaces that are in contact with plasma. Although FH deficiency has been shown to lead to similar diseases in man and mice (membranoproliferative glomerulonephritis or dense deposit disease) little is known about the similarity between the human and murine FH functions. We here characterize the interactions of murine FH (mFH) with C3b, glycosaminoglycans, and endothelial cells and compare these interactions with those of human FH (hFH). To achieve this we purified mFH and murine C3 from plasma, prepared murine C3b, and expressed recombinant mFH constructs containing domains 1-5 and 18-20 (mFH1-5 and mFH18-20). For comparisons, hFH, human C3b, and recombinant hFH1-5 and hFH18-20 were used. We demonstrate that mFH and mFH1-5 do act as cofactors for factor I-mediated cleavage of human C3b. Surface plasmon resonance analysis showed binding of mFH18-20 to murine C3b and weak binding to human C3b. The mFH18-20 construct bound to heparin in a manner comparable to hFH18-20. It was demonstrated by flow cytometry that mFH and mFH18-20 bind to human endothelial cells in a similar manner to hFH and hFH18-20. Taken together, locations of the key functions of mFH, i.e. complement regulation and surface recognition, are comparable to hFH. Recently, mutations in the carboxy-terminal end of hFH have been found to be associated with atypical hemolytic uremic syndrome (aHUS). Based on the results in this report it is conceptually attractive to establish a murine model for aHUS.

Animals↗
PubMed2006

Opsonic requirements for the respiratory burst of neutrophils against Giardia lamblia trophozoites.

BACKGROUND: Giardia lamblia is an important cause of parasitic diarrheal disease worldwide. Occasionally, polymorphonuclear neutrophils (PMNs) may participate as effector cells against Giardia lamblia. The present study was performed in order to examine the role of specific antibody and complement components in promoting the respiratory burst (RB) of PMNs against Giardia lamblia. METHODS: PMNs from human adult volunteers were incubated with Giardia trophozoites in the presence of non-immune (NS) or hyperimmune (HS) serum (anti-Giardia titer, >1:1024). Adherence was scored visually on coverslide after staining with Giemsa. The ability of Giardia to trigger the oxidative response of PMNs was measured by the anion superoxide (O2(-)) production using a cytochrome C reduction method and by the luminol amplified chemiluminescence (CL) assay. RESULTS: Incubation with NS or HS increased Giardia adherence to PMNs from 6.9 +/- 3.2% (basal adherence of Giardia incubated in buffer) to 39 +/- 18.6% (p <0.01) and 76 +/- 19.5% (p <0.001), respectively. In absence of serum, Giardia failed to trigger an oxidative response of PMNs. Opsonization with NS or HS increased the PMN O2(-) production from 3.9 +/- 0.92 nmol/2.5 x 10(6) PMNs/10 min to 9.04 +/- 1.68 (p <0.05) and 17.9 +/- 1.32 (p <0.001), respectively. A similar enhancement of the CL response was also observed. The inactivation of complement activity by heat as well as the elimination of specific antibodies by absorption produced a significant abrogation of the oxidative response but in the case of HS heat inactivation alone did not abolish the response. Similar findings (variable abrogation of the oxidative PMN response) were observed when PMNs were incubated with monoclonal antibodies directed against complement C3, C3b or the low-affinity Fc receptors (CR1, CR3 or FcRlo). CONCLUSIONS: These results show that complement components and specific antibodies influence in the Giardia-PMN interaction. Although components of complement can contribute to the RB of PMNs, specific antibodies are critical for an optimal oxidative PMN response.

Animals↗
PubMed2005

Localization of IL-8 and complement components in lesional skin of psoriasis vulgaris and pustulosis palmaris et plantaris.

BACKGROUND: Munro's microabscesses are a characteristic histopathologic feature of psoriasis vulgaris; however, the pathomechanisms underlying the migration of transepidermal leukocytes (PMNs) have not been fully elucidated yet. OBJECTIVE AND METHODS: Since the lesional scale extracts contain potent chemoattractants, such as IL-8 and C5a fragments, we studied their location in the lesions of psoriasis vulgaris and PPP with immunohistochemical techniques. RESULTS: Localization of IL-8 was not detected in the subcorneal keratinocytes but was demonstrated only in the basal keratinocytes together with migrating PMNs. In contrast, the presence of a complement fragment, C3b, was observed on the cell membranes of subcorneal keratinocytes, suggesting that these were the sites of complement activation. CONCLUSION: Such distinct localization of IL-8 and complement components suggests that the intraepidermal migration of PMNs takes place first according to the concentration gradient of IL-8, and thereafter they are guided by complement components to the final destination, the subcorneal portion of the lesional skin.

Cell Movement↗
PubMed1991

IgA-mediated inhibition of human leucocyte function by interference with Fc gamma and C3b receptors.

The inhibitory effects of IgA from human colostrum, and IgA1 and IgA2 from human serum on the chemiluminescence (CL) response and phagocytosis of polymorphonuclear leucocytes (PML) to Staphylococcus epidermidis and the CL response to formylmethionyl-leucyl-phenylalanine (FMLP) were studied. The dose-dependent inhibition of the luminol-mediated CL response of human PML to the bacteria was observed in the presence of more than 0.1 mg/ml IgA from both colostrum and serum. The preincubation of PML with a solution of IgA enhanced the suppressive effect of IgA on the cells. Removal of IgA from the reaction mixture after preincubation resulted in recovery, with time, of the response of PML to the bacteria. The bacteria treated with IgA did not give rise to any inhibition of the response. The CL response of PML to FMLP was not affected by the presence of IgA in the reaction mixture. The decrease of phagocytic activity of PML in the presence of IgA resulted in a decrease of NADPH oxidase activity of PML after stimulation with the bacteria as compared with the absence of IgA. The effect of IgA on the receptors of Fc and C3b (CR1) on the surface of PML was measured by monitoring erythrocyte-antibody (EA) or erythrocyte-antibody-complement (EAC) rosette formation and by direct and indirect immunofluorescence techniques using anti-CR1 antibody and Fc-specific antibodies. The presence of IgA in the reaction mixture led to a quantitative decrease in CR1 and the ability to bind IgG to the surface of PML.

Antigens, Differentiation↗