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PubMed1990

In vivo inhibition of the antibody response by a complement receptor-specific monoclonal antibody.

BALB/c mice were injected intravenously with three different monoclonal antibodies (mAbs) specific for complement receptor 1 (CR1). Two of the mAbs crossreacted with CR2. 24 h later, the mice were immunized with horse erythrocytes or keyhole limpet hemocyanin (KLH), and the primary antibody response was measured. One of the anti-CR antibodies, 7G6, suppressed greater than 99% of the direct plaque-forming cell response against horse red blood cells (HRBC). The same antibody markedly suppressed the serum antibody responses to both HRBC and KLH. To be optimally suppressive, the mAb had to be injected before suboptimal concentrations of antigen. The other two complement receptor-specific antibodies had very moderate, if any, effects on the antibody response. 7G6 was able to downregulate CR1 and CR2 on the surface of B cells and, in addition, to inhibit rosette formation with C3d-coated sheep erythrocytes (EC3d). One of the antibodies with a weak effect downregulated only CR1. The other downregulated both CR1 and CR2, although not as efficiently as 7G6, and was unable to inhibit EC3d rosette formation. We conclude that the reason 7G6 is outstanding in its suppressive capacity is that it is the only mAb tested that functionally blocks CR2. The data suggest that CR2 is of crucial importance in the initiation of a normal antibody response to physiological concentrations of antigen.

Animals↗
PubMed1990

A-431 cells and human keratinocytes synthesize and secrete the third component of complement.

Biosynthetic radiolabeling studies demonstrate that A-431 cells, a human epidermoid carcinoma cell line, and human keratinocytes synthesize and secrete C3 as two disulfide-linked polypeptide chains of 120 and 75 kD. Moreover, epithelial cell-derived C3 co-migrates in SDS-PAGE with that produced by HepG2 cells, a human hepatoma cell line previously used to elucidate complement component biosynthesis. Pulse-chase studies in A-431 cells demonstrate that epithelial cell-derived C3 is produced as a 195-kD precursor molecule, pro-C3, which is processed intracellularly by limited proteolysis into 120- and 75-kD C3 alpha and beta chains. Comparative studies demonstrate that A-431 cell-derived C3 is synthesized, processed, and secreted in parallel but in lower quantity than that produced by HepG2 cells. Treatment of biosynthetically labeled A-431 cell culture supernatants with normal human serum and zymosan produces C3 alpha chain cleavage and specific C3 fragments that are not present in control culture supernatants treated with heat-inactivated human serum and zymosan. Northern blot analysis of total cellular RNA extracted from A-431 cells, human keratinocytes, and HepG2 cells reveals quantitative identity of a 5.1-kb C3 mRNA species in these three cell types. Epithelial cell-derived C3 may play an important role in local inflammatory and immunologic reactions including such reactions in human skin. Moreover, epithelial cell C3 synthesis may have direct relevance to the recent demonstration of C3d,g within selected normal primate epithelial basement membranes, including epidermal basement membrane.

Blotting, Northern↗
PubMed1981

The effect of complement in adherent immune complexes on Fc and C3 receptor expression in human monocytes.

The effect of complement in surface-bound immune complexes on the expression of Fc and C3 receptors in membranes of adherent human monocytes was examined. Monocytes were isolated from mononuclear leucocyte preparations by adherence to substrates containing fibrin, fibrin with immune complexes (containing rabbit IgG antibodies), or fibrin with immune complexes and mouse complement. Fc or C3 receptors on the top or exposed surface of the monocytes were detected by rosette formation with sheep erythrocytes coated with IgG (EA) or IgM and complement (EAC). Monocytes adherent to surface-bound immune complexes exhibited an absence of EA rosette-forming ability without any change in EAC rosettes. This specific loss of Fc receptor function was induced more easily in freshly-isolated monocytes than in cells maintained in suspension culture for up to 7 days. The presence of complement in the immune complex substrates did not reverse the decrease in Fc receptors seen with freshly-isolated or cultured monocytes. Monocytes adherent to immune complexes and complement exhibited a decrease in C3 receptor function. This decrease was more readily induced in cells cultured for three days in the presence of serum than in freshly-isolated monocytes. Experiments performed with EAC or immune complex substrates relatively enriched in C3b or C3bi indicated that C3b in the substrate induced a decrease in monocyte C3b receptors and C3bi led to a decrease in C3bi receptors. No evidence was found for C3d receptors on the human monocytes although these receptors on a subpopulation of human lymphocytes appeared to be altered in a similar fashion.

Antigen-Antibody Complex↗
PubMed1985

Limited proteolysis of a chemically modified third component of human complement, C3, by cathepsin G of human leukocytes.

We have investigated the limited proteolysis of the third component of complement, C3, by a human leukocyte protease, cathepsin G, by using a chemically modified C3, which was prepared by treatment of C3 with methylamine and a fluorescent thiol reagent, N-(dimethylamino-4-methylcoumarinyl)-maleimide (DACM) and was thus named DACM-C3me. Although native C3 was hardly cleaved by cathepsin G, DACM-C3me was cleaved by cathepsin G into three major fragments, which were termed C3c-G (150,000 daltons, 150 kd), C3d-G (25 kd), and C3a-G (10 kd). C3c-G was composed of four disulfide-linked polypeptide chains of 75 kd, 35 kd, and two 25 kd. C3d-G and C3a-G were single-chain fragments derived from the alpha chain. The N-terminal sequence of C3d-G was determined as Thr-Glu-Asp-Ala-Val-, suggesting that cathepsin G released C3d-G by cleaving a Met-Thr peptide bond which is located at 19 residues toward the N-terminal from the cysteinyl residue forming an internal thiolester linkage in native C3. C3d-G, like C3d-K (a C3d fragment produced by the action of plasma kallikrein), was found to have bioactivities such as leukocytosis-inducing and immunosuppressive activities.

Amino Acid Sequence↗
PubMed1997

Bispecific antibodies overcome the opsonin-receptor mismatch of cystic fibrosis in vitro: restoration of neutrophil-mediated phagocytosis and killing of Pseudomonas aeruginosa.

Inflammation and infection associated with bacterial pathogens, primarily Pseudomonas aeruginosa (Pa), are the primary causes of morbidity and mortality for cystic fibrosis (CF) patients. CF patients may be predisposed to these bacterial infections by a defect in phagocytosis due to "opsonin-receptor mismatch," in which a complement receptor (CR1) and an important opsonin (iC3b) are destroyed by proteolytic enzymes. We show that opsonin-receptor mismatch can be mitigated in vitro using a bispecific Ab (bsAb) to cross-link neutrophils via the beta-chain of leukocyte integrins (CD18) to bacterial epitopes or C3d on opsonized Pa. Two chemically cross-linked bsAb were constructed with mAb specific for C3d (or the O-specific side chain of Fisher Devlin Immunotype 1 Pa) and CD18. Using an in vitro model of elastase-mediated opsonin-receptor mismatch, these bsAb specifically enhanced Pa phagocytosis and killing, with the anti-C3d-containing bsAb restoring the levels of phagocytosis to approximately those for the non-elastase-treated opsonic control. These results encourage the further investigation of bsAb as therapeutic agents for bacterial infection in the lungs of CF patients.

Antibodies, Bispecific↗
PubMedDate not supplied

Purification and use of the C3d subunit C3.

A method is described for the preparation of C3d from fresh-frozen CPD plasma. A redissolved EDTA euglobulin precipitate formed from defibrinated plasma is chromatographed on DEAE cellulose. The C3-rich peak is further chromatographed by stepwise elution from hydroxy-apatite. The highly purified C3 and C3b so obtained are then treated with commercial C3b inactivator. G-200 sephadex filtration of this material produces a low molecular weight peak containing C3d greater than 95 per cent purity. The purified C3d, which contains negligible carbohydrate, has a molecular weight of 28,000 (based on SDS polyacrylamide gel electrophoresis). When added at a final concentration of less than 70 mug per ml, it completely inhibits the anti-C3d hemagglutinin reactivity of a potent anti-C3d antiglobulin serum but does not inhibit sera with reactivities against C3c, C4b, and C5b. Two of three rabbits hyperimmunized with purified C3d produced antisera exhibiting only anti-C3d precipitin activity. After absorption of heterologous antibodies, the antisera strongly agglutinated C3d-coated RBC, failed to agglutinate RBC glutinated C3d-coated RBC, failed to agglutinate RBC coated only with C4, and reacted weakly against strongly anti-Rh coated RBC. The latter reactivity was readily absorbed by whole IgG. Preliminary studies with 125I-labeled C3d indicate its potential value in assessing potency of anti-C3d reagents.

Animals↗
PubMed1989

Reduced erythrocyte CR1 (CD 35) receptor function and complement opsonization in factor I-deficient patients is restored by plasma infusion.

Erythrocytes (E) from three factor I-deficient patients were investigated for surface-bound complement factors and CR1 (CD 35) expression and function. The E were coated with C4b, C3b, and factor H. Following plasma infusion or in vitro incubation of the patients' E with normal human serum (NHS) or purified factor I, cell-bound C4b and C3b could no longer be detected. The E now expressed C3d, and factor H was unaffected, indicating that factor H was bound to the C3d part of the C3b molecules, providing the co-factor for effective cleavage of E-bound C3b when purified factor I was added. The binding of monoclonal anti-CR1 antibodies (M710) to the patients' E was markedly reduced compared with control E, and was not normalized by treatment with NHS, probably because covalently bound C3d/factor H interfered with the binding of M710. By contrast, the reduced ability of the patients' E-CR1 to bind complement-opsonized immune complexes (IC) was normalized after plasma infusion. This shows that the impaired CR1 function was acquired and emphasizes the importance of performing functional CR1 assays. Complement opsonization of IC for binding to normal E was severely compromised in the patients' sera due to consumption of factor B and C3. After plasma infusion the opsonization capacity of the patients' sera was restored. Thus, two mechanisms of importance for normal clearance of IC were compromised in factor I-deficient patients: (1) the opsonization of IC for binding to E-CR1, and (2) the capacity of E-CR1 to bind opsonized complexes. Both dysfunctions were temporarily corrected by plasma infusion.

Adult↗
PubMedDate not supplied

Complement activation and adult respiratory distress syndrome during intermittent flow apheresis procedures.

We observed complement (C) activation during intermittent flow apheresis procedures (Haemonetics model 30) in four subjects, two of whom developed adult respiratory distress syndrome (ARDS). Actual C3 conversion during apheresis was illustrated by the finding of significantly elevated C3d levels (p less than 0.05) and of significantly increased alpha-1-antitrypsin/C3 ratios (p less than 0.05) in postapheresis serums. Similarly, marked granulocyte aggregating activity was found in these serums, indicative of the generation of significant amounts of the C-derived anaphylatoxin, C5a or C5a desarginine. A mean decrease of 59.75 percent in neutrophil count during the four procedures suggested sequestration of aggregated granulocytes in the pulmonary vasculature. Moreover, granulocytes activated by apheresis serums induced significant 51Cr leak from cultured human endothelial cells is vitro (p less than 0.001). We conclude that inflammatory C components produced during apheresis procedures may provoke granulocyte aggregation and embolization, leading to plugging of the pulmonary vasculature, and that apheresis-activated granulocytes may induce endothelial cytotoxicity, leading to the capillary leakage syndrome, characteristic of ARDS. Individual variability in C5a generation capacity or alterations in normal C5a clearing mechanisms may account for the low incidence of clinical C activation and true ARDS during apheresis. In these instances, high-dose steroids, which interfere with granulocyte-C interactions, may be beneficial.

Adult↗
PubMed1986

A family with complement factor I deficiency.

A family with inherited factor I deficiency is described. The proband was a 19-year-old Caucasian female with one episode of meningococcal meningitis and one episode of suspected septicaemia of unknown cause. Two obligate and two probable heterozygotes with factor I levels below the lower limit of the reference range were identified. None of these exhibited increased susceptibility to infectious diseases. The inheritance was autosomal codominant. In addition, molecular heterogeneity of factor H in plasma from the proband but not from any other family members was demonstrated by crossed immunoelectrophoresis. The migration of factor H component of fast electrophoretic mobility was retarded by antibodies to C3c and C3d, suggesting the presence of a fluid-phase complex between factor H and excess C3b generated by the uncontrolled activity of the amplification loop.

Adult↗
PubMed1987

[Evaluation of the biocompatibility of a new cellulose membrane by studying the complement system and histamine liberation].

This biocompatibility of the new cellulosic membrane hemophane (HE) is compared to that of cuprophane (CU) in ten maintenance hemodialysis (HD) patients dialyzed on the two types of membranes in randomized order, under otherwise similar technical conditions. Total white blood cell (WBC) and differential counts, blood concentrations of C3a, and C3d and histamine are determined at start of dialysis (TO) and 10, 20 and 180 minutes thereafter. HE is distinct from CU in exerting a minor effect on the generation of C3a, a minor drop of leucocytes during the course of dialysis (P less than 0.01) and also by a lesser increase in blood histamine concentration (P less than 0.05). Histamine liberation is observed on CU together with the generation of anaphylotoxin C3 and with a diminution of circulating basopolymorpho-nuclear cells. According to the variations observed for these three parameters (C3a, leucocytosis and blood histamine concentration), HE appears as being more biocompatible than CU.

Aged↗
PubMedDate not supplied

A study of the breakdown of the third component of human complement (C3).

This paper reports the description of fragments of human C3, purified from aged sera. Sera were incubated at different temperatures (4 degrees, 20 degrees and 37 degrees C) for 8 days or more. At 4 degrees C, the major isolated fragment was C3b which showed a heterogeneity of structure. Under reducing conditions, it liberated a 40,000 dalton fragment which reacted against an anti-C3d and which we called "C3d-like". At 20 degrees C, C3b was partially destroyed to yield the C3c- and C3d-fragments. At 37 degrees C, C3d and C3c were obtained. C3c presented a structural heterogeneity. It was made either of two chains of 75,000 daltons or of three chains with 75,000, 38,000 and 31,000 dalton MW. The comparison of these fragments with those obtained by tryptic hydrolysis led us to propose a pathway to the degradation of C3 and to bring some informations about the structure of this molecule.

Complement C3↗
PubMed1993

The CR2/CD19 complex on human B cells contains the src-family kinase Lyn.

The complement receptor 2 (CR2 or CD21) can be found in non-covalent association with the B lymphocyte specific CD19 complex at the surface of mature human B cells. Upon ligation of the B cell antigen receptor complex (BCR), members of the CR2-CD19 complex may associate with membrane immunoglobulin (mlg). Moreover, CD19 and CD21 ligands, either murine mAb, C3d fragments or Epstein-Barr virus, are known to have profound effects on B cell activation. We here show that CD19 is tightly linked to the non-receptor src kinase Lyn and that the CD19 glycoprotein itself serves as a substrate for a yet undefined serine/threonine kinase present within the complex. In the process of antigen recognition, mlg and the CR2-CD19 complex may bind different sites of a complement-opsonized antigenic particle. We hypothesize that in this process, approximation to the BCR allows CD19-associated Lyn kinase to phosphorylate potential substrates within the antigen-receptor complex, thereby effecting its coupling to the intracellular compartment.

Antigens, CD↗
PubMed1998

Origin and properties of soluble CD21 (CR2) in human blood.

By analysis with a panel of CD21 MoAbs it is shown that a large part of the soluble CD21 in human blood plasma is of the long isoform (CD21L), as judged by comparison with antigen produced by mouse L cells transfected with CD21L-cDNA and reactivity with the restricted CD21 MoAb R4/23. This is compatible with the hypothesis that soluble CD21 in the blood is mainly derived from follicular dendritic cells (FDC). Cells from a human keratinocyte cell line transfected with cDNA from the Burkitt lymphoma cell line Raji also produced soluble CD21L (sCD21L), whereas the short form of sCD21 (sCD21S) was the major component of sCD21 produced by the B lymphoblastoid cell line LICR-LON-HMy and the T cell line Jurkat. Confocal studies of FDC isolated from human tonsil revealed that CD21 was present in the cytoplasm. On gel filtration sCD21 from untreated serum has an apparent size considerably greater than the 130kD found by SDS-PAGE analysis. This may be partly accounted for by the non-globular shape of the molecule, but may also indicate, as reported by others, that in its native state sCD21 is complexed with other proteins. However, no evidence of complexing with sCD23 or C3d could be found.

Animals↗
PubMed1990

The human C3b receptor: function and role in human diseases.

The human C3b receptor (CR1) is a polymorphic glycoprotein which functions regulating the complement system by inhibiting the activation of C3 and C5, through its effect on their convertases, and serving as cofactor for factor I in mediating the degradation of C3b to its inactive fragment C3bi and further to C3d-g. The latter are then ligands for their respective receptors on leukocytes, CR3 and CR2. Additionally, CR1 on erythrocytes endows these cells with the capacity to deliver immune complexes (IC) to the reticuloendothelial system, resulting in their clearance from the circulation. On phagocytes, this receptor participates in the process of endocytosis of foreign particles. There is a wide inherited variation of CR1 expression on erythrocytes (CR1/E) of different individuals. Patients with diseases which feature elevated levels of IC, such as systemic lupus erythematosus, leprosy, and AIDS, have a marked decrease of CR1/E, which may result in an altered clearance. This reduction appears to be related to disease activity, and the most probable site for CR1/E loss is during the transfer of IC to macrophages. Healthy neutrophils increase tenfold their expression of CR1 in response to the effect of chemoattractant peptides. Neutrophils from patients with AIDS display an altered response to stimulation. This defect may be of relevance in the process of endocytosis.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Persistently circulating C3 nephritic factor (C3 NeF)-stabilized alternative pathway C3 convertase (C3 CoF) in serum of an 11-year-old girl with meningococcal septicemia--simultaneous occurrence with free C3 NeF.

Hemolytic complement was found to be absent in the serum of an 11-yr-old girl (R.N.) with meningococcal septicemia. C1, C4, and C2 were slightly decreased, C3 was absent, C5-C9 within the normal range. B levels immunochemically and electrophoretic mobility of B were normal. C3d was greater than 1000% of a pooled EDTA-plasma standard indicating hypercatabolism of C3. On incubation of the patient's serum with normal human serum activation of C3 occurred even in the presence of 0.04 M EDTA. The amount of C3b generated was, however, greater without any chelating agent or in Mg-EGTA. On gel filtration of the serum two protein containing peaks were found to be responsible for activation of C3: the IgG containing peak was able to activate C3 in normal human serum without chelating agents and in Mg-EGTA but not in the presence of EDTA. The IgM-containing peak activated the third component of complement even in the presence of EDTA. The factor responsible for this phenomenon was termed C3 converting factor (C3 CoF). The IgG fraction of the patients serum caused activation of C3 in Mg-EGTA. However, in the presence of EDTA no activation of C3 could be induced even if physiological concentrations of the patients IgG were added to normal human EDTA-plasma. Thus the activity of the patient's IgG did not differ from typical C3 nephritic factor. The decay of C2 in EAC42 intermediates in the presence of the patient's IgG was uninfluenced indicating that it did not carry autoantibody activity against the classical pathway convertase C4b,2a, an activity recently termed NFc.(ABSTRACT TRUNCATED AT 250 WORDS)

Child↗
PubMed1976

The Raji cell radioimmune assay for detecting immune complexes in human sera.

A sensitivie and simple procedure for the detection and quantitation of soluble complement (C)- fixing immune complexes in sera of patients with various disease states has been developed by utilizing C receptors on Raji cells. These cells lack membrane-bound immunoglobulin but have receptors for IgG Fc, C3b, C3d, and possibly with other C proteins. Uptake experiments showed that both aggregated human gamma globulin (AHG) and 7S IgG bound to receptors for IgG Fc; however, AHG reacted with C bound to cells only via receptors for C and this binding was much more efficient than via IgG Fc receptors. AHG was used as an in vitro model of human immune complexes and its uptake by Raji cells was quantitated by 125I-radiolabeled antihuman IgG. The limit of sensitivity of this test was 6 mug AHG/ml serum. The ability of Raji cells to detect AHG in serum depended on the amount of radioactive antibody used and the size of aggregates. The presence of an excess of C somewhat inhibited binding of AHG containing C to Raji cells. The efficient binding of AHG by receptors for C on Raji cells was used for the detection and quantitation of immune complexes in human sera. Raji cells were incubated with sera to be tested and then reacted with excess radiolabeled antihuman IgG; the amount of radioactivity bound to the washed cells was determined and referred to a standard curve of radioactive antibody uptake by cells previously incubated with increasing amounts of AHG in serum. Thereby immune complexes were detected and quantitated in serum hepatitis, systemic lupus erythematosus, vasculitis, subacute sclerosing panencephalitis, dengue hemorrhagic fever, and malignancies.

Antigen-Antibody Complex↗