Biomedical subjects

H V Marquart

Publications and source records attributed to H V Marquart.

8 recordsLinked to original sources

PubMed2001

CR2-mediated activation of the complement alternative pathway results in formation of membrane attack complexes on human B lymphocytes.

Normal human B lymphocytes activate the alternative pathway of complement via complement receptor type 2 (CR2, CD21), that binds hydrolysed C3 (iC3) and thereby promotes the formation of a membrane-bound C3 convertase. We have investigated whether this might lead to the generation of a C5 convertase and consequent formation of membrane attack complexes (MAC). Deposition of C3 fragments and MAC was assessed on human peripheral B lymphocytes in the presence of 30% autologous serum containing 4.4 mM MgCl2/20 mM EGTA, which abrogates the classical pathway of complement without affecting the alternative pathway. Blockade of the CR2 ligand-binding site with the monoclonal antibody FE8 resulted in 56 +/- 13% and 71 +/- 9% inhibition of the C3-fragment and MAC deposition, respectively, whereas the monoclonal antibody HB135, directed against an irrelevant CR2 epitope, had no effect. Blockade of the CR1 binding site with the monoclonal antibody 3D9 also resulted in a minor reduction in MAC deposition, while FE8 and 3D9, in combination, markedly reduced deposition of both C3 fragments (91 +/- 5%) and C9 (95 +/- 3%). The kinetics of C3-fragment and MAC deposition, as well as the dependence of both processes on CR2, indicate that MAC formation is a consequence of alternative pathway activation.

B-Lymphocytes↗

PubMed1997

Complement-activating ability of leucocytes from patients with complement factor I deficiency.

Previous studies from this laboratory have shown that normal peripheral blood B cells are capable of activating complement via the alternative pathway (AP), that the activation is associated with complement receptor type 2 (CR2) expression, and that erythrocytes at normal blood levels partially inhibit the activation. The purpose of the present study was to investigate whether factor I (FI) deficiency, which leads to continued formation of the AP convertase (C3bBb) resulting in the consumption of factor B and C3 and large scale generation of C3b fragments, affects the phenotype and/or function of the patients' B cells. Using flow cytometry, peripheral blood leucocytes (PBL) from two FI-deficient patients were investigated for expression of complement receptors and complement regulatory proteins, in vivo-deposited C3 fragments and in vitro complement-activating ability. CR1 levels on B cells were significantly lower in FI-deficient patients than in normal individuals, whereas CR2 levels were found to be reduced, although not to a significant extent. CR1 levels on monocytes and polymorphonuclear leucocytes (PMN) were found to be normal or slightly raised. All leucocyte subpopulations were found to be covered in vivo with C3b fragments. AP activation on B cells from FI-deficient patients in homologous serum was significantly reduced compared with that for normal individuals, whereas no in vitro activation was seen in autologous serum. In addition, the in vivo-bound C3b fragments were degraded to C3d,g when the patients' PBL were incubated in homologous serum containing EDTA. Finally, the patients, erythrocytes failed to exert any inhibition on AP activation in homologous serum.

Antigens, CD↗

PubMed1997

A comparative study of normal B cells and the EBV-positive Burkitt's lymphoma cell line, Raji, as activators of the complement system.

Contradictory reports regarding the ability of complement receptor type 2 (CR2,CD21) on normal B cells to activate complement (C') via the alternative pathway (AP), prompted us to compare the performance of human peripheral blood B cells and the Epstein-Barr virus-positive Burkitt's lymphoma cell line, Raji (a well characterized AP activator) by using flow cytometry. Measured in terms of the membrane deposition of C3 fragments per cell, Raji cells were significantly (6- to 26-fold) more effective as complement activators than were normal B cells. Raji cells were also found to express approximately four to five times as many CR2 as normal B cells. In addition, they distinguished themselves by displaying a greater Ca(2+)-dependent activation, with pooled normal human sera (NHS) as the complement source, and by degrading unprotected C3b fragments from iC3b to C3dg/C3d at a significantly lower rate than the B cells. The Ca2+ dependency of Raji cell activation was found to be partially a result of classical pathway (CP) triggering by specific antibodies in the NHS, although other triggering mechanisms may also be involved. If the influence of these variations between Raji cells and normal B cells was excluded, by relating deposition of anti-C3d-reactive fragments, during AP activation, to the number of CR2 expressed, the difference in performance between the two cell types was found to be insignificant.

Animals↗

PubMed1995

Complement receptor expression and activation of the complement cascade on B lymphocytes from patients with systemic lupus erythematosus (SLE).

It has previously been reported that the expression of the complement receptors, CR1 on erythrocytes and blood leucocytes and CR2 on B cells, is reduced in patients with SLE, and that the reduced expression of CR1 on erythrocytes is related to disease activity. We have earlier demonstrated that normal B cells are capable of activating the alternative pathway (AP) of complement in a CR2-dependent fashion. In this study we have investigated whether disturbances in this activity may be related to the altered phenotype of SLE B cells. Flow cytometry was used to measure expression of complement receptors and regulatory proteins on B cells from SLE patients, as well as the deposition of C3 fragments occurring in vivo or after in vitro AP activation. We have confirmed, for a proportion of the patients studied, reduced expression of CR1 and CR2 on B cells, and shown a consistency between low CR2 expression and reduced in vitro AP activation in the presence of homologous, normal serum. In addition, the B cells, like erythrocytes, bear raised levels of in vivo-deposited C3dg, but not C3b fragments, compared with normal B cells. The erythrocytes from SLE patients were unable to inhibit in vitro AP activation by B cells in homologous serum. Finally, we demonstrated an inverse relationship between SLE disease activity index (SLEDAI) and the expression of complement receptor 2 (CR2) on SLE B cells. Thus, determination of CR2 on B cells may emerge as an additional laboratory tool in the assessment of SLE activity.

Adult↗

PubMed1995

Complement activation by malignant B cells from patients with chronic lymphocytic leukaemia (CLL).

It has previously been reported that the expression of the complement receptors CR1 (CD35) and CR2 (CD21) on malignant B cells in CLL is reduced compared with the expression on normal B cells, while deposition of complement C3 fragments, as a consequence of alternative pathway (AP) activation of complement, is observed on mononuclear cells from patients with B CLL. Following our demonstration that normal B cells are capable of activating the AP of complement in a CR2-dependent fashion, we have chosen to re-examine the complement-activating ability of B CLL cells in relation to their altered phenotype with respect to CR2 and the complement regulatory membrane proteins, CR1, decay accelerating factor (DAF) (CD55) and membrane cofactor protein (MCP) (CD46). Flow cytometry was used to measure expression of complement receptors and regulatory proteins on CD5+ B cells from CLL patients, as well as the deposition of C3 fragments occurring both in vivo and after in vitro AP activation. We have confirmed the reduced expression of CR1 and CR2 on CLL cells and have shown that AP activation in the presence of homologous, normal serum was reduced on B CLL cells compared with normal B cells. The degree of AP activation correlated directly with CR2 expression. In addition, we observed that CLL cells bear in vivo-deposited C3d,g, although at a significantly lower level than normal B cells.

Animals↗

PubMed1994

CR2 is the primary acceptor site for C3 during alternative pathway activation of complement on human peripheral B lymphocytes.

Human cells infected with certain viruses acquire the ability to activate the alternative pathway (AP) of complement. Complement receptor 2 on EBV-infected lymphoblastoid cell lines has been reported to act as the covalent binding site for C3b during AP activation. Using flow cytometry, we investigated the ability of normal human peripheral blood leukocytes to activate the AP in homologous serum. Deposition of C3 fragments was determined as a measurement of complement activation on each of the subpopulations of the blood cells. Incubating human peripheral blood leukocytes with homologous or autologous serum resulted in C3 deposition on B cells and, to a lesser extent, on monocytes and polymorphonuclear leukocytes. Complement activation in the presence of Mg2+ ions and EGTA revealed major involvement of the AP in the case of B cells, and to a lesser extent for other leukocyte populations examined. Preincubation of the leukocytes with polyclonal anti-complement receptor 2 Ab markedly decreased the C3 fragment deposition, as a result of in vitro AP activation, on B cells, indicating that on normal human B cells this receptor may be involved in AP activation. Freshly isolated, normal human B cells also bear low but significant amounts of C3d,g fragments on their membranes, indicating that this AP activation also occurs in vivo. AP activation was partially decreased in the presence of autologous erythrocytes (RBC) suggesting that complement regulatory proteins on RBC play a role in limiting the AP activation in vivo.

Antigens, CD↗

PubMed1994

Interactions of opsonized immune complexes with whole blood cells: binding to erythrocytes restricts complex uptake by leucocyte populations.

The binding of opsonized, fluorescein-labelled bovine serum albumin (BSA)/rabbit anti-BSA complexes (IC) to washed human whole blood cells and isolated leucocytes in the presence of autologous serum was investigated by flow cytometry. In the presence of erythrocytes (E), the IC-binding to granulocytes (PMN), monocytes and lymphocytes was inhibited by up to 46%, 61% and 48%, respectively, depending on the incubation time and the IC-concentration tested. The E-mediated inhibition of the binding to PMN was found to correlate with the average numbers of CR1 per E during the initial 15 min of incubation. Thereafter, the difference between IC binding to PMN in absence and presence of E, decreased in accordance with decreasing binding to E. IC-uptake by PMN induced a drop in side-scatter characteristics, attributable to degranulation, which could be prevented by the presence of E. In contrast to the findings for PMN, the difference between IC-binding to monocytes in the absence and presence of E increased progressively over the 90 min observation period, suggesting that different mechanisms are involved in the late-phase IC uptake by monocytes and PMN. Lymphocytes were heterogeneous with respect to IC binding, the main contributors being B cells. E initially inhibited and then later enhanced the IC binding to lymphocytes, suggesting that E promote B cell uptake of C3d,g-covered IC via CR2. Our findings, that E can restrict the IC uptake by circulating leucocytes, and that an IC-induced degranulation of PMN may be prevented by E, indicate that E may act as a high capacity buffer limiting inappropriate activation of phagocytes by circulating IC.

Antigen-Antibody Complex↗

PubMed1988

Quantification of C3dg/Epstein-Barr virus receptors on human B cells and B cell lines.

The expression of C3dg/Epstein-Barr virus receptors (CR2) on human peripheral blood and tonsillar B lymphocytes and Raji cells was quantified by measuring binding of unlabeled monoclonal anti-CR2 antibody (OKB7 and HB-5) in an indirect immunoenzymatic assay. Scatchard analysis of saturation binding curves revealed that Raji cells on the average express about 22,000 and 17,000 binding sites, with mean affinity constants of 9.9 X 10(10) and 8.7 X 10(10) M-1 for OKB7 and HB-5, respectively. Tonsillar mononuclear cells (TMC) expressed 16,700 and 17,600 binding sites for OKB7 and HB-5, respectively, with a significantly lower affinity constant for HB-5 (3.2 X 10(10) M-1) than for OKB7 (9.0 X 10(10) M-1). On the average 34% of E- peripheral blood mononuclear cells (PBMC) from healthy donors and 49% of TMC expressed the CR2 antigen. When correcting for the fraction of CR2-positive cells, the mean CR2 density was 12,600 on E- PBMC (n = 10) and 34,000 on TMC (n = 4).

Animals↗