![]() ![]() ![]() Mannose and GlcNAc, whether employed alone or in combination with the AP-blocking anti-factor D, were completely without effect on either the deposition of C3 fragments or the formation of MAC (see Table 1), thereby precluding the LP as a contributor to the activation process. 12 B lymphocytes in the preparation were identified by inclusion of phycoerythrin (PE) -conjugated anti-CD19. The cells were then probed with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-human C3c or anti-human C3d antibody, or FITC-labelled mAb E11, which recognizes a neoepitope expressed by C9 incorporated in MAC. In order to assess the contribution of the LP to complement activation on human B lymphocytes, peripheral blood mononuclear cells (PBMC) from healthy volunteers were incubated with 30% autologous serum in the presence or absence of 50 m m mannose or 50 m m N-acetyl-glucosamine (GlcNAc), to block the activation of mannan-binding lectin and the L-/H-ficolins, respectively, 10, 11 and 5 μg/ml rabbit anti-human factor D (either alone or combined), or 20 m m ethylenediaminetetraacetic acid (EDTA). The purpose of the present study therefore was to establish unequivocally, which calcium-dependent pathway(s) (CP and/or LP) is(are) involved in the activation of complement on human B lymphocytes, and to examine directly their contribution to both C3-fragment deposition and MAC formation. from the observation that calcium chelation reduces slightly the extent of the activation seen, compared to that with untreated serum. The contribution of the CP/LP to complement activation on B lymphocytes has hitherto been established only by inference, i.e. Thus, CR1 appears to play a dual role at the B-lymphocyte surface: as a member of the ternary complex it supports complement activation while, as a free entity, it exerts a regulatory effect as cofactor in C3 fragment degradation. 4, 5, 8 Many of the deposited C3b fragments are subsequently degraded via iC3b to C3dg, in a process dependent on CR1's unique role as cofactor in the factor I-mediated cleavage of iC3b 6, 9 whilst others, by attaching to C3 convertases generated via CP/LP and/or AP, convert these to C5 convertases and thereby initiate MAC formation. 5 C3b fragments generated by the convertase then become covalently attached to CR2 itself and possibly to other acceptor molecules in the locality. 7 The bound C3i captures factor B (B) from the fluid phase to generate the alternative C3 convertase, upon factor D cleavage of B. ![]() 5 While CR2 is capable of initiating the AP in its own right 3, 6 CR1 assists this process (1) by rapidly binding C3i, generated in the fluid phase, for presentation to CR2, and (2) by stabilizing the C3i–CR2 interaction through forming a ternary complex with both molecules. 3 AP activation has been shown to be mediated primarily by CR2 1, 4 as a result of the receptor's ability to bind the hydrolysed form of C3 (C3i). 1, 2 Activation occurs both via calcium-dependent (classical/lectin, CP/LP) and calcium-independent (alternative, AP) pathways, where the latter appears to play a predominant role. Human B lymphocytes, by virtue of their expression of the complement receptors CR1 (CD35) and CR2 (CD21), are capable of activating the complement cascade, resulting in deposition of C3 fragments and membrane attack complex (MAC) formation at the cell surface. A model accounting for the effectiveness of the AP in both preserving C3 fragment integrity and initiating MAC is presented. The extent of MAC formation was also found to be highly pathway dependent, with the AP being about 15-fold more efficient at initiating this process than the CP. A marked difference was observed, however, in the nature of the fragments deposited as a result of CP and AP activation: C3b fragments deposited via the CP were extensively (∼ 90%) converted to the terminal degradation product, C3dg, whereas about 50% of those deposited by the AP persisted as C3b/iC3b fragments. While the total amount of C3 fragments deposited was relatively unaffected by blocking either pathway individually, deposition was virtually abrogated by their combined blockade. The contributions of the classical (CP) and alternative (AP) pathways of complement activation to the spontaneous deposition of C3 fragments and the formation of membrane attack complexes (MAC) on human B lymphocytes, were assessed by incubating peripheral blood mononuclear cells with autologous serum in the absence and presence of selective inhibitors of the AP and CP, respectively. ![]()
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