The glycosylation of the circulating immunoglobulin-gamma (IgG) antibody molecules changes in rheumatoid arthritis. The extent of the changes correlates with the disease severity and reverses in remission. We demonstrate here that the alteration in glycosylation associated with rheumatoid arthritis can create a new mode for the interaction of IgG with complement through binding to the collagenous lectin mannose-binding protein (MBP). Rheumatoid arthritis is associated with a marked increases in IgG glycoforms that lack galactose (referred to as G0 glycoforms) in the Fc region of the molecule and that terminate in N-acetyl glucosamine (GlcNAc). We show, using nuclear magnetic resonance (NMR) and X-ray data, that these terminal GlcNAc residues become accessible for MBP binding. We further demonstrate that multiple presentation of IgG-G0 glycoforms to MBP results in activation of the complement. This suggests that a contribution to the chronic inflammation of the synovial membrane could arise from the localization of the IgG-G0 glycoforms in the affected joint and from resulting activation of complement.

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A receptor binding to the C1q subcomponent of complement has been reported by many workers. In this paper we report for the first time that C1q receptor binds not only to C1q, but also to three other structurally similar ligands, namely mannan binding protein (MBP), conglutinin, and lung surfactant protein (SP-A). All these ligands have been reported to enhance removal of species bound to their globular domain from blood (MBP, conglutinin, C1q) or lung (SP-A) through phagocytosis. One of the possible roles for ligand-receptor binding may be initiation of phagocytosis.

SummaryRituxan, a chimeric anti-CD20 antibody, is the first antibody approved for immunotherapy in non-Hodgkin's B-cell lymphoma and other B-cell lymphoproliferative disorders. Additionally, efficacy of Rituxan treatment has been reported in nonmalignant autoimmune diseases such as rheumatoid arthritis. Crosslinking of CD20 molecules by Rituxan induces therapeutic Bcell depletion. CD20 is a B-lymphocyte specific integral membrane protein, proposed to function as a store-operated calcium channel, which is activated upon receptor-stimulated calcium depletion of intracellular stores. Crosslinking of CD20 by antibodies has been reported to induce a redistribution of CD20 molecules to specialized microdomains at the plasma membrane known as lipid rafts. Here, we report that in the absence of Rituxan, CD20 exhibits a low affinity to lipid rafts. However, binding of Rituxan significantly increases the affinity of CD20 for lipid rafts resulting in its redistribution to a fraction resistant to Triton X-100 solubilization. Furthermore, we demonstrate that disturbing the raft integrity by cholesterol extraction results in dissociation of CD20 from a Triton X-100 resistant fraction followed by complete inhibition of Rituxan-induced calcium entry and apoptosis. The integrity of lipid rafts seems to play a crucial role for CD20-induced caspase activation. These data show, for the first time, that Rituxan-induced translocation of CD20 to lipid rafts is important for increased intracellular Ca 2 + + + + levels and downstream apoptotic signalling.

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Earlier we reported the purification of C1q receptor (C1qR) from U937 cells and human tonsil lymphocytes (Malhotra, R. and Sim, R. B., Biochem. J. 1989. 218: 625) and showed that C1qR interacts with the ligands C1q, mannose-binding protein, conglutinin and lung surfactant protein A (SP-A) (Malhotra, R., Thiel, S., Reid, K. B. M. and Sim, R. B., J. Exp. Med. 1990. 172: 955). C1qR was characterized as an acidic glycoprotein, which, when solubilized, exists as a dimer of Mr 115,000 under non-denaturing conditions. In this article we provide evidence for binding of radioiodinated SP-A to U937 cells and show that binding of radioiodinated SP-A to U937 cells is specific, saturable, salt dependent and is inhibited by purified C1qR and by C1q. The interaction of SP-A with U937 cells was found to up-regulate the surface expression of C1qR. Incubation of SP-A with U937 cells at 37 degrees C for 80 min was found to increase the receptor number per cell. Increase in receptor number was inhibited in the presence of sodium azide and monensin. Incubation of cells with calcium ionophore A23187 induced increased surface expression in the absence of SP-A. The results indicate that interaction of SP-A with U937 cells triggers the expression of an intracellular pool of C1qR.

The pulmonary collectin, lung surfactant protein D (SP-D), plays a role in host defense mediated by the interaction of surface carbohydrates of inhaled pathogens with the lectin domains of SP-D. Respiratory syncytial virus (RSV), the most important viral pathogen of neonates and infants, encodes a highly glycosylated attachment protein, G. Binding studies were performed with G protein from RSV (human, A2 strain) and both native and recombi-nant human SP-D. The effect of recombinant trimeric SP-D lectin domains (rSP-D) on the interaction between RSV and host cells was determined by two methods: an infectivity study with monolayers of Hep-2C cells and in vivo infections in BALB/c mice. These studies show that full-length and recombinant SP-D bind to RSV G protein in a concentration-dependent manner. Both EDTA and mannan inhibited binding of full-length SP-D. These results indicate that binding occurs via the carbohydrate recognition domain of the SP-D. The recombinant SP-D inhibited RSV infectivity in cell culture in a dose-dependent manner, giving 100 % inhibition of replication. Intranasal administration of recombinant SP-D to RSV-infected mice inhibited replication of the virus in the lungs, reducing levels of lung virus by 80 %. These results suggest that SP-D plays a major role in clearing RSV from the lungs. Abbreviations: SP-D: Surfactant protein D CRD: Carbohydrate recognition domain RSV: Respiratory syncytial virus ffu: Focus-forming units