Presence of plasma complement regulatory proteins clusterin (Apo J) and vitronectin (S40) on circulating immune complexes (CIC)

Abstract

The complement regulatory (CR) proteins clusterin and vitronectin bind to the membrane attack complex (MAC) and thus prevent cytolysis. In this report, we demonstrate the presence of both of these CR proteins on MAC bound to circulating immune complexes (CIC). We measured the amount of clusterin and vitronectin on MAC in plasma, also referred to as soluble MAC (SMAC), as well as on MAC bound to CIC (MAC-CIC), using antibody directed to polymerized C9 in systemic lupus erythematosus (SLE) patients. We observed a strong correlation among the quantities of SMAC and MAC-CIC. The amount of both clusterin and vitronectin associated with MAC-CIC was two- to threefold higher in comparison to the SMAC. Patients with high levels of clusterin and vitronectin demonstrated renal involvement. We hypothesize that these complement regulatory proteins besides regulating the insertion of MAC play other critical roles, in disease pathogenesis.

Fig 1

Concentration of the complement regulatory proteins clusterin and vitronectin bound to circulating immune complexes (CIC) and the fluid phase soluble membrane attack complex (SMAC), measured in systemic lupus erythematosus (SLE) patients. The amount of vitronectin is presented on the left y axis (ng/ml). Clusterin is represented on the right y axis, measured as optical density at 450 nm. The amount of clusterin and vitronectin bound to CIC is two- to threefold higher in every patient, when compared to the vitronectin and clusterin bound to fluid phase SMAC.

Fig 2

Scatter-plot of the amount of membrane attack complex (MAC)-circulating immune complexes (CIC), soluble MAC (SMAC), clusterin (clust)–CIC and clust–SMAC. Monoclonal antibody aE11 directed to a neoepitope on polymerized C9 was used as capture reagent in an enzyme-linked immunosorbent assay (ELISA)-based assay to measure SMAC. A strong correlation was observed between the levels of the clusterin present on SMAC and MAC–CIC.

Fig 3

Complement proteins bound to circulating immune complexes (CIC) were measured in samples from 35 systemic lupus erythematosus (SLE) patients. Levels of C1q, C3, and C4 bound to CIC were measured using enzyme-linked immunosorbent assay (ELISA). The concentration of complement proteins present on CIC is represented as ng/ml. The C3 and C4 bound to CIC are represented on the left y axis while C1q bound to CIC are represented on the right y axis. The C3 levels were the highest on CIC followed by the C1q and C4 amounts.

Fig 4

Quantities of the C5 and membrane attack complex (MAC) present on circulating immune complexes (CIC) as measured using enzyme-linked immunosorbent assay (ELISA)-based assays. A strong correlation was observed between these late complement components. C5 levels were lower than the detection limit of the assay in 16 patients. The amount of C5 is represented on the left axis in ng/ml, while the amount of MAC on CIC is represented on the right axis as OD at 450 nm.

Fig 5

2D sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the circulating immune complexes (CIC) purified from systemic lupus erythematosus (SLE) patients. The first dimension isoelectric focusing (IEF) was carried out using immobilized pH gradient (IPG) immobiline strip pH 3–10 linear gradient. The second-dimension run was performed on 4–12% gradient SDS-PAGE gel. The spots for both heavy and light chains of human globulins are marked. Both the γ chain and µ chain, in addition to ι and κ light chains, were identified by comparison of gel images with National Biological Research Foundation (NBRF) 2D database. The identity of these spots was also confirmed using Western blot analysis and MASS analysis. 2D SDS-PAGE profile of heavy chains in one patient (a) demonstrated the presence of both IgG and IgM containing CIC, while in the other patient CIC were composed mainly of IgM isotype (b). The patient with CIC composed of both immunoglobulin isotype also demonstrated a broader light chain profile in comparison to CIC composed of only IgM isotype.