Complement regulatory activity of normal human intraocular fluid is mediated by MCP, DAF, and CD59

Abstract

Purpose: To identify the molecules in normal human intraocular fluid (aqueous humor and vitreous) that inhibit the functional activity of the complement system.

Methods: Aqueous humor and vitreous were obtained from patients with noninflammatory ocular disease at the time of surgery. Samples were incubated with normal human serum (NHS), and the mixture assayed for inhibition of the classical and alternative complement pathways using standard CH(50) and AH(50) hemolytic assays, respectively. Both aqueous humor and vitreous were fractionated by microconcentrators and size exclusion column chromatography. The inhibitory molecules were identified by immunoblotting as well as by studying the effect of depletion of membrane cofactor protein (MCP), decay-accelerating factor (DAF), and CD59 on inhibitory activity.

Results: Both aqueous humor and vitreous inhibited the activity of the classical pathway (CH(50)). Microcentrifugation revealed the major inhibitory activity resided in the fraction with an M(r) >/= 3 kDa. Chromatography on an S-100-HR column demonstrated that the most potent inhibition was associated with the high-molecular-weight fractions (>/=19.5 kDa). In contrast to unfractionated aqueous and vitreous, fractions with an M(r) >/= 3 kDa also had an inhibitory effect on the alternative pathway activity (AH(50)). The complement regulatory activity in normal human intraocular fluid was partially blocked by monoclonal antibodies against MCP, DAF, and CD59. Immunoblot analysis confirmed the presence of these three molecules in normal intraocular fluid.

Conclusions: Our results demonstrate that normal human intraocular fluid (aqueous humor and vitreous) contains complement inhibitory factors. Furthermore, the high-molecular-weight factors appear to be the soluble forms of MCP, DAF, and CD59.

Figure 1

Size determination of complement inhibitory activity of normal human intraocular fluid. Using Micron-3 microconcentrators, pooled aqueous humor (n = 3) and vitreous (n = 3) were separately divided into two fractions and the effect of both fractions (≥ 3 and < 3 kDa) on the classical pathway hemolytic activity was studied. Control for the < 3-kDa fraction consisted of GVB²⁺ buffer spun in a microconcentrator, whereas uncentrifuged buffer was used as the control for the ≥ 3-kDa fraction. Data points are an average of three individual experiments; * P < 0.05.

Figure 2

Elution profiles of human vitreous (—) and molecular weight standards (- - -) from a Sephacryl-S-100 HR column with PBS containing 0.05% CHAPS as the mobile phase. Pooled vitreous (10 samples) was fractionated by size exclusion chromatography, and protein concentration was measured by assessing absorbance at 280 nm. The figure shows the concentration of total protein in various fractions from the column. Fractions 5 to 8, 10 to 13, and 15 to 18 were combined as peaks 1, 2, and 3, respectively.

Figure 3

Effect of anti-MCP, anti-DAF (A), and anti-CD59 (B) antibodies on the complement inhibitory activity. Vitreous peak 1 was treated with monoclonal neutralizing antibodies to MCP, DAF, and CD59 or an irrelevant monoclonal antibody of the same isotype (MOPC-21 or UPC-10). Depleted and nondepleted vitreous peaks 1 were incubated with normal human serum and the lytic capacity of these serum samples were compared in the CH₅₀ assay using sensitized sheep erythrocytes as target cells. Reversal of inhibition of CH₅₀ by these antibodies was observed. The control represents NHS incubated with buffer alone. Results are the means of four individual experiments. Comparisons were made between samples treated with anti-MCP or anti-DAF antibodies and MOPC-21 (A); samples incubated with anti-CD59 and UPC-10 (B); *P < 0.05.

Figure 4

Immunoblotting analysis of sMCP, sDAF, and sCD59 in normal human intraocular fluid. Aqueous humor peak 1 (lanes 1, 3, 5, and 7) was subjected to SDS-PAGE under nonreducing conditions, and transblotted onto a PVDF membrane. 10% SDS-PAGE was run for MCP and DAF, whereas for CD59 a 14% gel was used. Normal human urine (lanes 2, 4, 6, and 8) was used as the positive control. Blots were probed with pooled monoclonal anti-MCP (A), anti-DAF (B), and anti-CD59 (C). (D) The control blot where equivalent concentrations of irrelevant monoclonal antibodies (MOPC-21 and UPC-10) were used. Molecular masses are indicated in the middle of the figure.