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

PURPOSE. To identify the molecules in normal human intraocular fluid (aqueo us humor and vitreous) that inhibit the functional activity of the compleme nt system. METHODS. Aqueous humor and vitreous were obtained from patients with noninf lammatory ocular disease at the time of surgery. Samples were incubated wit h normal human serum (NHS), and the mixture assayed for inhibition of the c lassical and alternative complement pathways using standard CH50 and AH(50) hemolytic assays, respectively. Both aqueous humor and vitreous were fract ionated by microconcentrators and size exclusion column chromatography. The inhibitory molecules were identified by immunoblotting as well as by study ing the effect of depletion of membrane cofactor protein (MCP), decay-accel erating factor (DAF), and CD59 on inhibitory activity. RESULTS. Both aqueous humor and vitreous inhibited the activity of the clas sical pathway (CH50). Microcentrifugation revealed the major inhibitory act ivity resided in the fraction with an M-r greater than or equal to 3 kDa. C hromatography on an S-100-HR column demonstrated that the most potent inhib ition was associated with the high-molecular-weight fractions (greater than or equal to 19.5 kDa). In contrast to unfractionated aqueous and vitreous, fractions with an M-r greater than or equal to 3 kDa also had an inhibitor y effect on the alternative pathway activity (AH(50)). The complement regul atory activity in normal human intraocular fluid was partially blocked by m onoclonal antibodies against MCP, DAF, and CD59. Immunoblot analysis confir med the presence of these three molecules in normal intraocular fluid. CONCLUSIONS. Our results demonstrate that normal human intraocular fluid (a queous humor and vitreous) contains complement inhibitory factors. Furtherm ore, the high-molecular-weight factors appear to be the soluble forms of MC P, DAF, and CD59.