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Tears contain the complement regulator CD59 as well as decay-accelerating factor (DAF)
E Cocuzzi1, L B Szczotka, W G Brodbeck
1Department of Pathology and Center For Vision Research, Case Western Reserve University, Cleveland, OH 44106-2622, USA.
Insights
Tear fluid contains membrane inhibitor of reactive lysis (MIRL or CD59), a protein that inhibits complement-mediated cell lysis. CD59 is functional in tears and its levels vary with tear type, suggesting a role in ocular surface immunity.
Area of Science:
- Ocular immunology
- Complement system regulation
Background:
- Decay-accelerating factor (DAF or CD55) regulates C3 activation in tears.
- The presence and function of other complement regulators in tears are not well understood.
Purpose of the Study:
- To investigate the presence and function of membrane cofactor protein (MCP or CD46) and membrane inhibitor of reactive lysis (MIRL or CD59) in human tears.
- To characterize the biochemical properties and functional activity of tear CD59.
Main Methods:
- Two-site immunoradiometric assays to quantify MCP and CD59 levels in tears.
- Western blotting to determine the molecular weight of tear CD59.
- Biochemical assays (Phenyl-Sepharose adsorption, Triton X-114 partitioning) to assess GPI anchor presence.
- Functional assays using complement-activated serum and erythrocytes to evaluate CD59's inhibitory activity.
Main Results:
- MCP was detected at low levels (42 ± 8 ng/ml) in tears.
- CD59 was present at significant levels (222 ± 78 ng/ml), comparable to DAF.
- CD59 concentrations were higher in closed eye tears and lower in reflex tears.
- Tear CD59 lacks GPI anchors but is functionally active, inhibiting C5b-9-mediated lysis.
- CD59 accounts for >85% of C9 inhibitory activity in tear fluid.
Conclusions:
- Tears contain functional MCP and CD59, in addition to DAF.
- CD59 is a major inhibitor of complement-mediated lysis in tears.
- The presence and regulation of these complement inhibitors suggest a role in ocular surface protection.
Abstract:
Previous studies have shown that DAF (or CD55), a cell surface inhibitor of autologous C3 activation, is present in tears and that > 90% of the C3 convertase regulatory activity in tear fluid resides in this protein (Lass JH et al., Invest Ophth Vis Sci 1990; 31:1136-48). This study investigated whether (i) the membrane cofactor protein (MCP or CD46), an additional factor that regulates C3 activation, and (ii) the membrane inhibitor of reactive lysis (MIRL or CD59), a cell surface regulator that acts to prevent formation of the membrane attack complex, are also present in tears, and if so, are functional. Two-site immunoradiometric assays showed that MCP is present in tears at low levels (42 + 8 ng/ml, n = 8) while CD59 is present at levels (222 + 78 ng/ml, n = 14) comparable to those of DAF (325 + 289 ng/ml, n = 12). The concentrations of CD59 (i) were increased two-fold or more in closed eye tears, and (ii) were decreased in reflex tears. Western blotting showed that CD59 protein in tears migrates with an apparent mol. wt similar to membrane CD59 protein. Phenyl-Sepharose adsorption and Triton X-114 partitioning of tear CD59 as well as of tear DAF however, showed that both proteins are devoid of GPI anchors. Assays using cobra venom factor-activated human serum and guinea pig erythrocytes showed that CD59 is functionally active in inhibiting autologous C5b-9-mediated lysis and, under constitutive conditions, accounts for > 85% of the C9 inhibitory activity in tear fluid.