Abstract
Purpose :
The complement cascade, best known for its role in host defense, has been shown to be strongly activated by immune complexes in many autoimmune diseases, suggesting that it may contribute to autoimmune dry eye disease (DED) such as Sjögren’s syndrome (SS). The ocular surface is known to have a functional complement system, though the exact site(s) of complement-protein production remain unknown. As part of a larger project interrogating complement’s role in SS-DED, we conducted a pilot study to assess whether complement proteins C3 and C4 were produced by immortalized human conjunctival epithelial cells (HCjE).
Methods :
HCjE were maintained in a 6-well plate in KSFM + 5 ng/ml EGF + 50 µg/ml BPE for 2-4 days. Cell lysates were prepared, and total protein was quantitated (Pierce BCA kit). Samples of lysate (0.625-40 µg of total protein) were loaded in duplicate into 96-well plates, and the amounts of C3 and C4 were measured by Human Complement C3 and C4 ELISA kits (Abcam). To assess C3 and C4 function, we measured formation of soluble C5b-9 (sC5b-9) from HCjE lysates treated with heat-aggregated IgG, a known activator of the classical pathway of complement (CH50 Eq EIA, Quidel). Briefly, 86 µl of IgG were added to 90 µg of HCjE total protein. The activated sample was diluted 1:2, 1:4, and 1:8 prior to performing an enzyme immunoassay targeting sC5b-9, a marker of the activated complement cascade.
Results :
Across all HCjE lysates (n=12), the total protein concentration was 982.1 ± 115.19 µg/ml (mean ± SD). Both C3 and C4 were detected in lysates with C3 being expressed at concentrations approximately 22-fold higher than C4. C3 was quantified at 0.152 ± 0.101 ng/µg of total protein and C4 at 0.007 ± 0.005 ng/µg of total protein. Upon activation by IgG, sC5b-9 was detected at dilutions of 1:2 (6.21 ± 1.20 CH50 U Eq/ml) and 1:4 (6.11 ± 0.45 CH50 U Eq/ml) but not at 1:8.
Conclusions :
HCjE produce complement C3 and C4, suggesting that these proteins may be produced locally at the ocular surface. Supplementing HCjE lysates with IgG results in formation of sC5b-9, confirming that HCjE-derived C3 and C4 are functional and suggesting that HCjE likely produce all proteins (C1 through C9) required for the formation of sC5b-9 via the classical pathway. Further research is needed to confirm these findings in primary cells and to determine complement’s role in SS-DE.
This is a 2021 ARVO Annual Meeting abstract.