Abstract
Purpose :
The complement system is an important part of the immune system reported to contribute to inflammation and local tissue damage in many autoimmune diseases, including Primary Sjögren’s syndrome (pSS). Prior to studying the complement’s role in MGD associated with pSS, we conducted a pilot study to assess whether immortalized human meibomian gland epithelial cells (HMGECs) produce and secrete functional complement proteins C3, C4, and Factor B (FB).
Methods :
HMGECs were seeded at a density of 3.0 x 105 cells per well and cultured in growth media (KSFM + 5 ng/ml EGF + 50 µg/ml BPE) for two days then differentiating media (DMEM/F12 + 10 ng/ml EGF + 50 µM rosiglitazone + 2% heat-inactivated fetal bovine serum) for two days. Cell lysates and supernatants were collected and assessed for full-length complement proteins C3 (Complement C3 Human ELISA Kit; Abcam), C4 (Complement C4 Human ELISA Kit; Abcam) and FB (Complement FB Human ELISA Kit; Abcam). To assess complement protein function, a MicroVue CH50 Eq EIA (Quidel, San Francisco) was performed. Experiments were conducted in three technical and three experimental replicates.
Results :
Complement proteins C3, C4, and FB were readily detected across all HMGEC cell lysates and cell culture supernatants. Complement C3 was the most abundantly detected complement component in both lysates and supernatants. For cell lysates, C3, C4 and FB were quantified at 25.67 ± 0.492, 1.85 ± 0.099 , and 8.92 ± 0.496 pg/ug of total protein, respectively. For supernatants, C3, C4 and FB were detected at concentrations of 73.03 ± 6.30, 0.148 ± 0.013, and 1.07 ± 0.100 ng/ml, respectively. Upon activation of the classical pathway with heat-aggregated IgG (CH50 Eq EIA), sC5b-9 was formed and detected in HMGEC cell lysates.
Conclusions :
To the best of our knowledge, this report represents the first description of C3, C4, and FB being produced by HMGECs. Further, the formation of C5b-9 following stimulation by IgG not only implies that all three proteins are biologically active, but that all complement components, C1 through C9, are produced by HMGECs in quantities sufficient to generate C5b-9, the terminal effector molecule of complement which is responsible for cellular lysis. If these results translate to the ocular surface, autoantibodies, such as those seen in pSS, may activate the complement system in the meibomian glands and contribute to poor gland function.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.