Glucose-Regulated Protein 78 Autoantibodies Are Associated with Carotid Atherosclerosis in Chronic Obstructive Pulmonary Disease Patients

Abstract

Atherosclerosis prevalence is increased in chronic obstructive pulmonary disease (COPD) patients, independent of other risk factors. The etiology of the excess vascular disease in COPD is unknown, although it is presumably related to an underlying (if cryptic) systemic immune response. Autoantibodies with specificity for glucose-regulated protein 78 (GRP78), a multifunctional component of the unfolded protein response, are common in COPD patients and linked to comorbidities of this lung disease. We hypothesized anti-GRP78 autoreactivity might also be a risk factor for atherosclerosis in COPD patients. Carotid intima-medial thickness (cIMT) was measured in 144 current and former smokers by ultrasound. Concentrations of circulating IgG autoantibodies against full-length GRP78, determined by ELISA, were greater among subjects with abnormally increased cIMT (p , 0.01). Plasma levels of autoantibodies against a singular GRP78 peptide segment, amino acids 246-260 (anti-GRP78 aa 246-260), were even more highly correlated with cIMT, especially among males with greater than or equal to moderate COPD (r s = 0.62, p = 0.001). Anti-GRP78 aa 246-260 concentrations were independent of CRP, IL-6, and TNF-a levels. GRP78 autoantigen expression was upregulated among human aortic endothelial cells (HAECs) stressed by incubation with tunicamycin (an unfolded protein response inducer) or exposure to culture media flow disturbances. Autoantibodies against GRP78 aa 246-260 , isolated from patient plasma by immunoprecipitation, induced HAEC production of proatherosclerotic mediators, including IL-8. In conclusion, anti-GRP78 autoantibodies are highly associated with carotid atherosclerosis in COPD patients and exert atherogenic effects on HAECs. These data implicate Ag-specific autoimmunity in the pathogenesis of atherosclerosis among COPD patients and raise possibilities that directed autoantibody reduction might ameliorate vascular disease in this high-risk population. ImmunoHorizons, 2020, 4: 108-118.

HLA-C and KIR permutations influence chronic obstructive pulmonary disease risk

“…PBMNC were characterized by flow cytometry, using methods detailed elsewhere ( 42 ). PBMNC (effector cells) were used in cytotoxicity assays after 2 days treatment in: (a) media control (baseline); (b) media supplemented with IL-2 at 6000 IU/mL; or (c) wells precoated with mixtures of an anti-KIR monoclonal antibody cocktail (MAB1848 and MAB2238, R&D Systems, Bio-Techne; and HP-MA4, BioLegend).…”

A Method for Checking Recombinant Protein Quality to Troubleshoot for Discordant Immunoassays

“…E. coli strain BL21(DE3) (Thermo Fisher, Waltham, MA), cultured in Luria-Bertani medium, was used as the recombinant host to express human GRP78. Following bacterial cell lysis, centrifugation, and washing, the rGRP78 was purified using nickel columns [30].…”

“…Recombinant human GRP78s purchased from StressMarq Biosciences or produced in the laboratory [30] were used as antigens in ELISA. The proteins were diluted to 0.5 µg/ml in PBS to coat in ELISA plates (100 µl/well) overnight at 4°C.…”