Prevalence and correlation of cytokine-specific autoantibodies with epidemiological factors and C-reactive protein in 8,972 healthy individuals: Results from the Danish Blood Donor Study

Abstract

Natural cytokine-specific autoantibodies (c-aAb) have been measured in healthy and diseased individuals, and have been considered as both endogenous immune-regulators and pathogenic factors. Overall, the etiology and potential pathology of c-aAb are still undefined. To further characterize the sero-prevalence, predictors and consequences of high c-aAb levels, we performed the largest population-based study of c-aAb to date, using participants and epidemiological data from the Danish Blood Donor Study. Using a validated bead-based multiplex assay we assessed plasma levels of IL-1α, IL-6, IL-10, IFNα and GM-CSF-specific c-aAb in 8,972 healthy blood donors. Trace levels of at least one of the investigated c-aAb could be measured in 86% of the participants. The presence of high levels of potentially inhibitory c-aAb was generally associated with increasing age and male or female sex, depending on the c-aAb in question. A negative correlation between high levels of IL-6-specific c-aAb and plasma levels of C-reactive protein was observed, indicating cytokine-neutralizing levels of c-aAb in healthy blood donors. There was no substantial correlation between high levels of the five individual c-aAb investigated in this study. These data suggest that autoimmunity against endogenous cytokines is a relatively common phenomenon in healthy individuals, and that predictive factors for high, potentially neutralizing c-aAb levels vary depending on the cytokine in question, and may differ from predictors of general c-aAb presence.

Fig 1. Distribution of MFI for five c-aAb in healthy blood donors.

Plasma samples from 8,972 healthy participants were diluted 10-fold and incubated with cytokine-conjugated MagPlex beads. High levels of c-aAb MFI signals were defined as the 99^th MFI percentile (black lines), as described in materials and methods. White lines indicate median with interquartile range. One screening was performed per sample.

Fig 2. IgG pool c-aAb dilution.

A pool of IgG derived from Danish blood donors (Privigen^®) was serially diluted in assay buffer followed by incubation with cytokine-conjugated MagPlex beads and analysis on a Luminex 100 platform. The IgG pool c-aAb levels were analyzed at a final dilution range of 0.625 to 50 mg/ml. The data shown represent averages with SD for experiments with 3 individual lots of Privigen^©.

Fig 3. Correlation between c-aAb positivity and epidemiological parameters.

Multivariate logistic regression analysis was used to investigate the predictors of high levels of individual c-aAb. Age, sex, obesity, smoking and CRP were used as independent variables and high levels of c-aAb as the dependent variable. For dichotomous variables high levels of c-aAb, female sex, active smoking and obesity were defined as “1”. Data are presented as OR with 95% confidence interval for age, sex, obesity, and smoking (panels A-D) as predictors of high levels of c-aAb. * denotes a p value of < 0.05, **p < 0.01, and *** P ≤ 0.001.

Fig 4. C-aAb as a predictor of CRP.

A: Log-transformed CRP was used as the dependent variable in a series of linear regression analyses, with sex, age, obesity, active smoking and high levels of c-aAb as independent variables. High levels of c-aAb were defined as MFI values above the 99^th percentile. Data are presented as regression coefficients with 95% confidence intervals. B: Participant status of being CRP-low (CRP < 0.1 mg/L) was used as the binary dependent variable in a series of logistic regression analyses, with sex, age, obesity, active smoking and high levels of c-aAb as independent variables. Data are presented as OR in favor of being CRP-low, with 95% confidence intervals. ***denotes a p value of ≤ 0.001.