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. 2018 Apr 4;13(4):e0195346.
doi: 10.1371/journal.pone.0195346. eCollection 2018.

Naturally Occurring Antibodies Against Serum Amyloid A Reduce IL-6 Release From Peripheral Blood Mononuclear Cells

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Free PMC article

Naturally Occurring Antibodies Against Serum Amyloid A Reduce IL-6 Release From Peripheral Blood Mononuclear Cells

Tadeja Kuret et al. PLoS One. .
Free PMC article

Abstract

Serum amyloid A (SAA) is a sensitive inflammatory marker rapidly increased in response to infection, injury or trauma during the acute phase. Resolution of the acute phase and SAA reduction are well documented, however the exact mechanism remains elusive. Two inducible SAA proteins, SAA1 and SAA2, with their variants could contribute to systemic inflammation. While unconjugated human variant SAA1α is already commercially available, the variants of SAA2 are not. Antibodies against SAA have been identified in apparently healthy blood donors (HBDs) in smaller, preliminary studies. So, our objective was to detect anti-SAA and anti-SAA1α autoantibodies in the sera of 300 HBDs using ELISA, characterize their specificity and avidity. Additionally, we aimed to determine the presence of anti-SAA and anti-SAA1α autoantibodies in intravenous immunoglobulin (IVIg) preparations and examine their effects on released IL-6 from SAA/SAA1α-treated peripheral blood mononuclear cells (PBMCs). Autoantibodies against SAA and SAA1α had a median (IQR) absorbance OD (A450) of 0.655 (0.262-1.293) and 0.493 (0.284-0.713), respectively. Both anti-SAA and anti-SAA1α exhibited heterogeneous to high avidity and reached peak levels between 41-50 years, then diminished with age in the oldest group (51-67 years). Women consistently exhibited significantly higher levels than men. Good positive correlation was observed between anti-SAA and anti-SAA1α. Both anti-SAA and anti-SAA1α were detected in IVIg, their fractions subsequently isolated, and shown to decrease IL-6 protein levels released from SAA/SAA1α-treated PBMCs. In conclusion, naturally occurring antibodies against SAA and anti-SAA1α could play a physiological role in down-regulating their antigen and proinflammatory cytokines leading to the resolution of the acute phase and could be an important therapeutic option in patients with chronic inflammatory diseases.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Levels of anti-SAA and anti-SAA1α antibodies.
(A) Boxplots show the median OD (A450) and IQR for anti-SAA and anti-SAA1α levels in the sera of 300 HBDs (220 male and 80 female). The number of samples in each group is indicated in brackets. Whiskers represent 5th and 95th percentile. Medians between groups were compared using Man Whitney U-test. *p <0.05, ** p <0.01 and *** p <0.001. (B) Shown are medians for anti-SAA and anti-SAA1α levels in HBDs sera based on age distribution (4 groups). The number of samples in each group is indicated in brackets. HBDs, healthy blood donors; SAA, serum amyloid A.
Fig 2
Fig 2. Positive correlation between levels of anti-SAA and anti-SAA1α antibodies.
Correlation between levels of anti-SAA and anti-SAA1α antibodies in sera of 300 HBDs is shown. Spearman coefficient (r), 95% confidence interval (CI) and p value are indicated. Ab, antibody; HBDs, healthy blood donors; SAA, serum amyloid A.
Fig 3
Fig 3
Immunoglobulin avidity of anti-SAA (A) and anti-SAA1α (B) antibodies. Avidity of IgG antibodies against SAA and SAA1α was determined in 6 HBDs samples (3 male, 3 female; as indicated in brackets) using increasing concentration of NaCl in sample dilution buffer. As control, 1% BSA in PBS+0.1% Tween-20 with the same NaCl concentrations, was used. BSA, bovine serum albumin; HBDs, healthy blood donors; PBS; phosphate buffered saline; SAA, serum amyloid A.
Fig 4
Fig 4. Anti-SAA and anti-SAA1α levels in IVIg.
Octagam IVIg was serially diluted in sample dilution buffer (dilution range of 1.56–50 μg/ml) and analyzed for the presence of anti-SAA and anti-SAA1α antibodies using in-house ELISA. IVIg, intravenous immunoglobulin; SAA, serum amyloid A.
Fig 5
Fig 5
Inhibition of IL-6 release by anti-SAA (A, B) and anti-SAA1α (C, D) isolated antibodies on hrSAA- and hrSAA1α- stimulated PBMCs. Dose-dependent effects of isolated anti-SAA and anti-SAA1α antibodies on IL-6 release from hrSAA-treated PBMCs (A, B) and hrSAA1α-treated PBMCs (C, D). Mean±SD from five HBDs is shown for each treatment in panels A and C, while corresponding individual graphs from HBDs are shown in panels B and D. * p<0.05, ** p<0.01. PBMCs from 5 HBDs were isolated and incubated for 5h with the indicated agents. HBDs, healthy blood donors; IVIg, intravenous immunoglobulin; PBMCs, peripheral blood mononuclear cells; SAA, serum amyloid A.

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Grant support

This work was supported by the Slovenian Research Agency (ARRS), National Research Program P3-0314.
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