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. 2012 May;23(5):934-47.
doi: 10.1681/ASN.2011070668. Epub 2012 Jan 26.

Serum amyloid A in uremic HDL promotes inflammation

Thomas Weichhart  1 , Chantal Kopecky, Markus Kubicek, Michael Haidinger, Dominik Döller, Karl Katholnig, Cacang Suarna, Philipp Eller, Markus Tölle, Christopher Gerner, Gerhard J Zlabinger, Markus van der Giet, Walter H Hörl, Roland Stocker, Marcus D Säemann
Affiliations

Serum amyloid A in uremic HDL promotes inflammation

Thomas Weichhart et al. J Am Soc Nephrol. 2012 May.

Abstract

Uremia impairs the atheroprotective properties of HDL, but the mechanisms underlying why this occurs are unknown. Here, we observed that HDL isolated from healthy individuals inhibited the production of inflammatory cytokines by peripheral monocytes stimulated with a Toll-like receptor 2 agonist. In contrast, HDL isolated from the majority of patients with ESRD did not show this anti-inflammatory property; many HDL samples even promoted the production of inflammatory cytokines. To investigate this difference, we used shotgun proteomics to identify 49 HDL-associated proteins in a uremia-specific pattern. Proteins enriched in HDL from patients with ESRD (ESRD-HDL) included surfactant protein B (SP-B), apolipoprotein C-II, serum amyloid A (SAA), and α-1-microglobulin/bikunin precursor. In addition, we detected some ESRD-enriched proteins in earlier stages of CKD. We did not detect a difference in oxidation status between HDL isolated from uremic and healthy patients. Regarding function of these uremia-specific proteins, only SAA mimicked ESRD-HDL by promoting inflammatory cytokine production. Furthermore, SAA levels in ESRD-HDL inversely correlated with its anti-inflammatory potency. In conclusion, HDL has anti-inflammatory activities that are defective in uremic patients as a result of specific changes in its molecular composition. These data suggest a potential link between the high levels of inflammation and cardiovascular mortality in uremia.

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Figures

Figure 1.
Figure 1.
Defective anti-inflammatory potency of HDL from ESRD patients. (A–C) Human monocytes were pretreated with HDL from ESRD patients (ESRD-HDL; n=27) or HDL from healthy controls (control-HDL; n=7) and then stimulated with SAC for 20 hours. The amount of (A) IL-12p40, (B) TNF-α, and (C) IL-10 was determined in the supernatant and is expressed relative to cells stimulated with SAC only. The individual values and the median are shown in the scatter plots. (D) Monocyte-derived dendritic cells were treated with 100 µg/ml HDL from 4 healthy controls (control-HDL) or 10 ESRD patients (ESRD-HDL) and then stimulated with LPS for 20 hours. The expression of the indicated surface markers was evaluated by flow cytometry and is expressed relative to cells stimulated with LPS only. *P<0.05, ** P<0.01, ***P<0.001.
Figure 2.
Figure 2.
Amount of oxidized apoA-I and apoA-II in HDL and its susceptibility to oxidation of ESRD patients and control subjects. HDL was tested from the replica cohort consisting of 14 ESRD patients and 12 controls. The amounts of (A) total apoA-I and -II, (B) oxidized apoA-I and -II, (C) total nonoxidized apoA-I/II and oxidized apoA-I/II, and (D) the ratio of oxidized apoA-I/total apoA-I and oxidized apoA-II/total apoA-II were determined by HPLC analysis from freshly isolated HDL (Fresh), HDL that was stored for 3 days at 4°C (3d), and HDL that was stored for 3 days at 4°C and then chemically oxidized by 2,2'-azobis-2-methyl-propanimidamide (3d+AAPH) for 2 hours. *P<0.05.
Figure 3.
Figure 3.
Gene ontology functional associations of HDL proteins. Total identified HDL proteins from 10 healthy controls and 10 ESRD patients were associated with biologic functions using gene ontology process annotations. HRP, haptoglobin-related protein; TTR, transthyretin; IGHG1, Ig γ-1 chain C region; PLTP, phospholipid transfer protein; A1BG, α-1B-glycoprotein; DBP, Vitamin D-binding protein; AHSG, α-2-HS-glycoprotein; VTN, vitronectin; AGP 2, α-1-acid glycoprotein 2; PEDF, pigment epithelium-derived factor; Ugrp2, secretoglobin family 3A member 1; PCYOX1, prenylcysteine oxidase 1; CP, ceruloplasmin; PBP, platelet basic protein; AGP 1, α-1-acid glycoprotein 1; AZGP1, zinc-α-2-glycoprotein; SP-B, surfactant protein B; CFD, complement factor D.
Figure 4.
Figure 4.
Relative abundance of proteins identified by MS from HDL of ESRD patients and healthy controls. Data are from 10 subjects with ESRD and 10 controls. The relative abundance of the HDL-associated proteins was assessed by the peptide index as described in Concise Methods. *P<0.05.
Figure 5.
Figure 5.
Immunodetection of HDL-associated proteins in both ESRD patients and healthy subjects. Shown are immunoblots of the indicated MS-identified HDL proteins in 10 µg HDL from the replication cohort consisting of 14 ESRD and 12 healthy controls (Table 1).
Figure 6.
Figure 6.
Immunodetection of MS-identified proteins in the plasma or HDL of healthy subjects and patients with CKD3, CKD4, or ESRD. (A) Shown are immunoblots for SP-B, PEDF, SAA, and transferrin in 10 µg whole plasma of six ESRD subjects and five healthy controls. (B–D) Immunodetection of HDL-associated proteins in healthy subjects and patients with CKD3 or CKD4. Immunoblot of (B) SP-B, (C) SAA, and (D) PEDF in 10-µg HDL samples.
Figure 7.
Figure 7.
SAA, but not SP-B, apoC-II, or AMBP, mediates the proinflammatory effects of ESRD-HDL. Human monocytes from three individual donors were treated with the indicated concentrations of SAA, SP-B, AMBP, or apoC-II (in ng/ml) without (A–D) or together with 20 µg/ml SAC (E–H) for 20 hours. The amount of (A and E) IL-12p40, (B and F) TNF-α, (C and G) IL-10, and (D and H) IL-6 was determined in the supernatant and is expressed as means ± SEM. *P<0.05, **P<0.01, ***P<0.001 compared with the respective SAC controls.
Figure 8.
Figure 8.
SAA reverses the anti-inflammatory effects of HDL. (A) Immunoblot of 3.5 µg HDL isolated from plasma of a healthy individual incubated with PBS (Ctrl-HDL) or SAA (SAA-HDL). As control, 5 ng SAA was loaded. (B and C) Human monocytes were pretreated with Ctrl-HDL or SAA-HDL (µg/ml) and then stimulated with 100 ng/ml LPS for 20 hours. The amount of (B) IL-12p40 and (C) TNF-α was determined in the supernatant and is expressed relative to cells stimulated with LPS only (n=3). *P<0.05, **P<0.01, ***P<0.001.
Figure 9.
Figure 9.
SAA in ESRD-HDL correlates with IL-12p40 expression. The expression values of human monocytes stimulated with 20 µg/ml SAC together with 10 µg/ml ESRD-HDL (Figure 1, A–C) were used to correlate SAA levels and the expression of (A) IL-12p40, (B) IL-10, and (C) TNF-α. Pearson’s correlation coefficient r was used as measure for the correlation analysis.
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