Pentameric CRP attenuates inflammatory effects of mmLDL by inhibiting mmLDL–monocyte interactions
Highlights
► We examine the effects of minimally modified LDL (mmLDL) and CRP isoforms on monocytes in vitro. ► MmLDL co-incubation leads to monocyte activation and adhesion via the activation of Mac-1. ► Pro-inflammatory mmLDL characteristics are blunted by low concentrations of pCRP. ► In contrast mCRP further enhances pro-inflammatory effects on monocytes. ► pCRP reduces the binding of mmLDL to monocytes thus reducing pro-inflammatory effects of mmLDL.
Introduction
C-reactive protein (CRP) is a highly conserved protein of the pentraxin family that consists of five non-covalently linked subunits. It is an acute phase protein and of importance in host defense and inflammation. Recently, CRP has been identified as an independent predictor of cardiovascular events [1], [2] in atherosclerosis. This disease is characterized by lipid deposition in the arterial wall and vascular inflammation leading to wall thickening and formation of atherosclerotic plaques [3].
Another important risk factor for atherosclerosis is an elevated level of plasma cholesterol, in particular low-density lipoproteins (LDL). Several studies have shown that the oxidized modifications of LDL influence the development of atherosclerotic lesions through the inflammatory response [4], and mildly oxidized (mm) LDL in particular has been shown to induce pro-inflammatory activities in monocytic cells [5].
Interestingly, recent studies have shown that these cardiovascular risk factors, LDL and CRP, directly interact with each other. CRP can bind to oxidized [6] and enzymatically modified LDL [7]. It has been suggested that this interaction can opsonize LDL for macrophages [8] and enhance LDL mediated complement activation [9]. The effects of the CRP–LDL interaction have been thoroughly investigated in regards to their effects on macrophage activity and uptake. However, the physiological relevance of the various LDL modifications and their interactions with circulating monocytes still remains elusive. In particular, the LDL interaction with CRP has yet to be sufficiently addressed in cell biological studies that aim to examine the functional consequence of this interaction in monocytes.
In light of a recent report, describing the interaction of oxidized LDL with circulating CRP [10], we aimed to investigate the relevance of this interaction for events involved in the pathological sequelae of plaque formation. Since the increased adhesion of monocytes to the inflamed endothelium and subsequent transmigration into the arterial wall is an early and crucial event in atherogenesis we examined the effects of mmLDL–CRP interaction on monocytes. As conformational changes of the CRP molecule (pentameric CRP (pCRP) dissociates to a monomeric form (mCRP)) can alter its biological effects [11], [12], [13], [14] and LDL binding characteristics [15], [16] we also compared the influence of these different CRP isoforms, p- and mCRP, on pro-inflammatory effects of mmLDL in various biological assays.
In the present study we report that mildly oxidized LDL (mmLDL) exerts pro-inflammatory effects on monocytes that lead to monocyte activation and adhesion. These effects can be blunted by pCRP via blocking mmLDL binding to monocytes Furthermore, we report that these protective pCRP properties are lost with the loss of pentameric symmetry of CRP, which occur in the vicinity of the inflamed endothelium in atherosclerosis [14] and on apoptotic or necrotic cells [17] in the early and late stages of atherosclerosis, as well as on circulating microparticles in myocardial infarction [18]. Our results elucidate the physiological relevance of the interactions of CRP isoforms with mmLDL and monocytes and furthermore confirm the importance of pCRP dissociation to mCRP as a localized inflammatory “activation” mechanism.
Section snippets
Reagents and antibodies
Sodium-azide free native, pCRP and recombinant mCRP were prepared as described previously [19]. mCRP was used in the soluble, citraconylated form, as described by Khreiss et al. [19]. Anti-CRP antibody clone 8 was obtained from Sigma. In awareness of potential problems with bacterial contamination in CRP preparations [20] all reagents were tested for LPS contamination with a Limulus assay (Sigma). The endotoxin level of all protein solutions was below the detection limit (0.125 U/ml or
pCRP and mCRP interact with mm- and maximally (CuSO4) oxidized oxLDL
MmLDL shows a shift in lipid gel electopheresis compared to plasma LDL reflecting the minimal oxidation (Fig. 1A). This was confirmed by the more sensitive conjugated diene measurements for the mmLDL preparations used for the experiments compared to freshly isolated LDL (Fig. 1B). In comparison maximally oxidized (CuSO4) oxLDL showed a marked increase in electrophoretic motility and in content of conjugated dienes.
In order to assess the binding and the direct interaction of oxidized forms of
Discussion
We demonstrate a direct interaction between pCRP/mCRP and mmLDL particles. This interaction strongly influences the pro-inflammatory properties of mmLDL. We thereby describe a novel differential effect on mmLDL by pCRP and mCRP and its potential role in atherosclerosis. Based on our findings we conclude: 1. pCRP and mCRP both bind to mmLDL, but pCRP shows a lower affinity to mmLDL than mCRP. 2. MmLDL has strong pro-inflammatory effects on monocytes and leads to monocyte activation and adhesion
Sources of funding
SUE was supported by a National Health and Medical Research Council (NHMRC) project grant # 472666, and a grant from the German Research Foundation (DFG) # EI 866/1-1, KP is a future fellow of the Australian Research Council.
Disclosures
The authors declare no conflict of interest relating to the manuscript.
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Both authors contributed equally.