Dissociation of pentameric to monomeric C-reactive protein localizes and aggravates inflammation: in vivo proof of a powerful proinflammatory mechanism and a new anti-inflammatory strategy.

BACKGROUND: The relevance of the dissociation of circulating pentameric C-reactive protein (pCRP) to its monomeric subunits (mCRP) is poorly understood. We investigated the role of conformational C-reactive protein changes in vivo. METHODS AND RESULTS: We identified mCRP in inflamed human striated muscle, human atherosclerotic plaque, and infarcted myocardium (rat and human) and its colocalization with inflammatory cells, which suggests a general causal role of mCRP in inflammation. This was confirmed in rat intravital microscopy of lipopolysaccharide-induced cremasteric muscle inflammation. Intravenous pCRP administration significantly enhanced leukocyte rolling, adhesion, and transmigration via localized dissociation to mCRP in inflamed but not noninflamed cremaster muscle. This was confirmed in a rat model of myocardial infarction. Mechanistically, this process was dependent on exposure of lysophosphatidylcholine on activated cell membranes, which is generated after phospholipase A2 activation. These membrane changes could be visualized intravitally on endothelial cells, as could the colocalized mCRP generation. Blocking of phospholipase A2 abrogated C-reactive protein dissociation and thereby blunted the proinflammatory effects of C-reactive protein. Identifying the dissociation process as a therapeutic target, we stabilized pCRP using 1,6-bis(phosphocholine)-hexane, which prevented dissociation in vitro and in vivo and consequently inhibited the generation and proinflammatory activity of mCRP; notably, it also inhibited mCRP deposition and inflammation in rat myocardial infarction. CONCLUSIONS: These results provide in vivo evidence for a novel mechanism that localizes and aggravates inflammation via phospholipase A2-dependent dissociation of circulating pCRP to mCRP. mCRP is proposed as a pathogenic factor in atherosclerosis and myocardial infarction. Most importantly, the inhibition of pCRP dissociation represents a promising, novel anti-inflammatory therapeutic strategy.

Circulating microparticles generate and transport monomeric C-reactive protein in patients with myocardial infarction.

AIMS: Elevated serum C-reactive protein (CRP) following myocardial infarction (MI) is associated with poor outcomes. Although animal studies have indicated a direct pathogenic role of CRP, the mechanism underlying this remains elusive. Dissociation of pentameric CRP (pCRP) into pro-inflammatory monomers (mCRP) may directly link CRP to inflammation. We investigated whether cellular microparticles (MPs) can convert pCRP to mCRP and transport mCRP following MI. METHODS AND RESULTS: MPs enriched in lysophosphatidylcholine were obtained from cell cultures and patient whole-blood samples collected following acute MI and control groups. Samples were analysed by native western blotting and flow cytometry. MPs were loaded with mCRP in vitro and incubated with endothelial cells prior to staining with monoclonal antibodies. In vitro experiments demonstrated that MPs were capable of converting pCRP to mCRP which could be inhibited by the anti-CRP compound 1,6 bis-phosphocholine. Significantly more mCRP was detected on MPs from patients following MI compared with control groups by western blotting and flow cytometry (P = 0.0005 for association). MPs containing mCRP were able to bind to the surface of endothelial cells and generate pro-inflammatory signals in vitro, suggesting a possible role of MPs in transport and delivery of pro-inflammatory mCRP in vascular disease. CONCLUSION: Circulating MPs can convert pCRP to pro-inflammatory mCRP in patients following MI, demonstrating for the first time mCRP generation in vivo and its detection in circulating blood. MPs can bind to cell membranes and transfer mCRP to the cell surface, suggesting a possible mCRP transport/delivery role of MPs in the circulation.

Amyloid plaques dissociate pentameric to monomeric C-reactive protein: a novel pathomechanism driving cortical inflammation in Alzheimer's disease?

Beta-amyloid (Aß) plaques and local inflammation are central to the pathogenesis of Alzheimer's disease. Although an association between circulating pentameric C-reactive protein (pCRP) and Alzheimer's disease has been reported no pathomechanistic link has been established. We hypothesized that Aß plaques induce the dissociation of pCRP to individual monomers (mCRP), which possess strong pro-inflammatory properties not shared with pCRP and localizing inflammation to Alzheimer's plaques. pCRP was incubated with Aß plaques generated in vitro and with non-aggregated Aß(42) peptide. pCRP dissociation to mCRP was found only when co-incubated with Aß plaques. Furthermore, sections of frontal cortex from brains of patients with and without Alzheimer's disease were stained with antibodies specific for mCRP and pCRP. There was significantly more mCRP in the cortex of Alzheimer's disease patients (P ≤ 0.01). In contrast, there was no significant difference in pCRP staining. These findings establish that Aß plaques possess a previously unrecognized potential to dissociate pentameric CRP to monomeric CRP. The existence of mCRP but not pCRP in the brains of Alzheimer's disease patients strongly indicates that this newly described biological effect of Aß plaques is relevant in Alzheimer pathobiology; potentially localizing and amplifying inflammation via the strong pro-inflammatory effects of locally generated mCRP.