Madathilparambil V. Suresh

The connection between C-reactive protein and atherosclerosis.

The connection between C‐reactive protein (CRP) and atherosclerosis lies on three grounds. First, the concentration of CRP in the serum, which is measured by using highly sensitive (a.k.a. ‘hs’) techniques, correlates with the occurrence of cardiovascular disease. Second, although CRP binds only to Fcγ receptor‐bearing cells and, in general, to apoptotic and damaged cells, almost every type of cultured mammalian cells has been shown to respond to CRP treatment. Many of these responses indicate proatherogenic functions of CRP but are being reinvestigated using CRP preparations that are free of endotoxins, sodium azide, and biologically active peptides derived from the protein itself. Third, CRP binds to modified forms of low‐density lipoprotein (LDL), and, when aggregated, CRP can bind to native LDL as well. Accordingly, CRP is seen with LDL and damaged cells at the atherosclerotic lesions and myocardial infarcts. In experimental rats, human CRP was found to increase the infarct size, an effect that could be abrogated by blocking CRP‐mediated complement activation. In the Apob100/100Ldlr‐/‐ murine model of atherosclerosis, human CRP was shown to be atheroprotective, and the importance of CRP‐LDL interactions in this protection was noted. Despite all this, at the end, the question whether CRP can protect humans from developing atherosclerosis remains unanswered.

Leukotriene B4 is a physiologically relevant endogenous peroxisome proliferator-activated receptor-α agonist

Peroxisome proliferator-activated receptors (PPARs) are nuclear transcription factors that play central roles in metabolism and inflammation. Although a variety of compounds have been shown to activate PPARs, identification of physiologically relevant ligands has proven difficult. In silico studies of lipid derivatives reported here identify specific 5-lipoxygenase products as candidate physiologically relevant PPAR-α activators. Subsequent studies show both in vitro and in a murine model of inflammation that 5-lipoxygenase stimulation induces PPAR-α signaling and that this results specifically from production of the inflammatory mediator and chemoattractant leukotriene B4 (LTB4). Activation of PPAR-α is a direct effect of intracellularly generated LTB4 binding to the nuclear receptor and not of secreted LTB4 acting via its cell-surface receptors. Activation of PPAR-α reduces secretion of LTB4 by stimulating degradation of this fatty acid derivative. We also show that the LTB4 precursors leukotriene A4 (LTA4) and 5-hydroperoxyeicosatetrenoic acid (5-HPETE) activate PPAR-α but have no significant endogenous effect independent of conversion to LTB4. We conclude that LTB4 is a physiologically relevant PPAR-α activator in cells of the immune system. This, together with previous findings, demonstrates that different types of lipids serve as endogenous PPAR-α ligands, with the relevant ligand varying between functionally different cell types. Our results also support the suggestion that regulation of inflammation may involve balancing proinflammatory effects of LTB4, exerted through cell-surface receptors, and anti-inflammatory effects exerted through PPAR-α activation.

Role of the Property of C-Reactive Protein to Activate the Classical Pathway of Complement in Protecting Mice from Pneumococcal Infection

C-reactive protein (CRP) is not an acute-phase protein in mice, and therefore, mice are widely used to investigate the functions of human CRP. It has been shown that CRP protects mice from pneumococcal infection, and an active complement system is required for full protection. In this study, we assessed the contribution of CRP’s ability of activating the classical pathway of complement in the protection of mice from lethal infection with virulent Streptococcus pneumoniae type 3. We used two CRP mutants, Y175A and K114A. The Y175A CRP does not bind C1q and does not activate complement in human serum. The K114A CRP binds C1q and activates complement more efficiently than wild-type CRP. Passively administered, both CRP mutants and the wild-type CRP protected mice from infection equally. Infected mice injected with wild-type or mutant CRP had reduced bacteremia, resulting in lower mortality and increased longevity compared with mice that did not receive CRP. Thus, the protection of mice was independent of CRP-mediated activation of the classical pathway of complement. To confirm that human CRP does not differentiate between human and mouse complement, we analyzed the binding of human CRP to mouse C1q. Surprisingly, CRP did not react with mouse C1q, although both mutant and wild-type CRP activated mouse C3, indicating species specificity of CRP-C1q interaction. We conclude that the mouse is an unfit animal for exploring CRP-mediated activation of the classical complement pathway, and that the characteristic of CRP to activate the classical complement pathway has no role in protecting mice from infection.

Human C-Reactive Protein Protects Mice from Streptococcus pneumoniae Infection without Binding to Pneumococcal C-Polysaccharide

Human C-reactive protein (CRP) protects mice from lethality after infection with virulent Streptococcus pneumoniae type 3. For CRP-mediated protection, the complement system is required; however, the role of complement activation by CRP in the protection is not defined. Based on the in vitro properties of CRP, it has been assumed that protection of mice begins with the binding of CRP to pneumococcal C-polysaccharide on S. pneumoniae and subsequent activation of the mouse complement system. In this study, we explored the mechanism of CRP-mediated protection by utilizing two CRP mutants, F66A and F66A/E81A. Both mutants, unlike wild-type CRP, do not bind live virulent S. pneumoniae. We found that passively administered mutant CRP protected mice from infection as effectively as the wild-type CRP did. Infected mice injected with wild-type CRP or with mutant CRP lived longer and had lower mortality than mice that did not receive CRP. Extended survival was caused by the persistence of reduced bacteremia in mice treated with any CRP. We conclude that the CRP-mediated decrease in bacteremia and the resulting protection of mice are independent of an interaction between CRP and the pathogen and therefore are independent of the ability of CRP to activate mouse complement. It has been shown previously that the Fcγ receptors also do not contribute to such CRP-mediated protection. Combined data lead to the speculation that CRP acts on the effector cells of the immune system to enhance cell-mediated cytotoxicity and suggest investigation into the possibility of using CRP-loaded APC-based strategy to treat microbial infections.

C-reactive protein-bound enzymatically modified low-density lipoprotein does not transform macrophages into foam cells

The formation of low-density lipoprotein (LDL) cholesterol-loaded macrophage foam cells contributes to the development of atherosclerosis. C-reactive protein (CRP) binds to atherogenic forms of LDL, but the role of CRP in foam cell formation is unclear. In this study, we first explored the binding site on CRP for enzymatically modified LDL (E-LDL), a model of atherogenic LDL to which CRP binds. As reported previously, phosphocholine (PCh) inhibited CRP-E-LDL interaction, indicating the involvement of the PCh-binding site of CRP in binding to E-LDL. However, the amino acids Phe66 and Glu81 in CRP that participate in CRP-PCh interaction were not required for CRP-E-LDL interaction. Surprisingly, blocking of the PCh-binding site with phosphoethanolamine (PEt) dramatically increased the binding of CRP to E-LDL. The PEt-mediated enhancement in the binding of CRP to E-LDL was selective for E-LDL because PEt inhibited the binding of CRP to another PCh-binding site-ligand pneumococcal C-polysaccharide. Next, we investigated foam cell formation by CRP-bound E-LDL. We found that, unlike free E-LDL, CRP-bound E-LDL was inactive because it did not transform macrophages into foam cells. The function of CRP in eliminating the activity of E-LDL to form foam cells was not impaired by the presence of PEt. Combined data lead us to two conclusions. First, PEt is a useful compound because it potentiates the binding of CRP to E-LDL and, therefore, increases the efficiency of CRP to prevent transformation of macrophages into E-LDL-loaded foam cells. Second, the function of CRP to prevent formation of foam cells may influence the process of atherogenesis.

The protective function of human C-reactive protein in mouse models of Streptococcus pneumoniae infection

Human C-reactive protein (CRP), injected intravenously into mice or produced inside mice by a human transgene, protects mice from death following administration of lethal numbers of Streptococcus pneumoniae. The protective effect of CRP is due to reduction in the concentration of bacteria in the blood. The exact mechanism of CRP-dependent killing of pneumococci and the partners of CRP in this process are yet to be defined. The current efforts to determine the mechanism of action of CRP in mice are directed by four known in vitro functions of CRP: 1. the ability of pneumococcal C-polysaccharide-complexed CRP to activate complement pathways, 2. the ability of CRP to bind to Fcgamma receptors on phagocytic cells, 3. the ability of CRP to bind to immobilized complement regulator protein factor H which can also be present on pneumococci, and, 4. the ability of CRP to interact with dendritic cells. CRP-treated dendritic cells may well be as host-defensive as CRP alone. An interesting condition for the protective function of CRP is that CRP must be given to mice within a few hours of the administration of pneumococci. CRP does not protect mice if given later, suggesting that CRP works prophylactically but not as a treatment for infection. However, full knowledge of CRP may lead to the development of CRP-based treatment strategies to control pneumococcal infection. Also, because CRP deficiency in humans has not yet been reported, it becomes important to investigate the deficiency of the mechanism of action of CRP in CRP-positive individuals.

Activation of hypoxia-inducible factor-1α in type 2 alveolar epithelial cell is a major driver of acute inflammation following lung contusion.

Objective:Lung contusion is a major risk factor for the development of acute respiratory distress syndrome. Hypoxia-inducible factor-1&agr; is the primary transcription factor that is responsible for regulating the cellular response to changes in oxygen tension. We set to determine if hypoxia-inducible factor-1&agr; plays a role in the pathogenesis of acute inflammatory response and injury in lung contusion. Design:Nonlethal closed-chest unilateral lung contusion was induced in a hypoxia reporter mouse model and type 2 cell–specific hypoxia-inducible factor-1&agr; conditional knockout mice. The mice were killed at 5-, 24-, 48-, and 72-hour time points, and the extent of systemic and tissue hypoxia was assessed. In addition, injury and inflammation were assessed by measuring bronchoalveolar lavage cells (flow cytometry and cytospin), albumin (permeability injury), and cytokines (inflammation). Isolated type 2 cells from the hypoxia-inducible factor-1&agr; conditional knockout mice were isolated and evaluated for proinflammatory cytokines following lung contusion. Finally, the role of nuclear factor-&kgr;B and interleukin-1&bgr; as intermediates in this interaction was studied. Results:Lung contusion induced profound global hypoxia rapidly. Increased expression of hypoxia-inducible factor-1&agr; from lung samples was observed as early as 60 minutes, following the insult. The extent of lung injury following lung contusion was significantly reduced in conditional knockout mice at all the time points, when compared with the wild-type littermate mice. Release of proinflammatory cytokines, such as interleukin-1&bgr;, interleukin-6, macrophage inflammatory protein-2, and keratinocyte chemoattractant, was significantly lower in conditional knockout mice. These actions are in part mediated through nuclear factor-&kgr;B. Hypoxia-inducible factor-1&agr; in lung epithelial cells was shown to regulate interleukin-1&bgr; promoter activity. Conclusion:Activation of hypoxia-inducible factor-1&agr; in type 2 cell is a major driver of acute inflammation following lung contusion.

Phosphoethanolamine-complexed C-reactive protein: A pharmacological-like macromolecule that binds to native low-density lipoprotein in human serum

BACKGROUND C-reactive protein (CRP) is an acute phase plasma protein. An important binding specificity of CRP is for the modified forms of low-density lipoprotein (LDL) in which the phosphocholine-binding sites of CRP participate. CRP, however, does not bind to native LDL. METHODS We investigated the interaction of CRP with native LDL using sucrose density gradient ultracentrifugation. RESULTS We found that the blocking of the phosphocholine-binding sites of CRP with phosphoethanolamine (PEt) converted CRP into a potent molecule for binding to native LDL. In the presence of PEt, CRP acquired the ability to bind to fluid-phase purified native LDL. Because purified native LDL may undergo subtle modifications, we also used whole human serum as the source of native LDL. In the presence of PEt, CRP bound to native LDL in serum also. The effect of PEt on CRP was selective for LDL because PEt-complexed CRP did not bind to high-density lipoprotein in the serum. CONCLUSIONS The pharmacologic intervention of endogenous CRP by PEt-based compounds, or the use of exogenously prepared CRP-PEt complexes, may turn out to be an effective approach to capture native LDL cholesterol in vivo to prevent the development of atherosclerosis.

Binding of the monomeric form of C-reactive protein to enzymatically-modified low-density lipoprotein: Effects of phosphoethanolamine

BACKGROUND The 5 subunits of native pentameric C-reactive protein (CRP) are dissociated to generate the monomeric form of CRP (mCRP) in some in vitro conditions, both physiological and non-physiological, and also in vivo. Many bioactivities of mCRP generated by urea-treatment of CRP and of mCRP generated by mutating the primary structure of CRP have been reported. The bioactivities of mCRP generated by spontaneous dissociation of CRP are largely unexplored. METHODS We purified mCRP generated by spontaneous dissociation of CRP and investigated the binding of mCRP to enzymatically-modified low-density lipoprotein (E-LDL). RESULTS mCRP was approximately 60 times more potent than CRP in binding to E-LDL. In the presence of the small-molecule compound phosphoethanolamine (PEt), at 37 degrees C, the binding of mCRP to E-LDL was enhanced <2-fold, while the binding of CRP to E-LDL was enhanced >10-fold. In contrast, PEt inhibited the binding of both CRP and mCRP to pneumococcal C-polysaccharide, another phosphocholine-containing ligand to which CRP and mCRP were found to bind. We have not investigated yet whether PEt alters the structure of CRP at 37 degrees C. CONCLUSIONS Combined data suggest that the targeting of CRP with the aim to monomerize CRP in vivo may be an effective approach to capture modified forms of LDL.

Toll-like receptor-9 (TLR9) is requisite for acute inflammatory response and injury following lung contusion

ABSTRACT Lung contusion (LC) is a significant risk factor for the development of acute respiratory distress syndrome. Toll-like receptor 9 (TLR9) recognizes specific unmethylated CpG motifs, which are prevalent in microbial but not vertebrate genomic DNA, leading to innate and acquired immune responses. TLR9 signaling has recently been implicated as a critical component of the inflammatory response following lung injury. The aim of the present study was to evaluate the contribution of TLR9 signaling to the acute physiologic changes following LC. Nonlethal unilateral closed-chest LC was induced in TLR9 (-/-) and wild-type (WT) mice. The mice were sacrificed at 5, 24, 48, and 72-h time points. The extent of injury was assessed by measuring bronchoalveolar lavage, cells (cytospin), albumin (permeability injury), and cytokines (inflammation). Following LC, only the TLR9 (-/-) mice showed significant reductions in the levels of albumin; release of pro-inflammatory cytokines IL-1&bgr;, IL-6, and Keratinocyte chemoattractant; production of macrophage chemoattractant protein 5; and recruitment of alveolar macrophages and neutrophil infiltration. Histological evaluation demonstrated significantly worse injury at all-time points for WT mice. Macrophages, isolated from TLR9 (-/-) mice, exhibited increased phagocytic activity at 24 h after LC compared with those isolated from WT mice. TLR9, therefore, appears to be functionally important in the development of progressive lung injury and inflammation following LC. Our findings provide a new framework for understanding the pathogenesis of lung injury and suggest blockade of TLR9 as a new therapeutic strategy for the treatment of LC-induced lung injury.